The Parathyroids, Second Edition: Basic and Clinical Concepts

Parathyroids Basic and Clinical Concepts SECOND E D I T I O N

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The Parathyroids Basic and Clinical Concepts SECOND E D I T I O N

Editor-in-Chief

John P. Bilezikian, M.D. Professor of Medicine and Pharmacology Chief, Division of Endocrinology Director, Metabolic Bone Diseases Program Departments of Medicine and Pharmacology College of Physicians and Surgeons Columbia University New York, New York Associate Editors R o b e r t Marcus, M.D. Professor of Medicine Department of Medicine Stanford University School of Medicine Stanford, California and Director, Aging Study Unit VeteransAffairs Medical Center Palo Alto, California

M i c h a e l A. L e v i n e , M.D. Professor of Pediatrics, Medicine, and Pathology Direct~ PediatricEndocrinology TheJohns Hopkins University School of Medicine Baltimore, Maryland

ACADEMIC PRESS A Harcourt Science and Technology Company

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This book is printed on acid-free paper. 0 Copyright © 2001, 1994 by John E Bilezikian, Robert Marcus, and Michael Levine M1 Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without the permission in writing from the publisher. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Inc., 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 A c a d e m i c Press A Harcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, US http://www, academicpress, com A c a d e m i c Press Harcourt Place, 32 Jamestown Road, London NW1 7BY, UK http://www.academicpress.com Library of Congress Catalog Card Number: 00-111700 International Standard Book Number: 0-12-098651-5 PRINTED IN THE UNITED STATES OF AMERICA 01 02 03 04 05 06 MM 9 8 7 6 5 4

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2

1

Contents Contributors

.................................................

Preface to the Second Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface to the First Edition

......................................

ix xv

xvii

Section I: Basic Elements of the Parathyroid System 1.

Parathyroids: Morphology and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . .

Virginia A. LiVolsi 2.

Parathyroid H o r m o n e Biosynthesis a n d Metabolism . . . . . . . . . . . . . . . . . . .

17

Henry M. Kronenberg, E Richard Bringhurst, Gino V. Segre, and John T. Potts, Jr. 3.

Parathyroid H o r m o n e - R e l a t e d Protein: Gene Structure, Biosynthesis, Metabolism, and Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31

William M. Philbrick 4.

Interactions of Parathyroid H o r m o n e a n d Parathyroid H o r m o n e - R e l a t e d Protein with Their Receptors . . . . . . . . . . . . . . . . . . . . . .

53

Michael Chorev, Joseph M. Alexander, and Michael Rosenblatt 5.

Receptors for Parathyroid H o r m o n e a n d Parathyroid H o r m o n e - R e l a t e d Protein: Signaling a n d Regulation . . . . . . . . . . . . . . . . . .

93

Robert A. Nissenson 6.

Nuclear Actions of P T H r P

.......................................

105

Andrew C. Karaplis and M. T. Audrey Nguyen 7.

............................

117

Receptors a n d Signaling for Calcium Ions . . . . . . . . . . . . . . . . . . . . . . . . . . .

127

Signal Transduction of P T H a n d P T H r P

Lee S. Weinstein and Michael A. Levine 8.

Edward M. Brown, Arthur Conigrave, and Naibedya Chattopadhyay 9.

Immunoassays for P T H a n d PTHrP: Clinical Applications . . . . . . . . . . . . . .

143

L. J. Deftos

Section II: Physiological Aspects of the Parathyroid 10. 11.

Physiology of Calcium Homeostasis

................................

167

Edward M. Brown Parathyroid H o r m o n e : Anabolic a n d Catabolic Effects on Bone a n d Interactions with Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Janet M. Hock, Lawrence G. Raisz, and Ernesto Canalis

183

vi

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12.

Cellular Actions of Parathyroid H o r m o n e on Osteoblast and Osteoclast Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199

Physiologic Actions of PTH and PTHrP: I. Skeletal Actions . . . . . . . . . . . . . .

213

Physiologic Actions of PTH and PTHrP: II. Renal Actions

227

Jane E. A ubin and Johan N. M. Heersche

13. 14.

15.

GordonJ. Strewler

E Richard Bringhurst

..............

E n d o c h o n d r a l Bone Formation: Regulation by Parathyroid Hormone-Related Peptide, Indian Hedgehog, and Parathyroid H o r m o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

245

Physiologic Actions of PTH and PTHrP: IV. Vascular, Cardiovascular, and Neurologic Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

261

Physiologic Actions of PTH and PTHrP: V. Epidermal, Mammary, Reproductive, and Pancreatic Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

275

Gino V. Segre and Kaechoong Lee

16.

Thomas L. Clemens and Arthur E. Broadus

17.

JohnJ. Wysolmerski, Andrew E Stewart, and John T. Martin

Section III: Clinical Aspects of Primary Hyperparathyroidism 18.

Parathyroid Growth: Normal and Abnormal . . . . . . . . . . . . . . . . . . . . . . . . .

19.

Molecular Basis of Primary Hyperparathyroidism

2O.

293

A. Michael Parfitt Andrew Arnold

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331

Clinical Presentation of Primary Hyperparathyroidism in the United States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

349

Clinical Presentation of Primary Hyperparathyroidism: Europe . . . . . . . . . .

361

Clinical Presentation of Primary Hyperparathyroidism: India, Brazil, and China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

375

23.

Clinical Course of Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . .

387

24.

Molecular Markers of Bone Metabolism in Parathyroid Disease . . . . . . . . . .

399

ShonniJ. Silverberg and John P. Bilezikian

21. 22.

Jonas Rastad, Ewa Lundgren, and Sverker LjunghaU

Ambrish Mithal, Francisco Bandeira, Xunwu Meng, ShonniJ. Silverberg, Yifan Shi, Saroj K. Mishra, Luiz Griz, Geisa Macedo, Gustav Celdas, Cristina Bandeira, John P. Bilezikian, and D. Sudhaker Rao

25.

ShonniJ. Silverberg and John P. Bilezikian

MarkusJ. Seibel Cytokines in Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . .

411

26.

H i s t o m o r p h o m e t r i c Analysis of Bone in Primary Hyperparathyroidism . . . .

423

27.

Nephrolithiasis in Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . .

437

Inaam A. Nakchbandi, Andrew Grey, Urszula Masiukiewicz, Maryann Mitnick, and Karl Insogna May Parisien, David W. Dempsteg, Elizabeth Shane, and John P. Bilezikian Vanessa A. Klugman, Murray J. Favus, and Charles Y. C. Pak

Contents

28.

Guidelines for the Medical and Surgical M a n a g e m e n t of Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael Kleerekopeg, Robert Udelsman, and Michael A. Levine

29.

Medical M a n a g e m e n t of Primary Hyperparathyroidism John L. Stock and Robert Marcus

30.

Preoperative Localization of Parathyroid Tissue in Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . John L. Doppman

................

459

475 487

31.

The Surgical M a n a g e m e n t of Hyperparathyroidism Samuel A. Wells,Jr. and Gerard M. Doherty

32.

Ectopic Locations of Parathyroid Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . Norman W. Thompson and Paul G. Gauger

499

33.

Parathyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elizabeth Shane

515

34.

Acute Primary Hyperparathyroidism Lorraine A. Fitzpatrick

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527

Multiple Endocrine Neoplasia Type 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . StephenJ. Marx

535

36.

Multiple E n d o c r i n e Neoplasia Type 2 Robert E Gagel

585

37.

Familial Forms of Primary Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . Lawrence Mallette and Robert Marcus

38.

Familial Benign Hypocalciuric Hypercalcemia and Neonatal Severe Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ghada E1-Hajj Fuleihan and Hunter Heath III

35.

...................

451

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601

607

Section IV: Secondary Hyperparathyroidism 39.

The Parathyroids in Renal Disease: Pathophysiology . . . . . . . . . . . . . . . . . . . Kevin J. Martin, Esther A. Gonzdlez, and Eduardo Slatopolsky

625

40.

Renal Bone Diseases: Clinical Features, Diagnosis, and M a n a g e m e n t . . . . . . Jack W. Coburn and Isidro B. Salusky

635

Section V: Special Considerations 41.

Evaluation of the Hypercalcemic Patient: Differential Diagnosis . . . . . . . . . David Heath

663

42.

Hypercalcemia Due to P T H r P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Richard Kremer and David Goltzman

671

43.

O t h e r Local and Ectopic H o r m o n e Syndromes Associated with Hypercalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gregory R. Mundy and Babatunde Oyajobi

691

Genetic Disorders Caused by Mutations in the P T H / P T H r P Receptor: Jansen's Metaphyseal Chondrodysplasia and Blomstrand Lethal Chondrodysplasia . . . . Caroline Silve and HaraldJi2ppner

707

44.

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vii

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45. 46.

Acute M a n a g e m e n t of Hypercalcemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

729

Jean E. Mulder and John P. Bilezikian Primary H y p e r p a r a t h y r o i d i s m a n d O t h e r Causes of Hypercalcemia in Children a n d Adolescents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

743

Emily L. Germain-Lee and Michael A. Levine

Section VI: The Hypoparathyroid States 47.

H y p o p a r a t h y r o i d i s m in the Differential Diagnosis of Hypocalcemia . . . . . . .

48.

Magnesium Deficiency in Parathyroid Function . . . . . . . . . . . . . . . . . . . . . . .

755

Robert W. Downs 763

Robert K. Rude 49.

T h e Molecular Genetics of H y p o p a r a t h y r o i d i s m . . . . . . . . . . . . . . . . . . . . . .

779

R. V. Thakker

50.

A u t o i m m u n e Hypoparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51.

Pseudohypoparathyroidism: Clinical, Biochemical, and Molecular Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

791

Michael P. Whyte 807

Suzanne M. Jan de Beur and Michael A. Levine

52.

T r e a t m e n t of Hypoparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

827

Marc K. Drezner

Section VII: The Parathyroids in Osteoporosis 53.

Parathyroid Function in the N o r m a l Aging Process . . . . . . . . . . . . . . . . . . . .

54.

Parathyroid Function and Responsiveness in Osteoporosis . . . . . . . . . . . . . .

55.

Parathyroid H o r m o n e and Growth H o r m o n e in the T r e a t m e n t of Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

835

Sundeep Khosla, L.J. Melton III, and B. L. Riggs 843

ShonniJ. Silverberg and John P. Bilezikian 853

Robert Marcus Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

865

Contributors Harvard Medical School Boston, Massachusetts 02114

Joseph M. Alexander (53)* Division of Bone and Mineral Metabolism Charles A. Dana and Thorndike Laboratories Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School Boston, Massachusetts 02215

Arthur E. Broadus (261) Section of Endocrinology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut 06510

Andrew Arnold (331) Center for Molecular Medicine and Division of Endocrinology and Metabolism University of Connecticut School of Medicine Farmington, Connecticut 06030

Edward M. Brown (127, 167) Endocrine-Hypertension Division Department of Medicine Brigham and Women's Hospital, and Harvard Medical School Boston, Massachusetts 02115

Jane E. Aubin (199) Department of Anatomy and Cell Biology and Department of Medical Biophysics Faculty of Medicine University of Toronto Toronto, Ontario, Canada M5S 1A8

Ernesto Canalis (183) Department of Medicine The University of Connecticut School of Medicine Farmington, Connecticut 06030; and Departments of Research and Medicine Saint Francis Hospital and Medical Center Hartford, Connecticut 06105

Cristina Bandeira (375) Endocrine Unit Hospital dos Servidores do Estado, and Hospital Agamenon MagalhSes Secretaria da Saude de Pernambuco University of Pernambuco Pernambuco, Brazil

Gustav Celdas (375) University of Brazil 0020-020 Recife-PE, Brazil

Francisco Bandeira (375) Endocrine Unit Hospital Agamenon MagalhSes Secretaria da Saude de Pernambuco University of Pernambuco Pernambuco, Brazil

Naibedya Chattopadhyay (127) Endocrine-Hypertension Division Department of Medicine Brigham and Women's Hospital, and Harvard Medical School Boston, Massachusetts 02115

John P. Bilezikian (349, 375, 387, 423, 729, 843) Departments of Medicine and Pharmacology College of Physicians and Surgeons Columbia University New York, New York 10032

Michael Chorev (53) Division of Bone and Mineral Metabolism Charles A. Dana and Thorndike Laboratories Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School Boston, Massachusetts 02215

E Richard Bringhurst (17, 227) Endocrine Unit Massachusetts General Hospital, and Department of Medicine *Numbers in parentheses indicate the pages on which authors'contributions begin. ix

x

/ Contributors

Thomas L. Clemens (261) Division of Endocrinology and Metabolism University of Cincinnati College of Medicine Cincinnati, Ohio 45267 Jack W. Coburn (635) Departments of Medicine and Pediatrics UCLA School of Medicine, and Nephrology Section West Los Angeles Veterans Affairs Medical Center Los Angeles, California 90095 Arthur Conigrave (127) Endocrine-Hypertension Division Department of Medicine Brigham and Women's Hospital, and Harvard Medical School Boston, Massachusetts 02115 L.J. Deftos (143) Department of Medicine University of California, San Diego, and San Diego VA Medical Center LaJoUa, California 92161 David W. Dempster (423) Department of Pathology College of Physicians and Surgeons Columbia University New York, New York 10032; and Regional Bone Center Helen Hayes Hospital West Haverstraw New York, New York 10993 Gerard M. Doherty (487) Department of Surgery Washington University School of Medicine St. Louis, Missouri 63110 John L. Doppman (475)* Diagnostic Radiology Department National Institutes of Health Bethesda, Maryland 20892

Murray J. Favus (437) Department of Medicine University of Chicago Pritzker School of Medicine Chicago, Illinois 60637 Lorraine A. Fitzpatrick (527) Division of Endocrinology, Metabolism, Nutrition, and Internal Medicine Mayo Clinic and Foundation Rochesteg, Minnesota 55905 Ghada EI-Hajj Fuleihan (607) Calcium Metabolism and OsteoporosisProgram American University of Beirut Medical Center Beirut 113-6044, Lebanon Robert F. Gagel (585)' Division of Internal Medicine University of Texas M.D. Anderson Cancer Center Houston, Texas 77030 Paul G. Gauger (499) Division of Endocrine Surgery Department of Surgery University of Michigan Ann Arbor, Michigan 48105 Emily L. Germain-Lee (743) Division of Pediatric Endocrinology Department of Pediatrics The Johns Hopkins University School of Medicine Baltimore, Maryland 21287 David Goltzman (671) Departments of Medicine and Physiology McGiU University, and Calcium Research Laboratory Royal Victoria Hospital Montreal, Quebec Canada H3A 1A1 Esther A. GonzAlez (625) Division of Nephrology St. Louis University, and Renal Division Washington University St. Louis, Missouri 63110

Robert W. Downs (755) Division of Endocrinology and Metabolism Department of Internal Medicine Virginia Commonwealth University School of Medicine Richmond, Virginia 23298

Andrew Grey (411) Department of Medicine University of Auckland 92019 Auckland, New Zealand

Marc K. Drezner (827) University of Wisconsin-Madison Madison, Wisconsin 53792

Luiz Griz (375) University of Brazil 0020-020 Recife-PE, Brazil

*Deceased

Contributors David Heath (663) Department of Medicine SeUy Oak Hospital Birmingham B29 6JD United Kingdom Hunter Heath III (607) United States Medical Division Eli Lilly and Company Indianapolis, Indiana 46285 Johan N. M. Heersche (199) Faculty of Dentistry University of Toronto Toronto, Ontario, Canada M5G 1G6 Janet M. Hock (183) Department of Periodontics Indiana University School of Dentistry Indianapolis, Indiana 46202

Karl Insogna (411) Department of Medicine Yale University School of Medicine New Haven, Connecticut 06520 Suzanne M. Jan de Beur (807) Division of Endocrinology Department of Medicine and Metabolism The Johns Hopkins University School of Medicine Baltimore, Maryland 2128 7 Harald Jiippner (707) Endocrine Unit Department of Medicine and Children's Service Massachusetts General Hospital, and Harvard Medical School Boston, Massachusetts 02114 Andrew C. Karaplis (105) Division of Endocrinology Department of Medicine Sir Mortimer B. Davis-Jewish General Hospital, and Lady Davis Institute for Medical Research McGiU University Montreal, Quebec Canada H3T 1E2 Sundeep Khosla (835) Mayo Clinic and Foundation Rochest~ Minnesota 55905 Michael Kleerekoper (451) Department of Medicine Wayne State University Detroit, Michigan 48201

Vanessa A. Klugman (437) West Suburban Hospital Oak Park, Illinois 60302 Richard Kremer (671) Department of Medicine McGiU University, and Calcium Research Laboratory Royal Victoria Hospital Montreal, Quebec Canada H3A 1A1 Henry M. Kronenberg (17) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Kaechoong Lee (245) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Michael A. Levine (117, 451,743, 807) Departments of Pediatrics, Medicine, and Pathology The Johns Hopkins University School of Medicine Baltimore, Maryland 21287

Virginia A. LiVolsi (1) Department of Pathology and Laboratory Medicine University of Pennsylvania Medical Center Philadelphia, Pennsylvania 19104 Sverker Ljunghall (361) Global Clinical Sciences AstraZeneca Research and Development S-431 83 M61ndal, Sweden Ewa Lundgren (361) Department of Surgery Endocrine Unit University Hospital S-751 85 Uppsala, Sweden Geisa Macedo (375) University of Brazil 0020-020 Recife-PE, Brazil

/

xi

xii

/ Contributors

Lawrence Mallette (601) Department of Medicine Stanford University School of Medicine, and Aging Study Unit VA Medical Center Palo Alto, California 94304 Robert Marcus (459, 601,853) Department of Medicine Stanford University School of Medicine, and Aging Study Unit VA Medical Center Palo Alto, California 94304 John T. Martin St. Vincent's Institute of Medical Research Fitzroy, VIC3065 Australia Kevin J. Martin (625) Division of Nephrology St. Louis University, and Renal Division Washington University St. Louis, Missouri 63110 Stephen J. Marx (535) Metabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892 Urszula Masiuldewicz (411 ) Department of Medicine Yale University School of Medicine New Haven, Connecticut 06520 L.J. Melton III (835) Mayo Clinic and Foundation Rochesteg, Minnesota 55905 Xunwu Meng (375) Peking University Medical College Hospital 100730 Beijing, China Saroj K. Mishra (375) Department of Surgery Sanjay Gandhi Post Graduate Institute of Medical Sciences 226 O14 Lucknow, India Ambrish Mithal (375) Indraprastha Apollo Hospital 110044 New Delhi, India Maryann Mitnick (411) Mineral Metabolism Laboratory

Yale University School of Medicine New Haven, Connecticut 06520 Jean E. Mulder (729) Department of Medicine College of Physicians and Surgeons Columbia University New York, New York 10032 Gregory R. Mundy (691) Medicine~Endocrinology University of Texas Health Science Center San Antonio, Texas 78284 Inaam A. Nakchbandi (411) Mannheim Faculty of Medicine University of Heidelberg 68135 Mannheim, Germany M. T. Audrey Nguyen (105) Division of Endocrinology Department of Medicine Sir Mortimer B. Davis-Jewish General Hospital, and Lady Davis Institute for Medical Research McGiU University Montrgal, Quebec Canada H 3 T 1E2 Robert A. Nissenson (93) Endocrine Unit San Francisco VA Medical Centeg, and Departments of Medicine and Physiology University of California, San Francisco San Francisco, California 94121 Babatunde Oyajobi (691) Medicine~Endocrinology University of Texas Health Science Center San Antonio, Texas 78284 Charles Y. C. Pak (437) Department of Internal Medicine University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75390 A. Michael Parfitt (293) Division of Endocrinology and Centerfor Osteoporosis and Metabolic Bone Disease University of Arkansas for Medical Sciences Little Rock, Arkansas 72205 May Parisien (423) Department of Pathology College of Physicians and Surgeons Columbia University New York, New York 10032

Contributors William M. Philbrick (31) Section of Endocrinology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut 06520 John T. Potts, Jr. (17) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Lawrence G. Raisz (183) Department of Medicine The University of Connecticut School of Medicine Farmington, Connecticut 06030 D. Sudhaker Rao (375) Bone and Mineral Metabolism Department of Medicine Henry Ford Health System Detroit, Michigan 48202 Jonas Rastad (361) Department of Surgery Endocrine Unit • University Hospital S-751 85 Uppsala, Sweden B. L. Riggs (835) Mayo Clinic and Foundation Rochester, Minnesota 55905 Michael Rosenblatt (53) Division of Bone and Mineral Metabolism Charles A. Dana and Thorndike Laboratories Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School Boston, Massachusetts 02115 Robert K. Rude (763) University of Southern California School of Medicine Los Angeles, California 90089 Isidro B. Salusky (635) Departments of Medicine and Pediatrics UCLA School of Medicine, and Nephrology Section West Los Angeles Veterans Affairs Medical Center Los Angeles, California 90095 Markus J. Seibel (399) Division of Endocrinology and Metabolism Department of Internal Medicine I

University of Heidelberg 69115 Heidelberg, Germany

Gino V. Segre (17, 245) Endocrine Unit Massachusetts General Hospital, and Department of Medicine Harvard Medical School Boston, Massachusetts 02114 Elizabeth Shane (423, 515) Department of Medicine College of Physicians and Surgeons Columbia University New York, New York 10032 Yifan Shi (375) Peking University Medical College Hospital 100730 Beijing, China Caroline Silve (707) INSERM U. 42 6 Facult~ de M~decine Xavier Bichat 75018 Paris, France Shormi J. Silverberg (349, 375, 387, 843) Department of Medicine College of Physicians and Surgeons Columbia University New York, New York 10032 Eduardo Slatopolsky (625) Division of Nephrology St. Louis University, and Renal Division Washington University St. Louis, Missouri 63110 Andrew E Stewart University of Pittsburgh Medical Center Pittsburgh, Pennsylvania 15213 John L. Stock (459) Department of Medicine University of Massachusetts Medical School Worcester, Massachusetts O1605; and Eli Lilly and Company Indianapolis, Indiana 46285 Gordon J. Strewler (213) VA Boston Healthcare System West Roxbury, Massachusetts 02132; and Department of Medicine Harvard Medical School Boston, Massachusetts 02114

/

xiii

xiv

/ Contributors

R. V. Thakker (779) Nuffield Department of Clinical Medicine University of Oxford Headington, Oxford OX3 9DU United Kingdom Norman W. Thompson (499) Division of Endocrine Surgery Department of Surgery University of Michigan Ann Arbor, Michigan 48105 Robert Udelsman (451) ~ Department of Surgery The Johns Hopkins University School of Medicine Baltimore, Maryland 21287 Lee S. Weinstein (117) Metabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases

National Institutes of Health Bethesda, Maryland 20892 Samuel A. Wells, Jr. (487) Department of Surgery Washington University School of Medicine St. Louis, Missouri 63110 Michael P. Whyte (791) Centerfor Metabolic Bone Disease and Molecular Research Shriners Hospitals for Children St. Louis, Missouri 63131; and Division of Bone and Mineral Diseases Washington University School of Medicine at Barnes-Jewish Hospital St. Louis, Missouri 63110 John J. Wysolmerski Yale University School of Medicine New Haven, Connecticut 06520

Current affiliation: Department of Surgery, Yale University, New Haven, Connecticut 06520.

Preface to the S e c o n d Edition The first edition of The Parathyroids was published in 1994. It marked a milestone in the field, carrying on the tradition of Albright and Reifenstein whose 1948 classic The Parathyroid Glands and Metabolic Disease established a key role for the parathyroids in calcium homeostasis and metabolic bone disease. In The Parathyroids, we assembled a body of knowledge that had been accumulating over a 30-year period. The spectacular pace of discovery placed the tiny parathyroid glands at an epicenter of an enormous research effort in metabolic bone disease. The first edition was used widely and filled an essential gap in reference literature. Over the past seven years, as this field has continued to grow, with newer and greater appreciation of the role of the parathyroids in the overall governance of calcium homeostasis, a second edition appears to be particularly apt. The second edition of The Parathyroids contains chapters that have been extensively revised and expanded and many new chapters as well. The chapters d o c u m e n t our new knowledge about virtually every facet of this field and reexamine classic precepts that have stood the test of time. We understand better than ever before the structure and function of the parathyroid h o r m o n e gene and protein as well as the regulatory control of parathyroid h o r m o n e synthesis and secretion, the physiological and pathophysiological aspects of parathyroid hormone-related protein (PTHrP), the mechanisms of parathyroid h o r m o n e and PTHrP action, and the cell biology of PTH and PTHrE With regard to primary hyperparathyroidism, we now appreciate a spectrum of clinical presentations according to where in the world it is detected. Information about the course of primary hyperparathyroidism with and without parathyroid surgery is also new, as are the molecular genetics, biochemical, and histomorphometric dynamics of primary hyperparathyroidism. Advances in preoperative localization of parathyroid tissue and newer operative approaches to parathyroid gland surgery are noteworthy. The hypoparathyroid disorders are understood better with regard to their molecular genetics, pathophysiology, and mechanism. Finally, newer information is available about how parathyroid h o r m o n e can be both a catabolic and anabolic h o r m o n e for bone. This newer knowledge has fueled provocative ideas about the pathophysiology of osteoporosis and is heralding a new era in the therapeutics of osteoporosis. The second edition, thus, is still a comprehensive examination of basic and clinical concepts of the parathyroids. It is intended for students, teachers, practitioners, and investigators. In light of these newer developments in the field, the second edition has been reorganized to provide the reader with information that follows best the changing scientific logic. Fifty-five chapters are divided into seven sections. In Section I, nine chapters are devoted to basic concepts of parathyroid h o r m o n e and PTHrP, covering embryology, anatomy, and pathology of parathyroid tissue; gene structure, biosynthesis, and metabolism of PTH and PTHrP; receptors, nuclear targeting, and signal transduction for PTH, PTHrP, and calcium ion; and a comprehensive review of the immunoassays for PTH and PTHrE In Section II, eight chapters are devoted to the physiological aspects of calcium metabolism and the anabolic and catabolic effects of PTH at the level of bone and bone cells. Five chapters cover in detail all aspects of PTH and PTHrP with regard to traditional and nontraditional target organs. In Section III, 21 chapters are devoted to clinical aspects of primary hyperparathyroidism. Chapters on the growth of normal and abnormal parathyroid cells and the molecular genetics of primary hyperparathyroidism are followed by three chapters that describe different clinical presentations of primary hyperparathyroidism t h r o u g h o u t the world. Detailed coverage of bone dynamics and stone disease is followed by information relevant to the medical and surgical m a n a g e m e n t of primary hyperparathyroidism. Also covered are other presentations of primary

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/ Preface to the Second Edition hyperparathyroidism: as a malignancy, as an acutely hypercalcemic disorder, and in association with the multiple endocrine syndromes I and II. A chapter on familial hypocalciuric hypercalcemia completes this section. Two chapters in Section IV cover the parathyroids in renal disease. These are followed by six chapters in Section V that focus on special considerations. The first three chapters review the differential diagnosis of hypercalcemia, including syndromes caused by the local and systemic production of hypercalcemic factors such as PTHrR Jansen's disease, the acute m a n a g e m e n t of hypercalcemia, and hypercalcemia in children are considered in separate chapters. In Section VI, the hypoparathyroid states are reviewed in six chapters, which cover molecular, ionic, and immunological defects in the hypoparathyroid states and the role of hypoparathyroidism in the differential diagnosis of hypocalcemia. In Section VII, the role of parathyroid function in osteoporosis is covered in three chapters describing changes in parathyroid function with aging, parathyroid function and responsiveness in osteoporosis, and the potential of parathyroid h o r m o n e as a therapy for osteoporosis. As was true for the first edition, we recognize that this book is not likely to be read from cover to cover. Thus, each chapter has been written to provide a body of knowledge that can stand alone. The chapters, however, are also liberally cross-referenced to help the reader continue reading more directly related material if desired. The first edition of this book was dedicated to the m e m o r y of Gerald D. Aurbach, whose untimely and tragic death was its catalyst and inspiration. Virtually all the principal authors of the first edition had known and worked with Jerry. We r e m e m b e r e d him then for his "wisdom, scientific acumen, investigative skills, and daring insights." We r e m e m b e r him now in m u c h the same way. We were and still are mindful of the role Jerry had not only for us but also for the entire field, which he helped to create. We were his scientific progeny. It is 10 years since Jerry's death, virtually a generation in the world of science. As a result, some of the leading figures in this field have e m e r g e d without having had the special privilege of working with or knowing Jerry. The authorship of the second edition has been broadened, therefore, to include the very best in our field, recognizing that although Jerry's legacy is still alive, it now extends to an even broader cross section of the field. We wish to thank Jasna Markovac of Academic Press, who was instrumental in both the first and current editions of The Parathyroids. Mica Haley of Academic Press was also most helpful in attending to the many details required to ensure a rapid t u r n a r o u n d time to final publication. Enjoy the book.

John P. Bilezikian Robert Marcus Michael A. Levine

Preface to the First E d i t i o n One of us (JPB), d r e a m e d of this book about five years ago. It seemed then that advances in our knowledge of the parathyroids represented nothing less than a 30-year revolution of spectacular progress. We gained knowledge over this period at an explosive pace with a concomitant new appreciation of the basic and clinical ramifications of these four tiny endocrine glands. The major secretory product, parathyroid h o r m o n e (PTH), was isolated, sequenced, assayed, and cloned. PTH became one of the first h o r m o n e s to be shown to utilize cAMP as a second messenger. Regulation of PTH synthesis and secretion by calcium and 1,25-dihydroxyvitamin D was appreciated, as well as the cellular effects of PTH on its two major target organs, bone and kidney. The discovery of parathyroid hormone-related protein (PTHrP) as a cause of hypercalcemia of malignancy and a more general appreciation of PTHrP and PTH as polypurpose factors with many diverse biological effects represent exciting new advances in our field. The recent cloning of a bona fide receptor for both PTH and PTHrP is a tremendous achievement, as is the thinking that both PTH and PTHrP may utilize more than one second messenger pathway, and perhaps interact with more than one receptor. At the clinical level, we have seen a remarkable evolution in the presentation of primary hyperparathyroidism and are beginning to understand molecular features of this disease. Pseudohypoparathyroidism is now appreciated, in its classical form, to be a G protein deficiency disease. A u t o i m m u n e and molecular features of hypoparathyroidism have been identified and studied. New knowledge of the pathophysiology of secondary hyperparathyroidism associated with renal failure has had direct impact on m a n a g e m e n t and clinical outcome. PTH is now appreciated to have important anabolic properties in bone that may have implications for its use as a therapeutic agent in osteoporosis. This incomplete summary argues persuasively for how fast and how far this field has advanced. This is not to say that we were in the dark ages before Aurbach isolated parathyroid h o r m o n e . Certainly, it was Fuller Albright who in 1948 correctly pointed out that "back in the dark ages of endocrinology, in the early 1920s, hyperparathyroidism was an u n k n o w n fact." It was also Albright who r e m i n d e d us of the work of Sandstrom, who in 1880, 40 years before the first known cases of hyperparathyroidism wrote, "The existence of a hitherto u n k n o w n gland in animals that have so often been a subject of anatomical examination called for a t h o r o u g h approach to the region a r o u n d the thyroid gland even in man. Although the probability of finding something hitherto unrecognized seemed so small that it was exclusively with the purpose of completing the investigations rather than with the hope of finding something new that I began a careful examination of this region, so m u c h the greater was my astonishment therefore when in the first individual I examined, I found on both sides at the inferior b o r d e r of the thyroid gland an organ of the size of a small pea, which j u d g i n g from its exterior, did not appear to be a lymph gland, or an accessory thyroid gland, and u p o n histological examination showed a rather peculiar structure." The first chapters on the parathyroids were indeed written by Albright and a band of spectacular clinical investigators of the 1920s, 1930s, and 1940s. These chapters are recorded in the Albright and Reifenstein classic The Parathyroid Glands and Metabolic Disease. We r e c o m m e n d this insightful 45-year-old book as important and provocative reading. The Parathyroids is designed to follow the Albright and Reifenstein text. Certainly all endocrinology reference texts routinely include a section on the subject matter of this book. O t h e r texts that are more focused on calcium metabolism provide more information than the standard endocrinology texts on the parathyroids. However, there is no book that is exclusively devoted to a comprehensive examination of basic and clinical concepts

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/ Preface to the First Edition of the parathyroids. As indicated by the size and scope of The Parathyroids, it is clear that a book devoted to this subject is worthy and long overdue. It is time for such a book to stand on the endocrine shelf near its anatomical partner, the thyroid gland, which in Werner and Ingbar's The Thyroid has had its own literary repository since 1955. This book is intended for students, teachers, practitioners, and investigators of this field. It covers in a current and concise yet complete m a n n e r virtually all that we know about the parathyroids. Thus, it is both a basic and a clinical text. The 51 chapters are divided into a presentation of basic knowledge of the parathyroids and the clinical disorders associated with dysfunction of these glands. Section I, Basic Concepts of the Parathyroids, consists of 22 chapters. Chapters 1-7 cover the embryology, anatomy, and pathology of the parathyroid glands; calcium homeostasis; regulation of parathyroid hormone by dietary calcium and vitamin D; anabolic and catabolic effects of parathyroid hormone; cellular actions of parathyroid hormone on osteoblast and osteoclast function; autocrine and paracrine functions of parathyroid tissue; and the chemistry and biology of parathyroid h o r m o n e secretory protein. In Chapters 8-16, parathyroid hormone is considered with respect to the discovery by Aurbach of one of its second messengers, cAMP; regulation of it biosynthesis and metabolism; the parathyroid hormone gene; structure-function analysis of parathyroid hormone and parathyroid hormone-related protein; measurement of parathyroid hormone in the circulation; parathyroid hormone and parathyroid hormone-related protein as polyhormones; receptors for parathyroid hormone and parathyroid hormone-related protein; G proteins as transducers of parathyroid hormone action; biochemical mechanisms of parathyroid hormone action. The book proceeds in Chapters 17-20 to a consideration of PTHrP: its structure, physiological processing, and actions; its causative role in hypercalcemia of malignancy; it skeletal and renal actions; and its measu r e m e n t in the circulation. Other causes of hypercalcemia, besides PTHrP, and the management of PTH and PTHrP-dependent hypercalcemia complete this section (Chapters 21-22). Section II, Clinical Concepts of the Parathyroids, begins with an 18-chapter section on primary hyperparathyroidism (Chapters 23-40). This segment is a full exploration of the hyperparathyroid state from theoretical aspects of parathyroid cell growth to the molecular basis of primary hyperparathyroidism. A discussion of the spectrum of parathyroid tumors leads to a consideration of its modern clinical presentations and the course of primary hyperparathyroidism. The change in clinical presentation of primary hyperparthyroidism from a disease of bones and stones and groans to a relatively asymptomatic disorder does not lose sight of a major clinical complication, nephrolithiasis, which is still seen in patients on a regular basis. A chapter devoted to newer markers of bone turnover in primary hyperparathyroidism is followed by a discussion of the histomorphometric features of the disease. Medical and surgical management of primary hyperparathyroidism and the role of preoperative localization techniques are covered completely. Unusual manisfestations of primary hyperparathyroidism include separate discussions of parathyroid carcinoma and acute primary hyperparathyroidism. The MEN syndromes I and II focus on the parathyroids, as does the chapter on familial hypocalciuric hypercalcemia. In Chapters 41 and 42, the parathyroids in renal disease are reviewed with respect to pathophysiology, clinical profile, and management. Chapters 43-47 cover the hypoparathyroid states with respect to differential diagnosis, autoimmune etiologies, molecular genetics, and a special consideration of the clinical, biochemical, and molecular features of pseudohypoparathyroidism. A separate chapter is devoted to the therapy of hypoparathyroidism. The last four chapters of the book, Chapters 48-51, cover unusual aspects of the parathyroids: parathyroid function in the pathophysiology of osteoporosis and parathyroid hormone as a potential therapy of osteoporosis. Parathyroid functions in Paget's disease of bone and in magnesium deficiency complete the treatise. We recognize that few readers will read this book from cover to cover, although many of the chapters are closely interrelated. In order to permit virtually all chapters to "stand alone" but also to be connected to the rest of the book, we have liberally included cross-references to other chapters where appropriate. The reader can thus easily refer to other chapters for more information on a given subject. This design also necessarily calls for some interdigitation between chapters so that the reader in not always required to refer to another chapter but, rather, can get a brief summary in the chapter being read of an area that is covered more completely elsewhere.

Preface to the First Edition If it was true that we n e e d e d a b o o k on this subject five years ago w h e n the idea was first germinating, why did it take so long to get it d o n e a n d what was the impetus for finally accomplishing the task? T h e first of these two questions has a simple answer. Ideas for books are r a t h e r easy to develop but it is quite a n o t h e r m a t t e r to mobilize an army of over 90 experts to bring that idea to reality. As is true for so m a n y things, this idea was p u t on the shelf to be a d m i r e d for its own sake a n d to be c o m p l e t e d later. T h e mobilizing impetus a n d the inspiration for this effort eventually did come. Regrettably, it came in the f o r m of a tragic event in o u r lives, the death of Gerald D. Aurbach. T h e death of Jerry on a street in Charlottesville, Virginia, on N o v e m b e r 4, 1991, was r a n d o m , senseless, a n d violent. At 64 years of age, J e r r y was still alive with love for his work, his family, a n d his friends. In a m o m e n t , we suddenly lost a m a n who g u i d e d the very definition of o u r field for over 30 years. We lost a m a n who was o u r t e a c h e r a n d o u r friend. We lost a brilliant scientist who was involved in most of the major advances in this field over the past three decades. We lost a m a n who trained an e x t r a o r d i n a r y n u m b e r of us for successful careers in basic a n d clinical investigation of the parathyroids. We lost a gentle m a n who consistently b r o u g h t out the best of us. A s u m m a r y of the m a n y a c c o m p l i s h m e n t s that came f r o m Jerry's laboratory a n d the trainees, collaborators, a n d associates who worked with h i m is depicted in the time-line on pages xxvi-xxvii of this book. It is an e x t r a o r d i n a r y legacy. T h e two IN MEMORIA, by Bilezikian (Journal of Bone and Mineral Research 7:ix-x, 1992) a n d by Potts a n d Spiegel (Journal of Clinical Endocrinology and Metabolism 75:1386-1388, 1992), speak volumes to his career, to his accomplishments, a n d to his persona. In a flash, the d r e a m shelved in the recesses of consciousness a n d relegated to "when I get to it" b e c a m e an u r g e n t need. The Parathyroids h a d to be written in the m e m o r y a n d h o n o r of Gerald D. Aurbach, a n d it s e e m e d altogether fitting that it be written by those who were close to Jerry. We who knew h i m so well a n d respected h i m so m u c h would write a volume for the field. Virtually all of the principal authors of this text fit into that category. Maurice Attie, who also belongs in this book, was tragically killed in a bicycle accident in Philadelphia only a few m o n t h s after Jerry's death. We r e m e m b e r Maurice a n d wish that he too were still with us. It is e x t r a o r d i n a r y that a b o o k designed to be as c o m p r e h e n s i v e as this could be assembled by a collective a u t h o r s h i p whose scientific roots were established by Jerry. His contributions to this field are r e p r e s e n t e d not only by his science b u t also by his scientific p r o g e n y who are the n e x t g e n e r a t i o n of investigators to study a n d write a b o u t it. We took up this task with time in mind. The Parathyroids h a d to be published with a short lag time because the b o o k is a timely dedication to Jerry's memory. It h a d to be published soon because this field is in "fast forward" a n d if one used the n o r m a l publication time for a b o o k of this m a g n i t u d e , it would r u n the risk of rapidly b e c o m i n g outdated. To the credit a n d thanks to all the authors, virtually all 51 chapters were submitted within a six-month p e r i o d of time. T h e dedication of the authors to this task is gratefully acknowledged by us. We also are grateful to J a s n a Markovac of Raven Press, who h e l p e d to ensure that the process ran as efficiently as possible a n d whose efforts also were i n s t r u m e n t a l in ensuring a rapid t u r n a r o u n d time to final publication.

John P. Bilezikian Robert Marcus Michael A. Levine

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Th e Parathyroids Basic and Clinical Concepts

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Para

thyr o l"ds

Morphology and Pathology

VIRGINIA A. LIVOLSI Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Centeg, Philadelphia, Pennsylvania 19104

INTRODUCTION

The morphologic abnormalities seen in the parathyroid glands are predominantly those related to hyperfunction, i.e., primary hyperparathyroidism. Thus the focus here is on this aspect of parathyroid pathology, because almost all surgical specimens of parathyroid lesions are derived from patients with hyperparathyroidism. Because morphologic abnormalities are an important factor in the surgical treatment of this disease, a review of parathyroid embryologic development, anatomy, and normal histology is included. A brief discussion of parathyroid pathology in h y p o p a r a t h y roidism is also included, as is discussion of the pathology of the glands in humoral hypercalcemia of malignancy.

D E V E L O P M E N T O F P A R A T H Y R O I D GLANDS In the 8- to 10-mm embryo, the parathyroids begin to develop from the third and fourth branchial pouches. The third branchial pouch gives rise to the thymus and the parathyroid complex. The parathyroids migrate to and remain at the lower poles of the thyroid. Thus, in the usual case, the inferior parathyroids migrating with the thymus come to rest below the parathyroid derived from branchial pouch four (1). Embryologic studies in animals have demonstrated that ablation of the ventral half of the third branchial arch leads to nonformation of the upper parathyroid gland (2). Hoxa3 mutant homozygotes show defects in development and migraThe Parathyroids, Second Edition

tion pathways of thymus, thyroid, and parathyroid glands; the molecular events underlying the actions of the Hoxa3 genes remain to be determined (3). The fourth branchial pouch, or the fourth-fifth pharyngeal complex, gives rise to the superior parathyroid glands and via the ultimobranchial body to the parafollicular or C cells in the lateral thyroid. The superior parathyroids lie adjacent to the upper poles of the thyroid.

A N A T O M Y O F P A R A T H Y R O I D GLANDS Both the number and the location of the parathyroid glands vary in normal individuals. Variation in location of the glands can lead to problems during surgical exploration of the neck. For example, there may be difficulty in locating the diseased, abnormal parathyroid tissue in patients with hypercalcemia; conversely, surgery on the neck for other reasons, such as thyroid or laryngeal disease, may inadvertently cause trauma or removal of parathyroid glands because of the normal variability in their anatomic position (1,4-7). A report by Lee et al. indicates that almost 12% of patients undergoing thyroid resection have one parathyroid gland removed inadvertently (8). Although from one to twelve parathyroid glands can be found, (1), 84% of normal adults have four parathyroids (4). From 1 to 7% of adults have three glands and 3 to 13% have five glands (1,4-7). The variability of the location of the parathyroid glands is usually greater in the lower parathyroids. The superior parathyroids may be found close to the thyroid capsule or actually within Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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the thyroid capsule, but they may also be located behind the pharynx or the esophagus, lateral to the larynx, or behind any part of the thyroid. The lower glands, which usually lie near the lower pole of the thyroid, may be found behind the thyroid, in the paratracheal area, or close to or within the thymus in the superior mediastinum. The glands tend to be bilaterally symmetrical in location, with approximately 75% of cases showing such symmetry (4,5). The parathyroid glands measure between 2 and 7 m m in length, 2 and 4 m m in width, and 0.5 and 2 m m in thickness. They are reniform, soft, and brown to rust in color. However, color varies with fat content, the degree of vascular congestion, and the n u m b e r of oxyphil cells present (5,9,10). Parathyroid tissue weight varies with sex, race, and overall nutritional status of the individual (11). The combined weight of all parathyroid tissues in a normal adult male is a r o u n d 120 mg; in females combined tissue weight is around 145 mg. Weights of individual glands range from 3 to 75 mg, with averages of a r o u n d 35 to 55 mg (5,9-11).

HISTOLOGY OF PARATHYROID GLANDS Microscopic examination shows that each parathyroid gland is invested by a thin connective tissue capsule that extends into the parenchyma as fibrous septae, dividing the gland into lobules. A rich capillary vascular network is s u r r o u n d e d by nests and cords of

parenchymal cells. Small clusters of cells are interspersed with foci of adipose tissue (Fig. 1). However, there is variability in the location and interrelationships between the fat and the parenchymal cells in the parathyroid gland, so that biopsies from specific areas of the parathyroid may be predominantly fat, predominantly parenchyma, or a mixture of these two. In the adult, the parathyroid is composed of chief and oxyphil cells, fibrous stroma that is usually thin and delicate, and variable amounts of fat. Historically, the ratio of 50:50 cells:fat has been accepted as normal for adults. However, numerous studies have indicated that individuals dying without h o r m o n a l dysfunction of any type have parathyroids in which the stromal fat content is significantly less than 50% in most cases. It may be as little as 10%. In fact, n u m e r o u s studies (11-14) have shown that an approximately 17% fat content is normal in an adult parathyroid gland. Indeed, cell:fat ratios in terms of stromal fat serve little purpose in microscopic interpretation of functional status. Densitometry measurements concur, indicating that parenchymal cell mass accounts for 74% of parathyroid weight (4,14). The cells that make up the parathyroid glands include chief cells, oxyphils, and clear cells (Fig. 2). These variable cell groups probably represent different morphologic expressions of the same parenchymal cell. The chief cell is polyhedral in shape, poorly outlined, and measures 6 to 8 nm in diameter (15). It has an amphophilic to slightly eosinophilic cytoplasm, a

FIG. 1 Normal parathyroid gland adjacent to thyroid (lower left). Note the cellularity of the gland and the relative paucity of fat (f clear spaces) in this section. Hematoxylin and eosin, x50.

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FIG. 2 Parathyroid chief cells (dark cells), oxyphils (larger cells), and fat in normal adult gland. Hematoxylin and eosin, × 150.

sharp nuclear membrane, and well-defined, abundant nuclear chromatin. Clear cells represent chief cells in which there is an excessive amount of glycogen in the cytoplasm. Oxyphils, which tend to be found initially around the time of puberty and rarely in childhood, apparently increase in n u m b e r with age and may form small micro-

scopic nodules. The oxyphil cell in the parathyroid, as in other organs, is large, measuring approximately 10 nm in diameter, has a well-demarcated cell membrane, and has eosinophilic granular cytoplasm (Fig. 3). This reflects a marked mitochondrial content (9,10,15). In contrast to stromal fat content, intracellular fat content may be helpful in defining functional status. Thus, in

FI6.3 Cluster of oxyphils in normal parathyroid gland. Hematoxylin and eosin, x250.

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chief cells, which are the predominant cells in the parathyroid, intracellular fat, i.e., intracytoplasmic fat, is found in the overwhelming majority of cells in the euparathyroid state (approximately 80% of cells) (11-14). Ultrastructurally, the chief cells undergo a cyclic process during synthesis and secretion of parathyroid hormones, with the hormone being synthesized on Golgi apparatus-associated membrane-bound secretory granules. These cells eventually secrete these particles of h o r m o n e into the surrounding milieu. Little lipid is present in the active parathyroid cell, which in the euparathyroid state is approximately 20% of the parenchymal cell population (15).

DISEASES OF THE PARATHYROIDmPATHOPHYSIOLOGY Surgical pathologists dealing with the parathyroids almost always evaluate parathyroid tissue in patients who have hypercalcemia. The predominant effect of parathyroid hormone, as noted, is to increase serum calcium. The usual clinical problem is not to distinguish normal from hypercalcemic patients, but rather to distinguish those who have hypercalcemia caused by hyperparathyroidism from those who have hypercalcemia arising from other causes. Primary hyperparathyroidism is defined as the disease in which, in the absence of a known stimulus, one or more parathyroid glands secrete excess parathyroid hormone, producing hypercalcemia. Serum calcium ranges from 11 to 18 mg/dl, with most asymptomatic patients found in the lower end of the spectrum (16). The prevalence of primary hyperparathyroidism in the United States is estimated to be 1-5 cases per 1000 adults (16). The etiology of the disease is unknown. In a certain number of individuals a history of irradiation to the head and neck may be found, although the magnitude and significance of this association are not clear (17,18). Prinz et al. (18) found that 67% of patients in their series with combined thyroid and parathyroid tumors gave a history of irradiation. In some patients, genetics plays a role [multiple endocrine neoplasia (MEN) syndromes; see also Chapter 19] (19-25). Mutations of the M E N - 1 gene (menin) have been identified in some irradiated patients with hyperparathyroidism (22-24).

Pathology of the Parathyroid Glands in Primary Hyperparathyroidism Three subgroups of pathologic lesions are found in patients with primary hyperparathyroidism: adenoma, multigland hyperplasia, and, rarely, carcinoma.

Parathyroid Adenoma The parathyroid adenoma is responsible for hyperparathyroidism in 30-90% of cases. The wide range of variation indicates both pathologic interpretation and surgical interpretation of the disease (9,10,26-29). Most researchers believe that 75-80% of primary hyperparathyroidism is caused by a solitary adenoma (9,10,26-29). Evidence supports a clonal origin for parathyroid adenomas. Although older studies using protein polymorphisms indicated that parathyroid adenomas were polyclonal (30,31), many studies (25,32-36) using the techniques of molecular biology show that sporadic parathyroid lesions are monoclonal neoplasms. Grossly, parathyroid adenomas tend to be located more commonly in the lower glands than in the upper glands. Typically, the adenoma is an oval red-brown nodule that is smooth, circumscribed, or encapsulated. The lesion, which often replaces one parathyroid gland, may show areas of hemorrhage and, if large, cystic degeneration. Occasionally in small adenomas, a grossly visible rim of normal yellow-brown parathyroid tissue may be seen. Weights of adenomas vary from 300 mg to several grams. The size ranges from 1 to over 3 cm (9,10,27,29). Microscopically, adenomas are usually encapsulated lesions composed of parathyroid chief cells arranged with a delicate capillary network, recapitulating endocrine tumors in general (Figs. 4 and 5). Rarely, lobules are seen, and sometimes nodules may be formed. Stromal fat is usually absent. Unless they are very large, about 50% of adenomas will appear to have a normal rim or even atrophic parathyroid tissue outside the adenoma capsule. The cells in the rim tend to be smaller and more uniform, with stromal and cytoplasmic fat abundant in the rim but absent in the adenoma (9,10,27,29,37). However, the absence of a rim does not preclude the diagnosis of adenoma, because large tumors may have overgrown the preexisting normal gland or the rim may have been lost during sectioning. In large tumors, zones of fibrosis may be found in addition to hemorrhage, cholesterol clefts, and hemosiderin, as well as occasional areas of calcification. Rarely, lymphocytes will be noted within an adenoma (38). Thymic tissue may be found in association with an adenoma or an adenoma may be found within the thymus. There may be atypical cells in an adenoma. Most cells comprising the lesion have relatively small, uniform, dark nuclei. Usually focally, bizarre multinucleated cells with dark, crinkled nuclei can be seen. These nuclei probably represent degenerative changes rather than malignant or premalignant potential. It has been stated that mitotic activity is never found in a parathyroid adenoma and that such activity should suggest the

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FIG. 4 Parathyroid adenoma. Almost all chief cells; no fat present. Hematoxylin and eosin, x200.

possibility of a malignant neoplasm. This particular diagnostic area, however, is fraught with difficulty and is u n d e r debate at the present time. The n o n a d e n o m a tous glands in a patient with a parathyroid a d e n o m a may show normal to increased cytoplasmic fat content and normal weight (9,10,13,14). In about 10% of cases microscopic examination of biopsies from "normal" glands will show areas of hypercellularity, so-called microscopic hyperplasia. Although

this may represent a true parenchymal cell increase, the difficulty in defining "normal," or more likely sampling errors, probably accounts for this (39-41). Oxyphilic or oncocytic adenomas do occur and can function. These tumors tend to be larger than chief cell adenomas and the serum calcium levels tend to be minimally elevated (42-47). Because of the embryologic migration patterns, parathyroid adenomas can occur in ectopic locations.

FIG. 5 Parathyroid adenoma with follicle formation; rarely, this is mistaken for thyroid tissue, especially on frozen section. Hematoxylin and eosin, x300.

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Thus, when hyperparathyroidism occurs in such an individual and no a d e n o m a or abnormal glands are identified in the neck, ectopic locations that should be considered include the mediastinum, with or without associated thymic tissue, b e h i n d the esophagus, or even intrathyroidal (48-54). Double adenomas, if they occur, are very rare (55-57). Most patients who have socalled double adenomas will, over a period of time, have recurrent hyperparathyroidism and in fact have four-gland hyperplasia. The diagnosis of double aden o m a can be made only if two glands are enlarged and histologically abnormal; the remaining glands are normal, there is no family history of parathyroid disease, and p e r m a n e n t cure of hypercalcemia follows excision of only two enlarged glands (55-65). Indeed, heterogeneous size of four glands in primary hyperplasia may account for some cases interpreted as "double adenomas" (65).

Primary Parathyroid Hyperplasia Primary parathyroid hyperplasia is divided into two main groups: chief cell hyperplasia, which is common, and water clear cell hyperplasia, which occurs less commonly (9,10,29). Chief cell hyperplasia accounts for 15% of hyperparathyroidism in most series, although some reports indicate that about half of primary hyperparathyroidism is produced by hyperplasia. The reasons for this probably lie in discrepancies in pathologic interpretation. About 30% of patients with chief cell hyperplasia have familial hyperparathyroidism or one of the syndromes of multiple endocrine neoplasia (9,10,29,66-73). Grossly, all four glands are enlarged equally or nonequally. If unequal in size, the lower glands are usually larger. Occasionally one gland will be much larger than the others and will convey the surgical impression of an adenoma. The weight of all four glands ranges from 150 mg to over 20 g, but usually is in the range of I to 3 g (9,10). Microscopically, diffuse chief cell hyperplasia may be characterized by solid masses of cells with minimal to no fat. Usually almost all cells are chief cells, with rare oxyphils. Nodular or pseudoadenomatous hyperplasia consists of circumscribed nodules of chief, transitional, or oxyphil cells, each nodule devoid of fat, and with there being little fat in the intervening stroma. Usually in hyperplasia there is no rim of normal tissue. Bizarre nuclei are rarely found in primary hyperplasia. Mitoses may occasionally, however, be identified (9). Therapy in this disease is directed to the removal of all parathyroid tissue, with or without autotransplantation. Clear cell (water clear cell) hyperplasia is very rare and is the only condition of the parathyroid in which the superior glands are larger than the lower. Total weights of such parathyroids always exceed 1 g and usu-

ally range from 5 to 10 g. The glands are irregular and show pseudopods and cysts; a distinct mahogany color is seen grossly. Histologically, the glands are composed of diffuse sheets of clear cells without any mixture of other cell types. No rim is present (9,10,74-76). An interesting association of clear cell hyperplasia with the blood group O allele has been reported (77).

Parathyroid Carcinoma Parathyroid carcinoma accounts for approximately 1% of primary hyperparathyroidism (78-94). There is clinically an unusual scenario with an almost equal sex ratio, which is u n c o m m o n in parathyroid adenomas and usual hyperplasias, in which women predominate. The incidence of benign hyperparathyroidism appears to increase with age; however, patients with parathyroid carcinoma tend to be somewhat younger and are almost always symptomatic with very high levels of serum calcium. Very rarely, parathyroid carcinoma can occur in the setting of familial endocrine disease (95-99) or as a complication of secondary parathyroid hyperplasia (100-104). Most of the latter cases occur in patients with renal failure (12 cases were d o c u m e n t e d in 1999) (104). Clinically, patients with parathyroid carcinoma show high calcium levels (up to 15 m g / d l ) . Many have polyuria, polydypsia, nausea, vomiting, weight loss, and constipation. They may also have bone pain, renal stones, and other symptoms related to hypercalcemia. An important clinical clue is the presence of a palpable mass in the neck on physical examination. The mass may be clinically thought to be an a d e n o m a of the thyroid (9,10,78-94). Parathyroid carcinomas tend to be large tumors (average weight 12 g) and characteristically show a histology with trabecular a r r a n g e m e n t of tumor cells divided by thick fibrous bands, with capsular and blood vessel invasion in the presence of mitotic figures (Fig. 6) (9,10,105). The cytology may be clear or rarely oxyphilic; nuclear atypia may be seen or may be absent (9,10,102,106). Because mitotic figures are almost never found in a benign parathyroid adenoma, their presence in tumor cells should raise the suspicion of malignancy. However, this has been called into question and parathyroid tumors with mitotic activity may in fact be benign. As a note of caution, long-term follow-up in the reported series is quite limited, and there is a long natural history to parathyroid carcinoma, so the answers are not all in yet (107,108). Mitotic activity in secondary hyperparathyroidism is not to be equated with malignancy, and mitotic activity may occasionally be found in primary hyperparathyroidism as well (9). The presence of capsular invasion is not equated with malignancy because large parathyroid adenomas

PARATHYROIDS: MORX'HOLOGYAND PATHOLOGY /

7

FI6. 6 Parathyroid carcinoma; note mitosis (+). This tumor recurred three times locally and eventually metastasized to the lungs. Hematoxylin and eosin, x300.

may have u n d e r g o n e prior hemorrhage, with consequent fibrosis and trapping of tumor cells within the capsule. Vascular invasion is difficult to define except if seen outside the vicinity of the neoplasm. An important clue to the diagnosis of parathyroid carcinoma is the surgical finding of adherence a n d / o r invasion into local structures, which should raise the suspicion of a carcinoma (9,10,78-94,102). Metastases at the time of presentation are unusual, but may be found in the regional lymph nodes. There may also be local invasion into nerves, soft tissue, and the esophagus. Rarely, nonfunctioning parathyroid carcinomas have been described. These lesions tend to be large and composed of clear or oxyphil cells (83,109,110). The prognosis of parathyroid carcinoma is usually one of an indolent malignancy. Metastases may occur in up to one-third of cases and are found in regional lymph nodes, bone, lung, and liver. Many patients survive long periods of time, however. Multiple recurrences are known to occur over a 15- to 20-year period (9,10,78-94,102). The severity of the symptoms due to metastatic disease is directly related to tumor burden, because this is related to parathyroid h o r m o n e p r o d u c e d (111). Some solitary parathyroid tumors show features that suggest carcinoma, such as large size, fibrous bands, etc., but not all of the characteristics of malignancy are present. We use the term "atypical adenoma" for these lesions; short-term follow-up suggests they are benign, but long-term studies are needed in this subset of lesions.

Multiple Endocrine Neoplasia Syndromes The syndromes of MEN-1 (Wermer's syndrome) and MEN-2 (Sipple syndrome) are associated with pathologic changes in the parathyroids. In MEN-l, pathologic changes similar to adenomatous or p s e u d o a d e n o m a t o u s chief cell hyperplasia as described above are found (9,10). I n MEN-2, the parathyroids tend to show a diffuse hyperplasia, but occasionally one gland is involved, suggesting an "adenoma." In this syndrome the hyperparathyroidism is considered to represent a genetically determined event and not a response to hypercalcitoninemia (9,10). Parathyroid abnormalities are m u c h less c o m m o n in other variants of MEN-2 syndromes. Familial hyperparathyroidism shows the pathologic alterations of chief cell hyperplasia similar to Wermer's syndrome; in familial hypercalciuric hypercalcemia, mild parathyroid hyperplasia has been described (9,10).

Unusual Lesions of the Parathyroid

Parathyroid Cysts Cysts of the parathyroid glands are unusual and may present and be misinterpreted clinically as thyroid nodules (112-124). They occur more frequently in women than in men, usually are large, ranging from 1 to 6 cm, and may be located in any parathyroid gland, although most are found in the lower glands. Occasionally they may be found in the mediastinum, mimicking super i o r / a n t e r i o r mediastinal masses (114,121,123).

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1

may be functional or nonfunctional and usually is circumscribed but rarely encapsulated. In unusual examples a rim of normal parathyroid tissue is present at the periphery. In some instances at least one other histologically normal parathyroid has been recognized. Also, in some, there is an unusual myxomatous stroma, and other mesenchymal elements including metaplastic bone may be found. Wolff and Goodman (125) suggest the term "parathyroid adenomas with stromal component." More than three-quarters of the reported cases functioned, although with relatively low levels of hypercalcemia. We have studied a woman presenting with an orbital brown tumor due to a parathyroid lipoadenoma that weighed over 10 g (128).

Grossly, these cysts are almost always unilocular and smooth walled and contain water fluid with a high parathyroid hormone content. Histologically, they are lined by one layer of clear epithelium containing glycogen. The cyst wall is fibrous, with fragments of smooth muscle and nests of normal parathyroid tissue. It is unclear how these cysts arise. Microcysts are found in about half of normal parathyroids and might possibly enlarge by accumulation of secretions, or may fuse and produce grossly visible cysts. The cysts may arise from embryologic remnants of pharyngeal pouches in the neck undergoing cystic degeneration and entrapping portions of parathyroid tissue. Many investigators believe, however, that parathyroid cysts represent degenerated parathyroid adenomas, and in some cases, in fact, that the cysts are associated with hyperparathyroidism (Fig. 7) (117-119). However, this is u n c o m m o n and only a few functional cysts have been reported. It may be that different parathyroid cysts have different origins, although pathologically they resemble one another. Cytologists may encounter parathyroid cysts during attempts to aspirate thyroid nodules (112,124). The cyst fluid can be assayed biochemically for parathyroid hormone to confirm the diagnosis.

Parathyromatosis In rare instances of hyperparathyroidism due to primary hyperplasia, nests of hyperplastic parathyroid cells are found in the neck, outside of hyperplastic glands (129-131). In the individuals for which this has been reported, these nests were discovered at the first neck exploration, so that spillage during prior surgery could be excluded. In each of these patients there was no evidence of malignancy. It has been postulated that during embryologic development nests of pharyngeal tissue containing parathyroid cells might be scattered throughout the adipose tissue of the neck and mediastinum. Normally these nests are inconspicuous. However, in the process of diffuse hyperplasia of the parathyroids, all functioning tissue may become hyperplastic and appear as separate fragments on histologic evaluation.

Lipoadenoma-Hamartoma of the Parathyroid These tumors present as masses that histologically are composed of parathyroid cells arranged in nests, similar to normal parathyroid but intimately associated with large areas of adipose tissue (125-128). The lesion

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FIG. 7 Parathyroid cyst. This tumor presented with hypercalcemia and extended from the lower neck to the upper mediastinum. It was 6 cm in size, but most of the lesion was cyst. However, about 10-15% was solid parathyroid tissue, making this a cystic adenoma. Hematoxylin and eosin, x 150.

PARATHYROIDS: MORPHOLOGYAND PATHOLOGY /

Infarction of Parathyroid Adenomas Twelve d o c u m e n t e d cases (132) of adenomas that spontaneously infarcted have been reported. This phen o m e n o n is associated with remission of hypercalcemia. The etiology of the infarction is unclear in most cases, although some have been associated with the intake of certain drugs that may predispose to vascular damage, thrombosis, or hemorrhage. Therapeutic infarction can also result in cure of the metabolic abnormalities (85).

INTRAOPERATIVE ASSESSMENT OF PARATHYROIDS--THE BANE OF THE SURGICAL PATHOLOGIST In normal parathyroid glands, 80% of the cells are in the nonsecretory phase and contain intracytoplasmic fat (12,13,15). Therefore, is the fat stain useful in distinguishing hyperplasia from adenoma, because all hyperfunctioning glands should be fat depleted? The advocacy of fat stains (Sudan IV or Oil Red O) on parathyroid tissue removed at surgery has come into vogue. The scenario is as follows. A sample of an enlarged parathyroid gland is sent for frozen section and by hematoxylin and eosin stain it is hypercellular with little or no stromal fat. Thus it either represents an a d e n o m a or a hyperplastic gland and is not normal. A biopsy of a second parathyroid is frozen and is normocellular or minimally hypercellular. Fat stain shows a b u n d a n t cytoplasmic fat in the latter biopsy; hence this is a normal gland. The enlarged gland, which shows minimal to no fat, represents an adenoma. Many authors have cautioned, however, that the fat stain cannot be the sole procedure on which to base a diagnosis, because although the fat stain is helpful, it is helpful in only about 80% of cases and must be considered as an adjunctive technique in light of gross findings, gland weight, and size, and cannot be relied on by itself (133-141). We have found it useful to perform a r a p i d (30-second) toluidine blue stain on frozen sections of parathyroid tissue. The intracellular fat is well defined by this stain and it is faster to perform and interpret compared to Oil Red O (Lyle S, et al., unpublished observations, 2000). Another rapid technique that may prove useful for intraoperative assessment is density gradient measurements (142). There is an almost linear relationship between density and parenchymal content of parathyroid tissue and thus such a technique can assess parenchymal cell mass. The technique is to take a sample of the gland and weigh it, and take a small piece from the center and a piece from the rim, determining their densities in a 25% mannitol solution. Abnormal parathyroid tissue sinks because of decreased fat and

9

high parenchymal mass. Wang and Ryder (142) have found that this is a simple test to be used by the surgeon in the operating room for distinguishing normal from abnormal glands. In the intraoperative assessment of parathyroid pathology it cannot be stated strongly enough that there must be close communication between the surgeon and pathologist during the operation. The pathologist needs to be apprised of the gross findings and cannot work in a vacuum. What is r e c o m m e n d e d is as follows: the largest parathyroid gland found is resected in toto, then the pathologist weighs it, measures it, and examines it histologically. If the gland shows diffuse growth of chief cells and perhaps a normal-appearing rim, a lack of fat, and bizarre nuclei, a diagnosis of presumed a d e n o m a can be rendered. If the histology is that of hypercellularity but criteria for a d e n o m a are not seen, biopsy of at least one more gland is needed, and, in fact, in many centers pathologists prefer to have the largest abnormal gland and at least a biopsy of one more gland. Weight ratio of parenchymal cells to fat, and normal or a b u n d a n t intracytoplasmic fat content in the second gland, strongly support that the first gland is an a d e n o m a (133-141). The success rate of identifying parathyroid tissue by frozen section is over 99% (143); distinguishing one-gland from multigland disease is much more problematic.

OTHER TYPES OF HYPERPARATHYROIDISM

Secondary Hyperparathyroidism Secondary hyperparathyroidism is usually due to renal disease and is relatively c o m m o n in the age of hemodialysis and renal transplantation. The role of the surgical pathologist in the evaluation of secondary hyperparathyroidism is basically to identify parathyroid tissue at the time of frozen section to allow for the surgeon to remove portions of this tissue for autotransplantation. Secondary hyperparathyroidism is really no different histopathologically from primary hyperparathyroidism (144-146). Mitotic activity may occasionally be found in such glands. Usually all four glands are enlarged, although one or two glands may be of very great size. Transplantation of parathyroid tissue is successful in the majority of cases and occasionally part of this tissue may be removed if hyperfunction again becomes a problem (147,148). Such lesions will have small nests and islands of vascularized parathyroid tissue growing in muscle or fat, usually having been implanted in the arm (149,150).

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Tertiary Hyperparathyroidism Although the existence of tertiary hyperparathyroidism has been questioned, most authors believe it represents the autonomous function of one parathyroid gland that develops in the face of long-standing secondary hyperparathyroidism (151). The pathology resembles that of secondary hyperparathyroidism, although one of the four glands is usually disproportionately enlarged.

Familial Hyperparathyroidism In addition to the multiple endocrine neoplasia syndromes, in which hyperparathyroidism is often a prominent clinical problem, familial parathyroid hyperplasia without other endocrine lesions has been reported. The lesions in all of these patients resemble those of primary chief cell hyperplasia (9,10,152-154), although a ribbon pattern of cell growth may be prominent in MEN-1.

Familial Hypocalciuric Hypercalcemia Familial hypocalciuric hypercalcemia, inherited as an autosomal dominant gene, is manifested clinically by familial occurrence, moderate to minimally elevated serum calcium, and reduced urinary calcium excretion (see Chapter 41). The parathyroid glands appear normal to mildly hypercellular, and subtotal parathyroidectomy fails to reverse the hypercalcemia. The defect appears not to be in the parathyroid glands (155,156).

SPECIAL STUDIES AND THE PARATHYROID Cytology Because most parathyroid lesions are not palpable, direct biopsy of a parathyroid tumor by fine-needle aspiration (FNA) is unusual. However, on occasion, parathyroid lesions present clinically as thyroid nodules or are large enough to be clinically evident. The FNA features of parathyroid adenoma include cellular fragments of epithelial cells arranged around vascular cores, an organoid or trabecular architecture, and microacini. Parathyroid chief cells contain uniform round nuclei; groups of oxyphilic cells are helpful in defining the tissue as parathyroid. If available, immunostains for parathyroid h o r m o n e may help (157).

Proliferative Markers Attempts at using immunocytochemical markers (158-164) of proliferation index (MIB1 for cell cycle-

associated Ki-67 antigen) for distinguishing between parathyroid adenomas and hyperplasia have met with varied success (159). Whereas statistically significant differences are found between normal (suppressed "rim") parathyroid tissue and hyperfunctioning glands, similar proliferative indices are noted between adenomas and hyperplasias (159,160). Loda et al. (160) identified higher numbers of labeled nuclei in adenomas than in hyperplasias by proliferating cell nuclear antigen (PCNA) immunostaining. The labeling index of individual cases of parathyroid tumors shows so much overlap that it cannot be used to distinguish benign from malignant lesions (161-164).

Flow Cytometry and the Parathyroid Several studies of DNA content have shown that aneuploidy may be found in parathyroid adenomas, and even in hyperplasia, as well as in carcinomas. Approximately 70% of parathyroid carcinomas, 30% of adenomas, and 30-50% of chief cell hyperplasia glands have aneuploid DNA populations (165-171). As in proliferations of other endocrine organs, the finding of aneuploid cell populations does not ensure a diagnosis of malignancy (169-173).

Clonality Modern molecular biology techniques, primarily using restriction fragment-length polymorphisms, have shown that most (if not all) parathyroid adenomas are monoclonal proliferations (25). In addition, about 40% of primary hyperplasias and 60% of secondary hyperplasia (secondary to chronic renal disease) are clonal. Different laboratories utilizing different probes as markers confirm these findings (25,174-176). The biologic meaning of these results is unclear.

Genetics The P R A D 1 oncogene has been implicated in parathyroid tumorigenesis. PRAD1 (for parathyroid adenoma), which encodes cyclin D1, results from a chromosome inversion that occurs as a dominant clonal event in some parathyroid adenomas. The inversion is created by a break in the vicinity of the parathyroid gene on the short arm of chromosome 11 (band 1 lp15), another break in the long arm (band 1 lq13), rotation of the center piece around the axis of the centromere, and rejoining (177). Cyclin D overexpression can be detected immunohistochemically in 18-38% of parathyroid adenomas, and in 91% of carcinomas (178,179). The retinoblastoma (Rb) gene is a tumor suppression gene that has growth inhibitory effects in the cell cycle. Inactivation of the Rb gene has been associated

PARATHYROIDS: MORPHOLOGY AND PATHOLOGY

with loss of an Rb allele by molecular analysis, and immunostaining for Rb protein may assist in the distinction between parathyroid adenomas and carcinomas (179-183). However, caution must be used in interpretation of the results, because some parathyroid carcinomas do not show loss of Rb protein and a few adenomas do (181,182). Studies of parathyroid neoplasms (benign and malignant) have not shown p53 mutations in such lesions (184). In another study of parathyroid tissues, there were significant differences between p27 protein expression in parathyroid hyperplasia, adenomas, and carcinomas, suggesting that this cell cycle protein may be useful in distinguishing between these two conditions (185,186).

HUMORAL HYPERCALCEMIA O F MALIGNANCY, O R ECTOPIC PARATHYROIDISM Hypercalcemia without bone metastasis in nonparathyroid malignancies may be found in association with a malignant tumor. Hypercalcemia is relieved by excision of the tumor and returns with its recurrence. This paraneoplastic endocrine syndrome is due in many cases to a peptide that resembles parathyroid hormone but is distinctly different. The factor responsible for the syndrome of humoral hypercalcemia of malignancy, which is due to parathyroid hormone-related protein (PTHrP), is discussed in other chapters in this volume. PTHrP binds to parathyroid h o r m o n e receptors on bone and kidney and mimics the actions of parathyroid hormone. The tumors most commonly associated with this syndrome include squamous carcinomas arising in a number of primary sites, including lung, vulva, esophagus, and head and neck, and clear cell cancers, especially of renal and ovarian origin (187-191). The parathyroid glands appear normal or atrophic histologically.

HYPOPARATHYROIDISM The most common parathyroid pathology found in patients with hypoparathyroidism is four normal glands. Unfortunately, they often have been surgically removed from the patient! Accidental excision of normal parathyroid glands during the course of neck surgery, especially thyroid surgery, is an u n c o m m o n but unfortunately not a rare event (8). In addition to actual excision of the glands, injury to their vascular supply may cause their infarction, or they may be so damaged that they become functionally absent.

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Infiltration Impaired parathyroid function caused by infiltration of parathyroid glands has been described in hemochromatosis, amyloidosis, and metastatic carcinomas. These are all rare causes of hypoparathyroidism (192).

Radiation Rarely, patients are reported who have developed hypoparathyroidism after radioactive iodine treatment for hyperthyroidism. The presumed mechanism is radiation damage to and fibrosis of the parathyroids (193).

Autoimmune Parathyroid Destruction Lymphocytic infiltration of parathyroid tissue, with subsequent autoimmune destruction of the glands, is probably the most common cause of hypoparathyroidism (noniatrogenic cause). It may occur as an isolated event or in association with autoimmune diseases of other endocrine organs, i.e., thyroid, adrenal, or ovary (194-197).

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41. Harrison TS, Duarte B, Reitz RE, et al. Primary hyperparathyroidism: Four to eight year postoperative follow-up demonstrating persistent functional insignificance of microscopic parathyroid hyperplasia and decreased autonomy of parathyroid hormone release. Ann Surg 1981;194:429-437. 42. McGregor DH, Lotuaio LG, Chu LH. Functioning oxyphil adenoma of parathyroid gland. An ultrastructural and biochemical study. A m J Patho11978;92:691-703. 43. Ordonez NG, Ibanez ML, MacKay B, et al. Functional oxyphil cell adenomas of parathyroid gland: Evidence of hormonal activity in oxyphil cells. A m J Clin Patho11982;78:681-689. 44. Rodriquez FH, Sarma DE Lunseth JH, Guileyardo JM. Primary hyperparathyroidism due to an oxyphil adenoma. Am J Clin Pathol 1983;80:878-880. 45. Bedetti CD, Dekker A, Watson CG. Functioning oxyphil cell adenoma of the parathyroid gland: A clinicopathologic study of ten patients with hyperparathyroidism. Hum Pathol 1984;15:1121-1126. 46. Jones SH, Dietler E Oxyphil cell adenoma as a cause of hyperparathyroidism. Am J Surg 1981 ;141:744-745. 47. Baloch ZW, LiVolsi VA. Oncocytic lesions of the neuroendocrine system. Semin Diagn Patho11999;16:190-199. 48. Nathaniels EK, Nathaniels AM, Wang CA. Mediastinal parathyroid tumors: A clinical and pathological study of 84 cases. Ann Surg 1970;171:165-170. 49. Russell CE Edis AJ, Scholz DA, et al. Mediastinal parathyroid tumors: Experience with 38 tumors requiring mediastinotomy for removal. Ann Surg 1981;193:805-809. 50. Russell CF, Grant CS, vanHeerden JA. Hyperfunctioning supernumerary parathyroid glands: An occasional cause of hyperparathyroidism. Mayo Clin Proc 1982;57:121-124. 51. Edis AJ, Purnell DC, vanHeerden JA. The undescended "parathymus": An occasional cause of failed neck exploration for hyperparathyroidism. Ann Surg 1979;190:64-68. 52. Sloane JA, Moody HC. Parathyroid adenoma in submucosa of esophagus. Arch Pathol Lab Med 1978;102:242-243. 53. Spiegel AM, Marx SJ, Doppmann JL, et al. Intrathyroidal parathyroid adenoma or hyperplasia. JAMA 1975;234:1029-1033. 54. Kobayashi T, Man IM, Shin E, et al. Hyperfunctioning intrathyroidal parathyroid adenoma: Report of two cases. Surgery Today 1999;29:766-768. 55. Schwindt WD. Multiple parathyroid adenomas. JAMA 1967;199:945-946. 56. Verdon CA, Edis AJ. Parathyroid "double adenomas." Fact or fiction? Surgery 1981;90:523-526. 57. Harness JK, Ramsbury SR, Nishiyama RH, Thompson NW. Multiple adenomas of the parathyroids; do they exist? Arch Surg 1979;114:468-474. 58. Seyfar AE, Sigdestad JB, Hirata RM. Surgical considerations in hyperparathyroidism: Reappraisal of the need for multigland biopsy. A m J Surg 1976;132:338-340.

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109. Merlano M, Conte P, Scarsi P, et al. Nonfunctioning parathyroid carcinoma. A case report. Tumori 1985;71:193-196. 110. Yamashita H, Noguchi S, Nakayama I, et al. Light and electron microscopic study of nonfunctioning parathyroid carcinoma. Acta PatholJpn 1984;34:123-132. 111. Zisman E, Buckle RM, Deftos LJ, et al. Production of parathyroid hormone by metastatic parathyroid carcinoma. Am J Med 1971 ;45:1068:619-623. 112. Wang CA, Vickery AL, Maloof E Large parathyroid cysts mimicking thyroid nodules. Ann Surg 1972;175:448-453. 113. Ginsberg J, Young JEM, Walfish PG. Parathyroid cysts. JAMA 1978;240:1506-1507. 114. Thacker WC, Wells VH, Hall ER. Parathyroid cysts of the mediastinum. Ann Surg 1971;174:969-975. 115. Hoehn JG, Beahrs OH, Woolner LB. Unusual surgical lesions of the parathyroid gland. A m J Surg 1969;118:770-778. 116. Troster M, Chiu HE McLarty TD. Parathyroid cysts: Report of a case with ultrastructural; observations. Surgery 1978;83:238-242. 117. Earll JM, Cohen A, Lundberg GD. Functional cystic parathyroid adenoma. Am J Surg 1969; 118:100-103. 118. Albertson DA, Marshall RB, Jarman WT. Hypercalcemic crisis secondary to a functioning parathyroid cyst. Am J Surg 1981;141:175-177. 119. Clark OH. Hyperparathyroidism due to primary cystic parathyroid hyperplasia. Arch Surg 1978;113:748-750. 120. SilvermanJE Khazanie PG, Norris T, Fore WW. Parathyroid hormone (PTH) assay of parathyroid cysts examined by fine needle aspiration biopsy. A m J Clin Patho11986;86:708-776. 121. Marco V, Carrasco MA, Marco C, Bauza A. Cytomorphology of a mediastinal parathyroid cyst. Acta Cyto11983;27:688-692. 122. Gough IR. Parathyroid cysts. Aust N Z J Surg 1999;69:404-406. 123. Shields TW, Immerman SC. Mediastinal parathyroid cysts revisited. Ann Thorac Surg 1999;67:581-590. 124. Shi B, Guo H, Tang N. Treatment of parathyroid cysts with fine needle aspiration. Lancet 1999;2:797-798. 125. Wolff M, Goodman EN. Functioning lipoadenoma of supernumerary parathyroid gland in the mediastinum. Head Neck Surg 1980;2:302-307. 126. Grimelius L, Johansson H, Lindquist B. A case of unusual stromal development in a parathyroid adenoma. Acta Chir Scand 1972; 138:628-629. 127. Ober WB, Kaiser GA. Hamartoma of the parathyroid. Cancer 1958; 11:601-606. 128. Perosio P, Brooks JJ, LiVolsi VA. Orbital brown tumor as initial manifestation of parathyroid lipoadenoma. Surg Pathol 1988;1:77-82. 129. Reddick RL, Costa JC, Marx sJ. Parathyroid hyperplasia and parathyromatosis. Lancet 1977; 1:549. 130. Fitko R, Roth SI, Hines JR, et al. Parathyromatosis in hyperparathyroidism. Hum Patho11990;21:234-237. 131. Kollmorgen CF, Aust MR, FerreiroJA, et al. Parathyromatosis: A rare yet important cause of persistent or recurrent hyperparathyroidism. Surgery 1994;116:111-115. 132. Kovacs KA, Gay JDL. Remission of primary hyperparathyroidism due to spontaneous infarction of a parathyroid adenoma: Case report and review of the literature. Medicine 1998;77:398-402. 133. Roth SI, Wang CA, Potts JT. The team approach to primary hyperparathyroidism. Hum Pathol 1975;6:645-658. 134. LiVolsi VA, Hamilton R. Introperative assessment of parathyroid gland pathology. A common view from the surgeons and the pathologist. A m J Clin Pathol 1994;102:365-373. 135. Dufour DR, Durkowski C. Sudan IV staining: Its limitations in evaluating parathyroid functional status. Arch Pathol Lab Med 1987;106:224-227.

136. King DT, Hirose FM. Chief cell intracytoplasmic fat used to evaluate parathyroid disease by frozen section. Arch Pathol Lab Med 1979;103:609-612. 137. Kasden EJ, Cohen RB, Rosen S, Silen W. Surgical pathology of hyperparathyroidism: Usefulness of fat stains and problems in interpretation. Am J Surg Pathol 1981 ;5:381-384. 138. Ljungberg O, Tibblin S. Perioperative fat staining of frozen sections in primary hyperparathyroidism. AmJPatho11979;95:633-642. 139. Dekker A, Watson CG, Barnes EL. The pathologic assessment of primary hyperparathyroidism and its impact on therapy: A prospective evaluation of 50 cases with oil-red-O stain. Ann Surg 1979;190:671-675. 140. Monchik JM, Farrugia R, Teplitz C, Brown S. Parathyroid surgery: The role of chief cell intracellular fat staining with osmium carmine in the intraoperative management of patients with hyperparathyroidism. Surgery 1983;94:877-886. 141. Bondeson AG, Bondeson L, Ljundberg O, Tibblin S. Fat staining in parathyroid disease mdiagnostic value and impact on surgical strategy. Hum Pathol 1985;16:1255-1263. 142. Wang CA, Ryder SV. A density test for the intraoperative differentiation of parathyroid hyperplasia from neoplasia. Ann Surg 1978;187:63-67. 143. Westra WH, Pritchett DD, Udelsman R. Intraoperative confirmation of parathyroid tissue during parathyroid exploration. Am J Surg Pathol 1998;22:538-544. 144. Roth SI, Marshall RB. Pathology and ultrastructure of human parathyroid glands in chronic renal failure. Arch Intern Med 1969;124:397-407. 145. Malmaeus J, Grimelius L, Johansson H, et al. Parathyroid pathology in hyperparathyroidism secondary to chronic renal failure. Scan J Urol Nephrol 1984;18:75-84. 146. Akerstrom G, Malmaeus J, et al. Histological changes in parathyroid glands in subclinical and clinical renal disease. ScandJ Urol Nephro11984; 18: 75-84. 147. Rattner DW, Marrone GC, Kasdon E, Silen W. Recurrent hyperparathyroidism due to implantation of parathyroid tissue. Am J Surg 1985;149:745-748. 148. Akerstrom G, Rudberg C, Grimelius L, Rastad J. Recurrent hyperparathyroidism due to preoperative seeding of neoplastic or hyperplastic parathyroid tissue. Acta Chir Scand 1988;154-219. 149. Jansson S, Tisell LE. Autotransplantation of diseased parathyroid glands into subcutaneous abdominal adipose tissue. Surgery 1987;101:549-556. 150. Max MH, Flint LM, Richardson JD, et al. Total parathyroidectomy and parathyroid autotransplantation in patients with chronic renal failure. Surg Obstet Gyneco11981;153:177-180. 151. Krause MW, Hedinger CE. Pathologic study of parathyroid glands in tertiary hyperparathyroidism. Hum Pathol 1985;16:772-784. 152. Jackson CE, Norum RA, Boyd SB, et al. Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: A clinically and genetically distinct syndrome. Surgery 1990;108:1006-1013. 153. Mallette LE, Malini S, Rappaport ME Kirkland JL. Familial cystic parathyroid adenomatosis. Ann Intern Med 1987;107:54-60. 154. Harach HR, Jasane B. Parathyroid hyperplasia in multiple endocrine neoplasia type 1. Histopathology 1992;20:305-313. 155. Law WM, Carney JA, Heath H. Parathyroid glands in familial benign hypercalcemia (familial hypocalciuric hypercalcemia). A m J M e d 1989;76:1021-1026. 156. Thorgeirsson U, Costa J, Marx SJ. The parathyroid glands in familial hypocalciuric hypercalcemia. Hum Pathol 1981; 12:229-237. 157. Abati A, Skarulis MC, Shawker T, Solomion D. Ultrasoundguided fine needle aspiration of parathyroid lesions. A mor-

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CHAPTER2 Parathyroid H o r m o n e Biosynthesis and Metabolism

HENRY M. KRONENBERG, E RICHARD BRINGHURST, GINO V. SEGRE, AND JOHN T. POTTS, JR. Endocrine Unit,

Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114

BI O SYNTHESIS OF PARATHYROID H O R M O N E

INTRODUCTION The parathyroid h o r m o n e gene has many jobs. It must encode a peptide that can bind to and activate receptors on target tissues. Equally importantly, the a m o u n t of p a r a t h y r o i d h o r m o n e (PTH) p r o d u c e d must be carefully controlled to maintain the blood level of calcium within a narrow range. Nature's solution to these problems has involved the specific synthesis of PTH primarily in the parathyroid chief cell, a cell designed to sense the blood level of calcium. In the chief cell, synthesis and secretion of the h o r m o n e can be carefully regulated. Furthermore, the structure of the h o r m o n e is designed for rapid metabolic degradation, even in the absence of receptor binding. In this way, the rapid turnover of the h o r m o n e can assure that blood levels of h o r m o n e change quickly in response to changes in h o r m o n e secretory rate. This rapid metabolism of h o r m o n e is required of a system designed to respond quickly to sudden changes in the amounts of calcium entering and leaving the bloodstream. Studies over the past two decades have shown that the sequences of PTH and its precursors are designed to steer the h o r m o n e through the chief cell's secretory pathway, to direct the h o r m o n e ' s binding to receptors, and to assure rapid metabolism of the hormone. More recent studies have begun to unravel the mechanisms whereby synthesis of PTH is regulated in the chief cell. Descriptions of the structure of the PTH gene and a summary of the current understanding of how this structure allows the gene to accomplish its multiple functions are presented in this chapter. The Parathyroids, Second Edition

PTH is synthesized as part of the larger precursor molecule, preproparathyroid h o r m o n e (preproPTH). Only trace amounts of this full-length precursor are found in parathyroid chief cells, because the "pre," or signal, sequence is cleaved from the amino terminus while the protein is being synthesized (see Fig. 1). As the signal sequence emerges from the ribosome, it binds to a signal recognition particle, an RNA-protein complex that recognizes signal sequences on most secreted proteins. The signal recognition particle then binds to a receptor on the rough endoplasmic reticulum (docking protein) and directs the nascent p r e p r o P T H molecule to a protein-lined channel, through which the p r e p r o P T H molecule is transported. A signal peptidase located on the inner surface of the m e m b r a n e of the endoplasmic reticulum then cleaves off the signal sequence, leaving the intermediate precursor, proparathyroid h o r m o n e (proPTH) in the cisternae of the endoplasmic reticulum. P r o P T H then travels via a series of vesicles to and through the Golgi apparatus (see Fig. 2). In the Golgi, the short, aminoterminal "pro" sequence is removed, leaving the mature PTH molecule. PTH is then concentrated in dense core secretory vesicles; these vesicles fuse with the plasma m e m b r a n e and release PTH in response to a decrease in extracellular calcium. The h o r m o n e secreted is predominantly the intact 84-residue PTH molecule, though a variable fraction made up of carboxy-terminal PTH fragments is secreted, as well.

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CHAPTER2

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®

O P

1. SRP

BINDING

O

2. DOCKING PROTEIN BINDING

4. MEMBRANE TRANSPORT, PEPTIDE CLEAVAGE

3. MEMBRANE INSERTION

5. TRANSPORT COMPLETED

,

I

FIG. 1 The signal, or pre, sequence directs the nascent polypeptide to the apparatus for transport across the membrane of the endoplasmic reticulum. SRP, Signal recognition particle.

Function of the Pre (Signal) Sequence The specific sequences of each of the three portions of the preproPTH molecule are responsible for directing the h o r m o n e through the complicated pathway of transport and cleavage. The known preproPTH sequences from h u m a n (1), bovine (2), rat (3), pig (4), chicken (5,6), and dog (7) tissues share a 25-residue pre sequence and a 6-residue pro sequence (see Fig. 3). Each pre sequence contains a hydrophobic stretch of

amino acids preceded by a positively charged residue. The signal sequence ends with a small amino acid at the last and third-to-last positions. These characteristics are typical of most signal sequences. The preproPTH signal sequence was first discovered (8) when parathyroid gland mRNA was translated in a cell-free extract devoid of endoplasmic reticulum. Directed mutations have demonstrated the importance of each of the regions of

AA, AAA

PTH "-------

FIG. 2 Multiple cleavages occur during the intracellular transport of PTH.

PTH BIOSYNTHESISAND METABOLISM /

human bovine porcine rat canine chicken

PRE $ PRO $ -31 -6 MIPAKDMAKVMIVNLAICFLTKSDG KSVKKR MMSAKDMVKVMIVNLAICFLARSDG KSVKKR MMSAKDTVKVMVVNLAICFLARSDG KPIKKR MMSASTMAKVMILMLAVCLLTQADG KPVKKR MMSAKDMVKVMIVMFAICFLAKSDG KPVKKR MTSTKNLAKAIVILYAICFFTNSDG RPMNKR

PTH +i +i0 SVSEIQLMHN AVSEIQFMHN SVSEIQLMHN AVSEIQLPIHN SVSEIQFMHN SVSEMQLMHN

human bovine porcine rat canine chicken

+20 +30 +40 +50 LGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG SQNPRK L G K H L S S M N R V E W L R K K L Q D V H N F V A L G A S IA Y R D G S S Q N P R K LGKHL S SLNNVEWLRKKLQDVHNFVALGAS IVHRDGG SQRPRK LGKHLASVERMQWLRKKLQDVH FVSLGVQMAAREGSYQNPTK L G K H L S S M N N V E W L R K K L Q D V H N F V A L G A P IA H R D G S S Q N P L K L G E H R H T V E N Q D W L Q M K L Q D V H . . S A L E ...... D A R T Q R P R N

human bovine porcine rat canine chicken

+60 +70 +80 DKADVNVLTKAKSQ KEDNVLVE...SHEKSLGEA K E D N V L V E . . . S H Q K S L G E A .......... D K A D V D V L I K A K P Q K E D N V L V E . . . S H Q K S L G E A .......... D K A A V D V L I K A K P Q K E E N V L V D . . . G N S K S L G E G .......... D K A D V D V L V K A K S Q K E D N V L V E . . . S Y Q K S L G E A .......... D K A D V D V L T K A K S Q KEDIVLGEIRNRRLLPEHLRAAVQKKSIDLDKAYMNVLFKTKP. .

.

.

.

.

.

.

.

.

.

FIG. 3 Amino acid sequences of preproPTH from mammalian and avian species. Residues -31 to - 7 constitute the pre sequences; residues - 6 to -1 constitute the pro sequences. Dots represent residues found in chicken PTH without corresponding residues in the mammalian sequences. Amino acids are indicated by the single-letter code: A, Ala; R, Arg; N, Asn; D, Asp; C, Cys; Q, Gin; E, Glu; G, Gly; H, His; I, lie, L, Leu; K, Lys; M, Met; F, Phe; P, Pro; S, Ser; T, Thr; W, Trp; Y, Tyr; V, Val.

the preproPTH signal sequence for normal signal function (9-11). Further, when a synthetic prepro peptide was added to a cell-free extract, it blocked the transport and cleavage of preproPTH by microsomal membranes (12). Most strikingly, a point mutation was found in the signal sequence of a preproPTH gene in a family with inherited hypoparathyroidism (13). A point mutation at residue 18 changed the cysteine to arginine and thereby inserted a charged residue into the hydrophobic core of the signal sequence. When this mutant preproPTH was expressed in cell-free extracts or in cultured cells, the precursor was inefficiently transported and cleaved (14).

Function of the Pro Sequence The signal sequence of preproPTH, thus, resembles the signal sequences of other secreted proteins and performs the important role of directing the protein across the membrane of the endoplasmic reticulum and into the secretory pathway. The function of the pro sequence is less well established. In all known preproPTH sequences, the pro sequence is six residues long. The first is always positively charged, the third is hydrophobic, and the last two residues are Lys-Arg. This pattern closely resembles that found in rat proalbumin

19

(Arg-Gly-Val-Phe-Arg-Arg) and that predicted to be present in the pro sequence of preproparathyroid hormone-related peptide (Arg-Arg-Leu-Lys-Arg). ProPTH was first discovered as a large PTH-related molecule that was the predominant form of the h o r m o n e found in parathyroid cells after pulse labeling with radioactive amino acids (15,16). Subsequent chase incubations demonstrated that the proPTH was converted to PTH in about 15 minutes; this correlated in time with transport to the Golgi (17). After this time, no trace of the pro peptide or possible fragments could be found in the cell or medium (18). These data strongly suggest that the pro sequence serves an exclusively intracellular function, probably involved in movement through the secretory pathway. Wiren et al. (19) tested this hypothesis by deleting the DNA sequences encoding the pro hexapeptide from cloned cDNA encoding h u m a n preproPTH and by subsequently expressing the cDNA in cell-free protein-synthesizing extracts and in intact rat pituitary GH4 cells. The mutant precursor functioned abnormally in both expression systems. The precursor crossed the membrane of the endoplasmic reticulum inefficiently, and, consequently, the subsequent cleavage of the signal sequence was inefficient. Cells secreted PTH but also secreted a molecule slightly bigger than PTH. Sequence analysis showed that the abnormal protein included the last two residues of the signal sequence. Thus, the removal of the pro sequence resulted in imprecise and inefficient function of the signal sequence. The pro sequence of preproPTH should be considered part of the functional unit responsible for transport and cleavage of the precursor on its entry into the secretory pathway. This result is not surprising. In other precursor proteins, the sequences immediately distal to the signal sequence can affect signal sequence function. One can speculate that the constraints on this region conflict with the constraints on the amino terminus of the mature PTH molecule. The PTH receptor, for example, requires very specific residues at the amino terminus of PTH for subsequent activation of adenylyl cyclase. The experiments of Wiren et al. show that these residues cannot be placed immediately distal to the signal sequence. The pro sequence can be considered a linker region that allows efficient signal sequence function and physically separates the signal sequence from the mature h o r m o n e sequence, which has its own and separate evolutionary constraints. The possibility that the pro sequence has additional functions, such as the promotion of proper folding of the PTH molecule in the endoplasmic reticulum, has not been rigorously examined. The enzyme responsible for cleavage of the pro sequence of proPTH has not yet been characterized, but a n u m b e r of arguments suggest that the protease, furin (or a close relative), is the cleavage enzyme (20).

20

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CI4AeTV.R2

Furin is a subtilisin-like enzyme that is located in the Golgi cisternae of probably all mammalian cells. The enzyme cleaves sequences like the pro sequence of rat proalbumin, which ends in dibasic residues and is preceded by other basic residues. Unlike the related PC2 and PC1 proteases, which are found in cells with secretory granules, furin cleaves precursors in cells like hepatocytes, which have no secretory granules. Cleavage by furin probably explains why proPTH, in contrast to proinsulin, for example, is cleaved normally when the hormone is synthesized in all sorts of cells, from parathyroid chief cells to fibroblasts and kidney cells (21,22). One can only speculate as to why proPTH, which is normally synthesized virtually exclusively in specialized parathyroid chief cells, uses an enzyme designed for cleavage of proteins secreted from nonendocrine cells. One plausible explanation is an evolutionary argument. The parathyroid hormone gene may well be derived from the gene encoding parathyroid hormone-related peptide (PTHrP). The PTHrP gene is widely expressed, both in cells with secretory granules, such as parathyroid chief cells and neurons, and in cells without secretory granules, such as smooth muscle cells. Therefore, it would be expected that the pro sequence of proPTHrP would be designed for cleavage by an enzyme expressed in most cells. The pro sequence of proPTH may well share this property because of its evolutionary heritage, even though proPTH is normally expressed only in cells with a secretory granule apparatus.

Intracellular Roles of the Mature PTH Sequence Like the prepro sequence, portions of the mature PTH molecule serve to facilitate intracellular handling of PTH (23). Shortened versions of preproPTH are not stable in transfected cells. When the h u m a n preproPTH cDNA was modified to encode preproPTH(1-40) (in which the numbers refer to the mature PTH sequence), the signal sequence functioned, and proPTH(1-40) was produced in transfected cells. The proPTH (1-40) was not further cleaved to PTH(1-40), however. Instead, it was degraded intracellularly; no PTH peptides were secreted from the cells. A similar, though less dramatic, defect in secretion was exhibited by preproPTH(1-52). These short precursors were long enough for the signal sequence to direct them into the secretory pathway, but they were unstable and were not transported through the entire pathway. These results may partly explain the role of the carboxy-terminal portion of the PTH molecule. One function of the full 84-residue protein may be to allow stable and efficient transport through the secretory apparatus. Because all secreted peptides are syn-

thesized as rather large precursors, this need for a minimal length of translation product may be a general one for secreted proteins. Of course, this "length" requirement for PTH does not preclude other functions for the carboxy-terminal portion of PTH, such as binding to a distinct PTH receptor (24). Even the 84-residue PTH molecule is not completely stable in the parathyroid chief cell. PTH(1-84) is concentrated in secretory vesicles and granules that contain the proteases, i.e., cathepsins B and H (25,26). This colocalization of proteases and PTH may explain the observation that the hormone secreted by calves in vivo under conditions of hypercalcemia consists largely of carboxy-terminal fragments of PTH (27). Secretion of fragments of PTH was studied in detail by Habener et al. (28) and Chu et al. (29). These workers noted that the degradation of newly synthesized PTH is influenced by the level of extracellular calcium. Few fragments were secreted when the gland was stimulated in vitro by medium containing low levels of calcium. In contrast, most of the hormone secreted under conditions of hypercalcemic suppression consisted of fragments. Thus, calcium regulated the amount of available intact PTH by causing the intracellular degradation of hormone. This effect could have been caused by the activation of a PTH-degrading pathway. Alternatively, the intracellular degradation rate might have been constant; the decrease in total degradation of PTH associated with low calcium levels might simply have resulted from rapid secretion of hormone and the concomitant shorter time of exposure to the intracellular degradation mechanism. Phorbol ester treatment of parathyroid cells in vitro has also been shown to result in the secretion of an increased fraction of PTH fragments, both in high and low calcium concentration conditions (30). Phorbols are either activating a proteolytic mechanism or may be selectively stimulating secretion from secretory granules containing a high proportion of PTH fragments. The physiologic correlate in vivo of this action of phorbol esters has not yet been established. In any case, the parathyroid gland has the capability of varying the fraction of PTH secreted as the biologically active, intact molecule. This seemingly wasteful capability makes it possible for the gland to vary quickly and dramatically the amount of biologically active hormone secreted. This regulatory capability provides a rationale for the intracellular instability of the hormone. To sum up, it can be seen that all portions of the preproPTH molecule have intracellular functions. The prepro region is required for efficient introduction of the hormone to the secretory pathway. The carboxyterminal region of the mature hormone is required for efficient and stable transport of PTH through the secre-

PTH BIOSYNTHESISAND METABOLISM / tory pathway. I n h e r e n t instability of even the full-length h o r m o n e provides a regulatory mechanism that allows extracellular calcium to alter rapidly the a m o u n t of active h o r m o n e available for secretion.

THE PARATHYROID HORMONE GENE The genomic DNAs encoding h u m a n (31), bovine (32), rat (33), and chicken (34) p r e p r o P T H have been cloned; the complete sequences of the h u m a n (35) and bovine (32) genes have been determined. Each gene contains three exons separated by two introns (see Fig. 4). The introns vary in size from species to species, though the first intron is invariably large, and the second intron in the human, bovine, and rat genes is about 100 base pairs in length. This length is close to the m i n i m u m length that can be recognized by the splicing machinery. The introns interrupt the sequences encoding mRNA at precisely the same locations in each species. The first exon contains most of the 5' n o n c o d i n g sequence. The second exon encodes most of the prepro sequence; the second intron comes in the middle of the triplet encoding the lysine residue that precedes the dibasic cleavage sequence Lys-Arg found at the end of the known pro sequences. The third exon encodes the Lys-Arg sequence, the mature PTH sequence, and the 3' noncoding region of the gene. The h u m a n and bovine genes are preceded by two functional TATA boxes that determine the two closely spaced start sites of the h u m a n and bovine transcripts. The rat and chicken genes are preceded only by one TATA box, found in a position equivalent to the second TATA box in the h u m a n and bovine genes. T h o u g h both start sites of transcription are used in the h u m a n and bovine genes, no conditions have been found that favor the use of one start site over the other. No data suggest that the two transcripts have importantly different stabilities or translatability, but such questions have not been exhaustively studied. The 5' n o n c o d i n g regions of each gene extend approximately 120 base pairs. The 3' n o n c o d i n g

iii !il!i iiiii!!i!iiiiii!iii! !iiiii!iiii!iiii!i~i!i~ii ii i liiii i!i iililiii ~~ii~!iiii

iiii!i i~i!iilililiiii i iiiiiii!illi!i!!ii!iiiiiii ii!i!iiiiiiiil iiliiiii ii!iil iiili!iiiiil!iiiiiiiiiii!i~iiiii!iiiiiil

ii

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!!!!!!!!!!!!! !!

!!!ii i!ii!iiiiiiii!iii !iili!i!iiiiiiiii!i!i!i!iiiiiii!iiiiiiiiiiiiiiilii i!iiiii!i!iiiii!!i!i!!!!!!ii i i i!i i~i~~ii~iii! !ill!if!!i~!~ ili i! i!ii! i! !i!i!iiii!ii!!iiiii!ii! i iiiiiii ii!iil FIG. 4 The parathyroid hormone gene. NC, Noncoding.

21

regions of each gene vary substantially in length, from the bovine at 227 base pairs to the chicken at more than 1600 base pairs. The 3' n o n c o d i n g region binds proteins that may regulate the stability of the p r e p r o P T H mRNA (36,37). The human, rat, and bovine PTH genes are represented only once in the haploid genomes of each species. The h u m a n PTH gene is located on the short arm of chromosome 11 at band 1 lp15 (38-40). A series of restriction fragment length polymorphisms (41,42) have made it possible to show that the h u m a n PTH gene is linked to the genes encoding catalase, calcitonin, H-ras, insulin, and [3-globin (43). Two other polymorphisms have been identified through the use of denaturing gel electrophoresis (44). All of these polymorphisms have proved useful in defining the inheritance of specific alleles of the PTH gene in families with calcium disorders (45). Several features of the PTH gene suggest that the gene is related to that encoding PTHrP (46-48). Most importantly, the major coding exon of both genes starts precisely at the same nucleotide, one base before the codons encoding the Lys-Arg residues of the pro sequences of each hormone. After the Lys-Arg sequences, the PTH and PTHrP amino acid sequences are identical in 8 of the next 13 residues. Further, the PTHrP gene is located on c h r o m o s o m e 12, a chromosome known to encode many genes that resemble genes on c h r o m o s o m e 11; for this reason, the chromosomes are t h o u g h t to have arisen by an ancient duplication event (49). One can speculate that the PTH gene may represent a variation of the PTHrP gene; the PTH h o r m o n e takes advantage of the PTHrP receptor in order to regulate calcium metabolism. If this hypothesis is correct, then the gene had to change in order to assure expression primarily in the parathyroid chief cell and to assure appropriate regulation by modulators such as extracellular calcium and 1,25-dihydroxyvitamin D [ 1,25 (OH) zOo]. A hypothalamic peptide called TIP39 (50) has been found to activate the PTH2 receptor and to be distantly related in sequence to PTH and PTHrE The structure of the TIP39 gene has not yet been reported. This gene may represent a third member of the PTH gene family.

REGULATION OF PTH BIOSYNTHESIS The minute-to-minute stability of the level of blood calcium depends on the regulation of PTH secretion by calcium. Longer term homeostasis depends on several other levels of control (see Fig. 5). The n u m b e r of parathyroid chief cells is carefully regulated; when appropriately stimulated, the parathyroid glands can

22

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C~a'TER2

TRANSCRIPTION/mRNA STABILITY

Ca, 1,25 D, PO4 Ca

Ca, 1,25 D

P H SECRETION PROLIFERATION

FIG. 5

Levels of parathyroid cell regulation.

increase in size dramatically. The parathyroid chief cell is uniquely designed to express the PTH gene; the state of differentiation of the chief cell can, therefore, influence the rate of PTH biosynthesis. Specific blood-borne signals, most notably calcium and 1,25(OH)zD3, regulate the activity of the PTH gene, as well. These several levels of regulation of PTH biosynthesis are examined in the following discussions (see also Chapter 18).

Regulation of Parathyroid Cell Number Little is known about the regulation of parathyroid cell number. The relatively uniform morphology of chief cells suggests that all chief cells have the potential to divide, if appropriately stimulated, but the alternative hypothesis that a subset of chief cells has the unique, stem-cell-like capability to proliferate has not been evaluated. Further, there is the general impression that parathyroid cells are long-lived, because mitoses are seldom seen in normal glands of mature animals, because the observed rate of apoptosis is low, and because hyperplastic glands only slowly decrease in size after stimulation. Nevertheless, specific studies to define potential modulators of chief cell longevity have not been performed. Despite this paucity of information, the dramatic hyperplasia of parathyroid cells in patients and animals with renal failure demonstrates the likely roles of calcium, phosphate, and 1,25(OH)2D 3 in regulating parathyroid cell proliferation. Dietary manipulation alone can similarly lead to chief cell hyperplasia. Naveh-Many and Silver (51), for example, used flow cytometry to count parathyroid cells and showed that 3 weeks of a calcium- and vitamin D-deficient diet fed to weanling rats led to a 1.7-fold increase in parathyroid cell number. These investigators subsequently studied the mechanism of the increase in parathyroid cell number caused by hypocalcemia, hyperphosphatemia, vitamin D deficiency, and uremia in vivo (52). They found that hypocalcemia and

uremia led to increases in parathyroid cell proliferation, whereas hypophosphatemia led to decreases in parathyroid cell proliferation. Administration of 1,25(OH)2D 3 for 3 days had no effect on parathyroid cell proliferation. None of these conditions led to changes in the rate of parathyroid cell apoptosis. Further studies of the effects of calcium in the uremic model suggest that calcium works by acting on the same calcium-sensing rector that mediates the actions of calcium on PTH secretion. The calcimimetic compound NPS R-568, like calcium, suppressed parathyroid cell proliferation in uremic rats (53). The possibly i n d e p e n d e n t roles of calcium and 1,25(OH)2D 3 in the regulation of parathyroid cell proliferation have not been studied extensively. In vivo, these variables are difficult to manipulate independently in the intact animal. One particularly instructive in vivo model, the vitamin D receptor knockout mouse, has been studied, however (54,55). These mice develop hypocalcemia, hypophosphatemia, and secondary hyperparathyroidism in the days and weeks after weaning. When the hypocalcemia and hypophosphatemia are prevented by a diet high in calcium, phosphate, and lactose, the hyperparathyroidism and parathyroid gland enlargement are prevented. Because these mice lack vitamin D receptors, they must be able to regulate parathyroid cell n u m b e r without using the genomic actions of 1,25 (OH) 2D~. Presumably, the direct effects of normal calcium and phosphate are sufficient to prevent parathyroid cell replication. In studies of cultured parathyroid chief cells, it has been possible to vary the levels of calcium and 1,25(OH)2D ~ separately. Several groups have shown that 1,25(OH)2D 3 can regulate parathyroid cell proliferation in vitro. Whether the cells were grown in the presence of serum (56,57) or serum-free growth factors (58), administration of 1,25(OH)2D 3 decreased their rate of proliferation. Studies of the effects of calcium on parathyroid cell proliferation in vitro have yielded differing results. Several studies (59-61) have shown that lowering of calcium leads to increased cellular proliferation. Other studies of dispersed, early-passage chief cells have demonstrated no effect of calcium on the rate of cell proliferation, however (57,58,62). Though extracellular levels of calcium and 1,25(OH)2D~ can be independently regulated in vitro, it is hard to be sure that parathyroid cells in culture respond to modulators of proliferation in this setting in the same way that they do in vivo. Thus, though the combined effects of low calcium and low levels of 1,25(OH)2D ~ to stimulate parathyroid cell proliferation are well established, the individual roles of calcium, phosphate, and 1,25 (OH)2D~ in vivo remain uncertain.

PTH BIOSYNTHESISAND METABOLISM /

Cell-Specific PTH Gene Expression Expression of the parathyroid h o r m o n e gene occurs almost exclusively in the parathyroid chief cell. [Expression has also been noted in the rat hypothalamus (63).] Thus, genes required for parathyroid chief cell differentiation are possible candidates for genes that might regulate the PTH gene as well. These genes, identified through the study of knockout mice, include hoxa3 (64,65), pax9 (66), and glial cells missing 2 (67). The hoxa3 and pax9 mutant mice lack a range of branchial arch derivatives, whereas the glial cell missing knockout mouse exhibits highly selective parathyroid cell deficit. When the chief cell is disrupted by neoplastic transformation, the regulation of PTH gene expression can be altered. For example, parathyroid cancers may stop synthesizing PTH completely (68). Presumably, specific DNA sequences associated with the PTH gene respond to the environment of the chief cell to activate gene expression. Because no well-differentiated cell line expressing the PTH gene has been established, it has been difficult to determine the sequences responsible for chief cell-specific PTH gene expression. Occasional "experiments of nature" have provided important clues, however. Very rarely, h u m a n nonparathyroid tumors have been found to produce PTH ectopically, for example. In one case that was studied carefully (69), the PTH regulatory region upstream from the gene was disrupted in tumor cells. Presumably, this gene r e a r r a n g e m e n t allowed the gene to be expressed in nonparathyroid cells by providing new regulatory signals or abolishing normal silencing mechanisms found upstream of the gene. Further, in a subset of parathyroid adenomas, the entire upstream portion of the PTH gene along with the first, noncoding exon are separated from the rest of the gene and rearranged adjacent to the PRAD1 gene (70). As a consequence of this rearrangement, the PRAD1 gene (encoding cyclin D1), a regulator of the cell cycle, is dramatically overexpressed. These observations suggest that the PTH gene upstream region contains sequences that stimulate gene transcription in parathyroid chief cells. Further analysis of the sequences that determine chief cell expression of the PTH gene m u s t await studies of transgenic animals or the establishment of welldifferentiated parathyroid chief cell lines.

Modulators of PTH Gene Expression The effects of calcium on PTH gene expression were first demonstrated in experiments using primary parathyroid cells in culture. Russell et al. (71) found that high levels of calcium resulted in a decrease in PTH mRNA levels over a several-day period. In those

23

studies, no difference was noted between the effects of low and normal levels of extracellular calcium. The decrease in PTH mRNA levels in response to high calcium levels could be reversed by lowering the calcium level; thus, the suppressive effect of calcium was not an irreversible, toxic effect. These in vitro observations have been confirmed by Brookman et al. (72), who noted a slight increase in PTH mRNA u n d e r low calcium level conditions at one time point. Subsequent studies by Russell et al. (73) showed that the rate of transcription of the PTH gene in nuclei of dispersed bovine parathyroid cells fell within 6 hours in response to high levels of extracellular calcium. The rate of transcription of the actin gene was unchanged; therefore, the effect of calcium was shown to be specific. The lack of parathyroid cell lines that produce PTH has h a m p e r e d the search for DNA sequences responsible for the transcriptional effects of calcium noted in cultured parathyroid cells. Okazaki et al. (74) have identified short sequences several thousand base pairs upstream from the start site of PTH gene transcription that may well be important for calcium regulation, however. These investigators identified the region by showing that several short sequences in the region could decrease gene transcription from many different promoters, including the PTH gene p r o m o t e r (75). Further, when the level of extracellular calcium was varied, after transfection of fusion genes containing a short oligonucleotide from this region, high calcium levels further suppressed transcription from genes containing the sequence but had no effect on control plasmids. Intriguingly, almost identical sequences were found in the gene encoding rat atrial natruiretic polypeptide, another gene negatively regulated by calcium. This DNA sequence could also confer calcium sensitivity to a fusion gene in fibroblast transfection experiments. T h o u g h these experiments are very suggestive, further studies will be required to show that the regulatory region can confer calcium sensitivity in its normal location far upstream from the PTH gene transcription start site. Ultimately, studies using welldifferentiated parathyroid cells will be required. Two groups have studied the acute effects of changes in blood calcium on PTH mRNA levels in the intact rat. Both showed that acute lowering of blood calcium (with phosphate, calcitonin, or EDTA) led to a p r o m p t increase in PTH mRNA levels (76,77). Elevations in blood calcium, in contrast, led to no change in PTH mRNA levels after 6 hours (76) and to a slight decrease in PTH mRNA levels after 48 hours (77). The parathyroid gland apparently, then, in the normal state, rests near the bottom of the calcium dose-response curve. The gland is well equipped to increase PTH production, but poorly prepared to decrease production in the

24

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CHAPTER2

face of hypercalcemia. Subsequent studies showed that changes in PTH mRNA caused by hypocalcemia in vivo are not caused by a transcriptional mechanism, but rather are caused by changes in mRNA stability (37). 1,25(OH)zD 3 has been shown to be an i m p o r t a n t regulator of PTH gene transcription in studies both in vitro and in vivo. Silver et al. (78) used primary parathyroid cells in culture to show that exposure to 1,25(OH)2D 3 led to a decrease in PTH mRNA levels. This work has been confirmed by studies of Karmali et al. (79) and Brown et al. (80). Russell et al. (81) then showed that 1,25(OH)zD 3 lowers the PTH gene transcription rate as early as 2 hours after exposure of cells to 1,25 (OH)zD 3. Similarly, in intact rats, intraperitoneal injections of 1,25(OH)zD 3 rapidly led to decreased transcription of the PTH gene and decreased PTH mRNA levels (82). The doses of 1,25(OH)zD 3 were so low that blood calcium did not change; the precise blood levels of 1,25(OH)zD 3 required to suppress PTH gene transcription acutely in vivo have not been established, however. The effects of low levels of 1,25(OH)zD 3 have not been studied extensively in intact animals. Such studies are difficult to interpret, because of confounding effects of vitamin D deficiency on blood calcium and parathyroid cell number. Weanling rats fed a vitamin Ddeficient diet for 3 weeks had a modest increase in their PTH mRNA levels (51). This increase occurred with no a p p a r e n t decrease in blood calcium levels. In the intact organism, calcium and 1,25(OH)zD 3 seld o m vary independently; consequently, the effects of changes in both parameters simultaneously have important physiologic relevance. When rats were made acutely hypocalcemic with phosphate and were at the same time given 1,25(OH)zD 3 intraperitoneally, the suppressive effect of 1,25(OH)2D 3 reversed the effect of hypocalcemia and led to a decrease in PTH mRNA (76). In contrast, when rats were fed a low-calcium diet for 3 weeks, blood calcium levels decreased and blood 1,25 (OH) 203 levels increased dramatically. In this setting, PTH mRNA levels rose severalfold; thus, the effects of low calcium levels were more influential than the effects of high 1,25(OH)zD 3 levels. The partial vitamin D resistance of the parathyroid gland in the setting of hypocalcemia makes sense physiologically: in that setting the action of vitamin D to increase intestinal calcium absorption is needed, but the action to inhibit PTH synthesis is not. Sela-Brown et al. (83) studied the mechanism of hypocalcemia-induced resistance to vitamin D action on the parathyroid gland. They showed that hypocalcemia in vivo induces nuclear accumulation of calreticulin, a calcium-binding protein, in parathyroid chief cells, and that calreticulin can interfere with the actions of the vitamin D receptor on a negative vitamin D response e l e m e n t in transfected cells in vitro.

In experimental uremia, the double stimulus of hypocalcemia and low levels of 1,25(OH)zD 3 has consistently led to increases in PTH mRNA (84,85). Administration of 1,25(OH)zD 3 could reverse this increase. This effect of 1,25(OH)zD 3 is likely to contribute importantly to the decrease in PTH blood levels seen in dogs with experimental uremia (86) and in dialysis patients (87). A series of transfection studies and DNA binding assays have been used to identify DNA sequences in the PTH gene responsible for modulating transcription of the PTH gene in response to 1,25(OH)2D 3. When a fusion gene containing 684 base pairs (bp) of DNA upstream of the h u m a n PTH gene was introduced stably into rat pituitary GH4 cells, expression of the gene was specifically suppressed by 1,25(OH)2D 3 (88). Three groups have identified DNA sequences upstream of the PTH gene that bind to 1,25(OH)zD 3 receptors in vitro. Filter binding assays showed that 1,25(OH)zD 3 receptors can bind to bovine PTH gene sequences between - 4 8 5 and - 1 0 0 bp upstream from the transcription start site (89). Subsequently, gel mobility-shift assays were used to identify a specific 26-bp sequence, located 125 bp upstream from the start site of transcription of the h u m a n PTH gene, that binds 1,25 (OH)203 receptors (90). When this short sequence was linked to a reporter gene and expressed in pituitary GH4 cells, 1,25(OH)zD 3 decreased expression of the reporter gene. This suppression of transcription was even greater when the n u m b e r of 1,25(OH)zD 3 receptors in the GH4 cells was increased by cotransfection of a 1,25(OH)zD 3 receptor expression vector. The h u m a n negative 1,25 (OH)203 (vitamin D) response e l e m e n t (VDRE) contains one copy of a motif found in two copies in the mouse osteopontin gene, a gene up-regulated by 1,25(OH)zD 3. Negative VDREs have also been identified in the chicken (91) and rat (92) PTH genes. These sequences closely resemble positive VDREs and have been shown to bind heterodimers of the vitamin D receptor and RXR, just as positive VDREs do. Subtle differences in binding interactions may explain why these particular VDREs in the PTH gene can act as negative VDREs with vitamin D receptor-RXR heterodimers (93). Until recently, the effects of phosphate on the parathyroid cell were t h o u g h t to be indirectly mediated by the hypocalcemia associated with increases in blood phosphate. The rapid actions of changes in phosphate levels in vivo on PTH secretion work through such a mechanism. However, studies using intact rat parathyroid glands in vitro demonstrate that changes in phosphate levels can, after several hours, lead to changes in PTH secretion (94,95). PTH mRNA did not change in these studies in vitro, but analogous studies p e r f o r m e d in intact rats d e m o n s t r a t e d that phosphate, in the setting of apparently constant levels of calcium and

PTH BIOSYNTHESISAND METABOLISM / 1,25(OH)2D ~, increases PTH mRNA by a posttranscriptional mechanism (96). Though calcium and 1,25(OH)zD 3 are certainly the most important physiologic regulators of PTH gene transcription, other circulating factors are likely to modulate PTH gene transcription as well. The PTH gene contains a consensus cyclic AMP response element that can function in the context of a fusion gene in transfection experiments (97). Thus, hormones that stimulate adenylyl cyclase may increase PTH gene transcription. Glucocorticoids have been shown to increase PTH mRNA in dispersed, hyperplastic h u m a n parathyroid cells (98) and to abolish the decrease in PTH mRNA in response to 1,25(OH)zD ~ in dispersed bovine parathyroid cells (79). These cell culture studies need to be confirmed by studies in vivo to determine their physiologic significance. In ovariectomized rats, estradiol administration led within 24 hours to a fourfold increase of PTH mRNA (99). Estrogen receptors were identified in rat parathyroids. These observations may have important implications for an understanding of postmenopausal osteoporosis and hyperparathyroidism. The possibility that the effect of estrogen on PTH mRNA levels is a direct effect on the parathyroid gland needs to be tested by studies using cultured parathyroid cells.

Peripheral Metabolism of PTH Intact PTH is rapidly cleared from the circulation with a disappearance half-time of approximately 2 minutes (100-103). Removal of PTH from the blood occurs mainly (60-70%) in the liver but also in the kidneys (20-30%) and, to a much lesser extent, in other organs (100,102,103). Clearance of PTH by the liver is mediated mainly by a high-capacity, nonsaturable uptake by Kupffer cells and is followed by rapid and extensive proteolysis (104). Renal clearance occurs almost entirely by glomerular filtration. The hormone is also reabsorbed by the renal tubules and then extensively degraded, so that little or no intact PTH appears in the final urine (102). A large membrane-bound protein, megalin, binds PTH (but not carboxy-terminal fragments of PTH) in the lumen of the proximal tubule to initiate this reabsorption (105). In both the liver and kidney, as in bone, some PTH is removed by high-affinity binding to cell surface receptors, but this constitutes only a small fraction (<1%) of overall PTH clearance (102,106,107). Thus, it appears that the main role of the liver and kidney in PTH metabolism is rapid removal and degradation of circulating biologically active hormone, assuring that the concentration of hormone available to receptors in target tissues is dictated exclusively by the rate at which PTH is secreted by the parathyroid glands.

25

This simple first-order model, in which secreted intact PTH that is not bound to specific high-affinity cellular receptors is rapidly cleared by peripheral organs, was found to be inadequate when it was recognized that multiple species of immunoreactive PTH molecules are present in the circulation (108,109). Early observations with region-specific immunoassays in vivo and in medium from perfused organs in vitro, followed later by direct analysis of the peripheral metabolism of intravenously administered radioactive PTH, confirmed that intact PTH is not only rapidly cleared from the blood but also is rapidly cleaved by endoproteases to a series of carboxyl-terminal (C) fragments, some of which reenter the circulation (100,102,103,108,110,111). The chemical identities of these fragments have not been established definitively. Microradiosequencing of radioactive fragments recovered from blood of rats following intravenous administration of [125I-Tyr-43]bPTH or [SH-Tyr-43]bPTH has shown that PTH peptides produced by cleavages between residues 33-34, 36-37, 40-41, and 42-43 are the predominant large "signature" C fragments (103,104,107,110). These fragments exhibit apparent molecular masses of 4000-7000 Da and it is not certain that they all extend entirely to the original C terminus (i.e., to residue 84) of the intact hormone. Also, because fragments shorter than PTH(43-84) would not have been detected by these radiosequencing analyses, it is possible that such shorter fragments also exist in blood. Other evidence, derived from detailed analysis by region-specific immunoassays of circulating forms of PTH, points to the presence in blood of shorter, "midmolecule" fragments, presumably derived from proteolysis within both the amino (N-) and C-terminal portions of the intact hormone (108,112,113). In vitro, isolated hepatic Kupffer cells, but not hepatocytes, generate C fragments that are chemically identical to the major circulating form (104,114). Also, liver ablation, but not nephrectomy, blocks the production of circulating C fragments that otherwise are produced by proteolysis of administered radiolabeled intact PTH (103). These findings suggest that hepatic Kupffer cells are responsible for both the rapid clearance and the extensive proteolysis of PTH that occur in the liver. Moreover, Kupffer cells appear also to be the source of the major circulating C fragments identified so far that result from postsecretory metabolism of PTH. In the kidney, extensive proteolysis of PTH occurs also, but the kidney does not contribute significantly to the circulating pool of PTH C fragments (100,103,110). Studies of the cellular enzymes responsible for these cleavages are most consistent with a role for lysosomal enzymes of the cathepsin B/D family (107,115). Quantitatively, less than 10-20% of secreted intact PTH is converted to circulating C fragments by

26

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CHAPTER2

peripheral metabolism (100,103,110). On the other hand, C fragments comprise between 50 and 90% of total circulating PTH immunoreactivity (102,107,108,112,113,116). This disparity results from at least two factors. First, the clearance of C fragments, which does not occur in the liver but rather proceeds mainly via glomerular filtration in the kidney, is significantly slower than that of intact PTH (102,117). Second, it is now clear that C fragments are secreted along with intact PTH by the parathyroid glands, which thus constitute an independent source of these fragments (115,116,118-120). Remarkably, the major C fragments secreted by the parathyroids are chemically identical (at their N termini) with the principal circulating products of peripheral metabolism of PTH (115,119). The impact of these factors is especially obvious in renal insufficiency, in which delayed renal clearance (up to 100-fold) of C fragments, combined with their accelerated generation, both within the hyperplastic parathyroid glands and during peripheral metabolism of overproduced intact hormone, leads to massive accumulation of C fragments vs. intact hormone in the circulation. Metabolism of PTH, both peripherally and within the parathyroid glands, involves cleavages within the region PTH(33-43), which, at least potentially, could generate biologically active N-terminal fragments. Accordingly, considerable interest has focused on the possibility that such circulating N fragments might result from PTH metabolism and, further, that the overall rate or pattern of PTH proteolysis might be regulated physiologically to modulate the production of such fragments. Circulating amino-terminal PTH fragments have been demonstrated occasionally, almost exclusively in the setting of renal failure or hyperparathyroidism, or both, but it has been difficult to exclude postcollection proteolysis as the explanation in these circumstances (107,108,121). Moreover, immunochemical analyses of normal plasma have not provided convincing evidence of circulating N-terminal PTH fragments. More recently, direct analysis of the fate of the N terminus of PTH has been possible using [~5S]methionine to radiolabel biologically active hormone to high specific activity within the N-terminal region of the molecule. These studies in normal rats have shown that N-terminal PTH fragments produced by isolated Kupffer cells in vitro or by the liver or kidney in vivo are rapidly degraded in situ and do not reenter the circulation, at least in concentrations above 50 fM (100). Similar investigations have provided evidence that peripheral metabolism of PTH is not regulated physiologically in response to alterations in serum or dietary calcium, vitamin D intoxication, or parathyroid status (101,122). In contrast, secretion of C fragments by the parathyroid glands is strikingly influenced by

extracellular calcium, in that hypercalcemia increases the ratio of secreted C fragments to intact hormone whereas the opposite occurs in hypocalcemia (108,116,118,119). These alterations in the intracellular proteolysis of PTH are reflected by corresponding changes in the predominant immunoreactive forms of PTH in the circulation observed during hypercalcemia and hypocalcemia (108,112). The extensive metabolism of PTH(1-84) to fragments of PTH that accumulate in the circulation but cannot activate the P T H / P T H r P receptor has created an obstacle to the measurement of biologically active PTH in the circulation by immunologic methods. A series of two-site immunoassays, in which antibodies to the amino-terminal region and carboxy-terminal region of PTH must both bind the ligand simultaneously to register as a PTH molecule, have revolutionized the clinical usefulness of PTH assays (123). However, later generation versions of these assays have demonstrated unanticipated complexity in the PTH fragments present in the circulation (124-126). Some two-site PTH assays, but not others, recognize a large PTH fragment, roughly the size of PTH(7-84). This fragment is normally only a small fraction of the circulating PTH, but it can represent as much as 50% of the PTH recognized by some two-site PTH assays in the presence of renal failure. The fraction of immunoreactive PTH represented by the large PTH fragments is also increased in hypercalcemic states. Though PTH fragments missing the amino terminus of PTH are not expected to activate the P T H / P T H r P receptor, the possibility that these fragments might antagonize the actions of PTH (1-84) or might have unique activities of their own is currently being explored. The precise structure, site of origin, and biologic activities of these fragments, thus, are important questions for the future. In summary, the extremely rapid peripheral clearance and proteolysis of intact PTH play an important role in limiting the duration of hormone action and in assuring that the secretory activity of the parathyroid glands is the overriding determinant of the circulating concentration of biologically active PTH. Whereas a small percentage of degraded PTH molecules reappears in the blood, these are exclusively composed of large carboxyl-region and midregion fragments that are devoid of classic PTH bioactivity. Additional fragments are released directly by the parathyroid glands, reflecting an intraglandular mechanism involved in calcium regulation Of hormone secretion, but these too are biologically inactive. It remains possible that such carboxylregion and midregion PTH fragments may exert novel biologic actions in some tissues via receptors other than those known to be activated by intact PTH or N-terminal PTH fragments, but there is little evidence at present in support of a critical physiologic role for such fragments.

P T H BIOSYNTHESIS AND METABOLISM

CONCLUSION The blood level of PTH is regulated at several levels, each designed to respond to different challenges to calcium homeostasis. Over short time frames, the regulation of PTH secretion by calcium, coupled to the rapid metabolism of the hormone, assures that the blood level of PTH can adjust to sudden changes in calcium flux. Turnover of PTH within the parathyroid gland decreases under hypocalcemic conditions; this adjustment provides rapid increases in available hormone. Over a longer time frame both calcium and 1,25(OH)zD 3 regulate PTH biosynthesis. It is, of course, reasonable that 1,25(OH)zD ~ should regulate PTH biosynthesis, because the need for PTH can be expected to be great in face of vitamin D deficiency, no matter what the instantaneous level of blood calcium. Though the physiologic studies are not extensive, the synthetic machinery seems designed particularly to respond dramatically to decreases in blood calcium and 1,25 (OH) 203 levels. Over a longer time period, calcium and, perhaps to a greater extent, 1,25(OH)zD ~ regulate the n u m b e r of parathyroid cells. This is a relatively crude and slow process. The slow turnover of parathyroid chief cells suggests that the parathyroid gland is not meant to rely on frequent changes in parathyroid cell number, but uses this method of amplifying its signal only when other alternatives prove insufficient. This perspective on parathyroid control, with its emphasis on multiple levels of regulation, provides a framework for understanding the alterations caused by disease and may suggest therapeutic strategies as well.

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25. Hashizume Y, Waguri S, Watanabe T, Kominami E, Uchiyama Y. Cysteine proteinases in rat parathyroid cells with special reference to their correlation with parathyroid hormone (PTH) in storage granules. J Histochem Cytochem 1993;41:273-282. 26. MacGregor RR, Hamilton JW, Shofstall RE, Cohn DV. Isolation and characterization of porcine parathyroid cathepsin B. J Biol Chem 1979;254: 4423-4427. 27. Mayer GP, Keaton JA, Hurst JG, Habener JE Effects of plasma calcium concentration on the relative proportion of hormone and carboxyl fragments in parathyroid venous blood. Endocrinology 1979;104:1778-1784. 28. Habener JE Kemper B, Potts JT, Jr. Calcium-dependent intracellular degradation of parathyroid hormone: A possible mechanism for the regulation of hormone stores. Endocrinology 1975;97:431-441. 29. Chu LLH, MacGregor RR, Anast CS, et al. Studies on the biosynthesis of rat parathyroid hormone and proparathyroid hormone: Adaptation of the parathyroid gland to dietary restriction of calcium. Endocrinology 1973;93:915-924. 30. Tanguay KE, Mortimer ST, Wood PH, Hanley DA. The effects of phorbol myristate acetate on the intracellular degradation of bovine parathyroid hormone. Endocrinology 1991;128:1863-1868. 31. Vasicek T, McDevitt BE, Freeman MW, et al. Nucleotide sequence of the human parathyroid hormone gene. Proc Natl Acad Sci USA 1983;80:2127. 32. Weaver CA, Gordon DE Kissil MS, et al. Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone. Gene 1984;28:319-329. 33. Heinrich G, Kronenberg HM, Potts JT, Jr, Habener JE Gene encoding parathyroid hormone: Nucleotide sequence of the rat gene and deduced amino acid sequence of rat preproparathyroid hormone. JBiol Chem 1984;259:3320. 34. Russell J, Olivera A, Liu S, Sherwood LM. Isolation and complete nucleotide sequence of the avian parathyroid hormone gene. Endocrine Society Program and Abstracts, 73rd Annual Meeting, June 19-22, 1991. 35. Reis A, Hecht W, Gr6ger R, B6hm I, Cooper DN, Lindenmaier W, Mayer H, Schmidtke J. Cloning and sequence analysis of the human parathyroid hormone gene region. Hum Genet 1990;84:119-124. 36. Moallem E, Silver J, Naveh-Many T. Post-transcriptional regulation of PTH gene expression by hypocalcemia due to protein binding to the PTH mRNA 3' UTR. J Bone Miner Res 1995;10(Suppl 1):S142. 37. Moallem E, Kilav R, Silver J, Naveh-Many T. RNA-protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate. J Biol Chem 1998;273 (9) :5253-5259. 38. Naylor SL, Sakaguchi AY, Szoka P, et al. Human parathyroid hormone gene (PTH) is on short arm of chromosome 11. Somatic Cell Genet 1983;9:609-616. 39. Mayer H, Breyel E, Bostock C, Schmidtke J. Assignment of the human parathyroid hormone gene to chromosome 11. Hum Genet 1983;64:283-285. 40. Zabel BU, Kronenberg HM, Bell GI, Shows TB. Chromosome mapping of genes on the short arm of human chromosome 11: Parathyroid hormone gene is at l lp15 together with the genes for insulin, c-Harvey-ras 1, and [3-hemoglobin. Cytogenet Cell Genet 1985;39:200-205. 41. Antonarakis SE, Phillips III, JA, Mallonee RL, et al. [3-globin locus is linked to the parathyroid hormone (PTH) locus and lies between the insulin and PTH loci in man. Proc Natl Acad Sci USA 1983;80:6615-6619. 42. Schmidtke J, Pape B, Krengel U, et al. Restriction fragment length polymorphisms at the human parathyroid hormone gene locus. Hum Genet 1984;67:428-431.

43. Kittur SD, Hoppener JWM, Antonarakis SE, et al. Linkage map of the short arm of human chromosome 11: Location of the genes for catalase, calcitonin, and insulin-like growth factor II. Proc Natl Acad Sci USA 1985;82:5064-5067. 44. Miric A, Levine MA. Analysis of the preproPTH gene by denaturing gradient gel electrophoresis in familial isolated hypoparathyroidism. J Clin Endocrinol Metab 1991 ;74:509-516. 45. Ahn TG, Antonarakis SE, Kronenberg HM, et al. Familial isolated hypoparathyroidism: A molecular genetic analysis of 8 families with 23 affected persons. Medicine 1986;65:73-81. 46. Suva LJ, Winslow GA, Wettenhall REH, Hammonds RG, Moseley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI. A parathyroid hormone-related protein implicated in malignant hypercalcemia: Cloning and expression. Science 1987;237:893-896. 47. Mangin M, Ikeda K, Dreyer BE, Broadus AE. Isolation and characterization of the human parathyroid hormone-like peptide gene. Proc Natl Acad Sci USA 1989;86:2408-2412. 48. Yasuda T, Banville D, Hendy GN, Goltzman D. Characterization of the human parathyroid hormone-like peptide gene: Functional and evolutionary aspects. J Biol Chem 1989;264: 7720-7725. 49. Comings DE. Evidence for ancient tetraploidy and conservation of linkage groups in mammalian chromosomes. Nature 1972;238:455-457. 50. Usdin TB, Hoare SRJ, Wang T, Mezey E, Kowalak JA. TIP39: A new neuropeptide and PTH2-receptor agonist from hypothalamus. Nat Neurosci 1999;2 (11):941-943. 51. Naveh-Many T, Silver J. Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat. J Clin Invest 1990;86:1313-1319. 52. Naveh-Many T, Rahamimov R, Livni N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure rats: The effects of calcium, phosphate and vitamin D. J Clin Invest 1995;96:1786-1793. 53. Wada M, Furuya Y, Sakiyama J, Kobayashi N, Miyata S, Ishii H, Nagano N. The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. J Clin Invest 1997;100(12):2977-2983. 54. Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, Demay MB. Targeted ablation of the vitamin D receptor: An animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci USA 1997;94(18):9831-9835. 55. Li YC, Pirro AE, Demay MB. Analysis of vitamin D-dependent calcium-binding protein messenger ribonucleic acid expression in mice lacking the vitamin D receptor. Endocrinology 1998; 139 (3) :847-851. 56. Nygren P, Larsson R, Johansson H, Ljunghall S, Rastad J, Akerstr6m G. 1,25(OH)zD ~inhibits hormone secretion and proliferation but not functional dedifferentiation of cultured bovine parathyroid cells. CalcifTissue Int 1988;43:213-218. 57. Kremer R, Bolivar I, Goltzman D, Hendy GN. Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinology 1989;125:935-941. 58. Ishimi Y, Russell J, Sherwood LM. Regulation by calcium and 1,25-(OH)zD ~ of cell proliferation and function of bovine parathyroid cells in culture. J Bone Miner Res 1990;5:755-760. 59. Lee MJ, Roth SI. Effect of calcium and magnesium on deoxyribonucleic acid synthesis in rat parathyroid glands in vitro. Lab Invest 1975;33:72-79. 60. Raisz LG. Regulation by calcium of parathyroid growth and secretion in vitro. Nature 1963;197:1115-1117. 61. Brandi ML, Fitzpatrick LA, Coon HG, Aurbach GD. Bovine parathyroid cells maintained for more than 140 population doublings. Proc Natl Acad Sci USA 1986;83:1707-1713.

P T H BIOSYNTHESIS AND METABOLISM 62. Leboff MS, Rennke HG, Brown EM. Abnormal regulation of parathyroid cell secretion and proliferation in primary cultures of bovine parathyroid cells. Endocrinology 1983;113:227-284. 63. Fraser RA, Kronenberg HM, Pang PK, Harvey S. Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus. Endocrinology 1990;127:2517-2522. 64. Manley NR, Capecchi MR. Hox group 3 paralogous genes act synergistically in the formation of somitic and neural crestderived structures. Dev Biol 1997;192 (2):274-288. 65. Manley NR, Capecchi MR. Hox group 3 paralogs regulate the development and migration of the thymus, thyroid, and parathyroid glands. Dev Bio11998;195(1) :1-15. 66. Peters H, Neubuser A, Kratochwil K, Balling R. Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities. Genes D e v 1998;12 (17):2735-2747. 67. Gunther T, Chen Z, Kim J, Priemel M, Rueger JM, Amling M, Moseley JM, Martin TJ, Anderson DJ, Karsenty G. Parathyroid hormone secretion in absence of parathyroid glands. J Bone Miner Res 1999;14(Suppl. 1) (Abstract 1008). 68. Baba H, Kishihara M, Tohmon M, et al. Identification of parathyroid hormone messenger ribonucleic acid in an apparently nonfunctioning parathyroid carcinoma transformed from a parathyroid carcinoma with hyperparathyroidism. J Clin Endocrinol Metab 1986;62:247-252. 69. Nussbaum SR, Gaz RD, Arnold A. Hypercalcemia and ectopic secretion of parathyroid hormone by an ovarian carcinoma with rearrangement of the gene for parathyroid hormone. N Engl J Med 1990;323:1324-1328. 70. Motokura T, Bloom T, Kim HG, et al. A BCLl-linked candidate oncogene which is rearranged in parathyroid tumors encodes a novel cyclin. Nature 1991;350:512-515. 71. Russell J, Lettieri D, Sherwood LM. Direct regulation by calcium of cytoplasmic messenger ribonucleic acid coding for preproparathyroid hormone in isolated bovine parathyroid cells. J Clin Invest 1983;72:1851-1855. 72. Brookman JJ, Farrow SM, Nicholson L, O'Riordan JLH, Hendy GN. Regulation by calcium of parathyroid hormone mRNA in cultured parathyroid tissue. J Bone Miner Res 1986;1:529-537. 73. Russell J, Sherwood LM. The effects of 1,25-dihydroxyvitamin D~ and high calcium on transcription of the pre-proparathyroid hormone gene are direct. Trans Assoc Am Physicians 1987;100:256-262. 74. Okazaki T, Zajac JD, Igarashi T, Ogata E, Kronenberg HM. Negative regulatory elements in the human parathyroid hormone gene. JBiol Chem 1991;266:21903-21910. 75. Okazaki T, Ando K, Igarashi T, Ogata E, Fujita T. Conserved mechanism of negative gene regulation by extracellular calcium. J Clin Invest 1992;89:1268-1273. 76. Naveh-Many T, Friedlaender MM, Mayer H, SilverJ. Calcium regulates parathyroid hormone messenger ribonucleic acid (mRNA), but not calcitonin mRNA in vivo in the rat. Dominant role of 1,25-dihydroxyvitamin D. Endocrinology 1989;125:275-280. 77. Yamamoto M, Igarashi T, Muramatsu M, Fukagawa M, Motokura T, Ogata E. Hypocalcemia increases and hypercalcemia decreases the steady state level of parathyroid hormone messenger ribonucleic acid in the rat. J Clin Invest 1989;83:1053-1058. 78. Silver j, Russell J, Sherwood LM. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA 1985;82:4270-4273. 79. Karmali R, Farrow S, Hewison M, Barker S, O'Riordan JLH. Effects of 1,25-dihydroxyvitamin D~ and cortisol on bovine and human parathyroid cells. JEndocrino11989;123:137-142. 80. Brown AJ, Ritter CR, FinchJL, MorrisseyJ, Martin KJ, Murayama E, Nishii Y, Slatopolsky E. The noncalcemic analogue of vitamin

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D, 22-oxacalcitriol, suppresses parathyroid hormone synthesis and secretion. J Clin Invest 1989;84:728-732. Russell J, Lettieri D, Sherwood LM. Direct suppression by 1,25(OH)zD 3 of transcription of the parathyroid hormone gene. Clin Res 1986;34:726A. Silver J, Naveh-Many T, Mayer H, Schmelzer HJ, Popovtzer MM. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat. J Clin Invest 1986;78:1296-1301. Sela-Brown A, Russell J, Koszewski NJ, Michalak M, Naveh-Many T, Silver J. Calreticulin inhibits vitamin D's action on the PTH gene in vitro and may prevent vitamin D's effect in vivo in hypocalcemic rats. Mol Endocrinol 1998;12:1193-1200. Shvil Y, Naveh-Many T, Barach P, Silver J. Regulation of parathyroid cell gene expression in experimental uremia. J Am Soc Nephrol 1990;1:99-104. Fukagawa M, Kaname S, Igarashi T, Ogata E, Kurokawa K. Regulation of parathyroid hormone synthesis in chronic renal failure in rats. Kidney Int 1991;39:874-881. Lopez-Hilker S, Galceran T, Chan YL, Rapp N, Martin KJ, Slatopolsky E. Hypocalcemia may not be essential for the development of secondary hyperparathyroidism in chronic renal failure. J Clin Invest 1986;78:1097-1102. Delmez JA, Tindira C, Grooms P, Dusso A, Windus DW, Slatopolsky E. Parathyroid hormone suppression by intravenous 1,25-dihydroxyvitamin D: A role for increased sensitivity to calcium. J Clin Invest 1989;83:1349-1355. Okazaki T, Igarashi T, Kronenberg HM. 5'-flanking region of the parathyroid hormone gene mediates negative regulation by 1,25(OH)z vitamin D3.JBiol Chem 1988;263:2203-2208. Farrow SM, Hawa NS, Karmali R, Hewison M, Walters JC, O'Riordan JLH. Binding of the receptor for 1,25-dihydroxyvitamin D3 to the 5'-flanking region of the bovine parathyroid hormone gene. JEndocrino11990;126:355-359. Demay MB, Kiernan MS, DeLuca HE Kronenberg HM. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D 3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D 3. Proc Natl Acad Sci USA 1992;89:8097-8101. Liu SM, Koszewski N, Lupez M, Malluche HH, Olivera A, Russell J. Characterization of a response element in the 5'-flanking region of the avian (chicken) parathyroid hormone gene that mediates negative regulation of gene transcription by 1,25dihydroxyvitamin D 3 and binds the vitamin D 3 receptor. Mol Endocrinol 1996;10:206-215. Russell J, Ashok S, Koszewski NJ. Vitamin D receptor interactions with the rat parathyroid hormone gene: Synergistic effects between two negative vitamin D response elements. JBone Miner Res 1999;14(11):1828-1837. Koszewski NJ, Ashok S, Russell J. Turning a negative into a positive: Vitamin D receptor interactions with the avian parathyroid hormone response element. Mol Endocrinol 1999; 13 (3) :455-465. Almaden Y, Canalejo A, Hernandez A, Ballesteros E, GarciaNavarro S, Torres A, Rodriguez M. Direct effect of phosphorus on PTH secretion from whole rat parathyroid glands in vitro. J Bone Miner Res 1996;11 (7):970-976. Slatopolsky E, Finch J, Denda M, Ritter C, Zhong M, Dusso A, MacDonald PN, Brown AJ. Phosphorus restriction prevents parathyroid gland growth. J Clin Invest 1996;97 (11 ) :2534-2540. KilavR, Silver J, Naveh-Many T. Parathyroid hormone gene expression in hypophosphatemic rats. J Clin Invest 1995;96:327-333. Rupp E, Mayer H, Wingender E. The promoter of the human parathyroid hormone gene contains a functional cyclic AMPresponse element. Nucleic Acids Res 1990;18:5677-5683. Peraldi MN, Rondeau E,Jousset V, et al. Dexamethasone increases preproparathyroid hormone messenger RNA in human hyperplastic parathyroid cell in vitro. EurJ Clin Invest 1990;20:392-397.

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99. Naveh-Many T, Almogi G, Livni N, Silver J. Estrogen receptors and biologic response in rat parathyroid tissue and C cells. J Clin Invest 1992;90:2434-2438. 100. Bringhurst FR, Stern AM, Yotts M, Mizrahi N, Segre GV, PottsJT, Jr. Peripheral metabolism of PTH: Fate of biologically active amino terminus in vivo. Am J Physio11988;255:E886-E893. 101. Fox J, Scott M, Nissenson RA, Heath H. Effects of plasma calcium concentration on the metabolic clearance rates of parathyroid hormone in the dog.JLab Clin Med 1983;102:70-77. 102. Martin KJ, Hruska KA, Freitag JJ, Klahr S, Slatopolsky E. The peripheral metabolism of parathyroid hormone. N Engl J Med 1979;302:1092-1098. 103. Segre GV, D'Amour P, Hultman A, Potts JT, Jr. Effects of hepatectomy, nephrectomy, and nephrectomy/uremia on the metabolism of parathyroid hormone in the rat.J Clin Invest 1981;67:439-448. 104. Bringhurst FR, Segre GV, Lampman GW, Potts JT, Jr. Metabolism of parathyroid hormone by Kupffer cells: Analysis by reverse-phase high-performance liquid chromatography. Biochemistry 1982;21:4252-4258. 105. Hilpert J, Nykjaer A, Jacobsen C. Megalin antagonizes activation of the parathyroid hormone receptor. J Biol Chem 1999;274:5620-5625. 106. Rouleau ME Warshawsky H, Goltzman D. Parathyroid hormone binding in vivo to renal, hepatic and skeletal tissues of the rat using a radioautographic approach. Endocrinology 1986; 118:919-931. 107. J/ippner HW, Gardella TJ, Brown EM, Kronenberg HM, Potts, JT, Jr. Parathyroid hormone and parathyroid hormonerelated peptide in the regulation of calcium homeostasis and bone development. In: Degroot LJ and Jameson JL, (eds), Endocrinology, 4th Ed., WB Saunders, Philadelphia, pp. 969-998. 108. Dambacher MA, Fischer JA, Hunziker WH, Born W, Moran J, Roth HR, Delvin EE, Glorieux FH. Distribution of circulating immunoreactive components of parathyroid hormone in normal subjects and in patients with primary and secondary hyperparathyroidism: The role of the kidney and of serum calcium concentration. Clin Sci 1979;57:435-443. 109. Berson SA, Yalow RS. Immunochemical heterogeneity of parathyroid hormone in plasma. J Clin Endocrinol Metab 1968;28:1037-1047. 110. Segre GV, D'Amour P, Potts JT, Jr. Metabolism of radioiodinated bovine parathyroid hormone in the rat. Endocrinology 1976;99:1645-1652. 111. Daugaard H, Egfjord M, Olgaard K. Metabolism of intact parathyroid hormone in isolated perfused rat liver and kidney. Am J Physiol 1988;254:E740-E748. 112. D'Amour P, PalardyJ, Bahsali G, Mallette LE, DeLean A, Lepage R. The modulation of circulating parathyroid hormone immunoheterogeneity in man by ionized calcium concentration. J Clin Endocrinol Metab 1992;74:525-532. 113. Roos BA, Lindall AW, Aron DC, Orf JW, Yoon M, Huber MB, PenskyJ, Ells J, Lambert PW. Detection and characterization of small midregion parathyroid hormone fragment(s) in normal and hyperparathyroid glands and sera by immunoextraction

and region-specific radioimmunoassays. J Clin Endocrinol Metab 1981;53:709-721. 114. Segre GV, Perkins AS, Witters LA, Potts JT, Jr. Metabolism of parathyroid hormone by isolated rat Kupffer cells and hepatocytes. J Clin Invest 1981;67:449-457. 115. MacGregor RR, Jilka RL, Hamilton JW. Formation and secretion of fragments of parathormone. Indentification of cleavage sites. JBiol Chem 1986;261:1929-1934. 116. D'Amour P, LaBelle E LeCavalier L, Plourde V, Harvey D. Influence of serum Ca concentration on circulating molecular forms of PTH in three species. AmJPhysiol 1986;251 :E680-E687. 117. D'Amour P, Lazure C, LaBelle E Metabolism of radioiodinated carboxy-terminal fragments of bovine parathyroid hormone in normal and anephric rats. Endocrinology 1985;117:127-134. 118. Flueck JA, DiBella FP, Edis AJ, Kehrwald JM, Arnaud CD. Immunoheterogeneity of parathyroid hormone in venous effluent serum of hyperfunctioning parathyroid glands. J Clin Invest 1977;69:1367-1375. 119. Hanley DA, Ayer LM. Calcium-dependent release of carboxylterminal fragments of parathyroid hormone by hyperplastic human parathyroid tissue in vitro. J Clin Endocrinol Metab 1986;63:1075-1079. 120. Tanguay KE, Mortimer ST, Wood PH, Hanley DA. The effects of phorbol myristate acetate on the intracellular degradation of bovine parathyroid hormone. Endocrinology 1991 ;128:1863-1868. 121. Goltzman D, Henderson B, Loveridge M. Cytochemical bioassay of parathyroid hormone. Characteristics of the assay and analysis of circulating hormonal forms. J Clin Invest 1980;65:1309-1317. 122. Bringhurst FR, Stern AM, Yotts M, Mizrahi N, Segre GV, PottsJT, Jr. Peripheral metabolism of [35S]parathyroid hormone in vivo: Influence of alterations in calcium availability and parathyroid status. J Endocrinol 1989;122:237-245. 123. Nussbaum SR, Zahradnik RJ, Lavigne JR, Brennan GL, NozawaUng K, Kim LV, Keutmann HT, Wang CA, Potts JT, Jr, Segre GV. Highly sensitive two-site immunoradiometric assay of parathyrin and its clinical utility in evaluating patients with hypercalcemia. Clin Chem 1987;33:1364-1367. 124. Brossard JH, Cloutier M, Roy L, Lepage R, Gascon-Barre M, D'Amour P. Accumulation of a non-(I-84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: Importance in the interpretation of PTH values. J Clin Endocrinol Metab 1996;81 (11) :3923-3929. 125. Lepage R, Roy L, Brossard JH, Rousseau L, Dorais C, Lazure C, D'Amour E A non-(I-84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples. Clin Chem 1998; 44 (4) :805-809. 126. John MR, Goodman WG, Gao P, Cantor TL, Salusky IB, Juppner H. A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: Implications for PTH measurements in renal failure. J Clin Endocrinol Metab 1999;84(11 ):4287-4290.

CHAPTER 3

Parathyroid Hormone-Related Protein Gene Structure, Biosynthesis, Metabolism, and Regulation

WILLIAM M. PHILBRICK Section of Endocrinology, Department of Intemal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520

INTRODUCTION

calcium and phosphate metabolism that occur in humoral hypercalcemia of malignancy (1-4,7-9). The similarity between PTHrP and PTH extends to their overall genomic organization as well. Their two genes share a similar organization and similar positioning of i n t r o n - e x o n boundaries (Fig. 1). Furthermore, the h u m a n PTHrP gene maps to the short arm of chromosome 12, where it is flanked by lactate dehydrogenase B and the K-ras protooncogene, and the PTH gene is located on a homologous region of chromosome 11 with lactate dehydrogenase A and H-ras (7-9). These two chromosomes are t h o u g h t to have arisen through a tetraploidization event that occurred some 200 to 300 million years ago (10). Both PTHrP and the P T H / P T H r P receptor sequences have been identified in teleost fish and the respective proteins have been found in the cartilaginous elasmobranchs (11-13), thus it seems quite possible that PTHrP represents the ancestral gene and that PTH evolved subsequent to the gene duplication, as a response to the demands placed on calcium regulation by the emergence of bony skeletons in fish or by the adaptation to a terrestrial envir o n m e n t by early amphibians.

Parathyroid hormone-related protein (PTHrP) was identified in the course of the search for the agent responsible for the paraneoplastic syndrome, humoral hypercalcemia of malignancy. Malignancy-associated hypercalcemia, recognized as a clinical entity since the mid-1920s, was originally assumed to be related to local erosion of the skeleton by resident metastatic lesions (1-4). However, in 1941, Fuller Albright proposed the elaboration of a humoral factor by a neoplasm as an alternative mechanism to explain the reversal of hypercalcemia and hyperphosphatemia after removal of the primary t u m o r in a patient with renal carcinoma (5). It is now recognized that local osteolytic mechanisms account for only approximately 20% of cases of malignancy-associated hypercalcemia and that in the majority of the remaining 80%, hypercalcemia results from elevated circulating levels of PTHrP (1-4,6). The biochemical purification of PTHrP and the subsequent cloning of the cDNA by several laboratories in the late 1980s revealed that PTHrP and parathyroid h o r m o n e shared striking similarity at the aminotermini of the respective mature peptides: 8 out of the first 13 amino acids are identical, a further 3 represent conservative changes, and the two proteins share considerable conformational similarity through residue 34 (Fig. 1). This extensive homology accounts for the ability of PTHrP to bind to and activate classic parathyroid h o r m o n e (PTH) receptors in bone and kidney and consequently to generate the paraneoplastic effects on

The Parathyroids, Second Edition

GENE STRUCTURE: P R O M O T E R ELEMENTS, SPLICE SITES, AND TRANSCRIPTIONAL TERMINATION SITES From their c o m m o n origin, the PTHrP and PTH genes have clearly diverged. In humans the PTHrP gene

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FIG. 1 Comparison of parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH). (A) Gene structure. The principal human PTHrP and PTH exons and introns are aligned relative to the coding regions. Exons are boxed, the 5' and 3' untranslated regions (UTRs) are white, the pre segment is hatched, the pro segment is shaded, and the mature peptide is black. Amino acid residues are numbered relative to the first residue of the mature peptide. (B) Amino acid sequence. The sequences of human PTHrP and PTH are aligned from the last two residues of the pro segment (encompassing the main cleavage site) through the first 13 residues of the mature peptide. Identity is indicated by vertical connecting lines and mismatches are scored according to the degree of evolutionary distance, with two vertical dots indicating greater similarity than one dot. In the rat and bovine PTH sequences, the first amino acid of the mature peptide is identical to that in PTHrP (Ala). From data in Mangin et aL (14).

is by far the larger and more complex of the two, spanning more than 15 kilobases (kb) and containing eight exons (Fig. 2; various exon n u m b e r i n g systems have been employed by different research groups; for the sake of simplicity, a sequential 1 through 8 nomenclature is used here). Three promoters have been functionally characterized (14-18). The first (P1) lies just upstream of exon 1 and contains a canonical TATA sequence; a second TATA-containing p r o m o t e r (P2) resides some 2.7 kb downstream within a small, 45-bp intron between exons 3 and 4. An additional p r o m o t e r element has been identified immediately upstream of exon 3, but does not contain a TATA sequence and instead resembles the GC-rich initiator element found in many housekeeping genes. In addition to the use of multiple promoters, the transcriptional diversity of PTHrP gene expression is also mediated by alternative splicing of primary transcripts (19-22). The 5' end of the gene contains four noncoding exons that may be variably incorporated into the mature, processed mRNA and the resultant splicing pattern appears to be largely d e p e n d e n t on p r o m o t e r usage. For transcripts initiating from P2, for example, the first exon (exon 4) is uniformly spliced to exon 5, whereas for transcripts initiating from the initiator element, the first exon (exon 3) is always spliced directly to exon 5, omitting exon 4. For transcripts initiating at P1, however, two possible patterns exist: exon 1 may be spliced to exon 2 (in which case splicing exon 2 to exon 3 and exon 3 to exon 5 is obligatory) or exon 1 may instead be spliced

directly to exon 3 (in which case splicing exon 3 to exon 5 is obligatory and exon 2 is omitted). However, because all of these 5' alternative exons represent untranslated regions (UTRs), the functional consequences of any particular splice choice are presently uncertain. Still further diversity of PTHrP mRNA expression in humans is generated by alternative splicing at the 3' end of the gene, which contains three possible terminal exons, each encoding a distinct carboxy terminus for the PTHrP protein (19-22). The splicing pattern here appears to be generated in large part by the site of transcription termination (Fig. 2). Transcripts terminating immediately 3' of exon 6 possess no 3' splicing alternatives and translation necessarily yields a 139residue mature peptide. Transcripts that terminate following exon 7 have only one possible 3' splice (exon 6 to exon 7), which, if available, will presumably be executed by default during mRNA processing. Translation of this mRNA product then yields a mature peptide of 173 amino acids. Only for transcripts terminating beyond exon 8 does there appear to be a splicing choice (either exon 6 to exon 7 or exon 6 to exon 8, with translation of the latter giving a 141-residue product), although complete removal of intronic sequences would necessarily render obligatory the exon 6 to exon 8 splice. The operative mechanism here, therefore, seems to be one of alternative transcription termination (and polyadenylation), rather than alternative splicing. In theory, the simplest means of generating equal amounts of each of the three alternative

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34

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CHAPTER3

3' mRNA termini (and, therefore, of the three alternative PTHrP peptides as well) would be to employ transcription terminator sequences of graded efficiencies, so that, for example, 33% of transcripts terminate after exon 6, 50% of those remaining terminate following exon 7, and 100% of the rest terminate beyond exon 8. Splicing would then be perfunctory, and not regulatory, in nature, although what regulatory mechanisms for PTHrP gene expression might exist at the level of transcription termination is presently unknown. The complexity in structure and processing evident in the h u m a n PTHrP gene seems to represent a relatively recent p h e n o m e n o n in evolutionary terms, because the organization of the gene is much simpler in lower species. The PTHrP gene in the rodent (both rat and mouse) appears to employ only a single TATA p r o m o t e r and contains only four exons (23-25) (Fig. 3). Exon 1 encompasses most of the 5' untranslated region, exon 2 encodes the "prepro" region from amino acids - 3 6 to - 8 , exon 3 encodes the r e m a i n d e r of the pro sequence (amino acids - 7 to - 1 ) and the bulk of the mature peptide (137 residues in the mouse and 139 in the rat), and exon 4 encodes the final two amino acids, the stop codon, and the 3' untranslated region. As seen in Fig. 3, one possible implication of the a r r a n g e m e n t of exons and introns in the colinear PTHrP genes from rodents and humans is that h u m a n exon 8 (rodent exon 4) represents the ancestral splicing pathway and that the emergence of the splice site defining h u m a n exon 7 occurred at a point in evolution distal to the rodent branchpoint. Interestingly, though the h u m a n exon 7 equivalent is also absent in chickens, the PTHrP gene in this species does display alternative termination 3' to exon 3 (the h u m a n exon 6 equivalent) and consequently generates a 139-residue peptide isoform, a p h e n o m e n o n not seen in rodents (26). This suggests either that the development of this termination site predated mammalian evolution and has been subsequently lost in m o d e r n rodents but retained in the human, or that this mechanism evolved independently in chickens and humans.

PROTEIN STRUCTURE AND POSTTRANSLATIONAL PROCESSING Translation of the three human PTHrP mRNAs, each with alternative 3' termini, generates three protein products with distinct carboxy termini (19-22). Each of these three protein isoforms have 139 amino acids in common; the isoform that undergoes translation termination at a stop codon within exon 6 ends with residue 139, whereas the isoform terminating in exon 8 adds two additional residues for a total of 141 and the isoform terminating in exon 7 extends the 139-amino acid trans-

lated product a further 34 amino acids for a total of 173 (Figs. 2B and 4). Rat PTHrP derives its carboxy terminus from the equivalent of h u m a n exon 8 and is also 141 amino acids in length, whereas the mouse protein, although similarly derived, is only 139 amino acids long due to the deletion of codons 130 and 131 (23-25) (Fig. 4). PTHrP coding sequences are extremely well conserved across species, with the region encompassing residues 1-111 of the mature peptide exhibiting approximately 98% homology between chickens and humans (26). The primary translation products of the human PTHrP gene (and from other species as well) share a c o m m o n 36-amino acid prepro sequence (residues - 3 6 to - 1 , encoded in the h u m a n by exons 3 and 4) that is composed of an N-terminal segment with the typical structural features of a signal peptide (the pre sequence), followed by a short leader peptide (the pro sequence; Fig. 1), the removal of which is required to allow ligand function (i.e., binding to and activation of the P T H / P T H r P receptor) (7-9). By analogy with other secreted proteins, the signal sequence presumably mediates the attachment of the nascent peptide to the endoplasmic reticulum, where it is then cotranslationally removed by signal peptidase as the growing chain is extruded through the membrane. The precise cleavage site is unknown, but is predicted to be in the region of residues - 8 to - 7 . The remaining pro sequence is then presumably cleaved at a tetrabasic site (RRXKR), spanning residues - 5 through - 1 , by the action of furin or a furinlike p r o h o r m o n e convertase within the endoplasmic reticulum or Golgi apparatus (27), thus generating the mature, secreted form(s) of the protein, PTHrP(1-139), PTHrP(1-141), and PTHrP(1-173) (7-9). The function of the pro sequence is unknown, but the sequence could be required for intracellular targeting or proper folding, or may simply serve as an inert spacer.

Amino-Terminal Peptides Early evidence for further posttranslational cleavage or fragmentation of native PTHrP emerged from the contradictory estimates of protein concentration obtained by standardized quantitative immunoassays that used antibodies generated against epitopes on specific regions of PTHrP (amino terminal, carboxy terminal, or midregion) versus two-site immunoassays designed to detect the complete PTHrP molecule. Inspection of the amino acid sequences of the PTHrP isoforms reveals a n u m b e r of potential sites for proteolytic cleavage, many of which appear to be functional in cultured cells or in vivo. There are frequent clusters of basic amino acids (arginine and lysine) across the length of the protein (Figs. 4 and Figs. 5); proteolytic processing at such sites is typical of neuroendocrine peptides

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FIG. 4 Amino acid sequence of the mature form(s) of parathyroid hormone-related protein. The top line is the sequence of the human 141-residue isoform, beginning with the first residue of the mature form and with the pre and pro segments removed. The alternative carboxy termini of the 139- and 173-isoforms are included for comparison. The sequences for mouse, rat, and chicken PTHrP are aligned with the human sequence. Amino acid substitutions are indicated, identity is designated by vertical lines, gaps or deletions are designated by dashes, and insertions are designated by an asterisk. From data in Mangin et aL (19,20,23), Suva et aL (21), Thiede et aL (22,26), Karaplis et aL (24), and Yasuda et aL (25).

such as insulin, proopiomelanocortin, gastrin, calcitonin, chromogranins, and many others (28,29). Cleavage at these sites is commonly carried out by prohormone convertases of the PC2, PC1/3 family located within the Golgi and in secretory granules. Another prominent cleavage site resides within the mature peptide adjacent to a monobasic arginine residue at position 37, thus generating a processed PTHrP peptide that consists of residues 1-36 (30). Although posttranslational processing of peptide hormones occurs more commonly at di- and multibasic sites, there are also numerous instances of cleavage at monobasic sites (31); among hormones processed in this manner are somatostatin, atrial natriuretic peptide, and glucagon. Mutagenesis of Arg-37 to Ala, Phe, or Lys effectively abolishes cleavage at this site (32). Using region-specific antibodies, aminoterminal species of PTHrP that do not contain carboxyterminal or midregion epitopes have been detected in media from cultured cell lines and in sera from hypercalcemic patients (8,9). More importantly, PTHrP(1-36) has been shown to be as potent as PTH (1-34) when analyzed in cell-based assays and in vivo (33). Keratinocytes appear to produce a longer, glycosylated, amino-terminal form of PTHrP (34); the core protein has an apparent molecular mass of 10 kDa; the fully glycosylated form has an apparent molecular mass of 18 kDa. This form has not been observed in other cell types thus far.

Midregion Peptides In addition to generating the 36-residue aminoterminal pepdde, cleavage of PTHrP at Arg-37 also gen-

erates a midregion peptide species with Ala-38 as its amino terminus (30) (Fig. 5). The amino acid sequence of PTHrP extending from residue 38 to 111 is remarkably well conserved among species, with only two substitutions in the h u m a n as compared to the rodent (23-25) (Fig. 4). This midregion peptide has been identified as an endogenous product in cultured cell lines from a n u m b e r of species and has been shown to be produced by rat insulinoma cell lines transfected with cDNAs encoding each of the three human PTHrP isoforms (35). Midregion PTHrP has been shown to be secreted in a regulated fashion by transfected neuroendocrine cells (36) and to be present in the circulation (37). The function of midregion PTHrP has not been well established, but studies in squamous epithelial cells provide evidence for a midregion-specific receptor that mediates the mobilization of cytosolic calcium and the formation of inositol phosphates through activation of the phospholipase C signaling pathway (38). Furthermore, studies in sheep have implicated a midregion peptide in establishing and maintaining the calcium gradient between the mother and fetus (39). This maternal-fetal calcium gradient is also abolished in PTHrP knockout mice, which have a hemochorial placenta as is found in humans. Placental calcium transport can be restored, however, by infusion of full-length or midregion fragments of PTHrP, but not by aminoterminal PTHrP or PTH (40). Finally, the natural secretory form(s) of midregion PTHrP have been only partially characterized; species with an amino terminus at residue 38 and carboxy termini at residues 94, 95, and 101 have been identified thus far (41), although

PTHrP:

G E N E STRUCTURE AND BIOSYNTHESIS

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FIG. 5 Parathyroid hormone-related protein domains. (A) Potential multibasic cleavage sites in the three human PTHrP isoforms. The positions of arginine (R) and lysine (K) residues are marked. Numbering is relative to the first amino acid of the mature peptide. (B) Functional domains and sequence homologies in PTHrP. The signal peptide (SP) and propeptide (P) are removed during intracellular processing. The PTH-like nature of the first 36 amino acids of the mature peptide reflects both the correspondence in primary structure in residues 1 through 13 and the conformational similarity that mediates receptor activation. The region between residues 36 and 111 is highly conserved among all species examined thus far. The carboxy-terminal segment from residues 111 to 139 or 141 is the least conserved region of the peptide and the portion extending beyond residue 141 has thus far only been found in humans. (C) Processed forms of PTHrP. Secreted products include the active, amino-terminal fragment (1-36), a glycosylated amino-terminal fragment found in keratinocytes, and the various midregion fragments defined thus far. Potential carboxy-terminal species include the purported osteostatin peptide and remain largely undefined. Adapted from Broadus AE, Stewart AF. Parathyroid hormone-related protein structure, processing, and physiological actions. In: Bilezikian JP, eds. The parathyroids. New York: Raven, 1994:259-294.

most experimental work testing for effects in cell culture and in vivo has been conducted with a peptide encompassing residues 67-86. Carboxy-Terminal Peptides A n u m b e r of multibasic proteolytic processing sites that may serve as substrates for furin/kexin can also be found in the carboxy terminus of PTHrP (at residues 88-91, 96-98, and 102-106) (42) and there is evidence that a carboxy-terminal species is generated as a native cellular product (Fig. 5). A carboxy-terminal

PTHrP fragment can be detected in the circulation of patients with renal failure (43) and is also found in the urine of normal h u m a n subjects (44). Similar fragments are secreted by h u m a n carcinoma cell lines and by cell lines transfected with PTHrP cDNAs (45). Based on the sequence conservation of PTHrP through amino acid 111 and t h e presence of the potential cleavage sites at residues 102-106, synthetic peptides containing amino acids 107-111 and 107-139 were tested for physiologic effects and were reported to inhibit osteoclastic bone resorption in vitro (46). To date, however, the effects of these

38

/

CHAPTER3

"osteostatin" peptides have not proved to be universally reproducible (47). Carboxy-terminal PTHrP has also been reported to inhibit the proliferation of cultured keratinocytes, whereas amino-terminal PTHrP had the opposite effect (48). Finally, immunoassays with antibodies directed against the unique 141-173 region of PTHrP that is derived from h u m a n exon 7 have suggested the presence of this fragment in fetal cord blood; h u m a n breast milk; cultured keratinocytes; renal, squamous and prostate carcinomas; and h u m a n amniotic m e m b r a n e s (49-51). However, as is the case with all carboxy-terminal species of PTHrP characterized thus far, the precise nature of the secretory form (s) has not been defined. Also contained within the multibasic proteolytic processing region that extends from residue 87 to residue 107 is a consensus bipartite nuclear processing signal, similar to that found in a n u m b e r of steroid h o r m o n e receptors and transcription factors (52-57). This targeting signal has been shown to be both necessary and sufficient for localization of PTHrP to the nucleus and nucleolus and similarly to mediate localization of heterologous peptides. Though the PTHrP signal sequence normally directs the nascent peptide into the secretory pathway via the endoplasmic reticulum, there appear to be circumstances u n d e r which this signal can be overridden and the resultant cytosolic PTHrP can then be directed into the nucleus (52-57). One possible mechanism by which such an "intracrine" pathway might be exercised entails the use of alternative translational initiation sites that effectively prevent the generation of a functional signal sequence. Another potential route by which PTHrP may be directed to the nucleus involves the receptor-mediated internalization of secreted peptide, a pathway that also appears to be d e p e n d e n t on the presence of the nuclear targeting signal and may be mediated by a receptor distinct from the classic type 1 P T H / P T H r P receptor (55-57). Evidence suggests that the biologic effects of nuclear PTHrP can be quite dissimilar to those transduced by interaction with the type 1 receptor, even within the same cell type (53).

POSTTRANSLATIONAL AND SECRETION

MODIFICATION

Although PTHrP contains no consensus sites for N-glycosylation, numerous serine and threonine residues are present that may serve as sites for O-glycosylation. As noted above, a heavily glycosylated fragment of PTHrP has been shown to be secreted by keratinocytes, but has not yet been demonstrated in other cell types (41). PTHrP also contains consensus sites at residues 87-91 and 94-97 for carboxy-terminal amidation by the enzyme peptidyl e~-monooxygenase

(28,29,58). Because peptide amidation is relatively c o m m o n and there is considerable overlap between the tissues that express PTHrP and those that express the amidating enzyme, this particular posttranslational modification of PTHrP seems likely to occur, but has not been formally documented. PTHrP is expressed by a wide variety of cell types, representing both the regulated and constitutive pathways of protein secretion. In n e u r o e n d o c r i n e cell types such as those found in the pancreatic islet, adrenal medulla, pituitary, parathyroid, and central nervous system, PTHrP is packaged into secretory granules, and in the parathyroid gland, evidence suggests that PTHrP is cosecreted with PTH (59). Also, in transfected rat insulinoma cells, midregion and carboxy-terminal fragments, as well as amino-terminal PTHrP, have been shown to colocalize with insulin and their release can be induced by secretagogues such as potassium chloride and leucine (36). In cell types such as keratinocytes, osteoblasts, chondrocytes, renal tubular cells, and smooth muscle cells, however, PTHrP is secreted via the constitutive movement of monensinsensitive secretory vesicles, so that its rate of release is d e p e n d e n t on its rate of synthesis (60). Interestingly, a comparison of the products secreted by cell lines transfected with cDNAs encoding the three h u m a n PTHrP isoforms (1-139, 1-141, and 1-173) and representing either the regulated (rat insulinoma cells) or the constitutive (Chinese hamster ovary cells) secretion pathways suggests that processing of PTHrP peptides can be both cell and isoform specific: though all six cell lines generated an amino-terminal (1-36) product, the CHO cell lines also contained a distinct amino-terminal peptide that was absent in the RIN lines, and both CHO and RIN lines expressing the 1-139 isoform secreted a unique carboxy-terminal peptide that was not secreted from the lines expressing other isoforms (35).

METABOLISM AND DEGRADATION Much is known about the metabolism and degradation of PTH, but this is not the case with PTHrP, although it is likely that there is some overlap in the pathways involved. PTH is predominantly cleared from the circulation by glomerular filtration, uptake by multifunctional endocytic receptors in the renal proximal tubules, and subsequent lysosomal degradation. Given the low circulating levels of PTHrP, this would seem unlikely to be a quantitatively significant disposal route u n d e r normal circumstances, but could well prove critical in conditions of PTHrP overexpression, such as humoral hypercalcemia of malignancy. In n o n h u m a n mammals, exogenous PTHrP fragments encompassing residues 1-34 and 1-86 were rapidly removed from the

PTHrP: GENE STRUCTUREAND BIOSYNTHESIS / circulation, with metabolic clearance rates in the range of 1.25-7.5 m l / m i n per kg, or only slightly slower than that of intact PTH in man (61). There is evidence, however, suggesting that the kidney may play a role in the normal disposal of PTHrE In patients with chronic renal failure, elevated levels of a carboxy-terminal PTHrP fragment can be found in the circulation, presumably due to impaired clearance (43). Further, studies in vitro have implicated the metalloprotease, meprin, in the degradation of carboxy-terminal PTHrP by the kidney (62). Another potential clearance mechanism for PTH is specific binding by the P T H / P T H r P type 1 receptor followed by receptor-mediated endocytosis (55, 57). Because most of the actions carried out by PTHrP appear to be restricted to the microenvironment surrounding its site of secretion, such a mechanism could well be an important means of turning off signal transduction. Unexpectedly, evidence has suggested that although PTHrP is a secreted protein, the precursor propeptide in the endoplasmic reticulum can gain access to the cytoplasm and there serve as a target for ubiquitin conjugation and sub-sequent proteosome-mediated degradation (63,64). Neither the pro sequence nor a carboxy-terminal PEST degradation motif appeared to be required as cis-acting determinants, although carboxy-terminal sequences have been implicated in the intracellular degradation of the fulllength peptide (65). In cell culture studies, both native PTHrP secreted into the medium and added synthetic PTHrP peptides undergo rapid degradation even in the presence of multiple protease inhibitors, suggesting that many cell types secrete proteases capable of progressive cleavage and inactivation of this peptide (66). PTHrP secreted into the extracellular milieu in vivo may also be labile, which would be in keeping with the short half-life of its mRNA. Overexpression of PTHrP at 10 to 20 times the normal level in the epidermis of transgenic mice does not lead to hypercalcemia nor does it generate detectable levels of the peptide in the circulation (67). Proteases known to cleave aminoterminal PTHrP and thus abolish bioactivity include kexin and prostate-specific antigen (68,69).

CONTROL ELEMENTS AND REGULATION OF GENE TRANSCRIPTION

As might be expected, given the size and complexity of the human PTHrP gene, the 5' controlling region contains numerous consensus regulatory sequences in addition to the two TATA-containing promoters and the GC-rich initiator element (14-18) (Fig. 6). Included among these are the following sites: (1) two enhancer sequences (CAAT boxes) within the long intervening sequence between exons 2 and 3, (2) multiple regions (5' to exon 1, within intron 2, and in exons 3 and 4) that contain potential binding sites for the transcription factors, Spl, AP-1 and AP-2, (3) sequences resembling cyclic AMP response elements 5' to exon 1 and within exon 4, and (4) sequences equivalent to glucocorticoid responsive half-sites, located both 5' to and within exon 1, and within intron 1, intron 2, and exon 3. It should be emphasized, however, that many of these sites have been identified on the basis of sequence alone, and not by functional assays. Information about the mechanisms of PTHrP gene regulation has been obtained principally with two complementary approaches: (1) by analysis of biological systems that display aberrant or dysregulated expression, such as cancer cells, and (2) by testing the activity of reporter genes (such as chloramphenicol acetyltransferase or luciferase) under the control of progressive deletions of PTHrP gene promoter regions in transfected cell lines. In adult T cell leukemia/lymphoma (ATLL), infection of T lymphocytes with the HTLV-1 retrovirus results in humoral hypercalcemia of malignancy through the production of PTHrP by tumor cells (70-76). A viral protein required for replication, Tax1, has been shown to transactivate PTHrP gene expression from the P2 promoter by acting at through an upstream binding site for the transcription factor Ets-1 ( - 7 3 to - 6 5 relative to the P2 transcriptional initiation site). Effective up-regulation was dependent on the presence of both Ets-1 and Taxl (70). Subsequent work has also established that P2 basal activity requires binding of the transcription factor Spl at an adjacent site,

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40

/

CHAPTER3

and that Ets-1 interacts directly with both Tax1 and Spl to form a cooperative ternary complex that interacts with the DNA binding site and then transcriptionally activates the promoter (71,72). Transactivation of the PTHrP gene was abolished by deletion of the carboxy terminus of the Tax protein and was also found to be d e p e n d e n t on an interaction between Ets-1 and a site within the amino terminus of Tax (72,73). Similarly, overlapping Ets-1 and Spl sites in the immediate upstream region of the mouse PTHrP gene ( - 8 8 to - 5 8 relative to the transcriptional start site) have been shown to be essential for retinoic acid-induced expression in embryonal carcinoma and embryonal stem cells (74). Additional studies with the h u m a n gene in this system have also shown up-regulation of expression by prostaglandin El, acting via a cAMP-dependent pathway, and by interleukin-2 (IL-2) and the transcription factor AP1/c-jun (73,75,76). Although PTHrP is a normal secretory product of a variety of squamous epithelia, including epidermal keratinocytes, only a subset of squamous carcinomas express the gene at levels sufficient to cause humoral hypercalcemia, raising the possibility that the nature of the transforming events could have an impact on PTHrP gene expression. Comparison of PTHrP expression levels with p53 functional status in a series of squamous carcinoma lines has revealed an association between the loss of p53 function and high levels of PTHrP mRNA (77). Evaluation of p53 isoforms with stabilizing mutations showed them to be capable of repressing PTHrP gene expression in a p53-negative squamous line and, correspondingly, inactivation of an endogenous, stabilized p53 gene product by the introduction of adenoviral E1B genes resulted in an increase in PTHrP expression. Conversely, mutant isoforms of p53 displaying a denatured, rather than stabilized, conformation were found to activate PTHrP gene expression (78). Both repression and activation of the human PTHrP gene by p53 appeared to occur primarily at the two TATA-based promoters. Finally, analysis of a spontaneously immortalized murine keratinocyte line trans-

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formed with adenoviral 12S EIA has shown certain key domains within this E1A gene product to effect marked repression of PTHrP gene expression by acting directly on the mouse TATA promoter (79). Similar analysis with a 13S E1A product identified an additional domain that served as a potent activator of PTHrP gene expression by acting through an Ets-1 site residing approximately 70 bp upstream of the transcriptional start site. Other oncogenes have also been found to affect PTHrP gene expression directly. The direct introduction of activated H- or K-ras oncogenes into a variety of cell lines has been shown to result in overexpression of PTHrP and transfection of cells with a constitutively activated tyrosine kinase oncogene, Tpr-Met, has been shown to enhance PTHrP transcription through the ras signaling pathway (80,81). Furthermore, the induction mechanism appeared to be dependent on ras processing events such as isoprenylation and farnesylation (81,82). In another system, analysis of a panel of renal carcinoma cell lines revealed a series of four CpG dinucleotide sites within the 5' flanking region of the human PTHrP gene that were consistently unmethylated in all PTHrP-expressing lines and methylated in all PTHrP-nonexpressing lines examined (83) (Fig. 7). Together these sites appear to constitute a minimal, methylation-free zone of approximately 550 bp, which resides approximately 1 kb upstream of the GC-rich initiator element and serves as a critical control switch for transcription from all three promoters. Furthermore, treatment of PTHrP-nonexpressing lines with the nucleoside analog, 5-azacytidine, effectively demethylated the identified critical sites and concurrently activated PTHrP gene transcription, thus strongly reinforcing the concept of a regulatory relationship. Also in this system, a 900-bp CpG island was identified in the proximal promoter region overlapping the 5' end of exon 3, which remained methylation-free regardless of PTHrP expression status (83). Work in a squamous carcinoma line from human lung, however, showed persistence of PTHrP gene expression despite

q~

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FIG. 7 Methylation pattern of the human PTHrP gene. Sites were identified in a panel of human renal carcinoma cells by using methylation-sensitive restriction enzymes. The half-filled circles correspond to CpG dinucleotides that were found to be unmethylated in cell lines expressing the PTHrP gene and methylated in cells not expressing the gene. Other CpG dinucleotides were found to be methylated (solid circle) or unmethylated (open circles), regardless of the status of PTHrP gene expression. The CpG island is indicated by a black bar. Modified from Holt et aL (83). Holt, EH., Vasavada, R., Broadus, AE., Philbrick, WM. Region-specific methylation of the PTH-related peptide gene determines its expression in human renal carcinoma lines. J Biol Chem 1993;268:20639-20645.

P T H r P : GENE STRUCTURE AND BIOSYNTHESIS

methylation of the distal two-thirds of the CpG island (84), leaving open the possibility that the methylation status of the 3' portion of the island may have regulatory implications. Control sequences that regulate the degree of basal expression have also been examined. Deletion experiments have identified a n u m b e r of negative regulatory elements operative in both normal and neoplastic cell lines in the regions from 3.8 to 2 kb and 1.1 to 0.35 kb upstream of exon 1 and 1.3 to 0.63 kb upstream of exon 3 (18). An additional regulatory element has been found within a segment from 0.63 to 0.34 kb upstream of exon 3 and appears to act as a repressor of the GC-rich initiator, because deletion of this region leads to increased levels of mRNA transcripts originating from this p r o m o t e r (85-87). Interestingly, this same region has been reported as a positive regulatory element when evaluated in constructs in which reporter gene expression is solely u n d e r the control of the downstream P2 p r o m o t e r (18), suggesting that this one region can differentially regulate two distinct promoters. Relative expression profiles of the three h u m a n PTHrP promoters both in context and in isolation suggest that the sequences within the GC-rich initiator element contribute to the activity of the two TATAcontaining promoters, as well as to the activity of the initiator element (86-88). Further dissection of the sequences within the GC-rich initiator element revealed a prototypical bipartite construction with an internal transcription initiation site s u r r o u n d e d by numerous Spl and AP-2 sites. The two halves of the core element appeared to be functionally equivalent, whereas a region extending from approximately 180 to 340 bp upstream of the GC-element transcriptional initiation site played a positive regulatory role (88). Despite the obvious potential for the generation of discrete, tissue-specific patterns of h u m a n PTHrP gene p r o m o t e r usage or alternative termination, this has not proved to be a c o m m o n finding in vivo. Examination of p r o m o t e r usage (P1 and P2) in a large n u m b e r of benign and malignant tissues (including esophagus, stomach, cecum, liver, pancreas, thyroid, parathyroid, adrenal, and kidney) found no consistent patterns (85,89). Relative differences in PTHrP p r o m o t e r usage do exist, however; in squamous carcinoma cells and keratinocytes, P1 was found to be more active than either P2 or the GC promoter; in renal carcinoma cells, the GC p r o m o t e r appears to be preferred; and in the uterus, P2 and the GC p r o m o t e r are used exclusively (87,88). Similarly, no consistent patterns in 3' exon usage have emerged, although exon 6 appears to be more prevalent in prostate cancer than in normal tissue (90), and has also been reported to be associated with breast cancer metastasis (91). Studies of PTHrP gene regulation have found two primary patterns of response, a rapid, transient

//

41

increase in PTHrP mRNA levels or a delayed, sustained up-regulation. Examples of the former include the response to serum and angiotensin II in smooth muscle cells (92,93), mechanical stimuli in aortic smooth muscle cells (94), serum factors in pancreatic islet cells (95) and keratinocytes (96), endotoxin in mouse spleen (97), and ischemia in rat kidney (98). In this response pattern, steady-state levels of PTHrP mRNA typically display a rapid increase within 1 or 2 hours after stimulation, reach a peak at 4 to 6 hours, and then quickly decline due to the short half-life of the mRNA. Examples of a delayed, sustained induction of PTHrP gene expression include the response to estrogen in rat myometrial cells (99); transforming growth factor [3 (TGF-[3) in h u m a n myometrial and endometrial cells in primary culture (100), mechanical stimuli in the bladder, rat uterus, and avian oviduct (101-104); and cAMP in embryonal cells and trophectoderm (105). The generation of this type of response would require a perpetuation of the transcriptional induction, an increase in the stability of the mRNA, or some combination of the two. A n u m b e r of hormones, cytokines, growth factors, and second messengers have been shown to induce PTHrP gene expression; more often then not, these effects tend to follow the rapid, transient response pattern (Table 1). Examples include estradiol in rat kidney, uterus, pituitary, and hypothalamus (106-108); estradiol and tamoxifen in the MCF-7 breast carcinoma cell line (109); calcitonin in lung carcinoma cell lines (110); TNFot and IL-113 in h u m a n umbilical vein endothelial cells (111); forskolin, cAMP, and IL-2 in HTLV-infected T cells (75,76); bradykinin, serotonin, endothelin, thrombin, and n o r e p i n e p h r i n e in smooth muscle cells (92,94); and prolactin in m a m m a r y tissue (112). As noted above, PTHrP gene expression has been shown to be induced by serum in a n u m b e r of cell types, including rat osteosarcoma (ROS) cells. Induction is rapid (peaking within 4 hours) and is mediated in part via a transcriptional mechanism (113), although a substantial effect on mRNA stability has also been observed in ROS cells (see Posttranscriptional Regulation, below). T h o u g h a prototypical serum response element of the c-fos/[3-actin type is not found in the rat PTHrP gene, the serumresponsive region has been localized to a segment extending from 0.3 to 1.05 kb upstream of the transcriptional start site (113). The effects of serum in this system may be mediated, at least in part, through insulin and epidermal growth factor, because both these factors have been shown to stimulate a rapid induction of PTHrP mRNA. Likewise, inhibition of the angiotensin II receptor greatly attenuated the seruminduced rise in PTHrP gene expression in vascular smooth muscle cells (94), suggesting a primary regulatory role for that factor. Phorbol esters, which activate

42

/

C~a'TF~R3

protein kinase C isozymes, have been reported to strongly up-regulate PTHrP mRNA in several cell types (114-116). Correspondingly, treatment of h u m a n myometrial smooth muscle cells with okadaic acid, an inhibitor of the serine/threonine protein phosphatases 1 and 2A, also effected a marked increase in PTHrP gene expression (116). In contrast to the more prevalent pattern of rapid induction, TGF-[3 has been found to provoke a slow, sustained rise in PTHrP mRNA levels that is maximal by 12 to 24 hours in several cell lines, including h u m a n keratinocytes, renal carcinoma cells, primary endometrial and myometrial cells, and mouse bone organ culture (100,117,118). Conversely, TGF-[3 has also been shown to decrease PTHrP gene expression in immortalized murine endochondral chondrocytes, a property shared by several bone morphogenetic proteins (BMP-2,-5 a n d - 7 ) and opposed by basic fibroblast growth factor (119). There are also examples of negative regulation of PTHrP gene expression (Table 1). The active vitamin D metabolite (1,25-dihydroxyvitamin D~) and two glucocorticoids (dexamethasone and triamcinolone) have been shown to decrease steady-state levels of PTHrP mRNA in a time- and dose-dependent m a n n e r in a human medullary thyroid carcinoma cell line and a lung carcinoid line (120,121). The glucocorticoid effect was completely blocked by the competitive antagonist, RU-486. Nuclear runoff and transcriptional inhibition experiments indicated that neither vitamin D nor glucocorticoids appeared to influence mRNA stability, but that these agents acted by repressing the rate of PTHrP gene transcription. Two noncalcemic analogs of vitamin D, EB1089 and 22-oxacalcitriol, were also found to suppress both basal and s e r u m - o r EGF-stimulated PTHrP gene expression in a lung squamous cancer cell line through a transcriptional mechanism, thus raising the possibility of therapeutic potential (122). In the rat PTHrP gene, two vitamin D-responsive elements (VDREs) have been localized approximately 1 kb upstream of the transcriptional start site; one element resembling a canonical positive regulatory VDRE was identified by both DNA-protein binding studies and functional assays, and a second element bearing similarity to the negative regulatory VDRE in the human PTH gene was identified by binding studies alone (123,124). Treatment with 9-cis-retinoic acid has also been shown to repress the transcriptional activity of the human PTHrP gene and mobility-shift experiments with the rat gene suggest that the effects of vitamin D may be mediated through the binding of a heterodimer composed of the vitamin D receptor and the retinoid X receptor (125). PTHrP has been localized to a n u m b e r of discrete cell populations in the central nervous system and evidence suggests that PTHrP gene expression in these neuronal cell types is up-regulated by excitation

(126,127). In primary cultures of cerebellar granule cells, for example, it has been shown that PTHrP gene expression can be induced by potassium ion-dependent membrane depolarization and the subsequent entry of extracellular C a 2+ into the cell through L-type voltagesensitive calcium channels; depolarization with sodium ionphores o r C a 2+ entry by other routes proved ineffective (128). The induction of PTHrP gene expression in this system appears to be mediated through the C a 2+ calmodulin kinase pathway in a m a n n e r similar to that for the c-fos gene. The L-type voltage-sensitive calcium channels (L-VSCCs) and the C a 2+ calmodulin kinase cascade also appear to be involved in excitation secretion coupling in neuroendocrine cells and in excitation-contraction coupling in skeletal, cardiac, and smooth muscle (129). Interestingly, the role played by PTHrP in the central nervous system appears to be neuroprotective in nature, based on experiments showing that the peptide serves as a highly effective inhibitor of L-VSCC-associated C a 2+ influx and consequent neuronal toxicity (see Chapter 16). PTHrP has also been shown to be expressed in smooth muscle types from a n u m b e r of organs, including the gastrointestinal tract, bladder, myometrium, vasculature, and chicken oviduct. In all of these sites, PTHrP gene expression appears to be induced by a variety of mechanical stimuli, such as balloon angioplasty in the aorta (94), atherosclerotic stenosis in the coronary arteries (130), stretch (131) or shear flow (94) in vascular smooth muscle cells in culture, distension of the uterus by fetal growth or balloon inflation (103), expansion of the chicken oviduct during egg transit (104), expansion of the bladder by urinary volume (101), and distension of the stomach by pyloric ligation and subsequent gastric filling (132) (Fig. 8). Accumulating evidence indicates that this mechanotransduction is mediated through the opening of stretch-activated cation channels, subsequent ion influx and membrane depolarization, and the resultant activation of L-type voltage sensitive calcium channels (133). PTHrP gene expression is thus induced and the secreted peptide then serves as a muscular relaxant and vasodilator, thereby constituting a feedback system to regulate muscular tone (see Chapter 16). The rapid induction-deinduction kinetics that typify PTHrP mRNA responses in many systems are reminiscent of the kinetics associated with so-called primary response or immediate early genes, which include many protooncogenes, cytokines, and growth factors (96,134). The transient nature of the induction serves to limit the translational yield of the resultant proteins and thus to restrict the biologic effects of these powerful regulatory molecules. The induction of PTHrP gene expression by 1713-estradiol in rat GH4C1 pituitary cells has been shown to display many of the features of the

PTHrP: GENE STRUCTURE AND BIOSYNTHESIS //

TABLE 1 Regulation of PTHrP Gene Expression a Stimulus

Physiologic Suckling Uterine occupancy Stretch Stretch Stretch Egg-laying cycle Differentiation Differentiation Differentiation Differentiation Pharmocologic Glucocorticoids Glucocorticoids Glucocorticoids Glucocorticoids 1,25(OH)2D 1,25(OH)2D 1,25(OH)2D 22-oxa-1,25(OH)2D Estrogen Estrogen Estrogen Estrogen Estrogen Serum Serum Serum Serum Growth factors EGF EGF IGF-I TGF-13 TGF-13 TGF-13 TGF-13 Prolactin Cycloheximide Cycloheximide Cycloheximide Tax Forskolin Calcitonin Phorbol ester Phorbol ester Endothelin-I Thrombin Angiotensin II

Tissue/cell type

mRNA/protein

Lactating breast (rat) Myometrium (rat) Myometrium (rat) Urinary bladder (rat) Amnion (human) Oviduct (chicken) Insulinoma (rat) Embryonal carcinoma Keratinocyte (human) Trophoectoderm (mouse)

1" 1" 1" 1" T 1" T 1" T 1"

Carcinoid (human) Insulinoma (rat) Aortic smooth muscle (rat) Keratinocyte (rat) Medullary carcinoma (human) Keratinocyte (human) Keratinocyte (rat) T cell (human) Uterus (rat) Pituitary/hypothalamus (rat) Myometrial cell (rat) Pituitary GH4C1 (rat) Kidney (monkey) Insulinoma (rat) Keratinocyte (human) Keratinocyte (rat) Aortic smooth muscle Keratinocyte (human) Keratinocyte (rat) Mammary epithelial (human) Mammary epithelium (human) Renal carcinoma (human) Myometrial (human) Endometrial (human) Keratinocyte (rat) Mammary gland (rat) Multiple (rat and human) Osteosarcoma (human) Insulinoma (rat) T cells (human) T cell MT-2 (human) Squamous carcinoma (human) Osteosarcoma (human) T cells (human) Aortic smooth muscle (rat) Aortic smooth muscle (rat) Aortic smooth muscle (rat)

,I, $ $ $ J, $ ,1, $ T T 1" T T 1" 1" T 1" T 1" 1" 1" T T 1" T 1" T T 1" T 1" T T 1" 1" T T

aModified from Broadus AE, Stewart AF. Parathyroid hormone-related protein structure, processing, and physiological actions. In: Bilezikian The parathyroids. New York: Raven, 1994:259-294.

43

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CHAPTER3

FIG. 8 Induction of PTHrP mRNA during gestation in rat uterus. Little PTHrP gene expression is detected in the nongravid uterus by Northern blot analysis. With progressive distention of the uterus during gestation, steady-state levels of PTHrP mRNA are markedly induced. After parturition on day 21, PTHrP gene expression declines precipitously and continues to fall throughout the postpartum period. When pregnancy is prolonged by the administration of progesterone, PTHrP mRNA levels continue to rise. Reproduced from Thiede et aL (103). Thiede MA, Daifotis AG, Weir EC, Brines ML, Burtis W J, Ikeda K, Dreyer BE, Garfield RE, Broadus AE. Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in pre-term rat myometrium. Proc Natl Acad Sci USA 87:6969-6973, 1990.

primary response pattern (134). Steady-state mRNA levels peaked at 1 to 2 hours due to a burst of transcription that was maximal at 20 to 40 minutes and declined thereafter. The 30-minute half-life of PTHrP mRNA in these cells, although unaffected by estradiol, was sufficiently short to mediate the rapid decline in steady-state mRNA levels. Inhibition of protein synthesis by treatm e n t with cycloheximide had no effect when used alone and failed to block the estradiol-mediated induction of PTHrP gene expression, but eliminated the transcriptional arrest, thus implicating the action of a labile transcriptional repressor protein that is estrogen inducible (134). As is the case for other primary response genes, the combination of a rapid post stimulation repression of gene transcription with a short mRNA half-life is essential to the generation of the transient response.

POSTTRANSCRIPTIONAL

REGULATION

A further measure of control over PTHrP gene expression appears to be exerted at the level of mRNA stability. The steady-state mRNA levels of all genes represent the p r o d u c t of both the rate of transcription and the rate of degradation. T h o u g h the transcription rate of the PTHrP gene is similar to that of the actin gene, steady-state levels of PTHrP mRNA are extremely low in most tissues (estimated to be from 0.001 to 0.01% of mRNA), as a result of rapid turnover (19,23,135-137). M e a s u r e m e n t of the half-life of PTHrP mRNA in a variety of tissues has ranged from 30 minutes to several

hours, kinetics that are similar to those for a n u m b e r of cytokines and protooncogenes. For such factors, a rapid rate of degradation serves to allow a rapid response to the transcriptional down-regulation of the gene, both in terms of mRNA levels and consequent biologic effects. The sequences responsible for mediating mRNA instability in many of these genes are AU-rich elements (AREs) typically found in the 3' untranslated regions of these genes (138). These motifs are also present in all PTHrP mRNAs characterized thus far; multiple iterations of the core element, AUUUA, can be found in all three alternative h u m a n PTHrP 3' transcriptional termini (exons 6, 7, and 8), as well as in the 3' untranslated regions (UTRs) of PTHrP mRNAs from the mouse, rat, and chicken. There is also some functional evidence to suggest that the relative stability of h u m a n PTHrP mRNAs is mediated through the 3' UTRs, with transcripts containing exon 8 typically displaying a more rapid degradation rate than those containing exons 6 or 7 (139,140). Transfection of fibroblasts or keratinocytes with each of the three alternative h u m a n PTHrP 3' UTRs fused to a luciferase reporter gene also showed transcripts containing exon 8 to be the most unstable, although this was d e p e n d e n t on the cell line used (141). A n u m b e r of studies looking at AU-rich elements in other genes, however, have indicated that the core AUUUA motif is insufficient by itself to mediate instability and that the m i n i m u m consensus element is an octamer or n o n a m e r (142,143), neither of which is found in PTHrP mRNAs. This implies that instability in this system may be mediated by non-AUUUA AREs or by elements other than AREs. Finally, there are data to suggest that the degradation rate of PTHrP mRNA is a regulated p h e n o m e n o n , because a n u m b e r of factors, including serum, TGF-[3 and epidermal growth factor (EGF), have been reported to affect stability (139,144-146). Preliminary evidence indicates that the TGF-[3-dependent stabilization of h u m a n PTHrP mRNAs may be mediated through cis-acting sequences that are not contained in the 3' UTR, but rather reside within the coding region (146). The inhibition of protein synthesis has been shown to result in the superinduction of PTHrP mRNA expression in a n u m b e r of h u m a n and rat cell lines (95,96,114,147). T h o u g h there is often a transcriptional c o m p o n e n t in this p h e n o m e n o n , the effect is mediated in large part at the posttranscriptional level and is presumed to reflect the reduced synthesis of critical components in the mRNA degradation/instability pathway.

DEVELOPMENTAL REGULATION The pattern of PTHrP gene expression in the adult is widespread and is even more so in the fetus (Table 2).

PTHrP: GENE STRUCTURE AND BIOSYNTHESIS /

TABLE 2

45

PTHrP Gene Expression during Embryogenesisa

Chicken

Mouse and rat

Human

Days 3-10 Viscera Allantois Yolk sac Chorioallantoic membrane Day 15 Brain Heart Lung Liver Gizzard Intestine Skeletal muscle

Day 3mcompacted morula Day 7.5mtrophoblast Days 8-12mplacental decidua Days 13-14 Epidermis, skin appendages Skeletal, cardiac muscle Vascular smooth muscle Liver parenchyma Renal tubular epithelium Bronchiolar epithelium Gastrointestinal epithelium Choroid plexus, spinal cord, dorsal root ganglia, and eye Days 15-16 Lung epithelium Perichondrium Dental lamina, inner ear Day 18 Salivary ducts Pancreatic ducts, islets Day 20.5--keratinocytes

Weeks 7-8 Trophoblastic layers of chorionic villi Lung epithelium Liver parenchyma Pancreatic acini Stomach epithelium Hindgut epithelium Kidney Perichondrium Epidermis Otic placode Tooth bud Choroid plexus, spinal cord, dorsal ganglia Weeks 18-20 Cardiac, skeletal muscle Vascular smooth muscle Endochondral and intramembranous bone

aModified from Broadus AE, and Stewart AF. Parathyroid hormone-related protein structure, processing, and physiological actions. In: Bilezikian JP, eds. The parathyroids. New York: Raven, 1994:259-294.

Cumulative data from a large n u m b e r of localization studies in fetal tissues allow the following generalizations: (1) PTHrP mRNA a n d / o r peptide can be found in almost every embryonic tissue examined, but are restricted to certain cell types within those tissues, (2) the types of tissues that express PTHrP encompass derivatives of all three germ layers, as well as extraembryonic sites, such as the amnion and trophoblast (148,149), (3) the levels a n d / o r locations of PTHrP gene expression are not static, but change as a function of developmental stage, and (4) at most sites of expression, both PTHrP mRNA and peptide are present in low abundance and require sensitive methods for detection. Indeed, it was precisely this pattern of near ubiquitous fetal expression, coupled with both spatial and temporal specificity, that first suggested that PTHrP was likely to be a factor involved in the regulation of growth and differentiation. The P T H / P T H r P type 1 receptor is also widely expressed during fetal life (150) and studies in the mouse suggest that PTHrP and the PTH1 receptor represent one of the earliest h o r m o n e receptor pairs operative in development (105). Mso, in most tissues, PTH and PTHrP are coordinately expressed in adjacent

cell layers (typically, PTHrP displays focal expression in the surface epithelium, but the receptor is expressed diffusely in the underlying mesenchyme), a pattern that is consistent with our emerging understanding of their paracrine interactions (150). Localization studies in preimplantation mouse embryos have shown that PTHrP can be detected as early as the compacted morula stage before the onset of e n d o d e r m a l differentiation (105) (Fig. 9) and that the peptide serves as an early marker for cells of the trop h e c t o d e r m lineage. PTHrP gene expression is also induced on differentiation of F9 embryonal carcinoma stem cells into parietal endoderm-like cells (137). Other studies with cultured cells, including keratinocytes and pancreatic islet tumor cells, have also shown up-regulation of PTHrP mRNA or protein when differentiation is stimulated (95,151). The temporal sequence of the tissue-specific acquisition of PTHrP gene expression during the fetal development of the rat has been carefully examined by in situ hybridization (150) (Fig. 10). By El5 to El6, PTHrP mRNA was found to be most highly expressed in the epidermis, hair follicles, and the enamel epithelium of the tooth

46

/

CHAPTER3

i~f ~ ~ ' "~....

~:~ ....

~:.:...~:~:~:,~...~a,,,

% ~ ~.......... ..

FIG. 9 Acquisition of PTHrP gene expression in preimplantation mouse embryos. Whole embryos were subjected to immunofluorescent staining with an anti-PTHrP antibody. (A) The 8-cell stage; (B) compacted morula; (C) blastocyst. Left panels: Nomarsky image. Right panels: fluorescent image. Preparations were viewed with confocal scanning laser microscopy. Reproduced from Van de Stolpe et aL (105), The Journal of Cell Biology, 1993, Vol. 120, pp. 235-243, by copyright permission of The Rockefeller University Press.

buds. Lower levels of expression were apparent in the epithelia of the inner ear and nasal cavity, the bronchial epithelium of the lung, the ependymal cells of the choroid plexus, and the endocardial cushion region of the heart. Diffuse mesenchymal expression could also be detected in some developing organ systems at this stage, but this expression diminished over time. Expression of PTHrP along the border of the intestinal epithelium could not be detected until E20. In the skeleton at E15-E16, hybridization was seen in the membranous bone forming within the mandible, in early chondrocytes of the cartilage primordia of endochondral bones such as the ribs and digits, and in the perichondrium of the sternum and the periosteum of the clavicle. By El8, expression could also be detected in the hypertrophic chondrocytes of the developing growth plates (150).

FIG. 10 Expression of PTHrP and the PTH/PTHrP receptor during development of the rat. Tissue sections were subjected to in situ hybridization and emulsion autoradiography. Darkfield views are shown. (Top) Whole rat fetus from day E15. (Bottom) Craniofacial portion of day E19 fetus. Labels: rP, PTHrP; R, PTH/PTHrP receptor; CP, choroid plexus; E, inner ear; TB, tooth bud; L, lung; H, heart; I, intestine; MC, Meckel's cartilage; Md, mandible; Mx, maxilla. Reproduced from Lee et aL (150). Lee K, Deeds JD, Segre GV. Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 1995; 136:453-463.

Longitudinal surveys have revealed a number of instances in which developmental progression is accompanied by changes in either the quantitative levels or the spatial patterns of PTHrP gene expression (150,152-158). In both the human and rat kidney, early expression of PTHrP is evident in the glomeruli and in the tubular epithelium of the mesonephros and metanephros. Soon after, however, glomerular expression begins to decline, and by midgestation, expression is limited to the proximal and distal tubules, the collecting duct, and the urothelium (152-155,158). As noted above, in the developing skeletal system of the rat, PTHrP is expressed in the early mesenchyme and cartilage primordia of the ribs, limbs, and vertebrae. As maturation proceeds, however, expression of the pep-

P T H r P : GENE STRUCTURE AND BIOSYNTHESIS

tide becomes increasingly restricted to hypertrophic chondrocytes, osteoblasts, and areas of the perichondrium and bone collar (150,152,154). Similar changes in PTHrP expression patterns have been observed to occur in the lung, liver, and dental lamina. The stage-specific acquisition of PTHrP gene expression in a number of organ systems, especially the developing epidermis, mammary gland, placenta, endochondral bone, tooth, and central nervous system, has been shown to correlate with distinct developmental functions, many of which are now becoming increasingly well understood. These varied roles of PTHrP in regulation of programmed differentiation and physiologic responses are detailed in succeeding chapters.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health grants DE12616, AR46032, and DK45735.

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hormone-related protein expression by oral cancer cells (HSC-3). J Endocrino11998;156:349-35 7. Weaver DR, Deeds JD, Lee K, Segre GV. Localization of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor mRNAs in rat brain. Mol Brain Res 1995;28:296-310. Weir EC, Brines ML, Ikeda K, Burtis WJ, Broadus AE, Robbins RJ. Parathyroid hormone-related peptide gene is expressed in the mammalian central nervous system. Proc Natl Acad Sci USA 1990;87:108-112. Holt EH, Broadus AE, Brines ML. Parathyroid hormone-related peptide (PTHrP) is produced by cultured cerebellar granule cells in response to L-type voltage-sensitive Ca 2+ channel flux via a CaZ+/calmodulin-dependent kinase pathway. J Biol Chem 1996;271:28105-28111. Moreno Davila H. Molecular and functional diversity of voltagegated calcium channels. Ann NYAcad Sci 1999;868:102-117. Nakayama T, Ohtsuru A, Enomoto H, Namba H, Ozeki S, Shibata Y, Yokota T, Nobuyoshi M, Ito M, Sekine I, Yamashita S. Coronary atherosclerotic smooth muscle cells overexpress human parathyroid hormone-related peptides. Biochem Biophys Res Commun 1994;200:1038-1045. Noda M, Katoh T, Takuwa N, Kumada M, Kurokawa K, Takuwa Y. Synergistic stimulation of parathyroid hormonerelated peptide gene expression by mechanical stretch and angiotensin II in rat aortic smooth muscle cells. J Biol Chem 1994;269:17911-17917. Ito M, Ohtsura A, Enomoto H, Ozeki S-I, Nakashiima M, Nakayama T, Shichijo K, Sekine I, Yamashita S. Expression of parathyroid hormone-related peptide in relation to perturbations of gastric motility in the rat. Endocrinology 1994;134:1936-1942. Calaghan SC, White E. The role of calcium in the response of cardiac muscle to stretch. Prog Biophys Mol Bio11999;71:59-90. Holt EH, Lu C, Dreyer BE, Dannies PS, Broadus AE. Regulation of parathyroid hormone-related peptide gene expression by estrogen in GH4C1 rat pituitary cells has the pattern of a primary response gene. J Neurochem 1994;62:1239-1246. Ikeda K, Mangin M, Dreyer BE, Webb AC, Posillico JT, Stewart AF, Bander NH, Weir EC, Insogna KL, Broadus AE. Identification of transcripts encoding a parathyroid hormonelike peptide in messenger RNAs from a variety of human and animal tumors associated with humoral hypercalcemia of malignancy. J Clin Invest 1988;81:2010-2014. Ikeda K, Weir EC, Mangin M, Dannies PS, Kinder B, Deftos LF, Brown EM, Broadus AE. Expression of messenger ribonucleic acids encoding a parathyroid hormone-like peptide in normal human and animal tissues with abnormal expression in human parathyroid adenomas. Mol Endocrino11988;2:1230-1236. Chan SDH, Strewler GS, King KL, Nissenson RA. Expression of a parathyroid hormone-like protein and its receptor during differentiation of embryonal carcinoma cells. Mol Endocrinol 1990;4:638-646. Sheng M, Greenberg ME. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 1990;4:477-485. Heath JK, Southby J, Fukumoto S, O'Keeffe LM, Martin TJ, Gillespie MT. Epidermal growth factor-stimulated parathyroid hormone-related protein expression involves increased gene transcription and mRNA stability. BiochemJ 1995;307:159-167. SouthbyJ, Heath JK, O'Keeffe LM, Martin TJ, Gillespie MT. The 3' untranslated sequence of the human PTHrP gene confers differential mRNA stability. J Bone Miner Res 1995;10 (Suppl. 1):$279. Campos RV, Zhang L, Drucker DJ. Differential expression of RNA transcripts encoding unique carboxy-terminal sequences

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of human parathyroid hormone-related peptide. Mol Endocrinol 1994;8:1656-1666. Knlys V, Huez G. Translational control of cytokine expression by 3' AU-rich sequences. Biochimie 1994;76:862-866. Zubiaga AM, Belasco JG, Greenberg ME. The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol Cell Bio11995;15:2219-2230. Zakalik D, Diep D, Hooks MA, Nissenson RA, Strewler GJ. Transforming growth factor beta increases stability of parathyroid hormone-related protein messenger RNA. J Bone Miner Res 1992;7:S118. Kirayama T, Gillespie MT, Glutz JA, Fukumoto S, Moseley JM, Martin TJ. Transforming growth factor beta stimulation of parathyroid hormone-related peptide: A paracrine regulator? Mol Cell Endocrinol 1993;92:55-62. Sellers RS, Tannehill-Gregg SH, Capen CC, Rosol TJ. Cis-acting elements in the 3' untranslated and coding regions of parathyroid hormone-related protein mRNA mediate transforming growth factor-J3 induced stability. J Bone Miner Res 1999; 14 (Suppl. 1) :$546. Ikeda K, Lu C, Weir EC, Mangin M, Broadus AE. Regulation of parathyroid hormone-related peptide gene expression by cycloheximide. J Biol Chem 1990;265:5398-5402. Beck E Tucci J, Senior PV. Expression of parathyroid hormonerelated protein mRNA by uterine tissues and extraembryonic membranes during gestation in rats. J Reprod Fertil 1993;99:343-352. Karperien M, Lanser P, DeLaat SW, Boonstra J, DeFize LHK. Parathyroid hormone-related peptide mRNA expression during murine postimplantation development: Evidence for involvement in multiple differentiation processes. Int J Dev Biol 1996;40:599-608. Lee K, Deeds JD, Segre GV. Expression of parathyroid hormonerelated peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 1995;136:453-463. Kremer R, Karaplis AC, Henderson J, Gulliver W, Banville D, Hendy GN, Goltzman D. Regulation of parathyroid hormonelike peptide in cultured normal human keratinocytes. J Clin Invest 1991;87:884-893. Burton PBJ, Moniz C, Quirke P, Malik A, Bui TD,J/ippner H, Segre GV, Knight DE. Parathyroid hormone-related peptide: Expression in fetal and neonatal development. JPath 1992;167:291-296. Campos RV, Asa SL, Drucker DJ. Immunocytochemical localization of parathyroid hormone-like peptide in the rat fetus. Cancer Res 1991;51:6351-6357. Moniz C, Burton PBJ, Malik AN, Dixit M, Banga JP, Nicolaides K, Quirke P, Knight PE, McGregor AM. Parathyroid hormonerelated peptide in normal human fetal development. J Mol Endocrinol 1990;5:259-266. MoseleyJM, HaymanJA, DanksJA, Alcorn D, Grill V, SouthbyJ, Horton MA. Immunochemical detection of parathyroid hormone-related protein in human fetal epithelia. J Clin Endocrinol Metab 1991;73:478-484. Senior PV, Heath DA, Beck E Expression of parathyroid hormonerelated protein mRNA in the rat before birth: Demonstration by hybridization histochemistry. J Mol Endocrino11991 ;6:281-290. Schermer DT, Chan SDH, Bruce R, Nissenson RA, Wood WI, Strewler GS. Chicken parathyroid hormone-related protein and its expression during embryo loci development. JBone Miner Res 1991;6:149-155. Dunne FP, Ratcliffe WA, Mansour P, Heath DA. Parathyroid hormone-related protein (PTHrP) gene expression in fetal and extra-embryonic tissues of early pregnancy. Hum Reprod 1994;9:149-156.

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CHAPTER 4

Interactions of Parathyroid Hormone and Parathyroid Hormone-Related Protein with Their Receptors

MICHAEL CHOREV, JOSEPH M. ALEXANDER, AND MICHAEL ROSENBLATT

Division of Bone and Mineral Metabolism, Charles A. Dana and Thorndike Laboratories, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

INTRODUCTION

plex protein structure occupying three distinct phases: the intracellular loops and carboxyl terminus (located in the cytoplasm), the seven hydrophobic membranespanning helices, and the extracellularly oriented N terminus and loops. The receptor is posttranslationally modified by N-glycosylation and disulfide bond formation, and u n d e r certain circumstances it can also be phosphorylated. Heterotrimeric G proteins are composed of a unique ot subunit, which binds GDP or GTP with high affinity, and which is associated with a [3y complex. H o r m o n e binding to the GPCR increases its affinity for the GDP-bound heterotrimeric G protein complex, which in turn activates it and causes a "GTP for GDP" exchange. The GTP-bound G protein separates from the receptor, and the Got-GTP subunit dissociates from the [3y complex. Both the G0t-GTP and the [3y dimer are able to interact with effectors such as adenylyl cyclase and phospholipase C. Hydrolysis of GTP to GDP by the GTPase activity of the e¢ subunit results in the dissociation of the oL subunit from the effector molecule, allowing its reassociation with the [3y dimer. G protein signaling is thus governed by the rates of GTP binding (catalyzed by the receptor) and GTP hydrolysis. This system is highly dynamic. The hormone has more diversified conformations when in solution than when membrane bound, and its conformation changes on interaction with the receptor. On ligand binding, the receptor may change conformation to allow global movements of transmembrane domains that lead to changes in the conformation of the cytoplasmic portions of the receptor. This, in turn, increases affinity

Obtaining a detailed understanding of structurefunction relations of a h o r m o n e - r e c e p t o r complex at the most fundamental level currently requires an interdisciplinary approach; state-of-the-art techniques in peptide and protein biochemistry, as well as cellular and molecular biology, must be utilized. Traditionally, most or all of the insights regarding the h o r m o n e - r e c e p t o r complex have been obtained by correlating the effects of structural modifications on function of either the hormone or the receptor molecule alone. The detailed mechanisms that explain how structural changes in either hormone or receptor can alter the h o r m o n e - r e c e p t o r bimolecular interaction or later in signaling events are only beginning to be understood. The complexity of the parathyroid hormone ( P T H ) - r e c e p t o r system presents a significant challenge, but investigations of this system have yielded novel insights, some of which may be generalizable to many members of the superfamily of G protein-coupled receptors (GPCRs). The P T H - r e c e p t o r system is composed of at least three major constituents: the linear peptide hormone, heptahelical transmembrane receptors, and heterotrimeric guanine nucleotide binding proteins (G proteins). PTH, a fully active form of which is a linear peptide 34 amino acids long, is a highly flexible molecule. Therefore, an assortment of low-energy conformations exists in fast dynamic equilibrium. Only a subset of these conformations is thought to be acceptable to the receptor for binding. The receptor is acomThe Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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toward the GDP-bound G protein. The formation of the hormone-receptor-G-protein ternary complex represents the initiation of the intracellular signaling cascade. In addition to h o r m o n e - r e c e p t o r - G protein interactions, there are important interactions of these components with other molecules, such as [3-arrestins, regulators of G-protein signaling (RGS) proteins that act as GTPase-activating proteins (GAPs), G proteincoupled receptor kinases (GRKs), and receptor activitymodifying proteins (RAMPs). Formation of GPCR homodimers and heterodimers may represent an additional mechanism for modulating receptor function. Several receptors, including the type 1 P T H / PTH-related protein (PTHrP) receptor (PTH1-Rc), can interact with more than one G protein (e.g., the PTH1Rc interacts with e i t h e r Gq o r G s proteins), but other GPCRs interact directly via their C-terminal domain with proteins containing primary decidual zones (PDZs) and Enable/vasodilator-stimulated phosphoprotein (VASP) homology-like domains. The complexity of the GPCR signaling system requires that studies of structure-function relations include direct analysis of multicomponent systems, such as the ligand-receptor complex, in order to achieve useful level of resolution. Analyzing the system at this level may provide insight into partially understood physiologic and pathophysiologic processes associated with the PTH/PTHrP-receptors system. These studies have the potential to identify new therapeutic targets and generate paradigms useful for the development of novel agents directed at ligand-receptor interactions. This chapter summarizes recent findings obtained from studies of either PTH ligands or receptors as single entities and the most recent data emerging from direct study of the complexes formed when PTH or PTHrP interact with the PTH1-Rc or PTH2-Rc. To provide a framework for understanding the most recent approach developed to study the PTH/PTHrP-receptors system, namely the "ligand-receptor-centered" approach, we review first the "ligand-centered" and "receptor-centered" approaches. Studying ligands and receptors separately also generates important insights. However, these concepts can only be validated when the hormone-receptor bimolecular interaction is examined directly.

LIGAND-CENTERED APPROACH Recent Advances in Structure-Activity Relations Extensive reviews have been published covering early work on structure-activity relations of PTH and PTHrP (1-3). It has been established for both these calciotrophic hormones that the N-terminal 1-34 amino

acid sequence of either is sufficient to induce the entire spectrum of in vitro and in vivo PTH1-Rc-mediated activities (4,5). PTH(1-34) and PTHrP(1-36) are equipotent for binding to the PTH1-Rc and for stimulating adenylyl cyclase and intracellular calcium transients in cells expressing the PTH1-Rc. Significant sequence homology is shared by residues 1-13 of both hormones (8 identical residues), though sequence homology is negligible for the 14-34 sequence. The assignment of "activation domain" to the homologous N-terminal sequences is based on demonstration that this region has a functional role in intracellular signaling, and that truncation of 2-6 residues from the N terminus converts an agonist peptide to an antagonist (6-8). The divergent mid- and C-terminal amino acid sequences contain the "binding domain" assigned to residues 14-34 (9-12). Based on these observations, it was hypothesized that N-terminal sequences comprising the activation and binding domains of both hormones share similar conformations despite their sequence differences (10,13). The early work on structure-activity relations of PTH and PTHrP has been reviewed previously (1,2); provided below is a summary of the more recent progress in this field.

Truncated Sequences Although amino-terminal fragments of PTH and PTHrP shorter than 1-27 were initially reported to be devoid of biological activity (14-17), recent efforts of Gardella and co-workers have focused on the activation domain represented by the amino terminus PTH(1-14) (18-20). In the search for small peptide and nonpeptide molecules with PTH activity as potential therapies for metabolic bone disorders, the marginally active PTH(1-14) was used as the starting point for structure-activity studies. The rationale was based on site-directed mutagenesis and chimera studies of PTH1-Rc (21-24), functional analysis of structural complementary between PTH1-Rc/calcitonin (CT) receptor chimera and P T H / C T hormone hybrids (25), and photoaffinity cross-linking studies between photoreactive PTH and PTHrP analogs and PTH1-Rc (26-30) or PTH2-Rc (31). These studies suggest that the activation domain of PTH interacts with the extracellular loops (ECLs) and the juxtamembrane portions of the transmembrane (TM) domains of receptor. These receptor sites are different than those involved in interacting with the binding domain of PTH, which is primarily within the receptor's N-terminal extracellular domain (N-ECD). Similar observations were reported for secretin (32,33), vasoactive intestinal peptide (VIP) (33,34), CT (35), and CT/glucagon chimera (36) receptors, all belonging to class II (or group B) of the GPCRs.

PTH/PTHrP/REcF~eTOR INTERACTIONS / PTH (1-14) stimulated cAMP levels with equipotency (EC50 ~ 100 IzM) via the intact rat (r) PTH1-Rc and the N-terminal truncated (missing residues 26-181 of the N-ECD) receptor (rANt), both transiently expressed in COS-7 cells. In contrast, PTH(1-34) was two orders of magnitude less potent in stimulating cAMP accumulation in the rANt than in the intact rPTH1-Rc (18). In addition, "Ala-scan" of PTH(1-14) revealed that the first nine N-terminal residues form the critical activation domain and are involved in ligand-receptor interaction rather than an intramolecular interaction with the C-terminal domain PTH(15-34), as was previously suggested (37-39). This study concludes that the N terminus of PTH interacts with binding determinants within the ECLs and the juxtamembrane portions of the TM domains of PTH1-Rc, Interestingly, some substitutions in the 10-14 sequence of the hormone were not only compatible with function, but also resulted in more potent peptides , such as [Ala3,10,12, Arg ~1] PTH (1-14) 1 and [Ala ~'1°, Arga1]PTH(1-11), which were 100- and 5-fold more active than PTH(1-14), respectively (19). In addition, increases in cAMP levels were observed following the insertion of His, a "Zn 2+ switch," into positions in the 10-13 sequence of PTH(1-14) (20). Taken together, Gardella and co-workers suggest that the C-terminal portion of PTH(1-14) contributes important interactions with the ECLs and TM domains, which are stabilized by complex formation with Zn 2+ salts. However, in the absence of demonstrable specific binding, the extremely small increases in cAMP production do not provide a high degree of confidence that the reported cAMP increases are mediated by a specific interaction between PTH(1-14) and PTH1-Rc. Substitutions within the Intact Sequence

Several independent studies comprehensively scanned either the entire or limited segment of PTH(1-34/36) by a multiplicity of substitutions. Extensive corroborating data were generated, as well as some new insights into the tolerance and significance of certain residues with regard to bioactivity (40-42). Both Gardella and Oldenburg and their co-workers used recombinant DNA methodologies to generate analogs either randomly mutated at codons 1-4 in hPTH (40) or with individual codon replacement with [ (A/G) (A/G)G] (coding for lysine, arginine, glummine, or glycine) (42). In addition, Gombert et al. used a parallel multisynthesis approach to generate D-Ala, L-Ala, and D-Xxx scans of hPTH (1-36) (41). 1To simplify the reference to amino acid residues in the ligand and the receptor, the amino acids of the ligand are denoted using the three-letter code, whereas the one-letter notation is used for the residues of the receptor.

55

For the D-Ala scan, the largest decrease in binding affinity was observed for the segments 2-8 and 20-28, accompanied by the largest loss in efficacy for the latter segment, especially for Arg-20, Trp-23, Leu-24, and Leu-28 substitutions (41). A high correlation between binding and adenylyl cyclase activation was observed in the L-Ala scan, with the segment 2-8 suffering the largest loss in activity (41). Only substitutions of Lys-13, Asn-16, and Glu-19 yielded slightly more active analogs. The D-Xxx scan resulted in an overall loss of affinity and efficacy, with the greatest loss at the putative amphiphilic helical domain (residues 23-29), and a slightly better tolerance at the C-terminal segment (32-36) (41). Mutations of the evolutionarily conserved first four N-terminal residues in PTH were carried out by Gardella and co-workers (40). Residues Glu-4 and Val-2 were less tolerant of substitution, suggesting that they contain important determinants for receptor binding and activation (40). Conversely, Ser-1 and Ser-3 were more tolerant of substitution, suggesting that they play less critical roles in h o r m o n e activity. The most intriguing finding of this study was the divergent activity displayed by [Arg2, Tyr~4]PTH(1-34)NH2 in two different cell lines, both expressing the wild-type PTH1-Rc (40). This analog binds to ROS 17/2.8 cells, a rat osteosarcoma cell line, with twofold higher affinity than do OK cells, an opossum kidney cell line (40). Nevertheless, it is a weak partial agonist for stimulation of adenylyl cyclase in ROS 17/2.8 cells, whereas it is a full agonist for cAMP increases in the OK cell system (40). It remains to be determined whether the differences in activity were related to potential tissue-and speciesspecific effects across the two cell types. A latter study addressed some of these questions and will be discussed below (43). The highly conserved residues Ser-3 and Gln-6 in PTH and PTHrP contribute importantly to binding and activation (39). Substitution of Ser-3 by either Phe or Tyr and of Gln-6 by Phe and Ser generated partial agonists. Both [Phe3]hPTH(1-34) and [Phe6]hPTH(1-34) were found to inhibit competitively bPTH(1-34)- and PTHrP(1-34)-stimulated adenylyl cyclase activity. Taken together, the findings that substitutions within the "activation domain" may convert full agonists into partial antagonists provide new tools to design potent full-length antagonists (1-34). It also suggests that structural perturbation of the ligand-receptor bimolecular interactions at the N terminus of PTH(1-34) may interfere with the conformational changes required for coupling the ligand-occupied receptor to G proteins, thus inhibiting induction of intracellular signaling. Some provocative observations were reported by Oldenburg and co-workers following an extensive

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CI-IAeTWk4

mutational study of h P T H ( 1 - 3 4 ) in UMR-106 rat osteosarcoma cells (42). Single nonconservative substitutions spanning the 11-30 sequence, Leu 11 Arg, Asn TM Lys, Glu 19 Arg, Glu 22 Arg, Asp 3° Arg, as well as a conserved one, Lys26 Arg, resulted in e n h a n c e m e n t of bioactivity. The C-terminal amino acid substitutions Asn 3~ Glu/Gly and Tyr 34 Lys/Glu also resulted in e n h a n c e d potency. One of the most interesting analogs is [Arg 19'22'3°, Lys 20, Hse34]hPTH(1-34), a peptide carrying a net charge of + 7 concentrated in the C-terminal amphiphilic helix. The analog is equipotent (EC50 --0.9 and Kd --- 1.5 nM) to bPTH. One possible explanation for the observation is that the amphiphilic nature of this helix is not critical per se, but rather the presence and disposition of the positive charges in this helix are critical for either intramolecular, ligand-receptor, or ligand-lipid interactions (42). Taken together, these results suggest that some favored paradigms, such as salt bridges stabilizing the bioactive conformation of PTH a n d / o r intramolecular stabilization between the amino- and carboxyl-terminal regions of PTH forming a U-shaped conformation, now need to be reexamined.

Receptor Subtype Specificity Switch The two homologous PTH receptors, PTH1-Rc and PTH2-Rc, display differing specifications for ligands: PTH1-Rc binds both PTH and PTHrP, whereas PTH2Rc binds only PTH. These two receptors provide a platform for probing the basis of molecular recognition of ligand and receptor (44,45). The finding that the N-truncated sequence, P T H r P ( 7 - 3 4 ) , can bind and weakly activate the PTH2-Rc suggests that the Nterminal sequence 1-6 of PTHrP must contain a structural element that disrupts P T H r P ( 1 - 3 4 ) - P T H 2 - R c interaction (45). Swapping the nonconserved residues in position 5 between PTH(1-34) and PTHrP(1-34) generates the single-point mutants [His 5, Nle s'ls, Tyr~4]bPTH(1-34)NH2 and [Ile5]PTHrP(1-34)NH2 (45). Indeed, in HEK293 cells stably transfected with

either hPTH1-Rc or hPTH2-Rc, the receptor specificity of these point hybrids is reversed when compared with their parent compounds. Therefore, His-5 is the specificity "switch" between these two highly homologous receptor subtypes (45). Gardella and co-workers conducted studies in COS-7 cells transiently expressing the PTH1-Rc and PTH2-Rc and reached a somewhat different conclusion (46). According to their study, two sites are responsible for the divergent specificity: position 5 determines signaling and position 23 determines receptor binding affinity. Swapping the residues in positions 5 and 23 between PTH and PTHrP results in [His 5, Phe 2~, Tyr34]PTH(1-34)NH2 (IC50 > 10,000 n M f o r both PTH1Rc and PTH2-Rc, and EC50 - 1.18 and > 1000 n M for PTH1-Rc and PTH2-Rc, respectively) and [Ile 5, Trp 2s, Tyr36]PTHrP(1-36)NH2 (IC50 = 16 and 10 nM, and EC50 = 0.21 and 0.5 n M for PTH1-Rc and PTH2-Rc, respectively). In that study [Trp 2~, Tyr36]PTHrP (1-36) NH 2 is an antagonist for the PTH2-Rc but a full agonist for the PTH1-Rc. The discrepancies between the two studies (45,46) may be related to differences in the experimental systems employed, such as stable versus transient transfections or homologous versus heterologous receptor-cell systems, and the use of different radioligands, e.g., rat- versus bovine-derived peptides. An endogenous ligand selective for PTH2-Rc, tuberoinfundibular peptide of 39 amino acids (TIP39), has been purified from bovine hypothalamic extracts (47). A homology search reveals that 9 out of the 39 residues of TIP39 are identical to b P T H (Fig. 1). Interestingly, TIP39 did not activate adenylyl cyclase in COS-7 cells transfected with either h u m a n or rat PTH1Rcs (47). The physiologic role of the TIP39-PTH2-Rc system remains to be established.

Search for the Putative Bioactive C o n f o r m a t i o n Identification of the peptide ligand conformation responsible for the recognition by, binding to, and activation of the GPCR is a major objective in structural

1

34

hPTH

SVSEIQLM

HNLGKHLNSM

~.RVEWLPKKL

QDVHNF ~~~

hPTHrP

AVSEHQLL

HDKGKSIQDL

RRRFFLHHLI

AEIHTA ~~~

SLALADDAAF

RERARLLAAL

~.RRHWLNSYM

HKLLVLDAP

TIP39

FI6. 1 Peptide Homology for human PTH, PTHrP, and TIP39. The functional N termini of hPTH and hPTHrP are shown [264-266], and are aligned with the complete human tuberoinfundibular peptide of 39 amino acids (TIP39) (47). Boldfaced amino acid residues shown in TIP39 have direct homology to the boldfaced ones shown in either PTH or PTHrP. The numbering at the top refers to PTH and PTHrP, and at the bottom, to TIP39.

PTH/PTHrP/REcEPTOR INTERACTIONS / biology. The ligand-receptor complex is the definitive system for study of the putative bioactive conformation. Unfortunately, for GPCRs this is currently an unattainable goal because no h o r m o n e - G proteincoupled receptor complex has been crystallized, probably because the receptor is embedded in the cell membrane. Based on GPCRs being embedded in the membrane, the hypothesis formulated by Schwyzer proposes that the initial conformation adapted by a ligand is induced by nonspecific interactions with the membrane (48-50). This membrane-induced conformation is the one recognized by the membrane-embedded GPCR. Therefore, study of conformations in the presence of membrane-mimetic milieu, like the micellar environment, is probably the best available approximation of the natural state. Secondary structure prediction methods (51-54) suggest that the N-terminal 1-34 sequences of both PTH and PTHrP assume helical structures at their N and C termini (39,55,56). These helical domains span residues 1-9 and 17-31 in PTH and 1-11 and 21-34 in PTHrP (39). Correlation between the receptor binding affinity and the extent of helicity was determined by circular dichroism (CD), a method that can assess the global conformational nature of a peptide (57). The same spectroscopic method estimated PTH(1-34) in water to have, on average, less than eight residues in the helical conformation. This n u m b e r was even smaller for PTHrP(1-34) (39,55,58-60). In the presence of 45% trifluoroethanol (TFE), a solvent that promotes secondary structure, the total helical content of bPTH(1-34) and hPTHrP(1-34) is 73% (39). Nevertheless, there is much controversy about the relevance of the conformation in TFE to the bioactive conformations. Early ~H nuclear magnetic resonance (NMR) studies in water demonstrate that PTH (1-34) is mostly random in structure, except for a short ordered region encompassing residues 20-24 (61-63). According to our recent findings, hPTH(1-34) in water is highly flexible, with some evidence of transient helical loops spanning the sequence 21-26 and 7-8 (64). Cohen and co-workers suggest that in TFE the amphiphilic helices located at the N and C termini of bPTH(1-34) and hPTHrP(1-34) interact to form a U-shaped tertiary structure with the hydrophobic residues facing inward to form a hydrophobic core. The hydrophilic residues orient outward and are exposed to the polar solvent (39). However, the lack of long-range interactions between the two helices in both hPTH(1-34) (65-67) do not support the notion of a U-shaped tertiary structure. Interestingly, the longrange p r o t o n - p r o t o n correlations between the two N-terminal helices (sequences 1-10 and 17-27) in full-

57

length recombinant hPTH(1-84) in aqueous TFE are d e p e n d e n t on interactions provided by the middle and C-terminal portion of the molecule (sequences 30-37 and 57-62, respectively) (68). In TFE, the low dielectric constant, which helps to stabilize helices, is also supposed to shield the side chains from hydrophobic interactions between the helices and, therefore, destabilizes alleged U-shaped tertiary structures. Marx and co-workers suggest that hPTH(1-37) in aqueous solution containing high salt concentration assumes a U-shaped structure (37). However, their reported long-range p r o t o n - p r o t o n correlations are limited to side chains of Leu-15 and Trp-23 located close to the bend forming the putative U-shaped structure, therefore leaving too much flexibility to define a stable U-shaped structure. The same researchers identified the loop region around Hisl4-Ser 17 and longrange p r o t o n - p r o t o n correlations between Leu 15 and Trp 23 found in hPTH (1-37) and in N-truncated analogs hPTH(2-37), hPTH(3-47), and hPTH(4-37) but did not interpret it to stabilize a U-shaped structure (69). Other studies of PTHrP analogs described interactions between the N- and C-terminal helical domains, in the presence of TFE, thus offering support for the U-shaped structure (70-74). However, current established understanding runs counter to the U-shaped structure as the predominant bioactive conformation of PTH(1-34) and PTHrP(1-34). In our studies (13,75) of PTHrP(1-34)-related analogs in aqueous solutions and in the presence of TFE, we could not confirm the presence of long-range helix-helix interactions (76). In addition, we studied a series of side chain to side chain-bridged monocyclic and bicyclic lactam-containing PTHrP analogs. These analogs are cyclized through side chain pairs AsplS-Lys17 and Lys26-Asps°, located at the putative N- and C-terminal helical domains, respectively (13,75,77-80). The i to i + 4 side chain to side chain cyclization is known to stabilize helical structures. Bioactivity in the agonist (1-34) and antagonist (7-34) series of lactamcontaining analogs was found to require well-defined N- and C-helical domains that are linked by two flexible hinges located around residues 12-13 and 19-20, the latter being associated with high bioactivity (Fig. 2) (13,75). Similar conclusions were reached by Gronwald and co-workers studying PTHrP(1-34) in water and in 50% TFE (81). In the presence of TFE, they observe two stable or-helical regions spanning residues 3 to 12 and 17 to 33, which are connected by a flexible linker. Their observations clearly exclude the possibility of any significant tertiary structure (81). Although Barden and Kemp mention the presence of a hinge at ArglO-Arg2° in [Ala°]PTHrP(1-34)NH2 and attribute to it a functional role in signal transduction, they also postulate

58 / CHAr'TR4 V, Antagonist C-Terminus

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FIG. 2 Superposition, using the heavy backbone atoms of residues 22-32, of agonist and antagonist containing (top) the midregion lactam, c[Lys13-Asp1~]PTH(1-34)NH 2 and c[Lys13-AsplT]PTH(7-34)NH2, respec26 30 26 30 tively, (middle) C-terminal region lactams, c[Lys -Asp ]PTH(1-34)NH 2 and c[Lys -Asp ]PTH(7-34)NH 2, 13 17 26 30 respectively, and (bottom) bicyclic mid- and C-terminal region lactams, c[Lys-Asp ,Lys-Asp ]13 17 26 30 PTH(1-34)NH 2 and c[Lys -Asp ,Lys -Asp ]PTH(7-34)NH 2, respectively. The structures were taken from the molecular dynamic trajectories to illustrate the consequences of the flexible hinge centered around Arg-19. Reprinted with permission from Ref. 13. Copyright 1997 American Chemical Society. (See color plates.)

PTH/PTHrP/Pd~CEPTOk INTERACTIONS / long-range interactions between side chains located on both sides of the turn Gln16Arg 19, implicating the presence of a tertiary structure (72). The CD and NMR studies of hPTH(1-34) in aqueous solutions (variable pHs and salt concentrations) and dodecylphosphocholine (DPC) micelles, coupled with distance geometry calculations, generate conformations that were refined by molecular dynamic simulations explicitly incorporating solvent (H20) (64). This study generated high-resolution conformational preferences of hPTH(1-34), which were then used in our building of an experimentally based model of the hormone-receptor complex. In benign aqueous solution and in the presence of DPC micelles, we observe conformational averaging that is fast on the NMR time scale. As anticipated, the two helical domains observed in aqueous solution, the N-terminal helix, comprising residues 6-14, and the C-terminal helix, comprising residues 19-23, are extended (4-17 and 21-33, respectively) and stabilized in presence of DPC micelles. A region of flexibility, which is centered around residues 15-16 in aqueous solutions and around residues 18-19 in the micellar system, separates both helices (Fig. 3). Therefore, in solution the helices adopt a range of orientations, none of which corresponds to a tertiary structure in which helix-helix inter-

59

actions can be observed (64). This observation is in complete accord with conformational studies of lactam-containing PTHrP analogs (13), PTH-PTHrP point-mutated hybrids (82), and a model amphiphilic oL-helix-containing PTHrP analog (83). The more hydrophobic and amphiphilic C-terminal sequence in PTHrP has a higher propensity for helicity than does the N-terminal domain, as demonstrated by the higher percentage of TFE needed for nucleation of an N-terminal helix (13). Similar conclusions were reached on the addition of lipids (55,58,84) or DPC micelles (64,83) to PTH- and PTHrP-derived sequences. Taken together, the conformational analyses allow us to postulate a dynamic model for ligand-receptor binding. Binding is initiated by complementary hydrophobic interactions between the hydrophobic face of the amphiphilic C-terminal helical domain of the ligand, including the principal binding domain, and the hydrophobic membrane. This hydrophobic ligand-membrane interaction allows the propagation of the C-terminal helix and formation of specific interactions with the extracellular portions of the PTH receptor. In the membrane environment, nucleation of the N-terminal helix occurs, either cooperatively with (in the antagonist) or independently from (in the agonist) the previously formed C-terminal

FIG. 3 Ribbon diagram of the two conformations resulting from the ensemble-based calculations. Averaging over this twomember ensemble fulfills all of the experimental observations. The different locations and extents of the (x-helices are highlighted in gray; the side chain of Trp-23, used to align the conformations, is shown in ball-andstick conformation (64).

60

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CHAPTER4

helical domain. Consequently, in the case of P T H / P T H r P agonists, the flexibility around hinges 12-13 and 19-20 allows the membrane-induced "message domain," to be correctly positioned within the receptor. Thus, specific message-receptor interactions leading to the conformational changes required for signal transduction can occur. In the case of P T H / PTHrP antagonists, which lack most of the "message" sequence, no conformational change in the receptor occurs and no signal transduction event is triggered. Absence of the critical hinge around position 19-20 or a shift in register of the hinge region results in reduced binding affinity and efficacy, as observed for the cyclic PTHrP analogs (13) and point-mutated PTH/PTHrP hybrids (82). This proposed dynamic model for ligandmembrane-receptor interaction provides a testable paradigm for future experiments.

cations that stabilize an element important in the putative bioactive conformation (13,77-79,85-87). Much attention has been drawn to the amphiphilic nature of the C-terminal helix comprising residues 20-34 of hPTH(1-34) and its role in receptor-binding (11,58). A Lys 27 to Leu substitution in PTH(1-34)NH 2 and PTH (1-31) NH 2 improved the amphiphilic character of the C-terminal helical sequence, resulting in about a fivefold and twofold increase in adenylyl cyclase activity over the corresponding nonsubstituted sequences (85,86). The substitution in PTHrP(1-34)NH 2 of the sequence 22-31 with a model amphiphatic peptide (MAP; Glul-Leu-Leu-Glu-Lys-LeuLeu-Glu-Lys-Leu-Lysl°), which is highly e~-helical when incorporated into short peptides (88), generates [ (MAP~_10)22-31]hPTHrP(l-34) NH 2 (RS-66271) (Fig. 4) (89). Important structural features, such as Leu-24 and Leu-27, are maintained in this analog; Ile-22 and Ile-31 are substituted conservatively by Leu. In aqueous buffer, RS-66271 displays eight- to ninefold higher helicity compared to the parent peptide. Detailed conformational analysis of RS-66271 in water, employing CD and 1H NMR spectroscopy, confirms the presence of an extensive helical structure encompassing residues 16--32 (83). The absence of a hinge element around Arg-19, considered to contribute to high biological activity, may explain the 6- and 10-fold lower adenylyl cyclase and binding affinity, respectively, in ROS 17/2.8 cells of RS-66271 as compared to the more flexible and less helical PTHrP(1-34) (88,90). Importantly, the preservation of significant in vitro potency despite multiple substitutions validates the rationale behind the design of RS-66271. Despite reduced binding affinity and efficacy in vitro, RS-66271 has significantly greater activity than either PTH (1-34) or PTHrP (1-34) in restoring lost trabecular and cortical bone in vivo in ovariectomized osteopenic

Conformation-Guided Design of PTH and PTHrP Analogs According to a generally accepted working hypothesis, only a small fraction of the ensemble of dynamically equilibrating conformations presented by linear sequences of native bioactive peptides display the putative bioactive conformation. Therefore, introduction of conformational constraints may stabilize the ligand in receptor-favored bioactive conformations by precluding a wide range of nonproductive conformations. Alternatively, enhancement of the amphiphilicity of a helix will increase potency if this structural feature is indeed important for stabilizing a favored secondary structure a n d / o r providing an important productive interaction with the receptor. Such modifications may favor conformations with the highest affinity for the receptor, resulting in enhancement of bioactivity. To this end, several studies incorporated structural modifi-

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the

PTH/PTHrP/RECF~PTOR INTERACTIONS rats (89,90). Another study confirmed the reduced in vitro activity of RS-66271, but observed anabolic activity in vivo that was similar to that of PTH (1-34) (91 ). Usdin and co-workers have suggested a different explanation to resolve the discrepancy between the low binding affinity of RS-66271 in vitro and its potent anabolic activity in vivo (92-94). Using a membrane-based binding assay, they demonstrated that hPTH(1-34) binds with high affinity (IC50 < 10 nM) to the PTH1-Rc whether it is coupled or uncoupled to G-protein (93). By contrast, RS-66271 binds with high affinity (IC50 = 16 pM) to the coupled receptor but with much lower affinity (IC50 > 100 nM) to the uncoupled receptor (94). Therefore, the uncoupled PTH1-Rc in the cell-based binding assay binds RS-66271 with lower affinity compared to hPTH(1-34). The lower affinity to the uncoupled receptor may result from either less favorable interactions between the MAP sequence and the receptor or a compromised conformation of RS-66271. It is generally accepted that PTH(1-34) and PTHrP(1-34) contain two helical domains spanning sequences 13-18 and 20-34 (66,67,95,96). Introduction of side chain to side chain cyclizations between residues that are four amino acids apart and located across a single helical pitch (residue i to residue i + 4) has been demonstrated to be an effective way to stabilize a helical structure (97-101). Therefore, we undertook replacement of a potential ion pair, participating in or-helical stabilization, by a covalent lactam bridge, in an attempt to further stabilize the helices in these regions. The initial application of this approach generated c[Lysl~-Asp17]PTHrP(7-34)NH2, which was about 10-fold more potent than the linear parent antagonist (Kb = 18 and 170 nM, Ki = 17 and 80 nM, respectively, in SaOS-2/B10 cells) (77). Rigidification of the C-terminal helix in c[LysZ6-Asp3°]PTHrP(7-34)NH2 did not improve antagonist potency (79). However, combination of two 20membered lactam bridges, in both the N- and C-terminal helices, generated c[Lysl~-Asp17, Lys 26Asp~°]PTHrP(7-34)NH2, a potent (Kb = 95 n M and Ki = 130 nM, in SaOS-2/B10 cells) (79) highly conformationally constrained PTHrP-derived antagonist and a valuable tool for conformational studies (75). The same approach applied to the agonist PTHrP(1-34)NH 2 yielded the mono- and bicyclic analogs, 13 17 26 c[Lys13-Asp17]PTHrP(1-34)NH2 and c[Lys-Asp , L y s 3O Asp ]PTHrP(1-34)NH 2, which were equipotent to the linear parent compound (Kb = 3.2, 2.1 and 1 nM, Km = 0.17, 0.22, and 0.57 nM, respectively, in SaOS-2/B10 cells) (79). A similar approach was also applied to the signalingselective analogs, hPTH (1-31)NH 2 and the more potent [Leu 27]hPTH (1-31 ) NH 2. Both of these analogs stimulate

/

61

adenylyl cyclase but not the PLC/PKC signaling pathway (85). Though i to i + 4 lactam bridge formation between Glu-22 and Lys-26, as in c[Glu 22LysZ6,Leu27]hPTH (1-31) NH 2, results in about a fourfold increase in adenylyl cyclase activity, as compared to the linear parent peptide (EC50 = 3.3 and 11.5 nM, respectively, in ROS 17/2 cells), similar cyclization between Lys-26 and Asp-30 or i to i + 3 lactam bridge formation between Lys27 and Asp-30 results in cyclic analogs that are less potent than the corresponding linear parent peptides (85). Interestingly, the higher adenylyl cyclase activity in vitro observed for c[ Glu22-LysZ6,Leu 27] hPTH (1-31 ) ~qH2 compared to the linear peptide results in greater anabolic effect on trabecular bone growth in ovariectomized rats (102) and affords more effective protection than hPTH(1-34) against loss of femoral trabeculae in the same animal model (103). The retention of full ability to activate PKC (in ROS 17/2 cells) by the extensively N-terminally truncated linear fragment, [LysZ7]hPTH(20-34)NH2, and the structurally related lactam-bridged analog, c[Lys 26Asp~°]hPTH(20-34)NH2, was consistent with the stabilization of the amphiphilic helix at the C-terminus, implicating the helix as an important functional motif for binding to the PTH1-Rc (104). Taken together, the above studies provide important insights regarding the structural nature of the hormones PTH(1-34) and PTHrP(1-34) and help to better characterize conformational features important for PTH binding and bioactivity.

Signaling-Selective Ligands Activation of PTH1-Rc evokes dual signaling pathways, increasing both adenylyl cyclase/PKA via GsOLand PLC/IP~-DAG/cytosolic transients of [CaZ+]i/PKC via Gq (43,105-111). Dual signaling is observed in homologous and heterologous receptor/cell systems, which include rat, opossum, mouse, porcine, and h u m a n receptors and cells. In general, maximal signaling intensity through both pathways increases with receptor density. However, a larger n u m b e r of PTH1-Rcs per cell is needed to activate the PLC-associated pathway than is needed to stimulate adenylyl cyclase. PTH modulates downstream activities in osteoblasts, leading to regulation of cell growth, proliferation, and differentiation (112,113). PTH affects osteoclasts indirectly through its direct action on osteoblasts (114). Subcutaneous administration of PTH results in an immediate and transient expression of c-fos mRNA in PTH1-Rc-bearing cells (chondrocytes, osteoblasts, and spindle-shaped stromal cells), followed by a delayed expression in the majority of stromal cells and osteoclasts (115). This observation provides further support

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for the indirect action of PTH on osteoclasts, which may be mediated by osteoblasts, a n d / o r a subpopulation of stromal cells. In UMR cells, PTH rapidly and dose-dependently induces transcription of c-fos (116,117). Pearman and co-workers reported that the cAMP response element (CRE) in the c-fos p r o m o t e r is required for PTH-induction of c-fos in UMR cells and that the CRE binding protein (CREB) binds to this site, apparently as a homodimer, and is phosphorylated in a PTH-inducible fashion at Ser-133 (118). Therefore, c-fos appears to have pleiotropic and essential effects in bone, including mitogenesis a n d / o r differentiation in the skeletal system, as well as inhibition of osteocalcin expression by binding to the AP-1 site in the osteocalcin promoter, thereby suppressing the mature osteoblast phenotype (119). PTH-induced c-fos p r o m o t e r activity was completely inhibited in a concentration-dependent m a n n e r by transfection of a heat-stable inhibitor of PKA (120). This finding provides strong evidence that PKA is the enzyme responsible for phosphorylation of CREB at Ser-133 in response to PTH and that PKA activity is required for PTH-induced c-fos expression. Nevertheless, the relationship between these signaling pathways and cellular and in vivo responses to PTH has not been completely elucidated. Understanding the role of cellular processes such as receptor inactivation, internalization, trafficking, and recycling in bone metabolism is only beginning to be elucidated. One of the major questions in the PTH field focuses on the mechanism responsible for catabolic versus anabolic actions of PTH induced by continuous versus intermittent administration of hormone, respectively. The linkage of one or both the signaling pathways to the anabolic activity of PTH remains to be established.

Search for the Ligand-Based Signaling Specificity Switch Much attention is directed toward identifying a signaling pathway that is specifically associated with the anabolic activity of PTH. One hypothesis is that this pathway or portions of it will be c o m m o n to all agents and treatments that have anabolic effects on bone. Studies carried out on bone cell and organ cultures suggest that residues 1-7 in PTH constitute the cAMP/PKA activation domain (121), whereas residues 28-34 in PTH comprise the PKC activation domain (122,123). The latter encompasses the region also associated with PTH mitogenic activity on cultured osteoblast-like cells (residues 30-34) (124,125). Cyclic AMP appears to be involved in the bone formation (126) and resorption activities (16) of PTH. PTH analogs that stimulate increases in cAMP levels have been shown either to inhibit (127-129) or stimulate (129-131) osteoblastic cell proliferation, d e p e n d i n g on species, the cell models used, and the experimental

conditions. However, N-terminally truncated fragments of PTH, which selectively activate PKC without affecting cAMP (121,132,133), are also mitogenic for osteoblastic cells (134). Because these truncated fragments do not stimulate bone resorption (16), they may be more effective "anabolic" analogs than are peptides with an intact N terminus. Truncation of two amino acids from the N terminus of PTH(1-34), e.g., PTH(3-34), reduces adenylyl cyclase activation without significantly affecting PKC activation or the mitogenic response in vitro (132). Similarly, the stimulation of TE-85 h u m a n osteosarcoma cell proliferation by PTH(1-34) is not associated with an increase in intracellular cAMP (135). Therefore, if stimulation of bone formation in vivo is related only to the mitogenic response in vitro, the bone formation response should be retained in the aminotruncated PTH fragments. Although PTH stimulation of bone resorption in vitro is mediated primarily through cAMP-dependent activation of PKA (136), it may not be the sole second messenger pathway involved in this activity (137,138). One of the current working hypotheses holds that dissociation between the two signaling pathways of PTH, adenylyl cyclase and PLC, will result in separation between the anabolic and catabolic activities of PTH in bone (14,126,132,139-143). If the stimulation of bone resorption in vivo is related to the bone resorption response in vitro, the in vivo response should be diminished in amino-terminal-truncated PTH fragments. However, neither PTH(3-34) nor PTH(3-38) (both PKC-selective, N-terminal-truncated analogs of PTH) are active in vivo as bone anabolic agents (4,122,126,143-146). Furthermore, desaminoPTH(1-34), which has drastically reduced ability to stimulate adenylyl cyclase but is equipotent to h P T H ( 1 - 3 4 ) in stimulating PKC, does not stimulate cortical or trabecular bone growth in ovariectomized rats (126). Surprisingly, hPTH (1-31 ) NH 2 (Ostabolin), an adenylyl cyclase-selective PTH agonist equipotent to PTH(1-34) in stimulating cAMP production in ROS 17/2 (57,123), strongly stimulates cortical and trabecular bone growth in ovariectomized rats (126,143,145-147). In this analog the putative PKC-signaling motif GlnZS-His32 is compromised by the elimination of His-32 (123). A second generation of adenylyl cyclase-selective analog, 22 26 27 c[Glu -Lys ,Leu ] hPTH (1-31) NH2, in which the helical nature of the C terminus was enhanced by the formation of a side-chain to side-chain lactam ring and the introduction of a hydrophobic residue at position 27, was 1.4to 2-fold stronger than the linear parent analog as a stimulator of femoral trabecular bone growth (102). Both 22 26 27 hPTH (1-31) NH 2 and c[Glu -Lys ,Leu ] hPTH (1-31)NH 2 were reported to prevent loss of vertebral trabecular bone in ovariectomized rats and to increase vertebral tra-

PTH/PTHrP/RECEPTOR INTERACTIONS / becular volume and thickness over those of control vehicle-injected sham-operated rats (147). The action of these analogs on vertebral bone was as effective as that of h P T H ( 1 - 3 4 ) N H 2. However, unlike h P T H ( 1 - 3 4 ) N H 2, their effects on pelvic BMD were equivocal. An alternative view has been offered regarding the structural determinants associated with signaling pathway activation. Replacement of Glu 19 --) Arg, a receptorbinding affinity-enhancing modification, generated [Arg19]PTH(1-28) as a potent and full stimulator of adenylyl cyclase and PLC. Interestingly, substituting 1 19 Gly-1 for Ala generated [Gly ,Arg ] h P T H ( 1 - 2 8 ) , which is an adenylyl cyclase-selective agonist (148). This study concluded that the extreme N terminus of h P T H constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially h P T H ( 2 8 - 3 1 ) , contributes to PLC activation through receptor binding, but the domain is not required for full PLC activation. The N-terminal determinants for adenylyl cyclase and PLC activation in h P T H ( 1 - 3 4 ) overlap but are not identical; subtle modifications in this region may dissociate activation of these two effectors. Another approach attempted to design target organspecific PTH analogs on the assumption that a boneselective analog would be a better bone anabolic agent. To this end, [HisS] - and [Leu~]hPTH(1-34) were generated and found to be partial agonists of adenylyl cyclase in a kidney cell line (50 and 20%, respectively), but full agonists in UMR-106 rat osteosarcoma cells (149). However, both analogs were less potent than native PTH(1-34) in vivo in the induction of bone formation. In the course of designing photoreactive PTHrP analogs for mapping the bimolecular ligand-receptor interface, we generated trB p a 1,Ile 5,Ar g 1113 ' ,T ry 361jPTHrP(1-36)MH 2 (29). This analog binds and stimulates adenylyl cyclase equipotently to the parent analog [Ile5,Arg11'a~,Try~6]PTHrP (1-36) NH 2 in HEK293/C-21 cells overexpressing the h u m a n PTH1-Rc (--400,000 receptors/cell), but does not elicit intracellular calcium transients. Moreover, it does not stimulate translocation of [3-arrestin2-green fluorescent protein (GFP) fusion protein, an effect that is PKC d e p e n d e n t (150). In summary, development of an effective and safe therapeutic modality that would stimulate the formation of new, mechanically competent bone and possibly reconstitute trabecular architecture in osteoporotic patients continues to be a worthy goal. This goal may be approached by analogs that interact with the PTH1-Rc in a signaling-selective manner.

Nuclear Localization o f PTHrP PTHrP has been shown to function in a second mode of action: as an intracrine factor with direct intracellular effects following translocation into the nucleus

63

a n d / o r nucleolus of the cell. Exogenous h u m a n PTHrP(1-108) is internalized specifically by UMR106.01 osteogenic sarcoma cells that express PTH1-Rc. The h o r m o n e accumulates in the nucleus and nucleolus (151). PTHrP contains a putative nuclear localization sequence (NLS) (residues 61-94) homologous to SV40 T antigen. Deletion of the NLS, or mutation of the conserved GxKKxxK motif within the NLS, effectively prevents both cell surface binding and n u c l e a r / n u c l e o l a r accumulation of PTHrP(1-141) (152). In contrast to proteins containing conventional NLS motifs, which are actively transported by importinet[3 heterodimers (members of a family of structural molecules that mediate nuclear import of proteins containing NLS motifs), PTHrP is recognized exclusively by importin-[3 and the small GTPase, Ran, which together actively transport PTHrP to the nucleus i n d e p e n d e n t of importin-e~ (151). Thus, PTHrP appears to be actively transported to the nucleus via a novel mechanism that is i n d e p e n d e n t of importin-ot, although the biologic significance of this alternate nuclear targeting pathway is currently not understood. Synchronized cell culture studies have demonstrated that PTHrP localizes to the nucleus at the G 1phase of the cell cycle and is transported to the cytoplasm on initiation of mitosis (153). Scanning mutagenesis reveals that T-85 adjacent to the NLS of PTHrP was phosphorylated by CDC2-CDK2 in a cell cycle-dependent manner. Mutation of PTHrP, [As5]PTHrP, results in nuclear accumulation of PTHrE Mutation to [E85], which mimics a phosphorylated threonine residue, results in localization of PTHrP predominantly to the cytoplasm. This study concludes that phosphorylation of T-85 results in decreased nuclear accumulation of PTHrP, whereas the unphosphorylated state (e.g., [A-85] mutant) is preferentially nuclear localized. However, a potential role for PTHrP in regulating cellular phenotype in a cell cycled e p e n d e n t m a n n e r is currently not known. Although the precise role of PTHrP translocation to the nucleus is currently unknown, it may participate in the regulation of cell proliferation, differentiation, and apoptotic cell death during development. Future studies that characterize the nuclear actions of PTHrP would add significantly to our understanding of the role of PTHrP during embryonic skeletal development and as an oncoprotein whose expression in ~ many tumors may correlate with increased tumor aggressiveness and metastatic potential.

RECEPTOR-CENTERED A P P R O A C H The physiologic effects of PTH and PTHrP are largely mediated through the glycosylated PTH1-Rc receptor. PTH1-Rc is encoded by a single-copy gene that

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CHAPTER4

is expressed in PTH target tissues, including kidney, intestine, and bone, where it is essential for maintaining proper mineral ion homeostasis (154). In kidney, PTH1Rc mediates PTH-induced calcium resorption directly by the distal nephrons (155,156). It also inhibits phosphate resorption in the brush border membrane of proximal tubules by targeted lysosomal degradation of the sodium-dependent cotransporter Npt2 (157). In kidney and intestine, PTH1-Rc indirectly mediates vitamin D-dependent calcium absorption by regulating 10t-hydroxylase activity as well as vitamin D receptor biosynthesis (158,159). In bone, PTH1-Rc mediates acute release of calcium from mineralized matrix by activation of osteoclasts. It also plays a major role in modulating more long-standing calcium metabolism by osteoblasts and indirectly by osteoclasts. PTH1-Rc is expressed on osteoblasts as well as in many osteosarcoma cell lines, where it has been demonstrated to signal PTH-mediated changes in gene expression for a number of critical factors in bone homeostasis, including osteocalcin and osteoprotegerin (160). PTH1-Rc has been cloned from several diverse species, including human, rat, mouse, opossum, Xenopus, and zebrafish (27,110,154,161-163). It is a m e m b e r of the class II G protein-coupled receptors that include heptahelical transmembrane receptors for peptide hormones such as secretin, glucagon, and calcitonin (Fig. 5) (164). The 85-kDa PTH1-Rc contains many of the hallmark structural features of class II GPCRs, including a large extracellular domain amino terminus (N-ECD), eight conserved extracellular cysteine residues, and a large (150-190 amino acid residues) cytoplasmic C terminus (Fig. 6). Class II receptors are also identified by conserved cysteines in the first and second extracellular loops as well as by several homologous N-glycosylation sites within the N-ECD. However, though highly conserved, N-glycosylation of PTH1-Rc appears to have little or no influence on receptor expression, ligand binding, and intracellular signaling (165). Like several characterized class II GPCRs, PTH1-Rc is capable of activating multiple intracellular signaling cascades. On binding ligand, PTH1-Rc rapidly up-regulates activity of two distinct intracellular pathways, G, ot/adenylyl cyclase/protein kinase A (PKA) and Gq/phospholipase C/protein kinase C (PKC) (111,166-169). The activation of these signaling cascades gives rise to increased cytosolic cAMP or calcium, respectively. In addition to multiple PTH receptor genes, differential mRNA exon splicing is another cellular mechanism for generating receptors that have altered ligand specificity a n d / o r signaling capacity. For example, Northern blot analyses of human squamous cell lines and keratinocytes demonstrate expression of multiple PTH1-Rc mRNA transcripts that differ in size from the cloned human receptor mRNA (170). Further analysis using a

GIP-Rc Gluc-Rc GLP1-Rc PTHR1 PTHR2 PACAP-Rc VIP2-Rc Sec-Rc VIP1-Rc GHRH-Rc

CRH-Rc

t

msDH-Rc CTR-Rc

FIG. 5 Phylogenetic dendrogram of the human class II GPCR gene family. Thirteen related receptors are shown. GIP-Rc, Gastric inhibitory polypeptide receptor; Gluc-Rc, glucagon receptor; PACAP-Rc, pituitary adenylate cyclase activiating peptide receptor; VIP1- and VIP2-Rc, vasoactive intestinal peptide type 1 and type 2 receptor; Sec-Rc, secretin receptor; GHRH-Rc, growth hormone releasing hormone receptor; CRH-Rc, corticotropin releasing hormone receptor; msDH-Rc, Manduca sexta diuretic hormone receptor; CTR-Rc, calcitonin receptor.

polymerase chain reaction (PCR)-based strategy in human kidney as well as SaOS-2 osteoblast cell lines detected two variants of the PTH1-Rc mRNA that are created by alternative splicing of exons coding for the N-terminal receptor domain (171). One alternatively spliced receptor, designated the S-N3-E2 isoform, juxtaposes exon 1 encoding the signal peptide (S) to an inframe alternative 3 acceptor site within the N3 intron. This splicing event produces a novel receptor with an additional 12 amino acids in the N-terminal extracellular domain of the receptor. In a second characterized PTH1Rc isoform, S-E2, an entire exon is deleted, causing a shift in the reading frame and premature translational truncation of receptor protein. However, an N-terminal truncated receptor may be produced by reinitiation of translation at a downstream initiation codon. A recombinant cDNA encoding the S-N3-E2 alternatively spliced receptor isoform exhibited weak signaling, inducing a two- to threefold increase in cAMP content, but not intracellular calcium, after stimulation with human PTH(1-34). A recombinant cDNA encoding the truncated S-E2 isoform failed to activate either signaling

PTH/PTHrP/R~cF~pTOR INTERACTIONS /

1

S i gnu_!. Sequence ..........................

....

PGLALLLCCP PSLALLLCCP WGWLMLGSCL CGWLILRSCL

60

hPTHR1 rPTHRI hPTHR2 rPTHR2

~~~MGTARIA .....MGAARIA MAGLGASLHV MPWLEALPYI

VLSSAYALVD VLS SAYALVD L.. .ARAQLD L ....VGAQLD

hPTHRI rPTHRI hPTHR2 rPTHR2

61 RPASIMESDK GWTSASTSGK P ~ K A S G K L YPESEEDKEA PTGSRYRGRP TAANIMESDK GWTPASTSGK PRKEKASGKF YPESKENKDV PTGSRRRGRP ITAQLQEGE ...................................... GN ...... ITAQFQEGE ...................................... GN .......

120 ~LP~HIL~ | t I l ~LP~NIV~ ~FPEWDGLI~ ~FPE~GLI~

hPTHRI rPTHR1 hPTHR2 rPTHR2

121 WPLGAPGEVV WPLGAPGEVV WPRGTVGKIS WPRGTAGKTS

180 SE~VKFLTNE S KFMTNE S LQPD SD~..FLQPD

AVP~PDYIYD I I AVP~PDYIYD I ! AVP~PPYIYD ~ P S ~

ADDVMTKEEQ ADD~~EQ SDGTITIEEQ SDGTITIEEQ

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iii

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rPTHRI TiP, ER~"~tFD h P T H ~ I S IGKQEFFE rPTHR2 INIGKQEFFE

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rPTHR2

36z hPTHR1 s

PGHNRTWANY PGhq~RTWANY HSLNK~ HGS~qK~ANY

TMI

rPTHR1 ~

hPT.RI

IFLLHRAQAQ ~EKRLKEVLQ IFLLHRAQAQ ~DKLLKEVLH IVLVLKAKVQ ~ ......~ IVLVMKAKMQ

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rPTHR1 S G ~ i ~ i ii~~ iiii~T~LRETNAGRCDTRQQYr~ii hPT~ AGDI~~ i~ iii~i ~I~=AV GHDTRKQ~i rP T H ~ A G D . ~ i i i i ~ ~ i ~ ~ I ' ~ T N A V GHDMRKQYN~

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FIG. 6 Alignment of amino acid sequences and assignment of TM domains (shaded segments) of human ' (h) and rat (r) PTH1-Rc and PTH2-Rc.

65

66

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CHAPTER4

pathway in response to ligand. However, studies utilizing either iodinated PTH or receptor-specific antibodies to evaluate the cell surface expression of receptor isoforms indicate that the low or absent responses to PTH stimulation for these alternatively spliced receptors were in part due to low surface expression of the S-N3-E2 and S-E2 isoforms. Therefore, these data suggest that exon E1 is critical for cell surface expression of PTH1-Rc, but the S-N3-E2 recombinant isoform lacking this exon is capable of PTH binding and ligand-induced intracellular signaling, albeit at low levels.

PTH1-Rc and Ligand Binding A great deal of information is available detailing the current model of PTH1-Rc-ligand interaction and the important regions of receptor that facilitate recognition and binding. Experimental approaches utilizing site-specific mutagenesis, receptor chimeras with other class II GPCRs, PTH and PTHrP ligand alterations as functional probes, and photoaffinity cross-linking of receptor-ligand interfaces all have contributed to the most current understanding of PTH1-Rc structure and function. Though each of these approaches have inherent limitations, the "receptor-centered" approach to PTH1-Rc structure/function continues to refine an overall working model of the receptor domains critical for both mediating ligand binding, pharmacology, and intracellular G protein activation. Moreover, structure-function analysis of PTH1-Rc using these techniques is especially important given that X-ray crystallography, which has been instrumental in dissecting enzyme-substrate interactions, is not available for GPCR transmembrane molecules because of the lack of suitable crystals for analysis.

Chimera Receptors and a General Model of Ligand-Receptor Interactions Studies examining the structure-function of recombinant chimeric receptors, cloned from two class II members, support a general model that relies on distinct extracellular interaction domains that act in concert to affect G protein binding and activation on the intracellular receptor surface. Cognate class II members are thought to be derived from a single ancestral precursor receptor, and therefore share a general mechanism for ligand binding and activation. In this model, the N terminus of the ligand binds to the extracellularjuxtamembrane regions of the transmembrane and extracellular loop regions of TM5, TM6, and TM7 as well as ECL3 and is responsible for G protein activation; the C terminus of the ligand is critical for specific binding to the receptor N terminus. Though class II GPGR receptor structure has diverged to allow for specificity of ligand binding and

receptor activation, chimeric receptor studies have revealed that the overall pattern of ligand-receptor interactions has remained similar for many members of this large receptor family of molecules. This "cognate receptor" model was directly tested using the porcine calcitonin receptor (CTR) and rat PTH1-Rc (25). Though CT and PTH share little homology, the N termini of both ligands have been shown to be critical for receptor activation, and the C termini for receptor-binding specificity. Though similar in structure, the CTR and PTH1-Rc class II receptor glycoproteins share only 42% homology and are selectively activated only by their respective ligands. Bergwitz et al. created reciprocal CT/PTH1-Rc chimeras in which the N-ECD was exchanged between the two receptors (25). Similarly, chimeric ligands were synthesized in which the ligand activation and binding domains of each ligand were exchanged to create C T / P T H hybrid peptides. Using a COS-7 mammalian expression system to assess ligand binding and cAMP accumulation, it was demonstrated that reciprocal hybrid ligands (CTl-11/PTH 15-34 and PTHl-13/CT12-32), which do not activate the normal CT or PTH1-Rc receptors, could activate P T H / C T and C T / P T H receptor chimeras, respectively. This interaction was dependent on the receptor N-ECD binding the appropriate ligand C terminus. Chimeric receptor was then activated by the common N terminus on each hybrid ligand. Similar studies using interspecies PTH1-Rc chimeras have defined receptor domains critical for ligand binding (21). The recombinant human PTH1-Rc binds several PTH ligands that lack the N-terminal activation domain, including bPTH(7-34), bPTH(15-34), and hPTH(10-34), with at least 50-fold higher affinity than does the rat PTH1-Rc homolog, whereas binding affinities for bPTH(1-34) are similar for both receptor homologs. Applying a similar approach to the CT/PTH1-Rc chimeric receptors, recombinant chimeric r a t / h u m a n PTH1-Rc receptors were cloned and expressed in COS-7 cells. All chimeras bound bPTH (1-34) with normal affinity. However, chimeras encoding the N-ECD of the hPTH1-Rc bound bPTH (7-34), bPTH ( 15-34), and hPTH (10-34) with high specificity, whereas chimeras expressing the rat N-ECD failed to bind those ligands. As in humans, the opossum PTH1-Rc homolog binds bPTH(7-34) with high specific affinity. Studies of rat/opossum PTH1-Rc chimeras confirm the importance of the N-ECD for bPTH (7-34) binding. Thus, studies utilizing chimeric receptors that are designed to exploit the differential binding of PTH analogs demonstrate consistently that a domain within the N-ECD region of the PTH1-Rc is a critical r e c e p t o r region in determining the binding affinity of amino-terminally truncated PTH analogs.

PTH/PTHrP/RECEPTOR I N T E R A C T I O N S Reciprocal receptor chimera studies have implicated the amino-terminal portion of each receptor in having a major role in observed differences in ligand binding affinity by specifically interacting with the C terminus of PTH(l-34). Another series of experiments investigating rat/opossum chimeras helped to elucidate the mechanism by which the N-terminally modified analog, [Arge]PTH(l-34), is an antagonist with rat PTH1-Rc but is an agonist when bound to opossum PTH1-Rc (24). Here, ligand activity was associated with extracellular juxtamembrane residues of TM5 and TM6 in the carboxyl terminus of PTH1-Rc. Site-specific mutagenesis further refined the residues critical for rat PTH1-Rc interactions with the Arg-2 sidechain a s S 370 and V~71 (TM5) and L 427 (TM6). Mutagenesis studies that replaced these residues in rat PTH1-Rc with corresponding residues in the opossum PTH1-Rc (S~7°A, V371I, and LazwT) resulted in an alteration in [ArgZ]PTH(1-34) binding toward that seen with wild-type opossum receptor, yet had no effect on the binding of PTH(1-34). One of these mutations in rat PTH1-Rc, $37°A, also conferred agonist activity to [ArgZ]PTH(1-34) in cAMP assays, whereas V371I 427 and L T failed to alter receptor activation by [ArgZ]PTH(1-34). Thus, these reciprocal mutations of specific residues confirmed results from chimeric receptors. In addition, specific mutagenesis pinpoints potential residues that are critical for local direct interactions with the amino terminus of the ligand as well as for the pharmacologic profile of [Arg 2]PTH(1-34). Thus, chimeric receptor studies have pointed to at least two distinct, independently functioning domains on the extracellular surface of the PTH1-Rc receptor: (1) the N-ECD, which largely determines binding specificity of ligand by interactions with the C terminus of PTH(1-34), and (2) the TM5/ECL3/TM6 region of the receptor, which interacts with the N-terminal activation domain in PTH. Taken together, receptor chimera-based studies indicate that these class II receptors share a similar overall structure with multiple functionally independent, ligand-specific domains. These domains are sufficiently different to permit synthetic hybrid ligands to bind and efficiently activate the complementary receptor chimeras.

Site-Specific Mutagenesis Identifies Residues Critical for Receptor-Ligand Interactions Chimera studies have provided a general model of PTH1-Rc-ligand interactions. Mutagenesis studies with intact receptors have provided more detailed insights into PTH1-Rc-ligand interactions (Fig. 7). Though receptor chimera studies have identified the juxtamembrane region composed of residues on TM5 and

/

67

TM6 as an important interaction domain for the extreme amino terminus of PTH and PTHrP, scanning mutagenesis of the N-ECD has identified an important binding region in the rat PTH1-Rc (residues 182-190). Specifically, F184A, Rla6A, L187A, and I19°A located at the base of the N-ECD were demonstrated to be important determinants for maximum binding of 125I-labeled bovine PTH(l-34) and 125I-labeled bovine PTH(3-34) (172). Homologous substitutions further revealed that hydrophobicity at positions occupied by F TM and L 187 in the PTH1-Rc plays an important role in determining functional interaction with the 3-14 portion of PTH. Conversely, deletion or epitope tag substitutions of more distal N-ECD domains are welltolerated by PTH1-Rc in terms of expression efficiency, ligand-binding affinity, and specificity. Mutagenesis strategies have also identified polar residues within the hydrophobic transmembrane domains of PTH1-Rc as important determinants of

IT33A,Q37A [

Extracellular

R233H, I234N R227A,

Intracellular

12

T33A, Q37A P132L R186A H223R R227A, R230A, R233H I234N L289I, I363Y $370A, V371I T410P M425L L427T W437L Q440L Q451K I458R

PTH COOH-terminus binding Bloomstrand chondrodysplasia PTH Bpzl3 crosslinking site Jansen's metaphyseal chondrodysplasia Disruption of ligand binding, signaling Critical for ligand specificity PTH position 5 (I/H) selectivity [Arg2]PTH binding Jansen's metaphyseal chondrodysplasia PTH Bpal crosslinking site [Arg2]PTH binding PTH[1-34] binding PTH[1-34] binding Disruption of binding, signaling Jansen's metaphyseal

FIG. 7 Overview of mutational analysis of residues involved in ligand binding and receptor activation.

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CHAPTER4

receptor function (22,173). Random mutagenesis of two highly conserved polar sites resulting in conservative polar residue substitutions in the TM regions of the rat PTH1-Rc, R 233 in TM2 and Q451 in TM7, causes 17- to 200-fold reductions in the binding affinity of the agonist peptide PTH(1-34). Yet these residue changes failed to alter the binding affinity of the antagonist/partial agonist PTH (3-34). Furthermore, the double-mutant receptor (R23~/Q451) displays a binding affinity for PTH (1-34) nearly equal to that of the wild-type receptor. However, the double-mutant receptor fails to activate either GsoLor G e~ signaling. Mutation of three residues, S227, R 230, and S2%, predicted to be aligned on the same face of TM2, resulted in blunted PTH(1-34)-stimulated adenylyl cyclase response and lower binding affinity for the agonist despite efficient cell surface expression (22). The same mutation at the corresponding sites in the secretin receptor, another member of the class II GPCRs, resulted in a similar reduction in adenylyl cyclase activity. Taken together, Turner and co-workers suggest that this region in TM2 participates in the mechanism of signal transduction that is common to the class II subfamily (22). Another series of experiments confirms the important role of TM regions in ligand recognition as well as receptor structure. Mutation of a single amino acid (N~92I) in the second TM of the secretin receptor to the corresponding residue in the PTH receptor led to PTH binding and functional signaling by secretin receptor (174). The reciprocal mutation in the PTH1-Rc (I2~4N) led to a PTH1-Rc that was responsive to secretin. Neither mutation significantly altered the response of the receptors to their own ligands. The results suggest a model of specificity wherein TM residues near the extracellular surface of the receptor function as selectivity filters that block access of the wrong ligands to sites involved in receptor activation (174). Recombinant expression of portions of PTH1-Rc has also offered insights into the molecular mechanisms of ligand recognition and receptor activation. The studies described above offer a model of PTH(1-34) receptor binding whereby the extreme N terminus of the peptide interacts with binding determinants within the extracellular loops/juxtamembrane region of the receptor, but more C-terminal residues of the ligand interact with the amino-terminal extracellular domain of PTH1-Rc. Studies by Luck et al. of binding of PTH(1-14) also support this paradigm (18). Other receptor deletion mutant studies have eliminated the second extracellular loop without affecting receptor function and have shown that PTH1-Rc can accommodate a heterologous epitope tag replacement of a portion of that region and retain full binding and signaling capacity (175).

PTH2-Rc

A second class II GPCR, designated PTH2-Rc, selectively binds PTH, but not PTHrE It has been cloned from rat and h u m a n cDNA libraries (176). Immunocytochemical and in situ hybridization studies have identified a n u m b e r of endocrine cells expressing PTH2-Rc, incuding thyroid parafollicular cells, pancreatic islet D cells, and a subset of gastrointestinal peptide-synthesizing cells (177). However, little is known about the endocrine role of PTH2-Rc in these tissues. Though its tissue distribution, in particular its lack of expression in kidney and bone, suggests it has a limited physiologic role in mineral metabolism, its ligand specificity has provided insight into the current model of PTH ligand-receptor interactions. Several lines of evidence suggest that PTH is unlikely to be a physiologically important endogenous ligand for PTH2-Rc: (1) different ligand rank order of intrinsic activity of a series of PTH analogs in the human and rat PTH2-Rcs, (2) considerable lower intrinsic activities and relative potencies of PTH-like ligands at the rPTH2-Rc than at the hPTH2Rc, and (3) the partial agonist effect of PTH-based peptides when compared to bovine hypothalamic extracts (178). Receptor chimera studies, in which the extracellular domains of PTH2-Rc are selectively replaced with homologous portions of PTH1-Rc, have identified several binding domains that regulate selectivity between PTH and PTHrE For example, Turner et al. demonstrated that chimeras in which the N-ECD of PTH1-Rc was fused to the remaining PTH2-Rc at the TM1 extracellular surface permitted binding and cAMP accumulation by PTHrP (177). Similarly, PTH2-Rc N-ECD fused to PTH1-Rc altered ligand specificity and disrupted binding of PTHrE In addition, mutational analysis of PTH2-Rc residues within TM3 and TM7 demonstrated that residue changes of I244L in TM3 and both C3°7Yand F4°°L in TM7 altered specificity of PTH2-Rc and increased binding of PTHrE Based on these data, it was postulated that the extracellular juxtamembrane portion of the transmembrane domain bundle functions as a selectivity filter or barrier that prevents PTHrP from interacting with the PTH2-Rc. In addition, another study using PTH2-Rc demonstrated that the N-ECD and the ECL3, specifically residues R3O4Q and Q44°R of human PTH2-Rc and PTH1-Rc, respectively, interact similarly with PTH and that both domains contribute to differential interaction with PTHrP (179). Other chimeric studies have identified three single amino acids in PTH2-Rc, I TM in TM3, y318 in TM5, and C 307 in TM7, as being involved in the specificity switch for PTH and PTHrP (180).

PTH/PTHrP/RECEPTOR INTERACTIONS / A Third PTH Receptor Subtype Three PTH receptor genes, including a novel PTH3Rc, were cloned by genomic PCR from zebrafish (z) DNA. The zPTH1-Rc and zPTH3-Rc receptors exhibited 69% similarity (61% identity), but less homology with zPTH2-Rc. Zebrafish PTH1-Rc and zPTH3-Rc showed 76 and 67% amino acid sequence similarity with hPTH1-Rc, respectively; but similarity with hPTH2-Rc was only 59% for both teleost receptors. Recombinant zPTH1Rc bound a variety of PTH and PTHrP ligands with a high apparent affinity (IC50, 1.2-3.5 nM), including [Tyr34] hPTH- (1-34) NH2 (hPTH), [Tyr36]hPTHrP(1-36) NH 2 (hPTHrP), and [AlaZ9,Glu3°,Ala~4,Glu35,Tyr36] fugufish PTHrP(1-36)NH 2 (fuguPTHrP). In addition, zPTH1-Rc was efficiently activated by all three peptides (EC50, 1.1-1.7 nM). PTH3-Rc exhibited higher affinity for hPTHrP and fuguPTHrP (IC50, 2.1-11.1 nM) than for hPTH (IC50, 118.2-127.0 nM) and adenylyl cyclase was more efficiently stimulated by fugufish and human PTHrP (ECs0 = 0.47 _+/0.27 and 0.45 _+0.16 nM, respectively) than by hPTH (EC50 = 9.95 _+1.5 nM). Finally, total inositol phosphate accumulation by zPTH1-Rc was observed to increase after agonist administration; however, zPTH3-Rc failed to activate this signaling pathway. These studies suggest that PTH and PTH-like peptides may exert their effects via as yet uncharacterized receptors.

Receptor Mutations and Human Disease

Jansen'sMetaphysealChondrodysplasia Jansen's metaphyseal chondrodysplasia (JMC) (also see Chapter X) is a rare form of short-limb dwarfism associated with abnormalities in endochondral skeletal development, hypercalcemia, hypophosphatemia, and normal levels of PTH and PTHrP. Originally, two missense mutations in the PTH1-Rc coding region were discovered in patients with the disease (181,182). These mutations, H223R and T41°P, resulted in constitutive activation of the cAMP signaling pathway and are both located at the cytoplasmic base of TM2 and TM6, respectively. A third novel missense mutation was found (I458R) in anotherJMC patient, and is located at the cytoplasmic juxtamembrane region of TM7 (183). In COS-7 cells expressing the human I458RPTH1-Rc, basal cAMP accumulation was approximately eight times higher than in cells expressing the recombinant normal receptor. Furthermore, the I458Rmutant showed higher activation by PTH than by the normal receptor in assays measuring accumulation of downstream effectors, adenylyl cyclase and phospholipase C. Like the HZZ3R and the T41°p mutants, the I458Rmutant does not constitutively activate basal inositol phosphate accumulation. Interestingly,

69

these mutations all occur at TM regions near the intracellular loops of PTH1-Rc that are hypothesized to interact and activate intracellular G proteins and the subsequent signaling cascade. These mutations in PTH1Rc also have been utilized to screen for identification of PTH and PTHrP analogs with inverse agonist activity. Two peptides, [Leu 11, D-Trp12]hPTHrP(7-34)NH2 and [D-Trp12,Tyr34]bPTH (7-34)NH 2, exhibited inverse agonist activity in COS-7 cells expressing either mutant receptor (H22~Rand the T41°P), and reduced cAMP accumulation by 30-50% with an EC50 of approximately 50 nM (184). Such inverse agonist ligands someday may be useful tools for exploring the different conformational states of the receptor as well as leading to new approaches for treating human diseases with an underlying etiology of receptor-activating mutations.

Blomstrand Chondrodysplasia Blomstrand osteochondrodysplasia (BOCD) (also see Chapter 44) is a rare lethal skeletal dysplasia characterized by accelerated endochondral and intramembranous ossification. The phenotype of BOCD is strikingly similar to PTH1-Rc knockout mice in which PTH1-Rc-ablated mice display prominent pathology in the growth plate (185). In both human disease and the PTH1-Rc-ablated mouse model, the growth plate is reduced in size due to a lack of columnar architecture of proliferating chondrocytes, as well as a greatly reduced zone of resting cartilage. This overall similarity of phenotype suggests an inactivating mutation of PTH1-Rc as a possible underlying genetic defect causing BOCD. To date, two types of inactivating mutations have been documented in BOCD (186,187). The first is a single homozygous nucleotide exchange in exon E3 of the PTH1-Rc gene. This alteration changes a proline residue to leucine at position 132 in the receptor's amino-terminal extracellular domain. Proline 132 is conserved in all mammalian class II G proteincoupled receptors. COS-7 cells expressing a green fluorescent protein-tagged mutant receptor do not accumulate cAMP in response to PTH or PTHrP and do not bind radiolabeled ligand, despite being expressed at levels comparable to GFP-tagged wild-type PTH1-Rc. Thus, while full-length PTH1-Rc is being synthesized, it lacks binding of ligand and is functionally inactive. At least one mutation in PTH1-Rc has also been detected in BOCD that causes a shift in the receptor mRNA open reading frame and thus generates truncated receptor fragments (188). Sequence analysis of all coding exons of the PTH1-Rc gene identified a homozygous point mutation in exon EL2 in which one nucleotide (G at position 1122) was absent. The

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CI-IAPTWR4

missense mutation produces a shift in the open reading frame, leading to a truncated protein after amino acid 364 in the second extracellular loop. The mutant receptor, therefore, lacked transmembrane domains 5, 6, and 7. These are precisely the regions thought to be critical for (1) interaction of PTH1-Rc with the activation domain at the extreme N terminus of PTH and PTHrP and (2) the activation of coupled G proteins at the intracellular surface of the receptor. Functional analysis of the m u t a n t receptor in COS-7 cells and of dermal fibroblasts obtained from the patient demonstrated that the mutation was inactivating. Neither the transiently transfected COS-7 cells nor the dermal fibroblasts increased cAMP accumulation in response to PTH or PTHrE

LIGAND-RECEPTOR BIMOLECULAR COMPLEX-CENTERED APPROACH One of the most effective ways of characterizing any ligand-acceptor system is to study the intact bioactive bimolecular complex under conditions that will not perturb their active conformation. Routinely, X-ray crystallography and NMR spectrometry are the tools of choice. These methods yield very detailed structures that have been utilized in rational drug design and have generated unprecedented leads for the development of novel therapeutic agents. Enzyme-substrate/inhibitor such as cathepsin K-inhibitor (189) and HIV protease-inhibitor (190), and soluble protein acceptor-ligand systems, such as the h u m a n growth h o r m o n e (hGH)-extracellular domain of the hGH receptor (191), erythropoietin (EPO)-EPO receptor (192), and ligand-FK506 binding protein (193) are just a few of a long list of successes demonstrating the power of studying the bimolecular complex and identifying intermolecular interfaces. Unfortunately, membrane-embedded proteins like the G protein-coupled receptors are not amenable for either NMR or X-ray analysis because of their large molecular weights and inability to form crystals. Two approaches, one ligand-centered and the other receptor-centered, have been pursued to further the understanding of ligand-PTH-Rc interactions and each has made important contributions (see preceding sections). The hormone-centered approach succeeded in mapping functional domains within the h o r m o n e for receptor-binding and activation. In some cases, structural features responsible for biologic properties have been identified down to the level of a single amino acid. However, this approach cannot be used to deduce the domains of the Rc that are in contact with the horm o n e across the interface. Furthermore, in many cases, the consequences of modifying the primary structure of

the h o r m o n e cannot be assumed to alter Rc interaction unambiguously. Although structural modifications of the h o r m o n e may alter directly the interaction with an important complementary structural feature of the Rc, some substitutions in the h o r m o n e may produce their effect on bioactivity through either local or global conformational changes within the h o r m o n e that prevent adaptation of an optimal "bioactive conformation." In essence, the hormone-centered approach is "blind" to the structure of Rc. The receptor-centered approach also has succeeded in providing valuable insights. PTH receptors with amino acid substitutions or chimeric structures (interspecies of PTH1-Rc, such as rat with opossum Rc, or interhormone receptors, such as PTH1-Rc with calcitonin or secretin Rcs) have been created, and information has been obtained regarding the importance of specific Rc domains and single amino acids necessary for Rc function. However, analysis of the consequences of modification of Rc structure alone cannot be used to deduce interacting complementary elements in the hormone. Furthermore, one usually cannot determine unambiguously whether a modification in the Rc disrupts function as a result of either a local change in an important "contact site," which affects directly the interaction with a site in the hormone, or an internal global conformational change in Rc leading to modified Rc topology, and thereby altered interaction with hormone, or both. Hence, despite the attractiveness of both lines of investigation and the importance of the contributions each makes, conclusions drawn from both the hormone-centered and the Rc-centered approaches have inherent limitations and are inferential at best. Therefore, the most appealing m e t h o d for identifying hormone-Rc interacting domains is a direct one, based on the analysis of cross-linking sites. Photoaffinity labeling has emerged as an effective methodology for studying interactions of biologic macromolecules with their ligands (194-197). It is now feasible to use a photocross-linked conjugate as a starting point for mapping "contact domains," and even "amino acid-to-amino acid contact points," between a biologically active comp o u n d and an interacting macromolecule (198-210). We and others have embarked on a challenging program to map the bimolecular interface between a large peptide h o r m o n e and a seven-transmembrane-spanning Rc. The approach, using photoaffinity scanning (PAS) to identify directly contact sites in the hPTH1-Rc responsible for h o r m o n e binding and signal transduction, relies on six parallel efforts: (1) the design and synthesis of bioactive PTH analogs that are resistant to certain kind of cleavage agents and enzymes, and that incorporate a photoreactive moiety and a radionucleide; (2) production of sufficient high quantities of functional

PTH/PTHrP/RECEPTOR INTERACTIONS /

tify at a highly localized level the structural elements critical for h o r m o n e - r e c e p t o r interaction. The mapping effort is interdisciplinary. Site-directed mutagenesis is used to generate new specific cleavage sites or to eliminate existing ones in an attempt to validate the digestion map generated from the wild-type receptor. Alternatively, mutagenesis is important to reduce the size of a cross-linked fragment in order to further delineate the contact site. Synthesized or expressed receptor sequences that include a contact site are conformationally analyzed, in the presence of micelles to mimic the m e m b r a n e milieu, to provide insight on the bioactive conformation of these domains. Finally, homology searches, computer modeling employing distance geometry, and molecular dynamics are used to merge the various inputs in an effort to generate a unified experimentally based model of the bimolecular ligand-GPCR complex. Indeed, this is an iterative process in which every new finding added to the data base results in the modification or confirmation of the emerging bimolecular model. Because of the nature of this approach it cannot yield molecular structures of the same resolution as those obtained by X-ray crystallography or NMR analysis. Nevertheless, u n d e r the current circumstances and with the technology available at h a n d it yields the best approximation of the actual ligand-receptor complex.

native and mutant hPTH-Rcs to permit cross-linking, exhaustive digestion, purification (epitope-tagged hRc), and subsequent analysis of fragments generated; (3) devising a strategy for a cascade of cleavages that identifies unambiguously hormone-binding sites within the hPTH-Rcs; (4) production of antibodies to various hRc extracellular epitopes for use in purification and analysis; (5) expressing receptor domains that contain the contact sites for conformational studies; and (6) integration of the cross-linking data with our Rc mutagenesis data, and eventually with conformational analysis and molecular modeling data to generate an experimentally based model of h o r m o n e - R c complex. Special design, and synthesis of PTH and PTHrP analogs containing photophores that are strategically and uniquely inserted along the h o r m o n e sequence permit the identification of h o r m o n e - r e c e p t o r interaction sites. These could be either interactions between an amino acid in the ligand and a contact site in the receptor, or more precisely between an amino acid in the ligand and an amino acid in the receptor, namely point-to-point interactions (Fig. 8). A radiolabeled h o r m o n e - r e c e p t o r photoconjugate thus generated is fragmented using enzymatic or chemical cleavage methods. The radiolabeled h o r m o n e or its fragment is covalently linked to a segment of the receptor containing a binding domain, and is subsequently isolated and characterized, thereby identifying small regions of horm o n e and receptor that are in contact with or in proximity to each other. By moving the photoreactive cross-linking moiety along the peptide sequence to certain discrete positions in PTH or PTHrP where bioactivity can be maintained, it will be possible to map precisely the binding sites of the receptor and to iden-

Radiolabe!ed Photoreactive

Receptor

Ligand

Ligand-Receptor Complex

71

Photoreactive Analogs The initial efforts to generate a photoreactive, radiolabeled, and biologically active analog of PTH aimed to identify the receptor as a distinct molecular entity (211-214). All of these studies used poorly character-

Ligand-Receptor Ligand-Receptor Conjugate ConjugatedFragment

2A

7

=/m i

_u

Binding

UV

3

• '

Radiolabef ~ - - Photoreactive moiety

j

FI6. 8 Schematic approach to photoaffinity scanning of PTH receptors. Photocross-linking is followed by fragmentation of the resultant radiolabeled hormone-receptor photoconjugate. Comparison of the fragmentation pattern elucidated by SDS-PAGE analysis with the theoretical restriction digestion map of the receptor identifies the putative contact site. Mass spectroscopic and microsequence analysis will identify the cross-linked residue in the receptor. (See color plates.)

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CI-IAeTWI~4

ized nitroarylazide-based photophores and reported molecular masses ranging between 28 and 95 kDa for the hormone-receptor complex. Shigeno and coworkers carried out a careful synthesis and characterization of the nitroarylazide-based photoligand and identified it to be [Nle8'18,Lysla (N~-(4-N~-2-NO2-phenyl),Tyra4] PTH(1-34)NH 2, a fully active analog in ROS 17/2.8 cells (Kd = 2.8 nM compared to 0.8 n M for the corresponding photoinactive radiolabeled PTH) (215). Using this photoaffinity ligand, they were able to identify in the same cells a plasma membrane glycoprotein corresponding to the PTH receptor that had the apparent molecular mass of 80 kDa (215,216). Nevertheless, more extensive characterization of the cross-linking site could not be carried out beyond this level with the arylazide-based photoreactive PTH analogs. Our strategy for the ligand/receptor-based approach was to introduce arylketone-based PAS methodology (27,217-220) into the field of calciotrophic hormones and their corresponding receptors (26,28,29,31, 220-222). In this section we summarize the major achievements in the design and development of benzophenone-containing PTH and PTHrP ligands, and their contribution to the mapping of the bimolecular ligand-receptor interface. The benzophenone moiety (which cross-links with >50% efficiency and has greater specificity than arylazides) was employed successfully in other systems (198-210). But in the mid-1990s the methodology was still at an early stage of development and was never applied to any of the calcium-regulating h o r m o n e / G proteincoupled Rc systems. The photogenerated triplet state of the benzophenone is capable of inserting into many types of C-H bonds, provided close proximity is achieved (223). This is in contrast to most other photoaffinity labels, e.g., azidoaryl functions, which generate highly reactive electrophilic species and therefore interact preferentially with nucleophilic groups on proteins (194). There are several advantages of benzophenones over other photophores. Relatively low-energy UV radiation is needed for photoactivation (224) and the reactive biradical is nearly nonreactive in water (225). Therefore, during cross-linking experiments, a large excess of photoaffinity label is unnecessary: efficiency of cross-linking is high because only a small amount is lost to hydrolysis. In addition, the photolabile moiety is compatible with solid-phase peptide synthesis methodology. Furthermore, synthesis, purification, and biological evaluation can be conducted in the laboratory under normal ambient light conditions. Radioiodination was chosen as the tagging method of choice because of its high specific radioactivity translating into high sensitivity of detection of the radiolabeled conjugated ligand-receptor complex and the fragments derived from it. The drawbacks are the regulatory constraints imposed on working with radioactive material

and the relatively short half-life of the radiolabeled material. Needless to say, radioiodination is not necessarily an innocuous modification. In several cases, radioiodination of otherwise bioactive benzophenone-containing PTH analogs resulted in a radiolabeled analog devoid of the binding affinity required for efficient photocross-linking. Because current technology has not optimized substitution of the benzophenone moiety with a radioiodine (226), these two features must be presented on two different amino acid moieties in the ligand. Therefore, successful PAS analysis requires maintaining the connectivity between the radiotag and the photophore throughout the controlled degradation of conjugated ligand-receptor complex. Modifications in PTH (1-34), which include Met 8 and 18 _...) Nle 8 and 18, Lysl~,26, and 27 .__) Lys13,26, and 27, and Yrp 2~ --+ 2-naphthylalanine 2~ (Nal), render the ligand resistant to the various chemical and enzymatic cleavage agents [i.e., CNBr, lysyl endopeptidase (Lys-C), and BNP-skatole, cleaving at the carboxyl side of Met, Lys, and Trp, respectively]. The premise of any photoaffinity cross-linking study is that analogs with similar pharmacologic profile share with the parent peptide hormone similar bioactive conformation and generate topochemically equivalent ligand-receptor complexes. The photoreactive benzophenone-containing analogs of PTH and PTHrP were designed specifically for PAS studies aimed at investigating the bimolecular interactions of the activation and binding domains of PTH and PTHrP with either the PTH1-Rc or the PTH2-Rc subtypes. Table 1 summarizes all bezophenone-containing analogs of PTH and PTHrP reported to date.

Identification o f Contact Sites We and others have identified contact sites for positions 1, 13, and 27 in PTH and positions 1, 2, and 23 in PTHrP using the PAS methodology (26-29,222,227). Two different photophores were used in different studies; p-benzoylphenylalanine (Bpa) (28,29,227) and Lys(N~-p-benzoylbenzoyl) (Lys(N~-pBz2)(26,27,29,222). The former has the benzophenone moiety attached to the peptide backbone through a [3-carbon while the latter is presented on a relatively long side chain removed by six atoms from the backbone. The differential positioning of the benzopheneone moiety relative to the backbone may play a limited role in selecting the cross-linking sites. Cross-Linking to Position 1 in PTH 1 Photocross-linking of [Bpa 1 ,Nle'8 1 8 ,Arg~'26'Z7,NalZ~,Tyr34]bPTH(1-34)NH2 (Bpal-PTH) to the human PTH1-Rc stably overexpressed (---400,000 Rcs/cell) in human embryonic kidney cell line 293 (HEK293/C-21)

PTH/PTHrP/R~cF.PToR INTERACTIONS / TABLE 1

Analog I

II III IV V Vl VII VIII IX X Xl Xll XlII XlV XV XVl XVll XVlII XlX XX XXl XXll XXlII XXiV XXV XXVl XXVll XXVlII XXlX XXXl XXXll XXXlII XXXlV XXXV

73

Benzophenone-containing analogs of PTH and PTHrP Analog (Ref.)

Position

[Bpa', Nle8'~8,Arg~3'26'2z,Na123,Tyr34]bPTH( 1-34)NH 2 (28) [Bpa2,Nle8'~8,Arg'3'26'27,Na123,Tyr34]bPTH(1-34)NH2 (28) [Bpa3, Nle8'~8,Arg~3'26'27,Na123,Tyr34]bPTH(1-34)N H2 (28) [Bpa4,Nle8'~8,Arg~3'26'27,Na123,Tyr34]bPTH(1-34)NH2 (28) [Bpa5, Nle8'~8,ArgO3'26'27,Na123,Tyr34]bPTH(1-34)N H2 (28) [Bpa6, Nle8"8,Arg'3'26'27,Na123,Tyr34]bPTH(1-34)NH2 (28) [Bpa7,NleS"8,Na123,Tyr34]bPTH(1-34)NH2 (217) [Arg2, Lys7(N'-pBz2),Tyr34]bPTH(1-34)NH 2 (217) [NleS"8,BpaZ,a-Nal'2, Na123,Tyr34]bPTH(7-34)NH2 (217) [Nle8'~8,Bpa~2,Na123,Tyr34]bPTH(1-34)NH2(217) [Nle8"8,Bpa'2,Na123,Tyr34]bPTH(7-34)NH2 (217) [NleS,,8,Lys,3(N,pBz2),Na123,Tyr34]bPTH(1_34)NH2 (217) [NleS,,8,D.Nal,2,Lys,3(N,.pBz2), Na123,Tyr34]bPTH(7_34) N H2 (217) [Nle8,,8,Lys,3(N,_p(3_l_Bz)Bz),Na123,Arg26,27,Tyr34]bPTH(1_34)NH2 (26) [Nle8,,8,Lys,3(N,.pBz2), Na123,Arg26,27,Tyr34]bPTH (1_34) N H2 (263) [Arg2, Lys '3(N'-pBz2),Tyr34]bPTH(1-34)N H2 (217) [NleS"8,Bpa23,Tyr34]bPTH(1-34)NH2 (217) [Nle8"8,D-NaI~2,Bpa23,Tyr34]bPTH(7-34)NH2 (217) [Nle8"8,D-NaI'2,Na123,Lys26(N'-pBz2),Tyr34]bPTH(7-34)NH2 (217) [Nle8"8,Na123,Lys26(N'-pBz2),Tyr34]bPTH(1-34)NH2 (217) [Nle8'~8,Arg'3'26,L-2-Na123,Lys27(N'-pBz2),Tyr34]bPTH(1-34)NH2 (222) [Bpa~, Ile~,Arg'l"3,Tyr36]PTHrP(1-36)NH 2 (29) [Bpa',IleS,Trp23,Tyr36]PTHrP(1-36)NH2 (227) [Bpa2,11e~,Arg~'~3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa2, IleS,Trp23,Tyr36]PTHrP(1-36)NH2 (227) [Bpa3,11eS,Arg~"~3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa3,11eS,Trp23,Tyr36]PTHrP(1-36)N H2 (227) [Bpa4,11e~,Arg~"3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa4, IleS,Trp2~,Tyr36]PTHrP(1-36)N H~ (227) [BpaS,Arg~"3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa5,Trp23,Tyr36]PTHrP(1-36) N H2 (227) [Bpa6,11e5,Arg'~"3,Tyr36]PTHrP(1-36)NH2 (29) [Bpa6, Ile~,Trp~3,Tyr36]PTHrP(1-36)NH2 (227) [lleS,Bpa23,Tyr36]PTHrP(1-36) N H2 (30)

1 2 3 4 5 6 7 7 7 12 12 13 13 13 13 13 23 23 26 26 27 1 1 2 2 3 3 4 4 5 5 6 6 23

generates an 87-kDa photoconjugate (28). Chemical digestions by CNBr and BNPS-skatole, which cleave at the carboxyl end of Met and Trp, respectively, and enzymatic digestions by lysyl endopeptidase (Lys-C) and endoglycosidase F/N-glycosidase F (Endo-F), which cleave at the carboxyl end of Lys and deglycosylate the aspargines at the consensus glycosylated sites, respectively, generate a digestion restriction map of the photoconjugated receptor. Although the resolving power of polyacrylamide gel electrophoresis is limited, the combination of consecutive cleavages (e.g., Endo-F followed by Lys-C followed by CNBr) carried out in

reversed order (e.g., Lys-C followed by BNPS-skatole and BNPS-skatole followed by Lys-C) is extremely powerful. It generates a reproducible pattern of digestions and produces a set of fragments delimited by specific end residues and the presence or absence of glycosylation sites. Comparing the putative digestion map of the hPTH1-Rc with the actual fragments identifies 125 the sequence of the smallest I-radiolabeled 1 Bpa-PTH-PTH1-Rc conjugated fragment (---4 kDa). This fragment includes the ligand (4489 Da) modified by a moiety contributed by a Met residue belonging to

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CHAPTER 4

the receptor (28). Two Met residues, 414 and 425, present at the midregion and the extracellular end of TM6, emerged as potential contact sites for position 1 in PTH. Contact between residue 1 in PTH and M 414 requires the N terminus of PTH to protrude into the seven helical and hydrophobic transmembrane domain bundle. In contrast, contact with M 425 can be achieved while the N terminus is dipping superficially into the transmembrane domain bundle. These biochemical methods can be supplemented by molecular biology to provide additional resolving power to the PSA. Transient expression of two point-mutated hPTH1-Rcs, [M414L] and [M425L], in COS-7 cells, generated fully active receptors (28). The 125I-labeled Bpa ~PTH lost its ability to photocross-link to [M425L] but not to [M414L], suggesting that position 425 is the putative contact site for position 1 in PTH. Cross-Linking to Position 13 in PTH

Biochemical analysis of the photocross-linking product of radiolabeled [NleS'lS,Lysl~(Aff-p(3-I-Bz)Bz) ,NalZ3,ArgZ6'ZW,Tyr~4]bPTH (1-34) NH 2 [Lys 13(PBz2)PTH] with hPTH1-Rc expressed in HEK293/C-21 cells identifies a glycosylated radioactive band of---6 kDa, which is delimited by Lys-C and CNBr cleavage sites at the N and C termini. The theoretical cleavage restriction map of hPTH1-Rc reveals the minimal 125 13 radiolabeled I-Lys (PBz2)-PTH-hPTH1-Rc conjugated fragment, corresponding to hPTH1-Rc(173-189) located at the C-terminal region of the extracellular N terminus (26). Site-directed mutagenesis within the 17 amino acid residues comprising hPTH1-Rc(173-189) combined with subsequent biochemical analysis further delineates the boundaries of the contact site for 125I-Lys13(pBz2)PTH to hPTH1-Rc(182-189), an 8-amino acid sequence (263). Several single site-mutated receptors were generated, which include a new Lys-C-susceptible cleavage site. The mutant [RlSlK]hPTH1-Rc was stably expressed in HEK293 cells (---200,000 Rcs/cell) and was fully functional. Compared to the wild-type receptor, Lys-C cleavage of the 125I-Lys13(pBz~)-PTH-[RlS1K] photoconjugate produces a smaller conjugated fragment (---18 vs. --~9 kDa, respectively), corresponding to a cleavage site upstream to the N-glycosylated Asn 176. Interestingly, the only functional mutations that failed to cross-link to 125I-Lys13(pBzz)-PTH were the [RlS6K/A] mutants (263). However, [R~S6K]hPTH1-Rc stably expressed in HEK293 cells cross-links effectively to 125IBpal-PTH and displays wild-type receptor-like cyclase activity and binding affinity similar to that in HEK293/C-21 cells. These findings suggest that R 186 participates in an interaction with the ligand that either

provides a contact site for position 13 in the ligand or provides an interaction that brings the ligand into the close spatial proximity required for cross-linking within the hPTH1-Rc(182-189) contact site (263). This interaction does not appear to be essential for a productive ligand-receptor interaction because [RlS6K] is fully functional and cross-links effectively with a25I_Bpaa_PTH. Cross-Linking to Position 27 in PTH

Though the previously mentioned studies address interaction between residues in the extended activation domain of PTH comprising residues 1-13, a similar approach was directed toward the principal binding domain (sequence 24-34). We have analyzed • • • 8 18 13 26 the blmolecular Interaction between [Nle' ,Arg ' ,L-223 27 34 27 Nal ,Lys (N~-PBz2),Tyr ]bPTH(1-34)NH 2 [Lys (pBzz)PTH], modified by a benzophenone-containing photophore at position 27, and hPTH1-Rc by employing a combination of biochemical analysis of the photoconjuate and site-directed mutagenesis (222). Analysis of the 5 27 I-Lys (pBzz)-PTH-PTH1-Rc photoconjugate by CNBr/ Endo-F and BNPS-skatole/Endo-F degradation pathways produced an overlapping sequence corresponding to L232-W 298. This contact domain includes part of TM2, ECL1, and the entire TM3. Secondary digestions of the CNBr- and BNPS-skatole-derived fragments by endoproteinase Glu-C, which predominantly cleaves at the carboxyl side of Glu, converged on an overlapping 38-amino acid sequence corresponding to L261-W298, which includes part of ECL1 and the entire TM3 (222). To further delineate and validate the sequence containing the cross-linking site for position 27 in PTH, three mutated receptors were generated and transiently expressed in COS-7 cells. All three receptors, [R262K], [LZ61M] a n d [LZ61A], were expressed and displayed characteristic binding affinity and PTHstimulated adenylyl cyclase activity compared to wildtype receptor. [RZ62K] a n d [LZ61M] were designed to modify the Lys-C and CNBr cleavage pattern, respectively. The [L26~A] was introduced to eliminate a favorable insertion site at position 261. Restriction digestion 125 27 262 analysis of the ' I-Lys (pBzz)-PTH-[R K] photoconjugate delineated the contact site to hPTH1-Rc(232-262). Taken together, the minimal contact sites (sequence 261-298) and (sequence 232-262) obtained from the analysis of the wild-type and m u t a n t [RZ62K] receptors, respectively, suggest either L 261 or R 262 as the contact site for Lys27 . Treatment of the 125I-Lys27(pBz2)-PTH-[LZ61M] photoconjugate with CNBr generated a conjugated fragment similar in size to the ligand. This result suggests position 261 in the receptor to be the contact

PTH/PTHrP/RECEPTOR INTERACTIONS / site for position 27 in the ligand. This was further confirmed by the elimination of effective cross-linking of I125-LysZ7(pBzz)-PTH to the mutated receptor [L261A] i n which a reactive insertion site such as Leu is replaced by Ala, a poor insertion site for the photoactivated benzophenone-derived biradical. Position 261, the contact site for position 27 in PTH, is located near the center of ECL1 (222). The identification of L 261 in hPTH1-Rc as a contact site in f o r Lysz7 in PTH provides important information for mapping the PTH-PTH1-Rc interface. The remoteness of position 27 from positions 1 and 13 in PTH, and that of L 261 f r o m R 186 and M e t 425 in hPTH1-Rc, generates an important additional structural constraint that can be used to refine the emerging experimentally based model of the PTH-PTH1-Rc complex. Based on conformational analyses and structure-activity studies of PTH(1-34) and PTHrP(1-34), the prevailing view argues that these two hormones interact very similarly if not identically with the PTH1-Rc. In line with this assumption, radioiodinated [Bpa1,Ile5,TrpZ3,Tyr36]PTHrP (1-36) NH 2 [lZ5IBpal-PTHrP] photocross-links to M 425 in hPTH1-Rc in the same fashion as the corresponding PTH analog, lz5I-Bpal-PTH (29). Cross-Linking to Position 23 in PTHrP

Another photoreactive analog of PTHrP, [Ile5,Bpa 2~, Tyr~6]PTHrP(1-36)NH2 [Bpa2~-PTHrP], modified by a benzophenone moiety incorporated at position 23, was reported by Mannstadt and co-workers to cross-link to Y23-L4°, located at the very N-terminal end of rat PTH1Rc (30). CNBr analysis of the 125I-Bpa2~-PTHrP-rPTH1Rc photoconjugate suggests that the contact site resides at the N terminus of the receptor, rPTH1-Rc(23-63). A combination of site-directed mutagenesis (single point mutation [M63I] and the double mutants [M63I,L4°M] and [M6~I,L41M]) and CNBr cleavages further delineates the contact site to span the sequence 23-40. Earlier findings demonstrated that the two mutant rPTH1-Rcs with deletions of residues 26-60 or 31-47 transiently expressed in COS-7 cells had little or no capacity to bind 125I-labeled PTH, therefore suggesting these regions to be important for ligand binding (228). Only two of the 31 32 33 four cassette mutant receptors ([V A,F A,T A, 35 36 37 38 K34A,Ea5A] and [E A,Q A,I A,F A]) spanning the 31-47 sequence displayed diminished 125I-labeled PTH binding capacity. Finally, in an Ala-scan of the 31-38 region, mutants [Ta3A] and [Q~WA] exhibited the largest loss in binding affinity of 125I-labeled PTHrP and complete loss of binding affinity toward the antagonist 11 12 [Leu ,D-Trp ]PTHrP(7-34)NH 2. Relying primarily on mutagenesis-based analysis, Mannstadt and co-workers

75

suggest that the first 18 amino acid residues of the PTH1Rc comprise the contact site for position 23 in PTH, and T 33 a n d Q37 are functionally involved in binding of the 7-34 region in PTH rather than the 1-6 region (30). The location of contact sites for two closely spaced residues in PTH/PTHrP (23 and 13) at both ends of the extracellular amino terminus of the receptor (within 23-40 and in proximity to R 186,respectively) is consistent with the current model of the ligand-receptor binding interface. The extensive length of the putative extracellular amino terminus of PTH1-Rc (---167 residues) allows for assumption of secondary and tertiary structures by the receptor that can accommodate simultaneously the above-mentioned bimolecular interactions. Cross-Linking of Position 1 of Agonist vs. Antagonist

A very interesting observation that directly distinguishes between the nature of the bimolecular interaction of an agonist versus antagonist with PTH1-Rc was recently reported by Behar and co-workers (29). Photoconjugation of radiolabeled [Bpa2,Ile5,Arg 11'13, Tyra6]PTHrP (1-36) NH 2 [BpaZ-PTHrP], a highly potent antagonist, to hPTH1-Rc was carried out in HEK293/ C-21 cells (29). Unlike the analog [BpaZ,Nle s'18, Ar g 132627 ' , , NalZ3,Tyra4]bPTH(1-34)NH2 [BpaZ-PTH], which is a full agonist (28) and cross-links t o M 425 in PTH1-Rc in a manner similar to 125I-Bpa1-PTH o r 125IBpal-PTHrP, 125I-BpaZ-PTHrP also cross-links to a proxi' mal site within the receptor domain p415-M425 (29). These results may reflect either differences between the binding modes of agonist and antagonist or differences in the interaction between the two consecutive positions in the PTHrP(1-36) sequence and PTH1-Rc. In an attempt to distinguish between these two possibilities, we utilized the agonist analog BpaZ-PTH, which carries the same photoreactive moiety at the same position as the antagonist BpaZ-PTHrE Analysis of 125I-Bpa2-PTH photoconjugates with wild-type [MalaL] and [M4Z5L] mutated hPTH1-Rcs indicates that this ligand cross-links only to the e-methyl of Met 4z5, similar to Bpal-PTHrP and to Bpal-PTH cross-linking (28). These results, therefore, provide strong support for the hypothesis that the differences observed between 2 the cross-linking of 125I-Bpa1- and 125 I-Bpa-PTHrP may reflect different interaction modes of an agonist versus an antagonist with the PTH1-Rc. Interestingly, two additional Bpa-containing PTHrP(1-36) analogs, [Bpa 2 ,Ile 5 ,Trp 23 ,Tyr36 ]-and [Bpa 4, 5 23 36 Ile ,Trp ,Tyr ]PTHrP(1-36)NH 2, were reported to preferentially antagonize and cross-link to hPTH1-Rc and hPTH2-Rc stably expressed in LLC-PK1 cells, respectively (227). However, in homologous systems composed of hPTH1- and hPTH2-Rcs expressed in a human cellular

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/

C~TF~k4

background (HEK293/C-21 and HEK293/BP-16, respectively), Bpa2-PTH is a full agonist and Bpa4-PTH is a very weak agonist with a slightly better affinity for the hPTH2-Rc (28,227). Similar to [Bpa4,Ile5,Trp2~,Tyr~6]PTHrP (1-36) NH 2 (227), [Bpa4,Ile5,Arg 11'13,TyrS6]PTHrP(1-36)NH 2 displays poor binding affinity and negligible efficacy in HEK293/C-21 cells expressing the hPTH1-Rc (29). Although PTH2-Rc may not be the physiologic target for PTH or PTHrP, its structural resemblance to PTH1-Rc, its high binding affinity, specific cross-linking, and effective coupling to the PTH-induced intracellular signaling pathways make it an attractive target for exploring structure-function relations in the P T H / PTHrP-PTH1-Rc system. Analysis of the photoconjug ates obtained on cross-linking of '25I-BpaI-PTH and 25I-Lys'~(PBz2)-PTH to hPTH2-Rc stably expressed in HEK293 cells (HEK293/BP-16, ---160,000 Rcs/cell) revealed that both hPTH1-Rc and hPTH2-Rc use analogous sites for interaction with positions 1 and 13 (31). The PAS methodology offers the only readily available experimental approach to study directly the bimolecular ligand-GPCR interface. To practice this methodology we introduce b e n z o p h e n o n e moieties, radioiodine, and substitutions that provide resistance to specific chemical and enzymatic cleavages. These modifications are tolerated as long as the modified photoreactive ligand binds to the receptor specifically and with high affinity, and stimulates adenylyl cyclase in a PTH-like manner. The photoinsertion site of the benzophenone moiety is dictated by spatial proximity. But it is also biased toward the more reactive insertion sites within its reactivity sphere. Last but not least, the cleavages employed and the level of resolution allowed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis limit analysis of the photoconjugate. The validation of a putative contact site by site-directed mutagenesis is not necessarily benign. It generates some degree of perturbation, which we accept as long as the mutated receptor is expressed and functions similarly to the wild type. Taken together, PAS is not the perfect method, but we believe it is the best available. Future improvement in separating the photoconjugated receptor a n d / o r fragment from the nonconjugated species, and the elimination or replacement of the radioactive tag with a nonradioactive tag will be very helpful in terms of allowing access to high-resolution instrumental techniques. The currently labor-intensive PAS methodology will become less time consuming and more robust as the technology evolves.

Experimentally Based Molecular Modeling The contact sites identified in the cross-linking studies described above are only a small fraction of the large

ensemble that forms the bimolecular interface. The cumulative effect of these multisite bimolecular interactions results in specific ligand recognition, binding affinity, and eventually a conformational change in the receptor that leads to specific intracellular signal transduction. In general, not all contact sites revealed by PAS methodology will have the same functional significance. However, all these contact sites will be part of the ligand-receptor interface and are therefore indispensable targets in mapping efforts. The objective of the PAS studies is to generate a series of constraints, which will be used in mapping the bimolecular interface. To this end, merging the information generated by the PAS studies with information about the conformation of the ligand and receptor domains, as well as molecular modeling, generates an integrated approach that results in an experimentally based ligand-receptor model. This integrated approach based on experimental data is in contrast to the more c o m m o n approaches that predict conformation and molecular models solely on a theoretical basis. The model for the PTH-PTH1-Rc complex is steadily evolving as new bimolecular contact sites are identified (28,229,230). The combination of hydrophobicity profile analysis and search of the Brookhaven Protein Data Bank (PDB) employing the Basic Logic Alignment Search Tool (BLAST) can identify and refine, respectively, the location of the TM helices (231,232). The identification of the TM domains of the PTH1-Rc is in good agreement with respect to the location of peptides containing TM helical regions as determined by high-resolution NMR (233,234). The arrangements of the TM heptahelical bundle in rhodopsin and bacteriorhodopsin (235-239) were used as templates for the initial arrangement of the putative TM helical domains of the PTH-Rcs. Rotating the helices about their long axis to orient the hydrophobic m o m e n t toward the membrane environment and optimize their helix-helix, helix-core, and helixmembrane interactions generated the putative core of our receptor model (Fig. 9) (234). The modeling of the extracellular and intracellular domains responsible for binding the ligand and coupling to the G protein and other adapter molecules is not as straightforward as that of the membraneembedded core. Unlike the high structural similarity for the arrangement of the seven TM domains bundle (240), the cytoplasmic and ectopic domains of the GPCR are extensively variable and no a pr/0r/ structure is available. The loops are constrained to some extent by the TM helical domains to which they are attached. Additional constraints are imposed by the three disulfide bridges at the extracellular N terminus and the disulfide bridge connecting the first and the second ECL. All of these cysteines are highly conserved in the class II GPCRs, of which PTH1-Rc is a member.

PTH/PTHrP/RECEPTOR INTERACTIONS /

TM1 TM5

IC3 FIG. 9 Depiction of the PTH1-Rc from a molecular dynamic simulation. The transmembrane oL-helices are depicted as cylinders. The regions of the receptor that have been experimentally determined are depicted as ribbons. The regions of the receptor that have been shown to crosslink with PTH analogs, PTH1-Rc(173-181) (26,27) and M42s (28), are depicted in gray (241). (See color plates.)

Unfortunately, the pattern of disulfide bridge formation in the ectopic part of the class II GPCRs is not known and therefore cannot be used in building the receptor model. Homology search with BLAST (232) helps to assign putative elements of secondary structure to regions in the receptor that are homologous to regions of protein for which a secondary structure is available. Once a secondary structure element is identified, it can be incorporated into the model. Practically, we focus only on the identification of ahelices, which can be studied in isolation from other secondary structural elements. Indeed, sequence homology searches identify conformational preferences of the C-terminal portion of the extracellular amino terminus proximal to TM1 of PTH1- and PTH2Rcs and the third extracellular loop of PTH1-Rc (28,229). These homology searches indicate that the third ECL adopts a helical conformation that is highly p r o n o u n c e d for T435-Y443 (229), and amphiphatic helices for K172-M 189 and L129-E 139 in PTH1- and PTH2Rcs, respectively (28,229). Unfortunately, such homology searches may not always result in the assignment of distinct secondary structure to a specific receptor sequence.

77

The modeling procedure for the receptor and receptor-ligand complex is described in detail by Mierke and Pellegrini (241). Briefly, the primary structure of the PTH1-Rc is embedded into a three-phase, H z O / d e c a n e / H 2 0 (40 A each) simulation cell. A multistep simulation is carried out in which either the heptahelical bundle a n d / o r cytoplasmic and extracellular domains are allowed to move freely. PTH, in its membrane-associated conformation, is added to the receptor model, applying the ligand/receptor distance constraints elucidated from the cross-linking experiments, and additional simulations are carried out. Indeed, additional constraints obtained via sitedirected mutagenesis, and chimera studies, PTH2-Rc and other class II GPCRs can be incorporated into the development of the model. The most direct way to identify the conformational features of the cytoplasmic and ectopic domains of the GPCR is by generating these receptor fragments and examining them by NMR in a membrane-mimetic system. Adding a small portion of the corresponding TMs to the otherwise flexible receptor-derived termini or loops provides anchors that partially reproduce the native orientation of the receptor domain relative to the membrane-mimicking milieu. Another design element useful in restraining an excised loop sequence from assuming extended conformations is the covalent binding of both ends of the sequence by a linker of---12 A, the approximate distance between two consecutive TM domains (235,237). To this end, we have characterized the conformational features of the third intracellular loop (ICL) of PTH1-Rc, which was constructed as a 29-amino acid peptide with the side chains of Cys residues in positions 1 and 28 bridged by an octamethylene linker (233,234). This linker also assists in the association of the peptide termini with the membranemimicking micelle. In the presence of either SDS or dodecylphosphocholine (DPC) micelles, the peptide assumes two helical domains composed of residues L e u 4 L e u 8 and Yyr22-Leu26, and [3-turns at Glul°-Ala 13 and Glya4-Asp 17. The hydrophobic residues of the N-terminal amphiphilic helix are embedded in the hydrophobic portion of the micelle, whereas the polar side chain protrudes into the aqueous phase. The two [3-turns point away from the membrane and are exposed to the aqueous solvent. The structure of the bridged peptide in the presence of micelles is distinct from the same peptide in the absence of micelles as well as from the linear form of the peptide. A similar approach was applied to characterize the conformational features of two ectopic domains found to photocross-link to Lys13 a n d Lys27 in PTH(1-34) (28,221,222). Position 13 in PTH was found to cross-link within an 8-amino acid domain,

78

/

C~a'TER4

PTH1-Rc(182-189), located at the C-terminal portion of the N-ECD proximal to the first TM helix (28,221). The combination of homology search and molecular dynamic calculations, using a two-phase simulation cell consisting of H20 and CC14 (to mimic a membrane-water interface), suggests that the segment R179-E-R-E-V-F-D-R-L-G-M189forms an amphipathic oL-helix whose axis is parallel to the membrane surface and points away from the heptahelical bundle (28,242). 1H NMR analysis of the synthetic peptide hPTH1-Rc(168-198) in presence of micelles (to provide the membranelike environment) was carried in combination with distance geometry and molecular dynamic simulation (Fig. 10) (242). The analysis identifies a C-terminal helix, hPTH1-Rc (190-196), corresponding to the ectopic portion of the first TM helix, which was perpendicularly embedded in the micelle. Two oL-helices, (180-188) and (169-176), lie on the m e m b r a n e surface. The polar residues in the linker, E 177 and R 170, and in the middle helix, R TM, E 182, D 185, and R 186, are exposed to the solvent while the hydrophobic residues, F 173, F TM,and L 187, are projecting

toward the hydrophobic membrane (242). Based on the finding that the contact site for Lys 1~ in PTH is within residues (182-189), which includes negatively charged amino acids, coulombic interaction between these charges and the positive charge on Lys is may function as one of the ligand/receptor-specific interactions. Nevertheless, this may not be an essential interaction because analogs in which the e-amino on Lys 1~is blocked by acylation maintain high affinity and efficacy. At this point one can bring together findings obtained from the cross-linking studies of position 13 in hPTH(1-34) with the putative bioactive conformation of PTH (64), and the conformational analysis of the receptor domain containing the contact site for position 13 in order to generate the first generation of an experimentally based model of the PTH-hPTH1-Rc complex (28). Using the cross-linking data as a docking cue to position the ligand (in its putative bioactive conformation) places its C-terminal amphiphilic helix parallel to the membrane-aligned portion of the receptor-derived peptide. This allows the formation of complementary coulombic interactions between the

A 8o

G188

¸¸¸i i Cytoplasm

B

FIG. 10 Structural features and topological orientation of PTH1-Rc (168-198) located at the C-terminal region of the extracellular N terminus followed by the ectopic portion of the first TM domain (241,242). (A) Schematic representation of the experimentally determined conformation. The structure consists of three o~-helices, two of which have been determined to lie on the surface of the membrane; the third, at the top of TM1, is membrane embedded. (B) The orientation of this peptide is shown with respect to the surface of the dodecylphosphocholine micelles used in the NMR study. The hydrophobicity of the molecule is indicated (blue, polar; red, hydrophobic). The decane molecules of the water/decane simulation cell used in the structure refinement are shown in green as CPK space-filling spheres. (See color plates.)

P T H / P T H r P / R E c E P T O R INTERACTIONS /

polar residues in the helix comprising the principal binding domain of the ligand and the polar residues E 177, R 179, R TM, E 182, D 185, and R 186 in the receptorderived peptide. Interestingly, this docking procedure brings only M 425, a n d not M 414, into sufficient proximity to permit cross-linking to position 1 in 125I-Bpa1-PTH. Therefore, these observations are in complete agreement with the results obtained through cross-linking studies (Fig. 11) (28). The structural features of the first ECL in the presence of dodecylphosphocholine micelles were revealed from the high-resolution NMR study, followed by distance geometry calculations and molecular dynamic simulations of a peptide, hPTH1-Rc(241-285), comprising the loop and few residues from the ectopic portions of TM2 and TM3 of the receptor (230). This peptide contains L 26], which was found to cross-link to Lys27 in hPTH(1-34) (222). The structure of this receptor fragment includes three or-helices, (241-244), (256-264), and (275-284). The first and the last helices correspond

79

to the ectopic pordons of TM2 and TM3, respectively. The topological orientation of the helices relative to the membrane surface was examined in the presence of 5and 12-doxylstearic acids, nitroxide radical-containing molecules that serve as reporters for the localization of amino acid residues in the membrane. The amino acids corresponding to the ectopic portion of the TMs are more strongly associated with the lipid micelle and may serve as membranal anchors. All of the hydrophobic residues in the partially ordered central helical portion (terminated by the unique helix-breaking sequence p258_p_p_p261) are projecting toward the lipid surface (230). The conformational analysis of the first ECL is very helpful in gaining important insights into the bimolecular ligand-receptor interaction revealed by crosslinking studies. The benzophenone moiety o n Lys27 in 125I-KZT(N~-pBzz)-PTH cross-links to L 261 in hPTH1-Rc (222). The long, amphiphilic C-terminal helix, which i n c l u d e s Lys27, was found to lie on the surface of the

W457

Extraeellu!ar

M414 s~

Intraeellu!ar Receptor

B TM3 ~.

TM2

i~:

N-Terminus .....

~

TM7

TM5 C.Te~in~

N~Te~ni:~s PTH Ligand

FIG. 11 Model for the binding of hPTH(1-34) to hPTH1-Rc. For clarity, only portions of the TM helices, N terminus, and the third extracellular loop are shown in blue (non-cross-linked domains) and green (contact domains hPTH1-Rc(173-189) and hPTH1-Rc(409-437)) (A, side view; B, top view). The amphipathic oL-helix of the extracellular N terminus of the receptor is projecting to the right, lying on the surface of the membrane. The high-resolution, low-energy structure of hPTH(1-34) determined by NMR in a micellar environment is presented in pink. Residues in cross-linking positions 1 and 13 of hPTH(1-34) are denoted in yellow. The C-terminal amphipatic oL-helix of hPTH(1-34) is aligned in antiparallel arrangement with the amphipatic cx-helix of the extracellular N-terminus hPTH1-Rc(173-189), contiguous with TM1 and encompassing the 17-amino acid contact domain (in green), to optimize the hydrophilic interactions. Side chains of residue M414 and M425 within the "contact domain" TM6-third extracellular loop (hPTH1-Rc(S"°9-W"37)) are shown (28). (See color plates.)

80

/

CHAPTER4

micelle with its hydrophobic face projecting into the lipid layer (64). We therefore propose that these two helices, the C-terminal helix in PTH and the central helix in the first ECL in PTH1-Rc, interact in an antiparallel fashion allowing exposed charged residues on both helices to form numerous intermolecular interactions. Integrating these findings into the PTH-PTH1-Rc model results in the enhancement and refinement of the overall bimolecular topology by positioning the C-terminal helix of PTH between the first ECL and the C-terminal helix of the N-ECD of hPTH1-Rc. This topological organization is consistent not only with the individual bimolecular contact sites between positions 1, 13, and 27 in PTH and the respective sites in PTH1-Rc (namely, M 425, a site in the proximity of R 186, and L261), but also accommodates the contact site between position 23 in PTHrP and YZ~-L4° in PTH1-Rc (Fig. 12). R61z and co-workers constructed the PTH1-Rc and PTH2-Rc as described previously and used the membrane-bound conformation of hPTH(1-34) (64) and the contact sites identified for positions 1 and 13 in PTH (26,28,221) to dock hPTH(1-34) to the receptors (229). Using these models, they identify interresidue contacts within the seven-transmembrane helical bundle (Fig. 13) and suggest explanations for ligand specificity (46,243), site-directed mutagenesis (23,173,177,179,180), constitutively activated receptors (182,244), cross-linking outcomes (28), substitutions

N-terminus 261

C-terminus FIG. 12 Schematic representation of the binding of PTH to its G protein-coupled receptor, PTH1-Rc. The locations of the contact points in PTH1-Rc identified by photoaffinity crosslinking are indicated (SerlmM 42s, Lys13--R 186,Trp23--T33/Q37, Lys2L--L261). The structural features of the PTH and fragments of PTH1-Rc are indicated (230).

C281-C351

. , a t e , TM2

TM1 TM4

TM7 TM6 FIG. 13 Illustration of some key residue-residue contacts within the seven-transmembrane helix bundle of PTH1-Rc. These contacts provide support that the model contains the correct topology of the seven-TM helices. Reprinted with permission from Ref. 229. Copyright 1999 American Chemical Society.

within the ligand (39,245), and signal transduction. These authors also suggest some mutations, which may reverse the specificity of the PTH1- and PTH2-Rcs for their respective ligands, P T H / P T H r P and PTH (229). An interesting study reported by Shimizu and coworkers has incorporated data generated by the ligandand receptor-centered approaches in a very innovative way to design a constitutive active ligand-tethered hPTH1-Rc (Fig. 14) (246). Four concepts were established: (1) A peptide as small as PTH(1-14) can stimulate weak cAMP formation with both wild-type and N-ECD-truncated rPTH1-Rc, rANt (18). (2) Residues 1-9 in PTH (1-14) are critical for interacting with the rANt (18). (3) Position 13 in PTH photocross-links in the proximity of R ]86 in PTH1-Rc (26,27). (4) The hydrophobic residues F TM and L 187 in PTH1-Rc are functionally important for the interaction with the 3-14 portion of PTH(1-34) (172). In this ligand-tethered hPTH1-Rc, the N-ECD was truncated from E 182, juxtaposed to the TM1 (AN-ECD-hPTH1-Rc), and was extended by a Gly4 spacer (Ga-AN-ECD-hPTH1-Rc) linked to PTH(1-9) (246). Transient expression of this construct in COS-7 cells resulted in 10-fold higher basal cAMP levels compared to the control, wild-type

PTH/PTHrP/REcEPTOR INTERACTIONS /

~NH2

[A1-V-S-E-I-Q-L-M-H9 ]

PTH[ 1-9]

[AI-V-S-E-I-Q-L-M-H-NI° I

PTH[ 1-10]

[AI-V-S-E-I-Q-L-M-H-N-L 11]

PTH[1-11]

[A'-V-S-E-I-Q-L-M-H-N-R 1']

Arg11PTH[1-11]

y

H2N~77-/'~G_G_G_G

HOOC ~

81

H2N

HOOC hPTH1-Rc

HOOC -

Tether-G4-AN-ECD-

AN-ECD-hPTH1-Rc

hPTH1-Rc FIG. 14 Schematics include the wild-type hPTH1-Rc, the AN-ECD-hPTH1-Rc, and the Tether-G4-AN-ECDhPTH1-Rc. Also listed are the different N-terminal sequences derived from PTH, which are tethered to E 182 (solid diamond) via a tetraglycine (G4) spacer. All the receptor constructs retain the 23-amino acid native hPTH1-Rc signal sequence. Therefore, the putative N-terminal residue in all the receptors is y23 generated on signal peptidase cleavage. Modified from Shimizu et aL (246).

hPTH1-Rc in the same expression system. T e t h e r i n g the extended and more potent [Arg11]PTH(1-11) resulted in 50-fold higher basal c-AMP levels than those seen with the wild-type hPTH1-Rc. Interestingly, similar to the PTH(1-14) (18) in which Val 2, Ile 5, and Met 8 were the most critical residues for activation they were also the most critical ones for the constitutive activity of the [Arg11]PTH(1-11)-G4-AN-ECD-hPTH1-Rc (246). The elegance of this study is in devising a unique way to specifically "immobilize" the principal activation domain of the ligand in the proximity to the contact sites critical for receptor activation. The correspondence between the substitutions in the PTH (1-11) that increase the efficacy of the free and the tethered peptide supports the notion that both exercise the same contact points responsible for receptor activation. The high effective molarity of the tethered ligand minimizes the role of binding affinity as it is known for the free ligand, thus allowing the identification of residues within the tethered ligand essential for induction of activity. However, the accessibility to the tethered ligand-receptor system is limited to the recombinant technology and therefore to coded amino acids, and the stringent requirements for efficient expression may turn out to be major obstacles in practicing and extending this approach in the future. It

remains to be shown whether the tethered ligand-receptor system may be a source for identifying structural constraints that can contribute to the refinem e n t of the experimentally based ligand-receptor model and to rational drug design. The elimination of most of the entropic c o m p o n e n t from the ligand-receptor interaction may generate contact interactions and produce activation mechanisms that differ from those involved in the interaction with a diffusable ligand. The quality of any model, namely, its capacity to represent ligand-receptor interactions realistically and predict the nature of the interface, is based primarily on the data and procedures used in construction of the model. A model can become highly speculative and thus only remotely relevant to biology if overloaded with data derived from indirect and circumstantial conclusions. It is important to avoid overinterpretation of model and r e m e m b e r the assumptions and approximations used in its construction. Last, any extrapolation derived from the model must be tested in order to validate its predictive potential. Therefore, the evaluation of any models for complexes of PTH and PTHrP with PTH1-Rc and PTH with PTH2-Rc should follow the above-mentioned principles.

82

/

CHAPTER4

FUTURE DIRECTIONS T h e most powerful insights into the nature of h o r m o n e - r e c e p t o r interactions are emerging from direct PAS studies of h o r m o n e - r e c e p t o r photoconjugates. Future experimentally based models of the bimolecular interface will be m o r e refined and better validated. As additional contact sites are demonstrated, other constraints are generated for the model, which further refines the entire model. W h e n sufficiently advanced, this experimentally based model of the PTH-PTH1-Rc interface will b e c o m e a powerful tool for u n d e r s t a n d i n g structure-based mechanisms responsible for differences in biologic activities of h o r m o n e agonists, signaling-selective agonists, antagonists, partial agonists, a n d inverse agonists. It will also provide the means to u n d e r s t a n d aberrant mechanisms underlying pathologic mutations of PTH1-Rc leading to the clinical disorders of Jansen's metaphyseal chondrodysplasia and Blomstrand's osteochondrodysplasia. Finally, the detailed and validated model of the h o r m o n e - r e c e p t o r complex will serve as a molecular template for design of therapeutically advantageous analogs of PTH and PTHrP. O n e area of research that has grown steadily in interest over the past decade is the potential utility of PTH- or PTHrP-derived agonists for the t r e a t m e n t of osteoporosis. It is very well established that low-dose intermittent administration of several forms of P T H stimulates b o n e formation, leading to an overall anabolic effect on b o n e (247,248). Further observations have b e e n m a d e in vivo in animals and in h u m a n studies (249-255). This beneficial effect on b o n e occurs despite the well-documented action of P T H in stimulating b o n e resorption via increased osteoclast n u m b e r and activity. Nevertheless, instigating osteoblasfic bone formation without concomitant activation of osteoclasts a n d resultant b o n e resorption remains an ultimate goal for t r e a t m e n t of osteoporosis. To this end, future focus on the developm e n t of signaling-selective PTH or PTHrP analogs is one of the m o r e promising directions for analog design. Unfortunately, the chronic n a t u r e of osteoporosis implies that long-term or even life-long t r e a t m e n t is required. This poses serious c o m p l i a n c e issues due to the fact that the administration of a peptide-based d r u g such as P T H is generally limited to p a r e n t e r a l routes. In the short a n d i n t e r m e d i a t e term, we anticipate the d e v e l o p m e n t of i m p r o v e d d r u g delivery systems that will greatly e n h a n c e the t h e r a p e u t i c potential of PTHa n d PTHrP-derived agonists a n d antagonists. However, in the long term, d e v e l o p m e n t of small n o n p e p t i d e PTH-mimetic drugs is of major interest. In the past few years a growing n u m b e r of small n o n p e p t i d e peptide-mimetic agonists for GPCRs have b e e n r e p o r t e d (256-262). This raises our expectations that either

through rational d r u g design (based on the P T H - P T H 1 - R c e x p e r i m e n t a l model), or high-throughp u t screening of collections of synthetic c o m p o u n d s , natural products, a n d culture broths, new lead molecules will be discovered. These "leads" can then be optimized chemically into n o n p e p t i d e PTH-rnimetic anabolic agents. To this end, studying the s t r u c t u r e activity relations a n d the degrees of structural tolerance of P T H ( 1 - 1 4 ) is a p r o m i s i n g initiative (18-20). Given the probability of substantial progress in developing a m o d e l for the h o r m o n e - r e c e p t o r complex, in analog design, in elucidating h o r m o n a l m e c h a n i s m s of action, a n d in peptide delivery systems, P T H or PTHrP agonists are likely to find clinical utility in t r e a t m e n t of disorders of calcium a n d b o n e metabolism.

REFERENCES

1. Chorev M, Rosenblatt M. Structure-function analysis of parathyroid hormone and parathyroid hormone-related protein. In: Bilezikian JP, Levine MA, Marcus R, eds. The parathyroids. New York: Raven, 1994:139-156. 2. Chorev M, Rosenblatt M. Parathyroid hormone: Structurefunction relations and analog design. In: BilezikianJP, Raisz LG, Rodan GA, eds. Principles of bone biology. San Diego, Academic Press, 1996:305-323. 3. PottsJT, Jr, Gardella TJ, Jfippner H, Kronenberg HM. Structure based design of parathyroid hormone analogs. J Endocrinol 1997;S15-$21. 4. Whitfield JF, Morley E Small bone-building fragments of parathyroid hormone: New therapeutic agents for osteoporosis. Trends Pharmacol Sci 1995;16:382-386. 5. Dempster DW, Cosman E Parisien M, Shen V, Lindsay R. Anabolic actions of parathyroid hormone on bone. Endocr Rev 1993;14:690-709. 6. Rosenblatt M. Peptide hormone antagonists that are effective in vivo: Lessons from parathyroid hormone. N Engl J Med 1986;315:1004-1013. 7. Rosenblatt M, Callahan EN, Mahaffey JE, Pont A, Potts JT, Jr. Parathyroid hormone inhibitors: Design, synthesis, and biologic evaluation of hormone analogues. J Biol Chem 1977;252:5847-5851. 8. Rosenblatt M, Chorev M, Nutt RF, Caulfield ME Horiuchi N, Clemens TL, Goldman ME, McKee RL, Caporale LH, FisherJE, LevyJJ, Reagan JE, Gay T, DeHaven E New directions for the design of parathyroid hormone antagonists. In: Massry SG, Fujita T, eds. New actions of parathyroid hormone. New York: Plenum, 1993:61-67. 9. Nussbaum SR, Rosenblatt M, Potts JT, Jr. Parathyroid hormone renal receptor interactions: Demonstration of two receptorbinding domains. J Biol Chem 1980;255:10183-10187. 10. Caulfield ME McKee RL, Goldman ME, Duong LT, Fisher JE, Gay CT, DeHaven PA, LevyJJ, Roubini E, Nutt RF, Chorev M, Rosenblatt M. The bovine renal parathyroid hormone (PTH) receptor has equal affinity for two different amino acid sequences: The receptor binding domains of PTH and PTH-related protein are located within the 14-34 region. Endocrinology 1990;127:83-87. 11. Gardella TJ, Wilson AK, Keutmann HT, Oberstein R, PottsJT,Jr. Kronenberg HM, Nussbaum SR. Analysis of parathyroid hor-

P T H / P T H r P / R E c F ~ V T O R INTERACTIONS

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

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233. Mierke DE Royo M, Pellegrini M, Sun H, Chorev M. Peptide mimetic of the third cytoplasmic loop of the PTH/PTHrP receptor. J Am Chem Soc 1996;118:8998-9004. 234. Pellegrini M, Royo M, Chorev M, Mierke DE Conformational characterization of a peptide mimetic of the third cytoplasmic loop of the G-protein coupled parathyroid hormone/parathyroid hormone related protein receptor. Biopolymers 1997;40: 653-666. 235. Schertler GF, Villa C, Henderson R. Projection structure of rhodopsin. Nature 1993;362: 770-772. 236. Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E, Downing KH. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 1990;213:899-929. 237. Schertler GF, Hargrave PA. Projection structure of frog rhodopsin in two crystal forms. Proc Natl Acad Sci USA 1995;92:11578-11582. 238. Grigorieff N, Ceska T, Downing K, Baldwin J, Henderson R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J Mol Bio11996;259:393-421. 239. Pebay Peyroula E, Rummel G, Rosenbusch JP, Landau EM. X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. Science 1997;277: 1676-1681. 240. Baldwin JM. The probable arrangement of the helices in G protein-coupled receptors. E M B O J 1993;12:1693-1703. 241. Mierke DE Pellegrini M. Parathyroid hormone and parathyroid hormone-related protein: Model systems for the development of an osteoporosis therapy. Curr Pharm Design 1999;5: 21-36. 242. Pellegrini M, Bisello A, Rosenblatt M, Chorev M, Mierke D. Binding domain of human parathyroid hormone receptor: From conformation to function. Biochemistry 1998;37:12737-12743. 243. Behar V, Nakamoto C, Greenberg Z, Bisello A, Suva LJ, Rosenblatt M, Chorev M. Histidine at position 5 is the specificity "switch" between two parathyroid hormone receptor subtypes. Endocrinology 1996;137:4217-4224. 244. Schipani E, Jensen GS, Pincus J, Nissenson RA, Gardella TJ, Jfippner H. Constitutive activation of the adenosine 3', 5'monophosphate signaling pathway by parathyroid hormone (PTH)/PTH-related peptide receptors mutated at the two loci for Jansen's metaphyseal chondrodysplasia. Mol Endocrinol 1997;11:851-858. 245. Rosenblatt M, Goltzman D, Keutmann HT, Tregear GW, Potts JT, Jr. Chemical and biological properties of synthetic, sulfurfree analogues of parathyroid hormone. J Biol Chem 1976;251:159-164. 246. Shimizu M, Carter PH, Gardella TJ. Autoactivation of type-1 parathyroid hormone receptors containing a tethered ligand. J Biol Chem 2000;275:19456-19460. 247. Howard GA, Bottemiller BL, Turner RT, Rader JI, Baylink DJ. Parathyroid hormone stimulates bone formation and resorption in organ culture: Evidence for a coupling mechanism. Proc Natl Acad Sci USA 1981;78:3204-3208. 248. Tam CS, Heersche JNM, Murray TM, Parsons JA Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: Differential effects of intermittent and continual administration. Endocrinology 1982;110:506-512. 249. Reeve J, Davies UM, Hesp R, Katz D. Treatment of osteoporosis with human parathyroid peptide and observations on effect of sodium fluoride. Br MedJ 1990;301:31 4-318. 250. Tada K, Yamamuro T, Okumura H, Kasai R, Takahashi H. Restoration of axial and appendicular bone volumes by hPTH(1-34) in parathyroidectomized and osteopenic rats. Bone 1990;11:163-169.

251. Hock JM, Gera I, Fonseca J, Raisz LG. Human parathyroid hormone-(1-34) increases bone mass in ovariectomized and orchidectomized rats. Endocrinology 1988;122:2899-2904. 252. Hodsman AB, Fraher LJ. Biochemical responses to sequential human parathyroid hormone (1-38) and calcitonin in osteoporotic patients. Bone Miner 1990;9:137-152. 253. Tsai K-S, Ebeling PR, Riggs BL. Bone responsiveness to parathyroid hormone in normal and osteoporotic postmenopausal women. J Clin Endocrinol Metab 1989;69:1924-1027. 254. Slovik DM, Rosenthal DI, Doppelt SH, Potts JT, Jr, Daly MA, CampbellJA, Neer RM. Restoration of spinal bone in osteoporotic men by treatment with human parathyroid hormone (1-34) and 1,25-dihydroxyvitamin D. J Bone Miner Res 1986;1:377-381. 255. Wronski TJ, Yen C-F, Qi H, Dann LM. Parathyroid hormone is more effective than estrogen or bisphosphonates for restoration of lost bone mass in ovariectomized rats. Endocrinology 1993;132:823-831. 256. Kivlighn SD, Huckle WR, Zingaro GJ, Rivero RA, Lotti VJ, Chang RSL, Schorn TW, Kevin N, Johnson RG, Greenlee WJ. Discovery of L-162, 313: A nonpeptide that mimics the biological actions of angiotensin II. A m J Physio11995;268:R820-R823. 257. Aquino CJ, Armour DR, Berman JM, Birkemo LS, Carr RAE, Croom DK, Dezube M, Dougherty RW, Ervin GN, Grizzle MK, Head JE, Hirst GC, James MK, Johnson ME Miller LJ, Queen KL, Rimele TJ, Smith DN, Sugg EE. Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity. 1. Optimization of the agonist "trigger."J Med Chem 1996;39:562-569. 258. Yang L, Berk SC, Rohrer SP, Mosley RT, Guo L, Underwood DJ, Arison BH, Birzin ET, Hayes ED, Mitra SW, Parmar RM, Cheng K, Wu TJ, Butler BS, Foor E Pasternak A, Pan Y, Silva M, Freidinger RM, Smith RG, Chapman K, Schaeffer JM, Patchett AA. Synthesis and biological activities of potent peptidomimetics selective for somatostatin receptor subtype 2. Proc Natl Acad Sci USA 1998;95:10836-10841. 259. Rohrer SP, Birzin ET, Mosley RT, Berk SC, Hutchins SM, Shen DM, Xiong Y, Hayes EC, Parmar RM, Foor E Mitra SW, Degrado sJ, Shu M, Klopp JM, Cai sJ, Blake A, Chan WWS, Pasternak A, Yang L, Patchett AA, Smith RG, Chapman KT, Schaeffer JM. Rapid indentification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry. Science 1998;282:737-740. 260. Tian SS, Lamb P, King AG, Miller SG, Kessler L, Luengo JI, Averill L, Johnson RK, Gleason JG, Pelus LM, Dillon SB, Rosen J. A small, nonpeptidyl mimic of granulocyte-colony-stimulating factor. Science 1998;281:257-259. 261. Hansen TK, Ankersen M, Hansen BS, Raun K, Nielsen KK, Lau J, Peschke B, Lundt BE Thogersen H, Johansen NL, Madsen K, Andersen PH. Novel orally active growth hormone secretagogues. J Med Chem 1998;41:3705-3714. 262. Zhang B, Salituro G, Szalkowski D, Li Z, Zhang Y, Royo I, Vilella D, Diez MT, Pelaez F, Ruby C, Kendall RL, Mao X, Griffin P, CalaycayJ, Zierath JR, HeckJV, Smith RG, Moller DE. Discovery of a small molecule insulin mimetic with antidiabetic activity in mice. Science 1999;284:974-977. 263. Adams A, Bisello A, Chorev M, Rosenblatt M, Suva L. Arginine 186 in the extracellular N-terminal region of the human parathyroid hormone 1 receptor is essential for contact with position 13 of the hormone. Mol Endocrinol 1998; 12:1673-1683. 264. Moseley JM, Kubota M, Diefenbach-Jagger H, Wettenhall REH, Kemp BE, Suva LJ, Rodda CP, Ebeling PR, Hudson PJ, Zajac JD, Martin TJ. Parathyroid hormone-related protein purified from a human lung cancer cell line. Proc Natl Acad Sci USA 1987 ;84:5048-5052.

P T H / P T H r P / R E C E P T O R INTERACTIONS 265. Suva LJ, Winslow GA, Wettenhall RE, Hammonds RG, Moseley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI. A parathyroid hormone-related protein implicated in malignant hypercalcemia: Cloning and expression. Science 1987;237:893-896.

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266. Horiuchi N, Caulfield ME Fisher JE, Goldman ME, McKee RL, Reagan JE, LevyJJ, Nutt RF, Rodan SB, Schofield TL, Clemens TL, Rosenblatt M. Similarity of synthetic peptide from human tumor to parathyroid hormone in vivo and in vitro. Science 1987;238:1566-1568.

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Receptors for Parathyroid Hormone and Parathyroid Hormone-Related Protein Signaling and Regulation ROBERT A. NISSENSON Endocrine Unit, San Francisco VA Medical Centeg, and Departments of Medicine and Physiology, University of California, San Francisco, California 94121

INTRODUCTION The endocrine effects of parathyroid hormone (PTH) and the paracrine actions of PTH-related protein (PTHrP) are initiated by the same intrinsic plasma membrane receptor (1,2). Several years ago, the sequence of the PTH/PTHrP receptor cDNA was obtained (3), and it was evident that the protein had a predicted topology similar to that of other known G protein-coupled receptors (GPCRs). In particular, the receptor is predicted to contain seven membrane-spanning helices, with a long amino-terminal extracellular domain, three extracellular loops, three intracellular loops, and a large carboxyterminal cytoplasmic tail (Fig. 1). Despite containing seven membrane-spanning segments, the PTH/PTHrP receptor does not share a number of the specific sequence motifs present in the largest subfamily of GPCRs (the class I family, which includes receptors for a diverse group of ligands ranging from photons to polypeptide hormones). Instead, the PTH/PTHrP receptor is a member of a second GPCR subfamily (class II) that includes receptors for calcitonin, glucagon, and a number of other polypeptide ligands (4). The evolutionary relationship between members of the class II GPCR subfamily is evident from the similarity of the intron/exon boundaries of their cognate genes, as well as the presence of a variety of conserved protein sequence motifs, particularly in their transmembrane domains (4,5). Moreover, these receptors generally utilize G s (coupling to adenylyl cyclase) and Gq (coupling to phospholipase C) for generating intracellular signals. Members of the class II GPCR subfamily presumably share a common The Parathyroids, Second Edition

NHz

COOH

FIG. 1 Structural representation of the three-dimensional topology of the PTH/PTHrP receptor, based on the model developed for other GPCRs. The large, glycosylated aminoterminal domain of the receptor is on the extracellular side of the membrane, and the large carboxyl-terminal tail is cytoplasmic. The seven transmembrane helices interact with one another and line a central polar cavity in the receptor.

basic mechanism of G protein activation, but have evolved determinants of specificity that permit binding and activation by only the appropriate peptide ligands. 93

Copyright © 2001 John P. Bilezikian, Robert Marcus, and Michael A. Levine.

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The molecular basis of ligand specificity and interaction with the P T H / P T H r P receptor is discussed elsewhere in this volume. What follows is a summary of the current understanding of molecular events that underlie activation of signal transduction following the binding of PTH or PTHrP to the P T H / P T H r P receptor; the mechanisms by which the P T H / P T H r P receptor is regulated following receptor activation, and diseases that are associated with abnormalities in P T H / P T H r P receptor expression and function.

SIGNAL TRANSDUCTION

PTH/PTHrP RECEPTOR

BY

THE

The G Protein-Coupled Receptor Superfamily Even before determination of the P T H / P T H r P receptor sequence, functional studies suggested that the P T H / P T H r P receptor was a member of the GPCR superfamily. For example, GTP and its analogs were found to regulate the affinity of PTH for the receptor, and to potentiate PTH-induced stimulation of adenylyl cyclase (6--11). This prediction was confirmed with the cloning of the cDNA encoding the P T H / P T H r P receptor (3), which revealed a predicted protein sequence containing seven putative membrane-spanning domains, a topology characteristic of members of the G protein-coupled receptor superfamily (12,13). There are well over 1000 known GPCRs, all of which appear to mediate agonistdependent G protein activation through a c o m m o n basic molecular mechanism (Fig. 2) (14,15). In brief, in GDP

Inactive

G protein

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GDP-(~y

GTP

GTPa+ ~y ~nGS ~L'~ GDP-(~

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FIG. 2 G protein activation by a GPCR. The rate-limiting step in G protein activation-dissociation of GDP from the G protein o~subunit is catalyzed by the agonist-activated receptor (HR*). This permits GTP to bind to the o~ subunit, resulting in subunit dissociation. Both the GTP-bound oL subunit and the 137 subunit dimer are capable of activating effectors such as adenylyl cyclase (AC) and phospholipase C (PLC), sometimes synergistically. Termination of signaling requires the hydrolysis of GTP to GDP by the intrinsic GTPase activity of the oL subunit. For some G proteins, the rate of GTP hydrolysis is enhanced by the action of a regulator of G protein signaling (RGS protein). The GDP-oL subunit complex then binds the 137 subunit dimer, regenerating the inactive heterotrimeric G protein.

the resting, inactive state, G proteins are plasma membrane-associated heterotrimers consisting of or, [3, and ~/subunits. The heterotrimeric form is stabilized by the binding of GDP to the guanylyl nucleotide binding site on the e~ subunit. Agonist binding to its GPCR induces the receptor to interact with the heterotrimeric G protein, producing a conformational change that results in the release of GDP from the e~ subunit. This allows GTP (which is more abundant than GDP in the cell) to bind to the oL subunit. Binding of GTP in turn induces a structural change in the G protein that results in G protein activation, i.e., dissociation of the e~ subunit-GTP complex from the [3y complex (the latter are tightly associated under all physiologic conditions). The free e~ subunit-GTP complex is able to regulate activity of specific effector systems (e.g., adenylyl cyclase, phospholipase C) that can produce a variety of second messengers. It has become clear that the [3y complex can also participate in regulation of effector activity, often (but not always) synergistically with the GTP-bound 0L subunit. Termination of signaling is effected by the intrinsic GTPase activity of the oLsubunit, resulting in the generation of a GDP--a subunit complex that rapidly reassociates with [3~/. In some cases, GTPase activity can be accelerated by the activity of a "regulator of G protein signaling" (RGS) protein (16). The inactive heterotrimeric G protein is now regenerated and poised to respond to another round of receptor activation. There are over 20 genes encoding G protein oL subunits, as well as multiple genes for [3 and ~/ subunits (17,18). Although there is evidence that the [3~/ complex participates in the specificity of G proteins for receptors and effectors, it is the oLsubunit that plays the p r e d o m i n a n t role in determining specificity. In the case of the P T H / P T H r P receptor, the major G proteins that can be activated are G s and Gq, which contain the e~ subunits % and C~q, respectively. Activation of G s leads to increased adenylyl cyclase activity, resulting in increased cellular levels of cyclic AMP and activation of Protein Kinase A (PKA). Activation of Gq results in stimulation of phospholipase C-[3 (PLC-[3) resulting in mobilization of intracellular calcium and activation of PKC. The relative activation of these pathways in a given cellular context presumably depends on the relative abundance of receptors as well as G s a n d Gq, and the relative affinity of the agonist-occupied receptor for these G proteins. In the case of the P T H / P T H r P receptor, signaling via G s appears to be preferred, probably due to greater affinity of the receptor for G s v e r s u s Gq. Thus, signaling by the P T H / P T H r P receptor through the cyclic AMP pathway can be detected at levels of PTH that occupy only a minute fraction of cellular receptors, which in part accounts for the ability to use urinary excretion of n e p h r o g e n o u s cyclic AMP as an in vivo bioassay for circulating levels of PTH (19) that are

RECEPTOR SIGNALING AND REGULATION

100--

---

Adenylyi cyclase

80--

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60--

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40--

,,.

m

20--

0"-0

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11

10

9

8

7

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FIG. 3 Relative potency of PTH in activating two signal transduction pathways in UMR-106 osteoblastic cells. In this system, PTH is at least an order of magnitude more potent in stimulating the adenylyl cyclase pathway versus the phospholipase C pathway (measured indirectly by the increase in cytosolic calcium). This result probably reflects a greater affinity of the activated PTH/PTHrP receptor for Gs as compared to Gq. Redrawn from Ref. 20; M Babich, H Choi, RM Johnson, KL King, GE Alford, RA Nissenson; Thrombin and parathyroid hormone mobilize intracellular calcium in rat osteosarcoma cells by distinct pathways. Endocrinology, Vol. 129, pp. 1463-1470, 1991. © The Endocrine Society.

in the picomolar range. Preference of the P T H / P T H r P receptor for the cyclic AMP signaling pathway is also suggested by studies on PTH target cells in vitro, where activation of adenylyl cyclase occurs at m u c h lower concentrations of added PTH than does activation of phospholipase C (Fig. 3) (20).

Transmembrane Signaling by the P T H / P T H r P Receptor To perform their physiological functions properly, the activation of GPCRs must be tightly regulated by the

BASAL III

VI

His

Inactive

+PTH III

95

binding of receptor agonists. In the absence of agonists, the p r e d o m i n a n t receptor conformation is one that does not interact productively with the G protein on the cytoplasmic surface of the plasma membrane. Binding of an agonist to extracellular a n d / o r transmembrane domains of the receptor stabilizes a receptor conformation that favors interaction with and activation of the target G protein. One of the most intriguing questions in the GPCR field is how binding of an agonist to sites in the extracellular and transmembrane regions of a GPCR alters the structure of the cytoplasmic domain in a way that promotes G protein activation. In the case of well-studied GPCRs such as rhodopsin, the inactive and active receptor conformations can be distinguished by differences in the interactions between residues in the transmembrane helical domains (21,22). Alterations in the relative orientation of the transmembrane domains that are induced by agonist binding result in changes in the three-dimensional structure of the intracellular loops. This exposes key amino acids that participate in the activation of the cognate G protein(s) on the cytoplasmic face of the plasma membrane. In the case of the visual receptor rhodopsin, there is evidence that one of the critical conformational changes that accompanies receptor activation is the relative m o v e m e n t of the cytoplasmic ends of transmembrane helices 3 and 6 away from one a n o t h e r (23,24). In addition, specific interactions between amino acids in t r a n s m e m b r a n e domains 2 and 7 are i m p o r t a n t in stabilizing the active conformation of the receptor (22). Available evidence suggests that the f u n d a m e n t a l conformational shift that occurs on agonist binding to GPCRs m a y be conserved across the receptor superfamily. To examine this issue for the P T H / P T H r P receptor, a study was carried out to d e t e r m i n e whether preventing the relative m o v e m e n t of t r a n s m e m b r a n e domains 3 and 6 would inhibit PTH-induced receptor activation (Fig. 4). This study took advantage of the ability of zinc ions to complex with histidine residues in proteins. The P T H / P T H r P receptor contains a

.... Cai ++ 0

/

+PTH VI

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Active

III

VI

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\z/

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FIG. 4 Activation of the PTH/PTHrP receptor may require the relative movement of the cytoplasmic ends of transmembrane domains 3 and 6. A mutated receptor in which histidine residues are present in the cytoplasmic end of these two transmembrane domains is fully functional in the present of PTH. However, the addition of zinc to coordinate the histidines, thereby constraining the relative movement of transmembrane helices 3 and 6, inhibits receptor activation by PTH. See the text for further details.

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histidine at the cytoplasmic end of transmembrane domain 3, and a second histidine residue was inserted at the cytoplasmic end of transmembrane domain 6 by mutagenesis. The modified P T H / P T H r P receptor was fully functional with respect to supporting PTHsimulated adenylyl cyclase, but activation of the G protein G s by PTH was blocked by the addition of zinc ions. Inhibition of receptor activation by zinc required the presence of both histidine residues, indicating that the mechanism of zinc inhibition involved coordination of the histidines by zinc ions. This coordination apparently prevented the movement away from one another of the cytoplasmic ends of transmembrane domains 3 and 6 of the P T H / P T H r P receptor. Mutagenesis studies have also demonstrated a probable interaction between transmembrane domains 2 and 7 in the activated P T H / P T H r P receptor (25), similar to what has been reported for rhodopsin. Much of what we know about the structural basis for activation of the P T H / P T H r P receptor has come from studies of receptor mutations. A variety of receptor mutations have been shown to result in diminished agonist-dependent receptor activation with retention of ligand binding. This might be due to effects of mutations on the agonist-induced conformational switch in the receptor that is required for activation. Indeed, replacement of any of three amino acids (serine, arginine, serine) along a polar face of transmembrane domain 2 of the P T H / P T H r P receptor result in diminished responsiveness to PTH (26) (Fig. 5). These mutations may disrupt the interaction of transmembrane domains 2 and 7, and indeed mutation of a glutamine residue in transmembrane domain 7 likewise diminished signaling through both the adenylyl cyclase and phospholipase C pathways (25). Mutation of specific cytoplasmic sequences in the receptor can also disrupt PTH-induced signaling by the P T H / P T H r P receptor, presumably by directly interfering with receptor-G protein interactions. The critical amino acids for agonist-

AC PLC

COOH PLC

stimulated activation of G, and Gq appear to lie in the second and third cytoplasmic loops of the receptor. A variety of mutations in the second cytoplasmic loop produce a reduction in phospholipase C activation, without major loss of adenylyl cyclase activation, indicating that this region is particularly crucial for efficient receptor coupling to Gq (27). A lysine residue in the third cytoplasmic domain (near the cytoplasmic end of transmembrane helix 5) was found to be essential for signaling both to G s and Gq, whereas mutations of nearby amino acids resulted in selective reduction in the signaling to either G s or Gq (28). Studies of a synthetic peptide mimetic of the third cytoplasmic loop indicate that these critical amino acids are within a domain capable of forming an oL-helix in a nonpolar environment (29). This is reminiscent of other GPCRs, whereby positively charged helical domains in the cytoplasmic loops are essential for G protein activation. Taken together, these results demonstrate that multiple sites in the cytoplasmic domain of the P T H / P T H r P receptor are involved in the activation of G proteins. There appear to be some sites that are important generically for G protein activation, presumably contacting structural features that are shared by G s and Gq. Other sites in the receptor are involved in the selective activation of either of these G proteins. It is clear that more information is needed concerning the threedimensional structure of the cytoplasmic domain of the P T H / P T H r P receptor in order to establish the molecular basis for the initiation of signal transduction. The precise role of the cytoplasmic tail of the P T H / P T H r P receptor in signaling is not entirely clear. In one study, truncation of the cytoplasmic tail of the rat P T H / P T H r P receptor was found to enhance PTH-stimulated adenylyl cyclase, but not phospholipase C activity (30). This finding is consistent with previous reports that the P T H / P T H r P receptor is able to couple to a third G protein (Gi) that inhibits activation of adenylyl cyclase (31), and that this may involve deter-

FIG. 5 Location in the PTH/PTHrP receptor of specific amino acids that are essential for agonist-stimulated signal transduction. Mutations shown to inhibit either adenylyl cyclase (AC) activation or phospholipase C (PLC) activation, or activation of both pathways, are specified. Note that these mutations are found in transmembrane helices 2 and 7, as well as in the second and third cytoplasmic loops. As discussed in the text, these receptor domains are important for G protein activation by many members of the GPCR superfamily.

RECEPTOR SIGNALING AND REGULATION

minants in the receptor's cytoplasmic tail (32). It is not yet clear whether the P T H / P T H r P receptor is able to activate G i in PTH-responsive bone and kidney cells. In a second study, truncation of the cytoplasmic tail of the opossum P T H / P T H r P receptor had no effect on the adenylyl cyclase response to PTH (33), indicating that there may be species-specific differences in P T H / P T H r P receptor-G protein coupling. The cytoplasmic tail of the P T H / P T H r P receptor also contains determinants of cell surface targeting and expression of the receptor (33). It is possible that some of the observed effects of receptor truncation on signaling in response to PTH are due to altered receptor targeting a n d / o r expression rather than to altered signal transduction.

REGULATION OF THE PTH/PTHrP RECEPTOR As with other GPCRs, signaling by the P T H / P T H r P receptor is tightly regulated by both homologous and heterologous mechanisms. Homologous regulation occurs in response to agonist binding whereas heterologous regulation occurs in response to factors acting though different pathways. Regulation can be manifest at multiple levels, including suppression of the ability of the agonist-occupied receptor to promote activation of cognate G proteins (desensitization) and physical removal of the receptor from the cell surface into an intracellular compartment (internalization/sequestration). Long-term regulation of receptor signaling is accomplished by agonist-induced changes in steadystate levels of expression of receptors, due to increased receptor catabolism following receptor internalization (down-regulation) and to changes in de n o v o receptor synthesis. Homologous regulation commonly involves all of these mechanisms, whereas heterologous regulation most often occurs through changes in steady-state levels of receptor expression.

PTH Receptor Phosphorylation and Desensitization Homologous regulation of P T H / P T H r P receptor signaling has been extensively d o c u m e n t e d . Treatment of cultured bone and kidney cells with PTH generally dampens the adenylyl cyclase and phospholipase C responses to a second addition of the hormone (34-43). Generally, desensitization of the PTH response occurs rapidly, within minutes of initial exposure to PTH, suggesting that the P T H / P T H r P receptor has become acutely uncoupled from its cognate G proteins. The mechanisms underlying acute desensitization have been explored in depth for GPCRs

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such as rhodopsin and [3-adrenergic receptors (44-46). A major mechanism underlying acute desensitization of these receptors is phosphorylation of the cytoplasmic domain of the receptor by a GPCR kinase (GRK) (46,47). Activation of rhodopsin or the [3-adrenergic receptor alters the conformation of the cytoplasmic domain in a way that favors the binding of a GRK to the receptor. There are at least six distinct GRK genes, encoding GRK1, specifically a rhodopsin kinase, and GRKs 2, 3, and 5, which are capable of phosphorylating other GPCRs (48). Once bound, the GRK phosphorylates serine a n d / o r threonine residues in the cytoplasmic tail or (less commonly) the third cytoplasmic loop of the receptor. Phosphorylation of the receptor by a GRK promotes the binding of arrestin proteins, which physically uncouple the receptor from its cognate G protein(s) (49) and also facilitate the entry of the receptor into clathrin-coated pits, thereby promoting receptor internalization (50). There is increasing evidence that similar mechanisms apply to the regulation of P T H / P T H r P receptor signaling. The P T H / P T H r P receptor is subject to phosphorylation in response to agonist binding (51,52), and this appears to occur largely if not exclusively on serine residues in the proximal portion of the cytoplasmic tail (52-54). Available evidence suggests that GRKs are largely responsible for agonist-stimulated phosphorylation of the P T H / P T H r P receptor. Thus, GRK2 is expressed in a variety of osteoblastic cell lines (55), and this GRK, and to a lesser extent GRKs 3 and 5, have been shown to phosphorylate the P T H / P T H r P receptor in isolated membranes (56). Moreover, the recombinant cytoplasmic tail of the P T H / P T H r P receptor is a substrate for phosphorylation by GRK2 (53). Overexpression of GRK2 in cells promotes the phosphorylation of the P T H / P T H r P receptor (54) and inhibits P T H / P T H r P receptor signaling (56). Interestingly, the latter effect was also seen with a C-terminally truncated form of the P T H / P T H r P receptor lacking the sites of phosphorylation by GRKs (51). This finding raises the interesting possibility that recruitment of GRKs to the receptor in response to agonist binding might suppress signal transduction by a mechanism distinct from receptor phosphorylation (e.g., steric interference with G protein activation). In further support for a role of GRKs in regulating PTH action, stable expression of a dominant-negative form of GRK2 in SaOS-2 cells was found to suppress PTH-induced desensitization of P T H / P T H r P receptor signaling (57). In the case of the [3-adrenergic receptor, agonistinduced phosphorylation can result from activation of a second messenger-dependent kinase (PKA) as well as from GRKs (58). These kinases phosphorylate different sites in the cytoplasmic domain of the [3-adrenergic

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receptor, but each can promote desensitization of receptor signaling. At least two second messengerd e p e n d e n t kinases are activated in response to P T H / P T H r P signaling, PKA and PKC. Studies with the recombinant cytoplasmic tail of the P T H / P T H r P receptor and purified kinases indicate that this domain is potentially a substrate of both of these kinases, and that the sites of phosphorylation are different from the sites phosphorylated by GRK2 (53). Moreover, exposure of cells expressing P T H / P T H r P receptors to either forskolin (to activate PKA) or phorbol esters (to activate PKC) resulted in increased receptor phosphorylation. However, studies using inhibitors of these kinases have given equivocal results. In h u m a n embyonic kidney (HEK293) cells expressing the opossum P T H / P T H r P receptor, inhibitors of PKA and PKC had little effect on PTH-induced receptor phosphorylation (51), suggesting that GRKs are responsible for receptor phosphorylation in that system. However, staurosporine (at a dose than inhibits both PKA and PKC, but not GRKs) was found to inhibit partially PTH-induced phosphorylation of the rat P T H / P T H r P receptor in COS-7, LLC-PK1, and ROS 17/2.8 cells (52). Thus, the precise role of PKA and PKC in the agonistinduced phosphorylation and desensitization of the P T H / P T H r P receptor remains to be fully defined.

Endocytosis and Down-Regulation of the PTH/PTHrP Receptor Chronic exposure of target cells to high levels of PTH or PTHrP results in a decrease in the number of cellular P T H / P T H r P receptors (down-regulation), and a corresponding reduction in the maximal signaling response to the hormone (42,59-62). This has been demonstrated in a large n u m b e r of studies in vitro, but receptor down-regulation may also have pathophysio-

459EVQ (-)

/

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logic relevance. For instance, vitamin D deficiency can be associated with target cell resistance to PTH (63-65). In animal studies, this resistance can be reversed by parathyroidectomy, suggesting that it is the secondary hyperparathyroidism that is responsible for target cell resistance (66). Infusion of PTH to levels typical of severe secondary hyperparathyoidism produces down-regulation of P T H / P T H r P receptors and a reduction in the adenylyl cyclase response to PTH (59). In chronic renal failure, factors other than hyperparathyroidism may also contribute to reduced target cell expression of P T H / P T H r P receptors (67). It is also possible that down-regulation of P T H / P T H r P receptors is one of the factors that contributes to the decreased anabolic response of the skeleton to high level continuous administration of PTH, as compared to intermittent treatment. For these reasons, there has been considerable interest in better defining the cellular and molecular bases for agonist-induced down-regulation of the P T H / P T H r P receptor. The initial step in down-regulation of P T H / P T H r P receptors appears to be agonist-induced accumulation of the receptor in plasma membrane clathrin-coated pits (68,69). These pits are endocytic organelles that pinch off from the plasma membrane, thus becoming endocytic vesicles. Once internalized, P T H / P T H r P receptors can be recycled to the plasma membrane, or can presumably progress further down the endocytic pathway to the lysosomes for degradation. The molecular mechanisms underlying the agonist-induced internalization of the P T H / P T H r P receptor are not entirely clear. The role of specific structural features of the receptor in the endocytic process has been investigated by mutagenesis (Fig. 6). The results of early studies demonstrated that a truncated P T H / P T H r P receptor lacking all but 16 amino acids in the cytoplasmic tail was capable of signaling but displayed only about 50% of the

FIG. 6 Amino acids in the PTH/PTHrP receptor that are important for regulating receptor endocytosis, identified by targeted mutagenesis. The positive endocytic signals include a tyrosine-based signal in the cytoplasmic tail, a lysine residue in the third cytoplasmic loop (also important for signal transduction), and an asparagine residue in the third transmembrane helix. A negative endocytic signal in the juxtamembrane region of the cytoplasmic tail is also indicated. The amino acids corresponding to endocytic signals are conserved among various species of PTH/PTHrP receptors. The position numbers are for the opossum PTH/ PTHrP receptor. See the text for more details.

RECEPTOR SIGNALING AND REGULATION

normal rate of agonist-stimulated receptor internalization (69). For some other GPCRs, receptor phosphorylation has been shown to promote internalization at least in part by allowing the binding of arrestins proteins and their subsequent interaction with clathrin (50). The truncated PTH/PTHrP receptor lacked the sites of GRK-mediated phosphorylation, and it was therefore logical to hypothesize that this was the basis for the reduced endocytosis. However, a PTH/PTHrP receptor mutated to eliminate selectively the sites of phosphorylation (leaving the bulk of the cytoplasmic tail intact) was not impaired in its ability to be internalized (54). Progressive truncation of the cytoplasmic tail of the P T H / P T H r P receptor revealed that mutation of a stretch of sequence containing the amino acids Tyr-GlyPro-Met resulted in impaired receptor endocytosis (69). This sequence fits the consensus sequence of endocytic motifs that have been demonstrated in the cytoplasmic domain of a large number of plasma membrane proteins (70). It appears that this sequence mediates the interaction of these proteins with the AP-2 protein complex of the clathrin-coated pit (71), and this is likely to be the major endocytic signal in the cytoplasmic tail of the P T H / P T H r P receptor. Interestingly, this same study identified a potential negative endocytic signal (Glu-Val-Gln) at the junction between the seventh transmembrane segment and the cytoplasmic tail. Mutation of this sequence resulted in enhancement of agoinst-dependent receptor internalization. Although the mechanism underlying the actions of negative endocytic signals is unclear, they may prevent internalization by interacting with proteins that are excluded from clathrin-coated pits. Despite these results, there is evidence that the GRK/arrestin system participates in P T H / P T H r P receptor internalization and down-regulation. Expression of a dominant-negative form of GRK2 resulted in diminished PTH-induced down-regulation of the P T H / P T H r P receptor in SaOS-2 cells (57). Whether this effect resulted from blockage of receptor phosphorylation is not clear, particularly in light of the finding (discussed earlier) that GRK2 can exert phosphorylation-independent actions on P T H / P T H r P receptor function. It has been demonstrated that activation of the P T H / P T H r P receptor resulted in translocation of arrestin from the cytoplasm to the cell membrane, with subsequent colocalization of the receptor and arrestin in intracellular vesicles (72). This elegant result suggests that arrestins play a role in facilitating receptor endocytosis, as has been seen with a number of other GPCRs. Whether agonist-induced phosphorylation of the receptor is required for this effect of arrestin remains to be established.

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99

Regulation of PTH/PTHrP Receptor Gene Expression Long-term regulation of PTH/PTHrP receptor levels can occur through changes in the expression of the receptor gene. Exposure to PTH is reported to decrease levels of PTH/PTHrP receptor mRNA in osteoblasts by a mechanism involving the cyclic AMP pathway (73,74). This may be due to direct regulation of transcriptional activity of the PTH/PTHrP receptor gene by transcription factors phosphorylated by PKA, but the details of this pathway have yet to be elucidated. Homologous control of PTH/PTHrP receptor expression appears to be target cell specific in that PTH reportedly does not reduce expression of the PTH/PTHrP receptor gene in the kidneys of rats with secondary hyperparathyroidism (67,75). Heterologous factors are also reported to regulate levels of PTH/PTHrP receptor expression in bone and kidney. The cytokine TGF-[3 up-regulates the expression of the PTH/PTHrP receptor in osteoblastic osteosarcoma cells (76), although the opposite effect is reported in primary cultures of fetal rat osteoblasts (77) and in OK cells (78). Dexamethasone treatment produces an increase in expression of the PTH/PTHrP receptor in osteoblastic cells, but not in kidney cells (79,80), whereas 1,25(OH) 2 vitamin D down-regulates expression of the PTH/PTHrP receptor gene (81). Many of these studies have been carried out in cultured bone and kidney cells in vitro, and further work is needed to establish the physiologic relevance of the changes in PTH/PTHrP receptor gene expression.

GENETIC DISORDERS OF THE PTH/PTHrP RECEPTOR H u m a n genetic diseases are associated with both loss-of-function and gain-of-function mutations in the P T H / P T H r P receptor (Fig. 7) (82). Homozygous loss of expression of functional P T H / P T H r P receptors is responsible for the rare familial disorder Blomstrand lethal chondrodysplasia (82-85). Blomstrand infants have abnormalities reflecting the lack of PTHrPdirected signaling during endochondral bone development. They display short-limbed dwarfism with increased bone density, accelerated skeletal maturation, and reduced numbers of proliferating growth plate chondrocytes. Two mutant P T H / P T H r P receptor alleles have been identified in such patients, both containing point mutations. One is in the coding region, and encodes a leucine residue rather than the normal proline at position 132 in the receptor's N-terminal extracellular domain (84,85). The other mutation produces a splice variant that encodes a

100

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CHAPTER5

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P T H / P T H r P receptor lacking 11 amino acids in the transmembrane helix (83). These mutations probably alter the conformation of the receptor in a way that precludes effective ligand binding. As discussed previously, activation of GPCRs ordinarily requires agonist binding to stabilize the active receptor conformation. However, a large number of mutations have been identified that allow partial stabilization of the active conformation of GPCRs in the absence of ligand. Such ligand-independent ("constitutive") activation of GPCRs is frequently associated with functional dysregulation and overt disease. Mutated P T H / P T H r P receptors with constitutive activity have been identified in patients with Jansen's metaphyseal chondrodysplasia. This is a rare, dominantly inherited disorder that is characterized by severe growth plate abnormalities, including a delay in chondrocyte differentiation, increased bone resorption, hypercalcemia, and hypophosphatemia (86--89). Circulating levels of PTH and PTHrP are normal or low in these individuals. Three mutated alleles of the P T H / P T H r P receptor have been identified in Jansen's chondrodysplasia. These encode receptors with single amino acid transversions toward the cytoplasmic end of transmembrane helix 2, 6, or 7 (90-92). Each of these mutant receptors displays constitutive activity for the adenylyl cyclase pathway. That is, these receptors promote activation of adenylyl cyclase even in the absence of PTH or PTHrE Constitutive signaling in developing cartilage presumably mimics the effect of excessive PTHrP, thus producing the growth plate phenotype, whereas constitutive signaling in bone and kidney reproduces the effect of excess PTH, resulting in the abnormalities in bone turnover and mineral homeostasis. It is of interest that these mutant receptors do not produce constitutive activation of phospholipase C when expressed in cultured cells, although it is possible that this pathway is activated in vivo. The structural basis for the constitutive signaling by these mutant P T H / P T H r P receptors is not established.

FIG. 7 Human PTH/PTHrP receptor mutations identified in patients with Blomstrand (B) and Jansen's (J) chondrodysplasias. Blomstrand mutations result in a loss of receptor function, whereas Jansen's mutations result in receptors that display ligand-independent (constitutive) activity.

As noted previously, the cytoplasmic ends of transmembrane domains 3 and 6 must separate for agonistinduced signaling to occur. The threonine at position 410 in transmembrane domain 6 may be crucial for preventing the separation of these regions in the absence of agonist. Mutation of this threonine to any of several amino acids, including proline, as seen in some patients with Jansen's chondrodysplasia, would allow separation of these domains and thus signal transduction in the absence of agonist binding. As discussed earlier, there is evidence that transmembrane domains 2 and 7 interact during the course of receptor activation. It is possible that the mutations in these regions in Jansen's patients (histidine to arginine at position 223 and isoleucine to arginine at position 458) stabilize this interaction even in the absence of agonist. Most antagonist analogs of PTH and PTHrP are neutral competitive antagonists. That is, they bind to the P T H / P T H r P receptor (and thereby competitively inhibit agonist binding), but they do not stabilize a particular receptor conformation. However, a few analogs have been shown to function as inverse agonists with respect to constitutively active P T H / P T H r P receptors (93). The binding of these analogs stabilizes the mutated receptor in an inactive conformation and thereby suppress its constitutive activity. It is conceivable that such inverse agonists will prove to be useful for treating individuals with Jansen's metaphyseal chondrodysplasia.

SUMMARY There has been great progress in understanding the molecular basis of activation of the G protein-coupled receptor for PTH and PTHrE Ligand binding to defined sites in the extracellular and transmembrane domains facilitates a conformational change in the receptor that appears to include the movement of the cytoplasmic ends of transmembrane helices 3 and

RECEPTOR SIGNALING AND REGULATION

6 away from one another. Specific cytoplasmic amino acids, particularly in the second and third cytoplasmic loops, have been found to mediate the activation of the cognate G proteins G s a n d Gq, resulting in activation of adenylyl cyclase and phospholipase C. Once activated, PTH/PTHrP receptors are subject to regulatory phosphorylation on serine residues in the cytoplasmic tail. Members of the GRK family, particularly GRK2, appear to be primarily responsible for phosphorylation, with a lesser role for PKC. Phosphorylation of the receptor followed by the binding of arrestin may participate in both desensitization and endocytosis of the P T H / P T H r P receptor, but this has yet to be demonstrated unequivocally. The receptor also contains other determinants of endocytosis (both positive and negative), and these are likely to regulate PTH/PTHrP receptor down-regulation during chronic exposure to agonists. Naturally occuring loss-of-function and gain-of-function mutations in the human PTH/PTHrP receptor have been identified, and these are associated with Blomstrand lethal chondrodysplasia and Jansen's metaphyseal chondrodysplasia, respectively. Further progress in understanding the structural basis of PTH/PTHrP receptor function will continue to provide insights into the control of cellular function by these essential regulatory polypeptides.

6.

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12.

13. 14.

15.

ACKNOWLEDGMENTS

16. 17.

I am grateful to Margaret Bencsik for skillful assistance in the preparation of this manuscript. Portions of the work discussed here were supported by NIH Grant DK35323 and by the Medical Research Service of the Department of Veterans' Affairs.

18. 19. 20.

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78. Law F, Bonjour JP, Rizzoli R. Transforming growth factor-beta: A down-regulator of the parathyroid hormone-related protein receptor in renal epithelial cells. Endocrinology 1994;134:2037-2043. 79. Urefia P, Iida-Klein A, Kong XE Jfippner H, Kronenberg HM, Abou-Samra AB, Segre GV. Regulation of parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid by glucocorticoids and PTH in ROS 17/2.8 and OK cells. Endocrinology 1994;134:451-456. 80. Yaghoobian J, Drfieke TB. Regulation of the transcription of parathyroid-hormone/parathyroid-hormone-related peptide receptor mRNA by dexamethasone in ROS 17/2.8 osteosarcoma cells. Nephrol Dial Transplant 1998;13:580-586. 81. Wald H, Dranitzki-Elhalel M, Backenroth R, Popovtzer MM. Evidence for interference of vitamin D with PTH/PTHrP receptor expression in opossum kidney cells. Pfluegers Arch EurJPhysiol

1998;436:289-294.

82. Nissenson RA. Parathyroid hormone (PTH)/PTHrP receptor mutations in human chondrodysplasia [editorial; comment]. Endocrinology 1998;139:4753-4755. 83. Jobert AS, Zhang P, Couvineau A, Bonaventure J, Roume J, Le Merrer M, Silve C. Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. J Clin Invest 1998;102:34-40. 84. Zhang P, Jobert AS, Couvineau A, Silve C. A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab 1998;83:3365-3368. 85. Karaplis AC, He B, Nguyen MT, Young ID, Semeraro D, Ozawa H, Amizuka N. Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia [see comments]. Endocrinology 1998;139:5255-5258. 86. Jansen M. Uber atypische chondrodystrophie (achondroplasie) und uber eine noch nicht beschriebene angeborene wachstumsstarung des knochensystems: Metaphysare dysostosis. Z Orthop Chir 1934;61:253-286. 87. Ozonoff MB. Metaphyseal dysostosis of Jansen. Radiology 1969;93:1047-1050. 88. Charrow J, Poznanski AK. The Jansen type of metaphyseal chondrodysplasia: Confirmation of dominant inheritance and review of radiographic manifestations in the newborn and adult. Am J Med Genet 1984;18:321-327. 89. Kruse K, Schfitz C. Calcium metabolism in the Jansen type of metaphyseal dysplasia. EurJPediatr 1993;152:912-915. 90. Schipani E, Kruse K, Jfippner H. A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 1995;268:98-100. 91. Schipani E, Langman CB, Parfitt AM,Jensen GS, Kikuchi S, Kooh SW, Cole WG, Jfippner H. Constitutively activated receptors for parathyroid hormone and parathyroid hormone-related peptide in Jansen's metaphyseal chondrodysplasia [see comments]. N EnglJ Med 1996;335: 708-714. 92. Schipani E, Langman C, Hunzelman J, Le Merrer M, Loke KY, Dillon MJ, Silve C, Jfippner H. A novel parathyroid hormone (PTH)/PTH-related peptide receptor mutation in Jansen's metaphyseal chondrodysplasia. J Clin Endocrinol Metab

1999;84:3052-3057.

93. Gardella TJ, Luck MD, Jensen GS, Schipani E, Potts JT, Jr, Jfippner H. Inverse agonism of amino-terminally truncated parathyroid hormone (PTH) and PTH-related peptide (PTHrP) analogs revealed with constitutively active mutant PTH/PTHrP receptors. Endocrinology 1996;137:3936-3941.

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CHAPTER 6

Nuclear Actions of PTHrP

ANDREW C. KARAPLIS AND M. T. AUDREY NGUYEN Division of Endocrinology, Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital, and Lady Davis Institute for Medical Research, McGill University, Montrial, Quebec, Canada H3T 1E2

INTRODUCTION

midregion fragment (amino acid residues 37-86) that is proposed to play a role in placental calcium transport (41); and a carboxyl-terminal fragment [PTHrP(107-139)] that is reported to inhibit osteoclastic bone resorption (13), stimulate osteoblast growth (12), and induce calcium transients in hippocampal neurons (17). As can be inferred from this discussion, PTHrP should be regarded as a prototypical polyhormone that encompasses several distinct, functional regions that mediate unique and independent biological processes under highly specific circumstances (54). Nevertheless, for the most part, the nature of these circumstances remains an open question.

Parathyroid hormone-related protein (PTHrP) was initially identified as the humoral factor responsible for hypercalcemia in malignancy. It is now recognized, however, that its role in calcium regulation represents only a fraction of the wide spectrum of its physiologic actions (41). Indeed, PTHrP controls a diverse range of developmental and homeostatic functions in a wide variety of tissues by acting primarily at the local or cellular level (68). The mature PTHrP sequence is preceded by a 36amino acid prepro segment, in which the first 20-30 amino acids likely represent a signal sequence critical in directing the nascent peptide from the cytosolic compartment to the rough endoplasmic reticulum (ER). It is presumed that the signal peptide is cleaved cotranslationally in the ER by signal peptidase, likely within the - 1 5 to - 5 region, although the exact site has not been rigorously proved. The presence of a prosequence is even less well characterized, although circumstantial evidence and parallels drawn from knowledge of PTH processing overwhelmingly support its existence (9). Cleavage of the propeptide either in the Golgi apparatus or in secretory granules yields the mature PTHrP form with alanine at position + 1. The PTHrP protein is generally thought to comprise several biologically active domains (Fig. 1A). These include the amino-terminal 1-36 peptide, which binds and activates the type 1 PTH cell surface receptor (PTHR1 or P T H / P T H r P receptor) and thereby influences cellular proliferation and differentiation in cartilage (35), bone (4), breast (85), and skin (16); a The Parathyroids, Second Edition

THE PTHrP N U C L E A R / N U C L E O L A R LOCALIZATION SEQUENCE The portion of the mature protein spanning amino acids 87-106 comprises two clusters of basic amino acids (88-91 and 102-106) that have been previously viewed as putative endoproteolytic processing sites (Fig. 1B). This sequence also bears structural homology to a nuclear localization sequence (NLS). The two best defined classes of nuclear import signals are the monopartite and bipartite NLSs, such as PKKKRKV found in the SV40 large T antigen (34), and the nucleoplasmin sequence KR(PAATKKAGQA)KKKK (72), that consists of two basic domains separated by 10 intervening "spacer" amino acids (indicated in parentheses), respectively. This observation formed the basis for the early studies that set out to investigate whether 105

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FIG. 1 PTHrP as a polyhormone. (A) PTHrP cDNA encodes a prepropeptide and mature forms of 139, 141, and 173 amino acids. The three isoforms are identical for the first 139 amino acids, which is the portion depicted here. Proposed biologically active domains within the protein are shown. SP, Signal peptide; P, propeptide. (B) The PTHrP bipartite nuclear localization sequence. This segment encompasses amino acids 88-106 of the mature form (indicated in the single-letter amino acid code) and comprises two basic clusters separated by 10 intervening "spacer" amino acids, resembling the Xenopus laevis nucleoplasmin nuclear localization sequence (72). Furthermore, this region conforms to the structural requirements for a nucleolar localization sequence, as described in key regulatory proteins of human retroviruses (HTLV-1 Rex, and HIV-1 Tat and Rev), consisting of an "arginine hinge" (KRK, in blue) and an adjacent Q inserted between two putative nuclear localization sequences (22). (C) Subcellular distribution of PTHrP in transfected COS-7 cells. Plasmid constructs encoding PTHrP forms having either an intact coding region, deletions within the coding region, or fused in frame to the Escherichia coli lacZ gene were expressed in COS-7 cells and the subcellular localization of the recombinant protein was determined by indirect immunofluorescence. SP, Secretory pattern; N, nucleolar, C, cytoplasmic. Size of lettering on the right-hand side is indicative of the levels of PTHrP immunoreactivity in the various subcellular compartments (24). (See color plates.)

the NLS in PTHrP is indeed functional (24). When preproPTHrP cDNA was transiently expressed in COS-7 cells, the subcellular localization of the protein, as determined by indirect immunofluorescence, was consistent with that of a secretory protein (Fig. 1C). Moreover, in these randomly cycling cell populations, approximately 10% of transfected cells also displayed nucleolar PTHrP staining, suggesting that the putative NLS could target PTHrP to the cell nucleus/nucleolus. When plasmid expressing an engineered mature form of PTHrP (i.e., lacking the prepro sequence) was transfected in COS-7 cells, this mature form, in striking contrast to the predominantly cytoplasmic accumulation of the expressed preproPTHrP eDNA, was targeted almost exclusively to the nucleus, where it was distributed in a nucleolar pattern. Removal of the putative NLS resulted in purely cytoplasmic staining. A similar deletion from the preproPTHrP eDNA construct elicited an exclusively secretory pattern for the expressed protein. These findings suggested that, in the absence of the prepro sequence, PTHrP is preferentially directed to the nuclear compartment and that deletion of the NLS effectively abolishes intranuclear localization of the recombinant protein. Interestingly, fusion of the PTHrP NLS to the cytoplasmic protein [3-galactosidase can target the fusion protein to the nucleus, consistent with the PTHrP NLS sequence being sufficient to target PTHrP to the nucleus. In addition to the foregoing studies, endogenous native PTHrP has also been shown, both in vitro as well as in situ, to localize to the nucleolus (Fig. 2). In osteoblasts, immunoelectron microscopy detected endogenous PTHrP over the dense fibrillar component of nucleoli, a subnucleolar structure and major site for transcription of rRNA genes (24). Nuclear/nucleolar PTHrP immunoreactivity has subsequently been described in keratinocytes (47), vascular smooth muscle (55), breast cancer (10), malignant melanoma (87), astrocytoma (77), and glial and neuronal cells (A. C. Karaplis and M. T. Audrey Nguyen, personal observation, 2000). It is becoming apparent, therefore, that nuclear translocation of PTHrP constitutes a means by which this peptide growth factor could modulate cell function via an intracrine mechanism of action. This unconventional and rather contentious view of PTHrP action is not unique to this protein. Over the past two decades, other molecules that bind to cell surface receptors, such as insulin (21); growth hormone (52); prolactin (71); somatostatin (59); nerve growth factor (26); fibroblast growth factors (FGFs) such as bFGF (7,8), aFGF (28), and FGF3 (36,37); plateletderived growth factor (PDGF) (53); angiogenin (27,60); and insulin-like growth factor-binding proteins (IGFBP)-3/IGFBP-5 (75,76) have been proposed to

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influence cellular function in a dual m a n n e r [for reviews, see (23,32)], first, by binding to cell surface receptors in their classic e n d o c r i n e / p a r a c r i n e mechanism of action, and second, by targeting of the protein to the nucleus a n d / o r nucleolus of the cell in an intracrine signaling pathway. Little is known about the events that govern the timing and extent of nuclear transport of these peptides or their nuclear actions in normal cell biology. In this review, we highlight the present knowledge of the PTHrP nuclear actions, concentrating on four specific issues: (1) How does PTHrP, a secreted protein, gain access to the cytosol? (2) Once in the cytosol, how is PTHrP transport to the cell nucleus regulated? (3) How does n u c l e a r / n u c l e o l a r PTHrP modulate cell functions? (4) What are the cellular consequences of nuclear signaling by PTHrP?

HOW DOES PTHrP, A SECRETED PROTEIN, G A I N A C C E S S TO THE C Y T O S O L ? In attempting to understand how PTHrP, a secreted pepdde, gains access to the cytosol for subsequent nuclear targeting, three distinct pathways should be considered (Fig. 3). First, depicted by pathway A in Fig. 3, PTHrP could be internalized after secretion. This may be a receptor d e p e n d e n t or i n d e p e n d e n t process. What evidence exists that secreted PTHrP utilizes this route to access the cell cytosol? Intuitively, one would assume that binding to the type 1 PTH receptor is the primary mode for PTHrP internalization. In concordance with this assumption, Lam et al. (45) have demonstrated that PTHrP(1-108), when added to culture medium, can be taken up specifically by receptor-expressing

FIG. 3 Potential pathways (A, B, and C) utilized by PTHrP to gain access to the cytosol. In pathway A, secreted PTHrP undergoes internalization at the cell surface in a "receptor"dependent manner. Endocytosis could be mediated by the type 1 PTH receptor (PTHR1) or a binding protein that is distinct and recognizes either the N-terminal domain or other regions of PTHrP (R). In pathway B, PTHrP, after entering the ER lumen, "dislocates" back to the cytosol via the Sec61p translocon, a key component of the mammalian cotranslational protein translocation system, which functions as a twoway channel shuttling proteins both into the ER and back to the cytosol. Ubiquination of preproPTHrP may serve as the signal for retrograde transport of the peptide. In pathway C, initiation of translation in PTHrP mRNA downstream from the initiator methionine generates a protein with a shorter signal peptide. Such a protein would fail to be targeted for secretion and remain in the cytosol for subsequent nuclear import. Experimental evidence supporting each of these pathways is illustrated in Fig. 4. (See color plates.)

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UMR-106.01 cells. This process is blocked by an excess of either PTHrP(1-108) or PTHrP(1-34), indicating that the endocytosis is mediated by the type 1 PTH receptor (Fig. 4A). Further support for type 1 PTH receptor-mediated uptake has come from studies showing receptor immunoreactivity in the nucleus of osteoblast-like cells at the time of DNA synthesis and mitosis (84). This, however, does not preclude involvement of the distinct amino-terminal PTHrP receptor, previously described in keratinocytes and squamous cell lines (67), or the novel splice variant of the type 1 receptor that lacks the signal peptide and displays low levels

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of cell surface expression (33), in the reuptake pathway. In contrast to the above reports, another study found nuclear import of the protein in both type 1 PTH receptor-positive and-negative cells, thus suggesting that a core motif within the PTHrP NLS is responsible for endocytosis and nuclear targeting of the protein (2). This argues for the existence of an as yet-unidentified cell surface receptor, distinct from the type 1 PTH receptor, that mediates ligand internalization. Second, PTHrP could be diverted away from the secretory route by retrograde translocation from the ER lumen to the cytosol (Fig. 3, pathway B). This novel

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PTHrP NUCLEARACTIONS / mechanism has emerged from studies on the dislocation and degradation of unfolded or misfolded proteins (40,69). Membrane and secretory proteins are inserted in the ER membrane or enter the lumen, respectively, in an unfolded state, where highly efficient quality control systems assure that they are folded in their appropriate conformation before arriving at their final destination. Misfolded proteins are retained in the ER and transferred back into the cytosol where they are ubiquitinated by ubiquitin-conjugating enzymes and subsequently degraded by the proteasome. Recognition and retention are mediated in part by the lumenal chaperone immunoglobulin heavy chain binding protein (BiP), whereas reverse transport occurs through the Sec61p translocon, a key component of the mammalian cotranslational protein translocation system, which functions as a two-way channel shuttling proteins both into the ER and back to the cytosol. Integral membrane proteins such as the h u m a n cystic fibrosis transmembrane conductance regulator protein (CFTR) and the yeast pleiotropic drug-resistance protein 5 (Pdr5), as well as soluble mutant proteins such as carboxypeptidase Y (CPY), the h u m a n oLl-proteinase inhibitor, and the yeast p h e r o m o n e ot factor, have been reported to dislocate from the ER, for presentation to the cytoplasmic ubiquitin-proteasome degradation machinery [for review, see Plemper and Wolf (69)]. Although in this limited number of available examples, improper folding serves as the major molecular signal that reverses the early steps that commit a protein to secretion, it is evident that additional signals must be involved in identifying proteins targeted for retrograde transport. First to be considered is the regulated degradation of ER proteins. For instance, the yeast 3-hydroxy-3-methylglutaryl-CoA reductase isoenzyme 2 (HMG-CoA-R2), a key regulatory enzyme in sterol biosynthesis that resides in the ER, is subject to endproduct inhibition and end-product-induced degradation by the ubiquitin-proteasome route (20). Therefore, not only mutated and hence incorrectly folded proteins are selected for reverse translocation; this pathway also subserves regulated proteolysis of native proteins. Second, not all dislocated proteins undergo degradation. Protein toxins such as ricin and Shiga, which enter the cell through endocytosis, have been shown to travel retrogradely, through the secretory path, to the ER lumen. Because these toxins inhibit protein synthesis in eukaryotic cells, they must transverse the ER membrane to get to their targets within the cytosol. In such a scenario, at least a fraction of the exported toxins has to escape ubiquitination and degradation in order to kill the cell. The details of the mechanism employed to elude degradation, however, remain obscure. What is the evidence that PTHrP in the ER lumen utilizes dislocation to enter the cytosol? Studies have

109

established that PTHrP is indeed a substrate of the ubiquitin and proteasome-mediated proteolytic system (Fig. 4B), making this path an attractive mechanistic alternative for cytosolic localization of the peptide (56,57). ProPTHrP in the ER binds to the lumenal chaperone BiP, which, together with Sec63p and ATE functions to reel proteins from the ER to the cytosol for degradation. Treatment of cells expressing preproPTHrP with proteasome inhibitors MG 132 or lactacystin results in the accumulation of proPTHrP in microsomal fractions. It is conceivable that a certain fraction of PTHrP could escape ubiquitination and proteasomal degradation so as to gain access to the cytosol and nuclear import system. Based on these findings, it was proposed that a carefully regulated ubiquitin-dependent mechanism modulates the halflife of PTHrP in the cytosol and in the nucleus, and thereby exerts a regulatory role on the peptide's intracrine effects. A third possible mechanism for translocation of PTHrP to the cytosol is diversion away from the default secretory route prior to its translocation to the ER lumen (Fig. 3, pathway C). Many secreted proteins accomplish this by using alternate sites for initiation of translation. With wild-type mRNAs, context-dependent leaky scanning, reinitiation, and possibly direct internal initiation permit escape from the first-AUG rule and allow access to other AUG codons, which often are in close proximity to the 5' end of the transcript (42). Although a rare event, alternate initiation of translation could also take place at non-AUG codons, usually upstream ACG, AUU, CUG, and GUG, in addition to the initiator AUG codon, as documented for several plant (79), viral (14,70), bacterial (38,74,78,83), Drosophila (39,80), and mammalian (15,37,81,86) mRNAs [for review, see Kozak (44)]. In the case of PTHrP mRNA, the absence of other AUGs suggests that translation might be initiated at alternate CUG and GUG codons. All of these are located downstream from the initiator AUG codon, some of which are e m b e d d e d within an optimal or favorable context for initiating translation, as defined by Kozak (43). As with other natural mRNAs, the utilization of alternate translation start sites in PTHrP mRNA could be explained by the lack of an optimal facilitating context of translation in the vicinity of the initiator codon and the presence of stabilizing secondary structures within the mRNA transcript. Additional findings support the contention that some nuclear forms of PTHrP arise from initiation of translation at alternate codons within the signal peptide that are downstream of the conventional initiator AUG codon (Fig. 4C) (66). How would such a process produce PTHrP species that localize to the cytosol? The resulting PTHrP forms would contain a truncated signal sequence that does not bind the signal recognition

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particle (SRP), a cytosolic ribonucleoprotein, GTPcontrolled complex that targets proteins to the ER. The nascent peptide would, therefore, remain sequestered within the cytosol for subsequentnuclear import. Although these three potential mechanisms for cytosolic relocation of PTHrPmendocytosis, ER dislocation, and utilization of alternate sites for initiation of translation--are presented here as mutually exclusive, it is equally plausible that they act in concert to bring about alterations in the intracellular routing of the peptide. Regardless of the pathway(s) used by the cell, once PTHrP enters the cytosolic compartment, it would be directed to the nucleus on the strength of its fully functional NLS.

ONCE IN THE CYTOSOL, H O W IS PTHrP T R A N S P O R T TO THE CELL NUCLEUS REGULATED? Because compartmentalization of proteins is required to regulate cell cycle progression and signaling pathways, among others, discriminatory controls for protein movement into the nuclear compartment are critical. Macromolecules travel back and forth between the cytoplasm and nucleus through the nuclear pore complexes (NPCs), which are large organelles in the nuclear envelope composed of at least 50 different proteins. Shuttling through the aqueous channel of the NPC can take place either by passive diffusion or by active transport, the latter being highly selective in the choice of substrates to be moved and occurring along distinct paths, many of which involve nuclear transport receptors [for review, see Gorlich and Kutay (18) ]. There are two distinct steps required for importing proteins into the nucleus. In the first step, signals are identified and bound by saturable transport receptors, and it is this interaction that brings the complex to the cytoplasmic side of the NPC. In the second step, there is translocation of the complex through the NPC by an energy-driven mechanism with accumulation and ensuing dissociation of the complex within the nucleoplasm. Part of the energy requirement for orchestrating this nuclear traffic arises from guanosine triphosphate hydrolysis by the 25-kDa protein Ran (Ras-related nuclear protein), one of the most abundant G proteins in the cell (58). Import into the nucleus is mediated by adapters and transport receptors such as importin ~ and [3 (also known as karyopherin a and b), transportin, hsp70, RanBP5, RanBP7, and p l0. Importin e~, presumably acting as an adapter (29), binds the classic NLS-containing proteins (SV40-type or bipartite NLS) in the cytoplasm and interacts with importin [3. In turn, importin [3 associates with the NPC, and in several Randependent steps, the trimeric NLS protein-importin

or/[3 heterodimer complex enters the nucleus. There, RanGTP binds importin [3 to displace the 0t subunit, the NLS protein dissociates from it, and importin ot and RanGTP-importin [3 are returned to the cytoplasm. The specificity of this interaction as well as the effectiveness of nuclear transport can be attributed to distinct binding activities of various karyopherin a isoforms and their differential expression (62). Alternatively, direct binding to importin [3 and receptors similar to it (transportin, RanBP5, and RanBP7) has been observed with nuclear proteins such as hnRNP A1, Nab2p and Nab4p, the ribosomal proteins L23a, $7, and L5 (30), and IGFBP-3/IGFBP-5 (75). Domains that interact with these transport receptors are, in general, very basic. Progress has been made toward clarifying the molecular mechanisms that regulate PTHrP nuclear import. Lam et al. (45) have demonstrated that in contrast to most other NLS-containing proteins, PTHrP is solely recognized by importin [3 and not importin e~ (also see Chapter 4). The sequence in PTHrP that interacts with importin [3 was mapped to amino acids 66-94, which includes an SV40 large T antigen NLS-like sequence, although residues amino terminal to this region were essential for high-affinity binding. In in vitro reconstitution assays, importin [3 and the GTP-binding protein Ran, in the absence of importin e~, were able to mediate efficient nuclear accumulation of PTHrE In general terms, nucleolar localization of a protein relies primarily on the capacity of its NLS to be carried into the nuclear compartment and then targeted to the appropriate subnuclear site. In essence, our knowledge of intranuclear protein sorting actually ends here, because the underlying molecular intricacies are only beginning to be elucidated. The first indication that definite protein domains could direct the respective protein to the nucleolus was derived from studies mapping the regions responsible for targeting key regulatory proteins of human retroviruses (HTLV-1 Rex, and HIV-1 Tat and Rev) to this subnuclear location (22). It was determined that a nucleolar localization sequence (NOS) consists of an "arginine hinge" and an adjacent glutamine inserted between two putative NLSs. As illustrated in Fig. 1B, the 87-106 region of PTHrP closely parallels these requirements and conforms with the prerequisite that this sequence is necessary and sufficient for translocating PTHrP and the heterologous cytoplasmic protein, [3-galactosidase, exclusively to the nucleolus (24). This implies that the PTHrP NOS constitutes a structure that is recognized both by the nuclear transport machinery and, once into the nucleus, by nucleolar targeting components. For other cellular nucleolar proteins, however, a NOS has not been unambiguously demonstrated given that multiple domains may be required for nuclear/nucleolar import

PTHrP NUCLF_~.RACTIONS / (6,19,50,82). Due to lack of a consensus sequence, nucleolar localization is believed to be mediated by a conformational feature common to nucleolar proteins, which is recognized and targeted by nucleoplasmic or other nucleolar proteins. Alternatively, for small basic molecules such as ribosomal proteins, it has been proposed that importin [3 may shield them from undesired interactions on the way from the NPC to the nucleolus, where they could be transferred from this receptor either to a chaperon or directly to rRNA. Given the very basic nature of PTHrP, this mechanism may operate to deliver the peptide to this subnuclear site. The subcellular partitioning of proteins is not a random event. Rather, it is a dynamic, well-defined and timed event, which occurs in response to intra- or extracellular stimuli. Nuclear uptake of most proteins is regulated by extracellular stimuli or by the cell cycle, suggesting that mechanisms exist to control the timing and extent of this process. Similarly, nuclear translocation of PTHrP has been directly linked to cell cycle progression. In cultured HaCaT keratinocytes, PTHrP localizes to the nucleus/nucleolus in G1, but it relocates to the cytoplasm when cells are actively dividing (47). In contrast, untransfected and PTHrP-overexpressing A-10 vascular smooth muscle cells contain PTHrP immunoreactivity in nuclei of cells that are dividing or completing cell division ( 6 2 o r M phase) (55). Progression through the cell cycle is often accompanied by a change in the phosphorylation status of selected proteins and their subsequent redistribution between cytoplasmic and nuclear compartments. One efficient means of controlling NLS accessibility for nuclear transport is through protein phosphorylation/dephosphorylation. In proteins such as SV40 large T antigen, p53, c-abl, and lamin A/C, phosphorylation proximal to the NLS by CDC2/cylinB kinase has been reported to regulate nuclear translocation (31). Cyclindependent kinases (CDKs) are critical for progression through the various phases of the cell cycle. Activation depends on their association with cyclins whereas deactivation results from the degradation of the cyclins. CDK4 and CDK6 associate with the D cyclins to progress the cell through the G 1 phase. CDK2 complexes with cyclins E and A to bring about the G 1 to S transition, and the complex formed by CDC2 and cyclin B gates the transition from the G 2 t o the M phase of the cell cycle. In PTHrP, a consensus motif (KTS5pGK) for phosphorylation by CDK2-CDC2 lies immediately upstream, and overlapping the NLS (24) (Fig. 5A). Its presence suggests that PTHrP could serve as substrate for CDK2-CDC2 and that phosphorylation at this position may affect PTHrP nuclear import. In vitro studies have provided convincing evidence that PTHrP is indeed a substrate for phosphorylation by CDK2-CDC2, and that

111

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FIG. 5 Regulation of PTHrP nuclear import. (A) Phosphorylation site for CDK2-CDC2 in PTHrP. The NLS (NOS) in PTHrP spans amino acids 88-106 of the mature protein (yellow-colored box) and is preceded by a consensus sequence (blue-colored box) for phosphorylation by CDK2-CDC2. The asterisk denotes the threonine (T) residue that undergoes phosphorylation. Below, the analogous region in the SV40 large T antigen is depicted for comparison. (B) Phosphorylation of PTHrP at T ss by cyclin-dependent kinases negatively regulates its nuclear translocation. The Gl-specific cyclin-CDKs do not phosphorylate PTHrP, which then localizes to the nucleolus. Phosphorylation at T 8~ by CDK2-CDC2 in the other phases of the cell cycle leads to the exclusion of the protein from the nucleus. Although phosphorylation of PTHrP is shown here to inhibit its interaction with importin [3 (113), this has not been vigorously demonstrated. (See color plates.)

phosphorylation occurs at residue T 85, and likely at residues S4~ and S128 (46,65). Site-directed mutagenesis of threonine at position 85 to alanine (T85A) causes an appreciable increase in the nuclear translocation of the protein in transfected COS-1 cells, suggesting that dephosphorylation at this site favors nuclear targeting. The T85A mutation, which precludes phosphorylation of PTHrP, has also been correlated with nuclear and nucleolar uptake of the protein in HaCaT cells, whereas a T85E alteration, functionally simulating phosphorylation, triggers nuclear exclusion of the peptide (46). PTHrP, therefore, demonstrates cell cycle-dependent phosphorylation and nuclear localization (Fig. 5B). This is the first report of an endocrine/paracrine factor that exhibits a phosphorylation-dependent regulation of partitioning between cytoplasm and nucleus, implying that indeed, the protein has definite nuclear roles.

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CI4AeTF~R6

HOW DOES NUCLEAR/NUCLEOLAR MODULATE CELL FUNCTIONS?

PTHrP

PTHrP immunoreactivity over the dense fibrillar component of the nucleolus, both in vitro and in situ (24), implies that PTHrP could modulate specific cellular activities by altering nucleolar function. Because nucleoli are sites of transcription and processing of rRNA and ribonucleoprotein complex formation prior to their export to the cytoplasm, it is conceivable that nucleolar PTHrP regulates cell function by influencing this process. Ribosome synthesis entails transcription of rDNA by RNA polymerase I, processing of pre-rRNA transcript, and assembly of rRNA with ribosome proteins. Nucleolar PTHrP may affect de novo ribosomal synthesis rate and distribution by modifying the activity of RNA polymerase I, as for FGF-2 (8), the protein product of the Rb gene (11), and the SV40 large T antigen (88), following localization to the nucleolus. PTHrP may also play a role, yet undefined, in pre-rRNA processing and ribosome assembly. This series of events relies on the presence and function of numerous nucleolar proteins that possess RNA-binding properties, a characteristic shared by PTHrP (2). Alternatively, the diffuse nucleoplasmic distribution of PTHrP in vascular smooth muscle cells (55) suggests that the peptide could have other intranuclear actions, such as regulation of DNA replication a n d / o r gene transcription, as reported for FGF (63) and angiogenin (27).

W H A T ARE T H E C E L L U L A R CONSEQUENCES OF NUCLEAR S I G N A L I N G BY PTHrP? Accruing evidence indicates that the nuclear forms of PTHrP have unique consequences on cell function, which are distinct from the paracrine/autocrine effects that result from binding and activation of the type 1 PTH receptor. Initial observations of nuclear effects of PTHrP came from in vitro studies. When localized to the nucleus, PTHrP enhanced the survival of serumdeprived CFK2 chondrocytic cells by inhibiting programmed cell death (24). Moreover, constitutive expression of preproPTHrP in the CFK2 cells stimulated mitogenic activity and inhibited differentiation, whereas expression of the nucleolar form impaired the differentiation process (25). In quiescent keratinocytes, nuclear PTHrP induced cell cycle arrest at G 1 (47), whereas in vascular smooth muscle cells, it promoted cell cycle activation (55). These apparent discrepancies may be related to the cell and tissue specificity of PTHrP nuclear bioactivity. The most convincing evidence for nuclear actions by PTHrP comes from in vivo studies using gene targeting technology. Mice homozygous for PTHrP gene disrup-

tion are born alive but die soon after birth because of a multitude of skeletal deformities that arise from diminished proliferation and inappropriately accelerated differentiation of chondrocytes in the developing skeleton (5,35). In contrast, animals homozygous for the type 1 PTH receptor-null allele exhibit a more severe phenotype characterized by early embryonic death (49). Although the n u m b e r of mutant fetuses meets mendelian expectations at embryonic (E) day 9.5, only 10% of the living ones are receptor null at E12.5, and almost all of them die by E14.5. The phenotypic severity of the receptor-negative mutants is very intriguing. Does this imply that circulating PTH partly compensates for the lack of PTHrP so as to alleviate the abnormalities of ligand-null systems? This is unlikely, because PTH expression does not normally occur before E15.5 (73), whereas PTHrP is widely expressed in the embryo proper by that time. These findings are relevant to the dual mode of action proposed for PTHrP (64). It would seem that normal cellular function necessitates the coordinated activity between the amino-terminal end of the protein signaling via the cell surface receptor and the intracellular form operating at the level of the nucleus/nucleolus. In the absence of the receptor, the unopposed nuclear/nucleolar effects of PTHrP would lead to dysregulated cellular differentiation and thereby early demise of the receptor-negative mutants. Conversely, ablation of the ligand would eliminate both receptormediated and intranuclear activities, producing a more "coordinated" cellular impairment, and hence a less severe phenotype. More recent genetic studies in mice provide additional in vivo insights into the potential nuclear actions of PTHrP during skeletal development. As already alluded to, PTHrP-deficient chondrocytes stop proliferating at an early stage, hypertrophy, and finally undergo apoptosis (Fig. 6A) (3,5). Accelerated bone mineralization ensues. In sharp contrast, chondrocytes from type 1 PTH receptor-negative mice become hypertrophic later and are replaced by invading blood vessels and osteoblasts more slowly than in wild-type mice (48). Consequently, the primary spongiosa, which is the first bone to form on a cartilage matrix, is greatly diminished in receptor-null mice, and this results in a decrease in secondary spongiosa, with less trabecular bone throughout the skeleton. None of these abnormalities are seen in bones from ligand-negative mice. To better understand the delay in cell differentiation specific to the receptor-null bones, bones of doublehomozygous (PTHrP- and type 1 PTH receptor-negative) mice were examined. In these animals, the pattern of vascular invasion and endochondral bone formation was more similar to that of wild-type than receptor-null specimens. This partial rescue of the type 1 PTH receptor-negative phenotype strongly advocates that some

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actions of PTHrP are i n d e p e n d e n t of receptor activation. PTHrP, perhaps through its intranuclear effects, may slow blood vessel and osteoblast invasion into hypertrophic cartilage. From the foregoing discussion, it is apparent that confirmation of a physiologic role for nuclear PTHrP signaling will require analysis in the intact animal. This in vivo approach demands the development of an animal model in which a strategically placed mutation in the PTHrP gene leads to synthesis of a PTHrP molecule that is unable to translocate to the nucleus but is able to signal normally through the cell surface type 1 PTH receptor. Generating mice expressing mutant forms of PTHrP is now feasible using standard knockin methodology, which offers the opportunity to introduce subtle

.

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FIG. 6 Abnormal endochondral bone development caused by deficiency of type 1 PTH receptor and PTHrP. (A) Contrasting effects of type 1 PTH receptor and PTHrP targeted disruption. Unlike in wild-type phalanges (a), blood invasion and bone replacement are delayed in the receptor-null bones (b), as illustrated by the persistence of proliferating chondrocytes. Conversely, in PTHrP-null phalanges (c), these processes are advanced and chondrocytes hypertrophy prematurely, leading to precocious ossification. (B) Partial rescue of the delay in vascular invasion and endochondral ossification in the type 1 PTH receptornull mice by further ablation of the PTHrP gene. The impairment in blood invasion and bone formation is partially rescued in receptor/ligand double-negative mutant bones (b), as demonstrated by the more timely appearance of the ossification zone, compared to the receptor-negative single mutant (a). This argues in favor of PTHrP actions that are independent of the type 1 PTH receptor. [Reproduced from (48), with permission.] (See color plates.)

mutations into the gene of interest without changing the rest of the murine g e n o m e (51,61). Such genetically modified mice with intact but mutated PTHrP alleles would be amenable for studies addressing the physiologic consequences associated with PTHrP nuclear actions.

CONCLUSION Initial analyses of PTHrP action were considered only in terms of the interaction of PTHrP with the type 1 PTH receptor. Studies now indicate that this peptide can influence fetal development via signaling pathways that extend beyond conventional secretion and cell

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surface receptor activation. Thus, PTHrP action must also be considered in terms of direct effects at the level of the nucleus/nucleolus. Therefore, it becomes essential to understand the mechanism whereby PTHrP translocates to the nucleus and influences cell function through its intracrine actions. Impairment of this process may critically alter the course of normal cell biology. This could have important implications for our appreciation of PTHrP as a signaling molecule in develo p m e n t and as an oncoprotein during the progressive stages of neoplasia.

ACKNOWLEDGMENTS We thank Minh Dang Nguyen for his work on the spinal cord extraction and immunohistochemistry. Work on PTHrP in the authors' laboratory has been supported in part by the Canadian Arthritis Network and the Medical Research Council (MRC) of Canada. M.T.A.N. and A.C.K. are recipients of MRC Doctoral Research and Scientist Awards, respectively.

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CHAPTER

7

Signal Transduction of PTH and PTHrP

LEE S. WEINSTEIN Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892

MICHAEL A. LEVINE Departments of Pediatrics, Medicine, and Pathology, TheJohns Hopkins University School of Medicine, Baltimore, Maryland 21287

INTRODUCTION

ubiquitously expressed, binds PTH and PTHrP with equal affinity, and is required for the endocrine and local actions of PTH and PTHrP, respectively (4-7). On binding of either PTH or PTHrP, the P T H I R receptor activates G s, leading to stimulation of AC and generation of cAMP, and at about 10-fold higher concentration of ligand, the receptor also a c t i v a t e s Gq, leading to stimulation of PLC and generation of IP3 and DAG. More recently a second receptor that binds PTH but not PTHrP has been identified (8). This receptor (the PTH2R receptor) has a more limited tissue distribution, and its natural ligand, identified as the hypothalamic neuropeptide TIP39 (9), is unrelated to PTH. There is also diversity of the effector enzymes, with at least nine distinct molecular forms of AC (10) and at least four forms of PLC that are directly activated by G proteins (11). The P T H / P T H r P receptors (see Chapter 5) and the second-messenger pathways involved in PTH action (see Chapters 5 and 12-14) are discussed in depth elsewhere in this volume. This chapter focuses on the structure and function of the G proteins, particularly those involved in P T H / P T H r P signaling, with special emphasis on more recent developments in the field.

Both parathyroid hormone (PTH) a n d p a r a t h y r o i d hormone-related protein (PTHrP) bind to a c o m m o n receptor (the P T H I R receptor) that activates at least two major second-messenger pathways. Chase and Aurbach initially showed that PTH stimulates generation of the second messenger cyclic adenosine monophosphate (cAMP) and that in pseudohypoparathyroidism (PHP), an inherited disorder in which affected individuals are resistant to PTH action, the defect resides proximal to cAMP generation (1): More recent studies have shown that, in both kidney (2) and bone (3), PTH also stimulates phosphoinositide breakdown with resultant generation of the dual second messengers diacylglycerol (DAG) and inositol triphosphate (IP3). PTH is a m o n g a large group of extracellular first messengers, including the peptide and glycoprotein hormones, neurotransmitters, growth factors, chemotactic agents, and sensory signals, that activate G protein-coupled pathways to generate second messengers such as cAMP, DAG, and IP3. The general mechanism of action of each of these first messengers involves binding to a specific receptor that in turn interacts with one or more G proteins; the G protein regulates the activity of specific effector proteins, such as enzymes of second-messenger metabolism or ion channels. The components of the P T H / P T H r P signaling pathway include one or more receptors that bind PTH a n d / o r PTHrP and the G proteins G s and Gq/11, which stimulate the respective enzymes adenylyl cyclase (AC) and phospholipase C (PLC). The PTH1R receptor is The Parathyroids, Second Edition

G E N E R A L FEATURES OF R E C E P T O R E F F E C T O R C O U P L I N G BY G P R O T E I N S The heterotrimeric G proteins are members of a superfamily of guanosine triphosphate (GTP)-binding proteins that also includes smaller m o n o m e r i c proteins such as ras and the ras-like proteins as well as initiation 117

Copyright © 2001 John R Bilezikian, Robert Marcus, and Michael A. Levine.

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and elongation factors in protein synthesis. All members of this superfamily bind guanine nucleotides with high affinity and specificity, possess intrinsic guanosine triphosphatase (GTPase) activity, and function as molecular switches; they are "on" in the GTP-bound conformation, and hydrolysis of GTP to guanosine diphosphate (GDP) leads to the "off," GDP-bound conformation (12). Members of the family are typically regulated by "exchange factors" that catalyze release of bound GDP, and may also be regulated by GTPaseactivating proteins (GAPs) that stimulate the intrinsic GTPase activity of the GTP-binding protein. The G proteins are composed of or, [3, and ~/ subunits, each the product of a separate gene. The c~ subunit is the GTP-binding homolog of other members of the superfamily. The [3 and ~/subunits bind tightly but noncovalently to form a heterodimer that dissociates from the e~ subunit when the latter is activated. There is substantial diversity in the G protein subset of the superfamily (13). At least 16 distinct mammalian genes encoding e~ subunits have been identified (Table 1). Additional diversity is created by alternative splicing of mRNAs encoding Gse~, Gocx, and Gi2~ subunits (14-17). More recently, substantial diversity among the [3 and

~/ subunits has also been recognized, with at least five [3 subtypes and eleven ~/subtypes (13). Therefore the number of potential G protein heterotrimers is very large. There is evidence that all three subunits of the heterotrimer may be important for specificity of receptor-effector coupling by G proteins (18-20). The most clearly defined function for G proteins is in transduction of information from extracellular "first messengers" to intracellular "second messengers." This function correlates with the location of most G proteins on the inner surface of the plasma membrane, juxtaposed to the transmembrane receptors for first messengers and to the various effector molecules. Posttranslational lipid modifications of cx and ~/ subunits appear to be critical in anchoring G proteins to the inner surface of the plasma membrane (21). The rod photoreceptor G protein transducin (Gt) is associated with the cytoplasmic surface of the specialized outer segment disk membrane. Other G proteins may also be localized to specialized intracellular compartments such as the Golgi complex and the apical surface of polarized epithelial cells. This may indicate a broader role for G proteins in transmembrane signaling and intracellular trafficking (22).

TABLE 1 Mammalian (x Subunit Diversity Family Gs

Gs

Golf Gi/o Gtl Gt2 6gust G~I

Gi2 Gi3

Go Gz

Effector

Expression

Toxin substratea CTX CTX

Ubiquitous Olfactory

TAdenylyl cyclase, Ca2+ channels TAdenylyl cyclase

PTX/CTX PTX/CTX PTX/?CTX PTX PTX PTX PTX

Retinal rod, taste cells Retinal cone Taste cells Neural > other tissues Ubiquitous Other tissues > neural Neural, endocrine Neural, platelets

l"cG M P-phosphodieste rase l"cGMP-phosphodiesterase

Gq Gq Gll

G14 615/16

G12/13 G12 G13

acmx, Cholera toxin;

'2

SAdenylyl cyclase; I"K + channels SAdenylyl cyclase; TK + channels SAdenylyl cyclase; I"K + channels $Ca 2+ channels

9

Ubiquitous Ubiquitous Liver, lung, kidney Blood cells

TPhospholipase TPhospholipase TPhospholipase TPhospholipase

Ubiquitous Ubiquitous

"2

PTX, pertussis toxin•

p 115RhoG EF

CI3 CI3 CI3 CI3

P T H / P T H r P SIGNAL TRANSDUCTION /

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T h e G T P a s e Cycle M1 G proteins have a similar cycle of activation and inactivation, referred to as the GTPase cycle (Fig. 1). In the inactive state G proteins are in the heterotrimeric form, with GDP b o u n d to the c~ subunit. On agonist binding, receptors act catalytically to release GDP from inactive heterotrimers and permit binding of ambient GTP to the c~ subunit. After binding GTP, the ot subunit undergoes a conformational change that leads to its activation and dissociation from the [3~/dimer. Activated G protein ot subunits bind to and regulate the activity of specific effector molecules. Free [3~/ dimers can also modulate the activity of certain effectors (23). The c~ subunit activation is terminated by an intrinsic GTPase activity that hydrolyzes b o u n d GTP to GDE After GTP hydrolysis, the GDP-bound c~ subunit reassociates with the [3~/dimer to reform the inactive heterotrimer.

The intrinsic GTPase activity of some ¢x subunits can be stimulated either by interaction with its specific effectors or by a recently discovered family of proteins ( n a m e d the RGS proteins, for regulators of G protein signaling) that act as GAPs for G protein ot subunits. U n d e r artificial conditions, G proteins can be persistently activated by binding of nonhydrolyzable GTP analogs or fluoride anions complexed with aluminum. The latter activates GDP-bound ot subunits by mimicking the ~/phosphate of GTE Covalent modification by cholera toxin of residue Arg 2°1 of the G s ot subunit (GsoL) inhibits its GTPase activity and thereby leads to persistent activation. Similarly, mutation of this residue or residue Gln 227 (or the analogous residues in other G protein c~ subunits) also inhibits GTPase activity, leading to constitutive activation. • PTX

• unc

mutation

GTP Agonist l lt

l|

l|

vvvk RECEPTOR

• CTX

• Activating G

GDP

@:,,/4 [ e H21a mutation I f

] EFFECTOR I FIG. 1 The G protein GTPase cycle. In their basal, inactive state, oLsubunits contain tightly bound GDP and are associated as a heterotrimer with the 137 dimer. Interaction with the intracellular portion of an agonist-bound, activated receptor (shown in the schematic with seven transmembrane-spanning domains) leads to release of bound GDP and binding of ambient GTP. Binding of GTP leads to dissociation of G protein from receptor and of oL subunit from 137.GTP-bound o~and free 137 subunits regulate effector activity. Intrinsic GTPase activity of the oLsubunit leads to hydrolysis of bound GTP to GDP, with liberation of inorganic phosphate. This causes dissociation of the c~ subunit from the effector and reassociation with 137. Bacterial toxins covalently modify o~ subunits and thereby alter signal transduction. Pertussis toxin (PTX) blocks signal transduction by several G proteins by uncoupling them from receptors. Cholera toxin (CTX) constitutively activates its substrate GsoL,causing agonist-independent c-AMP formation. Mutations identified in GsoLblock signal transduction by uncoupling it from receptors (unc) or preventing activation by GTP (H21a). Certain oLsubunit mutations can also lead to constitutive activation of the c~ subunit and effector pathway by inhibiting GTPase activity (reproduced from Spiegel AM, Shenker A, Weinstein LS. Receptoreffector coupling by G proteins: Implications for normal and abnormal signal transduction. E n d o c r i n e R e v i e w s 1992;13:536-565, with permission).

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G PROTEIN ~ SUBUNIT STRUCTURE AND FUNCTION Structural Basis o f G Protein ~ Subunit F u n c t i o n The mammalian oLsubunits are between 40 and 90% identical in amino acid sequence, and their overall length varies between 350 and 395 residues. The 16 eL subunits can be divided into four classes based both on their degree of primary sequence homology and on functional similarities (Table 1). Within the past several years the crystal structures of various G protein e~ subunits (bound to GDP, GTPTS, or aluminum fluoride) and of G protein heterotrimers have been defined (24-31). The G protein oL subunits are composed of a GTPase domain, which is very similar to the structure of the smaller ras-like proteins and other members of the GTPase superfamily, and a variable helical domain, which is less conserved and present only in the c~ subunits of heterotrimeric G proteins. The GTPase domain contains the conserved guanine nucleotide binding site and the domains necessary for effector activation. Guanine nucleotides sit within a cleft between the two domains. The helical domain appears to be important for maintaining guanine nucleotide in the binding pocket, because disruption of interactions between the two domains leads to an increased rate of GDP release in the basal state (32,33). Alternative splicing of Gse~ exon 3 produces long and short forms of Gse~ with helical domains of variable length (14). These different forms of Gse~ appear to have similar signaling properties. Comparison of the crystal structures of active GTP~/S-bound and inactive GDP-bound cx subunits has identified three so-called switch regions within the GTPase domain whose configuration is altered by the presence of the ~/phosphate of b o u n d GTP, resulting in an overall "active" conformation of the e~ subunit. In the inactive GDP-bound heterotrimer, residues within switch regions 1 and 2 form contacts with the top of the seven-blade propeller structure of the [3 subunit. On GTP binding, these regions swing inward toward the center of the e~ subunit to effect release of the c~ subunit from the [37 dimer. The switch 2 region swings toward the switch 3 region and forms polar interactions with switch 3 that are important for maintaining the active conformation. Mutations of mutually interacting residues within switch 2 (34) or switch 3 (35) prevent activation. Mutation of Gly226within the switch 2 region of Gse~ (H21a mutation) reduces the ability of the e¢ subunit to discriminate between binding of GDP or GTP to the guanine nucleotide binding pocket (36). Mutagenesis studies of several ot subunits and the crystal structure of GTP~/S-bound Gse~ complexed with the catalytic domain of AC (37) have identified regions within the GTPase domain facing away from the membrane that interact with effectors.

Several residues within the GTPase domain are critical for catalyzing the GTPase reaction. An arginine residue (Arg 2°1 in the 394 residue form of Gse¢) within the switch 1 region is the site of cholera-toxin-catalyzed ADP-ribosylation that leads to inhibition of GTPase activity. A glutamine residue within switch 2 (Gln 227 in Gsot, which is the equivalent of Gln 61 in ras p21, a known oncogenic "hot spot") is also a critical c o m p o n e n t of the GTPase reaction. Mutations of the switch 1 arginine or switch 2 glutamine lead to constitutive activation of ot subunits by inhibiting intrinsic GTPase activity (38). Other residues within the GTPase domain may also be important in regulating the GTPase activity. For example, substitution of residue Arg 258 within the switch 3 region of Gse~ leads to markedly increased GTPase activity (39). The RGS proteins act as GAPs for G i, G o, G z, and Gq by stabilizing the transition state of the GTPase reaction, thereby lowering the activation energy for this reaction (40). The RGS proteins preferentially bind to aluminum fluoride-bound e~ subunits, which mimics the transition state of the GTPase reaction. Crystal structure of an RGS-G~ complex indicates that the RGS protein does not contribute catalytically important residues, but rather stabilizes the transition state through interactions with the three switch regions of Got (41). RGS proteins do not interact with Gse~due to the presence of a specific residue unique to GsOt (Asp 229) that prevents interaction with RGS proteins (42,43). The amino-terminal region ( ~ 1 - 2 kDa) of the e~ subunit interacts with the side of the [3 subunit propeller structure and is important for membrane attachment, because it is the site of lipid modifications. Certain e~ subunits (G i, G o, Gz) u n d e r g o cotranslational myristoylation on the amino-terminal glycine (44,45), a modification required for m e m b r a n e attachment and [3~/binding (46). In addition, these G proteins, as well as Gsot, are palmitoylated at cysteines near the amino terminus. Palmitoylation of Gse~ is important for its m e m b r a n e attachment; on activation Gse~ is depalmitoylated and detaches from the plasma membrane (47,48). Distinct subtypes of Gse~ (49) and Gi2e~ (17) produced by alternative splicing have different amino or carboxyl terminal regions that target the G proteins to intracellular m e m b r a n e compartments. Several lines of evidence demonstrate the importance of the carboxyl terminus for specific receptor interaction. The Gie~ and Goe~ subunits have a cysteine in the fourth position from the carboxyl terminus, which is ADP-ribosylated in a reaction catalyzed by pertussis toxin. This modification leads to uncoupling of the G protein from receptor (Fig. 1). Mutations near the carboxyl terminus (50-52), or binding of specific peptide antibodies to this region (53,54), prevent receptor coupling to G protein or alter receptor-G protein specificity. Mutagenesis studies also implicate the

P T H / P T H r P SIGNALTRANSDUCTION / amino terminus and the e~4-[36 region as important for interactions with receptor (55). Based on crystal structures, all three regions of the (x subunit are predicted to be exposed and facing toward the membrane. It is unclear exactly how interaction with receptor leads to GDP release, because the receptor binding domains are located at a distance from the guanine nucleotide binding site.

Molecular and Functional Diversity o f G Protein ~ Subunits The earliest G proteins identified were those involved in retinal phototransduction (G t, transducin) and cAMP generation (Gs). G t is a highly specialized G protein expressed only in retina. In contrast, G s is ubiquitously expressed and couples many receptors to AC stimulation. Another G protein in the G s subfamily, Golf, also stimulates AC but has a much more limited tissue distribution and is believed to play a primary role in olfaction. In addition to stimulating AC, GsoLhas been shown to directly activate certain Ca 2+ channels (56), although the physiologic relevance of this action has not been defined. The h u m a n Gse~ gene (GNAS1) is located at 20q13 and the mouse ortholog Gnas is located in the syntenic region in distal chromosome 2. GNAS1/Gnas is an imprinted gene that produces multiple gene products through the use of alternative promoters and first exons that splice onto a c o m m o n downstream exon (57). GsoLis primarily expressed from the maternal allele in certain tissues, such as the renal proximal tubules, but is biallelically expressed in most other tissues (58). XLc~s, an isoform of GsoLwith a long amino-terminal extension, is expressed only from the paternal allele, whereas the chromogranin-like protein NESP55 is expressed only from the maternal allele (59-61). Both of these products are localized to neurosecretory granules, but their biologic functions are unknown. The G i family of proteins are capable of producing several downstream effects, including the inhibition of AC and the modulation of ion channels. GioL has been shown to be able to interact directly with some, but not all, AC subtypes. The exact in vivo role of some G proteins, such as G z, G o, and G12, are not well defined. G13 has been shown to interact with and activate p l l 5 R h o G E E which provides a mechanism for coupling of classical G protein-coupled receptors to Rho activation (62,63). Stimulation of PLC to generate the dual second messengers DAG and IP3 involves pertussis-sensitive G proteins in certain cells (neutrophils and monocytes) but is pertussis toxin insensitive in most cells. Pertussisinsensitive PLC activation is mediated by members of the Gq family of e~ subunits, which includes the ubiquitously expressed Gqot and GileS, as well as G16ot, which is

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only expressed in leukocytes (Table 1) (11). Assays utilizing transient expression of defined ot subunits have shown that all members of the Gq family can stimulate PLC of the [3 subtype but that there may be quantitative differences, d e p e n d i n g on the G protein and PLC-[3 subtype. For PLC-[31 stimulation, Gq = Gll > G16 > G14 (64), whereas for the PLC-[32 subtype, G16 appears to be most effective (64,65). Gq has also been shown to activate Bruton's tyrosine kinase (66).

G P R O T E I N [~ A N D 3, S U B U N I T S : STRUCTURE AND FUNCTION There are at least five distinct mammalian genes encoding [3 subunits of ~ 3 4 0 - 3 5 3 amino acids. The [31-[34 subtypes are 80-90% identical w h e r e a s the [35 subtype appears to be unique based on its sequence, tissue distribution, and functional properties (23,67). The amino termini of [3 and ~/subunits interact through the formation of a coiled-coil structure. The amino terminus of [35 is believed to form coiled-coil interactions with GGL (G~/ subunit-like) domains within certain RGS protein subtypes, rather than with G protein ~/subunits (68). In all [3 subunits the coiled-coil region is followed by internally repeated ~40-amino acid long segments called "WD-40" repeats because of a characteristic tryptophan-aspartate pair that forms a sevenblade propeller structure composed of four [3 sheets within each blade. The remainder of the ~/ subunit is stretched along the bottom of the [3 propeller, and the switch regions 1 and 2 of the e~ subunit interact with the top of the barrel. The side of the [3 propeller that is positively charged is believed to face the membrane. The diversity of ~/subunits is greater than that of the [3 subunits, with at least 11 distinct mammalian ~/genes identified. The ~/ subunits are ~ 7 0 amino acids in length and are more divergent in primary sequence (27-75% conserved) than are [3 subunits. Each terminates with a "CAAX" motif, which has been shown to u n d e r g o three sequential posttranslational modifications (69). These involve isoprenylation of the cysteine, proteolytic cleavage of the "AAX" tripeptide, and methylation of the cysteine carboxyl group. These modifications are required for m e m b r a n e association of the heterotrimer (70). The [3~/heterodimers are now known to interact with and modulate the activity of multiple effectors, including several subtypes of AC and PLC-[3, K + and Ca 2+ channels, one subtype of phosphoinositol-3-kinase, and the MAP kinase pathway. The [37 h e t e r o d i m e r stimulates Gsot-activated AC subtypes 2, 4, and 7, whereas it inhibits AC subtype 1. Heterodimers containing [35 are unique in that they also inhibit AC2 (67). There is little difference in the structure of free [3~/and [3~/bound to ot subunits, and therefore ot subunits may inhibit the

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interaction of [37 with effectors by binding to a common site on the [3 subunit. Evidence for this has been provided by cross-linking and peptide binding studies (71,72). Phosducin, a protein that disrupts signaling via transducin and possibly other G protein pathways by binding to [3y dimers, has been also shown to bind to the same site on the [3 subunit (73). Other potential effector interaction sites include the amino terminal coiled-coil region (74) and one of the sides of the [3 propeller (75,76). The diversity of [3y heterodimers may be limited by tissue-specific patterns of expression of both types of subunit or selectivity in the interactions of specific subtypes (77). There is also evidence for selectivity of association of ot subunits with different [3~/ heterodimers (78,79). There are now several examples of coupling of specific receptors to effectors by a specific G protein heterotrimer in situ, implicating e~, [3, and ~/subunits as all being important in determining receptor-coupling sensitivity (18-20).

RECEPTOR-G PROTEIN COUPLING G protein-coupled receptors have a common overall topology consisting of a single polypeptide with seven membrane-spanning domains, three extracellular and three intracellular loops, an extracellular amino-terminal portion, and an intracellular carboxylterminal tail. Ligand binding produces a change in conformation of the receptor that promotes highaffinity binding to heterotrimeric G protein(s) and initiates the signal transduction cascade by catalyzing release of GDP from the G protein ot subunit (80). Many studies implicate the intracellular loops and carboxyl-terminal tail, particularly the third intracellular loop, in G protein coupling and in defining the specificity of receptor coupling to a particular G protein subtype (81). Multiple distinct receptors may couple to the same G protein subtype and specific receptors may couple to one or more G proteins. In particular, the P T H I R (4,5), calcitonin (82), and other related receptors all couple to both G s a n d Gq/ll. Each of these receptors has been shown to stimulate both cAMP formation and phosphoinositide breakdown, the latter with an EC50 one order of magnitude higher than for AC stimulation. One study examining the labeling of specific G proteins by GTP-~/-azidoanilide in response to PTH1R receptor stimulation directly demonstrated coupling of PTH1R to G~ a n d Gq, and minimal coupling to Gil (83). Most G protein-coupled pathways display desensitization, defined as reduced response despite continued presence of agonist. Homologous (agonist-specific) desensitization involves the phosphorylation of receptor by a G protein receptor kinase (GRK) (84). GRKs

specifically bind to and phosphorylate agonist-bound receptors at serines and threonines located mostly, though not necessarily exclusively, on the carboxylterminal tail. Six distinct GRK subtypes have been identified to date; GRK1 (rhodopsin kinase) is expressed only in retina and GRK4 is expressed only in testis, whereas GRKs 2, 3, 5, and 6 are ubiquitously expressed. Some GRKs associate to membranes using lipid modifications. GRKs 2 and 3 have a [3~/binding site at their carboxyl terminus and are therefore targeted to the membrane by free [3~/ dimers produced by G protein activation. The latter mechanism therefore links receptor desensitization to G protein activation. There is little specificity in receptor-GRK interactions in in vitro assays, although there may be greater specificity in vivo. Reconstitution studies indicate that in most cases receptor phosphorylation per se is insufficient to inhibit receptor-G protein coupling (84). However receptor phosphorylation promotes the binding of the protein arrestin, which blocks receptor-G protein coupling and acts as an adapter between receptor and clathrin to promote internalization of receptor via clathrin-coated pits. Two retinal-specific and two ubiquitously expressed arrestins have been identified to date. In the case of the PTHIR receptor, studies suggest homologous desensitization and receptor internalization are not dependent on receptor phosphorylation, but only on binding of GRK to receptor (85-87). Receptors can also be desensitized by phosphorylation by other kinases, including the cAMP-dependent protein kinase (PKA) and PKC. In the [32-adrenergic receptor, which stimulates the Gs-cAMP-PKA pathway, phosphorylation of a specific residue within the third intracellular loop by PKA changes the coupling specificity of the receptor from G s to G i (88,89). This creates a negative feedback loop whereby activation of the downstream effector can turn off the signaling pathway at a proximal step. There is no evidence, however, that the PTHIR receptor is negatively regulated by its downstream effectors.

G PROTEIN-EFFECTOR COUPLING Effectors regulated by G proteins are diverse in structure and function. They include peripheral membrane proteins (cGMP phosphodiesterase and PLC) and integral membrane proteins (AC and various ion channels). For AC (10) and for PLC (11), substantial diversity exists at the molecular level. There are nine identified isoforms of AC, all of which are stimulated by Gs0t. Additional work suggests that Gsot may stimulate various AC isoforms through different mechanistic models, however. Gse~ stimulation of AC1 appears to involve a single high-affinity site, whereas stimulation of AC2 and

P T H / P T H r P SIGNAL TRANSDUCTION / AC6 appears to involve Gsc~binding to two Gse~ sites, one with high affinity and one with low affinity (90). In the presence of the diterpene forskolin, the affinity of Gs0L for AC is increased and stimulation is r e n d e r e d monotonic, indicating that both the low- and high-affinity binding sites are converted to a single higher affinity state (90). Some isoforms, such as AC4, AC7, and AC9, are expressed in many tissues, but others, such as AC1, AC8, and AC5, have a more restricted tissue distribution. The various isoforms also differ in their regulation by other factors: [37 stimulates AC2, AC4, and AC7, but inhibits AC1; Gie¢ directly inhibits AC1, AC3, AC5, and AC6; PKA phosphorylates and inhibits AC5 and AC6; Ca 2+ stimulates AC1, AC3, and AC8 in a calmodulind e p e n d e n t m a n n e r and inhibits AC5 and AC6 in a calmodulin-independent manner. The ACs are transm e m b r a n e proteins comprising two membranespanning regions, each containing six transmembrane helices, a cytoplasmic loop between the membranespanning regions, and a cytoplasmic carboxyl-terminal tail. The loop and tail form a pseudosymmetric dimer that contains the catalytic core. G~ot (and [37 in some isoforms) binds on the side opposite that where the substrate ATP binds (37), whereas Giot is believed to bind at a distinct site on the same side as ATP (91). Of the PLC subtypes, the 7 variety is regulated by tyrosine kinase-type receptors rather than G proteins. The [3 variety is regulated by G proteins. As discussed above, ot subunits of the Gq family are known to stimulate PLC-[3. It now appears that [37 subunits can also stimulate certain PLC subtypes, particularly the [32 variety (92). This may explain the pertussis toxin-sensitive PLC stimulation seen in certain cells, such as neutrophils. In this instance, activation of a pertussissensitive G protein (perhaps G~2) by receptors such as the chemotactic receptor f-Met-Leu-Phe receptor leads to dissociation of a [37 subunit capable of stimulating PLC. Certain effectors stimulate the intrinsic GTPase activity of the G protein e~ subunits to which they bind. This was initially shown in kinetic studies involving Gq and PLC-[31 (93) and for transducin and cGMP phosphodiesterase (94). Additional studies suggest that AC may act as a GAP for GsOt (95) and that the p115RhoGEF acts as a GAP for G12otand G13e~ (63). RGS proteins bind to and inhibit specific G protein e~ subunits by two mechanisms: increasing the intrinsic GTPase activity and blocking e~ subunit-effector interactions (40).

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short stature, brachydactyly, subcutaneous ossifications, and in some cases, mental deficits (96,97). Paternal transmission of Gsot mutations produces offspring with the physical features of AHO alone (termed pseudopseudohypoparathyroidism) whereas maternal transmission leads to offspring with AHO plus resistance to multiple h o r m o n e s (e.g., PTH, thyrotropin, gonadotropins) that activate Gs-coupled pathways in their target tissue (termed pseudohypoparathyroidism type la) (98). This variable presentation of AHO, d e p e n d i n g on parental inheritance, is likely to be due to tissuespecific imprinting of Gsot (57,58,99). Pseudohypoparathyroidism type l b, a disease characterized by renal PTH resistance in the absence of A H O or resistance to other hormones, was m a p p e d to 20ql 3, suggesting that this disease may also be due to a Gsot genetic defect (100). Pseudohypoparathyroidism is discussed in detail in Chapter 51. Constitutive activation of Gs can occur secondary to covalent modification of Arg 2°1 by cholera toxin. The resulting elevated cAMP levels in intestinal epithelial cells lead to the severe secretory diarrhea characteristic of intestinal cholera infection. Somatic activating Gse~ mutations encoding substitutions of either Arg 2°1 or Gln 227 are found in pituitary somatotroph tumors and a small n u m b e r of thyroid tumors (38,101). The McCune-Albright syndrome (MAS) is a sporadic disease characterized by caf6-au-lait skin pigmentation, polyostotic fibrous dysplasia, and hyperfunction of multiple endocrine glands. Patients with this syndrome harbor a mutation of Arg 2°1 of Gsot in a mosaic pattern, suggesting that in these patients a somatic mutational event occurs in early embryonic life (102,103). The resultant constitutive activation of cAMP formation explains the pleiotropic autonomous endocrine hyperfunction observed in this disease. The same mutation is present in cells isolated from fibrous dysplasia bone lesions (104,105), demonstrating that G s activation leads to increased proliferation and abnormal differentiation of osteoblastic precursor cells (106). Some MAS patients are hypophosphatemic (with resultant tickets or osteomalacia). It had previously been suggested that this metabolic disturbance may be secondary to either secretion of a phosphaturic substance from dysplastic bone or to hypersensitivity of the renal proximal tubules to PTH (107-109). Basal urinary cAMP levels were shown to be elevated in MAS patients, suggesting that renal proximal tubules are hypersensitive to PTH due to the presence of an activating Gs0t mutation (110).

CLINICakL C O N S I D E R A T I O N S Both acquired and genetic G protein defects can lead to clinical disease. Heterozygous loss-of-function mutations of GsOt produce Albright hereditary osteodystrophy (AHO), a disease characterized by obesity,

ACKNOWLEDGMENTS This work was supported by the NIDDK Intramural Research Program and by NIDDK Grant R01-DK34281.

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81. Wess J. G-protein-coupled receptors: Molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J 1997; 11:346-354. 82. Chabre O, Conklin BR, Lin HY, Lodish HE Wilson E, Ives HE, Catanzariti L, Hemmings BA, Bourne HR. A recombinant calcitonin receptor independently stimulates 3',5'-cyclic adenosine monophosphate and Ca2+/inositol phosphate signaling pathways. Mol Endocrino11992;6:551-556. 83. Schwindinger WF, Fredericks J, Watkins L, Robinson H, Bathon JM, Pines M, Suva LJ, Levine MA. Coupling of the PTH/PTHrP receptor to multiple G-proteins: direct demonstration of receptor activation of Gs, G q / l l , and Gi 1 by [a?2p]GTP-~/-azidoanilide photoafflnity labeling. Endocrine 1998;8:201-209. 84. Krupnick JG, Benovic JL. The role of receptor kinases and arrestins in G protein-coupled receptor regulation. A n n u Rev Pharmacol Toxicol 1998;38:289-319. 85. Malecz N, Bambino T, Bencsik M, Nissenson RA. Identification of phosphorylation sites in the G protein-coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization. Mol Endocrinol 1998;12:1846-1856. 86. Huang Z, Banbino T, Chen Y, Lameh J, Nissenson RA. Role of signal transduction in internalization of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein. Endocrinology 1999; 140:1294-1300. 87. Dicker E Quitterer u, Winstel R, Honold K, Lohse MJ. Phosphorylation-independent inhibition of parathyroid hormone receptor signaling by G protein-coupled receptor kinases. Proc Natl Acad Sci USA 1999;96:5476-5481. 88. Okamoto T, Murayama Y, Hayashi Y, Inagaki M, Ogata E, Nishimoto I. Identification of a Gs activator region of the [32-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation. Cell 1991 ;67:723-730. 89. Daaka Y, Luttrell LM, Lefkowitz RJ. Switching of coupling of the [32-adrenergic receptor to different G proteins by protein kinase A. Nature 1997;390:88-91. 90. Harry A, Chen Y, Magnusson R, Iyengar R, Weng G. Differential regulation of adenylyl cyclases by Gc~s. J Biol Chem 1997;272:19017-19021. 91. Dessauer CW, TesmerJJ, Sprang SR, Gilman AG. Identification of a Gie~ binding site on type V adenylyl cyclase. J Biol Chem

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CHAPTER 8

Receptors and Signaling for Calcium Ions

EDWARD M. BROWN, ARTHUR CONIGRAVE, AND NAIBEDYA CHATTOPADHYAY Endocrine-Hypertension Division,

Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115

INTRODUCTION

of C a 2+ o is also crucial for calcium's n u m e r o u s intracellular roles. T h e level of Ca2o+ is m a i n t a i n e d by a complex homeostatic m e c h a n i s m , which in m a m m a l s primarily involves the parathyroid glands, calcitonin (CT)secreting C cells, kidney, bone, and intestine (1,2)--as described in detail in C h a p t e r 10. By necessity there must be cells that are capable of recognizing and r e s p o n d i n g to (i.e., "sensing") changes in C a 2+ o in ways that restore n o r m a l levels-that is, that enable Ca2o+ homeostasis (1). It has long b e e n known that calcium ions move across cell m e m b r a n e s t h r o u g h various ion channels a n d other transport processes (4), but the actual m e c h a n i s m s t h r o u g h which C a 2+ o is "sensed" r e m a i n e d poorly u n d e r s t o o d for m a n y years. This chapter provides an u p d a t e on our c u r r e n t u n d e r s t a n d i n g of the molecular m e c h a n i s m (s) underlying the process of C a 2+ o sensing that have b e e n elucidated d u r i n g the past 5-10 years, particularly as it relates to the maintenance of C a 2+ o homeostasis. It is increasingly evident, however, that Ca 2+ o serves as a versatile extracellular first m e s s e n g e r that regulates n u m e r o u s processes in addition to those involved in m a i n t a i n i n g near constancy of C a 2+ o via interaction with specific calcium-sensing receptors on the cell surface [for review, see (5)]. Moreover, there are interesting interactions between various homeostatic systems crucial for the successful adaptation of c o m p l e x life forms to the terrestrial e n v i r o n m e n t m s u c h as those regulating water and f u e l / n u t r i e n t m e t a b o l i s m - - i n which the calcium-sensing r e c e p t o r (CaSR) likely participates, thereby providing new insights into the physiology of

Complex, free-living terrestrial organisms such as h u m a n s and other m a m m a l s m a i n t a i n their extracellular ionized calcium c o n c e n t r a t i o n (Ca2o+) within a narrow limit of about 1.1-1.3 m M (1,2). Calcium ions are of critical i m p o r t a n c e for n u m e r o u s vital body functions. Intra- (3) and extracellular (1) Ca > ions act in distinct but sometimes c o m p l e m e n t a r y ways to regulate these various biologic processes. T h e near constancy of C a 2+ o ensures the availability of Ca 2+ ions for their extracellular roles, such as serving as a cofactor for adhesion molecules, clotting factors, and o t h e r proteins and regulating cardiac contraction and n e u r o n a l excitability (1). F u r t h e r m o r e , salts of calcium and p h o s p h a t e form the mineral phase of bone, thereby providing both a rigid framework that affords protection of vital structures and enables locomotion a n d other bodily movements as well as serving as a large reservoir of these ions for times of n e e d (2). The cytosolic free calcium c o n c e n t r a t i o n (CaZi+), in contrast, is m a i n t a i n e d at a basal level of---100 n M but increases 10-fold or m o r e on cellular activation by extracellular signals acting on their respective cell surface r e c e p t o r s - - o w i n g to influx of C ao2+ a n d / o r release o f C a 2+ f r o m its i n t r a c e l l u l a r stores (3). C a 2+i plays pivotal roles in controlling cellular processes as diverse as muscular contraction, cellular motility, differentiation a n d proliferation, secretion of h o r m o n e s and o t h e r factors, and apoptosis (4). Because all intracellular C a 2+ ultimately originates from that p r e s e n t in the extracellular fluids (ECFs), the availability of a constant source The Parathyroids, Second Edition

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mammals and other free-living terrestrial organisms. This chapter also addresses these newly emerging relationships between Ca2o+ homeostasis and other physiologic regulatory mechanisms.

C L O N I N G OF THE CALCIUMSENSING R E C E P T O R Only a decade ago, the idea that there was a specific 2+ Ca o -sensing "receptor" was held by only a few investigators and was based largely on a body of indirect evi2+ dence in a very limited number of Cao-sensing cells, such as bovine parathyroid cells (1,6-8). The lack of direct evidence that this putative receptor actually existed necessitated undertaking a molecular cloning approach that utilized a bioassay detecting 2+ Ca o -sensing activitymnamely, expression cloning in Xenopus laevis oocytes. Racke et al. (9) and Shoback and co-workers (10) independently showed that X. laevis oocytes become responsive to Ca 2+ o -sensing receptor agonists after being injected with messenger RNA (mRNA) extracted from bovine parathyroid glands. Subsequently, Brown et al. used a similar assay to screen a bovine parathyroid cDNA library and isolated a single full-length, functional clone of the Ca 2+ o -sensing receptor (11). It was then possible to use traditional, hybridizaion-based methodologies to isolate cDNAs encoding CaRs from human parathyroid (12) and kidney (13), rat kidney (14), brain (viz. striatum) (15) and C cell (16), rabbit kidney (17), and chicken parathyroid (18) [for review, see (5)]. All are very similar in their predicted structures and are thought to represent the various tissue and species homologs of the same ancestral gene [for review, see (5)].

PREDICTED S T R U C T U R E A N D BIOCHEMICAL PROPERTIES OF THE CALCIUM-SENSING R E C E P T O R Predicted Structure o f the CaSR and Its H o m o l o g y to Other GPCRs The topology of the human parathyroid CaSR prot e i n ~ p r e d i c t e d from the nucleotide sequence of its cDNA--is shown in Fig. 1. It exhibits three principal structural domains. These are, respectively, its (1) large, 600-amino acid amino-terminal extracellular domain (ECD), (2) "serpentine" motif of seven membranespanning domains characteristic of the superfamily of G protein-coupled receptors (GPCRs), and (3) substantial carboxyl (C)-terminal tail of some 200 amino acids. Over the past decade, several different subfamilies of GPCRs have been identified that share the large

ECD exhibited by the CaSR as well as a modest (20-30%) degree of amino acid identity within their transmembrane domains (TMDs). These structurally related GPCRs are called the family C receptors (19) and contain three separate groups--the metabotropic glutamate receptors (mGluRs) (group I), the CaSR and a family of putative p h e r o m o n e receptors (group II), and the G protein-coupled receptors for y-amino butyric acid (GABA) or GABAB receptors (group III). The mGluRs are G protein-coupled receptors for glutamate, the principal excitatory neurotransmitter in the central nervous system (CNS) (20), whereas the GABAB receptors are the GPCRs for GABA, the central nervous system's principal inhibitory neurotransmitter (21,22). The putative pheromone receptors (VRs) within the group II GPCRs are found exclusively in neurons of the rat vomeronasal organ (VNO) that express the guanine nucleotide regulatory (G) protein, G% (23). The VNO is a small sensory organ regulating instinctual behavior via input from environmental pheromones (23). Additional GPCRs closely related to the CaSR a n d / o r VRs, which are, respectively, taste and putative odorant receptors have been identified in mammals (24) and fish (25). They may represent evolutionary precursors of the pheromone receptors identified in rats, and they also exhibit the characteristic topology of the family C GPCRs. Thus all of the family C GPCRs seem to have as ligands small molecules that serve as environmental cues (i.e., pheromones) or are extracellular messengers within the CNS (e.g., glutamate or GABA) or systemic extracellular fluid (ECF) (viz. Ca2o+).As described below, the CaSR (and probably the other family C GPCRs as well) bind their respective ligands within their ECDsmin contrast to many other GPCRs, whose small ligands (e.g., epinephrine or dopamine) bind within the respective receptors' TMDs a n d / o r extracellular loops (ECLs). The "sensing" function of the ECD of the family C GPCRs likely has its origin in a class of extracellular binding proteins in bacteria (26)rathe periplasmic binding proteins (PBPs), which are receptors for a variety of small ligands, including ions (although these ions apparently do not include Ca2o+) and amino acids (27). The PBPs regulate bacterial chemotaxis toward these environmental substances and can participate in their cellular uptake by coupling the binding of a given ligand to its respective PBP to subsequent transport of that ligand by an associated transport system (27). Thus the family C GPCRs can be thought of as fusion proteins comprising an extracellular ligand-binding, "sensing" motif (the ECD) and a signal-transducing (e.g., serpentine) motif that couples the sensing process to intracellular regulators of various cellular functions (i.e., G proteins and their associated effector

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elements). Interestingly, these functions include the same ones regulated by the PBPs, namely, chemotaxis [e.g., of monocytes toward high levels of Cao2+ (28) ] and cellular transport [i.e., of C a 2+ o by CaSR-regulated, Ca 2+permeable channels (29)]. Furthermore, as described later (see Possible Additional Ca 2+ Sensors), the CaSR binds not only Ca2o+ but also several other ligands, including various amino acids ( 3 0 ) - - f u r t h e r supporting its functional and evolutionary relationships to the other members of the family C GPCRs and, ultimately, to the PBPs.

Biochemical Properties of the CaSR Studies utilizing chimeric receptors that comprise the ECD of the CaSR coupled to the TMDs and C tail of the mGluRs (and vice versa) have shown that C a 2+ o binds to the CaSR ECD (31). Studies have suggested that

residues within the CaSR ECD (e.g., Ser-147 and Ser170) may participate in the binding of C ao2+• These residues correspond to key amino acids thought to be involved in the binding of glutamate and GABA to the mGluRs and GABA B receptors, respectively (32). Given that the CaSR likely binds several calcium ions, however, because of its apparent "positive cooperativity" and the resultant steep slope of the curve describing the activation of the receptor by its various polycationic agonists (e.g., C a 2+ o and Mg2o+) (1), further work is n e e d e d to define the nature of these Ca2+o binding site(s). Of interest in this regard, the CaSR resides on the cell surface principally as dimers (33,34) that are linked by disulfide bonds within its ECD involving cysteines at amino acid positions 129 and 131 (35). Furthermore, functional interactions occur between the m o n o m e r i c subunits of these CaSR dimers, because two individually inactive CaSRs harboring mutations

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within different domains (e.g., the ECD and C tail) can recover substantial biological activity when they form heterodimers after being cotransfected into h u m a n embryonic kidney (HEK293) cells (36). That is, even though the individual CaSRs are inactive, they can "complement" one another's deficiencies to form a partially active heterodimer, presumably via intermolecular interactions. It is possible, therefore, that some of the receptor's apparent positive cooperativitymwhich is crucial to ensure that the CaSR responds over the narrow range of Ca2o+ that regulates, for example, PTH secretion (see later)mresults from the presence of 2+ • Ca o -binding sites on both ECDs of the dimer a n d / o r even at the site(s) where the two ECDs within the dimer interact with one another. Ultimately, solving the three-dimensional structure of the CaSR ECD by X-ray crystallography will shed a great deal of light on how the CaSR binds C a 2+ o and its other polycationic ligands, including the locations, numbers, and interactions, if 2+ • any, between these Ca o -binding sites. The ECD of the cell surface form of the CaSR is extensively N-glycosylated with complex carbohydrates (37), and eight of the predicted N-glycosylation sites within the h u m a n CaSR ECD are efficiently glycosylated (38). Disruption of four to five of these sites reduces cell surface expression of the receptor by 50-90%. Thus, glycosylation of at least three of the sites is essential for cell surface expression, although glycosylation per se is not critical for CaSR binding of Ca~o+ and its subsequent activation of signal transduction (38). Within its intracellular loops and C-terminal tail, the h u m a n CaSR has five predicted protein kinase C (PKC) and two predicted protein kinase A (PKA) phosphorylation sites (12,39). The functional importance of the PKA phosphorylation sites is unknown. Activation of PKC inhibits CaSR-induced stimulation of phospholipase C (PLC), and studies using site-directed mutagenesis have revealed that phosphorylation of a single PKC site within the CaSR C-terminal (C) tail (Thr-888) accounts for most of the PKC-mediated modulation of the receptor's function (39). PKC-evoked phosphorylation of the CaSR C tail, therefore, may provide a mechanism for negative feedback control of its coupling to PLC, whereby PLC-mediated activation of protein kinase C m b y phosphorylating the receptor at Thr888mlimits further activation of this pathway.

I N T R A c E L L u L A R S I G N A L I N G BY T H E CaSR CaSR agonists activate phospholipases C, A 2 (PLA2), and D (PLD) in bovine parathyroid cells as well as in HEK293 cells stably transfected with the CaSR (40).

These actions are most likely mediated by the CaSR in both cell types, because high C a 2+ o no longer produces them in nontransfected HEK293 cells, which do not express an endogenous CaSR, or in parathyroid cells maintained in culture for 3-4 days, in which the level of CaSR expression diminishes by about 80% (41,42). In most cells, CaSR-elicited activation of PLC involves the pertussis toxin-insensitive G protein, Gq (43), although in some it occurs via pertussis toxin-sensitive pathways, likely involving one or more isoforms of the G protein, G i (44). In bovine parathyroid and CaSR-transfected HEK293 cells, activation of PLA 2 and PLD involve PKCd e p e n d e n t pathways that are activated by the CaSR, presumably via PLC (40). The high Ca 2+ • • o -elicited, transient rise in C a 2+ i in bovine parathyroid cells and CaSR-transfected HEK293 cells probably results from the activation of PLC (40) and the resultant IP~-mediated release of Ca 2+ from its intracellular stores (37). High Ca2o+ likewise evokes sustained increases in CaZi+ in both CaSR-transfected HEK293 cells (37) and bovine parathyroid cells (1) through incompletely defined CaZo+ influx pathways. We have shown using the patch-clamp technique that 2+ the CaSR enhances the opening of a Ca o -permeable, nonselective cation channel (NCC) in CaSR-transfected HEK cells (45). A similar NCC is present in bovine parathyroid cells and is activated by high Ca2o+, presumably through a CaSR-dependent pathway (29), suggesting that it could contribute to the high Cao2+-elicited, • sustained elevation in C ai2+ in these cells (46). High C a 2+ o markedly inhibits agonist-stimulated cAMP accumulation in bovine parathyroid cells (47). This action is thought to involve direct inhibition of adenylate cylcase via one or more isoforms of G i, because it is pertussis toxin sensitive (47). In other cells, however, high Ca 2+ o -evoked, CaSR-mediated inhibition of cAMP accumulation can involve indirect pathways, such as suppression of the activity of a 2+. Ca i-lnhibitable isoform of adenylate cyclase by the associated rise in CaZi+ (48). The CaSR also stimulates mitogen-activated protein kinase (MAPK) activity in several cell types, including rat-1 fibroblasts (49), ovarian surface cells (50), and CaSR-transfected but not nontransfected HEK293 cells (50a). As has been described with other GPCRs, the CaSR activates MAPK via PKC- and tyrosine kinase-dependent pathways involving c-Src-like cytoplasmic forms of the latter enzyme, which are downstream of Gq a n d / o r G i, respectively (49,50). That is, in CaSR-transfected HEK293 cells, PKC inhibitors reduce high Ca o2+_evoked, CaSR-mediated activation of MAPK by about 50%, and the remaining activation of the enzyme can be largely abolished by the further addition of pertussis toxin a n d / o r tyrosine kinase inhibitors (50a).

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THE CALCIUM-SENSING R E C E P T O R GENE A N D ITS R E G U L A T I O N Very little is currently known about the structure of the CaSR gene and the factors that control its expression. The h u m a n CaSR gene resides on the long arm of chromosome 3 as d o c u m e n t e d by linkage analysis (51) and in band 3q13.3-21 as determined by fluorescent in situ hybridization (52), whereas the rat and mouse CaSR genes, respectively, reside on chromosomes 11 and 16 (52). The h u m a n CaSR gene has seven e x o n s m the first encodes upstream untranslated regulatory regions, the next five code for various portions of the ECD, and the last encodes the r e m a i n d e r of the CaSRmfrom its first TMD to the C terminus (53). Understanding the upstream regulatory regions of the CaSR gene will be of substantial interest, because expression of the CaSR can change in a variety of circumstancesmsome of which are described below. Several factors are associated with increased expression of the CaSR gene. Both high Ca2+o and 1,25(OH)zD ~ can up-regulate expression of the CaSR gene in certain cell types [e.g., adrenocorticotropin h o r m o n e (ACTH)-secreting, pituitary-derived AtT-20 cells (44) and in both rat kidney and parathyroid (54), respectively]. Interleukin-l[3 modestly raises the level of CaSR mRNA in bovine parathyroid gland fragments, which could contribute to the associated decrease in PTH secretion (55). In the kidney there is substantial up-regulation of the CaSR during the peri- and postnatal periods, and the resultant higher level of CaSR expression persists t h r o u g h o u t adulthood (56). There is also a developmental increase in CaSR expression in the brain, but in contrast to that occurring in the kidney, the increase in the brain takes place about 1 week postnatally (57). Furthermore, the increased CaSR expression in the brain is transient, and it decreases severalfold about 2 weeks later, reaching a lower level that remains stable thereafter (57). The biologic significance of these developmental changes in CaSR expression are unknown. Conversely, CaSR expression decreases in several circumstances. Calf parathyroid cells show a rapid and marked (80-85%) decrease in CaSR expression after they are put in culture (41,42), which likely is a major factor contributing to the concomitant reduction in high 2+ Ca o -evoked suppression of PTH release. The level of expression of CaSR in the kidney is also decreased in chronic renal insufficiency induced in the rat by subtotal nephrectomy (58). This change in the level of CaSR expression could potentially contribute to the hypocalciuria that occurs in the setting of renal insufficiency, because reduced renal CaSR expression a n d / o r activity are associated with increased tubular reabsorption

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131

of C a 2+ (59). Because, as noted above, 1,25(OH)2D ~ increases renal CaSR expression (54), the decrease in the receptor's expression with impaired renal function could result, in part, from the associated reduction in circulating 1,25(OH)zD ~ levels (2). The detailed mechanisms underlying these changes in the expression of the CaSR gene, however, including the relative contributions of changes in gene transcription and posttranscriptional mechanisms, require additional investigation.

ROLES OF THE CALCIUM-SENSING R E C E P T O R IN TISSUES M A I N T A I N I N G Ca2o+ HOMEOSTASIS Parathyroid The parathyroid glands of humans (60), rats (61), mice (62), rabbits (17), and chickens (18) express a b u n d a n t CaSR mRNA and protein. Several lines of evidence support the CaSR's role as the key mediator of the inhibitory action of elevated C a 2+ o on PTH secretion, although the principal intracellular signaling mechanisms through which it exerts this action remain uncertain [for review, see (63)]. As described above, the reduction in CaSR expression in cultured parathyroid cells is associated with loss of inhibition of PTH secretion by high Ca o2+ (41). Furthermore, humans with familial hypocalciuric hypercalcemia (FHH), who are heterozygous for naturally occurring, inactivating mutations of the CaSR gene (59), or mice heterozygous for targeted disruption of this gene (62) show modest right-shifts in their relationships between C a 2+ o and inhibition of PTH secretion, indicative of "resistance" to Ca2o+. Humans and mice homozygous for such defects (59,62), in turn, show m u c h more severe i m p a i r m e n t of high Ca 2+. o -induced suppression of PTH release, docu m e n t i n g that this parathyroid "C a 2+ o resistance" is inversely related to the n u m b e r of normally functioning CaSR alleles. Thus mice with "knockout" of the CaSR gene as well as the naturally occurring CaSR knockout in humans prove the central role of the CaSR 2+ in Ca o -regulated PTH secretion. Another feature of parathyroid function that is likely CaSR regulated is PTH gene expression. Garrett et al. (64) showed in preliminary studies that the calcimimetic CaSR activator, NPS R-568, which activates the receptor by an allosteric mechanism involving an increase in the receptor's apparent affinity for Ca2o+, decreases the level of PTH mRNA in bovine parathyroid cells. Finally, the CaSR appears to inhibit parathyroid cellular proliferation tonically, because humans homozygous for inactivating CaSR mutations (59) or mice homozygous for targeted disruption of the CaSR gene (62) show m a r k e d

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parathyroid cellular hyperplasia. Moreover, treatment of rats with experimentally induced renal impairment with NPS R-568 prevents the parathyroid hyperplasia that otherwise occurs in this setting (65), providing additional evidence that CaSR activation inhibits parathyroid cellular proliferation.

C Cells In contrast to PTH release, CT secretion is stimulated by elevating Ca2o+, similar to the more classic, positive relationship between Ca 2+ and exocytosis in most other hormone-secreting cells (1,2). This was one of 2+ several lines of indirect evidence that the Ca o -sensing mechanism in C cells might differ fundamentally from that in parathyroid cells. Recent studies, however, have shown that rat, human, and rabbit C cells express CaSR mRNA and protein (16,17,66). Moreover, cloning of the CaSR gene from a rat C cell tumor line revealed that it was identical to that expressed in rat kidney (16). It is currently thought, therefore, that the CaSR is the mediator of the stimulatory action of high Ca2o+ on CT secretion, although studies have not been p e r f o r m e d in humans or mice with knockout of the CaSR gene, for example, to establish this point definitively. Tamir and co-workers have provided evidence that the following sequence of events underlies CaSR-stimulated CT secretion (67). High C a 2+ o initially activates phosphatidylcholine-specific PLC, which provides a source of diacylglycerol for PKC-induced activation of an NCC. The latter allows cellular uptake of Na + and Ca 2÷, thereby producing cellular depolarization and resultant activation of voltage-gated, principally L-type, C a 2+ channels, elevating CaZi + a n d stimulating exocytosis of 5-hydroxytryptamine- and CT-containing secretory vesicles. Kidney In the adult rat kidney, the CaSR is expressed in almost all segments of the nephron, with the highest levels at the basolateral m e m b r a n e of the cortical thick ascending limb (CTAL) (68), which reabsorbs divalent minerals in a regulated m a n n e r (69,70). The CaSR is also expressed basolaterally in the distal convoluted tubule (DCT), where the tubular reabsorption of CaZ+~like that in C T A L ~ i s known to be stimulated by PTH. Additional sites of expression of the receptor include the base of the brush border of the proximal tubule (68), the basolateral m e m b r a n e of the cells of the medullary thick ascending limb (MTAL) (68), and the luminal surface of the inner medullary collecting duct (IMCD) (71). None of these sites is directly involved in renal Ca2o+ handling, although CaSRs in these n e p h r o n segments may regulate the handling of

other solutes a n d / o r water. For instance, in the proximal tubule, it is conceivable that the CaSR mediates the direct phosphaturic action of raising Ca2o+ (72), which might contribute to the reduction in serum phosphate levels in patients with hypoparathyroidism treated with vitamin D and calcium supplementation (2). As will be described in more detail below, the CaSR in CTAL, in addition to regulating reabsorption of Ca 2+ and Mg 2+, also modulates renal handling of Na +, K +, and C1- (73). Finally, as is discussed later, the CaSR in the IMCD is thought to mediate the well-known action of high GaZeo to inhibit vasopressin-stimulated water reabsorption (71,74), which contributes to the defective urinary concentrating capacity in hypercalcemic patients (5,59). The localization of the CaSR in the basolateral membrane of the CTAL suggests that it could mediate the previously demonstrated inhibitory action of elevated peritubular but not luminal Ca2o+ on Ca 2+ and Mg 2+ reabsorption in perfused tubules from this n e p h r o n segment (70). Figure 2 illustrates schematically how the CaSR likely acts at a cellular level to inhibit PTHstimulated reabsorption of divalent cations in the CTAL. As shown in detail in Fig. 2, it acts in a "lasixlike" m a n n e r to inhibit the overall activity of the Na/K/2C1 cotransporter that generates the lumen positive potential normally driving the passive paracellular reabsorption of about 50% of NaC1 and nearly all of the C a 2+ and Mg 2+ in this n e p h r o n segment (75). Interestingly, persons with F H H have a markedly reduced capacity to up-regulate their urinary excretion of C a 2+ in response to hypercalcemiameven after total parathyroidectomy (76). Therefore, there is a PTH-independent, overly avid reabsorption of C a 2+ in F H H that likely results, at least in part, from a reduced n u m b e r of normally functioning CaSRs in the CTAL, thereby rendering the tubule "resistant" to Ca2o+ and limiting the capacity of elevated levels of Ca2o+ to reduce tubular reabsorption in this n e p h r o n segment (59). Furthermore, in normal persons the hypercalciuric action of hypercalcemia results from at least two distinct CaSR-mediated actionsm(1) inhibition of PTH secretion and (2) direct suppression of tubular reabsorption of C a 2+ in the CTAL. It is not currently known if the CaSR modulates PTH-stimulated Ca '~+ reabsorption in the DCT.

Intestine The intestine is an important participant in the maintenance of Ca2o+ homeostasis owing to its capacity for regulated absorption of dietary Ca2o+ in response to active metabolites of vitamin D (1,2). The d u o d e n u m is the major site for 1,25-dihydroxyvitamin D3-dependent intestinal Ca 2+ absorption, involving a process of active transport that most likely includes the vitamin D-dependent CaZ+-binding protein, calbindin. In con-

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trast, the j e j u n u m and ileum not only absorb lesser amounts of C a 2+ but also s e c r e t e Ca 2+, which may enable chelation of fatty acids and bile acids, thereby forming insoluble "calcium soaps" that mitigate potential damage to colonic epithelial cells from soluble, unchelated fatty acids and bile acids. Even though the major function of the colon in fluid and electrolyte metabolism is to absorb water and Na +, it nonetheless absorbs significant amounts of Ca 2+ by both vitamin D-dependent and -independent routes, particularly in its proximal segments (77). The CaSR is expressed in all segments of rat intestine and at the highest levels on the basal aspect of the absorptive cells of the small intestinal villi and the crypt cells of the small intestine a n d colon, and in the enteric nervous system (78). Does the CaSR in any of these locations participate in systemic Ca 2+ o homeostasis? The CaSR in the enteric nervous system, which controls gastrointestinal secretomotor functions, could conceivably mediate the known actions of high and low Ca2o+ (e.g., in hyper- and hypocalcemic individuals) to reduce and increase GI motility, respectively. Such an action on intestinal motility, however, even if CaSR-mediated, has no known relevance to systemic C a 2+ o homeostasis. Available evidence, however, is consistent with a role for the CaSR in regulating intestinal C a 2+ absorption. Hypercalcemia inhibits the absorption of dietary Ca 2+ (79). Furthermore, direct measurement of the level of Ca2o+ in the interstitial fluid beneath the small intestinal absorptive epithelial cells has revealed that Ca2o+ increases by nearly two fold when luminal C a 2+ o is elevated to the levels known to be reached following • . intake of C a 2 + -containing foods (80). This level of

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FIG. 2 Possible mechanisms by which the CaSR controls intracellular second messengers and ionic transport in CTAL. Hormones elevating cAMP (e.g., PTH) enhance paracellular Ca 2÷ and Mg 2÷ reabsorption by stimulating the NaK2CI cotransporter and an apical K ÷ channel and by increasing Vt. The CaSR, likewise on the basolateral membrane, stimulates PLA 2 (2), increasing free arachidonic acid, which is metabolized by the P450 pathway to an inhibitor of the apical K ÷ channel (4) and, perhaps, the contransporter (3). Both actions decrease overall cotransporter activity, reduce Vt, and, therefore, diminish paracellular divalent cation transport. The CaSR also inhibits adenylate cyclase (1) and, therefore, hormone-stimulated divalent cation reabsorption. Reprinted from B o n e , Vol. 20; Brown EM, Hebert SC. Calcium-receptor regulated parathyroid and renal function; pp. 303-309. Copyright 1997, with permission from Elsevier Science.

Ca2o+ would be more than sufficient to activate CaSRs resident on the basal aspect of the small intestinal absorptive cells. It is possible, therefore, that a homeostatically relevant, negative feedback control of intestinal C a 2+ absorption takes place via local increases in C a 2+ o occurring as a result of the absorptive process per se or increases in the systemic level of Ca2o+. It is not currently known whether the CaSR regulates the secretion of C a 2+ o and other solutes by the small intestinal a n d / o r colonic crypts, although hypercalcemia has been found to stimulate intestinal calcium secretion in some studies (79):

Bone and Cartilage The levels of C a 2+ o within the bony microenvironm e n t are likely to vary substantially owing to the regulated turnover of the skeleton through osteoclastic resorption of small portions of bone followed by their replacement by bone-forming osteoblasts~a process totally replacing the h u m a n skeleton about every 10 years (2). Indeed, the levels o f Ca2o+ beneath an actively resorbing osteoclast can be as high as 8-40 m M (81). Furthermore, Ca2o+ exerts a variety of actions on the functions of bone cells in vitro that may serve physiologically useful functions, although it has not yet been proved that these same actions take place in vivo. For instance, high Ca2+o enhances osteoblastic functions that could promote their recruitment to sites of future bone formation, such as chemotaxis and proliferation, a n d / o r p r o m o t e their differentiation to mature osteoblasts [for review, see (82,83)]. In addition, high Ca2o+ inhibits both the formation (84) and activity (85) of osteoclasts in vitro. Thus C a 2+ o exerts effects on

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osteoblasts and osteoclasts a n d / o r their precursors that are homeostatically appropriate. For instance, increasing Ca2+owould promote net movement of calcium into the skeleton by enhancing bone formation and inhibiting its breakdown in the short and longer term. Moreover, locally high levels of Ca o2+generated by osteoclasts at sites of bone resorption could potentially contribute to the "coupling" of bone resorption to the subsequent replacement of the missing bone by osteoblasts, by amplifying and recruiting the resident pool of preosteoblasts to such sites and promoting their differentiation to mature osteoblasts (82,83). As described below, however, the molecular nature of the 2+ Ca o -sensing mechanisms in bone cells remains uncertain, although the CaSR is expressed in at least some bone cells, and therefore, could potentially participate in this process. A substantial body of indirect evidence accumulated prior to and around the time of the cloning of the 2+ CaSR suggested that the Ca o -sensing mechanisms in osteoblasts and osteoclasts differed in their pharmacologic and certain other properties from those of the CaSR [for reviews, see (82,85)]. In addition, some investigators have failed to detect the expression of CaSRs in osteoblast- (86) and osteoclast-like cells (87). Other studies, however, have provided strong evidence that the CaSR is expressed by a variety of cells that originate from the bone and bone marrow, including various hematopoietic precursors (88), at least some osteoblast-like and osteoclast-like cell models in vitro, and cells of both lineages in situ within sections of bone [for review, see (83)]. The ST-2 stromal cell line (89), which is derived from the same mesenchymal stem cells that serve as progenitors for osteoblasts, expresses CaSR mRNA and protein, as do several osteoblast-like cell lines, including SaOS-2, MC-3T3-E1, UMR-106, and MG-63 cells (90-92). Moreover, Chang et al. have shown expression of both CaSR mRNA and protein in most osteoblasts in sections of murine, rat, and bovine bone (92). With regard to cells of the osteoclast lineage, most h u m a n peripheral blood monocytes, which are known to contain osteoclast precursors, express a b u n d a n t CaSR mRNA and protein (93). Preosteoclastlike cells formed in vitro also expresses the CaSR (84), as do osteoclasts isolated from rabbit bone (94). In murine, rat, and bovine bone sections, however, only a minority of multinucleated osteoclasts expressed CaSR mRNA and protein (92). Further studies are needed, therefore, to clarify whether only osteoclast precursors and not mature osteoclasts express the CaSR. Moreover, additional work is n e e d e d in which the activity of the CaSR in bone cells a n d / o r their precursors is blocked using genetic a n d / o r pharmacologic approaches in order to clarify whether the CaSR that has been found to be expressed in at least

some cells of both lineages is the actual mediator of some or even all the effects of high Ca2o+ on these cells. One study, which failed to detect the CaSR in transformed osteoblast-like cells derived from either wildtype mice or those with knockout of the CaSR, found that these cells still exhibited responses to Ca2o+ and 2+ A1~+, suggesting the presence of another Ca o -sensing receptormas has been postulated by the same workers in earlier studies (95). Although the cartilage-forming chondrocytes do not directly participate in systemic C a 2+ o homeostasis, they play a key role in the growth of the skeleton by providing a cartilaginous model of the future skeleton that is gradually replaced by bone. Moreover, the cartilaginous growth plate represents a site where longitudinal growth takes place until the skeleton is fully mature at the end of puberty. The availability of C a 2+ is known to be important to ensure proper growth and differentiation of cartilage cells and resultant skeletal growth in vivo (96,97). In addition, altering Ca2o+ modulates the differentiation a n d / o r other functions of cells of the cartilage lineage (98,99). The latter arise from the same mesenchymal stem cell, giving rise to osteoblasts, adipocytes, smooth muscle cells, and fibroblasts (100,101). It is of interest, therefore, that the rat cartilage cell line, RCJ3.1C5.18, expresses CaSR mRNA and protein (102). Furthermore, various types of cartilage cells within intact bone also express CaSR mRNA and protein, including the hypertrophic chondrocytes within the growth plate that participate importantly in long bone growth (92). Therefore, the CaSR could potentially mediate some or all of the previously demonstrated direct actions of Ca2o+ on chondrocytes and cartilage growth. Indeed, raising the level of Ca2o+ exerts several direct actions on RCJ3.1C5.18 cells, including dose dependently decreasing the levels of the mRNAs encoding a major proteoglycan in cartilage, aggrecan, as well as the ~x1 chains of types II and X collagen and alkaline phosphatase (102). Moreover, treating the cells for 48-72 hours with a CaSR antisense oligonucleotide lowered the level of the CaSR protein significantly and p r o m o t e d increased expression of aggrecan mRNA (102), consistent with a mediatory role of the CaSR in the regulation of this gene. These results indicate, therefore, that (1) Ca2o+ regulates the expression of several biologically important genes in this chondrocytic cell line, (2) cartilage cells express the CaSR, and (3) the receptor mediates some or all of these actions of Ca2o+ in the RCJ3.1C5.18 cartilage cell model. Thus the CaSR potentially not only regulates bone turnover a n d / o r the coupling of bone resorption to bone formation through effects on bone cells a n d / o r their precursors, but may also regulate skeletal growth through its actions on chondrocytes.

RECEPTORS/SIGNALING FOR Ca2o+ /

THE CALCIUM-SENSING RECEPTOR AND T H E I N T E G R A T I O N OF CALCIUM A N D WATER M E T A B O L I S M Hypercalcemic patients not infrequently have abnormally decreased urinary concentrating capacity and, occasionally, frank nephrogenic diabetes insipidus (103,104). The presence of the CaSR in several n e p h r o n segments participating in the urinary concentrating mechanism (68,71) has provided novel insights into the likely mechanism(s) underlying the long-recognized but poorly understood inhibitory effects of high C a 2+ o on this parameter of renal function. As noted above, high Ca2o+ levels, probably by activating CaSRs present on their apical membranes, reversibly inhibit vasopressin-elicited water flow by about 35-40% in the IMCD (71). The CaSR has been shown to be present within the same apical endosomes containing the vasopressin-regulated water channel, aquaporin-2 (71). This observation suggests that the CaSR could potentially reduce vasopressin-stimulated water flow in the IMCD by either stimulating the endocytosis or inhibiting the exocytosis of these endosomes out of or into the apical plasma m e m b r a n e , respectively (71). Moreover, inducing chronic hypercalcemia in rats by treatment with vitamin D decreases aquaporin-2 expression (74), which would further diminish vasopressinstimulated water flow in the terminal collecting duct. In addition to the mechanisms just described, high Ca o -elicited, CaSR-mediated inhibition of NaC1 reabsorption in the MTAL (105,106), by diminishing the magnitude of the medullary c o u n t e r c u r r e n t gradient, would be expected to reduce further the maximal urinary concentrating power of hypercalcemic persons (Fig. 3). What is the evidence that these various actions of high Ca2o+ are mediated via the CaSR? Interestingly, individuals with inactivating mutations of the CaSR concentrate their urine normally despite their hypercalcemia (107), probably because they are "resistant" to the usual inhibitory actions of C a 2+ o on the urinary concentrating mechanism. In contrast to persons with F H H or mice with targeted disruption of the CaSR gene, families have been defined in which activating mutations of the CaSR produce a form of autosomal d o m i n a n t hypocalcemia (59). Because parathyroid and kidney are "overresponsive" to the usual actions of C a 2+ o in such families, the Ca2+o homeostatic system is "reset" to maintain stable hypocalcemia in association with relative hypercalciuria (e.g., affected persons have hypercalciuric hypocalcemia). In contrast to persons with inactivating CaSR mutations, those with activating mutations can develop symptoms of diminished urinary concentrating capacity even at normal levels of C a 2+ o when treated with vitamin D and calcium supplementation, 2+

•

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probably because their renal CaSRs involved in urinary concentration are overly sensitive to Ca2o+ (108). Is there any physiologic relevance to the defective renal handling of water in hypercalcemic patients? We have previously postulated that it affords a mechanism for integrating the renal handling of divalent cations, particularly C a 2+, and water, thereby allowing appropriate "trade-offs" in how these aspects of renal function are regulated u n d e r specific physiologic conditions (73). For instance, when a systemic C a 2+ load must be disposed of, a CaSR-mediated increase in urinary C a 2+ excretion ensues owing to reduced tubular reabsorption of C a 2+ in the CTAL. The resultant increase in the luminal levels of Ca2o+ in the IMCD, especially in a dehydrated person, might predispose to forming Ca2+-containing renal stones if it were not for the associated high Ca 2+. o -induced, presumably CaSRmediated, inhibition of maximal urinary concentration. In addition, a b u n d a n t CaSRs are expressed in the subfornical organ (SFO) ( 1 0 9 ) m a n important thirst center (110)mwhich may provide an additional layer of integration of C a 2+ o and water homeostasis. That is, a Ca2o+ -induced, CaSR-mediated increase in drinking owing to activation of CaSRs in the SFO could prevent dehydration that might otherwise result from loss of free water in the kidney because of concomitant inhibition of the urinary concentrating mechanism (Fig. 3). Finally, prior studies have docum e n t e d the existence of a specific "calcium appetite" in rats (111) that could provide a mechanism for a physiologically appropriate modulation of the intake of calcium-containing food during hypo- and hypercalcemia. Some reduction in the intake of Ca2o+containing foods would presumably also result from activation of CaSRs in the area postrema of the b r a i n m a "nausea center" (109)mowing to the resultant anorexia/nausea. We postulate, therefore, that multiple layers of CaSR-mediated integration and coordination participate in the regulation of water and calcium metabolism, serving to optimize the capacity of terrestrial organisms to adapt to their intermittent access to dietary calcium and water (73). The modulation of vasopressin-mediated water flow in the IMCD can be thought of as an example of"local" C a 2+ o homeostasis, whereby Ca2o+ within a restricted microenvironment outside of the general ECF is only allowed to rise to a certain maximal level (5). Of interest, whereas alterations in Ca2o+ made by the system governing systemic C a 2+ o homeostasis are usually accomplished principally by adjusting the m o v e m e n t of C a 2+ into or out of the ECF (e.g., by intestine, kidney, or bone) (1), the adjustment of Ca2o+ in the IMCD takes place via alterations in the movement of water. It seems possible, however, that one function of the increased thirst in hypercalcemic patients,

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$

Ca2+-receptor activation /

"~ ~'NaCI re:bT~rpti°n ~

~ H20 Reabsorption by Collectingduct

~, Lumen positive voltage

~Countercurrent multiplication

~, Ca2+and Mg2* reabsorption

Urinary concentrating ability

Increased C a 2÷ and Mg 2~ excretion in a more dilute urine

/

)

DecreasedCa2÷ • Reabsorption, Increased Ca2+Excretion J • •

Decreased Urinary Concentration

icreased Urinal Ca2÷Excretion Without Risk of Stones Decreased Gut Motility, Increased H20 Absorption FIG. 3 Possible mechanisms that interrelate systemic Ca2o+ and water homeostasis (see text for further details). (a) Renal mechanisms through which the CaSR decreases maximal urinary concentrating ability. Reproduced with permission from Brown EM, Hebert SC. Novel insights into the physiology and pathophysiology of Ca 2+ homeostasis from the cloning of an extracellular Ca2+-sensing receptor. Regulatory Peptide Letter 1997;VI1:43-47. (b) Activating CaSRs in the SFO could enhance water intake, and mitigate loss of free water that otherwise would result from diminished urinary concentration. Reproduced with permission from Brown EM, Harris HW, Jr, Vassilev P, Hebert SC. The Biology of the extracellular Ca 2+sensing receptor. In: Bilezikian JP, Raiscz LG, Rodan GA, eds. Principles of bone biology, San Diego:Academic Press.

in a d d i t i o n to p r o v i d i n g m o r e free water to r e d u c e the level of C a 2+ o in the IMCD, is to dilute C a 2+ o in t h e ECF as well. T h e r e f o r e , p e r h a p s the i n t e g r a t i o n

o f calcium a n d water m e t a b o l i s m is n o t limited to regu l a t i n g the level of Ca2o+ within the u r i n e b u t also that in b l o o d .

RECEPTORS/SIGNALINGFORC a 2+ o / OTHER CALCIUM-SENSING RECEPTOR A G O N I S T S A N D M O D U L A T O R S - - T H E CaSR AS A N I N T E G R A T O R O F P H Y S I O L O G I C S I G N A L S A N D AS A " N U T R I E N T R E C E P T O R " Agonists and Activators o f the CaSR O t h e r T h a n C a 2+ o A variety of divalent cations (Sr2o+), trivalent cations (e.g., Gd~o+), and even organic polycations [i.e., neomycin and spermine (112) ] are effective agonists of the CaSR, probably interacting with the receptor's ECD [for review, see (5)]. Only a few of these, however, are likely to be present within biologic fluids at concentrations that would activate the receptor (112). In addition to Ca2o+, Mg2o+ and spermine are two such polycationic agonists of the CaSR. It is likely that in certain microenvironments, such as in the gastrointestinal tract and CNS, the levels of spermine are sufficiently high to activate the CaSR even at levels of C a 2+ o that would not by themselves do so (5,112). Indeed, all polycationic CaSR agonists potentiate one anothers' actions on the receptor, so that only small increases in any given agonist (i.e., spermine) may be n e e d e d to activate the CaSR in the presence of a sufficient level of another agonist that is present in the m i c r o e n v i r o n m e n t (e.g., Ca2o+) (5). Some support for the role of the CaSR in "setting" Mg2o+ comes from the observation that persons with inactivating mutations of the CaSR tend to have mild 2+ whereas those with activating mutaincreases in M go, tions can have reductions in Mg2o+(59). The potency of M go2+ is about twofold less than that of Ca 2+ o on a molar basis in activating the CaSR' (11,17). Because circulating levels of Mg2o+ are, if anything, lower than those for Ca2o+ (2) , one might wonder how M go2+ could regulate its own homeostasis via changes in PTH secretion. It appears m o r e likely that M go2+ acts via the CaSR in the CTAL to regulate its own level in the ECF, because Mg2o+ is reabsorbed to a lesser extent than other solutes in proximal n e p h r o n segments, resulting in 1.6- to 1.8-fold increases in Mg2o+ in the tubular fluid of the CTAL (70). The latter levels should be sufficiently high to inhibit Mg2o+ reabsorption in this n e p h r o n segment, because raising not only Ca2o+ but also Mg2o+ inhibits the reabsorption of both divalent cations in perfused CTAL (70). Another ionic factor that modulates the effects of Ca2o+ and other polycationic agonists on the CaSR is a change in ionic strength per se (e.g., via alterations in the concentration of NaC1) (113). Increases in ionic strength reduce, and decreases in ionic strength enhance, the sensitivity of the CaSR to activation by C a 2+ o . The impact of changes in ionic strength on the CaSR may be particularly relevant in specific microenvironments, such as in the urinary or gastroin-

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testinal tracts, where this p a r a m e t e r can vary over a wide range (113).

P o s s i b l e R o l e o f the CaSR as a "Nutrient-Sensing" Receptor Calcimimetics are prototypical "modulators" of the CaSR that activate the receptor only in the presence of C a 2+ o, as opposed to the polycationic agonists of the CaSR (e.g., Gdo3+),which can activate it even in the total absence of Ca O2+ (114) ° Recent studies have identified another class of endogenous modulators of the CaSRmnamely, various amino acids (30). Although individual amino acids only activate the receptor in the presence of ~>1 m M Ca2o+ and are of relatively low potency (e.g., acting in the range of 0.1-1 m M o r higher), a mixture of amino acids emulating that present in h u m a n serum u n d e r fasting conditions substantially shifts the receptor's sensitivity to C a o2+. Moreover, changes in the levels of the amino acid mixture above and below this fasting level have readily detectable effects on the function of the CaSR (30). Although the implications of the direct effects of amino acids on the CaSR are far from clear, they may help to explain several long-standing observations linking protein and C a 2+ o metabolism and suggest future avenues for research into the possible role of the CaSR as a "nutrient-sensing" receptor. For instance, highprotein diets substantially increase urinary calcium excretion (115). Although this effect has traditionally been ascribed to buffering of the acidic products of protein metabolism by bone and direct calciuric actions of the acid load (116), perhaps direct activation of CaRs in the CTAL contributes as well. Conversely, a reduction in dietary protein has been shown to increase serum PTH levels up to twofold in normal women (117). Could it be that the latter effect results from decreased inhibition of PTH secretion owing to reduced circulating levels of amino acids and that the reduced intake of dietary prot e i n that is a standard part of the therapy of patients with chronic renal insufficiency (2) contributes to the secondary hyperparathyroidism in the latter setting? Viewing the CaSR as not only a "homeostatic" receptor for C a 2+ o but as a nutrient receptor, sensing not only C a 2+ o and M go2+but also amino acids, may enable the formulation and testing of novel hypotheses directed at understanding, for instance, the link between the needs of the organism for both protein and mineral ions during growth. Skeletal growth in childhood involves laying down both a protein and a mineral phase in bone as well as growth of soft tissuesmall of which contain varying mixtures of mineral ions and protein. Indeed, in the GI tract, the presence of an amino acid receptor regulating secretion of gastrin, gastric acid, and cholecystokinin has been postulated, and the pharmacology for

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the effects of various a m i n o acids on these parameters is remarkably similar to that for the effects of the same amino acids on the CaSR (118-120). CaSRs in the gastrointestinal system could represent a particularly suitable target for the sensing of the intake of protein and mineral ions, which are, of course, generally ingested together (i.e., in milk). F u r t h e r studies are needed, therefore, to d e t e r m i n e w h e t h e r the CaSR, in fact, represents the putative a m i n o acid receptor, and w h e t h e r recognition by this receptor of amino acids in the context of C a 2+ o and M go2+ within the gastrointestinal tract and elsewhere represents the molecular basis for a physiologically relevant link between protein and mineral ion metabolism.

POSSIBLE ADDITIONAL

Ca2o+ S E N S O R S

As n o t e d above and described in detail elsewhere (82,85), there may be C a 2+ o sensors in addition to the CaSR in osteoblasts and osteoclasts. F u r t h e r m o r e , additional work has revealed that some mGluRs can sense Ca2o+ in addition to r e s p o n d i n g to glutamate as their principal physiologic agonist, although the physiologic relevance of this Ca2o+ sensing is not yet clear. Kubo et al. (121) d e m o n s t r a t e d that mGluRs 1, 3, and 5 sense Ca2o+ over a range of a b o u t 0.1 to 10 mM, whereas mGluR2 is considerably less responsive to changes in C a 2+ o . M1 three of the mGluRs that sense Ca2o+ have identical serines and threonines, respectively, at amino acid positions equivalent to residues 165 and 188 in m G l u R l a (32). These two residues play key roles in the binding of glutamate to the ECDs of the mGluRs (26). In contrast, t h o u g h mGluRs la, 3, and 5 have a serine at a position equivalent to residue 166 in m G l u R l a , mGluR2 has an aspartate in this position (121). Changing this serine to an aspartate in mGluRs la, 3, and 5 considerably reduces their capacities to sense Ca2o+, but replacing the aspartate in mGluR2 with a ser2+ ine increases its a p p a r e n t affinity for Ca o i n t o a level similar to those of mGluRs 1, 2, and 5 (121). Therefore, the serines at a m i n o acid position 166 in m G l u R l a and the equivalent positions in mGluRs 3 and 5 apparently play key roles in the capacities of these receptors to sense Ca2o+. No d o u b t f u r t h e r studies will reveal the capacity of additional cell surface proteins to sense Ca2o+ and, perhaps, o t h e r ions, probably not only GPCRs but also o t h e r integral m e m b r a n e proteins capable of m o d u l a t i n g cellular function (e.g., ion channels).

SUMMARY In conclusion, the discovery of the CaSR has provided a molecular m e c h a n i s m mediating many of the

known actions of Ca2o+ on the functions of cells and tissues involved in systemic Ca 2+ o homeostasis. Much remains to be learned, however, a b o u t the functions of the CaSR in these tissues as well as in n u m e r o u s other CaSR-expressing cell types that are not directly involved in systemic mineral ion homeostasis. In the latter, the receptor probably serves diverse roles, making it a versatile regulator of a wide variety of cellular functions such that it could serve as an i m p o r t a n t therapeutic target. F u r t h e r m o r e , the capacity of the CaSR to integrate and coordinate several types of signals may enable it to serve as a central homeostatic regulator, not only of mineral ion homeostasis but also of processes related to water, protein, and n u t r i e n t metabolism m o r e generally.

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mining ligand specificity for Ca 2+ receptors. Mol Pharmacol

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32. Brauner-Osborne H, Jensen AA, Sheppard PO, O'Hara P, Krogsgaard-Larsen E The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain. JBiol Chem 1999;274:18382-18386. 33. Bai M, Trivedi S, Brown EM. Dimerization of the extracellular calcium-sensing receptor (CAR) on the cell surface of CaRtransfected HEK293 cells. J Biol Chem 1998;273:23605-23610. 34. Ward DT, Brown EM, Harris HW. Disulfide bonds in the extracellular calcium-polyvalent cation-sensing receptor correlate with dimer formation and its response to divalent cations in vitro. J Biol Chem 1998;273:14476-14483. 35. Ray K, Hauschild BC, Steinbach PJ, Goldsmith PK, Hauache O, Spiegel AM. Identification of the cysteine residues in the aminoterminal extracellular domain of the human Ca ~2+) receptor critical for dimerization. Implications for function of monomeric Ca~2+~ receptor. J Biol Chem 1999;274:27642-27650. 36. Bai M, Trivedi S, Kifor O, Quinn sJ, Brown EM. Intermolecular interactions between dimeric calcium-sensing receptor monomers are important for its normal function. Proc Natl Acad Sci USA 1999;96:2834-2839. 37. Bai M, Quinn S, Trivedi S, Kifor O, Pearce SHS, Pollak MR, Krapcho K, Hebert SC, Brown EM. Expression and characterization of inactivating and activating mutations in the human 2+ Ca o -sensing receptor. J Biol Chem 1996;271:19537-19545. 38. Ray K, Clapp P, Goldsmith PK, Spiegel AM. Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction. JBiol Chem 1998;273:34558-34567. 39. Bai M, Trivedi S, Lane CR, Yang Y, Quinn sJ, Brown EM. Protein kinase C phosphorylation of threonine at position 888 in Ca 2+sensing receptor (CAR) inhibits coupling to Ca 2+ store release. J Biol Chem 1998;273:21267-21275. 40. Kifor O, Diaz R, Butters R, Brown EM. The Ca2+-sensing receptor (CAR) activates phospholipases C, A 2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells. JBone Miner Res 1997;12:715-725. 41. Mithal A, Kifor O, Kifor I, Vassilev P, Butters R, Krapcho K, Simin R, Fuller F, Hebert SC, Brown EM. The reduced responsiveness of cultured bovine parathyroid cells to extracellular Ca 2+ is associated with marked reduction in the expression of extracellular Ca2+-sensing receptor messenger ribonucleic acid and protein. Endocrinology 1995;136:3087-3092. 42. Brown AJ, Zhong M, Ritter C, Brown EM, Slatopolsky E. Loss of calcium responsiveness in cultured bovine parathyroid cells is associated with decreased calcium receptor expression. Biochem Biophys Res Commun 1995;212:861-867. 43. Hawkins D, Enyedi P, Brown EM. The effects of high extracellular Ca 2+ and Mg 2+ concentrations on the levels of inositol 1,3,4,5tetrakisphosphate in bovine parathyroid cells. Endocrinology 1989;124:838-844. 44. Emanuel RL, Adler GK, Kifor O, Quinn sJ, Fuller F, Krapcho K, Brown EM. Calcium-sensing receptor expression and regulation by extracellular calcium in the AtT-20 pituitary cell line. Mol Endocrino11996;10:555-565. 45. Ye C, Rogers K, Bai M, Quinn sJ, Brown EM, Vassilev PM. Agonists of the Ca~2+~-sensing receptor (CAR) activate nonselective cation channels in HEK293 cells stably transfected with the human CaR. Biochem Biophys Res Commun 1996;226:572-579. 46. Brown EM, Chen CJ, Kifor O, Leboff MS, E1-Hajj G, Fajtova V, Rubin LT. Ca~Z+~-sensingsecond messengers, and the control of parathyroid hormone secretion. Cell Calcium 1990;11:333-337. 47. Chen C, Barnett J, Congo D, Brown E. Divalent cations suppress 3',5'-adenosine monophosphate accumulation by stimulating a

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74. Sands JM, Flores FX, Kato A, Baum MA, Brown EM, Ward DT, Hebert SC, Harris HW. Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats. Am J Physiol 1998;274:F978-F985. 75. Hebert SC, Brown EM, Harris HW. Role of the Ca(Z+~-sensing receptor in divalent mineral ion homeostasis. J Exp Biol 1997;200:295-302. 76. Attie ME Gill J, Jr Stock JL, Spiegel AM, Downs RW, Jr Leone MA, Marx SJ. Urinary calcium excretion in familial hypocalciuric hypercalcemia. Persistence of relative hypocalciuria after induction of hypoparathyroidism. J Clin Invest 1983;72:667-676. 77. Favus MJ. Intestinal absorption of calcium, magnesium and phosphorus. In: Coe FL, Favus, MJ, eds. Disorders of bone and mineral metabolism. New York: Raven, 1992:57-81. 78. Chattopadhyay N, Cheng I, Rogers K, Riccardi D, Hall A, Diaz R, Hebert SC, Soybel DI, Brown EM. Identification and localization of extracellular Ca(2+)-sensing receptor in rat intestine. Am J Physiol 1998;274:G 122-G 130. 79. Krishnamra N, Angkanaporn K, Deenoi T. Comparison of calcium absorptive and secretory capacities of segments of intact or functionally resected intestine during normo-, hypo-, and hypercalcemia. Can J Physiol Pharmaco11994;72: 764-770. 80. Mupanomunda MM, Ishioka N, Bukoski RD. Interstitial Ca2+ undergoes dynamic changes sufficient to stimulate nerve-dependent Ca2+-induced relaxation. AmJPhysiol 1999;276:H1035-H1042.

RECEPTORS/SIGNALING FOR C a 2+ o / 81. Silver IA, Murrils RJ, Etherington DJ. Microlectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp Cell Res 1988;175:266-276. 82. Quarles LD. Cation-sensing receptors in bone: A novel paradigm for regulating bone remodeling? J Bone Miner Res 1997;12:1971-1974. 83. Yamaguchi T, Chattopadhyay N, Brown EM. G Protein-coupled extracellular Ca 2+ (Ca2+ o )-sensing receptor (CAR)" Roles in cell signaling and control of diverse cellular functions. Adv Pharmacol 1999;47:209-253. 84. Kanatani M, Sugimoto T, Kanzawa M, Yano S, Chihara I~ High Extracellular calcium inhibits osteoclast-like cell formation by directly acting on the calcium-sensing receptor existing in osteoclast precursor cells. Biochem Biophys Res Commun 1999;261:144-148. 85. Zaidi M, Adebanjo OA, Moonga BS, Sun L, Huang CL. Emerging insights into the role of calcium ions in osteoclast regulation. J Bone Miner Res 1999;14:669-674. 86. Pi M, Hinson TK, Quarles L. Failure to detect the extracellular calcium-sensing receptor (CasR) in human osteoblast cell lines. JBone Miner Res 1999;14:1310-1319. 87. Seuwen K, Boddeke HG, Migliaccio S, Perez M, Taranta A, Teti A. A novel calcium sensor stimulating inositol phosphate formation and [Ca2+]i signaling expressed by GCT23 osteoclastlike cells. Proc Assoc Am Physicians 1999;111:70-81. 88. House MG, Kohlmeier L, Chattopadhyay N, Kifor O, Yamaguchi T, Leboff MS, Glowacki J, Brown EM. Expression of an extracellular calcium-sensing receptor in human and mouse bone marrow cells. J Bone Miner Res 1997;12:1959-1970. 89. Yamaguchi T, Chattopadhyay N, Kifor O, Brown EM. Extracellular calcium (Ca~o~)-sensing 2+ receptor in a murine bone marrow-derived stromal cell line (ST2): Potential mediator of the actions of Ca~o 2+~ on the function of ST2 cells. Endocrinology 1998; 139:3561-3568. 90. Yamaguchi T, Chattopadhyay N, Kifor O, Butters RR, Jr, Sugimoto T, Brown EM. Mouse osteoblastic cell line (MC3T3El) expresses extracellular calcium (Ca2o+)-sensingreceptor and its agonists stimulate chemotaxis and prolif-eration of MC3T3E1 cells. JBone Miner Res 1998;13:1530-1538. 91. Yamaguchi T, Kifor O, Chattopadhyay N, Brown EM. Expression of extracellular calcium (Ca2+o)-Sensing receptor in the clonal osteoblast-like cell lines, UMR-106 and SAOS-2. Biochem Biophys Res Commun 1998;243:753-757. 92. Chang W, Tu C, Chen T-H, Komuves L, Oda Y, Pratt S, Miller S, Shoback D. Expression and signal transduction of calciumsensing receptors in cartilage and bone. Endocrinology 1999;140:5883-5893. 93. Yamaguchi T, Olozak I, Chattopadhyay N, Butters RR, Kifor O, Scadden DT, Brown EM. Expression of extracellular calcium (Ca 2+ o )-sensing receptor in human peripheral blood monocytes. Biochem Biophys Res Commun 1998;246:501-506. 94. Kameda T, Mano H, Yamada Y, Takai H, Amizuka N, Kobori M, Izumi N, Kawashima H, Ozawa H, Ikeda K, Kameda A, Hakeda Y, Kumegawa M. Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells. Biochem Biophys Res Commun 1998;245:419-422. 95. Quarles DL, Hartle II JE, Siddhanti SR, Guo R, Hinson TK. A distinct cation-sensing mechanism in MC3T3-E1 osteoblasts functionally related to the calcium receptor. J Bone Miner Res 1997;12:393-402. 96. Jacenko O, Tuan RS. Chondrogenic potential of chick embryonic calvaria: I. Low calcium permits cartilage differentiation. Dev Dyn 1995;202:13-26. 97. Reginato AM, Tuan RS, Ono T, Jimenez SA, Jacenko O. Effects of calcium deficiency on chondrocyte hypertrophy and type X

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stimulated secretion of cholecystokinin is calcium dependent. Am J Physio11995;268:G90-G94. 120. Taylor IL, Byrne WJ, Christie DL, Ament ME, Walsh JH. Effect of individual L-amino acids on gastric acid secretion and serum gastrin and pancreatic polypeptide release in humans. Gastroenterology 1982;83:273-278. 121. Kubo Y, Miyashita T, Murata Y. Structural basis for a CaZ+-sensing function of the metabotropic glutamate receptors. Science 1998;279:1722-1725.

CI-IAPI:F R9 Immunoassays for P T H and PTHrP Clinical Applications

L.J. DEFTOS Department of Medicine, University of California, San Diego, and San Diego VA Medical Cent~ LaJolla, California 92161

INTRODUCTION

ficity for the target peptides a n d / o r polypeptides. Either the antibodies or the peptide standard, depending on assay format, are labeled so that they can be quantified by one of a variety of detection systems. The most widely available detection systems use radioisotopes, colorimetry, or chemiluminescence. Most immunoassays for PTH and PTHrP can detect n a n o g r a m (ng) to picogram (pg) concentrations of the hormone. In the case of PTH, the circulating concentrations can be readily measured in both health and disease. But normal ranges for serum PTHrP are not yet well established, and the question of the circulation of PTHrP in normal individuals remains open. This chapter reviews the development of the wide variety of PTH immunoassays and the increasing n u m b e r of PTHrP assays that are currently available, commonly with Food and Drug Administration (FDA) approval, for clinical application in the United States. T h o u g h the detailed background in relevant physiology for a full understanding of PTH and PTHrP assays is provided in other chapters of this book, this chapter provides a synopsis of the biosynthesis, secretion, and metabolism of PTH and PTHrP that will focus on the rationale that has been used for assay development. This background synopsis is followed by a more detailed discussion of the clinical application of PTH immunoassays to specific disorders of calcium and skeletal metabolism. Developments in assay theory and practice are also discussed. The m e a s u r e m e n t of circulating levels of PTHrP has only approximated the accurate and precision available for PTH assays. However, the m e a s u r e m e n t of PTHrP is assuming increasing

This chapter discusses the development and clinical application of assays for circulating levels of parathyroid h o r m o n e (PTH) and parathyroid hormone-related protein (PTHrP). The accurate and precise measurement of circulating levels of PTH has revolutionized the clinical management of patients with calcium and skeletal disorders, and assays for PTHrP are beginning to have comparable clinical impact for the patient with hypercalcemia, especially hypercalcemia due to cancer. Sensitive and specific assays for PTH and PTHrP aid in the differential diagnosis of hypercalcemia, hypocalcemia, and a variety of other calcium and skeletal diseases. In addition to their usefulness in clinical management, PTH and PTHrP serum assays have helped to elucidate the pathophysiology of many disorders of calcium and skeletal metabolism. (In this chapter, the term "serum" will be generally used for convenience to connote measurements of circulating levels of PTH and PTHrP, because, in general, serum measurements are widely used and there are few substantial differences between serum and plasma measurements. However, in some instances there are differences, and when the distinction is important, it will be addressed.) The measurements of circulating serum or plasma concentrations of PTH and PTHrP are primarily based on immunoassay procedures that recognize different peptides of the linear sequence of the native molecules. Although bioassays are available for research purposes, they are not used clinically. These immunoassay procedures utilize one or two antibodies with defined speciThe Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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clinical importance. Thus, this chapter also reviews the expanding information on the application of PTHrP immunoassays to clinical diagnosis and management. In contrast to PTH, assays for PTHrP are in their early stages of development. Furthermore, they are complicated by the more complex biosynthesis, secretion, and metabolism of this polypeptide. New and improved research assay systems for PTH and PTHrP are continually developed and touted. However, it takes substantial clinical evaluation to establish the diagnostic value of an immunoassay procedure for clinical application and ultimate FDA approval. This chapter thus focuses on describing the general principles that underlie the development and application of immunoassays for PTH and PTHrP so that the clinician can chose the most appropriate assay.

PTH BIOSYNTHESIS, SECRETION, AND METABOLISM Overview PTH is an 84-amino acid polypeptide secreted by the parathyroid glands, primarily in the chief cells of the gland (1-9). Like other peptide hormones, PTH is originally synthesized as a larger precursor molecule; the PTH precursor, named preproparathyroid hormone, is 115 residues long (2). This intracellular species of PTH is processed, metabolized, and secreted primarily as the native 84-amino acid hormone, although some intracellular metabolism also occurs. On secretion, native PTH is metabolized to amino-terminal and carboxyterminal fragments by the liver, kidney, and other peripheral sites (6). The amino terminus of native PTH, approximately the first 27 to 34 amino acids, contains the classic biologic activity of the hormone. It binds to a PTH cell surface receptor that also recognizes PTHrP (5,6). This receptor had been named the P T H / P T H r P receptor, but is now known as the PTH1 receptor (Chapter 2). Other fragments of PTH are generally considered to be inactive. Measurement of biologically active PTH species would appear to have more clinical relevance than measurement of inert fragments of the hormone, but this is where theory and practice diverge (4,8). This divergence will be illuminated by considering some aspects of the biosynthesis, secretion, and metabolism of PTH that provide insights into the rationale for development and the application to clinical studies of the various PTH immunoassays that are available. Selected aspects of PTH biology are briefly reviewed throughout this chapter. More details about the cellular and molecular physiology of the h o r m o n e can be found elsewhere in this volume (see Chapters 13-17).

Biosynthesis of PTH The biosynthesis of PTH is controlled by its relatively simple gene located on the short arm of chromosome 11 at band 11p15 (3,6). The gene contains three exons separated by two introns, with the first exon conmining most of the 5' prime noncoding sequence, the second exon coding most of the prepro sequence, and the third exon coding the mature PTH sequence (2). Several factors can regulate PTH gene transcription or mRNA stability, with calcium and 1,25-dihydroxyvitamin D being the most important (3). Low ambient calcium stimulates gene transcription, and high ambient calcium suppresses gene transcription, but to a lesser extent. Exposure to the active vitamin D metabolite, 1,25-dihydroxyvitamin D, suppresses gene transcription (1,6). Preliminary studies of estrogen and ambient phosphorus and magnesium reveal that they can also regulate gene transcription (6). The regulation of PTH gene transcription is discussed in more detail in Chapter 2. The product of the h u m a n PTH gene is a 115-amino acid preproPTH containing the 84-residue native molecule and a 29-residue prepro moiety that undergoes several intracellular cleavage steps as it passes through cellular trafficking (1,6). As the signal sequence of the nascent peptide emerges from the ribosome, it is directed by a signal recognition particle to the endoplasmic reticulum (ER), where in the lumen a signal peptidase cleaves the 25-amino acid pre sequence, releasing the intermediate proPTH form (2). ProPTH then travels to and through the Golgi apparatus, where the six-amino acid pro sequence is cleaved by furin or a furinlike molecule, leaving the mature 1-84 native PTH. The mature molecule is then concentrated into secretory vesicles for secretion in response to ambient calcium and other regulators (2,6). In addition to the mature PTH molecule, there is evidence for the presence of other intracellular forms of the hormone. A m i n o - a n d carboxy-terminal peptides, especially the latter, are derived from intracellular processing a n d / o r metabolism of PTH, and they can be secreted as well (2,8). In fact, truncation of the carboxy terminus of PTH impairs its secretion (2). These observations about the intracellular journey of PTH have led to the assignment of a functional role for different PTH peptide regions (1,2), and as will be seen later, for the corresponding sequences in PTHrE The pre region directs the nascent molecule to the ER; the pro region is required for its introduction into a Golgi pathway and the carboxy-terminal region of the mature hormone is required for transportation through the secretory pathway that leads to secretory vesicles and, ultimately, to hormone secretion.

PTH AND PTHrP IMMUNOASSAYS /

Intracellular Metabolism o f P T H PTH is metabolized in the parathyroid gland to carboxy- and amino-terminal forms (Fig. 1). Most of the information regarding the intracellular metabolism of PTH comes from animal studies and in vitro studies of h u m a n hyperplastic and adenomatous parathyroid glands (2-8). It is notable that the intracellular and secreted carboxy-terminal forms of PTH are identical at the amino terminus to the carboxy-terminal fragments of the molecule generated by peripheral metabolism of PTH, and have the corresponding structures (discussed later) (4,8). In general, generation of both the cellular and the peripheral carboxyterminal fragments of PTHrP involves cleavages within P T H ( 3 3 - 43) (8,9). The intracellular degradation of the h o r m o n e appears to be regulated (2,8,9). When secretion is stimulated by low ambient calcium, most of the h o r m o n e is in the form of the native molecule (6,9). By contrast, when secretion is suppressed by high ambient calcium, most of the secreted h o r m o n e consists of fragments. Curiously, phorbol ester-stimulated

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II 21M ] ~

I////////////////71 E. Renal Clearance

secretion also results in an increased a m o u n t of PTHrP fragments, regardless of the ambient calcium concentration (2,9). T h o u g h a signal peptidase is responsible for cleavage of the pre sequence of preproPTH, the enzymes responsible for subsequent intracellular cleavage of PTH have not been conclusively identified (2). However, it is likely that furin or a furinlike molecule affects the cleavage of the pro sequence of proPTH. Furin, a subtilisin-like enzyme located within the Golgi cisternae of essentially all mammalian cells, cleaves proteins and peptides at the basic residues sites flanking pro sequences (6). The p r o h o r m o n e convertases (PCs) found in secretory granules, a m o n g them PC1 and PC2, are also candidates for intracellular processing of PTH (9). There is also evidence that cathepsins affect the intracellular metabolism of PTH. There is little evidence for the secretion of PTH precursors, as there is for other h o r m o n e s such as insulin and adrenocorticotropic h o r m o n e (ACTH) (2,6,9). The result of this intracellular metabolism of PTH is the intracellular development, with potential for secretion, of several species of the hormone, including intact, midregion, carboxyl, and amino-terminal forms (4,8,9). As discussed later, each of the species has the potential of being detected by immunoassay procedures based on antibodies that recognize their included PTH epitopes. But it is important to recall that only aminoterminal forms of defined length can exert biologic activity by activating the PTH receptor (5).

Secretion o f P T H

el (b)

t7 ..........................................e t (c)

D. Peripheral (Hepatic and Renal) Metabolism

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(d)

(e)

All Circulating Forms [(a) - (e)]

FIG. 1 Biosynthesis, secretion, and metabolism of PTH by the parathyroid gland (PG) and in the periphery. Schematic representation of the cellular biology of PTH and the resulting molecular heterogeneity of circulating forms that are detected by immunoassays. The peptides (a-e) represent the circulating forms of immunoassayable PTH, including (a) native PTH, (b) amino (N)-terminal and carboxy (C)-terminal PTH, and (d) midregion (M) PTH peptides; (c) the recently postulated amino-terminal deleted PTH peptide; and (e) the uncharacterized PTH peptides that result from further metabolism and/or degradation of all other PTH forms. Circulating PTH is a complex, immunochemically heterogeneous mixture of peptides a-e, with fragments predominating. See text for full discussion.

The secretion of PTH is regulated mainly by serum calcium concentration (1,2). In a homoeostatically appropriate response, increased extracellular calcium suppresses PTH secretion, and decreased extracellular calcium stimulates PTH secretion. The primary effect of increased extracellular calcium is to inhibit the secretion of preformed PTH from secretory granules by blocking their fusion with the cell m e m b r a n e (6). This contrasts to most other cells, wherein stimulation of exocytosis is inhibited by the depletion of calcium. Thus, the inverse relationship between ambient calcium and PTH secretion contrasts to the effect of calcium on the secretion of other hormones, including calcitonin, the biological antagonist of PTH (1,6). The relationship between serum calcium and PTH is sigmoidal, with the steepest portion of the curve corresponding to the normal range of serum calcium (9). It is also likely that these two signals correspondingly regulate the growth and proliferation of parathyroid gland cells and thus exert long-term effects on horm o n e secretion (6). The major secretory effects are on native PTH rather than on PTH fragments. In fact,

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carboxy-terminal truncation of the native molecule impairs its secretion, because this end of the molecule seems necessary for guiding PTH through the secretory apparatus (9). Ambient calcium mediates its effects on PTH secretion through the calcium sensor of the parathyroid gland, as discussed in detail in Chapter 8. The secretion of PTH can also be regulated to a lesser extent by ambient magnesium and by catecholamines (4,8). The effect of magnesium on PTH secretion is quantitatively similar to that of ambient calcium but physiologically less important. However, severe magnesium deficiency and hypomagnesemia can inhibit the secretion as well as the biologic activity of PTH (2). After secretion, native PTH is rapidly cleared from the circulation with a half-life of a few minutes (9,10). By contrast, PTH fragments are cleared with a half-life of several hours (10). So these PTH fragments, especially carboxyterminal fragments, accumulate and are readily measurable in the circulation when renal function is impaired (4). In contrast, amino-terminal fragments of PTH are difficult to demonstrate in blood (4,8). The secretion of PTH in humans is episodic (pulsatile) and rhythmic, although there is controversy about the nature of the patterns in health and disease (4,8). In studies of normal subjects, some investigators have reported hourly pulses of secretion that last for minutes, whereas others have described broad peaks that last for hours (4,7,10). In circadian studies, both nocturnal and biphasic peaks have been reported (4). Abnormal patterns of PTH secretion have been reported in several disease states, including osteoporosis and primary hyperparathyroidism (8). During induced perturbations of serum calcium, there can be hysteresis in the relationship between calcium perturbation and the recovery period (1,4,7). Most studies of patterns of PTH secretion have been conducted in small numbers of subjects, and it is difficult to draw firm conclusions about the results (4,8,9). Dynamic tests have been used, respectively, to stimulate and suppress serum PTH in order to assess the secretory status of the parathyroid gland. T h o u g h such studies have helped to define the relationship between the regulatory parameters and PTH secretion, the involved maneuvers are not suitable for clinical practice. For practical clinical purposes, the effects of patterns of basal and regulated PTH secretion on diagnostic studies can be circumvented by collecting blood samples for analysis at a consistent time, preferably in the morning after an overnight fast (9,10).

Peripheral Metabolism of PTH After the glandular PTH forms, which arise from biosynthesis and intracellular metabolism, are secreted into blood, they are also metabolized at several peripheral sites into peptide fragments (Fig. 1). The liver is

the most important metabolic site for PTH, with the kidney and skeleton following (2). Binding of PTH to specific receptors in target tissues does not contribute substantially to the metabolism of the h o r m o n e (5). As was the case for studies of intracellular metabolism, most of the information regarding peripheral metabolism of PTH comes from animal studies and in vitro h u m a n studies using both labeled and unlabeled native PTH (4,8). In their aggregate, these studies reveal that PTH is peripherally processed between residues 33 and 34, 36 and 37, 40 and 41, and 42 and 43, resulting in the corresponding amino- and carboxy-terminal peptides. Most of this metabolism seems to occur in the liver in Kuppfer cells (8). After passage through the liver, the PTH fragments are routed to the kidney, where they are cleared by glomerular filtration along with the relatively smaller amounts of circulating intact PTH (4). As will be seen later, the renal,clearance of PTH and its fragments plays an important role in regulating the concentrations and types of circulating PTHrP forms. This effect becomes especially important in renal failure, where it can confound immunoassay measurements. It is not clear if the peripheral metabolism of PTH is regulated by ambient calcium, as appears to be the case for the glandular metabolism of PTH (4,8). A homeostatically appropriate process would be for hypercalcemia to promote the peripheral degradation of PTH; however, studies of this p h e n o m e n o n have not been conclusive. In summary, as a result of the biosynthesis, secretion, and metabolism of PTH, the circulation contains several forms of the molecule (Fig. 1). This immunochemical heterogeneity of circulating PTH was discovered and d o c u m e n t e d by Berson and Yalow, the first developers of PTH immunoassay, who went on to win the Nobel Prize (11). The forms that comprise this immunochemically heterogenous collection of PTH species include primarily native PTH (1-84) and midregion and carboxy-terminal PTH fragments (6). Overall, 10-20% of circulating PTH immunoreactivity comprises the intact hormone, with the remainder being a heterogeneous collection of peptide fragments corresponding to the middle and carboxy regions of the molecule. Evidence for other circulating PTH species is inconclusive, although some studies indicate the presence in the circulation of the PTH amino terminus (5). It is important to reemphasize that only the amino terminus of PTH can bind to the PTH receptor and mediate its classic biologic effects that result in hypercalcemia. So only amino-terminal-containing PTH forms have the potential for biologic activity mediated through the PTH receptor. However, it should be kept in mind that each of the circulating forms of PTH, regardless of biologic activity, contain within them peptide sequences that can be recognized by a variety of antibodies (4,8).

PTH AND PTHrP IMMUNOASSAYS / The half-life of the relatively low concentrations of intact h o r m o n e and its amino terminal fragments can be measured in minutes, whereas the higher concentrations of the biologically inactive mid- and carboxyterminal peptides have half-lives of hours (8,11). As will be seen later, antibodies directed at the diverse bioactive as well as nonbioactive epitopes included within PTH(1-84) have been used to develop immunoassays for the h o r m o n e . In general, intact and non-aminoterminal fragments of the molecule circulate in blood. However, though intact forms of the molecule containing parts of the amino terminus can be measured in blood, amino-terminal fragments, themselves, are difficult to demonstrate. Certain technical considerations can affect immunoassay p e r f o r m a n c e (4,8,9-11). Although originally considered to be a labile h o r m o n e , in vitro losses of PTH immunoreactivity are less than 10% when whole blood or serum is left at room temperature for 4 hours. However, in vitro degradation may be increased in patients with pancreatitis and the resulting high levels of circulating proteases. In most circumstances, a reasonable delay in the separation and freezing of serum should not have a major effect on assay measurement. Nevertheless, blood samples should be processed within a reasonably short period of time, 30-60 minutes, to minimize h o r m o n e degradation. Some assay procedures utilize collecting tubes that contain enzyme inhibitors. And in some assay systems, there are differences between serum and plasma measurements (9-11). In any case, the ordering physician should comply with the sample-collecting instructions of the testing laboratory. Correct interpretation of PTH assay results requires a simultaneous serum calcium measurement, because, as detailed later, the relationship between the two can distinguish between a primary and secondary secretory disorder of the parathyroid glands (10). Total serum calcium will suffice for most purposes. However, because almost half of serum calcium is b o u n d to serum albumin, a correction is sometimes n e e d e d for abnormal, usually low, concentrations of albumin. T h o u g h there are relatively complex formulas for such a correction, a change of 1 g / d l in serum albumin will generally produced a corresponding change of 0.8 m / d l in measured total serum calcium. Measurements of ionized calcium can be used to circumvent the effects of serum albumin concentrations. However, such tests are not routinely available. Abnormalities in serum g a m m a globulin concentrations do not usually affect total serum calcium measurements. However, if they are markedly increased, as they can be in certain dysgammmaglobulinemias (e.g., Waldenstrom's), serum g a m m a globulin concentrations can also artifactually increase total serum calcium measurement. Spurious increases in serum calcium can be also caused by venousstasis-induced

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changes in local albumin and pH at the time of blood collection, as can the essentially obsolete use of calcium-containing cork-stopped blood collection tubes (4,8,10). The clinician should be aware of these possibilities when there is discordance between serum measurements and the clinical status of the patient.

PTH IMMUNOASSAYS Introduction The development of accurate and precise i m m u n o assays for circulating PTH has evolved from a discouraging beginning to a promising present (4,8). Early PTH immunoassays were based on uncharacterized antibodies and impure standards. Crude extracts of animal parathyroid glands were used for production of antibodies, and heterologous and impure preparations of PTH were used as standards. The clinical value of early immunoassays for PTH was suspect. It took the revolutionary discovery of Berson and Yalow of the i m m u n o c h e m i c a l heterogeneity of circulating PTH to lead to the correction of the clinically contentious course that PTH immunoassays had taken in the 1960s (11). Following the Nobel prize-winning contributions of these two pioneers, PTH immunoassays began to evolve in a rational manner. Immunoassays were developed based on defined PTH peptides and characterized antibodies, both polyclonal and monoclonal (11,13). Even polyclonal antibodies could be purified by peptide-specific immunoaffinity c h r o m a t o g r a p h y (12,13). Immunoassays subsequent to the first crude assay systems could thus be directed at specific forms of the h o r m o n e (4). The molecular targets of PTH assays could be predicted by basic studies that elucidated the complex nature of PTH in the circulation (8). The most prevalent forms of PTH in serum would be fragments of the molecule that contained midregion and carboxyterminal epitopes, whose molecular d o m i n a n c e was e n h a n c e d by relatively long half-lives (4,11). By contrast, assays directed at the amino terminus were not likely to be clinically successful because of the small amounts of this PTH form in the circulation and it short half-life. Intact PTH species, which revealed several epitopes, occupied an intermediary position, but were later to b e c o m e targets for two-site assays (11-14). Following the development of clinically useful midregion and carboxy-terminal PTH solution immunoassays based on an antibody to the desired epitope, solid-phase, two-site immunoassays were developed that were based on the simultaneous use of two antibodies of differing specificity for the PTH molecule (4,8,15,16). The specificity of the antibodies used in these two-site formats defined the PTH species

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being detected. These two-site assays were accompanied by the supplanting of radioiodine in radioimmunoassays (RIAs) by nonisotopic methods for detection, such as the use of colorimetry or chemiluminescence (17-20). These new technologies provided the newer immunoassays with descriptive acronymic titles, such as enzyme-linked immunosorbent assays (ELISAs), immuno-radiometric assays (IRMAs), and immunochemiluminometric or -fluorometric assays (ICMAs and IMFAs, respectively). The development of novel detection systems and improvements to assay automation continues (19-21).

PTH Assay Formats Overview

Detection systems aside, there are two general formats for PTH immunoassays, one-epitope-site solution immunoassays and two-epitope-site solid-phase immunoassays (4,16,22,23). Solution immunoassays, developed first, are based on the comparison of the displacement from an antibody, usually polyclonal, of labeled, usually radioiodinated, PTH or PTH peptide representing the PTH present in a blood sample and the PTH standard. As increasing quantities of the PTH in the unknown sample and PTH standard are added to the radiolabeled PTH-antibody reaction, there is a progressive competition by the sample and standard PTH for antibody binding by the radiolabeled PTH tracer. This competition by each known amount of PTH standard produces a progressive displacement of radiolabeled PTH tracer from the antibody in a manner that can be used to generate a standard curve. The unknown amount of PTH in the unknown sample has the same effect. By comparing the displacement produced by the PTH in the unknown sample to the known PTH standard, the amount of PTH in the sample can be calculated. The sensitivity of the assay can be enhanced by sequential (nonequilibrium) rather than simultaneous (equilibrium) addition of the unlabeled PTH standard (and sample) and labeled PTH tracer (16).

Solution Immunoassaysfor t ~ H In the earliest of PTH solution immunoassays, commonly referred to as radioimmunoassays, the standards were usually impure and often heterologous, and the antibodies were not well characterized, often being generated against only partially purified preparations of glandular PTH (4,8). Many technical advances followed these early PTH solution immunoassays. Standard preparations of PTH became progressively more pure and homologous, and PTH peptides were introduced as standards. Antibodies were raised against

fully characterized forms of PTH and PTH peptides, thus allowing the development of antibodies with welldefined specificities for PTH and its peptides. Although most antibodies used in solution immunoassays were polyclonal, occasional high-affinity monoclonal antibodies were identified (4,8,16). The combination of synthetic peptide standards and antibodies of known specificity allowed the development of PTH immunoassays that could measure defined regions of the molecule (11,12). Thus, immunoassays could be developed to detect specifically the circulating forms of PTH that contained different PTH epitopes, including amino-, mid-, and carboxyterminal PTH species (8,21-23). Although each assay so designed would measure intact PTH, it preferentially measures with appropriate specificity and respectively increased sensitivity the middle and carboxy-terminal regions of PTH that circulate at severalfold higher concentrations than intact PTH (23-31). Despite their limitations, these solution immunoassays proved clinically useful, and most of them are still in use (28-35). They specifically identify a large majority of patients with primary hyperparathyroidism and patients with secondary hyperparathyroidism in the absence of renal failure. Thus, both commercial and research immunoassays for PTH are still currently available that can detect midregions of the molecule using, for example, PTH(44-68) as standard and the tryrosinated form of the same peptide as tracer and for antibody generation, and carboxy-terminal regions of PTH using, for example, corresponding reagents for PTH (68-75) (4,8,28,33-38). The clinical value of solution immunoassays for nonamino-terminal PTH forms is also due to the fact that they can measure the relatively low concentrations of PTH that circulate in the serum of normal individuals, thus providing a basis for comparison with disease states, and because these assays, especially midmolecule assays, are notably sensitive (32,35). In addition to the inherent sensitivity of such assays, their cognate nonamino-terminal forms of PTH are predominant in the circulation because of their metabolic characteristics, discussed earlier. Furthermore, these assays can detect the small amount of circulating intact hormone conmining their epitopes (34-40). In contrast, assays designed to measure the amino terminus cannot readily detect this form in normal subjects because it circulates at such low concentrations and has a short half-life, although some exceptions have been reported (40-48). Despite their continuing clinical utility, radioimmunoassays for the midregion and carboxy regions of PTH do have their limitations, especially the latter (33,38,42). Both of these fragments, especially carboxyterminal fragments, accumulate disproportionately in

PTH AND PTHrP IMMUNOASSAYS / the patient with renal disease (4,8,45). Thus, it is difficult to assess accurately parathyroid gland secretory status using carboxy-terminal assays in such patients. However, it must be kept in mind that in renal disease there is an accumulation of all of the circulating forms of PTH, including all fragments and the intact hormone, too (4,8,47). As will be discussed in detail later, assessing parathyroid gland secretory activity remains a major problem in renal disease, especially in chronic renal failure.

Solid-Phase Immunoassays The two-site assay is based on two antibodies with different recognition sites for an antigen, in this case PTH (49,50). Although the principle of such assays, generally referred to as immunometric assays, had been recognized for years, the onset of monoclonal antibody production enhanced the development of the reagents requisite for these assays and their research and clinical application (6,19,20). Reactivity in two-site assay systems is dependent on the separate recognition of two antigenic sites in PTH by a pair of antibodies respectively directed against them. One antibody is attached to a solid matrix, usually beads or microtiter wells, and the other antibody is radioiodinated (or otherwise labeled) and in solution. The antigen, PTH, binds to the antibody on the solid phase (e.g., beads) according to the antigenic recognition site of that antibody. The labeled antibody binds the PTH antigen bound to the immobilized antibody according to its (the labeled antibody) different recognition site. The radioactivity remaining after washing the solid matrix is thus proportional to the amount of antigen having both antigenic determinants. The two-site assay has many advantages (15,26,27). It can directly measure specific and defined forms of PTH; the kinetics of the two-site assay permit an extraordinary increase in assay sensitivity, even with relatively low-affinity antibodies; and this assay system is remarkably free of nonspecific protein artifacts that have continually plagued immunoassays. This latter advantage is especially important, because two-site procedures can minimize the problems associated with immunoassay in protein-rich biologic fluids such as blood (15,26-29). The antibodies used in two-site immunoassays are usually generated against specific peptides of PTH (21). Monoclonal antibodies are favored over polyclonal antibodies for two-site immunoassays. Monoclonal antibodies of exquisite specificity can be developed in the relatively large amounts needed for the solid-phase component of two-site assays (16,19). Furthermore, monoclonal antibodies can be more readily purified for labeling. A disadvantage of m o n o -

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clonal antibodies is that they generally do not have the affinity of polyclonal antibodies (4,16). However, this property is not so important for the noncompetitive kinetics of two-site assays as it is for the kinetics and competition of solution immunoassays (8,16,21,49,50). Another approach for developing epitope-specific antibodies is to purify them from polyclonal antiserum using peptide-specific affinity chromatography (16,51). Epitope-specific polyclonal antibodies can then be used as described above for the two-site assay format; mixed monoclonal-polyclonal systems are also suitable (4,8,51). Using these general approaches and their variations, assay systems have been developed for peptides that span the linear sequence of PTH. Especially popular are two-site systems using amino- and carboxyterminal PTH antibodies in concert in order to detect in tact native PTH (9,15,52). Solid-phase immunoassays also have other technical advantages over solution immunoassays (4,8,15,21). They can be completed in hours rather than in the days usually needed for solution immunoassays. They are relatively free of the nonspecific serum effects that plague (solution immunoassays, because serum is not present during the critical incubation stage of the procedure. And they can be performed with a general technical ease that provides more accuracy and precision. Intact F I ' H

Immunoassays

Assays for intact PTH(1-84) have become the holy grail of the PTH immunoassay field, even though classic solution immunoassays, especially midmolecule assays, have sufficient accuracy and precision for most clinical indications (15,23,25,28,52-57). These assays use an antibody directed against the amino terminus of PTH in tandem with an antibody directed against the carboxy-terminal regions of PTH in order to detect circulating levels of intact PTH(1-84), a (synthetic peptide version of which is used as standard (28,29). Though intact assays generally provide excellent discrimination between parathyroid disease and nonparathyroid disease, these) assays still demonstrate some overlap in their normal range with disease states (22,23). However, even in these situations, the diagnosis can usually be clarified by considering the PTH concentration together with the serum calcium concentration (4). Thus, even if the serum PTH concentration is not absolutely different from the normal range, it is inappropriately different from the normal range in hyperparathyroid disease states (8,30). So the patient with hypercalcemia due to primary hyperparathyroidism will have a serum intact PTH that is absolutely elevated above the normal range or close to the top of the normal range, and, by contrast, the patient with nonparathyroid hypercalcemia will have a

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serum intact PTH that is close to or below the normal assay limit. These considerations, of course, apply to other PTH assays. Intact PTH assays have been widely used in renal failure to assess parathyroid gland secretory status, as will be discussed in more detail subsequently (22,23). The rationale behind this application is the well-known accumulation of PTH fragments that occurs in renal failure. Because these fragments do not necessarily reflect the secretory activity of the parathyroid gland and because they are generally biologically inactive, their m e a s u r e m e n t does not give a true assessment of parathyroid gland function (4,38,57). These fragments should theoretically escape detection in a two-site assay that is designed to measure only the intact molecule, based on antibodies to the amino-terminal and carboxy-terminal regions of PTH. However, studies demonstrate that this theoretical advantage of intact PTH immunoassays may not always apply (23,57). In the first place, like PTH fragments, intact PTH also accumulates in renal failure, although less so (22,23). However, more critical, currently available intact PTH assays seem also to measure certain PTH fragments, especially in renal failure (23). This is probably due to the fact that antibodies used for presumably intact PTH immunoassays are not, respectively, directed at the far carboxy and far amino termini of the native molecule (22,23,57). Thus, nonintact fragments that are truncated at termini the PTH will react in some putatively intact PTH immunoassays (22,23). Other studies suggest that PTH(7-84) is such a fragment (22,57). This PTH species accumulates in renal failure and may even be secreted by both normal and abnormal parathyroid glands (4,8,58-60). Further confounding clinical assessment, PTH(7-84) may act as an antagonist a n d / o r weak agonist to PTH at its receptor. Assays based on antibodies to the extreme amino- and carboxy-terminal regions of intact PTH may not be so c o n f o u n d e d and may thus be more clinically useful, especially in renal disease (4,8,22,23,60-63).

SPECIFIC CLINICAL APPLICATION

OF PTH IMMUNOASSAYS

Serum PTH immunoassays are invaluable in the differential diagnosis of hypercalcemia and hypocalcemia (1,4,8,10). In general, all well-characterized PTH immunoassays can serve this function, even though studies have focused on assays of the intact PTH (4,8). Most PTH immunoassays segregate patients with hypercalcemia and hypocalcemia into two respective categories, parathyroid disease and nonparathyroid disease (4,8,16,21). In the patient with hypercalcemia, an elevated serum PTH usually means primary hyperparathy-

roidism, and low or undetectable serum PTH usually means nonparathyroid disease. In the patient with hypocalcemia, an elevated serum PTH usually means secondary hyperparathyroidism, and a low or undetectable serum PTH usually means hypoparathyroidism. Direct comparisons of PTH fragment and intact immunoassays in the differential diagnosis of hypercalcemia show comparable clinical discrimination (4,8,38,45,47,53). In fact, midregion assays seem to identify the patient with primary hyperparathyroidism with more discrimination compared to the intact assay (4,10,35). For example, in one study of 36 patients with surgically proved primary hyperparathyroidism, a midmolecule assay recorded PTH values greater than twice normal in 28 patients, whereas an intact assay was similarly elevated in only 17 patients (42). However, in patients with nonparathyroid hypercalcemia such as malignancy, serum intact PTH is more completely suppressed than is serum midregion PTH, making that differential diagnosis easier (21,27). Furthermore, intact assays are less c o n f o u n d e d than PTH fragment assays, especially those of carboxy-terminal fragments, by the accumulation of biologically inert PTH fragments that takes place especially in renal failure, as detailed subsequently (25,28). And, as discussed elsewhere, intact assays have technical advantages over solution immunoassays (21 ). The improved quality of all contemporary PTH immunoassays makes them invaluable in the differential diagnosis of hypercalcemia (10,30). With many assays available, a practical approach to the differential diagnosis of calcium disorders is to use the PTH immunoassay that is readily available and interpretable to the physician. With most assays, the correct diagnosis will be obtained, although there is still the rare false, positive or false-negative result (55). In this respect, it should be kept in mind that some patients with primary hyperparathyroidism, especially those in the early course of the disease, may have serum PTH levels that fluctuate close to the normal range (10,16). In such case, multiple PTH measurements may be necessary to establish the correct diagnosis. For even more complicated cases, m e a s u r e m e n t by more than one PTH assay can help to resolve the differential diagnosis.

Primary Hyperparathyroidism Primary hyperparathyroidism (PHPT) is the most c o m m o n endocrine cause of hypercalcemia, and the most c o m m o n cause of PHPT is a single adenoma of the parathyroid glands (6,10,30). There are about 4 million cases of PHPT worldwide, so the disease has substantial impact (10,30). Rather than presenting to the physician with symptoms, as was the case in the last millennium, the patient with PHPT is now more likely to be asymptomatic and is seen by a physician because

PTH AND PTHrP IMMUNOASSAYS / of an elevated serum calcium detected by a multichannel screening (21,30). Parenthetically, it should be noted, however, that multichannel screening is becoming more limited by health maintenance organizations. When there are clinical manifestations of PHPT, they involve the skeleton, kidney, gastrointestinal tract, and central nervous system (64). In hospitals, PHPT is second only to malignancy as a cause of hypercalcemia; in the outpatient setting, PHPT is the most c o m m o n cause. Whereas malignancy dominates the clinical picture when it causes hypercalcemia, in PHPT the physician in the clinic is often faced with the challenge of establishing the cause of an elevated calcium that was detected in a multichannel screening rather than via a specific request, even though the advent of managed care has reduced the use of such screening tests (10,64). The availability of precise and accurate serum PTH assays has made the diagnosis of hyperparathyroidism relatively easy to establish, even u n d e r these circumstances (21). In fact, this is a most c o m m o n contemporary application of PTH immunoassays (21,64). PHPT is discussed in more detail in Chapters 18-23. PHPT usually can be distinguished from the other c o m m o n cause of hypercalcemia, malignancy, by a careful history and routine testing (10,21,30,64). In many patients with malignancy, the hypercalcemia is due to the production by the cancer of parathyroid hormonerelated protein (PTHrP) (10,65). As detailed later, immunoassays have been developed for this oncoprotein (65). T h o u g h their clinical application is discussed subsequently, it can be noted here that PTHrP does not cross-react in PTH assays. An elevated serum calcium and decreased fasting serum phosphorus support the diagnosis of PHPT. (To be diagnostically useful, the serum p h o s p h o r o u s must be fasting; otherwise, it may be perturbed by prandial excursions.) An elevated serum PTH establishes the diagnosis. By contrast, in hypercalcemia of malignancy, the serum phosphorus does not have a consistent pattern, and the serum PTH is usually suppressed by the hypercalcemia (10,30). Thus, when PHPT seems likely, the evaluation can often be completed by confirmation of the suspected diagnosis with an elevated serum PTH. The vast majority of patients with PHPT will have an elevated serum PTH in essentially all established immunoassay systems. The rare patient with PHPT due to parathyroid gland cancer will have exceptionally high serum PTH levels (Chapter 41) (10,66). Up to 95% of patients with PHPT will have elevated values in both intact and midregion assays, with a slightly higher percentage in the latter (33). Hypercalcemia and increased serum PTH are the signal manifestations of PHPT, although levels of both can fluctuate close to the u p p e r limits of normal early in the disease as it waxes and wanes (10,64,65). More than one m e a s u r e m e n t of serum calcium and sometimes PTH should be made for

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a combination of the technical and biologic reasons discussed earlier. Because PHPT waxes and wanes, especially during its early course, a single m e a s u r e m e n t might miss the period of increase. Serum PTH is now most commonly measured by a two-site assay designed to detect the intact molecule (4,21). This is the most widely used test, even though well-characterized midmolecule assays are at least equally accurate and precise in diagnosis (8,33). Amino- and carboxy-terminal terminal tests are not widely used because of the limitations discussed earlier (46-48). The u p p e r limit of normal for intact PTH is about 50-65 p g / m l . However, after the age of about 45 years, even intact PTH seems to increase in normal subjects, perhaps due to decline in renal function (64). But in the younger hypercalcemic patient, an intact PTH above the mid-40 p g / m l level should cause suspicion for PHPT (10). There are converse considerations with regard to serum calcium measurements (30). Most laboratories place the u p p e r limit of normal at 10.2-10.5 m g / d l . However, serum calcium slowly declines in the aging individual (10). Thus, after approximately age 50, a serum calcium above 10 m g / d l should also be regarded with appropriate clinical suspicion. The relationship between serum calcium and PTH m e a s u r e m e n t should also be considered. Because even in PHPT calcium feeds back on the secretion of PTH, a serum PTH approaching the u p p e r limits of normal may be considered as increased in the face of a serum calcium above normal (10,30). PTH immunoassays have been applied during parathyroidectomy in order to assess the success of the surgery (67,68). For this application, rapid assay formats have been developed so that results of the assay can be known before the surgical procedure ends (67). This becomes practical, because the half-life of some circulating species of PTH can be measured in minutes, especially intact PTH and amino-terminal fragments (4,8). In addition to enhancing the success of the surgery, some studies have suggested that the time of surgery can be decreased by intraoperative PTH assay (68).

Nonparathyroid Causes of Hypercalcemia The many nonparathyroid causes of hypercalcemia are discussed in Chapters 41-43. They include hyperthyroidism and hypoadrenalism; the diagnostically elusive familial hypocalciuric hypercalcemia (FHH); vitamin D, vitamin A, and lithium intoxication; thiazide diuretics; and several granulomatous disorders, notably sarcoidosis (10,69). However, the most important nonparathyroid cause is the hypercalcemia of malignancy, where overproduction of PTHrP, to be discussed subsequently, is a c o m m o n etiological culprit (65,69,70). It can be reemphasized here that there

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is essentially no cross-reactivity of PTHrP in PTH immunoassays (65). In reference to PTH assays, these nonparathyroid diseases are characterized by suppressed PTH secretion and low or absent serum concentrations (21,70). Thus, in these patients, the detection of a low or absent serum concentration of PTH by any of the reliable assay procedures discussed confirms the absence of parathyroid disease and points to a nonparathyroid cause for the hypercalcemia (65,67). F H H may be an exception to this rule in that a small percentage of patients can have increased serum PTH concentrations, although most have PTH levels that are normal but not suppressed by the hypercalcemia (65,70). Similarly, some patients with lithium-or thiazide-induced hypercalcemia can have increased concentrations of serum PTH (70). In all diagnostic situations, and especially in patients with nonparathyroid h o r m o n e hypercalcemia, it is important that the PTH measurement be made before calciumlowering therapy is instituted. Otherwise, the induced decrease in serum calcium can cause an increase in serum PTH, sometimes up to the hyperparathyroid range, and thus confound the diagnosis (10,70).

Hypocalcemia and Secondary Hyperparathyroidism Renal failure and hypoparathyroidism are the most c o m m o n etiologies of hypocalcemia, with hypoalbuminemia being a c o m m o n artifactual cause (71,74). Renal failure is by far most frequent a m o n g the causes of hypocalcemia, with the other causes accounting for only a small minority of cases (72,73). The etiologies of hypocalcemia can be generally classified as nonparathyroid and parathyroid, and the parathyroid diseases associated with hypocalcemia can be further classified as primary hypoparathyroidism and secondary hyperparathyroidism. The primary parathyroid causes of hypocalcemia are due to inadequate secretion of PTH (73,74). This type of primary hypoparathyroidism can follow neck surgery, including thyroidectomy and parathyroidectomy; it can occur as an isolated parathyroid disease; or it can be part of an a u t o i m m u n e endocrine deficiency syndrome that variably involves, a m o n g other tissues, the adrenal, thyroid, and pancreas (74). In renal failure, hypocalcemia develops because of abnormalities in phosphorous and vitamin D metabolism. The hypocalcemia leads to the development of secondary hyperparathyroidism. These disorders are detailed in Chapters 39 and 47. There are other less c o m m o n causes of secondary hyperparathyroidism, such as osteomalacia and rickets and other vitamin D and calcium disorders (discussed in Chapters 39 and 47. In addition to these chronic causes of hypocalcemia, acute failure of normal cal-

cium homeostasis can also cause hypocalcemia (73,74). Hyperphosphatemia that results from phosphate administration, rhabdomyolysis, or tumor lysis can produce severe hypocalcemia, especially in renal insufficiency. In acute pancreatitis, sequestration of calcium by saponification with fatty acids causes hypocalcemia. Rapid or excessive skeletal mineralization can cause hypocalcemia, as in the "hungry bone" syndrome and in osteoblastic metastases (10,30). These are discussed in Chapter 24. In primary hypoparathyroidism, PTH is low or absent and serum phosphorus is often increased because of the loss of the phosphaturic effect of PTH (8,73,74). In the secondary hyperparathyroidism seen in the nonparathyroid causes of hypocalcemia, the opposite occurs because of compensatory secondary hyperparathyroidism that increases the serum PTH and consequently decreases the serum phosphorus. An exception to this rule is pseudohypoparathyroidism, the genetic disease of end-organ resistance to PTH characterized by the biochemical features of hypoparathyroidism, a characteristic somatotype, and a secondary increase in serum PTH (75). Hypocalcemia related to hypomagnesemia can also present an unusual picture. Magnesium deficiency can cause hypocalcemia by impairing PTH secretion (1,73). So the PTH response to the hypocalcemia can be attenuated in the magnesium-depleted patient, with inappropriately low PTH levels in the presence of anatomically normal but functionally impaired parathyroid glands. As briefly discussed earlier, the calcium and skeletal abnormalities of renal failure directly lead to hypocalcemia, which, in turn, leads to secondary hyperparathyroidism. Consequently, the serum PTH and phosphorus are elevated and the serum calcium is low in these patients. Thus the measurement of serum PTH is a key procedure in differential diagnosis of hypocalcemia. With the exceptions discussed above, a decreased serum PTH identifies a parathyroid cause for the hypocalcemia (primary hypoparathyroidism) and an increased serum PTH identifies a nonparathyroid cause for the hypocalcemia accompanied by secondary hyperparathyroidism. As is the case for other parathyroid disorders, most well-characterized immunoassays for PTH will serve to distinguish a m o n g the parathyroid and nonparathyroid causes of hypocalcemia. Thus, the measurement of serum PTH is a most valuable test in the differential diagnosis of hypocalcemia.

Renal Osteodystrophy and Secondary Hyperparathyroidism Renal osteodystrophy is the name given to the complex of skeletal disorders that occur in renal failure

PTH AND PTHrP IMMUNOASSAYS / (71,72). Two abnormalities associated with declining renal function initiate this complex skeletal disease: increased serum phosphorus and decreased renal production of the active vitamin D metabolite, 1,25-dihydroxyvitamin D. The increased serum phosphorus causes hypocalcemia as does the decreased renal production of 1,25-dihydroxyvitamin D, which can also cause osteomalacia. These events lead to hypocalcemia, which in turn increases PTH secretion and, through this secondary hyperparathyroidism, causes the skeletal disease of PTH excess, osteitis fibrosa cystica (72,74). The parathyroid gland escapes the control of its mineral and hormonal regulators in part because of decreased expression of its calcium and vitamin D receptors (76,77). One or more glands can also undergo monoclonal expansion as the gland becomes hyperplastic. As the lowered calcium simulates PTH secretion by the parathyroid gland in renal disease, the increased serum phosphate concentration further increases hormone biosynthesis (71,76). Treatment is directed at reversing this process and returning the serum calcium, phosphorus, and PTH toward normal. Parathyroidectomy is reserved for those few patients whose medical m a n a g e m e n t has failed or whose disease has advanced to tertiary hyperparathyroidism. Renal transplantation is the ultimate treatment (74). The precise and accurate measurement of parathyroid gland secretory activity in renal failure is an important goal, because suppressing the hyperplastic gland toward normal secretory activity is a major end point of treatment (77-80). However, assessment of PTH secretion is complicated by the accumulation of all PTH fragments in renal failure, but especially carboxyterminal fragments that seem to more closely reflect creatinine clearance than parathyroid gland secretory activity (57-61). Assays that measure non-carboxy-terminal PTH fragments are thus easier to interpret in this context (4,8). The important clinical goal of assessing parathyroid secretory function in renal disease rather than measuring biologically irrelevant retained fragments of the hormone has been elusive (58,60). The advent of the intact PTH assay has been welcomed as a solution to this problem (4,8,21,81). However, comparison studies of the intact assay with other PTH assays shows them all to give spuriously elevated values because of their impaired renal clearance, a complex process that may involve the multifunctional clearance receptor, megalin (22,23,57,63). Furthermore, studies have demonstrated that some reportedly intact assays can be questioned regarding their assessment of gland secretion in that they also seem to measure PTH fragments that can be affected by impaired glomerular filtration (22,23). This may be due to the fact that their antibodies are

153

not directed against the far termini of PTH, so that less than full-length fragments of the h o r m o n e are recognized (23). In fact, a newly described PTH fragment, PTH (7-84), has been observed to accumulate in renal failure, and it is variably measured by assays that were presumed to measure intact PTH(1-84) (23,63). This fragment may account for up to half of the circulating PTH immunoreactivity in patients on dialysis (22,23,63). So the recently recognized accumulation of PTH (7-84) in renal failure continues the dilemma of PTH assays and renal disease. This dilemma is further c o m p o u n d e d by the possibility that PTH(7-87) may be a PTH antagonist or weak agonist (23). New assays using antigenic determinants in PTH(1-6) are being developed to address this issue (22,23,63). These assays identify only half of the circulating concentrations of PTH in renal failure, as well as in other conditions (63). However, all progress in this area should be evaluated with the realization that the clinical value of PTH immunoassays is especially complex as an assessment tool in the secondary hyperparathyroidism of renal disease because the immunochemical heterogeneity of the molecule is further complicated by the accumulation of PTH fragments, both biologically agonistic, antagonistic, and inert (23,63). As is the case for primary hyperparathyroidism, however, all PTH assays have some value in the clinical m a n a g e m e n t of the patient with renal failure as long as the clinician is familiar with the proper interpretation of assay results. Tertiary Hyperparathyroidism Tertiary hyperparathyroidism is the name applied to secondarily hyperplastic parathyroid glands of renal failure that escape from secretory control of PTH by calcium, secrete even more PTH, and thereby lead to hypercalcemia (9,77,78). It has also been observed in certain vitamin D disorders (77,78). However, because of successful approaches to medical management, tertiary hyperparathyroidism is rare in renal disease. If hypercalcemia does occur in this setting, it is usually due to overtreatment with calcium a n d / o r vitamin D administration (10). So the diagnosis of tertiary hyperparathyroidism should not be made unless predialysis hypercalcemia can be demonstrated after t h e discontinuation of vitamin D and calcium administration. This distinction is important, because true tertiary hyperparathyroidism, a commonly monoclonal expansion of abnormal parathyroid cells, usually requires parathyroidectomy. In addition to calcium and vitamin D, other causes of hypercalcemia should also be ruled out before the diagnosis of tertiary hyperparathyroidism is made in renal disease.

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Adynamic Renal Osteodystrophy This form of low-turnover skeletal disease can be a significant problem in renal failure (78,79). Initially attributed to deposition of aluminum from drugs (e.g., aluminum hydroxide) or dialysate fluid, it is now appreciated that adynamic bone disease can occur in dialysis patients who are never exposed to excessive amounts of aluminum from either aluminum-containing phosphate binders or dialysate aluminum (79,80). Iron deposits may play a role in some patients (72,79,80). Because serum PTH is lower in patients with adynamic bone disease than in other forms of renal osteodystrophy, risk factors still include those that will suppress PTH, such as the use of dialysis fluid with high calcium concentration, use of calcium-containing phosphate binders (e.g., calcium carbonate), as well as the now rare use of aluminum-containing drugs (79,80). This rare variant of renal osteodystrophy should be suspected in the patient with renal disease whose serum PTH is low relative to the increased concentrations generally seen in renal failure (72,79).

Other Skeletal and Mineral Disorders Serum PTH levels are generally normal in most common skeletal disorders such as osteoporosis, unless they are complicated by hyperparathyroidism. How-ever, there may be subtle abnormalities of PTH secretion in some skeletal diseases (81,82). Furthermore, there is a seemingly increased incidence of primary hyperparathyroidism in Paget's disease of bone, involving up to 10% of the patients in some studies (82). Furthermore, most patients with osteomalacia will have secondary hyperparathyroidism because of the accompanying hypocalcemia (73,74). If the responsive increase in PTH secretion substantially corrects the hypocalcemia, serum calcium levels may approximate the normal range. However, in the absence of renal disease, there will be a deceased serum phosphorus caused by the phosphaturic

TABLE 1

effect of the increased PTH (73). The combination of a low serum calcium and phosphorus and a high PTH is the signature of secondary hyperparathyroidism. Table 1 summarizes the c o m m o n laboratory findings that can be useful in establishing the correct diagnosis in the patient with an elevated PTH. The differential diagnosis of hypercalcemia and hypocalcemia is discussed in detail in Chapters 41 and 47.

PTHrP BIOSYNTHESIS, PROCESSING, AND SECRETION Overview Parathyroid hormone-related protein can be characterized as an oncofetal protein (83-86). Originally discovered as a product of breast and lung cancer cells that produced hypercalcemia, PTHrP is now known to be produced by many normal and malignant tissues, with and without hypercalcemia, and regulated by a variety of factors (83-85). The amino terminus of PTHrP reacts with the P T H / P T H r P receptor and has the potential to produce most of the biologic effects of native PTH, including hypercalcemia (84,86). Although multiple, the functions of PTHrP in malignant and normal tissues seem to be related to cell growth and proliferation (87,88). A variety of factors, many of them also growth regulatory, affect the production of PTHrR T h o u g h ambient calcium mediates its effects on PTHrP through the calcium sensor, as it does for PTH, the effects are more complex than for PTH and can be opposite, in that increased ambient calcium can increase PTHrP production (1,87). More details about the physiology and pathophysiology of PTHrP can be found in Chapter 3. Despite many studies demonstrating the high frequency of PTHrP expression in many malignant tumors, secretion studies of PTHrP in blood have had limitations (89). T h o u g h PTHrP expression is c o m m o n

Routine Serum Tests in the Differential Diagnosis of the Patient with an Increased Serum PTH a Diagnosis

Calcium

Phosphorus

BUN/creatinine

Primary hyperparathyroidism Secondary hyperparathyroidism Renal Nonrenal Pseudohypoparathyroidism Tertiary hyperparathyroidism

H

L/N

N

L L L H

H L H/N H

H N N H

all,

High; L, low; N, normal; BUN, blood urea nitrogen.

PTH AND PTHrP IMMUNOASSAYS

Biosynthesis of PTHrP The expression of PTHrP is controlled by a complex gene that resides on chromosome 12 and seems related in evolution to the PTH gene (82,84). The PTHrP gene sequence spans more than 15 kb and is composed of

--5.4 --2-1.

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three primary regions: a complex promoter region, a coding region, and a multiple 3' noncoding region (83). The gene's promoter region contains three promoter elements, designated P1, P2, and P3. P1 and P3 are "TATA box" like, and the P2 element is a GC-rich region (85). The PTHrP gene expresses three native forms of the polypeptide through alternate mRNA splicing, PTHrP(1-141), a truncated 139-residue form, and a 173-residue form expressed primarily in humans (Fig. 2). Whereas PTHrP(1-139) is quite similar to PTHrP(1-141), PTHrP(1-173) completely diverges from both at its own carboxy terminus (83,85). It is not surprising that the biosynthesis of PTHrP shares many features with the biosynthesis of PTH, because they both follow the biologic rules of polypeptide biosynthesis (85,86). Thus, PTHrP contains a leader or prepro sequence whose components take it through a cellular journey not unlike that of PTH (86). However, PTHrP differs from PTH in that three isoforms of the polypeptide are encoded by the h u m a n PTHrP gene, and each of these exhibits multiple processing sites that can release unique peptides (83). The biologic effects of PTHrP are mediated, at least in part, through the receptor that it shares with PTH, which is a m e m b e r of the seven membrane-spanning and G protein-coupled cell surface receptors (84). PTHrP also contains nuclear localizing sequences that

in cancer and hypercalcemia, it usually occurs in advanced disease (90,91) And many patients who have PTHrP-expressing tumors fail to demonstrate hypercalcemia and abnormally increased serum PTHrP concentrations (89,90). However, new PTHrP assays of improved sensitivity and specificity are being developed to address these clinical limitations; they are becoming important tools in the evaluation of the patient with hypercalcemia (89-92). As for PTH, a detailed background in relevant physiology for a full understanding of the role of PTHrP in cancer is contained in Chapter 41. The following synopsis of the biosynthesis, secretion, and metabolism of PTHrP focuses on the rationale that has been used for PTHrP immunoassay assay development for clinical application. This background synopsis is followed by a discussion of the application of PTHrP immunoassays to specific disorders of calcium and skeletal metabolism, especially hypercalcemia of malignancy.

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may be responsible for some of its biologic effects (93). The most clinically important pathophysiologic effect of PTHrP is hypercalcemia (see Chapter 13 for more details about PTHrP biosynthesis and effects).

Processing of PTHrP In addition to biosynthesis of the three isoforms, PTHrP(1-139), PTHrP(1-141), and PTHrP(1-173), processing of PTHrP into peptides is an important regulatory mechanism (85,93). The processing of PTHrP is complex, because there are many processing sites within each of the PTHrP isoforms (Fig. 2). Enzymes have been identified from mammalian tissues that seem to serve this function, including furin, PC2, and PC3 (86,88), although the specific enzymes involved in PTHrP processing have not been conclusively established. In addition to classic basic amino acid processing sites, PTHrP can also be processed at monobasic sites, such as Arg-36 (93). T h o u g h many peptides are predicted by the processing sites in PTHrP, the presence of only a few has been experimentally demonstrated or theoretically implied. In addition to P T H r P ( 1 - 3 6 ) , they include 1-38, 1-74, 1-94, 1-95, 37-74, 1-101, 67-86, 107-139, 126.144, and 141-173 (83-92). It has not been d e t e r m i n e d which of these peptides is the result of processing, protease degradation, or both. As is the case for PTH, the demonstration of these peptides has resulted from both in vitro and in vivo studies (91,92). But the situation is more complicated than for PTH, for which only one gland is the source of the hormone; PTHrP a can arise from a wide variety of cancers and is thus susceptible to tissue-specific regulation (87,88). Accordingly, most immunoassays for PTHrP have been developed along empirical lines (90,92). Distinct biologic properties have been attributed to the different PTHrP peptides (84,93). For example, P T H r P ( 1 - 3 4 / 3 6 ) mediates the growth-regulating and hypercalcemic effects of the molecule, PTHrP(35-94) promotes placental calcium transfer, and peptides included in PTHrP(109-141) inhibit osteoclast function (88,93). Preliminary studies of PTHrP(140-173) suggest that it also has growth-regulating effects (94,95). Although these PTHrP peptides appear to have distinct biologic properties, their structures do not necessarily conform to those predicted by processing of the native molecules (93). Furthermore, relatively little is known about the tissue-specific processing of PTHrP, but it is u n d e r intense study (89-95). In addition to its fundamental importance, identifying the peptides processed from the three native forms of PTHrP will help in the design of immunoassays with improved specificity, because PTHrP and its related peptides are

expressed and secreted by several c o m m o n cancers and they have the potential to serve as tumor and serum markers for such malignancies (89,90). The biochemical hallmark of the PTHrP-producing cancer is hypercalcemia, an effect mediated by the amino terminus of PTHrP through the receptor that it shares with PTH (84,88) (more details of PTHrP processing can be found in Chapter 3).

PTHrP and Cancer PTHrP and its related peptides are expressed and secreted by many cancers, and they have the potential to serve as tumor and serum markers for such malignancies (83-92). PTHrP is the most c o m m o n mediator of the hypercalcemia of malignancy (90,91). It has a been estimated that 80% of hypercalcemic patients with cancer have elevated serum levels of PTHrP (91,92). In some types of tumors, the percentage is even higher (89,92). T h o u g h PTHrP expression was initially noted to be c o m m o n in squamous cell cancers, it has been subsequently shown that many other cancer types can overexpress PTHrP (90,91 ). The biologic and clinical importance of PTHrP in breast cancer has become well established (87,96-98). PTHrP production and secretion by breast cancers are very common, occurring in 50-60% of cases, with an even higher incidence when the patient is hypercalcemic (87,91). Breast tumors that produce PTHrP are more likely to metastasize, and breast cancers that metastasize to bone are even more likely to produce PTHrP (89,90,92). Breast cancers and their bone metastases commonly express the PTHrP receptor, and breast cell lines and primary cultures also commonly express PTHrP and its receptor (88,96-99). PTHrP and its peptides are secreted into blood by such breast cancers, and they can often serve as tumor and serum markers for this cancer (96--101). Prostate cancers robustly express PTHrP (94,95, 102-104). In the prostate, as in other tissues, PTHrP is processed into distinct peptides that have unique biologic effects (95,103,104). Prostatic expression of PTHrP is associated with regulatory effects and interactions that are important in the development and progression of prostate cancer (94,104). Furthermore, studies provide evidence for a role of PTHrP expression in the development of bone metastases in prostate cancer (102). However, serum measurements of PTHrP are not yet useful for clinical application in prostate cancer, because levels are seldom elevated (95,105). Furthermore, patients with prostate cancer seldom have osteolytic bone metastases and hypercalcemia; in fact, they are more likely to be hypocalcemic with osteoblastic metastases (95,100,102).

PTH AND PTHrP IMMUNOASSAYS /

157

PTHrP is commonly expressed in lung cancer, but, as in breast cancer, increased serum concentrations can be detected only in late stages of the disease (89,106,107). Nevertheless, this oncoprotein has important implications for the pathogenesis, diagnosis, and treatment of lung cancer. Abnormal PTHrP production by lung cancer can be demonstrated with specific immunochemical and nucleic acid probes (91,97,93). PTHrP is often produced in those lung cancers that metastasize to bone, and it is a common causative humoral agent in the patients that have hypercalcemia of malignancy (87,88,106,107). Lung cancer is intermediate to prostate and breast cancer in the incidence of"humoral hypercalcemia" (87,95,106). PTHrP and PTHrP peptides can have profound effects on growth and function of lung cells (107,112). However, as with prostate cancer, the majority of patients with lung cancer, including those with hypercalcemia, do not commonly have increases in serum PTHrP (90,106-108).

terminus through the receptor that it shares with PTH (5,84). Most patients with hypercalcemia and malignancy have PTHrP-expressing cancers (81,92). Despite the common pattern of PTHrP expression in many cancers, clinical secretion studies have revealed that many patients with PTHrP-expressing tumors and eucalcemia do not have elevated serum levels of PTHrP (90,113, 114). In general, most serum PTHrP assays do not yet have the sensitivity a n d / o r specificity to make them a clinically useful biomarker for the early course of the cancer, a problem compounded by the poorly defined normal range of PTHrP, discussed subsequently (89,91,92). In many ways, the current status of the development of PTHrP immunoassays is reminiscent of the comparably early days of PTH assay development, although PTHrP assay development is occurring at a faster pace (89-92). Accordingly, there is substantial promise that clinically useful assays for PTHrP will be eventually developed (see Chapters 3 and 6) for more details about PTHrP secretion).

Secretion of PTHrP

PTHrP Immunoassays

Despite studies demonstrating the common pattern of PTHrP expression in many cancers, especially breast, lung, and prostate, secretion studies have limited impact in that elevated levels of PTHrP are discovered relatively late in the course of the patient with hypercalcemia and malignancy (90,109,111). And the comm o n occurrence of PTHrP expression in prostate cancer is seldom accompanied by either hypercalcemia or elevated serum PTHrP levels (95,105). Thus, many patents who have PTHrP-expressing tumors fail to demonstrate abnormally increased serum PTHrP concentrations that are clinically useful. Most serum PTHrP measurements, especially solution immunoassays, do not have the sensitivity a n d / o r specificity for cancer-produced PTHrP forms to make them valuable biomarkers for the early diagnosis of many malignancies (89-92). However, as for PTHrP, midregion assays seem to be the most sensitive (89,90,113,114). In contrast to PTH, for which serum assays can often detect early parathyroid disease, serum levels of PTHrP are likely to be increased only when the cancer is advanced and the patient is hypercalcemic (91,92,110,111). In summary, PTHrP is produced by many normal and malignant tissues, and in these tissues, PTHrP expression commonly produces abnormal growth and proliferation (84,87,92). Through complex gene expression, mRNA splicing, and peptide processing of PTHrP, the h u m a n PTHrP gene expresses three native forms of the polypeptide and multiple processed peptides with distinct biologic properties (85,93). The hypercalcemic effect of PTHrP is mediated by its amino

It took over 30 years of development for the clinical application of serum PTH assays to be substantially realized, thus it is not surprising that current serum PTHrP assays still have clinical limitations. Nevertheless, there has been promising activity in this area of assay development. To correspond to actual and potential PTHrP processing, immunoassays for PTHrP have been designed to recognize peptides across the linear sequence of the native molecules. Investigators have essentially traversed the PTHrP molecule, developing solution (one-site) immunoassays for PTHrP(1-34), 1-40, 33-67, 37-74, 50-69, 53-84, 67-86, 109-138, 109-141, 127-141, and 141-173, and solid-phase (two-site) immunoassays for PTHrP(1-67), 1-72, 1-74, 1-83, 1-84, and 1-86, all of which have been applied to a variety of clinical studies (115-157). These many assays systems reflect the fact that, because of the complexity of PTHrP biosynthesis and processing, most PTHrP immunoassays have been developed empirically (91,92). It should be noted that PTH, as is the obverse for PTHrP, does not cross-react in these assays systems, because the homology between the two molecules is primarily limited to the first 13 amino acids (83). Despite their format differences, there is substantial uniformity among the different PTHrP assays in their clinical applications and characteristics. Most of the PTHrP assays can identify the majority of patients with hypercalcemia and PTHrP-producing tumors, but only the minority of patients with eucalcemia and cancer, including such patients with tumors demonstrated to

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CrtAeTER9

express PTHrP (89,90,98). PTHrP assays also demonstrate that, as is the case for PTH, the circulating forms of PTHrP are immunochemically heterogeneous (89-91). And like PTH immunoassays, PTHrP immunoassays that detect the midregion of the molecule seem to be the most sensitive (91,92,113). Although current assays can identify many patients with PTHrP-producing cancer, they do not yet seem to be able to identify the early course of the malignancy, where they would be most clinically useful (89,92).

PTHrP Immunoassay Development Solution Immunoassays The development of PTHrP immunoassays has, in many ways, mirrored the course of the development of PTH immunoassays, with region-specific solution immunoassays developed first, followed by the development of solid-phase, two-site immunoassays (122,129,156,157). Among the first solution immunoassays were aminoterminal PTHrP assays based on PTHrP (1-34) (91,156). These were followed by solution immunoassays based on the carboxy terminus and the midregion of PTHrP (129,151,157). These regional peptide designations were based on PTHrP(1-141), not PTHrP(1-173), so the carboxy terminus meant a peptide in the vicinity of PTHrP(109-141), and the midregion meant a peptide in the vicinity of PTHrP(36-67) (93,151). In general, these solution immunoassays could identify over half of the hypercalcemic cancer population, although a higher percentage was reported for some of them, with midregion assays identifying the most (89,90). Like PTH, midregion PTHrP assays resulted in higher PTHrP values than did amino-terminal assays (90). These assays also varied in their ability to detect serum PTHrP in normal subjects, ranging from some measuring it in none to others measuring it in all (91,92).

Two-Site Immunoassays Two-site immunoassays first focused on the amino PTHrP region, including PTHrP(1-34), 1-67, 1-74, and 1-84 (89-92,132,140,147,155). They had all of the advantages of the two-site PTH immunoassays discussed earlier, including improved specificity, freedom from serum protein-related artifacts, and technical ease (89). Some of them had improved clinical application, distinguishing better than solution immunoassays between normal subjects and patients with hypercalcemia and malignancy (90-92). With some two-site assays, more than 80% of patients with malignancy-associated hypercalcemia have elevated circulating PTHrP values (90,91). However, assays with greater sensitivity had more false positives, and the assays with lesser sensitivity had fewer false positives (92). There was less overlap between the two groups with intact assays, but the normal PTHrP

ranges tended to be somewhat arbitrary (89,90). The apparently improved clinical performance of two-site assays and their technical advantages have resulted in their wide clinical application (89-93). False positives aside, increased assay sensitivity is desirable for monitoring the course of the patient with a PTHrP-producing tumor, because that patient's pretreatment concentration will serve as the control for evaluation (90,93).

Specific Clinical Application of PTHrP Immunoassays Hypercalcemia and Malignancy PTHrP assay is most commonly used to establish pathogenesis in the patient with hypercalcemia, usually with cancer (91,92). In general, most patients with hypercalcemia and solid tumors have elevated serum PTHrP levels regardless of tumor type (85-92). This picture has evolved from early studies that suggested that squamous cell tumors were more likely to be associated with increased serum levels of PTHrP (89,90). In contrast, most patients with malignancy and normal calcium levels do not have elevated serum levels of PTHrP (87,90). Although there are exceptions to this generality, it holds true for most PTHrP assay systems (91,92). PTHrP seems to play only a minor role in hematologic malignancy, with the exception of adult T cell l e u k e m i a / l y m p h o m a (91,140,141). Although there is variability, in approximately half of these patients the hypercalcemia is associated with increased PTHrP expression by the cancer cells (89,90,140,141). This contrasts to myeloma and hypercalcemia, for which increased serum PTHrP is rare, even when the patient is hypercalcemic (89,141). Incidentally, the same situation seems to pertain to sarcoidosis (158). In contrast to PTHrP production in cancer, PTH expression by malignancy is now known to be so rare that it is reportable (89,91,159-166). In fact, an elevated serum PTH in the patient with malignancy (and the absence of renal failure and secondary hyperparathyroidism) most likely reflects coexisting PHPT. Several studies of PTHrP and hypercalcemia also assessed the presence or absence of skeletal metastases (89,97,126,156). In general, there can be a dissociation among these clinical events. Thus, many tumors producing PTHrP contribute to the development of hypercalcemia regardless of the presence or absence of skeletal metastases (89,100). PTHrP can thus act as a "humor" in the hypercalcemia of malignancy and stimulate osteoclastic bone resorption through the increased circulating levels derived from tumor expression (89,92). Of course, i n d e p e n d e n t of PTHrP production, a metastatic tumor can have the same effect, referred to as local osteolytic hypercalcemia (LOH) to distinguish it from the humoral hypercalcemia of malignancy (160).

PTH AND PTHrP IMMUNOASSAYS / Parathyroid adenomas or hyperplastic glands secondary to renal disease commonly express PTHrP mRNA (167-169). However, most reports demonstrate increased circulating levels of PTHrP only in secondary hyperparathyroidism, where accumulation of fragments can contribute to the measurement (88,109,143). As is the case in the secondary hyperparathyroidism of renal failure, carboxy-terminal PTHrP fragments accumulate more than the other fragments (109,134,143,157). The most important clinical relationship between PTH and PTHrP is the dissociation between the two found in hypercalcemia and malignancy, where serum PTH is suppressed and serum PTHrP is commonly increased (88).

Malignancy and Eucalcemia A more complicated relationship between PTHrP and malignancy seems to be present in cancer patients who are not hypercalcemic, as exemplified by prostate cancer (95,105,114). Prostate cells commonly express PTHrP (103,104). Furthermore, there seems to be a direct relationship between PTHrP expression and abnormal growth in that there is a directed gradient of PTHrP expression from normal cancer cells, through hyperplastic prostate cells, to malignant prostate cells (102,103). Despite this relationship, prostate cancer is rarely associated w i t h hypercalcemia, even though the tumor often metastasizes to bone, where it commonly produces osteoblastosis rather than osteolysis (95,100,102). It is interesting to speculate that this clinical riddle could be due to the prostate-specific processing of PTHrP into a peptide that inhibits osteoclast function, like peptides contained in PTHrP (107-141) (95). Nevertheless, serum PTHrP levels are not elevated in patients with prostate cancer, regardless of the absence or presence of bone metastases (89,95). Despite the disappointing lack of clinical value of serum PTHrP in the early stages of malignancy, where assays would be most valuable, some studies suggest that with appropriate specificity, PTHrP assays could be useful in early diagnosis (90). In one such study, circulating PTHrP levels were examined with three different immunoassays in 48 eucalcemic breast cancer patients (98). These immunoassays were directed against different parts of the PTHrP molecule. The methods used were a radioimmunoassay with antibodies directed against PTHrP(63-78), an immunofluorometric assay with antibodies against PTHrP (1-34) and PTHrP(38-67), and an immunoradiometric assay with antibodies against PTHrP(1-40) and PTHrP (38-72). PTHrP was detected by immunohistochemistry in tumors from nearly all patients. T h o u g h most patients had PTHrP levels indistinguishable from normal when measured by all three methods, 10% had increased serum levels in the IFMA. The IFMA thus

159

identified increased serum PTHrP forms in some patients with PTHrP-expressing breast cancer who were not hypercalcemic and presumably in the early course of their disease. By contrast, two other assays failed to distinguish between normal and breast cancer subjects, one being a commercial assay measuring PTHrP(1-74) and the other a research assay based on PTHrP (63-78). A hypothesis-setting explanation for this finding is that the forms/species of PTHrP secreted by the early breast cancers corresponded more closely to the PTHrP(1-67) form. This could indicate that there is a circulating form of PTHrP extending from the amino terminus to residues 58 or 66, where cleavage sites of basic amino acid residues are situated. This view is supported by studies that found no measurable serum levels of PTHrP(1-86) in unselected patients with breast cancer prior to surgery, although most of them had tumors with positive staining of PTHrP (89,92,157). Because there are cleavage sites at amino acid residues 66 (arginine) and 58 (arginine), one of the assays could be detecting a fragment extending from the amino terminus to one of these amino acid residues (85). However, these results do not explain the absence of hypercalcemia in the patients, because this effect is presumably due to PTHrP(1-34). Thus, it could be valuable to measure a shorter amino-terminal fragment of PTHrP, especially considering that there are several cleavage sites in this peptide and that PTHrP(1-36) is contained in a secretory form of the molecule.

Normal Range of Serum PTHrP and Other Problems The clinical limitations of current PTHrP immunoassays are exemplified by the contradictory studies regarding the circulation of PTHrP in normal individuals (91,92). In some studies, detectable levels of PTHrP are present in only a few normal individuals, while in other studies detectable levels of PTHrP are detected in most individuals (89,90). To add to the confusion, intermediate results have also been reported (91,109). This variability is not a function of assay sensitivity, because conflicting results have been reported by PTHrP immunoassays with both relatively greater and lesser respective sensitivities (89-95). A well-defined normal range for PTHrP, as for any clinical measurement, is obviously critical for meaningful interpretation of assay results. Some of the problems with contemporary PTHrP immunoassays may have a technical basis. Immunologic PTHrP activity may be labile and sensitive to serum proteases, and impaired renal function, c o m m o n in cancer, made confound PTHrP measurements because of the accumulation of PTHrP fragments, especially, as is the case for PTH, carboxy-terminal fragments (91,134). Studies of the normal values of PTHrP may be further

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CHAPTER9 TABLE 2

Routine Serum Tests in the Differential Diagnosis of the Patient with a Decreased Serum PTHa Diagnosis

Primary hypoparathyroidism Humoral hypercalcemia of malignancy (HHM) Nonparathyroid, non-HHM hypercalcemia Adynamic renal osteodystrophy Hypomagnesemia (severe)

Calcium

Phosphorus

BUN/creatinine

L

H/N

H

H/L/N

N N/H

H

H/N

N

L (relative to the degree of renal failure)

H

H

L

H/N

N

all, High; L, low; N, normal; BUN, blood urea nitrogen.

complicated by its lability, especially because these measurements are close to assay detection limits (91,92,170). Accordingly, proteases are used for sample collection in some assays, and, in all cases, the sample should be processed as quickly as practicable, following the guidelines outlined adapted from PTH assays (90,171). With some dramatic exceptions, circulating PTH concentrations in lactating women are essentially within the normal range, even though there are substantial concentrations of PTHrP in breast milk (118,172). In summary, the m e a s u r e m e n t of serum PTHrP is beginning to provide clinically useful information for the m a n a g e m e n t of the patient with cancer, especially when complicated by hypercalcemia (88-93). However, many hurdles have to be overcome before serum PTHrP assays are regarded by the clinician with the same confidence now conferred to contemporary PTH assays. The robust research activity in the development of PTHrP immunoassays promises to address the limitations of current assay systems, just as research during the past several decades has provided PTH immunoassays of great clinical value. C o n t e m p o r a r y assays exhibit increased serum PTHrP in the majority of patients in whom the malignancy causes hypercalcemia. This is usually a late clinical event of some diagnostic value but has relatively little impact on patient m a n a g e m e n t . W h e n developments lead to PTHrP immunoassays that have the specificity and sensitivity to measure putative tumor-specific forms of the native molecules and their derived peptides early in the course of the cancer, the promise of the clinical value of PTHrP assays in clinical cancer m a n a g e m e n t as well as diagnosis may be fulfilled. Table 2 summarizes the c o m m o n laboratory findings that can be useful in establishing the correct diagnosis in the patient with a decreased PTH, a substantial number of whom have increased serum PTHrP due to a PTHrP-producing tumor. The differential diagnosis of hypercalcemia and hypocalcemia are discussed in detail in Chapters 41 and 47).

As assay development proceeds, the combination of accurate and precise PTHrP and PTH immunoassays can help to establish the correct diagnosis in the patient with calcium and skeletal disorders. FDA-approved tests can be found on the Internet at www.accessdata.fda.gov.

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121. Yang KH, deOaoo AE, Soifer NE, Dreyer BE, Wy TL, Porter SE, Bellantoni M, Burtis WJ, Insogna KL, Burtis WJ, Insogna KLM, Broadus AE, et al. Parathyroid hormone-related protein: Evidence for isoform and tissue-specific posttranslational processing. Biochemistry 1994;33:7460-7469. 122. Ratcliffe WA, Norbury S, Scott RA, Heath DA, Ratcliffe JG. Immunoreactivity of plasma parathyrin-related peptide: Three region-specific radioimmunoassays and a two-site immunoradiometric assay compared. Clin Chem 1991;37:1781-1787. 123. Ratcliffe WA, Norbury S, Heath DA, Ratcliffe JG. Development and validation of an immunoradiometric assay of parathyrinrelated protein in unextracted plasma. Clin Chem 1991;37:678-685. 124. Imamura H, Sato K, Shizume K, Satoh T, Kasono K, Ozawa M, Ohmura E, Tsushima T, Demura H. Urinary excretion of parathyroid hormone-related protein fragments in patients with humoral hypercalcemia of malignancy and hypercalcemic tumor-bearing nude mice. J Bone Miner Res 1991 ;6:77-84. 125. Kitazawa S, Fukase M, Kitazawa R, Takenaka A, Gotoh A, Fujita T, Maeda S. Immunohistologic evaluation of parathyroid hormonerelated protein in human lung cancer and normal tissue with newly developed monoclonal antibody. Cancer 1991 ;67:984-989. 126. Grill V, Ho P, BodyJJ, Johanson N, Lee SC, Kukreja SC, Moseley JM, Martin TJ. Parathyroid hormone-related protein: Elevated levels in both humoral hypercalcemia of malignancy and hypercalcemia complicating metastatic breast cancer. J Clin Endocrinol Metab 1991;73:1309-1315. 127. Pandian MR, Morgan CH, Carlton E, Segre GV. Modified immunoradiometric assay of parathyroid hormone related protein: Clinical application in the differential diagnosis of hypercalcemia. Clin Chem 1992;38:282-288. 128. Grill V, Murray RM, Ho PW, Santamaria JD, Pitt P, Potts C, Jerums G, Martin TJ. Circulating PTH and PTHRP levels before and after treatment of tumor induced hypercalcemia with pamidronate disodium (APD). J Clin Endocrinol Metab 1992;74:1468-1470. 129. Ratcliffe WA, Bowden SJ, Emly J, Hughes S, Ratcliffe JG. Production and characterization of monoclonal antibodies to the mid-region 37-67 sequence of parathyroid hormone-related protein. J Immunol Methods 1992;146:33-42. 130. Blind E, Raue F, Goltzman J, Schmidt-Gayk H, Kohl B, Ziegler R. Circulating levels of midregional parathyroid hormonerelated protein in hypercalcemia of malignancy. Clin Endocrinol. 1992;37:290-297. 131. Ratcliffe WA, Hutchesson AC, Bundred NJ, Ratcliffe JG. Role of assays for parathyroid-hormone-related protein in investigation of hypercalcemia. Lancet 1992;339:164-167. 132. Burtis WJ, Fodero JE Gaich G, Debeyssey M, Stewart AF. Preliminary characterization of circulating amino-and carboxyterminal fragments of parathyroid hormone-related peptide in humoral hypercalcemia of malignancy. J Clin Endocrinol Metab 1992;75:1110-1114. 133. Bucht E, Eklund A, Toss G, Lewensohn R, Granberg B, Sjostedt U, Eddeland R, Torring O. Parathyroid hormone-related peptide, measured by a midmolecule radioimmunoassay, in various hypercalcemic and normocalcemic conditions. Acta Endocrinol 1992; 127:294-300. 134. Savage MW, Fraser WD, Bodmer CW, Ginty AF, Gallagher JA, Robinson J, Williams G. Hypercalcemia due to parathyroid hormone-related protein: Long-term circulating levels may not reflect tumour activity. Clin Endocrino11993;39:695-698. 135. Fraser WD, Robinson J, Lawton R, Durham B, Gallacher sJ, Boyle IT, Beastall GH, Logue FC. Clinical and laboratory studies of a new immunoradiometric assay of parathyroid hormonerelated protein. Clin Chem 1993;39:414-419.

136. Blind E, Raue F, Meinel T, Pecherstorfer M, Rath U, Schmidt-GGayk H, Kohl B, Ziegler R. Diagnostic significance of parathyroid hormone-related protein in tumor patients with hypercalcemia. Dtsch Med Wochenschr 1993;118:330-335. 137. Blind E, Raue F, Meinel T, Bucher M, Manegold C, Ebert W, Vog-Moykopf I, Ziegleer R. Levels of parathyroid hormonerelated protein in hypercalcemia of malignancy: Comparison of midregional radioimmunoassay and two-site immunmoradiometric assay. Clin Invest 1993;71:31-36. 138. Mune T, Katakami H, Morita M, Noguchi S, Ushiroda Y, Matsukura S, Yasuda K, Miura K. Increased serum immunoreactive parathyroid hormone-related protein levels in chronic hypocalcemia. J Clin Endocrinol Metab 1994;78:575-580. 139. Levin GE, Nisbet JA. Stability of parathyroid hormone-related protein and parathyroid hormone at room temperature. Ann Clin Biochem 1994;31:497-500. 140. Ikeda K, Ohno H, Hane M, Yokoi H, Okada M, Honma T, Yamada A, Tatsumi Y, Tanaka T, Saitoh T, et al. Development of a sensitive two-site immunoradiometric assay for parathyroid hormone-related peptide: Evidence for elevated levels in plasma from patients with adult T-cell leukemia/lymphoma and B-cell lymphoma. J Clin Endocrinol Metab 1994;79:1322-1327. 141. Yamaguchi K, Kiyokawa T, Watanabe T, Ideta T, Asayama K, Mochizuki M, Blank A, Takatuski K. Increased serum levels of C-terminal parathyroid hormone-related protein in different diseases associated with HTLV-1 infection. Leukemia 1994;8:1708-1711. 142. Hutchesson AC, Hughes SV, Bowden SJ, Ratcliffe WA. In vitro stability of endogenous parathyroid hormone-related protein in blood and plasma. Ann Clin Biochem 1994;31:35-39. 143. Burtis WJ, Dann P, Gaich GA, Soifer NE. A high abundance midregion species of parathyroid hormone-related protein: Immunological and chromatographic characterization in plasma. J Clin Endocrinol Metab 1994;78:317-322. 144. Sagarra E, Villabona C, Bonnin R, Moliner R, Merino FJ, Sahun M, Soleer J. The value of the parathyrin-related protein (PTHRP) in the diagnosis of cancer-associated hypercalcemia. Med Clin 1995;105:450--454. 145. Ramirez MM, Fraher LJ, Goltzman D, Hendy GN, Matthews SG, Sangha R, Challis JR. Immunoreactive parathyroid hormonerelated protein: Its association with preterm labor. EurJ Obstet Gynecol Reprod Biol 1995;63:21-26. 146. Bucht E, Rong H, Bremme K, Granberg B, Rian E, Torring O. Midmolecular parathyroid hormone-related peptide in serum during pregnancy, lactation and in umbilical cord blood. E u r J Endocrinol 1995;132:438-443. 147. Minebois-Villegas A, Audran M, Lortholary A, Legrand E, Boux De Casson-Raimbeau F, Jallet E Performances of two kits for parathyroid hormone-related peptide (PTHRP) assay in the additional study of malignant hypercalcemias. Pathol Biol 1995;43: 799-805. 148. Caplan RH, Wickus GG, Sloane K, Silva PD. Serum parathyroid hormone-related protein levels during lactation. J Reprod Med 1995;40:216-218. 149. Seguara Dominguez A, Andrade Olivie MA, Rodriguez Sousa T, Terron Alvarez ML, Rodriguez Perez D, Alvarez Novoa R, Garcia-Mayor RV. Plasma parathyroid hormone related-protein levels in patients with cancer, normocalcemic and hypercalcemic. Clin Chim Acta 1996;244:163-172. 150. Sowers ME Hollis BW, Shapiro B, Randolph J, Janney CA, Zhang D, Schork A, Crutchfield M, Stanczyk F, Russell-Auleet M. Elevated parathyroid hormone-related peptide associated with lactation and bone density loss. JAMA 1996;276:549-554. 151. Nagasaki K, Otsubo K, Kajimura N, Tanaka R, Watanabe H, Tachimori Y, Kato H, Yamaguchi H, Saito D, Watanabe T, Adachi I, Yamaguchi K. Circulating parathyroid hormone-

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CHAPTER10 Physiology of Calcium H o m e o s t a s i s

EDWARD M. BROWN Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115

INTRODUCTION

briefly discussed is an e m e r g i n g body of data indicating that C a 2+ o homeostasis does n o t operate in isolation but is integrated with o t h e r homeostatic systems, including those regulating the metabolism of water, sodium chloride, and even protein metabolism.

Calcium i o n s ( C a 2+) play n u m e r o u s critical physiologic and biochemical roles, in b o t h the intra- and extracellular spaces. Ultimately, all intracellular C a 2+ originates from C a 2+ within the extracellular fluid. It is not surprising, therefore, that free-living terrestrial organisms, including h u m a n s and o t h e r mammals, have developed a c o m p l e x homeostatic system that maintains near constancy of the level of free extracellular calcium (Ca2o+). F u r t h e r m o r e , all calcium within the extracellular fluids (ECFs) and elsewhere in the body is ultimately derived from dietary calcium. Once ingested and absorbed, excess calcium can be stored within b o n e or lost in the urine w h e n m o r e C a 2+ is available than is n e e d e d for intra- or extracellular processes. Conversely, if absorbed dietary calcium is insufficient for the body's needs, it can be withdrawn from skeletal reserves a n d the loss of urinary calcium can be mitigated by appropriate physiological responses. Thus maintaining C a 2+ o homeostasis involves the carefully orchestrated control of calcium's m o v e m e n t s into and out of the body via the gastrointestinal (GI) tract and kidney, respectively, as well as into and out of bone, so as to ensure near constancy of C a 2+ o while at the same time providing the Ca 2+ n e e d e d for this ion's diverse intracellular and extracellular functions. The purpose of this chapter is to review the mechanisms underlying Ca2o+ homeostasis, particularly those t h r o u g h which changes in the level of C a 2+ o within the bodily fluids are sensed and transduced into changes in the functions of kidney, bone, and GI tract so as to normalize Ca 2+ Also o

The Parathyroids, Second Edition

BIOLOGIC ROLES OF CALCIUM Calcium is an essential e l e m e n t t h r o u g h o u t the phylogenetic tree by virtue of its myriad biologic roles (Table 1). C a 2+ functions as a critical intracellular seco n d messenger that regulates n u m e r o u s cellular functions, including processes as diverse as h o r m o n a l secretion, muscle contraction, n e u r o n a l excitability, glycogen metabolism, and cell division (1-3). Many of these functions are the result of the interaction of C a 2+ with its intracellular binding proteins, e.g., calmodulin, and the c o n s e q u e n t activation of enzymes and o t h e r intracellular effectors systems (1-3). The free cytosolic calcium concentration (CaZi+) in cells u n d e r resting conditions is on the o r d e r o f 100 nM. The level of 2+. Ca i as controlled by diverse channels, pumps, and o t h e r transport systems that regulate the movements of C a 2+ into and out of the cytosol and between various intracellular c o m p a r t m e n t s (1-3). Consistent with its role as an i m p o r t a n t intracellular second messenger, C a 2+ i can increase by as m u c h as lO0-fold (i.e., to a level of 1-10 ~zM) during cellular activation. Such rises in Ca i are the result of uptake of extracellular Ca 2+ t h r o u g h Ca2+-per meable plasma m e m b r a n e channels, release of Ca 2+ from its intracellular stores, such as the endoplasmic

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Copyright © 2001 John R Bilezikian, Robert Marcus, and Michael A. Levine.

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CHAPTER10 TABLE 1 Biologic Calcium Form Intracellular Insoluble

Location

Mass (% of total)

Function

Plasma membrane, endoplasmic reticulum, mitochondria, other organelles

9 g (0.9%)

Structural integrity, storage

Soluble

Cytosol, nucleus

0.2 mg

Action potentials, contraction and motility, metabolic regulation, cytoskeletal function, cell division, secretion

Extracellular Insoluble

Bones and teeth

1-2 kg (99%)

Protection, locomotion, ingestion of minerals and other nutrients, mineral storage

Soluble

Extracellular fluid

1 g (0.1%)

Blood clotting, kinin generation, regulation of plasma membrane potential, exocytosis, a contraction a

aThe activation of exocytosis and muscle contraction, in part, depend on cellular uptake of extracellular calcium.

reticulum, or both. In spite the importance of intracellular Ca 2+ in its various forms in controlling cellular metabolism, this compartment only represents about 1% of total bodily Ca 2+ (4). In contrast to C a 2+ i, Ca 2+ o is on the order o f l mM. It is carefully regulated by a complex homeostatic mechanism with two key elements: (1) cells that secrete the Ca 2+ o -regulating hormones, parathyroid h o r m o n e (PTH), calcitonin (CT), and 1,25-dihydroxyvitamin D [ 1,25 (OH) 2° ] mnamely, the chief cells of the parathyroid glands, thyroidal parafollicular cells, and proximal tubular cells of the kidney, respectively, as well as specialized Ca2+-transporting/translocating cells in the intestine, skeleton, and kidney (4-7). This system controls the flow of calcium ions into and out of the body as well as between various bodily compartments, especially between the skeleton and extracellular fluid. The rigid control of the level of Ca2o+ ensures a constant supply of C a 2+ in bodily fluids for the vital intracellular functions of this crucial divalent cation. Ca2o+ has several other key roles, which include maintaining intercellular adhesion, contributing to the integrity of the plasma membrane, and promoting the clotting of blood, further emphasizing the importance of maintaining near constancy of C a 2+ o . The total quantity of soluble C a 2+ i in the ECF, however, like intracellular C a 2+, o n l y repre-

sents a tiny fraction of total bodily calcium (about 0.1%; see Table 1). Most calcium in the body (>99%) is deposited as calcium phosphate salts within the skeleton, where it serves two key functions. First, the skeleton serves as a protective covering for vital, potentially vulnerable internal organs (i.e., within the cranium or thoracic cavity) and affords a rigid but articulated framework that facilitates locomotion and other bodily movements (6,7). Second, it provides an almost inexhaustible reservoir of calcium and phosphate ions for those times of need when intestinal absorption and renal reabsorption are not sufficient to ensure adequate levels of these ions within the ECF to support the body's numerous bodily functions that are d e p e n d e n t on them (6). Thus, though C a 2+ within all bodily compartments plays critical roles, the c o m p o n e n t that is most closely regulated by the mineral ion homeostatic system and, therefore, affects all other forms of calcium within the body is Ca2o+. A clear understanding of the mechanism involved in extracellular Ca 2+ homeostasis requires a discussion of the various forms of C a 2+ present in the blood and other bodily fluids, a consideration of how Ca 2+ moves between the organism and the envir o n m e n t and a m o n g various bodily compartments (i.e., overall C a 2+ balance), and, finally, the mecha-

PHYSIOLOGY OF C a 2+ HOMEOSTASIS

Calcium is needed for the growth of both soft and hard tissues (9). Therefore, during childhood, more calcium enters the body through the GI tract than is lost it through the kidneys, GI tract, and perspiration (loss of C a 2+ in the sweat is generally not significant). That is, overall calcium balance is positive. Calcium balance is only truly zero (e.g., the C a 2+ homeostatic system precisely balances intake and output o f Ca 2+) for about two decades after skeletal growth ceases, at about age 20 years. Beginning as early as age 30-40, total bodily calcium begins to decrease, primarily because of loss of skeletal C a 2+ in the absence of any significant change in serum ionized C a 2+ ( 5 , 6 ) . Figure 1 illustrates calcium balance in a hypothetical young adult in zero Ca 2+ balance. Of the 1000 mg of elemental calcium that this individual ingests on a daily basis, about 30% (300 mg) actually undergoes intestinal absorption. About 100 mg of C a 2+ is lost by intestinal secretion, so that net absorption is 200 mg. Approximately 500 mg of C a 2+ e n t e r s and leaves the skeleton daily as a result of bone formation and resorption, respectively. To maintain mineral balance, therefore, 200 mg of C a 2+ m u s t exit the body via the kidneys. The C a 2+ lost in the urine comprises only 2% of that filtered daily (i.e., 10 g), which speaks to the kidney's remarkable efficiency in reclaiming, via tubular reabsorption, C a 2+ filtered at the glomerulus (10).

F O R M S O F C A L C I U M IN B L O O D Although the level of Ca2o+ in the interstitial fluids bathing the various tissues of the body is perhaps most relevant to Ca2o+ homeostasis, it cannot be readily measured. Instead, the total or ionized s e r u m C a 2+ activity is the parameter that is determined. Of the serum total calcium concentration, about 47% is in an ionized or free form. An equivalent a m o u n t (---46%) is b o u n d to various plasma proteins, especially albumin, which contains about 75% of b o u n d serum calcium (with the rest being b o u n d to various globulins), and the r e m a i n d e r (5-10%) is complexed to small anions, comprising phosphate, citrate, bicarbonate, and others (8). The fraction of s e r u m C a 2+ that is ultrafilterable comprises both free and complexed Ca 2+ (e.g., that b o u n d to small anions), but only the first of these is metabolically active (i.e., available for uptake by cells).

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O V E R A L L Ca 2+ B A L A N C E D U R I N G T H E L I F E CYCLE

nisms that control these movements. A critical element within the last of these is the system by which the body recognizes and responds to (i.e., "senses") changes in Ca2o+ (7). This Ca 2+ o -sensing mechanism enables direct a n d / o r indirect [i.e., via alterations in PTH, 1,25 (OH)2 D, and CT] regulation of the effector systems in intestine, kidney, and bone that modulate C a 2+ transport into and out of the ECF so as to restore Ca2o+ homeostasis (7).

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FIG. 1 Overall Ca 2÷ balance in a hypothetical normal individual. Of the 1 g of elemental Ca 2÷ ingested daily, there is net absorption of 200 mg (300 mg gained by absorption, 100 mg lost by intestinal secretion). Balance is achieved, therefore, by renal excretion of 200 mg of Ca 2÷, because equal amounts of Ca 2÷ are laid down and removed from the skeleton on a daily basis in this person. ECF, Extracellular fluid. (Adapted from Brown EM, LeBoff MS. Pathophysiology of hyperparathyroidism. In: Rothmund M, Wells SA, Jr, eds. Progress in surgery. Parathyroid surgery, Vol 18. Basel:Karger, 1986:13-22.)

170

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CICAeTER10

Furthermore, only modest alterations in the percentage of the filtered calcium that is reabsorbed (i.e., a decrease to 1%, or an increase to 3%, for instance) can have a substantial impact on overall Ca 2+ balance (11). The increasing recognition that osteoporosis is a major public health p r o b l e m in later life has e n g e n d e r e d a great deal of interest in defining optimal levels of calcium intake as a function of age, h o r m o n a l status, and o t h e r factors. Currently designated adequate intakes (AIs) of calcium in the United States for various individuals are as follows: 500 mg, 1-3 years old; 800 mg, 4-8 years old; 1300 mg, 9-18 years old; 1000 mg, 19-50 years old; and 1200 mg, > 5 0 years old. For pregnant w o m e n u n d e r 19 years of age the AI is 1300 mg, and is 1000 mg thereafter (12). Intake of these quantities should not be considered as dietary supplements but rather a nutritional r e q u i r e m e n t for skeletal health.

HORMONAL CONTROL C a 2+ o HOMEOSTASIS

that maintains near constancy of Ca2o+ t h r o u g h cal2+. ciotropic h o r m o n e - i n d u c e d and direct, Ca o -induced changes in the GI, renal, a n d / o r skeletal handling of Ca 2+, as illustrated in Fig. 2 (6,7). Overall C a 2+ o homeostasis can be u n d e r s t o o d in terms of three general principles: (1) The first priority of the homeostatic m e c h a n i s m is that it maintains a n o r m a l level of Ca o2+. (2) W h e n there are m o d e r a t e stresses on the system, changes in the intestinal a n d / o r renal h a n d l i n g of Ca 2+ are usually sufficient to sustain Ca2o+ homeostasis without alterations in skeletal Ca 2+ balance. (3) In the presence of severe hypocalcemic stresses, skeletal Ca 2+ is " mobilized in order to m a i n t a i n Ca2o+ homeostasis. This loss of skeletal calcium, if it persists for a sufficiently long time (e.g., m o n t h s to years), eventually compromises the structural integrity of the skeleton. The Ca2o+ homeostatic system, as n o t e d previously, has two essential elements. The first is a group of several different cell types that are capable of sensing changes in Ca2o+ and r e s p o n d i n g with homeostatically 2+ relevant alterations in their o u t p u t of Ca o -regulating h o r m o n e s [PTH, calcitonin, and 1,25(OH)2D] (7). The second key c o m p o n e n t is the effector cells that control the renal, intestinal, and skeletal handling of

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FIG. 2 The hormonal regulation of calcium homeostasis by PTH and vitamin D (solid lines and arrows). Ca2o÷ regulates PTH secretion in an inverse manner. PTH, in turn, modulates renal handling of Ca2o÷and phosphate as well as renal production of 1,25(OH)2D. PTH and 1,25(OH)2D act synergistically to mobilize Ca2o÷ and phosphate from bone, whereas 1,25(OH)2D increases intestinal absorption of both ions. Also shown are direct actions of calcium and phosphate ions on tissues that participate in maintaining mineral ion homeostasis (dashed lines and arrows), showing the roles of these ions as extracellular first messengers. For additional details, see text. (Reproduced with permission from Brown EM, Pollak M, Hebert SC. Cloning and characterization of extracellular Ca2+-sensing receptors from parathyroid and kidney. Molecular physiology and pathophysiology of Ca 2+ sensing. Endocrinologist 1994;4:419-426.)

PHYSIOLOGYor Ca 2+ HOMEOSTASIS / C a 2+. As described in more detail below, the transloca-

tion of calcium and phosphate ions into or out of the extracellular fluid by these tissues is regulated by PTH, calcitonin, and 1,25 (OH)2D as well as by mineral ions. The homeostatic system operates as follows: In the example shown (Fig. 2), a slight decrement in Ca2o+ evokes a p r o m p t increase in the secretory rate for PTH by the parathyroid chief cells. This h o r m o n e has several key actions on the kidney, including promoting phosphaturia (6), increasing distal tubular C a 2+ reabsorption, and enhancing the biosynthesis of 1,25 (OH) 2° from 25-hydroxyvitamin D [25(OH)D] (13-15). The ensuing increase in the circulating 1,25(OH)zD level directly stimulates intestinal absorption of C a 2+ and phosphate by i n d e p e n d e n t transport systems (15). PTH and 1,25 (OH)2 ° also synergistically increase the net release of skeletal C a 2+ and phosphate (6). The increased movement of C a 2+ into the ECF from bone and intestine, coupled with PTH-induced retention of this ion by the kidney, restore the circulating level of Ca2o+ to normal, thereby reducing PTH release and closing the negative-feedback loop. 1,25(OH)zD, synthesized in response to PTH, also directly reduces PTH synthesis and secretion (16), and contributes in this m a n n e r to the negative-feedback regulation of parathyroid function by the homeostatic system. Excess phosphate mobilized from bone and intestine undergoes urinary excretion in response to the phosphaturic action of PTH. As indicated in Fig. 2, both C a 2+ and phosphate ions have direct effects on several of the cells and tissues that participate in mineral ion metabolism. For instance, Ca2o+ not only inhibits PTH secretion but also directly reduces the proximal tubular production of 1,25 (OH) 2o (17), enhances the function of osteoblasts (18), and inhibits osteoclastogenesis (19) and osteoclastic bone resorption (20). Phosphate ions diminish the 1-hydroxylation of 25(OH) vitamin D, promote bone formation, inhibit bone resorption (7), and also stimulate several aspects of parathyroid function (21,22), as described in more detail later. These actions may well play important roles in mineral ion homeostasis by enabling not only the hormone-secreting but also the effector elements of the system to sense changes in the local levels of these ions in the ECF and, therefore, to respond in a physiologically relevant way. Indeed, because of their direct effects on cells participating in mineral ion homeostasis, Ca 2+ and phosphate can be viewed as acting in hormonelike roles as extracellular first messengers (7). 2+ Though the cloning of the Cao-sensing receptor (CaSR), which mediates the direct actions of C a o2+ on the functions of parathyroid glands, kidney, and several other tissues, has clarified substantially the m a n n e r in which these cells sense C a 2+ o (23), the mechanisms underlying phosphate sensing remain obscure.

171

R E G U L A T I O N OF PARATHYROID H O R M O N E S E C R E T I O N BY Ca2o+ A N D O T H E R FACTORS The Overall Secretory R e s p o n s e o f the Parathyroid Cell to Alterations in Ca2o+ The parathyroid cell manifests a hierarchy of responses to decreases in Ca2o+ that permit it to m o u n t a progressively larger secretory response that is appropriate for the rapidity, magnitude, and duration of the hypocalcemic stress that elicited this response (24). The most rapid response is the release of preformed PTH stored within secretory granules. This response occurs within seconds and can persist for as long as 60-90 minutes before these stores are completely depleted. This immediate secretory response exhibits a steep inverse sigmoidal relationship between PTH secretion and the level of Ca o2+ (Fig. 3) (24). Such a curve can be described by four parameters--maximal secretory rate at low Ca2o+ (parameter A), slope at the midpoint (parameter B), midpoint or "set point" (parameter C, the level of Ca2o+ half-maximally suppressing PTH), and minimal secretory rate at high Ca2o+ (parameter D) (25). The steepness of the curve contributes importantly to the nearly constant level of C a 2+ o that is maintained u n d e r normal circumstances (e.g., the percent coefficient of variation of Cao2+ is on the order of 1.5% in normal persons). The set point is also a key parameter that is the major determinant of the level at which Ca2o+ is "set," thereby serving as one of the body's key "thermostats" for Ca2o+, or "calciostats" (24). There are several additional features of the acute secretory response of the parathyroid cell to changes in Ca2o+ that may have an impact on the target tissues of PTH and the homeostatic system's overall response to alterations in C a o2+ (24). That is, PTH levels in vivodepend not only on Ca2o+per se, but also on the rate of change of C a o2+, particularly when it is decreasing (26). This "rate-dependence" is apparent when Ca2o+ is falling rapidly, which elicits a more vigorous secretory response than when Ca2+o decreases slowly. This dependence of PTH output on rate of change of C a o2+ may permit the parathyroid cell to m o u n t a more vigorous response, which would be homeostatically appropriate, when C a o2+ is diminishing rapidly. There also appears to be a "direction-dependence" or hysteresis to the secretory response of PTH to changes in C a o2+ (24). This hysteresis is manifested by the higher levels of PTH that are observed when C a o2+ is falling than when it is rising. Moreover, similar to the rate dependence of PTH secretion, it may be homeostatically relevant because it engenders higher levels of PTH when those are needed (i.e., when C a o2+is falling) than when they are not (e.g. , when C a o2+ is increasing). The parathyroid cell exhibits several other adaptive changes that, by virtue of their occurrence in a graded

172

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CHAPTER10

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greater expression of the PTH gene, owing both to increased transcription and enhanced stability of the messenger RNA (mRNA) encoding preproPTH, which takes place within a time frame of several hours to about a day (16,24). The resultant increase in the mRNA level for preproPTH is probably also accompanied by a more general increase in the parathyroid cell's biosynthetic capacity, particularly when hypocalcemia persists for days or longer, which produces the anticipated morphologic alterations (e.g., greater prominence of organelles that participate in hormonal biosynthesis, such as the rough endoplasmic reticulum and Golgi apparatus) (24). Finally, over several days to weeks or longer, the initiation of parathyroid cellular proliferation increases the total number of parathyroid chief cells and, as a consequence, the total secretory capacity for PTH by manyfold. Increases rather than decreases in Ca2o+ produce exactly the opposite changes in these various aspects of parathyroid cell function, although the time course may not be identical. That is, though the inhibition of PTH secretion by a rise in C a 2+ o takes place essentially immediately, the parathyroid gland shows a rather sluggish and, in all likelihood, incomplete capacity to rid itself of excess parathyroid chief cells once a stimulus to chief cell hyperplasia has abated ( 27,28 ).

The Molecular Mechanism of Ca2o+ Sensing by Parathyroid, Kidney, and Other Cells The technique of expression cloning in Xenopus laevis oocytes permitted the isolation of a phosphoMINIMUM

FIG. 3 (A) Relationship between PTH secretion and Ca2o+ in normal human parathyroid cells. Dispersed parathyroid cells were prepared and incubated with the levels of Ca2o+indicated. PTH released into the medium was then determined by radioimmunoassay. (B) The four parameters that describe the relationship between PTH secretion and Ca2o+: maximal (parameter A) and minimal secretory rates (parameter D) at low and high Ca 2+ concentrations, respectively, slope of the curve at its midpoint (parameter B), and set-point (parameter C, the level of Ca2o+ producing half of the maximal inhibition of PTH release). [(A) From Brown EM. In: Brenner BM, Stein H, eds. Contemporary issues in nephrology, Volume 2. Divalent ion homeostasis. New York:Churchill-Livingstone, 1983.]

and sequential manner, increase its overall capacity to produce biologically active PTH (24). The first is decreased intracellular degradation of PTH, so that a larger fraction of the secreted h o r m o n e is intact, bioactive PTH(1-84), which occurs within 20-30 minutes after exposure to hypocalcemia. The next response is

inositide-coupled, Ca 2+ o -sensing receptor (CaSR) from bovine parathyroid that is thought to represent the molecular mechanism by which parathyroid, kidney, and other cells sense Ca2o+ (23). The CaSR has a large amino-terminal extracellular domain, which is the major site where the binding of Ca2o+ takes place. Seven membrane-spanning helices follow that are characteristic of the superfamily of G protein-coupled receptors, and, finally, a cytoplasmic carboxy (C)-terminal domain (Fig. 4). The intracellular portions of the CaSR transduce the Ca2o+ signal into alterations in various intracellular second-messenger systems (29) that regulate key cellular processes involved in Ca2o+ homeostasis, such as hormonal secretion or renal tubular calcium reabsorption. The calcium-sensing receptors play a key role in C a 2+ o sensing by parathyroid and kidney, as proved by the demonstration that the CaSR harbors inactivating or activating mutations in several human genetic diseases manifested by abnormal Ca2o+ sensing (30). In familial hypocalciuric hypercalcemia (FHH), persons with heterozygous inactivating mutations of the CaSR (31) exhibit mild to moderate hypercalcemia accompanied by inappropriately normal (e.g., nonsuppressed) circu-

PHYSIOLOGY OF C a 2+ HOMEOSTASIS

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lating PTH levels and normal or even frankly low levels of urinary C a 2+ excretion (32). In contrast, persons with homozygous FHH present clinically as neonatal severe hyperparathyroidism (NSHPT) and have severe hypercalcemia accompanied by marked hyperparathyroidism (32). Finally, families have been identified with an autosomal dominant form of hypocalcemia that is accompanied by relative hypercalciuria (e.g., inappropriately high for the prevailing level of Ca2o+), in which affected persons harbor activating mutations in the CaSR, providing further proof of its importance in C a 2+ o sensing in parathyroid and kidney (33). In addition to its presence in the parathyroid chief cell, the CaSR is also expressed along nearly the entire renal tubule (34,35). It is present at the highest levels in the cortical thick ascending limb (CTAL; a segment of the nephron that is probably responsible for the abnormal renal C a 2+ handling in FHH) (34,35). The calciumsensing receptor's presence in the kidney likely provides a molecular basis for several of the longrecognized but poorly understood direct actions of

C a 2+ o on renal function, including the impaired urinary concentrating capacity present in some hypercalcemic patients) (36). In the thyroid, the CaSR resides almost solely in the calcitonin-secreting (C) cells and likely mediates the stimulatory effect of high levels of C a 2+ o on CT secretion (37,38).

Other Factors Modulating Parathyroid Function In addition to Ca2o+, several other factors, including vitamin D metabolites (especially 1,25 (OH) 2D), catecholamines and other biogenic amines, prostaglandins and peptide hormones, and phosphate and monovalent cations (e.g., potassium and lithium), also modulate PTH secretion (16,39). Of these, the most physiologically relevant are probably 1,25 (OH) 2o and phosphate. 1,25(OH)zD is thought to play an important role in the longer term (over days or longer) control of parathyroid function, tonically reducing PTH secretion (40,41), diminishing expression of the PTH gene (16,42), and probably inhibiting parathyroid

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cellular proliferation (43). Thus actions of PTH on its target tissues produce negative-feedback regulation of parathyroid cellular function not only by raising Ca2o+ but also by enhancing the synthesis of 1,25 (OH)2 D , which then directly exerts negative feedback actions on parathyroid function. Recent studies in vitro have shown that elevating and decreasing the ambient phosphate concentration will increase or reduce, respectively, PTH secretion (possibly indirectly through alterations in PTH gene expression), PTH gene expression, and parathyroid cellular proliferation (16,22).

EFFECTS OF PTH O N Ca2o+-REGULATING TISSUES Control of Gastrointestinal Ca 2+ Absorption The net amount of Ca 2+ absorbed from the GI tract is the difference between the amounts moving from lumen to plasma (absorption) and plasma to lumen (secretion) (44). The latter, which is called endogenous fecal calcium, is about 100 m g / d a y and varies little as a function of Ca balance. Ca absorption is the result of both passive diffusion across the intestinal mucosa via the paracellular route and active, transcellular transport. The passive, paracellular diffusion of C a 2+ is concentration dependent and nonsaturable; it accounts for absorption of approximately 10-15% of dietary C a 2+ (i.e., 100-150 m g / d a y of ingested C a 2+ when dietary C a 2+ is 1000 mg/day). The active transcellular component of C a 2+ absorption is a saturable, carrier-mediated mechanism regulated by 1,25(OH)2D. It involves apical uptake of calcium by a CaZ+-permeable channel(s) (45), transcellular movement by an incompletely understood mechanism that likely involves the intracellular Ca2+-binding protein, calbindin DgK, and then eventual extrusion of calcium at the basolateral cell surface by the CaZ+-ATPase and, perhaps, the Na+-Ca 2+ exchanger (46). The highest density of sites of active C a 2+ absorption is in the proximal small intestine, i.e., d u o d e n u m (44,47). There is vitamin D-responsive C a 2+ absorption in more distal segments of the intestine as well, including both the small intestine (ileum > jejunum) and the proximal large bowel (44). Because these segments of the GI tract are much longer than the duodenum, they may well contribute significantly to overall C a 2+ absorption. After administering 1,25 (OH)2D to vitamin D-depleted animals, GI absorption of C a 2+ rises over the next several hours. This increase is paralleled, in general, by increases in the levels of several intestinal vitamin D-dependent proteins, including calbindin DgI~, alkaline phosphatase, and Ca -Mg -ATPase (44,47). 1,25(OH)zD appears to stimulate both the influx and the egress of C a 2+ from the intestinal epithelial cells (48,49). Therefore, it will be of •

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interest to examine the effects of vitamin D nutrition on the expression of the Ca -permeable influx channel thought to mediate uptake of Ca 2+ from the GI lumen into the absorptive cells (45). The bulk of phosphate absorption by the intestine takes place in the small bowel through a vitamin D-responsive mechanism, which is distinct from that for Ca 2+ (6). Even in vitamin D deficiency, nearly half of total dietary phosphorus is still absorbed. The less stringent control of phosphate absorption in the GI tract is consonant with the ubiquity of dietary phosphate and the looser regulation of the serum phosphate concentration. A key feature of the C a 2+ o homeostatic system is its capacity to adapt appropriately the efficiency of Ca 2+ absorption to dietary intake. Persons placed on a low-Ca2+ diet elevate their serum concentrations of 1,25 (OH) 2D by 50% within 24-48 hours, whereas exposure to a high-Ca 2+ diet causes a 50% reduction in the circulating level of this metabolite over the same period (50). In experimental animals, the low dietary CaZ+-evoked increase in the 1,25(OH)2D level is largely prevented by prior parathyroidectomy (51), indicating that dietary CaZ+-induced alterations in 1,25(OH)2D concentration are the result of changes in serum C a 2+ concentration, which, in turn, regulate vitamin D metabolism indirectly through alterations in PTH secretion. Nonetheless, decreasing or elevating the level of C a 2+ o also has been shown directly to stimulate or inhibit, respectively, the 1-hydroxylation of 25-hydroxyvitamin D (17). These direct actions of Ca2o+ on renal vitamin D metabolism could potentially be mediated by the CaSR in the proximal tubule (34,35), although this has not yet been proved. The CaSR is also expressed along the entire GI tract, but it remains to be determined whether it directly regulates mineral ion absorption (52,53) Because of the C a 2+ and PTH-elicited, 1,25(OH)zD-mediated modulation of the efficiency of intestinal C a 2+ absorption, the absorption of this ion varies less than does its content in the diet. Absorption of supplemental dietary Ca 2+ may occur principally through the vitamin D-independent, paracellular route. Phosphate intake also regulates the production of 1,25 (OH) 2D , in a physiologically relevant manner, with hypophosphatemia increasing and hyperphosphatemia reducing its renal synthesis (6). 2+

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PTH-induced and direct Ca o -induced changes in renal Ca 2÷ handling play key roles in the overall finetuning of Ca 2÷ balance (54-56). In contrast, vitamin D and its metabolites exert only minor direct effects on renal Ca 2÷ handling. Of the approximately 10 g of Ca 2÷ that are filtered daily by the kidney, about 65% is reabsorbed in the proximal tubule (56). C a 2+ reabsorption in this site is closely coupled to the bulk transport of solutes,

PHYSIOLOGYOF Ca 2+ HOMEOSTASIS / such as sodium and water, and PTH has little effect on Ca 2+ transport in this segment of the nephron. In fact, PTH modestly inhibits, in some studies, proximal tubular Ca 2÷ absorption, perhaps because the hormone reduces sodium reabsorption in this part of the nephron (56). In the more distal portions of the tubule, the descending and ascending thin limbs of Henle's loop transport only small quantifies of Ca 2+ (56). In contrast, the thick ascending limb of Henle's loop (57) and the distal convoluted tubule (DCT) reabsorb about 20 and 10% of filtered C a 2+, respectively. PTH rapidly increases the reabsorption of C a 2+ in both the TAL and the DCT in experimental animals (56). It exerts this effect, similar to its other biologic actions in the kidney, by interacting with its own G protein-coupled receptor that is linked to activation of both adenylate cyclase and phospholipase C (58). cAMP appears to play the dominant role in mediating PTH-induced alterations in renal C a 2+ handling. PTH-sensitive adenylate cyclase

•~

resides within the proximal tubule, cortical thick ascending limb, and portions of the DCT (59). The location of this enzyme in the proximal tubule mediates the well-known, PTH-induced phosphaturia. PTH-activated adenylate cyclase activity in more distal nephron segments is present in sites where the hormone enhances Ca 2+ reabsorption. Furthermore, exposing renal tubules to cAMP analogs mimics the actions of PTH o n C a 2+ transport, further supporting the mediatory role of cAME In the CTAL, PTH increase the overall activity of the Na/K/2C1 cotransporter that drives transcellular NaC1 reabsorption in this nephron segment (Fig. 5) (36,60). This increased transcellular salt transport elevates the lumen-positive, transepithelial potential difference that drives about 50% of the reabsorption of NaC1 and most of the reabsorption of Ca 2+ and Mg 2+ in the CTAL. In contrast, raising C a 2+ o , by activating the CaSR that resides in the same epithelial cells of the CTAL, decreases overall

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FIG. 5 Schematic diagram illustrating how the calcium-sensing receptor may regulate intracellular second messengers and pad passu the transport of NaCI, K ÷, Mg 2÷, and Ca2÷ in the CTAL. Hormones that stimulate cAMP accumulation, such as PTH, enhance the reabsorption of Ca 2÷ and Mg 2÷ through the paracellular pathway by increasing overall activity of the Na/K/2CI cotransport, thereby enhancing the lumen-positive transepithelial potential, Vte.The calcium-sensing receptor, also resident on the basolateral membrane, increases arachidonic acid (AA) production by activating phospholipase A 2 (PLA2) (2). AA is then metabolized via the P450 pathway to an active metabolite, probably 20-HETE, which reduces the activity of the apical K ÷ channel (4) and, perhaps, the Na/K/2CI cotransporter (3). Both actions reduce overall cotransporter activity, thereby diminishing Vte and, in turn, paracellular Ca2+ and Mg 2+ transport. The calcium-sensing receptor probably also inhibits adenylate cyclase (1), which decreases hormone- and cAMP-stimulated divalent cation transport. (Reprinted from Bone, Vol. 20; Brown EM, Hebert SC. Calcium-receptor regulated parathyroid and renal function, pp. 303-309. Copyright 1997, with permission from Elsevier Science.)

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cotransporter activity, probably both by inhibiting the cotransporter as well as by reducing the activity of an apical potassium channel that recycles K + back into the tubular lumen (Fig. 5) (36,61). The resultant decrease in the transepithelial potential gradient diminishes the paracellular reabsorption of both Ca 2+ and Mg 2+. In effect, Ca2o+, by acting in a m a n n e r analogous to the "loop" diurectics (e.g., furosemide), directly controls 2+ its own reabsorption (and that of Mg ) by a CaSRmediated action on the CTAL that antagonizes the effect of PTH on the same cells (36). Although the detailed cellular mechanisms by which PTH regulates Ca 2+ transport in the distal convoluted tubule remain incompletely understood, they likely involve a PTH-stimulated increase in the apical uptake of Ca 2+ through a recently cloned, Ca 2+-permeable channel (62,63). As in the intestine, the ensuing transcellular Ca 2+ transport is likely facilitated by a vitamin D-dependent, Ca2+-binding protein, which in this case is calbindin D2sK. The latter is expressed in the DCT and is distinct from the related Ca2+-binding protein, calbindin DgK, that is present in the intestinal epithelial cells that absorb Ca 2+ (64). An additional small quantity of Ca 2+ (about 5% of the filtered load) is reabsorbed in the collecting duct, but Ca 2+ transport at this site is not PTH regulated. In addition to being present in the CTAL, the CaSR also resides in the DCT (34,35), but its role, if any, in controlling tubular reabsorption of Ca 2+ in this n e p h r o n segment is unknown. The net effect of PTH on renal Ca 2+ handling is to decrease the quantity of Ca 2+ excreted at any given concentration of serum Ca 2+ (55). This relationship has been shown in vivo by measuring renal Ca 2+ excretion as a function of serum Ca 2+ in persons with underactive, normal, or overactive parathyroid function (Fig. 6) (55). In patients with primary hyperparathyroidism, even though the total quantity of urinary Ca 2+ that is excreted per 24 hours may be greater than normal, substantially less urinary Ca 2+ is excreted than in a normal person whose serum Ca 2+ concentration has been elevated to the same extent. In contrast, patients with primary hypoparathyroidism exhibit a renal Ca 2+ "leak," excreting greater than normal quantities of urinary calcium at any given level of serum Ca 2+. Thus when treating patients with hypoparathyroidism with vitamin D and dietary Ca 2+ supplementation, their total serum calcium concentration should be maintained in the range of 8 to 9 m g / d l to avoid hypercalciuria. Figure 6 illustrates the steep positive relationship between the serum and urinary levels of Ca 2+ in these various states, which is probably mediated by the CaSR. This relationship is "reset" d e p e n d i n g on the prevailing state of parathyroid function, thereby shifting to the right or left with chronic increases and decreases, respectively, in circulating PTH levels (55).

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FIG. 6 Urinary excretion of Ca 2+ expressed as a function of the serum Ca 2+ concentration in normal persons [area enclosed by dashed lines (mean +_ 2SD)] and in hypoparathyroid (A, &; solid triangles are basal values) and hyperparathyroid (°) subjects. The shaded area represents the normal physiologic situation. (From Nordin BEC, Peacock M. Role of the kidney in regulation of plasma calcium. Lancet 1969;2:1280.)

Along with its actions on renal C a 2+ handling in the CTAL and DCT, PTH also reduces phosphate reabsorption in both proximal and distal sites and increases the proximal tubular synthesis of 1,25(OH)2D(5,6 ). The first of these effects, similar to the actions of the horm o n e on the reabsorption o f Ca 2+, is thought to be cAMP mediated. PTH activates phospholipase C (58), an action that may participate in the PTH-mediated stimulation of 1,25(OH)2D synthesis (65). R o l e o f the S k e l e t o n in Ca 2+ H o m e o s t a s i s

During the ongoing remodeling of the skeleton, there is close coupling of bone resorption and formation (6). Osteoblasts play a key role in the generation of osteoclasts from their precursors (66). Osteoclastic resorption of bone, in turn, is tightly coupled to the subsequent replacement of the resorbed bone by osteoblasts. The constant turnover and renewal of bone is thought to play an important role in maintaining the structural integrity of the skeleton. The precision of the coupling between resorption and formation is illustrated dramatically in patients with Paget disease of bone, in whom increases in the rate of skeletal turnover of up to 10-fold are often unassociated with any change whatsoever in s e r u m C a 2+ concentration or overall C a 2+ balance. Studies have identified key mechanisms that participate in regulating the differentiation and function of osteoclasts and osteoblasts; these are briefly described below.

PHYSIOLOGY OF C a 2+ HOMEOSTASIS

PTH and other agents activating bone resorption [e.g., interleukin-11, prostaglandin E2, and 1,25(OH)2D ] stimulate osteoclast maturation and function indirectly by increasing the expression of an osteoclast differentiating factor, most commonly called RANKL (67). RANKL is expressed on the cell surface of osteoblasts and stromal cells. It activates osteoclast development and increases the activity of mature osteoclasts by interacting with its receptor (called RANK) on preosteoclasts, which then differentiate to mature osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) (67). Osteoclastic bone resorption, in turn, is thought to be coupled to the subsequent osteoblastic replacement of the missing bone, at least in part, through the release of skeletal growth factors such as transforming growth factor-J3 (TGF-[3) and insulin-like growth factor-I (IGF-I), which stimulate the recruitment and maturation of preosteoblasts, and, in some cases, the activity of mature, bone-forming osteoblasts (68). As noted earlier, the skeleton provides a virtually inexhaustible reservoir for calcium and phosphate ions (5,6). Because the content of Ca 2+ in the skeleton is 1000-fold higher than that in the ECF, this function of bone as a reservoir can be accomplished by the net movement of relatively small amounts of Ca 2+ into or out of the skeleton. After administering PTH to animals, alterations in the structure of osteoclasts, osteoblasts, and osteocytes (which are osteoblasts trapped within the calcified bone matrix) take place within minutes (5). Those morphologic alterations are accompanied by e n h a n c e d activity of osteoclasts and inhibition of the function of osteoblasts, producing an increase in net skeletal release of Ca 2+ within 2 to 3 hours (5,69). The PTH-elicited increase in the size of the lacunae within which osteocytes reside has also been considered indirect evidence for a role of these cells in promoting release of skeletal calcium. Continued exposure to PTH produces increases in the activity and n u m b e r of osteoclasts, which are ultimately accompanied by a coupled increase in osteoblastic activity, as noted above. The mechanisms by which PTH regulates bone cell function remain to be fully elucidated. Evidence indicates that a PTH-induced increase in the expression of RANKL by osteoblasts probably is an important mechanism through which this h o r m o n e promotes osteoclastogenesis and stimulates the activity of p r e f o r m e d osteoclasts (70). PTH activates adenylate cyclase in osteoblasts through the same G protein-coupled receptor via which it exerts its actions on the renal handling of calcium and phosphate ions (58,71), but it may also act through other second-messenger systems, including those downstream of phospholipase C. In addition to modulating skeletal turnover indirectly, e.g., by altering PTH secretion a n d / o r 1,25 ( O H ) 2 o production, changes in Ca2o+ also directly

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control bone cell function in vitro in ways that likely contribute to the control of bone turnover in vivo. The CaSR is expressed in osteoblasts, and high Ca2o+ is known to stimulate the chemotaxis and proliferation of osteoblasts (18,72) as well as the production of osteocalcin, a marker of differentiated osteoblasts. If these actions of Ca2o+ are mediated by the CaSR expressed in osteoblasts, then it is possible that Ca 2+ released during the resorption of bone by osteoclasts serves as one of the signals that are designed to ensure the availability of osteoblasts to replace the missing bone (73). Conversely, elevated levels of Ca2o+ directly inhibit osteoclastic function (20), which could provide a mechanism by which osteoclasts autoregulate their activity as a function of the a m o u n t they resorb of Ca 2+ that has been released into the local ECE Pharmacologic evidence has implicated a mechanism for C a 2+ o sensing by osteoclasts that differs from the CaSR (20). Further studies, however, have demonstrated that the CaSR can be expressed by both osteoclasts and their precursors (19,74). Thus further studies are n e e d e d to clarify the molecular mechanisms by which bone cells sense C a 2+ o. Changes in the level of extracellular phosphate also modulate bone turnover, as noted before. Elevations in phosphate enhance bone formation and inhibit bone resorption, whereas reductions in ambient phosphate concentrations produce the converse effects (75). As with the direct effects of phosphate on parathyroid gland function, the mechanisms underlying the sensing of extracellular phosphate ions by bone cells remain unknown.

P O S S I B L E R O L E S OF T H E CaSR IN I N T E G R A T I N G C a 2+ o METABOLISM W I T H O T H E R H O M E O S T A T I C SYSTEMS CaSR-Mediated Interactions b e t w e e n C a ~ and Water Metabolism In addition to regulating renal handling of Ca 2+ and Mg 2+, the CaSR likely mediates the known action of hypercalcemia to reduce urinary concentrating ability (76). It is thought to do so by two actions. First, by inhibiting NaC1 reabsorpdon in the TAL, it reduces the medullary hypertonicity n e e d e d for passive, vasopressinstimulated reabsorption of water in the collecting ducts. Second, in the inner medullary collecting duct, raising Ca o2+directly reduces vasopressin-stimulated water flow, probably by an action mediated by the CaSR in the apical m e m b r a n e of these cells (77,78). Finally, a b u n d a n t calcium-sensing receptors in the subfornical organ (SFO) (79), an important hypothalamic thirst center, may promote a CaSR-mediated increase in thirst that could minimize dehydration from accompanying renal water loss owing to reduced urinary concentrating

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ability. Thus the CaSR may provide a mechanism for integrating the renal handling of Ca 2+ and water, permitring appropriate "trade-offs" in how these parameters of renal function are regulated u n d e r specific physiologic conditions (76). For instance, when disposing of a systemic C a 2+ load, a CaSR-mediated increase in luminal C a 2+ o in the inner medullary collecting duct (IMCD), particularly in a dehydrated individual, could predispose to CaZ+-containing renal stones were it not for the concomitant CaSR-mediated inhibition of maximal urinary concentrating capacity. Thus there may be multiple layers of CaSR-mediated integration and coordination of water and calcium metabolism that optimize the ability of terrestrial organisms to adapt to intermittent dietary access t o C a 2+ and water (76).

P o s s i b l e C a S R - M e d i a t e d Interactions b e t w e e n Ca2o+ a n d S o d i u m / V o l u m e / Blood Pressure Control Figure 5 shows that activating the CaSR reduces transcellular NaC1 transport in the CTAL by inhibiting the Na/K/2C1 cotransporter and pari passu reducing paracellular NaC1 reabsorption by diminishing V~.Therefore, high C a 2+ o exerts a "loop diuretic-like" action that likely contributes to the volume depletion of severely hypercalcemic persons (e.g., via urinary loss of NaC1) (76). The action of C a 2+ o could also potentially participate in the salutary action of dietary calcium supplementation on certain forms of genetic hypertension in experimental animals (e.g., the spontaneously hypertensive rat) (80,81), and, perhaps, in treating pregnancy-induced hypertension (82) or preventing preeclampsia (83). Because of its exquisite sensitivity to changes in Ca2o+ resulting from alterations in dietary calcium intake, the CaSR in the kidney could potentially modulate NaC1 reabsorption and sensitize the kidney to other agents promoting diuresis. There are additional actions through which the CaSR could modulate blood pressure. It is expressed in perivascular sensory nerve endings in rat mesenteric artery (84) and other vascular beds (e.g., mesenteric branch artery > basilar artery = renal interlobar artery > main renal trunk artery > left anterior descending coronary artery) (85). Moreover, stimulating the CaSR in these nerve endings releases a vasodilatory substancemlikely an endogenous cannabinoid [e.g., N-arachidoylethanolamine (anandamide) ]--which then acts on a cannabinoid receptor in the vascular wall (86). It is possible that the inhibition of renin release from the juxtaglomerular apparatus (]GA) by high Ca2o+ involves the CaSR (87). Further studies, therefore, may reveal additional mechanisms through which the CaSR, by regulating renal fluid and electrolyte metabolism, vascular tone, and, perhaps, central vasomotor control, contributes to overall blood pressure regulation.

P o s s i b l e C a S R - M e d i a t e d Interactions b e t w e e n Ca2o+ a n d P r o t e i n M e t a b o l i s m Protein and Ca 2+ metabolism are linked at a fundamental level. For instance, reducing protein intake below a certain level in normal young women produces secondary hyperparathyroidism despite normocalcemia (88-90), and high dietary protein intake can promote substantial hypercalciuria (91). Studies have shown that the CaSR can "sense" not only polyvalent 2+ but also amino acids at cations , such as Ca2o+ and M go, levels that are not dissimilar from those present in vivo (92). Although a large n u m b e r of amino acids are effective allosteric activators of the CaSR (that is, they activate the receptor in the presence but not in the absence of Ca2o+), the CaSR shows a preference for L-aromatic amino acids (i.e., L-Phe, L-Trp, L-Tyr, and L-His) (92). This pharmacologic profile is similar to that for the stimulation of gastrin and gastric acid secretion from the stomach by amino acids (93), which are physiologic processes that are also stimulated by high Ca2o+, likely by a CaSR-mediated mechanism (94). The finding that the CaSR senses L-amino acids may clarify its widespread distribution in the upper GI tract, including the stomach (53,94), where it is exposed to dietary amino acids (and calcium) and could potentially contribute to physiologic responses, such as the release of gastric acid and pancreatic enzymes. Thus the CaSR may act as a "nutrient sensor" in the lumen of the proximal GI tract, responding to Ca2o+ and amino acids as coagonists that might function together to coordinate digestive responses to ingested nutrients. The pharmacologic profile for the actions of amino acids on the CaSR differs, however, from that of other metabolic actions of amino acids, such as the stimulation of insulin release (92). Therefore, there are probably additional amino acid sensors, one of which might be one or more purinergic receptors (92). It is likely that the calcium-sensing receptor's capacity to sense amino acids is relevant to additional physiologic interactions between Ca2o+ and protein metabolism. L-Amino acid mixtures that emulate those present in fasting h u m a n plasma activate the CaSR, and changes in the levels of this mixture equivalent to those occurring during the transition from the fed to the fasted state can modify the receptor's sensitivity to Ca2o+ significantly (92). As noted above, low-protein intake causes secondary hyperparathyroidism (88,89), whereas high-protein intake promotes hypercalciuria ( 9 1 ) ~ a c t i o n s that could potentially be mediated by the CaSR in the parathyroid and CTAL, respectively. It is of interest in this regard that substantial amounts of both Ca 2+ and protein are laid down during the growth of not only the skeleton, but also soft tissues. For instance, smooth muscle has a calcium content when expressed per wet weight that is about 8 m M / k g , nearly one-half

PHYSIOLOGY OF C a 2+ HOMEOSTASIS

of that in bone (95). These observations raise the possibility that the CaSR, known to be expressed in growth plate chrondrocytes (96) and osteoblasts (73,97), for instance, could integrate information about the availability of key nutrients n e e d e d for growth of cartilage and bone, respectively, in ways that would be relevant to the physiologic control of these processes.

SUMMARY A N D C O N C L U S I O N S Ca2o+ homeostasis requires the coordinated functions 2+ 2+ of both the Ca o -sensing cells that secrete Ca o -elevating o r - l o w e r i n g h o r m o n e s as well as the effector tissues that translocate calcium ions into or out of the ECF in kidney, bone, and intestine. Furthermore, the capacity of not only the cells secreting Ca 2+ o -regulating hormones but also the capacity of these effector tissues to sense C a 2+ o (as well as phosphate ions) add additional layers of regulatory control to the mineral ion homeostatic system. The G protein-coupled CaSR has a central role in systemic C a 2+ o homeostasis by enabling maintenance of near constancy of Ca2o+ via its coordinated actions on the various tissues involved in mineral ion homeostasis. As we learn more about the calciumsensing receptor's roles in the tissues directly participating in C a 2+ o homeostasis, it may turn out that it contributes to the regulation of other processes relevant to mineral ion homeostasis, such as controlling 1-hydroxylation of vitamin D or phosphate reabsorption in the proximal tubule. In any event, the calciumsensing receptor's exquisite sensitivity to even m i n u t e changes in Ca2o+ permits adjustments in the C a 2+ o homeostatic system's responses, for example, to increases or decreases in dietary Ca 2+ intake that produce barely detectable changes in Ca2o+. Finally, the CaSR is present not only in tissues directly involved in C a 2+ o homeostasis but also in those that are not. Thus this receptor may participate in coordinating interactions a m o n g several different homeostatic systems, such as those for regu2+ lating water, M go, Na + , extracellular volume, blood pressure, a n d / o r protein metabolism, which are usually t h o u g h t of as functioning largely i n d e p e n d e n t l y of mineral ion metabolism.

ACKNOWLEDGMENTS The author gratefully acknowledges the support of the following grants for work described in this chapter, as well as for salary support: NIH grants DK41415, DK48330, and DK52005; The National Dairy Council; The Cystic Fibrosis Foundation; The National Space Bioscience Research Institute (NSBRI); and the St. Giles Foundation and NPS Pharmaceuticals, Inc.

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cells: A single receptor stimulates intracellular accumulation of both cAMP and inositol phosphates and increases intracellular free calcium. Proc Natl Acad 'Sci USA 1992;89:2732-2736. Morel F, Chabardes D, Imbert-Teboul M. Multiple hormonal control of adenylate cyclase in distal segments of the rat kidney. Kidney Int 1982;11 (Suppl.):555-565. De Rouffignac C, Quamme GA. Renal magnesium handling and its hormonal control. Physiol Rev 1994;74:305-322. Wang WH, Lu M, Hebert SC. Cytochrome P-450 metabolites mediate extracellular Ca~Z+)-induced inhibition of apical K+ channels in the TAL. AmJPhysio11996;271:C103-C111. Hoenderop JG, van der Kemp AW, Hartog A, van de Graaf SF, van Os CH, Willems PH, Bindels RJ. Molecular identification of the apical Ca 2+ channel in 1,25-dihydroxyvitamin D3-responsive epithelia. J Biol Chem 1999;274:8375-8378. PengJB, Chen XZ, Berger UV, Vassilev PM, Brown EM, Hediger MA. A rat kidney-specific calcium transporter in the distal nephron. J Biol Chem 2000;275:28186-28194. Sonnenberg J, Pansini AR, Christakos S. Vitamin D-dependent rat renal calcium-binding proteins: Development of a radioimmunoassay, tissue distribution, and immunologic identification. Endocrinology 1984;115:640-648. Ro HK, Tembe V, Favus MJ. Evidence that activation of protein kinase-C can stimulate 1,25-dihydroxyvitamin D 3 secretion by rat proximal tubules. Endocrinology 1992;131:1424-1428. Rodan GA, Martin TJ. Role of osteoblasts in hormonal control of bone resorptionma hypothesis. Calcif Tissue Int 1981 ;33:349-351. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998;95:3597-3602. Canalis E, Pash J, Varghese S. Skeletal growth factors. Crit Rev Eukaryot Gene Expr 1993;3:155-166. Robertson WG, Peacock M, Atkins D. The effect of parathyroid hormone on the uptake and release of calcium by bone in tissue culture. Clin Sci 1972;43:715-718. Lee SK, Lorenzo JA. Parathyroid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures: Correlation with osteoclast-like cell formation. Endocrinology 1999;140: 3552-3561. Chase LR, Fedak SA, Aurbach GD. Activation of skeletal adenyl cyclase by parathyroid hormone in vitro. Endocrinology 1969;84: 761-768. Yamaguchi T, Chattopadhyay N, Kifor O, Butters RR, Jr, Sugimoto T, Brown EM. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+ o )-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells. JBone Miner Res 1998;13:1530-1538. Yamaguchi T, Chattopadhyay N, Brown EM. G protein-coupled extracellular Ca 2+ (Ca2o+)-sensing receptor (CAR): Roles in cell signaling and control of diverse cellular functions. Adv Pharmacol 2000;47:209-253. Kameda T, Mano H, Yamada Y, Takai H, Amizuka N, Kobori M, Izumi N, Kawashima H, Ozawa H, Ikeda K, Kameda A, Hakeda Y, Kumegawa M. Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells. Biochem Biophys Res Commun 1998 ;245:419-422. Raisz LG, Niemann I. Effect of phosphate, calcium, and magnesium on bone resorption and bone formation in tissue culture. Endocrinology 1969;85:446-452. Hebert SC, Brown EM, Harris HW. Role of the CaCZ+)-sensing receptor in divalent mineral ion homeostasis. J Exp Biol

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77. SandsJM, Naruse M, Baum M,Jo I, Hebert SC, Brown EM, Harris HW. Apical extracellular calcium/polyvalent cation-sensing receptor regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct. J Clin Invest 1997;99:1399-1405. 78. Sands JM, Flores FX, Kato A, Baum MA, Brown EM, Ward DT, Hebert SC, Harris HW. Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats. Am J Physiol 1998;274:F978-F985. 79. Rogers KV, Dunn CK, Hebert SC, Brown EM. Localization of calcium receptor mRNA in the adult rat central nervous system by in situ hybridization. Brain Res 1997;744:47-56. 80. McCarron DA, Hatton D, RoulletJB, Roullet C. Dietary calcium, defective cellular Ca 2+ handling, and arterial pressure control. Can J Physiol Pharmaco11994;72:937-944. 81. Oparil S, Chen YF, Jin HK, Yang RH, Wyss JM. Dietary Ca 2+ prevents NaCl-sensitive hypertension in spontaneously hypertensive rats via sympatholytic and renal effects. Am J Clin Nutr 1991 ;54:227S-236S. 82. Tomoda S, Kitanaka T, Ogita S, Hidaka A. Prevention of pregnancy-induced hypertension by calcium dietary supplement: A preliminary report. J Obstet Gynaeco11995;21:281-288. 83. Mattar F, Sibai BM. Prevention of preeclampsia. Semin Perinatol

1999;23:58-64.

84. Bukoski RD, Bian K, Wang Y, Mupanomunda M. Perivascular sensory nerve Ca 2+ receptor and CaZ+-induced relaxation of isolated arteries. Hypertension 1997;30:1431-1439. 85. Wang Y, Bukoski RD. Distribution of the perivascular nerve Ca2+ receptor in rat arteries. BrJPharmaco11998; 125:1397-1404. 86. Ishioka N, Bukoski RD. A role for N-arachidonylethanolamine (anandamide) as the mediator of sensory nerve-dependent Ca2+-induced relaxation. J Pharmacol Exp Ther 1999;289:245-250. 87. Fray J, Park C, Valentine N. Calcium and the control of renin secretion. Endocr Rev 1987;8:53-93. 88. Kerstetter JE, Svastisalee CM, Caseria DM, Mitnick ME, Insogna KL. A threshold for low-protein-diet-induced elevations in parathyroid hormone. A m J Clin Nutr 2000;72:168-173. 89. Kerstetter JE, Caseria DM, Mitnick ME, Ellison AF, Gay LE Liskov TA, Carpenter TO, Insogna KL. Increased circulating concentrations of parathyroid hormone in healthy, young women consuming a protein-restricted diet. Am J Clin Nutr 1997;66:1188-1196. 90. Kerstetter JE, Mitnick ME, Gundberg CM, Caseria DM, Ellison AF, Carpenter TO, Insogna KL. Changes in bone turnover in young women consuming different levels of dietary protein. J Clin Endocrinol Metab 1999;84:1052-1055. 91. Burtis WJ, Gay L, Insogna KL, Ellison A, Broadus AE. Dietary hypercalciuria in patients with calcium oxalate kidney stones. Am J Clin Nutr 1994;60:424-429. 92. Conigrave AD, Quinn sJ, Brown EM. L-amino acid sensing by the extracellular Ca2+-sensing receptor. Proc Natl Acad Sci USA 2000;97:481 4-4819. 93. IsenbergJI, Maxwell V. Intravenous infusion of amino acids stimulates gastric acid secretion in man. NEnglJMed 1978;298:27-29. 94. Ray JM, Squires PE, Curtis SB, Meloche MR, Buchan AM. Expression of the calcium-sensing receptor on human antral gastrin cells in culture. J Clin Invest 1997;99:2328-2333. 95. Robertson WG, Marshall RW. Ionized calcium in body fluids. Crit Rev Clin Lab Sci 1981;15:85-125. 96. Chang W, Tu C, Bajra R, Komuves L, Miller S, Strewler G, Sh0back D. Calcium sensing in cultured chondrogenic RCJ3.1C5.18 cells. Endocrinology 1999;140:1911-1919. 97. Chang W, Tu C, Chen T-H, Komuves L, Oda Y, Pratt S, Miller S, Shoback D. Expression and signal transduction of calciumsensing receptors in cartilage and bone. Endocrinology 1999;140:5883-5893.

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CHAPTER11 Parathyroid H o r m o n e A n a b o l i c and Catabolic Effects o n B o n e and Interactions with Growth Factors JANET M. H O C K Department of Periodontics, Indiana University School of Dentistry, Indianapolis, Indiana 46202 LAWRENCE G. RAISZ Department of Medicine, The University of Connecticut School of Medicine, Farmington, Connecticut 06030 ERNESTO CANALIS Department of Medicine, The University of Connecticut School of medicine, Farmington, Connecticut 06030; and Departments of Research and Medicine, Saint Francis Hospital and Medical Cent~ Hartford, Connecticut 06105

INTRODUCTION

W h e n the synthetic fragment h P T H ( 1 - 3 4 ) became available in the early 1970s, there was renewed interest in evaluating the anabolic effect of PTH as a potential therapeutic to restore bone loss caused by diseases such as osteoporosis (6-8). N u m e r o u s studies since then have showed that synthetic h P T H ( 1 - 3 4 ) , given intermittently, increased bone mass in a variety of animal models, of which the best studied is the rat (9-64). The r e c o m b i n a n t full-length h o r m o n e , h P T H (1-84), as well as a variety of PTH and PTH-related protein (PTHrP) amino-terminal analogs, all induce anabolic effects similar to those induced by h P T H ( 1 - 3 4 ) (6-8, 65). The identification of growth factors and cytokines in the mechanisms by which differing exposures to PTH may induce either anabolic or catabolic effects is predominantly based on in vitro cell and bone organ model systems from embryonic and neonatal rodents. The extent to which these models mirror the mechanisms of in vivo events in osteonal cortical and trabecular bone of diseased h u m a n skeletons remains to be determined. Despite a wealth of literature affirming the anabolic effect of PTH on rat skeletons, many of these studies were done on ovariectomized rats, and the confounding variables associated with ovariectomy were not separated from the mechanisms activated by PTH. Recent studies of ovariectomized monkeys treated for up to 18 m o n t h s with PTH showed that increased bone turnover after ovariectomy was associated with decreased bone mass and strength, whereas increased bone turnover after PTH was associated with increased bone mass and strength (66-68). PTH reversed the changes in serum calcium, phosphate, and calcitriol induced by

The concept that parathyroid h o r m o n e (PTH) has both catabolic and anabolic effects was first proposed in the early n i n e t e e n t h century. The major mechanism for the catabolic action is a selective stimulation of bone resorption. The mechanisms underlying the anabolic response are still not clearly understood, but are likely to involve a cascade of growth factors and cytokines that regulate or support bone formation. Low doses of crude preparations of parathyroid extract (PTE) increased trabecular bone density in rodents, guinea pigs, and rabbits, after an initial dose-dependent episode of resorption with some tissue necrosis, and transient hypercalcemia (1-5). The PTE preparations contained a mix of proteins, and it was not clear which events could be attributed directly to PTH and which represented an inflammatory response to the protein mix. However, the p h e n o m e n a gave rise to the perception that the stimulatory effects on formation had to be preceded by a resorption phase to generate the appropriate growth factors. More recent data have modified this hypothesis, as dose and duration of exposure have become recognized as critical factors in determining the outcome on bone, and differences in responses of the different bone envelopes have become better characterized through studies of large animals with osteonal bone skeletons. The molecular mechanisms and contributing factors that underlie the differences in skeletal responses between the pharmacologic actions of exogenous PTH and the pathologic actions of endogenous PTH in hyperparathyroidism have yet to be identified. The Parathyroids, Second Edition

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ovariectomy to match the values in sham monkeys, in addition to stimulating accumulation of bone on cortical and trabecular endosteal surfaces (67-69). Understanding how the multiple growth factors and cytokines activated by ovariectomy may be modified by exogenous PTH to improve biomechanical properties of bone, and to counteract the negative bone balance associated with ovariectomy, will require innovative approaches and new in vitro models.

SKELETAL G R O W T H FACTORS In vitro, the anabolic effect has been defined on the basis of surrogate markers for bone gain, such as an increase in the rate of type I collagen synthesis, increased bone cell proliferation, or increased formation of bone marrow fibroblastic cell colonies. Cells either briefly or intermittently exposed to PTH exhibit stimulatory effects on osteoblast differentiation (70) and function (71). The extent to which these outcomes may model the in vivo actions of PTH is not well understood yet, because other agents that are not anabolic in vivo induce similar in vitro p h e n o m e n a . Although PTH undoubtedly has direct effects on bone cells, in vitro studies have shown that some of its actions may be mediated by locally produced growth factors. Bone cells synthesize insulin-like growth factors (IGF-I and IGF-II), transforming growth factor-[~ (TGF[31,-[32, a n d - [ 3 3 ) , platelet-derived growth factors (PDGFs), fibroblast growth factors (FGF-1 and FGF-2), bone morphogenetic proteins (BMPs) or osteoinductive factors, and hepatocyte growth factor or scatter factor (72). Of these various factors, TGF-[3 is of particular interest because it both stimulates bone formation and inhibits bone resorption. This suggests it may be involved in the molecular mechanisms of the reversal phase of coupling, that is, inhibition of resorption followed by stimulation of formation. Skeletal cells also synthesize and respond to cytokines known to have primary effects on i m m u n e and hematologic cells, such as interleukin-1 a n d - 6 (IL-1, IL-6), leukemia inhibitory factor (LIF), tumor necrosis factor ot (TNFot), and colony-stimulating factors. Of these, PTH regulates IL-6 and LIF as immediate-early genes in vivo (73-76), but the downstream consequences of this transcriptional regulation are not known. Most recently, PTH was found to regulate transcription of RANKL/OPG, which has been linked to regulation of osteoclast differentiation as an immediate-early gene, after a single injection was given to young male rats (77). Immunohistochemistry showed localization of RANKL protein to cells in close proximity to osteoblasts and osteoblasts, within the primary spongiosa, including the zone enriched for proliferating cells subjacent to the

growth plate (77). A detailed description of the growth factors synthesized by skeletal cells is beyond the scope of this chapter, and our discussion is limited to a selected group of factors.

Insulin-like Growth Factors-I a n d - I I IGF-I a n d - I I are polypeptides with a molecular mass (Mr) of 7600. IGF-I a n d - I I have 66% amino acid sequence homology and have similar biologic activities. IGFs are present in the systemic circulation and are synthesized by skeletal and a variety of nonskeletal cells. The role of IGFs as systemic regulators of bone metabolism has not been demonstrated fully. Mice in which the IGF-I gene has been genetically deleted show abnormal skeletal development, growth retardation, and do not attain puberty (78). Significant abnormalities in serum levels of IGF-I or-II have not been shown in patients with various metabolic bone disorders. In mice in which liver production of IGF-I was genetically abrogated, skeletal growth of long bones did not differ from those of intact controls, suggesting that hepatic endocrine production of IGF-I is not required for normal skeletal development (78,79). It is believed that IGF-I and -II act as local regulators of musculoskeletal cell function (78) through activation of the IGF-I receptor (80). IGFs stimulate bone formation in vitro (81,82). They increase the replication of bone cells, primarily of preosteoblasts, and independently stimulate osteoblastic collagen synthesis and matrix apposition rates (Fig. 1). IGF-I and-II have similar effects, although IGFI is somewhat more potent than IGF-II. In addition to their effects enhancing collagen synthesis, IGFs decrease collagen degradation, probably because they decrease collagenase expression (83). Because of their important effects on bone cell replication and differentiation, it is believed that these polypeptides are major regulators of bone formation and are important in the maintenance of bone mass. Skeletal cells secrete the six known IGF binding proteins (IGFBPs), as well as two of the four known IGFBP-related proteins (IGFBP-rPs) (84-86). The exact role of IGFBPs in bone cell metabolism is not entirely known. It is postulated that IGFBPs are important for the storage of IGF and to prolong its half-life. They may regulate IGF activity by competing with cell surface receptors for IGF binding. Some IGFBPs, such as IGFBP-4, have mostly an inhibitory activity on osteoblastic function, whereas others, such as IGFBP-5, can enhance the anabolic effects of IGF-I (86). In addition to IGFBPs, osteoblasts express IGFBP-rP, the product of the mac25 gene, and IGFBP-rP-2 or connective tissue growth factor, although it is not known if they express IGFBP-rP-3 and-4. PTH induces IGFBP-rP-1 in osteoblasts by transcriptional mechanisms (86).

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Transforming Growth Factor-[[ and Related Polypeptides TGF-[3 is a dimeric polypeptide of M r 25,000. TGF-[3 is secreted as a homodimer. Five TGF-[3 isoforms have been describedmTGF-[3 1 , - 2 , - 3 , - 4 , a n d - 5 (87). Heterodimers of TGF-[3 also have been isolated from various tissues. Skeletal cells synthesize TGF-[31,-2, a n d - 3 and bovine bone has been shown to contain TGF-[31.2 and TGF-[32.3 heterodimers (88-90). The reason for the presence of various TGF-[3 isoforms in bone tissue is not clear, particularly because the various forms tested have virtually the same effects on bone cell function, with modest differences in potency (91). For the most part, studies using cultures of normal osteoblasts and intact calvariae have demonstrated that TGF-[3 stimulates cell replication as well as collagen and noncollagen protein synthesis (88). TGF-[3 enhances biochemical parameters of bone formation as well as matrix apposition rates (92). TGF-[3 is secreted as a complex of large molecular weight consisting of the polypeptide, a precursor, and a binding protein. TGF-[3 is present in the tissue matrix in an inactive form and it is activated by various mechanisms, including lowering of the pH, which could occur in the bone environment during the process of bone resorption (93). Thus, TGF-[3 may be important in the coupling of bone resorption to formation. In addition to the five TGF-[3 isoforms described, there are a n u m b e r of related polypeptides that share up to 30% amino acid sequence homology with TGF-[3 and may have similar biologic activities. These polypeptides include inhibins, activin, Mullerian inhibiting substance, the gene products of Drosophila decapenta-

plegic,

and a variety of BMPs or osteoinductive substances (94). Initially, the primary function of BMPs was considered to be the induction of e n d o c h o n d r a l bone formation following the implantation of demineralized bone matrix in soft tissues. Further studies have indicated that BMPs play a central role in inducing the differentiation of mesenchymal cells into cells of the osteoblastic lineage and e n h a n c i n g the differentiated function of the osteoblast (95,96).

Platelet-Derived Growth Factor Platelet-derived growth factor is a dimer of M r 50,000. PDGF is the product of two genes, PDGF A and B, which give rise to two distinct PDGF chains with 60% homology (97). PDGF exists as a h o m o d i m e r or h e t e r o d i m e r of these two chains, which can combine to form PDGF AA, BB, and AB. PDGF is stored in platelet granules and, as such, it can act as a systemic regulator of cell function. In humans, the circulating forms of PDGF are, for the most part, PDGF BB and AB (98). Osteoblasts and osteosarcoma cells express the PDGF A and B genes (99-101). The three PDGF isoforms stimulate cell replication in intact calvariae and in osteoblast cultures (102-104). PDGF BB is more potent than PDGF AA, and PDGF AB has an intermediate effect. As a consequence of its effects on cell replication, PDGF causes a small increase in bone protein synthesis. However, PDGF does not stimulate the differentiated function of the osteoblast and it is, to some extent, inhibitory (104). At present, the production of specific PDGF binding proteins by bone cells is uncertain, and it is believed that skeletal PDGF is secreted in a biologically active form.

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Fibroblast Growth Factors FGFs are a group of polypeptides with mitogenic activity. FGF-1 (acidic fibroblast growth factor) and FGF-2 (basic fibroblast growth factor) are secreted by osteoblastic cells (105,106). FGFs have a n M r of approximately 17,000, and FGF-1 and-2 have about 55 % amino acid sequence homology. FGFs are not secreted proteins and are likely to be b o u n d to the bone matrix, where they probably act as local regulators of skeletal cell function. FGFs are bone cell mitogens and do not appear to have potent direct stimulatory effects on osteoblastic differentiated function (107,108). Currently, it is unknown if bone cells also secrete specific binding proteins for these factors.

I N T E R A C T I O N S O F P T H A N D SKELETAL G R O W T H FACTORS IN VITRO PTH stimulates IGF-I synthesis by osteoblastic cells (107). This effect is observed at PTH concentrations of 0.1-10 n M and involves the transcriptional regulation of IGF-I synthesis. PTH increases both IGF-I mRNA and polypeptide levels about threefold (Fig. 2). The effect of PTH appears to be mediated, at least in part, by an increase in cyclic AME because other agents known to enhance cyclic AMP production by osteoblastic cells, such as prostaglandin E 2 (PGE2) and forskolin, also increase skeletal IGF-I synthesis (109,110). In contrast, the calcium ionophore (ionomycin) and phorbol esters do not alter IGF-I synthesis by osteoblastic cells. The effect of PTH on IGF-I production is mimicked by parathyroid hormonerelated peptide in bone cells (111). PTH and PGE 2 each increase IGF-I production and PTH can increase PGE 2 production in bone, suggesting possible mechanisms for the anabolic effect (107,112,113). However, in cell culture, indomethacin, which inhibits PGE 2 synthesis, has little effect on the ability of PTH to increase IGF-I production. Nevertheless, it is possible that u n d e r some circumstancesmfor example, in estrogen deficiency, when PGE 2 production is e n h a n c e d m t h a t an increase in PGE2-induced IGF-I could amplify the anabolic effect of PTH (114). FGF may also mediate some of the anabolic effects on bone formation because it has been implicated as a possible mediator for PTH and PGE 2 (115-117). FGF appears to be important in limb develo p m e n t and patterning, due to its effects on growth plate cartilage and linear growth of bones (118). Knockout FGF m u t a n t mice exhibit decreased bone formation (118). In vitro, PTH increases FGF-2 mRNA levels in osteoblastic cells, as well as FGF receptor 1 and 2 transcripts (118). FGF receptors 3 and 4 were neither detected nor regulated by PTH in these studies

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(118). The regulation of FGF by PTH supports the possibility of a complex amplification system. Not only is FGF production stimulated by prostaglandins, particularly PGE2~, but FGF increases PGE 2 production in bone cells (115,117). Although PTH stimulates IGF-I synthesis and IGF-I increases bone collagen synthesis, continuous exposure of bone cells to PTH results in a decrease in bone collagen production (70,111) (Fig. 3). This inhibitory effect seems to be the result of PTH overriding the stimulatory actions of IGF I. When calvarial explants are concomitantly exposed to PTH and IGF-I, only the inhibitory effect of PTH on collagen synthesis is observed. In contrast, transient exposure to PTH, which results in an induction of skeletal IGF-I, causes a stimulation of collagen synthesis (Fig. 4) (71). The stimulatory effect of PTH on collagen synthesis is blocked by IGF-I neutralizing antibodies, suggesting that IGF-I is at least in part responsible for the increase in bone collagen (119).

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In addition to changes in IGF-I synthesis, PTH also modifies the synthesis of IGFBPs and IGFBP-rPs. However, it does not alter IGF-II synthesis or the binding of IGFs to specific cell surface receptors (119). The synthesis of IGFBP-3,-4, a n d - 5 in bone cells is cyclic AMP d e p e n d e n t , and transcripts for IGFBP-4 have been shown to be elevated by PTH (85). It is a p p a r e n t that the induction of cyclic AMP in bone cells not only results in an increased production of IGF-I, but also of selected IGFBPs (85). Although the role of the binding proteins is not entirely known, it is possible that they are i m p o r t a n t in mechanisms regulating the exposure of bone cells to endogenous IGF-I. PTH and other agents that stimulate bone resorption increase the secretion of TGF-[3 activity in bone cultures during this process (93). It has been suggested that this p h e n o m e n o n is due to the activation of TGF-[3 in the osteoclastic microenvironment, possibly the result of lowering the pH. Because PTH does not increase TGF-[3 mRNA levels in osteoblastic cells, it most likely activates previously synthesized TGF-[3. TGF-[3 may play an i m p o r t a n t role in the local control of bone resorption, because it has b e e n shown to inhibit this process as a result of a decrease in the formarion of osteoclast-like cells (120,121). In addition to its effects on TGF-[3 activation, PTH regulates the binding and activity of TGF-[3 in osteoblast cultures (122). PTH increases the n u m b e r of apparent TGF-[3 receptors, but for reasons not entirely understood it opposes the activity of TGF-[3 on bone DNA and collagen synthesis in vitro. The contribution of TGF to PTH effects in more mature postnatal bones has not b e e n studied.

PTH does not modify the concentrations of PDGF AA or BB in bone cell cultures or the binding of PDGF to its bone cell receptors, suggesting that PDGF is not an important m e d i a t o r of PTH function in bone. In vivo, PDGF increases bone mass to the same extent as PTH in rats, but also accelerates maturation of the growth plate and a n u m b e r of extraskeletal side effects (123), suggesting a different pathway of activation than that induced by PTH. There are few studies on interactions between multiple growth factors and PTH. The possibility that the ability of PTH to alter the geometry and connectivity of trabecular bone may be d e t e r m i n e d by a spectrum of local growth factor responses has not been adequately studied, but could represent an i m p o r t a n t feature of the anabolic effect. New models, based on developmental biology models of patterning, could be applied to investigate this hypothesis.

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responses have been reported in mice (138-140), but the efficacy of PTH appears to vary in different strains of mice.

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FIG. 4 Effect of transient PTH treatment on [3H]proline incorporation into collagenase digestible protein CDP; (collagen) in calvariae treated for only the first 24 hours of culture, and then transferred to control medium for the remainder of the incubation as indicated. Values are expressed as percentage of control. Reproduced with permission from J Clin Invest 1989;83:60.

6 months increases bone mass by more than two- to fourfold in the spine and proximal tibia, and by more than 20% in the other long bones of ovariectomized rats (52,126). In animals in which osteonal bone is the dominating structure, bone mass gain is in the range of 8-15% in both ovariectomized monkeys after 18 months (66-68) and in osteoporotic women after 2-3 years (6,127-129). The greatest incremental increase in bone mass occurs early in treatment, usually within the first one to two turnover cycles (8,39,64,130,131) (Fig. 5). The anabolic effect of PTH is i n d e p e n d e n t of g e n d e r and sexual maturity; it occurs in middle-aged and osteoporotic male and female h u m a n s (6,127-129) and in intact and castrated osteopenic male and female rats (20,24, 32-34,42,45,47-50,54,60,126,132). PTH also increases bone mass in rats with osteopenia induced by denervation (53,133,134), immobilization (35-37,134,135), or discontinuation of exercise (44,53,136,137). Anabolic

A n a b o l i c E f f e c t s o f P T H in A n i m a l s with Osteonal Bone

In adult h u m a n s and animals with osteonal cortical bone, the response to PTH is d e p e n d e n t on both modeling and remodeling processes in bone. T h o u g h the net outcome is a gain in bone mass, as in rats, the mechanisms by which this is accomplished are apparently quite different, and the bone gain is always less than that observed in rats. W h e t h e r PTH induces similar molecular signaling pathways in bone of different species has not been explored. The doses in humans are considerably lower than those used in rats, and are limited by the need to avoid chronic hypercalcemia. In h u m a n s and large animals with osteonal bone, PTH stimulates modeling as de novo bone formation on trabecular and cortical endosteal surfaces, and stimulates remodeling by increasing activation frequency in both trabecular and intracortical bone (44,141-146). The stimulation of new trabecular bone formation by modeling is present as early as 2 weeks after starting treatm e n t in h u m a n s (147,148). The remodeling process in cortical bone of large animals and h u m a n s replaces intracortical matrix in situ, so bone mass may be maintained or slightly decreased in the course of treatment. Endocortical formation is stimulated but PTH has little or no effect on periosteal bone formation. The increase in intracortical bone turnover is necessarily associated with remodeling

PTH EFFECTS ON BONE ANt) GROWTH FACTORS / transients manifest as increased porosity, signaling the formation of new osteons (130,131). In some species of large animals with cortical osteonal bone, such as intact dogs and rabbits or ovariectomized ferrets and sheep, the increase in activation frequency predominantly reflects an increase in remodeling sites, and there is often no change in overall bone mass (6,125,130,131,149,150). However, in ovariectomized monkeys and osteoporotic humans, the composite response of an increase in trabecular bone mass due to the increase in u n c o u p l e d modeling formation and an increase in the a m o u n t of replaced matrix due to intracortical remodeling results in increased bone mass and resistance to fracture (6,69,130,131). The tissue events supporting the increase in trabecular bone change with time in animals with remodeling bones. In iliac trabecular bone of ovariectomized monkeys, the increase in trabecular bone volume after 6 m o n t h s is due to increased trabecular thickness attributable to new endosteal bone formation (6,69,124,130,131,151). After 15 m o n t h s of treatment, the increased trabecular volume is associated with increased trabecular number. The thickened trabeculae are modified by increased sites of tunneling, indicative of the increase in n u m b e r of r e m o d e l i n g sites, to reestablish connectivity in the trabecular framework and reduce trabecular thickness back to control values (124). The consequence of bone matrix renewal and restructuring by remodeling, c o m b i n e d with the increase in trabecular bone, is a significant improvem e n t in bone quality and biomechanical properties at clinically relevant sites in the spine and f e m u r neck (66-68,124). The growth factors and patterning genes that must be required to regulate the restructuring and r e m o d e l i n g of bone have not been e x a m i n e d in animal models in which osteonal bone structure and remodeling processes predominate.

A n a b o l i c E f f e c t s o f P T H on the S k e l e t o n o f Rodent Models The complex changes in bone structure induced by PTH in animals with osteonal skeletons are only partially r e p r o d u c e d in the rat. The rat lacks osteonal bone structure, and so PTH given at normocalcemic doses given for equivalent turnover cycles only stimulates accrual of bone t h r o u g h cumulative appositional bone growth on cortical and trabecular bone surfaces (61-63,152,153). In rats, PTH stimulates linear bone growth and increases trabecular bone mass by thickening trabeculae of the primary spongiosa and increasing the n u m b e r of osteoblasts per unit area (35,126,154). As the woven bone of the primary spongiosa acts as a template for secondary spongiosa, its increased mass and volume translates into increased bone mass of the secondary spongiosa, which is composed of mostly

189

lamellar bone. In PTH-treated rats, the trabecular osteoblasts stay active for a longer time, so the overall bone-forming surfaces in the metaphyses increase significantly. At the demarcation between metaphyseal bone and diaphyseal hematopoietic bone marrow, trabeculae are terminated by osteoclastic resorption. Although the rate of resorption at this location is not coupled to the formation events at the growth plate, the two processes are usually in equilibrium (35,126,141-144,154). PTH increases the rate of metaphyseal bone turnover so that the metaphyseal trabeculae extend in to the diaphyseal marrow as the osteoclastic resorption of the trabeculae fails to keep pace. This process, which represents a "bulking up" of e n d o c h o n d r a l osteogenesis, underlies the two- to threefold increases in bone mass seen in rats treated with PTH. The anabolic effects of PTH on trabecular bone are reversible when the h o r m o n e is withdrawn and normal e n d o c h o n d r a l osteogenesis resumes (16,47-50). The process by which this occurs in rats is quite different from the more complex response of humans, where remodeling of bone is the d o m i n a n t m e c h a n i s m controlling bone mass. In cortical bone of rats, radial growth occurs t h r o u g h o u t life as bone diameter is increased by periosteal formation, but cortical width is maintained by a parallel resorption at the endocortical surfaces (58,63). PTH may increase endocortical bone formation rate by as m u c h as 400% and periosteal rate by 60-70% (58,63,64). The continued surface accumulation of matrix on both periosteal and endosteal surfaces significantly thickened the cortex by 75% and increased cortical area by 25% in just 10 weeks in aged ovariectomized rats (62,155). The question remains whether in vitro models can differentiate these multiple responses. The models should be designed to determine the critical roles for growth factors specific for PTH induction, versus their role in mediating accelerated bone turnover and skeletal growth. The m a g n i t u d e of the bone response in rats is dose d e p e n d e n t (8,9,15) (Fig. 6). The threshold dose for an anabolic effect on bone in rats has not been determined, although increased bone has b e e n induced in rats after 3-6 months of h P T H (1-34) at low doses ranging from 1.5 to 8 Ixg/kg/day (10,45,134). In older rat models where growth is less, the increase in p e r c e n t bone-forming surfaces is of a m a g n i t u d e similar to that of younger animals, but there is less surface available for formation because bone has been lost with aging. Much of the anabolic effect in aged rats is therefore limited to the cortical e n d o s t e u m and periosteum. The increased bone mass and improved biomechanical properties are attributed to increased cortical width and cortical area (12,13,33,34,60,61,126,132). In adult rats, there appears to be a limit to the magnitude of the dose response in terms of bone mineral

190

/

CHAPTER11 RAT TRABECULAR

CALCIUM U

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FIG. 6 Anabolic effects of PTH on calcium content and dry weight of cortical and trabecular bone of distal femurs of young male rats treated with vehicle or hPTH(1-34) once daily for 12 days. Reprinted courtesy of Metab Dis Relat Res 1984.

density (BMD). A plateau effect, especially at higher doses of PTH, may be induced after 3 or more months of t r e a t m e n t (10,11,39). The consequences of continuing t r e a t m e n t with PTH once this plateau has been reached, and the interaction of this induced response with the catabolic changes of aging, have not been studied. Published data have only reported on treatment for up to 6 months, and then only in ovariectomized rats (52,126). Interactions with growth factors and cytokines that may be involved in these processes have not been investigated. A n a b o l i c vs. C a t a b o l i c E f f e c t s o f P T H o n B o n e in Vivo

In rats, the timing of exposure of PTH appears to be more critical than dose or peak concentrations in d e t e r m i n i n g whether formation or resorption predominates. The anabolic effect of PTH requires its intermittent administration (27,156-158). Increasing the n u m b e r of injections of PTH per day to 2 - 3 / d a y did not a u g m e n t the anabolic effect on either total body calcium in female rats (19), or hydroxyproline content of femurs of young female rats (158,159). PTH, given by continuous infusion at doses that were anabolic when given once by daily injection, induced hypercalcemia and death in rats a n d d o g s (27,44,137). W h e n PTH was infused continuously, or when repeated

injections were given over extended periods of time each day, the stimulatory effect of PTH on bone turnover was retained but the balance shifted from formation to resorption (27,160,161). The extent of boneforming surfaces decreased and the extent of bone resorption surfaces increased, resulting in decreased bone mass (27,160,161). The matrix metalloproteinase MMP13 (collagenase 3) appears to be required in mice because the resorbing effects of PTH were blocked in calvaria of m u t a n t mice carrying a targeted mutation in the collagen I gene that was resistant to collagenase cleavage of type I collagen (162). In addition, subtle changes were observed in proteins synthesized by the bone lining cells, which were m o r e consistent with e m e r g e n c e of a fibroblastic phenotype when compared to the osteoblast phenotype associated with intermittent t r e a t m e n t (163). Peritrabecular fibrosis, which was not observed in rats or monkeys treated with once-daily PTH, and focal bone resorption were observed when PTH was infused for 6 days in rats (160,163,164), but not when PTHrP was infused (164). Hypercalcemia and increased calcitonin were reported during the infusion period, suggesting additional systemic factors might influence the response (28,165). There was also a shift in IGF and IGF binding proteins. IGF-I, which was detected in osteoblasts of rats treated with once-daily PTH, became undetectable in the bone lining cells following PTH infusion, whereas the intensity of immunostaining for IGF-BP-3,-4, a n d - 5 increased following infusion for 7 days (153). IGF-I infusion in older ovariectomized rats stimulated resorption and impaired the anabolic effect of PTH on bone mass (28). These cumulative data suggest that additional molecular and cellular pathways may be invoked when there is prolonged exposure to PTH. The signaling pathways that result in a switch from bone accumulation to bone loss in rats are not understood. Rats given PTH as two or three injections/day at doses that sustain normocalcemia continued to exhibit an anabolic effect equivalent to that occurring with once-daily injections (19,158,159). When PTH was given at an equivalent daily dose of 80 txg/kg/day, divided into one or six injections, given either within 1 hour or over 6-8 hours, the anabolic effect of six divided injections in 1 h o u r / d a y was equivalent to that measured with one injection/day (166). However, when injections were spread over 6-8 hours, loss of bone mass occurred (166). Similarly, when PTH was infused for at least 2 hours, peritrabecular fibrosis and focal resorption, characteristic of a catabolic outcome, could be detected (160). This would suggest that stimulation of the resorptive effects of PTH is less a consequence of peak blood concentration, and more likely due to a critical cumulative duration of exposure (166). It has been shown that RGS-2 (a m e m b e r of a family of "regulator of G protein

PTH EFFECTS ON BONE AND GROWTH FACTORS // signaling" proteins) increased within 1 hour of an injection of PTH in young rats, suggesting a novel mechanism that can limit G protein signaling, subsequent to effects on the PTH1 receptor (167). In large animals, PTH, irrespective of regimen, stimulates bone resorption in intracortical bone, thereby stimulating a transient increase in remodeling space, manifest as increased porosity (69,130,131,146,149). Activation of resorption associated with porosity was observed mostly within the endosteal region of cortical bone. This regionalization, combined with new bone apposition on cortical endosteal surfaces to thicken cortical width, m e a n t that the increased porosity did not significantly alter bone biomechanical measures of strength (69,130,131,145,146,149). W h e t h e r PTH is anabolic or catabolic in skeletons with cortical osteonal bone depends on the response of the cortical endosteum. In ovariectomized monkeys and intact rabbits, intermittent PTH stimulated endocortical bone formation to increase cortical width and area, thus preserving bone mass and biomechanical measures of strength (69,130,131,145,146,149). In contrast, in dogs, continuous infusion of PTH did not stimulate formation on the cortical endosteum, so there was a transient loss of bone mass associated with porosity during treatment (168).

Reversal o f the A n a b o l i c E f f e c t o n With drawal o f P T H In young rats, the activation of bone formation by PTH was abrogated within 24-48 hours after the last PTH injection (16). This reversal was attributed to inactivation of forming surfaces, because there are m a r k e d decreases in percent calcein-labeled bone surface (DLS/BS) and percent osteoblast surface (ObS/BS), but little change in n u m b e r of osteoclasts and osteoclast surface (16). In a series of studies of spine and long bones of older ovariectomized rats, Shen et al. showed that loss of the bone gain over several weeks could be blocked by c o n c u r r e n t estrogen (47-50). This suggested that in older rats, in which growth is a less significant factor, the loss of bone following withdrawal of PTH may be due in part to activation of previously suppressed resorption (47-50). Large animal studies have provided additional insights in the more complex skeleton in which osteonal bone structure dominates. Ovariectomized (OVX) monkeys were treated once daily with h P T H ( 1 - 3 4 ) for 12 months; t r e a t m e n t was then discontinued and monkeys were given daily vehicle injections for 6 additional months before being euthanized (67,68). Bone gain after this t r e a t m e n t regimen was significantly higher than OVX controls, and similar to that of animals treated for the entire 18 months (67,68). H i s t o m o r p h o m e t r y showed that bone formation rates

191

in trabecular bone following t r e a t m e n t withdrawal r e m a i n e d equivalent to that of OVX controls (69,124). In cortical osteonal bone, bone formation rates decreased dramatically to the level of sham controls, and below those of OVX controls. Such a response implies that a significant decrease in turnover of cortical bone occurred once t r e a t m e n t was withdrawn. In this monkey study, as well as an earlier study of intact rabbits, the percent porosity in cortical bone declined after withdrawal of treatment, as the r e m o d e l i n g cycles completed and newly formed matrix mineralized (69,130,131). These events may explain the gain in bone mass and bone quality after withdrawal of treatm e n t in a small clinical study of p r e m e n o p a u s a l women (169). In hyperparathyroid h u m a n s following surgical removal of the parathyroid gland adenoma, adverse cortical changes were reversed and i m p r o v e m e n t in cortical bone mass was observed, especially the first year following surgery (170-172). However, whether these changes alter susceptibility of bones to fracture has still to be determined. Because these sequelae have not been observed in rats or reported in in vitro protocols, new experimental models are n e e d e d to study the local mediators and molecular mechanisms that regulate the different responses of trabecular and cortical osteonal bone following t r e a t m e n t withdrawal.

I N T E R A C T I O N S O F P T H A N D SKELETAL HORMONES AND GROWTH FACTORS IN VIVO E v i d e n c e o f R e q u i r e m e n t for M e d i a t o r s to I n d u c e the A n a b o l i c E f f e c t o f P T H in Vivo The early histologic studies of the bone effects of intermittent PTE in young rats (1-5,125) reported a resorptive phase in the first few days, later followed by a significant gain in bone density. I n vitro studies of avian bones treated by transient exposure to PTH (173,174) showed increases in resorption and the rate of hydroxyproline incorporation in parallel cultures. However, in vivo, blocking resorption with a short-term course of either calcitonin or a bisphosphonate did not modify the anabolic effect of PTH in either rats or h u m a n s (21,27,175). Even in the presence of a loss of body weight and inhibition of resorption associated with 12 days of calcitonin treatment, PTH continued to increase bone mass (21). W h e n PTH was given by continuous infusion and bone resorption was blocked with a bisphosphonate, the inhibition of bone formation associated with bisphosphonate was reversed by the PTH to control levels (21,36,37,41,42). Studies on the early response in h u m a n s also showed that de novo bone

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CHAPTER11

formation was stimulated by PTH in the absence of any measurable change in resorption (147,148). Although PTH increases the activation frequency of remodeling, the r e q u i r e m e n t for a regulatory mediator in osteonal bone has not been investigated in vivo. In cortical bone, bone formation increased the percent active osteonal surfaces in ovariectomized monkeys treated with h P T H ( 1 - 3 4 ) . Although the percentage of osteons exhibiting resorption increased, this did not appear to be a d o m i n a n t m e c h a n i s m of action, and bone mass was retained or increased in ovariectomized monkeys.

Interaction with IGF-I In vivo, IGF-I stimulates both bone formation and resorption. W h e n given by continuous infusion, IGF-I stimulated tunneling resorption of the diaphyseal cortex in aged female rats (28). IGF-I may stimulate osteoclastic bone resorption in vitro by regulating osteoclast differentiation (176). PTH has been shown to regulate not only IGF-I but also its binding proteins in vitro (82,177). It may be that, in vivo, intermittent PTH transiently modifies the IGF-binding proteins to activate IGF-I and initiate its anabolic action in bone, as suggested by the work of Watson et al. (153). Circulating IGF-I could also play a role in regulating the anabolic effects of PTH. Preliminary data, obtained from young hypophysectomized (HX) rats and aged female rats, showed that although PTH did not alter serum IGF-I, growth h o r m o n e (GH) increased IGF-I, and PTH given with GH partially inhibited this stimulatory effect of GH on serum IGF-I (14,23). Long-term energy restriction of rats results in a 50% decrease in circulating IGF-I (178). Rats, energy restricted in their dietary intake by 60% since weaning, showed an attenuated response in their f e m u r bone mass and tibia bone mineral when treated with PTH alone or in combination with GH (14).

Interaction with Growth H o r m o n e Early in vitro studies in which calvaria from HX rats were treated with high doses of PTE in vivo and then cultured showed less hydroxyproline incorporation than did calvaria from intact rats treated with PTE (179). This suggested the hypothesis that a pituitary h o r m o n e , most likely, growth h o r m o n e , might be required for the anabolic effect of PTH on bone (23). We found that hypophysectomized rats, s u p p l e m e n t e d with corticosterone and thyroxine and treated with the intermittent PTH showed a 50-70% inhibition of the anabolic effect. GH not only reversed this inhibition, but also e n h a n c e d the anabolic effect of PTH on bone (23). To d e t e r m i n e if this was due to GH stimulation of

systemic growth, young intact rats were given PTH and either pair-fed to the dietary intake of age- and weightm a t c h e d HX rats, or fed a 50% calorie-restricted diet to prevent the increase in body weight associated with growth. The PTH-induced increase in bone mass of distal femurs of the restricted rats was equivalent to that of rats fed ad libitum (23). GH secretion declines with age (180-182). If GH is required for the anabolic action of PTH, aged animals should show a loss of responsiveness to PTH in their bones. In a study of aged intact females rats, neither PTH nor GH alone increased bone mass, but the combination of PTH and GH increased femur trabecular bone mass and vertebral trabecular bone volume (25). In another study of aged hypophysectomized rats, PTH increased bone formation rate despite the absence of GH, but the net effect on bone mass, to confirm an anabolic outcome rather than an outcome in which bone turnover but not bone mass increased, was not assessed (183). Further studies are n e e d e d to better resolve the relative r e q u i r e m e n t for GH and IGF-I in the anabolic effects of PTH on bone.

Interaction with Prostaglandins PTH stimulates prostaglandin synthesis in vitro by initiating the synthesis of the inducible cyclooxygenase (COX-2) (112,113). This effect may be involved in the catabolic responses to PTH in vivo because injection of PTH in transgenic mice lacking the COX-2 enzyme show less hypercalcemia than do wildtype animals. Both PTH and PGE 2 can stimulate bone formation in rats (114). PGE 2 appears to produce greater periosteal stimulation c o m p a r e d to PTH and may also produce more woven bone on cortical endosteal surfaces (184-186). In vivo studies, using indomethacin given by injection or orally to block prostaglandins, did not modify the anabolic response to PTH in young rats (187). These experiments were limited because the highest doses produced gastric lesions and the lower doses may not have fully inhibited local prostaglandin production. It seems likely both from in vitro studies (188,189), in which PTH stimulation of bone resorption and mitogenesis are not completely blocked when prostaglandin production is inhibited, and from the in vivo studies cited above that prostaglandins are not essential for anabolic action of PTH. Nevertheless it is possible that prostaglandins as well as fibroblast growth factor, production of which is stimulated by both PTH and PGE 2, can enhance the response to PTH, and that this could be of importance in the therapeutic response. It will now be possible to examine this question more completely by using selective COX-2 inhibitors in humans and COX-2 and FGF knockout animals.

P T H EFFECTS ON BONE AND GROWTH FACTORS

Other Growth Factors The possibility remains that other growth factors, such as TGF-[3, which is known to alter PTH receptors in vitro (88,122,190,191), may also play a contributing role. A flow cytometry study of growth factor profiles of bone cells isolated from the metaphysis after 3 days of PTH treatment showed small but significant increases in the percentage of cells expressing IGF-I, TGF-[3, PDGF, and FGF receptors, whereas cells isolated from the diaphysis showed increases in the percentage of cells expressing IL-4 and EGF receptors (J.M. Hock and N. Falla, unpublished data). In limb development, upregulation of FGF receptors has been associated with patterning and induction phenomena. Although not studied from a molecular aspect, PTH has significant effects in altering the geometry and connectivity of trabecular bone. It may be that PTH regulation of growth factors is the mechanism controlling patterning and localized placement of bone matrix in de novo bone formation on endosteal surfaces.

SUMMARY The roles for the selected growth factors studied in vitro have mostly been in embryonic or fetal rodent bones, or in immortalized or transformed bone cells in which key cell cycle control genes are suppressed. Rodents as an in vivo model contribute to our understanding of the mechanisms of action of PTH on modeling and de novo bone formation, as well as on the growth and development of the skeleton. However, the insights gained from in vitro models need to be validated in vivo, and their context better understood. The latest preliminary data on RANKL should provide additional insights on potential cytokine mediators of the complex actions of PTH in bone. However, the most exciting developments have been in studies of large animal models and osteoporotic humans in which PTH effects on remodeling dominate. New in vitro and in vivo models are needed to provide further insight into how growth factors and cytokines interact with PTH to mediate its effects in osteonal bone.

REFERENCES 1. Burrows RB. Variations produced in bones of growing rats by parathyroid extract. Am J Anat 1938;62:237-290. 2. Jaffe H, Bodansky A, Blair J. The effects of parathormone and ammonium chloride on the bones of rabbits. J Exp Med 1932;55:695-701. 3. Jaffe, H. Hyperparathyroidism (Recklinghausen's disease of bone). Arch Patho11933;16:63-122. 4. Pugsley LI, Selye H. The histological changes in the bone responsible for the action of parathyroid hormone on the calcium metabolism of the rat. JPhysio11933;79:113-117.

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5. Selye H. On the stimulation of new bone formation with parathyroid extract and irradiated ergosterol. Endocrinology 1932;16:547-558. 6. Cosman F, and Lindsay R. Is parathyroid hormone a therapeutic option for osteoporosis? A review of the clinical evidence. Calcif Tissue Int 1998;62:475-480. 7. ReeveJ. PTH: A future role in the management of osteoporosis. J Bone Miner Res 1996;11:440-445. 8. Whiffield J, Morley E Anabolic treatments for osteoporosis. Boca Raton, Florida: CRC Press LLC, 1998. 9. Whitfield J, Morley P, Willick G, Ross V, MacLean S, Barbier J, Isaacs R, Ohannessian-Barry L. Comparison of the ability of recombinant human parathyroid hormone, rhPTH (1-84) and hPTH (1-31) NH2 to stimulate femoral trabecular bone growth on ovariectomized rats. Calcif Tissue Int 1997;60:26-29. 10. Gasser J. Preclinical studies and clinical experiences with parathyroid hormone and its analogues. Curr Opin Orthoped 1998;9:1-6. 11. Gasser J, Jerome c. Parathyroid hormone: A cure for osteoporosis? Triangle 1992. 12. Ejersted C, Andreassen T, Nilsson M, Oxlund H. Human parathyroid hormone (1-34) increases bone formation and strength in cortical bone in aged rats. EurJEndocrino11994;130:201-207. 13. Ejersted C, Andressen T, Hauge E, Melsen F, Oxlund H. Parathyroid hormone (1-34) increases vertebral bone mass, compressive strength and quality in old rats. Bone 1995;17:507-511. 14. Gunness M Hock J. Anabolic effect of parathyroid hormone is not modified by supplementation with insulinlike growth factor I (IGF-I) or growth hormone in aged female rats fed an energyrestricted or ad libitum diet. Bone 1995;16:199-207. 15. Gunness-Hey M, Hock JM. Increased trabecular bone mass in rats treated with synthetic parathyroid hormone. Metab Bone Relat Dis 1984;5:177-181. 16. Gunness-Hey M, HockJM. Loss of anabolic effect of parathyroid hormone on bone after discontinuation of hormone in rats. Bone 1990;10:447-452. 17. Gunness-Hey M, Hock JM, Gera I, Fonseca J, Poser J, Bevan J, Raisz LG. Human parathyroid hormone and salmon calcitonin do not reverse impaired mineralization produced by high doses of 1,25-dihydroxyvitamin D~. Calcif Tissue Int 1986;38:234-238. 18. Gunness-Hey M, Gera I, Fonseca J, Raisz LG, Hock JM. 1,25Dihydroxyvitamin D 3 alone or in combination with parathyroid hormone does not increase bone mass in young rats. Calcif Tissue Int 1988;43:284-288. 19. Hefti E, Trechsel U, Bonjour JP, Fleisch H, Schenk R. Increase in whole body calcium and skeletal mass in normal and osteoporotic adult rats treated with parathyroid hormone. Clin Sci 1982;62:389-396. 20. Hock JM, Gera I, FonsecaJ, Raisz LG. Human parathyroid hormone (1-34) increases bone mass in ovariectomized and orchidectomized rats. Endocrinology 1988;122:2899-2904. 21. Hock JM, H u m m e r t JR, Boyce R, Fonseca J, Raisz LG. Resorption is not essential for the stimulation of bone growth by human parathyroid hormone 1-34 in rats in vivo. J Bone Miner Res 1989;4:449-458. 22. Hock JM, Fonseca J, Gunness-Hey M, Kemp BE, Martin TJ. Comparison of the anabolic effects of synthetic parathyroid hormone-related protein (PTHrP) 1-34 and PTH 1-34 on bone in rats. Endocrinology 1989;125:2022-2027. 23. HockJM, FonsecaJ. Anabolic effect of human synthetic parathyroid hormone (1-34) depends on growth hormone. Endocrinology 1990; 127:1804-1810. 24. Hock JM, Wood RJ. Bone response to parathyroid hormone in aged rats. Cells Materials 1991;(Suppl.1):53-58. 25. Hock J, McOskerJ. The anabolic effect of parathyroid hormone on bone does not depend on suppression of endogenous PTH. Endocrinology 1991;127.

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150. Li X, Stevens M, Mackey M, Combs K, Tressler D, McOsker J. The ferret: Skeletal responses to treatment with parathyroid hormone. J Bone Miner Res 1994;8 (Suppl. 1):$258. 151. Jerome C, Vafai cj, Kaplan K, Bailey J, Capwell B, Fraser E Hansen L, Ramsay H, Shadoan M, Lees C, Thomsen J, Mosekilde L. Effect of treatment for 6 months with human parathyroid hormone (1-34) peptide in ovareictomized cynomolgus monkeys (Macaca fascicularis) . Bone 1999;25:301-309. 152. Qi H, Li M, Wronski T. A comparison of the anabolic effects of parathyroid hormone at skeletal sites with moderate and severe osteopenia in aged ovariectomized rats. J Bone Miner Res 1995;10:948-955. 153. Watson P, Lazowski D, Han V, Fraher L, Steer B, Hodsman A. Parathyroid hormone restores bone mass and enhances osteoblast insulin-like growth factor I gene expression in ovariectomized rats. Bone 1995;16:357-365. 154. Toromanoff A, Ammann P, Riond J. Early effects of short-term parathyroid hormone administration on bone mass, mineral content, and strength in female rats. Bone 1998;22:217-223. 155. Baumann B, Wronski T. Response of cortical bone to antiresorptive agents and parathyroid hormone in aged ovariectomized rats. Bone 1995;16:247-253. 156. Podbesek R, Eduoard C, Meunier PJ, Parsons JA, Reeve J, Stevenson RW, ZanelliJM. Effects of two treatment regimes with synthetic human parathyroid hormone fragment on bone formation and the tissue balance of trabecular bone in greyhounds. Endocrinology 1983;112:1000-1006. 157. Podbesek RD, Mawer EB, Zanelli GD, Parsons JA, Reeve J. Intestinal absorption of calcium in greyhounds: The response to intermittent and continuous administration of human synthetic para thyroid hormone fragment 1-34 (hPTH 1-34). Clin Sci 1984;:591-599. 158. Riond J, Fischer I, G-V, Kuffer B, Toromanoff A, Forrer R. Influence of the dosing frequency of parathyroid hormone (1-38) on its anabolic effect in bone and on the balance of calcium, phosphorus and magnesium. Z Ernahrungswiss 1998;37:183-189. 159. Riond JL. Modulation of the anabolic effect of synthetic human parathyroid hormone fragment (1-34) in the bone of growing rats by variations in dosage regimen. Clin Sci 1993;85:223-228. 160. Dobnig H, Turner R. The effects of programmed administration of human parathyroid hormone fragment (1-34) on bone histomorpometry and serum chemistry in rats. Endocrinology 1997;138:4607-4612. 161. Uzawa T, Hori M, Ejiri S, Ozawa H. Comparison of the effects of intermittent and continuous administration of human parathyroid hormone (1-34) on rat bone. Bone 1995;16:477-484. 162. Zhao W, Byrne M, Boyce B, Krane S. Bone resorption induced by parathyroid hormone is strikingly diminished in collagenaseresistant mutant mice. J Clin Invest 1999;103:517-524. 163. Watson P, Fraher L, Kisiel M, DeSousa D, Hendy G, Hodsman A. Enhanced osteoblast development after continuous infusion of hPTH (1-84) in the rat. Bone 1999;24:89-94. 164. Kitazawa R, Imai Y, Fukase M, Fujita T. Effects of continuous infusion of parathyroid hormone and parathyroid hormonerelated peptide on rat bone in vivo: Comparative study by histomorphometry. Bone Miner 1991;12:157-166. 165. Dobnig H, Turner R. Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 1995;136:3632-3638. 166. Frolik C, Black E, Cain R, Hock J, Osborne J, Satterwhite J. Pharmacokinetic profile of LY333334, biosynthetic human parathyroid hormone (hPTH) (1-34), and serum biochemistry after anabolic or catabolic injection protocols. J Bone Miner Res 1997;12(Suppl. 1):$319. 167. Miles R, Sluka J, Santerre R, Hale L, Bloem L, Boguslawski G, Thirunavukkarasu K, Hock J. Dynamic regulation of RGS2 in

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bone: Potential new insights into PTH signaling mechanisms. Endocrinology 2000;141:28-36. Malluche H, Sherman D, Meyer W, Ritz E, Norman A, and Massry S. Effects of long-term infusion of physiologic doses of 1-34 PTH on bone. AmJPhysio11982;242:F197-F201. Finkelstein J, Klibanski A, Arnold A, Toth T, Hornstein M, Neer R. Prevention of estrogen deficiency-related bone loss with human parathyroid hormone- (1-34). J Am Med Assoc 1998;280:1067-1073. Christiansen P, Steiniche T, Brixen K, Hessov I, Melsen F, Heikerdorff L, Moskilde L. Primary hyperparathyroidism: Short-term changes in bone remdoeling and bone mineral density following parathyroidectomy. Bone 1999;25:237-244. Christiansen P, Steiniche T, Brixen K, Hessov I, Melsen E Heickerndorff L, Mosekilde L. Primary hyperparathyroidism: Effect of parathyroidectomy on regional bone mineral density in Danish patients: A three-year follow-up study. Bone 1999; 1999:589-595. Duan Y, Luca VD, Seeman E. Parathyroid hormone deficiency and excess: Similar effects on trabecular bone but differing effects on cortical bone. J Clin Endocrinol Metab 1999;84: 718-722. Howard GA, Bottemiller BL, Baylink DJ. Evidence for the coupling of bone formation to bone resorption in vitro. Metab Bone Dis Relat Res 1980;2:131-135. Howard G, Bottemiller B, Turner R. Parathyroid hormone stimulates bone formation and resorption in organ cultures: Evidence for a coupling mechanism. Proc Natl Acad Sci USA 1981;78:3204-3208. Cosman E Nieves J, Woelfert L, Shen V, Lindsay R. Alendronate does not block PTH-induced stimulation of bone formation. J Bone Miner Res 1998. Mochizuki H, Hakeda Y, Wakatusuki N, Usui N, Akashi S, Sato T, Tanaka K, Kumegawa M. Insulin-like growth factor I supports formation and activation of osteoclasts. Endocrinology 1992; 131:1075-1080. McCarthy TL, Centrella M, Canalis E. Parathyroid hormone enhances the transcript and polypeptide levels of insulin-like growth factor I in osteoblast-enriched cultures from fetal rat bone. Endocrinology 1989;124:1247-1253. Breese C, Ingram R, Sonntag W. Influence of age and longterm dietary restriction on plasma insulin-like growth factor I, IGF-I gene expression and IGF-binding proteins. J Gerontol 1991;46:B180-B187. Johnston C, Deiss W. Some effects of hypophysectomy and parathyroid extract on bone matrix biosynthesis. Endocrinology 1965;76:198-202.

180. Deslauriers N, Gadreau P, Abribat T, Renier G, Petitclerc D, Brazeau E Dynamics of growth hormone responsiveness to growth hormone releasing factor in aging rats: Peripheral and central influences. Neuroendocrinology 1991 ;53:439-436. 181. Goya RG, Quigley KL, Takahashi S, Reichert R, Meites J. Effect of homeostatic thymus hormone on plasma thyrotropin and growth hormone in young and old rats. Mech Ageing Dev 1989;49:119-128. 182. Takahashi S, Gottshall PE, Quigley KL, Goya RG, Meites J. Growth hormone secretory patterns in young, middle-aged and old female rats. Neuroendocrinology 1987;46:137-142. 183. Schmidt I, Dobnig H, Turner R. Intermittent parathyroid hormone treatment increases" osteoblast number, steady state messenger ribonucleic acid levels for osteocalcin, and bone formation in tibial metaphysis of hypophysectomized female rats. Endocrinology 1995;136:5127-5134. 184. Jee W, Ueno K, Kimmel D, Woodbury D, Price P, Woodbury L. The role of bone cells in increasing metaphyseal hard tissue in rapidly growing rats treated with prostaglandin E2. Bone 1987;8:171-178. 185. Jee W, Ma Y, Li X. The immobilized adult cancellous bone site in a growing rat as an animal model of human osteoporosis. J Histotechno11997;20:201-206. 186. Mori S, Jee w, Li X, Chan S, Kimmel D. Effects of prostaglandin E2 on production of new cancellous bone in the axial skeleton of ovariectomized rats. Bone 1990; 11:103-113. 187. Gera I, Hock J, Raisz L, Gunness-Hey M, Fonseca J. Indomethacin does not inhibit the anabolic effect of parathyroid hormone in rats. Calcif Tissue Int 1988;40:206-211. 188. Klein-Nulend J, Pilbeam C, Harrison J, Fall P, Raisz L. Mechanism of regulation of prostaglandin production by parathyroid hormone, interleukin-1 and corticsol in cultured mouse parietal bones. Endocrinology 1991 ;128:2503-2510. 189. Vargas S, Raisz L. Simultaneous assessment of bone resorption and formation in cultures of 22-day fetal rat parietal bones: Effects of parathyroid hormone and prostaglandin E2. Bone 1990; 11:61-65. 190. Pfeilschifter J, Oechsner M, Naumann A, Gronwald RGK, Minne HW, Ziegler R. Stimulation of bone matrix apposition in vitro by local growth factors: A comparison between insulin-like growth factor I, platelet-derived growth factor, and transforming growth factor B. Endocrinology 1990;127:69-75. 191. Oursler M, Cortese C, Keeting F, Andersons M, Bonde K, Riggs B, Spelsberg T. Modulation of transforming growth factor-beta in normal human osteoblast-like cells by 17-beta estradiol and parathyroid hormone. Endocrinology 1991 ;129:3313-3320.

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Cellular Actions of Parathyroid H o r m o n e on Osteoblast and Osteoclast Differentiation

JANE E. AUBIN Department of Anatomy and Cell Biology and Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8

JOHANN. M.

HEERSCHE Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5G 1G6

EFFECTS OF PARATHYROID H O R M O N E ON OSTEOCLAST AND OSTEOBLAST DIFFERENTIATION A N D / O R ACTIVITY IN VIVO: STUDIES IN HUMANS, RATS, AND MICE

other therapeutic factors in humans [for discussion, see Hirano et al. (14) ]. In agreement with results summarized above, intermittent injection of PTH(1-34) increased cancellous bone in the secondary spongiosa of parathyroidectomized rats (15,16), an effect that was i n d e p e n d e n t of resorptive activity (15,17). Other in vivo data from humans (18) and rats have confirmed that intermittent PTH stimulated bone formation de novo, without a prior episode of resorption. Studies done with PTH in fracture healing models in both normal and osteoporotic bones of rats showed that daily subcutaneous injections of PTH(1-34) increased both the ultimate load that could be tolerated before breaking and the callus volume of rat tibial fractures (19). In an interesting gene therapy approach not directly mimicking an intermittent dose regime, degradable (gene-activated collagen matrix) (GAM) sponges were loaded with a plasmid containing PTH (1-34) cDNA and implanted into critical-sized defects made surgically in rat femurs or beagle femurs and tibias (20,21). The matrix carrier appeared to act as a scaffold into which fibroblasts migrated and became infected with the plasmid, and acted as in vivo "bioreactors," locally secreting PTH(1-34), which stimulated fracture healing and new bone formation, but without evidence for stimulation of osteoclast formation in the area (20,21). The authors argued that local PTH concentrations were probably below levels required to see catabolic activity. These and a large n u m b e r of other studies in ovariectomized rats and postmenopausal women have consistently

A prolonged increase in circulating levels of parathyroid h o r m o n e (PTH) is associated with increased bone turnover, i.e., increased osteoclastic bone resorption and increased osteoblastic activity (1-3). In severe hyperparathyroidism, this results in loss of both cortical and cancellous bone (4,5). However, mild hyperparathyroidism is associated with normal or increased bone mineral density and increased bone volume in areas that are primarily cancellous, such as vertebrae (6-9), but bone is still lost in cortical areas (6). Daily injections of h u m a n PTH(1-34) for 6-12 months also increase the cancellous bone area in iliac crest biopsies (10) and decrease femoral cortical bone density of osteoporotic patients (11). Thus, u n d e r certain conditions, PTH can affect cancellous bone and cortical bone differently, with a net increase in bone mass occurring in cancellous bone concomitant with a net loss of cortical bone. Of particular interest is the observation that the adverse effects of treatment with PTH on cortical bone density might be ameliorated by simultaneous treatment with estrogen (12) or calcitriol (13). However, unambiguous conclusions on the effects of PTH on cortical bone remain difficult and c o n f o u n d e d by issues such as differences in models (e.g., rodents versus rabbits or dogs) and the presence and kinds of

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d o c u m e n t e d increased bone mass with intermittent PTH t r e a t m e n t (22-24). It is also of interest to note that the anabolic effects of PTH on bone apposition were abolished in vitamin D-deficient rats and were restored by vitamin D supplementation (25). This interaction seemed to be d e p e n d e n t on the presence of growth h o r m o n e (26). The m e c h a n i s m whereby increased levels of PTH are t h o u g h t to affect bone metabolism in h u m a n s is an increase in remodeling resulting from a 50% increase in activation frequency (27). Why this should maintain or increase cancellous bone volume and decrease cortical bone volume remains to be determined. One explanation, suggested by Parfitt (28) is that the depth of osteoclastic resorption lacunae on the endocortical surface is greater than the d e p t h of the lacunae on cancellous bone surfaces. The reasons for this difference, however, are not known.

EFFECTS OF PARATHYROID H O R M O N E ON OSTEOBLAST AND OSTEOCLAST PROLIFERATION AND DIFFERENTIATION IN B O N E O R G A N C U L T U R E SYSTEMS Early studies with mouse long bone rudiments showed conclusively that PTH affected both osteoblast and osteoclast numbers and activity in a dose- and timed e p e n d e n t fashion (29). With culture in the presence of low concentrations of PTH (0.01-0.00 U / m l ) , decreased activity of osteoblasts was seen after 12-14 hours, whereas multinucleated osteoclasts increased in n u m b e r and became active after 24-28 hours. The three types of tissue represented in such bone rudiments, that is, bone, cartilage, and marrow stroma, all responded to PTH, but timing and dose responsiveness differed. Low concentrations of PTH (0.01-0.00 U / m l ) had no effect on cartilage or marrow stroma, whereas higher concentrations of PTH induced increased proliferation of cartilage and of connective tissue and had more p r o n o u n c e d effects on osteoblasts and osteoclasts. These early experiments uncovered three aspects of PTH action on bone-related tissues that have been topics of major interest over many years: the time delay between PTH effects on osteoblasts and osteoclasts (compatible with a cause-and-effect relationship), reduction of osteoprogenitor differentiation into osteoblasts and e n h a n c e d proliferation of a fibroblastlike cell type, and the effects of PTH on chondrocyte proliferation and differentiation. Progress on the underlying cellular and molecular mechanisms leading to these observations has been made in the past several years in all three of these areas.

EFFECTS OF PARATHYROID H O R M O N E ON OSTEOBLAST PROLIFERATION AND D I F F E R E N T I A T I O N I N VIVO A N D I N VITRO There is m u c h emphasis on d e t e r m i n i n g what the "target" cell in bone is that leads to the anabolic versus catabolic effects of the h o r m o n e . Published studies repeatedly d o c u m e n t the ability of PTH to increase very rapidly the n u m b e r of functional osteoblasts, perhaps m o r e consistent with an effect of PTH on the state of osteoblast differentiation and functional status rather than on proliferation followed by differentiation events. However, it has also been suggested that PTH specifically and bone anabolic agents generally may work, at least in part, through stimulation of cell proliferation (30,31). Both stimulatory and inhibitory effects on proliferation have been reported for PTH. These inconsistent results probably reflect the different sources and kinds of cells used in different studies [e.g., osteosarcoma-derived lines, primary bone marrow stromal populations, primary fetal or neonatal calvariaderived osteoblastic cells, primary trabecular bone (femoral spongiosa cells) cells, etc.] and the many different culture conditions used (e.g., without or with a high percentage of serum). In the UMR-106 osteosarcoma cell line, for example, PTH is known to inhibit proliferation via inhibition of cell cycle progression through a c A M P / p r o t e i n kinase A-mediated process (32) that has been linked to p27Kipl induction (33,34). On the other hand, in the TE-85 osteosarcoma line, PTH stimulates proliferation through stimulation of cdc2 expression via increased levels of free E2F (35). Interspecies differences have also been reported in activation of MAP kinases in different osteoblastic models (36). These differences may be related to true species differences a n d / o r to relative stage of differentiation of the different cell lines (e.g., more or less mature), a possibility that may also explain differences reported in osteoblastic populations isolated from fetal or neonatal animals versus postnatal and m a t u r e rodents. However, osteoblast-like osteosarcoma-derived cells may lack or may express m u t a t e d forms of key cell cycle regulatory genes, such as p53 and Rb, contributing to aberrant proliferation controls and discrepancies seen with multiple agents, including PTH. Thus, it seems crucial to ask whether there are data supporting the ability of PTH either to stimulate or to inhibit proliferation in vivo in any animal model. In young rats, proliferating cells in bone are located subjacent to the growth plate, the cortical endosteum of the metaphysis, and the cortical periosteum of the diaphysis, all locations in which PTH exerts stimulatory effects on bone formation, but without apparently stimulating proliferation [ (37,38); reviewed in (24) ].

PTH AND BONE CELL DIFFERENTIATION / Oniya et al. (38) showed that intermittent PTH treatm e n t appeared to target proliferating cells in the primary spongiosa of young rat distal femur metaphysis, resulting in an increased n u m b e r of osteoblasts, but via and down-regulation of cell proliferation and up-regulation of cell differentiation in trabecular bone with transient stimulation of the early response genes and interleukin-6 (IL-6). In mature rats, PTH stimulates lining cells on quiescent surfaces to function as osteoblasts also without inducing proliferation (39,40). On the other hand, Nishida and colleagues (41) reported that intermittent PTH administration was able to increase the total n u m b e r of fibroblast colony-forming units (CFU-F) and alkaline phosphatase-positive CFU-F recoverable from the bone marrow and capable of growth ex vivo. Similarly, Bikle and colleagues (42) found that PTH treatment of normally loaded, but not unloaded, rats caused a 2.5-fold increase in the n u m b e r of bone marrow stromal cells, with similar increases in alkaline phosphatase activity and mineralization, compared with cultures from vehicle-treated rats. Direct in vitro PTH challenge of stromal cells isolated from normally loaded bone failed to stimulate their proliferation and inhibited their differentiation, suggesting that the in vivo anabolic effect of intermittent PTH on stromal cells may be mediated indirectly by a PTH-induced factor. The authors speculated that the factor is insulinlike growth factor-I (IGF-I), which stimulated the in vitro proliferation and differentiation of stromal cells isolated from normally loaded bone, but not from unloaded bone. Taken together, these data suggest that more remains to be done to show conclusively whether and how much proliferation may contribute to the anabolic effects of PTH and to clarify the effects of PTH in vivo on proliferative responses in different subpopulations of osteoblastic cells u n d e r different conditions. The early data with respect to PTH effects on differentiation are as complex, but did provide some evidence for osteoblast differentiation stage-specific effects and differences between chronic versus intermittent exposure to PTH. For example, early data showed that continuous exposure of osteoblasts to PTH in organ culture models decreased osteoblast activity and differentiation of functioning osteoblasts [e.g., (43) ], but that a brief incubation with PTH followed by a return to control medium stimulated collagen synthesis in a m a n n e r d e p e n d e n t on IGF-I (44). In addition, PTH was found to suppress alkaline phosphatase activity in the relatively mature rat osteoblastic cell line ROS 17/2.8 (45), but to stimulate the enzyme in the preosteoblastic mouse osteoblastic cell line MC-3T3-E1 (46). However, as raised previously (31), the ability of osteosarcoma-derived and established/immortalized cell lines to reproduce faithfully a normal differenti-

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ated cell phenotype is not always certain and, more recently, considerable insight has resulted from use of primary cell culture models of normal osteoblasts. Much has been learned from approaches in vitro in which the nature of osteoprogenitors and their more differentiated progeny in primary cultures have been investigated by functional (the nature of the colonies they form, e.g., mineralized bone nodules), immunologic (e.g., immunocytochemistry, Western analysis), and molecular (Northern blots, polymerase chain reaction of various sorts, in situ hybridization) assays of the bone nodules formed in bone marrow and bonederived primary cell populations. The progenitor cells present in these populations u n d e r g o a proliferation-differentiation sequence leading to expression of tissue-specific macromolecules, including the bone matrix molecules (type I collagen, osteocalcin, osteopontin, bone sialoprotein, a m o n g others) and transcription factors that regulate them and commitment/differentiation events (e.g., Cbfal, AP-1 family members, Msx-2, Dlx-5) (47). Ultimately, the differentiated osteoblasts that form are morphologically essentially identical to their counterparts in vivo and the deposited matrix contains the major bone matrix proteins and mineralizes in a regulated manner. These models have become extensively used to investigate the regulation of osteoblast development and activity by hormones, cytokines, and growth factors (Fig. 1) [reviewed in (48,49)]. Part of the value of this model stems from the fact that the bone nodules represent the end product of the proliferation and differentiation of osteoprogenitors present in the starting cell population, their presence and differentiation status can be quantified, and the effects of agents of interest can be studied after either chronic exposure t h r o u g h o u t the developmental sequence or pulsatile exposure during either proliferation or differentiation stages. There are growing data investigating the effects of PTH in this model. O u r labs were the first to use the RC cell bone nodule model to investigate the mechanisms by which PTH might affect osteoprogenitors in vitro and at what developmental stages (50). Continuous exposure to PTH caused a dose-dependent inhibition of bone nodule formation, with half-maximal inhibition at 0.05 nM, and total inhibition at 1 nM, concentrations much lower than those required to elicit a significant cAMP response in RC cells, and without effect on cell growth or saturation density. T h o u g h continuous exposure to 1 n M PTH eliminated bone nodule formation, a single 48-hour pulse administered at any time during the 17-day culture period had no effect. When 1 n M PTH was added on day 1 and removed at different times during the culture period, a time-related release

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Limited proliferation

Limited self-renewal Extensive proliferation~

+

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~

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PTH receptor expression PTH effects on bone nodule formation in vitro

Chronic exposure Repeated 1 hr exposures Repeated 6 hr exposures

No progression past preosteoblaststage and no bone nodules Differentiation and bone nodule formation ~ Differentiation and bone nodule formation +

PTH effects on osteoblast and osteocyte apoptosis in vivo and in vitro

FIG. 1 A schematic of the osteoblast lineage based on cell culture models that recapitulate a proliferation-differentiation program in vitro (see Refs. 31 and 71). These models have suggested that PTH1R is expressed from early osteoprogenitor stages, but increases in levels and activity as osteoblasts mature. In addition, they have provided evidence for differentiation-stage-specific effects of PTH and for anabolic versus catabolic effects on osteoblast development depending on the time and duration of exposure to PTH (see text for further details).

from inhibition was observed. Cultures exposed to 1 n M PTH until nodules had developed in the corresponding control cultures and then switched to m e d i u m without a d d e d PTH rapidly formed clusters of differentiated osteoblasts and nodules within 3 days. PTH added at different times during the culture period and present continuously thereafter suppressed formation of new nodules, the magnitude of the effect being a function of the duration of exposure. These experiments suggest that PTH is a p o t e n t - - b u t reversible-suppressor of osteoblast differentiation and that its effect u n d e r this t r e a t m e n t regime occurs at a late stage in the differentiation of osteoprogenitor cells, probably preventing differentiation of preosteoblasts into osteoblasts (Fig. 1). It was, of course, also of interest to

know what signaling pathway might be coupled to the inhibitory effect of PTH. As m e n t i o n e d above, in our experiments, complete inhibition of bone nodule formation was seen at concentrations of PTH below those required to elicit a measurable cAMP response in RC cells (50). In other experiments, we also found that forskolin at low concentrations that did not affect cAMP or cell architecture were stimulatory, whereas higher concentrations that did increase cAMP and affect cell architecture significantly were inhibitory, whether they were present continuously or in repeated short (1-hour) pulses at each m e d i u m change (each 48 hours) during the entire culture period (51). Taken together, all these results suggest that intermittent elevations in intracellular cAMP have an inhibitory

PTH AYO BoNE CELL DIFFERENTIATION / effect on bone formation in vitro, but that osteoprogenitor cells may be stimulated to differentiate possibly through a non-cAMP-dependent process. That RC cells respond differently depending on their differentiation status and that continuous versus pulsatile exposure of RC cells to PTH may elicit different biologic responses coupled to different signaling pathways was confirmed by Yamaguchi and colleagues (52). These authors treated RC cells either continuously or cyclically with PTH(1-34) for the first few hours of each 48-hour incubation cycle. When cells were exposed to PTH only for the first h o u r of each 48-hour incubation cycle and then cultured for the remainder of the cycle without PTH, osteoblast differentiation was inhibited, as evidenced by suppression of alkaline phosphatase activity, bone nodule formation (Fig. 1), and mRNA expression of alkaline phosphatase, osteocalcin, and receptors for PTH (hereafter referred to as PTHIR, the P T H / p a r a t h y r o i d hormonerelated peptide receptor) (53,54). Experiments using inhibitors and stimulators of cAMP/protein kinase A (PKA) and CaZ+/protein kinase C (PKC) demonstrated that cAMP/PKA was the major signal transduction system in the inhibitory action of PTH. In contrast, when cells were exposed to intermittent PTH for the first 6 hours of each 48-hour cycle, osteoblast differentiation was stimulated. Both cAMP/PKA and Ca2+/PKC systems appeared to be involved cooperatively in this anabolic effect. These authors further investigated the possible downstream mediators of the PTH effect and found that although both cAMP/PKA and CaZ+/PKC were involved in the effect of continuous exposure to PTH, they appeared to act independently. They found that a neutralizing antibody against IGF-I blocked the stimulatory effect induced by the 6-hour intermittent exposure, but not the inhibitory effect induced by the 1-hour intermittent exposure, again suggesting that PTH catabolic versus anabolic effects are likely mediated through different signaling pathways (52). In a more recent study with the MC-3T3-E1 cell model, Howard and colleagues (55) reported that when continuous PTH treatment was initiated during approximately the first half of the month-long culture period, mineralization decreased, whereas continuous exposure later had little to no effect. However, a 5-day pulse in roughly the middle of the culture period increased mineralization, an effect that also occurred in primary cultures of murine and h u m a n osteoblastic cells. The differences in PTH effects on mineralization did not correlate with P T H I R expression, which was detected early and increased only marginally in the second half of the culture period, nor with the cAMP response to PTH, which increased markedly after day 10 and remained high to the end of the culture. These data confirm that there are differentiation stage-

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specific effects of PTH and that signaling pathways other than stimulation of adenylate cyclase are involved in PTH effects on osteoblast differentiation. Another mechanism has been suggested to contribute to the anabolic effect of PTH, and that is an apoptotic effect. Jilka and colleagues (56) found that daily PTH injections in mice with either normal bone mass (SAMR1) or osteopenia due to defective osteoblastogenesis (SAMP6) increased bone formation without affecting the generation of new osteoblasts and with no evidence for reactivation of lining cells as seen in at least some mature rat studies (39). Rather, in this murine model, PTH appeared to increase the life span of the osteoblasts by preventing their apoptosis (Fig. 1), thereby prolonging the time spent in performing their matrix-synthesizing activity. To determine whether the antiapoptotic effect of PTH was due to direct action of the h o r m o n e on osteoblasts and osteocytes, as opposed to indirect actions mediated by compensatory changes, the effect of PTH on apoptosis was examined in r o d e n t and h u m a n osteoblast and osteocyte models in vitro. Apoptosis, stimulated by glucocorticoids (57,58) in primary cultures of osteoblasts isolated from neonatal murine calvaria, the MC-3T3-E1 murine osteoblastic line, h u m a n osteoblastic MG-63 osteosarcoma cells, or murine MLO-Y4 osteocytic cells (59), was attenuated by PTH(1-34). In osteoblasts, induction of apoptosis by tumor necrosis factor (TNF) was not affected by PTH, which suggested an interesting interference with some, but not all, death pathways (56). However, in a 293 cell model overexpressing PTH1R, PTH induced apoptosis and the TNF receptor and PTH1R pathways appeared to converge (60), suggesting that there may be osteoblast-specific mediators required for the observed antiapoptotic effects in mouse bone. These results on the SAMP6 model are reminiscent of the antiapoptotic effect of PTHrP on chondrocytes during e n d o c h o n d r a l bone development (61,62). Given their putative role as mechanosensors and their abundance compared to other osteoblast lineage cells in adult bone, the observed antiapoptotic effect on osteocytes is also intriguing, but whether it also may contribute to an anabolic effect of PTH is not yet known. As m e n t i o n e d earlier for differentiation effects of PTH, whether the antiapoptotic effect of PTH on osteoblasts is mediated by cAMP or other post-PTH1R signaling events deserves more attention. In the SAMP6 mouse studies, the antiapoptotic effect of PTH was blocked by the P T H I R antagonist bPTH(3-34) and was mimicked by dibutyryl cAMP, suggesting that it was mediated through PTH1R and subsequent activation of adenylate cyclase (56). This is consistent with data showing that periosteal cell apoptosis is inhibited by prostaglandin E through cAMP-dependent stimulation of sphingosine kinase (63). However, in the

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293 overexpressing PTH1R, Turner et al. (60) found that PTH and P T H I R signaling induce apoptosis through Gq-mediated phospholipase C / C a 2+ signaling, rather than Gs-mediated cAMP signaling. Though these discrepancies require clarification, the elucidation of the antiapoptotic effects of PTH in the mouse model in vivo and the evidence that PTH antagonizes the proapoptotic effects of glucocorticoids in vitro provide a possible mechanistic explanation for the efficacy of daily subcutaneous injection of PTH for treatment of glucocorticoid-induced osteoporosis in humans (64).

E X P R E S S I O N OF T H E P T H R E C E P T O R BY CELLS OF T H E O S T E O B L A S T LINEAGE AND SIGNALING THRESHOLDS It seems obvious that a better understanding of when during osteoprogenitor cell differentiation PTH receptors begin to be expressed may help to clarify which osteoblast lineage cells may be targets of PTH activity. PTH receptors were demonstrated on the osteoblast and its immediate precursors in early binding studies (65) and by immunohistochemistry (66). However, in a later binding study, a distinct cell type, not a mature osteoblast but possibly an immediate osteoblast progenitor or preosteoblast, was reported to be the major PTH target (67). On the other hand, by in situ hybridization, Lee et al. found PTH1R mRNA expressed highest in growth plate chondrocytes, followed by osteoblasts (which were higher than preosteoblasts), mononuclear cells along cortical periosteal and endsoteal surfaces, some stromal cells, and plump mononuclear cells lining trabecular bone surfaces, but not flat osteoblasts or in cells embedded in bone matrix (68). Supporting not only mRNA but also PTH1R protein expression, subcutaneous administration of h u m a n PTH (1-84) induced rapid and transient expression of the protooncogene c-fos mRNA in osteoblasts, chondrocytes, and some stromal cells, consistent with the highest PTH1R mRNA expression; only later was c-fos mRNA expressed in the majority of stromal cells and in osteoclasts, implying the latter did not express PTH1R and were probably responding by an indirect mechanism. Other support for high PTH1R expression on mature osteoblasts comes from binding studies in rat calvaria cell cultures whereby PTH binding correlated better with osteocalcin expression than with alkaline phosphatase (69) and from a combined in situ hybridization-PTH binding study in young adult rats (70). Global amplification polymerase chain reaction (PCR) of replica-plated osteoprogenitor cell colonies undergoing differentiation to bone nodule-forming

cells showed that P T H I R is detectable at low levels in very primitive osteoprogenitor cells, but expression increases markedly as the cells mature to functional osteoblasts expressing markers such as osteocalcin (71). McCauley and colleagues also analyzed P T H I R expression in MC-3T3-E1 cells and primary rat calvarial cells undergoing differentiation to bone nodule-forming cells in vitro (72). Their findings indicated that PTH1R expression at the level of mRNA, protein, and biologic activity increased as cells matured and bone nodules formed. Using the same MC-3T3-E1 model, however, Schiller et al. (55) found that PTH1R mRNA was detected early but went through only a modest increase in expression later in the cultures, remaining relatively constant, but that the cAMP response to PTH varied markedly with no response early and a marked response as cells matured. These results point out just some of the inconsistencies in different studies even when similar reagents and cell lines have been used, probably reflecting the variationsmsometimes quite largemin precise culture conditions, medium additives, etc. that have been used. Nevertheless, taken together, they do suggest that PTH1R expression is already detectable on quite primitive osteoblast precursor cells but that levels of expression and cellular responsiveness increase as osteoblastic cells mature, in keeping with earlier data based on cAMP response to PTH in many osteoblast models in vitro (73). Given that PTH1R appears to be present from relatively early stages of differentiation, albeit at apparently lower n u m b e r and with lower consequent cAMP stimulation than in more mature cells, and that continuous versus pulsatile exposure to PTH elicits different biologic effects, one aspect of the molecular mechanism of PTH that deserves more attention is receptor-ligand signaling thresholds. For example, receptors with intrinsic or associated tyrosine kinase activity are known to elicit both proliferative and differentiation responses in factor-dependent cell lines, based on both the duration and the magnitude of extracellular signal-regulated kinase (ERK) activity (74). The fact that the same cytokine can elicit a different outcome simply by changing the relative expression of the corresponding receptor supports the view that the magnitude of signaling (e.g., ERK activation), and not receptor-specific signaling, may determine a biologic outcome (75). Importantly, threshold-dependent regulation may also extend to osteogenesis because both the magnitude and the duration of PTH supplementation modulate bone responses (22). These effects, which result at least in part from the differential stimulation of adenylyl cyclase and phospholipase C, have been shown to be dependent on the density of PTH receptor expression on the cell surface (76,77).

PTH AND BONE CELL DIFFERENTIATION The role of PTH1R in osteoblasts is now also being investigated in another way. A series of clonal murine calvarial osteoblastic cell lines conditionally immortalized, via expression of a transgene encoding the tsA58 temperature-sensitive SV40 large T antigen, and lacking both functional alleles of the P T H I R gene have been made from PTH1R - / - mice made earlier (78). U n d e r nontransforming conditions, these cells stop proliferating, express a series of characteristic osteoblastic genes, and produce mineralized bone nodules in a m a n n e r that is regulated by 1,25-dihydroxyvitamin D 3 but not by PTH(1-84). An unexpected but interesting observation is that osteocalcin expression is lower than expected in P T H I R - / - cells, an observation that suggests that P T H I R expression is required for normal levels of osteocalcin expression and confirms that lower than usual levels of osteocalcin do not inhibit bone formation and mineralization (79). It remains to be determined whether not only altered PTH response but also the low osteocalcin can explain, in part, the increase in osteoblast n u m b e r and matrix accumulation during intramembranous bone formation in the shafts of long bones, the decrease in trabecular bone formation in the primary spongiosa, and the delayed vascular invasion in the

Osteoblastic cell autocrine/paracrin~ feedb

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205

P T H I R - / - mice (80). These cells may also provide a useful model in which to investigate the role of receptor density via transfection of P T H I R to various levels in cells with an otherwise identical osteoblast background.

ARE P T H E F F E C T S O N O S T E O B L A S T DEVELOPMENT MEDIATED THROUGH OTHER FACTORS? It has been suggested at several points that PTH may induce a variety of factors that contribute to the anabolic and catabolic effects of PTH. T h o u g h it is beyond the scope of this chapter to review all of these in detail, mention of a few seems appropriate. Various osteogenic cell models in vitro and in vivo expressing PTH1R respond to PTH treatment by production of the PTHrP holoprotein or processed fragments (81-83) (Fig. 2). The p r o h o r m o n e of PTHrP or certain cleavage products [ones containing the 87-107 nuclear-targeting sequence (NTS)] are intrakines, i.e., peptides that do or do not enter cell nuclei d e p e n d i n g on the presence and activity of cyclin-dependent protein kinases (84,85) and contribute to cell activity, including stimulation

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of expression of the antiapoptosis protein Bcl-2 (61), among other proliferation and differentiation responses (86,87). Such activities of specific fragments of PTH and PTHrP are the basis for drug design of anabolic PTH analogs (88,89). In addition, in numerous models in vitro and in vivo PTH treatment induces production of IGF-I (44,52,90-93), IL-6 (94,95), TGF[3s (TGF-[31 a n d / o r - [ 3 2 , d e p e n d i n g on the signaling pathways stimulated in responding) (96), and FGF-2 (also known as bFGF) (97-99). As m e n t i o n e d earlier, IGF-I was reported to be involved in the stimulatory, but not the inhibitory, effects of PTH on bone formation resulting from pulses of different duration in two different culture models in vitro (44,52). IGF-I, like PTHrP, induces Bcl-2 (100), perhaps contributing to the antiapoptotic effect of PTH, but also may stimulate proliferation of osteoprogenitors and preosteoblasts (101). FGF-2, like PTHrP fragments, is an intrakine factor (102). It is possible to speculate that the combination of these factors induced by PTH acts through multiple mechanisms, e.g., FGF-2 stimulating osteoblast precursor proliferation while IGF-I is promoting the lifetime and activity of the osteoblasts (Fig. 2). There have been many published studies of investigations of the signaling pathways and roles of these multiple factors in mediating PTH effects in vitro, but definitive proof of their role in vivo is often lacking. The PTHrP knockout mice have been used to suggest that locally produced PTHrP is required for osteoblast develo p m e n t and for the skeletal effects of PTH (103). T h o u g h IL-6 is clearly up-regulated in vivo in bones of rats treated with an anabolic PTH schedule (38), it is not clear that IL-6 plays a role in the anabolic response, because neutralization of IL-6 in a mouse model markedly reduced PTH-induced bone resorption but had no effect on bone formation parameters (104), consistent with the lack of effect of IL-6 in the rat calvaria bone nodule system in vitro (105). On the other hand, transgenic mouse calvariae carrying fusion genes of the rat Collal promoter and the chloramphenicol acetyltransferase reporter have been used to show that the inhibitory effect of PTH on Collal expression is mediated mainly by the cAMP signaling pathway and that prostaglandins and IL-6 are not local mediators of the PTH response, at least in this model (106). Transgenic mice overexpressing IGF-I in osteoblasts via the osteocalcin promoter showed increased trabecular bone volume, but no evidence for increased osteoblastic proliferation, suggesting that IGF-I increased the activity of resident osteoblasts; these mice did, in addition, have an increase in osteocyte lacunae occupancy, suggesting that IGF-I may extend the osteocyte life span, consistent with the antiapoptotic effect reported for PTH (56).FGF-2 - / mice have markedly reduced bone mass and bone formarion rates (99), but their responsiveness to PTH chal-

lenge has not yet been reported. A growing number of transgenic and mouse knockout models for factors and mediators proposed to play roles in PTH responses are becoming available and these should provide useful tools to tease out critical players in the PTH pathways.

EFFECTS OF PARATHYROID HORMONE ON OSTEOCLAST DIFFERENTIATION When long bone rudiments or calvariae from embryonic or newborn rats or mice are explanted and maintained in culture for several days, osteoclast progenitors located in marrow spaces or the periosteum proliferate and differentiate to form osteoclasts (107,108). Their differentiation in these culture systems is stimulated by PTH, PGE 2, 1,25(OH)zD ~, and a variety of other bone resorption stimulating agents (29,109). Osteoclast-like cells also develop in bone marrow cultures containing stromal and hematopoietic cells, but generally 1,25(OH)zD 3 is required to obtain significant numbers of osteoclasts (110). Progenitors present in the spleen can also differentiate into osteoclasts, but only in the presence of bone marrow-derived stroma and not in the presence of spleen-derived stromal cells (111,112), indicating that osteoclast differentiation from hematopoietic progenitors is controlled by specific stromal cells present in the marrow cultures. With regard to the effects of PTH on osteoclast differentiation in cocultures of spleen cells and stromal cell lines, it is of interest to note that PTH-induced osteoclast-like cell formation was observed in cocultures with a cell line that responded to PTH with an increase in cAMP (KS-4 cells) (113), but not in cocultures with cell lines not responsive to PTH. However, stromal cell lines not responsive to PTH but responsive to PGE 2 or 1,25(OH)2D 3 did mediate PGE 2- or 1,25-(OH)zD 3induced osteoclast formation in spleen cell coculture systems (114), indicating that several types of stromal osteoblast-like or non-osteoblast-like cells that differ in h o r m o n e responsiveness could be involved in the regulation of osteoclast formation. From the evidence provided in the experiments described above, it has become clear that the effects of PTH, PGE2, and 1,25(OH)zD~ on osteoclast differentiation are mediated through the osteoblastic or stromal cell c o m p o n e n t of the cultures, and that osteoclast differentiation requires direct cell-cell contact between the activated osteoblastic/stromal cell population and the hematopoietic cells (Fig. 2). The m e m b r a n e - b o u n d mediator involved, n a m e d osteoclast differentiation factor (ODF), was subsequently cloned and identified (115); it proved to be identical to the previously identified protein osteoprotegerin ligand (OPGL) (116), which in turn is identical to the ligand for the receptor

PTH AND BONE CELL DIFFERENTIATION / activator of NF-KB (RANKL). The receptor on the osteoclast recognizing ODF (i.e., OPGL/RANKL) turned out to be the RANK receptor, and RANKL has now become the generally adopted term for this ligand (Fig. 2). That RANKL is an absolute requirement for osteoclast formation was proved conclusively by the generation of RANKL knockout mice, which had severe osteopetrosis due to a complete lack of osteoclasts (117). The osteoblastic/stromal cell origin of RANKL was confirmed by setting up cocultures of spleen cells from o p g l - / mice with normal osteoblasts, which generated functional osteoclasts, and cocultures of spleen cells from normal mice with osteoblasts from o p g l - / - mice, which did not form osteoclasts. Thus, lack of RANKL production in osteoblastic cells of R A N K L - / - mice was the cause of the osteoclast deficiency. In agreement with this view, ablation of NF-KB1 and NF-KB2 had the expected results: the mice were osteopetrotic, did not develop osteoclasts, but had an increased n u m b e r of macrophages (118). Virtually simultaneously with the discovery of OPGL (RANKL), it was found that osteoclast formation could be inhibited by a soluble receptor for RANKL, osteoprotegerin (OPG), which is secreted by a large variety of cells and organs, including fibroblasts, osteoblasts, lung, heart, kidney, and intestine (119). The OPGdeficient mice are severly osteoporotic whereas mice overexpressing OPG have an osteopetrotic phenotype (120). OPG acts by binding OPGL, thereby preventing interaction of OPGL with its receptor on osteoclast lineage cells and thus inhibiting osteoclast differentiation. The consensus view now is that osteoclast formation in the bone microenvironment is regulated by the interaction of RANKL and OPG, whereby the a m o u n t of u n b o u n d RANKL available to interact with the RANK receptor on osteoclasts or osteoclast precursors determines the rate of osteoclast formation. That RANKL is involved in the actions of many of the factors known to cause bone resorption and induce hypercalcemia in vivo is clearly shown by the experiments of Morony et al. (121), who found that recombinant h u m a n OPG inhibited the hypercalcemic effects of IL-113, TNFoL, PTH (Fig. 2), PTHrP, and 1,25(OH)2D 3. However, whether these factors directly affect RANKL production or whether their effects on RANKL production are mediated by other factors is not clear. Okada et al. (122) examined this issue by evaluating the effects of disrupting the prostaglandin G / H synthase genes on 1,25(OH)2D ~- and PTH-induced osteoclast formation. They found that both PTH- and 1,25(OH)zD ~- induced osteoclast-formation was reduced by 60-70% in marrow cultures from PGHS-2 ( - / - ) mice, indicating that PGE 2 is a major mediator of PTH-induced and 1,25(OH)zD~-induced increased osteoclastogenesis. In agreement with this, PGE2- , PTH-, and 1,25 (OH)zD3 -

207

induced osteoclast formation was found to be reduced by 86, 58, and 50%, respectively, in marrow cultures of mice in which the receptor for PGE 2 had been disrupted (EP2 - / - mice) (123). Thus, the effects of PTH, and those of many other ligands stimulating osteoclast formation, appear to be mediated via increased PGE 2 production and PGEz-induced RANKL formation in a variety of cell types, among which are the cells of the osteoblastic lineage. However, when the effects of PTH and PGE 2 on OPG production were investigated, it appeared that PTH did stimulate OPG mRNA expression in osteoblast lineage cells in rat femur metaphyseal bone but that PGE 2 did not (124), suggesting that differences between the effects of PTH and some of the other resorption stimulating agents may be caused by differential effects on OPG production. With regard to other mediators of the action of PTH on osteoclasts, IL-6 seems the most likely to play a significant role: IL-6 is clearly up-regulated in vivo in bone treated with an anabolic PTH schedule (38), treatment of mice in vivo with either PTH or PTHrP increases expression of IL-6 by osteoblasts (125), and PTHinduced stimulation of bone resorption both in vitro and in vivo can be blocked by either anti-IL-6-receptor antibody or neutralization of IL-6 (104,126). It is not known whether some or all of the effects of PTH on IL6-production by stromal and osteoblastic lineage cells are direct or indirect. In all likelihood, however, such effects are indirect and mediated to a significant degree by PTH-induced PGE 2 production. Compatible with this view are the observations that PGE 2 e n h a n c e d IL-6 production in ST-2 and MC-3T3-E1 cells (127) and that PGE 2 stimulates IL-6 production in osteoblast-like MC-3T3 cells (128).

CONCLUDING

REMARKS

In summary, though much progress has been made on the cellular and molecular bases of bone cell responses to PTH that lead to catabolic versus anabolic outcomes, much remains unclear and discrepancies continue to abound. It is evident from examples and issues raised in this chapter that continued t h o u g h t must be given to not only the species but the age of animals being used, the concentrations/doses of PTH utilized, the duration of treatment, and the presence or absence of other potential regulators for in vivo, ex vivo, and in vitro studies. It also seems clear that osteoblast lineage cells express PTH1R through multiple stages of their developmental lifetime, and, concomitantly, this implies that PTH response in bone will only be fully understood when the sum of effects on progenitors through apparently terminally differentiated bone cells have been unambiguously dissected.

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ACKNOWLEDGMENTS We thank our current and former lab m e m b e r s and many colleagues for their contributions over many years. This work was supported by grants from the Canadian Institutes of Health R e s e a r c h / C I H R (MT12390 to J.E.A. and MT-14655 to J.N.M.H.) and the Arthritis Society (].E.A.). We apologize to all those colleagues whose work we could not reference directly due to space limitations, but refer readers to many excellent papers summarized in various other reviews included here.

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70. Fermor B, Skerry TM. PTH/PTHrP receptor expression on osteoblasts and osteocytes but not resorbing bone surfaces in growing rats. JBone Miner Res 1995;10(12):1935-1943. 71. AubinJE. Molecular fingerprinting of osteoblast differentiation: From primitive osteoprogenitor to mature osteoblast. In: Potts JT, Ogata E, Kronenberg HM, eds. The molecular and cell biology of bone, vol. 5. Tokyo: International Bone and Calcium Institute Incorporation, 1996:54-59. 72. McCauley LK, Koh AJ, Beecher CA, Cui Y, Rosol TJ, Franceschi RT. PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis. J Cell Biochem 1996;61 (4):638-647. 73. Rodan GA, Rodan SB. Expression of the osteoblast phenotype. In: Peck WA, ed. Bone and mineral, vol. 2. Amsterdam:Elsevier, 1988:244-285. 74. Traverse S, Gomez N, Paterson H, Marshall C, Cohen E Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem J 1992;288 (Pt 2):351-355. 75. Traverse S, Seedorf K, Paterson H, Marshall CJ, Cohen P, Ullrich A. EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curt Bio11994;4(8):694-701. 76. Takasu H, Guo J, Bringhurst FR. Dual signaling and ligand selectivity of the human PTH/PTHrP receptor. J Bone Miner Res 1999;14(1) :11-20. 77. Guo J, Iida-Klein A, Huang X, Abou-Samra AB, Segre GV, Bringhurst FR. Parathyroid hormone (PTH)/PTH-related peptide receptor density modulates activation of phospholipase C and phosphate transport by PTH in LLC-PK1 cells. Endocrinology 1995; 136 (9) :3884-3891. 78. Divieti P, Lanske B, Kronenberg HM, Bringhurst FR. Conditionally immortalized murine osteoblasts lacking the type 1 PTH/PTHrP receptor. J Bone Miner Res 1998;13 ( 12):1835-1845. 79. Ducy P, Desbois C, Boyce B, et al. Increased bone formation in osteocalcin-deficient mice. Nature 1996;382:448-452. 80. Lanske B, Amling M, Neff L, Guiducci J, Baron R, Kronenberg HM. Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development. J Clin Invest 1999; 104 (4) :399-407. 81. Kartsogiannis V, Moseley J, McKelvie B, et al. Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model. Bone 1997;21 (5):385-392. 82. Walsh CA, Bowler WB, Bilbe G, Fraser WD, GallagherJA. Effects of PTH on PTHrP gene expression in human osteoblasts: Upregulation with the kinetics of an immediate early gene. Biochem Biophys Res Commun 1997;239(1):155-159. 83. Zhang RW, Supowit SC, Xu X, et al. Expression of selected osteogenic markers in the fibroblast-like cells of rat marrow stroma. Calcif Tissue Int 1995;56(4):283-291. 84. Aarts MM, Rix A, Guo J, Bringhurst R, Henderson JE. The nucleolar targeting signal (NTS) of parathyroid hormone related protein mediates endocytosis and nucleolar translocation. J Bone Miner Res 1999; 14 (9): 1493-1503. 85. Lam MH, House CM, Tiganis T, et al. Phosphorylation at the cyclin-dependent kinases site (Thr85) of parathyroid hormonerelated protein negatively regulates its nuclear localization. J Biol Chem 1999;274(26):18559-18566. 86. Goltzman D. Interactions of PTH and PTHrP with the PTH/PTHrP receptor and with downstream signaling pathways: Exceptions that provide the rules [editorial; comment] [see comments]. J Bone Miner Res 1999;14(2):173-177. 87. Karaplis AC, Vautour L. Parathyroid hormone-related peptide and the parathyroid hormone/parathyroid hormone-related peptide receptor in skeletal development. Curr Opin Nephrol Hypertens 1997;6(4):308-313.

88. Stewart AF. PTHrP(1-36) as a skeletal anabolic agent for the treatment of osteoporosis. Bone 1996;19 (4) :303-306. 89. Morley P, Whitfield JF, Willick GE. Design and applications of parathyroid hormone analogues. Curr Med Chem 1999;6 ( 11 ) :1095-1106. 90. PfeilschifterJ, Laukhuf F, Muller-Beckmann B, Blum WF, Pfister T, Ziegler R. Parathyroid hormone increases the concentration of insulin-like growth factor-I and transforming growth factor beta 1 in rat bone. J Clin Invest 1995;96(2):767-774. 91. Hill PA, Tumber A, Meikle MC. Multiple extracellular signals promote osteoblast survival and apoptosis. Endocrinology 1997; 138 (9) :3849-3858. 92. Watson PH, Fraher LJ, Kisiel M, DeSousa D, Hendy G, Hodsman AB. Enhanced osteoblast development after continuous infusion of hPTH(1-84) in the rat. Bone 1999;24(2):89-94. 93. Watson P, Lazowski D, Han V, Fraher L, Steer B, Hodsman A. Parathyroid hormone restores bone mass and enhances osteoblast insulin-like growth factor I gene expression in ovariectomized rats. Bone 1995;16(3):357-365. 94. Masiukiewicz US, Mitnick M, Grey AB, Insogna KL. Estrogen modulates parathyroid hormone-induced interleukin-6 production in vivo and in vitro. Endocrinology 2000;141 (7):2526-2531. 95. Sanders JL, Stern PH. Protein kinase C involvement in interleukin-6 production by parathyroid hormone and tumor necrosis factor-alpha in UMR-106 osteoblastic cells. J Bone Miner Res 2000;15(5) :885-893. 96. Wu Y, Kumar R. Parathyroid hormone regulates transforming growth factor betal and beta2 synthesis in osteoblasts via divergent signaling pathways. J Bone Miner Res 2000;15(5):879-884. 97. Hurley MM, Tetradis S, Huang YF, et al. Parathyroid hormone regulates the expression of fibroblast growth factor-2 mRNA and fibroblast growth factor receptor mRNA in osteoblastic cells. J Bone Miner Res 1999;14(5):776-783. 98. Liang H, Pun S, Wronski TJ. Bone anabolic effects of basic fibroblast growth factor in ovariectomized rats. Endocrinology 1999;140(12) :5780-5788. 99. Montero A, Okada Y, Tomita M, et al. Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation. J Clin Invest 2000;105(8):1085-1093. 100. Pugazhenthi S, Miller E, Sable C, et al. Insulin-like growth factor-I induces bcl-2 promoter through the transcription factor cAMP-response element-binding protein. J Biol Chem 1999;274 (39) :27529-27535. 101. Zhang W, Lee JC, Kumar S, Gowen M. ERK pathway mediates the activation of Cdk2 in IGF-l-induced proliferation of human osteosarcoma MG-63 cells. J Bone Miner Res 1999; 14 (4):528-535. 102. Amalric F, Baldin V, Bosc-Bierne I, et al. Nuclear translocation of basic fibroblast growth factor. Ann N Y Acad Sci 1991;638:127-138. 103. Amizuka N, Karaplis AC, Henderson JE, et al. Haploinsufficiency of parathyroid hormone-related peptide (PTHrP) results in abnormal postnatal bone development. Dev Bio11996;175(1 ):166-176. 104. Grey A, Mitnick MA, Masiukiewicz U, et al. A role for interleukin-6 in parathyroid hormone-induced bone resorption in vivo. Endocrinology 1999; 140 (10): 4683-4690. 105. Malaval L, Gupta AK, Liu E Delmas PD, Aubin JE. LIE but not IL-6, regulates osteoprogenitor differentiation: Modulation by dexamethasone. J Bone Miner Res 1998;13:175-184. 106. Bogdanovic Z, Huang YF, Dodig M, Clark SH, Lichtler AC, Kream BE. Parathyroid hormone inhibits collagen synthesis and the activity of rat collal transgenes mainly by a cAMP-mediated pathway in mouse calvariae. J Cell Biochem 2000;77(1):149-158. 107. Scheven BA, Kawilarang-De Haas EW, Wassenaar AM, Nijweide PJ. Differentiation kinetics of osteoclasts in the periosteum of embryonic bones in vivo and in vitro. Anat Rec 1986;214( 4):418-423. 108. Burger EH, Van der Meer JW, van de Gevel JS, Gribnau JC, Thesingh GW, van Furth R. In vitro formation of osteoclasts

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CHAPTF a 13

Physiologic Actions of PTH and PTHrP I. Skeletal A c t i o n s

GORDON J. STREWLER VA Boston Healthcare System, West Roxbury, Massachusetts 02132; and Department of Medicine, Harvard Medical School,

Boston, Massachusetts 02114

INTRODUCTION In this chapter the effects of Parathyroid h o r m o n e (PTH) and a PTH-related protein (PTHrP) on the biochemistry and metabolism of individual bone cell types are reviewed, including the most recent analyses of a n u m b e r of the effects of these two peptides. Of necessity, the content overlaps with that in the previous two chapters on osteoblast and osteoclast differentiation ~' and the anabolic and catabolic effects of parathyroid h o r m o n e on bone. The final sections include a synthesis of the cellular effects of the peptides into a more integrated analysis of the skeletal effects of PTH and PTHrE

PTHrP AND RECEPTORS FOR PTH A N D P T H r P IN B O N E Expression o f P T H r P in B o n e PTHrP is expressed and secreted by osteoblast-like osteosarcoma cells (1,2) and by rat long bone explants in vitro (3). Messenger RNA for PTHrP is detected in periosteal cells of fetal rat bones (4). In situ hybridization and immunohistochemistry have localized PTHrP mRNA and protein to mature osteoblasts on the bone surface of fetal and adult bones from mice and rats (5,6), and to flattened bone lining cells and some superficial osteocytes (5) in postnatal mice. In addition, the PTHrP gene is expressed in preosteoblast cells in culture, and in some studies its expression is reduced as The Parathyroids, Second Edition

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preosteoblasts u n d e r g o differentiation (2,7,8). PTHrP is also expressed in tissues adjacent to bone, including growth plate cartilage (5,6) and synovium (9), sites where the peptide could affect bone during endochondral bone formation or destructive rheumatoid arthritis, respectively.

R e c e p t o r s and S e c o n d Messenger Systems for P T H and P T H r P As discussed in detail in Chapter 5, a shared receptor (PTHIR) for PTH and PTHrP is present on bone cells; this is a G protein-coupled receptor that recognizes PTH and PTHrP equally well. The receptor couples its ligands to two cellular effector systems, the adenylyl cyclase/cAMP/protein kinase A pathway and the phospholipase C / p r o t e i n kinase C pathway (Chapters 5 and 7). As will become clear as the individual effects of PTH on bone are presented, PTH and PTHrP utilize cAMP for virtually every action in bone for which a second messenger has been identified. The P T H / P T H r P receptor is expressed widely in the osteoblast lineage. In addition to mature osteoblasts, which are on the trabecular, endosteal, and periosteal surfaces (5,6,10,11) and osteocytes (11,12), the receptor mRNA and protein are expressed in marrow stromal cells near the bone surface (5), a putatively preosteoblast cell population that had previously been shown to bind radiolabeled PTH (13,14). Considering the anabolic effect of PTH on bone formation, it will be important to understand at what point in the osteoblast lineage the receptors for PTH are first expressed. Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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Transcripts for the P T H / P T H r P receptor are absent or n o n a b u n d a n t in STRO-l-positive, alkaline phosphatase-negative marrow stromal cells (15,16), which are thought to represent relatively early osteoblast precursors, but P T H / P T H r P receptor expression can be induced by differentiation of stromal cells, MC-3T3 cells, or C 3 H 1 0 T 1 / 2 cells with dexamethasone or bone morphogenetic proteins (16-21). Other data suggest that PTH receptors are limited to a relatively mature population of osteoprogenitor cells that express the osteocalcin gene (22). It thus appears that the P T H / P T H r P receptor appears at a point in osteoblast differentiation when the cells are acquiring other markers of the mature osteoblast phenotype. Whether receptors for PTH or PTHrP are expressed on the osteoclast is controversial. Initial studies using receptor radioautography failed to demonstrate them (13,23). Further studies also have not identified P T H / P T H r P receptor mRNA or protein on mature osteoclasts (5,6,10). However, relatively low-affinity binding of radiolabeled PTH peptides to osteoclasts or preosteoclasts has been reported (24). The functional importance of such putative receptors is unclear. As discussed in detail below (see Effects on Osteoclasts) the presence of osteoblasts or stromal cells seems to be required to elicit effects of PTH on osteoclasts in vitro (25), and studies have found a requirement for the RANKL/RANK system of cytokines and receptors for bone resorption by PTH or PTHrP (26-29) consistent with the interpretation that stromal or osteoblastic cells expressing the cytokine RANKL are required for the induction of bone resorption by PTH. Both PTH and PTHrP have additional receptors besides the P T H / P T H r P receptor. The PTH2R is a G protein-coupled receptor closely related to the P T H I R (30,31); PTH2R recognizes the amino-terminal domain of PTH but not of PTHrE This receptor is expressed predominantly in brain and has yet to be demonstrated in bone. Evidence for actions of carboxyl-terminal PTH peptides on bone has been presented (32-34), as discussed elsewhere in this chapter, and evidence for a specific receptor for carboxyl-terminal PTH peptides has been presented (35). The polyhormone PTHrP is cleaved to produce a set of peptides: those that contain the amino terminus activate the shared P T H / P T H r P receptors, and additional peptides representing the midregion and carboxyl terminus of PTHrP appear to have distinct biologic actions mediated by their own receptors (36,37). Receptors that are specific for amino-terminal PTHrP and do not recognize PTH have been identified in brain (38) and other tissues (39,40), and midregion peptides of PTHrP have actions on placental calcium . transport that imply a distinct receptor (41,42), but

there is presently no evidence for either receptor in bone. Carboxyl-terminal PTHrP fragments [e.g., PTHrP (107-139)] are reported to inhibit bone resorption (43,44) and stimulate the growth of osteoblasts (45). It is thus likely that a specific receptor for this peptide is present on osteoblasts, and conceivably also on osteoclasts.

EFFECTS OF PTH AND PTHrP O N B O N E CELLS E f f e c t s o n O s t e o b l a s t P r e c u r s o r Cells

In view of the anabolic effects of PTH and PTHrP, evidence for a proliferative effect on osteoblast precursors has been sought. Administration of PTH in vivo does not increase mRNA for the proliferation marker histone H4 (46). Immediate-early gene expression is increased after in vivo administration of PTH in osteoblasts and osteocytes (17,47), but the immediateearly gene response is delayed in stromal cells, suggesting that they may respond secondarily to factors elaborated by osteoblasts (47). Effects on Osteoblasts

Transcription Factors PTH induces the expression of the immediate-early genes c-fos and c-jun in osteoblastic cell lines and in osteoblasts in vivo (47,48). The effect on c-fos is largest and best studied. PTH induces c-fos mRNA in a fashion that does not require protein synthesis and is mediated by phosphorylation of the transcription factor CREB by protein kinase A (49-51), to induce binding to a CRE in the c-fos p r o m o t e r (49,50). The protein kinase C signaling pathway does not appear to be involved in this response (49,52). Because many bone cell genes are regulated by PTH, as discussed below, interactions of PTH with osteoblastspecific transcriptional regulation are likely. A splice variant of the runt-domain transcription factor Cbfal called OSF2 is required for determination of the osteoblast phenotype and confers osteoblast-specific expression on the osteocalcin gene (53,54). Although it is not known how PTH interacts with Cbfal at the osteocalcin promoter, a Cbfal site in the collagenase-3 (MMP13) promoter is required along with an AP-1 site for stimulation of collagenase-3 gene transcription by PTH (55,56). It has also been reported that PTH and other agents that elevate cAMP levels in MC-3T3 cells reduce the level of Cbfal and the activity of Cbfal-dependent genes by activating the destruction of the transcription factor b y t h e ubiquitin-proteosome pathway (57).

SKELETALACTIONS OF PTH AND PTHrP

Cytokines Insulin-like Growth Factors Bone is a rich source of insulin-like growth factors (IGFs) secreted by osteoblasts, with IGF-I predominating in rodent bone and IGF-II in h u m a n bone (58). The secretion of IGF-I by rat (59) and IGF-I and IGF-II by mouse (60) osteoblasts in vitro and in vivo (61) is stimulated by PTH. PTH appears to utilize cAMP as the p r e d o m i n a n t intracellular second messenger to stimulate IGF gene expression, because its effects are mimicked by cAMP analogs or agents that increase cAMP, but not by calcium ionophores or phorbol esters (62). Two sets of results raise the possibility that effects of PTH on IGF-I secretion may be essential for its overall anabolic effect on bone. Continuous exposure to PTH, which has catabolic effects on bone in vivo, inhibited collagen synthesis by isolated rat calvariae; but exposure to PTH for the first 24 hours of a 72-hour experim e n t markedly increased collagen synthesis (63). The stimulation of collagen synthesis by PTH is blocked by antibodies to IGF-I, but the stimulation of [3H]thymidine incorporation is not (64). Moreover, treatment of intact rats with PTH u n d e r conditions where it has an anabolic effect on bone leads to an increase in mRNA for IGF-I (61) and the bone matrix content of both IGF-I and transforming growth factor-J31 (TGF-[31) (65). Finally, skeletal unloading leads to resistance to the anabolic effect of PTH, and also resistance in vitro to IGF-I, a result that was interpreted as suggesting that resistance to IGF-I may account for the resistance of the unloaded skeleton to PTH (66). PTH and PTHrP also affect the secretion of binding proteins for IGFs. There are six IGF binding proteins (IGFBP), and all are present in bone (58). IGFBP-4 inhibits IGF action, but IGFBP-5 seems to function predominantly to anchor IGFs to the extracellular matrix, and may in some circumstances have stimulatory effects on IGF action. Exposure of bone cells to PTH or PTHrP increases the secretion of IGFBP-4 (67) and IGFBP-5 (68) by cAMP-dependent mechanisms, and also increases the level of a related protein, IGFBP-rP-1 (69). Both IGFBPs are subject to proteolysis and there is limited evidence to suggest that IGFBP protease activity may be regulated by PTH (70,71). It is not clear whether the effects of PTH on IGFBP levels are biologically significant.

Transforming Growth Factor-J3 PTH and PTHrP increase both the secretion of TGF[3 by osteoblast-like bone cells and the release of TGF-[3 from calvarial explants, the latter may represent in part the release of preformed TGF-[3 during bone resorption (72-75). Intermittent PTH treatment of rats

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increases the bone matrix content of TGF-[31 as well as IGF-I (61), raising the possibility that the anabolic effects of PTH observed with intermittent administration could be mediated, at least in part, by increased secretion of this potent osteoblast growth and differentiation factor.

Interleukin-6 Family Cytokines The cytokines interleukin (IL-6 and IL-11), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), and cardiotropin 1 (CT1) bind to related receptors and share a signal transduction pathway (76,77). The pathway involves the c o m m o n receptor subunit gp130, binding of JAK family protein kinases, and phosphorylation and nuclear translocation of the STAT family of transcription factors. Of this cytokine family, three members are prominently stimulated by PTH and PTHrP in bone cells, IL-6 (78-81), IL-11 (53,82), and LIF (79). Both PTHrP(1-34) and PTHrP(107-139) are reported to induce the expression of IL-6 (83). The production of IL-6 is also increased by PTH in mouse calvaria (84) and in vivo (46,85). PTH activates transcription of the IL-6 gene (84,86) using cAMP as its principal signaling pathway (84,86,87). It has been suggested from neutralization experiments that the induction by PTH of osteoblast secretion of IL-6 (87,88) or IL-11 (89), both of which are boneresorbing cytokines, may be one mechanism by which the osteoblast transmits the bone-resorbing signal of PTH to the osteoclast. However, studies have shown that blockade of the intracellular signaling pathway, using dominant negative STAT factors (90) or an IL-6 receptor antagonist (91), fails to inhibit bone resorption by PTH, even though bone resorption by IL-6 is blocked. Compelling evidence is now available to indicate that the principal mediators of the bone-resorbing effect of PTH are another set of cytokines, RANKL and osteoprotegerin, or OPG. This issue is further discussed below (see RANK Ligand and Osteoprotegerin).

Other Cytokines and Prostaglandins PTH induces osteoblasts to secrete granulocytemacrophage colony-stimulating factor (GM-CSF) (92,93). PTH also stimulates the production of the prostaglandin PGE 2 by mouse calvarial osteoblasts (94-96). The direct target of PTH is the enzyme prostaglandin G / H synthase (PGHS-2) (97), the protein levels of which are increased by PTH. Another isoform, PGHS-1, is expressed constitutively but is not affected by PTH. The effect of PTH is mediated by cAMP as the d o m i n a n t second messenger (96). PGE 2, in turn, has diverse effects on bone (98).

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RANK Ligand and Osteoprotegerin One of the most important new insights into the regulation of bone metabolism in recent years has been the delineation of a new system for osteoblast-osteoclast cross-talk. It has three major elements. The first is a new m e m b e r of the tumor necrosis factor (TNF) family of cytokines that is expressed on the osteoblast and stromal cell surface; this cytokine is variously known as RANK ligand (RANKL), osteoprotegerin ligand (OPGL), osteoclast differentiation factor (ODF), and TNF-related activation-induced cytokine (TRANCE) (99). By binding to a receptor on osteoclast precursors, RANKL provides an essential feeder function for osteoclastogenesis, accounting for earlier observations that coculture of bone marrow cells and stromal cells is required for osteoclastogenesis (100); RANKL also activates bone resorption by mature osteoclasts and inhibits osteoclast apoptosis (26,101-104). RANKL is both necessary and, with M-CSF, sufficient for osteoclastogenesis; disruption of the RANKL gene leads to severe osteopetrosis (105). The second element of this system is the receptor for RANKL on the surface of osteoclast precursors and mature osteoclasts. This receptor is called RANK (receptor activator of NF-KB) or ODAR (osteoclast differentiation and activation receptor). Disruption of the receptor gene also produces severe osteopetrosis (106). The third element is a decoy receptor, osteoprotegerin or osteoclastogenesis inhibitory factor (OCIF) (104,107,108). Targeted deletion of OPG produces severe osteoporosis, (107,109) but overexpression leads to osteopetrosis (107). Both genetic and cell biologic approaches to this system have yielded decisive results. Following an early c o m m i t m e n t step u n d e r the control of M-CSF, binding of RANKL to RANK is both necessary and sufficient for osteoclastogenesis. The system has a second function to regulate the activity of the mature osteoclast, and exposure of osteoclasts to RANKL inhibits their apoptosis. The decoy receptor OPG must also be important to modulate the tone of the system, because elimination of OPG produces a severe form of osteoporosis. It is conceivable that a parallel system exists, because MCSF-dependent osteoclast formation from cultured mouse bone marrow cells is induced by TNF~ and is blocked by antibodies to its receptor, but not by OPG or antibodies to RANK (110). The bone resorbing effects of PTH, long known to require the intermediation of osteoblasts (25), appear to occur principally through activation of the RANKL/RANK system. Exposure to PTH increases the expression of RANKL in murine bone marrow cultures, cultured osteoblasts, and mouse calvariae (27,104,111), and simultaneously decreases the expression of OPG (27). Stimulation of osteoclastogenesis by PTH is

blocked by antibodies to RANKL (112) or by OPG (103,104). Infusion of OPG into animals blocks the hypercalcemic response to PTH or PTHrP (28,29). Bone resorption by mature osteoclasts in response to PTH has long been recognized as requiring coculture with osteoblasts or marrow stromal cells (25), but when purified cultures of isolated osteoclasts that were unresponsive to PTH were exposed to RANKL, the cytokine was sufficient to induce bone resorption (26). It thus appears that both the stimulation of new osteoclast formation and the activation of the mature osteoclast by PTH and PTHrP take place by binding of the ligand to receptors on osteoblasts, followed by simultaneous induction of the presentation of RANKL on the osteoblast surface and inhibition of secretion of OPG. It is conceivable that the effect of PTH on RANKL and OPG is indirect, involving other cytokines as intermediate steps. It is also possible that a parallel pathway exists, in which other cytokines such as IL-6 or IL-11 could mediate part of the effect of PTH on bone resorption, but if so it is likely to be of secondary importance.

Cell Proliferation and Apoptosis Continuous exposure to PTH(1-34) or PTHrP(1-34) exerts an antiproliferative action on osteoblast-like UMR-106 osteosarcoma cells (113-115). This effect is cAMP-mediated and results, at least in part, from increased levels of p27Kipl, a regulator of Gl-phase cyclin-dependent kinases (115). However, in some cell lines (73,116,117) and primary cultures (118) PTH appears to increase osteoblast or preosteoblast proliferation. In the preosteoblast cell line TE-85, the mitogenic response to PTH requires an increase in levels of the cyclin-dependent kinase cdc2, probably brought about by increased levels of E2F (117). Treatment of rats with intermittent injections of PTH is reported in some studies to increase the number of osteoprogenitor cells (66,119), but not the proliferation of osteoprogenitors (46,120). In an important study, continuous labeling of bone with [3H] thymidine during a period of intermittent treatment with PTH(1-34) resulted in no increase in labeled osteoblasts, despite a marked increase in osteoblast n u m b e r (121). This indicates that the anabolic effect of PTH does not require proliferation of osteoblast precursors or of mature osteoblasts on the bone surface. The large increase in osteoblast n u m b e r produced in this study by intermittent treatment with PTH was attributed to activation of preexisting bone lining cells to osteoblasts (121), but is also possible that PTH induces the commitment of late osteoprogenitors to the osteoblast lineage without a requirement for mitosis. Another alternative explanation for the increase in osteoblast n u m b e r with intermittent PTH treatment is

SKELETAL ACTIONS OF P T H AND P T H r P

provided by work that indicates that treatment of mice with intermittent PTH inhibits osteoblast apoptosis (122). Prolongation of the osteoblast life span by PTH could account for the observed increase in osteoblast number, although it is not clear how large a quantitative effect on osteoblast survival would result from the observed inhibition of apoptosis. The integrated effects of PTH on bone formation are further discussed below and in Chapters 11 and 55.

Effects on Ion Channels In several bone cell types, PTH induces multiphasic changes in membrane potential, most often depolarization followed by sustained hyperpolarization (123-126). Depolarization has been attributed to cAMP-dependent inactivation of quinine-sensitive K channels (124). Depolarization of bone cells induces calcium entry through L-type voltage-sensitive Ca channels (126-129). Sustained hyperpolarization may result in return from opening of Ca-sensitive K channels (130), which may be identical to stretch-activated cation channels also activated by PTH (131).

Effects on Cell Shape PTH treatment of cultured osteoblasts induces a marked retraction of the cell (132) and similar changes have been observed with treatment in vivo (133). The change in cell shape is cAMP mediated (134) and is associated with disassembly of actin stress fibers (135). It can be blocked by inhibitors of the protease calpain (136). The significance of changes in cell shape is unknown, but it has been suggested that osteoblast retraction could have a role in bone remodeling by baring portions of the bone surface in response to PTH.

Effects on Gap Junctions PTH increases intercellular communication of bone cells by increasing connexin-43 gene expression (137) and opening gap junctions (138,139). The significance of intercellular communication to the overall effects of PTH on osteoblasts is not clear, although it is reported that reduction of connexin-43 levels by transfection of antisense cDNA markedly inhibited the cAMP response to PTH (140).

Effects on Bone Matrix Proteins and Alkaline Phosphatase The most abundant protein of bone matrix is type I collagen. Given acutely, PTH consistently inhibits' collagen synthesis in cultured rat calvaria and in cultured bone cells (141,142) by decreasing transcription of the

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COL1A1 gene (143). PTH treatment of calvaria inhibits transcription of a 2.3-kb fragment of the COL1A1 promoter, indicating that at least one major cis-acting element required for inhibition of gene transcription resides in this portion of the promoter (144). PTHrP and agents that increase cAMP have effects similar to those of PTH (145,146). Acute infusion of PTH into humans also inhibits collagen synthesis (147). In contrast, treatment of calvaria with PTH intermittently can stimulate collagen gene expression (63). The stimulatory effect of PTH on collagen synthesis in calvaria is attributed to stimulation of IGF-I production because it is blocked by IGF-I antibodies (64). Moreover, when given intermittently in an anabolic regimen, treatment with PTH in vivo increases bone collagen gene expression (148). The reversal of direction of the PTH effect in vivo can probably be attributed, at least in part, to increased bone remodeling and increases in osteoblast n u m b e r induced by the chronic regimen. Treatment of osteosarcoma cells with PTH has a stimulatory effect on several other bone matrix proteins, including osteocalcin (bone Gla protein, BGP) (149-152); administration of PTH or PTHrP acutely inhibits osteocalcin release from isolated rat hindlimb, but chronic administration of PTH is stimulatory (153). Exposure to PTH stimulates bone sialoprotein gene expression in embryonic chick bone cells (154). PTH treatment inhibits expression of the osteopontin gene in rat osteosarcoma cells (155). Amino-terminal peptides derived from PTH can either stimulate or inhibit secretion of alkaline phosphatase from bone cells, depending on the cell line (156-161). It is reported that carboxyl-terminal PTH fragments can stimulate alkaline phosphatase (32,33), and PTHrP(107-139) is also reported to inhibit alkaline phosphatase (162). Treatment of women with anabolic regimens of intermittent PTH(1-34) injections increases alkaline phosphatase (163), presumably at least in part owing to an increase in osteoblast number. Effects on Proteases of Bone

PTH stimulates the secretion of a n u m b e r of proteases from osteoblasts (164,165). These include stromelysin (166), gelatinase B (166), and collagenase3 (MMP-13) (167-171). Stimulation of the collagenase3 promoter by PTH requires interactions of an AP-1 site and a binding site for runt-domain transcription factors such as OSF2; the effect of PTH is on AP-1 (55,56). Bone resorption by PTH is markedly abrogated in mice with a mutation in the COL1A1 gene that renders the helical domain of type I collagen resistant to cleavage by collagenase (172). It has been suggested that collagenase action on a hypomineralized layer of collagen on

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bone surfaces may be necessary for osteoblast attachment, although multiple other explanations for the observation are also possible. PTH treatment also increases secretion of the inhibitor TIMP (166). Finally, activity of the serine protease plasminogen activator is increased by PTH in bone cell cultures (173,174). There is controversy as to whether the plasminogen activator is urokinase or tissue-type plasminogen activator and whether the effect of PTH is to increase the level of the protease or decrease the level of its inhibitor PAI-1 (165,175,176).

Effects on Osteocytes As noted previously, PTHrP and the P T H / P T H r P receptor appear to be expressed on osteocytes (5,11,12). Exposure to PTH induces ultrastructural changes in osteocytes (177). Although it was long thought that osteocytes, together with bone lining cells, participate in the acute release of calcium from bone in response to PTH (178), this remains conjectural and seems unlikely in view of evidence that the RANK/RANKL system in osteoclasts is involved (28). The current view of osteocytes has for them a predominant role in mechanotransduction, and it is not known how PTH interacts with the mechanotransduction system (179).

INTEGRATED EFFECTS OF P T H A N D PTHrP O N B O N E PTH, PTHrP, and Bone Resorption Cellular Basis of t ~ H Action PTH and PTHrP increase bone resorption by stimulating both the appearance of new osteoclasts and the activity of existing osteoclasts. The mechanistic details of osteoclastogenesis (100) and osteoclast activation (180) are beyond the scope of this chapter, but have been summarized elsewhere; in neither case does PTH have a distinctive effectmrather, the distal cellular responses of osteoclast precursors and mature cells to all bone resorbing agents seem to represent a final common pathway. Both the stimulation of osteoclastogenesis and the activation of the mature osteoclast appear to require the participation of stromal cells or osteoblasts (25,100,181). To recapitulate what has been summarized in previous sections of this chapter, osteoclasts have not been shown to possess high-affinity P T H / P T H r P receptors (5,6,10,13,23), although several groups have identified low-affinity receptors (24). It appears that the effects of PTH are predominantly

mediated by increased expression of the cytokine RANKL (OPGL, ODF, TRANCE) on the cell surface of stromal cells (27,103,104,111,112), perhaps together with a decrease in expression of the decoy receptor OPG (104). The precise target cell in the osteoblast lineage that is responsible for mediating the bone resorbing effects of PTH and PTHrP has not been identified, but various marrow stromal cell lines will suffice in vitro (100) and bone resorption is still active when mature osteoblasts have been ablated (182). By binding to its cognate receptor (RANK) on osteoclast precursors and mature osteoclasts, RANKL stimulates both osteoclastogenesis and the activity of mature osteoclasts. Osteoclast activation by RANKL is apparently responsible both for bone resorption at the cellular level and for hypercalcemia, because both are blocked by the decoy receptor OPG (28,29). Although it was previously suggested that the early phase of the increase in the plasma concentration of ionized calcium, e.g., within 1-2 hours, might have an osteoclastindependent mechanism involving release of calcium by bone lining cells (178), even early responses to PTH in animal models are blocked by inhibiting the RANK/RANKL system (28).

Comparative Effects of PTH and PTHrP The bone-resorbing effects of amino-terminal PTH and PTHrP are essentially indistinguishable when studied using isolated osteoclasts (183,184), bone explant systems (185,186), or infusion into the intact animal (187,188). PTHrP may be somewhat less potent than equimolar infusions of PTH to induce hypercalcemia in humans, probably owing to differences in plasma half-life (189). As discussed in Chapter 3, PTHrP is a polyhormone, the precursor of multiple biologically active peptides. Carboxyl-terminal peptides that are predicted to arise from cleavage of PTHrP in the polybasic region PTHrP(102-106) have been synthesized and shown to inhibit bone resorption in several explant systems (43,190,191), although not all (192), and also in vivo (44). On this basis, the minimal peptide that inhibits bone resorption, PTHrP(107-111), has been called osteostatin.

Effects o f P T H and PTHrP on Bone Formation The anabolic effects of PTH and PTHrP have been discussed in Chapter 11, and their involvement in the pathogenesis of bone changes in primary hyperparathyroidism will be presented in Chapters 24 and 26. The following discussion is a synthesis of a view of the effects of PTH and PTHrP on bone formation from the perspective of the individual cellular actions of the

SKELETAL ACTIONS OF P T H AND P T H r P

h o r m o n e s that have been summarized in the preceding sections of this chapter. Continuous exposure to PTH leads to a coupled increase in bone formation and bone resorption, with a net loss of bone mass in most circumstances, whereas intermittent t r e a t m e n t with injections of PTH once daily, or less frequently, produces a net anabolic effect (193) (see Chapters 11 and 55 for a review). In contrast, the initial interpretation of bone histomorphometry in malignancy-associated hypercalcemia was that, unlike primary hyperparathyroidism, bone resorption was u n c o u p l e d from bone formation (194), raising the possibility that the effects of PTHrP on bone formation differed radically from the effects of PTH. However, in animal models of h u m o r a l hypercalcemia, increases in bone resorption were appropriately coupled to increases in bone formation (195). It has been shown that intermittent administration of PTHrP(1-36) in h u m a n s for 2 weeks leads to increases in biochemical markers of bone formation and a decrease in markers of bone resorption (196). Moreover, a carboxylsubstituted analog of PTHrP(1-34) also mimics the anabolic action of PTH in the rat (197,198). Thus, the anabolic effects of PTH and PTHrP, administered intermittently, appear similar. Any attempt to u n d e r s t a n d the cellular basis for the anabolic actions of PTH and PTHrP must take into account their histomorphologic effects. The increase in bone formation is best correlated with m a r k e d increases in bone formation surfaces and activation frequency (199-202). Thus, a major effect of PTH is to increase the n u m b e r of active, bone-forming osteoblasts. Increases in mineral apposition rate are also seen but tend to be smaller (199-202). The duration of the active bone formation phase is not prolonged in dogs treated with PTH (199) but is increased in primary hyperparathyroidism (200). An increase in the n u m b e r of active osteoblasts could occur in several ways, and PTH may not have the same effect in all circumstancesmits p r e d o m i n a n t effect on growing bone in a young r o d e n t may differ from its p r e d o m i n a n t effect in aged bone. First, PTH could increase the birth rate or proliferation of osteoblast precursors in bone marrow. In the rat, an anabolic regimen of PTH does not increase the proliferation of osteoblast precursors, based on the absence of an increase in labeled nuclei on the bone surface after continuous labeling with [~H]thymidine (121). This is compelling evidence against the view that a proliferative effect of PTH is decisive in increasing osteoblast number. However, intermittent exposure to PTH could increase h o m i n g to the bone surface of late, postmitotic osteoblast precursors in the bone marrow, which are recognized as having PTH receptors (5,13).

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Second, PTH t r e a t m e n t could activate bone lining cells to again become active osteoblasts. There is no direct evidence for or against this hypothesis. However, bone lining cells cover a relatively large bone surface per cell because of their flattened, spread shape, and it is not clear that the numbers of bone lining cells are adequate to account for the increase in osteoblast number that is observed with PTH treatment. Third, an anabolic PTH regimen could increase the life span of the active osteoblast. In the mouse, intermittent t r e a t m e n t with PTH reduces the rate of osteoblast apoptosis (122). However, it is not certain whether the reduction in cell death is quantitatively sufficient to account for the anabolic activity of PTH. If it were, increases not only in m e a n wall thickness but also in the duration of the active formation period would be expected. It is reasonably clear that m e a n wall thickness is increased by anabolic PTH regimens or in primary hyperparathyroidism, but whether the duration of the active formation period is also increased has not been fully resolved (199,200). In order to d e t e r m i n e the m e c h a n i s m by which PTH or PTHrP increases osteoblast number, and thereby has its anabolic effect, it will ultimately be necessary to learn the origin and fate of osteoblasts that participate in the anabolic effects by d e t e r m i n i n g their precise cellular kinetics.

PERSPECTIVES As evident from the previous discussion of anabolic effects of PTH and PTHrP, there is m u c h to be learned about how the individual effects of the h o r m o n e s on bone cells are integrated to produce the final effects of the h o r m o n e s on the physiology of the skeleton. Moreover, there is a large lacuna in our u n d e r s t a n d i n g of the skeletal role of PTHrP. Bone cells both secrete and respond to P T H r E PTHrP is a major regulator of cartilage (the precursor of e n d o c h o n d r a l bones). It is tantalizing to speculate that PTH evolved as a systemic h o r m o n e to overdrive the local regulation of bone metabolism by its sister peptide. However, the physiology of PTHrP in the skeleton has been refractory to study. Genetic models, so powerful in unraveling the role of PTHrP in the cartilaginous phase of endochondral bone formation (Chapter 15), have yielded little information about bone per se, because any changes observed in bone when the P T H r P / r e c e p t o r systems are p e r t u r b e d are potentially explained by perturbations in e n d o c h o n d r a l bone formation. To apply genetic methods to the study of PTHrP in bone, what is now necessary is tissue-specific targeting of PTHrP and its receptor in bone, and such studies are underway. By ablating PTHrP or the P T H / P T H r P receptor in bone

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only, and ultimately restoring sequence-specific tions of the polyhormone PTHrP to such animals, eventually be possible to determine what is the role of PTHrP in bone, and how PTH and PTHrP act as regulators of skeletal physiology.

porit will local inter-

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and other matrix metalloproteinases in response to osteotropic hormones and cytokines. J Cell Sci 1992;103 (Part 4):1093-1099. Walker DG, Lapiere CM, Gross J. A collagenolytic factor in rat bone promoted by parathyroid extract. Biochem Biophys Res Commun 1964;15:397-402. Partridge NC, Jeffrey jj, Ehlich LS, Teitelbaum SL, Fliszar C, Welgus HG, et al. Hormonal regulation of the production of collagenase and a collagenase inhibitor activity by rat osteogenic sarcoma cells. Endocrinology 1987; 120:1956-1962. Scott DK, BrakenhoffKD, ClohisyJC, Quinn CO, Partridge NC. Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis. Mol Endocrino11992;6:2153-2159. Winchester SK, Bloch SR, Fiacco GJ, Partridge NC. Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts. J Cell Physio11999;181:479-488. Quinn CO, Scott DK, Brinckerhoff CE, Matrisian LM, Jeffrey JJ, Partridge NC. Rat collagenase. Cloning, amino acid sequence comparison, and parathyroid hormone regulation in osteoblastic cells. J Biol Chem 1990;265:22342-22347. Zhao W, Byrne MH, Boyce BF, Krane SM. Bone resorption induced by parathyroid hormone is strikingly diminished in collagenase-resistant mutant mice. J Clin Invest 1999;103:517-524. Hamilton JA, Lingelbach S, Partridge NC, Martin TJ. Regulation of plasminogen activator production by boneresorbing hormones in normal and malignant osteoblasts. Endocrinology 1985; 116:2186-2191. Leloup G, Peeters-Joris C, Delaisse JM, Opdenakker G, Vaes G. Tissue and urokinase plasminogen activators in bone tissue and their regulation by parathyroid hormone. J Bone Miner Res 1991 ;6:1081-1090. Catherwood BD, Titus L, Evans CO, Rubin J, Boden SD, Nanes MS. Increased expression of tissue plasminogen activator messenger ribonucleic acid is an immediate response to parathyroid hormone in neonatal rat osteoblasts. Endocrinology 1994;134:1429-1436. Fukumoto S, Allan EH, Yee JA, Gelehrter TD, Martin TJ. Plasminogen activator regulation in osteoblasts: Parathyroid hormone inhibition of type-1 plasminogen activator inhibitor and its mRNA. J Cell Physio11992;152:346-355. Krempien B, Friedrich E, Ritz E. Effect of PTH on osteocyte ultrastructure. Adv Exp Med Biol 1978;103:437-450. Talmage RV, Doppelt SH, Fondren FB. An interpretation of acute changes in plasma 45Ca following parathyroid hormone administration to thyroparathyroidectomized rats. Calcif Tissue Res 1976;22:117-128. Burger EH, Klein-Nulend J. Mechanotransduction in b o n e - role of the lacuno-canalicular network. FASEB J 1999;13 (Suppl.):S101-$112. Duong LT, Rodan GA. The role of integrins in osteoclast function. J Bone Miner Metab 1999;17:1-6. Akatsu T, Takahashi N, Udagawa N, Sato K, Nagata N, Moseley JM, et al. Parathyroid hormone (PTH)-related protein is a potent stimulator of osteoclast-like multinucleated cell formation to the same extent as PTH in mouse marrow cultures. Endocrinology 1989;125:20-27. Corral DA, Amling M, Priemel M, Loyer E, Fuchs S, Ducy P, et al. Dissociation between bone resorption and bone formation in osteopenic transgenic mice. P r o c Natl Acad Sci USA 1998;95:13835-13840. Evely RS, Bonomo A, Schneider HG, Moseley JM, Gallagher J, Martin TJ. Structural requirements for the action of parathyroid hormone-related protein (PTHrP) on bone resorption by isolated osteoclasts. J Bone Miner Res 1991;6:85-93.

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184. Murrills RJ, Stein LS, Fey CP, Dempster DW. The effects of parathyroid hormone (PTH) and PTH-related peptide on osteoclast resorption of bone slices in vitro: An analysis of pit size and the resorption focus. Endocrinology 1990;127:2648--2653. 185. Yates AJ, Gutierrez GE, Smolens P, Travis PS, Katz MS, Aufdemorte TB, et al. Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents. J Clin Invest 1988;81:932-938. 186. Raisz LG, Simmons HA, Vargas SJ, Kemp BE, Martin TJ. Comparison of the effects of amino-terminal synthetic parathyroid hormone-related peptide (PTHrP) of malignancy and parathyroid hormone on resorption of cultured fetal rat long bones. Calcif Tissue Int 1990;46:233-238. 187. Thompson DD, Seedor JG, Fisher JE, Rosenblatt M, Rodan GA. Direct action of the parathyroid hormone-like human hypercalcemic factor on bone. Proc Natl Acad Sci USA 1988;85:5673-5677. 188. Kitazawa R, Imai Y, Fukase M, Fujita T. Effects of continuous infusion of parathyroid hormone and parathyroid hormonerelated peptide on rat bone in vivo: Comparative study by histomorphometry. Bone Miner 1991;12:157-166. 189. Fraher LJ, Hodsman AB, Jonas K, Saunders D, Rose CI, Henderson JE, et al. A comparison of the in vivo biochemical responses to exogenous parathyroid hormone-(1-34) [PTH(1-34)] and PTH-related peptide-(1-34) in man. J Clin Endocrinol Metab 1992;75:417-423. 190. Fenton AJ, Kemp BE, Hammonds RG, Jr, Mitchelhill K, Moseley JM, Martin TJ, et al. A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein; PTHrP[107-111]. Endocrinology 1991 ;129:3424-3426. 191. Fenton AJ, Martin TJ, Nicholson GC. Long-term culture of disaggregated rat osteoclasts: Inhibition of bone resorption and reduction of osteoclast-like cell number by calcitonin and PTHrP [ 107-139]. J Cell Physio11993;155;1-7. 192. Sone T, Kohno H, Kikuchi H, Ikeda T, Kasai RKY, Takeuchi R, et al. Human parathyroid hormone-related peptide-(107-111) does not inhibit bone resorption in neonatal mouse calvariae. Endocrinology 1992;131:2742-2746. 193. Tam CS, Heersche JNM, Murray TM, Parsons JA. Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: Differential effects of intermittent and continuous administration. Endocrinology 1982;110:506-512. 194. Stewart AF, Vignery A, Silverglate A, Ravin ND, LiVolsi V, Broadus AE, et al. Quantitative bone histomorphometry in humoral hypercalcemia of malignancy. J Clin Endocrinol Metab 1982;55:219-227. 195. Strewler GJ, Wronski TJ, Halloran BE Miller SC, Leung SC, Williams RD, et al. Pathogenesis of hypercalcemia in nude mice bearing a human renal carcinoma. Endocrinology 1986;119:303-310. 196. Plotkin H, Gundberg C, Mitnick M, Stewart AF. Dissociation of bone formation from resorption during 2-week treatment with human parathyroid hormone-related peptide-(1-36) in humans: Potential as an anabolic therapy for osteoporosis. J Clin Endocrinol Metab 1998;83:2786-2791. 197. Vickery BH, Avnur Z, Cheng Y, Chiou SS, Leafier D, Caulfield JP, et al. RS-66271, a C-terminally substituted analog of human parathyroid hormone-related protein (1-34), increases trabecular and cortical bone in ovariectomized, osteopenic rats. J Bone Miner Res 1996;11:1943-1951. 198. Frolik CA, Cain RL, Sato M, Harvey AK, Chandrasekhar S, Black EC, et al. Comparison of recombinant human PTH(1-34) (LY333334) with a C-terminally substituted analog of human PTH-related protein(I-34) (RS-66271): In vitro activity and in vivo pharmacological effects in rats [see comments]. J Bone Miner Res 1999;14:163-172.

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199. Boyce RW, Paddock CL, Franks AF, Jankowsky ML, Eriksen EE Effects of intermittent hPTH(1-34) alone and in combination with 1,25(OH)(2)D(3) or risedronate on endosteal bone remodeling in canine cancellous and cortical bone. JBone Miner Res 1996;11:600-613. 200. Dempster DW, Parisien M, Silverberg SJ, Liang XG, Schnitzer M, Shen V, et al. On the mechanism of cancellous bone preservation in postmenopausal women with mild primary hyperparathyroidism. J Clin Endocrinol Metab 1999;84:1562-1566.

201. Shen V, Dempster DW, Birchman R, Xu R, Lindsay R. Loss of cancellous bone mass and connectivity in ovariectomized rats can be restored by combined treatment with parathyroid h o r m o n e and estradiol. J Clin Invest 1993;91: 2479-2487. 202. Lane NE, Kimmel DB, Nilsson MH, Cohen FE, Newton S, Nissenson RA, et al. Bone-selective analogs of human PTH(1-34) increase bone formation in an ovariectomized rat model. J Bone Miner Res 1996;11:614-625.

CHAPTER14 Physiologic Actions of P T H and PTHrP II. Renal Actions

E RICHARD BRINGHURST Endocrine Unit, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114

PTHR EXPRESSION, SIGNALING, AND REGULATION IN THE KIDNEY

INTRODUCTION The kidney is the focal point for the physiologic regulation of mineral ion homeostasis by circulating parathyroid hormone (PTH). By directly controlling renal tubular reabsorption of calcium and phosphate and the synthesis of 1,25-dihydroxy vitamin D (1,25(OH)zD), PTH exerts control over both the intestinal absorption and the urinary excretion of these key mineral ions. Renal tubular responses to P T H deficiency, PTH or PTH-related protein (PTHrP) excess, or defects in function of the type 1 P T H / P T H r P receptor (PTHR) lead to alterations in blood calcium, phosphate, or 1,25 (OH)zD that are the hallmarks of numerous clinical disorders, described in Section III of this volume. This chapter reviews current understanding of the mechanisms whereby PTH (and PTHrP) control renal tubular epithelial function. The discussion focuses principally on the known actions of PTH, because relatively little is known of the possible physiologic actions of PTHrP in the kidney. Because the PTHR recognizes the active amino termini of both ligands equivalently, however, it is likely that the effects described for PTH would pertain to PTHrP as well. Expression and action of PTHrP in the kidney are discussed in the last section of this chapter. Whereas species of PTH or PTHrP receptors distinct from the PTHR have been discovered (see Chapter 5), the roles of these, if any, in normal renal physiology currently are unknown. Though not unequivocally proved in each case, it is likely that the effects of PTH and PTHrP described here are mediated by the PTHR. The Parathyroids, Second Edition

The PTHR is widely expressed within the kidney among cells with dramatically different physiologic roles. The responses to PTHR activation observed in individual renal cells are a complex function of the number and location of expressed PTHRs on the cell surface; the cell-specific expression of effectors capable of coupling to the PTHR; the cell-specific repertoire of PTHR-regulated genes; enzymes, channels, and transporters; the local concentrations of PTH or PTHrP ligand; exposure to other agents that regulate PTHR function heterologously; and the pattern of recent exposure to PTHR ligands.

PTHR Expression within the Kidney The PTHR is widely but not universally expressed by the various cell types that collectively comprise the mammalian nephron. Early work, based on measurements of regional cAMP responses (1-5) and PTH radioligand binding in vivo (6), indicated that PTHRs are expressed in glomeruli, proximal convoluted tubules (PCTs) and proximal straight tubules (PSTs), the cortical thick ascending loop of Henle (CTAL), and portions of the distal nephron, including the distal convoluted tubules (DCTs), connecting tubules (CNTs), and early portions of the cortical collecting ducts (CCDs). More recently, these functional observations have been confirmed by in situ hybridization of tissue sections or by reverse transcriptase and polymerase 227

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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chain reaction (RT-PCR) of microdissected nephron segments, using probes derived from the cloned PTHR cDNA (7-9). Minor disparities regarding PTHR expression in Henle's loop and CCDs arising from use of these sensitive molecular techniques likely reflect methodologic issues (7-9). Given that circulating PTH peptides may be filtered at the glomerulus and appear in the tubular urine, it is of interest that PTHRs are expressed on the apical (luminal) as well as the basolateral membranes of proximal tubular cells (10-12). On the other hand, these apical membrane receptors appear not to be coupled tightly, if at all, to adenylyl cyclase (10,11). Moroever, a high-capacity apical peptide-uptake mechanism, mediated by the multifunctional endocytic clearance receptor megalin (13), likely would limit access of filtered bioactive PTH peptides to these receptors. PTHRs also are expressed within the vasculature of the kidney, including peritubular (but not glomerular) endothelial cells and vascular smooth muscle cells (12). As discussed further below, such receptors may mediate local or systemic vascular effects of PTHrP and PTH, respectively. As described in more detail in Chapter 5, the PTHR gene incorporates multiple promoters and 5' untranslated exons and therefore can generate multiple transcripts via alternative promoter usage and different patterns of RNA splicing (12,14-17). It is of interest that certain promoters (i.e., P1 in mouse and P3 in human) seem to be used exclusively in kidney cells, whereas a different promoter (P2) is employed to generate those PTHR mRNAs that are widely expressed in other tissues and organs (12,16,17). Whether these differences simply reflect opportunities for tissue-specific gene regulation or lead to expression of structurally different forms of the PTHR (15,16) remains to be established.

PTHR Signal Transduction in Renal Cells The PTHR is known to couple to multiple intracellular signal transducers and effectors, including but perhaps not limited to G s and the Gq/G11 family of heterotrimeric G proteins (18) (see also Chapter 7). Administration of PTH in vivo leads to the rapid generation of nephrogenous cAMP (19,20) and to activation of protein kinase C (PKC) in basolateral renal cortical membranes (21). This signaling plurality via the PTHR has been abundantly confirmed and further characterized in extensive studies in vitro, which have involved isolated renal tubules or slices, primary renal cortical cell cultures, a widely employed established opossum kidney cell line with characteristics of PCTs (OK cells), immortalized immunoselected distal tubular cells, and various established epithelial cell lines of renal origin (i.e., COS-7, HEK293, LLC-PK1), devoid of endogenously expressed PTHRs, which have been transfected with cDNA encoding the cloned PTHR (18,22-45).

Collectively, these studies indicate that PTH can activate adenylyl cyclase, protein kinase A (PKA), phospholipase C (PLC), PKC, and cytosolic free calcium (CaZi+) transients, as well as phospholipase A 2 (PLA2) (46-48) and phospholipase D (PLD) (43,45). The repertoire of PTHR signaling appears to differ depending on the region of the nephron in which it is expressed. For example, cells of proximal tubular origin manifest an acute spiking C a 2+ i response that likely is triggered by inositol trisphosphate released via PLC activation. Cells of distal tubular origin, in contrast, exhibit a very delayed and sustained CaZi+ response (probably due to apical C a 2+ entrymsee below) and show PKC activation in the absence of PLC stimulation (43). The PKC response to PTH in these DCT cells may be mediated by PLD (45). The coupling of specific PTHR-generated signals to the various physiologic responses to PTH or PTHrP that occur in different renal epithelial cells has not yet been fully clarified and will be discussed further below.

Regulation of PTHR Signaling in Renal Cells As in other P T H / P T H r P target cells, the responsiveness of renal epithelial cells to PTH or PTHrP may be regulated, both by previous or chronic exposure to the homologous ligand and by other agonists that do not interact directly with the PTHR. Desensitization of renal cellular responsiveness during continuous exposure to high concentrations of PTH or PTHrP has been well documented and extensively studied. Chronic hyperparathyroidism (either primary or secondary to calcium or vitamin D deficiency) and acute infusion of PTH lead to PTH resistance in humans or animals, manifested by impaired cAMP and phosphaturic responses (19,21,49-51). In humans, the cAMP response may be more readily desensitized than the phosphaturic response at low doses of hormone (52). Similar desensitization is observed in cultured renal epithelial cells (23,53-56). Several factors may contribute to this renal resistance to PTHR activation, including a reduced number of cell surface PTHRs, persistent occupancy of PTHRs by ligand, and defective coupling between available PTHRs and the G proteins that mediate activation of effectors such as adenylyl cyclase or PLC (i.e., a "postreceptor" defect). The relative roles of these factors in causing PTHR desensitization appear to vary according to the specific situation and experimental system (49,57-61). As reviewed in more detail in Chapter 5, it is clear that PTHRs are rapidly internalized following ligand occupancy and activation, a response that lowers cell surface receptor expression and that is due to PTHR phosphorylation by both PTHR-dependent activation of "signal kinases" (PKA, PKC) and by the action (s) of generic G protein-coupled receptor kinases

RENAL ACTIONS OF P T H AND P T H r P

(62-64). The particular PTHR-generated signals that mediate PTHR desensitization in renal epithelial cells may be cell type specific. For example, in OK proximal tubular cells, homologous desensitization of the PTHR cAMP response is PKC dependent (53), whereas in PTHR-transfected LLC-PKa cells, desensitization is pathway specific--i.e., adenylyl cyclase is fully desensitized by cAMP-dependent signaling only whereas desensitization of the PLC response is linked to prior PLC activation (56). Control of receptor expression may be an important mechanism for modulating the relative, as well as the absolute, intensities of signaling along the various transduction pathways coupled to the PTHR. Thus, as shown in a series of PTHR-transfected LLC-PK1 renal epithelial cell subclones that comprised a broad range of receptor expression, the magnitude of the PLC response was much more strongly influenced by changes in cell surface PTHR density than was the adenylyl cyclase response (37). This was interpreted as evidence that the coupling between G s and the PTHR in these cells is more efficient than that between the PTHR and the Gq that presumably mediates PLC activation. In any event, it is clear that changes in PTHR expression may allow differential modulation of PTHR signaling responses in a given renal cell. Expression of PTHRs on the surface of kidney cells also is controlled by the rate of PTHR gene transcription, although current understanding of this process is incomplete. Hypoparathyroidism, induced by either parathyroidectomy or dietary phosphate depletion, strongly up-regulates PTHR mRNA levels in rat renal cortex (65). Curiously, the opposite effect, i.e., suppression of PTHR mRNA by exposure to high concentrations of PTH, has not been observed, either in vivo or in vitro (55,65). Renal PTHR mRNA expression is reduced in rats with renal failure, but this apparently is due to some aspect of uremia or renal disease other than secondary hyperparathyroidism per se, because it is not prevented by parathyroidectomy (66-68). In rats with secondary hyperparathyroidism due to vitamin D deficiency, renal cortical PTHR mRNA levels actually were found to be twice as high as normal, a change that could not be corrected by normalizing serum calcium (61). This experiment has been interpreted as evidence of a suppressive action of vitamin D on PTHR gene transcription in the proximal tubule, although this may not be true of all renal epithelial cells. For example, PTHR expression is upregulated severalfold by 1,25(OH)zD ~ in immortalized DCT cells (69). In cultured OK cells, TGF-[31 was shown to diminish PTHR mRNA expression, but the possible physiologic significance of this effect in vivo has not been clarified (70). PTHR mRNA expression was not affected by the mild secondary hypoparathyroidism induced by ovariectomy in rats nor by subsequent estrogen treatment (71).

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CALCIUM AND MAGNESIUM EXCRETION The action of PTH to maintain blood calcium was among the first to be described, and early observations in animals or patients with hypoparathyroidism or hyperparathyroidism clearly implicated abnormalities in renal calcium handling (see Chapters 27 and 47). Alterations in serum magnesium concentrations frequently are encountered also in patients with parathyroid disorders, which led to the understanding that PTH participates in magnesium homeostasis as well (see Chapter 48). The mechanisms whereby Ca 2+ and Mg 2+ are reabsorbed are similar and interrelated in some regions of the nephron but different in others.

Sites and Mechanisms of Calcium and Magnesium Reabsorption Calcium and Mg 2+ are reabsorbed at many sites along the nephron (72,73). Approximately 60% of illtered Ca 2+, but only 20% of filtered Mg 2+, is reabsorbed by the proximal tubule. Reabsorption here is almost entirely passive, driven by both the ambient lumen-positive voltage and the progressive concentration of these ions within the tubular urine as Na + and water are reabsorbed along the proximal segments (72-74) (Fig. 1). In the proximal tubule, the route of reabsorption for both Ca 2+ and Mg 2+ is almost entirely paracellular, and differences in permeability of the intercellular tight junctions for the two cations presumably account for the preferential reabsorption of Ca 2+ here. Both Ca 2+ and Mg2+ also are passively reabsorbed in the CTAL of Henle's loop, although here the permeability for Mg 2+ may be greater than that for Ca 2+, because 60% of Mg 2+ but only 20% of Ca 2+ is reabsorbed in this segment, The lumen-positive transepithelial voltage gradient that drives Ca 2+ and Mg 2+ transport in the CTAL is maintained by, and proportional to, the rate of Na+/K+/CI2 transport, which is dependent, in turn, on the activities of the NKCC2, C1C-Kb, and ROMK transporters (75). The calcium-sensing receptor (CaSR) also is especially strongly expressed in Henle's loop, and activation of this receptor by high peritubular Ca 2+ or Mg 2+ concentrations inhibits Ca 2+ and Mg 2+ reabsorption in the CTAL, presumably by reducing the transepithelial voltage gradient (76) (see Chapter 8). It also is possible that the CaSR may mediate inhibition by Ca 2+and Mg 2+ of the cAMP response to PTH (7%79). Paracellin-1, a novel member of the claudin family of tightzjunction proteins that is expressed only in Henle's loop and the DCT, was identified as the cause of an autosomal recessive renal magnesium' and Ca2+-wasting disorder (80). Though not yet demonstrated directly, it seems likely that expression of paracellin-1 may control the passive permeability of the CTAL for both Ca 2+ and Mg 2+. There is some evidence also for active, transcellular transport of Ca 2+ by the

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FIG. 1 Calcium and magnesium reabsorption in (left) the proximal convoluted tubule (PCT) and (right) the cortical thick ascending loop of Henle (CTAL). In the PCT, Ca 2+ and Mg 2÷ are passively reabsorbed via paracellular routes at rates driven by the lumen-positive transepithelial voltage and limited by the conductance of the intercellular junctions for these cations. The transpeithelial voltage, depicted as positive at the apical (Ap) relative to the basolateral (BI) side of the epithelium, is generated by paracellular diffusion of CI- ions, which, like Ca 2+ and Mg 2+ ions, are progressively concentrated along the lumen by active transcellular Na + reabsorption. Major mechanisms of Na + reabsorption shown include Na+/H ÷ exchange, Na+-dependent cotransport of anions (phosphate, amino acids, sulfate, etc.), and a small apical Na + conductance, all driven by the low intracellular Na + concentration established by the Na+/K +ATPase, which pumps three Na + ions out for each two K + ions that enter the cell. The stoichiometry of the basolateral electrogenic Na+/HCO~ cotransporter (one Na + per three HCO~ ions) allows for active basolateral extrusion of some Na + because of the negative intracellular potential (not shown) and favorable HCO~ concentration gradient that drive HCO~ exit. PTHRs expressed in PCTs inhibit Na + transport by multiple mechanisms (see text) and thereby moderately impair Ca 2+ and Mg 2+ reabsorption (dashed lines indicate responses about which some uncertainty exists). In the CTAL (right panel), Ca 2+ and Mg 2+ reabsorption will again occur mainly via voltage-dependent paracellular transport, although transcellular Ca 2+ transport, presumably mediated by apical Ca 2+ channels and basolateral Ca2+-ATPases, also has been described (question mark). Apical NKCC2 cotransporters and ROMK K + channels maintain the lumen-positive transepithelial voltage necessary for cation transport, which is inhibited by Ca2+/Mg2+-dependent activation of the CaSR and by the loop diuretic furosemide. Chloride exits across the basolateral membrane via one or more CI- channels, including CIC-Kb (not shown). The channel protein paracellin-1 appears to be critical for paracellular cation transport in the CTAL and could be a target for CaSRs and PTHRs, which, respectively, reduce and augment cation transport in this nephron segment.

CTAL (81). Calcium-sensitive cation channels have been found in CTAL apical membranes (82), as have Ca '2+ATPases that would be necessary for extrusion across the steep basolateral electrochemical gradient (83). Finally, small but critical fractions of filtered Ca2+and 2+ Mg uapproximately 5-10% eachmare reabsorbed in the distal nephron (i.e., the DCT, CNT, and early CCD) (Fig. 2). The mechanism of Mg 2+ reabsorption by the distal nephron is obscure, but it seems to be closely related to that of NaCI, in that both pharmacologic (thiazide diuretics) and genetic (Gitelman's syndrome) inhibition

of the thiazide-sensitive NaC1 cotransporter (TSC) impairs Mg 2+ reabsorption. In contrast, Ca 2+ reabsorption in the distal nephron, which involves transcellular active transport against an unfavorable electrochemical gradient (84-86), is promoted by TSC inhibition, which hyperpolarizes the apical cell membrane. Cells of the distal nephron express several proteins that are required for effective transcellular active Ca 2+ transport (73). Calcium enters the apical membrane via multiple C a 2+ channels (87,88), one of which, ECaC, has been cloned and shown to be expressed in the distal tubule, to be acti-

RENAL ACTIONS OF PTH AND PTHrP

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FIG. 2 PTH regulation of distal tubular calcium reabsorption. In the DCT, Ca 2+ reabsorption involves apical Ca 2+ entry via voltage-sensitive Ca 2+ channels and subsequent basolateral extrusion by Ca2+-ATPases and, uniquely, Na+/Ca 2+ exchangers driven by the Na÷/K+-ATPase. Multiple Ca 2+ channels may be expressed here, including the ECaC channel that is activated by hyperpolarizing voltages (increased I Vml). Inhibition of the thiazide-sensitive NaCI transporter, with continued basolateral CI- exit, hyperpolarizes the cell toward the K + equilibrium potential, which then increases Ca 2+ entry by ECaC and other channels activated by hyperpolarizing potentials. Calbindin-D28K binds and shuttles Ca 2÷ from the apical membrane to the basolateral sites of active Ca 2÷ extrusion, thereby buffering the cytoplasm from high concentrations of transported Ca 2+. Calbindin-D28K is induced by 1,25(OH)2D 3 and may directly activate apical Ca 2+ channels, which otherwise are inhibited by intracellular Ca 2+ ions. PTHR activation leads to insertion of additional apical Ca 2+ channels, hyperpolarization of the cell (question mark) via enhancing basolateral CI- exit, and, thus, activation of Ca 2÷ channels, increased calbindin-D28K expression, and stimulation of the basolateral Ca2+-ATPase. The routes and mechanisms of Mg 2+ reabsorption in the DCTs are unknown.

vated by hyperpolarizing voltages, and to be inactivated by intracellular C a 2+ (89,90). These cells also express the vitamin D-dependent calbindin-D28K calcium binding protein, which can transport Ca 2+ across the cytoplasm while buffeting the submicromolar cytosolic free C a 2+ concentration against the high mass flux of transported C a 2+ (91-93). Calbindin-D28K also may directly activate apical membrane C a 2+ channels (94). Extrusion of transported Ca 2+ across the basolateral membrane can occur via both a direct CaZ+-ATPase and a high-capacity Na+/Ca 2+ exhanger driven by the transmembrane Na + gradient.

PTH Regulation of Renal Calcium and Magnesium Excretion Administration of PTH in vivo increases the net renal reabsorption of both Ca 2+ and Mg 2+ (72,95-99).

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PTH augments Mg 2+ reabsorption in the CTAL (100-102) and possibly in the distal n e p h r o n as well (103), but the mechanisms involved are obscure. PTH may increase the transepithelial voltage that drives paracellular Mg 2+ (and Ca 2+) transport in the CTAL, but this is controversial (100,101) and, in any event, is unlikely to explain the magnitude of the PTH effect (72). Other experiments indicate that the PTH response probably is mediated by an increase in paracellular Mg 2+ permeability (104). In this regard, it will be of interest to learn if PTH up-regulates paracellin-1 expression or permeability. M t h o u g h PTH increases net renal C a 2+ reabsorption overall, it actually moderately inhibits C a 2+ reabsorption in the PCT (105-107). As will be discussed further below, this results from a PTH-induced reduction in Na + reabsorption (via inhibition of both NaP i cotransport and N a + / H + exchange) and of Na+/K+/ATPase activity, processes that otherwise support net solute and water reabsorption and thereby establish the elevated intraluminal concentrations of C a 2+ and C1- required for effective paracellular movement of C a 2+ in the PCT. In contrast, PTH augments C a 2+ reabsorption in the CTAL and in the distal n e p h r o n , especially in the CNT (84,106,108-110), and it is these actions that account for the overall positive effect of PTH on renal C a 2+ reabsorption. The mechanism of the PTH effect in the CTAL has not been intensively studied but likely proceeds via an increase in transepithelial voltage and e n h a n c e d paracellular C a 2+ transport (101), although some evidence suggests a c o m p o n e n t of transcellular transport as well (111). The distal n e p h r o n clearly is the major site at which PTH regulates C a 2+ transport. PTH exerts several specific actions in these cells that independently contribute to increased C a 2+ reabsorption. PTH increases C a 2+ uptake across apical membranes of distal tubular cells, an effect that can be observed in apical membrane vesicles isolated following PTH administration in vivo or to isolated tubules in vitro (112,113). In cultured cells obtained from the mouse CTAL and DCT, PTH induced a delayed (10 minutes) and sustained increase in cytosolic C a 2+ that was of extracellular origin, was blocked by dihydropyridine C a 2+ channel antagonists, and appeared to result from exocytosis of membranes harboring preformed but functionally latent intracellular C a 2+ channels (114). These channels were of low conductance and were activated by hyperpolarizing voltages (87), features also reported for the subsequently cloned ECaC channel (90) (although more information is needed to determine if the ECaC channel serves as the main route of regulated C a 2+ entry in distal tubular cells). Importantly, PTH acutely hyperpolarizes distal tubular cells, at least in part by increasing basolateral C1- conductance (115). This action would activate the ECaC channel (90) and increase both the

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driving force for apical membrane C a 2+ entry and the rate of Na+/Ca 2+ exchange at the basolateral membrane (116). Increased Na+/Ca 2+ exchange has been demonstrated following PTH administration in vivo and in vitro (117,118). Moreover, activation of Na+/Ca 2+ exchange is critical for the action of PTH to increase C a 2+ reabsorption, because this could be blocked completely in rabbit CNTs and DCT cells either by disrupting the Na + gradient that drives the Na+/Ca 2+ exchanger with ouabain or monensin or by removing extracellular C a 2+ f r o m the basolateral compartment (116,119). The fact that this exchanger is expressed only in the distal, and not the proximal, nephron may explain, at least in part, why distal and not proximal tubular cells can conduct transcellular C a 2+ transport (113,116,120,121). PTH also may increase C a 2+ e x t r u s i o n by activating the basolateral CaZ+-ATPase (122), although this is not observed in all systems (120). Finally, expression of the calbindin-D28K protein in renal cortex has been shown to decrease following parathyroidectomy and to increase following PTH infusion into intact rats (123). The powerful inductive effect of 1,25(OH)zD ~ on calbindin-D28K expression in the distal nephron (124,125) may be involved in mediating this action of PTH, given that PTH augments 1,25(OH)zD 3 synthesis (see below) and that 1,25(OH)zD ~ directly accelerates the distal tubular calcium reabsorptive response to PTH in vitro (126). Other evidence indicates that PTH can increase calbindinD28K independently of 1,25(OH)zD ~ or serum calcium, however (123).

PTHR Signal Transduction in Regulation of Calcium and Magnesium Excretion The particular PTHR-generated signals responsible for these various effects of PTH on components of the distal tubular CaZ+-reabsorptive response are not fully clarified. The initial entry of C a 2+ a c r o s s the apical membrane seems to require activation of both PKA and PKC in immortalized murine DCT cells (43,45). In many experimental systems, the PTH effect on distal Ca 2+ transport can be mimicked by cAMP analogs or phosphodiesterase inhibitors (112,119,127), although in isolated rabbit CNT/CCD tubules, in which this cAMP mimicry also is true, the effect of PTH was prevented by chelerythrine, a PKC inhibitor, but not by dideoxyadenosine, an adenylyl cyclase inhibitor that did block PTH-dependent cAMP accumulation (128). Further evidence implicated a Ca2+-independent ("atypical") PKC as a mediator of this PTH effect (128). Similarly, the ability of dibutyryl cAMP to promote C a 2+ transport in rabbit distal tubules was greatly potentiated by phorbol esters, which exerted no effect alone, and PKC inhibitors did block the effect of the combination

of phorbol and cAMP analog as well as that of the cAMP analog alone (129). PTH stimulation of Na+/Ca 2+ exchange, transepithelial hyperpolarization, and, in canine cells, CaZ+-ATPase also is reproduced by cAMP analogs (110,117,118,120,122), although, as just noted, such evidence clearly does not exclude a role for other PTHR messengers in these processes as well. Considering that PTH may have to orchestrate a series of independent "elemental responses" to achieve effective distal tubular C a 2+ reabsorption, including membrane hyperpolarization, increased exocytosis of latent C a 2+ channels, increased calbindin-D28K expression, increased Na+/Ca 2+ exchange [this possibly secondary entirely to the hyperpolarization and increased cytosolic free C a 2+ (73)], and increased CaZ+-ATPase activity, and that these responses may not all occur in the same cells, it is perhaps not surprising that some ambiguity persists regarding the roles of PKA versus PKC (or other PTHR-activated effectors) in controlling overall distal tubular C a 2+ transport. Apparent requirements for multiple effectors may reflect a convergence of several signals on a single mechanism, independent actions of different effectors on one or more of the elemental cellular responses that contribute to the overall Ca2+-reabsorptive response, or both.

PHOSPHATE EXCRETION Phosphaturia was one of the earliest recognized actions of PTH (130-133). With the advent of micropuncture analysis, it became clear that the effect of PTH to inhibit phosphate reabsorption occurs almost entirely in the proximal tubules, especially in the late portion of the PCT (105-107,134-137). Some evidence points to a small component of PTH-inhibitable phosphate reabsorption in the distal nephron as well (107,136,138-140).

Mechanisms of Proximal Tubular Phosphate Reabsorption Extensive experimentation with isolated perfused tubules, renal membranes, and membrane vesicles over the past 25 years, exhaustively reviewed by Murer and colleagues (141-143), has provided a clear picture of the mechanisms of proximal tubular phosphate reabsorption. Phosphate (Pi) is moved across the apical membrane of the cell, against a steep electrochemical gradient (due mainly to the negative intracellular potential), by Na+/Pi cotransporters driven by the transmembrane Na ÷ gradient (Fig. 3). Detailed biochemical analyses had indicated that multiple such Na+/Pi cotransporters, with distinct kinetic, allosteric, and physical properties, are located within the renal

RENAL ACTIONS OF PTH AND P T H r P

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dietary phosphate (150,154-158). Thus, regulation of NaPi-2 activity is the principal mechanism whereby PTH controls phosphate reabsorption in the PCT.

PTH Regulation of Proximal Tubular Phosphate Reabsorption

Pi'=I tnM

I I "~''PPTTHI4rP

Early work had demonstrated that PTH rapidly lowers the maximal rate of NaPi cotransport in brush border membrane vesicles and that recovery from this effect requires new protein synthesis, suggesting that PTH causes degradation of NaP~ cotransporters (141, 159-161). Recent functional and immunohistochemical analyses of NaP~-2 protein expression in rat kidney and in cultured OK cells have confirmed that PTH induces a rapid (15 minutes) movement of NaPe2 protein into the subapical endocytic apparatus, followed by a microtubule-dependent delivery to lysosomes and proteolytic degradation (154,156,158,162,163) (see Fig. 4). These effects of PTH on NaP~-2 protein are not associated with suppression of NaPi-2 mRNA expression, although parathyroidectomy does increase both NaPi-2 protein and mRNA severalfold (65,155,164,165).

FIG. 3 Phosphate reabsorption in the proximal tubule. Phosphate (P~) must be actively transported across the apical membrane of the PCT cell because of the strongly interiornegative potential and the fact that cytosolic P~ concentration (1 mM) is roughly 100-fold above equilibrium. This transport is accomplished by an electrogenic type II NaP~ cotransporter [stoichiometry = 3 Na ÷ ions per P~(mono- or dibasic) ion] that is energized by the steep transmembrane Na ÷ gradient established by the basolateral Na+/K+-ATPase. Activity of this cotransporter is reduced by PTHR activation. Mechanisms of basolateral P~ exit are not well understood, but an anion exchanger could allow P~to leave the cell passively.

PTHR Signal Transduction in Regulation of Phosphate Excretion

cortex (144,145). Some of these may be so-called housekeeping cotransporters, presumed to reside on the basolateral membranes, that are ubiquitously expressed by all cells and involved in maintaining intracellular P~ concentrations, whereas others are epithelial-specific and devoted to the specialized function of transepithelial phosphate transport (142). Three major classes (types I, II, and III) of Na+/Pi ("NaPi") cotransporters, products of different genes, have been cloned and shown to be expressed in PCT cells (146-149). Both the type I and type II cotransporters are localized to the apical brush border membrane of PCT cells (150). Type III NaP~ cotransporters, originally identified as cell surface virus receptors (Glvr-1 and Ram-l), are widely expressed (151) and, like type I cotransporters, are not regulated by PTH or dietary phosphate (152). Type III cotransporters are expressed by DCT cells and thus could play a role in phosphate reabsorption in the distal nephron (153). Type II cotransporters are 80- to 90-kDa glycoproteins that are predicted to span the membrane eight times, with both their amino and carboxyl termini oriented into the cytosol (148). These cotransporters are electrogenic and transport Na + and HzPO 4 in a molar ratio of 3:1 (148). Expression and activity of the type II NaP i cotransporters (NaPe2, in rat) are strongly regulated by both parathyroid status and

Determination of the PTHR signals involved in mediating regulation of NaP i activity has been extensively studied. Early experiments in vivo or with isolated renal membranes indicated a role for cAMP-dependent actions of PTH in regulating phosphate excretion, based mainly on mimicry of the PTH effect by cAMP analogs or cAMP phosphodiesterase inhibitors (106,141,160). Many of these studies were conducted before the cAMP-independent signaling features of the PTHR were recognized (166). Analysis of this question in vitro has been pursued almost exclusively using the OK opossum kidney cell line, which expresses both the type II NaP i cotransporter and the type 1 PTHR (167) and manifests PTHdependent inhibition of NaP i cotransport along with other features typical of PCT cells (34,142,168-172). There is general agreement that direct pharmacologic activation of either PKA or PKC can inhibit NaPi activity in OK cells. The importance of the cAMP response of the PTHR was highlighted by experiments in which expression of a dominant-negative inhibitor of PKA (mutant PKA regulatory subunit gene) in OK cells completely blocked NaPi down-regulation by PTH (170) and by the demonstration that NaP~ regulation by PTH was unaffected when PLC/PKC activation was completely inhibited by the drug U73122 (34). On the other hand, a role for PKC is suggested by findings that NaP i activity can be at least partially regulated by PTH analogs, such

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CHAPTER14

Na+ Pi" I.! Na+llPi"

X-~)

PlasmaMembrane Submembranesomes

X/~k~

~ \Microtubules N

"q~'qk~ PTHrP PTH FIG. 4 Regulation of NaP~ cotransport by PTH. Activation of PTHRs on the basolateral membrane of PCT cells stimulates PKA and PKC. PKC induces a rapid decrease in activity of NaP~-2 transporters expressed on the apical surface, an effect that is mimicked by PTH(3-34). This may involve phosphorylation of one or more intermediary proteins (X), because consensus PKC phosphorylation sites within the NaP~-2 protein can be eliminated without affecting this regulatory effect of PKC. Activation of PKA also impairs NaP~ cotransport, but this effect is more delayed and involves retrieval of surface NaPe2 cotransporters by a microtubule-dependent process of endocytosis, lysosomal fusion, and degradation. The responsible PKA substrates and details of their actions currently are unknown (question marks).

as PTH(3-34), at concentrations that do not activate adenylyl cyclase or PKA but that do stimulate PKC (169,172-174). Moreover, NaP i regulation in OK cells may be observed at concentrations of PTH(1-34) that also activate PKC but are too low to measureably stimulate PKA, and NaP i regulation by PTH can be blocked by pharmacologic inhibition of PKC (29,169,175,176). In fact, the NaPi-2 protein can be phosphorylated, it contains several consensus sites for PKC, and its activity is inhibited by pharmacologic PKC activation when it is expressed in Xenopus oocytes (150,177). Because mutation of these sites does not interrupt PKC-dependent down-regulation of NaPi-2 activity, however, it is possible that PKC-dependent phosphorylation of other proteins, which act to regulate NaPi-2, may be the direct mediator of this effect (150,177). It also seems that PKA and PKC activation may lead to temporally and qualitatively distinct changes in NaPi-2 protein expression and activity (171,172) (Fig. 4). For example, PTH(3-34) initially inhibited NaP i activity comparably to PTH(1-34) but did so with no, or much less, induced clearance of the protein from the cell surface, which suggested that the main effect of PKC was to reduce the activity of the cotransporter directly or indirectly, whereas that of PKA may relate more to the physical removal of the protein from the apical membrane via endocytosis (171,172). Similarly, a study in intact rats showed no internal redistribution of membrane NaPi-2

protein in response to PTH(3-34) under conditions in which the peptide was shown to be bioactive, whereas PTH(1-34) provoked an 18% redistribution within 1 hour (163). On the other hand, direct pharmacologic activation of PKC caused membrane retrieval of NaPi-2 transporters expressed in Xenopus oocytes (178). Thus, a coherent view has yet to emerge in this area, but it seems reasonable to conclude at present that though activation of PKA and PKC via the PTHR each can separately downregulate NaPi activity, these kinases likely exert distinct regulatory effects, and activation of both may be necessary to achieve the full response to the hormone.

SODIUM AND HYDROGEN EXCRETION Studies in vivo and with isolated renal tubules in vitro have established that PTH produces an acute natriuresis and diuresis and rapidly inhibits proximal tubular acid secretion (HCO~ reabsorption) (105,179-182). As illustrated in Fig. 1, Na + reabsorption in the PCT proceeds via both the active, transcellular route and the passive, paracellular pathway. These mechanisms account for roughly 60 and 40%, respectively, of Na + reabsorption (183). Much of the transcellular Na + reabsorption in PCTs involves Na+-dependent cotransport of anions such as phosphate, sulfate, and amino acids or the operation of apical N a + / H + exchangers.

RENAL ACTIONS OV PTH AND PTHrP

PTH Regulation of Proximal Tubular Sodium and Hydrogen Excretion Effective reabsorption of Na ÷ and HCO~ in the proximal tubule requires the concerted activities of apical type 3 N a + / H + exchangers (NHE3s), basolateral Na+/K+-ATPases (to maintain the transmembrane Na + gradient), and electrogenic basolateral Na+-3HCO~ cotransporters, among others (184). PTH exerts at least three or four independent actions that conspire to powerfully inhibit Na + and HCO~ reabsorption. These include inhibition of apical N a + / H + exchange, apical Na+/Pi - cotransport, basolateral Na+/K+-ATPase activity, and, possibly, basolateral Na+-HCO~ cotransport (see Fig. 1). PTH strikingly inhibits the activity of the amiloridesensitive NHE3 in proximal tubular apical brush border membranes and in OK cells (185-188), directly impairing both Na + reabsorption and H + excretion. Conversely, parathyroidectomy increases NHE3 exchanger activity (189). The possibility that PTH may inhibit basolateral base exit via regulation of Na+-3HCO~ cotransporters is unsettled, because this has been observed in proximal tubules of rats (190) but not of rabbits (191). On the other hand, in vivo or in vitro ~administration of PTH greatly reduces the activity of the basolateral Na +/K+-ATPase in rat proximal tubules (46-48,192).

PTHR Signal Transduction in Regulation of Sodium and Hydrogen Excretion The mechanisms whereby PTHRs regulate these various effectors of proximal tubular Na + and H + excretion are both different and complex. Within 30-60 minutes of PTH exposure in vivo, NHE3 is phosphorylated and inactivated, after which it is sequestered (but not destroyed) via a more delayed internalization to a highdensity intracellular membrane fraction (163,186,193). Experiments in OK cells also indicate that PTH reduces the sensitivity of the exchanger to the intracellular H + concentration (194). Recent functional analysis of expressed recombinant NHE3 exchangers (195) supports previous evidence (106,185,191,196) that NHE3 is a direct substrate for PKA. Involvement of the cAMP/PKA signaling cascade in PTH regulation of NHE3 was suggested by the demonstration that PTH(1-34), but not a PTH(3-34) analog devoid of PKA activity, induced NHE3 internalization in rat proximal tubules (163). Also, PTHrP (1-34) inhibited NHE3 activity in an OK cell subclone in which this peptide could increase cAMP but not cytosolic Ca2i+, PLC, or PKC (197). On the other hand, others have obtained clear evidence, using both kinase inhibitors and signal-selective PTH analogs in OK cells, for involvement of both the PKA and PKC PTHR signaling pathways in NHE3

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235

regulation (36,39,185,188). By analogy with mechanisms of PTH-regulated PCT phosphate and DCT calcium excretion, it is likely that these two PTHR signal kinases exert cooperative but distinct effects in controlling NHE3 expression and activity. In the case of the basolateral Na+/K+-ATPase, analysis of PTH regulation has disclosed a novel pathway of PTHR signaling. Administration of PTH(1-34) in vivo causes a rapid inactivation of proximal tubular basolateral Na+/K+-ATPase activity without inducing destruction or sequestration of the pump proteins (163). In this case, PTH(3-34) does mimic the action of PTH(1-34) by activating PKC (not PKA) (47,192). This occurs via PTHR coupling to a Gq/G11 family member and leads to a series of further responses, which include activation of PLA 2, generation of arachidonic acid, and metabolism of arachidonate via the P450 monooxygenase pathway to produce active eicosanoids, notably 20-hydroxy-eicosatetraenoic acid (20-HETE) (46-48,192). In a manner as yet unknown, 20-HETE then leads to inhibition of Na+/K +ATPase activity (48,192). This monooxygenase-dependent pathway accounts for most of the PTH regulation of Na+/K+-ATPase activity, although a portion of the response is attributable to cAMP/PKA activation (46).

PTH Regulation of Sodium and Hydrogen Excretion beyond the Proximal Tubule Though it is true that PTH strongly inhibits proximal tubular HCO~ reabsorption, this is compensated to some extent by its effect to increase HCO~ reabsorption in Henle's loop and H + secretion in the CD (180,198,199). Moreover, the phosphaturia induced by PTH also contributes to net acid secretion (200), and PTH actually can increase net renal acid secretion during metabolic acidosis (201). Similarly, in perfused mouse CTAL, PTH may exert an antinatriuretic effect, manifested as augmented paracellular transport driven by an increased transepithelial voltage (202). Thus, the overall effect of PTH on renal acid and sodium excretion may vary markedly depending on the particular physiologic state of the organism.

VITAMIN D METABOLISM Synthesis of 1,25(OH)2D ~ is increased by PTH and reduced by parathyroidectomy (203). This results from regulated expression, in proximal tubular cells, of the 25(OH)Ds le~-hydroxylase gene, the promoter for which is rapidly induced by PTH in vitro (204-206). This effect of PTH can be overridden in vivo by the direct suppressive action of hypercalcemia on le~-hydroxylase expression (207). It is variably impaired in older animals or humans, even though indices of

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CI-IAPTWR14

PTHR signaling per se remain normal (208,209). PTH induction of l e~-hydroxylase mRNA is transcriptional, additive to that of calcitonin, occurs in the genetic absence of the vitamin D receptor, and is antagonized by coadministration of 1,25(OH)zD 3, which directly inhibits expression when given alone (206). The signaling pathways employed by the PTHR to increase 1,25(OH)zD 3 synthesis have been examined extensively in vivo and in vitro. Involvement of cAMP is suggested by the fact that the PTH effect can be mimicked by cAMP analogs, forskolin or phosphodiesterase inhibitors (210-216). Moreover, in a transformed murine proximal tubular cell line, transcriptional induction of the l oL-hydroxylase occurred with either PTH or forskolin, and the effects of both were blocked by the PKA-selective inhibitor H89 (206). On the other hand, careful studies of the effects of added PTH in isolated perfused rat proximal tubules have correlated rapid (30-60 minutes) increases in 1,25(OH)zD ~ synthesis with PKC activation on the basis of (1) concentration dependence (PKC and 1,25(OH)zD ~ syntheses were increased at PTH concentrations 100- to 1000-fold lower than required for PKA activation), (2) selective inhibition by PKC inhibitors, and (3) activation by truncated PTH analogs [i.e., PTH(3-34), PTH(13-34)] that can trigger PKC but not PKA in this system (32,217). More information clearly is needed, but available data seem most consistent with both a predominant effect of cAMP/PKA on transcriptional regulation of lo~-hydroxylase gene expression and a more rapid, posttransciptional effect of PKC on l e~-hydroxylase enzyme activity. The 25(OH)D 24-hydroxylase also is regulated by PTH. In kidney homogenates, cultured proximal tubular cells, and certain proximal tubular cell lines, PTH inhibits 24-hydroxylase activity by mechanisms that may involve cAMP (214,215,218-220). It also antagonizes the inductive effect of 1,25(OH)zD 3 on both 24-hydroxylase and vitamin D receptor expression (221). Interestingly, PTH leads to opposite effects on 24-hydroxylase and vitamin D receptor expression in proximal and distal tubules. Thus, PTH augments 1,25(OH)zD3-dependent induction of 24-hydroxylase in DCT cells, possibly by increasing expression of the vitamin D receptor (222), whereas it inhibits expression of both the 24-hydroxylase and the receptor in proximal tubules, as noted above.

O T H E R R E N A L EFFECTS OF P T H A variety of other effects of PTH on renal metabolism, secretion, and membrane function have been described, the physiologic roles of which currently are less clear than those described elsewhere in this chapter. Examples include rapid microvillar shortening in cultured proximal tubular cells (223); increased renin

release from perfused rat kidneys (224); increased proximal tubular gluconeogenesis, ammoniagenesis, and phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression (225-227); activation of an apical C1- channel in rabbit proximal tubular cells (228); and stimulation of ecto-5'-nucleotidase activity in apical membranes of OK cells, an effect that is mimicked by PTH(3-34) but not by forskolin and which is blocked by PKC inhibitors (38).

RENAL EXPRESSION AND ACTIONS OF P T H r P PTHrP is expressed in the glomeruli, distal tubules, and collecting ducts of fetal kidneys and in PCTs, DCTs, and glomeruli of the adult kidney (229,230). In one study in rats, PTHrP mRNA was found in glomeruli, PCTs, and macula densa but not in CTAL, medullary thick ascending loop (MTAL), DCTs, or CDs (9). It is unlikely that PTHrP is critical for normal renal development, because the kidneys of mice missing functional PTHrP genes appear histologically normal. When tested, the active amino-terminal fragments of PTHrP generally exhibit renal actions identical to those of PTH, including stimulation of cAMP production and regulation of P~ transport, C a 2+ excretion, and 1,25(OH)zD ~ synthesis (99,231,232). On the other hand, longer PTHrP fragments may possess unique properties. For example, in an assay of HCO~ excretion by the perfused rat kidney, hPTHrP(1-34) was equipotent with hPTH(1-34), whereas hPTHrP(1-84), hPTHrP(1-108), and hPTHrP(1-141) each were less active than hPTH (1-34) (233). As discussed in Chapters 3 and 6, the PTHrP gene can generate multiple transcripts and protein products, some of which may undergo unique nuclear localization. It is quite possible, therefore, that locally expressed PTHrP may exert actions in the kidney that are not shared with PTH, although this has not yet been adequately addressed. A possible role for locally produced PTHrP in the renal response to ischemia has been suggested by findings that PTHrP expression is induced by ischemia or following recovery from ATP depletion (68,234,235). PTHrP is expressed in the intimal and medial layers of human renal microvessels and in the macula densa (236). PTHrP (like PTH) increases renin release from the juxtaglomerular apparatus and also stimulates cAMP in renal afferent and efferent arterioles, leading to vasodilation and enhanced renal blood flow (224,237-242). Evidence for involvement of both cAMP and nitric oxide in PTHrP-induced vasorelaxation in vitro has been derived from use of specific inhibitors (236). Thus, enhanced local PTHrP production induced by inadequate renal perfusion or ischemia may be involved in both local and systemic autoregula-

RENAL ACTIONS OF P T H AND P T H r P

tory mechanisms, whereby direct local vasodilatory actions are supplemented by systemic activation of angiotensinogen that increase arterial pressure and further sustain renal blood flow.

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17. Bettoun JD, Minagawa M, Hendy GN, et al. Developmental upregulation of human parathyroid hormone (PTH)/PTHrelated peptide receptor gene expression from conserved and human-specific promoters. J Clin Invest 1998;102:958-967. 18. Abou-Samra AB, Juppner H, Force T, et al. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: A single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium. Proc Natl Acad Sci USA 1992;89:2732-2736. 19. Tomlinson S, Hendy GN, Pemberton DM, O'Riordan JL. Reversible resistance to the renal action of parathyroid hormone in man. Clin Sci Mol Med 1976;51:59-69. 20. McElduff A, Lissner D, Wilkinson M, Cornish C, Posen S. A 6hour human parathyroid hormone (1-34) infusion protocol: Studies in normal and hypoparathyroid subjects. CalcifTissue Int 1987;41:267-273. 21. Bellorin-Font E, Lopez C, Diaz K, Pernalete N, Lopez M, Starosta R. Role of protein kinase C on the acute desensitization of renal cortical adenylate cyclase to parathyroid hormone. Kidney Int 1995;47:38-44. 22. Meltzer V, Weinreb S, Bellorin-Font E, Hruska KA. Parathyroid hormone stimulation of renal phosphoinositide metabolism is a cyclic nucleotide-independent effect. Biochim Biophys Acta 1982;712:258-267. 23. Henry HL, Cunningham NS, Noland TA, Jr. Homologous desensitization of cultured chick kidney cells to parathyroid hormone. Endocrinology 1983;113:1942-1949. 24. Teitelbaum AP, Strewler GJ. Parathyroid hormone receptors coupled to cyclic adenosine monophosphate formation in an established renal cell line. Endocrinology 1984;114:980-985. 25. Goligorsky MS, Loftus DJ, Hruska KA. Cytoplasmic calcium in individual proximal tubular cells in culture. Am J Physiol 1986;251 :F938-F944. 26. Hruska KA, Goligorsky M, Scoble J, Tsutsumi M, Westbrook S, Moskowitz D. Effects of parathyroid hormone on cytosolic calcium in renal proximal tubular primary cultures. Am J Physiol 1986;251:F188-F198. 27. Hruska KA, Moskowitz D, Esbrit P, Civitelli R, Westbrook S, Huskey M. Stimulation of inositol trisphosphate and diacylglycerol production in renal tubular cells by parathyroid hormone. J Clin Invest 1987;79:230-239. 28. Tamura T, Sakamoto H, Filburn CR. Parathyroid hormone 1-34, but not 3-34 or 7-34, transiently translocates protein kinase C in cultured renal (OK) cells. Biochem Biophys Res Commun 1989;159:1352-1358. 29. Quamme G, Pfeilschifter J, Murer H. Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: Generation of second messengers in the regulatory cascade. Biochem Biophys Res Commun 1989;158:951-957. 30. Coleman DT, Bilezikian JE Parathyroid hormone stimulates formation of inositol phosphates in a membrane preparation of canine renal cortical tubular cells. J Bone Miner Res 1990;5:299-306. 31. Nemani R, Wongsurawat N, Armbrecht HJ. Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates. Arch Biochem Biophys 1991 ;285:153-157. 32. Janulis M, Tembe V, Favus MJ. Role of protein kinase C in parathyroid hormone stimulation of renal 1,25-dihydroxyvitamin D 3 secretion. J Clin Invest 1992;90:2278-2283. 33. Bringhurst FR, Juppner H, Guo J, et al. Cloned, stably expressed parathyroid hormone (PTH)/PTH-related peptide receptors activate multiple messenger signals and biological responses in LLC-PK1 kidney cells. Endocrinology 1993;132:2090-2098. 34. Martin KJ, McConkey CL, Jacob AK, Gonzalez EA, Khan M, Baldassare JJ. Effect of U-73,122, an inhibitor of phospholipase

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178. Forster I, Traebert M, Jankowski M, Stange G, Biber J, Murer M. Protein kinase C activators induce membrane retrieval of type II Na+-phosphate cotransporters expressed in Xenopus oocytes. J Physiol 1999;517:327-340. 179. Schneider EG. Effect of parathyroid hormone secretion on sodium reabsorption by the proximal tubule. Am J Physiol 1975;229:1170-1173. 180. Bank N, Aynediian HS. A micropuncture study of the effect of parathyroid hormone on renal bicarbonate reabsorption. J Clin Invest 1976;58:336-344. 181. PuschettJB, Zurbach P, Sylk D. Acute effects of parathyroid hormone on proximal bicarbonate transport in the dog. Kidney Int 1976;9:501-510. 182. McKinney TD, Myers E Bicarbonate transport by proximal tubules: Effect of parathyroid hormone and dibutyryl cyclic AME Am J Physio11980;238:F166-F174. 183. Rector E Sodium, bicarbonate, and chloride reabsorption by the proximal tubule. AmJPhysio11983;244:F461-F471. 184. Mpern RJ. Cell mechanisms of proximal tubular acidification. Physiol Rev 1990;70:79-114. 185. Kahn AM, Dolson GM, Hise MK, Bennett SC, Weinman EJ. Parathyroid hormone and dibutyryl cAMP inhibit Na+/H + exchange in renal brush border vesicles. Am J Physiol 1985 ;248:F212-F218. 186. Hensley CB, Bradley ME, Mircheff AK. Parathyroid hormoneinduced translocation of Na-H antiporters in rat proximal tubules. Am J Physio11989;257:C637-C645. 187. Pollock AS, Warnock DG, Strewler GJ. Parathyroid hormone inhibition of Na+-H + antiporter activity in a cultured renal cell line. Am J Physiol 1986;250:F217-F225. 188. Helmle-Kolb C, Montrose MH, Murer H. Parathyroid hormone regulation of Na+/H + exchange in opossum kidney cells: Polarity and mechanisms. Pfluegers Arch 1990;416:615-623. 189. Cohn DE, Klahr S, Hammerman MR. Metabolic acidosis and parathyroidectomy increase Na+/H + exchange in brush border vesicles. A m J Physiol 1983;245:F217-F222. 190. Pastoriza-Munoz E, Harrington RM, Graber ML. Parathyroid hormone decreases HCOs reabsorption in the rat proximal tubule by stimulating phosphatidylinositol metabolism and inhibiting base exit. J Clin Invest 1992;89:1485-1495. 191. Sasaki S, Marumo E Mechanisms of inhibition of proximal acidification by PTH. AmJPhysio11991 ;260:F833-F838. 192. Ominato M, Satoh T, Katz M. Regulation of Na-K-ATPase activity in the proximal tubule: Role of the protein kinase C pathway and of eicosanoids. J Membr Bio11996;152:235-243. 193. Fan L, Wiederkehr MR, Collazo R, Wang H, Crowder LA, Moe OW. Dual mechanisms of regulation of N a / H exchanger NHE3 by parathyroid hormone in rat kidney. J Biol Chem 1999;274:11289-11295.

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194. Miller RT, Pollock AS. Modification of the internal pH sensitivity of the Na+/H + antiporter by parathyroid hormone in a cultured renal cell line. JBiol Chem 1987;262:9115-9120. 195. Zhao H, Wiederkehr MR, Fan L, Collazo RL, Crowder LA, Moe OW. Acute inhibition of N a / H exchanger NHE-3 by cAME Role of protein kinase a and NHE-3 phosphoserines 552 and 605. J Biol Chem 1999;274:3978-3987. 196. Weinman EJ, Dubinsky WP, Shenolikar S. Reconstitution of cAMP-dependent protein kinase regulated renal Na+-H + exchanger. J Membr Biol 1988;101:11-18. 197. Maeda S, Wu S, Green J, et al. The N-terminal portion of parathyroid hormone-related protein mediates the inhibition of apical Na+/H + exchange in opossum kidney cells. J Am Soc Nephrol 1998;9:175-181. 198. Bichara M, Mercier O, Paillard M, Leviel E Effects of parathyroid hormone on urinary acidification. Am J Physiol 1986;251 :F444-F453. 199. Paillard M, Bichara M. Peptide hormone effects on urinary acidification and acid-base balance: PTH, ADH, and glucagon. Am J Physiol 1989;256:F973-F985. 200. Mercier O, Bichara M, Paillard M, Prigent A. Effects of parathyroid hormone and urinary phosphate on collecting duct hydrogen secretion. AmJPhysiol 1986;251 :F802-F809. 201. Bichara M, Mercier O, Borensztein P, Paillard M. Acute metabolic acidosis enhances circulating parathyroid hormone, which contributes to the renal response against acidosis in the rat. j Clin Invest 1990;86:430-443. 202. Wittner M, Di Stefano A. Effects of antidiuretic hormone, parathyroid hormone and glucagon on transepithelial voltage and resistance of the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron. Pfluegers Arch 1990;415:707-712. 203. Fraser DR, Kodicek E. Regulation of 25-hydroxycholecalciferol1-hydroxylase activity in kidney by parathyroid hormone. Nature 1973;241:163-166. 204. Garabedian M, Holick ME Deluca HE Boyle IT. Control of 25hydroxycholecalciferol metabolism by parathyroid glands. Proc Natl Acad Sci USA 1972;69:1673-1676. 205. Kong XE Zhu XH, Pei YL, Jackson DM, Holick ME Molecular cloning, characterization, and promoter analysis of the human 25-hydroxyvitamin Dflalpha-hydroxylase gene. Proc Natl Acad Sci USA 1999;96:6988-6993. 206. Murayama A, Takeyama K, Kitanaka S, et al. Positive and negative regulations of the renal 25-hydroxyvitamin D~ lalpha-hydroxylase gene by parathyroid hormone, calcitonin, and lalpha,25(OH)zD 3 in intact animals. Endocrinology 1999;140:2224-2231. 207. WeisingerJR, Favus MJ, Langman CB, Bushinsky DA. Regulation of 1,25-dihydroxyvitamin D3 by calcium in the parathyroidectomized, parathyroid hormone-replete rat. J Bone Miner Res 1989;4:929-935. 208. FriedlanderJ, Janulis M, Tembe V, Ro HK, Wong MS, Favus MJ. Loss of parathyroid hormone-stimulated 1,25-dihydroxyvitamin D 3 production in aging does not involve protein kinase A or C pathways. J Bone Miner Res 1994;9:339-345. 209. Halloran BE Lonergan ET, Portale AA. Aging and renal responsiveness to parathyroid hormone in healthy men. J Clin Endocrinol Metab 1996;81:2192-2197. 210. Larkins RG, MacAuley SJ, Rapoport A, et al. Effects of nucleotides, hormones, ions, and 1,25-dihydroxycholecalciferon on 1,25-dihydroxycholecalciferol production in isolated chick renal tubules. Clin Sci Mol Med 1974;46:569-582. 211. Horiuchi N, Suda T, Takahashi H, Shimazawa E, Ogata E. In vivo evidence for the intermediary role of 3',5'-cyclic AMP in parathyroid hormone-induced stimulation of lalpha,25-dihydroxyvitamin D~ synthesis in rats. Endocrinology 1977;101:969-974. 212. Rost CR, Bikle DD, Kaplan RA. In vitro stimulation of 25hydroxycholecalciferol lalpha-hydroxylation by parathyroid

213.

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223.

224.

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226.

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hormone in chick kidney slices: Evidence for a role for adenosine 3',5'-monophosphate. Endocrinology 1981;108:1002-1006. Armbrecht HJ, Forte LR, Wongsurawat N, Zenser TV, Davis BB. Forskolin increases 1,25-dihydroxyvitamin D3 production by rat renal slices in vitro. Endocrinology 1984;114:644-649. Henry HL. Parathyroid hormone modulation of 25-hydroxyvitamin D~ metabolism by cultured chick kidney cells is mimicked and enhanced by forskolin. Endocrinology 1985;116:503-510. Shigematsu T, Horiuchi N, Ogura Y, Miyahara T, Suda T. Human parathyroid hormone inhibits renal 24-hydroxylase activity of 25hydroxyvitamin Ds by a mechanism involving adenosine 3',5'monophosphate in rats. Endocrinology 1986;118:1583-1589. Korkor AB, Gray RW, Henry HL, Kleinman JG, Blumenthal SS, Garancis JC. Evidence that stimulation of 1,25(OH)2D 3 production in primary cultures of mouse kidney cells by cyclic AMP requires new protein synthesis. JBone Miner Res 1987;2:517-524. Janulis M, Wong MS, Favus MJ. Structure-function requirements of parathyroid hormone for stimulation of 1,25dihydroxyvitamin D3 production by rat renal proximal tubules. Endocrinology 1993;133:713-719. Tanaka Y, DeLuca HE Rat renal 25-hydroxyvitamin D~ 1- and 24-hydroxylases: Their in vivo regulation. Am J Physiol 1984;246:E 168-E 173. Matsumoto T, Kawanobe Y, Ogata E. Regulation of 24,25dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 in a cloned monkey kidney cell line (JTC-12). Biochim Biophys Acta 1985;845:358-365. Shinki T, Jin CH, Nishimura A, et al. Parathyroid hormone inhibits 25-hydroxyvitamin D~-24-hydroxylase mRNA expression stimulated by 1 alpha,25-dihydroxyvitamin D3 in rat kidney but not in intestine. J Biol Chem 1992;267:13757-13762. Reinhardt TA, Horst RL. Parathyroid hormone down-regulates 1,25-dihydroxyvitamin D receptors (VDR) and VDR messenger ribonucleic acid in vitro and blocks homologous up-regulation of VDR in vivo. Endocrinology 1990;127:942-948. Yang W, Friedman PA, Kumar R, et al. Expression of 25(OH)Ds 24-hydroxylase in distal nephron: Coordinate regulation by 1,25(OH)zD 3 and cAMP or PTH. AmJPhysio11999;276:E793-E805. Goligorsky MS, Menton DN, Hruska KA. Parathyroid hormoneinduced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells. J Membr Biol 1986;92:151-162. Saussine C, Massfelder T, Parnin F, Judes C, Simeoni U, Helwig JJ. Renin stimulating properties of parathyroid hormone-related peptide in the isolated perfused rat kidney. Kidney Int 1993;44:764-773. Wang MS, Kurokawa K. Renal gluconeogenesis: Axial and internephron heterogeneity and the effect of parathyroid hormone. AmJPhysiol 1984;246:F59-F66. Chobanian MC, Hammerman MR. Parathyroid hormone stimulates ammoniagenesis in canine renal proximal tubular segments. A m J Physiol 1988;255:F847-F852. Watford M, Mapes RE. Hormonal and acid-base regulation of phosphoenolpyruvate carboxykinase mRNA levels in rat kidney. Arch Biochem Biophys 1990;282:399-403. Suzuki M, Morita T, Hanaoka K, Kawaguchi Y, Sakai O. A C1channel activated by parathyroid hormone in rabbit renal proximal tubule cells. J Clin Invest 1991;88:735-742. Philbrick WM, Wysolmerski JJ, Galbraith S, et al. Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol Rev 1996;76:127-173. Aya K, Tanaka H, Ichinose Y, Kobayashi M, Seino Y. Expression of parathyroid hormone-related peptide messenger ribonucleic acid in developing kidney. Kidney Int 1999;55:1696-1703. Yates AJ, Gutierrez GE, Smolens P, et al. Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium

RENAL ACTIONS OF P T H AND P T H r P

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homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents. J Clin Invest 1988;81:932-938. Pizurki L, Rizzoli R, MoseleyJ, Martin TJ, Caverzasio J, Bonjour JE Effect of synthetic tumoral PTH-related peptide on cAMP production and Na-dependent Pi transport. Am J Physiol 1988;255:F957-F961. Ellis AG, Adam WR, Martin TJ. Comparison of the effects of parathyroid hormone (PTH) and recombinant PTH-related protein on bicarbonate excretion by the isolated perfused rat kidney. JEndocrinol 1990;126:403-408. Soifer NE, Van Why SK, Ganz MB, Kashgarian M, Siegel NJ, Stewart AF. Expression of parathyroid hormone-related protein in the rat glomerulus and tubule during recovery from renal ischemia. J Clin Invest 1993;92:2850-2857. Garcia-Ocana A, Galbraith SC, Van Why SK, et al. Expression and role of parathyroid hormone-related protein in human renal proximal tubule cells during recovery from ATP depletion. J A m Soc Nephro11999;10:238-244. Massfelder T, Stewart AF, Endlich K, Soifer N, Judes C, Helwig JJ. Parathyroid hormone-related protein detection and interaction with NO and cyclic AMP in the renovascular system. Kidney Int 1996;50:1591-1603.

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237. Schor N, Ichikawa I, Brenner BM. Mechanisms of action of various hormones and vasoactive substances on glomerular ultrafiltration in the rat. Kidney Int 1981;20:442-451. 238. Nickols CA, Metz MA, Cline WH, Jr. Endothelium-independent linkage of parathyroid hormone receptors of rat vascular tissue with increased adenosine 3',5'-monophosphate and relaxation of vascular smooth muscle. Endocrinology 1986;119:349-356. 239. Musso MJ, Barthelmebs M, Imbs JL, Plante M, Bollack C, Helwig JJ. The vasodilator action of parathyroid hormone fragments on isolated perfused rat kidney. Naunyn-Schmiedeberg'sArch Pharmacol 1989;340:246-251. 240. HelwigJJ, Musso MJ,Judes C, Nickols GA. Parathyroid hormone and calcium: Interactions in the control of renin secretion in the isolated, nonfiltering rat kidney. Endocrinology 1991;129:1233-1242. 241. Simeoni U, Massfelder T, Saussine C, Judes C, GeisertJ, Helwig JJ. Involvement of nitric oxide in the vasodilatory response to parathyroid hormone-related peptide in the isolated rabbit kidney. Clin Sci 1994;86:245-249. 242. Endlich K, Massfelder T, Helwig JJ, Steinhausen M. Vascular effects of parathyroid hormone and parathyroid hormonerelated protein in the split hydronephrotic rat kidney. J Physiol 1995;483:481-490.

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CHAPTER 15

Endochondral

Bone Formation

Regulation by Parathyroid Hormone-Related Peptide, Indian Hedgehog, and Parathyroid Hormone GINO V. SEGRE AND KAECHOONG LEE Endocrine Unit, Massachusetts General Hospital, Department of Medicine, Harvard Medical School,

Boston, Massachusetts 02114

ENDOCHONDRAL

BONE DEVELOPMENT

Spatially, chondrocyte differentiation in the growth plate is oriented longitudinally (Fig. 1). The proliferating zone at the distal end of the developing bone consists of small, round chondrocytes that have a high

Development of the vertebrate skeleton is a highly regulated process that takes place by at least two distinct mechanisms: intramembranous and endochondral ossification. The axial and appendicular skeletons develop by endochondral ossification; membranous bones are largely restricted to the skull and parts of the mandible and clavicles. Membranous bones form directly from mesenchymal progenitors, whereas endochondral bones form after cartilaginous primordia are first generated. These primordia consist of aggregated undifferentiated mesenchymal cells, whose position, shape and size prefigure the future skeletal elements. Cells in the central cores of these condensates differentiate into chondrocytes, as evidenced by their synthesis and secretion of cartilage matrix proteins, such as type II collagen and specific proteoglycans, and the cartilage model is surrounded by a sheath of apparently poorly differentiated cells, the perichondrium. Proliferation of cells within the condensates and the perichondrium, and deposition of matrix proteins, account for the initial growth of these skeletal elements. Cells in the center mature into hypertrophic cells, a process characterized by cellular enlargement, the exit of these cells from the cell cycle, and the secretion and deposition of a distinct extracellular matrix that progressively calcifies. Generally cartilage differentiation and replacement by bone occur earlier in proximal bones of the appendicular skeleton than in distal bones, and they progress from cephalic to caudal portions of the axial skeleton. The Parathyroids, Second Edition

'roliferation -lypertrophy Vlineralization 31ood Vessel Invasion Primary Spongiosa

Secondary Spongiosa Bone Marrow (Hemopoietic cells)

FIG. 1 A schematic representation of the growth plate. Chondrocytes proliferate, mature, and organize into columns, and undergo hypertrophic differentiation. As the hypertrophic chondrocytes undergo programmed cell death, the cartilage matrix is invaded by blood vessels and is then resorbed by chondroclasts. The remnant longitudinal septae provide the scaffolding for osteoblasts to lay down bone matrix to form the primary spongiosa. Differentiation of growth plate chondrocytes drives the longitudinal growth of bone.

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capacity to divide. These cells mature and assume a columnar architecture, each column representing the expansion of a single clone. Examination of the distribution of bromodeoxyuridine (BrdU) in fetuses, after BrdU was given shortly before delivery to their mothers, first showed this clonal expansion (1). This has been confirmed by elegant lineage studies in which the distribution of [3-gal-expressing cells in the growth plate of chimeric mice was determined after introducing embryonic stem cells transfected with this gene into blastocysts from normal mice (2). The columnar appearance and clonal distribution are maintained in the hypertrophic zone, although the cells no longer divide. The matrix surrounding the maturest hypertrophic cells becomes mineralized, the cells undergo p r o g r a m m e d cell death, or apoptosis, and are replaced by bone. Although evidence suggests that hypertrophic cells may "transdifferentiate" into osteoblast-like cells, this must involve no more than a small minority of hypertrophic cells, if it occurs (3). The net result is lengthening of the bone, whereas the thickness of the growth plate remains relatively constant. With continued bone lengthening, proliferating chondrocytes and the growth plate become progressively restricted to the two ends of the skeletal element. The processes by which the cortices of axial and appendicular bones develop are called appositional ossification. Cortical bone formation in the shafts of endochondral bone initiates in perichondrial/ periosteal cells, directly apposed to hypertrophic cells. These cells differentiate into osteoblasts and secrete a matrix that undergoes calcification, giving rise to the "bone collar." Because appositional ossification is morphologically similar to intramembranous ossification, it

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Type X Collagen

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has been thought to develop by the same mechanisms. Studies show, however, that different factors, to be reviewed below, are involved at least in the initial stages of appositional ossification. Although chondrocyte differentiation can be seen to progress longitudinally, the growth plate is spatially organized into morphologically distinct horizontal z o n e s ~ z o n e s of proliferation, maturation, and h y p e r t r o p h y ~ e a c h of which is heterogeneous, as evidenced by the genes expressed in each zone. For example, parathyroid hormone-related protein (PTHrP) mRNA is most intensely expressed in subarticular chondrocytes. H4 histone mRNA, a proliferation marker that identifies cells at the S phase of their cell cycle, is expressed in these cells, but also in more centrally located chondrocytes that express PTHrP at much lower levels. P T H / P T H r P receptor mRNA and protein, although expressed at low levels in proliferating chondrocytes, are most intensely localized to cells at the interface of columnar and hypertrophic cells in prehypertrophic and early hypertrophic chondrocytes. Indian hedgehog (Ihh) mRNA expression partially overlaps that of the P T H / P T H r P receptor, but extends to more mature hypertrophic cells as well. Type II collagen is expressed throughout the growth plate, except in hypertrophic cells. As cells become hypertrophic, they abruptly lower or cease their expression of type II collagen. All hypertrophic cells, except the maturest, express types VI and X collagen. Terminally differentiated chondrocytes in and near the zone of mineralization, most of which are apoptotic, intensely express osteopontin and vascular endothelial growth factor (VEGF) (4-6) (Fig. 2). This "spatial synchronization" integrates the individual chondrocyte clones within the PTH/PTHrP Receptor

FIG. 2 Differentiation of growth plate chondrocytes. (A) Distribution of proliferation/differentiation markers in the growth plate (fetal mouse femur, embryonic day 15.5). Growth plate chondrocytes, except for hypertrophic cells, express type II collagen. When chondrocytes become hypertrophic, they express type X collagen and exit from the cell cycle, as evidenced by the disappearance of H4histone expression, a marker for the S phase of the cell cycle. Transcripts for PTH/PTHrP receptors are most highly expressed at the boundary between the proliferating and hypertrophic zones. H & E, Hematoxylin and eosin. (B) Distribution of PTHrP mRNA (a, mouse fetal hindlimb, embryonic day 16.5). PTHrP is expressed in the subarticular perichondrium/chondrocytes and in tendon insertions (b, arrowheads). [Modified from Ref. (5); Lee K, Lanske B, Karaplis AC, Deeds JD, Kohno H, Nissenson RA, Kronenberg HM, Segre GV. Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development. Endocrinology 137: 5109-5118; 1996. © The Endocrine Society.]

ENDOCHONDRALBONE FORMATION / growth plate, suggesting that chondrocyte differentiation is regulated by enzymes, signaling, and, perhaps, matrix molecules that are secreted by cells within highly defined regions in the growth plate. Work from Werb's laboratory provides an excellent example of a synchronized program by which local factors drive critical events in the process by which bone replaces cartilage. They found that homozygous mice with a null mutation in the matrix metalloproteinase-9 (MMP-9)/gelatinase B gene have lengthening of the hypertrophic zone and exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. Although hypertrophic chondrocytes develop normally, apoptosis, vascularization, and ossification are delayed, resulting in progressive overall lengthening of the growth plate. Transplantation of wild-type bone

marrow cells rescues vascularization and ossification in gelatinase B-null growth plates, indicating that these processes are mediated by cells of bone marrow origin that mature to become chondroclasts that secrete MMP-9/gelatinase B (7). In subsequent studies, Werb's group systemically administered a soluble VEGF receptor chimeric protein, Flt (1-3)-IgG, to young mice. This completely blocks blood vessel invasion of the cartilage, which concomitantly leads to expansion of the hypertrophic chondrocyte zone, impaired trabecular bone formation, and an overall shorter bone. Recruitment a n d / o r differentiation of chondroclasts and resorption of terminal chondrocyes decrease, although proliferation, differentiation, and maturation of chondrocytes are apparently normal (Fig. 3). VEGFmediated capillary invasion is an essential signal that

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FIG. 3 .MMP-9/gelatinase B and VEGF play crucial roles in the replacement of cartilage by bone. (A) Histologic analysis of the metatarsals of wild-type (upper panels) and MMP-9/gelatinase B ( - / - ) mice (lower panels) (sections of 3-week-old metatarsals). Magnified view (right lower panel) shows cortical bone surrounding the hypertrophic cartilage (hc) in MMP-9/gelatinase B ( - / - ) mice (boundaries are indicated by white arrowheads). Black arrowheads point to capillaries invading from the cortical bone; tb, trabecular bone. (B) Growth plates of MMP-9/gelatinase B ( - / - ) mice after bone marrow transplantation. Histologic sections of metatarsals from untransplanted and bone marrow (BM)-transplanted mice at 3 and 4 weeks after transplantation. Scale bar = 200 i~m. (C) Histologic sections of proximal tibia of mice treated for 2 weeks with control IgG (cont) (a,c) or a soluble VEGF receptor chimeric protein, mFIt(1-3)-IgG (mFIt) (hematoxylin and eosin staining) (b,d). hcz, Hypertrophic chondrocyte zone; stb, secondary trabecular bone; bm, bone marrow. Blood vessels are oriented parallel to the columns of hypertrophic zone in control mice, and fail to invade the hcz in mFIt-treated groups. Arrowheads indicate blood vessels. [Modified from Ref. (7), Cell 93; Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z; MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes, pp. 411-422. Copyright 1998, with permission from Elsevier Science; and from Gerber et aL (6), with permission.]

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regulates growth plate morphogenesis and triggers cartilage remodeling. VEGF, secreted by terminal hypertrophic chondrocytes, recruits endothelial cells and thus induces blood vessels. These blood vessels bring in not only nutrients, but also cells that become chondroclasts and osteoblasts. Because osteoblasts express Fltl, a VEGF receptor, and VEGF is chemotactic for osteoblasts in culture, it is reasonable to postulate an essential role for VEGF in recruiting osteoblasts to regions where terminal hypertrophic chondrocytes that were shown to express VEGF reside (6). Thus, VEGF is an essential coordinator of chondrocyte death, chondrocyte function, extracellular matrix remodeling, and angiogenesis, and it plays a vital role in the transition from cartilage to bone in the growth plate. Interestingly, mineralization of the matrix surrounding the maturest hypertrophic chondrocytes is unimpaired, indicating that cartilaginous mineralization is regulated by VEGF-independent mechanisms. Osteoblast precursors are attracted by VEGF secreted by hypertrophic cells, and after they mature into osteoblasts, they deposit bone matrix proteins, or osteoid, on the longitudinal septae, the vertical cartilaginous remnants left behind after apoptosis of the hypertrophic cells. This region of the primary ossification center is called the primary spongiosa. Resorption of the cartilaginous matrix by chondroclasts/osteoclasts continues, at least in part due to secretion by these cells of MMP-9/gelantinase B, and the bone matrix proteins elaborated by osteoblasts replace the cartilaginous matrix. When the longitudinal septae contain only bone matrix proteins from which all cartilaginous matrix has been removed, this area is called the secondary spongiosa. It consists of trabecular bone, which is remodeled by processes of bone resorption and subsequent formation throughout life to meet the demands of continued longitudinal growth, and in response to a variety of hormones, factors, and physical forces. The net result is that chondrocyte proliferation and hypertrophy largely drive longitudinal growth of endochondral bone. Interestingly, additional data suggest that at least postnatally, the activity of bone cells also may contribute to bone elongation (8). Many chondrodysplasias result from aberrant endochondral ossification. However, compared to chondrodysplasias caused by mutations in structural molecules, chondrodysplasias caused by mutations in regulatory molecules are relatively few (9). In this chapter, we concentrate on the roles of parathyroid hormone-related protein, parathyroid hormone, and Indian hedgehog, which by their dependent and independent actions play key roles in skeletal development. Our understanding of the roles these factors play in endochondral development has been greatly advanced by several novel approaches. These include determining the genetic basis of h u m a n chondrodysplasias and

deleting or overexpressing these genes in mice to study the development of skeletal abnormalities, and from studies conducted in vitro with fetal limbs from wildtype and mutant mice in which endochondral bone development can be manipulated. For those interested in roles of matrix molecules in endochondral bone development, excellent reviews (10,11) are available.

T H E A C T I O N S OF P T H r P A N D P T H

PTHrP was originally identified as a causative agent of hypercalcemia associated with malignancy (12). Both PTHrP and PTH bind to and activate the same receptor (13,14). PTH functions as a hormone to regulate the blood calcium concentration. PTHrP, however, functions mainly as a paracrine factor, although elevated circulating levels resulting from its synthesis and secretion by neoplastic tissue cause tumor-associated hypercalcemia. Its most well-defined physiologic roles are to regulate branching morphogenesis of the breast (15) and in endochondral bone formation. It is also thought to have more general developmental roles because its distribution and that of its cognate receptor are apposed across many epithelial-mesenchymal interfaces (5). Mutations in the P T H / P T H r P receptor cause two rare chondrodysplastic syndromes, thus providing insight into critical developmental roles played by this receptor. Jansen's metaphyseal chondrodysplasia is a rare form a short-limbed dwarfism characterized by hypercalcemia, normal or low circulating levels of PTH and PTHrP, and progressive metaphyseal changes, initially reminiscent of rickets or primary hyperparathyroidism (16). Elegant studies by Schipani et al. (17,18) linked this phenotype to mutant P T H / P T H r P receptors that were then showed to be constitutively active, when expressed in COS-7 cells. In these mutant receptors, histidine at position 223 is replaced by arginine, putatively at the border of the receptor's first intracellular loop and second transmembrane domain, or threonine at position 410 is replaced by proline in the receptor's sixth transmembrane helix. The phenotype of patients with Blomstrand chondrodysplasia, a lethal syndrome characterized by shortlimbed dwarfism, increased bone density, and remarkably advanced endochondral bone formation, is the mirror image of those in Jansen's chondrodysplasia (19). Blomstrand chondrodysplasia has been shown to be due to absence of functional P T H / P T H r P receptors. In their initial report, Jobert et al. (20) showed the syndrome to be due to a point mutation that leads to an l 1-amino acid deletion in the P T H / P T H r P receptor's fifth transmembrane domain. The same workers (21) and a second group (22) found the same phenotype to be caused by replacement of proline by leucine at position 132 of the receptor's amino-terminal extracellular

ENDOCHONDRAL BONE FORMATION / domain. Both mutations render the receptor unable to bind PTH or PTHrE The phenotypes of mice missing both copies of either the PTHrP or P T H / P T H r P receptor gene have many similarities, but phenotypic differences allow discrimination between characteristics due to absence of PTHrP from those due to failure to respond to either PTH or PTHrE Deletion of both copies of either gene leads to short-limbed dwarfism with an apparently accelerated differentiation of growth plate chondrocytes. Additionally, there is diminished polarity of hypertrophic chondrocytes, in which these cells are widely distributed throughout the cartilage (Fig. 4). This is particularly evident immediately before vascular invasion and the development of the primary ossification center of the developing bone (23-27). P T H / P T H r P receptor-deficient mice, however, exhibit two unique phenotypes not shared with the PTHrP-deficient mice. During appositional bone formation in the shafts of the long bones, only P T H / P T H r P receptor-deficient mice exhibit a dramatic increase in osteoblast n u m b e r and matrix accumulation (Fig. 5). Furthermore, the P T H / P T H r P receptor-deficient mice show a dramatic decrease in trabecular bone formation in the primary spongiosa

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and a delay in vascular invasion of the early cartilage model. The abnormal bone development is likely to result from the inability of the bone to respond to the actions of PTH, and is the expected opposite to that seen in clinical mild hyperparathyroidism. The delayed vascular invasion did not occur in PTHrP; P T H / P T H r P receptor doubly deficient mice, suggesting that PTHrP, perhaps through a distinct receptor or through its direct nuclear actions, slows vascular invasion (27). The role of PTHrP and its receptor in chondrocyte differentiation also has been explored in overexpression models: mice that express a transgene encoding either PTHrP or a constitutively active '~Jansen receptor" u n d e r the control of the rat collagen e~l (type II) promoter manifest an apparent delay of chondrocyte differentiation (28,29). Moreover, overexpression of the latter corrected at birth the growth plate abnormalities of the PTHrP-deficient mice (29). Treating murine limb in explant cultures with PTH or PTHrP mimics the apparent delay of chondrocyte differentiation (30). Cell-cell interactions in the developing endochondral bone have been elegantly addressed using chimeric mice that express various ratios of mutant and wild-type cells. In the growth plate of these

FIG. 4 Cartilage phenotype in PTHrP ( - / - ) and PTH/PTHrP receptor ( - / - ) mice. Hematoxylin and eosin staining (a-c) and type X collagen mRNA in situ hybridization (d-f) of wild-type (a, d), PTH/PTHrP receptor ( - / - ) (b, e), and PTHrP ( - / - ) (c, f) phalanges at embryonic day 16.5. There is a loss of polarity of the cartilage in both PTHrP ( - / - ) bone and PTH/PTHrP receptor ( - / - ) bones. In addition, PTH/PTHrP receptor ( - / - ) mice show a delay in chondrocytic differentiation. [Reprinted from Lanske et aL (27), with permission.]

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chimeric mice, chondrocytes missing the P T H / PTHrP receptor gene ectopically differentiate into hypertrophic cells and are surrounded by wild-type proliferating cells. Essentially all mutant chondrocytes became hypertrophic below a line approximately corresponding to the level at which the growth plate narrows, not at the level at which P T H / PTHrP receptor mRNA and protein are most intensely expressed (Fig. 6) (2). These ectopic cells express genes typical of hypertrophic chondrocytes; they also cease to proliferate and undergo apoptosis. Thus, the specific targets of PTHrP signaling are proliferative chondrocytes that express low levels of receptors. Differentiation of the PTH/PTHrPdeficient cells occurs independently of the surrounding normal proliferative cells and thus is cell autonomous. Interestingly, PTHrP protein localize most intensely to prehypertrophic cells that also express P T H / P T H r P receptors most abundantly (26). Cartilage mineralization, one of the final steps of chondrocyte differentiation, occurs ectopicially around hypertrophic cells at sites in the cartilage of chimeric mice with more than 60% contribution of mutant cells. Matrix mineralization, therefore, seems to require a critical mass of hypertrophic cells and thus is not cell autonomous.

FIG. 5 Increase in osteoblast number and cortical bone in PTH/PTHrP receptor ( - / - ) animals, von Kossa staining of methylmethacrylate sections at the level of the metaphyseal region of a tibia (a-c) and at the diaphyseal region of a phalanx (d-f)in wildtype (a, d), PTH/PTHrP receptor ( - / - ) (b, e), and PTHrP ( - / - ) (c, f) animals at embryonic day 18.5. PTH/PTHrP receptor mutant bones reveal an abnormal augmentation in osteoblast layers accompanied by an increased bone matrix (b, e) that does not mineralize, as demonstrated by the lack of von Kossa staining. In contrast, PTHrP ( - / - ) bones (c, f) look indistinguishable or somewhat advanced in terms of mineralization and replacement of cartilage by bone when compared with wild-type bones (a, d). [Reprinted from Lanske et aL, (27), with permission.]

THE ACTIONS OF INDIAN HEDGEHOG The hedgehog (hh) gene was first identified in

Drosophila as a segment polarity gene. Among the five reported vertebrate hh homologs, most attention has focused on Sonic hedgehog (Shh), which is absolutely critical to the patterning and development of many organs and organ systems in vertebrates. Shh is synthesized in cells in the notochord and controls development of the ventral neural tube (32-34). It also controls specification of sclerotomal cell fate in the somites (35), and the anteroposterior axis of the limb bud (36). It plays an important role in mediating epithelial-mesenchymal signaling in the gut (37), and in establishing left-fight asymmetry in the heart (38,39). The critical role of Shh in tissue patterning is made apparent by the multiple developmental defects in Shh-deficient mice; they die before birth and exhibit cyclopia, lack ventral cells in the neural tube, and have a degenerate notochord. They also lack a spinal column and most of the ribs and distal limb structures (40). Other members of the hedgehog protein family have also been implicated in tissue patterning: Desert hedgehog (Dhh) was shown to be essential for testes development, and zebra-fish-specific Echidna and Tiggywinkle hedgehog are involved in muscle cell specification and eye patterning, respectively (41-43).

ENDOClqOND~U~ BONE FORMATION /

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!i?iiiiiii ili:iiill iii! FI6. 6 Ectopic differentiation of PTH/PTHrP receptor ( - / - ) chondrocytes and elongation of chimeric growth plates. (A, B) Sections of the tibiae from embryonic day 17.5 wild-type embryos (A) and embryonic day 17.5 wild-type embryos containing cells with one 13-galactosidase transgene (stained dark) and cells with no transgene (B) were stained for 13-galactosidase activity and counterstained with nuclear fast red. Cells with and without the transgene behave indistinguishably, p, Proliferating layer; h, hypertrophic layer, including prehypertrophic chondrocytes. (C, D) Sections of the tibiae from embryonic day 17.5 chimeric embryos containing both wild-type and PTH/PTHrP receptor ( - / - ) cells were stained for 13-galactosidase activity and counterstained with nuclear fast red. PTH/PTHrP receptor ( - / - ) cells (stained dark) ectopically differentiate into hypertrophic-like cells in the middle of wild-type proliferating chondrocytes. All mutant cells below a line approximately corresponding to the level at which the growth plate narrows (arrowhead) appear hypertrophic. (E-H) Sections of the tibiae from wild-type (E) and chimeric embryos with various contributions of PTH/PTHrP receptor ( - / - ) cells (F-H) were stained for 13-galactosidase activity and counterstained with hematoxylin and eosin. Contributions of mutant cells are estimated 10% (F), 30% (G), and 60% (H). In proportion to the contribution of mutant cells, columns of wild-type proliferating chondrocytes elongate (two-headed arrows). Bar = 100 i~m. [From Chung et aL (2), Proc Natl Acad Sci USA 1998;95:13030-13035. Copyright 1998 National Academy of Sciences, U.S.A.]

The first clue to the functions of Indian hedgehog in endochondral bone development came from the work of Bitgood and McMahon (44), who localized expression of its gene to growth plate chondrocytes as well to the hindgut, tooth, hair, whiskers, vas deferens, urethra, and lung. Vortkamp et al. (30) then showed intense Ihh expression in a discrete portion of the growth plate in the chick. Cells expressing Ihh partially overlap with those that express P T H / P T H r P receptors, but expression also extends to early hypertrophic cells more

toward the center of the developing bone (Fig. 7). When Ihh is misexpressed in the developing chick using a replication-competent retroviral vector, the infected cartilage elements become broader and shorter and lack hypertrophic chondrocytes. Thus, Ihh misexpression results in a phenotype similar to that of mice overexpressing PTHrP under control of the collagen loL(II) promoter (28). The similar sites at which P T H / P T H r P receptor and Ihh are expressed, together with the effects of

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FIG. 7 Expression of Indian hedgehog (Ihh) and PTH/PTHrP receptors in developing endochondral bone (distal end of tibia, embryonic day 16.5). The expression domain of PTH/PTHrP receptor mRNA expression is different from that of Ihh, but the two domains overlap. [Modified with permission from Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613-622. Copyright 1996 American Association for the Advancement of Science.]

overexpression of Ihh, led to the hypothesis that Ihh and PTHrP might interact. Initial studies in fact showed that PTHrP expression in chick limbs was greatly increased by misexpressed Ihh. Functional relationships between PTHrP and Ihh were further explored in a model system in which fetal murine limbs are grown in explant culture u n d e r serum-free conditions. When PTHrP(1-34) or PTH(1-34) is added to the cultured normal limbs, formation of hypertrophic cells is completely suppressed, as evidenced by histologic examination and by the disappearance of cells that express type X collagen. As expected, addition of either peptide to limbs of PTHrP-deficient mice completely rescues the abnormal phenotype (30), but it does not affect limbs from P T H / P T H r P receptor-deficient mice (25). When the effect of synthetic Shh protein on endochondral bone development was tested (Ihh protein was not available, but its activities are indistinguishable from those of Shh), it did not affect limbs from PTHrPdeficient mice, whereas it repressed hypertrophic differentiation in limbs from normal mice (Fig. 8). Thus, PTHrP is required for the response to Shh to occur (30). Similar experiments were conducted with limbs of P T H / P T H r P receptor-deficient mice; neither PTHrP nor Shh was effective (25). Most importantly, both Ihh misexpression in chicks and Shh treatment of normal murine limbs markedly increased PTHrP expression in perichondrial/subarticular chondrocytes (Fig. 9). The receptor for h e d g e h o g proteins is a cell surface heterodimer, consisting of patched (ptc), which binds hedgehog, and s m o o t h e n e d (smo), which conducts the signal to inside the cell. In the unliganded state, ptc and smo are noncovalently linked and smo is inactive.

On binding hedgehog, smo is thought to dissociate from ptc, and smo then becomes constitutively active (45,46). By binding hedgehog, ptc also limits hedgehog diffusion and thus its range of direct actions. A transcription factor, gli-1, is activated by h e d g e h o g signaling. Both ptc and gli-1 are transcriptional targets of hedgehog. Therefore, high expression of their genes identifies cells that have responded to hedgehog (47,48). In the bones of normal mice fetuses at 13.5 days postcoitus (dpc), ptc and gli-1 are most intensely expressed in perichondrial cells next to hypertrophic cells that express Ihh. Later in fetal development, when the growth plate is more highly organized into discrete zones and the primary ossification center is formed, ptc continues to be expressed in perichondrial/subarticular cells, but it is now also expressed in the growth plate cartilage in a gradient; it is most intensely expressed in chondrocytes immediately distal to the site where Ihh is expressed, progressively less expressed in less mature chondrocytes, and is barely detectable in subarticular chondrocytes that most intensively express PTHrE Interestingly, it is also highly expressed in cells that reside in the primary spongiosa, at some distance from any obvious site of Ihh. Nonetheless, the high level of ptc expression in these cells raises the likelihood that these bone cells also are h e d g e h o g targets (Fig. 10) (2). Whether Ihh protein is stored in more mature hypertrophic chondrocytes where it is not expressed is currently not known. The lack of ptc expression in subarticular cells that express PTHrP and the relatively long distance between PTHrP-expressJng cells and hypertrophic cells that express Ihh make it very unlikely that subarticular cells are direct ta~rgets of Ihh.

ENI~OCI-IONI~RALBONE FORMATION /

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The mechanism by which Ihh regulates PTHrP expression has yet to be elucidated. Many of the roles of Ihh in skeletal development become clear from the phenotype of Ihh-deficient mice (49). Importantly, Ihh-deficient mice do not phenocopy either PTHrP- or P T H / P T H r P receptor-deficient mice. Therefore, a detailed comparative examination distinguishes which of the activities of Ihh are PTHrP d e p e n d e n t from those that are PTHrP independent. About half of the Ihh-deficient mice die between 10.5 and 12.5 days postcoitus. This is due most probably to circulatory anomalies in the yolk sac, which develops abnormally in Ihh-deficient mice and is a site where Ihh is normally expressed. Some mice die later in gestation, and the remainder die at birth. The perinatal lethality is likely due to extraordinary shortening of the ribs, which severely impairs respiration. The development of cartilage primordia is not affected, consistent with the lack of expression of Ihh in early cartilage condensates of normal mice. Although

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FIG. 8 Sonic hedgehog (Shh) requires PTHrP to block chondrocytic differentiation. Embryonic day 16.5 mouse hindlimbs from wild-type and PTHrP ( - / - ) mice were cultured for 4 days in control, PTHrP-, or Shhcontaining medium. (A) Col-X expression is repressed after PTHrP and Shh treatment in wild-type hindlimbs. In situ hybridization with a Col-X probe to sections of tibias from the control animals shows repression of the hypertrophic cartilage marker Col-X after PTHrP or Shh treatment, relative to untreated limbs. (B, C) PTHrP rescues the PTHrP ( - / - ) phenotype, whereas Shh has no effect on the cartilage. In PTHrR ( - / - ) animals the hypertrophy of the cartilage is advanced in all the bones, including the tibia (B) and the digits (C), as shown by Col-X expression (B) and hematoxylin and eosin staining (C). Treatment of the cultures with PTHrP not only rescues the wild-type phenotype but also induces the same repression of hypertrophic cartilage as observed in PTHrP treatment of normal limbs: CoI-X expression is repressed (B), and no hypertrophic chondrocytes form during culture (C). In contrast to the rescue of the PTHrP ( - / - ) phenotype by PTHrP, Shh treatment does not change the phenotype of the cartilage elements: Col-X expression is still advanced (B), and hematoxylin and eosin staining shows the premature hypertrophic cartilage (C). [Reprinted with permission from Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613-622. Copyright 1996 American Association for the Advancement of Science.]

the digits of Ihh-deficient mice do not appropriately segment, skeletal elements are for the most part present in the correct positions and numbers, thus demonstrafing that Ihh is not required for skeletal patterning. The most overt abnormality is failure of limb growth that is visible by 13.5 dpc, and which is so progressive that by birth forelimbs are only one-third as long as those of wild-type mice (Fig. 11). At birth, in addition to severe dwarfism, the Ihh-deficient mice show other stigmata of abnormal endochondral bone developmentmforeshortened snout and mandible and rounded skull. Most endochondral bones also are misshapen, whereas membranous bones are much less affected. BrdU labeling shows markedly reduced chondrocyte proliferation. The absence of ptc and gli-1 expression at any stage in the mutant skeleton confirms that Ihh signaling is required to induce expression of the genes. The high level of ptc expression in proliferating chondrocytes at sites adjacent to those of Ihh expression at all developmental stages in normal mice

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FIG. 9 Shh up-regulates PTHrP expression. Hindlimbs from wild-type mice (embryonic day 16.5) were cultured with control or Shh-containing medium for 4 days. PTHrP expression was increased in the subarticular perichondrium in Shh-treated hindlimbs. [Reprinted with permission from Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613-622. Copyright 1996 American Association for the Advancement of Science.]

suggests that proliferating chondrocytes are likely to be direct targets of Ihh signaling. Chondrocyte maturation also is abnormal in Ihh-deficient mice. Hypertrophic cells appear later than in normal mice and are fewer in number, smaller in size, and not well organized. There is an admixture of cells expressing type II and X collagens, similar to that seen in PTHrP- and PTH/PTHrP-receptor-deficient mice. Cartilaginous mineralization takes place in the Ihh-deficient mice, which because of the lack of proliferating cells and the

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presence of hypertrophic chondrocytes in regions where chondrocytes usually remain undifferentiated, extends closer to the articular ends of the bones than in normal mice. Vascular invasion occurs, although it is delayed and never extensive, but neither histologically identifiable bone nor mineralized bone collars develop. Importantly, there is no detectable expression of PTHrP anywhere in the developing bone, and PTH/PTHrP receptor expression, although present in the mutant chondrocytes, is absent from the perichon-

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drium/periosteum. Thus, it appears that Ihh is required for maintenance of PTHrP signaling and therefore Ihh indirectly regulates chondrocyte differentiation by controlling PTHrE The role of Ihh in skeletal development was further examined by assessing expression of transcripts typically associated with bone. In contrast to normal mice, OSF2/Cbfal, a transcriptional activator of bonespecific target genes (50) whose expression is essential for formation of osteoblasts (51,52), P T H / P T H r P receptors, and BMP3 are not expressed in the perichondrial/periosteal region of mutant mice, although the former two are expressed in chondrocytes. The absence of bone was further confirmed by the lack of expression of osteocalcin, currently considered the most specific marker of mature osteoblasts, in any endochondral bone in the mutant appendicular or axial skeleton. Interestingly, abundant osteocalcin expression takes place in mutant bones formed entirely or partially by intramembranous ossification, such as the flat bones of the skull, the mandible, and the clavicle. This indicates that the absence of Ihh signaling

255

affects osteoblast development only in endochondral, not membranous, bones, and provides clear evidence that the developmental programs directing intramembranous and appositional ossification differ. Further insights into the respective roles of PTHrP and Ihh in endochondral ossification and morphogenesis come from examining embryos generated by crosses between mice carrying various combinations of three alleles, Ihh- and PTHrP-null alleles and a transgene in which the constitutively active P T H / P T H r P receptor is expressed under control of the collagen oL1(II) promoter (53). At 18.5 dpc, the limb skeletons of I h h / P T H r P compound mutants are identical to Ihhnull mutants, suggesting that Ihh is necessary for PTHrP function. Although expression of the constitutively active P T H / P T H r P receptor in Ihh-null mice prevents premature chondrocyte hypertrophy, it does not rescue either the short-limbed dwarfism or decreased chondrocyte proliferation (Fig. 12). As expected, expression of the constitutively active P T H / P T H r P receptor rescues the phenotype of PTHrP-null mice. These experiments confirm that molecular mechanisms controlling chondrocyte differentiation are distinct from those that drive proliferation. Ihh up-regulates PTHrP, which then prevents chondrocyte hypertrophy and maintains a population of cells competent to proliferate. In contrast, Ihh promotes chondrocyte proliferation via pathways that are independent of PTHrP and which, in the absence of Ihh, cannot be rescued by PTHrE The pivotal roles of Ihh on chondrocyte proliferation and osteogenesis are confirmed and extended by comparing the skeletal phenotypes of chimeric mice, resulting from experiments in which embryonic stem cells missing the P T H / P T H r P receptor gene alone or both the P T H / P T H r P receptor and Ihh genes were introduced into wild-type blastocysts (31,54). As mentioned above, chondrocytes deficient in P T H / P T H r P receptors hypertrophy at an ectopic location relatively near the articular end of the developing bone in these chimeric mice. These hypertrophic cells express Ihh, which induces expression of ptc in neighboring cells and up-regulates the expression of PTHrP in subarticular cells. Interestingly, higher degrees of chimerism result in proportional elongation of the wild-type proliferating chondrocytes. This elongation is not seen in chimeric animals, when doubly deficient, P T H / P T H r P receptor Ihh-null embryonic stem cells were introduced into wild-type blastocysts. Although doubly deficient chondrocytes hypertrophy ectopically in these mice, hypertrophy is not associated with either expression of ptc in adjacent cells or up-regulation of PTHrP in subarticular cells. Cartilage mineralization in areas of ectopic hypertrophic cells takes place when the chimerism is high, however, confirming that this process is not cell autonomous and is regulated by

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Fill. 12 Effects of manipulating PTHrP signaling in Ihh mutants. (A, F) Wild type; (B, G) PTHrP ( - / - ) ; (C, H) Ihh ( - / - ) ; (D, I)Ihh (-/-);PTHrP ( - / - ) ; (E, J)Ihh (-/-);PTHrPR* [PTHrPR*-constitutively active PTH/PTHrP receptor expression is driven by a collagen e~l (11)promoter]. (A-E) 18.5-dpc skeletal preparations taken at the same magnification, and (F-J) corresponding histology taken at the same magnification. Loss of PTHrP in Ihh ( - / - ) elements results in limbs that are identical by skeletal staining (C, D) and histology (H, I). Activation of PTHrP signaling in Ihh ( - / - ) using PTHrPR* significantly decreases red staining, which is entirely absent in the tibia (arrows in C and E). This is confirmed by histology (H, J), which demonstrates reduced chondrocyte hypertrophy. Arrows mark the regions of nonhypertrophic chondrocytes. [From Karp et aL (53), with permission from the Company of Biologists Ltd.]

neither PTHrP nor Ihh. Dams pregnant with chimeric fetuses in which PTH/PTHrP receptor-deficient embryonic stem cells were injected into the blastocyst were given BrdU at 17.5 dpc. The percentage of BrdUlabeled cells in growth plate chondrocytes differed little, if at all, from those in wild-type mice (U. Chung, personal communication, 2000). Thus, elongation of the wild-type proliferative zone in mice with a high degree of chimerism with PTH/PTHrP receptor-deficient cells appears to be due to Ihh-induced increases in PTHrP, which then inhibits the exit of proliferative cells to postmitotic hypertrophic chondrocytes, rather than to Ihh-induced chondrocyte proliferation. Some direct contribution of Ihh cannot, however, be excluded. Comparing osteogenesis in the same two chimeric mice models confirms the unique role of Ihh in osteogenesis suggested by the phenotype of Ihh-deficient mice. Whereas PTH/PTHrP receptor-deficient hypertrophic chondrocytes near the perichondium/periosteum induce an ectopic bone collar, I h h / P T H / P T H r P receptor doubly deficient hypertrophic chondrocytes do not.

Other lines of investigation support a major osteogenic role for Ihh. Hedgehog proteins have been shown to stimulate alkaline phosphatase in mesenchymal cells, primary osteoblasts, and osteogenic cell lines (55-57), and chicken fibroblasts engineered to overexpress Shh induce ectopic bone formation (55) when injected into nude mice. Hedgehog signaling also is modulated by a membrane-anchored glycoprotein, Hip (hedgehog interacting protein). Hip interacts with the amino-terminal portion of all three mammalian hedgehogs with an affinity similar to that of patched. Like ptc, hip is expressed next to cells that express hh and is a transcription target of hedgehog signaling. Cartilagespecific overexpression of hip phenocopies Ihhdeficient mouse, except that the bone collar forms normally in the periosteum where hip is not expressed, thus confirming the unique role played by Ihh in apposition ossification (31). Excessive signaling via the hedgehog pathway is responsible for some human disease. Many patients with nevoid basal cell carcinoma syndrome have ptc haploinsufficency (58,59). This syndrome is character-

ENDOCHONDRAL BONE FORMATION / ized by a high incidence of basal cell carcinomas and other cancers, and, of particular importance to this discussion, by skeletal overgrowth (60). The syndrome can be essentially phenocopied in mice by overexpressing Shh (61). Thus, perhaps not surprisingly, Shh is oncogenic and the gene for ptc functions as a tumor suppressor gene.

C U R R E N T M O D E L OF E N D O C H O N D R A L BONE DEVELOPMENT Knowledge acquired over the past few years concerning the actions of Ihh, PTHrP, and PTH make it possible to construct a far more detailed model than the one we proposed for regulation of cartilage differentiation by PTHrP and Ihh in 1996 (30). One model that takes all of the current data into account is as follows. In growth plate cartilage, Ihh up-regulates expression of PTHrP, whose major site of synthesis is in perichondrial/subarticular chondrocytes, and Ihh independently promotes chondrocyte proliferation. The proliferative actions of Ihh are likely to be direct, as evidenced by high expression of ptc and gli-1 in cells immediately adjacent to the hypertrophic chondrocytes that express Ihh. It is important to note that indirect actions of Ihh on chondrocyte proliferation via other signaling molecules cannot be excluded. The lack of ptc expression in subarticular cells and the relatively long distance between Ihh- and PTHrP-expressing cells make it highly likely that Ihh regulates PTHrP indirectly. The signaling molecules downstream of Ihh that control this action have not been defined. PTHrP determines the position relative to the articular ends of the developing bone at which chondrocytes exit the cell cycle and initiate the hypertrophic chondrocyte differentiation program. This would be consistent with PTHrP acting as a patterning molecule, or morphogen, and the periarticular perichondrium/subarticular chondrocytes as an organizing center. In this model, the periarticular perichondrium/subarticular chondrocytes are the major source of PTHrP, which then diffuses centrally through the growth plate, in which a PTHrP gradient is established. High concentrations of PTHrP inhibit hypertrophy, but a threshold exists below which PTHrP no longer prevents the cells from initiating the hypertrophic program. The distance from the articular end at which this threshold is attained depends on the level of PTHrP gene transcription. The high level of P T H / P T H r P receptors expressed in prehypertrophic cells probably serve as a sink to prevent further diffusion of PTHrP, and thus a sharp transition is established whereby PTHrP concentrations fall abruptly to below threshold levels. That prehypertrophic cells act as a sink for PTHrP is supported by the finding that most of the cell-bound PTHrP protein is

257

detected on these cells. This is consistent with the finding that the PTHrP acts mainly on cells that are relatively close to the articular end, rather than on prehypertrophic chondrocytes. It also predicts that chondrocytes undergo hypertrophy closer to the articular end in the absence of PTHrP, without necessarily affecting the rate of chondrocyte differentiation. Initiation of the hypertrophic chondrocyte program includes the expression of Ihh, but it is not dependent on Ihh. Because PTHrP has no or very small effects on chondrocyte proliferation in vivo, the absence of PTHrP results in a smaller population of proliferating cells, with hypertrophy and ossification displaced closer to the articular ends of the developing bone. This model is also consistent with the effects of PTHrP overexpression in the mouse where the PTHrP concentration is sufficiently high to markedly inhibit hypertrophy. Ihh-induced proliferation lengthens the distance between hypertrophic and subarticular cells, thus resulting in lowered PTHrP expression. The PTHrP gradient is altered, moving the threshold level needed to prevent proliferating cells from exiting the cell cycle closer to the articular end. The polarity of growth plate chondrocytes, namely, the columnar organization and orderly progression of chondrocytes from type II collagen-expressing cells to type X collagen-expressing cells, appears to be mainly controlled by PTHrE PTHrP-independent actions of Ihh on this process, however, cannot be excluded. Although hypertrophic chondrocytes undergo a defined differentiation program, the controlling factors, including those leading to apoptosis, are not well understood. Cartilage mineralization, however, occurs non-cell autonomously at sites where a sufficient number of late hypertrophic chondrocytes collect. These most mature hypertrophic chondrocytes control the process by which bone replaces cartilage. They express VEGF, attracting capillaries that bring chondroclast and osteoblast precursors to the region of dying chondrocytes. Chondroclasts, in part by secreting proteases, including MMP-9/gelatinase B, into their local environment and by phagocytosis, dispose of cell debris and cartilaginous matrix. Osteoblasts, attracted both by VEGF and perhaps also by matrix proteins, then lay down osteoid on the remnants of cartilaginous matrix, which, after further resorption and remodeling, form the trabeculae of the secondary spongiosa. Consideration of factors that mediate vascular invasion are beyond the scope of this chapter, other than to point out that PTHrP, by mechanisms apparently independent of the P T H / P T H r P receptor, is critical. Normal appositional ossification requires both PTH and Ihh that is secreted by hypertrophic chondrocytes abutting the perichondrium/periosteum, but neither alone is sufficient. Osteoblasts do not develop in the perichondrium/periosteum in the absence of Ihh,

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which obviously accounts for the lack of cortical bone matrix and mineral deposition. The periosteum of P T H / P T H r P receptor-deficient mice is dramatically different; it contains an excess of osteoblasts, and a b u n d a n t matrix that, however, does not mineralize. PTH is required to form a mineralized bone collar, but whether this is a due to direct or indirect mechanisms is currently not clear. The absence of bone cells in the core of skeletal elements in Ihh-deficient mice and the high level of ptc expression in the primary spongiosa of normal mice provide very strong evidence for an essential role for Ihh in the development of trabecular bone. The evidence, however, is not yet absolutely conclusive. A plausible counter a r g u m e n t is that the anomalous cartilage development, minimal vascular invasion, a n d / o r the early lethality of Ihh-deficient mice preclude the development of a primary spongiosa. The source of Ihh that potentially drives osteogenesis also is not clear; hypertrophic chondrocytes are the most plausible source, although osteoblastic cells, or other cells b r o u g h t in with vascularization, also may be sources. Experiments to resolve this issue definitively might be to eliminate Ihh expression selectively in hypertrophic chondrocytes or osteoblasts, or to block the putative actions of Ihh in osteoblasts by selectively overexpressing hip in these cells.

REFERENCES 1. Farnum CE, Wilsman NJ. Determination of proliferative characteristics of growth plate chondrocytes by labelling with bromodeoxyuridine. Calcif Tissue Int 1993;52:110-119. 2. Chung U, Lanske B, Lee K, Li E, Kronenberg H. The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation. Proc Natl Acad Sci USA 1998;95:13030-13035. 3. Gentili C, Bianco P, Neri M, Malpedi M, Campanile G, Castagnola P, Cancedda R, Cancedda FD. Cell proliferation, extracellular matrix mineralization and ovotransferrin transient expression during in vitro differentiation of chick hypertrophic chondrocytes into osteoblast cells. J Cell Bio11993;122:703-712. 4. Lee K, Deeds JD, Chiba S, Un-no M, Bond AT, Segre GV. Parathyroid hormone induces c-fos expression in bone cells in vivo: In situ localization of its receptor and c-fos messenger ribonucleic acids. Endocrinology 1994;134:441-450. 5. Lee K, DeedsJD, Segre GV. Expression of parathyroid hormonerelated peptide and its messenger ribonucleic acids during fetal development of rats. Endocrinology 1995;136:453-463. 6. Gerber H-P, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat. Med 1999;5:623-628. 7. Vu TH, ShipleyJM, Bergers G, BergerJE, HelmsJA, Hanahan D, Shapiro SD, Senior RM, Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998;93:411-422. 8. Ducy P, Starbuck M, Priemel M, Shen J, Pinero G, Geoffroy V, Amling M, Karsenty G. A Cbfal-dependent genetic pathway con-

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ENDOCHONDRAL BONE FORMATION 25. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LHK, Ho C, Mulligan RC, Abou-Samra A-B, Jfippner H, Segre GV, Kronenberg HM. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996;273:663-666. 26. Lee K, Lanske B, Karaplis AC, Deeds JD, Kohno H, Nissenson RA, Kronenberg HM, Segre GV. Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development. Endocrinology 1996;137:5109-5118. 27. Lanske B, Amling M, Neff L, Guiducci J, Baron R, Kronenberg HM. Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development. J Clin Invest 1999; 104:399-407. 28. Weir EC, Philbrick WM, Amling M, Neff LA, Baron R, Broadus AE. Targeted overexpression of parathyroid hormonerelated peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Proc Natl Acad Sci USA 1996;93:10240-10245. 29. Schipani E, Lanske B, Hunzelman J, Luz A, Kovacs CS, Lee K, Pirro A, Kronenberg HM, Jfippner H. Targeted expression of constitutively active receptor for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormonerelated peptide. Proc Natl Acad Sci USA 1997;94:13689-13694. 30. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613-622. 31. Chuang P-T, McMahon AP. Vertebrate hedgehog signalling modulated by induction of a hedgehog-binding protein. Nature 1999;397:617-621. 32. Echelard Y, Epstein DJ, St-Jacques B, Shen L, MohlerJ, McMahon JA, McMahon AP. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 1993;75:1417-1430. 33. Krauss S, Concordet J-P, Ingham PW. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebraf'lsh embryos. Cell 1993; 75:1431-1444. 34. Roelink H, Augsburger A, Heemskerk J, Korzh V, Norlin S, Ruiz Altaba A, Tanabe Y, Placzek M, Edlund T, Jessell TM. Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell 1994;76:761-775. 35. Johnson RL, Laufer E, Riddle RD, Tabin C. Ectopic expression of Sonic hedgehog alters dorsal-ventral patterning of somites. Cell 1994;79:1165-1173. 36. Riddle RD, Johnson RL, Laufer E, Tabin C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 1993; 75:1401-1416. 37. Roberts DJ, Johnson RL, Burke AC, Nelson CE, Morgan BA, Tabin C. Sonic hedgehog is an endodermal signal inducing Bmp4 and Hox genes during induction and regionalization of the chick hindgut. Development 1995; 121:3163-3174. 38. Levin M, Johnson RL, Stern CD, Kuehn M, Tabin C. A molecular pathway determining left-right asymmetry in chick embryogenesis. Cell 1995;82:803-814. 39. Pag~in-Westphal SM, Tabin CJ. The transfer of left-right positional information during chick embryogenesis. Cell 1998;93:25-35. 40. Chiang C, LitingtungY, Lee E, Young KE, CordenJL, Westphal H, Beachy PA Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 1996;383:407-413. 41. Bitgood MJ, Shen L, McMahon AP. Sertoli cell signaling by Desert hedgehog regulates the male germline. Curr Biol 1996;6:298-304. 42. Currie PD, Ingham PW. Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish. Nature 1996;382:452-455.

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43. Ekker SC, Ungar AR, Greenstein P, von Kessler DE Porter JA, Moon RT, Beachy PA. Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr Biol 1995;5: 944-955. 44. Bitgood MJ, McMahon AP. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev Bio11995;172:126-138. 45. Stone DM, Hynes M, Armanini M, Swanson TA, Gu Q, Johnson RL, Scott MP, Pennica D, Goddard A, Phillips H, Noll M, Hooper JE, de Sauvage F, Rosenthal A. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 1996;384:129-134. 46. Marigo V, Davey RA, Zuo Y, Cunningham JM, Tabin CJ. Biochemical evidence that patched is the Hedgehog receptor. Nature 1996;384:176-179. 47. Goodrich LV,Johnson RL, Milenkovic L, McMahon JA, Scott ME Conservation of the hedgehog/patched signaling pathway from flies to mice: Induction of a mouse patched gene by hedgehog. Genes Dev 1996;10:310-312. 48. Marigo V, Scott ME Johnson RL, Goodrich LV, Tabin CJ. Conservation in hedgehog signaling: Induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development 1996; 122:1225-1233. 49. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1999;13:2072-2086. 50. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbal: A transcriptional activator of osteoblast differentiation. Cell 1997;89:747-754. 51. Komori T, Yagi H, Monura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao Y, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfal results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997;89:755-764. 52. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfal, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997;89:765-771. 53. Karp SJ, Schipani E, St-Jacques B, Hunzelman J, Kronenberg H, McMahon AP. Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone-related protein-dependent and -independent pathways. Development 2000;127:543-548. 54. Chung U, Wei W, Schipani E, McMahon AP, Kronenberg HM. Indian Hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J Bone Miner Res 2000;15:$192. 55. Kinto N, Iwamoto M, Enomoto-Iwamoto M, Noji S, Ohuchi H, Yoshioka H, Kataoka H, Wada Y, Gao Y, Takahashi HE, Yoshiki S, Yamaguchi A. Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation. FEBS Lett 1997;404:319-323. 56. Nakamura T, Aikawa M, Iwamoto-Enomata M, Iwamoto M, Higuchi Y, Pacifici M, Kinto N, Yamguchi A, Noji S, Kurisu K, Matsya T, Maurizio E Induction of osteogenic differentiation by hedgehog proteins. Biochem Biophys Res Commun 1997;237: 465-469. 57. Jemtland R, Divieti P, Lee K, Segre GV. Recombinant sonic hedgehog promotes differentiation of primary mouse calvarial osteoblasts and increases PTHrP mRNA expression. J Bone Miner Res 1999;14:$293. 58. Hahn H, Wicking C, Zaphiropoulous PG, Gailani MR, Shanley S, Chidambaram A, Vorechovsky I, Holmberg E, Unden AB, Gillies S, Negus K, Smyth I, Pressman C, Leffell DJ, Gerrard B, Goldstein AM, Dean M, Toftgard R, Chenevix-Trench G, Wainwright B,

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Bale AE. Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 1996;85: 841-851. 59. Johnson RL, Rothman AL, Xie J, Goodrich LV, Bare JW, Bonifas JM, Quinn AG, Myers RM, Cox DR, Epstein EH, Jr, Scott ME Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 1996;272:1668-1171.

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CHAPTER16 Physiologic Actions of P T H and PTHrP IV. Vascular, Cardiovascular, and Neurologic Actions

THOMAS L. CLEMENS Division of Endocrinology and Metabolism, University of Cincinnati Collegeof Medicine, Cincinnati, Ohio 45267 ARTHUR E. BROADUS Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510

VASCULAR A N D C A R D I O V A S C U I A R ACTIONS

through different mechanisms. Finally, PTH exerts both ionotropic and chronotropic effects on the heart (6). Although the cardiovascular effects of PTH are undisputed, their physiologic significance has been frequently debated. This is in part because the concentrations of PTH required to produce vasodilation (10-100 nM) are substantially above those that normally circulate (low picomolar). Consequently, it has been difficult to conceptualize how physiologic levels of this systemic hormone, which is synthesized only in the parathyroid gland, could function in the local control of vascular tone. Also enigmatic is the fact that patients with primary hyperparathyroidism and elevated circulating PTH levels often have high (not low) blood pressure that sometimes returns to normal after parathyroidectomy. A plausible explanation for the seemingly enigmatic regulatory effects of PTH on the cardiovascular system emerged with the discovery of PTH-related protein (PTHrP) in 1987. As discussed in Chapter 13 of this volume, PTHrP was identified as the factor responsible for the paraneoplastic syndrome, humoral hypercalcemia of malignancy. Almost immediately after its cloning, expression of PTHrP was detected in many normal fetal and adult tissues but was undetectable in the circulation, suggesting that the protein functioned in an autocrine/paracrine mode. Although many functions have been ascribed for PTHrP, three main physiologic themes have emerged. Observations in gene knockout mice have demonstrated that PTHrP is required for development of cartilage, for morphogenesis of the

Historical Perspectives The origins of PTH as a putative cardiovascular regulatory factor date to the early 1900s, when the calcemic properties of the hormone were first identified. In classic studies, Collip and Clark (1) demonstrated that systemic injection into dogs of extracts of parathyroid glands lowered systemic blood pressure (Fig. 1). The first formal characterization of the cardiovascular activity of parathyroid hormone (PTH) was conducted by Charbon and colleagues in the early 1960s (2-4). These investigators quantified the vasodilatory effects of a purified parathyroid extract in the rabbit and cat and also showed that a synthetic N-terminal fragment displayed similar actions in the dog. The relaxant activity was not blocked by pharmacologic antagonists of other known vasoactive agents, suggesting a direct action of the hormone. Since then numerous studies have unequivocally established the hypotensive/vasodilatory and cardiac effects of PTH (5), and these can be summarized broadly as follows: First, the hypotensive and vasorelaxant actions of PTH occur in the absence of a change in blood calcium and are mediated by PTH activation of the type 1 PTH/PTH-related protein receptor (PTHIR) expressed in the smooth muscle layer of the vessel wall. Second, although all vascular beds are relaxed by PTH, resistance vessels appear to be more responsive than conduit vessels. Third, PTH can reduce the pressor effects of other vasoactive agents that exert their action

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FIG. 1 Effects of extracts of parathyroid glands on blood pressure in thyroparathyroidectomized dogs (from Collip JB, Clark EP. J. BioL Chem. 1925; 64:485-507, with permission) (1).

m a m m a r y gland, and for tooth eruption (7). PTHrP also appears to participate in materno-fetal calcium transfer across the placenta. The third physiologic role for PTHrP is in smooth muscle, in which the protein functions to regulate contractility and proliferation. Although this chapter focuses on the physiology of PTHrP in vascular smooth muscle, it is relevant to begin with a brief review of its physiologic effects in other smooth muscle-containing tissues. In all smooth muscle cell beds studied to date, induction of PTHrP expression occurs in close association with normal physiologic stimuli. In the smooth muscle layer of the chicken oviduct, induction of PTHrP expression coincides temporally with egg movement and its arrival in the shell gland (8). In the rat uterus, PTHrP expression is localized to the myometrium and is markedly up-regulated by fetal occupancy (9) or by mechanical distention of the uterine horn using a balloon catheter (10). PTHrP expression is increased in prelabor h u m a n amnion and abruptly decreases with the onset of labor and rupture of the amniotic sac (11). In the urinary bladder, induction of PTHrP mRNA occurs during filling in proportion to bladder distension (12). Finally, as discussed in detail below, PTHrP is also expressed in vascular smooth muscle, in which it is induced by vasoconstrictor agents and mechanical stimuli. In each of these smooth muscle beds, application of PTHrP to precontracted smooth muscle preparations induces relaxant activity, precisely mimicking the actions previously described for PTH. It would therefore appear that PTHrP rather than PTH represents the physiologically important regulator of smooth muscle tone. Consequently, the remainder of this chapter

focuses primarily on the physiology of PTHrP in the cardiovascular system.

P T H r P in the Vasculature Vascular Anatomy and Contractile Mechanisms Blood vessels are composed of three principal cellular layers: (1) the intima, which consists of a single epithelial cell layer, (2) the muscularis layer, made up of vascular smooth muscle cells embedded in a connective tissue matrix, and (3) an outer adventitial layer, which receives input from the cholinergic and adrenergic nervous system. The relative composition and contribution of each of the cell types to vascular growth and tone will vary during development and among different vascular beds. For example, during development, blood vessels form initially as simple tubular structures consisting entirely of endothelial cells into which smooth muscle cells migrate to form the vascular wall. In the mature mammal, the large conduit vessels (e.g., aorta) are highly elastic to accommodate high-capacity blood flow, whereas resistance vascular beds (e.g., mesentery) typically contain more smooth muscle cells and are densely innervated. Changes in the cellular and connective tissue constituents within the vasculature occur with normal aging and in particular during pathologic conditions such as athlerosclerosis. The regulation of vascular growth, remodeling, and smooth muscle cell tone is achieved through a coordinated network of both systemic and local factors as well as input from adrenergic, cholinergic, peptidegic, and sensory neurons.

PTHrP REGULATIONOF EXCITABLECELLS / The mechanisms regulating vascular smooth muscle cell contractility have been studied in detail (13). The intracellular free calcium concentration is the major d e t e r m i n a n t of vascular tone. Depolarization of vascular smooth muscle cells opens L-type voltage-sensitive calcium channels (L-VSCCs), enabling calcium to enter the cell. These events trigger the release of much larger quantities of calcium from the sarcoplasmic reticulum. Alternatively, pharmacologic or ligand activation of G protein receptors (e.g., angiotensin II) activate phospholipase (PLC), which catalyzes phosphoinositol hydrolysis and causes calcium release from intracellular stores. The increases in cytoplasmic calcium achieved by either of these mechanisms activate myosin lightchain kinase through the calcium-calmodulin complex and phosphorylation of the 20-kDa regulatory light chain of myosin, with subsequent cross-bridge cycling and force development. The mechanisms of vascular smooth muscle cell relaxation are less well understood. In the most simple scheme, a reduction of cytoplasmic calcium with a decrease in myosin light-chain kinase activity would suffice to account for dephosphorylation of the regulatory light chain and relaxation. However, other mechanisms have been implicated in cyclic nucleotide-dependent relaxation in vascular and other smooth muscle tissues (14). The demonstration of the calcium-sensing receptor in vascular smooth muscle with pharmacologic properties similar to those of the parathyroid calcium-sensing receptor (discussed in Chapter 3) has p r o m p t e d speculation that it might also participate in the regulation of contractile events (15). Alterations of extracellular calcium over the physiologic concentration range depress contractility of precontracted vascular smooth muscle. This effect of extracellular calcium has been shown to be mediated by activation of a calciumd e p e n d e n t potassium channel and is associated with alterations in myofilament calcium sensitivity. These activities were mimicked by gadolinium, neomycin, and lanthanum, all factors that activate the calciumsensing receptor. However, the structure of this putative calcium-sensing receptor is unknown and it remains unclear whether it bears homology to the renal or parathyroid or kidney calcium-sensing receptor.

expressed predominantly in the smooth muscle layer of the vessel, although its expression has also been reported in cultured endothelial cells (22,23). The regulation of PTHrP mRNA expression has been studied in detail using cultured vascular smooth muscle cells. In primary rat aortic vascular smooth muscle cells, expression of PTHrP is rapidly (2-4 hours) but transiently induced by exposure of quiescent cells to serum (24) (Fig. 2). This mode of tight regulation is reminiscent of the behavior of the cytokine mRNAs and would appear to constitute a mechanism that would restrict the activity of PTHrP to a narrow window of time. Among the most potent inducers of PTHrP are vasoconstrictors, including angiotensin II, seritonin, endothelin, norepinephrine, bradykinin, and thrombin, each of which induces PTHrP mRNA and protein levels over the same time course as that observed for serum (25). The induction of PTHrP mRNA by angiotensisn II is d e p e n d e n t on protein kinase C activation and is mediated by both transcriptional and posttranscriptional mechanisms (25). Prior addition of saralaysin and captopril, which inhibit angiotensin II action or generation, respectively, inhibits the serum-induced increase in PTHrP in vascular smooth muscle cells. This finding suggests that the angiotensin II present in serum represents a significant c o m p o n e n t of the serum induction of PTHrP. PTHrP is also induced in vascular smooth muscle in response to mechanical stimuli. PTHrP mRNA is transiently increased in rat aorta following distension with a balloon catheter (18). Flow motion-induced mechanical events induced by rocking or rotation of monolayer cultures of rat aortic vascular smooth muscle cells result

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Expression and Regulation of PTHrP PTHrP is expressed in blood vessels in essentially all vascular beds from a broad range of species, including rodent and h u m a n fetal blood vessels (16), adult rat aorta (17,18), vena cava (17), kidney afferent arterioles, artery, and microvasculature (19), the arterial and venous supply of the m a m m a r y gland (20), the serosal arterioles in avian egg shell gland (8), and blood vessels of the rat penis (21). The protein appears to be

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FIG. 2 Time course of serum induction of PTHrP mRNA in aortic vascular smooth muscle cells (from Hongo T, ot al. d. Glin. Invos. 1991; 88:1841-1847, with permission) (24).

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in increased PTHrP mRNA expression (26). The inductive effects of mechanical stretch and angiotensin II on PTHrP mRNA appear to be synergistic, suggesting that they occur through distinct mechanisms (27). PTHrP mRNA is also p r o d u c e d in capillaries of slow-twitch soleus and fast-twitch skeletal muscle, and its expression is increased in response to lowfrequency stimulation (28). This maneuver was associated with e n h a n c e d capillarization of the muscle, indicating that PTHrP might function to promote new capillary growth in response to increased contractile activity.

Vascular Actions of PTHrP Shortly after the identification of PTHrP, a n u m b e r of studies demonstrated that synthetic N-terminal fragments of the peptide replicated many of the vascular actions of PTH, including its vasorelaxant actions in aorta (29), portal vein (30), coronary artery (31 ), renal artery (32,33), placenta (34,35), and m a m m a r y gland (36). In general, the vasodilatory potency of PTHrP when examined in organ bath systems is comparable to that of PTH. By contrast, in mouse portal vein preparations, PTHrP(1-34) was shown to be a more potent vasorelaxant than PTH(1-34) (30). In perfused rabbit kidney (33) and in rat aorta (31) the vasorelaxant effects of PTHrP do not appear to require the presence of an intact endothelium. However, in mouse aortic rings, endothelium denudatation markedly attenuates the relaxant activity of PTHrP (37), possibly reflecting a species difference. In addition to its effects on vascular tone, PTHrP also modulates vascular smooth muscle cell proliferation. The peptide decreases serum and platelet-derived growth factor-activated DNA synthesis in primary arterial vascular smooth muscle cells (24,38) and in A-10 vascular smooth muscle cells stably expressing the PTH1R (39). In both cell types, the antimitogenic effects require the PTH-like N-terminal portion of the molecule and are mimicked by dibutyryl cAMP or forskolin. The mechanism for the antiproliferative effect of PTHrP involves the induction of the cyclind e p e n d e n t kinase inhibitor, p27Kipl, and impairment of the retinoblastoma gene product (Rb), which results in cell cycle arrest in mid-G 1 phase (40). On the other hand, Massfelder et al. (41) reported that overexpression of PTHrP in A-10 vascular smooth muscle cells was associated with an increase in DNA synthesis coincident with an increased nuclear localization of the protein. However, in these studies, exogenous application of PTHrP inhibited A-10 cell growth, in agreement with the studies cited above. A putative nuclear targeting motif was found to be required for nuclear import of PTHrP in vascular smooth muscle cells, in accordance

with previous studies in chondrocytes (42). Therefore, the ability of PTHrP to influence proliferation of vascular smooth muscle cells either positively or negatively appears to d e p e n d on where the protein is trafficked in the cell. Cellular levels of PTHrP fluctuate during the cell cycle and reach their highest levels in Gz/M (43). It is possible that the protein is directed to the nucleus in the later stages of the cell cycle to participate in mitotic events. PTHrP also inhibits platelet-derived growth factor (PDGF)-directed migration of vascular smooth muscle cells in vitro (44). The antimigratory effects of PTHrP are mediated through a cAMP-dependent mechanism that leads to diminished PDGF signaling through the PI3 kinase cascade. The effects on vascular smooth muscle cell (VSMC) growth and migration in vitro are likely to be physiologically relevant to conditions u n d e r which VSMC growth and migratory behavior are altered in vivo. For example, Ozeki et al. (45) have reported that PTHrP protein and mRNA expression were markedly up-regulated in neointimal smooth muscle in rat carotid arteries following experimental balloon injury. Moreover, immunoreactive PTHrP is increased in h u m a n arterial tissue removed from patients undergoing angioplasty. In light of the possibility of opposing effects of PTHrP on vascular smooth muscle cell growth cited above, these observations can be viewed in one of two ways: either up-regulation of PTHrP is a primary stimulus for growth under these conditions or, alternatively, it represents an antiproliferative signal. Consistent with the latter possibility, the local administration of 3',5'-cyclic AMP or the phosphodiesterase inhibitors aminophylline or amrinone inhibits neointimal formation following experimental balloon injury in rat carotid arteries in vivo (46). Moreover, other studies using a similar model of arterial injury showed high levels of p27Kipl expression in the media within 2 weeks after angioplasty (47). The ability of PTHrP to modulate vascular smooth muscle cell growth suggests that the protein might function during the development of the cardiovascular system. Although the cardiovascular system appears to develop normally in the PTHrP knockout mouse, homologous deletion of the PTH1R results in a higher incidence of early fetal death at approximately embryonic day 10, coincident with the development of the heart and major blood vessels (48). Furthermore, transgenic mice expressing high levels of PTHrP and its receptor in vascular smooth muscle, created by crossing the ligand- and receptor-overexpressing mice, die at day E9.5, with severe thinning of the ventricle and disruption of ventricular trabeculae (49) (Fig. 3). Additional anecdotal evidence for a role of PTHrP in heart and vascular development is evident from

PTHrP REGULATIONOF EXCITABLECELLS /

265

These patients die prenatally with coarctation of the aorta and hydrops fetalis, the latter condition typically caused by high-output heart failure.

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FIG. 3 Overexpression of PTHrP and the PTHrP receptor disrupts heart development. (A) Whole mounts at E9.5 of double-transgenic (left) and wild-type (right) embryos. The double transgenic exhibits a greatly enlarged heart with pericardial effusion and vascular pooling (arrows). (B) Histologic sections of double-transgenic (left) and wild-type (right) embryos at E9.5. The trabeculae within the ventricular cavity (v) of the wild-type embryo are prominent (large arrows), whereas in the double transgenic, trabeculae are severely reduced or absent (asterisks). Prominent gaps are also evident between the cardiomyocytes in the double-transgenic hearts (small arrowheads); a, atria; bar = 100 ~m. (C) Left panel shows the localization of SMP8 lacZ transgene in 9.5-day embryo. Staining is apparent in heart, hindgut, and somites. Right panel is an unstained control (from Qian J, et al, Endocrinology 1999;140:1826-1833, with permission) (49). (See color plates.)

the abnormalities seen in patients with the rare fatal condition known as Blomstrand chondrodysplasia, caused by an inactivating mutation of P T H I R (50).

PTHrP exerts its vasodilatory actions by activating the PTHIR. This receptor is expressed in rat vascular smooth muscle beds (51) and relaxation of aortic preparations is accompanied by an increased accumulation of cAMP (52). Cultured rat aortic smooth muscle cells also express the P T H I R and respond to N-terminal PTHrP peptide fragments with an increase in cAMP formation (53). Moreover, relaxation responses to PTH in aortic strip preparations are potentiated by phosphodiesterase inhibitors and forskolin (54). Although the P T H I R appears to be coupled primarily to adenylate cyclase, linkage to calcium-phosphoinositol pathways is suggested by studies by Nyby et al., who demonstrated a transient increase in cytosolic calcium and cAMP in response to PTHrP (1-34) in primary arterial rat vascular smooth muscle cells (55). However, other studies using similar preparations of primary rat aortic smooth muscle cells showed that PTHrP consistently stimulated cAMP accumulation but had no effect on intracellular calcium (53). Furthermore, in A-10 embryonic aortic vascular smooth muscle cells stably expressing recombinant PTH1Rs, PTHrP induced large increases in cAMP accumulation but did not increase cytoplasmic calcium (39), despite the presence of detectable levels of expression of Gq, known to be required for functional coupling of the receptor to the PLC-phosphoinositide calcium pathway. Gq was overexpressed in these cells, However, when PTHrP evoked a calcium transient. It therefore appears that u n d e r most conditions the PTH1R couples preferentially to G s and adenylate cyclase to elevate levels of intracellular cAMP, which would be consistent with the established vasodilatory properties of this cyclic nucleotide. This does not, however, preclude the possibility that u n d e r certain physiologic conditions (or in specific vascular smooth muscle cell beds), PTHrP might also activate PLC, which could mediate other as yet unidentified activities of the protein. Vasorelaxation induced by cyclic nucleotides in arterial smooth muscle has also been reported to be associated with a reduction in intracellular calcium. In addition, in rat tail artery, PTH relaxes KCl-induced contraction; this effect is inhibited by nifedipine, suggesting an inhibition of the L-VSCC (56). Subsequent patch-clamp experiments (57) confirmed a decrease in L-type voltage-dependent calcium currents in vascular smooth muscle cells in response to PTH. Although not yet formally tested, it is likely that

266

/

CI-IAPTWR16

PTHrP also inhibits the L-VSCC activity in vascular smooth muscle, as is the case in cultured neuroblastoma cells (see later). As discussed in detail in Chapter 3, PTHrP is subject to posttranslational processing to produce both N-terminal peptides, midregion PTHrP fragments, and possibly also C-terminal forms. PTHrP peptides, which lack the PTH-like N-terminal region, likely activate receptors distinct from the P T H I R and would be expected to exhibit a biologic profile different from N-terminal PTHrP peptides. To date, however, there is no evidence that these midregion or C-terminal forms of PTHrP are biologically active either in cultured vascular smooth muscle cells (53) or in intact vessel preparations (37). Although PTHrP is capable of relaxing vascular preparations devoid of endothelium, studies in mouse aortic preparations suggest that the endothelial layer may serve to amplify relaxant effects of PTHrP and PTH (37). The mechanism accounting for the endotheliumd e p e n d e n t relaxant effects of PTH and PTHrP remains unclear but does not appear to require nitric oxide formation. The demonstration of expression of a novel PTH2 receptor (PTH2R; see below) in endothelial and smooth muscle cells in blood vessels and heart (58) suggests an additional pathway through which PTHrelated peptides could alter vascular reactivity. As with other G-coupled receptors, prolonged exposure of vessel preparations (55) or cultured aortic smooth muscle cells (59) to PTHrP is associated with desensitization. Angiotensin II, which induces PTHrP expression in cultured aortic vascular smooth muscle cells, also rapidly desensitizes cells to PTHrP and downregulates PTH1R mRNA expression (59), indicating cross-talk in the signaling circuitry a m o n g these vasoactive peptides. From the studies summarized above it is possible to construct a simple model for the mode of PTHrP action in vascular smooth muscle (Fig. 4). In response to mitogenic, vasoconstrictor, or mechanical signals, PTHrP is

Vasoconstrictors ~ , , ~ _ , i - - - ~ Mechanical Stimuli ~ , g P ~

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released and acts locally via a short feedback loop to activate the PTH1R and stimulate adenylate cyclase in adjacent cells. Effector pathways downstream of cAMP impact on specific sets of genes that function to oppose the pressor (contraction coupling) and mitogenic (cell cycle) events. As m e n t i o n e d above, induction of p27Kipl with consequent inhibition of Rb phosphorylation would represent one such target for cAMP-induced cell cycle arrest. With regard to relaxant activity, stimulation of cAMP-dependent protein kinase A (PKA) is associated with a reduction in cytoplasmic calcium and attenuated myosin lightchain kinase activity (14). If PTH and PTHrP activate the same receptor, how does the smooth muscle P T H I R distinguish between these two ligands? A likely possibility is that the sensitivity of the tissues to PTH or PTHrP is governed by the relative abundance of each ligand and the n u m b e r of PTH1R. For example, in tissues such as vascular smooth muscle, which express high levels of PTHrP but relatively low numbers of the PTH1R, the fraction of receptor occupancy must be high in order to achieve a response, thus favoring the local (PTHrP) regulator. By contrast, in bone cells, PTHrP expression is low and the receptor expression is high, enabling preferential receptor activation by PTH arriving from the systemic circulation.

P T H r P in the Heart PTHrP and the PTH1R are expressed in fetal and adult heart from a n u m b e r of different species (17). PTHrP has been immunolocalized to atrial natriuretic peptide-containing granules of rat atria. One interpretation of this finding is that PTHrP, like atrial natriuretic peptide, is released in response to stretch, but this concept has yet to be tested. Both PTH and PTHrP exert p r o n o u n c e d effects on cardiac function (61 ).

PTH.PTHrPReceptor

~~~----. ~ d k ~ ~ ~-"q~:---~-"Zllr~"icAMP" ';"" ~__...L'~--~ ImFt,4p"~dt L-VSCC i n h i b i t i o n

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FIG. 4 Model for PTHrP production and action in vascular smooth muscle cells (see text for description). (See color plates.)

PTHrP REGULATIONOF EXCITABLECELLS / Infusion of physiologic levels of N-terminal fragments of PTH and PTHrP induces hypotension and tachycardia in intact rats (5). In isolated perfused hearts, PTHrP induces chronotropic and ionotropic effects that are i n d e p e n d e n t of perfusion pressure (31). It has been established that the inotropic activity of PTHrP occurs indirectly in response to increased coronary blood flow (6). The mechanisms responsible for the chronotropic effects of PTH and PTHrP have been examined in cultured cardiomyocytes (60). In neonatal cardiomyocytes, PTH increases beating frequency through a cAMP-dependent pathway. These effects are associated with increased L-type calcium currents, precisely the opposite of what is observed in vascular smooth muscle cells. By contrast, in adult rat cardiac myocytes, both PTH (1-34) and PTHrP (1-34) increase the rate of spontaneous contraction, but only PTHrP was found to stimulate cAMP accumulation. The reason for this difference is unclear but may relate to the coupling of the P T H I R to different G proteins. PTH has also been shown to elicit a hypertrophic response in adult rat cardiomyocytes characterized by increases in protein synthesis, cell mass, and the reexpression of embryonic cardiac proteins. These effects, together with clinical observations of patients with elevated PTH and increased left ventricular mass, have been interpreted as evidence for a pathogenic role of PTH in ventricular hypertrophy. Finally, as discussed above, the timing (El0) of the embryonic death occurring in the PTHIR-null mice raises the possibility that PTHrP functions during heart development. Insight into the global actions of PTHrP in the cardiovascular system have come from studies in genetically manipulated mice. Transgenic mice overexpressing either PTHrP or the PTH1R in smooth muscle have reduced systemic blood pressure, consistent with the prediction that PTHrP acts as a local vasodilator (49). In aortic ring preparations from the PTHrP-overexpressing mice the relaxant effects of both PTHrP and acetylcholine seen in the nontransgenic mice were markedly attenuated in aortas from PTHrPoverexpressing mice. This finding suggests that local overexpression of PTHrP not only desensitizes the vasculature to PTHrP but also dampens relaxation to acetylcholine and perhaps other vasorelaxants. Thus it appears that prolonged stimulation of the PTH1R and the consequent increase in cAMP converge on signaling circuitry used by acetylcholine.

PTH-Related Proteins and Hypertensive States Several lines of circumstantial evidence suggest that PTH and PTHrP alter vascular tone in hypertensive humans and animals. For example, primary hyperparathyroidism is commonly associated with hyperten-

267

sion that may be corrected on removal of the parathyroid lesion (61). However, because alterations in circulating PTH also influence other regulators of vascular tone (e.g., ionized calcium), it is probable that the hypertension seen in long-term hyperparathyroidism is a secondary event. Alternatively, prolonged exposure to elevated PTH concentrations in these patients could desensitize vascular tissue to PTH or PTHrP and thereby increase vascular tone (55). A similar scenario appears to occur in two rat models of hypertension. For example, removal of the parathyroid glands in the spontaneously hypertensive (SH) rat and the DOCAsalt hypertensive rat attenuates the development of hypertension (62). Moreover, the PTH-induced changes in urinary cAME magnesium, calcium, and phosphorus responses are blunted in the SH rats, again suggesting a desensitization of the PTH1R. The apparent resistance to PTH and PTHrP in humans and rats with hypertension as described above p r o m p t e d Pang and co-workers to propose the existence of an additional "hypertensive" factor made in the parathyroid gland (63). This group has undertaken an extensive analysis of this factor, which they originally isolated from the serum of SH rats. Cross-transplantation of the parathyroid glands between SH rats and controls implicated the parathyroid gland as the source of the hypertensive factor. Furthermore, a polyclonal antibody raised against a partially purified factor reduced blood pressure in SH rats. However, despite over a decade of work on this putative hypertensive factor, its precise structure is still unknown.

NEUROLOGIC ACTIONS Introduction As already noted, interest in potential regulation of excitable cells by P T H / P T H r P began with Collip's demonstration in 1925 that parathyroid extracts had hypotensive effects in the dog. For the next 60 years, PTH was the focus of work in both vascular and nonvascular smooth muscle and in neurons. In smooth muscle, it now seems quite clear that the physiologic regulator is actually PTHrP, acting on the PTH1R. In the central nervous system (CNS), the best functional evidence also involves PTHrP acting on the PTH1R. In addition, there is evidence that PTH may influence pituitary function, and the description of TIP39 acting on the PTH2R may prove to be an important CNS regulatory system (see the following discussion). There are two aspects of the P T H - s m o o t h / c a r d i a c muscle literature that are relevant to PTHrP function in the CNS. The first is that the L-type voltage-sensitive calcium channel seems to be the pivotal target

268

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CI-IAPTV.k16

of P T H / P T H r P regulation. The second is that PTH a n d / o r PTHrP appear to be capable of either inhibiting or stimulating L-VSCC-mediated C a 2+ influx, d e p e n d i n g on the cell/tissue in question (56).

PTH/PTHrP Gene Family Expression in the CNS The CNS was one of the first sites to be examined in detail for PTHrP gene expression, and the gene was found to be widely expressed in neurons of the cerebral cortex, hippocampus, and cerebellum (64) (Fig. 5). This work was extended by a second survey that included the P T H I R as well as PTHrP (65). Both were found to be widely expressed, and they colocalized in a n u m b e r of sites (65). It was noted at the time that the hot spots for PTHrP gene expression are neuronal populations that have a n u m b e r of features in common, including high-density L-VSCC expression as well as high-density expression of excita-

ANTISENSE

tory amino acid receptors and a known susceptibility to excitotoxicity. T h o u g h there were early histochemical studies suggesting that PTH might be present in a n u m b e r of neuronal populations, the most careful and reproducible work has localized PTH to nuclei on the hypothalamus, with projections into the portal system (66,67). The implication is that PTH may regulate pituitary function, specifically including prolactin secretion (67). Usdin and colleagues identified the PTH2R in a cerebral cortical cDNA library by homology screening in 1995 (68). This receptor is highly sensitive to PTH(1-34) (EC50 about 1 nM) but is unresponsive to PTHrP(1-36). The PTH2R is most abundantly expressed in several basal forebrain nuclei and hypothalamic nuclei (58). Usdin et al. have also succeeded in purifying the natural ligand for this receptor using a staggering 50 pounds of bovine hypothalamus as starting material (69). This ligand is a small unmodified peptide of 39 amino acids, referred

SENSE

FIG. 5 In situ hybridization histochemistry of PTHrP mRNA in the CNS of the rat. A wide range of hybridization intensities was observed, with many positive neurons throughout all regions of the nervous system. In the hippocampus (hip; x35), dentate granule (g) cells, pyramidal (p) neurons, and large dentate interneurons (arrowhead) were densely labeled. In the section of cerebral cortex (ctx; cingulate cortex; x90) the intercerebral surface (s) is to the left. Many large pyramidal (p) neurons in the deep layers and granule (g) cells in layer II exhibited strong hybridization, in contrast to small neuronlike horizontal cells in the molecular (m) layer. In the cerebellum (crb; x35), granule (g) cell perikarya hybridized intensely, in contrast to weak signals observed for Purkinje cells (arrowheads) and basket and stellate neurons in the molecular (m) layer. Glia did not appear to stain positively (from Weir EC, et aL Proc Natl Acad Sci USA 1990;87:108, Copyright 1990 National Academy of Sciences, U.S.A.) (64).

PTHrP REGULATIONOF EXCITABLECELLS / to as tuberoinfundibular peptide 39 (TIP39), and it bears only nine of 39 amino acids that are identical to those of bovine PTH. Only limited structure-function work has been done, but TIP39 is at least as potent as PTH(1-34) at the PTH2R and may be one or two orders of magnitude more potent than PTH depending on the species of origin of the PTH2R (69). The sites of PTH2R expression imply potential TIP39 function in regulating the pituitary and in modulating pain sensitivity. Thus, three ligands and at least two receptors of the P T H / P T H r P gene family are expressed in the CNS. Two of the ligands (PTH and TIP39) are expressed in highly discrete locations, whereas PTHrP is widely expressed in neuronal populations throughout the brain.

Calcium Channels, Neuromodulation, and Signaling Microdomains Calcium Channels Calcium channels are heteromeric associations of four or five subunits (70). The oL1 subunit is the poreforming structure that is responsible for permeation as well as the gating function of the channel. There are a half-dozen classes of calcium channels, each defined by a specific e~1 gene. Given the n u m b e r of different genes for each subunit and alternate splicing of these gene products, the combinatorial possibilities are enormous (perhaps 1000). In brief, calcium channels are either L-type or nonL-type (e.g., N, P / Q , T, and R channels) (70). The Ltype channels mediate large and long-lasting (L) Ca 2+ fluxes and are composed of three subclasses, defined by their oL1 subunits as well as by the locations in which they were initially identified. These are S (skeletal; OLlS), C (cardiac; oL~c), and D (neuroendocrine; OLld). The L-channels are dihydropyridine sensitive, and there are a n u m b e r of classes of these widely used drugs (nifedipine, diltiazem). Virtually every class of calcium channel is expressed in the CNS (70). The N and P / Q channels are expressed in both pre- and postsynaptic locations and are involved in regulation of synaptic transmission. The L-channels are widely expressed in neurons t h r o u g h o u t the brain and are found only in postsynaptic locations, specifically on cell bodies and proximal dendrites (71). This localization is crucial to L-channel function. These channels appear to regulate cytosolic Ca 2+ levels in the soma and proximal dendrites of neurons as a function of the integrated excitatory synaptic input into these locations (71). Given the location and gating of these channels, it is quite clear that their C a 2+ c u r r e n t s are not involved in neurotransmission, but rather with fundamental aspects of neuronal cell biology such as regu-

269

lation of cellular signaling pathways and regulation of gene expression. Neuromodulation

The clustering of L-VSCCs on neuronal cell bodies is characteristic also of the location of n e u r o p e p t i d e / growth factor receptors. This clustering of receptors is strategically convenient to the nucleus as well as to the regulation of channels of all sorts and the capacity of peptides and growth factors to cross-talk with each other (72). This kind of short-range a u t o c r i n e / paracrine signaling to the soma and proximal processes of neurons is referred to as "neuromodulation" to emphasize that the regulation and signaling involved are very different from neurotransmission (72).

Signaling Microdomains Even a generation ago, it was clear that signal transduction corresponded to more than cells simply serving as bags of rising and falling tides of cyclic nucleotides a n d Ca 2+, but the biochemical details that account for the exquisite specificity of signal transduction have become clear only in the past decade. The work of Greenberg and co-workers has provided great insight into the specificity of neuronal C a 2+ signaling. Depending on the specific route of entry into a n e u r o n , C a 2+ has highly specific and differential effects on a wide variety of neuronal processes, such as gene expression, learning, and memory; modulation of synaptic strength; and CaZ+-mediated cell death (73). For example, C a 2+ entry via L-VSCCs elicits an entirely different response in terms of gene expression than does Ca 2+ entry mediated via NMDA receptors (73). Clearly, every calcium ion entering the cytosol of a n e u r o n is not perceived in the same way. Equally clearly, cAMP generated in a n e u r o n by a voltage-sensitive adenylate cyclase as opposed to a G protein coupled to a h o r m o n e receptor is not perceived by the cell in the same way. A major advance in understanding the specificity of signaling has come from the recognition that microdomains that exist at the cell surface cluster together the r e c e p t o r / c h a n n e l in question, the PKA a n d / o r PKC transducers, and the target to be modified. The key recent players that account for this clustering of specific signaling components are the A kinase anchoring proteins (AKAPs) and the receptors for activated C kinase (RACKs) (74). In certain cases, a single AKAP is capable of binding both PKA and PKC, thus serving as a scaffold that brings together all of the early components of a complex regulatory system. The net result of this tethering of signaling receptor, transducer, and target into a microdomain is a tremendous resolution in terms of specificity and speed.

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Cr~AeTF~k16

PTHrP Is Neuroprotective

KA

PTHrP Gene Expression in Neurons Is Regulated by L-VSCC Ca 2+ Influx

It turns out that the regulation of PTHrP gene expression in cerebellar granule cells is a classic example of the kind of specificity of C a 2+ signaling described in the previous section. Cerebellar granule cells are a hot spot of PTHrP and PTH1R expression in vivo (64, 65), and cultured cerebellar granule cells are a commonly used neuronal model system in vitro. PTHrP gene expression in these cells is a direct function of depolarization, which triggers L-VSCC C a 2+ influx that tracks to the PTHrP gene via the calmodulin/CaM kinase cascade (75, 76). C a 2+ entry into granule cells by any other means (e.g., veratridine treatment) has no influence whatsoever on the PTHrP gene. In granule cells, as in most other cells that express the PTHrP gene, PTHrP is a constitutive secretory product, so that the quantity of PTHrP secreted by the cell is a linear function of the level of PTHrP mRNA expression. PTHrP immunolocalizes principally to the granule cell soma (75), so that it is presumably secreted by the cell bodies, acting in the autocrine/paracrine fashion typical of a n e u r o m o d u l a t o r y peptide. PTHrP Inhibits L-VSCC Ca 2+ Influx, Defining a Protective Feedback Loop

Overstimulation can lead to neuronal injury or death, a process referred to as excitotoxicity. Excitotoxicity comes in two flavors. High concentrations of the excitory amino acid, glutamate, cause a generalized influx of cations and a collapse in mitochondrial function, leading to almost immediate necrosis (77). Lower concentrations of glutamate or exposure to other excitotoxins such as kainic acid trigger C a 2+ entry via L-VSCCs, and this leads to excitotoxicity characterized by a long latency (6-24 hours to death) (77,78). The granule cell system is subject to both the immediate and the latent forms of excitotoxicity. A low concentration of kainic acid produces about 50% granule cell death at 24 hours, and the calcium channel blocker, nitrendipine, is capable of fully protecting these cells, thereby defining the central importance of L-VSCC Ca '~+ influx in long-latency excitotoxicity (79). It will be recalled that PTH has been shown to inhibit

Neuron

EAA, K+, etc. ~

Depolarization

KA & PTHrP & antagonist

KA & PTHrP

FIG. 6 Cell death assessed by propidium iodide staining. Propidium iodide can bind to nuclear DNA only when the cell membrane is not intact; each bright dot therefore represents the nucleus of a dead cell. The left panel shows kainic acid (KA) alone, the middle panel shows KA plus PTHrP, and the right panel shows KA together with PTHrP and a 10-fold molar excess of a competitive antagonist of PTHrP binding. The percent kill (+_ SEM) under these three conditions was 23 _+ 3% (n = 10), 2 _ 2% (n = 11, P < 0.001 with respect to KA alone), and 23 +_ 2% (n = 10), respectively; bar = 25 IJ.M (Reprinted from Neurosci Lett, Vol. 274; ML Brines, Z Ling, AE Broadus. Parathyroid hormone-related protein protects against kainic acid excitotoxicity in rat cerebellar granule cells by regulating L-type channel calcium flux, pp. 13-16. © 1999, with permission from Elsevier Science) (79).

L-VSCCs in smooth muscle and neuroblastoma cells (56,80). This led to the working hypothesis that PTHrP might be capable of inhibiting L-VSCC C a 2+ influx in cerebellar granular cells, and this proved to be the case. PTHrP was found to be fully neuroprotective in kainic acid-treated granule cells (Fig. 6) and was as effective as nitrendipine in reducing kainic acid-induced L-VSCC C a 2+ influx (79). Pang's group used whole-cell patchclamp techniques to demonstrate that PTH is capable of inhibiting L-VSCC Ca 2+ influx in mouse neuroblastoma cells (80), and one of us (A.E.B.) has used patchclamp techniques to demonstrate the same findings with PTHrP in these cells. This effect is mediated by the PTH1R, but nothing is yet known of the mechanism by which the channel is actually regulated. Taken together, these findings indicate that PTHrP serves as an endogenous L-VSCC regulator that functions in a neuroprotective feedback loop of the sort depicted in Fig. 7. As shown, the L-VSCC is the fulcrum of this loop, and the rheostat is L-VSCC C a 2+ entry. This loop would provide neuroprotection to individual (autocrine) and neighboring (paracrine) neurons. The calcium hypothesis of neurodegeneration and neuronal aging holds that abnormalities in C a 2+ regu-

= VSCC ~ l t

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fl" PTHrP

1

Neuroprotection

FIG. 7 Scheme of autofeedback look in which PTHrP, triggered by L-VCSS Ca 2+ influx, feeds back via the PTH1R to dampen L-VCSS Ca 2÷ currents.

PTHrP REGULATION OF EXCITABLE CELLS / lation contribute to neuronal death and degenerative disorders such as Alzheimer and Parkinson diseases as well as to age-related vulnerability of neurons to such disorders (81). This hypothesis would predict that the absence of an endogenous neuroprotective p r o d u c t with the effects described above would predispose to excitotoxic neuronal damage and that this would likely be progressive. As described in Chapter 13, the PTHrP knockout mouse dies at birth as a result of a systemic chondrodystrophy. This mouse has been rescued by a genetic strategy, generating a mouse that is PTHrP-sufficient in chondrocytes but PTHrP-null in all other sites (82). There are no CNS abnormalities in the rescued mice before 4 months of age, but at 4 months and beyond, progressive neurodegenerative changes are seen in many regions of the cerebral cortex and in the posterior hippocampus (these data are noted only briefly because the work is yet unpublished). All of the involved areas are regions of known expression of L-VSCCs as well as of sensitivity to excitotoxic injury.

Other Potential Calcium Channel Effects PTHrP increases L-VSCC activity and thereby enhances d o p a m i n e secretion in PC-12 cells (83). PTH a n d / o r PTHrP have been reported to increase calcium channel-like activity in snail neurons (84) and in rat hippocampal neurons (85,86), but these effects are slow and perhaps involve channels other than the classic L-VSCC. UMR-106 osteoblast-like cells contain L-VSCCs that are stimulated by PTH treatment (87). L-VSCCs are also widely expressed in a great many other excitable and nonexcitable cells that have thus far not been examined with respect to PTHrP regulation.

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CI4AeI:F17 Physiolo gic Actions of PTH and PTHrP V. Epidermal, Mammary, Reproductive, and Pancreatic Tissues J O H N j. WYSOLMERSKI Yale University School of Medicine, New Haven, Connecticut 06520 ANDREW E STEWART University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213

JOHN T. MARTIN st.

Vincent's Institute of Medical Research, Fitzroy, VIC3065 Australia

INTRODUCTION

ple studies have confirmed that in tissue culture, rodent and h u m a n keratinocytes express the PTHrP gene and secrete bioactive PTHrP (2). PTHrP expression has also been examined in skin in vivo using both immunohistochemistry and in situ hybridization. During fetal development in rats and mice, PTHrP is principally expressed within the epithelial cells of developing hair follicles (3,4). In mature skin, PTHrP has been found at low levels throughout the epidermis from the basal layer to the granular layer. Some studies have suggested that PTHrP is more highly expressed in the superbasal keratinocytes (5,6), although not all studies have reported this pattern (7,8). A variety of factors have been reported to regulate PTHrP production by cultured keratinocytes (2). For example, glucocorticoids and 1,25(OH)2D have been shown to downregulate PTHrP production, whereas fetal bovine serum, matrigel, and as yet unidentified factors secreted from cultured fibroblasts have been shown to up-regulate PTHrP production. The up-regulation of PTHrP production by fibroblast-conditioned media is particularly interesting, because PTHrP, in turn, acts on dermal fibroblasts, suggesting that it may function in a short regulatory loop between keratinocytes and dermal fibroblasts (9,10). Finally, in vivo, PTHrP expression has been shown to be up-regulated at the margins of healing wounds in guinea pigs (11). Interestingly, in this study, PTHrP was also detected in myofibroblasts and macrophages, suggesting that keratinocytes may not be the only source of PTHrP in skin. It is now clear that keratinocytes do not express the type 1 P T H / P T H r P receptor (PTH1R), but dermal

Documentation of the skeletal abnormalities in mice that either overexpressed parathyroid hormone-related protein (PTHrP) in their skeletons or had the genes for PTHrP and the PTH receptor ablated by the techniques of homologous recombination provided an exciting impetus for the rapid accumulation of knowledge regarding the mechanisms by which PTHrP regulates bone and cartlilage development and physiology. These findings are reviewed in Chapters 13 and 15. Over the past 5 years, increasing evidence has accumulated that PTHrP and the P T H / P T H r P receptor family also contribute to the development and functioning of several nonskeletal organs. The data regarding actions of PTHrP in the vascular system and the central nervous system are reviewed in Chapter 16. Here, the last of the series of chapters on the physiologic actions of PTHrP, we review the data regarding the functions of PTHrP in several other nonskeletal sites. We first consider functions of PTHrP in skin. Next, we review its functions in the mammary gland, placenta, and other reproductive tissues. Finally, we examine its role in the endocrine pancreas.

SKIN PTHrP and PTHrP Receptor Expression in Skin Normal human keratinocytes were the first nonmalignant cells shown to produce PTHrP (1), and multiThe Parathyroids, Second Edition

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fibroblasts do (12,13). PTHrP has been shown to bind to skin fibroblasts and to elicit biochemical and biologic responses in these cells (9,10,14). In addition, studies utilizing in situ hybridization have demonstrated that in fetal skin, P T H I R mRNA is absent from the epidermis, yet abundant in the fetal dermis, especially in those cells adjacent to the fetal keratinocytes (3,4,15). There are fewer data concerning the expression patterns of the P T H I R in more mature skin, but in mice, it appears that the relative amount of PTH1R mRNA in dermal fibroblasts is reduced in adult as compared to fetal skin (J.E Zhang a n d J J . Wysolmerski, unpublished data). Although keratinocytes do not express the classical P T H / P T H r P receptor, studies have shown that these cells bind and respond to PTHrP by inducing calcium transients, suggesting that there may be other receptors for PTHrP expressed on these cells (13,16). However, to date, no such receptors have been isolated, so their existence remains uncertain.

Biochemistry of PTHrP in Skin As described in Chapters 3 and 4, during transcription, the PTHrP gene undergoes alternative splicing to generate multiple mRNAs, which in human cells give rise to three main protein isoforms. In addition, each of these isoforms is subject to posttranslational processing to generate a variety of peptides of varying length. H u m a n keratinocytes have been shown to contain mRNA encoding for each of the three main isoforms, although, as in other systems, no clearly defined or unique role has yet emerged for any of the three individual isoforms (2). Keratinocytes have also been shown to process full-length PTHrP into a variety of smaller peptides including PTHrP(1-36) and a midregion fragment beginning at amino acid 38 (17). These cells have also been shown to secrete a large (---10 kDa) aminoterminal form that is glycosylated (18). There is currently no specific information regarding the secretion of COOH-terminal peptides of PTHrP in skin, but keratinocytes are also likely to produce these peptides.

Function of PTHrP in Skin There have now been several studies suggesting that PTHrP is involved in the regulation of hair growth. As noted above, in embryonic skin the PTHrP gene is most prominently expressed in developing hair follicles and overexpression of PTHrP in the basal keratinocytes of skin in transgenic mice leads to a severe inhibition of hair follicle morphogenesis during fetal development (19). The effects of PTHrP overexpression appear to act early during hair follicle induction, implicating PTHrP in the regulation of epidermal patterning dur-

ing embryogenesis. However, any such function of PTHrP during hair follicle morphogenesis is not critical, because disruption of the PTHrP or P T H I R genes does not seem to affect hair follicle formation or patterning in mice (20-22). In addition to effects on hair follicle morphogenesis, it has also been suggested that PTHrP may participate in the regulation of the hair cycle. It has been reported that the systemic administration of PTH1R antagonists to young mice perturbs the hair cycle by prematurely terminating telogen, prolonging anagen growth, and inhibiting catagen (23). These findings imply that PTHrP acts to inhibit hair follicle growth by pushing growing hair follicles into the growth-arrested or catagen/telogen phase of the hair cycle. If this hypothesis were correct, one would expect PTHrP knockout mice to exhibit findings similar to the PTH1R antagonisttreated mice. However, this does not appear to be the case. In mice that lack PTHrP in their skin, the hair cycle appears to be normal (22). In fact, rather than a promotion of hair growth, these mice demonstrate a thinning of their coat over time. These conflicting results are difficult to rationalize at this point, but they raise the intriguing possibility that the PTH1R antagonist might be inhibiting the function of another m e m b e r of the PTH receptor family and that there may be ligands other than PTHrP for such a receptor in skin. In addition to its effects on hair follicles, PTHrP has been implicated in the regulation of keratinocyte proliferation a n d / o r differentiation. Although there have been studies in cultured cells suggesting that PTHrP either enhances or inhibits keratinocyte proliferation, data from studies in vitro have suggested that PTHrP promotes the differentiation of keratinocytes (2). Studies in vivo have also supported a role for PTHrP in regulating keratinocyte differentiation, but have suggested that it inhibits their differentiation (22). A careful comparison of the histology of PTHrP-null and PTHrP-overexpressing skin demonstrated reciprocal changes in keratinocyte differentiation. In the absence of PTHrP, it appeared that keratinocyte differentiation was accelerated, whereas in skin exposed to PTHrP overexpression, keratinocyte differentiation appeared to be retarded (22). Therefore, in a physiologic context, PTHrP appears to slow the rate of keratinocyte differentiation and to preserve the proliferative, basal compartment. Remarkably, these changes in the rate of keratinocyte differentiation are exactly analogous to those noted for chondrocyte differentiation in the growth plates of mice overexpressing PTHrP as compared to PTHrP-null and PTH1R-null mice (2) (see Chapter 15). Again, at present, it is difficult to rationalize the conflicting data regarding the effects of PTHrP

PHYSIOLOGYOF PTH AND PTHrP on keratinocyte proliferation and differentiation, but the studies in genetically altered mice clearly indicate that PTHrP participates in the complex regulation of these processes in vivo. Further research will be needed to understand its exact role. An important but still unresolved question is whether the effects of PTHrP on keratinocyte proliferation, differentiation, and hair follicle growth are the result of its effects on keratinocytes directly or via its effects on dermal fibroblasts. At present there are more data to support the paracrine possibility. The P T H I R is expressed on dermal fibroblasts in vivo and in culture (4,12). Dermal fibroblasts have been demonstrated to show biochemical and biologic responses to PTHrP (9,10,14). Furthermore, PTHrP has been shown to induce changes in growth factor and extracellular matrix production that could, in turn, lead to changes in keratinocyte proliferation a n d / o r differentiation and hair follicle growth (9,10,24). Of course, the autocrine and paracrine signaling pathways are not mutually exclusive, but any direct autocrine effects of PTHrP on keratinocytes, as discussed above, would require the presence of PTHrP receptors other than the P T H I R on these cells. Although preliminary biochemical evidence has suggested that this possibility exists, no such receptors have been identified on keratinocytes (13,16). An alternative possibility by which PTHrP might have cell autonomous effects on keratinocytes is via an intracrine pathway involving its translocation to the nucleus (2). Clearly, much research is needed to define the receptors and signaling pathways by which PTHrP acts in skin. Only when this information is available will we be able to understand the mechanisms leading to the skin phenotypes that have been observed in the various transgenic models discussed above.

Pathophysiology of PTHrP in Skin To date, PTHrP has not been implicated in any diseases of the skin. It has been suggested that the skin and skin appendage findings in the rescued PTHrPnull mice are reminiscent of a series of disorders collectively known as the ectodermal dysplasias (22), but PTHrP has not been formally linked to any of these diseases. The most c o m m o n tumors causing humoral hypercalcemia of malignancy (HHM) are those of squamous histology, but these tumors rarely arise from skin keratinocytes. In fact, the most c o m m o n skin tumors, basal cell carcinomas, do not overexpress PTHrP and are not associated with hypercalcemia (2). Although PTHrP appears to participate in the normal physiology of the skin it is not clear at this juncture if it will be involved in skin pathophysiology.

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MAMMARY GLAND Very soon after its discovery, PTHrP was reported to be expressed in mammary tissue and to be secreted into milk (25,26). It is now known that PTHrP is critically important for the proper development and functioning of the mammary gland throughout life. In addition, it has been implicated as an important modulator of the biologic behavior of breast cancer. The mammary gland develops in several discrete stages and only reaches its fully differentiated state during pregnancy and lactation. PTHrP appears to serve different functions during these different stages of m a m m a r y development and, therefore, we will organize our discussion around three principal stages of mammary development: embryonic development, adolescent growth, and pregnancy and lactation. For each stage, we will first outline the pertinent developmental events in rodents, because the data regarding the function(s) of PTHrP largely come from studies in mice and rats. Next, we will discuss the localization of PTHrP and PTHrP receptors and the regulation of the expression of PTHrP and its receptors. Finally we will address the function of PTHrE

Embryonic Mammary Development In mice, there are two phases of embryonic mammary development. The first involves the formation of five pairs of m a m m a r y buds, each of which consists of a lightbulb-shaped collection of epithelial cells surr o u n d e d by several layers of fibroblasts known as the m a m m a r y mesenchyme (27). After the formation of these buds, mouse m a m m a r y development displays a characteristic pattern of sexual dimorphism. In male embryos, in response to androgens, the m a m m a r y mesenchyme destroys the epithelial bud and male mice are left without m a m m a r y glands or nipples (27). In female embryos, however, the m a m m a r y buds remain quiescent until embryonic day 16 (E16), when they u n d e r g o a transition into the second step of embryonic development, the formation of the rudimentary ductal tree. This process involves the elongation of the m a m m a r y bud, its penetration out of the dermis and into a specialized stromal c o m p a r t m e n t known as the m a m m a r y fat pad, and the initiation of ductal branching morphogenesis. At the time of birth, the gland consists of a simple epithelial ductal tree consisting of 15-20 branched tubes within a fatty stroma (27). This initial pattern persists until puberty, at which time the mature virgin gland is formed through a second r o u n d of branching morphogenesis, regulated by circulating h o r m o n e s (discussed below). The PTHrP gene is expressed exclusively within epithelial cells of the mammary bud, soon after it begins

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to form. PTHrP mRNA continues to be localized to mammary epithelial cells during the initial round of branching morphogenesis, as the bud grows out into the presumptive mammary fat pad and begins to branch (15,28,29). At some point after birth, PTHrP gene expression is down-regulated, and in the adult virgin gland PTHrP mRNA is found only within specific portions of the duct system (discussed below) (29). In contrast to the PTHrP gene, the PTH1R gene appears to be expressed within the mesenchyme but its expression is widespread and not limited to the developing mammary structures. Transcripts for the P T H I R gene are found within the mammary mesenchyme but also throughout the developing dermis (15,28). It is not clear when the receptor gene is first expressed within the subepidermal mesenchyme. However, it appears already to be present

when the mammary bud begins to form and it continues to be expressed within fibroblasts surrounding the mammary ducts as they begin to grow out and branch (28,29). The epithelial expression of PTHrP and the mesenchymal expression of the PTH1R are not unique to the developing mammary gland, and this pattern has long led to speculation that PTHrP and its receptor might contribute to the regulation of epithelialmesenchymal interactions during organogenesis. There is now solid evidence that this is the case during embryonic mammary development, when PTHrP appears to serve as an epithelial signal that influences cell fate decisions within the developing mammary mesenchyme. The data supporting this notion come from studies in several genetically altered mouse models. First, in PTHrP or PTH1R knockout mice, there is a failure of the normal

FI6. 1 Failure of sexual dimorphism during embryonic mammary development. Mammary buds from PTHrP knockout (A and B) and wild-type (B and D) embryos at E15. Note the destruction of the mammary bud in normal male embryos (B) as evidenced by the mesenchymal condensation that has obliterated the bud stalk (arrowheads) and the degenerating epithelial remnant (arrows). In PTHrP knockout males (A), this process is completely absent. The destruction of the mammary bud is an androgen-dependent phenomenon and the mesenchymal cells are the targets for androgen action, as evidenced by the positive staining for androgen receptors seen in the mammary mesenchyme surrounding a normal female mammary bud in D. In PTHrP knockout mammary buds, this process fails due to the failure of androgen receptor expression in the mammary mesenchyme, as shown in C (modified from Ref. 15 with permission).

PHYSIOLOGY OF P T H AND P T H r P

androgen-mediated destruction of the mammary bud, due to the failure of the mammary mesenchyme to differentiate properly and to express androgen receptors (15) (see Fig. 1). Second, in PTHrP or PTH1R knockout mice, the mammary buds fail to grow out into the fat pad and initiate branching morphogenesis, again due to defects in the mammary mesenchyme (28,29). Finally, in keratin 14 (K14)-PTHrP transgenic mice that ectopically overexpress PTHrP within all the basal keratinocytes of the developing embryo, subepidermal mesenchymal cells that should acquire a dermal fate instead become mammary mesenchyme (15). As demonstrated by these studies, PTHrP signaling is essential for mammary gland formation in rodents. When the mammary gland begins to form, the PTH1R is expressed in all the mesenchymal cells underneath the epidermis, but PTHrP is expressed only within the mammary epithelial buds and not within the epidermis itself (3,25,28). As the mammary bud grows down into the mesenchyme, the PTHrP produced by the mammary epithelial cells interacts over short distances with the PTH1R on the immature mesenchymal cells closest to the epithelial bud, and triggers these cells to differentiate into mammary mesenchyme. In this way, PTHrP acts as a patterning molecule contributing to the formation of small patches of mammary-specific stroma around the mammary buds and within the surrounding sea of presumptive dermis. The process of differentiation set in motion by PTHrP signaling is critical to the ability of the mammary-specific stroma to direct further morphogenesis of the epithelium. In the absence of this signaling, the mesenchyme can neither destroy the epithelial bud in response to androgens nor trigger the outgrowth of the bud and the initiation of branching morphogenesis (15,28,29). Although the above model was developed from studies in mice, it appears that PTHrP is also critical to the formation of breast tissues in human fetuses. It has been demonstrated that a fatal form of dwarfism known as Blomstrand chondrodyplasia is a result of null mutations of the P T H I R gene (30) (see Chapter 44). Affected fetuses have skeletal abnormalities similar to those caused by deletion of the PTHrP and P T H I R genes in mice (see Chapter 15), and, in addition, lack breast tissue or nipples (31). In normal human fetuses, the PTHrP gene is expressed within the mammary epithelial bud, and the PTH1R gene is expressed in surrounding mesenchyme (31). Therefore, in humans, as in mice, epithelial-to-mesenchymal PTHrP-PTH1R signaling is essential to the formation of the embryonic mammary gland.

Adolescent Mammary Development Following birth, the murine mammary gland undergoes little development until the onset of puberty. At

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that point, in response to hormonal changes, the distal ends of the mammary ducts form specialized structures called terminal end buds. These structures serve as the sites of cellular proliferation and differentiation for a period of active growth that gives rise to the typical branched duct system of the mature virgin gland (32). Once formed, the ductal tree remains relatively unchanged until another round of hormonal stimulation during pregnancy induces the formation of the lobuloalveolar units that produce milk. Similar to the findings in the embryonic mammary gland, during puberty, PTHrP appears to be a product of mammary epithelial cells and the P T H I R appears to be expressed in stromal cells (29). However, the structure of the pubertal gland is more complex than that of the embryonic gland and, here, there are conflicting data regarding the localization of PTHrP and the PTH receptor. Although there is general agreement that PTHrP is expressed in epithelial cells in the postnatal gland, there is some disagreement regarding the specific epithelial compartments in which PTHrP is found. Studies employing in situ hybridization in mice have suggested that, after birth, the overall levels of PTHrP gene expression in mammary ducts were reduced except for in the terminal end buds during puberty (29). In these structures, there were appreciable amounts of PTHrP mRNA detected in the peripherally located cap cells. In other parts of the gland there was little, if any, specific hybridization for PTHrP. In contrast, studies looking at mature h u m a n and canine mammary glands using immunohistochemical techniques have suggested that PTHrP can be found in both the luminal epithelial and myoepithelial cells throughout the ducts (8,33). Furthermore, studies using cultured cells have suggested that PTHrP is produced by luminal and myoepithelial cells isolated from normal glands (34-36). There have been fewer reports looking at the localization of P T H I R expression in the postnatal mammary gland, but as is the case during embryologic development, it is expressed in the mammary stroma (29). As with PTHrP gene expression, in situ hybridization studies have found the highest concentration of P T H I R mRNA in the stroma immediately surrounding terminal end buds during puberty (29). This same study found lower levels of P T H I R mRNA distributed generally within the fat pad stroma, but very little expression in the dense stroma surrounding the more mature ducts. In addition, these investigators found no evidence of PTH1R mRNA in freshly isolated epithelial cells (29). However, there have been reports suggesting that this receptor is expressed in cultured luminal epithelial and myoepithelial cells (35,36) as well as in cultured breast cancer cell lines (37). In summary, it is clear that during puberty, the expression of PTHrP and its receptor appear to be localized

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predominantly to the terminal end buds, with PTHrP found in the epithelium and the P T H I R found in the stroma. It remains an open and interesting question whether at some time during mammary ductal development epithelial cells express low levels of PTH1R. Studies in transgenic mice have demonstrated that PTHrP is an important regulator of mammary morphogenesis during puberty. Overexpression of PTHrP in mammary epithelial cells using the K14 promoter results in an impairment of ductal branching morphogenesis (38). There are two aspects to the defect. First, the terminal end buds advance through the mammary fat pad at a significantly slower rate than normal. Second, there is a severe reduction in the branching complexity of the ductal tree. As seen in Fig. 2, this results in a spare and stunted epithelial duct system. Experiments altering the timing and duration of PTHrP overexpression in the mammary gland by using a tetracycline-regulated K14-PTHrP transgene have demonstrated that the two aspects of this pubertal phenotype appear to represent separate functions of PTHrE The branching (or patterning) defect results from embryonic overexpression of PTHrP, whereas the

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ductal elongation defect is a function of overexpression of PTHrP during puberty (39). These effects on ductal patterning provide further evidence of the importance of PTHrP as a regulator of embryonic mammary development. In addition, the localization patterns for PTHrP and the P T H I R during puberty combined with the effects of pubertal overexpression of PTHrP on ductal growth suggest that PTHrP also functions later in mammary development. During puberty it appears to modulate epithelial-mesenchymal interactions that govern ductal elongation at the terminal end buds.

Pregnancy and Lactation Mammary epithelial cells reach their fully differentiated state only during lactation. Under hormonal stimulation during pregnancy, there is a massive wave of epithelial proliferation and morphogenesis, giving rise to terminal ductules and lobuloalveolar units. During the later stages of pregnancy the epithelial cells fully differentiate and then begin to secrete milk during lactation. By the time lactation commences, the fatty stroma of the mammary gland is completely replaced by

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FIG. 2 Overexpression of PTHrP in the mammary gland of K14-PTHrP transgenic mice antagonizes ductal elongation and branching morphogenesis during puberty. (A, B) Typical whole-mount analyses of the fourth inguinal mammary glands from wild-type (A) and K14-PTHrP transgenic mice (B) at 6 weeks of age. The dark oval in the center of each gland is a lymph node. Growth of the ducts during puberty is directional and each gland is arranged so that the primary duct (the origin of the duct system) is toward the center of the figure. Note that overexpression of PTHrP results in an impairment of the elongation of the ducts through the fat pad as well as a dramatic reduction of the branching complexity of the ductal tree. (C, D) Higher magnifications of a portion of the ducts from the wild-type (C) and transgenic (D) glands, demonstrating the reduction in side branching caused by overexpression of PTHrP (modified from Ref. 38 with permission)

PHYSIOLOGYOF PTH AND PTHrP actively secreting lobuloalveoli. On the completion of lactation, there is widespread apoptosis of the differentiated epithelial cells and the gland remodels itself into a duct system similar to that of the virgin animal (32). Localization studies in humans, rodents, and cows have all noted epithelial cells to be the source of PTHrP in the m a m m a r y gland during pregnancy and lactation (33,36,40,41). Based on the assessment of whole-gland RNA, PTHrP expression appears to be up-regulated at the start of lactation u n d e r the control of both local and systemic factors (2,25,42-44). Thiede and Rodan originally reported that in rats, PTHrP expression is d e p e n d e n t on suckling and on serum prolactin concentrations (25,42). However, prolactin must serve only as a permissive factor, for additional studies have shown that the suckling response is a local one and that PTHrP levels increase only in the milked gland (43). Furthermore, PTHrP expression gradually increases over the course of lactation, and in the later stages its expression becomes i n d e p e n d e n t of serum prolactin levels (44). It is clear that much of the PTHrP made during lactation ends up in milk, in which levels of PTHrP are up to 10,000-fold higher than in the circulation of normal individuals and 1000-fold higher than in patients suffering from humoral hypercalcemia of malignancy (2). PTHrP concentrations in milk have generally been found to mirror RNA levels in the gland, increasing over the duration of lactation, and rising acutely with suckling (43,45-47). In addition, there is evidence that PTHrP levels correlate with the calcium content of milk in humans and cows, but not in rodents (45-49). Finally, in mice, PTHrP mRNA levels are promptly down-regulated during the early stages of involution and then increase to prelactation levels about a week into the remodeling process, (M.E. Dunbar and J.J. Wysolmerski, unpublished data). In contrast to PTHrP, there has been little study of the expression or regulation of PTHrP receptors during pregnancy and lactation. In early pregnancy, the P T H / P T H r P receptor is expressed at low levels in the stroma surrounding the developing lobuloalveolar units (29). Studies using whole-gland RNA demonstrate a reciprocal relationship between PTH1R and PTHrP mRNA levels. That is, as PTHrP expression rises during lactation, PTH1R mRNA levels decrease, and as PTHrP mRNA levels fall during early involution, PTH1R expression increases to its former level (M.E. Dunbar and JJ. Wysolmerski, unpublished data). This may represent active down-regulation of the receptor by PTHrP or may simply reflect the changing a m o u n t of stroma within the gland at these different stages. However, in a study of cells isolated from lactating rats, it was suggested that epithelial cells as well as stromal cells express this receptor (36), so the regulation of the expression of this receptor during pregnancy and lactation may be complex.

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The initial reports of the presence of PTHrP in the mammary gland and in milk p r o m p t e d a great deal of speculation regarding its functions in breast tissue during lactation. These proposals have revolved around four general hypotheses: (1) PTHrP may be involved in maternal calcium homeostasis and the mobilization of calcium from the maternal skeleton; (2) PTHrP may be involved in regulating vascular a n d / o r myoepithelial tone in the lactating m a m m a r y gland; (3) PTHrP may be involved in transepithelial calcium transport into milk; a n d / o r (4) PTHrP may be involved in neonatal calcium homeostasis or neonatal gut physiology. Although the true functions of PTHrP during lactation remain obscure, there is some experimental evidence addressing the first two of these possibilities. These data are considered in the following discussions. However, at this point, the latter two ideas remain simple speculation and will not be discussed further. The control of maternal mineral metabolism and the mobilization of skeletal calcium for milk production remain enigmatic. Although a significant proportion of the calcium transported into milk is derived from the maternal skeleton, neither of the established calciumregulating hormones, PTH nor 1,25(OH)2D, seem to be necessary or sufficient to account for this p h e n o m e n o n (49). Therefore, the finding that PTHrP was produced in the lactating breast aroused interest in this protein as the missing factor acting to mobilize calcium during lactation. Although not every study has concurred, in support of such a role the weight of evidence across species now suggests that PTHrP levels in the systemic circulation are elevated during lactation (49). In addition, circulating PTHrP levels have been shown to correlate with bone density changes in lactating humans (50) and it appears that suckling leads to transient increases in circulating PTHrP levels (51). Suckling has also been shown to lead to increases in urinary phosphate and cAMP excretion in rodents and in cows (52,53), changes that might be expected if PTHrP released from the mammary gland was acting in a systemic fashion. Of course, none of these data actually prove that the source of the PTHrP in the circulation of lactating humans or animals is the mammary gland. More significantly, passive immunization of lactating mice with anti-PTHrP antibodies has not been found to influence maternal calcium homeostasis or the calcium content of milk (54). Therefore, although PTHrP remains an appealing candidate regulator of maternal calcium homeostasis during lactation, such an action remains unproved. The second potential function of PTHrP during lactation concerns the regulation of vascular a n d / o r myoepithelial cell tone. As discussed in Chapter 16, PTHrP has been shown to modulate smooth muscle cell tone in a variety of organs, including the vascular tree, where it acts as a vasodilator. Consistent with these effects, two studies have shown that PTHrP increases

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m a m m a r y blood flow during lactation (55,56). The injection of amino-terminal fragments of PTHrP into the m a m m a r y artery of dried ewes was shown to increase m a m m a r y blood flow and to override the vasoconstrictive effects of endothelin (55). Thiede and colleagues have demonstrated that the nutrient arteries of the inguinal m a m m a r y glands of rats make PTHrP and that its production is responsive to suckling and prolactin (56). Myoepithelial cells in the breast are similar, in some ways, to vascular smooth muscle cells and are thought to participate in the control of milk ejection by contracting in response to oxytocin (32). Therefore, it is interesting that myoepithelial cells in culture have been shown to express the PTH1R, and to respond to PTHrP by elevating intracellular cAMP (35,36). Furthermore, mirroring the effects of PTHrP on endothelin-induced contraction of vascular smooth muscle, PTHrP has been shown to block the rise in intracellular calcium normally induced in response to oxytocin in myoepithelial cells (35). Although much more study is needed, the current data support speculation that PTHrP might have effects on m a m m a r y blood flow a n d / o r milk ejection.

Pathophysiology of PTHrP in the Mammary Gland Although PTHrP has not been directly implicated in the pathogenesis of any specific disease of the mammary gland, there are now several instances in which it appears to contribute to pathophysiology in the h u m a n breast. First, as noted previously, fetuses afflicted with Blomstrand chondrodystrophy lack nipples and breast tissue (31). Second, there have been two case reports in which lactational hypercalcemia was noted to be related to elevations in circulating levels of PTHrP (57,58). One of these cases was caused by massive breast hyperplasia, and the patient required reduction mammoplasty in order to ameliorate her hypercalcemia (57). Finally, the area with the most potential impact on h u m a n health is the relationship of PTHrP production to breast cancer. This is evolving into a complicated topic and will be addressed only briefly here. However, it will be reviewed in more depth in Chapter 43. It is well documented that PTHrP is produced by a n u m b e r of primary breast carcinomas and that this sometimes leads to classic humoral hypercalcemia of malignancy (59). A potentially more widespread role may be the involvement of PTHrP in the osteotrophism of breast cancer (60,61). Animal models have suggested that PTHrP production by breast tumor cells is important to their ability to form skeletal metastases (60,61). However, there is conflicting evidence as to whether PTHrP production by a primary breast tumor is predictive of bone metastases in patients (62,63). The largest and most carefully controlled study to date suggested

that PTHrP production by the primary tumor is actually a negative predictor, not a positive predictor, of skeletal metastases (63). It may be that PTHrP production does not enable a tumor cell to get into the skeleton, but once there, the ability of tumor cells to up-regulate PTHrP production within the bone microenvironment becomes important to their ability to grow in the skeleton (61). These are important issues and ongoing studies should provide us more information in the near future.

REPRODUCTIVE TISSUES PTHrP and Placental Calcium Transport Nearly all of the calcium, and a large proportion of the inorganic phosphate (85%) and magnesium (70%), transferred from the mother to the fetus is associated with development and mineralization of the fetal skeleton (64). The concentrations of both total and ionized Ca in all mammalian fetuses studied during late gestation have been observed to be higher than maternal levels. As a result of studies in which sheep were used extensively for the study of fetal calcium control, one of the first suggested physiologic roles of PTHrP was that of regulating the transport of calcium from m o t h e r to fetus in the mammal, thereby making calcium available for the growing fetal skeleton (65). Immunoreactive PTH levels were found to be low in fetal lambs, whereas PTH-like biological activity in serum was high (66), suggesting the presence of another PTH-like substance. Parathyroidectomy in the fetal lamb resulted in loss of the calcium gradient that exists between m o t h e r and fetus as well as impairment of bone mineralization, implicating the parathyroids as the source of the regulatory agent. Crude, partially purified, or recombinant PTHrP, but neither PTH nor maternal parathyroid extract that contained no immunoreactive PTHrP, restored the gradient (65). Thus, PTHrP appeared to be the active c o m p o n e n t of the fetal parathyroid glands responsible for maintaining fetal calcium levels and suppressing fetal PTH levels. In support of this hypothesis, immunoreactive PTHrP was found to be readily detectable in sheep fetal parathyroids from the time they form (67) and also was found in early placenta, suggesting that the latter tissue may be a source of PTHrP for calcium transport early in gestation. The portion of PTHrP that appears to be responsible for regulating placental calcium transport lies between residues 67 and 86 (68), but the responsible receptor has not yet been identified. T h o u g h the syncytiotrophoblasts are believed to be central in the transport of calcium to the fetus, the cytotrophoblasts (which differentiate to form the syncytium) are believed to be the calcium-sensing cells, and elevating the extracellu-

PHYSIOLOGYOF PTH AND PTHrP lar calcium concentration has been shown to inhibit PTHrP release from these cells (69). The calciumsensing receptor (CaSR) has been localized to cytotrophoblasts of h u m a n placenta (70) and the work of Kovacs et al. (71) has implicated it in placental calcium transport. Furthermore, a calreticulin-like calciumbinding protein has been isolated from trophoblast cells and its expression is increased by treatment with PTHrP(67-84) but not with N-terminal PTHrP (72). A working hypothesis for how the CaSR, midregion PTHrP, and a midregion PTHrP receptor might interact to regulate transplacental calcium transport is presented in Fig. 3. Although these observations are strongly suggestive of involvement of PTHrP and the CaSR, the mechanisms of placental calcium transport are still not fully understood. Support for the role of PTHrP also comes from the PTHrP gene knockout mouse in which placental calcium transport is severely impaired (73). In mice homozygous for deletion of the PTHrP gene, fetal plasma calcium and the maternal-fetal calcium gradient were significantly reduced. When fetuses were injected in utero with fragments of PTHrP or PTH, cal-

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FIG, 3 A potential model for the interactions of PTHrP and the calcium-sensing receptor in regulating placental calcium transport. A decrease in circulating calcium levels in the fetus activates the calcium-sensing receptor on the cytotrophoblast. This leads to PTHrP secretion by the cytotrophoblast, which may then act via the putative midmolecule PTHrP receptor to promote calcium transport from maternal to fetal circulation. The nature and cellular location of the midmolecule PTHrP receptor are unknown, but are likely to be on the syncytiotrophoblast implicated in calcium transport across the placenta. Midmolecule PTHrP signaling could involve several mechanisms as indicated and may include participation of a calcium-binding protein (CaBP) (modified from Bradbury, Ph.D. Thesis, University of Sydney 1999, with permission).

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cium transport was significantly restored only by treatment with a midmolecular region of PTHrP that does not act via the PTH1R. Thus, the conclusions from the murine studies are similar to those in the sheep, namely, that PTHrP contributes to fetal skeleton calcium supply by controlling maternal-fetal calcium transport through actions mediated by a midmolecule portion of the PTHrP molecule.

Uterus and Extraembryonic Tissues The uterus, both pregnant and nonpregnant, is another of the many sites of production and action of PTHrE The relaxing effect of PTH on uterine smooth muscle has been long recognized (74), and it is not surprising that PTHrP has the same effect (75). The finding that expression of mRNA for PTHrP in the myometrium during late gestation in the rat was controlled by intrauterine occupancy by the fetoplacental unit raised the possibility of a role for PTHrP in regulating uterine muscle tone (76). In studies in rats with or without estrogen treatment, protein and mRNA for PTHrP were localized not only in the myometrium, as had been shown in pregnancy (76), but also in the epithelial cells lining the e n d o m e t r i u m and endometrial glands. Indeed, the strongest PTHrP production appeared to be in these sites (77), suggesting that the e n d o m e t r i u m and endometrial glands might be the major uterine site of PTHrP production, and that PTHrP might be a local regulator of endometrial function and myometrial contractility. Estrogen treatment enhanced uterine production of PTHrP, but most significantly, the relaxing effect of PTHrP on uterine contractility in vitro was greatly enhanced by pretreatment of noncycling rats with estrogen. In keeping with this observation, uterine horns from cycling rats in proestrous and estrous phases of the cycle showed a greater responsiveness to PTHrP than did those from noncycling rats. These findings are consistent with a role for PTHrP as an autocrine a n d / o r paracrine regulator of uterine motility and function. Furthermore, they suggest that PTHrP belongs to a class of other locally acting peptides such as oxytocin, vasoactive intestinal peptide, and relaxin, for which pretreatment of animals with estrogen increases the response of the uterus (78-80). Further evidence for a specific and regulated role of PTHrP in the uterus during gestation comes from the observation of a temporal pattern in the relaxation response to PTHrP by longitudinal uterine muscle during pregnancy in the rat, with maximal responses at times when estrogen levels would be high. In contrast, the circular muscle did not respond at any stage during gestation (81). The inability of PTHrP to relax uterine muscle in the last stages of gestation does not support a direct role in the onset of parturition. It has been

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hypothesized that PTHrP may be involved in keeping the uterine muscle relaxed to accommodate the fetus during pregnancy, with the demonstration (76) that expression of mRNA was dependent on the presence of the fetus and that levels increased throughout pregnancy and decreased sharply after delivery. It seems likely, therefore, that the observed decrease in PTHrP level reflects the recontracted state of the uterine muscle, consistent with the observation in the bladder (82), and that the level of expression is functionally related to contractility. The temporal expression of PTHrP in the endometrial glands and blood vessels (81) also supports roles in other regulated functions that might include uterine growth during pregnancy and the regulation of uterine and placental blood flow (83). Placenta and Fetal Membranes

PTHrP mRNA and protein have been detected in rat and human placenta in various cell types (69,84,85). In addition, neoplastic cells of placental origin secrete PTHrP, including hydatidiform moles and choriocarcinomas in vitro (86). The presence of P T H / P T H r P receptor mRNA has been demonstrated in rat (87) and human (88) placenta, and infusion of PTHrP(1-34) into isolated h u m a n placental lobules stimulates cyclic AMP production (89). Three further sets of observations lend support to the hypothesis that PTHrP is involved in placental/uterine interactions and that its most likely role in the placenta and placental membranes is related to the growth and maintenance of the placenta during pregnancy. First, PTHrP production by cultured amniotic cells has been shown to be regulated by prolactin, h u m a n placental lactogen, transforming growth factor-J3 (TGF-[3), insulin, insulin-like growth factor, and epidermal growth factor (90). Second, PTHrP has been shown to regulate epidermal growth factor receptor expression in cytotrophoblast cultures (91), an event associated with placental development. Third, studies of vascular reactivity in isolated human placental cotyledons preconstricted with a thromboxane A2 mimetic showed PTHrP to be a very effective vasodilator (92). The narrow concentration range to which the tissue responded, together with the desensitization in response to repeated PTHrP infusions, seemed consistent with a paracrine a n d / o r autocrine action of PTHrP in human gestational tissues. Adequacy of the fetoplacental circulation is essential for the nutritional demands of the growing fetus, and both humoral and local factors are likely to be important in its control. It is possible that alterations of the expression, localization, a n d / o r action of PTHrP might contribute to the genesis of conditions such as preeclampsia and intrauterine growth retardation, in

which placental vascular resistance is increased (93). Another related and potentially interacting influence is angiotensin II, known to be a powerful enhancer of PTHrP production in the vasculature and in human placental explants (94). The ability of angiotensin II to stimulate estradiol production in h u m a n placental explants through actions on its AT 1 receptor (95) provides a further link with PTHrP control. The most likely source of the increased amniotic fluid PTHrP concentrations during pregnancy is the amnion, because PTHrP mRNA expression is also highest at term and greater in the amnion than in choriodecidua or placenta (96-98). In tissue from women with full-term pregnancies and not in labor, the concentration of N-terminal PTHrP has been found to be higher in amnion covering the placenta than in the reflected amnion covering the decidua parietalis (96). Nevertheless, the concentration of N-terminal PTHrP in reflected amnion (the layer apposed to the uterus) was inversely related to the interval between rupture of the membranes and delivery. The observation that PTHrP levels in the amnion decrease after rupture of the fetal membranes has led to the proposal that PTHrP derived from the membranes may inhibit uterine contraction, and that labor may occur following loss of this inhibition. Plasma levels of PTHrP increase during pregnancy and at 6 weeks postpartum (99,100), with the likely sources being placenta and breast, respectively. Human fetal membranes have been shown to inhibit contractions of the rat uterus in vitro (101), so this tissue does appear to produce factors that can modulate uterine activity. Furthermore, primary cultures of human amniotic cells secrete PTHrP into the medium (84). Thus, though the physiological functions of amnion-derived PTHrP are currently unknown, the preliminary evidence suggests that it may play a role in the regulation of the onset of labor. It is also possible that it is a source of PTHrP ingested by the fetus, with a growth factor role in lung a n d / o r gut development. In summary, although many functional studies remain to be completed, potential roles for PTHrP produced by fetal membranes and placenta include transport of calcium across the placenta, accommodation of stretch of membranes, growth and differentiation of fetal a n d / o r maternal tissues, vasoregulation, and the regulation of labor.

Implantation and Early Pregnancy Some physiologic functions other than control of myometrial activity were suggested by findings of Beck et al. (102), who identified PTHrP mRNA as being limited to epithelial cells of implantation sites. This pregnancy-related expression appeared at day 5. 5 in

PHYSIOLOGYOF PTH AND PTHrP the rat fetus in the antimesometrial uterine epithelium of implantation sites, raising the possibility of a further function of PTHrP, playing a part in the localization of implantation or initial decidualization. Decidual cells produced mRNA for PTHrP both in normal gestation and after the induction of deciduomata. Expression of the gene in these cells followed epithelial expression by 48 hours. It was concluded from this work that the location of PTHrP gene expression in the uterus, together with the time of its expression, suggests that it plays a part in implantation of the blastocyst. Further evidence for a function of PTHrP in the implantation process came from Nowak et al. (103), who showed that PTHrP and TGF-[3 were potent stimulators of trophoblast outgrowth by mouse blastocysts cultured in vitro. The TGF-[3 effect appeared to be mediated by PTHrP, which was acting through a mechanism distinct from the PTHIR. Thus, both the timing and localization of PTHrP gene expression suggested that it might play a part in the implantation of the blastocyst (102). On finding substantial levels of immunoreactive PTHrP in uterine luminal fluid of estrogen-treated immature rats, and because the P T H / P T H r P receptor was known to be expressed in rat uterus (87), Williams et al. (104) investigated the role of PTHrP acting through this receptor, in influencing early pregnancy in the rat. Infusion of either a PTHrP antagonist peptide or a monoclonal anti-PTHrP antibody into the uterine lumen during pregnancy resulted in excessive decidualization. The latter appeared to be the result of a decrease in the n u m b e r of apoptotic decidual cells in the antagonist infused horn. In p s e u d o p r e g n a n t rats, infusion of receptor antagonist into the uterine lumen resulted in increases in wet weight of the infused h o r n compared with the control side, indicating an effect on decidu o m a formation. These observations suggest that activation of the P T H / P T H r P receptor by locally produced PTHrP might be crucial for normal decidualization during pregnancy in rats, probably not by being involved in the initiation of the decidual reaction, but rather in the maintenance of the decidual cell mass. Summary The multiple roles of PTHrP in the reproductive tissues and cycle and in the placenta largely reflect its roles as a p a r a c r i n e / a u t o c r i n e / i n t r a c r i n e regulator. Of the many functions it exerts in these systems, probably the only endocrine role is that in which PTHrP in the fetal circulation regulates placental calcium transport. There remains much to be learned of the role of PTHrP in reproductive and placental physiology and pathology.

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THE END O CRINE PANCREAS PTHrP and Its Receptors in the Endocrine Pancreas The presence of PTHrP in the pancreatic islet became apparent shortly following the identification of PTHrP in 1987. Drucker, Asa, and colleagues (105) demonstrated that PTHrP was present in islet cells, and demonstrated that it was present in all four cell types of the islet, including the alpha, beta, delta, and PP cells. PTHrP mRNA was demonstrated to be present in isolated islet RNA as well (106), demonstrating that the peptide could be p r o d u c e d within the islet. Gaich and collaborators (107) confirmed these findings, demonstrating that PTHrP was indeed present in islet cells of all four types, and that it was also present in pancreatic ductular epithelial cells. The peptide is not present in adult pancreatic exocrine cells. Plawner and colleagues (108) demonstrated that PTHrP is present in individual beta cells in culture, and showed that PTHrP colocalized with insulin in the Golgi apparatus, as well as in insulin secretory granules. Interestingly, in a perifusion system employing a beta cell line, PTHrP was shown to be cosecreted with insulin from beta cells following depolarization of the cell (108). The secreted forms of PTHrP included amino-terminal, midregion, and carboxy-terminal forms of PTHrP (see below). With regard to receptors for PTHrP on beta cells, little direct evidence has been provided for the presence of the PTHIR, although its presence has not been rigorously sought. On the other hand, there can be no question as to the presence of some type of PTHrP receptor on the pancreatic beta cell, because it is clear that PTHrP(1-36) elicits p r o m p t and vigorous responses in intracellular calcium in cultured beta cell lines. For example, Gaich et al. have demonstrated that PTHrP(1-36) in doses as low a s 1 0 -12 M stimulates calcium release from intracellular stores (107). Interestingly, unlike events observed in bone and renal cell types in which PTHrP receptor activation is associated with activation of both the cAMP/PKA and the PKC/intracellular calcium pathways, only the latter is observed in cultured beta cells in response to PTH or PTHrP(1-36) (107). W h e t h e r this reflects the presence of a different type of receptor on beta cells or differential coupling of the P T H I R to subsets of specific G proteins or catalytic subunits in beta cells, as compared to bone and renal cells, has not been studied.

Regulation of PTHrP and PTHrP Receptors There is little information describing how or to what degree PTHrP or the PTH receptor family is regulated in the pancreatic islet. As will become clear from the

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following discussions, there are physiologic reasons why such regulation might occur u n d e r normal circumstances, but this area remains unexplored.

Biochemistry of PTHrP in the Endocrine Pancreas PTHrP undergoes extensive posttranslational processing as described in Chapters 3 and 4. Most of what is known or inferred regarding PTHrP processing is derived from studies in the rat insulinoma line, RIN1038 cells (17,109,110). These cells have served as a model of PTHrP processing because they have been shown to serve as a model for authentic processing of other h u m a n neuroendocrine peptides such as insulin, proopiomelanocortin, glucagon, and calcitonin. Using a combination of untransfected RIN-1038 cells, RIN-1038 cells overexpressing hPTHrP(1-139), hPTHrP(1-141), or hPTHrP(1-173), and a panel of region-specific radioimmunoassays and immunoradiometric assays, RIN cells have been shown to secrete PTHrP(1-36), PTHrP(38-94), PTHrP(38-95), and PTHrP(38-101) (17,109,110). In addition, RIN-1038 cells have been shown to secrete a form of PTHrP that is recognized by a PTHrP (109-138) radioimmunoassay (109), and another form that is recognized by a PTHrP (139-173) radioimmunoassay ( 111 ). As described above, PTHrP(1-36) stimulates intracellular calcium increments in cultured beta cells (107). PTHrP(38-94) has also been shown to stimulate intracellular calcium release in these cells (110). PTHrP(38-94) does not activate adenylyl cyclase in cultured beta cells, and other PTHrP species have not been explored in beta cells in functional terms.

Function of PTHrP in the Endocrine Pancreas Pancreas development in rodents begins at approximately days E9-E10, and by days E18-E19, clusters of beta cells have begun to coalesce and form immature islets (112). These islet cell clusters continue to increase in number, in size, and in density of beta cells in the week following delivery and then decline abruptly in n u m b e r through a wave of beta cell apoptosis (113). The role of PTHrP in beta cell development and function is poorly understood at present. The pancreas of PTHrP-null mice (20) develops normally in anatomic terms (R.C. Vasavada and A.E Stewart, unpublished observations), but nothing is known about the function of these islets. PTHrP-null mice die immediately after delivery, so nothing is known of islet function or development following birth in the absence of PTHrP. "Rescued" PTHrP mice do exist (28) and they survive to adulthood. These mice have normal-

appearing pancreata and islets (R.C. Vasavada and A.E Stewart, unpublished observations), but they have dental abnormalities, are undernourished, and they grow poorly. Therefore, it is difficult to characterize their islets in functional terms, because islet mass, proliferation, and function are heavily d e p e n d e n t on fuel availability. Steuker and Drucker have suggested that PTHrP may play a role in beta cell differentiation, because it is up-regulated in beta cell lines in the presence of the islet-differentiating agent, butyrate (114). In an effort to understand the role of PTHrP in the pancreatic islet, Vasavada and collaborators have developed transgenic mice that overexpress PTHrP under the control of the rat insulin-II p r o m o t e r (RIP) (115,116). RIP-PTHrP mice display striking degrees of islet hyperplasia and an increase in islet n u m b e r as well as in the size of individual islets. This increased islet mass is associated with increased function: RIP-PTHrP mice are hyperinsulinemic and hypoglycemic as compared to their littermates (115,116). They become profoundly and symptomatically hypoglycemic with fasting. Interestingly, RIP-PTHrP mice are also resistant to the diabetogenic effects of the beta cell toxin, streptozotocin. Following the administration of streptozotocin, normal mice readily develop diabetes, but RIP-PTHrP mice either fail to become diabetic or develop only mild hyperglycemia (116). The mechanisms responsible for the increase in islet mass in the RIP-PTHrP mouse remain undefined. There are two levels at which this question can be addressed: identification of the source of the cells responsible for the increase in islet mass and the signaling mechanisms that are responsible for the increase. With respect to the first, islet mass can, in theory, be increased by three pathways: (1) the recruitment of new islets from the pancreatic duct or its branches distributed t h r o u g h o u t the exocrine pancreas, in a process referred to as "islet neogenesis"; (2) induction of proliferation of existing beta cells within islets; a n d / o r (3) prolongation of the life span of existing beta cells. Of these options, there is clear evidence against the second possibility (115), suggesting that islet neogenesis [e.g., PTHrP is present in the normal pancreatic duct and is up-regulated during ductular differentiation into beta cells (107,117)] or inhibition of islet cell death (as occurs in the presence of PTHrP in other cell types) is the likely explanation. These processes are u n d e r active study. At the signaling level, little is known regarding the mechanism of action of PTHrP on beta cells. Though it is known that PTHrP can stimulate intracellular calcium in cultured beta cell lines (107,110), it is not known whether this occurs in vivo in normal, nontransformed beta cells within intact islets. Nor is it known if PTHrP stimulates adenylyl cyclase in normal beta cells in vivo, or if it participates in nuclear or intracrine sig-

PHYSIOLOGYOF PTH AND PTHrP naling in beta cells as it appears to in chondrocytes, osteoblasts, vascular smooth muscle cells, or other cell types (118-120) (see Chapter 6). These processes, too, are u n d e r study. Finally, and importantly, the results of overexpression studies do not demonstrate that PTHrP plays beta cell mass-enhancing roles in vivo u n d e r normal circumstances. In the absence of meaningful data from knockout or rescued knockout mice, it is difficult to be sure if PTHrP is important in normal islet biology. This question, too, must await further studies, such as examination of the conditional or islet-specific deletion of the PTHrP gene.

Pathophysiology of PTHrP in the Endocrine Pancreas From the preceding discussion, it is clear that the normal physiologic role of PTHrP in the pancreatic islet remains undefined. In contrast, PTHrP plays clear pathophysiologic roles in at least some pancreatic islet neoplasms. PTHrP overexpression with resultant development of humoral hypercalcemia of malignancy has been demonstrated on multiple occasions in multiple investigators' hands (105,121-123). In the only large series of studies of malignancy-associated hypercalcemia in which tumors have been fully subdivided based on histology (123), islet cell carcinomas, which are not particularly common, produce humoral hypercalcemia of malignancy fully as often as pancreatic adenocarcinomas, a very c o m m o n neoplasm. Historically, islet tumors were among the first in which PTHrP bioactivity was identified (121,122). Furthermore, patients with islet carcinomas regularly demonstrate increases in circulating PTHrP as determined by radioimmunoassay or immunoradiometric assay (21). When assessed by immunohistochemistry, these tumors also demonstrate increased staining for PTHrP (105,106). The significance of these findings for islet tumor oncogenesis is not known. Is this simply a r a n d o m derepression of the PTHrP gene? Or is it a specific up-regulation of the PTHrP gene? Is there a pathologic role for PTHrP in the development of pancreatic islet tumors, corresponding to the mass enhancing effects of PTHrP in the islets of the RIP-PTHrP mouse? These questions remain interesting but unanswered at present.

CONCLUSION Advances in mouse genetics and in transgenic technology have been a b o o n to the study of physiology. This has certainly been the case for the PTHrP field, wherein studies in genetically altered mice have provided a starting place for the study of the physiology of

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a protein that was discovered out of its natural context. In this chapter we have outlined the current state of knowledge regarding the physiologic roles of PTHrP in skin, the m a m m a r y gland, placenta, uterus, and pancreas. Much of this information (although not all) has come from studies performed in a variety of transgenic mice. These studies have shown that PTHrP is important to both the development and the physiologic functioning of these organs. However, at this point, we have only the rudiments of an understanding of the functions of PTHrP at these sites. Thus, we continue to have more questions than answers. There will be many challenges to be overcome before we truly c o m p r e h e n d all the nuances of the functions of PTHrP at these sites. There will also be new tools with which to investigate these questions (as this chapter is being written, several eagerly anticipated experiments ablating the PTHrP and P T H I R genes in organ-specific fashion are in the pipeline). The next several years promise to be an exciting time for the investigation of the nonskeletal effects of this remarkable molecule, and the next edition of this volume will certainly d o c u m e n t some upcoming surprises.

ACKNOWLEDGMENTS The authors would like to thank Dr. Jane Moseley for valuable discussions and advice. This work was supported by the National Health and Medical Research Council of Australia (to J.T.M.), the National Institutes of Health (DK47168 and DK55023 to A.ES.; DK55501 to j j . w . ) , the U.S. Department of Defense (DAMD17-96-16198 to j.j.w.), and the Juvenile Diabetes Foundation (to A.ES.).

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CHAPTER18 Parathyroid Growth Normal and Abnormal

A. M I C H A E L

PARFITT

Little Rock, Arkansas 72205

Division of Endocrinology and Centerfor Osteoporosis and Metabolic Bone Disease, University of Arkansas for Medical Sciences,

INTRODUCTION AND BACKGROUND

parathyroid gland. Yet another reason is that people are attracted to endocrinology by their fascination with the h o r m o n e s and hormonal mechanisms of secretion and action, rather with the morphology of their glands of origin. A neglect of gland size may be justified pragmatically if the aim is restricted to understanding normal physiology, but it involves neglect also of several interesting scientific questions. Why is the parathyroid gland so much smaller than many other endocrine glands? Why is total parathyroid weight a m u c h larger fraction of body weight in the chick (---10 m g / k g ) than in the rat (---1 m g / k g ) (3)? Such questions lie within the realm of evolutionary biology, but their answers might well provide information relevant to medical science. At a more practical level, short-term changes in h o r m o n e secretion are of lesser magnitude than those in other glands and are not sustainable indefinitely; long-term changes, whether they are expressions of adaptation or of disease, invariably require changes in cell n u m b e r as well as in individual cell function. Every parathyroid disorder considered in this book is a reflection of, or at least is associated with, characteristic changes in the n u m b e r of functioning parathyroid cells. Understanding the mechanisms whereby parathyroid cells are able to change their n u m b e r is essential to a full understanding of pathogenesis and is also relevant to both medical and surgical treatment. However, before these clinically important issues are approached, it is necessary to review concepts of normal organ and tissue growth, the regulation of cell n u m b e r as the balance between cell proliferation and cell death, and the normal growth

Why Study Parathyroid Growth? The functional performance of every endocrine gland requires delivery of the right n u m b e r of horm o n e molecules into the circulation during each successive interval of the appropriate time scale. Total h o r m o n e secretion comprises the aggregate contribution of each cell and so depends not only on the average secretion per cell, but also on the n u m b e r of contributing cells. The regulation of cell number, it seems, should receive as much attention as the regulation of individual cell behavior, but in practice it is almost entirely ignored. For example, in a 3000-page textbook of endocrinology (1), less than 1% of the material is concerned with the attainment and maintenance of gland size. Whether there are 100 or 1 billion cells in a gland appears not to matter, and cell n u m b e r is never considered explicitly in the description of feedback relationships. There are several reasons for this neglect. The rules of development normally ensure that each organ grows to the right size (2), so that adult cell n u m b e r varies only over a three- to fourfold range. By contrast, h o r m o n e secretion by many endocrine glands can vary acutely over as much as a 100-fold range, which must reflect changes in the performance of individual cells. Another reason is that the endocrine glands are among the smallest of organs. Only the thyroid and the gonads can be palpated; the other glands are inaccessible clinically, and estimation of their size by noninvasive methods is of varying precision, which is least for the The Parathyroids, Second Edition

293

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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CHAPTER18

and turnover of parathyroid tissue. A central theme will be the close interrelationship between the regulation of h o r m o n e secretion and the regulation of cell n u m b e r in the parathyroid glands.

Concepts of Growth There is more to biologic growth than increase in size; growth has been described as "the study of change in an organism not yet mature" (4), which includes the processes of development and the determination of form, both external and internal. Relative growth is most rapid in utero, although for some tissues and organs absolute growth is more rapid after birth. It is useful to extend the scope of growth to cover also maintenance by turnover, repair, and regeneration (5). Growth can be studied at different levels of organization, both functional and structural. A functional unit, the smallest structure that can carry out a specific function by itself, may correspond to any structural level: whole organisms, organ, tissue, cell, or subcellular entity. In the endocrine system, functional units may be either multicellular, such as thyroid and ovarian follicles, or unicellular, as in the anterior pituitary and the testis. All functional units can increase or decrease in size (hypertrophy or hypotrophy), but only some can increase or decrease in n u m b e r (hyperplasia or hypoplasia) (3,5,6). During early development, hyperplasia, whether of individual cells or of functional units, precedes hypertrophy (2,7). After birth there is overlap between these different modes of growth, although in most organs significant hypertrophy is absent after the first 2 weeks, and at all ages hyperplasia is the main determinant of increase in size (3). The basic instrument of growth is the cyclical process of cell replication and division. Despite spectacular advances in the understanding of this process (8), along with understanding of its stimulation by an ever larger n u m b e r of growth factors (8-10), knowledge concerning growth regulation at higher levels of organization remains incomplete (11,12). In species of finite life span and determinate size, a growth target for the whole body and for each organ has to be specified very early in development (2,5), and approach toward the target has to be monitored and controlled in accordance with a characteristic growth curve. Precisely how such a "sizostat" (13) could function remains a mystery, but its rules of operation must be encoded somewhere in the g e n o m e (11). Many general theories of growth have been proposed, but none has become established. Almost 200 years ago, J o h n H u n t e r concluded that growth was regulated by functional d e m a n d (3). This remains a sound principle of physiologic adaptation in mature organisms but cannot be the main explanation for developmental growth. During functional adapta-

tion, hypertrophy is the initial response and may remain the only response if the work of the cell is mainly physical or if the functional unit is multicellular and of sufficient complexity that new ones cannot develop after birth, as for n e p h r o n s or lung alveoli (5). However, if the work of the cell is mainly biochemical, as in the endocrine glands, eventually hyperplasia will also develop. It has been proposed that the default state of all normal cells is proliferation rather than quiescence (12). In accordance with this notion, each organ or tissue may secrete, in proportion to its size, an inhibitor of its own growth, referred to as its chalone (3,14). Much evidence for the existence of chalones has been assembled (15), including feedback regulation of intestinal crypt cell proliferation in a m a n n e r consistent with the chalone concept (16), but no chalone has been fully characterized (14). A currently more popular theme is external control by means of tissue-specific stimulators, but most of these remain unidentified, and most known growth factors are ubiquitous in their distribution and sites of action (8-12). In some organs the same growth factors may be involved in developmental growth, regeneration after injury, and functional adaptation (17,18). In the pituitary and probably also in the glands u n d e r its control, the same h o r m o n e s stimulate growth as well as h o r m o n e secretion (19), but at the appointed time growth stops, yet h o r m o n e secretion continues. There is evidence for central regulation of targeted whole-body growth by a non-growth-hormone-dependent mechanism in the brain (3,13), but individual organs are more likely to be controlled locally by some widely distributed system, such as cells derived from the neural crest, lymphoid tissue (3), or vascular endothelium (20). The same growth factor could be used by different tissues, target specificity depending on autocrine or paracrine mechanisms (9), or on the constraints of the local microcirculation, but this would not explain how a predetermined size was chosen and reached. The possible role of parathyroid endothelium (21) is mentioned below.

Patterns of Cell Renewal All cells can be classified, on the basis of their current relationship to the cell cycle (Fig. 1), as cycling or noncycling. Cycling cells are in one of the four stages of the cycle: G 1, S, Gz, or M. Noncycling cells may be in a resting state (Go) between one cycle and the next or no longer dividing because of terminal differentiation (14,22). Cells in G o have withdrawn from the cycle and differ from cells in G 1 in several respects (14), most obviously in that they are not enlarging in preparation for cytokinesis. From the standpoint of cell kinetics, all adult tissues have traditionally been classified into one

PARATHYROID GkOWrH: N O I ~ . L AND ABNORMAL /

295

G2 ¸

M

DIFF (NC)

S

" ~ Death

"1~ (shedding)

Discontinuous (low turnover) (GO) G2

M

v

DIFF (Go)

y

S

.,d

"1~ Death (apoptosis)

8

FI6. 1 Cell cycle in two types of tissue with respect to replication. The cell cycle comprises two periods of executionmDNA synthesis (S) and mitosis (M)--interrupted by two periods of preparationmgaps (G) 1 and 2. Durations (in hours) are chosen partly for convenience of illustration but are broadly representative of human nonneoplastic tissue (14). As well as duplication of the nucleus, the cycle involves duplication of all other constituents of the cell. The interrupted line between M and early G is traversed only by continuously cycling cells. Other cells enter a quiescent state with respect to proliferation (Go), the duration of which varies from days to decades in different tissues. Tissues in which replication is continuous (top) have high turnover, and a separate population of stem cells (ST). Periodically, a stem cell switches from G o to G1, and on average the mitosis leads to one new stem cell and one cell committed to differentiate. The commitment step is amplified by continuous cycling in a dividing transit (DT) compartment for a variable number of generations (two in this case). The differentiated cells (DIFF) are noncycling (NC) and are eventually lost by shedding, or by sequestration, as in bone. Tissues in which replication is discontinuous (bottom) have low turnover and no stem cell or dividing transit compartments. Periodically, a differentiated cell switches from G o to G1, and the result of the mitosis is two new differentiated cells. The addition of each new cell is balanced by loss of one old cell by apoptosis; it is this type of replication that occurs in the parathyroid gland.

of three major types (14,22). Tissues with a high rate of cell turnover (continuous replicators) have a high rate of mitosis, which occurs initially in a functionally (and often anatomically) separate population of stem cells (Table 1). These cells, usually in G 0, repeatedly traverse the cell cycle, each complete cycle on the average producing one new cell committed to differentiate in a particular direction and one new stem cell. Renewal of stem cells probably depends on a stochastic balance between two types of symmetric cell division rather than on each stem cell division being asymmetric (23). The committed daughter cells undergo clonal expansion in a transit c o m p a r t m e n t for a variable n u m b e r of generations (22,24). The n u m b e r of terminally differentiated, nondividing, but functionally active cells is maintained approximately constant by continuous shedding, either from the body (as in the skin or the intestinal mucosa) or into the circulation (as in the bone marrow).

TABLE 1

Contrasting Characteristics of Two Mechanisms for Maintenance of Adult Tissue Mass Type of replication

Aspect Cell function Cell life span Mitosis Potential Location Rate Cell loss Mechanism Rate

Continuous

Discontinuous

Poststem cells only Short

All cells Long

Stem cells and transit cells only Often segregated High

All cells

External b shedding High

Internal b deletion Low

Scattered LOWa

aCan increase sharply in response to various stimuli. bwith respect to the tissue.

296

/

CHAPTF~k18

By contrast, tissues with low cell turnover (discontinuous replicators or conditional renewal tissues) have a low rate of mitosis and no separate stem cell population (14,22) (Table 1). All cells of the tissue spend most of their time in G o carrying out normal function, but all have the potential for undergoing cell division. Periodically, a functioning cell enters the cell cycle (G o + G1), and the result of the mitosis is two new functioning cells. Cell balance is maintained by a few cells losing the capacity for division, eventually dying, and being removed in some way. The low basal rate of mitosis can be increased by the need to regenerate (as in the liver) or by increased functional d e m a n d (as in endocrine glands) and occurs at r a n d o m t h r o u g h o u t the tissue. Tissues such as brain and cardiac muscle used to be regarded as nonreplicators, the cells of which had permanently lost the capacity to divide, but it is now evident that at least some cells retain this capacity (25,26), although the great majority of adult cells in these tissues will not divide during a normal lifetime. Continuously and discontinuously replicating tissues differ in their relationship to the control of cell cycle progression, which depends on the sequential synthesis and destruction of different members of the cyclin family (27), and on passing several checkpoints, at which are verified successful completion of earlier stages and maintenance of genomic integrity (28). In the former, the duration of the cycle may be rate limitingma reduction from 36 to 24 hours could increase the rate of proliferation by 50%. But if the interval between successive divisions is more than 3 months, shortening the cycle can have only a trivial effect; tissue turnover depends only on the frequency with which quiescent cells enter the cycle; very little is known about how the switch from G o to G 1 is regulated (29), although protooncogenes such as c-myc may promote reassembly of the cell cycle machinery (8). What is the mechanism of cell loss in tissues with a low rate of turnover and no obvious means of shedding cells? Except in pathologic situations in which the blood supply is jeopardized, it does not occur by necrosis. In recent years a nonnecrotic mechanism of cell deletion has been identified, referred to as apoptosis (30). This process normally affects scattered single cells and has characteristic histologic and ultrastructural features, beginning with condensation and disintegration of the nucleus, followed by breaking up of the whole cell into m e m b r a n e - b o u n d fragments of varying size, which are rapidly subjected to phagocytosis by adjacent cells. The remaining cells simply close ranks so that no gap is left by the deleted cell. The distinction between necrosis and apoptosis may be likened to the distinction between m u r d e r and suicide. Apoptosis is an active process d e p e n d e n t on altered gene expression, of which the molecular mechanisms are u n d e r intense

investigation (31). Apoptosis is a convenient way of eliminating cycling cells with irreparable DNA damage (32) but more commonly occurs in noncycling cells that have outlived their usefulness (33). Particularly relevant to the present discussion is that whenever tissue that is hyperplastic as the result of endocrine stimulation undergoes involution, the process of cell loss occurs by apoptosis (3,30). For regulating the n u m b e r of functioning cells in tissues and organs with normally low turnover, apoptosis is as important as mitosis (30).

NORMAL PARATHYROID GROWTH

AND TURNOVER Methods of Study

The most straightforward m e t h o d of studying growth is to express some index of size, either weight or volume, as a function of age. Weight is preferred in that it is easier to measure accurately and is in general use in anatomy and pathology. Because of variation in tissue density, weight is a more accurate index of cell n u m b e r than is volume (= weight/density). A commonly used approximation is that 1 g of tissue contains 2 "~° (1.094 × 109) cells (34). This disregards variation in cell size but in the parathyroid gland gives results very similar to total DNA content (35). Parathyroid tissue contains fat cells and a vascular connective tissue stroma as well as the parenchyma consisting of chief cells and their derivatives (see Chapter 1). Parenchymal volume as a fraction of total volume can be measured by point counting on histologic sections (36), and parenchymal weight as a fraction of total weight can be derived from measurement of wholegland density, assuming a constant value for fat cell density (37). Estimated parenchymal weight provides a more accurate value for parenchymal cell n u m b e r than does total weight. Regrettably, even though "the best histological criterion (of diagnosis) is the weight of the gland" (3), weighing of parathyroid glands is frequently omitted, and only linear dimensions are recorded. The product of three dimensions (rectangular volume; RV) is significantly correlated with weight (r = 0.81) and can be used to estimate weight from the regression, Wt (g) = 0.585RV (cm ~) + 0.134 (38). The product of only two dimensions, measured in a representative section, also correlates quite well (r = 0.76) with weight (39). If the parathyroid glands are very small, as in the rat, measurement of weight is less accurate because complete removal of extraneous tissue is more difficult, and measurement of volume is a good alternative. This can be obtained by means of the Cavalieri principle from serial sections that cover the entire length of the e m b e d d e d gland. Volume can be calculated from the n u m b e r of sections, the average distance between the

PARATHYROIDGROWTH: NORMALAND ABNORMAL / sections, and the average cross-sectional area in the sections (36,40). For an unbiased estimate, it is necessary that the location of the first section with respect to the pole of the gland is selected randomly, but this precaution has usually been omitted. In the past, section area was measured by various planimetric and projection methods, but today point counting with systematic random sampling and an unbiased counting frame (36), digitization, or automated image analysis (41) would be used. The estimation of gland volume in this m a n n e r is essential for the most accurate distinction between hyperplasia (increase in cell number) and hypertrophy (increase in cell size). Average cell profile area can be measured in sections and average cell volume calculated on the basis of reasonable assumptions (42). More accurately, the n u m b e r of cells or cell nuclei per unit volume of tissue can be obtained through the disector m e t h o d (42). The distinction can also be approached biochemically, using total DNA as an index of cell number, and total protein as an index of total cell mass; the p r o t e i n / D N A ratio remains u n c h a n g e d in hyperplasia but increases in hypertrophy (3,14). Cell number, cell size, and tissue mass represent the "bottom line" of growth but give no information concerning mechanisms. For a complete description of cell cycle kinetics, the proportion of cycling cells (or growth fraction) and the durations of each phase of the cell cycle must be determined (14,22). Together these give the birth rate of new cells, from which can be calculated the rate of increase in tissue volume, usually expressed as potential doubling time (34). Comparison of this with the actual rate of tissue growth provides an indirect estimate of the rate of cell loss (22,30,34). The necessary methods are complex, are most appropriate for rapidly growing tissues, and have never been applied to the parathyroid gland. More generally useful is determination of the proportion of cells in one or more stages of the cell cycle (Table 2). The durations of S, G2,

TABLE 2

Cell Cycle Markers a

Aspect

Marker

Feature

Need either administration in vivo or cell survival ex vivo or in vitro Information already in nucleus

[3H]Thymidine BrDu

Accepted standard Non radioactive

Mitosis Ki-67 Cyclin/PCNA

First to be used Short half-life DNA repair as well as synthesis

aExcept for mitosis, these markers mainly label S phase but Ki-67 and cyclin/proliferating cell nuclear antigen (PCNA) label a greater fraction of the cycle.

297

and M phases (Fig. 1) vary between fairly narrow limits among different tissues (14,22), and, if a representative value is assumed, the birth rate of new cells can be estimated, provided the rate of proliferation is stable and growth is slow (34,38,40). The first cell cycle marker to be used was the change in nuclear morphology during mitosis (43). Prompt fixation for an adequate duration in fluid of the right pH is n e e d e d (44), and positive cells are much less frequent than with other cell cycle markers because the duration of mitosis is m u c h shorter (Fig. 1). If mitosis is arrested, mitotic figures will be more frequent, but cell birth rate can no longer be estimated. The most well established of such methods is identification of S-phase cells by autoradiography after administration of tritiated thymidine (14,22,34). Much less satisfactory is m e a s u r e m e n t of thymidine incorporation into acid-insoluble macromolecules, the results of which are influenced not only by the rate of DNA synthesis but also by thymidine pool size, activity of thymidine kinase, utilization of the salvage pathway for pyrimidine biosynthesis, and diffusion of thymidine into dead or dying cells (3,17,45). These pitfalls are m u c h more significant with acute in vitro experiments than with steady-state in vivo measurements, and in parathyroid adenomas there is a good correlation between thymidine incorporation and the proportion of S-phase cells (35,46). The S-phase cells can also be labeled by bromodeoxyuridine, a nonradioactive analog of thymidine that can be detected by immunostaining (3). Results can be obtained more quickly, but the m e t h o d is less useful for following the migration of labeled cells or the recognition of subsequent division by the dilution of label intensity. More widely applicable are methods for the immunologic identification of other cell cycle markers (43); because the information is already present in the nucleus, nothing has to be administered, and the methods can be applied to paraffin-embedded sections and so to archival material. Of those most commonly used, the MIB-1 antibody to the Ki-67 antigen has several advantages over proliferating cell nuclear antigen (PCNA). The latter has a long halflife so that expression persists for some time after the end of the cycle, expression can be induced in adjacent noncycling cells, and expression can occur in cells undergoing DNA repair as well as in cycling cells (40,47). Whatever m e t h o d is used, it is essential that an appropriately randomized sampling procedure is adopted, because biased sampling, which is often deliberate in diagnostic studies, can increase the a p p a r e n t prevalence of labeled cells by up to 50-fold (47). Cells can be sorted according to their stage in the cycle by flow cytometry based on their content of DNA, which doubles between G0/G 1 and Gz/M phases and is intermediate in S phase (48). Despite the very large n u m b e r of cells that can be counted, it is impossible to

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CHAPTER18

Cell Number, Proliferation, and Loss as

get accurate results when cell turnover is low and the growth fraction very small, because of cellular debris and other artifacts (49). In this circumstance, changes in DNA content are m u c h more likely to reflect changes in ploidy than changes related to the cell cycle. Cells with a normal n u m b e r of chromosomes are referred to as diploid. Cells with a diploid n u m b e r increased exactly by a power of 2 are polyploid, most commonly tetraploid, and occasionally octaploid or of higher ploidy (50). Polyploidy is a normal occurrence in the liver and the heart. The mechanism is unknown but presumably involves normal duplication of each chromosome, with failure of mitosis and cytokinesis. Such cells are, in effect, arrested in G2, a condition that may also represent a response to a growth stimulus that shares features of both hyperplasia and hypertrophy (14). Euploid cells can be either diploid or polyploid. Cells with an abnormal c h r o m o s o m e n u m b e r that does not differ exactly by a power of 2 from the diploid number are aneuploid, either h y p o - o r hyperdiploid. This is the result of loss or duplication of individual chromosomes, a cytogenetic abnormality that is not related to normal cell replication (50,51).

Cell No. (10 6)

Wt. (mg)

19.3

18 -

16.1

15 -

12.9

12 -

9.7

9-

6.6

6-

3.2

3-

0

0-

PRENATAL

_

0

8

-

F u n c t i o n s o f Age The parathyroid glands appear abruptly during the fifth week of gestation (52). Based on the very small a m o u n t of fat present during growth and a mean parenchymal cell density of 1.065 (37), the glands grow to a total parenchymal weight of---3 lxg at a crownr u m p length of---30 m m , corresponding to a gestational age o f - - - 8 weeks (3). Weight increases quite slowly from 3 to 6 txg between 8 and 12 weeks, but then increases m u c h more rapidly, growing to ---300 Ixg (0.3 mg) between 12 and 18 weeks, and ---4 mg, corresponding to ---4.3 × 1 0 6 cells at birth (3). Based on interpolation and smoothing of quite sparse data (3), parathyroid growth appears to follow a sigmoid curve through the end of the first year of life, or up to 92 weeks of conceptional age (Fig. 2). The conversion of weight to cell n u m b e r is only approximate, because in the rat parathyroid cell volume increases ---50% in the first 10 weeks of life (53). Similar sigmoid growth curves have been found for the adrenal, thyroid, and pituitary glands (54), but with the inflections occurring before

=B

•

I

•

I I

POSTNATAL

w

!

r

!

!

f

I

I

i

i

!

16

24

32

40

48

56

64

72

80

88

96

Age (weeks since conception)

FIG. 2 Parathyroid growth curve during the first 2 years of life. Total parenchymal weight and calculated cell number are plotted as a function of age (in weeks) since conception. The cell number scale assumes that 1 mg of tissue contains 1.074 x 106 cells (34) and disregards modest changes in parathyroid cell volume during growth (53). A continuous curve w a s constructed based on data in Refs. 3 and 52. The dashed line denotes time of birth (B); the dotted line indicates predicted outcome of continued exponential growth at the peak rate found at 16 weeks.

PARATHYROIDGROWTH: NORMALAND ABNORMAL / rather than after birth. If the interpretation given in Fig. 2 is correct, absolute weight gain is most rapid at "--20 weeks after birth, although relative weight gain is most rapid at ---16 weeks of gestational age. Parathyroid growth continues at the rate, established toward the end of the first year, of ---2.5-3.0 mg/year. The data are too few to demonstrate a second parathyroid growth spurt during adolescence, but such a spurt has been found in every organ and tissue for which sufficient data are available (54), and it seems reasonable to assume that such a spurt would also occur in the parathyroid gland (Fig. 3). Growth then occurs progressively more slowly until the mature parenchymal weight is reached at age ~-30 years (3,55). Total mature weight in white subjects has varied from "-85 mg (3,55,56) to "--95 mg (57,58), corresponding to a total cell n u m b e r of (90-100) × 10 6, with a coefficient of variation [CV; = (SD/mean)100] of---30% (55), but there is no significant further change with increasing age (3,56,57). In different series, parathyroid weight in m e n has been less than (3), the same as (55), or more than (56,58) that in women. In three series (56,58,59), parathyroid weight has been significantly greater in black than in white subjects of both sexes, to a greater extent than could be accounted for by differences in body weight. Also in blacks, in contrast to whites, there appear to be significant changes in parathyroid weight after attainment of maturity, with peak values in both sexes between the ages of 41 and 60 years (58).

Cell No. (106)

During embryonic development, in organs and tissues that change in location or shape, as well as in size, or in which provisional structures must be replaced, growth is partly offset by apoptotic deletion of unwanted cells (30), but there is neither evidence nor apparent need for this process in the developing parathyroid gland. Assuming the absence of apoptosis, the first derivative of the growth curve, or growth velocity, provides an estimate of the absolute frequency of mitoses associated with growth, which is about five times higher during the first year of life than at any other time (Fig. 3), presumably with a second, much smaller peak during the adolescent growth spurt. The absolute frequency of cell division depends on the n u m b e r of cells present at a particular time and on the relative rate of cell division, or specific cell birth rate; the latter rate determines how frequently successive divisions occur in the same cell line. Disregarding turnover and considering only growth, this rate is most rapid during the second trimester of embryonic development, which is the only time of life when the interval between successive divisions is <1 month. The peak rate (3400%/year) occurs at --- 16 weeks, when each cell is dividing about once every 10 days and the population doubling time is just over 1 week (Fig. 4). The specific birth rate then falls rapidly by more than 10-fold to ---300%/year at birth, with a doubling time o f - - - 3 months, continuing to fall to ---8%/year at age 9 years, with a doubling time of about 8 years. There is a

Rate of gain Wt. Cell No. (mg/y) 106/y

Wt. (mg)

96.7

90 -

85.9

45

48.3

80-

40

43.0

75.2

70-

35

38.6

64.4

60- !

30

32.2

53.7

50- 1~

25

26.9

20

21.5

15

16.1

10

10.7

43.0

II No. Attained

ii "' 40-

32.2

/

iT

- - ' - - Wt/Cell No. rate of Gain

Jl!T

21.5

eo-li

10.7

100

299

0B

~..:

-5

4

8

12

16

20

24

Age (years since conception)

28

32

36

5.4

FIG. 3 Parathryoid growth and velocity curves, from birth (B) to age 36 years. Total parenchymal weight and cell number, calculated as for Fig. 2, plotted as a function of age (in years) since conception (scales on the left). A continuous curve was constructed from data in Refs. 3, 52, and 55, assuming that there is an adolescent spurt centered at conceptional age 14 years. The dotted line is the first derivative of the growth curve and shows rate of absolute gain, or velocity, as a function of conceptional age (scales on the right). Note that the lifetime parathyroid growth curve consists of two successive sigmoid curves, the second one magnified on both axes.

300

/

CHAPTER18

BR DT

%/y (d) 10000

I ,ooo-

I

/

100

~

lO1'1,

'

I I

01'

;

~ ; ;

; ;lbl'll

l'al'415

Age (years since conception) FIG. 4 Parathyroid relative growth and inferred doubling time, from birth (B) to age 15 years. Cell birth rate (BR; %/year) and doubling time (DT, days) plotted on a logarithmic scale as functions of age (in years) since conception, derived from the continuous curves illustrated in Figs. 2 and 3. The values are based only on growth and take no account of turnover. The relationship between the two variables is DT (days) - log2 2/BR (%/year) x 100 x 365. The relationship is hyperbolic on a linear scale, but the curves are mirror images on a logarithmic scale. The solid straight line indicates the normal turnover rate in the adult gland, and the dashed straight line indicates the corresponding doubling time.

modest increase in specific birth rate and a decrease in doubling time during the adolescent growth spurt, after which the birth rate falls and the doubling time rises progressively toward the adult values, which are based solely on turnover, growth having ceased. From the standpoint of cell turnover, the parathyroid glands are the least extensively studied tissue in the body; they are not m e n t i o n e d in an otherwise comprehensive text (22). The rapid growth rate inferred from serial weights during embryonic development is supported by m e a s u r e m e n t s of mitotic prevalence in 35- to 50-day guinea pig embryos (60). Assuming a duration of mitosis of 45 min (14), the mean value of 22.2/104 cells corresponds to a population doubling time of "--10 days, very similar to the inferred value of 7 days in the human. The prevalence of mitosis falls steadily after birth, although in the mouse a brief burst of mitosis occurs ---3 weeks after birth (3). Cell turnover in the adult parathyroid gland has been measured using the MIB-1 antibody to Ki-67 and an assumed cycle duration of 24 hours. The values in %/y (geometric means with multiplicative standard deviations) in rats were 53.2 (1.95) at age 8-14 weeks and 39.7 (2.23) at age 22 weeks (40). In h u m a n s aged 18-76 years the value was 5.24 (2.54) with no effect of age, sex, or race (47). In

both species cycling cells were randomly distributed with no evidence for a separate stem cell population. The results firmly establish the adult parathyroid gland as a discontinuously replicating or conditionally renewing tissue (22), confirming the conclusion based on indirect evidence (3), with an average cell life span of about 2-3 years in the rat and about 20 years in humans. If cell divisions at the m e a n rate of 5.24% conform to a Polsson distribution in time, then during 50 years after maturity about 7% of cells will not divide at all, only 5 % will divide m o r e than five times, and no cell will divide more often than once every 3 years. Even though cell turnover is very low, it contributes more than 75% to the total lifetime n u m b e r of parathyroid mitoses (Fig. 5). Assuming that turnover-related mitoses occur at the same fractional rate t h r o u g h o u t life, their rate of accumulation will increase with increasing size, reaching a m a x i m u m slope when adult size is attained at age 30 years. Growth-related mitoses follow the growth curve exactly and obviously predominate in early life, but they no longer occur when growth stops, so that turnover-related mitoses predominate after age 45 years and b e c o m e a progressively larger fraction of the total with advancing age. In a life span of 80 years, the median growth-related mitosis occurs at age 9.5 years, the m e d i a n turnover-related mitosis at age 44.5 years, and the m e d i a n parathyroid mitosis of any kind at age 35 years (Fig. 5). Because adult parathyroid cell n u m b e r does not change, the very slow rate of cell gain must be balanced by a correspondingly slow

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Parathyroid lifetime mitoses. Accumulated number of mitoses as a function of age, growth related (short dashed line), turnover related (long dashed line), and total (combined; solid line). Note that growth-related mitoses predominate during the first 20 years but that turnover-related mitoses predominate after about age 45 years, The solid right-angle lines indicate that, for a life span of 80 years, the median parathyroid mitosis occurs at age 35 years, the median growth-related mitosis occurs at age 9.5 years, and the median turnover-related mitosis occurs at age 44.5 years. FIG. 5

PARATHYROIDGROWTH: NORMALAND ABNORMAL / rate of cell loss. Indeed, if this were not so, parathyroid size would increase about sevenfold between ages 30 and 70 years (1.054o = 7.04). Although there is no direct evidence for the occurrence of apoptosis in the parathyroid glands (40,61), the very brief duration of this process makes it as difficult to find as mitosis (30), and the evidence presented earlier indicates that apoptosis is the likely mode of cell death in all endocrine glands. By analogy with many other tissues and organs (5,14,22,30), cells loss is presumably the primary event, and parathyroid cells somehow are occasionally triggered from G o to G 1 (Fig. 1) in order to maintain a stable cell number. Such a compensatory response occurs in all endocrine glands u n d e r the control of the hypothalamus or the pituitary (1), but its occurrence in a gland that has no known trophic h o r m o n e is more difficult to understand. Perhaps signals from a dying cell induce a neighboring cell to divide.

Physiologic Influences on Parathyroid Growth For the thyroid, adrenal, and reproductive glands, an important c o m p o n e n t of growth control resides in the pituitary (1); the relevant h o r m o n e s are not only tropic but also trophic, and the same applies to the hypothalamic control of the pituitary (19). The parathyroid and pancreatic islets both lack a trophic hormone, and the variable they control (calcium and glucose, respectively) affects growth as well as h o r m o n e secretion. It is more difficult to establish the absence than the presence of something, but hypophysectomy has no effect on parathyroid size (62,63), any effects of growth h o r m o n e administration are mediated indirectly by increasing plasma phosphate (63), and the functions of individual pituitary cells are known in sufficient detail to leave no room for an undiscovered parathyrotrophic h o r m o n e (64). The association between pituitary and parathyroid disease in the multiple endocrine neoplasia type 1 (MEN-l) syndrome has suggested a pituitary origin for a novel parathyroid growth factor (65), but this is not a t r o p h i c h o r m o n e in the ordinary sense, and there is no evidence that it plays any role in normal physiology. The difference between the presence and the absence of a t r o p h i c horm o n e is illustrated by the responses to hemiresection. In the thyroid, compensatory hyperplasia is initiated promptly and briskly, with a 15-fold increase in mitotic prevalence within 2 days (66). In the parathyroid, however, the mitotic response is slow and of much smaller magnitude, with no more than a twofold increase (66,67), and may require simultaneous dietary calcium restriction for its unequivocal demonstration (66). A hyperplastic response to a t r o p h i c stimulus is usually preceded by hypertrophy (40) and by the ultra-

301

structural appearances of increased secretory activity (68). The sequence of increased parathyroid h o r m o n e (PTH) secretion, cell hypertrophy, increased DNA synthesis, and increased mitosis has been demonstrated most convincingly within the first 48 hours after total nephrectomy (3,69), but is probably the characteristic mode of parathyroid growth response. The n u m b e r of cells that contribute to total h o r m o n e secretion can also be increased by changing the relative durations of secretory activity and quiescence (70). The concept of a parathyroid secretory cycle, originally based on the ultrastructural distinction between dark and light chief cells (71), was challenged a few years ago as an artifact of the preparative m e t h o d (72) but has now been confirmed by the study of separated cells using the reverse hemolytic plaque m e t h o d (73) and by the restriction of PTH mRNA expression to chief cells with large vesicular nuclei (74). This concept has several implications for the study of parathyroid growth. The response to an increased d e m a n d for PTH consists of a hierarchy of mechanisms that occur on successively longer time scales (70): release of stored hormone, decreased intracellular degradation of hormone, increased h o r m o n e biosynthesis, and decreased duration of secretory quiescence. Only if these mechanisms collectively are unable to meet the d e m a n d does hyperplasia become necessary. The first three of these mechanisms are ways of increasing h o r m o n e secretion by each contributing cell, but the last two are ways of increasing the n u m b e r of contributing cells and represent the means whereby the parathyroid gland adds integral control to the proportional and derivative controls that suffice to meet most short-term needs (75). Alternation of cells between secretory activity and quiescence also provides an explanation for a central paradox of parathyroid growth regulation. With few exceptions, a cell either executes its differentiated function or divides, but does not do both at the same time (8,14). Consequently, when the stimulus to PTH secretion is most intense, some cells would have to disregard the stimulus, stop making PTH, and instead, get ready to divide. However, if secretion is cyclical, there will always be some cells able to respond to a proliferative stimulus without interrupting secretion. At the end of each quiescent period, the cell has the option of resuming secretion or entering the cell cycle (Fig. 6). As in other tissues with very long intercycle durations it is the frequency with which this option is exercised that determines the rates of cell division and tissue turnover. The calcium-sensing receptor (CaSR) appears to be expressed in all normal parathyroid cells (76), so that intermittency of h o r m o n e secretion is probably not the result of intermittency of CaSR expression, but there has been no direct examination of this question.

302

/

C~TF~R18

PROLI FERATIVE CYCLE

SEC RETORY CYCLE Active (dark)

Go I ~ Quiescent (light)

FIG. 6 Integration of proliferative cycle and secretory cycle in parathyroid cells. The chief cell alternates between periods of secretory activity and secretory quiescence (73) with characteristic ultrastructural appearances (71); there are other stages in the secretory cycle that are omitted for simplicity of illustration. At the end of the period of secretory quiescence, the cell has the option of resuming secretion or switching from G o to G1, to enter the proliferative cycle. The latter option is very rarely exercised in normal circumstances but is available if there is a need for more cells to contribute to hormone secretion, which cannot be met by a further increase in the duration of secretory activity relative to quiescence.

The notion that hypocalcemia stimulates parathyroid growth has a long history and is deeply e m b e d d e d in current concepts regarding pathophysiology, but the occurrence of hyperplasia as well as hypertrophy has been challenged (77,78). Most of the earlier papers reported only gland volume (79), wet weight (80), or dry weight (81). An increase in organ size out of proportion to the degree of hypertrophy has often been claimed (3,82) but inadequately documented. In the most histologically complete study, the increase in parathyroid volume in response to dietary calcium restriction in young rats was due mainly to hypertrophy rather than to hyperplasia (78). Interpretation of such experiments is complicated by the ability of the rat to adapt to a low calcium intake with a large increase in calcitriol production by a PTH-independent mechanism (83,84). Calcitriol has an effect, i n d e p e n d e n t of calcium, to inhibit parathyroid cell proliferation (3,21,85). It is unclear whether this effect is physiologically specific to the parathyroid gland or is simply part of a generalized antiproliferative action of calcitriol (86), but in either case it could mask the proliferative response to hypocalcemia (84). Conversely, prolonged calcitriol deficiency would likely increase parathyroid cell proliferation as well as h o r m o n e secretion (87). Consequently, if deficiency of vitamin D as well as calcium has been induced (88), it may be difficult to determine the relative contributions of hypocalcemia and calcitriol deficiency to parathyroid hyperplasia. However, dietary calcium restriction in another study led to moderate hypocalcemia and substantially

increased the proportion of cycling cells, despite a concomitant increase in serum calcitriol (61). In an in vivo study of experimental renal failure, parathyroid hyperplasia, inferred from increased weight and u n c h a n g e d p r o t e i n / D N A ratio, also developed concurrently with an increased serum calcitriol level (85). Phosphate administration to rats increased cell birth rate fourfold and caused directly measured hyperplasia with a sixfold increase in serum PTH and a 2.5-fold increase in serum calcitriol (40). Although hypocalcemia was the most likely stimulus for both h o r m o n e secretion and cell proliferation, both can be direct as well as indirect effects of hyperphosphatemia (87), as will be further discussed in relation to uremic secondary hyperparathyroidism (see also Chapter 39). The confounding effect of altered calcitriol biosynthesis in vivo can be circumvented by in vitro studies, but a major problem with cultured cells is their rapidity of growth, much faster even than the peak embryonic rate in vivo, with a doubling time of only 1-2 days (3,89). Cultured cells are driven to proliferate until confluence regardless of their tissue of origin, because they lack the restraint on proliferation imposed by normal tissue organization (12). Consequently, the inability of calcium to inhibit serum-stimulated growth of cultured parathyroid cells (27) and the even more paradoxical effect of calcium to stimulate growth (90) are unlikely to be of physiologic relevance. The most convincing results have been obtained with organ culture, in which lower ambient calcium concentrations have been accompanied by increased prevalence of both mitosis (91) and of S-phase cells labeled with tritiated thymidine (92). The failure to find this response consistently in vivo may reflect more than the suppressive effect of increased calcitriol levels. Why does parathyroid hyperplasia fail to occur (78), or to progress further (85), in hypocalcemic animals that are in serious need of more PTH? One possibility is that severe hypocalcemia has a generalized inhibitory effect on growth that includes the parathyroid gland, so that parathyroid cell number, although unchanged, may be increased in relation to body weight (78,84). Another possibility is that with severe hypocalcemia the duration of secretory quiescence is too short to permit the G o ~ G 1 switch to occur. In several in vitro studies, the effect of calcium has been biphasic, mild hypocalcemia stimulating and severe hypocalcemia inhibiting DNA synthesis (89,92,93), with a peak response occurring at --~0.5 mmol/liter. Perhaps hyperplasia has evolved as a defense against hypocalcemia that is mild and chronic, rather than severe and acute, and so is more easily demonstrated in older animals that are growing more slowly. Biologic plausibility and the weight of experimental evidence both support the traditional view that hypocal-

PARATHYROIDGROWTH: NORMALAND ABNORMAL / cemia stimulates cell division as well as cell growth in the parathyroid gland. The greater effectiveness of this mechanism in countering mild, rather than severe, hypocalcemia is exemplified by the responses to hemiparathyroidectomy (66,67) and to pregnancy (3) and by the inverse correlation between plasma calcium and parathyroid weight in normal subjects (56,58). The mechanism of the proliferative effect of hypocalcemia is becoming clearer (21). There is evidence for an autocrine mechanism, whereby hypocalcemia increases both the production of acidic fibroblast growth factor (FGF) by parenchymal cells and the n u m b e r of its surface receptors in the same cells (94). Less clear is a possible paracrine mechanism. Parathyroid capillary endothelial cells release a factor that stimulates thymidine incorporation in parenchymal cells (95), but the endothelial cells do not respond to hypocalcemia (M.L. Brandi, personal communication, 1994). The link between h o r m o n e secretion and proliferation may be different in each endocrine gland or may reflect separate responses to the same intracellular signaling mechanism (93,96), but it remains an attractive concept that entry into the cell cycle is triggered by a fall in intracellular h o r m o n e concentration below a critical level when biosynthesis fails to keep pace with release (46). Such a relationship, for which there is evidence in the prolactin-secreting cells in the pituitary (97), could provide a c o m m o n adaptive mechanism that was available to all endocrine cells.

ABNORMAL PARATHYROID GROWTH

Relationship between Cell Number, Hormone Secretion, and Plasma Calcium Unlike the pituitary and thyroid glands, in which abnormal growth can lead to space-occupying and pressure effects as well as h o r m o n a l effects, abnormal growth in the parathyroid gland is important only in that it contributes to abnormalities of h o r m o n e secretion. Implicit in the concept that parathyroid hyperplasia is the eventual long-term response to a sustained need for more PTH is the existence of an empirical, not just a formal, relationship between cell n u m b e r and total h o r m o n e secretion. Indeed, as I stated more than 30 years ago, "if the secretory behavior of each individual cell in relation to its chemical environment is unaffected by the total n u m b e r of c e l l s . . . " then PTH secretion must be proportional to the n u m b e r of contributing cells (98). Although an individual cell could communicate with adjacent cells by means of autocrine or paracrine mechanisms (21), the only way it could be influenced by the total n u m b e r of cells is by the result-

303

ant changes in the composition of the blood, so that the proposition stated above still appears to be logically unassailable. However, in normal subjects, parathyroid parenchymal cell n u m b e r has a CV of 30% (55), whereas plasma free calcium, and, by inference, the average secretory set point of parathyroid cells (which is the plasma calcium level that individual cells recognize as normal and attempt to defend), has a CV of < 3 % (75). Three main factors will obscure the relationship between total cell n u m b e r and plasma free calcium within a population, based on the reciprocal functions that underlie the regulation of calcium homeostasis (99). The first factor concerns the control of PTH secretion as a d e p e n d e n t variable by calcium. PTH secretion by the same total n u m b e r of cells will differ between subjects because of variations in set point (75), maximum PTH secretion per cell or secretory capacity (100), and proportion of cells contributing to secretion (73). The response to calcium will also be modified by several other factors of lesser importance (75). The second factor is the clearance of secreted intact PTH. The plasma concentration of PTH p r o d u c e d by the same secretion rate will differ between subjects because of variations in the metabolic clearance rate (MCR) and volume of distribution (Vdist) (1). The third factor concerns the control of calcium as d e p e n d e n t variable by PTH. The plasma free calcium produced by the same circulating level of PTH will differ between subjects because of variation in the responses of target cells in bone and kidney to PTH (75). Variation from all these sources is greater in patients with parathyroid disease than in normal subjects. However, in an individual, secretory set point and PTH secretory capacity are likely to remain reasonably stable, so that a change in active parathyroid cell n u m b e r can be assumed, at least as a first approximation, to lead to a proportional change in h o r m o n e secretion, in response to the same values for plasma free calcium and for all other relevant variables. Likewise, if PTH MCR and Vdist and target cell responses to PTH remain stable, the effect of a change in h o r m o n e secretion on plasma calcium will be predictable. In all forms of hyperparathyroidism, three major mechanisms contribute in varying degrees to PTH hypersecretion. First, the secretory set point is increased (75,100,101). Second, the proportion of active cells, reflecting the relative duration of secretory activity, is increased (74). Finally, the total n u m b e r of parenchymal cells is increased. Together these latter two changes will increase the n u m b e r of cells that contribute to h o r m o n e secretion at any time and so increase the aggregate PTH secretion by all glands proportionately at every level of plasma calcium. In terms

304

/

CHAPTER18

of the sigmoid logistic model relating steady-state PTH secretion to steady-state plasma free calcium concentration (102) (Fig. 7), this will increase m a x i m u m total secretion, provided that individual values for this parameter are included, rather than setting m a x i m u m secretion to 100%, as is often done for ease of comparison between studies (102). Any combination of changes in these two fundamental quantities can be symbolized in the same two-dimensional diagram, a change in set point by a linear displacement along the abscissa, and a change in m a x i m u m total secretion, representing the n u m b e r of secretory cells, by a proportional change along the ordinate (Fig. 7). To apply this

160

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model, it will initially be assumed that PTH MCR and Vdist are constant, so that there is a linear proportional relationship between PTH secretion and the circulating level of intact PTH. The relative importance of increased set point and increased n u m b e r of contributing cells can be estimated in terms of the reciprocal causality previously m e n t i o n e d (99). The effect of calcium on PTH has already been examined. To this must now be added the effect of PTH on calcium. Based on mean values for plasma albumin-adjusted calcium and intact PTH at various levels of parathyroid function, the relationship is curvilinear such that the same successive increment in PTH produces successively smaller increments in calcium (68,75,99), and is modeled in Fig. 8 by an exponential approach to an asymptotic value. For clarity, the large between-person variances have been omitted; each relationship should be represented by a band rather than by a single line. System equilibrium is defined by the intersection of these two functional relationships in which d e p e n d e n t and i n d e p e n d e n t variables are interchanged (99). Four versions of the sigmoid relationship between PTH and calcium are shown, taken from Fig. 7. They correspond to normal values for both set point and maximum secretion,

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FIG. 7 Sigmoid relationship between PTH secretion (as reflected by plasma concentration) and extracellular fluid (ECF) free calcium. The component not suppressible by calcium is assumed to be 5% of maximum secretion, indicated by the dashed lines. Four curves are shown, representing normal set point (SP) and normal number of secreting cells (lower solid line), increased set point and normal number of cells ( l o w e r d o t t e d line), normal set point and increased number of cells ( u p p e r solid line), and increased set point and increased number of cells ( u p p e r d o t t e d line). For clarity of depiction, the increase in set point (50%) is greater and the increase in number of cells (100%) is smaller than those usually found.

FIG. 8 Effect of increases in cell number and/or set point on steady-state values for extracellular fluid (EFC) free calcium and plasma PTH. Calcium (dependent variable) as a function of PTH (independent variable) is depicted by the solid curved monotonic line, based on published or personal values for adjusted total plasma calcium and intact PTH at various levels of parathyroid function (75). The dashed line represents the asymptotic value, estimated from the highest stable value for adjusted total plasma calcium found in patients with normal renal function of - 3 . 5 mmo/liter. Points A-D represent the intersection of this curve with the four versions of the sigmoid curve depicting plasma PTH (dependent variable) as a function of ECF free calcium (independent variable), taken from Fig. 7. The values for both variables corresponding to the points of intersection are given in the inset. For further details, see text.

PARATHYROID GROWTH: NORMALAND ABNORMAL /

increased set point with normal maximum secretion, increased maximum secretion with normal set point, and increased values for both determinants. The mean steady-state values for both variables corresponding to the four points of intersection are shown in the inset in Fig. 8, but the regions of intersection should be represented by areas rather than by points. The superimposition of the curves confirms the conclusion drawn from earlier, less rigorous models (6,98), and what has long been inferred from both clinical and experimental (103) observations, that a relatively larger increase in n u m b e r of secretory cells has a lesser effect on PTH and calcium than a relatively smaller increase in set point (6). Several points require emphasis. First, because the proportion of active cells, as well as the n u m b e r of cells, is increased, the increase in maximum secretion will be more than the increase in parathyroid size. Second, as cell n u m b e r increases without an increase in set point, each cell operates ever more closely to its m i n i m u m capacity and is less able to defend against a rise in plasma calcium (75). Third, a nonsuppressible c o m p o n e n t of PTH secretion (75,102) is not a prerequisite for an effect of increasing cell n u m b e r but increases the slope of that effect (6). Fourth, although an increase in set point alone will increase both plasma calcium and PTH levels, as set point increases without an increase in cell number, each cell will operate ever more closely to its maximum capacity and will be less able to defend against a fall in plasma calcium (75). With either abnormality alone, the biologic advantage of operating on the central steep portion of the curve relating PTH secretion to calcium is blunted. This biologic advantage is partly restored when an increase in set point is combined with an increase in the n u m b e r of secreting cells, as is found in most patients with primary hyperparathyroidism. With this theoretical background in mind, the empirical relationships between cell number, h o r m o n e secretion, and plasma calcium can be examined. In all studies that included an adequate n u m b e r of patients and covered a wide range of tumor size, there has been a highly significant correlation between tumor weight or volume and plasma calcium, with an average r value of about 0.6, ranging from 0.4 to 0.8 (3,104-107). The intervening correlations between weight (or volume) and plasma PTH level and between plasma PTH level and plasma calcium are equally or even more significant, although they are determined less frequently (3,105,108,109). The most impressive correlation (r = 0.98) was found between the acute increase in plasma PTH in response to a standard hypocalcemic challenge and parathyroid volume (110). A high correlation (0.87) was also found between a d e n o m a weight and the negative slope of PTH against calcium, as would be predicted from Fig. 7 (101). In other studies,

305

the scatter about the regression line has invariably been wide, with about two-thirds of the variance in the level of plasma calcium unexplained by the regression on parathyroid weight or volume. Consequently, although the relationship is of biologic and pathophysiologic importance, it has been of little practical value to surgeons attempting to predict the characteristics of the tumor from preoperative measurements (3,109). There are several reasons for the wide scatter. The slope relating plasma calcium to cell n u m b e r is quite shallow (6), and all the relationships in the model have wide confidence intervals. Many of the n u m e r o u s factors that disturb the relationship between cell n u m b e r and plasma calcium have already been described, but two additional mechanisms, altered maximum secretory capacity and altered suppressibility, were not included in the model in order to maintain a manageable degree of complexity. Maximum secretory capacity varies much more between subjects in patients with parathyroid adenomas than in normals, with almost a 10-fold range between the lowest and the highest values (100). Combining weight with various ultrastructural indices of h o r m o n e secretion significantly improved the correlation with PTH (108). In some patients, the slope relating PTH secretion to calcium is shallow and suppressibility is markedly impaired, such that the conventionally defined set point cannot be determined unless the ambient calcium concentration is raised to a much higher level than is usual (111). These abnormalities contribute to hypercalcemia in only a small proportion of patients (100) but contribute substantially to the variability between patients. Nevertheless, the empirical observations in patients with primary hyperparathyroidism support the conclusion from the theoretical analysis that, other things being equal, the more parathyroid cells, the greater the rate of h o r m o n e secretion and the higher the level of plasma calcium.

Primary Hyperparathyroidism: Disease Course as an Expression of Rate and Extent of Growth Hypersecretion of PTH is usually classified as secondary or primary. Not all definitions of these qualifying terms are equally clear; for the purposes of this chapter, the former is a response to a sustained increase in d e m a n d for PTH that is extrinsic to the glands, whereas the latter is the expression of an intrinsic abnormality (112). In secondary hyperparathyroidism, plasma calcium is usually low or normal, and parathyroid histology shows hyperplasia, whereas in primary hyperparathyroidism plasma calcium is usually high, and parathyroid histology usually shows a d e n o m a (see Chapter 1). However, classification according to the origin of the hyperparathyroid state makes more physiologic sense than classification according to the level of

306

/

CHAPTER18

plasma calcium or the structure of the glands at the time when the condition is recognized (3,112). The designation of hyperparathyroidism as "primary" is otherwise not restrictive with respect to etiology; some cases of "primary" hyperparathyroidism are "secondary" to some etiologic agent, whether genetic or envir o n m e n t a l (38), which is not directly related to the d e m a n d for h o r m o n e secretion, and the proportion of such cases is likely to increase as further knowledge is gained. Primary hyperparathyroidism has an extraordinarily wide range of clinical manifestations, encompassing severe, life-threatening hypercalcemia; moderate symptomatic hypercalcemia with osteitis fibrosa; mild hypercalcemia with nephrolithiasis; and even milder hypercalcemia in patients discovered fortuitously, many of whom are ostensibly asymptomatic and free of any obvious harmful consequences. In the 50 years prior to 1980 there was a progressive increase in the a p p a r e n t incidence of the disease, accompanied by a progressive change in the relative frequency of these different syndromes. Before about 1935, almost all patients, whether the diagnosis was made at autopsy or during life, had osteitis fibrosa (113), the specific bone disease of primary hyperparathyroidism (114). The subsequent changes occurred in two stages. First, following the work of Albright et al. (115), more cases were found a m o n g patients with nephrolithiasis; the magnitude of increase is uncertain, but was probably more than 10-fold (3). Second, following the widespread adoption of routine multichannel biochemical screening, more cases were found a m o n g patients who lacked any of the traditional indications for plasma calcium measurement. The magnitude of increase, after eliminating the backlog of undiagnosed patients, was about fourfold (116). At both stages, the increases were sustained and were accompanied by a rise rather than a fall in age at diagnosis (117) (Table 3). Consequently, only a small part of the fourfold increase in apparent prevalence was due to interception of the same type of patient at an earlier age, and most of the increase was due to the

TABLE 3

discovery of cases that previously were never diagnosed (117), except very rarely by accident (118-120). More recently a fall in incidence has b e e n reported from one center (121), but this has not been the general experience (107,122). During the same period when the disease appeared to become both m o r e c o m m o n and less severe, profound changes occurred in the characteristics of the tumors, particularly in their size (Table 4). There was almost a 10-fold decrease in geometric m e a n weight, and almost a 20-fold decrease in the weight of the largest tumor (104,107,109,113). This change provides a good explanation for the decline in disease severity, but because the age at diagnosis was rising at the same time as tumor size was falling, there must also have been a large reduction in the average rate of growth. Initially, only the most rapidly growing and consequently largest tumors were diagnosed, and, at each stage of increased case finding, the disease population was expanded by the addition of patients whose tumors were growing more slowly, and so were smaller, despite their longer course. The relationship between tumor weight and disease course was first examined in detail by Lloyd (104). He began by dividing the patients into those with osteitis fibrosa (type 1), those with nephrolithiasis (type 2), and those with neither of these disease manifestations (type 3). This classification corresponded to the three stages in the history of the disease defined by the two mechanisms of increased case finding described earlier. The classification was not logically exhaustive, because a patient could have had both osteitis fibrosa and nephrolithiasis. Such patients have become progressively less c o m m o n , and there were none in the particular series that was being studied. The use of osteitis fibrosa as a criterion of classification has frequently been criticized on the grounds that osseous effects of excess PTH can be found in almost all patients if sought with the right methods (114,123). However, increased bone turnover and increased rate of loss of cortical bone are nonspecific, whereas osteitis fibrosa is a qualitatively different bone disorder that is

Primary Hyperparathyroidism: Mean Age at Diagnosis

Authors

Ref.

Year

Clue

Patient age (years) at diagnosis

Norris

113

1947

Osteitis fibrosa

43

Helistrom and Ivemark

118

1962

Stones

49

Lloyd

104

1968

Stones

48

Mallette et aL

105

1974

Mixed

53

Mundy et aL

119

1980

Accidental

70

Rao et al.

107

2000

Accidental

62

PARATHYROIDGP.oWTrt: N o m v ~ AND ABNORMAL / TABLE 4

307

Historic Changes in Parathyroid Adenoma Weight

Parameter

Norris (113)

Lloyd (104)

Rutledge et al. (109)

Rao et aL (107)

Period Number Mean age (years) Geometric mean wt (g) Logarithmic SO b Range (calculated) c Range (observed)

1931-1945 69 a 43 6.44 3.2 0.61-68 0.40-120

1950-1965 98 48 1.26 3.4 0.11-14.6 0.15-26

1974-1984 68 56 0.58 2.2 0.12-2.9 0.10-2.6

1992-1997 148 62 0.74 2.7 0.10-5.3 0.1-8.7

aExcluding cases diagnosed at autopsy. bMultiplier corresponding to SD on a linear scale. CGeometric mean x/+(Iogarithmic SD) 2.

specific to excess PTH secretion (114), and its presence is both a logical and an unambiguous index of a particular degree of disease severity that has existed for several years. Regrettably, many observers have failed to appreciate this crucial distinction and have reported the presence or absence of "bone disease" without qualification. The major characteristics of types 1 and 2 are compared in Table 5. As would be expected from the preceding discussion, and as previously observed many times (3,105,106,124), every disease manifestation was more severe in type 1, with the exception of hypophosphatemia, which was masked by greater impairment of renal function. However, it was noted for the first time that the duration of symptoms was significantly shorter in type 1, indicating that it was not simply a more advanced form of type 2. The inverse relationship between weight of tumor and length of disease history, together with the substantial difference in tumor weights, immediately suggested that the fundamental difference between the two types was in the rate of

TABLE 5

tumor growth (104), an inference entirely consistent with the historic changes in the expression of the disease described earlier. The notion that tumor growth rate is a major determinant of disease course and m a n n e r of presentation, although a commonplace of oncology, has been slow to find general acceptance among endocrinologists, despite several examples in other endocrine tumors. In hyperparathyroidism, tumors growing more rapidly lead to greater h o r m o n e hypersecretion, more severe hypercalcemia, and osteitis fibrosa, and tumors growing more slowly lead to less severe hypercalcemia, h o r m o n e secretion rarely attaining the level n e e d e d to produce osteitis fibrosa. The idea can be further developed in several directions. Some patients present with severe, life-threatening hypercalcemia of recent onset, so-called acute hyperparathyroidism or parathyroid crisis (125,126) (see Chapter 34). Many such patients have had prior long-standing hyperparathyroidism of varying severity, often with osteitis fibrosa (acute superimposed

Two Types of Primary Hyperparathyroidism a

Parameter

Type 1b

Type 2 c

Number of cases Tumor weight (geometric mean) Range (observed) Range (predicted) e Length of history (years) Plasma Ca (mmol/liter)

44 3.74 (2.69) d

88 0.65 (2.64) d

0.7-26.0 0.5-27.1 3.6 (4.8) 3.34 (0.60)

0.15-3.5 0.10-4.5 6.7 (7.2) 2.91 (0.20)

aData from Ref. 104 and personal communication from the author. bpresentation with osteitis fibrosa. Cpresentation with nephrolithiasis. dMultiplier corresponding to SD on a linear scale. eGeometric mean x / + (logarithmic SD) 2.

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on chronic), but in others the crisis is the first manifestation of the disease, with a course too short for osteitis fibrosa to have developed (acute de novo). In either case, the hypercalcemia is of the progressive disequilibrium variety (127), often precipitated by intercurrent illness or surgery; the consequent immobilization and sodium depletion initiate several vicious circles that, uninterrupted, will lead to death. Extreme PTH hypersecretion is characteristic (125). The pathogenesis is complex (3,125-128), but, because the tumors are generally large (129,130), and the duration of symptoms can be measured in weeks or months rather than in years, parathyroid growth is likely to have been unusually rapid, either abruptly accelerated (acute on chronic) or rapid from the outset (acute de novo). The importance of rapid growth is strengthened by the close resemblance between acute hyperparathyroidism and parathyroid carcinoma with respect to disease course, frequent presence of both osteitis fibrosa and nephrolithiasis (otherwise an unusual combination), m a n n e r of presentation, and severity of hypercalcemia (125,131) (see Chapter 33). Yet another resemblance is that the frequency of both disorders has remained fairly stable, uninfluenced by the factors that have increased the frequency of diagnosis of hyperparathyroidism in general (125,132). In both conditions, geometric mean tumor weights are in the range of 4-10 g in representative series (129,130,133,134). The term atypical a d e n o m a has been coined for tumors that resemble carcinomas, not only in clinical expression, but in histologic appearance (135), and in rapidity of regrowth following incomplete excision (132). At the opposite end of the scale, in most patients in whom mild hypercalcemia is discovered fortuitously, indices of h o r m o n e hypersecretion and disease severity commonly remain stable for many years (136), as would be expected if such patients were rarely diagnosed before the introduction of multichannel biochemical screening (117). Type 3 disease, as originally defined by Lloyd (104), thus includes patients at both ends of the growth scale, and, if the term is to be retained as a useful r e m i n d e r of the historical evolution of the disease, it must be restricted to refer to patients who lack any of its traditional manifestations. Serial m e a s u r e m e n t of tumor size by noninvasive methods is obviously difficult and has never been accomplished in patients with primary hyperparathyroidism in whom surgical treatment has been withheld for any reason. Nevertheless, it is highly unlikely that the various determinants of horm o n e secretion would change proportionately in opposite directions, and the most reasonable explanation for long-term stability of h o r m o n e secretion is long-term stability of all its determinants, including cell number. Thus lack of disease progression implies that tumor

growth has markedly slowed down or has even ceased altogether. The infrequency with which patients are intercepted during the transitional period when PTH a n d / o r plasma calcium levels are increasing (136,137) implies that the duration of this period must, in most cases, be quite short (117). An important question is whether it is only our perception of the disease that has changed, for the reasons given earlier, or whether the disease itself has changed. Most observers have taken the former view (138,139); after all, recognizing that there is more to an iceberg than its tip does not change the iceberg. Prior to the introduction of multichannel screening, nephrolithiasis was found in 80% of a large representative series (3); a fourfold increase in the apparent prevalence of primary hyperparathyroidism should have reduced the proportion of patients with nephrolithiasis to 20%. The proportions of 18% and 22% found in two recent series (139,140) suggest that the absolute incidence of nephrolithiasis due to hyperparathyroidism has not changed. Applying similar calculations to osteitis fibrosa is more problematic. If the absolute incidence of this remains unchanged, and the frequency of diagnosis has increased 40-fold from the time when osteitis fibrosa was present in almost all patients, it should occur in at least 2% of patients seen in current practice. Experience at Henry Ford Hospital suggests that the current prevalence is < 1 % (107) (see Chapter 22); it was --~1% (1/97) and 1.7% (2/118) in two other recent series (139,140). These numbers are consistent with a modest reduction in the absolute frequency of osteitis fibrosa but are too small to draw this conclusion with confidence. It has been suggested that osteitis fibrosa is less c o m m o n than in the past, not just relatively but absolutely, as a result of improvement in vitamin D nutrition (141) (see Chapter 22), an issue considered in more detail below in the discussion of the pathogenesis of different growth patterns.

Primary Hyperparathyroidism: Patterns of Growth and Implications for Pathogenesis In his seminal paper, Lloyd (104) made three separate proposals. The first two, that there are large differences in growth rate between patients and that these differences contribute substantially to differences in clinical expression, are supported by all the available data and are consistent with accepted principles of clinical oncology (34,142). The third proposal, that the growth rates are bimodally distributed as a reflection of a basic difference in etiology, was more novel, has been more difficult to establish, and has largely been forgotten (or ignored) by contemporary students of the disease. Nevertheless, the proposal has been supported by

PARATHYROID GROWTH: NORMALANDABNORMAL / the discovery that, in most patients with the mildest disease, tumor growth has probably ceased altogether, which suggests a more fundamental difference, not just in the rate but in the pattern of growth (Fig. 9). The clinical course in these patients implies that growth is initially rapid but slows down progressively with time as an asymptotic value for tumor size is approached (35). This pattern of growth is often referred to as gompertzian, because the sigmoid curve can be described by the Gompertz function (143,144), but is referred to herein as asymptoticgrowth, the value of the asymptote differing between subjects. Asymptotic growth is a feature of many tumors (34,142-146) but is usually the result of mechanisms that only rarely apply to the parathyroids. In rapidly growing malignant tumors, neoangiogenesis may occur too slowly to maintain adequate perfusion, leading to nutritional deprivation, hypoxia, and sometimes necrosis (145). Parathyroid tumors very occasionally undergo infarction by a similar mechanism, leading to temporary biochemical remission (3,147), but this is

5

clearly not the explanation for growth retardation in patients with mild disease. In many tumors, benign as well as malignant, retardation and even cessation of growth are the result of increased cell loss (143,146), inferred because the observed doubling time is much longer than the potential doubling time deduced from cell kinetic studies (34,142). Most cell loss in tumors is due to apoptosis (146), but there is no evidence that this process is increased in parathyroid tumors (6,35), so that retardation of their growth is due to reduction in cell birth rate, as will later be demonstrated. According to the current paradigm for neoplasia, most tumors originate from a single cell that has acquired a competitive growth advantage as the result of a heritable change in gene function, and gives rise to a clone of cells which at first are genetically and phenotypically identical (8,148,149). The heritable change is generally assumed to be a somatic mutation, implying a change in nucleotide sequence (8), but may also be an epimutation (150), implying an epigenetic change

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FIG. 9 Hypothetical growth curves of parathyroid adenomas corresponding to two types of genetic dysfunction. In each case a solid line depicts the more typical behavior and an interrupted line depicts the less typical behavior. For type 2, the curves are based on gompertzian modeling (144) and represent cases in which the growth asymptote was above or below the geometric mean. The clinical threshold is the weight of tumor below which detection is unlikely; the value chosen is close to the normal mean total weight and corresponds to --~27 doublings of a single cell (34). Type 2 tumors will grow almost exponentially until the clinical threshold is reached, after which growth will be progressively retarded as the asymptotic value corresponding to the new secretory set point is approached; the level of the growth asymptote will depend on the degree of increase in set point. Type 1 tumors will grow exponentially at a rate depending on the extent of progression along the neoplastic pathway. Eventually growth will be slowed by the same kinds of factors as in other neoplasms (142-146), but will rarely cease altogether.

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in gene expression due to an altered pattern of DNA methylation (151,152). The concept of clonality is not so straightforward as is often assumed. All normal tissue consists of clones of adjacent cells that arose from a single cell in the primordial anlage, so that m o n o p h e n o typia implies monoclonality only if the tissue sample examined was large enough to contain many such "patches" (153,154). Furthermore, the definition of the founder cell of a clone may be ambiguous (148), and the founder cell of the population that has survived the process of clonal evolution (155) is not necessarily identical with the founder cell (or cells) from which the tumor originated (156). Despite these complexities, the concept of clonality has provided a useful starting point for the understanding of abnormal parathyroid growth. As with other benign endocrine tumors (3,157,158), parathyroid adenomas are usually monophenotypic, probably as the result of growth from a single genetically dysfunctional cell (159,160) (see Chapter 19). If the dysfunction is due to a mutation it may reflect a predisposition to one of several types of hereditary genetic abnormality (159,161) or be a consequence of exposure to external mutagens, such as therapeutic (3,38) or military (3,162) irradiation, but in most cases no

TABLE 6

such factor is present. Much less is known about the etiology of epimutations, but at least some are environmental in origin (163). Assuming an origin from a single genetically dysfunctional cell, it is possible to estimate the n u m b e r of doublings n e e d e d to produce a tumor of given weight (35), a useful first approach to studying the kinetics of growth even when doubling time is not constant. Dividing the required n u m b e r of doublings into the estimated age of the tumor gives an upper limit to the doubling time, from which can be calculated the minim u m cell birth rate needed to produce the tumor. This value will have little meaning in an individual case, but the mean value in groups of cases (Table 6) is five to eight times greater than the current cell birth rate at the time of t u m o r excision, both in sporadic (35,164) and in radiation-associated adenomas (38). The low cell birth rate has been demonstrated by three indep e n d e n t methods (35,38,164). Higher reported values reflect bias due to n o n r a n d o m sampling (164,165) or labeling cycling cells with PCNA (166), which is expressed also in cells undergoing DNA repair, rather than with Ki-67, which is not. At the time of excision, the cell birth rate is 10-20 times lower than in other

Age of Adenoma and Minimum Cell Birth Rate in Hyperparathyroidism Etiology a

Parameter Number of cases Age of onset (years) Age at operation (years) Age of tumor (years) Tumor weight (mg) Number of doublings (N) f Longest doubling time (years) g Minimum birth rate (%/year) h Current cell birth rate (%/year)

Radiation

Unknown

56 16.2 b (9.3)

63 23.3 c (6.1)

55.7 (9.5) 39.5 (10.6) 819 d (2.63) e

55.0 (15.1) 31.7 (9.1) 827 c (2.85) e

29.7 (1.40) 1.33 (0.36) 54.4 c (1.37) e 6.2 c (1.8) e

29.7 (1.51 ) 1.07 (0.33) 69.7 c (1.56) e 13.7 c (2.06)e

aStandard deviations are given in parentheses. Radiation data are from Ref. 38; unknown etiology data are from Ref. 35. t'Age at exposure to external radiation. Data in parentheses are mean SD. CAssuming that genetic dysfunctions have the same age distribution as mitoses (Fig. 5), so that age at median mitosis is the best estimate of age of onset (3). dGeometric mean. eMultiplier corresponding to SD on a linear scale. fLog e wt (g)/Log e 2 + 30. gAge of tumor/N. hLog e 2/DTrnax.100.

PARATHYROID

benign tumors such as meningiomas (38) and is only twice the normal value, with a very wide range and substantial overlap (164). Very low cell turnover appears to be a distinctive characteristic of parathyroid adenomas, not shared by any other tumor for which adequate data are available. According to a Gompertz model, the magnitude of reduction in cell birth rates during tumor growth is about 25-fold (144), which supports the conclusion previously drawn from the usual course of the disease in current practice. The reduction is even greater in the central (and presumably older) portion of the tumor, where the cell birth rate is no greater than in normal parathyroid tissue (164), making unlikely an origin from cells with abnormally high proliferative potential, as has been proposed for thyroid nodules (167). The preceding discussion highlights a central paradox in the pathogenesis of parathyroid adenomas. Another aspect of the prevailing paradigm of neoplasia is that abnormal cells accumulate a succession of mutations, each of which confers an additional growth advantage, a process known as clonal evolution (155). This paradigm is widely believed to apply to endocrine tumors in general and to parathyroid adenomas in particular (159,160) (also see Chapter 19). A high proportion of these tumors harbor at least one of a large n u m b e r of different mutations, including activation of the putative p r o t o o n c o g e n e cyclin D 1 (168), and allelic losses at multiple sites, each consistent with the loss or inactivation of a different tumor suppressor gene (169). However, during a process that supposedly leads to a progressive increase in the rate of cell division, the rate is progressively declining. Furthermore, the cells that have had the longest time to accumulate mutations have the lowest rate of cell division, a rate that is no longer significantly greater than normal. Another difficulty is that the expected consequence of the reported mutations would be more rapid traversal of the cell cycle, which is irrelevant to a tissue of such low turnover, rather than activation of the G o ~ G 1 switch. Finally, the only mutation studied in individual cells, rather than in tissue extracts, was never present in more than 70% of cells (168), indicating that it was not present in the founder cell of any of the tumors, and no more than one mutation has been found in the same cell. Despite their frequency, the mutations could be e p i p h e n o m e n o n a that are only rarely of pathogenetic significance (12,170,171), or could be having effects different from those that lead to neoplasia. These facts, together with the prolonged duration of latency (about 40 years in radiation-associated adenomas) (Table 6), and the rarity of malignant transformation suggest that many cases may arise by a nonneoplastic mechanism (35,38,164).

GROWTH: NORMALAND ABNORMAL /

311

Lloyd (104) suggested that type 2 tumors arose from hyperplastic foci of cells that were unusually responsive to the calcium demands of pregnancy and lactation, elaborating on a suggestion made earlier by Albright and Reifenstein (172). This concept was supported by evidence for polyphenotypia in parathyroid adenomas (157), but that evidence is open to an alternative interpretation and does not necessarily indicate polyclonality (3,154). The concept of focal hyperplasia can be reconciled with a monoclonal origin by means of the set-point hypothesis, which proposes that the initial genetic dysfunction in a single cell is decreased sensitivity to its ambient calcium concentration and consequent increase in secretory set point (35,38,164). Such a cell would respond to a normal extracellular fluid (ECF) calcium concentration in exactly the same way as a normal cell responds to a low concentration (75). It will maximize its rates of PTH biosynthesis and secretion, but to no avail. Sooner or later the abnormal cell will do the only other thing it is able to do in response to perceived hypocalcemia, which is to divide, and the new clone of cells will grow exponentially until it is large enough to begin increasing the rate of h o r m o n e secretion by the whole gland. As the patient's ECF calcium rises, the rate of growth will slow down, and when the value corresponding to the new set point is attained, there will be no stimulus to further growth (75). Because of the very long time interval between successive divisions, the decline in cell birth rate would be very slow and would have progressed further in older cells. The rate of cell division in the altered cells, rapid initially and slowing down with time, recapitulates on a longer time scale the changes that occur during normal development (Figs. 2-4). A parathyroid aden o m a resulting from an increase in set point, although descriptively a tumor, would not be a neoplasm but, at least initially, a uniglandular focus of hyperplasia. The increase in secretory set point that is characteristic of primary hyperparathyroidism was first discovered more than 20 years ago (100) but its mechanism has only recently been clarified. The response of parathyroid cells to changes in ambient calcium concentration is governed by the serpentine G protein-coupled calcium-sensing receptor (173,174) (Chapter 8) and the main determinant of the set point is the n u m b e r of CaSR molecules (173,175). No mutations in the CaSR gene have been found in parathyroid adenomas (176,177) but there is marked underexpression of the CaSR, for both message (178,179) and protein (76,180), which is the likely proximate cause of the increase in set point (175). Because reduced CaSR expression appears to be present in every cell (76,180), it could have been the initial abnormality in the founder cell, possibly due to hypermethylation of the

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CaSR gene (152). In some cases reduction in CaSR could be the result of underexpression of the vitamin D receptor (VDR) (180,181). Gompertz modeling has provided additional support for this hypothesis; the time from the initial genetic dysfunction to the first cell division is unusually long, the geometric m e a n ranging from 40 to 100 days d e p e n d i n g on the assumption made concerning t u m o r age (144). It seems unlikely that a cell with a primary mutation in a growth control gene would wait so long before d i v i d i n g ~ s u c h a duration seems more reasonable for a cell that will divide only as a last resort, as predicted by the set-point hypothesis. A link between set-point level and rate of cell proliferation is the effect of a calcimimetic drug that mimics an increase in CaSR expression to inhibit parathyroid proliferation in response to n e p h r e c t o m y (182). M t h o u g h not yet supported by direct evidence, the set-point hypothesis accounts for the current clinical characteristics of primary hyperparathyroidism, the main abnormality in PTH secretion, the low rates of cell proliferation at the time of surgical excision, the difference in cell birth rate between the central and peripheral regions, and the inferred pattern of asymptotic growth, all by means of a single m e c h a n i s m (164). Lloyd (104) suggested that type 1 tumors were true neoplasms, in which growth becomes slower than it was at first but is sustained, so that in the absence of treatm e n t the disease progresses in severity as the extent of growth increases in accordance with the rate of growth (Fig. 9). In such tumors the initial change in the founder cell would be dysfunction of a growth control gene that activated the G 0 -o G 1 switch. Few of the reported mutations have been consistently related to the pattern of growth, but this may indicate no more than the rarity of type 1 disease in current practice. Loss of the retinoblastoma t u m o r suppessor gene, a freq u e n t finding in parathyroid carcinoma (183), occurred also in a few benign but aggressive tumors (184,185), and extensive microsatellite instability was found in a n o t h e r (186). Many characteristics of parathyroid adenomas that are consistent with, but do not establish, a dual etiology have already been mentioned, and there are several more. Histologic features of neoplasia, such as greater variability in size and shape of cells and nuclei and the presence of giant, multiple, and hyperchromatic nuclei, were commonly observed when only more severe disease was recognized (3,106), but are now only occasional findings (see Chapter 1). Histochemical or flow cytometric evidence of polyploidy a n d / o r aneuploidy is found in a minority of patients but not in the majority (3,187); differences in nuclear diameter, greater than can be accounted for by proliferation or by ploidy (Table 7), suggest that type 1 tumors are more neoplastic than are type 2 tumors

TABLE 7

Type Normal Diploid a Tetraploid b Octaploid b Adenoma Type 2 (n = 45) Type 1 (n = 10) Carcinoma (n = 18)

Parathyroid Nuclear Diameter Diameter (l~m) (SD)

5.0 (3.30) 6.3 7.9 5.64 c 6.68 d 8.11 e (1.39)

aMeasured in 39 subjects (188). bCalculated. CSignificantly different from normal diploid (P < 0.01). dSignificantly different from type 2 (P < 0.001). eSignificantly different from type 1 (P < 0.001 ) (134). The increase in adenomas is much too great to be accounted for either by polyploidy or by the proportion of cycling cells.

(153,188). H o r m o n e secretion is relatively autonomous in a small n u m b e r of patients, both in vitro and in vivo, but is normally suppressible in most (100,189). In one large series, plasma calcium values were bimodally distributed, with a minority of cases having a modal value of 3.6 m m o l / l i t e r and a majority having a modal value of 2.8 m m o l / l i t e r (190). However, whether it is the same patients who are atypical for all these different characteristics is not known. Nevertheless, the classification based on the dual concept (Table 8) appears to be better able than the less complex previous scheme (104,112) to a c c o m m o d a t e the variation in clinical and pathologic expression of primary hyperparathyroidism and to provide a framework for attempting to understand its molecular basis. The preceding discussion implies that a parathyroid cell can acquire a growth advantage in two different ways, a dysfunction in secretory set-point control leading to a hyperplastic a d e n o m a with asymptotic growth, or a dysfunction in cell cycle control leading to a neoplastic a d e n o m a with continuous growth (Table 8; Fig. 9) This notion makes more sense from the available data than the current d o g m a that all parathyroid tumors are neoplasms, but three major questions remain to be answered. First, if a significant minority of cases do not have an initial set-point dysfunction, why is the set point increased in almost all cases? The usual answer is that rapid cell proliferation leads to underexpression of the CaSR (3,191), but the most rapid rate of cell division that has been measured at the time of t u m o r excision (44%/year) (35) is at least 300 times lower than the rate observed in cultured parathyroid cells (89). Consequently, rapid proliferation might lead to an increase in set point during the early phase of clonal expansion but is not a plausible explanation for

PARATHYROID GROWTH: NORMAL AND ABNORMAL /

TABLE 8

313

Modified Classification of Clonal Parathyroid Tumors Type a

Aspect

1

2 and 3

Nature of genetic dysfunction Manner of growth Expression of variability b Clinical presentation c

Cell cycle control Continuous Rate of growth Osteitis fibrosa (Other factors) Hypercalcemia

Set-point control Asymptotic Plasma calcium Nephrolithiasis (type 2) (Other factors) Fortuitous (type 3)

aFrom Ref. 104. bCharacteristic in which tumors of similar type may differ depending on how large is the effect of the genetic dysfunction. CMay be modified by environmental and life style factors as well as by characteristics of the tumor.

a sustained increase in set point. An alternative explanation is that underexpression of the CaSR is the first event in almost all parathyroid tumors, regardless of their behavior at the time of diagnosis and excision. The second question concerns how neoplastic transformation occurs in a tissue with such low turnover. DNA undergoes continuous damage and repair t h r o u g h o u t life (8), but the perpetuation of a genetic or chromosomal abnormality as a mutation usually occurs only as a result of mitosis (3,192,193). In bacteria and in some lower eukaryotic species mutation can be time d e p e n d e n t rather than replication d e p e n d e n t (194). A possible explanation is that the machinery for postreplicative mismatch repair can operate in circumstances in which the d a m a g e d and u n d a m a g e d DNA strands cannot be distinguished (195). However, this process has not been shown to occur in m a m m a l i a n cells. Aside from this possibility, the absolute frequency of any mutation is the p r o d u c t of the probability of its occurrence at each mitosis (the mutagenic risk) and the frequency of mitosis (192,193). Increased frequency of successive divisions within the same clone may further increase the risk of mutation by reducing the time available for DNA repair (8,196). Although increased cell proliferation by itself may not be mutagenic (197), it increases the risk of mutations due to other factors (192,196). W h e n the basal rate of cell division in a tissue is very low, as in the liver, the occurrence of mutations that induce neoplasia is often p r e c e d e d by an increase in cell proliferation (196,198). The parathyroid glands have even lower turnover than the liver, and appear to be even m o r e in need of increased cell proliferation prior to neoplastic transformation. In the normal parathyroid, the relative rate of cell division approaches that found in the dividing transit population of tissues with rapid turnover for only a brief

period a r o u n d 16 weeks of conceptional age (Fig. 4), during which there probably occur the mutations that lead to the rare parathyroid t u m o r that presents in childhood (199). At other times the necessary population of rapidly dividing cells might be created by the initial nonneoplastic increase in set point. With the shortest initial doubling time predicted by the Gompertz model (144) of 21 days, and p o s t p o n e m e n t of the gompertzian decline in proliferation rate until the new clone had grown to two standard deviations of the normal m e a n parathyroid cell weight (50 mg) (55), then total parathyroid cell mitosis would be increased by about eightfold for about 1.5 years. The increase would usually be less than this, but there would still be a modest increase in the risk of a mutation. In most cases, no mutation would occur, and growth would be asymptotic, and the value of the asymptote would d e p e n d on the magnitude of the increase in set point, but in a minority of cases, a neoplastic-type mutation would occur, leading to continuous growth, in some cases c o m p o u n d e d by one or more further mutations leading to parathyroid carcinoma. An alternative possibility is that neoplastic transformation reflects not mutagenesis, but rather the breakdown of constraints on proliferation normally imposed by communications of cells with each other and with their stromal matrix (12,170,171). The third and most clinically relevant question is whether variations in t u m o r growth rate and disease expression d e p e n d on differences not in pathogenesis, but in vitamin D nutrition (141). The evidence suggests that vitamin D deficiency can influence parathyroid growth in several different ways. In the United States there is a significant negative regression of log a d e n o m a weight on serum calcidiol level, an effect not mediated by either serum calcium or serum calcitriol and so probably indirect. A plausible explanation is that a low

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calcidiol level blunts the calcemic effect of PTH, so that a higher asymptotic value for tumor weight is needed to raise the serum calcium to the new set point (107). Although not found in the United States, in some European countries a low calcidiol level limits calcitriol production in patients with primary hyperparathyroidism (200,201 ). Calcitriol deficiency directly increases PTH secretion independent of other factors (202); calcitriol also has an antiproliferative effect on parathyroid cells (77,85), so that tumor growth rates could be enhanced by calcitriol deficiency. In France, patients with primary hyperparathyroidism are more likely to have osteitis fibrosa if their serum levels of calcidiol and calcitriol are low (200). However, in England, the serum calcitriol level was positively correlated with immunoreactive PTH and was normal or increased in each of 11 patients (17% of the total) with osteitis fibrosa, in whom the mean value tended to be higher than in patients with nephrolithiasis (203). Individual calcidiol levels were suboptimal by m o d e r n standards (less than 10 n g / m l ) in more than half the patients, but the results were not classified according to clinical features. In India, where vitamin D deficiency is endemic and almost all patients with primary hyperparathyroidism continue to have large tumors and osteitis fibrosa (204), tumor weight is substantially higher than predicted from the regression on serum calcidiol, and the same is true for the rare occurrence of osteitis fibrosa in the United States (see Chapter 22). As in England, serum calcitriol levels in these patients are not reduced. Some patients with concurrent parathyroid a d e n o m a and severe vitamin D deficiency are hypocalcemic (3,205), but this was not so for the patients in question. Thus, in the setting of endemic vitamin D deficiency, parathyroid adenomas grow larger than can be accounted for by current vitamin D nutrition, in the absence of either hypocalcemia or calcitriol deficiency. A plausible explanation is that lifelong deficiency of vitamin D and calcium leads to parathyroid hyperplasia in a substantial proportion of the population. Increased cell proliferation in all four glands should be a more potent p r o m o t e r of mutagenesis than increased proliferation confined to the progeny of a single cell. Direct evidence for this notion is that radiation-induced parathyroid adenomas occur more frequently in vitamin D-deficient than in normal rats (206). The same transition from nonneoplastic hyperplasia to neoplasia is observed in other endocrine glands (207) and especially in the thyroid (167). According t o the concept discussed, genetic dysfunctions in set-point control and in cell cycle control are not alternative but successive events, the former being a necessary preliminary to the latter, except when cell division is stimulated by some other means (Fig. 10), such as a germinal mutation (161), or by the

initiation of secondary hyperparathyroidism; the latter sequence is referred to as tertiary hyperparathyroidism, a condition already exemplified by the situation in India, and further discussed in the next section. In MEN-l, polyclonal hyperplasia would also be the initial response to the putative MEN growth factor (65,95), the later occurrence of a mutation or epimutation in cell cycle control (e.g., somatic mutation in the wildtype menin allele) accounting for asymmetry of glandular enlargement (208), and for monoclonality of the largest glands. A novel feature of the set-point hypothesis is that, in every other tissue in the body, a dysfunctional gene that tended to decrease the intracellular calcium concentration would inhibit rather than stimulate growth (209) and would likely lead to malfunction and possibly death of the cell. Such defects could be very common, but only in the parathyroid cell would they have a clinically detectable consequence. The new concept not only accounts for all the data suggesting nonneoplastic etiology but also answers each of the three questions previously posed. It explains why an increase in secretory set point is found in almost all cases, reconciles the occurrence of mutations in cell Set-point "Mutation"

Ca~ / Calcitriol ~

MEN 1 Factor

Germinal "Mutation"

Population of rapidly proliferating parathyroid -Monociona,

.......

i ........

onai----

Cells/

...........

Cell cycle No Set-point No "Mutation. . . . Mutation . . . . Mutation. . . . Mutation" Neoplastic Adenoma

Hyperplastic Seconoary Primary Adenoma Hyperplasia Hyperplasia

FIG. 10 Unified model of abnormal parathyroid growth. "Mutation" means any heritable genetic dysfunction, either a classic mutation or an epimutation. For reasons explained in the text, the first step is always the establishment of a population of rapidly proliferating cells, either monoclonal, as the result of a set-point "mutation" or polyclonal as the result of stimulation by the circumstances leading to secondary hyperparathyroidism, or by the MEN-1 growth factor, or as a consequence of one of the several genetic defects that can lead to primary hyperplasia. The rapidly proliferating cells are at increased risk of "mutation." A set-point "mutation" leading to monoclonal proliferation, followed by a cell cycle "mutation," leads to a neoplastic adenoma causing type 1 primary hyperparathyroidism, and occassionally parathyroid carcinoma. A set-point "mutation," followed by no further "mutation," leads to hyperplastic adenoma causing type 2 primary hyperparathyroidism. Polyclonal proliferation as the expression of secondary hyperparathyroidism with no "mutation" leads to secondary hyperplasia, either diffuse or nodular; hypercalcemia may develop because of an acquired increase in secretory set point. Diffuse or nodular hyperplasia, followed by "mutation" of either kind, leads to tertiary hyperparathyroidism.

PARATHYROIDGROWTH: NORMALAND ABNORMAL / cycle control of the kind that can lead to neoplasia with the extremely low rate of cell division in the normal parathyroid gland, and provides a means whereby vitamin D deficiency may contribute to pathogenesis.

Parathyroid Growth in Secondary and Tertiary Hyperparathyroidism Secondary hyperparathyroidism, as defined in the previous section, can occur in a wide variety of circumstances, usually identified on the basis of physiologic reasoning and a sustained increase in blood levels of PTH, sometimes supported by histomorphometric, radiographic, and densitometric evidence of the skeletal effects of PTH excess. The circumstances are numerous (112) and include normal aging (210), dietary deficiency or malabsorption of calcium, renal wasting of calcium, pregnancy and lactation, Paget's disease, pseudohypoparathyroidism (see Chapter 51), vitamin D depletion (both extrinsic and intrinsic) (211), and impaired vitamin D metabolism at multiple levels (86). In normal aging the pathogenesis of secondary hyperparathyroidism is multifactorial, and there is no evidence for a progressive age-related increase in parathyroid cell number (55). Presumably, the modest increase in PTH secretion (40% between ages 20 and 90 years) can be accommodated by the earlier members of the hierarchy TABLE 9

315

described above (see Physiological Influences on Parathyroid Growth). An alternative explanation is that values above the wide normal range in younger subjects, found in a small number of elderly persons (210), are the result of parathyroid microadenomas or hyperplastic nodules that occasionally occur in the absence of hypercalcemia (212). Qualitative evidence for parathyroid enlargement due to hyperplasia has been found in a few cases of rickets and osteomalacia at autopsy (3) and in a few cases of pseudohypoparathyroidism at diagnostic surgical exploration (3), but detailed information on abnormal parathyroid growth is available only for chronic renal failure (213) (see Chapter 39). In this condition, PTH hypersecretion and parathyroid hyperplasia are initiated by some combination of hypocalcemia, hyperphosphatemia, and calcitriol deficiency, each alone able to stimulate parathyroid cell proliferation (87,90,202), but other factors contribute to progression of the growth disorder in a minority of patients. In an unselected autopsy series of patients with renal failure published between 1926 and 1966 (213-216), before the wide availability of therapeutic regimens capable of substantially prolonging life, the degree of parathyroid enlargement was quite modest but was greater in patients dying from renal failure due to chronic glomerulonephritis than in those with less severe forms of renal disease (215) (Table 9).

Parathyroid Weights in Chronic Renal Failure

Authors

Ref. (year)

Pappenheimer and Wilens Castleman and Mallory Stanbury and Lumb

214 (1935) 215 (1937) 216 d (1966)

Sivula et aL

DeFrancisco et aL

219 (1979) 221 (1985)

Krause and Hedinger

222 (1985)

Lloyd et aL

223 (1989)

Feature

Unselected CGN b Unselected Rickets/OM t Osteitis fibrosa ~ PTX PTX Diffuse Nodular Total PTX Diffuse Nodular Total PTX

n

Geometric mean (SD)

Range (g)

27 12 119 10 26 34

0.148 (1.45) 0.301c (2.21) 0.184 (2.23) 0.158 (1.53) 3.517 (2.35) 0.915 (3.88)

0.067-0.433 0.12-0.863 0.045-0.891 e 0.071-0.281 e 0.78-12.0 0.071-19.7

1.36 2.74 1.67 1.44 31.9 8.3

17 44 61

0.824 (1.86) 2.014 (2.16) 1.570 (2.30)

0.280-2.56 0.126-9.32 0.126-9.32

7.5 18.3 14.3

17 22 39 16

1.509 3.523 2.434 3.920

0.2-7.5 0.9-10.1 0.2-10.1 0.91-17.2

13.7 32.0 22.1 35.6

(2.28) (1.93) (2.32) (1.96)

Relative

increase a

Assuming normal geometric mean (213) or median (55) of 0.110 g for all glands combined. CGN, Chronic glomerulonephritis, blood nonprotein nitrogen 105-300 mg/dl (normal 20-40). CSome values estimated from rectangular volume. dlncluding all cases previously published. eEstimated from histograms. tBased on skeletal histology at autopsy: OM, osteomalacia; PTX, parathyroidectomy, either total or subtotal.

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Individual values for combined parathyroid weight were log-normally distributed, and the geometric mean value was increased by--~65%; only --~40% of the values were >2 SD above the normal mean, whether arithmetic or geometric, although the highest values were far outside the normal range. Replacement of fat is an early stage of hyperplasia (213), but, even taking this into account, mean parenchymal weight could not have been increased by more than two- to threefold. In a more recent series, total parathyroid parenchymal weight was positively correlated with serum creatinine and was inversely correlated with renal weight, but hyperplasia could be detected histologically in patients without clinically manifest renal dysfunction (217). In patients with rickets or osteomalacia as the main skeletal abnormality, the geometric mean parathyroid weight was similar to, and individual values were in the same range as, that in the unselected cases (216). The data suggest that mild parathyroid hyperplasia occurs in most patients with chronic renal failure (87) and in the predialysis era the aggregate increase in cell number was equivalent to a small adenoma. In some dialysis patients PTH levels continue to increase, but the PTH assays commonly used probably exaggerate the degree of PTH hypersecretion in renal failure (218). In patients with osteitis fibrosa as the main skeletal abnormality, there was very little overlap with the unselected cases; the geometric mean weight was increased >30-fold, and both the mean and range were much the same as for single adenomas causing osteitis fibrosa in primary hyperparathyroidism (Table 5). In later years, parathyroid weights were published mainly for patients who were selected for surgical treatment, usually because of osteitis fibrosa or hypercalcemia or both (219-223) (Table 9). The values for total excised weight obviously reflect both historic changes and local variation in surgical policy but are in general quite similar to the total weights previously found at autopsy in patients with osteitis fibrosa. In patients with hyperparathyroidism that was of severity sufficient to merit either individual publication of autopsy findings or selection for surgical treatment, the increase in parathyroid weight was so much greater than in unselected patients with chronic renal failure or in those with defective bone mineralization alone (Table 9) that the difference is likely to be qualitative as well as quantitative. Although precise figures are not available, it seems reasonably certain that such severe hyperparathyroidism remains more frequent than in the prehemodialysis era, mainly because longer survival provides more time for the necessary parathyroid growth to occur. A good candidate for a factor that could lead to much more severe parathyroid growth in a minority of patients is an increase in PTH secretory set point; abnormal growth would then be driven by the same

mechanism as in primary parathyroid adenomas with a set-point dysfunction, with the difference that many cells would be affected, not just a single cell. Conceptually, the set point is a property of individual cells (75) and was originally determined by manipulation of PTH secretion in vitro (100). Studies of set point in vivo in patients with normal renal function have given consistent and physiologically intelligible results (75,101,189). However, in patients with renal failure the results have been inconsistent and often uninterpretable partly because of differences in experimental protocol (224) and perhaps partly because of unrecognized defects of current PTH assays in uremic patients (218). For example, intravenous calcitriol has been reported both to decrease (225) and to increase (226) the set point. With this reservation in mind, in unselected dialysis patients the secretory set point appears to be normal and not to increase with the severity of hyperparathyroidism (227). But in many patients needing surgical removal of parathyroid tissue, an increase in secretory set point has been found in dispersed cells obtained at surgery (228), an abnormality even more evident in cells harvested from nodular regions within the enlarged gland (229). More recently, reduced expression of the CaSR, the probable regulator of the set point, has been found in such patients (76,178). However, because of the many factors involved in chronic renal failure that impair the effectiveness of PTH in raising plasma calcium (see Chapter 39), the increase in total h o r m o n e secretion necessary to attain the new set point can be achieved only with an enormous increase in the n u m b e r of secretory cells. Consequently, the asymptotic value for total gland size is much more difficult to reach than in primary hyperparathyroidism. An increase in secretory set point due to reduced calcium receptor expression (76,178) also provides a plausible explanation for the development of hypercalcemia in a minority of patients with severe secondary hyperparathyroidism (223,230). This is often attributed to autonomy of h o r m o n e secretion, but in such patients, both parathyroid weight (220,230) and the rate of DNA synthesis (223) are positively correlated with both PTH levels and plasma calcium; indicating that it is not h o r m o n e secretion but rather growth that has become autonomous, because plasma calcium is behaving as the dependent, rather than as the independent, variable (75), a conclusion supported by the sustained restoration of normocalcemia after surgery (223,230). The birth rate of new cells is higher than in primary parathyroid adenomas, and does not slow down as in primary hyperparathyroidism, but remains as rapid as when the glands first began to enlarge soon after the onset of renal failure (223). Calcitriol increases CaSR expression in rat parathyroid tissue

PARATHYROID GROWTH: NORMALAND ABNORMAL /

(231 ), so that calcitriol deficiency could account for the increase in set point (223) as well as for the progressive resistance to the hypercalcemic effect of PTH. If so, the target of the parathyroid cells would continually recede instead of remaining stable, accounting for the difference in growth behavior between the two disorders (223). This p h e n o m e n o n would be augmented by the lower levels of calcitriol receptors in parathyroid cells from uremic patients (232), which would blunt the suppressive effect of calcitriol on both hormone secretion and cell proliferation, and possibly contribute further to CaSR underexpression (180). Further insight into the mechanism of the growth disorder is provided by the histologic distinction between diffuse and nodular hyperplasia (see Chapter 1). The nodules consist of cells that are more closely packed together with larger nuclei and a greater prevalence of cell cycle markers (221,233), more cycling cells by flow cytometry (234), higher expression of cyclin D1 (233), greater depletion of calcitriol receptors (232), higher secretory set point (229), and greater reduction of CaSR expression (76,178). These characteristics account for the significantly greater increase in total parathyroid weight (Table 9) and for the continued growth, often rapid, of many parathyroid autografts (235). Detailed histochemical and immunocytochemical studies indicate similarity in gene expression between the cells in each nodule but differences between nodules (236). Each nodule could have arisen from a different single cell or, more likely, from a group of adjacent cells that were the clonal descendants of a founder cell present during embryonic development and so constituted a patch (153). In this sense, nodular hyperplasia is multiclonal, in contrast to the polyclonality of diffuse hyperplasia and the monoclonality of adenomas. There can be as many as 100 nodules in four enlarged glands, so the likelihood that each nodule arose from a separate mutation is infinitesimally small. In some cases, parathyroid glands removed from such patients have been reported as monoclonal, implying an origin from a single mutant cell (237). As previously discussed, the biochemical determination of clonality, based on polymorphism for X-linked DNA sequences, is less rigorous than the morphologic determination of clonality, based on studying gene expression in individual cells (153,154). Consequently, the proportion of the original hyperplastic gland that contributed to the tumor, and the relationship between such a tumor and the hyperplastic nodules previously described, are unclear. The parathyroid growth response to chronic renal failure progresses through several stages. Diffuse secondary hyperplasia (polyclonal) is initiated by hypocalcemia, becomes more severe as the result of hyperphosphatemia and calcitriol deficiency, and leads

317

eventually to osteitis fibrosa in some patients (216). Because of the additional effects of CaSR underexpression to increase the parathyroid secretory set point and VDR underexpression to decrease responsiveness to calcitriol, continued growth can lead to hypercalcemia (223); the hyperplasia becomes nodular (multiclonal) and the glandular enlargement asymmetric (238). The next stage is the emergence of an adenoma in one, or occasionally more than one, of the nodules, as the expression of a genetic dysfunction in one of the cells undergoing the most rapid proliferation; the accumulation of 1.0 g of additional parathyroid tissue in 5 years requires a 50-fold increase in the frequency of mitosis. This sequence is referred to as tertiary hyperparathyroidism (3,239), a term often misapplied to patients with hypercalcemic secondary hyperparathyroidism but more accurately reserved for the disorder that combines in its etiology the hyperplasia of secondary hyperparathyroidism with the monoclonality of primary hyperparathyroidism (211,239). In addition to the usual histologic features of adenoma, nuclear diameter is increased to the same extent as in primary hyperparathyroidism (240) and a variety of allelic losses have been found in several different chromosomes, as in primary hyperparathyroidism (241-243) (see also Chapter 19). In other cases the dysfunction could lead to a further increase in secretory set point. The final and least common stage is malignant transformation leading to parathyroid carcinoma, an event that seems to occur with unusual frequency in patients on longterm hemodialysis (3,87,244,245), possibly because of defective DNA repair (246) as well as increased cell proliferation. Because long-term continued stimulation of parathyroid growth by chronic renal failure can culminate in neoplasia, either benign or malignant, the same would be expected in other circumstances. The occurrence of unusually large parathyroid adenomas in India was mentioned earlier (204). Hyperplasia in the nonadenomatous glands was reported in some cases but has not been sought systematically, so that the distinction between primary and tertiary hyperparathyroidism may be difficult (3,211). Hypercalcemic hyperparathyroidism developed in some vitamin D-deficient migrants from India to England (247) but its anatomic basis remains unclear. A parathyroid adenoma was found in two such patients (248), but whether the incidence is increased in this population has not been established. In intestinal malabsorption, secondary hyperparathyroidism has been demonstrated biochemically (211), but the histologic data are fragmentary (249). The apparent association between intestinal malabsorption and parathyroid adenomas has been described as tertiary hyperparathyroidism (3), but there was no evidence for hyperplasia of the other

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glands (250), as is found invariably in tertiary hyperparathyroidism due to renal failure. In the early stages of calcium malabsorption, serum calcitriol levels tend to be increased rather than decreased (211). This would delay the rise in secretory set point, which is probably necessary for the emergence of multiple foci of rapidly proliferating cells and increased mutagenic risk. Consequently, as has been suggested (250), growth would be stimulated only in the most responsive cells. A similar mechanism may underlie the rare case of parathyroid carcinoma in patients with intestinal malabsorption (245). There is also evidence for tertiary hyperparathyroidism in patients with hypophosphatemic osteomalacia given long-term treatment with supplemental phosphate, which induces intermittent slight decreases in plasma calcium and stimulation of PTH secretion (251). Hypercalcemic hyperparathyroidism has developed in at least 25 such patients (3,252-254), in whom parathyroid pathology encompassed the same spectrum of diffuse hyperplasia, nodular hyperplasia, and adenoma as in patients with chronic renal failure. Another similarity is that in three cases the secretory set point was increased (253); in contrast to renal failure, the serum calcitriol levels were normal, but they tend to be low in the disease and to be affected little by treatment with calciferol (211,251). One of the three cases was treated only with calcitriol, but this compound seems to be more effective than calciferol in preventing the progression of parathyroid growth (251). Another therapeutic regimen causing short-term stimulation of PTH secretion is sodium fluoride administration (255). The author has observed the development of hypercalcemic hyperparathyroidism during prolonged treatment with sodium fluoride and its cure by removal of a parathyroid adenoma, but whether this was more than a coincidence is not clear. In summary, tertiary hyperparathyroidism definitely occurs in chronic renal failure and probably also in chronic vitamin D deficiency and in phosphate-treated osteomalacia.

Therapeutic Implications of Parathyroid Growth In primary hyperparathyroidism, a sustained decrease in PTH secretion can at present be achieved only by ablation of surplus parathyroid tissue, usually by surgical resection (see Chapter 31). Short-term control of hormone secretion is possible with calcimimetic drugs (256) and their suitability for long-term management is currently under investigation, but there is currently no parathyroid counterpart to bromocriptine, which can produce shrinkage as well as reduced hormone secretion in many pituitary tumors (257). The rate of growth influences the timing of surgery, which is urgently needed in acute primary hyperparathyroidism,

is mandatory but not immediately required in many patients with progressive disease, and can be delayed, sometimes indefinitely, in mild nonprogressive disease (117). Regarding secondary hyperparathyroidism, however, there is a long-standing and pervasive assumption that, if the initial stimulus to cell proliferation could be removed, the parathyroid glands would eventually return to their previous size. There are several bases for this assumption. First, a noninvasive reduction in size has for many years been easy to observe in the thyroid gland (1). Second, classic nutritional experiments that demonstrated cross-sectional differences in parathyroid growth (79) have often been misquoted as evidence for parathyroid involution. Third, the ultrastructural changes that accompany suppression of hormone synthesis and release by hypercalcemia (258) have been misinterpreted as evidence for suppression of growth. Nevertheless, it is far from certain that a nondestructive increase in cell loss is possible in human parathyroid glands. The absence of a parathyrotrophic hormone may limit the capacity for involution as well as for growth, but even in the thyroid gland regression of hyperplasia is less frequent and less complete than is commonly assumed (167). The first indication that hyperplastic parathyroid glands regressed very slowly, if at all, came from the frequent persistence of PTH hypersecretion after renal transplantation (6). There are several causes for hypercalcemia in this situation, but in current practice the most common is hyperparathyroidism. Although sometimes this is described as tertiary hyperparathyroidism, in most patients only hyperplasia is found (259). Nevertheless, hypercalcemic patients had higher PTH levels before transplantation and their secondary set point is increased (260). Mild hypercalcemia can persist unchanged, with no tendency to resolve; if parathyroid involution is occurring in such patients, it is at a rate too low to be reflected by detectable changes in plasma calcium for up to 10 years (6). In primary hyperparathyroidism, the nonadenomatous parathyroid glands may show ultrastructural evidence of functional suppression (258), and a 50% reduction in cell proliferation (164), but the reduction in parenchymal cell mass is only--~10% (261), consistent with regression of hypertrophy without cell loss (40). In patients with nonparathyroid hypercalcemia, the parathyroid glands at autopsy are indistinguishable from normal glands in histologic appearance, in relative proportions of fat and parenchyma, and in size (39). For these reasons, if stable hypercalcemia lasts for more than 1 year after renal transplantation, its permanence should be assumed and surgery r e c o m m e n d e d unless there is a strong contraindication (87). The therapeutic implications of disorders in growth in untransplanted patients are more complex. In

PARATHYROIDGROWTH: NORMALAND ABNORMAL / primary hyperparathyroidism, cell birth rate in nonadenomatous glands falls only by about 50% (164). Even if, as a result of medical treatment, cell birth rate in patients with renal failure fell to zero, and apoptosis continued at the normal rate of 5%/year, parathyroid cell n u m b e r would fall by about 40% in 10 years and 3.0 g of surplus parathyroid tissue would shrink to 0.5 g in about 35 years. These extremely pessimistic calculations have several important implications. First, much more emphasis needs to be placed on the prevention of parathyroid hyperplasia in early chronic renal failure. Because parathyroid hyperplasia is always a later response than PTH hypersecretion to the same stimuli, if PTH levels are increased more than twofold for more than 6 months, it is safe to assume that some parathyroid hyperplasia has already occurred. Prevention of PTH hypersecretion, which is easy to monitor, will prevent parathyroid hyperplasia, which at present is impossible to monitor in its early stages. Second, once severe hyperparathyroidism has been allowed to occur, then however successful medical treatment may be in reducing PTH hypersecretion, then as after transplantation, in the absence of evidence to the contrary it should be assumed that there will be no significant reduction in parathyroid cell number. In one of the few prospective controlled clinical trials in this field, there was no significant difference in the response to oral and intravenous calcitriol given in the same dose, and after 36 weeks there was no change in ultrasonographically determined parathyroid size or dynamic indices of PTH hypersecretion, regardless of the route of administration (262). Implicit in the preceding discussion is that apoptosis of parathyroid cells continues at the normal rate of about 5%/year; an important unresolved issue is whether it is possible to stimulate apoptosis and so accelerate parathyroid involution. If calcitriol is a negative parathyroid growth factor, then a large abrupt rise in its concentration might have the same effect as a large abrupt fall in the concentration of a positive growth factor, which induces apoptosis in many endocrine tissues (30). There have been several reports that pulse administration of calcitriol in high dose (8 txg) may be followed by substantial reduction (about 40%) in parathyroid volume determined ultrasonographically; most of the shrinkage occurred in the first 4 weeks (263,264). Calcitriol may induce parathyroid apoptosis in rats (265), but no histologic verification has been reported in the clinical studies, and the shrinkage could reflect reductions in cell size and in vascularity, rather than in cell number. Nevertheless, the latter interpretation is supported by experiments in 3-month-old vitamin D-deficient chicks that received vitamin D replacement. There was good indirect evidence for a reduction in parathyroid cell number,

319

based on similar reductions in weight, protein, and DNA content (266); the change was detectable within 4 days and could have resulted only from apoptosis. In the controlled trial previously referred to (262), calcitriol was administered in pulse fashion, although the maximum dose of 4 txg may have been too small to achieve the effects reported with a higher dose. If induction of apoptosis by calcitriol can be confirmed in patients, a bromocriptine for the parathyroid glands may be at hand. Disordered parathyroid growth also has implications for the surgical m a n a g e m e n t of hyperparathyroidism in untransplanted patients (87). The first procedure carried out was subtotal parathyroidectomy, leaving only a portion of the smallest gland. Much later an alternative procedure was introduced, namely, total parathyroidectomy and autotransplantation of tissue into the forearm. The advantages claimed were that monitoring of the function of the autograft was easier because its venous drainage was accessible, and if the r e m n a n t grew rapidly e n o u g h for severe hyperparathyroidism to recur, reexploration would be easier (267). More recently, total parathyroidectomy without autotransplantation has been r e c o m m e n d e d (268). The prolonged maintenance of normocalcemia that sometimes follows this procedure is presumed to d e p e n d on parathyroid embryonic rests that were too small or too aberrant in location to be removed. Each of these procedures has its proponents and opponents, but more important than the choice between them is the timing, regardless of which procedure is adopted. Before the development of nodular hyperplasia, subtotal parathyroidectomy, followed by calcitriol to control the secretion and growth of the parathyroid remnant, should be effective and is rarely followed by recurrence of severe hyperparathyroidism. Conversely, if parathyroidectomy is delayed until the patient has severe osteitis fibrosa or hypercalcemia, then nodular hyperplasia or a d e n o m a formation can be presumed. If total parathyroidectomy is chosen, it makes sense to transplant only tissue harvested from the smallest and least nodular gland, or, if all glands are nodular, from an internodular region (267,269), because indiscriminate choice of transplanted tissue is more likely to be followed by aggressive and even invasive growth (270).

Parathyroid Growth in Hypoparathyroidism The parathyroid glands are difficult to find at autopsy even when they are normal in size, and there is only fragmentary information about their pathology when they are small. The available data in the various forms of idiopathic hypoparathyroidism are described in Chapters 1, 47, and 50. In surgical hypoparathyroidism, there has been almost no histologic examination, but an

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interesting paradox can be formulated on the basis of the course of parathyroid function in the absence of treatment. In many patients, the level of plasma calcium is higher than expected for the complete absence of PTH (271), there is a low but detectable level of intact PTH (272), and some recovery from EDTA-induced hypocalcemia is possible (273). Evidently some cells capable of secreting PTH remain, but depressed function persists. According to the model depicted in Figs. 7 and 8, because the relationship between cell number and plasma calcium is curvilinear, the decrease in plasma calcium is proportionally less than the decrease in cell number. Hormone secretion per cell is increased, and as cell number declines, each cell operates ever more closely to its upper limit with respect to hormone secretion (75). In these circumstances, why do not the remaining cells proliferate until normal function is restored? As mentioned earlier the effect of calcium on parathyroid cell proliferation is biphasic, and severe hypocalcemia is less effective than mild hypocalcemia. But this cannot be the main explanation for the paradox, because even mild hypoparathyroidism can remain unchanged in severity for many years (271). Except when all four glands are removed, which almost never happens with neck surgery for nonmalignant disease, there is no relationship between the development of hypocalcemia and the amount of parathyroid tissue in the surgical specimen (274). Rather than simple removal of some parathyroid tissue, there is interference with the blood supply of all four glands as the result of the surgical dissection and hemostasis (275). This occurs only in a small minority of patients, presumably because of individual differences in the precise location of the parathyroid glands and in the detailed anatomy of their blood supply. The parathyroid glands are also susceptible to fibrosis (276,277), and the progression of fibrosis is a likely explanation for the late occurrence of surgical hypoparathyroidism after a period of apparent recovery with normocalcemia (278). Parathyroid ischemia is a likely explanation for the development of apparently idiopathic hypoparathyroidism late in life (279); the susceptibility of parathyroid tissue to ischemia is indicated by uncertain survival after transplantation (235), and by spontaneous infarction in rapidly growing tumors (147). A subnormal degree of parathyroid function can be maintained by small islands of cells, but if separated by bands of fibrous tissue their viability will be precarious. Regeneration depends on neoangiogenesis as well as on chief cell proliferation (95), and both processes would be compromised by vascular injury, impaired circulation, and persistent fibrosis.

I N T E G R A T I O N OF PARATHYROID GROWTH A N D H O R M O N E SECRETION Increases in Set Point and in Cell P r o l i f e r a t i o n ~ Further Examples Both in primary and in hypercalcemic secondary hyperparathyroidism, cell proliferation is driven in large part by an increase in secretory set point, due to CaSR under expression for one or other reason. Three additional examples of this relationship merit brief discussion--normal embryonic development (280), familial hypocalciuric hypercalcemia (FHH) (see Chapter 38), and lithium-induced hypercalcemia (see Chapter 55). The fetal parathyroid gland secretes PTH-related peptide (PTHrP) rather than PTH, but its secretory activity is regulated by calcium in the same manner as the adult parathyroid gland, except that the secretory set point is higher than in the adult (280) most probably because of CaSR underexpression (281). The rapidity of embryonic parathyroid growth is consonant with a need to prevent fetal hypocalcemia (52) by supplying PTHrP to maintain normal placental calcium transport (282), a process dependent on CaSR expression (281). This is probably an evolutionary rather than an individual functional adaptation, but proliferation could be driven in part by the increase in set point. In FHH there is an inactivating mutation of the CaSR gene (174). As a result the parathyroid secretory set point determined in vivo is increased (101,189,283) and there is mild and nonprogressive hypercalcemia. Because of ineffective renal tubular CaSR, tubular resorption of calcium is increased; urinary calcium excretion is normal, but is low relative to the plasma level. Whether bone lining cells express the CaSR is unknown, but the blood-bone equilibrium is probably set at a higher level in FHH (75). Serum PTH levels are usually normal in childhood but increase slowly with time and are often above normal after age 30 (284). The geometric mean for parathyroid parenchymal area in histologic sections was increased approximately three fold, compared to a fivefold increase in other forms of primary hyperplasia, whether familial or sporadic (285). Parathyroid weights have been reported only in a few patients; the mean was increased about twofold, but few individual glands weighed more than 75 mg (286). The mild hyperplasia represents the polyclonal (or multiclonal) expression of a heterozygous germinal mutation, for which homozygosity leads to severe neonatal hyperparathyroidism (174). In terms of the set-point hypothesis, a lesser degree of parathyroid enlargement is needed to satisfy the increased secretory set point because the elevated renal tubular set point does not depend on increased PTH secretion (75).

PARATHYROID GROWTH: NORMALAND ABNORMAL / A double dose of the mutant gene, or a d o m i n a n t negative mutant, causes a higher set point (111,287) and a consequent greater drive to cell proliferation. Administration of lithium salts, usually for bipolar affective disorder, is in most patients followed within a few weeks by modest increases in plasma PTH and calcium levels, occasionally of sufficient magnitude to constitute hypercalcemia (288,289). Even a single dose of lithium carbonate increases the intact PTH level after 2 hours, with no change in ionized calcium (290). The early changes are reversible, but mild persistent hypercalcemia is found in about 6% of patients given prolonged treatment (291). The features of lithiuminduced hypercalcemia are similar to those of FHH (289,292). Serum PTH levels are nonsuppressed or moderately raised, and tubular reabsorption of calcium is increased. Both short-term and long-term clinical studies indicate that lithium administration increases the PTH secretory set point (292,293), a conclusion supported by the in vitro effects of lithium on cultured parathyroid cells, both bovine and h u m a n (293,294). As predicted by the set point hypothesis, lithium administration stimulates parathyroid growth. Exposure to lithium increases tritiated thymidine uptake by cells cultured from parathyroid adenomas (294), and there is a significant increase in parathyroid volume measured by ultrasound in patients on long-term treatment (295). In lithium-induced hypercalcemia of sufficient severity to need surgical treatment, both hyperplasia and aden o m a have been found (3,288,289,296). A d e n o m a tends to occur earlier than hyperplasia in the course of lithium treatment (297), suggesting that lithium promotes hyperplasia of normal parathyroid tissue and stimulates the growth of small adenomas already present (292).

A New Concept of Calcium Homeostasis The consistency of the association between increased secretory set point and increased cell proliferation suggests a new way of looking at plasma calcium homeostasis. According to this view, the controlled variable is not the plasma calcium, but rather the difference between the parathyroid secretory set point (using the physiologic definition given earlier) and the prevailing plasma calcium level, with the target value for this variable being zero (Fig. 11). If plasma calcium is below the set point, the well-known short-term feedback loop is initiated; based on the hierarchy of mechanisms described earlier (see above, Physiologic Influences on Parathyroid Growth, and Ref. 75), total horm o n e secretion by all glands is increased. Depending on the response of target cells in bone and in the renal tubule to higher PTH levels (75), plasma calcium rises

321

[Set Pt-P.Ca]

(I)I(~~

(6) (~ Cell Number (~. ~-(~)PTHSecretion

,(~ Target cells

(4) ® GO-----~ G~ FIG. 11 Short-term and long-term loops in calcium homeostasis. The controlled variable is the difference between the parathyroid secretory set point and the current level of plasma calcium, with a target value of zero. The signs indicate, for each step, the directional effect on the dependent variable of an increase in the independent variable. Steps 1, 2, and 3 constitute the short-term loop, and steps 4, 5, and 6 constitute the long-term loop. Step 4 in initiated when PTH hypersecretion by individual cells is prolonged because a positive value of the controlled variable persists, but the mechanism is unknown. (For further details, see text.)

and the deviation from the set point is eliminated. If this process is incompletely effective, there will be a sustained increase in d e m a n d for PTH, bringing into play a long-term feedback loop, whereby some parathyroid cells are triggered from the quiescent G o state to the G 1 stage of the cell cycle. The initiating signal may be a fall in PTH concentration in some critical intracellular compartment, augmented by the autocrine mechanism described earlier (see Physiologic Influences on Parathyroid Growth and Ref. 21). The result will be an increase in the n u m b e r of parathyroid cells, a consequent increase in total h o r m o n e secretion in response to the same stimulus, and a rise in plasma calcium toward the set point. The cycle will be repeated until cell n u m b e r has increased sufficiently to reduce the disturbing signal to trivial magnitude. As m e n t i o n e d earlier, the long-term loop adds integral control to the proportional and derivative controls that suffice for the short-term loop (75). This ensures that stimulated parathyroid cells need not long sustain m a x i m u m rates of h o r m o n e synthesis and secretion, in which state they cannot respond to an additional demand, but are returned to the steep central portion of the sigmoid curve where they can respond with greatest efficiency in either direction (75). The sustained increase in the controlled variable needed to initiate the long-term feedback loop can arise either because the plasma calcium is reduced or because the set point is increased. Chronic hypocalcemia (if not due to hypoparathyroidism) is the result of decreased efficacy of PTH in maintaining normocalcemia, for one of the reasons previously given (see above, Parathyroid

322

/

CHAPTWR18 TABLE 10

Comparison of Different Mechanisms of Increase in Set Point

Disorder

P rim a ry adenoma

Seco nda ry hyperplasia

FHH a

Lith iu m therapy

Set-point mechanism Affected cells PTH efficacy ~

Genetic dysfunction One clone Normal

Calcitriol deficiency Nodules Reduced c

Germinal mutation All Increased ~

Direct effects on cell All Increased a

Cell increase

Moderate

Severe

Mild

Mild

aFamilial hypocalciuric hypercalcemia: the situation in other forms of genetic hyperplasia is less clear. °In raising plasma calcium. CBecause of calcitriol deficiency and other effects of renal failure dBecause of increase in tubular reabsorption of calcium.

Growth in Secondary and Tertiary Hyperparathyroidism), leading to diffuse secondary parathyroid hyperplasia. Depending on its cause, an increase in secretory set point will affect a different population of cells and have different morphologic consequences (Table 10). If due to an epimutation, then only a single clone of cells will be affected, leading to primary or tertiary hyperparathyroidism (Fig. 10). If due to renal failure and calcitriol deficiency, some groups of cells may be affected more than others, leading to nodular secondary hyperplasia. If due to a germinal mutation or to an external agent such as lithium, all cells will be affected equally, leading to primary hyperplasia (Table 10). There are significant differences between individuals in mean plasma calcium level, reflecting small individual differences in parathyroid secretory set point (75), due to polymorphism of the CaSR gene (298), which also accounts for significant differences between families (75). According to the set-point hypothesis, there should be corresponding differences in cell number, and parathyroid weight should be positively correlated with plasma calcium level. But, in fact, the correlation is not positive but negative (56,58)! The patients available for sampling at autopsy probably encompassed a wide range of calcium and vitamin D nutrition, so that in this multiethnic population the variation in target cell responsiveness to PTH (107) was large enough to dominate the reciprocal feedback relationship, allowing the observations to be reconciled with the set-point hypothesis. Whatever the molecular basis of the link between cell proliferation and hormone secretion in the parathyroid gland turns out to be, the setpoint hypothesis, in conjunction with variation in the efficacy of PTH in raising plasma calcium (Table 10), accounts for the slowing down of growth in parathyroid adenomas, the persistence of rapid growth in secondary hyperplasia with hypercalcemia, and the small extent of growth in FHH. Integration of the

short-term loop governing hormone secretion and the long-term loop governing proliferation ensures that both in health and in disease, the parathyroid glands will attain the size needed to accomplish their biologic purpose.

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259. Kilgo MS, Pirsch JD, Warner TE Starling JR. Tertiary hyperparathyroidism after renal transplantation: Surgical strategy. Surgery 1998;124:677-683. 260. Messa P, Sindici C, Cannella G, Miotti V, Risaliti A, Gropuzzo M, Di Loreto PL, Bresadola F, Mioni G. Persistent secondary hyperparathyroidism after renal transplantation. Kidney Int 1998;54:1704-1713. 261. Ejerblad S, Grimelius L, Johansson H, Werner I. Studies on the non-adenomatous glands in patients with a solitary parathyroid adenoma. UppsalaJ Med Sci 1976;81:31-36. 262. Quarles LD, Yohay DA, Carroll BA, Spritzer CE, Minda SA, Bartholomay D, Lobaugh BA. Prospective trial of pulse oral versus intravenous calcitriol treatment of hyperparathyroidism in ESRD. Kidney Int 1994;45:1710-1721. 263. Fukagawa M, Okazaki R, Takano K, et al. Regression of parathyroid hyperplasia by calcitriol-pulse therapy in patients on longterm dialysis. N EnglJ Med 1990;323:421-422. 264. Hyodo T, Ono K, Koumi T, et al. Can oral 1,25(OH)zD, pulse therapy reduce parathyroid hyperplasia? Nephron 1991 ;59:171-172. 265. Fukagawa M, Wi H, Kurokawa K. Calcitriol induces apoptosis of hyperplastic parathyroid cells in uremia. J Am Soc Nephrol 1991;2:635. 266. Henry HL, Taylor AN, Norman AW. Response of chick parathyroid glands to the vitamin D metabolites, 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol. J Nutr 1977;107:1918-1926. 267. Tominaga Y, Tanaka Y, Sato K, Numano M, Uchida K, Falkmer U, Grimelius L, Johansson H, Takegi H. Recurrent renal hyperparathyroidism and DNA analysis of autografted parathyroid tissue. WorldJ Surg 1992; 16:595-603. 268. Kaye M, Rosenthal L, Hill RO, Tabah RJ. Long-term outcome following total parathyroidectomy in patients with end-stage renal disease. Clin Nephrol 1993;39:191-197. 269. Ohta K, Manabe T, Katagiri M, Harada T. Expression of proliferating cell nuclear antigens in parathyroid glands of renal hyperparathyroidism. WorldJ Surg 1994;18:625-629. 270. Abbona GC, Papotti M, Gasparri G, Bussolati G. Recurrence in parathyroid hyperplasias owing to secondary hyperparathyroidism is predicted by morphological patterns and proliferative activity values. Endocr Pathol 1996;7:55-61. 271. Parfitt AM. The spectrum of hypoparathyroidism. J Clin Endocrinol 1972;152-158. 272. Wilson P, Kleerekoper M, Lillich R, Parfitt AM. Immunoradiometric assay for intact parathyroid hormone in the diagnosis of hypoparathyroidism. J Bone Miner Res 1988;3(Suppl. 1):S131. 273. Parfitt AM. The study of parathyroid function in man by EDTA infusion. J Clin Endocrinol 1969;29:569-580. 274. GirlingJA, Murley RS. Parathyroid insufficiency after thyroidectomy. Br MedJ1967;l:1323. 275. Wade JSH, Goodall P, Deane L, et al. The course of partial parathyroid insufficiency after thyroidectomy. Br J Surg 1965;52:493. 276. Barakar AY, D'Albora JB, Martin MM, Jose PA. Familial nephrosis, nerve deafness, and hypoparathyroidism. J Pediatr 1977;91 (1):61-64. 277. Sentochnik DE, Hoffman GS. Hypoparathyroidism due to progessive systemic sclerosis. J Rheumato11988;15(4):711-713. 278. Parfitt AM. Delayed recognition of postoperative hypoparathyroidism. MedJAust 1967;1:702-708. 279. Parfitt AM. Idiopathic, surgical and other varieties of parathyroid hormone deficient hypoparathyroidism. In: DeGroot L, ed. Endocrinology, 2nd Ed. Philadelphia: Saunders, 1989:1049-1064. 280. Mallette LE. The parathyroid polyhormones: New concepts in the spectrum of peptide hormone action. Endocr Rev 1991;12:110-117.

PARATHYROID GROWTH: NORMAL AND ABNORMAL / 281. Kovacs CS, Ho-Pao CL, Hunzelrnan JL, Lanske B, Fox J, Seidman JG, Seidman CE, Kronenberg HM. Regulation of murine fetal-placental calcium metabolism by the calcium-sensing receptor. J Clin Invest 1998;101 (12)2812-2820. 282. Rodda CP, Caple IW, Martin TJ. Role of PTHrP in fetal and neonatal physiology. In: Halloran BE Nissenson RA, eds. Parathyroid hormone-related protein: Normal physiology and its role in canc~ Boca Raton, Florida:CRC Press, 1992:169-196. 283. AuwerxJ, Demeats M, Bouillon R. Altered parathyroid set point to calcium in familian hypocalciuric hypercalcemia. Acta Endocrinol 1984; 106:215-218. 284. McMurtry CT, Schranck FW, Walkenhorst DA, et al. Significant developmental elevation in serum parathyroid hormone levels in a large kindred with familian benign (hypocalciuric) hypercalcemia. A m J M e d 1992;93:247-258. 285. Thorgeirsson U, Costa J, Marx sJ. The parathyroid glands in familial hypocalciuric hypercalcemia. Hum Pathol 1981 ;12:229-237. 286. Law WM,Jr, CarneyJA, Heath III H. Parathyroid glands in familial benign hypercalcemia (familial hypocalciuric hypercalcemia). AmJMed 1984;76:1021-1026. 287. Bai M, Pearce SHS, Kifor O, Trivedi S, Stauffer UG, Thakker RV, Brown EM, Steinmann B. In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the Ca2+-sensing receptor gene: Normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia. J Clin Invest 1997;99:88-96. 288. Mallette LE, Eichhorn E. Effects of lithium carbonate on human calcium metabolism. Arch Intern Med 1986;146:770-776.

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289. Larkins RG. Lithium and hypercalcemia. Aust N Z J Med 1991;21:675-677. 290. Seely EW, Moore TJ, LeBoff MS, Brown EM. A single dose of lithium carbonate acutely elevates intact parathyroid hormone levels in humans. Acta Endocrinol 1989; 121:174-176. 291. Bendz H, Sjodin I, Toss G, Berglund K. Hyperparathyroidism and long-term lithium therapy--a cross-sectional study and the effect of lithium withdrawal. J Intern Med 1996;240(6):357-365. 292. Christiansen C, Baastrup PC, Transbol I. Development of "primary" hyperparathyroidism during lithium therapy: longitudinal study. Neuropsychobiology 1980;6:280-283. 293. Shen F, Sherrard DJ. Lithium-induced hyperparathyroidism: An alteration of the "Set-Point." Ann Intern Med 1982;96:63-65. 294. Saxe AW, Gibson G. Lithium increases tritiated thymidine uptake by abnormal human parathyroid tissue. Surgery 1991; 110:1067-1077. 295. Mallette LE, Khouri K, Zengotita H, Hopis BW, Malini S. Lithium treatment increases intact and mid region parathyroid hormone and parathyroid volume. J Clin Endocrinol Metab 1989;68:654-660. 296. Abdullah H, Bliss R, Guinea AT, Delbridge L. Pathology and outcome of surgical treatment for lithium-associated hyperparathyroidism. BrJ Surg 1999;86(1):91-93. 297. NordenstromJ, Strigard K, Perbeck L, et al. Hyperparathyroidism associated with treatment of manic-depressive disorders by lithium. EurJ Surg 1992;158:207-211. 298. Cole DE, Peltekova VD, Rubin LA, Hawker GA, Vieth R, Liew CC, Hwang DM, EvrovskiJ, Hendy GN. A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations. Lancet 1999;353(9147):112-115.

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CIeTWR 19 Molecular Basis of Primary Hyperparathyroidism

ARNOLD Centerfor Molecular Medicine and Division of Endocrinology and Metabolism, University of Connecticut School of Medicine, Farmington, Connecticut 06030

ANDREW

INTRODUCTION

feature of cancers is their clonal nature; in other words, cancers arise from a single precursor cell (i.e., "monoclonal") whose progeny of essentially identical daughter cells have a selective growth advantage. Thus, every neoplastic cell in the resulting clinically apparent tumor typically contains an identical detailed pattern of DNA damage to multiple key growth-regulating genes, identifying this clone uniquely. To varying degrees, subpopulations of such a clone may develop due to the overlay of further acquired DNA damage after the initial clone is established, conferring additional selective advantage in a process called clonal evolution. These later changes might be expected to be found in only a subpopulation of the tumor cells. Distinguishing the genetic changes important for initial clonal expansion versus later clonal evolution has been difficult to address, although recent advances in selecting small groups of tumor cells for separate analyses will allow major progress on this issue over the next several years. That said, from the viewpoint of the clinically apparent tumor, the pervasiveness of many specific mutations throughout its entire set of neoplastic cells makes it clear that many of the important underlying genetic events occurred early, before major proliferation or clonal expansion of affected cells to clinical significance. The clonality of tumors also indicates that the summ a t i o n / a c c u m u l a t i o n of factors n e e d e d to transform (or confer the neoplastic phenotype upon) a cell occurs only rarely in a large population of cells in a tissue. Within such a population of cells, the rare emergence of a transformed clone is consistent with the rare occurrence of mutations in certain key rate-limiting

General Principles in Molecular Oncology In recent years we have witnessed an explosion in our knowledge of the molecular basis of neoplasia. This revolution was spearheaded by work on various hematopoietic tumors, but major insights into solid tumor pathogenesis have become commonplace as well. Despite some notable advances, however, knowledge of the molecular mechanisms underlying the pathogenesis of endocrine tumors in general, and of parathyroid tumors i n particular, remains relatively undeveloped in comparison to that for neoplasms such as lymphomas, leukemias, colon, and breast cancers. That said, existing information does indicate that many of the well-established general themes in tumor biology are quite applicable to parathyroid tumorigenesis, in spite of the typically nonmalignant status of these tumors. In addition, one molecular insight into parathyroid tumor development, the discovery of the cyclin D1 (PRAD1) oncogene, has also proved to be of tremendous general significance in h u m a n molecular oncology and in basic cell cycle biology. It is now solidly established that cancer cells contain genetic damage that is central to the abnormal neoplastic phenotypes they characteristically exhibit. In addition to this damage to the actual base sequence in the DNA, certain "epigenetic" factors may also be important; these could include aberrant patterns of DNA methylation, hormonal influences on gene expression, or i m m u n e system stimulation. A critical The Parathyroids, Second Edition

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pathways a n d / o r a requirement for the accumulation within one cell of multiple different damaging DNA alterations. The clonality of tumors does not, however, exclude an important role for field effects or generalized proliferative stimuli directed at the particular tissue as one of several factors contributing to tumorigenesis. The molecular genetic heterogeneity underlying neoplasia should be emphasized. Though certain tumor types might exist in which damage to only one gene is necessary a n d / o r sufficient for transformation, the general rule appears to be that damage to multiple distinct genes, all within the same cell, must accumulate for the ultimate expression of the neoplastic phenotype (1-3). Certain genes may be implicated in tumors of only one or a few cell types (e.g., TSH receptor, RET) while others may be involved in many different types of tumors (e.g., p53, Rb, cyclin D1). For most (and perh a p s all) tumor types, however, it appears that no single genetic change will prove to be both a necessary and sufficient causative agent. More likely, disruption of certain biochemical pathways may be important unifying themes for the emergence of particular tumors, and different combinations of mutated genes may result in similar cellular and clinical consequences. Studies of oncogene additivity or cooperativity, and other functional studies, will help to shed light on these issues. There are two broad categories of normal cellular genes in which clonal DNA damage contributes to neoplasia; these are protooncogenes and tumor-suppressor genes. Protooncogenes often have roles in the normal physiologic control of cellular growth, proliferation, or differentiation, and may function by regulating protein phosphorylation, signal transduction, or gene transcription, for example. Damage to a protooncogene, converting or activating it to an "oncogene," usually causes a deregulation of expression of its normal protein product or the formation of an intrinsically abnormal product. In contrast, tumor suppressor genes normally function to restrain cell proliferation and contribute to neoplasia through their functional inactivation. The types of somatic DNA derangements that can activate protooncogenes include chromosome translocations or inversions, point mutations, proviral insertions, and gene amplification. Inactivation of tumor suppressor genes is often accomplished by internal mutation or deletion, and may be inherited or incurred somatically. The causes of these types of oncogenic "hits" are not well understood. Environmental carcinogens such as ionizing irradiation or chemicals play a direct role in some instances, and errors in normal chromosomal recombinatory mechanisms also appear to occur, perhaps randomly. Certain genetic changes tend to occur more commonly in mitotically active cells, and carcinogens may act either by direct

mutagenesis of DNA or through augmentation of the mitotic rate, which secondarily increases the likelihood of an oncogenic chromosome aberrancy. Still other mechanisms may yield genetic damage without the requirement for high mitotic activity in the cell type. Many excellent reviews are available to the reader interested in exploring these principles further (2-8).

Special Issues in Parathyroid Neoplasia Though the general principles described above are expected to hold true for the specific example of parathyroid neoplasia, a complete molecular description of parathyroid tumorigenesis will ultimately need to explain a number of special features unique to the parathyroid model. Among these mysteries are the increased incidence of parathyroid tumors after exposure to neck irradiation, the heightened frequency with which hyperparathyroidism is found in postmenopausal women, the rarity of parathyroid cancer as relative to the more typical development of benign parathyroid tumors, and the relationship between excess cellular proliferation and the misadjusted set point linking ambient calcium level with parathyroid hormone (PTH) secretion in the tumor cells. Also, evidence exists on several levels that the growth rate of many clinically detectable parathyroid adenomas is quite slow, and may have changed over time (9). Information relevant to these and other special issues is only beginning to be generated, but the continued application of modern methods certainly promises eventually to yield the required molecular/pathophysiologic synthesis.

CLONALITY OF PARATHYROID TUMORS Controversy and uncertainty exist regarding the pathologic etiologies and clinicopathologic categorization of hyperparathyroidism. Distinction between parathyroid adenoma and hyperplasia can be made on the basis of the n u m b e r of abnormal, hypercellular parathyroid glands found in the patient, with a single abnormal gland being defined as an "adenoma." It can, however, be difficult histologically to distinguish a normal from a mildly hypercellular gland, and histologic examination of a particular hypercellular gland offers no features solidly predictive of whether it is a solitary tumor (i.e., adenoma) or one of several (i.e., hyperplasia) in the patient (10,11). Multigland disease can affect an individual's parathyroid glands in a highly nonuniform and asynchronous fashion ("asymmetric hyperplasia"), even to the point of confusing parathyroid hyperplasia with adenoma when only

MOLECULARBASISOF HYPERPARATHYROIDISM / one enlarged gland is found at surgery. Whether "double adenomas" or "multiple adenomas" exist (i.e., together with at least one truly normal parathyroid gland) has therefore been controversial, but the preponderance of clinical evidence suggests this entity is indeed genuine (12-15). Early studies of the clonal status of parathyroid tumors were designed to address some of these uncertainties. Assessment of X-chromosome inactivation patterns using the glucose-6-phosphate dehydrogenase (G6PD) protein polymorphism had indicated that parathyroid adenomas (single-gland disease) were polyclonal as opposed to monoclonal growths (16,17). These data suggested that a parathyroid "adenoma" was really a highly asymmetric form fruste of multigland hyperplasia, and were taken to support the surgical practice of routine bilateral neck exploration a n d / o r subtotal parathyroidectomy for hyperparathyroidism. This conceptualization of the origins of parathyroid tumors certainly did not encourage a search for the types of DNA damage, discussed above, characteristic of monoclonal tumors. The clonal status of parathyroid adenomas was reevaluated several years later, again by X-chromosome inactivation analysis, but using a DNA polymorphismbased method that avoids many of the pitfalls of the protein polymorphism approach. It was determined that most and perhaps all parathyroid adenomas were in fact monoclonal tumors, i.e., that these glands were true neoplastic outgrowths of a single abnormal cell (18). Subsequent studies have confirmed the monoclonality of parathyroid adenomas (19-28). This finding has been taken to support to the surgical practice of resecting only the clearly enlarged gland (and not exploring the other side of the neck) if gross examination and biopsy of an identified ipsilateral gland are completely normal. This approach would, of course, fail if the contralateral side harbored another independent clonal adenoma (12,13,29). Parathyroid carcinomas, not surprisingly, are also monoclonal (25,30,31 ). Even if one puts aside the possibility of multiple adenomas arising de novo in a given patient, a finding of monoclonality in an enlarged parathyroid gland clearly cannot be viewed in isolation as diagnostic for singlegland disease. It has now been established that in various forms of parathyroid hyperplasia, which presumably begin with a stimulus for generalized (polyclonal) parathyroid cell proliferation affecting all the glands, monoclonal tumors can subsequently evolve in some or many of these glands. For example, monoclonality has been documented in parathyroid tumors from patients with nonfamilial (sporadic) primary parathyroid hyperplasia (32) and in the large majority of glands from patients with the

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refractory secondary (or "tertiary") hyperparathyroidism of uremia (32). The clinical or pathophysiologic significance of monoclonality in these settings remains to be determined; it is highly plausible, for example, that such tumors may exhibit a greater degree of pathological autonomy in PTH secretory function as well as higher growth rates than do any truly hyperplastic (polyclonal) neighboring glands in the same patient. Clonal expansion also characterizes the enlarged parathyroids of patients with multiple endocrine neoplasia type 1 (MEN-l) (20,21,33,34), but it is not known whether such outgrowths evolve from a preliminary stage of true polyclonal hyperplasia that might be driven by the inherited haploinsufficiency for the MEN1 gene.

SPECIFIC GENETIC A B N O R M A L I T I E S IN B E N I G N PARATHYROID T U M O R S The determination that parathyroid adenomas were monoclonal neoplasms conveyed the expectation that these tumors, albeit benign, result from some of the same types of genetic damage that characterize clonal malignancies (18). This expectation is now shared by a subset of tumors, also found to be monoclonal, in patients with various forms of multigland "hyperplasia," discussed above. Identification of the particular genes whose tumor-specific activation or inactivation results in these clonal expansions is critical, because building a detailed understanding of the molecular defects that underlie parathyroid neoplasia may ultimately lead to successes in diagnosis, pathologic classification, prevention, a n d / o r treatment. Identifying these genes may also point to particular biochemical pathways of importance in regulating parathyroid cell proliferation, thus speeding the discovery of other genes of similar importance. So far, only two specific genes, cyclin D1 (PRAD1) and MEN1, have been convincingly incriminated in the common sporadic forms of benign parathyroid neoplasia. For cyclin D1, this initial molecular insight has proved to have exceedingly broad implications regarding basic cell cycle biology and human cancer, which speaks well for parathyroid tumors as potentially generalizable models of benign or well-differentiated neoplasia. The principle of molecular heterogeneity predicts that still more parathyroid tumor genes exist, and studies have pointed to specific chromosomal regions that almost certainly contain such genes (22-24,35,36). These target areas for critical (recurrent and clonal) DNA damage, which together confer a selective growth advantage on the parathyroid cell in which they develop (i.e., the clonal precursor cell), are discussed in this section.

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Activation o f the cyclin D1 (PRAD1) O n c o g e n e Initial Cloning of PRAD1

DNA rearrangements, in the form of translocations and related chromosomal events, are among the best characterized types of clonal oncogenic genetic abnormalities (37). Very frequently, these rearrangements involve the tumor-specific juxtaposition of cellular protooncogenes with DNA sequences designed to regulate other genes; this may result in the overexpression or deregulated expression of the protooncogene, thus converting it to an oncogene. In fact, the cloning of DNA immediately adjacent to clonal chromosome breakpoints has proved to be a tremendously effective method for identification of new oncogenes. One example was the discovery of the BCL-2 oncogene in follicular lymphomas with the t(14;18) chromosome translocation. BCL-2 (on chromosome 18) is "activated" and overexpressed as a consequence of its relocation into the part of the immunoglobulin heavy-chain gene, on chromosome 14, that contains sequences responsible for immunoglobulin's strong transcriptional activity in B lymphoid cells (38). It is interesting to note that most oncogenes found to be activated by rearrangement in human cancer have been detected in hematopoietic tumors. Though it is likely that chromosomal translocations occur more frequently in these cell types, the situation may in part also reflect technical advantages in performing cytogenetic analyses on hematopoietic as compared with solid tumors. Improved cytogenetic methods may permit detection of more chromosome translocations in solid tumors, including parathyroid tumors, and thus provide searchlights for the identification of additional oncogenes. The key initial observation in the identification of PRAD1 was a band of abnormal size on a Southern blot of DNA from a parathyroid adenoma, probed with a

;~TH Coding

Break

~

P T H genomic DNA fragment (18,39). This band was not present in the same patient's normal leukocyte control DNA, and was thus tumor specific and clonal. Relative intensifies of this band compared with the remaining normal-size band suggested that the underlying DNA alteration affected one of the tumor's two P T H alleles, and that the abnormality was present in every cell in the tumor. It had, therefore, presumably occurred in the original clonal progenitor cell of the mature tumor, likely conferring a selective growth advantage, and hence appeared worthy of additional investigation for its possible pathogenetic importance. Southern analysis of this original adenoma revealed that the clonal alteration responsible for the observed abnormal band was a tumor-specific DNA rearrangement (39). The rearrangement separated the 5' regulatory region and noncoding exon 1 of the PTH gene from its coding exons, with different, non-PTH DNA placed adjacent to each P T H gene section (Figs. 1 and 2). In addition, the tumor possessed one intact P T H gene per cell, which appeared to be the source of PTH production by the adenoma. Though a DNA rearrangement with this structure could have been tumorigenic in a few possible ways, it was hypothesized that in analogous fashion to the immunoglobulin/ BCL-2 model, the P T H gene regulatory region, which strongly drives tissue-specific gene expression in the parathyroid cell, could be activating the expression of a protooncogene placed under its influence by the rearrangement. The DNA sequences of interest, lying adjacent to the PTH breakpoint, were then cloned from a genomic DNA library prepared from this adenoma. From the P T H gene-positive bacteriophage inserts, subclones were made that contained only the breakpoint-adjacent n o n - P T H D N A . This "anonymous" single-copy DNA was mapped using somatic cell genetics and in situ

PTH Coding

;~TH 5' Regulatory

,ntromere

PTH 5' Regulatory

Break

Cyclin DI/PRAD1

C:yclin DI/PRAD1

Normal

Inverted

FIG. 1 Schematic diagram illustrating the DNA rearrangement involving the PTH gene and the cyclin D1/PRAD1 gene in a subset of parathyroid adenomas. The chromosomal inversion event is deduced as the simplest cytogenetic mechanism consistent with the molecular details of this DNA rearrangement. Modified from Ref. 129, A Arnold. Genetic basis of endocrine disease 5 molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab, Vol. 77, pp. 1108-1112, 1993. © The Endocrine Society.

MOLWCULA~ BASIS OF HYPERPARATHYROIDISM /

11p15

pr~ees_

h'

335

11q13

Re~y2W_,v- Region

"="'

T

"C;clin Ol Gene

Exons

/

/

• Transcription starts with Cyclin 01 's first e~on, using its own promot,dr. • Active Cyclin D1 transcription is driven by ti~,sue-specificenhancer(F) from the 5' PTH gene region. ~ ~_ ~ /" Overexpressed cyclin D1 mRNA ~i!~i!i!i!!ii!iii!ii{!iiiiii ! !i!i!i~i~!!i!iiiiiii!~i] l with normal coding sequence |~!~!:i)~i~.~.~:~i~i!~ii~i~i~i~:~i~i~i!~i~!~!i~i~!~iii~i!i!:~i~:~`~iii~!~.~i~ O v e r e x p r e s s e d cyclin D1 protein, due to gene rearrangement or other activating m e c h a n i s m s ~ - 4 0 % of parathyroid adenomas

G1 ~S +G2 +M Cell cycle deregulation

Other o n c o g e n i c effects

hybridization, was found to be normally located on chromosome 11, band q 1 g, and was initially called D11 $287 (39). Because the normal chromosomal location of the PTH gene is also on chromosome 11, at 11p15, the simplest cytogenetic explanation for the observed tumor-specific DNA rearrangement was a pericentromeric inversion of chromosome 11 (Fig. 1). Although there were several oncogenes that had previously been mapped to 11q13, namely INT-2, HST-1, SEA, and the BCL-1 lymphoma translocation breakpoint, the newly cloned DllS287 region did not appear to be identical to any of these, as evidenced by restriction map comparisons and by direct clone to clone hybridization analysis. DllS287 subclones were then surveyed in attempts to detect an expressed gene or transcription unit in the region. One such probe, located about 1 kb from the breakpoint, detected a distinct 4.5-kb mRNA species on Northern blots (40). This mRNA species was present in normal h u m a n parathyroid tissue, parathyroid adenomas without the PTH/D11S287 rearrangement, normal thyroid, and normal placenta. Thus, the transcript's presence was immediately recognized as not being highly cell type specific. Moreover, the same h u m a n DllS287 probe hybridized to mRNA of the same approximate size, withstanding stringent washing, on RNA blots from other species and tissues, including bovine lymph node, muscle, thyroid, and parathyroid, and mouse heart and liver (41). Most importantly, this mRNA proved to be dramatically (15-fold) overexpressed in the original parathyroid adenoma from which the rearrangement had been cloned (40). Additional independent parathyroid adenomas containing similar PTH rearrangement breakpoints were

FIG. 2 Diagram of the directly observed molecular structure of the PTH/cyclin D1 (PRAD1)DNA rearrangement and its functional consequences. Modified from Ref. 129, A Arnold. Genetic basis of endocrine disease 5 molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab,Vol. 77, pp. 1108-1112, 1993. © The Endocrine Society.

then detected; they also had DllS287 region breakpoints (40,42) that were seen to vary by as much as 15 kb (40), and no other expressed genes were detectable in this 15-kb region. These additional cases also exhibited dramatic overexpression of the DllS287 transcription unit (40). Because of the involvement of DllS287 in clonal DNA rearrangements associated with gross abnormalities in its transcription, analogous to those in lymphoid neoplasia, this highly conserved sequence was considered further as a putative oncogene and was subsequently called PRAD1, for 12arathyroid adenomatosis 1.

PRAD1 Gene Product as a Novel Cyclin The normal cDNA for PRAD1 was cloned from a h u m a n placental cDNA library (41) and screened with the original breakpoint-adjacent genomic fragment from 1 l ql 3 that had hybridized to a distinct transcript. Sequence of the cDNA revealed one long open reading flame, encoding a 295-amino acid protein. The derived amino acid sequence indicated at that time that the PRAD1 protein was not closely related to any known protein or family. However, a weaker but consistent homology between PRAD 1 and the various members of the cyclin classes of proteins, which were known to have important roles in regulation of the cell cycle, was observed (41). The PRAD1 protein's region of maxim u m homology was an approximately 100-amino acid stretch (amino acids 55-160), which matched within the so-called "cyclin box," a 100- to 150-amino acid domain of greatest conservation between all the known cyclins. These other cyclins included A and B forms from yeast, clam, starfish, sea urchin, and Drosophila;

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PRAD1 had similar homology to their cyclin box regions, e.g., 28% identity with Drosophilacyclin A, and 31% identity with clam cyclin A. For comparison, h u m a n versus clam cyclins A are 71% identical in their cyclin boxes (and, as is typical, quite dissimilar throughout their other regions). These sequence comparisons suggested that PRAD1 was a novel cyclin, representing its own family distinct from the other families of cyclins (41). This suggestion was immediately reinforced by functional studies of the PRAD1 gene product, and then confirmed and expanded by countless studies over the past decade. The recognition that PRAD1 encoded a novel cyclin-type protein raised fascinating possibilities for its role in tumorigenesis, based on the role of cyclins in regulating progression through the cell division cycle. The cell cycle comprises stages G 1, S (DNA synthesis), G2, and M (mitosis). Progression through the cell cycle is regulated at two critical checkpoints, the G2/M border and a point in G 1 (called START in Saccharomyces and the "restriction point" in mammalian cells) that once traversed, enables the G1/S transition to proceed (43-47). Control of the G2/M transition is attained through a universal mechanism c o m m o n to all eukaryotic cells. The key feature of M phase is the activation of a s e r i n e / t h r e o n i n e protein kinase, designated cdc2, p34 c~c2,p34, M-phase kinase, and others, depending on the system of original study. Activation of cdc2 kinase induces mitosis through a process of phosphorylation of key proteins, leading to events such as chromosome condensation, cytoskeletal reorganization, nuclear envelope breakdown, and cell shape changes. The mitotic cyclins are another universal c o m p o n e n t of this system. First identified in dividing eggs of marine invertebrates, they are characterized by their accumulation t h r o u g h o u t interphase until the Gz/M transition, and then their rapid destruction during mitosis. Cyclin B, the prototypical mitotic cyclin, can be considered a regulatory subunit that must associate with cdc2, the catalytic subunit, to create an active holoenzyme complex. The disappearance of cyclin B in M phase, effected through the ubiquitin-proteasome degradation pathway, is necessary for kinase inactivation and exit from mitosis. The G1/S transition is also a critical checkpoint, and as the major regulatory point determining the cell's decision to attempt to divide, is an attractive site for attack by an oncoprotein. At the time of discovery of PRAD1, "G 1 cyclins" essential for this transition had been demonstrated only in yeast. These proteins, called CLN1, CLN2, and CLN3, have weak homology to cyclins A and B, and associate with cdc2 kinase specifically at G1/S (48-51). The discovery of PRAD1 thus raised the intriguing possibility that it might encode a h u m a n G 1 cyclin. Consistent with this possibility,

PRAD 1 mRNA and protein levels begin to rise and also peak within G 1, in 70N m a m m a r y epithelial cells (52) and fibroblasts (53) synchronized by serum starvation and subsequent addition of growth factors. The mouse homol0g of PRAD1 also acts in G 1, and was in fact cloned as a macrophage cDNA specifically induced in G 1 in response to colony-stimulating factor-1 (CSF-1) (54). Compelling evidence that the PRAD1 product is truly a functional cyclin was the demonstration that its cDNA could rescue mutant yeast deficient in G 1 cyclins; introduction of PRAD 1 cDNA released these yeast from cell cycle arrest in G 1 (55,56), and was an alternative route to the cloning of PRAD1. Because classic "mitotic cyclins" such as cyclin B can also rescue these G 1 cyclindeficient yeast, these data could not be taken as proof that the PRAD1 cyclin normally functions in G 1 phase. However, such proof has come, in overwhelming fashion, over the past several years and PRAD1 is indeed established as a critical mammalian G 1 cyclin (47). With the subsequent discovery of still other families of h u m a n cyclins, up to about a dozen, the picture has increased in complexity. Given the need for unifying nomenclature, the PRAD1 gene product is now commonly referred to as cyclin D1. In addition to the recognition of other cyclin families, two new cyclins quite closely related to PRAD1/cyclin D1 were isolated by low-stringency hybridization; these members of the Dtype cyclin family are known as cyclin D2 and cyclin D3 (52,57,58). To some extent, the expression of particular D cyclins is cell type specific, but though there may be some capacity for redundancy, most evidence suggests that these cyclins must have functionally distinct roles. Furthermore, multiple new cdc2-1ike kinases have been found, defining the general category of cyclind e p e n d e n t kinases (cdks), many of which have been matched up with specific cyclin partners. For example, the major cdk partner for cyclin D1 is cdk4 or cdk6, d e p e n d i n g on the differentiated cell type. The discovery of cyclin D also led to the identification of a class of proteins found in physical association with cyclin-cdk complexes (46,47,55,59,60), which proved to be inhibitors of cdk activity. These cdk inhibitors include p16, which has high specificity for blocking cyclin D - c d k 4 / 6 kinase activity. Importantly, the p16 gene has been solidly established as a human tumor suppressor gene, participating in a variety of neoplasms such as m e l a n o m a and squamous cell cancers. Unlike cyclin D 1, however, p l 6 does not appear to participate in parathyroid cell neoplasia (35), implying that cyclin D1 overexpression contributes to parathyroid tumorigenesis in ways that are not duplicated or precisely mimicked by p 16 inactivation. The cyclin D l - c d k 4 - p l 6 biochemical pathway also involves the protein product of the retinoblastoma tumor suppressor gene, Rb. The Rb protein (pRb) is an

MOLECULAR BASIS OF HYPERPARATHYROIDISM / important regulator of the G1/S transition of the cell cycle, and is phosphorylated in a cell cycle-dependent fashion (61,62). Early in G 1, the hypophosphorylated form of pRb is thought to bind and sequester a variety of transcription factors, such as those in the E2F family, maintaining the cell in G 1. Release of such factors later in G1, as pRb becomes highly phosphorylated, goes on to drive the cell into S phase. Thus, active (hypophosphorylated) pRb has a growth-restraining effect, which can be reversed or eliminated by inactivation through normal cell cycle-dependent hyperphosphorylation, or through germ-line or somatic mutational events. Much biochemical evidence has shown active pRb to be a major substrate target for cell cycle-dependent phosphorylation by active cyclin D l - c d k 4 / 6 complexes. It is thought, therefore, that overexpression or deregulated expression of cyclin D1 could quite conceivably accelerate the cell's progress through G 1 into S phase, bypassing normal regulatory controls in committing it to divide, and also be well tolerated by the cell during the remainder of the cycle. Such a mechanism would provide an appealing explanation for the benign nature of parathyroid adenomas, because it could yield excessive cellular proliferation without necessarily conferring the phenotypes of invasiveness or metastasis on the tumor cell. The measurable effects of cyclin D1 on proliferation of cells in culture appear to be mediated entirely through its ability to phosphorylate and thereby inactivate pRb; in fact, cyclin D1 becomes dispensible, in terms of its crucial and rate-limiting role at the G1-S checkpoint, in cells with deleted or otherwise genetically inactivated Rb. Rb is a classical example of a h u m a n tumor suppressor gene, involved in numerous tumors such as retinoblastoma, osteosarcoma, and breast and lung cancers. It appears that the cyclin D l - c d k 4 / 6 - p l 6 - R b pathway, viewed as a whole, has become aberrant in virtually every h u m a n cancer, highlighting it along with the p53 gene pathway as apparently essential targets for deregulation if neoplasia is to result. Cyclin D1 stands out as the only cyclin clearly implicated as a h u m a n oncogene (47,63,64). Because genetic abnormalities in many other G1-S regulators, including other cyclins and most cdk inhibitors, apparently do not confer a selective advantage on the cell (i.e., are not seen as clonal changes), it remains to be proved whether the oncogenicity of cyclin D1 is implem e n t e d entirely or at all by the shortening or acceleration of the G 1 phase of the cell cycle. Furthermore, it is plausible that cyclin D1 overexpression contributes to oncogenesis through mechanisms other than, or in addition to, its ability to activate cdk4 and phosphorylate pRb. In other words, tumorigenesis in the intact patient is a highly complex process not fully modeled by cultured cells, and it may be overly simplistic

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to assume that cyclin D1 acts solely via the D l - c d k 4 p16-Rb paradigm that we now understand. This caution seems especially apt in considering parathyroid neoplasia, given its typically benign nature and slowgrowth properties. It is hoped that newly developed animal models of cyclin D 1-driven parathyroid neoplasia will provide new insight into parathyroid tumorigenesis.

Further Analysis of the Structure of PTH/Cyclin D1 Rearrangements After the cloning and analysis of the full-length complementary DNA (cDNA) representing PRAD1 mRNA, discussed above, it was possible to define the complete i n t r o n / e x o n organization of the normal chromosomal PRAD1/cyclin D1 gene (65). The gene is oriented on l l q 1 3 such that it is transcribed in a centromeric to telomeric direction, and contains five exons and four introns spanning approximately 15 kb. In the parathyroid adenomas with PTH/cyclin D1 rearrangements that have been well characterized to date, the 1 lq13 breakpoints have occurred from 1 to 15 kb upstream of cyclin D1 exon 1, leaving its exon-containing region and immediate p r o m o t e r intact (Fig. 2). Attached in these rearrangements to the cyclin D1 structural gene is the 5' regulatory region, including n o n c o d i n g exon 1, of the P T H gene (Fig. 2). The remarkable up-regulation of cyclin D1 expression in these parathyroid tumors therefore appears to be driven by DNA sequences normally found in this upstream PTH gene vicinity. In fact, these rearrangements provided the strongest initial evidence for localizing the still-undefined tissue-specific enhancer(s) of the P T H g e n e to its 5' regulatory region, a c o m m o n but by no means universal site for such sequences. This general localization of the P T H gene's tissue-specific e n h a n c e r has been confirmed by its success in targeting expression of a cyclin D1 transgene to parathyroid tissue in mice (66). The presence of n o n c o d i n g exon 1 of the P T H gene in the DNA rearranged upstream of cyclin D1 raised the possibility that a P T H exon 1/cyclin D1 fusion mRNA might ensue, and be transcribed from the P T H gene's own promoter. However, this does not appear to be the case. Analysis of the overexpressed cyclin D1 transcript (cDNA) from one such parathyroid t u m o r revealed normal cyclin D1 sequence at its 5' end, with no contribution from the P T H gene (67). The rearranged cyclin Dl's use of its own p r o m o t e r could also predict that its overexpression might be insulated from signals such as 1,25-dihydroxyvitamin D 3 or hypercalcemia, which are inhibitory for PTH gene transcription (see Chapter 2). In addition, it appears that point mutations in cyclin D1 are not necessary for tumorigenesis, because the amino acid coding sequence of this overexpressed cyclin D1 cDNA was entirely normal (67).

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Frequency of Cyclin D1/PRAD1 Involvement in Parathyroid Neoplasia

examined by immunohistochemistry in at least three studies, showing cyclin D 1 overexpression in 20-40% of parathyroid adenomas (68-70). The fraction of parathyroid adenomas for which cyclin D1 overexpression is due to PTH-cyclin D1 rearrangement, or other cyclin DI rearrangements for that matter, remains uncertain. It is important to note that the original Southern blot screens for such rearrangements used probes of PTH or cyclin D1 in

Because cyclin D1 oncogene activation in h u m a n tumors occurs through a variety of mechanisms, of which gene r e a r r a n g e m e n t is but one, the best estimates to date of the frequency of the involvement of cyclin D1 in parathyroid adenomas come from assessm e n t of cyclin D1 expression at the protein level. This pathogenetic c o m m o n d e n o m i n a t o r (Fig. 3) has been

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FIG. 3 Example of cyclin Dl-positive parathyroid adenoma (anticyclin D1 immunoperoxidase staining). (A) Normal parathyroid gland, lacking immunoreactivity; (B) parathyroid adenoma from the same patient, showing strong nuclear immunoreactivity in the majority of cells. Reprinted from Ref. 68, E Hsi, L Zukerberg, W-I Yang, A Arnold. Cyclin D1/PRAD1 expression in parathyroid adenomas: An immunohistochemical study. J Clin Endocrinol Metab, Vol. 81, pp. 1736-1739, 1996. © The Endocrine Society.

MOLECUtAR BASIS OF HYPERPARATHYROIDISM / the vicinity of their coding regions, and with the use of a limited number of restriction endonucleases (18,39,40,42). However, rearrangement breakpoints on 11q13 associated with overexpression of cyclin D1 in B cell lymphomas frequently occur well over 100 kb upstream of the cyclin D1 gene (71), and parathyroid adenomas with rearrangement breakpoints about 60 kb and otter 200 kb upstream of cyclin D1 have been observed (M. E. Williams, Y. Hosokawa, S. Mallya, and A. Arnold, unpublished observations), and would have been missed in the original Southern blot screens, which documented a minimum of about 5% of adenomas bearing PTH-cyclin D1 rearrangements. It is quite conceivable that PTH gene breakpoints could also vary considerably and have eluded detection by the previous approach; indeed, cyclin D1 expression could be deregulated in some parathyroid tumors by rearrangement with actively transcribed genes other than the PTHgene. For all these reasons, more comprehensive approaches will be necessary, and are in progress, to give a better sense of the frequency at which cyclin D1 gene rearrangement is responsible for deregulated expression of the cyclin D1 oncoprotein in parathyroid neoplasia. The first few patients with parathyroid adenomas that were clearly documented to have PTH/cyclin D1 rearrangement and overexpression exhibited clinical features of symptomatic hyperparathyroidism and had unusually large adenomas (6-8 g), without any histologic features suggestive of malignancy (18,39,40,42). However, subsequent study demonstrated cyclin D1 overexpression (Fig. 3) across the entire spectrum of parathyroid adenoma-related phenotypes, including asymptomatic patients with modestly sized adenomas (68). Thus, it appears that deregulation of cyclin D1 is an important contributor to the development of many (20-40%) parathyroid adenomas, which cover the broad spectrum of clinical and pathologic severity characteristic of this disorder. Role of Cyclin D1 in Other Human Tumors

Interest in the role of cyclin D1 in oncogenesis has been intensified by its incrimination in other, nonparathyroid, human tumors (64). In mantle cell (or centrocytic) B cell lymphomas, the upstream cyclin D1 gene region is rearranged with the immunoglobulin heavy-chain gene enhancer on chromosome 14. This t(ll;14) translocation results in the overexpression of cyclin D1, in a fashion analogous to the deregulated expression of oncogenes c-MYC and BCL-2 by their translocation into the immunoglobulin region in other types of B cell lymphomas (72-74). The possibility that a different gene in this region could be the true target of such rearrangements was effectively excluded by

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genetic evidence demonstrating some breakpoints located very close to cyclin D1 (71,75). Thus, cyclin D1 is now established to be the so-called "BCL-I" oncogene, which had been presumed to exist but had eluded workers searching 11 ql 3 in the vicinity of these translocation breakpoints for many years. The difficulty they had was due to the large distance, approximately 120 kb, between the original BCL-1 translocation breakpoint and the first exon of cyclin D1. Detection of cyclin D1 rearrangement or overexpression has become part of the armamentarium of diagnostic pathology labs in distinguishing subtypes of B cell neoplasms. More recently, tumor translocation breakpoints involving cyclin D1 and a different part of the immunoglobulin locus have been described in multiple myeloma (76,77). In addition to being a B cell lymphoma oncogene, cyclin D1 has been implicated in many other types of h u m a n tumors, including breast cancer; squamous cell cancer of the head, neck, and esophagus; bladder cancer; and more. In 12-15% of breast cancers and in up to 40% of the squamous cell tumors, a large stretch of DNA on 11q13 is amplified (present in extra copy number), implying the existence of at least one key gene, a "driver oncogene," whose amplification confers a selective growth advantage on the cell. Though there appears to be some complexity in the patterns of amplification in this region (78,79), cyclin D1 is now considered to be the, or at least one, driver oncogene on the major 11q13 "amplicon." First, no other gene is more consistently present on 11q13 amplicons in breast and squamous cell cancers (80,81). Second, cyclin D1 is overexpressed in these tumors; in contrast, most other genes that may be coamplified along with cyclin D1 typically exhibit poor expression (80). Finally, mammarytargeted overexpression of cyclin D1 in transgenic mice causes mammary carcinoma (82); no other candidate oncogene on the 11q13 amplicon has demonstrated this oncogenic capacity. Further studies have raised fascinating new possibilities regarding the mechanisms through which cyclin D1 exerts its oncogenicity in breast cancer. Two groups determined that overexpressed cyclin D1 can bind to and activate the estrogen receptor in cultured mammary carcinoma cells, without the need for estrogen and without the need for cyclin D1 to bind cdk4 (83,84). Though this mechanism requires further investigation in vivo, it has the potential to explain why virtually all cyclin Dl-overexpressing breast cancers are estrogen receptor positive. Other data have also raised the possibility that cyclin D1 could have non-cdkd e p e n d e n t functions (62,85,86). Such emerging information reinforces the concept that an open mind must be maintained regarding the mechanism(s) by which cyclin D1 contributes to parathyroid neoplasia.

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Inactivation of the MEN1 Tumor Suppressor Gene Multiple Endocrine Neoplasia Type 1 In 1988 the gene responsible for the MEN-1 tumor predisposition syndrome was m a p p e d by linkage analysis to the long arm of chromosome 11, in the general vicinity of the muscle phosphorylase (PYGM) gene (87). The positive finding of linkage initiated an extensive search of this region of 11q13 for the specific "MEN1 gene" (see Chapter 35). It should be noted that though cyclin D1/PRAD1 also m a p p e d to 11q13, the tightest MENlqinked markers were located far upstream (centromeric) of cyclin D1, probably several million base pairs away (72,80,88-92). It was considered that MEN-1 might prove to be due to an inherited mutation in a tumor suppressor gene, akin to the paradigms of familial retinoblastoma and Li-Fraumeni syndromes, which involve inheritance of mutations in the Rb and p53 genes, respectively (61). Therefore, tumors from MEN-1 patients were examined for evidence of somatic genetic events that could have inactivated one allele of a gene in this region and thereby unmask the inherited constitutional MEN1 mutation. In support of this hypothesis, "allelic loss" (or loss of heterozygosity) of polymorphic marker DNAs from chromosome 11 was initially found in two insulinomas from brothers with MEN-1 (87) and was subsequently found in the large majority of MEN-l-associated parathyroid tumors (20,21,33,34,93,94). The n u m b e r of markers from different parts of chromosome 11 exhibiting allelic loss varied widely from tumor to tumor, but the region of overlap of these losses was consistent with the MEN1

region as d e t e r m i n e d by linkage analysis. Furthermore, when the parental origin of the tumor-specific, somatically lost allele was able to be determined it derived from the clinically unaffected parent (21,33); this would be the expected pattern in the event that the retained chromosome contained an inherited mutant tumor suppressor gene (Fig. 4). The positional cloning of MEN1 in 1997 (95) provided further evidence in support of a tumor suppressor function for its gene product. Specifically, many of the observed inherited MEN1 mutations would be expected to eliminate functionality of its gene product, called menin (see Chapter 35). This type of inherited mutation in one allele, together with the commonly found acquired deletion of the remaining (normal) MEN1 allele, would result in absence of menin protein in the resulting parathyroid tumors in this syndrome. The possibility that certain inherited missense mutations might not completely eliminate menin function does not alter the essential theme of MENI's tumor suppressor nature, but might cause a moderated tumorigenic phenotype in some instances. These issues require further investigation. It is important to note that tumor-specific allelic deletions from chromosome 11 are also markers of monoclonality of these MEN-l-associated parathyroid tumors, indicating that despite the usual multigland involvement ("hyperplasia") found in MEN-1 patients, their parathyroid tumors are often (and perhaps always) clonal. It should also be borne in mind that critical acquired DNA damage may need to develop at still other genetic loci in order for clinically significant parathyroid tumors to emerge in MEN-1. Additional

Chromosome 11

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mutant copy

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Mutant copy of MEN1 tumor suppressor gene on 11q13 is inherited in MEN-1 and present in all parathyroid cells Mutation of one allele of MEN1 gene can occur somatically in other patients present in specific parathyroid cell(s)

mutation of other genes?

benign ~ parathyroid neoplasia

Clonal progenitor cell lacks functional menin gene product: at least 12-17% of sporadic parathyroid adenomas

Other Chromosomes

normal copy

mutant copy

somatic deletion/mutation of remaining normal allele

Somatic mutation of one copy of relevant tumor suppressor qene: no adverse consequences to parathyroid cell

mutation of other genes?

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Clonal p r o g e n i t o r cell lacks functional tumor suppressor gene product

FIG. 4 Schematic diagram illustrating the established (for MEN1) and hypothesized roles of inactivation of classic tumor suppressor genes as contributory mechanisms in parathyroid neoplasia. Modified from Ref. 129, A Arnold. Genetic basis of endocrine disease 5 molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab, Vol. 77, pp. 1108-1112, 1993. © The Endocrine Society.

MOLECULARBASIS OF H~Em'ARAT~OmISM study will also hopefully address the interesting question of whether the presence of one mutant plus one normal copy of MEN1 confers an abnormal proliferative phenotype on a parathyroid cell and a preliminary state of generalized true hyperplasia. Finally, it has been hypothesized that some examples of familial primary hyperparathyroidism without other manifestations of MEN may well be variants of MEN-1. Mutational analyses of the MEN1 gene have provided an answer, namely, that most kindreds with familial isolated hyperparathyroidism have no detectable mutations and do not appear to be MEN-1 variants, although such variants do occur (see Chapters 35 and 37). Sporadic Parathyroid Adenomas

The possibility that a chromosome 11-based tumor suppressor gene was involved in sporadic (nonfamilial) parathyroid adenomas was raised by two i n d e p e n d e n t observations. In the well-characterized Rb tumor suppressor gene model, the sporadic (nonfamilial) counterpart of familial retinoblastoma results from the somatic inactivation/loss of both (initially normal) alleles of the Rb gene in the clonal precursor cell. It therefore seemed reasonable to hypothesize that some sporadic parathyroid adenomas might evolve from a cell in which both copies of the MEN1 tumor suppressor gene, m a p p e d to 11q13, became inactivated by somatic mechanisms (Fig. 4). The other key result that focused attention on chromosome 11 was the recognition of chromosome breakpoints, described aboved, involving 11 q13. Study of sizeable numbers of sporadic parathyroid adenomas has determined that allelic loss of chromosome 11 markers occurs in 25-40% of the tumors (21,23,24,94). Examination of polymorphic markers spanning chromosome 11 in one especially informative case revealed that one allele of each marker was lost in this adenoma, indicating that an entire copy of the chromosome was somatically deleted from the tumor genome (19). Careful gene dosage analysis confirmed that this was a true deletion, unaccompanied by reduplication of the remaining chromosome, showing that monosomy 11 can be tolerated by a parathyroid cell and that only a single P T H gene copy per cell is sufficient to allow hyperparathyroid function by the adenoma (19). These latter conclusions were confirmed subsequently by comparative genomic hybridization (24). Given that most (but not all) regions of allelic loss on chromosome 11 included marker loci closely linked to the MEN1 gene, it was expected that inactivating somatic mutations would be identified in the nondeleted MEN1 allele in these tumors, consistent with its hypothesized role as a classic tumor suppressor (Fig. 4). The subsequent cloning of MEN1 has allowed this

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hypothesis to be tested. Interestingly, acquired inactivation of both alleles of the MEN1 gene has been found in 12-16% of parathyroid adenomas (26-28), representing about half of the adenomas with 1 lq deletions. This finding certainly establishes the importance of MEN1 gene inactivation in 12-16% of parathyroid adenomas. However, the large percentage of adenomas with 11q loss but without detectable mutation in the nondeleted MEN1 allele raises the fascinating possibility that inactivation of a different tumor suppressor gene from this chromosome region may contribute to the proliferative phenotype in many of those adenomas. Pursuit of the mechanisms by which MEN1 inactivation can participate in parathyroid neoplasia is in its early stages. Menin, the protein product of the MEN1 gene, was shown to associate with JunD, supporting a role in transcriptional control (96). The normal tissue distribution for expression of MEN1 is essentially ubiquitous and clearly not confined to the tissues susceptible to MEN-l-related tumorigenesis (97). Sporadic adenomas with MEN1 mutations were reported to have a somewhat higher frequency of allelic losses and chromosomal imbalances compared with sporadic adenomas without MEN1 involvement. Although these differences were not statistically significant, they hint at a possible role for MEN1 in maintenance of chromosomal stability (98), a theme that has been raised previously (99,100). The possibility of a relationship or cooperation between cyclin D 1 and menin in the develo p m e n t of sporadic a n d / o r MEN-l-related parathyroid neoplasia will also need to be addressed. Finally, the contribution of MEN1 inactivation to other forms of sporadic parathyroid neoplasia appears to be minor. The 11q13 allelic loss was found in 2-3% of uremia-associated parathyroid tumors (36,101), with MEN1 mutation in a just a subset of these. Such data reinforce the concept that different forms of parathyroid neoplasia are likely to have distinct molecular pathogenetic origins.

O T H E R G E N E T I C A B N O R M A L I T I E S IN SPORADIC PARATHYROID TUMORS For sporadic parathyroid adenomas, there is strong evidence that genes in addition to cyclin D1 and MEN1 participate in their pathogenesis. Highly recurrent clonal allelic losses, which frequently and successfully highlight the genomic locations of tumor suppressor genes, have been observed on chromosomes 1, 6, 11, and 15 in sporadic parathyroid adenomas in molecular allelotyping studies (22-24). O t h e r chromosomes, such as 9 and 13, have also been so implicated, albeit with lower frequency (24,35). Studies of sporadic adenomas using comparative genomic hybridization, a molecular

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cytogenetic method, have generally confirmed these c o m m o n areas of acquired deletion, and have also identified areas of chromosomal gain that may signify the presence of new parathyroid oncogenes (24,98,102). In contrast, traditional cytogenetic analysis has not so far been very helpful in highlighting chromosomal target regions to be intensively searched for new parathyroid oncogenes or tumor suppressor genes. A cytogenetic translocation between chromosomes 1 and 5 has been reported in a single parathyroid aden o m a (103), but it is unclear whether this was an isolated r a n d o m occurrence or will be characteristic of a distinct subset of adenomas. A n u m b e r of specific candidate genes, some of which map to regions of frequent allelic loss, have been examined for clonal defects in parathyroid adenomas. These include genes encoding RAS (42); p53 (104-106); p15, p16, and p18 cdk inhibitors (35,107); the calciumsensing receptor (108); the vitamin D receptor (108a); RET (109-111); RAD51 (112); and RAD54 (113). No tumor-specific mutations that would implicate any of these candidates as an oncogene or tumor suppressor gene in parathyroid adenomatosis have been observed, and these genes do not appear to participate in a primary fashion in typical sporadic adenomas. Although the discussion here relates primarily to c o m m o n sporadic parathyroid tumors, brief mention should be made at this point of inherited syndromes other than MEN-1 that predispose to hyperparathyroidism. Hereditary hyperparathyroidism-jaw tumor syndrome (familial parathyroid adenomatosis with ossifying fibromas of the jaw, or HPT-JT) is clearly distinct genetically from either MEN syndrome (114,115), and the gene responsible for this predisposition has been mapped using linkage analysis to the long arm of chromosome 1 (116). Eventual cloning of the responsible gene will permit an assessment of its role in c o m m o n sporadic hyperparathyroidism. In familial isolated hyperparathyroidism, a subset of kindreds also show linkage to this l q region, and may be variants of the HPT-JT syndrome (117). Other examples of familial isolated hyperparathyroidism are MEN-1 variants (118) and still other causative loci may well exist. Hyperparathyroidism is a c o m p o n e n t of MEN-2A, although not as penetrant or relentless a feature in this syndrome as it is in MEN-1. Heritable mutations in the RET gene are responsible for virtually all cases of MEN2A (see Chapter 36), but such RET mutations have not been observed as acquired clonal defects in sporadic parathyroid adenomas (109-111). Familial hypocalciuric hypercalcemia (FHH) results in large part from germ-line-inactivating mutations, usually heterozygous, in the calcium-sensing receptor (see Chapter 38). Parathyroid cell proliferation is at best only minimally increased in FHH, but homozygous germ-line CaSR deft-

ciency causes neonatal severe hyperparathyroidism with markedly excessive parathyroid cellularity. However, acquired inactivating mutations that would implicate the CaSR as a tumor suppressor in the pathogenesis of sporadic hyperparathyroidism have been sought and not found (108,119). Thus, among the genes currently known to be responsible for inherited predisposition to hyperparathyroidism, only MEN1 has been shown to participate in the cognate sporadic disease. It may be inferred that acquired RET or CaSR mutations do not tend to confer an important selective advantage on normal parathyroid cells under usual circumstances.

M O L E C U L A R P A T H O G E N E S I S OF PARATHYROID CARCINOMA No gene has been d o c u m e n t e d to be a definite participant in the pathogenesis of parathyroid cancer. However, strong evidence indicates that a tumor suppressor gene important in malignant or especially aggressive parathyroid neoplasia is located on chromosome 13. Specifically, a large proportion of these tumors have clonal allelic losses on 13q (30,120,121), which typically cover a large region including the Rb gene. That Rb might be involved was suggested by an immunohistochemical analysis showing no evidence of pRb expression in carcinomas with 13q loss (30), but it is quite conceivable that a different tumor suppressor on 13q, instead of or in addition to Rb, is the pathogenetically relevant target of these deletions. Also, given that a small subset of benign parathyroid adenomas exhibit 13q deletions, an interesting question is whether identical or distinct putative 13q tumor suppressors become inactivated in malignant versus benign hyperparathyroidism. Also noteworthy is the increased incidence of parathyroid carcinoma in the HPT-JT syndrome, linked to l q as mentioned above. Other genetic abnormalities have been sought in parathyroid carcinoma using genomic analyses such as molecular allelotyping and comparative genomic hybridization (25,31). Certain recurrent abnormalities appear to occur preferentially or exclusively in carcinomas as opposed to adenomas, suggesting that genes in these regions contribute specifically to the malignant phenotype. Equally important, a set of chromosomal regions that are frequently lost in benign adenomas are rarely if ever lost in parathyroid cancer (31 ). This observation suggests that parathyroid cancers do not generally evolve from preexisting typical benign adenomas. Even prior to the definitive identification of the involved genes, these data raise the possibility that tumor genome analysis might provide diagnostic or prognostic guidance in assessing parathyroid tumors with "atypical" features, for example.

MOLECULARBASIS OF HYPERPARATHYROIDISM /

E C T O P I C S E C R E T I O N OF P T H Primary hyperparathyroidism is a biochemical diagnosis, and a rare cause of primary hyperparathyroidism is the ectopic secretion of PTH by nonparathyroid tumors. Older literature describing this as a c o m m o n entity was c o n f o u n d e d by poor specificity of assays for PTH fragments. Developing knowledge that led to the ultimate identification of PTHrP as the major cause of hypercalcemia of malignancy later placed the very existence of the true ectopic PTH syndrome in doubt. However, m o d e r n highly specific immunometric assays for intact PTH, combined with molecular biologic approaches, have confirmed the occurrence of this syndrome, and the molecular basis for ectopic PTH production in one such case has been identified. Because tumors may synthesize hormones without releasing them to cause an identifiable clinical syndrome, specific criteria have been recognized for documenting the diagnosis of a true ectopic h o r m o n e syndrome (122). These criteria were unequivocally fulfilled for the diagnosis of one case of the ectopic PTH syndrome (123). This patient had elevated serum PTH (but not PTHrP) levels, four normal parathyroid glands, and an ovarian carcinoma. PTH secretion from the carcinoma was demonstrated and was proved to be the cause of hypercalcemia by a sevenfold increase in PTH concentration in the tumor's venous effluent, an immediate decline in serum PTH level and the develo p m e n t of hypocalcemia after tumor resection, the production of PTH by the ovarian carcinoma cells in culture, and the presence of mRNA encoding PTH (but not PTHrP) in the tumor tissue (123). This ovarian cancer was the only nonparathyroid tumor responsible for hypercalcemia in over 300 consecutive patients with a biochemical diagnosis of hyperparathyroidism (123). In addition to this ovarian carcinoma, reasonable but less definitive support for the diagnosis of ectopic PTH syndrome has been presented through the description of a growing n u m b e r of cases (124-128). The tumor types involved have been metastatic small cell carcinoma, thymoma, neuroectodermal malignancy, squamous cell lung carcinoma, and papillary thyroid carcinoma. These tumors have p r o d u c e d PTH mRNA or protein, with or without PTHrP, and were associated with hypercalcemia and elevated serum PTH levels in the absence of detectable parathyroid gland tumors. Ectopic h o r m o n e production can be considered as an aberration in the tissue specificity of gene expression; it usually involves dysregulation of a normal hormone gene product. Tissue specificity of gene expression is controlled by DNA sequences called "enhancer" and "silencer" elements, often located in the upstream regulatory region of a h o r m o n e gene,

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interacting with the particular mix of DNA binding proteins characteristic of that tissue type. Ectopic h o r m o n e production in a tumor could therefore result from an alteration in that tumor cell type's DNA binding protein environment (activating the intact enhancer of the h o r m o n e gene), or from a change in the e n h a n c e r / s i l e n c e r region adjacent to the h o r m o n e structural gene (thereby conferring responsiveness to the DNA binding proteins typical of the tumor cell type). In the ovarian carcinoma that ectopically produced PTH, described above, a DNA rearrangement was present in the upstream regulatory region of the PTH gene (Fig. 5), replacing the gene's own control elements with DNA sequences that could interact with the ovarian cell's DNA binding proteins and thereby "inappropriately" activate PTH gene transcription (123). In addition, this PTH gene with its rearranged upstream regulatory region was amplified, i.e., present in severalfold extra copy number, in the ovarian cancer cells, which almost certainly heightened the level of PTH production caused by the DNA r e a r r a n g e m e n t (123). The molecular mechanisms underlying other examples of the ectopic PTH syndrome remain to be elucidated.

SUMMARY This is an exciting time for those interested in the molecular basis of the various forms of primary hyperparathyroidism. The cyclin D1/PRAD1 oncogene, discovered through its involvement in the pathogenesis of parathyroid adenomas, is having a broad impact in oncology and cell cycle biology. The MEN1 gene has been identified, also plays an important role in sporadic hyperparathyroidism, and the function of its protein product is being vigorously pursued. New tools will facilitate the identification of still other parathyroid tumor-provoking genes that, given recent evidence, are virtually certain to exist. Genetic causes for MEN-2A and familial hypocalciuric hypercalcemia have been determined, which will continue to yield information of basic and clinical importance in parathyroid and other endocrine diseases. Finally, there is reason to hope that the molecular basis of the relationship between abnormal parathyroid cell proliferation and abnormal hormonal regulatory function, plus other problems unique to parathyroid disease, may soon be elucidated.

ACKNOWLEDGMENT The author wishes to thank Pamela Vachon for her invaluable assistance in the preparation of this chapter.

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FIG. 5 Molecular pathology of the ectopic production of PTH by an ovarian cancer. Schematic diagram and restriction map of the rearranged and amplified PTH gene region (bottom) in the PTH-secreting ovarian tumor discussed in the text (123), compared with the normal PTH gene region (top). PTH exons I, II, and III are represented by open bars; normally present introns and flanking regions are denoted by solid bars, and the DNA placed upstream of PTH by the tumor-specific rearrangement are denoted by the cross-hatched bar. Restriction sites are labeled, and fragment sizes are shown in kilobases. For reasons of clarity and space, the map is not drawn to scale. The precise location of the breakpoint of the rearrangement was narrowed to a segment between the Hindlll site at -562 bp (562 base pairs upstream of the start of exon I) and the normally present Bglll site 150 bp further upstream (no longer present in the rearranged gene). Bam, BamHI; Msp, Mspl; Hind, Hindlll; Bgl, Bglll; Eco, EcoRl. Reprinted from Ref. 123, SR Nussbaum, RD Gaz, A Arnold. Hypercalcemia and ectopic secretion of parathyroid hormone by an ovarian carcinoma with rearrangement of the gene for parathyroid hormone. N Engl J Med 1990;323:1324-1328. Copyright © 1990 Massachusetts Medical Society. All rights reserved.

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MoLwcvlag BASIS OF HYPERPARATHYROIDISM / 104. Cryns VL, Rubio ME Thor AD, Louis DN, Arnold A. p53 abnormalities in human parathyroid carcinoma. J Clin Endocrinol Metab 1994;78:1320-1324. 105. Yoshimoto K, Iwahana H, Fukuda A, Sano T, Saito S, Itakura M. Role of p53 mutations in endocrine tumorigenesis: Mutation detection by polymerase chain reaction-single strand conformation polymorphism. Cancer Res 1992;52:5061-5064. 106. Hakim J, Levine M. Absence of p53 point mutations in parathyroid adenoma and carcinoma. J Clin Endocrinol Metab 1994;78:103-106. 107. Tahara H, Smith A, Gaz R, Zariwala M, Xiong Y, Arnold A. Parathyroid tumor suppressor on lp: Analysis of the p18 cyclindependent kinase inhibitor gene as a candidate. JBone Miner Res 1997;12:1330-1334. 108. Hosokawa Y, Pollak MR, Brown EM, Arnold A. Mutational analysis of the extracellular CaZ+-sensing receptor gene in human parathyroid tumors. J Clin Endocrinol Metab 1995;80:3107-3110. 108a.Brown SB, Brierley TT, Palanisamy N, Salusky IB, Goodman W, Brandi ML, Drueke TB, Sarfati E, Urena P, Chaganti RSK, Pike JW, Arnold A. Vitamin D receptor as a candidate tumor suppressor gene in severe hyperparathyroidism of uremia. J Clin Endocrinol Metab 2000;85:868-872. 109. Pausova Z, Soliman E, Amizuka N, et al. Role of the RET protooncogene in sporadic hyperparathyroidism and in hyperparathyroidism of multiple endocrine neoplasia type 2. J Clin Endocrinol Metab 1996;81:2711-2718. 110. Padberg B, Schroder S, Jochum W, et al. Absence of RET protooncogene point mutations in sporadic hyperplastic and neoplastic lesions of the parathyroid gland. Am J Pathol 1995;147:1600-1607. 111. Kimura T, Yoshimoto K, Tanaka C, et al. Obvious mRNA and protein expression but absence of mutations of the RET proto-oncogene in parathyroid tumors. EurJ Endocrino11996; 134:314-319. 112. Carling T, Imanishi Y, Gaz R, Arnold A. RAD51 as a candidate parathyroid tumor suppressor gene on chromosome 15q: Absence of somatic mutations. Clin Endocrino11999;51:403-407. 113. Carling T, Imanishi Y, Gaz R, Arnold A. Analysis of the RAD54 gene on chromosome l p as a potential tumor suppressor gene in parathyroid adenomas. I n t J Cancer 1999;83:80-82. 114. Mallette LE, Malini S, Rappaport ME Kirkland JL. Familial cystic parathyroid adenomatosis. Ann Intern Med 1987;107:54-60. 115. Jackson CE, Norum RA, Boyd SB, et al. Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: A clinically and genetically distinct syndrome. Surgery 1990;108:1006-1013. 116. Szabo J, Heath B, Hill VM, et al. Hereditary hyperparathyroidism-jaw tumor syndrome: The endocrine tumor gene HRPT2 maps to chromosome lq21-q31. A m J H u m Genet 1995;56:944-950.

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117. Teh BT, Farnebo E Twigg S, et al. Familial isolated hyperparathyroidism maps to the hyperparathyroidism-jaw tumor locus in 1q21-q32 in a subset of families. J Clin Endocrinol Metab 1998;83:2114-2120. 118. Teh B, Esapa C, Houlston R, et al. A family with isolated hyperparathyroidism segregating a missense MEN1 mutation and showing loss of the wild-type alleles in the parathyroid tumors. A m J Hum Genet 1998;63:1544-1549. 119. Cetani F, Pinchera A, Pardi E, et al. No evidence for mutations in the calcium-sensing receptor gene in sporadic parathyroid adenomas. J Bone Miner Res 1999;14:878. 120. Dotzenrath C, Teh BT, Farnebo F, et al. Allelic loss of the retinoblastoma tumor suppressor gene: A marker for aggressive parathyroid tumors? J Clin Endocrinol Metab 1996;81:3194-3196. 121. Pearce SHS, Trump D, Wooding C, Sheppard MN, Clayton RN, Thakker RV. Loss of heterozygosity studies at the retinoblastoma and breast cancer susceptibility (BRCA2) loci in pituitary, parathyroid, pancreatic and carcinoid tumours. Clin Endocrinol 1996;45:195-200. 122. Sherwood LM. Paraneoplastic endocrine disorders: Ectopic hormone syndromes. In: DeGroot LJ, ed. Endocrinology, 2nd Ed., Vol 3. Philadelphia:Saunders, 1989:2550-2599. 123. Nussbaum SR, Gaz RD, Arnold A. Hypercalcemia and ectopic secretion of parathyroid hormone by an ovarian carcinoma with rearrangement of the gene for parathyroid hormone. N EnglJ Med 1990;323:1324-1328. 124. Yoshimoto K, Yamasaki R, Sakai H, et al. Ectopic production of parathyroid hormone by small cell lung cancer in a patient with hypercalcemia. J Clin Endocrinol Metab 1989;68:976-981. 125. Iguchi H, Miyagi C, Tomita K, et al. Hypercalcemia caused by ectopic production of parathyroid hormone in a patient with papillary adenocarcinoma of the thyroid gland. J Clin Endocrinol Metab 1998;83:2653-2657. 126. Nielsen P, Rasmussen A, Feldt-Rasmussen U, Brandt M, Christensen L, Olgaard K. Ectopic production of intact parathyroid hormone by a squamous cell lund carcinoma in vivo and in vitro. J Clin Endocrinol Metab 1996;81:3793-3796. 127. Rizzoli R, Pache J, Didierjean L, Burger A, Bonjour J. A thymoma as a cause of true ectopic hyperparathyroidism. J Clin Endocrinol Metab 1994;79:912-915. 128. Strewler G, Budayr A, Clark O, Nissenson R. Production of parathyroid hormone by a malignant nonparathyroid tumor in a hypercalcemic patient. J Clin Endocrinol Metab 1993;76: 1373-1375. 129. Arnold A. Genetic basis of endocrine disease 5: molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab 1993;77:1108-1112.

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CI-IAPTFI20 Clinical Presentation of Primary Hyperparathyroidism in the United States

SHONNI J. SILVERBERG Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032 J O H N E BILEZIKIAN Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

INTRODUCTION

mary hyperparathyroidism does seem to present more along classic lines (1-3) (see Chapters 21 and 22). In addition to describing the clinical features of primary hyperparathyroidism in the United States, this chapter also considers the evolving clinical spectrum of primary hyperparathyroidism, with a retrospective view of what used to be more typical presentations of the disease. Frequent cross-references will facilitate easy access to other chapters in this volume for readers who want to explore some of these points in greater depth.

Primary hyperparathyroidism is a common endocrine disorder, characterized by the excessive and incompletely regulated secretion of parathyroid hormone (PTH) from one or more parathyroid glands. The major actions of PTH, to mobilize calcium from bone, to conserve calcium in the kidney, and indirectly to increase gastrointestinal calcium absorption, lead to one of the major biochemical hallmarks of the disease, hypercalcemia. Another major sign of the disorder is an elevated level of PTH, now readily detected by accurate assays for the hormone. Advances in evaluation of metabolic bone diseases by sensitive circulating and urinary markers of calcium metabolism have permitted a more detailed assessment of patients who do not appear to be suffering from overt clinical consequences of primary hyperparathyroidism. In addition, bone densitometry and analysis of bone by quantitative histomorphometry have provided direct insight into important current features of the disease. The result is a profile of primary hyperparathyroidism that not only is quite different from earlier historical descriptions but also requires consideration of a new set of issues insofar as the clinical m a n a g e m e n t of the disease is concerned. Because primary hyperparathyroidism is the major clinical disorder of the parathyroid glands, it is fitting that this disorder be the focus of an extensive discussion in this volume. This chapter describes major clinical features of primary hyperparathyroidism as it presents in the United States today. The clinical presentation of the disease differs in other parts of the world, where priThe Parathyroids, Second Edition

PREVALENCE A N D INCIDENCE OF PRIMARY HYPERTHYROIDISM It is remarkable that within the lifetimes of some currently practicing endocrinologists, primary hyperparathyroidism has been transformed from an extremely rare endocrine disorder to a relatively common one. In the 1930s and 1940s, primary hyperparathyroidism was appreciated virtually always in the context of a most unusual disorder with characteristic skeletal features known as osteitis fibrosa cystica. In fact, it was said in those days that "the X-ray findings proved to be so characteristic that chemical analysis was needed only for confirmation" (4). Other features of the historical summary of primary hyperparathyroidism by Oliver Cope in 1966 point out how these patients invariably deteriorated with a particularly pernicious bone disease. Studies of the famous sea captain Charles Martell by Bauer and colleagues (5,6) and the work of the Viennese surgeon Mandl (7) gave great impetus to 349

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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the correct idea that primary hyperparathyroidism is caused by abnormal function of parathyroid tissue and that removal of the offending a d e n o m a leads to correction of the hypercalcemia. Despite the fact that the disorder used to be rare and that the first series of patients with primary hyperparathyroidism described in 1934 included only 17 patients (8), it was evident even in those days that the incidence of the disease was, in part, a function of how high one's index of suspicion was for it. For example, Raymond Keating, whose work at the Mayo Clinic helped to establish m o d e r n concepts of the disease, was dispatched to the Massachusetts General Hospital in 1942 specifically to learn how the physicians there seemed to recognize patients fairly readily (Aub, Bauer, Albright, and Cope had seen 67 patients by this time); the experience at the Mayo Clinic was much more limited. After this tutorial in Boston, Keating returned to the Mayo Clinic and, with a clear intention to uncover the disease, saw more patients with primary hyperparathyroidism in the next year than he had seen in the preceding 15 years (9). This early experience makes a point that has been relived in the m o d e r n era. In the absence of serendipity or incidental discovery, the disorder is not readily diagnosed without a high index of suspicion. The dramatic change in the incidence of primary hyperparathyroidism occurred in the late 1960s and early 1970s, due primarily to the introduction of the multichannel autoanalyzer. Documentation of this change comes from the work of Heath and colleagues as well as from other groups (10-14). Incidence figures among residents of Rochester, Minnesota, increased nearly fivefold in the first year after the multichannel screening profile became routinely available (1974-1975). Thereafter, allowing for the catch-up detection factor in that first year (the "sweeping" effect), the incidence figures continued to show an impressive fourfold increase in comparison to the premultichannel autoanalyzer era (15). It is difficult to estimate true incidence figures for primary hyperparathyroidism (16), but the experience in Rochester, Minnesota, of 27.7 per 100,000 person years is essentially identical to figures from Sweden (12) and from Birmingham, England (11). On the basis on these figures, an estimate of approximately 100,000 new cases of primary hyperparathyroidism per year in the United States is likely to be accurate. The prevalence of primary hyperparathyroidism (the proportion of the population affected with the disease at a given point in time) is higher than earlier estimates of incidence (the n u m b e r of new cases diagnosed over a specified period of time). Prevalence estimates have been as high as 1 in 100 (17), but 1 per 1000 would appear to be closer to the true prevalence rate in the early autoanalyzer era (18).

A report from Rochester, Minnesota suggests that newly diagnosed cases of primary hyperparathyroidism have been declining continuously since the mid-1970s (19). This experience has not been clearly repeated in other American centers. It is possible that the particular demographics of Rochester, Minnesota, combined with the rather complete discovery of primary hyperparathyroidism in a population that receives virtually all of its care in one system (allowing for ideal epidemiologic surveillence), could account for declining numbers. More research in this area will be necessary to determine the direction of future changes in incidence figures (see later discussion). Primary hyperparathyroidism occurs throughout life, but the incidence peaks in the middle years. Women predominate over men by a 2:1-3:1 margin. The disease is recognized most commonly in women who are in the first postmenopausal decade, between ages 50 and 60 years. It is perhaps because of the effects of estrogens to oppose some of the skeletal actions of PTH that the disease may surface clinically when estrogen levels fall. There do not appear to be any well-established predisposing factors for the development of primary hyperparathyroidism, but a history of irradiation to the neck and u p p e r chest area in childhood is obtained in as many as 15-25% of patients with the disease (20,21). Primary hyperparathyroidism after radioactive iodine therapy for thyroid disease has been reported infrequently (22). Exciting new insights into the molecular bases of some cases of primary hyperparathyroidism are covered in Chapter 19 (23). Newer concepts of parathyroid cell growth properties in primary hyperparathyroidism are presented in Chapter 18 (24).

PATHOLOGY Most patients with primary hyperparathyroidism (80-85%) harbor a single adenoma; the other three glands are normal. In 2-4% of cases, multiple parathyroid adenomas have been described. Histologically the a d e n o m a is described as a confluence of parathyroid cells that may be associated with a rim of normal tissue at the margins. The average a d e n o m a is 0.5 g, although abnormal glands distinctly smaller or larger are seen. Even the smaller adenomas, <0.5 g, are usually much larger than normal parathyroid glands, which are 25-35 mg. Cystic elements in an adenomatous gland may call attention to a rare familial variant of primary hyperparathyroidism, cystic parathyroid adenomatosis (25). Details of the histopathology of primary hyperparathyroidism are covered in Chapter 1, and of unusual variants in Chapter 37.

CLINICAL PRESENTATION OF PHPT: UNITED STATES

Approximately 15-20% of patients with primary hyperparathyroidism have a pathologic process involving all four parathyroid glands. Four-gland hyperplasia may occur sporadically and is more likely to be seen in younger individuals. This pathology is also seen in conjunction with multiple endocrine neoplasia types 1 and 2A (see Chapters 35 and 36). The extent to which each parathyroid gland is involved may vary from a gland that is abnormal only by the most subtle histologic clues (i.e., diminished fat content) to others that are so grossly enlarged that they look like adenomas. The correct distinction between hyperplastic and adenomatous disease is important because the surgical approach is defined, in part, by the pathology (see Chapter 31). The success of this distinction depends on readily available, accurate histological appraisal at the time of parathyroid surgery. The rarest form of primary hyperparathyroidism is parathyroid carcinoma (26,27). In that primary hyperparathyroidism in the United States has changed in its clinical presentation from a disorder with invariable signs and symptoms to one of asymptomatic hypercalcemia, it is not surprising that the diagnosis of parathyroid carcinoma is made much less frequently relative to the hyperparathyroid population. Incidence figures place parathyroid carcinoma as a cause of primary hyperparathyroidism in well under 0.5% of all patients with primary hyperparathyroidism. For patients suspected of having parathyroid carcinoma from their rather distinctive clinical presentation (see Chapter 33), the gross appearance and the pathology of the parathyroid tissue removed become key elements in the diagnosis.

It is obvious that the clinical manifestations of primary hyperparathyroidism have changed dramatically over the past 40 years. A comparison of several series makes this point well. The series published by Cope

TABLE 1 Changing Profile of Primary Hyperparathyroidism

Neph rolithiasis Skeletal disease

Hypercalciuria Asymptomatic aNot reported.

Cope

351

(4), which is typical of the preautoanalyzer years, reviewed the experience of the first 343 cases at the Massachusetts General Hospital up to 1965. Heath and colleagues (10,15) and Mallette et al. (28) report an experience that straddles a 10-year period when the autoanalyzer became widely used. The experience of Silverberg and colleagues (29,30) exemplifies the more modern clinical presentation of primary hyperparathyroidism in the 1980s and 1990s. These comparisons are shown in Table 1. The frequency of specific radiologic manifestations of primary hyperparathyroidism dropped from 23% in the Cope series to 10-14% in the Heath and Mallette series, to a remarkably low 2% in the Silverberg series. In fact, hyperparathyroid bone disease is now so rarely seen that most clinicians dispense with routine radiologic assessment of primary hyperparathyroidism (31). Mso noteworthy is the drop in the incidence of nephrolithiasis among these series. In the Cope series, stone disease was very common, occurring in 57% of all cases. In the Heath and Mallette series, the incidences of stone disease were 51 and 37%, respectively. In the Silverberg series, the incidence dropped further to 19%, (29). Hypercalciuria was not noted in the Cope series, but it is interesting that the incidence of this feature of primary hyperparathyroidism did not change from an incidence of 36% reported by Heath and colleagues to 40% reported by Mallette et al. to 39% reported by Silverberg et al. (32). Other possible manifestations of note, such as pancreatitis and peptic ulcer disease, are too infrequent in any of the series to make meaningful comparisons or to detect any trends. It is thus clear that the profile of primary hyperparathyroidism in this country, insofar as the wellestablished clinical manifestations of the disease are concerned, has changed dramatically. If potential manifestations of primary hyperparathyroidism (hypertension, neuropsychiatric abnormalities, etc.) are excluded from consideration, simply because we do not yet know whether they are specific for the hyperparathyroid process, one reaches the conclusion that the vast majority of patients seen with primary

CLINICAL MANIFESTATIONS OF PRIMARY HYPERPARATHYROIDISM: T H E N A N D N O W

Symptom

/

(1930-1965)

(1965-1974)

Heath et aL

Mallette et aL (1965-1972)

Silverberg et aL (1984-2000)

57% 23% NRa 0.6%

51% 10% 36% 18%

37% 14% 40% 22%

17% 1.4% 39% 80%

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hyperparathyroidism today (approximately 80%) are asymptomatic. The definition of asymptomatic primary hyperparathyroidism was stated best by Heath et al. (19): "well-documented primary hyperparathyroidism in which there are neither complications nor symptoms that are clearly and commonly attributable to either hypercalcemia or parathyroid h o r m o n e excess."

Physical Findings The most noteworthy aspect of the physical examination in primary hyperparathyroidism is that the examination is not noteworthy. There are usually no abnormal physical findings specifically related to the disease. Hypertension, which is frequently seen, has not been established to be related directly to primary hyperparathyroidism (see below). Once a hallmark of the physical examination in classic primary hyperparathyroidism, calcium phosphate deposition in the medial and lateral limbic margins of the cornea (band keratopathy) is seen now only rarely and virtually only by ophthalmologic slit-lamp examinations. Enlarged parathyroid tissue is usually palpable only when parathyroid carcinoma is present. The neurologic examination, which used to be of interest in the days when the neuromuscular manifestations of primary hyperparathyroidism were often seen (see below), is similarly normal.

Symptoms and Signs The symptoms and signs of primary hyperparathyroidism are, in part, related to those of hypercalcemia per se (24,34), the clinical features of which are reviewed in Chapter 45. In this regard, the extent to which patients may be symptomatic is related directly to whether the hypercalcemia is in the range usually associated with specific symptomatology. It should be recalled that the symptoms of hypercalcemia are a function of the rate of rise of the serum calcium as well as the actual level (34). In addition, symptoms of hypercalcemia vary from patient to patient. Most patients with mild primary hyperparathyroidism and calcium levels within 1 m g / d l of the upper limits of normal do not have features that can readily be attributed to the hypercalcemia per se. Specific manifestations of primary hyperparathyroidism are those traditionally viewed as features of the disease. Along with the dramatic increase in the incidence of this disease, its clinical presentation has also undergone a major change. Nevertheless, primary hyperparathyroidism may still be associated with a n u m b e r of different clinical presentations (Table 2) (35).

TABLE 2 Clinical Presentations of Primary Hyperparathyroidism Asymptomatic hypercalcemia Bone or stone disease Other recognized complications (neuromuscular, gastrointestinal, articular, hematologic, CNS) Acute primary hyperparathyroidism Parathyroid carcinoma Familial primary hyperparathyroidism Familial cystic parathyroid adenomatosis Neonatal hyperparathyroidism Multiple endocrine neoplasia type 1 or 2

Skeletal Manifestations of Primary Hyperparathyroidism The classic bone disease of primary hyperparathyroidism is osteitis fibrosa cystica (Fig. 1). When symptomatic, osteitis fibrosa cystica is experienced by patients as bone pain. Pathologic fractures may occur. Overt bone resorption caused by excessive concentrations of PTH is associated with several typical radiologic signs. Subperiosteal bone resorption of the distal phalanges is the most sensitive radiologic sign of primary hyperparathyroidism. It is appreciated best on the radial side of the middle phalanges. Similar radiologic changes may be present in the skull in the form of a motheaten or saltand-pepper pattern. The distal one-third of the clavicles may appear to be tapered. Local destructive lesions, bone cysts, and "brown tumors" in the long bones and pelvis constitute other skeletal manifestations of the disease. Brown tumors are collections of osteoclasts intermixed with poorly mineralized woven bone. Nonspecific generalized skeletal demineralization is sometimes evident in the absence of these other features of hyperparathyroid bone disease. Both nonspecific demineralization and the specific radiologic manifestations outlined above reflect the catabolic skeletal actions of PTH. Although osteitis fibrosa cystica is distinctly unusual in patients who present with primary hyperparathyroidism in the United States, this does not imply that the skeleton is unaffected in those with asymptomatic disease. There is now ample evidence of skeletal involvement in the hyperparathyroid process. The availability of sensitive techniques to monitor the skeleton has given us an opportunity to address these issues in patients who have asymptomatic primary hyperparathyroidism. Bone Densitometry in Primary Hyperparathyroidism

Bone density has been measured at three sites to evaluate areas enriched in cortical bone (distal radius),

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353

FIG. 1 Radiographic representation of osteitis fibrosa cysctica in classic primary hyperparathyroidism. (A) Salt-and-pepper skull. (B) Cystic bone disease of the clavicle. (C) Subperiosteal bone resorption of the digits. (D) Cortical erosions.

cancellous bone (vertebral spine), and a mixture of both (the hip region) (29,31 ). At the distal radius, bone density was <80% of age- and sex-matched control values in 58% of our patients. The mean value at this site was only 79% of expected. In contrast, at the lumbar spine, bone mineral density was relatively well preserved. Only 13% of patients had lumbar bone density <80% of age- and sex-matched control values, and the mean value was within 5% of expected. The values for the hip region were midway between data obtained for the spine and those for the distal radius (Fig. 2). Pfeilschifter et al. have confirmed these findings (36). These results indicate that, in mild primary hyperparathyroidism, reductions in cortical bone density are seen regularly and that the cancellous bone is relatively well preserved. This is of particular importance for several reasons. First, it is consistent with the impression that PTH has effects to mobilize calcium from cortical sites before it impacts negatively on the cancellous skeleton. In patients with more marked elevations in PTH, reductions in bone density at cancellous sites would be expected to be seen. Second, significant reductions in cortical bone are detectable before any radiologic manifestations are apparent. Third, the preservation of cancellous bone may be of particular importance in that primary hyperparathyroidism is a disease that disproportionately affects postmenopausal women. Women in their postmenopausal )ears are at

risk for bone loss due to estrogen deficiency, which occurs first in the cancellous bone of the spine. Thus the effects of primary hyperparathyroidism on vertebral cancellous bone appear to be opposite to those of estrogen deficiency. Moreover, a characteristic pattern of bone densitometry in primary hyperparathyroidism

100 7

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l-i. ¢,D Ii~ Q. X

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90

o

80

70

SITE

FIG. 2 Bone densitometry in primary hyperparathyroidism. Data are shown in comparison to age- and sex-matched normal subjects. Divergence from expected values is different at each site (p = 0.0001.) r-I, Lumbar; [], femoral; I , radius. From Ref. 26, E Shane, JP Bilezikian. Parathyroid carcinoma. In: Williams CJ, Krikorian JC, Green MR, Raghavan D, eds. Textbook of uncommon cancer. Copyright © 1988 John Wiley & Sons Limited. Reproduced with permission.

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is the opposite of the early changes seen in postmenopausal osteoporosis, namely, more significant loss at a cortical site, exemplified by the distal radius, than at a cancellous site, such as the spine. These observations have several important implications. They support the significant body of data suggesting that the anabolic actions of PTH can be appreciated at the spine and that, in some protocols, this anabolic effect occurs at the expense of a loss of cortical bone. They also have implications for the use of parathyroid h o r m o n e as a therapeutic agent for postmenopausal osteoporosis (see Chapter 55). Although the vast majority of patients with primary hyperparathyroidism demonstrate preserved vertebral bone density, there is a group of patients who do not do so. In our longitudinal study, approximately 15% of patients had a reversal of the typical bone densitometry pattern, with evidence of significant loss of bone at the spine (37). These osteopenic or osteoporotic patients were heterogeneous in their demographic (i.e., sex and menopausal status) and biochemical features; there were no predictive indices that identified them. These patients are of interest with regard to their bone densitometry after parathyroidectomy, a subject which will be discussed in Chapter 23. Bone Histomorphometry in Primary Hyper~arathyroidism

The results obtained by bone densitometry have been supported by a direct analysis of the bone biopsy by quantitative histomorphometry (38-43). Bone biopsy results are consistent with a loss of cortical bone and preservation of cancellous bone. The measurements included both static and dynamic parameters of bone as well as a newer approach, strut analysis. The work of Parisien et al. has shown that static parameters of bone such as osteoid surface, osteoid volume, and eroded surface are all elevated in both men and women with primary hyperparathyroidism compared to normal values. Dynamic parameters of bone turnover (mineralizing surface and bone formation rate) are also elevated. The known catabolic effect of PTH on cortical bone has been d o c u m e n t e d on bone biopsy in patients with primary hyperparathyroidism. Cortical thinning is seen on biopsy, and PTH levels have been shown to correlate with cortical porosity (38,39). On the other hand, consistent with observations made via bone densitometry, indices that describe cancellous bone architecture are preserved in primary hyperparathyroidism. These parameters include cancellous bone volume, trabecular number, and trabecular separation. Indeed, cancellous bone volume is actually increased in primary hyperparathyroidism (42,43). In patients with this disease, trabecular plates do not show typical age-related changes. The striking maintenance of trabecular plates with age

further supports the idea that the hyperparathyroid process protects cancellous bone. By strut analysis, this preservation appears to be based on an architectural maintenance of connectivity between and among trabecular plates. In summary, the data obtained from histomorphometric studies support the idea that patients with primary hyperparathyroidism have increased bone turnover, thinning of cortical bone, and increased connectivity of cancellous bone (see Chapter 26). Fracture Incidence in Primary Hyperparathyroidism

The preceding observations might lead to certain predictions about fracture incidence in primary hyperparathyroidism. It is generally agreed that, as bone mass decreases, there is a rise in the incidence of fractures. Certainly, generalized osteopenia and fractures were a regular feature of the form of primary hyperparathyroidism seen many years ago (45). In the disease as it is seen today in the United States, however, the skeleton appears to be targeted selectively, with cortical sites being the focus of the catabolic actions of PTH, and cancellous sites being a focus of its anabolic actions. One might therefore expect that, in primary hyperparathyroidism, fracture incidence would not be increased at the spine, whereas fracture of the distal radius or other cortical sites might depend, in part, on the degree to which there has been a reduction in bone mineral density at those site(s). Unfortunately, information in this area is limited, with studies arguing for or against an increase incidence of fractures (46-49). Unfortunately, several studies of fracture incidence have methodologic flaws that make interpretation difficult. One study showing an increase in fractures at any site in hyperparathyroidism (48) was flawed not only by its small sample size, but also by the unusually high fracture incidence in both patients (48%) and control subjects (28%), and by the use of thyroid medication in a significantly greater n u m b e r of patients relative to control subjects. A population-based study of 407 patients with primary hyperparathyroidism in Rochester, Minnesota, found an increased incidence of vertebral, Colles', rib, and pelvic fractures (49). Unfortunately, the population in this study was not fully characterized with regard to bone density, making it unclear as to how typical a group they represent. Until a multicenter trial with sufficient statistical power is performed, the question of fracture incidence in primary hyperparathyroidism in the United States will not be resolved.

Stone Disease Kidney stones constitute another classic manifestation of primary hyperparathyroidism. Although the incidence of nephrolithiasis in primary hyperpara-

CLINICAL PRESENTATION OF PHPT: UNITED STATES

thyroidism has diminished along with the incidence of bone disease, stones nevertheless are still seen. Most series now place the incidence of kidney stones at 15-20% of all patients with primary hyperparathyroidism (see Chapters 35 and 36). Because nephrolithiasis is such an important complication of primary hyperparathyroidism and because primary hyperparathyroidism is such a c o m m o n disorder, it is still advisable to investigate the possibility of primary hyperparathyroidism in any patient who develops a kidney stone. Besides nephrolithiasis, the kidneys may be affected in other ways. Deposition of calcium phosphate crystals throughout the renal parenchyma, a process known as nephrocalcinosis, may occur. Nephrocalcinosis may or may not be associated with frank stones a n d / o r a reduction in creatinine clearance. Hypercalciuria, defined as a total urinary calcium excretion of >250 mg (women) or >300 mg (men), occurs in up to 35-40% of patients with primary hyperparathyroidism (32). The hypercalciuria is caused by the greater load of filtered calcium, which exceeds the capacity of the kidney to reabsorb it despite the conserving actions of PTH on renal calcium handling. Whether hypercalciuria is related to, or places patients at risk for, stone disease is discussed in Chapter 31. Some patients with primary hyperparathyroidism will show reduced creatinine clearance without stones, hypercalciuria, nephrocalcinosis, or other predisposing factors.

Bone and Stone Disease in Primary Hyperparathyroidism Older concepts of primary hyperparathyroidism included the teaching that concurrent bone and stone disease is rare (8,45,50-53). It was believed that patients who hyperabsorbed calcium from the gastrointestinal tract were predisposed to stones, whereas those who did not show gastrointestinal hyperabsorption of calcium were predisposed to bone disease. The hypothesis of two distinct pathophysiologic groupings of patients with primary hyperparathyroidism is difficult to test now because overt, radiologically evident bone disease has become rare. Thus concomitant overt skeletal disease and stone disease are seen usually only in the special situations of acute primary hyperparathyroidism and parathyroid cancer (see Chapters 33 and 34). However, data indicate that hyperparathyroid bone involvement can be detected in a large n u m b e r of patients with asymptomatic primary hyperparathyroidism when more sensitive methods (bone densitometry and quantitative bone histomorphometry) are employed, thus this hypothesis has been reconsidered. In our experience, cortical demineralization occurs to the same extent and with the same frequency in patients with and without nephrolithiasis (32).

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Moreover, the pattern of skeletal demineralization of cortical sites was similar in stone formers and in the entire cohort of hyperparathyroid subjects. Bone densitometric analysis provided further evidence against a pathophysiologic distinction between bone disease and stone disease in primary hyperparathyroidism. Urinary calcium excretion correlated negatively with forearm bone mineral density, suggesting that at least a component of urinary calcium excretion reflects events occurring in bone. These studies thus fail to confirm the classic teaching that bone disease and stone disease are mutually exclusive manifestations of primary hyperparathyroidism.

Involvement of Other Organs Primary hyperparathyroidism has the potential to involve organ systems besides the skeleton and the kidneys. The c o m m o n complaints of weakness and fatigue used to be associated with a particular neuromuscular syndrome characterized histologically by atrophy of type II muscle fibers (54,55). Current experience suggests that the weakness and easy fatigability sometimes ascribed to the hyperparathyroid syndrome are no longer associated with overt neurologic findings (55). However, in detailed electromyographical studies of muscles in primary hyperparathyroidism, abnormalities are still reported (56). In one study, carpal tunnel syndrome was seen in a higher proportion of patients than expected in the normal population (55). The significance of this finding remains unclear. The gastrointestinal tract may also appear to be a target of the hyperparathyroid state. Historically, peptic ulcer disease was regarded as a frequent complication, but is now seen predominantly with the multiple endocrine neoplasia type 1 (MEN-l) syndrome, in which primary hyperparathyroidism and peptic ulcer disease may coexist. Aside from this specific association, there is continuing debate over a pathophysiologic link between these two relatively c o m m o n disorders (57). Similarly, the association between primary hyperparathyroidism and acute pancreatitis, apart from that related to hypercalcemia per se, remains to be established (58). Gout or pseudogout may affect the articular system of those with primary hyperparathyroidism. Older patients with asymptomatic chondrocalcinosis of the knees and bones of the wrist may be at risk for the development of pseudogout (59,60). Anemia of primary hyperparathyroidism, characterized by normocytic and n o r m o c h r o m i c indices (61), is an unusual finding, occurring only when other systemic manifestations of primary hyperparathyroidism are also present. Cardiovascular effects of primary hyperparathyroidism remain the subject of debate. Hypertension has

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been thought for many years to be a complicating feature of primary hyperparathyroidism. The experience of Heath et al. (10), in which there seems to be a greater incidence of hypertension among those with primary hyperparathyroidism in comparison to a control population, is in agreement with the experience of others (62,63). However, most patients do not experience a significant reduction in the blood pressure after successful parathyroid surgery (63,64). Mthough hypercalcemia has been associated with numerous cardiac abnormalities (i.e., left ventricular hypertrophy, myocardial and valvular calcification), the effect of primary hyperparathyroidism on the heart in unclear (65,66). Much of the available data in this regard has come from European centers, describing patients with more severe disease than is usually seen in the United States. The applicability of these data to American patients who present with mild disease is controversial. The neuropsychiatric manifestations of primary hyperparathyroidism are the subject of great uncertainty (67-72). In part, this is because the symptomatology is exceedingly nonspecific. Affective disorders, anxiety, cognitive difficulties, and somadzadon exemplify the kinds of manifestations that have been ascribed to the hyperparathyroid syndrome. Certainly most clinicians who care for patients with primary hyperparathyroidism agree that such symptoms are sometimes elicited from or volunteered by their patients. Moreover, some reports are intriguing for an apparent reversal of these symptoms after successful parathyroid surgery. Ljunghall and colleagues (68-70) have investigated this problem in a series of impressive studies attempting to quantify this symptom complex using psychopathologic rating scales before and after surgery in a population of European patients with primary hyperparathyroidism. Solomon et al. reported on the improvement of a series of neuropsychiatric measures (using the SL-149 rating scale) after parathyroidectomy (72). These data remain difficult to interpret, because the control group (patients who underwent thyroid surgery) also improved. It is unclear to what extent patients with primary hyperparathyroidism have manifestations that can be ascribed with certainty to a neuropsychiatric complex specifically due to primary hyperparathyroidism, and the extent to which such symptoms may be reversible after parathyroidectomy.

B I O C H E M I C A L MANIFESTATIONS OF PRIMARY HYPERPARATHYROIDISM The presence of hypercalcemia is an essential part of the biochemical definition of primary hyperparathyroidism. In the United States, the serum calcium concentration is often within 1 m g / d l of the upper limits of normal. However, in marked contrast to the mild hyper-

calcemia characteristic of most patients with primary hyperparathyroidism, life-threatening hypercalcemia can still occur. This entity, which is known as acute primary hyperparathyroidism (also as parathyroid poisoning or parathyroid crisis) has a distinctive presentation (73) (see Chapter 34). On the other hand, there are rare patients with normal serum calcium values who have primary hyperparathyroidism. The designation normocalcemic primary hyperparathyroidism for these patients is probably not accurate, because hypercalcemia invariably surfaces in these patients over time or is seen intermittently. If both the total and ionized serum calcium concentrations are normal, it is exceedingly difficult to make the diagnosis of primary hyperparathyroidism. One possible exception is the patient with coexisting primary hyperparathyroidism and vitamin D deficiency. Despite their hyperparathyroid state, the absence of vitamin D could have a calcium-lowering effect, pushing levels into the normal range. Another important feature of the biochemical definition of primary hyperparathyroidism is elevated circulating levels of PTH. Immunoradiometric and immunochemiluminometric techniques have not only served to establish the diagnosis of primary hyperparathyroidism with certainty but have also served to exclude other causes of hypercalcemia. The assays in common use in the United States detect elevated levels of PTH in patients with primary hyperparathyroidism 85-90% of the time. If multiple samples are obtained in patients who eventually are shown to have primary hyperparathyroidism, the incidence of elevated levels is even higher. This subject is covered in greater detail in Chapter 9. The serum phosphorus level is usually in the lower range of normal. It is frankly below normal, <2.5 mg/dl, in only 25% of all patients. In the absence of significant renal insufficiency, it is distinctly unusual for the serum phosphorus to be >3.5 m g / d l in primary hyperparathyroidism. The serum chloride is usually > 103 mEq/liter (74). Lafferty has used serum chloride, calcium, and phosphorus as well as the hematocrit to derive a discriminant function to distinguish primary hyperparathyroidism from other causes of hypercalcemia. Similarly, Lind and Ljunghall (75) have employed serum chloride, alkaline phosphatase, and albumin. Mthough this approach is undoubtedly valid, the great diagnostic utility of currently available assays for PTH makes discriminant analysis useful only in those very unusual patients for whom the differential diagnosis of hypercalcemia is exceedingly difficult. It is used rarely now. Total urinary calcium excretion in most patients with primary hyperparathyroidism is at the upper limits of the normal range. The 25-hydroxyvitamin D level tends to be in the lower range of normal, but the 1,25-dihydroxyvitamin D [ 1,25 (OH) 2D] level tends to be in the upper range of

CLINICAL PRESENTATION OF P H P T : UNITED STATES

normal. In 30-35% of patients, the 1,25 (OH) 2° level is frankly elevated (76), illustrating a physiologic action of PTH to stimulate the production of 1,25 (OH) 2o . When hyperparathyroidism is associated with marked hypercalcemia, the 1,25(OH)2 ° level may be suppressed, mimicking the profile seen in hypercalcemia of malignancy. In this unusual situation, it would appear that the inhibitory effects of hypercalcemia on 1,25(OH)zD production override the stimulatory effects of PTH (77). Although widespread vitamin D deficiency is not seen in the United States (as it is elsewhere in the world), it has become clear that even in the United States vitamin D insufficiency or frank deficiency is common in patients with mild primary hyperparathyroidism. In our natural history study, 25-hydroxyvitamin D concentrations were below the 20 n g / m l level suggested for vitamin D "sufficiency" in over half of our patients (53%) (78). Those with lowest 25-hydroxyvitamin D had highest parathyroid hormone levels, suggesting a more active hyperparathyroidism in those with lowest vitamin D. In these patients, the effects of primary hyperparathyroidism on biochemical, densitometric, and histomorphometric indices are more pronounced. Because bone resorption and bone formation are increased by parathyroid hormone, markers of bone turnover can provide clues to the e x t e n t of skeletal involvement in primary hyperparathyroidism. Bone formarion is reflected by osteoblast products, including bone specific alkaline phosphatase activity, osteocalcin, and type I procollagen peptide. Despite the availability of these sensitive measurements of bone formation, the total alkaline phosphatase activity is still widely assessed in primary hyperparathyroidism. In primary hyperparathyroidism, levels can be mildly elevated, but in many individuals total alkaline phosphatase values are within normal limits. The bone specific isoenzyme of alkaline phosphatase is far more sensitive, and is clearly elevated in many patients with mild primary hyperparathyroidism (79,80). In a small study from our group, bone specific alkaline phosphatase correlated with parathyroid hormone levels and bone mineral density at the lumbar spine and femoral neck (79). Osteocalcin is also generally increased in patients with primary hyperparathyroidism (80-83). Markers of bone resorption include the osteoclast product, tartrateresistant acid phosphatase, and collagen breakdown products such as hydroxyproline, hydroxypyridinium cross-links of collagen, and N- and C-telopeptides of type I collagen (84). Once the only available marker of bone resorption, urinary hydroxyproline excretion no longer offers sufficient sensitivity or specificity to make it a useful tool in the assessment of patients with primary hyperparathyroidism. Although urinary hydroxyproline was frankly elevated in patients with osteitis fibrosa cystica, with mild, asymptomatic primary hyperparathyroidism, it is not typically normal.

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Hydroxypyridinium cross-links of collagen (pyridinoline and deoxypyridinoline), on the other hand, are often elevated in primary hyperparathyroidism, and return to normal after parathyroidectomy (85).

PRIMARY HYPERPARATHYROIDISM:

YESTERDAY, TODAY, AND TOMORROW How can we account for the reduction in the symptomatic presentation of this disease in the United States? In one view, this disorder is merely being detected earlier than it used to be. Thus, with the widespread use of the autoanalyzer, the old-fashioned presentation of primary hyperparathyroidism, with its specific symptomatology, does not have a chance to develop, and asymptomatic primary hyperparathyroidism predominates among the many other potential presentations of the disease. There are several problems with this theory. If primary hyperparathyroidism is simply being detected at an earlier stage, one might expect that the average age of the patient at diagnosis would be younger now than before. This is not clearly the case. However, the well-recognized trend toward an aging population may explain why this disease is discovered so frequently in older individuals and may confound the analysis of this point. Moreover, if the predominance of asymptomatic disease is due solely to earlier detection, one would predict that many of those asymptomatic patients who are followed conservatively without surgery would demonstrate progression to overt clinical disease. This also does not seem to be the case. With the emergence of longitudinal data in patients with asymptomatic primary hyperparathyroidism, it is now known that in an American population with mild disease, most patients show no evidence of disease progression over a decade of follow-up with no intervention (30) (see Chapter 23). And although some patients did have biochemical worsening (increasing hypercalcemia or hypercalciuria), or progressive declines in bone density, none of the patients in our recent report developed signs or symptoms of classic primary hyperparathyroidism in a decade of observation. Consideration of these questions about asymptomatic primary hyperparathyroidism raises another possibility, namely, that the disease has changed. Perhaps the predominance of asymptomatic disease in our population is a reflection of different etiologies or environmental or nutritional factors. One speculative example might suffice. Residents of many Western countries supplement their diet with vitamin D. Thus vitamin D stores are more likely to be replete in those countries today than in the past. Reports of primary hyperparathyroidism from emerging nations (India, Saudi Arabia, Brazil, Vietnam) show the disease in those

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countries to have the phenotype of classic primary hyperparathyroidism (86-89). Symptomatic patients were often afflicted by both primary hyperparathyroidism and vitamin D deficiency, with the latter condition possibly increasing the manifestations of the former. It is conceivable that the high normal or frankly elevated levels of 1,25 (OH)zD routinely seen in primary hyperparathyroidism in the United States could serve to limit the extent to which PTH levels are elevated by virtue of the effect of 1,25 (OH)2 D to control PTH gene transcription. Moreover, sufficient vitamin D might protect against some of the skeletal effects of vitamin D deficiency in conjunction with elevated levels of PTH. In France, primary hyperparathyroidism is more likely to present with osteitis fibrosa cystica if levels of vitamin D metabolites are low (90). This hypothesis thus suggests that the disease has changed along with development of a technology that has improved recognition. Whether we are in the midst of an evolving disease process in part responsible for the changing clinical profile of primary hyperparathyroidism or whether we are seeing merely a disorder that is showing its clinical features more accurately now than in the past are issues that await further study. Furthermore, it is possible that the clinical presentation of PHPT in the United States could change once again. The "incidental" finding of hypercalcemia, which has led to the diagnosis of primary hyperparathyroidism in many asymptomatic patients, may become a thing of the past due to federal health care financing mandates, restricting reimbursement for routine multichannel biochemical determinations. Thus, unless a physician suspects an abnormality in calcium homeostasis, the test may not be performed. Should this occur, the clinical profile of primary hyperparathyroidism could return to one in which only symptomatic patients come to medical attention. Furthermore, as vitamin D insufficiency becomes more commonplace in our society, more symptomatic primary hyperparathyroidism may once again manifest. As we look forward to the next 50 years, we can only speculate on the future evolution of this c o m m o n disorder of mineral metabolism.

SUMMARY In the United States today, primary hyperparathyroidism presents most frequently as an asymptomatic disease. This is a picture markedly different from descriptions of the disease from the era antedating the widespread use of the multichannel autoanalyzer, when symptoms and signs were the rule rather than the exception. In the United States, the disease no longer appears to be one of "stones, bones, and groans." The

research efforts of the past several decades have provided a portrait of the disease as it now presents, largely asymptomatic, yet with clear end-organ manifestations. Questions remain concerning some of the clinical effects of this mild hyperparathyroid process, including the vague constitutional complaints of many patients, the neuropsychological manifestations of the disease, and possible cardiovascular complications. Ongoing studies are addressing these important issues.

ACKNOWLEDGMENT Some of the information contained in this chapter was obtained with support from NIH grants DK32333 and AR39191.

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CIJAPTER21 Clinical Presentation of Primary Hyperparathyroidism Europe JONAS RASTAD *Department of Surgery, Endocrine Unit, University Hospital, S-751 85 Uppsala, Sweden EWA LUNDGREN Department of Surgery, Endocrine Unit, University Hospital, S-751 85 Uppsala, Sweden SVERKER LJUNGHALL Global Clinical Sciences, AstraZeneca Research and Development, S-431 83 M6lndal, Sweden

INTRODUCTION

treat PHPT at mild stages in patients lacking "traditional" symptoms. The following text elucidates what we believe characterizes PHPT today in a society with a fairly high degree of PHPT awareness, and some outlooks on its variability inside and outside Europe.

The clinical characteristics and treatment of primary hyperparathyroidism (PHPT) have created rather a strong controversy, despite that PHPT is a common disorder in risk groups, is familiar to most physicians, is easy to diagnose, and in principle can be actively treated by surgery only. The controversy is overt, particularly regarding the prevalence, symptomatology, and complications of PHPT, and from this follows that divergent ideas must exist also with respect to the indication for active treatment. Despite a Medline search showing that about 150 scientific reports on PHPT have been published annually during the past decade, few structured attempts have been made to settle these issues. PHPT throughout Europe does not necessarily have a common mode of clinical presentation, nor does presentation necessarily differ substantially among most parts of the industrialized world. Nevertheless there are indisputable geographic and ethnic peculiarities concerning clinically diagnosed PHPT in the Western Hemisphere in comparison to other regions. Hypothetically this could depend on previously undescribed heterogeneities in the genetic and metabolic alterations causing the disease or other risk factors influencing clinical presentation. However, our strong view is that most of the noted geographic differences are caused by a variation in the degree of awareness of PHPT among physicians in general and their ability and willingness to recognize and to

P H P T IN A H I S T O R I C A L P E R S P E C T I V E A historical perspective provides an interesting insight into the current heterogeneity in the incidence and clinical characteristics of PHPT. More than half a century ago this disease was diagnosed rarely in Europe and other parts of the Western Hemisphere, and it was characterized by threatening symptoms and considerable risks of pronounced hypercalcemia (1).

P H P T in the Past Most PHPT patients in the past were middle-aged and had severe skeletal symptoms due to generalized osteopenia and osteitis fibrosa cystica. The bone involvement was often accompanied by benign but locally destructive brown tumors. Fragility fractures, bone pain, and neurologic complications were a clinical reality, as were skeletal deformation and an altered body stature (2). Most patients had fairly p r o n o u n c e d hypercalcemia and substantially enlarged and often palpable parathyroid adenomas. It was soon recognized that recurrent renal stones also accompanied PHPT, with risks for an impaired renal function and the develo p m e n t of uremia (Table 1).

*To whom correspondence should be addressed. Present address: AstraZeneca Research and Development, S-431 83 M61ndal, Sweden.

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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TABLE 1

Historical Subgroups of PHPT a

Type

Presentation

HPT type 1

Middle-aged individuals, substantial hypercalcemia, large parathyroid adenomas, strongly symptomatic disease, severe bone involvement with osteopenia, osteitis fibrosa cystica, brown tumors, and fragility fractures Middle-aged individuals, moderate hypercalcemia and less substantially enlarged parathyroid glands, recurrent renal stones, risks of renal damage Elderly individuals, mild to moderate hypercalcemia, mild glandular enlargement, no or mild traditional (bone and stone) symptoms, cardiovascular complications and premature death, little risk for disease progression

HPT type 2

HPT type 3

aThis subgrouping is schematic and theoretical, and many patients presented with intermediate variants.

Patients with a propensity for renal stones exhibited less marked skeletal involvement and usually had fewer neurologic complications and a milder extent of hypercalcemia, suggesting that there might exist different disease subtypes (3). One subtype was characterized by more substantial glandular enlargement, bone disease, and a usually severe, symptomatic hypercalcemia. Another subtype included a more discrete increase in the glandular size, formation of renal stones, nephrocalcinosis, and impaired renal function. In more recent decades, these subtypes of PHPT have become increasingly rare in Europe. W h e n patients representing the first subtype do occur in the clinic, they are almost invariably immigrants from nutrient-deficient areas or Third World countries, in addition to the rare case with parathyroid cancer. Similarly, the second subtype is now infrequent, and the limited proportion of patients with renal stones due to PHPT are younger and have a generally mild degree of hypercalcemia (4). It can be assumed that this circumstance reflects the substantial awareness of the coupling of PHPT to an increased risk of renal calculi. Bearing in mind that HPT once was an apparently rare and strongly symptomatic disorder in the Western Hemisphere, it is interesting to note that crippling bone disease and renal dysfunction now characterize PHPT of the Third World (5-9). These patients display modest hypercalcemia, usually an exceptional parathyroid h o r m o n e (PTH) elevation, and large parathyroid glands with an increased incidence of parathyroid can-

cer. Although etiologic and pathogenic factors are beyond the scope of this chapter, it is noteworthy that there exists nutritional vitamin D and calcium deficiencies in many of these populations, and that changes in this respect may be one of the causes for the altered clinical characteristics of PHPT in the West. However there is growing support for the existence of vitamin D insufficiency, particularly in the elderly population, in the West (7,10). It also is noteworthy that no one would raise the question of conservative therapy in a patient who is b e d r i d d e n due to bilateral femoral fractures and severe proximal myopathy and with such severe osteopenia that the long bones are virtually invisible on plain radiographs.

PHPT Today C o n t e m p o r a r y series of PHPT are largely dominated by elderly women with mild to moderate hypercalcemia and few--if a n y m o f the above-mentioned "traditional" symptoms or signs of the disorder (Table 1). These patients exhibit apparently mild skeletal complications of PHPT and an essentially nonexistent incidence of renal stones. The natural course of this variant involves limited progression over time and rarely converts into other subtypes of PHPT (11,12). Much of the shift in the classic presentation of primary HPT occurred during the 1970s, when a u t o m a t e d serum calcium became more widely utilized. As would be expected from such screening on clinical grounds, the apparently altered disease expression was coupled to a substantial increase in incidence (13,14). The most dramatic rise in the diagnosis of PHPT occurred in postmenopausal women. It cannot be completely excluded, however, that there also was a change in true prevalence and clinical characteristics of the disorder due to essentially unknown factors. Furthermore, we see so few of the historic types of PHPT today that at least some of their etiologic factors possibly have been obviated. Indeed, studies on the incidence and prevalence of PHPT now suggest that this disorder may have reached its peak, and currently may be less c o m m o n in Sweden (and North America) than was the case some decades ago (15,16).

PREVALENCE The prevalence of PHPT rarely has been determined by population-based screening, and this probably contributes to notions of its geographically heterogeneous frequency. The diagnosis can be complicated when calcium concentrations are normal or mildly elevated, and require a set of biochemical investigations that are costly to apply at the population level. This difficulty has led to

CLINICAL PRESENTATION: Eukoew

various biochemical definitions of PHPT, with a diagnostic limit for total serum calcium ranging from 2.55 to 2.85 m m o l / l i t e r in population-based screenings in Europe (16,17). Another complicating factor is that PHPT today typically is a disease that is stable over time and lacks characteristic symptoms (11,12,18,19). Clinical recognition due to progression of diseasespecific symptoms thus should be expected to be rare. Moreover, hospital-based or other types of patient cohorts in Europe (and North America) seem to underestimate the prevalence of PHPT about 10 times even when liberal screening with total or ionized blood calcium determinations have been utilized (20-26). Series of patients routinely recruited in clinical settings are also h a m p e r e d by the fact that individuals come to medical attention for specific reasons. These often differ from the symptoms and complications commonly denoted as traditional in PHPT (27). Recurrent kidney stones, impaired renal function, and low-energy fractures consequently are rare today in PHPT in Europe. Most population-based studies on the prevalence of PHPT are Scandinavian. The investigated target population has been adults of different age and sex, postmenopausal women, and elderly individuals (16,28-31). Screening a m o n g 16,000 unselected citizens of a Swedish city revealed persistent hypercalcemia during two successive years in 1.07% of individuals over 25 years of age (28). Although the hypercalcemic cases were followed for decades and PHPT was a probable cause in all of them, the diagnosis was established unequivocally in only the 16 parathyroidectomized individuals. Another Swedish screening study substantiated a serum calcium concentration above 2.65 m m o l / l i t e r in 0.4-0.6% of g o v e r n m e n t employees 20-63 years old, and a single value above 2.78 m m o l / l i t e r was considered to be consistent with PHPT in 0.06% of the cohort (29). A recent screening with c o m b i n e d use of serum and urine calcium and intact serum PTH substantiated PHPT in 2.6% of w o m e n 55-75 years old (16,32). Merely two-thirds of them, however, were persistently or intermittently hypercalcemic on longitudinal follow-up (16). Studies of incidence and prevalence both indicate that the frequency of PHPT increases with age for both sexes, and that the disease is substantially more comm o n in females (16,21,28-31). In one of the population-based screenings in Sweden, PHPT was considered to be the cause of hypercalcemia in 0.3% of adult m e n and 1.6% of corresponding women (12,28). Indeed, the value rose to 3.0% for females above the age of 60 years. In another such study the corresponding values for m e n and women were 0.3 and 1.0%, and again an increase with age was noted (17). The differences between sexes and age groups have not been clarified. It has been suggested that a slight elevation in serum

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calcium values at m e n o p a u s e introduces an ascert a i n m e n t bias, which also can occur with thiazide therapy (28). The characteristic E u r o p e a n patient with PHPT today consequently is an elderly female, who is apparently asymptomatic and exhibits mild hypercalcemia (33). Only a single population-based screening study has a t t e m p t e d consistent operative verification of PHPT (16,34). At a m e a n age of 68 years, it was not surprising that only 61 of the 109 cases were considered eligible for parathyroidectomy for truly mild PHPT. All but one of them, however, demonstrated conventional histopathologic signs of the disorder. The remaining case had hypercalcemia and elevated PTH concentration probably caused by an ectopic parathyroid adenoma. Because there was no bias in serum calcium between the operated and u n o p e r a t e d individuals, it was concluded that the Swedish prevalence of 2.6% for PHPT was an underestimation; the extent of this is unknown and possibly relates to normocalcemic individuals in whom there is no recognized way to ascertain the diagnosis biochemically (32). A Swedish autopsy examination revealed 10% prevalence for parathyroid a d e n o m a and hyperplasia without consistent differences between sexes (35). Although no case had overt signs of functional renal impairment, the frequency of parathyroid gland e n l a r g e m e n t rose with age and with histologic evidence of nephrosclerosis. It could be argued that such autopsy findings are inconclusive evidence for the clinical existence of PHPT, and serum calcium values indeed were available for only a minority of cases. Together with the rapidly increasing n u m b e r of elderly people in Europe, the findings have led to expectations of a continuing increase in disease incidence. A study from the United States, however, suggested a recent decrease in the frequency of PHPT (15). This notion also has been supported by comparison of two population-based studies of postmenopausal females in Sweden (16,28). Moreover, the average serum calcium value was similar for the hypercalcemic individuals in the studies, which were carried out more than two decades apart. Any increased awareness of the existence of PHPT consequently has influenced the degree of hypercalcemia at recognition to a very limited extent (16).

DIAGNOSIS PHPT may be defined as a primary disturbance in the parathyroid cells leading to inappropriately elevated secretion of PTH in relation to the patient's ionized blood calcium activity. This definition involves an abnormal relationship between these biochemical variables, and not necessarily an elevation above the

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reference range for either of them. The exceptionally short half-life of intact PTH in the circulation (minutes) usually allows estimation of the h o r m o n e "secretion" by m e a s u r e m e n t of the basal value at steady state. Serum calcium must be corrected for albumin binding, but n e e d not be obtained in the fasting state or with other dietary restrictions. Analysis of ionized blood calcium seems to add little to the diagnosis whenever it relies on c o m b i n e d evaluation with serum PTH. In a strict sense, recognition of PHPT could include demonstration of an increased parathyroid cell mass, which necessitates morphologic examination after operative exploration. Very mild increases in the parathyroid cell mass can be difficult to establish, but the increase is usually diagnostic even in the mildest forms of HPT that currently can be recognized biochemically (34,36). Nevertheless, routine clinical diagnosis almost always is simple and uncomplicated. It requires awareness of the generally positive correlation between calcium and PTH concentrations in blood, which contrasts PHPT from euparathyroid individuals and those with hypercalcemia of nonparathyroid origin. By the use of repetitive sampling for less than a year (16), PHPT could be biochemically diagnosed and operatively verified in Swedish women with a mean serum calcium level of 2.42 m m o l / l i t e r (normal range 2.20-2.60 m m o l / l i t e r ) . Patients with PHPT may be arbitrarily divided into three categories. Those with severe PHPT have pron o u n c e d hypercalcemia and an unequivocal rise in serum PTH concentrations, as well as often potentially dangerous symptoms of PHPT (Table 2). The most c o m m o n variant of PHPT lacks such alarming features, and may be d e n o t e d as mild (to moderate). At least

TABLE 2 PHPT subtype Severe

Mild

Normocalcemic

75-80% of such patients with hypercalcemia have a serum PTH concentration above the reference interval. In the others, serum PTH may extend down into the m i d p o i n t of the n o r m a l range. This latter combination of calcium and PTH should be confirmed by repetitive sampling over a 6-month period (Table 2). Because the pattern of hypercalcemia with normal PTH (usually low normal) is typical for familial hypercalcemic hypocalcuria (FHH), it is necessary to exclude hypocalciuria, i.e., the 24-hour urine calcium should be above 2 m g / k g body weight on an unrestricted diet (37,38). Little is known about the progression of PHPT over time, although most studies on its natural course suggest the existence of an insidious and stable disorder (11,28,39). Often an elevated serum calcium value is disclosed many years before the diagnosis of even mild PHPT. It has been hypothesized that PHPT may progress in small steps due to coupling of the secretory calcium resistance to the rate of cell proliferation (40,41). Unless there is always stepwise elevation of the calcium concentration to levels above the normal range, variable periods of normocalcemia could be foreseen in possibly all patients (42-44). Only a single study, however, has systematically explored the prevalence of normocalcemic PHPT. This Swedish study substantiated that essentially one-third of postmenopausal females with PHPT can be recognized despite repeated serum calcium values in the reference range (16). It also was concluded that the n u m b e r of patients was underestimated, because histologic signs of the disorder were found in all the patients subjected to parathyroidectomy (34). The paucity of data indicates that this variant of PHPT still should be regarded as controversial.

Overview of Biochemical Criteria for Diagnosis of PHPT a Diagnostic analytes

High serum calcium, usually high serum PTH, and sometimes alarming symptoms of PHPT; serum calcium and PTH need not to be measured more than once Serum calcium and PTH are mildly to moderately elevated above the normal range; both variables need not to be measured more than twice Serum calcium but not PTH is above the normal range; both variables need to be measured two times over a 6-month period, and serum PTH must be above the midpoint of the normal range for the utilized assay; daily urine calcium should be above 2 mg/kg on an unrestricted diet Mean serum calcium is above 2.50 mmol/liter (normal range 2.20-2.60 mmol/liter) and serum PTH is above the normal range on three occasions; serum creatinine should be normal and plasma 25-OHD 3 should be above 20 ng/liter

aNote that this is not the entire workup that is necessary in the individual patient with suspected PHPT and that the necessity of repetitive sampling has not been tested critically.

CLINICAL PRESENTATION: EUROPE / Diagnostic separation of biochemically very mild HPT from overtly healthy individuals is complex. Various dynamic tests involving challenges of the calcium control of PTH release have been utilized for this purpose. These may involve calcium loading to suppress h o r m o n e secretion (45), calcium complexing with EDTA or citrate to stimulate secretion (46), or both (47,48). The idea of such tests naturally is to disclose a PHPT-specific decrease in the calcium control of PTH release. Little attention has been paid to the possibility of identifying normocalcemic PHPT by mere use of basal serum calcium and intact PTH values. It could be argued that this issue is clinically irrelevant because normocalcemic PHPT has been assumed to require no treatment unless recurrent renal stones exist (43-45). This assumption, however, is based on educated guesses rather than on prospective studies and has been challenged by surgeons in Europe in particular. In the group of patients with normocalcemic PHPT, histologically abnormal parathyroid tissue has been identified at surgery (32,34). Surgery restored PTH concentrations to normal and lowered the mean serum calcium level. With support from only one study, this group can be diagnosed on the basis of mean serum calcium (corrected for albumin binding) above 2.50 m m o l / l i t e r (10.0 m g / d l ) and serum PTH above the normal range on at least three occasions over a 6-month period (Table 2). To exclude secondary PHPT in these patients, chronic renal failure and vitamin D deficiency must be diagnosed. This requires the demonstration of a normal serum creatinine level and a plasma 25hydroxyvitamin D level above about 20 ng/dl.

CONTROVERSIAL SYMPTOMS IN PHPT A variety of confounding factors should be considered on analyzing symptoms of PHPT. Physicians' attitudes vary with regard to the m e a s u r e m e n t of serum calcium and how to treat an increased level. Attention consequently should be paid to the degree of hypercalcemia in published series. This variable indicates the degree of awareness of PHPT a m o n g physicians in general, and the applied indications for treatment. There seem to be considerable differences a m o n g European countries in these respects. It also indicates the expected degrees of PTH elevation and parathyroid gland enlargement and the chance for true positive localization procedures before surgery. Moreover, assessment of treatment outcome is influenced by expectations of both the therapist and the patient, particularly when treatment involves some degree of risk. Many evaluations of PHPT also lack appropriate controls. Ideally, studies on the frequency, severity, and

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handling of symptoms and signs of PHPT should utilize population-based recruitment of both cases and controls. The following discussions focus on some controversial issues in the symptomatology of PHPT, with emphasis on its "nontraditional" manifestations that have attracted considerable attention a m o n g European researchers.

Neurobehavioral and Muscular Dysfunction Patients with recognized neurobehavioral dysfunction of PHPT have increased in n u m b e r during the past decade, particularly when structured interviews and elaborate tests have been prospectively utilized (49). With few exceptions, studies reporting on psychiatric manifestations have found the prevalence to be significantly higher than in seemingly appropriate control groups. Affective and neurasthenic symptoms are the most c o m m o n of these disturbances. In particular, the patients have substantiated increased fatigue, weakness, and anxiety, as well as m o o d swings, irritability, and apathy (49-53). More recent studies substantiate significant decreases in the quality of life with influences on physical, emotional, and social functions, and body pain and vitality (Table 3) (54-57). Psychological symptoms and decreased quality of life are identified also in mild PHPT, but they rarely are volunteered and usually must be actively searched by a systematic approach. The relevance of neurobehavioral symptoms still is controversial, and such symptoms are best considered "nontraditional" manifestations of PHPT (Table 4) (27,58). Self-rating scales nevertheless have supported the existence of a psychological morbidity even in overtly asymptomatic females with normocalcemic to mildly hypercalcemic PHPT (59-61). Low m o n o a m i n e metabolites and increased calcium levels have been found in cerebrospinal fluid, and patients with PHPT consequently mimic patients with e n d o g e n o u s depression (62). There is no disagreement that severe hypercalcemia from any cause may be associated with p r o f o u n d mental changes that are reversible with correction of the hypercalcemia. Some studies have reported weak but statistically significant associations between abnormal psychometric tests and the degree of hypercalcemia in PHPT, but this has not been a universal finding. Muscular weakness in PHPT has been analyzed objectively with respect to strength and endurance of the thigh, arm, hand, and respiratory muscles (63-69). Also, structured interviews and various neurophysiologic tests have been applied. It is unclear if the tiredness and feeling of fatigue are psychological, neuromuscular, muscular, or a combination of these. Normal neuromuscular examinations consequently

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Summary of Recent Analyses on Quality of Life in PHPT

Number, follow-up

Control

Study variables and outcome

63 cases, 12 months

Goiter controls

56 cases, 2-6 months

Normal data

82-110 cases, 2-6 months 104 cases, 6 months

Normal data

53 cases, annual change

Randomized to surveillance

VAS-based questionnaire. Rapid improvement (p < 0.001) after parathyroidectomy. General health improved by 60% (p = 0.019) SF-36. Marked reduction in all domains. Improvement in emotions and body pain at 2 months and in all domains 6 months after parathyroidectomy SF-36. Marked preoperative impairment, 6/8 domains improved after parathyroidectomy SF-36. Marked improvement in 6/8 general health domains after surgery both below (n = 55) and above (n = 49) serum calcium of 10.9 mg/dl Mean serum calcium of 10.3 mg/dl. Scores of two domains of SF-36 differed between the groups, and both were in favor of parathyroidectomy

Normal data

TABLE 4

Overview of "Untraditional" Symptoms and Complications of PHPT and the Expected Influences of Parathyroidectomy

Psychic symptoms and complications Usually affective and neurasthenic in character Mainly involve fatigue, weakness, anxiety, mood swings, irritability and apathy, and a decreased quality of life Can be difficult to appreciate A dementia-like state may occur in the elderly Parathyroidectomy can reverse the affective derangement, the decreased quality of life, and dementia-like symptoms in the elderly, even in the presence of organic brain injuries Diabetes mellitus and glucose intolerance Overrepresented in PHPT Involves a peripheral insulin resistance Unrelated to hypertension, body weight, and vitamin D Overt diabetes mellitus may improve after parathyroidectomy Blood lipids and body fat Increased atherogenic index Normalized by parathyroidectomy Obesity and abnormal fat distribution are controversial Hypertension Overrepresented in biochemically unequivocal PHPT Treatment for hypertension is more common in PHPT Vascular smooth muscles may react inadequately to nitride oxide Parathyroidectomy rarely improves the blood pressure Cardiac derangements Involve left ventricle hypertrophy, increased enddiastolic volume, valvular calcifications, and electrocardiographic changes Increased signs of myocardial ischemia and arrythmia during exercise Usually improve after parathyroid surgery Premature death Verified in population-based and clinical assessments of PHPT in Europe Mainly relates to cardiovascular disorders and affects individuals below 70 years of age Occurs even in mild hypercalcemia, and increases with the extent of hypercalcemia Can be normalized with time after parathyroidectomy

Ref. 57 54 55 60 56

CLINICAL PRESENTATION:EUROPE /

have been described in fatigued patients (70). Casecontrol comparison also has shown that particularly the elderly individuals may experience musculoskeletal symptoms due to reasons that may be unrelated to HPT (71). Severe proximal muscle weakness, especially of the lower extremities, histologic abnormalities in the muscle fibers, and an abnormal electromyogram were primarily thought to be a neurogenic lesion. Further studies could not prove any disturbances in the neuromuscular transmission, and an impaired muscle contraction seems to be a possible explanation (69,72).

Expected Influences of Parathyroidectomy Long-lasting relief of neurobehavioral disturbances has been recognized after parathyroidectomy (49-51,73). This is controversial, however, because limited or no improvement also has been found in studies mainly from North America (74,75). The recorded improvement has ranged from affective derangements, such as fatigue, anxiety, m o o d swings, irritability, and apathy, to such dominant hemispheric functions as memory, learning, and cognition. Signs of organic brain syndrome (dementia-like presentation) may be alleviated, at least temporarily, in elderly patients particularly when such complications have been clinically appreciated during only a few years before surgery (53,76). There are also consistent data on the favorable influence of parathyroidectomy on the quality of life from North America (Table 3). These findings include one randomized comparison of parathyroidectomy with conservative surveillance in elderly individuals (50-75 years of age) with mild hypercalcemia (10.1-11.5 m g / d l ) and no overt symptoms of PHPT (56). Improved muscle function after parathyroidectomy has been noted especially when the tiredness and weakness have been regarded to be muscular in origin (50,64,66,68,77,78). O t h e r studies, however, have found no objective signs of postoperative recovery (65,72). Methodologic difficulties related to such observations entail large intraindividual variations, and training of the voluntary c o m p o n e n t of the muscle contraction on repeated evaluation (64,72). The need for carefully m a t c h e d controls has been stressed, because improvement also has been noted in controls (78). More p r o n o u n c e d improvement can occur in symptomatic patients with high serum calcium concentrations (79), although the treatment outcome mostly has been unrelated to the preoperative calcemia.

Nephrolithiasis The frequency of a renal stone disorder varies considerably a m o n g PHPT cohorts and has decreased during recent decades. Today it seems to affect only a small

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proportion of the patients (60,80). This traditional sign is clinically easy to recognize and an undisputed indication for parathyroidectomy (4,27). It consequently can be overrepresented in clinical reports on operatively treated subgroups (44). Nephrolithiasis occurs mainly in younger individuals, in whom PHPT is less c o m m o n (81,82). In fact, a history of such stones has been found to be equally u n c o m m o n in postmenopausal females with PHPT and their matched controls in Sweden (60). It has been suggested that this discrepancy between age groups may d e p e n d on age-related decreases in calcitriol production, which suggests a lessened urine calcium excretion, a decreased suppression of PTH, and perhaps even an increased risk to develop PHPT (35,83).

Expected Influences of Parathyroidectomy Patients with a history of nephrolithiasis should be referred for parathyroidectomy on this basis alone given the documentation that surgery reduces the rate of recurrent stone disease. Hypercalciuria is present in all PHPT patients with nephrolithiasis, and an elevated urine calcium excretion and recurrent renal stones have been used to identify patients with normocalcemic PHPT (43,44,84). Although the propensity for stone formation mostly is reduced after successful parathyroid surgery, postoperatively normocalcemic patients have substantiated recurrent stones at a frequency similar to idiopathic stone formers (80,85). This may relate to persistence of a renal calcium leak and to a decreased urinary concentrating capacity after operation (86).

Metabolic and Cardiovascular Complications A variety of disturbances in patients with PHPT have become increasingly evident to European researchers (Table 4). Many of these disturbances involve an increased risk for cardiovascular alterations and diseases and for p r e m a t u r e death, which also seem to comprise an u n d e r r a t e d consequence of even biochemically mild HPT (71,87,88). The causal coupling to HPT is complex, however, and some of the metabolic disturbances remain unaltered after parathyroidectomy. This observation suggests the need also to evaluate other treatment regimens, perhaps in combination with parathyroid surgery, to optimize the chances of improved morbidity and mortality in PHPT. Diabetes

MeUitus

PHPT has been associated with impaired glucose tolerance and diabetes mellitus due to peripheral insulin insensitivity (89-93). The age- and sex-adjusted prevalence of PHPT has been estimated to 0.82% in a

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large cohort of patients with diabetes mellitus (93), which was significantly greater than the reported prevalence in that community of 0.36%. Conversely, the prevalence of diabetes mellitus was 7.8% in clinical PHPT compared to 3.0% in consecutive patients without PHPT (91). Similarly, the incidence of diabetes mellitus was 22% in patients undergoing parathyroidectomy at an age older than 65 years compared to 8.6% in a matched population (94), and was about threefold higher (8.2%) in PHPT than in a matched control population in Sweden (89). Insulin resistance and impaired glucose tolerance of PHPT seem to be independent of other risk factors for diabetes mellitus, such as hypertension and obesity (90). The excess of PTH and calcium and the trend to hypophosphatemia in PHPT could contribute in this respect by directly stimulating insulin secretion independent of the glucose level, by reducing the peripheral glucose utilization, and by antagonizing insulin at the receptor level (94-98).

ExpectedInfluences of Parathyroidectomy After parathyroidectomy, fasting glucose concentrations and hemoglobin Alc values have remained essentially unchanged in nondiabetics. Lessened insulin hypersecretion, improved insulin sensitivity and glycemic control by lowered doses of therapy, decreased hepatic insulin extraction, and retarded progression of diabetes mellitus type II have been noted in those with this complication (98-100). In one study such improvement included more than one-third of patients with PHPT and diabetes mellitus types I and II and was most pronounced in those with low elevation of serum PTH levels (100). It should be recognized, however, that controlled studies are lacking and that lack of favorable influence of parathyroidectomy also has been reported (96).

Dyslipidemia and Body Fat Altered concentrations of serum cholesterol and triglycerides have been reported in PHPT (60,101,102). Density fractionation clarified that high-density (HD) and low-density (LD) lipoproteins were lowered and that very low-density (VLD) lipoprotein values were increased, whereas some studies showed increased serum VLDL or no changes at all. A populationbased case-control study of 97 postmenopausal women concluded that mild PHPT is proatherosclerotic (Hagstr6m, Hellman, Lundgren, and Rastad, 2000, unpublished data). These Swedish women all had asymptomatic PHPT and the mean serum calcium concentration was in the normal range (2.57 mmol/liter). Such mild PHPT was associated with decreased HDL cholesterol levels, and increased total triglycerides, VLDL triglycerides, and VLDL cholesterol, as well as

an elevated atherogenic index. The dyslipidemia seems to be more prevalent in women than in men with PHPT (60,92). A direct lypolytic effect of PTH was thought to be responsible for the abnormal lipid metabolism in PHPT in conjunction with findings in secondary HPT (103,104). The peripheral insulin insensitivity of PHPT might contribute by decreasing the enzymatic decomposition of VLD and enhancing the activity of hepatic HDL metabolism.

ExpectedInfluences of Parathyroidectomy HD and LD lipoproteins seem to normalize rapidly after parathyroidectomy, and the VLD lipoprotein values usually some months later (102,105). This improves the unfavorable ratio of LD to HD lipoproteins and atherogenic index, and thereby associated risks for cardiovascular complications. The prospective controlled study on postmenopausal females with mild PHPT underlines that parathyroidectomy effectively normalizes dyslipidemia, but only marginal changes were found in conservatively treated cases (Hagstr6m, Hellman, Lundgren, and Rastad, 2000, unpublished data). Patients with PHPT may exhibit an increased body mass index with abnormal body fat distribution (106,107). A report of postmenopausal females with mild HPT, however, failed to prove any difference in body weight and composition in comparison to ageand sex-matched controls (60). Markedly obese subjects can display moderately elevated PTH concentrations together with a slightly lowered serum calcium, which at least partly may relate to an increased complex binding of calcium to, e.g., free fatty acids. These derangements have been shown to normalize after successful weight reduction (108).

Cardiac and Vascular Complications An increased prevalence of hypertension has been shown in PHPT, which seems to affect 15-50% of the patients and to be less common in the normocalcemic than in the hypercalcemic patients (109-116). In a small but controlled study it was found that the vitamin D analog alphacalcidol reduced the blood pressure in hypertensive patients with mild PHPT (117). Theoretically, the association of PHPT with hypertension includes renal impairment, abnormalities in the noradrenergic and renin-angiotensin systems, and interaction with the cardiovascular PTH receptor (118-120). PTH has effects on the regulation of vascular tone and can directly affect cardiovascular functions (118,121-124). Acute PTH administration causes vasodilation, whereas longer infusion of physiologic doses raises the blood pressure in man (125,126). A parathyroid hypertensive factor, that has been partially purified alters vascular tone and reactivity (127,128).

CLINICAL PRESENTATION: EuRopE

The European focus on these aspects of PHPT is underlined by studies from Austria and Sweden, which show presence of vasodilatory dysfunctions related at least in part to nitride oxide (129-131). These dysfunctions may be one cause for the cardiovascular complication of PHPT, because they seem to predict development of arteriosclerosis. PHPT has been clinically associated with a variety of cardiac disturbances, including left ventricular hypertrophy and increased end-diastolic volume, myocardial and valvular calcifications, increased cardiac output, and electrocardiographic abnormalities (122,129,130, 132-137). An estimate of the significance of cardiovascular derangements has been obtained by analyzing sickness benefits prior to the recognition of biochemically mild HPT (71). In comparison to matched controls, postmenopausal females with normocalcemic or mildly hypercalcemic PHPT substantiated a significantly greater n u m b e r of days off work for cardiovascular diseases. In fact, the need for sick leave during more than half of the investigated 5-year period was 11 times greater in the cases than in controls.

Expected Influences of Parathyroidectomy Parathyroid surgery should not be expected consistently to affect hypertension (109,115,116,138), and the derangements can be indirectly associated (114,115,138). Many of the cardiac disturbances TABLE 5 Risk of death, reference material

172; mean 14 years

Increased p = 0.014, matched population

334; 1-25 years

Increased p < 0.05, surgical patients Cumulative relative survival below 1.0, matched population Increased p < 0.001, matched population

896; mean 13 years

4461; mean 4 years

Increased by 71%, matched population

340; mean 2.9 years

Increased p = 0.001, German population Not significant, matched population

1052; mean 12 years

435; mean 13 years

369

that have been noted in patients with PHPT may improve after parathyroidectomy (118,121-123,130, 139). Such improvement may involve left ventricle dysfunction, hypertrophy and increased end-diastolic volume, myocardial and valvular calcifications, increased cardiac output, and electrocardiographic abnormalities.

Premature Death and P a r a t h y r o i d e c t o m y Premature death has been described in four Scandinavian and one German series, which included more than 6500 hypercalcemic patients recruited in routine clinical settings and by population-based screening (87,88,140-146,152) (Table 5). Premature death has been found to occur mainly in patients less than 70 years of age, and the incidence was diminished after parathyroidectomy (88,144). The approximately 40% increase in the risk of dying was related to cardiovascular disorders and to some extent also to malignant neoplasms. Mortality correlated to the extent of hypercalcemia and the weight of parathyroid adenomas, whereby it has been hypothesized that the duration of PHPT might be important for the risk of dying (92,144,145). Mso, serum creatinine, glomerular filtration rates, and the tubular concentration capacity at the time of parathyroidectomy correlated unfavorably to a patient's survival (141).

Summary of Studies on Mortality in PHPT

Number studied; follow-up

441; mean 8 years

/

Not significant, matched population

Comment

Ref.

Mean serum calcium 2.72 mmol/liter from probable PHPT, population based. Increased mortality <70 years of age from cardiovascular diseases Clinical PHPT, mean serum calcium 3.08 mmol/liter. Increased mortality mainly from cardiovascular diseases Clinical PHPT, mean serum calcium 2.87 mmol/liter. Premature death in cardiovascular diseases (p = 0.06) with normalization 5-8 years after parathyroidectomy Clinical PHPT, mean serum calcium 2.82 mmol/liter. Premature death from cardiovascular and malignant diseases, normalized with time after surgery, and related to serum calcium Nationwide recruitment of PHPT due to adenoma; serum calcium not reported. Premature death in cardiovascular diseases Clinical PHPT, postoperative analysis only

88

Clinical PHPT, mean serum calcium 11.2 mg/dl, p < 0.058 for increased mortality in nonstone formers. Higher serum PTH independent predictor of mortality (p = 0.012) Clinical PHPT treated (29%) and untreated (71%) by PTX, mean highest serum calcium 10.9 mg/dl. Higher serum calcium independent predictor of mortality (p < 0.02)

142 146

87

141

143 150

151

370

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CHAPTF.R21

The coupling of PHPT to several risk factors, including hypertension, dyslipidemia, and insulin resistance, as well as to an increased need for sick leave for cardiovascular diseases, coincides with the notion of an increased risk of dying. O t h e r findings also argue for the existence of this risk. Population-based analyses of m e n have indicated a positive correlation between normal serum calcium values and the risk of dying from cardiovascular disorders (147,148). Another study has provided novel insight into the cardiovascular risk of PHPT (149). Besides substantiating the increased mass and diastolic dysfunction of the left ventricle by echocardiography, patients with PHPT u n d e r w e n t an e r g o m e t e r bicycle test. During such exercise PHPT was associated with higher blood pressure, more pron o u n c e d ST-segment depression, and an increased frequency of ventricular arrhythmias despite that m a t c h e d controls attained a similar workload. These derangements were improved partially or fully 1 year after parathyroidectomy. Studies from the United States have been unable to demonstrate an increased risk of mortality in PHPT (150,151), and the a p p a r e n t controversy between series has been debated (152). One suggestion for the discrepant findings relates to the extent of biochemical features of PHPT, which may have been milder in the North American than in the European studies (Table 4). However, higher serum calcium and PTH values were i n d e p e n d e n t predictors of death also in the American series. Moreover, the increased risk of dying almost reached statistical significance in one of these studies for the PHPT patients without a history of renal stones (150). It consequently may seem logical to conclude that, if patients with PHPT u n d e r w e n t elective parathyroidectomy at an early stage, none of the variables ( a d e n o m a weight, serum calcium, serum PTH) would reach high levels and the seriously increased risk of death would not occur.

CONCLUDING REMARKS This chapter addresses some controversial issues we believe should be emphasized in PHPT, and these contributions need not originate only from Europe. Indeed, there is a strong a r g u m e n t for operative intervention at even early stages of the disease, consistent with proposals by other investigators in this field (153,154). We fully appreciate that only portions of the a r g u m e n t for parathyroidectomy have been included in the text. O t h e r contributions to this book, however, deal with the compelling data from recent studies of bone mass and fragility fractures that generally speak in favor of accepting the idea of a liberal indication for parathyroidectomy (155,156). It also is fully appreci-

ated that the decision to treat actively to a large extent depends on expectations from alternative strategies as well as the natural course of the disease. Again we feel that this information argues for parathyroidectomy, and appreciate that this will be dealt with elsewhere in this volume.

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117. Lind L, Wengle B, Lithell H, Ljunghall S. Plasma ionized calcium and cardiovascular risk factors in mild primary hyperparathyroidism: Effects of long-term treatment with active vitamin D (alphacalcidol). Jlntern Med 1992;231:427-432. 118. Schlfiter KD, Piper HM. Left ventricular hypertrophy and parathyroid hormone: A causal connection? Cardiovasc Res 1998;39:523-524. 119. Schiffi H, Sitter T, Lang SM. Noradrenergic blood pressure dysregulation and cytosolic calcium in primary hyperparathyroidism. Kidney Blood Press Res 1997;20:290-296. 120. Bernini G, Moretti A, Lonzi S, Bendinelli C, Miccoli P, Salvetti A. Renin-angiotensin-aldosterone system in primary hyperparathyroidism before and after surgery. Metabolism 1999;48:298-300. 121. Lind L, Ljunghall S. Hypertension, primary hyperparathyroidism and the parathyroid hypertensive factor. Blood Pressure 1993;2:4-5. 122. Lind L, Ljunghall S. Preoperative evaluation of risk factors for complication in patients with primary hyperparathyroidism. Eur J Clin Invest 1995;25:955-958. 123. Rodriguez-PortalesJA, Fardella C. Primary hyperparathyroidism and hypertension: Persistently abnormal pressor sensitivity in normotensive patients after surgical cure. J Endocrinol Invest 1994;17:307-311. 124. Schlfiter KD, Piper HM. Trophic effects of catecholamines and parathyroid hormone on adult ventricular cardiomyocytes. Am J Physiol 1992;263:1739-1746. 125. Pang PK, Janssen HE Yee JA. Effects of synthetic parathyroid hormone on vascular beds of dogs. Pharmacology 1980;21:213-222. 126. Hulter HN, MelbyJC, Peterson JC, Cooke CR. Chronic continuous PTH infusion results in hypertension in normal subjects. J Clin Hypertens 1986;2:360-370. 127. Schlfiter H, Quante C, Buchholz B, Dietl KH, Spieker C, Karas M, Zidek W. A vasopressor factor partially purified from human parathyroid glands. Biochem Biophys Res Commun 1992;15: 323-329. 128. Pang PK, Benishin CG, Lewanczuk RZ. Parathyroid hypertensive factor, a circulating factor in animal and human hypertension. AmJHypertens 1991;4:472-477. 129. Stefenelli T, Abela C, Frank H, Koller-Strametz J, Globits S, Bergler-Klein J, Niederle B. Cardiac abnormalities in patients with primary hyperparathyroidism: Implications for follow-up. J Clin Endocrinol Metab 1997;82:106-112. 130. Stefenelli T, Abela C, Frank H, Koller-Strametz J, Niederle B. Time course of regression of left ventricular hypertrophy after successful parathyroidectomy. Surgery 1997;121:157-161. 131. Nilsson I-L, ~,berg J, Rastad J, Lind L. Endothelial vasodilatory dysfunctions in primary hyperparathyroidism is reversed after parathyroidectomy. Surgery 1999;126:1049-1055. 132. Symons C, Fortune F, Greenbaum RA, Dondona E Cardiac hypertrophy, hypertrophic cardiomyopathy, and hyperparathyroidism--an association. Br HeartJ 1985;54:539-542. 133. Niederle B, Stefenelli T, Glogar D, Woloszczuk W, Roka R, Mayr H. Cardiac calcific deposits in patients with primary hyperparathyroidism: Preliminary results of a prospective echocardiographic study. Surgery 1990; 108:1052-1056. 134. Stefenelli T, Pacher R, Woloszczuk W, Glogar D, Kaindl E Parathyroid hormone and calcium behavior in advanced congestive heart failure. Z Kardio11992;81:121-125. 135. Dahlberg K, Brodin LA, Juhlin-Dannfelt A, Farnebo LO. Cardiac function in primary hyperparathyroidism before and after operation. An echocardiographic study. Eur J Surg 1996;162:171-176. 136. Ohara N, Hiramatsu K, Shigematsu S, Hayashi Y, Ishihara F, Aizawa T, Niwa A, Yamada T, Hashizume I~ Effect of parathyroid hormone on left ventricular diastolic function in patients with primary hyperparathyroidism. Miner Electrolyte Metab 1995;21:63-66.

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137. Georgiannos SN, Jenkins BJ, Goode AW. Cardiac output in asymptomatic primary hyperparathyroidism: A stigma of early cardiovascular dysfunction? Int Surg 1996;81:171-173. 138. Davies M. Current therapy. Primary hyperparathyroidism: Aggressive or conservative treatment? Clin Endocrinol 1992;36: 325-332. 139. Ljunghall S, Joborn C, Palm6r M, Rastad J, Akerstr6m G. Primary hyperparathyroidism--the surgically cured patient. In: Kleerekoper M, Krane SM, eds. Clinical disorders of bone mineral metabolism. New York:Mary Ann Liebert, 1989:353-358. 140. Hedbfick G, Od6n A. Increased risk of death from primary hyperparathyroidism~an update. Eur J Clin Invest 1998;28: 271-276. 141. Hedbfick G, Od6n A. Death risk factor analysis in primary hyperparathyroidism. EurJ Clin Invest 1998;28:1011-1018. 142. Ronni-Sivula H, Sivula A. Long-term effect of surgical treatment on the symptoms of primary hyperparathyroidism. Ann Clin Res 1985;17:141-147. 143. Walgenbach S, Hommel G, Junginger T. Outcome after surgery for primary hyperparathyroidism: Ten-year prospective followup study. WorldJ Surg 2000;24:564-569. 144. Hedbfick G, Od6n A. The influence of surgery on the risk of death in patients with primary hyperparathyroidism. World J Surg 1991;15:399-407. 145. Hedbfick G, Od6n A, Tisell L-E. Parathyroid adenoma weight and the risk of death after treatment for primary hyperparathyroidism. Surgery 1995;117:134-139. 146. Palmer M, Adami H-O, Bergstr6m R, ~kerstr6m G, Ljunghall S. Mortality after surgery for primary hyperparathyroidism: A follow-up of 441 patients operated on from 1956 to 1979. Surgery 1987;102:1-7.

147. Leifsson BG, Ahr~n B. Serum calcium and survival in a large health screening program. J Clin Endocrinol Metab 1996;81: 2149-2153. 148. Lind L, Skarfors E, Berglund L, Lithell H, Ljunghall S. Serum calcium: A new, independent, prospective risk factor for myocardial infarction in middle-aged men followed for 18 years. J Clin Epidemiol 1997;50:967-973. 149. Nilsson IL, ~tberg J, Rastad J, Lind L. Left ventricular systolic and diastolic function and exercise testing in primary hyperparathyroidism--effects of parathyroidectomy. Surgery 2000; 128:895-902. 150. S6reide JA, van Heerden JA, Grant CS, Yau Lo C, Schleck C, Ilstrup DM. Survival after surgical treatment for primary hyperparathyroidism. Surgery 1997;122:1117-1123. 151. Wermers RA, Khosla S, Atkinson EJ, Grant CS, Hodgson SF, O'Fallon WM, Melton III LJ. Survival after the diagnosis of hyperparathyroidism: A population-based study. Am J Med 1998;104:115-122. 152. Hedbfick G, Od6n A. Survival of patients operated on for primary hyperparathyroidism. (letter). Surgery 1999;125:240-241. 153. Toft AD. Surgery for primary hyperparathyroidism--sooner rather than later. Lancet 2000;355:1478-1479. 154. Silverberg SJ, Bilezikian JP, Bone HG, Talpos GB, Horwitz MJ, Stewart AF. Therapeutic controversies in primary hyperparathyroidism. J Clin Endocrinol Metab 1999;84:2275-2285. 155. Khosla S, Melton LJ, Wermers RA, Crowson CS, O'Fallon W, Riggs BI. Primary hyperparathyroidism and the risk of fracture: A population-based study. J Bone Miner Res 1999;14:1700-1707. 156. Silverberg sJ, Shane E, Jacobs TP, Siris E, Bilezikian JE A 10-year prospective study of primary hyperparathyroidism with or without parathyroid surgery. N EnglJ Med 1999;341:1249-1255.

CI-IAPTEI 22 Clinical Presentation of Primary Hyperparathyroidism India, Brazil, and China AMBRISH MITHAL Indraprastha Apollo Hospital, 110044 New Delhi, India FRANCISCO BANDEIRA Endocrine Unit, Hospital Agamenon Magalhgtes, Secretaria da Saude de Pernambuco, University of Pernambuco, Pernambuco, Brazil

XUNWU MENG Peking University Medical CollegeHospital, 100730 Beijing, China SHONNI j. SILVERBERG Department of Medicine, Collegeof Physicians and Surgeons, Columbia University, New York, New York 10032 YIFAN SHI Peking University Medical CollegeHospital, 100730 Beijing, China SAROJ K. MISHRA Department of Surgery, Sanjay Gandhi Post Graduate Institute of Medical Sciences, 226 O14 Lucknow, India LUIZ GRIZ University of Brazil, 0020-020 Recife- PE, Brazil GEISA MACEDO University of Brazil, 0020-020 Recife- PE, Brazil GUSTAV CELDAS University of Brazil, 0020-020 Recife- PE, Brazil CRISTINA BANDEIRA Endocrine Unit, Hospital dos Servidores do Estado, and Hospital Agamenon Magalhges, Secretaria da Saude de Pernambuco, University of Pernambuco, Pernambuco, Brazil

J O H N E BILEZIKIAN Departments of Medicine and Pharmacology, Collegeof Physicians and Surgeons, Columbia University, New York, New York 10032 D. SUDHAKER RAO Bone and Mineral Metabolism, Department of Medicine, Henry Ford Health System, Detroit, Michigan 48202

INTRODUCTION

and the contemporary primary hyperparathyroidism of these other areas of the world appear to be largely related to the prevailing vitamin D and calcium nutritional status of these populations. In addition, an interesting analogy can be made between the endemic goiter of iodine deficiency and osteitis fibrosa cystica of primary hyperparathyroidism. Just as endemic goiters have virtually disappeared following iodination of common table salt, so has osteitis fibrosa cystica declined following fortification of milk with vitamin D. It will be of great interest to see whether such a change in clinical presentation of primary hyperparathyroidism occurs in parts of the world where it is still a symptomatic disease when better nutritional policies are implemented. In the interim, continued

Primary hyperparathyroidism is the third most common endocrine disorder after diabetes and thyroid disorders. Over the past half century, the pattern of presentation of the disease has changed in the West such that nearly 70-80% patients are asymptomatic and do not usually suffer from its skeletal or renal complications. In contrast, primary hyperparathyroidism still presents with its traditional manifestations in the East and in some regions of the Southern Hemisphere. In these regions, it is associated with more symptoms, large parathyroid tumors, and a higher incidence of parathyroid cancers. The striking similarities between the traditional primary hyperparathyroidism of the West

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observations and comparisons of patients with primary hyperparathyroidism from different parts of the world are likely to provide further insights into this everchanging disease.

H I S T O R I C A L PERSPECTIVE Since the classic descriptions of primary hyperparathyroidism by Fuller Mbright in the first half of the twentieth century (1), two major events have taken place. First, in the United States, United Kingdom, and in most European countries, the apparent incidence of primary hyperparathyroidism has risen dramatically (2) (see Chapters 18, 20, and 21). Second, there has been a major shift in its pattern of clinical presentation (2-5). The increase in the recognition of primary hyperparathyroidism over the past 75 years occurred in two steps: in 1934, when primary hyperparathyroidism was discovered to be a cause of nephrolithiasis (6), the incidence figures rose by about 10-fold (7,8). A further 4- to 5-fold increase emerged with the introduction of routine biochemical screening in the United States in the early 1970s (2). More recently, however, the Mayo Clinic has reported a 3-fold decline in the incidence of primary hyperparathyroidism (3), which can be best explained by the expected decrease in incidence that typically follows a period of accelerated rate of case finding after the introduction of any new screening policy. Other possible explanations include abandoning of the routine therapeutic use of head and neck radiation (an epidemiologic risk factor for parathyroid tumorigenesis) for benign disorders (9,10), increasing use of estrogen replacement therapy in postmenopausal women (11), and changes in vitamin D and calcium nutrition of the population (12,13). Similar swings in disease prevalence have not yet occurred in parts of the world such as China, the Indian subcontinent, the Middle East, island nations of the South Pacific, and some locations in South America, where routine biochemical screening is not practiced and suboptimal calcium and vitamin D nutrition are widely prevalent. Over the past two decades, as described in Chapters 18 and 54, there has also been a dramatic change in the pattern of clinical presentation of primary hyperparathyroidism in the West, with a significant decline in the prevalence of overt bone disease (2,4,5, 14-20). Most of this relative decline in the incidence of overt bone disease (osteitis fibrosa cystica) can be accounted for by the dilutional effect of a small number of patients with severe disease by a much larger number of patients with mild asymptomatic disease discovered by routine biochemical screening. Additionally, however, a real decline in the incidence of osteitis fibrosa cystica is likely.

In contrast, the clinical presentation of primary hyperparathyroidism has changed very little, if any, in countries such as Brazil, China, and India over the same period of observation (21-27). We have compiled the available information from these three countries as a representative cohort of patients with primary hyperparathyroidism from parts of the world with developing economies. Published and unpublished information on primary hyperparathyroidism from other countries such as Hong Kong, Malaysia, Pakistan, Singapore, and Saudi Arabia, as well as the experience with this disease in immigrant Indians and Pakistanis in the United Kingdom (21-34), appear to be similar to that seen in Brazil, China, and India (21,23-27,29,30). Insight into the clinical manifestations, response to parathyroidectomy, and parathyroid pathology in patients from developing countries might bring greater understanding of the changing pattern of presentation of primary hyperparathyroidism in the West. This comparative study might also help us understand why the disease is still seen in its traditional pattern in these and other parts of the world.

PREVALENCE A N D I N C I D E N C E Depending on the characteristics of the population analyzed, the methods used for case finding or disease screening, and the country of the study, the estimated prevalence of primary hyperparathyroidism varies from 1 in 200 to 1 in 1000 (see Chapters 20 and 21). As a result, primary hyperparathyroidism is now the third most common endocrine disorder in the West after diabetes mellitus and thyroid disorders (20,35). However, it must be recognized that most of the published literature on the prevalence and incidence rates of the disease is dominated by studies from western countries (2,3,14,15,36-39). In contrast, descriptions of primary hyperparathyroidism are seldom reported or published from the developing countries. For decades, for instance, it was widely believed that primary hyperparathyroidism does not occur in India, and that all cases of overt parathyroid disease were simply a reflection of more severe and prolonged vitamin D deficiency, so-called tertiary hyperparathyroidism (see Chapter 18). Therefore, it has been difficult to determine the true prevalence of "authentic" primary hyperparathyroidism in Brazil, China, and India because comprehensive health assessment or biochemical screening programs are not generally practiced. Because both symptomatic (the vast majority of patients) and asymptomatic primary hyperparathyroidism exist in these countries (21,25,27), it follows that the disease must be much more prevalent than is

HYPERPARATHYROIDISMIN INDIA, BRAZIL, AND CHINA / currently appreciated. Furthermore, there does not appear to be any biologic or genetic reason why primary hyperparathyroidism should be rare in these parts of the world. As health awareness programs become commonplace in these developing nations, we may learn that the true prevalence of the disease is much greater, and the pattern of presentation is different from that which is being reported today.

DEMOGRAPHIC

CHARACTERISTICS

Patients with primary hyperparathyroidism in the East (note: the term "East" is used for the sake of simplicity and clarity rather than in a true "regional" sense) are at least a decade or two younger at presentation compared to those in the West (Table 1). The average age at presentation in the West varies between 55 and 65 years (2,5,14,15,37-39) compared to 35 and 40 years in the East (23-27,40). It is generally assumed that the more frequent presentation of symptomatic primary hyperparathyroidism in the East is a reflection of delay in diagnosis, but the mean duration of symptoms in most patients is not excessively long (Table 1). On the contrary, the estimated duration of the disease is significantly shorter in the East (p = 0.032; Table 1). Gender distribution also differs in the two regions of the world. Women represent a disproportionately lower fraction of patients in the series from the East (Table 1). However, there does not appear to be any specific racial or ethnic predilection, because primary hyperparathyroidism has been reported in almost all peoples. These and other aspects of the disease will be discussed in detail later.

TABLE 1

PRESENTING MANIFESTATIONS Most patients in Brazil, China, and India present to clinicians with either symptomatic hypercalcemia or complications of the disease such as osteitis fibrosa cystica, kidney stones, or nephrocalcinosis. The asymptomatic primary hyperparathyroidism commonly seen in the West is either rare or not reported. The clinical manifestations of the disease are generally due either to hypercalcemia or to excess parathyroid h o r m o n e (PTH) secretion. Symptoms or complications related to hypercalcemia (see Table 1, and Chapters 20, 34, and 45) are really not specific to primary hyperparathyroidism, d e p e n d largely on the serum calcium concentration, and can occur in any condition that causes hypercalcemia. Hypercalcemic symptoms, therefore, do not distinguish different populations of patients with primary hyperparathyroidism. In contrast, symptoms and signs related to excess PTH secretion are more specific to primary hyperparathyroidism and d e p e n d on the serum PTH concentrations. It is these PTHmediated manifestations of the disease that vary between populations (40) and most likely within the same population over time (4,5). Despite a widely held view that PTH is directly responsible for some of the symptoms of primary hyperparathyroidism, considerable uncertainty exists about the nature, scope, and quantification of these features (see Chapters 20, 21, and 23).

Clinical Features A typical patient with modern-day primary hyperparathyroidism in the West is a postmenopausal woman in whom mild hypercalcemia is discovered during

Comparison of Demographic Characteristics of Patients with Primary Hyperparathyroidism from Different Countries

Characteristic Time period Latitude Total number Gender ratio (F:M) Age at presentation (mean +_ SD, years) Duration of disease or symptoms (mean _ SD, years)

377

United States (New Y o r k )

United States (Detroit)

China (Beijing)

India (North)

1984-1999 40°N 143 3.4:1 55__ 12

1989-1999 43°N 346 3.7:1 61 _+ 14

1958-1993 40°N 134 2.6:1 37+_ 13

1973-1999 28°N 93 1.8:1 3 5 _ 16

3.5 _+ 1.5

a

i

2.8 _+ 2.9 b

aCalculated from the computerized laboratory data base for the group with mild asYbrnptomatic PHPT (n = 105, D.S. Rao, unpublished data). Estimated from the clinical history.

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East-West Comparison of Relevant Clinical Manifestationsa

Variable

West (historic)

West (contemporary)

East (historic and contemporary)

Bone disease b Osteitis fibrosa cystica Palpable brown tumors Fractures Nephrolithiasis Nephrocalcinosis Palpable parathyroid

50-60% 25-50% Infrequent Frequent 50-80% 5-10% Not infrequent

10-20% <2% Almost never Infrequent 5-20% Almost never Almost never

80-100% >50% Not infrequent Frequent 10-40% 5-10% Not infrequent

a"East" and "West" are used for the sake of convenience rather than in a rigid territorial sense. Estimates for the East are limited to Refs. 21-34. t~Bone disease as detected by X-rays and/or bone mineral density.

routine biochemical screening (2,5,14,15,37-39). Consequently, in the West, attention has been shifted to consideration of asymptomatic disease and its nontraditional manifestations (see Chapters 20 and 21). Thus primary hyperparathyroidism appears to have changed from a disease with symptoms to a disease in search of symptoms! In contrast, patients with primary hyperparathyroidism in developing countries present with classic signs and symptoms of the disease (Table 2). In addition to the symptoms of hypercalcemia (see Chapters 20, 21, and 34), bone pain, skeletal deformities, and pathologic fractures are frequent (28,31,41). "Brown tumors," a radiologic manifestation of osteitis fibrosa cystica, are seen in about 50-90% of patients (24-27,40,42). Occasionally, a brown tumor in the rib

can mimic a lung mass (41). Palpable parathyroid tumors in the neck and palpable brown tumors of the skeleton are not u n c o m m o n (Figs. 1 a n d 2). Pseudoclubbing of the fingers due to resorption of the terminal digits is frequent and is almost always multiple and bilateral. Pathologic fractures through brown tumors and deformities even after healing of osteitis fibrosa cystica are c o m m o n (Figs. 3 and 4). In children and adolescents, associated vitamin D depletion can cause rickets (43), and severe hyperparathyroidism can lead to slipped capital epiphysis of the femur. Similarly, pseudofractures, a typical feature of osteomalacia, can occur in adults with primary hyperparathyroidism (30,44,45). Generalized muscle weakness and a more specific proximal myopathy are p r o n o u n c e d (46-48). The

FI6.1 Visible and palpable parathyroid tumor. At surgery a 9.2-g parathyroid adenoma was found and removed successfully. (See color plates.)

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FIG. 2 Swollen and deformed knee due to osteitis fibrosa cystica. The X-rays of this knee are shown in Fig. 3. (See color plates.)

patient who suffers a combination of severe bone disease and p r o f o u n d myopathy can be r e n d e r e d immobile and is often referred to as a "parathyroid cripple." The resultant immobilization further accelerates bone resorption and worsens existing hypercalcemia. The frequency of nephrolithiasis and nephrocalcinosis appears to be greater than that in the West (25-27). However, the prevalence of other conditions such as peptic ulcer, pancreatitis, gallbladder disease, and hypertension that are commonly reported to be associated with primary hyperparathyroidism do not appear to differ between patients in the East and West. Since the introduction of routine calcium measurements in Brazil about 16 years ago, the proportion of patients with asymptomatic primary hyperparathyroidism has risen to about 35% (21). A similar trend may follow in other parts of the world when such screening tests are adopted, although the prevalence of asymptomatic disease did not change in H o n g Kong after the introduction of routine biochemical screening (22).

Biochemical In general, the symptoms and signs of primary hyperparathyroidism in patients from developing countries show a stronger correlation with the degree of

FIG. 3 Brown tumor of the knee (see Fig. 2) before (top) and 6 months after (bottom) parathyroidectomy.

PTH elevation than with the level of serum calcium. In fact, the disease tends to be more severe than is reflected by the serum calcium concentration alone (Table 3). Despite more severe disease, patients with primary hyperparathyroidism in the East have lower serum calcium concentrations compared to patients in the West. Consequently, it is not u n c o m m o n for the total serum calcium concentration to be within the reference range, suggesting the diagnosis of so-called "normocalcemic primary hyperparathyroidism" (49-51). In one series from India, the serum calcium values were within the reference range in almost half the patients (27), reflecting the opposing effects of excess PTH secretion and the high prevalence of coexistent vitamin D depletion (26,27,40,42). The magnitude of hypercalcemia is, therefore, related both to a patient's calcium and vitamin D nutritional status and to the severity of the hyperparathyroidism. In contrast, serum concentrations of PTH and alkaline phosphatase activity are significantly higher and the prevalence of

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FIG. 4 Extensive osteitis fibrosa cystica with deformed femora before and 6 and 12 months after curative parathyroidectomy.

osteitis fibrosa cystica is greater in patients in the East (21,23,29,40,42). Consequently, there is a disproportionate elevation in serum PTH concentration in relationship to the serum calcium concentration (Table 3). Consistent with this more severe form of primary hyperparathyroidism, biochemical markers of bone turnover are markedly elevated, often in the range typically seen in patients with Paget's disease of bone. A vast majority of patients in the East have coexistent vitamin D depletion as reflected by low serum concentrations of 25-hydroxyvitamin D (40,42). Although nutritional deficiency is the most c o m m o n cause of vitamin D depletion, "acquired" deficiency may arise as a result of e n h a n c e d hepatic inactivation of 25-hydroxy vitamin D in primary hyperparathyroidism (13,52). Poor dietary calcium intake, a c o m m o n occurrence in developing countries, further amplifies the effect of vitamin D deficiency by independently augmenting TABLE 3

PTH secretion. Serum values of 1,25-dihydroxyvitamin D, on the other hand, are either normal or elevated d e p e n d i n g upon the status of 25-hydroxyvitamin D and PTH secretion (40,42,53). Despite poor calcium and vitamin D nutrition, hypercalciuria is more pron o u n c e d (Table 3) and c o m m o n than expected (33). This is related to the osseous effects of excess PTH, i.e., osseous hypercalciuria, due to mobilization of skeletal calcium, an observation that may partly explain the continued higher prevalence of nephrolithiasis and nephrocalcinosis in patients with primary hyperparathyroidism in the East (26,27).

Radiologic Findings Subperiosteal bone resorption of phalanges, symphysis pubis, and pubic rami, resorption of terminal digits resulting in pseudoclubbing of the fingers, resorption

Comparison of Relevant Biochemical Findings in Patients with Contemporary PHPT from Different Countries a

Measurement

North America (New York and Detroit)

South America and Far East (Brazil, China, and North India)

Calcium (mg/dl) 25-Hydroxyvitamin D (ng/ml) Alkaline phosphatase (IU/liter) PTH (ng/ml)

10.8 _ 0.60 20.0 _+ 10.0 110 _+ 60 128 _ 96

11.8 _ 1.04 11.1 _+ 6.2 1497 +_ 1894 1024 ___565 b

Urinary Ca (mg/dl)

249 _ 122

320 + 160

aApproximate representative values calculated from the available published (21-27) and unpbublished data. Values are given as mean _+ SD. Intact PTH values were available only in patients from Lucknow, India (n = 17).

HYPERPARATHYROIDISMIN INDIA, BRAZIL, AND CHINA / and lysis leading to tapering of the lateral ends of the clavicles, cortical thinning of long bones, and generalized osteopenia are c o m m o n radiologic findings in patients with primary hyperparathyroidism seen in Brazil, China, and India (21,23-27,29,30). The typical "salt-pepper" pattern visible on lateral skull X-rays is the rule rather than the exception. In severe cases, vertebrae may show the typical appearance of a "ruggerjersey" spine of renal osteodystrophy even in the absence of renal failure (see Fig. 5; see also Chapters 39 and 40). Paradoxically, vertebral fractures are relatively u n c o m m o n despite more severe hyperparathyroidism. Biconcave vertebral deformities ("codfish" vertebrae), on the other hand, are seen. In addition, most patients develop osteitis fibrosa cystica, manifested on X-rays as cystic expansile lesions or brown tumors that can sometimes be palpated clinically (Figs. 2 and 3). Occasionally, osteitis fibrosa cystica in the spine can result in paraplegia (54). Pathologic fractures resulting from severe cortical thinning or through brown tumors

FIG. ,5 "Rugger-jersey" spine due to severe hyperparathyroidism in the absence of renal failure.

381

are a c o m m o n presenting feature (Fig. 6). The lesions all heal completely with dense remineralization following parathyroidectomy, but the deformities are usually p e r m a n e n t (Figs. 2-4).

Bone Mineral Density As can be expected, bone density is frequently and more severely reduced in patients from developing countries with typical predominance of cortical bone loss. The magnitude of deficits far exceeds that seen in early postmenopausal osteoporosis when cancellous bone loss predominates. Nevertheless, the magnitude of increase in bone density following parathyroidectomy can be impressive (42,55-58) and is largely d e p e n d e n t on the base line bone density (15,59-62). M t h o u g h the average increases in bone density are of the order of 1-15% depending on the type and site of measurement in patients with primary hyperparathyroidism in the West (59), bone density can increase up to 500% of the base line value in patients in the East (42,55)! This spectacular increase in bone density

FIG. 6 Severe cortical thinning and pathologic fracture leading to a "parathyroid cripple."

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emphasizes the underlying potential for remineralization of bone tissue following surgery in severe cases of primary hyperparathyroidism (57,63,64). Even with these impressive increases in bone density, the recovery of cortical bone deficits is largely incomplete (59,65,66) and many patients are left with permanent deformities (see Figs. 2-4) and continued excess fracture risk for the rest of their lives (67).

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Parathyroid Pathology

35

The proportion of patients with single or multiple gland involvement at operation does not differ between patients in the West and in the East (23,27,33). However, compared to the western series the prevalence of parathyroid cancer is three- to fivefold among patients with primary hyperparathyroidism from Brazil, China, India, and Pakistan (23,27,29,33) (see also Chapter 1). Although a significant reduction in the mean weight of the parathyroid adenomas has been observed over the past 50 years in the West (see Chapter 18), such a decline in tumor weights has not occurred in patients operated on for primary hyperparathyroidism in the East (see Table 4 and Fig. 7). Though there are many reasons for these discordant observations, a change in calcium and vitamin D nutrition of the population is the most likely explanation. This is supported by the recent observation of a relationship between serum concentrations of 25-hydroxy vitamin D, the best index of vitamin D nutritional status, and parathyroid adenoma weight, the best index of the severity of primary hyperparathyroidism (40,53). Increased stimulation of growth of an existing ade-

~

40

45

50

55

60

Mean Age at Surgery (years)

FIG. 7 Relationship between parathyroid adenoma weight and age at the time of parathyroidectomy. The adenoma weight is shown (,,) for the United States patients with regression and 95% confidence interval lines. There are only limited available data from India (•). Note the declining tumor weights in the United States patients and increasing tumor weight in the Indian patients over time. The decline in adenoma weight was most marked between 1945 and 1950, somewhat less so between 1965 and 1974 and stable from 1974 to 1999 (see Chapter 18). Vitamin D was added to milk sold in the United States between 1933 and 1940. Also illustrated is the progressive decline in tumor weights over time in the United States patients despite an increase in age at surgery [data are compiled from published data, from Chapter 18 (this volume), and from D.S. Rao, unpublished; copyrighted by Dr. D. Sudhaker Rao].

noma by vitamin D and calcium malnutrition may predispose to the development of parathyroid cancers. This mitotic stimulus from prolonged vitamin D and calcium depletion may partly explain both the larger tumor size (Table 4; Fig. 7) and a higher prevalence of

TABLE 4 Comparison of Biochemical Findings and Parathyroid Gland Weights in Patients from the United States and India with Comparable Serum Levels of 25-Hydroxy Vitamin D Measurement Number of patients Age (years) 25-Hydroxyvitamin D (ng/ml) 1,25-Dihydroxyvitamin D (pg/ml) Parathyroid gland weight (g) Parathyroid gland weight (g)b PTH (pg/ml) Alkaline phosphatase (IU/liter) Adjusted calcium (mg/dl)

65

United States (Detroit)

India (Delhi and Lucknow)

p

51

27

m

62 _+ 14 9.29 _+ 3.79 63.5 _+ 21.9 1.66 +_ 1.75 1.05 _+ 2.69 172 _ 192 141 _+ 113 11.0 _+ 0.80

36 _+ 16 10.22 _+ 6.17 50.4 _+ 21.6 7.21 _+ 7.48 4.25 _+ 3.47 1024 _ 565 c 983 +_ 1456 11.5 _ 1.41

<0.001 0.412 a 0.014 <0.001 <0.001 <0.001 <0.001 0.049

avariable used to select patients. bGeometric mean and multiplicative SD. Clntact PTH values were available only in patients from Lucknow, India (n - 17).

HYPERPARATHYROIDISMIN INDIA, BRAZIL, AND CHINA / parathyroid cancers among patients with primary hyperparathyroidism in the East (23,27,29,33).

C O N T R A S T I N G FEATURES OF D I S E A S E F J ~ R E S S I O N : EAST V E R S U S WEST Several features of contemporary primary hyperparathyroidism as seen in the West and East deserve further discussion (Table 2). First, the differences in demographic, clinical, biochemical, and radiologic manifestations of the two patient populations highlight the differential expression of the disease in different parts of the world. Second, it is essential to recognize the temporal changes in disease expression that have taken place both within and between given geographical regions of the world. Third, the general assumption that severity of primary hyperparathyroidism in the developing regions of the world is due to delayed recognition is not well founded. Fourth, the differences in parathyroid gland mass and pathology between the two regions of the world do not simply reflect the prevailing surveillance patterns. A careful and systematic comparison of the disease characteristics of the two regions of the world leads to a different conclusion (Tables 1, 3, and 4). Over the past two or three decades the mean age of patients with primary hyperparathyroidism has not changed significantly in either region of the world and the difference in the mean age at presentation of the two populations has remained constant (21-34). If the basis for the greater prevalence of overt bone disease in the developing countries were due mainly to a delay in diagnosis or late presentation, one would have expected the age at presentation to be greater, rather than low~ than that for patients in the West. The lack of change in the mean age at presentation over the past two or three decades (2,4,5,14,15,39) and a much younger age of presentation in an earlier era within the West (1,6) implies that the pattern of disease expression rather than disease has changed (19). Furthermore, conservative follow-up of patients with mild asymptomatic primary hyperparathyroidism for as long as 10-20 years has not been associated either with progression of the disease or development of disease-specific complications in most patients (14,15,37,39,68). Another interesting feature is that the proportion of women in the series from Brazil, China, and India (21,23,25-27,42) is lower than in the West (14,15,39). This suggests that a more severe nature of disease presentation in the East has obscured the usual predominance of women seen in the series reported from the West (see Chapters 20 and 21). Although the hypercalcemic symptoms do not differ among patients from different parts of the world, the

383

disease-specific complications such as bone and stone disease are modified by factors that may be relevant in one, but not in the other regions of the world. Clinically a n d / o r radiologically manifest bone disease is a regular feature a m o n g patients in the East (21-34). In a recent comparison, we found that the prevalence of overt bone disease was nearly 100% in patients from India whereas it was < 1% a m o n g patients from Detroit; the only two patients with osteitis fibrosa cystica from the United States had severe vitamin D depletion (40). A similar observation was made when patients with primary hyperparathyroidism from New York were compared to patients from China (23). Other associated radiologic features, such as rickets and osteomalacia, the specific bone lesions of vitamin D deficiency, are frequently seen in patients with primary hyperparathyroidism from China and India (23,24,30,44). Such an occurrence is rarely, if ever, seen in patients from Europe and United States (see Chapters 18, 20, and 21). These comparative observations lead us to speculate that osteitis fibrosa cystica develops only in those patients with severe and prolonged vitamin D depletion, but not all patients with vitamin D depletion necessarily develop osteitis fibrosa cystica. The exceptions to this prediction would be patients with more aggressive tumors or parathyroid cancer. Severe generalized muscle weakness is another clinical feature that is more prevalent in the East and is likely a reflection of very high PTH concentrations in combination with severe vitamin D depletion (69). To the extent that the foregoing speculation is operative, it is reasonable to assume that a true change in the growth behavior of these tumors is possible. Indeed, the mean weight of the tumors in patients with contemporary primary hyperparathyroidism in India is similar to what was so c o m m o n in the West in 1930-1950 (1,6) (Fig. 7). Furthermore, for a comparable degree of vitamin D depletion, patients with primary hyperparathyroidism in India have larger tumors compared to their counterparts in the United States (40) (Table 4). The most important major public health policy that may have influenced parathyroid growth behavior as well as the disease expression is the addition of vitamin D to milk in the United States beginning in the late 1930s and early 1940s. A dramatic decrease both in parathyroid tumor weight (Fig. 7) and prevalence of osteitis fibrosa cystica followed implementation of this policy (53). Admittedly, it is difficult to separate the effect of routine biochemical screening programs introduced in the 1970s from the effect of fortification of milk with vitamin D initiated in the 1930-1940s on the disease severity or its expression. How vitamin D deficiency influences the growth of a parathyroid a d e n o m a is uncertain because most

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adenomas lack nuclear receptors for 1,25-dihydroxyvitamin D (70-72). We have demonstrated skeletal resistance to the calcemic action of PTH in patients with vitamin D depletion (53), and this may explain the larger tumor size. A potential alternative mechanism involving a membrane-bound vitamin D receptor is possible (73,74), but the existence of such a receptor in parathyroid gland has not yet been demonstrated. Nevertheless, it is tempting to draw an analogy with iodine deficiency and endemic goiter. Just as endemic goiters have virtually disappeared following iodination of common table salt, so has osteitis fibrosa cystica following fortification of milk with vitamin D. It will be of great interest to see whether such a change in clinical presentation of primary hyperparathyroidism occurs when better nutritional policies are implemented in the developing countries. Certainly, other factor(s) might conceivably influence the growth pattern of parathyroid tumors in patients with primary hyperparathyroidism from China, Brazil, and India, and perhaps from other developing countries. Such an exaggerated growth behavior of the abnormal parathyroid tissue might also explain a higher proportion of parathyroid cancers seen in patients with primary hyperparathyroidism from Brazil, China, and India.

RELEVANCE OF N I H G U I D E L I N E S F O R PATIENTS W I T H PRIMARY H Y P E R P A R A T H Y R O I D M IN T H E DEVELOPING COUNTRIES The 1991 National Institutes of Health (NIH) consensus guidelines were developed for the management of a growing numbers of patients with mild asymptomatic primary hyperparathyroidism (75). To the extent they are applicable to patients with this disease in other parts of the world, parathyroidectomy is appropriate for patients with symptomatic disease. However, to what extent these guidelines should be adopted for management of a potentially growing n u m b e r of patients with asymptomatic primary hyperparathyroidism in these countries is debatable (21). Because follow-up care is likely to be less rigorous, curative parathyroidectomy should be considered in all patients with primary hyperparathyroidism in the East. Lack of comprehensive monitoring programs in the developing nations and lack of suitable medical therapy make any conservative m a n a g e m e n t strategy less desirable. Until more information is available from long-term nonoperative followup of patients with asymptomatic primary hyperparathyroidism in the developing countries, parathyroidectomy should be r e c o m m e n d e d uniformly to all patients with this disease. However, access to expe-

rienced parathyroid surgeons in a country with low rates of recognition of the disease may dissuade many physicians and patients to take that option.

SUMMARY A N D CONCLUDING OBSERVATIONS Many unexpected insights have emerged from these comparative analyses. First, contrary to the popular belief, only the pattern of expression of primary hyperparathyroidism has changed significantly, but not the disease. Many of the similarities between traditional primary hyperparathyroidism of the West and contemporary primary hyperparathyroidism of the East can be explained by the differences in prevailing surveillance patterns and vitamin D and calcium nutrition of the populations. The higher prevalence of parathyroid cancers may be a reflection of an exaggerated growth behavior of abnormal parathyroid tissue partly influenced by vitamin D and calcium nutritional status of the individual. Based on the available information, it is reasonable to infer that more severe bone disease such as osteitis fibrosa cystica, brown tumors, and pathologic fractures are likely only when excess PTH secretion is accompanied by profound and prolonged vitamin D and calcium malnutrition. The only exception to this will be an occasional patient with parathyroid cancer. The effect of calcium and vitamin D nutrition of the population appears to have transcended the effect of age, gender, and menopausal status. Continued observations and comparisons of patients with primary hyperparathyroidism from different parts of the world are likely to provide even greater insights into this everchanging disease.

ACKNOWLEDGMENTS Partly supported by NIH grants DK 43858, DK 32333, AR 39191, and RR 00645. One of the authors (D. Sudhaker Rao) wishes to acknowledge the contributions of the following individuals: Dr. C. Gopalan, former Director of The National Institute of Nutrition, Hyderabad, AP, India, for introducing me to the field of vitamin D nutrition, Dr. S.ES. Teotia, former Head, Division of Endocrinology and H u m a n Metabolism, LLRM Medical College, Meerut, UP, India, for helpful discussions over the past 25 years with respect to primary hyperparathyroidism in India, and Dr. A. Michael Parfitt, for teaching me to pay attention to details and to question traditional views, and for helpful discussions during the preparation of this manuscript. Last, but not the least, the late Boy Frame, who nurtured me to be the best clinician that I can be.

HYPERPARATHYROIDISM IN

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5. Rao DS. Primary hyperparathyroidism: Changing patterns in presentation and treatment decisions in the eighties. Henry Ford Hospital MedJ 1985;33:194-197. 6. Albright F, Aub JC, Bauer W. Hyperparathyroidism: Common and polymorphic condition as illustrated by seventeen proven cases from one clinic. JAMA 1934;102:1276-1287. 7. Goldman L, Gordan GS, Chambers ELJ. Changing diagnostic criteria for hyperparathyroidism. Ann Surg 1957;146:407-416. 8. Keating RF, Jr. Diagnosis of primary hyperparathyroidism. JAMA 1961;178:547-555. 9. Rao DS, Frame B, Miller MJ, Kleerekoper M, Block MA, Parfitt AM. Hyperparathyroidism following head and neck irradiation. Arch Intern Med 1980;140:205-207. 10. Beard CM, Heath HI, O'Fallon M, AndersonJA, Earle JD, Melton LJI. Therapeutic radiation and hyperparathyroidism: A casecontrol study in Rochester, Minn. Arch Intern Med 1989; 149:1887-1890. 11. Belchetz EE Hormonal treatment of postmenopausal women. N EnglJ Med 1994;3302:1062-1071. 12. McKenna MJ. Differences in vitamin D status between countries in young adults and the elderly. Am J Med 1992;93:69-77. 13. Rao DS. Perspective on assessment of vitamin D nutrition. J Clin Densitometry 1999;2:457-464. 14. Parfitt AM, Rao DS, Kleerekoper M. Asymptomatic primary hyperparathyroidism discovered by multichannel biochemical screening: Clinical course and considerations bearing on the need for surgical intervention. J Bone Miner Res 1991 ;6:$97-S 101. 15. Rao DS, Wilson RJ, Kleerekoper M, Parfitt AM. Lack of biochemical progression or continuation of accelerated bone loss in mild asymptomatic primary hyperparathyroidism: Evidence for biphasic disease course. J Clin Endocrinol Metab 1988;67:1294-1298. 16. Wilson RJ, Rao DS, Ellis B, Kleerekoper M, Parfitt AM. Mild asymptomatic primary hyperparathyroidism is not a risk factor for vertebral fractures. Ann Intern Med 1988;109:959-962. 17. Parisien M, Silverberg SJ, Shane E, et al. The histomorphometry of bone in primary hyperparathyroidism: Preservation of cancellous bone structure. J Clin Endocrinol Metab 1990;70:930-938. 18. Silverberg sJ, Shane E, De La Cruz L, et al. Skeletal disease in primary hyperparathyroidism. JBone Miner Res 1989;4:283-291. 19. Silverberg sJ, Bilezikian JR Primary hyperparathyroidism: Still evolving ? JBone Miner Res 1997;12:856-862. 20. Rao DS. Primary hyperparathyroidism: Treatment issues. Mediguide 1997;4:1-7. 21. Bandeira F, Griz L, Caldas G, Macedo G, Bandeira C. Characteristics of primary hyperparathyroidism in one institution in northeast Brazil. Bone 1998;5:$380. 22. Cheung PSY, Boey JH, Wang CCL, Ma JTC, Lam KSL, Yeung RTT. Primary hyperparathyroidism: Its clinical pattern and results of surgical treatment in Hong Kong Chinese. Surgery 1988;103:558-562.

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44. Vaishnava H, Rizvi SN. Primary hyperparathyroidism associated with nutritional osteomalacia. A m J M e d 1969;46:640-644. 45. Gannage MH, Abikaram G, Nasr F, Awada H. Osteomalacia secondary to celiac disease, primary hyperparathyroidism, and Graves' disease. A m J M e d Sci 1998;315:136-139. 46. Turken SA, Cafferty M, Silverberg SJ, et al. Neuromuscular involvement in mild, asymptomatic primary hyperparathyroidism. A m J M e d 1989;87:553-557. 47. Joborn C,Joborn H, RastadJ, Akerstrom G, Ljunghall S. Maximal isokinetic muscle strength in patients with primary hyperparathyroidism before and after parathyroid surgery. Br J Surg 1988;75:77-80. 48. Joborn C, Rastad J, Stalberg E, Akerstrom G, Ljunghall S. Muscle function in patients with primary hyperparathyroidism. Muscle Nerve 1989;12:87-94. 49. Shieber W, Birge SJ, Avioli LV, Teitelbaum SL. Normocalcemic hyperparathyroidism with "normal" parathyroid glands. Arch Surg 1971;103:299-302. 50. Siperstein AE, Shen W, Chan AK, Duh QY, Clark OH. Normocalcemic hyperparathyroidism. Biochemical and symptom profiles before and after surgery. Arch Surg 1992;127:1157-1159. 51. Monchik JM. Normocalcemic hyperparathyroidism. Surgery 1995;118:917-923. 52. Clements MR, Davies M, Fraser DR, Lumb GA, Mawer EB, Adams PH. Metabolic inactivation of vitamin D is enhanced in primary hyperparathyroidism. Clin Sci 1987;73:659-664. 53. Rao DS, Honasoge M, Divine GW, et al. Effect of vitamin D nutrition on parathyroid adenoma weight: Pathogenetic and clinical implications. J Clin Endocrinol Metab 2000;85:1054-1058. 54. Sarda AK, Arunabh, Vijayaraghavan M, Kapur M. Paraplegia due to osteitis fibrosa secondary to primary hyperparathyroidism: Report of a case. Surg Today 1993;23:1003-1005. 55. Kulak CAM, Bandeira C, Voss D, et al. Marked improvement in bone mass after parathyroidectomy in osteitis fibrosa cystica. J Clin Endocrinol Metab 1998;83:732-735. 56. Block MA, Dailey GEI, Muchmore DE. Bone mineralization, a factor of increasing significance in the management of primary hyperparathyroidism. Surgery 1989;106:1063-1069. 57. Abugassa S, Nordenstrom J, Eriksson S, Mellerstrom G, Alveryd A. Skeletal remineralization after surgery for primary and secondary hyperparathyroidism. Surgery 1990;107:128-133. 58. Silverberg SJ, Locker FG, Bilezikian JE Vertebral osteopenia: A new indication for surgery in primary hyperparathyroidism. J Clin Endocrinol Metab 1996;81:4007-4012. 59. Kleerekoper M, Rao DS, Talpos GB, Bone HG. Management of asymptomatic primary hyperparathyroidism. Adv Endocrinol Metab 1991 ;2:205-220. 60. Antonelli R, Rao DS, Kleerekoper M, Nelson D, Parfitt AM. Incomplete recovery of bone mineral deficit after parathyroidectomy in primary hyperparathyroidism. Calcif Tissue Int 1990;46:144. 61. Nakaoka D, Sugimoto T, Kobayashi T, Yamaguchi T, Kobayashi A, Chihara K. Prediction of bone mass change after parathyroidec-

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69. Prabhala A, Garg R, Dandona E Severe myopathy associated with vitamin D deficiency in western New York. Arch Intern Med 1999;160:1199-1203. 70. Rao DS, Han Z-H, Phillips E, Palnitkar S, Parfitt AM. Loss of calcitriol receptor expression in parathyroid adenomas: Implications for pathogenesis. Clin Endocrino12000;in press. 71. Carling T, Rastad J, Akerstrom G, Westin G. Vitamin D receptor (VDR) and parathyroid hormone messenger ribonucleic acid levels correspond to polymorphic VDR alleles in human parathyroid tumors. J Clin Endocrinol Metab 1998;83:2255-2259. 72. Carling T, Rastad J, Szabo E, Westin G, Akerstrom G. Reduced parathyroid vitamin D receptor messenger ribonucleic acid levels in primary and secondary hyperparathyroidism. J Clin Endocrinol Metab 2000;85:2000-2003. 73. Nemere I, Schwartz Z, Pedrozo H, Sylvia VL, Dean DD, Boyan BD. Identification of a membrane receptor for 1,25-dihdroxyvitamin D 3 which mediates rapid activation of protein kinase C. J Bone Miner Res 1998;13:1353-1359. 74. Boyan BD, Sylvia VL, Dean DD, et al. 1,25-(OH),~D~ modulates growth plate chondrocytes via membrane receptor-mediated protein kinase C by a mechanism that involves changes in phospholipid metabolism and the action of arachidonic acid and PGE2. Steroids 1999;64:129-136. 75. Consensus Development Conference Panel. Diagnosis and management of asymptomatic primary hyperparathyroidism: Consensus Development Conference Statement. Ann Intern Med 1991;114:593-597.

CI4APTFa 23

Clinical Course of Primary Hyperparathyroidism

SHONNI

J.

SILVERBERG

New York, New York 10032

AND

JOHN

R BILEZIKIAN

Department of Medicine, College of Physicians and Surgeons, Columbia University,

INTRODUCTION

destined from their origin to grow rapidly and to produce a more severe disturbance of calcium homeostasis, whereas the rest were inherently less aggressive in their growth and clinical consequences. Patients with either type of primary hyperparathyroidism were easily identified at presentation and were routinely referred for parathyroidectomy at that time. These observations predated the introduction of automated biochemistry in medical practice, which heralded a new era for primary hyperparathyroidism (and many other disorders, such as hyperuricemia and hypercholesterolemia), in which a biochemical diagnosis could be firmly established in the absence of any relevant clinical symptoms. Today, the classic bone disease of primary hyperparathyroidism as described by Lloyd (type I) is quite rare. Many series also report a reduced prevalence of primary hyperparathyroidism a m o n g kidney stone formers (5), suggesting that type II primary hyperparathyroidism also has become less common. Previously, the evolution from the classic to the m o d e r n clinical presentations of primary hyperparathyroidism has been described. The question addressed in this chapter concerns what we know about the natural history of primary hyperparathyroidism as it presents today. Is the current p r e d o m i n a n t form of primary hyperparathyroidism simply a benign variant of early presentations (type I or II), or does it represent a new entity, as was postulated by Lloyd et al. (6), with its own natural history? One way to address this question is to characterize the typical presentation of the disease as it is seen today, along with its m o d e r n natural history.

The natural history of primary hyperparathyroidism as it used to be recognized is well d o c u m e n t e d in the early medical reports of this disease. Beginning with Mandl's Viennese tram conductor (1) and seaman Captain Charles Martel in Boston (2), the earliest patients came to medical attention invariably because of a severely progressive bone disease, osteitis fibrosa cystica. After the first 50 or so patients had u n d e r g o n e successful parathyroidectomy, Albright (3) observed that almost 80% of the patients also gave a history of recurrent nephrolithiasis. A diligent search in stoneforming patients uncovered many more patients with primary hyperparathyroidism who had no clear-cut evidence of osteitis fibrosa cystica or other metabolic bone disease. Serum calcium in these stone-forming patients tended to be lower than that seen in patients with the bone disease, and they were less prone to the neurologic complications of severe hypercalcemia. These observations led Lloyd (4) to classify primary hyperparathyroidism into two distinct types; type I, with a large gland, was associated with severe, symptomatic hypercalcemia, and with bone disease; type II, with a smaller gland, was associated with less severe hypercalcemia and with stone disease. Lloyd noted that, though many patients with type I disease gave a history of stones, there was apparently little overlap in the opposite direction. He postulated that the natural history of primary hyperparathyroidism was related to the growth of the tumorous parathyroid tissue; some tumors were

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CLINICAL COURSE OF CLASSIC MANIFESTATIONS OF PRIMARY HYPERPARATHYROIDISM

Biochemical Markers of Primary Hyperparathyroidism Calcium Levels without Surgery in

Primary Hyperparathyroidism

Not surprisingly, the mean level of serum calcium declined in the first several years after the introduction of automated biochemical profiling. Reports currently show most patients to have serum calcium levels within 1 m g / d l of the u p p e r limits of the normal range (6-8). As has been discussed in earlier chapters, primary hyperparathyroidism appears to result from a resetting of the level at which the parathyroid glands respond to ambient serum calcium. Once this has developed, further derangements of this set point do not generally occur. Serum calcium shows very minor fluctuations over many years of observation (Fig. 1) (8,9). The abnormal serum calcium is so well regulated in primary hyperparathyroidism that patients who develop a second illness that might independently be associated with hypercalcemia, e.g., thyrotoxicosis or granulomatous disease, do not appear to experience worsening of

-

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FIG. 1 Serum calcium and parathyroid hormone levels in 60 patients with primary hyperparathyroidism followed without intervention.

the hypercalcemia. Similarly, there is no apparent adverse effect of normal dietary calcium intake on the serum calcium levels in primary hyperparathyroidism (10). Tohme et al. (11) did report, however, that the rise in serum calcium in response to a fixed oral calcium load was slightly greater in primary hyperparathyroidism compared to normal, and there was less suppression of circulating levels of amino-terminal parathyroid h o r m o n e (PTH) fragment. Nonetheless, it is probably unwise to restrict dietary calcium intake in patients with primary hyperparathyroidism, in that this could result in a "secondary" hyperparathyroidism, with increased PTH synthesis and secretion. Though not aggravating the hypercalcemia, it could potentially exaggerate any adverse effect of the PTH on the skeleton, which, in the absence of adequate dietary calcium, becomes the major source of calcium in the circulation. Data from our group suggest that dietary calcium intake can be safely liberalized in those patients with normal levels of 1,25-dihydroxyvitamin D 3 (10). In patients with elevated levels of 1,25-dihydroxyvitamin D3, high dietary calcium intake can be associated with increasing urinary calcium excretion. T h o u g h intercurrent illness and dietary calcium intake have little effect on serum calcium in primary hyperparathyroidism, this is not the case for several therapeutic agents. The relationship of thiazide diuretics and lithium to the pathogenesis a n d / o r detection of primary hyperparathyroidism is well reported but poorly understood. Some studies have suggested that both these drugs cause the de novo development of PTH hypersecretion and primary hyperparathyroidism. Others have suggested that patients receiving these drugs have very mild primary hyperparathyroidism that is aggravated or "unmasked" by the therapy. Ideally, the diagnostic evaluation for possible primary hyperparathyroidism should be deferred until the patient has not taken thiazides or lithium for 3 months. This may not be practical for patients on lithium, for which there are fewer alternative medications. Thiazides cause a mild rise in the serum calcium, usually <0.05 mmol/liter, apparently by inhibiting urinary calcium excretion. This may be sufficient in some patients to unmask hypercalcemia. In patients with known primary hyperparathyroidism, it is appropriate to withhold thiazides whenever possible, although in one study it was not possible to detect any significant difference in either albumin-corrected or ionized calcium for periods in which patients were on or off thiazides (12). It should be added parenthetically that thiazides may in fact be no less risky for a patient with primary hyperparathyroidism than more potent loop diuretics, which have a greater tendency to cause dehydration and to worsen hypercalcemia, on that basis.

CLINICAL COURSE OF HYPERPARATHYROIDISM / A cause-and-effect relationship between lithium therapy for affective disorders and the development of hyperparathyroidism is more clearly established. Several series have shown that there is a durationdependent tendency for lithium-treated patients to develop hypercalcemic hyperparathyroidism, with the usual history being one of therapy for 10 years or more (13,14). Additionally, there is an apparently greater likelihood of parathyroid hyperplasia or multiple adenoma at parathyroidectomy in lithium-treated patients (13,15) than is seen in either sporadic primary hyperparathyroidism or radiation-induced primary hyperparathyroidism (16). Saxe and Gibson (17) demonstrated that lithium increases tritiated thymidine uptake in dispersed cells taken from patients with parathyroid adenoma (not treated with lithium prior to the diagnosis of primary hyperparathyroidism). Other studies using dispersed bovine parathyroid cells have indicated that lithium results in a decrease in low calcium-stimulated PTH release but a potentiation of PTH release at physiologic concentrations of extracellular calcium (18). In a perfusion experiment using normal parathyroid tissue from patients undergoing thyroid surgery and hyperplastic parathyroid tissue removed from patients with renal osteodystrophy, Birnbaum et al. (19) demonstrated an increased release of intact PTH under normocalcemic conditions. This could not be demonstrated in human parathyroid tissue obtained from patients with single-gland adenoma. Following a single oral dose of 600 mg lithium carbonate to nine normal subjects, Seely et al. (20) demonstrated a significant rise in serum levels of intact PTH but no significant change in the serum ionized calcium levels. Mallette et al. (21) demonstrated that, compared to normal subjects, patients with affective disorders treated with lithium for < 6 months had a higher mean serum ionized calcium, but there were no differences in PTH. In subjects treated with lithium for >3 years, plasma PTH and parathyroid gland volume were significantly higher than in controls. Thus chronic lithium therapy appears to stimulate parathyroid cell growth and PTH secretion, leading, after long-term therapy, to hypercalcemic parathyroid hyperplasia, i.e., primary hyperparathyroidism. However, as is the case with thiazide therapy, there is no evidence that lithium therapy per se alters the natural history of the disease in patients with sporadic adenomatous primary hyperparathyroidism.

Course of Other Biochemical Indices in

389

roidism patients (8,9). The serum phosphorus tends to be in the lower range of normal whereas frank hypophosphatemia is present in less than one-quarter of patients. Average total urinary calcium excretion is at the upper end of the normal range, with less than half of all patients having hypercalciuria. Serum 25-hydroxyvitamin D levels tend to be in the lower end of the normal range. Though mean values of 1,25-dihydroxyvitamin D~ are in the high normal range, approximately one-third of patients have frankly elevated levels of this important hormone. Data on long-term stability of PTH in this form of primary hyperparathyroidism were, until recently, quite limited and difficult to interpret because of frequent changes in the methods for measuring serum PTH. A decade of data now available using the highly sensitive immunoradiometric assay (IRMA) assay suggest that PTH levels are stable as well (Fig. 1). This could be predicted from the observed set-point change but the otherwise normal relationship between serum calcium and PTH synthesis and secretion and the long-term stability of serum calcium in this disease. As was discussed above, lithium-induced primary hyperparathyroidism may be an exception to this general observation, along with those rare cases of acute hypercalcemic crises complicating seemingly mild, asymptomatic primary hyperparathyroidism. There is no evidence that mild primary hyperparathyroidism is associated with progressive renal impairment, at least as measured by serum creatinine, blood urea nitrogen, or endogenous creatinine clearance. With regard to skeletal involvement, serum alkaline phosphatase, both total and bone specific, is also stable during several years of follow-up. This suggests lack of progression of PTH-mediated bone disease, which is discussed in detail below. It must be noted that although groups of patients with primary hyperparathyroidism exhibit remarkable stability of biochemical indices, a small proportion of patients do have evidence of disease progression over time (8) (Fig. 2). Although no clinically overt complication (i.e., fracture, kidney stone, etc.) occurred in a decade of observation, 4% of our asymptomatic patients developed substantial worsening of their hypercalcemia (serum calcium > 12mg/dl), and 15% developed marked hypercalciuria (urinary calcium excretion > 400 mg/day). There were no demographic or biochemical predictors of disease progression identified. Therefore, close follow-up of these parameters is necessary in all patients who do not undergo surgery.

Primary Hyperparathyroidism

Course of Biochemical Indices Following Surgery

There is ample documentation that the other biochemical abnormalities so characteristic of primary hyperparathyroidism are also stable during long-term follow-up of mild, asymptomatic primary hyperparathy-

Following parathyroidectomy, there is a prompt normalization of serum and urinary calcium levels, as well as a return of parathyroid h o r m o n e concentrations to normal. Several possible exceptions to this observation

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are noteworthy. One is the patient with significant hyperparathyroid bone disease. These individuals can develop a "hungry bone syndrome." After surgery calcium is rapidly deposited into the remineralizing skeleton. This situation may be associated with symptomatic hypocalcemia, requiring intravenous calcium administration. Though a common postoperative feature of classical primary hyperparathyroidism, this condition is very rarely seen in the United States today. A second situation in which calcium and PTH levels may not be normal following parathyroidectomy is that of the patient with coexisting primary hyperparathyroidism and vitamin D deficiency (22). Once the primary hyperparathyroidism is cured, these patients may become hypocalcemic and show persisting elevations in parathyroid hormone levels (secondary hyperparathyroidism). It is of interest that, despite the lack of overt skeletal manifestations, the total serum alkaline phosphatase concentration is often mildly elevated. In addition, more specific markers of bone formation (bone specific alkaline phosphatase and osteocalcin) as well as markers of bone resorption (collagen cross-links, pyridinoline, and deoxypyridinoline) can be elevated (23-28). Studies of bone markers in the longitudinal follow-up of patients with primary hyperparathyroidism are limited, but indicate a reduction in these markers of bone turnover following parathyroidectomy. Although the choice of markers in the individual studies differed, our group (29), Guo et al. ( 3 0 ) , and Tanaka et al. (31) all report declining levels of bone markers following surgery. Data are also available concerning the kinetics of change in bone resorption versus bone formation fol-

lowing parathyroidectomy. Markers of bone resorption decline rapidly following successful parathyroid surgery, but indices of bone formation decline more gradually (29). Urinary pyridinoline and deoxypyridinoline fell as early as 2 weeks following parathyroidectomy, preceding reductions in alkaline phosphatase. Similar data were reported from Tanaka et al. (31 ), who demonstrated a difference between changes in osteocalcin and urinary N-telopeptide following parathyroidectomy, and Minisola et al., who reported a decrease in bone resorption markers without any significant change in alkaline phosphatase or osteocalcin (32). The persistence of elevated bone formation markers coupled with rapid declines in bone resorption markers indicates a shift in the coupling between bone formation and bone resorption toward an anabolic accrual of bone mineral after surgery. Increases in bone density after surgery provide support for this idea.

Parathyroid Bone Disease Osteitis fibrosa cystica, the classic form of parathyroid bone disease, is rarely encountered in primary hyperparathyroidism today. However, patients with asymptomatic primary hyperparathyroidism do exhibit bone involvement if this is sought diligently. A small proportion of patients, well under 10%, will have radiographic evidence of hyperparathyroidism if hand radiographs are examined with a magnifying glass for subperiosteal bone resorption (33). Many more patients, possibly the majority, can be shown to have skeletal demineralization at sites containing cortical

CLINICAL C O U R S E OF HYPERPARATHYROIDISM

bone when investigated with the most sensitive noninvasive methods for measuring bone mass, namely, bone densitometry (34,35). Using bone densitometry in mild primary hyperparathyroidism, we and others have shown a preferential loss of cortical bone, with relative sparing of cancellous bone (36-40). These results support not only the notion that parathyroid h o r m o n e is catabolic at cortical sites, but also the view that parathyroid horm o n e is anabolic at cancellous sites. In postmenopausal women, in whom estrogen deficiency would normally lead to cancellous bone loss, the same pattern was observed (42,43). The relative preservation of cancellous bone in women who are vulnerable to cancellous bone loss by virtue of estrogen deficiency illustrates the anabolic potential of parathyroid h o r m o n e at cancellous sites. Histomorphometric studies of patients with mild primary hyperparathyroidism confirm these findings. Both static and dynamic parameters give a picture of cortical thinning, with maintenance of cancellous bone volumes (see Chapter 26). In addition, as expected from the available bone marker data, histom o r p h o m t e r i c analysis illustrates a very dynamic process associated with high turnover and accelerated bone remodeling. It should be noted that not all patients demonstrate the typical densitometric profile, in which there is relative preservation of skeletal mass at the vertebrae and diminution at the more cortical distal radius. Though this pattern is evident in the vast majority of patients, a small group of patients have evidence of vertebral osteopenia at the time of presentation. In our natural history study, approximately 15% of patients had a lumbar spine z-score less than - 1 . 5 at the time of diagnosis (41).

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Longitudinal Course Most patients with mild, asymptomatic primary hyperparathyroidism do not appear to have progressive loss of bone, if the disease is left untreated (8,9). Data from our group and others show remarkable stability in bone density over time in patients with mild disease (Fig. 3). Only 12% of patients had a progressive decline in bone mineral density to the point that they met National Institutes of Health Consensus Conference Guidelines for Surgery (radius z-score < - 2 ) . One possible explanation for this comes from histomorphometric data showing that patients with primary hyperparathyroidism do not show the expected age-related decline in indices of trabecular connectivity. Thus, despite advancing age, patients with primary hyperparathyroidism continue to have well-connected trabecular plates. Surgery, on the other hand, leads to marked increases in bone mass (8,42,43). Data are now

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C~TF~R23

available from our group spanning a decade of postoperative follow-up (8). Parathyroidectomy leads to a 10-12% rise in bone density at the lumbar spine and femoral neck (Fig. 3). The increase at the lumbar spine and femoral neck was prompt, with the greatest increment in the first postoperative year. The trend toward a further increase after year 1 was significant only at the femoral neck (paired t-tests; years 1 to 4, P = 0.02; years 1 to 7 and 1 to 10, P = 0.03). The increase at the lumbar spine and femoral neck sites, which contain a significant a m o u n t of cancellous bone, is sustained over a decade after surgery, despite the tendency of advancing age to decrease bone density over time. Lumbar spine and femoral neck bone mineral density increased to the same extent in the subgroup of 28 postmenopausal women. Postmenopausal women showed a similar pattern of increased cancellous bone density. This is a curious observation in view of the fact that the lumbar region, enriched in cancellous bone, appears to be relatively well protected by PTH in primary hyperparathyroidism. The higher turnover rate of cancellous bone and the filling in, postoperatively, of the expanded remodeling space at this region could account for at least part for these observations. In the group as a whole, there was no significant change in bone mineral density at the distal radius (Fig. 3). This is consistent with earlier data suggesting that, at least to some extent, PTH-mediated cortical bone loss may be irreversible. We have, however, found an increase in distal one-third radius bone mineral density in patients with greatest demineralization at this site (43). In patients with primary hyperparathyroidism who have vertebral osteopenia or osteoporosis at the time of diagnosis, the postoperative increase in vertebral bone density is even greater, averaging 20% after parathyroidectomy (41). The marked improvement seen in patients with low vertebral bone density argues for surgery in those who present with cancellous as well as cortical bone loss. The data on fracture incidence in primary hyperparathyroidism remain controversial. An early paper by Dauphine et al. reported an increased prevalence of vertebral fractures in patients with mild primary hyperparathyroidism, but several other studies failed to confirm this observation (44-47). When vertebral fracture is the starting point for case finding studies, primary hyperparathyroidism is rare, although measurement of serum calcium is r e c o m m e n d e d as part of the routine screening for newly diagnosed cases of osteoporosis. In one series including over 600 consecutive patients referred to a metabolic bone disease clinic for evaluation of bone loss with or without vertebral fracture (48), no case of primary hyperparathyroidism was discovered. Similarly, J o h n s o n et al. (49) did not find a single case of primary hyperparathyroidism in more than 300 consec-

utive cases of osteoporosis. It is reasonable to conclude that this form of primary hyperparathyroidism does not predispose patients to osteoporosis at the spine and may in fact offer some protection against it. Interestingly, Melton et al. (50) reported that patients with primary hyperparathyroidism had sustained more fractures prior to the diagnosis than did a control population, but during more than 1000 person-years of follow-up after the diagnosis of primary hyperparathyroidism was made, survival without new fracture was the same in the two groups. This is in keeping with the observation that bone loss appears to occur prior to the diagnosis of primary hyperparathyroidism but that, once the diagnosis is established, accelerated bone loss is not seen. One possible explanation for this observation is that the skeleton is the source of the extra calcium in the circulation as a new steady state is being established during the initial stages of primary hyperparathyroidism. According to this hypothesis, once primary hyperparathyroidism is established, bone mass is relatively well maintained by bone formation dynamics that more closely match those associated with resorption. There are insufficient data to form any valid conclusions about a relationship between PTH-mediated cortical bone loss and an increased occurrence of long bone or proximal femur fractures. It would seem logical to anticipate this consequence of cortical bone loss, but primary hyperparathyroidism is not a dominant feature in any series of hip fracture patients, and hip fractures are not a d o m i n a n t feature in any series of elderly patients with primary hyperparathyroidism. In a study that considered hip fracture, a populationbased prospective analysis (mean of 17 years duration; 23,341 person years) showed women with primary hyperparathyroidism in Sweden not to be at increased risk (47). This strongly suggests that, as is the case with primary hyperparathyroidism and vertebral fractures, long bone and proximal femur fractures are not seen commonly in untreated primary hyperparathyroidism. Another population-based study reported an increased incidence of vertebral, Colles', rib, and pelvic (but not hip) fractures in patients who were being followed with primary hyperparathyroidism in Rochester, Minnnesota (50a). The result with respect to vertebral fractures is somewhat unexpected, given the relative preservation of bone density at this site, based on densitometry testing. Selection bias in the determination of vertebral fractures could explain these fifldings. The skeleton of patients who carry the diagnosis of primary hyperparathyroidism is monitored far more carefully than most, and vertebral fractures might be diagnosed with increased frequency in this closely observed population, in whom back pain is more likely to lead to an X-ray. On the other hand, it is unlikely that a Colles' fracture, a more expected consequence of the hyper-

CLINICAL COURSE OF HYPERPARATHYROIDISM /

parathyroid process in bone, would be asymptomatic. Thus the increased incidence of forearm fractures, at a site containing more cortical bone, is consistent with these expectations.

Clinical Course of Stone Disease in Primary Hyperparathyroidism Stone disease in primary hyperparathyroidism is covered in Chapter 27. Kidney stones remain the principal clinical complication of this disease (51). Estimates in some studies place the incidence of kidney stones at 15-20% of all patients. The etiology of nephrolithiasis in primary hyperparathyroidism is certainly multifactorial (52-55). Other renal manifestations of primary hyperparathyroidism include hypercalciuria, which is seen in approximately 40% of patients, and nephrocalcinosis, the frequency of which is unknown. Nephrolithiasis is a well-accepted indication for surgery in primary hyperparathyroidism. Urinary calcium excretion is reduced following surgery. Surgery is also of clear benefit in reducing the incidence of recurrent nephrolithiasis. Over 90% of patients with stone disease and hyperparathyroidism do not form additional stones after parathyroid surgery. The 5-10% of patients who continue to form kidney stones after parathyroidectomy are thought to have a second, alternative cause for their stone disease, which persists despite cure of their hyperparathyroidism (8,56,57). Those patients with nephrolithiasis who do not undergo surgery are at risk for recurrent stone disease and progressive hyperparathyroidism. In our report (8) on a decade of follow-up of patients with primary hyperparathyroidism with and without surgery, 20 patients with kidney stones were included (Fig. 2). No recurrence of nephrolithiasis was reported in the 12 patients who underwent successful parathyroidectomy. The 8 patients who refused, or had undergone unsuccessful surgery, experienced a less sanguine course. Recurrent kidney stones were reported in 6 of the 8 patients, and the remaining 2 patients had other evidence o f worsening hyperparathyroidism.

COURSE OF N O N T R A D I T I O N A L SYMPTOMS AND SIGNS OF PRIMARY HYPERPARATHYROIDISM Primary hyperparathyroidism detected as part of a routine biochemical screening program is typically asymptomatic. For many subjects, however, symptoms not completely unrelated to primary hyperparathyroidism may be present. Thus asymptomatic primary hyperparathyroidism could be described as two entities:

393

(1) totally asymptomatic and (2) accompanied by manifestations not traditionally linked either to primary hyperparathyroidism or to hypercalcemia. The true prevalence of totally asymptomatic primary hyperparathyroidism can be determined only by measuring serum calcium and parathyroid hormone in all members of a selected cohort and ascertaining the proportion of identified patients who are asymptomatic. This may never be known. It would, however, be of interest to assess the natural history of the disease in this group separately from the natural history of the disease in patients diagnosed on the basis of symptoms.

Neuropsychiatric Manifestations Anecdotal information suggests that many patients with primary hyperparathyroidism discovered in the course of biochemical screening may have symptoms that are vague and nonspecific. Nonetheless, some authorities believe that they do result from primary hyperparathyroidism and are alleviated by parathyroidectomy. These nonspecific symptoms can be classified under the general rubric of neuropsychiatric disorders: pain, weakness, lassitude, anxiety, and depression (58). Because this has been a particularly difficult area to study in a quantitative manner, the literature has not led to clear conclusions. In 1987, Kleerekoper and colleagues reported on 34 patients with primary hyperparathyroidism. Formal psychiatric interview and neuropsychologic testing was performed (59). Follow-up observations were obtained in 10 patients 6 months after successful parathyroidectomy and after 6 months of conservative follow-up without surgery in 9 patients. Despite the expected significant reduction in serum calcium following parathyroidectomy, and improvement in some neurobehavioral abnormalities, they observed no improvement in functioning for those behavioral domains that were correlated with the serum calcium at the time of initial testing. Cogan et al. (60) made similar observations in seven subjects undergoing parathyroidectomy for primary hyperparathyroidism. Most had abnormal psychologic tests preoperatively, with no improvement when these patients were retested an average of 3.6 months after surgery. Joborn and colleagues (61) reported self-rated psychiatric symptoms in 30 patients with primary hyperparathyroidism referred for surgery, 38 subjects detected in a health screening with 15 years of mild hypercalcemia and presumed primary hyperparathyroidism, and 38 normocalcemic control subjects. Using the Hopkins Symptom Checklist (HSCL-56) scores improved 1 year postoperatively. In the nonoperated subjects and in the controls, 6 months of oral vitamin D (1 mg oL-calcidiol daily) or placebo had no effect on the HSCL score, despite a

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0.05 mmol/liter increase in serum calcium in the vitamin D-treated group (compared to placebo). McMlion and Paterson (62) reported reduced psychiatric morbidity 3 months after parathyroidectomy. An additional 21 patients with primary hyperparathyroidism of 2-7 years' duration, regarding whom an independent decision had been made to follow without surgery, underwent the same standardized psychiatric interview. The results were not different from the postoperative results in the I treated group. In the entire group of 37 unoperated patients with primary hyperparathyroidism, there was no relationship between the psychiatric findings and either serum calcium or parathyroid h o r m o n e levels. The study of Solomon et al. is perhaps the best attempt at studying this problem in a prospective manner (63). Using the SL-149 rating scale, she observed a constellation of neuropsychologic abnormalities, most of which improved after successful surgery. The control group, subjects who underwent neck surgery for removal of a thyroid nodule, had similar postoperative improvement. This study could, therefore, not clearly attribute the improvement in symptomatology to cure of hyperparathyroidism or to the effects of a successful surgical procedure. Overall, the prevalence of neuropsychiatric abnormalities in patients with primary hyperparathyroidism appears to be greater than the prevalence of similar complaints in outpatient medical settings (64). The reported prevalence varies widely, ranging from 1 to 65% in differing studies. It is clear that formal studies of these neurobehavioral aspects of primary hyperparathyroidism must become an integral part of planned studies evaluating the natural history primary hyperparathyroidism and the effect of parathyroidectomy. However, formal psychiatric consultation of psychometric testing for individual patients is not recommended outside the confines of such research studies. Furthermore, because in most expert opinion, we cannot be sure whether these nonspecific aspects of primary hyperparathyroidism will be improved with surgery, they should not constitute an indication for surgery by themselves.

Hypertension When primary hyperparathyroidism is complicated by renal impairment, or severe hypercalcemia, or is present as part of a multiple endocrine neoplasia (MEN) syndrome that includes either pheochromocytoma or hyperaldosteronism, hypertension is common. Hypertension has also been reported to be more prevalent in mild, asymptomatic primary hyperparathyroid patients than in appropriately matched control groups (65-67). There have been reports of amelioration of

hypertension following successful parathyroidectomy (65,66,68), but this has not been a universal observation (69-72). Moreover, it has been extremely difficult to show a cause-and-effect relationship between primary hyperparathyroidism and hypertension. Brinton et al. (73) reported a small series of subjects with primary hyperparathyroidism and hypertension with and without increased plasma renin activity. Amelioration of hypertension was observed only in the subset of patients in whom renin activity normalized. Ganguly et al. (74) could not confirm any association between the renin-angiotensin-aldosterone system and hypertension in primary hyperparathyroidism. Extensive experience suggests that hypertension is not cured after parathyroid surgery, nor is it easier to control. Therefore, recommendations for or against surgery should not be based on hypertension alone.

Cardiovascular Effects The effect of primary hyperparathyroidism on the formation of calcifications in the myocardium, heart valves, and coronary arteries parameters is unclear. Stefanelli et al. (76) prospectively studied 64 Austrian patients with primary hyperparathyroidism and found that myocardial calcifications were markedly increased in affected individuals (69% of patients vs. 17% of controls). Valvular calcifications were also seen in a significantly higher percentage of patients than controls (63% at the aortic, and 49% at the mitral valve). Regression in left ventricular hypertrophy was seen in nonhypertensive patients 1 year after successful parathyroid surgery. The results of this study, however, may have limited applicability to patients with the mild form of primary hyperparathyroidism observed most commonly in the United States today, because the population described by Stefanelli was more severely affected.

Other Nontraditional Aspects of Primary Hyperparathyroidism Attempts to link carbohydrate intolerance and frank diabetes mellitus to primary hyperparathyroidism have been made (76,77), but the association is even more tenuous than the association between hypertension and primary hyperparathyroidism (78). Peptic ulcer disease and pancreatitis do not appear to be part of the syndrome of mild primary hyperparathyroidism (79-85). The neuromuscular complications of classic primary hyperparathyroidism are not seen in the mild form of the disease. In a detailed neurologic study of 42 patients with a mean serum calcium of 11.1 _+ 0.1 mg/dl, Turken et al. (86) found no consistent pattern

CLINICAL COURSE OF HYPERPARATHYROIDISM /

of abnormalities either on physical examination or on electromyography. Joborn et al. (87) studied 18 randomly selected patients with primary hyperparathyroidism and concluded that, as a group, patients had slight but significant impairment of muscle function, a finding that the authors speculated might be responsible for the "fatigue" that is apparently so prevalent in this disease.

Asymptomatic Primary Hyperparathyroidism and Cancer There are several reports of an increased occurrence of nonparathyroid cancers in patients with primary hyperparathyroidism (88,89). Many are subject to selection bias. In patients with hypercalcemia detected unexpectedly on a biochemical profile, the most important cause of the hypercalcemia to exclude is hypercalcemia associated with malignancy. Thus the association between primary hyperparathyroidism and cancer may simply reflect the more diligent search for cancer in patients with hypercalcemia. Another possible mechanism for a chance association between primary hyperparathyroidism and cancer results from the frequency with which clinically silent thyroid malignancies are found during the neck exploration for parathyroidectomy (90,91). A study of survival following the diagnosis of primary hyperparathyroidism found no increased mortality in those diagnosed in the current era of mild hyperparathyroidism (92).

Acute Hypercalcemic Crisis Acute hypercalcemic crisis can occur in patients with long-standing, mild primary hyperparathyroidism. This rare complication is generally seen in the setting of a patient (often elderly) developing an acute, unrelated illness complicated by dehydration. Nonetheless, 25% of patients with acute primary hyperparathyroidism have a history of mild hypercalcemia, often of many years duration (93,94) (see also Chapter 34).

MORTALITY Data supporting an increase in mortality rates in patients with primary hyperparathyroidism come from a retrospective study of limited size from Scandinavia (95). These data have not been confirmed in a study from the United States (92). In a population-based study of hyperparathyroidism in Rochester, Minnesota over a 28-year period, no increase in mortality was seen in 435 patients with surgically confirmed hyperparathyo roidism, hypercalcemia with inappropriately elevated

395

parathyroid hormone levels, or unexplained hypercalcemia for more than a 1-year duration. It is possible that the change in clinical profile to a largely asymptomatic disease is responsible for the apparent reduction in mortality rates. Consistent with this notion, in the aforementioned survival study, higher maximal serum calcium level was found to be an independent predictor of mortality. Thus the very mild elevations in serum calcium found today could account for the improved survival rates.

SUMMARY AND CONCLUSIONS The earliest clinical descriptions of primary hyperparathyroidism point to an inexorably progressive, crippling, and sometimes fatal metabolic bone disease. Later reports highlighted nephrolithiasis as a common feature of this disease, and it subsequently became apparent that more patients with primary hyperparathyroidism in fact have stone disease without prominent skeletal involvement. Three decades ago, the introduction of automated, screening biochemical profiles that included the serum calcium concentration led to the "chance" discovery of primary hyperparathyroidism in large numbers of patients who had no clinical evidence of either metabolic bone or stone disease, so-called asymptomatic primary hyperparathyroidism. This review has developed the hypothesis that this constitutes a distinct clinical entity, with few clinical features in common in classic primary hyperparathyroidism, and little evidence of progression to this phenotype except in very rare cases. Asymptomatic patients appear to remain asymptomatic during many years of observation. In those patients with symptoms, the major clinical manifestation is nephrolithiasis. Many more patients, however, complain of a constellation of vague and nonspecific neurobehavioral symptoms that were not traditionally thought to be related to primary hyperparathyroidism. The prevalence of these symptoms is greater than in control populations, which may suggest a cause-andeffect relationship to primary hyperparathyroidism. However, most patients are not predictably improved by successful parathyroidectomy. Finally, although patients no longer have clinically overt bone disease, bone densitometry indicates that the skeleton remains an important target organ in primary hyperparathyroidism. The disease is characterized by disproportionate loss of cortical bone, with preservation of cancellous bone. The clinical consequences of this are unclear, because fracture data confirming protection from vertebral fractures or increases in long bone or hip fractures are not available to date.

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Interestingly, the clinical, biochemical, and bone densitometric manifestations of mild primary hyperparathyroidism appear to be present when the disease is first discovered in an individual patient, but show little evidence of progression if the disease remains untreated. Thus the natural history of primary hyperparathyroidism, once the diagnosis has been established, is one of very mild clinical manifestations, with little objective or subjective evidence of progression if left untreated. A small group of patients do have laboratory evidence of worsening disease, with gradually increasing levels of serum or urinary calcium, or decreasing bone mineral density. Because there are no predictors for patients at risk for progressive disease, all patients followed without surgery must have regular monitoring. Surgical cure of primary hyperparathyroidism, on the other hand, leads to biochemical normalization and increased bone density at sites of cancellous bone.

ACKNOWLEDGMENTS Supported in part by National Institutes of Health grants NIDDK 32333, NIAMS 39191, and RR 00645.

REFERENCES 1. Mandl E Therapeutiscle Versuch bei Ostitis fibrosa generalisata mittels Extirpation lines Epithelk6perchentumon. Wien Klin Wochenschr 1925;50:1343-1344. 2. Cope O. The story of hyperparathyroidism at the Massachusetts General Hospital. N FnglJ Med 1966;21:1174-1182. 3. Albright E Page out of history of hyperparathyroidism. J Clin Endocrinol Metab 1948;8:637-657. 4. Lloyd HM. Primary hyperparathyroidism: An analysis of the role of the parathyroid tumor. Medicine 1968;47:53-71. 5. Fischer JA, Bronner F Coburn J. Parathyroid hormone. In: Bronner, Coburn, eds. Disorders of mineral metabolism, Vol 2. New York:Academic Press, 1982:271-358. 6. Parfitt AM, Rao DS, Kleerekoper M. Asymptomatic primary hyperparathyroidism discovered by multichannel biochemical screening: Clinical course and considerations bearing on the need for surgical intervention. J Bone Miner Res 1991 ;6 (Suppl. 2):$97-S101. 7. Bilezikian JP, Silverberg SJ, Shane E, Parisien M, Dempster DW. Characterization and evaluation of asymptomatic primary hyperparathyroidism. J Bone Miner Res 1991 ;6 (Suppl. 2):585-$89. 8. Silverberg SJ, Shane E, Jacobs TP, Siris E, Bilezikian JE The natural history of treated and untreated asymptomatic primary hyperparathyroidism: A ten year prospective study. N Engl J Med 1999;341:1249-1255. 9. Rao DS, Wilson RJ, Kleerekoper M, Parfitt AM. Lack of biochemical progression or continuation of accelerated bone loss in mild asymptomatic primary hyperparathyroidism: Evidence for biphasic disease course. J Clin Endocrinol Metab 1988 ;67:1294-1298. 10. Locker 11. Tohme JE Bilezikian JR Clemens TL, Silverberg SJ, Shane E, Lindsay R. Suppression of parathyroid hormone secretion with

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CLINICAL COURSE OF HYPERPARATHYROIDISM 32. Minisola S, Romagnoli E, Scarnecchia L, et al. Serum CITP in patients with primary hyperparathyroidism: Studies in basal conditions and after parathyroid surgery. Eur J Endocrinol 1994; 130:587-591. 33. Sancho JJ, Rouco J, Riera-Vida R, Sitges-Serra A. Long-term effects of parathyroidectomy for primary hyperparathyroidism on arterial hypertension. WorldJ Surg 1992;16:732-736. 34. Bilezikian JP, Silverberg SJ, Shane E, Parisien M, Dempster DW. Characterization and evaluation of asymptomatic primary hyperparathyroidism. J Bone Miner Res 1991;6(Suppl. 1):585-589. 35. Silverberg SJ, Shane E, DeLaCruz L, et al. Skeletal disease in primary hyperparathyroidism. J Bone Miner Res 1989;4:283-291. 36. Parisien MV, Silverberg SJ, Shane E, de la Cruz L, Lindsay R, Bilezikian JP, Dempster DW. The histomorphometry of bone in primary hyperparathyroidism: Preservation of cancellous bone structure. J Clin Endocrinol Metab ] 990;70:930-938. 37. van Doorn L, Lips P, Netelenbos JC, Hackengt WHL. Bone histomorphometry and serum intact PTH (1-84) in hyperparathyroid patients. Calcif Tissue Int 1989;44S:N36. 38. Parisien M, Mellish RWE, Silverberg SJ, et al. Maintenance of cancellous bone connectivity in primary hyperparathyroidism: Trabecular strut analysis. J Bone Miner Res 1992;7:913-920. 39. Parisien M, Cosman F, Mellish RWE, Schnitzer M, Nieves J, Silverberg SJ, Shane E, Kimmel K, Recker R, Bilezikian JP, Lindsay R, Dempster DW. Bone structure in postmenopausal hyperparathyroid, osteoporotic and normal women. JBone Miner Res 1995;10:1393-1399. 40. Dempster DW, Parisien M, Silverberg SJ, Liang X-G, Schnitzer M, Shen V, Shane E, Kimmel DB, Recker R, Lindsay R, Bilezikian JE On the mechanism of cancellous bone preservation in postmenopausal women with mild primary hyperparathyroidism. J Clin Endocrinol Metab 1999;84:1562-1566. 41. Silverberg SJ, Locker FG, Bilezikian JE Vertebral osteopenia: A new indication for surgery in primary hyperparathyroidism. J Clin Endocrinol Metab 1996;81:4007-4012. 42. Martin P, Bermann P, Sillet C. Partially reversible osteopenia after surgery for primary hyperparathyroidism. Arch Intern Med 1986; 146:689-691. 43. Silverberg sJ, Gartenberg F, Jacobs TP, et al. Increased bone mineral density following parathyroidectomy in primary hyperparathyroidism. J Clin Metab 1995;80:729-734. 44. Dauphine RT, Riggs BL, Scholz DA. Back pain and vertebral crush fractures: An unrecognized mode of presentation for primary hyperparathyroidism. Ann Intern Med 1975;83:365-367. 45. Wilson RJ, Rao DS, Ellis B, Kleerekoper M, Parfitt AM. Mild asymptomatic primary hyperparathyroidism is not a risk factor for vertebral fractures. Ann Intern Med 1988;109:959-962. 46. Lafferty FW, Halsay CA. Primary hyperparathyroidism: A review of the long-term surgical and non-surgical morbidities as a basis for a rational approach to treatment. Arch Intern Med 1986;149:789-796. 47. Larsson K, Ljunghall S, Krusemo UB, Naessen T, Lindh E, Persson I. The risk of hip fractures in patients with primary hyperparathyroidism: A population-based cohort study with a follow-up of 19 years. Jlntern Med 1993;234:585-593. 48. Kleerekoper M, Peterson E, Nelson D, et al. Identification of women at risk for developing postmenopausal osteoporosis with vertebral compression fractures: Role of history and single photon absorptiometry. Bone Miner 1989;7:171-186. 49. Johnson BE, Lucasey B, Robinson RG, Lukert BE Contributing diagnoses in osteoporosis. Arch Intern Med 1989;149:1069-1072. 50. Melton III LJ, Atkinson EJ, O'Fallon WM, Heath III H. Risk of age-related fractures in patients with primary hyperparathyroidism. Arch Intern Med 1992;152:2269-2273. 50a.Khosla S, Melton LJ, Wermers RA, Crowson CS, O'Fallon WM, Riggs BL. Primary hyperparathyroidism and the risk of fracture: A population-based study. J Bone Miner Res 1999;14:1700-1707.

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51. Silverberg SJ, Shane E, Jacobs TP, Siris ES, Gartenberg F, Seldin D, Clemens TL, Bilezikian JE Nephrolithiasis and bone involvement in primary hyperparathyroidism, 1985-1990. Am J Med 1990;89:327-334. 52. Pak CYC, Ohata M, Lawrence EC, Snyder W. The hypercalciurias: Causes, parathyroid functions and diagnostic criteria. J Clin Invest 1974;54:387-391. 53. Kaplan RA, Haussler MR, Deftos LJ, Bone H, Pak CYC. The role of 1,25(OH)2D in the mediation of intestinal hyperabsorption of calcium in primary hyperparathyroidism and absorptive hypercalciuria. J Clin Invest 1977;59: 756-760. 54. Broadus AE, Horst RL, Lang R, Littledike ET, Rasmussen H. The importance of circulating 1,25(OH)zD in the pathogeneisis of hypercalciuria and renal stone formation in primary hyperparathyroidism. N EnglJ Med 1980;302:421-426. 55. Pak CYC, Holt K. Nucleation and growth of brushite and calcium oxalate in urine of stone formers. Metabolism 1976;25:665-673. 56. Deaconson TF, Wilson SD, Lemann J. The effect of parathyroidectomy on the recurrence of nephrolithiasis. Surgery 1987;215:241-251. 57. Kaplan RA, Snyder WH, Stewart A, et al. Metabolic effects of parathyroidectomy in asymptomatic primary hyperparathyroidism. J Clin Endocrinol Metab 1976;42:415-426. 58. Joborn C, Hetta J, Johansson H, Rastad J, Agren H, Akerstrom G, Ljunghall S. Psychiatric morbidity in primary hyperparathyroidism. WorldJ Surg 1988;12:476-481. 59. Brown GG, Preisman RC, Kleerekoper MD. Neurobehavioral symptoms in mild primary hyperparathyroidism: Related to hypercalcemia but not improved by parathyroidectomy. Henry Ford MedJ 1987;35:211-215. 60. Cogan MG, Covey CM, Arieff AI, Wisniewski A, Clark OH. Central nervous system manifestations of hyperparathyroidism. Am J Med 1978;65:963-970. 61. Joborn C, Hetta J, Lind L, Rastad J, Akerstrom G, Ljunghall S. Self-rated psychiatric symptoms in patients operated on because of primary hyperparathyroidism and in patients with longstanding mild hypercalcemia. Surgery 1989;105:72-78. 62. McAllion SJ, Paterson CR. Psychiatric morbidity in primary hyperparathyroidism. Postgrad Med J 1989;65:628-631. 63. Solomon BL, Schaaf M, Smallridge RC. Psychologic symptoms before and after parathyroid surgery. A m J M e d 1994;96:101-106. 64. Cavanaugh S, Wettstein RM. Prevalence of psychiatric morbidity in medical populations. In: Grinspoon L, ed. Psychiatry update, the American Psychiatric Association annual review, Vol 3. Washington, D.C.:American Psychiatric Press, 1984;187-215 and 279-281. 65. Diamond TW, Botha JR, Wing J, Meyers AM, Kalk WJ. Parathyroid hypertension. A reversible disorder. Arch Intern Med 1986;146:1709-1712. 66. Broulik PD, Horky K, Pacovsky V. Blood pressure in patients with primary hyperparathyroidism before and after parathyroidectomy. Exp Clin Endocrinol 1985;86:346-352. 67. Nainby-Luxmoore JC, Langford HG, Nelson NC, Watson RL, Barnes TY. A case-comparison study of hypertension and hyperparathyroidism. J Clin Endocrinol Metab 1982;55:303-306. 68. Ringe JD. Reversible hypertension in primary hyperparathyroidism-pre- and postoperative blood pressure in 75 cases. Klin Wochenschr 1984;62:465-169. 69. Jones DB, Jones JH, Lloyd HJ, Lucas PA, Wilkins WE, Walker DA. Changes in blood pressure and renal function after parathyroidectomy in primary hyperparathyroidism. Postgrad Med J 1983;59:350-353. 70. Bradley III EL, Wells JO. Primary hyperparathyroidism and hypertension. Am Surg 1983;49:569-570. 71. Rapado A. Arterial hypertension and primary hyperparathyroidism. Am J Nephrol 1986;6 (Suppl. 1) :49-50.

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72. Dominiczak AF, Lyall E Morton JJ, et al. Blood pressure, left ventricular mass and intracellular calcium in primary hyperparathyroidism. Clin Sci 1990;78:127-132. 73. Brinton GS, Jubiz W, Lagerquist LD. Hypertension in primary hyperparathyroidism: The role of renin-angiotensin system. J Clin Endocrinol Metab 1975;41:1025-1029. 74. Ganguly A, Weinberger MH, Passmore JM, et al. The renin angiotensin-aldosterone system and hypertension in primary hyperparathyroidism. Metabolism 1982;31:595-600. 75. Taylor WH. The prevalence of diabetes mellitus in patients with primary hyperparathyroidism and among their relatives. Diabet Med 1991 ;8:683-687. 76. Stefenelli T, Mayr H, Bergler-Klein J, Globits S, Woloszczuk W, Niederle B. Primary hyperparathyroidism: Incidence of cardiac abnormalities and partial reversibility after successful parathyroidectomy. Am J Med 1993;95:197-202. 77. Ljunghall S, Palmer M, Akerstrom G, Wide L. Diabetes mellitus, glucose tolerance and insulin response to glucose in patients with primary hyperparathyroidism before and after parathyroidectomy. EurJ Clin Invest 1983;13:373-377. 78. Bannon ME vanHeerden JA, Palumbo PJ, Ilstrup DM. The relationship between primary hyperparathyroidism and diabetes mellitus. Ann Surg 1988;207:430-433. 79. Linos DA, vanHeerden JA, Abboud CE Edis AJ. Primary hyperparathyroidism and peptic ulcer disease. Arch Surg 1978;113:384-386. 80. Watson RG, vanHeerden JA, Grant CS, Klee GG. Postoperative hypermylasemia, pancreatitis, and primary hyperparathyroidism. Surgery 1984;96:1151-1157. 81. Sitges-Serra A, Alonso M, deLecea C, Gores PF, Sutherland DE. Pancreatitis and hyperparathyroidism. BrJ Surg 1988;75:158-160. 82. Prinz RA, Aranha GV. The association of primary hyperparathyroidism and pancreatitis. Am Surg 1985;51:325-329. 83. vanLanschot JJ, Bruining HA. Primary hyperparathyroidism and pancreatitis. Netherlands J Surg 1984;36:38-41.

84. Paloyan D, Simonowitz D, Paloyan E, Snyder TJ. Pancreatitis associated with primary hyperparathyroidism. Am Surg 1982;48: 366-368. 85. Bess MA, Edis AJ, van Heerden JA. Hyperparathyroidism and pancreatitis. Chance or a causal association? JAMA 1980;243:246-247. 86. Turken SA, Cafferty M, Silverberg SJ, et al. Neuromuscular involvement in mild, asymptomatic primary hyperparathyroidism. AmJMed 1989;87:553-557. 87. Joborn C, Rastad J, Stalberg E, Akerstrom G, Ljunghall S. Muscle function in patients with primary hyperparathyroidism. Muscle Nerve 1989;12:87-94. 88. Wajngot A, Werner S, Granberg PO, Lindvall N. Occurrence of pituitary adenomas and other neoplastic diseases in primary hyperparathyroidism. Surg Gynecol Obstet 1980; 151:401-403. 89. Farr HW, Fahey TJ, Jr, Nash AG, Farr CM. Primary hyperparathyroidism and cancer. A m J Surg 1973;126:539-543. 90. Attie JN, Vardhan R. Association of hyperparathyroidism with nonmedullary thyroid carcinoma: Review of 31 cases. Head Neck 1993;15:20-23. 91. Kambouris AA, Ansari MR, Talpos GT. Primary hyperparathyroidism and associated neoplasms. Henry Ford Med J 1987;35:207-210. 92. Wermers RA, Khosla S, Atkinson EJ, Grant CS, Hodgson SF, O'Fallon M, Melton LJ. Survival after the diagnosis of hyperparathyroidism. AmJMed 1998;104:115-122. 93. Fitzpatrick LA, Bilezikian JE Acute primary hyperparathyroidism. A m J Med 1987;82:275-282. 94. Corsello SM, Folli G, Crucitti F, et al. Acute complications in the course of "mild" hyperparathyroidism. J Endocrinol Invest 1991;14:971-974. 95. Palmer M, Adami H-O, Bergstrom R, Akerstroom G, Ljunghall S. Mortality alter surgery for primary hyperparathyroidism: A follow-up of 441 patients operated on from 1956 to 1979. Surgery 1987;102:1-7.

CHAPTER

24

Molecular Markers of Bone Metabolism in Parathyroid Disease

MARKUS

Germany

j.

S E I B E L Division of Endocrinology and Metabolism, Department of Internal Medicine I, University of Heidelberg, 69115 Heidelberg,

Fo ati:on

INTRODUCTION

Parathyroid h o r m o n e (PTH), one of the principal regulators of bone metabolism, influences bone formation and bone resorption via both direct and indirect actions. It is therefore not surprising that acute or chronic disturbances of parathyroid gland function and PTH secretion result in more or less p r o n o u n c e d changes of bone turnover. In turn, these changes may lead to alterations in bone mass and structure and ultimately to the development of PTH-induced bone disease. In recent years, characterizations of cellular and extracellular c o m p o n e n t s of the skeletal matrix have led to the development of biochemical markers that specifically reflect either bone formation or bone resorption (Fig. 1). These new markers have greatly enriched the spectrum of analytes used in the assessm e n t of skeletal pathologies. In addition to the classic m a r k e r of bone formation, i.e., serum total alkaline phosphatase (sTAP), a host of other markers such as the bone-specific isoenzyme of alkaline phosphatase (sBAP), osteocalcin (sOC), and collagen propeptides and are now being used to assess osteoblast activity (Table 1). These newer markers exhibit a significantly higher degree of tissue specificity than does sTAP. Bone resorption, traditionally assessed by m e a s u r e m e n t of the urinary excretion of calcium and hydroxyproline, can now be m o n i t o r e d m o r e effectively by new serum and urine markers. A m o n g these, the most i m p o r t a n t ones are the pyri-

The Parathyroids, Second Edition

BAP Collage propept

dine dne-Glycosides ium crosslinks aked collagen tides

Osteoc~

Osteoblasts

Bone matrix

Osteoclasts

FIG. 1 Schematic representation of the currently used biochemical markers of bone metabolism. BAP, bone specific alkaline phosphatase; TRAP, tartrate-resistant acid phosphatase; BSP, bone sialoprotein (all measured in serum); OH-, hydroxy.

dinium cross-links and the cross-linked telopeptides of type I collagen. However, markers based on noncollageneous proteins, such as bone sialoprotein or tartrateresistant acid phosphatase, are gaining increasing relevance as specific markers of osteoclast activity (Table 2). This chapter reviews the currently used indices of bone formation and resorption and the effects of parathyroid disease on molecular markers of bone turnover.

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Marker (abbreviation)

Tissue of origin

Markers of Bone Formation Analytic specimen

Analytic method

Specificity

Bone, liver, intestine, kidney, placenta Bone

Serum

Colorimetric

Specfic for bone formation only in the absence of liver or biliary disease

Serum

Osteocalcin (OC, BGP)

Bone, platelets

Serum

Colorimetric, electrophoretic, precipitation, IRMA, EIA RIA, ELISA, CLIA

Carboxy-terminal propeptide of type I procollagen (PICP) Amino-terminal propeptide of type I procollagen (PINP)

Bone, soft tissue, skin Bone, soft tissue, skin

Serum

RIA, ELISA

Specific product of osteoblasts; some assays show up to 20% cross-reactivity with liver isoenzyme Specific product of osteoblasts; many immunoreactive forms in blood; some may be derived from bone resorption Specific product of proliferating osteoblasts and fibroblasts

Serum

RIA, ELISA

Total alkaline phosphatase (AP, TAP, total ALP)

Bone-specific alkaline phosphatase (BAR bone ALP)

MOLECULAR MARKERS OF B O N E METABOLISM The various markers of bone turnover include enzymes, nonenzymatic peptides, and mineral components, and they are usually classified according to the metabolic process they are considered to reflect (Fig. 1). For clinical purposes, therefore, markers of bone formation are distinguished from indices of bone resorption (Tables 1 and 2). Biochemical markers of bone turnover are noninvasive, relatively inexpensive, and, when applied and interpreted correctly, helpful tools in the assessment of metabolic bone disease as well as patient response and compliance with therapeutic interventions. It should be borne in mind, however, that some of these markers may reflect, at least to a certain degree, both bone formation and bone resorption. Furthermore, most if not all of these markers are present in tissues other than bone and may therefore be influenced by nonskeletal processes as well. Last, changes in biochemical markers of bone turnover are usually not disease specific, but reflect alterations in skeletal metabolism that are i n d e p e n d e n t of the underlying cause.

Markers o f B o n e Formation M1 markers of bone formation are direct products of active osteoblasts and are measured in serum or

Specific product of proliferating osteoblast and fibroblasts; partly incorporated into bone extracellular matrix

plasma. The most commonly used formation markers are alkaline phosphatase, osteocalcin, and the type I collagen propeptides. Mkaline phosphatase (AP) is a ubiquitous, membraneb o u n d enzyme that is located on the outer cell surface. The precise function of the enzyme is as yet unknown, but it certainly plays an important role in osteoid formation and mineralization. The total AP serum pool consists of several isoforms, coded by four gene loci, which are synthesized in various tissues: liver, bone, intestine, spleen, kidney, and placenta (1,2). Bone alkaline phosphatase is encoded by the tissue nonspecific alkaline phosphatase gene, which is also expressed in liver and kidney. In adults with normal liver function, approximately 50% of the total AP activity in serum is derived from the liver and 50% arises from bone (3). In children and adolescents the bone-specific isoenzyme predominates (up to 90%) because of skeletal growth (4). Utilizing the different physicochemical properties of the various AP isoenzymes, a n u m b e r of techniques have been developed to differentiate between the bone and liver isoforms (e.g., heat denaturation, electrophoresis, precipitation, selective inhibition, and immunoassays). However, all techniques show a certain degree of cross-reactivity between bone and liver AP. Therefore, in subjects with liver disease bone AP measurements may be artificially high due to elevated liver AP, and can lead to incorrect interpretations (5).

MOLF~CUk~ MARKERSOF BONE METABOLISM / TABLE 2

Marker (abbreviation)

Tissue of origin

Markers of Bone Resorption Analytic specimen

Analytic method

Hydroxyproline, total and dialyzable (OH-Pro, OHP)

Bone, cartilage, soft tissue, skin

U rine

Colorimetric, HPLC

Pyridinoline (PYD, Pyr)

Bone, cartilage, tendon, blood vessels Bone, dentin

Urine serum

HPLC, ELISA RIA

Urine serum

HPLC, ELISA RIA

Carboxy-terminal cross-linked telopeptide of type I collagen (ICTP)

Bone, skin

Serum

RIA

Carboxy-terminal cross-linked telopeptide of type I collagen (oL-CTX, 13-CTX) Amino-terminal cross-linked telopeptide of type I collagen (NTX) Hydroxylysine-glycosides

All tissues containing type I collagen All tissues containing type I collagen Bone, soft tissue, skin, serum complement

Urine (od13), serum (13 only) Urine, serum

ELISA, RIA

Urine

HPLC

Bone sialoprotein (BSP)

Bone, dentin, hypertrophic cartilage

Serum

RIA, ELISA

Tartrate-resistant acid phosphatase (TRAP)

Bone, blood

Plasma, serum

Colorimetric, RIA, ELISA

Deoxypyridinoline (DPD, d-Pyr)

401

Due to its wide availability, total serum AP is the most often used marker of bone formation. In the absence of severe liver disease, sTAP provides a good index of the extent of new bone formation and osteoblast activity (6,7). In parathyroid bone disease, however, measurement of the more sensitive bone-specific isoenzyme is increasingly preferred. Osteocalcin (OC), also known as bone Gla protein (BGP), is a hydroxyapatite-binding protein exclusively synthesized by osteoblasts, odontoblasts, and hypertrophic chondrocytes (8). A major feature of OC is three vitamin-K dependent, ~/-carboxyglutamic acid (Gla) residues, which are responsible for the calciumbinding properties of the protein (9). Although these and other functions imply a role for OC in the organi-

ELISA, RIA, ECLA

Specificity All fibrillar collagens and partly collagenous proteins, including Clq and elastin; present in newly synthesized and mature collagen Collagens, with highest concentrations in cartilage and bone; absent from skin; present in mature collagen only Collagens, with highest concentration in bone; absent from cartilage or skin; present in mature collagen only Collagen type I, with highest contribution probably from bone; may be derived from newly synthesized collagen Collagen type I, with highest contribution probably from bone Collagen type I, with highest contribution probably from bone Collagens and collagenous proteins; glycosylgalactosylOHLys in high proportion in collagens of soft tissues, and Clq; galyctosyI-OHLys in high proportion in skeletal collagens Synthesized by active osteoblasts and laid down in bone extracellular matrix; appears to reflect osteoclast activity Osteoclasts (5b), platelets, erythrocytes

zation of the extracellular matrix, the precise function of OC has yet to be determined. Despite this fact, OC is considered a specific index of osteoblast function (10). Serum levels of immunoreactive OC have been shown to correlate well with the bone formation rate as assessed by histomorphometry (11,12). However, the peptide is subject to rapid degradation in serum, so that both intact OC and fragments of various sizes are present in the circulation (13). Furthermore, because OC is incorporated into the bone matrix, some investigators have suggested that OC fragments may be released during bone resorption (14). The ensuing heterogeneity of OC fragments in serum results in considerable limitations in the clinical application of this a priori highly specific marker. Thus, the various assays used to

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measure OC in serum detect fragments of various sizes, and usually epitope specificity and antibody crossreactivity of the assays are ill defined. I n practice, different immunoassays have routinely yielded such varying results in measurements that interassay comparisons are not possible (15). The procollagen type I propeptides are fragments observed during the synthesis of type I collagen, the most commonly occurring collagen of bone (16). Like all fibrillar collagens, type I collagen is synthesized in the form of a preprocollagen, a precursor molecule that contains a short signal sequence and that undergoes processing to yield terminal extension peptides: the amino (N)-terminal propeptide (PINP) and the carboxy (C)-terminal propeptide (PICP) (17). After secretion of the procollagen into the extracellular space, the globular trimeric propeptides are enzymatically cleaved (18) and carried into the circulation (19). Because both PICP and PINP are generated from newly synthesized collagen in a stoichiometric fashion, the propeptides are considered quantitative measures of newly formed type I collagen. Both propeptides are currently measured by specific immunoassays that utilize polyclonal antibodies. Different studies have shown good correlations between serum PICP levels and the rate of bone formation or serum TAP activity (20). From a practical point of view, the thermostability of the propeptides is an advantage in that extended transport and frozen storage are well tolerated without significant loss of immunoreactivity. The propeptides share these properties with most of the parameters of collagen metabolism [e.g., cross-links (ICTP, NTx, and CTx; see Table 2), hydroxyproline].

Markers of Bone Resorption The majority of bone resorption markers are degradation products of bone collagen (i.e., hydroxyproline, galactosyl-hydroxylysine, type I collagen cross-links, type I collagen telopeptides). Exceptions to this rule are noncollageneous proteins such as bone sialoprotein (BSP) or osteoclast-specific enzymes such as tartrateresistant acid phosphatase (TRAP) (Fig. 1). Hydroxyproline (OHP) is formed intracellularly from the posttranslational hydroxylation of proline and constitutes 12-14% of the total amino acid content of mature collagen. Of the O H P liberated during degradation of bone collagen 90% is metabolized primarily in the liver (21). Subsequently, it is excreted in the urine, where it may be detected either as free or peptide-bound hydroxyproline by colorimetric or highperformance liquid chromatography (HPLC) methods (22). Urinary O H P is usually considered an index of bone resorption. However, it should be noted that sig-

nificant amounts of urinary OHP are derived from the degradation of newly synthesized collagens (23). In addition, hydroxyproline can be found in other tissues, such as the skin (24), and, moreover, is liberated from the metabolism of elastin and C l q (25). Because certain foodstuffs (e.g., meat) contain considerable amounts of OHP, measurements in urine are useful only when subjects adhere to a hydroxyproline-free diet. Urinary O H P is therefore considered a rather unspecific index of bone resorption and, consequently, has been largely replaced by more specific markers. Like hydroxyproline, the hydroxylysine-glycosides are formed during the posttranslational modification of collagen and are incorporated into the bone matrix. The component is liberated into the circulation as collagen is degraded and can be measured in the urine after appropriate derivatization (26). Hydroxylysine normally exists in two glycosylated forms, namely, glycosyl-galactosylhydroxylysine (GGHL) and galactosyl-hydroxylysine (GHL) (27). Whereas GGHL is present in skin and Clq, GHL is more specific for bone. Thus determination of the ratio of G G H L / G H L may enhance bone specificity. Importantly, the gycosylated forms are not metabolized and are not influenced by dietary factors (25,27). Although the hydroxylysines have potential as markers of bone resorption (28,29), the major limitation to their use is the lack of a convenient immunoassay format. The pyridinium cross-links of collagen, pyridinoline (PYD) and deoxypyridinoline (DPD), are formed during the extracellular maturation of fibrillar collagens (19,30,31). During bone resorption, cross-linked matrix collagens are proteolytically broken down, which leads to the release of the cross-link components into the circulation and from there into the urine. In contrast to OHP, concentrations of PYD and DPD in serum or urine are not influenced by the degradation of newly synthesized collagens and are not affected by dietary proteins (32). Both PYD and DPD specifically reflect the degradation of mature (i.e., cross-linked) collagens, and thus processes of collagen breakdown ("process specificity"). This theoretically predicted specificity has been confirmed experimentally by close correlations between cross-link excretion and histomorphometric or dynamic parameters of bone resorption (33). In addition, the pyridinium cross-links show a high specificity for skeletal tissues. PYD is found in cartilage, bone, ligaments, and vessels, whereas DPD is almost exclusively found in bone. Neither derivative is present in the collagen of skin or in other sources such as C l q or elastin (25,34,35). Because bone has a much higher turnover than cartilage, ligaments, vessels, or tendons, the amounts of PYD and DPD in serum or urine are mainly derived from the skeleton. Thus, the pyridinium cross-links are currently considered as excellent indices for assessing bone resorption.

MOLECULAR MARKERSOF BONE METABOLISM / Initially, PYD and DPD were quantified in urine by a reverse-phase ion-paired HPLC technique (36) (Fig. 2). Although this technique is somewhat labor intensive, it is still considered the gold standard for the measurem e n t of pyridinium cross-links. For routine purposes, however, direct immunoassays for the free (nonpeptide-bound) cross-links were developed and a number of studies have shown that these assays produce results that are similar to those provided by the HPLC technique (37,38). The aminoterminal or carboxyterminal telopeptides of type I collagen, i.e., the specific regions of the collagen molecule involved in cross-linking, have also been targets for assay development. Assays for both of these molecules are available: a radioimmunoassay (RIA) for the carboxyterminal type I collagen telopeptide (serum ICTP) (39); immunoassays that measure the carboxyterminal telopeptide of type I collagen in urine or serum (urinary/serum CTx) (40); and immunoassays measuring the cross-linked N-terminal telopeptide of type I collagen in serum or urine ( s e r u m / u r i n e NTx) (41). All immunoassays show reasonable correlations with each other, and the analytes seem to be stable at room temperature and during up to three freeze-thaw cycles (42). Bone sialoprotein is a phosphorylated glycoprotein with an apparent molecular mass of 70-80 kDa and accounts for 5-10% of the noncollageneous matrix of bone (43). The protein is a major synthetic product of active osteoblasts and odontoblasts, but is also found in osteoclast-like and malignant cell lines (44,45). Consequently, BSP or its mRNA is detected mainly in mineralized tissue such as bone, dentin, and at the interface of calcifying cartilage (45). Intact BSP contains an Arg-Gly-Asp (RGD) integrin recognition

Pyd

403

sequence, improves the attachment of osteoblasts and osteoclasts to plastic surfaces (46), binds preferentially to the % chain of collagen (47), nucleates hydroxyapatite crystal formation in vitro, and appears to enhance osteoclast-mediated bone resorption (48). The protein is therefore considered to play an important role in cell matrix adhesion processes and in the supramolecular organization of the extracellular matrix of mineralized tissues. Immunoassays have been developed that measure immunoreactive bone sialoprotein in serum (49,50). Children and adolescents exhibit greatly elevated values, particularly during the growth spurt (51). Based on clinical data and the rapid reduction of serum BSP levels following intravenous bisphosphonate treatment, it is assumed that serum BSP reflects processes mainly related to bone resorption (52). Tartrate-resistant acid phosphatase belongs to a large family of ubiquitous enzymes, of which at least five different isoforms are known. These isoforms are expressed by different tissues and cells such as prostate, bone, spleen, platelets, erythrocytes, and macrophages. All acid phosphatases are inhibited by (+)-tartrate, except band 5 which was therefore termed tartrateresistant acid phosphatase. Of the latter, two subforms, 5a and 5b, are known, and research has shown that TRAP-5b is characteristic for osteoclasts (53,54). Most assays for the m e a s u r e m e n t of TRAP in blood have been colorimetric and have detected both isoforms without differentiating between bands 5a and 5b. Specific immunoassays have been developed, however, and clinical results indicate that TRAP-5b may be useful to assess osteoclast activity (55).

BIO CHEMICAL MARKERS OF B O N E T U R N O V E R IN PRIMARY HYPERPARATHYROIDISM

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Elution time (min) FIG. 2 Reverse-phase ion-pair HPLC elution profile of a normal, unhydrolyzed urine sample. The three sequential peaks for glycosylated pyridinoline (GIc.GaI-Pyd), pyridinoline (Pyd), and deoxypyridinoline (Dpd) are shown. IS, Internal standard. (Courtesy of Dr. Simon Robins, Aberdeen, UK.)

Until some 20 years ago, primary hyperparathyroidism (PHPT) was usually diagnosed on the basis of clinical signs and symptoms, such as r e c u r r e n t kidney stones a n d / o r painful skeletal changes (osteitis fibrosa cystica). Since the introduction of multichannel analyzers and routine m e a s u r e m e n t of serum calcium concentrations, the diagnosis of PHPT has increasingly become based on the unexpected finding of hypercalcemia. Consequently, today most patients with newly diagnosed PHPT have few or no symptoms. Although evidence of skeletal changes are typically absent on conventional radiographs, bone densitometry and histom o r p h o m e t r y demonstrate significant cortical bone loss in many of these otherwise asymptomatic patients (56). It is therefore not surprising that many patients with oligosymptomatic or even asymptomatic PHPT

404

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CHAPTER24

exhibit moderate but significant changes in bone turnover when sensitive techniques are employed. As expected from the biologic actions of PTH, both bone formation and bone resorption are accelerated.

Bone Formation Alkaline Phosphatases Due to its limited skeletal specificity, serum total alkaline phosphatase is usually normal or only slightly elevated in cases of mild PHPT (56-59). In contrast, serum concentrations of the bone specific alkaline phosphatase (sBAP) are elevated in most cases, including patients with asymptomatic PHPT (58,60-61). However, direct comparisons and receiver operating characteristic (ROC) analysis between sBAP and sTAP have shown that sBAP has no greater diagnostic validity for PHPT than do measurements of sTAP (62,63). In one study of 16 patients with PHPT, z scores of sTAP were higher (5.93 _ 4.4) than those of sBAP (3.53 + 1.3) (64). In general, good agreements are found between sTAP and sBAP with r values between 0.4 and 0.8, d e p e n d i n g on the population studied. By comparison, correlations between AP and serum intact PTH are usually less pronounced. Some studies (59,104) have found inverse correlations between sTAP levels and bone mineral density in patients with PHPT (r = -0.49; p < 0.05), whereas others have not (56,64,65). One study found a significant correlation between serum BAP levels and ultrasound measures (bone ultrasound attenuation, speed of sound, stiffness) in a small group of patients with PHPT (104). Serum TAP and BAP decrease within 6 to 12 months after successful parathyroidectomy in patients with PHPT (66,105).

Osteocalcin Various authors have described elevated values of serum osteocalcin in patients with PHPT (59,61,64, 67-72). Some studies investigating a variety of metabolic bone diseases have described positive correlations between osteoblast activation (as reflected by sOC) and serum PTH levels (60,73,74). However, these findings, as well as covariation between sOC and bone mineral density (BMD) at several sites, were not confirmed in a larger prospective study of patients with PHPT (56,65,104). Like other specific markers of bone turnover, sOC decreases within 2-12 months after successful parathyroidectomy in patients with PHPT (74,105).

Procouagen Propep tides Few studies have analyzed type I collagen propeptides in patients with PHPT (75-78). Serum concentra-

tions of the carboxy-terminal propeptide of type I procollagen (sPICP) are usually elevated in PHPT, and rise transiently after successful parathyroidectomy (76,78). However, in two other studies of PHPT, levels of sPICP (79,104) and sPINP were found to be normal (104). Healthy subjects infused with h u m a n PTH (1-38) for 24 hours showed an increase in bone resorption markers, and a significant decrease in sPICP levels (79). The decrease in sPICP correlated with sBAP, but not with sOC, suggesting an a p p a r e n t dissociation between different osteoblast activities. In another study, 4 weeks of clodronate treatment was used to induce secondary hyperparathyroidism in nine healthy subjects. Although levels of NTX, a telopeptide marker of bone resorption, fell, sPICP did not show any change in response to short-term bisphosphonate treatment (80). In studies investigating a variety of metabolic bone diseases, sPICP levels correlated with mineral apposition and bone formation rates, as well as with activation frequency (r = 0.53-0.61) (81,82). Taken together, it appears that the serum levels of collagen propeptides show only modest changes in response to hyperparathyroidism. In the absence of direct comparisons between markers of osteoblast activity (TAP, BAP, OC) and collagen formation (PICE PINP), no conclusion is possible on the diagnostic validity of collagenbased markers in patients with mild or asymptomatic PHPT.

Bone Resorption Hydroxyproline and Hydroxylysine-Glycosides The urinary excretion of hydroxyproline (uOHP) is usually normal in mild PHPT, whereas in more severe cases this marker of total body collagen degradation may be frankly elevated. The few existing reports show no relationship between u O H P and serum calcium or parathyroid h o r m o n e levels (56,59,83,84). The urinary excretion of OHP decreases significantly 1-2 months after successful parathyroidectomy (105). A somewhat more sensitive technique may be the determination of urinary hydroxylysine-glycosides (uOHGly). In one study, 12 patients with PHPT had elevated levels of uOHGly, but only 8 had abnormal results for u O H P (83). Following intravenous bisphosphonate treatment, levels of u O H P and uOHGly fell by 31 and 55%, respectively. Similarly, the change in uOHGly was more significant than the reduction in u O H P following parathyroidectomy. In the same study, uOHGly but not u O H P correlated with serum calcium and parathyroid h o r m o n e levels (83). However, probably due to the lack of a convenient immunoassay format, uOHGly is not routinely used as a marker of bone resorption.

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Collagen Cross-Links In contrast to other bone markers, a large n u m b e r of studies have analyzed the usefulness of urinary pyridinium cross-links in PHPT and other metabolic bone diseases. Regardless of the techniques used for their determination, both total or free pyridinoline (uPYD) and total or free deoxypyridinoline (uDPD) are significantly elevated in patients with mild or asymptomatic PHPT (66,85-88) (fig. 3). In a small group of patients with osteoporosis or PHPT, uDPD levels were shown to correlate moderately with certain histomorphometric

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FIG. 3 Comparison of serum (S) and urinary (U) markers of bone resorption in various metabolic bone diseases. OPO, Osteoporosis; PHPT, primary hyperparathyroidism; PD, Paget's disease of bone; MM, multiple myeloma; B C - , breast cancer without bone metastases; BC+, breast cancer with bone metastases. Assays: DPD, deoxypyridinoline; BSP, bone sialoprotein; CTX, C-terminal telopeptide of type I collagen; NTX, N-terminal telopeptide of type I collagen. Shown are box-and-whisker-plots, where the horizontal line within the box represents the group median. Circles are individual values above and below the 95th percentile, respectively. All values are z values as compared to a healthy, agematched population. The zero (solid)line represents the mean of the control group; the dotted lines denote the upper and lower limit of normal, respectively (+_ 2 SD). Level of significance: p versus controls: ,, < 0.05; **, < 0.01; .... < 0.001. Reproduced from Ref. 88, J. Bone Miner. Res. 1999;14:792-801 with permission of the American Society for Bone and Mineral Research.

indices of bone resorption (89). A similar correlation (approximately r - 0.5) was observed in clinical studies between uDPD and serum PTH levels. Very similar findings have been reported for the "telopeptide" markers of bone resorption, sICTP and uNTX (Fig. 5). All three assays reveal accelerated bone resorption in patients with mild PHPT (79,80,90). In addition, an inverse correlation between uNTX and bone mineral density at the level of the l u m b a r spine and the ultradistal radius has been shown (90). Healthy subjects infused with h u m a n PTH(1-38) for 24 hours show a significant increase in sICTP levels that is corre-

406

/

CHAPTER24

lated with serum calcium levels and changes in other markers of osteoclast activity (79). Histomorphometric studies in patients with a variety of metabolic bone diseases (including PHPT) found moderate correlations between sICTP levels and volume-referent resorption rates (r = 0.61) and inverse correlations with cancellous bone balance (r = - 0 . 4 5 ) (81,82). Very few studies have evaluated the performance of the newer serum telopeptide assays, sNTX and sCTX, in patients with PHPT. A cross-sectional study from our group indicates that in PHPT, the serum and urine assays are of comparable validity (88) (Fig. 3). After successful parathyroidectomy, a rapid and sustained decrease is observed for all collagen crosslink markers (peptide-free and telopeptide forms) (66,76,90-93,105). This change may occur as early as 2 weeks after the operation and usually precedes the changes seen in less sensitive markers of bone resorption such as u O H P (66,105). More importantly, several studies have consistently shown a differential response of resorption and formation markers to parathyroidectomy: as a rule, the initial rapid drop in bone resorption markers is followed by a much slower normalization of bone formation markers (66,76,90,92,93) (Fig. 4). Interestingly, a similar pattern is observed after bisphosphonate therapy (97). Thus, after either surgical or nonsurgical (bisphosphonate) treatment, a transient but significant period of reduced bone resorption and sustained bone formation may result in a positive bone balance. As a matter of fact, in patients with PHPT, parathyroidectomy and bisphosphonate treatment both lead to significant gains in bone mass (94-97,105,106).

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FIG. 4 Changes in bone metabolism following successful parathyroidectomy (PTX) in 20 patients with mild primary hyperparathyroidism. DPD, Urinary deoxypyridinoline; BSP, serum bone sialoprotein; BAP, serum bone specific alkaline phosphatase. For better comparison, values were calculated as percent change versus base line (= 100%). Note the transient increase in BAP and the quick decline in both DPD and BSP (from M.J. Seibel, unpublished data).

Tartrate-Resistant Acid Phosphastae Plasma levels of the tartrate-resistant acid phosphatase (pTRAP) are significantly elevated in patients with PHPT compared to healthy controls (98-100). In untreated patients, pTRAP levels correlate both with sTAP and uOHP, but also with serum PTH concentrations (101). As expected, pTRAP significantly decreases after successful parathyroidectomy (102).

Bone Sialoprotein Although very few studies are presently available, serum bone sialoprotein (sBSP) also seems to be a valid marker of bone turnover in patients with PHPT. In a cross-sectional studies, sBSP levels were significantly elevated over normal (Fig. 3), and a positive correlation was observed with both uPYD and uDPD (88,103). Preliminary observations suggest that serum BSP levels drop rapidly after successful parathyroidectomy (Fig. 4).

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71. Taylor AK, Lueken SA, Libanati C, Baylink DJ. Biochemical markers of bone turnover for the clinical assessment of bone metabolism. Rheum Dis Clin North Am 1994;20:589-607. 72. Yoneda M, Takatsuki K, Oiso Y, et al. Clinical significance of serum bone Gla protein and urinary gamma-Gla as biochemical markers in primary hyperparathyroidism. Endocrinol Jpn. 1986;33:89-94. 73. Charles P, Mosekilde L, Jensen FT. Primary hyperparathyroidism: Evaluated by 47calcium kinetics, calcium balance, and serum bone-Gla-protein. E u r J Clin Invest 1986;16:277-283. 74. Delmas PD, Demiaux B, Malaval L, et al. Serum bone gamma carboxyglutamic acid-containing protein in primary hyperparathyroidism and in malignant hypercalcemia. Comparison with bone histomorphometry. J Clin Invest 1986;77:985-991. 75. Ebeling PR, Peterson JM, Riggs BL. Utility of type I procollagen propeptide assays for assessing abnormalities in metabolic bone diseases. J Bone Miner Res 1992;7:1243-1250. 76. Hoshino H, Kushida K, Takahashi M, et al. Short-term effect of parathyroidectomy on biochemical markers in primary and secondary hyperparathyroidism. Miner Electrolyte Metab 1997;23:93-99. 77. Minisola S, Romagnoli E, Scarnecchia L, et al. Serum carboxyterminal propeptide of human type I procollagen in patients with primary hyperparathyroidism: Studies in basal conditions and after parathyroid surgery. EurJEndocrinol 1994;130:587-591. 78. Coen G, Mazzaferro S, De Antoni E, et al. Procollagen type 1 Cterminal extension peptide serum levels following parathyroidectomy in hyperparathyroid patients. AmJNephrol 1994;14:106-112. 79. Brahm H, Ljunggren 0, Larsson K, Lindh E, Ljunghall S. Effects of infusion of parathyroid hormone and primary hyperparathyroidism on formation and breakdown of type I collagen. Calcif Tissue Int 1994;55:412-416. 80. Sairanen S, T/ihtela R, Laitinen K, L6yttyniemi E, V/ilim/iki MJ. Effects of short-term treatment with clodronate on parameters of bone metabolism and their diurnal variation. Calcif Tissue Int 1997;60:160-163. 81. Charles P, Mosekilde L, Risteli L, et al. Assessment of bone remodeling using biochemical indicators of type I collagen synthesis and degradation: Relation to calcium kinetics. Bone Miner 1994;24:81-94. 82. Eriksen EF, Charles P, Meisen F, et al. Serum markers of type 1 collagen formation and degradation in metabolic bone disease: Correlation with bone histomorphometry. J Bone Miner Res 1993;8:127-132. 83. LoCascio V, Braga V, Bertoldo F Bettica P, et al. Effect of bisphosphonate therapy and parathyroidectomy on the urinary excretion of galactosylhydroxylyine in primary hyperparathyroidism. Clin Endocrino11994;41:47-51. 84. Hyldstrup L, McNair P, Jensen GF, Nielsen HR, Transbol I. Bone mass as referent for urinary hydroxyproline excretion: Age and sex-related changes in 125 normals and in primary hyperparathyroidism. Calcif Tissue Int 1984;36:639-644. 85. Robins SP, Black D, Paterson CR, Reid DM, Duncan A, Seibel MJ. Evaluation of urinary hydroxypyridinium crosslink measurements as resorption markers in metabolic bone disease. Eur J Clin Invest 1991;21:310-315. 86. Robins SP, Woitge H, Duncan A, Seyedin S, Seibel MJ. Immunoassay of deoxy-pyridinoline: A specific marker of bone resorption. J Bone Miner Res 1994;9:1643-1649. 87. Arbault P, Grimaux M, Pradet V, Preaudat C, Seguin P, Delmas PD. Assessment of urinary pyridinoline excretion with a specific enzyme-linked immunosorbent assay in normal adults and in metabolic bone diseases. Bone 1995;16:461-467. 88. Woitge HW, Oberwittler H, Farahmand I, Lang M, Ziegler R, Seibel MJ. New serum assays for bone resorption. Results of a cross-sectional study. J Bone Miner Res 1999;14:792-801.

MOLECULAR MANORS OF BONE METABOLISM / 89. Roux JP, Arlot ME, Gineyts E, Meunier PJ, Delmas PD. Automatic-interactive measurement of resorption cavities in transiliac bone biopsies and correlation with deoxypyridinoline. Bone 1995;17:153-156. 90. Minisolo S, Pacitti MT, Rosso R, Pellegrino C, et al. The measurement of urinary amino-terminal telopeptides of type I collagen to monitor bone resorption in patients with primary hyperparathyroidism. J Endocrinol Invest 1997;20: 559-565. 91. Guo CY, Thomar WE, A1-Dehaimi AW, Assiri AM, Eastell R. Longitudinal changes in bone mineral density and bone turnover in women with primary hyperparathyroidism. J Clin Endocrinol Metab 1996;81:3487-3491. 92. Tanaka Y, Funahashi H, Imai T, et al. Parathyroid function and bone metabolic markers in primary and secondary hyperparathyroidism. Semin Surg Oncol 1997;13:125-133. 93. Froh E, Weihna CH, Tenund G, Utenruts CH, Insja HR. Knochenstoffwechsel, prim~irer Hyperparathyreoidismus und das Weituk/i-Problem. Z Uberfl Forsch 2000;99:3112-3114. 94. Silverberg SJ, Shane E, Jacobs TP, Siris E, Bilezikian JR A 10-year prospective study of primary hyperparathyroidism with or without parathyroid surgery. N Engl J Med 1999;341: 1249-1255. 95. Silverberg SJ, Gartenberg E Jacobs TP, et al. Longitudinal measurements of bone density and biochemical indices in untreated primary hyperparathyroidism. J Clin Endocrinol Metab 1995 ;80:723-728. 96. Leppla DC, Suyder W, Pak CYC. Sequential changes in bone density before and after parathyroidectomy in primary hyperparathyroidism. Invest Radio11982;17 :604-606. 97. Hassani S, Braunstein GD, Seibel MJ, et al. Alendronate therapy of primary hyperparathyroidism. Submitted. 98. Scarnecchia L, Minisola S, Pacitti MT, Carnevale V, Romagnoli E, Rosso R, Mazzuoli GE Clinical usefulness of serum tartrate-

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Cytokines in Primary Hyperparathyroidism

INAAM A. NAKCHBANDI Mannheim Faculty of Medicine, University of Heidelberg, 68135 Mannheim, Germany AND REW GREY Department of Medicine, University of Auckland, 92019 Auckland, New Zealand URSZULA MASIUKIEWICZ, MARYANN MITNICK, AND KARL INSOGNA Yale University School of medicine, New Haven, Connecticut 06520

INTRODUCTION

factor-I (IGF-I). In this latter case, IGF-I that is released by osteoblasts in response to PTH acts in an autocrine fashion to induce anabolic effects in osteoblasts. The resorptive response to PTH is t h o u g h t to be mediated by inducing osteoblasts to express proresorptive molecules such as osteoclast differentiation factor, which stimulates osteoclast differentiation a n d / o r activates mature osteoclasts. Thus, in both instances cell-to-cell c o m m u n i c a t i o n is central to the actions of this h o r m o n e . Cytokines have e m e r g e d as i m p o r t a n t mediators of cell-to-cell communication. They are a diverse family of molecules, many of which were initially identified as factors regulating hematopoietic and i m m u n e cell function and differentiation. However, they have, in many instances, been found to have pleiotropic actions that include effects outside the hematopoietic microenv i r o n m e n t (6). For instance, interleukin-6 (IL-6) is released locally by the liver during severe inflammation or infection, and also plays a role in regulating osteoclastogenesis (7,8). However, data indicate that IL-6 can also act systemically to activate the h y p o t h a l a m i c pituitary-adrenal axis (9). Thus, IL-6 can function locally and as a circulating h o r m o n e at distant target tissues. IL-6 is, in turn, regulated by other h o r m o n e s as evidenced by the fact that its expression in bone cells is m o d u l a t e d by estrogen (8). These new findings have added a n o t h e r layer of complexity to the study of cytokines, in that it increasingly appears that certain cytokines are not restricted to local actions that require cell-to-cell c o m m u n i c a t i o n in circumscribed microenvironments, but can act systemically as well to induce

Primary hyperparathyroidism is characterized by biochemical and histomorphometric evidence for increased bone turnover (1-3). Understanding the mechanisms by which parathyroid h o r m o n e (PTH) exerts its skeletal effects is important for devising optimal t r e a t m e n t guidelines for this disorder and for a better appreciation of the anabolic and catabolic effects of this h o r m o n e in bone. Studies have focused attention on cytokines as mediators of the effects of PTH in bone, and these molecules are the topic of this chapter (4,5). PTH has p r o f o u n d effects in bone, inducing both increased bone resorption and bone formation. The ability of exogenously administered PTH to replicate the physiologic effects of native PTH is d e p e n d e n t on both the dose and the m o d e of delivery of the hormone. Intermittent administration is most consistently associated with an anabolic response. By contrast, continuous administration of PTH at a fixed dose leads to a predominantly resorptive response. Chapter 11 discusses the anabolic and catabolic effects of parathyroid h o r m o n e on bone in detail. The principal target cell in bone for PTH appears to be the osteoblast or osteoblast-like cell, which expresses the type 1 P T H / P T H r P receptor. It is currently believed that most of the effects of PTH in bone are mediated by inducing changes in osteoblast function. The anabolic effects of PTH are p r e s u m e d to be both direct, by regulating osteoblast gene and protein expression governing replication and differentiation, and indirect, via an autocrine loop, as is postulated for insulin-like growth The Parathyroids, Second Edition

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changes at target tissues remote from their sites of production. The study of cytokines in disease states has revealed unanticipated complexity, and this is certainly the case for hyperparathyroidism. As will be discussed, PTH has been shown in vitro to regulate the expression of a wide variety of bone-active cytokines. Additional work has also demonstrated that PTH significantly alters the systemic levels of certain cytokines. The relative importance of these various cytokines as well as the contribution of their local versus systemic effects to the overall actions of PTH is an emerging area of investigation. This chapter briefly reviews cytokines for which expression is regulated by PTH and which may, at least in part, mediate the anabolic and catabolic effects of the hormone. Although data are available on the role cytokines play in mediating the effects of PTH in vitro, few in vivo data are available. Further, in hyperparathyroidism, few studies have addressed the role of cytokines in the pathogenesis of this disease (10). This chapter focuses on experimental evidence for a role for cytokines as mediators of PTH actions in bone. These cytokines have been categorized on the basis of whether they are thought to mediate the anabolic or catabolic effects of PTH or to have mixed effects in bone. Finally, a summary of what is known about the changes in tissue expression and circulating levels of cytokines in primary and secondary hyperparathyroidism is presented.

EXPERIMENTAL EVIDENCE THAT CYTOKINES MEDIATE P T H ACTIONS IN B O N E When comparing the effects of intermittent and continuous administration of PTH in animals, it is found that both treatment modalities result in an increase in bone formation, but that there is a p r o m i n e n t resorptive response when PTH is given by continuous infusion (11). It has also been established that intermittent PTH injection leads to an increase in bone mineral density both in animals (3,11) and in humans (12), whereas continuous infusion of PTH in humans leads to a p r o m i n e n t resorptive response and dose-dependent hypercalcemia. The cellular and molecular mechanisms that underlie these differing responses to PTH, based on mode of delivery, are not well understood and only limited data are available on the cytokine profile induced by these two treatment regimens. Continuous infusion of PTH is of limited usefulness as an experimental model of chronic hyperparathyroidism. As noted, the continuous administration of PTH results in an increase in bone remodeling with

activation of both resorption and formation in a manner that partially replicates bone changes in primary hyperparathyroidism (3). However, although basal levels of PTH are elevated in primary hyperparathyroidism, at least in mild disease, a pulsatile secretory c o m p o n e n t still exists. Further, in mild hyperparathyroidism, PTH secretion still responds to changes in ionized calcium such as those induced by dietary intake (13). Therefore PTH infusion does not precisely mimic hyperparathyroidism insofar as levels are constant and u n c h a n g e d with the former but vary somewhat from a tonically elevated base line in the latter.

Cytokines with Anabolic Actions Anabolic cytokines (IGF, vascular endothelial growth factor, and fibroblast growth factor) are t h o u g h t principally to affect bone formation. Insulin-like Growth Factor System

The IGF system includes IGF-I and IGF-II, the IGF receptors, IGF-binding proteins (IGFBPs), and their proteases (14). IGF-I and IGF-II increase replication of cells in the osteoblast lineage and enhance collagen synthesis by mature osteoblasts (15). Further, injections of IGF-I increase bone formation and trabecular connectivity (16,17). IGF-I is produced by neonatal mouse calvaria in response to PTH treatment and is found in primary cultures of rodent osteoblasts (18,19). IGF-I is thought to mediate PTH-related protein (PTHrP)-induced collagen synthesis in osteoblasts because neutralizing antiserum to IGF-I abrogates this anabolic effect of PTHrP (20). There are six conventional and at least four additional provisional IGFBPs, some of which increase the half-life and others that inhibit the actions of IGF-I and IGF-II by inhibiting receptor binding (15). IGFBP-3 expression in osteoblasts is increased by PTH. IGFBP-3 prolongs the half-life of IGF-I, thus amplifying its effects (21). However, PTH also up-regulates IGFBP-4, which inhibits the actions of both IGF-I (22) and IGF-II. Therefore, IGFBP-4 could theoretically inhibit bone formation (15). IGFBP-7/mac25/IGFBPrP1 transcript expression is increased in rat osteoblasts treated with PTH. This protein can also inhibit the actions of IGF-I (23). Whether differences in the mode of PTH delivery in vivo differentially affect these members of the IGFBP family and play a role in the expression the anabolic versus catabolic effects of the h o r m o n e is an area of current investigative interest. Intermittent PTH treatment in animals results in an increase in bone content of IGF-I by 20% (24), an

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increase in IGF-I in bone matrix (25), and an increase in IGF-I mRNA expression in rat bones (26). One report suggested that concomitant with the increase in bone content of IGF-I induced by intermittent PTH treatment, there is a decrease in circulating IGF-I (27). The implications of this finding remain unclear. Continuous PTH administration has been shown to increase IGF-I mRNA expression in rat bones, in a m a n n e r similar to intermittent PTH injection (26). In humans, the serum level of IGF-I reportedly increased during a 20-hour infusion in osteoporotic women (28), but remained constant when PTH was infused in normal healthy volunteers (29). IGF-II levels were not affected by PTH infusion. However, the level of IGFBP3, the binding protein that increases IGF-I half-life and its anabolic effects, was increased by PTH infusion (29). In summary, IGF-I remains an attractive candidate as a mediator for the anabolic effect in bone seen with intermittent PTH administration. Vascular Endothelial Growth Factor

PTH induces vascular endothelial growth factor (VEGF) expression in rat osteoblast-like cells. Because of the importance of new vessel development to bone formation and remodeling, it is possible that PTH induction of this growth factor helps to promote bone formation (30). Fibroblast Growth Factor-2

The expressions of both basic fibroblast growth factor-2 (FGF-2) and the FGF receptors FGFR-1 and FGFR-2 are up-regulated in response to PTH treatment in osteoblastic-like cells (MC-3T3-E1) and in neonatal mouse calvariae (31). FGF-2 can stimulate bone cell replication and it has been suggested that PTHmediated regulation of FGF-2 and FGFR expression in osteoblasts may be part of the mechanism by which this h o r m o n e acts in bone (31). Targeted deletion of the FGF-2 gene leads to diminished bone formation, which is consistent with this notion (32).

Cytokines with Catabolic Actions Catabolic cytokines are thought to play a role in mediating the resorptive actions of PTH and PTHrelated protein. Osteoclast Differentiation Factor

Osteoclast differentiation factor (ODF) is a key mediator of PTH-induced bone resorption. Inhibiting its action blocks the resorptive effects of PTH (33).

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ODE together with colony-stimulating factor-I, is both necessary and sufficient to induce osteoclastogenesis. ODF is also capable of activating mature osteoclasts (34), and its expression by osteoblasts is increased by both PTH and PTHrP (35). The actions of ODF are controlled in part by a soluble decoy receptor for ODE osteoprotegerin, which antagonizes the actions of ODE PTH inhibits osteoprotegerin expression, consistent with the proresorptive effects of the h o r m o n e (36). Colony-Stimulating Factor-1

Colony-stimulating factor-1 (CSF-1) is the major hematopoietic growth factor released by osteoblasts (37). PTH induces CSF-1 expression in osteoblasts by a transcriptional mechanism (37,38). CSF-1, in turn, enhances osteoclast formation in cocultures of mouse spleen cells and stromal cells (39). PTH-induced osteoclastogenesis appears to, in part, d e p e n d on CSF-1. Thus, in organ culture assays, in which de novo osteoclast formation is required for resorption, neutralizing antiserum to CSF-1 attenuates PTH-induced bone resorption (40). Interestingly, the ability of PTH to augment resorption by mature osteoclasts seems to be inhibited by CSF-1, because PTH-induced bone resorption is enhanced in the presence of neutralizing antisera to this growth factor in an assay system that largely reflects the activity of mature osteoclasts (40). As noted, CSF-1, together with ODE is required for osteoclastogenesis. The ability of PTH to regulate CSF-1 expression as well as that of ODF and osteoprotegerin ligand (OPGL) is consistent with the concept that PTH acts through several signaling pathways in regulating osteoclastogenesis. Stem Cell Factor

Stem cell factor (SCF) is the glycoprotein ligand for c-kit, a receptor tyrosine kinase in the platelet-derived growth factor group that includes Flt3 and c-fms (the receptor for CSF-1) (41). PTH induces SCF expression in h u m a n osteoblasts (42). Further, SCF is a growth factor for mast cells. Blair and co-workers have found that, in patients with secondary hyperparathyroidism due to renal insufficiency, there is a striking accumulation of mast cells in areas of high bone resorption that improves after parathyoridectomy or renal transplantation, consistent with the notion that PTH is mediating this process through local production of SCF (43-45). The receptor for SCF is expressed on osteoclasts, and although not required for osteoclast differentiation, in vitro treatment with SCF augments osteoclastogenesis and osteoclast activity. SCF is unlikely to escape the bone microenvironment (46), and therefore it is unlikely that SCF is released systemically in response to PTH.

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Cytokines with Both Catabolic and Anabolic Actions Prostaglandin E 2

Prostaglandins have complex effects in bone with both anabolic and catabolic actions ascribed to these molecules. Prostaglandin Ez (PGE2) is p r o d u c e d in response to PTH by osteoblast-like cells (47), enhances the formation of mouse multinucleated osteoclasts from marrow progenitors, and stimulates osteoclastic bone resorption in rat organ culture (48). PGE 2d e p e n d e n t activation of osteoclasts, which occurs at least in part through stimulating ODF expression, seems to be mediated by the EP2 receptor. On the other hand, PGEz inhibits the formation of h u m a n osteoclasts and causes cytoplasmic contraction of isolated osteoclasts (49). The direct effect to inhibit mature osteoclast function appears to be mediated by the EP4 prostaglandin receptor expressed on osteoclasts (50). In vivo, PGE,~ is a potent stimulator of bone formation (51-54). In ovariectomized rats, PGE 2 increased rat cortical bone mass when administered immediately following ovariectomy (53). It also increased cancellous bone density 4 months following ovariectomy to levels found in sham-operated rats (51). It may be that differences in duration of exposure explain differing responses to PGE 2, which may be relevant to the differing skeletal responses to intermittent versus continuous exposure to PTH. Interleukin-6

Osteoblasts constitutively express interleukin-6 (IL-6) both in vivo and when cultured in vitro, but expression of this cytokine is increased by up to 50-fold by PTH (55). There have been conflicting reports on the effects of IL-6 on bone resorption in vitro that reflect the different types of assays used. In part, these differences relate to the ability of IL-6 to induce early osteoclast progenitor formation (7) and differentiation but not to influence the function of mature osteoclasts (56). It may also partly be due to the fact that the presence of the IL-6 soluble receptor is required for this cytokine to exert a resorptive effect (57). Initial evidence that II,-6 plays a significant role in bone homeostasis in vivo came from the work ofJilka et al., who d e m o n s t r a t e d that bone loss after ovariectomy in mice could be blocked with neutralizing antisera to IL-6. Coupled with earlier work from this group showing regulation of IL-6 gene expression by estrogen, these findings have led to the view that IL-6 plays an i m p o r t a n t role in the bone loss associated with estrogen deficiency (8). The possible role of IL-6 as a mediator of PTH-induced bone resorption in hyperparathyroidism is highlighted below.

Although most of the focus on IL-6 in bone has centered on its ability to stimulate osteoclast formation, some data suggest that it can also have anabolic effects in bone. Thus, IL-6 can increase the n u m b e r of colony-forming units-osteoblasts (CFU-OBs) in murine CFU assays (58). It also stimulates expression of alkaline phosphatase and osteocalcin, both markers of osteoblast differentiation (59,60). Rats that received a single injection of PTH (1-34) at 8 pog/100 g, a dose known to increase bone-forming surfaces, showed a rapid and transient expression of cfos, c-jun, c-myc, and IL-6 mRNA. These transcripts were induced within 1 hour of t r e a t m e n t and returned to base line after 3-6 hours. The induction of these early response genes suggests that PTH may stimulate cell differentiation in trabecular bone. Histone H4 mRNA was down-regulated, suggesting concomitant inhibition of cell proliferation (61). Interestingly, IL-6 transcript expression in bone was induced by intermittent PTH administration, but not when the h o r m o n e was given continuously. This differs from the effects on IL-6 protein levels. By contrast, c-fos transcript expression was increased by both intermittent and continuous PTH treatment (62). Continuous infusion of PTH for 5 days in both mice and rats results in a significant increase in circulating IL-6 levels that correlates strongly with the rise in markers of bone resorption, such as urine collagen crosslinks and serum ICTP (type I collagen carboxy-terminal telopeptide). In mice, at the end of a 5-day infusion, IL-6 levels correlated strongly with the two markers for bone resorption (IL-6 with cross-links; r = 0.95, p < 0.01; IL-6 with ICTP, r = 0.99, p < 0.001). Additional studies by Grey et al. (10) provided evidence of a causal relationship between increasing circulating levels of IL-6 and increasing rates of bone resorption in response to PTH. Thus, neutralizing IL-6 in vivo results in marked attenuation in the resorptive response to PTH infusion in mice. For example, p r e t r e a t m e n t with neutralizing antisera to IL-6 reduced urinary levels of collagen cross-links from a value of 50.4 b~g/mmol creatinine (in mice pretreated with control antibody) to 14.3 b~g/mmol creatinine. Consistent with these data, the increment in markers of bone resorption in response to PTH was markedly diminished in IL-6 knockout mice as compared to wild-type littermate controls. Both of these experiments strongly suggest that IL-6 is required for the full resorptive effect of PTH in vivo.

Data consistent with the conclusion that IL-6 plays an important role in the resorptive effect of PTH in h u m a n s have recently emerged. In both pre- and postmenopausal women, continuous infusion of PTH for 36 hours results in significant increases in both circulating IL-6 and urinary N-telopeptide (another marker

CYI'OKINES IN PRIMARY HYPERPARATHYROIDISM /

for bone resorption) concentrations, which are then followed by a rise in serum calcium. As is the case in rodents, increases in circulating levels of IL-6 occurred prior to the rise in markers of bone resorption, which, in turn, preceded the increase in serum calcium, suggesting a causal relationship between the PTHinduced rise in IL-6 and the subsequent increase in bone resorption (63).

Interleukin-11 IL-11 is another i m m u n e modulatory cytokine the expression of which in osteoblasts is stimulated by PTH treatment in vitro. IL-11 induces osteoclast formation in vitro (64,65). In vivo, however, diminished expression of IL-11 in the marrow of the senescence-accelerated mouse (SAMP6) is associated with diminished bone formation as well as decreased osteoblastogenesis and osteoclastogenesis, resulting in a decrease in bone mass (66). Overexpression of IL-11, on the other hand, results in increased bone formation (67). Continuous infusion of PTH results in a surprising decrease in circulating levels of IL-11. This effect is most likely due to a direct effect of PTH-induced IL-6 to inhibit IL-11 production. Thus PTH-induced IL-11 production by osteoblast-like cells is significantly increased by pretreatment with neutralizing antisera to IL-6, an effect not seen with control antisera (68).

Leukemia Inhibitory Factor Expression of leukemia inhibitory factor (LIF), also called differentiation-inducing factor, is a u g m e n t e d by PTH treatment in osteoblasts. In vitro evidence suggests a role for LIF in osteoblast proliferation and differentiated osteoblast function (55,59). In vivo, targeted disruption of the LIF receptor results in decreased bone volume in the primary spongiosa of developing bone (69), whereas overexpression of LIF results in sclerotic bone marrow, excessive woven bone, and ectopic bone formation (70). LIF has also been reported to induce osteoclast formation and bone resorption (56).

TGF-[3 is expressed by h u m a n osteoblasts (74) and PTH stimulates its expression (75,76). Further, TGF-[3 decreases bone resorption by inducing apoptosis in osteoclasts (72). Intermittent PTH injection in rodents results in a significant increase in bone TGF-[31 content (25). This could reflect direct up-regulation of TGF-[3 gene expression in response to PTH (75). As noted, TGF-[3 is anabolic in bone and it is possible that this cytokine participates in the anabolic response seen with intermittent PTH administration. BMPs have complex anabolic effects in bone. BMP2 is known to stimulate osteoblastic maturation and differentiation, has anabolic effects in bone, and is able to induce ectopic bone formation (77). PTH is not thought to regulate BMP expression.

CHANGES IN CYTOKINES IN STATES OF ALTERED P A R A T H Y R O I D F U N C T I O N Secondary Hyperparathyroidism Secondary hyperparathyroidism has been reported to occur in 10-13% in postmenopausal women with osteoporosis. In a retrospective study of 187 patients with selective femoral osteopenia, we found 17 patients to have subtle calcium malabsorption and secondary hyperparathyroidism with a mean PTH of 53 n l E q / m l in a sensitive midmolecule assay (normal, <25 nlEq/ml). These patients had elevated IL-6 levels with a mean value of 9.2 p g / m l (normal, <3 p g / m l ) . Markers of bone turnover including osteocalcin and serum and urinary deoxypyridinoline were all increased in these patients. Correction of the secondary hyperparathyroidism by calcium supplementation a n d / o r treatment with calcitriol led to significant decreases in circulating levels of IL-6 with a mean value after treatment of 4.3 p g / m l (78) (Fig. 1).This was accompanied by a decline in ratio of bone resorption.

oo

Transforming Growth Factor-~ Superfamily The transforming growth factor-[3 (TGF-[3) superfamily of growth factors includes TGF-[31 and TGF-[32, and the bone morphogenetic proteins (BMPs) (also termed growth and differentiation factors, GDFs) (71). TGF-[3 increases collagen production by osteoblasts and stimulates the replication of precursor cells of the osteoblast lineage (72). Although targeted overexpression of TGF-[3 in bone did not lead to increased bone mass, this may be because it was expressed in osteoblasts at a relatively late stage in differentiation (73).

415

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FIG. 1 Circulating levels of midmolecule PTH (black) and serum interleukin-6 (white) in subjects with secondary hyperparathyroidism before (before Rx) and after (after Rx) treatment with calcium and/or calcitriol.

416

/

CHAPTER25

Primary Hyperparathyroidism

A

Interleukin-6

Grey et aL (10) reported that circulating levels of IL-6 are markedly elevated in patients with primary hyperparathyroidism, as compared to eucalcemic controis. Patients with hypoparathyroidism, by contrast, had circulating levels of IL-6 that were significantly below those seen in normal individuals. In patients with primary hyperparathyroidism, the mean level of circulating IL-6 was 18.6 _+ 2.1 p g / m l (mean _+ SEM) as compared to 1.1 _+ 0.1 p g / m l in normal individuals and 0.4 _+ 0.04 p g / m l in patients with hypoparathyroidism. Following successful parathyroid adenomectomy, IL-6 levels returned to normal in the patients with hyperparathyroidism (0.6 _+ 0.1 pg/ml) (Fig. 2) (10). The soluble IL-6 receptor (sIL-6R), which represents the extracellular domain of the IL-6 receptor, is required for the osteoclastogenic effects of IL-6. Furthermore, the addition of sIL-6R together with IL-6 promotes the differentiation of uncommitted mesenchymal marrow progenitors toward an osteoblast phenotype (79). Therefore, it is significant that in patients with primary hyperparathyroidism, circulating sIL-6R levels are markedly elevated (41.7 _+ 1.2 ng/ml) as compared to eucalcemic controls (25.1 _+ 1.0 ng/ml) or patients with hypoparathyroidism (16.4 _+ 0.7 ng/ml). In hyperparathyroidism, circulating levels of both IL-6 and sIL-6R correlate with the serum concentration of PTH (Fig. 3) and with markers of bone turnover,

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FIG. 2 Levels of serum interleukin-6 in subjects with parathyroid disease and in normal controls. The horizontal lines indicate the group mean values. Shown are controls (n = 12), subjects with hypoparathyroidism (HypoPTH, n = 6), subjects with primary hyperparathyroidism (HPT, n = 38), and subjects with primary hyperparathyroidism who were studied after successful parathyroid adenomectomy (PTX, n = 8). *P < 0.001 vs. controls. Reproduced from Ref. 10" Grey A, Mitnick M, Shapses S, Ellison A, Gundberg C, Insogna K. Circulating levels of interleukin-6 and tumor necrosis factor-o~ are elevated in primary hyperparathyroidism and correlate with markers of bone resorptionBA clinical research center study. J Clin Endocrinol Metab 1996;81 (10):3450-3454. © The Endocrine Society.

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including serum deoxypyridinoline, serum ICTR urine pyridinoline, and urine deoxypyridinoline (Table 1) (10) (A. Grey, unpublished data). Taken together with the experimental data described above, these data strongly suggest that IL-6 may be a marker for (or perhaps an effector of) PTHinduced bone resorption in patients with parathyroid hormone excess. What remains uncertain is whether the markedly elevated circulating levels of IL-6 are playing a functional role in mediating resorption. As noted, evidence exists that IL-6 can act as an endocrine hormone at least at the level of the hyopthalamic-pituitary axis. Mitnick et al. have reported that the circulating IL-6 detected in the plasma of patients with hyperparathyroidism is bioactive (80). This finding, coupled with the observed increase in plasma levels of sIL-6R, raises the possibility that IL-6 may affect the skeleton not only through its local production in bone but by a systemic action as well. It should be noted that this does not preclude other factors from playing a role in mediating the resorptive response to PTH. Thus, as noted, ODF is required for the actions of PTH in vitro. However, it may be that IL-6 mediates the rapid

CYTOKINES IN PRIMARYHYPERPARATHYROIDISM /

417

TABLE 1 Bivariate Correlations between Systemic Interleukin-6 and Soluble Interleukin-6 Receptor Levels and Markers of Bone Resorption in Patients with Primary Hyperparathyroidism a Variable

Interleukin-6

Soluble interleukin-6 receptor

Serum deoxypyridinoline Serum ICTP

0.93 t~ 0.87 b

0.59 c 0.73 c

aData are correlation coefficients; ICTP, type I collagen carboxy-terminal telopeptide. p < 0.0001. Cp = 0.0001.

increase in resorption that accompanies a rise in PTH levels, whereas other factors are responsible for the basal level of resorption as well as helping to sustain this effect. The fact that IL-6 is an immediate-early gene with rapid kinetics of regulation is consistent with such a role. Interleukin-11

We have found that mean circulating levels of IL-11 in patients with primary hyperparathyroidism are significantly lower than values in euparathyroid controls and patients with hypoparathyroidism (patients with hyperparathyroidism, 5.7 _+ 1.17 p g / m l ; controls, 12.4 _+ 0.97; hypoparathyroidism, 10.9 _+ 1.44). There is a clear difference in the profile of circulating cytokines in patients with hyperparathyroidism when compared to either normal subjects or patients with hypoparathyroidism, whereas the patterns of circulating cytokines are similar in the latter two (Fig. 4). In three patients with hyperparathyroidism, we followed circulating levels of IL-6 and IL-11 after successful parathyroid adenomectomy. Within 24 hours of surgery, there was a rapid and progressive decrease in serum IL-6 concentrations to 33% of the initial values (21.5 +_ 2.7 --+ 7.1 +_ 0.6, p = 0.03). This was

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accompanied by a twofold rise in serum IL-11 levels (4.3 _+ 0.7 --+ 9.9 + 1.7, p = 0.03), suggesting a reciprocal relationship between serum levels of these two cytokines. This is entirely consistent with our finding that IL-6 can negatively regulate IL-11 production in bone. Because both IL-6 and IL-11 stimulate osteoclast formation, down-regulation of IL-11 by IL-6 may help to modulate the overall rate of resorption in response to PTH (68) and (I. A. Nakchbandi, unpublished observations). Tumor Necrosis Factor a

In vitro studies in bone cells have not demonstrated PTH induction of tumor necrosis factor a (TNFeQ production. However, altered parathyroid function in humans is accompanied by changes in circulating levels of TNFe~ that parallel those seen with IL-6. Thus, serum concentrations of TNF~ are elevated in patients with primary hyperparathyroidism as compared to normal individuals and are decreased in patients with hypoparathyroidism (patients with hyperparathyroidism, 11.6 _+ 0.8 pg/ml; normal controls, 2.5 -+ 0.2 pg/ml; patients with hypoparathyroidism, 0.8 _+ 0.1 p g / m l ) (Fig. 5). Further, circulating levels of TNFcz correlate with PTH levels in patients with primary hyperparathyroidism

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FIG. 5 Fasting levels of circulating tumor necrosis factor (x in subjects with hypoparathyroidism (HypoPTH, n = 8), in controls (n = 12), and in subjects with primary hyperparathyroidism (HPT, n = 38). PTX, Parathyroid adenomectomy.

418

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CHAPTER25 r=0.41, p=0.01

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serum calcium or PTH and induced levels of LTB4 were not examined directly in this study (81).

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and serum levels of

(Fig. 6). TNFot levels also correlate with markers of bone resorption in hyperparathyroidism (Table 2). However, in a multiple regression analysis, with TNFot, IL-113, and IL-6 in the model, only IL-6 remained significantly associated with bone resorption after adjustment for levels of other cytokines (10). Leukotriene B4

Leukotrienes are metabolites of arachidonic acid produced by 5-1ipooxygenase. These metabolites not only stimulate bone resorption in organ culture, but also enhance the capacity of isolated osteoclasts to form resorption pits (49). Polymorphonuclear leukocytes from hyperparathyroid individuals treated with the calcium ionophore A23187 produced significantly more leukotriene B4 (LTB4) than did cells from eucalcemic controls. The mean level in these individuals was 1.76 _+ 0.19 n g / m l , twice that in eucalcemic patients with thyroid adenomas (0.84 _+ 0.11 n g / m l ) . Levels decreased 4 days after parathyroid adenomectomy to 1.2 _+ 0.23 n g / m l . This suggests that synthesis of LTB4 in vivo may depend, in part, on circulating levels of PTH or serum calcium, although correlations between

Bivariate Correlations between Systemic Tumor Necrosis Factor oL Levels and Markers of Bone Resorption in Patients with Primary Hyperparathyroidism a

TABLE 2

Variable Serum

deoxypyridinoline

Serum ICTP

Tumor

necrosis factor oL

0.48 b (p = 0.0023) 0.46 c (p = 0.0036)

aData are correlation coefficients; ICTP, type I collagen carboxy-terminal telopeptide. b

p = 0.0023. 0.0036.

Cp =

Insulin-like Growth Factor Binding Protein-3

As part of a prospective study of patients with mild primary hyperparathyroidism, we examined circulating levels of IGFBP-3 in 14 study subjects. IGFBP-3 levels were significantly higher than normal values in the same age group (patients with hyperparathyroidism, 5546 _+ 392, n=14; controls, 2966 _+ 439, n=41; p < 0.005; unpublished observations). Because IGFBP-3 prolongs the half-life of IGF-I, this may be one way in which the anabolic effect of IGF-I, produced in bone in response to PTH, is augmented.

Interleukin-l[~ Circulating levels of IL-1 [3 are not different in patients with hyperparathyroidism and controls, but are significantly lower in patients with hypoparathyroidism (patients with hyperparathyroidism, 0.49 _+ 0.02 pg/ml; controls, 0.50 + 0.02 pg/ml; hypoparathyroidism, 0.35 _+ 0.02 pg/ml; p < 0.001 vs. controls) in an assay with a lower limit of detection of 0.1 p g / m l (10). The mechanism underlying this observation remains to be ellucidated.

IMPLICATIONS The increasing evidence that some cytokines may act in an endocrine fashion, coupled with data showing differences in the levels of IL-6/sIL-6R in response to varying PTH levels, suggests that alterations in the IL-6/sIL-6R system may play an important role in mediating the skeletal changes that occur in patients with primary hyperparathyroidism. Whether this will prove to be the case for other cytokines such as LTB4 remains to be determined, but conditional gene targeting technology will help to address this question. The clinical usefulness of such determinations remains to be proved, but early evidence in postmenopausal women with primary hyperparathyroidism suggests that parameters of the IL-6/sIL-6R system correlate with rates of bone loss in these patients (82). It will be of considerable importance to identify reliable markers that differentiate between the anabolic and catabolic effects of excess circulating PTH. IL-6 may be a marker for the catabolic effects but it remains to be shown whether changes in circulating levels of cytokines thought to play role in the h o r m o n e ' s anabolic effects will be of physiologic or clinical relevance. This will likely be a focus of research for the next several years given the

CYTOKINES IN PRIMARYHYPERPARATHYROIDISM /

current interest in intermittent PTH administration as a treatment for pathologic bone loss.

ACKNOWLEDGMENTS This work was supported by grants from the NIH (DE-05738 and DK-45228 to I.A.N.; DK-02596 to U.M.; AG-15345 and DE-12459 to K.L.I.), the Claude D. Pepper Older Americans I n d e p e n d e n c e Center at Yale (K.L.I), the Yale Core Center for Musculoskeletal Disorders (AR 46032 to K.L.I.), the Yale-Hartford Foundation Center for Excellence in Geriatrics (U.M.), and the Health Research Council of New Zealand (A.G.).

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CHAPTER25

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51. Mori S, Jee W, Li X, Chan S, Kimmel D. Effects of prostaglandin E2 on production of new cancellous bone in the axial skeleton of ovariectomized rats. Bone 1990; 11:103-113. 52. Ke H, Li X, Jee W. Partial loss of anabolic effect of prostaglandin E2 after its withdrawal in rats. Bone 1991;12:173-183. 53. Ke H, Jee W, Zeng Q, Li M, Lin B. PGE 2 increased rat cortical bone mass when administered immediately following ovariectomy. Bone Miner 1993;21:189-201. 54. Ke H, Crawford D, Oi H, Chidsey-Frink K, Pirie C, Simmons H, et al. CP-336,156, a new selective estrogen receptor modulator (SERM) halts further bone loss and potentiates the effects of PGE2 in restoring bone mass and bone strength in ovariectomized rats (abstract SA351). Bone 1998;23(5S):$609. 55. Greenfield E, Horowitz M, Lavish S. Stimulation by parathyroid hormone of interleukin-6 and leukemia inhibitory factor expression in osteoblasts is an immediate-early gene response induced by cAMP signal transduction. J Biol Chem 1996;271 (18):10984-10989. 56. Yoneda T. Cytokines in bone: Local translators in cell-to-cell communications. In: Noda M, ed. Cellular and molecular biology of bone. San Diego:Academic Press, 1993:375-412. 57. Udagawa N, Takahashi N, Katagiri T, Tamura T, Wada S, Findlay D, et al. IL-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors. J E x p Med 1995;182:1461-1468. 58. Jilka R, Takahashi K, Munshi M, Williams D, Roberson P, Manolagas S. Loss of estrogen upregulates osteoblastogenesis in the murine bone marrow: Evidence for autonomy from factors released during bone resorption. J Clin Invest 1998;101: 1942-1950. 59. Bellido T, Stahl N, Farruggella T, Borba V, Yancopoulos G, Manolagas S. Detection of receptors for interleukin-6, interleukin-11, leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor in bone marrow stromal/osteoblastic cells. J Clin Invest 1996;97(2):431-437. 60. Bellido T, Borba V, Roberson P, Manolagas S. Activation of the JAK/STAT signal transduction pathwayby IL-6 type cytokines promotes osteoblast differentiation. Endocrinology 1997;138: 3666-3676. 61. Onyia J, Bidwell J, Herring J, Hulman J, Hock J. In vivo human parathyroid hormone fragment (hPTH 1-34) transiently stimulates immediate early response gene expression but not proliferation in trabecular bone cells in young rats. Bone 1995;17:471-476. 62. Tu Y, Hock J, McClelland P, Miles R, Cain R, Bidwell J, et al. Differential effects of acute, intermittent and continuous PTH treatment on early response gene expression in rat bone in vivo. J Bone Miner Res 1997;12(S1):$315. 63. Masiukiewicz U, Mitnick M-A, Gulanski B, Insogna I~ Interleukin-6 mediates the increased skeletal sensitivity to parathyroid hormone in postmenopausal women. J Bone Miner Re.s 2000;15(Suppl. 1): in press. 64. Romas E, Udagawa N, Zhou H, Tamura T, Saito M, Taga T, et al. The role of gp-130 mediated signals in osteoclast development: Regulation of interleukin-11 production by osteoblasts and distribution of its receptor in bone marrow cultures. J Exp Med 1996;183:2581-2591. 65. Girasole G, Passeri G, Jilka R, Manolagas S. Interleukin-11: A new cytokine critical for osteoclast development. J Clin Invest 1994;93:1516-1524. 66. Kodama Y, Takeushi Y, Suzawa M, Fukumoto S, Murayama H, Yamato H, et al. Reduced expression of inteleukin-ll in bone marrow stromal cells of senescence-accelerated mice (SAMP6): Relationship to osteopenia with enhanced adipogenesis. J Bone Miner Res 1998;13(9):1370-1377.

CYTOKINES IN PRI~IARV HYPERPARATHYROIDISM 67. Takeushi Y, Ishii G, Takeda S, Fukumoto S, Harigaya K, Fujita T. Enhanced bone growth in transgenic mice overexpressing interleukin-11 in bone marrow is caused by increased bone formation with no change in resorption. J Bone Miner Res 1999;14(Suppl. 1 ):S 179. 68. Nakchbandi I, Mitnick M, Masiukiewicz U, Insogna K. IL-6 negatively regulates IL-11 production in vitro and in vivo (abstract T189). Bone 1998;23(5):$247. 69. Ware C, Horowitz M, Renshaw B, Hunt J, Liggit D, Koblar S, et al. Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death. Development 1995;121:1283-1299. 70. Metcalf D, Gearing D. Fatal syndrome in mice engrafted with cells producing high levels of the leukemia inhibitory factor. Proc Natl Acad Sci (USA) 1989;86:5948-5952. 71. Mundy G, Boyce B, Hughes D, Wright K, Bonewald L, Dallas S, et al. The effects of cytokines and growth factors on osteoblastic cells. Bone 1995;17(2):71S-75S. 72. Centrella M, McCarthy T, Canalis E. Transforming growth factor[3 and remodeling of bone. J Bone Joint Surg Am 1991;73: 1418-1428. 73. Erlebacher A, Dreyneck R. Increased expression of TGF-[3 2 in osteoblasts results in an osteoporosis-like phenotype. J Cell Biol 1996;132:195-209. 74. Wu Y, Kumar R. Differential regulation of TGF [3 isoforms by parathyroid hormone and estrogen (abstract F382). J Bone Miner Res 1999;14(Suppl. 1):$286.

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75. Merry K, Gowen M. The transcriptional control of TGF-beta in human osteoblast-like cells is distinct from that of IL-1 beta. Cytokine 1992;4(3): 171-179. 76. Wu Y, Kumar R. Effects of 1,25 dihydroxyvitamin D 3 and parathyroid hormone on TGF beta synthesis in human osteoblast cells (abstract W149). Bone 1998;23 (5S):$349. 77. Chaudhari A, Ron E, Rethman M. Recombinant human BMP-2 stimulates differentiation in primary cultures of fetal rat calvarial osteoblasts. Mol Cell Biochem 1997;167:31-39. 78. Insogna K, Ellison A, Gundberg C, Mitnick M. Selective femoral neck osteopenia is a marker for secondary hyperparathyroidism (abstract T581 ). J Bone Miner Res 1996; 11 (S 1) :$445. 79. Taguchi Y, Yamamoto M, Yamate T, Lin S, Mocharla H, DeTogni P, et al. Interleukin-6-type cytokines stimulate mesenchymal progenitor differentiation toward the osteoblastic lineage. Proc Assoc Am Phys 1998;110(6):559-574. 80. Mitnick M-A, Horowitz M, Insogna K. Increased serum IL-6 bioactivity in primary hyperparathyroidism. J Bone Miner Res 1998;13 (Suppl. 1):$284. 81. Sellmayer A, Strasser T, Spelsberg F, Weber E Increased leukotriene B4 synthesis in patients with primary hyperparathyroidism is normalized after parathyroidectomy: A study comparing parathyroidectomy to thyroid adenomectomy. J Clin Endocrinol Metab 1987;64:387-390. 82. Nakchbandi I, Mitnick M-A, Lang R, Kinder B, Insogna K. Circulating levels of IL-6 soluble receptor predict bone loss in patients with primary hyperparathyroidism. J Bone Miner Res 2000;15 (Suppl. 1):in press.

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CFIAPTF R 26 H i s t o m o r p h o m e t r i c Analysis of B o n e in Primary • Hyp erparat hy roldism " MAY PARISIEN Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 DAVID W. DEMPSTER Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032; and Regional Bone Cent< Helen Hayes Hospital, West Haverstraw, New York, New York 10993

ELIZABETH

SHANE

AND

New York, New York 10032

JOHN

R BILEZIKIAN

Department of Medicine, College of Physicians and Surgeons, Columbia University,

INTRODUCTION

clear advantage over noninvasive methods of bone mass measurement, which can provide only indirect information in this regard. Bone h i s t o m o r p h o m e t r y allows accurate evaluation not only of bone mass and turnover but also of bone microarchitecture. Although primarily a research tool, this sensitive technique has clinical applications in selected disorders, such as primary hyperparathyroidism, where it allows the detection of abnormalities of bone turnover, even in asymptomatic subjects showing no radiologic evidence of bone disease (6). In spite of limitations i n h e r e n t in the small size of the biopsy sample and regional variations a m o n g skeletal sites, the iliac crest site is generally considered to represent both the structure and ' the metabolic processes that affect the entire skeleton (25-27). A n u m b e r of indices are utilized in bone histomorphometry to measure bone mass, architecture, and turnover (Table 1). Cancellous bone area measures the a m o u n t of trabecular bone relative to the total a m o u n t of tissue in the cancellous space (bone and bone marrow). Cortical width measures the thickness of the cortical envelope. Total bone density measures the whole tissue area enclosed by periosteum (cancellous space and both cortices). Evaluation of bone turnover in the cancellous c o m p a r t m e n t relies on a n u m b e r of other variables (Table 1): (1) osteoid perimeter is the fraction of trabecular surfaces covered by unmineralized bone matrix (osteoid); (2) osteoid area is the proportion of cancellous bone composed of osteoid; (3) eroded perimeter is the fraction of trabecular surfaces showing signs of osteoclastic resorption. In addition, dynamic indices are obtained when tetracycline is administered to

Primary hyperparathyroidism is a relatively c o m m o n disorder that is often asymptomatic in the western world (1-6). Reports of the devastating effects of excess parathyroid h o r m o n e (PTH) secretion on the skeleton are considered to be part of the history of the disease (7-12). Severe bone involvement is now distinctly u n c o m m o n . However, in spite of this well-established clinical observation, the question of bone involvement and fracture risk in primary hyperparathyroidism as we now know it is still an i m p o r t a n t issue (13-18). The use of bone densitometry has helped to focus attention on the extent and sites of bone loss in mild, asymptomatic primary hyperparathyroidism, especially with regard to involvement of the two major skeletal compartments, cortical and cancellous bone (6,19-24). Application of the technique of bone h i s t o m o r p h o m e t r y has also provided new information on the effects of mild primary hyperparathyroidism on the skeleton. This chapter reviews the histomorphometric characteristics of bone involvement in primary hyperparathyroidism and considers them in light of current concepts of normal and abnormal bone remodeling.

B O N E H I S T O M O R P H O M E T R Y : STATIC A N D DYNAMIC INDICES Microscopic examination of bone biopsied from the iliac crest after in vivo tetracycline labeling permits separate assessments of cortical and cancellous bone, a The Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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C~PTER 26

TABLE 1

Histomorphometric Indices of Bone Mass, Architecture, and Turnover

Index (abbreviation) Cancellous bone area (Cn BA/TA) Cortical width (Ct.wi) Total bone density (TBD) Trabecular number (Tb.N) Trabecular width (Tb.wi) Trabecular separation (Tb.Sp)

Marrow space star volume (Vm.space) Total strut length a (TSL) Node number a (N.Nd) Terminus number a (N.Tm) Node-to-node strut a (Nd.Nd) Node-to-terminus strut a (Nd.Tm) Node-to-terminus ratio a (N.Nd/N.Tm) Terminus-to-terminus strut a (Tm.Tm) Wall width (W.wi) Osteoid perimeter (OS/BS) Osteoid area (OA/BA) Osteoid width (O.wi) Eroded perimeter (ES/BS) Mineralizing perimeter (MS/BS) Bone formation rate, tissue level (BFR/BS) Mineral apposition rate (MAR) Adjusted apposition rate (AJ.AR) Activation frequency Mineralization lag time Osteoid maturation time Total formation period Active formation period

Resorption period

Remodeling period

Unit % ta,m % /mm t~m ~m mm 3 mm/mm 2 /mm 2 /mm 2 mm/mm 2 mm/mm 2 mm/mm 2 ~m % % #m % % i~m3/#m2/day iJ,m/day #m3/l~m2/day /year days days days days days days

aSee Fig. 4.

the patient in two time-spaced doses before the biopsy is obtained. Tetracycline is avidly taken up by bone and is deposited at sites of active bone formation, where it is readily visualized by fluorescence microscopy. Dynamic indices are as follows: (1) mineralizing perimeter is the extent of tetracycline-labeled surface relative to the total trabecular surface; (2) bone formation rate (tissue based) is the volume of mineralized bone formed per unit of bone surface per day (bone formation rate is derived from the measurement of the mineralizing perimeter and the calculation of the rate of mineral apposition); (3) adjusted apposition rate is the volume of mineralized bone formed per unit of osteoid-covered bone surface per day. Other variables of bone structure have been used to study cancellous bone architecture.

These variables include trabecular number, trabecular width, trabecular separation, and, more recently, marrow star volume (25,28-32), total strut length, node number, node-to-node strut, node-to-terminus and terminus-toterminus strut lengths, and node-to-terminus ratio (33-35) (Table 1). All these indices are obtained using manual stereologic methods or computer-assisted or automated image analysis technology. The values obtained are compared to those of age- and sex-matched controls from the relevant normal population.

BONE TURNOVER IN PRIMARY HYPERPARATHYROIDISM An increase in the rate of bone turnover is the hallmark of skeletal involvement in primary hyperparathyroidism (36). Among histomorphometric studies performed three decades ago, the classic studies of Meunier, Melsen, Mosekilde, and colleagues have contributed importantly to our present understanding of bone turnover in this disease (37-41). Studies conducted over the past 15 years have confirmed the accelerated rate of bone remodeling in primary hyperparathyroidism (42-50). In a group of subjects with mild primary hyperparathyroidism, there was a two- to threefold increase in the values of static and dynamic turnover indices (Fig. 1). Moreover, osteoid perimeter, osteoid area, mineralizing perimeter, and tissue-based bone formation rate correlated well with concentrations of PTH as determined by midmolecule radioimmunoassay. Eroded perimeter and mineralizing perimeter correlated well with serum calcium and urinary adenosine m o n o p h o s p h a t e (AMP) values, respectively (49). These correlations are highly reflective of the response of the skeleton to PTH, in spite of only minimal clinical manifestations and the complete absence of radiologic signs of bone disease (6,49). Further quantitative evidence of PTH action on bone was provided by correlations between PTH or 1,25dihydroxyvitamin D [1,25 (OH) zD ] concentrations and static and dynamic indices of bone turnover (44,50,51).

B O N E MASS I N PRIMARY HYPERPARATHYROIDISM Cortical B o n e Studies employing densitometric techniques to measure bone mass have shown a decrease in bone density at the appendicular skeleton in primary hyperparathyroidism (6,20-23). A reduction at this site, which is made up primarily of cortical bone, is in keeping with physiologic actions of PTH to act preferentially on cortical bone. Further documentation of cortical bone loss was

BONE MORPHOMETRY IN HYPERPARATHYROIDISM

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FIG. 1 (A) Low-power photomicrograph illustrating the extended osteoid perimeter (stained red) on mineralized trabecular surfaces (stained green). Original magnification, x6. (B) Extended eroded perimeter (ES) appearing as irregular scalloped surface covered by osteoclasts (OC). Original magnification, x25. (C) Extended osteoid perimeter (OS) seen at higher magnification. Peritrabecular fibrosis (arrows). Original magnification, x25. Goldner's trichrome. (D) Extended double tetracycline labels (DL) seen by fluorescence microscopy covering trabecular surfaces. Original magnification, x25. Unstained section. (See color plates.)

obtained by analysis of biopsy specimens of iliac crest bone in which several studies, including our own, have shown that cortices are thinner (Figs. 2 and 3) (49,52-54) or excessively porous in primary hyperparathyroidism (51,54). A catabolic effect of excessive PTH secretion on cortical bone was also suggested by van D o o m et al. (51), who observed a positive correlation between circulating intact PTH and cortical porosity.

Cancellous B o n e In spite of a substantial increase in bone turnover, cancellous bone volume is maintained and may even be higher than normal in primary hyperparathyroidism (23,41-45,47,49-51,53,55,56) (Fig. 2). The mean volume of cancellous bone in subjects with primary hyperparathyroidism exceeded control values by more than 2 SD in 32% of patients in the Delling (45) study and in 26% of patients in our study (49) (Fig. 3). An increase in cancellous bone volume was reflected in greater compressive strength, trabecular ash weight, and maxim u m stress in iliac biopsy cores (57). These observations support the view that modest elevations of endogenous PTH exert a dual action on bone, with a preferential loss of cortical bone and conservation of

cancellous bone (49). Clinical data from our work reinforce this concept, as shown by the general maintenance of bone mineral density in the spine, which is well endowed with cancellous bone, as compared to a substantial decline of bone mineral density at the distal radius, a site rich in cortical bone (6) (see Chapter 20). An important clinical aspect of these observations that remains controversial relates to the incidence of cancellous bone fracture in primary hyperparathyroidism. Although the few published reports have shown no increase in the rate of fracture after diagnosis and conservative treatment of primary hyperparathyroidism (16,17), an epidemiologic study has shown a substantial increase in the overall risk of fractures, particularly at the vertebrae, distal forearm, and proximal femur (58).

B O N E S T R U C T U R E IN PRIMARY HYPERPARATHYROIDISM Conventional Indices o f B o n e Structure With the recognition that bone structure, in addition to bone mass, is an important determinant of bone strength, quantitative analysis of bone microarchitecture

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CFIAPTER26

FIG. 2 Scanning electron micrographs of iliac core biopsies of a hyperparathyroid patient (top) and of a control subject (bottom). The sample on the top was obtained from a 38year-old woman with PHPT. The one on the bottom was obtained at autopsy from a 27-year-old woman who died suddenly without evidence of bone disease. Note the thinning of cortices in the PHPT sample as well as the maintenance of cancellous bone and trabecular connectivity. Samples were prepared as per Dempster et aL Reproduced from Ref. 49; Parisien M et al. The histomorphometry of bone in primary hyperparathyroidism: Preservation of cancellous structure. J Clin Endocrinol Metab 1990;70(4):930-938. © The Endocrine Society.

in iliac biopsy specimens has provided further insight to our understanding of metabolic bone disease. Through primary measurements of cancellous bone area and bone perimeter, our study of patients with primary hyperparathyroidism showed a greater n u m b e r of trabeculae with a reciprocal reduction in trabecular separation. In addition, trabecular plates were somewhat thinner than in normal subjects (49,56) (Fig. 3). When these indices were evaluated as a function of age, very different patterns between hyperparathyroid and control subjects were seen. Cancellous bone volume declined in both groups. However, trabecular n u m b e r did not decline with age in hyperparathyroid subjects, in contrast to normal individuals. Moreover, trabecular separation increased as a function of age in the normal subjects

but not in the patients with primary hyperparathyroidism. Finally, trabecular thickness did not change in the control group but showed an age-related decline in the hyperparathyroid subjects. These observations clearly suggest that modestly elevated levels of PTH can retard several normal processes of aging with respect to the microarchitecture of trabecular bone. The profile of structural changes that we observed with age in normal subjects conforms to that previously described by Wakamatsu and Sissons (28) and later by Parfitt et al. (29). This profile is the basis for the proposed mechanism of bone loss with aging in normal subjects, particularly in women. According to this mechanism, bone is lost by way of increased spacing between the trabeculae, but without significant reduction in plate thickness (28). Parfitt et al. (29) later (1983) refined this concept and postulated that, with age, cancellous bone is lost by the removal of entire trabecular units through enhanced resorption, resulting in increased intertrabecular separation without significant reduction in plate thickness. The age-related pattern of bone loss in subjects with primary hyperparathyroidism differs dramatically from that which occurs in normal subjects (30,49). In primary hyperparathyroidism, cancellous bone structure appears to be preserved through the maintenance of trabecular plates, which remain connected despite agerelated thinning (49). This concept is supported by the findings of Christiansen et al. (50). This pattern of bone loss is more consistent with the normal aging pattern observed in men as opposed to women (28,29,35, 59-61). However, although two i n d e p e n d e n t groups have now reported trabecular thinning in this disease, it should be noted that Delling (45) found an increase in trabecular thickness as the underlying structural c o m p o n e n t of the higher cancellous bone volume in primary hyperparathyroidism. In the study of Christiansen et al. (50) involving 69 patients with primary hyperparathyroidism, normal cancellous bone volume was observed with no disturbance of the normal cancellous network. Normal bone structure was clearly maintained in spite of apparently more severe disease in this population, as j u d g e d by higher values of serum calcium and the greater prevalence of complications than are reported in other studies of mild primary hyperparathyroidism (16,49). Quantitative analysis of bone structure indicated normal trabecular microarchitecture, with no decrease in cancellous bone volume or trabecular number, as shown in Table 2. The only abnormality was the presence of significantly thinner plates than normal, with no change in intertrabecular separation (50). Furthermore, there was an age-related decline of cancellous bone volume in both patients and controls, with preservation of the normal architecture in hyperparathyroid subjects but a contrasting loss of

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structure with age in the controls (50) (Fig. 4), confirming previous findings (28-30,49).

Indices o f Connectivity: Trabecular Strut Analysis Examination of iliac crest bone biopsies from patients with primary hyperparathyroidism suggests that the plates are remarkably well connected. Twodimensional trabecular strut analysis has provided support for this observation. This technique permits quantification of the n u m b e r of connecting points or nodes between trabecular segments, the free ends of unconnected trabeculae or termini, the relative lengths of trabecular struts showing various degrees of connectivity, and the total length of all the trabecular struts (33,34) (Fig. 5). As shown in Fig. 6, it can be appreciated that all indices of connectivity are greater in patients with primary hyperparathyroidism than in controls. These

Control

FIG. 3 Structural indices in primary hyperparathyroidism (see Table 1). Comparison between patients (PHPT) and controls. Values are expressed as mean _ SEM; NS, not significant. Reproduced from Ref. 49; Parisien M, et aL The histomorphometry of bone in primary hyperparathyroidism: Preservation of cancellous structure. J Clin Endocrinol Metab 1990;70(4):930-938. © The Endocrine Society.

findings are consistent with higher cancellous bone volume and greater degree of trabecular connectivity in primary hyperparathyroidism patients than in normal individuals. Moreover, total strut length, an index of the amount of cancellous bone, declined with age, confirming the age-related loss of cancellous bone volume in both hyperparathyroidism and control subjects. However, though a loss of trabecular connectivity was shown in the controls, no such decline was observed in the hyperparathyroid group (55) (Table 3).

MECHANISM OF MAINTENANCE OF BONE VOLUME AND STRUCTURE It now seems well established that in primary hyperparathyroidism, cancellous bone volume is normal or increased despite higher bone turnover. Possible mechanisms for the maintenance of normal bone balance are

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CRAPTF~k 26 TABLE 2

Parameters Characterizing the Iliac Trabecular Bone Structure in Primary Hyperparathyroidism Compared with Normal Controls Matched for Age and Sex Normal controls b

PHP patients ° Parameter a

All

Trabecular bone volume (BV/TV, %)

Males

20.6 _+ 7.1

Trabecular thickness (T.Th, i~m) Mean trabecular plate density (MTPD, i~m1)

124 +_ 33 1.33 _+ 0.36

Intertrabecular distance (IT.D, i~m) Marrow space star volume

648 _ 154 14.4 +_ 14.0

( Vm.space"mm 3)

Females

21.3 _+ 4.2 (20)

20.3 _+ 8.0 (47)

117 _ 21 (21)1 1.44 _ 0.29 (20)

127 _+ 48 (45) 1.28 _ 0.38 (44)

628 _+ 140 (20) 7.2 _+ 5.0 (11)

659 _+ 160 (40) 17.6 _+ 15.6 (24)

All

Males

21.5 _+ 8.7

Females

21.8 _ 7.2 (10)

139 _+ 32*

21.3 _+ 9.3 (20) 141 _ 32 (20)

135 _ 33 (10) 1.26 +_ 0.26 (10)

1.22 _ 0.42 632 ___156

1.20 _ 0.49 (2O) 623 _+ 172 (19)

650 _+ 126 (10) 13.8 _+ 12.2 (8)

15.9 _ 14.5

18.2 _ 17.5

(7)

aTrabecular bone volume (BV/TV) = trabecular bone area (BA/TA); trabecular thickness (T.Th) = trabecular width; mean trabecular plate density (MTPD) = trabecular number (Tb.N); intertrabecular distance (IT.D) = trabecular separation (Tb.Sp). Reprinted from Bone, Vol. 13, Christiansen et aL, Primary hyperparathyroidism: Iliac crest trabecular bone volume, structure, remodeling, and balance evaluated by histomorphometric methods, pp. 41-49. Copyright 1992, with permission from Elsevier Science (50). ~Number of males and females in each group shown in parentheses.

results in normal or slightly increased bone balance. Formation period is not significantly different from normal. According to this model, a smaller a m o u n t of bone is exchanged during the remodeling sequence, which is compensated for at the tissue level by increased bone turnover (Fig. 7). The trend toward a reduction in cancellous bone volume (reversible bone loss) is attributed to the expansion of the remodeling space (48). This mechanism contrasts with that observed in hyperthyroidism, in which normal erosion depth and decreased estimated completed wall width result in a net negative

still speculative. Delling (45) has suggested that intact coupling between erosion and formation together with increases in the activity and life span of osteoblasts will maintain bone structure. Baron and Magee (46) suggest a coupled and balanced increase in bone turnover. Through reconstruction of the remodeling sequence in primary hyperparathyroidism, Eriksen and co-workers (47,48) proposed that the activation frequency of remodeling cycles is increased, while the final erosion depth and wall width of completed structural units are decreased. This protective mechanism against bone loss

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FIG. 4 Mean trabecular plate density (trabecular number) and marrow space star volume in relation to age in women with primary hyperparathyroidism and normal subjects. A decrease in trabecular number and increase in marrow space star volume with age are observed in the control groups (dashed lines) but not in the patients. Modified from Bone, Vol. 13, Christiansen et al., Primary hyperparathyroidism: Iliac "crest trabecular bone volume, structure, remodeling, and balance evaluated by histomorphometric methods, pp. 41-49. Copyright 1992, with permission from Elsevier Science (50).

A

80

BONE M O R P H O M E T R Y

.,~

FIG. 5 Nodes, termini, and trabecular struts are drawn from the photomicrograph of the iliac crest biopsy of a subject with primary hyperparathyroidism. Abbreviations: Nd, node; Tm, terminus; Nd.Nd, node-to-node strut; Nd.Tm, node-toterminus strut. Reproduced from Ref. 55; J Bone Miner Res 1992;7:913-919 with permission from the American Society for Bone and Mineral Research.

balance between erosion and formation at each remodeling cycle. This process is c o m p o u n d e d in hyperthyroidism by the reduction in bone formation period and the increased activation frequency, resulting in increased trabecular perforation and irreversible bone loss (47,48). T h o u g h the data on cancellous bone volume in primary hyperparathyroidism are not disputed, those related to wall width of cancellous bone remain controversial. We have reported an increase in wall width as the underlying mechanism of conservation of cancellous bone in this disease (62), confirming previous findings (63). However, in our study, greater wall width was associated with thinner trabecular plates (62), in contrast with thicker plates observed in the Vogel et al. study (63). The reason for this discrepancy is not clear and, at present, we have no direct information on whether the greater final amount of bone is the result of a positive bone balance in each remodeling unit or that of de novo bone formation, because in neither study was depth of erosion measured (62,63). Furthermore, in our study, we found that the active formation period was longer in hyperparathyroid than in normal women. This indicates that in primary hyperparathyroidism, the osteoblasts 1.8 A N

E

1.5

E E

1,2

=

0.9

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,--

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429

spend a greater amount of time in the active mode, forming bone matrix, than in the inactive mode, resulting in greater adjusted apposition rate (62). The Christiansen et al. (50) study of primary hyperparathyroidism shows increased activation frequency with a corresponding decrease in the extent of the quiescent surface, which further documents expansion of the remodeling space. In that study, however, there was no difference in the final depth of erosion or in the final a m o u n t of bone deposited at the bone remodeling unit (wall width) between patients and controls and, hence, no change in bone balance or in cancellous bone volume (50). These observations give credence to the concept of "reversible bone loss" in primary hyperparathyroidism. In further support of this concept, an increase in cancellous bone volume in a small subset of patients following successful parathyroidectomy was attributed to normalization of bone turnover and, consequently, to the reduction of an expanded remodeling space (64). In a more recent study by the same investigators, a larger group of 24 hyperparathyroid subjects showed a decline in biochemical markers of bone turnover within 6 months of parathyroidectomy, indicating a return of bone remodeling to normal (65). These changes occurred in parallel with an increase in bone mineral density (BMD) at the spine and at the hip. T h o u g h short-term increases in bone density may reflect a decrease in the previously expanded remodeling space (reversible bone loss), this concept does not satisfactorily explain long-term increases in cancellous bone density, such as observed in follow-up investigations by our group (66,67). In the study by Silverberg et al., 61 patients treated by successful parathyroid surgery and followed for up to 10 years have shown sustained increases in bone mineral density at sites rich in cancellous bone, such as the spine and the hip, but not at sites rich in cortical bone. These postoperative increases, which were also seen in a subset of postmenopausal women with primary hyperparathyroidism, contrasted with the absence of any

1.4

-[-

IN HYPERPARATHYROIDISM

Control

C

0.2 0.0

PHPT

Control

FIG. 6 Bar graphs representing the values of (A) total strut length, an index of the amount of cancellous bone, (B) node number, and (C) node to node strut length; indices shown in B and C are two indices of trabecular connectivity in patients with primary hyperparathyroidism.

430

/

CHAPTER26 TABLE 3 Age PHPT r P Control r P

Correlations between Age and Variables Expressing Connectivity in PHPT and Controlsa

TSL (mm/mm 2)

N.Nd (/ram 2)

N.Tm (/ram2)

N.Nd/N.Tm

-0.38 0.020

-0.28 NS

0.014 NS

-0.08 NS

-0.44 0.030

-0.42 0.043

0.117 NS

-0.46 0.033

Nd.Nd (mm/mm 2)

Nd.Tm (mm/mm 2)

Tm.Tm (ram/ram 2)

-0.35 0.032

-0.0009 NS

0.114 NS

-0.43 0.033

-0.077 NS

0.205 NS

aN.Nd, Node number; N.Tm, terminus number; TSL, total strut length; N.Nd/N.Tm, node-to-terminus ratio; Nd.Nd, node-to-node strut length" Nd.Tm, node-to-terminus strut length; Tm.Tm, terminus-to-terminus strut length. Values are mean +_ SD; NS, not significant: Reproduced from Parisien M, et aL J Bone Miner Res 1992;7:913-919 with permission from the American Society for Bone and Mineral Research (Ref. 55).

substantial change in 60 subjects treated conservatively (67). In the light of the normal or even greater a m o u n t of cancellous bone universally observed in histomorphometric studies of mild primary hyperparathyroidism (41-45,47,49-51,53,55), it appears that the sustained, rather than transient, nature of the postoperative increase in bone density most likely reflects a true anabolic effect of parathyroid hormone. Such an effect may be sustained postoperatively in spite of lower levels of circulating hormone. Proposed mechanisms for this effect include restoration of normal pulsatility and circadian dynamics of parathyroid h o r m o n e as well as other possibilities such as increased mineralization density as a result of the decrease in activation frequency (see Chapter 23).

What is the cellular mechanism for the maintenance of trabecular connectivity, and how does one reconcile the paradox that trabecular plates become thinner but remain connected with the increased turnover state of primary hyperparathyroidism? The plate thinning reported by Christiansen et al. (50) and the age-related attenuation of plates observed in our study (49) could well be consequences of a twofold increase in activation frequency with substantial enlargement of the remodeling space. If one considers the stochastic model of Reeve (68), the increase in birth rate of teams of osteoclasts would greatly increase the probability of erosion on opposite sides of a plate and hence of plate perforation (59,69). The likelihood of such perforation might be further increased by the coincidental occurrence of

H ~=0

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FIG. 7 Complete remodeling sequences in primary hyperparathyroidism (H) (top) and age- and sex-matched normal controls (N) (bottom). Stippled area represents the thickness of mineralized bone. The different phases of bone resorption and bone formation are indicated: osteoclastic function period (I), mononuclear function period (11), preosteoblast function period (111),initial mineralization lag time (IV), and mineralization period (V). Reprinted from Bone, Vol. 7, Eriksen et aL, Trabecular bone remodeling and balance in primary hyperparathyroidism, pp. 213-221. Copyright 1986, with permission from Elsevier Science (47).

BoNw MORPHOMETRYIN HYPERPARATHYROIDISM / deep erosion on thin plates (70). This might be expected from the known aggressive behavior of the osteoclast in more severe forms of primary hyperparathyroidism. However, a lesser degree of osteoclastic activity is suggested in the milder, more c o m m o n form of the disease (47). Under these circumstances, the resulting reduction in the final depth of erosion might offer a mechanism whereby trabecular plates would be protected against perforation (69). This protective mechanism would be operative despite the predisposition to plate thinning caused by the greater activation frequency. A correlative mechanism of protection, inferred from the pattern of "tunneling resorption" of the more severe form of the disease, would be that osteoclasts alter their behavior under the influence of excessive endogenous PTH secretion. They appear to change direction, eroding in a direction parallel to the long axis of the trabecular plate rather than to penetrate in perpendicular fashion across the full thickness of the plate. In doing so, the osteoclasts create long furrows in the interior of the trabecular plate (Fig. 8) (12,36,71,72). This mechanism is compatible with Eriksen's finding of decreased erosion depth (47,48). Under conditions of moderate PTH excess, the change in osteoclast behavior would result in a more tangential pattern of "sweeping erosion," quantitatively translated by more shallow erosion lacunae, thereby allowing the plates to remain connected, although they are thinner than normal. Paradoxically, under the influence of PTH, osteoclasts are thought to exhibit more aggressive behavior along the corticomedullary junction, where they appear to erode more deeply and extensively. This is the presumed mechanism underlying the thinning of cortices observed in primary hyperparathyroidism (49,53). These findings suggest that the effects of PTH on osteoclast recruitment a n d / o r activity differ when cortical and cancellous

OF

~

~

PM

FIG. 8 Two morphologically different types of increased bone resorption in severe primary hyperparathyroidism (osteitis fibrosa; OF) and in the postmenopausal state (PM). In OF, the osteoclasts have eroded more deeply into the bone and then changed direction to undermine the surface by intratrabecular "dissecting" erosion. In PM, the osteoclasts have eroded a deeper than normal cavity on the way to coplate perforation of the trabecular plate. The predominant direction of osteoclast movement is perpendicular to the surface. Modified from Ref. 72, AM Parfitt, Relationship to the amount and structure of bone, and the pathogenesis and prevention of fractures. In: Riggs BL, Melton III LJ, eds. Osteoporosis, etiology, diagnosis, and management. New York: Raven, 1988:45-93, with permission of Lippincott.

431

envelopes are compared. The slower, more modest increments in bone mass or complete lack of gain at sites richer in cortical bone following parathyroidectomy (66,67,73) probably reflect the greater osteoclastic activity at the corticomedullary junction a n d / o r lower bone turnover rates of cortical bone compared to cancellous bone. An anabolic action of PTH on bone is suggested by histologic evidence of increased bone-forming surfaces and mineral apposition rate following intermittent administration of PTH to rats (74), an effect that is not altered by antiresorptive agents (75). Furthermore, though continuous exposure to PTH causes inhibition of collagen synthesis in cultured fetal rat calvariae (76,77), transient exposure to PTH stimulates collagen synthesis (78). This anabolic effect is presumably the basis for the increased bone density and calcium accretion observed after chronic administration of PTH to rats (79). In human subjects with primary hyperparathyroidism, the development of osteosclerosis in the axial skeleton and femora of adults (80-86) and in the long bone metaphyses of children (87-92) are further evidence of this anabolic response. An even more compelling argument is the greater bone density and increase in new bone accretion (by 47Cakinetic studies) as well as the histomorphometric evidence of increased cancellous bone volume following treatment of osteoporotic subjects with PTH(1-34) (93-97) (see Chapter 55). Convincing evidence of increased cancellous bone volume and trabecular connectivity was also shown in a rat experimental model of osteoporosis after administration of PTH (1-34) (98,99).

CLINIC.zkL R E L E V A N C E O F HISTOMORPHOMETRIC FINDINGS IN PRIMARY HYPERPARATHYROIDISM The relevance of the maintenance of cancellous bone mass as well as structural integrity in primary hyperparathyroidism with regard to sites of potential fracture deserves clinical consideration. The suitability of the iliac crest as a representative site to study cancellous bone compared to more directly relevant sites such as the spine has been previously noted (25,31,34,100-103). The maintenance or even increase of trabecular connectivity (55) and greater compressive strength and ash weight at the iliac crest in primary hyperparathyroidism (57), validated by the concordance between indices of bone mass and structure at these two sites in normal subjects (31,34), suggest the strong possibility that cancellous architecture and strength are maintained at the spine as well. This maintenance is further supported by the positive correlations found in women with primary hyperparathyroidism between conventional indices of bone mass and structure and bone density measurements at the

432

/

CI-~a~wl~26

lumbar spine, a site of high cancellous bone composition, but not at the hip and forearm (104,105).

Postmenopausal Women with Primary Hyperparathyroidism Data supporting the idea that cancellous bone is protected in primary hyperparathyroidism have particular relevance for postmenopausal women, who are at increased risk for cancellous bone loss due to estrogen deficiency. If mild primary hyperparathyroidism is a relative protector of the cancellous skeleton, will postmenopausal women with primary hyperparathyroidism show better indices of cancellous bone mass and architecture than normal postmenopausal women without primary hyperparathyroidism? To study this issue, we compared a group of postmenopausal women with primary hyperparathyroidism with a cohort of healthy postmenopausal women. When the two groups were matched by age and by n u m b e r of years after menopause, cancellous bone volume was greater in hyperparathyroid women than in women without primary hyperparathyroidism (106), in keeping with previous findings (107). Trabecular n u m b e r did not show the age-related decline observed in the normal population. This finding is of great clinical significance,

because trabecular n u m b e r was found to be a better discriminator than cancellous bone volume when distinguishing between those subjects with vertebral fractures and normal subjects (59). The demonstration of greater trabecular connectivity in a heterogeneous group of patients with primary hyperparathyroidism is strong evidence that cancellous integrity is maintained in primary hyperparathyroidism (55). To d o c u m e n t these findings more convincingly, we have compared three groups of postmenopausal women matched by n u m b e r of years since menopause: one composed of a subset of 16 women with primary hyperparathyroidism, the other two each composed of an equivalent n u m b e r of healthy and osteoporotic women. In this three-way comparison, cancellous bone volume and indices of connectivity were lowest in the osteoporotic group and highest in the hyperparathyroid subjects, The converse was true for indices of disconnectivity (Figs. 9 and 10). These findings indicate that in osteoporosis there is an irreversible loss of bone with deterioration of trabecular architecture due to loss of connectivity, whereas in primary hyperparathyroidism there is maintenance of the architectural integrity of cancellous bone, with a degree of plate connectivity similar to, or even greater than, normal, in spite of higher bone turnover (55,108) (Fig. 10).

A

"

i.........i!

£ FIG. 9 Photomicrographs of the iliac crest biopsies of three postmenopausal women: osteoporotic (A), hyperparathyroid (B), and normal (C). Reproduced from Ref. 108; J Bone Miner Res 1995;10:1393-1399 with permission from the American Society for Bone and Mineral Research.

BONE MORPHOMETRY IN HYPERPARATHYROIDISM

A

Bone Formation Rate A

p
0.14-

¢u

Cancellous Bone Volume 30-

10 0.12 E

0.10-

o~

E

o.o6-

t~ m

0.04 -

tr

0.02 -

i.i. m

20-

< !-

0.08-

15-

< m

10-

B

Normal

OP

~"~

p
PHPT

1.8 -

1.8 ~

1.5 -

E 0.9 "0 Z 0.6 Z 0.3 -

-

Normal

OP

Node/Terminus Ratio

Node Number

0.0

i

0

PHPT

~E 1.2

~ ~i ~%

5"

0

A

p<0.0001

25-

A

p<0.002 E

..........t ..........

1.5

-

1.2

-

p<0.003

0.9 -

z. 0.6 = 0.3 -

p~.ool PHPT

0.0

PHPT

OP

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p
I)<0,003 Normal

I

OP

Node to Node Strut Length 1.0

p<0.001

-

A

NEE 0.8 i

0.6

-

E

"o 0.4 Z

-,lI

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PHPT

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C 1.8 1.5 A I:: 1 . 2 -

"

~,'~.,,,~

I

0.9 -

///// ///i/

0.20 I

///././. ;';'~'~';"

-

0.3

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0.10

-

p
~='

///// .....

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i

E 0.05

///_,,-_/_/ ////

PHPT

I

¢~ E 0.15-

I-

//I//

0.0

p
E

p
"

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Terminus to Terminus Strut Length

p
E

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I:)<0.05

0.00

OP

PHPT

Normal

OP

FIG. 10 Histomorphometric indexes of bone turnover, mass, and structure in three groups of postmenopausal women: hyperparathyroid, osteoporotic, and normal. (A) Bone formation rate and cancellous bone volume. (B) Node number, node-to-terminus ratio, and node-to-node strut length. (C) Terminus number and terminus-to-terminus strut length. Values expressed as mean _+ SEM. Reproduced from Ref. 108; J B o n e M i n e r R e s 1 9 9 5 ; 1 0 : 1 3 9 3 - 1 3 9 9 with permission from the American Society for Bone and Mineral Research.

/

433

434

/

CHAPTER26

If bone mass and structure are predictive of bone strength, the evidence accumulated over the past two decades clearly supports the concept that mild primary hyperparathyroidism exerts a protective effect on cancellous bone. W h e t h e r postmenopausal women with this disease are less susceptible to vertebral fracture and whether there is truth in the logical assumption of a greater risk of peripheral fractures await confirmation by long-term, randomized, controlled, clinical trials.

16.

17.

18. 19.

ACKNOWLEDGMENTS 20.

Some of the work covered in this chapter was supported, in part, by NIH grants AR 41386, DK 32333, AR 39191, and RR 00645.

21.

22.

REFERENCES 1. Heath H, Hodgson SF, Kennedy MA. Primary hyperparathyroidism incidence, morbidity, and potential economic impact in a community. N EnglJ Med 1980;302:189-193. 2. Mundy GR, Cove DH, Fisken R. Primary hyperparathyroidism: Changes in the pattern of clinical presentation. Lancet 1980;1:1318-1320. 3. Mallette LE, Bilezikian JR Heath DA, Aurbach GD. Primary hyperparathyroidism: Clinical and biochemical features. Medicine 1974;53:127-146. 4. Lafferty FW. Primary hyperparathyroidism: Changing clinical spectrum, prevalence of hypertension, and discriminant analysis of laboratory tests. Arch Intern Med 1981; 141:1761-1766. 5. Sudhaker RD. Primary hyperparathyroidism: Changing patterns in presentation and treatment decisions in the eighties. Henry Ford Hosp MedJ1985;33:194-197. 6. Silverberg SJ, Shane E, De La Cruz L, et al. Skeletal disease in primary hyperparathyroidism. J Bone Miner Res 1989;4:283-291. 7. von Recklinghausen E Die fibrose oder deformirende Ostitis, die Osteomalacie und die carcinose in gengensitigen. In: Beziehungen, ed. Festschrifi Rudolf Virchow. Berlin:G.Reiner, 1891. 8. Mandl R. Therapeutisher verusch bei ostitis fibrosa generalizata mittels extirpation eines epithelkorperchen tumors. Wien Klin Wochenschr 1925;38:1383-1384. 9. Albright E AubJC, Bauer W. Hyperparathyroidism: A common and polymorphic condition as illustrated by seventeen proven cases from one clinic. J Am Med Assoc 1934; 102:1276-1287. 10. Albright F, Reifenstein EC. The parathyroid glands and metabolic bone disease. Baltimore:Williams & Wilkins, 1948. 11. Schajowicz E Tumors and tumor-like lesions of bones and joints. Berlin:Springer-Verlag, 1981. 12. Uehlinger E. Osteofibrosis deformans juvenilis. Virchows Arch 1940;306:255. 13. Dauphine RT, Riggs BL, Scholz DA. Back pain and vertebral crush fractures: An unemphasized mode of presentation for primary hyperparathyroidism. Ann Intern Med 1975:83:365-367. 14. Kochersberger G, Buckley NJ, Leight GS. What is the clinical significance of bone loss in primary hyperparathyroidism? Arch Intern Med 1987;147:1951-1953. 15. Peacock M, Horsman A, Aaron JE, Marshall DH, Selby PL, Simpson M. The role of parathyroid hormone in bone loss. In: Christiansen C, Arnaud CD, Nordin BEC, Parfitt AM, Peck

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33.

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52. Parfitt AM. Accelerated cortical bone loss: Primary and secondary hyperparathyroidism. In: Uhthoff H, Stahl E, eds. Current concepts of bone fragility. Berlin:Springer-Verlag, 1986:279-285. 53. Parfitt AM. Surface specific bone remodeling in health and disease. In: Kleerekoper M, ed. Clinical disorders of bone and mineral metabolism. New York:Mary Ann Liebert, 1989:7-14. 54. Brockstedt H, Christiansen P, Mosekilde L, Melsen E Reconstruction of cortical bone remodeling in untreated primary hyperparathyroidism and following surgery. Bone 1995;16:109-117. 55. Parisien M, Mellish RWE, Silverberg SJ, et al. Maintenance of cancellous bone connectivity in primary hyperparathyroidism: Trabecular strut analysis. J Bone Miner Res 1992;7:913-919. 56. Parisien M, Dempster DW, Shane E, Silverberg S, Lindsay R, Bilezikian JE Structural parameters of bone biopsies in primary hyperparathyroidism. In: Takahashi HE, ed. Bone morphometry (Proceedings of the Fifth International Congress on Bone Morphometry, Niigata, Japan). New York:Smith-Gordon, 1988:228-231. 57. Mosekilde LE, Mosekilde L. Iliac crest trabecular bone compressive strength and ash weight is increased in moderate primary hyperparathyroidism. In: Takahashi HE, ed. Bone morphometry (Proceedings of the Fifth International Congress on Bone Morphometry, Niigata, Japan). New York:Smith-Gordon, 1988:483. 58. Khosla S, Melton III J, Wermers RA, Crowson CS, O'Fallon M, Riggs BL. Primary hyperparathyroidism and the risk of fracture: A population-based study. JBone Miner Res 1999;14:1700-1707. 59. Kleerekoper M, Villaneuva AR, Stanciu J, Rao DS, Parfitt AM. The role of three-dimensional trabecular microstructure in the pathogenesis of vertebral compression fractures. Calcif Tissue Int 1985;37:594-597. 60. Parfitt AM. Trabecular bone architecture in the pathogenesis and prevention of fracture. A m J M e d 1987;82:68-72. 61. Mellish RWE, Garrahan NJ, Compston JE. Age-related changes in trabecular width and spacing in human iliac crest biopsies. Bone Miner 1989;6:331-338. 62. Dempster DW, Parisien M, Silverberg SJ, Liang X-G, Schnitzer M, Shen V, Shane E, Kimmel DB, Recker R, Lindsay R, Bilezikian JE On the mechanism of cancellous bone preservation in postmenopausal women with mild primary hyperparathyroidism. J Clin Endocrinol Metab 1999;84:1562-1566. 63. Vogel M, Hahn M, Delling G. Trabecular bone structure in patients with primary hyperparathyroidism. Virchows Arch 1995;426:127-134. 64. Christiansen P, Steiniche T, Mosekilde Le, Hessov I, Melsen E Primary hyperparathyroidism: Changes in trabecular bone remodeling following surgical treatment evaluated by histomorphometric methods. Bone 1990;11:75-79. 65. Christiansen P, Steiniche T, Brixen K, Hessov I, Melsen F, Heickendorff, Mosekilde LE. Primary hyperparathyroidism: Short-term changes in bone remodeling and bone mineral density following parathyroidectomy. Bone 1999;25:237-244. 66. Silverberg SJ, Gartenberg F, Jacobs TP, Shane E, Siris E, Staron RB, McMahon DJ, Bilezikian JE Increased bone mineral density after parathyroidectomy in primary hyperparathyroidism. J Clin Endocrinol Metab 1995;80:729-734. 67. Silverberg SJ, Shane E, Jacobs TP, Siris E, Bilezikian JP. A 10 year prospective study of primary hyperparathyroidism with or without parathyroid surgery. N Engl J Med 1999;341: 1249-1255. 68. Reeve J. A stochastic analysis of iliac trabecular bone dynamics. Clin Orthop 1986;213:264-278. 69. Parfitt AM. Implications of architecture for the pathogenesis and prevention of vertebral fracture. Bone 1992; 13:$41-$47. 70. Compston JE, Mellish RWE, Croucher P, Newcombe R, Garrahan NJ. Structural mechanisms of trabecular bone loss in man. Bone Miner 1989;6:339-350.

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71. Parfitt AM, Oliver I, Villanueva ADR. Bone histology in metabolic bone disease. Orthop Clin North Am 1979:10:329-345. 72. Parfitt AM. Relationship to the amount and structure of bone, and the pathogenesis and prevention of fractures. In: Riggs BL, Melton III LJ, eds. Osteoporosis, etiology, diagnosis, and management. New York:Raven, 1988:45-93. 73. Martin P, Bergmann P, Gillet C, Fuss M, Corvilain J, Van Geertruyden Jean. Long-term irreversibility of bone loss after surgery for primary hyperparathyroidism. Arch Intern Med 1990;150:1495-1497. 74. Tam CS, Heersche JNM, Murray TM, Parsons JA, Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: Differential effects of intermittent and continuous administration. Endocrinology 1982;110:506. 75. Hock JM, Hummert JR, Boyce R, Fonseca J, Raisz LG. Resorption is not essential for the stimulation of bone growth by hPTH-(1-34) in rats in vivo.JBone Miner Res 1989;4:449. 76. Kream BE, Rowe RW, Gworek SC, Raisz LG. Parathyroid hormone alters collagen synthesis and procollagen mRNA levels in fetal rat calvariae. Proc Natl Acad Sci USA 1980;77:5654. 77. Vargas SJ, Raisz LG. Simultaneous assessment of bone resorption and formation in cultures of 22-day fetal rat parietal bones: Effects of parathyroid hormone and prostaglandin E2. Bone 1990; 11:61. 78. Canalis E, Centrella M, Burch W, McCarthy TL. Insulin-like growth factor 1 mediates selective anabolic effects of parathyroid hormone in bone cultures. J Clin Invest 1989;83:60. 79. Kalu DN, Pennock J, Doyle FH, Foster GV. Parathyroid hormone and experimental osteosclerosis. Lancet 1970;27:1363. 80. Doyle FH. Some quantitative radiological observations in primary and secondary hyperparathyroidism. BrJRadiol 1966;39:161-167. 81. Ellis K, Hostim RJ. The skull in hyperparathyroid bone disease. Am J Roentgenol 1960;83: 732. 82. Jaffe HL. Hyperparathyroidism (Recklinghausen's disease of bone). Arch Patho11933;16:63. 83. Aitken RE, Kerr JL, Lloyd HM. Primary hyperparathyroidism with osteosclerosis and calcification in articular cartilage. A m J Med 1964;37:813-820. 84. Eugenidis N, Olah AJ, Haas HG. Osteosclerosis in hyperparathyroidism. Radiology 1972;105:265. 85. Genant HK, Baron JM, Straus FH, Paloyan E, Jowsey j. Osteosclerosis in primary hyperparathyroidism. AmJMed 1975;59:104. 86. van Holsbeeck M, Roex L, Favril A, Burssens D, Baert AL. Osteosclerosis in primary hyperparathyroidism. Fortschr Rontgenstr 1987;147:690-691. 87. Dresser R. Osteitis fibrosa cystica associated with parathyroid overactivity. Am J Roentgenol Radium Ther Nucl Med 1933;30:596. 88. Albright E Baird PC, Cope D, Bloomberg E. Studies on the physiology of the parathyroid glands IV. Renal complication of hyperparathyroidism. A m J Med Sci 1934;187:49. 89. Aub JC. Lack of effect of parathyroid gland on growth of bone. Case presentation. In: Association Proceedings 26th Annual Meeting Atlantic City June 8-9, 1942. Endocrinology 1942; (Suppl.): 1024. 90. Shallow TA, Fry KE. Parathyroid adenoma. Occurrence in father and daughter. Surgery 1948;24:1020-1025. 91. Adam A, Ritchie D. Hyperparathyroidism with increased bone density in the areas of growth. J Bone Joint Surg 1954;37B:257. 92. Lloyd HM, Aitken RE, Ferrier TM. Primary hyperparathyroidism resembling rickets of late onset. Br MedJ 1965;2:853. 93. Reeve J, Meunier PJ, Parsons JA, et al. Anabolic effect of human PTH on trabecular bone in involutional osteoporosis: A multicenter trial. Br MedJ 1980;280:1340.

94. Reeve J, Arlot M, Bernat M, et al. Calcium-47 kinetic measurements of bone turnover compared to bone histomorphometry in osteoporosis: The influence of human parathyroid fragment (hPTH 1-34) therapy. Metab Bone Dis Relat Res 1981:3:23-30. 95. Reeve J, Arlot M, Bernat M, et al. Treatment of osteoporosis with human parathyroid hormone fragment 1-34: A positive final tissue balance in trabecular bone. Metab Bone Dis Relat Res 1980; (Suppl. 2):355. 96. Slovik DM, Rosenthal DI, Doppelt SH, et al. Restoration of spinal bone in osteoporotic men by treatment with human parathyroid hormone (1-34) and 1,25-dihydroxyvitamin D. J Bone Miner Res 1986;1:377. 97. Neer RM, Slovik D, Doppelt S, et al. The use of parathyroid hormone plus 1,25-dihydroxyvitamin D to increase trabecular bone in osteoporotic men and postmenopausal women. In: Christiansen C, Johansen IS, Riis BJ, eds. Osteoporosis. Copenhagen:Osteopress, 1987:829-835. 98. Shen V, Dempster DW, Mellish RWE, Birchman R, Horbert W, Lindsay R. Effects of combined and separate intermittent administration of low-dose human parathyroid hormone fragment (1-34) and 17B-estradiol on bone histomorphometry in ovariectomized rats with established osteopenia. Calcif Tissue Int 1992;50:214-220. 99. Shen V, Dempster DW, Birchman R, Xu R, Lindsay R. Loss of cancellous bone mass and connectivity in ovariectomized rats can be restored by combined treatment with parathyroid hormone and estradiol. J Clin Invest 1993;91:2479-2480. 100. Meunier PJ, Courpron E Iliac trabecular bone volume in 236 controls: Representativeness of iliac samples. In: Jaworski ZFG, ed. Proceedings of the first workshop on bone morphometry. Ottawa:Univ. Ottawa Press, 1976:100-105. 101. PodenphantJ, Gotfredsen A, Nilas L, Norgaard H, Braendstrup O. Iliac crest biopsy: Representativity for the amount of mineralized bone. Bone 1986;7:427-430. 102. Mosekilde L, Viidik A, Mosekilde L. Correlation between the compressive strength of iliac and vertebral trabecular bone in normal individuals. Bone 1985;6:291-295. 103. Mosekilde L, Mosekilde L. Normal vertebral body size and compressive strength: Relations to age and to vertebral and iliac trabecular bone compressive strength. Bone 1986;7:207-212. 104. Parisien M, Dempster DW, Silverberg SJ, et al. Relationship between bone mass as assessed by densitometry and by histomorphometry in primary hyperparathyroidism. J Bone Miner Res 1990;5:137. 105. Parisien M, Cosman E Silverberg SJ, et al. Relationship between bone mass by densitometry and by histomorphometry in primary hyperparathyroidism and in osteoporosis. Bone Miner 1992; 17 (Suppl. 1) :214. 106. Parisien M, Recker R, Silverberg SJ, et al. Cancellous bone structure in postmenopausal women with primary hyperparathyroidism. In: Christiansen C, Overgaard K, eds. Third International Symposium on Osteoporosis, October 14-20, 1990. Copenhagen:Osteopress, 1990:1139-1140. 107. Courpron P, Meunier E Bressot C, Giroux JM. Amount of bone in iliac crest biopsy: Significance of the trabecular bone volume. Its values in normal and in pathological conditions. In: Meunier PJ, ed. Proceedings. Lyon: Second International Bone Histomorphometry Workshop, 1976:39-53. 108. Parisien M, Cosman F, Mellish RWE, Schhnitzer M, Nieves J, Silverberg SJ, Shane E, Kimmel D, Recker RR, Bilezikian JP, Lindsay R, Dempster DW. Bone structure in postmenopausal hyperparathyroid, osteoporotic, and normal women. J Bone Miner Res 1995;10:1393-1399.

CHAPTER 27

1 l a S 1" S I"n N e p h r o l'th" Primary Hyperparathyroidism

VANESSA A. KLUGMAN West Suburban Hospital, Oak Park, Illinois 60302 MURRAY

J.

F A V U S Department of Medicine, University of Chicago, Pritzker School of Medicine, Chicago, Illinois 6063 7

CHARLES Y. C. PAK Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390

INTRODUCTION

faces of renal papillae or within the urinary collecting system occurs. Anchored stones may become manifest with gross or microscopic hematuria alone. However, those that pass into the renal pelvis and urinary tract may obstruct the flow of urine and cause severe acute renal colic. The pain usually begins gradually in the flank or lower anterior abdomen and increases with time. As the stone moves, so does the location of the pain. Stones at the junction of the ureter and bladder cause dysuria and urinary frequency. Pain from stones located in the lower portion of the ureter can radiate to the ipsilateral testicle or vulva, mimicking genital disease. Stone passage, either spontaneous or by surgical removal, rapidly alleviates the pain.

The initial patient with primary hyperparathyroidism who underwent successful parathyroidectomy in the report by Mandl (1) may have had kidney stones as well as severe bone disease. Though patients had been previously described as having bone disease and nephrocalcinosis (2), it was not until 1930 that Barr and Bugler (3) called attention to the association between renal calculus and primary hyperparathyroidism. Severe primary hyperparathyroidism without clinical or radiographic evidence of bone disease was first described in 1937 by Albright, Sulkowitch, and Bloomberg (4). The primary clinical manifestation of these patients was a kidney stone, either with or without nephrocalcinosis. Although clinical experience is now dominated by a large subgroup of asymptomatic patients with mild disease, the renal stone remains the single most common presenting manifestation in patients with symptomatic primary hyperparathyroidism.

Clinical Presentation of Primary Hyperparathyroidism In the past, clinical descriptions of primary hyperparathyroidism have emphasized bone disease and nephrolithiasis as primary complications of the disease. Studies in 1948 by Albright and colleagues (5) showed that bone and stone diseases usually do not occur in the same patient. They proposed that differences in calcium intake might explain the two clinical presentations of the disorder. Furthermore, they suggested that bone disease might occur more commonly in patients with an inadequate calcium intake and predisposition to negative calcium balance, whereas stone disease might occur in patients with normal or excessive calcium intake and neutral or positive calcium balance.

CLINICa~ PRESENTATION

Stone Disease The clinical manifestations of stone formation and passage observed in patients with primary hyperparathyroidism are indistinguishable from those of other types of calcium nephrolithiasis. The patients are usually asymptomatic during the initial phase of stone formation, when the early growth of crystals on the surThe Parathyroids, Second Edition

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CHAPTW~27

Hodgkinson (6) confirmed Albright's finding that patients with primary hyperparathyroidism and stone disease maintain neutral external calcium balance. Conversely, Dent et al. (7) found no difference between the dietary calcium intake of patients presenting with bone disease and those with stone disease. They proposed that the parathyroid glands might produce two hormones, one that causes hypercalcemia and another that affects bone and is nephrotoxic. Mallette et al. (8) found that patients with osteitis fibrosa had a brief period of symptoms before diagnosis, high serum calcium, and immunoreactive parathyroid hormone (PTH) levels, and large tumors. In contrast, patients with nephrolithiasis tended to show moderate hypercalcemia, a longer duration of symptoms, and perhaps a more slowly growing tumor mass. Lloyd (9) also found that patients with clinically evident bone disease had higher serum calcium levels, lower urine calcium excretion, and a shorter duration of symptoms compared to those with kidney stones. Peacock (10) found higher intestinal calcium absorption rates in patients with kidney stones than in those without stones. Thus, it is possible that patients with stones were able to preserve bone mass through increased calcium absorption, whereas those without stones developed bone disease because of lower intestinal calcium absorption rates and increased bone resorption. Patron et al. (11) reported that, among 306 patients with documented primary hyperparathyroidism, those with overt bone disease had a mean serum PTH level four times greater than that in those who presented with kidney stones. Patients with bone disease also had lower circulating 1,25-dihydroxyvitamin D [1,25 (OH) zD] levels and diminished calciuric responses to a standard oral calcium load. These observations suggest that, in patients with bone disease, PTH hypersecretion might be due to lack of PTH suppression by the low to normal circulating 1,25(OH)2D levels. In contrast, D'Angelo et al. (12) found elevated serum 1,25(OH)zD in patients with predominantly bone or stone disease, and no difference in levels between the two groups. Relative vitamin D deficiency, also noted in patients with bone disease as indicated by low circulating 25-hydroxyvitamin D levels, could contribute to the low 1,25(OH)zD levels. In one patient

with severe primary hyperparathyroidism, osteitis fibrosa, and normal serum 1,25(OH)zD level, increasing daily doses of intravenous 1,25(OH)zD (calcitriol) over several days doubled circulating 1,25(OH)zD and decreased PTH levels by 46% without changing the serum calcium concentration. Thus serum calcitriol levels in patients with bone disease may be insufficient to suppress PTH hypersecretion by individual parathyroid cells independent of serum calcium.

Stone Types Renal calculi in patients with primary hyperparathyroidism are similar in composition to calculi from nonhyperparathyroid patients. These calculi may contain pure calcium oxalate, mixed calcium phosphate and calcium oxalate, or pure calcium phosphate (brushite) (Table 1). Patients with primary hyperparathyroidism differ from those with idiopathic hypercalciuria by having a greater percentage of calcium phosphate or brushite stones (16) (Fig. 1). Using chemical analysis of stones from 33 patients, Hellstrom and Ivemark (13) found that 15% contained only calcium oxalate, 48% were composed of a combination of calcium oxalate and calcium phosphate, and 36% were composed of pure calcium phosphate. Lagergren and Ohrling (14) found a similar frequency of pure calcium phosphate stones and calcium phosphate with a small admixture of calcium oxalate. Ten patients had calcium oxalate stones and two had a m m o n i u m magnesium phosphate stones. Thus twothirds of their patients had calculi composed purely or predominantly of calcium phosphate. Hodgkinson and Marshall (17) analyzed the composition of stones passed by 23 patients with primary hyperparathyroidism and found the portion of calcium phosphate to be considerably higher than in calcium-containing stones from stone formers without hyperparathyroidism. The increased phosphate content was thought to be due to the renal effects of PTH to increase urine phosphate excretion and raise urine pH. Peacock et al. (18) also found that most hyperparathyroid stone formers produced stones composed mainly of calcium phosphate (20-80%), with calcium oxalate and magnesium ammonium phosphate making up the remainder.

TABLE 1 Composition of Kidney Stones in Primary Hyperparathyroidism Series Lagergren (1959) Hellstrom (1962) Parks (1980)

NO. a

CaOX

CaPO4

CaOx/PO4

MgNH4PO4

CaOx/U

33 33 31

10 (30) 5 (15) 20 (64)

13 (40) 12 (36) 2 (6)

8 (24) 8 (48) 2 (6)

2 (6) -1 (3)

5(16)

aNo., number of stone formers with primary hyperparathyroidism. Values are number of each type of stone (percent of total). Ca, calcium; Ox, oxalate; U, uric acid.

NEPHROLITHIASIS IN HYPERPARATHYROIDISM

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FIG. 1 Chemical composition of 30 stones from patients with idiopathic hypercalciuria and 14 stones from patients with primary hyperparathyroidism. All patients are males. M.A.P., Magnesium ammonium phosphate. Note that no patient with primary hyperparathyroidism had a pure calcium oxalate stone. (Reprinted from Ref. 16, with permission.)

Maurice-Estepa et aL (19) found carbonate apatite was the most frequent crystalline phase of phosphate among 1148 patients who passed phosphate-containing stones. The carbonate content was less than 10% in stones from patients with primary hyperparathyroidism and other, noninfection stone-forming states, and was greater than 15% in stones that formed in the presence of urinary tract infection with urea-splitting bacteria. Brushite was also found in stones from patients with primary hyperparathyroidism. Thus, the presence of either carbonate apatite or brushite in stones is consistent with the presence of primary hyperparathyroidism. However, in a series of patients with primary hyperparathyroidism and renal stones reported by Parks et al. (15), the majority of patients (20 of 31 ) formed calcium oxalate stones. For the others, two had pure calcium phosphate, two had a mixture of calcium phosphate and oxalate, five had a mixture of calcium oxalate and uric acid, one had calcium oxalate and cystine, and one had a struvite (ammonium-magnesium phosphate) stone. Taken together, these series indicate that stones from patients with primary hyperparathyroidism vary from pure calcium phosphate to pure calcium oxalate or are a mixture of the two.

PREVALENCE OF STONES

Common Primary Hyperparathyroidism The frequency of stone disease in primary hyperparathyroidism varies among series (Table 2). The incidence has been noted to decrease since the condition was first described because of the increasing numbers

of asymptomatic patients discovered by routine biochemical screening. In 1948, Albright and Reifenstein (5) reported that 80% of their patients with primary hyperparathyroidism had renal calculi. A series of TABLE 2

Prevalence of Renal Stones in Patients with Primary Hyperparathyroidism

Series Keating (1961 ) Hodgkinson (1963) Cope (1966) Pyrah (1966) Lloyd (1968) Purnell (1971 ) Pratley (1973) Mallette (1974) Broadus (1979) Mundy (1980) Siminovitch (1981 ) Ranni-Sivula (1985) Deaconson (1987) Lafferty (1989) Silverberg (1990) Haddock (1998) Total

No. of patients

Percentage with stones a

380 50 343 68 138 171 60 57 50 111 448 289 258 100 62 128

64 68 57 40 59 51 78 39 42 7 41 11 28 18 18 56

2713

42

aThe percentages may reflect a small contribution of patients with nephrocalcinosis but without nephrolithiasis, in that some series did not clearly distinguish those findings. Adapted and reprinted from Ref. 20 with permission.

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171 patients with primary hyperparathyroidism evaluated at the Mayo Clinic (21) consisted of twice as many women as men. No clinical findings or biochemical tests distinguished those with stone disease from those without. Of this series, 59% developed urologic complications, including nephrolithiasis in 46%, nephrocalcinosis in 5%, and impairment of renal function in 8%. Calcification of the papillary tips was not included in this evaluation, so the incidence of nephrocalcinosis may have been underestimated. Nephrolithiasis accompanied all cases of nephrocalcinosis. Active stone disease as documented by new stone passage, appearance on X-ray, or passage of gravel within a year prior to parathyroidectomy was present in only 10.5% of patients. The prevalence of nephrolithiasis in 16 series of patients published since 1961 (Table 2) varied from 7 to 78%, with an average incidence of 42%. From these and other series, it appears that nephrolithiasis is currently the most frequent complication of primary hyperparathyroidism, The incidence of primary hyperparathyroidism in patients presenting with nephrolithiasis has, on the other hand, remained quite stable. The occurrence of primary hyperparathyroidism among all stone formers in series published since 1960 ranges from 3 to 13%, with an average of 7% (20). Thus, the frequency of primary hyperparathyroidism as a cause of stone disease in patients presenting with nephrolithiasis is rather low.

Mild Primary Hyperparathyroidism In recent years, routine screening of serum calcium levels with automated biochemical techniques has led to the increased diagnosis of asymptomatic primary hyperparathyroidism. For middle-aged adults, the annual incidence is 100-200 cases per 100,000 population, which accounts for approximately 60,000 new cases diagnosed each year in the United States (21). Primary hyperparathyroidism is being recognized more frequently, and the clinical presentation is changing. At the Mayo Clinic, the frequency of stone disease in patients with primary hyperparathyroidism has decreased from 51 to 4% following the advent of routine measurement of serum calcium in the late 1960s (22).

Multiple Endocrine Neoplasia Type 1 Multiple endocrine neoplasia type 1 (MEN-l) is an autosomal dominant disorder characterized by benign tumors of the parathyroid, pancreatic islet, and anterior pituitary cells. Among patients with this disorder, 95% present with hyperparathyroidism, and fewer than onethird have either gastrinoma or prolactinoma. The clinical manifestations of primary hyperparathyroidism in MEN-1 are very similar to those in sporadic cases of pri-

mary hyperparathyroidism, with a few important exceptions. Patients with MEN-1 tend to appear at a younger age (20-40 years), and the sex ratio does not favor females (23). In addition, parathyroid hyperfunction in MEN-1 is generally multiglandular, with hyperplasia of all four glands. In series of patients with primary hyperparathyroidism reported from academic medical centers, 50% had asymptomatic hypercalcemia, up to 50% nephrolithiasis, and <10% had clinical bone disease.

Multiple Endocrine Neoplasia Type 2A Multiple endocrine neoplasia type 2A (MEN-2A) syndrome is characterized by medullary carcinoma of the thyroid, pheochromocytoma, and parathyroid hyperplasia. This disorder is also inherited as an autosomal dominant trait. In contrast to the frequent appearance of hyperparathyroidism in MEN-l, hypercalcemia occurs in only 10% of patients with MEN-2A (24). Histologically proved parathyroid hyperplasia may be found in 40-50% of patients and remains clinically silent in most. Because of the mild nature of the primary hyperparathyroidism, patients with MEN-2A can best be managed conservatively, in contrast to the surgical approach recommended for MEN-1.

Familial Hypocalciuric Hypercalcemia Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant disorder characterized by hypercalcemia and relative hypocalciuria that begins in infancy or early childhood and follows a benign course (25). Patients with FHH are usually asymptomatic, lack typical manifestations of primary hyperparathyroidism, and maintain a stable hypercalcemia. The rate of nephrolithiasis in FHH is the same as in the general population.

Familial Hyperparathyroidism The occurrence of two or more cases of isolated primary hyperparathyroidism in one kindred, though less common than the familial MEN syndromes, is referred to as familial hyperparathyroidism. In a study of 29 families with presumed familial hyperparathyroidism (26), 83 of the 261 members studied had surgically proved hyperparathyroidism, with multiglandular parathyroid enlargement demonstrated in 42; 57% (47/83) had either nephrocalcinosis or nephrolithiasis. Inheritance consistent with an autosomal dominant pattern was found in most families, although a family has been described with an autosomal recessive pattern of inheritance and multiple recurrent large adenomas. Of 30 patients with familial hyperparathyroidism studied at the Mayo Clinic (27), 47% had nephrolithiasis. The authors

NEPHROLITHIASIS IN HYPERPARATHYROIDISM /

comment that the familial form of hyperparathyroidism was diagnosed at a young age (mean age 39 years; range was 13 to 78 years) and had a high incidence of nephrolithiasis. However, the frequency of nephrolithiasis among 16 series of primary hyperparathyroidism of 42% (Table 2) is not very different than the 57 or 47% reported in the two series of familial disease.

Medullary Sponge Kidney and Hyperparathyroidism The prevalence of medullary sponge kidney in patients with nephrolithiasis has been estimated at 3.6% (28). Using the criteria of medullary sponge kidney on excretory urograms as the presence of three or more linear or round collections of contrast material within one renal papilla, the frequency was 12% among patients with renal stones compared to 1% in patients without kidney stones (29). Metabolic disorders accounting for the cause of stones in medullary sponge kidney were present in 60% of patients and in 93% of patients with nephrolithiasis without medullary sponge kidney. No patient with primary hyperparathyroidism was found in one series of 17 patients (28), but three patients with both conditions were reported (30). For two subjects, stone passage was abolished following parathyroidectomy for primary hyperparathyroidism. The mechanism of stone formation may not be different from that in other stone formers, but the frequency of hypercalciuria was high (15 of 17) in one series (28). The results suggest that medullary sponge kidney is a cause of calcium nephrolithiasis and that primary hyperparathyroidism may be important in the pathogenesis of stone formation in some patients.

DIAGNOSIS Diagnosis of Primary Hyperparathyroidism The approach to the diagnosis of primary hyperparathyroidism is discussed in Chapter 20.

Differential Diagnosis of Hypercalcemia and Nephrolithiasis Primary hyperparathyroidism is the most common of the several diseases that may present with hypercalcemia and stone formation (Table 3). Other causes include vitamin D intoxication, sarcoidosis and other active granulomatous diseases, milk alkali syndrome, thyrotoxicosis, and immobilization. All can be distinguished from primary hyperparathyroidism by normal or suppressed serum PTH levels. All patients may have hypercalciuria unless renal failure supervenes.

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TABLE 3 Causes of Hypercalciuria and Nephrolithiasis Primary hyperparathyroidism Thyrotoxicosis (spontaneous and iatrogenic) Vitamin D intoxication Milk alkali syndrome Immobilization Sarcoidosis

PATHOGENESIS OF ABNORMAL U R I N E CHEMISTRY Parathyroid hormone excess may increase or decrease the urinary excretion of several ions and may alter the urinary saturation and inhibitor activity, resulting in urine that favors crystallization of stone-forming calcium salts (Table 4).

Hypercalciuria The pathogenetic risk factors responsible for stone formation in primary hyperparathyroidism remain unclear. Hypercalciuria (24-hour urine calcium excretion >250 m g / d a y for women and >300 m g / d a y for men) is frequently encountered in patients with hyperparathyroidism and has been implicated in the pathogenesis of renal stone formation (31). Excess calcium in the urine may come from increased intestinal absorption, increased bone resorption, or from both. As a result, ultrafilterable calcium increases and may be sufficient to exceed distal tubular reabsorptive capacity. Increased intestinal calcium absorption has been documented in primary hyperparathyroidism using various methods, including metabolic balance, double isotope, and fecal excretion of radiolabeled Ca (32). 1,25(OH)zD is the major stimulator of intestinal calcium absorption, and PTH stimulates its synthesis in the renal proximal tubule (33). In addition, low serum phosphate concentrations, caused by PTH inhibition of

TABLE 4 Metabolic Causes of Stone Formation in Primary Hyperparathyroidism Hypercalciuria Reduced inhibitory activity Increased urinary promoter activity Reduced urinary pyrophosphate Low urine citrate excretion Reduced urinary magnesium Hyperuricosuria Mild renal tubular acidosis

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proximal tubular phosphate reabsorption, may also stimulate 1,25(OH)2D synthesis (33). In support of a regulatory role of 1,25(OH)zD in the increased intestinal calcium absorption, Kaplan et al. (32) found that the mean plasma concentration of 1,25(OH)zD was greater in 18 patients with primary hyperparathyroidism than in normal subjects and was highly positively correlated with fractional calcium absorption. Increased intestinal calcium absorption and elevated serum calcitriol levels were found in some but not all hyperparathyroid stone formers. In a study by Peacock (10), patients with primary hyperparathyroidism and stone disease uniformly displayed an increase in fractional intestinal calcium absorption. Broadus et al. (34) also attributed stone formation to increased intestinal calcium absorption and calcitriol excess. Among 50 unselected patients with primary hyperparathyroidism, 22 had a history of one or more kidney stones. An abnormal response to a calcium load test (>0.20 mg calcium per 100 ml glomerular filtration) was associated with a greater serum 1,25(OH)zD level, a higher incidence of stones, and a higher 24-hour urinary calcium excretion compared to patients showing a normal response (<0.20 mg calcium per 100 ml glomerular filtration) to the calcium load test. Fasting urine calcium excretion was not different between the groups. Patients with greater calcium absorption and more marked hypercalciuria had greater parathyroid suppression as determined by suppression of nephrogenous cyclic adenosine monophosphate (cAMP). Broadus et al. concluded that patients with elevated serum 1,25(OH)zD levels, increased intestinal calcium absorption, and greater parathyroid suppression are predisposed to stone formation by their more marked hypercalciuria. In contrast, Pak et al. (35) found no significant difference in 1,25(OH)zD levels, intestinal calcium absorption, urinary calcium excretion, height of serum calcium and PTH concentrations, serum phosphate level, or bone density between stone-forming and nonstone-forming patients. Both groups had elevated fasting urinary calcium excretion and low bone mineral density. From these data, Pak et al. concluded that there was no unique pathophysiologic background for nephrolithiasis in primary hyperparathyroidism. The discrepancy between the studies of Pak et al. (35) and Broadus et al. (34) may have resulted in part from differences in patient classification. Broadus et al. initially separated patients with primary hyperparathyroidism on the basis of intestinal calcium absorption using the indirect method of incremental increase in urinary calcium after an oral calcium load. In contrast, Pak et al. compared data from stone formers with those from patients without stone disease. Among 62 patients with primary hyperparathyroidism, 18% of whom were stone formers, Silverberg et al. (36) found a strong positive correlation between

urinary calcium excretion and serum 1,25(OH)2D levels. However, as in the study ofPak et al. (35), stone formers and nonformers did not differ with respect to serum PTH, calcium, phosphorus, or 1,25(OH)zD or in urinary calcium excretion. Mild hypercalcemia may be associated with significant hypercalciuria and stone formation. Parks et al. (15) found that, in patients with surgically documented hyperparathyroidism, the serum calcium levels were between 10.2 and 10.8 mg/dl, just above the upper limit of normal of 10.1 mg/dl. However, urine calcium excretion exceeded normal in most patients and was markedly elevated in some patients in whom hypercalcemia was extremely mild. Thus one cannot assume that mild hypercalcemia is accompanied by only mild hypercalciuria. Elevated serum 1,25(OH)2D levels have been reported in some hyperparathyroid stone formers (11) but not in others (37). The latter study did not control calcium intake, whereas other studies that have reported serum 1,25(OH)zD levels have used a restricted calcium intake. Serum 1,25(OH)2D is sensitive to dietary calcium restriction in patients with primary hyperparathyroidism (38); diet, therefore, could account for the elevated 1,25 (OH)2D levels in some series reported. A second source of elevated urine calcium is bone resorption. PTH increases osteoclastic bone resorption, which results in the release of both mineral constituents and bone matrix protein degradation products. As a result, serum calcium concentration and urinary hydroxyproline excretion increase (39). In 26 patients, urinary calcium excretion exceeded net intestinal calcium absorption (40). In addition, fasting urinary calcium excretion is elevated in the majority of patients. Thus, hypercalciuria is partly the result of an excessive skeletal mobilization of calcium. Silverberg et al. (36) found elevated levels of urinary hydroxyproline and a negative correlation between urinary calcium excretion and forearm bone density in hyperparathyroid subjects with hypercalciuria, including stone formers (Fig. 2). Thus it appears that bone resorption contributes to the hypercalciuria observed in patients with hyperparathyroidism.

Inhibitors and Promoters Reduced urinary inhibitory activity a n d / o r increased promoter activity could contribute to the predilection for stone formation in primary hyperparathyroidism. Pak and Holt (41) found that the urine of patients with hyperparathyroidism was significantly more supersaturated with respect to calcium oxalate and brushite (CaHPO4.2H20) as expressed by the activity product ratio (APR) compared to normal subjects without stones. Urine APR was higher in patients with stones than in those without stones. The urinary formation

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product ratio (FPR), an inhibitor activity against spontaneous nucleation of brushite and calcium oxalate, was reduced in patients with primary hyperparathyroidism compared to values in normal subjects. Following parathyroidectomy, both urinary APR and FPR returned toward normal. These studies indicate that the urine of patients with primary hyperparathyroidism is supersaturated with respect to stone-forming constituents and shows an increased propensity for crystallization of stone-forming calcium salts. These findings are consistent with either excess urine promoters or reduced inhibitors leading to stone formation in primary hyperparathyroidism. However, physicochemical presentation in urine of stone-forming patients with primary hyperparathyroidism has not been rigorously compared to that of nonformers. Moreover, promoter excess or inhibitor deficiency has not been documented by direct analysis. Thus, the propensity to form stones cannot be understood on the basis of present knowledge of urinary inhibitors and promoters. Pyrophosphate is a naturally occurring urinary inhibitor of the crystallization of calcium oxalate and calcium phosphate. Measurements of urinary excretion of pyrophosphate in patients with hyperparathyroidism have been reported to be either not different from normal (42) or increased (43). Studies have found no significant change in pyrophosphate excretion following surgical removal of the adenoma (44). The reason

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for the difference between the findings in these studies remains obscure. Citrate is a natural urinary inhibitor of crystallization of calcium salts through its formation of soluble calcium citrate complexes. Citrate is also a direct inhibitor of crystal growth of calcium phosphate and nucleation of calcium oxalate. Alvarez-Arroyo et al. (45) and Smith et al. (46) reported that hyperparathyroid kidney stone formers showed lower urinary citrate excretion than did patients without stones, apparently due to increased tubular reabsorption of citrate. Magnesium inhibits calcium oxalate crystallization by complexing oxalate. Low urinary magnesium in relation to calcium is a common finding in hyperparathyroid stone formers and may contribute to the development of calcium oxalate stones (47). Others have found low urinary M g / C a ratios in primary hyperparathyroidism, which appear to return to normal postoperatively (48). However, normalization of the Mg/Ca ratio is a function of changes in urinary calcium excretion, because low urinary magnesium is u n c o m m o n in primary hyperparathyroidism. "Consumption" of normal urine inhibitors may occur with hypercalciuria. Zerwekh et al. (49) found that hypercalciuria induced by calcium supplementation in stone formers decreased the urinary formation product ratio, probably by complexing of negatively charged urinary inhibitors by calcium. Other evidence suggesting an alteration in crystal growth inhibitors comes from observations that urine of hyperparathyroid stone formers contains crystals that are larger due to enhanced aggregation rather than crystal growth (50). Thus, stone-forming urine may contain promoters of crystal growth or substances that block the effects of crystal growth inhibitors found in normal urine. Hyperuricosuria Hyperuricosuria has been linked to calcium oxalate crystal formation through induction of epitaxial growth of calcium oxalate crystals or adsorption of naturally occurring inhibitors by uric acid or sodium urate (51,52). There is, however, no convincing evidence that urinary uric acid excretion is elevated in patients with primary hyperparathyroidism. Broulek et al. (53) found that hyperparathyroid patients have lower urate clearance than do controls. Ljunghall and Akerstrom (54) found a reduction in the clearance of urate and a rise in serum urate concentrations that normalized postoperatively. The fasting urine excretion of urate was slightly higher in stone formers preoperatively, whereas there were no significant differences for the 24 hours before or after surgery. Thus disturbances of urate metabolism are not likely to be important in the pathogenesis of renal stones in primary hyperparathyroidism.

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Acidosis Hereditary, type I distal renal tubular acidosis (RTA) typically presents with a n o n a n i o n gap hyperchloremic acidosis, medullary nephrocalcinosis, calcium phosphate nephrolithiasis, and osteopenia. Pure calcium phosphate stones develop in this environment as a consequence of the alkaline urine, decreased citrate excretion, and hypercalciuria secondary to enhanced bone resorption and impaired renal tubular reabsorption of calcium. Because patients with primary hyperparathyroidism have an increased incidence of calcium phosphate stones, acidification defects have been considered in the pathogenesis of stones in this disorder. Primary hyperparathyroidism may be associated with a proximal RTA, which, unlike the distal RTA, has not been associated with stone formation. It is clear that a mild hyperchloremic acidosis may occur in patients with hyperparathyroidism, but its frequency is debated. Muldowney et al. (55) described four patients with primary hyperparathyroidism who had hyperchloremic acidosis; however, all the patients had impaired renal function. Because hypercalcemic n e p h r o p a t h y can impair renal acidification, the cause of acidosis in these patients is unclear. Among 13 patients with primary hyperparathyroidism studied by Coe (56), only two had metabolic acidosis. Blood chloride and CO 2 concentrations rose after parathyroidectomy, and the serum PTH level was not significantly correlated with either. Thus, PTH is unlikely to be an important regulator of renal acid excretion. Whether metabolic acidosis occurs in primary hyperparathyroidism was challenged by Hulter and Peterson (57), who found a mild transient acidosis, which subsequently was replaced by a sustained mild alkalosis associated with increased urine proton excretion. In addition, PTH does not typically alter urinary pH (41). Therefore, there is little evidence to favor sustained acidosis as an important factor in the pathogenesis of nephrolithiasis in primary hyperparathyroidism.

TREATMENT

Surgical Treatment of Hyperparathyroidism Stone Recurrence after Parathyroidectomy For asymptomatic patients, the decision whether to treat hyperparathyroidism medically or surgically is controversial (see Chapters 28, 29, and 31). However, there is agreement about the value of surgery in patients who form stones. McGeown (58) followed 56 patients for 1-5 years following parathyroidectomy. Prior to parathyroidectomy, 99 stones were formed in 401 patient-years, whereas, in the postparathyroidectomy period, only 8 stones were formed in 158 patient-

years. Deaconson et al. (59) also observed a decline in the rate of new stone formation after surgical correction of primary hyperparathyroidism, from 0.36 to 0.02 new stones formed per patient per year. Two of the four patients with recurrent stones had persistent hypercalcemia and evidence of persistent or recurrent hyperparathyroidism. Parks et al. (15) described similar findings in 48 patients with primary hyperparathyroidism and renal stones. The only patient with stone recurrence postoperatively was the only one who remained hypercalciuric following parathyroidectomy. On the other hand, Posen et al. (60) observed a 50% recurrence rate of new stone formation or passage of an existing stone after surgery among 77 hyperparathyroid patients followed for 7-8 years. Patients with no previous history of renal calculi were unlikely to form stones postoperatively even if they remained hypercalcemic and hypercalciuric. Thus, parathyroidectomy results in a virtual disappearance of first-onset stone formation. Resolution of stone disease is less certain in patients with parathyroid hyperplasia than in those with adenomas. Siminovitch et al. (61) observed 448 patients who underwent surgery for presumed hyperparathyroidism. Among the 72 patients with active stone disease, 48 were found to have adenomas, 18 had hyperplasia, and 6 had normal glands. Patients with adenomas had no recurrent calculi, whereas 50% of those with normal glands and 45% of those with hyperplasia had recurrent stone formation. Similarly, Johannsen et al. (62) found that patients with adenomas had no recurrent calculi after parathyroidectomy, whereas recurrent calculi developed in 25% of the patients with parathyroid hyperplasia and in 48% of those with normal parathyroid glands.

Metabolic Changes after Surgery Successful parathyroidectomy decreases serum calcium levels to normal, which, in the majority of patients, leads to resolution of hypercalciuria (63). It has been estimated that, of the 5-10% of patients who continue to form stones after surgery, 90% have some form of hypercalciuria (64). Occurrence of hypercalciuria after parathyroidectomy may be as high as 30% (65). Parks et al. (15) found that, in 66 patients with renal stones and primary hyperparathyroidism, urine calcium, urine oxalate, and urine calcium oxalate concentration product ratio (CPR) returned to normal after surgery in males. In contrast, in women, urine oxalate and the calcium oxalate CPR returned to normal, but the urine calcium levels did not. Hypercalciuria persisted in 7 of 17 women even though the serum calcium level was normal. The cause of this persistent hypercalciuria despite normocalcemia remains unknown, but it is probably

NEPHROLITHIASIS IN HYPERPARATHYROIDISM /

multifactorial. Breslau et al. (66) have suggested coexistence of absorptive (idiopathic) hypercalciuria as the underlying etiology of the recurrent hypercalciuria observed in some patients after surgery. In five such patients, serum PTH and calcium levels returned to normal, but serum 1,25(OH)zD remained elevated in four. Four of the patients had recurrent stones and persistent elevation in fractional intestinal radiocalcium absorption. Thus, absorptive hypercalciuria and primary hyperparathyroidism may occur together, and absorptive hypercalciuria may persist after surgery. Absorptive hypercalciuria was also suggested in a patient with persistent hypercalciuria postparathyroidectomy, in whom hypercalciuria was abolished with dietary calcium restriction (67). This would not be expected to lower urine calcium excretion if it were the result of a renal calcium leak or secondary to bone resorption. An alternative mechanism for persistent hypercalciuria after parathyroidectomy is so-called renal hypercalciuria. Bordier et al. (68) and Maschio et al. (69) described patients with apparent normocalcemic hyperparathyroidism, in whom persistent hypercalciuria after parathyroid surgery resulted from a primary renal calcium leak. This leak led initially to a state of secondary hyperparathyroidism and eventually to autonomous or tertiary hyperparathyroidism. It appears therefore that recurrent stones can occur despite cure of the clinical hyperparathyroidism. The etiology may be secondary to underlying absorptive or renal hypercalciuria. All patients should therefore have urinary calcium excretion evaluated after parathyroidectomy. Although successful surgery for hyperparathyroidism corrects serum and urine calcium abnormalities, Pak (70) observed that urinary calcium decreased significantly after parathyroidectomy without significant changes in urinary phosphate, oxalate, magnesium, sodium, potassium, uric acid, or pH. The urinary activity product ratio (state of saturation) of brushite (CaHPO4.2H20) and calcium oxalate decreased significantly due primarily to decreased urinary calcium. In addition, the urinary formation product ratio of calcium oxalate, the minimum supersaturation required for spontaneous nucleation, increased significantly after parathyroidectomy (Fig. 3). Therefore, surgery restores the normal urinary environment with respect to saturation and inhibitor a n d / o r promoter activity. Medical T r e a t m e n t o f H y p e r p a r a t h y r o i d i s m Curative surgery is indicated for patients with primary hyperparathyroidism and renal stones. Medical treatment outcome is less certain and should be reserved for patients who refuse surgery, who are unable to withstand general anesthesia, or who require a temporizing measure prior to surgery. Several med-

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ical therapies have been assessed, with few shown to be of clear value in reducing serum or urine calcium and diminishing stone formation (Table 5). Fluid Intake

Because low urine volume has been considered a risk factor in the development of renal stones (71), high fluid intake is recommended in patients with renal stones. Pak et al. (72) suggested that the high fluid intake may be effective through dilution of stone-forming constituents as well as decreased urinary saturation of stone salts. Urinary dilution by high fluid intake assessed both in vitro and in vivo in stone formers and control subjects revealed that both forms of urinary

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TABI.15 5 Medical Therapy or Primary Hyperparathyroidism and Kidney Stones High fluid intake Dietary restriction Sodium Protein Calcium Cellulose phosphate Diuretics Inhibitors of PTH secretion Propanolol Cimetadine Antiresorptive agents Estrogen Bisphosphonates

sis also increase urinary calcium excretion. A large epidemiologic study showed that urinary sodium excretion directly correlated with urinary calcium excretion (73). Dietary sodium intake may therefore be a risk factor for hypercalciuria and for calcium oxalate kidney stone formarion. Moreover, patients with recurrent stones and hypercalciuria were more sensitive to the hypercalciuric action of dietary sodium. Muldowney et al. (76) suggested that dietary sodium restriction may have a role in the management of hypercalciuria in mild hyperparathyroid patients when parathyroidectomy is contraindicated. For example, moderate dietary sodium restriction decreases 24-hour urine calcium excretion in primary hyperparathyroidism patients. Calcium Restriction

dilution resulted in a significant reduction of urinary APR (state of saturation) of calcium phosphate, calcium oxalate, and monosodium urate. In addition, the formation product ratio (minimum supersaturation needed to elicit spontaneous nucleation) of calcium oxalate significantly increased. Thus, the propensity for crystallization of calcium salts is decreased by dilution of the urine, resulting in a beneficial role of increased fluid intake in the management of nephrolithiasis.

Calcium restriction has been used commonly in patients with primary hyperparathyroidism to control hypercalcemia and reduce hypercalcuria. Though severe restriction of dietary calcium may reduce urine calcium excretion, it may also result in negative calcium balance (77) and hyperoxaluria by increasing intestinal oxalate absorption.

Dietary Protein Restriction

This resin binds dietary calcium in the intestinal lumen and prevents absorption. Cellulose phosphate decreases urinary calcium excretion and may thus be important in preventing stones (78). However, it increases urinary oxalate. Moreover, it may stimulate bone resorption because urinary hydroxyproline excretion tends to increase. Thus, long-term use of cellulose phosphate is contraindicated.

High dietary protein intake, estimated by excretion of urinary urea nitrogen, may be highly correlated with urinary calcium in some patients with recurrent nephrolithiasis (73,74). Moreover, stone formers appeared to be more sensitive to the calciuric action of protein, because dietary protein load resulted in a greater increment in urinary calcium in patients with recurrent nephrolithiasis than in control subjects. Urinary oxalate and uric acid may increase along with calcium excretion following ingestion of a protein load (75), and the increases may persist for as long as the increased dietary protein is maintained. Protein may also increase calcium oxalate stone formation through changes in the excretion of various promoters and inhibitors. Wasserstein et al. (73) found that protein ingestion results in hypocitrituria in patients with recurrent stones, whereas protein restriction increases urinary citrate excretion. Thus, the hypocitrituria often found in patients with recurrent nephrolithiasis may be the result of a high-protein diet. Sodium Restriction

The renal tubular handling of calcium and sodium is closely related such that factors that promote natriure-

Cellulose Phosphate

Diuretic Theraj~y

Diuretics should be used with caution in patients with primary hyperparathyroidism. Because of the reversible loss of urine concentrating ability due to the hypercalciuria, excessive fluid loss or decreased fluid intake could result in dehydration and lead to a further increase in blood calcium level. Thiazide diuretics particularly should be avoided; they may increase hypercalcemia by increasing distal tubular calcium reabsorption (79). Thiazide-induced hypercalcemia tends to occur in patients with nonsuppressible parathyroid glands, as in primary hyperparathyroidism. Loop diuretics such as furosemide, with adequate sodium intake, may facilitate urinary calcium excretion and therefore may be a useful short-term measure in decreasing serum calcium until surgery is performed.

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However, furosemide should not be used over the long term; it increases urinary calcium excretion, may increase the risk of further stone formation, and may worsen negative calcium balance.

Inhibitors of Parathyroid Hormone Secretion An agent that inhibits the secretion of parathyroid hormone would be extremely useful in primary hyperparathyroidism, eeAdrenergic catecholamines stimulate PTH secretion (80), and the beta blocker propranolol can block catecholamine-stimulated PTH secretion. Short-term in vivo studies in animals and humans have shown that [3-receptor agonists stimulate PTH secretion and that this response can be eliminated by propranolol. Caro et al. (81) observed a variable decrease in serum PTH and calcium levels with propranolol in eight patients with primary hyperparathyroidism, with a marked response in two patients and marginal or no response in the remainder. Other reports have failed to demonstrate a decline in serum calcium during propranolol administration (80,82). The stimulatory action of histamine to increase PTH secretion can be inhibited by the H2 receptor antagonist cimetidine. Sherwood et al. (83) reported a significant decrease in serum PTH levels with cimetidine in 12 patients with primary hyperparathyroidism. However, subsequent studies have not confirmed these initial findings (84,85). Thus, the therapeutic trials to date have failed to identify an inhibitor of PTH secretion that may be useful as medical therapy for primary hyperparathyroidism.

Antiresorptive Therapy Inhibition of osteoclastic bone resorption is another medical strategy to control the hypercalcemia and hypercalciuria of primary hyperparathyroidism.

Estrogens Estrogens are inhibitors of PTH-stimulated osteoclastic activity and can reduce bone resorption. Gallagher and Nordin (86) showed that estrogen administration successfully decreased serum calcium and urine calcium and hydroxyproline excretion in a group of women with primary hyperparathyroidism. They therefore attributed the effects of estrogen to the inhibition of PTH-induced bone resorption. Marcus et al. (87) observed a reduction in serum calcium and urinary calcium excretion for up to 2 years in 10 patients treated with estrogen. Temporary interruption of therapy was associated with return of hypercalcemia to untreated levels within 2-3 days, but this resolved quickly with resumption of estrogen therapy.

447

Urinary hydroxyproline and serum alkaline phosphatase decreased during estrogen therapy, suggesting a decrease in bone formation and resorption rates. Serum PTH and 1,25(OH)2D levels and urine cAMP excretion did not change with treatment. These data indicate that estrogen inhibits the skeletal actions of PTH but does not secondarily stimulate parathyroid function and, therefore, does not alter the course of the disease. None of the previous studies has addressed the effect of estrogen therapy on stone formation in particular. Therefore, estrogen therapy may provide an alternative to surgical cure for postmenopausal females who are unwilling or unable to undergo parathyroidectomy (see Chapter 29).

Bisphosphonate Therapy Bisphosphonates are related synthetic analogs of endogenous pyrophosphate. They bind with high affinity to bone crystal hydroxyapatite and inhibit its dissolution and growth. Perhaps more importantly, bone resorption is inhibited by a direct action to suppress osteoclastic activity (88). Ethane hydroxy-l,l-diphosphonate (EHDP) was the first bisphosphonate to be investigated in primary hyperparathyroidism. Serum calcium was not reduced by EHDP therapy, although 5 weeks of EHDP decreased urine calcium and hydroxyproline (89). Urine calcium increased after 6 months of therapy, suggesting that the decrease in bone resorption may be transient. Dichloromethylene diphosphonate (C12MDP, clodronate), which does not inhibit skeletal mineralization, decreased serum calcium and urinary calcium and hydroxyproline excretion in each of the 14 primary hyperparathyroidism patients treated (90). 3-Amino-1hydroxypropylidine-l,l-diphosphonate (APD) is similar to clodronate in that it inhibits bone resorption at doses that do not alter mineralization. APD rapidly reduced serum calcium to normal in two patients with primary hyperparathyroidism (91). Clearly, more studies are needed before a role, if any, for APD or other newer bisphosphonates can be established in treatment of hyperparathyroid stone disease.

Phosphate Therapy A decrease in urinary calcium following phosphate administration to hypercalciuric patients was first described by Mbright et al. (92). Subsequently, Goldsmith and Ingbar (93) confirmed that phosphate reduces urine calcium excretion and also noted that administration of intravenous or o r a l phosphate reduced hypercalcemia in 20 patients with hypercalcemia of diverse etiology. A n u m b e r of side effects, including renal failure and dystrophic calcification, have limited the use of this approach, however.

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Phosphate decreases serum calcium levels by shifting extracellular fluid calcium into bone and also by inhibiting PTH-mediated bone resorption and stimulating osteoblastic bone formation. Purnell et al. (94) treated 14 patients with primary hyperparathyroidism, who were surgical failures, with moderate doses (1000-2500 mg) of elemental phosphorus daily. They concluded that patients with moderate disease (mean serum calcium 12.5 m g / d l ) , particularly those with prior renal impairment, were not good candidates for phosphate therapy and were at risk for worsening of renal function and/or rapid progression of hyperparathyroidism. However, patients with mild primary hyperparathyroidism (mean serum calcium 10.8 mg/dl) had no side effects of therapy, and the metabolic activity of renal stone complications appeared to be controlled. Broadus et al. (95) found that 1 year of oral phosphate therapy in 10 patients with primary hyperparathyroidism and elevated serum 1,25(OH)zD levels significantly decreased circulating 1,25(OH)2D , calciuric response to an oral calcium tolerance test, and urine calcium excretion on an unrestricted calcium diet. Of the 10 patients, 6 patients had a history of renal stones, and none formed new renal stones during the course of the treatment. Although phosphate appears to reverse intestinal calcium hyperabsorption, serum PTH levels increase and may further stimulate bone resorption. The indications for phosphate therapy remain uncertain. Even though urinary calcium excretion is reduced, the efficacy in reducing stone formation has not been determined. In addition, phosphate may accelerate renal impairment in patients with serum calcium levels > 11.5 m g / d l and in those with established renal insufficiency. REFERENCES 1. Mandl E Therapeutischer versuch bei einem falle von ostitis fibrosa generalisata mittels extirpation eines epithelkorperchentumors. Z Chir 1926;53:260-264. 2. Davies-Colley N. Bones and kidneys from a case of osteomalacia in a girl aged 13. Trans Pathol Soc London 1884;35:285-297. 3. Barr DE Bulger HA. The clinical syndrome of hyperparathyroidism. A m J M e d Sci 1930;179:471-473. 4. Albright F, Sulkowitch HW, Bloomberg E. Further experience in diagnosis of hyperparathyroidism, including discussion of cases with minimal degree of hyperparathyroidism. Am J Med Sci 1937;193:800-881. 5. Albright E Reifenstein EC. The parathyroid glands and metabolic bone disease. Baltimore:Williams & Wilkins, 1948:393. 6. Hodgkinson A. Biochemical aspects of primary hyperparathyroidism: An analysis of 50 cases. Clin Sci 1963;25:231-236. 7. Dent CE, Hartland BV, Hicks J, et al. Calcium intake in patients with primary hyperparathyroidism. Lancet 1961;2: 330-338.

8. Mallette LE, Bilezikian JR Heath DA, et al. Primary hyperparathyroidism: Clinical and biochemical features. Medicine 1974;53:127-146. 9. Lloyd HM. Primary hyperparathyroidism: An analysis of the role of the parathyroid tumor. Medicine 1968;47:53-71. 10. Peacock M. Renal stone disease and bone disease in primary hyperparathyroidism and their relationship to the action of parathyroid hormone on calcium regulation. In: Calcium regulating hormones, proceedings of the fifth parathyroid conference. Amsterdam:Excerpta Medica, 1975:78. 11. Patron P, Gardin JP, Poullard M. Renal mass and reserve of vitamin D: Determinants in primary hyperparathyroidism. Kidney Int 1987;31:1176-1180. 12. D'Angelo A, Lodetti MG, Giannini S, et al. Hyperparathyroidism: Cause or consequence of recurrent calcium nephrolithiasis? Miner Electrolyte Metab 1992;18:359-364. 13. Hellstrom J, Ivemark BI. Primary hyperparathyroidism: Clinical and structural findings in 138 cases. Acta Chir Scand 1962;294:Suppl. 1. 14. Lagergren C, Ohrling H. Urinary calculi composed of pure calcium phosphate. Acta Chir Scand 1959;117:335-341. 15. Parks J, Coe F, Favus M. Hyperparathyroidism in nephrolithiasis. Arch Intern Med 1980;140:1479-1481. 16. Rose AG. Primary hyperparathyroidism. In: Wickham JEA, Buck AC, eds. Renal tract stone: Metabolic basis and clinical features. London:Churchill Livingstone, 1990:401-413. 17. Hodgkinson A, Marshall RW. Changes in the composition of urinary tract stones. Invest Uro11975; 13:131-135. 18. Peacock M, Marshall RW, Robertson WG, et al. Renal stone formation in primary hyperparathyroidism and idiopathic stone disease; diagnosis, etiology and treatment. In: Finlayson B, Thomas WC, eds. Colloquium on renal lithiasis. Gainesville: University Press of Florida 1976:339-355. 19. Maurice-Estepa L, Levillain P, Lancour B, et al. Crystalline phase differentiation in urinary calcium phosphate and magnesium phosphate calculi. ScandJ Urol Nephrol 1999;33:299-305. 21. Purnell DC, Smith LH, Scholz DA, et al. Primary hyperparathyroidism: A prospective clinical study. A m J Med 1971;50:670-678. 20. Broadus A. Nephrolithiasis in primary hyperparathyroidism. In: Coe E Brenner B, Stein J, eds. Nephrolithiasis. New York:Churchill Livingstone, 1980. 21. Heath III H, Hodgson SE Kennedy MA. PHPT: Incidence, morbidity and potential economic impact in a community. N EnglJ Med 1980;302:189-193. 22. Hodgson SE Heath III H. Asymptomatic primary hyperparathyroidism: Treat or follow? Mayo Clin Proc 1981;56:521-523. 23. Marx SJ, Vinek AI, Sanken RJ, et al. Multiple endocrine neoplasia type I: Assessment of laboratory tests to screen for the gene in a large kindred. Medicine 1986;65:226-261. 24. Scheinke RN. Genetic aspects of multiple endocrine neoplasia. Annu Rev Med 1986;35:25-31. 25. Marx SJ, Brandu ML. Familial primary hyperparathyroidism. In: Peck WA, ed. Bone and mineral research, Vol 5. Amsterdam:Elsevier, 1987:375-407. 26. Goldsmith RE, Sizemore G, Falme E, et al. Familial hyperparathyroidism. Ann Intern Med 1976;84:36-43. 27. Barry MK, van Heerden JA, Grant CS, et al. Is familial hyperparathyroidism a unique disease? Surgery 1997; 122:1028-1033. 28. O'Neill M, Breslau NA, Pak CY. Metabolic evaluation of nephrolithiasis in patients with medullary sponge kidney. JAMA 1981 ;245:1233-1236. 29. Ginalski JM, Portmann L, Jaeger E Does medullary sponge kidney cause nephrolithiasis? A m J Roentgenol 1990;155:299-302. 30. Rao DS, Frame B, Block MA, et al. Primary hyperparathyroidism. A cause of hypercalciuria and renal stones in patients with medullary sponge kidney. JAMA 1977;237:1353-1355.

NEPHROLITHIASIS IN HYPERPARATHYROIDISM 31. Coe FL, Parks JL, Asplin JR. The pathogenesis and treatment of kidney stones. N EnglJ Med 1992;327:1141-1152. 32. Kaplan RA, Haussler MR, Deftos LJ, et al. The role of 1,25-dihydroxyvitamin D in the mediation of intestinal hyperabsorption of calcium in primary hyperparathyroidism and absorptive hypercalciuria. J Clin Invest 1977;59:756-760. 33. Henry HL, Norman AW. Metabolism of vitamin D. In: Coe FL, Favus MJ, eds. Disorders of bone and mineral metabolism. New York:Raven, 1992:149-162. 34. Broadus AE, Horst RL, Lang R, et al. The importance of circulating 1,25-dihydroxyvitamin D in the pathogenesis of hypercalciuria and renal stone formation in primary hyperparathyroidism. N EnglJ Med 1980;302:421-425. 35. Pak CY, Nicar MJ, Peterson R, et al. A lack of unique pathophysiologic background for nephrolithiasis of primary hyperparathyroidism. J Clin Endocrinol Metab 1981 ;53:536-542. 36. Silverberg SJ, Shane E, Jacobs TP, et al. Nephrolithiasis and bone involvement in primary hyperparathyroidism. Am J Med 1990;89:327-334. 37. Thakker RV, Fraher LJ, Adami S, et al. Circulating concentrations of 1,25-dihydroxyvitamin D in patients with primary hyperparathyroidism. Bone Miner 1986;1:137. 38. Insogna KL, Mitnick ME, Stewart A, et al. Sensitivity of the parathyroid hormone 1, 25-dihydroxy vitamin D axis to variations, in calcium intake in patients with primary hyperparathyroidism. N E n g l J Med 1985;313:1126-1130. 39. Fitzpatrick LA, Coleman DT, BilezikianJE The target tissue actions of parathyroid hormone. In: Coe FL, Favus MJ, eds. Disorders of bone and mineral metabolism, Vol 6. New York:Raven, 1992:123-148. 40. Pak CY, Ohata M, Lawrence EC, et al. The hypercalciurias: Causes, parathyroid functions, and diagnostic criteria. J Clin Invest 1976;54:387-400. 41. Pak CYC, Holt K. Nucleation and growth of brushite and calcium oxalate in urine of stone formers. Metabolism 1976;25:665-673. 42. Russell RGG, Hodgkinson A. The urinary excretion of inorganic pyrophosphate in hyperparathyroidism, hyperthyroidism, Paget's disease and other disorders of bone metabolism. Clin Sci 1969;36: 435-443. 43. Avioli LV, McDonald JE, Singer RA. Excretion of pyrophosphate in disorders of bone metabolism. J Clin Endocrino11965;25:912-915. 44. Lewis AM, Thomas WC, Tomita A. Pyrophosphate and the mineralizing potential of urine. Clin Sci 1966;30:389-397. 45. Alvarez-Arroyo MV, Traba ML, Rapado A, et al. Role of citric acid in primary hyperparathyroidism with renal lithiasis. Urol Res

1992;20:88-90.

46. Smith LH, Vandenberg CJ, Wilson DM, et al. Urolithiasis in primary hyperparathyroidism. Abstr Am Soc Nephro11977;109A. 47. Sutton RAL, Watson L. Urinary excretion of calcium and magnesium in primary hyperparathyroidism. Lancet 1969;1000-1003. 48. Johannson G, Danielson BG, Ljunghall S. Magnesium homeostasis in mild to moderate primary hyperparathyroidism. Acta Chir Scand 1980;146:85-91. 49. Zerwekh JE, Hawing TIS, Poindexter J, et al. Modulation by calcium of the inhibitor activity of naturally occurring urinary inhibitors. Kidney Int 1988;33:1005-1008. 50. Koide T, Yoshioka T, Oka T, et al. Promotive effect of urines from patients with primary hyperparathyroidism calcium oxalate crystal aggregate. In: Walker V, Sutton RAL, Cameron EC, Pak CYC, Robertson WA, eds. Urolithiasis. New York:Plenum, 1989:109-111. 51. Lonsdale K. Human stones. Science 1968;159:1199-1207. 52. Pak CYC, Holt K, ZerwekhJK. Attenuation by sodium urate of the inhibitory effect of glycosaminoglycans on calcium oxalate nucleation. Invest Uro11979; 17:138-141. 53. Broulek PD, Stepan JJ, Pacovsky V. Primary hyperparathyroidism and hyperuricemia are associated but not corre-lated with indicators of bone turnovers. Clin Chim Acta 1987; 170:195-200.

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54. Ljunghall S, Akerstrom G. Urate metabolism in primary hyperparathyroidism. UroUnt 1982;37:73-78. 55. Muldowney JP, Carroll DV, Donohoe JF, et al. Correction of renal bicarbonate wastage by parathyroidectomy. Q j Med 1971; 160:487-498. 56. Coe FL. Magnitude of metabolic acidosis in primary hyperparathyroidism. Arch Intern Med 1974;134:202-205. 57. Hulter HN, Peterson JC. Acid-base homeostasis during chronic PTH excess in humans. Kidney Int 1985;28:187-192. 58. McGeown MG. Effect of parathyroidectomy on the incidence of renal calculi. Lancet 1961;1:586-587. 59. Deaconson TF, Wilson SD, Lemann J. The effect of parathyroidectomy on the recurrence of nephrolithiasis. Surgery 1987;102:910-912. 60. Posen S, Clifton-Bligh P, Reeve TS, et al. Is parathyroidectomy of benefit in primary hyperparathyroidism? Q j Med 1985;215:241-251. 61. Siminovitch JMP, Caldwell BE, Straffen RA. Renal lithiasis and hyperparathyroidism: Diagnosis, management and prognosis. J Uro11981;126:720-722. 62. Johannsen H, Thoren L, Werner I, et al. Normocalcemic hyperparathyroidism, kidney stones and idiopathic hypercalciuria. Surgery 1975;77:691-696. 63. Kaplan RA, Snyder WH, Stewart A, et al. Metabolic effects of parathyroidectomy in asymptomatic primary hyperparathyroidism. J Clin Endocrinol Metab 1976;42:415-426. 64. Siminovitch JMP, James RE, Esselsytne CBJ, et al. The effect of parathyroidectomy in patients with normocalcemic calcium stones. J Uro11980;123:335-337. 65. Fabris A, Ortalda V, D'Angelo A, Giannin S, Maschio G. Biochemical and clinical studies after parathyroidectomy in primary hyperparathyroidism. In: Walker V, Sutton RAL, Camer EC, Pak CYC, Robertson WG, eds. Urolithiasis. New York:Plenum, 1989:637-640. 66. Breslau NA, Pak CYC. Combined primary hyperparathyroidism and absorptive hypercalciuria: Clinical implication. In: Walker V, Sutton Rai, Cameron EC, Pak CYC, Robertson WG, eds. Urolithiasis New York:Plenum, 1989:627-630. 67. Muir JW, Baker LRI. Hypercalciuria and recurrent urinary stone formation despite successful surgery, for primary hyperparathyroidism. Br MedJ 1978;738:1-3. 68. Bordier P, Ryckewort A, Gueris J. On the pathogenesis of so-called idiopathic hypercalciuria. A m J M e d 1977;63:398-408. 69. Maschio G, Vecchioni R, Tessitore N. Recurrence of autonomous hyperparathyroidism in calcium nephrolithiasis. Am J Med

1980;68:607-609.

70. Pak CYC. Effect of parathyroidectomy on crystallization of calcium salts in urine of patients with primary hyperparathyroidism. Invest Uro11979;17:140-148. 71. Robertson WG. Risk factors in calcium stone disease. In: Brackis G, Finlayson B, eds. International urinary stone conference. Nettleton, Massachusetts: PSG Publ., 1979:12. 72. Pak CYC, Sakhaee K, Crowther C, et al. Evidence justifying a high fluid intake in treatment of nephrolithiasis. Ann Intern Med 1980;93:36-39. 73. Wasserstein AG, Stolley PD, Soper KA, et al. Case-control study of risk factors for idiopathic calcium nephrolithiasis. Miner Electrolyte Metab 1987;13:85-95. 74. Goldfarb S. Dietary factors in the pathogenesis and prophylaxis of calcium nephrolithiasis. Kidney Int 1988;34:544-555. 75. Robertson WG, Heyburn PJ, Peacock M. The effect of high animal protein intake on the risk of calcium stone-formation in the urinary tract. Clin Sci 1979;57:285-288. 76. Muldowney FP, Freaney R, Muldowney EP, et al. Hypercalciuria in parathyroid disorders: Effect of dietary sodium control. Am J Kidney Dis 1991;17:323-329.

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77. Coe FL, Favus MJ, Crocket T, et al. Effects of low calcium diet on urine calcium excretion, parathyroid function and serum 1,25(OH)zD levels in patients with idiopathic hypercalciuria and in normal subjects. A m J M e d 1982;72:25-32. 78. Pak CYC, Delea CS, Bartter FC. Successful treatment of recurrent nephrolithiasis with cellulose phosphate. N Engl J Med 1976;290:175-180. 79. Duarte CG, Winnaker JL, Becker KL, et al. Thiazide induced hypercalcemia. N EnglJ Med 1971;15:828-830. 80. Kukreja SC, Johnson PA, Ayala G, et al. Role of calcium and betaadrenergic system in control of parathyroid hormone secretion. Proc Soc Exp Biol Med 1976;151:320-328. 81. Caro JF, Castro JH, Glennon JA. Effect of long term propranolol administration on parathyroid hormone and calcium concentration in primary hyperparathyroidism. A n n Intern Med 1979;91: 740-741. 82. Monson JP, Beer M, Boucher BJ, et al. Propranolol in primary hyperthyroidism. Lancet 1979;1:884. 83. Sherwood JK, Ackroyd FW, Garcia M. Effect of cimetidine on circulating parathyroid hormone in primary hyperparathyroidism. Lancet 1980;1:616-620. 84. Palmer FJ, Sawyers TM, Wierzbinski SJ. Cimetidine and hyperparathyroidism. N EnglJ Med 1980;302:692. 85. Awoke S, Lawrence GD. Cimetidine and hyperparathyroidism. Lancet 1980; 1:1134.

86. Gallagher JC, Nordin BEC. Treatment with estrogens of primary hyperparathyroidism in post-menopausal women. Lancet 1972;1:503-507. 87. Marcus R, Modvig P, Crim M, et al. Conjugated estrogens in the treatment of post menopausal women with hyperparathyroidism. Ann Intern Med 1984;100:633-640. 88. Fleisch H, Felix R. Diphosphonates. Calcif Tissue Int 1979;27:91-94. 89. Kaplan RA, Gero WB, Poindexter C. Metabolic effects of diphosphonate in primary hyperparathyroidism. J Clin Pharmacol 1977;17:410-419. 90. Shane E, Baquiran DC, Bilezikian JE Effect of dichloromethylene diphosphonate on serum and urinary calcium in primary hyperparathyroidism. Ann Intern Med 1981;95:23-27. 91. Mundy GR, Wilkinson R, Heath DA. Comparative study of available medical therapy for hypercalcemia of malignancy. A m J M e d 1983; 74:421-432. 92. Albright E Bauet W, Claflin D, et al. Studies in parathyroid physiology. J Clin Invest 1932;11:411-435. 93. Goldsmith RS, Ingbar SH. Inorganic phosphate treatment of hypercalcemia of diverse etiologies. N EnglJ Med 1966;274:1.94 94. Purnell DC, Scholz DA, Smith LH, et al. Treatment of primary hyperparathyroidism. A m J Med 1974;56:800-810. 95. Broadus AE, Magee JS, Mallette LE. A detailed evaluation of oral phosphate therapy in selected patients with primary hyperparathyroidism. J Clin Endocrinol Metab 1983;56:953-961.

C TF R 28 Guidelines for the Medical and Surgical Management of Primary Hyperparathyroidism MICHAEL KLEEREKOPER Department of Medicine, Wayne State University, Detroit, Michigan 48201 ROBERT UDELSMAN Department of Surgery, TheJohns Hopkins University School of Medicine, Baltimore, Maryland 21287 MICHAEL A. LEVINE* Departments of Pediatrics, Medicine, and Pathology, TheJohns Hopkins University School of Medicine, Baltimore, Maryland 21287

INTRODUCTION

patients if the total serum calcium level is >1 m g / d l above the u p p e r limit of normal range; if there is evidence of overt bone disease (e.g., osteitis fibrosa cystica) or a decrease in cortical bone mineral density more than 2 standard deviations below the age- and sexadjusted mean value; if there is reduced renal function, stone disease (nephrolithiasis or nephrocalcinosis), or significant hypercalciuria (>400 m g / d a y ) ; and if there has been an episode of acute PHPT. Last, patients who are young (age u n d e r 50 years) should be considered for surgery even in the absence of other indications, because younger patients will have PHPT for a longer period of time and thus have a greater theoretical risk of complications. T h o u g h only about 20% of patients with PHPT are symptomatic at presentation, these guidelines will identify an additional 30-40% of asymptomatic patients who are candidates for surgery. Thus, about one-half of patients with PHPT will meet criteria for surgery (1,2). The 1990 Consensus Conference concluded that PTX is the treatment of choice for patients with PHPT, but also acknowledged that in many patients the disease is sufficiently mild and free of obvious adverse health effects (i.e., asymptomatic PHPT) that cautious monitoring might be a reasonable plan. Given the incomplete knowledge of the long-term consequences of untreated PHPT, the Consensus Conference findings were proposed as guidelines, with the expectation that clinical use of the guidelines as well as controlled study of PHPT would lead to the development of optimal m a n a g e m e n t algorithms. What has been the impact of these recommendations on the clinical care of patients

Prior to the introduction of the multichannel autoanalyzer approximately 30 years ago, primary hyperparathyroidism (PHPT) was typically associated with symptoms and parathyroid surgery was regularly performed. Since that time, widespread use of the autoanalyzer has facilitated the routine determination of serum calcium concentration in otherwise healthy patients, and consequently the proportion of patients with PHPT who apparently lack signs or symptoms of parathyroid h o r m o n e (PTH) excess or hypercalcemia has increased. This remarkable shift in the presentation of PHPT not only e n g e n d e r e d the somewhat awkward term "asymptomatic PHPT" to describe this ambiguous condition, but more importantly raised new questions about the appropriate m a n a g e m e n t of all patients with PHPT. O n the one hand, it seems unwise to recomm e n d parathyroidectomy (PTX), with its attendant risks, to all patients when many will have no features of metabolic bone disease or risk of renal stone disease. On the other hand, long-term data about the potential adverse effects of asymptomatic PHPT are only now beginning to emerge. A National Institutes of Health (NIH) Consensus Development Conference considered these issues and others in 1990, and proposed a set of guidelines (Table 1) to distinguish patients who might be suitable candidates for surgical treatment for PHPT (1,2). Surgery was generally r e c o m m e n d e d for

*To w h o m c o r r e s p o n d e n c e s h o u l d be addressed.

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TABLE 1 Guidelines for the Surgical Management of Patients with Primary Hyperparathyroidism Serum calcium concentration >1 mg/dl above the upper limit of normal Evidence of any end-organ complication of primary hyperparathyroidism, such as overt bone disease or nephrolithiasis An episode of acute primary hyperparathyroidism Hypercalciuria in excess of 400 mg/day Evidence of reduced cortical bone mass at the distal radius as determined by bone densitometry (i.e., >2 SD below age- and sex-matched controls) Young age (i.e., less than 50 years)

with PHPT? In 1997, an extensive survey in the United States of endocrinologists and endocrine surgeons who care for patients with PHPT was undertaken to determine the current standard of practice for m a n a g e m e n t of PHPT. Surprisingly, this study indicated that many of these clinical specialists were unaware of these published guidelines, and that the majority did not follow the conference recommendations (3,4). On the other hand, carefully controlled research studies have tested the reliability of the Consensus Conference guidelines. The results of a 10-year prospective study of a 121 patients with PHPT who were followed with or without PTX has been reported (5). Of the subjects, 50% underwent surgery, including 49 with symptomatic disease and 12 who met no guideline for surgery. After surgery these patients exhibited clear evidence of cure of PHPT, with resolution of hypercalcemia, no recurrence of nephrolithiasis, significant and prolonged improvement in bone mineral density (BMD), and return of accelerated skeletal turnover to a normal rate. The remaining subjects did not have surgery, either because they refused (8 subjects) or they met no guideline for PTX (52). During the next 10 years all 8 symptomatic patients showed evidence of progression of disease, most often manifested by recurrent nephrolithiasis. In addition, 14 of the patients who were initially considered asymptomatic (27%), based on the NIH Consensus Conference guidelines, also showed progression of PHPT, as evidenced by the develo p m e n t of one or more new criteria for PTX. By contrast, most (77%) of the patients who met the NIH guidelines for careful monitoring without immediate need for PTX did well. They did not have resolution of any of the manifestations of PHPT but did not have any evidence of disease progression either. The only guideline criterion associated with a significantly increased risk of worsening disease in subjects with "asymptomatic PHPT" was young age. This was particularly true for

those women who became menopausal during the course of the study and who experienced a progressive decrease in BMD coincident with the development of estrogen deficiency. Although the results of this prospective study provide important confirmation that the initial NIH recommendations had substantial merit, they also emphasize that the guidelines cannot predict which asymptomatic patients will have progressive disease. The NIH consensus statement also recognized that there is a growing interest in nonconventional manifestations of PHPT that range from premature cardiovascular death (6-12) to vague neurobehavioral symptoms of being unwell (13-17). In 1990 it was felt that there was insufficient evidence to include these as definite manifestations of PHPT, and no specific recommendations were made for m a n a g e m e n t of patients with unusual or poorly characterized features of PHPT. Regrettably, the important 10-year prospective study did not consider any of these potential manifestations of PHPT, and this issue remains largely unaddressed except for epidemiologic data collected in crosssectional studies. A small controlled clinical trial of PTX versus careful monitoring indicated that PTX resulted in a significant improvement in quality of life, assessed using a validated questionnaire. Results of this study have been published only in abstract form (18) as of this writing, so full details are not yet available. Based on current evidence, it seems reasonable to conclude, as did the NIH conference 10 years ago, that even though PTX is the treatment of choice for PHPT, surgery will be neither necessary nor beneficial for all patients. It is equally important to acknowledge that the three important studies cited above have not really advanced our ability to predict which patients with mild or asymptomatic PHPT at initial diagnosis will ultimately require PTX. As the risks of untreated, mild PHPT become more clearly defined, and the benefits of early treatment are rigorously established, it will become possible to develop evidence-based guidelines for the m a n a g e m e n t of PHPT. On the other hand, advances in surgical m a n a g e m e n t of PHPT now make definitive, surgical correction of PHPT an increasingly attractive option for most patients, regardless of whether they meet any of the guidelines for surgery proposed by the NIH Consensus Developm e n t Conference. Thus, some physicians may recomm e n d PTX to all patients, and some asymptomatic patients may elect surgical treatment because they find the prospect of long-term, continuous monitoring of PHPT to be inconvenient or worrisome. In the remainder of this chapter we describe several new approaches to medical and surgical treatment of PHPT that have already begun to impact traditional

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m a n a g e m e n t algorithms, and which will continue to influence our assessment of the risks and benefits of treating PHPT.

ADVANCES IN T H E S U R G I C A L M A N A G E M E N T OF P H P T A large n u m b e r of published studies have demonstrated that major advances have been made in surgical approaches to the cure of PHPT (19-43). The prevailing opinion in 1990 was that preoperative localization procedures lacked sufficient sensitivity and specificity to r e c o m m e n d their routine use. It is now clear that scintigraphy with technetium-99m sestamibi, either alone (19-22) or combined with ultrasonography (23-25), has markedly improved this sensitivity and specificity such that most experienced parathyroid surgeons now routinely obtain these studies preoperatively. High-quality sestamibi scans, when combined with single-photon emission computed tomography (SPECT), can demonstrate a three-dimensional image of an enlarged parathyroid gland with enhanced anatomic resolution and relationship to adjoining structures (26,27). The sestamibi scan is p e r f o r m e d as a two-phase test in order to take advantage of the observation that sestamibi accumulated by the thyroid gland is rapidly washed out, but uptake by parathyroid tissue is retained and visible with a 2-hour image. This precise anatomic localization has been a major advance in assisting surgeons in performing outpatient PTX through minimal incisions. This has been shown in some but not all studies to decrease significantly the operative time and costs of PTX (28-30). An adjunct of this preoperative localization has been the intraoperative use of either ultrasonography or scintigraphy (31-36). This may further improve sensitivity and specificity and has the potential to further reduce operative time. A related intraoperative procedure is m e a s u r e m e n t of circulating concentrations of parathyroid h o r m o n e in peripheral venous blood, while the neck is surgically exposed and the patient is still anesthetized. A base line level of PTH is first determined, and subsequent PTH levels are obtained after resection of abnormal parathyroid gland(s). A greater than 50% reduction in peripheral venous PTH occurs within 15 minutes of the resection of hyperfunctioning parathyroid tissue, and is indicative of curative procedure. Moreover, use of intraoperative PTH measurement can be an adjunct, or a replacement, to frozen-section histology for confirmation that parathyroid tissue has been resected. U n d e r unusual circumstances, blood can be obtained from exposed neck veins and assayed for PTH in order to afford

453

"real-time" localization of an ectopic or missing parathyroid gland. The assay procedures can be modified to reduce sensitivity without affecting specificity, providing assay results within 12-14 minutes of blood sampling (37-39). In appropriate patients, localization of a parathyroid a d e n o m a by technetium-99m sestamibi scintigraphy makes it reasonable to r e c o m m e n d minimally invasive parathyroidectomy rather than a standard bilateral neck exploration in which all of the parathyroid glands (normal and abnormal) are to be identified. Moreover, intraoperative m e a s u r e m e n t of PTH can enhance the likelihood of success of this more limited surgical procedure. It is now estimated that 85% of all patients with PHPT who are referred for surgery are candidates for minimally invasive outpatient PTX (see Chapter XX). This minimally invasive approach appears to result in durable cure rates that are at least as good as those obtained with conventional bilateral cervical exploration u n d e r general anesthesia. In addition, the avoidance of general anesthesia and hospital admission results in a mean hospital charge that is 40% less than the traditional approach. Many patients with the most mild PHPT can now experience PTX as a totally outpatient procedure, spending 6 hours or less in the surgical environment (40-43). This approach may not be suitable for every patient with PHPT, however, particularly those with more advanced disease. T h o u g h these improvements lower the expense of surgery through reduced need for hospitalization, the cost of pre- and intraoperative localization and intraoperative PTH assays can be substantial. This makes it imperative to develop guidelines to determine who will derive objective benefit from minimally invasive parathyroidectomy. Nor does this lessen the need to complete appropriate studies to d o c u m e n t carefully just which patients with PHPT are least likely to benefit from surgical cure, as well as the most cost-effective monitoring for those not referred for PTX at the time of diagnosis. Thus, PTX in the year 2001 differs substantially from the conventional surgical approach of 1990, with the important proviso that the experience of the parathyroid surgeon is still paramount.

ADVANCES IN MEDICAL MANAGEMENT OF P H P T Patients with asymptomatic PHPT who do not meet criteria for surgery (above) as well as patients who refuse initial recommendations for surgery must be monitored regularly in order to detect complications that would justify p r o m p t referral for surgery. Studies have demonstrated a notable lack of biochemical

454

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CHAPTER28

progression or continued loss of bone density in many patients who are followed conservatively without treatm e n t (5), suggesting that the natural history of mild PHPT can be benign. Current recommendations for medical surveillance include measurement of serum calcium every 6 months, and measurement of urinary calcium and creatinine every 12 months. Although most studies indicate that bone density remains stable in patients with mild PHPT, in the absence of specific guidelines it nonetheless seems p r u d e n t to monitor bone mineral density (including the distal radius, an important site of cortical bone) in asymptomatic patients by densitometry at periodic (every 1 to 3 years) intervals. Medical m a n a g e m e n t is also appropriate for symptomatic patients who are unfit for surgery, who refuse surgery, or who have inoperable parathyroid cancer. Urgent medical treatment of hypercalcemia is often necessary to stabilize symptomatic patients prior to surgery. Several general guidelines can be r e c o m m e n d e d to all patients with hypercalcemia who will be followed medically. Patients should be instructed to avoid diuretics, particularly thiazides, and to maintain a liberal fluid intake. Ambulation should be encouraged. The appropriate dietary intake of calcium has not been determined. A high calcium intake could theoretically aggravate hypercalcemia, whereas a low intake might stimulate further secretion of PTH. Until more information is available, it is reasonable to r e c o m m e n d that patients moderate their dietary calcium intake and avoid calcium or vitamin D supplements. Safe and effective therapeutic agents are still lacking for the chronic medical treatment of PHPT, and studies of medical therapy for PHPT have not made as much progress as our understanding of the natural history and the surgical advances (44). There is great interest in calcimimetic agents, which bind to a calcium-sensing receptor. This cell surface receptor (see Chapters XX) is most abundantly expressed in parathyroid tissue and in distal segments of the nephron, with smaller amounts present in brain and other tissues. The calcium-sensing receptor enables parathyroid cells to monitor the ionized calcium concentration in extracellular fluid, and is a critical c o m p o n e n t of the signaling pathway that regulates calcium-responsive secretion of PTH (see Chapter XX). Calcimimetics interact with this receptor and amplify the effect of b o u n d calcium, thereby tricking the sensor into thinking that the prevailing calcium concentration is higher than it actually is (45,46). The effect of this charade is to decrease synthesis and secretion of PTH by the parathyroid cell. A report (47) detailing the results of a short-term dosefinding study with an experimental calcimimetic agent, R-568, provided the clinical p r o o f of this principle for this class of agents, and demonstrated an almost 80%

reduction in serum levels of PTH in patients receiving the highest dose of the drug. There was an associated decrease in both serum and urine calcium. Another pilot study of a longer acting calcimimetic, AMG 073, in patients with mild PHPT showed an approximately 45% decrease in serum levels of intact PTH and a dosed e p e n d e n t reduction of serum calcium levels to normal over 15 days (48). W h e t h e r the efficacy of these agents will persist with chronic use is being actively investigated. Moreover, the effects of these drugs on clinical and laboratory manifestations of PHPT, particularly the effect on BMD, are also u n d e r study. These agents offer the hope of an important therapeutic option, but they are still several years from being widely available. The ability of intravenous bisphosphonates to lower the serum calcium concentration rapidly and safely in patients with severe hypercalcemia is well established, and these agents are now the standard of care for this clinical situation (49,50). These observations led several groups to evaluate the potential of oral bisphosphonate therapy in patients with PHPT who have milder degrees of hypercalcemia. Although these studies have produced seemingly encouraging results, unfortunately none of these trials has yet to be published in full and the only available information is from published abstracts. In one such 2-year study, oral alendronate (10 mg, every other day) significantly reduced markers of bone resorption and appeared to be as effective as PTX in increasing BMD of hip, spine, and radius (51). Shortly after beginning alendronate therapy serum levels of calcium decreased and serum levels of PTH increased, but these changes were transient and both calcium and PTH returned to pretreatment levels within a few months. Epidemiologic studies have shown PHPT is most prevalent in the fifth and sixth decades of life and is two to three times more c o m m o n in women than men. These observations have led several investigators to assess the role of estrogen replacement in the management of postmenopausal women with PHPT (52-54). One such study (54) was a rigorous, 3-year prospective, randomized, placebo-controlled clinical trial, and produced a n u m b e r of important findings. First, at all measurement sites estrogen was significantly more effective than placebo at maintaining or increasing BMD. Second, there was a significant reduction in serum calcium, serum PTH, and biochemical markers of bone turnover. This is a very encouraging observation and both confirms and extends earlier reports. It can safely be concluded that estrogen is effective therapy for postmenopausal women with PHPT, at least for up to 3 years. However, a disturbing aspect of this report was the progressive decline in BMD in the group receiving placebo only. This is in sharp contrast to the observational study m e n t i o n e d above that concluded

GUIDELINES FOR P H P T MANAGEMENT

that the BMD wasstable in u n t r e a t e d mild PHPT for up to 10 years (5). The authors of the estrogen trial analysis raised concerns about the validity of the BMD measurements obtained in the earlier observational study, particularly as the densitometer originally used to monitor BMD was replaced during the course of the study. There were also substantial differences between the study populations enrolled in the two studies, which included m e n and women in the observational study but was obviously restricted to women in the estrogen study. It should be r e m e m b e r e d that a small segment of subjects in the observational study did experience progressive bone loss and that these were primarily w o m e n who entered m e n o p a u s e during the observation period. Based on these conflicting experimental observations and continued uncertainties about the natural history of u n t r e a t e d PHPT, it would seem both logical and appropriate to r e c o m m e n d that bone mass in women with u n t r e a t e d PHPT be m o n i t o r e d closely, particularly in those who become menopausal after the diagnosis of PHPT has been made. These patients appear to be at increased risk of rapid bone loss, and may need PTX or estrogen replacement. I m p o r t a n t questions that remain unanswered include the duration of estrogen's beneficial effect in these patients, and how late after the m e n o p a u s e will initiation of estrogen replacement be effective. These issues are of increasing relevance because the incidence of PHPT continues to increase with age, again more so in women than in men, and concerns regarding the safety of long-term estrogen r e p l a c e m e n t therapy (e.g., breast cancer and exacerbation of underlying heart disease) continue to grow. These issues have h e i g h t e n e d interest in the potential usefulness of selective estrogen receptor modulators (SERMs) in postmenopausal women with PHPT. To date the only information available about this application of SERMs is an anecdotal report of one woman treated with tamoxifen and a second woman treated with raloxifene (55). In both cases, there were encouraging early responses. Because the assumption is that estrogen is effective in PHPT owing to a direct skeletal effect, and both of these SERMs are estrogen receptor agonists with respect to the skeleton, these results are not surprising and should encourage more formal clinical trials.

SUMMARY A N D C O N C L U S I O N S PTX remains the t r e a t m e n t of choice for those patients with PHPT who require treatment, and the reco m m e n d a t i o n s first proposed by the NIH Consensus Development Conference continue to provide a useful, if dated, algorithm for identifying patients for whom surgery is indicated (Table 1). Despite advances in our

/

455

u n d e r s t a n d i n g of the natural history of u n t r e a t e d PHPT, it remains certain that some patients will have such mild disease that no specific intervention will be necessary. Advances in minimally invasive surgery assure that there will be fewer patients in whom surgery is contraindicated, but we must intensify efforts to develop rigorous, evidence-based guidelines that will identify all patients in whom surgery is likely to be beneficial. A role for estrogen therapy in postmenopausal women with PHPT is b e c o m i n g m o r e clearly defined. In such women with disease sufficiently mild that curative PTX is not immediately indicated and in whom there is no contraindication to estrogen, this would seem a preferred approach over simple noninterventional observation. For m e n with PHPT and w o m e n who are not candidates for estrogen, very preliminary but nonetheless encouraging studies suggest that an effective medical therapy (calcimimetic, bisphosphonate) will soon be forthcoming. W h e n this happens it will be more feasible to conduct the definitive studies comparing no intervention to either medical or surgical therapy. The analyses and discussions presented here are certain to present very different perspective in the future.

REFERENCES 1. Proceedings of the NIH Consensus Development Conference on diagnosis and management of asymptomatic primary hyperparathyroidism. Bethesda, Maryland, October 29-31, 1990. JBone Miner Res 1991;(Suppl 2):$1-$166. 2. NIH conference. Diagnosis and management of asymptomatic primary hyperparathyroidism: Consensus development conference statement. Ann Intern Med 1991;114:593-597. 3. SosaJA, Powe NR, Levine MA, Udelsman R, Zeiger MA. Profile of a clinical practice: Thresholds for surgery and surgical outcomes for patients with primary hyperparathyroidism: A national survey of endocrine surgeons. J Clin Endocrinol Metab 1998;83:2658-2665. 4. Sosa JA, Powe NR, Udelsman R, Zeiger MA, Levine MA. Clinical management of patients with primary hyperparathyroidism: A national survey of endocrinologists. Unpublished observations. 5. Silverberg SJ, Shane E, Jacobs TP, Sifts E, Bilezikian JE A 10-year prospective study of primary hyperparathyroidism with or without parathyroid surgery. N EnglJ Med 1999;341:1249-1255. 6. Barletta G, De Feo ML, Del Bene R, Lazzeri C, Vecchiarino S, La Villa G, Brandi ML, Franchi E Cardiovascular effects of parathyroid hormone: A study in healthy subjects and normotensive patients with mild primary hyperparathyroidism. J Clin Endocrinol Metab 2000;85:1815-1821. 7. Smith JC, Page MD, John R, Wheeler MH, Cockroft JR, Scanlon ME Davies JS. Augmentation of central arterial pressure in mild primary hyperparathyroidism. J Clin Endocrinol Metab 2000;85:3515-3519. 8. Silverberg SJ. Cardiovascular disease in primary hyperparathyroidism. J Clin Endocrinol Metab 2000;85:3513-3514. 9. Kosch M, Hausberg M, Vormbrock K, Kisters K, Gabriels G, Heinz Rahn K, Barenbrock M. Impaired flow-mediated vasodilation of the brachial artery in patients with primary hyperparathyroidism improves after parathyroidectomy. Cardiovasc Res 2000;47:813-818.

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10. Neunteufl T, Heher S, Prager G, Katzenschlager R, Abela C, Niederle B, Stefenelli T. Effects of successful parathyroidectomy on altered arterial reactivity in patients with hypercalcaemia: Results of a 3-year follow-up study. Clin Endocrino12000;53:229-233. 11. Nilsson IL, Aberg J, Rastad J, Lind L. Endothelial vasodilatory dysfunction in primary hyperparathyroidism is reversed after parathyroidectomy. Surgery 1999;126:1049-1055. 12. Barna I, Varadi A, Lakatos E Metabolic abnormalities related to cardiovascular risk in primary hyperparathyroidism: Effects of surgical treatment. J Intern Med 1999;245:311-313. 13. Burney RE, Jones KR, Christy B, Thompson NW. Health status improvement after surgical correction of primary hyperparathyroidism in patients with high and low preoperative calcium levels. Surgery 1999;125:608-614. 14. Lundgren E, Ljunghall S, Akerstrom G, Hetta J, Mallmin H, Rastad J. Case-control study on symptoms and signs of "asymptomatic" primary hyperparathyroidism. Surgery 1998;124:980-985. 15. Burney RE, Jones KR, Peterson M, Christy B, Thompson NW. Surgical correction of primary hyperparathyroidism improves quality of life. Surgery 1998;124:987-991. 16. Pasieka JL, Parsons LL. Prospective surgical outcome study of relief of symptoms following surgery in patients with primary hyperparathyroidism. WorldJ Surg 1998;22:513-518. 17. Lundgren E, Szabo E, Ljunghall S, Bergstrom R, Holmberg L, Rastad J. Population based case-control study of sick leave in postmenopausal women before diagnosis of hyperparathyroidism. Br Med J. 1998;317:848-851. 18. Rao D, Phillips ER, Divine GW, Talpos GB. Randomized controlled clinical trial of surgery versus no surgery in patients with mild asymptomatic primary hyperparathyroidism: Final report. JBone Miner Res 2000;15(Suppl. 1):S164. 19. Dillavou ED, JenoffJS, Intenzo CM, Cohn HE. The utility of sestamibi scanning in the operative management of patients with primary hyperparathyroidism. J Am Coll Surg 2000;190:540-545. 20. Rubello D, Saladini G, Casara D, Borsato N, Toniato A, Piotto A, Bernante P, Pelizzo MR. Parathyroid imaging with pertechnetate plus perchlorate/MIBI subtraction scintigraphy: A fast and effective technique. Clin Nucl Med 2000;25:527-531. 21. Greene AK, Mowschenson P, Hodin RA. Is sestamibi-guided parathyroidectomy really cost-effective? Surgery 1999; 126:1036-1040. 22. Jofre j, Gonzalez P, Massardo T, Zavala A. Optimal imaging time for delayed images in the diagnosis of abnormal parathyroid tissue with Tc-99m sestamibi. Clin Nucl Med 1999;24(8):594-596. 23. Casara D, Rubello D, Piotto A, Pelizzo MR. 99mTc-MIBI radio-guided minimally invasive parathyroid surgery planned on the basis of a preoperative combined 99mTc-pertechnetate/ 99mTc-MIBI and ultrasound imaging protocol. EurJ Nucl Med 2000;27:1300-1304. 24. De Feo ML, Colagrande S, Biagini C, Tonarelli A, Bisi G, Vaggelli L, Borrelli D, Cicchi P, Tonelli F, Amorosi A, Serio M, Brandi ML. Parathyroid glands: Combination of (99m)Tc MIBI scintigraphy and US for demonstration of parathyroid glands and nodules. Radiology 2000;214:393-402. 25. Purcell GP, Dirbas FM, Jeffrey RB, Lane MJ, Desser T, McDougall IR, Weigel RJ. Parathyroid localization with high-resolution ultrasound and technetium Tc 99m sestamibi. Arch Surg 1999;134:824-828. 26. Moka D, Voth E, Dietlein M, Larena-Avellaneda A, Schicha H. Technetium 99m-MIBI-SPECT: A highly sensitive diagnostic tool for localization of parathyroid adenomas. Surgery 2000;128:29-35. 27. Chen H, Sokoll LJ, Udelsman R. Outpatient minimally invasive parathyroidectomy: A combination of sestamibi-SPECT localization, cervical block anesthesia, and intraoperative parathyroid hormone assay. Surgery 1999;126:1016-1021. 28. Goldstein RE, Blevins L, Delbeke D, Martin WH. Effect of minimally invasive radioguided parathyroidectomy on efficacy, length

29.

30.

31.

32. 33.

34.

35.

36.

37.

38.

39.

40.

41.

of stay, and costs in the management of primary hyperparathyroidism. Ann Surg 2000;231 (5):732-742. Denham DW, Norman J, Cost-effectiveness of preoperative sestamibi scan for primary hyperparathyroidism is dependent solely upon the surgeon's choice of operative procedure. JAm CoU Surg 1998;186:293-305. SosaJA, Powe NR, Levine MA, Bowman HM, Zeiger MA, Udelsman R. Cost implications of different surgical management strategies for primary hyperparathyroidism. Surgery 1998;124:1028-1035. Dackiw AP, Sussman JJ, Fritsche HA, Jr, Delpassand ES, Stanford P, Hoff A, Gagel RF, Evans DB, Lee JE. Relative contributions of technetium Tc 99m sestamibi scintigraphy, intraoperative gamma probe detection, and the rapid parathyroid hormone assay to the surgical management of hyperparathyroidism. Arch Surg 2000;135:550-555. Flynn MB, Bumpous JM, Schill K, McMasters KM. Minimally invasive radioguided parathyroidectomy JAm Coll Surg 2000;191:24--31. Udelsman R, Osterman F, Sokoll LJ, Drew H, Levine MA, Chan DW. Rapid parathyroid hormone measurement during venous localization. Clin Chim Acta 2000;295:193-198. Udelsman R, Donovan PI, Sokoll LJ. One hundred consecutive minimally invasive parathyroid explorations. Ann Surg 2000;232:331-339. Ng WD, Cheng PW. Minimally invasive surgery for primary hyperparathyroidism: A systematic review: Comment. Aust N z J Surg 2000;70:743-745. Smit PC, Rinkes IH, van Dalen A, van Vroonhoven TJ. Direct, minimally invasive adenomectomy for primary hyperparathyroidism: An alternative to conventional neck exploration? Ann Surg 2000;231:559-565. Mengozzi G, Baldi C, Aimo G, Mullineris B, Salvo R, Biasiol S, Pagni R, Gasparri G. Optimizing efficacy of quick parathyroid hormone determination in the operating theater. Int J Biol Markers 2000; 15:153-160. Gordon LL, Snyder III WH, Wians E Jr, Nwariaku E Kim LT. The validity of quick intraoperative parathyroid hormone assay: An evaluation in seventy-two patients based on gross morphologic criteria. Surgery 1999;126:1030-1035. Garner SC, Leight GS,Jr. Initial experience with intraoperative PTH determinations in the surgical management of 130 consecutive cases of primary hyperparathyroidism. Surgery 1999;126:1132-1137. Inabnet WB, Fulla Y, Richard B, Bonnichon P, Icard P, Chapuis Y. Unilateral neck exploration under local anesthesia: The approach of choice for asymptomatic primary hyperparathyroidism. Surgery 1999;126:1004-1009. Lo Gerfo E Bilateral neck exploration for parathyroidectomy under local anesthesia: A viable technique for patients with coexisting thyroid disease with or without sestamibi scanning. Surgery

1999;126:1011-1014.

42. Willeke F, Willeke M, Hinz U, Lorenz D, Nitschmann K, Grauer A, Senninger N, Klar E, Herfarth C. Effect of surgeon expertise on the outcome in primary hyperparathyroidism. Arch Surg 1998;133:1066-1070. 43. Utiger RD. Treatment of primary hyperparathyroidism. NEnglJ Med 1999;341:1301-1302. 44. Strewler GJ. Medical approaches to primary hyperparathyroidism. Endocrinol Metab Clin North Am 2000; (3):523-539. 45. Nemeth EF, Fox J. Calcimimetic compounds: A direct approach to controlling plasma levels of parathyroid hormone in hyperparathyroidism. Trends Endocrinol Metab 1999;10:66-71. 46. Rosen HN, Lim M, Garber J, Moreau S, Bhargava HN, Pallotta J, Spark R, Greenspan S, Rosenblatt M, Chorev M. The effect of PTH antagonist BIM-44002 on serum calcium and PTH levels in hypercalcemic hyperparathyroid patients. Calcif Tissue Int 1997;61:455-459. 47. Silverberg SJ, Bone III HG, Marriott TB, Locker FG, Thys-Jacobs S, Dziem G, Kaatz S, Sanguinetti EL, Bilezikian JE Short-term

GUIDELINES FOR P H P T MANAGEMENT

48.

49. 50.

51.

inhibition of parathyroid hormone secretion by a calciumreceptor agonist in patients with primary hyperparathyroidism. N EnglJ Med 1997;337:1506-1510. Shoback DM, Bilezikian J, Binder TA, Graves T, Turner SA, Peacock M. Calcimimetic AMG 073 normalizes total serum calcium in patients with primary hyperparathyroidism. Annual Meeting of the American Society for Bone and Mineral Research, 2000. Abstract F116, pS210 (abstract). Russell RG, Rogers MJ. Bisphosphonates: From the laboratory to the clinic and back again. Bone 1999;25(1):97-106. Gerrard GE, Dodwell DJ, Vail A, Watters J, Overend MA. An audit of the management of malignant hypercalcaemia. Clin Oncol (R Coll Radiol) 1996;8(1):39-42. Bertoldo E Furlan E Gasperi E, Colapietro E Lorenzi T, Lo Cascio V. Two years treatment of primary hyperparathyroidism with oral alendronate. Annual Meeting of the ASBMR, 1999, p. S143 (abstract 1043).

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52. Diamond T, Ng AT, Levy S, Magarey C, Smart R. Estrogen replacement may be an alternative to parathyroid surgery for the treatment of osteoporosis in elderly postmenopausal women presenting with primary hyperparathyroidism: A preliminary report. Osteoporos Int 1996;6(4):329-333. 53. McDermott MT, PerloffJJ, Kidd GS. Effects of mild asymptomatic primary hyperparathyroidism on bone mass in women with and without estrogen replacement therapy. J Bone Miner Res 1994;9 (4 ) :509-514. 54. Orr-Walker BJ, Evans MC, Clearwater JM, Horne A, Grey AB, Reid IR. Effects of hormone replacement therapy on bone mineral density in postmenopausal women with primary hyperparathyroidism: Four-year follow-up and comparison with healthy postmenopausal women. Arch Intern Med 2000;160:2161-2166. 55. Shaw I, Osehobo P, Sarcedote A. Treatment of primary hyperparathyroidism with selective estrogen receptor modulators. Abstract 1730, Endocrine Society Annual Meeting, Toronto,June 2000.

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CHAPTER 4, FIGURE 2 Superposition, using the heavy backbone atoms of residues 22-32, of agonist and antagonist containing (top) the midregion lactam, c[Lys13-Asp17]PTH(1-34)NH2 and c[Lys13-Asp17]PTH(7-34)NH2 , respectively, (middle) C-terminal region lactams, c[Lys26 -Asp 30 ]PTH(1-34)NH 2 and c[Lys26 -Asp 30 ]PTH(7-34)NH 2, respectively, and (bottom) bicyclic mid- and C-terminal region lactams, c[Lys 13-Asp 17 ,Lys26 -Asp 30 ]PTH(1-34)NH 2, and c[Lys13-AsplF,Lys26-Asp3°]PTH(7-34)NH2 , respectively. The structures were taken from the molecular dynamic trajectories to illustrate the consequences of the flexible hinge centered around Arg-19. Reprinted with permission from Ref. 13. Copyright 1997 American Chemical Society.

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CHAPTER 4, FIGURE 4 Helical wheel representations of hPTHrP(22-31), RS-66271(22-31), and hPTH(22-31), illustrating the amphiphilic nature of the helices. Hydrophobic (red) and hydrophilic (blue) amino acids are shown. Reprinted with permission from Ref. 83. Copyright 1997 Amercan Chemical Society.

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IC3 CHAPTER 4, FIGURE 9 Depiction of the PTH1-Rc from a molecular dynamic simulation. The transmembrane oL-helices are depicted as cylinders. The regions of the receptor that have been experimentally determined are depicted as ribbons. The regions of the receptor that have been shown to cross-link with PTH analogs, PTH1Rc[173-181] (26,27) and M 425 (28), are depicted in gray (241).

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PTH Ligand C H A P T E R 4, FIGURE 10 Structural features and topological orientation of PTH1-Rc[168-198] located at the C-terminal region of the extracellular N terminus followed by the ectopic portion of the first TM domain (241, 242). (A) Schematic representation of the experimentally determined conformation. The structure consists of three (x-helices, two of which have been determined to lie on the surface of the membrane; the third, at the top of TM1, is membrane embedded. (B) The orientation of this peptide is shown with respect to the surface of the dodecylphosphocholine micelles used in the NMR study. The hydrophobicity of the molecule is indicated (blue, polar; red, hydrophobic). The decane molecules of the water/decane simulation cell used in the structure refinement are shown in green as CPK space-filling spheres.

CHAPTER 4, FIGURE 11 Model for the binding of hPTH(134) to hPTH1-Rc. For clarity, only portions of the TM helices, N terminus, and the third extracellular loop are shown in blue (non-cross-linked domains) and green (contact domains hPTH1 -Rc[173-189] and hPTH1 -Rc[409-437]) (A, side view; B, top view). The amphipathic o~-helix of the extracellular N terminus of the receptor is projecting to the right, lying on the surface of the membrane. The high-resolution, low-energy structure of hPTH(1-34) determined by NMR in a micellar environment is presented in pink. Residues in cross-linking positions 1 and 13 of hPTH(1-34) are denoted in yellow. The C-terminal amphipatic oL-helix of hPTH(1-34) is aligned in antiparallel arrangement with the amphipatic oL-helix of the extracellular N-terminus hPTH1-Rc[173-189], contiguous with TM1 and encompassing the 17-amino acid contact domain (in green), to optimize the hydrophilic interactions. Side chains of residue M 414 and M 425 within the "contact domain" TM6-third extracellular loop (hPTH1-Rc[S4°9-W437]) are shown (28).

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CHAPTER 6, FIGURE 1 PTHrP as a polyhormone. (A) PTHrP cDNA encodes a prepropeptide and mature forms of 139, 141, and 173 amino acids. The three isoforms are identical for the first 139 amino acids, which is the portion depicted here. Proposed biologically active domains within the protein are shown. SP, Signal peptide; P, propeptide, (B) The PTHrP bipartite nuclear localization sequence. This segment encompasses amino acids 88-106 of the mature form (indicated in the single-letter amino acid code) and comprises two basic clusters separated by 10 intervening "spacer" amino acids, resembling the Xenopus laevis nucleoplasmin nuclear localization sequence (72). Furthermore, this region conforms to the structural requirements for a nucleolar localization sequence, as described in key regulatory proteins of human retroviruses (HTLV-1 Rex, and HIV-1 Tat and Rev), consisting of an "arginine hinge" (KRK, in blue) and an adjacent Q inserted between two putative nuclear localization sequences (22). (C) Subcellular distribution of PTHrP in transfected COS-7 cells. Plasmid constructs encoding PTHrP forms having either an intact coding region, deletions within the coding region or fused in frame to the Escherichia coil lacZ gene were expressed in COS-7 cells and the subcellular localization of the recombinant protein was determined by indirect immunofluorescence. SP, Secretory pattern; N, nucleolar, C, cytoplasmic. Size of lettering on the right-hand side is indicative of the levels of PTHrP immunoreactivity in the various subcellular compartments (24).

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CHAPTER 6, FIGURE 2 Nuclear/nucleolar localization of endogenous PTHrP in vitro and in situ. (A) Human keratinocyte HaCaT cell line. [Reproduced from Gillespie M. Role of phosphorylation of parathyroid hormone-related protein (PTHrP). International Bone Forum 1997; 30 with permission.] (B) Cultured osteoblast-like cells harvested form calvariae of newborn mice. Insert: immunogold labeling of an osteoblast nucleolus in a section from a fetal mouse tibia (N, nucleus; Nu, nucleolus). [Reproduced and modified from (24), with permission.] (C) Neurons (arrow) and glial cells (arrowhead) in the ventral horn of a normal mouse.

CHAPTER 6, FIGURE 4 (A) Nuclear/nucleolar uptake of PTHrP by UMR-106.01 osteoblasts. Cells incubated with exogenous PTHrP(1-108) conjugated to a chromophore show intranuclear distribution of labeled PTHrP after 50 minutes, which is blocked by an excess of unlabeled ligand (pathway A). [Reproduced from (45), with permission.] (B) In COS-7 cells transiently expressing ubiquitin and PTHrP, ubiquitinated forms of PTHrP were present in total cell lysates treated with MG 132, a proteasome inhibitor, but not with vehicle DMSO, calpain inhibitor II, or cysteine proteinase inhibitor E-64 (pathway B). [Reproduced from (56) with permission.] (C) COS-1 cells transfected with a PTHrP cDNA in which the unique initiator ATG is altered to an ATC. Non-AUG-initiated PTHrP forms localized exclusively to the nucleolar compartment (pathway C). Regardless of the path utilized (A, B or C in Fig. 3), once PTHrP relocates to the cytosolic compartment, it would bind to importin 13and be directed to the nucleus/nucleolus on the strength of its fully functional NLS

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CHAPTER 6, FIGURE 3 Potential pathways (A, B, and C) utilized by PTHrP to gain access to the cytosol. In pathway A, secreted PTHrP undergoes internalization at the cell surface in a "receptor"-dependent manner. Endocytosis could be mediated by the type 1 PTH receptor (PTHR1) or a binding protein that is distinct and recognizes either the N-terminal domain or other regions of PTHrP (R). In pathway B, PTHrP, after entering the ER lumen, "dislocates" back to the cytosol via the Sec61p translocon, a key component of the mammalian cotranslational protein translocation system, which functions as a two-way channel shuttling proteins both into the ER and back to the cytosol. Ubiquination of preproPTHrP may serve as the signal for retrograde transport of the peptide. In pathway C, innitiation of translation in PTHrP mRNA downstream from the initiator methionine generates a protein with a shorter signal peptide. Such a protein would fail to be targeted for secretion and remain in the cytosol for subsequent nuclear import. Experimental evidence supporting each of these pathways is illustrated in Fig. 4.

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CHAPTER 6, FIGURE 5 Regulation of PTHrP nuclear import. (A) Phosphorylation site for CDK2-CDC2 in PTHrP. The NLS (NOS) in PTHrP spans amino acids 88-106 of the mature protein (yellow-colored box) and is preceded by a consensus sequence (blue-colored box) for phosphorylation by CDK2-CDC2. The asterisk denotes the threonine (T) residue that undergoes phosphorylation. Below, the analogous region in the SV40 large T antigen is depicted for comparison. (B)Phosphorylation of PTHrP at T 85 by cyclin-dependent kinases negatively regulates its nuclear translocation. The G-specific cyclin-CDKs do not phosphorylate PTHrP, which then localizes to the nucleolus. Phosphorylation at T 85 by CDK2-CDC2 in the other phases of the cell cycle leads to the exclusion of the protein from the nucleus. Although phosphorylation of PTHrP is shown here to inhibit its interaction with importin 13 (113),this has not been vigorously demonstrated.

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CHAPTER 6, FIGURE 6 Abnormal endochondral bone development caused by deficiency of type 1 PTH receptor and PTHrP. (A) Contrasting effects of type 1 PTH receptor and PTHrP targeted disruption. Unlike in wild-type phalanges (a), blood invasion and bone replacement are delayed in the receptor-null bones (b), as illustrated by the persistence of proliferating chondrocytes. Conversely, in PTHrP-null phalanges (c), these processes are advanced and chondrocytes hypertrophy prematurely, leading to precocious ossification. (B) Partial rescue of the delay in vascular invasion and endochondral ossification in the type 1 PTH receptor-null mice by further ablation of the PTHrP gene. The impairment in blood invasion and bone formation is partially rescued in receptor/ligand double-negative mutant bones (b), as demonstrated by the more timely appearance of the ossification zone, compared to the receptor-negative single mutant (a). This argues in favor of PTHrP actions that are independent of the type 1 PTH receptor. [Reproduced from (48), with permission.]

CHAPTER 16, FIGURE 3 Overexpression of PTHrP end the PTHrP receptor disrupts heart development. (A) Whole mounts at E9.5 of double-trensgenic (left) end wild-type (right) embryos. The double trensgenic exhibits 8 greatly enlarged heart with pericardia1 effusion end vascular pooling (arrows). (B) Histologic sections of double-transgenic (left) end wild-type (right) embryos at E9.5. The trebeculee within the ventriculer cavity (v) of the wild-type embryo ere prominent (large arrows), whereas in the double trensgenic, trebeculee ere severely reduced or absent (asterisks). Prominent gaps ere also evident between the cerdiomyocytes in the double-trensgenic hearts (smell arrowheads); 8, atria; bar = 1001~m. (C) Left panel shows the localization of SMP8 lacZtrensgene in 9.5-day embryo. Staining is apparent in heart, hindgut, end somites. Right panel is an unstained control (from Qien J., et al., Endocrinology 1999; 140:1826-1833, with permisison)(49).

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CHAPTER 22, FIGURE 1 Visible and palpable parathyroid tumor. At surgery a 9.2-g parathyroid adenoma was found and removed successfully.

CHAPTER 22, FIGURE 2 Swollen and deformed knee due to osteitis fibrosa cystica. The X-rays of this knee are shown in Fig. 3.

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CHAPTER 26, FIGURE 1 (A) Low-power photomicrograph illustrating the extended osteoid perimeter (stained red) on mineralized trabecular surfaces (stained green). Original magnification, x6. (B) Extended eroded perimeter (ES) appearing as irregular scalloped surface covered by osteoclasts (OC). Original magnification, x25. (C) Extended osteoid perimeter (OS) seen at higher magnification. Peritrabecular fibrosis (arrows). Original magnification, x25. Goldner's trichrome. (D) Extended double tetracycline labels (DL) seen by fluorescence microscopy covering trabecular surfaces. Original magnification, x25. Unstained section.

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CHAPTER 44, FIGURE 5 Schematic representation of the role of PTHrP and the PTH/PTHrP receptor during mammary gland and tooth development. Upper panel: Branching morphogenesis depends on the interaction between mammary epithelium and the mesenchyme that condenses underneath (dense mesenchyme). PTHrP is produced by the mammary epithelium, and acts through the PTH/PTHrP receptor, which is expressed in the dense mesenchyme (72). Lower panel: During tooth development, PTHrP is expressed in the bone surrounding the tooth and in the dental mesenchyme. Stimulation of bone resorption following PTH/PTHrP receptor activation by PTHrP is requriedd for normal tooth eruption (71).

CHAPTER 44, FIGURE 9 Section of the upper tibia end from a fetus affected by Blomstrand lethal chondrodysplasia (BLC) (A) and an age-matched control (B). Note the severely reduced size of the growth plate, the irregular boundary between the growth plate and the primary spongiosa, and the increased cortical bone thickness (from Ref. 163, with permission, and Anne-Lise Delzoide, personnal collection).

CHAPTER 50, FIGURE 1 A young boy with APS1. This boy with APS1 has severe mucocutaneous candidiasis. There is striking hyperkeratosis from infection of the skin of the hands and face in addition to involvement of the oropharynx and fingernails.

C a'TEk 29 Medical Management of Primary Hyperparathyroidism J O H N L. STOCK Department of Medicine, University of Massachusetts Medical School, Worcest~ Massachusetts 01605; and Eli Lilly and Company, Indianapolis, Indiana 46285

ROBERT MARCUS Department of Medicine, Stanford University School of Medicine, and Aging Study Unit, VA Medical Cent~ Palo Alto, California 94304

INTRODUCTION

of any etiology (12-14). Patients with PHPT may be volume depleted due to decreased oral intake and increased renal losses of free water. Immediate rehydration and treatment of nausea and vomiting followed by saline diuresis usually result in a prompt decrease in the serum calcium concentration. The acute treatment of hospitalized patients with intravenous saline and furosemide has been well studied (12), but the value of salt and water loading and chronic oral furosemide in the outpatient setting is not well documented. Potential complications of this therapy include congestive heart failure if salt loading is too vigorous, prerenal azotemia with worsening of hypercalcemia if excessive diuretics are used, and other electrolyte abnormalities. Thiazides and related diuretics, including metolazone and indapamide, actually decrease calcium excretion at distal tubular sites and should be discontinued in patients with PHPT. Lithium carbonate may also decrease urinary calcium excretion (15), and, by direct effects of the drug on the parathyroid glands, magnify hypercalcemia. Short-term administration of lithium carbonate decreases the sensitivity of parathyroid cells to inhibition by calcium; chronic administration of this drug may predispose to development of parathyroid adenomas (15). At the least, lithium toxicity, if present, should be corrected in any patient with PHPT, but the discontinuation of this psychotropic drug is often problematic and depends on the underlying psychiatric diagnosis and other available options for therapy. Immobilization is known to result in hypercalciuria and hypercalcemia as a consequence of increased bone resorption and decreased bone formation (16), and

Surgery is the treatment of choice for patients with symptomatic or complicated primary hyperparathyroidism (PHPT) (1-3). Although the role of medical management for asymptomatic PHPT is still controversial (3,4), there are subsets of symptomatic patients who may benefit from medical rather than surgical intervention. These include patients who refuse surgery, do not have access to an experienced parathyroid surgeon, are too ill for surgery, have had unsuccessful previous neck explorations (5,6), or have inoperable parathyroid carcinoma (7-9). Occasionally, a trial of medical therapy to normalize the serum calcium concentration can assist the patient and physician in deciding whether symptoms are related to hypercalcemia (10). Patients may require stabilization of severe hypercalcemia prior to surgery (5). In its most extreme form, this has been called parathyroid crisis or acute PHPT (see Chapter 34) (11). This chapter describes the nonsurgical modalities available for the treatment of PHPT. After an introduction to the general principles of medical care for patients with PHPT, specific agents that decrease parathyroid h o r m o n e (PTH) action a n d / o r decrease PTH secretion are discussed.

GENERAL PRINCIPLES The general principles of medical treatment of symptomatic hypercalcemia in PHPT are the same as those for the treatment of symptomatic hypercalcemia The Parathyroids, Second Edition

459

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

460

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CHAPTER29

patients with PHPT should be instructed to avoid sustained bed rest and to increase their general levels of activity. Although the hypercalcemia of PHPT is predominantly related to increased bone resorption, a c o m p o n e n t of it may respond to changes in diet. Some patients with PHPT show hyperabsorption in a calcium tolerance test, hypercalciuria, and elevated serum 1,25-dihydroxyvitamin D [1,25 (OH) 2D] concentrations (17). However, in a study of 18 unselected patients with PHPT, a high-calcium diet (1000 mg) suppressed PTH and 1,25(OH)2D concentrations in some subjects (18). Thus, the benefits of a low-calcium diet might include a lower urinary calcium excretion in certain patients, but should be balanced by a concern about aggravating the hypersecretion of PTH, with its possible skeletal consequences. The benefit of a higher calcium diet might be to suppress parathyroid function and thus theoretically delay progression of disease in a subset of patients, but could be offset by an increase in urinary calcium excretion in some of these subjects. Because no long-term studies currently allow one to tailor dietary r e c o m m e n dations, we suggest a m o d e r a t e dietary calcium intake for most patients with PHPT. SPECIFIC PHARMACOLOGIC

serum calcium or creatinine values. The mechanisms of the effects of oral phosphate therapy were investigated in a series of 10 patients with PHPT and elevated serum levels of 1,25 (OH) 2D who received 1500 mg of elemental phosphorus daily for 1 year (22). Phosphate treatm e n t led to decreases in serum 1,25 (OH) 2D levels, calciuric responses to oral calcium loading, and urinary calcium excretion, suggesting a decrease in calcium hyperabsorption in these subjects (Fig. 1).

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Albright et al. (19) described the use of oral phosphate for the t r e a t m e n t of PHPT in 1932. In elegant metabolic studies of three patients, they attributed the decrease in serum calcium concentrations and urinary calcium excretion with phosphate therapy to the increase in the Ca × P product in the serum. They also predicted the theoretical dangers of such therapy: "parathyroid poisoning," or the deposition of calcium deposits in the kidney and other organs with resultant uremia, as well as the risk of producing phosphate kidney stones. In 1966 Goldsmith and Ingbar (20) described the short-term use of oral phosphate in four patients with PHPT, with a resultant decline in the serum calcium concentration in all and actual normalization of the serum calcium concentration in one subject. Purnell gave 14 subjects with persistent or recurrent PHPT 1.0-2.5 g of elemental phosphorus daily (orally) for up to 51 months (21). In seven patients with serum calcium concentrations >11.0 m g / d l , calcemia normalized in one, decreased in four, and was u n c h a n g e d in two after 1 m o n t h of therapy. Although these effects were generally sustained with longer term treatment, an increase in serum creatinine concentrations was noted in three patients. In those patients affected by nephrolithiasis, there appeared to be a palliative effect on disease progression. In patients with milder disease and a serum calcium concentration <11.0 m g / d l , phosphate t r e a t m e n t had no effect on

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FIG. 1 Effect of oral phosphate (1500 mg elemental phosphorus daily) on the plasma 1,25(OH)2D concentration, calciuric response to an oral calcium tolerance test, and 24-hour calcium excretion (mg/kg/day) in 10 patients with PHPT. Subjects consumed a 1000-mg calcium diet. The hatched bars adjoining the ordinates represent normal ranges, and the circles with slashes are the mean values. From Broadus AE, et aL A detailed evaluation of oral phosphate therapy in selected patients with primary hyperparathyroidism. J Clin Endocrinol Metab 1983;56:953-961, by permission of Blackwell Science Ltd.

MEDICAL MANAGEMENT OF HYPERPARATHYROIDISM

No untoward clinical events were noted, but circulating immunoreactive PTH and n e p h r o g e n o u s cyclic adenosine m o n o p h o s p h a t e (cAMP) excretion increased. No definitive skeletal effects were d o c u m e n t e d other than a trend toward a reduction in bone turnover in those patients with increased bone resorption before therapy. These results suggest that the effect of phosphate therapy to lower serum calcium is more complex than just a lowering of the Ca × P product and includes also a decrease in calcium absorption mediated by the decrease in 1,25(OH)zD and probable complex effects on bone metabolism. The conclusion from these studies is that oral phosphate therapy may have a role in the treatment of a few, select patients with PHPT, but its long-term consequences on renal function and bone metabolism are still not well established. An additional concern is the possibility of ectopic calcification in soft tissues such as lung, gastric, mucosa, blood vessels, and myocardium (23). Estrogens, Selective Estrogen Modulators, and Progestins

Receptor

Other pharmacologic therapies that lower the serum a n d / o r urinary calcium excretion in patients with PHPT may be divided into two categories: those that act primarily by inhibiting the effects of PTH on bone resorption or those that decrease PTH secretion. Drugs or hormones that inhibit the effects of PTH on bone resorption have been effective in treating PHPT. A salutary effect on calcium metabolism may be noted in postmenopausal women with PHPT treated with estrogen replacement therapy. In 1972 Gallagher and Nordin (24) gave 10 such women ethinyl estradiol (50 lxg/day, 3 weeks out of 4) for up to 1 year and noted gradual decreases in fasting plasma calcium concentra-

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tion and in urinary calcium and hydroxyproline excretion, suggesting an inhibitory effect of estrogen on PTH-mediated bone resorption (Fig. 2). A follow-up study of 8 women confirmed these findings and also d o c u m e n t e d an improved calcium balance and a decrease in bone mineralization and resorption rates in most subjects (25). I n 1984 Marcus et al. (26) treated 14 postmenopausal women with conjugated estrogen (0.625-2.5 m g / d a y ) for up to 1 year. Four women also received cyclic medroxyprogesterone acetate for uterine protection. Ten subjects responded with a decrease in serum calcium concentrations. There were no changes noted in serum PTH or 1,25(OH)2D concentrations, or in urinary cAMP excretion. Decrements in the serum alkaline phosphatase activity and urinary hydroxyproline and calcium excretion suggested that estrogen inhibited the effects of PTH on bone turnover. Iliac crest bone biopsy specimens in subjects responding to estrogen showed maintenance of bone volume but no significant change in hyperosteoidosis after 1 year of therapy. A few subjects demonstrated typical minor side effects of estrogen, including breast tenderness, vaginal bleeding, and mild increases in blood pressure. Tenyear follow-up data on these subjects have also been reported (27). Of the 10 initial responders, 7 continued to have normocalcemia and urinary calcium excretion, although 3 elected to have parathyroidectomy because of their desire to stop regular surveillance. One subject stopped estrogen because of concerns about malignancy, and two were lost to follow-up. Selby and Peacock (28) reported a series of 17 postmenopausal women with PHPT t r e a t e d with ethinyl estradiol (30 txg/day) for 3 weeks. Despite a decrease in serum calcium concentrations and in fasting urinary calcium and hydroxyproline excretion, there were no

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changes in serum concentrations of PTH, calcitonin, or calculated free 1,25 (OH) 2° o r in urinary cAMP excretion. Except for a small increase in the calculated free 1,25 (OH)2 D concentration, these metabolic changes were maintained for a 3-month follow-up period. These results confirmed the efficacy of estrogen in the treatment of the biochemical abnormalities of PHPT and suggested that their action was mediated by resetting the threshold for PTH secretion, although estrogen receptors have not been documented in normal or abnormal parathyroid tissue (29,30). The synthetic stilbestrol derivative cyclofenil was given to six women and two men with PHPT for 5-13 weeks and reductions in serum calcium and urinary calcium and hydroxyproline were noted (31). Methallenestril was given to six postmenopausal women with PHPT for 5-24 weeks with similar results (32). The selective estrogen receptor modulator (SERM) tamoxifen (10 mg three times daily) was given to a postmenopausal patient with PHPT and breast cancer, and a decrease in the activity of alkaline phosphatase and serum ionized calcium and an upward trend in PTH concentration were noted (33). In a preliminary report of 11 postmenopausal women with mild PHPT treated with the SERM raloxifene (60 mg daily), the mean serum calcium concentration declined by 0.7 m g / d l over an average follow-up period of 7 months (34). Further investigation of SERMs in the treatment of postmenopausal women with PHPT is warranted. Some progestins may also have beneficial effects on bone and mineral metabolism in patients with PHPT. Selby and Peacock (28) treated 11 postmenopausal women with the androgenic progestin norethindrone (5 mg/day) and found the same beneficial effects as noted above in their group treated with ethinyl estradiol. Horowitz et al. (35) treated 20 postmenopausal women with PHP with the same regimen of norethindrone for 3 months, with no apparent side effects, and confirmed the decrease in serum calcium and urinary calcium excretion. There was no change in calcium absorption, and forearm mineral density increased. A 2year follow-up was reported for 15 of these women. Fatcorrected forearm bone mineral content measured by single-photon absorptiometry (SPA) increased 1.9% per year, with most of the gain occurring in the first 6 months (36). The beneficial effects of progestins may be limited to those agents with androgenic properties. Marcus (27) reported that serum calcium and phosphorus concentrations, alkaline phosphatase activity, and urinary calcium and hydroxyproline excretion were unchanged in six women and two men treated with the nonandrogenic C-21 progestin medroxyprogesterone acetate (10 m g / d a y for 2 months followed by 20 m g / d a y for 2 months).

Bone mineral density (BMD) has also been a primary end point in studies of hormonal therapy in patients with PHPT. In a retrospective study, Block et al. (37) found retrospectively that a lumbar spine BMD <0.9 g / c m 2 measured by dual-photon absorptiometry (DPA), fracture, or height loss were present in patients with PHPT who had received chronic estrogen therapy prior to parathyroidectomy. However, others have documented beneficial effects of hormone replacement therapy (HRT) on BMD in postmenopausal women with PHPT. In a cross-sectional study of 59 postmenopausal women with mild asymptomatic primary hyperparathyroidism and 129 healthy controls (38) BMD (measured by SPA and DPA) was lower in peripheral and central sites in patients with PHPT compared with normal controls. Those with PHPT who received estrogen replacement therapy or HRT for a mean of 15 years had BMD values that were greater than those with PHPT not receiving hormones, but lower than hormone-treated controls. Grey et al. measured BMD by dual-energy X-ray absorptiometry (DXA) (39) in a randomized, double-blind, placebo-controlled trial of 33 postmenopausal women with mild PHP who completed 2 years of daily treatment with conjugated equine estrogen (0.625 mg) and medroxyprogesterone (5 mg) or placebo. Total body and proximal forearm BMD decreased compared to base line in the placebo group whereas total body, lumbar spine, and femoral neck BMD increased compared to base line in the treatment group (Fig. 3). In this study, although HRT decreased biochemical markers of bone turnover and urinary calcium excretion, there were no significant changes in serum ionized calcium activity or PTH concentrations. In a nonrandomized study of 33 postmenopausal women with mild to moderate PHPT and 33 controls followed for 2 years, Guo et al. found that BMD of the total body, lumbar spine, and femoral neck measured by DXA increased similarly in the groups receiving surgical correction or hormone therapy, compared to a loss of total body and femoral neck BMD in the control group (40). Diamond et al. found that 1 year of treatment with conjugated equine estrogens or parathyroidectomy followed by calcium or calcitriol therapy was equally effective in increasing BMD in older postmenopausal women with PHPT and osteoporosis (41). The risk-benefit equation for determining the utility of HRT is complex. Estrogen replacement therapy does not reduce PTH concentrations in patients with PHP (26,28), so that any theoretical concerns regarding the effects of chronically elevated levels of PTH would not be ameliorated by this treatment. Unopposed estrogen increases the risk of endometrial hyperplasia and carcinoma and HRT may cause vaginal bleeding. Estrogen treatment increases the risk of venous thromboem-

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bolism (42) and the effect of estrogen on the incidence of breast cancer is controversial (43). In addition to the reduction in calcemia and urinary calcium excretion and the increases in BMD, benefits of estrogen replacement therapy may include reduction of symptoms of estrogen deficiency, such as hot

/

463

flashes and vaginal atrophy and an improvement in the lipid profile. The effect of estrogen replacement on the risk of ischemic heart disease remains controversial and may be complicated by factors such as presence of preexisting ischemic heart disease or concomitant use of progestins (44-46). Given this complexity, the use of h o r m o n e therapy in patients with PHPT must be individualized, based on an assessment and discussion with the patient of risk and benefit.

Bisphosphonates Bisphosphonates are pyrophosphate analogs that bind to hydroxyapatite and inhibit osteoclastic bone resorption. These drugs have been used in the treatment of Paget's disease, in the prevention and treatment of osteoporosis, and in the m a n a g e m e n t of hypercalcemia of malignancy (47). Etidronate (EHDP, Didronel), the first widely available bisphosphonate, was given to six patients with PHPT by Kaplan et al. in 1977 (48). After 5 weeks of a relatively high oral dose (20 m g / k g / d a y ) , the serum calcium concentration decreased in only one subject, and this was not maintained after 6 months of treatment. The urinary calcium excretion decreased in three patients, and this was maintained i n two of them after 6 months of treatment. There was a significant decrease in urinary hydroxyproline excretion in all subjects, which was more sustained at 6 months. There were no significant changes in serum PTH levels, urinary cAMP excretion, fractional calcium absorption, or bone density of the distal radius measured by singlep h o t o n absorptiometry. Due to its lack of efficacy in correcting hypercalcemia and its potential for causing osteomalacia, there has been little further interest in using this agent to manage PHPT, other than a case report of a patient who responded to intermittent administration of a lower dose (5 m g / k g ) (49). Clodronate (ClzMDP), a more potent bisphosphonate with little effect on bone mineralization, is currently available in Europe for the treatment of hypercalcemia. Shane et al. (50) treated 14 patients with PHPT with clodronate (orally, 1.6 g/day) for 12 weeks in a double-blind, placebo-controlled, cross-over design. The mean serum calcium concentration d r o p p e d significantly from 11.5 to 10.8 m g / d l during treatment and remained below initial pretreatment levels 3 months after drug was discontinued (Fig. 4). There were also significant declines in urinary excretion of calcium and hydroxyproline, and no changes in serum PTH or urinary cAMP excretion were observed. Douglas et al. (51) treated nine patients with clodronate (orally, 1.0-3.2 g/day) for 2-32 weeks in an open study design. There were significant decreases in calcemia to the u p p e r normal range and in urinary calcium and

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FIG. 4 Effects of clodronate (1.6 g/day, orally) on the serum calcium concentration and urinary calcium excretion in 14 patients with PHPT. The values shown represent the means (+_ SEM) for the entire group of patients. Mean treatment values for serum calcium (P < 0.001) and urinary calcium excretion (P < 0.01) and mean posttreatment serum calcium levels were significantly different from base line. The horizontal line indicates the upper limits of normal for serum calcium concentration (10.7 mg/dl). From Shane E, et al. Effects of dichloromethylene disphosphonate on serum and urinary calcium in primary hyperparathyroidism. Ann Intern Med 1981 ;95:23-27.

hydroxyproline excretion into the normal range. The decrease in serum calcium concentration was not sustained in three subjects who were followed for more than 19 weeks. Similar results were found by H a m d y et al. (52), who treated 20 PHPT patients with clodronate (orally, 0.8-1.6 g/day) for 8 weeks. As in the previous study, the serum calcium concentrations t e n d e d to increase over time in the 12 patients who were treated for 12 weeks, despite a sustained decrease in the parameters of bone resorption. Adami et al. (53) also noted a temporary and inconsistent effect of clodronate orally or parenterally for up to 180 days. The drug a p p e a r e d most effective in those subjects with marked bone resorption, and prolonged therapy was associated with an increase in serum PTH concentrations. These studies consistently suggest a partial short-term effect of clodronate on serum calcium levels related to an inhibition of bone resorption. The incompleteness of this effect may relate to an increase in renal tubular calcium resorption due to the rise in circulating PTH (52,53). Thus, clodronate does not appear to be very useful in the long-term managem e n t of hypercalcemia of PHPT, although its partial short-term effects on serum calcium levels suggest a possible role for the intravenous use of this drug in the acute m a n a g e m e n t of life-threatening hypercalcemia before surgery. Similar short-term effects of lowering the serum calcium concentrations in PHPT have been noted with the bisphosphonate pamidronate (Aredia, previously known as APD). The serum calcium concentration declined into the normal range in six of eight patients

with PHPT treated by van Breukelen et al. (54) after 2 weeks of orally administered pamidronate. Schmidli et al. (10) treated 10 patients with mild PHPT with a single intravenous dose of pamidronate (30 mg) in a randomized single-blind, placebo-controlled, cross-over design. The serum calcium concentration decreased in 9 of the 10 subjects, and a decrease in urinary calcium excretion and an increase in circulating PTH were noted after 7 days. Although there were no changes in hypercalcemia symptoms, muscle strength, cognitive function, or blood pressure in these patients with mild PHPT, this approach might be useful in surgical candidates for whom it is not clear whether their symptoms are related to the hypercalcemia. Jansson et al. (55) treated nine elderly patients with PHPT with a single intravenous infusion of pamidronate (15-60 mg). Six of the patients had severe hypercalcemia, and three were in hypercalcemic crisis, due in one instance to parathyroid carcinoma. After 1 week, seven patients were normocalcemic or mildly hypocalcemic and two patients had serum calcium values at the u p p e r limits of normal (Fig. 5). Monthly infusions in two patients for up to 9 months maintained serum calcium concentrations at the u p p e r limit of normal. Ishimura et al. (56) treated five patients with PHPT with a single intravenous dose of p a m i d r o n a t e (30 mg) and noted a transient mild decrease in the serum calcium concentration 1-2 days later. Although there were decreases in biochemical markers of bone turnover, an increase in serum P T H and 1,25(OH)zD concentrations was thought possibly to be contributing to the limited efficacy of this treatment. There have been other reports

MEDICAL MANAGEMENTOF HYPERPARATHYROIDISM / Mean 3.6-

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of the successful acute treatment of parathyroid crisis using intravenous infusion of pamidronate (57). Although transient elevations in body temperature and decrease in lymphocyte counts are described after pamidronate treatment of patients with hypercalcemia of malignancy, minimal side effects were noted in subjects with PHPT. There are few studies investigating the chronic use of pamidronate in PHPT, although animal experiments suggest a protective effect on bone loss (58). However, chronic use of pamidronate will be limited because it is unlikely to become available as an oral agent. Other potent bisphosphonates have also been tested in patients with PHE Reasner et al. (59) treated 19 patients with mild or asymptomatic PHPT with oral risedronate (Actonel) (20-40 mg/day) for I week. Fasting, but not postprandial, serum calcium concentrations fell, and this limitation of efficacy was t h o u g h t possibly related to a rise in serum PTH concentration and an e n h a n c e d renal tubular resorption of calcium. LoCascio et al. (60) treated 12 women with five consecutive daily intravenous infusions of alendronate (2.5 m g / d a y ) . At the end of the infusion period, the serum calcium concentration had declined toward the normal range, but it increased after an additional 25 days of observation. As noted with other bisphosphonates, the serum PTH concentrations increased and biochemical markers of bone turnover decreased. Adami et al. (61) noted a similar transient mild decline in serum calcium concentrations in six patients with PHPT 1 week after treatment with a single intravenous infusion of alendronate (5 rag). Although orally administered alendronate (Fosamax) has been similarly ineffective in the long-term normalization of calcemia in several other preliminary studies (62-65), it showed potential effi-

465

cacy in increasing BMD. Parker et al. (62) administered alendronate (10 m g / d a y ) to 14 patients with PHPT and osteoporosis and found an increase in lumbar spine BMD compared with an untreated control group of 19 patients with normal BMD or osteopenia. Rossini et al. (63) treated 10 elderly women with osteoporosis and mild PHPT with alendronate (10 mg every other day) and d o c u m e n t e d an increase in lumbar spine and femoral neck BMD after 1 year compared to base line. Hassani (64) treated 7 patients with PHPT with alendronate (10 m g / d a y ) and after 6 months found an increase in lumbar spine BMD but no change in femoral neck or wrist BMD compared to base line. The ionized serum calcium activity actually increased in the patients receiving alendronate treatment in this study. Bertoldo et al. (65) compared 19 patients with PHPT receiving alendronate for 2 years (10 m g / d a y ) to 12 control patients treated with parathyroidectomy. Increments in BMD were measured at the lumbar spine, femoral neck, and proximal radius in both medically and surgically treated patients. Of the currently available bisphosphonates, intravenously administered pamidronate appears to be the most effective in the acute m a n a g e m e n t of the hypercalcemia associated with PHPT. The long-term benefits of other bisphosphonates in the medical m a n a g e m e n t of the bone loss associated with PHPT are promising but not yet proved. Their limitations in the long-term m a n a g e m e n t of the hypercalcemia associated with PHPT may relate to p o o r gastrointestinal drug absorption as well as a rise in PTH levels with secondary increased renal tubular resorption and gastrointestinal absorption of calcium. It remains to be proved whether oral or parenteral administration of more potent bisphosphonates now u n d e r development will be able to overcome those limitations.

Calcitonin The peptide h o r m o n e calcitonin inhibits osteoclastic bone resorption and increases renal calcium excretion. H u m a n calcitonin and the more potent fish-derived calcitonins have been successfully used to treat hypercalcemia, particularly in patients with malignancy, taking advantage of their rapidity of action and good safety profile (12). However, their general use has been limited by low potency and short-lived effects. Torring et al. (66) treated 13 patients with histologically confirmed PHPT with salmon calcitonin by either intranasal or intramuscular administration. Intramuscular calcitonin (100 IU) lowered the mean blood ionized calcium activity for up to 12 hours but three subjects did not respond. Intranasal administration of up to 400 IU calcitonin led to only minimal decreases in blood ionized calcium activity. Stone et al.

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CI-IAeTF.~29

(67) administered salmon calcitonin to 20 patients with biochemical evidence for PHPT, either by a single intramuscular injection (100 IU) or by daily intravenous infusion over 5 days (100 I U / d a y ) . The serum calcium concentrations were slightly but significantly lower after 5 days in both groups. The response to the intravenous infusions was greater than that to the single intramuscular injection, possibly because of the e n h a n c e d renal effects of intravenous calcitonin. The expected attenuation of the hypocalcemic response with time was noted after only 2 days of infusions. These studies suggest that the chronic use of calcitonin for the treatment of PHPT would be limited by its low potency, short-lived efficacy, and inconvenient routes of administration.

Pharmacologic Agents That Decrease PTH Secretion Pharmacologic agents that decrease PTH secretion should theoretically correct most of the manifestations of PHPT. This approach is more disease-specific than the maneuvers described earlier, which deal primarily with the consequences of PTH action on bone a n d / o r kidney. Based on animal models suggesting sympathetic nervous system control of PTH secretion, Kukreja et al. (68) showed that injections of the [3-adrenergic antagonist propranolol decreased circulating PTH, although did not significantly affect serum calcium concentrations. Brown et al. (69), working in Dr. Aurbach's laboratory, demonstrated the presence of [3-adrenergic receptors in parathyroid cells obtained from h u m a n parathyroid adenomas and hyperplastic glands. Most subsequent studies failed to show any consistent effects of propranolol on serum concentrations of PTH or calcium in patients with PHPT (70-72) or in normal individuals (72). The lack of importance of the sympathetic nervous system in parathyroid regulation in vivo (72) or pathologic changes in PHPT causing a loss of responsiveness to [3-adrenergic agents (70) have been offered as explanations for these consistently negative results. Documentation of functional H2 receptors for histamine in pathologic parathyroid tissue (73) led to attempts to treat PHPT with H2 receptor antagonist cimetidine. Although a preliminary study of 12 patients showed a normalization of the serum PTH and a variable decrease in the serum calcium concentration (74), subsequent efforts have shown consistently negative results (75-79). Similarly, although there is in vitro evidence for serotonergic modulation of parathyroid function, treatment of PHPT with the serotonin antagonist cyproheptadine has not been successful (80). The demonstration that 1,25(OH)zD directly inhibits PTH gene transcription (81) led to a clinical trial of the analog l e~-hydroxyvitamin Ds in 31 patients with mild

PHPT (82). Circulating concentrations of PTH were only transiently lowered when this agent was given in high doses and the serum calcium concentration increased. In a preliminary report, 1,25(OH)2D 3 (3-6 flog/week by intravenous injection for 8-10 weeks) was administered to nine postmenopausal women with PHPT (83). Circulating PTH decreased in all patients and the serum calcium concentrations decreased by 20-22% in the six patients who responded. Decrements in urinary excretion of calcium and deoxypyridinium cross-links were also noted. Vitamin D and 1,25(OH)zD ~ were effective in lowering serum concentrations of PTH in a patient with parathyroid carcinoma (84). These preliminary results suggest that 1,25(OH)2D 3 might be effective in decreasing parathyroid gland function and lowering the serum calcium levels in PHPT. Analogs of 1,25(OH)zD such as 22-oxacalcitriol (85) and 19-nor-l,25(OH)zD 2 (86), which suppress PTH secretion but do not have calcemic activity, offer another potential approach to the treatment of PHPT. In 1983 Glover et al. (87) demonstrated that the radioprotective organic thiophosphate agent WR-2721 inhibited PTH release from dispersed bovine parathyroid cells in vitro. This drug administered intravenously has been used successfully in the treatment of hypercalcemia or malignancy, including parathyroid carcinoma, where its use is accompanied by a decrease in PTH levels (87-89). In a preliminary report, several patients with PHPT received a single dose of WR-2721 (400 mg by intravenous injection), and the mean serum calcium concentration d r o p p e d significantly from 11.6 to 10.6 m g / d l after 24 hours, but in the absence of a significant change in circulating PTH (90). Thus, in addition to its possible effects on PTH secretion, WR-2721 may cause a PTH-independent inhibition of renal tubular calcium resorption (89). Attie et al. (91) also demonstrated direct inhibitory effects of WR-2721 on bone resorption. The multiple sites of action of this drug may explain its increased efficacy compared with agents that only inhibit PTH secretion. However, it is unlikely that WR-2721 will ever become an attractive medical approach to PHPT because of debilitating side effects, including nausea, vomiting, somnolence, and hypotension (87). Direct antagonism of the activity or binding of PTH would be a more specific approach to decreasing its target organ effects. The immunologic inactivation of PTH was tested by Bradwell and Harvey (92). In a case report of a 62-year-old female with metastatic parathyroid carcinoma resistant to standard therapy, PTH antibodies were induced by the intradermal administration of various h u m a n and bovine PTH peptides. The free PTH became undetectable and the serum calcium concentration declined toward normal range with clinical

MEDICAL MANAGEMENTOF HYPERPARATHYROIDISM / improvement that was maintained for 6 months. Side effects included the development of skin nodules and lymphadenopathy, possibly related to the use of Freund's adjuvant, and transient fever. Using a different approach, Goldman et al. (93) found that [Nle 8'1s, D-Trp 12, Tyr34] bPTH (7-34)NH 2 (BIM-44002) was highly active in vitro, binding competitively to the P T H / P T H r P receptor and inhibiting adenylate cyclase activity. Dresner-Pollak et al. (94) demonstrated that this antagonist infused into rats was effective in inhibiting the rise in serum calcium caused by an agonistic PTH peptide infused 1 hour before. Rosen et al. (95) then infused BIM-44002 into three patients with primary hyperparathyroidism at increasing doses for 36 hours, but found no effect on serum ionized calcium activity or PTH concentration. In the previous animal experiments, the antagonist had been effective when infused prior to the agonist, and it may be that the same antagonist was ineffective clinically because it could not be infused prior to the onset of primary hyperparathyroidism. The most promising approach to pharmacologically decreasing PTH secretion in patients with primary hyperparathyroidism is the use of calcimimetic agents. These agents mimic or potentiate the action of calcium at the level of the calcium-sensing receptor, which regulates the secretion of PTH (96). The concept for this approach arose from the work of Edward Brown in his studies of the regulation of PTH secretion during his training in Dr. Aurbach's laboratory. Brown then went on to identify and clone the calcium-sensing receptor on parathyroid cells. This seven-transmembrane domain G protein-coupled receptor enables parathyroid cells to detect elevations in extracellular calcium and to translate them into increases in intracellular calcium and decreases in PTH secretion (97,98). Not only is this receptor critical in regulation of parathyroid and other cells on which it has been identified, but mutations that inactivate the calcium-sensing receptor may result in familial hypocalciuric hypercalcemia (FHH) (see Chapter 38) and those that overactivate the receptor may cause familial hypoparathyroidism (see Chapter 38) (98). Several calcimimetics were initially described to increase cytosolic calcium concentrations in bovine parathyroid cells with a corresponding decrease in PTH secretion, which was also documented in cells from a h u m a n parathyroid adenoma (96). Administration of one of these, R-568, an organic molecule with a molecular weight of 303, acted sterospecifically in rats to cause a rapid, dose-dependent decrease in plasma PTH concentrations with a parallel decline in plasma calcium concentrations (99). Clinical studies by Heath et al. documented the ability of R-568 to safely decrease serum PTH and calcium

467

concentrations in normal postmenopausal women (100). Subsequently, Silverberg et al. studied 20 postmenopausal women with mild primary hyperparathyroidism, selected because they did not meet National Institutes of Health (NIH) guidelines for parathyroidectomy or had refused or had had unsuccessful parathyroidectomy (101). They were enrolled in a randomized, double-blind, placebo-controlled study of the oral administration of increasing doses of R-568 ranging from 4 to 160 mg, with monitoring of serum calcium and PTH concentrations for 36 hours. A dose-related decline in serum PTH concentration was found, reaching a nadir 2 hours after drug administration at the higher doses and normalizing by 8 hours. The 160-mg dose decreased the serum PTH concentration 51 +_ 5 % after 2 hours, and the serum calcium level significantly decreased 4% in these subjects (Fig. 6). As expected, the urinary calcium excretion subsequently increased two- to threefold. The drug was well-tolerated in this short-term trial, which provides proof of principle that calcimimetics can inhibit PTH secretion in patients with primary hyperparathyroidism. The other clinical experience with R-568 is described by Collins et al. in a case report of a 78-year-old man with long-standing metastatic parathyroid carcinoma and refractory hypercalcemia (102). After hospitalization for severe hypercalcemia and mental status changes and 17 days of unsuccessful conventional treatment, he received R-568 in doses up to 400 mg daily. During the subsequent 22 days of hospitalization, clinical improvement was paralleled by a dose-related decrease in serum PTH and calcium concentrations. The drug was continued on an outpatient basis for 600 days at doses up to 600 mg daily and the patient remained clinically improved with stable hypercalcemia despite gradually increasing serum PTH concentrations. The interest in calcimimetics continues with ongoing studies and interest in the development of new more potent and longer acting drugs. The class of drug holds the best promise for a successful medical management of primary hyperparathyroidism.

SPECIFIC SITUATIONS Parathyroid Crisis Although most patients with PHPT have chronic mild hypercalcemia, the concomitant dehydration, immobilization, other intercurrent illnesses, or drug therapy occasionally lead to severe, potentially lifethreatening hypercalcemia (11) (see Chapter 34). The approach to lowering the serum calcium concentration in a patient with parathyroid crisis is similar to that

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for life-threatening hypercalcemia of any etiology (see Chapter 45). In addition, parathyroid surgery should follow correction of the hypercalcemia as soon as the patient's condition permits. Mthough the usual nonspecific measures of rehydration and diuresis are helpful and should be part of any initial therapeutic approach to severe hypercalcemia, specific pharmacologic agents are also beneficial in decreasing the effects of PTH on bone a n d / o r kidney. Intravenous infusions of pamidronate have been used successfully to lower the serum calcium concentration in several patients with parathyroid crisis (55,57). As was previously discussed, parenteral administration of salmon calcitonin might safely achieve a rapid, albeit short-lived and incomplete, improvement of hypercalcemia (103), although more prolonged responses have been described (104). The intravenous administration of gallium nitrate inhibits PTH-mediated bone resorption. This drug has been used to treat

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severe hypercalcemia associated with parathyroid carcinoma (105). Experience with gallium nitrate, however, as well as its availability, is extremely limited. The potential of this agent to produce nephrotoxicity necessitates maintenance of hydration and avoidance of concomitant use of other nephrotoxic drugs (12). Plicamycin (mithramycin) administered intravenously also decreases PTH-mediated bone resorption by inhibiting osteoclast function. Plicamycin has been effective in the treatment of parathyroid crisis (106) and in the control of hypercalcemia associated with parathyroid carcinoma (107), but its prolonged use is limited by the potential for nephrotoxicity and thrombocytopenia (12). Phosphate administered intravenously has also been used to treat parathyroid crisis but is dangerous because of the risk of ectopic calcification in blood vessels, lungs, and kidney, which may lead to organ failure and fatal hypotension (12).

MEDICAL MANAGEMENTOF HYPERPARATHYROIDISM /

Pregnancy Clinically apparent PHPT is unusual during pregnancy (108), but it has been described in association with maternal pancreatitis and hypercalcemic crisis as well as with neonatal hypocalcemia and tetany (109,110). Parathyroidectomy during the second trimester has been suggested for women with progressive symptoms (110,111) and by others for all patients with PHPT (112,113). Medical therapy has been proposed for patients with mild hypercalcemia, although the specific risks to the fetus of untreated mild maternal hypercalcemia have not been well documented. The use of oral phosphate has been described in several pregnant women who were not surgical candidates (114). In one case report, intravenous administration of magnesium to treat preeclampsia in a patient with PHPT and acute pancreatitis also normalized the serum calcium concentration (109). Most of the usual pharmacologic agents used to treat hypercalcemia have not been studied during pregnancy. Anecdotal experience suggests that a nonsurgical, nonpharmacologic approach to mild PHPT in pregnancy is safe and well tolerated.

SUMMARY AND CONCLUSIONS There is currently no ideal long-term medical management for PHPT. The best studied treatment has been estrogen, which remains an excellent option for selected postmenopausal women. Bisphosphonates may be useful to provide skeletal protection and to treat osteoporosis associated with PHPT. Development of a potent, long-acting calcimimetic holds the best promise for an effective treatment in the future. There are currently several potential options for short-term medical treatment of PHPT in patients who need therapy before surgery, in parathyroid crisis, or with parathyroid carcinoma, including intravenous infusion of pamidronate or the parenteral administration of calcitonin. Even if an effective long-term therapy is developed for PHPT, issues of cost and compliance must be addressed given the relatively inexpensive and effective surgical treatment available. There have been no prospective randomized clinical trials assessing the clinical benefit, cost-benefit, or cost-effectiveness of any surgical or medical treatment for PHPT (3,115). Most of the cost estimates of this disease have not included a specific cost for a potential pharmacologic therapy. The expense of follow-up testing, combined with drug treatment, would be interesting to compare to the expense of surgery (115,116). Such a cost-benefit analysis would also have to take into consideration the potential risks of surgery, the availability of expert parathyroid surgeons, and the prospects of unsuccessful parathyroid

469

surgery in hospitals that are not fortunate e n o u g h to have those with requisite surgical expertise. The effects of managed care on referral patterns and cost estimates would further complicate this analysis (4). As a minimum, patients whose PHPT would ordinarily lead to surgery but who do not u n d e r g o parathyroidectomy should be followed similarly to patients with mild, asymptomatic PHPT (3). This would include semiannual follow-up until lack of disease progression has been established and then annual follow-up evaluations. M o n g with interval history and physical examination, serum calcium and creatinine concentrations, creatinine clearance, 24-hour urinary calcium excretion, and bone mineral density determinations are reco m m e n d e d . More frequent monitoring for potential side effects should be tailored to the individual and to the pharmacologic therapy, if any, being used. Compliance with long-term medical follow-up is also an issue. No long-term prospective data have satisfactorily addressed this point, yet, in most reported series of patients with asymptomatic PHPT, a small but significant percentage of subjects are lost to follow-up. Heath and Heath (117) found that 13 of 122 of their conservatively managed, mostly asymptomatic patients with PHPT seen over a 10-year period in Birmingham, England, were lost to follow-up. Of 142 patients with mild, asymptomatic PHPT in Rochester, Minnesota, who were followed prospectively for 10 years by Scholz and Purnell (118), 10 refused follow-up evaluations and 9 others could not be traced. Marcus reported that only 4 of the 10 estrogen-responsive patients originally reported (26) were controlled on therapy 5 years later (27). Three well-controlled patients elected to u n d e r g o surgery because they wanted to stop regular surveillance, one subject discontinued estrogen because of concerns about malignancy, and two patients were lost to follow-up. W h e n data on compliance become available, it will be important to consider whether an expected "dropout" rate is acceptable and who exactly is "dropping out." If it is likely that asymptomatic patients who show no manifestations of disease except mildly abnormal biochemical indices or low BMD are the ones most likely to be lost to follow-up, studies will be n e e d e d to ascertain whether this lack of compliance is detrimental. One could argue that this conservative, nonsurgical approach to patients with asymptomatic PHPT can tolerate a certain d r o p o u t rate because, after all, before the multichannel analyzer, these patients were all unknown or undiscovered and were not followed. Presumably, the great majority of these patients who never came to medical attention never suffered adverse consequences of PHPT. Definitive recommendations for long-term medical m a n a g e m e n t of PHPT must await well-controlled,

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prospective studies of specific medical therapies compared to surgery. The development of a potent, longacting calcimimetic (101), the clinical application of specific PTH antagonists (119) and vitamin D analogs (85), and development of other more potent pharmacologic agents that block the action of PTH at multiple sites hold the greatest promise for future approaches to the medical management of PHPT.

REFERENCES 1. Clark OH, Wilkes W, Siperstein AE, Duh QY. Diagnoses and management of asymptomatic hyperparathyroidism: Safety, efficacy, and deficiencies in our knowledge. J Bone Miner Res 1991;6(Suppl. 2):S135-$152. 2. Wells SA, Jr. Surgical therapy of patients with primary hyperparathyroidism: Long-term benefits. J Bone Miner Res 1991;6(Suppl. 2):S143-S149. 3. Consensus Development Conference Panel. Diagnosis and management of asymptomatic primary hyperparathyroidism: Consensus Development Conference Statement. Ann Intern Med 1991;114:593-597. 4. Silverberg SJ, Bilezikian JP, Bone HG, Talpos GB, Horwitz MJ, Stewart AF. Therapeutic controversies in primary hyperparathyroidism. J Clin Endocrinol Metab 1999;84:2275-2285. 5. Editorial. Medical management of primary hyperparathyroidism. Lancet 1984;2:727-728. 6. VeldhuisJD, NortonJA, Wells SA, Jr, Vinik AI, Perry RR. Surgical versus medical management of multiple endocrine neoplasia (MEN) type I. J Clin Endocrinol Metab 1997;82:357-364. 7. McCance DR, Kenney BO, Sloan JM, Russell CFJ, Hadden DR. Parathyroid carcinoma: A review. J R Soc Med 1987;80:505-509. 8. Shane E, Bilezikian JE Parathyroid carcinoma: A review. Endocr Rev 1982;3:218-226. 9. Silverberg SJ, Bilezikian JE Evaluation and management of primary hyperparathyroidism. J Clin Endocrinol Metab 1996;81:2036-2040. 10. Schmidli RS, Wilson I, Espiner EA, Richards AM, Donald RA. Aminopropylidine diphosphonate (APD) in mild primary hyperparathyroidism: Effects on clinical status. Clin Endocrinol 1990;32:293-300. 11. Fitzpatrick LA, Bilezekian JE Acute primary hyperparathyroidism. A m J M e d 1987;82:275-282. 12. Bilezekian JP. Management of acute hypercalcemia. N E n g l J Med 1992;326:1196-1203. 13. Zahrani AA, Levine MA. Primary hyperparathyroidism. Lancet 1997;349:1233-1238. 14. Shane E. Medical management of asymptomatic primary hyperparathyroidism. J Bone Miner Res 1991 ;6 (Suppl. 2) :S 131-S 134. 15. Mallette LE, Eichhorn E. Effects of lithium carbonate on human calcium metabolism. Arch Intern Med 1986;146:770-776. 16. Stewart AF, Adler M, Byers CM, Segre GV, Broadus AE. Calcium homeostasis in immobilization: An example of resorptive hypercalciuria. N EnglJ Med 1982;306:1136-1140. 17. Broadus AE, Horst RL, Lang R, Littledike E, Rasmussen H. The importance of circulating 1,25-dihydroxyvitamin D in the pathogenesis of hypercalciuria and renal stone formation in primary hyperparathyroidism. N EnglJ Med 1980;302:421-426. 18. Insogna KL, Mitnick ME, Stewart AF, Burtis WJ, Mallette LE, Broadus AE. Sensitivity of the parathyroid hormone-1,25-dihydroxyvitamin D axis to variations in calcium-intake in patients with primary hyperparathyroidism. N EnglJ Med 1985;313:1126-1130.

19. Albright E Baver W, Claflin D, Cockrill JR. Studies in parathyroid physiology. III. The effect of phosphate ingestion in clinical hyperparathyroidism. J Clin Invest 1932;11:411-435. 20. Goldsmith RS, Ingbar SH. Inorganic phosphate treatment of hypercalcemia of diverse etiologies. N EnglJ Med 1966;274:1-7. 21. Purnell DC, Scholz DA, Smith LM, et al. Treatment of primary hyperparathyroidism. A m J Med 1974;56:800-809. 22. Broadus AE, Magee JS, Mallette LE, et al. A detailed evaluation of oral phosphate therapy in selected patients with primary hyperparathyroidism. J Clin Endocrinol Metab 1983;56:953-961. 23. Vernava III AM, O'Neal LW, Palermo V. Lethal hyperparathyroid crisis: Hazards of phosphate administration. Surgery 1987; 102:941-948. 24. Gallagher JC, Nordin BEC. Treatment with oestrogens of primary hyperparathyroidism in postmenopausal women. Lancet 1972; 1:503-507. 25. Gallagher JC, Wilkinson R. The effect of ethinyloestradiol on calcium and phosphorus metabolism of post-menopausal women with primary hyperparathyroidism. Clin Sci Mol Med 1973;45:785-802. 26. Marcus R, Madvig P, Crim M, Pont A, KosekJ. Conjugated estrogens in the treatment of postmenopausal women with hyperparathyroidism. Ann Intern Med 1984;100:633-640. 27. Marcus R. Estrogens and progestins in the management of primary hyperparathyroidism. J Bone Miner Res 1991;6(Suppl.

1):S125-$129.

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58. Mitlak BH, Rodda CP, von Deck MD, Dobrolet NC, Neer RM, Nussbaum SR. Pamidronate reduces PTH-mediated bone loss in a gene transfer model of hyperparathyroidism in rats. J Bone Miner Res 1991;6:1317-1321. 59. Reasner CA, Stone MD, Hosking DJ, Ballah A, Mundy GR. Acute changes in calcium homeostasis during treatment of primary hyperparathyroidism with risedronate. J Clin Endocrinol Metab 1993;77:1067-1071. 60. LoCascio V, Braga V, Bertoldo F, et al. Effect of bisphosphonate therapy and parathyroidectomy on the urinary excretion of galactosylhydroxylysine in primary hyperparathyroidism. Clin Endocrinol 1994;41:47-51. 61. Adami S, Zamberlan N, Mian M, et al. Duration of the effects of intravenous alendronate in postmenopausal women and in patients with primary hyperparathyroidism and Paget's disease of bone. Bone Miner 1994;25: 75-82. 62. Parker CR, Blackwell PJ, Hosking DJ. Alendronate in the treatment of primary hyperparathyroidism. Bone 1998;23(Suppl. 5) :$478 (abstract). 63. Rossini M, Bianchini D, Gatti D, Viapiana O, Braga V, Adami S. Effect of oral alendronate in elderly patients with osteoporosis and mild primary hyperparathyroidism. Bone 1998;23(Suppl. 5):$285 (abstract). 64. Hassani S. Alendronate therapy for primary hyperparathyroidism. Program and Abstracts of the Endocrine Society's 80th Annual Meeting, June 24-27, 1998, New Orleans, Louisiana, 495 (abstract). 65. Bertoldo E Furlan E Gasperi E, Colapietro F, Lorenzi T, LoCascio V. Two years of treatment of primary hyperparathyroidism with oral alendronate. J Bone Miner Res 1999;14(Suppl. 1) :$143 (abstract). 66. Torring O, Bucht E, Sjostedt U, Sjoberg HE. Salmon calcitonin treatment by nasal spray in primary hyperparathyroidism. Bone 1991;12:311-316. 67. Stone MD, Marshall DH, Hosking DJ, Garcia-Himmelstine CG, White DA, Worth HG. Comparison of low dose intramuscular and intravenous salcatonin in the treatment of primary hyperparathyroidism. Bone 1992;13:265-271. 68. Kukreja SC, Hargis GK, Bowser EN, Henderson WJ, Fisherman EW, Williams GA. Role of adrenergic stimuli in parathyroid hormone secretion in man. J Clin Endocrinol Metab 1975;40:478-481. 69. Brown EM, Gardner DG, Windeck RA, Hurwitz S, Brennan ME Aurbach GD. [3-Adrenergically stimulated adenosine 3',5'-monophosphate accumulation in and parathyroid hormone release from dispersed human parathyroid cells. J Clin Endocrinol Metab 1979;48:618-626. 70. Caro JF, Castro JH, Glennon JA. Effect of long-term propranolol administration on parathyroid hormone and calcium concentration in primary hyperparathyroidism. Ann Intern Med 1979;91:740-741. 71. Kukreja SC, Williams GA, Vora NM, Hargis GK, Bowser EN, Henderson WJ. Parathyroid hormone secretion in primary hyperparathyroidism: Retained control by calcium with impaired control by [3-adrenergic system. Miner Electrolyte Metab 1980;3:98-103. 72. Ljunghall S, Rudberg C, Akerstrom G, Wide L. Effects of betaadrenergic blockade on serum parathyroid hormone in normal subjects and patients with primary hyperparathyroidism. Acta Med Scand 1982;211:27-30. 73. Brown EM. Histamine receptors on dispersed parathyroid cells from pathologic human parathyroid tissue. J Clin Endocrinol Metab 1980;51:1325-1329. 74. Sherwood JK, Ackroyd FW, Garcia M. Effect of cimetidine on circulating parathyroid hormone in primary hyperparathyroidism. Lancet 1980;1:616-619.

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75. Williams GA, Longley RS, Bowser EN, et al. Parathyroid hormone secretion in normal man and in primary hyperparathyroidism: Role of histamine H 2 receptors. J Clin Endocrinol Metab 1981;52:122-127. • 76. Robinson ME Hayles AB, Heath III H. Failure of cimetidine to affect calcium homeostasis in familial primary hyperparathyroidism (multiple endocrine neoplasia type I). J Clin Endocrinol Metab 1980;51:912-914. 77. Fisken RA, Wilkinson R, Heath DA. The effects of cimetidine on serum calcium and parathyroid hormone levels in primary hyperparathyroidism. B r J Clin Pharmaco11982;14: 701-705. 78. Wiske PS, Epstein S, Norton JA, Jr, Bell NH, Johnston CC, Jr. The effects of intravenous and oral cimetidine in primary hyperparathyroidism. Horm Metab Res 1983;15:245-248. 79. Kristoffersson A, Dahlgren S, Jarhult J, Wahlby L. Cimetidine does not correct circulating calcium and parathyroid hormone in primary hyperparathyroidism. J Endocrinol Invest 1983;6: 489-491. 80. Gedik O, Usman A, Telatar F, Adalar N, Koray Z. Failure of cyproheptadine to affect calcium homeostasis and PTH levels in primary hyperparathyroidism. Horm Metab Res 1983;15:616-618. 81. Silver J, Naveh-Many T, Mayer H, Schmelzer HJ, Popovtzer MM. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat. J Clin Invest 1986;78: 1296-1301. 82. Lind L, Wengle B, Sorenson OH, Wide L, Akerstr6m G, Ljunghall S. Treatment with active vitamin D (alpha calcidol) in patients with mild primary hyperparathyroidism. Acta Endocrinol 1989;120:250-256. 83. Wassif W, Nyan O, Moniz C, Parsons V. Intravenous 1,25 dihydroxy-cholecalciferol reduces serum calcium in primary hyperparathyroidism. J Bone Miner Res 1991;6(Suppl. 1):S176 (abstract). 84. Palmieri-Sevier A, Palmieri GMA, Baumgartner CJ, Britt L. Case report: Long-term remission of parathyroid cancer: Possible relation to vitamin D and calcitriol therapy. Am J Med Sci 1993;306:309-312. 85. Brown AJ, Ritter CR, Finch JL, et al. The normocalcemic analogue of vitamin D, 22-oxacalcitriol suppresses parathyroid hormone synthesis and secretion. J Clin Invest 1989;84:728-732. 86. Slatopolsky E, Finch J, Ritter C, et al. A new analog of calcitriol, 19-nor-l,25-(OH)zD2, suppresses PTH secretion in uremic rats in the absence of hypercalcemia. J Bone Miner Res 1995;10(Suppl. 1):S167 (abstract). 87. Glover D, Riley L, Carmichael K, et al. Hypocalcemia and inhibition of parathyroid hormone secretion after administration of WR-2721 (a radioprotective and chemoprotective agent). N EnglJMed 1983;309:1137-1141. 88. Glover DJ, Shaw L, Glick JH, et al. Treatment of hypercalcemia or parathyroid cancer with WR-2721. S-2-(3-aminopropylamino) ethyl-phosphorothioic acid. Ann Intern Med 1985;103:55-57. 89. Hirschel-Scholz S, Jung A, Fischer A, Trechsel U, Bonjour JE Suppression of parathyroid secretion after administration of WR-2721 in a patient with parathyroid carcinoma. Clin Endocrino11985;23:313-318.

90. Morita M, Higashi K, Tajiri J, Sato T. S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) in primary hyperparathyroidism. Ann Intern Med 1985;103:961 (letter). 91. Attie ME Fallon MD, Spar B, WolfJS, Slatopolsky E, Goldfarb S. Bone and parathyroid inhibitory effects of S-2(3-aminopropylamino)ethylphosphorothioic acid. J Clin Invest 1985;75: 1191-1197. 92. Bradwell AR, Harvey TC. Control of hypercalcemia of parathyroid carcinoma by immunisation. Lancet 1999;353:370-373.

93. Goldman ME, McKee RL, Caulfield MP, et al. A new highly potent parathyroid hormone antagonist: [D-Trp 12, Tyr34] bPTH(7-34) NH 2. Endocrinology 1988;123:2497-2599. 94. Dresner-Pollak R, Yang QM, Behar V, Nakamoto C, Chorev M, Rosenblatt M. Evaluation in vivo of a potent parathyroid hormone antagonist: [Nle s'18, D-Trp 12, Tyr34] bPTH (7-34) NH 2. J Bone Miner Res 1996; 11:1061-1065. 95. Rosen HN, Lim M, Garber J, et al. The effect of PTH antagonist BIM-44002 on serum calcium and PTH levels in hypercalcemic hyperparathyroid patients. Calcif Tissue Int 1997;61:455-459. 96. Steffey ME, Fox J, Van Wagenen BC, Delmar EG, Balandrin MJ, Nemeth EE Calcimimetics: Structurally and mechanistically novel compounds that inhibit hormone secretion from parathyroid cells. JBone Miner Res 1993;8(Suppl. 1):S175 (abstract). 97. Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 1993;366:575-580. 98. Brown EM, Pollak M, Seidman CE, et al. Calcium-ion-sensing cell-surface receptors. N E n g l J Med 1995;333:234-239. 99. Fox J, Hadfield S, Petty BA, Nemeth EE A first generation calcimimetic compound (NPS R-568) that acts on the parathyroid cell calcium receptor: A novel therapeutic approach for hyperparathyroidism. JBone Miner Res 1993;8 (Suppl. 1);S 181 (abstract). 100. Heath III H, Sanguinetti EL, Oglesby S, Marriott TB. Inhibition of human parathyroid hormone secretion in vivo by NPS-R-568, a calcimimetic drug that targets the parathyroid cell-surface calcium receptor. Bone 1995;16(Suppl.):85S (abstract). 101. Silverberg SJ, Bone III HG, Marriott TB, et al. Short-term inhibition of parathyroid hormone secretion by a calcium-receptor agonist in patients with primary hyperparathyroidism. N EnglJ Med 1997;337:1506-1510. 102. Collins MT, Skarulis MC, Bilezikian JP, Silverberg SJ, Spiegel AM, Marx SJ. Treatment of hypercalcemia secondary to parathyroid carcinoma with a novel calcimimetic agent. J Clin Endocrinol Metab 1998;83:1083-1088. 103. Sjoberg HE, Hjern B. Acute treatment with calcitonin in primary hyperparathyroidism and severe hypercalcemia of other origin. Acta Chir Scand 1975;141:90-95. 104. Minisola S, Romagnoli E, Scarnecchia L, et al. Parathyroid storm: Immediate recognition and pathophysiological considerations. Bone 1993;14:703-706. 105. Warrell RP, Jr, Issacs M, Alcock NW, Bockman RS. Gallium nitrate for treatment of refractory hypercalcemia from parathyroid carcinoma. Ann Intern Med 1987;107:683-686. 106. Perlia CP, Gubisch NJ, Wolter J, Edelberg D, Dederick MM, Taylor III SC. Mithramycin treatment of hypercalcemia. Cancer 1970;25:389-394. 107. Trigonis C, Cedermark B, Willems J, Hamberger B, Granberg PO, Parathyroid carcinoma--problems in diagnosis and treatment. Clin Onco11984;10:11-19. 108. Ficinski ML, Mestman JM. Primary hyperparathyroidism during pregnancy. Endocr Practice 1996;2:362-367. 109. Rajala B, Abbasi RA, Hutchinson HT, Taylor T. Acute pancreatitis and primary hyperparathyroidism in pregnancy: Treatment of hypercalcemia with magnesium sulfate. Obstet Gynecol 1987;70: 460-462. 110. Kelly TR. Primary hyperparathyroidism during pregnancy. Surgery 1991;110:1028-1034. 111. Croom III RD, Thomas CG, Jr. Primary hyperparathyroidism during pregnancy. Surger 1984;96:1109-1118. 112. Kristoffersson A, Dahlgren S, Lithner E JarhultJ. Primary hyperparathyroidism in pregnancy. Surgery 1985;97:326-330. 113. Carella MJ, Gossain VV. Hyperparathyroidism and pregnancy. J Gen Intern Med 1992;7:448-453.

MEDICAL MANAGEMENT OF HYPERPARATHYROIDISM 114. Montoro MN, Collea JV, Mestman JH. Management of hyperparathyroidism in pregnancy with oral phosphate therapy. Obstet Gynecol 1980;55:431-434. 115. Melton III LJ. Epidemiology of primary hyperparathyroidism. JBone Miner Res 1991;6(Suppl. 2) :$25-$29. 116. Heath III H, Hodgson SF, Kennedy MA. Primary hyperparathyroidism: Incidence, morbidity, and potential economic impact in a community. NEnglJ Med 1980;302:189-193.

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C a 'TF k 30 Preoperative Localization of Parathyroid Tissue in Primary Hyperparathyroidism

J O H N L. DOPPMAN Diagnostic Radiology Department, National Institutes of Health, Bethesda, Maryland 20892

INTRODUCTION

sestimibi/Tc pertechnetate (MIBI) subtraction scintigraphy, computerized tomography (CT), and magnetic resonance imaging (MRI)mare performed first. Ultrasound is best for eutopic lesions in (3) and near the thyroid gland, whereas CT and MRI are more effective in detecting ectopic parathyroids in the anterior mediastinum and tracheoesophageal groove. MIBI subtraction scintigraphy has emerged as the single most sensitive study for both perithyroidal and ectopic lesions, detecting more than 90% of adenomas and up to 60% of hyperplastic glands in most large series (4-12). Two groups of investigators reported the use of 2(fluorine-18)-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) to localize parathyroid adenomas. Neumann et al. (13) found the technique helpful but Sisson et al. (14) failed to image any adenomas. Further series have not been reported, and PET scanning will not be further considered until more studies are completed. Because of the n u m b e r of false-positive studies (15-18%) based on noninvasive techniques, we require inclusion of a patient in two positive noninvasive studies at the same site for definitive localization. In a previous report, one positive study included 70% of patients, but only 30% of patients were included in two such positive studies (15). More recent studies show approximately 60% of patients will be included in two positive noninvasive studies, the difference being the contribution of sestamibi scintigraphy in confirming ectopic locations detected by CT/MRI scanning. The sequence of performing the noninvasive studies is not

In patients with primary hyperparathyroidism and failed initial surgical explorations to locate parathyroid tissue, localization studies have made a significant contribution to the success of reoperations. In a review of a large series of patients from the Mayo Clinic reported in 1975, prior to the use of preoperative localization studies (1), the success of reoperative surgery was only 62% compared to a 95% success rate in patients undergoing initial surgery. Relying heavily on localization studies, the success rate in 107 consecutive patients at the National Institutes of Health (NIH) undergoing reoperative surgery was 97%, comparable to the results in patients undergoing initial surgery (2). There is unanimous agreement concerning the need for preoperative localization in patients undergoing reoperation. The first section of this chapter will describe an imaging strategy for such patients and the expected results. Controversy exists concerning the need to perform preoperative localization in patients undergoing their initial operation for hyperparathyroidism. In the second section of this chapter the advantages and disadvantages of localization in unoperated patients are considered.

LOCALIZATION STUDIES IN PATIENTS U N D E R G O I N G REOPERATIVE SURGERY The need for localization studies in patients undergoing reoperative parathyroid surgery is undisputed. The noninvasive studies--ultrasound, 99mTc-labeled The Parathyroids, Second Edition

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critical. Because sestamibi scanning and ultrasound are least invasive and expensive, we perform them first. The principle of 99mTc-labeled sestamibi/Tc pertechnetate subtraction scanning is based on the fact that Tc pertechnetate labels thyroid: the images of scanned thyroid are then subtracted from the sestamibi scan, which includes labeled thyroid and parathyroid glands (Fig. 1). Shortly after sestamibi was introduced (16) as a parathyroid scanning agent, Taillefer et al. (6) suggested the use of sestamibi alone with delayed-phase imaging as an alternate technique to the dual-isotope subtraction technique. Delayed-phase imaging is based on the fact that sestamibi elutes from thyroid tissue at a faster rate than it elutes from parathyroid tissue; parathyroid is thus distinguishable from thyroid in the 2- to 3-hour delayed image. A number of publications (11,17,18) have compared the dual-isotope subtraction technique with the single-agent double-phase study, and in almost all instances the dual-isotope subtraction technique yielded slightly but significantly improved detection. Several reasons account for the improved performance of sestamibi scanning over the previously recommended thallium/technetium scanning. Sestamibi images are acquired at 140 kV, compared to thallium at 60 kV, therefore permitting better penetration of overlying soft tissue structures, a factor explaining its better performance in the detection of mediastinal adenomas. In addition, technetium has a shorter half-life (6 hours) compared to thallium (72 hours). Therefore, it can be given in a larger dose, permitting singlephoton emission computed tomography (SPECT) (19). SPECT not only increases the sensitivity of planar scintigraphic imaging, but allows localization of parathyroid adenomas in three dimensions. This is particularly important when a positive planar scan indicates a mediastinal adenoma, because pathology within the anterior mediastinum (intrathymic) must be distinguished from adenomas in the aortopulmonary (AP) window (20). In our series of 10 patients with AP window adenomas, 6 had undergone sternotomy and thymectomy based on the planar sestamibi images. None of the patients was cured. SPECT imaging at the time of sestamibi scanning discloses the midmediastinal location of these tumors and should be performed whenever intrathoracic lesions are detected on the planar scans. Glands in the aortopulmonary window are preferably approached by a left thoracotomy, especially if the patient has previously undergone an exploration of the anterior mediastinum through a standard sternotomy incision. Figure 2 illustrates an example of such a midmediastinal location, and its false localization when only planar imaging is performed. Ultrasound is particularly sensitive to the presence of intrathyroidal parathyroid glands, a diagnosis difficult to confirm by the other noninvasive studies (3). When

an intrathyroidal parathyroid gland is suspected, aspiration for PTH levels under ultrasound control (21,22) is recommended in order to avoid arteriography and venous sampling (Fig. 3). None of the noninvasive studies will establish the diagnosis of hyperplasia with any reliability, each generally visualizing only the dominant hyperplastic gland. However, Jeanquillaume, et al. (23) have reported some value of sestamibi scanning in patients with secondary hyperparathyroidism. Computer tomography of the neck and mediastinum should always be performed during a dynamic injection (2-3 ml/second) of iodinated contrast material. Nonionic contrast material should be used because it is less likely to cause patient motion due to burning, nausea, and vomiting. Modern scanners generally allow scanning from the base of the skull to the level of the carina during maximal contrast enhancement. The common procedure of commencing the CT scan at the angle of the mandible will fail to detect some undescended glands (24-26). These tumors may occur as high as the wall of the nasopharynx. We have encountered adenomas within the vagus nerve just below the base of the skull (27). Such undescended glands fail to migrate from their site of origin in the third and fourth branchial pouches. Commonly they lie within the carotid sheath (undescended inferior gland) (Figs. 4 and 5) or in the wall of the oronasopharynx (Fig. 6). Undescended adenomas are very difficult to palpate from a classic thyroid incision: their preoperative detection is critical to successful surgery. Using magnetic resonance imaging, parathyroid adenomas have an intermediate signal intensity similar to that of muscle on Tl-weighted images and are bright on T2-weighted and short-tau inversion recovery (STIR) sequences (28-32). Although parathyroid adenomas enhance following gadolinium dithylenetriamine pentaacetic acid (Gd-DPTA) (31) (Magnavist, Berlex, Wayne, NJ), use of this expensive paramagnetic contrast material is generally not necessary when fatsuppressed pulse sequences are available. In our experience, STIR or other fat-suppressed sequences have been particularly helpful in detecting parathyroid adenomas in ectopic locations. Saturation pulses should be used to eliminate the bright flow-related artifacts from vascular structures. Intrathymic adenomas are readily imaged in older patients in whom thymus has been completely replaced by fat. The detection of mediastinal adenomas is more difficult in young patients with residual thymic tissue. In patients less than 30 years old, detection of mediastinal adenomas is not possible because thymic tissue and parathyroid glands have the same MR imaging characteristics (Fig. 7). CT similarly fails in this group of patients. Because ultrasound cannot image the mediastinum, sestamibi scanning may be the only positive

PR~OPERATIW L O C A L I Z A T I O N OF T I S S U E

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FIG. 1 A sestamibi/pertechnetate subtraction scintigraphic study consists of (A) a sestamibi scan labeling thyroid and parathyroid, (B) a pertechnetate scan labeling thyroid, and (C) the subtracted image, identifying a right inferior parathyroid adenoma (arrow). Note that sestamibi and pertechnetate label and therefore subtract the salivary glands, an important contribution when looking for undescended parathyroid adenomas. When iodine is used to label thyroid, salivary gland uptake is more variable.

study. U n d e r such circumstances, we confirm the aden o m a by arteriography because this group of patients comprises prime candidates for transcatheter ablation, which can be p e r f o r m e d at the same time. When ultrasound or CT shows a suspicious lesion not confirmed by a second noninvasive technique, direct aspiration to measure PTH levels will often elim-

inate the necessity of proceeding to arteriography and venous sampling (21,22). A 23-gauge needle is passed u n d e r ultrasound or CT control back and forth through the suspicious mass; on withdrawing the needle, 1 ml of albumin is aspirated through the needle and the PTH level in the specimen is measured. PTH levels are usually astronomically high and the diagnosis

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FIG. 2 This 64-year-old man with primary hyperparathyroidism underwent sternotomy and total thymectomy after a planar sestamibi scan demonstrated a mediastinal adenoma (arrows, A). The patient remaining hypercalcemic. Repeat sestamibi scanning showed persistence of the mediastinal adenoma, which on coronal (arrows, B) and sagittal (arrow, C) SPECT scans is demonstrated to lie in the aortopulmonary window or midmediastinum. CT scan demonstrates the adenoma (arrows, D) between the ascending and descending aorta just above the main pulmonary artery. At left thoracotomy, the adenoma was successfully resected from the aortopulmonary window.

is very specific. A single PTH measurement is also less expensive than cytology. Although an experienced cytologist can distinguish thyroid from parathyroid gland, the distinction between thyroid and parathyroid adenomas cannot always be made cytologically. In a review of our last 40 aspirations, the sensitivity was 75% with no false positives (22). A negative aspiration means nothing because 25% of surgically proved parathyroid

adenomas had a negative aspiration. We have aspirated lesions in the tracheoesophageal groove and the anterior mediastinum under CT control with no morbidity. In a single instance, a postaspiration intraadenomal hemorrhage led to remission of hyperparathyroidism (Fig. 8). However, the deliberate attempt to ablate parathyroid tissue by direct injection of ethanol into the adenoma (33,34) is not routinely recommended.

PREOPERATIVE LOCALIZATIONOF TISSUE /

479

FIG. 3 There is a suspicious mass just to the left of the trachea below the left thyroid lobe (black arrows, A) in a very obese patient with previous unsuccessful surgery. Neither ultrasound nor Th/Tc scanning identified the mass, probably because of the marked obesity. The barium markers (white arrows, A) on the anterior neck assist in directing the needle. Needle aspiration (B) was performed for PTH level. Measurement revealed very high levels of PTH in the aspirate and a left paratracheal adenoma was subsequently removed.

It is difficult to control the spread of alcohol into the surrounding tissues, which contain the recurrent laryngeal nerve and other vital structures. The direct percutaneous injection of ethanol into cervical adenomas has been associated with recurrent nerve injuries and is not justified in p a t i e n t s w i t h well-localized adenomas and no previous surgery. Injection of ethanol into intrathyroidal parathyroid adenomas can be safely p e r f o r m e d because of the protective effect of surrounding thyroid tissue (35). Direct ethanol injection may be an acceptable technique to palliate secondary hyperparathyroidism in severely ill patients with chronic renal failure (36), but as a primary treatment for cervical adenomas its effectiveness and safety have not been demonstrated. Parathyroid arteriography and selective venous sampling are the ultimate invasive studies for parathyroid localization in reoperative patients. In our experience (37) about 35-40% of patients will require arteriography and approximately 20% will require venous sampling (an unequivocally positive parathyroid arteriogram in a patient with known a d e n o m a does not require venous sampling). Parathyroid arteriography requires selective injection of the internal m a m m a r y arteries, the thyrocervical trunk, and the c o m m o n carotid arteries. The internal m a m m a r y arteries and thyrocervical trunk are injected first because there is a slight but definite risk when performing carotid artery injections in this middle-aged population. W h e n the thyrocervical and internal m a m m a r y arteriograms are negative, c o m m o n carotid injections are p e r f o r m e d to exclude an u n d e s c e n d e d parathyroid gland. Contrary

to a widely held opinion, there is no risk to the cervical spinal cord when injecting the thyrocervical trunk. Blood supply to the cervical spinal cord arises from the costocervical, not the thyrocervical, trunk, and the two should not be confused by an experienced angiographer. In over 2000 selective injections of the thyrocervical trunk, I have seen only a single instance of blood supply to the cervical spinal cord and have never had a cord injury. We have recently introduced a modification of parathyroid arteriography (38) that, in some instances, renders subsequent venous sampling unnecessary. Replacement of normal blood by selective arterial injection of a noncalcium containing fluid during arteriography provides a hypocalcemic stimulus to release of parathyroid h o r m o n e from any parathyroid a d e n o m a that is perfused by this injection. Our initial studies indicated that nonionic contrast m e d i u m used for selective parathyroid arteriography was slightly more effective than normal saline as a provocative stimulus for parathyroid h o r m o n e release. The hypertonicity, and the presence of a chelating agent, EDTA, in contrast media may account for the superiority of contrast media over normal saline for this application. Prior to arteriography, a catheter is placed via the femoral vein in the superior vena cava, because this vessel receives the venous drainage of all potential parathyroid pathology in the neck or mediasfinum. Parathyroid h o r m o n e is measured in samples of blood collected immediately before and at 20, 40, and 60 seconds following each selective arteriogram. A 1.4-fold increase in

A

~110 I

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118

149

FIG. 4 This 35-year-old man had undergone three previous neck operations and one mediastinal exploration without correcting his hypercalcemia. On ultrasound, CT, and MRI, no lesion was seen, although multiple anterior cervical "lymph nodes" were noted bilaterally. (A) Parathyroid venous sampling showed increasing levels (underlined) of PTH ascending the left internal jugular and left vertebral veins, indicating a left-sided undescended parathyroid gland. Review of the CT revealed an enhancing mass between the left internal carotid artery and the left jugular vein (black arrow, B). Note that this lesion enhances as compared to the lymph node anterior to the left internal jugular vein (white arrow, B). With MRI, both the parathyroid adenoma (white arrow, C) and the lymph node lateral to the internal jugular vein (black arrow, C) appear bright and cannot be distinguished, but the results of parathyroid venous sampling assured us that the lesion was high in the left neck. A repeat ultrasound examination demonstrated a hypoechoic mass within the carotid sheath between the carotid artery and jugular vein (white arrows, D). The patient was cured by a simple vertical incision just anterior to the left midsternocleidomastoid muscle, thus avoiding the heavily scarred original operative site.

P~OPERATIW LOCALIZATIONOF TISSUE /

481

FIG. 5 The undescended adenoma (arrows, A) immediately anterior to the carotid artery is at the level of the lateral pharyngeal pouch from which parathyroid glands arise. Lateral left carotid arteriography demonstrates the "undescended" position of the adenoma (arrows, B) at the level of the carotid bifurcation.

PTH following arteriography identifies the vessel supplying the a d e n o m a and thereby localizes the a d e n o m a to the right or left neck (positive thyrocervical and com-

m o n carotid injections) or the mediastinum (positive internal m a m m a r y artery injections). In about half of our cases, stimulated venous sampling following selec-

FIG. 6 Note the flattened enhancing undescended parathyroid adenoma in the wall of the right piriform sinus. Such glands lie in the submucosal or muscular layers of the nasopharynx and oropharynx and represent undescended superior glands.

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FIG. 7 This 19-year-old woman underwent unsuccessful neck exploration for hypercalcemia. Selective left internal mammary arteriogram demonstrates a densely enhancing 1-cm parathyroid adenoma in the anterior mediastinum (arrow, A) supplied by the thymic branch of the internal mammary artery. Delayed films identify venous drainage (arrowheads, B) into the left innominate vein, but venous sampling would not be necessary with such a pathognomonic arteriogram. (C) Note that this gland is not visible by CT because it is contained within a water-density thymic gland. The adenoma was successfully treated by staining with water-soluble contrast agent.

tive arteriography is positive when the arteriogram fails to demonstrate an adenoma. Overall, we see a positive response of PTH to intraarterial stimulation with hypocalcemic contrast agents in 60% of the cases. We are currently evaluating use of calcium chelating agents

as more potent hypocalcemic stimulants that potentially may improve the sensitivity of this test. At arteriography, parathyroid glands appear as areas of h o m o g e n e o u s staining (Figs. 5 and 7). Adenomas in the anterior mediastinum may have a descending blood

FIG. 8 Computerized tomography demonstrates needle aspiration of a typical ectopic superior adenoma in the right tracheoesophageal groove (A). The patient developed discomfort in the neck and hypocalcemia following the positive aspiration, probably due to hemorrhage within the adenoma. Two years later the adenoma has disappeared (B) and the patient remains normacalcemic.

PREOPERATIVELOCALIZATIONOF TISSUE / supply from the thyrocervical trunk or may be fed by the thymic branch of the internal mammary artery. Glands with a blood supply from the internal mammary artery are deeper and more difficult to extract by cervical thymectomy. Such glands are particularly suited for transcatheter ablation (39-41). If the catheter can be wedged into the artery feeding the adenoma, a prolonged injection of contrast agent results in dense staining of the gland. We use ionic contrast material and repeat the injection three or four times until a longlasting dense opacification is achieved (Fig. 9). Persistence of the stain on a CT examination 24 hours later has, in our experience, been associated with long-term remission of the hyperparathyroidism. Only minor discomfort is associated with staining and it avoids sternotomy. We have stained over 60 glands with only one morbidity, a severe vasovagal reaction. Unsuccessful staining does not compromise subsequent surgery, unlike direct alcohol injection, which tends to fix glands to surrounding critical structures and renders subsequent surgical excision difficult. Venous sampling for elevated levels of PTH (42) remains the final and often the only positive study in patients with persistent hyperparathyroidism and multiple, previous operations. A positive venous sampling often leads to detection of an adenoma on reviewing the noninvasive studies (Fig. 4). Venous sampling does not demonstrate the adenoma but directs the surgeon to the region (right neck, left neck, mediastinum) in which the adenoma lies. Successful venous sampling is compromised by the previous performance of a thyroidectomy, because access to parathyroid venous

483

drainage is d e p e n d e n t on the larger thyroid veins. When three normal glands are identified at the initial exploration, unilateral thyroidectomy is frequently performed in hope of removing an occult intrathyroidal parathyroid adenoma. The incidence of intrathyroidal parathyroid adenomas is less than 5% in our experience, and these adenomas can be excluded with 100% accuracy by the use of intraoperative ultrasound (3). There is little reason for performing a hemithyroidectomy at the time of an unsuccessful parathyroid exploration. Following thyroidectomy, venous drainage of cervical parathyroid glands is diverted into the vertebral veins, which should always be sampled in difficult cases. Parathyroid venous sampling requires experience for competent performance and interpretation: such difficult cases should be referred to institutions performing this study. At NIH, we obtain venous gradients in over 90% of patients sampled. Undescended parathyroid glands constitute approximately 10% of all reoperative cases, but are particularly common among referrals from experienced parathyroid surgeons (24-26). They can be visualized by ultrasound, CT, and MR, provided these studies are performed high enough in the neck. Sestamibi scanning can image undescended adenomas but they are often obscured by uptake of isotope in the submandibular and salivary glands, especially when 123I is used in dual-isotope scanning and only planar images are obtained. 99mTCpertechnetate goes to salivary as well as thyroid tissue (unlike l~lI) and on subtraction studies is more likely to disclose undescended parathyroid adenomas. SPECT will also separate the more anterior salivary glands from the

FIG. 9 Right internal mammary arteriogram demonstrates a parathyroid adenoma in the anterior mediastinum (arrow, A). Computerized tomography scanning was positive (arrow, B) but the patient elected to undergo staining rather than sternotomy. At 24 hours (C) note the persistent staining and swelling of the adenoma. Such findings at 24 hours are almost always associated with permanent ablation, as in this patient (follow-up was for 6 years).

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posterior undescended adenomas. Single-agent delayed scanning often fails because accelerated elution from salivary glands has not been demonstrated. Noninvasive studies must be carried above the angle of the mandible to visualize undescended glands. Distinguishing them from lymph nodes is particularly difficult. Both undescended parathyroid glands and cervical lymph nodes appear bright on T2-weighted and STIR sequences and are often indistinguishable by ultrasound. The presence of multiple nodules or a fatfilled hilum favors a diagnosis of lymph nodes, but direct aspiration for parathyroid hormone levels or venous sampling can confirm the diagnosis of an undescended parathyroid gland. In summary, the noninvasive techniques--dualisotope sestamibi subtraction scintigraphy with SPECT, ultrasound, CT, and MRI--should be performed initially for parathyroid localization in reoperative cases. For definitive localization, two positive studies at the same site are preferable because of the high incidence (15-18%) of false-positive studies, especially in ultrasound, CT, and MRI. If only a single noninvasive study is positive, the diagnosis can often be confirmed by direct aspiration of the suspicious mass for PTH under control of the appropriate imaging modality. When noninvasive studies are negative (30-40% of reoperative cases in our experience), parathyroid arteriography and venous sampling should be performed. Using this algorithm, we have achieved a success rate of 97% in a large series of consecutive reoperative cases (43). Blind thyroidectomies, and sternotomies following a negative cervical exploration, are no longer justified in these days of sophisticated localization techniques. A close collaboration between endocrinologist, radiologist, and surgeon is essential to assure a successful outcome.

LOCALIZATION STUDIES IN PATIENTS U N D E R G O I N G INITIAL SURGERY F O R HYPERPARATHYROIDISM Prior to the introduction of sestamibi as a scanning agent for parathyroid scintigraphy, there was a sharp

difference of opinion concerning the need for localization studies in patients prior to their initial operation. In preparation for the 1991 NIH Consensus Conference dealing with asymptomatic hyperparathyroidism (44), I reviewed 15 large series (>25 subjects) of patients undergoing noninvasive localizing studies (ultrasound, thallium technetium scanning, computed tomography, and magnetic resonance imaging) before successful surgery. Table 1 summarizes the true-positive and falsepositive rates of the four rnodalities as reported. The sensitivities varied between 60 and 70%, with a falsepositive rate of all studies of approximately 15%. Although some investigators reported a higher sensitivity with a specific modality, it often occurred at the expense of an increased false-positive rate. A few institutions reported superior sensitivity using a modality with which they had extensive experience, but the purpose of the study was to evaluate the results that could be expected in institutions with broad experience in parathyroid localization but without dedication to a single modality. The NIH experience was not included because very few patients are seen at this institution before initial surgery. The sensitivity of 60-70%, with a false-positive rate of 15%, for all noninvasive localizing studies contrasted with the widely reported surgical success rates of 90-95 % in patients undergoing initial operations (1). In addition, studies that looked at operating room times or surgical success rate did not establish a significant difference between patients without and patients with preoperative localization (45-49). For these reasons, the following statement summarized my conclusion: "There has never been a well-controlled study which showed a decrease in operating time or improvement in surgical success, attributable to preoperative localization in patients undergoing initial surgery." This controversy concerning preoperative localization on nonoperated patients has been put to rest by the introduction of a truly sensitive technique, MIBI scintigraphy with SPECT. Several studies have shown that the dual-isotope technique improves the sensitivity when compared with the single-isotope delayed-scanning technique (17) and that SPECT increases the sensitivity as well as provides the more precise anatomic localiza-

TABLE 1 Sensitivity and False-Positive Rate of Noninvasive Localizing Studies a Study

Sensitivity (%)

False-positive rate (%)

Ultrasound Thallium/technetium scintigraphy

65 55

12.5 13.5

74

18

Computerized tomography Magnetic resonance imaging

63

aAdapted from Ref. 44; JL Doppman, DL Miller. Localization of parathyroid tumors in patients with asymptomatic hyperparathyroidism and no previous surgery. J Bone Miner Res 1991 ;6:$153-$158.

PREOPERATrVE LOCALIZATION OF TISSUE

tion (19). Using the procedure as described above, detection rates of adenomas have been over 95% in the hands of many experienced investigators (7-12). The sensitivity for detecting all hyperplastic glands drops to 60-70%, but in many patients more than one gland is imaged, thereby establishing a diagnosis of hyperplasia. As in all localization studies, the sensitivity is dependent on the size of the adenoma, but we (18), as well as others, have demonstrated that sestamibi detects smaller lesions than thallium/technetium scanning did. Finally, we have a test that outperforms an experienced parathyroid surgeon, and although there remains a difference of opinion (50), most patients now undergo sestamibi scanning prior to their initial surgery. The widespread application of preoperative localization with sestamibi scintigraphy has been fueled by the interest in shortening hospitalization for patients undergoing parathyroidectomy. Although there are proponents for the conservative or nonoperative treatment of such patients (51), most authorities remain convinced that surgery should be performed in all patients with documented hyperparathyroidism (52), principally because of the long-term effects on bone density. The accuracy of sestamibi scanning has led to the routine performance of targeted surgical explorations based on sestamibi localization, often under local anesthesia on an outpatient basis (53-55). The surgeon removes the previously localized adenoma and initiates closure without an effort to identify the other three glands. A rapid PTH determination is performed 10 minutes after surgical excision of the adenoma and demonstrates a greater than 50% decline of PTH levels in patients who are cured. The application of fast PTH assay in the operating room (56-58) has enabled the surgeon to abandoned the classic routine of identifying the remaining three parathyroid glands, surgical detection of which often requires more time than removal of the adenoma. Although large series have not been reported, a 50% decrease in PTH levels at 10 minutes postexcision has proved to correlate well with surgical cure (56). In the 3-5% of patients with negative sestamibi studies, a classic four-gland exploration will be required.

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5. Wei JR Burke GJ, Mansburger AR. Prospective evaluation of the efficacy of technetium 99m sestamibi and iodine 123 radionuclide imaging of abnormal parathyroid glands. Surgery 1992;112:1111-1117. 6. Taillefer RM, Boucher Yvan, Potvin C, Lambert R. Detection and localization of parathyroid adenomas in patients with hyperparathyroidism using a single radionuclide imaging procedure with technetium-99m-sestamibi (double-phase study). J Nucl Med 1992;33:1801-1807. 7. Johnston LB, Carroll MJ, Britton KE, Lowe DG, Shand W, Besser GM, Grossman AB. The accuracy of parathyroid gland localization in primary hyperparathyroidism using sestamibi radionuclide imaging. J Clin Endocrinol Metab 1996;81:346-352. 8. Lee VS, Wilkinson RH, Jr, Leight GS, Jr, Coogan AC, Coleman RE. Hyperparathyroidism double-phase techentium 99m sestamibi imaging. Radiology 1995;197:627-633. 9. Hindie E, Melliere D, Simon D, Perlemuter L, Galle E Primary hyperparathyroidism: Is technetium-99m-sestamibi/iodine-123 subtraction scanning the best procedure to locate enlarged glands before surgery? J Clin Endocrinol Metab 1995;80: 302-307. 10. Hindie E, Melliere D, Jeanguillaume C, Perlemuter L, Chehade F, Galle E Parathyroid imaging using simultaneous doublewindow recording of technetium-99m-sestamibi and iodine-123. J Nucl Med 1998;39:1100-1105. 11. Neumann DR, Esselstyn CB, Jr, Go RT, Wong CO, Rice TW, Obuchowski NA. Comparison of double-phase 99m Tc-sestamibi with 123-I 99m Tc-sestamibi subtraction SPECT in hyperparathyroidism. AmJ Roentgenol 1997;169:1671-1674. 12. Caixas A, Berna L, Hernandez A, Tebar FJ, Madariaga P, Vegazo O, Bittini AL, Moreno B, Faure E, Abos D, Piera J, Rodriquez JA, Farrerons J, Puig-Domingo M. Efficacy of preoperative diagnostic imaging localization of technetium 99m-sestamibi scintigraphy in hyperparathyroidism. Surgery 1997;121:535-41. 13. Neumann DR, Esselstyn CB, Jr, MacIntyre wJ, Chen EQ, Go RT, Kohse LM, Licata AA. Primary hyperparathyroidism: Preoperative parathyroid imaging with regional body FDG PET. Radiology 1994;192:509-512. 14. Sisson JC, Thompson NW, Ackerman RJ, Wahl RL. Use of 2-[F-18]-fluoro-2deoxy-D-glucose PET to locate parathyroid adenomas in primary hyperparathyroidism (letter to the Editor). Radiology 1994;192:280-280. 15. Miller DL, Doppman JL, Krudy AG, et al. Localization of parathyroid adenomas in patients who have undergone surgery. Part I. Non-invasive imaging methods. Radiology 1987; 162:133-137. 16. Coakley J, Kettle AG, Wells CP, O'Doherty MJ, Collings REC. Technetium-99m-sestamibi--a new agent for parathyroid imaging. Nucl Med Commun 1989; 10:791-794. 17. Chen CC, Holder LE, Scovill WA, Tehan AM, Gann DS. Comparison of parathyroid imaging with technetium-99mpertechnetate/sestamibi subtraction, double-phase technetium99m-sestamibi and technetium-99m-sestamibi SPECT. J Nucl Med 1997;38:834-839. 18. Chen CC, Skarulis MC, Fraker DL, Alexander HR, Marx SJ, Spiegel AM. Technetium-99m-sestamibi imaging before reoperation for primary hyperparathyroidism. JNucl Med 1995;36:2186-2191. 19. Billotey C, Sarfati E, Aurengo A, Duet M, Mundler O, Toubert M-E, Rain J-D, Najean Y. Advantages of SPECT in technetium99m-sestamibi parathyroid scintigraphy. J Nucl Med 1996;37: 1773-1778. 20. Doppman JL, Skarulis MC, Chen CC, Chang R, Pass HI, Fraker DL, Alexander HR, Niederle B, Marx SJ, Norton JA, Wells SA, Spiegel AM. Parathyroid adenomas in the aortopulmonary window. Radiology 1996;201:456-462. 21. Doppman JL, Shawker TH, Krudy AG, et al. Aspiration of enlarged parathyroid glands for parathyroid hormone assay. Radiology 1983;148:31-35.

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22. MacFarlane DL, Fraker DL, Shawker TH, Doppman JL, Chang RA, Skarulis MC, Marx SJ, Spiegel AM, Alexander HR. Use of preoperative fine-needle aspiration in patients undergoing reoperation for primary hyperparathyroidism. Surgery 1994;116:959-965. 23. Jeanguillaume C, Urena PK, Hindie E, Prieur P, Petrover M, Menoyo-Cologne V, Janin A, Chiappini-Briffa D, Melliere D, Boulahdour H, Galle E Secondary hyperparathyroidism: Detection with 1-123 Tc-99m-sestamibi subtraction scintigraphy versus US. Radiology 1998;207:207-213. 24. Doppman JL, Shawker TH, Krudy AG, et al. Parathymic parathyroid, CT, US, and angiographic findings. Radiology 1985;157:419-423. 25. Fraker DL, Doppman JL, Shawker TH, Marx SJ, Spiegel AM, Norton JA. Undescended parathyroid adenoma: An important etiology for failed operations for primary hyperparathyroidism. WorldJ Surg 1990;14:342-348. 26. Billingsley KG, Fraker DL, Doppman JL, Norton JA, Shawker TH, Skarulis MC, Marx SJ, Spiegel AM, Alexander HR. Localization and operative management of undescended parathyroid adenomas in patients with persistent primary hyperparathyroidism. Surgery 1994;116:982-989. 27. Doppman JL, Shawker TH, Fraker DL, Alexander HR, Skarulis MC, Lack EE, Spiegel AM. Parathyroid adenoma within the vagus nerve (case report). AmJRoentgenol 1994;163:943-945. 28. Peck WW, Higgins CB, Fisher MR, et al. Hyperparathyroidism; comparison of MR imaging with radionuclide scanning. Radiology 1987;163:415-420. 29. Kneeland JB, Krubsack AJ, Lawson TL, et al. Enlarged parathyroid glands; high resolution local coil MR imaging. Radiology 1987;162:143-146. 30. Auffermann W, Gooding GAW, Okerlund MD, et al. Diagnosis of recurrent hyperparathyroidism; comparison with MR imaging with other imaging techniques. AmJ Roentgenol 1988; 150:1027-1033. 31. Seelos KC, DeMarco R, Clark OH, et al. Persistent and recurrent hyperparathyroidism; assessment with gadopentate dimeglumine-enhanced MR imaging. Radiology 1990;177:373. 32. Lee VS, Spritzer CE, Coleman RE, Wilkinson RH, Jr, Coogan AC, Leight GS, Jr. The Complementary roles of fast spin-echo MR imaging and double-phase 99mTc-sestamibi scintigraphy for localization of hyperfunctioning parathyroid glands. Am J Roentgeno11996;167:1555-1562. 33. Karstrup S, Transbol I, Holm HH, Glenthoj A, Hegedus L. Ultrasound-guided chemical parathyroidectomy in patients with primary hyperparathyroidism: A prospective study. Br J Radiol 1989;62:1037-1042. 34. Karstrup S, Holm HH, Granthoj A, et al. Non-surgical treatment of primary hyperparathyroidism with sonographically guided percutaneous injection of ethanol: Results in a selected series of patients. Am J Roentgenol 1990; 154:1087-1090. 35. Charboneau JW, Hay ID, van Heerden JA. Persistent primary hyperparathyroidism: Successful ultrasound-guided percutaneous ethanol ablation of an occult adenoma. Mayo Clin Proc 1988;63:913-917. 36. Solbiati L, Giangrande AL, DePra L, et al. Percutaneous ethanol injection of parathyroid tumors under US guidance: Treatment for secondary hyperparathyroidism. Radiology 1985; 155:607-610. 37. Miller DL, Doppman JL, Krudy AG, et al. Localization of parathyroid adenomas in patients who have undergone surgery. Part II. Invasive procedures. Radiology 1987;162:138-141. 38. Doppman JL, Skarulis MC, Chang R, Alexander HR, Bartlett D, Libutti SK, Spiegel A. Hypocalcemic stimulation and nonselective venous sampling for localizing parathyroid adenomas: Work in progress. Radiology 1998;208:145-151. 39. Doppman JL. The treatment of hyperparathyroidism by transcatheter techniques. Cardiovasc Interventional Radiol 1980;3:268-281.

40. Miller DL, Doppman JL, Chang R, et al. Angiographic ablation of parathyroid adenomas; Lessons from a 10-year experience. Radiology 1987;165:601-607. 41. Doherty GM, Doppman JL, Miller DL, et al. Results of the multidisciplinary strategy in managing mediastinum parathyroid adenoma as the cause of persistent primary hyperparathyroidism. Ann Surg 1992;215:101-1061 42. Sugg SL, Fraker DL, Alexander R, Doppman JL, Miller DL, Chang RC, Skarulis MC, Marx SJ, Spiegel AM, NortonJA. Prospective evaluation of selective venous sampling for parathyroid hormone concentration in patients undergoing reoperations for primary hyperparathyroidism. Surgery 1993:114 (6):1004-1010. 43. Jaskowiak N, Norton JA, Alexander HR, Doppman JL, Shawker T, Skarulis M, Marx S, Spiegel A, Fraker DL. A prospective trial evaluating a standard approach to reoperation for missed parathyroid adenoma. Ann Surg 1996;224:308-322. 44. Doppman JL, Miller DL. Localization of parathyroid tumors in patients with asymptomatic hyperparathyroidism and no previous surgery. JBone Miner Res 1991; (6) :S153-S158. 45. Wilson SD, Hoffmann RG, Cerletty JM, et al. Parathyroidectomy for primary hyperparathyroidism; the influence of preoperative localizing studies on cure rate and operating time. Presented at the 1991 Annual Meeting Society for Endocrine Surgeons, San Jose, California, March 1991. 46. Serpel JW, Campbell PR, Young AE. Preoperative localization of parathyroid tumors does not reduce operating time. B r J Surg 1991;78:589-590. 47. Thompson N. Localization studies of patients with primary hyperparathyroidism. Br MedJ 1988;75:97-98. 48. Bruining HA, Birkenhager JC, Ong GL, Lamberts SWJ. Causes of failure in operations for hyperparathyroidism. Surgery 1987;101:562-565. 49. Levin KE, Clark OH. The reasons for failure in parathyroid operations. Arch Surg989;124:911-915. 50. Shen W, Sabanci U, Morita ET, Siperstein AE, Duh Q-Y, Clark OH. Sestamibi scanning is inadequate for directing unilateral neck exploration for first-time parathyroidectomy. Arch Surg 1997;132:969-976. 51. Silverberg SJ, Shane E, Jacobs TP, Siris E, Bilezikian JP. A 10-year prospective study of primary hyperparathyroidism with or without parathyroid surgery. NEnglJ Med 1999;341:1249-1255. 52. Utiger RD. Treatment of primary hyperparathyroidism (editorial). N EnglJ Med 1999;341:1301-1302. 53. Hindie E, Melliere D, Perlemuter L, Jeanguillaume C, Galle E Primary hyperparathyroidism: Higher success rate of first surgery after preoperative Tc-99m sestamibi-I123 subtraction scanning. Radiology 1997;204:221-228. 54. Borley NR, Collins REC, O'Doherty M, Coakley A. Technetium99m sestamibi parathyroid localization is accurate enough for scandirected unilateral neck exploration. BrJ Surg 1996;83:989-991. 55. Sfakianakis GH, Irvin III GL, FossJ, Mallin W, Georgiou M, Deriso GT, Molinari AS, Ezuddin S, Ganz W, Serafini A, Jabir A, Chandarlapaty SKC. Efficient parathyroidectomy guided by SPECTMIBI and hormonal mea~surements. J Nucl Med 1996;37:798-804. 56. Irvin III GL, Sfakianakis G, Yeung L, et al. Ambulatory parathyroidectomy for primary hyperparathyroidism. Arch Surg 1996; 131:1074-1078. 57. Carty SE, Worsey J, Virgi MA, Brown MA, Watson CG. Concise parathyroidectomy: The impact of preoperative SPECT 99m-Tc sestamibi scanning and intraoperative quick parathormone assay. Surgery 1997;122:1107-16. 58. Proye CAG, Goropoulos A, Franz C, Carnaille B, Vix M, Quivreux J'L, Couplet-Lebon G, Racadot A. Usefulness and limits of quick intra-operative measurements of intact (I-84) parathyroid hormone in the surgical measurement of hyperparathyroidism. Sequential measurements in patients with multiglandular disease. Surgery 1991;110:1035-1042.

CHAPTER

31

The Surgical Management of Hyperparathyroidism

SAMUEL A. WELLS, JR. AND GERARD M. DOHERTY Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110

H I S T O R Y OF PARATHYROID S U R G E R Y

The patient was a streetcar conductor, whose condition transiently improved following removal of a parathyroid tumor; however, the hypercalcemia recurred and no other parathyroid tissue was found at repeat operation or at autopsy performed after the patient succumbed from complications of hyperparathyroidism. The first operation performed for hyperparathyroidism in the United States involved a sea captain, Charles Martel, who was treated in 1926 at the Massachusetts General Hospital. However, the disease was not corrected until the seventh operation 7 years later (6). In the meantime, the first successful operation for hyperparathyroidism was performed at Barnes Hospital in 1929, when I.Y. Olch removed an enlarged parathyroid gland from a patient with bone disease and kidney stones (7). The term hyperparathyroidism was first used in the report of this case. Since the first operations for parathyroid surgery over 75 years ago we have learned a great deal about parathyroid gland physiology and now understand at the molecular level many bone and mineral diseases associated with parathyroid gland disorders. The surgical management of patients with hyperparathyroidism has also changed over time and has particularly evolved recently with the introduction of sophisticated preoperative imaging techniques and the use of "minimally invasive surgery." The current practice for the operative management of patients with hyperparathyroidism is the topic of this chapter.

The parathyroid glands were discovered in 1880 by Ivar Sandstrom, a Swedish student, working as an assistant in a histology laboratory at the University of Uppsala in Sweden (1). He identified the parathyroid glands in several species of animals, including humans, and his manuscript includes both gross and microscropic descriptions of the structures. Sandstrom's observation was one of the last great anatomic discoveries. However, his report was accepted for publication only in the periodical published by his medical school, and went largely unnoticed until 10 years later, when the Frenchman, Gley, rediscovered the parathyroid glands and showed in experimental animals that their removal led to tetany (2). Von Recklinghausen described osteitis fibrosa cystica generalisata, the bone disease associated with hyperparathyroidism (3). Other pathologists had also reported the association of bone disease and enlarged parathyroid glands, but it was generally accepted that the parathyroid enlargement was secondary to the skeletal pathology. The theory was not challenged until 1915, when Schlagenhaufer reasoned that it would be unlikely for secondary hyperparathyroidism to affect only one gland and predicted that in some cases the parathyroid tumor was the primary abnormality and the bone disease was secondary (4). The first parathyroidectomy for hyperparathyroidism was performed in 1925 by Felix Mandl of Vienna (5).

The Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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CLINICAL FEATURES OF PRIMARY HYPERTHYROIDISM Clinically, there are three types of hyperparathyroidism. Primary hyperparathyroidism (PHPT) defines patients whose hyperparathyroidism results from de novo enlargement and overactivity of one or more parathyroid glands. In secondary hyperparathyroidism (SHPT) the parathyroids are generally enlarged in reaction to the chronic hypocalcemia caused by disease in another organ system, most commonly the kidney or the gastrointestinal tract. Tertiary hyperparathyroidism (THPT) refers to the condition of autonomous parathyroid hyperfunction that occasionally develops in patients with SHPT. Although each type of hyperparathyroidism is managed operatively, most of our discussion will concern patients with primary hyperparathyroidism. The clinical presentation and methods of laboratory and radiologic diagnosis of PHPT have been discussed in previous chapters, but it is relevant to address certain facets of the clinical disease that are of particular concern to the surgeon. The classic signs and symptoms of PHPT relate to kidney stones and bone disease. Patients who present with bone disease have a more severe form of hyperparathyroidism than do those who present with renal stones, because the serum calcium concentration is higher, the onset of PHPT is more abrupt, and the resected parathyroid tumors are larger. Furthermore, patients with radiologic evidence of bone disease, or those with an elevated alkaline phosphatase preoperatively, are at increased risk of prolonged hypocalcemia following parathyroidectomy (i.e., "hungry bone" syndrome) and may require calcium and vitamin D therapy for several months before the serum level of calcium returns to normal. Patients with renal stones are often cured by parathyroidectomy, even though some may continue to have hypercalciuria or later excrete stones that were in situ at the time of parathyroidectomy.

PATIENT HISTORY The majority of patients with PHPT are now either asymptomatic at the time of diagnosis or they have mild and nonspecific symptoms such as muscle weakness, fatigue, lethargy, constipation, urinary frequency, or loss of memory. Indeed, if one carefully questions patients with PHPT, most will be found to have one or more of these symptoms. Often patients present to a physician with complaints of weakness, tiredness, and loss of energy, but these symptoms are common in older patients and the diagnosis of PHPT is not sus-

pected. This is unfortunate because the signs or symptoms of PHPT, though having minimal, if any, effect on younger patients, may significantly alter the life quality of older patients. Furthermore, the onset of the signs and symptoms of PHPT are subtle and insidious. Many older patients think their symptoms are due to increased age, only to find that their sense of well being and their energy and strength improve markedly after their parathyroid tumor is removed. The past history and family history are very important parts of the evaluation in patients with PHPT. Patients should be questioned about a history of external beam radiotherapy to the neck. Years ago it was common for infants and young children who presented with upper respiratory symptoms, incorrectly thought to be due to an enlarged thymus gland, to be treated with low-dose radiotherapy. Approximately 30% of these children developed thyroid nodules subsequently, and about 30% of these were malignant. The incidence of PHPT in these children with X-ray exposure is also increased and the cause is almost always found to be due to a single enlarged gland. At the time of neck exploration for PHPT it is important to evaluate the thyroid gland for the presence of pathology and perform a thyroidectomy if a malignancy is found. Any patient with PHPT should be studied for the presence of tumors of the pancreatic islet system or the pituitary gland. If either of these is present the diagnosis of multiple endocrine neoplasia (MEN) type 1 must be excluded. The diagnosis of MEN-1 in a patient with PHPT should lead to a thorough evaluation of the extended family. The occurrence of medullary thyroid carcinoma (MTC) or pheochromocytoma in a patient with PHPT suggests the presence of MEN-2a and similarly calls for screening of the patient's family. A history of PHPT alone in a family suggests the presence of familial hypocalciuric hyperparathyroidism (FHH) or familial primary hyperparathyroidism. Each of these familial endocrinopathies is inherited in an autosomal dominant pattern, characterized by complete penetrance. In MEN-1 and MEN-2A there is variable expressivity of the component diseases. Virtually all patients with MEN-1 have PHPT at the time of diagnosis, 50% have islet cell tumors (most often a gastrinoma or an insulinoma), and less than 20% develop a pituitary tumor (most often a prolactinoma). In patients with MEN-2A, MTC is uniformly present, however, only 50% of patients develop pheochromocytomas and 25% develop PHPT. In patients who are less than 40 years of age at the time of presentation with PHPT, the examining physician should suspect MEN-l, MEN-2A or FHH. It is important to establish the diagnosis of familial hyperparathyroidism prior to operating for PHPT, for

SURGICAL MANAGEMENTOF HYPERPARATHYROIDISM /

two reasons. Patients with these familial endocrinopathies have generalized involvement of the parathyroid glands and the operation should be planned accordingly. Furthermore, and perhaps most importantly, patients with F H H are not candidates for parathyroidectomy. They develop neither renal stones nor bone disease although they have hypercalcemia. More importantly, their hyperparathyroidism is virtually impossible to cure surgically, unless one performs a total parathyroidectomy. It is also important to note that the offspring of parents who both have F H H have a 25% chance of inheriting a mutated allele from each parent. In such cases the child will develop neonatal severe hyperparathyroidism (NSHP), which is a medical emergency with an excessive morbidity and mortality. The children have extraordinarily high levels of serum calcium and are often dehydrated. Because it is difficult to reduce the serum calcium permanently by medical means, these children are candidates for parathyroidectomy. Occasionally, during an operation on a patient thought to have sporadic PHPT, the surgeon finds four enlarged parathyroid glands. The patient should have an appropriate operation and in the postoperative period the presence of either MEN-l, MEN-2A, or F H H should be excluded. Although the PHPT is not lifethreatening in these patients, the pancreatic islet cells tumors in patients with MEN-1 and the MTC and pheochromocytomas in patients with MEN-2A may prove lethal if untreated. If the diagnosis of MEN-1 is made first and patients are found to have a pancreatic gastrinoma, the hyperparathyroidism should be corrected first, because normalization of the serum calcium level will reduce gastric acid secretion. However, if an insulinoma is detected, it should be treated first, because there is no g o o d medical therapy for insulin o m a and the associated hypoglycemia may be lifethreatening. Virtually all patients with MEN-2A have MTC at the time of diagnosis and if a total thyroidectomy is p e r f o r m e d all four parathyroid glands should be identified and an appropriate operation performed. Failure to detect the presence of a p h e o c h r o m o c y t o m a in a patient with MEN-2A who is undergoing operation for PHPT or MTC may be catastrophic because severe hypertension may develop, either during induction of anesthesia, during the operative procedure, or in the postoperative period. The diagnosis of MEN-l, MEN-2A, and F H H has been greatly simplified because the genetic mutation for each has been identified. The m e n i n gene on chromosome 11 is mutated in patients with MEN-l, the RET protooncogene on c h r o m o s o m e 10 is mutated in MEN-2a, and the calcium-sensing receptor gene on chromosome 3 is mutated in F H H (8-11). Each of

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these mutations occurs in the germ line so they can be detected by direct DNA analysis of genomic DNA from a patient's white blood cells.

THE P H Y S I C A L E X A M I N A T I O N On physical examination of patients with PHPT one can rarely feel an enlarged parathyroid gland. A palpable neck mass in this setting most often represents a thyroid nodule or an enlarged lymph node. On the other hand, if a neck nodule is palpable and the serum calcium concentration is above 14 m g / d l one should suspect a parathyroid carcinoma (12). These patients often present with hoarseness and systemic manifestations of severe hypercalcemia, including nausea, vomiting, and dehydration. On physical examination the neck mass is firm and immobile and at operation the parathyroid tumor is usually fixed to the trachea or surrounding tissues. Occasionally, patients with a parathyroid carcinoma will require urgent surgery to control hypercalcemia, particularly when it is not possible to control the serum calcium concentration medically.

Laboratory Studies Most often surgeons see patients in referral from colleagues in internal medicine, endocrinology, or pediatrics, who have already established the diagnosis of PHPT biochemically. Thus the laboratory work need not be repeated unless there is some question about the results of a specific test.

Imaging Studies Most patients with PHPT will not require specific radiologic studies prior to surgery. Often bone densitometry studies are p e r f o r m e d to determine the degree of calcium loss from the skeleton. In patients who are being followed medically the clinician may choose to perform serial bone densitometry studies to monitor bone mass. Renal imaging studies may be helpful to determine whether a patient has occult renal stones, or to measure the stone b u r d e n in a known stone former. The use of imaging procedures to localize enlarged parathyroid glands was formerly applicable only to patients with a failed operation, who required repeat neck exploration. More recently, it has been shown that 99mTc-labeled sestamibipertechnetate (MIBI) scintigraphy combined with single-photon emission c o m p u t e d tomography successfully detects single parathyroid adenomas in approximately 95% of patients with PHPT (13). Unfortunately, the technique is not nearly as useful (60-70%) in patients with multiple-gland disease.

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However, the introduction of this technology has remarkably changed the surgical management of patients with PHPT and patients commonly have a sestamibi scan once the diagnosis of PHPT is established. Consequently, patients frequently present for surgical consultation bringing radiographic tests showing the location of an enlarged parathyroid gland. Unfortunately, there has been no prospective study of the indications for preoperative localization studies and their place in the management of patients with primary hyperparathyroidism. The combination of preoperative localizing studies and minimally invasive surgery has led to a dramatic change in the management of patients with PHPT, because the procedure can be performed on an outpatient basis at substantial cost savings. The discussion will be contined in the section on operative management of patients with PHE

Indications for Operative Intervention The decision, whether to operate on patients with PHPT, is influenced by several considerations and is somewhat controversial. The accepted indications for operation include the presence of either bone disease, kidney stones, or muscle weakness or tiredness of a degree that it limits the patient's function. A serum calcium above 12 m g / d l in a patient who has minimal or no symptoms is also an indication for neck exploration. Prolonged follow-up of patients with PHPT can become expensive, especially if bone densitometry studies and laboratory tests are performed repeatedly over time. It is the opinion of most surgeons that once the clinical diagnosis of PHPT is established in a medically fit patient, they should undergo a neck exploration, because the cure rate is high and the operation is usually simple. The indications for operative intervention have become liberalized since the introduction of new techniques for minimally invasive surgery. In a study by Silverberg and associates of 121 patients with PHPT (101 of whom were asymptomatic), 61 had parathyroidectomy and 60 were followed expectantly (14). Whether patients were symptomatic or asymptomatic, the parathyroidectomy resulted in correction of the hypercalcemia and an increase in bone mineral density of the lumbar spine and the femoral neck, but not the radius. Of the 52 asymptomatic patients not having a neck exploration, the serum calcium levels and the bone densitometry values did not change; however, 14 (23%) of them developed other indications for parathyroidectomy. Unfortunately, in patients with asymptomatic PHPT there have been no prospective randomized clinical trials comparing immediate parathyroidectomy to nonoperative management. This is an important issue that has significant clinical, scientific, and economic considerations.

CONSIDERATIONS IN THE SURGICAL TRF~TMENT OF PATIENTS WITH HYPERPARATHYROIDISM

Embryology and Anatomy of the Parathyroid Glands It is extremely important for the operating surgeon to have a clear understanding of the embryology and anatomy of the parathyroid glands. Although it is not commonly done today, many skilled parathyroid surgeons in the past spent hours in the autopsy suite, or the anatomy laboratory, searching for parathyroid glands and learning normal parathyroid anatomy, before they began to operate on patients with parathyroid or thyroid disease. This remains an important exercise because the normal parathyroid glands are small and may be difficult to recognize, even by the experienced eye.

Embryology Phylogenetically, the parathyroid glands first appear in Amphibia. In humans the upper parathyroids derive from the fourth pharyngeal pouch and the lower parathyroids originate from the third. During embryogenesis both sets of glands are intimately associated with the derivatives of their respective pouches, the lower glands with the thymus and the upper glands with the lateral thyroid complex. As the embryo matures the parathyroid glands descend to assume their normal positions (Fig. 1). The upper glands come to rest on the posterior surface of the midportion of the thyroid lobes, close to the point where the inferior thyroid artery enters the thyroid parenchyma. The lower parathyroid glands descend to reside close to the anterior-lateral surface of the lower thyroid pole. In some cases the migratory patterns are imperfect, which has important clinical implications. Either an upper or lower parathyroid gland may fail to migrate and remain embedded in the pharyngeal musculature. Conversely, a lower parathyroid gland may have an arrested descent and remain high in the neck as an "undescended parathymus." In this situation the thymus gland also fails to descend and remains as a thick cord of tissue medial and parallel to the carotid artery. The undescended parathyroid gland may reside adjacent to or within any portion of the undescended thymic tissue. Conversely, during migration a parathyroid gland may fail to separate from the thymus gland and descend into the anterior or deep mediastinum. On very rare occasions a portion of the thymus, containing an enlarged parathyroid gland, may descend to fill the space bounded by the aortic arch and the pulmonary artery, the so-called "middle mediastinal parathyroid adenoma."

SURGICAL MANAGEMENT OF HYPF~AgATm~omISM

FI6. 1 The migratory patterns of the parathyroid glands during embryogenesis. The upper parathyroid glands a r e derived from the fourth pharyngeal pouches and the lower parathyroid glands are derived from the third pharyngeal pouches. (Reprinted with permission from Langman J. Medical embryology, 3rd ed., Baltimore:Williams & Wilkins, 1975:266.)

Anatomy Usually, there are four parathyroid glands, but there may be more. It has been questioned whether there are ever less than four parathyroid glands. The phenomenon does appear to occur rarely. In Alveryd's report of 354 adults studied at autopsy, 90.6% had four glands, 3.7% had five glands, 5.1% had three glands, and 0.6% had only two glands (15). In only one of Alveryd's 18 patients with less than four identified parathyroid glands was the combined weight of the glands sufficient to suggest that none had been overlooked. Moreover, Norris serial sectioned 109 embryos from the base of the skull to the thoracic diaphragm and found at least four parathyroid glands in every specimen (16). Akerstr6m and associates performed autopsy studies on 503 cadavers and found four parathyroid glands in all but 18 specimens. There was a striking constancy to the location of the parathyroid glands, as shown in Fig. 2 (17). From these studies one can surmise two important facts. It is extremely unusual for a patient to have less than four parathyroid glands and most often the parathyroid glands are where they should be. It is often tempting for the inexperienced surgeon to assume, after an arduous neck exploration during which only

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two or three parathyroid glands are found, that the patient naturally has less than four. One must always assume that each patient has at least four parathyroid glands. During the process of neoplastic transformation the enlarged parathyroid glands may migrate to an ectopic site (Fig. 3). Bulky upper parathyroid glands tend to descend into the posterior neck or the upper posterior mediastinum. Enlarged lower glands usually descend into the anterior mediastinum, adjacent to the thymus gland. This inferior migration of enlarged parathyroid glands is due to an increasing weight and the intermittent, but persistent, negative pressure generated in the thoracic cavity. Even when parathyroid glands are in a "normal" position they may be difficult to find because of pathologic changes within the thyroid gland. A normal or enlarged parathyroid gland may be entrapped between thyroid nodules and hidden from view. If, during a neck exploration for PHPT in a patient with a multinodular goiter, three normal parathyroid glands are found but the fourth cannot be found, it may be necessary to remove the thyroid lobe ipsilateral to the missing gland. Often in this situation the pathologist will incorrectly interpret the parathyroid gland as being intrathyroidal. The normal parathyroid glands are flat and weigh less than 30-45 mg each. They are yellowish tan in color, although enlarged parathyroid glands may be deep reddish brown. The glands are usually ovoid in shape but they may be lobulated or stellate. When the glands enlarge, their shape will be influenced by the surrounding structures, but they often assume an elongated shape with little adherence to adjacent tissues.

Preoperative Preparation It is important that the surgeon explain the operative procedure to the patient. The surgeon should review the possible causes of hyperparathyroidism and discuss the specific surgical treatments for the various disease states, e.g., operations for single-gland disease and multiple-gland disease. Most importantly, the possible complications of parathyroid surgery should be explained, including damage to the nerves that control phonation, postoperative hypocalcemia, and failure to find a parathyroid tumor due to its ectopic location. Patients should also be told of complications that are common to all surgical procedures, such as wound infection and postoperative bleeding. The patient may wish to know more about expected outcomes because many will have already read a great deal about the disease and will be familiar with the various treatments available.

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FIG. 2 Locations of the superior (A) and inferior (B) parathyroid glands. The most common locations are indicated by the darker shading. [Reprinted with permission from Akerstr6m G, Malmaeus J, and Bergstro R: Surgical anatomy of human parathyroid glands. Surgery 1984;95: 14-21 (17).]

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SURGICAL MANAGEMENT OF HYPERPARATHYROIDISM //

Operative Technique The primary goal of surgery in patients with PHPT is to cure the hypercalcemia without intraoperative or postoperative complications. In the past seven years the surgical m a n a g e m e n t of patients with hyperparathyroidism has changed markedly, primarily due to three innovations: (1) sestamibi scanning has proved to be highly accurate in localizing enlarged parathyroid glands; (2) techniques of minimally invasive surgery have been designed, and in concert with sestambi localization and neck explorations these are frequently performed in an outpatient setting with either light general, regional, or local anesthesia; and (3) the development of rapid immunoassays for parathyroid hormone, which can be performed intraoperatively to document that hyperfunctioning parathyroid tissue has been completely resected. These techniques have been introduced only recently and there has been insufficient time and experience to clearly judge their worth in clinical practice. However, it appears clear that the new technologies will have an important place in the management of patients with PHPT. Regardless of the operative technique for patients with PHPT there are certain important factors that need to be considered. There is an extremely low mortality following parathyroidectomy and the morbidity of the operation relates to postoperative bleeding, wound infection, hoarseness, and hypocalcemia. Bleeding usually occurs within the first 24 hours postoperatively and is associated with a sudden increase in venous pressure due to the patient's straining or coughing. Postoperative wound infection is rare and usually occurs in patients who suffer from a chronic skin irritation. Hoarseness results from injury to either the recurrent laryngeal nerve or the external branch of the superior laryngeal nerve. It is important that these nerves be identified during the operation and that they be kept from harm's way.

General Technique of the Standard Operation In patients who either do not have a preoperative localization procedure, or in whom the findings are controversial, a standard open neck exploration is performed through a cervical incision u n d e r general anesthesia. The goal of operation is to identify each parathyroid gland and to remove those that are enlarged. The remaining normal-sized parathyroid glands should be confirmed as such by biopsy. Some surgeons have reported that biopsy of all four parathyroid glands is associated with an increased incidence of postoperative hypoparathyroidism, but we have not found this to be the case. It must be emphasized that

493

great care needs to be taken in performing the parathyroid biopsy because only a very small amount of tissue is taken from the portion of the parathyroid gland opposite to the arterial supply. Even though the terminologies of adenoma and hyperplasia are common descriptors of parathyroid pathology, we do not find the terms useful and prefer to characterize hyperparathyroidism as being either due to single-gland disease or multiple-gland disease. Nearly all patients who have a single enlarged parathyroid gland and three normal glands are cured following resection of the large gland. Patients who have two or three enlarged glands have a less good outcome. This disease state is commonly referred to as double or triple parathyroid adenomas, or early parathyroid hyperplasia. Little has been written about the long-term evaluation of patients with this condition following parathyroidectomy, however. Our group evaluated 375 consecutive patients with PHPT and found 85 (15%) who had enlargement (greater than 50 mg) of two or three parathyroid glands (18). At operation the enlarged parathyroid glands were removed and the remaining normal-sized glands were biopsied. Of these patients, 76 were followed from 12 to 140 months after surgery and 8 (10.5 %) had either persistent (2 patients at 1 and 4 months) or recurrent (6 patients from 45 to 133 months) hyperparathyroidism. When compared to age- and gender-matched patients with PHPT due to single-gland disease, patients with two-or three-gland disease had lower serum calcium concentrations and a lower combined weight of the resected parathyroid tissue. This indicated a mild form of PHPT with a higher incidence of persistent or recurrent hypercalcemia compared to patients with single-gland disease, but a much lower incidence of postoperative hypercalcemia compared to patients with four-gland disease. Patients with PHPT due to generalized parathyroid enlargement represent the most difficult m a n a g e m e n t problems. Surgery is not often curative for this disease, because regardless of the procedure performed there is a high rate of persistent or recurrent hyperparathyroidism. The original operation for this disease was radical, subtotal, 3g-gland parathyroidectomy. The strategy was to remove three of the four enlarged parathyroid glands and one-half or less of the fourth. An attempt is made to remove a substantial portion of the parathyroid mass so that the patient remains normocalcemic. The problem is that the forces that led to the hypertrophy and excess secretion of parathyroid hormone are still at play. There have been almost no long-term reports of patients with four-gland disease who have been managed by this operation, however, the few publications that have addressed this issue provide evidence that the failure rate exceeds 70%. An alternate

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approach to patients with four-gland disease is total parathyroidectomy with autotransplantation of fragments of the most normal-appearing parathyroid tissue to a heterotopic site, such as the brachioradialis muscle of the n o n d o m i n a n t forearm. This operation is associated with a transient period of hypocalcemia, while the graft is vascularized. There is also an appreciable likelihood that these patients will develop graft-dependent hyperparathyroidism, but this complication can be managed by removing a portion of the graft under local anesthesia. Placement of the autotransplant in the arm allows one to monitor graft function by comparing PTH concentrations in venous blood obtained from the two arms. A markedly elevated level of PTH in blood draining the graft excludes a fifth hyperfunctioning parathyroid gland in the neck or mediastinum. In patients who develop hypercalcemia after 3~gland parathyroidectomy a repeat neck exploration under general anesthesia is required. Most surgeons have a methodical way of exploring the neck, and in the process identify all four parathyroid glands. In most circumstances the operation can be performed in less than 1 hour. If one or more parathyroid glands cannot be identified, biopsies should be taken of the identified normal glands and their location marked with a black silk suture so that they can be readily identified at a subsequent operation after localization procedures have been performed. Above all, normal parathyroid glands should not be removed.

Minimally Invasive Parathyroid Surgery A variety of less invasive operative strategies have been developed recently to take full advantage of advances in parathyroid localization and testing. The

goal of these strategies has been to maintain the outstanding success rate of the conventional neck exploration in curing hyperparathyroidism, while decreasing the invasiveness, and potentially the cost, of the procedure. Sestamibi scanning and rapid PTH measurement have had substantial effects, whereas the application of videoscopic surgical techniques remains investigational. 99mTechnetium-labeled sestamibi was originally developed for cardiac imaging. It was found incidentally to image parathyroid tissue on delayed scans and has since been used extensively for noninvasive parathyroid imaging (Fig. 4) (19,20). This technique has advantages in lateral, oblique, and three-dimensional imaging compared to the formerly used technetiumthallium scanning because of its single-nuclide nature and short half-life, high-energy profile. Sestamibi scans can identify the site of abnormal tissue in 75-85% of patients, but is less useful in patients with small adenomas or multiple-gland disease. The success of sestamibi in identifying the site of abnormal parathyroid tissue has been an impetus for the reassessment of directed, unilateral strategies of parathyroidectomy. Techniques for the rapid, accurate measurement of serum PTH have been developed that allow the intraoperative assessment of the effect of parathyroidectomy (21,22). A two-site immunoassay for intact PTH with standard curves adjusted to the short incubation period is used. The time to PTH determination is typically 20-30 minutes from the blood draw, which makes it practical to perform the assay during the operation. Experience has demonstrated that if the PTH level decreases by at least 50% within 10 minutes after removal of the enlarged parathyroid gland, the patient will be normocalcemic postoperatively (Fig. 5). Thus a directed, unilateral approach to parathyroidectomy, without visualization of the other normal

FIG. 4 99mTechnetium-labeled sestamibi scan of a patient primary hyperparathyroidism due to a right lower parathyroid adenoma. The activity is distributed in the normal thyroid gland, salivary glands, and heart in the initial scan performed 10 minutes after radionuclide injection (white arrows in left panel). Two hours after injection, the radioactive tracer has washed out of the thyroid gland, but remains in a right-sided parathyroid gland that overlies the lower pole thyroid activity from the initial scan.

SURGICAL MANAGEMENT

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tion, except that the focus is limited to identifying the enlarged parathyroid gland identified on the preoperative scan. Ten minutes after resecting the abnormal gland, a second blood sample is taken to determine the PTH level; if this PTH level is 50% less than the base line value, then the operation is terminated (29). If, however, the PTH level does not decrease, the exploration is extended to a conventional exploration and the other parathyroid glands are identified.

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Sestamibi-Guided Parathyroidectomy Single gland disease

Multiple gland disease

FIG. 5 Intraoperative parathyroid hormone levels from patients who underwent conventional bilateral neck exploration for primary hyperparathyroidism. Of these 72 patients, 55 had single-gland disease and 17 had multiple-gland disease as defined by the gross parathyroid morphology and gland weights. A decrease to 50% of the base line intraoperative PTH level 10 minutes following parathyroid gland resection predicted successful parathyroidectomy in 93% (51/55) of patients; if the PTH remained >50% of base line, 76% (13/17) of patients had multiple-gland disease. (Data from Gordon LI, et aL Surgery 1999;126:1030-1035.)

glands, appears to be justified by the low failure rate reported from early experience with this procedure (23,24).

Concise Parathyroidectomy This is the most frequently used approach (25-28). The surgeon obtains a sestamibi scan in advance of the operative day in an attempt to identify the site of the abnormal parathyroid gland. If the sestamibi scan does not identify a gland, then the patient undergoes the conventional full-neck exploration. If the sestamibi scan identifies an enlarged parathyroid gland, the patient is taken to the operating room and a limited neck exploration and parathyroidectomy are performed. The operation can be performed u n d e r general anesthesia, cervical block, or local anesthesia with sedation. A transverse incision is made at the level of the thyroid isthmus, on the side of the midline ipsilateral to the abnormal parathyroid gland. Often this incision is less than 2.5 cm. Blood is drawn at the outset of the operation (often from the anterior or internal jugular vein) to determine the base line PTH level. The strap muscles are opened, either by separating them in the midline for a relatively anteriorly placed lower parathyroid gland, or by splitting the sternohyoid and sternothyroid muscles. Exploration proceeds as for the conventional opera-

In this approach, the sestamibi concentration in the parathyroid tissue is used to aid the surgeon in identification of the enlarged gland (30,31). The positioning and anesthetic requirements are the same as for concise parathyroidectomy. A sestamibi scan is obtained on the day of the procedure, within 2 hours of the operative start time. The operation is begun by using a handheld gamma probe to localize the site where the per-second counts are highest in the neck. A limited incision is fashioned over that site and dissection is carried directly to the parathyroid gland using the probe for directional guidance. The parathyroid gland is removed, and the operation terminated. Some surgeons use intraoperative PTH measurements to confirm resection of all hyperfunctioning parathyroid tissue, but many do not (32).

Videoscopic Cervical Exploration Some surgeons use a videoscopic approach to resect an abnormal parathyroid gland that has been identified on preoperative sestamibi scan (33,34). This is done using a variety of different work-space maintenance techniques (carbon dioxide gas pressure, or gasless) and anatomic approaches (lateral approach anterior to the sternocleidomastoid muscle or central approach from the suprasternal notch). The technique is still in its developmental stage, and the optimal approach remains to be determined. Investigators using this technique use a sestamibi scan in order to identify the abnormal gland preoperatively, and in general identify and resect only that gland. Given the use of a limited incision to identify and remove parathyroid glands in the directed, open approaches, m u c h of the advantage of a videoscopic approach has been nullified.

The Failed Operation In approximately 95% of patients with PHPT an operation will be successful. When the operation is unsuccessful it is usually due to one or more of several factors: inexperience of the operating surgeon, enlargement of more than one parathyroid gland,

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ectopic location of an enlarged parathyroid gland, or an erroneous diagnosis. The importance of patience and experience cannot be overemphasized, and as with most operative procedures the surgeon with the most experience with a given technique gets the best results. When the neck exploration has been unsuccessful one should allow the patient to convalesce from the procedure and then reconsider the diagnosis of PHPT by biochemical tests. In patients who were found to have more than one enlarged parathyroid gland it is important to exclude familial hyperparathyroidism, especially FHH. Other diagnoses should be considered in hypercalcemic patients in whom four normal-sized parathyroid glands were found. Once the biochemical diagnosis of PHPT is confirmed, attempts should be made to identify by localization techniques the site of the missed enlarged parathyroid gland. The noninvasive localization procedures should be performed first and only if they are unsuccessful should invasive localization procedures be performed. The specific radiographic localization procedures will not be mentioned here because they are discussed in Chapter 30. However, the importance of these procedures cannot be overemphasized because the specific identification of a lesion will markedly shorten the time of the repeat operative procedure and make the patient less susceptible to the complications attendant to repeat neck explorations when no hyperfunctioning parathyroid tissue has been identified. In almost every series of patients undergoing repeat neck exploration for PHPT the missed enlarged parathyroid gland is most often found in the neck, or it is retrievable through a cervical incision (Table 1) (35). The technique for repeat neck exploration is similar to that used for a standard neck exploration. The previous scar should be excised and if the parathyroid gland has been localized by imaging techniques, only that side of the neck need be explored if the patient has sporadic PHPT. Even if the enlarged parathyroid gland is in the deep anterior mediastinum close to the thymus gland it can be removed with the thymus through a cer-

TABLE 1

Location of Missed Parathyroid Glands Found during Reoperation a

Location

Persistent

Recurrent

Total

Cervical Mediastinal

208 20

35 1

243 21 264

aReprinted with permission from Brennan MF, Norton JA. Reoperation for persistent and recurrent hyperparathyroidism. Ann Surg 1985;201:40-44 (35).

vical excision (36). Rarely, it is necessary to perform a thoracotomy or a mediastinotomy to remove the enlarged parathyroid gland.

Renal Osteodystrophy and Secondary Hyperparathyroidism Most patients with advanced renal disease who are maintained on chronic dialysis have evidence of parathyroid-induced bone disease, with elevated serum levels of PTH. The management of patients with this disorder is discussed in Chapter 39. Due to improvements in the medical management of patients with chronic renal failure, the frequency of parathyroidectomy has decreased. Nevertheless, there are indications for parathyroidectomy in patients with secondary hyperparathyroidism due to chronic renal failure, or in patients with autonomous hyperplastic parathyroid glands (tertiary hyperparathyroidism), including hypercalcemia either in patients who are candidates for renal transplantation or in patients who have a wellfunctioning kidney, pruritis, bone pain, and extensive soft tissue calcification. Patients with secondary hyperparathyroidism almost always have generalized parathyroid gland enlargement and should be treated with either radical subtotal parathyroidectomy or total parathyroidectomy and parathyroid autotransplantation. Rothmund and associates performed a prospective randomized trial comparing these two procedures and found that total parathyroidectomy and autotransplantation afforded better results in that there was a lower incidence of postoperative hypoparathyroidism and a higher incidence of bone healing (37).

Postoperative Management After a standard neck exploration for PHPT the patient should have minimal ambulation on the day of the operation but can eat a normal diet on the day after surgery. Patients are usually discharged on the day after surgery but they may require prolonged hospitalization or careful evaluation as an outpatient if the serum calcium remains low and they have symptoms of hypocalcemia. Patients who have minimally invasive operations are usually discharged the same day of surgery, although they still require careful postoperative evaluation.

REFERENCES 1. Sandstrom I. O m e n ny k6rtel hos menniskan och fitakillga d/iggdjur. Uppsala Liika F6rh 1879;15:441. 2. Gley E. Sur les fonctions du corps thyroide. C R Soc Biol 1891;43:841.

SURGICAL MANAGEMENT OF HYPERPARATHYROIDISM 3. Von Recklinghausen FD. Die fibr6se oder deformierte Ostitis, die Osteomalacie und die osteoplastiche Carcinose in ihren gegenseitigen Beziehungen. Festschr Rud Virchow (Berlin) 1891 ;1:89. 4. Schlagenhaufer E Zwei Ffille von Parathyreoidea Tumoren. Wien Klin Wochenschr 1915;28:1362. 5. Mandl E Therapeutischer Versuch bei einem Falle von Ostitis fibrosa generalisata mittels Extirspation eines Epithelk6rperchentumors. Zentrabl Chir 1926a;5:260. 6. Cope O. The story of hyperparathyroidism at the Massachusetts General Hospital. N EnglJ Med 1966;21:1174-1182. 7. Barr DE Bulger MA. The clinical syndrome of hyperparathyroidism. Ann J Med Sci 1930;179:449. 8. Chandrasekharappa SC, Guru SC, Manickam P, et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 1997;276:404-407. 9. Donis-Keller H, Dou S, Chi D, et al. Mutation in the RET protooncogene are associated with MEN-2A and FMTC. Hum Mol Genet 1993;2:851-856. 10. Mulligan LM, Kwok jBj, Healey CS, et al. Germ-line mutations of the RETprotooncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458-460. 11. Pollak MR, Brown EM, Chou Y-HW, et al. Mutations in the CA2+sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297-1303. 12. Wang CA, Gaz RD. Natural history of parathyroid carcinoma: Diagnosis, treatment, and results. A m J Surg 1985;149:522-527. 13. Neumann DR, Esselstyn CB, Jr, Go RT, Wong CO, Rice TW, Obuchowski NA. Comparison of double-phase 99mTc-sestamibi with 123-I 99mTc-sestamibi subtraction SPECT in hyperparathyroidism. A m J Roentgeno11997;169:1671-1674. 14. Silverberg SJ, Shane E, Jacobs TP, Siris E, Bilezikian JE A 10-year prospective study of primary hyperparathyroidism with or without parathyroid surgery. N EnglJ Med 1999;341:1249-1255. 15. Alveryd A. Parathyroid glands in thyroid surgery. Acta Chir Scand 1968;389:1-120. 16. Norris EH. The parathyroid glands and the lateral thyroid in man: Their morphogenesis, histogenesis, topographic anatomy and prenatal growth. Contrib Embryo11937;26:47. 17. Akerstr6m G, Malmaeus J, Bergstro R. Surgical anatomy of human parathyroid glands. Surgery 1984;95:14-21. 18. Wells SA, Leight GS, Hensley M, et al. Hyperparathyroidism associated with the enlargement of two or three parathyroid glands. Ann Surg 1985;202:533-538. 19. Wei, JP, Burke GJ. Cost utility of routine imaging with Tc-99msestamibi in primary hyperthyroidism before initial surgery. 1997;1097-1101. 20. Bhatnagar A, Vezza PR, Bryan JA, Atkins FB, Ziessman HA. Technetium-99m-sestamibi parathyroid scintigraphy: Effect of P-glycoprotein, histology and tumor size on detectability, J Nucl Med 1998;39:617-620. 21. Irvin, GL, Dembrow VD, Prudhomme DL. Clinical usefulness of an intraoperative "quick parathyroid hormone" assay. Surgery 1993; 114:1019-1023.

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22. Nussbaum SR, Thompson AR, Hutchenson BA, Gaz RD, Wang C. Intraoperative measurement of parathyroid hormone in the surgical management of hyperparathyroidism. Surgery 1988;104:1121-1127. 23. Duh Q-Y, Uden P, Clark OH. Unilateral neck exploration for primary hyperparathyroidism: Analysis of a controversy using a mathematical model. WorldJ Surg 1992;16:654-662. 24. Shen W, Sabanci U, Morita ET, Siperstein AE, Duh Q-Y, Clark OH. Sestamibi scanning is inadequate for directing unilateral neck exploration for first-time parathyroidectomy. Arch Surg 1977;132:969-974. 25. Carry S, Worsey M, Virji, M, Brown M, Watson C. Concise parathyroidectomy: The impact of preoperative SPECTOomTc sestamibi scanning and intraoperative quick parathormone assay. Surgery 1997;122:1107-1116. 26. Boggs JE, Irvin GL, Molinari AS, Deriso GT. Intraoperative parathyroid hormone monitoring as an adjunct to parathyroidectomy. Surgery 1996;120:954-958. 27. Irvin GL, Sfakianakis G, Yeung L, Deriso, GT, Fishman LM, Molinari, AS, Foss JN. Ambulatory parathyroidectomy for primary hyperparathyroidism. Arch Surg 1996;131:1074-1078. 28. Chen H, Sokoll, LJ, Udelsman R. Outpatient minimally invasive parathyroidectomy: A combination of sestamibi-SPECT localization, cervical block anesthesia, and intraoperative parathyroid hormone assay. Surgery 1999;126:1016-1022. 29. Gordon LI, Snyder WH, Wians F, Nwariaku F, Kim LT. The validity of quick intraoperative parathyroid hormone assay: An evaluation in seventy-two patients based on gross morphologic criteria. Surgery 1999;126:1030-1035. 30. Norman J, Chheda H. Minimally invasive parathyroidectomy facilitated by intraoperative nuclear mapping. Surgery 1997; 122:998-1004. 31. Norman J, Chheda H, Farrell C. Minimally invasive parathyroidectomy for primary hyperparathyroidiam: Decreasing operative time and potential complications while improving cosmetic results. Ann Surg 1998;64:391-396. 32. Murphy C, Norman J. The 20% rule: A simple, instantaneous radioactivity measurement defines cure and allows elimination of frozen sections and hormone assays during parathyroidectomy. Surgery 1999;1126:1023-1029. 33. Miccoli P, Bendinelli C, Vignali, E, Mazzeo S, Cecchini G, Pinchera A, Marcocci C. Endoscopic parathyroidectomy: Report of an initial experience. Surgery 1998;124:1077-1080. 34. Henry JF, Defechereux T, Gramatica L, de Boissezon C. Minimally invasive videoscopic parathyroidectomy by lateral approach. Langenbecks Arch Surg 1999;384:298-301. 35. Brennan ME Norton JA. Reoperation for persistent and recurrent hyperparathyroidism. Ann Surg 1985;201:40-44. 36. Wells SA, Cooper JD. Closed mediastinal exploration in patients with persistent hyperparathyroidism. Ann Surg 1991 ;214:555-561. 37. Rothmund M, Wagner PK, Schark C. Subtotal parathyroidectomy versus total parathyroidectomy and autotransplanation in secondary hyperparathyroidism: A randomized trial. World J Surg 1991;15:745-750.

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CI4APTF32 Ectopic Locations of Parathyroid Glands

NORMAN

W. T H O M P S O N

Arbor, Michigan 48105

A N D P A U L G. G A U G E R

Division of Endocrine Surgery, Department of Surgery, University of Michigan, Ann

INTRODUCTION

The following discussion considers ectopic glands based on their embryologic origin.

A thorough knowledge of the location of ectopic parathyroid glands is of fundamental importance to the surgeon undertaking a neck or mediastinal exploration for either primary or secondary hyperparathyroidism. Although relatively infrequent, ectopic parathyroid glands are the most common cause of failure when the neck is explored by an experienced parathyroid surgeon. Because of the possible ectopic location of a hyperplastic parathyroid gland in any neck exploration, it is essential that as each gland is identified it is characterized as a superior (branchial pouch IV origin) or inferior (branchial pouch III origin) gland based on its anatomic location and relationships to other anatomic structures. Thus, when three normal glands have been found and the missing gland has not been identified after a careful search in the usual locations, a systematic exploration can be made in the known ectopic locations for that gland based on its embryologic origin. With a disciplined exploration, nearly all cervical and upper mediastinal glands can then be identified. Ideally, only deep mediastinal glands in either the anterior or the middle mediastinum (1-2%) should remain undetected after such a search (1). Rarely, the cause of hyperparathyroidism may be due to an ectopic supernumerary (fifth gland) determined by finding four normal glands. In this unusual situation, the surgeon's search cannot focus on the embryologic domain of one specific gland, but must extend to the entire neck and all known accessible ectopic sites before termination of the exploration. The Parathyroids, Second Edition

S U P E R I O R PARATHYROID G I A N D S The incidence of true superior parathyroid gland ectopia is rare. Normal superior parathyroid glands are more likely to be found in a relatively discrete and predictable area than are those glands designated as inferior. Nearly 95% of normal superior glands can be found within a 2-cm radius of the junction of the recurrent laryngeal nerve and the inferior thyroid artery. Only 2-4% of normal superior glands will be found caudal to the inferior thyroid artery within the tracheoesophageal groove and about the same percentage is found more cephalad along the superior pole of the thyroid gland (2,3). Less than 0.1% is even further cephalad in approximation to the pharyngeal wall (4). Nevertheless, abnormal parathyroid glands can descend to an ectopic location (pseudoectopia) in more than 40% of all patients with hyperparathyroidism due to superior gland disease (1). By far, the most common location is along the esophagus, often in the tracheoesophageal groove (Fig. 1). Pseudoectopia occurs when the enlarged gland descends u n d e r or posterior to the inferior thyroid artery. In this plane, unless retained by investing fascia or vessels, an enlarged gland can acquire an ectopic localization as it descends anterior to the prevertebral fascia, into the posterior mediastinum. The extent of descent is probably limited by the end artery supplying the tumor, which is always a 499

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

500

/

C-6

CHAPTER32

~ ..

,, ...::..

.. ~;.:,, ...

FIG. 1 Locations of enlarged superior parathyroid glands. Approximately 40% of superior gland adenomas are found in ectopic locations as a result of developmental mechanical forces (pseudoectopia).

branch from the inferior thyroid artery (3,5). These "acquired ectopic" superior parathyroid glands can always be removed through a cervical incision, even when they are fairly deep in the posterior mediastinum (Fig. 2). It is noteworthy that more than half of these pseudoectopic adenomas will not be visualized even after the initial mobilization of the thyroid lobe and its medial retraction. The caudally descended superior gland is always covered by a layer of fascia extending from the thyroid medially as the thyroid sheath, and laterally to the carotid artery where it becomes the carotid sheath. Caudally the pretracheal fascia extends laterally before evolving into the carotid sheath. This fascia lies anterior to an ectopic superior parathyroid gland as well as the recurrent laryngeal nerve as it ascends the neck toward the larynx. Because abnormal superior glands are so frequently found in a paraesophageal location in the lower neck or upper posterior mediastinum, these locations must be routinely evaluated whenever a parathyroid gland has not been detected in the usual anatomic area. Using a routine but disciplined approach to exploration for the superior gland, an enlarged gland in this ectopic location should never be overlooked (1,3,4,6,7). In our opinion, the key maneuver at exploration is initially to incise the fascia lateral to the upper thyroid pole, vertically proceeding caudally to the inferior thyroid artery. This allows exposure of the prevertebral fascia posteriorly, improved medial rotation of the entire thyroid lobe with appropriate retraction, and direct visualization caudally into the posterior mediastinum. An index finger can be inserted behind the inferior thyroid artery into

the tracheoesophageal groove and or behind the esophagus (Fig. 3). Rarely, a truly ectopic parathyroid gland may be found in the midline posteriorly, adherent to the esophageal wall rather than freely movable on a vascular pedicle. Such parathyroid tumors are covered by a thin layer of fascia fixing their position and establishing their embryonic heritage, rather than an acquired location. Occasionally, a large gland will be found behind the lower pharynx or upper esophagus, but completely free except for its vascular pedicle. Enlarged parathyroid glands along the upper pole of the thyroid gland are infrequent (2%) and are considered as embryologically ectopic because of their fixed position beneath the sheath of fascia covering the thyroid gland (Fig. 4). Even when very large, they may be overlooked because they can be molded around the underlying thyroid tissue, compressed within its sheath, and may be initially considered a portion of the upper pole. However, when the sheath has been incised, these abnormal glands may literally "spring out" of their confined space. These glands should not be considered intrathyroidal even when found compressed into a groove between thyroid lobulations or nodules, if present. In our experience with more than 3500 parathyroid explorations and observations in more than 5000 thyroidectomy specimens, we have found only one truly ectopic intrathyroidal superior gland beneath the true thyroid capsule, on the left side, within the Tubercle of Luckerkandl. As a result, we consider there to be little if any basis in ever performing a thyroid lobectomy for a missing superior parathyroid gland. We a b a n d o n e d this step in our explorations many years ago. During the past decade we came to appreciate that an abnormal superior parathyroid gland could be ectopically located in the region of the pharynx at the level of the pyriform sinus (4). This is the site of the fourth branchial pouch and the embryonic origin of the superior parathyroid gland, as well as a component of thymic tissue (5,8). This location is more than 2 cm cephalad to the upper thyroid pole and is medial to the carotid sheath. We have observed that several of these adenomas were actually covered by pharyngeal muscle, although external to the submucosa of the pharyngeal wall (Fig. 5). We consider these parapharyngeal parathyroid glands to be undescended superior glands (4). They are much rarer than undescended inferior glands, which may be found at the same level of the neck but in a more lateral location within the carotid sheath (9,10). Another extremely rare ectopic site where a superior parathyroid gland may be found is in the neck, but lateral to the carotid sheath. Only a few such cases have been described (11). We have e n c o u n t e r e d only one such case in 3500 patients, which presented as a large right superior gland approximately 4 cm above the

EcToeIc LOCATIONS OF PARATHYROID GLANDS /

501

FIG. 2 (A, B) Magnetic resonance imaging demonstrates a huge (6 x 5 cm) right superior parathyroid adenoma in the posterior mediastinum in a patient with severe hypercalcemia (serum calcium, 16 mg/dl). (C) Operative photo showing the adenoma from the same patient as in A and B. This is a right lateral view with the thyroid lobe retracted medially, causing tension on the inferior thyroid artery and its branch to the adenoma. The cephalic end of the adenoma is emerging from under the inferior thyroid artery. A finger was inserted laterally and posteriorly and with gentle pressure caused the adenoma to be easily delivered out of the posterior mediastinum.

502

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CIJaPTWI~32

FIG. 3 Drawing showing the key maneuver in detecting an occult parathyroid gland in the tracheoesophageal groove. Note that the pretracheal fascia has been incised vertically and the index finger inserted along the prevertebral fascia. This is done whenever a normal superior gland has not been identified in the area around the recurrent laryngeal nerve and inferior thyroid artery junction.

level of the clavicle. This was easily palpable and was initially considered to be a scalene lymph node involved by lymphoma. The patient, a 22-year-old woman, also had marked hypercalcemia and biochemical evidence of primary hyperparathyroidism. The surgical plan was to excise the node for definitive biopsy and to perform a parathyroid exploration. At surgery, three normal parathyroid glands were identified but the right superior gland was not detected after a preliminary exploration. With further dissection, a large arterial branch from the right inferior thyroid artery was noticed, crossing laterally and posterior to the carotid sheath and its contents. With retraction of the entire carotid sheath anteriorly, this artery was traced into the scalene fat pad where it terminated in a 3-cm parathyroid adenoma.

INFERIOR PARATHYROID GLANDS Ectopic inferior parathyroid glands are more comm o n than those arising from superior parathyroid glands if only embryologic factors are considered in their definition (5). Ectopic locations are not caused by mechanical forces influencing the movement of an enlarged gland. Ectopic sites of inferior parathyroid glands are determined during embryogenesis. The

majority (80%) of inferior parathyroid glands migrate with the thymus from branchial pouch III and, after separation, settle in an area within 2 cm of the inferior pole of the thyroid gland (1,3). Approximately 15% of inferior parathyroid glands will remain within the thyrothymic tract or upper pole of the thymic tissue but still within 2 to 4 cm of the inferior thyroid pole. Fewer than 5% of inferior parathyroid glands are located on the anterior or anterior lateral surface of the thyroid gland, most often beneath its sheath. These are all classic locations for normal or abnormal parathyroid glands and are sites that would routinely be explored during parathyroidectomy. Approximately 15% of inferior parathyroid gland adenomas will be found within thymic tissue that descends from the lower thyroid pole as thyrothymic tract (fascia, fat, vessels) or atrophic thymus gland (an encapsulated structure composed primarily of fat in older adults) (1,2,3,5,7). In children and young adults, thymic tissue can usually be identified within a capsule as grayish pink in color and may extend up to the inferior thyroid artery level. Although intrathymic parathyroid glands are considered to be ectopic, the great majority should be readily accessible through a standard cervical incision. Several maneuvers are needed for those more caudal than 4 to 6 cm below the thyroid gland. First, is the identification of the lateral edge of the thymus or tract after reflecting the sternothyroideus muscle laterally at the base of the neck. The fascia lateral to the thymus is then incised to allow finger dissection along and around the thymic horn or cervical thymus. At this point, the thyrothymic tract may be divided, maintaining a clamp on the caudal end to be used for cephalic traction as an index finger is used to gently free the thymus from surrounding tissue as its passes into the mediastinum anterior to the innominate vessels. A right-angle clamp is used to grasp the emerging thymic tissue at intervals as the traction pulls more and more thymic tissue into view (Fig. 6). Care must be taken to observe this tissue for a possible parathyroid adenoma before reapplying a potentially tumor-crushing clamp. Several points need to be emphasized. This dissection must be done gently or the thymus will tear off before much tissue can be mobilized. Nearly all intrathymic adenomas above the level of the aortic arch can be retrieved through a cervical incision. Bleeding is not a problem if this is done gently, even though occasionally the thymic vein may be torn off without its being ligated or clipped. Frequently, this specific vein will be seen posteriorly as the thymus is retracted anteriorly and can be divided between clips. Although we have performed cervical thymectomy in several h u n d r e d patients with primary hyperparathyroidism, in all patients with multiple endocrine neoplasia type 1 (MEN-l) and in all patients with secondary hyperparathyroidism (potential supernumerary glands),

ECTOPIC LOCATIONSOF PARATHYROIDGlaNr)s MEN-1 Hyperparathyroidism

................

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Right inferior gland totally i posterior tO ~I~!!i !i !! right inferior pole ~ ~

503

18 yr. WM Operati0.n 4/7/99

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undescended right superior gland

/

Largest gland est. 4-500 mg

iil !! i~i ~i~i~i~'i~~i~~i~i~~!i! ~~~~i I

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Subtotal parathyroidectomy 34/5 th glands Cervical thymectomy

D . . I 80 mg viable remnant !eft inferior gland with large clip

................... ~.:.~.:~ ....... ............. ...............

FIG. 4

Drawing showing location of right superior gland. This gland was beneath the

sheath of the thyroid but associated with some extrathyroidal thymic tissue that is occa-

sionally seen in children and teenagers. Thymic tissue is rarely found near adult superior parathyroid glands.

we have never encountered any significant hemorrhage. In young patients, in whom the thymus and its capsule may not be atrophic, it may be possible to tease out even deep mediastinal ectopic parathyroid adenomas, thus avoiding a sternal split (Fig. 7). Inferior parathyroid glands may be found entirely within the thyroid capsule in 1-4% of patients with

hyperparathyroidism (1,2,12). Nearly all intrathyroidal glands are within the lower one-third of the thyroid lobe, although some may extend into the middle third of the gland. These totally intrathyroidal abnormal glands can be palpated in only about 50% of cases but can be readily detected by intraoperative ultrasonography. There are several means by which enlarged

504

/

CI4APTW~.32

A Reoperative 1° HPT Sternocleido

Operation March 12, 1992

2.5 cm Right superior gland adenoma partially under pharyngeal muscles

Previous Operation Memphis, Tenn. Left superior excised - normal Left inferior Bx - normal Right inferior Bx - normal Ca++ 12.0 mg/dl Thallium Tc scan + Cephalic to upper lobe right thyroid

Strap muscles

Prevertebral fascia

Lateral approach medial to carotid sheath... between SCM and strap muscles

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Inferior thyroid A.

adenoma in wall of pharynx

B Reoperative Primary HPT Carotid bifurcation

z..d

Right superior adenoma adherent to pharyngeal wall - 3 cm above thyroid level of pyriform sinus

64 yr. WF

0peration Oct. 17, 1996 Undescended right superior gland adenoma 460 mg

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FIG. 5 (A) Drawing showing a patient's large undescended right superior parathyroid adenoma in the wall of the pharynx at the level of the pyriform sinus. This was localized by a thallium scintiscan following a failed exploration. (B) Drawing showing a patient's right superior parathyroid adenoma adherent to the pharynx but not covered by any of its muscle fibers. The reoperation was through a "lateral" approach between the strap muscles and sternocleidomastoid muscles. Localization was achieved by SVS of PTH. (C) Drawing showing undescended left superior parathyroid adenoma at reoperation through a lateral approach. During the first operation, a right superior gland weighing 150 mg was excised and a left superior gland was, in retrospect, mistakenly identified. Localization was by SVS and cervical ultrasound.

E C T O P I C L O C A T I O N S OF P A R A T H Y R O I D G L A N D S

C

/

505

Reoperative Hyperparathyroidism (1°) Operation Jan. 24, 2000 i

:

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Excision

Left superior para adenoma

Left superior adenoma from behind pharynx

Right superior excised at first operation wt. 150 mg

Lateral approach

Localization US and selv. sampling 2000 in Left high jug.

Left inferior parathyroid

Right inferior parathyroid .............~i~~iii~~il ..........

~::::~!iiii~.:::i~i~i~i~i~i:i|il

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Left inferior gland

FIG. 5 (continued)

Noted left superior at first operation NOT seen during procedure #2 :. mistaken identity

intrathyroidal glands can be detected besides preoperatively. The simplest noninvasive means of localization is by cervical ultrasound, which can detect nearly 100% of intrathyroidal parathyroids. Characteristically, these glands are hypoechoic and homogeneous. Specificity is not as high because some thyroid nodules may also have these features. Sestamibi scanning is considerably less sensitive in detection and even less specific because both benign and malignant thyroid neoplasms may concentrate and retain sestamibi. Localization studies are frequently n e e d e d because intrathyroidal parathyroid adenomas are frequently overlooked at initial exploration and require a second operative procedure for their excision. However, with a systematic plan of exploration for the inferior parathyroid gland, the ectopic intrathyroidal parathyroid gland should be detected unless it occurs as a s u p e r n u m e r a r y fifth gland

::

Undescended left superior parathyroid adenoma

or as a second adenoma. W h e n a n o r m a l or abnormal inferior gland has not been detected after a reasonable search in the usual locations, attention should be directed to the lower pole of the thyroid. This is done after an u n d e s c e n d e d or intrathymic gland parathyroid a d e n o m a has been ruled out. At one time, we routinely p e r f o r m e d a vertical thyroidotomy in the lower third of the gland and then enucleated the a d e n o m a when detected. In several cases, however, the capsule of an a d e n o m a was inadvertently incised in p e r f o r m i n g this maneuver. Although to our knowledge this has not resulted in subsequent parathyromatosis, we now prefer to resect the lower third of the thyroid gland. We do not routinely do this p r o c e d u r e when two other normal parathyroid glands are found as well as an adenoma. However, one of our patients did require a reoperation for a second a d e n o m a that was nonpalpable within the

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CHAeTER32 Reoperative 1° HPT 66 yr. WF Operation July 10, 1996

Age 61 Feb. 13, 1991 1st operation Petosky, MI 3 Glands excised 1. 2.3 cm LS. "Adenoma"

Right superi( gland not identi

2. Normal left inferior 3. Normal antimediastinal Right inferior(?) (surgeon's operative note)

2nd operation University of Michigan July 10, 1996

Operation Thyroid isthmus

1. Digital ant. mediastinal exploration 2. Cervical thymectomy 3. Transplantation 50 mg left 2 cm

marked with 3 clips

2 cm "adenoma" intrathymic Left Innominate A.

Thymus

Transplant site 60 mg

...... ~

~

~{}

.

Medial head _ left S.C.M. 3 clips 3 cm

FIG. 6 Drawing based on a surgeon's operative note during the first exploration, during which a left superior adenoma was excised. Reoperative drawing shows the procedure through previous incision. A right angle on the thymus allowed the gland to be teased from the anterior mediastinum. This contained another large adenoma that had been located behind the upper sternum. Regional localization was by SVS of PTH.

lower pole of the thyroid gland. In our experience, this is unlikely to ever happen again. Another important location for an ectopic inferior parathyroid gland is the area immediately around the carotid artery bifurcation within the carotid sheath. These glands are referred to as undescended inferior parathyroid glands and are nearly always associated with some thymic tissue, which may trail caudally to the base of the neck (Fig. 8) (1,3,5,7,9,10). In older adults, this tissue is usually so atrophic that it appears to be only a cord of fatty tissue. Although most undescended inferior ectopic glands are near the carotid bifurcation, rarely a gland may be found at an even higher level along the internal carotid artery behind the submandibular gland or in a more caudal location within the carotid sheath (Fig. 9). We have found hyperplastic glands as low as the base of the neck, where they are usually "molded" around the carotid artery by the carotid sheath and

therefore are not easily palpated or visualized until the sheath has been open (Fig. 10). For practical purposes, an undescended inferior gland cannot be excluded until the carotid sheath has been opened from the level of the clavicle to the internal carotid artery. We have observed that when an ectopic undescended inferior gland is present, there is invariably a trail of either thymic or fatty tissue from the cervical thymus (if present) or mediastinal thymus crossing laterally into the carotid sheath and then cephalically. We refer to this as the "yellow brick road" and when found and followed will lead to a parathyroid gland at its termination (Fig. 8). Only 1-2% of parathyroid adenomas are located in the deep mediastinum, a space defined as any site caudal to the aortic arch (1,2,5,6,13,14). M t h o u g h these adenomas are most commonly within the thymic tissue or adherent to its surface within the anterior mediastinum, an increasing n u m b e r of glands are being

ECTOPIC LOCATIONSOF PARATHYROIDGLANDS /

B

507

36 yr. WF Deep mediastinal parathyroid a d e n o m a excised through cervical incision Cervicomediastimal

thymectomy

!iiii~i~¸~il!!

~,i~ ¸....... Thyrothymic tract Thymus

i~!~

Intrathymic left inferior a d e n o m a

~'

3 x 1.5 cm

Pericardial level .

FIG. 7

(A) Operative photograph during primary operation in a young woman with an intrathymic 2-cm a d e removed from the deep mediastinum by cervical thymectomy. Note the bilateral intact thyrothymic tracts. The adenoma was 12 cm caudal to the manubrium. (B) Surgeon's drawing. noma

reported within the middle mediastinum (15,16). There is good evidence to support the theory that all ectopic glands in the anterior mediastinum represent either inferior parathyroid glands or supernumerary glands derived from branchial pouch III tissue (2,5). Deep anterior mediastinal ectopic inferior glands most commonly receive their blood supply from the internal mammary artery (anterior thoracic) rather than the inferior thyroid artery (Fig. 11) (14). Options for treatment include a traditional median sternotomy and thymectomy, embolization after identification of the adenoma by selective angiography (internal mammary artery), and thorascopic excision after precise anatomic localization with imaging studies (Fig. 12). The embryonic origin of middle mediastinal parathyroid glands has not been clearly established, although there is some evidence to suspect the superior parathyroid gland because of a close relationship of the evolving great vessels and heart and superior parathyroid glands (16). However, ectopic middle mediastinal adenomas are usually not associated with a missing superior gland and are most often classified as supernumerary glands. The two most common locations for middle mediastinal

adenomas are in the aortopulmonary window and behind either main pulmonary artery and anterior to either main stem bronchus or the tracheal carina (6,13,16). The rarest middle mediastinal location is within the pericardium. These adenomas are most commonly localized by sestamibi scanning but have been detected by computerized tomography (CT), magnetic resonance imaging (MRI), and selective arteriography. Although some middle mediastinal adenomas may receive their blood supply from the internal mammary artery, more often it is from a bronchial artery (14). Embolization is not a good option for these tumors because the vasonervosum of the left vagus nerve and left recurrent laryngeal nerve also may be supplied by the same artery. Unless clearly localized in the aortopulmonary window, a left thoracotomy offers the best surgical approach (6,15). Aortopulmonary window adenomas can be excised thoracoscopically or through a sternal split, although this may be difficult. We have excised middle mediastinal tumors through all three of these .approaches and favor a left thoracotomy especially when the patient has had a previous sternal split, which is often the case (Fig. 13).

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Primary Hyperparathyroidism Undescended Right Inferior Parathyroid Adenoma Operation March 20, 1999

Ca bifur Left superior gland

F su g q

lormal left inferior gland

"C

Thyro tn

Following the "yellow brick road" Atrophic thymic fat

B

Reoperative Parathyroidectomy

Right inferior parathyroid 1.4 grams within carotid sheath "Undescended" inferior gland

Distinct thymic t r a c ~ ~ ~ ; i in carotid sheath ....~,~.~,~:.: Vagus nerv

"'

~::i~

Thyrocervic

' ~)

::;i~~':

| 1 t

Recurrent " I~ ~ .~i~ill ~:i:~ ...... laryngeal nerve ...................... , ~ :i .... ~ ...... Rim of thymic tissue ~ ~ .:: ..... along pleura ::::

.............i:::~....

40 yr. BM 14 yr Hx renal dialysis PTH > 1,450 Renal osteodystrophy Operation Aug. 26, 1992

1st operation: Roland Gandy, MD Toledo Ohio July 6, 1992 Excism of left inferior Bx right superior

FIG. 8 (A) Drawing depicting the primary exploration in a patient with an undescended right inferior gland adenoma at the carotid bifurcation. Note the trail of thymic fat within the carotid sheath, which was first seen rising from the thyrothymic tract during the exploration. (B) Drawing showing the undescended right superior gland adenoma during reoperation. Thymic tissue with fat within the carotid sheath led to its detection. Localization was determined to be regional using SVS, which showed an increased PTH level that was elevated in the mid- and high jugular vein samples.

ECTOPIC LOCATIONS OF PARATHYROIDGLANDS /

Reoperation Persistant 1° HPT Parathyroid "adenoma" with surrounding fat...? posterior to jugular veins atrophic thymus ? undescended right inferior gland

65 yr. WF Operation July 8, 1997

360 mgm

Cervical reexploration Lateral approach to level above carotid bifurcation

Venus brar source of 1" I

300 mg adenoma found at level just below angle of jaw...posterior to high jugular vein after opening carotid sheath above level of bifurcation

Approached througt carotid sheath Initial expl. ne

Localization: SVS highest level right superior thyroid V 1300/pg VS peripheral 100 FIG. 9 Drawing showing a very high undescended right inferior gland adenoma at reoperation. A sestamibi scintiscan was negative but the adenoma was posterior to the submandibular gland, which concentrated sestamibi. SVS of PTH regionalized the missing gland to the high neck.

Reoperative Primary Hyperparathyroidism 43 yr. WF !st operation: Feb. 7, 1995 excision left inferior gland within upper thymus left superior- not identified excision right superior gland Right inferior not identified

i~~:III:IIIII~II!I I~I~!:II~II~ i~:......

Thymic ton(, within carotid she~ }Iml

Right inferior parathyroid adenom~

I/ml

115 pg/ml Left mid mominate 125 pg/m! Superic ca~ PTH 150 pg/ml

2nd operation: Dec. 20, 1995 Cervical approach excision of thymus and tongue within carotid sheath right with parathyroid adenoma 250 mg Abnormal descent Right inferior gland N__Qsternal split

~'ised)

FIG. 10 Drawing showing a partially descended right inferior parathyroid gland adenoma at reoperation within grossly apparent thymic tissue that could be seen within the carotid sheath. The adenoma was neither palpable nor visualized until the carotid sheath was opened.

509

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CHAPTER32

FI6. 11 Selective internal mammary arteriogram using a subtraction technique. The arrow points to the adenoma in the anterior mediastinum.

FI6. 12 Chest X-ray of a patient on the day following hyperosmotic dye injection and selective arterial occlusion of the internal mammary arterial branch to a deep mediastinal adenoma. Dye retention as noted here implies necrosis of the adenoma and a good result. This patient's serum calcium returned to normal and remained there during a 3-year follow-up.

ECTOPIC LOCATIONSOF PARATHYROIDGLANDS /

511

Middle Mediastinal Parathyroid Adenoma Left Lateral Thoracotomy

2 previous operations in Leiden, Holland January, 1996 Ligamentum arteriosum ~ f / / a g u s nerve AortopulmonarYwindow"~ ~ Esophagus

~.~

~:ii :i:~2~!~

left nerve

..

,~

,,...

nerve ~ :.~ : i ) ~ ~

I

. ,~;........ Left lung ''II)>;II ..... ;"

Left pulmonary artery Left Lateral View of Mediastinum and Aortopulmonary Windows Divided ligamentum arteriosurn ,\, ~ Vagusnerve ~! .!i>~~ /Left main stem bronchus Anthracotic ,,=,, n o d e s ~ ~

i~ "

Hyperplastic parathyroid pulmonary ~:,~i artery

SUPERNUMERARY PARATHYROID GLANDS In anatomic studies, 5-6% of individuals are found to have a fifth (supernumerary) gland (2). However, in studies specifically searching for small supernumerary glands, the incidence is greater than 10% (3). This has particular clinical relevance in patients with hyperparathyroidism due to multiple gland disease, particularly those with MEN-1 and secondary hyperplasia, in whom growth factors and other stimuli may cause these rudimentary glands, which otherwise might be insignificant, to enlarge and cause persistent or recurrent hyperparathyroidism after a subtotal or total parathyroidectomy. However, occasionally a supernumerary gland may be the only abnormal gland causing primary hyperparathyroidism. Most supernumerary glands are in an ectopic location. The most common site for a supernumerary gland is the cephalic portion of the thymus. (1-3). Because of this fact, cervical thymectomy should be routinely performed in all patients with sec-

FIG. 13 Drawing showing a left lateral thoracotomy approach to a middle mediastinal parathyroid adenoma located posterior to the left pulmonary artery and anterior to left main stem bronchus. This patient had previously undergone both cervical and anterior mediastinal explorations.

ondary hyperparathyroidism and in those with MEN-1 (Fig. 14). A secondary benefit in MEN-1 is the reduction of the possibility of the development of a malignant thymic carcinoid. In our experience, supernumerary glands, as the sole cause of primary hyperparathyroidism, have been most common in the cervical thymus or deep mediastinum. Intrathyroidal supernumerary glands are also a relatively common occurrence. Other rare sites, such as the nasopharynx, have been reported. We have found hyperplastic parathyroid glands within the vagus nerve in two patients (Fig. 15). Of interest is that both were supernumerary, as were the 12 cases of intravagal parathyroid glands reported in the literature (17). It would appear that the only time a surgeon should be concerned about this possible site is after an initial exploration reveals only four normal parathyroid glands. Fortunately, this is a very infrequent occurrence in patients with biochemically proven primary hyperparathyroidism.

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MEN-1 Hyperparathyroidism 55 yr. WF 1 HPT + ZES Bilobed ~ft superior gland Right superior glan(

Operation Feb. 23, 1995 ST parathyroidectomy leaving 60 mg right inferior gland partial thymectomy

Ectopic thryoid tissue Intrathymic right inferior glan~

Left inferior gland

~ernumerary 15th) gland t +

-v ~-

inferior gland

:::....

]i~------- Metal clip on edge of remnant

FIG. 14 Drawing showing supernumerary gland location in the left lobe of the thymus in a patient with the MEN-1 syndrome and hyperparathyroidism. Both inferior glands were also within the cervical thymus. Cervical thymectomy is routinely done in MEN-1 patients because of the 15-20% incidence of supernumerary glands, most of which are within the thymus.

lntra Vagal Parathyroid Adenoma 5th gland (4 normal glands location) Reoperation

43 yr. WM

~i;~~"'::;;:)~:i::,~+(....

:J!~i:!~;

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elsewhere

~i~,~i 4 normal glands Bx right thyroid Iobectomy and excision 2 normal glands

~/:i.+ 1

Operation Oct. 9,1997 .

.

.

.

i :

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~'~: :i,

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1.4 x 1.0 cm u~/:,.~:J One small nerve branch ran within adenoma

,:fll

/l~i,¢: ~

1. Sel. venous sampling 2. Cervical V.S.

FIG. 15 Drawing showing location of an ectopic left intravagal parathyroid adenoma at reoperation in a patient found to have four normal glands at initial exploration. Great care must be taken in dissecting intravagal adenomas from the nerve or ipsilateral (recurrent laryngeal nerve) dysfunction will result.

ECTOPIC LOCATIONS OF PARATHYROID GI~aXl)S

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REFERENCES 1. Thompson NW, Eckhauser FE, Harness JK. The anatomy of primary hyperparathyroidism. Surgery 1982;92:814-822. 2. Gilmour JR. The gross anatomy of the parathyroid glands. J Pathol 1938;46:133-143. 3. Akerstrom G, Malmaeus J, Bergstrom R. Surgical anatomy of human parathyroid glands. Surgery 1984;95:14-21. 4. Simeone DM, Sandelin K, Thompson NW. Undescended superior parathyroid gland: A potential cause of failed cervical exploration for hyperparathyroidism. Surgery 1995:949-956. 5. Gilmour JR. The embryology of the parathyroid glands, the thymus and certain associated rudiments. J Pathol 1937;45:507-519. 6. Bondeson AG, Thompson NW. Mediastinal parathyroid adenomas and carcinomas. In: Shields TW, ed. Mediastinal surgery. Philadelphia:Lea & Febiger, 1991:289-316. 7. Wang CA. Parathyroid re-exploration: A clinical and pathological study of 112 cases. Ann Surg 1977;186:140. 8. Joseph ME Nodol JB, Pilch BZ, Goodman ML. Ectopic parathyroid tissue in the hypopharyngeal mucosa (pyriform sinus). Head Neck Surg 1982;5:70-74. 9. Edis AJ, Purnell DC, van HeerdenJA. The undescended "parathymus"; An occasional cause of failed neck exploration for hyperparathyroidism. Ann Surg 1979;190:64-68.

10. Billingsley, KG, Fraker DL, Doppman JL, et al. Localization and operative management of undescended parathyroid adenomas in patients with persistent primary hyperparathyroidism. Surgery 1994; 116:982-990. 11. Udekwu AO, Kaplan EL, Wu T, Arganini M. Ectopic adenoma of the lateral triangle of the neck: Report of two cases. Surgery 1987;101:114-118. 12. Wheeler MH, Williams ED, Wade JSH. The hyperfunctioning intrathyroidal parathyroid gland: A potential pitfall in parathyroid surgery. WorldJSurg 1987;11:110-114. 13. Shields TW. Mediastinal parathyroids. In: Shields TW, ed. Mediastinal surgery. Philadelphia:Lea & Febiger, 1991:19-22. 14. Doppman JL, et al. The blood supply of mediastinal parathyroid adenomas. Ann Surg 1977;185:488-494. 15. McHenry C, et al. Resection of parathyroid tumors in the aortopulmonary window without prior neck exploration. Surgery 1988;104:1090-1094. 16. Curly I, Wheeler M, Grant C, Thompson NW. The challenge of the middle mediastinal parathyroid. WorldJSurg 1988;12:818-823. 17. Pawlik TM, Richards M, Giordano TJ, Burney RE, Thompson NW. Intravagal parathyroid adenoma. A report of two cases. Arch Surg 2000 accepted.

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CI-IAPTVR 3 3

Parathyroid Carcinoma

ELIZABETH SHANE Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032

INTRODUCTION

National Cancer Data Base reported 286 cases of parathyroid carcinoma, the largest series to date (26). In most series, this entity accounts for less than 1% of patients with primary hyperparathyroidism (1-3,11, 17,27-31). The disease may be somewhat more common in Japan than in Western countries, accounting for 5% of patients with primary hyperparathyroidism (18,32-34), and in an Italian study 5.2% of patients operated on for primary hyperparathyroidism were eventually found to have parathyroid carcinoma (15).

Parathyroid carcinoma is an u n c o m m o n cause of parathyroid hormone-dependent hypercalcemia. The collective published experience with this rare neoplasm has provided a distinctive clinical profile that differs in a n u m b e r of respects from that of benign primary hyperparathyroidism (1-3). The distinguishing features of parathyroid carcinoma assume even greater prominence when viewed within the current context of primary hyperparathyroidism, which commonly presents today as a mild asymptomatic disease (4-8). In this chapter, the clinical features, natural history, and prognosis of parathyroid cancer are reviewed. Surgical approaches to parathyroid cancer are outlined as well as medical therapies of the hypercalcemia that accompanies recurrent or metastatic disease. Because the ultimate prognosis depends to a major extent on successful resection of the tumor at the time of the initial operation, major emphasis is placed on those features of parathyroid carcinoma that help to differentiate it from primary hyperparathyroidism due to benign adenomatous or hyperplastic disease.

ETIOLOGY The etiology of parathyroid cancer is unknown. No clear pattern of predisposing factors has emerged in the cases described thus far. However, there are a number of clinical situations that may predispose to the development of parathyroid carcinoma. Several cases of parathyroid carcinoma have been reported in patients with a history of neck irradiation (35-37). There have also been a n u m b e r of reports of carcinoma occurring within an adenoma or a hyperplastic parathyroid gland (38-45), and there is a report of parathyroid carcinoma occurring in a patient with prolonged secondary hyperparathyroidism secondary to celiac disease (10). Despite these associations, Shantz and Castleman (28), in an extensive review of 70 cases, found no evidence for malignant transformation of previously pathologic tissue.

INCIDENCE Approximately 290 cases of parathyroid carcinoma were reported in the English literature between 1930 and 1992. Since 1992, more than 100 additional cases have been reported (9-25). Moreover, in 1999 the

The Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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End-Stage Renal Disease Parathyroid carcinoma has been described in several patients with end-stage renal disease. Miki et al. (16) reviewed 12 such cases, originally published between 1982 and 1996, of patients with parathyroid carcinoma on maintenance hemodialysis. All demonstrated hyperplasia of other parathyroid glands (16,37,38,46-50) and one had a history of prior neck irradiation (37). The diagnosis was made an average of 6 years after the start of hemodialysis. In all cases, parathyroid carcinoma was diagnosed during or after parathyroidectomy on the basis of local invasion (n = 5), tumor pathology (n = 4), or distant metastases (n = 2). The average age of the patients was 49 years. Only 50% of these patients presented with signs of hypercalcemia; the mean serum calcium level was 10.8 m g / d l with a range of 8.5 to 12.6 m g / d l , considerably lower than serum calcium levels generally observed in patients with parathyroid carcin o m a (see below). Parathyroid h o r m o n e levels were more than twice the u p p e r limit of normal in all patients, not an unusual finding in patients on maintenance hemodialysis. Although the tumor recurred in one-third of the patients, only one died of hypercalcemia due to recurrent disease. The authors of the review concluded that no preoperative features distinguished hemodialysis patients with parathyroid carcin o m a from those with parathyroid hyperplasia and that the clinical course may be more benign because of the tendency for renal insufficiency to lower serum calcium levels.

Familial Hyperparathyroidism Carcinoma has been reported in association with familial hyperparathyroidism (9,51-56), particularly in the autosomal d o m i n a n t form with isolated hyperparathyroidism that is not part of the multiple endocrine neoplasia type 1 (57). In one such family, there was no evidence of antecedent hyperplasia in unaffected glands, and chromosomal abnormalities commonly observed in other solid tumors were identified (a reciprocal translocation between chromosomes 3 and 4, trisomy 7, and a pericentric inversion in chromosome 9) (55). Analyses of tumor DNA from one family m e m b e r with parathyroid carcinoma showed no evidence of ras gene mutations, PTH gene arrangement, or allelic loss from chromosome 11q13, the locus of the gene for multiple endocrine neoplasia type 1. In addition, a greatly increased risk of parathyroid carcinoma is associated with the hereditary hyperparathyroidism-jaw tumor syndrome (52,56,57), recently localized to chromosome lq21-31 (58).

PATHOGENESIS Over the past decade, evidence has accumulated for the involvement of mutations of both protooncogenes and tumor suppressor genes in the development of parathyroid tumors. In this regard, a chromosomal inversion activating one copy of the PRAD1 (parathyroid a d e n o m a l ) gene by placing it next to the regulatory region of the PTH gene has been reported in 5% of parathyroid adenomas (59,60). Cell cycle regulators have also been implicated in the pathogenesis of parathyroid carcinoma. The retinoblastoma (Rb) gene is a tumor-suppressor gene on chromosome 1 lq13. A variety of h u m a n neoplasms are related to inactivation of the Rb gene. In 1994, Cryns et al. studied nine parathyroid cancers for evidence of loss of Rb gene DNA and for altered expression of Rb protein. The cancers were compared to 21 parathyroid adenomas (61). All 11 parathyroid cancers lacked an Rb allele and most had complete absence of nuclear staining for the Rb protein. In contrast, only one parathyroid adenoma lacked the allele and none had abnormal staining for the Rb protein. Subramaniam et al. used a mouse monoclonal antibody to detect Rb gene expression immunohistochemically in three parathyroid carcinomas and 11 benign adenomas. Evidence of Rb gene inactivation was observed in two of the three cancers and only one of 11 adenomas (62). Pearce et al. observed allelic deletions of the 13q12-14 region, involving both the Rb gene and the hereditary breast cancer susceptibility gene in 3 of 19 parathyroid adenomas, all of which had aggressive clinical or histopathologic features, and in 1 of 1 parathyroid cancers. Yoshimoto and colleagues demonstrated allelic deletions on chromosome 13q in parathyroid adenomas from two members of a family with isolated primary hyperparathyroidism, one who also had parathyroid cancer and one with only an adenoma (9). Allelic losses of Rb or D13S71 at 13q14 in a parathyroid cancer were also reported by Dotzenrath (63). In addition, Cryns and colleagues have found evidence for involvement of another cell cycle regulator, the p53 tumor suppressor gene (31). There was both p53 allelic loss and abnormal p53 protein expression in some cases of parathyroid cancer, suggesting a role for p53 in the pathogenesis of some of these tumors. Both wild-type Rb and p53 proteins halt progression through the cell cycle. Abnormal function at this point related to mutations in either gene could therefore predispose to the clonal development of parathyroid carcinoma.

PARATHYROIDCARCINOMA / CLINICAL FEATURES The clinical features of parathyroid carcinoma (1-3,11,12,14,15,17,26-31,34) are due primarily to the effects of excessive secretion of parathyroid hormone by the functioning tumor, rather than to infiltration of vital organs by tumor mass. Thus, signs and symptoms of hypercalcemia often dominate the clinical picture with contributions from typical hyperparathyroid bone disease and features of renal involvement, such as nephrolithiasis or nephrocalcinosis. The challenge to the clinician rests on differentiating between hyperparathyroidism due to parathyroid carcinoma and that due to its much more common benign counterpart. It is of great importance that parathyroid carcinoma be considered in the differential diagnosis of parathyroid hormone-dependent hypercalcemia, because the morbidity and mortality associated with this diagnosis are substantial and optimal outcomes are associated with complete resection of the tumor at the time of the initial operation (1-3,11,12,14,15,17,26-31,34,64). All too often the diagnosis of parathyroid carcinoma is made in retrospect when hypercalcemia recurs due to local spread of tumor or distant metastases. Several presenting features of a patient with primary hyperparathyroidism, when present, should suggest a malignant rather than a benign etiology. There is no association of gender with parathyroid carcinoma. The ratio of affected women to men is 1:1 in most series, compared to primary hyperparathyroidism, where there is a marked female predominance (3-4:1). Most investigators have noted the average age of the patient with parathyroid carcinoma to be in the fifth decade, approximately 10 years younger than typical patients with primary hyperparathyroidism, who most often present in their riffles or sixties. In contrast, a review of the Mayo Clinic experience (30) and that of the National Cancer Data Base indicated that the average age of their patients was somewhat greater (26), in the middle fifties. In any case, considerations of gender and age are of little help in evaluating the individual patient. Since the advent of the multichannel autoanalyzer in the late 1960s, the clinical profile of primary hyperparathyroidism due to benign adenomatous or hyperplastic disease has changed. Today, primary hyperparathyroidism usually presents with mild hypercalcemia (within 1 m g / d l above the upper limit of normal) that is frequently asymptomatic and often discovered during a routine evaluation or during the investigation of an unrelated complaint (4-6). In contrast, the serum calcium level of most patients with parathyroid carcinoma is much higher, generally above 14 mg/dl, or 3-4 m g / d l above the upper limit of

517

normal (1-3,11,12,14,15,17,26-31,34). Moreover, this more severe hypercalcemia is almost invariably associated with the typical signs and symptoms of hypercalcemia. The most frequent complaints are fatigue, weakness, weight loss, anorexia, nausea, vomiting, polyuria, and polydipsia. Other common presenting symptoms, characteristic of a severely hyperparathyroid state, include bone pain, fractures, and renal colic. When reported, parathyroid hormone levels have ranged from 3 to 10 times above the upper limit of normal for the assay employed. Extremely high levels of parathyroid h o r m o n e are unusual in primary hyperparathyroidism, for which circulating concentrations are commonly less than twice normal. Alkaline phosphatase is also higher in patients with parathyroid carcinoma than in those with primary hyperparathyroidism, for which levels are generally in the vicinity of the upper limit of the normal range (7). Patients with parathyroid carcinoma may have elevated levels of ot and [3 subunits of human chorionic gonadotrophin whereas patients with primary hyperparathyroidism do not (65). A palpable neck mass has been reported in from 30 to 76% of patients with parathyroid carcinoma. This important clinical finding constitutes another striking difference between benign and malignant parathyroid disease, because a palpable neck mass is distinctly unusual in primary hyperparathyroidism (66). In addition, recurrent laryngeal nerve palsy in a patient with primary hyperparathyroidism who has not had previous neck surgery is also very suggestive of parathyroid cancer. The classic target organs of parathyroid hormone, kidney and skeleton, are affected with greater frequency and severity in parathyroid carcinoma (1,2,11,12,27,30,32,33) than is commonly observed in the modern presentation of benign primary hyperparathyroidism. Most recent series of primary hyperparathyroidism report the prevalence of renal involvement, including nephrolithiasis, nephrocalcinosis, and impaired glomerular filtration, to be less than 20% (4,7). In contrast, renal colic is a frequent presenting complaint of parathyroid carcinoma. The prevalence of nephrolithiasis was 56% and the prevalence of renal insufficiency was 84% in one recent series (30). These figures are somewhat higher than previous reports in which the prevalence of renal involvement generally has ranged from 32 to 60%. Bone pain and pathologic fractures are also common features of parathyroid cancer. Overt radiologic signs of hyperparathyroid skeletal disease, such as osteitis fibrosa cystica, subperiosteal bone resorption, "salt and pepper" skull, and absent lamina dura, as well as less specific signs such as diffuse spinal osteopenia, are

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commonly seen in parathyroid carcinoma (44-91%). In contrast, patients with benign primary hyperparathyroidism rarely have skeletal complaints and specific radiologic signs are found in less than 5% (4,5,7). It is also important to note the high incidence of concomitant bone and stone disease that occurs in parathyroid cancer, whereas simultaneous renal and overt skeletal involvement is distinctly unusual in primary hyperparathyroidism. In addition to the kidneys and the skeleton, other organs are frequently affected. Recurrent severe pancreatitis, peptic ulcer disease, and anemia occur with greater frequency in patients with malignant disease than in those with benign primary hyperparathyroidism. Parathyroid carcinoma shares many clinical features with acute primary hyperparathyroidism (parathyroid crisis; see Chapter 34). In view of the marked elevations of serum calcium and parathyroid h o r m o n e that are c o m m o n in parathyroid crisis, the diagnosis of parathyroid cancer should be considered. T h o u g h the distinction between these two entities is not possible preoperatively, it is important to bear the diagnosis in mind because the surgical approach differs. A summary of features that might lead one to suspect parathyroid cancer in a patient with hypercalcemia and elevated parathyroid h o r m o n e levels is shown in Table 1. It should be noted however, that some patients with benign primary hyperparathyroidism present with more severe disease than is commonly seen today. In such patients, the distinction between benign and malignant disease may be even more difficult on clinical grounds because profound hypercalcemia, renal disease, and osteitis fibrosa or diffuse osteoporosis may occur and even concomitant kidney and bone disease

TABLE 1

may be present (64). However, it is preferable to have a high index of suspicion for parathyroid carcinoma when these features are present than to miss the opportunity for surgical cure by failing to consider it in the differential diagnosis.

PATHOLOGY Several operative findings have been described; when present, these help to distinguish benign parathyroid adenomas from parathyroid carcinoma. The typical parathyroid a d e n o m a is usually of soft consistency, r o u n d or oval in shape, and of a reddish-brown color. In contrast, parathyroid carcinoma is frequently described as a lobulated, firm to stony-hard mass. In about 50% of cases it is surrounded by a dense fibrous greyish-white capsule that adheres tenaciously to adjacent tissues and makes the tumor difficult to separate from contiguous structures. If there is gross infiltration of adjacent thyroid, nerve, muscle, or esophagus or obvious cervical node metastases, the diagnosis of carcinoma is not difficult. However, any one or all of these operative findings may be absent and the examination of frozen sections is of little value in distinguishing benign from malignant disease. As is the case with many endocrine neoplasms, the histopathologic distinction between benign and malignant parathyroid tumors is difficult. In 1973 Shantz and Castleman, based on an analysis of 70 cases of parathyroid carcinoma, established a set of criteria for the pathologic diagnosis of this malignancy (28). These histologic features are (1) uniform sheets of (usually chief) cells arranged in a lobular pattern separated by

Parathyroid Carcinoma and Benign Primary Hyperparathyroidism" Typical Features

Feature

Parathyroid carcinoma

Female:male ratio Average age (years) Asymptomatic Serum calcium

1:1 48 <5% >14 mg/dl

Parathyroid hormone Palpable neck mass Renal involvement a Skeletal involvement b Concomitant renal and skeletal disease

Markedly elevated Common 32-80% 34-91% Common

Primary hyperparathyroidism 3.5:1 55 >80% _< 1 mg/dl above upper limit of normal Mildly elevated Rare 4-18% <5% Rare

alncludes nephrolithiasis, nephrocalcinosis, and impaired renal fuction in absence of any other etiology. blncludes osteitis fibrosa, subperiosteal resorption, "salt and pepper" skull, and diffuse osteopenia on plain radiographs.

PARATHYROIDCARCINOMA / dense fibrous trabeculae, (2) capsular or vascular invasion, and (3) mitotic figures within tumor parenchymal cells, which must be distinguished from endothelial cell mitoses. Unfortunately, none of these features is pathognomonic of parathyroid carcinoma. Several featuresmnamely, dense fibrous trabeculae, trabecular growth pattern, mitoses, and capsular invasion--have been found in parathyroid adenomas (66). Capsular and vascular invasion appear to correlate best with subsequent tumor recurrence (see Chapter 1). Several other histologic techniques have been investigated in order to improve further the accuracy of diagnosing parathyroid carcinoma. Electron microscopy of parathyroid cancer tissue reveals nuclear and mitochondrial alterations and evidence of increased secretory activity but does not appear to be of value in distinguishing benign from malignant tumors (67-69). Nuclear diameter appears to be greater in parathyroid carcinomas than in adenomas (28,67-71), but this index is not very useful in the individual case. Measurement of nuclear deoxyribonucleic acid content by flow cytometry may be of some value both in establishing the diagnosis of parathyroid carcinoma and in predicting the invasive potential of the tumor. Mean nuclear DNA content is greater and an aneuploid DNA pattern is more common in parathyroid carcinoma than in adenomas; when present, aneuploidy appears to be associated with a poorer prognosis (33,64,72). Unfortunately, however, aneuploidy occurs too frequently in parathyroid adenomas to be of great use in differentiating benign from malignant parathyroid lesions (34,72,73). Some experts believe that the overall histologic pattern is more useful than any single feature in the differentiation of parathyroid carcinoma from benign disease, and the presence of more than one in a lesion should raise the index of suspicion (32,34). Bondeson and colleagues consider that cellular atypia, including nuclear pleomorphism and enlargement, and macronucleoli are associated with greater likelihood of malignancy (74). Immunohistochemical staining of Rb protein may also prove useful in distinguishing benign from malignant parathyroid tumors. Cryn and colleagues were the first to report that staining for Rb protein with polyclonal antibodies was commonly absent in parathyroid carcinomas and almost always present in parathyroid adenomas (61). Other investigators have corroborated their results (62). In contrast, Farnebo et al., who used monoclonal antibodies directed against the Rb protein, did not find immunostaining of Rb protein to be useful in distinguishing between benign and malignant parathyroid tumors (75). However, these investigators did observe a trend for more intense immunostaining in parathyroid cancers of the cell cycle-associated anti-

519

gen Ki-67, a marker for proliferative activity. In their bands, the technique was not useful for distinguishing carcinomas from adenomas. (75), although Abbona et al., however, did find significant differences in Ki-67 staining between benign and malignant parathyroid disease (76). Further studies are necessary to assess the value of immunohistochemical stains for the differential diagnosis of parathyroid lesions. Invasive growth of various neoplasms may be facilitated by tumoral secretion of proteolytic enzymes. One such enzyme is gelatinase A. Farnebo et al. have reported that gelatinase A mRNA was detected in 14 of 18 unequivocal and in 4 of 13 equivocal parathyroid cancers (77). The strongest signal was detected in the fibroblasts and macrophages at the tumor border, rather than in the tumor cells. This new technique may provide additional support for the diagnosis of malignancy.

NATURAL HISTORY Parathyroid carcinoma is an indolent, albeit tenacious, tumor with rather low malignant potential. It tends to recur locally at the operative site and spread to contiguous structures in the neck. Metastases occur late in the course of the disease and spread via both lymphatic and hematogenous routes. Cervical nodes (30%) and lung (40%) are involved most commonly, followed by liver (10%). Occasional involvement of bone, pleura, pericardium, and pancreas has been reported.

MANAGEMENT

Surgery The single most effective therapy for parathyroid carcinoma is complete resection of the primary lesion at the time of the initial operation, when extensive local invasion and distant metastases are less likely (1,18,27,29,34,66). For this reason both preoperative suspicion and intraoperative recognition are of param o u n t importance. Patients whose clinical presentation is suggestive of parathyroid carcinoma warrant thorough exploration of all four parathyroid glands, because parathyroid carcinoma has been reported to coexist with benign adenomas or hyperplasia (10,78). When the gross pathologic findings suggest malignancy, the following steps should be taken: en bloc removal of the lesion together with the ipsilateral thyroid lobe and isthmus, and skeletonization of the trachea and removal of any contiguous tissues to which the tumor adheres. Great care must be exercised to avoid rupture of the capsule of the gland, which increases the likelihood of local seeding of the tumor.

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If the recurrent laryngeal nerve is involved with tumor, it must be resected. Tracheoesophageal, paratracheal, and u p p e r mediastinal lymph nodes should be excised, but an extensive lateral neck dissection is indicated only when there is spread to the anterior cervical nodes. The situation becomes more complex when the diagnosis is made in the early postoperative period on the basis of pathology. This is particularly so in view of the controversy that exists regarding the histopathologic diagnosis of parathyroid carcinoma. If the gross characteristics of the lesion were typical of a parathyroid cancer, and if the subsequent pathology appears to be aggressive with extensive vascular or capsular invasion or if the patient remains hypercalcemic, reexploration of the neck is indicated. The structures adjacent to the tumor site should be resected in the m a n n e r described above. If none of these features is present, but the diagnosis is made on the basis of the microscopic characteristics, immediate reoperation may not be necessary because a simple complete resection of the tumor is often curative. Such a patient must be observed carefully, with frequent measurements of parathyroid h o r m o n e and serum calcium levels. The postoperative m a n a g e m e n t of a patient with parathyroid cancer must include careful attention to the serum calcium level. Because calcium and phosphorus are deposited into the skeleton, symptomatic hypocalcemia ("hungry bone syndrome") may ensue and should be regarded as a sign that the surgery has been successful. The hypocalcemia may be severe and protracted, requiring large doses of intravenous calcium. Sufficient supplemental calcium and calcitriol should be prescribed to maintain the serum calcium at the low end of the normal range. As the bones heal and the remaining parathyroid glands recover, the requirem e n t for calcium will decrease, permitting gradual reduction of the doses of calcium and calcitriol. After this point serum calcium and parathyroid h o r m o n e levels should be monitored every 3 months. The m a n a g e m e n t of recurrent or metastatic parathyroid carcinoma reflects the rather indolent biology of this cancer, and in contrast to many other tumors, is primarily surgical (1,11,15,18,19,26,29,30,33,79). Because even very small tumor deposits may produce sufficient PTH to cause severe hypercalcemia, significant palliation may result from resection of lesions in the neck, lymph nodes, lungs, or liver (18). Many situations have been described in which resection of such lesions has resulted in periods of normocalcemia that range from months to years. Even surgery is only palliative; amelioration of the hypercalcemia may also result making its control more amenable to medical therapies. In patients with recurrent hypercalcemia, localization studies should be p e r f o r m e d prior to reoperation.

Careful palpation of the neck should be performed, because recurrence occurs earliest and most often at the original site and such tumors are frequently palpable. Thallium-201technetium-99m scanning is useful in locating tumors in the neck and u p p e r mediastinum (80-82). Technetium-99msestamibi used concurrently with a hand-held, gamma-detecting probe may also prove to be useful for the intraoperative localization of abnormal parathyroid tissue (22). Thallium-201 is also helpful for situations in which the thyroid has been partially or completely resected or when pulmonary metastases are suspected. Computerized tomography and magnetic resonance imaging are useful adjuncts to ultrasonography in evaluation of the neck and are superior for detection of distant metastases in the chest or abdomen. If noninvasive testing does not yield results, arteriography or selective venous catheterization may be useful. Fine needle aspiration biopsy in conjunction with ultrasonic localization and immunoperoxidase confirmation may be useful in localizing parathyroid tissue in patients with recurrent hyperparathyroidism (83). However, biopsy must be used with caution, if at all, in parathyroid carcinoma to avoid seeding the needle track with deposits of malignant tissue. Recurrent carcinoma in the neck should be treated with wide excision of the involved area, including the regional lymph nodes and other involved structures. Accessible distant metastases should also be resected when possible.

Radiation Therapy Parathyroid carcinoma is not a radiosensitive tumor. The use of radiation therapy to control tumor growth and decrease h o r m o n e production has been ineffective in the majority of cases in which it has been attempted (3,28). In the occasional situation, radiation to the neck after surgery for recurrence may be helpful in preventing tumor regrowth (23,66). An apparent cure (10 years) of locally invasive parathyroid carcinoma by radiation therapy was reported by Wynne and colleagues (30). In addition, six patients who received adjuvant radiation therapy for microscopic residual disease have been followed for 12 to 156 months without recurrence (23).

Chemotherapy Because of the rarity of parathyroid carcinoma, few investigators have sufficient numbers of patients to permit large-scale clinical research trials. Thus complete investigations of the utility of a given therapy do not exist and experience is usually limited to scattered case reports. It is with these unavoidable limitations in mind that the following comments should be interpreted. Attempts to control tumor b u r d e n with chemotherapy have been disappointing. Several regimens (nitro-

PARATHYROIDCARCINOMA / gen mustard; vincristine, cyclophosphamide, and actinomycin D; Adriamycin, cyclophosphamide, and 5-fluorouracil; and Adriamycin alone) have been ineffective (78,84,85). Two patients have been treated with synthetic estrogens with some success (86,87). A single patient with pulmonary metastases responded to treatm e n t with dacarbazine, 5-fluorouracil, and cyclophosphamide with a decrease in PTH and normalization of serum calcium for 13 months (88). Another patient responded to dacarbazine alone with a brief but significant decline in her serum calcium level (89). An 18m o n t h remission with regression of a mediastinal mass and pleural effusion was induced in a patient with a nonfunctioning parathyroid carcinoma by a regimen consisting of methotrexate, doxorubicin, cyclophosphamide, and lomustine (90). Such approaches warrant further investigation.

Management of Hypercalcemia When parathyroid carcinoma has become widely disseminated and surgical resection is no longer effective, the prognosis is poor. However, even at this j u n c t u r e relatively prolonged survival is possible. The therapeutic goal at this point is to control the hypercalcemia, which, because of the extremely elevated PTH levels and the intensity of the associated bone resorption, may be a difficult and frustrating task. The acute hypercalcemia of parathyroid carcinoma is treated in the same way as hypercalcemia due to any other cause (see Chapter 45) (91,92). Management includes infusion of saline to restore fluid volume and enhance urinary calcium excretion and loop diuretics to further increase calciuresis. Such measures rarely suffice, however, and addition of agents that interfere with osteoclast-mediated bone resorption is always necessary.

Bisphosphonates The bisphosphonates are a group of drugs that inhibit osteoclast-mediated bone resorption. Several of these drugs have shown some promise in the therapy of parathyroid carcinoma. Dichloromethylene diphosphonate (clodronate, ClzMDP) lowers serum calcium in parathyroid carcinoma when administered intravenously (93-95). It is widely available in Europe and the United Kingdom, but it is not available in the United States. Etidronate has also been shown to lower serum calcium transiently in parathyroid cancer patients (96). It is administered intravenously over a 2h o u r period at a dose of 7.5 m g / k g and may be repeated daily or until the serum calcium falls to normal for a m a x i m u m of 7 days. Although the drug is available in an oral form, it is not effective in patients

521

with parathyroid carcinoma and even the intravenous preparation may not normalize the serum calcium. A more potent bisphosphonate, pamidronate, is now widely available for intravenous use. When infused for periods ranging from 2 to 24 hours and in doses ranging from 45 to 90 mg per day, pamidronate has been at least transiently effective in lowering serum calcium levels in several patients with parathyroid cancer (18,20,21,97-99). New and more potent bisphosphonates (ibandronate, zoledronate) are being investigated actively in the United States and soon may become available for the treatment of hypercalcemia of malignancy, including that due to parathyroid cancer.

Plicamycin Plicamycin (mithramycin), another specific inhibitor of bone resorption, lowers serum calcium levels in parathyroid carcinoma (100). It is administered intravenously at a dose of 25 p~g/kg over 4 to 8 hours and may be repeated at daily intervals for up to 7 days until the serum calcium falls into an acceptable range (91,92). Unfortunately, complete normalization of the serum calcium is often not achieved and the effectiveness of the drug is not only transient but diminishes with repeated courses. Conversely, the toxic effects of plicamycin on the liver, kidneys, and bone marrow increase with the n u m b e r of exposures. Plicamycin therapy should be reserved for therapy of life-threatening hypercalcemia, unresponsive to intravenous bisphosphonates, while surgically accessible metastases are sought or for those patients whose hypercalcemia can be controlled in no other way.

Calcitonin This agent both inhibits osteoclast-mediated bone resorption and increases urinary calcium excretion. However, it lowers serum calcium transiently, if at all, in most patients with parathyroid carcinoma (1,88, 93,101-103). It has been effective in a single patient when used in doses of 200"-600 MRC units per day in combination with glucocorticoids (300 mg of hydrocortisone) (104) and in a few patients when used alone (105).

Gallium Gallium nitrate appears to inhibit bone resorption by preventing dissolution of hydroxylapatite crystals (106). Gallium nitrate lowered serum calcium in two patients with parathyroid carcinoma (106) and was later reported to be effective in four of five patients (19). It is administered as a continuous 5-day infusion at a dose of 200 mg/m2/day. Significant toxicities

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include elevation of the serum creatinine that is potentiated by volume depletion and the concomitant use of potentially nephrotoxic drugs. It remains unclear whether gallium nitrate will prove useful in the management of chronic hypercalcemia due to parathyroid cancer. Calcimimetics

U n d e r normal circumstances, parathyroid h o r m o n e secretion is mediated by a cell surface calcium-sensing receptor and this regulatory response is generally retained in benign parathyroid tumors. An allosteric modulator of the calcium receptor with calcimimetic properties has been shown to lower serum parathyroid h o r m o n e and calcium concentrations in patients with primary hyperparathyroidism (107). This same agent was used to treat a patient with parathyroid carcinoma. Serum calcium was controlled for 2 years without adverse effects (24). Such agents show promise in the m a n a g e m e n t of parathyroid cancer. WR-2721

WR-2721 [ S-2-(3-aminopropylamino) ethylphosphorothioic acid], is a hypocalcemic agent that acts by inhibiting PTH secretion and bone resorption. It has been shown to lower PTH levels and serum calcium levels in parathyroid carcinoma (108). Severe toxicities limit its use (see Chapter 29). Octreotide

The long-acting somatostatin analog, octreotide, has also been reported to inhibit parathyroid horm o n e secretion in woman with parathyroid carcinoma metastatic to bone (109). Immunization

Finally, a novel approach to therapy of hypercalcemia due to parathyroid cancer has been published in case report form (25). A patient with parathyroid carcinoma metastatic to lungs and pleura had severe hypercalcemia that was resistant to oral clodronate, intravenous pamidronate, octreotide, 5-fluorouracil, and streptozotocin. She was immunized with h u m a n and bovine PTH peptides, followed with booster doses at 4 and 11 weeks. Antibodies against PTH were detected at 4 weeks. Before therapy, serum calcium varied between 3.5 and 4.2 mmol/liter. Serum calcium levels remained significantly lower (2.5 to 3.0 mmol/liter) t h r o u g h o u t the 6 months of observation. There was rapid improvement in her clinical condition and no significant adverse effects were observed. If confirmed,

this would seem to be a novel and relatively simple approach to the control of hypercalcemia in patients resistant to other measures.

PROGNOSIS The prognosis of parathyroid carcinoma is quite variable. No one characteristic correlates predictably with outcome. Early recognition and complete resection at the time of the initial surgery carries the best prognosis. The average time between surgery and the first recurrence is approximately 3 years, although intervals of up to 20 years have been reported. Once the tumor has recurred, complete cure is unlikely although prolonged survival is still c o m m o n u n d e r these circumstances with palliative surgery. Survival rates of 5 years vary from 40 to 86%. The National Cancer Data Base survey has reported 10-year survival to be approximately 49% (26).

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57. Wassif WS, Moniz CE Friedman E, Wong S, Weber G, Nordenskjold M, Peters TJ, Larsson C. Familial isolated hyperparathyroidism: A distinct genetic entity with an increased risk of parathyroid cancer. J Clin Endocrinol Metab 1993;77 (6) :1485-1489. 58. Szabo J, Heath B, Hill VM, Jackson CE, Zarbo RJ, Mallette LE, Chew SL, Besser GM, Thakker RV, Huff V, et al. Hereditary hyperparathyroidism-jaw tumor syndrome: The endocrine tumor gene HRPT2 maps to chromosome 1q21-q31. AmJHum Genet 1995;56(4):944-950. 59. Arnold A, Kim HG, Gaz RD, Eddy RL, Fukushima Y, Byers MG, Shows TB, Kronenberg HM. Molecular cloning and chromosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. J Clin Invest 1989;83(6):2034-2040. 60. Arnold A. Genetic basis' of endocrine disease 5. Molecular genetics of parathyroid gland neoplasia [published erratum appears in J Clin Endocrinol Metab 1994;Feb;78(2):492-493]. J Clin Endocrinol Metab 1993;77(5):1108-1112. 61. Cryns VL, Thor A, Xu HJ, Hu SX, Wierman ME, Vickery AL, Jr, Benedict WF, Arnold A. Loss of the retinoblastoma tumor-suppressor gene in parathyroid carcinoma [see comments]. NEngl JMed 1994;330(11):757-761. 62. Subramaniam P, Wilkinson S, Shepherd JJ. Inactivation of retinoblastoma gene in malignant parathyroid growths: A candidate genetic trigger? Aust N ZJSurg 1995;65(10):714-716. 63. Dotzenrath C, Teh BT, Farnebo F, Cupisti K, Svensson A, Toell A, Goretzki P, Larsson C. Allelic loss of the retinoblastoma tumor suppressor gene: A marker for aggressive parathyroid tumors? J Clin Endocrinol Metab 1996;81 (9):3194-3196. 64. Levin K, Chew K, Ljung B, Mayall B, Siperstein A, Clark O. Deoxyribonucleic acid flow cytometry helps identify parathyroid carcinomas. J Clin Endocrinol Metab 1988;67:779-784. 65. Stock J, Weintraub B, Rosen S, Aurbach G, Spiegel A, Marx S. Human chorionic gonadotropin subunit measurement in primary hyperparathyroidism. J C l i n Endocrinol Metab 1982;54:57-63. 66. Levin K, Galante M, Clark O. Parathyroid carcinoma versus parathyroid adenoma in patients with profound hypercalcemia. Surgery 1987;101:647-660. 67. de la Garza S, de la Garza E, Batres E Functional parathyroid carcinoma: Cytology, histology and ultrastructure of a case. Diag Cytopathol 1985;1:232. 68. Holck S, Pedersen N. Carcinoma of the parathyroid gland. A light and electron microscopic study. Acta Pathol Microbiol Scand 1981;89:297-302. 69. Smith J, Coombs R. Histological diagnosis of carcinoma of the parathyroid gland. J Clin Pathol 1984;37:1370-1378. 70. Jacobi J, Lloyd H, Smith J. Nuclear diameter in parathyroid carcinomas. J Clin Pathol 1986;39:1353-1354. 71. Lloyd H, Jacobi J, Cooke R. Nuclear diameter in parathyroid ademomas. J Clin Patho11979;32:1278-1281. 72. Sandelin K, Auer G, Bondeson L, Grimelius L, Farnebo LO. Prognostic factors in parathyroid cancer. A review of 95 cases. WorldJ Surg 1992;16(4):724-731. 73. Mallette LE. DNA quantitation in the study of parathyroid lesions. A review. AmJ Clin Patho11992;98(3):305-311. 74. Bondeson L, Sandelin K, Grimelius L. Histopathological variables and DNA cytometry in parathyroid carcinoma. A m J Surg Patho11993;17 (8 ) :820-829. 75. Farnebo E Auer G, Farnebo LO, Teh BT, Twigg S, Aspenblad U, Thompson NW, Grimelius L, Larsson C, Sandelin K. Evaluation of retinoblastoma and Ki-67 immunostaining as diagnostic markers of benign and malignant parathyroid disease. WorldJ Surg 1999;23(1 ):68-74. 76. Abbona GC, Papotti M, Gasparri G, Bussolati G. Proliferative activity in parathyroid tumors as detected by Ki-67 immunostaining. Hum Patho11995;26(2):135-138.

77. Farnebo F, Svensson A, Thompson NW, Backdahl M, Grimelius L, Larsson C, Farnebo LO, Sandelin K. Expression of matrix metalloproteinase gelatinase A messenger ribonucleic acid in parathyroid carcinomas. Surgery 1999; 126 (6) :1183-1187. 78. Anderson B, Samaan N, Vassilopoulou-Sellin R, Ordonez N, Hickey R. Parathyroid carcinoma: Features and difficulties in diagnosis and difficulties in diagnosis and management. Surgery 1983;94:906-915. 79. Sandelin K. Parathyroid carcinoma. Cancer Treat Res 1997;89:183-192. 80. Fujimoto Y, Obara T, Ito Y, Kodama T, Nobori M, Ebihara S. Localization and surgical resection of metastatic parathyroid carcinoma. WorldJ Surg 1986;10:539-547. 81. Johnston LB, Carroll MJ, Britton KE, Lowe DG, Shand W, Besser GM, Grossman AB. The accuracy of parathyroid gland localization in primary hyperparathyroidism using sestamibi radionuclide imaging. J Clin Endocrinol Metab 1996;81:346-352. 82. Obara T, Fujimoto Y, Tanaka R, Ito Y, Kodama T, Yashiro T, Kanaji Y, Yamashita T, Fukuuchi A. Mid-mediastinal parathyroid lesions: Preoperative localization and surgical approach in two cases. Jpn J Surg 1990;20:481-486. 83. Sardi A, Bolton JS, Mitchell WT, Jr, Merritt CR. Immunoperoxidase confirmation of ultrasonically guided fine needle aspirates in patients with recurrent hyperparathyroidism. Surg Gynecol Obstet 1992;175:563-568. 84. Golden A, Canary J, Kerwin D. Concurrence of hyperplasia and neoplasia of the prathyroid gland. Am J Med 1965;38:562-578. 85. Grammes C, Eyerly R. Hyperparathyroidism and parathyroid carcinoma. South Med J 1980;73:814-816. 86. Sigurdsson G, Woodhouse N, Taylor S, Joplin G. Stilboestrol diphosphate in hypercalcemia due to parathyroid carcinoma. Br MedJ 1973;1:27-28. 87. Goepfert H, Smart C, Rochlin D. Metastatic parathyroid carcinoma and hormonal chemotherapy; case report and response to Hexestrol. Ann Surg 1966;164:917-918. 88. Bukowski R, Sheeler L, Cunningham J, Esselstyn C. Successful combination chemotherapy for metastatic parathyroid carcinoma. Arch Intern Med 1984;144:399-400. 89. Calandra D, Chejfec G, Foy B, Lawrence A, Paloyan E. Parathyroid carcinoma: Biochemical and pathologic response to DTIC. Surgery 1984;96:1132-1137. 90. Chahinian A, Holland J, Nieburgs H, Marinescu A, Geller S, Kirschner E Metastatic nonfunctioning parathyroid carcinoma: Ultrastructural evidence of secretory granules and response to chemotherapy. Am J Med Sci 1981 ;282:80-84. 91. Bilezikian J. Management of acute hypercalcemia. NEnglJMed 1992;326:1196-1203. 92. Shane E. Hypercalcemia: Pathogenesis, clinical manifestations, differential diagnosis and management. In: Favus M, ed. ASBMR primer on metabolic bone disease, 4th Ed. Philadelphia:LippincottRaven, 1999:183-186. 93. Jungst D. Disodium clodronate is effective in management of severe hypercalcemia caused by parathyroid carcinoma. Lancet 1984;1:1043. 94. Jacobs T, Siris E, Bilezikian J, Baquiran D, Shane E, Canfield R. Hypercalcemia of malignancy: Treatment with intravenous dichloromethylene diphosphonate. Ann intern Med 1981;94:312-316. 95. Shane E, Jacobs T, Siris E, Steinberg S, Stoddart K, Canfield R, Bilezikian J. Therapy of hypercalcemia due to parathyroid carcinoma with intravenous dichloromethylene diphosphonate. AmJ Med 1982;72:939-944. 96. Jacobs T, Gordon A, Gundberg C, Silverberg S, Shane E, Reich L, Clemens T. Neoplastic hypercalcemia: Physiologic response to intravenous etidronate. AmJMed 1987;82:42-52.

PARATHYROID CARCINOMA 97. Mann K. Oral bisphosphonate therapy in metastatic parathyroid carcinoma. Lancet 1985; 1:101-102. 98. Sandelin K, Thompson N, Bondeson L. Metastatic parathyroid carcinoma: Dilemmas in management. Surgery 1992;110:978-988. 99. Weinstein R. Parathyroid carcinoma associated with polycythemia vera. Bone 1991;12:237-239. 100. Singer FR, Neer RM, Murray TM, Keutmann HT, Deftos L, Potts JT, Jr. Mithramycin treatment of intractable hypercalcemia due to parathyroid carcinoma. N Engl J Med 1970;283: 634-636. 101. Dubost C, Jehanno C, Lavergne A, Charpentier Y. Successful resection of intrathoracic metastases from two patients with parathyroid carcinoma. WorldJ Surg 1984;8:547-551. 102. Lake M, Kahn S, Favus M, Bermes E. Case report: Clinical pathological correlations in a case of primary parathyroid carcinoma. Ann Clin Lab Sci 1984;14:458-463. 103. Trigonis C, Cedermark B, Willems J, Hamberger B, Granberg P. Parathyroid carcinomamProblems in diagnosis and treatment. Clin Onco11984;10:11-19. 104. Au W. Calcitonin treatment of hypercalcemia due to parathyroid carcinoma: Synergistic effect of prednisone on long-

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107.

108.

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term treatment of hypercalcemia. Arch Intern Med 1975;135: 1594-1597. Edelson G, Kleerekoper M, Talpos G, Zarbo R, Saeed-Uz-Zafar M. Mucin-producing parathyroid carcinoma. Bone 1992;13:7-10. Warrell R, Isaacs M, Alcock N, Bockman R. Gallium nitrate for treatment of refractory hypercalcemia from parathyroid carcinoma. Ann Intern Med 1987;197:683-686. Silverberg SJ, Bone III HG, Marriott TB, Locker FG, Thys-Jacobs S, Dziem G, Kaatz S, Sanguinetti EL, Bilezikian JE Short-term inhibition of parathyroid hormone secretion by a calciumreceptor agonist in patients with primary hyperparathyroidism. NEnglJMed 1997;337:1506-1510. Glover DJ, Shaw L, Glick JH, Slatopolsky E, Weiler C, Attie M, Goldfarb S. Treatment of hypercalcemia in parathyroid cancer with WR-2721, s-2-(3-aminopropylamino)ethyl phosphorothioic acid. Ann Intern Med 1985;103(1):55-57. Koyano H, Shishiba Y, Shimizu T, Suzuki N, Nakazawa H, Tachibana S, Murata H, Furui S. Successful treatment by surgical removal of bone metastasis producing PTH: New approach to the management of metastatic parathyroid carcinoma. Intern Med 1994;33(11):697-702.

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CHAPTER 3 4

Acute Primary Hyperp ar athy rol"dism

LORRAINE A. FITZPATRICK Division of Endocrinology, Metabolism, Nutrition, and Internal Medicine, Mayo Clinic and Foundation, Rochest~

Minnesota 55905

ACUTE PRIMARY HYPERPARATHYROIDISM

10 patients out of 325 consecutive cases of surgically proved primary hyperparathyroidism developed acute primary hyperparathyroidism. Nine of these patients had a single adenoma (4). Of 90 cases of primary hyperparathyroidism treated surgically between 1975 and 1985 in Athens, Greece, one case of hyperparathyroid crisis was identified (5). The risk of developing hypercalcemic crisis in patients with untreated primary hyperparathyroidism is low. Of a group of 47 patients followed over a 5-year interval, only one developed acute primary hyperparathyroidism (6). In a prospective series of patients at the Mayo Clinic followed for 10 years, only 1 of 142 patients, or 0.7%, developed acute primary hyperparathyroidism (7). Neither series was able to identify a specific index to predict accurately the likelihood of progression to severe hypercalcemia. In 121 patients with primary hyperparathyroidism followed longitudinally for 10 years, 27% showed progression but none developed severe hypercalcemia (8).

Prior to the development of multichannel autoanalyzers, primary hyperparathyroidism (HPT) was an infrequent diagnosis and was often associated with clear manifestations of end-organ disease (1,2). Older clinical descriptions of primary HPT were associated with symptoms related to the effects of parathyroid hormone (PTH) on the kidney and bone. Acute, severe hypercalcemia (parathyroid poisoning, parathyroid intoxication, or parathyroid crisis) was described as a possible complication of primary HPT. Primary HPT now presents as asymptomatic hypercalcemia. However, severe life-threatening, symptomatic hypercalcemia is still described (3). Acute primary hyperparathyroidism is associated with high rates of morbidity and mortality. A review of 48 cases of acute primary hyperparathyroidism emphasized the life-threatening nature of the hypercalcemia (3). Invariably, marked signs and symptoms were present. Some of the highest serum calcium levels reported in the literature have been attributed to acute primary HPT. Although rare, the n u m b e r of cases in the medical literature and the curable nature of the disorder indicate that this disease must be considered in the differential diagnosis of severe hypercalcemia.

DEMOGRAPHICS In one series, the age at the time of clinical presentation of acute primary hyperparathyroidism was the same or slightly younger than the average age of patients with primary HPT (Table 1) (3,8,9). This is in contrast to expected findings in which patients who are elderly, immobile, and with a higher likelihood of compromised renal function might be expected to be at increased risk for the development of acute primary

RISK OF D E V E L O P I N G A C U T E PRIMARY HYPERPARATHYROIDISM The incidence of acute primary hyperparathyroidism is difficult to assess. At Loyola Medical Center, The Parathyroids, Second Edition

527

Copyright © 2001 John R Bilezikian, Robert Marcus, and Michael A. Levine.

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CHAPTER34 TABLE 1

Acute Primary Hyperparathyroidism vs. Benign Primary Hyperparathyroidism Acute

Parameter Period of review

Benign

Fitzpatrick and Bilezikian (Ref. 3)

Heath et aL (Ref. 9)

Silverberg et aL (Ref. 8)

1974-1981

1974-1976

48 1.1:1.0 55 17.5 69 53 0

51 3.6:1 62 10.8 4 20 51

10-year longitudinal study a 121 3:1 55 10.6(?) 17 1.7 b 83

Number of cases Female/male ratio Average age (years) Serum calcium (mg/dl) Renal involvement (%) Skeletal involvement (%) Asymptomatic (%)

aof asymptomatic patients, 27% showed disease progression over the 10-year period of observation but none developed hypercalcemic crisis. bDefined as clinical bone disease or fractures. If patients with bone mineral density <80% of normal are excluded, then 1-2% of patients have skeletal involvement (8).

HPT. Ages ranged from 27 to 82 years and distribution by decade was fairly constant. No correlation was noted between serum calcium concentrations and age (R = 0.11, P > 0.05). The ratio of affected women to men (1.1:1.0) was similar to the distribution in parathyroid carcinoma (Table 2) (10-13) but markedly different from the gender ratio in recent series of primary HPT (8,9).

LABORATORY EVALUATION The average level of serum calcium is markedly elevated and in one series averaged 17.5 m g / d l (3). Serum

TABLE 2

calcium levels as high as 26.3 m g / d l have been reported. In another series, the serum calcium concentrations ranged from 15.0 to 17.6 m g / d l (4). These levels of serum calcium are striking in contrast to those noted in traditional presentations of primary hyperparathyroidism (Table 1). Serum calcium levels were indistinguishable from levels noted in patients with parathyroid carcinoma (Table 2) (3,10-12,15). Serum phosphate levels were in the low-normal range and severe hypophosphatemia was not c o m m o n (3). Of particular note were the strikingly elevated levels of PTH, averaging 20 times normal. Extremely high levels of PTH in association with marked hypercalcemia are strongly suggestive of acute primary hyperpara-

Acute Primary Hyperparathyroidism vs. Parathyroid Carcinoma

Acute primary hyperparathyroidism

Period of review Number of cases Female/male ratio Average age (years) Serum calcium (mg/dl) Renal involvement (%) Skeletal involvement (%) Asymptomatic (%)

Parathyroid carcinoma

Fitzpatrick and Bilezikian (aef. 3)

Shane et aL (Ref. 11)

Wang (Ref. 12)

Wynne et aL (Ref. 10)

Favia et aL (Ref. 14)

1974-1984 48 1.1:1.0 55 17.5 69 53 0

1968-1981 62 1.2:1.0 48 15.5 60 55 2

1948-1983 28 1:1 45 13.7 64 46 4

1920-1990 43 1:1 54 14.6 56 91 7

1980-1996 16 1.0:2.2 61 13.5 50 75 19

ACUTE PRIMARYHYPERPARATHYROIDISM / thyroidism (3) or parathyroid carcinoma (10,14). Hypercalcemia associated with nonparathyroid malignancy or other etiologies is notable for suppressed levels of PTH (15). In asymptomatic primary HPT, a twoto three-fold elevation of serum PTH is common (16), and in parathyroid carcinoma, PTH levels at presentation are much higher (10 times the upper limit of normal) (10,14). Thus, parathyroid h o r m o n e levels can be helpful in distinguishing malignancy-associated or asymptomatic primary HPT from acute primary HPT, but these markedly elevated levels of PTH do not rule out the possibility of parathyroid carcinoma. The distinction between acute HPT due to a benign adenoma and parathyroid carcinoma is important because the surgical approach to the patient depends on the nature of the lesion. An antecedent history of mild hypercalcemia within 10 years in up to 25% of patients presenting with acute primary HPT has been noted (3). This history is extremely useful in the differential diagnosis of the disorder. Intervening illness, perhaps with associated bed rest, may stimulate mobilization of calcium from bone and play a role in the development of the acute parathyroid crisis. Among intervening events, 11 patients had undergone surgery, 2 had viral illnesses, 2 had pneumonia, and 1 each presented with diabetic ketoacidosis, trauma, or urinary tract infection (3). Another reported case was thought to be precipitated by herpes zoster infection (17), and in another report a 50-year-old woman developed acute hypercalcemia associated with HPT after head and intraabdominal trauma (18).

TARGET O R G A N MANIFESTATIONS Most subjects with acute primary HPT have some manifestation of renal involvement. In 29 cases in which clinical documentation was adequate, 20 (69%) subjects had nephrolithiasis or nephrocalcinosis (3). In a recent series of 16 patients, 50% had nephrolithiasis or renal failure (14). This is in contrast to the low incidence of renal involvement (17%) in patients presenting with primary hyperparathyroidism (Table 1). Patients with parathyroid carcinoma, however, have a high incidence of renal disease, similar to that noted in patients with acute primary hyperparathyroidism (Table 2). Radiologic criteria of primary hyperparathyroidism included subperiosteal resorption of the distal phalanges, overt osteitis fibrosa cystica, "salt and pepper" skull, or diffuse activity on bone scan. Of 36 patients, 19 (53%) had documented skeletal disease (3) and in a smaller series, 75% had osteopenia (14). The percentage of patients with skeletal involvement is markedly

529

increased compared to current estimates of overt skeletal manifestations in primary hyperparathyroidism (0-2%) (8,19). In association with the skeletal findings, alkaline phosphatase activity averaged twice the upper limit of normal in patients with acute primary HPT. This is unusual for the typical patient with HPT in whom the average serum alkaline phosphatase is only minimally elevated. A high frequency (50%) of both renal and skeletal involvement was observed in 10 of 20 patients for whom sufficient information was available (3). The presentation of concurrent renal and radiologic skeletal disease is unusual in most patients with HPT. The death of a patient with acute primary HPT has revealed further information regarding the calcium dynamics of the disorder (20). Based on the urinary calcium excretion, the amount of calcium loss from bone during the period of acute parathyroid crisis was estimated to be approximately 2 g per day. Histologic appearance of bone was remarkable for the low level of bone formation despite a massive increase in osteoclastic activity. Trabecular osteoid surface was at the lower limit of normal and osteoid consisted of only one lamella. No active osteoblasts were noted and there was no woven bone visible. Trabecular bone volume was slightly reduced. In this particular patient, immunoreactive parathyroid h o r m o n e was markedly elevated, as was serum calcium (5.1 m m o l / 1 ) . The histologic appearance of bone in this patient suggested marked activation of basic multicellular units of bone. Subperiosteal erosions of the phalanges and clavicles were absent in this patient, although the length of time necessary for these characteristic radiographic signs of hyperparathyroid bone disease to develop is unknown. The authors emphasized that there was no compensatory increase in bone formation indicating a marked dissociation between bone resorption and bone formation in this patient (20). This pathologic appearance distinguishes the bone histology from a patient with acute HPT from the histologic appearance of bone in routine primary HPT. In primary HPT, bone formation and bone resorption are increased (see Chapter 55). If this case report is representative of bone dynamics in acute HPT, one might speculate that the inhibition of bone formation contributes to the marked hypercalcemia.

OUTCOME In a series of patients studied between 1960 and 1986, there were no preoperative deaths, a gratifying result of medical m a n a g e m e n t including rapid rehydration and correction of electrolyte disorders (21). Three perioperative deaths (6%) in 48 cases (2) mark a great improvement from the 59% mortality observed

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CI4aeTw~34

prior to 1970. This decrease in mortality may be due to earlier recognition, refined preoperative medical management, and improved surgical techniques. A nonoperative approach to ablate a parathyroid adenoma in a patient with acute HPT has been described. A single parathyroid adenoma was treated by direct instillation of 96% ethanol u n d e r ultrasonic guidance. Recurrence of the hypercalcemia required repeat injections 1 year later (22). Further studies will be necessary to assess the feasibility and safety of this technique, which has also been used successfully in HPT.

PATHOLOGY Review of the pathology of tissue obtained from patients with acute HPT revealed parathyroid adenomas exclusively except in one patient who appeared to have multiple gland involvement (3). In another series of 57 patients with serum calcium greater than 13.0 m g / d l , 28% of the patients had multiglandular involvement (23). At Loyola Medical Center, 1 in 10 patients with acute primary HPT had multiglandular involvement at surgery (4). Although cysts are not commonly noted on pathologic examination of the parathyroid glands (see Chapter 1), two cases of parathyroid cysts were noted in patients with acute primary HPT (24). In an additional report, Albertson and co-workers described hypercalcemic crisis due to the presence of a large functioning parathyroid cyst (25). Ultrastructural analyses of the parathyroid gland associated with acute HPT revealed features of marked hyperactivity. The cell nucleus is enlarged, with increased pores and abundant rough endoplasmic reticulum. Conspicuous interdigitations of the plasma membrane, increased n u m b e r of mitochondria, and extensive Golgi apparatus are also present. These features are consistent with the markedly increased function of the parathyroid cell associated with acute HPT (26).

PATHOPHYSIOLOGY This unusual presentation of parathyroid disease is marked by extreme hypercalcemia and associated target tissue manifestation. The mechanisms by which a seemingly "ordinary" case of primary hyperparathyroidism develops into a life-threatening medical emergency remain enigmatic. However, several factors may be gleaned from the various case studies and provide evidence for the pathophysiology of this disorder. One hallmark of parathyroid crisis is the extraordinary elevation of parathyroid hormone. Levels 20 times

normal values have been described (3) and are much greater than the 1.5- to 2.0-fold elevation of PTH measured in patients with primary hyperparathyroidism. Although one might predict that renal impairment may contribute to the increased circulating levels of PTH, intact hormone levels may be as high as the C-terminal fragment (3). This observation suggests that the secretion of intact PTH from the abnormal parathyroid gland is enhanced. As described, the pathology of the parathyroid gland reflects this increased secretory rate (26). With rare exceptions, the single adenoma found at surgery has not been compromised by infarction or rupture leading to massive release of PTH into the systemic circulation. The physiologic effects of excessive PTH production are revealed in the kidney by mal~ked hypercalciuria, and in bone, by massive osteoclast activity. The failure to deposit calcium in bone, as shown in the case report (26) with reduced bone formation, fuels the development of further hypercalcemia.

A C U T E P A R A T H Y R O I D CRISIS A S S O C I A T E D WITH PREGNANCY Management of maternal hyperparathyroidism during pregnancy is usually based on the patient's symptoms, severity of the disease, and gestational age of the fetus (see Chapter 29). A few cases of acute parathyroid crisis have been described during pregnancy (27-29). Although most patients underwent surgical removal of a parathyroid adenoma, acute primary HPT has been described in which medical m a n a g e m e n t resulted in a favorable outcome (28). In an unusual case, a 34-year-old female developed acute primary hyperparathyroidism several days postpartum (29). She was admitted to the hospital at 39 weeks of gestation due to polyhydramnios, and spontaneously delivered a healthy 3070-mg infant at 40 weeks of gestation. Postpartum, she developed progressive weakness and nausea. On the third postpartum day, serum calcium was >5 mmol/liter and phosphate was 1.9 mmol/liter. Further evaluation revealed subperiosteal resorption on hand radiographs and a "pepper pot" appearance and resorption around the teeth on skull X-rays. Her condition deteriorated rapidly, and she was treated medically with intravenous fluids, hydrocortisone, and phosphates. Hemodialysis was performed on day 5, and respiratory failure was managed with intermittent positive pressure ventilation. Neck exploration revealed a 3-g parathyroid tumor in the left pole of the thyroid gland, and laparotomy was performed due to gross distension of the abdomen. Acute pancreatitis was treated with drainage. On the following

ACUTE PRIMARYHYPERPARATHYROIDISM / day, shock intervened, and resuscitation was unsuccessful. On pathologic examination, two normal parathyroid glands were identified. Metastatic calcification was noted in kidney tubules, myocardium, pulmonary vessel walls, and bronchial walls. A serum sample obtained at 16 weeks of gestation was retrieved, and the patient was found to have had an elevated serum calcium (2.91 mmol/liter). The authors suggest that the increased need of the fetus for calcium, and physiologic hypoalbuminemia, may have protected the patient in the peripartum period. In an animal model, advanced pregnancy has provided protection against administration of PTH in toxic doses; this protection did not extend to the postpartum period (30). A similar situation may have occurred in this unfortunate woman with acceleration of calcium mobilization and increased serum concentration of calcium in the postpartum period.

OTHER ASSOCIATIONS WITH ACUTE PRIMARY H Y P E R P A R A T H Y R O I D I S M An extraordinarily rare combination of endocrine emergencies, acute primary HPT, and thyrotoxicosis has been described in one unfortunate patient (31). Asymptomatic hyperparathyroidism in a patient with thyrotoxicosis is a relatively u n c o m m o n condition; other authors have described an incidental occult parathyroid adenoma in a patient with a preexisting thyroid disorder (32,33). This 32-year-old female was diagnosed with Graves' disease and treated with radioactive iodine. After 3 months, she was admitted to a psychiatric hospital with the diagnosis of depression. Her medical evaluation revealed emesis, constipation, perioral and digital numbness, and a serum calcium of 14 mg/dl. On transfer to a medical facility, serum calcium rose to 15 m g / d l and diffuse demineralization was noted on hand radiographs. Serum calcium remained markedly elevated in spite of treatment with intravenous fluids (400 m l / h o u r ) , prednisolone (30 mg bid), and phosphorus (750 mg tid). For her thyroid disorder (T 4 = 19.1 dg/dl, T~ = 361 n g / d l ) , the patient was treated with propranolol, saturated solution of potassium iodide, and propylthiouracil. On hospital day 11, she underwent neck exploration and a 2 × 2 × 3-cm right upper parathyroid adenoma weighing 6 g was removed; bilateral subtotal thyroidectomy was also performed. Postoperatively, the patient required intravenous calcium gluconate when her serum calcium reached a nadir of 4.6 mg/dl. The authors emphasize the need to render the patient euthyroid prior to surgical treatment of hyperparathyroidism (33).

531

ANIMAL MODELS OF ACUTE PRIMARY H Y P E R P A R A T H Y R O I D I S M In 1926, the administration of large amounts of parathyroid extract to dogs produced anuria, renal failure, and death. Calcium deposits were present in soft tissues throughout the animal (34). These experiments, which provided insight into the pathophysiology of primary hyperparathyroidism, were extended by Hulter and colleagues (35), who proposed that chronic alterations in plasma calcium concentrations would alter the response of plasma calcitriol concentrations to PTH. Intact dogs underwent continuous intravenous PTH infusion for 12 days with sustained hypercalcemia and hypophosphatemia. Plasma calcitriol concentrations decreased significantly. Nussbaum and colleagues have developed an in vivo model of hyperparathyroidism (36). A cDNA encoding h u m a n p r e p r o P T H was cloned into a replication-defective retroviral vector. The retrovirus containing the PTH gene was then transfected into a retroviral packaging cell line. Harvested virus was used to infect RAT-1 fibroblasts. The RAT-1 fibroblasts were injected into the peritoneal cavity of syngeneic Fisher rats. A severe and progressive rise in h u m a n PTH levels and a rise in serum calcium occurred in these animals. This model allowed histologic evaluation of bone and kidney in severe hyperparathyroidism. Marked osteoclastic bone resorption and extensive osteitis fibrosa cystica were observed. Nephrocalcinosis was present on histologic examination of the kidneys. This model mimics acute primary hyperparathyroidism in the rapidity of the development of hypercalcemia and may help to provide further insight into the pathogenesis of acute parathyroid disease.

TREATMENT Definitive treatment of acute HPT is surgical extirpation of the abnormal parathyroid gland or glands. However, treatment of severe, life-threatening hypercalcemia must first be instituted, an approach similar to therapy of acute hypercalcemia of other causes (37) (see Chapter 45). In two reported cases, acute hypercalcemia occurring several days after cholecystectomy (38) or splenectomy (18) was successfully ameliorated with the administration of intravenous pamidronate prior to surgical removal of the parathyroid adenoma (see Figs. 1 and 2). The use of intravenous phosphates in acute HPT is contraindicated. Extensive systemic calcinosis precipitated by phosphates can occur in patients pre- or postoperatively (39,40) (see Fig. 3). Intravascular

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FIG. 1 Preoperative course of a patient with acute primary hyperparathyroidism. Two doses of plicamycin (25 ~g/kg) 12 hours apart were administered for life-threatening hypercalcemia. The serum calcium levels remained stable until the diagnosis of primary hyperparathyroidism was confirmed and the patient underwent successful parathyroidectomy. The shaded area represents the normal range of the serum calcium concentration.

precipitation of calcium-phosphate salts may cause pulmonary insufficiency, vascular calcification, hypotension, and death. Several reports have d o c u m e n t e d rapid accretion of bone mass after surgical removal of the parathyroid a d e n o m a in patients with hyperparathyroid crisis. In patients with primary hyperparathyroidism, prospective studies have indicated a rapid and continuous increase in bone mineral density (BMD) postsurgery over a 10year period (8). These gains are in the range of approximately 11 to 15% over 10 years. In one 40-year-old woman with hyperparathyroid crisis~a serum calcium of 20.1 m g / d l and severe ostitis fibrosa cystica on a d m i s s i o n ~ B M D of the lumbar spine was 0.434 g / c m 2. At 11 months postoperatively, BMD had increased to 0.536 g / c m 2 (lumbar spine), a gain of 24% (41). In a premenopausal women with acute primary hyperparathyroidism, BMD increased 25% in the hip and 22% in the lumbar spine 1 year after surgical removal of the parathyroid a d e n o m a (42).

SUMMARY Acute primary hyperparathyroidism is a reversible, curable, but life-threatening disorder. Although the pathophysiology is not well understood, several factors are c o m m o n to many reported cases. Age and gender are not risk factors. An antecedent history of primary hyperparathyroidism has been confirmed in several cases (3,29), although the risk of developing acute tox-

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FIG. 2 Postoperative course of the same patient shown in Fig. 1 following successful parathyroidectomy. The decline in N-terminal PTH (top)levels was monitored in sequence with the postoperative decline in serum calcium levels (bottom). Calcium (1 g daily) and 1,25(OH)2D (0.25 i~g three times daily) were administered over the times indicated. Shaded areas in both panels indicate the normal range of the N-terminal PTH and calcium concentration, respectively.

icity in the asymptomatic hyperparathyroid population is low (6,7). An incapacitating illness, perhaps associated with bed rest, is frequently associated with acute primary HPT. The immobilization that occurs during intercurrent illness may foster mobilization of calcium from bone, aggravating the hypercalcemic state. Remarkable elevations of parathyroid h o r m o n e suggest that the hallmark of the disorder is uncontrolled PTH secretion. PTH levels are elevated to a much greater extent than can be accounted for by impairment of kidney function alone. Surgical cure of the disorder results in rapid gain of bone mass. Recognition of acute HPT, rapid diagnosis, aggressive medical management, and successful surgery can result in a favorable outcome. The fact that the diagnosis can be readily distinguished from other causes of hypercalcemia and the fact that the disorder is curable mean that this disorder must be considered in any patient with life-threatening hypercalcemia.

ACUTE PRIMARYHYPERPARATHYROIDISM /

FIG. 3 Pulmonary calcinosis in a patient with acute primary hyperparathyroidism. Hypercalcemic crisis occurred due to a large mediastinal parathyroid adenoma. (Reproduced from Khafif RA et aL, Arch Intern Med 1989;149:682. Copyright 1989, American Medical Association.)

REFERENCES 1. Fitzpatrick LA. Is surgery necessary for the asymptomatic patient with primary hyperparathyroidism? Gitnick F, Barnes HV, Duffy TP, Lewis RP, Winterbauer RH, eds. Debates in medicine, 4th Ed. Chicago:Mosby Year Book, 1991:114-157. 2. Fitzpatrick LA, Bilezikian JE Primary hyperparathyroidism. In: Becker KL, ed. Principles and practice of endocrinology and metabolism. Philadelphia:Lippincott, 1990:430-437. 3. Fitzpatrick LA, Bilezikian JE Acute primary hyperparathyroidism. AmJ Med 1987;82:275-282. 4. Maselly MJ, Lawrence AM, Brooks M, Barbato A, Braithwaite S, Oslapas R, Paloyan E. Hyperparathyroid crisis. Successful treatment of ten comatose patients. Surgery 1981;90:741-760. 5. Nudelman IL, Deutsch AA, Reiss R. Primary hyperparathyroidism due to mediastinal parathyroid adenoma. Int Surg 1987;72:104-108. 6. Corlew DS, Bryda SL, Bradley EL, DiGirolamo M. Observations on the course of untreated primary hyperparathyroidism. Surgery 1985;98:1064-1071.

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7. Scholz DA, Purnell DC. Asymptomatic primary hyperparathyroidism: 10-year prospective study. Mayo Clin Proc 1981 ;56:473-478. 8. Silverberg SJ, Shane E, Jacobs TP, Siris E, Bilezikian JP. A 10-year prospective study of primary hyperparathyroidism with or without parathyroid surgery. N EnglJ Med 1999;341:1249-1255. 9. Heath H, Hodgson SF, Kennedy MA. Primary hyperparathyroidism: Incidence, morbidity, and potential economic impact in a community. NEnglJMed 1980;4:189-193. 10. Wynne AG, van Heerden J, Carney JA, Fitzpatrick LA. Parathyroid carcinoma: Clinical and pathologic features in 43 patients. Medicine 1992;71:197-205. 11. Shane E, Bilezikian JE Parathyroid carcinoma: A review of 62 patients. Endocr Rev 1982;3:218-226. 12. Wang C, Gaz RD. Natural history of parathyroid carcinoma: Diagnosis, treatment and results. AmJ Surg 1979;149:522-527. 13. Hundahl SA, Fleming ID, Fremgen AM, Menck HR. Two hundred eighty-six cases of parathyroid carcinoma treated in the U.S. between 1985-1995: A National Cancer Data Base Report. Cancer 1999;86:538-544. 14. Favia G, Lumachi F, Polistina F, D'Amico DE Parathyroid carcinoma: Sixteen new cases and suggestions for correct management. WorldJ Surg 1998;22:1225-1230. 15. Martin TJ, Grill, V. Hypercalcemia in cancer. J Steroid Biochem Mol Bio11992;43:123-129. 16. Kao PC, Jiang N, Klee G, Purnell DC. Development and validation of a new radioimmunoassay for parathyrin (PTH). Clin Chem 1982;28:69-74. 17. Phillips P. Letter to the editor: Herpes zoster-induced acute hyperparathyroid crisis. Clin Infect Dis 1992;14:1270-1271. 18. Canivet JL, Damas P, Lamy M. Posttraumatic parathyroid crisis and severe hypercalcemia treated with intravenous bisphosphonate (APD). Case report. Acta Anaesthesiol Belg 1990;41:47-50. 19. Bilezikian JP, Silverberg SJ, Shane E, Parisien M, Dempster D. Characterization and evaluation of asymptomatic primary hyperparathyroidism. J Bone Miner Res 1991;6 (Suppl. 2) :$85-$90. 20. Wang CA, Gaz RD. Bone metabolism in acute parathyroid crisis. Clin Endocrino11985;22:787-793. 21. Sarfati E, Desported L, Gossot D, Dubost C. Acute primary hyperparathyroidism: Experience of 59 cases. Br.J Surg 1989;76:979-981. 22. Karstrup S, Lohela P, Apaja-Sarkkinen M, Borgmastars H, Holopeinen O. Non-operative inactivation of a parathyroid tumour in a patient with hypercalcaemic crisis. Acta Med Scand 1988;224:187-188. 23. Bizard JP, Qui6vreux, Carnaille B, Proye C. Formes toxiques de l'hyperparathyroidie primaire (57 observations) R6appr6ciation de leur d6finition et de leur substratum anatomopathologique. Lyon Chir 1992;88:117-122. 24. Calandra DB, Shah KH, Prinz RA, Sullivan H, Hofmann C, Oslapas R, Ernst K, Lawrence AM, Paloyan E. Parathyroid cysts: A report of eleven cases including two associated with hyperparathyroid crisis. Surgery 1983;94:887-892. 25. Albertston DA, Marshall RB, Jarman WT. Hypercalcemic crisis secondary to a functioning parathyroid cyst. Am J Surg 1981;141: 175-177. 26. Thiele J. The human parathyroid chief cellma model for polypeptide hormone producing endocrine unit as revealed by various functional and pathological conditions. A thin section and freeze-fracture study. J Submicrosc Cyto11986;18:205-220. 27. Clark D, Seeds JW, Cefalo RC. Hyperparathyroid crisis and pregnancy. Am J Obstet Gyneco11981;140:840-842. 28. Thomason JL, Sampson MB, Farb HE Spellacy WN. Pregnancy complicated by concurrent primary hyperparathyroidism and pancreatitis. Obstet Gyneco11981 ;57:34S-36S. 29. Matthias GSM, Helliwell TR, Williams A. Postpartum hyperparathyroid crisis. Case report. BrJ Obstet Gyneco11987;94:807-810.

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30. Lehr D, Krukowski M. Prevention of myocardial necrosis by advanced pregnancy. J A m Med Assoc 1961;1781:823-826. 31. Piccione W, Selenkow HA, Cady B. Management problems in coexisting parathyroid crisis and florid thyrotoxicosis. Surgery 1984;96:1009-1014. 32. Attie JN, Estrin J, Khafif RA, Dweck E Parathyroid adenomas discovered incidentally during explorations of the thyroid. AmJSurg 1967;114:538-542. 33. Frame B, Durham RH. Simultaneous hyperthyroidism and hyperparathyroidism. Am J Med 1959;27:824-828. 34. Albright F, Reifenstein EC. The parathyroid glands and metabolic bone disease. Baltimore:Williams & Wilkins, 1948. 35. Hulter HN, Halloran BE Toto RD, Peterson JC. Long-term control of plasma calcitriol concentration in dogs and humans. Dominant role of plasma calcium concentration in experimental hyperparathyroidism. J Clin Invest 1985;76:695-702. 36. Wilson JM, Grossman M, Thompson AR, Lupassikis C, Rosenberg A, Potts JT, Kronenberg HM, Mulligan RC, Nussbaum SR. Somatic

gene-transfer in the development of an animal-model for primary hyperparathyroidism. Endocrinology 1992;130:2947-2954. 37. Bilezikian JE Management of acute hypercalcemia. N E n g l J Med

1992;326:1196-1203.

38. Evans RA. Aminohydroxypropylidene diphosphonate treatment of hypercalcemic crisis due to primary hyperparathyroidism. Aust N z J Med 1987;17:58-59. 39. Khafif RA, Delima C, Silverberg A, Frankel R, Groopman J. Acute hyperparathyroidism with systemic calcinosis. Report of a case. Arch Intern Med 1989;149:681-684. 40. Vernava AM, O'Neal LW, Palermo V. Lethal hyperparathyroid crisis: Hazards of phosphate administration. Surgery 1987; 102:941-948. 41. Ishikawa S, Ozaki T, Kawai A, Inoue H, Doihara H. Hyperparathyroid crisis in a patient with a giant brown tumor of the iliac bone: A case report. HiroshimaJMed Sci 1998;47:27-30. 42. Tritos NA, Hartzband E Rapid improvement of osteoporosis following parathyroidectomy in a premenopausal woman with acute primary hyperparathyroidism. Arch Intern Med 1999; 159:1495-1498.

CI4AeT:V 35 Multiple Endocrine Neoplasia Type 1

STEPHEN J. MARX Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892

D E F I N I T I O N S OF MULTIPLE E N D O C R I N E

Multiple e n d o c r i n e neoplasia (MEN) is a b r o a d term that, i n t e r p r e t e d literally, encompasses any case or family p r e s e n t i n g with neoplasia in m o r e than one potentially h o r m o n e - s e c r e t i n g tissue. Some cases have a r a n d o m coincidence of several c o m m o n tumors. Some have a c o m m o n extrinsic cause, such as n e c k radiation leading to thyroid a n d p a r a t h y r o i d tumors. Some have one sporadic t u m o r that directly causes a second, such as a first t u m o r oversecreting A C T H or GHRH. 1 Some MEN t u m o r patterns occur repeatedly. T h e first two r e c o g n i z e d r e p e a t i n g MEN patterns are now t e r m e d multiple e n d o c r i n e neoplasia type 1 (MEN-l) a n d type 2 (MEN-2). Prior n a m e s for MEN-1 have included multiple e n d o c r i n e a d e n o m a t o s i s type 1 a n d W e r m e r syndrome. All or most cases of MEN-1 are

caused by m u t a t i o n in the MEN1 gene 2, all or most cases of MEN-2 are caused by m u t a t i o n in a s e c o n d a n d very different gene, the RET gene. MEN-1 causes tumors in m a n y tissues with only partial p e n e t r a n c e in each tissue; therefore, no simple definition could e n c o m p a s s all cases or all families. Still, a simple definition is useful in practice. MEN-1 is operationally d e f i n e d as a case with tumor(s) in two of the following three tissues: parathyroid cells, gastrin cells, a n d lactotrope(s). Similarly, familial MEN-1 is defined as a family with a case of MEN-1 a n d at least one firstd e g r e e relative showing one or m o r e of the m a i n three tumors of MEN-1. T h e above definitions are based on clinical phenotypes. T h e y have b e e n e n h a n c e d by genebased definitions, m a d e possible by testing for MEN1 mutations. T h e clinical a n d genetic definitions differ mainly in asymptomatic MEN1 m u t a t i o n carriers.

1Abbreviations for hormones: ACTH, adrenocorticotropic hormone; GH, growth hormone; GHRH (also GRF), growth hormone-releasing hormone; IGF-I, insulin-like growth factor type-I; PTH, parathyroid hormone; PTHrP, PTH-related peptide; PP, pancreatic polypeptide; TSH, thyrotrophin; VIP, vasoactive intestinal peptide. 2By convention, the acronym for the name of a syndrome is in roman upper case; the gene name is in italicized upper case. Thus MEN-1 is a syndrome and MEN1 is a gene. The following nomenclature is used in this chapter to indicate genes: CaSR,

calcium-sensing receptor; CCND1, cyclin D1; EXT1 and EXT2, multiple exostosis syndrome genes; GK, glucokinase gene; GLUD1, glutamate dehydrogenase; GNAS1, Gs0t; Kir6.2, inward rectifying, ATPbinding subunit of pancreatic islet potassium channel; MEN1, multiple endocrine neoplasia type 1; MET, a transmembrane tyrosine kinase; PYGM, DNA marker in or near muscle phosphorylase gene at 11q13; RET, a transmembrane tyrosine kinase, also gene for MEN-2 and some cases of Hirschsprung disease; RB1, retinoblastoma gene; SUR1, sulfonyluria-binding subunit of pancreatic islet potassium channel; TP53, Li-Fraumeni cancer family syndrome gene.

NEOPLASIA TYPE 1

The Parathyroids, Second Edition

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Copyright © 2001John E Bilezikian, Robert Marcus, and Michael A. Leone.

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BRIEF H I S T O R Y OF MULTIPLE E N D O C R I N E NEOPIakSIA TYPE 1 Erdheim is generally credited with the first report of MEN-1. In 1903, he described an autopsy showing acromegaly, eosinophilic tumor of the pituitary, and four enlarged parathyroid glands (1). Rossier and Dressler first reported similar features in a kindred in 1939 (2). Reports in 1953-1954 by Underdahl (3), Moldawer (4,5), and Wermer (6) further established MEN-1 as a distinctive familial syndrome. Ulcer diathesis had been recognized early as an integral feature of MEN-l, but the connection to gastrinoma became possible only after delineation of sporadic gastrinoma in 1955-1961 (7-9). Similarly, prolactinoma could be assigned to MEN-1 only after identification of prolactin in 1971 (10). Around 1961 MEN-2 began to be recognized as a distinct familial syndrome (thyroidal C cell cancer, pheochromocytoma, hyperparathyroidism, and occasionally a mucosal neuroma phenotype) (11). Advances in MEN-1 management overlap with advances in much of clinical endocrinology: blood testing (hormone immunoassays and gene mutation testing), pharmacology (hormone secretion blockers and hormone action blockers), tumor localization [CT ~, MRI, US, angiography, radioactive scans, basal and stimulated hormone sampling protocols (aided by catheter, fine needle aspiration, or endoscopy)], and surgery (pituitary microsurgery, parathyroid cryopreservation, intraoperative tumor imaging, rapid assay of hormones intraoperatively). In 1988 Larsson reported that the MEN1 gene was on chromosome 11 and that both MEN1 gene copies were probably inactivated in MEN-1 tumors (12). These findings led to further genetic studies in kindreds and in tumors. A decade later, the MEN1 gene was identified in 1997 (13), initiating another phase of accelerated clinical and basic insights. MEN-1 was recognized early as a possible source for understandings of several topics in pathophysiology, particularly in oncology and endocrinology. For this reason, SAbbreviations: BAO, basal gastric acid output; CaSR, calciumsensing receptor; Cg-A, chromogranin-A; CGH, comparative genome hybridization; CT, computerized tomography; ECL, enterochromaffin-like (cell); EST, expressed sequence tag--a fragment of DNA sequence derived from messenger RNA, thereby representing an expressed gene; Gscx, activating subunit of the heterotrimeric G protein that regulates adenylyl cyclase; FISH, fluorescent in situ hybridization; FHH, familial hypocalciuric hypercalcemia; HPT-JT, hyperparathyroidism and jaw tumors; LOH, loss of heterozygosity; MEN, multiple endocrine neoplasia (MEN-1 is type 1; MEN-2A is type 2A; MEN-2B is type 2B); MRI, magnetic resonance imaging; NLS, nuclear localization signal; PHHI, persistent hyperinsulinemic hypoglycemia of infancy; TSC, tuberous sclerosis; US, ultrasound; ZES, Zollinger-Ellison syndrome.

more has been written about MEN-1 than would be predicted from its low prevalence. Additional studies have justified this emphasis; in particular, somatic mutation of the MEN1 gene often contributes to several commonvariety endocrine and nonendocrine neoplasms including sporadic parathyroid adenoma.

PREVALENCE OF MEN-1 IN S U B G R O U P S There is no comprehensive population-based analysis of the prevalence of MEN-1. An autopsy series estimated a prevalence of 2.2 per 1000 (14). Biochemical surveys have suggested a far lower prevalence of 0.01-0.175 per 1000 (15-18). MEN-1 population prevalence can also be estimated from the fraction of MEN-1 cases among a group with one endocrine tumor that is common in MEN-1. The MEN-1 fraction in primary hyperparathyroidism (HPT) was variously estimated at 1-18% (16); 2-3% seems more likely (19,20a,b). In Japan, germ line MEN1 mutation was found in 3 of 64 cases of sporadic hyperparathyroidism (20b). In Japan, familial involvement with MEN1 may be particularly difficult to determine by routine questioning (see following), increasing the frequency of undiscovered familial MEN-1. Combined with an HPT annual incidence of 0.5 per 1000 (21,22), this gives an estimated MEN-1 annual incidence of 0.015 per 1000 and thus an accumulated prevalence 10- to 20-fold higher (0.15-0.3 per 1000). MEN-1 also represents a substantial fraction (16-38%) of all cases with Zollinger-Ellison syndrome (ZES) (23-26), a lower fraction (5% or less) among nonfunctioning enteroendocrine tumors (27) 4, and a smaller fraction (1-5 %) of all cases with pituitary tumor (28-30) [and higher (3-14%) in the prolactinoma subgroup (31,32) ]. The majority of MEN-1 cases is familial. In a large MEN-1 series from the United Kingdom, 36 sporadic and 220 affected cases occurred among 62 families (33). The National Institutes of Health (NIH) series has approximately 30 sporadic and 500 affected cases in 80 families (S. J. Marx, unpublished). Two series from Japan showed a different distribution with 61 sporadic and 45 affected cases from 15 families (34) and with 19 sporadic cases plus 41 mutation carriers from 16 families (35,36). This difference likely reflects reluctance to discuss personal illness in Japan. Some MEN-1 cases labeled as sporadic undoubtedly have unrecognized

4Nonfunctional tumors are defined here as those that do not make a known hormone, make a hormone but do not secrete enough to cause a syndrome, or make and secrete a peptide (such as PP or calcitonin) that does not cause a syndrome.

MULTIPLE ENDOCRINE NEOPLS~SIATYPE 1 familial MEN-l, unless this has been excluded by detailed analyses (including DNA testing to verify parent assignment) (37). C L I N I C A L E X P R E S S I O N S O F MEN-1 WITHIN A TISSUE

Benign Mass or Malignancy as a Major Expression of MEN-1 The commonest endocrine expressions of MEN-1 are hormonal, the results of high concentrations of parathyroid h o r m o n e (PTH), gastrin, and prolactin. Excellent treatments are available for each of these states. Less frequently, a mass effect from benign tissue becomes a problem in MEN-l, as with compression of the optic nerve or of the normal pituitary tissue (34,38). On the other hand, some but not all tumors in MEN-1 have a substantial malignant potential. With the increased life expectancy that has resulted from prevention of the lethal hormonal features of ZollingerEllison syndrome or of HPT (38,39), MEN-1 malignancy must be expected to contribute increasingly to morbidity and mortality in MEN-1 (40,41). In one MEN-1 series, malignancy caused one-third of MEN-1 deaths, with a 2:1 ratio between death from enteropancreatic malignancy and carcinoid malignancy (41). In an unrelated gastrinoma series (sporadic and MEN-l),

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half of deaths were from malignant expression of gastrin o m a and none were from problems caused by excess gastric acid (42). The financial and emotional burdens of MEN-1 are thus a result of combined endocrinopathy and malignancy.

Parathyroid Glands in MEN-1 High Penetrance of HFF in MEN-1 Primary HPT is the commonest endocrinopathy in MEN-1 (34,38,39,43--49) (Table 1), with frequencies between 80 and 100% among series of MEN-1 cases. This variation generally reflects the methods of patient selection (variable ages, variable symptoms, and case reports or autopsy reports) and the methods of parathyroid gland testing (symptom-driven, nonsystematic testing, and regular screening, yielding progressively greater detection). MEN1 mutation analysis now allows measuring trait penetrance based on a genetic denominator (33). Not only is HPT the most c o m m o n endocrine expression, but it is usually the earliest endocrine expression of MEN-1. Occasional exceptions exist (47,50). The earliest expressions of HPT in MEN-1 have been during ages 5-8 years, with rapidly rising penetrance: 50% at ages 20-30 years and 95% by ages 40-50 years (Fig. 1) (15,33,43,48,51). Rare MEN1 mutation carriers do not express HPT or any other MEN-1 endocrine t u m o r even beyond age 65 (52).

TABLE 1 Featuresof Multiple Endocrine Neoplasia Type 1 among Adultsa Endocrine features

Nonendocrine features

Parathyroid adenoma (95%) Enteropancreatic Gastrinoma (40%) Insulinoma (10%) Nonfunctioning,b including pancreatic polypeptide-oma (20%)c Other: glucagonoma, VIPoma, somatostatinoma, etc. (each <2%) Foregut carcinoid Thymic carcinoid nonfunctioning (2%) Bronchial carcinoid nonfunctioning (4%) Gastric enterochromaffin-like tumor nonfunctioning (10%) Anterior pituitary Prolactinoma (25%)Other: nonfunctioning (10%), growth hormone + prolactin, growth hormone (both 5%), ACTH (2%), TSH (rare)

Facial angiofibromas (85%) Collagenoma (70%) Lipoma (30%) Ependymoma (< 1%) Leiomyoma ( 1%?)

Adrenal

Cortex nonfunctioning (20%) Pheochromocytoma (< 1%)

aEstimated average penetrance shown in parentheses. Underlines indicate tumor types with substantial (above 25% of cases) malignant potential. bMany "nonfunctioning" MEN-1 tumors synthesize a peptide hormone or other factors (such as small amine), but do not oversecrete enough to produce a hormonal expression. COmits nearly 100% prevalence of nonfunctioning and clinically silent tumors, some of which are detected incidental to enteropancreatic surgery in MEN-1.

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Clinical Features of HPT That Differ in MEN-1 versus in Sporadic Cases Many clinical features of primary HPT are similar in MEN-I (15,48,53) and sporadic cases (54). These include a prolonged early phase of asymptomatic

TABLE 2

Primary HPT in MEN-1 has been recognized several times at age 8 years (15,43,48). The average age of onset in MEN-1 is 25 years (48,49), in distinct contrast to the higher onset age (55 years) in sporadic HPT (21,22) and the much lower (i.e., neonatal) onset age in familial hypocalciuric hypercalcemia (Table 2). The generally early onset age for HPT in MEN-1 implies a potentially long duration of adverse effects from high circulating PTH and calcium concentrations; for example, early urolithiasis is common in MEN-1 (53). Surprisingly, there has been little documentation of chronic skeletal effects of HPT during adolescence and young adulthood. Such effects could be deleterious or even beneficial (55,56). In women with MEN-l, HPT is usually associated with osteopenia already between ages 20 and 30 years (57). In fact, no morbidity of any type from HPT has been documented before age 15 years in MEN-I (58).

Distinguishing Features in the Three Categories of Primary Hyperparathyroidisma

Feature Heredity Hypercalcemia onset age (years) Sex ratio (F:M) Calcium in urine PTH in serum Non-PTH tumors Gastrinoma interact Parathyroid pathology Surgical result c Immediate cure (%) Persistence (%) Hypopara (%) c Late recur (%)

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aThe estimated frequency of these diagnoses among 100 unselected adults is 85:2:2, with sporadic multigland tumors in most others. ~Neonatal severe primary hyperparathyroidism is rare and has been seen with a double or single dose of calcium-sensing receptor gene mutation. It should be considered as a separate and much more aggressive syndrome (273). CThese are estimated results for a team using appropriate preoperative evaluation of the patient and family and with an experienced parathyroid surgeon. ~Hypoparathyroidism is scored per se and again as a "cure" though less desirable than euparathyroidism. A delayed parathyroid autograft without hyperparathyroidism in MEN-1 would also be scored as a cure. eNA, Not applicable, because virtually zero operations lead to transient normocalcemia in FHH.

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Female:Male Gender Ratio Unlike sporadic parathyroid adenoma, with a 3:1 female:male gender ratio (21,22), HPT in MEN-1 has a similar penetrance and thus a similar prevalence in females and males (46).

Interaction with Gastrinoma HPT exacerbates the expressions of gastrinoma. Treatment of HPT in MEN-1 can decrease gastrin levels, decrease the dosage requirement for antiacidsecretory drugs, and occasionally cause remission of all biochemical features of Zollinger-Ellison syndrome (59,60). However, because of excellent gastric acid control by antiacid-secretory medications, concomitant ZES is rarely a sufficient indication for parathyroidectomy in the HPT of MEN-1.

Tumor Multiplicity The commonest parathyroid finding in sporadic primary HPT is single adenoma. In contrast, MEN-1 HPT is associated with multiple parathyroid tumors. The tumors are asymmetric; in fact, one, two, or rarely three glands may be of normal size (Fig. 2) (61).

Three Problematic Outcomes of Parathyroidectomy The frequencies of outcomes of parathyroidectomy differ in important ways between MEN-1 and other diagnoses (Table 2) (62-68). First because more than one gland is usually overactive in MEN-l, there is an increased rate of persistent HPT postoperatively, reflecting unexpected or hard to locate tumors. The incidence of postoperative persistent HPT can reach 40-60% with inexperienced surgeons (64) and is up to 25% even with experienced parathyroid surgeons (63-68). Second, because of the need for identification

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I

18

FIG. 3 Cumulative recurrent primary hyperparathyroidism after "successful" initial parathyroidectomy in 41 cases with MEN-1. The criterion for a successful operation was normocalcemia at least 3 months after surgery. Hyperparathyroidism recurred progressively (64).

and removal of most parathyroid glands, the prevalence of postoperative hypoparathyroidism is relatively high at 0-25% (63-67). Third, the incidence of postoperative recurrent HPT is strikingly high in MEN-1 to the point of being a clue to undiagnosed MEN-1 (64,66-69). The time for 50% postoperative recurrence of HPT in MEN-1 has been 8 and 12 years after "successful" subtotal parathyroidectomy (Fig. 3) (64,67). High recurrence rate is also a feature with parathyroid autografts in MEN-1 (70). Recurrence probably comes from neoplastic parathyroid tissue remaining postoperatively or from mutational genesis of new neoplastic parathyroid clone(s). These three problematic surgical outcomes, relatively specific to MEN-l, have led to modified approaches to parathyroid surgery in MEN-1 (see below). Like MEN-1 parathyroid tumors, sporadic multigland parathyroid tumors 5 show asymmetry of parathyroid tumor size (71); of course, some "sporadic" cases have unrecognized MEN-1. Recurrent hyperparathyroidism may be less common in multigland parathyroid tumors of sporadic cases than of MEN-1 cases (72).

Enteropancreatic Tumors in MEN-1 Multiplicity of Enteropancreatic Tumors Unlike sporadic enteropancreatic tumors, those in MEN-1 are virtually always multiple (44,73-75b). MEN-1 cases may oversecrete several hormones from

5"Parathyroid hyperplasia" is sometimes applied to all states with multiple parathyroid tumors. Often it is based on histologic features. Because the multiple parathyroid tumors are all or mostly monoor oligoclonal, this is best considered functionally as multiple adenomas, not a diffuse polyclonal process.

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one enteropancreatic tumor or from several independent tumors. For the same reason, an enteropancreatic t u m o r that stains immunologically for a given peptide need not be the one that is causing high serum concentrations of that peptide. In the past, MEN-1 enteropancreatic tumors became evident at a later age, compared to HPT. However, with screening at presymptomatic stages (Table 3), enteropancreatic tumors are now occasionally recognized before HPT in MEN-1 (47,50,76). Because an oversecreted enteropancreatic h o r m o n e can cause symptoms and early tumor recognition, the nonfunctional tumors (see footnote 4), if recognized at all, are noticed at later stages and with high frequency of malignancy. Gastrinoma is, by far, the most c o m m o n symptomatic enteropancreatic tumor in MEN-1 (Table 1). However, multiple unrecognized nonfunctional enteropancreatic tumors probably have similar frequency, with nearly 100% prevalence at surgery (73,77) or at autopsy (44). Gastrinoma in MEN-1

Symptoms The gastrinoma syndrome is ZES (78-81). The comm o n features are abdominal pain, reflux symptoms, and diarrhea. However, dangerous oversecretion of gastric acid may be present with no symptoms. Gastrinomas cause symptoms and morbidity mainly by gastrin-dependent oversecretion of gastric acid. Ulcers in atypical locations and intestinal perforation characterize ZES in its severe expressions; with earlier recognition and ready availability of potent antiacid-secretory medications, the ulcers do not usually have these features. Still, the delay to diagnosis of ZES averages 4 years in MEN-l, just as in sporadic cases (60). And 10% may suffer intestinal perforation before diagnosis is made (in a nonfamilial setting without periodic screening) (82). Symptoms from gastrinoma malignancy are also substantial.

Gastrinoma Multiplicity MEN-1 gastrinomas are usually multiple and originate mainly as small nodules (most below 1 cm in diameter) in the duodenal submucosa, with fewer in the pancreatic islets; this may not be a real difference from the distribution of sporadic gastrinoma, which, however, is solitary (74,81). Efforts to remove surgically the largest gastrinoma(s) in MEN-1 almost always fail to lower serum gastrin concentrations significantly (78-81). This contrasts with one-third surgical success in sporadic gastrinoma (81). In MEN-l, gastrinoma is usually associated also with multiple enteropancreatic neuro-endocrine tumors that are not gastrinomas. In one gastrinoma surgery series, the largest tumor removed was not a gastrinoma in 11 of 28 MEN-1 cases (81).

Onset Age for Tumor Expression ZES typically begins a r o u n d age 35 years in MEN-l, 10 years earlier than in sporadic cases (49,60). It has not been seen earlier than age 12 years in MEN-1 (15,43,58, R.C.Jensen, personal communication).

Exacerbation by HPT ZES in MEN-1 is usually accompanied by HPT due to earlier onset and higher penetrance of HPT (60). Elevated serum calcium concentrations exacerbate release of gastrin from gastrinomas (see above, ClinicalFeatures of HPT

That Differ in MEN-1 versus in Sporadic Cases).

Malignancy of Gastrinoma in MEN-1 ZES in MEN-1 generally follows an indolent tumor course, with about 15% of cases experiencing aggressive features of rapid tumor growth or progression of metastases (83,84). At the time of recognition, more than half of MEN-1 gastrinomas have local metastases (81 ). Hepatic metastases at surgery are particularly ominous. With these, 10-year survival is 20%; without these, it is near 100% (81,85). Gastric acid secretion can be and usually is well controlled; still, half of deaths among cases with ZES are from malignant features of their tumors, mainly from cachexia (42). No test reliably predicts whether a gastrinoma will progress to aggressive malignancy. In particular, serum concentrations of gastrin or chromogranin-A are weak indices of gastrinoma mass (86). Two groups have suggested that gastrinomas (sporadic and MEN-1 pooled) greater than 3 cm are the most likely to metastasize (83,87). Another study of 43 MEN-1 operations for gastrinoma and other enteropancreatic tumors found no relation between size of the primary t u m o r and likelihood of local metastases at surgery; however, hepatic metastases were seen only with primary tumors greater than 2 cm (88). The largest tumor in MEN-1 is almost never the only source of high circulating gastrin concentrations (81); similarly, it may not be the only or the main source of metastases. Gastrinoma features that predict decreased survival are larger primary tumor, pancreatic primary tumor, and metastases in nodes, liver, or bone (42).

Insulinoma in MEN-1 Insulinoma is expressed in 10-30% of MEN-1 cases (38,39,45,48,49,76). Fewer than 20% of all insulinoma cases have MEN-l, but most insulinoma patients with multiple islet tumors have MEN-1 (89-91). Typical onset age of insulinoma seems younger in MEN-1 than in sporadic cases (30 vs. 45 years) (48,49). Because of its low penetrance in MEN-l, insulinoma is rarely the first expression of MEN-l, but MEN-1 insulinoma onset has been noted as early as age 6 years (92). Presentation is

MULTIPLE ENOOCeaNE NEOPI~S~ TYPE 1 TABLE 3

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541

A Program of Tests and Test Schedules to Screen for Endocrine Tumor Expressiona

Tumor

Age to begin tests (years)

Parathyroid adenoma Gastrinoma

5 20

Insulinoma and other enteropancreatic

5

Anterior pituitary Thymic or bronchial carcinoid c

5 20

Biochemical tests yearly Calcium (mainly ionized), PTH Gastrin, gastric acid output; secretin-stimulated gastrinb Glucose, chromogranin-A Prolactin None

Imaging tests every 3 years None None Somatostin receptor scintigraphy; CT or MRI MRI CT

aScreening in known or highly likely (near 100%) carriers of the MEN1 mutation (identified from MEN1 mutation or other criteria). Further characterization (such as tumor localization or metastasis search) is often ind!cated after the tumor is diagnosed (see text). From Ref. 75b. °Gastric acid output measured if gastrin is high; secretin-stimulated gastrin measured if gastrin is high or if gastric acid output is high. CStomach best evaluated for carcinoids ("ECLomas") by gastric endoscopy. Thymus removed at parathyroidectomy in MEN-1.

similar to that in sporadic cases with symptoms of neuroglycopenia, mainly while fasting. Most studies suggest that insulinoma syndrome in MEN-1 results from insulin hypersecretion by a single tumor. However it is almost always associated with gastrinomas a n d / o r multiple coexistent nonfunctional pancreatic islet tumors (see footnote 4). (75,93-96), some of which may even show immunologic staining for insulin. The main or symptomatic insulinoma is generally small (0.2-3 cm) and anywhere in the pancreas. The insulinoma postoperative recurrence rate may be higher in MEN-1 than in sporadic insulinoma (20 vs. 5% at 5 years) (97). Enteropancreatic Tumors Other Than Gastrinoma o r Insulinoma in MEN-1

Enteropancreatic tumors overall in adults with MEN-1 are highly p e n e t r a n t and multiple (44,73,77) (Table 1). Their very frequency implies that most such tumors are hormonally silent (see footnote 4). As a result, such "silent" tumors may not become evident at all or only after they attain the malignant features of a large mass or of metastases. Insulinoma is the only symptomatic MEN-1 enteropancreatic tumor that has a low frequency of malignancy. Glucagonoma in MEN-1

Though many islet tumors in MEN-1 synthesize glucagon (73,75,98-100), glucagonoma is a rare cause of metabolic or malignant features in MEN-1 (58) and few glucagonoma patients have MEN-1. Glucago-

n o m a can cause a characteristic syndrome of dermatitis [migratory necrolytic erythema (101) ], weight loss, glucose intolerance, and anemia (101,102). Glucagonomas are usually large and metastatic at diagnosis (98,99). VIPoma in MEN-1

Vasoactive intestinal peptide (VIP) is occasionally oversecreted by an enteropancreatic tumor (i.e., a VIPoma). This can be associated with severe watery diarrhea ("pancreatic cholera"), hypokalemia, and hypochlorhydria (103-107). Other terms for the VIPoma syndrome are Verner-Morrison syndrome or watery diarrhea/ hypochlorhydria/achlorhydria (WDHA). Most VIPomas are sporadic, and they are rare in MEN-1 (108). MEN-1 VIPomas are in the pancreas, whereas sporadic VIPomas occasionally occur also in ganglioneuromas in children at ages 2-4 years (109,110), in intestinal carcinoids, or in pheochromocytomas (110). Up to half ofVIPomas show hypercalcemia from overproduction of PTH-related protein (PTHrP) (109,111), but coexistent true HPT is even more frequent in MEN-1. Like glucagonoma in MEN-l, VIPomas are usually large and metastatic at presentation. GHRHoma in MEN- 1

Rare tumors oversecrete growth hormone-releasing factor, and are thus called a G H R H o m a (112,113). W h e n present, a G H R H o m a is associated with MEN-1 in half of cases (114-117) and causes polyclonal e n l a r g e m e n t of the pituitary and oversecretion of growth h o r m o n e (116,118). GHRHomas occur in lung, pancreas, or small intestine. They are often large or metastatic at presentation (116).

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Somatostatinoma Somatostatinoma is a rare tumor, sometimes associated with a specific syndrome of mild diabetes mellitus, gall bladder disease, weight loss, and anemia (119,120). Most somatostatinomas are in the pancreatic head, fewer in the proximal d u o d e n u m or Ampulla of Vater (121,122). At presentation, they are usually large and metastatic; somatostatinoma syndrome has been rare in MEN-1 (123).

Other Enteropancreatic Tumor Secretions and Syndromes Malignant enteropancreatic tumors may oversecrete ACTH. This is occasional with sporadic gastrinoma, but rare in MEN-1 gastrinoma (124,125). Oversecretion of PTHrP is less c o m m o n with other enteropancreatic malignancies than with VIPoma (see above) (111,126,127). Some enteropancreatic tumors oversecrete calcitonin, but this causes no syndrome (128). Furthermore, this must not be confused with the calcitonin overproduction by C cell neoplasias in MEN-2. Few tumors oversecrete neurotensin, and no clear syndrome is associated (122).

Nonfunctional Tumors and PPomas Pancreatic islet tumors that synthesize a n d / o r secrete pancreatic polypeptide (i.e., PPomas) and other nonfunctional tumors are c o m m o n in MEN-1 (76,129-131) (Table 1). These tumors are often multiple in MEN-1 (73,75,77) and recognized at surgery or autopsy. For similar reasons, multiple pancreatic islet tumors, including functional ones, should always be considered as highly suggestive of MEN-1 (73,77). Nonfunctional tumors may cause symptoms from enlargement or metastasis.

Foregut Carcinoid Tumors in MEN-1 Carcinoid Tumors of the Thymus or Bronchus Sporadic carcinoid tumor occurs in derivatives of foregut, midgut [the principal location without MEN-1 (132)], and hindgut (133). In contrast, MEN-1 carcinoid is virtually limited to foregut derivatives (thymus, bronchus, stomach, pancreas, d u o d e n u m ) (134). Thymic or bronchial carcinoid each occur in 2-4% of MEN-1 cases (Table 1). Thymic carcinoid occurs predominantly in males in either MEN-1 or sporadic cases (135); bronchial carcinoid occurs predominantly in females in MEN-1 versus in a 1:1 gender ratio in sporadic cases (136). MEN-1 thymic carcinoid has been commonest a m o n g cigarette smokers (135). Certain MEN-1 families may have a higher penetrance for carcinoids (137) (see below). Average age at presentation of thymic carcinoid in MEN-1 is 45 years, i.e., later than other MEN-1 tumors (135,137). Later recognition could reflect, in part, the asymptomatic stages of carcinoid tumor in MEN-1.

Thymic carcinoid may be discovered incidental to chest imaging, incidental to routine thymectomy, or, more commonly, at an advanced stage as a symptomatic and locally invasive mass. If widely metastatic, usual sites are bone, lung, and liver. Bronchial carcinoid can present incidental to imaging, as bronchial obstruction, or as metastases. Thymic carcinoid in MEN-1 is more aggressive (60-90% malignant) than bronchial carcinoid (10-20 % malignant) (135,137,138,139). Sporadic thymic carcinoids often (30-40%) secrete ACTH (140,141) and occasionally secrete GHRH (142). MEN-1 carcinoid tumors of thymus or bronchus rarely oversecrete ACTH or GHRH, rarely cause carcinoid syndrome, and rarely oversecrete serotonin; thus, they can usually be considered to be "nonfunctioning." They may oversecrete histamine, which is metabolized to 5-hydroxyindoleacetic acid and easily measurable in urine (80). Gastric ECLomas

Histamine-secreting enterochromaffin-like cells (ECL cells) are prominent among the endocrine cells of the h u m a n gastric oxyntic mucosa. In sporadic cases they can give rise to tumors sometimes unrelated to hypergastrinemia. On the other hand, these tumors seem gastrin induced in a setting of hypochlorhydria (143). In MEN-l, ECLomas (gastric carcinoids) are associated with hypergastrinemia (144,145). Hypergastrinemia may stimulate ECL proliferation and thus ECLoma formation (143-149). ECLomas have been recognized in up to 15% of MEN-1 cases as an incidental finding during endoscopy (150), but they are not associated with sporadic gastrinoma. Gastric ECLomas do not cause a hormonal syndrome, and their course is unknown. Some have substantial malignant potential (151 ).

Duodenal Carcinoid in MEN-1 Carcinoid tumors are structurally indistinguishable from enteropancreatic tumors (152); the term carcinoid is usually reserved for those enteropancreatic islet tumors not containing gastrin or a normal islet hormone. Duodenal carcinoid, unlike duodenal gastrinoma, is u n c o m m o n in MEN-1 (153). Sporadic carcinoid of the midgut or hindgut has no apparent association with MEN-1. Metastasis from carcinoid of the terminal ileum is the commonest cause of the carcinoid syndrome (154); this primary tumor location or carcinoid syndrome is not a feature of MEN-1 (80).

Anterior Pituitary Tumors in MEN-I Symptoms and signs of pituitary tumor are present in 10-30% of symptomatic MEN-1 cases. The symptoms, signs, presenting age, and h o r m o n e distribution

MULTIPLE ENDOCRINE NEOPLASIATn'E 1

are similar to those in sporadic pituitary tumors, excepting for an increased fraction with prolactinoma in MEN-1 (28,31,34,39,48,49,155). Similarly, a substantial portion of MEN-1 pituitary tumors are "nonfunctional." The nonfunctional category includes pituitary tumors producing and even oversecreting subunits of glycoprotein h o r m o n e s (156,157). Like the sporadic counterpart, MEN-1 pituitary tumors can give mass effects such as visual compromise, hypopituitarism, or pituitary apoplexy (34,36,76).

Other Endocrine Tumors in MEN-1 Primary Adrenocortical Neoplasms in MEN-1 Silent adrenal cortical enlargement in MEN-1 in up to 40% of cases can be unilateral or bilateral (158). Most are recognized by external imaging without surgical confirmation. Rare MEN-1 cases have shown hypercortisolism, hyperaldosteronism, or adrenocortical cancer (43,90,158-160).

Pheochromocytoma in MEN-1 Pheochromocytoma has been reported six times (representing a total of seven cases) in familial MEN-1 (48,49,76,161-162c). Documentation of many features has been incomplete in each of the seven tumors. Each has been unilateral, most have been incidental or chemically silent, and one was malignant (76). A chromosome 11q136 loss of heterozygosity (LOH) was documented in two MEN-1 pheochromocytomas, each associated with a different germ line MEN1 mutation (162a,b). Based on this evidence for "two hits" at the MEN1 locus (see following), pheochromocytoma seems a valid, albeit rare, expression of MEN-1.

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lation has made this association difficult to use in diagnosis of MEN-1. Lipoma was found in one-third of MEN-1 cases versus in 6% of controls (163). Lipoma in MEN-1 is generally dermal and small, often multiple. Rarely it is recognized as large visceral lipoma (164).

Facial Angiofibromas Multiple facial angiofibromas have been found in 40-88% of MEN-1 cases but in no controls (163,163a). Half of MEN-1 cases have five or more angiofibromas. The angiofibromas are similar to but smaller than those in tuberous sclerosis, excepting that they may extend across the vermillion b o r d e r of the lips in MEN-1 (163).

Collagenoma Collagenomas were observed in 72% of MEN-1 cases and in no controls (163). These are whitish papular lesions about the trunk, but sparing the face and neck.

Other Nonendocrine Tumors Additional cutaneous lesions have been seen (confettilike hypopigmented trunk macules and multiple gingival papules) but not yet proved specific for MEN-1 (163), nor even proved to be clonal neoplasms. Several muscle tumors have been noted (37,43,162a,163b). Four cases of spinal or cerebellar e p e n d y m o m a have been noted in MEN-1 (52,92,165,166a,b). Rare association of MEN-1 with malignant m e l a n o m a has been suggested (166b). Several large clinical series have not established other rare associations with MEN-1 (38,43).

PHENOTYPES OF MEN-1 IN FAMILIES

Thyroid Follicular Neoplasm in MEN-1

Importance of Large Kindreds

Thyroid follicular adenoma has been mentioned since the earliest MEN-1 reviews (43), but r a n d o m association with a c o m m o n thyroid neoplasm remains a possible explanation. The thyroid gland receives unusual attention because of the high frequency and extensiveness of parathyroid exploration in MEN-1 (49).

Some hereditary syndromes, such as MEN-l, can virtually always be attributed to mutation in one gene (167,168). Others, such as familial hypocalciuric hypercalcemia, MEN-2 (169), or neurofibromatosis (170), have distinctive variants that can arise from mutation in one or few underlying genes. Such variants are also termed subtypes or disease phenotypes. Phenotypes can also be caused by mutation-specific features, interaction with complex genetic background, or nongenetic factors (including ascertainment bias). MEN-1 is remarkably polymorphic. Thus a small MEN-1 family could show "atypical" features by r a n d o m chance. Several reports based on small families have called attention to unusual familial features of uncertain generality to other MEN-1 families: high prevalence of ACTH-producing tumor in a family (171), several insulinomas in a family (47), several carcinoids in a family (137), or clusters of aggressive gastrinoma within part of

Nonendocrine Tumors in MEN-1 ipoma

Lipoma has been associated with MEN-1 for over three decades (43). Its frequency in the general popu6 S h o r t h a n d for a c h r o m o s o m a l locus: NcB, w h e r e "N" is c h r o m o s o m a l n u m b e r (1-23, X, or Y), "c" is character(s) (p = short arm, q = long arm, cen = c e n t r o m e r e , tel = telomere), a n d "B" is n u m e r i c designation of traditional cytologic band. T h u s 11q13 is c h r o m o s o m e 11, long arm, b a n d 1.3.

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a kindred (47,172). The optimal clinical data to establish a distinctive variant should come from one or more large, well-studied kindreds.

with regard to lactotrope function. Two twin pairs showed prolactinoma in each m e m b e r (187,188a) and one pair had prolactinoma in neither case (188b).

Familial Isolated Hyperparathyroidism as an MEN-1 Expression

Homozygous M E N - l - - D o e s It Exist?

HPT is the earliest and also the commonest expression of MEN-1. Thus, familial isolated HPT will sometimes be an expression of MEN-1. In fact, several kindreds reported with this diagnosis were subsequently shown to express more typical MEN-1 (45,173). Furthermore, approximately one-fifth of kindreds with familial isolated HPT have M E N 1 mutation. Two kindreds with familial isolated HPT and MEN1 mutation were large (7 and 13 affected members), increasing the possibility of a durable MEN-1 phenotype. The M E N 1 mutations in these two kindreds were similar and not reported in typical MEN-l, giving further support for this possibility (174-176) (see below). When no MEN1 mutation is identified in familial isolated HPT, the likely syndrome causes are (1) an undiscovered MEN1 mutation, (2) hyperparathyroidism-jaw tumor syndrome, (3) familial hypocalciuric hypercalcemia, and (4) other syndromes (see below). The differential diagnosis relies on clinical data, genetic linkage analysis, and mutation analysis. When no specific diagnosis is available, a family member must be followed with this heterogeneous list of syndromes in mind.

Prolactinoma Variant Three large MEN-1 kindreds (9-83 affected members per family) have shown a high penetrance of HPT (90%) and ofprolactinoma (35-65%) but low penetrance of gastrinoma (3-11%) (177-184). Four families in Newfoundland were descended from a c o m m o n founder (183), and thus treated together as the single largest family. This largest family also showed 14% carcinoid tumors (181). The trait in this variant has been termed the prolactinoma or Burin variant of MEN-1. HPT may have a later onset age in the prolactinoma variant than in typical MEN-1 (184). It seems that each of the three families has an MEN1 mutation with no other common mutation feature (37). A fourth kindred with similar phenotype is difficult to assess because many affected members are too young to express ZES (185). Three other observations have suggested that prolactinoma expression in MEN-1 could d e p e n d on other genetic background factors. First, in a large Tasmanian MEN-1 family, prolactinoma had high penetrance only in selected branches (186). Second, three pairs of identical twins have been described in three different MEN-1 families. T h o u g h each pair was discordant for some MEN-1 features, each was concordant

Germ line inactivation of both MEN1 alleles seems rare, if not lethal in utero. One daughter from parents with heterozygous MENI~u,~" had galactorrhea at age 23 (presumed prolactinoma) and died before 1980 at age 30 of an invasive thymic carcinoid, without known expression of HPT (178). Her MEN1 allele or mutation status was not known. In another kindred, three offspring of unrelated parents, both with MEN-l, were characterized by haplotype analysis about the MEN1 locus; two offspring were judged to be compound heterozygotes for MEN1 mutations and one a simple heterozygote (189a). MEN1 mutation has been identified thus far in only one side of this family (189b), so compound heterozygosity for MEN1 mutation in not proven. One presumed compound heterozygote was a 40-year-old female with HPT and nonfunctional adrenal cortical adenoma, the other a 35-year-old male with HPT. Their endocrine tumors were typical of MEN1 heterozygotes. However, each had unexplained infertility, which might thus have been caused by compound heterozygosity for mutant MEN1. Endocrine tumors from both siblings showed 11q13 LOH (190). According to a two-hit mechanism (see below), 11q13 LOH is usually an index of somatic inactivation of a normal MEN1 copy; therefore, this finding also causes uncertainty as to whether either is an MEN1 germ line compound heterozygote.

DIFFERENTIAL DIAGNOSIS Because of its polymorphic features, MEN-1 is associated with a large n u m b e r of states to consider in differential diagnosis (Table 4).

Nonhereditary Neoplasia of One Endocrine Tissue MEN-1 characteristically begins as HPT alone. Thus HPT is usually the central feature in the MEN-1 differential diagnosis (Table 4). At the same time, approximately 5-10% of MEN-1 cases first present with only prolactinoma, only gastrinoma, or only insulinoma (50,58,76). Usually a history of familial involvement of MEN-1 is easy to elicit (26). Conversely, the likelihood of MEN-1 among all endocrine tumors is variable: about 2-3% in HPT, 20% in gastrinoma, 10% in insulinoma, 5% in nonfunctioning enteropancreatic tumor, and 5% in prolactinoma (16-32,90,97). Some features that increase the likeli-

MULTIPLE ENDOCRINE NEOPIASIA TYPE 1

TABLE 4

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Syndromes and States with Important Relations to MEN-1 a Hereditary multiple hyperfunction

Hereditary single hyperfunction

Multiple neoplasia, of which one is endocrine Hyperparathyroid-jaw tumor Hereditary breast/ovarian cancer Cowden syndrome P53, b, TSC, FAP, etc. Multiple neoplasia with multiple endocrine neoplasia Multiple endocrine neoplasia type 2A von HippeI-Lindau syndrome Carney complex Neurofibromatosis type 1

Single endocrine neoplasia Familial isolated hyperparathyroid Familial isolated pituitary tumor Familial isolated adreocort tumor Islet, carcinoid, or thyroid Single endocrine hyperfunction that is not neoplastic Familial hypocalciuric hypercalcemia PHHI ~ Testotoxicosis Neonatal thyrotoxicosis

Nonhereditary hyperfunction Single endocrine neoplasia Parathyroid "hyperplasia" Gastrinoma Prolactinoma Insulinoma Multiple endocrine hyperfunction Random cooccurrence Postradiation to neck Tumor secretes ACTH, TSH, or GHRH McCune-Albright syndrome

aFor consideration in differential diagnosis or in relation to mechanisms. One state that can frequently include hyperparathyroidism is at the top of each category. bP53 - Li-Fraumeni cancer family syndrome from TP53 mutation; TSC, tuberous sclerosis; FAP, familial adenomatous polyposis of the colon; PHHI, persistent hyperinsulinemic hypoglycemia of infancy.

hood of MEN-1 in any of these apparently sporadic cases are tumor multiplicity and early age of tumor onset.

One Endocrine Neoplasm as Cause of a Second Endocrine Hyperfunction

MEN-1 is but one (albeit the most striking one) among a list of causes for sporadic endocrine neoplasms in several tissues.

Endocrine tumor products that can cause a downstream endocrine tumor include ACTH (203,204), GHRH (28,113,117), and TSH (155). The downstream tumor is likely to be polyclonal or hyperplastic. Among these endocrinopathies, only GHRH oversecretion from neuroendocrine tumor is suggestive of MEN1 gene mutation (117).

Random Concurrence of Two Sporadic Neoplasms

McCune-Albright Syndrome

Because sporadic endocrine neoplasias are not uncommon, the more common sporadic endocrine neoplasias will occasionally be paired with another endocrine tumor. Case reports have also documented less common associations, such as intriguing elements of MEN-1 and MEN-2 in a single patient (161,191-197). When the association is one otherwise typical of MEN-l, then additional MEN-1 features must be carefully sought. The "sporadic" association of HPT and ZES is usually caused by MEN1 germ line mutation (37); in contrast, the sporadic association of HPT and acromegaly is usually not associated with MEN1 mutation (36).

McCune-Albright syndrome includes fibrous dysplasia of bone and multiple caf~-au-lait spots. The main endocrine expressions are GH-secreting pituitary tumor and ovarian tumor (oversecreted estrogen causing precocious puberty). The syndrome is caused by activating mutation of the GNAS1 gene that encodes Gsot, the activating subunit of the heterotrimeric G protein that regulates adenylate cyclase and other effectors (205). The mutation may be lethal to the embryo because all or most cases have arisen postzygotically, resulting in a mosaic status (205,206). The mutated Gse~ contributes to growth and h o r m o n e secretion in certain tissues (207). Such mutation occasionally might cause parathyroid neoplasia (208). Fibrous dysplasia and perhaps other associated pathologic tissues are mosaic, with likely depending upon admixture from the mutant G~ot clone and from the normal cells (209). The embryonic mutant clone, per se, does not show clear neoplastic features. Malignancy (osteosarcoma, chondrosarcoma, etc.) is rare (205). The hyperfunctional tissues have not been tested for clonal evolution.

N o n h e r e d i t a r y H y p e r f u n c t i o n o f Several Endocrine Tissues

Thyroid and Parathyroid Neoplasms Following Radiation Radiation is an established cause of thyroid neoplasia (198,199). A role of radiation in parathyroid neoplasia also seems highly likely (199-202). The postradiation thyroid neoplasms have high malignant potential; the parathyroid neoplasms may be multiple but are virtually always benign.

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Hereditary Multiple Neoplasias, of Which One Neoplasm Is Endocrine The multiple neoplasia syndromes, comprising a broad group, have received intense scrutiny because of their potential and realized contributions to cancer pathophysiology (210). A single endocrine tumor in some cases varies between being a major or a minor component.

Hyperparathyroidism-Jaw Tumor Syndrome The hyperparathyroidism-jaw tumor syndrome (HPT-JT) combines parathyroid tumors (benign or malignant), cemento-ossifying fibromas of the jaw, multiple kidney cysts, Wilms tumor, kidney hamartomas, and nephroblastoma (211-213). HPT is the commonest expression with about 80% penetrance by age 40 years; thus, HPT-JT can occur as familial or sporadic isolated HPT (214,215). Typically, HPT first appears around age 30 years, with slightly lower penetrance in females. Parathyroid tumors may present as asynchronous parathyroid adenomas. The parathyroid may show numerous cysts of all sizes (216) and increased Ki-67 immunostaining, suggesting increased nuclear proliferation (217). In fact, parathyroid cancer occurs in a higher fraction (10%) of cases in this syndrome than in any other setting. The gene for HPT-JT is at chromosome lq24 (212) and is likely a tumor suppressor gene, because loss of heterozygosity at l q has been seen in some parathyroid tumors and renal hamartomas from the syndrome (213,215,218).

Other Hereditary Multiple Neoplasias with One Endocrine Neoplasm Additional syndromes with one endocrine neoplasm include Cowden syndrome (thyroid neoplasms associated) (219), Li-Fraumeni syndrome (adrenocortical cancer associated rarely) (220,221), tuberous sclerosis (TSC) (functioning or nonfunctioning pancreatic islet tumor rarely associated) (222), adenomatous polyposis of the colon (thyroid neoplasms associated) (223,224), and breast cancer (ovarian neoplasms associated) (225).

Hereditary Multiple Neoplasia Syndromes with Multiple Endocrine Neoplasms By definition, each MEN syndrome is also part of the broader category of multiple neoplasia syndromes. Interestingly, most MEN syndromes include nonendocrine neoplasms, varying from a major to a minor feature.

Multiple Endocrine Neoplasia Type 2 MEN-2A is a complex of C cell neoplasms of the thyroid with high malignant potential, pheochromocytoma, and HPT. A less aggressive variant (familial

medullary carcinoma of the thyroid) has isolated medullary thyroid cancer, and a more aggressive variant (MEN-2B) also has no HPT but includes mucosal and intenstinal neuromas and a marfanoid habitus (169). The thyroidal C cell tumors secrete calcitonin and other products. Though hypercalcitoninemia evokes no metabolic effect, it can be a highly convenient marker of C cell tumors. HPT has lower and later penetrance than the C cell and adrenomedullary features, but it may reach 80% by age 70 years in MEN-2A (226). MEN-2 rarely would resemble MEN-l, and it has not presented as familial isolated HPT, though this is remotely possible. Though there is little clinical overlap between MEN-2 and MEN-l, there is an overlap in syndrome nomenclature that leads to major confusion between these disorders. Efforts should be expended to assure that biochemical and even genetic MEN1 testing is not done in MEN-2 families and vice versa. Furthermore, the strategy of high priority for genetesting and malignancy intervention is applicable to MEN-2 but not to MEN-1 families (see below).

Other Multiple Neoplasias Causing Multiple Endocrine Tumors Other MEN syndromes have names that do not highlight an endocrine component. Furthermore, in clinical practice, they cannot be confused with MEN-1. Their relevance here lies in the unknown pathobiology that results in targeting neoplasms to multiple endocrine tissues.

von Hippel-Lindau Syndrome The major morbidities from von Hippel-Lindau syndrome include renal clear cell carcinoma, retinal angiomatosis, and hemangioblastoma of the brain (227). The endocrine features are pheochromocytoma and pancreatic islet cell cysts and tumors. The latter occasionally stain for insulin but do not oversecrete this hormone (228). The syndrome is caused by a known tumor suppressor gene (227).

Carney Complex Carney complex includes myxomas (notably intracardiac) and skin lentigines. The endocrine features are pigmented nodular adrenocortical hyperfunction with hypercortisolism and hormone nonsecreting tumors of thyroid, sertoli, and Leydig cells (229-231). Genetic linkage studies have implicated two different loci at 2p15 and 17q2 (232,233a). The gene at 17q has been discovered to encode a known regulatory subunit of protein kinase-A (cyclic AMP dependent protein kinase) (233b).

Neurofibromatosis Type 1 Neurofibromatosis type 1 includes peripheral neurofibromas and caf6-au-lait spots (170). Pheochromocytoma

MULTIPLE ENDOCRINE NEOPLAStA T~E 1

is common. Rare associations include HPT (234) and duodenal somatostatinoma (235). The syndrome is caused by a known tumor suppressor gene (170).

Hereditary Neoplasia of Only One Endocrine Tissue Hereditary neoplasia in only one tissue is rare.

Isolated HPT Familial isolated HPT has been described in many small kindreds, generally with autosomal d o m i n a n t transmission (236). D e p e n d i n g on selection criteria, approximately one-fifth have MEN1 mutation (37,45,174-176) (see below). Perhaps a similar fraction have familial hypocalciuric hypercalcemia (see below), and some have H P T - J T (see above). There could be subgroups of familial isolated HPT from other genes that have not yet been delineated, such as a gene causing solitary parathyroid a d e n o m a (236-238). One kindred had features of autosomal recessive transmission (239). At the same time, it is also possible that after full delineation of the above three principal causes, few families will remain unclassified. A rare and u n r e p o r t e d etiology would be MEN-2A.

Isolated Pituitary Tumor Isolated autosomal d o m i n a n t pituitary tumor, mainly somatotropinoma, has been seen in approximately 20, mostly small, families (240-244a,b). Isolated prolactinoma has been d o c u m e n t e d incompletely in six small families (245,246). Another particularly unusual family had one prolactinoma, two somatotropinomas, and one case of hyperparathyroidism (247). Because no member expressed tumors in two tissues, this family would not m e e t the simplified definition of MEN-1 (see above). Approximately 15 isolated pituitary tumor families have been tested without any revealing MEN1 mutation (244a,248-249). An 11q13 L O H was found in pituitary tumors of two of these families, and the retained allele was possibly mutated based on haplotype analysis in one (243,244a). Genetic linkage analysis in the largest somatotropinoma family showed linkage to 11q13 and, surprisingly, also to 2p (250). No MEN1 mutation was found in it (244a), suggesting undiscovered mutation of the MEN1 gene or mutation of another gene near it at 1 l q l 3.

Other Solitary Endocrine Tumors There are two reports of familial isolated insulinoma (251,252). There have been several reports of familial clustering of isolated carcinoid (253-257), including one family with three cases of duodenal carcinoid (257). There have also been familial clusters of isolated

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adrenocortical tumors (258,259) or of isolated thyroid oxyphil tumors (260,261 ).

Hereditary Single Endocrine Tissue Hyperfunction That Is Not Neoplastic At least one major hereditary syndrome in the MEN-1 differential diagnosis has no neoplastic component (262).

Familial Hypocalciuric Hypercalcemia Familial hypocalciuric hypercalcemia (FHH) or familial benign hypocalciuric hypercalcemia is expressed as autosomal dominant hypercalcemia with few if any of the usual symptoms of HPT (263) (Table 2). Hypercalcemia is expressed continuously from birth and with near 100% penetrance. Urine calcium excretion rate is not proportional to the hypercalcemia (i.e., it shows the distribution of normal urine calciums), so the rate of nephrolithiasis is not increased over the population background. PTH concentrations typically are in the normal range, with mild elevation in 10% of cases (264). Subtotal parathyroidectomy in F H H inevitably fails to cure hypercalcemia (263): this and similar prevalence to MEN-1 make F H H a particularly important syndrome in differential diagnosis of MEN-1 (Table 2). The parathyroids in F H H are only mildly hyperplastic and are usually interpreted as normal (265). F H H and MEN-1 likely have similar population prevalence, and both syndromes are likely to be recognized at routine screening as asymptomatic hypercalcemia of adolescents or young adults. F H H is caused by an inactivating mutation of the calcium-sensing receptor (CaSR) gene (CaSR) at chromosome 3q (266,267). In two families, genetic linkage has excluded chromosome 3q but implicated two genes at 19p and 19q (268,269). The main function of the CaSR is mediation of serum Ca 2+ regulation of PTH secretion (270). CaSR inactivation shifts the parathyroid cell set point for PTH secretory inhibition to a higher extracellular ionized calcium (271). Similar dysfunction in the renal distal tubule CaSR likely explains the lack of hypercalciuria despite increased filtered calcium load in F H H (272). The nonneoplastic nature of the abnormal parathyroids in F H H is emphasized by the consequences of a homozygous CaSR-inactivating mutation. In both humans and mice, homozygous CaSR inactivation causes m u c h more severe dysregulation of PTH secretion and massive parathyroid hyperplasia by infancy but no recognized a d e n o m a or even nodularity of the parathyroid (262,273,274). Submicroscopic parathyroid features in FHH, such as clonality, have not been tested directly in F H H heterozygotes or homozygotes to allow more definitive statements about possible clonal evolution (262).

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Persistent Hyperinsulinemic Hypoglycemia of Infancy Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a syndrome of severe infantile hypoglycemia (275). One variant has later onset (276). Inheritance can be at.'tosomal dominant or autosomal recessive. Four genes, accounting for the majority of familial cases, have been identified (276-281). These genes are SUR1 (encodes pancreatic islet beta cell potassium channel subunit), Kir6.2 (encodes another subunit of the potassium channel), GLUD1 (encodes mitochondrial glutamate dehydrogenase), and GK (encodes cytoplasmic glucokinase). Each encodes a protein in the pancreatic islet beta cell pathway of glucose sensing/insulin exocytosis (262,275). The pancreatic beta cells show only subtle histologic abnormality with no neoplastic features (282). Subtotal removal of the pancreas is ineffective in these familial variants. Certain defects (that do not disrupt the islet potassium channel) can be controlled with diazoxide (262). Most PHHI cases are sporadic and not caused by the above mutations. One-third of these have a focal pancreatic abnormality, focal adenomatous hyperplasia (282,283). This latter process is a clonal neoplasm that can be cured by focal pancreatic resection. The origin of the tumor clone often is heterozygous germ line inactivation of SUR1 with inactivation the other SUR1 copy through 11 p LOH (284,285). Classification of focal adenomatous hyperplasia of pancreatic islets is difficult. The heterozygous mutation is hereditary but not yet recognized in several members of a family; thus, focal adenomatous hyperplasia has extremely low penetrance.

Other Hereditary Endocrine Hyperfunctions

without Neoplasia

Other nonneoplastic endocrine hyperfunctions, including PHHI (see above), cannot be confused with MEN-1 but have mutated genes that are mechanistically relevant. Autosomal dominant endocrine hyperfunction can arise with activating mutation of the TSH receptor (286,287) or the LH receptor (288). In these cases receptor activation can lead to not only hormone hypersecretion (iodothyronines or androgens, respectively) but also presumed polyclonal proliferation of the mutant receptor-carrying cells; among these four nonneoplastic syndromes, accumulation of overactive hormone-secreting cells is least notable or absent in the parathyroids of FHH (262,265). Clonal endocrine neoplasia has been reported from somatic mutation of the TSH receptor in the thyroid or the LH receptor in the ovary (287,289,290) but not the CaSR in the parathyroid (291,292). To summarize, certain disorders need often to be distinguished from MEN-1 (Table 4). The non-familial

disorders include sporadic multiple parathyroid adenomas, sporadic gastrinoma, and sporadic occurrence of tumors in two tissues, particularly hyperparathyroidism and somatotropinoma. Familial disorders include isolated hyperparathyroidism, particularly from HPT-JT, FHH, and other familial hyperparathyroidism. Other relevant considerations are familial somatotropinoma or familial prolactinoma. Within this list, the disorders often most relevant to differential diagnosis of MEN-1 have HPT as their main expression, just as does MEN-1.

SHARED ASPECTS A M O N G MEN-1 AND O T H E R MULTIPLE NEOPLASIAS Features of the Age of Onset of Tumors

Postnatal Delay until Onset of Tumors Many dominant hereditary syndromes are expressed fully at birth, even with only one mutated allele. This feature is termed a gene dosage effect, haploinsufficiency, or sometimes a dominant negative effect. Obvious examples include collagen mutations (causing osteogenesis imperfecta) and keratin mutation (causing epidermolytic skin disorders) (293). Certain endocrine hyperfunction syndromes have such a dosage effect: FHH and PHHI (see above) (262). These endocrine syndromes are nonneoplastic, fully expressed at birth, and not generally associated with stepwise or other progression of dysfunction in the endocrine tissue. In contrast, the hereditary neoplasias almost always have a postnatal delay until the first tumors become evident. For example, in MEN-1 the earliest ages for recognition of the principal tumors have been as follows: parathyroid tumor, 5-8 years; gastrinoma, 12-21 years; prolactinoma, 5-10 years; and insulinoma, 6-9 years (see above).

Average Agefor Onset of Tumor Is Younger in MEN-1 Than in Sporadic Single Tumor Many types of neoplasia, despite their postnatal delay until onset, typically begin earlier in familial than in sporadic cases. This includes C cell neoplasia (169); pheochromocytoma in von HippelLindau syndrome, MEN-2, or neurofibromatosis type 1 (169,170,227); retinoblastoma (294), and colon cancer (227). In MEN-1 this age disparity is striking for HPT onset (25 years in MEN-1 vs. 55 years in sporadic cases) but less impressive for gastrinoma (35 vs. 45 years) or insulinoma (30 vs. 45 years); there is no apparent age differential for prolactinoma (35 vs. 35 years) (49).

MULTIPLE ENDOCRINE NEOP~SIn T ~ E 1

Tumor Multiplicity Multiplicity of Tumors within a Tissue Hereditary neoplasias typically express multiple tumors in the affected organs. This can result in bilaterality of tumors in a paired organ, such as the eye [with retinoblastomas (294)] or adrenal medulla [with pheochromocytomas in MEN-2, von H i p p e l - L i n d a u syndrome, or neurofibromatosis type 1 (169,170,227) ]. A major characteristic of MEN-1 is multiplicity of neoplasms within a tissue: the parathyroids, enteropancreatic tissues, including gastrinoma and ECLoma, and dermis. Bilaterality has not been apparent in MEN-1 tissues with lower penetrance for tumors: thymus, bronchus, anterior pituitary, or pheochromocytoma. Tumor multiplicity can result in tumor recurrence after an apparent cure involving partial removal of the tissue as in the colon (with polyps for FAP) (223) or the kidney (with clear cell cancers for VHL) (227). Recurrence of HPT and possibly of hyperinsulinism in MEN-1 is another expression of this (64-67,97).

Multiplicity of Tissues ExpressingNeoplasia Approximately 30 highly p e n e t r a n t familial neoplasia syndromes have been identified; most express neoplasms in multiple tissues (210). MEN-1 is perhaps the most dramatic in tissue multiplicity, with neoplasms of at least 15 hormone-specific tissues, fewer if the tumors are pooled and grouped as enteropancreatic or anterior pituitary (Table 1).

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lation (APUD) system (297). A subsequent and related suggestion was a c o m m o n origin from ectoderm of neural crest (298). It is now believed that gut endocrine cells are not derived from ectoderm of the neural crest but from e n d o d e r m (299). Thus, even a m o n g the endocrine tumors of MEN-l, there is no c o m m o n embryologic derivation (299,300). The mesenchymal tumors in MEN1 also diverge from this pattern. One interesting speculation about the puzzling tissue distribution is that the underlying tumor suppressor (such as menin protein, encoded by MEN1) is part of a redundant process. For each disorder (such as MEN-l) of a tumor suppressor gene, the redundancy may exclude only the affected tissues (such as the parathyroid and the pancreatic islet) that are thereby specifically susceptible to tumorigenesis from MEN1 gene inactivation.

Multiple Neoplasia Syndrome Generally Has Malignant Potential In each multiple neoplasia syndrome there is a multiplicity of benign neoplasms [for example, in intestinal polyposis (223) or nevoid basal cell nevus syndrome (301) ]. In most syndromes, a fraction of neoplasms can become malignant and thereby dominate the disorder. This is seen in MEN-1 with enteropancreatic or foregut carcinoid malignancies. The few syndromic exceptions with little or no malignancy are Carney complex and several other syndromes that may not be neoplastic in essence (Table 4, and see above) (162,205,230,231).

Puzzling Combination of Tissues with Neoplasms

The Two-Hit Hypothesis~An Oncogenesis Mechanism from Epidemiology

The pattern of tissue involvement in the multiple neoplasia syndromes usually does not provide insight into the underlying disorder. An exception is the multiple exostosis syndrome arising from mutations in tumor suppressor genes encoding either of three homologs that interact with glycosyl transferase enzymes; such enzymes have a cartilage matrix-specific role (295,296). Often the different tumors within a multiple neoplasia syndrome are from tissues without known developmental relation. For example, von Hippel-Lindau syndrome includes renal clear cell cancer and cerebellar hemangiblastoma (227), Li-Fraumeni syndrome includes endometrial cancer, glioblastoma, and leukemia (220), and HPT-JT includes parathyroid neoplasms, jaw fibromas, and Wilms tumor (211). Similiarly, in the case of MEN-l, there is no unifying developmental or other known relation a m o n g the many endocrine tumors and the several mesenchymal tumors. Early theories suggested that affected endocrine tissues in MEN-1 had c o m m o n embryologic origins in the amine precursor uptake and decarboxy-

Knudson in 1971 analyzed some of the unusual features in the epidemiology of hereditary versus sporadic retinoblastoma. He hypothesized that these neoplasms developed by two events (two hits) (302). It is now clear that most hereditary tumors are caused by sequential inactivation of both copies (two hits) of one gene (sequential inactivation as precursor to neoplasia is the definition of a tumor suppressor gene), initiating release of one or a few tumor precursor cells from restraints on clonal expansion (303). Sporadic tumor can occur if the first hit is a somatic mutation at that gene. Sporadic tumor can also be initiated by a nonmutational (epigenetic) first hit, such as gene inactivation by methylation (304). Because hereditary cases have the first hit in all cells at birth as an inherited germ line mutation, any second hit alone may be sufficient to initiate a neoplasm. Thus, the patient, receiving one mutation through germ line inheritance, is primed to develop tumors early, in multiple tissues, and at multiple loci of any susceptible tissue (305) (Fig. 4). Most tumors evolve in a more complex manner, with multiple steps and with involvement of several genes (306).

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1st Hit

2nd Hit

Patient"

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1st Hit

2nd Hit

Patient without

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MEN1

I I Chromosome 11 in Germline Nucleus

MEN1Gene O Normal ~" Inactivated

I

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Chromosome 11 Changes in Somatic Nucleus

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Tumor Clone

FI6. 4 Neoplasia contribution from a tumor suppressor gene, such as MEN1. The two copies of chromosome 11 on the left show the inherited DNA pattern (germ line nucleus). Middle portions show changes in a clone precursor cell (somatic cell nucleus). The first hit to the MEN1 gene is generally small, such as a point mutation; though it inactivates one MEN1 gene copy it may have no immediate biologic effect on the cell. The first hit can be inherited with the germ line, thereby being identical in every cell (MEN1 carrier, upper row). Alternately, the first hit can be a rare and silent event in a somatic cell nucleus (patient without MEN-l, lower row). The second hit in a somatic cell typically involves a much larger portion of the other, normal chromosome. It, thereby, inactivates the remaining active copy of MEN1. Thus, the two brakes on growth of the susceptible cell (such as a parathyroid cell) a r e removed, and the cell can proliferate as a neoplastic clone (305).

The two-hit hypothesis applies not only to tumor suppressor genes but also to growth-promoting genes, also known as oncogenes or direct-acting oncogenes. Inheritance of a mutated MET oncogene causes multiple papillary kidney cancers (227). The MET gene, like RET, encodes a transmembrane tyrosine kinase that becomes oncogenic by an activating mutation (227). The second hit at the gene locus might be amplification of the mutated MET gene (307). Hereditary tumors caused by mutated oncogenes, such as METand its h o m o l o g RET, show tumor multiplicity, early tumor onset, tumor malignancy, and other two-hit-related features just like the hereditary tumors, caused by inactivation of tumor suppressor genes (303,305). Broad Importance a Rare Disorder

of the Gene for

Genes for rare hereditary disorders may be uniquely accessible to identification through molecular genetics. Their identification can have far-reaching implications.

Gene for a Rare Neoplasia Syndrome Has Implications in Common Diseases Most tumor suppressor genes, mutated in rare diseases, have important roles in c o m m o n disorders. These roles may be through mutation or other types of dysregulation. For example, an activating mutation of the RET gene is implicated in sporadic C cell cancer and, through splicing mutations, in much of commonvariety papillary thyroid cancer (169); an inactivating RET mutation is implicated in a small fraction of cases with aganglionosis of the colon (308). Either TP53 or RB1 are inactivated in many c o m m o n neoplasms, especially high-grade malignancies, including in the parathyroids ( 309,310 ). Somatic mutation of the MEN1 gene has been implicated as frequent in the following c o m m o n sporadic tumors outside of any MEN-1 context: parathyroid adenoma, gastrinoma, insulinoma, pituitary tumor, bronchial carcinoid tumor, angiofibroma, and lipoma (see below).

MULTIPLE ENDOCRINE NEOPLASIATYPE 1

Gene for a Rare Neoplasia Syndrome Is Important in Normal Cells The genes for many rare hereditary neoplasia syndromes have provided unique insights in cell biology (210). For example, the retinoblastoma protein is central in cell cycle regulation of all cells (294). The Li-Fraumeni gene-encoded protein (p53) regulates cell birth and cell death (220,311). The MEN-lencoded protein, menin, interacts with the important j u n / f o s AP-1 transcription factors (312).

PATHOLOGY (MACROSCOPIC, MICROSCOPIC, AND CHROMOSOMAL) IN MEN-1 Criteria for Neoplasia and for Clonality The essence of neoplasia is inappropriate accumulation of cells, reflecting increased cell birth, decreased cell death, or a combination of the two. It may be benign or malignant. The neoplastic process generally develops in steps until it is overgrown by one or several dominating clone(s) (306). This contrasts with a diffuse or polyclonal overgrowth, sometimes equated with hyperplasia. A major test for such a process is its behavior in situ. But tissues can also be analyzed for their neoplastic morphology and for other indices of neoplasia. For example, certain nuclear antigens [Ki-67, or proliferating cell nuclear antigen (PCNA)] related to cell cycling can give an index of aggressiveness of neoplasia (217,313,314). Indices of clonality also have a high correlation with neoplasia and have been widely used as the criterion for neoplasia in MEN-1 and in other states (315-317.). Clonality can be assessed in tissue from a female by X chromosome inactivation analysis. Independently of this, clonality can be evaluated by testing for loss of heterozygosity at any chromosomal markers, on comparing DNA between a heterozygous germ line and a t u m o r specimen. Any pattern of DNA loss must be in many or most cells to allow its detection against a background of normal DNA; if a large fraction of cells has the same DNA change, this population is presumably a clonal descendant of one mutated cell. Several clones may arise independently with 11q13 LOH; this may appear as one DNA change population. Therefore, L O H such as X inactivation analysis does not distinguish between mono- and oligoclonality. Rather than analyze L O H at selected chromosomal markers, it is also possible to quantitate tumor DNA across all normal chromosomes at metaphase, giving data about not only loss of DNA segments but also gain

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of DNA segments. This is comparative genome hybridization (CGH). Data from L O H and CGH methods generally correlate closely with each other for a given subchromosomal locus, and either can be similarly an index of clonality.

Parathyroids in MEN-1 Clonal Character

The parathyroid gland abnormality in MEN-1 has previously been classified as hyperplasia or chief cell hyperplasia (62). This term is better reserved for a process that is diffuse, symmetric, and polyclonal (262,318-320) (see footnote 5). Macroscopically, the parathyroid tumor sizes are quite asymmetric in MEN-l, suggesting stochastic processes (Fig. 2) (61). Furthermore, there is cytologic variation a m o n g glands and within glands (303,321). Chief cells dominate, but other types (dark and clear, small or large) can also be identified. Cells may be arranged in cords, sheets, acini, or follicles. The basic pattern may be nodular, diffuse, or intermediate. Nodules are commonly multiple and variable in size. The capsule of any nodule varies from well-defined to none. Foci of normal parathyroid tissue have smaller cells and high adipocyte content; these may be interspersed or form a rim about nodules (321). A totally normal gland is rare at time of parathyroidectomy in MEN-1. Parathyroid h o r m o n e and chromogranin-A are present, each with variable distribution but with little correlation to each other (322,323a,b). Clonality (i.e., neoplasia) is present in virtually all MEN-1 parathyroids (315,321,324,325); however, an 11q13 L O H was less c o m m o n in small than in large parathyroid tumors in MEN-l, suggesting a polyclonal neoplasia precursor stage in small MEN-1 parathyroids (326).

Rarity of Parathyroid Cancer Parathyroid cancer is extremely rare in MEN-1. Two cases were reported in a sporadic MEN-1 setting (one with nonfunctioning pituitary a d e n o m a and one with insulinoma and multifocal islet tumor) without analysis for MEN1 germ line mutation (328,329). A third was alluded to in a m e m b e r of a large MEN-1 kindred (330a). However, that parathyroid specimen was subsequently j u d g e d to be an atypical parathyroid a d e n o m a (J. J. Shepherd, personal communication). One case of parathyroid cancer was reported in a patient with MEN1 germ line mutation but without other tumors of MEN-1 (330b). This rarity of parathyroid cancer contrasts with the frequency of malignancy in enteropancreatic and carcinoid tissues of MEN-1.

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Unproved Role for a Tumor Precursor Stage (Part I) With the lack of clear evidence for polyclonal proliferation in MEN-1 parathyroids, any role for a MEN-1 parathyroid adenoma precursor stage remains unknown (326,331). The same applies to any role for an MEN-l-specific circulating growth factor that might stimulate polyclonal growth of the parathyroids (332) (see below). On the other hand, the near universality of clonality in parathyroid tumors of MEN-1 justifies naming the pathologic stage of the MEN-1 hyperparathyroid process as "multiple parathyroid adenomas."

Enteropancreatic Tumors Excepting Carcinoids in MEN-1 Unproved Role for a Tumor Precursor Stage (Part II) Nesidioblastosis is neogenesis of pancreatic islet or acinar cells by budding from pancreatic ducts (333). It occurs mainly during embryogenesis. It was once thought to be widespread and fundamental in the adult islets of MEN-1 (334). It is now j u d g e d to be rare or occasional in MEN-1 and not specific to MEN-1 (73,75). Similarly, diffuse islet hyperplasia is not characteristic of MEN-l, except in the mouse model of MEN-1 (see following).

Clonal Character The characteristic MEN-1 change is multiple macroscopic and microscopic (0.03-0.5 cm) neoplasms in the duodenal submucosa and pancreatic islets (73-75,321). The duodenal tumors are mainly in the proximal portion of the d u o d e n u m , and the islet tumors are throughout the pancreas. The growth pattern is trabecular or solid, sometimes with a distinct capsule or a distinct stroma. Cell distribution is random, unlike the organization of normal islets (321). By the criterin of 11q13 LOH, all or most MEN-1 islet tumors are clonal (12,325,335,336).

Enteropancreatic Hormones Most enteropancreatic tumor cells in MEN-1 contain peptide hormones, and sometimes multiple cell/ hormonal types are in one nodule, excepting gastrin o m a (73,75,77). Each tumor is classified according to the principal h o r m o n e type. Duodenal tumors frequently contain gastrin (74) and occasionally somatostatin (337,338). Among two large series totaling 300 MEN-1 pancreatic tumors, the order of dominant hormonal cell type (with frequency in parentheses) was glucagon (24-45%), insulin (23-32%), pancreatic polypeptide (21-26%), and no h o r m o n e and no PP (6-18%) (73,75). Duodenal gastrinomas were not

included in those studies. Most tumors contain cells normally found in those tissues. Ectopic h o r m o n e types (ACTH, PTHrP, and calcitonin) are sometimes present with larger (>0.5 cm) and malignant neuroendocrine lesions (111,128,339). A h o r m o n a l syndrome is unlikely with an enteropancreatic tumor less than 0.5 cm. The frequent exception is ZES from duodenal gastrinomas, which are usually below 0.6 cm. The multiplicity of associated MEN-1 tumors makes it impossible to use histologic criteria alone to predict an associated h o r m o n a l syndrome for any one and, similarly, which, if any, tumor is oversecreting its hormone. Claims of one hormonal syndrome arising from two or more enteropancreatic tumors in MEN-1 (89,90) have so far not been fully proved; such a process is possible, but massive oversecretion, dominated by one tumor, is more likely with the possible exception of ZES.

Enteropancreatic Malignancy Enteropancreatic tumors in MEN-l, with the exception of insulinoma, have a substantial malignant potential. As with most endocrine tumors, the only reliable histologic criteria for malignancy are capsular invasion and distant metastasis. In general, malignant behavior (in sporadic tumors) also correlates with nuclear pleiomorphism, nuclear atypia, positivity for Ki-67 antigen, and vascular invasion (340).

Gastrinoma Malignancy Gastrinoma merits special consideration. Among MEN-1 cases, gastrinoma is the most frequent enteropancreatic tumor, the most frequent enteropancreatic tumor to cause endocrine symptoms (ZES), and the most frequent cause of malignant complications (40,41). Gastrinoma shows similar malignant potential in MEN-1 and sporadic cases (60). Gastrinoma is malignant in over half of MEN-1 cases, mainly by virtue of spread to local nodes at time of surgery (81). The nodal metastases may sometimes be considerably larger than the primary (341), and they may have an indolent course. Liver metastasis occurs in about 20% and late; it implies a poor prognosis (42).

Other Enteropancreatic Malignancies Less than 10% of insulinomas in MEN-1 are malignant. Most MEN.1 enteropancreatic tumors do not produce a syndrome and are thus nonfunctional. Tumors that produce mainly glucagon, VIE or GHRH will occasionally produce that h o r m o n e excess syndrome, mainly after they have become malignant and reached a large tumor mass. Similarly, a nonfunctional tumor may only become evident after it has well-developed malignant features. These events have been recognized in a small fraction of MEN-1 cases (17,49,77) (Table 1). But because most

MULTIPLE ENDOCRINE NEOPI_ASIATYPE 1 MEN-1 cases have many enteropancreatic tumors, the cumulative enteropancreatic tumor (excluding gastrinoma) malignant potential during an MEN-1 patient lifetime is substantial (81). This prevalence, perhaps 25% by age 50 years, is hard to estimate because of little available data and because of the confounding malignancy of gastrinoma or foregut carcinoid.

Foregut Carcinoids in MEN-l--Location Specificity of Features The histologic appearance of foregut carcinoid is similar to that of other enteropancreatic tumors in MEN-l, excepting for the absence or sparsity of enteropancreatic hormones (152). These two tumor types are part of a single enteroendocrine spectrum (152). Thus "duodenal carcinoid" is a term sometimes applied to a gastrinoma, a somatostatinoma, or a nonfunctioning tumor. MEN-1 carcinoids have unique sex ratios [thymic mostly in males and bronchial mostly in females (see above) ] and site-specific rates of malignancy. Thymic and bronchial carcinoids have high malignant potential. In particular, thymic carcinoids usually are locally invasive when discovered (135,137). Gastric carcinoid (ECLoma) seems less aggressive, but with notable exceptions (151). More information about its prognosis is needed. In MEN-l, gastric carcinoids are multifocal and associated with diffuse hyperplasia of ECL cells ("carcinoidosis"), from which the carcinoids likely arise (342,343). It has been suggested that these cells are responding to hypergastrinemia, a known stimulant to ECL cells (144-149). However similar ECL hyperplasia is not found with sporadic gastrinoma, so this is likely a hyperplastic process specific to MEN-1. MEN-1 gastric carcinoid tissue is clonal with 11q13 LOH (345). A series of MEN-1 thymic malignant carcinoids was tested for clonality. Two of three showed clonality by virtue of LOH at chromosome l p; surprisingly, in the same study none showed LOH at 11q13 (137). This seems to contrast with other studies, showing frequent 11q13 LOH in MEN-l-associated gastric carcinoids (345) and in sporadic bronchial carcinoids (344). The overall data suggest that all MEN-1 carcinoids are clonal. The nature of the "second hit" in MEN-1 thymic carcinoid needs further evaluation; is it at the MEN1 gene, at another gene, at several loci, or even nonexistent? Neuroendocrine markers, such as chromogranin-A, synaptophysin, pancytokeratin, and neuron-specific enolase, are present in all carcinoids (152). Immunostaining for ACTH is usually negative in thymic carcinoid in MEN-l, unlike in sporadic cases (135,137,140).

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Pathology of Other Tumors in MEN-1 Anterior Pituitary The pituitary tumor in MEN-1 has been generally similar to that in sporadic disease with the usual spectrum of function, nonfunction, and size. There is slight overrepresentation of prolactinoma in MEN-1 (Table 1). By immunologic staining, surgical specimens in the 1980s most commonly showed prolactin a n d / o r growth hormone (28). Even so-called "nonfunctioning" sporadic pituitary tumors usually synthesize subunits of glycoprotein hormones (156,157). There are insufficient data to evaluate whether MEN-1 pituitary adenoma arises from a hyperplastic precursor stage. MEN-1 pituitary tumor at the time of surgery is mono- or oligoclonal, by the criterion that virtually each shows 11q13 LOH (346). Adrenocortical Tumors A spectrum of adrenocortical tumors has been seen in MEN-l, from functional adenoma to carcinoma (158,160). Most common is a diffuse or nodular hyperplasia without hypersecretion of a steroid hormone (158,160). MEN-1 benign adrenal tumors appear polyclonal, because no 11q13 LOH was detected (158). Dermal Tumors

Angiofibroma and collagenoma are the most common and specific skin tumors in MEN-1 (163). Their histologic features are similar to those in the same tumors without MEN-l, including angiofibroma in tuberous sclerosis (163). Angiofibroma and collagenoma contain prominent extracellular collagen, and angiofibroma has more prominent blood vessels. Each lesion type in MEN-1 is likely a true clonal neoplasm by virtue of showing loss of one l l q 1 3 allele by fluorescent in situ hybridization (FISH) (316). Clusters of perivascular cells of MEN-1 angiofibroma, separated by microdissection, had 11q13 allelic loss (347). This suggested that these or similar cells could contribute clonal precursors for the angiofibroma (348). Lipoma (generally in the dermis) is a somewhat less common MEN-1 tumor; no detailed analysis of its histologic features has been made in MEN-1. MEN-1 dermal tumors are rarely or never malignant. There is one report of liposarcoma (349). Other Tumors

Other tumors may not be specific for MEN-1 (follicular adenoma of the thyroid is still uncertain) or have been too rare [pheochromocytoma, including malignant (76); leiomyoma, ependymoma] to allow generalizations about pathologic features.

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MANAGEMENT OF TUMORS IN MEN-1 Tumor Multiplicity~The Special Challenges in MEN-1 Management Multiplicity of tumors is a central feature in MEN-1. This occurs as multiplicity of tumors in one tissue, as multiplicity of tissues with neoplasia (Table 1), and as tumors that can recur after subtotal removal of a tissue (64,67). Tumor multiplicity presents special challenges for tumor localization and for tumor removal. Many methods have been developed to address aspects of these problems. These include hormonespecific tumor localization methods (such as h o r m o n e assay after selective venous sampling with or without h o r m o n e provocation, or h o r m o n e assay after fine needle aspiration). Another highly successful application of h o r m o n e analysis to MEN-1 is the use of rapid intraoperative m e a s u r e m e n t of h o r m o n e s (PTH or insulin, in particular) to know during surgery if any residual tumor continues to oversecrete that h o r m o n e (350,351). Pharmacotherapy, particularly that for ZES, can effectively address similar tumors in multiple locations.

Testing for Endocrine Tumors in MEN-I Pandora's Box, or the Good and Bad of Tumor Humors Many of the products released by MEN-1 tumors are active hormones. The high concentrations of circulating h o r m o n e s can cause toxic metabolic effects. However, sometimes these tumor-humors can be turned into assets. Their metabolic effects may be found or even sought as early indicators of a developing neoplasm (Table 3), and the signs may be evident long before the neoplasm develops malignant properties. Benefit can also accrue if it is possible to measure a biologically active or even inactive peptide (such as chromogranin-A) as a marker of tumor mass (352-354) (see below). Earliest Ages for Tumor Expression in MEN-1 Data on earliest reported age of MEN-1 tumor expression and tumor morbidity can assist decisions about when and how to start screening MEN1 carriers for each tumor (Table 3). The earliest ages for MEN-1 tumor recognition have been at ages 5-8 years with possible HPT (15,43,48), 12-21 years with probable ZES (15,43,58, R. C. Jensen, personal communication), 5-10 years with probable prolactinoma (58,75b,355a,b), 6 years with somatotropinoma (34), and 6-9 years with insulinoma (52,92,97). Earliest morbidity has been at age 15 years with probable ZES (58), 5 years with prolactinoma (75b), and

6 years with insulinoma (92). Early morbidity from primary hyperparathyroidism has not been clearly docum e n t e d in MEN-1 but seems possible with increasing age, between 10 and 20 years in unusual cases.

Distinct Objectives in Testing for Endocrine Tumors Tumor testing can be complex and expensive in MEN-1 because of the many potential tumors, the many possible tests, and the sometimes expensive tests. Selection of appropriate testing methods also depends on testing purpose, such as a tumor m a n a g e m e n t subcategory or ascertainment of MEN-1 diagnosis.

Tumor Testing Purpose #1: Periodic Screening for Early Tumor Recognition in a Likely MEN1 Mutation Carrier Likely (near 100%) MEN1 mutation carriers can be identified by several processes [MEN1 mutation, position a n d / o r disease expression in a MEN-1 family, genetic linkage analysis in a kindred, biochemical ascertainment (see below, Testing Purpose #4)]. Periodic tumor screening is done in likely MEN1 mutation carriers in the hopes that early tumor recognition could guide early intervention and, thereby, result in decrease of certain types of morbidity. The efficacy of treatments for most of the MEN-1 endocrinopathies is clear, and the likelihood of patient benefit is high after treatment of gastrinoma, insulinoma, and, to a lesser degree, HPT (40,41). It has not been proven, however, that early recognition and treatment causes an improved outcome. Systematic prevention or cure of malignancy in MEN-l, analogous to the prevention or cure of C cell cancer in MEN-2 (169), would be highly desirable. This has not been achieved in MEN-l, and it seems unlikely in the near future (see below). Highly likely MEN1 mutation carriers should be screened biochemically every 1-3 years for parathyroid tumor, gastrinoma, insulinoma, other enteropancreatic tumors, and prolactinoma (Table 3). Similarly they should be screened less frequently with tumor imaging methods than with biochemical tests (75b); imaging is particularly relevant for nonfunctioning tumors. Selection and frequency of biochemical tests and imaging tests must represent a balance between utility, availability, and cost. 1. Screening for parathyroid adenoma. For HPT, testing total serum calcium alone has given satisfactory results in MEN-1 (357,358); this is partly because maximal sensitivity to earliest parathyroid tumor expression is not a central issue. Where greater sensitivity is sought [for example, if earliest possible MEN-1 ascertainment is desired or earliest parathyroid tumor treatment is intended (see below)], the combined use of ionized calcium and PTH assay seems preferable (Table 3).

MULTIPLE EYDOCVdNENEOPIaSlA T~E 1 2. Screening for gastrinoma. For gastrinoma screening, the fasting gastrin concentration is central (Table 3) (80). Patients must also be educated about early ZES symptoms because these may appear in an interval between tests. Annual gastrinoma screening in highly likely MEN1 mutation carriers should begin at age 20 (Table 3). Gastrin should be measured during at least 2 days at each test. Two thirds of ZES cases show a gastrin concentration of more than 10-fold above the upper normal limit. The commonest false positives are hypochlorhydria, causing high gastrin, and idiopathic peptic disease, causing high basal gastric acid output (BAO). Hypochlorhydria can be caused by acidblocking drugs, atrophic gastritis, chronic renal failure, or vagotomy (80). False positives also occur with retained gastric antrum after Billroth II gastrectomy (80). Major variations in basal calcium concentrations from management of parathyroid disease have parallel effects on gastrin (60). Suggestive symptoms or a high serum gastrin value should lead to measurement of BAO [normal below 15 mEq/hour, or, after acid lowering surgery, below 5 m E q / h o u r (80)]. Unfortunately, gastric acid output is not measured in

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the test should be carried to 72 hours. Glucose, insulin, C peptide, and proinsulin fraction or concentration should be measured at the time of (fast-induced or other) hypoglycemia (362). 4. Screening for nonfunctioning or functioning enteropancreatic tumor. For nonfunctioning enteropancreatic tumor, widely different approaches are under evaluation, from testing few peptides, to testing many hormones without (363) or with imaging tests, to using endoscopic ultrasound alone. Unfortunately, few reports examine any of the methods systematically in MEN-1. The biochemical screening test with the highest true positive rate for enteropancreatic tumors in MEN-1 is chromogranin-A (Cg-A) (352-354) (Table 3). It was very high in all MEN-1 cases with radiologically detectable enteropancreatic tumors and moderately high in 3 of 9 MEN-1 cases without evidence of enteropancreatic tumor (354). In contrast, pancreatic polypeptide has given low utility in screening (46,354), unless a prohibitively high cutoff value was used (364,365). Insulin C peptide has been promising as a general screen because its positives were often different from

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excludes ZES, but a lower reading does not establish the diagnosis (80). Either basal gastrin or BAO is insufficient alone for a diagnosis of ZES. Additional confirmatory tests include a pairing of gastrin and BAO, or secretin-stimulated gastrin level (an abnormal result is a provoked rise of more than 200 p g / m l above base line) (80,359). Confirmation, albeit less reliable, can also be derived by testing maximal acid output after pentagastrin infusion (80); however, pentagastrin is not currently available in the United States. Endoscopy, which is not a substitute for gastric acid measurement, offers the chance to search for mucosal ulcers and to evaluate MEN-l-related gastric and duodenal tumors [carcinoids (enterochrommaffin-like cell tumors) and gastrinomas, respectively] (80). Somatostatin receptor scintigraphy is not recommended as a principal screen for gastrinoma but may provide useful information incidental to screening for or pursuing enteropancreatic tumors in general (360,361) (Table 3). 3. Screening for insulinoma. Fasting glucose concentrations should be included as a screen for insulinoma. Serum insulin values provide confirmation if there is a suspicion of insulinoma. The single most useful indicator is an insulin concentration above 6 m U / m l during hypoglycemia; however, this does not exclude antibodies against the insulin receptor or surreptitious use of insulin or sulfonylureas. If diagnostic confirmation is needed, the best test is a supervised fast. Most affected patients develop symptomatic hypoglycemia (plasma glucose < 40 mg/dl) by 48 hours of fasting, but, lacking this,

serial measurements of pancreatic polypeptide and gastrin has been proposed (366), but the test is complex, and confirmation of utility is not available (354). Imaging tests, though expensive, can be effective in programs to screen for several types of MEN-1 enteropancreatic tumor (Table 3). Somatostatin receptor scintigraphy is the most effective noninvasive imaging method for pancreatic islet tumors (361). Positive scintigraphic findings may arise before a tumor can be confirmed by any other nonoperative method (77). Tissue findings associated with such sensitive images are not yet available; thus, such findings should not yet be used as the sole reason for intervention. MRI or CT scans have similar yields in this setting, and either is complementary to somatostatin receptor scintigraphy. Spiral CT is an enhancement beyond prior CT methods (367-369). Endoscopic ultrasound with needle biopsy may be the most sensitive and specific method for sporadic enteropancreatic tumor (370,371). However, it is not widely available. For the forseeable future it will require considerable skill and experience; it will probably be limited to specialized roles. 5. Screening for foregut carcinoid tumor: For MEN-1 carcinoid tumor, no biochemical screening test has shown adequate sensitivity to be useful (Table 3). CT is the most sensitive imaging method for mediastinal and bronchial carcinoid. Somatostatin receptor scintigraphy also should have good yield (372). Gastric and duodenal carcinoids can be recognized and biopsied during endoscopy for ZES (150).

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6. Screening for pituitary tumor. Biochemical diagnosis of prolactinoma is based on fasting prolactin 20-fold the upper normal limit (32). Lesser elevations are difficult to distinguish from results of stalk compression. Pregnancy, lactation, or dopamine antagonists may give false positive results. GH a n d / o r IGF-I can be measured with only occasional elevations expected. ACTH, TSH, and gonadotropin subunits should be measured only if there are more specific indications. MRI is currently the most sensitive pituitary imaging test (373) (Table 3), but its clinical applicability is limited by expense and a modest clinical consequence.

Tumor Testing Purpose #2: Further Delineation of a Tumor after Tumor Recognition After abnormal test results have been validated (repeated if needed) and false positives excluded, a newly diagnosed tumor should receive further evaluation appropriate for its management (see below). For example, enteropancreatic tumors in MEN-1 may warrant complex pre- a n d / o r intraoperative tumor localization methods. In contrast, hyperparathyroidism may justify initial parathyroidectomy without any tumor imaging.

Tumor Testing Purpose #3: Periodic Follow-up of a Tumor That Is Not a Current Candidate for Surgery Procedures in this category are standard. The management is not specific for MEN-l, aside from the fact that it is pursued alongside periodic screening for other MEN-1 tumors.

Tumor Testing Purpose #4: Ascertainment of MEN1 Genetic Diagnosis MEN1 mutation testing is now the gold standard for MEN-1 genetic diagnosis (see below). However, there remain many situations where biochemical screening (for tumors) is the only option for MEN-1 genetic ascertainment, even though it incurs the inconvenience of periodic repetition following normal results, the inaccuracies of biochemical testing, and occasional false positive readings, such as from sporadic HPT within a MEN-1 family (374). 1. No MEN1 mutation found in this family. In 10-30% of typical MEN-1 families, no MEN1 mutation is found in the index case (33,37,92,249,375-377). Most or all such families likely have a MEN1 mutation undetectable with the method used (see below). 2. MEN1 mutation test not available. The test is done in few centers, though this is likely to change in the next few years. Another DNA-based option, testing of haplotype or genetic linkage, is rarely available (see below). 3. Patient refuses MEN1 mutation test. Some patients object to mutation testing for legal, religious,

or other reasons. They may still want testing by other methods. With these three circumstances, periodic biochemical screening for MEN-1 tumors becomes the best available testing option for ascertainment of the MEN-1 diagnosis. An effective panel for MEN-1 ascertainment screening is calcium (preferably ionized), PTH, and prolactin every 3 years beginning at age 5 years (356). Depending on resources, other tests such as gastrin and Cg-A can be included after age 20 years.

Hyperparathyroidism Management in MEN-1 Medical Supervision Unless MEN-1 patients are fortuitously rendered hypoparathyroid, it is likely that they will have HPT for many years, representing a combination of unrecognized, recognized but unoperated, unlocalizable, and recurrent parathyroid tumors. The consequences of mild HPT for many decades in MEN-l, including during adolescent growth, are not known (57).

Pharmacotherapy Current pharmacologic approaches for hyperparathyroidism have limited efficacy and are reviewed elsewhere (378). New pharmacologic approaches are under development for hyperparathyroidism. The calcium-sensing receptor agonists, in particular, show promise as agents to inhibit directly PTH secretion by, and perhaps growth of, the parathyroid glands (379). The difficulties of parathyroid surgery could make them very appropriate treatment for hyperparathyroidism in MEN-1.

If and When to Do Parathyroid Surgery The threshold for surgical intervention in sporadic HPT remains controversial (380). There are additional controversies in MEN-1. Most groups delay parathyroid surgery in MEN-1 to await stronger indications than in sporadic adenoma. This is because the MEN-1 operation is more difficult and has higher rates of persistent HPT, postoperative hypoparathyroidism, and recurrent postoperative HPT (Table 2). Furthermore, if HPT is recognized early through screening in MEN-l, the tumors may be small and more difficult to find. Others, however, feel that parathyroidectomy should be done early in MEN-l, even if HPT is asymptomatic and recognized during screening (357). One reason is the hope that, just as parathyroidectomy ameliorates high gastrin and high Cg-A in MEN-1 (60,81,381,382), parathyroidectomy might also delay or prevent some

MULTIPLE ENDOCPdNE NEOPt~SIA T~E 1 other MEN-1 n e u r o e n d o c r i n e expressions, even malignancy. This speculation has not been evaluated.

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Many details, relatively specific to MEN-l, require attention at initial parathyroidectomy.

tumor bioassay to establish a point when no hyperfunctioning parathyroid tissue remains. Experienced parathyroid surgeons had as high as 95% success rate at initial operation before this measurement was available (68), so the potential for yet higher success rate is limited. However, breadth of the operative field, operation time, and morbidity could decrease with its use (388).

Preoperative Planning

A utografi with Fresh Parathyroid Tissue

There must be a readiness to make the diagnosis of MEN-1 in the setting of HPT. Attention to family history and to unusual patient features, such as dermal lesions and early age at tumor onset (163), is critical. Preoperative parathyroid gland imaging is not recomm e n d e d before initial parathyroid surgery, whether or not a diagnosis of MEN-1 has been made. Ultrasound or 99mTc-sestamibi will usually show some but not all parathyroid tumors in HPT of MEN-1 (96). Thus, it is not required.

Deliberate total parathyroidectomy with autograft of fresh parathyroid tissue has been used for HPT from multiple adenomas (389,390). This has been associated with a substantial rate (30-50%) of graft failure in MEN-1 (65,390). The successful operation is followed by total hypoparathyroidism for 4-6 weeks before graft establishment. If hypoparathyroidism does not occur within several days, the patient has residual functioning parathyroid tissue in the neck or chest. After autograft success, late r e c u r r e n t HPT should have a similar likelihood to that after other methods of subtotal parathyroidectomy in MEN-1 (64-68,70).

Initial Parathyroideetomy--Optimizing Outcome

Experienced Surgeon A surgeon with considerable experience in parathyroidectomy is likely to have a better outcome in MEN-1 parathyroidectomy than ~n in ,~vporl on cod ~no (a4~

Extensive Exploration at Initial Parathyroid Surgery The surgeon should try to identify all four parathyroid glands, preferably with biopsy confirmation in MEN-1 (64,68,383). Unilateral exploration will have a high failure rate.

Resection of Parathyroid Tissue Some surgeons prefer not to remove normal-appearing parathyroid glands and to minimize the amount of trauma caused by a biopsy (384). Most remove three and one-half glands, leaving 50 mg of the most normalappearing tissue. The remnant may stay attached to its natural pedicle or be autografted to another site (see below). A minority of surgeons attempt to do a total parathyroidectomy.

Transcervical Thymectomy Transcervical near-total thymectomy should be attempted during parathyroidectomy in all patients with MEN-1 and no prior neck exploration. The thymus may contain parathyroid adenoma, carcinoid tumor, or precursor tissue for either tumor.

Intraoperative Parathyroid Tumor Testing Intraoperative ultrasound can improve the outcome in difficult parathyroid operations. It is particularly helpful to locate an intrathyroidal parathyroid adenoma (380,385). It may not be needed at initial parathyroidectomy, but it should be available in MEN-1. Intact PTH has a circulating half-life of 2-5 minutes (386,387). Thus, rapid intraoperative measurement of PTH is possible for

Cryopreservation of Parathyroid Tissue Parathyroid tissue can be cryopreserved with relatively simple methods (389,390). It can then be stored at - 7 0 ° C indefinitely. It can be used as an autograft if the patient becomes hypoparathyroid, whether or not this outcome had been intended (see below). U n i n t e n d e d hypoparathyroidism is sufficiently c o m m o n in MEN-1 operations that parathyroid cryopreservation should be done during all MEN-1 parathyroidectomies. Unfortunately, cryopreservation methods have not usually resulted in 100% viable tissue. Typical rates for success of a cryopreserved autograft are only 50-70% (390).

Parathyroid A utograft with Cryopreserved Tissue If postoperative hypoparathyroidism seems somewhat likely, a fresh autograft should be used because of its expected higher success rate than with a cryopreserved tissue. If postoperative hypoparathyroidism ensues, a late autograft with the cryopreserved tissue can be considered (390). Hypoparathyroidism should be well documented before the late parathyroid autograft procedure. This can be done effectively 1-3 months postparathyroidectomy by partially withdrawing calciotropic medications to allow demonstration of inappropriately low PTH at a time of mild hypocalcemia.

Parathyroid Reoperation in MEN-l--Optimizing Outcome The m a n a g e m e n t of parathyroid reoperation is similar, whether or not the patient has MEN-1. Aspects of unusual importance in MEN-1 will be emphasized here. The cause of HPT can be postoperative persistence or recurrence. Persistence reflects unsought or hard to find parathyroid tumor. High postoperative recurrence

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is a feature, highly characteristic of the hyperparathyroidism in MEN-1 (see above) (64,66-68).

Review Information from Prior Operation(s) Efforts should be made to review surgery notes, histologic slides, and pathology reports. Even with these efforts, it is often inaccurate to predict the number and location of parathyroid tumors that are present.

Preoperative Imaging of Parathyroid Tumors All MEN-1 patients undergoing parathyroid reoperation should have preoperative parathyroid tumor imaging. This requires selection among available noninvasive and invasive tests (391-394). This is less essential if the first operation was a limited one, under the mistaken impression of solitary adenoma. It is rarely possible to image more than one parathyroid tumor preoperatively in a MEN-1 reoperation (96).

Intensification of Tumor Localization Methodsfrom Initial Parathyroidectomy Intraoperative parathyroid tumor evaluation methods are particularly helpful during reoperation. These include intraoperative ultrasound and ultrarapid PTH assay (see above) (350,385-388,394). There is limited experience with intraoperative isotope scanning at parathyroid reoperations (395).

Parathyroid Autografi Tissue cryopreservation for possible delayed autograft is often helpful. Because of damage to other parathyroid glands at prior operations and during a successful operation, 5-20% of successful parathyroid reoperations result in hypoparathyroidism (380,396). Furthermore, the inability to cause hypoparathyroidism deliberately in a MEN-1 reoperation renders fresh autograft a less appealing option. A combination approach is to attempt to induce hypoparathyroidism and do a fresh parathyroid autograft. Success at induction of hypoparathyroidism at parathyroid reoperation in MEN-1 is always uncertain. If hypoparathyroidism does not develop within a week postoperatively, viable tissue parathyroid (presumably normalfunctioning) remains; the autograft can be retained or removed.

Management of Hormone Excess Syndromes from Enteropancreatic Tumors in MEN-I Management of enteropancreatic tumors in MEN-1 focuses in part on the hormonal syndrome because of its usually reversible but sometimes severe metabolic effects. A second, and sometimes more life-threatening, problem is the potentially malignant aspect of the tumors. The endocrinopathy and the malignant potential are gener-

ally handled separately, excepting with insulinoma, where both are usually addressed at the same time by surgery.

Gastrinoma~Gastric Acid Release Inhibitors for ZES Gastric acid hypersecretion can be controlled in virtually all gastrinoma cases with a histamine Hz-receptor antagonist (397) or, preferably, with an H+,K+-ATPase ("pump") inhibitor (398,399). The pump inhibitors are the preferred therapy because of higher potency, longer duration of action, and fewer side effects. Treatment of MEN-1 cases should begin with a high dose, such as omeprazole, 60 mg, twice daily. It should then be adjusted to maintain gastric acid output below 10 m E q / h o u r in the last hour before the next dose (398). After initial control of acid output, drug efficacy improves and, therefore, dose requirement decreases (400). When a patient with ZES undergoes surgery for any purpose, an intravenous form of acid secretioninhibitory medication should be substituted. Excess dose of pump inhibitor should be avoided not only because of expense but also over concern about hypochlorhydria as a stimulant of ECLoma. Long-term admistration of omeprazole to rats resulted in gastric ECLomas, some of which were invasive (144). ECLomas are an intrinsic feature of MEN-1 (146-150), but there is disagreement over whether pump inhibitors accelerate their development or progression in MEN-1 (148,149).

Insulinoma See below.

Secretion of Multiple Neuroendocrine Hormones Can Be Decreased by Somatostatin Analogs Enteropancreatic enteroendocrine cells and carcinoid cells express somatostatin receptors (401). As a consequence, somatostatin analogs have been explored in these and other tissues as radioactive scanning agents, as hormone secretion blockers, and as tumor growth suppressors. The information below is mainly from sporadic tumors. Long-acting analogs of somatostatin (octreotide, lantreotide) are well tolerated with subcutaneous administration. In gastrinoma, they decrease gastrin output and also directly inhibit gastric acid output (402,403). They are rarely used for this purpose because H+-pump inhibitors have fewer long-term side effects. Though somatostatin analogs a-'e also rarely needed for insulinoma, hyperinsulinemia responds in 65% of cases of insulinoma (402). Most metabolic expressions of glucagonoma respond well (402-405). Plasma VIP concentrations and diarrhea in VIPoma usually respond dramatically (402-405), as do glucagon

MULTIPLE ENDOCRINE NEOPLASIATYPE 1 concentrations and glucagonoma syndrome (402,403, 405). Somatostatin analogs are also effective in G H R H o m a (406,407), and this may be mainly by direct action on the pituitary (406,408). They are highly effective for carcinoid syndrome (402,403), but MEN-1 carcinoids rarely if ever cause this. These medications are not r e c o m m e n d e d for somatostatinoma or for nonfunctioning neuroendocrine tumors, and they do not provide effective blockade of tumor growth proportional to the suppression of tumor secretion (409). Side effects do not usually warrant discontinuation of treatment. There is occasional postprandial hyperglycemia. Steatorrhea is c o m m o n and may be a problem at higher doses. Gallbladder dysfunction is c o m m o n with potential to form gallstones.

Management of the Neoplastic Aspect of Enteropancreatic Tumors in MEN-1 Determination of Tumor Extent The intensity of tumor evaluation (i.e., tumor size and metastases) for gastrinoma and other enteropancreatic tumors in MEN-1 should be determined largely by the philosophy concerning surgery. These philosophies vary dramatically among teams and a m o n g tumor types, from no surgery to surgery in all cases excepting those with advanced metastases (see below). Prior to an enteropancreatic operation, any MEN-1 patient needs evaluation both for coexisting gastrinomas, for multiple pancratic tumors, and for metastases. Certain serum markers correlate moderately well with enteropancreatic tumor burden. These are gastrin and Cg-A for gastrinoma (86,354); Cg-A (354), PP (365), and others to a lesser degree can be used (352,353) for enteropancreatic neuroendocrine tumors. None is an effective predictor of potential for surgical benefit, but some may be used as an index of t u m o r progression or of efficacy of treatment. Endoscopy should be done preoperatively to evaluate activity of acid-related mucosal disease, to identify, biopsy, or remove gastric ECLomas, and to identify, biopsy or remove small duodenal gastrinomas (80,150). Preoperative imaging methods (somatostatin receptor scintigraphy, MRI, endoscopic ultrasound with needle biopsy, and angiographies) that are effective for solitary pancreatic islet tumors are less sensitive to multiple small gastrinomas of the d u o d e n u m (360,410,428), the usual gastrinoma expression in MEN-1. Because of t u m o r multiplicity and possible metastasis, MRI or CT combined with somatostatin receptor scintigraphy can give complementary, albeit incomplete, information (91,361). Helical CT may be one important improvem e n t (367,368). Ultrasound and MRI are less valuable in the a b d o m e n because of bowel gas and motion arti-

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facts, respectively. Selective arterial infusion of secretin with m e a s u r e m e n t of gastrin in the hepatic vein can achieve regional localization in half of sporadic ZES cases (411,412), and it can include hepatic arterial infusion to screen for liver metastases. This has replaced selective portal venous sampling, which was more difficult, more morbid, and less useful (413); however, in MEN-1 it has been largely replaced by somatostatin receptor scintigraphy (414,415).

Search for Metastasis Enteropancreatic enteroendocrine tumors first spread to local nodes and from there to the liver. Patients with hepatic metastases have a p o o r prognosis and are much less likely to benefit from removal of enteropancreatic tumors (85). MEN-1 gastrinoma is as likely to metastasize to the liver as is sporadic gastrinoma (85,416). Metastases to bone may occur late (417) and to lungs, brain, and other sites rarely. The most useful imaging methods for enteropancreatic tumor metastases in liver are somatostatin receptor scintigraphy and MRI (414,415).

Insulinoma Management Insulinoma is the syndrome from enteropancreatic tumor in MEN-1 that usually responds best to surgery. This is because it is usually caused by one benign aden o m a oversecreting insulin, albeit in a setting of multiple enteropancreatic neoplasms. The insulinoma may be difficult to locate even with endoscopic ultrasound and somatostatin receptor scintigraphy (91,361,418). Insulinoma is the only MEN-l-related enteropancreatic h o r m o n a l syndrome not frequently associated with high Cg-A (based on data from a series of sporadic tumors) (353). Because of the multiplicity of associated tumors in MEN-l, it can be helpful to localize the oversecreting insulinoma with a function-based test, such as fine needle aspiration with h o r m o n e assay or selective calcium infusion with peripheral sampling of evoked insulin (419). Some groups have advised resection of the pancreatic tail in MEN-1 insulinoma (90), but this may no longer be appropriate when ultrasound is used intraoperatively (420-422). Even without preoperative localization of MEN-1 insulinoma, high operative cure rates have been predicted to be likely with guidance from intraoperative ultrasound (422), which can identify 90% or greater of sporadic insulinomas. Rapid intraoperative measurment of insulin can be an effective adjunct (351). Pharmacotherapy should be attempted preoperatively to establish the potential for such treatment. Unresponsive patients become candidates for more aggressive surgical exploration if no lesion is detected

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initially. The mainstay of medical treatment is diazoxide (423,424). Somatostatin analogs and other medications have less efficacy (425,426).

Gastrinoma~Controversial Roles of Surgery Knowledgeable groups advocate widely differing approaches to gastrinoma in MEN-1. Because of the multiplicity of MEN-1 gastrinomas, several groups have even advocated pancreaticoduodenotomy (Whipple's procedure) (412,427), but favorable outcomes of this high-risk procedure have not been documented in MEN-1 ZES. Several groups advocate extensive efforts to find all gastrinomas, stating 70-90% success rates j u d g e d by postoperative basal gastrin (428). The majority report that, using a similar surgical approach, the cure rate for ZES was disappointingly low (78,429). In a large surgical series, 34% of sporadic gastrinoma cases were disease free at 10 years postsurgery versus 0% of MEN-1 gastrinoma cases (81). Thus, most recommend against attempts at cure of MEN-1 gastrinoma outside of a research setting. If gastrinoma surgery is undertaken, duodenotomy and bimanual palpation, perhaps with endoscopic transillumination, should be part of the abdominal surgical procedure in a MEN-1 case (81,420,428,431). Because of multiplicity and small size of most MEN-1 gastrinomas, intraoperative ultrasound is not effective for ZES in MEN-1 (420). Any gastrinoma treatment (surgical or other) should be preceeded by an assessment for liver metastases.

Nonfunctioning Enteropancreatic Tumors-Roles of Surgery An inherent problem is the difficulty to predict which tumors have high malignant potential. No circulating markers have been helpful in this regard (see above). Tumor size has received the most attention. Two studies show that, for sporadic gastrinoma, a size of 3 cm correlates with liver metastasis (83,87). Groups have adopted widely differing approaches to nonfunctioning enteropancreatic tumors in MEN-1. The arguments for aggressive intervention are that (1) some pancreatic tumors will become malignant even before any test is abnormal and (2) removal at earliest stages has the best possibility of cure (77). The earliest tumor stage for an aggressive approach may be variably defined as the lower detection limit of radiologic methods (about 1 cm by MRI or spiral CT), as a biochemical diagnosis alone (77), or as any tumor proved by biopsy with guidance from endoscopic ultrasound. The arguments for a conservative or delaying strategy are that (1) pancreatic surgery has substantial morbidity, (2) only the largest enteropancreatic tumor size (3 cm)

may correlate with malignant potential, (3) most such tumors have an indolent course, (4) early surgery may not preclude development of further tumors, and (5) subsequent operation on the pancreas will be rendered more difficult by "early" surgery. Surgery seems contraindicated if a patient has few symptoms and is likely to outlive the tumor, and also if the tumor is widely metastatic. Some groups have advocated routine removal of the pancreatic tail during any abdominal procedure in MEN-l, because it will contain additional tumors (90,428). Patients should receive pneumococcal vaccine in anticipation of possible splenectomy. With intraoperative ultrasound, blind removal of the pancreatic tail may not be needed (420). Because of the uncertain tumor course, an intermediate approach is advised. Multiple small tumors should be followed; they are generally too numerous to allow removal by a conservative procedure. Some of the larger ones (below 3 cm diameter) may be removed endoscopically. The largest or less accessible tumors should be removed surgically. This same strategy is applicable to gastric carcinoid tumors.

Surgery for Uncommon Functioning Enteropancreatic Tumors Glucagonoma, VIPoma, and GHRHoma are MEN-Irelated but very rare. At the time of diagnosis any of these is likely to be large and metastatic. Their hormonal syndromes all respond well to somatostatin analogs (402-407). Operative intervention is dependent on the extent of tumor; often there is good possibility for palliation by surgery (430).

Surgical and Regional Management of Metastatic Disease The role of surgery for metastatic enteropancreatic disease is controversial. Occasional cure may be possible with local or wide hepatic resections (80,432,433). Diverse methods for "debulking" are u n d e r exploration (434,435). Palliation is possible in some patients, such as with hepatic artery embolization a n d / o r transarterial chemotherapy (434,437)

Chemotherapyfor Enteropancreatic Tumor Available data are quite limited. In all series, case numbers are small, tumor types are pooled, and MEN1 is not handled as a distinct group. In general these well-differentiated malignancies have responded poorly to all agents. There may, in fact, be hormonal type differences. Streptozotocin gave 5-40% responses in gastrinoma (436-439) but as high as 90% in VIPoma (438). Streptozotocin with doxorubicin is one favored regimen (436,440). Combining etopiside and cisplatin is another (438). This regimen has several toxicities,

MULTIPLE ENOOCVaNENEOPL~SIAT~E 1 and most would not give such a regimen unless the patient was symptomatic or the tumor was progressing. T h o u g h the somatostatin receptor antagonists have been highly effective in inhibiting h o r m o n e release by these tumors (see above), they alone have had little effect on tumor mass (409). Interferon showed 77% response in pancreatic islet tumors (441), but another series showed no benefit from interferon in gastrinoma (442). Interferon may be more effective in combination with a somatostatin receptor antagonist (443).

Management of Foregut Carcinoid Tumors Thymic and to a lesser degree bronchial carcinoid tumors have high malignant potential in MEN-1. The main treatment is surgical, including resection of tumors and local nodes. Some centers have used postoperative radiation for all thymic carcinoids, but this has had little efficacy (135,444a). No chemotherapeutic agents have been effective in reducing tumor mass. ECLoma in MEN-1 can remit following treatment with a somatostatin receptor blocker (octreotide), perhaps secondary to its lowering of serum gastrin, a likely ECLoma stimulant (444b).

Treatment of Other Tumors Pituitary Tumors

Treatment of pituitary tumor is the same in MEN-1 as in sporadic cases (444c). In particular, most MEN-1 prolactinomas respond well to dopamine agonists (445).

Prevention or Cure of Malignancy; Unreached Goals in MEN-1 There are many priorities in treatment of MEN-l: ascertainment of MEN-1 diagnosis, early recognition of tumors, cure of endocrinopathy, cure of malignancy, palliation of malignancy, and prevention of malignancy. Recognition and cure of endocrinopathy in MEN-1 are effective, albeit with possibility of late recurrence. Cure of malignancy in MEN-1 is rarely possible, but palliation may be possible. Prevention of malignancy in MEN-1 would be desirable for gastrinoma, other enteropancreatic tumors, and foregut carcinoids. At present there are no good options for malignancy prevention in MEN-1. Prophylactic thymectomy is suggested during initial parathyroidectomy, but its efficacy is not proved. Most MEN-1 malignancies have a high tendency to be multifocal, and their organs (unlike the thyroid in MEN-2) cannot be ablated without high morbidity. Thus, current programs are directed to attempts to detect and remove tumors at early stages, but not always before they become malignant. The lack of measures to prevent or cure malignancies in MEN-1 causes MEN1 germ line mutation testing to take on lower urgency than RET germ line mutation testing in MEN-2 (see below).

GENES AND MOLECULES

Adrenal cortical lesions should be handled similarly to other nonfunctioning adrenal tumors (446-448). Tumors causing a steroid h o r m o n e excess syndrome, those larger than 3 cm diameter, or those growing rapidly should be removed.

Identification of the M E N 1 Gene

A lipoma should not be removed unless it causes a cosmetic problem. Rare Associated Tumors

Pheochromocytoma (always unilateral in MEN-l) or e p e n d y m o m a will occur rarely in MEN-1 and should be treated as in sporadic cases.

Treatment of Familial Variants of MEN-1 With no g e n o t y p e / p h e n o t y p e correlation established, members of families with the prolactinoma MEN-1 variant or even with familial isolated HPT and a

561

MEN1 mutation should be followed similarly to members of other MEN-1 families.

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The DNA-based stages in MEN1 gene identification were initiated by Larsson et al. in 1988 (12). They showed that the MEN-1 trait in several large families was linked to the P Y G M m a r k e r at chromosome 11q13; they also found 11q13 L O H in two MEN-1 insulinomas, suggesting tumorigenesis by MEN1 gene inactivation (a two-hit mechanism (see above)). The MEN1 gene identification process then advanced slowly for several years because the gene maps of 11q13 were incomplete and partly incorrect, because there were few 11q13 gene markers available, and, most importantly, because none of the known genes in this interval was ultimately the MEN1 gene. Ironically, the MEN1 gene locus was within 70,000 bases of P Y G M (13,449). It could have been identified by the gene-walking methods of 1988, but there was insufficient reason to adopt that strategy at the time or later. MEN1 was identified by positional cloning (450), a strategy of narrowing the candidate interval (its chromosomal "position") and then testing as many genes as

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necessary in the minimized interval, until the correct gene is found. The successful MENl-cloning process was a combination of evolving technologies (449-453) and detailed exploitation of patient DNA samples. Because the technologies are i n h e r e n t in a positional cloning project, particularly one directed at a t u m o r suppressor gene, they will not be reviewed. P a t i e n t DNA was an essential tool at three steps of the process. Step 1: Patient Germ Line DNA Allowed Analysis of Meiotic Recombination in MEN-1 Family Members to Narrow the Candidate Gene Interval

First, DNA from MEN-1 kindreds was analyzed against polymorphic 11q13 markers to identify MEN-l-affected members with an informative ancestral 11q13 meiotic recombination (crossing over during gametogenesis), deduced in one parental gamete. Such a recombination establishes one boundary within the 11q13 region. All DNA on one side of the boundary can be traced to an unaffected grandparent, and that entire chromosomal zone can thus be excluded from the MEN1 candidate interval. Affected cases from approximately 150 MEN-1 kindreds, some quite large, were analyzed (168,454). Tested with newly developed DNA markers in the candidate interval (455), the rare and most informative genetic recombinations allowed the MEN1 candidate interval to be narrowed to about 3 million bases (168,454). Step 2: Patient Tumor DNA Helped to Map Chromosome 1 lq13 LOH to Narrow the Candidate Gene Interval

Second, LOH in tumors can also lead to definition of boundaries of a candidate gene interval. LOH analysis takes advantage of DNA changes near the normal MEN1 copy (second hit), unlike meiotic recombination analysis, which focuses on DNA changes near the mutated MEN1 copy (first hit). But the purpose of either analysis in a positional cloning context is the s a m e m t o minimize the MEN1 candidate interval. Because clonal tumor cells are interspersed with n o n t u m o r cells that could obscure the clonal LOH pattern, tumor-enriched cells were obtained by microdissection (452,453). Approximately 200 MEN1-related tumors were analyzed for l lq13 LOH boundaries (335,336). One particularly informative gastrinoma had an 11 q 13 LOH boundary that narrowed the working MEN1 candidate interval to 0.3 million bases (325). This was narrow enough to encourage attempted analysis of every gene in the interval. Step 3:MEN-1 Index Cases' Germ Line DNA Gave a Test Panel to Establish Which MEN1 Candidate Gene Had Mutations in MEN-1

Most hereditary disorders are caused by small inactivating mutations that may occur at different points in

one gene. In order to decrease the likelihood of not recognizing MEN1 mutations, a panel was assembled with DNA from index cases from 15 different kindreds, each with well-characterized MEN-1. The 11q13 haplotype analysis had suggested that two pairs of index cases descended from two different c o m m o n founders, but many or all of the other index cases likely would have unique mutations (456). All or most (n = 8) of the genes in the narrowest 1 l q13 candidate interval were identified. Genes were identified by sequencing most of the 0.3 million-base interval. T h e n c o m p u t e r searches m a t c h e d the new sequence against expressed sequence tags (ESTs), cDNA fragments previously cloned from messenger RNA and stored in the worldwide data base; alternately, c o m p u t e r searches predicted an i n t r o n / e x o n boundary (a second criterion to recognize a new gene) (450,451). All exons of the open reading frame were identified, and mutations were then sought in the MEN-1 test panel with dideoxyfingerprinting (ddF), a powerful DNA segment-screening method. Eight newly identified genes in this narrowest interval were tested; one, initially t e r m e d Mu, showed mutations in 14 of 15 index samples but not in many normals, establishing this as the MEN1 gene (13). This identification was confirmed and extended by similar testing in other MEN-1 index cases (37,375,457,458).

M E N 1 m R N A and Menin Protein MEN1 mRNA

The MEN1 gene is 10 kb long. It encodes messages of 2.7 and 3.1 kb (451,457). Both forms of the mRNA are ubiquitously expressed, with highest levels in CNS, thymus, and testis. MEN1 mRNA predicts a 610-amino acid protein, termed menin. Menin had no related proteins or obvious signature domains. The mouse Menl gene encodes an alternate splice site for intron 1, causing a longer 5' untranslated portion but virtually identical e n c o d e d protein (459-462). The fly Menl gene is 50% homologous to the m a m m a l i a n gene (463a). No M_EN1 gene has been found in S. cerevisiaeor C. elegans despite evaluation of those complete genomes (463b). Menin Protein

Menin is mainly a nuclear protein. It has two C-terminal nuclear localization sequences at amino acids 479-497 and 588-608 (464). Each NLS has two possible interpretations, a basic lysine-rich core or a bipartite basic region (464). During the cell cycle, m e n i n concentration varies only mildly and its compartmentalization is stable (465). Many further details of menin function will undoubtedly be recognized shortly.

MULTIPLE ENDOCeaNE NEOPLASIATYPE 1

Tumor Development in MEN-1

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563

(transformed) after stable transfection with a mutated oncogenic RAS gene. Those transformed cells were then stably transfected with MEN1 to overexpress menin. Partial normalization of oncogenic properties in vivo in nude mice and in vitro supported MEN1 function as a tumor suppressor (467).

MEN1 gene identification may contribute to understanding the process of MEN-1 tumor formation. However, alternate approaches may be even more informative. M E N 1 as a Tumor Suppressor Gene

Menin Molecular Pathways

The initial finding of 11q13 L O H in MEN-1 tumors suggested that MEN-1 tumors develop by sequential inactivation of both MEN1 alleles (12) (Fig. 4). This is the mechanism that defines a tumor suppressor gene (see above). The pattern of germ line and somatic MEN1 mutations in MEN-l-related tissues, highly suggestive of inactivating mutation as a first hit, strongly supported this (Fig. 5). Two-thirds of the MEN1 mutations predict a truncated or otherwise shortened menin protein or no menin at all. Such changes would almost certainly be deleterious (i.e., inactivating). An alternate and unlikely interpretation would be that these mutations lead to an activation of a very small aminoterminal menin fragment. Because at least one MEN1 mutation causes deletion of the entire mRNA, this interpretation is excluded (466). Further support for MEN1 as a tumor suppressor would be reversion of oncogenicity by menin expression in MENI-null endocrine tumor cells. MENI-null cells did not exist, so a surrogate cell line was used. Murine NIH 3T3 cells had been r e n d e r e d malignant

The menin protein must interact with at least one other cellular element, be it protein, DNA, lipid, etc. One likely important menin-interacting partner is the j u n D protein (312,468a). The j u n D protein is a member of the j u n / f o s family of AP-1 transcription factors (312). Menin binds to j u n D but not to other members of the j u n / f o s family. The AP-1 transcription factors form homo- or heterodimers through their C-terminal basic/leucine zipper domain; that domain binds to a small response element [triphorbol-ester acetate responsive element (TRE)] in the p r o m o t e r of many genes. Growth stimulators c-jun and c-fos each can contribute to tumorigenesis. The normal roles and targets of j u n D are not known, but they seem quite different from those of c-jun. Unlike the growth-promoting c-jun, j u n D may have mild growth-suppressive properties (312). Menin binding to j u n D involves the middle (amino acids 139-242 from total of 610) of menin and the amino terminus (aa 8-70 from a total of 347) of junD. Menin binding causes inhibition of the j u n D transcriptional activity in artificial systems. Missense

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Codon Change FIG. 5 Germ line and somatic mutations of the MEN1 gene, depicting unique germ line MEN1 mutations in families and in sporadic cases. Repeating mutations are shown only once but with a number in parentheses. Somatic MEN1 mutations in diverse tumors without MEN-1 are shown separately along upper and lower borders. MEN1 messenger RNA is diagrammed with exons numbered; untranslated regions are cross-hatched. Truncating mutations [frameshift mutations, splice error, and nonsense (stop codons) mutations] are shown above the diagram. Codon shift mutations (missense mutations or small in-frame deletions) are shown below. NLS, Nuclear localization sequence. The critical domain for binding to junD is dotted. One large deletion, likely of the entire MEN1 gene, is not shown (466).

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(amino acid codon change) mutations can point to critical functions or domains of a protein. Missense MEN1 mutants, derived from MEN-1 cases, generally cause loss or even reversal of menin's inhibition of junDmediated transcriptional activation (312). Preliminary data suggested that transcriptional inhibition may involve menin recruitment of histone deacetylase (468a). Because menin and junD each have growth inhibitory properties, this inhibitory property might depend on synergism between the two (468b).

Physiologic Pathways Knowledge about the mutational process (i.e., gene inactivation during oncogenesis is synonymous with tumor suppressor) and molecular pathway (i.e., menin nuclear protein interacts with a junD transcription factor) has not yet clarified the physiologic process disturbed by MEN1 mutation. In fact, the possibilities are many. For example, an inactivating mutation of other tumor suppressor genes accelerates cell birth (RB1) (294), decreases cell death (TP53) (311), decreases extracellular matrix differentiation (EXT1) (295,296), or decreases genome stability [at least five DNA mismatch repair genes (hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 (GTBP)for nonpolyposis colon cancer] (223,469). Several studies have suggested genome instability in MEN-1 cells. Initial studies suggested some MEN-1 cases with an increased rate of chromosomal breakage in phytohemagglutinin-stimulated lymphocytes (470,471). Another study found increased chromosomal breakage and double minutes (or extrachromosomal small copies of chromosomal DNA) in cultured lymphocytes and fibroblasts (472). The same group confirmed the increase of double minutes in MEN-1 (473). A separate study found MEN-1 to be associated with doubling of the rate of premature centromere division after exposure of cultured lymphocytes to an alkylating agent (474a). Defects in responses to DNA damage have been reported (474b,474c). During meiotic prophase, menin was localized to telomeres (475). A circulating growth factor has been identified in plasma from MEN-1 patients (332). The initial assay was based on a mixed population of bovine parathyroid cells. MEN-1 plasma showed a distinctive mitogenic activity on those cells. The activity was not seen in plasma from normals or several control groups. Similar activity was also seen on bovine parathyroid endothelial cells, suggesting that the endothelium might be the proximal target of the circulating factor(s) (321,476, 477). The activity was not found in parathyroid tumor supernatant fractions or in parathyroid gland effluent. Activity was lower in plasma from MEN-1 patients previously treated for pituitary tumor, suggesting that the

pituitary had released it or caused it to be released from elsewhere (478). Partial characterization of the factor(s) showed physicochemical and epitope properties similar to basic fibroblast growth factor (FGF-2) (477-479a). At this point, it is not known if this activity has any role in the parathyroid or other tumors of MEN-1. One alternative is that it is an e p i p h e n o m e n o n of MEN-1 progression.

Animal Models of MEN1 Homozygous inactivation of the murine Menl gene causes death during late gestation. Heterozygous Menl inactivation causes features with striking resemblance to h u m a n MEN-1. At maturity, the mice develop endocrine tumors, particularly parathyroid tumors, insulinomas, and prolactinoma. Interesting differences from h u m a n MEN-1 include a milder degree of parathyroid hyperfunction, no gastrinomas, and hyperplastic pancreatic islets as an insulinoma precursor lesion. Furthermore, the mice develop bilateral pheochromocytoma and adrenocortical cancer (479b). Another mouse model of MEN-1 arose from inactivation of two types of cyclin dependent kinase inhibitor, p18 IuK4c and p27 ram. Coexpressed knockouts of both genes cause tumors in the parathyroid, anterior pituitary, pancreatic islets, and d u o d e n u m (similiar to MEN-l) (479c) plus C-cell cancers and pheochromocytoma (like MEN-2). Because both inactivated genes are in the G1 phase of the cell cycling pathway, this model raises the possibility that MEN-1 a n d / o r MEN-2 involve important disruption in this pathway. Mice have been developed with homozygous inactivation of junD. Menin and junD interact directly with each other, suggesting a pathway that is critical for endocrine tumorigenesis (312); however, the only phenotype from junD inactivation was male infertility, decreased body size, and decreased growth hormone in serum (479d). Furthermore, the effects ofjunD knockout in cultured fibroblasts from the mice were puzzlingminhibition of cell accumulation in primary fibroblast cultures but stimulation of accumulation in immortalized cells (479e).

Tumor Analyses about the MEN1 Locus MEN1 Mutation Testing in Tumors

MEN1 mutation is common in sporadic tumors of the type found in MEN-1. Frequency of MEN1 Somatic Mutation by Sporadic Tumor Type The prevalence of MEN1 mutations has been 10-22% in sporadic parathyroid tumors (480-484) and

MULTIPLE ENDOCV.INENEOPIaSIA T~E 1 radiation-associated parathyroid tumors (484), 25% in gastrinoma (485-487), 10-20% in insulinoma (485,488), 50% in VIPoma (485,488), and 25-35% in bronchial carcinoid (344,488). Among sporadic gastrinomas the presence or type of MEN1 mutation did not correlate with any major clinical phenotypic subgroup (487). In contrast, the MEN1 mutation rate has been low (0-5%) in sporadic pituitary tumors (355,489-491) or in follicular thyroid tumors (492). MEN1 mutation is rare in sporadic adrenocortical tumors that are benign (0-5%) or malignant (0%) (493-495). MEN1 mutation is also rare in the clonal parathyroid tumors of uremic secondary hyperparathyroidism (496-499) and in parathyroid cancer (500). A m o n g n o n e n d o c r i n e tumor types, MEN1 mutation has been found in some sporadic dermal tumors, of the same types as seen in MEN-1 [2 of 19 sporadic angiofibromas (501) and and 1 of 6 lipomas (502) ]. MEN1 mutation has not been found in m e l a n o m a (503) or in leukemia even though 1 lq13 L O H was found in 35 % of those leukemias (504). Except in bronchial carcinoid (344), 11q13 LOH has been twice as frequent as MEN1 mutation in a tumor category, raising the possibility of mechanisms other than mutation for the first hit at MEN1 [for example, undocu m e n t e d promoter methylation (505)] or for a first hit at other chromosome 11 genes (480,481,485,488). Adrenocortical cancer has an even more disproportional ratio (above 10:1) between frequencies of 11q13 L O H and of MEN1 mutation; 11q13 L O H here seems to be a subset of LOH at many loci (i.e., genome instability in adrenocortical cancer) (493-495). Occasional Small MEN1 Second Mutations in MEN- 1 Tumors Even MEN-1 tumors from tissues with generally clonal tumors occasionally do not show 11q13 LOH; a m o n g eight such tumors selected from a large group of MEN-1 tumors, four had a second hit as a small mutation of the normal MEN1 allele (506). MEN1 as One of Several Genes Implicated in Parathyroid Adenoma MEN1 is one of many genes sometimes mutated in endocrine tumors (507-509), including those in tumors from MEN-1. MEN-1 tumors have revealed L O H at loci beyond 11q13 (510) as well as zones of DNA loss or amplification by comparative g e n o m e hybridization (CGH) analysis (162c, 511), suggesting that inactivation of both MEN1 alleles is not always sufficient to cause parathyroid neoplasm. MEN1 may be the gene most commonly mutated in nonfamilial, common-variety (non-MEN-l) parathyroid adenoma. An undiscovered gene at l p is also clearly important based on frequent l p L O H (512,513);

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other unknown genes have also been tentatively implicated in parathyroid tumors by L O H or CGH (484,512,514,515). Certain known genes can contribute in a varying fraction of parathyroid tumors by activating mutation (CCND1, RET) (169,507,508) or inactivating mutation (CaSR, RB1, TP53) (270,309,310,516,517). Mutation of RET or CaSR has been implicated in hereditary parathyroid hyperfunction [presumably polyclonal hyperparathyroidism in a CaSR germ line mutation (262)] but not implicated in sporadic parathyroid adenoma (291, 292,518-520). A different spectrum of mutated genes is implicated in any other sporadic tumor type, for example, nonfunctioning enteropancreatic tumors (521). LOH and Other l l q 1 3 Rearrangements in Tumors The 11q13 L O H analysis had a central role in early understanding and search for the MEN1 gene. Even now, this analysis can c o m p l e m e n t analysis of MEN1 mutation in tumors in several ways. Predict Which Tumors Have MEN1 Mutation The 11 q 13 L O H can be a screening m e t h o d in research to look for sporadic tumors likely to harbor a MEN1 mutation. Such use predicted long ago that many types of sporadic endocrine tumor would harbor a MEN1 mutation (315,522,523). 11q13 L O H has also been useful in pointing to inactivation of the second MEN1 allele in certain interesting tumors (pheochromocytoma, leiomyoma) of MEN-1 cases and, thereby, pointing to a tumor suppressor role of the MEN1 gene in development of these neoplasms (162b,163b). An Indicator of Tissue Clonality The 11q13 L O H was the first p r o o f that parathyroid and other tumors in MEN-1 are fundamentally monoor oligoclonal, not diffusely polyclonal (315,324,524). In a similar manner, loss of 1 l q13 alleles by FISH supported clonality and the role of the MEN1 gene in three types of MEN-l-associated dermal lesions: angiofibroma, collagenoma, and lipoma (316). A Clue to Possibly Novel Dysfunction at l l q 1 3 When 11q13 LOH is found in a sporadic tumor but no MEN1 mutation is found, it raises the possibility of MEN1 inactivation without MEN1 mutation [for example, MEN1 promoter methylation is being sought in a fraction of parathyroid adenomas (505)] a n d / o r of a different tumor suppressor gene in the 11q13 zone (502,525,526a). The core interval with 11q13 LOH in follicular thyroid tumors includes the MEN1 gene but is not associated with somatic mutations within the MEN1 gene (526b). Four of five hibernomas, a tumor related to lipoma, lost DNA markers encompassing both MEN1 copies (527).

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A Predictor of Aggressive Tumor Behavior The 11q13 LOH in sporadic adrenocortical tumors correlates with malignancy, apparently as an expression of genome instability and not of underlying MEN1 mutation (see above) (493-495). Also in sporadic pituitary tumors, one study found that 11q13 LOH correlated with aggressive histology (528). This last correlation has not been found with regard to aggressiveness of sporadic parathyroid neoplasms (480-483,500) or gastrinomas (485,487). This parallels the finding that a MEN1- inactivating mutation is less common in sporadic parathyroid cancer than in sporadic parathyroid adenoma (see above).

l lq13 Rearrangements Other Than LOH The 11q13 amplification and r e a r r a n g e m e n t are comm o n in n o n e n d o c r i n e tumors. These changes generally reflect activation of genes other than MEN1. Specifically, CCND1 is an 11q13 gene, amplified or overexpressed in many neoplasms including parathyroid a d e n o m a (507,508,529). Other potential oncogenes at 11 q13, such as fgf3 and fgf4, have not been conclusively implicated to be mutated(530). MEN1 mRNA and Protein in Tumors

Menin could have direct or secondary roles in certain tumors. There is currently little information about MEN1 mRNA or menin protein in tumors.

Germ Line Analyses about the M E N 1 Locus ( l l Q 1 3 ) Genetic Linkage Analysisat 11 q13 Linkage testing to chromosome 11q13 to ascertain the MEN1 cartier state was shown to be feasible in MEN-1 families in 1988 (12). Sufficient markers were soon available to give greater than 99% predictive yield in some families (531a). Data from over 100 families established that, for virtually all MEN-1 families large enough to allow linkage analysis, linkage of the MEN-1 trait to 11q13 could be confirmed (167,168). Only one family with very atypical features failed to show this linkage, and this family can now be classified as having a familial isolated pituitary tumor (see above) (247,249). Because MEN1 mutation testing usually has greater predictive power and is easier to perform, l lq13 linkage and haplotype analysis has been largely replaced. However, in the substantial fraction of familial cases in which MEN1 mutation testing cannot be offered, linkage analysis is still a consideration. MEN1 Mutation Frequency in Germ Line DNA from MEN-1 Cases

MEN1 mutation has been found in 60-100% of families by groups testing 20 or more index cases from typ-

ical MEN-1 families (33,37,92,249,375-377). Because all large MEN-1 families have shown linkage to 11q13 (167,168), all or most MEN-1 families without detected MEN1 mutation likely have MEN1 mutation, unrecognized with current methods. In such cases, likely explanations include technical limitations, such as deliberate omission of testing portions of the MEN1 gene (5' untranslated region, 3' untranslated region, and all or most introns) or failure of the polymerase chain reaction (PCR) to amplify the mutated amplicon. For example, a deletion of one entire copy of the MEN1 gene would not be recognized by a PCR step, which would amplify only the remaining normal allele. The MEN1 mutation rate among sporadic cases has been lower and more variable, 30-100%, probably reflecting greater clinical diversity among this diagnostic group than among the familial cases (36,92,246,249,377). M E N 1 Mutation Patterns

Genotype-Phenotype Relations in MEN1 No clear genotype-phenotype relation has yet emerged from analysis of germ line or somatic mutations in the MEN1 gene (33,37,484,487). Two-thirds of MEN1 mutations predict truncation or loss of the encoded protein. In general, the somatic and germ line mutations show a remarkably similar distribution about the MEN1 mRNA (Fig. 5). Missense mutations in many genes cluster in a critical domain. There is no obvious clustering of MEN1 missense mutations about either the NLS or the other locus (Fig. 5). Virtually all missense mutations are at codons, conserved between h u m a n and fish (463). An occasional missense MEN1 "mutation" could be a rare, linked benign polymorphism. Only a validated test of "mutant" gene function can distinguish this definitively. Three large families with the prolactinoma variant of MEN-1 have been examined. Two different MEN1 mutations were found; in the third family, no MEN1 mutation was found but linkage to 11 q13 was established. Thus, it seemed that three unrelated MEN1 mutations were present (37). A MEN1 germ line mutation has been found in only about one-fifth of tested kindreds with isolated HPT (37,249). Of twelve identified mutations in familial isolated HPT, five were likely truncating mutations [359de147 (531b), 764 splice change (531c), Y353X 7Mutation coding is as follows: mutations causing a simple amino acid codon change are abbreviated with the n u m b e r (position from the N-terminus) of the amino acid codon(s) changed. The normal codon is designated first by a one-letter abbreviation, and the new mutated codon is so designated afterward; a new stop codon is shown as "X." A frameshift or a large insertion or deletion is listed with the number of the first involved nucleotide base (numbered from the 5' initiator ATG), followed by "ins" or "del" for insertion or deletion and then the number of bases inserted or deleted. These conventions and related conventions for more complex mutations are used widely (533).

MULTIPLE ENOOCeaNE NEOPLAStAT~E 1 (532,533), E366X (534), and 1483de14 (533b). There were six nontruncating mutations. [V184E (535), E225K (536a), Q260P (175,176), L267P (246), G305D (536b), L414del (537a), and a seventh possibly nontruncating mutation [369ins18 (537b)]. M E N 1 Mutation Causes and Recurrences

Most of the M E N 1 mutations have been small mutations (base substitutions; insertions or deletions of 1 to 3 bases). The mutations occur at positions of C p G / C p N p G or at short tracts of 1- to 3-base repeats (538). These positions, like the common mutation sites in other genes, are susceptible to mutation by 5-methylcytosine deamination or DNA polyrnerase slippage, respectively (538). At least one M E N 1 mutation was a deletion encompassing all or most of the M E N 1 gene (466). Approximately 10% of M E N 1 mutations arise de novo (33,37). This has implications of absent inheritance risk in such siblings and 50% risk in offspring. De novo mutation cannot be deduced only from lack of a positive family history. Proof of de novo mutation comes from testing DNA of both parents, proving no mutation in either, and documenting a very low likelihood of

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misidentification of a parent (37). Because M E N 1 is a large gene, susceptible to inactivation by point (and other) mutations across its open reading frame, unique mutations will continue to be found indefinitely. Approximately half of M E N 1 mutations are repeating mutations (Fig. 5). In some cases the same mutation was seen de novo in somatic tissue as in an unrelated germ line setting. Analysis of the affected haplotype can reveal if a repeating germ line mutation reflects a comm o n ancestor (founder effect) (Fig. 6) or, by exclusion, a mini-hotspot of mutation (538). About half of repeating M E N 1 mutations have been from founder effects, meaning that the repeat reflects descent from a remote and unknown c o m m o n ancestor. Founder effects are often mutation specific for a geographic region, as has been the case in MEN-1. Geography-specific founder M E N 1 mutations have been reported in the United States (416delC or 512delC) (538), Canada (R460X) (183) (Fig. 6), France (1650insC and likely 1650delC) (92), Northern Europe (D172Y) (246), and Finland (1466de112) (249). Through geneology, a c o m m o n founder has been identified tentatively for a large group of MEN-1 families in Tasmania (330) and confirmed by a c o m m o n mutation (374).

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FIG. 6 Haplotype analysis about the MEN1 gene locus at chromosome 1 lq13 shows a founder effect among four nuclear families (each representative of four larger families) with the prolactinoma variant of MEN-l, or MEN-1gur~n (183). Sixteen alleles are tabulated according to their ordering in the chromosome 11 "map". Shaded zone is a core haplotype shared by the affected members in each family. The likelihood of sharing so many alleles by chance is extraordinarily low; by induction, a remote common ancestor with the core haplotype and MEN-1 should account for the observations.

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Some Issues in Genetic Counseling Much of this chapter and many other publications are relevant to genetic counseling about MEN-1. There is confusion in the patient, paramedic, and physician community about similarities between MEN-1 and MEN-2. In particular, m u c h confusion comes from the similar syndrome names. Education about their overriding differences should be offered. Some central issues about MEN1 mutation analysis are outlined here. 1. MEN1 mutation testing in endocrine tumors, t h o u g h often abnormal, has little or no clinical use. The information is not generally helpful in tumor staging, unlike data for a few other genes, such as TP53. Tissue (such as leukocyte or buccal scraping) more representative of germ line is generally more appropriate for a screen of germ line MEN1 mutations. 2. MEN1 germ line testing can be offered to the following groups: (a) An index case in a MEN-1 family--the test can confirm the diagnosis and direct the design of simplified (i.e., limited to the mutationcarrying PCR fragment) mutation testing in other family members. (b) Affected or unaffected relatives in a MEN-1 family with known MEN1 familial mutation. The test can give diagnosis confirmation to clinically affected members, and a far more important lifetime "yes or no" answer to asymptomatic members. (c) Sporadic MEN-1 phenocopies. Sporadic cases with HPT (mainly appropriate for those with multigland disease and particularly cases below age 30 years) or gastrinoma. Germ line MEN1 mutation rate will be lower in cases with sporadic prolactinoma, insulinoma, foregut carcinoid, and other nonfunctioning enteroendocrine tumors. M1 cases with a tumor in two or more of these tissues (particularly if one c o m p o n e n t is multiple parathyroid tumors) can be tested. (d) Familial MEN-1 phenocopies. An index m e m b e r of a family with a MEN-1 phenocopy, such as isolated hyperparathyroidism or isolated pituitary tumor. 3. M1 or virtually all MEN-1 families have a MEN1 mutation. Thus, a MEN-1 family with no MEN1 mutation found probably has an undiscovered MEN1 mutation. This may be more easily diagnosed in the future. In the meanwhile, MEN1 ascertainment may be possible by genetic linkage analysis or by periodic biochemical screening for tumors. 4. MEN1 mutation testing in the germ line provides useful information to the patient and physician. Unlike the RET test for MEN-2, the MEN1 test does not lead to an intervention to prevent or cure malignancy. Thus the clinical imperative for testing is usually lower in MEN-1 than in MEN-2. 5. Because aggressive prolactinoma in familial MEN-1 has been noted before age 5 (355b), testing of

children for MEN1 ascertainment can begin by age 5. As this guideline is based on only one case, many physicians might begin testing children for MEN-1 at a later age. The general guideline in the USA is that gene testing deprives children of their ability to make a life-affecting decision and should not be offered to children unless it can lead to an important intervention (539), as it would in about half of children in an MEN-2 family. 6. A MEN-1 patient information booklet is available as an internet page (540). 7. New inactivating mutations in MEN1 will continue to be uncovered anywhere in the open reading frame, in contrast to the few activating mutations in susceptible parts of the RET gene. Thus the MEN1 mutation test will be more d e m a n d i n g technically than the RET mutation test. 8. The MEN1 mutation test is not p e r f o r m e d as widely as the RET test because of its lesser urgency and higher complexity. It is mainly available from research laboratories. This might carry a disadvantage of slow t u r n a r o u n d time, but an advantage of association with skilled genetic counseling. In the near future, the scarcity and cost will probably decrease.

M E N - I ~ A N EYE TO THE FUTURE At this time, several years after the MEN1 gene was first identified, much new information has been obtained. Knowledge about the pathophysiology of the MEN-1 syndrome and the MEN1 gene is undergoing rapid expansion. Some future advances are predictable; many will be unexpected. In the next 5 years, it is likely that MEN1 mutation detection will improve and become more widely available. This will increase the need for genetic counseling. Normal and pathologic regulation of menin will be clarified in tumors and other states. Over a longer time frame, it is likely that cellular and animal models of MEN1 inactivation and of forced overexpression of MEN1 will contribute to understanding and to exploration of new treatments of hereditary and sporadic tumors. Patient care will be improved from further research on the menin metabolic pathways and the MEN1 gene.

ACKNOWLEDGMENTS The studies of MEN-1 at the National Institutes of Health began and developed u n d e r the direction of the late Gerald Aurbach. His contributions were numerous and profound. Many of the ideas and much of the data cited herein arose from collaborations at the National Institutes of Health and elsewhere. Some of the principal collaborators have been Sunita K. Agarwal,

MULTIPLE ENDOCRINE NEOPraSIA TYeE 1

H. Richard Alexander, Gerald D. Aurbach, Allen E. Bale, Sherri J. Bale, Mark S. Boguski, Maria-Luisa Brandi, A. Lee Burns, Settara C. Chandrasekharappa, Francis S. Collins, Judy S. Crabtree, Thomas N. Darling, Larisa V. Debelenko, Qihan Dong, John L. Doppman, Michael R. Emmert-Buck, Eitan Friedman, Paul K. Goldsmith, Jane S. Green, Joseph E. Green III, Sirandalahalli C. Guru, Christina Heppner, Robert C. Jensen, Mary-Beth Kester, Young C. Kim, Stephen K. Libutti, Lance A. Liotta, Irina A. Lubensky, Pachiappan Manickam, ShodimuEmmanuel Olufemi, Bruce A. Roe, William E Simonds, Monica C. Skarulis, Allen M. Spiegel, Lee S. Weinstein, Jane Weisemann, and Zhengping Zhuang. I am also indebted to additional participants in the NIDDK/ NICHD Interinstitute Endocrine Training Program and to many patients and their families. The organization and portions of the text of this chapter are based partly on two multiauthored companion chapters about MEN-1 from the previous edition of this volume (80,321).

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32. Tortosa E Chico A, Rodriguez-EspinosaJ, RuscalledaJ, de Leiva A, MEN1 Study Group. Prevalence of MEN1 in patients with prolactinoma. Clin Endocrino11999;50:271-272. 33. Bassett JHD, Forbes SA, Pannett AAJ, Lloyd SE, Christie PT, Wooding C, Harding B, Besser GM, Edwards CR, Monson JP, Sampson J, Wass JAH, Wheeler MH, Thakker RV. Characterization of the mutations in patients with multiple endocrine neoplasia type 1. A m J H u m Genet 1998;62:232-244. 34. Yoshimoto K, Saito S. Clinical characteristics in multiple endocrine neoplasia type 1 in Japan: A review of 106 patients. Folia Endocrinol 1991 ;67: 764-774. 35. Hai N, Aoki N, Matsuda A, Mori T, Kosugi S. Germline MEN1 mutations in sixteen Japanese families with multiple endocrine neoplasia type 1 (MEN1). EurJEndocrino11999;141:475-80. 36. Hai N, Aoki N, Shimatsu A, Mori T, Kosugi S. Clinical features of multiple endocrine neoplasia type 1 (MEN1) phenocopy without germline MEN1 gene mutations: Analysis of 20Japanese sporadic cases without MEN1. Clin Endocrinol 2000;52:509-518. 37. Agarwal SK, Kester MB, Debelenko LV, Heppner C, EmmertBuck MR, Skarulis MC, Doppman JL, Kim YS, Lubensky IA, Zhuang Z, Green JS, Guru SC, Manickam P, Olufemi SE, Liotta LA, Chandrasekharappa SC, Collins FS, Spiegel AM, Burns AL, Marx SJ. Germline mutations of the MEN1 gene in familial multiple endocrine neoplasia type 1 and related states. Hum Mol Genet 1997;6:1169-1177. 38. Eberle E Grun R. Multiple endocrine neoplasia, type I (MEN1). Ergeb Inn Med 1981;46:76-149. 39. Vasen HFA, Lamers CBHW, Lips CJM. Screening for multiple endocrine neoplasia syndrome type I: A study of 11 kindreds in the Netherlands. Arch Intern Med 1989;149:2717-2722. 40. Wilkinson S, Teh BT, Davey KR, McArdleJP, Young M, Shepherd JJ. Cause of death in multiple endocrine neoplasia type 1. Arch Surg 1993;128:683-690. 41. Doherty GM, Olson JA, Frisella MM, et al. Lethality of multiple endocrine neoplasia type 1. WorldJ Surg 1998;22:581-585. 42. Yu E Venzon DJ, Serrano J, Goebel SU, Doppman JL, Gibril F, Jensen RT. Prospective study of the clinical course, prognostic factors, causes of death, and survival in patients with ling-standing Zollinger-Ellison syndrome. J Clin Oncol 1999;17:615-630. 43. Ballard HS, Frame B, Hartsock RJ. Familial multiple endocrine adenoma-peptic ulcer complex. Medicine 1964;43:481-516. 44. Majewski JT, Wilson SD. The MEA-I syndrome: An all or none phenomenon? Surgery 1979;86:475-484. 45. Marx SJ, Spiegel AM, Levine MA, Rizzoli RE, Lasker RD, Santora AC, Jr, Downs RW, Aurbach GD. Familial hypocalciuric hypercalcemia: The relation to primary parathyroid hyperplasia. N EnglJ Med 1982;307:416-426. 46. Marx SJ, Vinik AI, Santen RJ, Floyd JC, Jr, Mills JL, Green III JE. Multiple endocrine neoplasia type I: Assessment of laboratory tests to screen for the gene in a large kindred. Medicine 1986;65:226-241. 47. Skogseid B, Eriksson B, Lundqvist G. Multiple endocrine neoplasia type 1: A 10-year prospective screening study in four kindreds. J Clin Endocrinol Metab 1991 ;73:281-287. 48. Trump D, Farren B, Wooding C, PangJT, Besser GM, Buchanan KD, Edwards CR, Heath DA, Jackson CE, Jansen S, Lips K, Monson JP, O'Halloran D, Sampson J, Shalet SM, Wheeler MH, Zink A, Thakker RV. Clinical studies of multiple endocrine neoplasia type 1 (MEN1). QJMed 1996;89:653-669. 49. Skarulis MC: Clinical expressions of MEN1 at NIH. Multiple endocrine neoplasia type 1: Clinical and genetic topics. Ann Intern Med 1998;129:484-494. 50. Benya RV, Metz DC, Venzon DJ, Fishbeyn VA, Strader DB, Orbuch M, Jensen RT. Zollinger-Ellison syndrome can be the initial endocrine manifestation in patients with multiple endocrine neoplasia-type I. AmJMed 1994;5:436-444.

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510. Williamson C, Pannett A, PangJT, McCarthy M, Sherppard MN, Monson JP, Clayton RN, Thakker RV. Localisation of a tumour suppressor gene causing endocrine tumours to a four centimorgan region on chromosome 1. Prog Abstracts Endocrinol Soc 1996;961 (abstract). 511. Kytola S, Makinen MJ, Kahkonen M, Teh BT, Leisti J, Salmela P. Comparative genomic hybridization studies in tumors from a patient with multiple endocrine neoplasia type 1. Eur J Endocrino11998;139:202-206. 512. Tahara H, Smith AP, Gaz RD, Cryns VL, Arnold A. Genomic localization of novel candidate tumor suppressor gene loci in human parathyroid adenomas. Cancer Res 1996;56:599-605. 513. Williamson C, Pannett AA, Pang JT, Wooding C, McCarthy M, Sheppard MN, et al. Localisation of a gene causing endocrine neoplasia to a 4 cM region of chromosome l p35-p36. J Med Genet 1997;34:617-619. 514. Agarwal SK, Schrock E, Kester MB, Burns AL, Heffess CS, Reid T, Marx sJ. Comparative genome hybridization analysis of human parathyroid tumors. Cancer Gene Cytogenet 1998;106:30-36. 515. Palanisamy N, Imanishy Y, Rao PH, Tahara H, Chaganti RSK, Arnold A. Novel chromosomal abnormalities identifed by comparative genomic hybridization in parathyroid adenomas. J Clin Endocrinol Metab 1998;83:1766-1770. 516. Dotzenrath C, Teh BT, Farnebo T, Cupisti K, Svensson A, Toell A, Goretzki P, Larsson C. Allelic loss of the retinoblastoma tumor suppressor gene: A marker for aggressive parathyroid tumors? J Clin Endocrinol Metab 1996;81:3194-3196. 517. Pearce SH, Trump D, Woodling C, Sheppard MN, Clayton RN, Thakker RV. Loss of heterozygosity studies at the retinoblastoma and breast cancer susceptibility (BRCA2) loci in pituitary, parathroid, pancreatic and carcinoid tumors. Clin Endocrinol 1996; 45:195-200. 518. Thompson DB, Samowitz WS, Odelberg S, Davis RK, Szabo J, Heath III H. Genetic abnormalities in sporadic parathyroid adenomas: Loss of heterozygosity for chromosome 3q markers flanking the calcium receptor locus. J Clin Endocrinol Metab 1995;80:3377-3380. 519. Kifor O, Moore FD, Jr, Wang P, Goldstein M, Vassilev P, Kifor I, Hebert SC, Brown EM. Reduced immunostaining for the extracellular Ca2+ -sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 1996;81:1598-1606. 520. Pausova Z, Soliman E, Amizuka N, Janicic N, Konrad EM, Arnold A, Goltzman D, Hendy GN. Role of the RET protooncogene in sporadic hyperparathyroidism and in hyperparathyroidism of multiple endocrine neoplasia type 2. J Clin Endocrinol Metab 1996;81:2711-2718. 521. Bartsch D, Hahn SA, Danichevski KD, Ramaswamy A, Bastian D, Galehdari H, et al. Mutations of the DPC4/Smad4 gene in neuroendocrine pancreatic tumors. Oncogene 1999;18: 2367-2371. 522. Bale AE, Norton JA, Wong EL, FryburgJS, Maton PN, Oldfield EH, Streeten E, Aurbach GD, Brandi ML, Friedman E, Spiegel AM, Taggart RT, Marx SJ. Allelic loss on chromosome 11 in hereditary and sporadic tumors related to familial multiple endocrine neoplasia type 1. Cancer Res 1991;51:1154-1157. 523. Thakker RV, Pook MA, Wooding C, Boscaro M, Scanarini M, Clayton RN. Association of somatotrophinomas with loss of alleles on chromsome 11 and with gsp mutations. J Clin Invest 1993;91:2815-2821. 524. Dong Q, Debelenko L, Chandrasekharappa S, Emmert-Buck MR, Zhuang Z, Guru SC, Manickam P, Skarulis M, Lubensky IA, Liotta LA, Collins FS, Marx SJ, Spiegel AM. Loss of heterozygosity at 1 lq13: Analysis of pituitary tumors, lung carcinoids, lipomas, and other uncommon tumors in familial multiple endocrine neoplasia type 1. J Clin Endocrinol Metab 1997;82:1416-1420.

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525. Eubanks PJ, Sawicki ME Samara GJ, Gratti R, Nakamura Y, Tsao D, Johnson C, Hurwitz M, Wan YJ, Passaro E. Putative tumorsuppressor gene on chromosome 11 is important in sporadic endocrine tumor formation. A m J Surg 1994;167:180-184. 526a. Chakrabarti R, Srivatsan ES, Wood TF, Eubanks PJ, Ebrahimi SA, Gatti RA, Passaro E, Sawicki ME Deletion mapping of endocrine tumors localizes a second tumor supressor gene on chromosome band 11q13. Genes, Chromos Cancer 1998;22:130-137. 526b.Nord B, Larsson C, Wong FK, Wallin G, Teh BT, Zedenius J: Sporadic follicular thyroid tumors show loss of a 200-kb region in llq13 without evidence for mutations in the MEN1 gene. Genes Chromos Cancer 1999;26:35-39. 527. Gisselson D, Hoglund M, Mertens F, dal Cin P, Mandahl N. Hibernomas are characterized by homozygous deletions in the multiple endocrine neoplasia type 1 region. Am J Pathol 1999;155:61-66. 528. Bates AS, Farrell WE, Bicknell EJ, McNicol AM, Talbots AJ, Broome JC, Perrett CW, Thakker RV, Clayton RN. Allelic deletion in pituitary adenomas reflects aggressive biological activity and has potential value as a prognostic marker. J Clin Endocrinol Metab 1997;82:818-824. 529. Hosokawa Y, Arnold A. Mechanism of cyclin D1 (CCND1, PRAD1) overexpression in human cancer cells: Analysis of allele-specific expression. Genes Chromos Cancer 1998;22:66-71. 530. Wong KF. l l q 1 3 is a cytogenetically promiscuous site in hematologic malignancies. Cancer Genet Cytogenet 1999;113:93-95. 531a.Larsson C, Shepherd J, Nakamura Y, Blomberg C, Weber G, Werelius B, et al. Predictive testing for multiple endocrine neoplasia type 1 using DNA polymorphisms. J Clin Invest 1992;89:1344-1349. 531b.Karges W, Jostarndt K, Maier S, Flemming A, Weitz M, Wissman A, et al. Multiple endocrine neoplasia type 1 (MEN1) gene mutations in a subset of patients with sporadic and familial primary hyperparathyroidism target the coding sequence but spare the promoter region. J Endocrino12000;166:1-9. 531c. Osthus RC, Reyes Y, Glynn MW, Bale AE: Mutations in typical and variant MENI kindreds: Splice site mutation in familial isolated hyperparathyroidism. Am J Hum Genet 1998;63 (Suppl) :A80 (Abstract). 532. Shimizu S, Tsukada T, Futami H, Ui K, Kameya T, Kawanaka M, et al. Germline mutations of the MEN1 gene in Japanese kin-

dred with multiple endocrine neoplasia type 1. J p n J Cancer Res 1997;88:1029-1032. 533. Beaudet AL, Tsui L-C. A suggested nomenclature for designating mutations. Hum Mutat 1993;2:245-288. 534. Takami H, Shirahama S, Ikeda Y, et al. Familial hyperparathyroidism. Biomed & Pharmacother 2000;54 Suppl 1:21-24. 535. Fujimori M, Shirahama S, Sakurai A, Hashizumi K, Hama Y, Ito K, et al. Novel V184E MEN1 germline mutation in a Japanese kindred with familial hyperparaathyroidism. Am J Med Genet 1998;80:221-222. 536a.Teh BT, Esapa CT, Houlston R, Grandell U, Farnebo F, Nordenskjold M, et al. A family with isolated hyperparathyroidism segregating a missense MEN1 mutation and showing loss of the wild type alleles in the parathyroid tumors. A m J H u m Genet 1998;63:1544-1549. 536b. Honda M, Tsukada T, Tanaka H, et al. A novel mutation of the MEN1 gene in a Japanese kindred with familial isolated primary hyperparathyroidism. EurJ Endocrino12000;142:138-143. 537a. Ohye H, Sato M, Matsubara S, Miyauchi A, Imachi H, Murao K, Takahara J. Germline mutation of the multiple endocrine neoplasia type 1 (MEN1) gene in a family with primary hyperparathyroidism. EndocrJ 1998;45:719-723. 537b. Bergman L, Teh B, Cardinal J, et al. Identification of MEN1 gene mutations in families with MEN1 and related disorders. BritJ Canc 2000;83:1009-1014. 538. Agarwal SK, Debelenko LV, Kester MB, Guru SC, Manickam P, Olufemi SE, Skarulis MC, Heppner C, Crabtree JS, Lubensky IA, Zhuang Z, Kim YS, Chandrasekharappa SC, Collins FS, Liotta LA, Spiegel AM, Burns AL, Emmert-Buck MR, Marx SJ. Analysis of recurrent germline mutations in the MEN1 gene encountered in apparently unrelated families. Hum Murat 1998;12: 75-82. 539. American Society of Human Genetics Board of Directors and the American College of Medical Genetics Board of Directors: Points to consider: Ethical, legal, and psychosocial implications of genetic testing in children and adolescents. A m J H u m Genet 1995;57:1233-1241. 540. Marx SJ. Familial multiple endocrine neoplasia type 1. NIH Publication No. 96-3048, 1997. An updated version is available through NIH/NIDDK. Internet address: http://www.niddk.nih. gov/health / endo / pubs / fme n 1/ fmen 1.h tm

CI-IAP3:FR 3 6

Multiple Endocrine Neoplasia Type 2

ROBERT E GAGEL Division of Internal Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

INTRODUCTION

MULTIPLE E N D O C R I N E NEOPLASIA TYPE 2

Multiple endocrine neoplasia type 2 (MEN-2) is an autosomal dominant hereditary syndrome with diverse clinical manifestations. A unifying feature is the derivation of most of the components from the neural crest. The association of thyroid carcinoma and pheochromocytoma was first noted 40 years ago (1), although it is clear that some variants of the syndrome were described at least 50 years earlier (2,3). The components of the syndrome that have garnered the most attention are medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Other features of variant syndromes include mucosal and alimentary neuromas, skeletal abnormalities, a rare form of cutaneous amyloidosis, and Hirschsprung disease. The derivation of most of the neuroendocrine and neuronal cell types from the neural crest led to the recognition that the causative gene was likely to be involved in differentiation of the neural crest. The subsequent recognition that mutations of the RET protooncogene, a tyrosine kinase receptor for glial cell-derived neurotrophic factor (GDNF), cause MEN-2 and its several variants confirmed prior scientific hunches and defined a unique receptor signaling system important in neural crest differentiation. This chapter integrates a rapidly evolving literature regarding the molecular abnormalities in MEN-2 and neural crest development with the clinical features of MEN-2. The clinical use of RET genetic testing will also be addressed. Clinical management of specific MEN-2 manifestations will not be addressed because of extensive review elsewhere (4-9).

An association between thyroid carcinoma and pheochromocytoma was first described by Sipple in 1961 (1), although an understanding of the syndrome, its heritable components, and its differentiation from multiple endocrine neoplasia type 1 (MEN-l) evolved over the subsequent decade (10,11). Although these syndromes were first synthesized in their current form less than 40 years ago, there is unequivocal evidence based on geneologic studies of affected kindreds that the mutant gene existed in the eighteenth century or earlier (12). Williams and co-workers (13) are generally credited with the reidentification of the mucosal neuroma syndrome (MEN-2B) first described by Froboese (3) and the recognition that this clinical syndrome is associated with MTC and pheochromocytoma. Additional variants of MEN-2 include familial medullary thyroid carcinoma (FMTC) with no other manifestations of MEN-2 (14), MEN-2A in association with cutaneous lichen amyloidosis (15,16), and MEN-2A in association with Hirschsprung disease (17) (Table 1).

The Parathyroids, Second Edition

MULTIPLE E N D O C R I N E NEOPLASIA TYPE 2A The term Sipple's syndrome is most correctly applied to the association of MTC, pheochromocytoma, and parathyroid neoplasia (18) (Table 1), now known as MEN-2A. Our thinking about this syndrome has evolved over the past several decades. During the first decade after Sipple's report, the three major 585

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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CI-IAeTF.R36 TABLE 1

Multiple Endocrine Neoplasia Type 2 Classification

MEN-2A MTC Pheochromocytoma Parathyroid neoplasia Variants of MEN-2A MEN-2A in association with Hirschsprung disease MEN-2A in association with cutaneous lichen amyloidosis Familial MTC only (FMTC) MEN-2B MTC Pheochromocytoma Mucosal and aliamentary tract ganglioneuromatosis Marfanoid features Absence of parathyroid neoplasia

components of the syndrome were characterized (10,19). The convergence of several observations led to the use of provocative tests for diagnosis of early MTC. These included the identification of the peptide hormone calcitonin (20-22), the identification of the C cell (thyroid parafollicular cell) as the transformed cell type in MTC (23), and the development of radioimmunosaays for calcitonin (11,24). The observation that either intravenous calcium or pentagastrin could be used to stimulate calcitonin release (25-28) provided Melvin and co-workers with a tool to identify early C cell abnormalities. This included C cell hyperplasia, a histologic lesion thought to represent a premalignant form of MTC (29). This provocative test was subsequently used by others to screen MEN-2 kindreds for early C cell abnormalities (30-32). In parallel studies, hyperplasia of the adrenal medulla was identified as a precursor lesion (31,33,34). Implementation of prospective screening studies over the next two decades resulted in the routine identification of MTC and pheochromocyoma at early developmental stages. It is now reasonable to believe that death related to either of these manifestations can be prevented by surgical intervention at an appropriate point during the natural history of this clinical syndrome (30,35).

T H E N A T U R A L H I S T O R Y OF T H E T H R E E N E O P L A S T I C M A N I F E S T A T I O N S O F MEN-2 C Cell H y p e r p l a s i a a n d MTC The parafollicular cells are distributed within the thyroid gland in a characteristic pattern (Fig. 1A). Their greatest concentration occurs in the upper portion of each lobe in a central location intersecting the upper one-third and lower two-thirds of each lobe.

~

C

FIG. 1 Distribution of C cells in the thyroid gland. (A) Reconstruction of the distribution of C cells in the thyroid gland. The greatest concentration of C cells occurs at the junction of the upper one-half and lower-two thirds of the gland. This distribution explains the characteristic location of hereditary medullary thyroid carcinoma. (Modified from Wolfe HJ, Voelkel EF, Tashjian AH, Jr. Distribution of calcitonin containing cells in the normal adult human thyroid gland: A correlation of morphology with peptide content. J Clin Endocrinol Metab 1974;38:688-694. © The Endocrine Society.) (B) Hereditary medullary thyroid carcinoma is almost always bilateral, although the extent of involvement may not be equal. (C) Sporadic medullary thyroid carcinoma is most commonly a unilateral process that may develop at any location within the thyroid gland.

Transformation of the C cell is thought to be a random, clonal event (36) and it is therefore not surprising that most examples of hereditary MTC develop in this anatomic location where the concentration of C cells is greatest (Fig. 1). Hyperplasia of the C cells is the earliest abnormality observed in hereditary MTC. This lesion most commonly develops in childhood in MEN-2A (Fig. 2A) (12,30,35). Continued growth (or a transforming event) leads to the development of microscopic carcinoma (Fig. 2B). It is not clear at what point along this pathway of progression that metastasis occurs, although it has been found in individuals with microscopic carcinoma (37-39). It is common to find separate foci within the thyroid gland at different developmental points along the pathway from normal to carcinoma. Metastasis to central lymph nodes of the neck occurs commonly in patients with carcinoma detectable by the human eye at the time of examination. The fact that 25% of patients with lymph node metastasis without evidence of other metastatic disease can be cured by neck lymph node dissection (35,40) suggests that spread to local lymph nodes occurs first, with later development of distant metastatic disease. A considerable literature has developed regarding secretory or nonsecretory proteins produced by MTC, collectively categorized as tumor markers. Genes that are normally expressed by the C cell include those encoding calcitonin, somatostatin (41), chromogrannin A (42), and DOPA decarboxylase (8,41,43-47). Their expression in MTC is normal, although the level of expression and subsequent processing events may be altered. Less is known about the expression of

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FIG. 2 Progression of histologic changes in hereditary medullary thyroid carcinoma. There is a progression of histologic findings from a normal distribution of parafollicular cells to nodular hyperplasia to microscopic carcinoma. (A) Nodular hyperplasia with displacement of a thyroid follicle by parafollicular cells (× 100) (reprinted with permission from Ref. 4; JF Moley, WG Dilley, MK DeBenedetti. Improved results of cervical reoperation for medullary thyroid carcinoma. Ann Surg 1997;225:734-740.). (B) Microscopic medullary thyroid carcinoma, which stains positive for calcitonin by immunohistochemical staining (x400) (reprinted with permission from Ref. 12; M Telenius-Berg, B Berg, B Hamberger, S Tibblin, LE Tisell, LYsander, G Welander. Impact of screening on prognosis in the multiple endocrine neoplasia type 2 syndromes: Natural history and treatment results in 105 patients. Henry Ford Hosp Med J 1984; 32:225-231 ).

carcinoembryonic antigen, insulin-like growth factors (IGF-I and IGF-II), and fibroblast growth factor, and their receptors (48). There is abundant evidence that one or more variants of the somatostatin receptor is present in normal and transformed C cells (49-51). The utilization of each of these expressed gene products for diagnostic or therapeutic purposes has been explored and several, including calcitonin, carcinoembryonic antigen, and somatostatin, have proved useful. The fully formed MTC is characteristically multifocal. Each tumor appears as a whitish-yellow circumscribed lesion on a background of thyroid tissue. The characteristic histologic features of the tumor include a nested

endocrine-type appearance or sheets of cells interspersed with amyloid, which is derived at least in part from calcitonin gene products (52).

Hyperplasia and Pheochromocytoma

Adrenal Medullary

Pheochromocytomas occur in approximately 50% of gene carriers and are bilateral in approximately onehalf (10,35). It is reasonable to believe that adrenal medullary abnormalities of a lesser degree occur in a higher percentage of patients and occur in parallel with the hyperplasdc events affecting the C cell.

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Several features differentiate pheochromocytomas associated with the MEN-2 from sporadic pheochromocytomas. The most characteristic is the type of catecholamine produced. Almost all pheochromocytomas associated with MEN-2 develop in the adrenal medulla and express the enzyme phenylethanolamine-N-methyltransferase, the enzyme that catalyzes the methylation of norepinephrine to epinephrine. Studies from multiple kindreds have demonstrated an increase in the relative production of epinephrine in MEN-2-associated pheochromocytomas. The first abnormality observed in a MEN-2 gene carrier is an absolute increase in the 24-hour urine or an exercise-induced plasma epinephrine concentration (31,35,53,54) or an increase in plasma normetanephrine or metanephrine values (55). Palpitations, nervousness, jitteriness, and the absence of hypertension, the earliest clinical signs and symptoms in MEN-2 patients who are subsequently documented to have adrenal medullary hyperplasia or pheochromocytoma, are thought to relate directly to the increased epinephrine production. In patients whose adrenal medullary abnormalities remain undetected, there may be a subsequent development of hypertension. A second characteristic feature is the invariate location of the pheochromocytoma in association with the adrenal medulla. The few examples of extraadrenal pheochromocytoma in this syndrome are thought to have arisen from adrenal rest tissue or represent recurrence of a pheochromocytoma in the anatomic region of the adrenal gland (56,57).

Parathyroid Abnormalities It has beeen difficult to study the natural history of hyperparathyroidism in MEN-2 because of its low incidence (5-20%) and the frequent late onset of clinical features of hyperparathyroidism. Nonetheless, several interesting points have emerged. First, in some kindreds hyperparathyroidism occurs with greater frequency than others. A second observation, made in several families, is the lack of hyperparathyroidism in gene carriers who were thyroidectomized for early C cell abnormalities, despite follow-up periods of 15-25 years (35,58). Whether this observation has pathophysiologic significance or has resulted from the inevitable loss of parathyroid tissue during total thyroidectomy is not clear and will require additional investigation. The clinical syndrome of hyperparathyroidism does not appear to differ from that observed with sporadic hyperparathyroidism. Hypercalcemia and nephrolithiasis are the most common presenting manifestations found in MEN-2 patients. Pheochromocytoma may rarely cause hypercalcemia, possibly related to production of parathyroid hormone-related protein, and should be excluded prior to surgical exploration (35).

Hyperplasia of multiple parathyroid glands is the most common abnormality found in hyperparathyroidism associated with MEN-2, although many reports emphasize the occurrence of multiple parathyroid adenomas in individuals who present with the fully developed MEN-2 syndrome, generally after the age of 35 years.

FAMILIAL MEDULLARY THYROID CARCINOMA In approximately 10-15% of families hereditary MTC occurs without other manifestations of MEN-2A (FMTC). The pattern of inheritance and clinical features of MTC in this variant do not differ from those found in MEN-2A except that the MTC tends to develop later and is, in general, a clinically less virulent form of the disease (14). Categorization of a family as having FMTC should be made only after a careful examination of the clinical spectrum of the disease in several generations of family members. The penetrance of pheochromocytoma and parathyroid disease in MEN-2A is 50% or less and categorization of a small family with apparent FMTC may result in inadequate screening for pheochromocytoma.

HIRSCHSPRUNG'S DISEASE IN ASSOCIATION WITH MEN-2A A small number of families have been described in which classic Hirschsprung disease has been observed in association with MEN-2A. In the MEN-2A/Hirschsprung disease variant, aganglioneuromatosis may be found throughout the colon or in smaller segments (17). This contrasts with MEN-2B, whereby there are oral mucosal neuromas and neuromas throughout the entire gastrointestinal tract. The gastrointestinal clinical features of Hirschsprung disease and those associated with MEN2B may overlap and include megacolon, obstruction, and colonic atony. Clinical differentiation between this variant and MEN-2B based solely on gastrointestinal symptomatology may be difficult, especially in those patients who do not have other features of MEN-2B (see section on MEN-2B below).

MEN-2A WITH CUTANEOUS LICHEN A M Y L O I D O S I S In 15-20 kindreds a unilateral or bilateral pruritic skin lesion located over the upper back (dermatomes C6-T5) has been associated with MEN-2A (Fig. 3) (16,59). The clinical features of MEN-2A in these families do not differ in any recognizable way from the

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FIG. 3 Characteristic skin lesion associated with cutaneous lichen amyloidosis associated with MEN-2A. The lesion has been found unilaterally or bilaterally over the upper back in all individuals affected with this variant.

more common MEN-2A except for the presence of the skin lesion and a relative paucity of parathyroid disease (60-63). Patients with this variant describe intermittent periods of intense pruritus during which they scratch the upper back. These symptoms begin in early childhood in some families and may precede the developm e n t of MTC (15). In other kindreds the skin lesion develops later. Biopsy of more advanced forms of this lesion has demonstrated typical findings of cutaneous lichen amyloidosis (16), whereas amyloid is found infrequently in early skin lesions (15,62,64). Several explanations have been considered to explain the presence of amyloid. One possibility, that the amyloid consists of calcitonin gene products, has been excluded experimentally (16,59,65). Immunohistochemical studies indicate the amyloid consists of keratin, analogous to more common varieties of hereditary or sporadic cutaneous lichen amyloidosis (66). A dermatome-like distribution for the skin lesion that corresponds to the normal expression of RET (67) (Fig. 3) combined with a clinical history of intense pruritis preceding the development of the skin lesion suggests that the skin lesion is caused by RET-induced neuronal firing causing pruritus (68). The finding of abnormal cutaneous innervation in RET knockout mice provides additional support for the hypothesis that this clinical finding is directly related to RET overexpression.

MULTIPLE E N D O C R I N E NEOPLASIA T Y P E 2B The association of MTC, pheochromcytoma, mucosal neuromatosis, skeletal abnormalities suggestive of

Marfan syndrome, and the absence of hyperparathyroidism has been categorized as multiple endocrine neoplasia type 2B (MEN-2B). The most striking characteristic of this clinical syndrome is the presence of mucosal neuromas on the tip of the tongue, on the conjunctival surface of the eye lids, and throughout the gastrointestinal tract. The most common presenting manifestation in children are gastrointestinal manifestations that include diarrhea, intestinal obstruction, megacolon, and crampy abdominal pain (69). The marfanoid-like manifestations include an altered u p p e r / l o w e r body ratio, pectus abnormalities, long and thin arms and fingers, hyperextensibility of joints, and epiphyseal abnormalities (13,70,71). Other manifestations of Marfan syndrome such as lens and aortic manifestations are not seen. The penetrance of medullary thyroid carcinoma in MEN-2B approaches 100% and is evident at an early age. Metastastatic disease has been found in the first year of life and is uniformly present in cases diagnosed after the age of 10 years. If the disease is not diagnosed prior to development of metastasis, the tumor pursues a virulent course in about 50% of patients. Greater longterm experience with MEN-2B indicates the prognosis is not uniformly poor (72,73) and a n u m b e r of multigenerational families exist (73). Pheochromocytomas are found in approximately 50% of affected individuals and do not differ substantially from those found in MEN-2A. Rarely MTC is the only identifiable manifestation, suggesting that the penetrance of clinical features in MEN2B is variable. One example is a mother with MEN-2B and two children with MTC, only one of whom had the ganglioneuromatosis phenotype (74). The author has seen two additional patients (with proved molecular

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abnormalities of MEN-2B) with variable penetrance, one with ganglioneuromatosis without other skeletal findings of MEN-2B and another with complete absence of skeletal manifestations and minimal evidence of ganglioneuromatosis. Hyperparathy-roidism occurs rarely (75). A syndrome consisting of mucosal neuromas without other features of MEN-2B and no identifiable genetic abnormality has been identified (76).

MAPPING THE MEN-2 LOCUS The availability of large and well-defined kindreds with MEN-2 made it an early candidate for application of DNA polymorphism-based genetic linkage approaches during the early 1980s. The gene was m a p p e d to centromeric chromosome 10 in 1987 (77,78). Although progress was h a m p e r e d by low rates of recombination a r o u n d the centromeric locus (79), contributions by multiple groups gradually narrowed the genetic region and in 1993 mutations of the RET p r o t o o n c o g e n e were identified (80,81).

GFRo~-I (Glial Cell-Derived Neurotrophic Factor Receptom Glial Cell-Derived / Neurotrophic Factorl~

Interaction between GDNF a n d R E T / G F R ( x - 1 leads to: - Branching of collecting ducts into kidney - Movement of neurons into gastrointestinal tract

RET Tyrosine Kinase Recept

- Normal dorsal sympathetic development - Possible role for migration of C cells into thyroid gland

FIG. 4 The RET receptor complex. Glial cell-derived neurotrophic factor (GDNF) is a small peptide that promotes neuronal survival. It interacts with a receptor complex formed by the RET tyrosine kinase receptor (RET) and the glial cellderived neurotrophic factor receptor (GFR(x-1). Activation of the receptor by GDNF causes autophosphorylation of RET and activation of ERK and JNK kinase pathways. GDNF and RET/GFR(x-1 interactions promote development of the collecting system of the kidney, normal migration of neurons from the neural crest into the gastrointestinal tract, and normal sympathetic nervous development in those neurons derived from the first six somites.

The RET Receptor System The 8 years since the initial identification of RET mutations in MEN-2 has brought maturity and clarity to the field. Not only have a broad spectrum of RET mutations been identified, but the components of the receptor system, their normal physiologic and developmental functions, and the mechanisms by which some of the mutations cause transformation have been elucidated. These are highlighted in this section. The RET receptor complex is defined by three components (Fig. 4). The first is the RET tyrosine kinase receptor, first identified in 1985 (82,83). In a series of remarkable observations over the past 4 years, two other components of this receptor system, a ligand (glial cell-derived neurotrophic factor) and a second extracellular c o m p o n e n t of the receptor system [glial cell-derived neurotrophic factor receptor e~-I (GFRe~1)] have been identified (Fig. 4) (84). The elucidation of the receptor system has also led to an understanding of the function of the receptor complex in normal physiologic development. The first step was the targeted disruption of the RET protooncogene. Mice homozygous for the targeted disruption have a distinctive phenotype characterized by a failure of normal kidney development, a Hirschsprung-like neuronal phenotype in the gastrointestinal tract (disordered neuronal development), and abnormal dorsal sympathetic development (67,85). The identification of an identical phenotype in a mouse knockout of GDNF, a small peptide that promotes neuronal survival (86),

first raised the possibility that RET might be a receptor for GDNE Mice with targeted disruption of both alleles of the GDNF gene develop a phenotype identical to that observed in RET knockout mice (87-89). These observations were followed very quickly by the demonstration that GDNF is a ligand for RET (90,91). The third observation, cloning of GFR~-I, resulted from a separate and parallel effort to define a receptor for GDNF (92). These efforts identified a protein that is tethered to the extracellular membrane, binds GDNF, but contains no transmembrane or intracellular domain. Within a short period of time it became clear that RET and GFR~-I together form the receptor complex for GDNF (Fig. 4) (93). Over the past several years several additional GRFc~-I variants have been identified along with at least three additional ligands, forming an important multicomponent receptor system involved in neuronal differentiation and function (84). Somewhat surprisingly, there have been no mutations of either GDNF or GFRc~-I identified in MEN-2, but both have been found to be expressed in MTC (94).

Mutations Causative for MEN-2 Genotype-Phenotype Correlation The mutations of the R E T p r o t o o n c o g e n e that cause hereditary MTC affect either the extracellular cysteinerich dimerization domain or the intracellular tyrosine kinase domain (Fig. 5). Most are single amino acid sub-

MULTIPLE ENDOCRINE NEOPLASIA TYPE 2

MEN2A/Sipple

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CLA

FIG. 5 Mutations of the RET protooncogene in hereditary MTC. There is a high degree of genotype-phenotype correlation between specific RET mutations and clinical phenotype (MEN-2A/Sipple, multiple endocrine neoplasia type 2A, or Sipple syndrome; FMTC, familial medullary thyroid carcinoma; MEN-2A/Hirschsprung, the coexpression of MEN-2A and Hirschsprung disease; MEN-2B, multiple endocrine neoplasia type 2B). The codons shown to be mutated account for over 98% of all known mutations in hereditary MTC. A more complete listing of mutations is available at Human Gene Mutation Database Cardiff at http://www.uwcm.ac.uk/uwcm/ m g/search/120346, html.

stitutions (missense mutations), although some small deletions have been described. Here we present the most common mutations; additional mutations continue to be reported and can be accessed through a Web data base. 1 The extracellular domain mutations affect highly conserved cysteine residues important for the regulation of receptor dimerization. The mutations associated with MEN-2 or familial MTC affect codons 609, 611, 618, 620, 630, and 634 (Fig. 5). Codon 634 mutations account for approximately 80% of all mutations in MEN-2, and a single substitution (TGC to CGC; cysteine to arginine) accounts for approximately 50% of all RET mutations found in hereditary MTC. The extracellular domain mutations cause transformation by promoting dimerization of the RET receptor complex and activation of downstream tyrosine kinase pathways in the absence of GDNE Intracellular mutations affect codons 768, 790, 791, 804, 891, 883, and 918. Mutations of codon 918 cause transformation by activation of downstream kinase pathways without causing receptor dimerization. The specific mechanism by

1Online Mendelian Inheritance in Man provides an overview of RET mutations at h t t p : / / w w w . n c b i . n l m . n i h . g o v / h t b i n - p o s t / Omim/dispmim? 164761. Additional information can be found at the H u m a n Gene Mutation Database Cardiff at http://www.uwcm.ac.uk/

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which other intracellular mutations cause transformation has not been evaluated. There is a specific genotype-phenotype relationship between specific codon substitutions and the clinical phenotype (Fig. 5). Any amino acid substitution at codon 634 is most commonly associated with classic MEN-2A, or Sipple syndrome (1,95); indeed Sipple's original kindred has a germ line codon 634 mutation. In addition, MEN-2A has been associated with mutations at codons 609, 611, 618, 620 (80,96), 791 (97), and V804L (98). Two unique variants of MEN-2A also have specific genotype-phenotype correlations. All patients with the MEN-2A/cutaneous lichen amyloidosis variant have a substitution at codon 634 (Fig. 5) (95). Individuals with MEN-2A and Hirschsprung disease have been described with mutations at codons 609, 618, and 620 (Fig. 5) (96,99,100). Familial MTC, a variant in which MTC is the only manifestation, is most commonly associated with RETmutations at codon 609, 611,618, or 620 (80,96), although substitutions at these codons have also been associated with MEN-2A (Fig. 5). Mutations at codons 768 (101), 790 (97), V804M (95,99,102-106), and 891 (107,108) have been associated only with FMTC, although the total n u m b e r of families reported is small for each of these codons (Fig. 5). The rarity of these latter mutations suggests that periodic screening for pheochromocytoma should continue in these families unless there is a clearly defined family history of MTC without other manifestations of MEN-2A. The most common mutation associated with MEN-2B is at codon 918 (109,110), although a handful of cases with mutations at codon 883 (111,112) have been identified (Fig. 5). Of interest is that the majority of cases of MEN-2B occur as de novo mutations (neither parent has a germ line mutation) and there is compelling evidence that most are derived from the paternal allele, suggesting the mutations are acquired during spermatogenesis (113). It is important to recognize that there are families with hereditary MTC in whom no RET mutation has been identified (1-2% of all kindreds); therefore, this compilation of mutations will probably enlarge over the next 5 years.

Involvement of RET in Other Malignancies A curious p h e n o m e n o n is that mutations of the RET protooncogene are also responsible for papillary thyroid carcinoma (PTC), the most common neoplasm of the thyroid follicular epithelium (114-118). These rearrangements bring the tyrosine kinase domain of RET under the regulatory control of promoter sequences of other genes through physical rearrangement [named P T C oncogene(s)]. Each of

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these rearrangements excludes the extracellular and transmembrane sequences from the final protein, leaving the intracellular tyrosine kinase domains expressed under the control of a ubiquitously expressed "housekeeping" gene. The P T C oncogene is found in 20-35% of papillary thyroid carcinomas, including the pediatric papillary thyroid carcinomas caused by radiation exposure resulting from the Chernobyl disaster, and is thought to be important in the development or progression of this tumor (119-125). Rearrangements of the RET protooncogene have been described in other tumors (117,126,127). In each of these cases, expression of the rearranged protooncogene is driven by promoter sequences thought to be constitutively expressed in the cell type and do not represent normal functioning of the receptor. Expression of the RETprotooncogene has been found frequently in neuroblastoma cell lines and tumors, evidence that it is expressed in malignant cells of neural crest origin (128,129).

H o w the RET Mutations Found in MEN-2 Cause Transformation There is evidence of enhanced expression of the RET protooncogene in hereditary and sporadic MTCs and pheochromocytomas derived from MEN-2 patients (116). These results were confirmed by Miya et al., who demonstrated high levels of expression in 12 of 12 MTCs and in 6 of 8 pheochromocytomas (130), whereas there was no expression of RET in adjacent follicular cells. Moreover, it was demonstrated that there was mRNA of different sizes, 7.0, 6.0, 4.5, and 3.9 kb, consistent with a normally expressed and alternatively processed RET mRNA. In addition there is evidence for expression of P&'T in MEN-2A parathyroid tumors (131). There is now abundant evidence that the missense mutations of RETinitiate the transformation process by one of two different mechanisms. Expression of RET with an extracellular domain mutation of codon 634 causes transformation of NIH 3T3 cells, which do not normally express RET. Detailed studies by several groups have shown that these mutations cause dimerization and activation of the receptor, presumably in the absence of GDNF (132,133). In contrast, the most common intracellular domain mutation (at codon 918) causes transformation without dimerization of the receptor (132,133). Both types of mutations cause receptor autophosphorylation and phosphorylation of downstream proteins in the signaling cascade; the tyrosine at codon 1062 (an Shc binding site) is autophosphorylated and mediates most of the transforming activity caused by mutations at either codon 634 or 918 (134-137). There is evidence that c-src interacts with the Shc binding site at tyrosine1062 (138). A JNK pathway is also acti-

vated by these mutations, providing a second potential pathway for mediation of transformation (139).

O T H E R GENETIC ABNORMALITIES IN HEREDITARY O R SPORADIC T U M O R S OF THE TYPE ASSOCIATED WITH MEN-2 Chromosome 1 A high percentage of MEN-2-associated tumors have a loss of heterozygosity on chromosome lp (140). Mapping studies have further defined the region between DIS15 and D1Z22 (141). There is also evidence of a loss of heterozygosity in sporadic pheochromocytoma at l p (142-145).

Chromosome 3 Studies by Taylor et al. document frequent abnormalities of chromosome 3 in human and rat MTC cell lines (146). In addition, studies by Khosla et al. in MTC and pheochromocytoma document loss of alleles on chromosome 3 in both of these tumors (142). Other studies document chromosomal loss at 3p in sporadic pheochromocytomas and paraganglionomas (143,144).

Chromosome 22 Reports from several sources provide evidence of a loss of heterozygosity on chromosome 22. Takai et al. have reported a loss of heterozygosity in one of nine MTCs and two of five pheochromocytomas taken from MEN-2 patients (147). Cooley et al. have characterized two human and three rat MTC cell lines and found evidence for chromosome 22 abnormalities, most commonly deletion, in all of these cell lines (146). Finally, Khosla and co-workers have demonstrated evidence for loss of alleles from chromosome 22q in 31% of pheochromocytomas (142).

A Multistep Model of Carcinogenesis It is unclear whether activating mutations of the RET protooncogene are sufficient for transformation. The finding of somatic loss of chromosomal DNA at lp, 3p, and 22q suggest that additional genetic events are involved in progression of MTC (Table 2). Other findings suggest that yet another mechanism may contribute to transformation. A high percentage of hereditary MTCs have somatic duplication of the mutant chromosome 10 or loss of the normal chromosome 10. This suggests that homodimerization of mutant receptor may be required for transformation (148).

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TABLE 2

Mechanisms for Transformation in MEN-2-Related Tumors

Primary genetic event Germ line missense mutations of the RET protooncogene Secondary genetic events Somatic reduplication of the mutant chromosome 10 in a single cell; may cause homodimerization of the mutant RET receptor Somatic loss of the normal chromosome 10 in a single cell; may cause homodimerization of the mutant RET receptor Somatic loss of Chromosomes lp, 3p, and 22q Additional mutational events not yet identified

THE RET PROTOONCOGENE: AN IMPORTANT REGULATOR OF NEURAL CREST DIFFERENTIATION Multiple endocrine neoplasia is a syndrome in which each of the affected tissues is a likely derivative of the neural crest. The C cells of the thyroid, the adrenal medullary cells, the parathyroid cells, the neuromas in MEN-2B, the cartilaginous tissue leading to marfanoidlike features, and the neuronal dysplasia associated with Hirschsprung disease involve cell types that are likely to originate in the neural crest. As a beginning, it may be instructive to sketch the development of neural crest tissue in broad strokes to provide a framework for subsequent discussion of individual cell types and relevant information relating to their development. It is important to recognize that what follows is an overview of very

A

DORSOLATERAL MIGRATION PATHWAY FOR MELANOCYTES AND ENTERIC NEURONS

VENTRAL MIGRATION PATHWAY FOR C CELLS, PARATHYROID G LANDS, SYMPATHETIC NEURONS AND ADRENAL GLANDS

B ADRENAL MEDULLA

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complex events and the account given is an attempt to orient the reader to current investigation related to embryologic development. There is very little in this rapidly evolving field that can be accepted as dogma at the present time. There is evidence showing that rostral-to-caudal segmentation of mammalian species is directed by a series of genes closely related to the Hox family of genes, which direct segmentation of Drosophila melanogast~ Studies in mammalian species indicate these genes are conserved and are likely to play a similar role in higher organisms. Targeted recombination of these genes in mice results in defective rostral-to-caudal segmentation and provides evidence for a similar role in mammalian species (149,150). Neural crest development proceeds within the developmental pattern provided by the Hox genes, but follows a developmental pattern of its own. The neural crest coalesces around the neural tube during development (Fig. 6). What has most fascinated several generations of investigators about the neural crest is its potential for development into many tissue types and the migratory behavior of its cells. Cells migrate from the neural crest in clearly defined patterns (Fig. 6) to form a variety of tissues. Migration of cells occurs in rostral-to-caudal and dorsal-to-ventral directions. These migratory patterns are observed most commonly within specific segments most likely developed u n d e r Hox gene control, defined as somites. Within these recurring units there is a pattern of organization (Fig. 6). For example, the neural crest cells destined to become the dorsal root ganglion migrate in a dorsal-to-ventral direction within the most rostral portion of each somite (151).

FIG. 6 (A) Cross-section of developing embryo showing patterns of neural crest migration. The pluripotential neural crest cells can either migrate in a ventral direction to form precursors for the sympathetic nervous system, adrenal medulla, C cells, and parathyroid glands, or laterally to develop into melanocytes or enteric neurons. Scl, Sclerotome; No, notochord; Ao, aorta; DM, dermatomyotome; NT, neural tube. (B) Impact of somite organization on neural crest migration patterns. The neural crest migration is tighly regulated, resulting in a segmental pattern of migration within the confines of a particular somite. There is a specific organization of sympathetic neurons and other neural crest-derived structures within this organizational pattern. Abbreviations: Scl, sclerotome; No, notochord; Ao, aorta; DM, dermatomyotome; NT, neural tube. Modified from Ref. 112.

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Migratory Pathways of Neural Crest Cells Involved in MEN-2 Derivation of the C Cell

The C (calcitonin-producing) cell type was among the first of the neural crest cells to be mapped. Studies by Le Douarin and co-workers (152,153), performed by grafting of quail neural crest cells with distinctive nuclear structure, demonstrated unequivocally that C cells are derived from neural crest and migrate in a ventral direction to their ultimate resting place in the ultimobranchial body (Fig. 6) in avian species (or the thyroid gland in mammalian species). Derivation and Differentiation of the Adrenal Medulla

The neural crest cells destined to become the adrenal medulla are derived from somites 18-24 (154). These pluripotential neural crest cells migrate in a ventral direction (Fig. 6) to form the progenitor cells for either the sympathetic nervous system or the adrenal medulla (151). What specifies a ventral route of migration of neural crest cells has not been determined, although it seems clear that a series of molecular processes that include expression of growth factors, glycoproteins, and their receptors provide primary signals. Positive reinforcement of the sympathoadrenal phenotype appears to be provided by fibroblast growth factor expression, which in turn stimulates expression of the trk A protooncogene (nerve growth factor receptor). Basic fibroblast growth factor (bFGF) causes the cell to differentiate to a neuronal phenotype, which includes the development of neuritic processes and the expression of receptors for, and a dependence on the presence of, nerve growth factor (NGF). Expression of NGF further reinforces the neuronal phenotype and commits the cell to a sympathetic phenotype (155). Development of the neuronal phenotype can be suppressed by high concentrations of glucocorticoid present in the developing adrenal gland. Glucocorticoids have been shown experimentally to prevent NGFinduced differentiation toward a sympathetic neuronal phenotype. This effect is thought to be mediated by positive or negative effects of glucocorticoid on transcription (155).

ROLE OF THE RET P R O T O O N C O G E N E IN NEURAL CREST DIFFERENTIATION The GDNF/RET/GFRe~-I receptor system is an ancient system. Studies in the zebrafish demonstrate that RET is expressed in primary motor and sensory neurons, a subset of neural crest cells and cranial ganglia, and in the enteric nervous system (156). A sub-

stantial amount of evidence has accumulated over the past 5 years to indicate that the GDNF/RET/GFRcx-1 receptor system is responsible for normal neuronal migration, differentiation, and development in somites 1-6 and in the developing gastrointestinal tract. This understanding has evolved from several different experimental approaches. Figure 7 shows the normal distribution of RET expression as it migrates from the neural crest to a more ventral position anterior to the developing spinal cord. In addition, there is a branching of RET immunoreactivity into the developing gastrointestinal tract with further migration of this plume of RETpositive cells into the gastrointestinal tract during embryonic development (Fig. 7). Further evidence for the important role of this receptor system comes from targeted deletion of the GDNF (87-89), RET (67,85), and GFRcx-1 genes in mice. The phenotypic abnormalities in these animals include diminished ganglionic development and lack of normal neural innervation (a Hirschsprung-like d i s o r d e r ) o f the gastrointestinal (GI) tract. It is therefore reasonable to conclude that GDNF expression in the gastrointestinal tract entices RET-positive cells into the GI tract, thereby causing normal gastrointestinal innervation. A question that remains unanswered is why C cells migrate from the neural crest to the ultimobranchial body in fish and birds, whereas in mice, rats, humans, and most other mammals the C cells are distributed throughout the thyroid gland. Observations by Belluardo et al. provide the first potential insight into this question. Studies of GFRc~-I and RET expression in the thyroid gland show that RET is expressed by the C cell, whereas GFRoL-1 is expressed on thyroid follicular cells (157). There is no GDNF expressed in the fully developed thyroid gland (157). These findings, if confirmed, suggest that there may be a unique relationship between the two major cell types in the thyroid gland that dictates their spatial organization, although more detailed embryologic studies will be needed to understand this process better.

USE OF GENETIC INFORMATION IN THE CLINICAL MANAGEMENT OF MEN-2 During the years since the discovery of RET mutations in MEN-2 there has been a remarkable coalescence of opinion and practice regarding the use of RET testing in the management of MTC, the most common manifestation of MEN-2 or FMTC. Indeed, the use of genetic testing in MEN-2 has quickly become one of the more successful uses of genetic testing for management of cancer, especially because there is little consensus regarding the use of genetic testing for clinical decision making in the m a n a g e m e n t of breast or colon cancer

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Expressioh

Somite

Branching of RET into developing gastrointestinal tract

Further movement of RET in gastro-

intestinal tract

FIG. 7 RET expression in the developing embryo. Initially RET expression is limited to the neural crest. During development there is a plume of RET-expressing neurons that migrate into the developing gastrointestinal tract. RET is also expressed in the developing urogenital bud that invades the mesonephros to form the collecting system of the kidney (not shown).

for which molecular causation has been known for a comparable period of time. There are several reasons for the rapid acceptance of genetic testing for m a n a g e m e n t of hereditary MTC. The first is that the known h o r m o n a l products produced by the thyroid gland can be easily replaced in children, although noncompliance with thyroid horm o n e can affect normal brain development. Second, thyroidectomy is well tolerated in young children, although development of hypoparathyroidism or recurrent laryngeal nerve damage remains a concern. Finally, an earlier experience has demonstrated the usefulness of early thyroidectomy in MEN-2. Studies initiated in the early 1970s based on provocative tests of calcitonin release were successful for the identification of early MTC or, in some cases, preneoplastic abnormalities (32,158,159). More importantly, thyroidectomy p e r f o r m e d at an average age of 13 years was well tolerated and more than 85% of these individuals remain without evidence of disease more than 25 years later (35). Thus the application of early detection strategies

for identification of hereditary MTC has given clinicians working in this field assurance that early treatm e n t will not only be well tolerated but has a high probability of success. It is important to recognize, however, that not all children who received early thyroidectomy (mean age of 13 years) were cured of their disease. Recurrent disease has been reported in 15-25% of individuals treated by thyroidectomy before the age of 20 years. The finding of metastatic MTC in children as young as 6 years of age and a few cases in younger children has led clinicians who care for these children to advocate earlier thyroidectomy than was possible when identification of gene carrier status was based on provocative calcitonin testing. Although incomplete, and certainly imperfect, experience with families with specific mutations over the past decade suggests there is a correlation between mutations at specific codons and MTC aggressiveness. In addition, there is a growing in vitro body of information describing the ability of specific mutations to cause transformation in in vitro assays.

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For example, there is evidence that a mutation at codon 634 has greater transforming capacity than does a mutation at codon 609, 611, 618 or 620 (160,161). Based on the c o m b i n e d clinical and in vitro experience there has been a stratification into three broad groups of aggressiveness. MTC caused by a mutation at codon 918 (MEN-2B) is generally considered to be the most aggressive form of this disease. Children with this mutation should have a total thyroidectomy and central node dissection performed during the first m o n t h of life or at the earliest time of detection. Cure is difficult because these children frequently come to attention between the ages of 5 and 10 years. In children with gross MTC an extensive lymph node dissection will improve the chances of a surgical cure (4,162). Children with mutations at codons 611,618, 620, 634 are considered to have a high risk of developing aggressive MTC. In these children there is a general recomm e n d a t i o n for thyroidectomy at the age of 5-6 years of age, although reports of metastatic MTC in children aged 2-4 suggest that earlier thyroidectomy may be required to cure most children. A total thyroidectomy with central lymph node dissection is generally considered to be appropriate therapy for these children (39,163,164). A minority of surgeons perform a total thyroidectomy, central neck dissection, and transplantation of parathyroid tissue to the n o n d o m i n a n t arm to lower the risk of hypoparathyroidism (5,39). Finally, children with a mutation at codon 609, 768, 790, 791,804, or 891 are considered to have an intermediate risk of aggressive disease. Deaths from MTC have been reported in kindreds with all of these mutations except for a mutation at codon 790 or 791 and there are kindreds in which metastasis has occurred in family members before the age of 30 years (106). It is also clear there are some kindreds with one of these mutations in which death from MTC has never been reported. It is difficult to convince a parent with one of these mutations and a clearly defined family history of benignity to have a young child with a m u t a n t RET gene undergo early thyroidectomy. As a result, clinicians caring for kindreds with these mutations have continued provocative testing for calcitonin abnormalities, delaying a thyroidectomy until the development of an abnormal test result. This type of approach may delay thyroidectomy until the mid-teenage years or later. A total thyroidectomy with central lymph node dissection is generally appropriate therapy for these cases. There is at present no evidence that earlier intervention will further improve outcomes in children with germ line mutations. Initial reports suggest that early intervention is associated with a high rate of success (5,39,163,164). However, this enthusiasm must be tempered by the reality that the genetic abnormality in MEN-2 affects all cells. Any remaining C cell has the

potential for subsequent transformation. This underscores the importance of completeness of the thyroidectomy and the n e e d for long-term follow-up. Most of these children are u n d e r the age of 5 years at the time of thyroidectomy and will be u n d e r the age of 30 years at the time of a 25-year follow-up. Use of genetic testing has had little impact on the m a n a g e m e n t of either p h e o c h r o m o c y t o m a or parathyroid disease in this clinical syndrome, except to exclude family m e m b e r s with a normal RET analysis from screening. Routine annual m e a s u r e m e n t of plasma or urine catecholamines or m e t a n e p h r i n e s permits routine detection of p h e o c h r o m o c y t o m a s before they become a significant clinical problem (9). Genetic testing is readily available through several commercial laboratories in the United States and through national testing programs in many countries in Europe. A partial listing of these sources can be found at http://endocrine.mdacc.tmc. Information about genetic testing outside the United States can be found at h t t p : / / m c r c r 2 . m e d . n y u . e d u / m u r p h p 0 1 / l a b s 2 . h t m .

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CHAPTER37 Familial Forms of Primary Hyperparathyroidism

LAWRENCE

MALLETTE

AND

ROBERT

MARCUS

VA Medical Centeg, Palo Alto, California 94304

Department of Medicine, Stanford University School of Medicine, and Aging Study Unit,

INTRODUCTION

14.7 mg/dl) due to a single parathyroid adenoma. Three members of this family developed recurrent hypercalcemia from a second parathyroid adenoma 6-10 years later, whereas the fourth had become hypoparathyroid after his first operation. Review of the pathologic specimens showed that each parathyroid tumor was a cystic adenoma. Cysts were also present in the normal parathyroid glands removed from these same patients, an unusual finding in people under age 35 years. The inheritance pattern suggested but did not prove an autosomal dominant trait. Mallette et al. termed this syndrome cystic parathyroid adenomatosis, using the term adenomatosis to emphasize the tendency to develop multiple adenomas, usually in a metachronous fashion (2). In the originally described cohort, urinary calcium excretion was elevated in all four patients, and one originally presented with nephrolithiasis. Three other first-degree relatives of the affected subjects were hypercalcemic. They were not available for detailed study, but two proved to be hypocalciuric, suggesting a possible relationship of this syndrome to benign familial (hypocalciuric) hypercalcemia (see Chapter 38). Three of the four adenoma patients also had undergone resection of ossifying fibromas of the mandible or maxilla. Even though they were resected while the patients were severely hyperparathyroid, these jaw tumors did not have the appearance of classic brown tumors of this condition because they contained no osteoclasts. The authors suggested that the tumors might be an integral part of the syndrome, but cautioned that data from additional affected kindreds were needed before this suggestion could be definitively accepted.

Although the great majority of patients with primary hyperparathyroidism have developed this condition as a sporadic event, several interesting and distinct familial syndromes have been described in which hyperparathyroidism represents the dominant or sole manifestation. At least three of these have been shown to be genetically distinct, multiple endocrine neoplasia types 1 and 2 (MEN-l, MEN-2) and cystic parathyroid adenomatosis; in two cases (MEN-1 and MEN-2) the affected gene has been unequivocally identified since the first edition of this volume. In the last of this triplet of syndromes, important progress toward gene identification has been reported. Similarly, the genetic basis for most cases of the hyperparathyroidism mimic syndrome familial hypocalciuric hypercalcemia (FHH; also called familial benign hypercalcemia) has been resolved. Other hereditary endocrine syndromes and variants only sporadically involve the parathyroid gland (1). The primary focus of this chapter is familial hyperparathyroidism. The syndromes of multiple endocrine neoplasia types 1 and 2 are comprehensively discussed in Chapters 35 and 36 and will not be considered here.

CYSTIC PARATHYROID ADENOMATOSIS WITH FIBROOSSEOUS JAW TUMORS In 1987, Mallette et al. (2) described a father and three sons who at early ages developed severe hypercalcemia (total serum calcium values ranging as high as The Parathyroids, Second Edition

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It turns out that several years earlier, a syndrome of familial parathyroid adenomas with fibro6sseous jaw tumors had been described (3,4). At Dr. Mallette's suggestion, the parathyroid histology of that family was reexamined and the cystic nature of the adenomas was confirmed (5). Thus the two presumably unrelated families probably had the same syndrome, of which the fibro6sseous jaw tumors can be regarded as an integral part. A literature search revealed five other families who may have the same trait, although the parathyroid histology was not described adequately to determine whether the adenomas were cystic (6-9). Three of these families included at least one patient with a parathyroid carcinoma, suggesting that definition of the syndrome may require expansion. The cellular basis for the development of these adenomas, for their cystic nature, and for their possible occasional malignant transformation must be established. However, p e n d i n g further evidence, the parathyroid adenomas in this syndrome should be considered surgically to be premalignant, so care must be taken to resect them intact and to avoid using them for grafts when possible. The p r o p e r surgical m a n a g e m e n t of this syndrome is still evolving, but a few tentative principles can be listed. At least in the kindred of Mallette et al. (2), the parathyroid adenomas seemed more often to involve inferior parathyroid glands (10). Furthermore, sporadic parathyroid carcinomas seem to occur almost exclusively in the inferior parathyroid glands (11,12). Thus, it appears sensible to r e c o m m e n d bilateral inferior parathyroidectomy (and transcervical thymectomy) at the first operation. Total parathyroidectomy with autogenous grafting of one or both superior parathyroid glands might also be considered. Preliminary genetic analysis of this syndrome indicated that the responsible gene(s) were not located near the respective loci for MEN-2 or MEN-1 on chromosomes 10 or 11 (13). Subsequent work by Hobbs et al. (5a), based on genetic analysis of two affected families, indicates mapping of this syndrome to chromosome 1 between lq25 and lq31. Further study of this syndrome revealed that some families also have renal involvement, described variously as Wilms tumors, nephroblastomas, and hamartomas. Wassif et al. (14) described a large kindred with apparent isolated familial primary hyperparathyroidism in which the risk for parathyroid carcinoma was increased. Genetic analysis of this family revealed linkage to the same region described for the hyperparathyroidism-jaw tumor syndrome, leading the authors to pursue further clinical screening of the family members. Two members of the family were found to have jaw lesions, whereas one m e m b e r was shown to have polycystic kidney disease.

A large, previously u n r e p o r t e d Dutch kindred with hyperparathyroidism-jaw tumor syndrome has been studied (14a). Additional clinical features of this kindred included cystic kidney disease, pancreatic carcinoma, renal cortical adenomas, papillary renal cell carcinoma, testicular mixed germ cell tumor, and Hfirthle cell thyroid adenoma. Careful genetic analysis showed showed complex mapping to the lq25 region as described above. When combined with previous reports, the authors concluded that they had delineated the gene location within a 14 centimorgan region.

D OMINANT ISOLATED FAMILIAL PARATHYROID ADENOMAS, HYPERPLASIA, OR CARCINOMA

Adenomas Parathyroid adenomas with apparently dominant transmission but without accompanying jaw tumors or other endocrinopathy have been reported in a few families, although usually only in two consecutive generations. In at least two such families, the parathyroid adenomas were cystic (15,16), suggesting a variant of the familial cystic parathyroid adenomatosis syndrome in which there is low penetrance of the jaw fibroma component. This hypothesis may become testable when the genetic locus of the cystic a d e n o m a j a w fibroma syndrome has been found. In at least one family, the adenomas were not cystic (17), so a separate syndrome may exist.

Hyperplasia Several families have been described who seemed to show only primary parathyroid hyperplasia. Unless the family is large, it is difficult to exclude the presence of MEN-l, and at least three of these families have later been found to have MEN-1 (18,19).

Carcinoma Primary hyperparathyroidism occurred in three siblings and their uncle (20). The siblings' mother had died at age 31 years with features suggesting severe hyperparathyroidism. There was no evidence for parathyroid hyperplasia, hypercalcemia being relieved by resection of parathyroid carcinomas in two patients and an atypical a d e n o m a in two. No other endocrine tumors and no evidence of parathyroid hyperplasia were found. Chromosomal abnormalities were identified in one of the carcinomas (reciprocal translocation

FAMILIALPRIMARYHPT between chromosomes 3 and 4, trisomy 7, and a pericentric inversion in chromosome 9). Tumor DNA from one carcinoma and one adenoma showed no loss of heterozygosity of chromosome 11, no evidence of ras gene mutations, and no PTH gene rearrangements.

BENIGN FAMILIAL (HYPOCALCIURIC) HYPERCALCEMIA WITH HYPERPARATHYROIDISM Benign familial (hypocalciuric) hypercalcemia is an autosomal dominant syndrome marked by the lifelong elevation of free and total serum calcium values and relative hypocalciuria (21,22). A comprehensive account of this syndrome is detailed in Chapter 38. PTH concentrations are normal in most patients with FHH (22,23) as are sonographic estimates of parathyroid size (24). In some families, however, elevated serum intact PTH concentrations occur in a high percentage of affected members, the incidence rising with age (25,26). To explain this variant, one might hypothesize that the increased renal retention of calcium has not elevated serum calcium concentrations quite enough to overcome the decreased parathyroid sensitivity to calcium, so that generalized parathyroid hyperplasia occurs. These patients may develop a hypophosphatemic osteomalacia, which can serve as an indication for parathyroid surgery (26). The molecular basis of FHH was resolved with the discovery and cloning of a cell surface calcium-sensing receptor (27). This classic G protein-coupled receptor enables parathyroid cells to translate elevations in extracellular calcium into increases in intracellular calcium and decreases in PTH secretion (27,28). Inactivating mutations of this receptor produce FHH whereas activating mutations are a cause of familial hypoparathyroidism (see Chapter 38).

NEONATAL SEVERE PRIMARY HYPERPARATHYROIDISM Infants homozygous for the FHH gene may be born with severe hypercalcemic hyperparathyroidism, with both life-threatening hypercalcemia and severe bone disease. This syndrome differs from the neonatal hyperparathyroidism seen in infants born to hypocalcemic mothers, whereby the hyperparathyroidism develops in utero as a compensatory response to hypocalcemia. The latter syndrome is manifested postnatally as transient mild or moderate hypercalcemia that spontaneously resolves as the parathyroid hyperplasia involutes.

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Clinical Manifestations Serum calcium values in severe neonatal primary hyperparathyroidism have ranged from ---13 to >20 mg/dl. Serum PTH values measured in several cases were found to be high, although not always as elevated as might be expected. Affected infants also have shown slightly elevated or high-normal serum magnesium concentrations and low urinary calcium excretions. Clinical signs include lethargy, muscular hypotonia, and severe skeletal deformities, including a narrow thorax, spontaneous rib fractures, bowed long bones, craniotabes, and radiographic osteopenia. As often occurs when hyperparathyroidism develops while the epiphyses are open, the radiographic changes of rickets are found. Subperiosteal resorption of bone is observed, and histologic examination of bone may show typical osteitis fibrosa cystica (29). As with rickets, morbidity often derives from respiratory complications of the thoracic deformity. The syndrome is often fatal, especially if parathyroid surgery is delayed in the face of severe muscular weakness or increasing serum calcium values.

Etiology The earliest reports found that the syndrome was associated with parathyroid hyperplasia but not adenoma (30). Marx et al. (29) recognized that many of these patients were members of families affected with FHH. In one of their cases, both parents were affected members of separate FHH kindreds, suggesting that homozygosity for FHH could be a cause of the syndrome. Cooper et al. (31) reported another patient who was apparently homozygous for FHH because of parental consanguinity and demonstrated that the infant's parathyroid glands in vitro showed poor suppresibility by calcium. In other cases, however, only one parent has had FHH, suggesting that another genetic (presumably recessive) or environmental factor must be acting synergistically with the FHH trait to generate the severe degree of hyperparathyroidism. Such factor(s) remain unidentified but must be operative in only a small percentage of FHH births. In some reported cases, including three of the four patients reported by Marx et al. (29), the father was the affected parent, the mother being normocalcemic. Here the usual activity of the placental calcium pump would place the fetal serum calcium concentration below the presumably higher fetal parathyroid set point and produce parathyroid hyperplasia. In this model, however, the hyperplasia and the bone disease that had developed in utero should resolve gradually after birth, leaving the infant with only the usual features of FHH. This situation is analogous to hyperparathyroidism developing in

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the normal fetus of a hypocalcemic mother. Marx et al. (29) found no evidence of excess fetal wastage in FHH pregnancies of normocalcemic mothers, suggesting that this mechanism alone is not the cause of the severe neonatal hyperparathyroid syndrome. How then would one explain the occasional case of severe neonatal hyperparathyroidism in this situation? One might postulate that another factor prevented the placental calcium p u m p from increasing the calcium gradient sufficiently. Studies of placental weight and histology would be of interest but may not be technically feasible, because the syndrome may be recognized hours to days after birth. Another hypothesis would be that some other factor, such as a subclinical form of F H H in the mother, had shifted the fetal parathyroid set point even further from normal. Detailed study of the calcium inhibitory set point of parathyroid glands from severely affected, but apparently heterozygous, neonates and dynamic in vitro studies of maternal parathyroid function would be of great interest. Other causes of severe neonatal hyperparathyroidism appear to be unrelated to FHH. Several reported cases seemed to show recessive transmission or represented the sporadic occurrence of a similar neonatal syndrome (29).

Treatment In neonates with severe hypercalcemia and marked skeletal deformities, aggressive respiratory support and treatment can be lifesaving, because the skeletal deformity may eventually resolve. Parathyroidectomy can be lifesaving in the most severe cases and should not be delayed (32). Repeated parathyroid operations have sometimes been required because of early recurrence of severe hypercalcemia after subtotal parathyroidectomy. Thus, some authors have advocated deliberate total parathyroidectomy (29,31), with the understanding that lifelong treatment of hypoparathyroidism would then be needed. A possible alternative approach would be total parathyroidectomy with parathyroid autogenous grafting. This seemed satisfactory in one case, but only 6 weeks of postoperative follow-up was available, and later graft-dependent recurrence was a concern (31 ). The presence of skeletal hyperparathyroid changes in F H H offspring is itself not an indication for parathyroid exploration, unless accompanied by severe hypercalcemia. At least two patients with FHH-associated neonatal hyperparathyroidism with significant skeletal changes but mild hypercalcemia (11.2 and 11.4 m g / d l ) have been reported to survive and their skeletal deformities to heal without parathyroid surgery (33,34). These patients, however, clearly did not have the full syndrome of severe neonatal primary hyperparathyroidism.

Histology Histologic examination of the parathyroid glands in severe neonatal hyperparathyroidism usually shows chief cell hyperplasia (29), but one patient had water clear cell hyperplasia of four glands (35).

PRIMARY HYPERPARATHYROIDISM IN FAMILIAL SYNDROMES OF RECESSIVE OR UNCERTAIN INHERITANCE Recessive Isolated Familial Parathyroid Adenomas or Hyperplasia Adenomas

Apparently recessive familial parathyroid adenomas were reported in each of three offspring of a nonconsanguineous marriage. There was no other evidence of endocrine tumors and no hypercalcemia or hyperparathyroidism in other members of a large extended family (36). Hyperplasias

Several reports have described recessive syndromes of isolated primary parathyroid hyperplasia with other associations not including endocrine tumors. A familial syndrome of primary parathyroid hyperplasia with nephropathy and neural deafness was reported in five of six children born to a marriage between first cousins (37). Three of the five had primary parathyroid hyperplasia, three had renal failure, and all five had sensorineural deafness. Female patients were equally as severely affected as males, and the absence of hematuria distinguished the syndrome from Alport's syndrome, which is usually an X-linked illness. The hyperparathyroidism was of early onset, being diagnosed at ages 9, 18, and 22 years. A familial syndrome of primary parathyroid hyperplasia with a tendency for intrathyroidal location of one or more parathyroid glands has been reported (38). A mother and two of her six children were affected. The age of onset was early in the two sons, at "~22 years of age, and was uncertain in the mother, who presented with staghorn calculi at age 49 years. In two of the three affected patients, one of the hyperplastic parathyroid glands was located within the substance of the thyroid. A literature review of large series of hyperparathyroidism in which location of the abnormal gland (s) was specified suggested that an intrathyroidal location was more c o m m o n in familial (10.4%) than in sporadic cases (4.2%). The family was not large e n o u g h to allow the mode of inheritance to be determined or to exclude the possibility of an associated endocrinopathy.

FAMILIALPRIMARYHPT Primary parathyroid hyperplasia was associated with polyps and carcinoma of the colon in two brothers (39). The colon malignancy appeared 12 and 36 years before the hypercalcemia. A sister also had u n d e r g o n e resection of a colon carcinoma but refused calcium measurement. Both parents had expired of a malignancy (lung or breast) in the fifth or sixth decade. This association was probably more than a chance occurrence. Data from three large series found that 7% of 244 patients with primary hyperparathyroidism also had a history of colon cancer; matched controls had a 5% incidence of colon cancer, not a significant difference (39). Colonic polyposis is also associated with familial papillary thyroid cancer (40-42), but those families have not been found to have a high incidence of parathyroid hyperplasia. Mallette et al. (43) reported a family a m o n g whom parathyroid hyperplasia occurred in three siblings, without evidence of other endocrine tumors, with one of the siblings also having a parathyroid carcinoma. A pancreatic carcinoma, visualized in the m o t h e r at laparotomy but not biopsied, had followed a rapid clinical course, making it unlikely to have been of islet cell origin. The parathyroid carcinoma presented as a hard nodule fixed to the thyroid, with severe hypercalcemia (14.7 m g / d l ) . In addition to thyroid invasion, it showed fibrosis, a trabecular growth pattern, and venous invasion. Persistent hypercalcemia, which worsened progressively, led to three further operations at which, respectively, were resected (1) a hyperplastic right inferior parathyroid gland, (2) a hyperplastic right superior parathyroid gland displaced to the posterior superior mediastinum, and (3) a large mass of nodules adherent to the trachea at the site of the initial thyroid resection plus multiple nodules studded along the entire left paratracheal area from u p p e r thyroid cornu to u p p e r thymic pole. These nodules microscopically contained numerous mitoses but more importantly had invaded muscle, confirming that this was a local recurrence of a carcinoma rather than parathyromatosis. A similar syndrome of parathyroid hyperplasia and carcinoma has been reported in at least two other families, each with two affected siblings (44,45). The parathyroid carcinoma did not metastasize in any of the five patients, the diagnosis of carcinoma being based on local recurrence of tumor or histologic findings or both. Only one of the five parathyroid carcin o m a patients in these three families died during the reported follow-up period. He succumbed to severe hypercalcemia hyperparathyroidism, the cause of which was presumed to be further recurrence of carcinoma, but neither left-side parathyroid gland was ever identified. The inheritance of this syndrome is uncertain. Two families could not be screened adequately, but in one family both parents and two other siblings

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were normocalcemic (44). Thus a d o m i n a n t trait has not been excluded. The syndrome is rare e n o u g h that it may take many years to track down its genetic basis. It is possible that one or more of these families had a variant of the cystic parathyroid adenomatosisfibro6sseous jaw tumor syndrome, which may also be associated with parathyroid carcinoma (see above). Two of the four patients examined via mandibular radiographs had mandibular tumors (43,44), although biopsy of one lesion showed increased osteoclastic activity that excluded an ossifying fibroma. There was at least one recurrence of hypercalcemia in each patient at a time that would have been compatible either with appearance of a metachronous adenoma or with progression of hyperplasia in a previously minimally affected gland. The published histologic studies of the carcinomas showed mild cystic changes in only one of the five cases (41), but the cysts in the original a d e n o m a might have been lost after a carcinoma developing in situ had spread t h r o u g h o u t the gland. In the future, we should carefully examine all parathyroid tissue for cystic change in such families. Once such a family has been identified, the surgical treatment of this syndrome should probably employ the same approach used for the cystic adenomatosis jaw tumor syndrome. Because any enlarged parathyroid gland might represent or harbor a carcinoma, biopsy of enlarged parathyroids should be avoided and enlarged parathyroid glands handled with extreme caution. Bilateral inferior parathyroidectomy should probably be routine. Autogenous parathyroid grafting of the most normal-appearing superior parathyroid gland might be considered in preference to subtotal parathyroidectomy, even with the increased chance of later malignant change. It is not clear whether metastasis would be more likely to occur from a gland fragment left at the original site in the neck or from a transplant in the forearm, but the forearm site would be more accessible should the parathyroid r e m n a n t develop aggressive behavior.

REFERENCES 1. Mallette LE. Hyperparathyroidism: The spectrum of parathyroid tumors in primary and secondary hyperparathyroidism. In: Mazzaferri EL, Samaan NA, eds. Endocrine tumors. Boston: Blackwell Scientific, in press. 2. Mallette LE, Malini S, Rappaport ME Kirkland JL. Familial cystic parathyroid adenomatosis. A n n Intern Med 1987;107:54-60. 3. Jackson CE. Hereditary hyperparathyroidism associated with recurrent pancreatitis. A n n Intern Med 1958;49:829-836. 4. Jackson CE, Boonstra CE. The relationship of hereditary hyperparathyroidism to endocrine adenomatosis. A m J M e d 1967;43:727-734. 5. Jackson CE, Norum RA, Boyd SB, et al. Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: A clinically and genetically distinct syndrome. Surgery 1990; 108:1006-1013.

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5a. Hobbs MR, Ole AR, Pidwirny GN, Rosen IB, Zarbo RJ, et al. Hyperparathyroidism-jaw tumor syndrome: The HRPT2 locus is within a 0.7 cm region on chromosome lq. Am J Hum Genet 1999;64:519-525. 6. Kennett S, Pollick H. Jaw lesions in familial hyperparathyroidism. Oral Surg 1971;31:502-510. 7. Dinnen JS, Greenwood RH, Jones JH, Walker DA, Williams ED. Parathyroid carcinoma in familial hyperparathyroidism. J Clin Patho11977;30:966-975. 8. Rosen IB, Palmer JA. Fibro6sseous tumors of the facial skeleton in association with primary hyperparathyroidism: An endocrine syndrome or coincidence? A m J S u r g 1981;142:494-498. 9. Warnakulasuriya S, Markwell BD, Williams DM. Familial hyperparathyroidism associated with cementifying fibromas of the jaws in two siblings. Oral Surg 1985;59:269-274. 10. Mallette LE, Malini S, Rappaport MR Kirkland JL. Familial cystic parathyroid adenomatosis. Ann Intern Med 1987; 107:54-60. 11. Flye MW, Brennan ME. Surgical resection of metastatic parathyroid carcinoma. Ann Surg 1981;193:425-435. 12. Cohn K, Silverman M, Corrado J, Sedgewick C. Parathyroid carcinoma: The Lahey Clinic experience. Surgery 1985;98:1095-1100. 13. Hobbs MR, Ole AR, Pidwirny GN, Rosen IB, Zarbo RJ, Coon H, Heath III H, Leppert M, Jackson CE. Hyperparathyroidism-jaw tumor syndrome: The HRPT2 locus is within a 0.7 cM region on chromosome lq. A m J H u m Genet 1999;64:519-525. 14. Wassif WS, Farnebo E The BT, Moniz CE Li FY, Harrison JD, Peters TJ, Larsson C, Harris P. Genetic studies of a family with hereditary hyperparathyroidism-jaw tumour syndrome. Clin Endocrinol 1999; 50:191-196. 14a.Haven CJ, Wong FK, van Dam EWCM, van der Luijt R, van Asperen C, Jansen J, Rosenberg C, de Wit M, Roijers J, Hoppener J, Lips CJ, Larsson C, The BT, Morreau H. A genotypic and histopathological study of a large Dutch kindred with hyperparathyroidism-jaw tumor syndrome. J Clin Endocrinol Metab 2000;85:1449-1454. 15. Grevsten S, Grimelius L, Thordn L. Familial hyperparathyroidism. UppsalaJMed Sci 1974;79:109-115. 16. Sandler LM, Moncrieff MW. Familial hyperparathyroidism. Arch Dis Child 1980;55:146-147. 17. Mlo M, Thompson NW. Familial hyperparathyroidism caused by solitary adenomas. Surgery 1982;92:486-490. 18. Jung RT, Davie M, Grant AM, Jenkins D, Chalmers TM. Multiple endocrine adenomatosis (type 1) and familial hyperparathyroidism. Postgrad MedJ 1978;54:92-94. 19. Marx SJ, Attie ME Levine MA, Spiegel AM, Downs RW, Lasker RD. The hypocalciuric or benign variant of familial hypercalcemia: Clinical and biochemical features in fifteen kindreds. Medicine 1981 ;60:397-412. 20. Streeten EA, Weinstein LS, Norton JA, et al. Studies in a kindred with parathyroid carcinoma. J Clin Endocrinol Metab 1992;75: 362-366. 21. Marx sJ. Familial hypocalciuric hypercalcemia. N Engl J Med 1980;303:810-811. 22. Heath III H. Familial benign (hypocalciuric) hypercalcemia. Endocrinol Metab Clin North Am 1989; 18:723-740. 23. Marx SJ, Spiegel AM, Brown EM, et al. Circulating parathyroid hormone activity: Familial hypocalciuric hypercalcemia versus typical primary hyperparathyroidism. J Clin Endocrinol Metab 1978;47:1190-1197. 24. Law WM,Jr, James EM, CharboneauJW, Purnell DC, Heath III H. High-resolution parathyroid ultrasonography in familial benign hypercalcemia (familial hypocalciuric hypercalcemia). Mayo Clin Proc 1984;59:155-159. 25. Gilbert F, D'Amour P, Gascon-Barns M, et al. Familial hypocalciuric hypercalcemia: Description of a new kindred with emphasis

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on its difference from primary hyperparathyroidism. Clin Invest Med 1985;8:78-84. McMurtry CT, Schranck FW, Walkenhorst DA, et al. Significant developmental elevation in serum parathyroid hormone levels in a large kindred with familial benign (hypocalciuric) hypercalcemia. A m J M e d 1992;93:247-258. Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 1993;366:575-580. Brown EM, Pollak M, Seidman CE, et al. Calcium-ion-sensing cellsurface receptors. N EnglJ Med 1995;333:234-239. Marx SJ, Attie ME Spiegel AM, Levine MA, Lasker RD, Fox M. An association between neonatal severe primary hyperparathyroidism and familial hypocalciuric hypercalcemia in three kindreds. N EnglJ Med 1982;306:257-264. Mihlethaler JR Scharer K. Antener 1. Akuter hyperparathyreoidismus bei primarer nebenschilddriisenhyperplasie. Helv Pediatr Acta 1967;22:529-557. Cooper L, Wertheimer J, Levey R, et al. Severe primary hyperparathyroidism in a neonate with two hypercalcemic parents: Management with parathyroidectomy and heterotopic autotransplantation. Pediatrics 1986;78:263-268. Marx SJ, Spiegel AM, Levine MA, et al. Familial hypocalciuric hypercalcemia: The relation to primary parathyroid hyperplasia. N EnglJ Med 1982;307:416-426. Eftekhari E Yousefzadeh DK. Primary infantile hyperparathyroidism: Clinical, laboratory, and radiographic features in 21 cases. Skel Radiol 1982;8:201-208. Page LA, HaddowJE. Self-limited neonatal hyperparathyroidism in familial hypocalciuric hypercalcemia. J Pediatr 1987;111:261-264. Steinmann B, Gnehm HE, Rao VH, Kind HE Prader A. Neonatal severe primary hyperparathyroidism and alkaptonuria in a boy born to related parents with familial hypocalciuric hypercalcemia. Helv Pediatr Acta 1984;39:171-186. Law WM,Jr, Hodgson SE Heath III H. Autosomal recessive inheritance of familial hyperparathyroidism. N Engl J Med 1983;309: 650-652. Edwards BD, Patton MA, Dilly SA, Eastwood JB. A new syndrome of autosomal recessive nephropathy, deafness, and hyperparathyroidism. J Med Genet 1989;26:289-293. Colon-Zorba GE, Aguilo E Jr, Vazquez-Quintana E. A syndrome of familial intrathyroidal primary parathyroid hyperplasia: Case reports and critical review of literature. PR Health Sci J 1986;555-563. Feig DS, Gottesman IS. Familial hyperparathyroidism in association with colonic carcinoma. Cancer 1987;60:429-432. Delamarre J, Capron JP, Armand A, Dupas JL, Deschepper B, Davion T. Thyroid carcinoma in two sisters with familial polyposis of the colon. Case reports and review of the literature. J Clin Gastroenterol 1988;10:659-662. Herrera L, Carrel A, Rao U, Castillo N, Petrelli N. Familial adenomatous polyposis in association with thyroiditis. Report of two cases. Dis Colon Rectum 1989;32:893-896. Reed MW, Harris SC, Quayle AR, Talbot CH. The association between thyroid neoplasia and intestinal polyps. Ann R Coll Surg England 1990;72:357-359. Mallette LE, Bilezikian JP, Ketcham AS, Aurbach GD. Parathyroid carcinoma in familial hyperparathyroidism. Am J Med 1974;57:642-648. Frayha RA, Nassar VH, Dagher F, Salti IS. Familial parathyroid carcinoma. Geb MedJ 1972;25:299-309. Leborgne J, Neel L, Buzelin E Malvy P. Cancer familial des parathyroides. L'angiographie dans le diagnostic des recidives loco- regionales. Considerations a propos de deux cas. J Chir 1975;109:315-326.

CI-IAPXV 3 8

F a m i l •i a"l and

B e n i g•n

Neonatal

Hyp Severe

" " ocalclurlc

Hyp ercalcemla "

Hyperparathyroidism

GHADA EL-HAJJ FULEIHAN Calcium Metabolism and Osteoporosis Program, American University of Beirut Medical Centeg, Beirut 113-6044, Lebanon HUNTER HEATH III United States Medical Division, Eli Lilly and Company, Indianapolis, Indiana 46285

H I S T O R I C A L PERSPECTIVE AND NOMENCLATURE

glands did not normalize the serum calcium. Seventeen other members with hypercalcemia were discovered in the family, spanning three generations. The hypercalcemia seemed to be inherited in an autosomal dominant pattern. The authors reported the disorder as "Hereditary Hypercalcemia without Definite Hyperparathyroidism" and recognized it as an entity different from hyperparathyroidism: "The lack of firm evidence for hypersecretion of parathyroid h o r m o n e in any of the hypercalcemic members of this family suggests that this condition may be a new entity which certainly differs from that observed in the other 5 families with hyperparathyroidism . . . . " Other terms occasionally used to describe the syndrome include familial parathyroid hyperplasia and familial hypercalcemia. However, the terms familial benign hypercalcemia (FBH) and familial hypocalciuric hypercalcemia (FHH) predominate, being about equally used in the literature. In 1989, Heath coined the encompassing term familial benign hypocalciuric hypercalcemia (FBHH) that was subsequently used in several papers (4-6), and which was later suggested by Strewler as a unifying term containing the key features of the syndrome and as a means to end the confusing division in the literature describing this rare syndrome (7). In this chapter, we use the abbreviation FBHH. FBHH is distinct from other inherited hypercalcemic syndromes usually associated with primary hyperparathyroidism, such as multiple endocrine neoplasia (MEN) types 1 and 2A (8,9), isolated familial primary hyperparathyroidism (10,11) and the hyperparathyroidism-jaw tumor syndrome (12). Indeed,

In 1972, T.E Foley and colleagues described in their paper "Familial Benign Hypercalcemia" (FBH) the key features of the syndrome: asymptomatic hypercalcemia inherited in an autosomal dominant pattern, normal serum PTH levels in the presence of hypercalcemia, low or low normal urinary calcium excretion, normal or slightly elevated serum magnesium level, normal or slightly decreased phosphate level, and normal parathyroid gland pathology in the proband (1). The serum PTH level of the proband only decreased by 29% during an intravenous calcium infusion. The authors interpreted the abnormal pathophysiology of the disease as "an inappropriate requirement of an unusually high concentration of calcium to suppress the production of parathormone." A few years later, investigators at the National Institutes of Health reported the same syndrome as "familial hypocalciuric hypercalcemia" (FHH), in recognition of the unexpectedly low urinary excretion of calcium relative to the hypercalcemia of affected persons (2). However, Jackson and Boonstra had described the syndrome without labeling it as such in 1966, when they reported familial hypercalcemia in a kindred wherein the proband was discovered during a routine serum calcium screening survey (3). The patient had hypercalcemia (12.1 mg/dl, or 3.0 mmol/liter) and a hypernephroma. The serum calcium was not normalized by excision of the renal tumor. Neck exploration revealed four-gland hyperplasia, and removal of three and one-half parathyroid The Parathyroids, Second Edition

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FBHH is characterized by lack of cure after subtotal parathyroidectomy, and the absence of other associated endocrine neoplasias. A disorder closely related to FBHH, neonatal severe hyperparathyroidism (NSHPT), was first described by Landon three decades earlier (13). Philips first noted a case of NSHPT arising from a consanguinous marriage (14), and Hillman commented on the familial occurrence (15). Spiegel et al. first linked this entity to FBHH in 1977; an infant with NSHPT was later found to have a n u m b e r of relatives diagnosed with familial hypocalciuric hypercalcemia (16). Most subsequent publications have described the NSHPT syndrome as occurring in infants belonging to kindreds having FBHH (17-23). NSHPT is characterized by severe hypercalcemia, failure to thrive, respiratory distress, and skeletal anomalies. In some cases, total parathyroidectomy was effective in alleviating the symptoms. Years before the demonstration that mutations in the parathyroid calcium receptor gene can cause FBHH and NSHPT, investigators had proposed that NSHPT is the homozygous form of a genetic abnormality in calcium metabolism, whereas the heterozygous state presents only a mild disorder (17,23,24). However, the genetic basis of NSHPT is more complex than originally thought, as outlined in detail below (see Genetics of Neonatal Hyperparathyroidism, Including Neonatal Severe Hyperparathyroidism).

GENETICS OF FBHH Inspection of many pedigrees convincingly demonstrates that FBHH is inherited as an autosomal dominant condition (1-4,25-30) with equal sex distribution and over 90% penetrance. In two studies of five kindreds evaluating a total of 170 individuals, 42% were affected, 39% were unaffected, and in 19% the status was unclear (31,32). The biochemical abnormalities are present at birth in affected children. Reports of new mutations causing FBHH are extremely rare (33) and very difficult to verify in the absence of a family history and biochemical or genetic testing (see below). Because of the paucity of symptoms associated with this bland syndrome, isolated cases may often go undetected, making it less likely to detect new FBHH mutations. In contrast to FBHH, there have been three well-documented cases of NSHPT associated with de novo mutations (see below). An early report suggested an association of FBHH with h u m a n leukocyte antigen (HLA) haplotypes, but this was not validated in other studies (34-37). Several candidate genes involved in calcium metabolism have been examined with negative findings, including genes for the MEN-l, MEN-2, and parathyroid h o r m o n e (PTH) (31,36,38). An FBHH disease gene was first m a p p e d to chromosome 3 (band

q21-24) by Chou et al. (39) using linkage analysis in four large FBHH families (FBHH~q). Subsequent investigations validated these findings in over 90% of families suitable for genetic linkage analyses (32,40). Two additional genetically distinct forms of FBHH have been described in single families, one linked with markers on the short arm of chromosome 19 (FBHH19p) (32). Another FBHH kindred with atypical features (see clinical characteristics below) from Oklahoma was linked to chromosome 19q (FBHHoK) (41,42).

Molecular Basis of FBHHsq: Mutations in the Calcium Receptor At the same time as the linkage of the FBHH trait to chromosome 3, Brown et al. had also cloned the bovine parathyroid gland calcium-sensing receptor (CaSR) (43) and shortly thereafter Pollak et al. demonstrated that the h u m a n h o m o l o g was close to the site of linkage of FBHH (44). This novel receptor is present in many tissues, including intestine, lung, and various regions of the brain, but is most heavily expressed in the parathyroid glands, thyroid C cells, and kidneys (43). The CaSR shares sequence homology with the metabotropic glutamate receptors that are highly expressed in the central nervous system. The CaSR has three major domains: a large 612-amino acid (aa) extracellular amino-terminal region, a 250-aa domain with seven predicted membrane-spanning segments characteristic of the G protein-coupled receptor superfamily, and a 222aa intracytoplasmic sequence. Because of abnormal calcium sensing by the parathyroids and kidneys in FBHH, the newly cloned CaSR was obviously the next candidate gene to pursue. Pollak et al. used molecular probes for the bovine CaSR to demonstrate three distinct missense mutations in the CaSR gene in three separate FBHH families previously shown to have the FBHH trait linked to chromosomal locus 3q13.3-q21 (44). Different single nucleotide base substitutions were present in each kindred: Arg185Gln, GluZ97Lys, and ArgV95Trp. These sequence variations were not found in genomic DNA of 50 normocalcemic unaffected individuals. Several groups have since then identified dozens of point mutations (missense, nonsense) in the CaSR gene that cosegregate with the FBHH trait (40,45-48). Most of the mutations causing FBHH have been missense mutations occurring in three regions of the receptor sequence: (1) about two-thirds occur within the first 300 aa of the extracellular domain (Fig. 1); (2) proximal to the first transmembrane segment; and (3) within the transmembrane segments and intraor extracellular loops. The first point mutation located in the cytoplasmic tail of the receptor was described in a Swedish family with FBHH having some atypical features (see below, Clinical Characteristics of FBHH).

FBHH AND NSHPT /

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Whereas most mutations described have been missense ones, several additional types of mutations have been described. Pearce et al. described in one kindred a nonsense mutation just proximal to the transmembrane domain, producing a stop codon resulting in a truncated and presumably inactive receptor (40). The same group identified in another kindred a single base deletion and nucleotide transversion leading to premature termination of the receptor protein (40). Janicic et al. described in a Nova Scotian family the insertion of a repetitive Alu sequence (46), also predicting a truncated receptor. However, only about two-thirds of families demonstrated to have FBHH linked to chromosome 3 have demonstrable mutations in the coding region of the CaSR gene. In the remaining onethird, the syndrome is presumably due to mutations in introns, or upstream or downstream regulatory regions modulating gene function. Finally, apparently silent benign polymorphisms (without associated abnormalities in calcium metabolism) have been described in the

carboxy-terminal part of the receptor in up to one-third of 100 unaffected subjects (47). However, Cole et al. reported subtle changes in serum calcium in members with these "benign" polymorphisms (49). Perhaps normal h u m a n variation in serum calcium concentration results partly from variations in intrinsic calcium-sensing activity related to genetic polymorphisms (49). Some inactivating mutations of the CaSR have been expressed in appropriate cell lines, yielding insights into the mechanisms through which receptor mutations reduce CaSR activity (50). Reduced functional activity of the receptor could potentially be due to several factors: 1. Reduced affinity for the agonist calcium ion 2. Prevention of formation of the fully glycosylated, biologically active calcium receptor 3. Failure of receptor coupling with and activation of the respective G proteins 4. "Dominant negative" interactions of mutated with normal CaSR protein

610

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CI-I~TF~R38

Whereas two out of three known missense mutations producing truncated CaSR result in an inactive protein, few missense mutations seem to result in full-length nonglycosylated receptor with marked reduction in biologic function. The majority of missense mutations examined have resulted in apparently normal size glycosylated receptors, which have modest reduction in ligand (calcium) binding affinity and receptor activity. One such mutation, RlS5Q, showed a prominent "dominant negative effect" on the coexpressed wild-type receptor, which may account for the unusually marked hypercalcemia in affected members of the FBHH family harboring that mutation (50). The exact mechanism for the negative dominant effect has not been elucidated, although it may be related to receptor dimerization. Electrophoretic evidence suggests the existence of nonglycosylated and glycosylated monomeric CaSR as well as a CaSR dimer formed through disulfide bonds (50,51). The addition of divalent and trivalent cations to solubilized CaSR shifts its electrophoretic mobility from a mainly monomeric to a dimeric form in a manner similar to their rank order of potency for CaSR activation (51). One mechanism for the dominant negative action may be the formation of heterodimers between the wild-type and mutant receptors, which may reduce the activity of the wild-type partner in the heterodimeric complexes, thereby reducing the total n u m b e r of normally functioning receptors on the cell surface.

In summary, the FBHH syndrome is genetically heterogeneous. In >90% of affected families the FBHH trait links to the region of chromosome 3 containing the CaSR gene. In two-thirds of chromosome 3-1inked families, there are discrete inactivating mutations of the CaSR gene that are almost certainly the cause of the hypercalcemia. The remainder may have CaSR gene mutations outside the coding region. Further understanding of the cation-CaSR structure-function relationship will allow insight into the clinical and biochemical heterogeneity of FBHH syndromes (see below).

Gene Knockout Models of FBHH

Key Characteristics

Ho et al. used targeted disruption of the CaSR gene to create mice that were heterozygous for inactivated CaSR (52). There was a 50% reduction of calcium receptor protein expression in the parathyroid gland and kidney of the heterozygous mice compared with the wild-type animals. These mice shared many phenotypic and biochemical features with h u m a n FBHH: they looked and behaved normally, and had normal fertility and life span. They had mild hypercalcemia (mean serum calcium, 10.4 mg/dl, or 2.6 mmol/liter), nonsuppressed serum PTH levels, higher serum magnesium levels compared to the wild-type mice, and reduced urinary calcium compared to the normal mice. In the mutant mice there was a mild (10%) elevation in the apparent set point for calcium-regulated PTH release, similar to what has been reported in FBHH families (see below). Their skeletal films were normal. Thus, at least three different genetic syndromes result from abnormalities in the CaSR. Inactivating mutations of the CaSR gene result in FBHH and NSHPT (see below), and activating mutations cause autosomal dominant hypocalcemia (53) (see Chapter 49).

FBHH is a rare hypercalcemic syndrome inherited in an autosomal dominant pattern, equally distributed between the sexes, the true prevalence of which is unknown. It is characterized by asymptomatic and usually uncomplicated lifelong hypercalcemia (1,2). The hypercalcemia is mild to moderate, usually <12 m g / d l (<3.0 mmol/liter), in the majority of the large kindreds described (2,27-32), although values of 12-13 m g / d l (3.0-3.25 mmol/liter) may occur. Longevity is normal as demonstrated in large studies from the National Institutes of Health, the Mayo Clinic, and others (25-28,30). Other key features of the syndrome include unexpectedly normal or low urinary calcium excretion, absence of nephrolithiasis, inappropriately normal and sometimes low PTH levels, usually normal parathyroid pathology, and the inability of subtotal parathyroidectomy to normalize serum calcium (1-3, 25-30). It was estimated that among patients referred to the National Institutes of Health in the 1970s after failed parathyroid surgery for suspected primary hyperparathyroidism (PHPT), FBHH was the correct diagnosis in about 9% (55).

Molecular Basis of FBHH19 p and FBHHoK Hypercalcemic disorders not clearly distinguishable from FBHH3q have been linked to two distinct loci on chromosomes 19p (32) and 19q (41,42). Because FBHH19 p and FBHH19 q have each been reported only in single families, the genes responsible for these phenocopies of FBHH3q are unknown. The region mapped for FBHHI9 p contains the guanine nucleotide binding protein subunit gene, GNA11, which encodes a G stimulatory protein expressed in parathyroid tissue (54). It is possible that the FBHH loci on chromosome 19 may encode transcription factors regulating the expression of the calcium receptor.

C L I N I C A L C H A R A C T E R I S T I C S OF FBHH

FBHH AND NSHPT /

Symptoms and Complications FBHH is characterized by a paucity of clinical features and complications. Despite the hypercalcemia, affected subjects have few if any of the classic signs, symptoms, or complications associated with hypercalcemia. One series suggested that fatigue, weakness, m e n t a l problems, polydipsia/polyuria, arthralgia, chondrocalcinosis, and headache were symptoms more likely to be present in 95 hypercalcemic subjects than in the 79 unaffected members of FBHH kindreds (30), but these findings have not been confirmed subsequently (25,27,28). Law and Heath conducted a structured interview study in affected and unaffected members of 15 FBHH families (27). They asked 14 questions regarding symptoms, and 27 medical diagnoses were sought (including pancreatitis, gallstones, ulcer disease, cardiac disease, and hypertension). Whereas nocturia, arthritis, gallstones, and arterial hypertension were commoner in the index cases than in unaffected members, among cases found by screening only, the only disease with increased frequency in FBHH family members was gallstones (27). The authors concluded that detection bias caused the seeming association of FBHH with various symptoms. That is, the initial finding of hypercalcemia may be falsely linked to any concomitant symptoms. The bone turnover in patients with FBHH may be mildly increased (56) as suggested by iliac crest bone biopsy in the limited number of patients studied (28,56). However, there appears to be no increased incidence of fractures in FBHH, and the skeletons of these patients are normal by histomorphometric, radiologic, and densitometry criteria (25-30,57-59). Unfortunately, FBHH does not provide immunity against age-related bone loss: elderly patients with FBHH may have osteoporosis indistinguishable from that expected for their age.

Phenotype-Genotype Associations There are several reports of patients with FBHH presenting with pancreatitis, which in some cases was fatal (29,60-66). However, it is unclear whether pancreatitis represents a true complication of the syndrome, or a spurious association secondary to ascertainment bias (patients presenting with pancreatitis are commonly examined for hypercalcemia). Stuckey et al. reviewed 10 published cases, and in 8 of 10 there were potential confounders such as alcohol use (5 cases) and biliary pathology (3 cases) (65). The issue is clouded by the report of Toss et al. that a male nonabuser of alcohol had seven episodes of acute pancreatitis before subtotal parathyroidectomy and none after (29). An increased risk for pancreatitis in FBHH may be biologically plau-

611

sible on grounds other than an adverse effect of hypercalcemia. Bruce et al. demonstrated expression of the CaSR in the pancreatic ducts (67), wherein the receptor might play a role in composition of the ductal fluid. An abnormally functioning CaSR might favor intraductal calcification and precipitate pancreatitis. Some FBHH kindreds do seem to have an increased incidence of pancreatitis, and in their study of three such kindreds, Pearce et al. identified three novel heterozygous missense mutations in the extracellular domain of the CaSR that were not present in the 55 unrelated normocalcemic controls (68). At this point, it is impossible to make a definitive statement, but pancreatic risk may represent one of several potential genotype-phenotype associations for CaSR mutations. FBHH is generally characterized by histologically normal parathyroid glands and relative hypocalciuria, with rare if any renal complications, and differing from the classic profile of primary hyperparathyroidism (see below). However, parathyroid gland enlargement (hyperplasia and adenoma) and nephrolithiasis have been described in some cases of FBHH (28-30). Carling et al. reported a large Swedish kindred with a novel mutation in the cytoplasmic tail of the CaSR, with clinical and biochemical features between those of FBHH and primary hyperparathyroidism. Of 20 affected members, 2 had nephrolithiasis, and the mean 24-hour urinary calcium and the C a / C r clearance ratio were elevated compared to nonaffected family members (69). Nine individuals underwent radical subtotal parathyroidectomy: seven had chief cell hyperplasia, one had an adenoma, and in one the findings were equivocal. In all but two of those who had hypercalcemia and hypercalciuria, the latter was reversed postoperatively (69). This family's clinical and biochemical presentation is a hybrid between typical hyperparathyroidism and FBHH. Perhaps it represents differential signal coupling in parathyroid and kidney, depending on the site of the CaSR mutation. In this instance, the mutation in the cytoplasmic tail appears to have little effect on renal tubular reabsorption of calcium: in the reported family, urinary calcium excretion is elevated due to the increased filtered load of calcium. The same CaSR mutation may lead to parathyroid cell proliferation and hyperplasia. Consistent with this hypothesis is the example from another FBHH kindred exhibiting an inserted Alu repetitive sequence with truncation of the CaSR. Of 36 carriers undergoing parathyroid surgery, 3 had parathyroid gland enlargement (46,70). Another interesting association include the peculiar clinical presentation with osteomalacia and more than expected elevations in PTH levels in the Oklahoma kindred, the exact pathophysiology etiology of which is still unclear (41).

612

/

Cr~eTwR38 serum magnesium in FBHH was verified in subsequent studies, with clustering of serum magnesium levels in the u p p e r normal or mildly elevated range (27,28,30,72), whereas serum magnesium tends to be low in primary hyperparathyroidism. T h o u g h there was a significant negative correlation between serum calcium and magnesium levels in hyperparathyroidism (R = - 0 . 3 2 ) , the relation was significantly positive between these cations in FBHH (R = 0.5-0.8) (27,72), an early hint for a c o m m o n cation-handling mechanism, now established to be the calcium receptor (73).

LABORATORY AND DYNAMIC STUDIES IN FBHH Serum

Calcium

Hypercalcemia is the cardinal feature of FBHH. It is generally mild to moderate, ranging from 10.0 to 14.6 m g / d l (25,27-30,71). The distribution of serum calcium levels in affected family members from two major studies is illustrated in Fig. 2 (mean _+ SD, 11.4 ___ 1.4 m g / d l , or 2.85 ___0.35 mmol/liter). The low serum calcium values in five family members in one study were explained by concomitant low serum protein (Fig. 2) (71). It has been suggested that the hypercalcemia in FBHH19p is the mildest, but this experience arose only from one small kindred (32). In contrast, a specific missense mutation, R185Q, showed a p r o m i n e n t "dominant negative effect" on the coexpressed wild-type receptor, which may account for the unusually great hypercalcemia in affected members of an FBHH family harboring that mutation (50). The hypercalcemia in FBHH is similar to that of hyperparathyroidism, in that both the total and ionized calcium are elevated, and the hypercalcemia is constant with age (27,28,30,72).

Serum Phosphorus

Affected individuals have serum inorganic phosphorus levels intermediate between those of normal persons and those of patients with primary hyperparathyroidism (1,2,27,28,30,72). Specifically, in one large series the serum phosphorus in affected family members was 3.1 _+ 0.1 m g / d l (mean ___ SE), and 3.6 + 0.1 m g / d l in unaffected relatives (27). Occasionally, serum phosphorus is frankly low in FBHH. Indexes

Older immunoreactive PTH assays detected intact PTH and N- and C-terminal fragments of the molecule. With these methodologic limitations, serum PTH levels were normal in 92 out of 106 subjects (87%) with FBHH from 16 separate studies prior to the mid-1980s (74). The mean PTH levels in patients with FBHH are

Serum Magnesium

The first reported patients with FBHH were noted to have elevated serum magnesium; both the proband and his m o t h e r had serum magnesium levels at the u p p e r limit of normal, 2.5 m g / d l (1). This elevation of

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FBHH undistinguishable from those of normal subjects and clearly lower than values registered by patients with primary hyperparathyroidism, whether using older assays or those measuring the intact PTH moiety (74-76). Intact PTH levels were 23.4 _+ 1.7 p g / m l (2.43 + 0.2 pmol/liter, mean +_ SE) in 20 normal subjects, 28.3 +_ 3.6 p g / m l (2.98 _+ 0.4 pmol/liter) in 26 subjects with FBHH, and 80 _+ 1.5 p g / m l (8.4 _+ 1.6 pmol/liter) in 12 patients with hyperparathyroidism (77). Even when "normal," however, serum PTH levels in FBHH are inappropriately so in view of the elevated serum calcium values, suggesting abnormal calcium sensing by the parathyroid glands, because PTH is not suppressed. Some patients (up to 15%) with FBHH have serum intact PTH values above the upper limit of normal (Fig. 3) (76,77). Similarly, about 15% of patients with primary hyperparathyroidism may have normal PTH values (Fig. 3) (76,77). Therefore, an individual presenting with mild hypercalcemia and serum intact PTH that is normal or mildly elevated may have either primary hyperparathyroidism or FBHH (although the odds would favor the former). Additional clinical, biochemical, and family characteristics may help differentiate the two entities. An exception to the rule of (inappropriately) normal PTH levels in FBHH is the Oklahoma kindred (FBHHoK), in which substantial elevations of intact PTH levels were noted [65 + 17 p g / m l (mean _+ SEM) in affected members (N = 15), and 28 +_ 4 p g / m l in nonaffected members (N = 26)] (6). The PTH levels were

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613

highest in older individuals, although there seemed to be no trend for a concomitant increase in serum calcium levels with age. The pathophysiology of these developmental increments in PTH levels is unclear.

Renal Function I n d e x e s

Urinary Calcium Early reports noted relative hypocalciuria, i.e., lack of hypercalcemia, in people affected with FBHH, the failure of parathyroid surgery to normalize serum calcium, and the persistence of relatively low urinary calcium levels after parathyroidectomy, all pointing to a central role for the kidney in the path•physiology of FBHH (1,29,30,72,78-84). About 75% of persons having FBHH excrete less than 100 mg of calcium daily (27); however, some individuals excrete as much as 250 m g / 2 4 hours (27,30). Although the absolute 24-hour urinary calcium excretion may not be decreased compared to normal (27,28,82), the lack of hypercalciuria in response to hypercalcemia and increased filtered load of calcium suggests avid tubular reabsorpfion of calcium. This renal abnormality may be made more apparent by calculating the calcium/creatinine clearance ratio (27-30,79,83) or by plotting urinary calcium excretion as a function of serum calcium (81-84). The calcium/creatinine excretion ratio, or fractional excretion of calcium, is calculated as follows: [UCa × SCr]/[SCa × UCr], where UCa is the urinary calcium concentration, SCr is the serum creatinine, SCa is the serum calcium concentration, and UCr is the urinary creatinine concentration, all in mg/dl. A cutoff of <0.01 has been suggested to differentiate between patients with FBHH and those with hyperparathyroidism (30). Two studies showed that 70-80% of FBHH subjects had a C a / C r clearance ratio of less than 0.01, and that the majority of patients with primary hyperparathyroidism exceeded that value (29,30). However, others have demonstrated that in many FBHH patients C a / C r clearance values can exceed 0.01, and that there is substantial overlap between values in FBHH and PHPT (79) (Fig. 4). This ratio is most helpful as supportive evidence for the diagnosis of FBHH in a family, but is not consistent enough to be diagnostic in individuals. Calcium infusion has also been used to distinguish renal calcium handling in FBHH from that in primary hyperparathyroidism (Fig. 5) (81-84). In primary hyperparathyroidism, a rising filtered load of calcium increases urinary calcium excretion more than occurs in FBHH. This enhanced renal tubular reabsorption of calcium excretion has persisted in the few FBHH subjects studied after total parathyroidectomy (81,82,84). Thus, hypocalciuria after parathyroidectomy in FBHH

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is not PTH dependent, consistent with an intrinsic abnormality of renal tubular calcium reabsorption. The avid tubular reabsorption of calcium is corrected with loop diuretics such as ethacrinic acid (80,84), pointing to the thick ascending limb as the site of the abnormality in calcium handling. Studies have confirmed expression of the CaSR in all segments of the nephron, with greatest expression of the transcripts in the cortical thick ascending limb (85). It is possible that the spectrum of abnormal renal calcium handling in FBHH may mirror the large variety of mutations; those in the cytoplasmic domain of the CaSR may be least likely to result in hypocalciuria (69).

FIG. 5 Calcium clearance as a function of plasma calcium in (A) patients with familial benign hypocalciuric hypercalcemia and patients with primary hyperparathyroidism under base line conditions (adapted from Stuckey et a/; Fasting calcium excretion and parathyroid hormone together distinguish familial hypocalciuric hypercalcemia from primary hyperparathyroidism. C/in Endocrino/1987;27:525, with permission from Blackwell Science Ltd.) and (B) during an infusion of calcium chloride in two patients with FBHH and three patients with hypoparathyroidism (adapted from Attie et a/. JCI 1983;72:667-676) in comparison to the calcium clearance curve derived from eight healthy men studied during a calcium-PTH infusion (adapted from EI-Hajj Fuleihan et a/, JCEM 1998;83:2366).

Urinary Magnesium FBHH is characterized by relative hypomagnesuria, with increased tubular reabsorption of magnesium (72,82). The CaSR affects both calcium and magnesium handling, with an inactivated receptor enhancing tubular reabsorption of both calcium and magnesium. Close correlation between urinary calcium and magnesium excretion has been noted in several studies of FBHH (72).

Renal Function and Concentrating Ability Renal function (creatinine clearance) decreases as expected with aging in FBHH, and is comparable to

normal (28,30). Marx et al. compared urinary concentrating ability after an 18- to 24-hour dehydration test in 10 patients with FBHH and 40 patients with PHPT having comparable serum calcium levels and renal function. In contrast to patients with PHPT, subjects with FBHH had no obvious impairment in urinary concentrating ability (86). Other Calciotropic Hormones The vitamin D metabolites 25-hydroxyvitamin D [25 (OH) D ] and 1,25-dihydroxyvitamin D [ 1,25 (OH) 2D ] have been measured in several studies and found to be

FBHH AND NSHPT / TABLE 1

Characteristics of Patients with Familial Benign Hypocalciuric Hypercalcemia and Primary Hyperparathyroidism

Variable Age of onset Symptoms Serum levels Calcium Magnesium Phosphorus Intact PTH 1,25(OH)2D Calcitonin Urinary excretion Cyclic AMP Calcium Ca/Cr clearance Magnesium

615

FBHH

HPT

At birth Usually none

Usually >40 years Asymptomatic in 80%; cortical bone loss; urolithiasis <20%

Elevated Normal to elevated Normal to slightly low Normal, 80-85%; elevated, 15-20% Normal Normal

Elevated Variable Normal to very low Elevated, >80%

Normal to increased Normal to low Usually <0.01 Normal to low

Elevated Normal to elevated Usually >0.02 Normal to elevated

normal in patients with FBHH (27,87-91). This is in sharp contrast to patients with PHPT, wherein serum 1,25(OH)zD is elevated in 40-60% of patients (92). In parallel to elevated calcitriol levels in primary hyperparathyroidism is enhanced intestinal calcium absorption, whereas calcium absorption is normal in FBHH (27,28,89). Thyroid C cells normally express the CaSR and increase calcitonin secretion in response to elevations in serum calcium. The inappropriately normal serum calcitonin concentrations in patients with FBHH might therefore reflect abnormal calcium sensing (27,28,77) and a shift to the right in the calcium-calcitonin curve. However, basal plasma calcitonin levels are also normal in chronic primary hyperparathyroidism, and calcitonin responses to secretagogues are normal in FBHH (77). Table 1 summarizes the major characteristics of FBHH and PHPT.

Dynamic Studies of PTH Regulation Steady-state serum calcium and PTH levels in patients with FBHH are consistent with abnormal calcium sensing by the parathyroid glands, because the "normal" PTH levels are inappropriate in view of the hypercalcemia. Total parathyroidectomy yields hypocalcemia in FBHH, further evidence for the central role of the parathyroid glands in the hypercalcemia of FBHH. In their original description of the syndrome, Foley et al. suggested that "the primary defect is an abnormally high parathyroid gland reference input value for extracellular fluid calcium ion concentration (1). The responsiveness of the parathyroid glands to changes in

Normal to elevated Normal

extracellular ionized calcium is preserved in FBHH, although somewhat altered. In the limited number of subjects studied, EDTA-induced hypocalcemia resulted in PTH increments that were similar in FBHH patients and healthy controls (93,94), and calcium infusions suppressed PTH variably (1,27,77,94,95). The regression curve relating PTH to calcium in these infusion studies was shifted to the right in FBHH compared to control subjects, suggesting an altered set point (94,95). However, the definition of a set point depends on the determination of C a / P T H curve derived over a spectrum of calcium values, for example, derived from consecutive EDTA and calcium infusions in the same patient, which was not done in previous studies. Our group is systematically evaluating PTH dynamics in FBHH patients with such a protocol, and there is the predicted shift to the right in the C a / P T H (Fig. 6), reflecting partial resistance of the parathyroid gland to extracellular calcium.

Parathyroid Glands in FBHH There is some controversy as to the histologic findings in the parathyroid glands of FBHH patients. Most case and single-family reports have described grossly and histologically unremarkable parathyroid glands (1,3,60,79), findings that were corroborated in larger studies (27-29). With high-resolution parathyroid ultrasonography, there is uniform absence of parathyroid gland enlargement in patients with FBHH, in contrast to patients with PHPT, whose gland enlargement is generally detectable by this technique (96). However, systematic histologic examination of FBHH parathyroid

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CHAPTER38 • Normal(N=24) • FBHH pt KV

100-

[ -r

I .

50-

0 3.5

410 415 510 515 610 615 710 715 Ca i (mgldl) • Normal (N=24) • FBHH pt KV

150 E 100-

I-Q.

50.-,..... 0

3.s 4'.0 4'.s s'.o s'.s 6'.0 6'.s 710 Ca i (mgldl)

FIG. 6 Inverse sigmoid curve in response to consecutive citrate and calcium infusions in patient KV with FBHH (Haden ST, Brown EM, EI-Hajj Fuleihan G, unpublished observation, 1997) in comparison to the Ca/PTH curve derived from 48 healthy subjects (adapted from Haden ST, et aL, Clin Endocrinol 2000;52:329-338). There is a clear abnormality of PTH dynamics in the patient with FBHH compared to normal controls: a shift in the Ca/PTH curve to the right, increased PTH levels in response to hypocalcemia, and decreased suppression in response to hypercalcemia. The set point, the calcium concentration at which there was 50% suppression in PTH levels, was 4.89 mg/dl in the curve derived from 24 young healthy controls and 5.65 mg/dl in the patient with FBHH.

glands has revealed some deviation from normal (97,98). Twenty-eight glands from 23 patients were compared with 82 glands from 47 control patients; whereas the majority of FBHH glands were within extreme normal limits for weight, 15-20% exceeded normal size (97). However, the parenchymal area in FBHH glands was slightly but significantly less than normal, 30 _+ 3% versus 21 + 2% percent, respectively, and the percent fat was higher in FBHH glands (97). Those authors concluded that parathyroid hyperplasia is not a feature of FBHH. In contrast, another study reported that variable degrees of parathyroid hyperplasia were c o m m o n in FBHH (98). In 55 parathyroid glands from 18 patients with FBHH, the average parathyroid parenchymal area was three times that in normal subjects and 13 of

18 patients had one enlarged gland or more (98). The reason for the discrepancies in the histologic findings of the two large studies above is unclear (97,98), but may relate to the different populations studied, with different inactivating mutations in the CaSR having divergent effects parathyroid cell cycle and proliferation. Specifically, alterations in the cytoplasmic tail of the CaSR may promote parathyroid cell proliferation (see earlier, Phenotype-GenotypeAssociations).

DIAGNOSIS AND MANAGEMENT OF FBHH Diagnosis The genetic and molecular bases of FBHH are now clear in most cases. However, the diagnosis of FBHH must still be based on clinical judgment. Genetic linkage studies are impractical because of cost and the necessity for family sampling. It is also impractical to consider specific molecular diagnosis, for several reasons. The n u m b e r of known and suspected CaSR mutations is large, and technology to screen for them is not available; at least a third of FBHH3q kindreds do not have detectable mutations in the coding region of the CaSR gene; and a given family might have one of the rare chromosome 19 variants of FBHH. The diagnosis of FBHH can be straightforward in an asymptomatic hypercalcemic patient with a family history of hypercalcemia, a personal or family history of failed neck exploration, normal serum PTH, and low urinary calcium excretion (<100 m g / 2 4 hours, C a / C r clearance ratio <0.01). In that instance, education of the patient and family about this benign condition is very important. This would avoid unnecessary and expensive monitoring and the morbidity from unnecessary parathyroid exploration in the patient and h i s / h e r relatives. The differentiation between FBHH and PHPT is more difficult in the absence of a family history of hypercalcemia, and if the C a / C r clearance ratio is <0.01 (29) (Fig. 5). Indeed, patients with PHPT are now usually asymptomatic, have normal PTH levels in up to 20% of cases, and may have low urinary calcium excretion (2) (Fig. 5). In that instance a low calcium diet and vitamin D deficiency should be considered, and thiazide a n d / o r lithium intake should be excluded. Correction of any of these abnormalities will lead to hypercalciuria if the patient has hyperparathyroidism. To complicate matters further, patients with FBHH may have urinary calcium levels above 100 m g / 2 4 hours (30) or C a / C r clearance ratios >0.01 (29,69) (Fig. 5). In most cases, the age at diagnosis of hypercalcemia and family history are crucial. Detection of asympto-

FBHHAyD NSHPT / matic hypercalcemia before the age of 40 years or so should lead the physician to consider the diagnosis of FBHH. Obtaining serum calcium values from firstdegree relatives in the absence of a family history is also of great value. Another helpful clue is that patients with FBHH seldom have C a / C r ratios >0.02, whereas this is quite c o m m o n in PHPT (29) (Fig. 5). Finally, in case of inability to differentiate the two conditions, one should simply observe the patient over time. Patients with FBHH do not develop complications from their disorder, and the c a l c i u m / P T H levels are usually stable over years. An exception to the above rule is one reported large kindred with FBHH wherein serum PTH levels were found to increase with aging (6). Similarly the majority of as),anptomatic patients with PHPT can be safely observed for many years without untoward effects.

Management Because FBHH is usually a benign disorder with normal longevity, it is r e c o m m e n d e d not to intervene except for reassurance and education of the patient and family. An exception to that rule is the case of an adult FBHH patient with recurrent pancreatitis lacking a secondary cause. In that instance total parathyroidectomy may be indicated in an effort to reduce the risk of further attacks of pancreatitis. However, the physician must recognize the limited, almost anecdotal nature of evidence to support this approach. Family screening and education are m a n d a t o r y to avoid unnecessary surgery in hypercalcemic members. Pregnancy in an FBHH carrier or in the spouse of an FBHH carrier requires special consideration. The unaffected offspring of a m o t h e r with FBHH may experience transient hypocalcemia (due to fetal parathyroid suppression from maternal hyperalcemia) that may last up to 2 m o n t h s (99). The affected offspring of an affected m o t h e r will be hypercalcemic, possibly severely (see below, Neonatal Severe Hyperparathyroidism). The affected offspring of an unaffected m o t h e r with a carrier husband may have severe neonatal hypercalcemia due to intrauterine secondary hyperparathyroidism, but the severe hypercalcemia may be self-limited (see below).

GENETICS OF NEONATAL HYPERPARATHYROIDISM, INCLUDING NEONATAL SEVERE HYPERPARATHYRODISM Neonatal hyperparathyroidism can be mild and represent the neonatal expression of FBHH, wherein the child inherits a single dose of the abnormal gene (because the disease is inherited as autosomal domi-

617

nant with nearly 100% penetrance) or can be more severe (NSHPT) and result from inheritance of a double dose of an abnormal gene.

NSHPT as the Homozygous Form of FBHH Many reported neonates with NSHPT were encountered in families with consanguinous marriage of two hypercalcemic individuals, seemingly reflecting the phenotypic expression of a double dose of FBHH genes (17,18,23,30,100). Indeed, in 15 families with FBHH, three children in two kindreds had NSHPT, suggesting that in some cases NSHPT represents the homozygous form of FBHH (17,23,30). Further evidence for that possibility comes from a study by Pollak et al., who d e m o n s t r a t e d in a study of 11 families with an abnormal copy of the CaSR gene m a p p i n g to c h r o m o s o m e 3q that consanguineous marriage of affected m e m b e r s in 4 of these families p r o d u c e d children with NSHPT (100). Subsequent studies of families in which FBHH arose from mutations in the CaSR gene confirmed that the inheritance of two copies of an abnormal CaSR gene resulted in NSHPT (46,48,70).

NSHPT as the Compound Heterozygous Form of FBHH A case of NSHPT resulted from a marriage of two individuals with FBHH caused by two distinct mutations in the CaSR (101). Because offspring with either homozygous FBHH or c o m p o u n d heterozygous FBHH have no normal CaSR genes, they usually exhibit severe hyperalcemia (please see clinical manifestations of NSHPT below) and may require parathyroidectomy to prevent or reverse skeletal anomalies and symptomatic hypercalcemia (see below, Management of Neonatal Hyperparathyroidism) .

Heterozygous Form of N H P T Many reported cases of N H P T or NSHPT occurred either sporadically or in FBHH families with only one affected p a r e n t (16,22,102-105). Several explanations can be offered for this observation: 1. The offspring may be c o m p o u n d heterozygotes of two distinctly abnormal CaSR alleles as outlined above, wherein one of the alleles may produce mild intermittent hypercalcemia that was not evident in one of the parents. 2. Alternatively, the offspring may have inherited a m u t a t e d CaSR from one of the parents as well as another gene that has been associated with FBHH (gene linked to either 19p or 19q). There is no d o c u m e n t a t i o n of such cases to our knowledge.

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3. The offspring may have been from a normal mother and a father with FBHH. It has been suggested that the fetus in that instance would respond to the relatively low maternal serum placental calcium by secondary hyperparathyroidism superimposed on abnormal calcium sensing due to the inherited FBHH gene from the father (17,22,106). 4. The offspring may experience a negative dominant effect of an abnormal CaSR gene on the remaining normal allele (106). Much of the CaSR on the cell surface exists as a dimer (50). Some FBHH mutations, such as R185Q and Rwos~v,produce abnormal receptors that, when coexpressed with the wild-type CaSR, reduce its apparent affinity for calcium, possibly due to the formation of abnormally functioning heterodimers (50). Thus, the severity of hypercalcemia is increased due to interference with function of the normal CaSR (44,50,106). 5. Finally, the offspring may have de n o v o heterozygous mutations in the CaSR with unaffected parents (40,106).

Mouse "Knockout" Models of NHPTH Ho et al. used targeted disruption of the CaSR gene to produce mice homozygous for mutations of the CaSR gene, providing an animal model for NSHPT. There was complete absence of CaSR protein in the parathyroids and kidneys of the homozygous mice. The phenotypic and biochemical profile of homozygous mice was comparable to that of NSHPT (51). The mice grew poorly due to poor feeding. They had severe hypercalcemia, with serum calcium averaging 14.8 m g / d l (3.7 mmol/liter), serum PTH levels 10 times higher than in normal litter mates, and slightly elevated serum magnesium levels. Despite severe hypercalcemia, the urinary excretion of calcium was lower than in the normal mice. Skeletal X-rays showed multiple abnormalities, including demineralization and bowing of the long bones. Most of the mice died within 2 to 4 weeks (51).

CLINICAL CHARACTERISTICS OF NEONATAL HYPERPARATHYROIDISM

Neonatal Severe Hyperpathyroidism Due to the severity of its clinical manifestations, the NSHPT syndrome was described several decades before FBHH was recognized (13-16,107-112). NSHPT is often diagnosed within a week of birth and may carry a very high mortality rate unless recognized and treated promptly. Salient features include anorexia, constipa-

tion, failure to thrive, muscular hypotonia, respiratory distress, and skeletal anomalies. Physical abnormalities may include deformed chest, dysmorphic facial features, and anovaginal and rectovaginal fistulas (113-115). Skeletal X-rays typically show severe demineralization, widening of the metaphyses, fractures, osteitis fibrosa cystica, and occasionally changes suggestive of rickets (17,113). The hypercalcemia is usually severe, ranging from 14 to 20 m g / d l (3.5 to 5.0 mmol/liter). Serum calcium as high as 30.8 m g / d l (7.7 mmol/liter) has been reported (116). Serum magnesium is elevated, and despite the severity of the hypercalcemia relative hypocalciuria may be observed. Serum PTH levels are elevated 5- to 10-fold (24) and the parathyroid glands are grossly enlarged, due to chief cell or water clear cell hyperplasia (17,18,20,24,117). We are unaware of any reports of parathyroid adenoma causing NSHPT. The severity of the hypercalcemia, bone disease, and high PTH levels is most likely explained by the lack of any normal calcium receptor in the homozygous and compound heterozygous forms of NSHPT, and by the abnormally functioning CaSR in the case of a dominant negative effect. The dramatic increase in parathyroid gland mass suggests a negative trophic effect of the normal CaSR on parathyroid cell growth and proliferation. The rest of the phenotypic features of the syndrome, including dismorphic facies and anorectal and anovaginal fistulas, suggest an as-yet unrecognized role of the CaSR in morphogenesis.

Neonatal Hyperparathyroidism Cases with a milder course than NSHPT that is selflimited may be termed neonatal hyperparathyroidism (NHPT). The neonates may present with respiratory distress, metaphyseal widening, and bone demineralization; osteitis fibrosa cystica has not been reported (22,102-104). Follow-up at 4-17 months may document normal growth and development, and resolution of the metaphyseal widening and demineralization (22,102-104). The serum calcium elevations are usually more modest than in NSHPT (<14 m g / d l or <3.5 mmol/liter), and hypercalcemia is detected in only one parent. This milder syndrome probably represents the phenotypic expression of FBHH in neonates, although this has not been proved by genetic testing. It is obvious that most persons with FBHH had unremarkable gestation and postnatal life (27). It is not clear why some infants heterozygous for a CaSR mutation have clinical manifestations of hypercalcemia, which in few may be self-limited, but the most likely explanation is heterogeneity in the dysfunction of CaSR protein.

FBHH AND NSHPT /

M A N A G E M E N T OF N E O N A T A L HYPERPARATHYROIDISM Cases of NSHPT reported before about 1980 that were managed medically had a very poor prognosis and patients usually expired within 2-4 weeks of birth (15-18), whereas patients who underwent parathyroidectomy had a much better prognosis (17,20,24). When subtotal parathyroidectomy was performed there was a high rate of persistent or recurrent hypercalcemia necessitating reoperation (15,17); total parathyroidectomy resulted in hypocalcemia and the need for calcium and vitamin D supplementation (16,18). Total parathyroidectomy with autotransplantation of parathyroid tissue was first proposed by Thompson et al. (I 18) and adopted by others, resulting in long-term normocalcemia in the majority of subjects, albeit with variable periods of transient hypocalcemia (20,24,117). The autotransplantation can be either immediate (20,24) or delayed, using cryopreserved parathyroid tissue (117). Postoperatively, there is usually a dramatic improvement in the child's sympt0matology, as well as rapid healing of the skeletal lesions, even with persistent mild hypercalcemia (17,20,117). Since the 1980s, there has been a broadening in the range of severity of clinically diagnosed neonatal hyperparathyroidism, probably due to increased availability of calcium measurement and increased recognition of the syndrome. The course of NHPT and NSHPT can be self-limited and healing of the bone lesions may occur despite the lack of surgical treatment (22,102,104). Due to improvements of medical m a n a g e m e n t over the past 15 years, conservative m a n a g e m e n t is indicated first in all cases of NHPT. Aggressive hydration, possibly the use of skeletal antiresorptive therapy, and mechanical ventilation should be pursued in symptomatic cases. Prompt surgical intervention should be performed in patients who deteriorate despite aggressive medical therapy. The procedure may include total parathyroidectomy and immediate or delayed parathyroid autotransplantation as described above. The CaSR represents a potentially important therapeutic target for disorders in which the receptor is underactive, such as NHPT or NSHPT. Initial attempts have been made to use CaSR-based therapeutics in the treatment of primary hyperparathyroidism (119), but not to our knowledge in NHPT. In primary hyperparathyroidism, the use of CaSR agonist was complicated by severe hypercalciuria (119), but this should be less of an issue in the case of NHPT or NSHPT. This strategy would therefore constitute an attractive alternative therapy in NSHPT that may afford medical therapy as the sole and successful treatment even in severe cases. In that case evidence for residual CaSR activity in

619

the patient must be present for the calcimimetic agents to have any therapeutic effect. This, however, is yet to be proved. Over the past six decades, we have gone from clinical descriptions of NSHPT and FBHH, originally as separate then as related clinical syndromes, to biochemical and physiologic studies in individual patients suggesting abnormal calcium-sensing in the parathyroid glands and kidneys, and finally to a molecular explanation for the FBHH syndromes. The rapidly evolving technology of genetic linkage analysis, and the intuitive pursuit of a candidate gene in an "experiment in nature" for abnormal calcium-sensing, have established that inactivating mutations in the CaSR are responsible for both FBHH and NSHPT. FBHH is caused by heterozygous inactivating mutations in the majority of cases manifesting as mild to moderate resistance of the parathyroid gland and kidney to calcium, mild hypercalcemia, and relative hypocalciuria. NSHPT results from more severe cellular and tissue resistance to calcium due to the presence of either heterozygous, compound heterozygous, or homozygous mutations in the calcium receptor. Further structure-function experiments in animal models incorporating various forms of mutated calcium receptors will shed further light on the pathophysiologic mechanism for the wide clinical spectrum in FBHH and NHPT, and for some of the anomalies described in NSHPT that are not readily explained by abnormalities in the CaSR.

ACKNOWLEDGMENTS The authors wish to recognize Dr. Edward M. Brown for his pioneer work on the calcium receptor that allowed the elucidation of the molecular basis of FBHH and NSHPT in many subjects. The authors thank Dr. Brown for his critical reading of the manuscript, for stimulating discussions, and for insightful comments and suggestions.

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5. Heath III H. The familial benign hypocalciuric hypercalcemia syndromes. In: BilezikianJ, Raisz L, Rodan G, eds. Principles of bone biology, 2nd Ed. San Diego:Academic Press, 1996, pp. 769-782. 6. McMurtry CT, Schranck FW, Walkenhorst DA, Murphy WA, Kocher DB, Teitelbaum SL. Significant developmental elevation in serum parathyroid hormone levels in a large kindred with familial benign (hypocalciuric) hypercalcemia. Am J Med 1992;93:247-258. 7. Strewler GJ. Familial benign hypocalciuric hypercalcemiamfrom the clinic to the calcium sensor. WestJMed 1994;160:579-580. 8. Gagel R. Multiple endocrine neoplasia type II. In: Bilezikian J, Marcus R Levine M, eds. The parathyroids: Basic and clinical concepts, 2nd Ed. San Diego:Academic Press: 2001, pp. 585-600. 9. Friedman E, Larsson C, Amorosi A, Brandi ML, Bale A, Metz D, Jensen R, Skarulis M, Eastman R, Nieman L, Norton J, Marx S. Multiple endocrine neoplasia type I: Pathology, pathophysiology, molecular genetics, and differential diagnosis. In: Bilezikian J, Marcus R, Levine M, eds. The parathyroids: Basic and clinical concepts, 2nd Ed. Academic Press; 2000. 10. Law WM Jr, Hodgson SF, Heath III H. Autosomal recessive inheritance of familial hyperparathyroidism. N Engl J Med

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11. Wassif WS, Moniz CF, Friedman E, Wong S, Weber G, Nordenskjold M, Peters TJ, Larsson C. Familial isolated hyperparathyroidism: A distinct genetic entity with an increased risk of parathyroid cancer. J Clin Endocrinol Metab 1993;77:1485-1489. 12. Szabo J, Heath B, Hill VM, Jackson CE, Zarbo RJ, Mallette LE, Chew SL, Besser GM, Thakker RV, Huff V, Leppert MF, Heath III H. Hereditary hyperparathyroidism-jaw tumor syndrome; The endocrine tumor gene HRPT2 maps to chromosome lq21q31. A m J Hum Genet 1995;56:944-950. 13. LandonJE Parathyroidectomy in generalized osteitis fibrosa cystica. Report of a case in child two and one half years of age. J Pediatr 1932;1:544-554. 14. Philips RN. Primary diffuse hyperplasia in an infant of four months. Pediatrics 1948;2:428-434. 15. Hillman DA, Scriver CR, Peduis S, Schragowitch I. Neonatal familial primary hyperparathyroidism. NEnglJ Med 1964;270:483-490. 16. Spiegel AM, Harrison HE, Marx SJ, Brown EM, Aurbach GD. Neonatal primary hyperparathyroidism with autosomal dominant inheritance. J Pediatr 1977;90:269-272. 17. Marx SJ, Attie ME Spiegel AM, Levine MA, Lasker RD, Fox M. An association between neonatal severe primary hyperparathyroidism and familial hypocalciuric hypercalcemia in three kindreds. N EnglJ Med 1982;306:257-264. 18. Matsuo M, Okia K, Takemine H, Fujita T. Neonatal primary hyperparathyroidism in familial hypocalciuric hypercalcemia. AmJDis Child 1982;136:728-731. 19. Lillquist K, Illum N, Jacobsen BB, Lockwood K. Primary hyperparathyroidism in infancy associated with familial hypocalciuric hyperalcemia. Acat Paediatr Scand 1983; 72 (4) :625-9. 20. Cooper L, Wertheimer J, Levey R, Brown E, Leboff M, Wilkinson R, Anast CS. Severe primary hyperparathyroidism in a neonate with two hypercalcemic parents: Management with parathyroidectomy and heterotropic autotransplantation. Pediatrics 1986;78:263-268. 21. Steinman B, Gnehm HE, Rao VH, Kind HE Prader A. Neonatal severe primary hyperparathyroidism and alkaptonuria in a boy born to related parents with familial hypocalciuric hypercalcemia. Helv Paediatr Acta 1984;39:171-186. 22. Page LA, Haddow JE. Self-limited neonatal hyperparathyroidism in familial hypocalciuric hyperpcalcemia. J Pediatr 1987;111:261-264. 23. Marx SJ, Fraser D, Rapoport A. Familial hypocalciuric hypercalcemia. Mild expression of the gene in heterozygotes and severe expression in homozygotes. AmJMed 1985;78:5-22.

24. Fujimoto Y, Hazama H, Oku K. Severe primary hyperparathyroidism in a neonate having a parent with hypercalcemia: Treatment by total parathyroidectomy and simultaneous heterotropic autotransplantation. Surgery 1990; 108:933-938. 25. Heath DA. Familial benign hypercalcemia. Trends Endocrinol Metab 1989; 1:6-9. 26. Heath III H. Familial benign hypercalcemia--from clinical description to molecular genetics. WestJMed 1994;160:554-561. 27. Law WM, Jr, Heath III H. Familial benign hypercalcemia (hypocalciuric hypercalcemia). Clinical and pathological studies in 21 families. Ann Intern Med 1985;102:511-519. 28. Menko FH, Bijvoet OL, Fronen JL, Sandler LM, Adami S, O'Riordan JL, Schopman W, Heymen G. Familial benign hypercalcemia. Study of a large family. QJ Med 1983;52:120-140. 29. Toss G, Arnqvist H, Larsson L, Nilsson O. Familial hypocalciuric hypocalcemia: A study of four kindreds. J Intern Med 1989;225: 201-206. 30. Marx SJ, Attie ME Levine MA, Spiegel AM, Downs RW, Jr, Lasker RD. The hypocalciuric or benign variant of familial hypercalcemia: Clinical and biochemical features in fifteen kindreds. Medicine 1981 ;60:397-412. 31. Heath III H, Leppert ME Lifton RP, Penniston JT. Genetic linkage analysis in familial benign hypercalcemia using a candidate gene strategy I. Studies in four families. J Clin Endocrinol Metab 1992; 75:846-851. 32. Heath III H, Jackson CE, Otterud B, Leppert ME Genetic linkage analysis in familial benign (hypocalciuric) hypercalcemia: Evidence for locus heterogeneity. Am J Hum Genet 1993;53: 193-200. 33. Ujihara M, Sato K, Ohashi T, Tomori N, Kasono K, Tsushima T, Demura H. A case of hypocalciuric hypercalcemia without a family history. EndocrinolJpn 1991 ;38:689-692. 34. Sopwith AM, Burns C, Grant DB, Taylor GW, Wolf E, Besser GM. Familial hypocalciuric hypercalcemia: Association with neonatal primary hyperparathyroidism, and possible linkage with HLA haplotype. Clin Endocrinol 1984;21:57-64. 35. Paterson CR, Leheny W, O'Sullivan AF. HLA antigens and familial benign hypercalcemia. Clin Endocrinol 1985;23:111-113. 36. Menko FH, Bijvoet OL, Khan PM, Nijenhuis LE, Loghem EV, Schreuder I, Bernini LF, PronkJC, Madan K, Went LN. Familial benign hypercalcemia: Linkage studies in a large Dutch family. Hum Genet 1984;67:452-454. 37. Kowalska G, Peacock C, Davies M, Dyer P. Absence of linkage between familial hypocalciuric hypercalcemia and the major histocompatibility system. Tissue Antigens 1987;30:91-95. 38. Almahroos GM, Docherty K, Fletcher JA, Webb T, Heath DA. Studies of parathyroid hormone gene in normal subjects, in subjects with primary hyperparathyroidism and familial benign hypercalcemia. J Endocrinol 1987; 115:183-186. 39. Chou YH, Brown EM, Levi T, Crowe G, Atkinson AB, Arnquist HJ, Toss G, Fuleihan GE, SeidmanJG, Seidman CE. The gene responsible for familial hypocalciuric hypocalcemia maps to chromosome 3 in four unrelated families. Nat Genet 1992;1:295-300. 40. Pearce S, Trump D, Wooding C, Besser G, Chew S, Grant DB, Heath D, Hughes LA, Paterson CR, Whyte ME Calcium-sensing receptor mutations in familial bengnin hypercalcemia and neonatal hyperparathyroidism. J Clin Invest 1995;96:2683-2692. 41. Trump D, Whyte MB, Wooding C, Pang JT, Pearce SH, Kocher DB, Thakker RV. Linkage studies in a kindred from Oklahoma with familial benign (hypocalciuric) hypercalcemia (FBH) and developmental elevations in serum parathyroid hormone levels, indicate a third locus for FHH. Hum Genet 1995;96:183-187. 42. Lloyd SE, Pannett AA, Dixon PH, Whyte ME Thakker RV. Localization of familial benign hypercalcemia, Oklahoma variant (FBHok), to chromosome 19q13. A m J H u m Genet 1999;64: 189-195.

FBHH AND NSHPT / 43. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger HA, Lytton J, Herbert SC. Cloning and characterization of an extracellular Ca(Z+)-sensing receptor from bovine parathyroid cells. Nature 1993;366:577-580. 44. Pollak MR, Brown EM, Chou YH, Hebert SC, Marx SJ, Steinmann B, Levi T, Seidman CE, SeidmanJG. Mutations in the human Ca 2+sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297-1303. 45. Aida K, Koishi S, Inoue M, Nakazato M, Tawata M, Onaya T. Familial hypocalciuric hypercalcemia associated with mutation in the human CaZ+-sensing receptor gene. J Clin Endocrinol Metab 1995;80:2594-2598. 46. Janicic N, Pausova Z, Cole DE, Hendy GN. Insertion of an Alu sequence in the Ca2+-sensing receptor gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Am J Hum Genet 1995;56:880-886. 47. Heath III H, Odelberg S, Jackson CE, Teh BT, Hayward N, Larsson C, Buist NR, Kvapcho KJ, Hung BC, Capuano IV, Garett JE, Leppert ME Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains. J Clin Endocrinol Metab 1996;81:1312-1317. 48. Chou YH, Pollak MR, Brandi ML, Toss G, Arnqvist H, Atkinson AB, Papapoulos SE, Marx S, Brown EM, Seidman JG. Mutations in the human Ca(2+)-sensing receptor gene that causes familial hypocalciuric hypercalcemia. AmJHum Genet 1995;56:1075-1079. 49. Cole D, Peltekova V, Rubin L, Hawker G, Vieth R, Liew C, Hevang DH, Evrovski J, Hendy GN. A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations. Lancet 1999;353:112-115. 50. Bai M, Quinn S, Trivedi S, Kifor O, Pearce SH, Pollak M, Kvapcho K, Herbert SC, Brown EM. Expression and characterization of inactivating and activating mutations in the human CaZ+-sensing receptor. J Biol Chem 1996;271:19537-19545. 51. Ward DT, Brown EM, Harris HW. Disulfide bonds in the extracellular calcium-polyvalent cation-sensing receptor correlate with dimer formation and its response to divalent cations in vitro. J Biol Chem 1998;273:14476-14483. 52. Ho C, Conner DA, Pollak MB, Ladd DJ, Kifor O, Warren HB, Brown EM, Seidman JG, Seidman CE. A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat Genet 1995;11:389-394. 53. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert S, Seidman CE, Seidman JG. Autosomal dominant form of hypocalcemia caused by Ca2+-sensing receptor gene mutation. Nat Genet 1994;8:303-307. 54. Varrault A, Pena MS, Goldsmith PK, Mithal A, Brown EM, Spiegel AM. Expression of G protein e~-subunits in bovine parathyroid. Endocrinology 1995;136:4390-4396. 55. Marx SJ, StockJL, Attie ME Downs RW, Jr, Gardner DG, Brown EM, Spiegel AM, Doppman JL, Brennan ME Familial hypocalciuric hypercalcemia: Recognition among patients referred after unsuccessful parathyroid exploration. Ann Intern Med 1980;92:351-356. 56. Kristiansen JH, Rodbro P, Christiansen C, Johansen J, Jensen JT. Familial hypocalciuric hyeprcalcemia III: Bone mineral metabolism. Clin Endocrinol 1987;26:713-716. 57. Alexandre C, Chappard D, Riffat G. Bone histomorphometric analysis in familial hypocalciuric hypercalcemia. J Clin Pathol 1983;36:1319-1320. 58. Law WM, Jr, Wahner HW, Heath III H. Bone mineral density and skeletal fractures in familial benign hypercalcemia (hypocalciuric hypercalcemia). Mayo Clin Proc 1984;59:811-815. 59. Abugassa S, Nordenstrom J, Jarhult J. Bone mineral density in patients with familial hypocalciuric hypercalcemia (FHH). EurJ Surg 1992;158:397-402.

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60. Damoiseaux P, Tafforeau M, Henkinbrant A. Episode unique de pancreatite aigue revelant une hypercalcemie hypocalciurique familiale. Acta Clin Belg 1985;40:247-250. 61. Davies M, Klimiuk PS, Adams PH, Lumb GA, Large DM, Anderson DC. Familial hypocalciuric hypercalcemia and acute pancreatitis. Br MedJ Clin Res Ed 1981;282:1023-1025. 62. Falko JM, Maeder MC, Conway C, Mazzafferi EL, Skillman TG. Primary hyperparathyroidism: Analysis of 220 patients with special emphasis on familial hypocalciuric hypercalcemia. Heart Lung 1984;13:124-131. 63. Robinson PJ, Corrall RJ. The importance of distinguishing familial hypocalciuric hypercalcemia from asymptomatic primary hyperparathyroidism prior to neck exploration. Clin Otolaryngol 1990;15:141-146. 64. Doumith R, Ulmann A, Biclet P, Rieu M, Dubost C. Syndrome d'hypercalcemie-hypocalciurie: Une cause meconnue d'hypercalcemie. Nouv Presse Med 1980;9 (16) :1157-9. 65. Stuckey BG, Gutteridge DH, Kent GN, Reed WD. Familial hypocalciuric hypercalcemia and pancreatitis: No causal link proven. Aust N ZJMed 1990;20:718-719 and 725. 66. Van-Haeften TW, Hoogenberg K, van-Essen LH. Acute pancreatitis in a patient with familial benign hypercalcemia. NethJMed 1994; 45:110-113. 67. Bruce JI, Yang x, Ferguson CJ, Elliot AC, Steward MC, Case RM, Riccardi D. Molecular and functional identification of a Ca 2+ (polyvalent cation)-sensing receptor in rat pancreas. JBiol Chem 1999;274:20561-20568. 68. Pearce SH, Wooding C, Davies M, Tollefsen SE, Whyte ME Thakker RV. Calcium-sensing receptor mutations in familial hypocalciuric hypercalcemia with recurrent pancreatitis. Clin Endocrino11996 ;4 5 :6 7 5-680. 69. Carling T, Szabo E, Bai M, Ridefelt P, Westin G, Gustavsson P, Trivedi S, Hellman P, Brown E, Dahl N, RastadJ. Familial hypercalcemia and hyperpcalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor. J Clin Endocrinol Metab 2000;85:2042-2047. 70. Cole D, Janicic N, Salisbury S, Hendy G. Neonatal severe hyperparathyroidism, secondary hyperparathyroidism, and familial hypocalciuric hypercalcemia: Multiple different phenotypes associated with inactivating Alu insertion mutation of the calciumsensing receptor gene. Am J Med Genet 1997;71 (2) :202-210. 71. Rajala MM, Heath III H. Distribution of serum calcium values in patients with familial benign hypercalcemia (hypocalciuric hypercalcemia): Evidence for a discrete genetic defect. J Clin Endocrinol Metab 1987;65:1039-1041. 72. Marx SJ, Spiegel AM, Brown EM, Koelhler JO, Gardner DG, Brennan ME Aurbach GD. Divalent cation metabolism. Familial hypocalciuric hypercalcemia versus primary hyperparathyroidism. A m J M e d 1978;65:235-242. 73. Brown EM, Pollak M, Seidman CE, Seidman JG, Chou YH, Riccardi D, Herbert SC. Calcium-ion-sensing cell-surface receptors. N EnglJ Med 1995;333:234-240. 74. Law WM, Bollman S, Kumar R, Heath III H. Vitamin D metabolism in familial benign hypercalcemia (hypocalciuric hypercalcemia) differs from that in primary hyperparathyroidism. J Clin Endocrinol Metab 1984;58: 744-747. 75. Marx SJ, Spiegel AM, Brown EM, Windeck K, Gardner DG, Downs RW, Jr, Attie M, Aurbach GD. Circulating parathyroid hormone activity: Familial hypocalciuric hypercalcemia versus typical primary hyperparathyroidism. J Clin Endocrinol Metab 1978; 47:1190-1197. 76. Firek AF, Kao PC, Heath III H. Plasma intact parathyroid hormone (PTH) and PTH-related peptide in familial benign hypercalcemia: Greater responsiveness to endogenous PTH than in primary hyperparathyroidism. J Clin Endocrinol Metab 1991 ;72: 541-546.

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77. Rajala MM, Klee GG, Heath III H. Calcium regulation of parathyroid and C cell function in familial benign hypercalcemia. JBone Miner Res 1991;6:117-124. 78. Gunn IR, Wallace JR. Urine calcium and serum ionized calcium, total calcium and parathyroid hormone concentrations in the diagnosis of primary hyperparathyroidism and familial benign hypercalcemia. Ann Clin Biochem 1992;29(Part 1):52-58. 79. Paterson CR, Gunn A. Familial benign hypercalcemia. Lancet 1981;2:61-63. 80. Watanabe H, Sutton RA. Renal calcium handling in familial hypocalciuric hypercalcemia. Kidney Int 1983;24:353-357. 81. Davies M, Adams PH, Lumb GA, BerryJL, Loveridge N. Familial hypocalciuric hypercalcemia: Evidence for continued enhanced renal tubular reabsorption of calcium following total parathyroidectomy. Acta Endocrinol 1984; 106:499-504. 82. Kristiansen JH, Brochner-Mortensen J, Pedersen KO. Familial hypocalciuric hypercalcemia I: Renal handling of calcium, magnesium and phosphate. Clin Endocrinol 1985;22:103-116. 83. Stuckey BG, Kent GN, Gutteridge DH, Pullan PT, Price RI, Bhagat C. Fasting calcium excretion and parathyroid hormone together distinguish familial hypocalciuric hypercalcemia from primary hyperparathyroidism. Clin Endocrino11987;27:525-533. 84. Attie ME Gill JR, Jr, Stock JL, Spiegel AM, Downs RW, Jr, Levine MA. Urinary calcium excretion in familial hypocalciuric hypercalcemia. Persistance of relative hypocalciuria after induction of hypoparathyroidism. J Clin Invest 1983;72:667-676. 85. Riccardi D, Park J, Lee W-S, Gamba G, Brown EM, Hebert SC. Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci USA 1995;92:3161-3165. 86. Marx SJ, Attie ME Stock JL, Spiegel AM, Levine MA. Maximal urine-concentrating ability: Familial hypocalciuric hyperpcalcemia versus typical primary hyperparathyroidism. J Clin Endocrinol Metab 1981 ;52: 736-740. 87. Davies M, Adams PH, Berry JL, Lumb GA, Klimiuk PS, Marver EB. Familial hypocalciuric hypercalcemia: Observations on vitamin D metabolism and parathyroid function. Acta Endocrinol 1983;104:210-215. 88. Gilbert E D' Amour P, Gascon-Barre M, Boutin JM, Havramkova J, Betanger R, Belanger R, Matte R. Familial hypocalciuric hypercalcemia: Description of a new kindred with emphasis on its difference from primary hyperparathyroidism. Clin Invest Med 1985;8:78-84. 89. Kristiansen JH, Rodbro P, Christiansen C, Brocher JM, Carl J. Familial hypocalciuric hypercalcemia II: Intestinal calcium absorption and vitamin D metabolism. Clin Endocrino11985;23:511-515. 90. Law WM, Jr, Bollman S, Kumar R, Heath III H. Vitamin D metabolism in familial benign hypercalcemia (hypocalciuric hypercalcemia) differs from that in primary hyperparathyroidism. J Clin Endocrinol Metab 1984;58:744-747. 91. Lyons TJ, Crookes PE Postlethwaite W, Sheridan B, Brown RC, Atkinson AB. Familial hypocalciuric hypercalcemia as a differential diagnosis of hyperparathyroidism: Studies of a large kindred and a review of surgical experience in the condition. BrJ Surg 1986;73:188-192. 92. Silverberg S, Shane E, Jacobs T, Siris E, Bilezikian J. A 10-year prospective study of primary hyperparathyroidism with or without parathyroid surgery. N EnglJ Med 1999;341:1249-1255. 93. Heath III H, Purnell DC. Urinary cyclic 3',5' adenosine monophosphate responses to exogenous and endogenous parathyroid hormone in familial benign hypercalcemia and primary hyperparathyroidism. J Lab Clin Med 1980;96:974-984. 94. AuwerxJ, Demedts M, Bouillon R. Altered parathyroid set point to calcium in familial hypocalciuric hypercalcemia. Acta Endocrinol 1984;106:215-218.

95. Khosla S, Ebeling PR, Firek AF, Burritt MM, Kao PC, Heath III H. Calcium infusion suggests a "set-point" abnormality of parathyroid gland function in familial benign hyperpcalcemia and more complex disturbances in primary hyperparathyroidism. J Clin Endocrinol Metab 1993;76:715-720. 96. Law WM, Jr, James EM, Charboneau JW, Purnell DC, Heath III H. High-resolution parathyroid ultrasonography in familial benign hypercalcemia (familial hypocalciuric hypercalcemia). Mayo Clin Proc 1984;59(3):153-155. 97. Law WM,Jr, CarneyJA, Heath III H. Parathyroid glands in familial benign hypercalcemia (familial hypocalciuric hypercalcemia). A m J M e d 1984;76:1021-1026. 98. Thorgeirsson U, Costa J, Marx sJ. The parathyroid glands in familial hypocalciuric hypercalcemia. Hum Pathol 1981 ;12:229-237. 99. Powell B, Buist N. Late presenting, prolonged hypocalcemia in an infant of a woman with hypocalciuric hypercalcemia. Clin Pediatr 1990;29:241-243. 100. Pollak M, Chou Y, Marx S, Steinmann B, Cole D, Brandi M, Papapoulos SE, Menko FH, Hendy GN, Brown EM, et al. Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Effects of mutant gene dosage on phenotype. J Clin Invest 1994;93:1108-1112. 101. Kobayashi M, Tanaka H, Tsuzuki K, Tsuyukim M, Igaki H, Ichinose Y, Aya K, Nishioka N, Seino Y. Two novel missense mutations in calcium-sensing receptor gene associated with neonatal severe hyperparathyroidism. J Clin Endocnnol Metab 1997;82:2716-2719. 102. Harris S, D'Ercole A. Neonatal hyperparathyroidism: The natural course in the absence of surgical intervention. Pediatrics 1989;83:53-56. 103. Orwoll E, Silbert J, McClung M. Asymptomatic neonatal familal hypercalcemia. Pediatrics 1982;69:109-111. 104. Wilkinson H, James J. Self-limiting neonatal primary hyperparathyroidism associated with familial hypocalciuric hypercalcemia. Arch Dis Child 1993;69:319-321. 105. Powell BR, Blank E, Benda G, Buist NR. Neonatal hyperparathyroidism and skeletal demineralization in an infant with familial hypocalciuric hypercalcemia. Pediatrics 1993;91:144-145. 106. Bai M, Pearce SH, Kifor O, Trivedi S, Stauffer UG, Thakker RV, Brown EM, Steinmann B. In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the CaZ+-sensing receptor gene: Normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia. J Clin Invest 1997;99:88-96. 107. Anspach WE, Clifton WM. Hyperparathyroidism in children: Report of two cases. AmJDis Child 1939;58:540-557. 108. Pratt EL, Geren BB, Neuhauser EBD. Hypercalcemia and idiopathic hyperplasia of the parathyroid glands in an infant. J Pediatr 1947;30:388-399. 109. Hillman DA, Scriver CR, Pedvis S, Shragovitch I. Neonatal familial primary hyperparathyroidism. NEnglJMed 1964;270:483-490. 110. Goldbloom RB, Gillis DA, Prasad M. Hereditary parathyroid hyperplasia: A surgical emergency of early infancy. Pediatrics 1972;49:514-523. 111. Rhone DE Primary neonatal hyperparathyroidism: Report of a case and review of the literature. A m J Clin Patho11975;64:488-499. 112. Proesmans W, Dhondt E Logghe N. Congenital hyperparathyroidism: Case report and review of the literature. Acta Paediatr Belg 1977;30:45-52. 113. Eftekhari E Yousefzadeh DK. Primary infantile hyperparathyroidism: Clinical, laboratory and radiographic features in 21 cases. Skel Radiol 1982;8:201-208. 114. Garcia-Banniel R, Kutchemeshgi A, Brandes D. Hereditary hyperparathyroidism. The fine structure of the parathyroid gland. Arch Patho11974;97:399-403.

FBHH AND NSHPT / 115. Nguyen VC, Sennot WM, Knox GS. Neonatal hyperparathyroidism. Radiology 1974;112:175-176. 116. Corbeel L, Casaer P, Malvaux P, Lormans J, Bourgeois N. Hyperparathyroidie congenitale. Arch Fran¢ Ped 1968;25:879-891. 117. Lutz P, Kane O, Pfersdorff A, Seiller F, Sauvage P, Levy JM. Neonatal primary hyperparathyroidism: Total parathyroidectomy with autotransplantation of cryopreserved parathyroid tissue. Acta Paediatr Scand 1986;75:179-182.

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118. Thompson NW, Carpenter LC, Kessler DL, Nishiyama RH. Hereditary neonatal hyperparathyroidism. Arch Surg 1978;113: 100-103. 119. Silverberg SJ, Bone III, HG, Marriott TB, Locker FG, Thys Jacobs S, Dziem G, Kaatz S, Sanguinetti E, Bilezikian J. Shortterm inhibition of parathyroid hormone secretion by calciumreceptor agonist in patients with primary hyperparathyroidism. NEnglJMed 1997;337:1506-1510.

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CI4APTF R 39 The Parathyroids in Renal Disease

Pathophysiology

KEVIN J. MARTIN, ESTHER A. GONZALEZ, AND EDUARDO SLATOPOLSKY Division of Nephrology, Saint Louis University, and Renal Division, Washington University, St. Louis, Missouri 63110

INTRODUCTION

malities are considered below separately, it should be emphasized t h a t all these factors are closely interrelated, and one or more of these factors may predominate at different stages of renal insufficiency and may vary in importance according to the clinical circumstances.

The association between renal failure, bone disease, and hyperplasia of the parathyroid glands has been recognized for more than 50 years. Since that time, considerable effort (1-4) has been devoted to the elucidation of the pathogenesis of secondary hyperparathyroidism. An understanding of the factors involved in the initiation and the maintenance of the state of disordered parathyroid h o r m o n e (PTH) secretion can form the basis of a rational approach to the treatment of this important and c o m m o n complication of renal insufficiency. Hyperplasia of the parathyroid glands and elevated levels of PTH in blood are a m o n g the earliest alterations of mineral metabolism seen in patients with decreased renal function. Elevated levels of PTH have been reported in patients with only mild decreases in renal function (5,6). Additional evidence of parathyroid overactivity was provided by the observation that patients with decreased renal function exhibit a greater than normal increase in serum PTH in response to hypocalcemia (7). U n d e r normal circumstances the principal determinant of PTH secretion is the concentration of ionized calcium in blood, but in the presence of renal insufficiency there is a constellation of factors that contribute to alter the regulation of the secretion of PTH. The main factors involved in the pathogenesis of hyperparathyroidism in chronic renal disease are illustrated in Fig. 1 and include phosphorus retention, decreased levels of calcitriol, abnormal parathyroid gland function, hypocalcemia, and skeletal resistance to the calcemic action of PTH. T h o u g h these abnorThe Parathyroids, Second Edition

ROLE OF P H O S P H A T E R E T E N T I O N The importance of phosphate retention as a factor in the pathogenesis of the secondary hyperparathyroidism of renal insufficiency has been continuously emphasized by Slatopolsky and colleagues over a number of years (8-13). It was originally proposed that a transient small increase in serum phosphorus would occur early in the course of renal insufficiency as a consequence of decreased ability of the failing kidney to excrete phosphorus (8,9). This transient episode of h y p e r p h o s p h a t e m i a would result in a small decrease in the levels of ionized calcium in blood, which would consequently result in an increase in PTH secretion to restore the serum calcium to normal. The increased levels of PTH would decrease phosphorus reabsorption by the proximal tubule of the kidney and thus cause phosphaturia and restore the elevated serum phosphorus to normal. This new steady state would be maintained at the expense of higher circulating levels of PTH. Support for a role of phosphorus in the genesis of hyperparathyroidism was provided by the observation that a diet high in phosphorus results in parathyroid hyperplasia (14,15). More compelling, however, are the 625

Copyright © 2001 J o h n P. Bilezikian, Robert Marcus, and Michael A. Levine.

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CI-IApTWR39 $ Renal Mass

Y [PhosphorusRetention I 1" Phosphorus $ Renal Mass ? acidosis ? other

Hypocalcemia

J, Calcitriol

--->

[ LowCalcitriolLevels [

Calcitriol resistance 1" Set-point

[ AIteredand FunctioGrowth nPTG

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$ VDR PTG Hyperplasia 1" PTG Mass J, Ca Receptor 1" Phosphorus

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I HyperparathyroidismI 1" Phosphorus $ Calcitriol

1" Phosphorus

Desens. to PTH

Calcitriol Resistance Skeletal Resistance PTG :

Parathyroid Gland

VDR : V i t a m i n D

studies of Rutherford et al., who demonstrated that restriction of dietary phosphate in proportion to the decrease in glomerular filtration rate (GFR) (thereby negating the initial stimulus for the series of events outlined above to increase the secretion of PTH) was successful in preventing the development of hyperparathyroidism in dogs with renal insufficiency (16). Subsequent studies by Llach and Massry (17) confirmed these findings in h u m a n subjects. T h o u g h there is general agreement that phosphorus retention plays an important role in the genesis of the hyperparathyroldism of renal insufficiency, the mechanism by which this effect occurs is complex and is somewhat controversial. Several potential mechanisms need to be considered, including phosphorus-induced hypocalcemia, phosphorus-induced decreased levels of calcitriol, direct effects of phosphorus on the parathyroid gland, and skeletal resistance to the calcemic actions of PTH and possibly other unknown factors. Again, it is important to point out that these mechanisms are closely interrelated and are not mutually exclusive.

Phosphorus Retention and Hypocalcemia That an increase in serum phosphorus level can evoke an increase in PTH secretion was shown many years ago by Reiss et al. (18), who demonstrated that an oral phosphorus load led to an increase in serum phosphorus, a decrease in ionized calcium, and an increased level of PTH in normal h u m a n subjects. Whether this sequence of events occurs in early renal failure has been questioned because fasting or even postprandial levels of serum phosphorus are not consistently elevated in early renal insufficiency (19,20). In fact, low levels of serum phosphorus are not uncommon. Furthermore, in early renal failure, although a

Receptor

J, PTH -R ? J, Calcitriol ? Calcitriol resistance

FIG. 1 Diagrammatic representation of the multiple factors involved in the pathogenesis of secondary hyperparathyroidism in renal insufficiency.

trend toward a decrease in the levels of plasma ionized calcium has been demonstrated by Llach and Massry (17), hypocalcemia is not demonstrable in many patients (21). Portale et al. (19) also examined the possibility that intermittent hypocalcemia may occur after phosphate loading but could not demonstrate hypocalcemia despite careful monitoring of the serum ionized calcium concentration. Thus, it appears that the mechanism of the effect of phosphate retention may not be exerted exclusively through the induction of hypocalcemia. This concept was further tested directly by Lopez-Hilker et al. (22). In these studies, hypocalcemia was prevented in dogs after the induction of uremia by the administration of a high-calcium diet. In these animals, which actually developed a mild increase in the serum calcium concentrations, increased levels of PTH nonetheless occurred. These studies clearly demonstrated that hypocalcemia is not essential for the develo p m e n t of secondary hyperparathyroidism in chronic renal failure. These observations also demonstrate that other factors need to be considered to explain the effects of phosphorus retention in the pathogenesis of secondary hyperparathyroidism.

Phosphorus Retention and Calcitriol Because phosphorus plays a major role in the regulation of the production rate of 1,25-dihydroxyvitamin D (calcitriol) by altering the activity of the enzyme loLhydroxylase (23), it is possible that phosphorus retention leads directly to a decrease in the production of calcitriol. Conversely, the beneficial effects of phosphorus restriction on ameliorating the development or in decreasing established hyperparathyroidism could be explained by an increase in the levels of calcitriol. Evidence has been presented in support of a role of calcitriol deficiency from the studies of Lopez-Hilker

PARATHYROIDS IN RENAL DISEASE / Calcium

Calcitriol

Phosphorus

627

PTH

8

400 350 300 250

m

-o ~1 E

.

3

200 150 100 -t 50 .1

0

0 ---

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FIG. 2 The effect of reduction in dietary phosphorus on the serum levels of calcium, calcitriol, phosphorus, and PTH in dogs with chronic renal failure. The solid bars show the data on a 1.6% calcium, 0.96% phosphorus diet; the open bars show data on 0.6% calcium, 0.6% phosphorus diet. Modified from Ref. 24, with permission. et al., in which the administration of calcitriol immediately after the induction of renal insufficiency was successful in preventing the development of hyperparathyroidism (22).

Direct Effects of Phosphorus on the Parathyroid Gland Further studies on the m e c h a n i s m of the phosphorus effect were carried out by Lopez-Hilker et al. (24) in chronically uremic dogs with severe hyperparathyroidism, in which dietary phosphorus was restricted in a progressive fashion. Simultaneously, dietary calcium was adjusted to maintain serum ionized calcium levels. Calcitriol concentrations r e m a i n e d low and did not change despite lowering of dietary phosphorus, presumably because total renal mass, reflecting 1c~-hydroxylase activity, was reduced. As shown in Fig. 2, u n d e r these circumstances, with no change in the levels of ionized calcium or calcitriol, the decrease in serum phosphorus was associated with a remarkable decrease in the levels of PTH. These data d e m o n s t r a t e d the possibility that dietary phosphorus a n d / o r the levels of serum phosphorus, was directly or indirectly affecting the secretion of PTH. Accordingly, evidence for a direct effect of phosphate on PTH secretion was e x a m i n e d in studies in vitro, and two groups successfully demonstrated that elevated m e d i u m phosphorus concentration was associated with significantly increased PTH secretion u n d e r circumstances in which the concentration of ionized calcium was unchanged, as shown in Fig. 3 (13,25). These observations have been confirmed in h u m a n parathyroid tissue in vitro (26), and clinical observations in patients with renal failure have confirmed the correlations of serum phosphorus with the

levels of PTH (27,28). The m e c h a n i s m by which phosphorus increases PTH secretion is not known. T h o u g h some investigators have reported that high-phosphorus diets result in increased levels of PTH mRNA (29), others have not d e m o n s t r a t e d any change (13). These a p p a r e n t contradictory results may be reconciled by the fact that the studies indicating an increase in PTH mRNA utilized thyroparathyroid tissue rather than isolated parathyroid tissue; therefore, an increase in parathyroid growth would lead to an a p p a r e n t increase in PTH mRNA, c o m p a r e d to [3-actin mRNA, in the total thyroparathyroid tissue, even if there was no increase in PTH mRNA per parathyroid cell, whereas no such

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FIG. 3 Time course of PTH secretion by intact rat parathyroid glands in low (0.2 mM, dashed line) or high (2.8 mM, solid line) phosphorus-containing media. Concentrations of ionized calcium were identical in both media. Redrawn from Ref. 13, with permission.

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increase in PTH mRNA would be evident in isolated parathyroid tissue. The data from both perspectives are, however, consistent with the interpretation that the stimulatory effect of high phosphorus concentrations on PTH secretion is posttranscriptional, as demonstrated in nuclear run-on studies by Kilav et al. (29), and thus, the focus of investigation has turned to the role of phosphorus in regulating PTH mRNA stability (30). Additional important effects of phosphorus on the parathyroid have also been uncovered. Thus, NavehMany et al. have demonstrated that dietary phosphorus has a major effect on parathyroid cell growth (31). Using proliferating cell nuclear antigen (PCNA) staining of parathyroid tissue from rats, these investigators showed that a high-phosphorus diet accelerated parathyroid growth whereas a low-phosphate diet prevented parathyroid hyperplasia (31). Similar results were also obtained by Y1 et al. (32). Studies in experimental animals with renal failure in vivo by Denda et al. have also shown that parathyroid hyperplasia is regulated by dietary phosphorus and added the important observation that parathyroid hyperplasia occurs very rapidly (within days) following the induction of renal failure (Fig. 4) (33). The mechanism of the direct effects of phosphorus on parathyroid growth remains to be determined, but dietary phosphate induced changes in the cell cycle regulator p21 and the expression of transforming growth factor-e~ (TGF-c~) appear to be involved. Thus, it has been shown that following the induction of uremia, a low-phosphate diet is associated with an increase in the cyclin-dependent kinase inhibitor p21 at both mRNA and protein levels and is associated with prevention of parathyroid hyperplasia (34). Additional studies have revealed a potential role for TGF-e~ in that a high-phosphorus diet is associated with a marked increase in the expression of TGF-~ after 5 days of experimental uremia (35). Mso of consider-

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FIG. 4 The changes in parathyroid weight, total protein, and DNA after induction of renal failure in rats fed a highphosphorus diet. Modified from Ref. 33, with permission.

able interest is the recent demonstration that a type III phosphate transporter, PiT-l, is present in the parathyroid and that the levels of its mRNA are regulated by calcitriol and by changes in dietary phosphorus (36). Further studies are required to delineate the exact mechanisms of the effects of phosphorus on the parathyroid gland.

Other Effects of Phosphorus It is also possible that there are other consequences of alterations in dietary phosphorus that indirectly affect parathyroid function, for example, contributing to skeletal resistance to the calcemic effects of PTH, which is discussed below in detail.

ROLE OF DECREASED SYNTHESIS OF CALCITRIOL The kidney is the major site for the production of calcitriol, thus it follows that a decrease in renal mass as renal disease progresses may lead to a decrease in the ability of the diseased kidney to produce calcitriol, an important metabolite of vitamin D. The consequences of calcitriol deficiency on mineral metabolism contribute importantly to the development of secondary hyperparathyroidism. Substantial evidence has been presented in support of the role of abnormal vitamin D metabolism in the pathogenesis of hyperparathyroidism in renal Insufficiency. In general, adults with chronic renal insufficiency have levels of calcitriol in the normal range until the glomerular filtration rate (GFR) falls to <50 m l / m i n u t e (37-40). Some investigators, however, have found lower plasma levels of calcitriol with creatinine clearances between 50 and 80 m l / m i n u t e (20). Mthough blood levels of calcitriol may be normal, it is important to point out that these values still may be inappropriately low because of the fact that levels of PTH are already elevated at this stage of renal insufficiency, and therefore the stimulus to raise calcitriol levels above normal is present. It is possible that, in this situation, phosphate retention is counteracting the stimulating actions of PTH on calcitriol production. An additional consideration is that the development of metabolic acidosis with renal failure decreases the levels of calcitriol and therefore prevents an appropriate increase with PTH stimulation. This issue is somewhat controversial, because acidosis has been reported to decrease, to increase, or not to change levels of calcitriol (41-45). Some evidence suggests that the critical determinant of the changes in calcitriol during metabolic acidosis might be the effects of acidosis on phosphorus homeostasis (46). It is also possible that other factors that either accumulate in plasma in renal insufficiency (47), or that occur as a conse-

PARATHYROIDS IN RENAL DISEASE

quence of decreased GFR, such as decreased delivery of 25-hydroxyvitamin D~-bound vitamin D binding protein (DBP) to the proximal tubule uptake mechanism, could potentially limit 1-ot-hydroxylase activity in the kidney (48). Such factors may be more relevant in more advanced renal insufficiency. As renal disease advances and renal mass becomes limiting, the levels of calcitriol unquestionably fall, and this becomes a major factor in the pathogenesis of hyperparathyroidism. Decreases in the level of calcitriol may lead to impaired intestinal calcium absorption, which in turn contributes to hypocalcemia, and, importantly, low levels of calcitriol may directly lead to abnormal function of the parathyroid glands.

RO LE O F ALTERED PARATHYROID FUNCTION

Role of Hypocalcemia If hypocalcemia occurs during the course of renal insufficiency there is a powerful stimulus to PTH secretion and parathyroid growth. Hypocalcemia is not an invariable finding however, in patients with renal failure (21), and it has been shown clearly experimentally that hypocalcemia is not essential for the development of hyperparathyroidism (22). Evidence has accumulated to indicate that there may be intrinsic abnormalities that develop at the level of the parathyroid gland in renal failure, leading to disordered calcium-regulated PTH secretion. It has been shown that the enzyme adenylate cyclase in parathyroid cell membranes prepared from hyperplastic parathyroid glands from patients undergoing dialysis for renal failure is less susceptible to inhibition by calcium than that in membranes prepared from normal parathyroid tissue (49). Subsequent studies revealed that this difference could also be demonstrated for calcium-regulated PTH secretion in vitro, using dispersed cells from parathyroid glands from patients with renal failure, similar to previous findings in parathyroid adenomata (50,51). These data demonstrated that the set point for calcium was elevated in hyperplastic parathyroid glands; that is, a higher calcium concentration was required to decrease PTH secretion by 50%. Though these data suggested an intrinsic abnormality in calcium-regulated PTH secretion, the mechanism of the abnormal response of the parathyroid cell was not addressed.

Role of Calcitriol Current evidence indicates that calcitriol is a major factor in the direct regulation of PTH secretion. The findings of cytoplasmic and nuclear binding components for calcitriol in chick parathyroid glands by

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629

Brumbaugh et aL (52) first demonstrated that the parathyroid glands may be an important target tissue for calcitriol. These observations were followed by the demonstration that calcitriol decreased PTH secretion in vitro (53). Despite the controversial nature of the initial reports (54), it was subsequently demonstrated by Cantley et al. (55), with more prolonged experimental protocols, that calcitriol clearly suppresses PTH secretion. This observation was confirmed by others (56) and was further detailed by Silver et al. (57) and Russell et al. (58), who demonstrated that the effect of calcitriol was at the level of transcription of the PTH gene. These data were subsequently confirmed in studies in vivo by Silver et al. (59). Other investigators have extended these observations and identified a region in the 5' flanking region of the PTH gene that appears to mediate the inhibition of PTH gene transcription by calcitriol (60-62). These studies clearly show that calcitriol has a direct effect on the synthesis and secretion of PTH. The mechanisms by which low levels of calcitriol may directly and indirectly alter PTH secretion are illustrated diagrammatically in Fig. 5. In addition to the indirect effects of calcitriol to increase intestinal absorption of calcium and possibly to improve skeletal resistance to PTH, there are several direct effects of calcitriol on parathyroid function. Thus, as indicated above, calcitriol directly decreases preproPTH gene transcription. Observations by Korkor have indicated that the n u m b e r of receptors for calcitriol in parathyroid glands appears to be reduced in patients with chronic renal failure compared to either patients undergoing renal transplantation or patients with primary hyperparathyroidism (63). These observations suggest that a decrease in parathyroid vitamin D receptor n u m b e r may contribute to the pathogenesis of hyperparathyroidism by reducing the ability of calcitriol to inhibit PTH secretion on this basis. Similar

Effects of Calcitriol on Parathyroid Function Direct Effects Indirect Effects

Decreased PTH Secretion

Increased Serum Calcium

Control of Hyperparathyroidism

<2~

FIG. 5 The direct and indirect effects of calcitriol on parathyroid hormone secretion.

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observations have been made by Merke et al. (64) and Brown et al. (65), who also found a decreased n u m b e r of calcitriol receptors in the parathyroid glands from uremic rats and dogs, respectively. Further studies by Naveh-Many et al. (66) have shown that the administration of calcitriol led to a dose-dependent increase in the n u m b e r of vitamin D receptors in the parathyroid glands of normal rats. These data are all consistent with the view that calcitriol may regulate its own receptor n u m b e r in the parathyroid cell. An additional effect of calcitriol on parathyroid function is suggested by the studies of Kremer et al. (67), who showed that exposure of quiescent bovine parathyroid cells in culture to serum or serum substitute results in an increase in tritiated thymidine incorporation, followed by an increase in cell number. These changes were preceded by an increase in c-myc and c-fos protooncogene mRNA levels. Mterations in medium calcitriol concentration had no effect on the growth rate of quiescent parathyroid cells. However, calcitriol, when added with serum or serum substitute, blocked the increase in c-myc mRNA levels, and the expected increase in parathyroid cell n u m b e r failed to occur. These results indicated that calcitriol may directly modulate parathyroid cell proliferation by altering the expression of specific replication-associated oncogenes. Thus, if calcitriol is also an important growth regulator of parathyroid cells, as is suggested by these data, low levels of calcitriol may allow parathyroid cells to proliferate. Conversely, the administration of calcitriol may suppress proliferation of parathyroid cells. It is important to keep in mind, however, that recent studies in vitamin D receptor (VDR) knockout mice have shown that if serum concentrations of calcium and phosphorus are normalized by the use of a rescue diet, parathyroid hyperplasia does not occur (68).

Role of Abnormal Parathyroid Growth The process and consequences of parathyroid growth are extremely important factors in the disordered parathyroid function of renal failure. Fukuda et al. observed that although some enlarged parathyroid glands resected at parathyroidectomy had a relatively h o m o g e n e o u s appearance, some glands had numerous nodules of varying size (69). The nodules demonstrated marked decreases in immunostaining for the vitamin D receptor. Subsequent studies demonstrated that these nodules represented monoclonal expansions of parathyroid cells (70). Additional studies demonstrated that the nodules also had a marked decrease in the expression of the calcium receptor (71,72). These data suggest that this nodular hyperplasia represents a severe manifestation of secondary hyperparathyroidism and raise the question of whether the loss of the VDR

a n d / o r the calcium-sensing receptor (CaSR) leads to accelerated growth or whether the accelerated growth leads to a loss of the receptors. Some insight into these issues has been provided by Ritter et al., who, using immunohistochemical techniques, d e m o n s t r a t e d that parathyroid cell proliferation, assessed by staining for PCNA, appears to precede the loss of the calcium receptor in parathyroid glands from uremic rats (73). There is also evidence that signaling at the parathyroid calcium receptor may be involved in the regulation of parathyroid cell growth, based on the demonstration that the administration of the calcimimetic agent, NPS R-568, appeared to prevent the development of parathyroid hyperplasia in the 5 / 6 nephrectomized uremic rat model (74). The additional observations that calcitriol as well as phosphate may regulate the expression of the calcium receptor in the parathyroid lend support for the possibility that the calcium receptor may play a role in the regulation of parathyroid cell growth (75). Clinical Correlations These findings explain the now well-known clinical observation of the effects of calcitriol on PTH secretion in patients with renal failure. Thus, Slatopolsky et al. (76) showed that the intravenous administration of calcitriol to patients with end-stage renal disease maintained on chronic hemodialysis resulted in substantial suppression of PTH levels. Although serum calcium levels gradually rose during the treatment with calcitriol, it appeared that the levels of PTH in serum began to decrease prior to the elevation in serum calcium. The findings have been confirmed in multiple studies subsequently and extended to the use of calcitriol analogs (77,78). There is also the potential for calcitriol to alter the set point for calcium-regulated PTH secretion in vivo; however, this has not been consistently demonstrated, possibly reflecting the degree of hyperparathyroidism and the extent of the parathyroid hyperplasia in the patients studied. In summary (Fig. 5), the effects of calcitriol on parathyroid gland function are complex and may occur at many levels. Calcitriol deficiency may lead to hypocalcemia principally by decreasing intestinal calcium absorption. Low levels of calcitriol may also play a role in initiating parathyroid cell hyperplasia by an effect on the regulation of parathyroid cell growth. Low levels of calcitriol have a profound effect on p r e p r o P T H synthesis by a direct effect on the regulation of PTH gene transcription. Evidence has also been presented that low levels of calcitriol may decrease the expression of vitamin D receptor and the calcium receptor in parathyroid tissue and consequently may influence the responsivity of the parathyroid gland to

PARATHYROIDS IN RENAL DISEASE / changes in calcitriol and calcium. Decreased levels of calcitriol may potentially also play a role in the regulation of the set point for calcium-regulated PTH secretion. These abnormalities in parathyroid gland function as a consequence of decreased levels of calcitriol may contribute to the generation and maintenance of hyperparathyroidism in the course of renal insufficiency and provide a sound basis for the therapeutic effects of calcitriol in the t r e a t m e n t of hyperparathyroidism in uremia.

ROLE OF HYPOCALCEMIA Hypocalcemia is not u n c o m m o n during the course of u n t r e a t e d renal insufficiency and, if present, provides a powerful stimulus for parathyroid h o r m o n e secretion. The potential mechanisms involved in the development of hypocalcemia are several. The retention of phosphorus leading to h y p e r p h o s p h a t e m i a may result in the formation of complexes with calcium. Hypocalcemia may occur as a result of deposition of these complexes in soft tissue a n d / o r bone. Because a major action of calcitriol is to regulate intestinal absorption of calcium, it follows that, as levels of calcitriol decline with progressive renal disease, the intestinal absorption of calcium also declines. Decreased intestinal absorption of calcium may contribute to the develo p m e n t of hypocalcemla, which in turn would provide a stimulus for increased PTH secretion. Studies of the m e a s u r e m e n t of intestinal calcium absorption at various levels of renal function have revealed results similar to the relationship between calcitriol and renal function, and reflect the major role of calcitriol in the regulation of intestinal calcium absorption. There is also evidence to suggest that uremic toxins, yet to be definitively identified, might attenuate the actions of calcitriol by interfering with the binding of the vitamin D receptor to its response elements on DNA (79). An additional cause of hypocalcemia is that of skeletal resistance to the calcemic actions of PTH (see below).

R O L E O F SKELETAL RESISTANCE T O PARATHYROID HORMONE Resistance of the skeleton to the hypercalcemic action of PTH is a n o t h e r potential cause of hypocalcemia and hyperparathyroidism in patients with renal insufficiency. It has been d e m o n s t r a t e d that the increase in serum calcium in response to infusion of PTH is significantly less in hypocalcemic patients with renal failure than in normal subjects (80,81). Additional observations have indicated that recovery from induced hypocalcemia in patients with mild renal

631

insufficiency is delayed c o m p a r e d to results from normal subjects, despite a .greater augmentation of PTH levels in those with renal insufficiency. These findings could be interpreted to indicate that the skeleton is resistant to the calcemic actions of PTH (82). In experimental acute renal failure, it has been shown that treatm e n t with calcitriol results in the partial correction of the blunted calcemic response to PTH (83). In studies in isolated perfused bone from uremic animals, it has been shown that there is a blunted release of cyclic adenosine m o n o p h o s p h a t e (cAMP) in response to PTH. However, if parathyroidectomy is p e r f o r m e d prior to the perfusion of isolated bone, the cAMP response to PTH is restored to normal (84,85). These studies indicate the possibility that desensitization or down-regulation of the PTH receptor-adenylate cyclase system in bone, as a result of high levels of circulating PTH, leads to a resistance to further infusions of PTH. Further studies in vivo by Galceran et al. (86) have d e m o n s t r a t e d that parathyroidectomy prior to the study of the calcemic response to PTH results in a normalization of the response, whereas, when parathyroid glands were intact in the animal, a blunted response to PTH was observed. These observations provide further support for the concept of desensitization of the PTH receptor-effector mechanisms and minimize a direct role for calcitriol, because the levels of calcitriol remain low in both groups of animals. Similar results were obtained in uremic rats by Rodriguez et al. (87). An additional m e c h a n i s m for skeletal resistance has been suggested from studies in organ culture in which high levels of m e d i u m phosphorus result in blunted release of calcium on bone in response to PTH (88). Additional observations in rats have also shown that hyperphosphaternia may play an i m p o r t a n t role in skeletal resistance (89,90). These abnormalities at the level of bone can, therefore, contribute to the pathogenesis of hyperparathyroidism in uremia.

R O L E OF A L U M I N U M O N PARATHYROID FUNCTION As is discussed above, central to the control of hyperparathyroidism is the control of serum phosphorus. This was initially accomplished by the use of aluminumcontaining antacids in order to bind dietary phosphorus and prevent its absorption. T h o u g h it was initially believed that such t r e a t m e n t was safe, following the demonstration of a l u m i n u m toxicity in patients with end-stage renal disease as a result of high a l u m i n u m content of the dialysis water (91), the issue of alum i n u m accumulation as a result of the ingestion of aluminum-containing antacids was reevaluated and was found to result in significant toxicity (92,93).

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Characteristics of the syndrome of aluminum toxicity are related mainly to the skeleton and include bone pain, easy fracturability, hypercalcemia (especially if vitamin D analogs are administered), relatively low levels of PTH in serum, and a characteristic appearance of bone biopsy (see Chapter 40). Special staining of the bone biopsy may show aluminum deposition in bone, often localized at the mineralization front (94). The low levels of PTH in blood can be explained by the observation that aluminum can accumulate in the parathyroid gland (95) and directly decrease PTH secretion in vitro (96). Because it has been shown that cessation of aluminum ingestion is associated with a gradual reduction in the levels of plasma aluminum in patients with end-stage renal disease, it is now recomm e n d e d that aluminum-containing antacids should be avoided if possible, that aluminum levels be carefully monitored, and that phosphate binding be achieved with calcium salts, and thus aluminum toxicity has become a diminishing clinical problem (97).

SUMMARY The pathogenesis of hyperparathyroidism in chronic renal disease is multifactorial. The major factors involved are the retention of phosphorus and the decreased levels of calcitriol, which set in motion a series of events resulting in a state of disordered calcium-regulated PTH secretion. Though each of these factors may contribute to the development of hyperparathyroidism in a variety of ways, the relative role of each of these mechanisms in the development of hyperparathyroidism may vary from patient to patient. It may also vary as a result of the particular nature of the renal disease (tubulointerstitial or glomerular/vascular), overall vitamin D status of the patient, and individual dietary habits with respect to the intake of calcium, phosphorus, and protein. The elucidation of the pathogenetic factors involved in the genesis of secondary hyperparathyroidism in renal failure provides the basis for a rational therapeutic approach to its treatment.

REFERENCES 1. Albright F, Drake TG, Sulkowitch HW. Renal osteitis fibrosa cystica: Report of case with discussion of metabolic aspects. Johns Hopkins MedJ 1937;60:377. 2. Follis RHJ, Jackson DA. Renal osteomalacia and osteitis fibrosa in adults. Johns Hopkins MedJ 1943;72:232. 3. Pappenheimer AM, Wilens SL. Enlargement of the parathyroid glands in r e n a l disease. Am J Patho11935; 11:73. 4. Pappenheimer AM. Effect of an experimental reduction of kidney substance upon parathyroid glands and skeletal tissue. JExp Med 1936;64:965.

5. Reiss E, Canterbury JM, Kanter A. Circulating parathyroid hormone concentration in chronic renal insufficiency. Arch Intern Med 1969;124(4):417-422. 6. Arnaud CD. Hyperparathyroidism and renal failure. Kidney Int 1973;4:89-95. 7. Llach F, Massry SG, Singer FR, Kurokawa K, Kaye JH, Coburn JW. Skeletal resistance to endogenous parathyroid hormone in pateints with early renal failure. A possible cause for secondary hyperparathyroidism. J Clin Endocrinol Metab 1975;41:339-345. 8. Slatopolsky E, Caglar S, Pennell JP, Taggart DD, Canterbury JM, Reiss E, et al. On the pathogenesis of hyperparathyroidism in chronic experimental renal insufficiency in the dog. J Clin Invest 1971 ;50:492-499. 9. Slatopolsky E, Caglar S, Gradowska L, Canterbury J, Reiss E, Bricker NS. On the prevention of secondary hyperparathyroidism in experimental chronic renal disease using "proportional reduction" of dietary phosphorus intake. Kidney Int 1972;2:147-151. 10. Slatopolsky E, Bricker NS. The role of phosphorus restriction in the prevention of secondary hyperparathyroidism in chronic renal disease. Kidney Int 1973;4:141-145. 11. Slatopolsky E, Delmez J. Alteration of mineral metabolism in chronic renal failure. Transplant Proc 1991 ;23:1816-1817. 12. Slatopolsky E, Delmez JA. Pathogenesis of secondary hyperparathyroidism. A m J Kidney Dis 1994;23:229-236. 13. Slatopolsky E, Finch J, Denda M, Ritter C, Zhong M, Dusso A, et al. Phosphorus restriction prevents parathyroid gland growth. High phosphorus directly stimulates PTH secretion in vitro. J Clin Invest 1996;97:2534-2540. 14. Laflamme GH, Jowsey J. Bone and soft tissue changes with oral phosphate supplements. J Clin Invest 1972;51:2834-2840. 15. JowseyJ, Reiss E, CanterburyJM. Long-term effects of high phosphate intake on parathyroid hormone levels and bone metabolism. Acta Orthop Scand 1974;45:801-808. 16. Rutherford WE, Bordier P, Marie P, Hruska K, Harter H, Greenwalt A, et al. Phosphate control and 25-hydroxycholecalciferol administration in preventing experimental renal osteodystrophy in the dog. J Clin Invest 1977;60:332-341. 17. Llach F, Massry SG. On the mechanism of secondary hyperparathyroidism in moderate renal insufficiency. J Clin Endocrinol Metab 1985;61:601-606. 18. Reiss E, Canterbury JM, Bercovitz MA, Kaplan EL. The role of phosphate in the secretion of parathyroid hormone in man. J Clin Invest 1970;49:2146-2149. 19. Portale AA, Booth BE, Halloran BE Morris RCJ. Effect of dietary phosphorus on circulating concentrations of 1,25-dihydroxyvitamin D and immunoreactive parathyroid hormone in children with moderate renal insufficiency. J Clin Invest 1984;73:1580-1589. 20. Wilson L, Felsenfeld A, Drezner MK, Llach E Altered divalent ion metabolism in early renal failure: Role of 1,25(OH)2D. Kidney Int 1985;27:565-573. 21. Martinez I, Saracho R, Montenegro J, Llach E The importance of dietary calcium and phosphorous in the secondary hyperparathyroidism of patients with early renal failure. AmJKidney Dis 1997;29:496-502. 22. Lopez-Hilker S, Galceran T, Chan YL, Rapp N, Martin KJ, Slatopolsky E. Hypocalcemia may not be essential for the development of secondary hyperparathyroidism in chronic renal failure. J Clin Invest 1986;78:1097-1102. 23. Tanaka Y, Deluca HE The control of 25-hydroxyvitamin D metabolism by inorganic phosphorus. Arch Biochem Biophys 1973;154: 566-574. 24. Lopez-Hilker S, Dusso AS, Rapp NS, Martin KJ, Slatopolsky E. Phosphorus restriction reverses hyperparathyroidism in uremia independent of changes in calcium and calcitriol. Am J Physiol 1990;259(3 Part 2):F432-F437. 25. Almaden Y, Canalejo A, Hernandez A, Ballesteros E, GarciaNavarro S, Torres A, et al. Direct effect of phosphorus on PTH

PARATHYROIDS IN RENAL DISEASE secretion from whole rat parathyroid glands in vitro. JBone Miner Res 1996;11:970-976. 26. Almaden Y, Hernandez A, Torregrosa V, Canalejo A, Sabate L, Fernandez Cruz L, et al. High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro.JAm Soc Nephro11998;9:1845-1852. 27. Fournier AE, Arnaud CD, Johnson WJ, Taylor WF, Goldsmith RS. Etiology of hyperparathyroidism and bone disease during chronic hemodialysis. II. Factors affecting serum immunoreactive parathyroid hormone. J Clin Invest 1971;50:599-605. 28. Indridason OS, Pieper CE Quarles LD. Predictors of short-term changes in serum intact parathyroid hormone levels in hemodialysis patients: Role of phosphorus, calcium, and gender. J Clin Endocrinol Metab 1998;83:3860-3866. 29. Kilav R, Silver J, Naveh-Many T. Parathyroid hormone gene expression in hypophosphatemic rats. J Clin Invest 1995;96:327-333. 30. Moallem E, Kilav R, Silver J, Naveh-Many T. RNA-Protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate. J Biol Chem 1998; 273:5253-5259. 31. Naveh-Many T, Rahamimov R, Livni N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate, and vitamin D. J Clin Invest 1995;96:1786-1793. 32. Y1H, Fukagawa M, Yamato H, Kumagai M, Watanabe T, Kurokawa K. Prevention of enhanced parathyroid hormone secretion, synthesis and hyperplasia by mild dietary phosphorus restriction in early chronic renal failure in rats: Possible direct role of phosphorus. Nephron 1995;70:242-248. 33. Denda M, Finch J, Slatopolsky E. Phosphorus accelerates the development of parathyroid hyperplasia and secondary hyperparathyroidism in rats with renal failure. Am J Kidney Dis 1996;28:596-602. 34. Dusso A, Naumovich L, Pavlopoulos T, Finch J, Morrissey J, Slapopolsky E. Phosphorus regulation of parathyroid cell growth in renal failure: A role for the cyclin-dependent kinase inhibitor p21. J A m Soc Nephro11998;9:564A. 35. Dusso A, Lu Y, Pavlopoulos T, Slatopolsky E. A role for enhanced expression of transforming growth factor alpha (TGF-~) in the mitogenic effects of high phosphorus on parathyroid cell growth. J A m Soc Nephro11999;10(617A). 36. Tatsumi S, Segawa H, Morita K, Haga H, Kouda T, Yamamoto H, et al. Molecular cloning and hormonal regulation of PiT-l, a sodium-dependent phosphate cotransporter from rat parathyroid glands. Endocrinology 1998; 139:1692-1699. 37. Mason RS, Lissner D, Wilkinson M, Posen S. Vitamin D metabolites and their relationship to azotaemic osteodystrophy. Clin Endocrinol 1980;13:375-385. 38. Christiansen C, Christensen MS, Melsen F, Rodbro P, DeLuca HE Mineral metabolism in chronic renal failure with special reference to serum concentrations of 1.25(OH)2D and 24.25(OH)2D. Clin Nephro11981;15:18-22. 39. Juttmann JR, Buurman cJ, De Kam E, Visser TJ, Birkenh/iger JC. Serum concentrations of metabolites of vitamin D in patients with chronic renal failure (CRF). Consequences for the treatment with 1-alpha-hydroxy-derivatives. Clin Endocrinol 1981;14: 225-236. 40. Tessitore N, Venturi A, Adami S, Roncari C, Rugiu C, Corgnati A, et al. Relationship between serum vitamin D metabolites and dietary intake of phosphate in patients with early renal failure. Miner Electrolyte Metab 1987;13:38-44. 41. Gafter U, Kraut JA, Lee DB, Silis V, Walling MW, Kurokawa K, et al. Effect of metabolic acidosis in intestinal absorption of calcium and phosphorus. Am J Physio11980;239:G480-G484. 42. Bushinsky DA, Favus MJ, Schneider AB, Sen PK, Sherwood LM, Coe FL. Effects of metabolic acidosis on PTH and 1,25(OH)2D 3 response to low calcium diet. AmJPhysio11982;243:F570-F575.

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43. Kraut JA, Gordon EM, Ransom JC, Horst R, Slatopolsky E, Coburn JW, et al. Effect of chronic metabolic acidosis on vitamin D metabolism in humans. Kidney Int 1983;24:644-648. 44. Bushinsky DA, Riera GS, Favus MJ, Coe FL. Response of serum 1,25(OH)2D 3 to variation of ionized calcium during chronic acidosis. AmJPhysio11985;249(3 Part 2):F361-F365. 45. Langman CB, Bushinsky DA, Favus MJ, Coe FL. Ca and P regulation of 1,25(OH)zD 3 synthesis by vitamin D-replete rat tubules during acidosis. AmJPhysio11986;251 (5 Part 2):F911-F918. 46. Krapf R, Vetsch R, Vetsch W, Hulter HN. Chronic metabolic acidosis increases the serum concentration of 1,25-dihydroxyvitamin D in humans by stimulating its production rate. Critical role of acidosisinduced renal hypophosphatemia. J Clin Invest 1992;90:2456-2463. 47. Hsu CH, Patel S. Uremic plasma contains factors inhibiting 1 alpha-hydroxylase activity. J Am Soc Nephro11992;3:947-952. 48. Nykjaer A, Dragun D, Walther D, Vorum H, Jacobsen C, Herz J, et al. An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D 3. Cell 1999;96:507-515. 49. Bellorin-Font E, Martin KJ, Freitag JJ, Anderson C, Sicard G, Slatopolsky E, et al. Altered adenylate cyclase kinetics in hyperfunctioning human parathyroid glands. J Clin Endocrinol Metab 1981;52:499-507. 50. Brown EM, Brennan ME Hurwitz S, Windeck R, Marx SJ, Spiegel AM, et al. Dispersed cells prepared from human parathyroid glands: Distinct calcium sensitivity of adenomas vs. primary hyperplasia. J Clin Endocrinol Metab 1978;46:267-275. 51. Brown EM, Wilson RE, Eastman RC, Pallotta J, Marynick SE Abnormal regulation of parathyroid hormone release by calcium in secondary hyperparathyroidism due to chronic renal failure. J Clin Endocrinol Metab 1982;54:172-179. 52. Brumbaugh PE Hughes MR, Haussler MR. Cytoplasmic and nuclear binding components for 1alpha25-dihydroxyvitamin D~ in chick parathyroid glands. Proc Natl Acad Sci USA 1975;72:4871-4875. 53. Chertow BS, Baylink DJ, Wergedal JE, Su MH, Norman AW. Decrease in serum immunoreactive parathyroid hormone in rats and in parathyroid hormone secretion in vitro by 1,25-dihydroxycholecalciferol. J Clin Invest 1975;56:668-678. 54. Golden P, Greenwalt A, Martin K, Bellorin-Font E, Mazey R, Klahr S, et al. Lack of a direct effect of 1,25-dihydroxycholecalciferol on parathyroid hormone secretion by normal bovine parathyroid glands. Endocrinology 1980;107:602-607. 55. Cantley LK, Russell J, Lettieri D, Sherwood LM. 1,25Dihydroxyvitamin D 3 suppresses parathyroid hormone secretion from bovine parathyroid cells in tissue culture. Endocrinology 1985;117:2114-2119. 56. Chan YL, McKay C, Dye E, Slatopolsky E. The effect of 1,25-dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells. Calcif Tissue Int 1986;38:27-32. 57. Silver J, Russell J, Sherwood LM. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA 1985;82:4270-4273. 58. Russell J, Lettieri D, Sherwood LM. Suppression by 1,25(OH)zD ~ of transcription of the pre-proparathyroid hormone gene. Endocrinology 1986;119:2864-2866. 59. Silver J, Naveh-Many T, Mayer H, Schmelzer HJ, Popovtzer MM. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat.J Clin Invest 1986;78:1296-1301. 60. Okazaki T, Igarashi T, Kronenberg HM. 5'-Flanking region of the parathyroid hormone gene mediates negative regulation by 1,25-(OH)2 vitamin D 3. JBiol Chem 1988;263:2203-2208. 61. Demay MB, Kiernan MS, DeLuca HE Kronenberg HM. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 1992;89:8097-8101.

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62. Mackey SL, Heymont JL, Kronenberg HM, Demay MB. Vitamin D receptor binding to the negative human parathyroid hormone vitamin D response element does not require the retinoid x receptor. Mol Endocrino11996; 10:298-305. 63. Korkor AB. Reduced binding of [SH] 1,25-dihydroxyvitamin D 3 in the parathyroid glands of patients with renal failure. NEnglJMed 1987;316:1573-1577. 64. Merke J, Hfigel U, Zlotkowski A, Szab6 A, Bommer J, Mall G, et al. Diminished parathyroid 1,25(OH)zD 3 receptors in experimental uremia. Kidney Int 1987;32:350-353. 65. Brown AJ, Dusso A, Lopez-Hilker S, Lewis-Finch J, Grooms P, Slatopolsky E. 1,25-(OH)zD receptors are decreased in parathyroid glands from chronically uremic dogs. Kidney Int 1989;35: 19-23. 66. Naveh-Many T, SilverJ. Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat. J Clin Invest 1990;86:1313-1319. 67. Kremer R, Bolivar I, Goltzman D, Hendy GN. Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinology 1989;125:935-941. 68. Li YC, Amling M, Pirro AE, Priemel M, Meuse J, Baron R, et al. Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets, and osteomalacia, but not alopecia in vitamin D receptor-ablated mice. Endocrinology 1998;139:4391-4396. 69. Fukuda N, Tanaka H, Tominaga Y, Fukagawa M, Kurokawa K, Seino Y. Decreased 1,25-dihydroxyvitamin D 3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest 1993;92:1436-1443. 70. Arnold A, Brown ME Urena P, Gaz RD, Sarfati E, Drueke TB. Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia. J Clin Invest 1995;95:2047-2053. 71. Gogusev J, Duchambon P, Hory B, Giovannini M, Goureau Y, Sarfati E, et al. Depressed expression of calcium receptor in parathyroid gland tissue of patients with hyperparathyroidism. Kidney Int 1997;51:328-336. 72. Kifor O, Moore FD, Jr, Wang P, Goldstein M, Vassilev P, Kifor I, et al. Reduced immunostaining for the extracellular CaZ+-sensing receptor in primary and uremic secondary hyperparathyroidism [see comments]. J Clin Endocrinol Metab 1996;81:1598-1606. 73. Ritter CS, Finch JL, Slatopolsky E, Brown AJ. The decrease in the calcium receptor in parathyroid glands of uremic rats is an early event in the development of secondary hyperparathyroidism. J Am Soc Nephro11999;10:623A. 74. Wada M, Furuya Y, Sakiyama J, Kobayashi N, Miyata S, Ishii H, et al. The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. Control of parathyroid cell growth via a calcium receptor. J Clin Invest 1997;100:2977-2983. 75. Brown AJ, Ritter CS, Finch JL, Slatopolsky EA. Decreased calcium-sensing receptor expression in hyperplastic parathyroid glands of uremic rats: Role of dietary phosphate. Kidney Int 1999;55:1284-1292. 76. Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin KJ. Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxy-cholecalciferol in uremic patients. J Clin Invest 1984;74:2136-2143. 77. MacDonald PN, Ritter C, Brown AJ, Slatopolsky E. Retinoic acid suppresses parathyroid hormone (PTH) secretion and PreproPTH mRNA levels in bovine parathyroid cell culture. J Clin Invest 1994;93: 725-730.

78. Martin KJ, Gonzfilez EA, Gellens M, Hamm LL, Abboud H, Lindberg J. 19-Nor-1-0L-25-Dihydroxyvitamin O 2 (Paricalcitol) safely and effectively reduces the levels of intact PTH in patients on hemodialysis. J A m Soc Nephrol 1998; 10:1427-1432. 79. Patel SR, Ke HQ, Vanholder R, Koenig RJ, Hsu CH. Inhibition of calcitriol receptor binding to vitamin D response elements by uremic toxins. J Clin Invest 1995;96:50-59. 80. Evanson JM. The response to the infusion of parathyroid extract in hypocalcaemic states. Clin Sci 1966;31:63-75. 81. Massry SG, Coburn JW, Lee DB, Jowsey J, Kleeman CR. Skeletal resistance to parathyroid hormone in renal failure. Studies in 105 human subjects. Ann Intern Med 1973;78:357-364. 82. Somerville PJ, Kaye M. Resistance to parathyroid hormone in renal failure: Role of vitamin D metabolites. Kidney Int 1978;14:245-254. 83. Massry SG, Stein R, Garty J, Arieff AI, Coburn JW, Norman AW, et al. Skeletal resistance to the calcemic action of parathyroid hormone in uremia: Role of 1,25 (OH)2 D~. Kidney Int 1976; 9:467-474. 84. Olgaard K, Schwartz J, Finco D, Arbelaez M, Korkor A, Martin K, et al. Extraction of parathyroid hormone and release of adenosine 3',5'-monophosphate by isolated perfused bones obtained from dogs with acute uremia. Endocrinology 1982;111: 1678-1682. 85. Olgaard K, Arbelaez M, Schwartz J, Klahr S, Slatopolsky E. Abnormal skeletal response to parathyroid hormone in dogs with chronic uremia. Calcif Tissue Int 1982;34:403-407. 86. Galceran T, Martin KJ, Morrissey JJ, Slatopolsky E. Role of 1,25dihydroxyvitamin D on the skeletal resistance to parathyroid hormone. Kidney Int 1987;32:801-807. 87. Rodriguez M, Felsenfeld AJ, Llach E Calcemic response to parathyroid hormone in renal failure: Role of calcitriol and the effect of parathyroidectomy. Kidney Int 1991;40:1063-1068. 88. Raisz LG, Niemann I. Effect of phosphate, calcium and magnesium on bone resorption and hormonal responses in tissue culture. Endocrinology 1969;85:446-452. 89. Somerville PJ, Kaye M. Evidence that resistance to the calcemic action of parathyroid hormone in rats with acute uremia is caused by phosphate retention. Kidney Int 1979;16:552-560. 90. Rodriguez M, Martin-Malo A, Martinez ME, Torres A, Felsenfeld AJ, Llach E Calcemic response to parathyroid hormone in renal failure: Role of phosphorus and its effect on calcitriol. Kidney Int 1991 ;40:1055-1062. 91. Platts MM, Goode GC, Hislop JS. Composition of the domestic water supply and the incidence of fractures and encephalopathy in patients on home dialysis. Br MedJ 1977;2:657-660. 92. Kaehny WD, Hegg AP, Alfrey AC. Gastrointestinal absorption of aluminum from aluminum-containing antacids. N Engl J Med 1977;296:1389-1390. 93. Ott SM, Maloney NA, Coburn JW, Alfrey AC, Sherrard DJ. The prevalence of bone aluminum deposition in renal osteodystrophy and its relation to the response to calcitriol therapy. NEnglJMed 1982;307:709-713. 94. Sherrard DJ, Andress DL. Aluminum-related osteodystrophy. Adv Intern Med 1989;34:307-323. 95. (;ann CE, Prussin SG, Gordan GS. Aluminum uptake by the parathyroid glands. J Clin Endocrinol Metab 1979;49:543-545. 96. Morrissey J, Slatopolsky E. Effect of aluminum on parathyroid hormone secretion. Kidney Int Supp11986;18:$41-$44. 97. Gonzfilez E, Martin K. Aluminum and renal osteodystrophy: A diminishing clinical problem. Trends Endocrinol Metab 1992;3:371-375.

CHAPTER 40

Renal B o n e Diseases Clinical Features, Diagnosis, and Management

JACK W. COBURN AND ISIDRO B. SALUSKY Departments of Medicine and Pediatrics, UCLA School of Medicine, and Nephrology Section, West Los Angeles Veterans Affairs Medical Center, Los Angeles, California 90095

INTRODUCTION

even to subnormal bone turnover, adynamic bone, the latter condition arising, in part, from "oversuppressed" parathyroid glands.

The prevention and management of secondary hyperparathyroidism are major challenges to physicians who care for patients with renal failure, particularly insofar as the lives of these patients are extended with improved conservative management and various modes of dialysis. The development of secondary hyperparathyroidism is ubiquitous in patients with advanced renal failure (1), and it was once believed that its occurrence was almost inevitable as renal function decreased (2). However, findings in a crosssectional study of bone biopsies from 249 Canadian dialysis patients suggested that there had been a major change in the distribution of the types of bone diseases that were encountered during the 3-4 years prior to the study in dialysis patients compared to the distribution 10-15 years previously (3). This chapter considers the types of bone disorders that occur in patients with renal failure, the various syndromes and clinical features encountered, the methods for recognition and diagnosis of these disorders, and the clinical management of uremic secondary hyperparathyroidism. (Pathophysiologic mechanisms are covered in Chapter 39.) It must be emphasized that the diagnosis of secondary hyperparathyroidism differs from the recognition of primary hyperparathyroidism. Thus there is a qualitative difference between patients with primary hyperparathyroidism compared to normal individuals, whereas there is a continuum in renal insufficiency, with the condition varying from profound secondary hyperparathyroidism to "mild" disease and The Parathyroids, Second Edition

CLASSIFICATION OF R E N A L B O N E DISEASES The renal bone diseases are classified based on bone biopsy features, including the bone formation rate as measured with double tetracycline labeling and the surface staining with aluminum. The disorders can be divided, from a pathogenic standpoint, into a broad group with increased bone turnover, compared to those with a subnormal rate of bone turnover (4). The groups with high turnover include patients with osteitis fibrosa, "mild, hyperparathyroid disease, and mixed bone disease. The other major type of disorder includes patients with low rates of bone turnover. These include osteomalacia and "aplastic" bone. Nearly all symptomatic renal patients with these types of lesions used to be secondary to aluminum intoxication and were identified by significant surface-stainable aluminum (5). However, the prevalence of aluminum-related bone disease has markedly diminished due to the widespread use of the different calcium-containing phosphate binding agents (3). At present, the aplastic form of renal osteodystrophy, which lacks significant aluminum staining (also called aplastic bone or adynamic bone), is very common in patients treated with maintenance dialysis and those with advanced renal failure (3,6). The differentiation between osteomalacia and "aplastic" bone is determined by the presence of widened or increased 635

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osteoid in the former. Mixed bone disease exhibits markedly increased osteoid plus peritrabecular fibrosis, features of both osteitis fibrosa and osteomalacia.

M A N I F E S T A T I O N S AND

SYNDROMES OBSERVED The symptoms and signs of severe secondary hyperparathyroidism and the other forms of renal osteodystrophy are usually nonspecific, and the laboratory and radiographic abnormalities generally antedate the appearance of any clinical manifestations. It must be emphasized that most renal patients with immunoreactive parathyroid hormone (IPTH) levels and bone biopsies that show severe osteitis fibrosa have no symptoms that can be clearly attributed to overactivity of the parathyroid glands. In adult patients with adynamic bone a higher prevalence of vertebral fractures has been described (7), and more severe growth retardation was found in pediatric patients that developed such lesions after intermittent calcitriol therapy (8). Some specific symptoms and certain syndrome complexes occur in dialysis patients; these are described below.

Bone and/or Joint Pain Bone pain is a common manifestation of severe bone disease in patients with advanced renal failure. It is usually insidious in appearance and is often aggravated by weight bearing or a change in posture. Physical findings are often absent. Pain is most common in the lower back, hips, and legs, but it may occur in the peripheral skeleton. Deep, generalized bone pain is more common and often much worse in patients with aluminumrelated bone disease than in those with osteitis fibrosa; the pain is perceived by the patient as being more deeply seated than in joints or muscles (9-11). On rare occasions, acute, severe, localized bone pain can develop in patients with marked secondary hyperparathyroidism. The sudden appearance of pain around the knee, ankle, or heel can suggest an acute arthritis; such pain is usually not relieved by massage or local heat. Radiographs may or may not show localized subperiosteal erosions; a bone scan may show increased uptake in an area corresponding to the symptoms. The disappearance of the pain after PTH levels diminish with calcitriol therapy or following parathyroidectomy is the feature indicating that the pain arose from secondary hyperparathyroidism. Acute periarthritis that is associated with periarticular calcium deposition and crystals characteristic of hydroxypapatite was not u n c o m m o n in the past in dialysis patients with marked hyperphosphatemia and a significantly elevated Ca × P product in serum

(Ca × P product >75 mg2/dl 2 for prolonged periods). This condition is manifested by acute pain, redness, and swelling, leading to a suspicion of gout or pseudogout. The discomfort often responds to therapy with nonsteroidal antiinflammatory drugs, and it usually disappears completely after parathyroidectomy (4). This problem is almost never seen in dialysis patients with reasonable control of serum phosphorus levels. In long-term dialysis patients, severe arthralgias develop in association with the disposition of a unique amyloid made up of [32-microglobulin; this type of amyloid is characteristically deposited in articular and periarticular structures (12). The joint pain may be asymmetrical and the joints most commonly affected are the shoulders, knees, wrists, and small joints of the hand. The symptoms are typically worse at night or after a period of inactivity, and the pain is reduced or relieved by moving about (13,14). The presence of such pain in association with thin-walled bone cysts, which are erroneously described as brown tumors, may lead to an erroneous diagnosis of secondary hyperparathyroidism. Other syndromes associated with dialysis amyloidosis are described below.

Proximal Myopathy Muscle weakness can be marked in patients with advanced renal failure. Symptoms appear so gradually that they are often ignored until the patient is limited to crutches or a wheelchair. The gait is commonly abnormal, so that the patient waddles from side to side with a typical "penguin gait." Patients note difficulty in climbing stairs or rising from a low chair, and they can have difficulty in holding their arms elevated to comb their hair. This proximal muscle weakness resembles that observed with nutritional vitamin D deficiency and that reported in primary hyperparathyroidism. Plasma levels of muscle enzymes are usually normal, and electromyographic changes are nonspecific. The pathogenesis of such myopathy is not known; the mechanisms implicated include secondary hyperparathyroidism (15), phosphate depletion (16), abnormal vitamin D metabolism (17), and aluminum intoxication (18). Improvement in the gait posture has been observed in azotemic children after treatment with 1,25-dihydroxyvitamin D 3 [1,25(OH)zD 3] (19), and muscle weakness can improve rapidly after this treatment in affected adults (20). The expression of vitamin D receptors in muscle may account for these findings (21). Improvement in proximal muscular strength has occurred also after treatment with 25hydroxyvitamin D [25 (OH)D 3] following subtotal parathyroidectomy, successful renal transplantation, and after chelation therapy of aluminum toxicity using desferrioxamine (18). The favorable response of

RENAL BONE DISEASES / some uremic patients to treatment with 25(OH)D~ or 1,25(OH)zD ~ suggests that a therapeutic trial with an active vitamin D sterol is warranted in uremic patients with myopathy. Pruritus Severe itching, a common symptom in patients with advanced renal failure, sometimes improves or disappears when regular dialysis is initiated. Pruritus is particularly common in dialysis patients with severe or overt secondary hyperparathyroidism; moreover, this symptom often improves substantially or even vanishes within a few days after total or subtotal parathyroidectomy (22). The mechanism whereby secondary hyperparathyroidism leads to pruritus is unclear. Unfortunately, this symptom is not at all specific, and many dialysis patients have troublesome pruritus but lack significant secondary hyperparathyroidism. Parathyroid surgery should never be done for pruritus unless there is objective evidence of secondary hyperparathyroidism (see below, Diagnosis of Secondary

Hyperparathyroidism) .

Spontaneous Tendon Rupture Spontaneous tendon rupture occurs in patients with advanced renal failure, and this occurrence is almost always associated with evidence of marked secondary hyperparathyroidism (4). The mechanism for tendon rupture is uncertain; it has been postulated to arise due to alterations in collagen metabolism affecting the structure of tendons or secondary to microfractures in bone at the site of the tendon insertion (4). When a patient with end-stage renal disease (ESRD) has a tendon rupture, there should be a careful search for features of secondary hyperparathyroidism, and aggressive therapy should be directed to correct the overactive parathyroid glands.

Deformities of Bone Bone deformities are particularly common in uremic patients with severe aluminum toxicity, but they can also occur as a consequence of secondary hyperparathyroidism. Bone deformities are common in children with renal failure and secondary hyperparathyroidism, almost certainly related to the high rates of bone growth, modeling, and remodeling that exist in the immature skeleton; both the axial and the appendicular skeletons are involved in children. In adults, bony deformities arise due to fractures or to remodeling, with the axial skeleton most commonly affected (23). Thus rib deformities and kyphoscotiosis commonly produce a "funnel chest" abnormality.

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Enlargement of the distal tufts of the fingers due to osteitis fibrosa produces an appearance that is appropriately termed pseudoclubbing. In children younger than 10 years of age, bowing of long bones, genu valgum, and ulnar deviation of the wrist are frequent. Slipped epiphyses occur most commonly in the preadolescent period and are particularly common in patients with long-standing congenital renal disease (24). The radiographic features of a "ricketslike" lesion observed in uremic children include histologic features of osteitis fibrosa and secondary hyperparathyroidism rather than vitamin D deficiency or osteomalacia (25). Calciphylaxis This is an unusual syndrome that occasionally develops in patients with advanced renal failure, those treated with regular dialysis, and those with functioning kidney transplants; it is characterized by spontaneous ischemic necrosis of the skin, muscles, a n d / o r subcutaneous fat (26). The pathogenesis of this syndrome is unknown. Extensive medial calcification of the arteries is present, but such calcification commonly exists without causing gangrene or ulcerations; thus it is not certain that the vascular calcification, per se, is the cause of the ischemic necrosis. There are two rather distinct types of the syndrome, proximal calciphylaxis, with the thighs, abdomen, and chest wall affected, and the acral variety that involves sites distal to the knees and elbows, e.g., toes, fingers, and ankles (27). The former has a terrible prognosis, with death occurring in more than 80-90% of affected patients; this syndrome may be limited to white patients and morbid obesity is common; also, hypoalbuminemia is observed. With acral or distal calciphylaxis, many patients have severe secondary hyperparathyroidism and the great majority have a history of severe and uncontrolled hyperphosphatemia (28,29). A significant percentage of such patients have shown clinical improvement within a few days after parathyroidectomy (26). However, calciphylaxis has been described in patients with adynamic bone (30). In some patients and in an animal model of this syndrome, glucocorticoid therapy may predispose to the development of the lesion. Patients with calciphylaxis often die from secondary infection. Because of this poor prognosis, urgent parathyroidectomy is often r e c o m m e n d e d in patients with secondary hyperparathyroidism. Patients with diabetes mellitus and renal failure also develop ischemic lesions, medial vascular calcification, and a similar clinical picture. However, the lesions observed in diabetic patients rarely improve following parathyroidectomy, and parathyroid surgery should be reserved for patients with definite evidence of overt secondary hyperparathyroidism.

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Growth Retardation Delayed growth is c o m m o n in children with chronic renal failure. Chronic acidosis, malnutrition, secondary hyperparathyroidism, and low levels of somatomedin are factors suggested to cause this complex problem (31-34). The correction of certain of these abnormalities has been associated with improved growth velocity; however, this does not occur in all cases. Improved or even catch-up growth has been observed in a few children following treatment with calcitriol (35); however, the n u m b e r of patients studied was small, and subsequent reports have not confirmed the original findings (36). Moreover, diminished linear growth has been demonstrated after intermittent calcitriol therapy in children with bone biopsy proved secondary hyperparathyroidism (8). Greatest reductions in height were observed in those patients who developed adynamic bone after intermittent use of calcitriol (8). Overall, such findings suggest that the therapeutic administration of calcitriol can directly affect epiphyseal growth plate chondrocytes. Recombinant growth h o r m o n e has been introduced for the m a n a g e m e n t of the growth retardation found in uremic children, and such reports demonstrated increased growth velocity, particularly in prepubertal patients with stable renal insufficiency (37). The response to growth horm o n e therapy is diminished in patients treated with maintenance dialysis compared to those with stable renal failure (38).

Dialysis Amyloidosis Dialysis-related amyloidosis can lead to certain clinical syndromes that resemble secondary hyperparathyroidism or other types of renal osteodystrophy; moreover, such amyloidosis often exists concurrently with o n e of the metabolic bone conditions. Dialysis amyloidosis arises from the deposition in bone and periarticular structures of a specific type of amyloid made up of [32-microglobulin (39). The frequency of its clinical presentation increases substantially in patients treated with regular dialysis for more than 5-10 years, and it is much more c o m m o n in patients who start dialysis after age 50 years (40). The blood levels of [32-microglobulin are strikingly elevated in all patients with ESRD. The clinical manifestations can include (1) carpal tunnel syndrome, which is the most c o m m o n feature; (2) destructive or erosive arthropathy involving the large and medium-sized joints, with shoulder, knee, hip, or back pain the most c o m m o n manifestations; (3) spondyloarthropathy, most commonly affecting the cervical spine; (4) subchondral, thin-walled cysts of bone, most commonly affecting the carpal bones,

humoral and femoral heads, disc radius, acetabulum, and tibial plateau (Fig. 1); (5) local tendonitis, particularly of the hand; and (6) joint effusions and tendon sheath cysts. The involvement of the tendons of the hand can cause trigger fingers, swan-neck deformities, and camptodactly. The subendochondral cysts probably represent amyloidomas infiltrating the cavity of the bone at a site of a tendon insertion. They are at times confused with brown tumors of secondary hyperparathyroidism. However, their location and multiple occurrences differ from brown tumors, which are usually solitary and occur most commonly in the rib or mandible (41). Nonetheless, one must consider this syndrome as a potential cause of musculoskeletal and periarticular symptoms that are encountered in a longterm dialysis patient. One pitfall to avoid is to recomm e n d parathyroid surgery for a dialysis patient whose musculoskeletal symptoms do not improve after lowering of PTH levels with calcitriol therapy; the symptoms may be due to dialysis-related amyloidosis. A specific diagnosis of the latter can be made from the biopsy demonstration of amyloid made up of [32-microglobulin. However, invasive procedures are rarely indicated to establish the diagnosis, which can generally be made from the clinical presentation (41), the finding of multiple thin-walled cysts on skeletal radiographs, a n d / o r the demonstration of thickened shoulder tendons by ultrasound examination of the shoulders (42). The m a n a g e m e n t of this syndrome is largely unsatisfactory. Successful renal transplantation leads to rapid normalization of plasma levels of [32-microglobulin, the symptoms often disappear (41), and there may be no further

FI6. 1 Thin-walled cysts characteristic of dialysis amyloidosis in the proximal humerus of a long-term dialysis patient.

RENAL BONE DISEASES / progression of the bony cysts on subsequent radiographs (43); however, the histologic features of amyloid may persist.

Destructive Spondyloarthropathy The frequency of reports suggests that destructive spondyloarthropathy is more common in patients with uremia, particularly those undergoing hemodialysis (44). The cervical spine may be more commonly involved. The underlying pathogenic mechanisms include secondary hyperparathyroidism both with (45) and without brown tumors (46), dialysis amyloidosis (43,46), and microcrystalline deposits (hydroxylapatite) (44); in yet other cases, no etiologic diagnosis has been established (J. W. Coburn and I. B. Salusky, personal observations). In many patients the manifestations are merely radiographic, but in others spinal cord compression with severe neurologic sequelae follows. The prognosis for such patients, particularly those who are symptomatic, has not been good, although neurologic improvement has been reported following spinal cord decompression and parathyroidectomy (46). The finding of this clinical syndrome in a dialysis patient should lead to a search for both secondary hyperparathyroidism and dialysis amyloidosis.

Low-Turnover Bone without Aluminum: Idiopathic Aplastic or Adynamic Bone This condition, which is defined above, is a histologic diagnosis from bone biopsy. It is included because it may be related to "oversuppressed" PTH levels and because its presence may impact on the maneuvers used to prevent and treat renal bone disease. The histologic features of adynamic bone without aluminum deposition cannot be distinguished from those of corticosteroid-induced osteoporosis or either age-related or postmenopausal osteoporosis. Several reports have noted the finding of "aplastic" or "adynamic" bone without aluminum staining in dialysis patients (3,6,47-49). They were identified not because of symptoms or biochemical features but because bone biopsies were done in cross-sectional studies of dialysis patients. This lesion has occurred in both children (47) and adults (3,6,48,49). The percentage of patients with this lesion has varied from 10% (48) to 31% (47) and to as high as 48% (3) among those treated with continuous ambulatory peritoneal dialysis (CAPD), and it has been seen in up to 35% of hemodialysis patients (3). They may be more prone to development of hypercalcemia, and serum PTH levels [immunoradiometric assay (IRMA)] are in general below 150 pg/ml, and low levels of bone specific alkaline phosphatase have been described in a large cohort of patients treated with maintenance hemodialysis (50).

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The factors associated with the occurrence of this disorder include the ingestion of large doses of calcium carbonate/calcium acetate as phosphate binders, the use of continuous ambulatory peritoneal dialysis as the dialysis modality, the presence of diabetes mellitus, and the use of dialysate calcium concentrations of 3.0-3.5 mEq/liter. Another factor that may predispose to this lesion is therapy with calcitriol, particularly when serum PTH levels are not significantly elevated (51,52). There has been no difference between IPTH levels in patients with the idiopathic disorder compared to those with the low-turnover state and significant aluminum staining (3), and the dynamics of parathyroid function during acute hypocalcemia and hypercalcemia did not differ (53). On the theory that PTH levels were oversuppressed by the flux of calcium from dialysate, Hercz et al. (54) lowered the calcium concentration in peritoneal dialysate from 3.5 to 2.0 mEq/liter for several months; this was followed by a fourfold rise in the mean intact (IRMA) PTH from 38 to 139 pg/ml, indicating that the high dialysate calcium contributes to the suppressed PTH levels. The long-term consequences of this "disorder," when not attributable to aluminum toxicity, remain to be determined, but Atsumi et al. have demonstrated increased prevalence of vertebral fractures in adult male patients treated with hemodialysis and serum PTH levels consistent with adynamic renal osteodystrophy (7). In addition, impaired linear growth was pronounced in those pediatric patients that developed adynamic bone after therapy with calcitriol (8). Furthermore, the development of soft tissue and vascular calcifications may be enhanced by the repeated episodes of hypercalcemia. Until more is known about the natural history of this condition, it would seem prudent that IPTH levels in dialysis patients should be maintained at > 1.5- to 2.0-fold the upper normal limits (see below).

DIAGNOSIS OF THE RENAL BONE DISEASES Because more than one pathologic process is involved in producing the "renal bone diseases," it is important to identify the type of disorder and its cause. Serum levels of immunoreactive IPTH were initially believed to hold the key to recognizing secondary hyperparathyroidism in patients with renal failure. However, the heterogeneity of IPTH and its fragments in the plasma (55) and the fact that renal failure, per se, leads to retention of certain PTH fragments have led to difficulty with the interpretation of a given IPTH value in recognizing significant or overt secondary hyperparathyroidism. Several studies have reported correlations between serum IPTH levels and various histologic features of secondary hyperparathyroidism

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observed on bone biopsy (3,47,56-58), but most do not provide information on the sensitivity and specificity of an IPTH level for a diagnosis. A l u m i n u m toxicity, which causes bone disease by a different process and requires totally different m a n a g e m e n t (59), must be distinguished from secondary hyperparathyroidism. Three questions must be answered: Does the patient have significant secondary hyperparathyroidism? Is a l u m i n u m toxicity present? Does the patient have features consistent with low bone turnover without aluminum? This chapter considers the procedures and laboratory tests that can assist in the diagnosis of the specific type of renal bone disease present. Clinicians also e n c o u n t e r many dialysis patients with musculoskeletal symptoms that arise from dialysis amyloidosis (13); the diagnosis of this disorder is generally based on clinical features, radiographs, and ultrasonography. There are no laboratory tests that aid in the recognition of this problem. Except for a brief description of its clinical features, this condition is beyond the scope of this review.

Bone Biopsy The evaluation of bone histology provides (1) a diagnosis of the specific type of renal bone disease present, (2) a m e t h o d for understanding the pathophysiology of renal bone disease, and (3) a guide to its p r o p e r management. Currently, bone biopsy procedures can be done on an outpatient basis with minimal morbidity in both adult and pediatric patients (3,47,58). Double tetracycline labeling c o m b i n e d with quantitative histom o r p h o m e t r y permits evaluation of bone formation rate and is the m e t h o d to identify defective mineralization (9,60). For this, tetracycline is given on two occasions, separated by a specific time interval, usually 10-17 days. The width of separation of the two fluorescent bands of tetracycline observed on the biopsy and the length of bone surface showing such labels permit calculation of the bone formation rate (BFR); the finding of subnormal bone turnover is critical in identifying certain disorders of bone. The disorders with high turnover include osteitis fibrosa and mild hyperparathyroidism. The bone in osteitis fibrosa shows increased bone turnover, with increased numbers of osteoblasts and osteoclasts and variable degrees of peritrabecular fibrosis. Such bone shows increased amounts of woven osteoid, with a haphazard a r r a n g e m e n t of collagen fibers, in contrast to the usual lamellar pattern of osteoid in normal bone. Mild hyperparathyroidism has increased formation, but there is minimal or no peritrabecular fibrosis. With these lesions, the major pathogenic factor is increased levels of PTH (4). The other major category of alteration in uremic bone is the state of low bone turnover, with either

osteomalacia or a state of reduced bone formation without osteomalacia. Osteomalacia is characterized by the presence of wide osteoid seams, increased numbers of osteoid lamellae, increased trabecular surface covered with osteoid, and diminished rate of bone formation, as assessed by double tetracycline labeling. Peritrabecular fibrosis is absent or minimal (61). Biopsies that show normal osteoid volume, the absence of fibrosis, and yet a reduced bone formation rate, as measured via double tetracycline label, are classified as aplastic or adynamic bone lesion (47,61). Patients with > 2 5 % surface staining with a l u m i n u m along the bone surfaces and a paucity of osteoblasts and osteoclasts often present all the clinical features of aluminum-related bone disease (61). Patients with the aplastic or adynamic lesion but without significant surface-stainable a l u m i n u m are believed to represent a different process, which is reviewed above. When osteitis fibrosa coexists with osteomalacia, the pattern is called a mixed lesion (61). Mixed lesions, as observed in the past, were often associated with significant hypocalcemia and elevated serum PTH levels; the disorder responded readily to calcitriol therapy (60). Such a lesion is more commonly seen as a transition from osteitis fibrosa to aluminum-related bone disease or the reverse. The reader is referred elsewhere for a more complete description of bone histology and histomorphologic findings in the renal bone diseases (3,58).

SERUM CALCIUM, PHOSPHORUS, AND ALKALINE PHOSPHATASE The m e a s u r e m e n t of serum calcium and phosphorus levels in a large n u m b e r of ESRD patients often reveals differences between groups with different types of bone disease (3,62); however, these levels are of little value to separate individual patients. Significant hypercalcemia is not u n c o m m o n in patients with severe osteitis fibrosa or aluminum-related bone disease (63,64); it is more c o m m o n in those with the lowturnover bone condition without a l u m i n u m (aplastic or adynamic bone) (3,65). The m e a s u r e m e n t of ionized calcium increases the sensitivity to recognize hypercalcemia, particularly in patients undergoing peritoneal dialysis (66), because their serum albumin levels are often below normal and are generally lower than those observed in hemodialysis patients. Serum phosphorus levels are commonly higher in a group of patients with osteitis fibrosa than in those with alum i n u m bone disease (64), but serum phosphorus levels are not discriminating in individual patients. The serum alkaline phosphatase levels are commonly elevated in patients with osteitis fibrosa; normal

RENAL BONE DISEASES / levels are also common, however. A slow progressive rise of alkaline phosphatase, even within the normal range, may indicate worsening secondary hyperparathyroidism. This is because the bone isoenzyme of alkaline phosphatase represents a small fraction of total alkaline phosphatase. Thus the bone isoenzyme of alkaline phosphatase was above normal in 30% of dialysis patients who had normal total alkaline phosphatase levels (67). More recently, Coutteneye et al. found low levels of bone alkaline phosphatase as predictors of adynamic osteodystrophy in European patients treated with hemodialysis (50). Patients with severe aluminumrelated osteomalacia that arose from aluminum-contaminated dialysate were reported to have normal total alkaline phosphatase levels (68), whereas other patients with a similar disorder that developed slowly from the ingestion of aluminum gels often had elevated levels (69). From this, one could conclude that alkaline phosphatase levels are not as specific for separation of osteitis fibrosa from osteomalacia as was once believed. The serial evaluation of alkaline phosphatase levels is of value, however, in following the course of a dialysis patient both over a period of many months and also during specific therapy, e.g., with calcitriol.

Parathyroid Hormone Assays One major goal of internists and endocrinologists who use an immunoassay for PTH is the differential diagnosis of hypercalcemia, separating primary hyperparathyroidism from other causes. The interpretation of results in patients with renal insufficiency is more complex. Biologically active PTH in humans and other mammals with normal renal function circulates predominantly as a 1-84 amino acid peptide. PTH fragments of varying length arise either from metabolism of the intact hormone within the parathyroid glands or in peripheral tissues, such as the liver, where cleavage occurs predominantly between residues 33 and 37 (70,71). The resulting carboxyl-terminal peptides, and larger fragments that lack only a few amino-terminal residues (72,73), are eliminated mainly by glomerular filtration and subsequent tubular degradation, and this process may involve megalin, a multifunctional clearance receptor (74). Excess amounts of PTH fragments are retained in patients with renal failure, and this has made it difficult to interpret PTH measurements obtained with conventional radioimmunoassays, with antisera directed against epitopes within the m i d - o r carboxyl-terminal regions of PTH (72). I m m u n o r a d i o m e t r i c assays that utilize two distinct antibodies directed against carboxyl- and aminoterminal portions of the h o r m o n e thus detecting only longer peptides such as intact PTH, have been widely used for the diagnosis and m a n a g e m e n t of the differ-

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ent renal bone diseases (3,62,75). However, chromatographic studies in patients with end-stage renal disease have demonstrated the presence of a heterogeneous group of carboxyl-terminal PTH fragments, some of which exhibit an elution profile on high-performance liquid chromatography (HPLC) that it is indistinguishable from that of synthetic PTH (7-84). This may therefore overestimate the concentration of biologically active full-length PTH, particularly in patients with ESRD, and provide a potential explanation to the skeletal resistance to the calcemic action of PTH in patients with renal failure (72). The clinician who evaluates a dialysis patient must ask whether the PTH level is high e n o u g h to cause osteitis fibrosa a n d / o r to produce the clinical symptoms of secondary hyperparathyroidism (proximal myopathy, hypercalcemia, etc.) Cohen-Solal et al. (75) used three different PTH assays, "intact" PTH using an IRMA assay, midregion PTH assay, and carboxylterminal PTH assay, in 24 dialysis patients who had never received aluminum gels and whose dialysate alum i n u m levels were very low. The intact PTH showed the best correlation with bone formation rate (r = 0.63) and provided the best separation between groups. Patients with "severe" hyperparathyroidism (increased resorption and high BFR) had intact PTH levels that were 1.2-5 times the u p p e r limit of normal. In separating this group with hyperparathyroidism from a small group of patients with subnormal bone formation rate but no aluminum staining (adynamic or aplastic bone), the intact PTH had a sensitivity of 100% and a specificity of 70%. Quarles et al. (76) reported the relationship between intact PTH using an IRMA and bone biopsy features of secondary hyperparathyroidism in 39 dialysis patients, although data from the biopsies from 17 additional patients with a low BFR, aluminum staining, or both features were excluded from the analysis. The correlation between the intact PTH assay and BFR was high over a very wide range of IPTH levels (r = 0.84), and linear regression analysis was used to evaluate the relation between IPTH and various histologic indices of secondary hyperparathyroidism. In those with an average PTH 2.6 times the u p p e r limit of normal, the BFR was above normal and there was minimal fibrosis; woven osteoid was present when the mean PTH level exceeded the normal limit by 3.2-fold. Histologic findings of severe hyperparathyroidism were invariable when the levels averaged 7.5 times normal. At the other extreme, the patients with PTH levels averaging 1.5-fold the u p p e r normal limit had normal bone formation and resorption surfaces and lacked both peritrabecular fibrosis and woven bone. The results were not reported in a way that indicates the sensitivity or specificity of the PTH assay for the identification of secondary hyperparathyroidism.

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Segre et al. and Sherrard et al. have summarized the IRMA results of intact PTH in relation to histologic findings on bone biopsy in 259 ESRD patients from Toronto (3); these results are shown in Fig 2. In those with a PTH level >7.7-fold, the upper limit of normal (500 p g / m l ) , there was 100% specificity for identification of severe osteitis fibrosa, but the sensitivity was only 46%. In those with PTH lowered to 4.6-fold the upper normal value (300 p g / m l ) , the sensitivity was 85% and the falsepositive rate was 5%. Finally, at a PTH value below 3.1-fold normal (200 p g / m l ) , 99% of those with severe osteitis fibrosa were excluded. Thus, in summary, double-antibody immunoradiometric serum PTH assays are presently utilized to separate patients with secondary hyperparathyroidism from those with adynamic lesions of bone in patients treated with no vitamin D or daily calcitriol therapy (3). In pediatric patients undergoing maintenance dialysis without vitamin D therapy a n d / o r receiving small daily doses of calcitriol, bone evidence of secondary hyperparathyroidism is found when serum PTH levels are above 250-300 pg/ml. In contrast, values below 150 p g / m l are associated with adynamic renal osteodystrophy (62). On the other hand, in patients with stable chronic renal failure and a glomerular filtration rate of 65 m l / m i n u t e / 2 4 hours, mean PTH levels of 160 p g / m l are associated with bone biopsy findings of osteitis fibrosa in adult patients (77). A new immunoradiometric assay for hPTH that uses a radiolabeled detection antibody raised against h P T H ( 1 - 6 ) (S-IRMA), developed by Scantibodies Laboratories, Inc. (Santee, CA) selectively detects h u m a n PTH, and when compared to the IRMA assay,

2000

Intact P T H ( p g / m l )

1500

1000

500

[---J Osteitis Fibrosa Mild

Mixed

High/Normal Turnover

Aplastic '

.

L-

Osteomalacia

Low Turnover

I

FIG. 2 Intact PTH levels (IRMA) in 259 dialysis patients from Toronto with iliac crest bone biopsies and classification of the type of bone disease. The entire ranges of PTH and mean values __ SEM for each group are given. The numbers of patients with each of the disorders are given: osteitis fibrosa and mild, 112; mixed, 18; aplastic, 128; and osteomalacia, 11. (Modified from Ref. 3, by permission of Blackwell Science, Inc.)

values were markedly diminished (78); indeed, the IRMA assay yielded consistently higher values than those obtained by the S-IRMA. This difference may be due to the presence of substantial amounts of large amino-terminally truncated PTH fragments, possibly hPTH(7-84). This discrepancy provided by the two IRMAs may have significant implications for the diagnosis and treatment of the different subtypes of renal bone diseases. Prospective studies are needed to determine whether PTH concentrations determined by SIRMA provide a better prediction of the different types of renal osteodystrophy.

Considerations Regarding Aluminum-Related Bone Disease A lengthy description of aluminum-related bone disease is beyond the scope of this chapter. However, the distinction between hyperparathyroidism and aluminum-related bone disease is not always simple, and there are several reasons why the methods used to make a diagnosis of aluminum toxicity or aluminum loading are included. For example, an aluminumloaded patient with osteitis fibrosa will often convert to symptomatic aluminum-related bone disease after parathyroidectomy. It is more difficult to treat aluminum-related bone disease after parathyroidectomy when serum PTH levels remain very low (79); thus it is generally r e c o m m e n d e d that the aluminum overload be treated before parathyroidectomy is performed in patients who have aluminum overload combined with severe secondary hyperparathyroidism (18,80). Patients with aluminum-related bone disease who u n d e r g o parathyroidectomy often become worse, sometimes markedly so (81), and so the correct diagnosis is important. Sherrard et aL (82) coined the term pseudohyperparathyroidism to describe patients with bone pain, fractures, variable degrees of hypercalcemia, elevated PTH levels (usually with a C-terminal or MM assay), and skeletal radiographs that show subperiosteal erosions. Parathyroidectomy usually led to worsening of the symptoms, and bone biopsies disclosed features of aluminum-related bone disease. The radiographic finding of erosions represents earlier osteoclastic erosions that had not "healed" or mineralized but were filled with unmineralized osteoid as a consequence of the superimposed aluminum loading. Because of the tendency for substantial worsening after parathyroidectomy (80,81), aluminum toxicity should be excluded as a possible diagnosis before parathyroid surgery is undertaken (82). This matter is much less of a problem now that fewer and fewer dialysis patients are receiving aluminum gels for control of hyperphosphatemia. The "gold standard" for recognizing aluminum-related bone disease is a bone biopsy,

RENAL BoNE DISEASES / which must be seriously considered in a patient who has ingested large doses of a l u m i n u m gels in the past. The value of assessing plasma aluminum, the change in plasma a l u m i n u m after desferrioxamine, and the serum IPTH level is briefly considered below. PTH levels are lower in patients with a l u m i n u m toxicity than in a usual dialysis population (64,69), and such levels are of value in identifcation of these patients. Among a study of 40 patients with severe alum i n u m toxicity referred for therapy with desferrioxamine, Norris et al. (69) found PTH levels to average 10-fold the u p p e r limit of normal c o m p a r e d to a m e a n value of 120-fold normal in 21 patients with symptomatic osteitis fibrosa. Plasma a l u m i n u m levels are believed to represent evidence of recent exposure to a l u m i n u m rather than evidence of a l u m i n u m toxicity (83), and the levels will fall over 2-4 months in an aluminum-loaded dialysis patient after all exposure to a l u m i n u m has been withdrawn (84). A combination of the PTH level and the i n c r e m e n t in serum a l u m i n u m after a desferrioxamine infusion has been suggested as a useful method. With use of the IRMA intact PTH assay, Pei et al. (85) used a "cut-off' intact PTH level <3.1-fold the u p p e r normal value (200 p g / m l ) c o m b i n e d with an i n c r e m e n t in plasma a l u m i n u m after a desferrioxamine (DFO) infusion to identify dialysis patients with aluminum-related bone disease a m o n g 259 Canadian dialysis patients who u n d e r w e n t biopsies. The combination of intact PTH <200 p g / m l and an i n c r e m e n t in plasma a l u m i n u m after DFO infusion of 150 Ixg/liter provided the best positive predictive value (95%) in both peritoneal dialysis and hemodialysis patients (85); however, the sensitivity was only 35-45%. The authors evaluated cut-off increments in plasma a l u m i n u m of 100 pog/liter and 50 p g / l i t e r c o m b i n e d with the same limit for PTH level. There was a modest reduction in positive predictive value, but the sensitivity rose substantially. Pei et al. noted that the sensitivity of these methods, as indicated by a change in false-negative rates, was substantially lower in patients who had had a l u m i n u m gels withdrawn for > 6 months c o m p a r e d to patients still ingesting a l u m i n u m gels. They considered the usefulness of these tests u n d e r circumstances when the prevalence of a l u m i n u m loading is reduced, as would occur with decreased use of a l u m i n u m gels (84); u n d e r such circumstances, a bone biopsy would m u c h more likely be required. These authors suggest that these evaluations, done at various intervals, would be of most value in dialysis patients who continue to require a l u m i n u m gels for the control of serum phosphorus. From our experience, it seems likely that monitoring serum a l u m i n u m per se is also useful; as long as serum a l u m i n u m levels remain < 3 0 - 4 0 lxg/liter, it is unlikely that such a patient could develop a l u m i n u m loading and toxicity

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de novo. If the levels rise progressively in a patient, there should be concern about a l u m i n u m loading, and the clinician should explore the possibility of a l u m i n u m loading from a new source, either the breakdown of a water-treatment system or markedly a u g m e n t e d alum i n u m absorption due to the ingestion of citrate (86).

Other Biochemical Tests Several other biochemical tests have been utilized in uremic patients to identify the degree of activity of bone disease; these include osteocalcin or bone Glaprotein (BGP) and insulin-like growth factor-I (IGF-I). In two studies that reported levels of both IPTH and osteocalcin (3,87), the correlations between bone histologic features and either IPTH levels or osteocalcin levels were quite similar. The osteocalcin levels of all uremic patients exceeded normal, most likely due to renal retention of this peptide in a m a n n e r similar to that in the MM PTH assay. A n o t h e r report showed similar correlations between bone formation rate and osteoclast n u m b e r and either PTH levels or levels of IGF-I (II 0).Jehle et al. d e m o n s t r a t e d that IGFBP-5 may have a potential role as a m a r k e r for osteopenia and low bone turnover in patients treated with long-term dialysis (88). However, the lack of general availability of these determinations limits their clinical usefulness. W h e n persistent and u n e x p l a i n e d hypercalcemia develops in a patient with renal failure, the measurem e n t of serum calcitriol [1,25(OH)zD 3] may aid in identification of a granulomatous process, such as sarcoidosis or tuberculosis, as a cause of extrarenal generation of the active vitamin D sterol. U n d e r usual circumstances, the serum calcitriol levels are either undetectable or below the normal range in patients with ESRD; thus the finding of a value that is in the u p p e r range of normal or higher would suggest the existence of such a problem.

Radiologic Evaluation of Secondary Hyperparathyroidism Radiographic Features Subperiosteal erosions are the most consistent radiographic feature of secondary hyperparathyroidism (23,89). The degree of subperiosteal erosions can correlate with serum PTH and alkaline phosphatase levels, but radiographs are quite insensitive and can be normal in patients with histologic features of m a r k e d osteitis fibrosa on bone biopsy (56). A m o n g pediatric patients, metaphyseal changes (i.e., growth zone lesions that are t e r m e d ricketslike lesions) are c o m m o n (23,24). Radiographic features of secondary hyperparathyroidism are best detected by h a n d radiographs, with

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several techniques used to enhance their sensitivity. Meema et al. (90) use fine-grain films and then magnify them six- to sevenfold with a hand lens. Direct magnification X-rays can also be employed. Subperiosteal erosions occur as well in the distal ends of clavicles, at the surface of the ischium and pubis, at the sacroiliac joints, and at the junction of the metaphysis and diaphysis of long bones (91,92). Subperiosteal erosions can also be observed in patients with aluminum-related bone disease (93), so they are not always specific. This occurrence represents the residual manifestations of previous secondary hyperparathyroidism with osteitis fibrosa, as was noted above. Aluminum toxicity prevents remineralization of bone and prevents normalization of the radiograph when secondary hyperparathyroidism is replaced by aluminum loading (93); the term pseudohyperparathyroidism has been used to describe such patients (82). The radiographic abnormalities of the skull in secondary hyperparathyroidism (92) can include (1) a diffuse "ground glass" appearance, (2) a generalized mottled or granular appearance, (3) focal radiolucencies, and (4) focal sclerosis. Among children with renal failure, abnormalities of the growth zone are common. The radiographic changes arising from secondary hyperparathyroidism resemble true rachitic abnormalities. Mehls (94) demonstrated that the histologic features of these epiphyseal lesions in uremic children are those of osteitis fibrosa and noted radiographic features that are distinct from those of rickets due to vitamin D deficiency. Brown tumors do develop in renal patients with secondary hyperparathyroidism, but their occurrence is unusual, even in patients with bone biopsy findings of severe osteitis fibrosa. They are most commonly seen in the jaw or ribs, and they are generally single. The radiographic features of dialysis amyloidosis, noted above, can be misinterpreted as brown tumors. Indeed, serial skeletal radiographs are presently of the most value in both detecting the appearance and following the progress of the cystic lesions of dialysis amyloidosis.

bones leads to a feature that has been termed the superscan. In contrast to this finding, there is generally a diffuse reduction of the uptake in renal patients with aluminum-related bone disease and osteomalacia (97,98). Karsenty et al. (96) were able to differentiate between patients with osteitis fibrosa and those with osteomalacia from aluminum intoxication by the uptake on bone scan. However, patients with mixed lesions on biopsy could not be identified. Hodson et al. (99) concluded from their results that bone scans did not provide e n o u g h useful information on either the type or the severity of renal osteodystrophy in their comparison of scintiscan and results on bone biopsy. The bone scintiscan will also detect nondisplaced fractures (pseudofractures) in the ribs and elsewhere in patients with osteomalacia (23); it is also useful to detect ectopic calcification. Thus the bone scan is a noninvasive diagnostic method that can establish the severity or type of bone disease in some but not all patients. A bone biopsy is still required, however, to establish the specific type of renal bone disease in many cases.

Bone Scintiscan

A major therapeutic goal is to avoid phosphate retention and hyperphosphatemia in renal failure. As is noted in Chapter 39, phosphate retention is a major factor leading to the development of secondary hyperparathyroidism in renal patients (100). Thus, in subjects with mild to moderate renal failure, a reduction in dietary phosphorus was associated with reductions in serum PTH values and improvement in the calcemic response to parathyroid h o r m o n e (101,102). In advanced renal failure, the degree of hyperphosphatemia correlates with the degree of secondary hyperparathyroidism (103), and marked hyperphosphatemia can block the effect of calcitriol to suppress serum IPTH levels (104).

The scintiscan of bone, using technetium-99-1abeled diphosphonate, will detect osteitis fibrosa, and this procedure can be used to estimate the severity of skeletal disease in patients with advanced renal failure (95); also, the response to a specific treatment can be followed by serial bone scans (96). In one study, the scintiscans were abnormal in 13 of 14 dialysis patients, with symmetrically increased uptake over the skull, mandible, sternum, shoulders, vertebrae, and distal aspects of the femur and tibia. This symmetrically increased uptake of the diphosphonate by the axial skeleton and a r o u n d the epiphyseal areas of the long

TREATMENT OF

SECONDARY HYPERPARATHYROIDISM

For the prevention and m a n a g e m e n t of secondary hyperparathyroidism arising with renal failure, the specific goals are (1) to maintain the blood levels of calcium and phosphorus as near to normal as possible, (2) to prevent hyperplasia of the parathyroid glands and to suppress PTH secretion appropriately if secondary hyperparathyroidism is already present, and (3) to avoid the development of extraskeletal calcification. In addition, it is important to prevent or minimize other disorders affecting bone that can develop in advanced renal failure, namely, aluminum toxicity and the idiopathic aplastic or adynamic bone disorder.

Modification of Dietary Phosphorus and Calcium

RENAL BONE DISEASES / Dietary phosphorus is derived in large part from meat and dairy products, and phosphorus intake commonly ranges from 1.0 to 1.8 g / d a y in adults in the United States and Western Europe. To prevent hyperphosphatemia, the dietary intake of phosphorus should be reduced to < 1000 m g / d a y by sharply restricting the intake of dairy products in patients with moderate to advanced renal failure, but further decreases in dietary phosphorus content may compromise nutrition, particularly protein intake (105). In patients with ESRD, one relies on the dialysis procedure to remove a substantial amount of phosphate (106); however, the excess phosphate that accumulates during the interval between two dialysis treatments has a space of distribution well beyond the extracellular fluid. Because of the slow movement of such phosphate into the extracellular fluid, the serum phosphorus levels fall very rapidly during the first 30-60 minutes of a hemodialysis procedure; subsequently, there is a very small gradient for phosphate diffusion from the blood into the dialysis fluid, limiting the net amount of phosphate that is removed by dialysis (107). The ingestion of a highly phosphate-restricted diet (e.g., <600 mg/day) would be very useful; however, such a diet is highly unpalatable, particularly to patients who are accustomed to the typical high-protein diet consumed in North America (105). Therefore, a reasonable "target" for dietary phosphate is 800-900 m g / d a y in patients with ESRD. With such a diet, phosphate-binding agents must be given to most dialysis patients to prevent the development of hyperphosphatemia, and such phosphatebinding agents are indicated in most predialysis patients with advanced renal failure and creatinine clearances of < 15-20 ml/minute. If the dietary intake of phosphate increases, the required dosages of phosphate binders become very large, so attention must be paid to both dietary phosphate restriction and the intake of adequate doses of phosphate binders (Fig. 3). Control of hyperphosphatemia is important for the prevention of soft tissue calcifications and secondary hyperparathyroidism as well as mortality (108,109). There has been a recommendation that the calcium intake should be augmented in patients with advanced renal failure. This is done by adding calcium salts, usually calcium acetate or calcium carbonate. There are two reasons for this. First, calcium salts, either calcium carbonate or calcium acetate, are effective for the intestinal binding of ingested phosphate in patients with ESRD (110-112), and, second, calcium supplements are indicated because calcium absorption is impaired in uremia and because the calcium intake is suboptimal in most patients with ESRD (113). Because of the importance of reducing the intake of dairy products to limit the phosphate intake, the amount of calcium in the diet is often as low as 400-700 m g / d a y in patients with renal failure (113). Furthermore, studies of net

645

CaC03 Intake (g/day)

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800 looo 12oo Dietary Phosphorus (mg/day)

14oo

FIG. 3 Relation between total daily dose of calcium carbonate and dietary phosphate intake in 21 compliant patients who exhibited good control of their serum phosphate levels. (Modified from Ref. 135, by permission of Blackwell Science, Inc.)

intestinal calcium absorption in uremic patients indicate that a neutral or positive calcium balance can be achieved when the dietary calcium is increased to >1.5 g / d a y with calcium carbonate, calcium citrate, or calcium lactate (114). However, other evidence implicates the dosage of calcium carbonate with the presence of vascular calcifications ( Vide infra) (139,140).

Phosphate-BindingAgents Because dietary phosphate restriction alone cannot control the hyperphosphatemia that exists in almost all patients undergoing hemodialysis, the intake of phosphate-binding agents is required in 90-95% of dialysis patients. The aluminum-containing gels, aluminum hydroxide and aluminum carbonate, were used in the past to reduce phosphate absorption and to control the serum phosphorus levels in patients with far advanced renal failure and in those treated with dialysis (115). However, it was recognized that the ingestion of aluminum-containing gels is a major risk factor for the development of aluminum intoxication, particularly that causing osteomalacia and other symptomatic "lowturnover" disorders of bone (116-121). Kaehny et al. (122) and Recker et al. (123) found that small amounts of aluminum were absorbed and excreted in the urine following the ingestion of large doses of aluminumcontaining gels by normal men. When aluminum is absorbed by patients with renal failure, it cannot be excreted, and it accumulates in the body. Thus both dialysis encephalopathy and aluminum-related bone disease have been reported prior to the initiation of dialysis in azotemic adults and children who were ingesting aluminum gels (116-121). It was shown that plasma aluminum levels correlated with the amount of oral aluminum intake from the

646

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CHAPTER40

phosphate-binding agents (121). Such observations implied a role of aluminum absorption from the gastrointestinal tract as a source of aluminum loading and toxicity. The observations that plasma aluminum levels fall strikingly (84,112) and that aluminum-related bone disease will improve or reverse after the total withdrawal of aluminum gels (84) provide proof that the oral intake of aluminum gels can be responsible for aluminum intoxication. Guidelines for "safe" doses of aluminum hydroxide were proposed, with safe maximum doses considered to be 30 m g / k g / d a y for children (124) and 4-6 tablets/day of aluminum hydroxide for adult patients treated with hemodialysis (125). However, when this r e c o m m e n d e d dose was prospectively evaluated in pediatric patients undergoing peritoneal dialysis, there was a progressive increase in the body burden of aluminum as judged by increments in plasma aluminum levels, increases in plasma aluminum levels after a desferrioxamine infusion test, and by histologic evidence of aluminum deposition in bone (126). Thus, aluminum-containing drugs should be avoided in the vast majority of patients with renal failure. In some dialysis patients, if aluminum gels are needed to control hyperphosphatemia, the gels can be combined with the calcium salts, with the "recomm e n d e d dosages" of aluminum gels not exceeded in such cases, and should be given for short period of time, i.e., few months. In patients receiving aluminum gels, there should be caution concerning the intake of medications that can augment aluminum absorption. Among the factors that enhance aluminum absorption, the most potent is citrate, as citric acid or a salt (127). The simultaneous ingestion of citrate with an aluminum-containing gel markedly augments aluminum absorption, e.g., produces a 20- to 50-fold increase (128). Fatal cases of acute aluminum toxicity have occurred in patients with advanced renal insufficiency (129,130) due to the simultaneous intake of aluminum hydroxide for hyperphosphatemia and the prescription of Shohl's solution or Bicitra to control metabolic acidosis. Other sources of citrate should be avoided with advanced renal failure as well. Thus calcium citrate is an effective phosphatebinding agent (131), but calcium citrate should be avoided because of the potential risk for aluminum intoxication if aluminum gels are coincidentally ingested (127,128). Another drug, AlkaSeltzer, which is often ingested by patients with dyspepsia, contains citric acid and led to fatal aluminum toxicity in a hemodialysis patient (132). Calcium carbonate has proved to be an effective phosphate-binding agent in at least 80-90% of adult and pediatric dialysis patients. Calcium carbonate should be ingested together with a meal, both to maximize its phosphate-binding efficiency and to minimize

the absorption of calcium (133). The required dosage of calcium carbonate varies from patient to patient, but the initial doses have averaged 4-7 g/day. In individual patients, the dose is adjusted empirically according to the levels of serum phosphorus (84,111,112,134). Hypercalcemia is the major side effect, occurring either with or without concurrent vitamin D therapy (84,111,112,135,136). The use of dialysate solutions with a calcium concentration of 2.5 mEq/liter has been very useful in patients treated with hemodialysis (135,136). When calcium carbonate was given as the sole phosphate binder in adult continuous ambulatory peritoneal dialysis patients who used dialysate with 3.5 mEq/liter calcium, the "standard" peritoneal dialysate calcium concentration for several years, hypercalcemia occurred in as many as 44% of patients, and many required the addition of aluminum gels (137). Evidence is accumulating, on the other hand, that disturbances in mineral metabolism contribute to the development of cardiovascular disease and to overall mortality in patients with end-stage renal disease (108,138-141). Block et al. reported that elevated serum phosphorus levels were an independent risk factor for death in adults undergoing dialysis even after adjusting for established cardiovascular risks and other comorbid conditions (108). The mechanisms that underlie this association remain uncertain, but hyperphosphatemia has long been recognized as an important determinant of soft tissue and vascular calcification in patients with chronic renal failure. Several studies suggest that vascular calcification, due at least in part to hyperphosphatemia a n d / o r the dosage of calcium carbonate, represents one pathway by which alterations in mineral metabolism can adversely affect clinical outcomes both in adult and in pediatric patients with ESRD (138-140,142,143). In this context, hyperphosphatemia in patients undergoing long-term dialysis should be managed by implementing alternative methods that do not entail the use of very large oral doses of calcium. Dietary calcium intake should be maintained at levels that are sufficient to satisfy daily requirements, but the administration of additional calcium as calcium-containing, phosphate-binding agents should be avoided. The new phosphate-binding agent, sevelamer (Renagel), approved by the Food and Drug Administration (FDA) in the United States, is an ionexchange resin that binds phosphorus in the intestinal lumen and prevents its absorption. It does not contain either calcium or aluminum, and it has been shown to be effective in managing phosphate retention both in short-term and in long-term studies of patients undergoing hemodialysis (144-147). The cholesterollowering properties of this compound also make it an appealing therapeutic alternative for use in a subgroup

RENAL BONE DISEASES / of patients who are known to be at risk for developing cardiovascular disease. In addition to sevelamer, other iron-containing compounds in different phases of drug development, such as stabilized polynuclear iron hydroxide and ferric polymaltose complex, have been shown to be effective in controlling serum phosphorus levels in short-term studies in adults and rats with chronic renal failure (148,149). Another agent, lanthanum chloride hydrate, also decreases intestinal phosphorus absorption in experimental animals, and clinical trials using this compound are currently underway (150). However, the long-term safety of new phosphate-binding agents containing iron or other heavy metals such as lanthanium remain to be established; until then such drugs should be considered only in the experimental phase. We should not forget the consequences of aluminum intoxication in patients with renal failure. Whether the more stringent control of serum phosphorus levels and the avoidance of hypercalcemia using therapeutic strategies, as advocated by Block and Port, will favorably affect the development and progression of vascular calcification and cardiovascular disease in the ESRD population (Fig. 4) remains to be determined (109). Evidence is accumulating, however, that phosphate retention a n d / o r the conventional therapeutic interventions aimed at managing this consequence of chronic renal failure can aggravate soft tissue and vascular calcifications (139,140). As such, maintaining serum calcium and phosphorus levels and values for the Ca × P ion product in serum within the ranges seen in persons with normal renal function, rather than at the higher levels previously considered to be acceptable in those with chronic renal failure,

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FIG. 4 Coronary artery calcification scores as measured by electron beam computed tomography according to age in 39 children and young adults with end-stage renal disease treated by dialysis. Reprinted from Ref. 139, WG Goodman, J Goldin, BD Kuizon, C Yoon, B Gales, D Sider et aL Coronary artery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med 2000;342:1478-1483. Copyright © 2000 Massachusetts Medical Society. All rights reserved.

647

seems prudent. In addition, the dialysate calcium concentration of 2.5 mEq/liter should be the standard in the majority of patients, particularly for adults ingesting large doses of calcium-containing, phosphate-binding agents (135,136). Active V i t a m i n D Sterols

Despite dietary phosphate restriction, the intake of phosphate-binding agents, the use of an appropriate level of calcium in dialysate, and an adequate intake of calcium, a significant n u m b e r of uremic patients develop progressive osteitis fibrosa. Appropriate therapy with an active vitamin D sterol can halt or retard the progression of the bone disease in patients with overt secondary hyperparathyroidism. Treatments employing vitamin D~ or D 2 in pharmacologic doses (1), dihydrotachysterol (151), 25-hydroxyvitamin D~ (calcifediol) (152,153), lct-hydroxyvitamin D 3 (alfacalcidol) (77), or 1,25(OH)zD 3 (calcitriol) (20,154-156) have all been associated with improved symptoms and correction of certain biochemical and radiologic features of secondary hyperparathyroidism. Certain data suggest that vitamin D~ or D 2 can produce more normal mineralization of bone than can calcium carbonate (157). In doses of 50,000-200,000 IU/day, vitamin D 3 can improve secondary hyperparathyroidism in uremic patients (1). The use of vitamin D, however, can be accompanied by hypercalcemia that persists for weeks after the drug is stopped. Calcifediol has also been employed in the management of renal osteodystrophy. Considerable data exist to indicate that both adult and pediatric renal patients respond favorably to this sterol in doses of 25-100 ~ g / day (152,153). The results from a six-center study of therapy with 25(OH)zD ~ in dialysis patients demonstrated a reversal of bone pain and tenderness, a decline in the serum alkaline phosphatase activity, and a decrease in the extent of osteitis fibrosa. Several clinical trials have documented the efficacy of 1,25 (OH)zD ~for the treatment of patients with symptomatic renal osteodystrophy (20,35,154,155,158). The results of these studies can be summarized as follows. With regard to symptoms and signs, there has been a decrease in bone pain, improvement in proximal muscle weakness, and improvement in gait posture. An increase in growth velocity of uremic children was shown in patients with very severe secondary hyperparathyroidism (35); other groups failed to confirm significant improvement in height velocity in children, although plasma alkaline phosphatase levels and serum PTH levels decreased toward normal (154). Studies of bone histology have demonstrated improved osteitis fibrosa (155,159,160). The doses of oral 1,25(OH)2D ~ used in these trials have ranged from 0.25 to 1.5

648

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CHAPTER40

Ixg/day. The major side effect was the appearance of hypercalcemia. The increments in serum calcium levels were sometimes rapid and marked, and they were more c o m m o n in two situations: after many weeks or months of treatment in patients with osteitis fibrosa who had experienced a favorable response to therapy, and after only a few weeks of calcitriol treatment in patients receiving relatively low doses and with no clinical response to treatment. In the latter group, who developed hypercalcemia while receiving calcitriol in dosages of 0.25-0.5 pg/day, aluminum-related bone disease or low bone turnover of other cause should be suspected (160). In patients with secondary hyperparathyroidism, the development of hypercalcemia can often be anticipated by a decline in serum alkaline phosphatase into or toward the normal range. Thus the response to treatment with 1,25(OH)zD 3 may suggest the type of renal bone disease that is present (60,160). Increments in serum phosphorus or a greater requirem e n t for a phosphate-binding agent are also observed, probably due to the action of 1,25(OH)zD 3 to augment intestinal absorption of phosphate as well as that of calcium (Fig. 5). l e~-Hydroxyvitamin D~, or alfacalcidol, is the active vitamin D sterol that has been widely used for the m a n a g e m e n t of ESRD patients in Europe, Canada, and Japan (77,161). This sterol undergoes hepatic 25-hydroxylation to 1,25(OH)zD3; the effects of alfacalcidol are very similar to those of 1,25(OH)zD3, but the required dosage is generally 50-75% greater. Patients who receive anticonvulsant therapy concomitantly may fail to respond, perhaps due to impaired hepatic 25-hydroxylation (162).

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Changes in net absorption of calcium (solid bars)

and phosphorus (hatched bars) in patients with advanced renal failure during therapy with either calcitriol or le~(OH)2D3 as determined from metabolic balance studies for 14-28

days. The data for the two sterols are combined because of

trivial differences between the two. Data are mean _+ SE.

(Modified from Ref. 218, with permission.)

Intravenous Calcitriol a n d "Pulse" Oral Dosing

A major advance in the therapeutic approach with vitamin D sterols has been the introduction of parenteral calcitriol and pulse-dose oral therapy. Slatopolsky et al. (163) first reported the marked effect of intravenous 1,25(OH)2D 3 to suppress serum PTH levels in hemodialysis patients. These studies showed a suppression of PTH that was significantly greater than that observed with calcium carbonate and was more marked than that previously observed with the daily oral administration of 1,25 (OH) 203. Moreover, there was a 20% inhibition of PTH release before serum ionized calcium rose (as shown in Fig. 4 in Chapter 39). This effect of 1,25(OH)2D 3 is probably due to the action of calcitriol in suppressing the synthesis of mRNA for p r e p r o P T H by the parathyroid cells (164,165). Slatopolsky et al. (163) postulated that the higher blood levels of calcitriol after intravenous administration may allow a direct effect on the parathyroid glands and bypass part of the effect of augmenting intestinal calcium absorption. Since the initial use of intravenous calcitriol, other reports have documented that calcitriol, given intravenously two or three times weekly at the time of regular dialysis, was effective in reversing features of uremic secondary hyperparathyroidism (166-171). Andress et al. (166) gave intravenous calcitriol for 1 year or longer in thrice weekly doses of 1.0-2.5 txg per dialysis to 12 hemodialysis patients with severe secondary hyperparathyroidism; all previously received daily oral calcitriol, but the dose was limited by hypercalcemia. Bone biopsies, carried out before and after 12 months of therapy, showed substantial improvement in bone formation rate, osteoblastic osteoid, and the extent of peritrabecular fibrosis (Fig. 6); there was also a substantial reduction in amino-terminal PTH and in alkaline phosphatase levels. During this same period, other reports have shown substantial suppression of serum IPTH levels following the administration of large oral doses of calcitriol given twice weekly. Thus the pulse oral doses of calcitriol, 3.0-5.0 Ixg given twice weekly (172-174), led to reductions in serum IPTH over a period of 4-8 weeks with only slight increments or no change in serum calcium. A comparison of serum calcitriol levels after a single oral dose versus an intravenous dose in the same patients revealed serum levels that exceeded the normal range for a period up to 24 hours by both routes (175). Over the first 3 hours after dosing, the blood levels were higher after the intravenous dose than after the oral dose, but they were no different thereafter. The overall area u n d e r the curve after intravenous calcitriol was 62% greater than that after oral dosing. Fukagawa et al. (172) demonstrated the intermittent pulse oral calcitriol, in doses of 4.0 txg twice weekly, lowered the PTH levels and also reduced the size of the parathyroid

RENAL BONE DISEASES / 4

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FIG. 6 Change in bone formation rate, double tetracycline label length, and mineral apposition rate in 10 patients with severe osteitis fibrora. The values are from bone biopsies obtained before and after 11 months of therapy with intravenous calcitriol. The horizontal line indicates the mean, and the normal range is indicated by the shaded area. Reprinted from Ref. 166, DL Andress, KC Norris, JW Coburn, EA Slatopolsky, DJ Sherrard. Intravenous calcitriol in the treatment of refractory osteitis fibrosa of chronic renal failure. N Engl J Med 1989;321:274-279. Copyright © 1989 Massachusetts Medical Society. All rights reserved.

649

glands by 41% as measured via ultrasound in hemodialysis patients with hyperparathyroidism (172). Tsukamoto et al. (173) studied 19 long-term hemodialysis patients who had previously received calcitriol or alphacalcidol for 4 years, with the dose limited by hypercalcemia. With pulse dose, 4.0 Ixg twice weekly, PTH was lowered by 52-59% in 16 patients treated for 6 months; three other patients discontinued the pulse treatment because of hypercalcemia. Similar reductions in serum IPTH levels after pulse oral doses of calcitriol in patients undergoing CAPD have been reported by Martin et al. (174). Furthermore, Salusky et al. demonstrated that the skeletal lesions of secondary hyperparathyroidism improved with either oral or intraperitoneal intermittent calcitriol administration in patients with bone biopsy-proved high bone turnover. Adynamic bone occurred in 33% of the patients (52). It is interesting to note that growth retardation was more severe in those patients that developed adynamic bone after intermittent calcitriol therapy (8). Thus, intermittent calcitriol therapy reverses many of the histologic abnormalities of secondary hyperparathyroidism in pediatric patients undergoing regular peritoneal dialysis (52). The question of whether pulse-dose calcitriol can lead to regression of the hyperplastic glands, as suggested by Fukagawa et al. (172), is not entirely certain. Most studies have shown a rather rapid return of serum IPTH levels to pretreatment levels within a short time after the calcitriol has been discontinued, an observation suggesting little regression in gland size. The effectiveness of intermittent intravenous doses of lc~-hydroxyvitamin D 3 in lowering serum PTH levels (176) has also been shown in patients treated with hemodialysis. But there have been no studies of 1,25(OH)2D 3 (given either in pulsed oral doses or intravenously) and lcx-hydroxyvitamin D 3 that permit a comparison of the relative effectiveness of the two sterols. Regardless of the route of calcitriol administration, the data suggest that the use of intermittent (pulse) therapy with calcitriol may have advantages over daily dosing in the control of IPTH levels in these patients. It seems likely that supranormal plasma levels of calcitriol may have effects on the parathyroid glands that are not achieved with normal levels. It is of interest that such supranormal plasma levels of 1,25 (OH) 203 are observed during the recovery period when patients with nutritional vitamin D deficiency are given moderate doses of vitamin D; this observation suggests that "recovery" from secondary hyperparathyroidism may involve serum calcitriol levels that exceed the normal range. Summary of Calcitriol in ESRD: A Current View

Several changes in the evaluation and m a n a g e m e n t of patients with ESRD affect the use of calcitriol and other vitamin D sterols in uremic patients. These

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CI4AeTWR40

include the substantial effectiveness of pulse calcitriol in suppressing serum IPTH levels; the widespread use of calcium salts rather than aluminum gels as phosphate in most ESRD patients, resulting in substantially higher calcium intake; the use of dialysate calcium levels that do not raise the total or ionized calcium levels, thereby giving greater safety with use of calcium salts, and perhaps calcitriol as well; the recognition of the syndrome of idiopathic low bone turnover, which arises in part from oversuppression of PTH; (e) the use of intact PTH measurements, with their close correlations with the histologic types of renal bone disease (this may permit better prediction of the renal bone disease present and the treatment needed in these patients); and the availability of a new phosphate binding agent, sevelamar (Renagel) that it is calcium and aluminum free and may have an important effect in the reduction of soft tissue and vascular calcifications by reducing the exogenous calcium load. On the basis of these changes, it seems reasonable that intact PTH levels will be used as guides to therapy with calcitriol and other active vitamin D sterols, particularly in adults. With intact PTH levels more than six- to eightfold greater than normal, calcitriol therapy should almost always be initiated. With intact PTH levels of three to six times normal, therapy might be considered, particularly if serial levels of PTH are showing an increase. With intact PTH levels between one and onehalf and three times normal, calcitriol is not indicated, in that it might lower PTH levels further, with a risk of inducing a "low-turnover" state. With intact PTH levels within the normal range or up to 1.5-fold greater than normal, there is a strong likelihood that such a patient has subnormal bone turnover, and there is no reason to add calcitriol.

Calcitriol in Early and Predialysis Renal Failure Because reductions in calcitriol synthesis by the kidney are important in the pathogenesis of secondary hyperparathyroidism in renal failure, and because some patients have already developed significant hyperparathyroidism by the time they begin dialysis therapy, there is a question of whether calcitriol therapy be given in the predialysis phase of renal failure. Several well-controlled studies have been done with daily calcitriol administration (156,177); the results are in general agreement, and these data are reviewed in detail elsewhere (178,179). From some of the very early reports, there has been concern that the l e~hydroxylated vitamin D3 sterols may accelerate the rate of progression of renal disease in such patients (179,180). However, when either 1,25(OH)zD 3 or loLhydroxyvitamin D~ has been given in daily doses of 0.25-0.50 Ixg, the occurrence of hypercalcemia, hyper-

phosphatemia, or any impairment of renal function has been very rare (178,179). There is some evidence that calcitriol can impair creatinine secretion by the renal tubule, accounting for the reversible change in serum creatinine and creatinine clearance reported in studies using calcitriol at doses of 0.50 Ixg/day (181). When the daily doses rarely exceeded 0.25 Ixg/day, calcitriol therapy reversed secondary hyperparathyroidism, as j u d g e d from both biochemical findings and histologic features found on bone biopsy (177). Such therapy may be particularly valuable in patients, such as children and those with very slowly progressive tubulointerstitial renal disease, who are at high risk of developing progressive secondary hyperparathyroidism as their renal disease slowly worsens (182). The large double-blinded multicenter study of alfacalcidol in patients with moderate renal failure provides further evidence of the safety and effectiveness of alfacalcidol over 2 years of follow-up. In addition, the data demonstrated that the developm e n t of adynamic bone is extremely rare in these patients (77).

N e w Vitamin D Sterols Despite the widespread availability of calcitriol and alfacalcidol, the m a n a g e m e n t of secondary hyperparathyroidism remains a problem for many patients with end-stage renal disease. Daily and intermittent administration of calcitriol and alfacalcidol commonly cause hypercalcemia a n d / o r aggravate hyperphos' phatemia. Moreover, the use of these vitamin D sterols is risky and probably contraindicated in the face of significant hyperphosphatemia. There are several newer vitamin D sterols that may be less likely to induce hypercalcemia a n d / o r aggravate hyperphosphatemia but still act to reduce IPTH levels, and a few of these have reached clinical use. The goal has been to identify sterols that exert less calcemic and phosphatemic effects and yet can reduce PTH secretion and exert antiproliferative and cell-differentiating effects. This section includes the newer vitamin D analogs that have been evaluated in clinical trials in uremic patients with secondary hyperparathyroidism (183); these include 22-oxacalcitriol (maxacalcitol), falecalcitriol, 19-Nor (paricalcitol) (184), and loL-hydroxyvitamin O 2 (185). In animal models, these sterols suppressed serum IPTH levels as effectively or more effectively than calcitriol and yet exerted less calcemic and phosphatemic actions. The chemical structures of calcitriol and these four analogs are shown in Fig. 7. Paricalcitol (186) and doxercalciferol (187) are FDA-approved for treating secondary hyperparathyroidism in dialysis patients in the U.S., and 22-oxacalcitriol and falecalcitriol are awaiting approval for treating secondary hyperparathyroidism in Japan.

RENAL BONE DISEASES / 22-Oxacalcitriol

adynamic bone and osteomalacia improved in two patient (189). No data are available that compare OCT with intravenous calcitriol of alfacalcidol. This sterol is awaiting approval for the treatment of secondary hyperparathyroidism in Japan.

The 22-oxacalcitriol (OCT) analog differs from calcitriol by the substitution of an oxygen atom for the methylene group at carbon 22 of the A ring (Fig. 7). OCT was the first sterol identified to have high activity in causing in vitro differentiation of a leukemia cell line and yet have little calcemic action. The efficacy of OCT was reported in dialysis patients with secondary hyperparathyroidism and baseline IPTH levels ranging from 620 to 2730 pg/ml; they received intravenous OCT after each dialysis for 12 weeks; the dose was increased if the IPTH was not reduced 30% and yet serum calcium and phosphorus levels remained acceptable (188). The IPTH suppression ranged from 35 to 51% of pretreatment values; serum calcium levels rose in all patients, with the highest values being 9 . 9 - 11.6 mg/dl; hypercalcemia responded to reductions in the dose of OCT; serum phosphorus levels were below 6 m g / d l throughout. Serum alkaline phosphatase activities fell with treatment. Preliminary reports of the effect of OCT on bone histology of 11 hemodialysis patients disclosed significant improvement of osteitis fibrosa in those affected;

Falecalcitriol

Fale calcitriol ( 26,26,26,27,27,27-hexafluoro-1,25dihydroxyvitamin D 3 differs from calcitriol by fluorine substitution for the six hydrogens at carbons 26 and 27 (Fig. 7). The metabolism of falecalcitriol does not involve the usual hydroxylation at carbon 24, but instead hydroxylation occurs at carbon 23. Falecalcitriol was one-third as potent as calcitriol in binding to the intestinal vitamin D receptor but had two- to fourfold greater transcriptional activity than calcitriol (190), features that may explain its greater biologic activity. Two clinical studies have evaluated falecalcitriol. A placebo-controlled, double-blinded study compared oral falecalcitriol and placebo in 121 hemodialysis patients with secondary hyperparathyroidism (191).

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652

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CI4APTWk40

After a 4-week washout, 0.3 ixg of falecalcitriol or placebo was given daily for 8 weeks. At the end of this time, serum IPTH fell 35 _+ 4 % from the base line of 437 + 31.2 p g / m l ; in contrast, IPTH rose by 12.4 _+ 4.9 % from base line with placebo. Serum calcium rose during falecalcitriol treatment but it varied within 10% of base line; the serum calcium exceeded 11.0 m g / d l in 16% of patients receiving falecalcitriol but in none of the placebo patients. Serum phosphorus levels were unchanged; total alkaline phosphatase and osteocalcin levels fell with falecalcitriol but were u n c h a n g e d with placebo. In an open-label cross-over study, the efficacy of falecalcitriol was compared to alfacalcidol in 25 patients who were stable on a constant dose of alfacalcidol (192). For the first 8 weeks, the same dose of alfacalcidol was continued; 13 patients were then randomly assigned to falecalcitriol and the others continued on alfacalcidol for 24 weeks (Period 1); later the groups crossed over to the other drug for another 24 weeks (Period 2). The doses of both drugs were adjusted to maintain serum calcium levels as near to base line as possible. The initial doses were 0.25 - 0.50 txg/day for alfacalcidol and 0.15 - 0.3 Ixg/day for falecalcitriol. At base line before Period 1, the biochemistries of the groups were similar except serum phosphorus was higher in the group given falecalcitriol. During Period 1, serum phosphorus fell more from base line with falecalcitriol than with alfacalcidol; serum calcium did not differ between the groups. Serum PTH levels, measured with three different antisera, fell slightly with falecalcitriol treatment but increased with alfacalcidol. Wilcoxon's test using discriminatory analysis of changes of the three IPTH assays indicated that falecalcitriol was superior to alfacalcidol. The actions of falecalcitriol were reproducible, as j u d g e d from results of the group that received falecalcitriol during Period 1 compared to those during Period 2. The mechanism whereby falecalcitriol may induce less phosphatemia than alfacalcidol is uncertain. These results comparing the effects of falecalcitriol with alfacalcidol represent the only published data that compare a new sterol with one of the standards (calcitriol or alfacalcidol) in dialysis patients (192). Falecalcitriol awaits approval for the m a n a g e m e n t of secondary hyperparathyroidism in Japan. Paricalcitol

Intravenous paricalcitol (19-nor-l,25-dihydroxyvitamin D2) has been approved by the FDA for the management of secondary hyperparathyroidism in dialysis patients. This sterol has a vitamin D 2 side chain, and carbon 19, with its exocyclic double bond, is absent

(Fig. 7). In several studies in uremic rats, paricalcitol was less calcemic than calcitriol but appeared equally effective in reducing IPTH levels (184,193). Paricalcitol and placebo were compared in a 12-week study of 78 hemodialysis patients with moderate to severe secondary hyperparathyroidism and entry IPTH of 785 +_ 66 and 745 _+ 52 p g / m l (_+ SE), respectively (186). Only calcium-based phosphate binders were utilized. After 4 weeks of washout, placebo or paricalcitol (0.04 Ixg/kg) was started; the dose was increased every 2 weeks if PTH levels were not lowered by 30% from base line and serum calcium and phosphorus levels were adequately controlled. After 12 weeks of paricalcitol, IPTH levels fell by approximately 60% to 406 +_ 106 p g / m l (range, 40 to 2388 p g / m l ) , while IPTH levels did not change in placebo patients. Among the five paricalcitol-treated patients with entry IPTH values above 1200 pg/ml, two showed no suppression. Serum calcium rose from 9.24 + 0.12 to 9.56 _+ 0.15 m g / d l in the paricalcitol group (p < 0.02) but values were unchanged in the placebo group; serum calcium exceeded 12.0 m g / d l in 2 of 414 measurements. The average mean serum phosphorus was not different between the paricalcitol and placebo groups except during 3 of 12 weeks, when values were higher in paricalcitol patients than in placebo groups. On completion of the study, the paricalcitol dose averaged near 7 Ixg with each hemodialysis. Serum alkaline phosphatase fell significantly with paricalcitol treatment but was u n c h a n g e d with placebo. The reason paricalcitol suppresses IPTH but has had less effect on elevating serum calcium and phosphorus is uncertain. Paricalcitol has one-third the affinity to both the vitamin D receptor and vitamin D-binding protein compared to calcitriol; whether these small differences could account for the differences is uncertain. The favorable effects of paricalcitol need to be confirmed by long-term studies. To date, there are no published data d o c u m e n t i n g that paricalcitol, when given in doses that effectively suppress serum IPTH levels, exerts less calcemic effect than intravenous calcitriol in dialysis patients with secondary hyperparathyroidism. Unpublished data, available from the FDA though the Freedom of Information Act, failed to document a superiority of paricalcitol over calcitriol in causing hypercalcemia in hemodialysis patients with secondary hyperparathyroidism.

Doxercalciferol The oral and intravenous forms of doxercalciferol [ 1-hydroxyvitamin 0 2 (1oLD2) ] have been FDA-approved for the treatment of secondary hyperparathyroidism in dialysis patients. Doxercalciferol (Fig. 7) lacks the 25-hydroxy group and has a vitamin 0 2 side chain, with a 22-23 diene b o n d and a 28-carboxyl group attached to

RENAL BONE DISEASES / carbon 24. lorD 2 undergoes hepatic hydroxylation at carbon 25 to form the active sterol, 1,25-dihydroxyvitamin O2, in a m a n n e r similar to the conversion of lc~D3 (alfacalcidol) to 1,25-dihydroxyvitamin D 3. Doxecalciferol is one-fifth to one-tenth as toxic in animals compared to alfacalcidol (194), a sterol widely used for managing secondary hyperparathyroidism in Europe, Asia, and Canada. It is uncertain why larger doses of 1o~D2 have less calcemic effects than lcxD3 does and why lower doses are equally potent. Clinical trials with doxercalciferol in patients with secondary hyperparathyroidism were undertaken because of its lesser toxicity in animals and because daily oral lorD 2 doses, from 1.0 up to 5.0 Ixg/day, were not associated with hypercalciuria in women with postmenopausal osteoporosis (195). In a first trail, daily oral lorD 2 was given to hemodialysis patients with moderate to severe secondary hyperparathyroidism (base line serum IPTH, 672 -+ 70 p g / m l ) (_+ SE) and receiving only calcium-based phosphorus binders (196). After 8 weeks of washout, oral l o~D2 was given daily; the initial dose of 4.0 txg was adjusted over 12 weeks to maintain the serum IPTH between 130 and 250 p g / m l , levels associated with normal or near-normal bone formarion and little or no features of osteitis fibrosa (187). In individual patients, IPTH fell by 48-96% (mean, 75.4%), with the IPTH averaging 51% lower than base line at the end 12 weeks; 87% of patients reached the target PTH range. There were modest increases in serum calcium, with the mean rising from 8.8 _+ 0.2 to 9.5 _+ 0.2 m g / d l after 12 weeks; serum phosphorus levels did not change significantly. Subsequent studies showed that treatment with 10 Ixg thrice weekly had similar effects to 4.0 txg daily (196). A larger study of doxercalciferol included 99 hemodialysis patients with moderate to severe secondary hyperparathyroidism (IPTH, 442 to 3644 p g / m l ) in a prospective, multicenter, double-blind, placebocontrolled trial (197). After 8 weeks of washout, there was 16 weeks of open-label treatment with l oLD2 followed by 8 weeks of randomized, double-blinded treatm e n t with either continued lorD 2 or placebo. The doses of calcium-based phosphate binders did not differ between the two study groups. The initial dose of l oLD2 was 10 Ixg after each hemodialysis, with the doses adjusted to maintain serum IPTH within a target range of 150-300 p g / m l . Serum IPTH fell more than 50 % in 92% of the patients, and 83% of patients reached the target PTH range (Fig. 8). When the patients entered into double-blinded treatment, IPTH levels rose quickly to values no different from base line by 4 weeks after conversion to placebo. The dose of l ecD2 was reduced substantially from the first week to the end of the study. Serum calcium levels increased slightly but significantly from base line in the l oLD2 treatment group;

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FIG. 8 Plasma immunoreactive IPTH levels (mean _+ SE) of 99 per-protocol patients divided into three groups according to base line IPTH levels: group I, IPTH less than 600 pg/ml; group II, 600 to 1200 pg/ml; and group III, greater than 1200 pg/ml. Dotted lines encompass the target IPTH range (150-300 pg/ml), a, Group I differs from group II; b, group I differs from group III; c, group II differs from group III; each P < 0.05. (Reprinted from Ref. 197, with permission.)

mean values ranged from 9.25 to 9.81 m g / d l throughout treatment. Serum calcium levels exceed 12.0 m g / d l in 0.67% of 2398 measurements. The asymptomatic hypercalcemia was readily corrected by temporarily discontinuing l oLD2 and subsequent dose reduction. Serum phosphorus levels rose slightly when patients began l oLD2 treatment, but serum phosphorus levels did not differ between the placebo- and l oLD2-treated groups during double-blinded treatment. Thus, doxercalciferol was highly effective in reducing IPTH, even in patients with very severe secondary hyperparathyroidism. There were small but statistically significant incidence of mild hypercalcemia and hyperposphatemia. No data are available to compare doxercalciferol with either calcitriol or alfacalcidol in dialysis patients with secondary hyperparthyroidism.

Calcimimetic Agents The discovery and cloning of the extracellular calcium-sensing receptor (CaSR) and clarification its role in calcium metabolism represent a major scientific advance during the past decade. (198,199). This lowaffinity, G protein-coupled receptor is found in high concentrations on the surface of parathyroid cells, on the calcitonin-secreting C cells of the thyroid gland, and at various sites along the n e p h r o n (199). Its activation by small changes in extracellular ionized C a (Ca 2+) accounts for the rapid suppression of PTH secretion that occurs as s e r u m C a 2+ increases (198) and the steep rise in urinary calcium excretion that occurs as serum calcium levels increase above a threshold level. These effects permit fight regulation of both calcium

654

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CHAPTER40

homeostasis and blood calcium. Calcimimetic compounds that enhance the affinity of the CaSR for C a 2+ and consequently reduce PTH secretion (200,201) are being explored as potential therapies for secondary hyperparathyroidism. Altered function or regulation of the CaSR may play a role in the pathogenesis of secondary hyperparathyroidism. Thus, the parathyroid glands obtained from uremic patients undergoing parathyroidectomy for severe secondary hyperparathyroidism have shown reduced expression of the CaSR on the parathyroid cells (202,203). Changes in the CaSR on parathyroid cells could account for the shift to the right of the calcium/parathyroid set point or the calcium concentration required to suppress PTH secretion by 50% in patients with severe secondary hyperparathyroidism (63). Data on whether calcitriol or vitamin D deficiency affects the regulation of the CaSR are conflicting, and two studies in vitamin D-deficient rats have yielded conflicting results, although both showed no regulation of the mRNA for CaSR by changes in C a 2+ (204,205). One report found no effect of calcitriol on the mRNA for the CaSR, but a second study of vitamin D-deficient rats found a 40% reduction of mRNA for the CaSR that could be restored by calcitriol treatment (205). Further data are needed to define the role of alterations of the CaSR in secondary hyperparthyroidism.

Potential Use of Calcimimetic Agents Calcimimetic agents (such as R-568 by NPS Pharamaceuticals) that modify the CaSR to make it more sensitive t o C a 2+ and consequently reduce PTH secretion have the potential for effective medical treatm e n t of hyperparathyroidism, both secondary and primaIT. In uremic rats, the administration of R-568 caused reversible suppression of PTH levels and substantially reduced the proliferation of parathyroid cells that occurred in untreated uremic animals (206). Other trials in animals with uremia of longer duration showed substantial improvement of osteitis fibrosa and bone density, and more normal bone stiffness following administration of R-568 (207). The results of initial trials documenting the ability of R-568 to reduce IPTH levels in patients with both primary and secondary hyperparathryoidism are reviewed elsewhere (200,201,208). In these studies, serum calcium levels fell slightly, but only after IPTH levels had been lowered substantially; such observations indicate a fall in serum calcium occurs as a consequence of the reduced IPTH levels. Available data on both primary and secondary hyperparathyroidism indicate that the average percentage reductions of IPTH from pretreatm e n t values varied from 63 to 73% after the administration of larger doses of R-568 (200,201). Such

suppression occurred independently of the pretreatment IPTH levels in patients with primary and secondary hyperparathyroidism (200,201,208). The trials in dialysis patients with secondary hyperparathyroidism indicated that the duration of action of R-568 was up to 24 hours, considerably longer than the 4 to 6 hours observed in patients with primary hyperparathyroidism (200). When the calcimimetic drug was given daily for up to 15 days, the effects (lower serum IPTH) persisted even in the face of much lower pretreatment IPTH levels and somewhat lower serum calcium levels (Fig. 9) (201). These relatively short-term studies suggest that R-568 can suppress IPTH levels and may be safe; a case report d o c u m e n t e d the safe control of symptomatic hypercalcemia over a period of nearly 2 years in a patient with parathyroid carcinoma managed with R-568. However, R-568 has very low bioavailability and its metabolism is largely restricted to a single P450 enzyme (CYP 2D6), resulting in high potential for untoward interactions with other drugs. Therefore, clinical trials are now underway utilizing a new, second-generation calcimimetic agent that has greater bioavailability and a more favorable metabolic profile.

Parathyroidectomy Certain laboratory and clinical features of severe secondary hyperparathyroidism indicate the necessity for parathyroidectomy. The presence of hyperplasia a n d / o r hypertrophy of the parathyroid glands should be d o c u m e n t e d by the finding of very high levels of serum PTH; the documentation of osteitis fibrosa by bone biopsy is required if a potential role for aluminum intoxication is considered. The indications for parathyroid surgery in the presence of elevated serum PTH levels include persistent hypercalcemia (serum calcium levels > 11.0 m g / d l ) , particularly when the hypercalcemia is symptomatic; intractable and severe pruritus that fails to respond to dialysis or other medical treatment; progressive extraskeletal calcification when the Ca × P product in serum exceeds 70-75 mgZ/dl 2 despite appropriate phosphate restriction; severe skeletal pain, fractures, skeletal deformities, or tendon rupture; and the syndrome of calciphylaxis. When the patient does not have significant hypercalcemia or hyperphosphatemia that is refractory to management, or progressive calciphylaxis, a therapeutic trial with intravenous or pulse-oral administration with one of the different vitamin D analogs is indicated prior to resorting to parathyroidectomy. Persistent hypercalcemia can occur in patients with aluminum-related bone disease or those with low bone turnover lacking aluminum (69,160,179), and aluminum toxicity must be excluded by bone biopsy prior to parathyroid sur-

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gery. O t h e r causes of hypercalcemia, such as sarcoidosis, malignancy-related hypercalcemia, intake of calcium supplements, and presence of adynamic/aplastic bone lesion not related to a l u m i n u m should also be considered (4,58). When a decision to perform parathyroid surgery has been made, it is essential to avoid a marked postoperative decrease in serum calcium levels due to the "hungry bone" syndrome. Because of the severity of the bone disease, this hypocalcemia is often m o r e m a r k e d and more prolonged than that following parathyroidectomy for primary hyperparathyroidism. Renal patients should receive oral calcitriol, 0.5-1.0 I~g/day, or intravenous calcitriol, 1.5-2.0 ~g per hemodialysis treatment, for 2-6 days before the parathyroid surgery to stimulate intestinal calcium absorption during the postoperative period and to maximize the effectiveness of oral calcium salts. Following surgery, serum calcium and potassium levels should be m o n i t o r e d every 8-12 hours and serum phosphorus and magnesium should be measured daily. For 24-36 hours up to 7-10 days after surgery, m a r k e d hypocalcemia, with serum calcium decreasing to levels below 6-7 m g / d l , may be seen; this can be associated with serious symptoms, including severe convulsive seizures that can cause major fractures and t e n d o n avulsions. Such seizures may occur within 2-3 days up to 3-4 weeks after the surgery. For uncertain reasons, these seizures most commonly occur during the last 1-2 hours of the hemodialysis procedure or shortly afterward. To reduce the risk of this serious and avoidable problem, an infusion containing calcium gluconate should be initiated when the serum calcium falls below 7.5-8.0 m g / d l ; enough ampules of 10% calcium gluconate (calcium content, 110 m g / 1 0 - m l ampule) should be a d d e d to an intravenous infusion to provide approximately 100 mg calcium ion per hour, and the infusion should be con-

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FIG. 9 Plasma PTH levels during 15 days of treatment with R-568 or placebo and during a 9-day posttreatment interval. Blood samples were obtained after hemodialysis but before drug administration on days of regularly scheduled dialysis treatments. Values are mean _ SE. There was a significant overall difference between patients treated with R-568 and those given placebo.*, P < 0.05. (Reprinted from Ref. 201, by permission of Blackwell Science, Inc.)

tinued for 8-12 hours or longer if serum calcium does not rise. During this infusion, the serum calcium should be measured every 4-6 hours; if the serum calcium continues to fall, the infusion of calcium should be increased to deliver 200 mg calcium per hour. R e c o m m e n d e d doses of infused calcium have also been based on the degree of preoperative elevation of serum alkaline phosphatase (209). Oral calcium carbonate, in doses that provide up to 2-3 g of ionic calcium and divided into five or six doses daily should be used; high doses of one of the different forms of calcitriol administration should be added if hypocalcemia persists. The length of time for which intravenous calcium is required varies greatly; some patients require it for only 2 or 3 days, but severe hypocalcemia can persist for several weeks or even months, and a p e r m a n e n t central catheter may be required for daily h o m e infusions of 800 to 1000 mg of calcium ion. W h e n the serum calcium begins to rise toward normal, intravenous calcium can be discontinued, the calcitriol t r e a t m e n t can be r e d u c e d or stopped, and the dosage of oral calcium salts can be reduced and then adjusted to control hyperphosphatemia. Serum phosphorus levels often fall to normal or even subnormal levels postoperatively; any phosphate t r e a t m e n t will markedly aggravate the hypocalcemia, and patients should not receive phosphate unless the serum phosphorus falls to very low levels, e.g., below 1.5-2.0 m g / d l . Serum phosphorus levels are ideally maintained between 2.5 and 4.0 m g / d l to avoid a risk of aggravating the hypocalcemia. Calcium carbonate or calcium acetate should be the phosphate-binding agent of choice, and A1 (OH)3 should be avoided after surgery unless it is absolutely necessary because of the increased susceptibility to a l u m i n u m toxicity after parathyroidectomy (81,82).

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CHAPTER

40

ACKNOWLEDGMENTS This work was supported by U.S. PHS grants DK35423 and RR-00865, by research funds from the U.S. Department of Veterans Affairs and the Casey Lee Ball Foundation.

REFERENCES 1. Dent CE, Harper CN, Philpot GR. Treatment of renal-glomerular osteodystrophy. Q J Med 1961;30:1-31. 2. Slatopolsky E, Bricker NS. The role of phosphorus restriction in the prevention of secondary hyperparathyroidism in chronic renal disease. Kidney Int 1973;4:141-145. 3. Sherrard DJ, Hercz G, Pei Y, Maloney N, Greenwood C, Manuel A, et al. The spectrum of bone disease in end-stage renal failure~Arl evolving disorder. Kidney Int 1993;43:436-442. 4. Goodman WG, Coburn JW, Slatopolsky E, Salusky IB. Renal osteodystrophy in adults and children. In: Favus MJ, ed Primer on the metabolic bone diseases and disorders of mineral metabolism. Philadelphia:Lippincott, Williams & Wilkins, 1999:347-363. 5. Andress DL, Maloney NA, Coburn JW, Endres DB, Sherrard DJ. Osteomalacia and aplastic bone disease in aluminum-related osteodystrophy. J Clin Endocrinol Metab 1987;65:11-16. 6. Torres A, Lorenzo V, Hernandez D, Rodriguez JC, Concepcion MT, Rodriguez AP, et al. Bone disease in predialysis, hemodialysis, and CAPD patients: Evidence of a better bone response to PTH. Kidney Int 1995;47:1434-1442. 7. Atsumi K, Kushida K, Yamazaki K, Shimizu S, Ohmura A, Inoue T. Risk factors for vertebral fractures in renal osteodystrophy. Am J Kidney Dis 1999;332:287-293. 8. Kuizon BD, Goodman WG, J/ippner H, Boechat I, Nelson P, Gales B, et al. Diminished linear growth during treatment with intermittent calcitriol and dialysis in children with chronic renal failure. Kidney Int 1998;53:205-211. 9. Sherrard DJ. Renal osteodystrophy. Semin Nephro11986;6:56-67. 10. Coburn JW, Henry DA. Renal osteodystrophy. Adv Intern Med 1984;30:387-424. 11. Nebeker HG, Coburn JW. Aluminum and renal osteodystrophy. A n n u Rev Med 1986;37:79-95. 12. Noel LH, ZingraffJ, Bardin T, Atienza C, Kuntz D, Dr/ieke T. Tissue distribution of dialysis amyloidosis. Clin Nephrol 1987;27: 175-178. 13. Kleinman KS, Coburn JW. Amyloid syndromes associated with hemodialysis. Kidney Int 1989;35:567-575. 14. Hampl H, Lobeck H, Bartel-Schwarze S, Stein H, Eulitz M, Linke RP. Clinical, morphologic, biochemical, and immunohistochemical aspects of dialysis-associated amyloidosis. ASAIO Trans 1987;33:250-259. 15. Mallette LE, Patten BM, Engel WK. Neuromuscular disease in secondary hyperparathyroidism. Ann Intern Med 1975;82:474-483. 16. Baker LR, Ackrill P, Cattell WR. Iatrogenic osteomalacia and myopathy due to phosphate depletion. Br MedJ1974;3:150-150. 17. Smith R, Stern G. Myopathy, osteomalacia and hyperparathyroidism. Brain 1967;90:593-602. 18. Coburn JW, Nebeker HG, Hercz G, Milliner DS, Ott SM, Andress DL, et al. Role of aluminum accumulation in the pathogenesis of renal osteodystrophy. In: Robinson RR, ed. Nephrology, Vol 2. New York:Springer-Verlag, 1984:1383-1395. 19. Kanis JA, Cundy T, Earnshaw M, Henderson RG, Heynens G, Maik R, et al. Treatment of renal bone disease with l ethydroxylated derivatives of vitamin D3: Clinical, biochemical, radiographic and histological responses. QJMed 1979;48:289-322.

20. Brickman AS, Sherrard DJ, Jowsey J, Singer FR, Baylink DJ, Maloney N, et al. 1,25-Dihydroxycholecalciferol: Effect on skeletal lesions and plasma parathyroid hormone levels in uremic osteodystrophy. Arch Intern Med 1974;134:883-888. 21. Zanello SB, Collins ED, Marinissen MJ, Norman AW, Boland RL. Vitamin D receptor expression in chicken muscle tissue and cultured myoblasts. Horm Metab Res 1997;29:231-236. 22. Massry SG, Popovtzer MM, Coburn JW, Makoff DL, Maxwell MH, Kleeman CR. Intractable pruritus as a manifestation of secondary hyperparathyroidism in uremia. Disappearance of itching following subtotal parathyroidectomy. N Engl J Med 1968;279:697-700. 23. Wright RS, Mehls O, Ritz E, Coburn JW. Musculoskeletal manifestation of chronic renal failure, dialysis and transplantation. In: Bacon P, Hadler N, eds. Renal manifestations in rheumatic disease. London:Butterworth, 1982:352-352. 24. Mehls O, Ritz E, Krempien B, Gilli G, Link K, Willich E, et al. Slipped epiphyses in renal osteodystrophy. Arch Dis Child 1975;50:545. 25. Mehls O, Krempien B, Ritz E, Scharer K, Schuler HW. Renal osteodystrophy in children on maintenance hemodialysis. Proc EDTA 1973;10:197-201. 26. Gipstein RM, Coburn JW, Adams JA, Lee DBN, Parsa KP, Sellars A, et al. Calciphylaxis in man: A syndrome of tissue necrosis and vascular calcification in 11 patients with chronic renal failure. Arch Intern Med 1976;136:1273-1280. 27. Bleyer AJ, Choi M, Igwemezie B, de le Torre E, White WL. A case control study of proximal calciphylaxis. Am J Kidney Dis 1998i32:376-383. 28. Hafner J, Keusch G, Wahl C, Sauter B, Hurlimann A, Van Weizsacker F, et al. Uremic small-artery disease with medial calcification and intimal hyperplasia (so-called calciphylaxis): A complication of chronic renal failure and benefit from parathyroidectomy. J Am Acad Dermatol 1999;33:954-962. 29. Straumann E, Meyer B, Misteli M, Blumberg A, Jenzer HR. Aortic and mitral valve disease in patients with end stage renal failure on long-term haemodialysis. Br Heart J 1992;67:236-239. 30. Mawad HW, Sawaya BE Sarin R, Malluche HH. Calcific uremic arteriolopathy in association with low turnover uremia bone disease. Clin Nephrol 1999;52:160-166. 31. McSherry E, Morris RC. Attainment and maintenance of normal status with alkali therapy in infants and children with classic renal tubular acidosis (RTA). J Clin Invest 1978;61:509-527. 32. Challa A, Krieg RJ, Jr, Thabet MA, Veldhuis JD, Chan JC. Metabolic acidosis inhibits growth hormone secretion in rats: Mechanism of growth retardation. Am J Physiol 1993;265: E547-E553. 33. Mehls O, Tonshoff B, Blum WF, Heinrich U, Seidel C. Growth hormone and insulin-like growth factor I in chronic renal failure--Pathophysiology and rationale for growth hormone treatment. Acta Paediatr Scand Suppl 1990;370:28-34. 34. Stickler GB, Bergen BJ. A review: Short stature in renal disease. Pediatr Res 1973;7:978-982. 35. Chesney RW, Moorthy AV, EismanJA, Tax DK, Mazess RB, DeLuca HE Increased growth after long-term oral 1,25-vitamin D~ in childhood renal osteodystrophy. N EnglJ Med 1978;298:238-242. 36. Bulla M, Delling G, Offermann G. Renal bone disorders in children: Therapy with vitamin D:~ or 1,25 dihydroxycholecalciferol. In: Norman AW, Shaefer K, Herrath DV, eds. Basic research and its clinical application. Berlin:de Gruyter, 1979:853-858. 37. Tonshoff B, Mehls O, Heinrich U, Blum WF, Ranke MB, Schauer A. Growth-stimulation effects of recombinant human growth hormone in children with end-stage renal disease. J Pediatr 1990;116:561-566. 38. Wuhl E, Haffner D, Nissel R, Schaefer E Mehls O, German Study Group for Growth Hormone Treatment in Chronic Renal

RENAL BONE DISEASES /

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178. Goodman WG, Coburn JW. Calcitriol in early and pre-dialysis renal failure: What are the risks and benefits? In: Norman AW, Shaefer K, v. Herrath D, Grigoleit H-G, eds. Vitamin D: Chemical, biochemical and clinical endocrinology of calcium metabolism. Berlin: de Gruyter, 1991. 179. Goodman WG, Coburn JW. The use of 1,25-dihydroxyvitamin D in early renal failure. Ann Rev Med 1992;43:227-237. 180. Christiansen C, Rodbro P, Christensen MS, Hartnack B, Transbol I. Deterioration of renal function during treatment of chronic renal failure with 1,25-dihydroxycholecalciferol. Lancet 1978;2: 700-702. 181. Bertoli M, Luisetto G, Ruffatti A, Urso M, Romagnoli G. Renal function during calcitriol therapy in chronic renal failure. Clin Nephrol 1990;33:98-102. 182. Cundy T, Hand DJ, Oliver DO, Woods CG, Wright FW, KanisJA. Who gets renal bone disease before beginning dialysis? Br Med J 1985;290:271-275. 183. Brown AJ, FinchJL, Lopez-Hilker S, Dusso A, Ritter C, Pernalete N, et al. New active analogues of vitamin D with low calcemic activity. Kidney Int 1990;29:$22-$27. 184. Takahashi E Finch JL, Denda M, Dusso AS, Brown AJ, Slatopolsky E. A new analog of 1,25-(OH)zD~, 19-nor-I, 25-(OH)zD 2, suppresses serum PTH and parathyroid gland growth in uremic rats without elevation of intestinal vitamin D receptor content. A m J Kidney Dis 1997;30:105-112. 185. Israel O, Gips S, Hardoff R, Rudoy J, Frajewicki V, Iosilevsky G, et al. Bone loss in patients with chronic renal disease: Prediction with quantitative bone scintigraphy with SPECT. Radiology

1995; 196:643-646.

186. Martin KJ, Gonzalez EA, Gellens M, Hamm LL, Abboud H, Lindberg J. 19-Nor-l~-25-dihydroxyvitamin D2 (paricalcitol) safely and effectively reduces the levels of intact parathyroid hormone in patients on hemodialysis. J Am Soc Nephrol 1998;9:1427-1432. 187. Tan AU, Jr, Leone BS, Mazess RB, Kyllo DM, Bishop CW, KnutsonJC, et al. Effective suppression of parathyroid hormone by 1 alpha-hydroxy-vitamin Dz in hemodialysis patients with moderate to severe secondary hyperparathyroidism. Kidney Int 1997;51:317-323. 188. Kurokawa K, Akizawa T, Suzuki M, Akiba T, Ogata E, Slatopolsky E. Effect of 22-oxacalcitriol on hyperparathyroidism of dialysis patients: Results of preliminary study. Nephrol Dial Transplant 1996;11:121-124. 189. Tsukamoto Y, Hanaoka M, Matsuo T, Saruta T, Nomura M, Takahashi Y. Effect of 22-oxacalcitriol on bone histology of hemodialyzed patients with severe secondary hyperparathyroidism. Am J Kidney Dis 2000;35:458-464. 190. Sasaki H, Harada H, Handa Y. Transcriptional activity of a fluorinated vitamin D analog on VDR-RXR-mediated gene expression. Biochemistry 1995;34:370-377. 191. Morii H, Ogura Y, Koshikawa S. Efficacy and safety of oral falecalcitriol in reducing parathyroid hormone in hemodialysis patients with secondary hyperparathyroidism. J Bone Miner Metab 1998;16:34-43. 192. Akiba T, Marumo F, Owada A. Controlled trial of falecalcitriol versus alfacalcidol in suppression of parathyroid hormone in hemodialysis patients with secondary hyperparathyriodism. Am J Kidney Dis 1998;32:238-246.

RENAL BONE DISEASES 193. Slatopolsky E, Finch J, Ritter C, Denda M, MorrisseyJ, Brown A, et al. A new analog of calcitriol, 19-nor-l,25-(OH) 2D2, suppresses parathyroid hormone secretion in uremic rats in the absence of hypercalcemia. Am J Kidney Dis 1995;26:852-860. 194. Sj6den G, Smith C, Lindgren JU, DeLuca HE l c~-hydroxyvitamin D 2 is less toxic than le~-hydroxyvitamin D3 in the rat. Proc Soc Exp Biol Med 1985;178:432-436. 195. Gallagher JC, Bishop CW, Knutson JC, Mazess RB, DeLuca HE Effects of increasing doses of lc~-hydroxyvitamin O 2 on calcium homeostasis in postmenopausal osteopenic women. J Bone Miner Res 1994;9:607-614. 196. Hansson T, Roos B. The relation between bone mineral content, experimental compression fractures, and disc degeneration in lumbar vertebrae. Spine 1981;6:147-153. 197. Frazao JM, Elangovan L, Chesney RW, Archiado SR, Bower JD, Kelly BJ, et al. One-alpha-hydroxyvitamin 02 (doxercalciferol) effectively and safely suppresses intact parathyroid hormone levels in hemodialysis patients with moderate to severe secondary hyperparathyroidism: Results of a multicenter double-blinded, placebo-controlled study. Am JKidney Dis 2000;36:550-561. 198. Brown EM, Pollak M, Hebert SC. The extracellular calciumsensing receptor: Its role in health and disease. A n n u Rev Med 1998;49:15-29. 199. Brown EM. Physiology and pathophysiology of the extracellular calcium-sensing receptor. Am J Med 1999;106:238-253. 200. Brown EM, Hebert SC. The First Annual Bayard D. Catherwood Memorial Lecture. Ca2+-receptor-mediated regulation of parathyroid and renal function. Am J Med Sci 1996;312:99-109. 201. Goodman WG, Frazao JM, Goodkin DA, Turner SA, Liu W, Coburn JW. A calcimimetic agent lowers plasma parathyroid hormone levels in patients with secondary hyperparathyroidism. Kidney Int 2000;58:436-445.

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202. Kifor O, Moore FD, Jr, Wang P, Goldstein M, Vassilev P, Kifor I, et al. Reduced immunostaining for the extracellular Ca+2-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 1996;81:1598-1606. 203. Gogusev J, Duchambon P, Hory B, Giovannini M, Goureau Y, Sarfati E, et al. Depressed expression of calcium receptor in parathyroid gland tissue of patients with hyperparathyroidism. Kidney Int 1997;51:328-336. 204. Rogers KV, Dunn CK, Conklin RL, Hadfield S, Petty BA, Brown EM, et al. Calcium receptor messenger ribonucleic acid levels in the parathyroid glands and kidney of vitamin D-deficient rats are not regulated by plasma calcium or 1,25-dihydroxyvitamin D 3. Endocrinology 1995;136:499-504. 205. Brown AJ, Zhong M, Finch J, Ritter C, McCracken R, Morrissey J, et al. Rat calcium-sensing receptor is regulated by vitamin D but not by calcium. Am J Physio11996;270:F454-F460. 206. Wada M, Furuya Y, SakiyamaJ, Kobayashi N, Miyata S, Ishii H, et al. The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. Control of parathyroid cell growth via a calcium receptor. J Clin Invest 1997;100:2977-2983. 207. Wada M, Ishii H, Furuya Y, Fox J, Nemeth EF, Nagano N. NPS R568 halts or reverses osteitis fibrosa in uremic rats. Kidney Int 1998;53:448-453. 208. Silverberg SJ, Bone III HG, Marriott TB, Locker FG, ThysJacobs S, Dziem G, et al. Short-term inhibition of parathyroid hormone secretion by a calcium-receptor agonist in patients with primary hyperparathyroidism. N Engl J Med 1997;337: 1506-1510. 209. Dawborn JK, Brown DJ, Douglas MC, Eddey HH, Heale WF, Thomas DP, et al. Parathyroidectomy in chronic renal failure. Nephron 1983;33:100-105.

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CI4aPTEk41 Evaluation of the Hypercalcemic Patient Differential Diagnosis

DAVID HEATH Department of Medicine, Selly Oak Hospital, Birmingham B29 6JD, United Kingdom

INTRODUCTION

clinical practice the condition is infrequently complicated by renal stones and rarely by overt parathyroid bone disease. The majority of patients are either asymptomatic or have vague, nonspecific symptoms (1). For this reason primary hyperparathyroidism is by far the commonest cause of hypercalcemia in nonhospitalized patients. With the sensitive two-site PTH assays, PTHis usually above the upper limit of the normal range, occasionally in the upper half of the normal range, and only rarely in the lower half of the normal range (2-4). A small number of surgically proved cases of primary hyperparathyroidism have been reported with low-normal or suppressed PTH concentrations (5-6).

There are many known causes of hypercalcemia, although in the vast majority of clinical situations the differential diagnosis is usually quite narrow. In fact, except in very specialized clinical centers, most patients encountered will have either primary hyperparathyroidism or malignancy as the cause of the hypercalcemia. This suggests that the most expedient approach to the diagnosis of hypercalcemia is initially to perform a limited set of investigations directed at these two conditions. Should these initial investigations fail to identify the diagnosis, further investigations can then be ordered to disclose the less common causes of hypercalcemia. The advent of two-site immunometric assays for parathyroid hormone (PTH) that can measure concentrations throughout the normal range, and even below normal, has greatly facilitated investigations of hypercalcemia. These PTH assays will usually distinguish the parathyroid from the nonparathyroid causes of hypercalcemia without difficulty (see Chapter 9). This chapter reviews the causes of hypercalcemia in adults and provides a suggested approach to investigating the patient with hypercalcemia.

Tertiary Hyperparathyroidism Prolonged stimulation of the parathyroid gland by hypocalcemia leads to parathyroid gland hyperplasia-secondary hyperparathyroidism. This typically occurs in untreated vitamin D deficiency and chronic renal impairment. In a percentage of these patients typically one, but often more than one, parathyroid gland will develop into an autonomous tumor, producing hypercalcemia with suppression of the nonautonomous glands. This is known as tertiary hyperparathyroidism. Progression from secondary hyperparathyroidism is often not documented but has been seen most frequently in the context of chronic renal failure. This condition is discussed more extensively in Chapters 18, 39, and 40.

PARATHYROID HORMONE-INDUCED CAUSES OF HYPERCALCEMIA Primary Hyperparathyroidism Primary hyperparathyroidism is one of the two most common causes of hypercalcemia. In current general The Parathyroids, Second Edition

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Familial Hypocalciuric Hypercalcemia This condition is discussed extensively in Chapter 38. It is mentioned here because PTH mediates the hypercalcemia in the context of markedly increased renal reabsorption of calcium, an increase due to the heterozygous inactivating mutation in the calciumsensing receptor gene that reduces sensitivity of the "calciostat." Younger patients usually have normal PTH concentrations but in older patients the serum PTH may be slightly elevated (7). Homozygous mutations and some heterozygous mutations are associated with severe neonatal primary hyperparathyroidism in which both the serum calcium and PTH are markedly elevated (8). Three cases of late recognition of severe neonatal primary hyperparathyroidism have now been reported; in two patients concentrations of PTH have been high (9-11).

Hypercalcemia Associated with Thiazide Diuretics Many authorities list thiazide diuretics as a cause of hypercalcemia. It is much more likely that hypercalcemia in patients on thiazide therapy represents disclosure of an underlying defect in mineral metabolism, most notably primary hyperparathyroidism (12). It may well be that thiazide therapy may cause a worsening of the hypercalcemia. In the past a test was described whereby thiazides were prescribed to patients with borderline hypercalcemia. If the serum calcium rose to definitely abnormal values this was interpreted to indicate primary hyperparathyroidism (13). Conversely, patients found to be hypercalcemic on thiazide diuretics almost invariably have elevated PTH concentrations. It is also exceptional for the serum calcium to return completely to normal on stopping the diuretics, and the serum PTH remains elevated.

Hypercalcemia Due to Lithium Hypercalcemia has been reported in a minority of patients receiving lithium therapy for manic depression (14). As with thiazide diuretics, evidence suggests that lithium potentiates preexisting hyperparathyroidism. In vitro, lithium can be shown to stimulate PTH secretion by cultured parathyroid cells (15). In the clinical context various situations have been documented. Serum concentrations of PTH are typically elevated in patients receiving lithium. After discontinuation of lithium, several responses are possible: (1) both calcium and PTH may remain elevated, (2) the calcium may fall but PTH remains elevated, or (3) both calcium and PTH may return to the normal range. If these patients are subjected to a neck exploration, in the majority of cases a parathyroid a d e n o m a is found, even

when the serum calcium level returned to normal after stopping the lithium. After a successful parathyroidectomy, lithium can be reinstituted without causing hypercalcemia (16).

NONPARATHYROID HORMONE-INDUCED CAUSES O F HYPERCALCEMIA Malignancy Once primary hyperparathyroidism has been excluded, malignancy is the other c o m m o n cause of hypercalcemia encountered in clinical practice. Hypercalcemia is a c o m m o n complication of malignancy, but its prevalence varies considerably among different malignancies as well as at different stages of the malignant process. Hypercalcemia most frequently complicates advanced malignancy and in the vast majority of cases knowledge of the malignant disorder precedes the development of hypercalcemia. It is unusual for the malignancy to be at a surgically curable stage, presumably because a large tumor burden is usually required for hypercalcemia to occur. As a consequence the disease is very frequently metastatic, though not necessarily to the bones. Patients with malignancy and associated hypercalcemia are usually obviously unwell from a combination of the malignant process and the superimposed symptoms of hypercalcemia. In a retrospective study of 219 cases of malignancyassociated hypercalcemia, in only 4 was the hypercalcemia identified before the malignancy, and in 3 of these 4 the malignant process was rapidly identified (17). Hypercalcemia found by chance in an otherwise well patient is almost never due to malignancy. The malignancies most commonly associated with hypercalcemia are carcinoma of the lung and breast. Though in part it is a reflection of the commonness of these specific tumors, there is generally an overrepresentation of tumors that arise from squamous epithelial cells. The understanding of the etiology of the basis for malignancy-associated hypercalcemia has evolved rapidly over the past 10 to 15 years. It is now thought that a humoral agent is responsible for the majority of cases, with parathyroid hormone-related protein (PTHrP) being the most commonly implicated agent. A n u m b e r of the various cytokines may also be involved in isolation or in conjunction with PTHrE Although the concept of ectopic PTH production was used in the past to explain this syndrome of malignancy-associated hypercalcemia, we now know that genuine PTH production by nonparathyroid tumors is exceedingly rare (18-21). PTHrP mimics most, if not all, actions of PTH because it acts on the same receptor (i.e., the P T H / P T H r P receptor). This explains why many of the

HYPERCALCEMIA: DIFFERENTIALDIAGNOSIS / biochemical changes in hypercalcemia of malignancy and hyperparathyroidism are similar (e.g., serum and urinary phosphate, and urinary cyclic AMP excretion). Fortunately, although PTH and PTHrP act on the same receptor, the peptides are quite distinct structurally so that contemporary immunoassays for PTH do not crossreact with PTHrE This means that in malignancyassociated hypercalcemia, PTH concentrations are usually suppressed. As mentioned earlier, very rarely PTH can be secreted by nonparathyroid tumors when, in the few cases reported, PTH concentrations have been markedly elevated. Because primary hyperparathyroidism is a relatively c o m m o n disorder, especially in the elderly, occasional coincidental occurrence of malignancy and primary hyperparathyroidism in hypercalcemic patients is not surprising. Patients with hypercalcemia due to malignancy have, on the whole, a very poor prognosis, and many patients, despite treatment of their malignancy and the hypercalcemia, die within 6 months. As a result, long-term consequences of the hypercalcemia are very rare. Because PTHrP is considered the most c o m m o n humoral agent responsible for hypercalcemia in malignancy, and because it acts on the c o m m o n P T H / P T H r P receptor, there is, in theory, the possibility that renal stones a n d / o r parathyroid bone disease could occur. This has been reported but is extremely rare (22). The reason for this is that very few patients live long enough to develop these complications. For it to occur there needs to be prolonged P T H / P T H r P receptor stimulation, probably for a matter of years. Certain malignancies are typically very slowly progressive and may have a relatively good prognosis even when metastatic. A n u m b e r of endocrine tumors fall into this category, e.g., islet cell tumors, thyroid tumors, carcinoids, and pheochromocytomas. Some of these tumors have been known to be metastatic for many years and the patient has a good quality of life. Many of these tumors can secrete PTHrP and thereby cause hypercalcemia and, very rarely, radiologic signs of osteitis fibrosa cystica. PTHrP production by benign tumors (23) and mammary hyperplasia (24) has also been reported and would be anticipated to mimic the clinical picture of primary hyperparathyroidism.

Vitamin D Intoxication Calciferol administration in physiologic doses, e.g., 500 IU units/day, only exceptionally causes hypercalcemia (25). Pharmacologic doses of calciferol in doses of 50,000-200,000 units/day are used to treat hypoparathyroidism and unless carefully monitored can cause hypercalcemia. Use of such large doses of calciferol has been replaced, by many clinicians, with the

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active vitamin D metabolitesmalfacalcidol and calcitriol. Normal replacement doses in hypoparathyroidism are between 0.5 and 2 Ixg/day and both agents in such doses can cause hypercalcemia. The same two drugs have also found extensive use in chronic renal disease to reduce the incidence of renal osteodystrophy, and their use is one of the causes of hypercalcemia complicating chronic renal failure. Vitamin D analogs are being used as topical treatments of acne and can cause hypercalcemia (26). When hypercalcemia complicates vitamin D therapy, it almost always is associated with a physician's prescription of the vitamin and should readily be revealed by a careful drug history. Very occasionally the vitamin D can come from other sources, such as supplemented health foods and the overzealous use of nonprescription vitamin preparations. Vitamin A poisoning also causes hypercalcemia and has been seen with excessive vitamin A consumption or in patients eating large amounts of liver from carnivores (e.g., polar bear) (27). Retinoic acid analogs have also been used to treat severe acne and can lead to hypercalcemia (28).

Calcium Ingestion The ingestion of normal amounts of calcium in the diet or the use of calcium supplements in the amounts r e c o m m e n d e d for osteoporosis treatment do not cause hypercalcemia. For many years aluminum hydroxide was used to bind phosphate in the gut in patients with chronic renal disease. Because of occasional problems with aluminum toxicity there was a change in the use of oral calcium carbonate. Although this avoided alum i n u m toxicity, it was at a cost of a not insignificant incidence of hypercalcemia, perhaps because most of the patients were also receiving vitamin D metabolites (29). The milk-alkali syndrome was originally associated with therapy of peptic ulceration with large quantities of milk together with high doses of calcium-containing antacids. The advent of highly effective alternative treatments of peptic ulceration has made this cause of hypercalcemia virtually nonexistent. It is, however, seen occasionally in people who abuse nonproprietary indigestion remedies that have high calcium and bicarbonate contents, as well as other remedies that give a very high calcium intake (30,31).

Sarcoidosis and Other Granulomatous Disease Hypercalcemia is a very well-documented complication of sarcoidosis and the mechanism has been convincingly shown to be due to the extrarenal conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D by

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the sarcoid granulomatous cells. This conversion is blocked by corticosteroid administration. The incidence of hypercalcemia in sarcoidosis varies considerably in reported series, but probably occurs in less than 10% of cases (32). In some cases it has been recurrent and associated with exposure to ultraviolet light, usually sunlight. Although clinical evidence of sarcoidosis is usually present this is not always the case. When hypercalcemia is due to sarcoidosis, endogenous PTH secretion is suppressed. However, sarcoidosis and hyperparathyroidism have been reported to occur in the same patient in what is almost certainly a coincidental finding. Such cases are readily identified by the finding of an elevated rather than a suppressed PTH and a failure of the hypercalcemia to be corrected by steroid administration. Hypercalcemia has also been reported as a complication of other granulomatous diseases. There remains considerable debate as to whether hypercalcemia is a genuine complication of tuberculosis (33). Undoubtedly hypercalcemia has been reported but there is very considerable variation between series. There is some evidence to suggest that those series in which hypercalcemia has been found the patients are also receiving vitamin D supplementation or are in countries where endogenous vitamin D concentrations are naturally higher (34). Hypercalcemia with elevated circulating levels of 1,25-dihydroxyvitamin D has recently been reported as a complication in Crohn's disease (35). Treatment with prednisolone corrected the hypercalcemia. A similar situation has been described in Wegener's granulomatosis (36). A patient with hypercalcemia with methotrexate-induced pneumonitis and pulmonary granulomata has been reported (37). Beryliosis, as well as silicone, can cause a similar granulomatosis reaction that leads to hypercalcemia (38,39).

Thyrotoxicosis In thyrotoxicosis there is increased bone turnover with both increased bone formation and bone resorption. In a group of patients with thyrotoxicosis the mean serum calcium is higher than euthyroid controls, although the vast majority of patients have a normal serum calcium. The prevalence of hypercalcemia in recorded series of patients varies considerably, with figures as high at 20% being quoted. General experience suggests that hypercalcemia is, in fact, a relatively uncomm o n complication of hyperthyroidism and is only seen in patients with severe thyroid overactivity. When it occurs serum PTH concentrations are suppressed. The coincidental association of hyperthyroidism and hyperparathyroidism is well recorded and would be associated with elevated PTH concentrations. The hypercalcemia of

thyrotoxicosis resolves as the thyroid overactivity is controlled, but may take a n u m b e r of weeks (40).

Immobilization Immobilization is a well-documented cause of hypercalcemia and hypercalciuria. The prerequisite for it to develop is a generalized increase in bone turnover, together with immobilization of much of the body. It therefore typically occurs in adolescents following tetraplegia or immobilization of both legs within a hip spica. Serum PTH concentrations are reduced and the hypercalcemia resolves rapidly once the patient is mobilized (41).

Recovery Phase of Acute Renal Failure Hypercalcemia can occur for a variety of reasons in chronic renal failure. It also occurs in acute renal failure, typically during the polyuric recovery phase following acute renal failure associated with extensive rhabdomyolysis (42). The etiology is thought to be due to the release of calcium from damaged muscle. PTH concentrations are suppressed.

Parenteral Nutrition With the development of parenteral nutrition, reports soon emerged of its association with hypercalcemia, although the exact mechanism was unclear (43).

Adrenocortical Insufficiency Hypercalcemia has been reported in acute adrenocortical failure. Its exact incidence is not clear but it is likely to be an u n c o m m o n complication. The exact mechanism is unclear and PTH concentrations are normal or low. It is rapidly corrected by steroid therapy (44).

Infective Causes Various infections have been reported, usually as case reports, to be causes of hypercalcemia. The possibility of tuberculosis being complicated by hypercalcemia has been mentioned earlier. Other infections include Pneumocytis carinii p n e u m o n i a (45), brucellosis (46), cat scratch fever (47), coccidiomycosis (48), histoplasmosis (49), Nocardia asteroides (50), candidiasis (51) and leprosy (52).

Other Reported Causes Other reported causes of hypercalcemia include fat necrosis (53), Gaucher's disease (54), and hypereosinophilic syndrome (55).

HYPERCALCEMIA: DIFFERENTIALDIAGNOSIS /

A N A P P R O A C H T O T H E PATIENT WITH HYPERCALCEMIA Although there are many causes of hypercalcemia, the vast majority of causes need not be considered when dealing with cases of hypercalcemia typically encountered in practice. Outside the context of specialized units, e.g. renal clinics, studies have shown that about 97% of cases of hypercalcemia encountered in hospital or outpatient practice either have primary hyperparathyroidism or malignancy as a cause of the hypercalcemia. Of the rarer causes of hypercalcemia, many can be determined by taking a careful history and making a detailed clinical examination. The two c o m m o n causes, hyperparathyroidism and malignancy, usually have quite different presentations. Patients with hyperparathyroidism are rarely very ill. Many are asymptomatic, or if symptomatic, in a very nonspecific way. Consequently, patients who are found to be hypercalcemic and referred for outpatient investigation usually have hyperparathyroidism and uncommonly malignancy. In hypercalcemia due to malignancy, as discussed earlier, the hypercalcemia usually complicates already clinically apparent malignancy. The patients are ill from their malignant state with superadded symptoms from the hypercalcemia. Weight loss, anemia, hypoalbuminemia, and abnormal liver function tests are common, all of which would be unusual in hyperparathyroidism. Hence, on first meeting the patient the clinician is usually able to make a fairly accurate prediction of the likely cause of the hypercalcemia. If advanced or extensive malignancy is known to be currently present, it is probably not necessary to investigate the patient further for other causes of hypercalcemia. Efforts should be made to treat the malignancy, to control the hypercalcemia, and then to review. If, on controlling the malignant process, the hypercalcemia persists, then further investigations will be necessary and it is likely that the patient will be found to have coincidental hyperparathyroidism. If there is a history of mild malignancy or past malignancy, it is mandatory to investigate the patient further, because malignancy is probably not the cause, and once again coincidental hyperparathyroidism may well be present. If there is no evidence of malignancy or any other obvious cause of the hypercalcemia, a serum PTH measurement should be measured and further investigations delayed until the result is available. Three PTH results are possible: elevated, normal, and suppressed.

Elevated P T H Elevated PTH makes the diagnosis of primary hyperparathyroidism very likely. Another possibility is FHH,

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in which a small percentage of patients with this diagnosis will have elevated PTH values. In the absence of documentation of life-long asymptomatic hypercalcemia, the differentiation between the two conditions can be difficult. Although patients with F H H have, on average, lower urine calcium excretion values and higher average serum magnesium concentrations, these measurements do not reliably differentiate the two conditions. Patients with F H H do not have symptoms and complications of their disease. Patients with hyperparathyroidism also often do not have symptoms and complications of the disorder. If an asymptomatic patient is thought to have hyperparathyroidism and reco m m e n d e d to u n d e r g o a parathyroidectomy if the PTH is only slightly elevated, the possibility of FHH should be actively excluded wherever possible by careful review of past clinical chemistry records and by screening immediate members of the family for hypercalcemia. A third possibility for an elevated PTH are the very rare cases of true ectopic PTH production by nonparathyroid tumors.

Normal PTH Hyperparathyroidism can be associated with normal PTH concentrations, but in this situation values are usually a r o u n d the u p p e r end of the normal range. The other c o m m o n cause of hypercalcemia with a normal PTH is FHH. In this situation, the diagnosis of FHH must be actively pursued and wherever possible this should include family screening. If other hypercalcemic family members are found, also with normal PTH concentrations, then F H H would almost certainly be the correct diagnosis, especially if hypercalcemic children are identified. Obviously, familial hypercalcemia can occur in multiple endocrine neoplasia types 1 and 2. Here, other features of the disease may be present in some family members, PTH concentrations are usually elevated, and hypercalcemic children are exceptional.

Low P T H Although one or two cases of proved hyperparathyroidism have been reported with low PTH concentrations, this is exceptionally rare. It is the typical situation in malignancy and all the other non-PTH-mediated causes of hypercalcemia. If there is no evidence of malignancy then it is still possible that malignancy could still be the cause. Occult malignancies causing hypercalcemia are more likely to be low-grade hematological or lymphoid malignancies. Bone marrow examination and white cell marker studies may be indicated. Of the other nonmalignant, nonparathyroid causes of hypercalcemia, most will give some clue to the

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diagnosis; a possible exception is sarcoidosis. If this is a possibility a steroid suppression test can give a clue, because the hypercalcemia should be completely corrected by 10 days of steroid therapy, e.g., prednisolone (20 mg daily)--suppression may also occur in hematologic and lymphoid malignancies and other granulomatous diseases. Following a positive steroid suppression test a liver biopsy may be indicated, looking for evidence of hepatic granulomatas. Although most cases of hypercalcemia can be rapidly and correctly diagnosed, there are always difficult and atypical cases. Failure to make a diagnosis quickly should lead to referral of the case to a clinician with a special interest in calcium disorders.

REFERENCES 1. Mundy GR, Cove DH, Fisken R, Heath DA, Somers S. Primary hyperparathyroidism: Changes in the pattern of clinical presentation. Lancet 1980; 1:1317-1320. 2. Nussbaum SR, Zahradmk RJ, Lavigne JR, et al. Highly sensitive two-site immunoradiometric assay of parathyrin and its clinical utility in evaluating patients with hypercalcemia. Clin Chem 1987;33:1364-1367. 3. Ratcliffe WA, Heath DA, Ryan M, Jones SR. Performance and diagnostic application of a two-site immunoradiometric assay for parathyrin in serum. Clin Chem 1989;35:1957-1961. 4. Mischis-Troussard C, Goudet P, Verges B, Cougard P, Tavernier C, Maillefert J-F. Primary hyperparathyroidism with normal serum intact parathyroid hormone levels. QJ Med 2000;93:365-367. 5. Hollenberg AN, Arnold A. Hypercalcemia with low-normal serum intact PTH: A novel presentation of primary hyperparathyroidism. A m J Med 1991;91:547-548. 6. Glendenning P, Gutteridge DH, Retallack RW, et al. High prevalence of normal total calcium and intact PTH in 60 patients with proven primary hyperparathyroidism: A challenge to current diagnostic criteria. Aust N ZJMed 1998;28:173-178. 7. McMurtry CT, Schranck FW, Walkenhorst DA, et al. Significant developmental elevation in serum parathyroid hormone levels in a large kindred with familial benign (hypocalciuric) hypercalcemia. A m J M e d 1992;93:247-258. 8. Marx J, Attie ME Spiegel AM, Levine MA, Lasker RD, Fox M. An association between neonatal severe primary hyperparathyroidism and familial hypocalciuric hypercalcemia in three kindreds. N E n g l J Med 1982;306:257-264. 9. Cole DEC, Forsythe CR, DooleyJM, Grantmyre EB, Salisbury SR. Primary neonatal hyperparathyroidism: A devastating neurodevelopmental disorder if left untreated. J Craniofac Genet Dev Biol 1990; 10:205-214. 10. Arda K, Koishi S, Inove M, Nakazato M, Tawata M, Onaya T. Familial hypocalciuric hypercalcemia associated with mutation in the human CaZ+-sensing receptor gene. J Clin Endocrinol Metab 1995;80:2594-2598. 11. Chikatsu N, Fukomoto S, Suzawa M, Tanaka Y, Takeuchi Y, Takeda S, Tamura Y, Matsumoto T, Fujita T. An adult patient with severe hypercalcemia and hypocalciuria due to a novel homozygous inactivating mutation of calcium-sensing receptor. Clin Endocrinol 1999;50:537-543. 12. Farquhar CW, Spathis GS, Barron JL, Levin GE. Failure of thiazide diuretics to increase plasma calcium in mild primary hyperparathyroidism. Postgrad MedJ 1990;66:714-716.

13. SodeJ, SabolJJ, Meloni CR, CanaryJJ. Thiazide challenge in primary hyperparathyroidism. Clin Res 1969;17:550. 14. Mallette LE, Eickhorn E. Effects of lithium carbonate on human calcium metabolism. Arch Intern Med 1986;146:770-776. 15. Birnbaum J, Klandorf H, Guiliano A, Van Herle A. Lithium stimulates the release of human parathyroid hormone in vitro. J Clin Endocrinol Metab 1988;66:1187-1191. 16. Christensson TAT. Lithium, hypercalcemia and hyperparathyroidism. Lancet 1976;2:144. 17. Fisken RA,' Heath DA, Bold AM. Hypercalcemia--a hospital survey. QJ Med 1980;49:405-418. 18. Yoshimoto K, Yamasaki R, Sakai H, et al. Ectopic production of PTH by small cell lung cancer in a patient with hypercalcemia. J Clin Endocrinol Metab 1989;68:976-981. 19. Nussbaum SR, Gaz RD, Arnold A. Hypercalcemia and ectopic secretion of parathyroid hormone by an ovarian carcinoma with rearrangement of the gene for PTH. N Engl J Med 1999;323; 1324-1328. 20. Strewler GJ, Budayr AA, Ruce RJ, Clark OH, Nissenson RA. Secretion of authentic PTH by a malignant tumour. Clin Res 1990;38:462A. 21. Iguchi H, Miyagi C, Tomita K, et al. Hypercalcemia caused by ectopic production of parathyroid hormone in a patient with papillary adenocarcinoma of the thyroid gland. J Clin Endocrinol Metab 1998;83:2653-2657. 22. Loveridge N, Kent GN, Heath DA, Jones EI. Parathyroid hormone-like bioactivity in a patient with severe osteitis fibrosa cystica due to malignancy: Renotropic actions of a tumour extract as assessed by cytochemical bioassay. Clin Endocrinol 1985;22: 135-146. 23. Knecht TP, Behling CA, Burton DW, Glass CK, Deftos LJ. The humoral hypercalcemia of benignancy. A newly appreciated syndrome. Am J Clin Patho11996;105 :487-492. 24. Khosla S, van Heerden JA, Gharib H, et al. Parathyroid hormonerelated protein and hypercalcemia secondary to massive mammary hyperplasia. N EnglJ Med 1990;322:1157. 25. Jansen TL, Janssen M, de Jong AJ. Severe hypercalcemia syndrome with daily low-dose vitamin D supplementation. Br J Rheumatol 1997;36:712-713. 26. Hardman KA, Heath DA, Nelson HM. Hypercalcemia associated with calcipotriol (Dovonex) treatment. Br Med J 1993;306: 1344-1345. 27. Katz CM, Tzagournis M. Chronic adult hypervitaminosis A with hypercalcemia. Metabolism 1972;21:1171-1176. 28. Valente JD, Elias AN, Weinstein GD. Hypercalcemia associated with oral isotretinoin in the treatment of severe acne. JAMA 1983;250:1899. 29. Greaves I, Grant AJ, Heath DA, Michael J, Adu D. Hypercalcemia: Changing causes over the past 10 years. Br MedJ 1992;304:1284. 30. Muldowney WP, Mazbar SA. Rolaids-yogurt syndrome: A 1990s version of milk-alkali syndrome. Am J Kidney Dis 1996;27: 2709-2712. 31. Wu KD, Chuang RB, Wu FL, Hsu WA, Jan IS, Tsai KS. The milk-alkali syndrome caused by betelnuts in oyster shell paste. J Toxicol Clin Toxico11996;34:741-745. 32. Sharma OE Vitamin D, calcium, and sarcoidosis. Chest 1996;109:535-539. 33. Kelestimur F, Guven M, Ozesmi M, Pasaoglu H. Does tuberculosis really cause hypercalcemia? J Endocrinol Invest 1996;19: 678-681. 34. Chan TY. Differences in vitamin D status and calcium intake: Possible explanations for the regional variations in the prevalance of hypercalcemia in tuberculosis. Calcif Tissue Int 1997;60:91-93. 35. Bosch X. Hypercalcemia due to endogenous overproduction of 1,25 dihydroxyvitamin D in Crohn's disease. Gastroenterology 1998;114:1061-1065.

HYPERCALCEMIA: DIFFERENTIAL DIAGNOSIS 36. Bosch X, Lopez-Soto A, Morello A, Olmo A, Urbano-Marquez A. Vitamin D metabolite-mediated hypercalcemia in Wegener's granulomatosis. Mayo Clin Proc 1997;72:440-444. 37. Cook NJ, Lake FR. Hypercalcemia with methotrexate pneumonitis, possible association with pulmonary granulomata. Aust N Z J Med 1996;26:715. 38. Waldron HA, Scott A. In Raffle PAB, Adams PH, Baxter PJ, Lee WR, eds. Hunter's diseases of occupations, 8th Ed. London:Arnold, 118. 39. Kozeny GA, Barbato AL, Bansal VK, Vertuno LL, Hano JE. Hypercalcemia associated with silicone-induced granulomas. N EnglJ Med 1984;311:1103-1105. 40. Twycross RE, Markes V. Symptomatic hypercalcemia in thyrotoxicosis. Br MedJ 1970;2:701-703. 41. Stewart AF, Adler M, Byers CM, Segre GV, Broadus AE. Calcium homeostasis in immobilisation: An example of resorptive hypercalcemia. N EnglJ Med 1982;306:1136-1140. 42. Akmal H, Bishop JE, Telfer N, Norman AW, Kassry SE. Hypocalcemia and hypercalcemia in patients with rhabdomyolysis with and without acute renal failure. J Clin Endocrinol Metab 1986;63:137-142. 43. Hurley DL, McMahon MM. Long-term parenteral nutrition and metabolic bone disease. Endocrinol Metab Clin North Am 1990;19:113-131. 44. Muls E, Bouillon R, BoelaertJ, et al. Etiology of hypercalcemia in a patient with Addison's disease. Calcif Tissue Int 1982;34:523-526. 45. Ahmed B, Jaspan JM. Case report: Hypercalcemia in a patient with AIDS and Pneumocystis carinii pneumonia. Am Med Sci 1993; 306:313-316.

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46. Malik GM. High serum calcium in human brucellosis: A case control study Am Trop Med Hyg 1998;59:397-398. 47. Bosch X. Hypercalcemia due to endogenous overproduction of active vitamin D in identical twins with cat-stratch disease. JAMA 1998;279:532-534. 48. Lee JC, Catanzaro A, Parthemore JG, Roach B, Deftos LJ. Hypercalcemia in disseminated coccidiomyosis. N Engl J Med 1977;297:431-432. 49. Steele CJ, Kleiman MB. Disseminated histoplasmosis, hypercalcemia and failure to thrive. Pediatr Infect Dis 1994;13: 421-422. 50. Dockrell DH, Poland GA. Hypercalcemia in a patient with hypoparathyroidism and Nocardia asteroides infection: A novel observation. Mayo Clin Proc 1997;72:757-760. 51. Kantarijian HM, Saad ME Estey EH, Sellin RV, Samaan NA. Hypercalcemia in disseminated candidiasis. Am J Med 1983;74: 721-724. 52. Hoffman VH, Korzeniowski OM. Leprosy, hypercalcemia and elevated serum calcitriol levels. Ann Intern Med 1986;105: 890-891. 53. Kruse K, Irle U, Uhlig R. Elevated 1,25-dihydroxyvitamin D serum concentrations in infants with subcutaneous fat necrosis. JPediatr 1993;122:460-463. 54. Byrne CD, Bermann L, Constant C, Cox TM. Pathological bone fractures preceded by sustained hypercalcemia in type 1 Gaucher's disease. J Inherit Metab Dis 1997;5: 709-710. 55. Lacotte L, Delwait V, Sadoun A, et al. Hypercalcemia in essential eosinophilic syndrome. Ann Med Intern 1996;147: 595-596.

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CAeTWR 42

H y p e r c a l c e m i a D u e to P T H r P

RICHARD KREMER Department of Medicine, McGill University, and Calcium Research Laboratory, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1

DAVID GO LTZMAN Departments of Medicine and Physiology, McGiU University, and Calcium Research Laboratory, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1

INTRODUCTION

chemical alterations mimicked that seen in primary hyperparathyroidism. Despite the presence of these in vivo biologic indices of increased parathyroid hormone (PTH)-like activity, a reduction in circulating concentrations of immunoreactive PTH was the predominant finding. A n in vitro cytochemical bioassay, initially developed for identification of PTH, was successful at measuring what we now know to be PTHrP in the plasma of patients with malignancy-associated hypercalcemia (5). This assay, based on stimulation of glucose-6-phosphate dehydrogenase activity (G6PD) in renal distal tubule cells, is exquisitely sensitive. Not only did it permit the initial measurement of PTHrP in h u m a n plasma, but also facilitated the demonstration that the PTH-like bioactivity detected in the plasma of hypercalcemic cancer patients was chromatographically and immunologically distinct from PTH (Fig. 1). This bioassay was later used to identify and to characterize PTHrP in extracts of tumors associated with hypercalcemia in vivo (6). In subsequent studies, both renal (7,8) and skeletal (9) in vitro adenylate cyclase bioassays were shown to detect PTH-like bioactivity in extracts of tumors associated with this disorder and also in the conditioned medium of tumors grown in tissue culture. The activity in these in vitro assays could be inhibited by the syn8 18 34 thetic PTH antagonist [Nle' ,Tyr ]bPTH(3-34) NH 2, suggesting that the tumor-derived material was acting via PTH receptors despite the fact that it was not PTH. In vivo PTH-like bioactivity of partially purified material extracted from h u m a n and rodent tumors, or from conditioned medium of rodent tumors, was then

In this chapter, we begin by discussing the history of the chemical identification of parathyroid hormonerelated peptide (PTHrP). We next examine the nature of the circulating forms of PTHrP and available information on the metabolism of this molecule. We then describe important aspects of the biology of PTHrP in studies in vitro and in in vivo animal models of hypercalcemia, emphasizing pathophysiologic and pharmacologic information that is of relevance for understanding the role of PTHrP in humans. Finally, we summarize available data on h u m a n disorders, emphasizing malignancy, but also referring to other clinical conditions that have been investigated.

I D E N T I F I C A T I O N OF P T H r P Following the initial association of hypercalcemia with cancer, the major hypercalcemic factor, parathyroid hormone-related peptide, was identified by careful observation and interpretation of in vivo and in vitro biologic data accumulated over several decades (1-4). The search for a pathogenetic agent in malignancyassociated hypercalcemia was facilitated by recognition that biochemical abnormalities besides hypercalcemia occurred frequently in these patients. These other features included hypophosphatemia, increased renal phosphate clearance, decreased fractional calcium excretion, and increased nephrogenous cyclic AMP (NcAMP) excretion. Overall, this constellation of bioThe Parathyroids, Second Edition

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Froction number FIG. 1 Gel filtration analysis of cytochemical bioactivity in plasma from a patient with primary hyperparathyroidism (A) and from a patient with malignancy-associated hypercalcemia (B). Vertical arrows from left to right denote, respectively, the elution position of the void volume (Vo), labeled intact PTH(1-84), labeled active PTH(1-34), and salt. Note that in contrast to primary hyperparathyroidism, in malignancy-associated hypercalcemia heterogeneous bioactive forms were observed. (From Goltzman D, Bennett HPJ, Koutsilieris M, Mitchell J, Rabbani SA, Rouleau MF. Rec Prog Horm Res 1986;42:665-703, with permission.)

demonstrated (10). These extracts, when infused into parathyroidectomized rats, induced phosphaturia, increased urinary cyclic AMP excretion, and prevented the decrease in serum calcium that occurred after parathyroidectomy. These in vivo bioassays therefore confirmed the PTH-like nature of the material identified first by in vitro assays. It was then possible, using the cytochemical bioassay, to identify PTH-like bioactivity in the conditioned m e d i u m of cultured Xenopus oocytes that had been microinjected with polyadenylated messenger RNA isolated from several different h u m a n and rodent tumors. This observation demonstrated that the bioactive material was indeed a secreted peptide (11). Ultimately, in vitro adenylate cyclase bioassays were successfully employed to monitor the biochemical purification of tumor-derived material, and a short NHz-terminal fragm e n t of PTHrP was isolated and sequenced (7,12,13). The sequence was then employed, using molecular biologic techniques, to clone cDNAs encoding PTHrP

(14,15).

The deduced amino acid sequence of the initial PTHrP that was cloned from PTHrP cDNAs included a leader sequence, a "pro" sequence of approximately 36 amino acids, and a mature peptide of 141 residues. A high degree of homology with PTH was observed within the first 13 NH2-terminal amino acids. This homology appeared to account for the PTH-like bioactivity of PTHrP, resulting in the PTH-like biochemical abnormalities of malignancy-associated hypercalcemia. Further evidence to account for the similar bioactivities of these peptides was provided by the cloning of a comm o n P T H / P T H r P receptor (i.e., type 1 PTH receptor) with which both ligands interact (16). The P T H / P T H r P receptor was found to be a seven-transmembranespanning, G protein-coupled receptor. It is linked to both the adenylate cyclase and the phospholipase C transducdon systems, although to date, the bulk of existing evidence implicates cyclic AMP as the major mediator of the action of PTH and PTHrP. The receptor binds the NH2-terminal regions of both PTH and PTHrP with approximately equal affinity. A second G proteincoupled receptor, termed PTH receptor type, 2 localized predominantly in brain, pancreas, and testis, was subsequently identified (17); it binds the NH2-terminal end of PTH with considerably higher affinity than it does the NH2-terminal domain of PTHrP. More recently a gene encoding a third form of PTH receptor was identified in zebrafish (17a). This receptor appears to bind only PTHrP. The function of these additional receptors is currently uncertain. The P T H / P T H r P receptor has been found in both bone and kidney and is therefore in the appropriate site to transduce the classic biologic functions of the Nterminal domains of these entities in osseous and renal tissues. In bone, however, the receptor has been localized to stromal or preosteoblastic cells as well as to mature osteoblasts. This is consistent with previous hypotheses and in vivo studies examining the binding sites for PTH and PTHrP in bone. The localization of P T H / P T H r P receptors to osteoblastic cells suggests that the capacity of PTHrP and PTH to resorb bone must be indirect, requiring the release of mediators that then stimulate osteoclast formation and action. The tumor necrosis factor (TNF)-like molecule RANK (receptor activation of NF-KB) ligand (RANKL), also called osteoclast differentiation factor, or ODF (18), has been implicated as the likely mediator. This entity, after release from osteoblastic cells in response to PTHrP or PTH stimulation, can bind to its cognate receptor, RANK, on osteoclast precursors and enhance osteoclast development (18,19-21). A naturally occurring decoy soluble receptor, termed osteoprotegerin (OPG) (22,23), can also be released by osteoblastic stromal cells and bind RANKL, preventing its access to preosteoclast receptor sites and thereby inhibiting

PTHrP AND HYPERCALCEMIA / osteoclastic bone resorption. This system appears also to be involved in mediating the excessive bone resorption associated with hypercalcemia. Finally, evidence that the P T H / P T H r P receptor is responsible for transducing the majority of the bioactivity of PTH and PTHrP in humans was provided by the demonstration that an activating mutation of the receptor results in Jansen's metaphyseal chondrodystrophy (24), a disorder characterized by hypercalcemia and hypophosphatemia typical of hyperparathyroidism, as well as by osteochondrodystrophy typical of a developmental anomaly associated with excess PTHrP in the fetus. Despite the similar bioactivities of the NHz-terminal domains of PTH and PTHrP, differences between the manifestations of primary hyperparathyroidism and of malignancy-associated hypercalcemia are apparent. Osteoclastic bone resorption is enhanced in both disorders, but diminished bone formation, resulting in "uncoupling" of resorption and formation, is seen in malignancy-associated hypercalcemia (25-27). Additionally, in h u m a n subjects with malignancy-associated hypercalcemia, plasma 1,25(OH)203 concentrations are often decreased (4), whereas in primary hyperparathyroidism 1,25 (OH) 203 concentrations are in the upper range of normal or are actually elevated (28). The NHz-terminal bioactive region of PTHrP can, however, increase l e~-hydroxylase activity when infused into human subjects (29). To account for these observations, it has been suggested that other regions of the PTHrP molecule may modify the capacity of the NH zterminal region to increase l~-hydroxylase activity and that other factors released by the tumor may inhibit le~hydroxylase activity (30). Finally, the severe hypercalcemia associated with the malignancy itself may inhibit 1ot-hydroxylase activity. Cloning of cDNAs encoding rat PTHrP, and subsequently other species of PTHrP, disclosed considerable amino acid sequence homology with the h u m a n form of PTHrP up to approximately residue 110 (31-33). The highly conserved sequence between residues 13 and 110 may therefore be of functional importance, although the precise biologic role or roles of this region are at present uncertain. A midregion domain has been implicated in transplancental calcium transport. Additionally, a functional nuclear localization sequence has been identified within the (87-107) region that may direct PTHrP into the nucleolus (34,35) and that may account for the ability of PTHrP to inhibit apoptosis in target cells. Furthermore, carboxyl-terminal fragments induce calcium transients in hippocampal neurons (36), and a small carboxyl-terminal pentapeptide of PTHrP, PTHrP(107-139), also called osteostatin, has been reported to act as a potent inhibitor of osteoclastic bone resorption in vitro

673

(37-39). Whether such a small peptide acts as a systemic factor or is produced a n d / o r metabolized locally by bone cells from a larger precursor is unknown. In subsequent work, the gene structure of PTHrP in several species was elucidated (40-43) and revealed several important features. The gene was found to be a complex transcriptional unit spanning over 15 kb of DNA. The PTHrP (12p) and PTH (11p) genes share a common exonic organization, with separate exons encoding corresponding functional domains such as the leader and pro sequence. Together with other observations this organizational format provided evidence that the PTHrP and PTH genes probably arose from a common ancestral gene. The human PTHrP gene consists of at least seven exons and is driven by several promoter sequences at the 5' end. At the 3' end, alternative splicing may occur, resulting in three potential peptide isoforms of 139, 141, and 173 amino acids, each with a different COOH-terminal sequence. Consequently, peptide heterogeneity and messenger RNA transcript heterogeneity may result from both alternative splicing and alternative promoter utilization. These studies therefore defined the precise chemical features of PTHrP, an essential step in the development of immunologic methods of measurement. They proceeded from careful analysis of the biologic issues related to malignancy-associated hypercalcemia, through the development of bioassays for in vivo and in vitro measurement of the pathogenetic entity, to the application of biochemical and molecular biologic techniques for final identification of the novel mediator.

METABOLISM OF PTHrP Considerable effort has been made to define the molecular forms of PTHrP that may be produced, secreted, and metabolized.

In Vitro Studies: PTHrP Synthesis, Processing,

and Secretion

In addition to alternative splicing, which may lead to the production of three isoforms of h u m a n PTHrP, there is evidence to suggest that heterogeneous forms of PTHrP may also arise from posttranslational processing. Extracts of tumors contain multiple PTHrP molecules of different sizes (44,45). Parathyroid tissue appears to produce a single immunoreactive PTHrP species that migrates with PTHrP(1-84) (46). The lactating mammary gland also produces PTHrP, which (47), although not generally detected in the plasma of nursing mothers (48,49), is secreted in high levels into milk (48), from which it has been purified. The NH zterminal forms of PTHrP in milk have been found to

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CHAPTER42

have molecular masses ranging from 9 to 21 kDa (50,51), which may represent different species ranging from the full-length molecule to smaller fragments. A detailed analysis of the predicted amino acid sequences of the three major PTHrP isoforms reveals multiple potential sites of proteolysis. There are also two potential amidation consensus sites (52) as well as regions rich in serine and threonine residues that are potential sites for 0-glycosylation, as reported in h u m a n keratinocytes (53). Soifer et al. studied (54) the posttranslational processing of PTHrP in human renal carcinoma and rat insulinoma cells that were stably transfected with a h u m a n PTHrP cDNA encoding amino acids 1-141. Both cell lines produced at least three immunoreactive PTHrP species containing (1-36), (37-74), and (1-74) epitopes plus a novel midregion fragment starting at amino acid 38 (approximate molecular mass, 7 kDa) and distal cleavage sites at around amino acids 96-98 and 102-106. This midregion fragment was also shown to be secreted by normal h u m a n keratinocytes. Over 80% of the secreted material described by Soifer et al. was composed of the NH zterminal and midregion fragment whereas only a small fraction contained (1-74) immunoreactivity. These results are in good agreement with experiments examining the processing of endogenous, internally labeled PTHrP in rat Leydig tumor cells in culture (55). The latter studies demonstrated the presence of three molecular forms of PTHrP comigrating with PTHrP(1-36), PTHrP(1-86), and PTHrP(1-141) on high-pressure liquid chromatography (HPLC), and comprising approximately 63, 30, and 7% of newly synthesized PTHrP, respectively. These studies demonstrate that the half-life of intact PTHrP(1-141) is extremely short and suggest the presence of multiple secretory forms ranging from short NHz-terminal fragments to the intact molecule. Whether the multiple forms described are generated in a tissue-specific fashion is not yet known. However, this extraordinarily intricate processing can potentially generate many different forms of PTHrP, and may confound the development and interpretation of specific immunoassays. To add to this complexity, it is also likely that peripheral metabolism of PTHrP occurs and gives rise to additional forms. Not studied yet is the issue of metabolic clearance, which may also be highly variable for each of these different forms of PTHrE

In Vivo Studies: Circulating Forms of PTHrP There appear to be major differences in the circulating forms of PTH and PTHrE Considerably more is known about the secretion and metabolism of the PTH molecule that is uniquely produced by parathyroid glands. The principal form of PTH is PTH(1-84),

which is also believed to be the major if not the only bioactive form in the circulation. However, in contrast to PTH, secretion and metabolism of PTHrP is much less well understood and potentially far more complicated. First, in contrast to the human PTH gene, which encodes a single mature peptide, the h u m a n PTHrP gene has the potential to express three different isoforms. Each of these isoforms in turn has the potential to undergo complicated posttranslational processing and subsequently may be subject to further breakdown and metabolic clearance in sites outside the cell of synthesis. Additionally, in contrast to PTH, which is synthesized exclusively in parathyroid cells, PTHrP is produced by a wide variety of normal and malignant cells, each of which may exhibit tissue-specific expression and unique posttranslational processing. To add to this inordinately complex scheme, it is not known to what degree tumors of the same cell type express and process similar molecular forms. Initial in vivo studies in cancer patients used the cytochemical bioassay to demonstrate elevated plasma levels of PTH-like bioactivity in the absence of detectable immunoreactive PTH (5). Gel filtration analysis of bioactivity revealed a heterogeneous profile, suggesting the presence of multiple bioactive fragments (56) (Fig. 1). Immunoassays specific for selected epitopes of the molecule have confirmed this apparent heterogeneity. Burtis et al. (57), using region-specific immunoassays, identified both NH 2- and COOH-terminal moieties in the circulation of cancer patients. Using a two-site immunoradiometric assay (IRMA), a protein that concomitantly reacts with both PTHrP(1-36) and PTHrP(37-74) antisera was detected together, along with a COOH-terminal (109-136) fragment that was present in equimolar concentrations. The full-length molecule, PTHrP(1-141), appeared to be absent. Henderson et al. (58) reported the presence of PTHrP entities of approximately 6-7 kDa with a radioimmunoassay based on an NHz-terminal antibody raised against PTHrP(1-34). In addition, this study and that of Burtis et al. reported species larger than the predicted full-length protein, suggesting that fragments or the intact form may aggregate in complexes with each other or with other proteins in the circulation, resulting in species of abnormally high molecular weight. Studies of gel filtration patterns of circulating PTHrP u n d e r denaturing conditions may help to resolve this issue. Carboxyl-terminal fragments of PTHrP are metabolized by the kidney (59) and circulating levels of the carboxyl-terminal PTHrP sequence 109-136 have been found to be elevated in patients with renal insufficiency (57). Because malignancy-associated hypercalcemia is characterized by the presence in the circulation of bioactive forms of the hormone, and in view of the fact

PTHrP AND HVeWRCaLCEMIA / that structure-function studies of synthetic fragments indicated such material must contain the aminoterminal region of the molecule, two-site, noncompetitive immunoradiometric assays have been developed for P T H r E This technique increases specificity for the intact molecule and improves sensitivity. However, it must be r e m e m b e r e d that other regions of the molecule may exhibit yet undefined biologic actions that could contribute to the biochemical manifestations of malignancy-associated hypercalcemia or that could carry out noncalcemia related changes and would therefore be of interest to measure. Furthermore, an unidentified subset of tumors may secrete forms of the peptide with novel properties, because histologically identical tumors may or may not be associated with hypercalcemia. Production of other non NHz-terminal molecular forms of PTHrP may also in theory provide useful information concerning the origin of a particular cancer or for monitoring response to treatment, i.e., as a tumor marker. Indeed, radioimmunoassays (RIAs) measuring midregion and inert COOH-terminal fragments of PTH have provided useful information in the past regarding the overall secretory activity of the parathyroid gland as well as the status of renal clearance mechanisms for PTH. These assessments therefore point to the presence of heterogeneous forms of PTHrP in extracellular fluids; they will no doubt be refined as increased knowledge of the chemical nature of these forms becomes available.

R E G U L A T I O N OF PTHrP P R O D U C T I O N IN VITRO In contrast to PTH, which is expressed only in parathyroid tissue, PTHrP is expressed in a wide variety of normal and neoplastic tissues. Using N o r t h e r n blot hybridization and in situ hybridization techniques, mRNA encoding PTHrP has been identified in normal h u m a n keratinocytes (60,61), normal h u m a n cervical epithelial cells (62), normal islet cells (63), lactating m a m m a r y glands (47,64), rat (65) and h u m a n (66) m a m m a r y epithelial cells, brain, fetal liver (60), normal h u m a n melanocytes (67), fetal parathyroid (68), a variety of smooth muscle tissues (69-71), urinary bladder (72), and stromal cells of the spleen and other organs (73). PTHrP is also expressed in a wide variety of neoplastic tissues (46,60,74) and in epithelial tumor cell lines (75), including squamous cell cancers, renal carcinoma (68,76), skin cancers (77), breast cancers (78), m e l a n o m a (67), and parathyroid adenomas (46), as well as in HTL V-l-transformed lymphocytes (79), h u m a n osteosarcoma cells (80), and n e u r o e n d o c r i n e tumor cells (44). This wide tissue distribution is compatible with an a u t o c r i n e / p a r a c r i n e role for the pep-

675

tide, a function that is likely to predominate over its endocrine role in normal tissues. Such a role has been suggested by experiments in h u m a n epithelial cells in which PTHrP acted as a potent antiproliferative (62,81) and prodifferentiating (82) factor. In normal animals, it appears to play no role as an endocrine factor in mediating calcium homeostasis after birth, (83) although it may subserve such a function during fetal life. In adults it would appear that only during an extraordinary situation, such as cancer development and progression, does PTHrP play an endocrine role leading to hypercalcemia. The development of RIAs for PTHrP greatly facilitated the study of its regulation in both normal and cancer cells in culture. The control of PTHrP released into conditioned m e d i u m by normal and neoplastic cell lines was initially monitored using bioassays. RIAs have also been used in conjunction with the traditional bioassay systems to monitor the PTHrP response to various stimuli (61,65,66,83-85). Positive regulation of PTHrP expression and secretion by mitogenic stimuli such as epidermal growth factor (EGF) and fetal bovine serum (FBS) (61,75), as well as by inhibition by 1,25(OH)zD 3, has been demonstrated in h u m a n keratinocytes using these methods (Fig. 2). Studies have shown that the p r o m o t e r of the PTHrP gene contains a vitamin D response element (VDRE) (86) that inhibits gene transcription when it interacts with the vitamin D receptor 1,25(OH)2D 3 complex. Dexamethasone and testosterone (83) have also been shown to reduce PTHrP secretion. In addition, as opposed to the inhibitory influence of calcium on PTH secretion by parathyroid glands, PTHrP secretion by keratinocytes is e n h a n c e d by extracellular calcium. Calcium also stimulates PTHrP production in transformed h u m a n keratinocytes (75), in rat Leydig tumor cells (87), and in a lung carcinoma cell line (88), but inhibits PTHrP expression and secretion in a rat parathyroid cell line (89). A variety of cell lines have been shown to increase PTHrP secretion in vitro in response to a n u m b e r of factors, including phorbol esters (44,80) cyclic AMP (90), calcitonin (91,92), the product of the Tax gene (93), transforming growth factor-[~ (TGF-[3) (04), and angiotensin II (95). It should be emphasized that peptide growth factors are generally positive regulators of PTHrP production. This is exemplified in normal h u m a n keratinocytes, in which EGF is essential for growth and is a potent positive stimulus for PTHrP production. In normal h u m a n m a m m a r y epithelial cells, in sulin-like growth factor-I (IGF-I), rather than EGF, is an absolute r e q u i r e m e n t for cell growth and is also more potent than EGF in stimulating PTHrP production (66). In view of the fact that PTHrP is p r o d u c e d by many normal cells, its secretion by many histologic types of

676

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tumor cells undoubtedly represents eutopic overproduction of PTHrP in the cells as they undergo malignant transformation rather than ectopic production. The mechanism whereby PTHrP is produced in excess by tumor cells is, however, not well understood at the present time but may be a function of abnormal gene regulation. This p h e n o m e n o n has recently been studied in a model of tumor progression (75), in which PTHrP gene regulation and secretion were analyzed in the transition from the normal to the malignant phenotype. Normal human keratinocytes were established as a keratinocyte cell line following infection with h u m a n papilloma virus type 16 (HPKIA). This cell line

FIG. 2 In vitro regulation of PTHrP secretion in normal and malignant states. The upper panel depicts sites of regulation of PTHrP production that may be targets for intervention to diminish PTHrP release from tumor cells. Growth factors (GF) act via the Ras pathway to stimulate mitogen-activated protein kinase (MAPK) and increase PTHrP gene transcription. 1,25-Dihydroxyvitamin D (1.25) can bind to the vitamin D receptor (VDR) to inhibit transcription. After transcription and translation, preproPTHrP is converted to proPTHrP, which must then be processed to the active form of PTHrP before it is secreted. The lower panel compares the regulations of PTHrP in immortalized keratinocytes (HPK1A) versus malignant keratinocytes (HPK1Aras). (A) The time course of PTHrP secretion in the absence of exogenous mitogenic growth factors, demonstrating that HPK1Aras cells secrete far more PTHrP than do HPK1A cells. (B) The effects of 1,25(OH)2D 3 on EGF-stimulated PTHrP secretion in HPK1A and HPK1Aras cells, showing that 1,25(OH)2D3 is much more effective in inhibiting PTHrP secretion in HPK1A than in HPK1Aras cultures. Significar,t difference from HPK1A (*p < 0.01). (Redrawn from Henderson J, Sebag M, Rhim J, Goltzman D, Kremer R. Cancer Res 1991 ;51:5621-5628.)

was subsequently converted to the malignant phenotype (HPKIAras) using an activated r a s oncogene (75,96,97). In contrast to the established cells (HPKIA), which continued to produce PTHrP in a regulated manner, HPKIAras cells expressed and secreted PTHrP in a constitutive fashion, i.e., in the absence of exogenous mitogenic factors such as EGF, and displayed resistance to the inhibitory effect of 1,25 (OH) 2D~ (Fig. 2). This resistance p h e n o m e n o n was shown to be secondary to phosphorylation of the retinoid X receptor (RXR) on a specific MAP kinase consensus sequence by ras-raf-MAP kinase activation pathway (98). Whether the unregulated production of

PTHrP AND H~F~kCALCWMIA / PTHrP demonstrated in vitro by cultured tumor cells occurs in vivo and can explain the elevated circulating concentrations of PTHrP noted in patients with squamous carcinoma remains to be elucidated. Significant progress has been made from in vitro studies in examining the regulation of PTHrP production by both normal and malignant cells in culture. Studies of this nature have the potential of disclosing critical mechanisms that might also operate in vivo.

nephrogenous cAME Similar results obtained by Henderson et al. (113) using the rat Leydig cell model also demonstrated the important role played by the renal action of PTHrP in contributing to hypercalcemia of malignancy. These studies therefore strongly implicated PTHrP as the humoral factor responsible for hypercalcemia in these models and provided convincing evidence for a pathogenetic role for PTHrP in the syndrome of hypercalcemia associated with malignancy (Fig. 3).

STUDIES OF MALIGNANCY-ASSOCIATED HYPERCALCEMIA IN ANIMALS

Several animal models of malignancy-associated hypercalcemia have been developed over the years in an effort to define the pathogenesis of the human syndrome. These models include both spontaneous and induced tumors in rodents and dogs as well as h u m a n tumors transplanted into athymic mice. One of the most widely used, and therefore best defined model, is the Fischer rat bearing the Rice H500 Leydig cell tumor. This tumor, from which the rat PTHrP cDNA was cloned (32), arises spontaneously in aged Fischer rats but can be successfully passaged by subcutaneous transplantation in younger animals. The nonmetastatic tumor grows rapidly in association with hypercalcemia, hypophosphatemia, increased urinary cAMP, renal phosphate wasting, and suppressed immunoreactive PTH (99-102). This constellation of biochemical abnormalities therefore closely reproduces the syndrome of h u m a n malignancy-associated hypercalcemia. The Walker 256 carcinosarcoma, originating from a rat mammary gland, has also been shown to secrete PTHlike bioactivity, in the absence of PTH immunoreactivity, into conditioned medium when maintained in culture (103-105). Adenocarcinoma of the anal sac in dogs (106) is also associated with hypercalcemia in vivo and appear to produce a factor with PTH-like bioactivity in vitro. Tumors of h u m a n and animal origin that have been transplanted into athymic mice include h u m a n squamous carcinomas (107,108) and a n u m b e r of h u m a n renal carcinomas (109,110), and a melanoma cell line (67). Although all of these models demonstrated to some degree the biochemical abnormalities associated with malignancy-associated hypercalcemia, direct measurement of circulating PTHrP has been accomplished in only some (67,84,108,111). Once antisera directed against the human (h) PTHrP molecule had been developed for use in PTHrP RIAs they were also applied to passive immunization studies in rodents. Using athymic mice beating human squamous carcinomas, Kukreja et al. (107) infused an antiserum directed against NH2-terminal hPTHrP, which quickly reversed the hypercalcemia and the elevation of

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FIG. 3 Effect of passive immunization with PTHrP and PTH antisera on plasma calcium of normal rats and rats with hypercalcemia of malignancy. In normal rats (A) a moderate and transient decrease in plasma calcium was observed after injection of PTH antiserum (/k) but not after injection of PTHrP antiserum (o) or normal preimmune rabbit serum (©). In contrast, hypercalcemic rats implanted with the Leydig cell tumor H500 (B) sustained a prolonged reduction in plasma calcium after injection of PTHrP antiserum (°) but not of PTH antiserum (/k) or normal preimmune rabbit serum (©). Consequently PTH but not PTHrP appears to be the major modulator of plasma calcium homeostasis in the normal animals, whereas PTHrP is the major pathogenetic mediator in the hypercalcemia ofmalignancy. (From Henderson J, Bernier S, D'Amour P, Goltzman D. Effects of passive immunization against parathyroid hormone (PTH) -like peptide on PTH in hypercalcemic tumor bearing rats and normocalcemic controls. Endocrinology, Vol. 127, pp. 1310-1318, 1990. © The Endocrine Society.)

678

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C~Tv.R42

Animal models have also b e e n useful for examining the therapeutic efficacy of various agents in malignancyassociated hypercalcemia. We have taken advantage of our previous in vitro studies demonstrating the inhibitory effect of 1,25(OH)zD ~ and vitamin D analogs on PTHrP gene expression and secretion (61,75,112) to study their potential usefulness in reducing serum calcium levels in animal models in vivo. A 1,25(OH)2D 3 analog, EB1089, was found to have very low calcemic potency relative to 1,25 (OH)zD s when infused into control animals (84,108,114). In addition, when analyzed in vitro, this analog was 10-100 times more potent than 1,25(OH)2D 3 in inhibiting PTHrP production in cancer cells (112) and was therefore chosen as a candidate for in vivo studies with hypercalcemic, tumor-bearing rats. After continuous infusion into Fischer rats bearing the Rice Leydig cell tumor H500, a significant reduction in circulating PTHrP concentrations was demonstrated using an NHz-terminal PTHrP RIA, with a concomitant reduction in plasma calcium levels (Fig. 4). These studies were further extended in a nude mouse model of h u m a n squamous cancer producing PTHrP in which established hypercalcemia was reversed by infusion of EB1089 directly into the tumor (108). Other approaches have targeted additional loci known to be important for PTHrP production. In view of the critical

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stimulatory role of growth factors in PTHrP production, and in view of the important role of the Ras oncogene signaling pathway, Ras inhibitors have been employed. The inhibitor we used prevents farnesylation of Ras, a necessary step in facilitating binding to cell membranes and therefore permitting signal transduction. We thus showed that transplantation of a cell line overproducing PTHrP into nude mice could be inhibited from producing hypercalcemia after treatment of the mice with a small-molecule organic inhibitor of Ras farnesylation (115). In another approach, stable transfection of H500 Leydig tumor cells with antisense PTHrP was used to diminish intracellular PTHrP production. When these cells were implanted into normal Fischer rats, hypercalcemia did not occur (115). Finally, inasmuch as proper processing of proPTHrP to PTHrP has been shown to be critical for production of fully bioactive PTHrP, and in view of the fact that this process appears to be mediated by the convertase furin, an antisense furin cDNA was stably transfected into H500 Leydig cells. Implantation of these modified cells into Fischer rats resulted in reduced hypercalcemia, diminished tumor growth, and prolonged survival relative to implantation of native cells (116). In the future these and other approaches may yield useful therapies against hypercalcemia of malignancy.

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PTHrP AND HYPERCALCEMIA /

HYPERCALCEMIA D U E T O P T H r P P R O D U C T I O N IN H U M A N S With the development of accurate immunologic techniques for the measurement of PTHrP in humans, considerable progress was made in defining the pathogenetic mechanisms underlying malignancy-associated hypercalcemia as well as assessment of the therapeutic modalities to be used in its treatment.

P T H r P in Cancer Patients Classification of Cancer Patients

One criterion commonly used to define subgroups of patients with malignancy-associated hypercalcemia has been the presence or absence of bone metastases (4). One group, whose underlying mechanism was presumably local osteolytic hypercalcemia (LOH), was composed mainly of patients harboring hematologic malignancies or metastatic solid tumors (e.g., breast cancer). A second group, whose underlying mechanism of hypercalcemia was believed to be secretion by the tumor of a humoral factor with hypercalcemia activity (HHM), was composed mainly of patients with epithelial and renal cancers (3,4,25,117-119). Although this distinction was initially useful in the conceptual approach, which ultimately resulted in the purification and identification of PTHrP, it is less useful in the pathogenetic and clinical sense. The occurrence of bone metastases (i.e., LOH) does not exclude a pathogenetic role for PTHrP that might act systemically (i.e., endocrine) or locally (i.e., paracrine). In fact, animal studies (120,121) have demonstrated a role for PTHrP as a paracrine mediator of bone resorption in metastatic cancer. Thus, it seems likely that all malignancyassociated hypercalcemia, whether associated with a systematic circulating factor or not, is humoral in origin and could be called HHM. Additionally, several studies have reported a poor correlation between the presence or extent of bone metastases and the occurrence of hypercalcemia (122). The terminology "LOH" may also be inaccurate because it is uncertain whether hypercalcemia is ever due solely to osteolysis in the absence of altered renal handling of calcium. A more relevant pathogenetic consideration of malignancy-associated hypercalcemia (MAH), therefore, may be the presence or absence of elevated n e p h r o g e n o u s cyclic AMP, as a reflection of the overproduction and systemic action of PTHrP by these tumors. In this respect it has been estimated that approximately 80% of unselected patients with malignancy-associated hypercalcemia (4) have such an abnormality. Using two-site immunometric assays, several groups have confirmed that between 50

679

and 90% of these patients with solid tumors (57,123,124) and 20 and 60% of patients with hematological tumors (125-127) had elevated circulating concentrations of PTHrP. Similar results were obtained with a midregion PTHrP assay (128). The most frequent solid tumors associated with increased circulating levels of PTHrP are squamous cell cancers of lung, head, and neck, as well as cancer of the kidney and ovary. With the advent of sensitive and specific immunoassays for PTHrP it became possible to redefine the classification of hypercalcemic cancer patients. This is particularly true for patients with breast cancer and hypercalcemia, who often have elevated PTHrP levels, even in the presence of extensive bone metastatic lesions (129), indicating that the humoral and local osteolytic mechanisms frequently occur simultaneously. Indeed, the "humoral" nature of the hypercalcemia due to breast cancer had previously been predicted on the basis of studies of urinary phosphate and cyclic AMP excretion (130,131). It has been suggested that PTHrP may enhance the ability of breast cancer cells to invade bone, and that PTHrP production by metastatic tumor cells is increased by cytokines produced locally in the bone microenvironment. Thus, there occurs a vicious cycle of mutually activating factors (120). Additionally, hematologic malignancies may not readily fall u n d e r an L O H classification. Thus certain types of lymphomas have been shown to produce both PTHrP (79,125) and 1,25(OH)zD ~ (28,132), adding to the complexity of potential underlying mechanisms of hypercalcemia in this condition. As the sensitivity and specificity of PTHrP immunoassays improve, it will be possible to classify MAH patients more quickly and more accurately as PTHrP related or PTHrP unrelated, based on measu r e m e n t of the causative agent per se. As improved methods of treating PTHrP overproduction are developed, such improved diagnostic accuracy should have therapeutic as well as pathogenetic importance.

Relationship of Skeletal Metastasis to PTHrP Concentrations in Patients with Solid Tumors and Hypercalcemia Employing RIAs for the NH2-terminal assay of PTHrP, analyses of patients with malignancy-associated hypercalcemia revealed no more than a modest increase in the percentage of patients with elevated values when patients with bone metastases were excluded from the series studies (Table 1). Thus, in the study of Budayr et al. (133), 51% of patients with MAH had elevated PTHrP values regardless of the presence of bone metastases. The n u m b e r rose to about 85% when patients with bone metastases were excluded. In the study of H e n d e r s o n et al. (58), 50% of unselected

680

/

CHAPTER42 PTHrP Levels in Different Histologic Types of Malignancies

TABLE 1

Occurrence of elevated PTHrP in cancer patients (%) Hypercalcemic subjects Tumor type

BM ( - and +)

BM (+)

BM (-)

Normocalcemic subjects

Ref.

51

N/A a

53 48 60 85 c 100

N/A 33 65 b 0 N/A

85 N/A 53 100 N/A N/A

10 18 0 0 9 N/A

Budayr et al. (133) Henderson et aL (58) Kao et al. (134) Grill et aL (135) Burtis et aL (57) Ratcliffe et aL (123)

85 50 100

N/A N/A N/A N/A

N/A N/A 66 100

N/A 10 N/A N/A

Budayr et al. (133) Henderson et aL (58) Kao et al. (134) Grill et al. (135)

60 100 N/A ~

N/A N/A N/A

N/A N/A 25

N/A N/A N/A

Budayr et al. (133) Henderson et aL (58) Kao et al. (134)

50 60 N/A d

N/A N/A 16

N/A N/A 100

N/A 29 N/A

Budayr et aL (133) Henderson et aL (58) Kao et al. (134)

33

N/A d

N/A d

8

Henderson et aL (58)

0 80 62.5

N/A N/A N/A

N/A N/A N/A

N/A N/A 23

Budayr et al. (133) Kao et al. (134) Kremer et al. (61)

33 16

N/A N/A

N/A N/A

N/A N/A

Budayr et al. (133) Kao et al. (134)

Solid tumors Mixed

Squamous cancers

N/A d

Renal cell cancer

Breast cancer

Hematologic tumors Mixed Lymphoma

Multiple myeloma

aN/A, Information not available. bSolid tumors other than breast. dHypercalcemia of malignancy with I"NcAMP. Patients with (+) or without ( - ) bone metastases (BM).

patients with MAH, harboring a wide variety of histologic tumor types, had elevated PTHrP values. Grouping patients according to the presence or absence of bone metastases did not significantly alter those results. In the study of Kao et al. (134), approximately 48% of unselected patients with MAH had elevated PTHrP values, a percentage that increased to about 53% when patients with bone metastases were

excluded. Finally, in the study of Grill et al. (135), 100% of a group of patients with various solid tumors, excluding breast cancers, having no evidence of bone metastases, had elevated values of PTHrP (ranging from 2.8 to 51.2 pmol/liter). In a second group that included patients with solid tumors of the same type, but having radiologic evidence of metastases, roughly 60% had elevated PTHrP levels ranging from 4.9 to 47.5 pmol/liter.

PTHrP AND HYPERCALCEMIA / In this study all patients with squamous cancer, with or without bone metastases, had elevated PTHrP levels, whereas approximately 60% of hypercalcemic patients with breast cancer, almost all of whom had bone metastases (19/20), had elevated PTHrP values (ranging from 3.9 to 61.6 pmol/liter). Results are somewhat more conflicting when two-site IRMAs are employed. Burtis et al. (57), using a two-site assay specific for PTHrP (1-74), found that 85% of 30 hypercalcemic cancer patients classified as HHM on the basis of elevated nephrogenous cyclic AMP had elevated PTHrP values (mean level, 20.9 -+ 21.8 pM/liter). In contrast, a group of patients represented by four breast cancers, three multiple myelomas, and one undefined lung cancer were classified as LOH on the basis of normal nephrogenous cAMP and extensive bone involvement, and had normal PTHrP levels. On the other hand Ratcliffe et at (123), using a two-site assay specific for PTHrP (1-86), found that all patients in an unselected group with MAH of various histologic types and with advanced metastatic disease had elevated PTHrP levels. These studies indicate that a larger n u m b e r of tumors produce PTHrP than was predicted, and that PTHrP may contribute to the development of hypercalcemia regardless of the presence or absence of skeletal metastases (Table 1). Whether different NHz-terminal species of PTHrP are produced by tumors that differ histologically or have varying metastatic behavior is currently unknown.

associated biochemical abnormalities strongly indicate the presence of a circulating factor with PTH-like bioactivity (138). The demonstration of elevated PTHrP expression by T lymphocytes in culture after infection with HTLV-1 supports this hypothesis (79). Henderson et al. (58) reported 33% of patients with hematologic malignancies had elevated PTHrP values, whereas Kao et al. (134) found elevated levels of PTHrP in four out of five patients with lymphoma and one out of six patients with multiple myeloma. Burtis et al. (57) showed that two patients with lymphomas and elevated nephrogenous cAMP had increased PTHrP levels whereas one patient with multiple myeloma and normal nephrogenous cAMP had a normal PTHrP value. Another group has reported elevated concentrations of PTHrP in two patients with hypercalcemia and nonHodgkin's lymphoma without bone metastases (139). As is the case with breast cancer, evaluation of patients with various hematologic malignancies reveals that PTHrP is frequently elevated in these conditions (Table 1). Another study has confirmed and extended these observations in patients with diverse hematologic malignancies (125). In this study, which included a large n u m b e r of patients with non-Hodgkin's lymp h o m a classified according to disease stage and grade, elevated PTHrP levels were most often found in patients with late-stage disease and high-grade pathology (Table 2). After chemotherapy in several patients

TABLE 2 Distribution of PTHrP in Non-Hodgkin's Lymphoma, According to Disease Stage and Gradea

P T H r P Concentrations in Hematologic Malignancies Associated with Hypercalcemia

Hematologic malignancies that frequently cause hypercalcemia include lymphoma, chronic myeloid and lymphoblastic leukemia, multiple myeloma, and adult T cell leukemia. Extensive bone destruction is common in multiple myeloma, and over 30% of patients develop hypercalcemia (136). The mechanism that has been postulated to explain the hypercalcemia associated with this disorder is production by the plasma cells of a group of cytokines collectively termed "osteoclast-activating factor" (OAF). These include interleukin-one (IL-1) and tumor necrosis factors ~ and [3 (TNFe~ and TNF[3), all of which are potent stimulators of osteoclastic bone resorption. Hypercalcemia is less common in lymphomas (both Hodgkin's and nonHodgkin's). Circulating 1,25(OH)2D ~ is elevated in a number of lymphoma patients who are hypercalcemic without skeletal metastases, and likely plays a pathogenetic role (28,137). By contrast, there is a high incidence of hypercalcemia in patients with adult T cell leukemia/lymphoma. This disorder is caused by infection of T cells with the h u m a n T cell lymphotropic virus type 1 (HTLV-1). In addition to hypercalcemia, other

681

Lymphomas Stage IV

I to III

PTHrP (1")

[PTHrP] (pmol Eq/Liter)

Grade

n

n

Mean _+ SD

H

14

8

I

7

2

52.5 _ 22.5 2 5 _+ 15

L

9

I

H

9

2

13_+5

I

5

0

<7.5

L

7

1

--

b

c

aMean parathyroid hormone-related peptide (PTHrP) concentrations of both normo- and hypercalcemic patients measured with NH2-terminal assay, expressed in picomole equivalents of PTHrP(1-34)/liter (pmol Eq/Liter). Normal range was <7.5 pmol Eq/Liter). Grade classification: H, high; I, intermediate; L, low. (Reprinted from Am J Med, Vol. 100; R Kremer, C Shustik, T Tabak, V Papavasiliou, D Goltzman; Parathyroid hormone related peptide in hematologic malignancies, pp. 406-411. Copyright 1996, with permission from Elsevier Science.) bAll values <7.5 except for one value of 62.5 pmol Eq/Liter. CAll values <7.5 except for one value of 37.5 pmol Eq/Liter.

682

/

CHAeTER42

with B cell lymphomas, circulating PTHrP levels decreased concomitantly with reduced tumor burden. It appears, therefore, that despite frequent and extensive osseous metastases, many hematologic malignancies are associated with overproduction of PTHrP, suggesting a multifactorial basis for the hypercalcemia associated with those diseases.

Response to Treatment and Prognosis in Hypercalcemic Cancer Patients with Elevated I~HrP Values Henderson et al. (58) reported that a reduction in the serum calcium concentration in a patient with malignancy-associated hypercalcemia did not significantly affect plasma immunoreactive PTHrP concentrations. However, reduction of tumor mass by chemotherapy in two other patients with malignancy-associated hypercalcemia did decrease circulating PTHrP levels (Fig. 5). Bisphosphonates in general and and pamidronate in particular are the treatment of choice in MAH and can normalize calcium levels in up to 85% of unselected patients (138-140). An inverse relationship between

A

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A

PTHrP status and response to pamidronate has been demonstrated by several groups (142-144). In general, treatment of hypercalcemia in patients with elevated circulating PTHrP levels requires a higher dose of pamidronate and takes longer to achieve normocalcemia. Moreover, hypercalcemia recurs more rapidly following an initial response (140-143). Poor responsiveness to pamidronate may indicate that a substantial component of hypercalcemia is due to the elevated renal reabsorption of calcium induced by PTHrE The development of hypercalcemia in a patient with cancer is a signal of impending death, with a reported average survival from the onset of hypercalcemia of only 27 to 90 days (142-144). Correlation between overall survival and PTHrP status was studied retrospectively by Pecherstorfer et al. (144) in 59 patients who demonstrated a twofold increase in mortality rate as compared to patients with low or undetectable circulating PTHrP levels. A recent prospective study (145) confirmed and extended these results, demonstrating that PTHrP was the most important prognostic indicator in MAH, independent of other variables.

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O

TIME

FIG. 5 Effect of antitumor therapy on circulating PTHrP and calcium concentrations in malignancy. (A) Time course of PTHrP and calcium concentrations after treatment of hypercalcemia in a patient with squamous carcinoma using saline, furosemide, and mithramycin. (B and C) Time courses of PTHrP and calcium concentrations in two hypercalcemic patients with breast cancer treated with chemotherapy, which reduced tumor mass. All patients displayed a substantial lowering of serum calcium but only the patients who sustained a reduction in tumor burden (B and C) had a concomitant decrease in plasma PTHrP. (From Henderson JE, Shustik C, Kremer R, Rabbani SA, Hendy GN, Goltzman D. J Bone Miner Res 1990;5:105-113, with permission.)

P T H r P AND HYPERCALCEMIA /

The prognostic significance of PTHrP in cancer has also been examined in tissue samples obtained from breast cancer patients. In one study PTHrP positivity was associated with a more favorable long-term outcome (146), but similar results were not observed in other studies (147,148).

PTHrP Concentrations in Cancer Patients w i t h o u t Hypercalcemia There is still some controversy regarding the significance of an elevated level of PTHrP in normocalcemic cancer patients. Thus, all NHz-terminal RIAs reported to date have found elevated circulating PTHrP concentrations in a great proportion of patients with cancer and hypercalcemia. Several studies have also reported elevated NHz-terminal PTHrP concentrations in some normocalcemic cancer patients (Table 3). However, the prevalence of elevated PTHrP concentrations in normocalcemic cancer patients varies from assay to assay. Discrepancies a m o n g RIAs in the detection of NH2-terminal PTHrP moieties in normocalcemic subjects may reflect differences in antibody sensitivity a n d / o r epitope specificity (Table 3). Budayr et al. (133) and Kao et al. (134), using NH zterminal RIAs specific for PTHrP(1-34), detected elevated levels in over 50% of hypercalcemic patients and about 10% of normocalcemic cancer patients. H e n d e r s o n et al. (58), using a similar assay, detected elevated PTHrP levels in 40-50% of hypercalcemic

TABLE 3

patients and 10-30% of normocalcemic patients, whereas Grill et al. (135), also using an NH2-terminal RIA, did not detect elevated PTHrP in normocalcemic patients. In contrast to the NH2-terminal RIAs, all studies using IRMAs (57,123) published to date have found PTHrP levels in normocalcemic cancer patients to be similar to levels in normal controls. Nevertheless, the occurrence of elevated PTHrP levels in normocalcemic cancer patients (58,133,134), may have practical implications. It is possible that during the progression of malignancy, PTHrP secretion would increase to a point at which a critical threshold is attained, above which hypercalcemia ensues. In theory, therefore, PTHrP might be secreted long before hypercalcemia occurs. Specific and sensitive PTHrP assays could then be used to detect the presence of tumors at an early stage and conceivably predict those with the potential for developing hypercalcemic complications. Alternatively, the finding of elevated PTHrP concentrations in the absence of hypercalcemia might be due to the production of biologically inert fragments by tumors. Detection of such fragments might still be of clinical interest and utility in that they may serve as tumor markers to follow disease progression. It is also possible that other factors, in concert with PTHrP, are important in the overt expression of hypercalcemia in malignancy. Whether the next generations of PTHrP assays will be able to identify these and other forms of PTHrP as tumor markers remains to be determined.

Concentrations of PTHrP in Normal Subjects and Cancer Patients PTHrP concentrations (pM/Liter)a

Type of assay

683

Controls

1"Ca2+

---->Ca2+

Ref.

0-2.5 < 15 2-5 <2 0.35-5.7

2-49 15-245 2-85 2.8-51.2 2.7-41.3

0-7.5 15-105 2-3.7 <2 N/A ~

Budayr et aL (133) Henderson et aL (58) Kao et aL (134) Grill et aL (135) Ratcliffe et aL (123)

<2

2.1-74

2-20

270-680

150-1570

N/A

Ratcliffe et aL (123)

1-5.1 <0.23

1.7-1 03 0.46-26.5

<5.1 N/A

Burtis et aL (57) Ratcliffe et aL (123)

N-Terminal RIA

C-Terminal RIA Burtis et aL (57)

Midregion RIA IRMA

aCancer patients with hypercalcemia (1" Ca 2+) and normocalcemia (-->Ca2+). ON/A, Information not available.

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PTHrP during Pregnancy and Lactation PTHrP has been detected in human placental tissue (47) and rat trophoblastic cells (149), raising the possibility that PTHrP may play a physiologic role during pregnancy. It has been reported that PTHrP may promote maternal calcium transfer across the placenta to maintain the developing embryo hypercalcemic relative to the mother (150). Allgrove et al. (151) had previously demonstrated PTH-like bioactivity without PTH immunoreactivity in human umbilical cord blood and it has been subsequently shown that immunoactive PTHrP is increased in cord blood (152). These findings may explain the apparent absence of skeletal defects in Di George syndrome, a disorder characterized by congenital absence of parathyroid glands. It is plausible that production of PTHrP by the placenta may help to maintain fetal calcium homeostasis. Expression of PTHrP mRNA has also been demonstrated in lactating mammary tissue (47), and PTHrP mRNA levels increase in response to suckling and to infusion of prolactin in nonlactating breast tissue (153). PTHrP was subsequently found to be present in high concentrations in milk (48,50,51,64,154,155), where biochemical characterization revealed heterogeneity (50,51). It has been suggested, therefore, that PTHrP may play a role in the maturation of the newborn (or infant) digestive tract. However, no direct evidence in support of this thesis has yet been provided. The observation that PTHrP levels in bovine milk correlate with milk calcium concentration also suggests a local role for PTHrP in the transport of calcium across mammary epithelia (154). Increased circulating PTHrP concentrations have been documented in a lactatingwomen with massive breast hypertrophy and hypercalcemia, with normalization of the biochemical abnormalities following mastectomy (155). However, in general, circulating PTHrP concentrations in lactating women are essentially within the normal range (48,49), suggesting that PTHrP is not released into the maternal circulation under ordinary circumstances and therefore plays no endocrine role in maintaining normal blood calcium concentrations during lactation.

hyperparathyroidism, only one report to date has revealed elevated PTHrP concentrations in a small n u m b e r of these patients (58). On the other hand, two reports have demonstrated elevated levels of PTHrP in hyperparathyroidism secondary to chronic renal failure (57,58). PTH measurements are also clinically useful in malignancy-associated hypercalcemia inasmuch as, with rare exception, they demonstrate suppressed intact PTH concentrations. The presence of suppressed PTH is therefore clearly important in the differential diagnosis of primary hyperparathyroidism and the hypercalcemia of cancer. Primary hyperparathyroidism and malignant hypercalcemia can, however, coexist in perhaps 10% of patients with a primary diagnosis of cancer ( 157).

Idiopathic Infantile Hypercalcemia Elevation of PTHrP in children with idiopathic infantile hypercalcemia (IHH) has been reported (158) using an NH2-terminal RIA. However, William's syndrome, a cause of idiopathic infantile hypercalcemia that is usually associated with aortic arch and facial defects, is not characterized by elevated PTHrP levels.

SUMMARY Considerable progress has been made since the elucidation of the primary structure of PTHrE These developments have resulted in significant advances in our knowledge of the regulation of gene expression and metabolism of this hypercalcemic factor, as well as of its central pathogenetic role in malignancy-associated hypercalcemia. Additional progress is required to define the exact nature of circulating molecular forms of PTHrP, and such knowledge will be important in enhancing the utility of PTHrP as a marker for prognosis, diagnosis, and therapeutic monitoring in hypercalcemic disorders.

ACKNOWLEDGMENTS PTHrP and Hyperparathyroidism PTHrP mRNA is readily detectable in parathyroid tissue of patients with primary adenomas or with hyperplasia secondary to renal failure (46,77). Immunohistochemical localization of PTHrP in human fetal parathyroids has also been demonstrated (156). Although several attempts have been made to measure PTHrP in the circulation of patients with primary

We thank Shafaat A. Rabbani and Geoffrey N. Hendy for important collaboration in parts of this work and Michael Sebag and Janet Henderson for essential contributions. The research described in this article was funded by grants MT-10839 (R.K.) and MT-5775 (D.G.) from the Medical Research Council of Canada (MRC), by the National Cancer Institute of Canada, and by the Dairy Farmers of Canada.

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123. Ratcliffe WA, Norbury C, Stott RA, Heath DA, Ratcliffe JG. Immunoreactivity of plasma parathyrin-related peptide: Three region specific radioimmunoassays and a two-site immunoradiometric assay compared. Clin Chem 1991;37: 1781-1787. 124. Rabbani SA, Gladue J, Harakidas P, Jamison B, Goltzman D. Over-production of parathyroid hormone-related peptide results in increased osteolytic skeletal metastasis by prostate cancer cells in vivo. I n t J Cancer 1999;80:257-264. 125. Kremer R, Shustik C, Tabak T, Papavasiliou V, Goltzman D. Parathyroid hormone related peptide in hematologic malignancies. A m J Med 1996;100:406-411. 126. Firkin E Seymour JE Watson AM, Martin TJ. Parathyroid hormone related protein in hypercalcemia associated with haematological malignancy. BrJ Haematol 1996;94:486-492. 127. Nakamura Y, Bando H, Shintari J, Yokogoshi Y, Saito S. Serum parathyroid hormone related protein concentrations in patients with hematologic malignancies on solid tumors. Acta Endocrinol 1992;127:324-330. 128. Blind E, Rame E G6tzmann J, Schmidt-Gayk H, Kohl B, Ziegler R. Circulating levels of midregional parathyroid hormonerelated protein in hypercalcemia of malignancy. Clin Endocrinol 1992;37:290-297. 129. Bundred NJ, Ratcliffe WA, Walker RA, Coley S, Morrison JM, Ratcliffe JG. Parathyroid hormone related protein and hypercalcemia in breast cancer. Br MedJ 1991;303:1506-1509.

P T H r P AND HYPERCALCEMIA 130. Percival RC, Yates AJP, Grey RE, Galloway J, Rogers K, Neal FE, Kanis JA. Mechanism of malignant hypercalcemia in carcinoma of the breast. Br MedJ1985;291:776-779. 131. Ralston, SH, Fogelman I, Gardiner MD, Boyle IT. Relative contribution of humoral and metastatic factors to the pathogenesis of hypercalcemia in malignancy. Br MedJ 1984;288:1405-1408. 132. Rosenthal NR, Insogna KL, Godsall JW, Smaldone L, Waldron J, Stewart AF. Elevations in circulating 1,25dihyroxyvitamin D in three patients with lymphoma associated hypercalcemia. J Clin Endocrinol Metab 1985;60:29-33. 133. Budayr AA, Nissenson RA, Klein RE, Pun KK, Clark OH, Diep D, Arnaud CD, Strewler GJ. Increased serum levels of a parathyroid hormone-like protein in malignancy-associated hypercalcemia. Ann Intern Med 1989;111:807-812. 134. Kao PC, Klee GG, Taylor RL, Heath III H. Parathyroid hormone-related peptide in plasma of patients with hypercalcemia and malignant lesions. Mayo Clin Proc 1990;65:1399-1407. 135. Grill V, Ho P, BodyJJ, Johanson N, Lee SC, Kukreja SC, Moseley JM, Martin TJ, Parathyroid hormone-related protein: Elevated levels in both humoral hypercalcemia of malignancy and in hypercalcemia complicating metastatic breast cancer. J Clin Endocrinol Metab 1991 ;73:1309-1315. 136. Mundy GR, Martin TJ. The hypercalcemia of malignancy: Pathogenesis and management. Metabolism 1982;31:1247-1277. 137. Adams JS, Fernandez M, Gacad MA, Gill PS, Endres DB, Rasheed S, Singer FR. Vitamin D metabolite-mediated hypercalcemia and hypercalciuria patients with AIDS- and non-AIDSassociated lymphoma. Blood 1989;73:235-239. 138. Fukumoto S, Matsumoto T, Ikeda K, Yamashita T, Watanabe T, Yamaguchi K, Kiyokawa T, Takatsuki K, Shibuya N, Ogata E. Clinical evaluation of calcium metabolism in adult T-cell leukemia/lymphoma. Arch Intern Med 1988;148:921-925. 139. Dodwell DT, Abbas SK, Morton AR, Howell A. Parathyroid hormone-related protein and response to pamidronate therapy for tumor induced hypercalcemia. EurJ Cancer 1991;77:1629-1633. 140. Walls J, Ratcliffe WA, Howell A, Bundred NJ. Response to intravenous bisphosphonates therapy in hypercalcemic patients with and without bone metastases: The role of parathyroid hormone related protein. BrJ Cancer 1994;70:169-172. 141. Gurney H, Grill V, Martin TJ. Parathyroid hormone related protein and response to pamidronate in tumor-induced hypercalcemia. Lancet 1993;341:1611-1613. 142. Grill V, Murrary RM, Ho PW, SantamariaJD, Pitt P, Potts C,Jerums G, Martin TJ, Circulating PTH and PTHRP levels before and after treatment of tumor induced hypercalcemia with Pamidronate Disodium (APD). J Clin Endocrinol Metab 1992;74:1468-1470. 143. Wimalawana SJ. Significance of plasma PTHrP in patients with hypercalcemia of malignancy treated with bisphosphonates. Cancer 1993;73:2223-2230. 144. Pecherstorfer M, Schilling T, Blind E, Zimmer-Roth I, Baumgartner G, Ziegler R, Raue E Parathyroid hormone-

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CHAPTER 4 3

Other Local and Ectopic Hormone Syndromes Associated with Hypercalcemia

GREGORY R. MUNDY AND BABATUNDE OYAJOBI Medicine~Endocrinology, University of Texas Health Science Centeg, San Antonio, Texas 78284

INTRODUCTION

in the preceding chapter. However, many of the points that are relevant to humoral hypercalcemia may also be relevant here, and it should be noted that in some patients with local osteolysis, tumor burden in bone and subsequent production of PTHrP may be great enough that the h o r m o n e appears in the systemic circulation and causes the HHM syndrome superimposed on local osteolysis.

Hypercalcemia associated with malignancy may be part of the syndrome of humoral hypercalcemia of malignancy (HHM), in which elevated calcium is mediated by a circulating hormone and is responsible for a systemic increase in bone resorption and other disturbances in calcium homeostasis that overwhelm the homeostatic compensatory mechanisms. Malignancyassociated hypercalcemia may also be due to local tumor cell production of bone resorbing factors in the bone microenvironment, leading to adjacent bone destruction and, as a consequence, hypercalcemia. Formerly, these two syndromes were separated and known as humoral hypercalcemia and local osteolysis associated with hypercalcemia. However, this distinction has been blurred since it became clear that the mediator in many solid tumors that metastasize to bone is parathyroid hormone-related peptide (PTHrP), which is the same factor responsible for many cases of the humoral hypercalcemia syndrome. Distinctions between humoral hypercalcemia and osteolytic hypercalcemia have been made in the past based on measurements of nephrogenous cyclic AMP (1), a parameter of PTHrP production. However, PTHrP is not the only factor associated with the hypercalcemia of malignancy, and some patients with humoral hypercalcemia do not have increased nephrogenous cyclic AMP. Moreover, as noted above, PTHrP is often secreted by metastatic tumor cells associated with osteolytic lesions. In this chapter we focus on hypercalcemia associated with local osteolysis. The HHM syndrome is considered The Parathyroids, Second Edition

HYPERCakLCEMIA IN OSTEOLYTIC B O N E DISEASE D U E T O SOLID T U M O R S Hypercalcemia occurs commonly in patients with metastatic bone disease caused by solid tumors, and particularly in patients who develop osteolytic lesions as a result. It is most commonly caused by lung and breast cancer. In the past, approximately 30% of patients with breast cancer developed hypercalcemia at some time during the course of the disease, and usually late in the disease (2). The n u m b e r is probably less now, in part due to the widespread use of bisphosphonates in cancer bone disease. Hypercalcemia is almost always due primarily to an increase in bone resorption, which is caused in turn by the production of tumor cell factors that stimulate osteoclastic bone resorption. However, in most patients, there is also associated impairment of the kidney's capacity to compensate by excreting the increased filtered load of calcium. Renal tubular calcium reabsorption is increased almost universally in patients with the hypercalcemia of malignancy, and even in patients with hypercalcemia 691

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due to myeloma (3). It had previously been recognized that renal tubular calcium reabsorption was typically increased in patients with HHM-like syndromes (4), but it was widely t h o u g h t that hypercalcemia associated with osteolytic lesions or with myeloma was not usually associated with increased renal tubular calcium reabsorption. That conclusion has now been refuted by the studies of Tuttle et al. (3) and the earlier studies of Percival et al. (5). Gallacher et al. (6) also showed that in patients with breast cancer there was frequently an increase in reabsorption of calcium in the renal tubules that could not be readily accounted for by circulating P T H r E Tuttle et al. (3) carefully measured glomerular filtration rate as well as hydration status, and c o m p a r e d renal tubular calcium reabsorption with that of control patients who were m a t c h e d for i m p a i r m e n t of renal function. The mechanisms responsible for this increase in renal tubular calcium reabsorption are not entirely clear. PTHrP is likely responsible in some patients. In those patients with tumors that do not produce PTHrP, the m e c h a n i s m is likely related to other unexplained factors. Harinck et al. (7) suggested that calcium reabsorption may be due to changes in volume status caused by chronic hypercalcemia, so that subsequent volume depletion increased sodium reabsorption in the proximal tubules, and this is associated with increased calcium reabsorption. However, Tuttle et al. (3) showed that increased renal tubular calcium reabsorption occurred independently of hydration status and extracellular fluid volume.

Pathophysiology of the Metastatic Process The metastasis of t u m o r cells to specific sites in the skeleton is not a simple r a n d o m event d e t e r m i n e d solely by blood flow. Metastasis is instead a complex, directed, and multistep process that is d e p e n d e n t both on specific properties of the t u m o r cells and on factors in the bone m i c r o e n v i r o n m e n t that favor metastasis. The distribution of t u m o r cells in distant organs can be predicted by the anatomic distribution of blood flow from the primary site in 30% of cases. However, there are also specific properties at the metastatic site that d e t e r m i n e whether the metastasis will become established (8). Over 100 years ago, this n o n r a n d o m concept for t u m o r metastasis was recognized by Paget (9) as the "seed and soil" hypothesis of t u m o r spread. Because t u m o r metastasis is a multistep process involving separate discrete steps, interruption of any one or more of these steps can inhibit the metastatic process. Each of these discrete steps is due to specific molecular determinants of both the t u m o r and the tissue of the metastatic site (8,10). The steps involved in the shedding of t u m o r cells from the primary site

include d e t a c h m e n t of t u m o r cells from adjacent cells, followed by invasion of adjacent n o r m a l tissue. The cells then enter t u m o r capillaries (stimulated by specific angiogenesis factors p r o d u c e d by the tumor) and via these capillaries reach the general circulation (11). The mechanisms responsible for t u m o r cells entering blood vessels at the primary site are apparently similar to those involved in exit from the vasculature in the bone marrow cavity. These include a t t a c h m e n t of the t u m o r cells to basement membranes, secretion of proteolytic enzymes that disrupt the b a s e m e n t m e m b r a n e , and then migration of the t u m o r cells through these b a s e m e n t m e m b r a n e disruptions (12-14). A t t a c h m e n t of t u m o r cells to other cells and to extracellular structures is an i m p o r t a n t part of the metastatic process. Cell adhesion molecules (CAMs) such as laminin and E-cadherin play key roles in cancer cell invasion and metastasis. CAMs mediate cell-to-cell and cell-to-substratum communications. Cancer cell adhesion to normal host cells and to the extracellular matrix through C&Ms has been shown to regulate tumor cell invasiveness and proliferation (15). Loss of CAMs at the primary site promotes the d e t a c h m e n t of cancer cells from the primary tumor site and leads to local invasion and eventually to the develo p m e n t of a metastasis. In contrast, at the metastatic site, elevated expression of CAMs might be a prerequisite for cancer cells to arrest locally t h r o u g h attachment to the extracellular matrix. Expression of CAMs may then be important if reduced expression is necessary to free cancer cells from direct contact-mediated regulation by host i m m u n e cells. Studies have demonstrated that metastatic breast and ovarian cancers show heterogeneous expression of E-cadherin (16), and E-cadherin expression in these cancer cells may be reversibly modulated according to culture conditions in vitro (17) and environmental factors in vivo (18). Therefore, cancer cells may express either decreased or increased levels of ~s, d e p e n d i n g on the stage of metastasis developm e n t and sites of metastasis. We think it likely that the CAMs E-cadherin and laminin are involved in bone metastasis (see below). Integrins are the most a b u n d a n t CAMs and are responsible for a variety of cell-cell and cell-matrix interactions (19), and have been implicated in h e m a t o g e n o u s dissemination of t u m o r cells (20). In cancer metastasis, integrins have been shown to mediate cancer cell a t t a c h m e n t to vascular endothelial cells and to matrix proteins such as laminin and fibronectin, which underlie endothelium, an initial step in tumor colonization (15). For example, it has been demonstrated that A375 h u m a n m e l a n o m a cells express high levels of the CZv[33integrin (vitronectin receptor) on the cell surface when they bind to and invade the basement m e m b r a n e matrix matrigel (21).

OTHER SYNDROMESASSOCIATED WITH HYPERCALCEMIA /

Laminin Using a model based on earlier studies of Arguello et al. (23), we have found that synthetic antagonists to laminin inhibit osteolytic bone metastasis formation by A375 h u m a n m e l a n o m a cells in nude mice (22). We inoculated these h u m a n melanoma cells into the left ventricle of nude mice and examined the formation of osteolytic metastases by radiographs and histology. This process was prevented by a synthetic antagonist to laminin.

E-Cadherin E-Cadherin (Uvomorulin) is a 120-kDa cell surface glycoprotein involved in calcium-dependent epithelial cell-specific cell adhesion. E-Cadherin has homophilic properties in cell-cell adhesion and thus causes homotypic cell aggregation, which may be important in regulation of embryogenesis and morphogenesis (24). Evidence has shown that E-cadherin expression is related to cancer invasion and metastasis. Treatment of epithelial noninvasive Madin-Darby canine kidney (MDCK) cells with monoclonal antibodies to E-cadherin r e n d e r e d these cells more invasive (25). Overexpression of the E-cadherin gene in highly invasive cancer cells dramatically suppresses their invasiveness, and, conversely, introduction of E-cadherinspecific antisense RNA r e n d e r e d noninvasive epithelial cells invasive (26). E-Cadherin expression is increased in populations of MCF-7 breast cancer cells with reduced invasiveness, whereas relatively low levels of Ecadherin have been detected in the highly invasive h u m a n breast cancer cells, MDA-231 (27). We have examined the capacity of the h u m a n breast cancer cell lines MCF-7 (high E-cadherin expression) and MDA-231 cells (low E-cadherin expression) to form bone metastases (28). We have found that MDA-231 cells with low E-cadherin expression are much more effective at forming osteolyfic bone lesions in vivo. W h e n MDA-231 cells were transfected with E-cadherin cDNA, their capacity to form osteolydc bone lesions was markedly reduced (28). Although MDA-231 cells form obvious osteolytic lesions by 4 weeks after inoculation into the left ventricle, MCF-7 cells take more than 8 weeks to form similar lesions.

Secretion of Proteolytic Enzymes Tumor cells produce proteolytic enzymes to degrade b a s e m e n t m e m b r a n e s and to traverse blood vessels to enter the tissue stroma. These proteolytic enzymes may be p r o d u c e d directly by tumor cells (type IV collagenase) or by host cells. Garbisa et al. (29) have emphasized the potential role of type IV collagenase and its

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potential to degrade the capillary basement membrane. Basset et al. (30) have shown that host cells such as fibroblasts and stromal cells associated with some invasive breast carcinomas expressed a gene that encodes a metalloproteinase. Expression of this metalloproteinase was stimulated by growth factors produced by the breast cancer cells such as platelet-derived growth factor (PDGF), transforming growth factor c~ (TGF-~), and basic fibroblast growth factor (bFGF).

Cell Motility Tumor cells may be attracted out of the vasculature toward bone surfaces by a n u m b e r of chemotactic factors. Bone contains multiple factors with chemotactic potential for tumor cells. These include fragments of type I collagen, which have been shown to cause unidirectional migration of tumor cells (31), and fragments of the bone protein osteocalcin, which may cause chemotaxis of tumor cells and monocytes (32). The conditioned media harvested from resorbing or remodeling bones contain chemotactic activities that stimulate the unidirectional migration of rat and h u m a n tumor cells (33,34). The nature of the chemotactic factor(s) responsible has not been identified, but may be TGF-[3, which is present in a b u n d a n t amounts in bone (35).

Other Mechanisms Tumor cells have varying capacity for metastasis. Primary tumors comprise heterogeneous populations of cells that have been shown in many studies to have differing invasive properties and different metastatic potential (36-39). Tumor cells are unstable phenotypically for reasons that are not completely understood, although this has been the subject of many studies (40,41). As tumors grow, they undergo rapid clonal diversification (42). Some of the possibilities for this variability in individual tumor cell characteristics include host selection, which may be due to some cells having the capability of surviving physiologic immune defense mechanisms, or the effects of treatment with either anticancer drugs or radiation therapy, which may lead to acquired genetic variability. It is now well recognized that the genetic comp o n e n t of tumor cells can greatly influence their metastatic potential as well as their invasive capabilities andtumorigenicity (8,29). In breast cancers, expression of the Her-2/Neu oncogene occurs in parallel with aggressive behavior of the cells (43). In some tumors, particularly melanoma, expression of the NM23 oncogene appears to inhibit the capability of the tumor to metastasize (14,44). As already noted, the expression of laminin receptors on tumor cells may cause enhanced metastatic capabilities. Deletions of chromosomal material on chromosome l l p has been noted in aggressive

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breast cancers (45) and deletions of chromosomes 17 and 18 may be found in colon carcinomas that arise from colonic polyps (46,47). There are multiple mechanisms that can protect against t u m o r metastasis. Current evidence suggests that less than 1% of t u m o r cells survive in the circulation (8). Tumors probably survive best in the circulation as aggregates, which may prevent the loss of individual cells in the circulation from mechanical shear forces or from anoxia. The blood coagulation m e c h a n i s m has been long t h o u g h t to be i m p o r t a n t in p r o m o t i n g t u m o r cell metastasis (48-50). Anticoagulants of the coumadin class have been tested over many years for their capacity to inhibit metastasis and p r o m o t e survival, although the data remain inconclusive and controversial. Once within the bone marrow cavity, t u m o r cells pass through wide-channeled marrow sinusoids. The cellular and molecular events involved in their passage from the marrow sinusoid to the bone surface may be similar to the events involved in their escape from the primary site. Again, the tumors must attach to basement membranes of blood vessels, disrupt these basement membranes by the production of proteolytic enzymes, and then migrate through the b a s e m e n t m e m b r a n e to invade the tissue stroma. An additional step in the pathophysiology of the bone metastasis is the destruction of bone. There is some controversy over the mechanisms by which this may occur (see below). We have studied several h u m a n tumors (A375 m e l a n o m a cells and cultured breast cancer cells) to determine their capability of growing metastases in bone using a modification of the technique described by Arguello et al. (23). We have used inhibitors of specific cell adhesion molecules to d e t e r m i n e their effects on the metastatic process. Tumor cells bind to b a s e m e n t m e m b r a n e s on endothelial cells at the metastatic site through specific cell adhesion molecules such as fibronectin and laminin. We have used synthetic antagonists of laminin that interfere with the binding of laminin to laminin receptors on the t u m o r cells to d e t e r m i n e the effects on the formation of the bone metastasis. We have found that a synthetic antagonist YIGSR totally inhibits this process in bone (22). A similar result was found when this antagonist was used in a lung metastasis model (51). There may be a 50-fold increase in laminin receptors on the surface of highly invasive and metastatic t u m o r cells c o m p a r e d with other t u m o r cells that do not have the capability of metastasis or invasion (52).

T u m o r Cell Mediation o f B o n e Destruction at the Metastatic Site The cellular m e c h a n i s m responsible for local destruction of bone by t u m o r cells remains controver-

sial. Although definitive p r o o f is still not available, it is likely that the p r e d o m i n a n t and possible sole mechanism for bone destruction is an increase in osteoclast activity. Tumors produce factors that stimulate osteoclasts, which then in turn are responsible for the resorption of bone. An alternative possibility is that tumor cells may destroy bone directly without the addition of osteoclasts. The evidence in favor of osteoclastic bone resorption being the p r e d o m i n a n t mechanism is twofold. First, when looked for carefully using techniques such as scanning electron microscopy, osteoclasts are invariably found to be present adjacent to t u m o r deposits (53). Moreover, distinctive osteoclast resorption lacunae are universally present. Such studies show no evidence of smaller resorption lacunae corresponding to the size of m o n o n u c l e a r tumor cells. Second, drugs that effectively inhibit osteoclast activity, such as the bisphosphonates, plicamycin, and gallium nitrate, work very effectively both in the hypercalcemia of malignancy, which is due predominantly to increased bone resorption caused by tumors, as well as in localized osteolysis without hypercalcemia. These data suggest that because these drugs work through the inhibition of osteoclast function, osteoclasts are major (possibly sole) mediators of the bone destruction. Nevertheless, there is in vitro evidence that suggests that breast cancer cells have the capacity to resorb bone in vitro. W h e n breast cancer cells have been added to devitalized bone, they cause both mineral release and matrix degradation (54).

M e c h a n i s m s o f Osteoclast Stimulation at the Metastatic Site As indicated above, the major cellular mechanism for bone destruction in osteolytic bone disease is osteoclastic. Consequently, there is interest in determining the mediator that is responsible for this increase in osteoclast activity. Studies suggest that the factor responsible in breast cancer is PTHrP, the tumor peptide that has been associated with humoral hypercalcemia of malignancy. This is so even although many of these patients do not have increased plasma PTHrP or increased n e p h r o g e n o u s cyclic AMP. Thus, the absence of these latter parameters does not mean that PTHrP is unimportant in causing osteolysis and subsequent hypercalcemia. Studies using immunohistochemistry have shown that there is increased expression of PTHrP in bone sites compared with either soft tissue metastases or primary tumors in patients with carcinoma of the breast (55). O n e example we studied involved inoculation of the h u m a n breast cancer cell line MDA-MB-231 into the left cardiac ventricle of nude mice; the development of osteolytic lesions was followed over the following 4 to 6 weeks, and it was been found that there is an increase in

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PTHrP expression in the tumor cells that metastasize to bone. This is associated with the development of typical osteolytic bone lesions that are seen in patients with the disease. When tumor-beating nude mice are treated with neutralizing antibodies to PTHrP, not only is there a decrease in the development of the osteolytic bone lesions, but there is also a decrease in the tumor burden in bone (56). The most likely explanation for the increase in PTHrP production in the bone microenvir o n m e n t is that bone provides a fertile environment for the growth of tumor cells and also enhances the production of PTHrE Subsequent studies have shown that a likely mechanism responsible for this effect is production of TGF-[3, which is released from bone in active form when bone resorbs (57). Studies by Yln et al. (58) have shown that when MDA-MB-231 cells are transfected with a dominant-negative type II TGF-[3 receptor, which makes the cells unresponsive to TGF-[3, PTHrP production is not enhanced in the bone microenvironm e n t and the development and progression of osteolytic bone lesions is significantly less compared with this metastasis caused by parental MDA-MB-231. It is also likely that there are other mechanisms for bone destruction associated with tumor cells that metastasize to bone. These may involve production by the tumor cells or by host immune cells in the bone microenvironm e n t of mediators such as TGF-e~, interleukin-lot, tumor necrosis factor, and interleukin-6, each of which, acting alone or in combination with PTHrP, represents a strong stimulus to increase bone resorption (59).

Bone-Derived T u m o r Growth Factors at the Metastatic Site Bone provides a very favorable niche for tumor cells and it is clear that many tumors grow very well, in this microenvironment. In part, the reason may be that bone is a large repository for growth regulatory factors (35). In particular, bone is rich in TGF-[3, but also stores other growth regulatory factors that may act as tumor growth factors, including bone morphogenetic proteins, heparin-binding fibroblast growth factors, platelet-derived growth factors, and insulin-like growth factors (IGF-I and IGF-II). These factors are presumably the reason that bone is resorbed so avidly in metastases. They may be made available locally through bone resorption. This has been shown particularly in the case of TGF-[3. TGF-[3 may alter the behavior of many tumor cells, and in particular breast cancer cells, to enhance the production of parathyroid hormone-related protein (60). The mouse metastasis model, which was described above, provides a very useful approach for studying osteolytic bone metastasis (61). The bone lesions are typical of patients with the disease. The t u m o r cells can be modified by transfection with genes

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that are likely involved in the metastastic process, or by determining whether a specific mechanism is responsible by transfecting the cells with genes that interfere with this function, such as tissue inhibitor of matrix metalloproteinase (TIMP) or dominant-negative type II TGF-[3 receptors (58). These approaches can provide considerable insights into the molecular mechanisms responsible for osteolytic bone metastasis. Another factor that may be important in tumor cell behavior in the bone microenvironment is calcium. Calcium released by resorbing bone may alter tumor cell proliferation (62). Unfortunately, there is still limited information on this subject. One of the major difficulties that has limited the study of bone metastasis has been the lack of suitable animal models. Although this has retarded the accumulation of knowledge in the pathophysiology of bone metastasis, it has not been a problem in investigating other types of metastases, and in particular lung metastases (38). Arguello et al. (23) devised a technique whereby tumor cells are injected directly into the left ventricle of mice. This leads to the colonization of bone in regions containing hematopoietic bone marrow by tumor cells with potential to metastasize to bone. This process leads to lytic lesions resembling those seen in patients with cancer. In these models, bone metastasis occurs when tumor cells have ready access to the arterial circulation. We have modified this model by the use of h u m a n tumor cells in nude mice (22). By inoculating h u m a n breast cancer cells into the left ventricle of nude mice, we have been able to characterize a n u m b e r of steps involved in osteolytic bone metastasis. By treating the animals with antagonists or antibodies to specific molecules involved in the process (for example, E-cadherin, PTHrP) or by transfecting the tumor cells with specific genes (for example, E-cadherin, TIMP-2, src, and PTHrP), one can determine their role in this process. Moreover, by treating the animals with agents (for example, bisphosphonates, growth factors) that regulate normal bone cell function, one can determine the interaction between bone cells and tumor cells in the bone microenvironment. With a special modification of this technique, Yoneda et al. (63) have developed a m e t h o d for specifically examining metastasis to the calvarium to give a better picture of the progressive development of bone metastasis. Shevrin et al. (64) used prostate cancer cells to induce bone metastases following intravenous injection and occlusion of the inferior vena cava. Pollard et al. (65,66) used rat prostate adenocarcinoma cells in L o b u n d Wistar rats to show that these tumors cause a profound local change in bone formation. When this tumor is injected adjacent to a bone surface where the periosteum is mildly damaged by scratching with a needle tip, the tumor stimulates adjacent new woven bone formation.

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Specific Treatments for Metastatic Osteolytic Bone Disease The best form of treatment for metastatic bone disease is ablative therapy for the tumor. Unfortunately, as already indicated, in the vast majority of patients this is not feasible. Most attention at the present time is being focused on drugs that inhibit osteoclast activity, such as the bisphosphonates. Over the past 20 years there have been many studies examining the effects of bisphosphonates in patients with osteolytic bone metastases, and there is accumulating evidence that bisphosphonates are extremely effective at inhibiting skeletal complications (pathologic fractures, need for radiation or surgery for osteolytic bone disease, prevention of spinal cord compression due to vertebral collapse, and prevention of episodes of hypercalcemia). The most recent of these studies have been double-blind randomized and controlled. Most of them have been performed with pamidronate used intravenously by infusion every month. Pamidronate clearly reduces the n u m b e r of skeletal-related events and also delays the onset of the first skeletal-related event in breast cancer patients with osteolytic bone metastases (67). There are varying reports of the effects of pamidronate on tumor mass in soft tissue or bone. Our findings in the model described above, in which MDA-MB-231 cells are inoculated into the left ventricle of nude mice, suggests that bisphosphonates not only reduce osteolytic bone lesions, but also markedly reduce tumor burden in bone (61). This is likely due to the decreased production of growth factors in the bone microenvironment by inhibition of bone remodeling. It remains unclear whether this is also associated with effects of bisphosphonates to increase or alter tumor burden in other metastatic sites, although this seems unlikely from recent studies (68). Bisphosphonates do not have any direct effect on tumor mass, as has been shown in a n u m b e r of studies (69,70). It is h o p e d that a new generation of bisphosphonates that may be administered orally will be useful not only in the treatment of patients with established metastases, but also in the prevention of the development of new metastases. Although information is not currently available in this regard, it seems likely that inhibitors of osteoclastic bone resorption may be even more effective in the prevention of new metastases than in the treatment of established metastases. Theoretically, inhibition of continued resorption would leave the patient with a residual lytic lesion in bone (although occasionally patients may show some sclerosis of the healing lesion). On the other hand, in those patients who have malignancies with a predilection for the skeleton, such as breast cancer, preventive treatment with an inhibitor of osteoclastic bone resorption may inhibit the initial development of an osteolytic metastasis.

As indicated earlier, attention should not be focused solely on the osteoclasts. Drugs such as laminin antagonists that prevent binding of tumor cells to basement membranes, inhibitors of proteolytic enzyme disruption of the basement membrane, or t u m o r cell chemotaxis may also turn out to be useful in the prevention or treatment of established metastases. Laminin antagonists prevent tumor metastasis in an experimental model. We have used neutralizing antibodies of PTHrP to prevent progression of osteolytic bone lesions in animal models of h u m a n metastastic breast cancer (56) and also transfected the h u m a n t u m o r cells with inhibitors of the matrix metalloproteinases, namely, tissue inhibitor of matrix metalloproteinase-2, and have shown a decrease in osteolytic bone lesions (71). Models such as these may have predictive value prior to the initiation of clinical trials with experimental therapies such as laminin antagonists and inhibitors of proteolytic enzyme digestion.

PATHOGENESIS OF HYPERCakLCEMIA IN MYELOMA Hypercalcemia in myeloma is due primarily to increased bone resorption. However, the pathogenesis is more complex than just a simple increase in bone resorption. Only about 20 to 40% of patients with myeloma develop hypercalcemia (72). This figure is now declining, likely due to the widespread use of bisphosphonates in patients with multiple myeloma. Moreover, although hypercalcemia occurs in those patients who have the largest tumor volume, not all patients with the largest tumor burdens develop hypercalcemia. In addition, we have found that measurements of total body myeloma cell n u m b e r and production of bone resorbing activity by bone marrow myeloma cells in vitro do not correlate closely with the production of hypercalcemia (73). However, there is a highly significant correlation (P < 0.001) between the amounts of bone resorbing activity produced by cultured marrow myeloma cells and the extent of bone destruction as determined by radiologic lesions in the patient. Thus, other factors in addition to production of bone resorbing activity must be involved in the pathogenesis of the hypercalcemia. Impaired renal function is c o m m o n in myeloma for a n u m b e r of reasons. These include, in addition to hypercalcemia, uric acid nephropathy, amyloid nephropathy, myeloma kidney due to Bence-Jones protein excretion, and chronic infections. Because hypercalcemia occurs almost always in patients with impairment of renal function, we suggest that this is an important contributing factor in the pathophysiology of hypercalcemia. Increased bone resorption in myeloma leads to increased entry of calcium into the extracellular fluid, but many patients with

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normal homeostatic control mechanisms (i.e., normal renal function) are able to compensate for this increase in extracellular fluid calcium and maintain the serum calcium in the normal range by increasing urine calcium excretion. However, in patients with impaired renal function, this increase in bone resorption overwhelms the kidney's capacity to compensate and these patients develop hypercalcemia. This is more likely to occur in patients who produce Bence-Jones protein or who are predisposed to impaired renal function for other reasons (72). In many cases, impairment of renal function may also be due to the direct effects of hypercalcemia on the kidney. Because the mechanisms responsible for hypercalcemia in patients with myeloma are likely different from those in patients with solid tumors, there are also subtle differences in laboratory test findings at the time of diagnosis. Because renal impairment is frequent, many patients with myeloma have increased serum phosphorus rather than a decreased serum phosphorus, which is c o m m o n with other types of malignancy. In addition, serum alkaline phosphatase, a marker of osteoblast activity, is usually not increased in patients with myeloma because there is little active new bone formation. For similar reasons, bone scans are also typically negative. Most available evidence suggests that the cellular mechanism of bone destruction in myeloma is due to an increase in osteoclast activity. This was first studied systematically on autopsy and biopsy samples of bone from a series of 37 patients with myeloma. Active osteoclastic bone resorption was correlated with the presence of infiltrations of more than 20% myeloma cells in the adjacent marrow cell population. In biopsy specimens of bone from myeloma patients that contained few or no myeloma cells, little osteoclast activity or evidence of active bone resorption was observed. This suggested that the myeloma cells caused an increase in local osteoclast activity that led to increased bone resorption. There have been some suggestions that osteoclasts in patients with myeloma may not be as large as the osteoclasts in patients with primary hyperparathyroidism or even solid tumors associated with bone destruction. The association between increased osteoclasts on bone resorbing surfaces and areas of heavy myeloma cell infiltration was confirmed in a later study using quantitative bone h i s t o m o r p h o m e t r y on transiliac bone biopsies from 118 patients (74). Abnormalities in bone formation were also confirmed. Bone-forming surfaces were increased away from the areas of heavy myeloma cell infiltration but the osteoid seams were reduced in thickness and had a lowered calcification rate. The authors concluded that the activity of individual osteoblasts was reduced in many patients with myeloma. More detailed studies on bone formation rates in early and overt myeloma were reported by

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Bataille et al. (75). These workers suggested that bone formation rates were markedly suppressed in patients with advanced disease, but surprisingly were increased earlier in the disease course. Further evidence that the mechanism of bone resorption in myeloma is primarily osteoclastic is provided by data that show drugs that are relatively specific inhibitors of osteoclast activity, such as the bisphosphonates, plicamycin, and calcitonin, are usually very effective in lowering the serum calcium in patients with myeloma (76-78). Bone scans in myeloma often show no abnormality. Isotopic bone scans are p e r f o r m e d with labeled bisphosphonates that are taken up at sites of mineral deposition and reflect increased activity of bone-forming cells. Myeloma is characterized by discrete lytic lesions with little increase information and this is probably the explanation for the difference in appearance between the bone scans in myeloma and the bone scans in breast cancer, in which osteoblast activity is frequently increased. Similarly, serum alkaline phosphatase, which also reflects osteoblast activity, is usually not increased in patients with myeloma, unless they have coexistent pathologic fractures. In a few patients with myeloma (probably less than 1% in the experience of most clinicians), osteosclerosis rather than osteolytic bone destruction is the major radiologic finding (79). Valentin-Opran et al. (74) reported morphologic bone biopsy data from 118 patients, 3 of whom had increased trabecular bone volume, suggesting that these biopsies represented the osteosclerotic variant. The molecular mechanism responsible for the increase in osteoblast activity is unknown. These patients frequently have peripheral neuropathy and they may or may not have other multisystem features that include polyneuropathy, organomegaly, endocrinopathy, N protein, and skin changes (designated by the acronym, POEMS syndrome) (80). Hypercalcemia has not been described in this condition.

Pathophysiology of Myeloma Bone Disease Osteolytic bone lesions are a p r o m i n e n t feature of multiple myeloma (81,82). Of these patients, 80% suffer from extensive bone destruction, which may present as intractable bone pain, pathologic fractures, spinal cord compression, and life-threatening hypercalcemia. Although the bone lesions can occur as diffuse osteopenia, they are more commonly present as localized areas of osteolysis adjacent to "nests" of myeloma cells. This juxtaposition of the myeloma cells and the lytic lesions p r o m p t e d early reports that myeloma cells are capable of resorbing bone. However, it is now evident that this is not the case, based on histologic analyses of bone lesions that clearly indicating that bone destruction is a direct result of increased osteoclastic resorption (82).

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Although the observation that local cellular mechanisms were largely responsible for the bone destruction in myeloma was first made 25 years ago, there are still unresolved questions as to the nature of the local mediator(s) responsible for the increased osteoclast recruitm e n t and activation. In vitro studies show that h u m a n and murine myeloma cells secrete tumor necrosis factors (TNFet, TNF[3), IL-6, RANK ligand, PTHrP, IL-1, and IL-17, all of which can stimulate osteoclast formation and activity, acting independently or in concert (81-83). However, it has not been possible to show convincingly so far that any of these cytokines are responsible for the bone lesions in vivo. The situation resembles solid tumors and osteoblastic metastases, wherein the lack of good in vivo models has made it difficult to determine what the responsible mediator is. Current evidence suggests that myeloma cells behave differently in the marrow microenvironment and that cell-cell a n d / o r cell-extracellular matrix (ECM) interactions between tumor cells and marrow stroma are critical, not only for the growth and survival of the tumor cells, but may also initiate and maintain the aggressive bone destruction. Because multiple myeloma cannot presently be cured by standard chemotherapeutic approaches or by stem cell transplantations, and because patients with the disease may survive for some years, it will be necessary to understand the pathophysiology of the bone destruction in order to significantly reduce patient morbidity.

Importance of Cell-Cell Interactions in Myeloma Cell-cell and cell-ECM adhesion events have been increasingly implicated in the etiology and pathogenesis of many diseases, including cancer (83-85). H u m a n and murine myeloma cells express very late antigen 4 (VLA4) (83,84,86,87), a heterodimeric complex of 0/-4 and [31 integrin subunits. All three known VLA4 ligands, VCAM-1, fibronectin, and osteopontin, are present in the marrow and of these VCAM-1 is expressed by bone marrow stromal cells constitutively. There is evidence that indicates that myeloma cells use VIA4 not only to h o m e to bone, but also to adhere preferentially to bone marrow stromal cells through VC&M-1 as well as the ECM (83,84,86--88). Data from other studies, including ours, provide compelling evidence that these cellular interactions are important not only in the growth of myeloma in bone, but also in initiating the sequence of events that lead to bone destruction. For example, our data indicate a link between VCAM-1/VI_A4 and osteoclast formation via induction of RANKL following direct cell-cell contact between bone marrow stromal cells and myeloma cells. 5TGM1 myeloma cells exhibit tight adherence to the mouse marrow stromal cell line ST2 in vitro, and

contact between the two cell types increased RANKL mRNA expression in comparison to either cell type alone. This interaction also stimulated production of a soluble factor(s) by the myeloma cells capable of enhancing RANKL expression in ST2 cells and inducing resorption in fetal rat long bones (89,90). The effect of the cell-cell interaction was mimicked by treating the myeloma cells with recombinant soluble VCAM-1 (90) and conversely, the production of the soluble activity was blocked by neutralizing antibodies to either et4 or to VC&M-1 (89). Similar data showing that an anti-et 4 antibody blocks osteoclast formation in vitro in a coculture of the h u m a n ARH77 myeloma cell line and bone marrow stromal cells have been reported (91 ).

Macrophage Inflammatory Protein-1 ct and Receptors Macrophage inflammatory protein (MIP-let) is a likely potential candidate for the soluble osteoclastactivating factor (OAF) produced by myeloma cells. MIPlet belongs to a family of small (8-12 kDa) proteins that are chemoattractive for leukocytes (92-94). These proteins, called chemokines, are classified into two major groups, cysteine-cysteine (CC) or cysteine-X-cysteine (CXC), depending on the position of the first two invariant cysteine residues. Whereas CC chemokines preferentially attract and activate T lymphocytes and monocytes/macrophages, CXC chemokines mainly attract and activate neutrophils (93,94). Chemokines also activate leukocyte integrins, increasing their affinity for their cognate ligands (94). MIP-let, a CC chemokine, originally purified from endotoxinstimulated mouse peritoneal macrophages (95), is produced primarily by leukocytes and not by stromal cells. As with their conserved structural residues, chemokines and MIP-let share other properties, including lack of constitutive secretion by normal cells in vivo (93,94) and extremely low or undetectable circulating levels in plasma of normal healthy people. By in situ hybridization, MIP-let mRNA was detected only in eosinophil precursors in normal human bone marrow (96).

MIP-lct and Osteoclastogenesis in Vitro Although its biologic functions have not been fully elucidated, our data and the limited literature available on MIP-let so far suggest that it may play a role in osteoclastic bone resorption (91,96-99). The earliest identifiable stage in the osteoclast lineage is the g r a n u l o c y t e / m a c r o p h a g e colony-forming unit (CFUGM), the progenitor that proliferates and differentiates into committed precursors for the lineage (100). MIP-let was reported to enhance formation of CFU-GM

OTHER SYNDROMESASSOCIATEDWITH HYPERCALCEMIA / colonies in vitro and ex vivo (101). Perhaps of greater significance was the finding that MIP-le¢ enhanced formation of CFU-GM colonies in CCR1 + / + but not CCR1 - / - mice (101), demonstrating that these effects are mediated solely through the CCR1 chemokine receptor and that they represent nonredundant functions. Other in vitro studies show that human MIP-loL is chemotactic for human osteoclast precursors as well as for isolated rat osteoclasts (99). Human MIP-le~ was also shown to stimulate 1,25 (OH) 2D3-independent formation of TRAP+ multinucleated cells (MNCs) from rat bone marrow cells cultured on dentin (96). In addition, a neutralizing antibody to rat MIP-lc~ inhibited formation of MNCs in rat and porcine bone marrow cultures (98).

MIP-I~ and Multiple Myeloma Although various tumor cells have been shown to secrete chemoattractant factors for monocytes (92), there is a paucity of information on chemokines as they relate to myeloma. Work by Roodman and colleagues has implicated MIP-le~ in multiple myeloma (97). Using competitive polymerase chain reaction (PCR), MIP-I~ mRNA was shown to be elevated in bone marrow plasma of multiple myeloma patients compared to normal controls. Furthermore, elevated levels of MIPlc~ were detected in marrow supernatants from a majority (eight of nine) of multiple myeloma patients with active disease by enzyme-linked immunoassay (ELISA) but not in any of nine normal controls. An anti-MIP-lc~ neutralizing antibody blocked the stimulatory effect of bone marrow supernatants from multiple myeloma patients on osteoclast formation in h u m a n bone marrow cultures, but had no effect on control levels of osteoclast formation (97). Other workers have also reported elevated MIP-lot levels in four out of eight aspirates from multiple myeloma patients (91). MIP-lotproducing myeloma cell lines markedly enhanced formarion of TRAP+ MNCs when cocultured with rabbit bone marrow cells, and this was abrogated by an antiVI_A4 antibody (91). We have found also that MIP-lot is expressed by human and murine myeloma cell lines, including 5TGM1 cells. Importantly, MIP-le~ level is increased in a 5TGM1/ST2 coculture system implying a role for VI2k4-VCAM-1 interaction in regulating MIP-le~ expression. Because MIP-le~ is known to promote cellular adhesion by up-regulating expression of integrins such as % (93,94), MIP-lot could potentially increase adherence of myeloma cells to marrow stromal cells by up-regulating % and consequently activating VLA4. This increased cellular adhesion would, in turn, augment MIP-lot production, resulting in recruitment of monocytes/macrophages that are precursors for osteoclasts (100).

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5T Model of Myeloma Bone Disease Research on multiple myeloma has been hindered by a lack of appropriate animal models that faithfully replicate the disease in humans. However, several models of myeloma bone disease are now available, including the murine 5T model, which we and others have characterized (87,102,103). This model was originally described by Radl and colleagues, who propagated myelomas occurring spontaneously in aged inbred C57BL/KaLwRij mice primarily by intravenous transfers into young recipients of the same substrain (104). The murine 5T model shares a n u m b e r of clinical, histologic, and immunologic features with multiple myeloma in humans (87,102-104).

Effects of Bone and Bone Cell Products on Myeloma Cells Since the observation that myeloma cells cause bone destruction by producing factors that stimulate osteoclasts to resorb bone, the attention of the field has been focused on the identification of the responsible mediators. However, the interactions between myeloma cells and bone cells may be much more complicated than simple excess production by myeloma cells of a factor that stimulates osteoclasts. Data now suggest the hypothesis that the avidity with which myeloma cells grow in bone compared with other hematologic malignancies is likely influenced by products produced as a consequence of osteoclastic bone resorption, and in particular the cytokine interleukin-6, which is a major growth regulatory factor for myeloma cells. Thus, bone may not be simply a passive bystander in this disease, but may rather act to amplify the growth of myeloma cells in bone. If this concept is correct, then a vicious cycle may exist between myeloma cells and osteoclastic bone resorption whereby myeloma cells stimulate osteoclasts to resorb bone by the production of osteotropic cytokines such as tumor necrosis factor [3, interleukin-l[3, and interleukin-6, but during the process of osteoclastic bone resorption the cytokine interleukin-6 is generated in prodigious amounts by cells involved in the resorption process, and this enhances the growth of myeloma cells in bone. This vicious cycle could mean that the greater the bone destruction, the more aggressive the behavior of myeloma cells, which then may cause even greater bone destruction. This concept is based on the information that osteoclasts produce considerably more interleukin-6 compared to any other cell. The only cells that produce comparable amounts of IL-6 are endometrial cells. However, it should be recognized that these data do not indicate which cell in bone is the most abundant

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source, because osteoclasts are much fewer in n u m b e r than are other cells. Nevertheless, interleukin-6 production by bone organ cultures and by osteoclasts is reduced by treatment with calcitonin, suggesting that osteoclast production of interleukin-6 is clearly regulated by factors that control osteoclast function. It is certainly possible that production of IL-6 in the bone microenvironment could result from direct cellular interactions between myeloma cells and other cells, such as stromal cells, osteoblasts, or even osteoclasts (105). Studies have shown that cell-cell attachment molecules may be important in the production of bone resorbing activity in the bone microenvironment. Michigami et al. (89) found that myeloma cells attach to stromal cells in the marrow microenvironment by mechanisms that involve the integrin %[31 expressed on myeloma cells and VCAM-1 bone marrow stromal cells.

Treatment of Myeloma Bone Disease We consider that all patients with hypercalcemia require specific treatment to inhibit bone resorption. As in other malignant diseases, hypercalcemia due to increased bone resorption is likely to worsen very rapidly. This is particularly true for myeloma, in which renal function is so tenuous because of complicating factors that are deleterious to the kidneymBence-Jones proteinuria, hyperuricemia, urinary tract infection, and, in a few patients, amyloid. Hypercalcemia and increased bone resorption are improved in many patients with myeloma by treatment with cytotoxic drugs. This often leads to a decrease in bone pain and a lowering of the serum calcium. This is particularly likely to occur if corticosteroids, which also inhibit bone resorption in myeloma, are part of the treatment regimen. Even in patients with myeloma who respond to cytotoxic drug therapy, bone lesions are unlikely to recalcify (72), possibly because there is usually only about a 1 log reduction in myeloma cell mass. The primary goal of therapy for the hypercalcemia of myeloma should be the inhibition of osteoclastic bone resorption. A n u m b e r of therapeutic agents that inhibit osteoclastic bone resorption are available. These include lowering serum calcium by drugs such as corticosteroids, calcitonin, plicamycin, and the bisphosphonates. In patients with myeloma bone disease, the most effective of these are the new-generation bisphosphonates, one of which (pamidronate) was approved by the Food and Drug Administration (FDA) in 1995 in the treatment of myeloma bone disease. The bisphosphonates are pyrophosphate analogs that suppress bone resorption by mechanisms that are still not clearly defined, but probably involve binding of the drug to the bone surface as well as direct

inhibition of osteoclast activity (76). The agent 3-amino-l-hydroxypropane-l,l-diphosphonate (pamidronate) has been shown to be particularly effective in patients with secondary osteolytic lesions due to breast cancer and myeloma, and has also been extremely effective in lowering serum calcium (106). Unfortunately, pamidronate and other amino bisphosphonates may cause upper gastrointestinal mucosal ulcers when used in oral form, which limits its usefulness when taken over prolonged periods, but it is extremely effective over short periods when used parenterally. Other bisphosphonates, such as clodronate, are also very effective in lowering serum calcium, relieving bone pain, and reducing bone turnover in patients with myeloma (107). The older bisphosphonate, ethane-l-hydroxy-1, 1-diphosphonate (EHDP), or etidronate, has been used intravenously in the treatment of hypercalcemia (108). Its effects are possibly slower and less reliable than the newer bisphosphonates, and its current use is not recommended. Clodronate was withdrawn from testing in the United States because of potential toxicity. The use of bisphosphonates to treat myeloma bone disease without hypercalcemia will be considered below.

Corticosteroids Corticosteroids are frequently effective in controlling osteoclastic bone resorption in myeloma (109). As already indicated, they are often part of the initial cytotoxic regimen. The inhibitory effects of corticosteroids on bone resorption are d e p e n d e n t on the stimulus. They are much less effective against PTH than they are against other factors. Corticosteroids inhibit the bone resorbing activity produced by activated peripheral blood leukocytes in low concentrations (110,111 ), and this is possibly why they are very effective in lowering the serum calcium in many patients with myeloma and related diseases. The mechanism by which they exert this effect may be due to impairment of formation of new osteoclasts, as has been shown in a feline marrow cell culture system for generation of cells with osteoclast characteristics (112). Although most patients with myeloma and hypercalcemia respond to corticosteroid therapy, some do not (77), particularly those late in the course of their disease.

Plicamycin (Mithramycin) Plicamycin is a powerful inhibitor of bone resorption, acting possibly by exerting a cytotoxic effect on bone resorbing cells (113-115). It is frequently used as a treatm e n t of hypercalcemia and is usually effective (78,116). However, we do not advocate its use as a primary agent in myeloma (or any other situation) because it is directly nephrotoxic and its side effects are more frequent in patients with impaired renal function (76). Because

OTHER SYNDROMESASSOCIATEDWITH HYPERCALCEMIA /

many patients with myeloma and hypercalcemia have impaired renal function before treatment, we think it should be used as a third-line drug for therapy. Calcitonin and Corticosteroids Calcitonin and corticosteroids used in conjunction are almost always effective in patients with hypercalcemia due to myeloma (77). These patients may be extremely difficult to treat because they frequently do have impaired renal function, which makes the use of other agents that lower the serum calcium (such as oral phosphate or plicamycin) undesirable to use. Combined corticosteroid/calcitonin therapy is not harmful when used for short periods in patients with impaired renal function. Although the effects may be relatively short-lived, we find that withdrawal of calcitonin for several days followed by its reintroduction almost always results in return of the serum calcium into the normal range or into a range that is not associated with symptoms due to hypercalcemia. We have treated several patients for a n u m b e r of months with this form of therapy in situations where no other agent was suitable (76). Moreover, the effects of combined therapy are very rapid, with lowering of serum calcium usually occurring within 12 hours. The combination is more effective than either agent used alone and seems to be more effective in myeloma than in patients with solid tumors and hypercalcemia (77). Calcitonin and glucocorticoids are also very effective inhibitors of bone resorption in vitro (114). Calcitonin treatment alone leads to the escape p h e n o m e n o n , characterized by loss of effectiveness despite the continued presence of calcitonin (117). Glucocorticoids prevent the escape p h e n o m e n o n both in vitro and in vivo (76,77,118). Whether the effects of the combination are more directed at bone, as suggested by these data, or at the kidney, as proposed by Ralston et al. (119), is unclear.

Other Agents Other cytotoxic drugs, such as gallium nitrate (120) and cisplatinum (121), have been used in the therapy of hypercalcemia associated with malignant disease. Gallium nitrate, a very effective inhibitor of osteoclastic bone resorption, is apparently useful both in the treatm e n t of hypercalcemia of malignancy and in patients with myeloma. It seems to have a spectrum of activity similar to that of the new-generation bisphosphonates and to mithramycin. Promotion of Renal Calcium Excretion As in other forms of hypercalcemia, promotion of a saline diuresis in myeloma may transiently lower the

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serum calcium and improve renal function because sodium and calcium handling by the renal tubules are linked. This can be achieved with the use of saline infusions. Some workers also use furosemide, although at typical doses it produces only marginal additional benefits to saline infusion alone (76,122) and may result in dehydration and reduced renal calcium excretion. Great care must be taken in the use of large volumes of intravenous fluids in myeloma, because frequently the impairment of renal function is fixed and large volumes of fluids will lead to the rapid onset of congestive cardiac failure. For this reason, administration of large volumes of fluids to some patients with myeloma may require central venous pressure monitoring.

Treatment of Painful Bone Lesions without Hypercalcemia For a n u m b e r of years, clinicians have attempted to devise therapeutic approaches in myeloma that would relieve disabling symptoms due to skeletal destruction. This was first attempted with the use of fluoride and later calcium and fluoride, although this combination was ineffective and, in fact, detrimental because of the associated side effects. Several groups have now shown that the newer generation bisphosphonates will relieve bone pain, and produce a rapid, sustained, and significant decrease in the urinary excretion of calcium and hydroxyproline, indicating decreased bone turnover (106,107). This was shown first with pamidronate (106) and then with clodronate (107). The FDA approved the use of pamidronate for myeloma bone disease in 1995. Pamidronate has been shown to reduce skeletal events, to reduce the need for radiation therapy, and to reduce episodes of hypercalcemia and pathologic fracture when given in doses of 90 mg over 4 hours by intravenous infusion monthly for 9 months (123). The n u m b e r of skeletal events was almost halved in patients taking pamidronate. Moreover, an objective assessment of the quality of life showed a beneficial effect of bisphosphonates. Similar studies in Finland and Great Britain involving hundreds of patients have also suggested that potent bisphosphonates such as clodronate are effective in improving performance status, reducing vertebral fractures, reducing the progress of osteolytic bone lesions, and preventing hypercalcemic episodes (124). The bisphosphonates seem to be more effective in patients with minimal disease, but they are appropriate for essentially all patients. The results are comparable with either parenteral or oral agents. A reasonable conclusion from the current data is that all patients should be treated, and the real issues are cost, which bisphosphonate, and which route of administration. It is not clear as yet whether the use of bisphosphonates to inhibit

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bone resorption will also reduce the tumor burden in bone, but that is possible. For example, by reducing the production of cytokines, such as interleukin-6, which promotes myeloma cell growth and is produced during states of increased bone resorption, it could be expected that this reduction in growth stimulus for myeloma cells could lead to a decrease in total myeloma cell burden (123).

ACKNOWLEDGMENTS The authors wish to thank Nancy Garrett for secretarial assistance in the preparation of this manuscript. This work was funded in part by grants RR-01346 and CA-40035 from the National Institutes of Health and a grant from the International Myeloma Foundation.

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75. Bataille R, Chappard D, Marcelli C, Dessauw P, Baldet P, SanyJ, Alexandre C. Recruitment of new osteoblasts and osteoclasts is the earliest critical event in the pathogenesis of human multiple myeloma. J Clin Invest 1991 ;88:62-66. 76. Mundy GR, Martin TJ. The hypercalcemia of malignancy: Pathogenesis and management. Metabolism 1982;31:1247-1277. 77. Binstock ML, Mundy GR. Effects of calcitonin and glucocorticoids in combination in hypercalcemia of malignancy. Ann Intern Med 1980;93:269-272. 78. Mundy GR, Wilkinson R, Heath DA. Comparative study of available medical therapy for hypercalcemia of malignancy. Am J Med 1983;74:421-432. 79. Bardwick PA, Zvaifler NJ, Gill GN, Newman D, Greenway GD, Resnick DL. Plasma cell dyscrasia with polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes: The POEMS syndrome. Medicine 1980;59:311-322. 80. Farhangi M, Merlini G. The clinical implications of monoclonal immunoglobulins. Semin Onco11986;13:366-379. 81. Bataille R, Manolagas SC, Berenson JR. Pathogenesis and management of bone lesions in multiple myeloma. Hematol Oncol Clin North Am 1997;11:349-361. 82. Mundy GR. Myeloma bone disease. Eur J Cancer 1998;34: 246-251. 83. Teoh G, Anderson KC. Interaction of tumor and host cells with adhesion and extracellular matrix molecules in the development of multiple myeloma. Hematol Oncol Clin North Am 1997;11:27-42. 84. Van Riet I, Bakkus M, De Greef C, Faid L, Van Camp B. Homing mechanisms in the etiopathogenesis of multiple myeloma. Stem Cells 1995;13 (Suppl. 2):22-27. 85. VarnerJA, Cheresh DA. Integrins and cancer. Curt Opin Cell Biol 1996;8:724-730. 86. Faid L, Van Riet I, De Waele M, Falcon T, Schots R, Lacor P, Van Camp B. Adhesive interactions between tumour cells and bone marrow stromal elements in human multiple myeloma. E u r J Haemato11996;57:349-358. 87. Vanderkerken K, De Raeve H, Goes E, Van Meirvenne S, Radl J, Van Riet I, Thielemans K, Van Camp B. Organ involvement and phenotypic adhesion profile of 5T2 and 5T33 myeloma cells in the C57BL/KaLwRIj mouse. BrJ Cancer 1997;76:451-460. 88. Pellat-Deceunyck C, Barrille S, Puthier D, Rapp MJ, Harroseau JL, Bataille R, Amiot M. Adhesion molecules on human myeloma cells: Significant changes in expression related to malignancy, tumor spreading and immortalization. Cancer Res 1995;55:2647-3653. 89. Michigami T, Dallas SL, Mundy GR, Yoneda T. Interactions of myeloma cells with bone marrow stromal cells via a4bl integrinVCAM-1 is required for the development of osteolysis. J Bone Miner Res 1997;12(Suppl.):101. 90. Oyajobi BO, Traianedes K, Yoneda T, Mundy GR. Expression of RANK ligand (RANKL) by myeloma cells requires binding to bone marrow stromal cells via an a4bl-VCAM-1 interaction. Bone 1998;23 (Suppl.) :S180. 91. Abe M, Hiura K, Wilde J, Ogasahara K, Inoshita T, Kosaka M, Wakatsuki S, Inoue D, Moriyama K, Matsumoto T. Critical role of CC chemokines, macrophage inflammatory protein (MIP)-la and [3, in development of osteolytic lesions in multiple myeloma. J Bone Miner Res 1999;14(Suppl 1):1122. 92. Rollins BJ. Opdenakker G, Van Damme J, eds. Chemokines and cancer. Novel monocyte chemoattractants in cancer. In: Totowa, New Jersey:Humana, 1999:51-69. 93. Rollins BJ. Chemokines. Blood 1997;90:909-928. 94. Zlotnik A, Morales J, HedrickJA. Recent advances in chemokines and chemokine receptors. Crit Rev Immunol 1999; 19:1-47. 95. Wolpe, SD, Davatelis G, Sherry B, Beutler B, Hesse DG, Nguyen HT, Moldawer LL, Nathan CF, Lowry SF, Cerami A. Macrophages secrete a novel heparin-binding protein with inflamma-

96.

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99. 100. 101.

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112.

113.

114.

115.

tory and neutrophil chemokinetic properties. J Exp Med 1988; 167:570-581. Kukita T, Nomiyama H, Ohmoto Y, Kukita A, Shuto T, Hotokebuchi T, Sugioka Y, Miura R, Iijima T. Macrophage inflammatory protein-la (LD78) expressed in human bone marrow: Its role in regulation of hematopoiesis and osteoclast recruitment. Lab Invest 1997;76:399-406. Alsina M, Choi SJ, Cruz j c , Chung H, Roodman DG. Overexpression of the osteoclast stimulatory factor (OSF), macrophage inflammatory protein-l-alpha (MIP-la) in multiple myeloma. Blood 1998;92:680a (abstract 405). Scheven BAA, Milne JS, Hunter I, Robins SE Macrophageinflammatory protein-la regulates preosteoclast differentiation in vitro. Biochem Biophys Res Commun 1999;254:773-778. Votta BJ, James IE, Eichman C, Lee-Rykaczewski CE White E, White JR, Gowen M. JBone Miner Res 1996;11 (Suppl.) :M414. Roodman GD. Cell biology of the osteoclast. Exp Hematol 1999;27:1229-1241. Broxmeyer HE, Cooper S, Hangoc G, Gao JL, Murphy PM. Dominant myelopoietic effector functions mediated by chemokine receptor CCR1. JExp Med 1999;189:1987-1992. Dallas SL, Oyajobi BO, Garrett IR, Dallas MR, Boyce BE Bauss F, RadlJ, Mundy GR. Ibandronate reduces osteolytic lesions but not tumor burden in a murine model of myeloma disease. Blood 1999;93:1697-1705. Garrett IR, Dallas SL, Radl J, Mundy GR. A murine model of human myeloma bone disease. Bone 1997;20:515-520. RadlJ, Croese JM, Zurcher C, van den Enden-Vieveen MHM, De Leeuw AM. Animal model of human disease: Multiple myeloma. Am J Patho11988;132:593-597. Lokhorst HM, Lamme T, de Smet M, Klein S, de Weger RA, van Oers R, Bloem AC. Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures. Blood 1994;84:2269-2277. Van Breukelen FJM, Bijvoet OLM, Van Oosterom AT. Inhibition of osteolytic bone lesions by (3-amino-l-hydroxypropylidene)-l, 1-bisphosphonate (A.ED.). Lancet 1979;1:803-805. Siris ES, Sherman WH, Baquiran DC, Schlatterer JP, Osserman EF, Canfield RE. Effects of dichloromethylene diphosphonate on skeletal mobilization of calcium in multiple myeloma. NEngl J Med 1980;302:310-315. Garattini S. Use of intravenous etidronate disodium in the treatment of hypercalcemia. In: Ryzen E, Rude RK, Elbaum N, Singer FR, eds. Bone resorption, metastasis and disphosphonates New York:Raven; 1983:99-108. Lazor MZ, Rosenberg LE. Mechanisms of adrenal-steroid reversal of hypercalcemia in multiple myeloma. N Engl J Med 1964;270:749. Mundy GR, Rick ME, Turcotte R, Kowalski MA. Pathogenesis of hypercalcemia in lymphosarcoma cell leukemia--Role of an osteoclast activating factor-like substance and mechanism of action for glucocorticoid therapy. AmJMed 1978;65:600-606. Strumpf M, Kowalski MA, Mundy GR. Effects of glucocorticoids on osteoclast-activating factor. J Lab Clin Miner 1978; 92:772-778. Suda T, Testa NG, Allen TD. Effects of hydrocortisone on osteoclasts generated in cat bone marrow cultures. Calcif Tissue Int 1983;35:82-86. Minkin C. Inhibition of parathyroid hormone stimulated bone resorption in vitro by the antibiotic mithramycin. Calcif Tissue Res 1973;13:249-257. Raisz LG, Trummel CL, Wener JA, Simmons H. Effect of glucocorticoids on bone resorption in tissue culture. Endocrinology 1972;90:961-967. Raisz LG, Trummel CL, Simmons H. Induction of bone resorption in tissue culture: Prolonged response after brief exposure to

OTHER SYNDROMES ASSOCIATED WITH HYPERCALCEMIA

116. 117.

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parathyroid hormone or 25-hydroxycholecalciferol. Endocrinology 1972;90:744-751. Stamp TCB, Child JA, Walker PG. Treatment of osteolytic myelomatosis with mithramycin. Lancet 1975;1:719-722. Wener JA, Gorton SJ, Raisz LG. Escape from inhibition of resorption in cultures of fetal bone treated with calcitonin and parathyroid hormone. Endocrinology 1972;90:752-759. Au WYW. Calcitonin treatment of hypercalcemia due to para-thyroid carcinoma-synergistic effect of prednisone on long-term treatment of hypercalcemia. Arch Intern Med 1975;135:1594-1597. Ralston SH, Gardner MD, Dryburgh FJ, Jenkins AS, Cowan RA, Boyle IT. Comparison of aminohydroxypropylidene diphosphonate, mithramycin and corticosteroids/calcitonin in treatment of cancer-associated hypercalcemia. Lancet 1985;2:907-910. Warrell RP, Bockman RS, Coonley CJ, Isaacs M, Staszewski H. Gallium nitrate inhibits calcium resorption from bone and is effective treatment for cancer-related hypercalcemia. J Clin Invest 1984;73:1487-1490.

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CHAPTER 44

Genetic Disorders Caused by Mutations in the PTH/PTHrP Receptor Jansen's Metaphyseal Chondrodysplasia and Blomstrand Lethal Chondrodysplasia CAROLINE SILVE INSERM U. 426, Faculti de Mddecine Xavier Bichat, 75018 Paris, France H A R A L D JIJPPNER Endocrine Unit, Department of Medicine and Children's Se~ice, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114

INTRODUCTION

ing the last trimester of pregnancy, is characterized by premature and accelerated chondrocyte differentiation, as well as other developmental abnormalities. This disorder is most likely caused by c o m p o u n d heterozygous or homozygous mutations that lead to mutant P T H / P T H r P receptors with severely impaired functional properties. Thus, the most striking consequences of P T H / P T H r P receptor mutations are abnormalities in endochondral bone formation that result, in both diseases, in severe skeletal abnormalities. The evaluation of affected patients has furthermore provided new insights into the role of the P T H / P T H r P receptor in other aspects of normal human development and, at a more basic level, into the structure/function relationships of the P T H / P T H r P receptor and other members of this receptor family. To better comprehend the biologic consequences of P T H / P T H r P receptor mutations, we first review the actions of PTH and PTHrP that are mediated through this receptor.

Despite limited amino acid sequence homology, which is largely restricted to the first 13 residues, parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind to and activate a common receptor (1-3). This P T H / P T H r P receptor (also referred to as type 1 P T H / P T H r P receptor, or PTH1R) is abundantly expressed in bone and kidney (4,5). In these tissues, the receptor mediates the endocrine effects of PTH, thereby playing a critical role in the regulation of extracellular calcium and phosphorus concentration. The development and analysis of various genetically manipulated mouse models showed that the P T H / P T H r P receptor has, in addition to its role in mineral ion homeostasis, a pivotal role in endochondral bone formation (6-10). These insights from transgenic and gene-ablated animals suggested that the P T H / P T H r P receptor might be implicated in the pathogenesis of some forms of chondrodysplasia in humans. In fact, several different activating and inactivating mutations in the P T H / P T H r P receptor have been identified as the most plausible cause of two genetic disorders, Jansen's metaphyseal chondrodysplasia (]MC) and Blomstrand lethal chondrodysplasia (BLC), respectively. JMC, an autosomal dominant disorder characterized by short-limbed dwarfism due to decelerated chondrocyte differentiation and agonistindependent hypercalcemia, is caused by P T H / P T H r P receptor mutations that lead to constitutive, agonistindependent cAMP accumulation. BLC, an autosomal recessive disease that typically results in fetal death durThe Parathyroids, Second Edition

PARATHYROID H O R M O N E PTH, along with 1,25-dihydroxyvitamin D 3 [1,25 (OH)2D3], is the most important endocrine regulator of extracellular calcium homeostasis in mammals (11,12). The hormone is expressed almost exclusively in the parathyroid glands [low protein and mRNA levels were also found in the hypothalamus and in the thymus of rodents (13,14)]. Synthesis and secretion of PTH is regulated by the extracellular concentration of calcium that is monitored by a calcium-sensing receptor 707

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Leone.

708

/

CHAPTER44

that is abundantly expressed in the parathyroid glands (15,16); other regulators of PTH production include 1,25 (OH) 2D~and phosphate (17-19). PTH acts primarily on kidney and bone, where it binds to cells expressing the P T H / P T H r P receptor, and initiates a series of processes that maintain blood calcium and phosphate concentrations within narrow limits (Fig. 1). In kidney, the mRNA encoding the P T H / P T H r P receptor is expressed in the convoluted and straight proximal tubules, the cortical portion of thick ascending limb, and the distal convoluted renal tubules (20-22), i.e., in those renal segments that respond to PTH with an increase in cAMP accumulation (23-25). Clonal cell lines derived from proximal and distal portions of rat renal tubules show PTH-dependent cAMP accumulation, but the response of other second messengers appears to be strikingly different. For example, the increase in intracellular free calcium in these "proximal" tubular cells has been shown to depend entirely on the release of this second messenger from intracellular stores, whereas its increase in "distal" tubular cells relies largely on extracellular calcium entry (26,27). This indicates that distinct portions of the nephron may respond differently to challenge with PTH. In kidney, PTH stimulates the production of 1,25(OH)zD ~ and the reabsorption of calcium, and it inhibits in the proximal tubule the reabsorption of phosphate [for reviews see (28-32)]. PTH-dependent inhibition of renal phosphate reabsorption is achieved by reducing apical sodium-phosphate cotransport in both the proximal and the distal tubules, but the main hormonal effect is in the proximal straight tubules (pars recta). The inhibition of this renal action of PTH is associated with a reduction in the abundance of type II sodium-phosphate cotransporter (Npt2, also

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referred to as NaPi-2a) on the apical surface of tubular cells (33). This reduction in Npt2 depends, at least partially, on the second messenger cAMP, which increases internalization and subsequent lysosomal degradation of Npt2 (34-38) [for review see also (31) ]. The stimulation of l ot-hydroxylase activity, an action that appears to be at least partially cAMP dependent (39-41), is largely restricted to the proximal convoluted tubule. The resulting increase in 1,25(OH)zD 3 production enhances the absorption of calcium and phosphate from the intestine. PTH furthermore stimulates, possibly through other second messengers, the reabsorption of calcium in the distal convoluted tubule (26,27), decreases the glomerular filtration rate, inhibits the proximal reabsorption of bicarbonate and amino acids, and stimulates gluconeogenesis (30,42). The effects of PTH on bone are complex and often difficult to study. As outlined below, the peptide can influence, either directly or indirectly, the proliferation and differentiation of several bone cell precursors. Furthermore, the effects resulting from PTH stimulation of mature osteoblasts depend on the intensity and duration of the stimulus, and the hormonal effects observed in vitro often fail to reflect the conditions in vivo. For example, the continuous administration of PTH causes predominantly osteoclastic bone resorption, whereas intermittent doses of the hormone elicit anabolic effects (43,44); [for review see (45)]. Rapid changes in blood PTH concentration may occur in vivo, because small changes in ionized calcium can generate significant variations in PTH secretion (16) and these may contribute to the observed circadian rhythm of PTH concentration in the circulation (46-48). Along with other endocrine factors, notably 1,25(OH)zD~, PTH modulates the responsiveness of bone cells (49,50).

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FIG. 1 Biologic roles of the PTH/ PTHrP receptor in the regulation of calcium and phosphate ion homeostasis, and in the regulation of cellular proliferation and differentiation during development.

MUTATXONSIN P T H / P T H r P RECEPTOR / The initial effect of PTH on bone is a rapid release of calcium from those areas of the matrix that allow rapid exchange with the extracellular fluid (ECF). After a delay of several hours, PTH-induced changes in bone cell metabolism become apparent. PTH directly stimulates osteoblast activity and thus bone formation (51). Activated osteoblasts, in turn, increase the activity of osteoclasts, which results in increased bone resorption. This "coupling" between bone formation and bone resorption is thought to depend, at least in part, on the ability of PTH to induce changes in the synthesis a n d / o r activity of several osteoblast-specific proteins, including insulin-like growth factor-I (IGF-I) (52). However, the most important of these "coupling" factors appears to be osteoclast differentiation factor (ODF; also known as RANKL, TRANCE, or osteoprotegerin ligand), a tumor necrosis factor (TNF)-related protein that facilitates, in the presence of macrophage colony-stimulating factor (M-CSF), the differentiation of osteoclast precursors into mature bone-resorbing cells (53,54).

PARATHYROID H O R M O N E RELATED PEPTIDE PTHrP was first discovered as the major cause of humoral hypercalcemia of malignancy syndrome (55-59). Within its amino-terminal portion, PTHrP shares partial amino acid sequence homology with PTH, and as a result of these limited structural similarities, amino-terminal fragments of both peptides have largely indistinguishable biologic properties, at least with regard to the regulation of mineral ion homeostasis (60-63). Additional midregion/carboxyl-terminal fragments of either peptide are generated through alternative splicing a n d / o r posttranslational processing. These PTH and PTHrP fragments are unlikely to be important for adult mineral ion metabolism, and their actions appear to be mediated through different, as yet unidentified receptors (64). Shortly after its initial isolation from several different tumors, PTHrP and its mRNA were found in large variety of fetal and adult tissues, suggesting that this peptide has an important biologic role throughout life (65-67). In fact, two principal roles of PTHrP in mammalian development have emerged from observations in genetically manipulated mice (see Fig. 1). These include the regulation of chondrocyte proliferation and differentiation during the process of endochondral bone formation (6-10), and in epithelial-mesenchymal interactions during organogenesis of some epithelial organs, including skin, mammary gland, and teeth (68-71) [for review see (72,73)]. During fetal development, expression of mRNA transcripts encoding

709

PTHrP or the P T H / P T H r P receptor is closely linked, both spatially and temporally, implying that the ligand and its receptor are involved in paracrine/autocrine signaling events at these sites (6,70,71,74). These observations are consistent with the hypothesis that the P T H / P T H r P receptor mediates most actions of PTHrE Other PTHrP-dependent effects are likely to involve non-amino-terminal portions of the peptide and distinct, only incompletely characterized cell surface receptors a n d / o r direct interactions with the nucleus (75-80).

THE P T H / P T H r P RECEPTOR: A R E C E P T O R FOR TWO DISTINCT LIGANDS The cloning of the P T H / P T H r P receptor cDNA from opossum kidney (OK) and rat osteoblast-like (ROS 17/2.8) cells, and the subsequent characterization of cDNAs encoding this receptor from other organs of several different species, confirmed and extended three key insights into the mode of action of PTH and PTHrP: (1) the recombinant P T H / P T H r P receptor binds amino-terminal fragments of PTH and PTHrP with similar or indistinguishable affinity; (2) both ligands stimulate with similar potency the formarion of at least two second messengers, cAMP and inositol phosphate; and (3) identical receptors are expressed in renal tubular cells and in osteoblasts (1-3). Similar to the widely expressed PTHrP, it was furthermore shown that the mRNA encoding the P T H / P T H r P receptor is found in a surprisingly large variety of fetal and adult tissues (4,5,20,21,74,81), and it is particularly abundant in prehypertrophic chondrocytes of metaphyseal growth plate (6,8-10). P T H / P T H r P receptor belongs to the class B family of heptahelical G protein-coupled receptors (GPCRs), which also comprises the receptors for secretin, calcitonin, glucagon, and several other peptide hormones (82,83) (Fig. 2). These h o r m o n e receptors share no homology with other G protein-coupled receptors such as class A and C receptors, and the organization of genes encoding the members of each of these receptor families is distinctly different. All class B receptors are characterized by an amino-terminal, extracellular domain that consists of approximately 150 amino acids and by eight conserved extracellular cysteine residues; numerous other conserved amino acids are dispersed throughout the amino-terminal domain, the membrane-embedded helices, and the connecting loops. The organization of the P T H / P T H r P receptor gene, which in mammals contains 14 coding exons, appears to be similar in all vertebrates (84,85), possibly including fish (86). Transcripts encoding identical P T H / P T H r P receptors are derived from at least three different promoters, and are initiated in one of

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FIG. 2 Schematic representation of the organization of the human PTH/PTHrP receptor gene (A) and of the encoded amino acid sequence (B). (A) Untranslated and translated exons are shown as black and open boxes, respectively, and introns as solid lines. The mRNAs derived from the P1 or the P2 promoter are indicated by the solid and dotted lines, respectively, above the untranslated exons; the transcript derived from the P3 promoter starts within exon U4/S. Three partial nucleotide sequences are shown: (1) Wild-type exons EL2 and M5 (splice-donor and splice-acceptor sites are in italic and underlined; nucleotides in introns are in lower case; nucleotides in exons are in upper case). (2) The nucleotide change in exon M5 (nucleotide 1176 of the human cDNA), which was identified in one patient with BLC (169), introduces a novel splice-acceptor site leading to the deletion of amino acid residues 3 7 3 - 3 8 3 in the human PTH/PTHrP receptor; these deleted residues are shown in black in panel B. (3) The deletion of nucleotide 1122 in exon EL2 was identified in

MUTATIONS IN

TABLE 1

P T H / P T H r P RECEPTOR

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Principal Characteristics of the PTH/PTHrP Receptor Gene Promoters Promoter P1

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aThe designations P1 and P2 promoter are interchanged according to another nomenclature (84). t'Abbreviations: VSMC, vascular smooth muscle cell; PTEC, peritubular endothelial cell; TC, tubular cell; ND, not determined; 0, no splice variant detected.

three alternative untranslated first exons (84,85,87,88) (Table 1 and Fig. 2). Little is known about the factors that control the activity of these promoters, some of which appear to be species and tissue specific (89,90), and their activity may be different throughout development and adult life (90,91). Transcripts derived from the P1 and the P2 promoters have been observed in rodents and humans (85,92,93). In rodents, the P1 activity appears to be restricted mainly to vascular smooth muscle and peritubular endothelial cells in adult kidney (89), whereas the activity of the ubiquitous P2 promoter is detected in numerous fetal and adult tissues, including cartilage and bone (91). In contrast to the equivalent P2 promoter activity observed in humans and mice, P1 activity is only weak or absent in humans. A third promoter, P3, appears to be specific for humans and it controls P T H / P T H r P receptor expression in a number of tissues, including kidney and bone (88,90). The --~2.5-kb transcript encoding the fulllength P T H / P T H r P receptor is the predominant mRNA species in many different tissues (4,5), but several larger and smaller transcripts have also been

detected, suggesting the presence of splice variants of the P T H / P T H r P receptor. However, to date, alternatively spliced mRNAs that give rise to functionally active P T H / P T H r P receptors have not been identified

(93,94). A second PTH receptor, type 2 PTH receptor (PTH2R), isolated by Usdin et al. (95), belongs to the same family of G protein-coupled receptors as the PTH/PTHrP receptor. This novel receptor is less broadly expressed, with little if any expression in kidney and bone (95,96). The human PTH2R is efficiently activated by PTH, but not by PTHrP (97-99), and the rat receptor homolog is not activated by either of these two peptides (100). Interestingly, a novel hypothalamic pepfide, TIP39, which shows only very limited amino acid sequence homology with PTH and PTHrP, efficiently and potently activates the rat and the human PTH2R, but not the P T H / P T H r P receptor. It is therefore likely that TIP39 represents the primary agonist for the PTH2R (96,101). Due to this ligand specificity, and due to its restricted expression, the PTH2R does not appear to mediate any of the PTHrP-dependent actions (6,8).

another patient with BLC (167); due to the resulting shift in open reading frame after glutamine at position 364, the protein sequence diverges from the amino acid sequence of the wild-type PTH/PTHrP receptor; the novel amino acids are underlined. (B) Schematic representation of the amino acid sequence of the human PTH/PTHrP receptor. The locations of mutations are indicated (0); these lead either to constitutive activation of the PTH/PTHrP receptor (white numbers/letters in black box) (122,127,129,130) or to loss of function (black numbers/letters in white box) (170,171); the arrow indicates the glutamine after which the amino acid sequence is changed due to frame-shift mutation (167).

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ROLE OF PTHrP A N D THE P T H / P T H r P RECEPTOR IN E N D O C H O N D R A L B O N E FORMATION The most prominent biologic role of PTHrP, besides its importance in the humoral hypercalcemia of malignancy syndrome, was revealed when both alleles of its gene were ablated through homologous recombination in mice (6) and when expression of the peptide was targeted in transgenic animals to the growth plate (10). Homozygous PTHrP gene-ablated animals die during the perinatal period and show striking skeletal changes, which include domed skulls, short snouts and mandibles, and disproportionately short extremities, yet no obvious developmental defects in other organs (Fig. 3). These skeletal changes are caused by a dramatic acceleration of chondrocyte differentiation that leads to premature growth plate mineralization (6) (Fig. 4). Animals lacking only one copy of the PTHrP gene show normal growth and development and are fertile, but develop, despite apparently normal calcium and phosphorus homeostasis, mild osteopenia later in life (102). Growth plate abnormalities that are, in many aspects, the opposite of those found in PTHrP-ablated mice are observed in animals that overexpress PTHrP under the control of the ot~(II) collagen promoter (10). Throughout life these animals are smaller in size than their wild-type litter mates and show a disproportionate foreshortening of limbs and tail, which is most likely

due to a severe delay in chondrocyte differentiation and endochondral ossification (Fig. 4). Thus, too little or too much PTHrP leads to short-limbed dwarfism, although through entirely different mechanisms. From these and other studies, it is now well established that PTHrP facilitates the continuous proliferation of chondrocytes in the growth plate, and that it postpones their programmed differentiation into hypertrophic chondrocytes. Consistent with this role of PTHrP in endochondral bone formation, earlier in vitro studies had shown that PTH (used in these studies instead of PTHrP) affects chondrocyte maturation and activity (103,104). Subsequent studies confirmed these findings by showing that PTH and PTHrP stimulate, presumably through cAMP-dependent mechanisms (105), the proliferation of fetal growth plate chondrocytes, inhibit the differentiation of these cells into hypertrophic chondrocytes, and stimulate the accumulation of cartilage-specific proteoglycans that are thought to act as inhibitors of mineralization (106-108). In the absence of these cartilage-specific PTHrP effects, growth plates of homozygous PTHrP gene-ablated mice have a thinner layer of proliferating chondrocytes, whereas the layer of hypertrophic chondrocytes is relatively normal in thickness, but somewhat disorganized. Taken together these findings suggest that the lack of PTHrP accelerates the normal differentiation process of growth plate chondrocytes, i.e., resting and proliferating chondrocytes undergo fewer

FIG. 3 Macroscopic findings in a homozygous PTHrP-ablated mouse (day 18.5 of embryonic development). Wild-type fetus (left) and a PTHrP-ablated litter mate (right). Note the osteochondrodysplasia characterized by a domed skull, short snout and mandible, protruding tongue, and disproportionately short limbs (from Ref. 6, with permission).

MUTATIONS IN P T H / P T H r P RECWPTOR /

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FIG. 4 Low-magnification photomicrographs of proximal tibiae of a wild-type mouse (A) and a homozygous PTHrP-ablated mouse (B) (embryonic day 18.5). Note that the tibial epiphysis of the PTHrP-ablated mouse is shortened and the zones of "resting" and proliferative chondrocytes are markedly reduced. (Reproduced from Ref. 7, The Journal of Cell Biology, 1994, Vol. 126, pp. 1611-1623, by copyright permission of The Rockefeller University Press.)

cycles of cell division and differentiate prematurely into hypertrophic cells, which then undergo apoptosis before being replaced by invading osteoblasts. Most of the autocrine/paracrine actions of PTHrP that result in slowed chondrocyte differentiation are mediated by the PTH/PTHrP receptor. This has been demonstrated, as outlined above, through the generation of mice that lack either PTHrP or the PTH/PTHrP receptor (6,8). Furthermore, mice missing either PTHrP or its receptor are resistant to the actions of Indian hedgehog (Ihh), a developmentally important protein that is most abundantly expressed in growth plate chondrocytes that are about to differentiate into hypertrophic cells. Ihh binds directly to patched, a membrane receptor, which interacts with and thereby suppresses the constitutive activity of smoothened (109,110). The ectopic expression of Ihh in chicken wing cartilage stimulates the production of PTHrP and thereby blocks the normal chondrocyte differentiation program (9); conversely, PTHrP represses Ihh expression. In the growth plate, the PTH/PTHrP receptor thus mediates directly the actions of PTHrP, but indirectly it mediates also the actions of Ihh, and this feedback loop is important for the continuous regulation of chondrocyte growth and differentiation (8,9). PTHrP and Ihh are thus critically important components of normal bone growth and elongation. However, not all actions of PTHrP appear to be mediated through the PTH/PTHrP receptor, because the ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to subtle, but distinctly different, abnormalities in early

bone development (77), suggesting that some actions of PTHrP in bone involve either distinct receptors or the peptide's direct nuclear actions (78-80). R O L E OF P T H r P IN REGULATING EPITHELIALMESENCHYMAL INTERACTIONS

Studies with transgenic mice, in which PTHrP expression is targeted through a human keratinocyte-specific promoter (K14) to the developing epidermis and mammary gland, demonstrated that PTHrP also plays a critical role in hair follicle development and branching morphogenesis of the mammary gland (68,69) (Fig. 5, upper panel). These conclusions were further supported by findings in PTHrP-null mice that had been rescued from neonatal death by targeting PTHrP expression to chondrocytes through the % (II) collagen promoter (10). These rescued mice lack mammary epithelial ducts, due to a failure of the initial round of branching growth that is required for transforming the mammary bud into the primary duct system; ablation of both copies of the PTH/PTHrP receptor gene leads to phenotypic changes similar to those observed in PTHrPablated animals (70). Using similar approaches, it was furthermore demonstrated that PTHrP is required for normal tooth eruption (Fig. 5, lower panel). Teeth develop normally in rescued PTHrP knockout mice, but become trapped in surrounding bone and undergo progressive impactation (71). In both tissues, breast and

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CHAPTER44

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mandible, PTHrP mRNA was identified by in situ hybridization in epithelial cells, whereas the P T H / PTHrP receptor mRNA was found in mesenchymal or stromal cells. These observations led to the concept that the communication between epithelium and mesenchyme involves the P T H / P T H r P receptor, and that PTHrP signaling is essential for normal development of these tissues (72,74,81).

JANSEN'S METAPHYSEAL CHONDRODYSPLASIA JMC, first described in 1934 (111), is a rare autosomal d o m i n a n t form of short-limbed dwarfism associated with laboratory abnormalities that are typically observed only in patients with either primary hyperparathyroidism or with the humoral hypercalcemia of malignancy syndrome [reviewed in (112,113)]. These biochemical changes, i.e., hypercalcemia, renal phosphate wasting, and increased urinary cAMP excretion, occur despite low or undetectable concentrations of circulating PTH and PTHrE Marked hypercalcemia,

FIG. 5 Schematic representation of the role of PTHrP and the PTH/PTHrP receptor during mammary gland and tooth development. Upper panel: Branching morphogenesis depends on the interaction between mammary epithelium and the mesenchyme that condenses underneath (dense mesenchyme). PTHrP is produced by the mammary epithelium, and acts through the PTH/PTHrP receptor, which is expressed in the dense mesenchyme (72). Lower panel: During tooth development, PTHrP is expressed in the bone surrounding the tooth and in the dental mesenchyme. Stimulation of bone resorption following PTH/PTHrP receptor activation by PTHrP is required for normal tooth eruption (71 ). (See color plates.)

which is often asymptomatic, and hypophosphatemia had been noted in Jansen's first patient (114), and in a subsequently described child with the same disorder (115). It was not until the description of a third patient, however, that the association between the abnormalities in endochondral bone formation and in mineral ion homeostasis was formally considered (116). At that time the biochemical abnormalities could not be readily distinguished from those observed in primary hyperparathyroidism, but the surgical exploration of the patient revealed no obvious abnormalities of the parathyroid glands. It was therefore concluded that the changes in mineral metabolism were either "secondary to the underlying bone defect" or "related to an undefined metabolic disorder that gave rise to both metaphyseal and biochemical changes" (116). Subsequent studies showed that PTH levels in patients with JMC are either low or undetectable (117-122), and that the concentration of PTHrP is not increased (123). Most reported cases of JMC are sporadic, but the description of two unrelated affected females who each gave birth to affected daughters (124-126) suggested an autosomal dominant m o d e of inheritance; this conclusion was

MUTATIONS IN P T H / P T H r P RECEPTOR

subsequently confirmed for one of these families at the molecular level (127). At birth some patients with JMC have dysmorphic features, which can include high skull vault, flattening of the of the nose and forehead, low-set ears, hypertelorism, high-arched palate, and micro- or retrognathia [for a review, see (112)]. Although body length is within normal limits at birth, growth becomes increasingly abnormal, eventually leading to the develo p m e n t of short stature. Additional signs may include kyphoscoliosis with a bell-shaped thorax and widened costochondral junctions, metaphyseal enlargement of the joints, waddling gait, prominent supraorbital ridges, and frontonasal hyperplasia. The legs are usually bowed and short, but the arms are relatively long. One female patient with JMC was reported to be unable to breast-feed and to have, similar to her affected daughter, a dry and scaly skin (127). As discussed above, PTHrP and the P T H / P T H r P receptor are expressed in breast and skin, and this ligand/receptor system appears to have an important role in these two tissues. Thus, the described clinical findings may be related to the expression of constitutively activated P T H / P T H r P receptors in dermis and mammary epithelial cells. Tooth development and enamel formation appear normal in patients with JMC. Radiologic studies have shown considerable, aged e p e n d e n t differences in the osseous manifestations of JMC. In younger patients, severe metaphyseal changes, especially of the long bones, are present (Fig. 6). The metaphyses are enlarged and expanded, giving a clublike appearance to the ends of the long bones with a wide zone of irregular calcifications. Patches of partially calcified cartilage that protrude into the diaphyses are also present and appear relatively radiolucent. These findings, which are characteristically observed throughout early childhood, are similar to the lesions observed in rickets. However, disdnct from the findings in rickets, metacarpal and metatarsal bones are also involved. In addition, sclerosis and thickening of the base of the skull and of the calvaria are noted in most cases. Loss of the normal cortical outline, areas of subperiosteal bone resorption, and generalized osteopenia are reminiscent of the changes seen in hyperparathyroidism (113). Histologically, the growth plates show a severe delay in endochondral ossification of the metaphyses, including a lack of the regular columnar arrangement of the maturing cartilage cells, a lack of excess osteoid (which is usually indicative of active rickets or osteomalacia), little or no vascularization of cartilage, and no evidence for osteitis fibrosa (115,128). Later in childhood, the changes are no longer reminiscent of rickets. Until the onset of puberty, almost all tubular bones show irregular patches of partially calci-

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715

fled cartilage that protrude into the diaphyses; the spine and vertebral bodies show no obvious abnormalities (115,117,119-123,126). After adolescence, the cartilaginous tissue in the metaphyses gradually disappears and turns into bone, leading to bulbous deformities (Fig. 6). The ends of most tubular bones remain expanded, deformed, and radiolucent, but a more normal trabecular pattern gradually emerges. The base of the skull remains hyperostotic, which may lead to cranial auditory and optical nerve compression later in life. Intelligence appears to be normal in all reported cases (115,117,119-123,126). Most laboratory findings in JMC are reminiscent of those observed in patients with primary hyperparathyroidism or with the syndrome of humoral hypercalcemia of malignancy. In the newborn, blood phosphorus levels are typically at the lower end of the normal range, but alkaline phosphatase activity is almost invariably elevated. Hypercalcemia is usually absent at birth, developing during the first months of life and persisting throughout life, but is more pronounced during infancy and childhood. Hypercalciuria is usually present and can be associated with nephrocalcinosis. 1,25 (OH)zD 3 levels have been reported to be normal or at the upper end of the normal range. Serum alkaline phosphatase activity and osteocalcin concentration are elevated throughout life, indicating that osteoblast activity is increased; compatible with an increased osteoclastic activity, urinary hydroxyproline excretion is elevated (122,123).

Jansen's Disease Is Caused by Activating PTH/PTHrP Receptor Mutations Because of the findings in the various genetically manipulated mice described above, and because of the abundant expression of the P T H / P T H r P receptor in the three organs that are most obviously affected in JMC (i.e., kidney, bone, and metaphyseal growth plate), activating receptor mutations were considered as the cause of this rare disease. Indeed, in several unrelated patients with this disorder, a heterozygous nucleotide exchange, which changes a histidine at position 223 to arginine, was identified in exon M2 of the P T H / P T H r P receptor gene (122,127,129,130). Subsequently, two additional heterozygous nucleotide exchanges were identified, changing threonine at position 410 to proline (exon M5), or isoleucine at position 458 to arginine (exon M7), respectively (122,127) (see Fig. 2). The three involved amino acids are predicted to be located at or close to the intracellular surface of the cell membrane and are strictly conserved in all mammalian members of this receptor family (82,83), suggesting an important functional role for these three residues. With

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FIG. 6 Radiographs of a patient with Jansen's disease. Note that the metaphyseal ends are irregular at 5 weeks of age (upper panel) and have an appearance that resembles rickets; at age 10 weeks (lower left panel) there are irregular calcifications, the metaphyseal areas are significantly enlarged and expanded; at age 22 weeks (lower right panel) the abnormalities in zones of calcification have completely disappeared (from Ref. 117, with permission).

MUTATIONS IN P T H / P T H r P RECEPTOR

the exception of one family, in which a mother-todaughter transmission of the H223R mutation was documented (127), each of the three mutations was excluded in the healthy parents and siblings, and in genomic DNA from a significant number of unrelated healthy individuals. This suggests that JMC is usually caused by de novo mutations. To date, the T410P and the I458R mutation was found in one patient each; the H223R mutation was identified in eight patients and is thus the most frequent P T H / P T H r P receptor mutation in JMC. To test in vitro the functional consequences of the identified missense mutations in JMC, each of the three different nucleotide exchanges was introduced into the cDNA encoding the wild-type human P T H / P T H r P receptor (122,127,129,130). COS-7 cells transiently expressing P T H / P T H r P receptors with either the H223R, the T410P, or the I458R mutation showed significantly higher basal accumulation of cAMP, compared to cells expressing the wild-type P T H / P T H r P receptor (Fig. 7). Cells expressing P T H / P T H r P receptors with either of the three point mutations showed no evidence for increased basal accumulation of inositol phosphate (IP), indicating that this signaling pathway is not constitutively activated (122,127,129). Interestingly, an asparagic acid to histidine mutation at position 578 of the luteinizing hormone receptor, which is at a position equivalent to the T410P mutation in the P T H / P T H r P receptor (see Fig. 2), led to constitutive

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activation of both the cAMP and IP signaling pathways (131). It is therefore conceivable that the lack of constitutive IP generation by three activating mutations in the P T H / P T H r P receptor could be related to poor sensitivity of the methods that were used to explore this second-messenger system. When challenged with increasing concentrations of either PTH or PTHrP, cells expressing the mutant H223R and T410P receptors showed, in comparison to cells expressing the wild-type receptor, reduced maximal cAMP accumulation. In contrast, cells expressing the I458R mutant showed the same maximal cAMP accumulation as cells transfected with the wild-type receptor (122). Agonist-dependent IP accumulation was observed with COS-7 cells expressing the I458R and the T410P mutant, but not with cells expressing the H223R mutant. Despite the differences in the in vitro response to PTH or PTHrP, patients with either of the three P T H / P T H r P receptor mutations showed no obvious differences in their clinical a n d / o r biochemical presentation. Activating mutations in other G protein-coupled receptors have been implicated in several other human diseases. These disorders include rare forms of retinitis pigmentosa or congenital stationary blindness (activating mutations in rhodopsin) (132,133), thyroid adenomas or nonautoimmune hyperthyroidism (activating TSH receptor mutations) (134-143), gonadotropinindependent male precocious puberty (144--147), Leydig cell tumors (activating mutations in the luteinizing hormone receptor) (131), and autosomal dominant forms of familial hypocalcemia (activating calcium-sensing receptor mutations) (148-155). Cell membrane receptors exhibiting constitutive signaling have also been described in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas (constitutive signaling of the Kaposi's sarcoma herpes virus-G protein-coupled receptor (KSHVGPCR) via activation of phosphoinositide-specific phospholipase) (156,157). To prove that the growth plate abnormalities in Jansen's disease are indeed caused by constitutively active P T H / P T H r P receptors, transgenic mice were generated that express the H223R mutant under the control of the rat ot1(II) collagen promoter, thereby targeting receptor expression to the layer of proliferating chondrocytes (158). Two transgenic mouse lines were established, both of which showed delayed mineralization and decelerated differentiation of proliferative chondrocytes into hypertrophic chondrocytes, a delay in vascular invasion and a prolonged presence of hypertrophic chondrocytes. In one of these mouse lines, the defect in endochondral bone formation was apparent only at the microscopic level, whereas the second line

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CHAPTER44

showed shortened and deformed limbs, i.e., findings that are reminiscent of those in patients with Jansen's disease. Based on these results in transgenic mice it appears likely that the growth abnormalities in JMC are caused by the expression of mutant, constitutively active P T H / P T H r P receptor in growth plate chondrocytes. In an attempt to prolong the survival of homozygous PTHrP-ablated mice, the transgenic "Jansen" animals were crossed with heterozygous "PTHrP knockout" mice. I n b r e d offspring that lack both copies of the PTHrP gene, yet express the Jansen receptor transgene, showed no obvious developmental defect at birth. Subsequently, however, these rescued mice fail to grow normally, and die at the age of about 2 months; the cause of their death is currently unknown (158). These findings, which are similar to those observed in PTHrPdeficient animals rescued through the PTHrP transgene (10), demonstrate that the early lethality of mice lacking both copies of the PTHrP gene is caused by the dramatically accelerated process of endochondral bone formation. In an attempt to better understand how the P T H / P T H r P receptor can modulate bone developm e n t and turnover, transgenic mice were generated in which the h u m a n P T H / P T H r P receptor with the H223R mutation is expressed u n d e r the control of the type I collagen promoter. When compared to control litter mates, long bones of transgenic mice were reduced in length. The histologic analysis of these bones revealed a thinner and more porous cortex in the diaphysis, an increased trabeculation of the metaphysis, and a reduction in bone marrow space; these findings are reminiscent of the skeletal abnormalities in patients with hyperparathyroidism (159).

corrected for gestational age, appears to be normal, but may be overestimated because most infants are hydroptic; also the placenta can be immature and edematous. Nasal, mandibular, and facial bones are hypoplastic; the base of the skull is short and narrow; the ears are low set; the thoracic cage is hypoplastic and narrow with short, thick ribs and hypoplastic vertebrae. In contrast, the clavicles are relatively long and often abnormally shaped, the limbs are extremely short, and only the hands and feet are of relatively normal size and shape. Internal organs show no apparent structural or histologic anomalies, but preductal aortic coarctation was observed in most published cases. The lungs are hypoplastic and the protruding eyes typically show cataracts. Radiologic studies of patients with BLC reveal pronounced hyperdensity of the entire skeleton and markedly advanced ossification (Fig. 8). As mentioned above, the long bones are extremely short and poorly modeled, show markedly increased density, and lack metaphyseal growth plates. Endochondral bone formation is dramatically advanced, with premature fusion of the epiphyseal and metaphyseal ossification centers. The zones of chondrocyte proliferation and of column formation are lacking, and the zone that normally comprises the layer of hypertrophic chondrocytes is poorly defined, narrow, and irregular (Fig. 9). Capillary ingrowth, bone resorption, and bone formation are reported by some authors as being unaltered (162), whereas others describe these bone remodeling events as deficient (163). A defect in m a m m a r y gland development, previously overlooked, is illustrated by the absence of nipples in two fetuses with BLC; in both of these cases, computed tomography (CT) scans of mandibular and maxillary bones revealed evidence for abnormal tooth development (168).

BLOMSTRAND LETHAL

CHONDRODYSPLASIA

Blomstrand lethal chondrodysplasia (BLC) is an autosomal recessive h u m a n disorder characterized by early lethality, advanced bone maturation, accelerated chondrocyte differentiation, and most likely severe abnormalities in mineral ion homeostasis. The first patient was described by Blomstrand and colleagues in 1985 (160); descriptions of several other patients followed (161-167). The disorder was shown to occur in families of different ethnic backgrounds and appears to affect males and females equally. Most affected infants are born to consanguineous parents [only in one instance were unrelated parents reported to have two offspring that were both affected by Blomstrand disease (163) ], suggesting that BLC is an autosomal recessive disease. Infants with BLC are typically born prematurely and die shortly after birth. Birth weight, when

Blomstrand Disease Is Caused by Inactivating PTH/PTHrP Receptor Mutations Four different mutations in the P T H / P T H r P receptor gene have been described in genomic DNA from patients affected by BLC (see Fig. 2). The first reported case, a product of nonconsanguineous parents, was shown to have two distinct abnormalities in the P T H / P T H r P receptor gene (169). T h r o u g h a nucleotide exchange in exon M5 of the maternal P T H / P T H r P receptor allele, a novel splice acceptor site was introduced, which led to a m u t a n t mRNA encoding an abnormal receptor that lacks a portion of the fifth membrane-spanning domain [amino acids 373 to 383; (A373-383)]. This receptor m u t a n t fails, despite seemingly normal cell surface expression, to respond to PTH or P T H r P with an accumulation of cAMP and inositol phosphate. For yet unknown reasons, the

MUTATIONS IN P T H / P T H r P RECEPTOR /

FI6.8 Radiologic findings in two fetuses affected by Blomstrand lethal chondrodysplasia (BLC). Anterioposterior (A) and lateral (B) views of a male fetus at 26 weeks of gestation; upper (C) and lower (D) limbs of a female fetus with BLC at 33 weeks of gestation. Particularly striking is the dramatic acceleration of endochondral bone formation of all skeletal elements. No secondary ossification centers of ossification are seen in long bones. The limbs are coarsely shaped and extremely short, but carpal and tarsal bones have a comparatively normal shape and size. Note also that the clavicles are relatively long, but show abnormal bending. (From Ref. 163, Familial Blomstrand chondrodysplasia with advanced skeletal maturation: Further delineation. A Loshkajian, J Roume, V Stanescu, AL Delezoide, F Stampf, P Maroteaux; Am J Med Genet; Copyright 1997 John Wiley & Sons, Inc. Reprinted by permission of Wiley-Liss Inc., a subsidiary of John Wiley & Sons, Inc.)

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FIG. 9 Section of the upper tibia end from a fetus affected by Blomstrand lethal chondrodysplasia (BLC) (A) and an age-matched control (B). Note the severely reduced size of the growth plate, the irregular boundary between the growth plate and the primary spongiosa, and the increased cortical bone thickness. (From Ref. 163, Familial Blomstrand chondrodysplasia with advanced skeletal maturation: Further delineation. A Loshkajian, J Roume, V Stanescu, AL Delezoide, F Stampf, P Maroteaux; Am J Med Genet; Copyright 1997 John Wiley & Sons, Inc. Reprinted by permission of Wiley-Liss Inc., a subsidiary of John Wiley & Sons, Inc.; and from Anne-Lise Delezoide, personal collection). (See color plates.)

paternal P T H / P T H r P receptor allele from this patient is very poorly expressed, suggesting an unidentified mutation in one of the different promoter regions or in a putative enhancer element. A second patient with BLC, the product of a consanguineous marriage, was shown to have a nucleotide exchange that leads to a proline-to-leucine mutation at position 132 (P132L) (170,171). This residue in the amino-terminal, extracellular domain of the P T H / P T H r P receptor is invariant in all mammalian members of this family of G protein-coupled receptors, indicating that the identified mutation is likely to have significant functional consequences. Indeed, COS-7 cells expressing this mutant P T H / P T H r P receptor showed, despite apparently normal cell surface expression, dramatically impaired binding of radiolabeled PTH and PTHrP analogs, greatly reduced, although detectable, agonist-stimulated cAMP accumulation, yet showed no measurable increase of IP (Fig. 10). Furthermore, it is important to note that cells expressing the P132L mutant receptor showed reduced, but detectable, specific binding of radiolabeled PTHrE A homozygous deletion of the nucleotide at position 1122 (exon EL2) was identified in a third case of BLC (167). This mutation led to a shift in the open reading frame, which resulted in a truncated protein that completely diverged from the wild-type receptor sequence after amino acid 364, and thus lacked transmembrane domains 5, 6, and 7, the connecting intraand extracellular loops, and the cytoplasmic tail (A365-593). Functional analysis of the A365-593 mutant in COS-7 cells demonstrated a total absence of PTH-stimulated accumulation of intracellular cAME which was confirmed in studies performed with the patient's dermal

fibroblasts (167). As for the other cases of BLC, these findings provided a plausible explanation for the severe abnormalities in endochondral bone formation. The abnormalities in mammary gland and tooth development furthermore support the conclusion that the P T H / P T H r P receptor has, in humans and mice, identical roles in the development of these organs. It is also worth noting that abnormalities in skeletal development in the fetus carrying the P132L mutation (which inactivates the P T H / P T H r P receptor incompletely) are less severe than those observed in most other cases, particularly with regard to the bones of the lower limbs (161,167). Taken together these findings suggested that BLC is the human equivalent of the P T H / P T H r P receptor knockout in mice (8). Inactivating mutations have been described in other G protein-coupled receptors [reviewed in (172) and (173) ]. For example, genetic forms of growth hormone deficiency were shown to be caused by mutations in the growth hormone-releasing hormone receptor (174,175); mutations in the thyrotropin receptor are the cause of some inherited forms of hypothyroidism (176-178), and mutations in the calcium-sensing receptor are the cause of familial hypocalciuric hypercalcemia and neonatal severe primary hyperparathyroidism [reviewed in (179)].

CONCLUSIONS Genetic linkage studies and positional cloning strategies have led with increasing frequency to the identification of molecular defects, often in unforeseen

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FIG. 10 Functional evaluation of the wild-type PTH/PTHrP receptors and receptors with the P132L mutation expressed in COS-7 cells. Cells were transfected with the indicated doses of plasmid DNA coding wild-type (©, @) or mutant (A, A) PTH/PTHrP receptors: cAMP (A) and IP accumulation (C) in response to 10 -7 M hPTH(1-34); specific binding of radiolabeled ligand (E) [total binding of 12Sl-labeled PTHrP(1-36) minus binding observed in the presence of 5 x 10 -7 M unlabeled PTH]; cAMP (B) and IP accumulation (D) by cells expressing wild-type (circles) or mutant (triangles) PTH/PTHrP receptors that were incubated in the absence or presence of increasing concentrations of hPTH(1-34) (@, A) and PTHrP(1-34) (©, A); inhibition of 12Sl-labeled PTHrP(1-36) binding by the indicated concentrations of unlabeled hPTH(1-34) and PTHrP(1-34) (F). (From Ref. 170; P Zhang, AS Jobert, A Couvineau, C Silve. A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab Vol. 83, pp. 3365-3368, 1998; © The Endocrine Society.)

candidate gene or in novel genes with yet uncharacterized biologic roles. Similar advances in understanding the physiologic importance of a variety of proteins have been achieved through gene ablation techniques and through the targeted expression of genes using tissuespecific promoters, because these studies often provided unanticipated results that frequently redirected the search for abnormalities in mammalian disorders.

The findings in PTHrP-ablated and P T H / P T H r P receptor-ablated mice predicted that human disorders caused by mutations in either of these proteins would present with severe abnormalities in endochondral bone formation, and these insights from genetically manipulated animals led to the identification of P T H / P T H r P receptor mutations in patients with Jansen's and Blomstrand disease, respectively, thus

722

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providing a molecular explanation for two puzzling genetic disorders in humans, and confirming the importance of the PTH/PTHrP receptor in mammalian development. Furthermore, the evaluation of cells expressing constitutively active mutant PTH/ PTHrP receptors has provided new insights into the structure/function relationship and the signal transduction properties of this receptor, and has prompted similar functional studies with other members of this family of G protein-coupled receptors (180-185).

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14.

15.

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145. Latronico AC, Anasti J, Arnhold IJP, Mendonca BB, Domenice S, Albano MC, Zachman K, Wajchenberg BL, Tsigos C. A novel mutation of the luteinizing hormone receptor gene causing male gonadotropin-independent precocious puberty. J Clin Endocrinol Metab 1995;80:2490-2494. 146. Kraaij R, Post M, Kremer H, Milgrom E, Epping W, Brunner HG, GrootgoedJA, Themmen APN. A missense mutation in the second transmembrane segment of the luteinizing hormone receptor causes familial male-limited precocious puberty. J Clin Endocrinol Metab 1995;80:3168-3172. 147. Shenker A. Disorders caused by mutations of the lutropin/ choriogonadotropin receptor gene. In: Spiegel AM, ed. G proteins, receptors, and disease Totowa, New Jersey:Humana, 1998. 148. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert SC, Seidman CE, Seidman JG. Autosomal dominant hypocalcaemia caused by a CaZ+-sensing receptor gene mutation. Nat Genet 1994;8:303-307. 149. Pearce S. Calcium-sensing receptor mutations in familial benign hypercalcaemia and neonatal hyperparathyroidism. J Clin Invest 1995;96:2683-2692. 150. Pearce SHS, Brown EM. Calcium-sensing receptor mutations: Insights into a structurally and functionally novel receptor. J Clin Endocrinol Metab 1996;81:1309-1311. 151. Baron J, Winer K, Yanovski J, Cunningham A, Laue L, Zimmerman DG, CutlerJ. Mutations in the Ca 2+ sensing receptor cause autosomal dominant and sporadic hypoparathyroidism. Hum Mol Genet 1996;5:601-606. 152. Pearce SH, Williamson C, Kifor O, Bai M, Coulthard MG, Davies M, Lewis-Barned N, McCredie D, Powell H, Kendall-Taylor P, Brown EM, Thakker RV. A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor. NEnglJMed 1997;335:1115-1122. 153. Brown EM, Pollak M, Bai M, Hebert SC. Disorders with increased or decreased responsiveness to extracellular Ca 2+ owing to mutations in the Ca2+-sensing receptor. In: Spiegel AM, ed. G proteins, receptors, and disease. Totowa, New Jersey:Humana, 1998:181-204. 154. Watanabe T, Bai M, Lane CR, Matsumoto S, Minamitani K, Minagawa M, Niimi H, Brown E, Yasuda T. Familial hypoparathyroidism: Identification of a novel gain of function mutation in transmembrane domain 5 of the calcium-sensing receptor. J Clin Endocrinol Metab 1998;83:2497-2502. 155. Okazaki R, Chikatsu N, Nakatsu M, Takeuchi Y, Ajima M, Miki J, Fujita T, Arai M, Totsuka Y, Tanaka K, Fukumoto S. A novel activating mutation in calcium-sensing receptor gene associated with a family of autosomal dominant hypocalcemia. J Clin Endocrinol Metab 1999;84:363-366. 156. Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone arrow stromal cells. J Immunol 1999;163:2017-2022. 157. Burger M, Burger J, Hoch R, Oades Z, Takamori H, Schraufstatter I. Point mutation causing constitutive signaling of CXCR2 leads to transforming activity similar to Kaposi's sarcoma herpesvirus-G protein-coupled receptor. Blood 1999;94:3658-3667. 158. Schipani E, Lanske B, Hunzelman J, Kovacs CS, Lee K, Pirro A, Kronenberg HM, Jfippner H. Targeted expression of constitutively active PTH/PTHrP receptors delays endochondral bone formation and rescues PTHrP-less mice. Proc Natl Acad Sci USA 1997;94:13689-13694. 159. Hunzelman J, Saxton JM, Schipani E. In vitro targeted expression of constitutively active PTH/PTHrP receptors in osteoblasts. J Bone Miner Res 1999;14(Suppl. 1): 160. Blomstrand S, Cla6sson I, Sfive-S6derbergh J. A case of lethal congenital dwarfism with accelerated skeletal maturation. Pediatr Radio11985;15:141-143.

MUTATIONS IN 161. Young ID, Zuccollo JM, Broderick NJ. A lethal skeletal dysplasia with generalized sclerosis and advanced skeletal maturation: Blomstrand chondrodysplasia. J Med Genet 1993;30:155-157. 162. Leroy JG, Keersmaeckers G, Coppens M, Dumon JE, Roels H. Blomstrand lethal chondrodysplasia. Am J Med Genet 1996;63: 84-89. 163. Loshkajian A, Roume J, Stanescu V, Delezoide AL, Stampf E Maroteaux E Familial Blomstrand chondrodysplasia with advanced skeletal maturation: Further delineation. Am J Med Genet 1997;71:283-288. 164. den Hollander NS, van der Harten HJ, Vermeij-Keers C, Niermeijer ME Wladimiroff JW. First-trimester diagnosis of Blomstrand lethal osteochondrodysplasia. Am J Med Genet 1997;73:345-350. 165. Oostra RJ, Baljet B, Dijkstra PE Hennekam RCM. Congenital anomalies in the teratological collection of museum Vrolik in Amsterdam, The Netherlands. II: Skeletal dysplasia. Am J Med Genet 1998;77:116-134. 166. Galera M, de Silva Patricio F, Lederman H, Porciuncula C, Lopes Monlleo I, Brunoni D. Blomstrand chondrodysplasia: A lethal sclerosing skeletal dysplasia. Case report and review. Pediatr Radiol 1999;29:842-845. 167. Karperien MC, van der Harten HJ, van Schooten R, Farih-Sips H, den Hollander NS, Kneppers ALJ, Nijweide P, Papapoulos SE, and L6wik CWG. M. A frame-shift mutation in the type I parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand lethal osteochondrodysplasia. J Clin Endocrinol Metab 1999;84:3713-3720. 168. Wysolmerski JJ, Roume J, Silve c. Absence of functional type 1 PTH/PTHrP receptors in humans is associated with abnormalities in breast and tooth development. J Bone Miner Res 1999;14(Suppl. 1). 169. Jobert AS, Zhang P, Couvineau A, Bonaventure J, Roume J, LeMerrer M, Silve C. Absence of functional receptors parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. J Clin Invest 1998;102:34-40. 170. Zhang P, Jobert AS, Couvineau A, Silve C. A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab 1998;83:3365-3368. 171. Karaplis AC, Bin He MT, Nguyen A, Young ID, Semeraro D, Ozawa H, Amizuka N. Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. Endocrinology 1998;139:5255-5258. 172. Spiegel AM. Introduction to G-protein-coupled signal transduction and human disease. In: Spiegel AM, ed, G proteins, receptors, and disease. Totowa, New Jersey:Humana, 1998. 173. Spiegel AM. Hormone resistance caused by mutations in G proteins and G protein-coupled receptors. JPediatrEndocrinol Metab

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174. Wajnrajch ME Gertner JM, Harbison MD, Chua SC, Jr Leibel RL. Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse. Nat Genet 1996; 12:88-90. 175. Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins NA, Mayo K. E. GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nat Genet 1993;4:227-232. 176. Stein SA, Oates EL, Hall CR, Grumbles RM, Fernandez LM, Taylor NA, Puett D, Jin S. Identification of a point mutation in the thyrotropin receptor of the hyt/hyt hypothyroid mouse. Mol Endocrino11994;8:129-138. 177. Gu WX, Du GG, Kopp P, Rentoumis A, Albanese C, Kohn LD, Madison LD, Jameson JL. The thyrotropin (TSH) receptor transmembrane domain mutation (Pro556-Leu) in the hypothyroid hyt/hyt mouse results in plasma membrane targeting but defective TSH binding. Endocrinology 1995; 136:3146-3153. 178. Biebermann H, Sch6neberg T, Schulz A, Krause G, Gr/iters A, Schultz G, Gudermann T. A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G-protein coupling. FASEB J 1998;12:1461-1471. 179. Brown EM. Physiology and pathophysiology of the extracellular calcium-sensing receptor. Am J Med 1999;106:238-253. 180. Gardella TJ, Luck MD,Jensen GS, Schipani E, PottsJT, JrJ~ppner H. Inverse agonism of amino-terminally truncated parathyroid hormone (PTH) and PTH-related peptide (PTHrP) analogs revealed with constitutively active mutant PTH/PTHrP receptors linked to Jansen's meta-physeal chondrodysplasia. Endocrinology 1996;137:3936-3941. 181. Heller RS, Kieffer TJ, HabenerJE Point mutations in the first and third intracellular loops of the glucagon-like peptide-1 receptor alter intracellular signaling. Biochem Biophys Res Commun 1996;223:624-632. 182. Tseng CC, Lin L. A point mutation in the glucose-dependent insulinotropic peptide receptor confers constitutive activity. Biochem Biophys Res Commun 1997;232:96-100. 183. Cohen DP, Thaw CN, Varma A, Gershengorn MC, Nussenzveig DR. Human calcitonin receptors exhibit agonist-independent (constitutive) signaling activity. Endocrinology 1997;138: 1400-1405. 184. Hjorth SA, Qrskov C, Schwartz TW. Constitutive activity of glucagon receptor mutants. Mol Endocrino11998;12:78-86. 185. Gaudin P, Maoret JJ, Couvineau A, Rouyer-Fessard C, Laburthe M. Constitutive activation of the human vasoactive intestinal peptide 1 receptor, a member of the new class II family of G protein-coupled receptors. J Biol Chem 1998;273: 4990-4996.

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CHAPTER 4 5

Acute Management of Hypercalcemia

JEAN

E. M U L D E R

New York 10032

AND

JOHN

E BILEZIKIAN

Department of Medicine, College of Physicians and Surgeons, Columbia University, New York,

INTRODUCTION

or other factors. However, the approach to any severely hypercalcemic individual, independent of etiology, is similar. Outpatient treatment of mild hypercalcemia due to asymptomatic primary hyperparathyroidism is considered in Chapter 29.

Hypercalcemia is a nearly universal manifestation in patients with primary hyperparathyroidism and is a common event in the later stages of some cancers. The clinical spectrum ranges from a few patients with symptomatic and perhaps life-threatening hypercalcemia that requires immediate treatment to a larger number of patients whose hypercalcemia is milder and frequently asymptomatic. Widespread use of the multichannel screening test now accounts for the observation that a large percentage of hypercalcemic patients present without symptoms of this metabolic abnormality. The approach to management of hypercalcemic patients requires full knowledge of the principles of calcium homeostasis, a topic covered in Chapter 10. The approach to the patient also requires knowledge of the differential diagnosis of hypercalcemia and associated pathophysiologies (Chapter 41) and the clinical judgment to know when and how to administer appropriate therapy. This chapter deals with the management of hypercalcemic patients who require immediate attention in the hospital. It focuses on the management of acute hypercalcemia due to excess production of parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), or other osteolytic factors, such as prostaglandins, interleukins, or tumor necrosis factor. Because acute hypercalcemia requiring urgent therapy is now uncommonly due to primary hyperparathyroidism (see Chapter 34), this discussion is most applicable to cancer-associated hypercalcemia due to PTHrP The Parathyroids, Second Edition

DIFFERENTIAL DIAGNOSIS OF HYPERCALCEMIA A classification of the differential diagnosis of hypercalcemia is provided in Table 1. The overwhelming majority (>90%) of hypercalcemic patients will be shown to have either primary hyperparathyroidism or a malignancy. Hypercalcemia requiring urgent therapy is caused most often by cancer, but severe primary hyperparathyroidism (acute parathyroid crisis) and the other etiologies noted in Table 1 require consideration at times. Thus the most simple and direct differential diagnosis of hypercalcemia is a distinction between the two most common causes, primary hyperparathyroidism or malignancy. It is important not to assume that severe hypercalcemia is due to cancer, because when acute primary hyperparathyroidism is the cause it can be a clinical "lookalike." The distinction between cancer-related hypercalcemia and primary hyperparathyroidism is usually not difficult and is readily confirmed by measurements of the intact serum parathyroid hormone concentration. Most patients with primary hyperparathyroidism have elevated concentrations of intact PTH, which, in patients with acute hyperparathyroid crisis, are remarkably elevated (see Chapter 34). 729

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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TABLE 1

Differential Diagnosis of Hypercalcemia

Primary hyperparathyroidism Sporadic (adenoma, hyperplasia, or carcinoma)

Familial

Isolated Cystic Multiple endocrine neoplasia type 1 or 2

Malignancy

Parathyroid hormone-related protein Excess production of 1,25(OH)2D Other factors (cytokines, growth factors) Nonparathyroid endocrine disorders Thyrotoxicosis Pheochromocytoma Acute adrenal insufficiency Vasointestinal polypeptide hormone-producing tumor Granulomatous diseases [1,25(OH)aD excess] Sarcoidosis Tuberculosis Histoplasmosis Coccidiomycosis Leprosy Medications Thiazide diuretics Lithium Estrogens/antiestrogens, testosterone in breast cancer Milk-alkali syndrome Vitamin A or D toxicity Familial hypocalciuric hypercalcemia Immobilization Parenteral nutrition Aluminum excess Acute and chronic renal disease

In hypercalcemia of malignancy due to PTHrP and other nonparathyroid causes of hypercalcemia, the inhibition of parathyroid glandular activity is a normal physiologic response of parathyroid tissue. Virtually all patients with cancer-associated hypercalcemia have low or suppressed concentrations of intact PTH (1,2). Thus a good assay for parathyroid h o r m o n e will measure low or undetectable PTH in the circulation in patients with malignancy unless the patient has both cancer and primary hyperparathyroidism. If the PTH value is suppressed when hypercalcemia is present, the diagnosis of primary hyperparathyroidism becomes extremely unlikely. Practically, it is excluded. However, although suppressed PTH in hypercalcemia rules out primary hyperparathyroidism, it does not rule in malignancy, because a great many other causes of hypercalcemia are associated with suppression of parathyroid gland activity. Having ruled out primary hyperparathyroidism, one focuses more intently on malignancy in view of the fact that it is by far the most c o m m o n cause of PTH-

i n d e p e n d e n t hypercalcemia. The many other potential causes of hypercalcemia are considered only after malignancy has been ruled out or unless the history or physical examination suggests that a n o t h e r explanation for the hypercalcemia is likely. In general, other endocrine disorders associated with hypercalcemia exhibit classic findings of the endocrine disorder. The structure of PTHrP, which has limited peptide homology to PTH, probably explains why the available immunoradiometric assays (IRMAs) for PTH do not detect it in the circulation. The only area of similarity between PTHrP and PTH is the first 13 amino-terminal amino acids, where great sequence homology exists. Thereafter, PTHrP diverges extensively from PTH (see Chapter 4). The rest of the polypeptides of PTH and PTHrP diverge completely from each other. The available radioimmunoassays for PTH, even those with amino-terminal specificity, recognize epitopes of the PTH molecule that are carboxy terminal to the first 13 amino acids. Thus they do not detect PTHrP in the circulation but register the normal suppression of native PTH. Assays for PTHrP are now available with which the presence of this cause of cancer-related hypercalcemia can be confirmed sometimes by elevated concentrations in the circulation (3-7) (see Chapter 9). PTHrP concentrations are not elevated in primary hyperparathyroidism but, with some assays, elevation of this protein may be found if renal failure is present (5). The remaining cases of hypercalcemia, constituting only 10% of the total cases, are distributed among a great variety of other potential etiologies (Table 1). This is not just an academic discussion; in the occasional patient, one must search diligently among these many other possible etiologies. It is important to bear in mind these other disorders, especially in the patient who becomes a diagnostic problem. An important clinical observation, which is quite useful in the differential diagnosis of symptomatic hypercalcemia, is the rapidity with which hypercalcemic symptoms a n d / o r hypercalcemia develop. A normocalcemic state d o c u m e n t e d several weeks previously points to malignancy as a likely presenting cause of hypercalcemia. Review of prior calcium determinations in patients with primary hyperparathyroidism often reveals subtle hypercalcemia even over a period of years.

P A T H O P H Y S I O L O G Y OF ACUTE HYPERCALCEMIA The dominant process leading to severe hypercalcemia is the acceleration of calcium mobilization from bone due to activation of the osteoclast, a multinucleated, calcium-resorbing bone cell (8). The osteoclast

MANAGEMENT OF

can be activated by both local a n d / o r systemic effects of various substances such as PTHrP. Osteoclast activation is a seminal pathophysiologic feature of virtually all cases of marked hypercalcemia. Excessive absorption of calcium from the gastrointestinal tract is usually not a major factor, although it can contribute to hypercalcemia caused by exogenous or endogenous vitamin D excess. Hypercalcemia occurs when calcium resorbed from bone into the extracellular space exceeds the homeostatic mechanisms that maintain normocalcemia. One of these, the suppression of PTH secretion by calcium, is obviously negated when the cause of increased bone resorption is PTH. In PTHrP-associated hypercalcemia, PTH secretion is suppressed, but PTHrP acts similarly to accelerate osteoclast-mediated bone resorption. When bone resorption is accelerated, the kidney becomes the major defense against hypercalcemia (9-11). When renal mechanisms are operating normally, the tendency for the serum calcium to rise is attenuated by greater urinary calcium excretion. Severe hypercalcemia can be defined as a marked elevation in the serum calcium concentration (i.e., > 1 4 m g / d l ) , which is usually associated with clinical features of hypercalcemia (Table 2). It is a manifestation of a series of pathophysiologic events. Initially, factors that induce osteoclast-mediated bone resorption, such as PTH and PTHrP, also stimulate renal tubular

TABLE 2 Signs and Symptoms of Hypercalcemia General Weakness Dehydration Corneal and other ectopic calcification Central nervous system Impaired concentration Depression Psychosis Altered consciousness (confusion, lethargy, stupor, coma) Gastointestinal tract Polydipsia Anorexia Nausea Vomiting Abdominal pain (pancreatitis, peptic ulcer) Constipation Renal system Polyuria Low urinary specific gravity Reduced glomerular filtration rate Flank pain (nephrolithiasis) Neph rocalcinosis Cardiovascular system Hypertension Shortened QT interval Digitalis sensitivity

H~FgCALCFMIA /

731

reabsorption of calcium (12). E n h a n c e d conservation of calcium impairs the efficiency of the kidneys to excrete the increased filtered load of calcium that is simultaneously released from bone. Second, hypercalcemia per se interferes with the renal mechanisms for reabsorption of sodium and water, leading to polyuria. The ensuing polyuria may not be matched by commensurate oral fluid intake because of anorexia and nausea, frequent symptoms of hypercalcemia. The result is extracellular volume depletion and a reduction in the glomerular filtration rate, which further increases the serum calcium concentration by increasing renal tubular calcium reabsorption. Hypercalcemia may also be exacerbated by immobilization of the severely ill patient, superimposing another stimulus for bonerelated calcium loss. To a greater or lesser extent, these pathophysiologic mechanisms are operative in virtually all patients who require hospitalization for hypercalcemia.

CLINICAL FF~TURES OF HYPERCALCEMIA An appreciation of the clinical features of hypercalcemia apart from its etiology is important, because this helps to determine decisions regarding instituting therapy for the hypercalcemia per se as well as possibly directing therapeutic approaches to the underlying problem. The signs and symptoms of hypercalcemia are listed in Table 2 and should not be confused with those of the underlying disorder. In the absence of clear-cut features of hypercalcemia, measures to reduce the serum calcium concentration may not be indicated, and therapy of hypercalcemia may distract the clinician from the underlying disorder. It is true that hypercalcemia is not always associated with a typical set of signs and symptoms that dictate whether the patient would improve with treatment. Quite commonly, various nonspecific complaints such as weakness and lethargy challenge our ability to define the specific cause of the symptomatology. Despite this, an attempt should be made to conclude whether the signs and symptoms of hypercalcemia are present in a given patient. If it is not possible to be certain, empiric but judicious therapy for the hypercalcemia is appropriate. The clinical manifestations of hypercalcemia reflect disturbances in central nervous system, gastrointestinal, renal, and cardiovascular function (Table 2). Central nervous system dysfunction is most threatening to survival and ranges from mild i m p a i r m e n t of cognitive function to coma. Gastrointestinal symptoms include anorexia, nausea and vomiting, constipation, and, rarely, abdominal pain due to pancreatitis. The symptoms of renal dysfunction are polyuria and polydipsia; patients who develop kidney stones may experience

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acute flank pain. The cardiovascular manifestations include hypertension, if intravascular volume is maintained, a shortened QT interval on the electrocardiogram, and greater sensitivity to digitalis. Not all patients have all of these symptoms. A n u m b e r of factors account for the great clinical variability of hypercalcemic patients. These include the age of the patient, the presence of other medical problems, the duration of the hypercalcemia, and the rate of rise and the absolute level of the serum calcium. Therefore, in patients with moderate hypercalcemia, therapy should be based not only on the serum calcium concentration but also on the extent of symptomatology. A particularly difficult situation is presented by the patient in whom hypercalcemia is not in the range that one would ordinarily treat aggressively (<12 m g / d l ) but who exhibits central nervous system features that may be associated with hypercalcemia such as apathy, drowsiness, obtundation, or even coma. It is not possible to be certain that these changes in the sensorium are due to hypercalcemia, particularly in older patients with moderate hypercalcemia. This may be even more vexing in the younger patient who, in general, is more likely to tolerate the hypercalcemia. In this situation, it is important to seek other factors contributing to the symptomatology before attributing altered central nervous system function to hypercalcemia. On the other hand, it is entirely appropriate to suspect, when the serum calcium value is in the moderately elevated range, 12-14 m g / d l , that altered central nervous system function is due to hypercalcemia. This is especially relevant in the individual who may also have other reasons for altered mental status. Correction of the hypercalcemia should improve central nervous system function if hypercalcemia is responsible for the symptomatology.

P R I N C I P L E S OF T H E R A P Y FOR HYPERCALCEMIA Hypercalcemia is a treatable metabolic abnormality, which need not always be directly treated. It often precedes the diagnosis of an underlying disorder that should be given more initial attention than the hypercalcemia. At the other end of the spectrum, hypercalcemia may present as a life-threatening disorder that demands vigorous therapy i n d e p e n d e n t even of knowledge of the cause of the underlying disorder. Thus principles of therapy relate importantly to two elements; the underlying etiology and the severity of the hypercalcemia. When hypercalcemia is asymptomatic, attention should be directed to defining the etiology. In most patients, this would lead to a diagnosis of primary hyperparathyroidism. On the other hand, severe hyper-

calcemia associated with central nervous system symptoms demands that the hypercalcemia be treated first. An exception to this rule may be the patient with an incurable, disseminated malignancy in whom prolongation of life may be unkind.

THERAPY OF ACUTE HYPERCALCEMIA The magnitude of the hypercalcemia is a key consideration when the decision to treat is imminent. If the serum total calcium concentration is > 1 4 m g / d l (the u p p e r limit of normal varies a m o n g laboratories but is generally less than 10.4 m g / d l ) , a therapeutic plan to reduce the serum calcium is indicated, regardless of the presence of symptoms. The value of 14 m g / d l is based on the assumption that the serum albumin concentration is normal, but in hypercalcemic patients the serum albumin concentration may be increased because of dehydration or reduced because of chronic illness. A convenient rule of thumb is to adjust the serum total calcium concentration by 0.8 m g / d l for each 1 g / d l by which the serum albumin is increased or decreased (13). If the serum albumin concentration is increased, the serum total calcium concentration is adjusted downward; if the serum albumin concentration is reduced, the serum total calcium concentration is adjusted upward. When the serum calcium concentration is only moderately increased to 12-14 mg/dl, the clinical manifestations of hypercalcemia should dictate the type of therapy necessary and the dispatch with which it should be administered. There are four basic goals in the therapy of hypercalcemia: to correct dehydration, to enhance renal excretion of calcium, to inhibit accelerated bone resorption, and to treat the underlying disorder. A summary of reco m m e n d e d approaches is provided in Table 3.

General Measures

Hydration Intravenous administration of isotonic saline is the first step in the acute m a n a g e m e n t of hypercalcemia. This can be done safely in the great majority of patients, because congestive heart failure is rare in this clinical setting. When the depleted intravascular volume is restored to normal, the serum calcium concentration will decline at least by the degree to which the calcium concentration was elevated by the dehydration. The decrease in serum calcium usually amounts to 1.6-2.4 m g / d l , a substantial amount, but rarely does hydration alone lead to normalization of marked hypercalcemia (14). Expansion of intravascular volume is also helpful in that it begins to increase renal calcium clearance.

MANAGEMENT OF HVeWRCALCEMIA /

TABLE 3

Management of Hypercalcemia

Repletion of extracellular fluid volume Saline infusion Induction of calciuresis Saline infusion with modest doses of furosemide Inhibition of bone resorption Frequently used Bisphosphonates Calcitonin Rarely used Plicamycin Gallium nitrate (not recommended) Miscellaneous Glucocorticoids (lymphoma, sarcoid, vitamin D intoxication) Intravenous phosphate (not recommended) Renal failure Dialysis Bisphosphonates (reduced dose, slow infusion) Parathyroidectomy

First, the improved glomerular filtration rate leads to greater filtration of calcium. Second, increased proximal tubular sodium and calcium reabsorption, which has been mediated, in part, by the reduction in gromerular filtration rate (GFR), is returned toward normal. Third, as more sodium and water are presented to distal renal tubular sites, an obligatory calciuresis ensues. The rate of saline administration should be based on the severity of the hypercalcemia, on the extent of dehydration, and on j u d g m e n t of the cardiovascular tolerance to volume loading. A widely used plan of action is to administer 2.5-4 liters of isotonic saline daily, recognizing the need to adjust the rate of administration or to use judicious therapy with loop diuretics if symptoms and signs of fluid overload appear. The observation of an increasing urinary output during the initial hours of fluid repletion is an important guide to the adequacy of the fluid load and the ability of the patient to handle it.

Loop Diuretics

(Furosemide)

In addition to hydration with saline, adjunctive diuretic therapy with furosemide has been effectively used in patients. There were two reasons to consider the use of a loop diuretic in the m a n a g e m e n t of hypercalcemia before safe and effective agents were available to control bone resorption. The first was to facilitate further urinary calcium excretion. Loop diuretics, such as furosemide and ethacrynic acid, increase the calciuric effects of volume expansion by inhibiting calcium reabsorption in the thick ascending limb of the loop of Henle. Thiazide diuretics are contraindicated in this situation because they increase distal tubular reab-

733

sorption of calcium and may even exacerbate hypercalcemia. Volume repletion must precede use of furosemide because the drug's effect is d e p e n d e n t on delivery of calcium to the ascending limb. It is a key principle of diuretic therapy for hypercalcemia that normal hydration must first be established. It was reported in 1970 by Suki et al. (15) that intensive administration of furosemide, 80-100 mg every 1-2 hours, with fluid (10 liters or more in 24 hours) and electrolyte replacement based on urinary losses are an effective regimen for the acute treatment of hypercalcemia (15). Such an aggressive approach will lead to marked hypercalciuria, but it requires frequent measu r e m e n t of water and electrolyte excretion. An already marginally compensated patient can be destabilized further if losses of fluid and electrolytes, except for calcium, are not matched by adequate replacement therapy. Such intensive therapy with furosemide is not necessary in most patients. Concerns about volume overload are particularly relevant in older patients in whom cardiovascular function may be marginal. U n d e r these circumstances, it is reasonable to administer modest doses of furosemide, for example, 10-20 mg intravenously every 6-12 hours, depending on the patient's capacity to handle the volume load. If fluid tolerance is not a major concern, furosemide should not be used but rather held in reserve should signs of fluid overload become apparent. In the patient with a history of cardiac disease, it is probably best not to exceed 2-3 liters of intravenous saline in a 24-hour period.

Inhibition of Bone Resorption Saline does not affect the major pathophysiologic feature of hypercalcemia, which is excessive calcium mobilization from bone. Thus, it is usually necessary to use specific therapy to inhibit osteoclast-mediated bone resorption if the patient has severe symptoms of hypercalcemia or even if the serum calcium concentration remains moderately elevated after volume expansion. Such specific therapy is particularly indicated in a symptomatic patient whose initial serum calcium concentration is ---14 m g / d l . Well-controlled studies of the treatment of severe hypercalcemia are difficult, so some studies of osteoclast inhibitors are difficult to interpret because the extent of preceding saline and diuretic therapy was not always stated. The specific regimens found in the literature have been developed mainly in patients with hypercalcemia of cancer and acute primary hyperparathyroidism, disorders of greatest concern here. It is likely that other etiologies of hypercalcemia would be associated with similar responses to these agents. It is obvious, but should be remembered, that the specific means of inhibiting

734

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CHAPTER45

osteoclast-mediated bone resorption are not directed toward treating the underlying cause of the hypercalcemia. Management of hypercalcemia, thus, should not interdict the use of therapy for the cause of the hypercalcemia per se. It is also important to consider use of the least toxic agents to control bone resorption in patients who may also receive cytotoxic cancer therapies.

Bisphosphonates The bisphosphonates are a family of compounds structurally related to a normal product of metabolism, pyrophosphate. Unlike the P - O - P b o n d in pyrophosphate, the bisphosphonates are characterized by a P - C - P backbone that renders them resistant to phosphatases. They bind to hydroxyapatite in bone and inhibit crystal dissolution (16,17). Their great affinity for bone surfaces and resistance to metabolic degradation produce an extremely long half-life in bone. They are excreted u n c h a n g e d by the kidney. The major property shared by all bisphosphonates that has made them valuable agents for hypercalcemia is their inhibitory effect on osteoclasts. Bisphosphonates can be divided into two classes with distinct mechanisms of action. Less potent bisphosphonates, such as etidronate, clodronate, and tiludronate, are metabolized by osteoclasts to metabolites that exchange with the terminal pyrophosphate moiety of ATE The resulting molecule is an ATP that cannot be used as a source of cellular energy. Osteoclasts become dysfunctional and can undergo apoptosis (18). The more potent bisphosphonates, containing amino substituents (pamidronate, alendronate, risedronate), also interfere with osteoclast function but by a different mechanism. By inhibiting the mevalonate pathway, amino bisphosphonates block the production of geranylgeranyl diphosphate, which is important for protein prenylation, a process essential to osteoclast function (18). By either mechanism of action, the bisphosphonates potently inhibit bone resorption and thus reduce serum calcium levels. Clodronate was one of the first bisphosphonates used to treat patients with malignant hypercalcemia (19-22). It can be administered intravenously in a dose of 4-6 m g / k g in 2-5 hours daily over 3-5 days or as a single infusion for 2-9 hours (23,24). Daily infusion may be associated with more prolonged normocalcemia. Oral clodronate is also quite effective and has few side effects, but the intravenous route is preferred in the setting of acute hypercalcemia. After intravenous administration, the serum calcium concentration declines at a rate typical for the bisphosphonates, with the initial substantial change occurring after 2 days and normocalcemia frequently occurring by 7 days. Similarly to pamidronate, clodronate has been reported to have the potential to reduce progression of skeletal metastases and to prevent

the onset of hypercalcemia in patients with cancer who are at risk (25-28). Potential nephrotoxicity is avoided by slow infusion over a period of at least 2 hours. Clodronate is not available in the United States, but it is used in many other countries (22). Etidronate was the first bisphosphonate available in the United States. It is approved for administration in a daily dose of 7.5 m g / k g intravenously over 4 hours for 3 days. The drug is much more effective if given for up to 7 days. The serum calcium concentration begins to fall within 2 days after the first dose and reaches its nadir within 7 days. The nadir is within the normal range in 33-80% of patients (29-35). Patients who are well hydrated before etidronate and who continue to receive intravenous fluids respond better (30,33). Whether etidronate should be given for a full 7 days depends on the level of the hypercalcemia after hydration and the response of the patient to the drug. Cessation of therapy is reasonable after 3 days if the patient shows a decline in serum calcium concentration after the first two or three doses by > 2 - 3 m g / d l or if the serum calcium is close to the normal range. Persistent daily administration of etidronate until the serum calcium concentration has normalized theoretically could lead to a period of unwanted hypocalcemia. Single-dose etidronate, a more convenient mode of administration, has been described (36). Intravenous etidronate at 25-30 m g / k g over 24 hours results in normalization of serum calcium concentrations in 38-67% of patients within 7 days (36). In patients in whom parenteral administration of etidronate lowered the serum calcium concentration, oral administration given subsequently has not been nearly as effective as the intravenous therapy (30,33,35,37,38). Etidronate treatment of hypercalcemia is safe, the only reported adverse effects being transient increases in serum creatinine and phosphate concentrations. T h o u g h chronic administration of high doses of etidronate can impair bone formation and cause osteomalacia (39), short-term use apparently does not. Despite the fact that intravenous etidronate is clearly efficacious, it is used infrequently now that more potent bisphosphonates are available. Pamidronate is the most frequently used bisphosphonate in the United States for the treatment of severe hypercalcemia of malignancy. It is the bisphosphonate of choice for this purpose. Several studies have demonstrated the efficacy of intravenous pamidronate in correcting hypercalcemia (40). Pamidronate is administered intravenously in doses of 45-90 mg over a 4- to 24-hour interval. The serum calcium concentrations begin to decline in 1-2 days. This regimen leads to normalization of serum calcium concentrations over 3 to 4 days in 60-100% of patients (41,42). A dose-response relationship exists in patients with high base line calcium concentrations (>13.5 mg/dl), with the 90-mg dose of pamidronate showing the greatest efficacy (41) (Fig. 1).

MANAGEMENT OF HYPVgCALCEMIA /

Symptoms of hypercalcemia resolve in the majority o f patients whose hypercalcemia remits. The decrease in calcium concentration is due specifically to a reduction in bone resorption, as evidenced by a reduction in biochemical markers of bone resorption (41). The duration of normocalcemia after intravenous pamidronate

In our experience, one should reserve the 90-mg dose for those whose serum calcium is in the life-threatening range (i.e., >16 m g / d l ) . When the 90-mg infusion is used with lesser degrees of hypercalcemia, symptomatic hypocalcemia has occurred. For this reason, we tend most commonly to use the 60-mg dose.

Complete Response Rate Mean Corrected Serum Calcium by Day

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20% 0% N=9

N=10

N=8

Day 7 * Number of responders statistically greater (p<0.05) for: Pamidronate 90 mg versus 30 mg Pamidronate 90 mg versus 60 mg (unadjusted)

FIG. 1 Mean corrected serum calcium concentrations and response rates after 24-hour infusion of 30, 60, or 90 mg of pamidronate in patients with a base line serum calcium level of less than 13.5 mg/dl (top) or greater than 13.5 mg/dl (bottom) (reprinted from Ref. 41, Am J Med 95; SR Nussbaum, J Younger, CJ VandePol, et aL; Single dose intravenous therapy with pamidronate for the treatment of hypercalcemia of malignancy: Comparison of 30-, 60-, and 90-mg dosages; pp. 297-304, Copyright 1993, with permission from Excerpta Medica, Inc.).

736

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FIG. 2 Mean corrected serum calcium concentrations (_ SEM) after treatment with pamidronate, 60 mg (m) or etidronate, 7.5 mg/kg/day for 3 days (A). *p < 0.05 (from Ref. 44; R Gucalp, P Ritch, PH Wiernik, et aL Comparative study of pamidronate disodium and etidronate disodium in the treatment of cancer-related hypercalcemia. J Clin Oncol 1992; 10:134-142.).

therapy varies between 9 and 30 days, depending on the underlying disease process (41,43). Intravenous pamidronate has been demonstrated to be more effective than both intravenous clodronate and etidronate (Fig. 2) for the treatment ofhypercalcemia of malignancy (43,44). In two separate studies, clodronate, etidronate, and pamidronate normalized calcium concentrations in 31-37%, 31-41%, and 70-88% of patients, respectively (43,44). In addition, patients treated with pamidronate had a longer duration of remission (28 days vs. 14 days with clodronate). Repeat infusions of pamidronate may be administered as needed, depending on the magnitude of the initial response (40). Along with hypocalcemia, other adverse effects of pamidronate include a mild, transient increase in body temperature, transient leukopenia, and a small reduction in serum phosphate levels. Some patients may also experience a local phlebitis at the intravenous site. Besides hypercalcemia of malignancy, pamidronate has been demonstrated to be effective in treating hypercalcemia secondary to acute primary hyperparathyroidism (45), immobilization (46,47), vitamin D intoxication (48), and sarcoidosis (49). Pamidronate and clodronate have been shown to have additional benefits in reducing the progression of skeletal metastases in normocalcemic patients with breast cancer and multiple myeloma (50,51). Several new bisphosphonates, not yet available in the United States, are showing promise for the treatment of hypercalcemia. Intravenous ibandronate (infused over 2 hours) has been demonstrated to normalize serum calcium concentrations in 44-77% of patients with hypercalcemia of malignancy (52), whereas zoledronate resulted in a normalization of serum calcium levels in 93% of patients with hypercalcemia of malignancy (53). In addition, zoledronate has a rapid effect (2-3 days)

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FIG. 3 Mean corrected serum calcium concentrations (_ SEM) after intravenous zoledronate, 0.02 mg/kg (©) and 0.04 mg/kg (m). Hatched area represents normal serum calcium levels (reproduced from J Bone Miner Res 1999;14:1557-1561, with permission of the American Society of Bone and Mineral Research).

and a long duration of action (Fig. 3). Furthermore, zoledronate may be administered safely as a 30-minute infusion (53). Side effects are mild and include transient hypophosphatemia, transient hypocalcemia, and low-grade fever. Thus, zoledronate appears to have excellent therapeutic efficacy and is more potent and longer acting than pamidronate. All bisphosphonates are eliminated unmetabolized via the kidneys. Early reports of nephrotoxicity in animal studies raised concerns about administering bisphosphonates to patients with renal insufficiency. Furthermore, case studies reported that rapid intravenous administration of etidronate or clodronate resulted in renal failure in a small number of patients (54). However, subsequent reports suggest that many patients with hypercalcemia and mild renal insufficiency can be successfully treated with bisphosphonates. A retrospective study of 33 patients from Memorial SloanKettering Cancer Center revealed that intravenous pamidronate could be administered successfully to patients with renal insufficiency (creatinine -->1.5 mg/dl). Renal function improved or remained stable in 25 patients, but renal function deteriorated transiently in 8 patients (55). In all 8 patients, the rise in serum creatinine concentration preceded pamidronate administration or could be attributable to other causes. Bisphosphonates have been administered to patients on dialysis without ill effects (56,57). However, bisphosphonates should be administered slowly (over 24 hours) in patients with renal insufficiency, and a reduced dose should be considered (30-45 mg, rather than 60 mg).

MANAGEMENT OF H~F.kCALCV.MZA /

Calcitonin A relative disadvantage of all bisphosphonates is the time it takes (1-2 days) to reduce hypercalcemia. In many patients, it is desirable to begin to lower the serum calcium concentration more rapidly. The advantage of calcitonin is that it acts much more rapidly than any of the bisphosphonates. In combination with rehydration, it is useful for the initial m a n a g e m e n t of severe hypercalcemia. The speed of calcitonin's action may be more related to its effect to facilitate urinary calcium excretion (58) than to its antiosteoclast properties. The recomm e n d e d dose is 4-8 MRC units/kg administered subcutaneously every 6-12 hours for 2-3 days. Serum calcium concentrations may decrease within 2 hours of administration (59). The calcemic nadir is reached within 12-24 hours but is often followed by a return toward initial hypercalcemic levels within 24-72 hours despite continued administration (60,61). Thus, the major disadvantage of calcitonin is that its effects are short-lived and relatively weak. Moreover, many patients develop tachyphylaxis to repeated dosing (62). Because of its limited duration of action, calcitonin is most effective when combined with hydration and a bisphosphonate (Fig. 4). The combination of calcitonin and a bisphosphonate results in a rapid decrease in serum calcium concentration (calcitonin effect), which is sustained for a more prolonged period (bisphosphonate effect) (63-65). Calcitonin is a safe antihypercalcemic agent. Side effects include mild, transient nausea, abdominal cramps, and flushing. True allergic reactions to salmon calcitonin, the preparation that has been used most widely, are quite rare. H u m a n calcitonin, which is less potent than salmon calcitonin, has seldom been stud-

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ied in the m a n a g e m e n t of hypercalcemia. Thus, shortterm use of calcitonin should be considered in the cancer patient with severe hypercalcemia because of its rapid onset of action and its safety profile. Simultaneous administration of a bisphosphonate would make likely more prolonged control of the hypercalcemic state. Another feature of calcitonin that should be borne in mind is the suggestion that it has potent analgesic properties (66). It has been reported to provide impressive relief in some patients with painful skeletal metastases.

Glucocorticoids Glucocorticoids can be effective calcium-lowering agents in limited groups of patients with sarcoidosis, vitamin D toxicity, hematologic malignancies associated with increased circulating 1,25-dihydroxyvitamin D [1,25 (OH) zD] , and certain other malignant states (i.e., some breast cancers). For this purpose, 200-300 mg of hydrocortisone, or its equivalent, is given intravenously daily for 3-5 days. Glucocorticoids inhibit gastrointestinal calcium absorption, but their efficacy to reduce hypercalcemia may relate to other properties. They may act to inhibit directly the growth of neoplastic lymphoid tissue (67). In patients with lymphomas associated with increased 1,25(OH)2 O, glucocorticoids reverse hypercalcemia by lowering the concentration of the vitamin D metabolite (68). The same is true in sarcoidosis (69). In vitamin D intoxication, they may act on the target organ. Because excessive bone resorption also occurs in patients with vitamin D toxicity, bisphosphonates additionally may be of benefit in decreasing calcium concentrations in such patients. In general, patients with nonhematologic cancers do not respond to glucocorticoids (70). Primary hyperparathyroidism also is classically unresponsive to glucocorticoid administration (71).

3.0

Plicamycin (Mithramycin) 2.5

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01234

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14

Days FIG. 4 Mean corrected serum calcium concentrations (_+ SEM) in two groups of patients with hypercalcemia of malignancy treated with either a single infusion of pamidronate alone (©) or with a single infusion of pamidronate combined with suppositories of salmon calcitonin (O). *p < 0.01, **p < 0.005 (from Ref. 63; D Thiebaud, F Jacquet, P Burckhardt. Fast and effective treatment of malignant hypercalcemia. Arch Intern Med 1990;150:2125-2128. Copyrighted 1990, American Medical Association).

Plicamycin, an inhibitor of osteoclast RNA synthesis, is a potent therapy for hypercalcemia that has been in clinical use for more than 25 years (72-75). It is given intravenously in a dose of 15-25 Ixg/kg of body weight over 4-6 hours. The dose can be repeated several times, although a single dose may normalize the serum calcium concentration. The serum calcium concentration begins to decrease as early as 6 hours after administration of the drug. The maximal reduction occurs in 48-72 hours. In one study, 45% of patients randomized to plicamycin, compared to 86% of patients randomized to pamidronate (60 mg), achieved normocalcemia within 7 days (76). The duration of normocalcemia after a single dose of plicamycin is usually a few days and depends on the rate of ongoing bone resorption.

738

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CHAPTER45

Plicamycin has several adverse side effects. Nausea is c o m m o n and can be minimized by slow intravenous infusion. Care should be taken to avoid local extravasation of the drug, because irritation and cellulitis can result. Hepatic toxicity, manifested most often as transiently elevated serum aminotransferase activity, occurs in approximately 20% of patients (77). Nephrotoxicity (increased blood urea concentration, creatinine, and proteinuria) and thrombocytopenia can also occur (78), the latter especially in patients who have received previous chemotherapy or radiotherapy. Because of the availability of bisphosphonates, which have greater efficacy and fewer side effects, plicamycin is rarely used now for the treatment of hypercalcemia. One situation in which plicamycin may be useful is for the treatment of patients who have hypercalemia that is refractory to therapy with bisphosphonates. Contraindications to the use of plicamycin are overt hepatic or renal dysfunction, thrombocytopenia, or any coagulopathy. G a l l i u m Nitrate Gallium nitrate binds to bone mineral surface and may reduce hydroxyapatite crystal solubility, thus inhibiting bone resorption (79-81). In addition, a direct inhibitory action of gallium on osteoclasts has been observed (82). After gallium nitrate administration, reductions in urinary calcium and hydroxyproline excretion are found, confirming its action as an inhibitor of bone resorption (83). Gallium is administered as a continuous intravenous infusion, 200 m g / m 2 in 1 liter of fluid daily for 5 days. It has been demonstrated to normalize serum calcium concentration in some patients with hypercalcemia of malignancy (84,85). However, the mean duration of normocalcemia in patients who received gallium was only 8 days, which is much shorter than the duration of normocalcemia in patients treated with pamidronate. In addition, the rate of decline of the serum calcium was slow, with the nadir calcium values occurring 8-10 days after the initiation of the gallium infusion. A potential toxicity of gallium nitrate is impaired renal function, manifested by an increase in serum creatinine, which is especially concerning in patients with underlying renal disease or dehydration. Thus, gallium is contraindicated in renal insufficiency. Because of its slow onset and short duration of action, as well as its potential renal toxicity, gallium is no longer used for the treatment of hypercalcemia.

Miscellaneous Therapy

Phosphate Intravenous administration of sodium or potassium phosphate can produce a profound and rapid reduction in serum calcium concentrations. This treatment, however, is potentially very dangerous because of

the possibility of deposition of calcium phosphate complexes in blood vessels, lungs, and kidneys. Precipitation of these complexes has produced severe organ damage and even fatal hypotension in patients rapidly infused with high doses (86-88). The use of intravenous phosphate should be restricted, therefore, to patients with extreme, life-threatening hypercalcemia who are hypophosphatemic and in whom all other measures have failed. Oral phosphate is of little value in the emergency therapy of hypercalcemia, because its calcium-lowering activity is modest, and amounts >2 g daily are often associated with diarrhea. Oral phosphate should be reserved for settings of mild to moderate hypercalcemia associated with serum concentrations of phosphate that are frankly low or in the lower range of normal. The best rationale for its oral use is in patients with mild hypercalcemia due to primary hyperparathyroidism or in special circumstances surrounding the management of the secondary hyperparathyroidism of renal insufficiency (see Chapters 29 and 40).

Dialysis Hypercalcemia may occur in patients with either acute or chronic renal failure. A careful investigation for the cause of hypercalcemia is necessary in all patients with renal failure. Patients with hypercalcemia and renal failure pose a unique therapeutic dilemma. In general, hydration facilitates the renal excretion of calcium. However, in patients with renal failure, enhancing the renal excretion of calcium is not possible. In patients with significant renal failure, dialysis may be very effective in lowering the serum calcium concentration (89,90). Utilizing a low-calcium dialysate, either peritoneal dialysis or hemodialysis may be successfully performed. Reducing the dose of calcium-containing phosphate-binding agents may also be effective for long-term m a n a g e m e n t (91). Pamidronate and clodronate have been used successfully to treat hypercalcemia in a handful of patients on chronic dialysis (56,57). Some patients with advanced secondary hyperparathyroidism and severe hypercalcemia require parathyroidectomy for definite treatment of hypercalcemia.

Mobilization Bed rest is associated with a significant increase in the rate of bone resorption as well as reduced bone formation. Therefore, patients should be encouraged to ambulate as soon as possible so that this contribution to the hypercalcemic state can be prevented.

Choice of Agent The wide clinical spectrum of acute hypercalcemia prevents the use of a single therapeutic regimen for all

MANAGEMENT OF HYPF~RCALCENIA /

hypercalcemic patients. It is necessary to tailor the therapy based on a consideration of the cause of the hypercalcemia, the clinical symptomatology of the patient, and the m o d e of action and potential side effects of the various agents. With mild hypercalcemia (serum calcium concentration <12 m g / d l ) , hydration with intravenous saline may be adequate therapy. Even in the presence of severe hypercalcemia, hydration with saline is the first step in management. If hypercalcemia is potentially life-threatening (>16 m g / d l ) and is associated with clear symptomatology, more vigorous therapy is required along with saline. In this situation, the most rapidly acting osteoclast inhibitor, calcitonin, becomes a valuable drug. Because calcitonin alone seldom fully reverses hypercalcemia, immediate concurrent therapy should be considered. Based on safety profiles and efficacy, pamidronate is the treatment of choice. If the hypercalcemic state is likely to be sensitive to steroids, the concurrent administration of glucocorticoids is worthy of consideration. There are times when, despite the presence of marked hypercalcemia, the clinical appraisal does not lead to the same urgency to treat as in other situations. For example, in a patient whose serum calcium is high, > 1 4 m g / d l , but who has only modest signs or symptoms of hypercalcemia and is otherwise stable, one might use a bisphosphonate, along with modest saline administration. Finally, there is the rare patient in whom the serum calcium concentration is >20 m g / d l . Such a patient requires the most aggressive approach, with high rates of saline infusion, a bisphosphonate, calcitonin, and perhaps hydrocartisone as well if the patient has a hematologic malignancy.

T H E R A P Y OF T H E UNDERLYING DISORDER In most hypercalcemic patients, successful managem e n t of acute hypercalcemia is followed by reappearance of hypercalcemia if definitive therapy of the underlying disorder is not possible. The availability of potent bisphosphonates now allows more long-term control of hypercalcemia even if definitive treatment fails. This is of considerable importance in that the patient whose serum calcium is now normal is still very much subject to the same pathophysiologic mechanisms that originally p r o d u c e d the hypercalcemia. In patients with primary hyperparathyroidism, parathyroidectomy is nearly always successful in preventing recurrent hypercalcemia. Because most hypercalcemic cancer patients have advanced disease, a successful outcome for cancer therapy is much less likely. Nevertheless, satisfactory m a n a g e m e n t of acute hypercalcemia allows time to plan a more definitive approach to the underlying disease.

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24. Douglas DL, Russell RGG, Preston CJ, et al. Effect of dichloromethylene diphosphonate in Paget's disease of bone and in hypercalcaemia due to primary hyperparathyroidism or malignant disease. Lancet 1980;1043-1047. 25. Siris ES, Sherman WH, Baquiran DC, et al. Effect of dichloromethylene diphosphonate on skeletal mobilization of calcium in multiple myeloma. N E n g l J M e d 1980;302:310315. 26. Delmas PD, Charhon S, Chapuy MC, et al. Long-term effects of dichloromethylene diphosphonate (CIZMDP) on skeletal lesions in multiple myeloma. Metab Bone Dis Relat Res 1982;4:163-168. 27. Jung A, Chantraine A, Donath A, et al. Use of dichloromethylene diphosphonate in metastatic bone disease. N EnglJ Med 1983;308: 1499-1501. 28. Elomaa I, Blomqvist C, Porkka L, et al. Diphosphonates for osteolytic metastases. Lancet 1985;1:1155-1156. 29. Ryzen E, Martodam RR, Troxell M, et al. Intravenous etidronate in the management of malignant hypercalcemia. Arch Intern Med 1985;145:449-452. 30. Hasling C, Charles P, Mosekilde L. Etidronate disodium in the management of malignancy-related hypercalcemia. Am J Med 1987;82(Suppl. 2A) :51-54. 31. Kanis JA, Urwin GH, Gray RES, et al. Effects of intravenous etidronate disodium on skeletal and calcium metabolism. Am J Med 1987;82(Suppl. 2A):55-70. 32. Meunier PJ, Chapuy M-C, Delmas P, et al. Intravenous disodium etidronate therapy in Paget's disease of bone and hypercalcemia of malignancy: Effects on biochemical parameters and bone histomorphometry. A m J M e d 1987;82(Suppl. 2A):71-78. 33. Jacobs TP, Gordon AC, Silverberg SJ, et al. Neoplastic hypercalcemia: Physiologic response to intravenous etidronate disodium. A m J M e d 1987;82(Suppl. 2A):42-50. 34. Singer FR. Role of the bisphosphonate etidronate in the therapy of cancer-related hypercalcemia. Semin Oncol 1990;2 (Suppl. 5) :34-39. 35. Singer FR, Ritch PS, Lad TE, et al. Treatment of hypercalcemia of malignancy with intravenous etidronate. Arch Intern Med 1991; 151:471-476. 36. Flores JE Rude RK, Chapman RA, et al. Evaluation of a 24 hour infusion of etidronate disodium for the treatment of hypercalcemia of malignancy. Cancer 1994;73:2527-2534. 37. Ringenberg QS, Ritch PS. Efficacy of oral administration of etidronate disodium in maintaining normal serum calcium levels in previously hypercalcemic cancer patients. Clin Ther 1987;9:1-8. 38. Schiller JH, Rasmussen P, Benson AB, et al. Maintenance etidronate in the prevention of malignancy-associated hypercalcemia. Arch Intern Med 1987;147:963-966. 39. Mautalen C, Gonzalez D, Blumenfeld EL, et al. Spontaneous fractures of uninvolved bones in patients with Paget's disease during unduly prolonged treatment with disodium etidronate. Clin Orthop 1986;207:150-155. 40. Coukell AJ, Markham A. Pamidronate: A review of its use in the management of osteolytic bone metastases, tumor-induced hypercalcaemia and Paget's disease of bone. Drugs Aging 1998;12: 149-168. 41. Nussbaum SR, Younger J, VandePol CJ, et al. Single dose intravenous therapy with pamidronate for the treatment of hypercalcemia of malignancy: Comparison of 30-, 60-, and 90 mg dosages. A m J Med 1993;95:297-304. 42. Mannix KA, Carmichael J, Harris AL, et al. Single high-dose (45 mg) infusion of aminohydroxypylidene diphosphonate for severe malignant hypercalcemia. Cancer 1989;64:1358-1361. 43. Ralston SH, Gallagher SJ, Patel U, et al. Comparison of three intravenous bisphosphonates in cancer-associated hypercalcaemia. Lancet 1989;2:1180-1182. 44. Gucalp R, Ritch P, Wiernik PH, et al. Comparative study of pamidronate disodium and etidronate disodium in the treatment of cancer-related hypercalcemia. J Clin Oncol 1992;10:134-142.

45. Jansson S, Tisell LE, Lindstedt G, et al. Disodium pamidronate in the preoperative treatment of hypercalcemia in patients with primary hyperparathyroidism. Surgery 1991;110:480-486. 46. Mark S. Hypercalcaemia in an immobilized patient with pneumonia. B r J Clin Pract 1995;49:327-329. 47. McIntyre, Cameron DE Urquhart SM, et al. Immobilization hypercalcaemia responding to intravenous pamidronate sodium therapy. Post Grad MedJ 1989;65:244-246. 48. Selby PL, Davies M, Marks JS, et al. Vitamin D intoxication causes hypercalcaemia by increased bone resorption which responds to pamidronate. Clin Endocrino11995;43:531-536. 49. Gibb cJ, Peacock M. Hypercalcaemia due to sacoidosis corrects with bisphosphonate treatment. Postgrad MedJ 1986;62:937-938. 50. Hortobagy GN, Theriault RL, Porter L, et al. Efficacy of pamidronate in reducing skeletal complications in patients with breast cancer and lytic bone metastases. N E n g l J Med 1996;335: 1785-1837. 51. Berenson JR, Lichtenstein A, Porter L, et al. Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. N E n g l J Med 1996;334:488-493. 52. Pecherstorfer M, Herrmann Z, Body JJ, et al. Randomized phase II trial comparing different doses of the bisphosphonate ibandronate in the treatment of hypercalcemia of malignancy. J Clin Oncol 1996;14:268-276. 53. Body JJ, Lortholary A, Romieu G, et al. A dose-finding study of zoledronate in hypercalcemic cancer patients. J Bone Miner Res 1999; 14:1557-1561. 54. Bounameaux HM, Schifferli J, Montani JP, et al. Renal failure associated intravenous diphosphonates. Lancet 1983;1:471 (letter). 55. Machado CE, Flombaum CD. Safety of pamidronate in patients with renal failure and hypercalcemia. Clin Nephro11996;45:175-179. 56. Yap AS, Hockings GI, Fleming SJ, et al. Use of aminohydroxypropylidene bisphophonate (AHPrBE "APD") for the treatment of hypercalcemia in patients with renal impairment. Clin Nephrol 1990;34:225-229. 57. Hamdy NAT, McCloskey EV, Brown CB, et al. Effects of clodronate in severe hyperparathyroid bone disease in chronic renal failure. Nephron 1990;56:6-12. 58. Hosking DJ, Gilson D. Comparison of the renal and skeletal actions of calcitonin in the treatment of severe hypercalcaemia of malignancy. Q J Med 1984;53:359-368. 59. Silva O, Becker KL. Salmon calcitonin in the treatment of hypercalcemia. Arch Intern Med 1973;132:337--339. 60. Binstock ML, Mundy GR. Effect of calcitonin and glucocorticoids in combination on the hypercalcemia of malignancy. Ann Intern Med 1980;93:269. 61. Warrell RP, Israel R, Frisone M, et al. Gallium nitrate for acute treatment of cancer-related hypercalcemia: A randomized, doubleblind comparison to calcitonin. Ann Intern Med 1988;108:669-674. 62. Ralston SH. Medical management of hypercalcaemia. B r J Clin Pharmacol 1992;34:11-20. 63. Thiebaud D, Jacquet E Burckhardt E Fast and effective treatment of malignant hypercalcemia. Arch Intern Med 1990;150:2125-2128. 64. Ralston SH, Alzaid AA, Gardner MD, Boyle IT. Treatment of cancer associated hypercalcemia with combined aminohydroxypropylidene diphosphonate and calcitonin. Br MedJ 1986;292:1549-1550. 65. Fatemi S, Singer FR, Rude RK. Effect of salmon calcitonin and etidronate on hypercalcemia of malignancy. Calcif Tissue Int 1992;50:107-109. 66. Wisnecki LA. Salmon calcitonin in the acute management of hypercalcemia. Calcif Tissue Int 1990;46(Suppl.):526-530. 67. Goodwin JS, Atluru D, Sierakowski S, et al. Mechanism of action of glucocorticosteroids: Inhibition of T cell proliferation and interleukin 2 production by hydrocortisone is reversed by leukotriene B4. J Clin Invest 1986;77:1244.

1VIANAGEMENT OF HYPERCALCEMIA 68. Breslau NA, McGuire JL, Zerwekh JE, et al. Hypercalcemia associated with increased serum calcitriol levels in three patients with lymphoma. Ann Intern Med 1984;100:1-7. 69. Sandler LM, Winearls CG, Fraher LJ, et al. Studies of the hypercalcemia of sarcoidosis: Effect of steroids and exogenous vitamin D, on the circulating concentrations of 1,25-dihydroxyvitamin D. Q J Med 1984;53:165-180. 70. Percival RC, Yates AJP, Gray RES, et al. The role of glucocorticoids in the management of malignant hypercalcemia. Br Med J 1984;289:287. 71. Bilezikian JP. Hypercalcemic states. In: Coe FL, Favus MJ, eds. Disorders of bone and mineral metabolism. New York:Raven, 1992: 1493-522. 72. Stewart AF. Therapy of malignancy-associated hypercalcemia. Am J M e d 1983;74:475. 73. Perlia CP, Gubisch NJ, Cootter J, Edelberg D, Dederick MM, Taylor SG. Mithramycin treatment of hypercalcemia. Cancer 1970;25:389. 74. Minkin C. Inhibition of parathyroid hormone stimulated bone resorption in vitro by the antibiotic mithramycin. Calcif Tissue Res 1973;13:249-257. 75. Kiang DT, Loken MK, Kennedy BJ. Mechanism of the hypocalcemic effect of mithramycin. J Clin Endocrinol Metab 1979;48:341. 76. Thurlimann B, Waldburger R, Senn HJ, et al. Plicamycin and pamidronate in symptomatic tumor-related hypercalcemia: A prospective randomized crossover trial. Ann Oncol 1992;3: 619-622. 77. Green L, Donehower RC. Hepatic toxicity of low doses of mithramycin in hypercalcemia. Cancer Treat Rep 1984;68: 1379-1381. 78. Slavik M, Carter SK. Chromomycin AZ, mithramycin and olivomycin: Antitumor antibiotics of related structure. Adv Pharm Chem 1975;12:1-15. 79. Warrell RP, Jr, Bockman RS, Coonley CJ, et al. Gallium nitrate inhibits calcium resorption from bone and is effective treatment for cancer-related hypercalcemia. J Clin Invest 1984;73: 1487-1490.

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80. Bockman RS, Boskey AL, Alcock N, et al. Gallium nitrate increases bone calcium and crystallite perfection of hydroxyapatite. Calcif Tissue Int 1986;39:376--381. 81. Warrell RP, Bockman RS. Gallium in the treatment of hypercalcemia and bone metastases. In: Important advances in oncology. Philadelphia:Lippincott, 1989:205-220. 82. Hall TJ, Chambers TJ. Gallium inhibits bone resorption by a direct action on osteoclasts. Bone Miner 1990;8:211-216. 83. Warrell RP, Alcock NW, Buckman RS. Gallium nitrate inhibits accelerated bone turnover in patients with bone metastases. J Clin Onco11987;5:292-298. 84. Warrell RP, Israel R. Gallium nitrate for acute treatment of cancer-related hypercalcemia. Ann Intern Med 1988;108: 669-674. 85. Warrell RP, Murphy WK, Schulman P, et al. A randomized doubleblind study of gallium nitrate compared with etidronate for acute control of cancer-related hypercalcemia. J Clin Oncol 1991;9: 1467-1475. 86. Shackney S, Hasson J. Precipitous fall in serum calcium, hypotension and acute renal failure after intravenous phosphate therapy for hypercalcemia. Ann Intern Med 1967;66:906-916. 87. Vernava AM, O'Neal LW, Palermo V. Lethal hyperparathyroid crisis: Hazards of phosphate administration. Surgery 1987;102: 942-948. 88. Carey RW, Schmitt GW, Kopald HH. Massive extraskeletal calcification during phosphate treatment of hypercalcemia. Arch Intern Med 1968;122:150-155. 89. Cardella CJ, Birkin BL, Rapoport A. Role of dialysis in the treatment of severe hypercalcemia: Report of two cases successfully treated with hemodialysis and review of the literature. Clin Nephro11979; 12:285-290. 90. Heyburn PJ, Selby PL, Peacock M, et al. Peritoneal dialysis in the management of severe hypercalcaemia. Br MedJ 1980;280:525-526. 91. Goodman WG, Coburn JW, Slatopolsky E, et al. Renal osteodystrophy in adults and children. Flavus MJ, ed. Primer of the metabolic bone diseases and disorders of mineral metabolism. New York:LippincottRaven, 1996:341-360.

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CHAPTER 4 6

Primary Hyperparathyroidism and Other Causes of Hypercalcemia in Children and Adolescents

EMILY L. GERMAIN-LEE AND MICHAEL A. LEVINE Division of Pediatric Endocrinology, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287

INTRODUCTION

the child, and degree of hypercalcemia. Children may be more adaptive to hypercalcemia c o m p a r e d to adults, and infants and children with mild hypercalcemia (11-13 m g / d l or 2.75-3.25 m m o l / l i t e r ) may not have any symptoms (8). With m o d e r a t e to severe hypercalcemia, symptoms such as anorexia, vomiting, and constipation (rarely diarrhea) may occur. If hypercalcemia is chronic, there may be failure to thrive, which in the infant may be the only physical sign. Dehydration can occur rapidly because of the small size of an infant or child, and renal complications such as nephrocalcinosis, nephrolithiasis, or h e m a t u r i a may be the earliest clinical manifestation of hypercalcemia. Hypertension and increased cardiovascular tone may develop, as well as heart block and shortening of the ST segment. The neurologic symptoms can range from drowsiness or irritability to confusion; in the extreme cases, stupor and coma can ensue. T r e a t m e n t is aimed at the specific etiology, and is often complicated by the n e e d to consider the impact of therapy on growth and development. For infants with mild hypercalcemia, a low-calcium diet or low-calcium formula for the infant may be all that is necessary or practical. If emergency intervention is necessary because of severe hypercalcemia, the usual methods of t r e a t m e n t of hypercalcemia in adults are also used in children, although large, controlled studies showing efficacy and safety are often lacking (9-11). In this chapter, we place primary hyperparathyroidism within the context of the other disorders that cause hypercalcemia in children. We distinguish the neonate and infant from the older child and adolescent because

Hypercalcemia is far less c o m m o n l y detected in children than in adults. This is due in part to the relative infrequency with which serum calcium levels are measured in otherwise well children, but also because of the lower incidence of malignancy and primary hyperparathyroidism in the young. O n the other hand, the failure of many laboratories to report age-adjusted normal values for serum total and ionized calcium concentrations may lead to overdiagnosis of true hypercalcemia, because the u p p e r limit of normal calcium levels is slightly higher in children c o m p a r e d to adults (Table 1) (1-7). Hypercalcemia in neonates and early infancy is defined as a total serum calcium concentration consistently greater than 11.3 m g / d l ; ages 1-4 years, greater than 10.8 m g / d l ; ages 6-12 years, greater than 10.3 m g / d l ; and thereafter equivalent to adult normal ranges. The mechanisms of hypercalcemia in children are similar to those that occur in adults, but the smaller size and relative immaturity of the skeleton and kidney make children particularly sensitive to factors that affect renal handling of calcium and bone remodeling. An increase in net calcium mobilization from the skeleton is most often the cause of hypercalcemia, although excess intestinal absorption of calcium can also lead to hypercalcemia. Regardless of the underlying pathophysiology, hypercalcemia will occur when excessive transport of calcium from the skeleton a n d / o r gut into the extracellular fluid exceeds the ability of the kidney to excrete the increased filtered load. The clinical features are d e p e n d e n t on the underlying disorder, age of The Parathyroids, Second Edition

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TABLE 1 Representative Normal Values for Concentrations of Serum Total Calcium a Group Infants Children Men

Women

Age (years)

Serum total calcium (mg/dl)

0-0.25 1-5 6-12 20 50 70 20 50 70

8.8-11.3 9.4-10.8 9.4-10.3 9.1-10.2 8.9-10.0 8.8- 9.9 8.8-10.0 8.8-10.0 8.8-10.0

aRevised from Portale, AA, Chapter 19, Blood Calcium, Phosphorus, and Magnesium, p. 116 in Primer on the

Metabolic Bone Diseases and Disorders of Mineral Metabolism, Fourth Edition, Editor-in-Chief: Favus, MJ, 1999, Lippincott, Williams, & Wilkins.

the differential diagnoses of hypercalcemia in children are age dependent. Because of the strong association of genetic defects or epigenetic factors during pregnancy with childhood hypercalcemia, a thorough evaluation of hypercalcemia in the neonate, infant, and older child will generally require evaluation of mineral metabolism status in the biologic parents and siblings. Hypercalcemia that is chronic or inadequately treated can lead to special problems in children, including defects in growth and intellectual development, which can have a profound impact on the rest of the child's life. Thus, failure to recognize hypercalcemia in infants and children can lead to significant morbidity, even mortality, and accurate definition of the etiology of the hypercalcemia is critical in determining the prognosis and treatment.

DIAGNOSIS OF HYPERCALCEMIA IN NEONATES AND INFANTS The differential diagnosis of hypercalcemia in neonates and infants up to age 2 years is listed in Table 2 (10,12). Hypercalcemia in the neonatal period or during infancy not only leads to significant morbidity, but can also be life-threatening (10,13). Marked dehydration can occur rapidly in infants who develop polyuria as a consequence of renal resistance to vasopressin. The direct vasoconstrictive effect of calcium can lead to hypertension. Severe hypercalcemia can affect the nervous system and cause lethargy and seizures. In addition, there can be damage to the kidneys from nephrocalcinosis (8,10,12). The urinary excretion of calcium in children differs significantly from that in adults (14). On a diet con-

TABLE 2 Differential Diagnosis of Hypercalcemia in Neonates and Infants (up to 2 Years of Age) latrogenic Phosphate depletion Premature infants on human milk or standard formula Parenteral nutrition Hyperparathyroidism Congenital parathyroid hyperplasia Maternal hypoparathyroidism Inactivating mutations in Ca2+-sensing receptor gene Familial hypocalciuric hypercalcemia (familial benign hypercalcemia) Neonatal severe hyperparathyroidism Jansen's metaphyseal chondrodysplasia Persistent PTHrP Hypervitaminosis D Subcutaneous fat necrosis Williams syndrome/idiopathic infantile hypercalcemia Other inborn metabolic disorders Blue diaper syndrome Lactase deficiency Bartter syndrome Hypophosphatasia IMAGe Down syndrome Severe congenital hypothyroidism Maternal hypercalcemia Vitamin A intoxication

taining approximately 1400 m g / d a y calcium, the average daily calcium output of a child u n d e r 4 years of age is approximately 25-50 mg/day, and for ages 5-14 approximately 75-100 mg/day. On a weight basis this corresponds to a urinary calcium excretion of 2-4 m g / k g / d a y . In infants and younger children it is usually very difficult to obtain a 24-hour urine collection. In these cases a random urine sample can be collected and the calcium:creatinine ratio is determined. These values are age d e p e n d e n t and decline gradually in the first several years of life (15). The u p p e r limits of normal for ratios that have been calculated from molar (gravitometric) concentrations of calcium and creatinine are as follows: less than 7 months, 2.42 (0.86); 7-18 months, 1.69 (0.60); 19 months to 6 years, 1.18 (0.42); and adults, 0.61 (0.22). In neonates, symptoms and signs of hypercalcemia are difficult to detect, and hypercalcemia is typically discovered when a chemistry panel is obtained to evaluate failure to thrive. Hypercalcemia is frequently secondary to iatrogenic causes, such as excessive calcium supplementation or use of extracorporeal membrane oxygenation in the critically ill infant. Phosphate deple-

HVeVgCaLCWMtAIN CHILDREN / tion can result from human milk feeding in preterm, very-low-birth weight infants (10,12,16). This can cause not only mild hypercalcemia, but may also lead to an unusual condition termed "breast milk-induced rickets of prematurity" (10,12,17). This has become rare since the introduction of breast milk fortifiers that contain 30-40 m g / k g / d a y of phosphorus as disodium phosphate (18). Feeding preterm infants a regular-term infant formula also causes hypophosphatemia. Finally, a common cause of phosphate depletion in the hospitalized neonate is from inappropriately supplemented parenteral nutrition (19). Phosphate deficiency activates bone resorption and impairs bone formation. In addition, hypophosphatemia stimulates synthesis of 1,25-dihydroxyvitamin D [ 1,25 (OH) 2o ], which increases intestinal absorption of calcium.

NEONATAL HYPERPARATHYROIDISM Neonatal hyperparathyroidism is quite rare. It is usually due to parathyroid chief cell hyperplasia rather than to parathyroid adenoma as in older patients (20). Hyperparathyroidism may be sporadic or inherited, and both autosomal dominant and autosomal recessive patterns of transmission have been reported (21,22). Infants with this condition have very high serum levels of parathyroid hormone (PTH) and calcium along with low serum levels of phosphate and either normal or elevated alkaline phosphatase levels. At birth there may be evidence of severe bone deformities secondary to inadequate mineralization, as well as multiple fractures. Respiratory difficulties may arise if the rib cage is affected. There may be destructive lesions of metaphyseal ends of long bones as well as poor mineralization of the lateral ends of the clavicles. Hepatosplenomegaly and anemia may be present. The conventional treatment of neonatal hyperparathyroidism is subtotal parathyroidectomy, and failure to normalize the serum calcium level can have profound developmental implications (23). Infants with neonatal hyperparathyroidism secondary to maternal hypocalcemia (most commonly from maternal hypoparathyroidism) usually are not as hypercalcemic as those infants with primary hyperparathyroidism. In fact, only 25% of the cases have hypercalcemia (24). Transient neonatal hyperparathyroidism has also been reported in association with maternal pseudohypoparathyroidism (25) as well as maternal renal tubular acidosis (26,27). Treatment of secondary or adaptive neonatal hyperparathyroidism usually consists of no more than providing an appropriate calcium and phosphorus supply in the milk. Hyperparathyroidism typically resolves within a few weeks.

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The most common cause of neonatal hyperparathyroidism is related to genetic defects that cause familial hypocalciuric hypercalcemia (FHH) [also termed familial benign hypercalcemia (FBH)], an autosomal dominant trait characterized by moderate hypercalcemia and relative hypocalciuria (see Chapter 38). There is virtually 100% penetrance for hypercalcemia among heterozygotes for the FHH gene, and hypercalcemia can occur as early as the first week of life. By contrast, other autosomal dominant syndromes associated with parathyroid hyperplasia, such as multiple endocrine neoplasia (MEN) type 1 or MEN type 2 (see Chapters 35 and 36), are unlikely to cause neonatal hypercalcemia because of the later age of onset of hyperparathyroidism in these disorders. Inactivating mutations in the CaZ+-sensing receptor gene, localized to chromosome 3q, can lead to both neonatal severe hyperparathyroidism (NSHPT) and FHH (28-34). In many families, NSHPT and FHH are the respective homozygous and heterozygous manifestations of the same genetic defect (28,30,32,33). NSHPT can also result from heterozygous offspring born to affected fathers but unaffected normocalcemic mothers, or in neonates with an apparent de n o v o heterozygous muta2+ tion in the Ca -sensing receptor gene (35). Mutations that inactivate the CaZ+-sensing receptor in patients with NSHPT and FHH have been found scattered throughout the gene, and most families have private mutations. Genetic testing is now available, but requires molecular analysis of the entire gene. Loss of 50% of CaZ+-sensing receptors decreases the sensitivity of the parathyroid cells to extracellular Ca 2+, and leads to mild parathyroid hyperplasia and elevated circulating levels of PTH. Decreased receptor activity in the kidney is thought to account for relative hypocalciuria, the hallmark of the disorder. Although FHH and NSHPT have been linked to the CaZ+-sensing receptor gene on 3q in nearly all families, the disorder has been also linked to the long (36) and short (37) arms of chromosome 19, suggesting genetic heterogeneity for this disorder. In NSHPT, the PTH is quite high and the associated hypercalcemia is severe enough to be life-threatening. In addition, affected infants have hypophosphatemia and osteopenia at birth (34) and may die within a few days if the hypercalcemia is not treated aggressively (38). Children who survive NSPHT but who remain hypercalcemic are at risk of significant impairment in cognitive development (23). Jansen's metaphyseal chondrodysplasia resembles primary hyperparathyroidism in many respects, but levels of circulating PTH are suppressed. This unusual syndrome is caused by heterozygous mutations in the P T H / P T H r P receptor (39), found in the kidney, bone, and growth plate, that lead to constitutive (i.e., ligand independent) activation of the receptor

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(see Chapter 44). The activation of P T H / P T H r P receptor signaling increases bone resorption, leading to hypercalcemia, and impairs chondrocyte differentiation, leading to growth plate defects that cause postnatal short-limbed dwarfism. The skeletal abnormalities are seen on radiologic evaluation from the time of birth and include irregularities of the metaphyses of the long bones and rachitic changes. However, the physical appearance of infants with Jansen's syndrome is normal, including body length. During late childhood, however, the characteristic features gradually appear, including hypertelorism, mandibular hypoplasia, and short-limbed dwarfism. The pathophysiology of hypercalcemia in Jansen's syndrome shares many features with primary hyperparathyroidism (40), but the suppressed serum PTH levels, metaphyseal defects, and growth delay are conclusive distinguishing characteristics. Persistent expression of PTHrP has been reported as a cause of neonatal hypercalcemia (38,41), but description of additional cases will be required to confirm this pathophysiology.

common clinical sign associated with subcutaneous fat necrosis, which is associated with a surprisingly high 15% mortality (44). Williams syndrome is a sporadic disorder associated with hypercalcemia in approximately 15 % of cases. The hypercalcemia typically occurs during infancy and resolves between 2 and 4 years of age (47,48). There are cases, however, of older children and adults who have persistent hypercalcemia (49). There may also be associated nephrocalcinosis and soft-tissue calcifications. Children with Williams syndrome have a characteristic appearance at or soon after birth, which first suggested that there was a problem during intrauterine development (8). The physical features consist of an "elfin" facies (secondary to poor development of the facial bones) with epicanthal folds, hypertelorism, strabismus, bitemporal depressions, periorbital prominence, full cheeks, prominent nasal tip, long philtrum, prominent lips and mouth, and dolicocephaly (Fig. 1). They also have clinodactyly of the fifth fingers and hypoplas-

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N O N P A R A T H Y R O I D CAUSES OF HYPERCALCEMIA Neonates whose mothers ingested excessive amounts of vitamin D a n d / o r its derivatives during pregnancy can develop hypercalcemia (8,25). This usually occurs during treatment of the mother for a hypocalcemic disorder, but can occur by self-medication. Rarely, an infant will be given excessive vitamin supplements over a long period of time that leads to hypervitaminosis D. Approximately 2000 units/kg body weight per day as vitamin O 2 o r vitamin D~ over a period of many months will cause hypercalcemia and hypercalciuria in most patients, and 20,000-40,000 units per day has led to fatal hypercalcemia in infants (8). The earliest evidence of vitamin D intoxication may be the development of renal complications such as polyuria, hematuria, or nephrocalcinosis. Subcutaneous fat necrosis is common in neonates with a complicated delivery and may lead to hypercalcemia within days or weeks of birth. Hypercalcemia results from excess circulating 1,25(OH)zD that is produced by macrophages present within the granulomatous reaction to the necrotic fat. The hypercalcemia is also compounded by calcium release from fat tissues and increased prostaglandin E activity (42-44). The macrophages express ectopic 25 (OH) D~-l-0t-hydroxylase activity that is not regulated by PTH, calcium, phosphorus, or 1,25(OH)zD but that is responsive to glucocorticoids (45,46). Subcutaneous fat necrosis is found in areas of direct trauma that occur during a difficult birth process, such as with forceps or vacuum extraction. These infants often have a history of birth asphyxia as well. Failure to thrive is the most

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FIG. 1 Williams syndrome. An 8-year-old boy with characteristic facial features including epicanthal folds, hypertelorism, bitemporal depressions, periorbital prominence, full cheeks, prominent lips and mouth, and long nasal philtrum. Photograph from Aarskog D and Harrison H, Chapter 17, Disorders of Calcium, Phosphate, PTH, and Vitamin D, p. 1087 in Wilkins' the diagnosis and treatment of endocrine disorders in childhood and adolescence, Fourth edition, Editors: Kappy M, Blizzard RM, and Migeon CJ; 1994, Charles C. Thomas.

HYPERCALCEMIA IN CHILDREN

tic nails. They may have kyphoscoliosis, pecmm excavatum, and an abnormal gait. There are associated cardiac abnormalities, the most typical of which is supravalvular aortic stenosis (30% of affected patients), as well as valvular aortic stenosis, pulmonic stenosis, atrial septal defect, and ventricular septal defect. Many children with Williams syndrome grow poorly from very early in infancy and have general developmental delay. Interestingly, they are quite sociable ("cocktail personality") and do not appear delayed in linguistic abilities (50-53). Williams syndrome has been associated with loss of genetic material at 7q11.13, and likely represents a contiguous gene deletion that typically includes the elastin gene (ELN) (54-56). Hemizygosity of the E L N gene likely accounts for the associated cardiac defects, but cannot explain the hypercalcemia or phenotypic features (57,58). Williams syndrome has also been associated with other chromosomal abnormalities, including an interstitial deletion of chromosome 6(q22.2q23) (59), a terminal deletion of chromosome 4146,XX,del(4)(q33)] (60), as well as chromosomal translocations (61,62). However, the definitive basis remains unknown (63). In most cases, fluorescent in situ hybridization (FISH) using E L N is diagnostic (55,64). Patients with this syndrome have an exaggerated response to pharmacologic doses of vitamin D 2 (65) and a blunted calcitonin response to calcium loading (50). Elevated plasma concentrations of 1,25(OH)zD have been reported in some patients despite circulating levels of PTH that are low or normal (66,67). However, studies have failed to show any consistent abnormality in the metabolism of vitamin D that might explain these features. Some children with hypercalcemia show similar disturbances in vitamin D sensitivity but lack other phenotype features of Williams syndrome and do not have a 7q11.13 deletion. This condition, which may be familial, has been termed idiopathic infantile hypercalcemia (IIH) (68,69). The hypercalcemia in IIH usually resolves within the first few years of life, but persistent hypercalciuria is common. Clinical evaluation and genetic testing provide the ability to differentiate between Williams syndrome and IIH in more than 90% of cases.

I N B O R N ERRORS OF METABOLISM THAT CAUSE HYPERCALCEMIA Many inborn disorders of metabolism are associated with hypercalcemia. Blue diaper syndrome is caused by a defect in tryptophan metabolism (70). The block in tryptophan metabolism leads to urinary excretion of excessive amounts of indole derivatives, including a derivative called "indican" that gives the urine-soaked diaper a blue tint. The mechanism of hypercalcemia in this disorder is unknown.

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Congenital lactase deficiency can cause hypercalcemia during the first few months of life. Seven out of ten infants evaluated with this condition had hypercalcemia, and five of the seven had medullary nephrocalcinosis. The hypercalcemia resolved after initiation of a lactose-free diet, but later in childhood (ages 2 to 10 years) one of the patients still had hypercalciuria and three of the patients had nephrocalcinosis (71). The etiology of the hypercalcemia is unclear, but is thought to be related to metabolic acidosis a n d / o r an increase in intestinal calcium absorption secondary to increased gut lactose (71). Bartter syndrome, due to homozygous inactivation in the gene for either the furosemide-sensitive Na/K/2C1 cotransporter NKCC2 (SLC12A1) or the inwardly rectifying potassium channel ROMK (KCNJ1) (72,73), is a rare cause of neonatal hypercalcemia, but is more commonly associated with hypercalciuria (74,75). The neonate typically presents with vomiting, diarrhea, fever, and resultant failure to thrive. The mothers of these infants are often found to have intrauterine polyhydramnios, thought secondary to fetal polyuria, and deliver prematurely. Hypophosphatasia, an inherited condition that results from deficient bone alkaline phosphatase activity, can also cause hypercalcemia (76-80). Hypophosphatasia is classified into four forms: perinatal hypophosphatasia is the most severe form, and can be lethal in utero or shortly after birth because of inadequate thorax and skull formation. Infants live for a few days at most. Infantile hypophosphatasia presents before age 6 months and can cause pronounced hypercalcemia. Calcium is deposited inadequately into bone, which leads to hypercalcemia with hypercalciuria and nephrocalcinosis. Severe rickets ensues. The diagnosis can be confirmed by finding a very high level of phosphoethanolamine in the urine as well as radiographic evidence of severe bone demineralization and endochondral ossification defects. Although there is no known treatment for this condition, one patient improved after serum transfusions, presumably secondary to a circulating factor that activates alkaline phosphatase at the posttranscriptional level (80). The IMAGe syndrome consists of intrauterine growth retardation, metaphyseal dysplasia, and adrenal hypoplasia congenita (81). The three patients reported with this syndrome also had hypercalcemia a n d / o r hypercalciuria that led to nephrocalcinosis in one patient and prenatal liver and spleen calcifications in another. DAX1 gene mutations, which are associated with congenital adrenal hypoplasia, were not found in these three patients, however. Neither the molecular defect nor the basis for hypercalcemia in this newly recognized syndrome has been identified. There are several reports of hypercalcemia, hypercalciuria, and nephrocalcinosis in infants and toddlers with

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Down syndrome (81a-d). The etiology of the hypercalcemia was initially thought to be secondary to overingestion of cow's milk. However, this has not been found in all cases. The hypercalcemia is now thought to be specifically associated with the genetic defect(s) of Down syndrome, although the mechanism remains unclear.

O T H E R CAUSES OF H Y P E R ~ C E M I A THE NEONATE AND INFANT

IN

Severe congenital hypothyroidism (athyreotic cretinism) is rarely seen in the United States because of the initiation of newborn screening programs. The hypercalcemia in severe congenital hypothyroidism is associated with hypercalciuria, and renal calculus formation has been described. Radiologic studies do not reveal skeletal demineralization, which suggests that the hypercalcemia is secondary to hyperabsorption of calcium. The growth failure of these infants may contribute to the hypercalcemia as well because there is little skeletal uptake of bone mineral. Maternal hypercalcemia can result in transient neonatal hypercalcemia. Unusual causes of maternal hypercalcemia include medications such as lithium or thiazide diuretics as well excessive use of calcium supplements, thyroid hormone, or vitamins D or A. Vitamin A intoxication is usually the result of vitamin supplementation but can also arise from the ingestion of fish oils with a high concentration of vitamin A (82). There have been cases of vitamin A intoxication with resulting hypercalcemia in infants fed chicken liver (83).

HYPERCALCEMIA IN OLDER CHILDREN The differential diagnosis of hypercalcemia in children and adolescents (Table 3) is similar to, but broader than, the differential diagnosis for infants. Because children and adolescents are growing rapidly, the first sign of the hypercalcemia may be poor weight gain a n d / o r poor linear growth. Children with medical conditions requiring calcium supplementation may have excessive calcium intake. Phosphate depletion can occur in any child with an acute or chronic illness and can occasionally lead to hypercalcemia. Children on parenteral nutrition are also at a greater risk for develo p m e n t of hypophosphatemia.

HYPERPARATHYROIDISM IN CHILDREN AND ADOLESCENTS Primary hyperparathyroidism is usually acquired in the child or adolescent, and is nearly always due to a

TABLE 3 Hypercalcemia in Children (over 2 Years of Age) and Adolescents Excessive calcium intake Phosphate depletion Parenteral nutrition Hyperparathyroidism Aquired primary Adenoma Hyperplasia Genetic primary Autosomal dominant/recessive Familial MEN types 1 and 2A (2B) Familial hypocalciuric hypercalcemia Autonomous (tertiary) Hypervitaminosis D Excessive intake Granulomatous diseases: cat scratch fever, sarcoidosis, tuberculosis, histoplasmosis, coccidiomycosis, leprosy Chronic inflammatory disorders Williams syndrome/idiopathic infantile hypercalcemia Immobilization Malignancy-associated hypercalcemia Primary bone tumors Metastatic tumors with osteolysis Tumors secreting PTHrP, prostaglandins, cytokines, growth factors Hepatic disease Hyperthyroidism Adrenal insufficiency Pheochromocytoma Vasoactive intestinal polypeptide-secreting tumor Drugs (thiazides, lithium, systemic retinoid derivatives, theophylline, acetosalicylic acid) Milk-alkali syndrome/calcium gluttony

single a d e n o m a of the parathyroid glands (see Chapter 20). The age range is from 3 to 15 years with a mean of 12.8 years and an equal sex incidence (20,50). Primary hyperparathyroidism is far less c o m m o n in children and adolescents than in adults. From a review of several studies, only 7 out of 514 patients with primary hyperparathyroidism were less than 19 years old (84). Primary hyperparathyroidism may also be genetic, and is often the presenting manifestation of MEN type 1 and less commonly MEN type 2 (see Chapters 35 and 36). Older children with asymptomatic hypercalcemia may have FHH. Hypercalcemia can develop in patients with chronic renal failure who have developed autonomous (so-called tertiary) hyperparathyroidism. This can also occur in children with hypophosphatemic rickets who have been treated with phosphate supplements and inadequate calcitriol (85).

HYPERCALCEMIA IN CHILDREN /

N O N P A R A T H Y R O I D CAUSES OF HYPERCALCEMIA IN O L D E R CHILDREN AND ADOLESCENTS Hypervitaminosis D can result from ingestion of excessive amounts of vitamin D (or its metabolities) for medical conditions such as hypoparathyroidism and rickets, or for nutritional supplementation. Granulomatous disease is associated with ectopic expression of 25(OH) D~-l-ot-hydroxylase activity in activated macrophages (see above, neonatal subcutaneous fat necrosis) (46,86). Infectious diseases such as cat scratch fever (87) as well as histoplasmosis, coccidiomycosis, leprosy, and tuberculosis have all been associated with hypercalcemia in children. Tuberculosis is showing a resurgence in the United States and is especially important to consider in children who live in or emigrate from countries with higher incidences. The usual source of infection is someone close to the children in their home, but there have been recent outbreaks of childhood tuberculosis in schools (nursery, elementary, and secondary), day care centers, school buses, and sports teams. Sarcoidosis occasionally has its onset in childhood, although hypercalcemia is very unusual in the younger age groups (50). Chronic inflammatory diseases such as collagen vascular diseases, including systemic lupus erythematosus and rheumatoid arthritis, can lead to hypercalcemia, as can the h u m a n immunodeficiency virus.

IMMOBILIZATION Immobilization is a very important and c o m m o n cause of hypercalcemia in children and adolescents. Immobilization of a rapidly growing child will lead to a marked decrease in osteoblastic bone formation and a dramatic increase in osteoclastic bone resorption. This imbalance in bone remodeling causes increased movement of calcium (and phosphorous) out of the skeleton with a consequent net loss of bone mass that is termed disuse osteoporosis (88). Hypercalciuria can develop within a few days of immobilization, and hypercalcemia may follow within 1 to 3 weeks. The most typical scenario is the active adolescent boy who has suffered a femur fracture. The sudden transition from an active physical life to complete immobilization, especially if both extremities are immobilized, can cause severe hypercalcemia. In one study, 6 of 12 children who were immobilized following fracture of a single weight-bearing bone developed hypercalcemia (89). Infants and children who have any disorder causing limited mobility, especially those who are wheelchairb o u n d or bedridden, are at high risk for developing immobilization hypercalcemia. As continued improve-

749

ments in chronic care now make long-term survival possible for more children with spinal cord injuries or severe head trauma, neuromuscular disorders, cerebral palsy, and spina bifida, it is likely that immobilizationinduced skeletal problems will be more easily recognized. For now, however, it is all too c o m m o n for hypercalcemia to be overlooked in an immobilized child. The child's anorexia, nausea, weight loss, lethargy, and depression might be attributed to hospitalization and immobilization rather than hypercalcemia. The recent demonstration that bisphosphonates can rapidly reverse the hypercalcemia and hypercalciuria of immobilization provides additional justification to monitor young patients for the development of these complications (90,91).

MALIGNANCY-ASSOCIATED HYPERCALCEMIA Malignancy associated hypercalcemia occurs in less than 1% of children with cancer (92). Hypercalcemia has been associated with many kinds of cancer in children, including leukemia, lymphoma, myeloma, neuroblastoma, hepatocellular carcinoma, hepatoblastoma, rhabdomyosarcoma, and brain and ovarian tumors (92-99). There are two mechanisms that account for the development of hypercalcemia with nonparathyroid tumors: (1) direct invasion of the skeleton by tumor cells and (2) tumor secretion of humoral factors such as PTHrP (see Chapter 42), prostaglandins, interleukin-1 and interleukin-6, transforming growth factor-e¢, tumor necrosis factor, or calcitriol, which activate osteoclastic activity.

O T H E R CAUSES OF HYPERC&LCEMIA Liver diseases such as hepatitis and hepatic failure can cause hypercalcemia in children (100-102). Although this can occur in the infant, it is more comm o n in the older child and adolescent. The basis of the hypercalcemia in these conditions is not clear. Endocrine disorders, including hyperthyroidism (50), adrenal insufficiency, pheochromocytomas, and vasoactive intestinal polypeptide-secreting tumors, have also been d o c u m e n t e d to cause hypercalcemia in children, but are more c o m m o n in adults (see Chapter 41). A variety of drugs other than calcium and vitamin D (and its metabolites) can cause hypercalcemia in older children. Most importantly, systemic retinoic acid derivatives used to treat acne in the adolescent can lead to vitamin A intoxication and hypercalcemia. Theophylline and acetylsalicylic acid can also raise serum calcium levels (46,103). Milk-alkali syndrome and calcium gluttony, most commonly due to the

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excessive intake of calcium-containing antacids or calcium supplements, can lead to hypercalcemia, hypercalciuria, and systemic alkalosis.

TREATMENT The principles of treatment of hypercalcemia in children are similar to those used in the m a n a g e m e n t of adults. Parathyroid surgery, the primary treatment modality for primary hyperparathyroidism in adults, is also the preferred treatment for symptomatic primary hyperparathyroidism in infants and young children. Indeed, the younger age of pediatric patients provides an even more compelling justification for recommending surgery for most patients. Of course, hypercalcemic children with FHH will rarely require any intervention unless they have neonatal severe hyperparathyroidism, in which event urgent parathyroidectomy may be necessary. Due to the low frequency of hypercalcemia in children, comprehensive clinical trials on the safety and efficacy of new medical treatments such as the bisphosphonates are lacking, although recent small studies have reported promising results (9,11,91,102,104,105). Medical therapy of hypercalcemia in children requires special consideration of the long-term effects of many of these agents (such as glucocorticoids and bisphosphonates) on growth and development of the skeleton, as well as on other organ systems. Calcitonin tends to be used more frequently in children because it has no long-term sequelae, whereas steroids are used less frequently than in adults because they result in poor linear growth and osteoporosis. Children with Williams syndrome or idiopathic infantile hypercalcemia have mildly elevated serum levels of 1,25-dihydroxyvitamin D, and a low-calcium formula in the infant or reducedcalcium diet in the older child may be all that is needed to treat the hypercalcemia a n d / o r hypercalciuria, particularly when long-term treatment will be necessary. CalciloXD (Ross Laboratories, North Chicago, IL), a low-calcium infant formula without vitamin D, is commonly used. As the hypercalcemia improves, the CalciloXD can be gradually mixed with regular formula or breast milk. The infants and children on the lowcalcium diet need to be followed closely, however, for the possible development of hypocalcemia and rickets. In children, unlike adults, growth is a very valuable clinical parameter to monitor for efficacy of treatment.

SUMMARY Primary hyperparathyroidism and other causes of hypercalcemia occur far less commonly in children than in adults. Hypercalcemia can have a subtle clinical

presentation, with failure to thrive as the only sign. The etiology of hypercalcemia in children is age d e p e n d e n t and includes a broad differential diagnosis. Although these conditions are not common, it is nevertheless important not to overlook them, as untreated hypercalcemia can have a p r o f o u n d impact on a child's growth and development.

ACKNOWLEDGMENTS This work has been supported in part by grants from the National Institutes of Health (DK-34281 and DK56178 and GCRC M01-RR00052).

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50. Aarskog D, Harrison HE. Disorders of calcium, phosphate, PTH and vitamin D. In: Kappy MS, Blizzard RM, Migeon CJ, eds. Wilkins' the diagnosis and treatment of endocrine disorders in childhood and adolescence. Springfield, Illinois:Thomas, 1994:1068-1069. 51. Dilts CV, Morris CA, Leonard CO. Hypothesis for development of a behavioral phenotype in Williams syndrome. AmJMed Genet Suppl 1990;6:126-131. 52. Jones W, Bellugi U, Lai Z, Chiles M, ReillyJ, Lincoln A, Adolphs R. Ii. Hypersociability in Williams Syndrome. J Cogn Neurosci 2000;12(Suppl. 1):30-46. 53. Morris CA, Carey JC. Three diagnostic signs in Williams syndrome. A m J Med Genet Supp11990;6:100-101. 54. Ewart AK, Morris CA, Atkinson D, Jin W, Sternes K, Spallone P, Stock AD, Leppert M, Keating MT. Hemizygosity at the elastin locus in a developmental disorder, Williams syndrome. Nat Genet 1993;5:11-16. 55. Lowery MC, Morris CA, Ewart A, Brothman LJ, Zhu XL, Leonard CO, CareyJC, Keating M, Brothman AR. Strong correlation of elastin deletions, detected by FISH, with Williams syndrome: Evaluation of 235 patients. Am J Hum Genet 1995;57:49-53. 56. Meng X, Lu X, Li Z, Green ED, Massa H, Trask BJ, Morris CA, Keating MT. Complete physical map of the common deletion region in Williams syndrome and identification and characterization of three novel genes. Hum Genet 1998;103: 590-599. 57. Urban Z, Peyrol S, Plauchu H, Zabot MT, Lebwohl M, Schilling K, Green M, Boyd CD, Csiszar K. Elastin gene deletions in Williams syndrome patients result in altered deposition of elastic fibers in skin and a subclinical dermal phenotype. Pediatr Dermato12000; 17:12-20. 58. Zhang J, Kumar A, Roux K, Williams CA, Wallace MR. Elastin region deletions in Williams syndrome. Genet Test 1999;3: 357-359. 59. Bzduch V, Lukacova M. Interstitial deletion of the long arm of chromosome 6(q22.2q23) in a boy with phenotypic features of Williams syndrome [letter]. Clin. Genet. 1989;35:230-231. 60. Jefferson RD, Burn J, Gaunt KL, Hunter S, Davison EV. A terminal deletion of the long arm of chromosome 4 [46,XX,del(4) (q33) ] in an infant with phenotypic features of Williams syndrome. J Med Genet 1986;23:474-477. 61. Telvi L, Pinard JM, Ion R, Sinet PM, Nicole A, Feingold J, Dulac O, Pompidou A, Ponsot G. De novo t(X;21)(q28;ql 1) in a girl with phenotypic features of Williams-Beuren syndrome. J Med Genet 1992;29:747-749. 62. von Dadelszen P, Chitayat D, Winsor EJ, Cohen H, MacDonald C, Taylor G, Rose T, Hornberger LK. De novo 46,XX,t(6;7) (q27;qll;23) associated with severe cardiovascular manifestations characteristic of supravalvular aortic stenosis and Williams syndrome. Am J Med Genet 2000;90:270-275. 63. Peoples R, Franke Y, Wang YK, Perez-Jurado L, Paperna T, Cisco M, Francke U. A physical map, including a BAC/PAC clone contig, of the Williams-Beuren syndrome---deletion region at 7ql 1.23. A m J H u m Genet 2000;66:47-68. 64. Dewan K, Borgaonkar DS, Bartoshesky LE, Tuttle D. Micro-deletion detected by fluorescent in situ hybridization for Williams syndrome. Del MedJ 1999;71:467-469. 65. Taylor AB, Stern PH, Bell NH. Abnormal regulation of circulating 25-hydroxyvitamin D in the Williams syndrome. NEnglJMed 1982;306:972-975. 66. Garabedian M, Jacqz E, Guillozo H, Grimberg R, Guillot M, Gagnadoux ME Broyer M, Lenoir G, Balsan S. Elevated plasma 1,25-dihydroxyvitamin D concentrations in infants with hypercalcemia and an elfin facies. N Engl J Med 1985; 312:948-952.

67. KnudtzonJ, Aksnes L, Akslen LA, Aarskog D. Elevated 1,25-dihydroxyvitamin D and normocalcaemia in presumed familial Williams syndrome. Clin Genet 1987;32:369-374. 68. Martin ND, Snodgrass GJ, Cohen RD, Porteous CE, Coldwell RD, Trafford DJ, Makin HL. Vitamin D metabolites in idiopathic infantile hypercalcaemia. Arch Dis Child 1985;60:1140-1143. 69. McTaggart SJ, Craig J, MacMillan J, Burke JR. Familial occurrence of idiopathic infantile hypercalcemia. Pediatr Nephrol 1999;13:668-671. 70. Drummond KN, Michael AF, Ulstrom RA, Good RA. The blue diaper syndrome: Familial hypercalcemia with nephrocalcinosis and indicanuria. Am J Med 1964;37:928-948. 71. Saarela T, Simila S, Koivisto M. Hypercalcemia and nephrocalcinosis in patients with congenital lactase deficiency. J Pediatr 1995;127:920-923. 72. Amirlak I, Dawson KP. Bartter syndrome: An overview. QJMed 2000;93:207-215. 73. Bettinelli A, Ciarmatori S, Cesareo L, Tedeschi S, Ruffa G, Appiani AC, Rosini A, Grumieri G, Mercuri B, Sacco M. et al. Phenotypic variability in Bartter syndrome type I. Pediatr Nephrol 2000; 14:940-945. 74. Seyberth HW, Rascher W, Schweer H, Kuhl PG, Mehls O, Scharer K. Congenital hypokalemia with hypercalciuria in preterm infants: A hyperprostaglandinuric tubular syndrome different from Bartter syndrome. JPediatr 1985; 107:694-701. 75. Shoemaker L, Welch TR, Bergstrom W, Abrams SA, Yergey AL, Vieira N. Calcium kinetics in the hyperprostaglandin E syndrome. Pediatr Res 1993;33:92-96. 76. Fedde KN, Blair L, SilversteinJ, Coburn SP, Ryan LM, Weinstein RS, Waymire K, Narisawa S, Millan JL, MacGregor GR, et al. Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. J Bone Miner Res 1999;14:2015-2026. 77. Mornet E. Hypophosphatasia: The mutations in the tissuenonspecific alkaline phosphatase gene. Hum Mutat. 2000;15: 309-315. 78. Mochizuki H, Saito M, Michigami T, Ohashi H, Koda N, Yamaguchi S, Ozono K. Severe hypercalcaemia and respiratory insufficiency associated with infantile hypophosphatasia caused by two novel mutations of the tissue-nonspecific alkaline phosphatase gene. EurJ Pediatr 2000;159:375-379. 79. Teree TM, Klein LR. Hypophosphatasia: Clinical and metabolic studies. J Pediatr 1968; 72:41-50. 80. Whyte ME Magill HL, Fallon MD, Herrod HG. Infantile hypophosphatasia: Normalization of circulating bone alkaline phosphatase activity followed by skeletal remineralization. Evidence for an intact structural gene for tissue nonspecific alkaline phosphatase. J Pediatr 1986;108:82-88. 81. Vilain E, Le Merrer M, Lecointre C, Desangles E Kay MA, Maroteaux P, McCabe ER. IMAGe, a new clinical association of intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies. J Clin Endocrinol Metab 1999;84:4335-4340. 81a. Cobefias C, Spizzirri F, Zanetta D. Another toddler with Down Syndrome, nephrocalcinosis, hypercalcemia, and hypercalciuria. Pediatr Nephrol 1998;12:432. 8lb. Manz E A toddler with Down Syndrome, hypercalcaemia, hypercalciuria, medullary nephrocalcinosis and renal failure. Pediatr Nephrol 1996;10:251. 81c. Andreoli SP, Revkees S, Bull M. Hypercalcemia, hypercalciuria, medullary nephrocalcinosis and renal insufficiency in a toddler with Down syndrome. Pediatr Nephro11995;9:673. 81d. Proesmans W, DeCock P, Eyskens B. A toddler with Down Syndrome, hypercalcaemia, hypercalciuria, medullary nephrocalcinosis and renal failure. Pediatr Nephro11995;9:112-114.

HYVERCALCE~IA IN CHILDREN 82. Doireau V, Macher MA, Brun P, Bernard O, Loirat C. Vitamin A poisoning revealed by hypercalcemia in a child with kidney failure. Arch Pediatr 1996;3:888-890. 83. Mahoney CP, Margolis MT, Knauss TA, Labbe RF. Chronic vitamin A intoxication in infants fed chicken liver. Pediatrics 1980;65:893-897. 84. Mundy GR. Primary hyperparathyroidism. In: Mundy GR, ed. Calcium homeostasis: hypercalcemia and hypocalcemia. London: Martin Dunitz, 1990;137-167. 85. Rivkees SA, Hajj-Fuleihan G, Brown EM, Crawford JD. Tertiary hyperparathyroidism during high phosphate therapy of familial hypophosphatemic rickets. J Clin Endocrinol Metab 1992;75: 1514-1518. 86. Fuss M, Pepersack T, Gillet C, Karmali R, Corvilain J. Calcium and vitamin D metabolism in granulomatous diseases. Clin Rheumatol 1992;11:28-36. 87. Bosch X. Hypercalcemia due to endogenous overproduction of active vitamin D in identical twins with cat-scratch disease. JAMA 1998;279:532-534. 88. Stewart AF, Adler M, Byers CM, Segre GV, Broadus AE. Calcium homeostasis in immobilization: An example of resorptive hypercalciuria. N EnglJ Med 1982;306:1136-1140. 89. Rosen JF, Wolin DA, Finberg L. Immobilization hypercalcemia after single limb fractures in children and adolescents. AmJDis Child 1978;132:560-564. 90. Kedlaya D, Brandstater ME, Lee JK. Immobilization hypercalcemia in incomplete paraplegia: Successful treatment with pamidronate. Arch Phys Med Rehabi11998;79:222-225. 91. Massagli TL, Cardenas DD. Immobilization hypercalcemia treatment with pamidronate disodium after spinal cord injury. Arch Phys Med Rehabi11999;80:998-1000. 92. McKay C, Furman WL. Hypercalcemia complicating childhood malignancies. Cancer 1993;72:256-260. 93. Arase Y, Endo Y, Hara M, Kumada H, Ikeda K, Yoshiba A. Hepatic squamous cell carcinoma with hypercalcemia in liver cirrhosis. Acta PatholJpn 1988;38:643-650.

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94. Florell SR, Bruggers CS, Matlak M, Young RH, Lowichik A. Ovarian small cell carcinoma of the hypercalcemic type in a 14 month old: The youngest reported case. Med Pediatr Oncol 1999;32:304-307. 95. Giebel SC, Stanhope CR, Malkasian GD,Jr, Schray ME Heath III H, Gaffey TA. Humoral hypercalcemia associated with a dysgerminoma. Mayo Clin Proc 1992;67:966-968. 96. Leone N, Debernardi-Venon W, Marzano A, Massari M, Rizzetto M. Hypercalcaemia secondary to hepatocellular carcinoma. Ital J Gastroenterol Hepatol 1999;31:604-606. 97. Schleef J, Wagner A, Kleta R, Schaarschmidt K, DockhornDworniczak B, Willital G, Jurgens H. Small-cell carcinoma of the ovary of the hypercalcemic type in an 8-year-old girl. Pediatr Surg Int 1999;15:431-434. 98. Yamashita F, Iwao T, Torimura T, Tanaka M, Hirai K, Abe M, Toyonaga A, Sugihara S, Kojiro M, Tanikawa K. Sclerosing hepatocellular carcinoma with hypercalcemia--a case report. Kurume Med J 1992;39:113-116. 99. Yen TC, Hwang SJ, Wang CC, Lee SD, Yeh SH. Hypercalcemia and parathyroid hormone-related protein in hepatocellular carcinoma. Liver 1993;13:311-315. 100. CadranelJE Cadranel J, Buffet C, Ink O, Pelletier G, Bismuth E, Etienne JE Hypercalcaemia associated with chronic viral hepatitis. Postgrad Med J 1989;65:678-680. 101. Ford DJ, Reid IR. Hypercalcaemia associated with viral hepatitis. Lancet 1990;336:181. 102. Attard TM, Dhawan A, Kaufman SS, Collier DS, Langnas AN. Use of disodium pamidronate in children with hypercalcemia awaiting liver transplantation. Pediatr Transplant 1998;2: 157-159. 103. Pont A. Unusual causes of hypercalcemia. Endocrinol Metab Clin North Am 1989; 18: 753-764. 104. Tezer KM. Use of bisphosphonates in hypercalcemia associated with childhood cancer. J Clin Oncol. 1999;17:1960. 105. Lteif AN, Zimmerman D. Bisphosphonates for treatment of childhood hypercalcemia. Pediatrics 1998;102:990-993.

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Hyp oparat hy roldlsm . . in. the. Differential . Diagnosis of Hypocalcemia

W . D O W N S Division of Endocrinology and Metabolism, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298

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INTRODUCTION

for every 1 g / d l change in albumin concentration below the normal range (1):

Calcium is present in extracellular fluid in three forms: b o u n d to protein (40-45%), complexed with inorganic anions (5-10%), and ionized (45-50%). Clinically significant hypocalcemia involves a decrease in the concentration of the physiologically i m p o r t a n t ionized calcium. It is the free calcium ion concentration that is important for normal cellular function, and it is this free calcium ion concentration that is sensed and regulated by the parathyroid glands.

corrected calcium = total calcium + 0.8 (4 - serum albumin). For example, a patient with a measured total serum calcium of 8.0 m g / d l (normal range 8.9-10.5) and a measured albumin of 2.5 g / d l (normal range 3.7-4.9) would have an albumin concentration that is 1.5 g / d l below the normal value of 4.0 g/dl. Multiplying 1.5 by 0.8 provides a correction in the calcium of 1.2 m g / d l , which added to the measured value of 8.0 gives a corrected serum calcium of 9.2 m g / d l [calculated as 8.0 + 0.8(4 - 2.5)]. The corrected serum calcium in this case predicts that the ionized calcium, if measured, would be normal and that the decline in total calcium is attributable to the low concentration of albumin-bound calcium. Unfortunately, these formulas do not always correctly identify patients with true ionized hypocalcemia (2), because other factors, including p H and the concentration of other anions, contribute to changes in ionized calcium. Therefore, for patients who have symptoms that may be caused by hypocalcemia and in critically ill patients in whom ionized calcium is m o r e commonly abnormal (3), direct m e a s u r e m e n t of ionized calcium is advisable if there is any uncertainty about the diagnosis of hypocalcemia. M e a s u r e m e n t of ionized calcium requires attention to careful specimen collection and p r o m p t handling by a qualified laboratory.

D E T E R M I N A T I O N OF H Y P O C A L C E M I A Because total serum calcium is most often measured in clinical practice, rather than the free or ionized fraction, a low total serum calcium concentration can be found in patients who do not have low ionized calcium and who do not have any disorder of calcium metabolism. Albumin is the most a b u n d a n t calcium-binding serum protein, and hypoalbuminemia is responsible for most of the reduced total serum calcium concentration found in chronically ill, malnourished, and hospitalized patients. A n u m b e r of rule-of-thumb correction formulas have been proposed to determine which patients n e e d further evaluation for true hypocalcemia and which are likely to have normal ionized calcium with low serum protein as the cause for a low total serum calcium concentration. The most widely used of these corrections adjusts total serum calcium by 0.8 m g / d l The Parathyroids, Second Edition

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S Y M P T O M S A N D SIGNS OF H Y P O C A L C E M I A The clinical manifestations of hypocalcemia may be quite variable a m o n g patients. Those who have acute decreases in serum ionized calcium from normal can have p r o m i n e n t symptoms, whereas those with chronic hypocalcemia may be relatively symptom free. Most of the symptoms of hypocalcemia are related to the important role of calcium ions in neuromuscular function, and are manifestations of increased neuromuscular excitability (Table 1). Patients with mild hypocalcemia may have some perioral numbness, tingling paresthesias of the distal extremities, and occasional muscle cramping. With moderate hypocalcemia, some patients will notice a general sense of irritability and anxiety, and more severe muscle cramps can occur. Severe hypocalcemia is accompanied by tetanic muscle cramps, carpopedal spasm, and may progress to lifethreatening laryngospasm, seizures, and coma. In addition to the commonly recognized symptoms of skeletal muscle cramping, hypocalcemia can be associated with other manifestations of abnormal neuromuscular function. A prolonged QT interval is often observed on the electrocardiogram, and reversible congestive heart failure has been reported in patients with severe hypocalcemia (4). Nonspecific electroencephalographic changes can occur (5,6). Abnormal smooth muscle function of the gastrointestinal and biliary tracts appears to account for abdominal pain and malabsorptive symptoms in patients rarely (7,8). The classic physical findings of hypocalcemia are also manifestations of enhanced neuromuscular irritability. Chvostek's sign is elicited by tapping over the facial nerve just anterior to the ear, producing contraction of the facial muscles. Slightly positive reactions, such as minimal twitching at the corner of the mouth, occur in some normal individuals (9). However, more marked muscle contraction, including movement of the u p p e r lip, nasal fold, and periorbital muscles,

TABLE 1

Clinical Manifestations of Hypocalcemia

Manifestations of impaired neuromuscular signaling Paresthesias, perioral numbness Prolonged QT interval on EKG Muscle cramps, carpopedal spasm, tetany Laryngeal stridor, convulsions Mental changes Physical signs of hypocalcemia Chvostek's sign Trousseau's sign Calcium precipitation Cataract Basal ganglia calcification

indicates significant hypocalcemia. Trousseau's sign is elicited as carpal spasm after inflation of a blood pressure cuff above systolic pressure for 3-5 minutes. These signs of latent tetany are not present in every patient who has hypocalcemia, however. Chronic hypocalcemia can be accompanied by other signs. Dry skin, coarse hair, and brittle nails are common. Dental abnormalities, including enamel hypoplasia and absence of adult teeth, may indicate that hypocalcemia has been present since childhood (10). Calcification of the basal ganglia occurs in all forms of hypoparathyroidism and can be recognized with computed tomographic imaging even when plain X-rays are normal (11). The reason for calcification in this particular area of the brain is not known. Cataracts can occur with long-standing hypoparathyroidism (12).

PATHOPHYSIOLOGIC MECHANISMS OF HYPOCALCEMIA The parathyroid glands sense and regulate the concentration of ionized calcium by secreting parathyroid h o r m o n e (PTH). PTH has effects on bone, through osteocytes and osteoclasts, to shift calcium from mineral storage sites to the extracellular space. PTH also has effects on the kidney to enhance distal renal tubular calcium reabsorption, proximal renal tubular phosphate excretion, and production of 1,25-dihydroxyvitamin D [ 1,25 (OH) 2o ] , which promotes intestinal calcium absorption and stimulates the differentiation and development of functional osteoclasts. The multiple actions of PTH are reviewed in detail elsewhere in this volume (Chapters 11-17). Defects in any of these processes can result in abnormalities of calcium regulation. The specific causes of hypocalcemia can be divided into those in which parathyroid h o r m o n e secretion is impaired or abnormal and those in which the parathyroid glands are normal (Table 2). Clinical disorders causing hypocalcemia occur as a result of disordered calcium sensing; absence, damage, or dysfunction of the parathyroid glands; and abnormal target organ response to parathyroid hormone. When the parathyroid glands are normal, hypocalcemia caused by other mechanisms (such as deficiency or disordered metabolism of vitamin D) induces parathyroid h o r m o n e secretion and the physiologic effects of parathyroid h o r m o n e can be observed as reactive secondary hyperparathyroidism.

HYPOPARATHYROIDISM In hypoparathyroidism, the clinical findings are those expected as a result of low PTH, including low total and

HYPOCALCEMIA / TABLE 2 Causes of Hypocalcemia Hypoparathyroidism/abnormal PTH secretion Surgical hypoparathyroidism Transient postsurgical hypoparathyroidism Autoimmune (idiopathic) hypoparathyroidism Activating mutations of the calcium-sensing receptor Developmental abnormalities of parathyroid glands (DiGeorge syndrome) Transient neonatal hypocalcemia Infiltrative damage of parathyroid glands (hemochromatosis, Wilson's disease, sarcoidosis, amyloidosis, metastatic cancer) Irradiation Severe magnesium deficiency Hypermagnesemia Pseudohypoparathyroidism/PTH resistance Pseudohypoparathyroidism Type la, with Albright osteodystrophy and multiple hormone resistance Type lb, with resistance confined to PTH target tissues Type 2, defective phosphaturic response to PTH Severe magnesium deficiency Disorders of vitamin D metabolism Acquired vitamin D deficiency Inadequate sunlight and dietary vitamin D Malabsorption Renal insufficiency Anticonvulsants Hereditary disorders of vitamin D metabolism 1oL-hydroxylase deficiency Resistance to vitamin D action Altered bound calcium Hyperphosphatemia (rhabdomyolysis, tumor lysis, phosphate infusion) Massive blood transfusion (citrate) Acute severe illness (pancreatitis, sepsis, etc.) Increased osteoblastic activity Postparathyroidectomy, "hungry bones" syndrome Osteoblastic tumor metastasis Hypocalcemic drugs

ionized calcium, and an increase in serum phosphorus concentration. PTH concentrations are low, but even in modern assays may still remain detectable. Patients with hypoparathyroidism have increased renal calcium clearance because of the absence of a PTH effect to enhance distal tubular calcium reabsorption.

Surgical Hypoparathyroidism Despite the best efforts of experienced endocrine surgeons, hypoparathyroidism occurs in patients who have parathyroid or thyroid surgery and particularly after radical surgery for carcinoma involving structures in the neck. Surgical series have indicated that the incidence of transient hypoparathyroidism after thyroid

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surgery may be as high as 10-15%; the incidence of permanent hypoparathyroidism is about 1-4% (13). Permanent hypoparathyroidism can be caused not only by inadvertent removal of parathyroid tissue, but also by damage to the blood supply to the parathyroid glands. Experienced surgeons are aware of the location of the parathyroid glands in relation to their blood supply and will autotransplant threatened parathyroid tissue when necessary (14).

Transient Postsurgical Hypoparathyroidism Transient hypoparathyroidism occurs when the damage to the parathyroid glands is incomplete, but other reasons for hypocalcemia after thyroid surgery intervene. After surgery for thyrotoxicosis, for example, elevated bone turnover during the hyperthyroid state and increased ionized calcium may cause mild parathyroid gland suppresion. Temporary hypocalcemia after surgery for primary hyperparathyroidism can occur due to suppression of normal parathyroid glands by hypercalcemia. In the mild primary hyperparathyroidism characteristic of the western world, postoperative hypocalcemia has become uncommon. However, if normal parathyroid glands are suppressed, they usually recover quickly and the serum calcium concentration returns to normal within days. More prolonged hypocalcemia suggests damage to normal parathyroid tissue or significant uptake of calcium into remineralizing "hungry bones," a situation that can be distinguished from hypoparathyroidism by a persistently low phosphorus concentration and an appropriately elevated PTH concentration.

Autoimmune (Idiopathic) Hypoparathyroidism Hypoparathyroidism occurs as part of a polyglandular endocrine deficiency syndrome (type 1), characterized most commonly by hypoparathyroidism, adrenal insufficiency, and mucocutaneous candidiasis (15). The syndrome usually presents during childhood. Not all patients express the complete triad, and some affected patients have other autoimmune disorders, such as alopecia, pernicious anemia, thyroid disease, diabetes mellitus, autoimmune hepatitis, and gonadal failure. Patients have circulating cytotoxic antibodies against parathyroid tissue (16), and the parathyroid glands are typically atrophic. The immunologic and genetic aspects of autoimmune hypoparathyroidism are reviewed in detail in Chapter 50. Idiopathic hypoparathyroidism also occurs sporadically in adults and is associated with antiparathyroid antibodies occasionally. Some of these cases may simply be related to incomplete penetrance of the familial polyglandular syndrome type 1. It is also possible that

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some may be related to relatively asymptomatic hypoparathyroidism associated with activating mutations of the calcium-sensing receptor.

Activating Mutations of the Calcium-Sensing Receptor The calcium-sensing receptor is an extracellular G protein-coupled receptor that senses the extracellular ionized calcium concentration, and whose normal action in the setting of an increase in extracellular calcium causes a decrease in PTH gene transcription and PTH secretion (17) (see Chapter 8). Inactivating mutations of this receptor have been recognized to cause autosomal d o m i n a n t hypercalcemia associated with low urinary calcium [familial hypocalciuric hypercalcemia (FHH) ]. It has been recognized that activating mutations of the calcium-sensing receptor will lower the set point for regulation of serum calcium. Serum ionized calcium activity is consequently decreased, and PTH synthesis and secretion are reduced even at relatively low calcium concentrations. Although some patients may be symptomatic of hypocalcemia, these tend to be more mild and intermittent than might be expected. Many affected individuals are asymptomatic and are detected only on family screening once an affected p r o b a n d is identified (18). The disorder is inherited as an autosomal dominant trait. One hallmark of this type of familial hypoparathyroidism is increased urinary calcium excretion, just the opposite of what happens in FHH. Thus, affected individuals who are treated with calcium and vitamin D could be predisposed to marked increases in urinary calcium excretion, nephrocalcinosis, and renal insufficiency when serum calcium is normalized (18). Treatment of asymptomatic individuals with calcium and vitamin D is therefore not advisable, rather only when symptoms are present.

Developmental Abnormalities of the Parathyroid Glands Disorders in which there is abnormal or absent formation of the parathyroid glands are associated with hypocalcemia. The DiGeorge syndrome occurs as a result of abnormal development of the third and fourth branchial pouches, which give rise to the parathyroid glands, and is characterized by a group of findings now clustered u n d e r the term CATCH 22 (cardiac defects, abnormal facies, thymic hypoplasia/aplasia, cleft palate, h_ypocalcemia, and 2 2 q l l chromosomal microdeletion) (19). Because of an associated T cell abnormality, early mortality from infection is common. Other forms of congenital and familial hypoparathyroidism have been described less frequently (20,21). One interesting family appears to have an abnormality

of the preproparathyroid h o r m o n e signal peptide, which results in defective intracellular processing of PTH (22). The molecular genetics of hypoparathyroidism are described in detail in Chapter 49.

Transient Neonatal Hypocalcemia Transient hypocalcemia in the newborn period is much more c o m m o n than the genetic disorders that lead to p e r m a n e n t hypoparathyroidism. There is a n expected decrease in serum calcium concentration after birth, and some infants have an exaggeration of this process with development of hypocalcemia. Neonatal hypocalcemia is self-limited, and tends to occur more commonly in premature and low-birthweight infants, perhaps because of deficient PTH secretion from less mature parathyroid glands. More severe neonatal hypercalcemia occurs in infants born to mothers with primary hyperparathyroidism or other forms of hypercalcemia.

Infiltrative Damage to Parathyroid Glands Parathyroid gland function can be impaired by a variety of disease processes in which there is infiltrative involvement. Hypoparathyroidism is recognized to occur in patients with hemochromatosis and iron overload. In a large study of patients with thalassemia, 3.6% of 1861 patients were found to have hypoparathyroidism (23). Parathyroid gland infiltration can also occur in patients with Wilson's disease, sarcoidosis, tuberculosis, amyloidosis, and metastatic cancer, but there are only a few reports of hypocalcemia in such cases (24-26).

Radiation-Induced Damage to Parathyroid Tissue Exposure to external radiation and to the doses of internal radiation used in the treatment of Graves disease with iodine-131 is only rarely reported in association with hypocalcemia. In those who receive large doses of radioactive iodine for the treatment of thyroid cancer, however, some evidence for diminished parathyroid function after treatment has been documented (27). However, such patients have usually had previous thyroid surgery, which may have contributed to diminished parathyroid reserve.

Magnesimn Deficiency Moderate declines in serum magnesium concentration can stimulate PTH secretion slightly, but severe magnesium deficiency results in a reversible abnormality of PTH secretion (28). This is actually one of the more c o m m o n causes of acute hypocalcemic tetany in malnourished alcoholic patients. The tendency to

HYPO~CFMIA hypocalcemia cannot be treated simply with calcium, but is readily reversible with magnesium replacement. With profound magnesium depletion, there is evidence that cellular resistance to the actions of PTH can occur as well (29).

Hypermagnesemia Hypermagnesemia is associated with declines in serum calcium, usually during magnesium infusion in obstetric practice. Studies have shown suppression of PTH secretion and an increase in urinary calcium excretion during magnesium infusion (30). Hypocalcemia in this setting is rarely symptomatic, perhaps because the excess magnesium tends to blunt neuromuscular irritability.

PARATHYROID HORMONE RESISTANCE Pseudohypoparathyroidism Pseudohypoparathyroidism represents the classic syndrome of resistance to the actions of PTH. Patients with pseudohypoparathyroidism can have symptoms of hypocalcemia, and laboratory studies show hypocalcemia and hyperphosphatemia, just as in primary hypoparathyroidism. However, measurement of PTH reveals elevated rather than decreased h o r m o n e concentrations. The parathyroid glands are hyperplastic. Chase et al. showed that PTH infusion failed to induce an expected increase in urinary cyclic AMP in such patients (31), locating the biochemical defect to the PTH receptor-adenylate cyclase complex. Patients with defective urinary cyclic AMP production are now classified as pseudohypoparathyroidism type 1. Another group of patients with hypocalcemia has been shown to have a normal urinary cyclic AMP response to PTH, but a defective phosphaturic response. This second group, termed pseudohypoparathyroidism type 2 (32), is a heterogeneous group of disorders, and in many patients the abnormal phosphaturic response is reversible with normalization of serum calcium. Pseudohypoparathyroidism type 1 can be further classified by clinical features and biochemical defect. Patients with pseudohypoparathyroidism type la have hypocalcemia and PTH resistance associated with other endocrine deficits, including hypothyroidism and oligomenorrhea, and they have a characteristic body habitus, termed Albright hereditary osteodystrophy, with shortened metacarpals, short stature, round face, and obesity. These patients have a genetic disorder with abnormalities of the G protein signal-transducing protein that couples receptor activation with stimulation of adenylate cyclase (33,34). Because G proteins couple receptor-adenylate cyclase activity for multiple hormones, this explains the multiple hormone resistance.

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Some family members of patients with typical pseudohypoparathyroidism have abnormal G protein, but may have normal serum calcium and a normal urinary cyclic AMP response to PTH infusion; such individuals are said to have pseudopseudohypoparathyroidism (35). Patients with pseudohypoparathyroidism type l b have hypocalcemia and PTH resistance, but hormone resistance is confined to PTH target tissues. In fact, some patients with this form of pseudohypoparathyroidism have significant PTH-induced bone disease even though there is clear renal resistance to PTH. There has been speculation that the biochemical defect in pseudohypoparathyroidism type l b would be in the PTH receptor, but there is now good evidence that the PTH receptor is normal in these patients (36,37). There are some patients who have hypocalcemia, PTH resistance, multiple hormone deficiencies, and Albright hereditary osteodystrophy, who nevertheless appear to have normal G protein function. Some evidence for an adenylate cyclase catalytic unit defect was identified in one such patient (38), but this has not been confirmed by others. If a catalytic unit defect does exist, this would be a separate form of pseudohypoparathyroidism (type lc). The clinical presentation and biochemical mechanisms of pseudohypoparathyroidism are summarized in Chapter 51.

Severe Magnesium Deficiency As noted above, severe magnesium deficiency can cause resistance to the target organ effects of PTH (29), as well as abnormal PTH secretion.

DISORDERS OF VITAMIN D METABOLISM The active metabolite of vitamin D [1,25 (OH) 2D] plays a critically important role in normal calcium homeostasis, by enhancing calcium absorption and by promoting the normal differentiation and development of osteoclasts. Deficiencies in the supply of vitamin D precursors, abnormalities in conversion of precursors to the active metabolite, and resistance to the action of 1,25(OH)zD can all cause hypocalcemia. Inadequate sunlight exposure and inadequate dietary vitamin D are common even in temperate latitudes where sunlight exposure might be considered adequate. Malabsorption of vitamin D because of intestinal or biliary disease also leads to vitamin D deficiency. Some drugs, notably anticonvulsants, accelerate the clearance of vitamin D metabolites. In patients with renal failure, synthesis of active metabolites in the kidney is abnormal. Genetic disorders can also result in deficient renal l e~-hydroxylase activity and inherited resistance to the effects of 1,25 (OH) 2O .

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In all of these disorders, the normal parathyroid glands attempt to compensate for low serum calcium concentrations, and secondary hyperparathyroidism occurs. These disorders, unlike those characterized by hypoparathyroidism or pseudohypoparathyroidism, are likely to be associated with low serum phosphate concentrations because of increased renal phosphate excretion and with evidence for increased parathyroid h o r m o n e action on bone.

OTHER CAUSES OF HYPOCALCEMIA Alterations in Bound Calcium There are a n u m b e r of situations in which increases in organic anions or changes in calcium binding cause a shift in the equilibrium between b o u n d and ionized calcium. For example, an increase in phosphate concentration will bind calcium into complexes and decrease the ionized calcium activity. C o m m o n circumstances in which this occurs include rhabdomyolysis and tumor lysis syndrome. Phosphate infusion can also cause hypocalcemia, and phosphate enemas have been reported to cause hypocalcemia (39), particularly in children. Citrate infused during massive blood transfusion can also complex calcium and decrease the concentration of ionized calcium. Acute illness is associated with a high incidence of hypocalcemia, and it is suspected that release of free fatty acids binds calcium in this situation (3,40).

Increased Osteoblastic Activity Bone resorption and formation are usually very tightly coupled, but in certain situations, bone formation can proceed so briskly that hypocalcemia occurs. This type of "hungry bone syndrome" can occur during the postoperative period after surgical treatment for severe hyperparathyroidism (41,42). Early hypocalcemia in such patients is certainly due to suppression of any remaining normal parathyroid tissue, but healing bone can utilize such a large a m o u n t of calcium that hypocalcemia occurs despite recovery of suppressed parathyroid glands. Affected patients generally have preexisting evidence of hyperparathyroid bone disease, and hypocalcemia persists despite elevated circulating PTH concentrations until the bone is healed. Patients may require treatment with calcium and vitamin D for several days or weeks, and some patients with severe bone disease have required treatment for months. One sign that the bone is healing is a reduction in bone specific alkaline phosphatase activity, a readily available marker for bone formation. Certain cancers can form osteoblastic metastases in bone. Those cancers most commonly reported to be

associated with osteoblastic metastases and hypocalcemia are prostate cancer and breast cancer (43).

Drug-Induced Hypocalcemia A n u m b e r of medications cause hypocalcemia. Some inhibit osteoclast-mediated bone resorption (bisphosphonates, plicamycin, calcitonin, gallium nitrate), some decrease PTH secretion (calcimimetic agents), and some complex calcium and magnesium (phosphate, foscarnet) (44-46).

AN APPROACH TO THE PATIENT WITH H Y P O C A L C E M I A A careful history and examination are important in evaluating and managing patients who have hypocalcemia. Many of the disorders that cause hypocalcemia can be identified with a t h o r o u g h knowledge of the patient's medical and family history. The clinical setting will usually make readily apparent those patients who are likely to have transient hypocalcemia. The examination will determine the severity of hypocalcemia so that therapy can be planned, and will identify other endocrine disorders and skeletal or dental abnormalities. Laboratory evaluation should include measurement of serum proteins to identify those patients who have apparent hypocalcemia with normal corrected calcium, serum creatinine to assess renal function, and serum phosphorus. Magnesium concentrations should also be assessed, particularly in those at risk for magnesium deficiency. PTH measurement is now a routine clinical procedure that will help to narrow the relevant differential diagnosis. Basic clinical and laboratory data will guide the care of the patient who presents with hypocalcemia.

REFERENCES 1. Editorial. Serum calcium. Lancet 1979;1:858-859. 2. Ladenson JH, Lewis JW, Boyd JC. Failure of total calcium corrected for protein, albumin, and pH to correctly assess free calcium status. J Clin Endocrinol Metab 1978;46:986-993. 3. Zaloga GE Hypocalcemia in critically ill patients. Crit Care Med 1992;20:251-262. 4. Rowell WG, Kreisberg RA. Hypocalcemic congestive heart failure. South MedJ 1987;80:396-398. 5. Swash M, Rowan AJ. Electroencephalographic criteria of hypocalcemia and hypercalcemia. Arch Neuro11972;26:218-228. 6. Fukuyama Y, Hayashi M. Sleep electroencephalograms and sleep stages in hypoparathyroidism. Eur Neurol 1979; 18:38-48. 7. Heubi JE, Partin JC, Schubert WK. Hypocalcemia and steatorrhea--clues to etiology. Dig Dis Sci 1983;28:124-128. 8. Reracchi M, Bardella MT, Conte D. Late-onset idiopathic hypoparathyroidism as a cause of diarrhea. Eur J Gastroenterol Hepatol 1998; 10:163-165.

HVeOCALCEMIA / 9. McGreal GT, Kelly JL, Hehir DJ, Brady ME Incidence of false positive Chvostek's sign in hospitalised patients. Ir J Med Sci 1995;164:56. 10. Nikiforuk G, Fraser D. The etiology of enamel hypoplasia: A unifying concept. J Pediatr 1981 ;98:888-893. 11. Posen S, Clifton-Bligh P, Cromer T. Computed tomography of the brain in surgical hypoparathyroidism. Ann Intern Med 1979;91:415-417. 12. Blake J. Eye signs in idiopathic hypoparathyroidism. Trans Ophthalmol Soc UK 1976;46:448-451. 13. Bergamaschi R, Becouarn G, RoncerayJ, ArnaudJP. Morbidity of thyroid surgery. A m J Surg 1998;176:71-75. 14. Shaha AR, Jaffe BM. Parathyroid preservation during thyroid surgery. Am J Otolaryngol 1998; 19:113-117. 15. Betterle C, Greggio NA, Volpato M. Clinical review 93: Autoimmune polyglandular syndrome type 1. J Clin Endocrinol Metab 1998;83:1049-1055. 16. Brandi ML, Aurbach GD, Fattorossi A, Quarto R, Marx SJ, Fitzpatrick LA. Antibodies cytotoxic to bovine parathyroid cells in autoimmune hypoparathyroidism. Proc Natl Acad Sci USA 1986;83:8366-8369. 17. Brown EM. Physiology and pathophysiology of the extracellular calcium-sensing receptor. Am J Med 1999;106:238-253. 18. Pearce SH, Williamson C, Kifor O, Bai M, Coulthard MG, Davies M, Lewis-Barned N, McCredie D, Powell H, Kendall-Taylor P, Brown EM, Thakker RV. A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor. N EnglJ Med 1996;335:1115-1122. 19. Sergi C, Serpi M, Muller-Navia J, Schnabel PA, Hagl S, Otto HE Ulmer HE. CATCH 22 syndrome: Report of 7 infants with followup data and review of the recent advancements in the genetic knowledge of the locus 22q 11. Pathologica 1999;91:166-172. 20. Trump D, Dixon PH, Mumm S, Wooding C, Davies KE, Schlessinger D, Whyte ME Thakker RV. Localisation of X linked recessive idiopathic hypoparathyroidism to a 1.5 Mb region on Xq26-q27. J Med Genet 1998;35:905-909. 21. Kelly TE, Blanton S, Saif R, Sanjad SA, Sakati NA. Confirmation of the assignment of the Sanjad-Sakati (congenital hypoparathyroidism) syndrome (OMIM 241410) locus to chromosome 1q42-43. J Med Genet 2000;37:63-64. 22. Sunthornthepvarakul T, Churesigaew S, Ngowngarmratana S. A novel mutation of the signal peptide of the preproparathyroid hormone gene associated with autosomal recessive familial isolated hypoparathyroidism. J Clin Endocrinol Metab 1999;84: 3792-3796. 23. Italian Working Group on Endocrine Complications in Nonendocrine Diseases. Multicentre study on prevalence of endocrine complications in thalassemia major. Clin Endocrinol 1995;42: 581-586. 24. Carpenter TO, Larves DL, Anast CS. Hypoparathyroidism in Wilson's disease. NEnglJ Med 1983;309:873-877. 25. Dill JE. Hypoparathyroidism in sarcoidosis. South Med J 1983; 76:414. 26. Ellis HA, Mawhinney WH. Parathyroid amyloidosis. Arch Pathol Lab Med 1984;108:689-690. 27. Glazebrook GA. Effect of decicurie doses of radioactive iodine 131 on parathyroid function. A m J Surg 1987;154:368-373. 28. Anast CS, Mohs JM, Kaplan SL, Burns TW. Evidence for parathyroid failure in magnesium deficiency. Science 1972; 177:606-608. 29. Estep H, Shaw WA, Watlington C, Hobe R, Holland W, Tucker SG. Hypocalcemia due to hypomagnesemia and reversible

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parathyroid hormone unresponsiveness. J Clin Endocrinol Metab 1969;29:842-848. 30. Cholst IN, Steinberg SF, Tropper PJ, Fox HE, Segre GV, Bilezikian JR The influence of hypermagnesemia on serum calcium and parathyroid hormone levels in human subjects. N Engl J Med 1984;310:1221-1225. 31. Chase LR, Melson GL, Aurbach GD. Pseudohypoparathyroidism: Defective excretion of 3',5'-AMP in response to parathyroid hormone. J Clin Invest 1969;48:1832-1844. 32. Drezner M, Neelon FA, Lebovitz HE. Pseudohypoparathyroidism type II: A possible defect in the reception of the cyclic AMP signal. N EnglJ Med 1973;289:1056-1060. 33. Levine MA, Downs RW, Jr, Moses AM, Breslau NA, Marx SJ, Lasker RD, Rizzoli RE, Aurbach GD, Spiegel AM. Resistance to multiple hormones in patients with pseudohypoparathyroidism: Association with deficient activity of guanine nucleotide regulatory protein. AmJMed 1983;74:545-556. 34. Levine MA. Pseudohypoparathyroidism: From bedside to bench and back. J Bone Miner Res 1999;14:1255-1260. 35. Levine MA, Jap TS, Mauseth RS, Downs RW, Spiegel AM. Activity of the stimulatory guanine nucleotide-binding protein is reduced in erythrocytes from patients with pseudohypoparathyroidism and pseudopseudohypoparathyroidism: Biochemical, endocrine, and genetic analysis of Albright's hereditary osteodystrophy in six kindreds. J Clin Endocrinol Metab 1986;62:497-502. 36. Jan de Beur SM, Ding CL, LaBuda MC, Usdin TB, Levine MA. Pseudohypoparathyroidism l b: Exclusion of parathyroid hormone and its receptors as candidate disease genes. J Clin Endocrinol Metab 2000;85:2239-2246. 37. BettounJD, Minagawa M, Kwan MY, Lee HS, Yasuda T, Hendy GN, Goltzman D, White JH. Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTHrelated peptide receptor gene: Analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type lb. J Clin Endocrinol Metab 1997;82:1031-1040. 38. Barrett D, Breslau NA, Wax MB, Molinoff PB, Downs RW, Jr. New form of pseudohypoparathyroidism with abnormal catalytic adenylate cyclase. AmJPhysio11989;257:E277-E283. 39. Campisi P, Badhwar V, Morin S, Trudel JL. Postoperative hypocalcemic tetany caused by fleet phospho-soda preparation in a patient taking alendronate sodium: Report of a case. Dis Colon Rectum 1999;42:1499-1501. 40. Lind L, Carlstedt F, Rastad J, Stiernstrom H, Stridsberg M, Ljunggren O, Wide L, Larsson A, Hellman P, Ljunghall S. Hypocalcemia and parathyroid hormone secretion in critically ill patients. Crit Care Med 2000;28:93-99. 41. Brasier AR, Nussbaum SR. Hungry bone syndrome: Clinical and biochemical predictors of its occurrence after parathyroid surgery. Am J Med 1988;84:654-660. 42. Savazzi GM, Allegri L. The hungry bone syndrome: Clinical problems and therapeutic approaches following parathyroidectomy. EurJ Med 1993;2:363-368. 43. Riancho JA, Arjona R, Valle R, Sanz J, Gonzalez-Macias J. The clinical spectrum of hypocalcaemia associated with bone metastases. J Intern Med 1989;226:449-452. 44. Schussheim DH, Jacobs TP, Silverberg SJ. Hypocalcemia associated with alendronate. Ann Intern Med 1999;130:329. 45. Jacobson MA, Gambertoglio JG, Aweeka FT, Causey DM, Portale AA. Foscarnet-induced hypocalcemia and effects of foscarnet on calcium metabolism. J Clin Endocrinol Metab 1991 ;72:1130-1135. 46. Gearhart MO, Sorg TB. Foscarnet-induced severe hypomagnesemia and other electrolyte disorders. Ann Pharmacother 1993;27:285-289.

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CHAPTER 4 8

Magnesium Deficiency in Parathyroid Function

ROBERT

K. R U D E

University of Southern California School of Medicine, Los Angeles, California 90089

INTRODUCTION

M A G N E S I U M METABOLISM

Magnesium (Mg) is one of the most plentiful elements on earth. In vertebrates it is the fourth most abundant cation and the second most abundant intracellular cation. Therefore, it is not surprising that Mg is involved in numerous biologic processes and is essential for life (1,2). Mg was involved in early evolution as a means harnessing energy from the sun. Chlorophyll is the Mg chelate of porphyrin. T h r o u g h the process of photosynthesis, ATP is formed, providing energy for the synthesis of carbon dioxide and water into carbohydrate and oxygen. In animal cells as well as in plant cells in absence of the sun, stored chemical energy is utilized to maintain life. This chemical energy is released by Mg-dependent oxidative phosphorylation in which ATP is again formed. Mg has evolved to become a required cofactor in literally hundreds of enzyme systems (1-3). Examples of the physiologic role of Mg are shown in Table 1. Mg may be required for substrate formation. For example, all enzymes that utilize ATP do so as the metal chelate, MgATE Free Mg 2+ also acts as an allosteric activator of numerous enzyme systems as well as playing a role in ion currents and in membrane stabilization. Mg is therefore critical for a great n u m b e r of cellular functions, including oxidative phosphorylation, glycolysis, DNA transcription, and protein synthesis.

The normal adult total body Mg content is approximately 25g, of which 50-60% resides in bone. One-third of skeletal Mg is exchangeable and this fraction may serve as a reservoir for maintaining a normal extracellular Mg concentration. Extracellular Mg accounts for about 1% of total body Mg. The normal serum Mg concentration is 0.71-0.91 mmol/liter (1.7-2.2 m g / d l ) . About 70-75% of plasma Mg is ultrafilterable, of which the major portion is ionized. The remainder is protein bound, chiefly to albumin. The concentrations of Mg within cells are on the order of (1-3) X 10 -~ mol/liter, of which 0.5-5% is ionized or free (4,5). The kidney is the principal organ involved in Mg homeostasis (6). During Mg deprivation, the kidney avidly conserves Mg and less than 1 mEq is excreted in the urine per day. Conversely, when excess Mg is taken, it is rapidly excreted into the urine. The major sites of Mg reabsorption in the n e p h r o n are the proximal convoluted tubule (5-15%) and the thick ascending limb of Henle (50-60%), and the distal tubule (10%) (7). The factors that regulate renal Mg homeostasis are unknown. PTH, when given in large doses in humans or other species, will decrease urinary Mg excretion. Patients with either primary hyperparathyroidism or

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CI-IAeTER48 TABLE 1 Physiologic Role of Magnesium

Enzyme substrate (ATP-Mg, GTP-Mg) Kinase (hexokinase, creatine kinase, protein kinase) ATPase or GTPase (Na+/K+-ATPase, Ca2+-ATPase) Cyclases (adenylate cyclase, guanylate cyclase) Direct enzyme activation Phosphofructokinase Creatine kinase 5-Phosphoribosyl-pyrophosphate synthetase Adenylate cyclase Phospholipase C Na+/K+-ATPase Influence membrane properties Nerve conduction Calcium channel activity Potassium transport

hypoparathyroidism usually have normal serum Mg levels, however, suggesting that PTH is not an important physiologic regulator of Mg homeostasis (8). Studies have suggested that the concentration of calcium a n d / o r Mg in the extracellular fluid may regulate absorption of Mg in the thick ascending limb of Henle by activation of the CaZ+-sensing receptor in this segment of the nephron (7,9). Intestinal Mg absorption is inversely proportional to the amount ingested. Under normal dietary conditions in healthy individuals, approximately 30-50% of ingested Mg is absorbed (10). Mg is absorbed along the entire intestinal tract, including the large and small bowel, but the sites of maximal Mg absorption appear to be the ileum and distal jejunum (11). There exists both a passive and active transport system for Mg. A principal factor regulating intestinal Mg transport has not been described. Vitamin D and its metabolites, 25hydroxyvitamin D and 1,25-dihydroxyvitamin D, have been found in some studies to enhance intestinal Mg absorption but to a much lesser extent than they do calcium absorption (12,13).

MAGNESIUM DEFICIENCY Magnesium deficiency is more prevalent than previously appreciated. Approximately 10% of patients admitted to large city hospitals are hypomagnesemic (14). This incidence may increase to as high as 65% in a medical intensive care unit (15). Because Mg is ubiquitous in food, moderate to severe degrees of Mg depletion are most unusual in healthy individuals with a normal caloric intake. Clinically apparent hypomagnesemia a n d / o r Mg deficiency are usually due to losses of Mg from either the gastrointestinal tract or the kidney.

Causes of Mg deficiency are shown in Table 2 (for review see Refs. 3, 5, and 16). The Mg content of upper intestinal tract fluids is approximately 1 mEq/liter. Vomiting and nasogastric suction, therefore, may contribute to Mg depletion. The Mg contents of diarrheal fluids and fistulous drainage are much higher (up to 15 mEq/liter). Consequently, Mg depletion is common in acute and chronic diarrhea, regional enteritis, ulcerative colitis, and intestinal and biliary fistulas. Malabsorption syndrome due to nontropical sprue, radiation injury resulting from therapy for disorders such as Whipple's disease and carcinoma of the cervix, and intestinal lymphangiectasia may result in Mg deficiency, presumably due to intestinal mucosal damage. Steatorrhea may also cause or contribute to Mg malabsorption through formation of nonabsorbable magnesium-lipid salts. Resection or bypass of the small bowel, particularly the ileum, for obesity, enteritis, or vascular infarction, also often results in Mg deficiency.

TABLE 2 Causes of Magnesium Deficiency Gastrointestinal disorders Prolonged nasogastric suction Malabsorption syndromes Extensive bowel resection Acute and chronic diarrhea Intestinal and biliary fistulas Protein-calorie malnutrition Acute hemorrhagic pancreatitis Primary hypomagnesemia (neonatal) Renal loss Chronic parenteral fluid therapy Osmotic diuresis Glucose (diabetes mellitus) Mannitol Urea Hypercalcemia Alcohol Drugs Diuretics (furosemide, ethacrynic acid) Aminoglycosides Cisplatin Cyclosporin Amphotericin B Pentamidine Cardiac glycosides (possible) Metabolic acidosis (starvation, ketoacidosis, alcoholism) Renal diseases Chronic pyelonephritis, interstitial nephritis, and glomerulonephritis Diuretic phase of acute tubular necrosis Postobstructive nephropathy Renal tubular acidosis Postrenal transplantation Primary hypomagnesemia

MAGNESIUM IN PARATHYROIDFUNCTION / Excessive excretion of Mg into the urine underlies the basis of Mg depletion in many patients. Proximal tubular Mg reabsorption is proportional to tubular fluid flow and sodium reabsorption. Therefore, chronic parenteral fluid therapy, particularly with sodiumcontaining fluids and volume expansion states, such as primary aldosteronism, may result in Mg deficiency. Similarly, osmotic diuresis due to glucosuria (diabetes mellitus) (17), mannitol, and urea will result in urinary Mg wasting. Disorders causing an increase in the serum calcium concentration will also lead to renal magnesium wasting, presumably due to activation of the Ca 2+sensing receptor in the thick ascending limb of Henle, which results in a decrease in the transepithelial voltage and subsequent decrease in calcium and magnesium reabsorption (7,9). Exceptions are familial hypocalciuric hypercalcemia, due to an inactivating mutation of the CaZ+-sensing receptor, and lithium ingestion, in which urinary magnesium excretion is decreased. Certain drugs are becoming recognized as common causes of renal Mg wasting and Mg depletion (18). Diuretics acting at the loop of Henle (e.g., furosemide, bumetamide, and ethacrynic acid) have been shown by micropuncture studies and clinical studies to result in marked Mg wasting. The effect of thiazide diuretics is controversial; some studies demonstrate a Mg wasting effect, but others do not. The commonly used aminoglycosides have been shown to cause a reversible renal lesion that results in hypermagnesuria and hypomagnesemia (18). Similarly, amphotericin B therapy has been reported to result in renal Mg wasting. Cisplatin is a chemotherapeutic agent used in the treatment of epithelial neoplasms. Renal Mg wasting resulting in hypomagnesemia has been reported in up to 100% of patients receiving this agent. Cyclosporin is an immunosuppressive agent that also results in nephrotoxicity and renal Mg wasting. Pentamidine has been reported to result in renal Mg loss in AIDS patients (18). A rising blood alcohol level has been associated with renal Mg wasting and is one factor contributing to Mg deficiency in chronic alcoholism. Metabolic acidosis due to diabetic ketoacidosis, starvation, or alcoholism also causes renal Mg wasting. Some renal tubular, glomerular, or interstitial diseases have been associated with renal Mg wasting. There may be other accompanying tubular abnormalities, and reduced glomerular filtration rate may or may not be present. Diabetes mellitus is probably the most common disorder associated with Mg deficiency (17). The incidence of hypomagnesemia in diabetes mellitus has been reported to vary from 25 to 39% (3,5,16). The serum Mg concentration correlates inversely with the serum glucose concentration and the degree of glucosuria. The mechanism for the Mg depletion is probably

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mostly due to the glucosuria (osmotic diuresis). Patients with ketoacidosis may also waste Mg into the urine during the acidosis per se. Hypomagnesemia can be found in association with a number of other endocrine disorders, as shown in Table 3. The mechanism leading to the hypomagnesemia most frequently involves urinary Mg wasting. Phosphate depletion has been shown experimentally in humans and rats to result in urinary Mg wasting and hypomagnesemia (19). Excessive urinary Mg wasting and hypomagnesemia can be seen in severe primary hyperparathyroidism, treated hypoparathyroidism, and thyrotoxicosis; the urinary losses may be due to the hypercalcemia as discussed above a n d / o r hypercalciuria occurring in these states (7,9). The hypomagnesemia that may be seen in primary hyperaldosteronism has been related to plasma volume expansion and subsequent renal Mg wasting. Hypomagnesemia may also accompany the hungry bone syndrome, a phase of rapid bone mineral accretion in subjects with hyperparathyroidism or hyperthyroidism following surgical treatment. Bartter syndrome and Gitelman syndrome are autosomal recessive disorders characterized by hypokalemic alkalosis. Hypomagnesemia due to renal Mg wasting has been reported. Elucidation of the molecular defect of these disorders has clarified the clinical and biochemical classification (20). Gitleman syndrome has been found to be due to a genetic mutation of the thiazide-sensitive NaC1 cotransporter of the distal convulted tubule (chromosome 16) and is characterized by hypokalemic alkalosis, absence of hypertension, hypocalciuria, hypomagnesemia, and presentation after 8 years of age (20,21). Bartter syndrome has been found to be due to a genetic defect of the N a / K / 2 C L cotransporter in the thick ascending limb of Henle (20,22). It usually occurs in the neonatal period to offspring of consanguineous marriage and is characterized by salt wasting, hypovolemia, activation of the renin-antiotensin system, and hypolkalemic alkalosis. TABLE 3 Endocrineand Metabolic Disorders Associated with Magnesium Deficiency Diabetes mellitus Phosphate depletion Primary hyperparathyroidism Hypoparathyroidism Hyperthyroidism Primary aldosteronism Hungry bone syndrome Excessive lactation Gitelman syndrome

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Hypercalciuria occurs in conjunction with the Na loss and nephrocalcinosis often occurs. Hypomagnesemia is not often observed.

MANIFESTATIONS OF M A G N E S I U M DEFICIENCY Because Mg deficiency is usually secondary to another disease process or to a therapeutic agent, the features of the primary disease process may complicate or mask Mg deficiency. A high index of suspicion is therefore warranted. Frequent manifestations of moderate to severe Mg deficiency are shown in Table 4 (for review see Refs. 3, 5, 16, and 23). Neuromuscular hyperexcitability is often the presenting complaint. Latent tetany, as elicited by a positive Chvostek's and Trousseau's sign, or spontaneous carpal pedal spasm may be present. Frank generalized seizures may also occur. Though hypocalcemia may contribute to the neurologic signs, hypomagnesemia without hypocalcemia has been reported to result in neuromuscular hyperexcitability (24). Other signs may include vertigo, ataxia, nystagmus, and athetoid and choreiform movements as well as muscular tremor, fasciculation, wasting, and weakness. Electrocardiographic abnormalities of Mg deficiency in man include prolonged PR interval and QT interval.

TABLE 4

Manifestations of Moderate to Severe Magnesium Deficiency

Neuromuscular Positive Chvostek's and Trousseau's sign Spontaneous carpal-pedal spasm Seizures Vertigo, ataxia, nystagmus, athetoid and choreiform movements Muscular weakness, tremor, fasciculation, and wasting Psychiatric: depression, psychosis Cardiac arrhythmia EKG: prolonged PR interval and QT interval, U waves Atrial tachycardia, premature contractions, and fibrillation Junctional arrhythmias Ventricular premature contractions, tachycardia, fibrillation Sensitivity to digitalis intoxication Torsades de pointes Biochemical Hypokalemia Renal potassium wasting Decreased intracellular potassium Hypocalcemia Impaired PTH secretion Renal and skeletal resistance to PTH Resistance to vitamin D

Mg deficiency may also result in arrhythmias. Supraventricular arrhythmias, including premature atrial complexes, atrial tachycardia, atrial fibrillation, and junctional arrhythmias, have been described (25). Ventricular premature complexes, ventricular tachycardia, and ventricular fibrillation are more serious complications (26). A common laboratory feature of Mg deficiency is hypokalemia. During Mg deficiency there is intracellular potassium depletion as well as an inability of the kidney to conserve potassium. Attempts to replete the potassium deficit with potassium therapy alone are not successful without simultaneous Mg therapy (27-29). This biochemical feature may be a contributing cause of the electrocardiologic findings and cardiac arrhythmias discussed above. Soon after the observation that Mg deficiency may cause neuromuscular hyperexcitability, it was noted that hypocalcemia was also a common finding in moderate to severe Mg deficiency (30). Correction of the hypocalcemia was possible only with Mg therapy. The relationship of Mg with mineral homeostasis in normal physiology as well as the pathophysiologic events leading to hypocalcemia soon followed.

EFFECT OF MAGNESIUM ON PARATHYROID HORMONE SECRETION Calcium is the major regulator of PTH secretion. A n u m b e r of in vitro and in vivo studies, however, have demonstrated that Mg can modulate PTH secretion in a similar manner. Perfusion of isolated parathyroid glands of goats and sheep with varying concentrations of Mg showed that acute elevations of Mg inhibited PTH secretion but that acute reductions stimulated PTH secretion (31). These findings were confirmed in studies of bovine and rat parathyroid glands in vitro in which an increase in media Mg concentration inhibited release of PTH but low-media Mg stimulated PTH release (32,33). Studies have also demonstrated that hypermagnesemia will inhibit PTH secretion in humans (34-36). Mg could therefore be a physiologic regulator of PTH secretion. Mg, however, has only approximately 30-50% of the effect of calcium on either stimulating or inhibiting PTH secretion (35-39). The finding in humans that a 5% (0.03 mM) decrease in serum ultrafilterable Mg did not result in any detectable change in intact serum PTH concentration whereas a 5.5% (0.07mM) decrease in ionized calcium resulted in a 400% increase in serum PTH supports this concept (40). The inhibitory effects of Mg on PTH secretion may vary, depending on the extracellular calcium concentration (40). At physiologic calcium and Mg concentrations, these divalent cations were found

MAGNESIUM IN PARATHYROID FUNCTION

to be relatively equipotent at inhibiting PTH secretion from dispersed bovine parathyroid cells (40). At a low calcium concentration (0.5 mM) however, a threefold greater Mg concentration was required for similar PTH inhibition. Altering the Mg concentration did not diminish the ability of calcium to inhibit PTH secretion. Differences have also been noted in the effect of Mg and calcium on the biosynthesis of PTH in vitro. Changes in calcium over the range of 0 to 3.0 m M resulted in increased PTH synthesis as assessed by amino acid incorporation (41-43) or DNA synthesis (43), whereas changes in Mg over the range of 0 to 1.7 m M had no effect. The modulation of PTH secretion by extracellular calcium appears to be mediated by calcium binding to a specific cell surface receptor, the CaZ+-sensing receptor (44), which leads to activation of phospholipase C and subsequent accumulation of inositol 1,4,5-trisphosphate (45). This leads to release of calcium from intracellular stores (45) and subsequent decrease in PTH secretion. The CaZ+-sensing receptor is activated by other cations, including Mg (46). The affinity of this receptor for Mg is less than calcium, but does result in a rapid increase in intracellular calcium (47). It is probable, therefore, that the effect of Mg on PTH secretion may be mediated through the CaZ+-sensing receptor. It has also been suggested that Mg might alter the influx of extracellular calcium through ion channels (47). Based on the above, it is apparent that acute changes in the serum Mg concentration may modulate PTH secretion and should be considered in the evaluation of the determination of serum PTH concentrations.

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D therapy will not correct the hypocalcemia (for review see Refs. 3, 5, 16, and 30). Even mild degrees of Mg depletion, however, may result in a significant decrease in the serum calcium concentration, as demonstrated in experimental h u m a n Mg depletion (51). One major factor resulting in the decrease in the serum calcium is impaired parathyroid gland function. Low concentrations of Mg in the media of bovine or rat parathyroid cell cultures impair PTH release in response to a low media calcium concentration (52,53). Determinations of serum PTH concentrations in hypocalcemic hypomagnesemic patients have shown heterogeneous results, as shown in Fig. 1. The majority of patients have low or normal serum PTH levels (30,55-59). Normal serum PTH concentrations are thought to be inappropriately low in the presence of hypocalcemia. Therefore, a state of hypoparathyroidism exists in most hypocalcemic Mg-deficient

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EFFECT OF MAGNESIUM DEFICIENCY O N PARATHYROID GLAND FUNCTION Though acute changes in the extracellular Mg concentrations will influence PTH secretion qualitatively similar to that of calcium, it is clear that Mg deficiency markedly perturbs mineral homeostasis (30). Hypocalcemia is a prominent manifestation of Mg deficiency in humans (30). This has also been found to be true in most species, including monkey, cow, sheep, pig, dog, chick, and guinea pig (48). The rat, however, will develop hypercalcemia when Mg depleted while maintained on a normal calcium diet (48-50). On a low-calcium diet, however, the rat will become hypocalcemic (48). In humans, Mg deficiency must become moderate to severe before symptomatic hypocalcemia develops. A positive correlation has been found between the serum Mg and calcium concentrations in hypocalcemic hypomagnesemic patients (30). Mg therapy alone will restore serum calcium concentrations to normal in such patients within days (30). Calcium a n d / o r vitamin

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patients. Some patients, however, have elevated levels of PTH in the serum (30,55-59). The administration of Mg will result in an immediate increase in the serum PTH concentration, regardless of the basal PTH level (30,54,58,59). As shown in Fig. 2, 10 mEq of Mg administered intravenously over 1 minute caused an immediate marked rise in the serum PTH in patients with either low, normal, or elevated basal serum PTH concentrations. This is distinctly different than the effect of a Mg injection in normal subjects, in whom, as discussed above, Mg will cause an inhibition of PTH secretion (51). The ability of Mg to stimulate the rise in PTH appears to be specific for Mg depletion because Mg injection does not result in an increase in PTH in primary or secondary hyperparathyroidism (58). The serum PTH concentration will gradually fall to normal within several days of therapy as the serum calcium concentration normalizes (30,54,57-59), as shown in Fig. 3.

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FIG. 2 The effect of an intravenous injection of 10 mEq Mg on the serum concentrations of calcium, magnesium, and immunoreactive parathyroid hormone (IPTH) in hypocalcemic magnesium-deficient patients with undetectable (O), normal (©), or elevated (/k) levels of IPTH. Shaded areas represent the range of normal of each assay. The dashed line for the IPTH assay represents the level of detectability. The magnesium injection resulted in a marked rise in PTH secretion within 1 minute in all three patients (taken from Ref. 134).

The i m p a i r m e n t in PTH secretion appears to occur early in Mg depletion. Normal h u m a n subjects experimentally placed on a low-Mg diet for only 3 weeks showed similar, but not as marked, changes in the serum PTH levels (51). In this study there was a decrease in both the serum calcium and the PTH concentrations in 20 of 26 subjects at the end of a 3-week dietary Mg deprivation period. The administration of intravenous Mg at the end of the Mg depletion period resulted in a significant increase in the serum PTH concentration, whereas a similar Mg injection suppressed PTH secretion prior to the low-Mg diet. In this study, as with hypocalcemia hypomagnesemic patients, some subjects had elevations in the serum PTH concentration. The heterogeneous serum PTH values may be explained based on the severity of Mg depletion. As hypomagnesemia develops, the parathyroid gland will react normally with an increase in PTH secretion. As intracellular Mg depletion develops, however, the ability of the parathyroid to secrete PTH is impaired, resulting in a decrease in the serum PTH levels, with a

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FIB. 4 Mean and standard deviations of serum calcium concentration and urinary hydroxyproline and phosphate excretion in hypocalcemic magnesium-deficient patients before (O) and after (A) 3 days of parenteral magnesium therapy (taken from Ref. 61).

resultant decrease in the serum calcium concentration. This concept is supported by the observation that the change in serum PTH in experimental h u m a n Mg depletion is positively correlated with the decrease in red blood cell intracellular free Mg 2+ (51). A slight decrease in red blood cell Mg 2+ resulted in a increase in PTH. However, a greater decrease in red blood cell Mg 2+ correlated with a progressive decrease in serum PTH concentrations. It is conceivable that either PTH synthesis a n d / o r PTH secretion may be affected, given the wide requirem e n t of Mg for energy generation and protein synthesis. The immediate increase in PTH following the administration of intravenous m a g n e s i u m to Mgdeficient patients strongly suggests that the defect is in secretion, because biosynthesis of PTH is estimated to take approximately 45 minutes in vitro (41).

as illustrated in Fig. 4 (30,59). This also suggests skeletal resistance to PTH because exogenous PTH administered to hypoparathyroid patients causes a rise in the serum calcium within 24 hours (60). A n u m b e r of clinical studies, in which exogenous PTH was administered to hypocalcemic Mg-deficient patients, have demonstrated that PTH had little effect in increasing the serum calcium concentration (30,61-64). In one such study, shown in Fig. 5, parathyroid extract did not result in an elevation in the serum calcium concentration or urinary hydroxyproline excretion in hypocalcemic

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"-~ 'The above discussion strongly supports the notion that impairment in the secretion of PTH in Mg deficiency is a major contributing factor in the hypocalcemia. However, the presence of normal or elevated serum concentrations of PTH in the face of hypocalcemia (30,58,59) suggests that there may also be endorgan resistance to PTH action, such as exists in pseudohypoparathyroidism. In hypocalcemic Mgdeficient patients treated with Mg, the serum calcium concentration does not rise appreciably within the first 24 hours, despite elevated serum PTH concentrations,

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FI6.5 The effect of an intravenous injection of 200 units of parathyroid extract (PTE) on the excretion of urinary cyclic AMP in a magnesium-deficient patient before (o--o) and after (°---,) 4 days of magnesium therapy. Urine was collected for four consecutive 1-hour periods, two before and two after the PTE injection. Though Mg deficient, the patient had minimal rise in urinary cyclic AMP in response to PTH, but following Mg therapy the response was normal (taken from Ref. 30).

770

/

CI~WTwR48

hypomagnesemic patients (61). Following Mg repletion, however, a clear response to PTH was observed. PTH has also been shown to have a reduced calcemic effect in Mg-deficient dogs, chicks, and rats (65-69). The ability of PTH to resorb bone in vitro is also greatly diminished in the presence of low media Mg (70). In one in vivo study of isolated perfused femur in the dog, the ability of PTH to simulate an increase in the venous cyclic AMP was impaired during perfusion with low-Mg fluid, suggesting skeletal PTH resistance (71). Not all h u m a n studies have shown skeletal resistance to PTH, however (72-75). It appears likely that skeletal PTH resistance may be observed in patients with more severe degrees of Mg depletion. In h u m a n studies, patients in whom a normal calcemic response to PTH was demonstrated were subjects who had been on recent Mg therapy (72-75). Patients who have been found to be resistant to PTH have, in general, not had prior Mg administration (30,61-64). Consistent with this notion is that in the Mg-depleted rat, normal responses to PTH were observed when the serum Mg concentration was 0.95 m g / d l (76); however, in another study rats with a mean serum Mg of 0.46 m g / d l were refractory to PTH (65). In addition, a longitudinal study of Mg deficiency in dogs demonstrated a progressive decline in responsiveness to PTH with increasing degrees of Mg depletion (68). Calcium release from the skeleton also appears to be d e p e n d e n t on physicochemical processes as well as cellular activity (77,78). Low Mg will result in a decrease in calcium release from bone (77,78) and may be another mechanism for hypocalcemia in Mg deficiency. The renal response to PTH has also been assessed by determining urinary excretion of cyclic AMP a n d / o r phosphate (Figs. 4 and 5) in response to exogenous PTH. In some patients, a normal effect of PTH on urinary phosphate and cyclic AMP excretion has been noted (54-56,62,74,75). In general, these were the same subjects in which a normal calcemic effect was also seen (54,74,75). In other studies of more severely Mg-depleted patients, an impaired response to PTH has been observed (30,61,79). Shown in Fig. 5 is the effect of PTH on urinary cyclic AMP in one hypocalcemic Mg-deficient patient prior to and following Mg therapy. As shown, prior to Mg treatment, PTH did not increase urinary cyclic AMP excretion. Following Mg repletion, however, the rise in cyclic AMP was normal. A decrease in urinary cyclic AMP excretion in response to PTH has also been described in the Mg-deficient dog and rat (68,69).

EFFECT OF M A G N E S I U M D E F I C I E N C Y O N VITAMIN D M E T A B O L I S M A N D A C T I O N Mg may also be important in vitamin D metabolism a n d / o r action, as suggested by a n u m b e r of clinical observations(for review see Refs. 30, 80, and 81).

Patients with hypoparathyroidism who have been resistant to therapeutic doses of vitamin D have been reported to become more responsive to vitamin D after Mg therapy (82-84). The treatment of malabsorption syndromes with vitamin D may not be effective until Mg is simultaneously administered (85,86). Rickets, thought to be secondary to vitamin D resistance, have healed with Mg therapy (87). Patients with hypocalcemia and Mg deficiency have been reported to be resistant to pharmacologic doses of vitamin D (85,88,89), lc~-hydroxyvitamin D (90,91), and 1,25dihydroxyvitamin D (92). Similarly, impaired calcemic response to vitamin D has been found in Mg-deficient rats (93-95), lambs (96), and calves (97). Intestinal calcium transport in animal models of Mg deficiency has been found to be reduced in some (98,99) but not all (94,100) studies. Calcium malabsorption was associated with low serum levels of 25hydroxyvitamin D in one study (93), but not another (100), suggesting that Mg deficiency may impair intestinal calcium absorption by more than one mechanism. The exact nature of altered vitamin D metabolism a n d / o r action in Mg deficiency is unclear. Patients with Mg deficiency and hypocalcemia frequently have low serum concentrations of 25-hydroxyvitamin D (101-104) and therefore nutritional vitamin D deficiency may be one factor. Therapy with vitamin D, however, results in high serum levels of 25-hydroxyvitamin D without correction of the hypocalcemia (85), suggesting that the vitamin D deficiency is not the prime reason. In addition, conversion of radiolabeled vitamin D to 25-hydroxyvitamin D was found to be normal in three Mg-deficient patients (105). Serum concentrations of 1,25-dihydroxyvitamin D have also been found to be low or low normal in most hypocalcemic Mgdeficient patients (101-104). Magnesium-deficient diabetic children, when given a low-calcium diet, did not exhibit a normal rise in serum 1,25-dihydroxyvitamin D or PTH (106). The response returned to normal following Mg therapy, supporting the notion of altered vitamin D metabolism in Mg deficiency. Because PTH is a major trophic factor for 1,25-dihydroxyvitamin D formation, the low serum PTH concentrations could explain the low 1,25-dihydroxyvitamin D levels. In support of this is the finding that some hypocalcemic Mgdeficient patients treated with Mg have a rise in serum 1,25-dihydroxyvitamin D to high normal or to frankly elevated levels (see Fig. 6) (101). Most patients, however, do not have a significant rise within 1 week after institution of Mg therapy, despite a rise in serum PTH and normalization of the serum calcium concentration, as shown in Fig. 6 (101). These data suggest that Mg deficiency in humans also impairs the ability of the kidney to synthesize 1,25-dihydroxyvitamin D. This is supported by the observation that the ability of exogenous administration of h u m a n PTH(1-34) to normal sub-

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FI6.6 Serum concentrations of calcium and 1,25-dihydroxyvitamin D in hypocalcemic magnesium-deficient patients before and after 5-8 days of parenteral magnesium therapy. The dashed lines represent the upper and lower limits of normal for serum 1,25-dihydroxyvitamin D and the lower limit of normal for the serum calcium. Adapted from Ref. 101; RK Rude, JS Adams, E Ryzen, DB Endres, H Niimi, RL Horst, JF Haddad, FR Singer; Low serum concentrations of 1,25-hydroxyvitamin D in human magnesium deficiency. J Clin Endocrinol Metab 1985;61:933-940. © The Endocrine Society. I Ca2+ ~

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jects after 3 weeks of experimental Mg depletion resulted in a significantly lower rise in serum 1,25dihydroxyvitamin D concentrations than before institution of the diet (51). It appears, therefore, that the renal synthesis of 1,25-dihydroxyvitamin D is sensitive to Mg depletion. T h o u g h Mg is known to support the 25-hydroxy-lot-hydroxylase in vitro (107,108), the exact Mg requirement for this enzymatic process is not known. The association of Mg deficiency with impaired vitamin D metabolism and action therefore may be due to several factors. In some cases vitamin D deficiency may contribute (80,81,101-104). The major reasons, however, appear to be due to a decrease in PTH secretion with resultant decreased trophic effect on 1,25-dihydroxyvitamin D synthesis as well as a direct effect of Mg depletion on the ability of the kidney to synthesize 1,25-dihydroxyvitamin D (30,54-59,80,81,101-104). In addition, Mg deficiency may impair intestinal calcium absorption by resulting in low levels of vitamin D metabolites (30,101-104) or by a direct mechanism (98,99). Skeletal resistance to vitamin D and its metabolites may also play an important role (90-97). It is clear, however, that restoration of normal serum 1,25-dihydroxyvitamin D concentrations is not required for normalization of the serum calcium level. Most Mgdeficient patients who receive Mg therapy exhibit an immediate rise in PTH followed by normalization of the serum calcium prior to any change in serum 1,25-dihydroxyvitamin D concentrations (101-104). An overall view of the effect of Mg deficiency on calcium homeostasis is shown in Fig. 7.

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FIG. 7 Disturbance of calcium metabolism during magnesium deficiency. Hypocalcemia is caused by a decrease in PTH secretion as well as renal and skeletal resistance to the action of PTH. Low serum concentrations of 1,25-dihydroxyvitamin D may result in reduced intestinal calcium absorption. (From Ref. 135, RK Rude. Disorders of magnesium metabolism. In: RD Cohen, B Lewis, KGMM Alberti, AM Denman, Eds. The metabolic and molecular basis of acquired disease. Copyright © 1990 Bailliere Tindall, London.)

772

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CIIAeTWa48

M E C H A N I S M OF IMPAIRED MINERAL H O M E O S T A S I S IN M A G N E S I U M D E F I C I E N C Y The mechanism for impaired PTH secretion and action in Mg deficiency remains unclear. It has been suggested that a high serum PTH concentration is more important in causing PTH resistance than is the Mg deficiency, because PTH administration has been demonstrated to result in diminishing peripheral effects of PTH (59). In the Mg-deficient patient, however, as Mg deficiency worsens serum PTH concentrations fall (30,54-59). PTH resistance has been demonstrated in patients who had undetectable low PTH levels, suggesting that there are other operative mechanisms (30,61-64). Mg depletion may alter the function of secondmessenger systems. PTH is thought, in part, to exert is biologic effects through the intermediary action of cyclic AMP (109). Adenylate cyclase requires Mg for cyclic AMP generation both as a component of the substrate (Mg-ATP) and as an obligatory activator of enzyme activity (110). There are two Mg 2+ binding sites within the adenylate cyclase complex: one resides on the catalytic subunit and the other on the guanine nucleotide regulatory protein, Gs (111,112). The requisite role which Mg 2+ plays in adenylate cyclase function suggests that factors that would limit the availability of Mg 2+ to this enzyme could have significant effects on the cyclic nucleotide metabolism of a cell and hence on overall cellular function. It is clear that some patients with severe Mg deficiency have a reduced urinary excretion of cyclic AMP in response to exogenously administered PTH (30). In addition, PTH had a blunted effect in causing a rise in cyclic AMP from isolated perfused tibiae in Mg-deficient dogs (71). These observations correspond well with the impaired calcemic and phosphaturic effects of PTH in Mg-deficient patients and animals, as discussed above. Nine isoforms of adenylate cyclase have been identified; their activities are modulated by both Mg '2+ and C a 2+ (113). Though Mg 2+ is stimulatory, Ca 2+ may inhibit or activate enzyme activity (113). In plasma membranes from parathyroid, renal cortex, and bone cells, Ca '~+ was observed to inhibit competitively Mg z+activated adenylate cyclase activity (114-117). Thus, the ambient Mg 2+ concentrations can markedly affect the susceptibility of this enzyme to the inhibitory effects of Ca 2+. Because total intracellular calcium has been observed to rise during Mg depletion (118,119), the combination of higher intracellular C a z+ and increased sensitivity t o C a 2+ inhibition due to Mg depletion could explain the defective PTH action in Mg deficiency. Though cyclic AMP is an important mediator of PTH action, current data do not support an important role in mediating Ca 2+ -regulated PTH secretion (120).

The effect of C a 2+ o n PTH secretion appears to involve the Ca-sensing receptor and G protein activation of the phospholipase C second-messenger system (120,121). PTH activation of phospholipase C leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol1,4,5-triphosphate (IP~) and diacylglycerol. IP 3 binds to specific receptors on intracellular organelles (endoplasmic reticulum, calciosomes), leading to an acute transient rise in cytosolic C a 2+ with subsequent activation of calmodulin-dependent protein kinases. Diacylglycerol activates protein kinase C. Mg depletion could perturb this system via several mechanisms. First, Mg 2+ -dependent guanine nucleotide regulating proteins (e.g., G 8 or Gll ) are also involved in activation of phospholipase C (122,123). Mg 2+ has also been shown to be a noncompetitive inhibitor of IPs-induced Ca 2+ release (124). A reduction of Mg 2+ from 300 to 30 IxM increased C a 2+ release in response to IP~ by two- to threefold in mitochondrial membranes obtained from canine cerebellum (124). In these same studies Mg 2+ also was found to inhibit IP s binding to its receptor. Magnesium, at concentrations of 0.5 mM, decreased maximal IP s binding threefold (124). These Mg 2+ concentrations are within the estimated physiologic intracellular range (200-500 IxM) and therefore Mg 2+ may be an important physiologic regulator of the phospholipase C second-messenger system. Second-messenger systems are widely distributed enzymes in the body and if the above hypothesis were true, the secretion and action of other hormones might also exhibit impaired activity in Mg deficiency. The action of ACTH, TRH, GnRH, and glucagon, however, have been demonstrated to be normal in hypocalcemic Mg-deficient patients (125). It was hypothesized that the adenylate cyclase enzyme in the target tissues of those hormones might have a lower Mg 2+ requirement or be less inhibitable by Ca 2+ compared to the parathyroid, kidney, and bone, which would afford selective protection from the effects of Mg depletion. Specific forms of adenylate cyclase have distinct activities in the presence of Mg 2+(126). Prior investigations have suggested that Mg affinity for adenylate cyclase is higher (lower KaMg ) i n liver (127), adrenal (128), and pituitary (129) than in parathyroid (114). In one study, investigation of Ka Mg and /~ ca in tissues from one species (guinea pig) demonstrated that under agonist stimulation the Ka Mg from liver < thyroid < kidney = bone, and the/q. Ca 2+ for liver > renal > kidney = bone (117) These data suggest that adenylate cyclase regulation by divalent cations varies from tissue to tissue and may explain the greater propensity for disturbed mineral homeostasis in Mg deficiency. It is clear that the effect of Mg depletion on cellular function in terms of the second-messenger systems is

MAGNESIUM IN PARATHYROIDFUNCTION / most complex, potentially involving substrate availability, G protein activity, release of and sensitivity to intracellular Ca 2+, and phospholipid metabolism.

D I A G N O S I S OF M A G N E S I U M DEFICIENCY As m e n t i o n e d above, Mg is principally an intracellular cation. Less than 1% of the body Mg content is in the extracellular fluid compartments. The serum Mg concentration, therefore, may not reflect the intracellular Mg content. A serum Mg concentration that will result in impaired PTH secretion or resistance cannot be predicted. Nevertheless, measurement of the serum Mg concentration is the most available and commonly employed test to assess Mg status. The normal serum Mg concentration ranges from 1.7 to 2.2 m g / d l (0.71-0.91 mM); a value -<1.7 m g / d l usually indicates Mg deficiency (3-5). Ion-specific electrodes have become available for determining ionized magnesium in serum or plasma (130). Results suggest that this may be a better index of magnesium status than the total serum magnesium concentration. Differences in ionize&magnesium analyzers must be resolved, however, before use in routine patient care (131,132). The total Mg content or concentration in a n u m b e r of tissues, including red blood cells, skeletal muscle, bone, and peripheral lymphocytes, has been assessed as an index of Mg status. The Mg content of the peripheral lymphocyte has been u n d e r investigation and it has been found to correlate with skeletal and cardiac muscle Mg content (3-5). This has been used in a n u m b e r of research studies and, in general, seems to be a more accurate indicator of Mg status than the serum Mg concentration. A great deal of overlap with the normal range is seen, however, and therefore lymphocyte Mg content is probably not a sufficiently discriminatory test to diagnose Mg deficiency in any given patient. The

TABLE

5

773

Mg tolerance test has been used for many years and appears to be an accurate means of assessing Mg status (3-5). Retention of a parenterally administered Mg load is greater than normal in both hypomagnesemic patients and normomagnesemic patients at risk for Mg deficiency. A suggested protocol for the Mg tolerance test is shown in Table 5. Using this protocol, 23 normal controls retained 14 +_ 4% (mean +_ SEM) of the Mg load; 15 hypomagnesemic patients retained 85 _+ 3% (133). Twenty-four patients at risk for Mg deficiency (chronic alcoholics) also retained a significantly greater percentage of the Mg load than did the normal controls, 51 +_ 5%. These data suggest that the Mg tolerance test is a more sensitive m e t h o d to detect Mg deficiency than is the serum Mg concentration. T h o u g h this test does appear to be quite discriminatory in patients with normal renal function, its usefulness may be limited if the patient has a renal Mg leak or is on a medication that induces renal Mg wasting.

TREATMENT OF MAGNESIUM DEFICIENCY Patients who present with signs and symptoms of Mg deficiency should be treated with Mg. These patients will usually be hypomagnesemic a n d / o r have an abnormal Mg tolerance test (3,5). These circumstances usually indicate treatment by parenteral administration. An effective treatment regimen is the administration of 2 g of MgSO4"7H20 (200 mg elemental Mg) as a 50% solution every 8 hours intramuscularly. These injections can be painful and a continuous intravenous infusion of 600 mg elemental Mg over 24 hours therefore may be preferred and is better tolerated. Either regimen will usually result in a normal or slightly elevated serum Mg concentration. The restoration of a normal serum Mg concentration does not indicate repletion of body Mg stores, however, and therapy should be continued

Suggested Protocol for Clinical Use of Magnesium Tolerance

Test •

Procedure Collect base line urine (spot or timed) for magnesium/creatinine ratio Infuse 0.2 mEq (2.4 mg) elemental magnesium per kg lean body weight in 50 ml 5% dextrose over 4 hours Collect urine (starting with infusion) for magnesium and creatinine for 24 hours Calculation Percentage magnesium retained is given by the following formula: 1-

Postinfusion 24-hour urine magnesium - Preinfusion urine magnesium/creatinine × Postinfusion urine creatinine Total elemental magnesium infused

Criteria for Mg deficiency >50% retention at 24 hours -- definite deficiency >25% retention at 24 hours - probable deficiency

x

100

774

/

CHAPTER48

for approximately 3-7 days. By this time symptoms should resolve and biochemical abnormalities such as hypocalcemia and hypokalemia should correct. Patients who are hypomagnesemic and have seizures or an acute arrhythmia may be given 100-200 mg Mg as an intravenous injection over 5-10 minutes, followed by 600 mg/day. Ongoing Mg losses should be monitored during therapy. If the patient continues to lose Mg from the intestine or kidney, therapy may have to be continued for a longer duration. Once repletion has been accomplished, patients usually can maintain a normal Mg status on a regular diet, provided the reason for the Mg deficiency has been corrected. If repletion is accomplished and the patient cannot eat, a parenteral maintenance dose of 100 mg of Mg should be given daily. Patients who have chronic Mg loss from the intestine or kidney may require continued oral Mg supplementation. Magnesium salts in the form of sulfate, lactate, hydroxide, oxide, chloride, and glycerophosphate are available. An initial daily dose of 300 mg, to as high as 600 mg, of elemental Mg may be used. The Mg is given in divided doses three or four times a day to avoid its cathartic effect. Caution should be taken in Mg therapy in patients with any degree of renal failure. If a decrease in glomerular filtration rate exists, the dose of Mg should be halved, and the serum Mg concentration must be monitored daily. If hypermagnesemia ensues, therapy must be stopped.

REFERENCES

11. Kayne LH, Lee DBN. Intestinal magnesium absorption. Miner Electrolyte Metab 1993;19:210-217. 12. Krejs GJ, Nicar MJ, Zerwekh JE, Normal DA, Kane MG, Pak CY. Effect of 1,25-dihydroxyvitamin D 3 on calcium and magnesium absorption in the healthy human jejunum and ileum. AmJMed 1983;75:973-976. 13. Hodgkinson A, Marshall DH, Nordin BEC. Vitamin D and magnesium absorption in man. Clin Sci 1979;57:121-123. 14. Wong ET, Rude RK, Singer FR, Shaw ST. A high prevalence of hypomagnesemia and hypermagnesemia in hospitalized patients. A m J Clin Patho11983;79:348-352. 15. Ryzen E, Wagers PW, Singer FR, Rude RK. Magnesium deficiency in a medical ICU population. Crit Care Med 1985;13:19-21. 16. Rude RK. Magnesium metabolism. In: Becker KL, ed. Principles and practice of endocrinology and metabolism, 2nd Ed. Philadelphia: Lippincott, 1995:616-622. 17. Tosiello L. Hypomagnesemia and diabetes mellitus. A review of clinial implications. Arch Intern Med 1996;156:1143. 18. Shah GM, Kirschenbaum MA. Renal magnesium wasting associated with therapeutic agents. Miner Electrolyte Metab 1991 ;17:58--64. 19. Dominquez JH, Gray RW, Lemann J, Jr. Dietary phosphate deprivation in women and men: Effects on mineral and acid balances, parathyroid hormone and the metabolism of 24-OH-vitamin D. J Clin Endocrinol Metab 1976;43:1056-1068. 20. Simon DB, Lifton RP. The molecular basis of inherited hypokalemic alkalosis: Bartter's and Gitelman's syndromes. Am J Physiol 1996;271:F961-F966. 21. Simon DB, Karet FE, HamdanJM, DePietro A, Sanjad SA, Lifton RP. Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2C1 cotransporter NKCC2. Nat Genet 1996;13:183-188. 22. Simon DB, Nelson-Williams C, Bia MJ, Ellison D, Karet F, Molina AM, Vaara I, Iswata F, Cushner HM, Koolen M, Gainza FJ, Gitelman HJ, Lifton RP. Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-C1 cotransporter. Nat Genet 1996;12:24-30. 23. Whang R, Hampton EM, Whang DD. Magnesium homeostasis and clinical disorders of magnesium deficiency. Ann Pharmacol

1994;28:220.

1. Frausto da Silva JJR, Williams RJP. The biological chemistry of magnesium:phosphate metabolism. In: The biological chemistry of the elements. Oxford:Oxford Univ. Press, 1991:241-267. 2. Birch NJ. Magnesium and the cell. New York:Academic Press, 1993. 3. Rude RK. Magnesium. In: Stipanuk MH, ed. Biochemical and physiological aspects of human nutrition. Philadelphia:Saunders, 2000:671-685. 4. Elin R. Assessment of magnesium status. Clin Chem 1987;33: 1965-1970. 5. Rude RK. Magnesium disorders. In: Kokko JP, Tannen RL, eds. Fluids and electrolytes, 3rd Ed. Philadelphia:Saunders, 1996: 421-445. 6. Quamme GH. Magnesium homeostasis and renal magnesium handling. Miner Electrolyte Metab 1993:19:218-225. 7. Quamme GA. Renal magnesium handling: New insights in understanding old problems. Kidney Int 1997:52:1180. 8. Rude RK, Ryzen E. TmMg and renal Mg threshold in normal man in certain pathophysiologic conditions. Magnesium 1986;5: 273-281. 9. Brown EM, Hebert SC. A cloned Ca ++-sensing receptor: A mediator of direct effects of extracellular Ca ++ on renal function. J A m Soc Nephro11995;6:1530-1540. 10. Fine KD, Santa Ana CA, Porter JL, Fortran JS. Intestinal absorption of magnesium from food and supplements. J Clin Invest 1991 ;88:396-402.

24. Wacker WEC, Moore FD, Ulmer DD, Vallee BL. Normocalcemic magnesium deficiency tetany. JAMA 1962;180:161-163. 25. Iseri LT, Fairshter RD, Hardemann JL, Brodsky MA. Magnesium and potassium therapy in multifocal atrial tachycardia. Am Heart J 1985; 110: 789-794. 26. Dyckner T, Wester PO. Magnesium deficiency contributing to ventricular tachycardia. Acta Med Scand 1982;212:89-91. 27. Whang R, Oei TO, Aikawa JK, Watanabe A, Vannatta J, Fryer A, Markanich M. Predictors of clinical hypomagnesemia. Arch Intern Med 1984;144:1794-1796. 28. Shils ME. Experimental human magnesium depletion. Medicine

1969;48:61-82.

29. Ryan ME Interrelationships of magnesium and potassium homeostasis. Miner Electrolyte Metab 1993; 19:290-295. 30. Rude RK, Oldham SB, Singer FR. Functional hypoparathyroidism and parathyroid hormone end-organ resistance in human magnesium deficiency. Clin Endocrinol 1976;5:209-224. 31. Buckle RM, Care AD, Cooper CW, Gitelman HJ. The influence of plasma magnesium concentration on parathyroid hormone secretion. JEndocrinol 1968;42:529-534. 32. Sherwood LM, Herrman I, Bassett CA. Parathyroid hormone secretion in vitro: Regulation by calcium and magnesium ions. Nature 1970;225:1056-1057. 33. Oldham SB, Fischer JA, Capen C, Sizemore GW, Arnaud CD. Dynamics of parathyroid hormone secretion in vitro. A m J Med 1971 ;50:650-657.

MAGNESIUM IN PARATHYROID FUNCTION 34. Cholst IN, Steinberg SE Troper PJ, Fox HE, Segre GV, Bilezikian JP. The influence of hypermagnesemia on serum calcium and parathyroid hormone levels in human subjects. N Engl J Med 1984;310:1221-1225. 35. Ferment O, Garnier PE, Touitou Y. Comparison of the feedback effect of magnesium and calcium on parathyroid hormone secretion in man. JEndocrino11987;113:117-122. 36. ToffalettiJ, Cooper DL, Lobaugh B. The response of parathyroid hormone to specific changes in either ionized calcium, ionized magnesium, or protein-bound calcium in humans. Metabolism 1991;40:814-818. 37. HabenerJF, PottsJT, Jr. Relative effectiveness of magnesium and calcium on the secretion and biosynthesis of parathyroid hormone in vitro. Endocrinology 1976;98:197-202. 38. Mayer GP, Hurst JG. Comparison of the effects of calcium and magnesium on parathyroid hormone secretion rate in calves. Endocrinology 1978;102:1803-1807. 39. Wallace J, Scarpa A. Regulation of parathyroid hormone secretion in vitro by divalent cations and cellular metabolism. J Biol Chem 1982;257:10613-10616. 40. Brown EM, Thatcher JG, Watson EJ, Leombruno R. Extracellular calcium potentiates the inhibitory effects of magnesium on parathyroid function in dispersed bovine parathyroid cells. Metabolism 1984;33:171-176. 41. Hamilton JW, Spierto FW, MacGregor RR, Cohn DV. Studies on the biosynthesis in vitro of parathyroid hormone. J Biol Chem 1971 ;246:3224-3233. 42. Raisz LG. Effects of calcium on uptake and incorporation of amino acids in the parathyroid glands. Biochim Biophys Acta

1967:148;460-468.

43. Lee MJ, Roth SI. Effect of calcium and magnesium on dioxyribonucleic acid synthesis in rat parathyroid glands in vitro. Lab Invest 1975;33:72-79. 44. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC. Cloning and characterization of an extracellular Ca 2+ sensing receptor from bovine parathyroid. Nature 1993;366:575-580. 45. Nemeth EE Ca 2+ receptor-dependent regulation of cellular function. News Physiol Sci 1995;10:1-5. 46. Hebert SC. Extracellular calcium-sensing receptor: Implications for calcium and magnesium handling in the kidney. Kidney Int

1996;50:2129-2139.

47. Miki H, Maercklein PB, Fitzpatrick LA. Effect of magnesium on parathyroid cells: Evidence for two sensing receptors or two intracellular pathways? AmJPhysio11997;272:El-E6. 48. Shils ME, Magnesium, calcium and parathyroid hormone interactions. Ann NYAcad Sci 1980;355:165-180. 49. Anast CS, Forte LE Parathyroid function and magnesium depletion in the rat. Endocrinology 1983;113:184-189. 50. Rude RK, Kirchen ME, Gruber HE, Meyer MH, Luck JS, Crawford DL. Magnesium deficiency-induced osteoporosis in the rat: Uncoupling of bone formation and bone resorption. Magnesium Res 1999;4:257-267. 51. Fatemi S, Ryzen E, Flores J, Endres DB, Rude RK. Effect of experimental human magnesium depletion on parathyroid hormone secretion and 1,25-dihydroxyvitamin D metabolism. J Clin Endocrinol Metab 1991 ;73:1067-1072. 52. Targovnik JH, Rodman JS, Sherwood LM. Regulation of parathyroid hormone secretion in vitro: Quantitative aspects of calcium and magnesium ion control. Endocrinology 1971;88: 1477-1482. 53. Mahaffee DD, Cooper CW, Ramp WK, Ontjes DA. Magnesium promotes both parathyroid hormone secretion and adenosine 3',5'-monophosphate product in rat parathyroid tissues and reverses the inhibitory effects of calcium on adenylate cyclase. Endocrinology 1982;110:487-495.

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54. Anast CS, MohsJM, Kaplan SL, Burns TW. Evidence for parathyroid failure in magnesium deficiency. Science 1972;177:606-608. 55. Suh SM, Tashjian AH Jr, Matsuo N, Parkinson DK, Fraser D. Pathogenesis of hypocalcemia in primary hypomagnesemia: Normal end-organ responsiveness to parathyroid hormone, impaired parathyroid gland function. J Clin Invest 1973;52: 153-160. 56. Chase LR, Slatopolsky E. Secretion and metabolic efficacy of parathyroid hormone in patients with severe hypomagnesemia. J Clin Endocrinol Metab 1974;38:363-371. 57. Wiegmann T, Kaye M. Hypomagnesemic hypocalcemia. Arch Intern Med 1977;137:953-955. 58. Rude RK, Oldham SB, Sharp CF, Jr, Singer FR. Parathyroid hormone secretion in magnesium deficiency. J Clin Endocrinol Metab 1978;47:800-806. 59. Allgrove J, Adami S, Fraher L, Reuben A, O'Riordan JLH. Hypomagnesaemia: Studies of parathyroid hormone secretion and function. Clin Endocrinol 1984;21:435-449. 60. BethuneJE, Turpin RA, Inoui H. Effect of parathyroid hormone extract on divalent ion excretion in man. J Clin Endocrinol Metab 1968;28:673-678. 61. Estep H, Shaw WA, Watlington C, Hobe R, Holland W, Tucker SG. Hypocalcemia due to hypomagnesemia and reversible parathyroid hormone unresponsiveness. J Clin Endocrino11969;29:842--848. 62. Muldowney FP, McKenna TJ, Kyle LH, Freaney R, Swan M. Parathormone-like effect of magnesium replenishment in steatorrhea. N EnglJ Med 1970;281:61-68. 63. Connor TB, Toskes P, Mahaffey J, Martin LG, Williams JB, Walser M. Parathyroid function during chronic magnesium deficiency. Hopkins Med J 1972; 131:100-117. 64. Woodard JC, Webster PD, Carr AA. Primary hypomagnesemia with secondary hypocalcemia, diarrhea and insensitivity to parathyroid hormone. Digest Dis 1972;17:612-618. 65. MacManus J, Heaton FW, Lucas PW. A decreased response to parathyroid hormone in magnesium deficiency. J Endocrinol 1971;49:253-258. 66. Reddy CR, Coburn JW, Hartenbower DL, Friedler RM, Brickman AS, Massry SG, JowseyJ. Studies on mechanisms of hypocalcemia of magnesium depletion. J Clin Invest 1973;52:3000-3010. 67. Brietenbach RP, Gonnerman WA, Erring WL, Anast CS. Dietary magnesium, calcium homeostasis, and parathyroid gland activity of chickens. AmJPhysio11973;225:12-17. 68. Levi J, Massry SG, Coburn JW, Llach E Kleeman CR. Hypocalcemia in magnesium-depleted dogs: Evidence for reduced responsiveness to parathyroid hormone and relative failure of parathyroid gland function. Metabolism 1974;23:323-335. 69. Forbes RM, Parker HM. Effect of magnesium deficiency on rat bone and kidney sensitivity to parathyroid hormone. J Nutr 1980;110:1610-1617. 70. Raisz LG, Niemann I. Effect of phosphate, calcium and magnesium on bone resorption and hormonal responses in tissue culture. Endocrinology 1969;85:446-452. 71. FreitagJJ, Martin KJ, Conrades MB, Bellorin-Font E, Teitelbaum S, Klahr S, Slatopolsky E. Evidence for skeletal resistance to parathyroid hormone in magnesium deficiency. J Clin Invest 1979;64:1238-1244. 72. SaletJ, Polonovski CL, DeGouyon F, Pean G, Melekian B, Fournet JE Tetanie hypocalcemique recidivante par hypomagnesemie congenitale. Archives Francaises de Pediatrie 1966;23:749-767. 73. Stromme JH, Nesbakken R, Normann T, Skjorten F, Skyberg D, Johannessen B. Familial hypomagnesemia. Acta Paediat Scand 1969;58: 433-444. 74. Suh SM, Tashjian AH, Matsuo N, Parkinson DK, Fraser D. Pathogenesis of hypocalcemia in primary hypomagnesemia: Normal end-organ responsiveness to parathyroid hormone, impaired parathyroid gland function. J Clin Invest 1973;52:153-160.

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75. Chase LR, Slatopolsky E. Secretion and metabolic efficacy of parathyroid hormone in patients with severe hypomagnesemia. J Clin Endocrinol Metab 1974;38:363-371. 76. Hahn TJ, Chase LR, Avioli LV. Effect of magnesium depletion on responsiveness to parathyroid hormone in parathyroidectomized rats. J Clin Invest 1972;51:886-891. 77. Pak CYC, Diller EC. Ionic interaction with bone mineral. Calcif Tissue Res 1969;4:69-77. 78. MacManus J, Heaton FW. The influence of magnesium on calcium release from bone in vitro. Biochim Biophy Acta 1970;215: 360-367. 79. Medalle R, Waterhouse C. A magnesium-deficient patient presenting with hypocalcemia and hyperphosphatemia. Ann Intern Med 1973;79:76-79. 80. Carpenter TO. Disturbances of vitamin D metabolism and action during clinical and experimental magnesium deficiency. Magnesium Res 1988;1:131-139. 81. Leicht E, Biro G. Mechanisms of hypocalcaemia in the clinical form of severe magnesium deficit in the human. Magnesium Res 1992;5:37-44. 82. Homer L. Hypoparathyroidism requiring massive amounts of medication, with apparent response to magnesium sulfate. J Clin Endocrinol Metab 1961;21:219-223. 83. Jones KH, Fourman E Effects of infusions of magnesium and of calcium in parathyroid insufficiency. Clin Sci 1966;30:139-150. 84. Rossler A, Rabinowitz D. Magnesium induced reversal of vitamin D resistance in hypoparathyroidism. Lancet 1973;1:803-806. 85. Medalle R, Waterhouse C, Hahn TJ. Vitamin D resistance in magnesium deficiency. A m J Clin Nutr 1976;29:854-858. 86. Heaton FW, Fourman E Magnesium deficiency and hypocalcemia in intestinal malabsorption. Lancet 1965;2:50-52. 87. Reddy V, Sivakumar B. Magnesium-dependent vitamin D-resistant rickets. Lancet 1974;1:963-965. 88. Leicht E, Biro G, Keck E, Langer HJ. Die hypomagnesiaemiebedingte hypocalciaemie: funktioneller hypoparathyreoidismus, Parathormon-und Vitamin D resistenz. Klin Wochenschr

1990;68:678-684.

89. Coenegracht JM, Houben HGJ. Idiopathic hypomagnesemia with hypocalcemia in an adult. Clin Chim Acta 1974;50:349-357. 90. Selby PL, Peacock M, Bambach CE Hypomagnesaemia after small bowel resection: Treatment with 1 e~-hydroxylated vitamin D metabolites. BrJSurg 1984;71:334-337. 91. Ralston S, Boyle IT, Cowan RA, Crean GP, Jenkins A, Thomson WS. PTH and vitamin D responses during treatment of hypomagnesaemic hypoparathyroidism. Acta Endocrinol 1983;103:535-538. 92. Graber ML, Schulman G. Hypomagnesemic hypocalcemia independent of parathyroid hormone. Ann Intern Med 1986;104: 804-806. 93. Lifshitz E Harrison HC, Harrison HE. Response to vitamin D of magnesium deficient rats. Proc Soc Exp Biol Med 1967;125:472-476. 94. Rayssiguier Y, Carre M, Ayigbede O, Miravet L. Activite du 1-25 dihydroxycholecalciferol chez le rat carence en magnesium. C R Acad Sci Paris 1975;281:731-734. 95. Miravet L, Ayigbede O, Carre M, Rayssiguier Y, Larvor E Lack of vitamin D action on serum calcium in magnesium deficient rats. In: Cantin M, Seelig MS, eds. Magnesium in health and disease. New York:Spectrum, 1980:281-289. 96. McAleese DM, Forbes RM. Experimental production of magnesium deficiency in lambs on a diet containing roughage. Nature 1959; 184:2025-2026. 97. Smith RH. Calcium and magnesium metabolism in calves. Biochim J 1958; 70:201-205. 98. Winnacker JL, Anast CS. Vitamin D metabolism in magnesium and nutritional deficiency. 56th Annual Meeting of the Endocrine Society, Atlanta, GA, 1974;A179.

99. HiguchiJ, Lukert B. Effects of magnesium depletion on vitamin D-metabolism and intestinal calcium transport. Clin Res 1974;22:617. 100. CoburnJW, Reddy CR, Brickman AS, Hartenbower DL, Friedler RM. Vitamin D metabolism in magnesium deficiency. Clin Res 1975;23:3933. 101. Rude RK, Adams JS, Ryzen E, Endres DB, Niimi H, Horst RL, Haddad JF, Singer FR. Low serum concentrations of 1,25-dihydroxyvitamin D in human magnesium deficiency. J Clin Endocrinol Metab 1985;61:933-940. 102. Fuss M, Cogan E, Gillet C, Karmali R, Guerts J, Bergans A, Brauman H, Bouillon R, Corvilain J. Magnesium administration reverses the hypocalcaemia secondary to hypomagnesaemia despite low circulating levels of 25-hydroxyvitamin D and 1,25dihydroxyvitamin D. Endocrinology 1985;22:807-815. 103. Fuss M, Bergmann P, Bergans A, BagonJ, Cogan E, Pepersack T, VanGossum M, Corvilain J. Correction of low circulating levels of 1,25-dihydroxyvitamin D by 25-hydroxyvitamin D during reversal of hypomagnesaemia. Clin Endocrinol 1989;31:31-38. 104. Leicht E, Schmidt-Gayk H, Langer HJ, Sniege N, Biro G. Hypomagnesaemia-induced hypocalcaemia: Concentrations of parathyroid hormone, prolactin and 1,25-dihydroxyvitamin D during magnesium replenishment. Magnesium Res 1992;5:1,33-36. 105. Lukert BE Effect of magnesium depletion on vitamin D metabolism in man. In: Cantin M, Seelig MS, eds. Magnesium in health and disease. New York:Spectrum, 1980:275-279. 106. Saggese G, Federico G, Bertelloni S, Baroncelli GI, Calisti L. Hypomagnesemia and the parathyroid hormone-vitamin D endocrine system in children with insulin-dependent diabetes mellitus: Effects of magnesium administration. J Pediatr 1991;

118:220-225.

107. Gray RW, Omdahy JL, Ghazarian JG, DeLuca HE 25Hydroxycholecalciferol-l-hydroxylase. J Biol Chem 1972;247: 7528-7532. 108. Fisco F, Traba ML. Influence of magnesium on the in vitro synthesis of 24,25-dihydroxyvitamin D3 and l cx, 25-dihydroxyvitamin D3. Magnesium Res 1992;5:5-14. 109. Neer EJ. Heterotrimeric G proteins: Organizers of transmembrane signals. Cell 1995;80:249-257. 110. Northup JK, Smigel MD, Gilman AG. The guanine nucleotide activating site of the regulatory component of adenylate cyclase: Identification by ligand binding. JBiol Chem1982;257:11416-11423. 111. Maguire ME. Hormone-sensitive magnesium transport and magnesium regulation of adenylate cyclase. Trends Pharmacol Sci 1984;5:73-77. 112. Cech ST, Broaddus WC, Maguire ME. Adenylate cyclase: The role of magnesium and other divalent cations. Mol Cell Biochem 1980;33:67-92. 113. Sunahara RK, Dessauer CW, Gilman AG. Complexity and diversity of mammalian adenylyl cyclases. Annu Rev Pharmacol Toxicol 1996;36:461-480. 114. Oldham SB, Rude RK, Molloy CT, Lipson LG. The effects of magnesium on calcium inhibition of parathyroid adenylate cyclase. Endocrinology 1984;115:1883--1890. 115. Rude RK. Renal cortical adenylate cyclase: Characterization of magnesium activation. Endocrinology 1983; 113:1348-1355. 116. Rude RK. Skeletal adenylate cyclase: effect of Mg2+, Ca2+, and PTH. Calcif Tissue Int 1985;37:318-323. 117. Rude RK, Oldham SB. Hypocalcemia of Mg deficiency: Altered modulation of adenylate cyclase by Mg ++ and Ca ++ may result in impaired PTH secretion and PTH end-organ resistance. In: Altura BT, Durlach J, Seelig M, eds. Magnesium in ceUularprocesses and medicine. Basel:Karger, 1985:183-195. 118. George GA, Heaton FW. Changes in cellular composition during magnesium deficiency. BiochemJ 1975;152:609-615.

MAGNESIUM IN PARATHYROID FUNCTION 119. Ryan MD, Ryan ME Lymphocyte electrolyte alterations during magnesium deficiency in the rat. IsrJMed Sci 1979;148:108-109. 120. Brown EM. Extracellular Ca 2+ sensing, regulation of parathyroid cell function, and role of Ca 2+ and other ions as extracellular (first) messengers. Physiol Rev 1991;71:371-411. 121. Dunlay R, Hruska K. PTH receptor coupling to phospholipase C is an alternate pathway of signal transduction in bone and kidney. Am J Physio11990;258:F223-F231. 122. Babich M, King KL, Nissenson RA. G protein-dependent activation of a phosphoinositide-specific phospholipase C in UMR-106 osteosarcoma cell membranes. J Bone Miner Res 1989;4:549-556. 123. Litosch I. G protein regulation of phospholipase C activity in a membrane-solubilized system occurs through a Mg2+- and timedependent mechanism. J Biol Chem 1991 ;266:4764-4771. 124. Volpe P, Alderson-Lang BH, Nickols GA. Regulation of inositol 1,4,5-trisphosphate-induced Ca 2+ release. I. Effect of Mg2+. A m J Physiol 1990;258:C1077-C1085. 125. Cohan BW, Singer FR, Rude RK. End-organ response to adrenocorticotropin, thyrotropin, gonadotropin-releasing hormone, and glucagon in hypocalcemic magnesium deficient patients. J Clin Endocrinol Metab 1982;54:975-979. 126. Smit MJ, Iyengar R. Mammalian adenylyl cyclases. In: Cooper DMF, ed. Advances in second messenger and phosphoprotein research, Philadelphia:Lippincott-Raven, 1998:1-21. 127. Londos C, Preston MS. Activation of the hepatic adenylate cyclase system by divalent cations. J Biol Chem 1977;252:5957-5961.

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128. Glynn P, Cooper DMF, Schulster D. Modulation of the response of bovine adrenocortical adenylate cyclase to corticotropin. Biochemistry 1977;168:277-282. 129. Porier G, DeLean A, Pelletier G, Lemay A, Labrie E Purification of adenohypophyseal plasma membranes and properties of associated adenylate cyclase. J Biol Chem 1974;249:316-322. 130. Altura BT, Shirey TL, Young CC, Hiti J, Dell'Orfano K, Handwerker SM, Altura BM. A new method for the rapid determination of ionized Mg in whole blood, serum and plasma. Methods Find Exp Clin Pharmacol 1992;14:297-304. 131. Mimouni FB. The ion-selective magnesium electrode: A new tool for clinicians and investigators. J Am CollegeNutr 1996;15:4-5. 132. Cecco SA, Hristova EN, Rehak NN, Elin RJ. Clinically important intermethod differences for physiologically abnormal ionized magnesium results. Am J Clin Pathol 1997;108: 564-569. 133. Ryzen E, Elbaum N, Singer FR, Rude RK. Parenteral magnesium tolerance testing in the evaluation of magnesium deficiency. Magnesium 1985;4:137-147. 134. Rude RK. Parathyroid function in magnesium deficiency. In: Itokawa Y, Durlach J, eds. Magnesium in health and disease. London: John Libbey, 1989:317-321. 135. Rude RK, Oldham SB. Disorders of magnesium metabolism. In: Cohen RD, Lewis B, Alberti KGMM, Denman AM, eds. The metabolic and molecular basis of acquired disease. London:Bailliere Tindall, 1990:1124-1148.

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CHAPTV 4 9

The Molecular Genetics of Hypoparathyroidism

R. V. THAKKER Nuffield Department of Clinical Medicine, University of Oxford, Headington, Oxford OX3 9DU, United Kingdom

INTRODUCTION

of these forms of hypoparathyroidism has been investigated and this has helped to elucidate further the mechanisms involved in the control of extracellular calcium homeostasis. Extracellular calcium ion concentration is tightly regulated through the actions of PTH on kidney and bone. The intact peptide is secreted by the parathyroid glands at a rate that is inversely proportional to the prevailing extracellular calcium ion concentration. Theoretically, hypocalcemic disorders can be classified according to whether they arise from a deficiency of PTH, a defect in the PTH/PTH-related protein receptor (i.e., the P T H / P T H r P receptor), or an insensitivity to PTH caused by defects downstream of the P T H / P T H r P receptor (Fig. 1). Application of developments in molecular biology to the study of hypoparathyroid disorders has enabled characterization of some of the mechanisms involved in the regulation of parathyroid gland development, of PTH secretion, and of PTH-mediated actions in target tissues (1,2). Thus, mutations in the calcium-sensing receptor gene have been reported in patients with autosomal dominant hypocalcemia with hypercalciuria. In addition, mutations in the PTH gene and the mitochondrial genome have been demonstrated to be associated with some forms of hypoparathyroidism, mutations in the P T H / P T H r P receptor gene have been identified in patients with Blomstrand chondrodysplasia, and mutations in the alpha chain of the stimulatory G protein (Gsot) have been found in individuals with pseudohypoparathyroidism type la and pseudo-pseudohypoparathyroidism. Furthermore, the gene that causes the

In recent years, important advances in endocrinology have resulted from the application of the methods of molecular biology (1). These advances have helped not only to demonstrate the roles of mutant genes in the etiology of some inherited disorders, but have also helped to identify the chromosomal locations of susceptibility genes that predispose individuals at risk for a particular disorder. The results of such studies in the molecular genetics of the hypoparathyroid disorders (Table 1) have helped to elucidate some of the underlying pathways that are involved in calcium homeostasis (Fig. 1). Hypoparathyroidism is an endocrine disorder in which hypocalcemia and hyperphosphatemia are the result of a deficiency in parathyroid h o r m o n e (PTH) secretion or action. There are a variety of causes (2) of hypoparathyroidism (Table 1) and the disorder may occur as part of a pluriglandular autoimmune disorder or as a complex congenital defect, as for example in the DiGeorge syndrome or in association with other developmental anomalies involving dysmorphic features, nephropathy, sensorineural deafness, lymphedema, and cortical thickening of tubular bones. In addition, hypoparathyroidism may develop as a solitary endocrinopathy, and this form has been called isolated or idiopathic hypoparathyroidism. Familial occurrences of isolated hypoparathyroidism have been reported and autosomal dominant (3,4), autosomal recessive (3-6) and X-linked recessive (7-9) inheritances have been established (10). The molecular genetic basis for each The Parathyroids, SecondEdition

779

Copyright © 2001 John R Bilezikian, Robert Marcus, and Michael A. Levine.

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CIJaeXEk49 TABLE 1

The Hypoparathyroid Disorders and Their Chromosomal Locations

Disease

Isolated hypoparathyroidism

Hypocalcemic hypercalciuria Hypoparathyroidism associated with complex congenital syndromes DiGeorge

Hypoparathyroidism associated with Kearns-Sayre and mitochondrial encephalopathy, strokelike episodes, and lactic acidosis (MELAS) Blomstrand lethal chondrodysplasia Kenney-Caffey Barakat Lymphedema Nephropathy, nerve deafness Nerve deafness without renal dysplasia Dysmorphology, growth failure Hypoparathyroidism associated with polyglandular autoimmune syndrome (APECED) Pseudohypoparathyroidism (type la) Pseudohypoparathyroidism (type lb)

Inheritance

Gene Product

Chromosomal location

Autosomal dominant Autosomal recessive X-Linked recessive Autosomal dominant

PTH PTH Unknown CaSR

11 p 15a 11 p 15a Xq26-27 3ql 3-21

Autosomal dominant

rnex40 b nex2.2-nex3 b UDFIL b

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Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal Autosomal

Mitochondrial genome

recessive dominant c recessiveC recessive dominant c dominant recessive recessive

Autosomal dominant, paternally imprinted Autosomal dominant, paternally imprinted

PTH/PTH rPR Unknown Unknown Unknown Unknown Unknown Unknown AIRE

3p21.1-p22

9 ? ? 9 lq42-43 21q22.3

GNAS1

20q13.2-13.3

GNAS1

20q13.3-13.3

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aMutations of the PTH gene identified only in some families. t'Most likely candidate genes. CMost likely inheritance shown. ~?,Location not known.

polyglandular autoimmune syndrome has been characterized and candidate genes have been identified for the DiGeorge syndrome and for pseudohypoparathyroidism type lb. These molecular genetic studies have provided unique opportunities to elucidate the pathogenesis of the hypoparathyroid disorders and these advances, together with the structure and function of the PTH gene, are reviewed in this chapter.

PTH GENE STRUCTURE AND FUNCTION The PTH gene is located on chromosome 11 p15 and consists of three exons that are separated by two introns (11). Exon 1 of the PTH gene is 85 bp long and is untranslated (Fig. 2), whereas exons 2 and 3 encode the l l5-amino acid preproPTH peptide. Exon 2 is 90 bp long and encodes the initiation (ATG) codon, the p r e h o r m o n e sequence, and part of the prohormone

sequence. Exon 3 is 612 bp long and encodes the remainder of the p r o h o r m o n e sequence, the mature PTH peptide, and the 3' untranslated region (12). The 5' regulatory sequence of the human PTH gene contains a vitamin D response element 125 bp upstream of the transcription start site, which down-regulates PTH mRNA transcription in response to vitamin D receptor binding (13,14). PTH gene transcription (as well as PTH peptide secretion) is also dependent on the extracellular calcium and phosphate (15-17) concentrations, although the presence of specific upstream "calcium or phosphate response element(s)" has not yet been demonstrated (18,19). The mature PTH peptide is released from the parathyroid chief cell as an 84-amino acid peptide via a secretory pathway that is regulated through a G protein-coupled calcium-sensing receptor (CaSR). However, the PTH mRNA is first translated as a preproPTH peptide. The pre sequence consists of a 25-amino acid signal peptide (leader sequence) that is

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TARGET CELL e.g. Kidney, Bone, or Cartilage

responsible for directing the nascent peptide into the endoplasmic reticulum to be packaged for secretion from the cell (20) (see Chapter 2). The pro sequence is 6 amino acids long and, although its function is less well defined than that of the pre sequence, it is also essential for correct PTH processing and secretion (20). After the 84-amino acid mature PTH peptide is secreted from the parathyroid cell, it is cleared from the circulation (with a short half-life of about 2 minutes) via nonsaturable hepatic uptake and renal excretion. PTH shares a receptor with PTH-related protein (also known as PTHrH, PTH-related hormone) (21), and this receptor (Fig. 1) is a member of a subgroup of G protein-coupled receptors. The P T H / P T H r P receptor gene is located on chromosome 3p21-p24 (22) and is highly expressed in kidney and bone, where PTH is its predominant agonist (23). However, the most abundant expression of the P T H / P T H r H receptor occurs in chondrocytes of the metaphyseal growth plate, where it mediates predominantly the autocrine/paracrine actions of PTHrP (24,25). Five polymorphisms of the PTH gene have been reported and two of these are associated with restriction fragment length polymorphisms (RFLPs) (26), another two are the result of single base changes that are not associated with RFLPs (27), and one is due to a variation in the length of a

FIG. 1 Schematic representation of some of the components involved in calcium homeostasis. Alterations in extracellular calcium are detected by the calcium-sensing receptor (CaSR), which is a 1078-amino acid G protein-coupled receptor. The PTH/PTHrP receptor is also a G protein-coupled receptor. Thus, Ca 2+, PTH, and PTHrP involve G protein-coupled signaling pathways, and interaction with their specific receptors can lead to activation of Gs, Gj, and Gq. Gs stimulates adenylyl cyclase (AC), which catalyzes the formation of cAMP from ATP. Gi inhibits AC activity, cAMP stimulates PKA, which phosphorylates cell-specific substrates. Activation of Gq stimulates PLC, which catalyzes the hydrolysis of phosphoinositide (PIP2) to inositol triphosphate (IP3), which increases intracellular calcium, and diacylglycerol (DAG), which activates PKC. These proximal signals modulate downstream pathways, which result in specific physiologic effects. Abnormalities in several genes and encoded proteins in these pathways have been identified in patients with hypoparathyroid disorders. Adapted from Ref. 110, Thakker RV. Parathyroid disorders: Molecular genetics and physiology. In Morris PJ, Wood WC, eds. Oxford Textbook of Surgery, 1999, by permission of Oxford University Press.

microsatellite repetitive sequence in intron 1 (28). These polymorphisms are inherited in a mendelian m a n n e r and are thus useful as genetic markers in family studies. Mutations involving the genes that encode PTH, the CaSR, the P T H / P T H r P receptor, and G~ot all affect the regulation of calcium homeostasis (Fig. 1) and are associated with the hypoparathyroid disorders and hypocalcemia (Table 1).

ISOLATED HYPOPARATHYROIDISM

PTH Gene Abnormalities A Form of Autosomal Dominant Hypoparathyroidism DNA sequence analysis of the PTH gene (Fig. 2), from one patient with autosomal dominant isolated hypoparathyroidism, has revealed a single base substitution (T --> C) in exon 2 (29), which resulted in the substitution of arginine (CGT) for cysteine (TGT) in the signal pepdde. The presence of this charged amino acid in the midst of the hydrophobic core of the signal pepdde impeded the processing of the mutant preproPTH, as demonstrated by in vitro studies. These revealed that the mutation impaired the interaction with the nascent protein and the translocadon

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t

Mir2

T

Pstl

~--231-.~ .~-..---152-.-..--~ -,t--- 212 ---~ .q~-.--- 1821------~

FIG. 2 Schematic representation of the PTH gene. The PTH gene consists of three exons and two introns; the peptide is encoded by exons 2 and 3. The PTH peptide is synthesized as a precursor that contains a pre- and a prosequence. The mature PTH peptide, which contains 84 amino acids, and larger carboxyterminal PTH fragments are secreted from the parathyroid cell. The polymorphic sites associated with the PTH gene are indicated. Two restriction fragment length polymorphisms are associated with the PTH gene, and the Taql polymorphic site is within intron 2 and the Pstl polymorphic site is 1.7 kbp downstream in the 3' direction of the gene (20). Two other polymorphisms (21) of the PTH gene, Mirl and Mir2, are located in intron 1 and exon 3, respectively, and the tetranucleotide (AAAT)n polymorphism is in intron 1 (22). The distance between the tetranucleotide (AAAT)n polymorphism and the Mirl polymorphism is 231 bp, that between the Mirl polymorphism and the Taql RFLP is 152 bp, that between the Taql RFLP site and the Mir2 polymorphism is 212 bp, and that between the Mir2 polymorphism and the Pstl RFLP is 1821 bp. Linkage disquilibrium between the (AAAT)n, Taql, and Pstl polymorphic sites has been established (22). Adapted from Parkinson and Thakker (31), Nat. Genet., with permission.

machinery, and that cleavage of the mutant signal sequence by solubilized signal peptidase was ineffective (29,30). Ineffective cleavage of the p r e p r o P T H sequence results in a molecule that does not proceed successfully through the subsequent intracellular steps required for ultimate delivery of PTH to secretory granules. The parathyroid cell, therefore, cannot respond to hypocalcemia with the secretion of native, biologically active PTH. A Form o f A u t o s o m a l Recessive Hypoparathyroidism

Another abnormality of the PTH gene, involving a d o n o r splice site at the exon 2 and intron 2 boundary, has been identified in one family with autosomal recessive isolated hypoparathyroidism (31). This mutation involved a single base transition (g + c) at position 1 of intron 2. The effect of this alteration in the invariant gt dinucleotide of the 5' d o n o r splice site consensus on mRNA processing was assessed by an analysis of the non-tissue-specific transcription of the normal and mutant PTH genes. This non-tissue-specific expression of genes has been estimated to be at the rate of one molecule of correctly spliced mRNA per 1000 cells (32,33). Although the physiologic relevance of this low level of non-tissue-specific (or illegitimate) transcription is not known, it is of clinical importance. Easily accessible peripheral blood lymphocytes can be used to

detect abnormalities in mRNA processing, thereby avoiding the requirement for tissue that may be obtainable only by biopsy. Use of these methods revealed that the d o n o r splice site mutation resulted in exon skipping, in which exon 2 of the PTH gene was lost and exon 1 was spliced to exon 3. The lack of exon 2 would lead to a loss of the initiation codon (ATG) and the signal peptide sequence (Fig. 2), which are required, respectively, for the proper c o m m e n c e m e n t of PTH mRNA translation and for the translocation of the PTH peptide. Thus, the patients' parathyroid cells would not contain any translated PTH products.

X-Linked Recessive Hypoparathyroidism Hypoparathyroidism with an X-linked recessive transmission pattern has been reported in two multigenerational kindreds (34-37). Only males were affected, suffering from infantile epilepsy and hypocalcemia. The hypoparathyroidism is due to a defect in parathyroid gland development. Linkage studies utilizing X-linked RFLPs in these two families assigned the mutant gene to chromosome Xq26-q27 (34). A novel approach utilizing mitochondrial DNA analysis established a common ancestry in these two X-linked hypoparathyroid kindreds (35). A c o m m o n ancestry for these two kindreds from eastern Missouri in the United States had been suspected, but it could not be established despite five gen-

MOLECULARGENETICSOF HYPOPARATHYROIDISM / erations of extensive genealogic records. The mitochondrial genes are transmitted through the maternal line exclusively. If relatedness among the two kindreds involved the maternal lines, analysis of mitochondrial genetic markers would reveal common features. The DNA sequence of the mitochondrial (mt) D loop was compared among individuals in both kindreds. The mt DNA sequence was identical among affected males and their maternal lineage for individuals in both kindreds, but differed at three to six positions when compared with the mitochondrial DNA of the fathers. These results demonstrated that the two kindreds with Xlinked recessive hypoparathyroidism are indeed related and that an identical gene defect is likely to be responsible for the disease. Additional studies have refined the location of this gene to be between factor IX (FIX) and DXS 1205 (36). Analysis of a yeast artificial chromosome (YAC) contig of this region (37) indicates that the region is 1.5 million base pairs in size and contains at least three candidate genes. The specific gene defect responsible for this form of X-linked hypoparathyroidism has yet to be identified. The results of this study also demonstrate that the mitochondrial genetic approach may be of importance in detecting common ancestry in other X-linked diseases.

COMPLEX SYNDROMES ASSOCIATED WITH HYPOPARATHYROIDISM Hypoparathyroidism may occur as part of a complex syndrome that may either be associated with a congenital developmental anomaly or with an autoimmune syndrome.

Congenital Syndromes Hypoparathyroidism has been reported to occur in association with the congenital developmental anomalies of the DiGeorge, the Kenney-Caffey, and the Barakat syndromes and also in syndromes associated with either lymphedema, or renal dysplasia and deafness, or with dysmorphic features and growth failure (Table 1). The inheritance of these congenital disorders, which has been reported in a few patients or a single family, has sometimes not been fully established. However, an autosomal dominant inheritance for the DiGeorge syndrome, which has been investigated by the methods of molecular genetics, is established.

DiGeorgeSyndrome Patients with the DiGeorge syndrome (DGS) typically suffer from hypoparathyroidism, immunodefi-

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ciency, congenital heart defects, and deformities of the ear, nose, and mouth (2). The disorder arises from a congenital failure in the development of the derivatives of the third and fourth pharyngeal pouches with resulting absence or hypoplasia of the parathyroids and thymus. Most cases of DGS are sporadic but an autosomal dominant inheritance of DGS has been observed and an association between the syndrome and an unbalanced translocation and deletions involving 22q11.2 have also been reported (38). In some patients, deletions of another locus on chromosome 10p have been observed in association with DGS (39). Mapping studies of the DGS deleted region on chromosome 22q11.2 have defined a 250-kb minimal critical region (40), and cloning of the translocation breakpoint on 22q11.21 from a DGS patient (41) has revealed that there are probably two genes (rnex40 and nex2.2-nex3), transcribed in opposite directions, that are disrupted by this breakpoint (42). The coding region of one of these genes, rnex40, has homology to the mouse and rat androgen receptors and contains a leucine zipper motif, suggesting that the DGS candidate gene may be a DNA-binding protein. Eleven nucleotides of the rnex40 gene are deleted at the translocation junction, making it likely that loss of function of this gene is responsible for at least part of the DiGeorge phenotype (42). Another partial transcript, referred to as nex2.2-nex3, was also identified from this breakpoint. Both rnex40 and nex2.2-nex3 are deleted in all DGS patients with 2 2 q l l deletions and studies aimed at assessing the presence of hemizygosity and mutations in these two genes in DGS patients who do not have detectable 2 2 q l l deletions are required to demonstrate the role of these genes in the etiology of the DGS. Such studies have been performed for a h u m a n homolog of a yeast gene, referred to as UDF1L, that encodes a protein involved in the degradation of ubiquinated proteins, (43). UDF1L is located on 2 2 q l l and has been found to be deleted in all of 182 patients with the 2 2 q l l deletion syndrome (43), which includes patients with the DGS, the velo-cardio-facial syndrome (VCFS), and the conotruncal anomaly face syndrome (CAFS) (38,40). However, a smaller deletion of approximately 20 kb that removed exons 1 to 3 of UDF1L was detected in one patient (43). This patient, who had a de novo deletion resulting in haploinsufficiency of UDF1L, suffered from neonatal-onset cleft palate, small mouth, low-set ears, broad nasal bridge, an interrupted aortic arch, a persistent truncus arteriosus, hypocalcemia, T lymphocyte deficiency, and syndactyly of her toes (43). These results indicate that abnormalities of the UDF1L gene are likely to contribute to the etiology of early-onset DGS. Patients with late-onset DGS (44,45) develop symptomatic hypocalcemia in childhood or during adolescence with only subtle phenotypic

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abnormalities. These late-onset DGS patients have similar microdeletions in the 22ql I region, and the molecular definition of these variants of the DiGeorge syndrome may well provide additional insights into the regulation of PTH secretion a n d / o r parathyroid gland development.

Mitochondrial Disorders Associated with Hypoparathyroidism Hypoparathyroidism has been reported to occur in three disorders associated with mitochondrial dysfunction: the Kearns-Sayre syndrome (KSS), the MELAS syndrome, and a mitochondrial trifunctional protein deficiency syndrome. KSS is characterized by progressive external ophthalmoplegia and pigmentary retinopathy before the age of 20 years, and is often associated with heart block or cardiomyopathy. The MELAS syndrome consists of a childhood onset of mitochondrial encephalopathy, lactic acidosis, and strokelike episodes. In addition, varying degrees of proximal myopathy can be seen in both conditions. Both KSS and the MELAS syndrome have been reported to occur with insulin-dependent diabetes mellitus and hypoparathyroidism (46,47). A point mutation in the mitochondrial gene tRNA leucine (UUR) has been reported in one patient with the MELAS syndrome who also suffered from hypoparathyroidism and diabetes mellitus (48). Large deletions, consisting of 6741 and 6903 base pairs and involving >38% of the mitochondrial genome, have been reported in other patients who suffered from KSS, hypoparathyroidism, and sensorineural deafness (47,49). Rearrangements (50) and duplication (51) of mitochondrial DNA have also been reported in KSS. Mitochondrial trifunctional protein deficiency is a disorder of fatty acid oxidation that is associated with peripheral neuropathy, pigmentary retinopathy, and acute fatty liver degeneration in pregnant women who carry an affected fetus. Hypoparathyroidism has been observed in one patient with trifunctional protein deficiency (52). The role of these mitochondrial mutations in the etiology of hypoparathyroidism remains to be further elucidated.

Kenney-Caffey Syndrome Hypoparathyroidism has been reported to occur in over 50% of patients with the Kenney-Caffey syndrome, which is associated with short stature, osteosclerosis and cortical thickening of the long bones, delayed closure of the anterior fontanel, basal ganglia calcification, nanophthalmos, and hyperopia (53,54). Parathyroid tissue could not be found in a detailed postmortem examination of one patient (55) and this suggests that hypoparathy-

roidism may be due to an embryologic defect of parathyroid development. A molecular genetic analysis using PTH gene RFLP analysis revealed no abnormalities (56), and mutations at other locimfor example, in developmental genesmneed to be investigated.

Additional Familial Syndromes Single familial syndromes in which hypoparathyroidism is a component have been reported (Table 1). The inheritance of the disorder in some instances has been established and molecular genetic analysis of the PTH gene has revealed no abnormalities. Thus, an association of hypoparathyroidism, sensorineural deafness, and renal dysplasia has been observed in one British family, in whom an autosomal dominant inheritance of the disorder was established (57). An analysis of the PTH gene in this family revealed no abnormalities. Autosomal recessive inheritance of hypoparathyroidism in association with renal insufficiency and development delay has been reported in one Asian family (58), and a similar analysis of the PTH gene revealed no abnormalities (22). The occurrence of hypoparathyroidism, nerve deafness, and a steroid-resistant nephrosis leading to renal failure, which has been referred to as the Barakat syndrome (59), has been reported in four brothers from one family, and an association of hypoparathyroidism with congenital lymphedema, nephropathy, mitral valve prolapse, and brachytelephalangy has been observed in two brothers from another family (60). Molecular genetic studies have not been reported from these two families. A syndrome in which hypoparathyroidism was associated with severe growth failure and dysmorphic features has been reported in 12 patients from Saudi Arabia (61). Consanguinity was noted in families of 11 of the 12 patients, the majority of which originated from the western province of Saudi Arabia. This syndrome, which is inherited as an autosomal recessive disorder, has also been identified in families of Bedouin origin, and homozygosity and linkage disequilibrium studies have located this gene to chromosome lq42-q43 (62). Molecular genetic investigations of these disorders will help to identify additional genes that regulate the development of the parathyroid glands.

Blomstrand Disease Blomstrand chondrodysplasia is an autosomal recessive h u m a n disorder characterized by early lethality, dramatically advanced bone maturation, and accelerated chondrocyte differentiation (63). Affected infants, who usually have consanguineous unaffected parents (64-68), develop pronounced hyperdensity of the

MOLECULAR GENETICS OF HYPOPARATHYROIDISM / entire skeleton with markedly advanced ossification, which results in extremely short and poorly modeled long bones. Mutations of the P T H / P T H r P receptor that impair its function are associated with Blomstrand disease (69-71). Thus it seems likely that affected infants will, in addition to the skeletal defects, also have abnormalities in other organs, including secondary hyperplasia of the parathyroid glands, presumably due to hypocalcemia.

Pluriglandular Autoimmune Hypoparathyroidism Hypoparathyroidism may occur in association with candidiasis, pernicious anemia, allopecia, vitiligo, and a u t o i m m u n e Addison's disease, and the disorder has been referred to as either the a u t o i m m u n e polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome or the polyglandular autoimm u n e type 1 syndrome (72). This disorder has a high incidence in Finland, and a genetic analysis of 58 patients from 42 Finnish families indicated autosomal recessive inheritance of the disorder (73). In addition, the disorder has been reported to have a high incidence a m o n g Iranian Jews, although the occurrence of candidiasis was less c o m m o n in this population (74). Linkage studies of 14 Finnish families m a p p e d the APECED gene to chromosome 21q22.3 (75). Further positional cloning studies led to the isolation of a novel gene from chromosome 21q22.3. This gene, referred to as MRE (autoimmune regulator), encodes a 545-amino acid protein that contains motifs suggestive of a transcriptional factor and includes a nuclear localization signal, two zinc-finger motifs, a proline-rich region, and three LXXLL motifs (76,77). Six MRE mutations have been reported in the APECED families and a codon 257 (Arg ---> Stop) mutation was the p r e d o m i n a n t abnormality in 82% of the Finnish families (76,77). The identification of the genetic defect causing APECED will not only facilitate genetic diagnosis but will also enhance the elucidation of the mechanisms causing a u t o i m m u n e disease.

CALCIUM-SENSING

RECEPTOR ABNORMALITIES The CaSR is a G protein-compled receptor that is located in the plasma m e m b r a n e of the cell (Fig. 1); this is a critical site to enable the parathyroid cell to recognize changes in extracellular calcium concentration. Thus, an increase in extracellular calcium leads to CaSR activation of the G protein signaling pathway, which in turn increases the free intracellular calcium concentration and leads to a reduction in trascription of the PTH gene. The CaSR is also expressed in the dis-

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tal renal tubule, and activation in response to hypercalcemia can increase renal calcium excretion. CaSR mutations that result in a loss of function are associated with familial benign hypercalcemia with hypocalciuria (FHH) (78-83). However, CaSR missense mutations that result in a gain of function (or added sensitivity to extracellular calcium) lead to hypocalcemia with hypercalciuria (84-88). These hypocalcemic individuals are generally assymptomatic and have serum PTH concentrations that are in the low-normal range, and because of the insensitivities of previous PTH assays in this range, such patients have often been diagnosed to be hypoparathyroid. In addition, such patients may have hypomagnesemia. Treatment with Vitamin D or its active metabolites to correct the hypocalcemia in these patients results in marked hypercalciuria with attendent risk of nephrocalcinosis, nephrolithiasis, and renal impairment. Thus, these patients need to be distinguished from those with true hypoparathyroidism.

PSEUDOHYPOPARATHYROIDISM Patients with pseudohypoparathyroidism (PHP) are characterized by hypocalcemia and hyperphosphatemia due to PTH resistance (2,89-91) (also see Chapter 51). Instead of PTH deficiency these patients have elevated levels of serum PTH that are biochemically and biologically normal. Resistance to PTH, demonstrated by little or no increase in urinary excretion of n e p h r o g e n o u s cyclic AMP and phosphate after PTH infusion, is referred to as PHP type 1. The occurrence of PHP type 1 with the somatic features of Albright's hereditary osteodystrophy (AHO) is referred to as PHP type l a, whereas t h e presence of biochemical features without somatic features is referred to as PHP type lb. The occurrence of somatic features (AHO) without the biochemical abnormalities is referred to as pseudo-pseudohypoparathyroidism (PPHP). The absence of a normal rise in urinary excretion of cyclic AMP excretion after an infusion of PTH in PHP type la and PPHP indicates a defect at some site of the PTH receptor-adenylyl cyclase system. This receptor system is regulated by at least two G proteins, one of which stimulates (Gsot) and another which inhibits (Gi0t) the activity of the m e m b r a n e - b o u n d enzyme that catalyzes the formation of the intracellular secondmessenger cyclic AME Inactivating mutations of the Gsc~ gene (referred to as GNAS1), which is located on chromosome 20q13.2, have been identified in PHP type la and PPHP patients (89-91). A mutational "hot-spot" in exon 7 that consists of a 4-bp deletion (A GACT) of codon 189 and the first nucleotide of codon 190 has been identified (89).

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These mutations, which are heterozygous, lead to an ~ 5 0 % reduction in GsoLexpression or activity, and are thought to explain, at least partially, the resistance to PTH and other hormones [e.g., thyroid-stimulating h o r m o n e (TSH), gonadotrophins, and glucagon] that mediate their actions through G protein-coupled receptors (89-91). However, a similar reduction in GsoL activity/protein is also found in patients with PPHP, who show the same physical features as individuals with PHP-la, but lack the endocrine abnormalities. Thus, GNAS1 mutations do not fully explain the PHP-la or PPHP phenotypes (89-94), and studies of PHP-la and PPHP that occurred within the same kindred revealed that the h o r m o n a l resistance is paternally imprinted. Thus, PHP-la occurred in a child only when the mutation was inherited from a m o t h e r affected with either PHP-la or PPHP (95,96). These findings in humans gained support by observations in heterozygous Gnas knockout mice that lacked exon 2. Mice that had inherited the mutant allele from a female showed undetectable Gsct protein in the renal cortex and decreased blood calcium concentration due to PTH resistance. In contrast, offspring that had inherited the mutant allele, lacking exon 2, from a male showed no evidence of endocrine abnormalities (97). T i s s u e - o r cell-specific GsoLexpression is thus likely to be involved in the pathogenesis of PHP-la and PPHP, and this may also help to explain the dominant phenotype that arises from heterozygous GNAS1 mutations. Expression of the GNAS1 gene has been shown to be further complicated by alternative splicing, which results in several different mRNAs, some of which are derived only from either the paternal or the maternal allele. This complexity of the GNAS1 gene may contribute to the unique phenotypic abnormalities observed in patients with PHP because the alternative splicing gives rise to at least three different gene products that are transcribed either from the maternal or the paternal allele, or from both alleles (98-100). The first of these products is Gset, which is encoded by exons 1 through 13 of the GNAS1 gene and mediates the biologic functions of a large variety of G protein-coupled receptors, including the P T H / P T H r P receptor. The second product is XL~s, which is encoded by a novel first exon (XL) spliced onto exons 2 through 13. The encoded XL~s, which is a ~92-kDA protein, shares considerable amino acid sequence identity with the carboxyl-terminal portion of Gset (101), but does not appear to function as a stimulatory G protein. XL~s expression occurs at numerous sites and is particularly high in endocrine and n e u r o e n d o c r i n e cells (101). Furthermore XL~s appears to be transcribed only from the paternal allele (98-100). The third product of the GNAS1 gene is NESP55 (102), which is transcribed only from the maternal allele (99,100). NESP55 is encoded by yet another exon of the GNAS1 gene, which is located upstream of exon XL and the GsoL-specific exon

1. The NESP55 exon is also spliced to exons 2 through 13, but the NESP55 protein, which is thought to act as a n e u r o e n d o c r i n e secretory protein (102), shares no amino acid sequence homology with either XL~s or Gs0L. The complexity of the GNAS1 gene and its use of different allele-specific promoters makes it plausible to postulate that mutations in the Gsct-specific exons 1 to 13 (89-94) will affect not only the functional properties of GsoL, but also those of XL~s and of NESP55. GNAS1 mutations have not been detected in PHP type l b (PHP-lb), which had originally been considered to be due to a defect of the P T H / P T H r P receptor. However, it is important to note that PHP-lb patients generally have a normal or increased osseous response to PTH, as assessed by bone turnover and osteoclastic resorption (89,90,103,104), and the normal growth plate development in these patients is consistent with a normal chondrocyte response to PTHrE These observations made it unlikely that defects in the P T H / P T H r P receptor were the cause of PHP-lb, and indeed studies of the P T H / P T H r P receptor gene and mRNA in PHP-lb patients have failed to identify mutations (105-108). In order to identify the location of the PHP-lb gene a genome-wide search was therefore undertaken in four unrelated kindreds, and this m a p p e d the PHP-lb locus to chromosome 20q13.3, a location that also contains the GNAS1 gene (109). In addition, paternal imprinting of the genetic defect was observed and this is similar to the findings in kindreds with PHP type la a n d / o r PPHE Two possible explanations for these observations have been proposed. First, PHP-lb may be due to a defect in a tissue-or cellspecific enhancer, or promoter of the GNAS1 gene, and this may affect, directly or indirectly, the expression levels of the Gset-specific transcripts a n d / o r the transcripts encoding XL~s and NESP55; or second, PHP-lb may be caused by a defect in a gene close to the GNAS1 gene that is transcribed only from the maternal allele and affects P T H / P T H r P receptor or Gset expression a n d / o r function in some renal cells.

CONCLUSION Application of the methods of molecular genetics to the study of the hypoparathyroid disorders has resulted in considerable advances that have identified some genes and their encoded proteins that are involved in the regulation of PTH synthesis and secretion, and in mediating PTH actions in different target issues. In addition, the identification of mutations has helped to provide molecular explanations and insights into a variety of familial and sporadic disorders of calcium homeostasis and bone development. Genetic mapping studies have also helped to identify the chromosomal locations of some hypoparathyroid disorders, and

MOLECULARGENETICS OF HYPOPARATHYROIDISM / future positional cloning approaches, which will progress with greater rapidity owing to the Human Genome Sequencing project, will provide additional insights into the mechanisms that regulate PTH action and calcium homeostasis.

ACKNOWLEDGMENTS I am grateful to the Medical Research Council (United Kingdom) for support, to B. Harding for preparing the figures, and to Julie Allen for typing the manuscript and expert secretarial assistance. Note added in proof." Hypoparathyroidism, deafness, and renal dysplasia (HDR) is an autosomal dominant syndrome (57) associated with haploinsufficiency of the GATA3 gene (111), which is located on chromosome 10p15. GATA3 belongs to a family of zinc-finger transcription factors that are involved in vertebrate embryonic development, and these findings (111) in HDR patients indicate an important role for GATA3 in parathyroid development.

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33. Sarkar G, Sommer SS. Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science 1989;244:331-334. 34. Thakker RV, Davies KE, Whyte MP, Wooding C, O'Riordan JLH. Mapping the gene causing X-linked recessive idiopathic hypoparathyroidism to Xq26-Xq27 by linkage studies. J Clin Invest 1990;86:40-45. 35. Mumm S, Whyte ME Thakker RV, Buetowk H, Schlessinger D. mtDNA analysis shows common ancestry in two kindreds with X-linked recessive hypoparathyroidism and reveals a heteroplasmic silent mutation. A m J H u m Genet 1997;1:153-159. 36. Trump D, Mumm S, Dixon PH, Wooding C, Davies KE, Schlessinger D, Whyte MP, Thakker RV. Localisation of X-linked recessive idiopathic hypoparathyroidism to a 105Mb region on Xq26-q27. J Med Genet 1998;25:905-909. 37. Zucchi I, Mumm S, Pilia G, MacMillan S, Reinbold R, Susani L, Weissenbach J, Schlessinger D. YAC/STS map across 12 Mb of Xq27 at 25 Kb resolution, merging Xq26-qter. Genomics 1996;34:42-54. 38. Scambler PJ, Carey AH, Wyse RKH, et al. Microdeletions within 22qll associated with sporadic and familial DiGeorge syndrome. Genomics 1991;10:201-206. 39. Monaco G, Pignata C, Rossi E, et al. DiGeorge anomaly associated with 10p deletion. A m J M e d Genet 1991;39:215-216. 40. Gong W, Emanuel BS, Collins J, et al. A transcription map of the DiGeorge and velo-cardio-facial syndrome minimal critical region on 22q11. Hum Mol Genet 1996;5:789-800. 41. Augusseau S, Jouk S, Jalbert P, et al. DiGeorge syndrome and 22qll rearrangements. H u m Genet 1986;74:206. 42. Budarf ML, Collins J, Gong W, et al. Cloning a balanced translocation associated with DiGeorge syndrome and identification of a disrupted candidate gene. Nat Genet 1995; 10:269-278. 43. Yamagishi H, Garg V, Matsuoka R, et al. A molecular pathway revealing a genetic basis for human cardiac and craniofacial defects. Science 1999;283:1158-1161. 44. Scire G, Dallapiccola B, Iannetti P, et al. Hypoparathyroidism as the major manifestation in two patients with 22ql 1 deletions. A m J Med Genet 1994;52:478-482. 45. Sykes K, Bachrach L, Siegel-Bartelt J, et al. Velocardio-facial syndrome presenting as hypocalcemia in early adolescence. Arch Pediatr Adolesc Med 1997;151:745-747. 46. Moraes CT, DiMauro S, Zeviani M, et al. Mitochondrial deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome. N EnglJ Med 1989;320:1293-1299. 47. Zupanc ML, Moraes CT, Shanske S, et al. Deletions of mitochondrial DNA in patients with combined features of Kearns-Sayre and MELAS syndromes. Ann Neurol 1991 ;29:680-683. 48. Morten KJ, Cooper JM, Brown GK, et al. A new point mutation associated with mitochondrial encephalomyopathy. Hum Mol Genet 1993;2:2081-2087. 49. Isotani H, Fukumoto Y, Kawamura H, Furukawa K, Ohsawa N, Goto Y, Nishino I, Nonaka I. Hypoparathyroidism and insulindependent diabetes mellitus in a patient with Kearns-Sayre syndrome harbouring a mitochondrial DNA deletion. Clin Endocrinol 1996;45:637-641. 50. Wilichowski E, Gruters A, Kruse K, Rating D, Beetz R, Korenke GC, Ernst BE Christen HJ, Hanefeld E Hypoparathyroidism and deafness associated with pleioplasmic large scale rearrangements of the mitochondrial DNA. A clinical and molecular genetic study of four children with Kearns-Sayre syndrome. Paediatr Res 1997;41:193-200. 51. Abramowicz MJ, Cochaux P, Cohen LHE Vamos E. Pernicious anaemia and hypoparathyroidism in a patient with Kearns-Sayre syndrome with mitochondrial DNA duplication.Jlnherit Metab Dis 1996;19:109-111.

52. Dionisi-Via C, Garavaglia B, Burlina AB, Bertini E, Saponara I, Sabetta G, Taroni E Hypoparathyroidism in mitochondrial trifunctional protein deficiency. JPaediatr 1996;129:159-162. 53. Fanconi S, Fischer JA, Wieland P, Atares M, Fanconi A, Giedion A, Prader A. Kenny syndrome: Evidence for idiopathic hypoparathyroidism in two patients and for abnormal parathyroid hormone in one. JPediatr 1986;109:469-475. 54. Franceschini P, Testa A, Bogetti G, Girardo E, Guala A, LopezBell G, Buzio G, Ferrario E, Piccato E. Kenny-Caffey syndrome in two sibs born to consanguineous parents: Evidence for an autosomal recessive variant. Am J Med Genet 1992;42:112-116. 55. Boynton JR, Pheasant TR, Johnson BL, Levin DB, Streeten BW. Ocular findings in Kenny's syndrome. Arch Ophthalmo11979;97: 896-900. 56. Bergada I, Schiffrin A, Abu Srair H, Kaplan P, DornanJ, Goltzman D, Hendy GN. Kenny syndrome: Description of additional abnormalities and molecular studies. H u m Genet 1988;80:39-42. 57. Bilous RW, Murty G, Parkinson DB, Thakker RV, Coulthard MG, Burn J, Mathias D, Kendall-Taylor E Autosomal dominant familial hypoparathyroidism, sensineural deafness and renal dysplasia. N E n g l J Med 1992;327:1069-1084. 58. Shaw NJ, Haigh D, Lealmann GT, Karbani G, Brocklebank JT, Dillon MJ. Autosomal recessive hypoparathyroidism with renal insufficiency and development delay. Arch Dis Child 1991;66: 1191-1194. 59. Barakat AY, D'AlboraJB, Martin MM,Jose PA. Familial nephrosis, nerve deafness, and hypoparathyroidism. JPediatr 1977;91:61-64. 60. Dahlberg PJ, Borer WZ, Newcomer KL, Yutuc WR. Autosomal or X-linked recessive syndrome of congenital lymphedema, hypoparathyroidism, nephropathy, prolapsing mitral valve, and brachytelephalangy. Am J Med Genet 1983;16:99-104. 61. Sanjad SA, Sakati NA, Abu-Osba YK, Kaddoura R, Milner RD. A new syndrome of congenital hypoparathyroidism, severe growth failure, and dysmorphic features. Arch Dis Child 1991;66:193-196. 62. Parvari R, Hershkovitz E, Kanis A, et al. Homozygosity and linkage-disequilibrium mapping of the syndrome of congenital hypoparathyroidism, growth and mental retardation, and dysmorphism to a l cM interval on chromosome lq42-43. A m J Hum Genet 1998;63:163-169. 63. Blomstrand S, Cla6sson I, S~ve-S6derbergh J. A case of lethal congenital dwarfism with accelerated skeletal maturation. Pediatr Radio11985;15:141-143.

64. Young ID, Zuccollo JM, Broderick NJ. A lethal skeletal dysplasia with generalized sclerosis and advanced skeletal maturation: Blomstrand chondrodysplasia. J Med Genet 1993;30:155-157. 65. LeroyJG, Keersmaeckers G, Coppens M, et al. Blomstrand lethal chondrodysplasia. A m J Med Genet 1996;63:84-89. 66. Loshkajian A, Roume J, Stanescu V, et al. Familial Blomstrand chondrodysplasia with advanced skeletal maturation: Further delineation. A m J Med Genet 1997;71:283-288. 67. den Hollander NS, van der Harten HJ, Vermeij-Keers C, et al. First-trimester diagnosis of Blomstrand lethal osteochondrodysplasia. A m J Med Genet 1997;73:345-350. 68. Oostra RJ, Baljet B, D!ikstra PF, et al. Congenital anomalies in the teratological collection of museum Vrolik in Amsterdam, The Netherlands. II: Skeletal dysplasia. Am J Med Genet 1998;77:116-134. 69. Jobert AS, Zhang P, Couvineau A, et al. Absence of functional receptors parathyroid hormones and parathyroid hormonerelated peptide in Blomstrand chondrodysplasia. J Clin Invest 1998;102:34-40. 70. Zhang P, Jobert AS, Couvineau A, Silve C. A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab 1998;83:3373-3376.

MOLECUI~a~ GENETICS OF HYPOPARATHYROIDISM / 71. Karaplis AC, Bin He MT, Nguyen A, et al. Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. Endocrinology 1998; 139:5255-5258. 72. Ahonen P, Myllarniemi S, Sipila I, et al. Clinical variation of autoimmune polyendocrinopathy-candidiasis ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med 1990;322:1829-1836. 73. Ahonen E Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED): Autosomal recessive inheritance. Clin Genet 1985;27:535-542. 74. ZlotogoraJ, Shapiro MS. Polyglandular autoimmune syndrome type 1 among Iranian Jews. J Med Genet 1992;29:824-826. 75. Aaltonen J, Bjorses P, Sandkuijl L, et al. A n autosomal locus causing autoimmune disease: Autoimmune polyglandular disease type 1 assigned to chromosome 21. Nat Genet 1994;8:83-87. 76. Nagamine K, Peterson P, Scott HS, et al. Positional cloning of the APECED gene. Nat Genet 1997;17:393-398. 77. The Finnish-German APECED consortium: An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc finger domains. Nat Genet 1997;17:399-403. 78. Pollak MR, Brown EM, Chou YWH, Herbert SC, Marx sJ, Steinmann B, Levi T, Seidman CE, Seidman JG. Mutations in the human CaZ+-sensing receptor gene cause familial hypocalciuric hypercalcaemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297-1303. 79. Chou YWH, Pollak MR, Brandi ML, Toss T, Arnqvist H, Brew Atkinson A, Papapoulos SE, Marx S, Brown EM, Seidman JG, Siedman CE. Mutations in the human Ca2+-sensing receptor gene that cause familial hypocalciuric hypercalcaemia. Am J Hum Genet 1995;56:1075-1079. 80. Pearce SHS, Trump D, Wooding C, Besser GM, Chew SL, Grant DB, Heath DA, Hughes IA, Paterson CR, Whyte ME Thakker RV. Calcium-sensing receptor mutations in familial benign hypocalcaemia and neonatal hyperparathyroidism. J Clin Invest 1995;96:2683-2692. 81. Janicic N, Pausova Z, Cole DEC, Hendy GN. Insertion of an Alu sequence in the CaZ+-sensing receptor gene in familial hypocalciuric hypercalcaemia and neonatal severe hyperparathyroidism. Am J H u m Genet 1995;56:880-886. 82. Aida K, Koishi S, Inoue M, Nakazato M, Tawata M, Onaya T. Familial hypocalciuric hypercalcaemia associated with mutation in the human CaZ+-sensing receptor gene. J Clin Endocrinol Metab 1995;80:2594-2598. 83. Heath III H, Odelberg S, Jackson CE, The BT, Hayward N, Larsson C, Buist NRM, Krapcho KJ, Hung BC, Capuano IV, Garrett JE, Leppert ME Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcaemia suggest receptor functional domains. J Clin Endocrinol Metab 1996;81:1312-1317. 84. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert SC, Seidman CE, Seidman JG. Autosomal dominant hypocalcaemia caused by a calcium-sensing receptor gene mutation. Nat Genet 1994;8:303-307. 85. Finegold DN, Armitage MM, Galiani M, Matise TC, Pandian MR, Perry YM, Deka R, Ferrell RE. Preliminary localisation of a gene for autosomal dominant hypoparathyroidism to chromosome 3q13. Paediatr Res 1994;36:414-417. 86. Perry YM, Finegold DN, Armitage MM, Ferrell RE. A missense mutation in the Ca-sensing receptor causes familial autosomal dominant hypoparathyroidism. Am J Hum Genet 1994;55:A17. 87. Pearce SHS, Williamson C, Kifor O, Bai M, Coulthard MG, Davies M, Lewis-Barned N, McCredie D, Powell H, KendallTaylor P, Brown EM, Thakker RV. A familial syndrome of hypocalcaemia with hypocalciuria due to mutations in the calcium-sensing receptor gene. N EnglJ Med 1996;335:1115-1122.

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88. Baron J, Winer KK, Yanovski JA, et al. Mutations in the Ca 2+sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism. Hum Mol Genet 1996;5:601-606. 89. Yu S, Yu D, Hainline BE, BrenerJL, Wilson KA, Wilson LC, OudeLuttikhuis ME, Trembath RC, Weinstein LS. A deletion hot-spot in exon 7 of the GsoLgene (GNAS1) in patients with Albright hereditary osteodystrphy. Hum Mol Genet 1995;4:2001-2002. 90. van Dop C. Pseudohypoparathyroidism: Clinical and molecular aspects. Semin Nephrol 1989;9:168-178. 91. Weinstein LS. Albright hereditary osteodystrophy, pseudohypoparathyroidism, and Gs deficiency. In: Spiegel AM, ed. G proteins, receptors, and disease. Totowa, New Jersey:Humana,

1998;23-56.

92. Schuster V, Eschenhagen T, Kruse K, et al. Endocrine and molecular biological studies in a German family with Albright hereditary osteodystrophy. E u r J Pediatr 1993; 152:185-189. 93. Miric A, Bechio JD, Levine MA. Heterogeneous mutations in the gene encoding the oL-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy. J Clin Endocrinol Metab 1993;76:1560-1568. 94. Weinstein LS, Gejman PV, Friedman E, et al. Mutations of the Gs o~-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1990;87:8287-8290. 95. Davies AJ, Hughes HE. Imprinting in Albright's hereditary osteodystrophy. J Med Genet 1993;30:101-103. 96. Wilson LC, Oude-Luttikhuis MEM, Clayton PT, et al. Parental origin of GsoLgene mutations in Albright's hereditary osteodystrophy. J Med Genet 1994;31:835-839. 97. Yu S, Yu D, Lee E, et al. Variable and tissue-specific hormone resistance in heterotrimeric Gs protein a-subunit (GsO0 knockout mice is due to tissue-specific imprinting of the GsOL.Proc Natl Acad Sci USA 1998;95:8715-8720. 98. Hayward B, Kamiya M, Strain L, et al. The human GNAS1 gene is imprinted and encodes distinct paternally and biallelically expressed G proteins. Proc Natl Acad Sci USA 1998;95:10038-10043. 99. Hayward BE, Moran V, Strain L, Bonthron DT. Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins. Proc Natl Acad Sci USA 1998;95:15475-15480. 100. Peters J, Wroe SF, Wells CA. A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2. Proc Natl Acad Sci USA 1999;96:3830-3835. 101. Kehlenbach RH, MattheyJ, Huttner WB. XLoLsis a new type of G protein. Nature 1994;372:804-809. 102. Ischia R, Lovisetti-Scamihorn P, Hogue-Angeletti R, et al. Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity. J Biol Chem 1997;272:11657-11662. 103. Murray T, Gomez Rao E, Wong MM, et al. Pseudohypoparathyroidism with osteitis fibrosa cystica: Direct demonstration of skeletal responsiveness to parathyroid hormone in cells cultured from bone. J Bone Miner Res 1993;8:83-91. 104. Farfel Z. Pseudohypohyperparathyroidism-pseudohypoparathyroidism type Ib. JBone Miner Res 1999;14,1016. 105. Schipani E, Weinstein LS, Bergwitz C, et al. Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene. J Clin Endocrinol Metab 1995;80:1611-1621. 106. Bettoun JD, Minagawa M, Kwan MY, et al. Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene: Analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 1997;82:1031-1040.

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107. Suarez F, Lebrun JJ, Lecossier D, et al. Expression and modulation of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in skin fibroblasts from patients with type Ib pseudohypoparathyroidism. J Clin Endocrinol Metab 1995;80:965-970. 108. Fukumoto S, Suzawa M, Takeuchi Y, et al. Absence of mutations in parathyroid hormone (PTH)/PTH-related protein receptor complementary deoxyribonucleic acid in patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 1996;81: 2554-2558.

109. Jfippner H, Schipani E, Bastepe M, et al. The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc Natl Acad Sci. USA 1998;95:11798-11803. 110. Thakker RV. Parathyroid disorders: Molecular genetics and physiology. In: Morris PJ, Wood WC, eds. Oxford:Oxford Textbook of Surgery, 2nd Edition, Oxford Univ. Press, 2000; 1121-1129. 111. Van Esch H, Groenen P, Nesbit MA, et al. GATA3 haplo-insufficiency causes human HDR syndrome. Nature 2000;406:419-422.

CHAPTF R 50

A u t o l•m m u n e

Hyp

oparat h y roldlsm " "

M I C H A E L P. W H Y T E Center for Metabolic Bone Disease and Molecular Research, Shriners Hospitals for Children, St. Louis, Missouri 63131; and Division of Bone and Mineral Diseases, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110

INTRODUCTION

a pathogenetic factor (4-10). In this chapter, the autoimmune aspects of hypoparathyroidism are reviewed.

Hypoparathyroidism may be transient or permanent, and the causes for either type are many. Mthough in most cases hypoparathyroidism is sporadic and acquired, in some cases hypoparathyroidism is lifelong and results from a genetic disorder that can exhibit autosomal recessive, X-linked recessive, or autosomal dominant transmission (see Chapter 49). Hypoparathyroidism occurs as a component of an autosomal recessive disorder that features hypofunction of one or more endocrine glands as well as a variety of additional problems due to autoimmune disease (1) (MIM #240300). Initially termed polyglandular autoimmune syndrome, type 1 or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) to reflect the diverse tissues that are affected, it is now often designated as autoimmune polyendocrinopathy syndrome, type 1 (APS1) (1). The major features of APS1 are hypoparathyroidism, adrenal insufficiency, and chronic mucocutaneous candidiasis (moniliasis), which have led to the acronym HAM. The pathogenesis of APS1, i.e., the autoimmune destruction of endocrine tissues, is now understood at a molecular level and the defective gene has been identified (1). Although immune dysfunction also occurs in the DiGeorge anomaly (III-1V brachial pouch dysembryogenesis), dysembryogenesis rather than autoimmunity causes the associated hypoparathyroidism (2,3). However, there are other less well-characterized syndromes with hypoparathyroidism in which autoimmunity may be

The Parathyroids, Second Edition

HISTORY

At the beginning of the twentieth century, pituitary failure was considered to be the basis for hypofunction of multiple endocrine glands in one person. In 1908, however, Claude and Gougerot (11) described a second possible cause when they reported infiltration of lymphocytes and fibrosis within several endocrine tissues of individuals, a condition they termed pluriglandular endocrine atrophy (11). Four years later, Falta (12) suggested that a mild form of this syndrome could lead to partial atrophy of endocrine glands. Indeed, this mechanism for endocrine disease was documented in 1926 when Schmidt described two patients with adrenal insufficiency together with thyroid hypofunction due to chronic lymphocytic thyroiditis (13). Reports of endocrine deficiencies with candidiasis then began to appear in the medical literature. In 1929, Thorpe and Handley (14) described a child with both hypoparathyroidism and oral moniliasis. In 1943, familial occurrences of idiopathic hypoparathyroidism with Addison's disease (15) and with candidiasis (16) were published, offering the possibility of a single heritable defect. The first description of hypoparathyroidism, candidiasis, and idiopathic adrenal insufficiency also appeared that year (17). By 1946, adrenal insufficiency had become the first endocrinopathy associated

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pathogenetically with idiopathic hypoparathyroidism (18). In 1955, Craig and co-workers (19) emphasized the familial occurrence of chronic candidiasis with hypoparathyroidism or nontuberculous Addison's disease. One year later, Whitaker and colleagues (20) called attention to the syndrome of "familial juvenile hypoadrenocorticism, hypoparathyroidism, and superficial moniliasis." Further insight concerning the potential pathogenesis for this syndrome came in 1956 when Roitt and co-workers discovered circulating thyroglobulin autoantibodies in patients with lymphocytic (Hashimoto's) thyroiditis (21). They proposed that organ-specific immunoglobulins might cause endocrine atrophy (21). In 1957 and 1962, Anderson et al. (22) and Blizzard et al. (23), respectively, hypothesized that autoantibodies could account for some cases of nontuberculous Addison's disease. During the early 1960s, the clinical spectrum of APSlminvolving hypoparathyroidism, Addison's disease, and mucocutaneous candidiasismbroadened. In 1962, Gass (24) reported the syndrome of keratoconjunctivitis, superficial moniliasis, idiopathic hypoparathyroidism, and Addison's disease. In 1963, Kunin and colleagues (25) noted an association of steatorrhea, macrocytic anemia, and posthepatitic cirrhosis with moniliasis, hypoparathyroidism, and Addison's disease. In 1966, recognizing that approximately onethird of patients with these combined endocrine deficiencies also had moniliasis, Taitz and co-workers (26) coined the acronym HAM (hypoparathyroidAddison's-monilia) to describe this syndrome. By 1972 (27), eight patients with this condition had been reported (15,16,19,20,28-30). Further progress in defining APS1 and elucidating its pathogenesis was made during the early 1980s. In a series of publications spanning 1980 and 1981, Neufeld

TABLE 1

and colleagues (31-33) characterized a group of disorders they called polyglandular autoimmune (PGA) disease. Three principal clinical forms, types I, II, and III, and possibly an additional type IV, were delineated. As summarized in Table 1, PGA disease, type I (APS1) includes hypoparathyroidism, adrenocortical insufficiency, a n d / o r chronic mucocutaneous candidiasis that appear during childhood. PGA disease, type I could also be complicated by insulin-dependent diabetes mellitus, primary hypogonadism, autoimmune thyroid disease, pernicious anemia, chronic active hepatitis, steatorrhea with malabsorption, alopecia, a n d / o r vitiligo (see below) (33). PGA disease, type II is not associated with hypoparathyroidism, but does include adrenocortical insufficiency as well as autoimmune thyroid disease a n d / o r insulin-requiring diabetes mellitus (33,34). Onset of PGA disease, type II is usually during adult life, and women are more commonly affected than men (32). PGA disease, type III features autoimmune thyroid disease with insulin-dependent diabetes mellitus, pernicious anemia, or vitiligo a n d / o r alopecia, or additional organ-specific autoimmune diseases (e.g., hepatic dysfunction) (33). Finally, Neufeld and Blizzard (33) defined PGA disease, type IV as the presence of two or more organ-specific autoimmune diseases that do not fall into types I, II, or III. Additionally, Neufeld and colleagues (32) reported a 20% incidence of antiparathyroid antibodies in patients with isolated Addison's disease. This and subsequent advances in our knowledge concerning the pathogenesis of PGA disease, type I (APS1) are discussed later. Several hundred patients with APS1 have been described, often reported using other labels (17). In 1956, APS1 was called familial juvenile hypoadrenocorticism, hypoparathyroidism, and superficial moniliasis (20) and, in 1966, as noted above, HAM syndrome (26). The terms multiple endocrine deficiency, autoimmune

Classification of Polyglandular Autoimmune Disease a

Type

Features

II III-A III-B III-C IV

Candidiasis, hypoparathyroidism, Addison's disease and two or three of the following: insulin-dependent diabetes mellitus, primary hypogonadism, autoimmune thyroid disease, pernicious anemia, chronic active hepatitis, steatorrhea (malabsorption), alopecia (totalis or areata), and vitiligo ~ Addison's disease + thyroid autoimmune disease and/or insulin-dependent diabetes mellitus Thyroid autoimmune disease + insulin-dependent diabetes mellitus Thyroid autoimmune disease + pernicious anemia Thyroid autoimmune disease + vitiligo and/or alopecia and/or other organ-specific autoimmune disease Two or more organ-specific autoimmune diseases not falling into types I, II, or III

aFrom Ref. 33, with permission. ~Adapted from Ref. 32.

AUTOIMMUNE HYPOPARATHYROIDISM / candidiasis (MEDAC) syndrome, type I polyendocrine autoimmune disease, and autoimmune polyendocrinecandidiasis syndrome (APECS) were introduced later (35). APS1 gained favor more recently (1). Idiopathic hypoparathyroidism associated with yet additional disorders has also been hypothesized to have an autoimmune basis (10). Hypoparathyroidism (and occasionally endocrine disturbances such as hypothyroidism and diabetes mellitus) can occur in patients with Kearns-Sayre syndrome (oculocraniosomatic neuromuscular disease with mitochondrial myopathy) (6). In one patient, hypoparathyroidism complicated Down syndrome and autoimmune hyperthyroidism (4). Primary hypoparathyroidism has accompanied acute interstitial nephritis and uveitis (5). An elderly man with hypoparathyroidism, and probably incidental Paget disease of bone, had parathyroid glands that showed severe lipomatosis and a diffuse lymphocytic infiltration with atrophy of the endocrine cells (8). An elderly woman with postsurgical hypoparathyroidism had spuriously elevated serum parathyroid hormone (PTH) levels in a radioimmunoassay that used antiserum specific for the carboxy-terminal region of human PTH. She appeared to have an immunoglobulin G (IgG) that was cross-reactive with the assay antiserum (7). Another elderly patient with acquired hypoparathyroidism had detectable serum levels of PTH owing to the presence of antiidiotypic PTH autoantibodies (9). Finally, in other disorders believed to have an autoimmune basis, hypoparathyroidism can apparently result from fibrosis of the parathyroid glands [e.g., progressive systemic sclerosis (36) and Riedel's thyroiditis (37) ]. Despite the existence of a seemingly growing number of autoimmune (or autoimmune-like syndromes) that manifest with hypoparathyroidism, distinctive clinical features can be recognized that distinguish APS1. These problems are detailed below.

C L I N I C A L FEATURES O F APS 1 The incidence of autoimmune disease increases significantly with age. Syndromes in which multiple tissues are affected are possibly more prevalent than disorders involving only a single tissue. In newly diagnosed children and adolescents with an autoimmune disorder, additional conditions will frequently be detected. In early infancy and childhood, the most common association of endocrine and nonendocrine autoimmune disease is APS1 (17). APS1 is an autosomal recessive disease characterized by (1) autoimmune polyendocrine deficiencies (hypoparathyroidism, adrenocortical failure, diabetes mellitus, gonadal failure, and hypothyroidism), (2) chronic mucocutaneous candidiasis, and (3) ectodermal dystrophies (vitiligo, alopecia, keratopathy and dys-

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trophy of dental enamel, nails, and tympanic membranes) (38,39). In addition, there can be pernicious anemia, hepatitis, intestinal malabsorption, splenic atrophy, or gallstones, and a large proportion of patients develop squamous-cell carcinoma of the oral mucosa (38,40). Two of the classical triad of manifestations (hypoparathyroidism, mucocutaneous candidiasis, and adrenocortical insufficiency) are required for the diagnosis of APSI (31-33). Most reported cases of APS1 have been familial. Genetic analysis of affected families indicates an autosomal recessive mode of inheritance (1,41,42). APS1 is reported worldwide, but is exceptionally prevalent among the Finnish population (incidence 1:25,000) and Iranian Jews (incidence 1:9000) (38). Consanguinity has been recorded (41,43). The age of onset does not differ between familial and sporadic cases (17,41 ). APS1 occurs with equal frequency in girls and boys (17,41). The average age of first symptoms is ---8 years. A few individuals have apparently developed the condition after 10 years of age (32,44). The sequence for presentation of the three major components is typically chronic mucocutaneous candidiasis, hypoparathyroidism, and then Addison's disease. In one review, these disorders manifested, on average, at 5, 9, and 14 years of age, respectively (35). Of interest, within a single family, one individual with APS1 may have hypoparathyroidism, candidiasis, and adrenal insufficiency, whereas other affected siblings may develop only one or two of these problems (20,41,45). Hypoparathyroidism or Addison's disease may seem to occur independently within a sibship (42). Because hypoparathyroidism almost invariably precedes Addison's disease, very few patients who have isolated Addison's disease will later develop hypoparathyroidism (32,41). As discussed below, many patients with two or all three of these major components of APS1 manifest additional "atrophic" problems, including pernicious anemia, alopecia, premature ovarian failure, diabetes mellitus, vitiligo, and autoimmune thyroid disease (32,35). The autoimmune thyroiditis is indistinguishable from the isolated disorder (46). In 1966, Blizzard and co-workers (47) described 32 patients with idiopathic hypoparathyroidism who had one or more of the following associated conditions: moniliasis (66%), Addison's disease (56%), pernicious anemia (22%), thyroid disease (19%), alopecia totalis (13%), premature menopause before the age of 25 (6%), and juvenile cirrhosis (6%). Review of a large series of patients in 1969 by Fanconi (48) disclosed that moniliasis occurred in 72% (mean age 3 years), Addison's disease in 58% (mean age 11 years), steatorrhea in 26% (mean age 8 years), and pernicious anemia in 9% (mean age

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17 years). The complete APS1 syndrome affected 32%, whereas hypoparathyroidism occurred with moniliasis alone in 40% and with Addison's disease alone in 28%. In 1990, A h o n e n and co-workers reported their followup of 68 patients (48a). Clinical manifestations varied greatly, but all had candidiasis at some time. The majority of patients had three to five manifestations; all required lifelong follow-up (48a). The major and less c o m m o n clinical features of APS1 are discussed below.

Idiopathic Hypoparathyroidism Patients with APS1 have an especially interesting and challenging form of "idiopathic" hypoparathyroidism. The considerable n u m b e r of associated disorders diversifies its clinical presentation and offers important diagnostic and therapeutic hurdles (27,35). Idiopathic hypoparathyroidism with or without Addison's disease a n d / o r mucocutaneous candidiasis manifests between ages 6 months and 20 years (24). In APS1, symptoms of hypoparathyroidism begin, on average, 4 years after candidiasis is noted, with a mean age of onset from 6 to 9 years (35), d e p e n d i n g on the reported population. Importantly, hypoparathyroidism almost invariably precedes Addison's disease (32,41). Hypoparathyroidism in patients with APS1 seems not to differ clinically or biochemically from isolated idiopathic hypoparathyroidism. As with other forms of PTH deficiency, high serum levels of creatine kinase can accompany episodes of hypocalcemia (49).

Addison's Disease By 1974, idiopathic and tuberculosis-related Addison's disease were recognized to be distinct entities (50). In contradistinction to Addison's disease due to tuberculosis, --~14% of patients with idiopathic Addison's disease have additional problemsm hypoparathyroidism, diabetes mellitus, thyroid disease, pernicious anemia, or gonadal insufficiency (50). Addison's disease, on an a u t o i m m u n e basis, is a comp o n e n t of both PGA disease, types I (APS1) and II (Table 1). However, each disorder has a different mean age of onset, apparent genetic basis, and pathogenesis (32). In APS1, Addison's disease may rarely occur before hypoparathyroidism (32,45,51), but it never appears prior to moniliasis. Addison's disease is noted, on average, 5 years after hypoparathyroidism, at a mean age of 14 years (35). If unrecognized and untreated (see below), Addison's disease can be fatal (18). Rarely, selective involvement of the adrenal cortex may limit destruction to the zona glomerulosa, with development of isolated hypoaldosteronism (52). The unusually high mortality rate of adrenal insufficiency relates to the early age of onset, difficulty in managing hypoadrenalism,

and additional clinical problems, including hypocalcemia (29,53). It is critical to appreciate that untreated Addison's disease can conceal the presence of coexisting hypoparathyroidism (see below).

Candidiasis Chronic mucocutaneous candidiasis is a c o m m o n complication of immune deficiency conditions (54). Included are disorders with morphologic abnormalities of the thymus and thymus-dependent tissues that lead to profound deficiencies of cell-mediated immunity (e.g., DiGeorge anomaly), and those associated with both defective cellular and humoral immunity (e.g., Swiss-type agammaglobulinemia and thymic dysplasia) (54). In fact, clinicians should consider chronic mucocutaneous candidiasis to be a manifestation of an underlying disorder, rather than a primary disease (54). Chronic mucocutaneous candidiasis is usually accompanied by hypoparathyroidism, but Addison's disease etc. may also occur (55). Infection with Candida albicans occurs in --~14% of patients with idiopathic hypoparathyroidism. Candidiasis is usually the initial manifestation of APS1 and generally appears in early childhood. It develops 1-4 years before there is overt evidence of hypoparathyroidism or other endocrine failure (17,29). It is more apt to occur in patients who subsequently develop adrenocorticoid insufficiency or pernicious anemia. Candidiasis may be limited in distribution, but occasionally can involve almost the entire body (Fig. 1). Mucocutaneous candidiasis invariably affects the oral cavity. Oral lesions are diffuse and commonly cause per16che (cracks at the corners of the mouth) and fissures of the lip. The next most frequently involved site is the fingernails. The toenails are only sometimes infected. The entire width of the nail plate is affected, and the nails become thickened and dystrophic. There may be associated paronychia. Affected nails are typically pitted and friable m features that may mistakenly be attributed to hypocalcemia from associated hypoparathyroidism or hepatobiliary or gastrointestinal disease (16). The vagina can also be infected. Rarely, the skin of the hands and feet becomes hyperkeratotic and disfigured (51). Patients with chronic mucocutaneous candidiasis are predisposed to other skin infections (54). By contrast, systemic candidiasis is rare and occurs only if there is another predisposing factor that impairs immune defense mechanisms (e.g., diabetes mellitus). It is important to recognize the association between hypoparathyroidism and candidiasis, although the basis of the relationship is unknown (see below). Many individuals who suffer from chronic mucocutaneous candidiasis will later develop endocrine dysfunction, but such patients are clinically indistinguishable from those who will not (54,55).

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FI6. 1 A young boy with APS1 and severe mucocutaneous candidiasis. There is striking hyperkeratosis from infection of the skin of the hands and face in addition to involvement of the oropharynx and fingernails. (See color plates.)

Other Endocrine Disease Other endocrinopathies that may occur with hypoparathyroidism in APS1 include insulin-dependent diabetes mellitus, Hashimoto's thyroiditis (43,46,56), hypothyroidism (57), and ovarian failure (58-62). Several APS1 patients have developed severe, multiple

endocrine gland hypofunction after moniliasis, in some cases involving the parathyroid, adrenal, and thyroid glands as well as ovaries in succession (43,58). The pituitary gland has not been directly affected, although hypopituitarism due to a pituitary tumor has been reported and growth hormone deficiency has been described.

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Ovarian failure associated with antiovarian antibodies has been well documented in APS1 (58). In cases of primary amenorrhea, ovarian histopathology may resemble gonadal dysgenesis (43). Otherwise, menses are typically regular until the onset of secondary amenorrhea and premature menopause. Affected young women can, however, be fertile. As in other forms of primary gonadal failure, serum levels of follicle-stimulating h o r m o n e are elevated to a greater extent than those of luteinizing hormone.

Nonendocrine Diseases Nonendocrine problems, in addition to candidiasis, occur frequently in patients with APS1. Pernicious anemia is the most common (27,28,43,44,53,61,63,64). It usually manifests 5-10 years after hypoparathyroidism (27), and occasionally presents during adult life (44). Despite its early onset, the pernicious anemia is the "adult type," which is otherwise rare before age 20 years (27). There is acquired gastric atrophy, with loss of all secretory components. Typically, antibodies to parietal cells and intrinsic factor can be detected in the serum. This contrasts with '~juvenile type" pernicious anemia in which there is selective deficiency of intrinsic factor and absence of antibodies. Distinction should be made between cases of true pernicious anemia, in which intrinsic factor is deficient, and those cases in which absorption of vitamin B12 is impaired by steatorrhea or nonspecific gastrointestinal defects. Ikkala and colleagues (64) showed in 1964 that these latter changes may also occur in cases of postoperative hypoparathyroidism. Indeed, in other types of hypoparathyroidism, successful treatment of the hypocalcemia occasionally leads to improved absorption of fat and vitamin B12. Corneal abnormalities in a patient with idiopathic hypoparathyroidism were first described in 1929 (65), but initially, phlyctenular keratoconjunctivitis was thought to be an infrequent complication of APS1 (16,18,66). However, in 1962, a review by Pohjola indicated the prevalence was 10% (67). That same year, Gass (24) reported 12 cases of keratoconjunctivitis, all with moniliasis a n d / o r endocrinopathies. Keratoconjunctivitis is now recognized to occur in as many as 50% of patients with APS1 (24). In fact, it is one of the earliest presenting manifestations (24). In most patients, the symptoms are often chronic and disabling. However, they can also be minimal. There may also be long periods of remission (24). In mild cases, slight redness of the eyes, photophobia, and blepharospasm can occur. Intense photophobia and excessive lacrimation manifest during the acute phase of the keratitis (18,45,66). In severe cases, there is ulceration, scarring, and vascularization that can opacify the cornea and impair vision (24). Conjunctival biopsy specimens

may disclose heavy subepithelial infiltration of lymphocytes and plasma cells (68). Bilateral superficial keratitis accompanies corneal vascularization (66,69,70). Keratoconjunctivitis has been postulated to be a hypersensitivity response to the candidiasis, rather than an independent component of the syndrome. However, this complication has occurred in the absence of Candida infection (24). In 1987, Wagman and coworkers (68) studied 16 cases of APS1 and identified four children with bilateral, self-limited keratitis that began between 2 to 9 years of age (68). It preceded endocrinopathy in two of these patients and caused some impairment of vision. The authors concluded that the keratitis was not caused by the hypoparathyroidism or candidiasis (68). Nevertheless, others have noted that keratitis may improve when serum calcium levels are well controlled (66). Other reported ocular abnormalities of APS1 include strabismus, loss of eyebrows and eyelashes, recurrent blepharitis, keratic precipitates, retinitis pigmentosa, exotropia, pseudoptosis, cataracts, and papilledema (66,68). The cataracts and papilledema are probably related to the hypocalcemia and hyperphosphatemia that occur in untreated hypoparathyroidism (68). In 1999, a patient was reported with an extremely rapid evolution of typical hypocalcemic cataracts during acute idiopathic hepatic and renal failure, and serum calcium and phosphorus were unbalanced (71). Patients with APS1 and idiopathic hypoparathyroidism also have ectodermal problems that include dry, rough skin (18), coarse and brittle hair, and lusterless, somewhat hypoplastic, distally split nails (18,56,65,72). Alopecia totalis is another frequently mentioned complication (18,47,55,56,62). There can also be alopecia areata, (16,18,30,56,62,65), piebaldism (65), or vitiligo (56,63). Alopecia occurs in about 30% of affected individuals and may include loss of eyebrows and eyelashes (66). There can be dental abnormalities in APS1 (18,30), including partial anodontia, enamel hypoplasia, and delayed eruption of teeth (44,53,66,73). Dental dysplasia may occur before or in the absence of hypocalcemia. In a small series of patients, enamel hypoplasia affected canines most severely among maxillary and mandibular teeth, but all tooth types were involved (74). There may also be darkly pigmented skin (18,30) with hyperkeratosis (16). Pigmented nevi have been described (20). One patient with prominent ectodermal dysplasia has been reported (72). Occasionally, there is cutaneous infection with other fungi, such as Trichophytonrubrum (62,65). Recurrent staphylococcal infections of the skin are also common (54). Intracranial calcification (18,53,72) may occur with longstanding hypoparathyroidism, and some affected individuals can be mentally deficient (53). Papilledema

AUTOIMMUNE HYPOPARATHYROIDISM / can occur from raised intracranial pressure (16,18). Patients with APS1 may also develop a variety of intraabdominal disorders. Hepatitis that can progress to cirrhosis is well established (19,20). Indeed, it has been speculated that the hypoparathyroidism and Addison's disease could be sequellae (25). Chronic active hepatitis (45), '~juvenile cirrhosis" (47), posthepatitic cirrhosis (20,24,25,73), pancreatic cystic fibrosis or mucoviscidosis (53,63), and steatorrhea (45,61,63,75,76) have been described. Steatorrhea can lead to overt malabsorption (60,61). The steatorrhea of APS1 patients with idiopathic hypoparathyroidism has been explained as an independent feature, a direct effect of the hypoparathyroidism and hypocalcemia, a complication of intestinal candidiasis (77-81), or a result of abnormal liver function (25,82). There may be predisposition to severe bouts of viral hepatitis, measles, and encephalomyelitis (65).

PATHOGENESIS DiGeorge syndrome shares several clinical problems with APS1, specifically hypoparathyroidism and candidiasis. In DiGeorge syndrome these features result from gene defects that affect parathyroid and thymic development (3,4). Absence of the thymus leads to diminished T lymphocyte function, which seems to explain the predisposition to candidiasis (3,4). The pathogenesis of immune dysfunction in APS1, is less clear however. APS1 is due to defects in a gene that regulates immune function (83) (see below). The hypothesis that the endocrine dysfunction of APS1 is pathogenetically related to the associated candidiasis led to early (and correct) proposals of an autoimmune pathogenesis for the disorder. Subsequently, an autoimmune basis was considered as the cause for idiopathic hypoparathyroidism in several additional syndromes (4-9). By contrast, in 1961, Morse and co-workers (77) postulated inheritance of a gastrointestinal defect that impaired absorption of a factor necessary for parathyroid and adrenal function. In 1963, Kunin and colleagues (25) reviewed cases of APS1 with posthepatitic cirrhosis, and considered the possibility of a viral etiology that could present with hepatitis. In 1966, Sj6berg (61) found high serum titers of antibodies against C. albicans in three patients. Earlier reports had identified a potent, heat-stable, inhibitory factor(s) in serum that prevented the growth of C. albicans (84) that was deficient in patients with chronic moniliasis (85). However, in 1967, Esterly and coworkers (86) showed that lack of this inhibitory substance was not an explanation for the fungal disease. Until the 1970s, it was hypothesized that candidiasis could explain the hypoparathyroidism and other

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endocrine deficiencies of APS1 (77,87,88). However, autopsy studies have never shown invasion of the viscera or parathyroid glands by C. albicans (20,43,63). As an alternative to direct infection by Candida, in 1966 Sj6berg (61) and in 1970 Windorfer (89) postulated that this fungus elaborated a toxin that impairs endocrine gland function. In fact, in 1971, it was proposed that should a causal relationship between candidiasis and endocrinopathy be documented, it would be rationale to amputate fingertips because of the high mortality rate from Addison's disease (90). However, such a toxin has not been demonstrated, and candidiasis does not cause the endocrinopathies of APSl (55). In 1956, Whitaker and colleagues (20) had proposed that the candidiasis emanated from the other dystrophic lesions of the skin, mucosa, and nails due to the hypoparathyroidism. This hypothesis seems incorrect, however, because candidiasis precedes idiopathic hypoparathyroidism by an average of 5 years. Additionally, the dystrophic ectodermal changes improve when hypocalcemia is corrected, whereas the fungal disease usually does not (16). An autoimmune pathogenesis for APS1 was suspected from histopathologic and serologic studies of patients (50). Early on, postmortem investigations disclosed fibrous and lymphocytic infiltration of endocrine glands that replaced healthy tissue and presumably caused gradual destruction of epithelial cells (65). In the parathyroids, fatty replacement, atrophy with atypical cells, and various degrees of lymphocytic accumulation were documented at autopsy (24). Remnants (19,43) or complete absence of parathyroid tissue (18,20,91) were observed, suggesting that the parathyroid lesion was an acquired atrophy, rather than developmental aplasia or hypoplasia (20). The adrenal cortex also appeared atrophic and was replaced by dense fibrous connective tissue infiltrated by lymphocytes (18,24). The role of autoimmunity in APS1 became established in the 1960s. In 1966, the notion that autoantibodies could cause disease of the parathyroid glands was given considerable support by Blizzard, Chee, and Davis (47), who used both normal and pathologic human parathyroid tissue to demonstrate antiparathyroid antibodies in the sera of 38% of patients with idiopathic hypoparathyroidism, 26% with Addison's disease, 12% with Hashimoto's thyroiditis, and 6% of healthy controls. These autoantibodies seemed to be parathyroid specific. The antigen was detected in some, but not all, parathyroid adenomas. However, these investigators were hindered by limited availability of normal parathyroid tissue. Therefore, it was not possible to learn as much about the incidence, characteristics, and significance of parathyroid autoantibodies as was known about antibodies against other endocrine

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tissues (47). These researchers also found that many children and some adults with idiopathic hypoparathyroidism had antiadrenal antibodies (10%) and antigastric parietal cells antibodies (7%) (47). The antibodies did not react with C. albicans. Later, in 1981, a 20% incidence of antiparathyroid antibodies was reported in patients with isolated idiopathic adrenocortical insufficiency (32). In 1967, Wuepper and Fundenberg (45) identified immunologic abnormalities in family members of patients with hypoparathyroidism and speculated that the autoimmune pathogenesis probably had a genetic basis. Their patients showed delayed hypersensitivity to Candida (positive skin test), but lacked an anti-Candida factor detected in normal serum (45). The following year, Blizzard and Gibbs (55) reported that 40% of 44 patients with mucocutaneous moniliasis had an associated disease with a presumed autoimmune basis, and that most had antibodies that were reactive with endocrine glands or stomach. The presence of such antibodies was believed to reflect focal or generalized lymphocytic infiltration and at least subclinical involvement of the various tissues. A direct relationship between the candidiasis and the endocrinopathies could not be established, however (55). There was no cross-antigenicity between C. albicans and adrenal or thyroid tissue (92). In 1969, Irvine and Scarth found IgG antibodies against parathyroid oxyphil cells in only one of nine patients with idiopathic hypoparathyroidism (93), but those with "autoimmune" hypoparathyroidism were not tested. That same year, Spinner and co-workers (94) studied parents and siblings for antibodies to endocrine and gastric tissue and provided additional evidence to support their genetic/clinical classification of individuals with hypoparathyroidism a n d / o r Addison's disease (41). Beginning in the 1960s, several groups of investigators were able to show lymphocytic infiltration and even atrophy of parathyroid glands in animals that were immunized with parathyroid tissue (95,96). In 1968, Lupulescu and co-workers (97) produced lymphocytic and plasma cell infiltration and atrophy of the parathyroid glands in dogs that had been immunized with extracts of allogeneic parathyroid tissue. Moreover, similar changes were noted in adrenal cortex as well. In 1974, passive immunization of rats with antiserum against their parathyroid tissue caused a marked immunoparathyroiditis, but hypoparathyroidism did not ensue (78). Actually, there is little correlation between the presence of antibodies and clinical manifestations of APS1 even among members of a single kindred. The occurrence of antibodies in affected individuals, however, may be a harbinger of eventual endocrine deficiency, but the significance of antibodies found in apparently healthy family members is unclear.

In 1969, Hermans and co-workers (65) suggested that viral infection, in a genetically predisposed individual, might play a pathogenetic role in APS1 and that "immunologic deficiencies, especially those related to delayed hypersensitivity, appear to be of importance in the development of chronic mucocutaneous candidiasis." In 1971, Kirkpatrick and co-workers (54) and Block and colleagues (98) summarized evidence for various abnormalities in cell-mediated immunity in APS1. A woman with hypoparathyroidism, moniliasis, Addison's disease, and primary ovarian failure was described as lacking delayed hypersensitivity in vivo to C. albicans, although there was evidence of immune cellular activity in vitro (98). That same year, Levy et al. (99) reported impaired cellular immunity in a 17-year-old man with chronic mucocutaneous moniliasis, Addison's disease, pernicious anemia, and gastrointestinal malabsorption in whom thrush had cleared after transplantation of fetal thymus tissue. In a preliminary report in 1974, Rao and co-workers (100) noted that cell-mediated immunity to C. albicans could be impaired in idiopathic hypoparathyroidism. In 1975, patients with APS1 and hypoparathyroidism were shown to have abnormalities of T cell function (101). APS1 was postulated to be caused by a defect in T lymphocyte action. In 1979, abnormal suppressor T lymphocyte function, low circulating levels of IgA, and deficient cell-mediated immunity to C. albicans were reported (102). By that time, however, autoantibodies to endocrine glands, intrinsic factor, and the parietal cells of the stomach had been described. In the late 1970s, some evidence suggested that the disorders deemed to be polyglandular failure syndromes were HLA-B8 associated (103,104). Thus, immunologic dysfunction involving genes or chromosome 6 could be a factor in their pathogenesis (104,105). However, other studies of HLA type showed no consistent haplotypes in APS1 (32,102,105), although PGA disease, type II is indeed associated with HLA-B8 (DW3) (32,104,105). Discovery of the molecular basis for APS1 clarified this issue (see below). In 1977, Swana and co-workers (106) reported that reactivity against parathyroid tissue in patients with hypoparathyroidism was due to an antimitochondrial autoantibody. Yet, in 1981, Doniach and Bottazzo (107) noted that organ-specific parathyroid autoantibodies occurred rarely in patients with autoimmune polyendocrinopathy a n d / o r hypoparathyroidism. In 1983, Haspel and co-workers (108) discovered that mice infected with retrovirus type I can develop an autoimmune polyendocrine disease in which autoantibodies are organ specific. Subsequently, in 1985, Betterle and co-workers (109) described an IgG-class mitochondrial antibody that reacted with a 46-kDa mitochondrial membrane pro-

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tein in 31% of patients with APS1, but this antibody was not specific for parathyroid tissue. Furthermore, antibodies could appear before endocrine dysfunction (110). In 1986, Posillico and co-workers (111) described three patients with idiopathic hypoparathyroidism in whom sera contained autoantibodies that inhibited secretion of PTH from dispersed h u m a n parathyroid cells. These antibodies were reactive with epitopes on the extracellular surface of the parathyroid cell and possibly interfered with signal recognition/transduction mechanisms for calciumregulated PTH secretion (111). In 1986, Brandi and co-workers (112) developed a 51Cr release assay using bovine parathyroid cells in culture and showed that complement-dependent cytotoxic antibodies were present in sera from seven patients with autoimmune hypoparathyroidism, but not in 15 healthy individuals or 41 patients with other diverse conditions associated with immune dysfunction (112). Absorption of the sera with parathyroid or adrenal tissue caused a marked decrease in this effect. In 1988, Fattorossi and co-workers (113) extended this work and used fluorescence flow cytometry and tissue immunohistology to show IgM in patient sera that reacted with cultured endothelial cell membranes and tissue sections from bovine parathyroid glands. The antibody was not, however, completely species or organ specific. Two major bands (130 and 200 kDa) were associated with the parathyroid endothelial membranes and perhaps the principal targets of the antibody. An intriguing postulate is that these immunoglobulins disturb an important physiologic relationship between endocrine and epithelial cells (113). Mice with autoimmune polyendocrine disease also have antibodies of the IgM class. In 1992, Wortsman and co-workers (114) found generalized T cell activation to be a novel feature of adultonset idiopathic hypoparathyroidism. This observation suggested that an immune disturbance, possibly related to autoimmunity, could cause this type of hypoparathyroidism as well (114). Although the pathogenesis of the chronic bilateral keratoconjunctivitis in APS1 is unclear, hypoparathyroidism appears not to be the cause. This ophthalmic disorder is not a feature of other types of hypoparathyroidism and can occur without hypoparathyroidism (68). Although hypothesized to be an allergic reaction to C. albicans protein (24), for similar reasons phlyctenular keratitis is unlikely to reflect a hypersensitivity to candidiasis (68). Laboratory studies have not confirmed an autoimmune basis for the keratitis (68). In 1996, to characterize the antibody responses to C. albicans, Peterson and co-workers screened a candidal cDNA expression library with patient sera (115). The highest antibody reactivity was found with enolase (80% of patients reactive), but significant serological responses were also found with heat-shock protein 90

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(67%), pyruvate kinase (63%), and alcohol dehydrogenase (64%) (115). Overall, 96% of APS1 patients had detectable antibodies to at least one of these proteins. Hence, the four abundant candidal proteins are the major antigens and can be used as accurate markers of candidiasis in APS1 (115). Evidence reported in 1996 suggested that acquired hypoparathyroidism is associated with autoantibodies to the extracellular domain of the calcium-sensing receptor (116). Patients with APS1 have autoantibodies against several endocrine and nonendocrine organs (117,118). A new autoantigen related to this syndrome, tyrosine hydroxylase, was identified in sera from patients with alopecia areata through immunoscreening of a scalp cDNA library (117). Immunoreactivity against in vitro-expressed tyrosine hydroxylase was found in 41 (44%) of 94 APS1 patients studied (117). These findings stress the importance of enzymes involved in neurotransmitter biosynthesis as important targets in APS1 (117). Antibodies to glutamic acid decarboxylase occur frequently (119). Alopecia areata occurs in about one-third of APS1 patients, usually in the more severely affected individuals (118). Many patients have high-titer autoantibodies directed against the anagen matrix, cuticle, and cortex keratinocytes and a melanocyte nuclear antigen (118). Also, hair follicle keratinocyte immunostaining accompanies alopecia, especially alopecia totalis. Alopecia is a common feature in APS1, affecting 29-37% of the patients and occurring between ages 3 and 30 years (17). Tryptophan hydroxylase is an intestinal autoantigen in APS1 (120). Antibodies associated with gastrointestinal dysfunction in APS1 are specific for this disease and not present in patients with other bowel disorders or healthy controls (120). Intermittent gastrointestinal dysfunction occurs in 25-30% of APS1 patients, consisting of therapy-resistant steatorrhea, diarrhea, or constipation (120). Measurement of tryptophan hydroxylase antibodies is a valuable diagnostic tool to identify these patients, often young children presenting with atypical gastrointestinal disease, as having APS1 (120). There is as yet no animal model for APS1.

ETIOLOGY APS1 is the only systemic autoimmune disease with an established monogenic background, and the first autoimmune disorder localized outside the major histocompatibility complex region on chromosome 6 (1). In 1994, the founder effect in Finnish APS1 kindreds enabled the genetic mapping of APS1 to chromosome 21q22.3 (121). In 1997, the defective gene in APS1 was isolated and characterized using positional cloning, and was named AIRE-1, denoting an autoimmune regulator (83).

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AIRE-1 encodes a 545-amino acid, proline-rich protein with a molecular mass of 58 kDa. The amino acid sequence includes a putative nuclear targeting signal between amino acids 113 and 133 (122). APS1 protein is mainly localized, both in vitro and in vivo, to the cell nucleus, (122). The protein contains two zinc fingers of plant homeodomain (PHD) type-motifs that are found in many nuclear proteins (122), which implies a role in the regulation of gene expression (122,123). Mutations in the AIRE-1 gene cause APS1 (123). A total of 16 different mutations have been identified spread throughout the coding region (122). A common haplotype spanning the locus carrying a 964de113 mutation suggests a founder effect in the British population (40). Genotype-phenotype correlations for APS1 remain difficult, suggesting that other genetic or environmental factors, or both, influence the clinical presentation and disease progression in individual patients (124).

TREATMENT One must consider that children with hypoparathyroidism or Addison's disease may develop fullblown APS1. Furthermore, clinicians need to know that Addison's disease can conceal coexisting hypoparathyroidism (91), and that glucocorticoid therapy alone can lead to a fatal outcome (18-20,30,82,124a). Serum calcium concentrations rise in adrenocortical insufficiency, but may suddenly decrease after corticosteroid therapy is begun, because of both diminished gastrointestinal absorption and increased renal excretion of calcium (20). Similarly, estrogen replacement therapy for ovarian failure can diminish serum calcium levels (61). Patients with hypoparathyroidism and such additional disorders are, therefore, more likely to show fluctuations of serum calcium concentrations, and require especially careful regulation of doses of vitamin D sterols, etc. (30). This is discussed further below.

Hypoparathyroidism Management of hypoparathyroidism in patients with APS1 is essentially as described in Chapter 52 for patients with isolated idiopathic hypoparathyroidism. However, APS1 is a far more complex disorder than idiopathic hypoparathyroidism, and many potentially interacting medical problems will likely complicate therapy. A major factor, noted above, is Addison's disease. Another is exemplified by a case of a child with candidiasis and hypoparathyroidism, who seemed to have defective 25-hydroxylation of vitamin D due to concomitant giant cell hepatitis and severe cirrhosis (125). Here, 1,25-dihydroxyvitamin D 3 was considered

to be the best form of vitamin D therapy (125). Steatorrhea could also determine the type of vitamin D that would be most efficacious. As discussed below, the presence or absence of Addison's disease is an especially important consideration.

Addison's Disease Among the major confounding factors to consider when treating hypoparathyroidism in APS1 is the likelihood of Addison's disease. Prior to the 1950s, adrenocortical insufficiency was a fatal disorder, and more than 80% of patients were dead 2 years after receiving this diagnosis (65). In 1969, one-third of patients succumbed (48). Now, conventional replacement therapy using glucocorticoids and mineralocorticoids enables long-term survival. Addison's disease and its treatment both impact significantly on the manifestations of hypoparathyroidism. When hypoadrenalism supervenes in APS1, the clinical and biochemical features of hypoparathyroidism can diminish considerably, but will rapidly return when glucocorticoid replacement therapy is begun (30). Glucocorticoids can lower blood calcium levels by decreasing dietary absorption of calcium, and by enhancing renal excretion of calcium because glomerular filtration rate is increased. Accordingly, hypoparathyroidism must be excluded before glucocorticoid therapy is started for Addison's disease. If hypocalcemia is present, it may be rapidly and severely exacerbated by glucocorticoid treatment. An example is case 2 described in 1961 by Morse and colleagues (53). This child with hypoparathyroidism and a serum calcium concentration of 5.5 m g / d l experienced a spontaneous "remission" with a serum calcium level of 10.3 m g / d l as Addison's disease developed. When glucocorticoid replacement treatment (without additional vitamin D therapy) was initiated, the serum calcium level abruptly fell to 4.7 mg/dl. Similar clinical scenarios have been described by Leonard (18), Leifer and Hollander (126), Papadatos and Klein (30), and Quichaud and colleagues (127). Conversely, in 1964, Kenny and Holliday (46) reported that the onset of Addison's disease in a patient with well-controlled hypoparathyroidism was followed by marked hypercalcemia of 14.5 mg/dl.

Candidiasis The ectodermal changes that result from hypoparathyroidism per se, including brittleness of nails, respond to successful treatment of the mineral disturbances. However, the candidiasis often will not (16). Intractable superficial candidiasis involving mucous membranes, skin, and nails is one of the most perplexing and frustrating problems associated with

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autoimmune hypoparathyroidism. Candidiasis, once established, may be difficult to eradicate even when serum calcium concentrations are maintained in the normal range. Although treatment for mucocutaneous candidiasis had been disappointing generally, success was reported for a significant n u m b e r of patients who received transfer factor and amphotericin B. Transfer factor was prepared from lymphocytes of healthy individuals immunized against C. albicans. Kirkpatrick and Greenberg (128), however, studied 19 patients with chronic mucocutaneous candidiasis, the majority of whom had abnormalities in cell-mediated immunity. All had normal numbers of circulating T and B lymphocytes and normal lymphocyte responses to phytohemagglutinin and concanavalin A. Nevertheless, most had negative skin tests for C. albicans. Transfer factor administered for several months (with only local treatment with antifungal agents) was ineffective. Amphotericin B alone given intravenously, however, induced remissions in most of the patients (54). Nevertheless, complete and lasting eradication of all clinical evidence of the disease proved to be an elusive goal (88). Face and scalp lesions cleared most readily and relapsed least frequently. Thrush often reoccurred and remained the most symptomatic lesion (54). Local hypochlorite treatment has been suggested, because candidiasis of skin and nails improves in patients who swim frequently in chlorinated pools. Infected nails have been avulsed, but recurrence during nail regrowth ensues unless the candidiasis is controlled elsewhere (54). Long-term therapy with oral ketoconazole is now considered the treatment of choice for mucocutaneous candidiasis (129). Ketoconazole, 200 to 400 m g / d a y orally, is very effective for extensive and resistant disease (130,131). Nystatin applied topically as well as administered orally (to reduce intestinal colonization) may prevent the spread of the fungal lesions, but will rarely eradicate the infection.

Additional Disorders Absence of intrinsic factor production by gastric mucosa can lead to pernicious anemia in patients with APS1. The treatment is vitamin B12, as in any other form of intrinsic factor deficiency. However, steatorrhea is also a complication of autoimmune hypoparathyroidism. Patients with steatorrhea, hypocalcemia, and megaloblastic anemia have, on occasion, mistakenly been thought to have folate deficiency. When steatorrhea is severe, with its potential for vitamin D malabsorption, etc., hypoparathyroidism can be especially difficult to treat. Calcitriol is more water soluble than

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other forms of vitamin D, and thus may be the treatment of choice. It may be necessary to give calcium intravenously for a period of time (60). A diet enriched with medium-chain triglycerides has been reported to be helpful, perhaps by decreasing calcium loss due to saponification (60). One patient with APS1 who suffered recurrent episodes of severe intractable diarrhea, steatorrhea, and hypocalcemia was successfully treated only when given immunosuppression using intravenous high-dose methylprednisolone and oral methotrexate maintenance therapy (132). Of interest, a 10-year-old girl with APS1 who developed pure red cell aplasia resistant to conventional therapy showed hematologic remission after intramuscular injections of gamma globulin (13). The investigators postulated an idiotype-antiidiotype interaction in which specific suppression by antiidiotype antibodies corrected the hematologic disease (133). Patients with features of APS1 must be screened regularly for the associated abnormalities (32). Genetic testing for mutations in the AIRE-1 gene may confirm the diagnosis of APS1 and support this process, and can facilitate screening of healthy siblings. In the absence of genetic testing, these children should be examined for endocrine defects during at least the first decade of life (134). Because ---13% of individuals with APS1 will develop chronic active hepatitis, liver function tests and assays for smooth muscle and mitochondrial antibodies should be included (32). Medical therapy for keratoconjunctivitis can include corticosteroid eye drops. Surgical treatment involves keratectomy or corneal transplantation (66,68). Wagman and colleagues (68) r e c o m m e n d medical m a n a g e m e n t of the corneal disease without surgical intervention (68). The active phase is helped by topical antibiotic/corticosteroid medication; systemic corticosteroids or immunosuppression were not required (68). Of importance, these authors noted an apparent transition from an active to quiescent phase --~10 years after the onset. Interestingly, cimetidine has also been reported to have some efficacy, perhaps acting as an immunomodulator (135). Ward and colleagues reported complete resolution of photophobia and considerable hair regrowth, with minimal side effects, in a girl who received oral cyclosporine for severe APSl-associated exocrine pancreatic failure and keratoconjunctivitis (136).

ACKNOWLEDGMENTS This work was supported by Grant 8480 from Shriners Hospitals for Children. The author is grateful to Becky Whitener, CPS, for expert secretarial help.

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CHAPTER 51

P se u d o h y p o p ara thyr o i dis m Clinical, Biochemical, and Molecular Features

SUZANNE

M.

JAN

D E B E U R Division of Endocrinology, Department of Medicine and Metabolism, The Johns Hopkins University School of Medicine,

Baltimore, Maryland 21287

MICHAEL A. LEVINE Departments of Pediatrics, Medicine, and Pathology, TheJohns Hopkins University School of Medicine, Baltimore, Maryland 21287

INTRODUCTION

PATHOPHYSIOLOGY

Pseudohypoparathyroidism (PHP) is an u n c o m m o n metabolic disorder characterized by biochemical hypoparathyroidism (i.e., hypocalcemia and hyperphosphatemia), increased secretion of parathyroid hormone (PTH), and target tissue unresponsiveness to the biologic actions of PTH. Thus the pathophysiology of PHP differs fundamentally from true hypoparathyroidism, in which PTH secretion rather than PTH responsiveness is defective (1). In addition to functional hypoparathyroidism, many patients with PHP exhibit a distinctive constellation of developmental and skeletal defects, collectively termed Albright's hereditary osteodystrophy (AHO) (2), which includes a round face, short stature, obesity, brachydactyly, heterotopic ossification, and mental retardation (Fig. 1). The relationship between the metabolic abnormalities of PHP and the various features of AHO remains unknown. Indeed, in certain families some affected members manifest both AHO and PTH resistance (i.e., PHP) whereas other family members have AHO without evidence of any endocrine dysfunction, a variation termed "pseudopseudohypoparathyroidism" (pseudo-PHP) (3) Several forms of PHP have been described, which has led to the development of a diagnostic classification that is based on clinical, biochemical, and genetic characteristics (Table 1)

The first insights into the molecular basis for PHP emerged from the parallel observations that cAMP mediates many of the actions of PTH on kidney and bone, and that administration of PTH to normal subjects markedly increases the urinary excretion of nephrogenous cAMP and phosphate (4). Although the PTH infusion test remains a reliable test for the diagnosis of PHP, more important perhaps is the ability of the test to distinguish between several variants of the syndrome (Fig. 2). Patients with PHP type 1 fail to show an appropriate increase in urinary excretion of both nephrogenous cAMP and phosphate (4), suggesting that an abnormality in the renal PTH receptoradenylyl cyclase complex that produces cAMP is the basis for impaired PTH responsiveness (Fig. 3). This early hypothesis was subsequently confirmed by administration of dibutyryl cAMP to patients with PHP type 1. These patients showed a normal phosphaturic response to the infused cAMP analog, indicating that the renal response mechanism to cAMP was intact (5). These studies have led to the conclusion that proximal renal tubule cells are unresponsive to PTH. By contrast, cells in other regions of the nephron appear to respond normally to PTH, as evidenced by reduced urinary calcium excretion in PHP type 1 patients compared to those with hormonopenic hypoparathyroidism (6,7). Furthermore, renal handling of calcium (and sodium)

The Parathyroids, Second Edition

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Copyright © 2001 John R Bilezikian, Robert Marcus, and Michael A. Levine.

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A

B

D i

C

FIG. 1 Typical features of Albright hereditary osteodystrophy. (A) A young woman with characteristic features of AHO; note the short stature, disproportionate shortening of the limbs, obesity, and round face. (B) Radiograph of the AHO patient showing marked shortening of fourth and fifth metacarpals. (C) Archibald sign, the replacement of "knuckles" with "dimples" due to the marked shortening of the metacarpal bones. (D) Brachydactyly of the hand; note thumb sign (Murderer's thumb or potter's thumb) and shortening of the fourth and fifth digits.

TABLE 1 Feature Physical appearance Response to PTH Urine cAMP Urine phosphorous Serum calcium level Hormone resistance GsoL activity Inheritance Molecular defect

in response to exogenous PTH is normal in PHP type 1 (8). These observations suggest that the distal renal tubule is responsive to PTH in subjects with PHP type 1, and imply that adequate amounts of cAMP can be produced in these cells or that other second messengers (e.g., cytosolic calcium or diacylglycerol) may mediate these PTH responses. Administration of PTH to subjects with the less comm o n form of the disorder, PHP type 2, produces a normal increase in urinary cAMP but fails to elicit an appropriate phosphaturic response (9). This pattern of response has suggested that PTH resistance in PHP type 2 results from a biochemical defect that is either distal or unrelated to the PTH-stimulated generation of cAME It has b e e n generally assumed that bone cells of patients with PHP type 1 are innately resistant to PTH, based primarily on the observation that patients with PHP type 1 are hypocalcemic and that administration of PTH does not increase the plasma calcium level. In fact, cultured bone cells from a patient with PHP type 1 have been shown to increase intracellular cAMP normally in response to PTH treatment in vitro (10). Moreover, many patients with PHP type 1 develop radiologic (Fig. 4) or histologic evidence of significant bone resorption and hyperparathyroid bone disease (11). Patients with PHP may develop additional abnormalities in bone metabolism resulting from excessive PTH or deficient 1,25-dihydroxyvitamin D~ [1,25(OH)zD3], including osteomalacia (11), rickets (12), renal osteodystrophy (13), and osteopenia (14). One possible explanation for the variable responsiveness of bone to PTH is the existence of two distinct cellular systems in bone on which PTH exerts action: the remodeling system and mineral homeostasis. In

Classification of the Various Forms of Pseudohypoparathyroidism Based on Clinical, Biochemical, and Genetic Features

PHP type la

PseudoPHP

PHP type 1b

PHP type lc

PHP type 2

Albright hereditary osteodystrophy typical, but may be subtle or absent

Normal

Albright hereditary osteodystrophy

Normal

Defective Defective Low or (rarely) normal Generalized

Defective Defective Low or (rarely) normal Limited to PTH target tissues Normal Autosomal dominant (most cases) 20q13.3

Defective Defective Low

Normal Defective Low

Generalized Normal Unknown

Limited to PTH target tissues Normal Unknown

Unknown

Unknown

Reduced

Normal Normal Normal Absent

Reduced Autosomal dominant

Heterozygous mutations in the GNAS 1 gene

PSEUDOHYPOPARATHYROIDISM /

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FIG. 2 Urinary cAMP excretion in response to an infusion of bovine parathyroid extract (300 USP units). The peak response in normal subjects (A) as well as those with pseudoPHP (not shown) is 50- to 100-fold times basal. Subjects with PHP type la (©) or PHP type 1b (O) show only a 2- to 5-fold increase. Urinary cAMP is expressed as nanomoles per 100 ml of GF, UoAMP(nanomoles per 100 ml GF) = UoAMP (nanomoles/dl) x Scr e ( m g / d l ) / U c r e (mg/dl). Reprinted with permission from Ref 23; MA Levine, TS Jap, RS Mauseth, RW Downs, AM Spiegel. Activity of the stimulatory guanine nucleotide-binding protein is reduced in erythrocytes from patients with pseudohypoparathyroidism and pseudopseudohypoparathyroidism: Biochemical, endocrine, and genetic analysis of Albright's hereditary osteodystrophy in six kindreds. J Clin Endocrinol Metab 1986;62:497-502. © The Endocrine Society.

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FIG. 3 Cell surface receptors for PTH are coupled to two classes of G proteins. G s mediates stimulation of adenylyl cyclase (AC) and the production of cAME which in turn activates protein kinase A (PKA). Gq stimulates phospholipase C (PLC) to form the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from membrane-bound phosphatidylinositol 4,5-bisphosphate. IP3 increases intracellular calcium (Ca2+) and DAG stimulates protein kinase C (PKC) activity. Each G protein consists of a unique oL chain and a 13",/dimer.

FIG. 4 Photograph and radiograph of hands of a patient with marked hyperparathyroid bone disease. Marked periosteal bone erosion in terminal phalanges has resulted in "pseudoclubbing" From Levine MA, Parfrey NA, Feinstein RS. Pseudohypoparathyroidism. Johns Hopkins Medical Journal (1982), 151:137-146. © The John Hopkins University Press. Reprinted with permission of The John Hopkins University Press.

PHP type 1, the bone remodeling system appears to be more responsive to PTH than the homeostatic system. This variability may reflect that the remodeling system is less d e p e n d e n t on normal plasma levels of 1,25(OH)zD 3. Plasma levels of 1,25(OH)zD s are low in hypocalcemic patients with PHP type 1 (15), a finding that could explain the concurrence of hypocalcemia and increased skeletal remodeling in many of these patients. Hypocalcemia leads to a compensatory overproduction of PTH, which could eventually overcome the 1,25(OH)2D s dependency for remodeling but not for PTH-stimulated calcium mobilization. A role for 1,25(OH)zD ~ in modulating the responsiveness of the calcium homeostatic system to PTH is suggested by several observations. First, the calcemic response to PTH is deficient not only in patients with PHP type 1, but also in patients with other hypocalcemic disorders in which plasma levels of 1,25(OH)zD 3 are low. Moreover, normalization of the plasma calcium level in patients with PHP type 1 by administration of physiologic amounts of 1,25(OH)zD s or pharmacologic amounts of vitamin D restores calcemic responsiveness

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to PTH (16). Second, patients with PHP type 1 who have normal serum levels of calcium and 1,25(OH)2D a without vitamin D treatment (so-called normocalcemic PHP) show a normal calcemic response to administered PTH (16). These findings suggest that 1,25(OH)zD S deficiency is the basis for the lack of a calcemic response to PTH in hypocalcemic patients with PHP type 1, and challenge the premise that bone cells are intrinsically resistant to the actions of PTH. Subjects with PHP type 1 have increased serum levels of phosphate owing to an inability of PTH to decrease phosphate reabsorption in the proximal renal tubule. Hypocalcemia per se may also contribute to the develo p m e n t of hyperphosphatemia, secondary to impaired renal phosphate clearance by very low levels of intracellular calcium. Accordingly, restoration of plasma calcium levels to normal by chronic treatment with calcium and vitamin D can reduce elevated levels of serum phosphorous. Similar therapy has been shown to reverse the defective phosphaturic response to administered PTH in certain patients with PHP type 1, although the urinary cAMP response remains markedly deficient (17). Therefore, persistence of a blunted urinary cAMP response to PTH in PHP type 1 patients in whom chronic vitamin D therapy has led to normalization of plasma calcium levels and restoration of a phosphaturic response need not imply, as has been at least suggested (17), that there is no relationship between cAMP production and phosphate clearance. A circulating inhibitor of PTH action has been proposed as a cause of PTH resistance on the basis of studies showing an apparent dissociation between plasma levels of e n d o g e n o u s immunoreactive and bioactive PTH in subjects with PHP type 1. Despite high circulating levels of immunoreactive PTH, the levels of bioactive PTH in many patients with PHP type 1 have been found to be within the normal range when measured with highly sensitive renal (18) and metatarsal (19) cytochemical bioassay systems. Furthermore, plasma from many of these patients has been shown to diminish the biologic activity of exogenous PTH in these in vitro bioassays (20). Currently, the nature of this putative inhibitor or antagonist remains unknown. The observation that prolonged hypercalcemia can remove or reduce significantly the level of inhibitory activity in the plasma of patients with PHP has suggested that the parathyroid gland may be the source of the inhibitor. In addition, analysis of circulating PTH immunoactivity after fractionation of patient plasma by reversed-phase high-performance liquid chromatography has disclosed the presence of aberrant forms of immunoreactive PTH in many of these patients (21). Although it is conceivable that a PTH inhibitor may cause PTH resistance in some patients with PHP, it is more likely that circulating antagonists of PTH action arise as a

consequence of the sustained secondary hyperparathyroidism that results from the primary biochemical defect. The identification of circulating fragments of PTH that may act as competitive antagonists of intact PTH in uremic patients with secondary hyperparathyroidism now provide at least a theoretical basis for this hypothesis (22). The overall evidence indicates that the disturbances in calcium, phosphorous, and vitamin D metabolism in patients with PHP type 1 result directly or indirectly from reduced responsiveness of both bone and kidney to PTH. Hypocalcemia results from impaired mobilization of calcium from bone, reduced intestinal absorption of calcium [via deficient generation of 1,25(OH)2D3], and urinary calcium loss. Of these defects, the diminished movement of calcium out of bone stores into the extracellular fluid probably has the greatest role in producing hypocalcemia. Intensive treatment with calcitriol [1,25 (OH) zD3] or other vitamin D analogs improves intestinal calcium absorption and bone calcium mobilization, restores plasma calcium to normal, and reduces circulating PTH levels. Although resistance of the proximal renal tubule to PTH appears to be the primary biochemical defect, the major abnormalities in mineral metabolism found in patients with PHP type 1 can be largely explained on the basis of deficiency of circulating 1,25(OH)zD s.

PSEUDOHYPOPARATHYROIDISM

TYPE la

PHP type 1 can be subclassified into two apparently distinct disorders based on several important clinical and biochemical characteristics: (1) the absence or presence of AHO, (2) h o r m o n e resistance that is specific (PTH alone) or that is more generalized, and (3) normal or reduced tissue activity of the G protein (G~) that couples heptahelical receptors to stimulation of adenylyl cyclase (see Chapters 5 and 7). Subjects with the type la variant have an approximately 50% reduction in expression or activity of the ot chain of G s (Gs0t) in membranes from a wide variety of cells and tissues (23) (Fig. 5). The generalized deficiency of Gsot may impair the ability of PTH, as well as other hormones and neurotransmitters, to activate adenylyl cyclase, thereby accounting for the multihormone resistance that occurs in these patients. In addition to h o r m o n e resistance, patients with PHP type la also manifest AHO (Fig. 1) (1). By contrast, patients with PHP type l b lack features of AHO, have h o r m o n e resistance that is limited to PTH, and have normal levels of Gsot protein in cell membranes (Fig. 5). Early studies of PHP type la led to the identification of families in which some individuals had signs of AHO but lacked apparent h o r m o n e resistance (i.e.,

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FIG. 5 Gso~activity of erythrocyte membranes. GsoLis quantified in complementation assays with $49 cyc- membranes, which genetically lack Gse~ but retain all other components necessary for hormone-response adenylyl cyclase activity. Activity is reduced approximately 50% in patients with AHO subjects with either PHP type l a or pseudoPHP, but is normal in patients with PHP type lb.

pseudoPHP). The observation that PHP type la and pseudoPHP can occur in the same family first suggested that these two disorders might reflect variability in expression of a single genetic lesion. Support for this view comes from studies indicating that within a given kindred, subjects with either pseudoPHP or PHP type la have equivalent functional Gse~ deficiency (Fig. 5) (23,24), and that a transition from h o r m o n e responsiveness to h o r m o n e resistance may occur (25). It therefore seems appropriate to apply the term AHO to both of these variants in acknowledgment of the clinical and biochemical characteristics that patients with PHP type l a and pseudoPHP share (2). M o l e c u l a r D e f e c t in A l b r i g h t

Hereditary Osteodystrophy

The discovery that Gsot deficiency results from inactivating mutations in the GNAS1 gene, located at 20q13.2 --+ 13.3 (26), provided confirmation of autosomal d o m i n a n t transmission in AHO and resolved longstanding controversies regarding the inheritance of this disorder (2,27-29). GNAS1 is a complex gene (30) composed of at least 16 exons, including 3 alternative first exons (Figure 6) (31-33). Alternative splicing of nascent transcripts derived from exons 1-13 generates four mRNAs that encode GsOt. Deletion of exon 3 results in the loss of 15 codons from the mRNA, whereas use of an alternative splice site in exon 4 results in the insertion of a single additional codon into the mRNA. This produces two Gse~ proteins with apparent molecular masses of 45 kDa and two isoforms with apparent molecular masses of 52 kDa (30) that exhibit specific patterns of tissue expression (34). Both long and short forms of Gs0t can stimulate adenylyl cyclase

Nesp551NESP

2-13

Maternal

Gnasxl [ XL~s

2-13

Paternal

Gsot-t

2-13

Paternal

2-13

Biallelic

Gsot

L1A I 1

FIG. 6 The structural organization of the GNAS1 gene in schematic representation. The 13 exons that comprise transcripts for the 52-kDa forms of Gso~protein are labeled 1-13; exon 3 is not present in transcripts that encode the smaller, 45-kDa forms of GsoL protein (see text). The three alternative first exons that are present in transcripts encoding NESP55, Gnasxl, and the truncated (GsoL-t) proteins are also indicated. The transcripts that are derived from maternal, paternal, or both (biallelic) parental alleles are noted.

and open calcium channels (35), but biochemical characterization of these isoforms has revealed subtle differences in the binding constant for GDP, the rate at which the forms are activated by agonist binding, efficiency of adenylyl cyclase stimulation, and the rate of GTP hydrolysis. The significance of these differences remains unknown (35-37), but these distinctions imply the existence of as-yet undefined roles for these G proteins (38). Additional complexity in the processing of the GNAS1 gene arises from the use of alternative first exons that generate novel transcripts (Fig. 6). Because these proteins lack amino acid sequences encoded by exon 1, which are required for interaction of Gscx with G[3~/ and attachment to the plasma membrane, it is unlikely that these proteins can function as transmembrane signal transducers. In one case, a Gsc~ transcript is p r o d u c e d with an alternative first exon (exon 1A) that lacks an initiator ATG; thus, a truncated, nonfunctional Gsot protein is conceivably translated from an inframe ATG in exon 2 (33,39). These transcripts encode a protein of 45 or 42 kDa, d e p e n d i n g on the presence or absence of exon 3, and are most highly expressed in the retina and brain. The function of this Gsc~ chain is unknown. In three other instances unique transcripts are generated using additional coding exons that are present upstream of the exon 1 used to generate functional Gse~ protein. The more 5' of these exons encodes the n e u r o e n d o c r i n e secretory protein NESP55, a chromogranin-like protein, and is generated from a transcript that contains sequences derived from exons 2-13 of GNAS1 in the 3' nontranslated region (40,41). Accordingly, NESP55 shares no protein homology with Gsot. Eleven kilobases further downstream, a second

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alternative exon, XLots, when spliced in-frame to exons 2-13, results in a transcript of a larger, 51-kDa Gse~ isoform (42). Both NESP55 and XLots have been implicated in regulated secretion in neuroendocrine tissues. Molecular studies of DNA from subjects with AHO have disclosed inactivating mutations in the GNAS1 gene (43-57) that account for a 50% reduction in expression or function of Gse~ protein (Table 2) (181-185). All patients are heterozygous and have one normal GNAS1 allele and one defective allele. Mutations in the GNASI gene are heterogeneous, including missense mutations (46,48-50,55), point mutations that disrupt efficient splicing (47) or terminate translation prematurely (53), and small deletions (47,48,51,52,57,58). Although novel mutations have

TABLE 2 Mutation M1V Q12X Y37X Q31X GA/gtaagt L99P (AAA___GGAG)--> (AAAGG) AA/gtacgt -> AA/gtacat P115(CCC) ~ (_CC) P115S L153(CTGT) --> ( C T ) R165C ATT GAC TGT --->ATTGT CAG GCT GAC --->CAGGC Y190N AG/gtgt --->AG/gcgt CAG GTG GAC --> CAG AC G206 (GGA) --> ( G ) R231H $250R R258W E259V E267(CAG) --> (CCAG) L271(CTC) --> (_TC) AG/gt ~ AG/ct R385H A366S z~1382 m

been found in nearly all of the kindreds studied, a fourbase deletion in exon 7 has been detected in multiple families (56,58-60) (54,61) and an unusual missense mutation in exon 13 (A366S; see below) has been identified in two unrelated young boys (62), suggesting that these two regions may be genetic "hot spots." Most gene mutations lead to reduced expression of GsoL mRNA (24,63), but in some subjects the mutant allele produces normal levels of Gse~ mRNA (24,63,64) that encodes dysfunctional GsOt proteins (49,50,55,65). The replacement of arginine by histidine at codon 385 in the carboxyl-terminal tail of Gsot selectively "uncouples" G s from receptors and prevents receptor activation (49). Substitution of arginine by histidine at position 231 also prevents receptor activation of Gs,

Mutations in the GNAS1 Gene

Location

Type

Exon 1 Exon 1 Exon 1 Exon 1 Exon 1 Intron 2 Exon 4 Exon 4 Exon 4 Intron 4 Exon 5 Exon 5 Exon 6 Exon 6 Exon 6 Exon 7 Exon 7 Intron 7 Exon 8 Exon 8 Exon 9 Exon 10 Exon 10 Exon 10 Exon 10 Exon 10 Intron 10 Exon 13 Exon 13 Exon 13

Missense Nonsense Nonsense Nonsense Deletion 38 bp Deletion 2 bp Missense Deletion 2 bp Deletion 43 bp Substitution Deletion 1 bp Missense Deletion 2 bp Missense Deletion 4 bp Deletion 4 bp Missense Substitution Deletion 4 bp Deletion 2 bp Missense Missense Missense Missense Insertion 1 bp Deletion 1 bp Substitution Missense Missense Deletion 3 bp

Effect

Ref.

Initiator codon Truncated protein Truncated protein Truncated protein Frameshift Donor splice site

(46) (181 ) (181 ) (181 ) (52) (182)

--

(48)

Frameshift Frameshift Donor splice site Frameshift

(57) (45) (182) (51 )

Frameshift

(182)

Frameshift Frameshift

(182) (54,56,58-61,183) (182) (182) (48) (57) (50) (184) (184) (56) (51 ) (47) (47) (49) (44) (185)

Donor splice site Frameshift Frameshift 13"yinteraction Stability Stability Stability Frameshift Frameshift Donor splice site Receptor coupling GDP release Defective coupling to PTH 1R

(56)

(48)

PSEUDOHYPOPARATHYROIDISM

but by an entirely different mechanism (50). The replacement of Arg-231 hinders binding of GTP to the e~ chain, and thereby inhibits receptor-induced dissociation of Gse~ from G~7. Likewise, substitution of arginine at position 258 with tryptophan leads to increased GDP release and impaired receptor-mediated activation (55).

Multiple Hormone Resistance in Pseudohypoparathyroidism Type la Although biochemical hypoparathyroidism is the most commonly recognized endocrine deficiency in PHP type l a, early clinical studies described additional hormonal abnormalities, such as hypothyroidism (66,67) and hypogonadism (68). Because available evidence suggests that Gse~ is present in all tissues, generalized deficiency of this protein could be the basis for not only PTH resistance, the hallmark of PHP type la, but could also explain the decreased responsiveness of diverse tissues (e.g., kidney, thyroid gland, gonads, and liver) to hormones that act via activation of adenylyl cyclase (e.g., PTH, TSH, gonadotropins, and glucagon) (7,69,70). Primary hypothyroidism occurs in most patients with PHP Type la (69). Typically, patients lack a goiter or antithyroid antibodies and have an elevated serum TSH with an exaggerated response to TRH. Serum levels of Y 4 may be low or low normal. Hypothyroidism may occur early in life prior to the development of hypocalcemia, and elevated serum levels of TSH are not uncommonly detected during neonatal screening (71-73). Unfortunately, early institution of thyroid hormone replacement does not seem to prevent the development of mental retardation (72). Reproductive dysfunction occurs commonly in subjects with PHP type la. Women may have delayed puberty, oligomenorrhea, and infertility (69). Plasma gonadotropins may be elevated but are more commonly normal (74). Some patients show an exaggerated serum gonadotropin response to GnRH (68,75). Features of hypogonadism may be less obvious in men with variable serum testosterone levels ranging from normal to frankly reduced. Testes may show evidence of a maturation arrest or may fail to descend normally. Fertility appears to be decreased in men with PHP type l a. Deficiency of prolactin secretion (basal and in response to secretagogues such as TRH) had been reported in some patients with PHP type 1 (76), but later studies have not confirmed these early findings (69). Obesity is common in subjects with PHP type la and pseudohypoparathyroidism, and may reflect a defective lipolytic response to hormonal stimulation due to Gsc~ deficiency (77,78). Others have hypothesized that Gsc~ deficiency results in reduced signaling through the

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melanocortin 4 receptor, which might account for the disinhibition of satiety and hyperphagia observed in many patients with PHP la (79). Abnormal h o r m o n e responsiveness may occur in some tissues without obvious clinical sequellae. For example, the hepatic glucose response to glucagon is normal although plasma cAMP concentrations fail to increase normally (69,80). In other tissues significant hormone resistance does not occur despite the apparent reduction in GsoL. Diabetes insipidus is not a feature of AHO, and urine is concentrated normally in response to vasopressin in patients with PHP type la (81). Although there is a report of adrenal insufficiency in a single individual with PHP type la (82), hypoadrenalism is not a typical feature of PHP type la and adrenocortical responsiveness to ACTH is normal (69).

Neurosensory Defects in Pseudohypoparathyroidism Type la Patients with PHP type la frequently manifest distinctive olfactory (83), gustatory (84), and auditory (85) abnormalities that are apparently unrelated to endocrine dysfunction. The molecular basis of these neurosensory deficits has become more obscure with the discovery of unique G proteins that regulate signal transduction pathways related to vision (86,87), olfaction (88), and taste (89). Mild to moderate mental retardation is common in patients with PHP type la. Farfel and Friedman assessed intelligence in 25 patients with PHP type 1 whose GsOt activity had been determined (90). The authors found an association between mental deficiency and GsOt deficiency, and speculated that reduced cAMP levels in cortical tissue may lead to mental retardation. Other factors that might contribute to mental retardation in patients with PHP type la include hypothyroidism and hypocalcemia; however, early detection and treatment have not prevented cognitive dysfunction in all patients, suggesting that Gs0t deficiency may cause a primary abnormality of neurotransmitter signaling.

The Somatic Phenotype of Albright Hereditary Osteodystrophy AHO is characterized by a unique constellation of developmental defects, including short stature, obesity, a round face, shortening of the digits (brachydactyly), subcutaneous ossification, and dental hypoplasia (Fig. 1) (1,3). Considerable variability occurs in the expression of these features even among affected members of a single family, and all of these features may not be present in every individual (91). Sometimes it may

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not be possible to detect any features of AHO in an individual with Gs~ deficiency (47,48). Although patients with AHO may be of normal height and weight, approximately 66% of children and 80% of adults are below the tenth percentile for height. This reflects a disproportionate shortening of the limbs, and arm span is less than height in the majority of patients. Obesity is a c o m m o n feature of AHO and about one-third of all patients with AHO are above the ninetieth percentile of weight for their age, despite their short stature (92) (Fig. 1A). Patients with AHO typically have a r o u n d face, a short neck, and a flattened bridge of the nose. Numerous other abnormalities of the head and neck have also been noted. Ocular findings include hypertelorism, strabismus, nystagmus, unequal pupils, diplopia, microphthalmia, and a variety of abnormal findings on funduscopic exam that range from irregular pigmentation to optic atrophy and macular degeneration. Head circumference is above the ninetieth percentile in a significant minority of children (92). AHO subjects with long-standing hypocalcemia (i.e., PHP type la) frequently develop dental abnormalities that include dentin and enamel hypoplasia, short and blunted roots, and delayed or absent tooth eruption (93). Brachydactyly is often the most obvious clinical sign of AHO, and may be a useful criterion to distinguish AHO from other causes of obesity or short stature. Brachydactyly can be symmetrical or asymmetrical and involve one or both hands or feet (Fig. 1B). Shortening of the distal phalanx of the thumb is the most c o m m o n abnormality; typically, the ratio of the width of the nail to its length is increased (so-called Murder's thumb or potter's thumb; Fig. 1D). Shortening of the metacarpals causes shortening of the digits, particularly the fourth and fifth, frequently recognized on physical exam as dimpling over the knuckles of a clenched fist (Archibald sign; Fig. 1C). A definitive diagnosis requires careful examination of radiographs of the hands and feet (Fig. 1B). Severe shortening of the distal phalanx of the thumb and third through fifth metacarpals is the most specific pattern of brachydactyly in AHO (94,95), distinguishing it from other syndromes with brachydactyly, such as familial brachydactyly, Turner syndrome, and Klinefelter syndrome (94). In addition to brachydactyly, several other skeletal abnormalities occur in subjects with AHO. Numerous deformities of the long bones have been observed, including a short ulna, bowed radius, deformed elbow or cubitus valgus, coxa vara, coxa valga, genu varum, and genu valgus deformities (92). The most c o m m o n abnormalities of the skull are hyperostosis frontalis interna and a thickened calvarium. Rarely, spinal cord compression has been reported in AHO (96). The bone age is advanced by 2-3 years in the majority of

patients (92). It should be noted that the skeletal abnormalities of AHO may not be apparent until a child is 5 years old (97). Patients with A H O develop heterotopic ossifications of the soft tissues or skin (osteoma curls) that are frequently mistaken for subcutaneous calcifications. However, these lesions are unrelated to abnormalities in serum calcium or phosphorous levels and represent true, ectopic bone consisting of spicules of mineralizing osteoid and calcified cartilage. Osteoma cutis is present in 25 to 50% of patients and is typically noted in infancy or early childhood. Occasionally, ossification of the skin is the presenting feature of AHO (98,99). Blue-tinged, stony hard papular or nodular lesions that range in size from pinpoint up to 5 cm in diameter often occur at sites of minor trauma and may appear to be migratory on serial examinations (98). More extensive and progressive ossification that affects the deep connective tissues occurs in subjects with progressive osseous heteroplasia (POH), a rare genetic disorder with apparent autosomal d o m i n a n t inheritance (100). POH patients do not share any other phenotypic features with AHO or display hormonal resistance. A mutation in GNAS1 has been identified in a girl with POH-like heterotopic ossification, no h o r m o n e resistance, and no features of AHO except mild brachydactyly of the fourth and fifth metacarpals (101). The discovery of GNAS1 mutations in an overlap syndrome of AHO and POH lead to the analysis of GNAS1 in several POH kindreds. Heterozygous inactivating mutations in GNAS1, including the 4-bp deletion in exon 7 that is a mutational hot spot in AHO, were identified (102). The mechanism by which heterozygous loss-of-function mutations in GNAS1 lead to heterotopic bone formation is unclear. The association of POH with mutations in GNAS1 further widens the phenotypic expression of GsCX deficiency, and the molecular modifiers that modulate the phenotypic expression of identical gene defects are a subject of active investigation One or more of the developmental and skeletal abnormalities of AHO can occur in subjects who do not have AHO or a defect in the GNAS1 gene, and thus need not imply that the subject has PHP type l a or pseudoPHP. Features of AHO, particularly shortened metacarpals or metatarsals, may be present in normal subjects, and have been described in patients with h°rm°ne-deficient hypoparathyroidism (103-106), renal hypercalciuria (107), and primary hyperparathyroidism (108). Furthermore, obesity, round face, brachydactyly, and mental retardation are also present in several other genetic disorders (e.g., Prader-Willi syndrome, acrodysostosis, Ullrich-Turner syndrome, Gardener syndrome). In some instances overlapping clinical features between AHO and other syndromes may cloud the diagnosis. For example, an AHO-like

PSEUDOHYPOPARATHYROIDISM / syndrome has been described in a mother and her daughter who have a proximal 15q chromosomal deletion that resembles that found in Prader-Willi syndrome (109). Terminal deletion of 2q37 [del(2) (q37.3)] is a consistent karyotypic abnormality that has been documented in patients with an AHO-like syndrome (110,111). In contrast to AHO, these patients have normal endocrine function and normal GsOLactivity (111). Thus, high-resolution chromosome analysis, biochemical/molecular analysis, and careful physical and radiologic examination are essential in discriminating between these phenocopies and AHO.

Phenotypic Variability in AHO: The Paradox of Pseudohypoparathyroidism Type la and Pseudopseudohypoparathyroidism Molecular studies have provided a basis for Gs0Ldeficiency, but they do not explain the striking variability in biochemical and clinical phenotype. Why do some GsoLcoupled pathways show reduced hormone responsiveness (e.g., PTH, TSH, gonadotropins) whereas other pathways are clinically unaffected (ACTH in the adrenal gland and vasopressin in the renal medulla)? Perhaps even more intriguing is the paradox that GsoL deficiency can be associated with hormone resistance and AHO (PHP type la), AHO only (pseudoPHP), or no apparent consequences at all (48). These observations, when considered in the context of studies showing that the n u m b e r of G s molecules in cell membranes greatly exceeds the number of either receptor or adenylyl cyclase molecules (112), raise issue with the hypothesis that a 50% deficiency of GsoL can impair h o r m o n e responsiveness. Indeed, in vitro studies of tissues and cells from subjects with PHP type la have often demonstrated normal hormonal responsiveness despite a 50% reduction in GsoLexpression (113). Although the basis for the variable expression of GsOL deficiency remains unknown, several observations provide important insights. First, clinical genetic studies have documented that PHP type la and pseudoPHP frequently occur in the same family, but are not present in the same generation. Second, analysis of published pedigrees has indicated that in most cases maternal transmission of GsoL deficiency leads to PHP type l a whereas paternal transmission of the defect leads to pseudoPHP (23,54,114,115). These findings are inconsistent with models in which chance determines phenotype or in which a second gene is interactive with the defective GNAS1 gene, because both PHP type la and pseudoPHP would be expected to occur with equal frequency and in the same sibship. By contrast, these observations first suggested the possibility of genomic imprinting of the GNAS1 gene locus as an explanation for the variable phenotypic expression of a single

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genetic defect (115). Genomic imprinting is an unusual mode of regulation of gene expression that results in the expression of only one parental allele in somatic tissues. Thus, genomic imprinting can account for functional differences that arise as a consequence of the parental origin of a gene allele. Studies have indeed confirmed that the GNAS1 gene is imprinted, but in a far more complex m a n n e r than had been anticipated. Two upstream promoters, each associated with a large coding exon, lie 35 kb upstream of GNAS1 exon 1 (Fig. 6). These promoters are only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5' of these exons encodes NESP55, which is expressed exclusively from the maternal allele. By contrast, the XLoLs exon is paternally expressed (31,32). Exon 1A is a third upstream exon that apparently consists only of untranslated sequence that is spliced to exons 2-13 (33). This exon is also imprinted and is derived exclusively from the paternal allele (116). Despite the simultaneous imprinting in both the paternal and maternal directions of the GNAS1 gene, expression of GsOLappears to be biallelic in all h u m a n tissues that have been examined thus far (31,32,117). Moreover, the lack of access to relevant tissues (i.e., kidney) from subjects with PHP type la has prevented direct analysis of GsoL expression in patients with this disorder. To overcome these difficulties, murine models of PHP type la have been developed through disruption of a single Gnas gene in embryonic stem cells (118,119) Although these mice have reduced levels of GsoLprotein, they lack many of the features of the h u m a n disorder. Biochemical analyses of these heterozygous Gnas knockout mice suggest that Gs0Lexpression may derive from only the maternal allele in some tissues (e.g., renal cortex) and from both alleles in other tissues (e.g., renal medulla) (118,120). Accordingly, mice that inherit the defective Gnas gene maternally express only that allele in imprinted tissues, such as the PTH-sensitive renal proximal tubule, and therefore have no functional GsOLprotein. By contrast, the 50% reduction in Gsc~ expression that occurs in nonimprinted tissues, which express both Gnas alleles, may account for more variable and moderate h o r m o n e resistance in these sites (e.g., the thyroid). Thus, variable hormonal responsiveness implies that haploinsufficiency of GsoLis tissue specific; that is, in some tissues a 50% reduction in GsOLis still sufficient to facilitate normal signal transduction. Confirmation of this proposed mechanism in patients with AHO will require demonstration that the h u m a n GsoL transcript is paternally imprinted in the renal cortex. In AHO, inherited GNAS1 gene mutations reduce expression or function of GsoL protein. By contrast, in the McCune-Albright syndrome, postzygotic somatic mutations in the GNAS1 gene enhance activity of the

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protein (121,122). These mutations lead to constitutive activation of adenylyl cyclase and result in proliferation and autonomous hyperfunction of hormonally responsive cells. The clinical significance of Gse~ activity as a determinant of h o r m o n e action is emphasized by the description by Iiri et al. (44) of two males with both precocious puberty and PHP type la. These two unrelated boys had identical GNAS1 gene mutations in exon 13 (A366S) that resulted in a temperature-sensitive form of Gse~. This GsoL is constitutively active in the cooler environment of the testis, while being rapidly degraded in other tissues at normal body temperature. Thus, different tissues in these two individuals could show h o r m o n e resistance (to PTH and TSH), h o r m o n e responsiveness (to ACTH), or h o r m o n e - i n d e p e n d e n t activation (to LH).

PSEUDOHYPOPARATHYROIDISM

TYPE lb

Subjects with PHP type 1 who lack features of AHO, manifest h o r m o n e resistance that is limited to PTH target organs (Table 1), and have normal Gse~ activity (Fig. 5) (69) are variants of PHP termed PHP type lb (123). Although patients with PHP type lb fail to show a n e p h r o g e n o u s cAMP response to PTH, they often manifest osteopenia or skeletal lesions similar to those that occur in patients with hyperparathyroidism, including osteitis fibrosa cystica (Fig. 4) (124). Cultured bone cells from one patient with PHP type l b and osteitis fibrosa cystica were shown to have normal adenylyl cyclase responsiveness to PTH in vitro (10). These observations have suggested that at least one intracellular signaling pathway coupled to the PTH receptor may be intact in patients with PHP type lb. Specific resistance of target tissues to PTH, and normal activity of Gsc~, had implicated decreased expression or function of the P T H / P T H r P receptor as the cause for h o r m o n e resistance. In addition, cultured fibroblasts from some, but not all, PHP type 1b patients were shown to accumulate reduced levels of cAMP in response to PTH (125) and to contain decreased levels of mRNA encoding the P T H / P T H r P receptor (126). Several lines of evidence suggest that the primary defect in PHP type l b is not in the gene encoding the P T H / P T H r P receptor, however. First, pretreatment of cultured fibroblasts from subjects with PHP type l b with dexamethsone was found to normalize the PTHinduced cAMP response and to increase expression of P T H / P T H r P receptor mRNA (126). Second, molecular studies have failed to disclose mutations in the coding exons (127) and p r o m o t e r regions (128) of the P T H / P T H r P receptor gene or its mRNA (129). Furthermore, linkage studies have demonstrated discordance between inheritance of PHP type lb and alleles for the P T H / P T H r P receptor, thus excluding

defects even in regions of the gene that have not yet been examined by sequence analysis (130). Third, mice ( 131 ) and humans (132) that are heterozygous for inactivation of the gene encoding the P T H / P T H r P receptor do not manifest PTH resistance or hypocalcemia. And finally, inheritance of two defective P T H / P T H r P receptor genes results in Blomstrand chondrodysplasia, a lethal genetic disorder characterized b y advanced e n d o c h o n d r a l bone maturation (132). Thus, it is likely that the molecular defect in PHP type l b resides in other genes that regulate expression or activity of the P T H / P T H r P receptor. Although most cases of PHP type l b appear to be sporadic, familial cases have been described in which transmission of the defect is most consistent with an autosomal dominant pattern (133,134). In some of these kindreds, there appears to be a pattern of inheritance consistent with imprinting as observed in PHP type la. Studies have used gene mapping to identify the location of the PHP type lb gene (135). The PHP type lb gene was mapped to a small region of chromosome 20ql 3.3 near the GNAS1 gene, thus raising the possibility that some patients with PHP type l b have inherited a defective promoter or enhancer that regulates expression of GsoL in the kidney (135). Furthermore, an imprinting defect that impairs the switching of the paternal-to-maternal imprint in the female germ line, resulting in offspring with two paternally imprinted, transcriptionally silent genes, could result in renal Gse~ deficiency in PHP type lb. Alternatively, the chromosomal region demonstrating the greatest linkage to PHP type l b is centromeric to GNAS1, thus raising the intriguing possibility that a second gene responsible for mineral homeostasis resides close to GNAS1 and may be defective in PHP type 1b (135).

PSEUDOHYPOPARATHYROIDISM

TYPE lc

Resistance to multiple hormones has been described in several patients with AHO who have do not have a demonstrable defect in G s or G~ (Table 1) (69,99,136). This disorder is termed PHP type l c. The nature of the lesion in such patients is unclear, but it could be related to some other general c o m p o n e n t of the receptor-adenylyl cyclase system, such as the catalytic unit (137). Alternatively, these patients could have functional defects of G s (or Gi) that do not become apparent in the assays presently available.

PSEUDOHYPOPARATHYROIDISM

TYPE 2

PHP type 2 is the least c o m m o n form of PHE This variant of PHP is typically a sporadic disorder, although one case of familial PHP type 2 has been reported

PSEUDOHYPOPARATHYROIDISM / (138). Patients do not have features of AHO. Renal resistance to PTH in PHP type 2 patients is manifested by a reduced phosphaturic response to administration of PTH, despite a normal increase in urinary cAMP excretion (Table 1) (9). These observations suggest that the PTH receptor-adenylyl cyclase complex functions normally to increase cyclic AMP in response to PTH, and are consistent with a model in which PTH resistance arises from an inability of intracellular cAMP to initiate the chain of metabolic events that result in the ultimate expression of PTH action. Although supportive data are not yet available, a defect in cAMP-dependent protein kinase A has been proposed as the basis for this disorder (9). Alternatively, the defect in PHP type 2 may not reside in an inability to generate a physiologic response to intracellular cAMP: a defect in another PTH-sensitive signal transduction pathway may explain the lack of a phosphaturic response. One candidate is the PTH-sensitive phospholipase C pathway that leads to increased concentrations of the intracellular second messengers inositol 1,4,5-trisphosphate and diacylglycerol (139,140) and cytosolic calcium (141-144). In some patients with PHP type 2 the phosphaturic response to PTH has been restored to normal after serum levels of calcium have been normalized by treatm e n t with calcium infusion or vitamin D (145). These results point to the importance of Ca 2+ as an intracellular second messenger. Finally, a similar dissociation between the effects of PTH on generation of cAMP and tubular reabsorption of phosphate has been observed in patients with profound hypocalcemia due to vitamin D deficiency (146), suggesting that some cases of PHP type 2 may in fact represent vitamin D deficiency.

DIAGNOSIS Natural History The natural history of PHP is quite variable. Although PHP is congenital, hypocalcemia is not present from birth, and the biochemical defects arise gradually during childhood. The initial manifestations of tetany typically occur between 3 and 8 years of age, but the significance of these findings may not be appreciated and the diagnosis of hypocalcemia may be delayed for months or even years. A progressive decline in serum calcium, preceded by increasing levels of serum phosphate, PTH, and 1,25(OH)2D ~, has been documented in one child as he advanced from 3 to 31⁄2 years of age (147). In a second report serial PTH infusions were used to evaluate h o r m o n e responsiveness in an infant from an AHO family. This child was shown to have a normal cAMP response at age 3 months when serum levels of calcium, phosphorous, and PTH were normal, but was found to have an abnormal cAMP

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response when retested at age 2.6 years after he had developed tetany and was found to be hypocalcemic (25). At the time of his second PTH infusion, the child had markedly elevated serum concentrations of phosphorous and PTH and was receiving thyroxine for recently diagnosed primary hypothyroidism (25). Some affected children show few symptoms of tetany, and the diagnosis of PHP is recognized only later in life after hypocalcemia is discovered serendipitously or when features of AHO become obvious. Hypocalcemia may not always provide a clue to the clinical diagnosis of PHP, however, because some PHP patients are able to maintain a normal serum calcium level without treatment (i.e., normocalcemic PHP) (16).

General Physical, Laboratory, and Radiographic Features The diagnosis of PHP should be considered in any patient with hypocalcemia and hyperphosphatemia. A low serum calcium level may be found during an evaluation of unexplained paresthesias or seizures, or may be discovered after multichannel analysis of a blood specimen obtained as part of a routine examination. PHP should be strongly suspected if the serum concentration of PTH is elevated, although occasionally serum levels of PTH are "inappropriately" normal in subjects with PHP owing to confounding hypomagnesemia (133) or other factors (148). Hypocalcemia may be precipitated or worsened during times of "stress" on calcium homeostasis, such as during early pregnancy, lactation, or during an episode of acute pancreatitis. Although hypocalcemia is present in most patients with hypoparathyroidism b y the end of the first decade of life, this biochemical finding may go undetected for many years. Cataracts and intracranial calcification, particularly of the basal ganglion, are c o m m o n in patients with all forms of chronic hypoparathyroidism. Thus, the presence of these ectopic or metastatic calcifications does not help to discriminate a m o n g the various causes of hypocalcemia and hyperphosphatemia (149). Intracranial calcifications are readily detected when CT scanning is employed (150,151), and may occasionally be associated with symptoms such as Parkinson's disease (152). Unusual presenting manifestations of PHP include neonatal hypothyroidism (71,72), unexplained cardiac failure (153), Parkinson's disease (152) and spinal cord compression (154). A diagnosis of PHP should be suspected in any patient with hypocalcemia and elevated serum level of phosphorous and PTH, particularly when clinical features of AHO are present. Further corroboration of the diagnosis of PHP requires demonstration of normal renal function and normal serum levels of magnesium and 25-hydroxyvitamin D. The presence of AHO a n d / o r manifestations of m u l t i h o r m o n e resistance,

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such as hypothyroidism or hypogonadism, favors a diagnosis of PHP type la (69). When most or all of these features are present, more sophisticated tests may not be necessary to confirm the clinical diagnosis. Serum calcium levels can fluctuate in patients with PHP, and may spontaneously change from low to normal and vice versa, thus contributing to the confusion regarding the distinction between PHP and pseudoPHP (16,155). However, the abnormal cAMP response to administered PTH (see below) does not become normal in PHP patients who become normocalcemic with or without treatment. Thus, the PTH infusion remains the most reliable test to distinguish between these two variants (Fig. 2).

Specialized Tests The biochemical hallmark of PHP is the failure of the target organs--bone, and kidney--to respond normally to PTH. Additional tests have been developed to identify subjects with PHP type la; these research tests, which are based on analysis of G~ot protein or the GNAS1 gene, are only rarely indicated u n d e r typical clinical circumstances. The classic tests of Ellsworth and Howard, and of Chase, Melson, and Aurbach, involved the intravenous infusion of 200-300 USP units of bovine parathyroid extract (parathyroid injection, USP; Lilly) and subsequent measurement of urinary excretion of n e p h r o g e n o u s (or total) cAMP (Fig. 2) and phosphate. This relatively crude PTH preparation is no longer available, and has been replaced by synthetic peptides corresponding to the amino-terminal region of h u m a n PTH (e.g., hPTH(1-34) (156-158). A standard protocol involves the infusion of synthetic hPTH(1-34) peptide, 200 units in an adult and 3 u n i t s / k g body weight (200 units maximum) in children over the age of 3 years, intravenously over 10 minutes (156,159). Test subjects should be in a fasting state and active urine output should be initiated and maintained by the ingestion of 200 ml of water per hour, 2 hours prior to the infusion of PTH and continuing through the study. A base line urine collection should be made in a 60-minute period preceding the PTH infusion. Starting at time 0, urine should be collected in separate collections at three time periods: 0-30, 30-60, and 60-120 minutes. Blood samples should be obtained at time 0 and at 2 hours after the start of PTH infusion for m e a s u r e m e n t of serum creatinine and phosphorus concentrations. Urine samples should be analyzed for cAMP, phosphorus, and creatinine concentrations. The preferred unit for expression of urinary cAMP is n m o l / 1 0 0 ml (or per liter) of glomerular filtrate ( n m o l / d l GF). The cAMP response during the first 30 minutes after the start of PTH infusion best differentiates patients with PHP type 1 from those with

hypoparathyroidism and from normal subjects, compared to other parameters of cAMP metabolism (159). Normal subjects and patients with h o r m o n o p e n i c hypoparathyroidism usually display a 10- to 20-fold increase in urinary cAMP excretion, whereas patients with PHP type 1 (types la and lb) show a markedly blunted response regardless of their serum calcium concentration. The urinary cAMP response to infusion of synthetic h P T H fragments in patients with PHP type 1 is unrelated to serum calcium levels, but may be related to endogenous serum PTH levels. The maximal urinary cAMP response to PTH increases after suppression of endogenous PTH in patients with PHP type 1, but nevertheless does not reach that of the normal range (8). Thus, this test can distinguish patients with so-called "normocalcemic" PHP (i.e., patients with PTH resistance who are able to maintain normal serum calcium levels without treatment) from subjects with pseudoPHP (who will have a normal urinary cAMP response to PTH (4,23) (Fig. 2). Subjects with PHP type 2 typically manifest a normal urinary cAMP response to infused PTH but fail to demonstrate an appropriate phosphaturic response. Several metabolic abnormalities, such as hypo- and hypermagnesemia and metabolic acidosis, may interfere with the renal generation and excretion of cAMP in response to PTH (160-163). These abnormalities should be corrected if possible, but probably do not interfere with the interpretation of the test. Calculation of the phosphaturic response to PTH as the percent decrease in tubular maximum for phosphate reabsorption (percent decrease in TmP/GFR) during the first hour after PTH infusion yields the best separation between normal subjects and patients with PHP or hypoparathyroidism (159). However, distinction between groups is also possible when the results are expressed as the decrease in percent tubular reabsorption of phosphorus (decrease in % TRP). A nomogram has been developed that facilitates calculation of T m P / G F R (164). T m P / G F R is elevated in patients with PHP and hypoparathyroidism. Patients with hormonedeficient hypoparathyroidism have a steep decrease in T m P / G F R during the first h o u r after beginning the infusion of PTH. This decrease does not occur in patients with PHP [for further details see references by Mallette et al. (156,159)]. Although a normal phosphate response may occur in PHP type 1 patients with serum calcium or PTH levels in the normal range (8), in patients with PHP type 2 the phosphaturic response to PTH is not changed despite at least a 10-fold increase in cAMP excretion. Unfortunately, interpretation of the phosphaturic response to PTH is often complicated by r a n d o m variations in phosphate clearance, and it is sometimes not possible to classify a phosphaturic response as normal or subnormal regardless of the

PSEUDOHYPOPARATHYROIDISM / criteria employed. More perplexing yet is the observation that biochemical findings that resemble PHP type 2 have been found in patients with various forms of vitamin D deficiency (146). In these patients, marked hypocalcemia is accompanied by hyperphosphatemia due presumably to an acquired dissociation between the a m o u n t of cAMP generated in the renal tubule and its effect on phosphate clearance. The plasma cAMP response to PTH can also be used to differentiate patients with PHP type 1 from normal subjects and from patients with hypoparathyroidism (158,165,166). Patients with PHP type 2 can be expected to have normal responsiveness, however. This test offers few advantages over protocols that assess the urinary excretion of cAMP, because changes in plasma cAMP in normal subjects and patients with hypoparathyroidism are much less dramatic than changes in urinary cAMP, and urine must still be collected if one wishes to assess the phosphaturic response to PTH. One reasonable indication for measuring the plasma cAMP response to PTH is the evaluation of patients in whom proper collection of urine is not possible, such as young children (166). The plasma 1,25(OH)zD ~ response to PTH has been used to differentiate between hormone-deficient and hormone-resistant hypoparathyroidism (157,167). In contrast to normal subjects and patients with hypoparathyroidism, patients with PHP had no significant increase in circulating levels of 1,25(OH)zD 3. This proposed test readily demonstrates the difference in the pathophysiology between hypoparathyroidism and PHE Its clinical relevance is probably limited to distinguishing type 1 from type 2 PHP, whereby the expected increase in the latter form of PHP might be a more reliable parameter than the phosphaturic response to PTH.

TREATMENT The treatment of hypocalcemia and hyperphosphatemia in patients with PHP is based on the guidelines r e c o m m e n d e d for treatment of patients with other forms of hypoparathyroidism (see Chapter 52). Urgent treatment of acute or severe symptomatic hypocalcemia in patients with PHP is best accomplished by the intravenous infusion of calcium. The goal is alleviation of symptoms and prevention of laryngeal spasm and seizures. The long-term treatment of hypocalcemia in patients with PHP requires administration of oral calcium and vitamin D or analogs. The goals of therapy are to maintain serum ionized calcium levels in the normal range, to avoid hypercalciuria, and to reduce elevated levels of circulating PTH. Patients with hypoparathyroidism have increased urinary calcium

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excretion in relation to serum calcium and are therefore prone to hypercalciuria (168). By contrast, patients with PHP have significantly lower urinary calcium in relation to serum calcium (168,169) and can tolerate serum calcium levels that are within the normal range without developing hypercalciuria (6). Once normocalcemia has been attained attention should be directed toward suppression of PTH levels to normal. This is important because elevated PTH levels in patients with PHP are frequently associated with increased bone remodeling. Hyperparathyroid bone disease, including osteitis fibrosa cystica (97,124,170) and cortical osteopenia (Fig. 4) (14), can occur in patients with PHP type lb. These subjects may have elevated serum levels of alkaline phosphatase (170) and urine hydroxyproline (14,171). In this regard calcitriol has a theoretical advantage over other vitamin D preparations (see below) because it may inhibit PTH release directly (172). Oral calcium is usually administered in amounts of 1-3 g of elemental calcium per day in divided doses. To assure optimal absorption and to maximize binding of dietary phosphorous, oral calcium supplements should be taken with water or other fluids, and with meals (173). Many considerations are involved in the selection of a calcium supplement, and none are unique to the treatment of PHP (see Chapter 52). Calcium carbonate is an inexpensive form of calcium that is very convenient owing to its high content of elemental calcium (40%). W h e n taken with food, absorption of calcium from calcium carbonate is adequate even in patients who are achlorhydric. All patients with PHP who are hypocalcemic will require vitamin D or analogs in addition to calcium. Calcitriol, the active form of vitamin D, is the most physiologic treatment choice. Patients with PHP require about 75% as m u c h calcitriol to maintain normocalcemia as do patients with hypoparathyroidism (174). Almost all patients with hypoparathyroidism or PHP can be effectively treated with calcitriol in the a m o u n t of 0.25 txg twice a day to 0.5 txg four times a day. Because of the expense of calcitriol and the need to administer the drug several times per day, other vitamin D preparations may be preferred. Patients with all forms of hypoparathyroidism and PHP will respond to pharmacologic doses of ergocalciferol and calcifidiol. Ergocalciferol is the least expensive choice for vitamin D therapy, and provides a long duration of action (with corresponding prolonged potential toxicity). Patients with PHP require lower doses of vitamin D than do patients with hypoparathyroidism (174), an observation that reflects the response of bone and renal distal tubular cells to endogenous PTH (175). Treatment with calcium and vitamin D usually decreases the elevated serum phosphate to a high normal level because of a

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favorable balance between increased urinary phosphate excretion and decreased intestinal phosphate absorption. In general, phosphate-binding gels such as alum i n u m hydroxide are not necessary. Estrogen therapy and pregnancy have particularly interesting effects on calcium homeostasis in patients with PHR Estrogen therapy can reduce serum calcium concentrations in women with PHP type 1 (176) as well as women with hypoparathyroidism (177). In contrast, at the time of the menses, when estrogen levels are low, some well-treated hypoparathyroid women may develop symptomatic hypocalcemia with the cause remaining unknown (178). The same p h e n o m e n o n occurs occasionally in women with PHR Hypocalcemic symptoms are relieved in 30-60 minutes by ingestion of 200-400 mg of elemental calcium. Paradoxically, during the high-estrogen state of pregnancy, the patients of Zerwekh and Breslau remained normocalcemic without therapeutic amounts of calcium and vitamin D (179). During pregnancy, serum 1,25(OH)2D3 concentrations increased two- to threefold but the PTH levels were nearly half of what was present before pregnancy. After delivery, serum calcium and 1,25(OH)2D 3 levels decreased and PTH rose (176). Because placental synthesis of 1,25-dihydroxyvitamin D is not compromised in patients with PHP (179), the placenta may have contributed to the maintenance of normocalcemia during pregnancy in these patients. In contrast, patients with hypoparathyroidism may require treatment with larger amounts of vitamin D and calcium in the latter half of pregnancy (180). Patients with AHO may require specific treatment for unusual problems related to their developmental and skeletal abnormalities. Patients with PHP type la should be treated for their associated hypogonadism and hypothyroidism. Ectopic ossification occurs in about 30% of patients with AHO (92), but rarely causes a problem. At times, large extraskeletal osteomas may occur that will require surgical removal to relieve pressure symptoms (98).

CONCLUSION The evolving characterization of the signal transduction pathways that regulate PTH secretion and action has led to a new understanding of the molecular basis of some forms of hypoparathyroidism and PHE H a n d in h a n d with this experimental approach have come unexpected insights gained through careful study of patients with disorders of mineral metabolism. Patients with defects in the GNAS1 gene provide us with the unanticipated opportunity to explore genomic imprinting as a genetic mechanism, and to test this model for regulating parental contributions to the developing fetus as an explanation for the functional differences

between subjects with PHP type la and pseudoPHP. Continuing work to define the molecular genetic defect in PHP type l b will no doubt provide equally exciting and novel insights that further illuminate PTH receptor signaling pathways in classic target tissues such as bone and kidney.

ACKNOWLEDGMENTS This work has been supported in part by grants from the National Institutes of Health (DK-34281 and DK56178, and GCRC M01-RR00052).

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125. Silve C, Suarez F, el Hessni A, Loiseau A, Graulet AM, Gueris J. The resistance to parathyroid hormone of fibroblasts from some patients with type Ib pseudohypoparathyroidism is reversible with dexamethasone. J Clin Endocrinol Metab 1990;71:631-638. 126. Suarez E Lebrun JJ, Lecossier D, Escoubet B, Coureau C, Silve C. Expression and modulation of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in skin fibroblasts from patients with type Ib pseudohypoparathyroidism. J Clin Endocrinol Metab 1995;80:965-970. 127. Schipani E, Weinstein LS, Bergwitz C, Iida-Klein A, Kong XF, Stuhrmann M, Kruse K, Whyte ME Murray T, Schmidtke J, et al. Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene. J Clin Endocrinol Metab 1995;80:1611-1621. 128. BettounJD, Minagawa M, Kwan MY, Lee HS, Yasuda T, Hendy GN, Goltzman D, White JH. Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTHrelated peptide receptor gene: Analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type lb. J Clin Endocrinol Metab 1997;82:1031-1040. 129. Fukumoto S, Suzawa M, Takeuchi Y, Kodama Y, Nakayama K, Ogata E, Matsumoto T, Fujita T. Absence of mutations in parathyroid hormone (PTH)/PTH-related protein receptor complementary deoxyribonucleic acid in patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 1996;81: 2554-2558. 130. Jan de Beur SM, Ding CL, LaBuda MC, Usdin TB, Levine MA. Pseudohypoparathyroidism lb: Exclusion of parathyroid hormone and its receptors as candidate disease genes. J Clin Endocrinol Metab 2000;85:2239-2246. 131. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LK, Ho C, Mulligan RC, et al. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996;273:663-666. 132. Jobert AS, Zhang P, Couvineau A, Bonaventure J, Roume J, Le Merrer M, Silve C. Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. J Clin Invest 1998;102:34-40. 133. Allen DB, Friedman AL, Greer FR, Chesney RW. Hypomagnesemia masking the appearance of elevated parathyroid hormone concentrations in familial pseudohypoparathyroidism. Am J Med Genet 1988;31:153-158. 134. Winter JSD. Hughes IA. Familial pseudohypoparathyroidism without somatic anomalies. Can Med AssocJ 1980;123:26-31. 135. Juppner H, Schipani E, Bastepe M, Cole DE, Lawson ML, Mannstadt M, Hendy GN, Plotkin H, Koshiyama H, Koh T, et al. The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc Natl Acad Sci USA 1998;95: 11798-11803. 136. Farfel Z, Brothers VM, Brickman AS, Conte F, Neer R, Bourne HR. Pseudohypoparathyroidism: inheritance of deficient receptor-cyclase coupling activity. Proc Natl Acad Sci USA 1981;78: 3098-3102. 137. Barrett D, Breslau NA, Wax MB, Molinoff PB, Downs RW, Jr. New form of pseudohypoparathyroidism with abnormal catalytic adenylate cyclase. AmJPhysiol 1989;257:E277-E283. 138. Van Dop C. Pseudohypoparathyroidism: Clinical and molecular aspects. Semin Nephro11989;9:168-178. 139. Civitelli R, Reid IR, Westbrook S, Avioli LV, Hruska KA. PTH elevates inositol polyphosphates and diacylglycerol in a rat osteoblast-like cell line. A m J Physiol 1988;255:E660-E667. 140. Dunlay R. Hruska K. PTH receptor coupling to phospholipase C is an alternate pathway of signal transduction in bone and kidney. Am J Physio11990;258:F223-F231.

141. Gupta A, Martin KJ, Miyauchi A, Hruska KA. Regulation of cytosolic calcium by parathyroid hormone and oscillations of cytosolic calcium in fibroblasts from normal and pseudohypoparathyroid patients. Endocrinology 1991 ;128:2825-2836. 142. Civitelli R, Martin TJ, Fausto A, Gunsten SL, Hruska KA, Avioli LV. Parathyroid hormone-related peptide transiently increases cytosolic calcium in osteoblast-like cells: Comparison with parathyroid hormone. Endocrinology 1989; 125:1204-1210. 143. Reid IR, Civitelli R, Halstead LR, Avioli LV, Hruska KA. Parathyroid hormone acutely elevates intracellular calcium in osteoblastlike cells. Am J Physiol 1987;253:E45-E51. 144. Yamaguchi DT, Hahn TJ, Iida-Klein A, Kleeman CR, Muallem S. Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. J Biol Chem 1987;262:7711-7718. 145. Kruse K, Kracht U, Wohlfart K, Kruse U. Biochemical markers of bone turnover, intact serum parathyroid horn and renal calcium excretion in patients with pseudohypoparathyroidism and hypoparathyroidism before and during vitamin D treatment. EurJ Pediatr 1989;148:535-539. 146. Rao DS, Parfitt AM, Kleerekoper M, Pumo BS, Frame B. Dissociation between the effects of endogenous parathyroid hormone on adenosine 3',5'-monophosphate generation and phosphate reabsorption in hypocalcemia due to vitamin D depletion: An acquired disorder resembling pseudohypoparathyroidism type II. J Clin Endocrinol Metab 1985;61:285-290. 147. Tsang RC, Venkataraman P, Ho M, Steichen JJ, WhitsettJ, Greer E The development of pseudohypoparathyroidism. Am J Dis Child 1984;138:654-658. 148. Attanasio R, Curcio T, Giusti M, Monachesi M, Nalin R, Giordano G. Pseudohypoparathyroidism. A case report with low immunoreactive parathyroid hormone and multiple endocrine dysfunctions. Minerva Endocrinol 1986;11:267-273. 149. Litvin Y, Rosler A, Bloom RA. Extensive cerebral calcification in hypoparathyroidism. Neuroradiology 1981 ;21:271-271. 150. Sachs C, Sjoberg HE, Ericson K. Basal ganglia calcifications on CT: Relation to hypoparathyroidism. Neurology 1982;32: 779-782. 151. Korn-Lubetzki I, Rubinger D, Siew E Visualization of basal ganglion calcification by cranial computed tomography in a patient with pseudohypoparathyroidism. Isr J Med Sci 1980;16: 40-41. 152. Pearson DWM, Durward WF, Fogelman I, Boyle IT, Beastall G. Pseudohypoparathyroidism presenting as severe Parkinsonism. Postgrad MedJ 1981;57:445-447. 153. Miano A, Casadel G, Biasini G. Cardiac failure in pseudohypoparathyroidism. Helv Paediatr Acta 1981;36:191-192. 154. Cavallo A, Meyer III WJ, Bodensteiner JB, Chesson AL. Spinal cord compression: An unusual manifestation of pseudohypoparathyroidism. A m J Dis Child 1980;134:706-707. 155. Breslau NA, Notman D, CanterburyJM, Moses AM. Studies on the attainment of normocalcemia in patients with pseudohypoparathyroidism. Am J Med 1980;68:856-860. 156. Mallette LE. Synthetic human parathyroid hormone 1-34 fragment for diagnostic testing. Ann Intern Med 1988;109:800-804. 157. McElduff A, Lissner D, Wilkinson M, Cornish C, Posen S. A 6hour human parathyroid hormone (1-34) infusion protocol: Studies in normal and hypoparathyroid subjects. CalcifTissue Int 1987;41:267-273. 158. Furlong TJ, Seshadri MS, Wilkinson MR, Cornish CJ, Luttrell B, Posen S. Clinical experiences with human parathyroid hormone 1-34. Aust N ZJMed 1986;16:794-798. 159. Mallette LE, Kirkland JL, Gagel RF, Law WM, Jr, Heath III H. Synthetic human parathyroid hormone-(1-34) for the study of pseudohypoparathyroidism. J Clin Endocrinol Metab 1988;67: 964-972.

PSEUDOHYPOPARATHYROIDISM 160. Rude RK, Oldham SB, Singer FR. Functional hypoparathyroidism and parathyroid hormone end-organ resistance in human magnesium deficiency. Clin Endocrino11976;5:209-224. 161. Slatopolsky E, Mercado A, Morrison A, Yates J, Klahr S. Inhibitory effects of hypomagnesemia on the renal action of parathyroid hormone. J Clin Invest 1976;58:1273-1279. 162. Beck N. Davis BB. Impaired renal response to parathyroid hormone in potassium depletion. AmJPhysio11975;228:179-183. 163. Beck N, Kim HE Kim KS. Effect of metabolic acidosis on renal action of parathyroid hormone. AmJPhysiol 1975;228:1483-1488. 164. Walton RJ, Bijvoet OLM. Nomogram for derivation of renal threshold phosphate concentration. Lancet 1975;309:310. 165. Sohn HE, Furukawa Y, Yumita S, Miura R, Unakami H, Yoshinaga K. Effect of synthetic 1-34 fragment of human parathyroid hormone on plasma adenosine 3',5'-monophosphate (cAMP) concentrations and the diagnostic criteria based on the plasma cAMP response in Ellsworth-Howard test. EndocrinolJpn 1984;31:33-40. 166. Stirling HE DarlingJA, Barr DG. Plasma cyclic AMP response to intravenous parathyroid hormone in pseudohypoparathyroidism. Acta Paediatr Scand 1991;80:333-338. 167. Miura R, Yumita S, Yoshinaga K, Furukawa Y. Response of plasma 1,25-dihydroxyvitamin D in the human PTH(1-34) infusion test: An improved index for the diagnosis of idiopathic hypoparathyroidism and pseudohypoparathyroidism. Calcif Tissue Int 1990;46:309-313. 168. Litvak J, Moldawer ME Forbes AP, Henneman PH. Hypocalcemic hypercalciuria during vitamin D and dihydrotachysterol therapy of hypoparathyroidism. J Clin Endocrinol Metab 1958;18:246-252. 169. Yamamoto M, Takuwa Y, Masuko S, Ogata E. Effects of endogenous and exogenous parathyroid hormone on tubular reabsorption of calcium in pseudohypoparathyroidism. J Clin Endocrinol Metab 1988;66:618-625. 170. Kolb FO, Steinbach HL. Pseudohypoparathyroidism with secondary hyperparathyroidism and osteitis fibrosa. J Clin Endocrinol Metab 1962;22:59-64. 171. Tollin SR, Perlmutter S, AloiaJE Serial changes in bone mineral density and bone turnover after correction of secondary hyperparathyroidism in a patient with pseudohypoparathyroidism type Ib. JBone Miner Res 2000;15:1412-1416. 172. Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin KJ. Marked suppression of secondary hyperparathyroidism by

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intravenous administration of 1,25-dihydroxycholecalciferol in uremia patients. J Clin Invest 1984;74:2136-2143. 173. Shangraw RF. Factors to consider in the selection of a calcium supplement. Public Health Rep Supp11989;104:46-50. 174. Okano K, Furukawa Y, Morii H, Fujita T. Comparative efficacy of various vitamin D metabolites in the treatment of various types of hypoparathyroidism. J Clin Endocrinol Metab 1982;55:238-243. 175. Breslau NA. Pseudohypoparathyroidism: Current concepts. Am J Med Sci 1989;298:130-140. 176. Breslau NA, Zerwekh JE. Relationship of estrogen and pregnancy to calcium homeostasis in pseudohypoparathyroidism. J Clin Endocrinol Metab 1986;62:45-51. 177. Verbeelen D, Fuss M. Hypercalcemia induced by estrogen withdrawal in vitamin D-treated hypoparathyroidism. Br Med J 1979;1:522-523. 178. Mallette LE. Case report: Hypoparathyroidism with mensesassociated hypocalcemia. Am J Med Sci 1992;304:32-37. 179. Zerwekh JE. Breslau NA. Human placental production of lalpha,25-dihydroxyvitamin D3: Biochemical characterization and production in normal subjects and patients with pseudohypoparathyroidism. J Clin Endocrinol Metab 1986;62:192-196. 180. Caplan RH, Beguin EA. Hypercalcemia in a calcitriol-treated hypoparathyroid woman during lactation. Obstet Gynecol 1990; 76: 485-489. 181. Jan de Beur SM, Deng Z, Ding C, Levine MA. Amplification of the GC-rich exon 1 of GNAS1 and identification of three novel nonsense mutations in Albright's hereditary osteodystrophy. Endocr Soc 1998 (abstract). 182. Jan de Beur SM, Ding C, Levine MA. GNAS1 gene mutations. Unpublished work, 2000. 183. Takeda K, Yokoyama M, Hashimoto K, Hiromatsu Y, Yamanaka H, Shimizu T, Sasaki M. Mutations in exon 7 of the GTP-binding protein Gs alpha were not a common cause of pseudohypoparathyroidism with Albright's hereditary osteodystrophy in Japanese. EndocrJ 1997;44:621-625. 184. Warner DR, Gejman PV, Collins RM, Weinstein LS. A novel mutation adjacent to the switch III domain of G(S alpha) in a patient with pseudohypoparathyroidism. Mol Endocrinol 1997;11:1718-1727. 185. Wu W-I, Schwindinger WF, Aparicio LF, Levine MA. Selective resistance to parathyroid hormone caused by a novel uncoupling mutation in the carboxyl terminus of CotS: A cause of pseudohypoparathyroidism type lb. J Biol Chem 2001 ;276:165-171.

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CI-IAeTV52 Treatment of Hypoparathyroidism

MARC K. DREZNER University of Wisconsin-Madison, Madison, Wisconsin 53792

INTRODUCTION

can demonstrate increased intracranial pressure, frequently in association with papilledema, subcapsular cataracts, and calcification of the basal ganglia (6). Cardiovascular manifestations of a decreased serum calcium level include arrhythmias, bradycardia, hypotension, and impaired cardiac contractility (7). The hypotension is typically unresponsive to the use of fluids and pressors until calcium is administered. Similarly, impaired cardiac contractility responds poorly to inotropic agents unless the hypocalcemia is first corrected. Serum calcium concentrations in affected individuals vary from a very low value, 5.5 mg/dl, to low-normal levels, depending on the degree of PTH deficiency. Serum phosphorus concentrations usually are increased to values as high as 7.5 mg/dl.

The serum calcium concentration is normally mainmined within a very narrow range by an exquisitely sensitive homeostatic system (1). In the absence of the calcium-maintaining effects of parathyroid hormone, the hypoparathyroid disorders cause hypocalcemia in the subset of patients with this biochemical abnormality (2). The clinical manifestations in affected patients at the time of initial presentation often suggest the presence of these diseases, confirmation of which is usually easily achieved by appropriate laboratory tests. For example, a history of recent neck surgery may suggest injury to the parathyroid glands and surgical hypoparathyroidism, and the coexistence of ancillary endocrine disorders may indicate an autoimmune process that accounts for the hypoparathyroidism (3,4). The hypocalcemia secondary to these parathyroid disorders varies in its clinical presentation from an asymptomatic biochemical abnormality to a severe life-threatening condition. Patients with hypoparathyroidism generally experience an insidious onset of symptoms, with a slow increase in episodes related to the neuromuscular and neurologic systems. Common complaints include paresthesias (particularly in the oral area), muscle spasm, carpopedal spasm, facial grimacing, and, in extreme cases, laryngeal spasm and convulsion. In association, irritability, depression, impaired memory, and psychosis may be seen (5). Often, the symptoms are precipitated or intensified by physical or emotional stress-induced hyperventilation and consequent alkalosis. With long-standing hypocalcemia, patients The Parathyroids, Second Edition

MANAGEMENT Successful treatment of the hypoparathyroid disorders should lead to an increase in the extracellular fluid ionized calcium concentration sufficiently to abolish hypocalcemic symptoms and to prevent the long-term complications of hypocalcemia. Therapy is primarily dictated by the presence of acute severe symptoms of hypocalcemia, which demands use of intravenous calcium salts to restore normocalcemia, or the existence of relatively asymptomatic hypocalcemia, which requires only titration of orally administered vitamin D (or its analogs) a n d / o r calcium preparations in sufficient amounts to gradually achieve and maintain normocalcemia without undue complications. 827

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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CHAPTER52

Emergency Treatment The signs and symptoms of acute hypocalcemia primarily result from enhanced neuromuscular irritability (8). Sensations of numbness and tingling involving the fingertips, toes, and circumoral region are early symptoms. Subsequently, muscle cramps most commonly involve the lower back, legs, and feet. With severe hypocalcemia, the muscle cramps may progress to spontaneous carpopedal spasm (tetany). The carpal spasm often progresses to the classic "main d'accoucheur" posture. The hyperventilation commonly associated with tetany induces hypocapnea and increased epinephrine secretion, which worsen the muscle spasm. If the muscle spasm progresses, laryngospasm a n d / o r bronchospasm can follow. Seizures may also occur. Prolongation of the QT interval on the electrocardiogram, and rarely congestive heart failure, may also occur secondary to the acute hypocalcemia Hypokalemia, hypermagnesemia, metabolic acidosis, uremia, or hypophosphatemia may mask the tetany. Precipitation of acute hypocalcemia usually manifests in untreated patients with idiopathic or surgical hypoparathyroidism secondary to an inciting event, such as corticosteriod-induced malabsorption of calcium, diuretic-induced hypercalciuria, estrogenmediated inhibition of bone resorption, pregnancy, menstruation, infection, or withdrawal of thyroid medication. Alternatively, at presentation patients with neonatal hypocalcemia (early or late) secondary to hypoparathyroidism, those with surgical hypoparathyroidism and the hungry bone syndrome, and those with profound and progressive idiopathic hypoparathyroidism may spontaneously develop acute hypocalcemia. Although the presence of symptoms primarily reflects the magnitude of the hypocalcemia, a rapid decrease of the serum calcium concentration a n d / o r the concomitant presence of alkalosis, which enhances the binding of ionized calcium to albumin, may also precipitate severe signs and symptoms.

Therapy Any patient with hypocalcemic tetany or a relatively asymptomatic patient with serum calcium of less than 7.5 m g / d l must be treated aggressively with intravenous calcium administration. Such therapy may be necessary to treat neonatal hypocalcemia due to hypoparathyroidism in premature infants and in term infants (9) or to manage severe hypocalcemia in adults with hypoparathyroidism (10).

Newborns Serum calcium concentrations of 5-6 m g / d l in premature infants and less than 7.5 m g / d l in full-term

infants with neonatal hypocalcemia and hypoparathyroidism demand emergency therapy. Treatment at higher levels of the serum calcium concentration is often necessary if specific signs of hypocalcemia are present. Affected patients with severe hypocalcemia often present with convulsions, heightening the need for acute management. Though neonatal hypocalcemia is due to a variety of disorders, including perinatal asphyxia, preeclampsia, maternal diabetes, and maternal hyperparathyroidism, inherited forms of hypoparathyroidism (e.g., DiGeorge syndrome, familial hypoparathyroidism) often present as either "early" (<3 days postpartum) or "late" (5-10 days of life) neonatal hypocalcemia. The object of treatment is to relieve symptoms and forestall or ameliorate laryngeal obstruction a n d / o r convulsive seizures. Appropriate emergency therapy consists of a slow intravenous infusion (<1 ml/minute) of calcium gluconate (9% elemental calcium) in a 10% (w/v) solution (11). The appropriate dose is approximately 2 m g / k g of elemental calcium or about 0.2 ml of the 10% calcium gluconate per kilogram. A well-functioning indwelling intravascular catheter is essential to avoid extravasation. Intramuscular administration of the calcium is contraindicated because of local tissue toxicity. During the acute infusion of calcium careful observation of cardiac monitoring is important to assure resolution of the prolonged QT interval and to recognize the occurrence or persistence of 2:1 heart block a n d / o r ventricular arrhythmias. A total infusion of 1-3 ml will usually relieve laryngeal obstruction and arrest convulsions, and, in general, no more than 2 mg of elemental calcium per kilogram body weight should be administered as a single dose. Such bolus infusions may be repeated up to four times in a 24-hour period. If severe hypocalcemia persists, however, use of a long-term calcium gluconate infusion (20-50 mg of elemental calcium per kilogram body weight infused over 24 hours) is generally more effective (9). Calcium chloride is not used for therapy because it is more irritating than calcium gluconate. Neither bicarbonate nor phosphate should be coadministered with the calcium in order to prevent precipitation of the calcium salt. Coexistence of hypomagnesemia, although rare in neonatal hypocalcemia, can inhibit restoration of normocalcemia. When present, therefore, slow administration of magnesium sulfate (MgSO 4 • 7H20), 5-10 mg elemental magnesium per kilogram body weight, is required (11). Because hypocalcemia is likely to persist in infants with neonatal hypocalcemia secondary to hypoparathyroidism, initiation of therapy with 1,25-dihydroxyvitamin D [1,25(OH)zD] and calcium supplements is necessary. The approach to such treatment is discussed below.

TREATMENT OF HYPOPARATHYROIDISM / Older Children

Hypocalcemia due to hypoparathyroidism is similarly treated in older children. Acute intervention consists of intravenous infusion of 10% calcium gluconate, at a dose of I ml/kg, administered at a rate of 1 m l / m i n u t e . This dose may be delivered up to a maximum of 20 ml of calcium gluconate per bolus infusion. For a longer term infusion, 4-6 ml of 10% calcium g l u c o n a t e / k g / 24 hours may be used, providing 36-54 mg of elemental calcium per kilogram. When hypocalcemia due to hypoparathyroidism is exacerbated by an associated gastrointestinal disorder, nocturnal nasogastric supplementation of calcium carbonate or calcium gluconate is an effective therapy, delivering up to 20 mg of elemental calcium per kilogram body weight per night. As in infants, hypomagnesemia may result in refractoriness to correction of the hypocalcemia. In affected patients, up to 2.4 mg of elemental magnesium per kilogram body weight is administered over a 10-minute period (to a m a x i u m u m of 180 mg) to achieve a normal serum concentration. Alternatively, a continuous infusion of 576 mg of elemental magnesium over 24 hours may be administered. The duration of the intravenous magnesium requirement varies substantially, and in some cases transition to oral magnesium supplementation is necessary. Adu/ts In adults with symptomatic presentation of acute hypocalcemia or a sustained serum calcium concentration of less than 7.5 m g / d l , emergency m a n a g e m e n t consists of infusing 1-2 ampuls of calcium gluconate, diluted in 50-100 ml of 5% dextrose (180 mg of elemental calcium), over a 1 0 - t o 15-minute period (9). This procedure is repeated as necessary to control symptomatic hypocalcemia. Persistent or less severe hypocalcemia may be managed by administration of more dilute calcium solutions over longer periods. A practical approach to such therapy is to administer 10 ampuls of calcium gluconate in 1 liter of 5% dextrose, infused at a rate of 50 m l / h o u r (45 mg of elemental calc i u m / h o u r ) ; if the serum calcium does not normalize, the rate of infusion may be titrated. In general, however, 15 m g / k g elemental calcium infused over a period of 4-6 hours will raise the serum calcium concentration by 2-3 m g / d l (8). When administration of excessive volume is a concern, the concentration of the solution may be increased to as much as 200 mg of elemental calcium per 100 ml without u n d u e irritation to the veins, and in the event of extravasation, soft tissues. In patients treated with digitalis, the calcium infusion must occur with caution, because hypercalcemia increases sensitivity to the adverse effects of this drug, particularly arrhythmias. Indeed, for all patients receiv-

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ing a calcium infusion, the serum calcium concentration should be monitored frequently. Obviously, as with infants, there are certain additives that are incompatible with calcium solutions, including phosphate and bicarbonate. In addition, the calcium solutions are likewise irritating in adults and maintenance of a patent intravenous catheter is essential. Because hypocalcemia is likely to persist in patients with hypoparathyroidism (or pseudohypoparathyroidism), therapy should be initiated early with oral calcium supplementation and a vitamin D preparation (see below). With the occurrence of acutely exacerbated calcium malabsorption, as may often occur in patients with a u t o i m m u n e hypoparathyroidism with associated gastrointestinal disorders, m a n a g e m e n t of acute hypocalcemia over prolonged periods has been accomplished with nocturnal nasogastric supplementation with calcium carbonate or calcium gluconate, providing up to 20 mg of elemental calcium per kilogram of body weight per 8 hours (9). Such therapy generally maintains the serum calcium concentration at a safe level and is continued until the underlying intestinal disturbance is resolved. Similar to infants, the adult patient with hypocalcemia, who has coincident hypomagnesemia, will require treatment of the hypomagnesemia before the hypocalcemia will completely resolve. Moreover, in the acutely hypocalcemic patient in whom magnesium deficiency is clinically likely (e.g., alcoholic patients), adding magnesium to the treatment regimen before laboratory confirmation of the deficit is appropriate. Acute treatment of the magnesium deficiency requires administration of up to 2.4 m g / k g body weight of elemental magnesium infused over a 10-minute period (to a m a x i m u m of 180 mg). Alternatively, a continuous infusion of 576 mg of elemental magnesium may be administered over 24 hours. The length of therapy should be individualized. Once repletion has been accomplished, patients usually maintain a normal magnesium status on a regular diet. However, if repletion is accomplished and the patient cannot eat or continues to lose magnesium secondary to diarrhea, fistulous drainage, or other abnormalities, a daily oral dose of 300 to 600 mg of elemental magnesium in divided doses is often required. In patients with renal failure, the a m o u n t of magnesium required is considerably less and therapy should begin with one-half of the recomm e n d e d dose. The serum magnesium concentration should be monitored daily during the period of acute therapy. However, the serum magnesium may return to normal well before the total body deficit is corrected. Therefore, the effects of magnesium therapy on the serum calcium concentration may be delayed up to 3 to 7 days.

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CI-IAeTV.R52

Chronic Treatment The major goal of chronic therapy in all patients with hypoparathyroidism is to restore the serum calcium and phosphorus to levels as close to normal as possible without encountering u n d u e complications of the therapeutic intervention. Interestingly, there are wide variations in treatment regimens used around the world, which to some extent d e p e n d on the available preparations of vitamin D and its analogs and metabolites. Nevertheless, though there is no single best way to achieve stable normocalcemia in affected patients, the combination of a vitamin D metabolite with calcium supplements is generally preferred. In infants, youths, and adults with hypoparathyroidism, the principles of therapy are similar. However, the dosages of the medicines employed are sufficiently different that treatment protocols will be considered separately.

a significant problem, a serum calcium of 7.8 to 8.5 m g / d l is often considered adequate. As in adults (see below), the medical regimen for chronic management of the various forms of hypoparathyroidism consists of combination therapy with a vitamin D preparation and oral calcium supplementation (13,14). Although vitamin D, 25(OH)D (Calderol), and dihydrotachysterol (DHT) have been used, the preferred treatment at initiation of therapy in a child is 1,25(OH)2D (Rocaltrol); this preparation is more consistently effective at nontoxic doses, has a shorter halflife, and allows more rapid correction of hypercalcemic complications after withdrawal, should these develop (11). The various formulations of vitamin D available are variable and the typical doses are somewhat variable. However, current standards are shown in Table 1. The dose of calcium supplementation employed varies but is initiated at levels of 30 to 50 m g / k g body weight and titrated as necessary to levels as high as 1500 mg/day.

Infants and Youths The transition from acute management of hypocalcemia to chronic maintenance in the newborn period is highly dependent on the individual clinical situation. In the absence of hypoparathyroidism, oral calcium supplementation is generally initiated within a few days of intravenous therapy. Because usually no specific cause of transient hypocalcemia is identified, the course of such treatment is generally improvement, permitting gradual diminution of the oral calcium until none is required. In the case of hypoparathyroidism, particularly in the case of DiGeorge syndrome, it is possible that only partial hypoparathyroidism will persist (12). U n d e r these conditions, affected children may tolerate treatm e n t with oral calcium supplementation initially and ultimately maintain a normal serum calcium without treatment. However, their parathyroid reserve is so limited that hypocalcemia may recur when they are restricted to nothing by mouth for as little as 12 hours or when an episode of gastroenteritis intervenes. Therefore, monitoring of therapy is essential after oral calcium supplementation is discontinued. Indeed, it is important to establish a periodic schedule to reevaluate such children to d o c u m e n t appropriate maintenance of normocalcemia and parathyroid h o r m o n e reserve. The latter may require provocative testing with citrate or ethylenediaminetetraacetic acid (EDTA). When unambiguous hypoparathyroidism prevails in youths, the goal of treatment is obviously to maintain the serum calcium concentration at a level that results in no symptoms. T h o u g h optimally this level is in the low normal range, the absence of the calcium-retaining properties of parathyroid h o r m o n e permits a greater degree of calciuria at normal serum calcium levels. Thus, in order to protect the kidneys, if hypercalciuria is

Adu/ts A few patients with mild hypoparathyroidism or diminished parathyroid reserve may maintain a normal serum calcium level during treatment with oral calcium supplementation alone, but most patients require ancillary treatment with vitamin D. However, the vitamin D does not effectively regulate intestinal calcium absorption and serum calcium concentrations may fluctuate without provision of supplemental calcium.

TABLE 1

Vitamin D and Related Agents for Management of Hypoparathyroidism in Children Drug

Vitamin D Drisdol

25-Hydroxyvitamin D Calderol Dihydrotachysterol

1,25-Dihydroxyvitamin D Rocaltrol Calcijex

a] #g vitamin D = 40 IU.

Formulationa

Typicaldose

8000 IU/ml 25,000 IU tablet 50,000 IU tablet

2000 IU/d 1 tablet/day 1 tablet/day

20-#g tablet 0.2 mg/5 ml 0.125-mg tablet 0.2-mg tablet 0.4-mg tablet

1 tablet/day 0.5 mg/day 0.5 mg/day 0.5 mg/day 0.4 mg/day

0.25-#g capsule 0.50-1~g capsule 1.0 #g/ml 2.0 #g/ml

0.5 #g/day 0.5 #g/day

TREATMENT OF HYPOPARATHYROIDISM /

Since such fluctuation may occur, high-dose oral calcium is generally prescribed for patients with hypoparathyroidism. Although titration in individuals is required, the average dose administered is 10002000 mg of elemental calcium daily. Such high dosage permits the patient to maintain an appropriate serum calcium concentration by absorbing a large relatively constant a m o u n t of ingested calcium. The calcium is administered in divided doses with meals. Many oral calcium preparations are available and all are effective when administered in the correct dosage. Indeed, Heaney et al. (15) reported that patients taking calcium carbonate and calcium citrate with meals absorb equal amounts of elemental calcium. T h o u g h calcium carbonate is preferred by many, because it contains the highest content of elemental calcium, epigastric bloating and irritation often complicate administration of this drug. Moreover, there is some evidence that patients with atrophic gastritis may not absorb calcium well when it is administered as calcium carbonate (16). In such cases choice of an alternative is preferable. Several forms of vitamin D are available for ancillary therapy. Ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3) are effective and inexpensive in generic form and have been used frequently worldwide for the long-term m a n a g e m e n t of hypoparathyroid disorders (17). T h o u g h such treatment is not contemporary, uneventful and successful long-term m a n a g e m e n t may dictate maintenance of these drugs. Appropriate dosage of these agents varies in general from 25,000 to 200,000 U (1.25 to 10 m g ) p e r day. These drugs have a long duration of action because they distribute in body fat, thus a response to dose change is slow and toxicity is prolonged. Indeed, the stable effects of an altered dose on the serum calcium concentration will not manifest for several weeks. Any change in dose is also limited by the available dosage in pills or capsules. In this regard, changes in ergocalciferol are restricted to 50,000 U and when limited effects are required may necessitate alternate day dosage of 50,000 and 100,000 U or other variations. During chronic management, m e a s u r e m e n t of the serum calcium and phosphorus concentrations, urinary calcium, and creafinine clearance should be repeated every 3-4 months. If mild hypercalcemia (<10.6 m g / d l ) develops without impaired renal function, reduction in the dose of vitamin D ( a n d / o r calcium) may be sufficient to restore a stable low-normal serum calcium concentration within 1-2 months. Alternatively, if the hypercalcemia is more severe a n d / o r renal function is compromised, treatm e n t should be stopped until normocalcemia is reestablished. If the toxicity occurs in a patient receiving a high dose of calcium, serum calcium generally promptly returns to normal after discontinuation of the drugs. However, if the hypercalcemia persists or the cal-

831

cium is severely elevated, more aggressive therapy with volume expansion and glucocorticoids may be necessary. In either case, the calcium supplements likely will require limitation for a prolonged period until the vitamin D preparation is cleared from fat stores. Alternative vitamin D therapies include oral administration of 1,25(OH)zD 3 (calcitriol), 0.25 to 1.0 Ixg/day; le~-hydroxyvitamin D, 0.50 to 2.0 Ixg/day; and dihydrotachysterol, 0.2 to 1.2 mg/day. Dihydrotachysterol, however, is rarely used today and when available calcitriol is likely the treatment of choice. Indeed, calcitriol should be used in all patients beginning treatment for hypoparathyroidism and in those on other forms of vitamin D who have experienced poor control of the serum calcium concentration a n d / o r complications of treatm e n t requiting cessation of therapy. The initial dose of calcitriol employed is 0.5 Ixg/day twice daily and the maintenance dose varies from 0.5 to 1.0 Ixg/day. At inception of therapy, however, the calcitriol requirem e n t may be greater. Thus, titration of dose is essential, with a change in a m o u n t possible every 3-4 days as dictated by the serum calcium concentration. Over the first m o n t h of treatment, the required dose often escalates in the first 1-2 weeks to as much as 3.0 Ixg/day and thereafter decreases to maintenance levels. Once treatm e n t has been stabilized, serum calcium and renal function should be assessed every few months. Measurement of urinary calcium is also appropriate. However, because patients with hypoparathyroidism do not have parathyroid h o r m o n e to enhance renal tubular calcium reabsorption, even a serum calcium concentration in the middle of the normal range is associated with significant hypercalciuria. Thus, the treating physician must tolerate the hypercalciuria provided that stone formation, nephrocalcinosis, or compromised renal function does not supervene. To ensure that stone formation or nephrocalcinosis is not developing, a plain film of the a b d o m e n or ultrasound should be obtained every 1-2 years. In the event that a complication of hypercalciuria develops or the urine calcium exceeds 450 m g / 2 4 hours, a reduction in the dose of calcium supplements a n d / o r calcitriol is necessary. To maintain the serum calcium concentration in the low-normal range u n d e r such circumstances, addition of hydrochlorthiazide or chlorthaladone to the treatment regimen increases the renal reabsorption of calcium. Hypercalcemia, when it develops during therapy with calcitriol, usually resolves within 3-4 days after discontinuing the calcitriol (and calcium supplements), although an occasional patient maintains an elevated serum calcium concentration for as long as 1 week (18). Alternatively, if hypocalcemia manifests and therapeutic requirements unexpectedly increase, the possibility of magnesium deficiency should be considered.

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CHAPTER52

Such deficiency may render a patient relatively resistant to treatment and occurs most often secondary to steatorrhea, which occurs in the a u t o i m m u n e forms of the disorder. A n u m b e r of commonly used drugs may affect the serum calcium concentration in patients with hypoparathyroidism treated with vitamin D or its metabolites and calcium supplements. As noted above, thiazide diuretics enhance renal tubular calcium reabsorption and may precipitate hypercalcemia when added to a therapeutic regimen that has maintained the serum calcium level in the midrange or u p p e r range of normal. Conversely, loop diruetics may increase calcium excretion and precipitate hypocalcemia. Glucocorticoids antagonize the effects of vitamin D and decrease gastrointestinal absorption of calcium. Additionally, some agents, such as bile acid sequestrants (cholestyramine), prevent vitamin D absorption and others, such as aluminum and magnesium hydroxide, precipitate calcium, limiting gastrointestinal absorption. Because no compensatory mechanisms exist in the hypoparathyroid patient to offset such pertubations, careful monitoring of the serum calcium concentration is essential when adding new medications to the treatment regimen. The therapeutic approach in patients with pseudohypoparathyroidism is similar to that in primary hypoparathyroidism (19). However, hypercalciuria occurs less often because the existent parathyroid h o r m o n e acts on the distal renal tubule to enhance calcium reabsorption. In addition, deviations from normocalcemia occur infrequently once a stable regimen is achieved. Nevertheless, during initial treatment high-dose therapy may be required because untreated patients frequently manifest osteomalacia, increasing the calcium d e m a n d to restore mineralization. As the bone lesions heal, dose requirements decrease, often signaled by a decline in the serum alkaline phosphatase level and a rise in the serum calcium concentration. In the near future alternative forms of treatment may become readily available for patients with hypoparathyroidism. For example, studies of the active form of parathyroid h o r m o n e are underway to determine if parenteral administration of this c o m p o u n d is a viable therapeutic alternative. Further, several companies are investigating if structural modifications of parathyroid h o r m o n e may render it effective as an orally administered medication. In addition, early studies have revealed that allotransplantation into the brachioradialis muscle of parathyroid tissue, enclosed in semipermeable microcapsules, has successfully regulated the serum calcium concentration (20).

Complications of Treatment Use of large doses of vitamin D (and its analogs) a n d / o r calcium supplementation may result in severe complications, most commonly related to hypercalcemia and hypercalciuria. Prolonged occurrence or frequent bouts of either may have long-term negative sequelae on renal function. Therfore, after initial dosing with these compounds, serum and urinary calcium and creatinine determinations are p e r f o r m e d within 2 weeks and after all adjustments of dose. Once a stable dose is achieved, these tests should be repeated every 3 to 6 months. If the serum calcium becomes greater than 11 m g / d l or the urinary calcium/creatinine ratio is greater than 0.35 ( m g / m g ) , adjustments in dose are warranted. If severe hypercalcemia develops, medications should be discontinued and specific measures for the m a n a g e m e n t of hypercalcemia instituted.

REFERENCES 1. Brown EM. Extracellular Ca 2+ sensing, regulation of parathyroid cell function, and role of Ca 2+ as extracellular (first) messengers. Physiol Rev 1991 ;71:371-411. 2. Parfitt AM. The spectrum of hypoparathyroidism. J Clin Endocrinol Metab 1972;34:152-158. 3. Nusynowitz ML, Frame B, Kolb FO. The spectrum of the hypoparathyroid states: A classification based on physiologic principles. Medicine 1976;55:105-119. 4. Schneider AB, Sherwood LM. Pathogenesis and management of hypoparathyroidism and other hypocalcemic disorders. Metabolism 1975;24:871-878. 5. Frame B. Neuromuscular manifestations of parathyroid disease. In: Klawans HL, ed. Handbook of clinical neurology, Vol. 27. Amsterdam:North-Holland Publ. 1976:283-320. 6. Parfitt AM. Surgical, idiopathic and other varieties of parathyroid hormone-deficient hypoparathyroidism. In: DeGroot LJ, ed. Endocrinology, 2nd Ed. Philadelphia:Saunders, 1989:1049-1064. 7. Nagant de Deuxchaisnes C, Krane SM, Hypoparathyroidism. In: Avioli LV, Krane SM, eds. Metabolic bone disease, Vol. 2. New York:Academic Press, 1978. 8. Downs RW, Jr. Hypocalcemia and hypoparathyroidism. In: Bardin CW, ed. Current therapy in endocrinology and metabolism, 4th Ed. Philadelphia:Decker, 1991:440-444. 9. Carpenter TO, Insogna KL. The hypocalcemic disorders: Differential diagnosis and therapeutic use of vitamin D. In: Feldman D, Glorieux FH, Pike JW, eds. Vitamin D. San Diego: Academic Press, 1997:923-936. 10. Eastell R, Heath III H. The hypocalcemic states: Their differential diagnosis and management. In: Coe FL, Favus MJ, eds. Disorders of bone and mineral me.tabolism. New York:Raven, 1992:571-585. 11. Carpenter TO. Hypocalcemia and tetany. In: Burg FD, Wald ER, IngelfingerJR, Polin RA, eds. Current pediatric therapy. Philadelphia: Saunders 1999:776-780. 12. Gidding SS, Minciotti AL, Langman CB. Unmasking of hypoparathyroidism in familial partial DiGeorge syndrome by challenge with disodium edetate. N EnglJ Med 1988;319:1589-1591. 13. Russell R, Smith R, Walton R, Preston C, Basson R, Henderson R. 1,25-Dihydroxycholecalciferol and l ot-hydroxycholecalciferol in hypoparathyroidism. Lancet 1976;2:14-17.

TREATMENT OF HYPOPARATHYROIDISM 14. Neer R, Holick M, DeLuca, H, Potts J. Effects of let-hydroxyvitamin D 3 on calcium and phosphorus metabolism in hypoparathyroidism. Metabolism 1975;24:1403-1413. 15. Heaney RP, Dowell MS, Barger-Lux MJ. Absorption of calcium as the carbonate and citrate salts, with some observations on method. Osteoporosis Int 1999;9:19-23. 16. Recker R. Calcium absorption and achlorhydria. N Engl J Med 1985;313:70-73. 17. Okano K, Furukawa Y, Morii, H, Fujita T. Comparative efficacy of various vitamin D metabolites in the treatment of various types of hypoparathyroidism. J Clin Endocrinol Metab 1982;55:238-243.

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18. KanisJ, Russel RCG. Rate of reversal of hypercalcemia and hypercalciuria induced by vitamin D and its l et-hydroxylated derivatives. Br M e d J 1977;1:78-81. 19. Lewin IG, Papapoulos, SE, O'RiordanJLH. 10t-hydroxyvitamin D 3 in the long-term management of hypoparathyroidism and pseudohypoparathyroidism Clin Endocrinol 1977;7(Suppl.): 203S-207S. 20. Goltzman D, Cole DEC. Hypoparathyroidism. In: Favus MJ, ed. Primer on the metabolic bone diseases and disorder of mineral metabolism, 4th Ed. Philadelphia:Lippincott Williams & Wilkins, 1999: 226-230.

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CHAeTV R 53 Parathyroid Function in the Normal Aging Process

SUNDEEP KHOSLA, L.J. MELTON III, AND B. L. RIGGS Mayo Clinic and Foundation, Rochesteg, Minnesota 55905

INTRODUCTION

AGE-RELATED C H A N G E S IN PTH SECRETION

The syndrome of involutional osteoporosis has been subdivided by Riggs and Melton (1) into that of type I and type II osteoporosis. Type I osteoporosis presents during the first 15-20 years after menopause, is characterized by rapid and disproportionate loss of cancellous c o m p a r e d to cortical bone, and results in acute vertebral compression fractures and distal forearm (Colles') fractures. In contrast, type II osteoporosis affects m e n as well as women, corresponds to the slow phase of age-related bone loss seen in both sexes, and involves comparable losses of both cancellous and cortical bone. The two phases of bone loss in w o m e n and the differing overall patterns of bone loss in w o m e n versus m e n are shown schematically in Fig. 1. It is clear that the rapid phase of bone loss in w o m e n following m e n o p a u s e is due to the direct consequences of estrogen deficiency on the skeleton. The slow phase of bone loss in both genders has been attributed to a n u m b e r of factors, chief a m o n g which are agerelated secondary hyperparathyroidism and decreased bone formation (2). In this chapter, we review the changes in parathyroid function with aging, the relationship of the changes in parathyroid function to agerelated increases in bone turnover and bone loss, the potential causes of the age-related increase in serum parathyroid h o r m o n e (PTH), and possible means of preventing or treating age-related secondary hyperparathyroidism.

The Parathyroids, Second Edition

Soon after the introduction of assays for PTH, serum PTH concentrations were found to increase with age both in women and m e n (3-9). Because the initial studies used assays directed against the midregion or carboxy terminus of PTH, some of the a p p a r e n t increase with age could have b e e n due to retention of these inactive fragments as a consequence of reduced glomerular filtration rate in the elderly. However, a n u m b e r of studies using assays directed against the amino terminus or against intact PTH have n o t e d similar age-related increases in circulating PTH in both sexes (8,10,11) (Fig. 2). Moreover, urinary nephrogenous cyclic AMP was also found to increase with age (6), consistent with an age-related increase in biologically active PTH. T h o u g h most studies have depicted the increase in serum PTH with age as a continuous relationship, as shown in Fig. 2, more detailed analysis has revealed that the major increase in w o m e n occurs after age 70 years. Thus, Koh et al. (12) analyzed serum PTH concentrations from 12,238 w o m e n aged 55-81 years from the Fracture Intervention Trial and found that the p e r c e n t change in serum PTH per 10-year rise in age between ages 55 and 69 years was - 1.9% [95% confidence intervals (C.I.), - 4 . 2 to 0.4%]. In contrast, the change in serum PTH per 10-year rise in age between 70 and 81 years was 3.8% (95% C.I., 1.0 to 6.7). Similarly, Prince et al. (13) m e a s u r e d serum PTH levels in

835

Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Leone.

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CHAPTER53

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655 pre- and postmenopausal women, and noted that the age-related increase in serum PTH became evident approximately 20 years after the menopause (Fig. 3),or about age 70 years, given an average age of menopause at approximately 50 years. There are limited data at present on potential ethnic differences in the age-related changes in serum PTH concentrations. A n u m b e r of studies have demonstrated higher circulating PTH values in premenopausal African-American compared to Caucasian women (14,15). Perry et al. (16), however, compared circulating PTH in young (mean age, 36 years) and elderly (mean age, 71 years) African-American versus Caucasian women and found that, though serum PTH levels were slightly higher in the young African-

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American women, these differences were even greater in the elderly women (Table 1). Thus, age-related increases in serum PTH secretion may be even greater in African-American than in Caucasian women. Serum PTH also has a circadian rhythm, with peak concentrations in the midafternoon and at night (17,18). This circadian pattern is present in both young and elderly subjects, but Eastell et al. (17) demonstrated that the age-related increases in serum PTH are, in fact, greatest at night. Thus, because most studies have measured PTH in the morning, they may have underestimated the magnitude of the age-related increase in PTH secretion. In order to better characterize the age-related changes in parathyroid function, Ledger et al. (19) performed detailed studies assessing PTH secretory dynamics in young (age range, 27-34 years) versus elderly (range, 71-77 years) women. Using sequential infusions of calcium and EDTA, they defined the entire PTH secretory curve as a function of ionized calcium activity in the two groups (Fig. 4). As is evident in Fig. 4, the elderly women had an exaggerated response

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PARATHYROID FUNCTION IN AGING / TABLE

837

Serum PTH Concentrations in African-American and Caucasian Womena

1

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PTH b

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to i n d u c e d hypocalcemia and also had a higher nonsuppressible c o m p o n e n t of PTH secretion (elderly, 0.8 p m o l / l i t e r ; young, 0.4 pmol/liter; P < 0.001). Of note, the abnormal PTH secretory curve of the elderly women could be made identical to the PTH secretory curve in the young women following 1 week of 1,25-dihydroxyvitamin D 3 [1,25 (OH) zD~] therapy. The set point, or ionized calcium value at which halfmaximal PTH secretion was achieved (representing the sensitivity of each individual parathyroid cell to changes in serum ionized calcium), did not differ between the young a n d elderly women. These investigators interpreted their findings as demonstrating in the elderly women "functional" parathyroid hyperplasia that was reversible by short-term 1,25(OH)zD ~ therapy. 16

RELATIONSHIP OF AGE-RELATED INCHES IN SERUM PTH TO INCREASED BONE TURNOVER AND BONE LOSS

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Similar alterations in the maximal and minimal components of the PTH secretory curve were reported by Portale et al. (20) in young (mean _+ SEM, 39 +_ 1 years) versus elderly (74 __ 2 years) men. However, in contrast to the study by Ledger et al. (19), Portale et al. (20) did note a significant increase in the set point for PTH secretion in the elderly men. This difference may reflect a true gender difference in PTH secretory dynamics or possibly the use of different experimental protocols to define PTH secretory dynamics in the two studies. Overall, however, the data from both studies are consistent with functional parathyroid hyperplasia in elderly women and men, and this notion is supported by an autopsy study that found a trend toward parathyroid hyperplasia in elderly subjects (21).

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As noted earlier, age-related increases in serum PTH concentrations have been postulated to contribute to age-related bone loss in m e n and women, primarily by increasing bone turnover. In the setting of an agerelated i m p a i r m e n t in osteoblast generation or function, increased bone turnover leads to bone loss. Indirect evidence for this comes from observations showing correlations between serum PTH and bone turnover markers in elderly w o m e n (10,22) (Table 2). However, correlation does not prove causality. To test directly the hypothesis that secondary hyperparathyroidism m e d i a t e d the age-related increase in bone resorption, Ledger et al. (18) used a 24-hour calcium infusion to suppress PTH secretion in young (age range, 24--35 years) versus elderly (range, 71-78 years) women and then assessed the changes in bone resorption markers in the two groups. Specifically, they postulated that if bone resorption were more d e p e n d e n t on

838

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CHAPTER53

TABLE 2

Changes in Serum PTH and in Biochemical Markers of Bone Turnover a Spearman correlation coefficients

Variableb

Increase with age

vs. age

vs. PTH

PTH BSAP OC fPYD NTx

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serum PTH in the elderly compared to the young women, then the decrease in bone resorption markers following suppression of PTH secretion by an intravenous calcium infusion should be greater in the elderly women. Figure 5 shows the changes in the bone resorption marker, urinary N-telopeptide of type I collagen (NTx), in the young and elderly women. As is evident, suppression of PTH secretion eliminated the differences in NTx excretion between the two groups, and the mean decrement in NTx excretion following PTH suppression in the elderly women (7.5 + 1.9 n m o l / m m o l Cr) was significantly greater than that in the young women (4.1 _+ 1.5 n m o l / m m o l Cr; P < 0.05). These experiments, therefore, provide a causal link between age-related increases in PTH secretion and the corresponding increases in bone resorption. Consistent with these data, several (23-25) but not all (26) studies have also found associations between the age-related increases in serum PTH and changes in bone density in elderly subjects. Some of the conflicting data may relate to the fact that PTH likely has preferential effects in causing cortical bone loss with minimal (or perhaps even protective) effects on cancellous bone (27). Thus, studies using standard dual-energy X-ray absorptiometry (DXA), which provides an integrated measure of cancellous and cortical bone at the various skeletal sites, are unable to dissect these differing effects of PTH on bone. To address this issue, Boonen et al. (25) used peripheral quantitative computed tomography (pQCT), which allows the separate determination of cortical and cancellous bone mineral density (BMD) in the peripheral skeleton, to test the

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relationship between compartmental BMD and a number of variables, including serum PTH, in 129 women aged 70-87 years. By multiple regression analysis, they found that serum PTH was a negative predictor of cortical, but not cancellous, BMD at the wrist. Conversely, Shukla et al. (28) found that hypoparathyroid subjects (mean age, 57 years) had little or no change in vertebral cancellous BMD compared to age-matched controls as assessed by vertebral c o m p u t e d tomography (z scores, +0.5 to + 1.8), but they had marked increases in combined cancellous and cortical BMD as assessed by dual-photon absorptiometry (z scores, + 1.4 to +6.2). Collectively, therefore, the data are consistent with the notion that the age-related increases in PTH secretion contribute to increased bone resorption and that this, in turn, results in at least cortical bone loss. Clearly,

PARATHYROIDFUNCTIONIN AGING / however, further longitudinal studies are needed that relate PTH to cancellous versus cortical bone loss in elderly women and men to provide a more rigorous test of this hypothesis.

E T I O L O G Y OF T H E S E C O N D A R Y H Y P E R P A R A T H Y R O I D I S M OF A G I N G A number of factors have been postulated to contribute to the age-related increase in serum PTH (2). Chief among these are vitamin D deficiency, impaired renal 1,25 (OH)2 D production or action, and, more recently, estrogen deficiency. There is no doubt that vitamin D deficiency leads to secondary hyperparathyroidism (29). However, serum PTH concentrations have been shown to increase in populations in which there have been no age-related changes in 25-hydroxyvitamin D status (30). Though vitamin D deficiency may contribute in certain populations to secondary hyperparathyroidism, the latter is clearly present even in populations in which there is no evidence for vitamin D deficiency. Thus, other primary factors must be involved. A subset of elderly persons also have a decline in renal l ot-hydroxylase activity associated with a reduction in glomerular filtration rate (31). This results in low circulating 1,25(OH)2D concentrations, which, in turn, leads to impaired intestinal calcium absorption as well as a potential loss of feedback suppression of PTH secretion by 1,25 (OH) 2D, thus causing secondary hyperparathyroidism. However, Eastell et al. (10) found that in a population of relatively healthy women, serum PTH concentrations increased despite an agerelated increase (rather than decrease) in circulating 1,25(OH)zD. In contrast, intestinal calcium absorption did not change with age. Based on these data, Eastell et al. postulated that intestinal resistance to 1,25(OH)2D action accounted for the increase in serum 1,25(OH)zD concentrations with aging, which in turn were maintained by elevated circulating PTH. Consistent with this hypothesis, Ebeling et al. (32) found a decrease in intestinal vitamin D receptor concentrations in elderly women, although this issue remains controversial (33). Finally, Pattanaungkul et al. (34) recently used an in vivo dose response to 1,25 (OH)2 o in young versus elderly women to show that the slope of the relationship between intestinal calcium absorption and the molar ratio of 1,25(OH)2D to vitamin D binding protein [the "free" 1,25(OH)zD index], representing intestinal sensitivity to 1,25(OH)zD, was significantly greater in the young compared to the elderly women. Thus, though vitamin D deficiency or impaired renal function leading to deficient l ot-hydroxylase activity may contribute to secondary hyperparathyroidism in

839

some elderly individuals, the age-related increase in serum PTH is present even in the absence of both abnormalities. Overall, the data are consistent with impaired intestinal calcium absorption resulting from intestinal resistance to 1,25(OH)2D action as being a significant contributing factor to age-related secondary hyperparathyroidism. Finally, aging may also be associated with impaired renal calcium conservation, which would also contribute to secondary hyperparathyroidism. Thus, Ledger et al. (18) found that elderly women had a significantly higher fractional excretion of calcium, despite higher PTH values than young women. Collectively, then, the data are consistent with agerelated alterations in intestinal calcium absorption and in renal calcium handling as the proximate causes of the secondary hyperparathyroidism in elderly persons. Thus, the elderly must consume more dietary calcium to prevent negative calcium balance. By combined calcium balance and kinetic methods, Heaney et al. (35) found that premenopausal women required 1000 mg/day, whereas postmenopausal women required 1500 m g / d a y to maintain calcium balance. This figure considerably exceeds the average calcium intake of only 700 m g / d a y among postmenopausal women in the United States (36). To test directly for these differences in calcium requirement, McKane et al. (37) performed intensive metabolic studies in a group of young adult premenopausal women on their usual calcium intake, a group of elderly women on their usual calcium intake, and a group of elderly women who had received a high-calcium supplement for 3 years (Table 3). As shown in the table, the unsupplemented elderly women had the expected increases in serum PTH and in bone resorption markers, whereas the calcium-supplemented women had values for serum PTH and bone resorption markers that were indistinguishable from the (unsupplemented) young women. These data support the hypothesis that a failure of elderly women to increase their calcium intake to a level high enough to offset the age-related increase in net calcium loss contributes substantially to their development of secondary hyperparathyroidism and increased bone resorption. Somewhat surprisingly, evidence indicates that longterm estrogen deficiency may be a key permissive factor for the development of the age-related changes in calcium homeostasis and the ensuing secondary hyperparathyroidism. Thus, McKane et al. (38) studied three groups of women to assess the relative contributions of "aging" versus estrogen deficiency toward the secondary hyperparathyroidism and increased bone resorption in elderly women: young adult premenopausal women (Group A), untreated elderly women (Group B), and elderly women who were receiving long-term estrogen

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CHAPTER53 TABLE 3

Comparative Effects of Age and Calcium Intake in Womena Elderly women c

Variableb

Young women

Usual Ca diet

High-Ca diet

Number in group Age, years Dietary Ca, mg/day Serum PTH, pmol/liter Urine PYD, nmol/mmol Cr DPD, nmol/mmol Cr

12 30.1 _+ 1.3 918 _ 56 2.01 _ 0.16

15 71.0 _ 0.7 815 _+ 75 3.41 _ 0.27*

13 68.3 _+ 0.6 2414 _ 72* 2.18 +__0.17

43.7 _+ 1.8 10.9 _ 0.4

51.7 _+ 3.0 t 14.2 + 0.9*

43.1 +_ 2.6 10.7 _ 0.7

aData from Ref. 37; adapted from Riggs et al. (2), with permission. bSerum intact PTH is a 24-hour integral of samples obtained every 2 hours. Urine pyridinoline (PYD) and deoxypyridinoline (DPD) were measured by fluorometric detection after highperformance liquid chromatography and were expressed as ratio to creatinine (Cr). All results are mean ___SEM. CFor difference from the young group:*, P < 0.05; t, P < 0.005.

replacement therapy (Group C). Because Groups B and C were of comparable age, but differed in estrogen status, and Groups A and C were comparable with respect to estrogen status, but differed in age, the effect of age and estrogen deficiency could be dissociated. As shown in Table 4, after estrogen deficiency was corrected, there were no effects on serum PTH and bone resorption levels that could be attributed to aging per se. Consistent with these observations, Khosla et al. (39) found that serum PTH and bone turnover increased progressively in untreated postmenopausal women, whereas there were no age-related increases in postmenopausal women who had been on long-term estrogen therapy. The most plausible explanation for the ability of estrogen to prevent the age-related increase in serum

TABLE 4

PTH levels is the potential effect of estrogen on extraskeletal calcium homeostasis, namely, on intestinal calcium absorption (40), renal calcium handling (41,42), and possibly directly on PTH secretion (43). Aging m e n also have increases in serum PTH concentrations that are very similar to those seen in women (11), despite the absence of menopause. It is possible that the age-related decreases in bioavailable (or nonsex-hormone binding globulin bound) estrogen a n d / o r testosterone that have been observed (11) lead to alterations in calcium homeostasis in aging men similar to those found in elderly women, resulting in the eventual development of secondary hyperparathyroidism. However, this hypothesis needs to be tested directly.

Comparative Effects of Age and Estrogen Status in Women a Group c

Variable b Number in group Serum PTH, pmol/liter Urine NTx, nmol/mmol Cr PYD, nmol/mmol Cr DPD, nmol/mmol Cr

Premenopausal

Postmenopausal untreated

Postmenopausal treated

30 2.7 _+ 0.2

30 3.6 _+ 0.3*

30 2.5 _+ 0.2

28.8 _+ 2.3 45.6 _+ 1.6 11.9 __ 0.5

42.9 _+ 3.5 t 61.2 _+ 3.2 t 16.2 _+ 1.0 t

24.6 __ 2.3 40.7 _+ 1.6 9.4 _+ 0.5

aData from Ref. 38; adapted from Riggs et al. (2), with permission. bSerum intact PTH was fasting morning value. Bone resorption markers were measured by ELISA kit for N-telopeptide of type I collagen (NTx) and by fluorometric detection after HPLC for pyridinoline (PYD) and deoxypyridinoline (DPD). All results are mean ___SEM. CFor difference from premenopausal group: *P, 0.05; t, P < 0.005.

PARATHYROID FUNCTION IN AGING

SUMMARY A N D C O N C L U S I O N S Serum PTH concentrations clearly increase with age in a parallel m a n n e r in women and in men. At a functional and anatomic level, this is due to some degree of parathyroid hyperplasia in elderly persons. Indirect and direct evidence implicates the age-related increase in serum PTH in mediating at least part of the age-related increase in bone turnover and bone loss. T h o u g h vitamin D deficiency and impaired renal l et-hydroxylase activity resulting in deficient 1,25(OH)zD production may contribute to the secondary hyperparathyroidism in some elderly individuals, age-associated changes in calcium homeostasis, related to long-term sequelae of sex steroid deficiency, are a major cause of the secondary hyperparathyroidism of aging. Further studies are needed to better define the extraskeletal effects of sex steroids on calcium homeostasis in w o m e n and in m e n and the contribution of increased PTH concentrations toward cancellous versus cortical bone loss in the elderly.

REFERENCES 1. Riggs BO, Melton III LJ. Involutional osteoporosis. [Review]. N EnglJ Med 1986;314:1676-1686. 2. Riggs BL, Khosla S, Melton III LJ. A unitary model for involutional osteoporosis: Estrogen deficiency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men. JBone Miner Res 1998;13:763-773. 3. Wiske PS, Epstein S, Bell NH, Queener SF, Edmondson J, Johnston CC. Increases in immunoreactive parathyroid hormone with age. NEnglJMed 1979;300:1419-1421. 4. Gallagher JC, Riggs BL, Jerpbak CM, Arnaud CD. The effect of age on serum immunoreactive parathyroid hormone in normal and osteoporotic women. J Lab Clin Med 1980;95:373-385. 5. Insogna KL, Lewis AM, Lipinski BA, Bryant C, Baran DT. Effect of age on serum immunoreactive parathyroid hormone and its biological effects. J Clin Endocrinol Metab 1981 ;53:1072-1075. 6. Marcus R, Madvig P, Young G. Age-related changes in parathyroid hormone and parathyroid hormone action in normal humans. J Clin Endocrinol Metab 1984;58:223-230. 7. Epstein S, Bryce G, Hinman JW, Miller ON, Riggs BL, Hui SL, Johnston CC, Jr. The influence of age on bone mineral regulating hormones. Bone 1986;7:421-425. 8. Young G, Marcus R, MinkoffJR, Kim LY, Segre GV. Age-related rise in parathyroid hormone in man: The use of intact and midmolecule antisera to distinguish hormone secretion from retention. J Bone Miner Res 1987;2:367-374. 9. Forero MS, Klein RF, Nissenson RA, Nelson K, Heath III H, Arnaud CD, Riggs BL. Effect of age on circulating immunoreactive and bioactive parathyroid hormone levels in women. J Bone Miner Res 1987;2:363-366. 10. Eastell R, Yergey AL, Vieira NE, Cedel SL, Kumar R, Riggs BL. Interrelationship among vitamin D metabolism, true calcium absorption, parathyroid function, and age in women: Evidence of an age-related intestinal resistance to 1,25-dihydroxyvitamin D action. JBone Miner Res 1991;6:125-132. 11. Khosla S, Melton III LJ, Atkinson EJ, Klee GG, O'Fallon WM, Riggs BL. Relationship of serum sex steroid levels with bone min-

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eral density in aging women and men: A key role for bioavailable estrogen. J Clin Endocrinol Metab 1998;83:2266-2274. 12. Koh LKH, Bauer DC, Forsyth BA, Gore LR, Vogt MT, Cummings SR. PTH in postmenopausal osteoporotic women. J Bone Miner Res 1997;12:S166. 13. Prince RL, Dick I, Devine A, Price RI, Gutteridge DH, Kerr D, Criddle A, Garcia-Webb P, St. John A. The effects of menopause and age on calcitropic hormones: A cross-sectional study of 655 healthy women aged 35 to 90.JBone Miner Res 1995;10:835-842. 14. Dawson-Hughes B, Harris S, Kramich C, Dallal G, Rasmussen HM. Calcium retention and hormone levels in black and white women on high- and low-calcium diets. J Bone Miner Res 1993;8:779-787. 15. Bell NH, Yergey AL, Vieira NE, Oexmann MJ, Shary JR. Demonstration of a difference in urinary calcium, not calcium absorption, in black and white adolescents. J Bone Miner Res 1993;8:1111-1115. 16. Perry III HM, Horowitz M, Morley JE, Fleming S, Jensen J, Caccione Pg, Minner DK, Kaiser FE, Sundarum M. Aging and bone metabolism in African American and Caucasian women. J Clin Endocrinol Metab 1996;81:1108-1117. 17. Eastell R, Simmons PS, Colwell A, A_ssiri AM, Burritt ME Russell RGG, Riggs BL. Nyctohemeral changes in bone turnover assessed by serum bone Gla- protein concentration and urinary deoxypyridinoline excretion: Effects of growth and ageing. Clin Sci 1992;83:375-382. 18. Ledger GA, Burritt ME Kao PC, O'Fallon WM, Riggs BL, Khosla S. Role of parathyroid hormone in mediating nocturnal and agerelated increases in bone resorption. J Clin Endocrinol Metab 1995;80:3304-3310. 19. Ledger GA, Burritt ME Kao PC, O'Fallon WM, Riggs BL, Khosla S. Abnormalities of parathyroid hormone secretion in elderly women that are reversible by short-term therapy with 1,25-dihydroxyvitamin D~. J Clin Endocrinol Metab 1994;79:211-216. 20. Portale AA, Lonergan ET, Tanney DM, Halloran BE Aging alters calcium regulation of serum concentration of parathyroid hormone in healthy men. AmJPhysio11997;35:E139-E146. 21. Akerstrom G, Rudberg C, Grimelius L, Bergstrom R, Johansson H, Ljunghall S, Rastad J. Histologic parathyroid abnormalities in an autopsy series. Hum Patho11986;17:520-527. 22. Delmas PD, Stenner D, Wahner HW, Mann KG, Riggs BL. Increase in serum bone gamma-carboxyglutamic acid protein with aging in women. Implications for the mechanism of agerelated bone loss. J Clin Invest 1983;71:1316-1321. 23. Khaw K-T, Sneyd MJ, Compston JE. Bone density, parathyroid hormone, and 25-hydroxyvitamin D concentrations in middle aged women. Br MedJ 1992;305:273-277. 24. Murphy S, Khaw K-T, Prentice A, Compston JE. Relationships between parathyroid hormone, 25-hydroxyvitamin D, and bone mineral density in elderly men. Age Ageing 1993;22:198-204. 25. Boonen S, Cheng XG, Nijs J, Nicholson PH, Verbeke G, Lesaffre E, AerssensJ, DequekerJ. Factors associated with cortical and trabecular bone loss as quantified by peripheral computed tomography (pQCT) at the ultradistal radius in aging women. Calcif Tissue Int 1997;60:164-170. 26. Flicker L, Lichtenstein M, Colman P, Buirski G, Kaymakci B, HopperJL, WarkJD. The effect of aging on intact PTH and bone density in women. J A m Geriatr Soc 1992;40:1134-1138. 27. Bilezikian JE Estrogens and postmenopausal osteoporosis: Was Albright right after all? JBone Miner Res 1998;13:774-776. 28. Shukla S, Gillespy TI, Thomas WC. The effect of hypoparathyroidism on the aging skeleton. J A m Geriatr Soc 1990;38:884-888. 29. Chapuy MC, Schott AM, Garnero P, Hans D, Delmas PD, Meunier PJ. Healthy elderly French women living at home have secondary hyperparathyroidism and high bone turnover in winter. J Clin Endocrinol Metab 1996;81:1129-1133.

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30. Sherman SS, Hollis BW, Tobin JD. Vitamin D status and related parameters in a healthy population: The effects of age, sex, and season. J Clin Endocrinol Metab 1990;71:405-413. 31. Tsai KS, Heath III H, Kumar R, Riggs BL. Impaired vitamin D metabolism with aging in women. Possible role in pathogenesis of senile osteoporosis. J Clin Invest 1984;73:1668-1672. 32. Ebeling PR, Sandgren ME, DiMagno EP, Lane AW, DeLuca HE Riggs BL. Evidence of an age-related decrease in intestinal responsiveness to vitamin D: Relationship between serum 1,25-dihydroxyvitamin D 3 and intestinal vitamin D receptor concentrations in normal women. J Clin Endocrinol Metab 1992;75:176-182. 33. Kinyamu HK, GallagherJC, Prahl JM, DeLuca HE Petranick KM, Lanspa SJ. Association between intestinal vitamin D receptor, calcium absorption, and serum 1,25-dihydroxyvitamin D in normal and elderly women. J Bone Miner Res 1997;12:922-928. 34. Pattanaungkul S, Riggs BL, Yergey AL, Vieira NE, O'Fallon WM, Khosla S. Relationship of intestinal calcium absorption to 1,25dihydroxyvitamin D levels in young versus elderly women: Evidence for age-related intestinal resistance to 1,25-dihydroxyvitamin D action. J Clin Endocrinol Metab 2000;85:4023-4027. 35. Heaney RP, Recker RR, Saville PD. Menopausal changes in calcium balance performance. J Lab Clin Med 1978;92:953-963. 36. Looker AC, Harris TB, Madans JH, Sempos CT. Dietary calcium and hip fracture risk: The NHANES I epidemiologic follow-up study. Osteoporos Int 1993;3:177-184.

37. McKane WR, Khosla S, Egan KS, Robins SP, Burritt ME Riggs BL. Role of calcium intake in modulating age-related increases in parathyroid function and bone resorption. J Clin Endocrinol Metab 1996;81:1699-1703. 38. McKane WR, Khosla S, RisteliJ, Robins SP, MuhsJ, Riggs BL. Role of estrogen deficiency in pathogenesis of secondary hyperparathyroidism and bone turnover abnormalities in elderly women. Proc Assoc Am Physicians 1997;109:174-180. 39. Khosla S, Atkinson EJ, Melton III LJ, Riggs BL. Effects of age and estrogen status on serum parathyroid hormone levels and biochemical markers of bone turnover in women: A populationbased study. J Clin Endocrinol Metab 1997;82:1522-1527. 40. Gennari C, Agnusdei D, Nardi P, Civitelli R. Estrogen preserves a normal intestinal responsiveness to 1,25-dihydroxyvitamin D 3 in oophorectomized women. J Clin Endocrinol Metab 1990;71: 1288-1293. 41. Nordin BEC, Need AG, Morris HA, Horowitz M, Robertson WG. Evidence for a renal calcium leak in postmenopausal women. J Clin Endocrinol Metab 1991 ;72:401-407. 42. McKane WR, Khosla S, Burritt ME Kao PC, Wilson DM, Ory SJ, Riggs BL. Mechanism of renal calcium conservation with estrogen replacement therapy in women in early postmenopausema clinical research center study. J Clin Endocrinol Metab 1995;80:3458-3464. 43. Cosman E Nieves J, Horton J, Shen V, Lindsay R. Effects of estrogen on response to edetic acid infusion in postmenopausal women. J Clin Endocrinol Metab 1994;78:939-943.

CHAPTER54 Parathyroid Function and Responsiveness in Osteoporosis

SHONNI j. SILVERBERG Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032 J O H N E BILEZIKIAN Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

INTRODUCTION

sis have been a subject of active investigation in recent years. Vitamin D has also been implicated. This chapter reviews the evidence that parathyroid hormone (PTH) plays an important role in the pathophysiology of osteoporosis. In osteoporosis, the normally fight balance between bone formation and bone resorption is lost. When this balance is at its most efficient, the a m o u n t of bone resorbed during the initial phase of bone remodeling is completely replaced by new bone formed in the subsequent stage of the remodeling sequence. With age, the efficiency of this process is reduced so that the amount of bone formed does not quite match the amount of bone lost. Over many years, this inefficiency leads to gradual loss of bone. It is this process of gradual bone loss in which PTH and vitamin D have been investigated as possible etiologic factors. For example, intestinal calcium absorption, a function d e p e n d e n t in part on vitamin D, has long been known to decrease with advancing age and may be reduced to a greater extent in osteoporotic subjects (9-11). A likely candidate to account for this age-related reduction in calcium absorption is the active metabolite of vitamin D, 1,25-dihydroxyvitamin D [ 1,25 (OH) 2o ]. The rationale for focusing on parathyroid hormone as a potential causative agent in osteoporosis stems in part from its pivotal role in bone remodeling. Parathyroid hormone is important in the activation of the remodeling cycle, acting indirectly to stimulate the osteoclast through intercellular signals generated by its direct target cells, osteoblasts or osteoblast-like cells (12-14). In diseases of parathyroid h o r m o n e excess,

As the incidence and prevalence of osteoporosis increases, with any attendant rise in economic and social costs, efforts to delineate the pathophysiologic basis of this disorder have intensified. Although estrogen deficiency characterizes the menopause in all women, and is considered to be an important pathophysiologic feature, only 10-20% of postmenopausal women develop osteoporotic fractures within 20 years of cessation of menses (1). Despite some supporting data (2-4), most evidence argues against the hypothesis that postmenopausal women who develop osteoporosis are relatively more estrogen deficient than their counterparts who do not develop osteoporosis (5,6). Even studies that have noted a difference in sex steroid levels among those who develop osteoporosis have implicated additional factors in the pathogenesis of osteoporotic bone disease. Identifiable risk factors such as family history, racial and ethnic background, and peak bone mass achieved in young adulthood, as well as smoking, alcohol, and lack of physical exercise, are all believed to be important (7,8). In conjunction with estrogen deficiency, these other risk factors, knowledge of which is based on epidemiologic studies, give a more satisfactory accounting of bone loss in the postmenopausal years. However, the epidemiologic approach does not address the mechanisms by which bone is lost. It is reasonable to consider the possibility that calcium-regulating hormones play a mechanistic role in the development of osteoporosis. For example, parathyroid function and responsiveness in osteoporoThe Parathyroids, Second Edition

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Copyright © 2001 John E Bilezikian, Robert Marcus, and Michael A. Levine.

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such as primary hyperparathyroidism, bone remodeling is accelerated and characterized by a rate of bone resorption that exceeds the rate of bone formation. The inherent inefficiency of bone remodeling in adults would lead to bone loss in this setting. Another hypothesis, first proposed by Heaney in the 1960s (15), rests on the idea that PTH in postmenopausal women becomes more catabolic for bone because of enhanced skeletal sensitivity. This catabolic action of PTH would surface in postmenopausal women because estrogens, which normally counteract the actions of parathyroid hormone, are reduced. Thus, the skeleton of the postmenopausal woman would become more sensitive to the same circulating level of PTH. Recognition that PTH is catabolic under pathologic conditions has tended to obscure its physiologic role as an anabolic peptide for bone (see Chapters 11 and 55). This physiologic effect to maintain bone mass has led to efforts to reveal abnormalities in PTH regulation in osteoporosis. This argument focuses on the anabolic role of parathyroid hormone, not its catabolic effect. The hypothesis states that if parathyroid hormone is impaired in its anabolic capacity, osteoporosis will result. The potential importance of the anabolic actions of PTH on skeletal metabolism is well illustrated in another disorder of mineral metabolism, primary hyperparathyroidism. In the mild presentation of the disease most commonly seen today, there is preservation of cancellous bone, which is normally adversely affected by postmenopausal bone loss (see Chapter 26) (16-20). Despite data suggesting a possible role for PTH in the development of osteoporosis, definitive answers remain elusive. Many early studies in this area were flawed by the lack of an appropriate control group, or by use of pharmacologic doses of PTH to assess physiologic responses. Improvements in assays for PTH have required reconsideration of older studies whose validity was limited by assay methodology. In addition, it is necessary to distinguish between age-related changes in mineral metabolism that do not necessarily lead to osteoporosis and those that do.

PARATHYROID HORMONE AND 1 , 2 5 - D I H Y D R O X Y V I T A M I N D LEVELS I N OSTEOPOROSIS Advances in immunoassay techniques (see Chapter 9) have aided in the characterization of basal and stimulated levels of PTH in osteoporosis. Basal levels of PTH are clearly shown to increase with age in most studies, as measured by both radioimmunoassay and bioassay (21-25). However, in postmenopausal osteoporosis,

basal levels of PTH have been reported to be increased, normal, or decreased relative to those in age-matched controls (5,21,26,27). No difference was reported between osteoporotic and normal women using the immunoradiometric assay (IRMA) for parathyroid hormone (4,28), whereas the even more sensitive immunochemiluminometric assay (ICMA) has shown reduced basal PTH levels in osteoporotic patients (4,29). The effect of advancing age on levels of 1,25-dihydroxyvitamin D is less controversial. There is a decrease in circulating levels of 1,25(OH)zD in the elderly (11,22,29,30). Individuals with osteoporosis have 1,25(OH)zD, levels that are generally lower than those in age- and sex-matched normal individuals (11,31). This reduction is accompanied by a reduction in calcium absorption (11).

Pathophysiologic Implications of Parathyroid Hormone in Osteoporosis It is likely that osteoporosis is not a single entity but instead a descriptive end point for a n u m b e r of distinct pathways. With regard to parathyroid hormone, four different mechanisms leading to osteoporosis have been postulated (see Table 1). For each hypothesis, the primary abnormality is placed at a different physiologic "level." In hypothesis 1, the abnormality in PTH activity is due to a primary disturbance in vitamin D metabolism. In hypothesis 2, the skeleton has become abnormally sensitive to PTH. In hypothesis 3, unidentified factor(s) lead to secondary suppression of normally functioning parathyroid glands. In hypothesis 4, abnormalities in responsiveness of the parathyroid glands are accountable for the development of osteoporosis.

Hypothesis 1" Altered Vitamin D Metabolism Decreased intestinal calcium among the elderly has been reported in studies of fractional calcium absorption and metabolic balance, with an even greater decrease in calcium absorption noted in osteoporotic subjects (9,11). Bone loss resulting from reduced calcium absorption could be due to a primary abnormality

TABLE 1 Pathophysiologyof Osteoporosis Hypotheses Postulated primary mechanism

PTH change

Altered vitamin D metabolism Altered skeletal sensitivity to PTH Suppression of parathyroid gland Altered parathyroid gland responsiveness

Increase Normal/decrease Decrease Normal/decrease

PTH FUNCTION IN OSTEOPOROSIS / in vitamin D metabolism, with reduced levels of 1,25-dihydroxyvitamin D. The decrease in intestinal calcium absorption would lead to a subtle reduction in serum calcium concentration, which might not be detected. However, the hypocalcemic signal would induce a secondary increase in PTH. Though most studies to test this hypothesis have implicated a defect in 1,25(OH)2D production, a potential abnormality in metabolism of the precursor to 1,25(OH)2 D, 25-hydroxyvitamin D, has not escaped notice. Villareal et al. (32) found that 9.1% of their population of midwestern United States women with postmenopausal osteoporosis had low (<38 nmol/liter; mean, - 2 SD of their normal population) 25-hydroxyvitamin D levels. In this group vertebral bone density correlated positively with levels of 25-hydroxyvitamin D and inversely with PTH levels, which were increased. Multivariate analysis demonstrated that the rise in PTH was best correlated with the decrease in bone density. Early studies used pharmacologic doses of parathyroid extract to investigate whether decreased 1,25 (OH)2 ° levels in postmenopausal osteoporosis were due to a decrease in renal l e~-hydroxylase activity. A similar rise in 1,25(OH)zD was observed in normal and osteoporotic individuals when high doses of PTH were administered. Although normal renal responsiveness could thus be demonstrated under these pharmacologic conditions, it remained possible that an abnormality existed in renal responsiveness to more physiologic levels of PTH (27,33). Subsequent studies suggested the inability of the kidney to generate 1,25 (OH)2 O, in response to PTH decreases with advancing age, with a further unexplained impairment in a group with age-related osteoporosis (30). However, the two older groups of subjects in this study, one with and one without osteoporosis, were not directly comparable because of differences in renal function. It remained possible that the differences observed between the two older groups of subjects were due primarily to differences in renal function and not to a particular abnormality in the osteoporotic group. Using more physiologic doses, Slovik et al. showed a blunted rise in 1,25(OH)zD to PTH osteoporotic patients (34). Unfortunately, the control group in this study consisted of normal young individuals, making it impossible to know whether the observed abnormality in PTH responsiveness was due merely to advancing age or to the osteoporotic process. In addition to impaired formation of 1,25(OH)zD, other factors that could contribute to altered 1,25(OH)zD metabolism in osteoporosis include a decrease in 1,25(OH)zD receptors in the small intestine (35,36). In 44 healthy women aged 20-87 years, a decrease in the concentration of duodenal 1,25(OH)zD receptors was shown to be a function of age. Reduced

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calcitriol receptors could account for a reduction in gastrointestinal tract calcium absorption, offering an explanation for the PTH concentration. A third mechanism to account for altered vitamin D metabolism in osteoporosis involves altered binding of 1,25(OH)zD to its receptors (37), rather than a decrease in receptor number. Fourth, a postreceptor defect in 1,25 (OH) 2o responsiveness may be proposed. Evidence for this possibility comes from a study of normal young women who had undergone oophorectomy (38). Six months after oophorectomy, a reduction in fractional calcium absorption and vertebral bone density was seen. Calcium absorption in response to 1,25(OH)zD administration was blunted. The subnormal response, which could be prevented by estrogen replacement, was thought to be consistent with endorgan resistance to 1,25 (OH) 2o.

Hypothesis 2: Altered Skeletal Sensitivity to Parathyroid Hormone According to this hypothesis, originally proposed by Heaney in 1965, enhanced sensitivity to PTH in osteoporosis leads to greater bone resorption at any given level of circulating hormone (4,5,11,15). Despite normal circulating concentrations, parathyroid h o r m o n e could still induce bone loss if sensitivity is enhanced in the postmenopausal state. One point in support of this idea comes from the literature on primary hyperparathyroidism, a clinical disorder that is often unmasked as a function of declining estrogen levels in women during the first decade after the menopause (39). Furthermore, it is generally accepted that when postmenopausal patients with primary hyperparathyroidism are given estrogen, calcium levels decline somewhat without any change in the circulating concentration of parathyroid h o r m o n e (40,41). In the setting of the normocalcemic individual, however, McKane et al. have demonstrated that within 6 months of estrogen replacement therapy in early postmenopausal women, and despite no change in circulating serum calcium concentration, parathyroid hormone levels increase by 38% (42). These observations together argue for enhanced sensitivity to the skeletal effects of parathyroid hormone when women become estrogen deficient, and for a need for increased circulating parathyroid hormone when estrogen is provided to the early postmenopausal woman. Early data from Riggs et al. (5) provided indirect support for the hypothesis of increased skeletal sensitivity to parathyroid hormone in osteoporosis, in the form of histologic evidence of increased bone resorption in the face of normal or low serum PTH immunoactivity. This group has since published histomorphometric data supporting their earlier conclusions (4). Indices of

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bone remodeling were analyzed with regard to PTH levels by both immunoradiometric and immunochemiluminometric assays in normal and osteoporotic postmenopausal women. Measures of bone turnover, including bone resorption rate, bone formation rate, activation frequency, and rate of bone loss, all correlated with PTH levels in osteoporotic but not in normal individuals. For each picomole/liter increase in PTH, as measured in the immunochemiluminometric assay, there was greater activation frequency (1.3%/year), higher bone resorption rate (3.9%/year), and more cancellous bone loss (2.8%/year) in the women with postmenopausal osteoporosis. These conclusions, however, were not supported by Tsai et al. (43) in a study employing pharmacologic doses of PTH to assess biochemical, rather than histologic, markers. Bovine PTH(1-34), 400 U/day, was administered for 3 days to premenopausal and postmenopausal normal women and to women with postmenopausal osteoporosis. Although the osteoporotic women had evidence for increased bone turnover at base line, the hormone-induced increase in both serum calcium concentration and bone markers was similar in all three groups. The authors concluded that these data speak against enhanced sensitivity to PTH in osteoporosis. Limitations of this study, noted by the authors, include a nonphysiologic dose of PTH, the relative insensitivity of measured indices of increased resorption (urinary hydroxyproline), and inadequate statistical power inherent in the study design. In Hypotheses 1 and 2, alterations in parathyroid hormone associated with aging or menopause are etiologic in the development of osteoporosis. The mechanistic hypotheses (see below), on the other hand, describe changes in parathyroid gland functioning that are adaptive, serving to protect the skeleton from the causes of age-related changes in bone mass.

Hypothesis 3: Suppression of Parathyroid Gland Function A third hypothesis implicating PTH in the pathophysiology of osteoporosis argues for a secondary suppression of parathyroid gland function. The hypothesis recognizes the importance of PTH action as anabolic for bone. This concept differs from the first two hypotheses in invoking a process that, independent of PTH, increases bone resorption and leads ultimately to osteoporosis. The increase in bone resorption leads to a subclinical rise in ionized calcium, with a compensatory suppression of PTH secretion. In this model, the associated decrease in intestinal calcium absorption would be a secondary phenomenon, due to lack of action of PTH to stimulate 1,25(OH)zD formation. The list of agents potentially responsible for this suppression of PTH include interleukin-1, which is produced at

an enhanced rate by activated monocytes from postmenopausal osteoporotic women (44). In vitro interleukin-1 is a most potent stimulus to bone resorpfive activity when assessed on a molar basis (45). Interleukin-6, another potent cytokine, has been implicated specifically in the enhanced bone resorption that occurs in conjunction with estrogen deficiency (46,47). Other local factors that may stimulate bone resorption include lymphotoxin, tumor necrosis factor, transforming growth factor, and certain prostaglandins. Among studies supporting the concept of parathyroid suppression in osteoporosis, the work of Ebeling et al. (48) is noteworthy. These authors compared biochemical indices and bone markers in normal and osteoporotic women before and after calcium deprivation. At base line, the osteoporotic cohort had lower PTH and higher bone turnover markers (osteocalcin and hydroxypyridinium collagen cross-link levels) than normal women. After calcium deprivation, no difference was noted among the two groups in the decrement of serum calcium concentration or in the rise of PTH, 1,25(OH)2D, or hydroxypyridinium cross-link levels. The base line data were interpreted to be consistent with a non-PTH-mediated increase in bone resorption, with a secondary suppression of PTH. The similar response to a hypocalcemic stimulus in both groups was considered to be evidence against any enhanced skeletal sensitivity to PTH or any change in parathyroid gland responsiveness.

Hypothesis 4: Altered Parathyroid Gland Responsiveness This hypothesis suggests that an abnormality in parathyroid gland responsiveness is important in the development of osteoporosis. Using oral phosphate administration as a hypocalcemic stimulus, Silverberg et al. (49) studied the effects of the ensuing mild hypocalcemia on PTH and 1,25(OH)2D levels. Using a protocol of 5 days of oral phosphate administration, three groups were studied: postmenopausal healthy and osteoporotic women, and young healthy individuals. Despite a similar increase in inorganic phosphorus concentration in response to the oral phosphate challenge, and a similar decrease in serum calcium concentration among the groups, hormonal responses varied. Parathyroid hormone levels rose by a modest 43% in those with osteoporosis, a change similar to the 53% increase in young normal subjects. This modest response differed considerably from the more marked 2.5-fold rise in circulating parathyroid hormone seen in the older control group (see Fig. 1). Despite these markedly different responses, 1,25(OH)2D levels were unchanged. In those with osteoporosis, whose response was modest and similar to that of young normal subjects, 1,25(OH)zD levels fell by 50%. Normal older

PTH FUNCTION IN OSTEOPOROSIS 160

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women thus appeared to require greater PTH reactivity than their younger counterparts to overcome the suppressive effects of phosphate on 1,25(OH)2D formation. The data suggest that there is an age-related decline in the formation of 1,25 (OH) 2D, and that, as part of the normal aging process, postmenopausal women maintain their 1,25(OH)zD levels only by mounting a more vigorous PTH response to hypocalcemia than is required at a younger age. In osteoporosis, the age-appropriate rise in PTH responsiveness is not seen, suppressive effects of elevated phosphate prevail, and 1,25(OH)zD levels fall. The results support the concept of abnormal PTH responsiveness in osteoporosis, superimposed on a universal age-related decline in 1,25(OH)zD production. This possibility could also be consistent with suppression of parathyroid gland activity due to a nonparathyroid mechanism for activation of bone resorption (Hypothesis 3). Another study assessing parathyroid gland responsiveness avoided potential confounding effects of phosphorus on 1,25(OH)2D metabolism (28). Suppression of endogenous parathyroid hormone secretion was accomplished by infusing PTH(1-34) at physiologic doses (0.55 U / k g / h o u r for 24 hours). Intact PTH(1-84) was measured to assess endogenous hormone secretion. The extent of the response to PTH infusion, as reflected in both absolute and fold increase in serum calcium and decrease in endogenous PTH, was indistinguishable among premenopausal and postmenopausal normal women, as well as between untreated and estrogen-replaced postmenopausal women with osteoporosis. However, for any given increment in serum calcium concentration, osteoporotic individuals showed less suppression of endogenous PTH secretion compared to healthy women. Abnormal PTH suppressibility in osteoporotic subjects was not

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847

improved by estrogen-replacement therapy. Age or menopause did not seem to affect the result. The authors concluded that skeletal sensitivity to PTH is not altered by aging or osteoporosis. Rather, in osteoporosis there is an alteration in parathyroid gland responsiveness, characterized by a change in the set point for hormone secretion. Parathyroid insensitivity to circulating calcium is consistent with the blunted stimulatory response to hypocalcemia reported by Silverberg et al. (49). Altered responsiveness could also underlie changes in circadian rhythmicity of parathyroid hormone secretion. Daily parathyroid hormone secretion follows a biphasic profile, with peaks at approximately 1800 and 0200 hours (50,51). Presumably, the larger nocturnal peak is secondary to mild hypocalcemia induced by overnight fasting. Calvo et al. reported that women exhibited a blunted parathyroid hormone peak compared to that of men and, subsequently, a less dramatic decline in nighttime urinary calcium excretion (52). Overnight urinary calcium excretion declined in men by 34%, whereas in women, it decreased by only 17%. This nocturnal calcium wasting could, over the years, contribute to the greater bone loss seen in women. Postmenopausal osteoporotic women showed a further blunting of their nocturnal parathyroid hormone peak, with no change in fractional excretion of calcium at night (53). The inefficient renal calcium conservation thus documented could contribute to the osteoporotic process. The cause of this blunted parathyroid hormone response to nocturnal fasting is unknown. More sophisticated pulsatility studies by Prank et al. have shown that osteoporotic women demonstrate poorly predictable time series of pulses and patterns of parathyroid h o r m o n e secretion. Creating a discriminating statistic by fitting a time series model to the pooled data from normal subjects, normal and osteoporotic subjects could be distinguished from each other (54,55). In contrast, Samuels et al. could not demonstrate any differences in amplitude or frequency, or pulsatile parathyroid h o r m o n e secretory parameters among osteoporotic and normal subjects. The lack of a difference was not influenced by the presence of estrogens (56).

RELATIONSHIP OF PARATHYROID HORMONE TO CLASSIC DESIGNATIONS OF OSTEOPOROSIS

In 1947, Albright et al. (57) divided those with osteoporosis into two populations: a postmenopausal group and one with senile (age-related) osteoporosis. This concept was refined by Riggs and Melton (58), who suggested a different nomenclature, type I and type II

848

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CHAPTER

54

osteoporosis. Because this nomenclature has enjoyed lingering popularity, these "types" are described here in an attempt to place them into the mechanistic framework outlined above. Type I osteoporosis (postmenopausal osteoporosis) overwhelmingly affects women and develops within 15 years of menopause. Skeletal sites of primarily cancellous bone (the thoracic and lumbar vertebrae) are involved initially. Parathyroid h o r m o n e levels tend to be decreased in this group. Suppressed PTH levels fit with Hypothesis 3 (Table 1). Type II osteoporosis is the clinical end point of age-related bone loss. It is recognized later than type I, after age 70 years, and affects females more frequently than males. However, type II osteoporosis is seen in both women and men. Biochemically, this group is characterized by an increase in PTH concentration, generally thought to be a secondary response to decreased calcium absorption. From a mechanistic viewpoint, type II osteoporosis could be explained by Hypothesis 1. Patients with postmenopausal (type I) disease have an abnormality of vitamin D metabolism, suggesting that the pathogenesis of this disorder may be even more complicated than the simple designation would suggest.

P A R A T H Y R O I D H O R M O N E IN O S T E O P O R O S I S : A G E N E T I C BASIS? One avenue for investigation of a definitive role for parathyroid h o r m o n e in the pathogenesis of osteoporosis has been centered on a possible genetic basis for a connection between the disease and alterations in this important h o r m o n e of calcium homeostasis. Investigations into candidate genes to explain the significant genetic contibution to the development of osteoporosis has yielded suggestive data, linking parathyroid receptor type 1 to osteoporosis (59). A second study found polymorphism in the PTH gene to be a marker for low bone mineral density in a population of postmenopausal Japanese women (60). Although very interesting, an etiologic relationship between genetic alterations in PTH and the develo p m e n t of osteoporosis must remain speculative at this point.

ESTROGEN AND PARATHYROID HORMONE A full discussion of the interaction between estrogen and parathyroid h o r m o n e is beyond the scope of this chapter (see Chapter 55). Clinical observations supporting the idea of interactions between PTH and estrogen are appropriately considered within the concepts of this chapter. Increased bone resorptive activity,

a skeletal effect of PTH, emerges in menopause when estrogen deficiency develops. Furthermore, the clinical disorder primary hyperparathyroidism is often unmasked by menopause (18). In addition, use of estrogen in postmenopausal women with primary hyperparathyroidism is associated with a reduction in serum calcium levels (see Chapter 29) (40,41). It has thus been postulated that estrogen is somehow antagonistic to PTH. It is believed by some that estrogen deficiency, in menopause, potentiates PTH-mediated postmenopausal bone loss. Current understanding of the interplay between estogen deficiency and parathyroid function in either healthy or osteoporotic menopause remains limited. Available information often comes mostly from studies describing the effects of estrogen repletion in the menopause. The relative contributions of estrogen loss and subsequent changes in PTH as altered mineral metabolism develops in the menopause can only be inferred. In addition, data obtained from normal postmenopausal women are not necessarily applicable to postmenopausal women with osteoporosis. Early studies noted that menopause was associated with a state of negative calcium balance (61). Decreased intestinal calcium absorption contributed to the negative balance. This abnormality was more pron o u n c e d in osteoporotic women than in normal women and could be reversed by estrogen-replacement therapy. Data from Gallagher et al. (62) suggested that estrogen treatment increased calcium absorption in postmenopausal osteoporosis by increasing PTH levels. The ensuing stimulation of renal l e~-hydroxylase activity due to PTH would lead to a rise in 1,25(OH)zD, and hence to the positive effect on the gastrointestinal tract. Additional studies led to a revision of this formulation. Estrogen deficiency does seem to be associated with a small d e c r e m e n t in total and ionized serum calcium, which can be reversed by replacement therapy. The reported effect of this mild hypocalcemia on PTH levels has been variable, with a decrease or no change in normal postmenopausal women and an increase or no change reported in osteoporotic individuals (63-65). Free as well as total 1,25 (OH) 2o and vitamin D binding protein levels rise when normal postmenopausal women are given estrogen replacement (66,67). Several studies have proposed that estrogen deficiency may alter the set point for PTH stimulation by circulating calcium. Estrogen replacement reportedly leads to a decrease in set point for PTH secretion in normal women, but in osteoporotic women, blunted parathyroid gland sensitivity to circulating calcium was also found (28). In addition, estrogen treatment of the postmenopausal woman with osteoporosis leads to reduced bone resorptive (but not bone formative) activity in response to PTH administration (68).

PTH FUNCTION IN OSTEOPOROSIS / The concept of parathyroid hormone as being pathophysiologically involved in postmenopausal osteoporosis has been promulgated even more boldly by Riggs and associates. They have recently proposed a "unitary model for involutional osteoporosis" (69). In this hypothesis, Riggs et al. advance the notion that not only is parathyroid hormone involved in the early postmenopausal mechanisms of estrogen deficiency--a relative suppressionmbut that increases in parathyroid h o r m o n e become important later in the setting of agerelated bone loss. They propose that in the second phase of bone loss (type II-related or age-related bone loss), the actions of estrogen deficiency are predominant in nonskeletal sites, such as the gastrointestinal tract and the kidneys. Such extraskeletal actions lead to increased urinary calcium loss and reduced calcium absorption. The resulting secondary increase in parathyroid hormone is key in this explanation of the continued loss of bone mass with aging. Although Riggs and co-workers cite a number of studies in support of this hypothesis (70-72) our knowledge is admittedly still limited. We know little of the extraskeletal actions of estrogens with regard to renal calcium handling and to gastrointestinal absorption of calcium. Also vexing is the hypothesis that late-term estrogen deficiency is associated with an increase in parathyroid hormone. If one can account for these two separate actions of estrogens, which are dichomotous in time, the secondary increase in parathyroid hormone occurring in this later phase would help to account for the more apparent cortical bone loss and an amelioration of the earlier rapid cancellous bone loss. Such observations gain support from data on primary hyperparathyroidism in which increased parathyroid hormone levels are associated with preservation of cancellous bone at the expense of cortical bone (73). In fact, this and other observations have led many investigators to consider parathyroid hormone as a potential therapy for postmenopausal osteoporosis (74). Other confounding elements to the hypothesis put forth by Riggs et al. are noted by Bilezikian (75).

SUMMARY Although estrogen deficiency clearly plays a significant role in the development of postmenopausal osteoporosis, other factor(s) clearly contribute to the pathogenesis of this disorder. Evidence reviewed in this chapter implicates the parathyroid axis in the pathophysiologic abnormalities in osteoporosis. Several pathophysiologic mechanisms to explain possible relationships between PTH or parathyroid gland function and osteoporosis have been reviewed. Whether perturbations in PTH are a primary or a reactive mechanism

849

remains unclear. Alterations at the level of the parathyroid glands are described in the hypothesis of modified parathyroid responsiveness leading to osteoporosis. Among the hypotheses that describe a secondary change in PTH are those based on alterations in 1,25(OH)zD generation, enhanced skeletal sensitivity to the catabolic effects of PTH, and suppression of the parathyroids in the face of bone resorption driven by other agents. Recent data support further investigation into possible alterations in the parathyroid hormone gene, or parathyroid hormone receptor gene polymorphism, as potential mechanisms underlying the development of osteoporosis. The current state of our understanding leaves us with two sides of the argument. Parathyroid hormone could either be the culprit in the process of bone loss, or alternatively, parathyroid hormone may contibute to protective adaptations against osteoporosis. Ongoing research efforts should help further elucidate the role of parathyroid hormone in the development of this important disorder of skeletal metabolism.

ACKNOWLEDGMENTS Supported in part by National Institutes of Health grants NIDDK 32333, NIAMS 39191, and RR 00645.

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32. Villareal D, Civitelli R, Chines A, Avioli L. Subclinical vitamin D deficiency in postmenopausal women with low vertebral bone mass. J Clin Endocrinol Metab 1991;72:628-634. 33. Riggs B, Hamstra A, DeLuca H. Assessment of 25-hydroxyvitamin D 1-alpha hydroxylase reserve in postmenopausal osteoporosis by administration of parathyroid extract. J Clin Endocrinol Metab 1981;53:833-835. 34. Slovik DM, Adams JS, Neer RM, Holick ME Potts JT, Jr. Deficient production of 1,25-dihydroxyvitamin D in elderly osteoporotic patients. N E n g l J Med 1981 ;305:372-374. 35. Gallagher J. The pathogenesis of osteoporosis. Bone Miner 1990; 9:215-227. 36. Ebeling P, Sandgren M, DiMango E, Lane A, DeLuca H, Riggs B. Evidence of an age-related decrease in intestinal responsiveness to vitamin D: Relationship between serum 1,25-dihydroxyvitamin D and intestinal vitamin D receptor concentrations in normal women. J Clin Endocrinol Metab 1992;75:176-182. 37. Francis R, Peacock M, Taylor G, Storer J, Nordin B. Calcium malabsorption in elderly women with vertebral fractures: Evidence for resistance to the action of vitamin D metabolites on the bowel. Clin Sci 1984;66:103-107. 38. Gennari C, Agnusdei D, Nardi P, Civitelli R. Estrogen preserves a normal intestinal responsiveness to 1,25-dihydroxyvitamin D in oophorectomized women. J Clin Endocrinol Metab 1990;71: 1288-1293. 39. Bilezikian JP, Silverberg SJ, Gartenberg E Kim TS, Jacobs T, Siris E, Shane E. Clinical presentation of primary hyperparthyroidism. In: Bilezikian JP, Marcus R, Levine MA, eds. The parathyroids. New York:Raven, 1994:457-470. 40. Marcus R, Madvig P, Crim M, Pont A, Kosek J. Conjugated estrogens in the treatment of postmenopausal women with hyperparathyroidism. Ann Intern Med 1984;100:633-640. 41. Selby P, Peacock M. Ethinyl estradiol and norethindrone in the treatment of primary hyperparathyroidism in postmenopausal women. N EnglJ Med 1986;314:1481-1485. 42. McKane WR, Khosla S, Burritt ME Kao PC, Wilson DM, Ory SJ, Riggs BL. Mechanism of renal calcium conservation with estrogen replacement therapy in women in early postmenopause. J Clin Endocrinol Metab 1995;80:3458-3464. 43. Tsai K, Ebeling P, Riggs B. Bone responsiveness to parathyroid hormone in normal and osteoporotic postmenopausal women. J Clin Endocrinol Metab 1989;69:1024-1027. 44. Pacifici R, Rifas L, Teitelbaum S, et al. Spontaneous release of interleukin-1 from human blood monocytes reflects bone formation in idiopathic osteoporosis. Proc Natl Acad Sci USA 1987;84:4616-4620. 45. Gowen M, Mundy G. Actions of recombinant interleukin-1, interleukin-2 and interferon-gamma on bone resorption in vitro. J Immuno11986;136:2478-2482. 46. Jilka R, Girasole G, Passeri G, et al. Increased osteoclast development after estrogen loss: Mediation by interleukin-6. Science 1992;257:88-91. 47. Roodman GD. Interleukin-6: An osteotropic factor? J Bone Miner Res 1992;7:475-478. 48. Ebeling P, Jones J, Burritt M, et al. Skeletal responsiveness to endogenous parathyroid hormone in postmenopausal osteoporosis. J Clin Endocrinol Metab 1992;75:1033-1038. 49. Silverberg S, Shane E, de la Cruz L, Segre G, Clemens T, Bilezikian J. Abnormalities in parathyroid hormone secretion and 1,25-dihydroxvitamin D 3 formation in women with osteoporosis. N EnglJ Med 1989;320:277-281. 50. Tohme JF, Cosman E Lindsay R. Osteoporosis. In: Becker KL, ed. Principles and practice of endocrinology and metabolism, 2nd Ed. Philadelphia:Lippincott, 1995:491-503. 51. Calvo MS, Eastell R, Offord KP, Bergsalh EJ, Burritt ME Circadian variation in ionized calcium and intact parathyroid hormone: Evidence for sex differences in calcium homeostasis. J Clin Endocrinol Metab 1991 ;72: 77-82.

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65. Boucher A, D'Amour P, Hamel L, et al. Estrogen replacement decreases the set point of parathyroid hormone stimulation by calcium in normal postmenopausal women. J Clin Endocrinol Metab 1989;68:831-836. 66. Marcus R, Villa M, Cheema M, Cheema C, Newhall K, Holloway L. Effects of conjugated estrogen on the calcitriol response to parathyroid hormone in postmenopausal women. J Clin Endocrinol Metab 1992;74:413-418. 67. Cheema C, Grant B, Marcus R. Effects of estrogen on circulating "free" and total 1,25-dihydroxyvitamin D and on the parathyroidvitamin D axis. J Clin Invest 1989;83:537-542. 68. Cosman F, Shen V, Xie F, Seibel M, Ratcliffe A, Lindsay R. Estrogen protection against bone resorbing effects of parathyroid hormone infusion. Ann Intern Med 1993;118:337-343. 69. Riggs BL, Khosla S, Melton III LJ. A unitary model for involutional osteoporosis: Estrogen deficiency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men. J Bone Min Res 1998;13: 763-773. 70. McKane WR, Khosla S, Risteli J, Robins SP, Muhs JM, Riggs BL. Role of estrogen deficiency in pathogenesis of secondary hyperparathyroidism and increased bone resorption in elderly women. Proc Assoc Am Phys 1997;109:174-180. 71. Riggs BL, O'Fallon WM, Muhs J, O ' C o n n o r MK, Kumar R, Melton III LJ. Long-term effects of calcium supplementation on serum parathyroid hormone level, bone turnover, and bone loss in elderly women. JBone Miner Res 1998;13:168-174. 72. Khosla S, Atkinson EJ, Melton III LJ, Riggs BL. Effects of age and estrogen status on serum parathyroid hormone levels and biochemical markers of bone turnover in women: A populationbased study. J Clin Endocrinol Metab 1997;82:1522-1527. 73. Parisien M, Dempster DW, Shane E, Bilezikian JE Histomorphometric analysis of bone in primary hyperparathyroidism. In: Bilezikian JP, Marcus R, Levine MA, eds. The parathyroids. New York:Raven, 1994;567-574. 74. Finkelstein JS Pharmacological mechanisms of therapeutics: Parathyroid hormone. In: Bilezikian JP, Raisz LG, Rodan GA, eds. Principles of bone biology. San Diego: Academic Press, 1996; 993-1006. 75. Bilezikian JE Estrogens and postmenopausal osteoporosis: Was Albright right after all? JBone Miner Res 1998;13:774-776.

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CI-IAP:rV, 55

Parathyroid Hormone and Growth Hormone in the Treatment of Osteoporosis

ROBERT MARCUS Department of Medicine, Stanford University School of Medicine, and Aging Study Unit, VA Medical Cent~ Palo Alto, California 94304

90% of bone surfaces are inactive, covered by a thin layer of lining cells. Remodeling is initiated by hormonal or physical signals that cause precursor cells in

INTRODUCTION Recent years have seen considerable progress in our understanding of the normal processes by which peak adult bone mass is acquired and maintained, the physical, nutritional, and h o r m o n a l factors that regulate bone mass t h r o u g h o u t adult life (1-5), and the critical role of reproductive h o r m o n a l replacement for maintaining bone mass within the early years after menopause (6). During this same period, various therapeutic agents have been shown to minimize bone loss and confer protection against osteoporotic fracture (6-9). Almost exclusively, these agents act to reduce the rate of bone resorption. Each shows significant positive effects on bone mass and exerts a substantial influence on fracture risk (9), but none of these agents restores bone mineral density (BMD) of osteoporotic patients to normal levels. Thus, achieving major increases in bone density and strength remains a formidable research challenge. The discussion here concerns two classical peptide hormones, parathyroid h o r m o n e (PTH) and growth h o r m o n e (GH), which have provoked considerable interest as potential osteotropic factors. Reviewed for each h o r m o n e are pertinent animal data as well as the results of studies in humans. Because effective treatments require an understanding of factors that regulate bone mass and the mechanisms by which they operate, the central role of bone remodeling in skeletal maintenance is first summarized. Remodeling is a continuous cycle of destruction and renewal that is carried out by individual, i n d e p e n d e n t "bone remodeling units," illustrated in Fig. 1. Normally, The Parathyroids, Second Edition

FIG. 1 The remodeling cycle. (a) Resting trabecular surface; (b) multinucleated osteoclasts excavate a cavity of approximately 20 i~m; (c) completion of resorption to 60 i~m by mononuclear phagocytes; (d) recruitment of osteoblast precursors to the base of the resorption cavity; (e) secretion of new matrix by osteoblasts; (f) continued secretion of matrix, with initiation of calcification; and (g) completion of mineralization of new matrix. Bone has returned to quiescent state, but a small deficit persists. (With permission, from the Annual Review of Medicine, Volume 38, © 1987, by Annual Reviews, www.Annual Reviews.org.)

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Copyright © 2001 John R Bilezikian, Robert Marcus, and Michael A. Levine.

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the marrow to cluster on the bone surface, where they fuse to become multinucleated osteoclasts and excavate a cavity into the bone. In cortical bone this appears as a resorption tunnel within a Haversian canal. On trabecular surfaces it is a scalloped area called a Howship's lacuna. The resorption front leaves a cavity about 60 Ixm deep. The extent of deepest resorption appears as a thin c e m e n t line, a region of poorly organized collagen fibrils, as opposed to normal bone, which shows lamellar collagen deposition. Coupled to resorption, bone formation ensues when local release of chemical mediators attracts preosteoblasts into the resorption cavity. These cells mature into osteoblasts that replace the missing bone by secreting new collagen and matrix constituents. Matrix production is initially rapid, the new osteoid seam approaching a thickness of 20 Ixm in 30 days. At that point, mineral deposition begins. Mineralization catches up to matrix deposition with time, and the new bone becomes fully mineralized. Resorption and formation are complete within 8-12 weeks, several additional weeks being required to complete mineralization. If the remodeling cycle were completely efficient, bone would be neither lost nor gained. Each remodeling unit would be associated with complete replacem e n t of the packet of bone that was initially lost. However, remodeling, like most biological processes, is not entirely efficient, so that a small bone deficit persists after completion of each cycle. The consequence of this inefficiency is age-related bone loss, a normal, predictable p h e n o m e n o n , that begins shortly after cessation of linear growth. Alterations in remodeling activity represent the final pathway through which diverse stimuli, such as dietary or h o r m o n a l insufficiency, affect the rate of bone loss. Although it is not intuitively obvious, any stimulus that increases the rate of bone remodeling will increase the rate of bone loss. A change in overall bone remodeling rate can reflect distinct perturbations in individual c o m p o n e n t s of the remodeling cycle. These c o m p o n e n t s include a change in the activation, or birthrate, of remodeling units, in the resorptive capacity of individual units, or in the vigor of bone formation. Even a small change in activation rate can exert a greater effect on overall bone loss than on changes in r e m o d e l i n g balance within existing remodeling units. [A detailed discussion of bone remodeling can be found in a review by Ott (10).] Several points directly relevant to osteoporosis therapy can be inferred from this description. Resorption and formation are coupled, so drugs that act by decreasing either the activation of remodeling osteons or the formation of osteoclasts will eventually reduce overall bone formation rate. Furthermore, there is at any given time a transient deficit in bone mass representing bone that was previously resorbed but has not

yet been replaced. This so-called remodeling space (11) constricts if activation slows, and measured bone mass will rise until a new steady-state remodeling space has been achieved. This increase accounts for the relatively modest rise in bone mass that occurs during the first months of antiresorptive therapy. By contrast, agents that activate remodeling will e x p a n d the remodeling space and decrease bone mass. It is important to realize that any agent that stimulates osteoblast proliferation or function may not increase bone mass in the early months of therapy if it simultaneously increases the remodeling space through a c o n c u r r e n t effect on activation of new remodeling units. I m p o r t a n t questions n e e d to be addressed before a therapeutic role can be clarified for drugs that initiate remodeling, including, as will be discussed, (PTH) and (GH). For example, as both h o r m o n e s activate remodeling, the potential need to limit the degree of initial resorption in order to maximize subsequent gains in bone due to stimulation of formation requires consideration. However, current information does not permit us to know whether the bone formation response is contingent on a fully expressed resorptive phase. If so, combination therapy might prove ineffective.

PARATHYROID HORMONE The structural features and biological actions of PTH are reviewed in other chapters of this volume. The devastating skeletal consequences associated with sustained hypersecretion of parathyroid h o r m o n e have been recognized for several decades, and comprehensive reviews of the actions of PTH have been published (12,13) (see also Chapters 20-22). In accord with predictions based on clinical experience, continuous infusion of PTH reliably decreases bone mass in animals, an effect that is due primarily to increased bone resorption as well as to osteoblast suppression. However, a substantial body of evidence provides a rationale for considering PTH an osteotropic agent when administered u n d e r appropriate circumstances. As early as the 1930s, Selye (14) and Shelling et al. (15) induced osteosclerosis in experimental animals following injection of parathyroid extract, a p h e n o m e n o n that was later confirmed in other laboratories (16-18). Walker (16) demonstrated a profound age-dependent effect of daily PTH administration on metaphyseal trabeculae in rats. Initiation of treatment before birth resulted in substantial increases in matrix formation and obliteration of the medullary canal; if PTH was started 8 days postpartum, the increase in matrix was reduced by 50%, and treatment after 60 days of age had no effect. Parsons and Robinson (19) demonstrated that administration of PTH was rapidly followed by a shift of calcium from

TREATMENT OF OSTEOPOROSIS /

plasma into the skeleton and that low doses of PTH induced calcium retention in dogs (20). These results were attributed to an anabolic effect of PTH on bone. Several laboratories have addressed this problem using a synthetic h u m a n PTH, P T H ( l - 3 4 ) , in welldefined animal models. Hermann-Erlee et al. (21) showed that hPTH(1-34) induces anabolic responses in embryonic rabbit bone in vitro. With PTH administration, Tam et al. (22) stimulated bone apposition rate in parathyroidectomized rats. In that study the critical element was intermittent h o r m o n e administration. When PTH was given by continuous infusion, no increase in mineral apposition was observed. Similarly, Canalis et al. (23) reported that continuous exposure of rodent calvariae to PTH inhibited collagen production, whereas episodic exposure stimulated collagen synthesis, an effect apparently mediated by local production of insulin-like growth factor-I (IGF-I) (24). Guiness-Hey and Hock (25) reported that treatment with PTH for 12 days increased trabecular, but not cortical, bone mass of weanling rats. In a second study using a similar protocol, they showed an increase in both trabecular and cortical bone, and, in addition, showed disappearance of this effect after discontinuing h o r m o n e treatment (26). Other laboratories have confirmed the fundamental observation that daily injections of hPTH(1-34) at doses that maintain normal blood calcium concentrations promote bone mass in animals (18,27-29). Ibbotson et al. (30) showed a striking increase in bone mineral content of the distal femur and trabecular bone volume of lumbar vertebrae of rats 250

A

that had been oophorectomized for a year and then treated with daily injections of PTH for 14 days (Fig. 2). To explore more fully the role of intermittent PTH administration, Podbesek et al. (29) administered h P T H ( l - 3 4 ) to mature greyhound dogs either by single daily subcutaneous injection or by continuous infusion. Daily injections of 50 txg of hPTH(1-34) led to peak circulating PTH concentrations of about 4 n g / m l . Elevated values were maintained for no more than 3 hours each day. Continuous infusions were given at a daily dose of 0.5 txg/kg, which maintained blood calcium in the normal range. Daily hPTH (1-34) p r o d u c e d highly significant increases in the rate of bone formation as determined by 47Ca kinetics, in plasma alkaline phosphatase activity, and in several parameters of bone formation as determined directly by histomorphometric analysis of iliac crest bone biopsies. After 4 months of h o r m o n e treatment, trabecular bone volume increased by 30% and osteoid surfaces increased by 18%. Increased resorptive activity was also observed. By contrast, calcium accretion and alkaline phosphatase activities of dogs treated by continuous infusion did not change. In those animals from which adequate paired biopsies were taken, no change in trabecular bone volume was observed, and resorption surfaces appeared to increase substantially. This study as well as an earlier report by Malluche et al. (31) suggest that continuous administration of h P T H ( 1 - 3 4 ) does not increase iliac crest trabecular bone mass. It is apparent that several different actions may contribute to an overall anabolic effect. These include 30

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FIG. 2 Effects of 14 days of treatment of oophorectomized (Ovex) rats on (A) bone mineral content (BMC) of the distal femur, and (B) mineralized bone volume (MBV), expressed as percentage of total bone tissue in lumbar vertebrae. Sham, Intact animals; Ovx con, Ovex rats treated with no active drug; PTH, Ovex animals treated with hPTH(1-34); IGF-I, Ovex rats treated with recombinant human IGF-I; IGF-I + PTH, Ovex rats treated with both hormones, hPTH(1-34) increased both the BMC and MBV of Ovex rats, whereas IGF-I did not increase either variable. (Reproduced from Ref. 30, J Bone Miner Res 1992;7:425-432, with permission of the American Society for Bone and Mineral Research.)

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recruitment of marrow stromal cells to an osteoblastic phenotype, enhancing the biosynthetic capacity of individual osteoblasts, and inhibiting p r o g r a m m e d osteoblastic cell death (32). For approximately the past decade, considerable additional work has defined the skeletal response to PTH in animals. In particular, the oophorectomized (OVX) rat has emerged as a reasonable model for bone loss associated with estrogen deprivation (33-35). Using this model, several laboratories confirmed the ability of PTH both to prevent and to restore OVXassociated bone loss (36-39). In another OVX animal, the cynomolgus monkey, J e r o m e et al. (40) showed that administration of PTH(1-34) thrice weekly for 6 months increased spine BMD and also enhanced bone strength at the femoral neck and spine. An important aspect of this study was clear demonstration of increased trabecular BMD without jeopardizing the cortical skeleton. This body of information has now been supplemented by careful biomechanical analysis. Results with both intact and OVX rats clearly indicate that intermittent PTH treatment leads to a rise in vertebral (41,42) and femoral bone strength (43-45). Once again, in light of concerns regarding cortical bone status of PTHtreated animals, compressive tests on the femoral cortices showed increase bone strength

Clinical Studies Encouraged by results in animals, work in several laboratories has examined the effects of PTH on bone mass in patients with osteoporosis. Early forays into this area were carried out primarily by one group using hPTH(1-34) peptide prepared and standardized for clinical use. More recently, commercial preparations of hPTH(1-34) have been employed, as has a slightly larger peptide, h P T H ( 1 - 3 8 ) . Although clinical studies with these peptides have been ongoing since the 1970s, the results of most should be regarded as suggestive at best, because they suffer from poor generalizability, lack of randomization, poorly standardized participants, and the confounding effects of previous or concurrent therapy. Nevertheless, they remain of interest. Reeve et al. (46) administered synthetic hPTH (1-34) to four postmenopausal women with osteoporosis and monitored calcium balance, iliac crest biopsies, and 47Cakinetics over a period of 18 months. Bone mineral density of the distal femoral shaft was also monitored by single energy p h o t o n absorptiometry, but no measurement of trabecular bone was made. Calcium and phosphorus balance improved in three women, and was attended by a two- to threefold increase in estimates of bone accretion rate by tracer kinetics. Iliac crest bone biopsies showed a doubling of osteoid surfaces with no

change in resorption. Urinary hydroxyproline excretion did not change, confirming that bone resorption was not increased. Calcium absorption increased with little change in urinary or endogenous fecal calcium excretion. Bone mineral density did not change in this experiment. In a multicenter follow-up study, Reeve et al. (47) used hPTH(1-34) to treat 16 women and 5 men with osteoporosis. Approximately 500 U (100 Ixg) were administered per day by subcutaneous injection for 6 months. In this study bone density of the midradius was assessed in 14 subjects, and femoral shaft density was monitored in 8 subjects. Treatment produced no overall changes in plasma calcium concentration. Biochemical markers of bone turnover rose. There was no significant change in calcium balance. Major increases were seen in bone formation rate and in trabecular osteoid surfaces on iliac crest biopsies, and trabecular bone volume increased from 9.3 to 17.3%. By contrast, no significant changes were observed in bone mineral density at either of the monitored cortical sites. The authors concluded that the marked increases in trabecular bone could only be partially attributed to exchange with cortical bone. Furthermore, they suggested that lack of a therapeutic response in cortical bone reflected a failure of PTH to increase calcium absorption and balance, perhaps because the hormonal stimulus was insufficient to activate calcitriol production. Finally, they proposed that PTH (1-34) might best be used in combination with an agent to limit bone resorption. In a study published soon thereafter, Hesp et al. (48) showed significant loss of cortical bone from the femoral shaft in osteoporotic subjects given PTH (1-34) as monotherapy. In a subsequent report, Reeve et al. (49) incorporated an antiresorptive drug into their treatment regimen, hPTH(1-34) was administered for 1 year to 12 patients with vertebral osteoporosis; 9 subjects also received estrogen and 3 were treated with the anabolic steroid, nandrolone. Estrogen was initiated 4 months into the treatment schedule, whereas the timing of nandrolone was variable. Bone mineral status was assessed by both computed tomography (CT) and dual photon absorptiometry (DPA) at entry and yearly for 2 years of protocol. Treatment with the parathyroid peptide markedly increased trabecular bone mass, representing an increase of approximately 50% over base line values. The authors estimated the effect of treatment on the cortical portion of vertebral bone by comparing the DPA results, which include both the trabecular and cortical elements of vertebrae, with those of CT, which is selective for trabecular bone. They concluded that vertebral cortical bone had not changed. Similarly, no increase in bone density of the forearm, a site of predominantly cortical bone, was observed. Supplementary results showed confirmation

TREATMENT OF OSTEOPOROSIS

by bone biopsy of increased trabecular volume and improved calcium balance (50). Although it was encouraging that vertebral mineral increased substantially in the absence of a deleterious effect on cortical bone, nonrandomization of subjects and the lack of separate estrogen or nandrolone control groups limit interpretation of this study. As Reeve and co-workers (49) pointed out, "there is uncertainty whether hPTH (1-34) provided sufficient additional benefit when added to long-term h o r m o n e replacement therapy." They concluded that a randomized, controlled trial is warranted. A slightly different strategy was employed by Slovik et al. (51). These authors had reported previously (52) that elderly osteoporotic women were deficient in their production of calcitriol following a PTH challenge, and therefore they added exogenous calcitriol to the treatment regimen. Accordingly, a combined daily dose of hPTH(1-34) (400-500 U), calcium, and 1,25(OH) 2 vitamin D (calcitriol, 0.25 Ixg/day) was administered for 1 year to eight m e n with idiopathic osteoporosis. Bone mass was assessed at the lumbar spine by CT, and at the radius by single p h o t o n absorptiometry. In all four patients in whom spine mineral was measured, large increases in trabecular bone density were observed, averaging a 198% increase over base line. Four other patients had either completed the protocol before CT measurements became available or could not be evaluated because of vertebral fractures. Cortical bone density did not change. Calcium balance improved by 120 m g / d a y in four patients who were studied, and this was attended by an increase in calcium absorption. The authors proposed that combination therapy with calcitriol is essential to avoid cortical bone loss and to promote calcium retention. Because all subjects received both calcitriol and the PTH peptide, it is possible that some of the change in trabecular bone mass was due to calcitriol alone. This is not likely to be the case, because the modest dose of calcitriol that was used is well below that found to increase bone mass in other studies (8,53). Hesch et al. (54) studied the effects of a slightly longer parathyroid peptide, h P T H ( l - 3 8 ) , in eight osteoporotic patients who received a complex cyclic regimen of 70 daily injections of PTH peptide interspersed with two separate 14-day periods with intranasal salmon calcitonin, 200 U/day. Vertebral mineral density, assessed by CT, increased sharply over the 14-month study, but forearm BMD showed no change. The stated rationale of this protocol was to administer PTH over the functional life span of the osteoblast and to give CT according to the life span of osteoclasts. In the absence of control groups treated with either h P T H ( 1 - 3 8 ) or cyclical calcitonin alone, it is impossible to determine from this report what the contribution of calcitonin may have been to the results, and the rel-

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atively abstract theoretical rationale for this particular protocol cannot be evaluated. Subsequent to those reports, completion of several properly controlled clinical trials now leaves no doubt that PTH peptides achieve substantial BMD gains in postmenopausal women with osteoporosis. These gains are most impressive at the lumbar spine and have been associated with lesser gains, but n o t losses, at cortical sites. Largely out of concern for protecting the cortical skeleton, these controlled trials have employed PTH in combination with an effective antiresorptive agent, usually estrogen. In the most methodologically secure study reported to date, Lindsay et al. (55) conducted a 3-year randomized controlled trial in 34 women who were already taking stable doses of estrogen replacement therapy. Half of the women were randomized to receive a daily injection of 400 U of PTH (1-34), and the other half continued to receive only estrogen. Participants receiving both estrogen and PTH(1-34) achieved significant gains in vertebral BMD of 13% over 3 years and also achieved significant rises in hip BMD of 2.7% and in whole-body bone mineral content (BMC) of 8.0% (Fig. 3). This study was u n d e r p o w e r e d for detecting a PTH effect on vertebral fracture, but when a liberal criterion for fracture was employed (15% reduction in vertebral height), a significant decrease was observed. A cyclic strategy was employed by H o d s m a n et al. (56), who gave PTH(1-38), 800 U daily for 28 days, followed by salmon calcitonin (or its placebo) for another 42 days, and repeated every 3 months for 2 years. With this regimen, an increase in average lumbar spine

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FIG. 3 Lumbar spine bone mineral density response to PTH of estrogen-treated postmenopausal women. Reproduced from Ref. 55; R Lindsay, J Nieves, C Formica, E Henneman, L Woelfert, V Shen et aL Randomized controlled study of effect of parathyroid hormone on vertebralbone mass and fracture incidence among postmenopausal women on oestrogen with osteoporosis. Lancet, Vol. 350, pp. 550-555. © by The Lancet Ltd. 1997.

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BMD of 10.2% was observed with PTH alone and of 7.0% in the group that also received calcitonin. Fujita et al. (57) administered hPTH(1-34) to 220 patients as a single subcutaneous injection each week for 48 weeks. Three doses were employed, 50, 100, or 200 U. Lumbar spine BMD increased in a dose-responsive fashion, by 0.6, 3.6, and 8.1%, respectively. By contrast, metacarpal BMD and radiogrammetric cortical thickness did not change at any dose of PTH. As of this writing, confirmatory results have also been obtained in other studies (which have not yet appeared as full manuscripts) (58,59). In all of these, PTH has been well tolerated with few adverse experiences. In particular, hypercalcemia and hypercalciuria have not emerged as important problems. In addition to its potential efficacy in postmenopausal osteoporosis, PTH has been shown to ameliorate bone loss due to estrogen deprivation from gonadotropin-releasing h o r m o n e (GnRH) treatment and has shown dramatic results in restoring bone to patients with glucocorticoid-associated osteoporosis. Finkelstein and colleagues reported two trials of PTH(1-34) in the treatment of women with endometriosis as they initiated treatment with a GnRH analog. The first trial showed that women who were r e n d e r e d hypogonadal by the GnRH analog sustained a 2.8% loss of lumbar spine BMD by 6 months, but that simultaneous treatment with PTH(1-34), 500 U/day, prevented this loss. BMD loss from the hips was modest and similar in both groups (60). In a follow-up yearlong study (61), GnRH analog alone produced a 4.9% decrease in lumbar spine BMD, and losses at the hip were about 4%. Coadministration of PTH(1-34) increased spine BMD by 2.1% and prevented loss from the hip. Corticosteroid-induced osteoporosis remains a comm o n and vexing cause of secondary osteoporosis. Remodeling abnormalities secondary to corticosteroids include a variable rise in bone resorption accompanied by sometimes profound suppression of osteoblastic bone formation. Thus, if any clinical entity seems ripe for an anabolic agent, it is corticosteroid osteoporosis. Lane et al. (62) conducted a controlled trial of PTH (1-34) in older osteoporotic women who were taking corticosteroids as well as estrogen replacement therapy. Therapeutic response was assessed by two modalities: dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT). Women treated with PTH experienced lumbar spine mineral increases of 35% (DXA) and 11% (QCT), respectively, whereas those treated with estrogen only experienced corresponding changes of 1.7 and 0%. Intergroup differences at the spine were highly significant, whereas changes at the hip and forearm did not differ between or within groups. The authors noted that early in the course of PTH treatment markers of

bone formation rose nearly 150%, whereas markers of bone resorption increased only 100%. They interpreted this result to suggest an early uncoupling of bone turnover in favor of formation. This study permits reasonable optimism that PTH may offer dramatic benefit for patients who have suffered corticosteroid-induced bone loss. In a report by Kurland et al. (63), an 18-month placebo-controlled clinical trial of 23 men with idiopathic osteoporosis randomized to receive PTH(1-34), 400 U / d a y or placebo showed that PTH treatment produced a marked 13.% improvement in lumbar spine BMD, accompanied by a smaller, but significant 2.9% increase at the femoral neck. In that study, careful attention to bone turnover markers and indices of calcium homeostasis were closely monitored, and the authors observed no significant changes in serum or urinary calcium or in circulating calcitriol concentrations. All markers of bone turnover increased in patients receiving PTH, and the combined use of total pyridinolines at base line plus circulating osteocalcin at 3 months was a robust predictor of BMD response to PTH. The ultimate test for PTH as a skeletal anabolic therapy will be whether it reduces the incidence of fracture. Although none of the studies reported above had adequate statistical power to address this issue, a recently completed clinical trial offers substantial evidence in this regard. As of this writing, a formal manuscript has not been published, but the results have been presented in abstract form (64). In this randomized controlled trial, 1637 postmenopausal women with one or more prevalent vertebral fractures at base line were treated with calcium and vitamin D plus 20 or 40 ~ g / d a y of PTH(1-34) or placebo. After an average of 21 months follow-up, vertebral BMD increased by 8.6 and 12.6% at the 20- and 40-1~g dose, and femoral neck BMD increased by 3.6 and 4.6%. At the same time, the incidence of new vertebral fractures decreased by 65 and 69%, and the incidence of nontraumatic nonvertebral fractures decreased by 53 and 54%, in the 20- and 40-b~g groups, respectively (64). With further confirmation of this impressive outcome, it should not be very long before PTH will be available to physicians as the first approved anabolic treatment for osteoporosis.

GROWTH HORMONE Pituitary growth h o r m o n e is a classic peptide horm o n e with profound effects on somatic growth and body composition. Circulating concentrations of GH decline with advancing age, as do GH secretion rates and pituitary GH responsiveness to a variety of provocative stimuli (65-68). These declines are accompanied by reduced levels of insulin-like growth factors (IGFs),

TREATMENT OF OSTEOPOROSIS / the putative mediators of many of the actions of GH. Rudman (69) called attention to the fact that changes in body composition associated with normal h u m a n aging also characterize patients with GH deficiency. These changes include increased adiposity, reduced muscle mass and strength, and loss of bone mineral. Thus it seems reasonable to ask whether some agerelated changes in body composition are directly related to relative GH deficiency, and, further, whether GH therapy might have clinical utility in reversing them. The major actions of GH on skeletal growth in children are well described and will not be reviewed here. Less well defined are the skeletal actions of GH in adults. Although traditional models of GH action were predicated on an intermediary role for circulating IGFs, recent evidence indicates that GH directly stimulates IGF production in osteoblasts by activating cell surface receptors in cultured osteoblast-like cells to increase IGF-I production (70-72). In murine osteoblasts, GH induces expression of the oncogenes c-jun and c-fos by a mechanism that involves activation of the protein kinase C transduction complex (73). In osteoblasts raised in serum-free medium, GH stimulates proliferation and increases the relative a m o u n t of newly synthesized type I collagen, an effect that is obliterated by anti-IGF-I immunoglobulin (74). There appears to be redundancy in the control of IGF-I production in osteoblasts, because PTH and estradiol also promote accumulation of this somatomedin (23). In vivo, GH and IGF-I both increase bone turnover. In a classic experiment, Harris and Heaney (75) showed that administration of GH to adult dogs increased bone mass. More recently, recombinant h u m a n GH has been shown to maintain trabecular bone mass in hypogonadal primates (76). Such in vivo demonstrations, combined with an increasing volume of in vitro evidence, invite the conclusion that GH or IGF-I might have the potential to activate osteoblast proliferation and function to repair bone mineral deficits in osteoporosis.

Clinical Studies Until the 1980s, GH treatment of other than GHdeficient children was restricted by h o r m o n e supply. Limited experience with GH derived from h u m a n pituitaries gave interesting results suggesting a possibly useful role for this h o r m o n e in humans (77,78). Aloia et al. (78) described a 2-year intervention trial comparing the effect of GH followed by calcitonin (CT) to that of CT alone in 14 osteoporotic women. The combination of GH plus CT increased whole-body bone mineral by 2.3% per year, whereas CT alone p r o d u c e d no change. The effect of GH in this limited study seemed to be progressive, that is, without the plateau that would have

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occurred if treatment merely condensed the remodeling space. Unfortunately, the authors did not report blood levels of IGF-I, so it was not possible to j u d g e dose adequacy. Finally, concerns about the relationship between pituitary-derived GH and the development of Jakob-Creutzfeld disease led to cessation of clinical trials with this agent (79). With the availability of recombinant h u m a n GH (rhGH), therapy of adults became feasible. Marcus et al. (80) reported the effects of 7 days of rhGH administration to 16 healthy older men and women, rhGH produced a brisk rise in circulating IGF-I associated with striking increases in nitrogen and sodium retention and alterations in the parathyroid-vitamin D axis. Assayed 24-hour urinary nitrogen and sodium excretion decreased by 38 and 50%, respectively, whereas urine calcium excretion markedly increased. Thus rhGH uncoupled the usual tight relationship between urinary sodium and calcium excretion. Significant increases were observed in circulating osteocalcin and in urinary hydroxyproline, suggesting that bone remodeling had been activated. In this regard, Brixen et al. (81) also showed that several daily injections of rhGH in young m e n initiated a p r o m p t and very sustained elevation in circulating concentrations of osteocalcin. The most widely publicized study of GH was reported by R u d m a n et al. (82). In a randomized, placebo-controlled intervention trial in 21 elderly men, rhGH (0.03 m g / k g , three times per week for 6 months) p r o d u c e d significant increases in lean mass by 4°K analysis and an increase of 1.6% in lumbar spine bone mineral density. In addition, the authors stated that "the effects of 6 months of hGH on lean body mass and skin thickness were equivalent in magnitude to the changes incurred during 10 to 20 years of aging." This statement was extrapolated subsequently by the nonmedical community, especially the news media, to suggest that GH reverses aging. Although the results of this experiment were provocative, several concerns must be raised. The 4°K data provide convincing evidence of a true increase in lean mass. However, the stated changes in skin-fold thickness were undoubtedly c o n f o u n d e d by fluid retention. Further, the changes in BMD were marginally significant at best. Papadakis et al. (83) conducted another 6-month trial of rhGH in older men, observing a 0.9% average rise in lumbar spine BMD. Thus, these two studies provide little evidence for a skeletally anabolic effect of GH, although one must keep in mind that the BMD response was limited by the short duration of treatment. Holloway et al. (84) reported a randomized, placebocontrolled 1-year intervention trial of rhGH (0.025 m g / k g / d a y ) in 23 healthy elderly women. Compared to the placebo group, circulating IGF-I values in the

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active treatment group remained elevated t h r o u g h o u t the trial, rhGH stimulated a persistent increase in bone turnover. Sustained elevations were observed in circulating osteocalcin, type I procollagen peptide, and bone alkaline phosphatase, as well as in urinary excretion of hydroxyproline. These changes reverted to base line values by 3 months after stopping rhGH treatment. Despite clear changes in markers of bone metabolism, no significant changes were observed in bone mineral density at either the lumbar spine or the proximal femur. However, it is of interest that BMD at the femoral trochanter and Ward's triangle decreased significantly in the placebo group. Thus, although rhGH did not increase BMD, it may have permitted skeletal maintenance at the hip. No changes in lean body mass or adiposity were observed by hydrostatic weighing, although an increase in lean mass was suggested by skin-fold thickness. Rosen and colleagues (C.J. Rosen, personal communication) conducted a 12-month interim analysis of a 2-year GH trial in frail elders. Using a lower GH dose than had been given in any published trials, the results gave no evidence that anabolic effects on bone had been achieved. In fact, BMD was significantly lower than base line in treated subjects. This result probably indicates an expanded remodeling space. Finally, Holloway et al. (85) reported the results of a clinical trial in postmenopausal women, in which 2-month cycles of 7 days of rhGH, followed by 5 days of salmon calcitonin, were maintained for 2 years, with follow-up bone density assessment at 3 years. Groups receiving GH plus calcitonin or GH plus placebo significantly increased lumbar spine BMD at 2 years by 2.7 and 1.7%, respectively (Fig. 4). Significant increases in total hip BMD measurements of 1-2% were observed for the GH groups, but no significant change in femoral neck BMD was observed. Women taking replacement estrogen had the same BMD response as those who were estrogen deficient. The osteotropic actions of GH suggest a rationale for considering this h o r m o n e to increase bone mass in patients with osteopenia. Unfortunately, published experience permits the conclusion that rhGH, given either as daily m o n o t h e r a p y or in combination with antiresorptive medication, does not offer clinically meaningful improvement in skeletal status of older men and women. Although a small rise in lumbar BMD (85) or maintenance of BMD at the hip may have occurred (84), several antiresorptive agents currently offer similar protection, and it would be hard to justify the use of an expensive, injectable protein h o r m o n e to achieve a similar result. Because the doses employed are close to maximally tolerated levels, it is unlikely that an upward adjustment of dose will make the therapy more attractive.

*'1"

c

•~ 2 IZl

E 2

1

"

0

,,c

i

O -1

GH + CT

GH + placebo

placebo +CT

placebo + placebo

FIG. 4 Effect of cyclic growth hormone (GH) and calcitonin (CT) on lumbar spine BMD. Subjects received bimonthly cycles of GH + CT, GH + CT placebo, GH placebo + CT, or double placebo for 12 (solid boxes) or 24 (shaded boxes) months. Results are given as percent changes from base line (means - SEM). *p < 0.05, **p < 0.01 vs. base line; 1/:1: indicate significantly different from placebo/placebo group (reproduced, with permission, from Ref. 83).

REFERENCES 1. Dequeker J, Nijs J, Verstraeten A., Geusens P, Gevers G. Genetic determinants of bone mineral content at the spine and radius: A twin study. Bone 1987;8:207-209. 2. Seeman E, HopperJL, Bach LA, Cooper ME, Parkinson E, McKay J, Jerens G. Reduced bone mass in daughters of women with osteoporosis. N EnglJ Med 1988;320:554-558. 3. Marcus R, Carter DR. The role of physical activity in bone mass regulation. Adv Sports Med Fitness 1988; 1:63-82. 4. Marcus R. Calcium intake and skeletal integrity: Is there a critical relationship? J Nutr 1987; 117:631-635. 5. Longcope C, Baker RS, Hui SL, Johnston CC, Jr. Androgen and estrogen dynamics in women with vertebral crush fractures. Maturitas 1985;6:309-318. 6. Lindsay R, Hart DM, Forrest C, Baird C. Prevention of spinal osteoporosis in oophorectomised women. Lancet 1980;2:1151-1154. 7. Black D, Cummings SR, Karpf DB, Cauley JA, Thompson DE, Nevitt MC, Genant HK, Haskell WL, Marcus R, Ott SM, Torner JC, Quandt SA, Reiss TE Ensrud KE, for the Fracture Intervention Trial Research Group. Alendronate reduces the risk of fractures in women with existing vertebral fractures: Results of the Fracture Intervention Trial. Lancet 1996;348:1535-1541. 8. Chapuy MC, Arlot ME, Duboeuf E Brun J, Crouzet B, Arnaud S, Delmas PD, Meunier PJ. Vitamin D:~ and calcium to prevent hip fractures in elderly women. N EnglJ Med 1992;327:1637-1642. 9. Ettinger B, Black DM, Mitlak BH, Knickerbocker RK, Nickelsen T, Genant HK, Christiansen C, Delmas PD, Zanchetta JR, Stakkestad J, Glfier CC, Krueger K, Cohen FJ, Eckert S, Ensrud KE, Avioli LV, Lips P, Cummings SR. for the Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene. Results from a 3-year randomized clinical trial. JAMA 1999;282:637-645.

TREATMENT OF OSTEOPOROSIS 10. Ott SM. Bone remodeling: Theoretical and methodological approach. In: Bilezikian JP, Raisz LG, Rodan GA, eds. Principles of bone biology. San Diego:Academic Press, 1996:231-241. 11. Jaworski ZFG. Parameters and indices of bone resorption. In: Meunier PJ, ed. Bone histomorphometry, 2nd Int. Workshop, Paris: Armour Montague, 1976. 12. Parfitt AM. The actions of parathyroid h o r m o n e on bone: Relation to bone remodeling and turnover, calcium homeostasis and metabolic bone disease. Part III of IV Parts: PTH and osteoblasts, the relationship between bone turnover and bone loss, and the state of the bones in primary hyperparathyroidism. Metabolism 1976;25:1033-1069. 13. Fitzpatrick LA, Bilezikian JE Actions of parathyroid hormone. In: Belizikian JP, Raisz LG, Rodan GA, eds. Principles of bone biology. San Diego:Academic Press 1996:339-346. 14. Selye H. On the stimulation of new bone formation with parathyroid extract and irradiated ergosterol. Endocrinology 1932;16: 547-558. 15. Shelling DH, Asher DE, Jackson DA. Calcium and phosphorus studies. VII The effects of variations in dosage of parathormone and of calcium and phosphorus in the diet on the concentrations of calcium and inorganic phosphorus in the serum and on the histology and chemical composition of the bones of rats. Bull Johns Hopkins Hosp 1933;53:348-389. 16. Walker DG. The induction of osteopetrotic changes in hypophysectomized thyroparathyroidectomized and intact rats of various ages. Endocrinology 1971;89:1389-1406. 17. McGuire JL, Marks SL. The effects of parathyroid h o r m o n e on bone cell structure and function. Clin Orthop 1974;100: 392-405. 18. Kalu DN, PennockJ, Doyle FH, Foster GV. Parathyroid h o r m o n e and experimental osteosclerosis. Lancet 1970; 1:1363-1366. 19. Parsons JA, Robinson CJ. Calcium shift into bone causing transient hypocalcemia after injection of parathyroid hormone. Nature 1971;230:581-582. 20. Parsons JA, Reit B. Chronic response of dogs to parathyroid hormone infusion. Nature 1974;250:254-257. 21. Hermann-Erlee MPM, Heersche JNM, Hekkelman JW, Gaillard PJ, Treagear GW, Parsons JA, Potts JT, Jr. Effects on bone in vitro of bovine parathyroid h o r m o n e and synthetic fragments representing residues 1-34, 2-34, and 3-34. Endocrinol Res Commun 1976;3:21. 22. Tam CS, HeerscheJNM, Murray TM, ParsonsJA. Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: Differential effects of intermittent and continuous administration. Endocrinology 1982;110:506-512. 23. Canalis E, McCarthy TL, Centrella M. Differential effects of continuous and transient treatment with parathyroid hormone related peptide (PTHrP) on bone collagen synthesis. Endocrinology 1990; 126:1806-1812. 24. Canaliis E, Centrell M, Burch W, McCarthy T. Insulin-like growth factor I mediates selective anabolic effects of parathyroid hormone in bone cultures. J Clin Invest 1989;83:60-65. 25. Guiness-Hey M, Hock JM. Increased trabecular bone mass in rats treated with h u m a n synthetic parathyroid hormone. Metab Bone Dis Relat Res 1984;5:177-182. 26. Guiness-Hey M, Hock JM. Loss of the anabolic effect of parathyroid h o r m o n e on bone after discontinuation of h o r m o n e in rats. Bone 1989;10:447-452. 27. Hock JM, Gera I, Fonseca J, Raisz LG. H u m a n parathyroid hormone (1-34) increases bone mass in ovariectomized and orchidectomized rats. Endocrinology 1988; 122:2899-2904. 28. Hefti E, Trechsel U, Bonjour JP, Fleisch H, Schenk R. Increase of whole body calcium and skeletal mass in normal and osteoporotic adult rats treated with parathyroid hormone. Clin Sci 1982;62:389-392.

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29. Podbesek R, Edouard C, Meunier PJ, Parsons JA, Reeve J, Stevenson RW, ZanelliJM. Effects of two treatment regimens with synthetic h u m a n parathyroid h o r m o n e fragments on bone formation and the tissue balance of trabecular bone in greyhounds. Endocrinology 1983;112:1000-1006. 30. Ibbotson KJ, Orcutt CM, D'Sousa SM, Paddock CL, Arthur JA, Jankowsky ML, Boyce RW. Contrasting effects of parathyroid hormone and insulin-like growth factor I in an aged ovariectomized rat model of postmenopausal osteoporosis. J Bone Miner Res 1992;7:425-432. 31. Malluche HH, Sherman D, Meyer W, Ritz E, Norman AW, Massry SG. Effects of long-term infusion of physiological doses of 1-34 PTH on bone. AmJPhysio11982;242:197. 32. Jilka RL, S WR, Bellido T, Robertson P, Parfitt AM, Manolagas SC. Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 1999;104:439-446. 33. Wronski TJ, Dann LM, Scott KS, Cintron M. Long-term effects of ovariectomy and aging on the rat skeleton. Calcif Tissue Int 1989;45:360-366. 34. Wronski TJ, Dann LM, H o r n e r SL. Time course of vertebral osteopenia in ovariectomized rats. Bone 1989;10:295-301. 35. Wronski TJ, Yen C-F, Qi H, Dann LM. Parathyroid hormone is more effective than estrogen or bisphosphonates for restoration of lost bone mass in ovariectomized rats. Endocrinology 1993;132:823-831. 36. Hori M, Uzawa T, Morita K, Noda T, Takahashi H, Inoue J. Effect of h u m a n parathyroid h o r m o n e (PTH ( [ 1-34] ) on experimental osteopenia of rats induced by ovariectomy. Bone Miner 1988;3: 193-199. 37. Tada K, Yamamuro TH, Okamura R, Takahashi HE. Restoration of axial and appendicular bone volumes by hPTH(1-34) in parathyroidectomized and osteopenic rats. Bone 1990;11:163-169. 38. Liu CC, Kalu DN. H u m a n parathyroid h o r m o n e (1-34) prevents bone loss and augments bone formation in sexually mature ovariectomized rats. J Bone Miner Res 1990;5:973-982. 39. Liu CC, Kalu DN, Salerno E, Echon R, Hollis BW, Ray M. Preexisting bone loss associated with ovariectomy in rats is reversed by parathyroid hormone. JBone Miner Res 1991;6:1071-1080. 40. Jerome CO, Johnson CS, Vafai HT, et al. Effect of treatment for 6 months with h u m a n parathyroid h o r m o n e (1-34) peptide in ovariectomized cynomolgus monkeys ( Macaca fascicularis) . Bone 1999;25:301-309. 41. Mosekilde Li, Sogaard CH, Danielsen CC, Torring O, Nilsson MHL. The anabolic effect of human parathyroid hormone (hPTH) on rat vertebral body mass are also reflected in the quality of bone assessed by biomechanical testing: A comparison study between hPTH ( 1-34 ) and hPTH (1-84). Endocrinology 1991 ;129:421-428. 42. Mosekilde Li, Sogaard CH, McOsker JE, Wronski TJ. PTH has a more p r o n o u n c e d effect on vertebral bone mass and biomechanical competence than antiresorptive agents (estrogen and bisphosphonate) assessed in sexually mature, ovariectomized rats. Bone 1994;15:401-408. 43. Sogaard CH, Wronski TJ, McOskerJE, Mosekilde LI. The positive effect of parathyroid h o r m o n e on femoral neck bone strength in ovariectomized rats is more p r o n o u n c e d than that of estrogen or bisphosphonates. Endocrinology 1994;134:650-657. 44. Toromanoff A, A m m a n n P, Riond J-L. Early effects of short-term parathyroid h o r m o n e administration on bone mass, mineral content and strength in female rats. Bone 1998;22:217-223. 45. Mosekilde LI, Danielsen CC, Sogaard CH, McOsker JE, Wronski TJ. The anabolic effects of parathyroid h o r m o n e on cortical bone mass, dimensions and strength assessed in a sexually mature ovariectomized rat model. Bone 1995;16:223-230. 46. Reeve J, Williams D, Hesp R, Hulme P, Klenerman L, Zanelli J, Darby AJ, Tregear GW, Parsons JA. Anabolic effect of low doses of a fragment of h u m a n parathyroid h o r m o n e on the skeleton in postmenopausal osteoporosis. Lancet 1976;1:1035-1038.

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47. Reeve J, Meunier PJ, Parsons JA, Bernat M, Bijvoet OLM, Courpron P, Edouard C, Klenerman L, Neer RM, Renier JC, Slovik D, Visman FJ, Potts JT, Jr. Anabolic effect of human parathyroid hormone fragment (hPTH 1-34) therapy on trabecular bone in involutional osteoporosis: Report of a multi-centre trial. Br Med J 1980;280:1340-1344. 48. Hesp R, Hulme P, Williams D, Reeve J. The relationship between changes in femoral bone density and calcium balance in patients with involutional osteoporosis treated with human parathyroid hormone fragment (hPTH 1-34). Metab Bone Dis Relat Res 1981;2: 331-334. 49. Reeve J, Davies UM, Hesp R, McNally E, Katz D. Treatment of osteoporosis with human parathyroid peptide and observations on effect of sodium fluoride. Br M e d J 1990;301:314-318. 50. Reeve J, Bradbeer JN, Arlot M, Daview UM, Green JR, Hampton L, Edouard C, Hesp R, Hulme P, Ashby JP, Zanelli JM, Meunier PJ. hPTH 1-34 treatment of osteoporosis with added hormone replacement therapy: Biochemical, kinetic and histological responses. Osteoporosis Int 1991;1:162-170. 51. Slovik DM, Rosenthal DI, Doppelt SH, Potts JT, Jr. Daly MA, Campbell JA, Neer RA. Restoration of spinal bone in osteoporotic men by treatment with human parathyroid hormone (1-34) and 1,25-dihydroxyvitamin D. J Bone Miner Res 1986;1:377-381. 52. Slovik DM, Adams JS, Neer RM, Holick ME Potts JT, Jr. Deficient production of 1,25-dihydroxyvitamin D in elderly osteoporotic patients. N EnglJ Med 1981 ;305:372-374. 53. Ott SM, Chesnut III CH. Calcitriol treatment is not effective in postmenopausal osteoporosis. Ann Intern Med 1989;110:267-274. 54. Hesch R-D, Busch U, Prokop M, Delling G, Rittinghaus E-E Increase of vertebral density by combination therapy with pulsatile 1-38hPTH and sequential addition of calcitonin nasal spray in osteoporotic patients. Calcif Tissue Int 1989;44:176-180. 55. Lindsay R, Nieves J, Formica C, Henneman E, Woelfert L, Shen V, et al. Randomized controlled study of effect of parathyroid hormone on vertebral-bone mass and fracture incidence among postmenopausal women on oestrogen with osteoporosis. Lancet 1997;350:550-555. 56. Hodsman AB, Fraher LJ, Watson PH, Ostbye T, Stitt LW, Adachi JD, Taves DH, Drost D. A randomized controlled trial to compare the efficacy of cyclical parathyroid hormone versus cyclic parathyroid hormone and sequential calcitonin to improve bone mass in postmenopausal women with osteoporosis. J Clin Endocrinol Metab 1997;82:620-628. 57. Fujita T, Inoue T, Morii HM, Morita R, Norimatsu H, Orimo H, Takahashi HE, Yamamoto K, Fukunaga M. Effect of an intermittent weekly dose of human parathyroid hormone (1-34) on osteoporosis: A randomized double masked prospective study using three dose levels. Osteoporosis Int 1999;9:296-306. 58. Roe EB, Sanchez SD, del Puerto GA, et al. Parathyroid hormone (1-34) and estrogen produce dramatic bone density increases in postmenopausal osteoporosismresults from a placebo-controlled randomized trial. J Bone Miner Res 1999;14:S137 (abstract). 59. Reeve J, Mitchell A, Tellez M, et al. Treatment with parathyroid peptides and oestrogen replacement for severe postmenopausal vertebral osteoporosis: Long-term effects on spine and femur and determinants of magnitude of response. J Bone Miner Res 2000; in press. 60. Finkelstein JS, Klibanski A, Schaefer EH, et al, Parathyroid hormone for the prevention of bone loss induced by estrogen deficiency. N E n g l J M e d 1994;331:1618-1623. 61. FinkelsteinJS, Klibanski A, Arnold AL, et al. Prevention of estrogen deficiency-related bone loss with human parathyroid hormone(1-34). A randomized controlled trial. JAMA 1998;280:1067-1073. 62. Lane NE, Sanchez S, Modin GW, Genant HK, Pierini E, Arnaud CD. Parathyroid hormone treatment can reverse corticosteroidinduced osteoporosis: Results of a randomized controlled clinical trial. J Clin Invest 1998;102:1627-1633.

63. Kurland ES, Cosman E McMahon DJ, Rosen CJ, Lindsay R, Bilezikian JE Parathyroid hormone as a therapy for idiopathic osteoporosis in men: Effects on bone mineral density and bone markers. Lancet 2000;in press. 64. Marcus R, Gaich G, Satterwhite J, Myers S, Wang O, Mitlak B. Effects of baseline BMD, age, and prevalent vertebral fractures on the response of osteoporotic patients to LY333334 [rhPTH(1-34)]. Presented at the American Society for Bone & Mineral Research Annual Meeting, Toronto, September 2000. 65. Rudman D, Kutner MH, Rogers M, Lubin ME Fleming GA, Baine RP. Impaired growth hormone secretion in the adult population. J Clin Invest 1981;67:1361-1369. 66. Ho KY, Evans WS, Blizzard RM, Veldhuis JD, Merriam GR, Samojlik E, Furlanetto R, Rogol AD, Kaiser DL, Thorner MO. Effects of sex and age on the 24-hour profile of growth hormone secretion in man: Importance of endogenous concentrations. J Clin Endocrinol Metab 1987;64:51-58. 67. Franchimont P, Urbain-Choffray D, Lambelin P, Fontaine M-A, Frangin G, Reginster J-Y Effects of repetitive administration of growth hormone-releasing hormone on growth hormone secretion, insulin-like growth factor I, and bone metabolism in postmenopausal women. Acta Endocrinol 1989;120: 121-128. 68. Pyka G, Wiswell RA, Marcus R. Age-dependent effect of resistance exercise on growth hormone secretion in people. J Clin Endocrinol Metab 1992;75:404-407. 69. Rudman D. Growth hormone, body composition, and aging. J A m Geriatr Soc 1985;33:800-807. 70. Stracke H, Schultz A, Moeller D, Rossol S, Schatz H. Effect of growth hormone on osteoblasts and demonstration of somatomedin C/IGF-1 in bone organ culture. Acta Endocrinol 1984;107:16-24. 71. Chenu C, Valentin-Opran A, Chavassieux P, Saez S, Meunier PJ, Delmas PD. Insulin like growth factor I hormonal regulation by growth hormone and by 1,25(OH)zD~ and activity on human osteoblast-like cells in short-term cultures. Bone 1990;11: 81-86. 72. Barnard R, Ng KW, Martin TJ, Waters MJ. Growth hormone (GH) receptors in clonal osteoblast-like cells mediate a mitogenic response to GH. Endocrinology 1991;128:1459-1464. 73. Slootweg MC, de Groot RP, Herrmann-Erlee MPM, Koornneef I, Kruijer W, Kramer YM. Growth hormone induces expression of c-jun andjun B oncogenes and employs a protein kinase C signal transduction pathway for the induction of c-fos oncogene expression. J Mol Endocrinol 1991;6:179-188. 74. Ernst M, Froesch ER. Growth hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I. Biochem Biophys Res Commun 1988;151: 142-147. 75. Harris WH, Heaney RP. Effect of growth hormone on skeletal mass in adult dogs. Nature 1969;273:403-404. 76. Mann DR, Rudman CG, Akinbami MA, Gould KG. Preservation of bone mass in hypogonadal female monkeys with recombinant human growth hormone administration. J Clin Endocrinol Metab 1992; 74:1263-1269. 77. Aloia JE Zanzi I, Ellis K, Jowsey J. Effects of growth hormone in osteoporosis. J Clin Endocrinol Metab 1976;43:992-999. 78. Aloia JE Vaswani A, Kapoor A, Yeh JK, Cohn SH. Treatment of osteoporosis with calcitonin, with and without growth hormone. Metabolism 1985;34:124-129. 79. Brown P, Gajdusek DC, Gibbs CJ, Jr, Asher DM. Potential epidemic of Creutzfeld-Jakob disease from human growth hormone therapy. N EnglJ Med 1985;313:728-731. 80. Marcus R, Butterfield G, Holloway L, Gilliland L, Baylink DJ, Hintz RL, Sherman BL. Effects of short-term administration of recombinant human growth hormone to elderly people. J Clin Endocrinol Metab 1990;70:519-527.

TREATMENT OF OSTEOPOROSIS 81. Brixen K, Nielsen HK, Mosekilde L, Flyvbjerg A. A short course of recombinant human growth hormone treatment stimulates osteoblasts and activates bone remodeling in normal human volunteers. J Bone Miner Res 1990;5:609-618. 82. Rudman D, Feller AG, Nagraj HS, Gergans GA, Lalitha PY, Goldberg AF, Schlenker RA, Cohn L, Rudman IW, Mattson DE. Effects of human growth hormone in men over 60 years old. N EnglJ Med 1990;323:1-6. 83. Papadakis MA, Grady D, Black D, et al. Growth hormone replacement in healthy older men improves body composi-

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tion but not functional ability. Ann Intern Med 1996;124: 708-716. 84. Holloway L, Butterfield G, Hintz RL, et al. Effect of recombinant human growth hormone on metabolic indices, body composition, and bone turnover in healthy elderly women. J Clin Endocrinol Metab 1994;79:470-479. 85. Holloway L, Kohlmeier L, Kent K, Marcus R Skeletal effects of cyclic recombinant human growth hormone and salmon calcitonin in osteopenic postmenopausal women. J Clin Endocrinol Metab 1997;82:1111-1117.

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Index

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Index A

Acute primary hyperparathyroidism animal models, 531 demographics, 527-528 development from mild primary hyperparathyroidism, 395, 527 differential diagnosis, 529 incidence, 527 laboratory evaluation, 528-529, 532 management, 467-468 outcome, 529-530 pathology, 530 pathophysiology, 530 pregnancy association, 530-531 renal involvement, 529 skeletal involvement, 529 thyrotoxicosis association, 531 treatment, 531-532 Acute renal falure, hypercalcemia differential diagnosis, 666 Addison's disease, autoimmune hypoparathyroidism association, 794 treatment, 800 Adenoma, see Parathyroid adenoma Adenylate cycle, magnesium deficiency effects, 772 Adrenal medulla, derivation and differentiation, 594 Adrenocortical insufficiency, hypercalcemia differential diagnosis, 666 Adrenocortical neoplasm, multiple endocrine neoplasia type 1,543, 553, 561 Aging calcium, aging effects on metabolism, 839 estrogen deficiency and calcium metabolism, 839-840, 843, 848 growth hormone secretion changes, 858-859 parathyroid hormone secretion changes, 835-837 skeletal sensitivity, 844, 845-846 secondary hyperparathyroidism etiology, 839-840 vitamin D deficiency, 839, 843 A I R E - 1 , mutation in autoimmune hypoparathyroidism, 799-801 Albright's hereditary osteodystrophy, see Pseudohyperparathyroidism Alendronate, hyperparathyroidism management, 465 Alfacalcidol, secondary hyperparathyroidism treatment, 648 Alkaline phosphatase bone formation marker and hyperparathyroidism, 400-401,404 renal bone disease, serum levels, 640-641 Aluminum absorption from phosphate-binding agents, 645-647 bone toxicity aluminum levels in plasma, 643 hyperparathyroidism differential diagnosis, 642-643 parathyroid hormone levels, 643 parathyroidectomy effects, 642-643

parathyroid toxicity, 631-632 Angiofibroma, multiple endocrine neoplasia type 1,543, 553 Athyreotic cretinism, hypercalcemia association in children, 748 Autoimmune hypoparathyroidism AIRE-1 gene mutations, 799-801 classification of polyglandular autoimmune disease, 792-793 clinical features Addison's disease, 794 atrophic problems, 793-794 candidiasis, 794 corneal abnormalities, 796 dental abnormalities, 796 diagnostic criteria, 793 endocrine disorders, 794-795 hepatitis progression, 796-797 idiopathic hypoparathyroidism, 11, 757-758, 791 onset of symptoms, 793 pernicious anemia, 796 skin manifestations, 796 heredity, 793 historical perspective of research, 791-793 idiopathic hypoparathyroidism with complex syndromes, 793 nomenclature, 791-793 overview, 11,757-758, 785, 791 pathogenesis autoimmune response, 797-799 Candida role, 797-799 cell-mediated immunity abnormalities, 798-799 treatment Addison's disease, 800 candidiasis, 800-801 hypoparathyroidism, 800 pernicious anemia, 801 skin manifestations, 801 B

Barakat syndrome, hypoparathyroidism association, 784 Bartter syndrome hypercalcemia association in children, 747 magnesium deficiency association, 765-766 BCL-2, translocation in cancer, 334, 339 Bisphosphonates, see also specific drugs classes, 734 hypercalcemia management clodronate, 734 etidronate, 734 ibandronate, 736 pamidronate, 734-736 zoledronate, 736 hyperparathyroidism management alendronate, 465 clodronate, 463-464 etidronate, 463 overview, 454 pamidronate, 464-465 risedronate, 465

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867

mechanism of action, 734 metastatic osteolytic bone disease treatment, 696 myeloma bone disease management, 700-702 nephrolithiaSis management, 447 parathyroid carcinoma, hypercalcemia management, 521 renal metabolism, 736 Blomstrand chondrodysplasia clinical features, 70, 248, 718 heredity, 718 hypoparathyroidism association, 784-785 parathyroid hormone receptor mutations, 70, 99-100, 248, 718, 720 Blood pressure, primary hyperparathyroidism effects, 356, 368-369, 380, 394 Blood vessel calcium-sensing receptor expression in smooth muscle, 263 calcium signaling and tone, 263, 265-266 hypertension, parathyroid hormone resistance, 267 layers, 262 parathyroid hormone effects blood pressure reduction, 261-262 receptor selectivity, 266 vasodilation concentrations, 262 parathyroid hormone-related protein amino terminal fragment activity, 266 calcium current effects, 265-266 desensitization, 266 endothelial layer role in relaxation, 266 expression endothelium, 263 induction, 263-264 receptor, 265 smooth muscle, 262-263 signaling, 265 smooth muscle cell effects migration, 264 proliferation, 264 transgenic mouse phenotype, 264-265 vasorelaxation, 264 Blue diaper syndrome, hypercalcemia association in children, 747 Bone acute primary hyperparathyroidism manifestations, 529 age-related changes in parathyroid hormone secretion and effects, 835-839 biopsy for secondary hyperparathyroidism, 640 calcium homeostasis role, 176-177 cells, see Osteoblast; Osteoclast; Osteocyte endochondral bone formation, see Chondrocyte formation markers alkaline phosphatase, 400-401,404 osteocalcin, 401-402, 404

868

/

Index

Bone ( c o n t i n u e d ) overview, 399-400 primary hyperparathyroidism and parathyroidectomy effects, 404 procollagen propeptides, 402, 404 growth factors fibroblast growth factors, 186 insulin-like growth factors, 184 parathyroid hormone regulation, 184-185 transforming growth factor-[3, 184 metastasis, see Malignancy-associated hypercalcemia osteoporosis, see Osteoporosis parathyroid hormone effects anabolic effects in animal models bone mass gain, 187-188, 412, 854-855 cellular basis of action, 218-219, 856 dog, 855 growth factor mediation, 191-192 osteonal bone response, 188-189 ovariectomized animals, 183-184, 188, 191,856 reversal on withdrawal, 191 rodent models, 189-190, 855 anabolic versus catabolic effects, 183, 190-191 cell effects, see Chondrocyte; Osteoblast; Osteoclast; Osteocyte cytokine mediation, 411-419 extract studies, 183, 191 growth hormone interactions, 192 insulin-like growth factors binding protein regulation, 187 insulin-like growth factor I stimulation, 186, 192, 411-413 interleukin-6 interactions and bone resorption, 41 4-415 interleukin-11 interactions, 415 leukemia inhibitory factor interactions, 415 prostaglandin Eu interactions, 192, 414 transforming growth factor-J3 activation, 187, 415 parathyroid hormone-related protein expression, 213 primary hyperparathyroidism skeletal disease association trends, 308, 351,376, 383 bone mineral density measurement, 352-354, 381-382, 391-392, 430 concurrent stone disease, 355 fracture incidence, 354, 392-393 geographic distribution, 378 histomorphome try aging changes, 427-428 cancellous bone maintenance, 428-431 cancellous bone mass, 425 cortical bone mass, 424-425 indices, 423-424 overview, 354, 423 postmenopausal women, 432, 434 structural indices, 425, 427 trabecular connectivity maintenance mechanism, 430-431

trabecular strut analysis, 428 trabecular unit number, 427 turnover of bone, 424 longitudinal course, 391-393 markers, 357, 403-405 osteitis fibrosa cystica, 308, 351,375 parathyroidectomy effects on bone metabolism, 390, 392 radiology, 380-381,390 remodeling overview, 853-854 units, 853-854 renal bone disease, see Renal bone disease resorption markers bone sialoprotein, 403, 405 deoxypyridinoline, 402-403, 405 hydroxylysine glycosides, 402, 404 hydroxyproline, 402, 404 overview, 400-401 primary hyperparathyroidism and parathyroidectomy effects, 404-405 pyridinoline, 402-403, 405 tartrate-resistant acid phosphatase, 403, 405 Bone sialoprotein (BSP), bone resorption marker and hyperparathyroidism, 403, 405 BSP, see Bone sialoprotein C CalciloXD, indications for formula use, 750 Calciphylaxis, renal bone disease association, 637 Calcitonin acute hypercalcemia management, 736-737, 739 growth hormone combination therapy for osteoporosis, 859-860 myeloma bone disease management with corticosteroids, 701 parathyroid carcinoma, hypercalcemia management, 521 parathyroid crisis management, 468 hyperparathyroidism management, 465-466 safety for children, 750 Calcitriol, see also Vitamin D parathyroid effects in secondary hyperparathyroidism, 629-630 secondary hyperparathyroidism treatment and dosing, 647-650 synthesis kidney and secondary hyperparathyroidism, 628-629 phosphate effects, 626-627 Calcium, see a l s o Hypocalcemia; Hypercalcemia aging effects on metabolism, 839 balance during life cycle, 169 estrogen deficiency and calcium metabolism, 839-840, 843, 848 extracellular concentration regulation, 127, 135-136, 168-169 forms in blood, 169 glutamate receptor sensing, 138

homeostasis, see a l s o Calcium-sensing receptor gastrointestinal absorption, 174 hormonal regulation, 169-171 overview, 167 parathyroid secretory set point concept, 321-322 renal excretion control, 174-176 skeleton role, 176-177 hypercalciuria in nephrolithiasis, 441-442 hypoparathyroidism management, 828-832 intake recommendations, 169 levels familial hypocalciuric hypercalcemia levels dynamic studies, 615 serum, 612 urine, 613-614 intracellular concentration, 127, 167-168 nephrolithiasis, 438 primary hyperparathyroidism changes over time, 388 hypercalciuria, 355-356, 380, 389 intake recommendations, 388 parathyroidectomy effects, 389-390 serum, 4, 349, 351,356, 363, 380, 389 renal bone disease, serum levels, 640 normal serum values, 743 parathyroid cell number regulation, 22 parathyroid hormone renal calcium excretion effects, 174-176, 231-232 secretion regulation by calcium, 23-24, 27, 145-146, 171-174 physiological functions, 127, 167-169 pseudohyperparathyroidism treatment, 819-820 restriction for nephrolithiasis, 446 serum assay, 147 supplementation for secondary parathyroidism, 645, 647 vascular signaling and tone, 263, 265-266 Calcium channel L-type voltage-sensitive channel central nervous system clustering, 269 parathyroid hormone-related protein inhibition of calcium influx, 270-271 regulation of" parathyroid hormonerelated protein expression, 270 signaling domains, 269 parathyroid hormone inhibition in vasorelaxation, 265-266, 268 parathyroid hormone-related protein stimulation, 271 types, 269 Calcium-sensing receptor (CaSR) calcimimetic agents secondary hyperparathyroidism treatment, 653-654 therapeutic targeting, 454, 467, 522 calcium-binding sites, 129 dimerization, 129-130

Index dimerization, 129-130 extracellular calcium concentration regulation, 127, 135-136 familial hypocalciuric hypercalcemia defects, 320, 342, 608-611,616 gene cloning, 128 locus, 131 regulation of expression, 131 structure, 131 glycosylation, 130 G protein-coupled receptor homology, 128 hypocalcemia and activating mutations, 758 hypoparathyroidism abnormalities, 785 integration of calcium and sodium/volume/blood pressure control, 178 integration of calcium and water metabolism, 135-136, 177-178 integration of calcum and protein metabolism, 178-179 ligands and agonists, 129, 137 mutation and disease, 172-173 nutrient sensing role, 137-138 parathyroid adenoma expression, 311-313 phosphorylation, 130 placental expression, 283 signal transduction, 130 structure, 128, 172 tissue distribution and function bone and cartilage, 133-135, 177 C cell, 132 intestine, 132-133, 137-138 kidney, 132, 135-136, 172-173 parathyroid, 131-132, 172-173 topology, 128 cAMP, s e e Cyclic AMP Candidiasis, s e e Autoimmune hypoparathyroidism Carcinoma, s e e Parathyroid carcinoma Carney complex, multiple endocrine neoplasia type 1 differential diagnosis, 546 CaSR, s e e Calcium-sensing receptor C cell calcium-sensing receptor expression, 132 derivation, 594 hyperplasia in multiple endocrine neoplasia type 2A, 586-587 Cellulose phosphate, nephrolithiasis management, 446 Central nervous system calcium channels L-type voltage-sensitive channel clustering, 269 parathyroid hormone-related protein inhibition of calcium influx, 270-271 regulation of parathyroid hormonerelated protein expression, 270 signaling domains, 269 types, 269 parathyroid hormone

expression, 268-269 receptor expression, 268 parathyroid hormone-related protein expression, 268-269 neuroprotection, 270-271 TIP39 in cerebral cortex, 268-269 c-Fos, parathyroid hormone induction, 61-62 Chief cell cell number regulation, 22 parathyroid gland histology, 2-4 parathyroid hormone synthesis, 22-23 primary parathyroid hyperplasia, 6 Cholera toxin, G protein activation, 123 Chondrocyte calcium-sensing receptor expression, 134-135 endochondral bone formation collagen expression, 246 differentiation in growth plate, 245-246 Indian hedgehog role, 251-257 matrix metalloproteinase-9 role, 247-248, 257 modeling, 256-258 vascular endothelial growth factor role, 246-248, 257, 413 parathyroid hormone receptor mutation effects, s e e Blomstrand chondrodysplasia; Jansen's metaphyseal chondrodysplasia parathyroid hormone-related protein differentiation role, 249, 712-713 expression, 246 Indian hedgehog interactions, 251-252, 254-257, 713 receptor expression, 249-250 knockout effects, 255, 257 Cimetidine, parathyroid hormone secretion inhibition, 447, 466 Clodronate hypercalcemia management, 734 hyperparathyroidism management, 463-464 Collagenoma, multiple endocrine neoplasia type 1,543, 553 Colony-stimulating factor-1 (CSF-1), parathyroid hormone interactions, 413 Computed tomography (CT) inferior parathyroid gland, 507 multiple endocrine neoplasia type 1 tumors, 559 parathyroid imaging, 475-477, 483-484 Corticosteroids acute hypercalcemia management, 737 myeloma bone disease management, 700-701 osteoporosis induction, s e e Osteoporosis Crohn's disease, hypercalcemia differential diagnosis, 666 CSF-1, s e e Colony-stimulating factor-1 CT, s e e Computed tomography Cyclic AMP (cAMP) parathyroid hormone expression regulation, 25

/

869

pseudohypoparathyroidism diagnosis with parathyroid hormone response test, 807-808, 818-819 Cyclin D gene, s e e P R A D 1 D

Deoxypyridinoline, bone resorption marker and hyperparathyroidism, 402-403, 405 Desert hedgehog (Dhh), tissue patterning role, 250 Dhh, s e e Desert hedgehog Diabetes magnesium deficiency association, 765 primary hyperparathyroidism association, 367-368, 394 Dialysis amyloidosis clinical manifestations, 638 management, 638-639 renal bone disease association, 638-639 DiGeorge syndrome, hypoparathyroidism association, 783-784 Diuretics, nephrolithiasis management, 447 Down syndrome, hypercalcemia association in children, 747-748 Doxercalciferol, secondary hyperparathyroidism treatment, 652-653 Dyslipidemia, primary hyperparathyroidism association, 368 E

Ectopic parathyroid hormone syndrome molecular mechanisms, 343 ovarian carcinoma, 343 parathyroid adenoma locations, 5-6 Ependymoma, multiple endocrine neoplasia type 1,543 Estrogen hyperparathyroidism management bone mineral density effects, 462 cyclofenil, 462 ethinyl estradiol, 461-462 overview, 454-455 risk-benefit analysis, 462-463 nephrolithiasis management, 447 osteoporosis deficiency and calcium metabolism, 839-840, 843, 848 parathyroid hormone antagonism, 848-849 replacement therapy and parathyroid hormone effects, 848 parathyroid hormone expression regulation, 25 parathyroid hormone-related protein regulation, 43-44 Etidronate hypercalcemia management, 734 hyperparathyroidism management, 463 F

Falecalcitriol, secondary hyperparathyroidism treatment, 651-652 Familial hyperparathyroidism nephrolithiasis prevalence, 440-441 parathyroid carcinoma association, 516

870

/

Index

Familial hypocalciuric hypercalcemia (FHH) calcium levels dynamic studies, 615 serum, 612 urine, 613-614 calcium-sensing receptor defects, 320, 342, 608-611,616, 745 clinical manifestations, 610-611 complications, 611 diagnosis, 616-617 gene mapping, 608, 610 genotype-phenotype correlations, 611 heredity, 608 hypercalcemia differential diagnosis, 664 knockout mouse models, 610 magnesium levels serum, 612 urine, 614 management, 617 multiple endocrine neoplasia type 1 differential diagnosis, 547 neonatal severe primary hyperparathyroidism clinical manifestations, 603, 618, 745 etiology, 603-604 histology, 604 historical perspective, 608 homozygous versus heterozygous forms, 617-618 knockout mouse model, 618 mild form, 618 treatment, 604, 619 nephrolithiasis prevalence, 440 nomenclature, 607-608 parathyroidectomy contraindication, 488-489 parathyroid function, 10, 320 parathyroid gland features, 615-616 parathyroid hormone levels, 612-613, 615 phosphorous levels, 612 renal function, 614 vitamin D levels, 614-615 Familial parathyroid hyperplasia, features, 10 Fat necrosis, neonatal hypercalcemia association, 746 FGFs, see Fibroblast growth factors FHH, see Familial hypocalciuric hypercalcemia Fibroblast growth factors (FGFs) bone growth fhctors, 186 parathyroid hormone stimulation of FGF-2, 413 Flow cytometry analysis of parathyroid tissue ploidy, 10 parathyroid cell analysis, 297-298 Furin parathyroid hormone prohormone cleavage, 19-20 parathyroid hormone-related protein cleavage, 37 Furosemide, hypercalcemia management, 733

G Gallium nitrate acute hypercalcemia management, 738 parathyroid carcinoma, hypercalcemia management, 521-522 parathyroid crisis management, 468 Gastrinoma, multiple endocrine neoplasia type 1 age of onset, 540 exacerbation by hyperparathyroidism, 539-540 malignancy, 540, 552 management, 558-560 multiplicity, 540 screening, 554-555 symptoms, 540 GH, see Growth hormone Glucagonoma, multiple endocrine neoplasia type 1,541 Glucocorticoid, parathyroid hormone expression regulation, 25 G N A S 1 , mutations in pseudohyperparathyroidism, 785-786, 810-813, 815-816 G protein alpha subunit functional diversity, 121 homology, 120 mammalian diversity, 118 protein interactions, 120-121 switch regions, 120 beta-gamma subunit heterodimers, 121-122 beta subunits, 121 clinical implications of dysfunction, 123 effector coupling, 122-123 gamma subunits, 121 G,et mutations in pseudohyperparathyroidism, 785-786, 810-813, 815-816 GTPase cycle, 119 parathyroid hormone receptor coupling, 53-54, 93-94, 96, 117-118 receptor coupling, 122 Growth hormone (GH) aging effects on secretion, 858-859 osteoporosis clinical trials bone mineral density response, 859-860 calcitonin combination therapy, 859-860 parathyroid hormone interactions in bone, 192 skeletal actions, 859 Growth hormone-releasing hormonesecreting tumors, multiple endocrine neoplasia type 1,541 Growth retardation, renal bone disease association, 638 H

Heart parathyroid hormone effects, 267 parathyroid hormone-related protein expression and effects, 266-267

Hepatitis, hypercalcemia association in children, 749 Hirschsprung's disease, multiple endocrine neoplasia type 2 association, 588 Humoral hypercalcemia of malignancy, see also Malignancy-associated hypercalcemia animal models mouse, 677 passive immunization studies, 677 rat, 677 therapy evaluation antisense studies, 678 Ras inhibitors, 678 vitamin D analogs, 678 classification of hypercalcemia in cancer patients, 679, 691 hypercalcemia differential diagnosis, 664-665, 667, 730 parathyroid hormone-related protein immunoassay, 158-159 role, 11, 31,691 Hydrogen, parathyroid hormone and renal excretion effects, 234-235 Hydroxylysine glycosides, bone resorption markers and hyperparathyroidism, 402, 404 Hydroxyproline, bone resorption marker and hyperparathyroidism, 402, 404 Hypercalcemia, see also Calcium; Familial hypocalciuric hypercalcemia; Humoral hypercalcemia of malignancy; Idiopathic infantile hypercalcemia; Myeloma bone disease acute hypercalcemia, see also Acute primary hyperparathyroidism clinical manifestations, 731-732 pathophysiology, 730-731 children clinical presentation, 743-744 diagnosis, 744-745 differential diagnosis, 744 immobilization as cause, 749 inborn errors of metabolism, 747-748 malignancy-associated hypercalcemia, 749 neonatal hyperparathyroidism, 745-746 nonparathyroid causes, 746-747, 749 normal calcium levels, 743 older children, 748-749 prevalence of hypercalcemia, 743 treatment, 750 differential diagnosis acute renal falure, 666 adrenocortical insufficiency, 666 calcium ingestion, 665 Crohn's disease, 666 familial hypocalciuric hypercalcemia, 664 humoral hypercalcemia of malignancy, 664-665, 667 immobilization, 666 infection, 666 lithium toxicity, 664 overview, 729-730

Index parathyroid hormone levels elevated, 667 low, 667-668 normal, 667 parenteral nutrition, 666 primary hyperparathyroidism, 663 sarcoidosis, 665-666 tertiary hyperparathyroidism, 663 thiazide diuretics, 664 thyrotoxicosis, 666 tuberculosis, 666 vitamin D intoxication, 665 parathyroid carcinoma management bisphosphonates, 521 calcimimetics, 522 calcitonin, 521 gallium nitrate, 521-522 octreotide, 522 parathyroid hormone immunization, 522 plicamycin, 521 WR-2721,522 severe hypercalcemia calcium levels, 731 therapy ambulation, 738 bisphosphonates, 734-736 bone resorption inhibition, 733-734 calcitonin, 736-737, 739 corticosteroids, 737 dialysis, 738 furosemide, 733 gallium nitrate, 738 phosphate, 738 plicamycin, 737-738 principles, 732 saline infusion, 732-733, 739 serum calcium adjustment goals, 732 underlying disorder, 739 Hyperparathyroidism, see Primary hyperparathyroidism; Pseudohyperparathyroidism; Secondary hyperparathyroidism; Tertiary hyperparathyroidism Hyperparathyroidism-jaw tumor syndrome case reports, 601-602 linkage analysis, 602 multiple endocrine neoplasia type 1 differential diagnosis, 546 Hypertension, parathyroid hormone resistance, 267 Hyperuricosuria, nephrolithiasis pathogenesis, 444 Hypocalcemia, see a l s o Hypoparathyroidism bound calcium alterations, 760 classification of disorders, 779 clinical manifestations, 756 determination, 755 management, 760 osteoblast activity increase, 760 parathyroid growth regulation, 302-303 parathyroid hormone resistance, 759 pathophysiology, 756 phosphate induction, 626 vitamin D metabolism disorders, 759-760

Hypoparathyroidism, see a l s o Autoimmune' hypoparathyroidism; Hypocalcemia causes

accidental resection of parathyroids, 11 autoimmune idiopathic hypoparathyroidism, 11,757-758, 791 calcium-sensing receptor activating mutations, 758, 785 drug induction, 760 hypermagnesemia, 759 infiltrative damage to parathyroid glands, 11,758 magnesium deficiency, 758-759 overview, 756-757 parathyroid developmental abnormalities, 758 radiation-induced damage, 11,758 surgical hypoparathyroidism, 319-320, 757 transient neonatal hypocalcemia, 758 transient postsurgical hypoparathyroidism, 757 chronic management adults, 830-832 children, 830 neonates, 830 clinical manifestations, 827-828 complex syndrome association autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy, 785 Barakat syndrome, 784 Blomstrand chondrodysplasia, 784-785 DiGeorge syndrome, 783-784 Kenney-Caffey syndrome, 784 mitochondrial disorders, 784 complications of treatment, 832 disorders and gene loci, 779-780 emergency treatment adults, 829 children, 829 neonates, 828 parathyroid hormone gene abnormalities autosomal dominant hypoparathyroidism, 782 autosomal recessive hypoparathyroidism, 782 X-linked recessive hypoparathyroidism, 782-783 I

Ibandronate, hypercalcemia management, 736 Idiopathic infantile hypercalcemia (IHH), parathyroid hormone-related protein levels, 684 IGFs, s e e Insulin-like growth factors Ihh, s e e Indian hedgehog IHH, see Idiopathic infantile hypercalcemia IL-[3, see Interleukin-[3 IL-6, s e e Interleukin-6 IL-11, see Interleukin-ll IMAGe syndrome, hypercalcemia association in children, 747

/

871

Immobilization, hypercalcemia association in children, 749 differential diagnosis, 666 Immunoassay aplastic or adynamic bone levels, 639 clinical applications of parathyroid hormone assay adynamic renal osteodystrophy, 154 hypercalcemia from nonparathyroid causes, 151-152 overview, 143, 150 primary hyperparathyroidism, 150-151 secondary hyperparathyroidism hypocalcemia, 152 renal osteodystrophy, 152-153 skeletal disorders, 154 tertiary hyperparathyroidism, 153 familial hypocalciuric hypercalcemia, parathyroid hormone levels, 612-613, 615 parathyroid hormone antibody specificity, 147-148 circulating forms, 146-147 formats intact hormone assays, 149-150 overview, 148 solid-phase assays, 149 solution assays, 148-149 historical perspective, 147 sample degradation, 147 parathyroid hormone-related protein circulating forms, 158, 675 historical perspective, 157 humoral hypercalcemia of malignancy, 158-159 malignancy and eucalcemia, 159 normal values, 159 sensitivity, 157-158 solution assays, 158 technical limitations, 159-160 two-site assays, 158, 675 renal bone disease diagnosis, 639-643 sensitivity, 143 specificity, 730 Importin or, parathyroid hormone-related protein import role, 110 Indian hedgehog (Ihh), endochondral bone formation role chick studies, 251 hip signaling, 256 knockout mouse studies, 252-254 parathyroid hormone-related protein interactions, 251-252, 254-257 receptor expression, 252 Insulin-like growth factors (IGFs) binding proteins, 184, 187, 412 bone growth effects, 184 insulin-like growth factor I stimulation by parathyroid hormone, 186, 192, 411-413 parathyroid hormone interactions in osteoblast development, 206, 215 types, 184 Insulin-like growth factor binding protein-3, primary hyperparathyroidism levels, 418

872

/

Index

Insulinoma, multiple endocrine neoplasia type 1,540-541,555, 558-559 Interleukin-l[3 (IL-I[3), primary hyperparathyroidism levels, 418 Interleukin-6 (IL-6) hormone actions, 411 levels primary hyperparathyroidism, 416-418 secondary hyperparathyroidism, 415 parathyroid hormone interactions bone cell development osteoblast, 206, 215, 414 osteoclast, 207, 414 hyperparathyroidism and bone resorption, 41 4-415 Interleukin-11 (IL-11), parathyroid hormone interactions in bone, 415

J

Jansen's metaphyseal chondrodysplasia (]MC) clinical features, 69, 100, 248, 714-715, 746 laboratory findings, 715 parathyroid hormone levels, 745-746 parathyroid hormone receptor mutations, 69-70, 100, 248, 715, 717-719, 746 radiographic findings, 715 treatment, 100 JMC, see J a n s e n ' s metaphyseal chondrodysplasia K

Kearns-Sayre syndrome, hypoparathyroidism association, 784 Kenney-Caffey syndrome, hypoparathyroidism association, 784 Kidney acute primary hyperparathyroidism manifestations, 529 bone disease, see Renal bone disease calcium-sensing receptor expression and function, 132, 135-136, 172-173 integration of calcium and water metabolism, 135-136, 177-178 coronary artery calcification in dialysis patients, 647 magnesium metabolism, 764 parathyroid hormone calcium excretion effects, 174-176, 231-232 clearance, 25-26 miscellaneous effects, 236 phosphate reabsorption and secretion regulation, 233-234 receptor calcium and magnesium excretion regulation, 232 desensitization, 228-229 distribution of expression, 227-228 phosphate excretion regulation, 233-234 regulation of expression, 229 signal transduction, 228, 232-235

sodium and hydrogen excretion regulation, 235 vitamin D metabolism regulation, 235-236 sodium and hydrogen excretion effects, 234-235 parathyroid hormone receptor calcium and magnesium excretion regulation, 232 desensitization, 228-229 distribution of expression, 227-228, 708 phosphate excretion regulation, 233-234 regulation of expression, 229 signal transduction, 228, 232-235 sodium and hydrogen excretion regulation, 235 vitamin D metabolism regulation, 235-236, 708 parathyroid hormone-related protein expression and effects, 236-237 reabsorption calcium and magnesium, 229-231 phosphate, 232-233 renal insufficiency, see Secondary hyperparathyroidism stones, see Nephrolithiasis L Lactase deficiency, hypercalcemia association in children, 747 Lactation, see Mammary gland Laminin antagonists, 696 bone metastasis role, 692-694 Leukemia inhibitory factor (LIF), parathyroid hormone interactions in bone, 415 Leukotriene B4, primary hyperparathyroidism levels, 418 LIE see Leukemia inhibitory factor Lipoma, multiple endocrine neoplasia type 1,543, 561 Lithium hypercalcemia differential diagnosis, 664 parathyroid growth and hormone secretion, 321 primary hyperparathyroidism association, 388-389, 459 L-type voltage-sensitive calcium channel, see Calcium channel M

Macrophage inflammatory protein-10t (MIP-10t), myeloma bone disease role activation of osteoclasts, 698-699 myeloma expression studies, 699 Magnesium body distribution, 763-764 deficiency Bartter syndrome association, 765-766 clinical manifestations, 766 diabetes association, 765 diagnosis, 773 drug induction, 765 excretion excess, 765

malabsorption syndrome, 764 metabolic disorders, 765 mineral homeostasis impairment mechanism, 772-773 parathyroid gland function effects, 767-769 parathyroid hormone action effects, 769-770 prevalence, 764 treatment, 773-774 vitamin D metabolism and action effects, 770-771 familial hypocalciuric hypercalcemia levels serum, 612 urine, 614 functional overview, 763 hypoparathyroidism causes deficiency, 758-759 hypermagnesemia, 759 intestinal absorption, 764 parathyroid hormone secretion regulation, 146, 766-769 renal metabolism, 764 Magnetic resonance imaging (MRI) inferior parathyroid gland, 507 multiple endocrine neoplasia type 1 tumors, 559 parathyroid imaging, 475-476, 483-484 superior parathyroid gland, 500-501 M A H , see Malignancy-associated hypercalcemia Malignancy-associated hypercalcemia (MAH), see a l s o Humoral hypercalcemia of malignancy; Myeloma bone disease children, 749 classification of hypercalcemia in cancer patients, 679, 691 hypercalcemia differential diagnosis, 664-665, 667, 730 metastatic bone disease bone-derived growth factors, 695 bone destruction mediation by tumor cells, 694 hypercalcemia prevalence, 691 metastasis pathophysiology blood coagulation mechanism, 694 cell motility and chemotaxis, 693 E-cadherin role, 692-693 integrin role, 692 laminin role, 692-694 oncogenes, 693-694 proteases, 693 steps, 692 osteoclast stimulation mechanisms at metastatic site, 694-695 renal tubular calcium reabsorption, 691-692 treatment, 696 tumor types, 691 parathyroid hormone-related protein levels antitumor therapy effects, 682 hematological malignancies, 681-682 non-hypercalcemic patients, 683

Index prognostic value, 682-683 skeletal metastasis relationship, 679-681 Mammary gland adolescent development, 279-280 embryonic development, 277-279 lactation changes, 280-281 parathyroid hormone-related protein calcium mobilization in milk production, 281 expression and signaling adolescence, 279-280 embryo, 277-279 lactation, 280-282, 684 knockout mouse phenotype, 278-279, 713 pathophysiology, 282 receptor expression, 278-282 transgenic mouse overexpression, 28O vascular effects, 281-282 Matrix metalloproteinases (MMPs) MMP-9, endochondral bone formation role, 247-248, 257 osteoblast expression and parathyroid hormone regulation, 217-218 McCune-Albright syndrome G protein mutations, 123 multiple endocrine neoplasia type 1 differential diagnosis, 545 Medullary sponge kidney, nephrolithiasis prevalence, 441 Medullary thyroid carcinoma (MTC), multiple endocrine neoplasia type 2 association with type 2A, 585-586 association with type 2B, 589 C cell hyperplasia, 586-587 familial disease, 588 management, 594-596 MELAS, see Mitochondrial encephalopathy, strokelike episodes, and lactic acidosis M E N 1 , see Multiple endocrine neoplasia type 1 MEN-l, see Multiple endocrine neoplasia type 1 MEN-2, see Multiple endocrine neoplasia type 2 Menin, see Multiple endocrine neoplasia type 1 Metastasis, see Malignancy-associated hypercalcemia MIP-lot, see Macrophage inflammatory protein-10t Mithramycin, see Plicamycin Mitochondrial encephalopathy, strokelike episodes, and lactic acidosis (MELAS), hypoparathyroidism association, 784 MMPs, see Matrix metalloproteinases M R I , see Magnetic resonance imaging MTC, see Medullary thyroid carcinoma Multiple endocrine neoplasia type 1 (MEN-l) clinical phenotype overview, 535 variation in families, 543-544 clonality of parathyroid hyperplasia, 333 differential diagnosis Carney complex, 546

familial hypocalciuric hypercalcemia, 547 hereditary multiple neoplasias, 545-546 hereditary neoplasia of one endocrine tissue, 546-547 hyperparathyroidism-j aw tumor syndrome, 546 McCune-Albright syndrome, 545 multiple endocrine neoplasia type 2, 546 neurofibromatosis type 1,546 nonhereditary hyperfunction of endocrine tissues, 544-545 nonhereditary neoplasia of one endocrine tissue, 544 persistent hyperinsulinemic hypoglycemia of infancy, 547-548 radiation-induced neoplasm, 545 von Hippel-Lindau syndrome, 546 genetic counseling, 566-568 growth factor identification, 564 historical perspective of research, 536 hyperparathyroidism age of onset, 538 differential diagnosis, 538 etiology, 544 gastrinoma interaction, 539-540 management medical supervision, 556 parathyroidectomy, 539, 556-558 pharmacotherapy, 556 penetrance, 537, 544 sex ratio, 539 MEN1

cloning, 340, 561-562 genotype-phenotype relationships, 566 germ line inactivation, 544 inactivation in parathyroid tumors, 340-342 linkage analysis, 565 locus, 340, 536, 561 menin product function, 562 protein interactions, 341,550, 563 models for functional analysis, 563-564 mutation frequency in germ line, 565 origins, 566 testing, 556, 564-568 tumors, 550 transcript features, 562 tumor suppression, 562-563 two-hit hypothesis of oncogenesis, 549-550, 562 nephrolithiasis prevalence, 440 parathyroidectomy, 488-489, 539 parathyroid pathology, 4, 7 prevalence, 536 screening, 488 tumors adrenocortical neoplasms, 543, 553, 561 age of onset, 548 burden evaluation, 558-559 chemotherapy, 560 clonality, 551-552

/

873

collagenoma, 543, 553 endocrinopathy, 537 enteropancreatic tumor multiplicity, 539-540, 552 ependymoma, 543 facial angiofibroma, 543, 553 foregut carcinoid tumors duodenal carcinoid, 542 gastric ECLoma, 542 malignancy, 552-553 management, 560 screening, 555 thymic carcinoid, 542 gastrinoma age of onset, 540 exacerbation by hyperparathyroidism, 539-540 malignancy, 540, 552 management, 558-560 multiplicity, 540 screening, 554-555 symptoms, 540 glucagonoma, 541 growth hormone-releasing hormonesecreting tumors, 541 insulinoma, 540-541,555, 558-559 lipoma, 543, 561 loss of heterozygosity, 551,562, 565 malignancy, 537, 549, 552 management challenges, 553 metastasis evaluation, 559 miscellaneous hormone secretion, 542 octreotide management of hormone secretion, 558 pancreatic polypeptide-secreting tumors, 542 parathyroid adenoma, 539, 554 pheochromocytoma, 543, 553, 561 pituitary tumors, 542-543, 553, 555, 561 prevention of malignancy, 561 prolactinoma, 544 screening, 554-556 somatostatinomas, 541 surgical management, 559-560 thyroid follicular adenoma, 543 tissue multiplicity, 548-549 vasoactive intestinal peptide-secreting tumors, 541 Multiple endocrine neoplasia type 2 (MEN-2) chromosomal abnormalities, 592 clinical phenotype, 535, 585 hyperparathyroidism features, 588, 596 medullary thyroid carcinoma association with type 2A, 585-586 association with type 2B, 589 C cell hyperplasia, 586-587 familial disease, 588 management, 594-596 nephrolithiasis prevalence, 440 pheochromocytoma features, 58%588, 596 RET genotype-phenotype correlation, 590-591

874

/

Index

Multiple endocrine neoplasia type 2 (MEN-2) ( c o n t i n u e d ) locus, 590 molecular pathogenesis, 592 neural crest differentiation role, 593-594 overview of mutations, 342, 489, 535, 585 papillary thyroid carcinoma mutations, 591-592 receptor complex, 590 testing for mutations, 567-568, 594-596 type 2A cutaneous lichen amyloidosis association, 588-589 features, 585-586 Hirschsprung's disease association, 588 type 2B features, 589-590 Multiple myeloma, s e e Myeloma bone disease Muscle weakness primary hyperparathyroidism, 355, 365, 367, 378, 394-395 renal failure patients, 636-637 vitamin D therapy, 636-637 Myeloma bone disease adhesion molecules and cell-cell interactions, 698 hypercalcemia pathogenesis, 696-697 interleukin-6 production by osteoclasts, 699-700 laboratory findings, 697 macrophage inflammatory protein-lot role activation of osteoclasts, 698-699 myeloma expression studies, 699 murine 5T model, 699 osteolytic bone lesions, 697-698 renal function impairment, 696-697 treatment bisphosphonates, 700-702 calcitonin with corticosteroids, 701 corticosteroids, 700 cytotoxic drugs, 700-701 plicamycin, 700-701 saline infusion, 701

pathogenesis in hyperparathyroidism hypercalciuria, 441-442 hyperuricosuria, 444 renal tubular acidosis, 444 urinary inhibitors of crystallization, 443-444 urine activity product ratio, 442-443 urine formation product ratio, 443 prevalence studies common primary hyperparathyroidism, 439-440 familial hyperparathyroidism, 440-441 familial hypocalciuric hypercalcemia, 440 medullary sponge kidney, 441 mild primary hyperparathyroidism, 440 multiple endocrine neoplasia type 1,440 type 2A, 440 primary hyperparathyroidism age effects, 367 association trends, 308, 351,354-355 calcium levels, 438 clinical presentation, 437-438 composition of stones, 438-439 concurrent bone disease, 355 geographic distribution, 378, 380 historical perspective, 437 parathyroidectomy effects, 367, 387, 393, 444-445 vitamin D levels, 438, 442 Nervous system, see Central nervous system Neurofibromatosis type 1, multiple endocrine neoplasia type 1 differential diagnosis, 546 NLS, s e e Nuclear localization sequence NPC, s e e Nuclear pore complex Nuclear localization sequence (NLS), parathyroid hormone-related protein, 105-106 Nuclear pore complex (NPC), parathyroid hormone-related protein import, 110-111 Nucleolar localization sequence, parathyroid hormone-related protein, 110

N Neonatal severe primary hyperparathyroidism, see Familial hypocalciuric hypercalcemia Nephrolithiasis differential diagnosis, 441 management with hyperparathyroidism antiresorptive therapy, 447 bisphosphonate therapy, 447 calcium restriction, 446 cellulose phosphate, 446 dietary protein restriction, 446 diuretics, 447 estrogen therapy, 447 fluid intake, 446 parathyroid hormone secretion inhibitors, 447 phosphate therapy, 448 sodium restriction, 446

O Octreotide multiple endocrine neoplasia type 1, management of hormone-secreting tumors, 558 parathyroid carcinoma, hypercalcemia management, 522 ODE s e e Osteoclast differentiation factor Ostabolin, signaling-selective ligand, 62 Osteitis fibrosa cystica, see Bone Osteoblast calcium-sensing receptor expression, 133-134, 177 hypocalcemia and activity increase, 760 parathyroid hormone effects alkaline phosphatase expression, 217 animal model studies of differentiation, 199-200 apoptosis, 216-217

bone matrix protein expression, 217 cell shape, 217 gap junctions, 217 growth factor interactions, 206, 215 insulin-like growth factors and binding protein expression, 206, 215 interleukin-6 expression, 206, 215 ion channels, 217 matrix metalloproteinase and inhibitor expression, 217-218 precursor cells, 214 proliferation and differentiation animal model studies, 200-201, 203-204 bone organ culture studies, 200 MC-3T3-E1 cells, 201,203 MG-63 cells, 203 MLO-Y4 cells, 203 RC cell bone nodule model, 201-203 TE-85 cells, 200 UMR-106 cells, 200, 216 transcription factor expression, 214 transforming growth factor-[~ expression, 215 parathyroid hormone receptor expression and signaling thresholds, 204-205, 213-214 Osteocalcin bone formation marker and hyperparathyroidism, 401-402, 404 renal bone disease, serum levels, 643 Osteoclast calcium-sensing receptor expression, 133-134, 177 macrophage inflammatory protein-lot activation in myeloma bone disease, 698-699 parathyroid hormone effects animal model studies of differentiation, 199-200 cellular basis of action, 218 comparison with parathyroid hormonerelated protein effects, 218 differentiation, 206-207 growth factor interactions, 206 proliferation and differentiation animal model studies, 200-201, 203-204 bone organ culture studies, 200 MC-3T3-E1 cells, 201,203 MG-63 cells, 203 MLO-Y4 cells, 203 RC cell bone nodule model, 201-203 TE-85 cells, 200 UMR-106 cells, 200 RANKL expression induction, 176-177, 207, 216, 218 recruitment, 431 parathyroid hormone receptor expression, 214 stimulation mechanisms at metastatic site, 694-695 Osteoclast differentiation factor (ODF), parathyroid hormone interactions, 413 Osteocyte, parathyroid hormone effects, 218 Osteomalacia, bone biopsy, 640

Index Osteoporosis age-related changes in parathyroid hormone secretion, 835-837 estrogen deficiency and calcium metabolism, 839-840, 843, 848 parathyroid hormone antagonism, 848-849 replacement therapy and parathyroid hormone effects, 848 growth hormone clinical trials bone mineral density response, 859-860 calcitonin combination therapy, 859-860 parathyroid hormone clinical trials bone mineral density response, 856-858 calcium and calcitriol combination therapy, 857 corticosteroid-induced osteoporosis, 858 estrogen combination therapy, 856-857 fracture incidence studies, 858 idiopathic osteoporosis, 858 regimens, 857-858 gene mutation searching, 848 hyperparathyroidism protective effects in postmenopausal women, 432, 434 levels, 844 pathophysiologic hypotheses parathyroid gland altered responsiveness, 846-847 parathyroid gland suppression, 846 skeletal sensitivity alterations, 844, 845-846 vitamin D metabolism alterations, 844-845 prevalence, 843 risk factors, 843 therapeutic targeting of parathyroid hormone receptor, 83 types, 835, 847-848 22-Oxacalcitriol, secondary hyperparathyroidism treatment, 651 P

p27, expression in parathyroid tumorigenesis, 11 p53 parathyroid carcinoma mutation, 516 parathyroid hormone-related protein expression regulation, 39-40 Pamidronate hypercalcemia management, 734-736 hyperparathyroidism management, 464-465 metastatic osteolytic bone disease treatment, 696 parathyroid crisis management, 468 Pancreas, parathyroid hormone-related protein developmental function, 286 expression distribution, 285

knockout mouse phenotype, 286-287 pathophysiology, 287 posttranslational processing and effects of fragments, 286 receptor expression, 285 signaling, 286-287 transgenic mouse overexpression, 286 Pancreatic polypeptide-secreting tumor, multiple endocrine neoplasia type 1, 542 Papillary thyroid carcinoma, RET mutations, 591-592 Parathyroid adenoma, see also Primary hyperparathyroidism birth rates of cells, 310-311 calcium-sensing receptor expression, 311-313 classification, 312-313 clonality analysis, 10, 331-333 double adenoma, 6 ectopic locations of glands inferior parathyroid glands, 502-503, 505-507 overview, 499 superior parathyroid glands, 499-500, 502 supernumary parathyroid glands, 507, 511 ectopic locations, 5-6 ethanol injection, 479 familial forms dominant, 602 recessive, 604 flow cytometry analysis of ploidy, 10 genetics candidate gene searching, 341-342 M E N 1 inactivation, 340-342 p27 expression, 11 PRAD1

overexpression, 338-339 rearrangements, 10, 333-335, 337-339 retinoblastoma gene mutations, 10-11 gross features, 4, 350 growth rates, see histology, 4-5, 350 imaging, see Parathyroidectomy infarction, 9 microscopic features, 4-5 molecular oncology overview, 331-332 multiple endocrine neoplasia type 1,539, 554 pathogenesis, 311 ploidy and nuclear diameter, 12 proliferative markers, 10 thyroid adenoma distinguishing from parathyroid adenoma, 478 weight, historic trends, 306-307 X-chromosome inactivation analysis, 333 Parathyroid carcinoma calcium levels in serum, 6, 517 clinical features, 517-518 end-stage renal disease patients, 516 epidemiology, 6, 351, 515 etiology, 515-516 familial forms, 602-603

/

875

familial hyperparathyroidism association, 516 histology, 6-7, 518-519 invasion, 6-7, 519 management chemotherapy, 520-521 hypercalcemia bisphosphonates, 521 calcimimetics, 522 calcitonin, 521 gallium nitrate, 521-522 octreotide, 522 parathyroid hormone immunization, 522 plicamycin, 521 WR-2721, 522 radiation therapy, 520 surgery, 519-520 markers, 519 molecular pathogenesis, 342, 516 natural history, 519 palpable neck mass, 517 primary hyperparathyroidism, comparison of presentation, 518 prognosis, 7, 519, 522 Parathyroid crisis, see Acute primary hyperparathyroidism Parathyroid cyst adenoma relationships, 8 gross features, 7-8 localization, 7 Par athyroide cto my acute primary hyperparathyroidism management, 531-532 aluminum-related bone toxicity effects, 642-643 anatomy, 491 bone marker effects formation markers, 404 resorption markers, 404-405 chronic renal failure patients, 496 ectopic locations of glands inferior parathyroid glands, 502-503, 505-507 overview, 499 superior parathyroid glands, 499-500, 502 supernumary parathyroid glands, 507, 511 embryology, 490 evaluation of patients clinical features, 488 laboratory studies, 489 patient history, 488-489 physical examination, 489 failure rates and avoidance, 495-496 familial hypocalciuric hyperparathyroidism as contraindication, 488-489 historical perspective, 487 imaging initial surgery patients, 484-485 intraoperative imaging, 453 reoperative surgery arteriography, 479, 482-483

876

/

Index

Parathyroidectomy ( c o n t i n u e d ) computed tomography, 475-477, 483-484 magnetic resonance imaging, 475-476, 483-484 MIBI subtractive scintigraphy, 475-476, 483-484 positron emission tomography, 475 single photon emission computed tomography, 476, 483 ultrasound, 475-476 undescended glands, 483-484 sestamibi scan, 453, 475-476, 484-485, 489-490 indications, 451-452, 490 long-term outcomes, 452, 488 medullary thyroid carcinoma and thyroidectomy, 488-489 multiple endocrine neoplasia type 1 management, 539, 556-558 parathyroid carcinoma, 519-520 parathyroid growth implications in hyperparathyroidism management, 318-319 surgical hypoparathyroidism, 319-320 parathyroid hormone intraoperative monitoring, 453, 485 specimen testing, 477-478 venous sampling, 483, 494 postoperative management, 496 preoperative preparation, 491 primary hyperparathyroidism symptom effects bone metabolism, 390, 392 calcium levels, 389-390 cardiovascular complications, 369 diabetes, 368 dyslipidemia, 368 muscle weakness, 367 nephrolithiasis, 367, 387, 393, 444-445 neurobehavioral disturbance, 367 reoperative procedure success, 475 secondary hyperparathyroidism treatment, 654-655 technique concise parathyroidectomy, 495 minimally invasive surgery, 453, 494-495 overview, 493 reoperation, 496 sestamibi-guided parathyroidectomy, 495 standard operation, 493-494 videoscopic cervical exploration, 495 thyroid adenoma distinguishing from parathyroid adenoma, 478 Parathyroid glands anatomic variations location of glands, 1,491 number of glands, 1-2 anatomy, 491 apoptosis, 301, 319 calcium-sensing receptor expression, 131-132, 301 color, 2 development, 1,298-299, 320

ectopic locations inferior parathyroid glands, 502-503, 505-507 overview, 499 superior parathyroid glands, 499-500, 502 supernumary parathyroid glands, 507, 511 embryology, 490 familial hypocalciuric hypercalcemia features, 615-616 fat distribution, 2-4 fine needle aspiration, 10 growth abnormal growth, see Primary hyperparathyroidism; Secondary hyperparathyroidism; Tertiary hyperparathyroidism cell cycle markers, 297-298 cell renewal patterns, 294-296, 300 concepts of growth, 294 doubling time of cell division, 299-300, 313 growth curves, 298-299 rationale for study, 293-294 regulation hypocalcemia, 302-303 trophic stimuli and hormone synthesis, 301 turnover-related mitosis, 300 volume measurement, 296-297 weight measurement, 296 histology chief cell, 2-4 clear cell, 2 oxyphil, 2-3 imaging, see Parathyroidectomy infiltrative damage, 758 intraoperative assessment density gradient measurements, 9 fat stains, 9 pathologist role, 9 morphology, 2 primary hyperplasia, 6 radiation-induced damage, 758 vasculature, 2 weight, 2 Parathyroid hamartoma, features, 8 Parathyroid hormone (PTH) age-related changes in secretion, 835-837 antibody therapy tor hyperparathyroidism management, 466-467 carboxy-terminal role in stability, 20-21 clearance C fragments, 25-26 kidney, 25-26 kinetics, 25 liver, 25-26 conformational flexibility, 53 gene mutation, see Hypoparathyroidism polymorphisms, 21,782 regulation of expression, 23-25, 144 structure, 21,144, 780 TATA box, 21 heart effects, 266-267

immunization in parathyroid carcinoma, hypercalcemia management, 522 immunoassay, see Immunoassay kidney effects, see Kidney magnesium deficiency effects on action, 769-770 neurologic actions, see Central nervous system osteoporosis treatment, see Osteoporosis parathyroid hormone-related protein homology, 21, 31 phorbol ester induction of secretion, 20 preprohormone signal sequence cleavage, 17, 145, 780, 782 homology between species, 18-19 mutations, 18-19 trafficking, 17 prohormone signal sequence cleavage, 17, 19-20, 144 function, 19, 782 trafficking, 17 proteolytic fragments, 26, 145-146 pulsatility of secretion in osteoporosis, 847 receptors, see Receptors, parathyroid hormone regulation of levels calcium, 23-24, 27, 145-146, 171-174 gene expression modulation, 23-25, 707-708 levels of regulation, 21-22 magnesium, 146, 766-769 metabolism, 25-26, 145 parathyroid cell numbers, 22, 172 phosphate, 24, 627-628 steroid effects, 25 vitamin D, 24, 27, 173-174 secretion patterns, 146 vascular effects blood pressure reduction, 261-262, 267 vasodilation concentrations, 262 Parathyroid hormone-related protein (PTHrP) cell translocation mechanisms alternate translation start sites and trafficking, 109-110 receptor-mediated endocytosis, 107-108 retrograde translocation from endoplasmic reticulum, 108-109 developmental roles, 709 endochondral bone formation and chondrocyte effects differentiation role, 249, 712-713 expression, 246 Indian hedgehog interactions, 251-252, 254-257, 713 receptor expression, 249-250 receptor knockout effects, 255, 257 epithelialmesenchyme interaction regulation, 713-714 fetal secretion, 320 functional overview, 262

Index gene CpG islands, 40 locus, 31 promoters, 32, 39-41 regulatory regions, 39-40 species differences, 34 structure, 31-32, 34, 155, 673 glycosylation, 38 heart effects, 266-267 historical perspective of research, 671-672 homology between species, 673 humoral hypercalcemia of malignancy role, see Humoral hypercalcemia of malignancy hyperparathyroidism levels, 684 idiopathic infantile hypercalcemia levels, 684 immunoassay, see Immunoassay kidney expression and effects, 236-237 knockout and transgenic mouse phenotypes, 712-713 localization in embryos, 44-46 metabolism and degradation, 38 neurologic actions, see Central nervous system nuclear/nucleolar localization dual-action hormone examples, 106-107 importin [3 role, 110 nuclear localization sequence, 105-106, 673 nuclear pore complex, 110-111 nucleolar immunoreactivity, 106 nucleolar localization sequence, 110 overview, 63 phosphorylation-dependent regulation, 111 prospects for study, 113-114 ribosomal synthesis rate regulation, 112 transgenic mouse studies of functions, 112-113 osteoclast stimulation at metastatic site, 694-695 pancreas effects, see Pancreas parathyroid hormone homology, 21, 31, 672, 730 processing of prohormone, 20, 34, 36-37, 105, 673-674 proteolysis amino-terminal peptides, 34, 36-37, 105, 156, 673-674 carboxy-terminal peptides, 37, 105, 156, 674 functions of different peptides, 156, 214 midregion peptides, 37, 105, 156, 674 prohormone convertases, 36, 156 receptors, see Receptors, parathyroid hormone regulation of expression developmental regulation, 44-46 DNA methylation, 40 inducers, 41-42, 675 inhibitors, 41, 43, 675 kinetics of induction, 41, 43 neuroexcitation, 43 p53, 39-40

posttranscriptional regulation, 44 tissue-specific promoters, 40-41 transcription factors, 39 tumors, 675-677 reproductive tissue effects, see Mammary gland; Placenta; Uterus secretion, 38 skin effects, see Skin species distribution, 31 splice variants, 32, 34, 155, 673 structure, 34 tissue distribution of expression, 675 tumor expression antitumor therapy effects, 682 breast cancer, 156 gene regulation, 675-677 hematological malignancies, 681-682 lung cancer, 157 non-hypercalcemic patients, 683 overview, 154 prognostic value, 682-683 prostate cancer, 156-157 skeletal metastasis relationship, 679-681 vascular effects, see Blood vessel Parathyroid hormone resistance pseudohypoparathyroidism, 759, 785-786, 807-810 severe magnesium deficiency, 759 Parathyroid lipoadenoma, features, 8 Parathyromatosis, features, 8 Parenteral nutrition, hypercalcemia differential diagnosis, 666 Paricalcitol, secondary hyperparathyroidism treatment, 652 Pernicious anemia, autoimmune hypoparathyroidism association, 796 treatment, 801 Persistent hyperinsulinemic hypoglycemia of infancy, multiple endocrine neoplasia type 1 differential diagnosis, 547-548 PET, see Positron emission tomography Pheochromocytoma multiple endocrine neoplasia type 1,543, 553, 561 multiple endocrine neoplasia type 2, 587-588, 596 Phosphate acute hypercalcemia management, 738 binding agents aluminum toxicity in gels, 645-646 calcium carbonate, 646 sevelamer, 646-647 dietary reduction for secondary parathyroidism, 644-645 familial hypocalciuric hypercalcemia levels, 612 gastrointestinal absorption, 174 hyperparathyroidism management, 460-461 kidney excretion regulation, 233-234 reabsorption, 232-233 nephrolithiasis management, 448

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877

parathyroid cell number regulation, 22, 628 parathyroid crisis management, 468 parathyroid hormone expression regulation, 24, 627-628 renal bone disease, serum levels, 640 retention role in secondary hyperparathyroidism and renal insufficiency calcitriol production effects, 626-627 evidence, 625-626 hypocalcemia induction, 626 parathyroid cell growth effects, 628 parathyroid hormone secretion regulation, 627-628 skeletal resistance to parathyroid hormone, 628, 631 serum levels in hyperparathyroidism, 356 sodium/phosphate cotransporters, 232-233 vitamin D level effects, 460-461 Phospholipase C (PLC) G protein coupling, 122-123 subtypes, 123 Phosphorous, see Phosphate Pituitary tumor, multiple endocrine neoplasia type 1,542-543, 553, 555, 561 P K A , see Protein kinase A PKC, see Protein kinase C Placenta calcium-sensing receptor expression, 283 knockout mouse phenotype, 283 parathyroid hormone-related protein calcium transport regulation, 282-283 expression distribution, 284, 684 regulation of expression, 284 PLC, see Phospholipase C Plicamycin acute hypercalcemia management, 468, 737-738 myeloma bone disease management, 700-701 parathyroid carcinoma, hypercalcemia management, 521 Positron emission tomography (PET), parathyroid imaging, 475 PRAD1

B cell lymphoma rearrangements, 339 breast cancer role, 339 cell cycle role, 336-337 cloning, 334-335 cyclin-dependent kinase partners, 336 locus, 337 oncogenicity, 337, 339 parathyroid adenoma mutations overexpression, 338-339 rearrangements, 10, 333-335, 337-339 point mutations, 337 protein homology analysis, 335-336 Pregnacy, see a l s o Placenta; Uterus acute primary hyperparathyroidism association, 530-531

878

/

Index

Pregnacy, see also Placenta; Uterus ( c o n t i n u e d ) primary hyperparathyroidism management, 469 pseudohypoparathyroidism management, 82O Primary hyperparathyroidism, see also Multiple endocrine neoplasia; Parathyroid adenoma; Parathyroid carcinoma acute disease, see Acute primary hyperparathyroidism age at diagnosis developing countries, 383 peak, 350 trends, 306 asymptomatic disease, 352, 357, 395, 451 blood pressure effects, 356, 368-369, 380, 394 calcium levels changes over time, 388 hypercalciuria, 355-356, 380, 389 intake recommendations, 388 parathyroidectomy effects, 389-390 serum, 4, 349, 351,356, 363, 380, 389 cardiovascular effects, 356, 368-369, 394 chloride levels, 356 clinical manifestations by region developing countries, 375-384 Europe, 361-370 United States, 349-358 course, 364, 387-396 definition, 305-306 diabetes association, 367-368, 394 diagnostic criteria, 364-365 dyslipidemia, 368 etiology, 4 familial forms, see also Familial hypocalciuric hypercalcemia adenoma, 602,604 carcinoma, 602-603 hyperparathyroidism-j aw tumor syndrome, 601-602 hyperplasia, 602, 604-605 hypercalcemia differential diagnosis, 663 incidence trends, 349-350, 376 insulin-like growth factor binding protein-3 levels, 418 interleukin-l[3 levels, 418 interleukin-6 levels, 416-418 interleukin-11 levels, 417 leukotriene B4 levels, 418 lithium association, 388-389 management, see also Parathyroidectomy; specific d r u g s

compliance, 469 diet, 454, 459-460 exercise, 459-460 medical management principles, 459-460 medical surveillance, 454 National Institutes of Health guidelines, 384, 451-452 pregnancy, 469 prospects, 469-470 mortality, 395

multiple endocrine neoplasia type 1 age of onset, 538 differential diagnosis, 538 etiology, 544 gastrinoma interaction, 539-540 management medical supervision, 556 parathyroidectomy, 539, 556-558 pharmacotherapy, 556 penetrance, 537, 544 sex ratio, 539 multiple endocrine neoplasia type 2, 588, 596 muscle weakness, 355, 365, 367, 378, 394-395 nephrolithiasis, see Nephrolithiasis neuropsychiatric manifestations and surgical outcomes, 356, 365, 367, 393-394 nonparathyroid cancers, 395 parathyroidectomy effects on symptoms, 367-369 parathyroid growth, see also Parathyroid adenoma clonality of tumors, 310 disease course as function of growth rate and extent, 305-308 patterns of growth asymptotic growth, 309 birth rates of adenoma cells, 310-311 gompertzian growth, 309 growth curves, 308-309 pathogenesis implications, 311-315 relationship between cell number, hormone secretion, and calcium, 303-305 therapeutic implications, 318-319 vitamin D deficiency effects, 313-315 parathyroid hormone immunoassay, 150-151,356, 380, 389 pathology in developing countries, 382-383 phosphorous levels, 356, 389 physical examination, 352 premature death, 369-370 prevalence developing countries, 376-377 Europe, 362-363 United States, 4, 350 primary parathyroid hyperplasia chief cell, 6 clear cell, 6 gland numbers, 351 screening, 363, 378 sex differences, 363, 377 skeletal disease, see Bone symptoms and signs, 352, 355-356 tumor necrosis factor-et levels, 417-418 types, 307-308, 361-362, 364, 387 vitamin D levels, 356-358, 362, 376, 383-384 Procollagen propeptides, bone formation markers and hyperparathyroidism, 402, 404 Progestins, hyperparathyroidism management, 461

Prolactinoma, multiple endocrine neoplasia type 1,544 Propranolol, parathyroid hormone secretion inhibition, 447, 466 Prostaglandin E2, parathyroid hormone interactions in bone growth, 192, 215, 414 Protein kinase A (PKA), parathyroid hormone receptor, ligand activation domain, 62-63 Protein kinase C (PKC), parathyroid hormone receptor, ligand activation domain, 62 Pruritis, renal bone disease association, 637 Pseudohyperparathyroidism clinical features, 642, 785, 807 diagnosis laboratory findings, 817-818 natural history, 817 parathyroid hormone infusion test, 807-808, 818-819 physical examination, 817 parathyroid hormone resistance, 759, 785-786, 807-810 pathophysiology calcitriol role, 809-810 hormone inhibitors, 810 type 1,807-810 type 2, 808 treatment calcium, 819-820 pregnancy considerations, 820 vitamin D, 819-820 types overview, 785-786, 807-808, 810 type la Albright's hereditary osteodystrophy, 807, 810, 813-815 differential diagnosis, 814-815 G protein mutations, 785-786, 810-813, 815-816 multiple hormone resistance, 813 neurosensory defects, 813 obesity, 813-814 phenotype, 813-815 phenotypic variability, 815-816 type 1b features, 816 type 1c features, 816 type 2 features, 81 6-817 PTH, see Parathyroid hormone PTH 1-Rc, see Receptors, parathyroid hormone PTH2-Rc, see Receptors, parathyroid hormone PTHrP, see Parathyroid hormone-related protein Pyridinoline, bone resorption marker and hyperparathyroidism, 402-403, 405 R

R-568, hyperparathyroidism management, 467, 654 RANKL, expression induction and function in osteoclasts, 176-177, 207, 216 Rb, see Retinoblastoma gene Receptors, parathyroid hormone

Index activation domain interactions with receptor, 54 antagonists for hyperparathyroidism management, 467 bone cell expression osteoblasts and signaling thresholds, 204-205, 213-214 osteoclasts, 214 function, 672 conformation of ligand cyclic analogs, 61 design of analogs, 60-61 long-range helix-helix interactions, 57 membrane-induced conformation, 57 nuclear magnetic resonance, 57, 59 parathyroid hormone-related protein helicity, 59 rationale for study, 56-57 RS-66271, 60-61 secondary structure prediction, 57 trifluoroethanol solvent studies, 57, 59 disease mutations Blomstrand chondrodysplasia, 70, 99-100, 247, 707, 718, 720 Jansen's metaphyseal chondrodysplasia, 69-70, 100, 247, 707, 715, 717-718 knockout mouse phenotype, 249, 721 signaling defects, 100 down-regulation endocytosis, 98 induction, 98 mutant receptor studies, 98-99 phosphorylation role, 99 G proteins alpha subunit functional diversity, 121 homology, 120 mammalian diversity, 118 protein interactions, 120-121 switch regions, 120 beta-gamma subunit heterodimers, 121-122 beta subunits, 121 clinical implications of dysfunction, 123 effector coupling, 122-123 gamma subunits, 121 GTPase cycle, 119 receptor coupling, 122 types, 53-54, 93-94, 96, 117-118 gene expression regulation, 99, 709, 711 kidney calcium and magnesium excretion regulation, 232 desensitization, 228-229 distribution of receptor expression, 227-228, 708 phosphate excretion regulation, 233-234 regulation of expression, 229 signal transduction, 228, 232-235 sodium and hydrogen excretion regulation, 235 vitamin D metabolism regulation, 235-236, 708 message domain, 59-60 molecular modeling

cross-linking data incorporation, 78-80, 82 evaluation principles of model quality, 83 extracellular domains, 77-78 ligand interactions, 78-80, 82 overview, 76 sequence homology searching, 77-78 transmembrane helix identification, 76-77, 79 mutagenesis studies of ligand sequences, 55-56 osteoporosis, therapeutic targeting, 83 phosphorylation desensitization role, 97, 122 inducers, 97-98 kinases, 97, 101,122 photoaffinity labeling studies contact sites position 1 of agonists versus antagonists, 75-76 position 1 of parathyroid hormone, 74 position 13 of parathyroid hormone, 74 position 23 of parathyroid hormonerelated protein, 75 position 27 of parathyroid hormone, 74-75 mutagenesis studies, 72 photoaffinity spanning principle, 71 photoreactive ligands benzophenone analogs, 72-73 design, 63, 71-72 radioiodination, 72 rationale, 70-71 sequence alignment, 65 signal transduction, see also G protein G protein-coupled receptors, 94, 117-118 overview, 61-62, 709 second messengers, 117 tail signaling, 96-97 transmembrane signaling, 95-97 signaling-selective ligands anabolic versus catabolic activity, 62 Ostabolin, 62 protein kinase A activation domain, 62-63 protein kinase C activation domain, 62 subtype specificity switching, 56 TIP39 binding, 56, 268-269, 711 topology, 53, 93 truncated ligand studies, amino terminal activity, 54-55 type ! receptor activating mutations, 717-718 calcitonin receptor chimera studies, 66 gene structure, 709, 711 glycosylation, 64 inactivating mutations, 718, 720 interspecies chimera studies, 66-67 ligand specificity, 672, 707, 709 mutagenesis studies, 67-68, 717 phylogenetic analysis of receptor gene family, 64, 93 promoters, 711

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879

splice variants, 64 tissue distribution, 63-64 type II receptor chimera studies, 69 ligand specificity, 68 ligand specificity, 672, 711 mutagenesis studies, 69 tissue distribution and function, 68-69 type III receptor, 69, 214 Renal bone disease, see also Bone; Secondary hyperparathyroidism aluminum-related bone disease differential diagnosis, 642-643 classification, 635-636 clinical manifestations aplastic or adynamic bone, 639 bone pain, 636 calciphylaxis, 637 deformity of bone, 637 destructive spondyloarthropathy, 639 dialysis amyloidosis, 638-639 growth retardation, 638 joint pain, 636 proximal myopathy, 636-637 pruritis, 637 tendon rupture, 637 diagnosis bone biopsy, 640 osteocalcin marker, 643 parathyroid hormone immunoassay, 639-643 serum levels alkaline phosphatase, 640-641 calcium, 640 phosphorous, 640 vitamin D levels, 643 parathyroid hormone immunoassay, 152-154 radiography bone scintiscan, 644 X-ray, 643-644 treatment calcimimetic agents, 653-654 calcium supplementation, 645, 647 parathyroidectomy, 496, 654-655 phosphate-binding agents, 645-647 phosphorous reduction in diet, 644-645 vitamin D sterols active agents, 647 alfacalcidol, 648 calcitriol and dosing, 647-650 doxercalciferol, 652-653 falecalcitriol, 651-652 22-oxacalcitriol, 651 paricalcitol, 652 structures, 650-651 Renal insufficiency, see Secondary hyperparathyroidism Renal tubular acidosis, nephrolithiasis pathogenesis, 444 R E T , see Multiple endocrine neoplasia type 2 Retinoblastoma gene (Rb) cell cycle role, 336-337 inactivation in parathyroid tumorigenesis, 10-11

880

/

Index

Retinoblastoma gene immunostaining of protein in parathyroid tumors, 519 parathyroid carcinoma mutation, 516 Risedronate, hyperparathyroidism management, 465 S

Sarcoidosis, hypercalcemia differential diagnosis, 665-666, 749 SCF, see Stem cell factor Secondary hyperparathyroidism aging in etiology, 839-840 bone disease, see Renal bone disease calcitriol synthesis in kidney, 628-629 causes, 315 definition, 305-306 etiology, 9 hypocalcemia role in renal insufficiency, 631 interleukin-6 levels, 415 parathyroid function in renal insufficiency abnormal gland growth, 630 aluminum toxicity, 631-632 calcitriol role, 629-630 clinical correlations, 630-631 hypocalcemia role, 629 parathyroid growth calcium regulation, 316-317 calcum-sensing receptor underexpression, 31 6-317 hyperplasia, 625 parathyroid hormone secretory set point, 316 relationship between cell number, hormone secretion, and calcium, 303-305 renal failure patients, 315-317 therapeutic implications, 318 parathyroid hormone immunoassay hypocalcemia, 152 renal osteodystrophy, 152-153 parathyroid transplantation, 9 phosphate retention role in renal insufficiency calcitriol production effects, 626-627 evidence, 625-626 hypocalcemia induction, 626 parathyroid cell growth effects, 628 parathyroid hormone secretion regulation, 627-628 skeletal resistance to parathyroid hormone, 628, 631 radiography bone scintiscan, 644 X-ray, 643-644 treatment calcimimetic agents, 653-654 calcium supplementation, 645,647 parathyroidectomy, 654-655 phosphate-binding agents, 645-647 phosphorous reduction in diet, 644-645 vitamin D sterols active agents, 647

alfacalcidol, 648 calcitriol and dosing, 647-650 doxercalciferol, 652-653 falecalcitriol, 651-652 22-oxacalcitriol, 651 paricalcitol, 652 structures, 650-651 Selective estrogen receptor modulators (SERMs), hyperparathyroidism management, 455, 462 SERMs, see Selective estrogen receptor modulators Sestamibi guided parathyroidectomy, 495 inferior parathyroid gland imaging, 505, 507 parathyroid scan, 453, 475-476, 484-485, 489-490 reoperative parathyroid surgery imaging with MIBI subtractive scintigraphy, 475-476, 483-484 Shh, see Sonic hedgehog Single photon emission computed tomography (SPECT), parathyroid imaging, 476, 483 Skin, parathyroid hormone-related protein function expression localization, 275 regulation, 275 fibroblast effects, 277 hair growth, 276, 713 isoforms, 276 keratinocyte differentiation and proliferation, 276-277 pathophysiology, 277 receptor expression, 275-276 Sodium, parathyroid hormone and renal excretion effects, 234-235 Somatostatinoma, multiple endocrine neoplasia type 1,541 Sonic hedgehog (Shh) oncogenicity, 256 tissue patterning role, 250 SPECT, see Single photon emission computed tomography Stem cell factor (SCF), parathyroid hormone interactions, 413 T Tartrate-resistant acid phosphatase (TRAP), bone resorption marker and hyperparathyroidism, 403, 405 Tendon rupture, renal bone disease association, 637 Tertiary hyperparathyroidism features, 10 hypercalcemia differential diagnosis, 663 parathyroid growth calcium malabsorption effects, 317-318 hypophosphatemic osteomalacia patients, 318 relationship between cell number, hormone secretion, and calcium, 303-305 parathyroid hormone immunoassay, 153

TGF-[3, see Transforming growth factor-[3 Thiazide diuretics hypercalcemia association, 388, 664 hyperparathyroidism avoidance, 459 Thyroid follicular adenoma, multiple endocrine neoplasia type 1,543 Thyrotoxicosis, hypercalcemia differential diagnosis, 666 TIP39 cerebral cortex expression, 268-269 parathyroid hormone receptor binding, 56, 268-269, 711 Transforming growth factor-J3 (TGF-[3) bone growth factor, 184, 215 parathyroid hormone activation, 187, 415 tumor growth factor in bone metastasis, 695 TRAP, see Tartrate-resistant acid phosphatase Tuberculosis, hypercalcemia differential diagnosis, 666, 749 Tumor necrosis factor-or, primary hyperparathyroidism levels, 417-418 U Ultrasound inferior parathyroid gland, 505 parathyroid imaging, 475-476 Uterus, parathyroid hormone-related protein expression distribution, 283 fetal membrane effects, 284 implantation role, 284-285 motility role, 283 muscle relaxation fimction, 283-284 V Vascular endothelial growth factor (VEGF), endochondral bone formation role, 246-248, 257, 413 Vascular smooth muscle cell, see Blood vessel Vasoactive intestinal peptide-secreting tumors multiple endocrine neoplasia type 1,541 VEGF, see Vascular endothelial growth factor Vitamin A intoxication, hypercalcemia association in children, 748 Vitamin D, see a l s o Calcitriol calcium gastrointestinal absorption effects, 174 calcium homeostasis regulation, 171 complications of therapy, 832 deficiency aging association, 839, 843 effects on parathyroid growth, 313-315 familial hypocalciuric hypercalcemia levels, 614-615 hyperparathyroidism management with analogs, 466 hypocalcemia and metabolism disorders, 759-760 hypoparathyroidism management, 828-832 intoxication and hypercalcemia differential diagnosis, 665, 746, 749

Index magnesium deficiency effects on metabolism and action, 770-771 metabolism regulation in kidney by parathyroid hormone, 235-236 multiple endocrine neoplasia type 1 differential diagnosis, 546 muscle weakness treatment, 636-637 nephrolithiasis levels, 438, 442 osteoporosis and metabolism alterations, 844-845 parathyroid cell number regulation, 22 parathyroid hormone expression regulation, 24, 27, 173-174 parathyroid hormone-related protein regulation, 41, 43 phosphate therapy effects on levels, 460-461

phosphorous effects on metabolism, 847 primary hyperparathyroidism levels, 356-358, 362, 376, 380, 383-384 pseudohyperparathyroidism treatment, 819-820 renal bone disease, serum levels, 643 secondary hyperparthyroidism management active sterols, 647 alfacalcidol, 648 calcitriol and dosing, 647-650 doxercalciferol, 652-653 falecalcitriol, 651-652 22-oxacalcitriol, 651 paricalcitol, 652 structures, 650-651 von Hippel-Lindau syndrome

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881

W

Williams syndrome clinical manifestations, 746-747 gene mutations, 747 hypercalcemia in children, 746 WR-2721 hyperparathyroidism management, 466 parathyroid carcinoma, hypercalcemia management, 522 X

X-chromosome inactivation, parathyroid adenoma analysis, 333 Z

Zoledronate, hypercalcemia management, 736

ISBN

0-12-098651-5 90038

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20 986