Metabolic Bone Disease and Clinically Related Disorders, Third Edition

Metabolic Bone Disease Third Edition

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Metabolic Bone Disease and C1inically Re1at ed Disorders

Edited by

LOUISV. AVIOLI Washington University Medical Center St. Louis, Missouri

STEPHEN M. KRANE Harvard Medical School Massachusetts General Hospital Boston, Massachusetts

ACADEMIC PRESS San Diego

London

Boston

New York

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Toronto

This book is printed on acid-flee paper.

Copyright 9 1998, 1990, 1977 by ACADEMIC PRESS All 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 permission in writing from the publisher. Academic Press a division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http ://www. apnet.com Academic Press Limited 24-28 Oval Road, London NW 1 7DX, UK http ://www.hbuk.co.uk/ap/ Library of Congress Card Catalog Number: 97-074394 International Standard Book Number: 0-12-068700-3

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Contents

Contributors Preface

xi

The Nature of the Mineral Phase in Bone" Biological and Clinical Implications

CHAPTER 2

xv

MELVIN J. GLIMCHER

II. III.

CHAPTER 1

Embryology and Cellular Biology of Bone

LAWRENCE G. RAISZ AND GIDEON A. RODAN

2 I~ Embryonic Skeletal Development II. Limb Development and Pattern Regulation 2 III. Bone Morphogenetic Proteins and Development 4 IV. The Role of Parathyroid Hormone-Related Peptide in Development 4 V~ Fibroblast Growth Factors and Skeletal Development 4 5 VI. Cells of the Osteoblast Lineage 9 VII. The Osteoclast VIII. Cell-Cell Interaction in Bone Remodeling 12 Colony-Stimulating Factors and Bone 13 IX. X. The Transforming Growth Factor [3 Family 13 XI. Other Growth Factors (FGE VEGE PDGF, and HGF) 15 16 XII. Cell-Matrix Interactions References 17

IV. V.

Biological Functions of the Mineral Phase 23 The General Nature of the Mineral Phase in Bone and the Changes That Occur with Time 28 Postulated Phases Other Than Apatite as the Initial Solid Ca-P Mineral Phase Deposited in Bone 29 Crystal Size and Shape 32 Recent Studies of the Structure of Bone Apatites and the Applications of These Data to Clinical and Experimental Abnormalities and Diseases of Bone 36 References 46

CHAPTER 3 Parathyroid Hormone and Parathyroid Hormone-Related Peptide in Calcium Homeostasis, Bone Metabolism, and Bone Development: The Proteins, Their Genes, and Receptors JOHN T. PoTrs, JR. AND HARALD JUPPNER

I. Introduction: Regulators of Mineral Ion Homeostasis 52 53 II. Parathyroid Hormone 67 III. Parathyroid Hormone-Related Peptide IV. Receptors That Mediate Analogous and Distinct Molecular Actions of PTH and PTHrP 73 V~ Summary: Overall Biological Roles of PTH and PTHrP and Their Cloned Receptors 82 References 83

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Contents

CHAPTER 4

CHAPTER 7

Calcitonin

Z. J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

Io Nature of Calcitonin

II. III. IV. V. VI. VII. VIII.

96

Chemistry 98 Biosynthesis 99 Secretion and Metabolism 101 Actions of Calcitonin 102 Calcitonin Receptor 108 Calcitonin in Clinical Medicine Summary 113 References 114

Disorders of Phosphate Homeostasis

KEITH HRUSKAAND ANANDARUPGUPTA I. Phosphate Homeostasis II. Hypophosphatemia III. Hyperphosphatemia References 230

CHAPTER 8

113

207 216 226

Bone Biopsies: A Modern Approach

MARIE=CLAUDE MONIER-FAUGERE, M. CHRIS LANGUB, AND HARTMUT H. MALLUCHE

Io Function and Structure of the Skeleton

CHAPTER 5 Vitamin D Metabolism and Biological Function MICHAEL F. HOLICK AND JOHN S. ADAMS

I. II. III. IV. Wo

VI. VII. VIII. IX. X.

XI. XlI.

History of Vitamin D 124 Photobiology of Vitamin D3 127 Intestinal Absorption of Vitamin D 131 Metabolism of Vitamin D to 25-Hydroxyvitamin D 132 Metabolism of 25-Hydroxyvitamin D to 1,25-Hydroxyvitamin D 135 Altemative Metabolism of 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D 139 Metabolism of Vitamin D2 141 Biological Actions of 1,25(OH)2D 142 Biological Actions of 1,25(OH)2D in Tissues Regulating Calcium Balance 144 Actions of Vitamin D Metabolites and Analogs in Nonclassical Target Tissues 145 Assays for Vitamin D and Its Metabolites 148 Conclusion 155 References 156

237

II. III. IV. V. VI.

Bone Biopsies 244 247 Mineralized Bone Histology Techniques Molecular Bone Histology 249 Evaluation of Bone 251 Indications For and Information Derived from Bone Biopsies 256 VII. Information Derived from Molecular Histology 267 References 269

CHAPTER 9

Noninvasive Assessment of Bone

MARTIN UFFMANN, THOMAS e. FUERST, MICHAEL JERGAS, AND HARRY K. GENANT

I. II. III. IV.

Introduction 275 Radiation-Based Assessment of Bone 277 Assessment of Bone without Radiation 292 Standardization and Quality Assurance in Dual X-Ray Absorptiometry 298 V. Clinical Applications 301 References 303

CHAPTER 10 Biochemical Markers of Bone Turnover CLAUS CHRISTIANSEN, CHRISTIAN HASSAGER, AND BENTE JUEL RIIS

CHAPTER 6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption ROBERTO CIVITELLI, KONSTANTINOS ZIAMBARAS, AND RATTANA LEELAwATTANA

I. Calcium II. Phosphate III. Magnesium References

165 183 190 195

I. Introduction 313 II. Bone Matrix, Minerals, and Cells 314 III. Bone Tumover: Modeling and Remodeling 314 IV. Biochemistry of Bone Turnover 315 V. Markers of Bone Formation 316 VI. Markers of Bone Resorption 318 VII. 24-Hour Variation in Markers of Bone Tumover 321

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Contents VIII. Potential Use of Biochemical Markers IX. Conclusions 324 References 324

321

CHAPTER 14

EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ Io

CHAPTER 11

Osteomalacia and Related Disorders

IV. Wo

VI. VII. VIII.

Introduction

443

II. Secondary Hyperparathyroidism and Osteitis Fibrosa 443 III. Osteomalacia

451

IV. Bone Histology

A. M. PARFITT

II. III.

Renal Osteodystrophy

Bone Mineralization and the Mechanisms of Osteoid Accumulation 328 Manifestations of Osteomalacia 338 Etiological Classification and Pathogenesis of Osteomalacia 349 Osteomalacia Resulting from Abnormal Vitamin D Metabolism 354 Vitamin D and Age-Related Osteoporosis 360 Osteomalacia Resulting from Abnormal Phosphate Metabolism 361 Osteomalacia with Normal Vitamin D and Phosphate Metabolism 367 Therapeutic Intervention in Osteomalacia 370 References 374

451

V. Clinical and Biochemical Features of Altered Divalent-Ion Metabolism 453 VI. Radiographic Features of Renal Osteodystrophy 455 VII. Extraskeletal Calcifications

456

VIII. Therapeutic Approach to Renal Osteodystrophy 456 References

460

CHAPTER 15 Surgical Treatment for Hyperparathyroidism GERARD M. DOHERTY AND SAMUEL A. WELLS, JR. I. History of Surgery for Hyperparathyroidism

465

II. Primary Hyperparathyroidism: Preoperative Evaluation 466

CHAPTER 12

Osteoporosis Pathogenesis and Therapy

MICHAEL KLEEREKOPER AND LOUIS V. AVIOLI

I. II. III. IV. V.

Definition 387 Physiological Osteoporosis Diagnostic Aids 390 Classification 395 Management 397 References 406

389

III. Primary Hyperparathyroidism: Conduct of the Operation 469 IV. Primary Hyperparathyroidism: Management of Patients with Persistent or Recurrent Hyperparathyroidism 472 V. Operative Management of Patients with Parathyroid Carcinoma 475 VI. Operative Management of Patients with Renal Osteodystrophy 475 VII. Postoperative Management References

CHAPTER 13 Primary Hyperparathyroidism JOHN T. POTTS, JR. I. Introduction 411 II. Etiology and Pathology 412 III. Clinical Features: Changing Clinical Presentation 418 IV. Diagnosis and Differential Diagnosis 426 V. Medical Management of Hypercalcemia and Hyperparathyroidism 431 VI. Summary 435 References 435

475

477

CHAPTER 16 Familial Benign Hypocalciuric Hypercalcemia and Other Syndromes of Altered Responsiveness to Extracellular Calcium EDWARD M. BROWN, MEI B AI, AND MARTIN POLLAK I. Introduction

479

II. Syndromes of Extracellular Calcium Resistance 480 III. Autosomal Dominant HypocalcemiamA Syndrome of Increased Responsiveness of Target Tissues to Ca o2+ 493

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Contents

IV. Summary and Conclusions References 496

496

CHAPTER 20

Sarcoidosis and Related Disorders NORMAN H. BELL

CHAPTER 17 Hypoparathyroidism and Pseudohypoparathyroidism MICHAEL A. LEVINE

I. II. III. IV. V. VI. VII. VIII.

Introduction 501 Pathophysiology of Hypocalcemia Signs and Symptoms of Hypocalcemia Specific Causes of Functional Hypoparathyroidism 505 Pseudohypoparathyroidism 510 Diagnosis 519 Treatment 521 Conclusion 522 References 522

I. Granulomatous and Infectious Diseases II. Lymphoma and Solid Tumors 613 III. Miscellaneous Diseases 614 References 615

607

502 504

CHAPTER 21 Bone Disease in Rheumatological Disorders STEVEN R. GOLDRING AND RICHARD P. POLISSON

I. II. III. IV. V.

CHAPTER 18 Bone Disease in Hyperthyroidism

Introduction 621 Rheumatoid Arthritis 622 626 Seronegative Spondyloarthropathies 628 Systemic Lupus Erythematosus 628 Juvenile Rheumatoid Arthritis References 633

DOUGLAS S. R o s s

531 I~ Grades of Hyperthyroidism 532 II. Overt Hyperthyroidism III. Endogenous Subclinical Hyperthyroidism 534 IV. Exogenous Subclinical Hyperthyroidism: Thyroid Hormone Suppressive Therapy 535 539 V~ Thyroid Hormone Replacement Therapy Treatment and Prevention of Thyroid HormoneVI. Mediated Bone Loss 539 541 VII. Conclusions References 541

CHAPTER 22 Hypercalcemia of Malignancy GREGORY R. MUNDY

I. Pathophysiology 637 II. Clinical Features 642 III. Treatment 643 References 646

CHAPTER 23 CHAPTER 19

I~ Introduction

545 Incidence and Epidemiology 545 Histopathology 547 Focal Manifestations 554 Local Complications 562 Metabolic Aspects of Paget's Disease Systemic Complications and Associated Diseases 578 581 VIII. Drug Treatment IX. Surgery 592 593 X. Etiology References 596

II. III. IV. V.

I~ Historical Aspects

II. III. IV. V. VI. VII.

DAVID W. ROWE AND JAY R. SHAPIRO

Paget's Disease of Bone

FREDERICK R. SINGER AND STEPHEN M. KRANE

Osteogenesis Imperfecta

VI. 569 VII. VIII. IX.

651 Perspective: Clinical Introduction 651 Clinical Features 657 Pathophysiology 663 Collagen Biochemistry and Bone Cell Biology as Related to OI 666 Biochemical and Molecular Tools for the Identification of Mutations in Patients with OI 670 Molecular Pathophysiology of OI 673 Therapy 678 Future Diagnostic and Therapeutic Directions 681 References 683

Contents

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CHAPTER 24 Skeletal Disorders Characterized by Osteosclerosis or Hyperostosis MICHAEL P. WHYTE

I~ Introduction

II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII.

697 Osteopetrosis 698 Carbonic Anhydrase II Deficiency 703 Pycnodysostosis 705 Osteomesopyknosis 707 Progressive Diaphyseal Dysplasia (CamuratiEngelmann Disease) 707 Endosteal Hyperostosis 710 Osteopoikilosis 713 Osteopathia Striata 715 Melorheostosis 716 Mixed Sclerosing Bone Dystrophy 718 Fibrodysplasia Ossificans Progressiva 719 Axial Osteomalacia 722 Fibrogenesis Imperfecta Ossium 726 Fluorosis 728 Pachydermoperiostosis 729 Hepatitis C-Associated Osteosclerosis 731 Other Disorders 731 References 732

CHAPTER 25 Kidney Stones: Pathogenesis, Diagnosis, and Therapy CHARLES Y. C. PAK I. Introduction 739 740 II. Hypercalciuria 747 III. Hyperuricosuria 748 IV. Hyperoxaluria 750 V. Hypocitraturia 752 VI. Gouty Diathesis 753 VII. Cystinuria VIII. Infection with Urea-Splitting Organisms 755 IX. Conservative Management References 756

CHAPTER 26

755

Metabolic Bone Disease in Children

FRANCIS H. GLORIEUX, GERARD KARSENTY, AND RAJESH V. THAKKER

I. Introduction 759 II. Skeletal Development III. Rickets and Osteomalacia References 777

Index

785

759 764

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Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Gerard M. Doherty (465) Section of Endocrine and Oncologic Surgery, Washington University School of Medicine, St. Louis, Missouri 63110 D. M. Findlay (95) St. Vincent's Institute of Medical Research and The University of Melbourne Department of Medicine, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia Thomas P. Fuerst (275) Osteoporosis & Arthritis Group, Department of Radiology, University of California, San Francisco, San Francisco, California 94143 Harry K. Genant (275) Osteoporosis & Arthritis Research Group, Department of Radiology, University of California, San Francisco, San Francisco, California 94143 Melvin J. Glirneher (23) Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopaedic Surgery, Harvard Medical School, Children's Hospital, Boston, Massachusetts 02115 Francis H. Glorieux (759) Genetics Unit, Shriners Hospital for Children and Departments of Surgery and Pediatrics, McGill University, Montr6al, Qu6bec H3G 1A6, Canada Steven R. Goldring (621) Department of Medicine Harvard Medical School, Medical Services (Arthritis Unit), Massachusetts General Hospital, Boston, Massachusetts 02215; Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115; and New England Baptist Bone and Joint Institute, Boston, Massachusetts 02215

John S. Adams (123) University of California, Los Angeles, Los Angeles, California 90048 Louis V. Avioli (387) Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri 63110 Mei Bai (479) Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115 Norman H. Bell (607) Departments of Medicine and Pharmacology, Medical University of South Carolina and Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, South Carolina 29401 Edward M. Brown (479) Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115 Claus Christiansen (313) Center for Clinical and Basic Research, Ballerup, Denmark Roberto Civitelli (165) Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri 63110 James A. Delmez (443) The Renal Division and Chromalloy American Kidney Center, Washington University School of Medicine, St. Louis, Missouri 63110

xi

xii

Anandarup Gupta (207) Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110 Christian Hassager (313) Center for Clinical and Basic Research, Ballerup, Denmark Michael F. Holick (123) Boston University Medical Center, Boston, Massachusetts 02118 Keith Hruska (207) Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110 Michael Jergas (275) Department of Radiology, St. Josef Hospital, Ruhr-University of Bochum, Bochum, Germany Harald Jiippner (51) Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 Gerard Karsenty (759) Molecular Genetics, The University of Texas, MD Anderson Cancer Center, Houston, Texas 77030 Michael Kleerekoper (387) Department of Medicine, Wayne State University, Detroit, Michigan 48201 Stephen M. Krane (545) Department of Medicine, Harvard Medical School and Arthritis Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 M. Chris Langub (237) Division of Nephrology, Bone and Mineral Metabolism, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky 40536 Rattana Leelawattana (165) Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri 63110 Michael A. Levine (501) Division of Endocrinology and Metabolism, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Hartmut H. Malluche (237) Division of Nephrology, Bone and Mineral Metabolism, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky 40536 T. J. Martin (95) St. Vincent's Institute of Medical Research and The University of Melbourne Department of Medicine, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia

Contributors

Marie-Claude Monier-Faugere (237) Division of Nephrology, Bone and Mineral Metabolism, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky 40536 J. M. Moseley (95) St. Vincent's Institute of Medical Research and The University of Melbourne Department of Medicine, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia Gregory R. Mundy (637) University of Texas Health Science Center, San Antonio, Texas 78284 Charles Y. C. Pak (739) University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 A. M. Parfitt (327) Division of Endocrinology and Center for Osteoporosis and Metabolic Bone Disease, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Richard P. Polisson (621) Department of Medicine Harvard Medical School, Medical Services (Arthritis Unit), Massachusetts General Hospital, Boston, Massachusetts 02215; Department of Medicine, Beth-Israel Deaconess Medical Center, Boston, Massachusetts 02215; and New England Baptist Bone and Joint Institute, Boston, Massachusetts 02215 Martin Pollak (479) Renal Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115 John T. Potts Jr. (51, 411) Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 Lawrence G. Raisz (1) Division of Endocrinology and Metabolism, University of Connecticut Health Center, Farmington, Connecticut 06030 Bente Juel Riis (313) Center for Clinical and Basic Research, Ballerup, Denmark Gideon A. Rodon (1) Department of Bone Biology/Osteoporosis, Merck Sharp & Dohme Research Labs, West Point, Pennsylvania 19486 Douglas S. Ross (531) Thyroid Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 David W. Rowe (651) Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut 06032

Contributors

P. M. Sexton (95) St. Vincent's Institute of Medical Research and The University of Melbourne Department of Medicine, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia Jay R. Shapiro (651) Department of Medicine, Johns Hopkins Medical School, Baltimore, Maryland 21224 Frederick R. Singer (545) John Wayne Cancer Institute, Santa Monica, California 90404 Eduardo Slatopolsky (443) The Renal Division and Chromalloy American Kidney Center, Washington University School of Medicine, St. Louis, Missouri 63110 Rajesh V. Thakker (759) MRC Molecular Endocrinology Group, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 0NN, United Kingdom

xiii

Martin Uffmann (275) Osteoporosis & Arthritis Research Group, Department of Radiology, University of California, San Francisco, San Francisco, California 94143 Samuel A. Wells, Jr. (465) Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110 Michael P. Whyte (697) Divisions of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri 63110; and Metabolic Research Unit, Shriners Hospital for Children, St. Louis, Missouri 63131 Konstantinos Ziambaras (165) Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri 63110

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Preface

and monocytes, whereas when c-fos is knocked out, monocyte-macrophages are produced but osteoclasts are not. Since the knockout of c-src surprisingly resulted only in osteopetrosis with osteoclasts present in bone but functionally defective with no ruffled border, we await the elucidation of the precise role of c-src in osteoclast function. Knowledge of how osteoclasts utilize their unique, specialized cellular machinery to resorb bone, dramatically demonstrated in several laboratories, will be useful not only in understanding bone resorption but also in developing unique therapeutic agents to modulate bone resorption in patients. In this regard, we should also recognize that a peptidomimetic antagonist of the osteoclast OLv[33integrin has already been used to inhibit bone resorption in animal models of osteoporosis. We have also acquired an abundance of knowledge about osteoblasts, how they differentiate from precursor mesenchymal stem cells, and how several hormones, cytokines, and growth factors control their biological activities. The most striking set of observations appeared in the May 30, 1997, issue of Cell, too late to be discussed in this book. These observations relate to a transcription factor CBFA1, a member of the core-binding factor family. Deletion of the gene in mice results in a total lack of bone with retention of the partially calcified cartilagenous skeleton. Furthermore, most of the features of the human syndrome of cleidocranial dysplasia can be accounted for by mutations in the CBFA1 gene. These findings provide new insights into mechanisms of differentiation of cells of the osteogenic lineage. There has been extensive research in an effort to define those factors that control skeletal differentiation. The bone morphogenic proteins (BMPs) were first cloned in 1988, but their importance in skeletal biology in particular and biology in general has only recently

Twenty years have now elapsed since the first edition of Metabolic Bone Disease and Clinically Related Disorders was published in two volumes, and 7 years have elapsed since the second edition was published in a single volume. Since the appearance of the last volume there has been enormous progress in our field, much of which can be ascribed to the development of the powerful technologies of molecular genetics. As a result of these recent advancements we are much more cognizant of bone biology and the pathophysiology of several human skeletal disorders. New animal models that serve to increase our understanding of bone diseases have also been developed. The availability of new diagnostic tools enables us to measure bone mass with a precision that permits the identification of patients with osteoporosis who are at risk for fracture and the assessment of the efficacy of treatment. Furthermore, more specific serum and urinary assays have been developed to measure markers of bone formation and resorption that improve our ability to evaluate pathological alterations in bone turnover and to measure responses to therapy. Finally, new therapeutic agents with proven efficacy in retarding bone loss of osteoporotic individuals and decreasing fracture incidence have been introduced. With respect to the basic biology of bone, a great deal of knowledge about the genesis and differentiation of bone cell populations has been accumulated. The stages of differentiation of osteoclasts from pluripotent stem cells have been better defined, based primarily on findings from studies of mutations in mice and rats and the capacity to introduce null mutations into the mouse genome. It has been established that the stem cell precursor of osteoclasts is also the precursor of monocyte-macrophages and that these developmental pathways are well defined. A null mutation in the gene for spi-1, a transcription factor, results in depletion of osteoclasts XV

xvi been appreciated. New members of the BMP family of secreted signaling molecules are constantly being discovered. Spontaneous mutations in mice such as the short ear mouse have been shown to be due to mutations in BMPs. Similarly, the GDF genes related to BMPs have also been the sites of spontaneous mutations in mice as well as in humans. The extraordinary phenotypes of mouse and human brachypodism, ascribable to mutations in GDF5 genes, are excellent examples. Many other genes have now been found to play major roles in skeletal development. These include the basic helixloop-helix family of transcription factors, the hox genes, the pax genes, fibroblast growth factors, hedgehogs, wnt, and noggin. Elucidation of the roles of many of these important genes in regulating skeletal development in mammals has been possible because of important observations in Drosophila and chicks. Identification of the mutation in the FGF receptor-3 in achondroplasia, the most common form of human dwarfism, has been a major triumph. From observations of human diseases such as osteogenesis imperfecta, we recognized that mutations, even those involving single base substitutions, could have profound effects on skeletal development and remodeling. Since publication of the second edition of Metablic Bone Disease and Clinically Related Disorders in 1990, mutations in other genes that encode proteins of the extracellular matrix have also been identified in humans and animals. These include mutations in the genes that encode the fibrillar components of the cartilagenous matrix such as type II, type IX, and type XI collagens and result in abnormal structure of cartilage on articular surfaces and epiphyses as well as defects in metaphyseal bone remodeling. Another example is the recent identification of mutations in the cartilage oligomeric protein (COMP) in pseudoachondroplasia. We have been the beneficiaries of knowledge derived from manipulation of the mouse genome. Examples include the introduction of null mutations in the BMP-7 gene. The results of introducing a null mutation in the osteocalcin gene were indeed surprising, since deletion of the gene resulted in a skeleton that not only was normally mineralized but also contained an excessive amount of bone. Thus, the absence of osteocalcin results in increased bone formation without affecting bone resorption. On the other hand, deletion of the matrix GLA protein had no effect on the skeleton but did result in extraordinary ectopic calcification of blood vessel walls. Our understanding of the pathophysiology of human disorders has also advanced considerably since the last edition of this volume. Much of this progress can be ascribed to the identification of receptors for the major calcitropic hormones, parathyroid hormone (PTH) and calcitonin, and the characterization of the calcium sensor

Preface initially identified on the surface of parathyroid cells. A new PTH-2 receptor, which, unlike the PTH/PTHrP receptor, interacts almost exclusively with PTH and not with PTHrP, has been characterized. We now recognize that a mutation in the PTH/PTHrP receptor gene is responsible for the phenotype (hypercalcemia, low circulating PTH, abnormal skeletal growth) in patients with Jansen metaphyseal chondrodysplasia and results from constitutive activation of the receptor. Similarly, mutations in the calcium sensor can now explain the clinical abnormalities in some patients with so-called familial benign (hypocalciuric) hypercalcemia. What a surprise to discover that the mechanism of X-linked hypophosphatemic tickets in osteomalacia is due not to a mutation in the phosphate transporter gene (not on the X chromosome) but to a mutation in another gene (i.e., PEX) located on the X chromosome. All evidence indicates that PEX is a membrane-bound zinc metalloprotease and possibly an ectoenzyme. We are aware of several tantalizing observations on the potential genetic contribution to the most common disease that we deal with, osteoporosis. The significance of polymorphisms in the vitamin D receptor gene and other genes is yet to be established, but it is likely that the genetic underpinning of osteoporosis will soon be clarified. The alternative strategies being developed to decipher genetic determinants of osteoporosis (e.g., those that employ inbred strains of mice) provide much greater statistical power to assess the relationships between genes and complex phenotypes than do twin studies or association studies. Although the use of dual-energy X-ray absorptiometry is now commonplace, there was only brief mention of this technique in our last edition. Much attention has been directed toward the use of various bone markers in evaluating patients with osteoporosis and identifying individuals at risk and following therapy. These include measurement of free pyridinolines, measurement of the pyridinoline-derived crosslinks, and development of convenient assays for bone-specific alkaline phosphatase. It is likely that even more useful markers will soon be developed. For our patients, newly available therapies, such as potent bisphosphonates, are strikingly effective in preventing bone loss and even result in a gain of bone mass. It is clear that the prevention of bone loss by these agents results in decreased propensity to fracture, and their availability should prove to be invaluable in the longterm management of patients with osteoporosis. Calcitonins are still being used, and the availability of preparations that can be given by routes other than injection certainly represents an advance. Nevertheless, the administration of calcitonin preparations in disorders such as osteoporosis and Paget's disease will probably be largely replaced by potent oral and parenteral bisphos-

Preface phonates. Evaluation of the therapeutic efficacy and potential pitfalls of estrogen therapy in osteoporosis continues. Most recently, the development of selective estrogen receptor modulators (SERMs), stimulated in part by the observations on the use of tomoxifen in patients with breast cancer, now offers new promise in preventing bone loss in postmenopausal women without concerns of developing breast cancer or uterine cancer. In fact, the SERM may also prove effective in minimizing cardiovascular morbidity and mortality because of their estrogen-like effects on circulating lipids and other determinants of cardiovascular risk. In this third edition of Metabolic Bone Disease, we continue our attempts to present a correlated view of metabolic bone diseases and related topics emphasizing the relationships among genetics, molecular biology, biochemistry, pathology, and clinical manifestations. We have once again attempted to accomplish this in a single volume and recognize that the approach, emphasis, and style differ in each chapter. We have, however, selected major contributors to our field as authors of these chapters while still recognizing these differences in style and approach. Whereas the previous edition contained 24 chapters, the new edition has 26 chapters. The contents

xvii of the majority of the chapters have been updated either by the same authors or by others who graciously agreed to accept the challenge. Although the chapters "Metabolic Bone Disease in Patients with Gaucher's Disease" and "The Diagnosis and Management of Bone Tumors" that appeared in the previous edition of Metabolic Bone Disease and Clinically Related Disorders have been deleted from this new edition, new chapters including the "Surgical Treatment for Hyperparathyroidism," "Familial Benign Hypocalciuric Hypercalcemia and Other Syndromes of Altered Responsiveness to Extracellular Calcium," and "Hypoparathyroidism and Pseudohypoparathyroidism" have been added. We have been fortunate that Academic Press, which published our first edition in 1977, has reassumed the role as publisher for this third edition. Finally, we are particularly grateful to Dr. Jasna Markovac and her staff for their patience, professionalism, and support, which they generously expressed during the preparation of this volume. LOUIS V. AVIOLI STEPHEN M. KRANE

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2HAPTER

Embryology and Cellular Biology of Bone LAWRENCE G.

RAISZ

GIDEON m. RODAN

Division of Endocrinology and Metabolism, University of Connecticut Health Center, Farmington, Connecticut 06030 Department of Bone Biology/Osteoporosis, Merck Sharp & Dohme Research Labs, West Point, Pennsylvania 19486

I. II. III. IV.

Embryonic Skeletal Development Limb Development and Pattern Regulation Bone Morphogenetic Proteins and Development The Role of Parathyroid Hormone-Related Peptide in Development V. Fibroblast Growth Factors and Skeletal Development VI. Cells of the Osteoblast Lineage

Evolution of the mineralized skeleton was a key factor in the emergence of terrestrial vertebrates. Furthermore, mobility on dry land and erect posture, which freed the upper limbs for other tasks, are both skeletal functions that were crucial for human evolution. Normal skeletal functions are one of the foundations of health and well-being, and their compromise, caused for example by osteoporosis or osteoarthritis, is a major and sometimes early manifestation of aging. The development of the skeleton as an organ system that can successfully fulfill its three functionsmmechanical support of soft tissues, ion homeostasis, and housing of hemopoiesisminvolves complex and finely tuned processes, which start early in embryonic life and continue into adulthood. Spontaneous and experimentally targeted gene mutations that affect the skeleton have provided increasing molecular insights into these processes. Following is a very brief description of skeletal development and illustrative examples of specific genes that control it. METABOLIC BONE DISEASE

VII. VIII. IX. X. XI. XII.

The Osteoclast Cell-Cell Interaction in Bone Remodeling Colony-Stimulating Factors and Bone The Transforming Growth Factor 13 Family Other Growth Factors (FGF, VEGF, PDGF, and HGF) Cell-Matrix Interactions References

Four types of overlapping phenomena are involved in development: (1) cellular migration; (2) cellular differentiation, whereby cells start making the specific proteins, carbohydrates, and lipids that characterize cartilage or bone; (3) patterning, the organization in space of various cells, which give the organ the intended shape; and (4) acquisition of the ability for feedback responses, which integrate the organ into the organism, as exemplified by bone remodeling. As stated, these are interconnected and each is governed by a large number of molecular signals. Current knowledge for the molecular basis of the regulation of skeletal development is derived in part from the effects of disruption (loss of function) or overexpression (gain of function) of specific genes, which cause genetic abnormalities in humans or animals. These can occur spontaneously or are experimentally induced. The products of these genes do not act in isolation but interact closely with multiple other factors, each contributing either intracellularly or extracellularly in an Copyright 9 1998 by Academic Press. All fights of reproduction in any form reserved.

2 autocrine, paracrine, or matricrine manner to differentiation or pattern formation. At the anatomical level, we recognize two skeletal tissues: cartilage and bone. Each of them can be divided into subtypes with various cellular composition, dependent on location and function (see below).

I. E M B R Y O N I C SKELETAL D E V E L O P M E N T Bones are derived from three embryonic structures: the neural crest, which through the branchial arches gives rise to the craniofacial bones; the sclerotomes, which give rise to the axial skeleton; and the lateral plate mesoderm, which gives rise to the appendicular skeleton (limbs). The axial (skull, vertebrae, fibs, and pelvis) and appendicular (limbs) skeleton starts embryologically with a cartilage model in which ossification centers appear. These are characterized by vascular invasion, differentiation of cells into osteoblasts, the bone-forming cells, and deposition of bone matrix and its mineralization. At the periphery of the cartilage model, a similar sequence of events forms a bony envelope that becomes the periosteum. At the two ends of the bones, the cartilage specializes to form a growth zone called the epiphyseal growth plate, where at least three types of cartilage cells can be identified: (1) proliferating cells; (2) differentiated chondrocytes, which elaborate the cartilagespecific matrix; and (3) hypertrophic chondrocytes, which are larger and are surrounded by mineralizing matrix. The composition of the cartilage matrix changes during chondrocyte maturation, the major cartilage collagen is type 2, a homotrimer. A characteristic collagen of hypertrophic chondrocytes is type 10. Mutations in cartilage collagens have been associated with various diseases (e.g., type 2 mutations with chondrodysplasias, type 10 with spondylometaphyseal dysplasia and metaphyseal chondrodysplasia, and type 11 with spondyloepiphyseal dysplasia ([Stickler's syndrome]). 1Proteoglycans are also prominent components of the cartilage matrix and sulfation defects can also produce malformations (e.g., diastrophic dysplasia). 2 Mutations in the aggrecan gene were shown to cause cartilage matrix deficiency. 3 The mineralized cartilage matrix serves as a scaffold for the deposition of bone, following vascular invasion, and is subsequently removed (resorbed) by large cells, chondroclasts, which are morphologically similar to the bone-resorbing cells, the osteoclasts. The area where bone replaces cartilage is called the primary spongiosa and the process is endochondral ossification. Farther towards the center of the bone where no cartilage remnants

LAWRENCE G. RAISZ AND GIDEON A. RODAN

are left, we find in the interior of the bone spicules in a honeycomb arrangement, forming the spongiosa or cancellous bone. This area and the rest of the interior of the bone is filled with bone marrow, where the constituents of the hemopoietic system are made. Two processes are involved in providing the shape and architecture of bone: modeling and remodeling. Modeling is the formation of bone on surfaces where bone had not been previously removed by osteoclastic resorption as seen, for example, in periosteal bone growth. In remodeling, which accounts for most but not all bone formation in adult life, bone is deposited on surfaces from which bone was previously removed by osteoclasts. This form of bone formation does not involve cartilage replacement. The craniofacial bones are also formed by a process which does not involve cartilage replacement, called membranous bone formation. Within membranous structures that precede the skull and facial bones, following cellular condensation, cells differentiate into osteoblasts and start the bone-forming process. These bones will also develop a periosteal structure surrounding cancellous bone and some bone marrow. To summarize this section, the neural crest gives rise to the bones of the face and the skull through de novo (membranous) bone formation. The sclerotomes and the lateral plate mesoderm give rise to the vertebral column and limbs, respectively, through endochondral bone formation, where cartilage is replaced by bone and bone growth occurs at the epiphyseal growth plate. Molecular aspects of the control of these processes are discussed below.

II. LIMB D E V E L O P M E N T AND PATTERN REGULATION A feature observed by experimental embryologists long ago was that cells acquire a sense of their position in the organism or in an organ relative to the cephalocaudal, anteroposterior, or lateral orientation (left vs. right) and dorsoventral. Recently, genetic studies, especially in Drosophila, started unraveling the molecular basis for pattern formation, which strongly impacts on skeletal development. Relatively high up in the regulatory hierarchy are the mammalian Hox genes, which correspond to the Drosophila HOM-C homeotic genes. 4 These genes appeared early in evolution and increased in number through gene duplication. In mammals, there are 38 Hox genes organized in four clusters, Hoxa to Hoxd, up to 13 genes per cluster ( 1 - 1 3 paralogs, not each cluster has all 13 paralogs). Each Hox protein has a 6 0 - a m i n o acid "homeo

CHAPTER 1 Embryology and Cellular Biology of Bone domain," which binds with high affinity the A/T-rich elements in DNA, thus acting as transcription regulators, most likely of multiple genes. In each cluster, the Hox genes are expressed sequentially from the 3' to the 5' end of the chromosome, as a function of the progress of development, the 3' genes being expressed in the anterior structures and the 5' genes in the posterior ones. This relationship named by Lewis "co-linearity" is believed to be one of the mechanisms for the control of the anteroposterior axis of the embryo. Indeed, it was found in the mouse that loss of function mutations in Hoxc-8, Hoxb-4, Hoxa-2, Hoxd-3, or Hoxd-13 cause anterior transformations in the axial skeleton, recognized by changes in vertebral morphology. Loss of function mutations in Hoxa-1 and Hoxa-3, for example, produce effects on cranial and mesenchymal neural crest and cause changes in hind-brain segmentation. 4 Mutations in Hoxd-13 were shown to cause synpolydactyly (fusion of digits). 5 0 v e r e x p r e s s i o n of Hoxd-6 produces an extra digit in the chick wing. 6 Mice lacking Hoxa-11 and H o x d - l l completely lack the radius and ulna. 7 The upstream regulators of Hox gene expression and the downstream genes they control started to be identified only recently. The mixed lineage leukemia (MLL) gene was shown to be required for the expression of Hoxa-7 and Hoxc-9, and its absence produced homeotic transformations of the axial skeleton and sternal malformations. 8 Inactivation of the CDX1 gene by homologous recombination abolished the expression of the Hoxa-7 gene. 9 Krox-20 is an up-regulator of Hoxb-2. 4 Retinoic acid (RA) has long been known to produce changes in limb or palate development, and RA response elements have been identified in the Hoxb-1 gene. ~~ In limb development, sonic hedgehog also seems to be upstream of Hox genes (see below). Another upstream gene shown to regulate Hox expression is bmi-1, the absence of which was shown to cause posterior transformation of the axial skeleton, whereas its overexpression produces the opposite phenotype. ~1 Other homeobox genes, which are not part of the Hox clusters, were shown to have effects on skeletal development. Mutation of an msx-2 gene is associated with the Boston-type craniosynostosis. ~2 MHox deletion was shown to produce in mice shortening of the skull, face, and l i m b s . 13'14 Recent experimental studies on limb development have offered significant insight into the mechanism of pattern formation and development and the factors involved. It has been known for almost 20 years that in the chick limb bud there are groups of cells that control limb development, the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). ~5 If ZPA, which is on the posterior edge of the limb bud, is transplanted

3 to the anterior edge of the other limb bud, it produces a mirror image set of digits. Retinoic acid, endogenously present on the posterior side, can duplicate this phenomenon when applied at the anterior site, instead of the ZPA. 16 Various genes have been shown by in situ hybridization to be expressed in the limb bud. Genes of the Hoxd complex are centered on the posterior side, genes of the Hoxa complex in a proximodistal orientation, and other homeobox-containing genes are found in the distal tip of the limb bud. The sonic hedgehog gene (SHH) co-localizes the ZPA and is also capable of producing duplications. 17 The bone morphogenetic proteins (BMP) 2 and 4 of the transforming growth factor [3 (TGF-f3) family also co-localize with ZPA, as well as fibroblast growth factor 4 (FGF-4) and FGF-8, which are found in the posterior half of the apical ectodermal ridge, and can replace its function. 18-2~ The data support the following model for the interaction between these various factors, based on their endogenous expression and the effect of their exogenous application to chick limb buds. FGF-8 initiates limb bud outgrowth and participates in the activation of SHH expression and its maintenance in the lateral plate mesoderm. SHH induces the production of FGF-4. 21 FGF-4 is the endogenous signal that controls limb outgrowth and maintains the ZPA. SHH, which can mimic ZPA activity is the ZPA signal and it also up-regulates FGF expression, establishing a positive-feedback loop. 22 The growth factor wnt-7a, which is expressed in the dorsal ectoderm, stimulates SHH expression. 23 Thus, the major factors which constitute the ZPA are FGF-4, FGF-8, wnt-7A, and SHH. The Engrailed-1 gene is a homeodomain-containing transcription factor expressed in the ventral limb ectoderm. Its absence results in dorsal transformation, probably as a result of lack of suppression of Wnt-7a expression. 24 SHH 25 also induces the local expression of BMP-2, TGF-[3, and Hoxd-13, the mutation of which produces anterior transformation of the sacral vertebra. BMP-2 and BMP-4, acting either as homodimers or heterodimers, are found in the polarizing region and can be induced by retinoic acid, but BMP-2 beads do not cause digit duplication. ~5 It has been suggested that the BMPs participate in epithelial mesenchymal interaction. BMP-4 has also been implicated in determining the dorsal ventral axis of the early embryo, probably via the transcription factor Mix.1. 26 Thus, experimental development biology studies in chick limb bud have suggested ways in which FGFs, BMPs, Hox and hedgehog molecules, as well as retinoic acid, may interact to control tissue patterning. As already stated, based on mutations, each of these families of molecules have been implicated in skeletal development. Further examples follow.

4

III. BONE MORPHOGENETIC PROTEINS AND DEVELOPMENT BMPs were discovered based on their ability to induce bone when injected subcutaneously or intramuscularly. BMPs are expressed during development in skeletal and other tissues 27 and two spontaneous genetic defects in mice were shown to be associated with B MP mutations. The short ear mutation (SE) is characterized by changes in the shape of the sternum and the external ear cartilage. These defects are found to be caused by a mutation in BMP-5. 28 Another mouse mutation, brachypodism, is characterized by the shortening of some, but not all, long bones. This defect is caused by a mutation in the growth and differentiation factor 5 (GDF-5). 29 BMP-5 is expressed during development in the condensing mesenchymal cells of the ear cartilage and GDF5 in the cartilage of the affected limbs. The remarkable selectivity of this defect, similar to those produced by changes in individual Hox genes, illustrates the high degree of local control in development.

IV. THE ROLE OF PARATHYROID HORMONE-RELATED PEPTIDE IN DEVELOPMENT Parathyroid hormone-related peptide (PTHrP) was discovered as the agent that is released from certain tumors and is responsible for hypercalcemia of malignancy by acting on the PTH/PTHrP receptor, mimicking PTH action. In attempts to define the physiological role of PTHrP, the gene was deleted in mice through homologous recombination in embryonic stem cells. The mice died soon after birth and the major defect was in the growth cartilage where an accelerated maturation into hypertrophic chondrocytes was observed, suggesting that PTHrP delays chondrocyte differentiation. 3~ Mutation of the PTH/PTHrP receptor was embryonically lethal by day 14.5 and embryos were small. The crossing of PTH/ PTHrP receptor ( + / - ) heterozygotes with black Swiss mice increases survival of PTH/PTHrP receptor ( - / - ) embryos until birth. The phenotype was similar to that of PTHrP ( - / - ) and was consistent with that of Jensen osteochondrodystrophy in patients who have a point mutation in the PTH/PTHrP receptor gene. 3~'32 PTHrP was recently found to be co-expressed with Indian hedgehog (IHH), a member of the hedgehog family mentioned above in limb development, and its misexpression delayed chondrocyte differentiation into hypertrophic cells. 33 This led to the hypothesis that PTHrP may mediate the hedgehog signal. Since IHH and SHH have similar biological activities, either SHH or PTHrP were

LAWRENCE G. RAISZ AND GIDEON A. RODAN

added to limb explants from normal or PTH/PTHrP receptor ( - / - ) mice. Both agents retarded chondrocyte differentiation in limbs of normal mice, but not in mutant [PTHrP receptor ( - / - ) ] mice. Furthermore, SHH had no effect on chondrocyte differentiation in PTHrP ( - / - ) mice, supporting the hypothesis that PTHrP is the downstream mediator of the IHH effect. The hedgehog proteins play additional roles in skeletal development. There are at least three hedgehog genes: SHH, mentioned above in the context of limb development; desert hedgehog (DHH); and IHH, mentioned above as inducing PTHrP expression. SHH was also implicated in determining the left/right symmetry and the specification of sclerotome cell lines. ~8'34 As mentioned above in limb development, SHH also induces the expression of BMP-2, as well as FGF-4. Recently, the 92-kDa serine/threonine kinase fused has been implicated in the signal transduction pathway of HH. 35

V. FIBROBLAST GROWTH FACTORS AND SKELETAL DEVELOPMENT FGFs modulate growth and differentiation of bone and cartilage cells in vitro and are expressed, along with FGF receptors, in skeletal tissues during development. Nine FGFs and four FGFRs have been reported. Their features and effects on bone cells are summarized later in this chapter. In the context of skeletal development, FGFs act as mitogens, morphogens, and suppressors of differentiation. FGF expression patterns during development suggest that they might be involved in the proximal distal patternings of the limb. As mentioned above, FGF-2, FGF-4, and FGF-8 are expressed in the AER. FGF-2 and FGF-4 support limb outgrowth and can substitute for AER in limb buds. ~80verexpression of FGF-2 on the anterior aspect of the chick limb bud results in duplication of digits. Overexpression of FGF-1, FGF-2, or FGF-4 in the flank produces an extra limb. 19 The putative target cells of FGFs are chondrocytes, and it was shown that FGF-1 and FGF-2 promote the repair of damaged cartilage, 36 while FGF-2 inhibits the terminal differentiation of rabbit chondrocytes in culture. 37 However, the most compelling evidence for the role of FGF in skeletal development comes from mutations in FGF receptor genes, which cause skeletal abnormalities. A mutation in FGFR-1 causes Pfeiffer's syndrome. 38 A mutation in FGFR-2 results in Crouzon's syndrome, characterized by craniosynostosis and skeletal abnormalities in the limbs. 39 Mutations of various domains of FGFR-3 cause achondroplasia, thanatophoric dysplasia, and hypochondroplasia. 4~ Achondroplasia, a common cause of dwarfism, 42 is caused by a gain of

CHAPTER 1 Embryology and Cellular Biology of Bone function mutation, which causes constitutive activation of the receptor associated with suppression of osteogenesis, consistent with inhibition of osteoblastic differentiation in culture. 43 This was further confirmed by the disruption of the FGFR-3 gene in mice by the method of homologous recombination in embryonic stem cells. 44 Shortening of the limbs, which is observed in transgenic mice with overexpression of FGF-2, is also consistent with FGF-2 inhibiting bone growth. 45 The lack of FGFR-3 caused an expansion of the proliferating and hypertrophic zones in the epiphyseal cartilage, which further indicates that activation of FGFR-3 controls bone development by exerting a negative effect on osteogenesis. The above discussion focused on regulatory molecules, such as growth and differentiation factors and transcription factors. To the latter, one can add AP-2, recently shown to be essential for cranial closure, its absence causing severe dysmorphogenesis of the face and skull, as well as defects in the bones of the trunk region 46 and in the central nervous system. 47 Other genes required for cranial closure are the paired genes Pax-3 48 and twist. 49 Activating transcription factor 2 (ATF-2) is another general transcription factor belonging to the ATF/CREB family, the absence of which caused in mice chondrodysplasia and severely stunted growth with reduced spine length and shortened extremities. 5~ Yet another transcription factor, SOX-9, was found to be responsible in humans for the syndrome of campomelic dysplasia, usually a lethal syndrome characterized by severe skeletal abnormalities, defects in cartilage formation, as well as autosomal sex reversal. The SOX genes are sex-determining-region Y (SRY)-related genes. SOX-9 contains a HNG DNA-binding box of 78 amino acids, which is 71% similar to that of SRY. 51'52 SOX-9 was shown to be expressed during chondrogenesis in mouse embryos. Mutations in genes coding for extracellular matrix constituents have been known for some time to cause skeletal anomalies. Mutations in type 1 collagen, primarily in the procollagen region, are responsible for most cases of osteogenesis imperfecta. 1 As mentioned above, mutations in type 10 and 11 collagen cause severe anomalies in cartilage development and a mutation in a sulfate transporter, which probably affects the production of the sulfate proteoglycans, causing diastrophic dysplasia. 2 The deletion of the osteocalcin genes in mice was recently shown to increase cortical thickness as a result of increased bone formation, 53 suggesting that this gene regulates osteoblastic bone formation. To summarize this section, we have entered the era when one of the big biological riddlesmunderstanding how an organism develops from a single c e l l m h a s started unraveling. The players are gradually being iden-

5 tiffed through spontaneous or experimental gene mutations, which lead to loss or gain of function. For the skeleton, these include, among others, the Hox genes, FGFs and their receptors, hedgehogs (IHH, SHH, and DHH), BMPs, Wnt, PTHrP, retinoids, and the respective receptors. This list does not include all the genes that have been implicated in skeletal development, and their number is likely to continue to grow. In addition, the much more complicated task that lies ahead is to figure out how these genes interact and how they influence the number, position, and phenotypic expression of the cells responsible for the structure and function of the skeletal tissues.

VI. CELLS LINEAGE

OF THE

OSTEOBLAST

(Fig. 1 - 1) A. T h e O s t e o b l a s t P h e n o t y p e

Osteoblasts are anatomically/histologically defined as the plump cuboidal cells, organized in continuous layers along osteoid, the nonmineralized bone matrix on bone surfaces. The osteoblasts elaborate that matrix, which is 90% collagen, and possess at the ultrastructural level the characteristic features of protein-producing and-secreting cells: an abundance of endoplasmic reticulum and Golgi structures polarized towards the matrix side of the cell. These cells also have a distinct, large, oval nucleus. Osteoblasts are clearly polarized and seem to work in tandem, since the bone matrix is organized in extracellular collagen fibrils that exceed the dimension of single cells. The secretory osteoblast is therefore a cell which synthesizes and elaborates large amounts of type I collagen, as well as the noncollagenous matrix proteins found in bone: bone sialoprotein, osteopontin, osteonectin, osteocalcin, certain proteoglycans, and others. About 10 days after its elaboration, the matrix mineralizes. The alkaline phosphatase ectoenzyme, present at high levels in osteoblasts, is essential for this process, since its absence in the genetic disorder hypophosphatasia produces severe rickets. 54 To carry out their function in the context of the integrated tissue and organism, osteoblasts respond to autocrine, paracrine, and matricrine stimuli and are thus endowed with appropriate receptors. They also elaborate specific cytokines and factors for these regulatory functions. Although none of the osteoblastic features or products, including the bone matrix proteins, its receptors, or cytokines are totally unique for this cell, their combination and their level of expression define the osteoblastic phenotype. Following is a brief enumeration and description of these "osteoblastic markers."

6

LAWRENCE G. RAISZ AND GIDEON A. RODAN

FIGURE 1-1 Morphology of osteoblasts. A, In the low-power electron micrograph, a layer of osteoblasts that are tightly connected to each other forming a syncytium is seen on the upper (forming) side of the bone; on the lower side, cells that may represent either inactive osteoblasts or precursor cells are less tightly connected to each other so that there is no true syncytium on the bone surface. A relatively inactive osteocyte is seen within the bone. B, A higher power view shows one of the processes extending from an osteoblast toward the mineralized bone and connecting through gap junctions with a process from an internal osteocyte. The unmineralized osteoid contains collagen bundles of increasing size. Mineralization is at first spotty, but ultimately there is a dense and continuous mineralization front. (Courtesy of Dr. M. Holtrop.)

Collagen is the most abundant protein in the organism; 19 types have been described, 5 of which are helical (I, II, III, V, and XI). 1 The osteoblasts synthesize type 1 collagen and do not make type 2, which is produced by chrondrocytes, or type 3, the product of connective tissue, skin, or tendon fibroblasts. Type 1 is also abundantly present in those tissues, although its supramolecular organization is tissue specific, which may account in part for the selective mineralization of bone. Type 1 collagen in bone forms unique intra- and intermolecular crosslinks between lysine residues, which generate pyridinoline- and deoxypyridinoline-containing peptides, the excretion of which is today the best marker of bone resorption (degradation). 55 Osteocalcin or bone-gla protein, 56'57 although not always present in bone, is the most bone-specific protein. The only other cell where osteocalcin production was reported is the megakaryocyte. Osteocalcin appears to be absent in woven bone and more abundant in bones

from older animals. 58 Except for Paget's disease, there is generally a reasonably good correlation between the rate of bone formation and the circulating levels of osteocalcin, one of the biochemical markers of bone formation. 59 The normal serum levels are about 10 ng/ml. 6~ Like for other noncollagenous proteins, the function of osteocalcin is not firmly established. Recent "knockout" experiments in mice suggest that it suppresses osteoblastic bone formation. 53 Bone sialoprotein (BSP) is a 320-amino-acid glycoprotein 61 found in bone, dentin, cementum, and hypertrophic cartilage. 62-64 It contains an RGD sequence, which binds to integrins, the cell attachment receptors. Like osteopontin (see below), it is both sulfated and phosphorylated. 58'65'66 It is present at the onset of bone f o r m a t i o n , 67 but its precise function is not known. Osteopontin is also a glycoprotein with a protein backbone of about 32 kDa, produced and secreted by osteoblasts. It is found in many other tissues, where it was indepen-

CHAPTER 1 Embryologyand Cellular Biology of Bone dently discovered and named (2ar, SPP-1, uropontin, Eta-l). 68-71 In bone, it is synthesized also by osteocytes and osteoclasts. 72'73 Like BSP, it contains an RGD sequence and binds to the integrin vitronectin receptor, serving as a substrate for the attachment of osteoblasts or osteoclasts in vitro. Osteopontin is associated with inflammation, restenosis, and tumor invasion. TM Its precise function in bone is not known; it is found on the reversal line where osteoclastic resorption has stopped and osteoblastic bone formation started. 75 The two proteoglycans found in bone are decorin and biglycan. TM They are also found in articular and epiphyseal cartilage as well as in the subperichondral regions and in the periosteum. 77 Although, strictly speaking, bone formation cannot be observed in vitro, many of these proteins are produced and secreted by osteoblastic cells in culture. Such ex vivo experiments helped define the osteoblastic phenotype and study of the regulation of the production of these osteoblastic products by hormones, cytokines, and growth factors and the dependence of their expression on osteoblast maturation/ differentiation. 78-8~ In vivo, the expression of these osteoblastic genes can be followed by in situ hybridization and immunochemistry. Both types of studies show that the expression of osteoblast phenotypic genes is sequential, rather than synchronous, and may vary as a function of the type of osteoblast and osteogenesis (see below). Alkaline phosphatase (ALP) is a 65-kDa ectoenzyme, which requires zinc and dimerization for cleaving nonspecifically organophosphates at a neutral pH. The bone/ liver/kidney-ubiquitous (tissue nonspecific) isoenzyme is one of the three expressed in humans, the others being the intestinal and the placental one. 54 The bone enzyme differs in its glycosylation pattern from that of the liver and kidney. This made it possible to develop bone specific monoclonal antibodies, and skeletal ALP in the circulation is now a biochemical marker for bone formation. Although many other cells contain this ectoenzyme in their membrane, its level is at least 100 times higher in osteoblasts. The absence of this enzyme in hypophosphatasia causes tickets and the accumulation of phosphoethanolamine and pyridoxal-5-phosphate. 8~ In vitro and in vivo studies have identified a large number of factors which can regulate osteoblastic proliferation, differentiation, and activity, including factors produced by osteoblasts themselves. In vitro studies have shown that osteoblasts have receptors for many factors, not all of which may play a role in vivo. This list includes PTH; PTHrP; estrogens; androgens; progestins; glucocorticoids; 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]; retinoids; prostanoids (primarily PGE and PGF2~); (IGF-1) and IGF-2, insulin; FGFs, TGF-[3s and BMPs; EGF; PDGF-a and -b; interleukins (IL) -1, - 3 , - 4 , - 8 , and-11; TNF-ot; LIF; ANF; endothelin; and

7 colony-stimulating factors. Factors shown to be produced by osteoblasts include: IGF-1 and-2; PGE2 and PGF2~; I L - 1 , - 3 , - 6 , - 8 , and-11; B M P - 2 , - 4 , - 6 , and-7; TGF-[31 and TGF-[33; FGF-2; VEGF; LIF; MCSF; and granulocyte-macrophage colony-stimulating factor (GMCSF). These lists are incomplete. Some factors stimulate the production of other factors; for example, agents which up-regulate cyclic adenosine monophosphate (cAMP) such as PTH, PTHrP, or PGE stimulate IGF-1 and VEGF synthesis and secretion. 82-85 TGF-[3, BMPs, IL-1, and other agents stimulate PGE2 production, 86-89 which in turn is down-regulated by glucocorticoids. FGF stimulates TGF-13 synthesis and secretion. IL-1 and IL-6 stimulate IL-11 secretion, which acts on osteoclasts. Estrogens suppress IL-6 production. Not all osteoblastic cells produce and respond to all factors. For example, it has been observed in situ that only certain osteoblastlineage cells with a specific morphology were rich in PTH receptors. 9~ Taken together, these observations suggest that: (1) cells of the osteoblastic lineage (see below regarding osteocytes and lining cells) act as receivers and transmitters of bone remodeling signals, and (2) that there are hierarchial feedback loops and cascades, some factors inducing other factors. Additional cells, including osteoclasts, vascular endothelial cells, and possibly macrophages and other hemopoietic cells, may participate in these interactions (see Section VIII). Cell-cell interaction between osteoblasts is also mediated by gap junctions, which may explain their apparently coordinated function. Osteoblasts express the gap junction proteins connexin-43 and connexin-45 9~'92 and connexin-43 up-regulation was associated with stimulation of osteoblastic differentiation. 93 To summarize, the family of osteoblastic cells includes cells that produce the extracellular proteins in the bone matrix: type 1 collagen and the noncollagenous proteins osteopontin, BSP, osteocalcin, osteonectin, decorin, biglycan, and others. Specific osteoblastic cells possess receptors for stimuli of bone remodeling, including systemic hormones [PTH, sex steroids, glucocorticoids, 1,25(OH)2D3, insulin, thyroid hormone], local factors (prostanoids and cytokines, interleukins), etc. Osteoblastic cells also secrete many regulatory factors, such as IL-6, PGE, IGF-1, TGF-[3, and others, which generates a complex system of interacting arrays that coordinate the three bone functions: mechanical support, ion homeostasis, and hemopoiesis.

B. O s t e o b l a s t D i f f e r e n t i a t i o n As mentioned above, bone is derived embryologically either from the neural crest, from branchial arches, or from sclerotomes. In all cases, osteoblasts originate from

8

mesenchymal cells, which can give rise to other cell types: fibroblasts, myoblasts, chondroblasts, adipocytes, and tendon cells. It is not clear if this spectrum of differentiation potential is present in a single given stem cell. Calvaria-derived clonal cells were shown to give rise to adipocytes, myoblasts, chondroblasts, and osteoblasts. 94 Periosteal cells can probably differentiate into bone and cartilage cells following fracture. Mesenchymal cells in the bone marrow were proposed to have both osteogenic and adipogenic potential. 95 The transition of bone to tendon in Sharpey's fibers and the ossification of some tendons (at least in the turkey) is consistent with the common cellular origin for bone and tendon. Ectopic bone formation induced by "calciphylaxis" or BMPs indicates that mesenchymal cells with osteogenic potential are present in muscle and dermis. Pathological stimulation of such cells to become osteogenic is probably occurring in fibrodysplasia ossificans progressiva. 96 The stimuli required for osteogenic differentiation of mesenchymal cells probably differ for different types and stages of "osteogenic commitment" of such cells. Friendenstein many years ago distinguished between "committed" and "inducible" osteogenic cells, based on the stimuli required for the formation of bone in diffusion chambers implanted in animals. 97 The differentiation program is likely to be triggered by the induction of upregulation of transcription factors, which in turn induce the phenotype (osteoblast) -specific genes. The best paradigm for the up-regulation of such transcriptional control genes, sometimes called "master" genes, is the differentiation of muscle where myo-D, myogenin, and myf-5 play such a role. 98'99 Since myoblasts are also derived from mesenchymal cells, it was expected that analogous transcription factors, known as helix-loop-helix (HLH) factors, based on their structure, would initiate the differentiation of osteoblasts, chondroblasts, adipocytes, etc. No osteoblast-specific HLH transcription factors have been found so far. A key transcription factor involved in adipocyte differentiation was found to be the peroxisome proliferator activated receptor ~/ (PPAR-~/), which belongs to the family of steroid, retinoid, and vitamin D receptors. 1~176 External signals, which induce the transcription factors, should include growth and differentiation factors, as described for limb development: fibroblast growth factors, BMPs, hedgehog, etc. It was shown, for example, that B MP can induce osteoblastic differentiation in certain cell types ~~ and at the same time suppress myoblast differentiation in myogenic competent cells. ~~ Thus, although the details are not fully known, one can summarize as follows. Osteoblasts are derived from mesenchymal cells, which can have different levels of commitment for osteogenic differentiation. External stimuli, growth and differentiation factors, hormones,

LAWRENCE G. RAISZ AND GIDEON A. RODAN

and cell-cell and cell-matrix interactions induce in precursor cells transcription factors which "turn on" the osteoblastic phenotype. These factors are both nonspecific (such as early genes, fog, egr, etc.) and "tissuespecific" factors, which remain to be described, Differentiated cells are defined by the proteins they make, which include receptors, enzymes, etc., responsible for their tissue-specific function. As mentioned above, osteoblasts have very few tissue-specific proteins, may be osteocalcin and bone sialoprotein. However, the combination and abundance of proteins made by osteoblasts are clearly characteristic of this phenotype. Not all "phenotypic" genes are expressed in all osteoblastic cells. There is strong indication that osteoblasts in cortical bone are different from those in cancellous bone and they may differ somewhat from bone to bone. For example, parathyroid hormone was reported to be a stronger stimulator of bone formation in cancellous than in cortical bone. ~~ In a given bone, osteoblasts also differ from each other; for example, it was shown that PTH receptors are present in some osteoblasts and absent in others. 9~In vitro, cells of osteoblastic origin show a very wide spectrum of features with respect to the proteins they produce and the receptors they have. 73 Osteoblastic cells produce growth and differentiation factors, including TGF-[3, bFGF, PTHrP, IGF and IGF-binding proteins, BMP-4, and CSF-1. Osteoblasts also possess cyclooxygenase, both type 1 and type 2 (inducible), and produce PGE and other prostanoids. Osteoblasts are thus well endowed to participate in autocrine and paracrine signaling, and osteoblasts are believed to play a central role in the regulation of bone remodeling (see below). As mentioned above, not all osteoblastic cells exhibit all phenotype-related genes, and the genes they exhibit are sequentially expressed. The sequence of expression may differ according to the type of bone or osteoblast. In rat calvaria and metatarsal bones, alkaline phosphatase and BSP seem to be early markers, judging by the distance of cells which express these mRNAs, from the osteoid, v20steopontin and osteocalcin seem to be expressed only in the cell layer contiguous to osteoid, while collagen is expressed in all cell layers. The sequence is roughly similar to that observed in rat calvariaderived osteoblastic cells in culture, g~ Such cells, under appropriate conditions, form nodules that mineralize in the presence of [3-glycerophosphate. As observed in other differentiating cells, there is a reciprocal relationship in osteoblasts as well between proliferation and the expression of differentiation-related genes. Histologically, some osteoblasts appear to differentiate either into lining cells, which cover the mineralized osteoid at the end of the formation period, or into osteocytes, which are incorporated into the matrix. Only recently have osteocytes been isolated using specific an-

CHAPTER 1 Embryology and Cellular Biology of Bone tibodies. 1~ have long projections, which traverse the matrix and most likely form a communicating network throughout bone. Electric conductivity through bone is consistent with communication between osteocytes via gap junctions. ~~ Osteocytes and lining cells are ideally located to perceive strain changes in the matrix and were proposed to mediate the response of bone to mechanical loads. 1~ Mechanical stimulation was shown to increase the production of PGE2 by osteoblast lineage c e l l s 1~ and PGE2 stimulates both bone resorption and bone formation. Indomethacin, an inhibitor of PGE2 production, was shown to block mechanical effects on bone remodeling. ~~ TGF-[3 inhibits osteoclast formation and activity 1~ and stimulates bone formation when injected next to the periosteum in v i v o . 1~~ Macrophage colony-stimulating factor (M-CSF) and IL-1, -6, and -11 will be discussed below in the context of osteoblast/osteoclast interaction. PGI2 has also been implicated in the mechanical effects on b o n e . TM Growth and differentiation factors can also modulate each other's production and that of various receptors, which can generate feedback loops and signal cascades. We are dealing therefore in this system with multiple interactions generated by a diversity of osteoblastic lineage cells, each responding to specific stimuli and emitting various signals, which upon integration yield the marvelous performance of functional bone. To summarize this section, the osteoblastic lineage contains the matrix-producing cells and "signaling" cells, likely to include the osteocytes and lining cells. Osteoblastic cells differentiate from mesenchymal cells in response to growth and differentiation factors. The

9 factors responsible for osteoblastic differentiation during normal bone remodeling in the adult have not been established. Such factors are likely to stimulate the induction of up-regulation of transcription factors, which participate in turning on the osteoblastic phenotype. These transcription factors remain to be identified.

VII. THE

OSTEOCLAST

(Fig. 1 - 2 )

Osteoclasts are the bone resorbing (degrading) cells. They are large (up to 100 txm), multinucleated (usually two to five nuclei per cell but sometimes many more), and are formed by the fusion of mononuclear cells, which already contain many of the features of the mature osteoclasts and are also capable of normal resorption. Actively resorbing osteoclasts are polarized, the part of the cell involved in resorption consisting of an actincontaining membrane ring firmly attached to bone, which circumscribes a highly convoluted membrane, the ruffled border, which is the resorbing organ of the cell (see below). 112'113 Histologically, osteoclasts can be seen often in groups acting in cancellous bone on the bone surface and in cortical bone digging conical (haversian) canals. The lifespan of an osteoclast is estimated from histological studies at 3 to 4 weeks and the demise of the osteoclast is probably by apoptosis. TM Many bone diseases, including osteoporosis, are characterized by loss of bone, often due to excessive osteoclastic bone resorption due either to an increase in osteoclast number or activity. The osteoclast has therefore been a major target of therapeutic approaches to bone diseases.

FIGURE 1--2 Morphologyof osteoclasts. A, An osteoclast from PTH-stimulated bone in vivo. Notice the many nuclei, the large vacuoles at the site of resorption on the left, and the abundant mitochondria. B, A high-power view of the ruffled border in the act of degrading a spicule of bone. It is surrounded by a clear zone that attaches the osteoclast to the bone and separates the ruffled border area from the surrounding extracellular fluid. (Courtesy of Dr. M. Holtrop.)

10

LAWRENCE G. RAISZ AND GIDEON A. RODAN

A. Osteoclast Origin and Differentiation There is ample evidence that osteoclasts are of hemopoietic origin, they are related to monocytes/macrophages and probably derive from granulocyte-macrophage colony forming unit (CFU-GM) cells. Early osteoclast precursors have not been isolated using cell sorting or other methods, but osteoclast progenitors were reported to be present in the circulation, both in mice and humans, in the cellular fraction, which contains the monocytes. 115 In an experimental system, which uses fetal mouse metatarsal bones prior to osteoclast development to identify tissues which contain osteoclastogenic precursors, 116 those were found in bone marrow, as well as in liver and spleen, sites of hemopoiesis in the embryo. Earlier evidence demonstrating the hemopoietic origin of osteoclasts is based on the ability of bone marrow transplants to cure certain types of osteopetrosis, based on absence or malfunctioning of osteoclasts in animals and sometimes in humans. 117'118 During the last 10 years, experimental osteoclastogenesis has been produced in vitro by co-culturing bone marrow or spleen cells, usually from mouse, with a supportive feeder layer. 119 The cells required for the differentiation of osteoclastic cells could be either calvaria-derived osteoblasts, some but not all osteoblastic cell lines from calvaria, and mesenchymal, so-called stromal cells from bone marrow, which were selected for this capability. 12~ Using such a system, osteoclasts can be generated in vitro from bone marrow from various species in the presence of 1,25(OH)2D3. Direct contact between the living cells of the feeder layer and osteoclast progenitors is necessary for osteoclast generation and function. 121 Various stimuli promote or enhance osteoclast formation in this system, probably by acting on the "inducing" cells. These include, in addition to 1,25(OH)ED3: PTH, tumor necrosis factor ct (TNF-o0 PGE2, interleukin-1, interleukin-ll, and interleukin-6 in the presence of soluble interleukin-6 receptor. 119'122 Some feeder cells also require glucocorticoids. Like in the differentiation of other cell types, the sequence of events should start with extracellular signals, which induce transcription factors, initially early genes and then factors which turn-on the phenotypespecific genes. There are probably several signals conveyed by feeder layer cells to osteoclast progenitors. Among the genes up-regulated by 1,25(OH)2D3, C3 complement was shown to promote osteoclastogenesis. 122 CSF-1 is another important gene expressed by osteoblastic cells, both as a secreted and membrane-bound form demonstrated to be necessary for osteoclast formation by its absence in the osteopetrotic mutation op/op, lz3'124 Hepatocyte growth factor (scatter factor) was reported to promote osteoclastogenesis in vitro, its role

in vivo remains to be established. 125-127 TGF-[3 was shown to inhibit osteoclast formation. 128 Three of the transcription factors involved in osteoclast differentiation were revealed by osteopetrotic mutations. Deletion of src by homologous recombination (src-k.o.) produced an osteopetrotic phenotype in mice, characterized by the lack of ruffled border in osteoclasts. 129'13~The kinase activity of c-src is apparently not required to rescue, at least partially, the osteopetrotic phenotype. TM Another experimental mutation, which produced osteopetrosis, is the deletion of the early gene c-fos. 132 An osteopetrotic phenotype in mice was also shown to be due to the lack of a transcription factor PU-1.133 The downstream targets of these mutations are not yet known. For human osteopetrosis, only one mutated gene has been identified so far, carbonic anhydrase ( C A - I I ) TM and it was reported that bone marrow-derived osteoclast-like cells from a patient with craniometaphyseal dysplasia lacked the vacuolar proton pump. 135 Several genes are considered to be " m a r k e r s " of the osteoclastic phenotype. Osteoclasts have over 1 million receptors for calcitonin, the hormone that inhibits osteoclast activity. 136 Osteoclasts express very high levels of the vitronectin receptor integrin (]~v~3, the epitope of a monoclonal antibody thought initially to be osteoclast specific. 137 Tartrate-resistant acid phosphatase is highly abundant in osteoclasts. ~38 Another lysosomal hydrolytic enzyme, cathepsin K, was also cloned initially from osteoclasts, where its levels are extremely high. 139'14~Osteoclasts also express several proteins involved in the acidification process, which are not unique to this cell. These include a vacuolar ATPase and the associated chloride channel, 141-143 CA-II, 144 chloride bicarbonate exchanger, 145 sodium proton antiporter, 146 and sodium/ potassium A T P a s e . 147 Osteoclasts also produce the extracellular matrix osteopontin, which contains the RGD sequence and could be a ligand for the C~vl33receptor. Another structure reported to be present in osteoclasts is the potassium proton ATPase. 148 Many of these so-called phenotypic markers were shown to be present in the mononuclear cells prior to f u s i o n . 149 To summarize this section, osteoclasts are cells of hemopoietic origin related to monocyte/macrophages. Their differentiation from progenitor cells, which have not been isolated, can be reproduced in culture in the presence of osteoblasts or bone marrow stromal feeder cells. The growth and differentiation factors, which promote osteoclast differentiation in vivo, have not been identified but several agents, such as PTH, PGE2, and IL-1, which stimulate bone resorption, enhance osteoclast formation in culture. Osteopetrotic mutations have identified several genes essential for osteoclast development and function: CSF-1, src, fos, PU-1, and CA-II.

CHAPTER 1 Embryology and Cellular Biology of Bone B. O s t e o c l a s t F u n c t i o n Multinucleated osteoclasts are probably formed on or close to the surface of mineralized bone. This surface has probably been prepared for resorption by lining cell removal of a thin layer of matrix. 15~ Direct contact with mineral is probably important for osteoclast activation, since nonmineralized matrix is not readily resorbed. This is apparent in tickets and osteomalacia, where matrix accumulation is observed in cartilage and bone. Integrins, the heterodimeric cell attachment receptors, have been implicated in osteoclast attachment and/or activation, particularly the vitronectin receptor Otv[33,which also binds osteopontin and bone sialoprotein. 137 Osteopontin is an extracellular matrix glycoprotein produced by osteoblasts and other cells, as well as osteoclasts. TM Inhibitors of OLv[33, such as snake venom echistatin and blocking antibodies, were shown to inhibit bone resorption. 152-~54 Significant morphological changes occur during osteoclast activation. The cell becomes highly polarized and develops three clearly distinguishable membrane domains. 155 In fight contact with the bone surface is an organelle-free actin-rich ring through which the cell attaches tightly to bone. This ring, called the sealing or clear zone, separates the two other membrane domains. Facing the bone in a circle of about 500 ixm2, delineated by the clear zone, is the highly convoluted membrane called the ruffled border, produced by the insertion of vesicles with lysosomal characteristics. This membrane, the ruffled border, contains the structures that carry out the bone resorption function. Outside the clear zone is the rest of the osteoclast membrane called, by analogy to other polarized cells, the basolateral surface, which differs both in appearance and composition from the ruffled border. Bone resorption has two major components: (1) dissolution of the mineral, caused by acidification within the resorption lacuna and (2) digestion (hydrolysis) of the matrix proteins by cysteine proteinases, cathepsins, metalloproteinases, and collagenases. The osteoclast is endowed with several structures to acidify the lacuna to a pH of 4 or less. In the ruffled border, the proton pump, a vacuolar ATPase, has been localized by immunochemistry TM and the proton pump inhibitor bafilomycin A was shown to inhibit bone resorption. T M This is a multicomponent, ubiquitous pump present in the internal vacuoles of all cells, but it was suggested that it may differ somewhat in osteoclasts as a result of alternative splicing. 158 In the cytoplasm, acidification is aided by the presence of carbonic anhydrase, which catalyzes the conversion of bicarbonate into CO2 and protons. Sulfonamides, which inhibit this enzyme, reduce bone resorption, and a genetic defect of CA-II deficiency causes osteopetrosis. T M In the baso-

11 lateral membrane, there is a bicarbonate chloride exchanger, and its inhibition reduced resorption as well. 145 In the ruffled border chloride, transported by a chloride channel, is a counter ion for the proton pump. 159 The osteoclast has a number of other ion channels, including inward and outward potassium-rectifying channels, 16~ calcium channels, and others, yet to be described, as well as very abundant sodium-potassium ATPase in the basolateral membrane, 147 which contribute to the regulation of the membrane potential and the ionic homeostasis in this actively acidifying cell. To serve the energy needs for this hard work, the osteoclast is endowed with a large number of mitochondria, localized primarily above the ruffled border. It has recently been suggested that calcium phosphate is crossing the cell following mineral solubilization. In vitro, it was shown that low pH (e.g., 6.8) increases osteoclast activity and high calcium (>2.5 mM) decreases it. 163-166 There is not full agreement on the enzymes involved in matrix degradation. Among the cysteine proteinases, cathepsin K seems to be the most abundant one 167 (based on mRNA) and also has the ability to degrade nondenatured type 1 collagen. Cathepsin L and S are also present in osteoclasts. A cathepsin L-deficient (knock-out) mouse had no apparent bone defects. ~68 However, congenital cathepsin K deficiency in humans causes picnodysostosis, characterized by osteopetrosis and bone anomalies. ~4~ It was reported that osteoclasts produce neutral collagenase (MMP1), although there is no consensus on this assertion. Collagenase is also produced by osteoblasts and probably deposited in the matrix along with collagen. Inhibitors of collagenase suppress bone resorption in vitro; however, the role of this enzyme in vivo is not fully established. In vitro, osteoclasts resorb a certain site for several hours and then move to a separate site and may act similarly in Y i v o . 169 A single osteoclast may be involved in the resorption of more than one lacuna. In vivo, one often sees several osteoclasts resorbing side-by-side, suggesting the possibility of positive feedback, whereby an active osteoclast recruits additional ones analogous to leukocytes during inflammation. The lifespan of osteoclasts has been estimated histologically at 2 to 4 weeks and there is evidence that osteoclasts undergo apoptosis. T M It is not known what controls the termination of osteoclast resorption at a particular site in vivo, nor is it known if osteoclasts require sustained activation in order to resorb bone. In vitro, in certain systems, the presence of osteoblast lineage cells seems to be necessary for osteoclast activity. TM Systemic physiological agents, which increase osteoclast number and possibly activity, include parathyroid hormone, thyroid hormone, and the lack of estrogen, as discussed elsewhere. Local factors include IL-1 and TNF-ct, possibly involved in the bone destruction associated with

12 inflammation in periodontal disease and rheumatoid arthritis. The systemic hormone that inhibits osteoclast activity is calcitonin, but its role in the adult in calcium homeostasis or bone metabolism is questionable. Neither calcitonin deficiency nor excess calcitonin, produced endogenously by thyroid tumors, has an effect on calcium levels or bone mass. Recently, it was reported that mechanical strain in the matrix, to which osteoclasts are attached, may control osteoclast activity. 17~ This is an interesting observation, since it would explain in part the adaptation of bone structure to mechanical loads. To summarize this section, osteoclasts are activated when they come in contact with the prepared mineralized bone surface. Integrins may play a role in this process. Active osteoclasts form a resorption lacuna delineated by the tightly sealing clear zone. The ruffled border in the lacuna secretes protons, which solubilize the mineral, and proteases, including cathepsins, which digest the matrix. The lifespan of osteoclasts is about 2 weeks.

VIII. C E L L - C E L L INTERACTION IN BONE REMODELING Bone remodeling is initiated by signals that prepare the bone surface for resorption and attract osteoclasts to carry out this function. It is known that elevated PTH or thyroid hormone and reduced levels of estrogen in women and testosterone in men increase the number of sites at which remodeling occurs; however, the mechanism for specifying a particular site for bone remodeling is not well understood. Cytokines that increase remodeling include IL-1, TNF-c~, PGE, and TGF-[3; their local generation, such as in periodontal disease or rheumatoid arthritis, could be the initiating factor in those cases. The only perturbation shown experimentally to initiate remodeling at specific sites is mechanical stimulation. ~72 It is possible that mechanical function indeed specifies the sites at which remodeling will normally occur and the hormonal background in the organism determines its intensity. This could explain the more than additive effects of immobilization and calcium deficiency or estrogen deficiency on bone lOSS. 173 Similarly, local elevation of cytokines, such as PGE2, could specify remodeling sites, especially since PGE2 has also been implicated in mediating mechanical effects on bone. The cells that perceive mechanical perturbations and those that secrete cytokines are good candidates for initiating the cell-cell interactions that set remodeling into motion. The cells best situated to detect mechanically induced deformation of the matrix are the osteoblast lineage-derived osteocytes and lining cells. Biochemical changes, including elevations in PGI2, have indeed been observed in oste-

LAWRENCE G. RAISZ AND GIDEON A. RODAN

ocytes soon following mechanical stimulation. 111 The way osteocytes and lining cells may perceive matrix deformation could be via the integrins through which they attach to the matrix. Integrins are heterodimeric macromolecular receptors, which in addition to their attachment function also participate in signal transduction. 174 Cytokine-producing cells include local monocyte/ macrophages, as well as osteoblast lineage cells shown to produce IL-6 and IL-1 1 in response to IL-1, for example. Over 15 years ago, it was proposed on the basis of the responsiveness of osteoblast lineage cells to parathyroid hormone, prostaglandins, and other resorptioninducing agents that osteoblastic cells mediate the effect of those agents on osteoclastic bone resorption. 175 The following observations support this notion. Preparation of the resorption surface, as mentioned above, seems to require collagenase activity, which removes a matrix protective layer and uncovers the mineral in bone. This function is probably carried out by osteoblast-lineage cells. Collagenase production and secretion in osteoblastic cells was shown to be stimulated by parathyroid h o r m o n e . 176'177 The interaction of osteoblast-lineage and other cells with osteoclasts can occur at several levelsmosteoclast formation, osteoclast activity, or osteoclast s u r v i v a l m a n d may involve diffusible or membrane-associated regulatory factors. The best documented interaction so far is the requirement of osteoblastic or stromal lineage cells for osteoclast generation in culture, reviewed above under osteoclast differentiation. Essentially, in vitro, direct contact with living feeder cells is required for osteoclast generation. Recently, it was shown, at least for osteoclasts generated in culture, that contact with osteoblasts is also required for bone resorption. ~21 No soluble factors that are secreted by osteoblasts and activate the osteoclast directly have been purified or cloned so far. Osteoclasts may be activated by contact with mineralized bone, the way macrophages are activated by phagocytic particles. It is also possible that fusion of mononuclear osteoclasts to multinucleated cells occurs on the bone surface. The chemotactic signal, which attracts osteoclasts or osteoclast precursors to the bone surface during remodeling, is not known. Collagen fragments have been shown to be chemotactic for many cell types. A specific case of osteoblast lineage/stromal cell control of osteoclast generation is the proposed action of estrogen via suppression of cytokine production. 178 Another aspect of cell-cell interaction during remodeling is initiation of the bone formation sequence following bone resorption. The molecular basis for this process, called "coupling," has not been elucidated, lv90steoblast differentiation probably starts several cell layers away from the surface of the matrix, where osteoblastic markers such as elevated alkaline phosphatase can be

CHAPTER 1 Embryology and Cellular Biology of Bone detected. It has been proposed that growth factors such as IGF-2 or TGF-[3 released or activated during bone resorption initiate the bone formation that follows; however, direct in vivo information for this mode of coupling has not yet been obtained. At any rate, locally, bone formation follows bone resorption in most cases. Systemically, as estimated by biochemical markers of resorption and formation, the two processes also change in tandem, suggesting communication or coordination between bone-forming and bone-resorbing cells. Further insights into ~J~e molecular basis of this phenomenon could offer new therapeutic approaches to diseases such as osteoporosis.

IX. COLONY-STIMULATING FACTORS AND BONE Bone is the site of hemopoiesis, and the close relationship between bone and bone marrow has suggested: (1) that bone cells or their precursors may provide the environment and factors needed for hemopoiesis and (2) if an expansion of bone marrow is necessary, due to hypoxia for example, bone resorption will occur in response to factors secreted by hemopoietic cells. 18~ The association between bone and bone marrow seems to be part of a built-in genetic program, since even in ectopic bone, formed in muscle or skin by the administration of bone morphogenetic proteins, ossicles are colonized by bone marrow. In vitro, growth and differentiation of hemopoietic cells of the different lineages require colony-stimulating factors. Some of these were shown to be produced by osteoblastic cells and some were shown to influence the formation of the hemopoietically derived osteoclasts (reviewed above). IL-3 and the kit ligand or stem cell factor and the FLT3/FLK2 ligand act on early precursors of the hemopoietic lineage. ~81'182 IL-3, as well as GM-CSF and macrophage colony-stimulating factor (M-CSF or CSF-1), were shown to be produced by mouse calvafia. ~83 M-CSF promotes the differentiation of monocytes into macrophages and is of special interest to bone, since a mutation in this gene, which interfered with normal protein expression in mice, caused osteopetrosis due to osteoclast absence. Macrophages are also reduced in these mice but not equally in all tissues and there is a measure of recovery of the osteopetrotic phenotype with age. TM The involvement of CSF-1 in the osteopetrotic phenotype has convincingly been demonstrated, not only by the gene defect but by the curative effect of exogenous 185 CSF-1 treatment. CSF-1 seems to be required at multiple stages of osteoclast maturation and, under certain conditions, can increase osteoclast generation. 186 .~87 However, under other conditions, both GM-CSF and

13 CSF-1 reduce osteoclast generation from mouse bone marrow in vitro, possibly by diverting the differentiation pathway into the monocyte/macrophage direction. ~88 CSF-1 was shown to be produced by osteoblasts, in both a secreted and a bound f o r l T l . 189 Mice that lack GM-CSF expression due to disruption by homologous recombination were normal, fertile, and had no apparent defect in hemopoiesis, but had a high incidence of pulmonary infections. ~9~Mice lacking both CSF-1 and GM-CSF had osteopetrosis, similar to the CSF-1 ( - / - ) mice. 191 In vitro, GM-CSF stimulates the generation of osteoclastic cells from mouse bone marrow. 192 Overexpression of granulocyte colony-stimulating factor (G-CSF) in mice caused an increase in osteoclast number and osteopenia. 193 In summary, colony-stimulating factors were discovered based on their requirement for in vitro hemopoiesis. Some of them, such as IL-3 and CSF-1, are expressed by osteoblasts. The role of CSF-1 in osteoclastogenesis, at least in mice, has clearly been demonstrated by the osteopetrotic phenotype of CSF-1 ( - / - ) mutants. Its role and that of other colony-stimulating factors in bone resorption and formation in humans remains to be determined.

X. THE TRANSFORMING GROWTH FACTOR [~ FAMILY Members of the TGF-[3 family, including the BMPs, are homo- or heterodimeric proteins of about 25 kDa, generated by cleavage from larger inactive precursors of about 100 kDa. TGF-[3 is initially associated with a latency-binding protein of 130 kDa in platelets and 190 kDa in osteoblasts, and significant activation steps are necessary for producing active factor. In mammals, there are three independently coded proteins: TGF-[31, TGF-[32, and TGF-[33, which have slightly different functions. TFG-[3s are produced by platelets, osteoblasts, and other cells, but bone is the largest repository of TGF[3 in the body. Interestingly, the bone loss produced by lack of mechanical load was shown to be prevented by TGF-[3 administration. TM When locally injected into bone or next to the periosteum, TGF-[3 stimulates bone formation, 11~ as well as resorption, but the balance is positive. TGF-[3 is elevated in fracture callus and has been implicated in fracture healing. 195 Unlike BMPs, TGF-[3 does not stimulate bone formation when injected into muscle or skin, but was reported to augment the osteogenic effect of demineralized bone matrix. 196 TGF-[3 acts on multiple targets in vivo, the most pronounced effect being immunosuppression, observed pri-

14 marily with TGF-[31, which limits its potential for pharmacological use. In vitro, TGF-[3s were shown to have multiple effects on mesenchymal cells. They stimulate the production of extracellular matrix proteins, including fibronectin, collagen, osteonectin, osteopontin, proteoglycans, etc., and their respective receptors, and inhibit the production of procollagenase and plasminogen activator, thus having an anabolic effect. In many cells, TGF-[3 inhibits proliferation, which is the basis of its bioassay in mink lung fibroblasts. However, in some cells including some bone cells, TGF-[3 can be mitogenic. 197 In the MC3T3 mouse calvaria-derived cells, TGF-[3 inhibits osteoblastic maturation. 198 In vitro, TGF-[3 inhibits osteoclast formation from bone marrow stromal cells. 1~ Taken together, these observations indicate that the action of TGF-[3 depends on cell type and state of differentiation. Attempts to deduce the physiological function of TGF-[3 in vivo were made using targeted gene disruption experiments in mice. TGF-[31 knock-out produced mice that were normal until weening, when they died from massive infections. 199 Prior to that, they were apparently protected by TGF-[3 present in the milk. This emphasized again the important role of TGF-[3 in the immune system. TGF-[33 knock-outs had a cleft palate, indicating a role in palate fusion. 2~176 The fact that TGF-[3 is considered to play an important role in the regulation of bone remodeling is demonstrated by the observation of osteoporosis and increased bone turnover in transgenic mice with osteoblast-specific overexpression of TGF-[3. 2~ Receptors for this family of growth/differentiation factors have recently been identified. 2~ There are basically three types of TGF-[3 receptor: type 3 is a proteoglycan ([3-glycan), which does not seem to be involved in signal transduction. Types 1 and 2 form heterodimers in the presence of ligand and possibly tetramers. Both type 1 and type 2 receptors are threonine/serine kinases. According to the current model the ligand binds first to type 2, which then activates type 1, and type 1 phosphorylates downstream targets that feed into the MAP kinase cascade. 2~ The same receptors or type of receptors are also mediating the signals of BMPs. So far, five type 1 receptors have been cloned from mammalian systems. These are ActR-1, ActR-1B, BMP-R1A, B MP-R1B, and T[3R-1. The four type 2 receptors are BMPR-2, ActR-2, ActR-2B, and T[3R-2. A type 2 receptor for mifllerian inhibiting substance, which belongs to this family, has also been cloned. Recently, it was shown that among the downstream mediators of TGF-[3 action are the MAD (mothers against DPP) proteins related to the Drosophila MAD proteins, which mediate the BMP-related decapentaplegic (DPP) signal in the fly. 2~ Furthermore, the analogous Xenopus XMAD2 was shown to recognize the activin response element and

LAWRENCE G. RAISZ AND GIDEON A. RODAN

form a complex with the activin-dependent early gene transcription factor or Mix-2. 2~ The BMPs belong to this family and were discovered based on their ability to induce bone formation when injected subcutaneously or intramuscularly via a process that follows the stages of endochondral bone formation. It has been proposed that ectopic bone formation of fibrodysplasia progressiva ossificans is due to abnormal BMP production. 96 To date, about 13 BMPs have been cloned which, based on their homology, can be divided into subfamilies. They are structurally related to the DPP protein in Drosophila, which controls wing development, and to Vg-1 protein in Xenopus. As mentioned above, BMPs play an important role in limb development and other aspects of development. 2~ When injected into bone or near to bone, BMPs are active stimulators of bone formation. At least two BMPs, BMP-2 and BMP-7, are being developed for clinical use in the repair of fractures and bone defects. The role of B MP-5 and GDF-5 in skeletal development, revealed by spontaneous deletion of these genes in mice, was discussed above. Recently, experimental deletion of the BMP-7 gene was shown to cause severe defects in the development of the kidneys, where it is highly expressed, as well as the eye. It also altered skeletal patterning. 2~ Similar deletion of BMP-2 or BMP-4 caused early embryonic lethality before skeletal formation. 27 The expression of BMPs in limb development was reviewed above. MAD proteins were shown to also mediate downstream effects of BMPs; for example, Smadl was shown to translocate to the nucleus in response to BMP-4 and to mimic the effects of BMP-4 in Xenopus embryo. 2~ Interestingly, BMP-4, as well as BMP-2 and BMP-7, are expressed in the interdigital region in developing chick limb bud and seem to be required for apoptosis, which was inhibited by dominant negative type 1 BMP receptor. 2~ In vivo, BMP-2 and BMP-4 were immunolocalized in osteoblasts during fracture repair, 2~ as well as BMPR-1A and BMPR-1B, 21~the receptors for BMP-4 and BMP-7. In vitro, BMPs stimulate osteoblast and chondrocyte differentiation and suppress myogenic differentiation in appropriate precursor c e l l s . 211'212 In summary, BMPs are expressed during embryogenesis and control the development of many organs, including the skeleton. In the adult organism, they induce osteogenesis when injected into muscle or dermis or next to bone. BMPs are being developed for the healing of nonunion fractures and repair of bone defects. B MP mRNA is expressed in adult bone and healing fractures. The role of BMPs in bone remodeling and their therapeutic potential for osteoporosis is not known. TGF-[3s the first proteins discovered in this family, also stimulate bone formation when injected locally in bone, but effects

CHAPTER 1 Embryologyand Cellular Biology of Bone on other systems, such as immunosuppression, may preclude its therapeutic use.

XI. OTHER GROWTH FACTORS (FGF, VEGF, PDGF, AND HGF) FGFs are a family of proteins, members of which were shown, as mentioned above, to be involved in skeletal development and potentially in bone formation. To date, nine FGFs, products of different genes, have been characterized. FGF-1 (acidic FGF) and FGF-2 (basic FGF) lack a signal sequence for secretion. FGF-3 or oncogene int2; FGF-4 or oncogene hst; FGF-5, also discovered as an oncogene; FGF-6, oncogene hst2; FGF-7, keratinocyte growth factor (KGF); FGF-8, androgen-induced growth factor; and FGF-9, gliaactivating factor which, like FGF-1 and FGF-2, lacks a signal sequence. The molecular weight of these singlepeptide chains is around 20 kDa; for FGF-2 and FGF-3 multiple spliced variants were reported. Major reported target cells for FGF-1 and -2 are endothelial and neural cells; for FGF-3, mouse mammary carcinoma; for FGF-4, Kaposi's sarcoma and limb development; for FGF-5, inhibition of hair elongation; for FGF-7, epidermis, wound healing, and branching morphogenesis; for FGF-8, androgen-dependent tumors; and for FGF-9, glial cells. Four human FGF receptors with 55% to 72% identity have been identified. 2~3They differ, both with respect to ligand affinity and tissue distribution. The FGFRs are subject to alternative splicing, which generates receptors with different numbers of extracellular Ig-like domains involved in ligand binding. The intracellular domains contain the tyrosine kinase, which is activated following receptor dimerization and phosphorylates downstream targets in the MAP kinase cascade. Alternative splicing of FGFR-1 a n d - 3 is tissue specific and generates significant tissue selectivity for FGF responses. 214 Virtually all receptor forms respond to FGF-1, the B exon of the receptor is expressed in epithelial tissue and the C exon in mesenchymal tissue. FGF-3 stimulates the B form of FGF receptor 1 and 2 and FGF-7, the B form of FGF receptor 2. These highly interesting recent observations provide fascinating insight into specificity achieved for this family of factors by alternative receptor splicing. In addition to these signal transduction receptors, FGFs also bind with low affinity to heparan sulfate containing proteoglycan-binding sites, which contribute to FGF interaction with its signaling receptors. 215 The role of various FGFs in skeletal development, deduced from its expression and mutations in FGF receptors, was reviewed above. FGF was shown to be expressed during fracture repair and to accelerate callus formation when exogenously applied. 36'216'217 It was re-

15 cently shown that systemic administration of FGF-1 or FGF-2 into rats increases bone formation. 218'219 In vitro, FGFs are potent mitogens for osteoblastic cells and in some systems suppress the expression of phenotype-related genes, 43,aa~but FGF also increases the formation of mineralized nodules from bone marrow stromal cells. TM Since FGF is produced by endothelial cells, as well as osteoblasts, and was shown to upregulate the expression of TGF-[3222 in osteoblastic cells, it could play a role in cell-cell communication associated with bone formation. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is produced in four isoforms of 206, 189, 165, and 121 amino acids. The 165 AA is the predominant form. These molecules act as dimers and, like FGF, bind to sulfated proteoglycans. VEGFs are among the most potent and selective angiogenic factors. 223 VEGFs are up-regulated in response to hypoxia, providing an in vivo feedback loop for increased tissue oxygen perfusion. 224 VEGF has been implicated in the hypervascularization associated with retinopathies and tumor growth. 225'226 In bone cells, VEGF was shown to be up-regulated by PGE2 via cAMP-mediated mechanism, 85 possibly participating in the osteogenic effect of prostanoids. Glucocorticoids suppressed VEGF expression in bone cells. Two types of VEGF receptors have been identified. The c-fms-like tyrosine kinase, fit-1227 and the kinase domain-containing receptors K D R . 228 B o t h are about 1300 amino acids long and have seven extracellular Ig-like domains. Deletion of either receptor by homologous recombination in mice is lethal by day 8 or 10 of embryonic life. KDR knock-out causes total absence of vascular endothelial cells; whereas, fit knock-out results in poor organization of blood vessels. 229-231 The production of VEGF by osteoblastic cells and the known importance of angiogenesis for osteogenesis suggests that this growth factor may play an important role in bone metabolism. Platelet-derived growth factor (PDGF) is a 30-kDa polypeptide. There are two forms, A and B, and only the dimeric chains AA, B B, or AB are biologically active. Similarly, there are two PDGF receptors, et and [3, single-peptide chains with 44% identity. These transmembrane receptors have a tyrosine kinase cytoplasmic domain, related to the CSF-1 receptor, and to the kit oncogene. PDGFA binds primarily to receptor et and PDGFB to both. Like for other growth factors, receptor activation is induced by dimerization in the presence of ligand. PDGFA is expressed in rat osteoblasts and its expression is up-regulated by TGF-[31 or PDGE Both PDGF AA and BB are potent mitogens for osteoblastic cells in culture. As expected from the reciprocal relationship be-

16

LAWRENCE G. RAISZ AND GIDEON A. RODAN

tween proliferation and differentiation, they suppress collagen synthesis and matrix production. PDGF levels are elevated during fracture repair. The high abundance of PDGF in the clot following fracture makes it a strong candidate for action during the initial phases of callus formation. There is no knowledge if it plays a role in later bone formation and remodeling. 232 Hepatocyte growth factor (HGF) or scatter factor is a heterodimer consisting of a 60-kDa oL and a 30-kDa [3 chain connected by a disulfide bond. HGF has multiple types of effects on many tissues in the body, including proliferation, motility, inhibition of growth, effects on differentiation, adhesion, etc. 233 The HGF receptor is the m e t protooncogene, a heterodimer of a 50-kDa extracellular and 145-kDa transmembrane peptide with a cytoplasmic tyrosine kinase domain, both produced from a single-peptide chain by proteolytic cleavage. TM HGFR is expressed in normal epithelium melanocytes, endothelial cells, microglial cells, neurons, hematopoietic cells and, of course, liver cells. HGF levels increase markedly during liver regeneration following ablation. HGF was shown to play a role in organogenesis and embryonic development and its deletion causes embryonic lethality. During development, it is expressed in lung, kidney, and the central nervous system. An HGFlike receptor called RON was shown to be highly expressed in hematopoietic stem cells and monocytes. It was recently reported that HGF promotes osteoclast formation.125-127 Except for this observation, not much is known so far on the role of HGF and skeletogenesis.

XII. C E L L - M A T R I X INTERACTIONS In multicellular organisms, all cells are connected by cell-cell and cell-matrix contacts. For tissues such as bone, where matrix is not only very abundant but important for one of its major functions, mechanical support, cell-matrix interaction has particular importance. There are several ways in which the matrix is likely to influence cell function. During development, the matrix has been shown to play an instructive function in cell migration and differentiation. In vitro, nontransformed mesenchymal cells require attachment to matrix for proliferation and similar dependence may operate in vivo. It was shown that the signals induced by fibronectin attachment lead to the expression of cylin D1,17~ a molecule required for cell cycling. The matrix can store growth factors" FGFs, IGFs, PDGF, and TGF-[3 have all been purified from bone matrix. And last but not least, in bone the matrix probably influences cell behavior by deforming under mechanical loads and transmitting this information to the cells. The primary adhesion receptors through which cells attach to the matrix are the inte-

grins. 174 Integrins are heterodimeric, transmembrane receptors. They contain an oL chain of 120 to 150 kDa and a [3 chain of 90 to 210 kDa, connected by disulfide bridges. Both have very short intracellular domains, which interact with the cytoskeleton via cytoskeleton attachment proteins, such as paxillin, vinculin, tallin, and oL-actinin, and participate in signal transduction. There are at least nine oL and a similar number of [3 chains coded by separate genes and the pairing between o~ and 13 chains, which is not unique, determines the preference for ligand binding. For example, oL2131for type 1 collagen; c~5131for fibronectin; OL4[~1 for laminin; O[v~l, O~v~3, OLv[35for vitronectin; and so on. Binding requires calcium and ligand specificity is not unique; for example, OLv[33 also binds fibronectin, osteopontin, bone sialoprotein, etc. The integrins present in osteoblasts include a2131, the collagen receptor; 0L5131, the fibronectin receptor; and 0~v[35, the vitronectin receptor. 137 Integrins are probably involved in the deposition of the extracellular matrix and may carry out the highly organized deposition of collagen fibers in bone. Beta1 integrins were shown to mediate gel retraction in fibroblasts in vitro. 235 Conversely, the integrins probably mediate the perception of matrix deformation in response to mechanical stress, which was shown to correlate with bone formation: reduced bone formation under conditions of immobilization or weightlessness and increased bone formation with mechanical stimulation. In smooth muscle cells, which also respond to mechanical strain, that response was blocked by integrin antibodies. 236 The major osteoclast integrins are OLv[33and av[31, the vitronectin receptors which bind osteopontin and bone sialoprotein, and OL2[~1 the collagen receptor. 137 Osteoclast precursors may also have some of the [32 integrins found in monocyte/macrophages, such as MAC-1. A role for integrins, especially OLv[33,has been strongly implicated in osteoclast function (see above). Antibodies against OLv[33,as well as snake venoms which bind preferentially to this receptor, echistin and kistrin, were shown to inhibit osteoclast activity in vitro and bone resorption in v i v o . 153"237 The precise mechanism for this inhibition has not been determined. It could be due either to interference with osteoclast attachment, osteoclast activation, or osteoclast migration. It was shown in the osteoclast that the addition of OLv[33ligands can produce a surge in intracellular calcium. 238 Osteoclast activity was also reported to be influenced by mechanical function, but it is not known if this is a direct or indirect effect. To summarize this section, cell-matrix interaction is likely to play an important role in osteoblast and osteoclast differentiation and function. Given the mechanical function of the bone matrix and the effects of mechanical loads on bone remodeling, cell-matrix interaction

CHAPTER 1

Embryology and Cellular Biology of Bone

s h o u l d p l a y a r o l e in t h i s p r o c e s s . I n t e g r i n s , t h e c e l l u l a r a d h e s i o n r e c e p t o r s , a r e l i k e l y to m e d i a t e t h a t i n t e r a c t i o n .

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~HAPTER

The Nature of the Mineral Phase in Bone: Biological and Clinical Implications M E L V I N J. G L I M C H E R

Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopaedic Surgery, Harvard Medical School, Children's Hospital, Boston, Massachusetts 02115

I. Biological Functions of the Mineral Phase II. The General Nature of the Mineral Phase in Bone and the Changes that Occur with Time III. Postulated Phases Other than Apatite as the Initial Solid Ca-P Mineral Phase Deposited in Bone

IV. Crystal Size and Shape V. Recent Studies of the Structure of Bone Apatites and the Applications of these Data to Clinical and Experimental Abnormalities and Diseases of Bone References

The subjects that will be presented in this chapter are: (1) The nature of the mineral phase in bone; that is, the chemical composition and crystal structure of the solid calcium-phosphate (Ca-P) mineral phase in bone, and the changes that occur in the mineral phase p e r se with time and maturation; (2)the relationship of these maturational changes which occur with time to the biological, physiological, and mechanical functions of the crystals and to Ca and P mineral metabolism; and (3) the ultrastructural location of the mineral phase and its relationship to the basic underlying physical chemical mechanism responsible for the initiation of calcification.

extent on the exact size, shape, chemical composition, and crystal structure of the mineral crystallites. The mineral phase acts on the one hand as an ion reservoir and on the other hand as an excellently designed structural material that determines in large part the mechanical properties of b o n e s u b s t a n c e (the structural material), and consequently of bone tissue and of bone as an organ. The importance of the role of bone mineral as an ion reservoir can be appreciated from the fact that about 99% of the total body calcium, about 85% of the total body phosphorus, and from --~ 90% and --~ 50%, respectively, of the total body sodium and magnesium are ass o c i a t e d with bone crystals, which consequently serve as the major source for the transport of these ions to and from the extracellular fluids. As a result, the bone crystals play a critical role in maintaining the concentrations of these and other ions in the extracellular fluid (homeostasis), which are critical for a variety of physiological functions (e.g., nerve conduction, muscle contraction)

I. BIOLOGICAL FUNCTIONS OF THE MINERAL PHASE The solid Ca-P mineral phase in bone performs two major functions, both of which depend to a significant METABOLIC BONE DISEASE

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MELVIN J. GLIMCHER

and many other important biochemical reactions. For some of the ions like Ca, the critical physiological concentration of this ion must be maintained in the extracellular fluids within a very limited narrow range, not only for normal functioning of a variety of tissues such as the nervous system, but for the viability of the organism. From the structural standpoint, the mechanical properties of the bone substance result from the impregnation of the otherwise soft, pliable organic matrix of bone tissue by the rock-like but brittle Ca-P crystals of apatite which converts the soft organic matrix to a relatively hard, rigid material (bone substance). The bone substance (cells, organic matrix and bone mineral) is therefore a material that scientists and engineers refer to as a two-phase or multiple-phase composite material whose combined properties are quite different than the simple algebraic sum of its components. In the case of bone, the Ca-P crystals are extremely brittle, and the collagen is very flexible with little to no compressive strength but very high tensile strength. When the collagen and the mineral phase are combined in the very special way as they exist in bone at the molecular and ultrastructural level (see later sections), it creates an essentially twophase, composite material or substance with mechanical properties quite unlike a simple addition or mixture of the two components. Instead, the two-phase substance is particularly tailored to most efficiently provide the structural material or fabric from which the microscopic and gross elements are fashioned, viz., the trabecular bony, lace-like network of cancellous bone and the osteones (and other architectural microscopic structures in nonosteonal bone) of compact bone (Fig. 2 - 1 , see Color Plate). As in any gross structure, the three-dimensional

disposition of the individual building elements is critical and enables the assembly of the structural units to withstand the particular forces and stresses applied to the structure as a whole. The same is true of the arrangements of the osteones of compact bone with respect to the three-dimensional shape of the gross bone (skull, long bones), and indeed of the disposition and orientation of the collagen fibrils and fibers in individual lamellae of the osteones, and the axial deviation of the collagen fibrils and fibers from one lamella to another. Moreover, recent evidence has demonstrated that this plywood-like structure also rotates around the long axis of the osteone ("twisted plywood sheets") elegantly described by Boulignand and Giraud-Guille 1-3 (Fig. 2 - 2 ) . All of these elegant three-dimensional (3-D) relationships of the microscopic architectural elements of bone substance (for example, the 3-D spatial orientation of the long axes of the osteones with respect to the long axis of a long bone, the 3-D spatial orientation and disposition of the orientations of collagen fibers and mineral components of the individual lamellae of the osteones, and the 3-D relationship of the orientations of collagen fibers of one lamella and its adjacent lamellae) vary from section to section in the same bone depending on the kind, intensity, and distribution of the stresses on the particular volume of bone sampled. As will be pointed out later in the chapter, the mineral crystallites of apatite are also elegantly ordered, spatially distributed, and oriented with respect to the individual collagen fibril within which they are located. 4 Even at an intermediate level of the anatomical hierarchy, the collagen fibrils in individual collagen fibers

FIGURE 2--2 Schematicdemonstrating that there is an additional axial rotation of the collagen fibers in each of the lamellae of an osteon. (Courtesy of Dr. Marie-Madeleine Giraud-Guille.)

CHAPTER 2

The Nature of the Mineral Phase in Bone

25

FIGURE 2 - 3 Electron micrograph of compact lamellar bone of chick diaphysis. Note that the apatite crystals are not only distributed axially along the long axis (horizontal in the photograph) of the individual collagen fibrils but that they are also in lateral register (vertical dimension of photograph) as well.

are arranged in lateral register over relatively large distances and v o l u m e s of the bone (Fig. 2 - 3 ) . At the next higher level of the a n a t o m i c a l hierarchy, the 3-D organization and spatial distribution of bone as a tissue is equally well organized and ordered to m e e t the m e c h a n i c a l forces applied, viz., the 3-D spatial distribution of the cancellous trabecular plates and sheets so clearly o b s e r v e d in the femoral head and n e c k (Fig. 2 - 4 ) . The same is true of the shape, contour, and distribution of bone as an organ. As examples, note the femur, where there is a specific and appropriate angle of the femoral head and neck to the shaft of the bone to m a x i m i z e hip function and the efficiency of the muscles which supply the p o w e r for m o t i o n and the distribution of forces onto the shaft, and the changing crosssectional profile and variable thickness of the hollow shaft of a f e m u r (see Fig. 2 - 5 ) and other long bones which are reflections of the intensity and distribution of the local stresses applied to the bone at that particular position along the length of the diaphysis. The diaphyseal long bones are hollow rather than solid cylinders, because the m a x i m u m stresses on a cylinder subjected to bending forces occur in the structure farthest f r o m the center or centroid. Thus the m o s t efficient way to distribute the bone mass and use the least a m o u n t of material while also decreasing the weight of the cylinder is to concentrate the bone substance at the outer circumferential periphery of the cylinder. Thus bone, at all levels of the a n a t o m i c a l hierarchy from the gross shape of a bone as an o r g a n , its organ-

FIGURE 2--4 Human femoral head and neck. Note the highly specific organized distribution of the elements of the cancellous tissue (trabecular plates) which correspond very closely to the distribution of mechanical stresses imposed on mechanical loading of the femoral head. Note also the increased mass of the bone substance along the medial wall of the cortex of the upper portion of the femoral neck which is subjected to a great deal more compressive force than the lateral wall due to bending stresses.

26

MELVIN J. GLIMCHER

FIGURE 2--5 A, Cross-sectional slices of the femoral shaft showing different overall profiles and different mass distribution of bone substance within the profiles. This is felt to be regulated by the intensity and nature of the stresses induced by the forces applied to the femur. B, Young lamb tibia. Note flaring of upper end, which articulates with lower end of femur, and marked differences in cross-sectional profiles of lower and upper ends of the bone. (Courtesy of Nicholas Nehamas, Princeton, NJ.)

ization as a tissue, and the manner in which the molecular components are combined to form a multiple-phase composite structural material (bone substance), is very efficiently designed to meet both its mechanical and its multiple biological functions as well.

The distribution of the cancellous bone tissue (e.g., femoral head and neck) and bone substance in various locations along a diaphyseal shaft results in a marked economy in the amount of bone substance needed to meet the mechanical stresses to which it is subjected.

CHAPTER 2 The Nature of the Mineral Phase in Bone Even the flaring out and broadening of the ends of the long bones is an excellent design to increase the surface of the articular cartilage at the ends of bones where they articulate with the articular cartilage of the other bone of the joint. Thus by increasing the surface area of the articular cartilage which bears the load, the stress (force per unit area) applied to the cartilage is decreased significantly. All of these highly ordered geometrical and spatial characteristics of bone as a substance, a tissue, and an organ emphasize how the overall skeletal system has been biologically fashioned in order for bone to function most effectively as a structural substance, as a tissue (cancellous and compact bone), and as an organ (femur, rib) to meet its many physiological and specific mechanical functions (Fig. 2 - 6 ) . Later we will also point out that many of these characteristics as well as the size, shape, specific area, and overall area of the inorganic crystals as well as their location and relationship to the collagen fibrils have also been extremely well designed to meet their biological functions as an ion reservoir for homeostasis of the extracellular fluids, to facilitate their interaction with specific organic components of the matrix and certain bone cells (see below), and to fashion bone substance as an outstanding and efficient structural material. As noted above, recent evidence has indicated that the bone crystals are not only important biologically as an ion reservoir maintaining the ionic homeostasis of the extracellular fluids, and as a major structural element in determining the mechanical properties of bone substance, but they may also play important local roles in cell and matrix interactions that help regulate bone matrix synthesis and resorption 5-8 (Fig. 2 - 6 ) . An example of a crystal-matrix protein-cell signal interaction is the experiment that demonstrated that when osteocalcin (a non-collagenous bone matrix protein) was bound to apatite crystals and implanted in the subcutaneous tissues of an experimental animal, it chemotactically induced a migration of large monocytic cells to the crystal-osteocalcin complex where the cells proceeded to differentiate to active osteoclasts and resorbed the crystals. When the crystals alone were implanted, or when they were coated with albumin, a serum protein found in relatively large concentrations in bone and which probably is bound to the crystals, they initiated a typical macrophage inflammatory response. The cells did not differentiate to osteoclasts and the crystals were not resorbed. 6 These data may explain in part a characteristic feature of tickets, characterized by a marked diminution in the amount of apatite crystals deposited in the organic matrix of bone and cartilage, and in which the rate of the endochondral sequence of ossification is decreased. This occurs despite the fact that the normal sequence (but not the rate) of the cellular

27

FIGURE 2--6 Venndiagram showing the interactions between the internal and surface receptors of the cells and their gene expressions, of the constituents of the organic matrix, and the inorganic crystals of apatite. (Courtesy of Dr. Peter Hauschka.)

differentiation of the cartilage cells of the growth plate is maintained. However, the diminution or failure to calcify the hypertrophic zone of the cartilage matrix is accompanied by a very marked decrease in the invasion of the hypertrophic cartilage by blood vessels and osteoclasts (chondroclasts), and the subsequent formation of bone on the surfaces of the calcified cartilage by osteoblasts accompanying the blood vessels, possibly in part, because of the very significant decrease or absence of the mineral phase in the matrix of the hypertrophic zone of the growth plate. Consequently, there is a marked decrease in the amount of, or even a lack of, osteocalcin bound to the mineral phase. Similarly, it may also help explain why the more mineralized portions of bone are preferentially resorbed by osteoclasts during the normal turnover and remodeling of bone. Although not a direct function of bone mineral or of bone tissue, bone as an organ does provide for another important general physiological function: the marrow spaces, especially of cancellous bone, act as a host or depository for the precursors of the blood cells as well as the progenitor cells of bone. Conceptually, it is not surprising that the crystal structure and the fine, short-range 3-D order of the crystals and the environment of the reactive groups, especially on their surfaces, determines to a large extent their physical chemical reactive properties, and thus their potential biological functions. This dependence of physiological and biological function on the 3-D spatial distribution of the ion and atom constituents of the crystals is similar to what has been found for other biological components such as proteins. In addition, a very important physiological poifit to note is that like some of the other com-

28 ponents in bone such as the collagen, for example, significant changes occur in the composition, short-range order, and surface chemistry of the crystals as a function of the time that the crystals are present in the tissue (i.e., the age of the crystals). These maturational changes significantly alter their interaction properties and consequently their biological, physiological, and structural functions. Thus a " y o u n g " apatite crystal not only differs in its composition from an " o l d " apatite crystal, but also in its short-range order, surface composition, and structure. Importantly, the environment, lability, and reactivity of the surface constituents of the crystals, especially of the HPO4 and CO3 groups, are also significantly different. All of these changes in the crystals change their interaction properties and therefore their potential participation in a variety of biological and chemical functions. Thus any change in the relative rates of bone formation and bone resorption that significantly alters the age distribution of the crystals in bone will also directly alter their role in both mineral metabolism and in the mechanical properties of bone as a substance and as a tissue. It is precisely because the biological functions of the bone mineral ultimately depend on the precise chemical composition, physical chemical properties, and crystal structure of the mineral phase that so much attention has recently been directed at elucidating and clarifying these characteristics. 9-43 It is important to keep in mind that significant changes in both the chemical composition and structure occur in the mineral phase with time (i.e., the mineral phase changes with time after its initial deposition in the tissue). As will be discussed in some detail in a subsequent section, these changes in the mineral phase that occur after their initial deposition in the tissue (the maturation of the crystals) must be kept in mind when attempting to understand and correctly interpret the overall changes in mineral metabolism in vivo either as a function of the age of the organism and more importantly when trying to explain the serum and other changes (e.g., 45Ca uptake and disappearance) observed in normal subjects and especially in patients with metabolic bone diseases, in which the rates of bone formation and resorption have been significantly altered. In these latter instances, the amount and proportion of new bone and old bone, and therefore of the proportion of young crystals and older crystals, has also been markedly changed. This in tum significantly alters the population distribution of bone mineral as a function of bone mineral age and consequently of their physical chemical interaction properties and their biological functions. This makes it impossible to derive a direct relationship between the rate of new bone formation or resorption and new mineral deposition using the uptake, rate of uptake, or rate of disappearance of 45Ca from the serum after

MELVIN J. GLIMCHER

infusing it into the bloodstream and comparing these data with "normal" values or previous values of the patient or of normal standards: the solid mineral phase that is interacting with the ions in the extracellular fluids such as 45Ca is not the same mineral phase that was present under normal circumstances before the rates of bone formation and/or resorption and new mineral deposition were altered by disease or metabolic changes. In the case of an increase in new bone formation, for example, the very young crystals, now making up a larger proportion of the crystals exposed to the extracellular fluids containing 45Ca than existed under normal physiological conditions, are also more reactive and, therefore the 45Ca will not only be more rapidly incorporated into the newly forming crystals of the new bone, but the newly formed crystals will continue to exchange with 45Ca more readily than the older crystals even after their initial deposition. Thus the uptake rate and disappearance rates represent both the fact that there are more crystals being formed and the fact that the new crystals formed and for some time thereafter will continue to exchange more rapidly with 45Ca. Unfortunately, such considerations are rarely taken into account in clinical studies, which may in part account for some of the significant discrepancies noted in metabolic studies between predicted bone and bone mineral accretion and tumover values and those actually observed and measured directly.

II. THE GENERAL NATURE OF THE MINERAL PHASE IN BONE AND THE CHANGES THAT OCCUR WITH TIME The bone mineral has been known by chemical analyses to contain calcium and phosphorus as its principal constituents for over 150 years and since 1894 to be a calcium phosphate carbonate. 44 The first reports of its crystal structure were published by DeJong 45 and Roseberry et al. 46 Both groups of investigators identified the bone mineral as a hydroxyapatite (HA) based on the reflections generated by x-ray diffraction. Unfortunately, progress in identifying in detail the exact chemical composition and specific spatial arrangement of its constituents at any stage of its development (i.e., from its initial deposition to the final mature mineral) has been very slow. Indeed, these parameters are still not known in detail more than 70 years after the bone mineral was first identified as an apatite similar to HA by DeJong. 45 The obstacles that have prevented a definitive resolution of the problem are manymbiological, crystallographic, and technical (for a review, see Elliott29). In the first place, the apatite phase in bone is very poorly crystal-

29

CHAPTER 2 The Nature of the Mineral Phase in Bone line, generating only a few broad and poorly resolved xray diffraction reflections which by themselves do not permit one to assign to it a unique crystal structure or composition (i.e., one cannot differentiate by x-ray diffraction and chemical composition between a number of similar apatitic or apatite-like structures). Indeed, a number of what appear to be closely related but distinct chemical and structural Ca-P compounds give the same or very similar apatitic x-ray diffraction patterns. The poor x-ray diffraction pattern generated by the bone mineral has also precluded the detection of small amounts of Ca-P compounds other than and in addition to apatite. Chemical and physical analyses of bone mineral has also demonstrated that the bone mineral contains small but significant amounts of ions not present in pure HA, such as HPO 2- and CO3, both of which play highly significant roles in the physical, chemical, and interaction properties of the crystals and thus in determining their potential biological and mechanical functions. The ideal stoichiometry and the Ca-P molar ratio of hydroxyapatite, 1.67, is also rarely found in bone, especially in very young bone mineral which has a Ca-P ratio significantly less than 1.67. The bone mineral has also been shown to contain very strongly bound and/or ultrastructurally trapped water; but water is not a true constituent of the pure crystals or HA which has a constituent unit cell and overall formula, Cal0(PO4)6(OH)2. As will be described in detail later, bone apatite is not hydroxyapatite. It is a Ca-deficient apatite, either due to a substitution of Na or Mg for Ca, 47 or a true deficiency of Ca ions not substituted for by another cation, in which electrical neutrality is accomplished by the addition of protons to form HPO4 groups accompanied by the creation of crystal vacancies, 48 or both. The crystals contain both HPO4 and CO3 ions in various different environments and locations within the lattice structure and on the surface of the crystals, which change as a function of time. The crystals do not contain O H groups: they are not an hydroxyapatite. The critical ratio of the important calcium ions in bone mineral is therefore Ca Ca 1217 Ca P + CO-----------~'not -p-. ' The p + CO3 ratio provides an in-

the initial Ca-P solid phase formed in bone has been postulated by several investigators to be a Ca-P solid phase structurally and chemically different than apatite: a precursor solid phase that undergoes a solid state transformation to apatite or dissolves and is recrystallized and replaced by apatite. The two most prominent candidates postulated are (1) an amorphous (non-crystalline) Ca-P solid referred to as amorphous calcium-phosphate ( A C P ) 49-52 and (2) a crystalline calcium-phosphate, octocalcium phosphate (OCP) [Ca8(HPO4)2(PO4)4 9 5 H 2 0 ] , 53'54 which has many crystallographic and compositional features similar to apatite, but importantly contains crystalline water as an integral constituent of its lattice structure. 55-64 The conceptual and experimental basis for the proposition that the initial Ca-P solid phase formed during the calcification of bone is an amorphous and not a crystalline solid was introduced in 1966 by Eanes, Termine, Posner, and colleagues 49-52 (Fig. 2 - 7 , see Color Plate). Their proposal was based on the fact that the x-ray diffraction pattern generated by bone from the youngest animals contained only a few and more poorly resolved broader diffraction peaks than the diffraction patterns generated by bone from older animals--indeed, there is a progressive increase in the overall quality of the diffraction patterns generated, sharper, less broadened, and better resolved reflections, viz., the crystals are "more crystalline" with increased age (Fig. 2 - 8 ) . These inves-

8

dex of the vacancies in the crystal lattice.

III. POSTULATED PHASES OTHER THAN APATITE AS THE INITIAL SOLID CA-P MINERAL PHASE DEPOSITED IN BONE A. A m o r p h o u s C a - P Although the Ca-P crystals of even young bone generate an x-ray diffraction pattern of apatite, the nature of

,~Z)~

45 ~

40 ~

3,5~

30 ~

2.5~

20 ~

FIGURE 2--8 X-ray diffraction patterns generated from the midportion of the diaphysis of chick bone as a function of age. A, Seventeen-day embryonic (periosteal scrapings). B, Five-week postnatal chick. C, Two-year-oldpostnatal chick. D, Standard, highly crystalline synthetic hydroxyapatite. (From Bonar LC, Rouffose AH, Sabine WK, et al: X-ray diffraction studies of the crystallinity of bone mineral in newly synthesized and density fractionated bone. Calcif Tissue Int 35: 202-209, 1983.)

30 tigators reasoned that this phenomenon could arise not only from the fact that the younger crystals were smaller but also because there was more disorder in the structure of the individual crystals. Indeed, they postulated that if one extrapolated this relative disorder in the crystal lattice to the initial Ca-P solid phase formed, it was possible that the initial solid phase was completely disordered, viz., it had no long range order at all, it was amorphous (Fig. 2 - 7 , see Color Plate). They also postulated that with time the amorphous phase was replaced or underwent a solid phase transition to apatite. They based their hypotheses on the concepts outlined and on a series of in vitro precipitation experiments, in which they followed the structural and chemical composition of the Ca-P solids formed in vitro from solutions of Ca and P at neutral or slightly alkaline conditions as a function of the time elapsed after a solid phase was formed. They found that the first solid phase formed was indeed amorphous by x-ray diffraction. With time, the amorphous Ca-P solid phase in contact with the original mother liquor progressively converted to typical, poorly crystalline apatite similar in its x-ray diffraction characteristics and chemical composition to bone apatite. A similar set of experiments using rat bone from animals varying in age from very young postnatal animals to mature animals revealed, as already noted, very poorly crystalline apatite assessed by x-ray diffraction from the very youngest animals, which gradually improved (more "crystalline") with increasing age and maturity. Using an indirect method based on the ratios of the intensities of the apatite reflections and the total Ca-P contents of the samples compared with similar ratios from a series of standards composed of various proportions of synthetic amorphous Ca-P and crystalline apatite, they calculated that a very significant amount of the Ca-P solid phase of the very young bone was not contributing to the x-ray diffraction reflections (i.e., this portion of the Ca-P mineral phase was therefore "amorphous"). Moreover, the amorphous fraction (ACP) decreased with increasing age of the animals. They concluded from these data and their calculations that the very major Ca-P solid phase in the bone matrix of the youngest animals consisted of A CP. Importantly, because the percentage of ACP diminished only very slowly as a function of age, viz., the rate of ACP deposition was greater than the rate at which ACP was converted to poorly crystalline apatite, they concluded that not only was ACP the initial Ca-P solid phase formed, but it remained the major constituent of the mineral phase throughout the continued development and maturation of bone and the accretion of additional mineral phase almost to the point of complete mineralization. The ACP theory was a very attractive hypothesis: It accounted for the progressive change in chemical com-

MELVIN J. GLIMCHER

position with age and maturation as the proportions of ACP and poorly crystalline apatite changed. It explained the increasing intensity of the x-ray diffraction intensities with time as more and more of the ACP (which does not generate or contribute to the intensities of the x-ray diffraction reflections) was converted to poorly crystalline apatite. The ACP formed in vitro also had a low Ca-P ratio, contained tightly bound or crystalline H20, and had other characteristics of newly deposited bone mineral. It is not surprising, therefore, that the ACP theory of the nature of the initial deposits of Ca-P in bone and the changes that occur in the mineral during maturation received very wide international acceptance for more than 20 years. However, as more and more experimental data were compiled, and other structural and compositional factors were taken into account, such as the fact that the bone mineral contains carbonate and HPO4, constituents which change in concentration and in their environment with time and significantly alter the x-ray diffraction characteristics, and the fact that bone apatite is calcium deficient either due to replacement with other cations such as Na and/or M g 47 and/or a true deficiency of Ca ions creating spatial vacancies 48 or both, all of which were found experimentally to change the x-ray diffraction patterns of synthetic apatite standards, some doubt was cast on the validity of the indirect method of determining whether ACP was present in bone mineral and, if so, the quantitative proportion of ACP relative to the total Ca-P mineral phase present. When all of these and other factors were taken into account, the calculated proportions of ACP in bone mineral markedly decreased. Indeed, the ACP content of some samples of young bone previously calculated to contain 70% or more of ACP was now recalculated to be less than one-half of this value. Similarly, a sample of mature bone previously found to contain a significant amount of ACP was now calculated to contain less than 10% ACE an amount indistinguishable experimentally from samples containing no ACE

B. O c t o c a l c i u m - P h o s p h a t e ( O C P ) In the case of OCP, the choice of this crystalline component as the initial Ca-P mineral phase deposited in bone was based on the very close similarity in crystal structure and composition to HA; the very easy and rapid conversion of OCP to HA in vitro; theoretical and experimental thermodynamic and solubility studies; and the presence of water in bone apatites even at very high elevated temperatures which could, at least in part, be accounted for by the presence of water in OCP, particularly in young bone. In the case of both ACP and OCP as well as apatite, however, the obstacles that prevented

CHAPTER 2 The Nature of the Mineral Phase in Bone a clear-cut solution to the problem of defining the exact nature of the initial Ca-P solid phase deposited in bone were both biological and technical. From the biological point of view, there are clear data that significant changes occur in both the chemical composition and structure of the bone mineral with time after its initial deposition in the tissue. Consequently, the amount and proportion of new and old bone and therefore of young and older bone crystals also changes significantly as a function of the rate of bone turnover. During normal aging there is a progressive decrease in the rate of bone turnover and therefore there is a steady progression of a shift in the distribution of crystals as a function of the age of the crystals: there is a larger proportion of " o l d e r " crystals with increasing age of the animal and therefore a significant change in their reactivity and their ability to carry out their potential biological functions. Indeed, after the crystals reach a certain maturity, for example, little exchange of the carbonate groups is possible, even in vitro. 48 The relative rates of bone formation and resorption during the continuous remodeling of bone that normally occurs therefore results in an inhomogeneity of the crystals with respect to their age in any given gross or even microscopic sample of bone. Therefore, to experimentally determine the nature of the initial Ca-P solid phase and any transitional stages as the Ca-P solid phase matures to its final apatitic state, and the pathways and extent of the maturational changes in bone apatite, it becomes necessary to devise methods to obtain samples of bone that are homogeneous with respect to the age of the crystals. This is especially critical in the study of the nature of the initial crystals formed. Failure to recognize these biological impediments, and the use of whole bone samples to characterize the mineral phase provides data reflecting only the average properties of a heterogeneous sample of bone mineral containing various proportions of crystals ranging in age from the very youngest crystals to the very oldest crystals as a function of the age of the animal, has accounted for a good deal of the difficulty in characterizing the bone mineral and the changes that occur with time, and, most especially, in defining the nature of the initial CaP mineral phase deposited. The exception to this occurs in very young embryos such as embryonic chicks in which at the very earliest stages of bone growth and development, the rate of bone remodeling is extremely rapid and the total bone mass is turned over in 10 to 15 hours or less, and in slightly older embryos in 24 hours or less. Also, in very mature, old bone in which there is a very slow rate of turnover, 80% to 90% of the bone mineral was found to be in one density fraction when subjected to differential centrifugation. Thus, the errors due to heterogeneity of the age of the bone crystals are very much reduced but not eliminated if one uses whole

31 bone from very young embryos or from very mature postnatal animals. The third requirement was that one must utilize direct methods to detect the presence of ACP and/or crystal phases other than apatite, and use a variety of special techniques to characterize the solid phase. The problem of obtaining more homogenous samples with respect to the age of the crystals to study the initial very earliest deposits of the bone mineral as well as charting the changes that occur with time and maturation was solved in several different ways. First, by using the very youngest chick embryos as noted, and also in the very same embryos, scraping the very outer layer of the periosteal bony cuff of very young (i.e., 1 1- to 12-day) chick embryos in order to obtain the youngest and most recently deposited bone containing the youngest crystals. Third, it was also possible to obtain much more homogenous samples of embryonic and postnatal bone by differential centrifugation of finely milled bone in organic solvents. We note that we very early learned that exposure of bone crystals to water, for even very short periods of time, especially in the case of very young and highly reactive crystals, very quickly resulted in changes in the structure and composition of the crystals. This led to our development of non-aqueous methods to prepare sections of bone for electron microscopy and for structural analyses. 65 Indeed, for the spectral studies used to characterize the structure of the bone crystals, we extended the non-aqueous techniques to the cleaning and dissection of the bone. This is an important point, especially when dealing with the very youngest specimen in which the solid mineral phase is extremely labile and reactive and in which changes are rapidly induced when exposed to water. With regard to the physical chemical measurements, the bone samples were studied for the presence of ACP directly, using the technique of x-ray radial distribution function analysis (RDF) and high-resolution x-ray diffractometry. 66 In the case of OCP, there are several specific major x-ray reflections and specific spectral peaks when examined by Fourier transform infrared spectroscopy (FT-IR) and 31p nuclear magnetic resonance (NMR), which are unique to OCP and which can be used to identify definitively if this crystalline compound (OCP) is present. These x-ray diffraction reflections and unique spectral peaks of OCP were not present in bone mineral. 48 RDF analyses also failed to detect ACP in the mineral phase. The only crystalline Ca-P solid phase detected was apatite. Another obstacle in studying the very earliest bone mineral formed is the fact that in such samples containing the first crystals deposited, the overwhelming proportion of organic matrix present in the bone sample relative to the bone mineral can seriously alter the x-ray

32 and spectral characteristics of the mineral phase. Indeed, in the very youngest embryonic bone that we have studied to date, the presence of such an overwhelming proportion of organic matrix constituents completely obstructed the detection and characterization of the mineral phase by x-ray diffraction, FT-IR, and 31p NMR. Thus in order to examine the very earliest bone mineral, it was also necessary to develop techniques that would remove the organic matrix constituents from the bone, leaving only the bone mineral, and to accomplish this in a fashion that did not alter the crystal structure, chemical composition, or size of the crystals. This was only recently accomplished. 65 This not only permitted the direct visualization of the bone crystals free of organic matrix constituents by transmission electron microscopy (TEM), but also allowed us to characterize the composition of the very youngest crystals by x-ray diffraction, FT-IR, and 31p NMR spectroscopy. The method of isolating native crystals from biologically calcified tissues has also been successfully applied to progressively older bone and to other tissues such as calcified cartilage, 67 enamel, dentine, and to very old fossil samples of bone. It has also allowed us to chart changes in the size, shape, composition, and structure of the crystal as a function of age and maturation of the crystals. Using this technique, we were able to isolate the youngest bone crystals examined to date, obtained from the periosteal cuff of 7to 8-day-old chick femora. The concentration of the solid phase (i.e., the mineral content in the original tissue) varied from less than 1% to 2% (compared to ---66% in fully mineralized bone). When the isolated crystals were examined by electron microscopy, small crystals typical of bone apatite crystals were observed containing calcium and phosphorus identified by electron microscopic microprobe analysis, and identified structurally as apatite crystals by electron diffraction, x-ray diffraction, FT-IR, and 31p NMR spectroscopy. No evidence for the presence of ACP or OCP was obtained. The only crystalline solid phase detected was a poorly crystallized apatite (unpublished data). However, as we have pointed out in the past, 48 the failure to detect an ACP solid phase or a crystalline OCP phase does not rule out the possibility that ACP or OCP (or other compounds) may be precursors of apatite which are so rapidly and immediately converted to apatite that only an undetectably small amount of the solid phase remains in the tissue at any one time. Considering the extremely small size of the early crystals of bone, especially their thickness, in many cases no more than one unit cell, it is possible that the initial biologically formed, solid Ca-P, precursor phase formed prior to the poorly crystalline apatite phase, will be found to be a bidimensional structure whose short range and atomic and ionic organization and order is different from any crys-

MELVIN J. GLIMCHER

talline 3-D solid. However, for the moment, the only solid Ca-P phase detected even in the earliest stage examined is a very poorly crystalline apatite, distinctly and importantly different than hydroxyapatite. It is important to note the major differences between the possible presence of such an initial transitory Ca-P solid phase different than apatite which so rapidly converts to apatite that it cannot be detected even in the very earliest Ca-P mineral phase deposited in bone examined to date, and the original, amorphous Ca-P theory, 49-52 which concluded that the transition of the ACP to apatite was so slow that ACP was the major CaP solid phase present in bone throughout the development and maturation of bone until final maturity was reached, at which time ACP was markedly diminished or absent. Indeed, in experiments in which a sample of very young bone mineral was examined by both the indirect technique of Termine, Posner, et al. and by RDF, we found that 95% to 100% of the mineral phase was calculated to consist of ACP by the indirect method, whereas no ACP was detected by RDF analysis--only a poorly crystalline apatite phase was identified. At the present time, therefore, the available data do not support the ACP theory that an amorphous Ca-P solid phase is the first solid Ca-P formed during the calcification of bone, and that it remains as a major, albeit diminishing solid phase as a function of the age of the mineral phase. Similarly, there is no direct structural evidence that OCP is the initial Ca-P mineral phase present in bone or that it at any time is present in bone mineral at concentrations detectable by methods currently available. Further, more difficult experiments attempting to obtain data from even younger stages of the mineral phase will be necessary to establish the exact nature of the first nucleated solid phase of Ca-P. Until that is accomplished, the data to date have identified the initial earliest Ca-P solid phase deposited as nanocrystals of a Ca-deficient apatite, most likely containing vacancies as a result of a calcium deficiency, as well as the fact that it contains no OH groups. 27'28'68-71 The bone apatites contain HPO4 and CO3 groups, whose location and environments vary with the age of the crystals. Moreover, 31p solid state NMR spectroscopy has revealed that the HPO4 groups in bone mineral are unique and unlike those present in OCP, brushite (CaHPO4 92HzO) or any other solid phase of Ca-P containing HPO4 including synthetic apatites. 21

IV. CRYSTAL

SIZE AND SHAPE

As noted, the presence of significant amounts of organic matrix constituents in bone has been a major obstacle not only in defining and characterizing the earliest

CHAPTER 2 The Nature of the Mineral Phase in Bone mineral phase formed, but also in the elucidation of the habit and size of the crystals and their internal fine structure. With regard to crystal shape (habit) and size, the problem is further compounded by the intimate relationship of the very small mineral crystals and the collagen fibrils within which the crystals are embedded and their dense accumulation in the tissue (Fig. 2 - 3 ) . However, when viewed in sections by high resolution electron microscopy, one observes regions where the crystals have extremely low electron densities over a relatively broad area, and others where the crystals are seen as very thin and very electron dense lines (Fig. 2 - 9 ) . Both observations are very suggestive and consistent with the conclusion that the crystal habit is that of very small, very thin plates (nanocrystals), first observed by Robinson and Bishop in 1950, 72 Robinson in 1952, 73 and later confirmed by others. Unfortunately, the dense packing of the crystals and their very low density when viewed approximately at fight angles or even obliquely to the surfaces of the putative flat crystal plates, both of which obscure the outlines of the individual crystals, as well as a lack of knowledge of the exact angle at which the crystals are being viewed, have precluded an accurate description of the exact size and shape of the crystals as determined by standard T E M of tissue sections, or of the changes in their size, shape, composition, and electron

33 diffraction characteristics that may occur with normal aging and maturation of the crystals, or in pathological conditions. This is especially true in the more heavily mineralized regions of bone. Nonetheless, the conclusion that the crystals are plate-like, based on the data noted above, is consistent with observations made on such sections when the specimens are tilted in a goniometer device in the electron microscope. 74-76 Recent topographic, 3-D computer reconstruction analyses of thick sections of calcified and ossified turkey tendon using high voltage electron microscopy 77 have not only vividly delineated the relationship between collagen fibrils and the inorganic crystals but have also provided very convincing evidence that the crystals are thin plates. On the other hand, results from early and more recent studies in which attempts have been made to determine the size and shape (habit) of the crystals in bone and other collagenous-rich tissues by indirect methods such as small angle x-ray scattering 78-85 have indicated that the crystals in bone are needle-like rather than thin plates. Similar conclusions have also been reached from T E M studies of young newly formed bone crystals in tissue sections. 86 The length of the crystals and their a/b size measurements have also been estimated on calculations based on the extent of line width broadening of the few resolved or partially resolved x-ray diffraction peaks. However,

FIGURE 2--9 Electron micrograph of an unstained, longitudinal section of young undecalcified embryonic chick bone. The dense mineral phase appears to "stain" the collagen fibril at regular intervals along its axial length. In some areas, the inorganic crystals can be seen on edge as dark lines. This may be the result of the plate-like crystals being viewed on edge or as bent or folded plates.67 Most of the mineral phase is not resolvable into individual crystals. The very low electron-dense regions consist of the plate-like crystals lying fiat on the grid. This indicates that the crystals are very thin.

34

MELVIN J. GLIMCHER

such measurements are probably equally or more dependent on the degree of disorder in the crystal structure than on their size. The results obtained by small angle x-ray scattering and modeling have been questioned on theoretical and conceptual grounds based on the fact that such measurements must be made on particles that do not interact with each other, a situation that does not exist in the case of bone containing relatively immobile, close packed, interacting bone crystals. Indeed, Wachtel and Weiner 87 have in an elegant experiment demonstrated that when bone crystals are examined by small angle x-ray scattering in bone tissue, the ratio of 1/w indicates that they are needles, whereas when isolated crystals are extracted and dispersed in ethanol and the small angle x-ray scattering measurements repeated, the 1/w ratio clearly indicates that they are plates. Importantly, one should also note that small angle x-ray scattering does n o t provide direct m e a s u r e m e n t s of the crystals in any dimension, but merely the r a t i o s that depict shape. We believe that the electron microscopic observations of isolated bone crystals and the 3-D observations of calcified and ossified turkey tendon have conclusively established that the crystals in bone of species as varied as chicken, fish, mouse, turkey, and bovine exist as small thin plates. The lack of correlation between the shape and size of the crystals as determined by direct TEM visualization of the individual isolated bone crystals and by indirect calculations made from either x-ray line broadening measurements or from small angle x-ray diffraction scattering data and modeling 78-85 as well as from TEM of young bone 86 clearly points out that all of the indirect methods (such as x-ray diffraction line broad-

ening, FT-IR, and small angle x-ray diffraction and modeling) as well as TEM of bone sections fail to provide accurate information about the precise size or shape of the crystals. For example, recent studies of stereograms of electron micrographs of very young isolated crystals in our laboratories from a variety of calcified tissues have indicated that the very thin plates of apatite crystals are curved, bent, and folded on themselves and on immediately adjacent crystals, giving the appearance of very electron dense thin lines and the illusion that very thin needle- or rod-like crystals are being viewed on edge. 69 This may account for many of the apparent needle-like rods observed in thin sections of young bone 86 and in cartilage by TEM. 88-93 However, some of the very electron dense lines may indeed be due to plates viewed on edge. It also means that the thickness of such dense lines may not be a true measurement of crystal thickness. This general phenomenon was pointed out by Hodge. 94 The most convincing evidence that the crystals in bone are plates are the studies of the habit and size distribution of isolated bone crystals by electron microscopy, 65 (Fig. 2 - 1 0 ) including those of the very newly formed bone from the periosteal cuff of 7- to 8-day-old chick embryonic femurs. No rod or rod-like crystals were observed at this or any other stage of crystal age or maturation. Similar findings were reported for the isolated crystals of calcified cartilage 67 and the crystals deposited during osteoblast cell culture. 95 Of interest, however, was the finding that a small proportion of the crystals from samples of the very youngest bone, also present but to a lesser extent in samples of older bone, contained crystals very much smaller than the average size of the crystals. The very smallest of these crystals

FIGURE 2-- 10 Electronmicrographs of isolated crystals from bovine bone free of organic constraints. Crystals are clearly plates. The very low electron density of the crystals lying fiat in the grid indicates that the crystals are very thin.

CI-IAr'X~ 2 The Nature of the Mineral Phase in Bone were somewhat irregularly shaped, and roughly square, whose dimensions ranged from 1 to 2 nm. The presence of such small crystals of that shape and size and ones somewhat larger, as well as the usual larger, more common and characteristic "rectangular" plates, may explain the concern raised by attendees at a recent meeting, 96 namely, the difficulty of being able to conceive, thermodynamically, how apatite "nuclei" could be formed in the size and shape of the characteristic, rectangular plate-like crystals (---200 A-400 A • ---35 to 75 A x --~10 to 40 A) observed by TEM of thin sections of even the very youngest bone. In the first place, we have already pointed out the difficulties of assessing the size and shape of bone apatite crystals by TEM of thin sections. However, 3-D reconstruction from high voltage electron microscopy when modified to highlight the electron density of the mineral phase and diminish or abolish the electron density contribution of the organic matrix, clearly shows a very wide range of crystal size, especially of their length, even correcting for foreshortening. This is consistent with our findings using isolated crystals. Secondly, there appears to be some confusion between the thermodynamically conceptual "nuclei" and the first solid phase that can be observed visually even by electron microscopy. Even the smallest crystals observed by T E M m l e t alone the "relatively large" characteristic, rectangular nanocrystals~do not represent the "thermodynamically conceptual nuclei" (i.e., that critical size cluster of atoms and ions representing the first transition of a solution phase of Ca and P to a solid phase). 4 Although the size and shape of the nuclei induced in bone by heterogeneous nucleation in vivo are not known, Katz 97 has calculated the number of atoms and ions in apatite nuclei heterogeneously nucleated by collagen fibrils in vitro (ranging from 11 to 13). Not only would it be unlikely that one could observe particles of that size and low electron density by TEM of sections of bone tissue or from preparations of isolated crystals, but it would also be unlikely because of the extraordinarily rapid growth of the crystals that occurs immediately after nuclei are formed. Our observations of samples of isolated bone crystals that contained some irregularly square shaped crystals, --~1 to 2 nm in size, however, suggests that after this stage of crystal growth has been reached, there is a continued, rapid, but preferential crystal growth along the c-axis of the crystals producing a thin, rectangular plate. The physiological and biomechanical consequences of such small crystals in bone are important in order for the crystals to carry out their various biological and mechanical functions. First, the surface area of the individual crystals and the number of the total ions and atoms that are located on the crystal surfaces as well as the total surface area of the mineral phase in the skeletal

35 system that are able to exchange or react with the atoms, ions, and other constituents of the extracellular fluid and of the organic matrix is extraordinarily high (several square miles). This permits for the very rapid and extensive exchange, addition, and dissolution of the mineral mass and the constituent ions and atoms in the lattice, on the surface, or in the hydration shell of the crystals such as calcium, magnesium, phosphorus, sodium, carbonate, etc. (i.e., its ion reservoir and homeostasis functions). Mechanically, the highly ordered location and orientation of the very small nanocrystals located within the collagen fibrils not only contribute significantly to the structural rigidity and strength of the bone substance but their nanocrystal size permits an acceptable range of flexibility without fracture or disruption of the bone substance. This would not be possible if the "hardness" of the bone were accomplished by the random distribution of the mineral phase into the organic matrix or even if very large, brittle apatite crystals were impregnated into the collagen fibrils. Indeed, even a simple mixture of collagen fibrils and apatite crystals similar in size and shape to those of bone, or collagen molecules in solution aggregated to a gel in the presence of apatite crystals, produce substances with almost none of the structural properties of bone, in which case the small nanocrystals are deposited within the collagen fibrils in specific ordered locations and in a highly oriented fashion 4'77'98-1~176 (Fig. 2 - 1 1 to 2-20). In this instance, electron microscopy of bone at various stages of mineralization starting from the initial deposition of crystals within the collagen fibrils to the last steps of full mineralization of the fibrils has not only provided visual observations of the intimate relationship between the inorganic crystals of apatite and the collagen fibrils but, very importantly, has provided the physical chemical basis that confirms the original hypothesis that calcification of the collagen fibrils m the event that converts the soft organic matrix in bone to a hard, structural substance or fabric m occurs by the heterogeneous nucleation of apatite crystals within the collagen fibrils in selected, spatially and physical chemical independent nucleation sites. The calcification of the collagen fibrils is the event that hardens the bone substance and provides its structural properties. While there are many ways in which the heterogeneous nucleation of apatite crystals within the collagen fibril can be regulated (changes in the metastability of the EC fluids, complexing with phosphoproteins, etc.), it is critical to distinguish between the basic physical chemical mechanism as to why the crystals are induced to form within the collagen fibrils (heterogeneous nucleation) and the many ways by which the system can be altered to regulate this basic physical chemical process. Purified collagens, aggregated into native type fibrils, are able to nucleate apatite crystals from

36

MELVIN

J. G L I M C H E R

FIGURE 2 - - 1 1 Electron micrograph of the middyaphysis of late embryonic chick bone during early stages of calcification. Similar findings were obtained from early postnatal chicks from the same region during the early stages of calcification of this later stage of chick bone development. Collagen fibrils are seen in cross section. Note mineralfree collagen fibrils (CF) and fibrils in varying stages of mineralization, that is, impregnated with a solid phase of calcium phosphate. The spaces between the fibrils are essentially free of mineral particles. The calcification of each of the fibrils and of the spatially separated sites within the fibrils along the axial length of a single fibril represent physical chemical independent sites of crystal nucleation. Two osteoblast processes (OP) are indicated. (From Landis WJ, Paine MC, Glimcher MJ: Electron microscopic observations of bone tissue prepared anhydrously in organic solvents. J U1trastruct Res 59:1, 1977.)

metastable solutions of Ca and P in v i t r o 1~176 and when implanted in the peritoneal cavities of animals, l~176 The induction time for nucleation to begin is greatly diminished if the collagen fibrils are complexed with the native resident phosphoproteins or other phosphoproteins such as phosvitin. Enzymatic removal of the phosphate groups from the collagen-phosphoprotein complexes eliminates the added advantage of the intact collagen-phosphoprotein complexes. These data demonstrate that the reduction of the induction time induced by the complexing of phosphoproteins to collagen is due to the phosphate groups per se. 101 - 103

V. RECENT STUDIES OF THE STRUCTURE OF BONE APATITES AND THE APPLICATIONS OF THESE DATA TO CLINICAL AND EXPERIMENTAL ABNORMALITIES AND DISEASES OF BONE Over the past 6 to 7 years there has been an increased awareness that there are important differences between

the biological apatites and the geological and synthetic apatites, including those precipitated from solutions simulating extracellular fluids. Moreover, it has also become clear that the crystals initially formed, either biologically in a tissue or during in vitro precipitation, undergo a series of compositional and structural changes as a function of the time they remain in the tissue or remain in contact with the solution from which they were precipitated. These changes are collectively referred to as "maturation." Importantly, the reactivity of the crystals (i.e., their ability to interact with the ions and proteins in the ECF and with cells, and therefore their ability to carry out their biological and structural functions) is directly related to the structure and composition of the crystals, particularly the surface components. To address these problems, attention has been directed at the fine structure, short range order of the crystals, and the environment of two of the minor but important ions that play a major role in determining the reactivity of the crystals, CO3 and HPO4. These studies have included computer-generated deconvolution of FT-IR spectra, with and without curve-fitting, solid state, magic-angle spinning 31p NMR spectroscopy, and FT-IR microscopy of

CHAPTER 2

The Nature of the Mineral Phase in Bone

37

FIGURE 2--12 A and B, Electron micrographs of cross-sections of a fully calcified intermuscular fish bone of a pickerel. In this flexible bone, the collagen fibrils are relatively widely spaced from one another making it possible to determine whether the crystals are located between, or within the collagen fibrils. It is clear from this electron micrograph, as well as electron micrographs from other species of fish, 98 that the crystals are located within the collagen fibers. Note that there are no matrix vesicles observed. No other intracellular or extracellular calcified structures are present in the intramuscular bones of this or many other fish species.98 Note that there are few if any matrix vesicles present (either calcified or uncalcified) in chick bone in the late stages of embryonic development or postnatally (Fig. 2-10). (See also Landin SJ, Paine MC, Hodgens KJ, Glimcher MJ: Matrix vesicles in embryonic chick bone: Considerations of their identification, number, distribution, and possible effects on calcification of extracellular matrices. J Ultrastruct Mol Struct Res 95:142-163, 1986.) The mineral phase is located at these stages of development within the collagen fibrils. The basic two-phase composite material of bone substance which determines the mechanical properties of the bone substance are the calcified collagen fibrils.

tissue sections. These have revealed a number of important new details about the structure and composition of bone crystals, the changes that occur during the maturation of the crystals, and some of the factors that regulate the process. These techniques, especially FT-IR TM and FTIR microscopy, 1~ have been used to study various diseases and experimentally produce abnormalities in bone. First, as we have already pointed out, it is important to note that the apatite crystals of bone are not hydroxyapatite. Not only do they not contain hydroxyl groups, but the short range order and organization is distinctly different than synthetic or geological apatites. Indeed, in a recent, as yet unpublished study we were able to distinguish synthetic apatites from bone apatites using a new experimental technique using solid state magic-

angle spinning 31p N M R . These fundamental differences between bone apatites, and particularly synthetic apatites, which are used in m a n y in vitro studies, and which attempt to relate the results of certain perturbations of the solution phase during the precipitation of synthetic apatite crystal in vitro, or their interaction properties with organic components, or changes induced in the physical structure and chemical composition of the crystals once the solid state has been achieved by the introduction of specific ions or organic constituents, must be taken into account if one attempts to postulate what might happen or what has happened under certain conditions in vivo, or to interpret the role of specific ions or organic constituents on the crystallographic structure of the mineral phase, the size of the crystals, the " c r y s t a l l i n i t y " of the

38

FIGURE 2-- 13

Higher magnifications of a section similar to those depicted in Figure 2 - 1 2 A and B. Electron micrograph of unstained fish bone which was not decalcified. The dense particles, identified by electron diffraction as Ca-P apatite crystals, are located essentially within the collagen fibrils, as observed in this electron micrograph in which collagen fibrils are seen primarily in cross-sectional profile. Inset shows two adjacent collagen fibrils in cross-sectional profile at higher magnification. Mineral particles are located within the collagen fibrils. The location of the crystals within the collagen fibrils and not in the extracellular spaces is an important point in trying to establish the actual mechanism as to how and why the crystals are nucleated within the collagen fibrils, and the possible role of other factors which may regulate the nucleation event within the collagen fibrils. Care must be directed at distinguishing between the mechanism wherein crystals are nucleated within the collagen fibrils and the possible physical, chemical, and other factors which may regulate the basic

mechanism.

mineral phase, the mechanism of the induction of the mineral phase, or the phase transitions which occur after the initial mineral phase is formed and matures to a final phase, etc. 117-121 Secondly, one should also be aware of the limitations of certain techniques used to evaluate such changes in the crystals either in vitro or in vivo. For example, because of the very small size of the nanocrystals of bone and apatite synthesized in vitro to sim-

MELVIN J. GLIMCHER

ulate bone crystals, x-ray diffraction analyses provide only very limited information. Indeed, it cannot distinguish between bone apatites and apatites synthesized to simulate bone apatite, even when the compositions of the two are markedly different or purposefully altered over a wide range of compositions. This is due to the fact that the small crystals of bone and of synthetic apatite crystals are also poorly crystalline, and therefore generate only a few x-ray reflections which, in addition, are also poorly resolved (Fig. 2-8). Thus, despite compositional and structural changes in the crystals as a function of the time that they spend in the tissue (maturation) or in the mother liquor from which they were precipitated in vitro (maturation), x-ray diffraction may not show any discemible change, or if some overall change does occur such as better or less well-resolved reflections, it will not be possible to discem what has taken place to have caused such changes, viz., change in crystal size, increased or decreased perfection of the short- and long-range order, or the presence of small amounts of non-apatitic solid phases of Ca-P which may also alter the x-ray diffraction pattem. Because of the frequency with which x-ray diffraction is used to explain the results of experiments that result in changes in the mineral phase, and the conclusions and implications drawn from such experiments, we reiterate that it is possible to synthesize crystals with a very broad range of compositions including Ca-P ratios, all of which generate an apatitic x-ray pattern similar to those generated by "normal" bone crystals. In particular, we note again that the composition, short-range order, local environments of the crystals change significantly with the age o f the crystals in vitro or in vivo and that these ages are reflected in changes in the "crystallinity" of the mineral phase as "deduced" by x-ray diffraction. Therefore, if a disease process or an experimentally induced perturbation alters the rates of bone resorption or both which alters the proportion of young and old crystals in the bone, it will result in significant changes in the x-ray diffraction pattern and possibly in other spectral analyses as well. However, we should not conclude, as some investigators have, that the perturbated processes per se have directly altered the composition or structure of the crystals. The perturbation may have simply changed the relative proportion of young and old crystals in the tissue or in the in vitro synthesized precipitate. Nor should one attempt to interpret the mechanism of such changes or to attribute these alterations to changes in crystal size or the perfection of the crystallographic order of its constituent ions and atoms from x-ray diffraction data alone. With respect to the term "crystallinity" or "index of crystallinity," 122 which is often used to characterize bone and other biological and synthetic apatites, and to note

CHAPTER 2 The Nature of the Mineral Phase in Bone

39

FIGURE 2-- 14

A series of high-voltage electron micrographs illustrating the successive stages of mineral deposition in herring bone tissue. Thick (1-1xm) cross sections of collagen fibrils were treated completely anhydrously for specimen preparation. A, The very first electron-dense deposits (arrows) are seen to occur within the boundary of the collagen fibril. B and C, As the mineralization progresses further, it is apparent that no mineral particles have been deposited in the extracellular spaces between the fibrils. The deposition of mineral particles in the adjacent collagen fibrils demonstrates that the nucleation of Ca-P crystals in each of the fibrils and in each of the hole zones is an independent physical chemical event.

changes in apatites that have occurred as a result of experimental perturbations in their preparation in vitro or as the result of in vivo alterations in bone metabolism, we note that "crystallinity" or "index of crystallinity" is a function of both crystal size and how well the atoms and ions of the crystals are ordered, that is, the perfection and extent of the ordered disposition of the individual ion and atom constituents in the crystals. "Crystallinity" or "index of crystallinity" is most commonly measured or evaluated from certain characteristics of the x-ray diffraction pattern generated, principally by the number of reflections, the resolution of the reflections, and the breadth of the reflections at a

particular height of the reflections. It has also been evaluated from FT-IR spectra by several different methods. For example, it is possible to prepare a series of separate samples of large, highly ordered crystals in which each separate sample consists of crystals that differ in size from the other separate samples. Under these circumstances, it would be possible to obtain relatively accurate data of the size of the crystals, or certainly of the relative size of the crystals in each of the separate samples by analyses of the characteristics of the x-ray diffraction patterns generated by the crystals, all of which would have many, well-resolved peaks that would vary somewhat in the breadth of the individual reflections.

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MELVIN J. GLIMCHER

FIGURE 2-- 15

A and B, High-voltage electron stereomicrographs of a pickerel fish to be prepared anhydrously seen in cross-sectional profile. A' and B', Higher magnification of the same regions illustrating the electron-dense Ca-P particles located within collagen fibrils. Three dimensional location of these mineral crystallites can be fully appreciated by stereoscopic examination, eliminating the possibility that these minerals are located on the surface of the section.

The FT-IR spectra, however, which depend more on internal order or disorder and vary much less on crystal size, would in this instance not provide a very good or possibly any information relative to the size of the crystals. Similarly, one could prepare a series of separate samples of large crystals of roughly the same size in which each of the separate samples contained crystals that differed in how well the atoms and other constituents were ordered (i.e., differences in the perfection of the short- and long-range order). Under these circumstances, it would be possible to detect these differences in crystal perfection by changes in the x-ray diffraction patterns and, depending on the nature of the disorder, by FT-IR and possibly 31p NMR spectroscopy as well. However, in the case of the very small nanocrystals of bone apatites and of the equally small, synthetic nanocrystals precipitated in vitro similar to bone apatites, the "crystallinity" or "index of crystallinity" will depend on both the size of the very small crystals and the fact that the ions or atom constituents of the crystals are not as well ordered, in part because so many of these atoms

and ions are located on the surfaces of the crystals, many in an ordered configuration unlike those in the internal lattice, especially the labile environments of CO3 and HPO4. Since both the small crystal size and the relative disorder of the crystal structure contribute to the characteristics of the x-ray diffraction patterns generated by such nanocrystals, especially in the very young crystals, and to the spectral characteristics of the crystals by FTIR and 31p NMR, it is not possible to separate or distinguish which of the variables (size or disorder) or what proportion of the spectra can be attributed to the size or the internal disorder of the crystals. However, since the average size of the apatite crystals of bone as determined by the direct visualization of isolated crystals by TEM does not increase very much as a function of the age of the crystals, 67 the very poor x-ray diffraction patterns generated by the youngest embryonic bone crystals, which improves with the age of the animal, is probably due in large measure to the degree of perfection of the short- and long-range order and very much less to changes in the average size of the crystals. Similarly,

CHAPTER 2 The Nature of the Mineral Phase in Bone

41

FIGURE 2--16 Schematic of the Hodge-Petruska model of type I collagen fibrils demonstrating that the fibrils contain a very large volume of highly organized space probably in the form of channels. There are also present smaller spaces (pores) between individual collagen macromolecules. The Hodge-Petruska model answered the perplexing question of how so much mineral could be accommodatedin the closely packed collagen fibrils without completelydisrupting and destroying the structure of the fibrils.

with the exception of the large apatite crystals of enamel, which increase in size significantly with age and time of maturation, changes in the x-ray diffraction patterns of bone apatites during maturation in vitro and in vivo, probably represent to a greater extent changes in the internal order of the crystals and less to their size. Although the x-ray diffraction characteristics and the FT-IR spectra generated by the nanocrystals of bone apatite used to characterize their "degree of crystallinity" for the most part reflect their lattice crystallographic disorder, especially at the lower end of the scale (viz., very poorly crystalline mineral phase such as occurs in the very youngest embryonic bone or newly synthesized bone at any stage of development), these characteristics probably also reflect to a lesser extent the increased proportion of the very smallest nanocrystals observed by TEM of preparations of isolated crystals 67 and by 3-D high-voltage electron microscopic tomography of calcifying and ossifying turkey tendon. 77 Therefore, one must view with caution the interpretation of in vitro experiments in which changes are introduced into the solution phase from which crystals are precipitated or from experiments in which crystals are incubated after precipitation with various ions and other components, and changes noted in their x-ray diffraction characteristics and/or the FT-IR spectra, or in their solubility and other characteristics, and these results interpreted as due to

changes in crystal size or changes in crystallographic perfection, especially if x-ray diffraction alone has been used to analyze the crystals.

Specific Ions 1. CO3 IONS There are two major sites in the crystal lattice into which CO3 ions can be introduced: the OH sites (type A carbonate apatite) and the PO4 sites (type B carbonate apatite). 123'~24 CO3 ions in these lattice positions are relatively stable and not very reactive or exchangeable.~3C NMR spectroscopy studies of synthetic carbonate aparites and enamel aparites, however, have identified five magnetically inequivalent CO3 ions (viz., three different environments in addition to the A and B sites). 13 These different sites may be the result, as already noted, of the different chemical environments in which they are located and/or geometric factors, such as shorter bond links between carbon and oxygen which would shield the carbon atom, or distortions of the planar structure possibly by rearranging the p-electrons in the bonding orbitals of the Type A carbonate. Further analysis of these data and other structural data in the literature suggest that the three additional carbonate sites or environ-

42

MELVIN J. GLIMCHER

FIGURE 2--18 FIGURE 2 - 1 7

Experimental proof that most of the crystals are indeed located within the hole zone channels of the collagen fibrils was provided by additional electron micrographs of very young chick bone in which both the mineral phase and the intraband period of the collagen fibril containing the crystals were photographed in a single section. The identification of the location of the mineral phase in bone collagen between the a 3 and c 3 bands places the crystals in the hole zone channels. (From Glimcher MJ, Krane SM: The organization and structure of bone, and the mechanism of calcifications. In Ramachandran GN, Gould BS (eds): Treatise on Collagen, vol 7B. New York, Academic Press, 1968.)

ments are labile and very reactive, whereas the other two stable lattice carbonate locations are stabile and very much less reactive. Similar findings have been observed in ~3C studies of bone crystals, but the very low intensifies and somewhat poorer resolution have precluded a detailed interpretation of the ~3C spectra in bone crystals. FT-IR studies of bone crystals have also identified labile environments for CO3 ions in both biological apatites from bone, calcified cartilage, dentine and enamel, and in synthetic apatites similar to bone apatites. It is likely that these labile, very reactive carbonate groups are located on the surface of the crystals and not in the lattice proper. These very reactive carbonate groups are very important as far as the biological and structural functions of the crystals are concerned.

Schematic to illustrate that the majority of the crystals initially deposited in the collagen fibrils are located in the hole zone (channels).

2. HPO4 IONS Although HPO4 ions have been demonstrated by chemical analysis, 14 F T - I R , 14'19 and most recently by 31p NMR solid state, magic-angle spectroscopy, 9-1~'2~ both in biological and synthetic apatites with or without the presence of carbonate groups, it is very likely that the HPO4 ions do not exist as single "entities" in specific sites. 21 It is much more likely that there exist regions within the crystal lattice and on the crystal surfaces in which the packing density of the protons is greater than in other regions and that in these regions the protons are spatially closer to particular P O 4 ions. 21 As in the case with CO3 ions, stable HPO4 "groups" are also present in the crystal lattice, and labile, very reactive HPO4 groups are present on the surface of the crystals. From kinetic studies it would appear that both labile CO3 and labile HPO4 groups occupy the same surfaces. These labile HPO4 groups, like the labile CO3 groups, are very reactive and, also like the labile CO3 groups, are probably responsible for a major share of the interaction properties of bone apatite crystals. Recent studies of synthetic and bone apatites and other calcium phosphate solids containing HPO4 groups using a modified technique of differential crosspolarization, solid state 31p NMR magic angle spinning

CHAPTER2 The Nature of the Mineral Phase in Bone

43 ing CO3 and HPO4 ions from synthetic or geological apatites containing CO3 and HPO4 ions. In addition to the labile and stable HPO4 groups and the major phosphorus constituent of apatite, stable PO4 ions, high-resolution computer-generated deconvoluted FT-IR studies have identified at least two environments of labile P04 ions that were first detected only in very y o u n g c r y s t a l s . 14'19 Curve fitting of the deconvoluted FTIR spectra have more clearly demonstrated the labile PO4 groups. The presence of such labile PO4 groups may account for the very slight irregularity of the PO4 peak observed in certain preparations of young bone crystals examined by cross-polarization, solid state 31p NMR spectroscopy.

3. CHANGES IN CO3 AND HPO4 IONS DURING MATURATION (AGING OF CRYSTALS)

FIGURE 2--19

Schematic to illustrate that further calcification of the collagen fibrils occurs both by primary heterogeneous nucleation and by secondary, tertiary nucleation, etc., that is, nucleation from crystals already formed either from heterogeneous nucleation, or from secondary nucleation and are propagated in the collagen pores, eventually filling all of the available space within the fibril, and converting the soft pliable fibrils and fibers into a continuous hard substance and fabric. High-voltage 3-D electron microscopy 98 has also demonstrated that there is expansion of the collagen fibrils in the region of the hole zones, which permits the deposition of additional crystals of apatite. At this point in the calcification of the collagen fibrils, the --~700-A axial period of the mineral phase reflecting their position within the hole zone regions of the collagen fibril, which do have an axial repeat of --~700 A, is lost due to the deposition of crystals throughout the available spaces including the pore spaces.

spectroscopy have for the first time been able to obtain the spectrum from the small number of HPO4 groups present in bone and synthetic apatites directly in the presence of the very major PO4 peak. Previous 31p NMR studies 9'1~have assumed the presence of HPO4 groups by comparing the 31p NMR spectroscopy patterns with model compounds containing HPO4 groups such as Ca HPO4" 2H20 (brushite). The studies that have directly analyzed the 31p NMR spectral characteristics of the HPO4 groups in bone crystals have shown that the HPO4 groups are unique and are unlike those present in octocalcium phosphate, amorphous calcium phosphate, and brushite as well as synthetic apatites precipitated from solution. 21 These data once again emphasize the significant differences between the biological apatites contain-

Studies of changes in the total content of CO3 and HPO4 ions and of the labile and stable environments of these ions in small synthetic crystals similar to bone apatites have revealed that the very earliest crystals formed have a high concentration of HPO4 ions and a very low concentration of CO3 ions. In both instances, a large proportion of these ions are in very labile, highly reactive environments. Most of these ions are located on the surface of the crystals as determined by the kinetics of dissolution experiments. With time there is a progressive increase in the CO3 concentration which occurs first on the surface as labile ions, and which later displace some of the surface HPO4 ions. During further stages of the maturation, CO3 ions are progressively incorporated from the surface into stable positions in the apatite lattice as HPO4 groups (H § ions) in the lattice are displaced. Thus the stable CO3 groups in the crystal lattice increase during maturation while those on the surface present as labile CO3 ions decrease during maturation. 1e'14'17'19'48 Both the surface labile HPO4 groups and the stable HPO4 groups in the lattice decrease during maturation. Additional important studies have now correlated reactivity and ease of exchange of the CO3 ions as a function of the stage of maturation. 48 The crucial role of CO3 ions in the maturation of apatite crystals was demonstrated using synthetic apatite crystals precipitated in vitro, in which CO3 crystals were precipitated from solutions containing high concentrations of CO3 ions and from solutions containing a very low concentration of CO3 ions. These experiments clearly demonstrated that the initial crystals formed were independent of the CO3 concentrations of the initial solutions from which the crystals were precipitated: the crystals formed from solutions containing high concentrations as well as low concentrations of CO3 ions were characterized by their very low concentration of CO3 ions and relatively high concentra-

44

MELVIN J. GLIMCHER

FIGURE 2--20 Schematic showing the progressive calcification of the collagen fibrils and the loss of the axial periodicity of the mineral phases at the stage of maximum calcification. Calcification of mitochondria has been reported in the cartilage and questionably in bone. A great deal of attention has been focused on the calcification of matrix vesicles, which are very likely to be portions of the cell and cell membrane "pinched off." We emphasize as we have in the past, that (1) calcification of the vesicles and of the individual fibrils and indeed of the specific hole zone regions in a single fibril are all independent sites for nucleation. There is no way that the solid crystals in the matrix vesicles or, indeed the crystals in one channel of a single fibril which is spatially separated from another channel, or the crystals from one fibril spatially separated from another fibril, can directly induce the nucleation of crystals in any other spatially distinct sites. It is very unlikely that the solid-phase crystals can " s w i m " from the matrix vesicles specifically to the hole zone regions of the collagen fibrils. [From Glimcher MJ: On the form and function of bone: From molecules to organs. Wolff's Law revisited. In Veis A (ed): The Chemistry and Biology of Mineralized Connective Tissues. New York, Elsevier North-Holland, 1981, pp 618-673.] Neither can they " s e e d " the collagen fibrils by the release of the crystals from the matrix vesicles and induce additional crystals to form in the extracellular fluids between the fibrils. If that occurred, one would easily observe that the extracellular spaces between the fibrils would be occupied by crystals and that this would occur before calcification was initiated in the crystals. It is quite clear from the electron micrographs presented that this does not occur. As noted, matrix vesicles are observed only in the early phases of embryonic development of bone. With maturation of the bone, the number of matrix vesicles rapidly declines and, eventually, they are not found in the extracellular matrix of the more mature, developing embryonic and postnatal bone. Thus, during the embryonic and early and late postnatal stages of development, the initial bone tissue containing the matrix vesicles is wholly resorbed and the new bone tissue that has formed to replace it during the progressive growth and development of the bone to its adult stage is mineralized in the absence of matrix vesicles. During all of these stages, calcification occurs in the collagen fibrils. The precise biological function of the matrix vesicles in the early stages of bone development, or in some cases, in very rapidly synthesized bone (e.g., during the repair of bone defects or fractures) is not clear. Their absence in the later stages of embryonic bone development and in the postnatal period and their absence in the intermuscular bones of a number of fish species, in which it is the collagen fibrils which are calcified, establishes that the calcification of matrix vesicles is not obligatory for the calcification of collagen fibrils, which is, as we have pointed out, the component that hardens bone tissue.

tions of HPO4 ions, both of which were in labile environments. These data are wholly consistent with the findings obtained from the very early crystals formed in bone, cartilage, dentine, and enamel, all of which were found to have very low concentrations of CO3 and high concentrations of HPO4, both of which, like the in vitro crystals, were in labile environments. Thus the concen-

tration of CO3 ions in the solutions from which the crystals are formed in vitro or in vivo has little or no effect on the nature of the initial crystals formed. However, the evolution and nature of the maturational changes were also found to be highly dependent on the concentration of the CO3 ions in the solutions with which the crystals were subsequently equilibrated. However, there were

CHAPTER2 The Nature of the Mineral Phase in Bone distinct changes in the nature and pattern of the maturation processes depending on whether the initial crystals formed from solution (independent of whether the crystals were formed from solutions containing high or low CO3 concentrations) were equilibrated shortly after their precipitation with CO3-rich or CO3-poor solutions. In the former case (CO3-rich solutions), the maturational changes observed were very similar to those found during the maturation of bone crystals in vivo. On the other hand, if the initial crystals formed (both those precipitated from solutions with high or low concentrations of CO3 ions) were then equilibrated with solutions with low concentrations of CO3 ions, the evolution of the maturation changes were distinctly different than those occurring in bone crystals in vivo, and more closely resembled the nature and pathway observed in enamel crystals in vivo: continued low concentrations of CO3 and HPO4 .48 Interestingly, the crystals that evolved with maturation changes similar to those observed in bone crystals did not incorporate OH ions, whereas those that followed the maturational changes similar to those observed during the maturation of enamel crystals did eventually incorporate OH ions which increased in extent with time of maturation (enamel does contain OH groups that progressively increase during maturation). Interestingly, the evolution and the maturational changes were also dependent on the length of time that elapsed after precipitation and before the crystals precipitated in CO3-free solutions were equilibrated in CO3poor solutions. If this time was prolonged, there was less and less tendency for the crystals formed in CO3-rich solutions or CO3-poor solutions to mature in the same fashion as bone crystals in vivo. Indeed, if the elapsed time between precipitation and reequilibration with CO3rich solution was prolonged, the crystals precipitated from solutions with rich CO3 concentrations, despite incubation and equilibration in CO3-rich solutions, followed the maturation pathway of enamel crystals and not those of bone crystals. It is clear from these experiments that it is the CO3 concentration in the equilibrium solution during maturation that regulates the nature and rate of the maturational changes observed, and that this critical, relatively high concentration of CO3 must be present shortly after the crystals are formed if the rate and, most importantly, the path of maturation observed in bone crystals in vivo is followed by in vitro synthesized crystals. The reactivity of the labile and stable CO3 groups has also been studied using 13C-~2C exchange experiments. These studies showed that labile CO3 ions on the surface of the crystals are exchanged very rapidly, but are not exchanged or are exchanged only very slowly once they are incorporated into stable positions in the apatite lattice. It was interesting that the last CO3 ions incorporated

45 into lattice positions were in regions of less "crystallinity" than the first CO3 ions incorporated into the lattice structure. Another interesting finding was the interdependence of CO3 and HPO4 ions in establishing the compositions and short-range order and their environments of the apatite crystals: apatite crystals that contained only a few labile phosphate ions had little capacity to mature and incorporate CO3 into lattice positions, and underwent very few of the normal maturational changes in the composition and environment of both the HPO4 and CO3 ions observed both in vitro and in vivo, including the fact that CO3 ions were not incorporated into the interior of the crystals in stable CO3 sites (substituting in OH positions and for PO4 ions). Conversely, if the apatite crystals are rich in both labile C O 3 and labile HPO4 ions, they show a great potential for undergoing the maturational changes observed in bone crystals in vivo: the CO3 ions that do substitute for HPO4 ions on the surface can later be incorporated into the interior lattice of the maturing crystals in the two main stabile CO3 sites. An overview of the maturation processes that occur in vivo and in CO3-rich equilibrium solutions in vitro, which are similar to those that occur in vivo, begins by an exchange of CO3 ions from the solution phase displacing the labile non-apatitic HPO4 ions on the surface of the crystals. These labile carbonated domains increase in number as the less stable initially formed crystals, which are rich in labile phosphate species, are replaced by a solid phase whose surface is rich in labile CO3 ions. Substitution of one divalent ion (HPO4) by another divalent ion (CO3) does not modify the charge balance or the number of calcium ions. The number of vacancies is not changed during the process, nor is there is an increase in crystal size of the crystals equilibrated in CO3rich solutions. 4. MG IONS

A fairly large number of other atomic elements are associated with the bone crystals in very low concentrations. The phrase "associated with the crystals" is used advisedly, since it is not known for certain whether such ions are located in the lattice structure of the crystals, adsorbed on the surfaces of the crystals, or whether they are present in the more peripheral "hydration shell" enveloping the crystals. Magnesium (Mg) is one such ion. Mg is the fourth most abundant cation in the body and the second most abundant cation in the intracellular fluid. 125 Because of the signal importance of Mg in bone metabolism 125 and as a vital component in other important biochemical processes and enzymatic reactions, 126-132 and because a major portion (--~50% of the total body Mg) is associated with the mineral phase of bone, we chose to use it as an example of the problems facing investigators seeking to uncover exactly how el-

46 ements such as Mg are associated with the bone crystals (viz., precisely where the Mg ions are located, since their location and their environment will, in large part, determine as they do for CO3 and HPO4, how easily they can participate in the regulation of homeostasis of Mg in the ECF and in other biological functions as well). Similarly, the postulated direct roles of Mg in the formation, size, crystallinity, solubility, and other characteristics of the crystals will also depend on the precise location of the Mg ions with respect to the lattice and surfaces of the crystals. Although it is commonly stated that two-thirds of the Mg ions present in bone are an integral part of the apatite lattice, 125 presumably replacing Ca atoms, there is little direct evidence to support this conclusion, and a great deal of evidence that suggests that the vast majority, or, indeed, all of the Mg atoms are either located on the surface of the crystals and/or in the more peripheral "hydrated shell" that envelopes the crystals. These latter conclusions were based on the conceptual framework developed by Neuman and Neuman ~33 and later by Holmes et al. TM who proposed that, because of the small nanocrystal size of the bone crystals and their very large surface area with respect to their size, as well as the fact that the crystal surfaces contain charged constituents, that ions such as Na and Mg cannot only be incorporated into the apatitic lattice but can also be associated with bone crystals as bound counterions directly on the surfaces of the crystals or in the peripheral hydration shell. The "hydration shell" described by Neuman and Neuman ~33 was conceived by Holmes et al. TM as 100- to 300-A intercrystalline "pores" containing water. Both concepts have the same implication (viz., water compartments between the nanocrystals in which specific ions are concentrated). Experimentally, Neuman and Mulryan, ~35 using small, synthetic crystals of apatite precipitated from solutions containing Mg under conditions simulating those in vivo, performed kinetic and equilibration experiments using 2SMg. They found from such studies that 90% or more of the Mg ions were very rapidly changeable and, therefore, were located either on the surface or in the peripheral hydrated shell. They found no convincing evidence for the presence of stable Mg in the structural lattice of the apatites. On the other hand, in similar experiments using apatite crystals simulating those crystals present in bone, other investigators 47'~36-~39 concluded that a few Mg ions present in the apatite precipitates were actually incorporated into the apatite lattice displacing Ca. However, these conclusions, which were based principally on changes in the apatitic lattice parameters measured by x-ray diffraction, were questioned because of the poor resolution of the x-ray diffraction patterns generated which precluded the precise and sufficiently accurate measurements nec-

MELVIN J. GLIMCHER

essary to calculate the very small differences noted in the lattice parameters. As has been pointed out 29 there are a number of other changes that could occur when precipitating apatite crystals in the presence of Mg that could account for the changes in lattice parameters. 29 Several authors have shown that the uptake of Mg could be increased by increasing the uptake of CO3 ions. 137 However, it has been argued that, rather than increasing the inclusions of Mg into the apatite lattice, the additional uptake of Mg by the crystals may have been due to complexing with CO3 ions on the surfaces of the crystals or in the hydration shell. Several authors have suggested that the changes in the lattice parameters may also be accounted for by alterations other than the introduction of Mg into the apatitic lattice, for example, by surface compositional changes or by the formation of small amounts of other Ca, Mg, and PO4 solid phases. Except for the few exceptions noted, attempts to incorporate Mg into the apatite lattice have been carried out on large, highly crystalline hydroxyapatite crystals synthesized in most cases at high temperatures, or under very severe hydrolytic conditions. Even under these circumstances, it has not been possible to definitively demonstrate by x-ray diffraction that Mg was indeed incorporated into the lattice structure of the apatites. Similarly, very detailed conceptual and experimental studies concluded that it was very unlikely from the theoretical crystallographic calculations and from the experimental data that Mg ions could be incorporated as an integral part of the apatite lattice. 14~ To solve and settle these important questions requires direct crystallographic structural analysis utilizing single crystals of the putative Mg-substituted apatites combined with spectral evidence. Unfortunately, it has not been possible to date to prepare single crystals of apatite containing Mg and there are currently no special spectral methods available to study the environment of Mg in preparations of apatite crystals that contain or which are associated with small amounts of Mg. The fact that Mg may not be an integral part of the apatitic lattice does not in any way preclude the Mg ions present on the surface or in the hydrated shell of the crystals from participating in the homeostasis functions of the mineral phase. Indeed, their location on the surfaces and in the hydration shell of the crystals would permit them to respond more readily, since total dissolution of the crystals would not be necessary to release Mg ions into the ECF nor new crystals formed to remove Mg ions from the ECF. References 1. Boulignand Y, Giraud-Guille MM: Spatial organization of collagen fibrils in skeletal tissue: Analogies with liquid crystals.

CHAPTER 2

The Nature of the Mineral Phase in Bone

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CHAPTER 2

The Nature of the Mineral Phase in Bone

83. Fratzl P, Fratzl-Zelman N, Klaushofer K, et al: Nucleation and growth of mineral crystals in bone studied by small-angle xray scattering. Calcif Tissue Int 48:407-413, 1991. 84. Fratzl P, Fratzl-Zelman N, Klaushofer K: Collagen packing and mineralization. Biophysics 64:260-266, 1993. 85. Fratzl P: Statistical model for the mineral structure in bone. J Stat Phys 77:124-144, 1994. 86. Traub W, Arad T, Weiner S: Origin of mineralized crystal growth in collagen fibrils. Matrix 12:251-255, 1992. 87. Wachtel E, Weiner S: Small-angle x-ray scattering study of dispersed crystals from bone and tendon. J Bone Miner Res 9: 1651-1655, 1994. 88. Arsenault AR, Hunziker EB: Electron microscopic analysis of mineral deposits in the calcifying epiphyseal growth plate. Calcif Tissue Int 42:119-126, 1988. 89. Arsenault AR, Grynpas MD: Crystals in calcified epiphyseal cartilage and cortical bone of the rat. Calcif Tissue Int 43: 219-225, 1988. 90. Bonucci E: Fine structure of early calcification. J Ultrastruct Res 20:33-50, 1967. 91. Bonucci E: Further investigation on the organic/inorganic relationships in calcifying cartilage. Calcif Tissue Int 3:38-54, 1969. 92. Bonucci E, Silvestrini G, Digrezia R: The ultrastructure of the organic phase associated with the inorganic substance in calcified tissues. Clin Orthop 233:243-261, 1988. 93. Bonucci E: Comments on the ultrastructural morphology of the calcification process: An attempt to reconcile matrix vesicles, collagen fibrils and crystal ghosts. Bone Miner 17:219-222, 1992. 94. Hodge AJ: In discussion of: Grynpas et al., The emergence and maturation of the first apatite crystals in an in vitro bone formation system. Connect Tissue Res 21:227-237, 1989. 95. Rey C, Kim H-M, Gerstenfeld L, Glimcher MJ: Structural, chemical characteristics, and maturation of the calcium-phosphate crystals formed during the calcification of the organic matrix synthesized by chicken osteoblasts in cell culture. J Bone Miner Res 10:1577-1588, 1995. 96. Rey C, Kim H-M, Gerstenfeld L, Glimcher MJ: Characterization of the apatite crystals of bone and their maturation in osteoblast cell culture: Comparison with native bone crystals. Connect Tissue Res 35:343-349, 1996. 97. Katz EP: The kinetics of mineralization in vitro. Biochim Biophys Acta 194:121 - 129, 1969. 98. Lee DD, Glimcher MJ: Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus and herring (Clupea harengus) bone. J Mol Biol 217:487-501, 1991. 99. Glimcher MJ: Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds. Phil Trans R Soc London B 304:479-508, 1984. 100. Glimcher MJ: A basic architectural principle in the organization of mineralized tissues. Clin Orthoped Relat Res 61:16-36, 1968. 101. Glimcher MJ, Hodge AJ, Schmitt FO: Macromolecular aggregation states in relation to mineralization: The collagen hydroxyapatite system as studied in vitro. Proc Natl Acad Sci USA 43:860-867, 1957. 102. Glimcher MJ: Molecular biology of mineralized tissues with particular reference to bone. Rev Mod Physics 31:359-393, 1959. 102a. Mergenhagen SE, Martin GR, Rizzo AA, et al: Calcification in vivo of implanted collagens. Biochim Biophys Acta 43:563565, 1960.

49 102b. Goldhaber P, Burr TJ Jr., Glimcher MJ: Calcification of purified, native type (---700 A axial repeat) reconstituted collagen and decalcified bone powder in Millipore filters implanted in the peritoneal cavity of rats. Presented at Gordon Conference, Meridian, NH (unpublished). 103. Glimcher MJ: Mechanism of calcification: Role of collagen fibills and collagen-phosphoprotein complexes in vitro and in vivo. Anat Rec 224(2):139-153, 1989. 104. Rey C, Lian J, Grynpas M, et al: Non-apatitic environments in bone mineral: FT-IR detection, biological properties and changes in several disease states. Connect Tissue Res 21:267273, 1989. 105. Boskey AL, Pleshko N, Doty SB, Mendelsohn R: Applications of Fourier transform infrared (FT-IR) microscopy to the study of mineralization in bone and cartilage. Cells Mater 2:290220, 1992. 106. Boskey AL, Camacho NP, Gadaleta S, et al: Applications of Fourier transform infrared microscopy to the study of biologic mineralization. L'Eurobiologiste 30:259- 267, 1996. 107. Gadaleta SJ, Camacho NP, Mendelsohn R, Boskey AL: Fourier transform infrared microscopy of calcified turkey leg tendon. Calcif Tissue Int 58:17-23, 1996. 108. Gadaleta SJ, Landis WJ, Boskey AL, Mendelsohn R: Polarized FT-IR microscopy of calcified turkey leg tendon. Connect Tissue Res 34:203- 211, 1996. 109. Paschalis EP, Jacenko O, Olsen B, et al: Fourier transform infrared microspectroscopic analysis identified alterations in mineral properties in bones from mice transgenic for type X collagen. Bone 19:151 - 156, 1996. 110. Paschalis EP, Jacenko O, Olsen B, et al: The role of type X collagen in endochondral ossification as deduced by Fourier transform infrared microscopy analysis. Calcif Tissue Int (in press) 1997. 111. Boskey AL, Camacho NO, Mendelsohn R, et al: FT-IR microscopic mappings of early mineralization in check limb bud mesenchymal cell cultures. Calcif Tissue Int 42:443-448, 1992. 112. Boskey AL, Pleshko N, Binderman, Mendelsohn R: Mineralization during in vitro calcification: An FT-IR microscopic mappings of early mineralization in chick limb bud mesenchymal cell cultures. Calcif Tissue Int 51:443-448, 1992. 113. Mendelsohn R. Hassenkhani A, DiCarlo E, Boskey AL: FT-IR microscopy of endochondral ossification at 10Ix spatial resolution. Calcif Tissue Int 44:20- 24, 1989. 114. Pleshko N, Mendelsohn R, Boskey AL: Developmental changes in bone mineral: An FT-IR microscopy study. Calcif Tissue Int 41:72-77, 1992. 115. Pleshko N, Mendelsohn R, Boskey AL: A novel IR spectroscopic method for the determination of crystallinity of hydroxyapatite minerals. Biophy J 60:786-793, 1991. 116. Paschalis EP, DiCarlo E, Betts F, et al: FT-IR microscopic analysis of human iliac crest biopsies from normal bone. Connect Tissue Res 34:287, 1996. 117. Aoba T, Moreno EC, Chimoda S: Competitive adsorption of magnesium and calcium ions onto synthetic and biological apatites. Calcif Tissue Int 41:143-150, 1992. 118. Bigi A, Foresti E, Gregorini R, et al: The role of magnesium on the structure of biological apatites. Calcif Tissue Int 40: 439-444, 1992. 119. Blumenthal NC, Betts F, Posner AS: Stabilization of amorphous calcium phosphate by Mg and ATE Calcif Tissue Res 23:245-250, 1977. 120. Boskey AL, Rimnac CM, Bansal M, et al: Effect of short-term hypomagnesemia on the chemical and mechanical properties of rat bone. J Orthop Res 10:774-783, 1992.

5D

121. Cohen L, Laro A, Kitzes R: Bone magnesium, crystallinity index and state of body magnesium in subjects with senile osteoporosis, maturity-onset diabetes and women treated with contraceptive preparations. Magnesium 2:70-75, 1983. 122. Bonar LC, Roufosse AH, Sabine WK, et al: X-ray diffraction studies of the crystallinity of bone mineral in newly synthesized and density fractionated bone. Calcif Tissue Int 35:202-209, 1983. 123. LeGeros RZ, LeGeros JP: Carbonate analysis of synthetic mineral and biological apatites. J Dent Res 62:259, 1983. 124. LeGeros RZ, Trautz OR, LeGeros JP, Klein E: Carbonate substitution in the apatitic structure. Bull Soc Chim Fr 4:17121718, 1968. 125. Rude RK: Magnesium homeostasis. In Bilezikian JP, Raissz LG, Rodan GA (eds): Principles of Bone Biology. San Diego, Academic Press, 1996, pp 2 7 7 - 293. 126. Rude RK, Oldham SB: Disorders of magnesium metabolism. In Cohen RD, Lewis B, Alberti KGMM, Denmon AM (eds): The Metabolic and Molecular Basis of Acquired Disease. London, Baillier Tindall, 1990, pp 1124-1148. 127. Wacker WEC, Parish AF: Medical progress: Magnesium metabolism. N Engl J Med 45:658-663, 1968. 128. Wacker WEC, Parish AF: Medical progress: Magnesium metabolism (continued). N Engl J Med 45:712-716, 1968. 129. Wacker WEC, Parish AF: Medical progress: Magnesium metabolism (concluded). N Engl J Med 45:772-776, 1968. 130. Elin RJ: Assessment of magnesium status. Clin Chem 33: 1965-1970, 1987. 131. Wallach S: Availability of body magnesium during magnesium deficiency. Magnesium 7:262-270, 1988. 132. Wallach S: Effects of magnesium of skeletal metabolism. Magnes Trace Elem 9:1 - 14, 1990.

MELVIN J. GLIMCHER 133. Neuman FW, Neuman MW: The nature of the mineral phase of bone. Chem Rev 53:1, 1953. 134. Holmes JM, Davies DH, Meath WJ, Beebe RA: Gas adsorption and surface structure of bone mineral. Biochemistry 3:20192023, 1964. 135. Neuman WF, Mulryan B J: Synthetic hydroxyapatite crystals: IV. Magnesium incorporation. Calcif Tissue Res 7:133-138, 1971. 136. LeGeros RZ, Daculsi G, Kijkowska R, Kerebel B: The effect of magnesium on the formation of apatites and whitlockites. In Itokawa K, Durlach J (eds): Proc Magnesium Symposiums, 1988. Magnesium in Health and Disease. New York, Libbey, 1989, pp 11 - 19. 137. LeGeros RZ, Kijkowska R, Bautista C, LeGeros JP: Synergistic effects of magnesium and carbonate on properties of biological and synthetic apatites. Connect Tissue Res 33:203-209, 1995. 138. LeGeros RZ, Miravite MA, Quirolgico GB, Curzon MEJ: The effect of some trace elements on the lattice parameters of human and synthetic apatites. Calcif Tissue Res 22:$362-$367, 1975. 139. Boulet M, Marier JR, Rose D: Effect of magnesium on formation of calcium phosphate precipitatives. Archs Biochem Biophys 96:629-636, 1962. 140. Driessens FCM, Verbeeck RMH, Heijligers HJM: Some physical properties of Na- and CO3-containing apatites synthesized at high temperatures. Inorganica Chim Acta 80:19-23, 1983. 141. Driessens FCM, Verbeeck RMH: Domomite as a possible magnesium-containing phase in human tooth enamel. Calcif Tissue Int 37:376-380, 1984. 142. Featherstone JDB, Mayer I, Driessens FCM, et al: Synthetic apatites containing Na, Mg, and CO3 and their comparison with tooth enamel mineral. Calcif Tissue Int 35:169-171, 1983.

_~HAPTER

Parathyroid Hormone and Parathyroid Hormone~ Related Peptide in Calcium Homeostasis Bone Metabolism and Bone Development: The Proteins, Their Genes, and Receptors JOHN T. POTTS, JR. AND HARALD JOPPNER Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

I. II. III. IV.

Introduction: Regulators of Mineral Ion Homeostasis Parathyroid Hormone Parathyroid Hormone-Related Peptide Receptors that Mediate Analogous and Distinct Molecular Actions of PTH and PTHrP

METABOLIC BONE DISEASE

V. Summary: Overall Biological Roles of PTH and PTHrP, and Their Cloned Receptors References

51

Copyright 9 1998 by AcademicPress. All rights of reproductionin any form reserved.

52

JOHN T. POTTS, JR. AND HARALDJOPPNER I. I N T R O D U C T I O N :

REGULATORS

OF MINERAL

ION HOMEOSTASIS Parathyroid hormone (PTH) and the active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)zD3], are the principal physiological regulators of calcium homeostasis for humans and all terrestrial vertebrates. ~'2 The actions of both hormones are coordinated, and each influences the synthesis and secretion of the other. 1,25(OH)zD3 is critical for the day-to-day and week-to-week calcium balance; PTH is vital for the minute-to-minute regulation of extracellular calcium concentration, and mediates its actions through a Gprotein-coupled receptor, the PTH/PTHrP receptor. 3'4 The PTH/PTHrP receptor also serves several other, still incompletely characterized functions, not directly related to the control of calcium homeostasis. Particularly important is the receptor's role in bone development, where it mediates the actions of parathyroid hormone-related peptide (PTHrP). PTHrP was first discovered as the most important cause of the humoral hypercalcemia of malignancy syndrome, and it is now known to be of considerable importance for chondrocyte proliferation and differentiation, and thus for bone elongation. 5'6 As discussed below, PTH and PTHrP share some structural and functional similarities but are distinct molecules that mediate their unique physiological roles through the common PTH/PTHrP receptor. Current evidence indicates that intact PTH and PTHrP, or proteolytic fragments of either peptide, interact also with other, not yet isolated, receptors. 7-~4'3~However, the biological functions mediated through these receptors are still unknown. Extracellular calcium concentration is maintained within narrow limits to ensure a multitude of important cellular functions. 15'16 Furthermore, an adequate supply of calcium is required to mineralize the skeleton, which contains over 99% of the body's calcium. Bone, in turn, through its continuous remodeling, serves as a calcium reservoir. Food intake is discontinuous; intestinal calcium absorption thus occurs only intermittently and calcium content of the diet may be low, even when isocaloric. Therefore, the maintenance of a constant blood calcium concentration constitutes a major homeostatic challenge for most terrestrial vertebrates, and requires control of urinary calcium losses, efficient intestinal calcium absorption, and, if necessary, the rapid mobilization of calcium from the skeletal reservoir. ~6By contrast, regulation of calcium in blood and tissue fluids of marine animals poses a different environmental challenge inasmuch as the concentration of calcium in seawater exceeds that in extracellular fluid so that calcium disposal

(and the conservation of phosphate) is the principal challenge, not the prevention of hypocalcemia. 17 Phosphate, at least in early evolutionary history, was scarce in seawater, by contrast to its abundant supply in diets of terrestrial animals. It is attractive to speculate that certain aspects of mineral ion metabolism in modem terrestrial vertebrates may reflect earlier evolutionary challenges, specifically the efficient gastrointestinal absorption and renal retention of phosphate and the less efficient intestinal absorption and renal conservation of calcium. To maintain extracellular calcium concentrations at a constant level, PTH acts directly on bone and kidney, and indirectly via 1,25(OH)zD3 on the intestine. 1'~5 Through these mechanisms, it increases the flow of calcium into the extracellular fluid and thus maintains normal blood calcium concentrations. ~6 PTH synthesis and secretion are stimulated by a decrease in extracellular calcium, and are, conversely, inhibited by increased concentrations of this mineral ion (Fig. 3 - 1 ) . This negative feedback regulation of PTH production by blood calcium, and the resulting modulation of its biological activity, is the most important homeostatic mechanism for controlling calcium concentrations in the extracellular fluid. PTH also has important effects on the regulation of inorganic phosphate concentration, principally through renal actions, but these effects are best understood as a secondary rather than a homeostatic action. Daily urinary phosphate clearance most closely correlates with dietary intake of phosphate, and is independent of changes in

FIGURE 3--1 Physiologicalactions of PTH. The hormone acts directly on receptors in bone and kidney and indirectly (through vitamin D) on gut to raise the concentration of calcium in the extracellular fluid (ECF) compartment. Calcium in the ECF in turn exerts a negative feedback inhibition on the parathyroid gland to reduce the rate of PTH secretion.

CHAPTER 3 Parathyroid Hormone and Parathyroid Hormone-Related Peptide PTH secretion. Phosphate is abundant in the food chain in terrestrial existence. Therefore, phosphate deficiency, unlike nutritional calcium deficiency, is rarely the result of an environmental challenge, but rather caused by urinary phosphate wasting due to rare genetic disorders or t u m o r s . 18'19 Whenever calcium is released by dissolution of bone through PTH-dependent mechanisms, which would serve as a defense, for example, in a period of starvation to defend blood calcium content (desirable), phosphate is simultaneously liberated, increasing its concentration in blood (undesirable). High blood phosphate levels p e r s e tend to lower calcium concentrations through multiple mechanisms; it is therefore beneficial to excrete any excess phosphate in urine and to minimize, at the same time, urinary calcium losses by enhancing its tubular reabsorption. PTH protects calcium homeostasis efficiently by promoting urinary phosphate excretion while lowering urinary calcium excretion; PTH thus functions effectively by its combined bone and renal actions. There have been considerable advances in the chemical and functional characterization of PTH and PTHrP, and their genes, as well as the isolation and/or characterization of different receptors (especially the common PTH/PTHrP receptor) that mediate the endocrine and paracrine/autocrine actions of both hormones. These advances achieved initially through traditional methods of peptide chemistry and synthesis, later combined with techniques of molecular, developmental, and cellular biology, have led to a much improved understanding of the structure and function of these ligands (PTH and PTHrP), their receptors, and their biological actions throughout fetal and adult life.

II. PARATHYROID H O R M O N E During evolution parathyroid glands first appear as discrete organs in amphibians (i.e., with the migration of vertebrates from an aqueous to a terrestrial existence). ~7'2~ Although parathyroid glands have not been identified in fish or invertebrate species, PTH-like immunoreactivity was detected in the pituitary of several fish species ~7'2~'22and a nucleofide sequence with significant homology to the mammalian PTH gene was identified in trout genomic DNA. 23 Other reports provided evidence for a PTHrP-like substance in several nonmammalian v e r t e b r a t e s p e c i e s , 24-26 suggesting that homologs of both peptides developed early in vertebrate evolution. In mammals, PTH was thought to be produced exclusively by the parathyroid glands. However, small amounts of its mRNA were recently detected in the rat hypothalamus, 27'28 indicating that PTH may be produced

53

in the central nervous system (CNS), where it could act either through the PTH/PTHrP receptor or the PTH-2 receptor, which are both expressed in various sections of the brain. 29-31 PTH shares structural and functional similarities with PTHrP, and possibly another PTH-like peptide that was recently purified from the hypothalamus. This putative PTH-like entity was shown to interact predominantly with the PTH-2 receptor and not at all, or very poorly, with the common PTH/PTHrP receptor that is used by PTH and PTHrP. 32 PTH-dependent regulation of mineral ion homeostasis is largely mediated through the PTH/PTHrP receptor, which belongs to a novel family of G-protein-coupled receptors33-35; the receptor is most abundantly expressed in the target tissues for PTH actions (i.e. kidney and b o n e ) . 36'37 The PTH/PTHrP receptor is, however, also found in a large variety of other fetal and adult tissues, and at particularly high concentrations in growth plate chondrocytes) 8-4~ It is likely that the PTH/PTHrP receptor mediates in tissues other than kidney and bone, the paracrine/autocrine actions of PTHrP, rather than the endocrine actions of PTH. Other receptors that interact with intact PTH or its fragments have also been described, but these novel, so far insufficiently characterized receptors probably mediate functions that are not involved in the control of calcium homeostasis.

A. Chemistry The first biologically active extracts of parathyroid hormone from bovine glands were made in 1925 by using hot 5% hydrochloric acid, which partially degraded the protein. 41 Further purification of the hormone was not achieved for 30 years, when Aurbach 42 and Rasmussen et al. 43 developed improved extraction procedures with denaturing solvents (phenol, urea) that did not degrade the hormonal polypeptide. Subsequent efforts led to the isolation and structural analysis of bovine, porcine, and human PTH. The bovine and porcine hormones were extracted and purified from parathyroid glands collected as a by-product of meat-packing c e n t e r s . 41'42'44-47 The limited supplies of human tissue, however, required the development of special procedures for the extraction and purification of peptides to increase yields of the human hormone. 48 Isohormonal forms of PTH were suspected to exist on the basis of fractions isolated during the final chromatography of extracts of parathyroid glands in urea. However, only one hormonal isoform has been purified and characterized structurally in the bovine. 47 The amino acid sequence of the major form of bovine PTH and that of the porcine and human hormones were determined by the then standard techniques of protein chemists; automated, stepwise removal and identification

54

JOHN T. POTTS, JR. AND HARALDJOr'r'NER

of amino acids from the amino-terminal end of the intact polypeptide and from peptide fragments prepared by proteolytic digestions of P T H . 49-54 With the advent of recombinant DNA cloning and sequencing techniques, peptide hormone sequences were deduced from nucleotide sequence without requiting isolation of the protein p e r se; these techniques were used to determine the sequence of rat, chicken, and dog PTH. 55-58 The primary structure of human PTH and those of other vertebrate species are shown in Figure 3 - 2 . Nucleotide sequence analyses of the bovine and human PTH genes confirmed the amino acid sequence of the hormones determined earlier by protein structural analysis. 59-61 Not shown in Figure 3 - 2 are the points of difference between findings of two groupsmglutamine rather than glutamic acid at position 22 in the bovine, porcine, and human hormones, and lysine and leucine rather than leucine and aspartic acid at positions 28 and 30, respectively, of the human hormone. 52 Despite extensive reinvestigation of the sequences by protein structural techniques, 62"63 no explanation for the discrepancy in results has been found. One explanation is the presence of isohormones in the bovine, porcine and human species that contain the sequence differences proposed for each molecule by Brewer et al., 52'62 but as yet no isohormones with these structural features have been reported. Inasmuch as synthetic peptides used in parathyroid research today are based largely, if not exclusively, on the sequences of bovine, human, and porcine hormone 5~ that were confirmed by nucleotide sequence analysis, 59-61 only these amino acid sequences are included in Figure 3 - 2 . There is extensive sequence homology among human, bovine, porcine, dog, and rat PTH; the hormone from each of these vertebrate species is a single-chain polypeptide of 84 amino acids, which is devoid of cysteines or substituted amino acid residues, and has a molecular

1

10

20

weight of approximately 9300. There is a preponderance of basic residues conferring an overall positive charge to the molecule. The middle portion of the molecule is quite hydrophobic and exhibits the greatest structural differences among species; this region is also the site of proteolytic attack in peripheral tissues (see Section II.C). Differences in the amino acid sequence can account for the reduced reactivity of hormone from one species with antisera raised against hormone from a second species. Chicken PTH departs significantly from the overall pattern seen with the mammalian hormones. It has two sequence deletions in the hydrophobic middle portion of the sequence and one larger addition of amino acids near the carboxyl-terminus (see Fig. 3-2). The aminoterminal region, associated with most known biological actions, shows high homology with PTH from other vertebrate species, but has several amino acid changes in the 1 3 - 2 2 region. Overall, this biologically important, amino-terminal region of PTH shows strong homology among all known vertebrate species; however, the degree of sequence preservation in PTH is less than that noted in PTHrP; furthermore, the middle and carboxylterminal regions show greater sequence variation in PTH than in PTHrP, including changes of amino acid charge (see Fig. 3 - 2 and later Fig. 3-10). The development of solid-phase methods for peptide synthesis was an important stimulus for the understanding of structure/activity relationships for PTH. 64 A series of peptide syntheses defined the 1 - 3 4 region of the molecule as essential and sufficient for full calciumhomeostatic activity in multiple bioassay systems, 65'66 and allowed comparisons of the biological activity of full-length PTH and synthetic peptide fragments (Table 3-1). The subsequent synthesis of hundreds of PTH and, later, of PTHrP analogues and fragments defined the minimal sequence of PTH that retained measurable cy-

30

40

human bovlne porolne dog rat ohloken 50

human bovlne porcine dog rat r162

70

60

D K A D V N V

L T

D K A --D V / V D K Ai~Ivt~v I D K A D VI,]V

LB

i

I

H L R A A V Q K K S I D L D K AII~N

80

K A K

S Q

K A KUQ LlUlK A KUQ L T K A K S Q i

V LN

KIWI KIIII~

FIGURE 3--2 Alignmentof the amino acid sequences of all known vertebrate PTH species. Conserved residues are shown in white letters on black background; numbers indicate the positions of amino acids in the mammalian peptide sequences.

CHAPTER3 ParathyroidHormone and Parathyroid Hormone-Related Peptide TABLE 3-- 1

55

Comparison of Biological Activity of Parathyroid Peptides from Different Species Potency, MRC U/mg a In Vitro Rat Renal Adenyl Cyclase Assay

In Vivo Chick Hypercalcemia Assay

Native hormones Bovine 1 - 8 4 Porcine 1 - 8 4 Human 1 - 8 4

3000 (2500-4000) 1000 (850-1250) 350 (275-425)

2500 (2100-4000) 4800 (3300-7000)

Synthetic fragments Bovine 1 - 34 Human 1 - 34 [Alal]-Human 1 - 3 4

5400 (3900-8000) 1700 (1400-2150) 4300 (3400-5400)

Peptide

10,000 (9060-13,400) 7700 (5200-11,100) 7400 (5200-9700)

aValues expressed as mean potency with 95% confidence items, based on Medical Research Council research standard A for parathyroid hormone. Reprinted, by permission, from Rosenblatt M, Kronenberg HM, Potts JT Jr: In DeGroot L (ed): Endocrinology. Philadelphia, WB Saunders Co, 1989, pp 848-891.

clic adenosine monophosphate (cAMP)- stimulating activity [PTH(1-27)] and demonstrated the critical importance of residues 1 and 2 for stimulating adenylate cyclase. 66-73 A series of PTH analogues with aminoterminal deletions led to the early concept of separable binding and activation domains within the PTH molecule. 67'68 The analysis of a series of amino-terminally truncated PTH analogs, PTH(3-34), PTH(5-34), PTH(7-34), PTH(10- 34), and PTH(15-34), 68 demonstrated a stepwise loss of binding affinity with progressive shortening of the amino-terminus. The peptide PTH(7-34) retained specific, although greatly reduced, binding affinity yet showed no significant agonist activity in most systems such that it could be used as an inhibitor of PTH action in vitro and in v i v o . 7~ Evolutionary conservation of the 2 5 - 3 4 region among human, 54 b o v i n e , 49'5~ porcine, 51 and rat P T H 55'74 sequences led to a series of synthetic peptides and their evaluation in radioreceptor assays. Analyses of these PTH analogues and of additional PTH fragments shortened at the amino-terminus, including PTH(20-34) and PTH(25-34), led to the concept that PTH(25-34) comprised a principal binding domain of the PTH(1-34) molecule; however, the markedly lower binding affinity of these peptides demonstrated the contribution of sequences from the amino-terminus for high-affinity binding. 67'68'75 The important contribution of position 12 in PTH antagonist design was evidenced by the 10- to 30fold greater potency of D-Trp substituted analogues as inhibitors of PTH(1-34) a c t i o n s . 68'75'76 Considerable information is accumulating which indicates that regions of PTH(1-84) other than the aminoterminal 34 residues may be responsible for novel bio-

logical actions, although the analysis of these effects is still limited to in vitro studies. For example, a series of synthetic peptides encompassing the central region of PTH delineated a putative chondrocyte mitogenic domain, which does not appear to involve cAMP activation, to a core region, PTH(30-34). 77'78 Furthermore, competition binding assays with renal plasma membranes and rat osteosarcoma cells have demonstrated binding sites for carboxyl-terminal PTH(53-84) which are discrete from those that bind PTH(1-34). 9'79-81 The observation that amino- and carboxyl-terminal fragments of PTH elicited contrasting effects on alkaline phosphatase activity, 82-84 and, in other systems, osteoclast activity, 85 supports the existence of a receptor with specificity for the carboxyl-terminal portion of PTH. Recent studies that led to the characterization of ~80- and ~30-kDa proteins, represent an important step in the effort to clone and define the chemical/functional properties of this novel receptor/binding protein which in turn may further clarify the biological role of midregional/ carboxyl-terminal PTH fragments which bind to and activate it. 1~ Conformational analysis of PTH presents a formidable challenge. Ideally the hormone-receptor complex would be co-crystallized to permit analysis by x-ray diffraction of the intermolecular interactions that are characteristic of the biologically active hormone-receptor complex. Such analysis, as has been done with growth hormone and its receptor 86 (an analysis made feasible because the soluble, extracellular domain of the growth hormone receptor is sufficient for high-affinity hormone binding), allowed molecular modeling of ligand/receptor interaction to proceed on a rational basis. Similar struc-

56 tural data would be desirable for a G-protein-linked receptor such as the PTH/PTHrP receptor with bound ligands, such as PTH or PTHrP. However, these receptors are embedded in a plasma membrane and have several membrane-spanning helices, and are thus likely to have a significantly more complex structure. Furthermore, binding of PTH or PTHrP to this complicated receptor molecule and its subsequent activation probably involves several domains, including extracellular loops as well as membrane-spanning domains, and not just the aminoterminal, extracellular domain as in the growth hormone receptor. Such a lipid-embedded multidomain structure cannot so far either be crystallized or subjected to multidimensional nuclear magnetic resonance (NMR) analysis. However, the three-dimensional structure of PTH and PTHrP as free ligands was analyzed using either P T H ( 1 - 8 4 ) or the active fragments of PTH and PTHrP by various physicochemical methods including NMR, and the results of these studies have been summarized in a recent review. 1 Of particular interest was the question whether some common structural features might be discerned for both ligands that would explain the equivalent binding of PTH and PTHrP to the common PTH/ PTHrP receptor. A two-dimensional NMR study of the 1 - 3 4 fragment of PTHrP revealed that in aqueous solution, pH 4.5, residues 3 - 9 are oL-helical, residues 10-13 and 1 6 - 1 9 form two types I [3-turns, and residues 2 0 - 3 4 form a nonhelical but ordered conformation. 87 Early NMR studies of P T H ( 1 - 3 4 ) in aqueous solution failed to detect an ordered structureSS; however, an ordered structure was found when the peptide was analyzed after being dissolved in the helix-promoting solvent trifluoroethanol (TFE). 89 A more recent study of P T H ( 1 - 3 7 ) in an aqueous solvent led to a model in which an oL-helix was detected between residues 5 - 1 0 , and residues 17 through at least residue 28. 90 Between these two helical regions there is a flexible link, residues 12 and 13, and a [3-turn, residues 14-17; the helical carboxyl-terminal end of the ( 1 - 3 7 ) molecule is bent back to become parallel with the helical region at the amino-terminus. Strong hydrophobic interactions detected between tryptophan 23 and leucine 15 stabilize the structure. Thus, the amino-terminal fragments of PTH and PTHrP have, even when studied under similar conditions, clearly distinctive secondary structures that may reflect their limited primary structural similarity (see Fig. 3-11). Since both peptides nonetheless bind to and activate the same PTH/PTHrP receptor with similar or indistinguishable affinity and efficacy, respectively, 91-94 it is conceivable that both ligands may develop a similar and highly specific conformation when part of an active

JOHN T. POTTS, JR. AND HARALD JOPPNER

hormone/receptor complex, a conformation not detected so far when the peptides are analyzed as free ligands in various solvents.

2. Regulation of Biosynthesis and Secretion 1. HISTORICAL PERSPECTIVE Accounts of the early studies that led to the elucidation of the biosynthetic pathways involved in the formation, cellular transport, and metabolism of PTH are provided in several reviews. 95-98 Studies of hormone biosynthesis in vitro, in which pulse-chase incubations of slices of parathyroid tissue with radioactive amino acids were used, led to the identification of the precursor preproPTH.99-104 PreproPTH was one of the first secreted proteins to have its initial translational product defined. The precursor includes a hydrophobic leader or signal sequence which, as reviewed below, plays a central role in directing export of the protein through the cell for secretion. 96-99 Highly sensitive protein-sequencing techniques were utilized to determine from PTH precursors, biosynthetically labeled with radioactive amino acids, the complete primary structure of both preproPTH (115 amino acids) and proPTH (90 amino acids). 99'1~176 PreproPTH and proPTH consists of P T H ( 1 - 8 4 ) with the addition of amino-terminal extension peptides that comprise 31 amino acids (pre-sequence) and 6 amino acids (pro-sequence), respectively. Specific labeling of preproPTH in cell-free systems containing radioactive methionine bound to the initiator methionyl transfer RNA established that the amino-terminal methionine at position - 3 1 of preproPTH is the first amino acid incorporated into the nascent peptide during ribosomal synthesis and indicated that preproPTH is not a cleavage product of an even larger precursor with additional amino-terminal extensions. 1~ The elucidation of the nucleotide sequence of the cDNA and the gene encoding preproPTH (see following) confirmed that preproPTH is the initial and complete hormonal product. The application of molecular cloning and nucleotide sequencing techniques provided complete cDNA sequences encoding several mammalian preproPTH species as well as the genes for the bovine, human, and rat P T H . 55'61'1~176 The presence of a termination codon immediately following the codon for glutamine at position 84 of PTH, plus evidence that there is only a single gene copy per haploid genome, ruled out the existence of additional precursors of PTH. Although the nucleotide sequence analysis also established as correct the assignment of glutamic acid to position 22, it necessitated a new assignment in human PTH, asparagine at residue

CHAPTER 3 Parathyroid Hormone and Parathyroid Hormone-Related Peptide

sequence serves to both bind to signal-recognition particles and direct its own cleavage (Fig. 3-3). A series of mutant preproPTH molecules has been expressed in GH4 cells using retroviral vectors. The functional analysis of these mutants revealed distinct functional domains within the leader sequence that showed that the first six amino acids of the aminoterminal region are dispensable, and that the hydrophobic core is vital for membrane transport. 117'118The region bordering the signal cleavage site influences the efficiency of membrane transport, and the efficiency and specificity of signal cleavage. Detailed molecular analysis of genomic DNA of a patient with familial hypoparathyroidism further illustrated the important role of the signal sequence for normal secretion. 119 A point mutation in the nucleotide sequence encoding the signal sequence changes residue - 1 4 , normally cysteine, to arginine and thereby inserts a charged residue into the hydrophobic core of the signal sequence. When this mutant preproPTH is expressed in vitro in cell-free extracts or in cultured cells, translocation across the endoplasmic reticulum, signal sequence cleavage, and subsequent secretion are all defective, and thus explain the clinical phenotype of affected individuals. Many secreted proteins have prohormone forms that are usually cleaved at dibasic residues during the maturation process. These prohormone-specific sequences can occur anywhere in the precursor protein, but their apparent functions can vary from protein to protein. For example, the dibasic residues of the corticotropin-[3-

76, instead of aspartic acid; the former being missed because of deamidation prior to protein sequence analysis. 2. FUNCTION OF THE PRECURSOR MOLECULES AND THE SIGNAL SEQUENCE The amino acid substitutions found in signal sequences are generally conservative ones. Like most other signal sequences found at the beginning of precursors of secreted proteins, the signal sequences of preproPTH (1) contain at least one positively charged amino acid near the amino-terminus, (2) contain an uninterrupted stretch of hydrophobic and nonpolar amino acids, and (3) end with small amino acids just before and at the third residue proximal to the " p r e " sequence cleavage sites. 111 This signal sequence (sometimes called leader sequence), found at the amino-terminus of secreted proteins, is instrumental in the initial binding to the endoplasmic reticulum of polyribosomes. 112'113 As the leader sequence emerges from the ribosome, it binds to an 1 IS particle called the signal-recognition particle. TM The signal-recognition particle subsequently binds to a receptor on the rough endoplasmic reticulum, thereby bringing the polyribosome to the endoplasmic reticulum. T M The polyribosome then leaves the signalrecognition particle-receptor complex and binds independently to the endoplasmic reticulum. As protein synthesis continues, the precursor protein is transferred across the membrane of the endoplasmic reticulum. Before synthesis of the protein is complete, the signal sequence is cleaved off by a signal peptidase. The signal

PTH Gene

DNA---m=:~:t~mmm--

<

mRNA o - ~

pro

Precursor mRNA

c

57

AAA

NUCLEUS

. pre

,,

,=~

PTH

$

Rough Endoplasmic Reticulum

pro PTH

_

Secrefory Granule

~ ~ " ~ J Golgi Apparatus

PT FIGURE 3--3 Schematicmodel illustrating sequential steps in biosynthesis of PTH in a parathyroid cell. Precursor PTH ( m ) is processed; PTH ( ~ ) is released into the circulation.

58

JOHN T. POTTS, JR. AND HARALD Jf)PPNER

lipotropin precursor serve to link together several hormones 12~ and direct intracellular cleavage of the precursor to generate individual peptides. In contrast to that, the connecting peptide (or C-peptide) of the insulin molecule links A and B chains of the mature prohormone. Furthermore, the C-peptide serves to promote a conformation of the prohormone that facilitates the formation of appropriate interchain disulfide bonds between A and B chains. 121 The prohormone region of proPTH, in contrast, which comprises six amino acid residues, does not link together bioactive peptides, but is vital for the intracellular maturation, packaging, and transport of PTH, 118 and functions to optimize the efficiency and accuracy of signalsequence cleavage. 3. PARATHYROID HORMONE GENE: STRUCTURE AND FUNCTION

Genomic DNA sequences encoding human, 61'11~ bovine, 1~ and rat 55 PTH have been characterized. The overall structures of all three characterized mammalian PTH genes are similar to one another (Fig. 3 - 4 ; see also Fig. 3 - 9 ) . They each contain three exons (two coding and one noncoding exon), and two intervening sequences that interrupt the mature mRNA sequence at precisely the same nucleotides. The first intervening sequence, always large, comes just five nucleotides before the beginning of the region encoding preproPTH. The second, small intron separates most of the nucleotide sequence encoding the signal peptide from the hexapeptide of the pro-sequence and from the sequence encoding the rest of the protein and the 3'-noncoding region of the mRNA. A similar pattern of intervening sequences has been found in a number of genes encoding hormones such as gastrin 122 and insulin, 123 and PTHrP (see below). Restriction endonuclease mapping of genomic DNA obtained from the human, bovine, rat, and chicken indicate the existence of a single copy of the preproPTH gene per haploid g e n o m e . 55-57'61'1~ T h e human PTH gene is located on the short arm of chromosome 11 along with

genes encoding calcitonin, catalase, C-Harvey-ras 1 protooncogene, insulin, and [3-globin. 124-126 A PstI restriction fragment length polymorphism (RFLP) was found in this cluster of genes. As many as 70% of humans carry a 2.8-Kb PstI restriction fragment and a 30% 2.2Kb fragment. Approximately 40% of individuals are heterozygous for these two patterns of PstI cleavage. 124-126 An additional TaqI polymorphism resulting from a point mutation within the second intron of the PTH gene was also detected. 127 These frequent polymorphic markers are useful for defining allele inheritance and have potential importance for linkage studies in families with disorders of calcium homeostasis (Table 3 - 2 ) . 4. REGULATION OF PARATHYROID HORMONE BIOSYNTHESIS

The parathyroid glands seem to have an unusual functional pattern among endocrine organs in several respects. Storage of hormone is low, and synthesis and secretion are therefore necessarily tightly coupled. Also, hypertrophy and hyperplasia of the gland occur as a very rapid response to the demands of persistent hypocalcemia. 128-~32 Both calcium and 1,25(OH)zD3 affect the rate of gene transcription. However, the physiological relevance of 1,25(OH)zD3 is unclear, 2'~33-~38 although pharmacological suppression of PTH synthesis by injected doses of 1,25(OH)zD3 is striking and clinically u s e f u l . 139-141 Much of the secreted or stored hormone is cleaved and destroyed intracellularly, when the glands (in vivo or in vitro) are exposed to protracted hypercalcemia. ~~176 This varying rate of hormone destruction is one of the somewhat unusual means of regulating biosynthesis and secretion. Since estimates are that the parathyroid gland can secrete PTH at a maximal rate for only 1.5 hours, hormone synthesis must increase rapidly to meet any sustained secretory demand. ~3~ Several studies indicate that extracellular calcium regulates the transcription of the PTH gene. 133-138 Acute lowering of blood c a l c i u m m w i t h phosphate, calcitonin, or ethylenediaminetetraacetic acid

FIGURE 3--4 Schematic of the PTH gene along several thousand base pairs (approximate length shown by scale marker for 500 bp). The 3 exons in the mRNA are represented as numbered rectangles. Control elements are identified in the 5' noncoding region (5' NC). A region responsive to vitamin D is within a few hundred base pairs of exon 1; far upstream are silencers involved in calcium regulation.

CHAPTER3 ParathyroidHormone and Parathyroid Hormone-Related Peptide

59

TABLE 3--2 Chromosomal Location of the Genes Encoding PTH, PTHrP, the Common PTH/PTHrP, and the PTH-2 Receptor, and Known Intragenic Frequent Polymorphisms in These Genes Gene

Chromosomal localization

Intragenic frequent polymorphisms

References

PTH

1lpl 5

1. PstI polymorphism 2. TaqI polymorphism

126, 127, 406

PTHrP

12p12.1-11.2

Dinucleotide repeat located 3' of exon 5

299, 308

PTH/FrHrP receptor

3p21.1-22

1. BsmI polymorphism; located 5' of exon S 2. BsrDI polymorphism; located within exon M7

3, 340, 355

PTH-2 receptor

2q33

Unknown

398

(EDTA)mleads to a prompt increase in PTH mRNA levels. 136'137 Elevations in blood calcium, in contrast, lead to no change in PTH mRNA levels after 6 h o u r s 136 and only to a modest decrease in its levels after 48 h o u r s . 137 The parathyroid gland is thus apparently more prepared to increase PTH synthesis and secretion through transcriptional mechanisms in response to hypocalcemia than to decrease the levels of PTH mRNA in response to hypercalcemia. This finding resembles the in vivo pattern of PTH secretion, where the gland responds dramatically to hypocalcemia, but decreases its activity only incompletely in response to hypercalcemia. Isolated changes in calcium or 1,25(OH)2D3 seldom occur in vivo. Although the interactions between effects of calcium and 1,25(OH)2D3 are important, they have not been analyzed thoroughly. When rats were made acutely hypocalcemic with phosphate and were at the same time given 1,25(OH)2D3 intraperitoneally, the suppressive effect of 1,25(OH)2D3 dominated over that of hypocalcemia, and PTH mRNA levels fell. 136 However, when rats were fed a low-calcium diet for 3 weeks, blood calcium fell and 1,25(OH)2D3 increased dramatically. In this case, PTH mRNA levels increased several-fold. 138 The differing results of the acute and chronic studies suggest that the net effect of changes in calcium and 1,25(OH)2D3 depends on the chronicity of the manipulation and the degree of the changes in each variable. Lack of a suitable cell line that is well differentiated yet continues to secrete PTH has made analysis of regulatory elements in the gene that responds to 1,25(OH)2D3 or calcium difficult, especially with regard to physiological significance of these agents in vivo. Two groups have identified DNA sequences upstream of the PTH gene that bind to 1,25(OH)2D3 receptors in vitro (see Fig. 3 - 4 ) and act as negative response element. Other studies suggest that additional sequences in the human PTH gene may contribute to negative regulation by 1,25(OH)2D3 as well. T M A DNA sequence far upstream from the human PTH gene regulates gene tran-

scription in response to changes in extracellular calcium as analyzed after the transfection of this sequence into fibroblasts. Another regulatory mechanism that may be particularly important under conditions of hypocalcemic stress results from the influence of extracellular calcium on the degradation of newly synthesized PTH. 1~176 When bovine glands were incubated in hypocalcemic medium, almost all the newly synthesized PTH was secreted intact. In contrast, when exposed to hypercalcemic medium, most of the newly synthesized PTH was degraded. Similar findings were made in vivo in calves where hormonal fragments rather than intact hormone represented, under conditions of hypercalcemia, the predominant secretion product. 142This supports the hypothesis that PTH degradation, noted in vitro, is not an artifact of cell culture conditions, and that parathyroid glands can control the amount of hormone available for secretion by regulating the fraction of hormone either secreted or degraded. The biosynthesis of PTH is thus controlled at several cellular and genetic levels. 5. SECRETION

The regulation of PTH secretion by calcium is an example of unusually precise cellular control. A decrease of a few per cent in serum calcium concentration (experimentally induced) elicits a nearly instantaneous response in PTH secretion, with the hormone concentration rising twofold or more. The cellular mechanism in the parathyroid cell responsible for recognizing and then coordinating small changes in the extracellular environment with brisk intracellular responses has long elicited the interest of cellular physiologists. Certain features of the disease familial benign hypocalciuric hypercalcemia (FBHH) (minimal gland pathology but persistent PTH secretion despite mild hypercalcemia and extremely low urinary calcium clearance) led to the prediction that there was a calcium-sensing receptor in the parathyroid cell and renal tubule with physi-

60

JOHN T. POTTS, JR. AND HARALD JOPPNER

ological importance for controlling of PTH secretion and for fine regulating of urinary calcium excretion; it was further reasoned that this sensor was mutated and functionally defective in FBHH. This speculation proved to be correct. In 1993, Brown and colleagues, who had also used genetic linkage studies in families with FBHH to identify the responsible genetic locus, 145reported the isolation of cDNAs encoding the bovine calcium-sensing receptor through expression cloning strategies146; hybridization techniques subsequently led to the identification of cDNAs encoding the sensor homolog from humans and other mammalian s p e c i e s . 147-149 Numerous inactivating mutations in the calcium sensor were identified in patients with FBHH, while gain-of-function mutations were found in patients with autosomal dominant hypocalcemia. 147,150-153

tion is observed even if blood calcium decreases further. Raising blood calcium above the normal range results only in a modest fall in PTH secretion, and even dramatic increases in blood calcium fail to inhibit PTH secretion completely. The maximal secretory response in calves usually occurs when blood calcium levels are at the lower end of the normal range (8.5 and 7.5 mg/dl) (i.e., the lower portion of the sigmoidal d o s e response curve 154) (Fig. 3-5), and similar results have been seen in humans when using immunoassays that detect only the intact hormone to prevent interference by circulating fragments that have a long half-time of clearance. 1 4 1 , 1 5 5 - 158 The effects of magnesium on the function of the parathyroid gland are complex. It was shown in acute studies in vil,'O 159 and in v i t r o 16~ that raising the level of extracellular magnesium inhibits, while lowering the level of magnesium stimulates, PTH secretion. Magnesium, mole for mole, however, is a severalfold less potent modulator of PTH secretion than is calcium. Because of this weaker effect and because the blood levels of magnesium in vivo are generally lower than those of calcium, changes in the levels of magnesium within the physiological range have little effect on PTH secretion in vivo. High levels of magnesium, sometimes seen in patients

Ionized calcium in extracellular fluid working through the calcium-sensing receptor is the major determinant of PTH secretion. Although other agents also affect the secretory rate, none of them have an established, quantitatively important role in the normal function of the parathyroid gland. PTH secretion increases dramatically when blood calcium falls below normal levels. It becomes maximal with blood calcium level of approximately 7.5 mg/dl, but no further increase in PTH secre-

10

HJ rr

5

5

7

I 11

4

w~

rr~ O=m U.I 00 "1ia..

3

2

1~11 10

17

|

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6

7

8

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9 10 11 12 13 1PLASMA CALCIUM, mg/100 ml

9 14

7

15

5

16

17

Secretory response of bovine parathyroid glands to induced alterations of plasma calcium concentration. The dots and vertical bars indicate secretory rate (mean _ SD). The number of calves and samples are indicated by numbers below and above the bars. Secretory rates from catheterized parathyroid veins were calculated from measured effluent plasma flow rates and arteriovenous difference of immunoreactivity. (From Mayer GP, Hurst JG: Sigmoidal relationship between parathyroid hormone secretion rate and plasma calcium concentration in calves. Endocrinology 102:1036-1042, 1978.)

CHAPTER 3 ParathyroidHormone and Parathyroid Hormone-Related Peptide with renal failure or in patients treated for eclampsia, however, may inhibit PTH secretion. Chronic, severe hypomagnesemia, found in malnourished alcoholics, primary magnesium-wasting syndromes, or in association with acquired renal disease as in cisplatinum therapy, however, causes a paradoxical decrease in PTH secretion rather than the expected increase. 162-164 Current evidence indicates that the intracellular magnesium deficiency seen in these patients interferes with intracellular secretory mechanisms and blocks PTH secretion. Replenishment of magnesium rapidly restores PTH secretion in most of these patients. 162-165 Although definitive experiments demonstrating the importance of catecholamines in PTH physiology are lacking, the responsiveness of normal parathyroid tissue to [3-adrenergic agonists is well established. 166-168 However, their role, if any, in parathyroid physiology or pathophysiology remains undefined. 1,25(OH)2D3 can decrease the secretion of PTH, but its major effect is indirect through the suppression of PTH gene transcription. 2'169-171 Phosphate administration is a powerful stimulator of PTH secretion in vivo; however, its effect can be explained largely by the fall in levels of ionized calcium that is associated with phosphate administration. 2'172 Aluminum inhibits PTH secretion in vitro173; this inhibition may actually occur in pa-

MA. Parathyroid Adenoma

20 E z -r- 10 I13_ r 5 .B

tients with end-stage renal failure who can harbor large aluminum loads. Histamine stimulates the release of PTH in vitro from bovine parathyroid s l i c e s 174 and from cells isolated from dispersed human parathyroid adenom a s , 175 and these effects can be blocked by cimetidine. 174 Other agents have been reported to stimulate PTH secretion (calcitonin, 176 glucagon, 177 growth hormone, lv8 PGE2,179 c o r t i s o l , 18~ secretin, 177'181) or to inhibit PTH secretion (somatostatin, 182'183and PGEl184). The putative effects of somatostatin have not been detected by all observers, 185 and the physiological relevance of the effects of all these agents has not been established (see review by Brown186).

C. M e t a b o l i s m ~ H e t e r o g e n e i t y

Adenoma Removed

1. INTRODUCTION: BIOLOGICAL SIGNIFICANCE

In 1968 it was first reported by Berson and Yalow that the hormone circulating in plasma differed from the hormone extracted from the glands. 187 Highly variable rates of hormone disappearance, outlined in Figure 3 - 6 , were determined with radioimmunoassays that use two different antisera to measure hormone. These discrepant

FI. Uremia with Secondary Hyperparathyroidism , Subtotal Parathyroidectomy

Total ~, Parathyroidectomy r

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,

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,

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i

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Time After Surgery

FIGURE 3--6

Disappearance of immunoreactive parathyroid hormone from plasma after parathyroidectomy in patients with primary or secondary hyperparathyroidism. Plasma samples were assayed with antiserum C329 and antiserum 273 using an extract of a normal human parathyroid gland as standard (hPTH N) and 125I-bPTH as tracer. Plasma concentrations of hormone are given as microliter equivalents of the plasma standard of hPTH (see text). (From Berson SA, Yalow RS: Immunochemical heterogeneity of parathyroid hormone in plasma. J Clin Endocrinol Metab 28:10371947, 1968).

62

JOHN T. POTTS, JR. AND HARALD JUPPNER

findings must reflect differences in the epitopes recognized by the two antisera; both antisera, in turn must be detecting different molecular forms with different rates of clearance. It was subsequently confirmed that some antisera reacted more strongly with epitopes in the biologically active amino-terminal region [and hence as shown later could detect intact hormone PTH(1-84)] and other antisera only with the mid- and carboxylterminal regions that have no calcium-elevating activity. It was concluded correctly that much of the circulating hormone was devoid of amino-terminal immunoreactivity and hence must represent fragments of the hormone that are cleared more slowly. 188'189 The existence of different chemical forms of PTH in blood obviously complicated efforts to derive clinically useful information from measurements of circulating immunoreactive PTH. The results implied that not all immunoreactive forms are involved in the regulation of blood calcium, nor are all predictably related to glandular secretion. Whether the metabolism of PTH might be important in the expression or overall regulation of hormone action needed to be considered. Structure/activity studies with PTH had indicated that the aminoterminal portion of the molecule could exert full biological activity on hormonal targets to raise calcium and to lower phosphate through its classical homeostatic actions. It was, therefore, conceivable that similar biologically active, amino-terminal fragments are generated when the intact hormone is cleaved into fragments; ei-

FIGURE 3--7

ther in the gland or, after secretion, in peripheral tissues. Physiological alterations in the rate of intraglandular or peripheral proteolysis of PTH were thus postulated to complement changes in the rate of PTH secretion to control levels of active hormone more precisely. Two decades of work by several laboratories have focused on this intriguing but methodologically difficult area regarding the heterogeneity of circulating forms of PTH. Several general conclusions are now possible (Fig. 3-7). Fragments of the hormone are produced in and released from the parathyroid gland (intraglandular origin of hormone fragments) and are also derived from circulating hormone after secretion by efficient, highcapacity systems in liver, kidney, and other peripheral sites. Most of the circulating fragments represent middle and carboxyl-terminal regions of the molecule rather than amino-terminal fragments that are of calciumhomeostatic importance. The latter, if present at all, are below the level of detectability in animals and humans, even when using extremely sensitive techniques. 19~ Since it is now known that the intact hormone is fully active without requiting cleavage, some of the intensity of this work slowed when it was appreciated that there were essentially no detectable circulating amino-terminal f r a g m e n t s . 19~

Most recent work, summarized in the later part of this chapter, indicates that there are, besides the PTH/ PTHrP receptor, other receptors for PTH and PTHrP, and that other, particularly carboxyl-terminal, fragments may

Scheme of PTH-secretion by the parathyroid gland and its interaction with the PTH/PTHrP receptor on target cells; cleavage of the intact hormone into amino-terminal (N) (t77~ and carboxyl-terminal fragments (C) ( ~ ) by the parathyroid gland and by peripheral organs.

CHAPTER 3 ParathyroidHormone and Parathyroid Hormone-Related Peptide exert biological activities on osteoclasts and possibly other bone cells. Therefore, since hormonal fragments other than those that include the amino-terminus may be of biological importance, the nature of the heterogeneity of circulating PTH forms, and the intraglandular or peripheral production of hormone fragments, may again become of interest and may require considerable reevaluation. At present, we conclude, at least from the perspective of calcium homeostasis, that the peripheral metabolism of PTH appears to be purely degradative, and is the main process that determines the rate of clearance of hormone from the circulation once secreted. The lack of evidence for circulating forms of biologically active aminoterminal fragments made it possible to introduce newer types of double-antibody immunoassays that detect only full-length PTH rather than hormonal fragments. 158 This greatly reduced problems previously associated with hormone assays in clinical conditions, particularly in renal failure. Recently, non-PTH(1-84) fragments were 192 described that are detectable even by double-antibody immunoassays. However, these PTH fragments are present only in small concentrations, and are thus likely to be of limited biological importance, at least when renal function is normal. It is not clear how many carboxyl-terminal fragments appear in the circulation, the gland, or peripheral tissues, but at least four closely related fragments were detected. 193'194 For simplicity, these fragments are hereafter referred to as carboxyl-terminal fragments. Some groups have reported evidence based on combined physical, chemical, and immunological s t u d i e s 188'193'195-198 for smaller immunoreactive forms comprising the amino-terminal region that are present at much lower concentrations than the fragments corresponding to the mid- and carboxyl-terminal regions of the molecule. Perfusion of liver or kidney in vitro with intact hormone leads to amino-terminal fragments that can be detected in t h e p e r f u s a t e , 195'199 which in one case were shown to be biologically active, 2~176 although this does not establish their presence in vivo. The conditions under which amino-terminal fragments were detected in vivo usually involved studies in patients with hyperparathyroidism and/or renal failure, or the administration of high doses of hormone to experimental animals, sometimes also in the presence of renal failure. Circulating amino-terminal fragments are generally not detected by most assays in blood samples analyzed from individuals with normal parathyroid or renal function2~176 biologically active fragments, however, of the size of aminoterminal fragments have been detected by cytochemical bioassay in hyperparathyroid patients with renal failure. 2~176 There is no independent evidence as to whether this material represents PTH fragments or another class

63

of substances with PTH-like biological activity, or whether it could even be due to nonspecific interference with the assay p e r se. 2. PARATHYROID GLAND ORIGIN OF CIRCULATING PTH FRAGMENTS Two seemingly contradictory lines of evidence developed regarding the origin of circulating fragments of hormones. After the original observation of Berson and Yalow, 187 another group 189 showed that most of the PTH found in the venous effluent of the parathyroid glands in cows or in humans with parathyroid tumors (the latter collected during venous catheterization studies used to localize the site of the abnormal parathyroid gland) contained hormone that was indistinguishable in size and immunological properties from that extracted from parathyroid glands. In contrast, immunoreactive hormone found in the circulation was smaller in size and lacked immunological determinants corresponding to the amino-terminal portion of PTH. These results were interpreted as evidence that PTH heterogeneity is a postsecretory event and that cleavage of the hormone occurs after its release from the gland. Shortly thereafter, however, Silverman and Yalow, ~88 and another group, 2~ reported that multiple immunoreactive forms of PTH can be found in crude extracts of human parathyroid glands; these fragments were similar to fragments found in the peripheral blood. Silverman and Yalow 188 showed that, at least in the presence of renal failure, carboxyl-terminal PTH fragments persisted in the circulation for much longer periods of time than did the intact hormone; such prolonged survival of immunoreactive PTH fragments might therefore result in the fragments becoming the dominant hormonal form in the circulation, even if their rate of secretion was much lower than that of the intact hormone. They suggested that all circulating immunoreactive forms of the hormone originate from secretion by the parathyroid glands rather than from peripheral metabolism; however, the significant contribution of the small quantity of secreted PTH fragments to the heterogeneity of circulating hormone had been overlooked. The general conclusion that seems most reasonable at present is that fragments arise from several sites, the parathyroid glands and peripheral organs, especially liver and kidney. 198'2~ Direct measurement of arterial and venous differences in parathyroid effluent blood (with vigorous conditions to present any ex vivo cleavage of hormone after sample collection) is the most unambiguous method of ascertaining that the parathyroid gland in vivo releases fragments, as well as intact hormone, into the general circulation. Studies performed in cattle indicate that carboxyl-terminal fragments and intact hormone are secreted into the circulation, and that the relative concen-

64 tration of carboxyl-terminal fragments is even higher under conditions of systemic hypercalcemia when overall secretion rates of intact hormone a r e l o w e r . T M Studies using parathyroid gland slices that were incubated in vitro for short periods (< 6 hours), corrected earlier observations 22~ and showed that the principal product released from glandular tissue was intact PTH, although carboxyl-terminal fragments also were detected. 221-224 Subsequently, extensive use has been made of in vitro studies with glandular tissue to examine hormone production and secretion, and the nature of the responsible proteolytic enzymes. A considerable body of work suggests that the enzymes responsible for hormonal fragmentation within the parathyroid gland are cathepsins. 224'225Direct analysis of the nature of the PTH cleavage by cathepsin, partially purified from parathyroid tissue, showed that cleavage occurs between residues 36 and 37, a site identical to that used during hepatic metabolism of PTH (see below). Although amino-terminal PTH fragments are detected during analyses of glandular processing in vitro or in g l a n d u l a r e x t r a c t s , 188'220-222'224'226 only carboxyl-terminal fragments have been, so far, reported to be detectable in parathyroid effluent in vivo. The extent to which glandular secretion of carboxyl-terminal fragments contributes quantitatively to the content of carboxyl-terminal fragments detected in the peripheral circulation is unclear, but is likely to be considerable, especially in renal failure. Multiple approximate estimates under various pathophysiological conditions, such as after parathyroidectomy in patients with uremia, 188 indicate that the halflife of carboxyl-terminal fragments is about 100 times greater than that of intact hormone. 3. PERIPHERAL METABOLISM OF P T H : HEPATIC AND RENAL ORIGIN OF CIRCULATING FRAGMENTS

The peripheral metabolism of PTH has been analyzed by injecting intact hormone into the circulation of test animals; such tests have not been performed in human subjects. The clearance of intact PTH from plasma was found to be very rapid (half-life, 2 to 4 minutes), 158'19~ the major sites of clearance being liver and kidney. 194'e1~ Clearance by the liver predominates over clearance by the kidney; the two organs together account for virtually all the clearance of intact hormone. Hepatic clearance of intact hormone has been estimated to be 40% to 75% and renal clearance 20% to 3 0 % . 212'216'227-229 Clearance half-time for fragments was at least 20 to 40 minutes with intact renal function; as in humans, renal failure prolonged the clearance halftime. 23~

One group established, by sequential Edman degradation of PTH fragments found after administration of radioiodinated intact hormone to rats or dogs, that the

JOHN T. POTTS, JR. AND HARALD JUPPNER

two major circulating carboxyl-terminal fragments resulted from cleavage of the hormone between residues 33 and 34, and between residues 36 and 37, respectively. 23~ Sequence analysis of labeled carboxylterminal PTH fragments extracted from organs showed that those generated within the liver were closely similar to the circulating carboxyl-terminal fragments. 232 In vitro studies with isolated hepatic cells demonstrated that Kupffer cells, but not hepatocytes, were responsible for proteolytic modification of PTH; the detected products were identical to those found in the circulation. 233 In other studies, a cathepsin was identified in liver that cleaved PTH at sites identical to at least one of the sites detected in vivo. 221'222 A similar but slightly different cathepsin that seems responsible for intrarenal cleavage of PTH also has been identified. TM Incubations with Kupffer cells and purified cathepsins showed that the cleavage of the hormone resulted in the generation of both amino- and carboxyl-terminal PTH fragments, as might be expected from the action of an endopeptidase, yet only carboxyl-terminal fragments reappear in the circulation. 221,222,233,234

In some more recent studies, PTH metabolism was evaluated in rats with biologically active intact hormone that had been labeled biosynthetically, at known sites, through incorporation in vitro of either tritiated or radioactive sulfur-containing amino acids to eliminate any artifacts due to use of radioiodinated h o r m o n e . T M Injection of this hormone into animals led to the predictable uptake of intact hormone, approximately 50% by the liver and 20% by the kidney; the rapid disappearance of intact hormone was followed by the appearance of carboxyl-terminal fragments in the circulation. No convincing evidence for reentry of amino-terminal fragments into the circulation was found, although hormone labeled to high specific activity in the amino-terminal portion of the molecule was infused continuously in rats for periods of 10 to 30 minutes under a variety of conditions, including partial or total nephrectomy. The high sensitivity and specificity of these approaches seem to argue strongly that if there are circulating amino-terminal fragments, as suggested in some hyperparathyroid patients with uremia, they do not originate from peripheral metabolism. Whether aminoterminal fragments, formed within tissues such as kidney and liver, have local biological effects within the tissue of origin remains a possibility, as yet untested. The overall studies combined with data concerning secretion of fragments from the gland indicate, however, that intact hormone and fragments of the mid- and carboxylterminal regions are the principal, if not sole, circulating forms of hormone. If amino-terminal fragments circulate, in addition to intact hormone and carboxyl-terminal fragments, their concentration must be extremely low,

CHAPTER 3 Parathyroid Hormone and Parathyroid Hormone-Related Peptide less than 10 -13 to 10 -14 M, raising considerable doubt as to their physiological significance. It is for this reason and as a practical matter in the differential diagnosis of hypercalcemia that immunoassays for clinical use are now frequently designed to detect intact hormone only and to largely ignore circulating fragments. 158'235

65

relate immediate hormone-mediated receptor events such as increased second messenger activities (see Section IV), which occur within seconds to a few minutes, to changes in transporter activity, which lag by minutes or hours.236 1. ACTIONS IN KIDNEY

4. SUMMARY

Certain features of the PTH metabolism are puzzling. The principal fate of secreted PTH is metabolic clearance and proteolytic degradation by the liver and kidney rather than specific uptake by receptors in target cells in the kidney and bone (see Fig. 3-7). There is no convincing evidence that peripheral metabolism of hormone is subject to physiological regulation (i.e., that an increase or decrease in the rate and efficiency of hormone uptake and cleavage occurs as a function of various physiological states). 191 Clearly, high rates of peripheral metabolism do affect the steady-state concentration of PTH and serve as an alternate route to receptor uptake of secreted hormone. Peripheral metabolism, however, does not seem to result in the generation of biologically active, amino-terminal PTH fragments, at least not any that reenter the general circulation; it is also unlikely that significant quantities of amino-terminal fragments arise from the gland. The nature of the responsible enzymes is still unclarified, as is the nature of the specific cellular uptake and mechanisms of intraorgan disposal of fragments that are generated by tissue peptidases. The hormone may be metabolized by tissue-specific degradation pathways that serve general catabolic functions and may not be of physiological significance for the action of PTH on calcium and phosphate metabolism p e r se (although the biological significance of the mid- and carboxyl-terminal fragments in other biological functions could be considerable, as is discussed later).

D. A c t i o n s in K i d n e y a n d B o n e The cloning of the PTH/PTHrP receptor has helped to define specific steps in hormone action on target cells in the renal tubule and throughout the bone. Work by physiologists over the past 50 years had defined a number of physiological actions of PTH. These actions, some of which are now attributable to PTHrP, rather than to PTH, can be analyzed with highly differentiated cells in vitro where receptor content and second-messenger responses can be determined quantitatively. Efforts are intensifying to understand the known physiological effects on calcium and phosphate transport in kidney and bone, in terms of specific changes in cellular enzyme activity, gene expression, and membrane transporter activation/ translocation. It is not yet possible, however, to directly

Not only calcium and phosphate transport but also the transport of magnesium, sodium, potassium, and bicarbonate are modified by PTH action on the kidney. In addition, PTH activates the renal 25(OH)la-hydroxylase, the enzyme that controls synthesis of the principal active metabolite of vitamin D, 1,25(OH)2D3 (see Chapter 5). Specific regions of the nephron involved in each of these actions have been defined, through in vivo micropuncture analysis and through in vitro studies with nephron segments that have been dissected from the remainder of the kidney. 1 As well as work with isolated tubule segments, studies have proceeded with intact cells, cultured cell lines that represent specific regions of the kidney. Interpretation of the results, however, particularly those seen with isolated cells, has to take into account the complexity of the organization of the kidney as an organ (e.g., the complex anatomical relationship involving different renal tubular segments spanning both cortex and medulla, and the effects of countercurrent distribution that modify solute and water transport), and certain of these in vivo features are not readily imitated in vitro.

The critical actions of parathyroid hormone on the kidney, concerned with its role as a physiological regulator of mineral ion homeostasis, are principally the stimulation of renal tubular calcium reabsorption, increased synthesis of 1,25(OH)2D, and inhibition of phosphate reabsorption. a. Calcium R e a b s o r p t i o n Much of the bulk reabsorption of calcium occurs in the proximal t u b u l e . 237-239 The physiologically important stimulation of renal calcium reabsorption by PTH occurs, however, almost entirely in the distal nephron. 24~ It is now recognized that the calcium-sensor also plays an important role in the adjustment of renal calcium reabsorption, which is probably independent of PTH actions. In distal nephron segments PTH stimulates active transport of calcium against a steep chemical gradient. Two types of pumps or transporters are involved. One is a primary calcium pump (CaZ+/MgZ+-ATPase) and the second is a Na+/ CaZ+-exchanger that in turn is regulated by Na+/K +ATPase(s) to maintain the transcellular Na + gradient. Studies performed with membrane vesicles from the distal region of the kidney show increased activity of the Na+/CaZ+-exchanger in response to PTH. T M PTH also seems to stimulate translocation of preformed calcium

66

JOHN T. POTTS,JR. AND HARALD JUPPNER

fect of PTH shows longer lag times than its effect on Ca 2§ transport; stimulation of 1,25(OH)2D3 synthesis occurs over several hours and requires the synthesis of new protein. 248'249 Along with this action, PTH decreases the activity of the renal 24-hydroxylase. 25~ Hypercalcemia, as would be generated by sustained increases in PTH directly, suppresses the activity of l oL-hydroxylase limiting in a homeostatic manner the overall extent of increased 1,25(OH)2D3 synthesis. Many other factors, including blood phosphate concentrations, influence the synthesis of the active vitamin D metabolite.

FIGURE 3--8

PTH triggers the translocation of preformed voltage-dependent calcium channels from sites of intracellular sequestration to the apical membrane, which also undergoes rapid morphological changes that greatly increase its surface area. Intracellular free calcium rises significantly, and increased net transepithelial calcium transport occurs, mainly via enhanced Na+/Ca 2+ exchange at the basolateral membrane, supported in turn by the Na +, Ka+-ATPase. Analogous events underlie PTH regulation of proximal tubular phosphate transport.

channels, sequestered within the interior of certain distal tubular cells to the apical surface of the renal tubular cell. 245 These calcium pumps translocate to the apical/ renal tubular luminal surface of the cell and stimulate cellular calcium uptake. Since PTH simultaneously enhances the activity of the Na+/CaZ+-exchangers in the basolateral (antiluminal) membrane, the overall process mediates transcellular calcium uptake from lumen to blood (i.e., apical surface to basolateral membrane). Certain studies suggest the process is facilitated by morphological changes in the apical surface of the target cells as outlined in Figure 3-8. b. Vitamin D Metabolite Synthesis PTH is a major inducer of the activity of the proximal tubular 25(OH)D3-1 oL-hydroxylase, the enzyme that synthesizes 1,25(OH)zD from the substrate 25(OH)D3. 246'247This ef-

c. Phosphate Reabsorption PTH exerts a major influence on renal tubular phosphate reabsorption. This hormonal action is important to its overall physiological role (i.e., the protection of normal blood calcium levels); PTH is not, however, the principal regulator of serum phosphate concentration. 1 Variations in dietary phosphate absorption bring about profound changes in renal phosphate reabsorption; high phosphate intakes result in increased renal phosphate clearance and dietary phosphate restriction causes greatly enhanced renal phosphate reabsorption, apparently quite independent of any changes in PTH secretion. The level of blood calcium also influences the urinary clearance of phosphate. From a teleological perspective the actions of PTH to increase phosphate excretion are important, since whenever the action of the hormone causes increased bone resorption (as might occur with prolonged dietary calcium deprivation), there are increased flows of both calcium and phosphate into blood from bone. While calcium is needed, phosphate is best excreted; this is accomplished by a PTH-stimulated increase in renal phosphate excretion. PTH-dependent reduction of renal phosphate reabsorption occurs both in the distal portions of the proximal tubule 241'252'253and also in more distal regions of the nephron. 252-255 However, details of the mechanism(s) whereby the hormone modifies phosphate transport remain incompletely defined. Na-Pi co-transporters, perhaps several, 256-258 are the principal PTH targets; the hormone rapidly inhibits the actions and synthesis of these transporters. One major mechanism seems to be the removal of functioning Na-Pi co-transporters from the apical (luminal) surface of the cell to the interior; this action inhibits the capacity of the cell to transport phosphate intracellularly from the renal tubular lumen. Thus, the current explanations of the bidirectional actions of PTH action (namely, increased renal reabsorption of calcium and inhibition of renal reabsorption of phosphate) may be explained (Fig. 3 - 8 ) by hormonedirected increase or reduction, respectively, in critical apical membrane transport channels.

CHAPTER 3 Parathyroid Hormone and Parathyroid Hormone-Related Peptide d. Summary PTH action on ion transport is best understood as mediating coordinated and specific effects on rapid translocation of membrane-bound transporters either toward (stimulation of cellular calcium transport) or away from the cell (inhibition of phosphate reabsorption). As illustrated schematically in Figure 3 - 8 , these effects are accompanied by rapid morphological changes in cell membrane topography that increase the surface availability of active transporters. 259 Similar mechanisms may explain other renal effects of PTH (i.e., inhibition of sodium, water, and bicarbonate reabsorption). With regard to inhibition of bicarbonate reabsorption, PTH may work by dual mechanisms, namely, inhibition of both apical surface bicarbonate uptake and basolateral bicarbonate exit. 1'26~ 2. ACTIONS ON BONE

Numerous studies have emphasized that PTH affects a wide variety of the highly specialized bone cells including osteoblasts, osteoclasts, and other stromal cells. Some effects reflect direct actions of PTH; other effects are indirect, mediated through cytokines released by cells with PTH/PTHrP receptors that regulate the activity of yet other cells that lack these receptors 261-263 (an example is the stimulatory action of PTH on osteoclasts, which is believed to be mediated indirectly through osteoblasts; see below). It is unclear at the present time which specific cellular actions explain the physiological actions known to follow PTH administration to animals in vivo. Administration of PTH is followed in minutes by a transient lowering of blood calcium due to uptake by bone c e l l s . 264 This is rapidly followed by an increased mobilization of calcium from bone that may be derived by actions of distinctive cell types such as osteocytes. 265 There continue to be numerous speculations and disputes regarding the cellular/anatomical basis of these rapid responses of PTH that may be distinct from osteoclast-driven bone resorption. The latter clearly involves multiple cellular changes involving activation of cellular transporters and pumps as well as secretion of bone active enzymes. a. Effects on Osteoclasts Since mature osteoclasts are believed not to have PTH/PTHrP receptors, 266"267current evidence suggests that PTH signals to osteoclasts indirectly through osteoblasts. The latter cells clearly have PTH/PTHrP receptors on their surface and the cellular activity of osteoblasts is dramatically influenced by P T H . 37'38'262'268-272 Some groups have reported evidence for PTH receptors, 273'274 at least on early, differentiating o s t e o c l a s t s . 275'276 However, most studies indicate an indirect effect, a paracrine signaling cascade from osteoblasts to recruit/activate osteoclasts. 261-263 Molecules that might be involved in this paracrine stimulation include

67

interleukin-6 (IL-6), granulocyte-macrophage colonystimulating factor (GM-CSF), and other peptides not yet fully c h a r a c t e r i z e d . 277-279 Other work suggests that there needs to be direct cell-cell contact between osteoblasts and osteoclasts to allow osteoclast activation. 28~ In any event, PTH has multiple indirect effects on osteoclasts. Osteoclast differentiation and activation involves a complex series of cellular transformations influenced by many signals other than PTH. Cellular responses involved in bone resorption include the development of a vitronectin-mediated anchorage of osteoclasts to bone surface, acidification of the circumscribed and sealed-off extracellular environment that is created between the osteoclast and bone, and, in addition the secretion of a variety of proteases and other enzymes. b. Effects on Osteoblasts Most evidence suggests that the cells richest in PTH/PTHrP receptors and the cell type most dramatically affected by PTH action are osteoblasts. Earlier in vitro studies indicated that PTH stimulates increased generation of cAME inositol triphosphate (IP3), and diacylglycerol (DAG); membrane potential and pH a l s o c h a n g e , 262'268-27~ and these findings were confirmed with cells expressing the cloned PTH/PTHrP receptor. 34'282'283 A remarkable number of cellular activities of osteoblasts are influenced by PTH including cellular metabolic activity, ion transport, cell shape, gene transcriptional activity, and secretion of multiple proteases (Table 3 - 3 ) . There is known to be much heterogeneity among osteoblasts, and the developmental cascade of cellular proliferation/differentiation events that result in mature osteoblasts may be principally affected by P T H . 266'284-286 Cellular effects on osteoblasts by PTH are variable depending on the cell line studied in v i t r o 284-287 and the pattern of exposure to hormone (i.e., continuous vs. intermittent). Continuous PTH administration in vivo results in decreased bone mass, while the intermittent administration of PTH leads to an increase. 1'288'289 The cellular mechanisms whereby intermittent PTH administration selectively enhances bone formation remains unknown. Correlations between in vivo and in vitro responses are not well understood at present, but are clearly of great interest for clarifying the integrated responses of bone to PTH with its potential for anabolic skeletal actions.

III. PARATHYROID HORMONE-RELATED PEPTIDE (PTHrP) No discussion of the fundamental features of PTH in calcium and bone metabolism, and no consideration of the molecular basis of its biological activity, can be complete without considering closely related or analogous

68

JOHN T. POTTS, JR. AND HARALD JUPPNER

TABLE 3--3

Osteoblast Functions Regulated by Parathyroid Hormone

Proliferation Cellular Metabolism Glucose and amino acid transport Citrate, omithine decarboxylation Creatine kinase activity Glycogen synthesis, glucose oxidation Synthesis of RNA, protein, lipids Ion Transport Calcium phosphate, hydrogen ions Na § K+-ATPase Cytoskeletal and Membrane Structure Actin, vimentin, tubulin, and actinin synthesis Phosphatidylethanolamine synthesis Hormonal Responsiveness PTH receptor expression EGF receptor expression 1,25-Dihydroxyvitamin D receptor expression

functions of PTHrP. The latter, discovered and characterized within the last decade, most probably shares an evolutionary origin with PTH. PTHrP has, at least in mammals, both chemical and functional overlaps with PTH (e.g., sharing the common PTH/PTHrP receptor), but its roles diverged from PTH in important aspects. PTHrP is an autocrine/paracrine rather than an endocrine factor, and it is a larger, more complex protein than PTH, that is synthesized at multiple sites in different organs and tissues. Much evidence clearly points to biological roles of PTHrP that are distinct from those of PTH. The still-evolving story of this protein and its proteolytic fragments is beyond the scope of this chapter (for comprehensive review, see references 290 through 293). However, novel features and selective functions that overlap with or are analogous to the actions of PTH are reviewed below. A substance with biological properties similar to those of PTH was first proposed in the early 1940s, when Fuller Albright discussed a patient with malignancyassociated humoral hypercalcemia. 294 The clinical and biochemical characterization of similarly affected patients subsequently established the humoral hypercalcemia of malignancy syndrome, 295 and led to the molecular cloning of PTHrP from several different t u m o r s . 296-299 It is now generally accepted that PTHrP is the major humoral cause of hypercalcemia in malignancies. 29~176176It interacts with the same receptor used by PTH, and when large amounts of PTHrP are released from certain tumors, it mimics some or all of the effects of excess PTH. However, PTHrP is also expressed in a remarkable variety of normal fetal and adult tissues, 29~176176 which suggested, already shortly after its discovery, that it has additional biological role(s) that are unrelated to

Matrix Protein Synthesis and Secretion Collagen Osteonectin Osteopontin Enzyme Synthesis and Secretion Alkaline phosphatase Collagenase Plasminogen activator/inhibitor Metalloproteinase inhibitor Release of Paracrine Growth Factors, Cytokines, and Other Factors IGF-I, IGF-II, IGF binding proteins TGF-[3 M-CSF Prostaglandin E2, others Interleukin-6 Unidentified "coupling factors"

calcium and phosphorus homeostasis, and that these role(s) are distinct from those mediated by PTH. One of its most prominent, only recently discovered functions is the regulation of chondrocyte proliferation and differentiation, and consequently of bone elongation and growth 5'6'3~ (see below).

A. Structure of the PTHrP Gene The human gene that encodes PTHrP is located on chromosome 12, within the region p 12.1-11.2, 299 which is analogous to the region p 15 on chromosome 11, where the PTH gene is l o c a t e d 126 (Table 3 - 2 ) . The coding exons of both genes have a very similar organization with equivalent positions for intron/exon boundaries, but overall the PTHrP gene is considerably more complex 2'299'3~ (Fig. 3-9A). Regarding similarities, the genes encoding PTH and PTHrP have 5' noncoding exons, a single exon encoding all but seven amino acids of the prepro sequence (i.e., amino acid residues - 3 6 to - 8 ) , and an exon that encodes the remainder of the pro-sequence (i.e., amino acid residues - 7 to - 1), the coding sequence of PTH or most of PTHrP, and also contains 3' noncoding region (Fig. 3 - 9 B ) . Due to the similarities in their protein sequence and in the structure of their genes as well as overlap in some of their biological functions, it appears likely that PTH and PTHrP are derived from a common ancestor, whose gene underwent an ancient duplication event. Subsequently, both genes diverged, however, and the PTH gene is now less complex than the PTHrP gene; the latter uses at least three different promoters that give rise to several different mRNA species encoding pep-

CHAPTER 3 Parathyroid Hormone and Parathyroid Hormone-Related Peptide

69

FIGURE 3--9 A, Schematic representation of the intron/exon organization of the genes encoding PTH and PTHrP. B, Coding and noncoding exons of the genes encoding PTH and PTHrP. The introns are represented by a solid line, the exons are boxed; numbers indicate amino acids. Met is the initiator methionine of the PreProPTH sequence, and + 1 is the first residue of the mature peptide.

tides with different carboxyl-terminal ends 29~ (Fig. 3 - 9 A ) . An intragenic dinucleotide repeat in the human PTHrP gene, which may prove to be important for genetic linkage studies, is located 3' to exon 5 (Table 3 2). 308

B. Chemistry of the Translated Protein, Its Posttranslational Modifications, and Its Role in Calcium Homeostasis So far, only the primary structure of several mammalian PTHrP species and of chicken PTHrP are known (Fig. 3 - 1 0 ) . However, antibodies against human PTHrP were recently used to detect PTHrP-like immunoreactivity in fish and frogs, confirming the predicted evolutionary conservation of this peptide. 24-26 When compared to each other, the known PTHrP species show strong amino acid sequence homology within the first 111 residues; there is, in fact, a considerably higher degree of amino acid sequence conservation than that observed for the known PTH species (see Fig. 3 - 2 ) . Although PTH and PTHrP are likely to be derived from a common precursor, the amino acid sequence homology between both peptide families is restricted to the amino-terminal portion, where 6 of the first 12 amino acid residues are

conserved in all PTH and PTHrP species (Fig. 3 - 1 1 ) ; the mid- and carboxyl-terminal regions of both peptides are quite different. This limited structural homology is sufficient to enable both peptides to activate in vitro the common PTH/PTHrP receptor with similar or indistinguishable e f f i c a c i e s . 33'34'91'93'282'309 Likewise, aminoterminal fragments of PTH and PTHrP have largely indistinguishable biological properties in vivo, at least with respect to their roles in regulating the extracellular concentrations of calcium and phosphorus. 31~ Interestingly, analogs of P T H ( 1 - 3 4 ) and P T H r P ( 1 34) that are truncated at the amino-terminus (i.e., residues 1 5 - 3 4 of either peptide) and thus share minimal amino acid sequence homology, also interact equivalently with the common PTH/PTHrP receptor. 314'315This is presumably due to the capacity of both peptides to adopt similar conformations when binding to the receptor. This structural and/or functional similarity between both peptides allowed the construction of hybrids between PTH and PTHrP, some of which had, when tested with cells expressing the common PTH/PTHrP receptor, similar or even higher biological potencies than the native ligands. 316 These overall findings indicate that biologically active amino-terminal fragments of both peptides display, despite a very limited number of conserved amino acid residues, a similar secondary structure, which

Sheet I 30

human

40

dog

rat mouse chicken

100

110

120

human

D~ M A

T

rat mouse chicken

G S

S ~ S ~

human

.

K

K

150

K

E

.

N

.

N

.

R

.

R

.

T

rf

.

H

H

M

160

Q

N

L

M

L

Y ~

I

S

L

F

L L

N

K

S

P

E E

V

170

L

D D

P P

Q L

L

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L

T

E

S

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L

S ~

130 T B T ~

D

A L L W G L K

H H

T T

P R

S S

H H

V

L

N ~

S V

140

I

FIGURE 3--10 Alignment of the amino acid sequences of all known vertebrate PTHrP species. Conserved residues are shown in white letters on black background; numbers indicate amino acid positions.

FIGURE 3--11 Alignment of the ( 1 - 3 4 ) amino acid sequences of all known vertebrate PTH and PTHrP species. Amino acid residues that are conserved in both peptide families are shown in white boxes; amino acids identical to those residues found in human PTH or human PTHrP are indicated by -; numbers indicate amino acid positions.

72

JOHN T. POTTS, JR. AND HARALDJOPPNER

is not restricted to the first 14 residues, but comprises also the 1 5 - 34 region. PTHrP is likely to undergo extensive posttranslational processing, resulting in peptide fragments some of which may have biological properties that are distinct from those involved in the regulation of extracellular calcium. Best studied is the amino-terminal PTHrP fragment, functionally homologous to PTH, that is derived through cleavage at amino acid residue Arg37. 317 The resulting P T H r P ( 1 - 3 6 ) interacts with the common PTH/PTHrP receptor, 33'34'91'282 and thus has an in vivo efficacy that is similar to that of P T H ( 1 - 3 4 ) . 313 Longer, glycosylated fragments of amino-terminal PTHrP were also described; however, these forms appear to be generated predominantly by skin-derived cells, and their biological role(s) remains to be established. 318 In addition to the biologically active amino-terminal PTHrP fragment, different carboxyl-terminal PTHrP fragments are generated, which accumulate in patients with end-stage renal disease 319 (Table 3 - 4 ) . A PTHrP fragment comprising the amino acids 107-139, and a shorter peptide, PTHrP(107-111), may be particularly relevant to the control of bone metabolism, since both fragments were shown to inhibit osteoclastic bone resorption. 32~ While other investigators, using somewhat modified experimental conditions, were unable to confirm these in vitro findings, 322 more recent data indicate that P T H r P ( 1 0 7 - 1 3 9 ) reduces in vivo the number of osteoclasts and inhibits bone resorption. 13 Taken together these findings suggest that carboxyl-terminal PTHrP fragments may have a role in regulating osteoclast formation and/or activity. Cleavage at amino acid residue Arg 37 also generates PTHrP(38-94)amide, a PTHrP fragment, which is likely

TABLE 3--4

PTH-specific receptors PTHrP-specific receptors

C. B i o l o g i c a l A c t i o n s o f P T H r P o n Bone Development PTHrP is expressed in a large variety of normal fetal and adult tissues (for comprehensive review, see references 290 through 293). This widespread expression of PTHrP made it necessary to explore novel approaches to evaluate and define its biological relevance, besides its role in the syndrome of humoral hypercalcemia of malignancy. These included the ablation of the PTHrP gene through homologous recombination and its targeted overexpression through the use of tissue-specific promotors. The ablation of both PTHrP alleles revealed the peptide's most prominent biological function. 3~ Mice with only one copy of the PTHrP gene are normal at birth, show normal behavior and fertility, but develop, later in life, osteopenia despite an apparently normal regulation of calcium and phosphorus homeostasis. 328 In contrast, animals that lack both alleles of the PTHrP gene die,

Characterization of Additional PTH- and/or PTHrP-Specific Receptors Ligand specificity

Bone, cartilage, parathyroid gland-derived fibroblasts Bone, cartilage

Carboxyl-terminal fragments of PTH, i.e., 19-84 and 39-84 Midregional fragments of PTH

Unknown

Unknown

10, 11, 78, 397

Cell proliferation

PL-C

77, 399-402

Placenta

Midregional fragments of PTHrP, i.e., (38-94)NH2 or (67-86)NH2 Midregional fragments of PTHrP, i.e., (38-94)NH2 or (67-86)NH2 PTHrP(107-139)NH2

Transplacental calcium transfer Unknown

Unknown

324, 326

Ca+, IP3

12

Inhibition of osteoclasts Unknown Vasopressinrelease

Unknown 13, 320, 321 Ca§ cAMP

403 14

Unknown

Ca+

7, 8

Hippocampus Supraoptic nucleus

PTHrP(107-139)NH2 Amino-terminal fragments of PTHrP; PTH is considerably less active

Pancreatic cells, squamous cell carcinoma, keratinocytes

Amino-terminal fragments of PTH or PTHrP

Function

Signaling Properties

Target tissue

Squamous cell carcinoma, keratinocytes Osteoclasts

Other receptors

of considerable importance for maintaining fetal calcium homeostasis. 323'324 This peptide is found in the circulation 325 and appears to interact with a distinct receptor that signals through changes in intracellular free calcium and is likely to be an important regulator of transplacental calcium t r a n s f e r . 12'323'324 Recent studies have shown that PTHrP(38-94)amide and P T H r P ( 6 7 - 8 6 ) a m i d e increase the blood calcium concentrations of parathyroidectomized fetal lambs and of PTHrP-ablated murine fetuses, respectively. 324'326 This confirmed earlier data that had provided first evidence for an important role of PTHrP in the regulation of fetal calcium homeostasis. 327

References

CHAPTER 3 Parathyroid Hormone and Parathyroid Hormone-Related Peptide despite their grossly normal size, at birth or shortly thereafter. 3~ Further examination revealed that these mice have striking skeletal changes that include misshapen skulls, short snouts and mandibles, and disproportionately short extremities, but no obvious effects on the morphological development of other major organs (Fig. 3-12). Similar but more severe abnormalities were found in animals in which both alleles of the PTH/ PTHrP receptor gene had been ablated. 6 Both findings provided compelling evidence for an important biological role of PTHrP in chondrocyte differentiation and bone elongation, and further confirmed the previous conclusion that the amino-terminal portion of PTHrP mediates its actions through the common PTH/PTHrP receptor. From these and subsequent studies, it now appears likely 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 presumed role of PTHrP in endochondral bone formation, it has been long known that PTH (which was used in all earlier studies instead of PTHrP that had not yet been discovered) affects chondrocyte maturation and activity in v i t r o . 329"33~ More recent studies confirmed these previous findings by showing that PTH and PTHrP stimulate, presumably through cAMP-dependent mechanisms, TM the proliferation of fetal growth plate chondrocytes, inhibit the differentiation of these cells into hypertrophic chondro-

73

cytes, and furthermore stimulate the accumulation of cartilage-specific proteoglycans that are thought to act as inhibitors of mineralization. 332-334 Due to the lack of the cartilage-specific functions of PTHrP, growth plates of homozygous PTHrP geneablated mice have a thinner layer of proliferating chondrocytes, while the layer of hypertrophic chondrocytes is relatively normal in thickness, but somewhat disorganized. These findings suggested that the lack of PTHrP accelerates the normal differentiation process of growth plate chondrocytes (i.e., resting and proliferating chondrocytes undergo fewer cycles of cell division and differentiate prematurely into hypertrophic cells which then undergo apoptosis before being replaced by invading osteoblasts). Further evidence for an important role of PTHrP in chondrocyte proliferation and differentiation was also derived from studies with transgenic mice that overexpress PTHrP under the control of the mouse type II procollagen promotor. 335 Already at birth, these animals are smaller in size and show a marked disproportionate foreshortening of limbs and tail. The transgenic animals grow at a rate comparable to that of their normal littermates, but maintain their phenotypic abnormalities throughout life. Further analysis of their skeleton revealed that this unusual form of short-limbed dwarfism is most likely due to a severe delay in chondrocyte differentiation and endochondral ossification. In fact, these growth plate abnormalities may well represent the opposite of the accelerated chondrocyte differentiation program found in PTHrP-ablated mice. 3~

IV. RECEPTORS THAT MEDIATE A N A L O G O U S AND DISTINCT M O L E C U L A R ACTIONS OF PTH AND PTHrP

FIGURE 3-- 12 Macroscopicfindings in mice homozygous for the ablation of the PTHrP gene. (From Karaplis AC, Luz A, Glowacki J, et al: Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 8:277-289, 1994.) A wild-type fetus (left) and a PTHrP-ablated littermate (right) were obtained by cesarean section at day 18.5 postcoitum and fixed in buffered formalin. The mutant exhibits a chondroskeletal phenotype characterized by a domed skull, short snout and mandible, protruding tongue, narrow thorax, and disproportionately short limbs.

The role of intact PTH (and PTHrP) in regulating extracellular calcium concentrations is mediated through actions on bone and kidney, and requires the activation of hormone-specific cell surface receptors on target cells such as osteoblasts and renal tubular cells. The diverse actions of PTH (and under certain circumstances of PTHrP) on kidney cells (i.e., its effects on calcium and phosphate transport), and the synthesis of 1,25(OH)2D3, and its pleotropic, direct and indirect, effects on bone cells, had raised the possibility that multiple receptors might mediate this multitude of biological actions. Surprisingly, however, the initial molecular cloning of cDNAs encoding the PTH/PTHrP receptor revealed that only a single G-protein-coupled receptor mediates these classical calcium-homeostatic and bone cell-specific ac-

74

JOHN T.

tions of PTH. Furthermore, the recombinant PTH/PTHrP receptor was shown to interact equivalently with PTH and P T H r P , 33'34'282 which confirmed earlier studies using different clonal cell lines or renal membrane preparations. 91'93'94 The PTH/PTHrP receptor thus mediates the biological roles of two distinct ligands, and is, therefore, important for the calcium-homeostatic actions of PTH, and for the actions of PTHrP in other systems, in particular on chondrocyte proliferation and differentiation. As discussed elsewhere, other receptors have been characterized that interact only with different proteolytic fragGene ments of either PTH o r P T H r P . 7'8'10'12'14'3~ "knock-out" techniques, however, confirmed that the common PTH/PTHrP receptor has a unique, nonredundant role in mediating the endocrine actions of PTH in calcium homeostasis and the autocrine/paracrine actions of PTHrP in growth plate development.

A. The PTH/PTHrP Receptor The PTH/PTHrP receptor, which belongs to a novel family of G-protein-coupled receptors, mediates the en-

POTTS, JR.

AND HARALD JOPPNER

docrine regulation by PTH of mineral ion homeostasis 35 (Fig. 3-13). The first cDNAs encoding mammalian PTH/PTHrP receptor species were isolated through expression cloning techniques from opossum kidney and rat osteoblast-like cell lines that express relatively high levels of these cell surface receptors. 33'34 Subsequently, cDSAs encoding h u m a n , e82'337'338 m o u s e , 339 rat, 34~ chicken, 5 porcine, 341 and frog PTH/PTHrP receptors 34e'343 were isolated through hybridization techniques from very different tissue sources (i.e., kidney, osteoblast-like cells, brain, and embryonic stem cells). Interestingly, the tetraploid African clawed frog Xenopus laevis expresses two nonallelic PTH/PTHrP receptor isoforms, xPPR-A and xPPR-B, which differ in their coding region by several amino acids and that both lack the region corresponding to the mammalian e x o n E 2 . 342'343 The molecular cloning of cDNAs that encode identical PTH/PTHrP receptors from human kidney, brain, and bone-derived cells was particularly revealing, 282'337'338 since distinct tissue-specific receptors had been previously suggested on the basis of distinct pharmacological, organ-specific characteristics. 72'344 However, the abun-

FIGURE 3--1 3 Schematicrepresentation of the human PTH/PTHrP receptor and structural comparison with other mammalian receptor homologs. Top, Amino acids as shown in single letter code; amino-terminus of the receptor at top; potential sites for N-linked glycosylation (Y'). Bottom, Membrane-spanning helices I through VII are indicated by I----7, highly conserved regions are shown by ~ , regions that are less conserved are shown by ~ , and the putative signal peptide is indicated.

CHAPTER 3 Parathyroid Hormone and Parathyroid Hormone-Related Peptide dant expression of a single PTH/PTHrP receptor in the two major target organs of PTH action, kidney and b o n e , 33'34'282 indicated that these pharmacological differences are due to distinctive cellular responses or due to species differences rather than to the tissue-specific expression of different receptors. 33'34'282 Different forms of PTH receptors that are expressed in either bone or kidney were also considered as an explanation for pseudohypoparathyroidism type Ib (PHPIb), since affected patients often present with the renal resistance towards PTH, yet have severe hyperparathyroid bone d i s e a s e . 345'346 However, only a single PTH/ PTHrP receptor is expressed in both tissues, and receptor mutations were excluded in all its coding and 5' noncoding exons, in all adjacent intronic nucleotide sequences, and in the two known promoter r e g i o n s . 347'348 F u r t h e r more, inactivating mutations and splice variants were excluded at the m R N A l e v e l , 349'35~ and studies with larger kindreds with autosomal dominant PHP-Ib excluded the genetic locus of the PTH/PTHrP receptor gene from being linked to the disease. TM The tissue-selectivity of resistance towards a single hormone, PTH, must therefore be explained through a defect in another protein.

75

Recent data indicate that PTH/PTHrP receptor expression is under the control of at least two different promoters, which give rise to distinct m R N A s with differently spliced 5' noncoding e x o n s . 352-354 It appears plausible that a selectively impaired use of the kidneyspecific promoter, combined with normal activity of promoter(s) in most other tissues, including bone, could provide a plausible molecular explanation for the organic-specific resistance towards PTH, but not PTHrP, in patients with PHP-Ib. However, at present, there are no data directly bearing on this possibility. The human gene encoding the PTH/PTHrP receptor is located on the short arm of chromosome 3, within the region 3p21.1-22. 34~ Its intron/exon structure has been analyzed in d e t a i l , 347'348 and was shown to have a similar organization as the rat and the m o u s e gene 356'357 (Fig. 3 - 1 4 ) . All three mammalian genes span at least 20 Kb of genomic DNA, and consist of 14 coding exons and at least three noncoding exons. The size of the coding exons in the human PTH/PTHrP receptor gene ranges from 42 bp (exon M7) to more than 400 bp (exon T); the size of the introns varies from 81 bp (intron between exons M6/7 and M7) to more than 400 bp (intron

FIGURE 3--14 Schematicrepresentation of the human PTH/PTHrP receptor gene. Top, Amino acids encoded by each of 14 coding exons are shown with different patterns; bars indicate the boundaries between exons; amino-terminus of the receptor at top; exon S encodes the putative signal peptide; potential sites for N-linked glycosylation (g'). Bottom, Intron/exon structure; coding exons are shown as boxes, their names and sizes (bp) are indicated (n.d., not determined); size of each intron is shown and the approximate locations of two frequent polymorphisms is indicated.

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JOHN T. POTTS, JR. AND HARALDJCIPPNER

between exons S and El). Two frequent polymorphisms were identified within the human PTH/PTHrP receptor gene (Table 3-2). These are an intronic B s m I polymorphism, located between the 5' noncoding exon U1 and the coding exon S, 358 and a silent B s r D I polymorphism, identified in exon M7 (nucleotide 1417 of human PTH/ PTHrP receptor cDNA). The molecular cloning of the PTH/PTHrP receptor, 33'34 along with the receptors for secretin 359 and calcitonin, 36~ established a distinct family of G-proteincoupled receptors. Members of this family share, despite a similar overall structure with seven membranespanning helices, virtually no amino acid sequence homology with other G-protein-coupled receptors. All members of this secretin/calcitonin/PTH receptor family, including some invertebrate receptors, share about 45 strictly conserved amino acid residues (Fig. 3-15). Furthermore, all receptors of this family have a relatively long amino-terminal, extracellular domain, and at least those receptors that have been studied in more detail use two different signal transduction pathways, adenylate cyclase and phospholipase C. 35 These related receptors,

furthermore, contain up to four sites for potential asparagine-linked glycosylation, six conserved extracellular cysteine residues that appear to be important for ligand/receptor interaction and/or proper receptor processing 361'362 and several other "signature" r e s i d u e s . 363 Two receptor proteins, initially identified as leukocyte antigens CD97 and EMR1, whose cDNAs were isolated through hybridization techniques, 364'365 also share significant homology with the members of the secretin/calcit o n i n ~ T H receptor family, which is particularly high in the membrane-embedded portion of the receptors. However, because of several unique structural features, including a particularly long amino-terminal, extracellular domain, comprising more than 600 amino acid residues and several EGF-like domains, these novel receptors may belong to yet another distinct subfamily of this family of G-protein-coupled receptors. 1. FUNCTIONAL CHARACTERISTICS OF THE P T H / P T H R P RECEPTOR

As noted earlier, despite their limited structural homology that is restricted to the first 13 amino acid resi-

FIGURE 3--15 Left, Schematic representation of the PTH/FI'HrP receptor and the amino acid residues that are conserved in all members of this family of G-protein-coupled receptors; single letter code; Y', potential sites for Nglycosylation. Right, Phylogenetic tree of the PTH/PTHrP receptors and other members of the same family of G-proteincoupled receptor, including two insect (Achata and Manduca) receptors for a diuretic hormone and the putative protein encoded by a genomic DNA clone from C. elegans.

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dues, PTH and PTHrP act through the same PTH/PTHrP receptor. 33'34'282'337'341'366'367When expressed in one of several different clonal cell lines, PTH/PTHrP receptors from different mammalian species bind (1-34) or ( 1 36) analogs of PTH or PTHrP with similar or indistinguishable affinity. Furthermore, analogs of both peptides stimulate with similar potency the formation of two second messengers, cAMP and inositol phosphate. These findings explain why PTH and PTHrP have a similar biological potency when tested in vivo in either rodents310.311 or h u m a n s . 312'313 However, analogs of PTH and PTHrP that are truncated at the amino-terminus show considerable differences in binding affinity when tested with cell lines from different mammalian species that express the native PTH/PTHrP receptors. 368-371 Studies with cloned rat, opossum, and human PTH/PTHrP receptors confirmed these differences. 33'34'282This made it feasible to explore, through the construction of receptor chimeras, which regions of the protein confer functional differences. Binding studies with rat/human PTH/PTHrP receptor chimeras and a P T H ( 7 - 3 4 ) analog, or other further truncated PTH analogs, suggested that the carboxyl-terminal portion of P T H ( 1 - 3 4 ) is likely to interact predominantly with the amino-terminal, extracellular domain of the receptor372; this prediction was recently confirmed through physicochemical techniques. 373 Similar experiments were performed with [Arg 2] hPTH(1-34), a PTH analog that had shown impaired activation when tested with the native and cloned rat PTH/PTHrP receptor, but not with the opossum receptor. 374'375 Chimeras between opossum and rat PTH/PTHrP receptors showed that the ligand's amino-terminus interacts primarily with the membrane-embedded receptor portion. 375 Equivalent conclusions with regard to ligand binding and receptor activation were drawn from experiments with PTH/PTHrP receptors in which smaller portions had been deleted or replaced with homologous sequences from other receptors, and from the pointmutational analysis of single amino acid residues. 361'376-378 Furthermore, comparable studies with native and modified receptors for secretin, calcitonin, and glucagon confirmed a similar architecture of ligand/receptor interaction for other members of this family of G-proteincoupled receptors (Fig. 3-16). 378-381 Recent observations with chimeras between the receptors for calcitonin (CT) and PTH/PTHrP, which are among the most distantly related members of this receptor family, were particularly revealing. 382 A hybrid CT-PTH peptide, CT(1 - 1 1)/PTH(15- 34), selectively activated a chimeric receptor that contained the amino-terminal, extracellular domain of the rat PTH/ PTHrP receptor which had been attached to the membrane-embedded portion and the tail of the porcine cal-

FIGURE 3--1 6

Putative interaction between PTH (or PTHrP) and its PTH/PTHrP receptor. Note that the carboxyl-terminus of the ligand appears to interact predominantly with the receptor's amino-terminal, extracellular domain, while the ligand's amino-terminus interacts with the membrane-spanning helices and the extracellular loops.

citonin receptor; the wild-type CT receptor, the wild-type PTH/PTHrP receptor, and the reciprocal receptor chimera were not activated by this ligand. Conversely, a reciprocal peptide, PTH(1-13)/CT(12-32), selectively activated the chimeric receptor in which the aminoterminal, extracellular domain of the calcitonin receptor had been attached to the membrane-embedded portion and the tail of the PTH/PTHrP receptor; again the two wild-type receptors and the reciprocal chimeric receptor were not activated. These data confirm that the aminoterminus of the respective ligand interacts with the membrane-embedded receptor region, while the ligand's carboxyl-terminus interacts with the receptor's aminoterminus. Furthermore, these findings suggest that PTH and CT, and their respective receptors, share a similar architecture with at least two functional domains that allow the specific interaction between the respective ligand/receptor pairs. These ligand/receptor domains are sufficiently independent to permit synthetic hybrid ligands to efficiently activate the appropriate receptor chimeras. CT and PTH, and their respective receptors, thus follow a common pattern of ligand/receptor interaction, and it is likely that this mode of ligand/receptor interaction exists also for other members of this family of G-proteincoupled receptors and their cognate ligands. 2. THE IMPORTANCE OF P T H / P T H R P RECEPTOR IN BONE DEVELOPMENT

Similar to the widely expressed PTHrP, the mRNA encoding the PTH/PTHrP receptor is found in a surpris-

Regulation of chondrocyte proliferation and differentiation by parathyroid hormone-related peptide (PTHrP) and Indian Hedgehog (lhh). (Modified from Wallis GA: Bone growth: coordinating chondrocyte differentiation. Current Biol 6:1577-1580, 1996.)

FIGURE 3--17

CHAPTER 3 ParathyroidHormone and Parathyroid Hormone-Related Peptide ingly large variety of fetal and adult tissues. However, PTH/PTHrP receptor expression is most abundant at three sites, in renal tubular cells and in osteoblasts, and in prehypertrophic chondrocytes of the metaphyseal growth plate. 36-4~ Mice in which both alleles encoding the PTH/PTHrP receptor were ablated, showed growth plate abnormalities that are similar to, but more severe than, those observed in PTHrP-ablated animals. 6'3~ These studies confirmed that the same receptor that mediates the endocrine effects of PTH mediates in the growth plate the autocrine/paracrine effects of PTHrP. More extensive investigations of PTHrP- and PTH/PTHrP receptor-ablated animals showed furthermore that the PTH/PTHrP receptor mediates also, although indirectly, actions of Indian Hedgehog (Ihh). 5'6 Ihh belongs to the hedgehog family of secreted proteins that are important for embryonic patteming 383 and acts through Patched (Ptc) (or a closely related homolog). Ptc is a receptor with 12 membranespanning helices, which associates physically with Smoothened (Smo) and thereby suppresses the constitutive activity of the latter protein. 384'385 In contrast to the mRNA encoding the closely related Sonic Hedgehog (Shh), the mRNA encoding Ihh is found only in later stages of embryonic limb development, and its expression is restricted to the transition zone within the growth plate, where proliferating chondrocytes differentiate into hypertrophic cells. 5 The ectopic overexpression of Ihh in chicken wing cartilage through a replication-competent retroviral vector blocked the normal chondrocyte differentiation program, and prevented the appearance of hypertrophic chondrocytes that express collagen type X and bone morphogenic protein-6 (Bmp). The aberrant expression of Ihh thus led to similar changes as those observed in transgenic mice that overexpress PTHrP under the control of the collagen type II promoter, 335 but are the opposite of those found in mice in which the genes encoding PTHrP or its PTH/PTHrP receptor had been disrupted through homologous recombination. 6'3~ Interestingly, Ihh-infected growth plates revealed in the perichondrium a strong up-regulation of the message encoding Ptc, while the PTHrP mRNA was up-regulated predominantly in the periarticular regions of infected limbs. 5 This finding provided initial evidence for a negative feedback loop within the growth plate that involves PTHrP and its PTH/PTHrP receptor, as well as Ihh and its receptor Ptc (Fig. 3-17). To explore these local regulatory mechanisms further, bone explants from either PTHrP- or PTH/PTHrP receptor-ablated mice were treated either with PTHrP or with Shh (which has biological properties that are indistinguishable from those of Ihh). Both ligands were able to repress the differentiation into hypertrophic chon-

79

drocytes in bone explants from normal mice. PTHrP treatment also repressed the differentiation into hypertrophic chondrocytes in bone explants from PTHrPablated animals, while Shh had no effect in these bones. This indicated that PTHrP is required for mediating the actions of Ihh in the growth plate. 5 Similar experiments with bone explants from PTH/PTHrP receptor-ablated mice showed that neither PTHrP nor Ihh was able to repress the chondrocyte differentiation program, 6 indicating that the PTH/PTHrP receptor mediates directly the actions of PTHrP, and indirectly at least some of Ihh. PTH/gFHrP receptors, therefore, have important additional roles that are independent from their PTH-dependent functions in the regulation of calcium homeostasis. 3. JANSEN'S METAPHYSEAL CHONDRODYSPLASIA: A DISORDER CAUSED BY MUTANT PTH/PTHRP RECEPTORS

The sharing of a single receptor for mediating the activity of two distinct ligands that have entirely different biological functions is uncommon. This made it difficult to search for disorders in humans, or other mammals, that are caused by either activating or inactivating mutations in the PTH/PTHrP receptor. Pseudohypoparathyroidism type Ib (PHP-Ib), an autosomal dominant disorder, 345'346'386 was long thought to be caused by inactivating mutations in the PTH receptor (e.g., the PTH/ PTHrP receptor), 387 but mutations that result in an inactive receptor were excluded. 347'349'35~Consistent with these findings, affected individuals show no discernible abnormalities at the level of the growth plates, implying that the actions of PTHrP are appropriately mediated. These findings led to the conclusion that the disease must be caused by a molecular defect in a gene that is distinct from that encoding the PTH/PTHrP receptor. In retrospect, these "negative" findings are in agreement with our current understanding of the biological importance of the common PTH/PTHrP receptor. Consequently, homozygous inactivating mutations that affect both alleles of the PTH/PTHrP receptor gene should lead, if mild enough to be compatible with life, not only to abnormalities in calcium and phosphorus homeostasis but also to abnormalities in the metaphyseal growth plates. PTH/PTHrP receptor mutations were, however, identified as the cause of Jansen's metaphyseal chondrodysplasia, an autosomal dominant disease that is characterized by short-limbed dwarfism, and severe hypercalcemia and hypophosphatemia, despite normal or undetectable levels of PTH and PTHrP in the blood 388 391 circulation (Fig. 3-18). As outlined above, PTH/ PTHrP receptors are abundantly expressed in kidney and bone, and they mediate in these tissues the endocrine actions of PTH to maintain the concentrations of extra-

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JOHN T. POTTS,JR. AND HARALDJOr'r'NEr~

FIGURE 3-- 18 Patientwith Jansen's metaphyseal chondrodysplasia at age 5 (left) and 22 (right). [From Frame B, Poznanski AK: Conditions that may be confused with tickets. In DeLuca HF, Anast CS (eds): Pediatric Diseases Related to Calcium. New York, Elsevier, 1980, pp 269-289.]

cellular calcium and phosphate within narrow limits. PTH/PTHrP receptors are, furthermore, abundantly expressed in growth plate chondrocytes, where they mediate the autocrine/paracrine actions of PTHrP and regulate the normal process of endochondral bone formation. The most abundant expression of PTH/PTHrP receptors is thus observed in those tissues that show the most striking functional abnormalities in patients with Jansen's disease. Recently, two different heterozygous mutations were identified in the genomic DNA of several patients with this interesting disorder, but not in the DNA of their unaffected siblings and parents 388-39~ (Fig. 3-19). Expression of the mutant PTH/PTHrP receptors in COS-7 cells resulted in constitutive, ligandindependent accumulation of cAMP, while the basal accumulation of inositol phosphates was not increased. These findings provided a plausible explanation for the severe changes in mineral ion homeostasis and in growth plate development. Furthermore, these data support the notion that the common PTH/PTHrP receptor mediates the actions of PTH and PTHrP, and that PTHrP is an important regulator of chondrocyte proliferation and differentiation, and consequently of bone elongation and growth.

B. T h e P T H - 2 R e c e p t o r Although the PTH/PTHrP receptor appears to be the most important receptor to mediate the actions of PTH

and PTHrP, there is considerable evidence for several other receptors that are activated by PTH or PTHrP, or both, and mediate yet unknown biological functions that are presumably unrelated to the control of calcium and phosphorus (see also below). Best characterized is the PTH-2 receptor, which was recently isolated and was shown to share 51% amino acid identity with common PTH/PTHrP receptor3~ its structural homology with other members of the secretin/ calcitonin/PTH receptor family is considerably lower (see Fig. 3-15). In contrast to the common PTH/PTHrP receptor, which is activated with similar efficacy by PTH and PTHrP, the PTH-2 receptor is activated almost exclusively by PTH, but not by P T H r P . 3~ PTH may thus be able to mediate its actions through at least two distinct receptors, the PTH/PTHrP receptor and the PTH2 receptor. However, it is conceivable that a recently described hypothalamic PTH-like peptide, rather than PTH, is the natural ligand of the PTH-2 receptor. 3z Expression of the PTH-2 receptor is restricted to relatively few tissues (i.e., placenta, pancreas, blood vessels, testis, and brain), and its mRNA levels are typically much less abundant than those encoding the PTH/PTHrP receptor. 3~ Biological function(s) that are mediated by the PTH-2 receptor in these and possibly other tissues are currently unknown. However, previous studies in parathyroidectomized dogs had shown that PTH increases the pancreatic secretion of bicarbonate without affecting the amylase output. 394 D u e to its significant expression in the exocrine pancreas, 31 it may well be that the PTH-2 receptor, rather than the common PTH/PTHrP receptor, regulates these exocrine pancreatic functions. This conclusion is further supported by findings in transgenic mice in which overexpression of PTHrP is targeted to the pancreatic islet cells. These animals show significant changes in glucose metabolism, but not in exocrine pancreatic functions, which suggests that these actions of PTHrP are mediated through the PTH/PTHrP receptor, and that the PTH-2 receptor is largely resistant to abundant local concentrations of PTHrP. 395 Due to its ligand specificity and due to its restricted expression levels in only few tissues, it thus appears unlikely that the PTH2 receptor mediates any of the autocrine/paracrine effects of PTHrP. Although the biological importance of the PTH-2 receptor thus remains to be established, its molecular cloning provided an important new tool to study of ligand/ receptor interaction. Initial functional characterization had shown that the PTH-2 receptor is activated by PTH, but not at all by PTHrP; these findings were attributed to the significant structural differences between both ligands. 3~ However, despite this lack of receptor activation, radioligand studies with transfected COS-7 or HEK cells revealed a weak interaction between PTHrP and the

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81

Top, Schematic presentation of the human PTH/PTHrP receptor; two amino acid substitutions (H223R and T410P) that were identified in patients with Jansen's metaphyseal chondrodysplasia are indicated. 388-39~ Bottom, Conservation of the equivalent of residues H223 and T410 in all mammalian members of this family of Gprotein-coupled receptors; numbers indicate the position of the residue within the amino acid sequence of the respective receptors.

FIGURE 3--19

PTH-2 receptor. Interestingly, a truncated analog of human PTHrP, [Leu 11, D-Trp12]PTHrP(7-34)amide, which was previously developed as a competitive inhibitor of PTH-stimulated cAMP accumulation at the PTH/PTHrP receptor, 396 had similar antagonist potency when tested with PTH-stimulated cells that express either PTH-2 or PTH/PTHrP r e c e p t o r s . 392'393 This suggested that some portions of PTHrP can interact with the PTH-2 receptor. Subsequent studies with PTHrP analogs revealed that phenylalanine at position 23 dramatically reduces the ligand's binding affinity, while histidine at position 5 prevents the productive interaction with the PTH-2 receptor, but not with the PTH/PTHrP receptor. 392

C. A d d i t i o n a l R e c e p t o r s for P T H a n d / o r P T H r P Complementary DNAs encoding other putative receptors have not yet been isolated. However, based on radioreceptor studies and other functional assays, it appears likely that additional receptors exist for mid-regional and carboxyl-terminal fragments of PTH

and PTHrP, but their biological importance remains to be established (Table 3-4). For example, intact PTH and/or larger carboxyl-terminal fragments were shown to interact with a novel receptor/binding protein that is expressed in several different clonal cell lines, including bone- and cartilage-derived cells in which the common PTH/PTHrP receptor had been deleted through homologous recombination. 1~ Although the biological role of this novel receptor remains to be established, some data suggest that it could be required for bone- and/or cartilage-specific f u n c t i o n s 78'82'83'397 and for the recruitment of osteoclasts. 85 Additional, yet uncloned receptors most likely mediate the actions of midregional PTHrP, which is important for placental calcium t r a n s p o r t 324'326 a n d may have some unknown biological functions in s k i n . 12 Likewise, the actions of the carboxyl-terminal PTHrP portion on osteoclasts and the central nervous system appear to be mediated through a specific receptor, 13'32~ and novel receptors were characterized that interact equivalently with the amino-terminal portions of PTH and PTHrP 7'8'336 or predominantly with amino-terminal PTHrp.14 The lat-

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ter PTHrP-selective receptor is abundantly expressed in the supraoptic nucleus, where it regulates vasopressin synthesis and release.

D. S t r u c t u r e - B a s e d D e s i g n of P T H and PTHrP Analogues Many critical residues on both ligand and receptor have been identified that influence receptor binding and activation, through the extensive use of site-directed mutagenesis of the ligand and receptor. The careful functional analyses of such mutant ligands and receptors resuited in an indirect and thus only partial understanding of the conformation of the active bimolecular complex between hormone and receptor. Nonetheless, steadily accumulating data from ligand/receptor studies are leading towards a composite picture that may eventually permit structure-based design of smaller and/or more potent analogs of PTH; this approach may be feasible soon, even without a three-dimensional model of the active hormone-receptor complex. Important insights into ligand/receptor interactions are derived from studies with receptor homologs from several vertebrate species that showed important differences in their binding affinity of PTH fragments and/or in their activation properties by PTH and PTHrP analogs. Peptide analogs, receptor hybrids, and mutant receptors derived from more conventional approaches such as deletional, homolog-scanning, and point mutational analysis were important tools in these efforts to gradually develop hypotheses about the orientation of ligand/receptor interaction; a concept has emerged which suggests that the carboxyl-terminal end of the ligand binds to the aminoterminal, extracellular domain, and to some extent, to the extracellular loops of the PTH/PTHrP receptor as well. The amino-terminal end of the ligand, on the other hand, appears to interact with transmembrane segments and/or immediately adjacent external loops of the receptor as discussed above. When a peptide as long as 34 amino acids interacts with a G-protein-coupled receptor, subtle but critical intramolecular and intermolecular interactions may determine whether a particular ligand binds/activates a particular receptor. There are multiple opportunities for evolution to confer receptor selectivity by substituting key amino acids in the ligand; for example, PTHrP contains histidine at position 5 which prevents the efficient interaction with the PTH-2 receptor. 392'393 Furthermore, the carboxyl-terminal portion of larger peptide molecules may anchor the hormone to the extracellular domain of the receptor, thus allowing delivery of the aminoterminal, activation-specific region of the ligand into the

POTTS, JR. AND HARALD JOPPNER

receptor's putative activation pocket which appears to be formed by the seven transmembrane-spanning helices (as current models of these G-protein-linked receptors predict). This mode of activation may also offer an explanation, in part, for the compression or folding-back upon itself that is evident at the amino-terminus of PTH and PTHrP, 316 another feature of ligand structure that may affect specificity. The work with hybrid ligands and hybrid receptors, 382 reviewed above, suggests that the key portion of the ligand that specifically activates a receptor may be much smaller than PTH(1-34); residues 1-13 are sufficient for the hybrid PTH/CT ligand to activate a chimeric CTPTH/PTHrP receptor. Of course, if only a small fragment of the original ligand acts as the activating sequence, intensive molecular design may be needed to optimize the binding/activation between peptide and receptor to provide a meaningful potency similar to that of the fulllength ligand. Such directions of work hold promise, however, to simplify the chemical structure of a large polypeptide such as PTH in order to concentrate on structure/activity within limited regions of the aminoterminal sequence. If a minimum chain length for activation is determined, saturation mutagenesis of such a short amino-terminal PTH (or PTHrP) sequence could lead to the development of more potent analogs that activate without the need for carboxyl-terminal anchoring. There is considerable interest in shorter PTH or PTHrP sequences that might be more readily administered by nonparenteral means; such molecules in turn could open feasibility of screening for small, nonpeptide equivalents of PTH that may prove critical for medical applications in osteoporosis and other areas. The ultimate goal of this work obviously is to understand the precise intermolecular interactions that are important for PTH/PTHrP receptor activation. However, an accurate three-dimensional model of the active hormone-receptor complex will probably await application of direct physicochemical methods, but probing their critical points of interaction can be carried a long way by systematic mutagenesis studies.

V. SUMMARY: OVERALL BIOLOGICAL ROLES OF PTH and PTHrP, AND THEIR CLONED RECEPTORS The actions of amino-terminal PTH and PTHrP fragments are mediated through the common PTH/PTHrP receptor, which belongs to a distinct family of G-proteincoupled receptors, which typically signal through two second-messenger pathways, cAMP and inositol phos-

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Parathyroid Hormone and Parathyroid Hormone-Related Peptide

FIGURE 3--20

Biological functions mediated by the PTH/PTHrP ( ~ )

phate. Similar to the widely expressed PTHrP, the mRNA encoding the PTH/PTHrP receptor is found in a large variety of fetal and adult tissues, but its expression is most abundant in three tissues: kidney and bone, where it mediates the calcium-homeostatic endocrine actions of PTH; and the metaphyseal growth plate, where it mediates the autocrine/paracrine actions of locally synthesized PTHrP. F r o m studies in mice in which the genes encoding either P T H r P or the P T H / P T H r P receptor had been ablated through h o m o l o g o u s recombination, we now know about the critical role that is played by PTHrP in embryonic and postnatal d e v e l o p m e n t of cartilage and bone. 5'6'3~ In the adult animal, the biological roles of this paracrine P T H r P - P T H / P T H r P receptor system remains incompletely understood and is the object of further studies, some of which may require conditional gene " k n o c k - o u t " or overexpression approaches. In contrast to the c o m m o n P T H / P T H r P receptor, the closely related PTH-2 receptor is activated only by PTH, but not by PTHrP, 3~ and it may have a distinctive primary ligand, the recently described hypothalamic PTH-like molecule. Expression of the PTH-2 receptor is restricted to relatively few tissues and its m R N A levels are typically much less abundant than those encoding the P T H / P T H r P receptor; its biological importance remains to be determined. Perhaps P T H is its ligand in some organs or tissues, and another ligand mediates other biological actions in other (or all) sites. Figure 3 - 2 0 schematically illustrates our present understanding of this complex endocrine/paracrine system which involves at least two different, but closely related, G-proteincoupled receptors, and two, possibly three, different poly-

and the PTH-2 receptor ( ~ ) .

peptide ligands that are likely to have distinct biological functions.

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~HAPTER z

Calcltonln T. J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON St. Vincent's Institute of Medical Research and The University of Melboume Department of Medicine, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia

I. II. III. IV. V.

Nature of Calcitonin Chemistry Biosynthesis Secretion and Metabolism Actions of Calcitonin

VI. Calcitonin Receptor VII. Calcitonin in Clinical Medicine VIII. Summary Acknowledgments References

When it became established that parathyroid hormone acted directly on bone to promote its resorption, the idea developed that parathyroid hormone was the major hormonal factor governing calcium homeostasis in the body. This was the basis of the McLean and Urist 1 hypothesis developed in the mid 1950s that the serum calcium was regulated by appropriate changes in the secretion rate of parathyroid hormone by a negative feedback control system. In 1961, Rasmussen 2 questioned this in suggesting that if the bone were the only means of regulating serum calcium level in conjunction with the parathyroids, the resulting feedback system would lead to wide fluctuations in serum calcium. It had been known for some time that parathyroid hormone lowered the urinary calcium, and Rasmussen made use of this in extending the McLean-Urist theory to involve the kidney. He considered that the kidney regulator was rapid to respond, sensitive to small fluctuations in hormone concentration, and of limited capacity; the bone regulator was slow to respond, relatively insensitive, but of nearly unlimited capacity. The renal action conserved serum calcium by rapidly inhibiting urinary secretion of calcium, the skeletal action by causing a less rapid dissolution of calcium from the bone matrix to the extracellular fluid. Although this seemed a more satisfactory METABOLIC BONE DISEASE

explanation of calcium control, Copp and his associates looked persistently for a better o n e . 3'4 In experiments in which they perfused the thyroparathyroid apparatus of dogs and sheep, they obtained evidence for the secretion, in response to a high calcium stimulus, of a factor that rapidly lowered systemic plasma calcium. 3 They called this calcitonin, and suggested that it was produced by the parathyroid glands. 4 After this discovery, Hirsch et al. looked afresh at some observations made in their laboratory. It had been noted that rats parathyroidectomized by cautery showed a more rapid and profound fall in serum calcium than rats parathyroidectomized by surgical excision. 5 Hirsch et al. then found that acid extracts of rat thyroid glands caused hypocalcemia when injected into r a t s . 6 They suggested that cautery of the thyroid gland during parathyroidectomy caused the release of a factor from the thyroid gland that provoked a greater fall in serum calcium than that which occurred following simple removal of the parathyroid glands (Fig. 4 - 1 ) . They called this activity "thyrocalcitonin" and showed that it could be extracted from the thyroid glands of several species, but not from other tissues. At the same time, Maclntyre's group provided support for Copp's work by demonstrating in thyroparathyroid perfusion studies in the dog that 95

Copyright 9 1998 by Academic Press. All fights of reproduction in any form reserved.

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FIGURE 4--2 Effect on systemic plasma calcium in the goat of high calcium perfusion of parathyroid (e) and of thyroid and parathyroid glands (o). (From Foster GV, Baghdiantz A, Kumar MA, et al: Thyroid origin of calcitonin. Nature 202:1303-1305, 1964. Copyright 1964, Macmillan Journals Limited.)

a potent, rapidly acting calcium-lowering hormone existed. 7 Subsequent perfusion studies in the goat showed that this activity was released from the thyroid gland. 8 In this species it was possible to perfuse either the external parathyroid alone or the thyroid plus parathyroid glands together (Fig. 4 - 2 ) . Furthermore, autogenous thyroid extract injected into the goats rapidly lowered the systemic serum calcium. It soon became clear that calcitonin and thyrocalcitonin were identical, were products of the thyroid gland in mammals, and probably represented an important new hormone involved in the regulation of calcium metabolism in the body.

logical assay contributed to the fact that calcitonins of several species were isolated and sequenced within a few years of its discovery. The recognition of calcitonin as a hypocalcemic hormone was hailed for several years as an answer to the tight control of plasma calcium. As information emerged about its action, however, it became apparent that this was not so, at least in the mature animal. The ability of calcitonin to inhibit osteoclastic bone resorption is beyond doubt, but the physiological significance of this seems likely to vary, for instance, during growth and development. Finally, the recognition of calcitonin's origin in the " C " or parafollicular cells of the mammalian thyroid drew attention to a second endocrine system in the thyroid gland, deriving its origin from the cells of the ultimobranchial bodies, which in birds and fish remain as discrete organs.

Comparison of the effects of thyroparathyroidectomy with parathyroidectomy by cautery and surgery in the rat. (From Hirsch PF, Gauthier GF, Munson PL: Thyroid hypocalcemic principle and recurrent laryngeal nerve injury as factors affecting response to parathyroidectomy in rats. Endocrinology 73:244-251, 1963.)

I. NATURE OF CALCITONIN Experiments in several species rapidly confirmed that calcitonin was a peptide released from the thyroid gland in response to a hypercalcemic stimulus and capable of rapidly lowering the plasma calcium and phosphorus levels. It was shown to produce this effect independent of the kidney or intestine, and it acted as an inhibitor of bone resorption. Evidence for this is summarized later in this chapter (see Section V.A). The calcium-lowering effect of calcitonin was the basis for its biological assay, and indeed the convenience and sensitivity of the bio-

A. Embryological Origin of Calcitonin-Producing Cells Much of the early work on the localization of calcitonin-producing cells comes from Pearse and his colleagues, who suggested that the "mitochondrion-rich" cells of the thyroid were responsible for calcitonin secretion. 9 When they observed electron microscopic

CHAPTER4 Calcitonin changes in the parafollicular cells of the dog thyroid following high calcium perfusion of the gland, they ascribed to these cells the function of either synthesizing or storing calcitonin, and gave them the name C cells. These cells are identical with the argyrophil parafollicular cells described in the dog by Nonidez, 1~ and in other species (e.g., the pig) they occupy epifollicular and follicular positions. 11 Pearse produced further evidence that the C cells may produce a polypeptide hormone by demonstrating their property of uptake of 5-hydroxytryptophan, 11 a faculty common to other polypeptide hormone-producing cells (e.g., the pancreatic islet cells) and pituitary corticotrophs and melanotrophs (APUD cells). At the same time he pointed out that there were two possibilities for the embryological origin of the C c e l l s n t h e ultimobranchial bodies and the neural crest h a n d he favored the former site of origin. Subsequent cytochemical studies confirmed the ultimobranchial origin of the parafollicular C cells in the rodent thyroid, 12 and it is considered that the ultimobranchial and C cells derived originally from the neural crest. The ultimobranchial body arises in the embryos of all vertebrates, with the exception of the cyclostomes, caudal to the last branchial arch, one on either side. Its fate in the adult varies across species. In lower vertebrates it remains as a distinct structure that may persist on both sides or unilaterally. In mammals it becomes fused with the thyroid during embryonic life and here gives rise to calcitoninsecreting C c e l l s . 13 No function had previously been ascribed to the ultimobranchial body, but seasonal changes in its structure had been observed in lizards, bats, and frogs. The presence of secretory material in the follicles of the ultimobranchial bodies of reptiles, amphibians, and fish suggested an endocrine function, but there was no indication as to its nature. 14-~6 It was thought that the ultimobranchial body might form reserves of parathyroid or thymic tissue in conditions of stress, or possibly that it had undergone regression during evolution and lost its original function. Observations of the teleost A s t y a n a x mexicanus revealed that the ultimobranchial body hypertrophied after the fish had been kept in the dark for periods of between 4 months and 2 years. TM These changes were associated with skeletal deformities, but the authors attributed them to a parathyroid function of the ultimobranchial body. Hypertrophy of the ultimobranchial body had also been observed in frogs 15 under conditions of calcium stress and during metamorphic climax when calcium is being transferred from stores in the paravertebral lime sacs for incorporation into bone. Thus there was some evidence for involvement of the ultimobranchial body in changes in calcium metabolism, but no specific mechanisms were known. Throughout their work, Pearse's group emphasized the variable ultimate fate of the ultimobranchial bodies

97 in different species. After originating from the neural crest, these bodies migrate forward during embryonic development. Although the ultimobranchial bodies remain as separate endocrine glands in birds, fish, and reptiles, in some submammalian species the cells can be found in other tissues of the neck and in the lung. In the lizard, for example, the main source of calcitonin is the lung, 16 although calcitonin-producing cells are present in other parts of the neck. Although in the rat the C cells are virtually all within the thyroid, this is not the case with several other mammals. In humans, for example, there is evidence for calcitonin-producing cells in the thymus and the lung, and it is therefore difficult to determine the result of calcitonin deficiency in mammals by experimentation or in humans by clinical observation. The association of calcitonin with granules in the parafollicular cells of the rat was suggested by experiments showing that administration of calcium to rats resuited in discharge from these cells of granules visible on electron microscopy. 17 In addition to the demonstration of the peptide hormone-secreting properties of C cells by histochemistry, calcitonin was identified in the C cells of the dog and the pig by immunofluorescence. This was later confirmed by in situ hybridization localization of calcitonin messenger ribonucleic acid (mRNA) to the C cells of the thyroid. TM

B. C a l c i t o n i n in Vertebrates a n d N o n v e r t e b r a t e s There has been considerable interest in the discovery that immunoreactive calcitonin-like material can be detected in the nervous systems of prevertebrate species including the sea squirt Ciona intestinalis, 19 the pond snail L y m n a e a stagnalis, 2~ a number of protochordates, and a cyclostome, Myxine. 21 These species lack bony skeletons, and the occurrence of human calcitonin-like molecules in their nervous systems suggests that the bone-regulating function of the calcitonins may be a later evolutionary development in vertebrates. Human calcitonin-like immunoreactivity (hCT-I) also exists in human cerebrospinal fluid, 22 although no direct evidence for the origin of this material is available. Low levels of hCT-I were found in extracts of postmortem human brain. 23"24 However, in addition to hCTI, low levels of material immunologically and chromatographically similar to salmon calcitonin sCT (sCT-I) also occurred. 24 The highest concentration of immunoreactivity was found in the hypothalamus, which is consistent with the hypothalamus containing the highest level of calcitonin receptors (see Section V.F). The concentrations of CT-I in the brain were ---10 times greater than those in serum and cerebrospinal fluid. 24 Similarly, extracts of rat brain diencephalon revealed the existence

98 of a biologically active sCT-like peptide 25 which was distinct from the low levels of hCT-I reported in earlier studies. 26 Although hCT-I has been detected in rat brain, there is only limited evidence for the presence of rat calcitonin (rCT) mRNA. In a Northern blot analysis of hypothalamus, a low level of rCT mRNA was observedZ7; however, S 1 nuclease assay of various brain regions failed to detect calcitonin mRNA expression, indicating that levels of calcitonin mRNA must be less than 0.2% of that of calcitonin gene-related peptide 28 (see Section II.B). The existence of multiple types of calcitonin within one species is sustained by the immunological detection of at least two forms of calcitonin in fish, reptiles, amphibia, mammals, and birds, 29-31 while the existence of a gene expressing a sCT-like peptide in humans is supported by hybridization of chicken calcitonin (cCT) cDNA to Northern blots of human medullary thyroid carcinoma tissue. 32 A role for a sCT-like peptide as a neurotransmitter or neuromodulator is supported by the presence of sCT-I within the central nervous system of lower vertebrates. In pigeon, the distribution and levels of peptide detected by a sCT-specific radioimmunoassay 33 are consistent with findings in rat b r a i n y with the highest amount detected in the hypothalamus followed by midbrain and low levels in brainstem. Immunohistochemical studies in lizard brain further demonstrated sCT-I, localized to varicosities and terminals in the diencephalon, although not in other brain regions. 34 Furthermore, the levels of calcitonin-like peptide detected in the rat diencephalon 25 were in accord with the reported levels of other neuropeptides, including CGRP and neuropeptide y,35.36 consistent with a potential role of a sCT-like peptide acting as a neurotransmitter or neuromodulator in mammalian brain. Intriguingly, the rat C lb isoform of the calcitonin receptor (CTR) (see Section VI-B) has very little interaction with the thyroidally derived form of calcitonin. 37'38 This receptor isoform is enriched in brain and may form a physiological target for endogenous sCT-like peptides. Furthermore, the C3-amylin receptor has high affinity for the teleost but not mammalian calcitonins in the rat, 39'4~ and as such this receptor phenotype is also a potential substrate for sCT-like peptides. Immunoreactivity equivalent to the thyroidally derived or ultimobranchial-derived form of calcitonin has also been found in the anterior pituitary of both mammals and lower vertebrates. 23'26'41-45 Although calcitoninlike immunoreactivity is also reported to occur in the intermediate pituitary, 41'43'44 this finding is not routinely reproducible. 42'44 The identity of the pituitary calcitonin remains to be determined. While reacting to specific hCT-antisera, the rat pituitary material does not react with all hCT-antisera, nor does preabsorption clearly

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

abolish pituitary staining. 43 This suggests that the pituitary calcitonin-like material is not equivalent to the thyroidal form of calcitonin. Consistent with this, Northern blot analysis of anterior pituitary, using cDNA to thyroidal calcitonin mRNA, failed to detect expression of calcitonin mRNA. 46'47 More recent data indicate that a sCT-like peptide may also be present in the anterior pituitary, with release of sCT-like peptide from cultured rat anterior pituitary cells. 48 Salmon CT-I also occurs in the mouse pituitary carcinoma cell line oL-TSH.49 The physiological significance of calcitonin-like immunoreactivity in the pituitary remains to be determined; however, calcitonin receptors are present in the intermediate pituitary 5~ and therefore the calcitonin-like material may act as a paracrine regulator of these receptors.

II. CHEMISTRY Several groups almost simultaneously published the sequence of pig calcitonin (pCT) 52'53 and within months pCT had been chemically synthesized. 54 It is a 32-aminoacid peptide with a carboxyl-terminal proline amide and a disulfide bridge between cysteine residues at positions 1 and 7. The sequence and synthesis of salmon ultimobranchial calcitonin were to follow shortly. 55 Copp had noted in making extracts of salmon ultimobranchial glands that the hormone was extremely potent. Indeed the pure or synthetic salmon hormone, at 3000 to 5000 MRC units/ mg, was 20 to 40 times more potent than calcitonins of mammalian origin. At the same time it was pointed out that medullary carcinoma of the thyroid was most likely a tumor of cells of ultimobranchial origin. This led to the extraction and purification of human calcitonin from medullary thyroid carcinoma tissue, 56 and subsequent synthesis of the human peptide. 57 Based on their amino acid sequence homologies, the different species (Fig. 4 - 3 ) have been classified into three groups as follows: 1. Artiodactyl, which includes porcine, bovine, and ovine, which differ by four amino acids. 2. Primate/rodent, which includes human and rat calcitonins, differing by two amino acids. 3. Teleost/avian, which includes salmon, eel, goldfish, and chicken, differing by four amino acids.

A. S t r u c t u r e / A c t i v i t y R e l a t i o n s h i p s The sequences of several mammalian and fish calcitonins are shown in Figure 4 - 3 . They are all 32-aminoacid peptides with a common 1 - 7 disulfide bridge and a proline amide at position 32, which is essential for

CHAPTER 4 Calcitonin 1 C G C C C C G

Human CT Porcine CT Bovine CT Dog CT Rat CT Rabbit CT Chicken CT Salmon CT Eel CT

Rat Amylin IIRat CGRP

99

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FIGURE 4--3

Amino acid sequence of CTs from mammals, birds, and fish. Conserved residues are boxed. Sequences of rat amylin and rat CGRP are also included with amino acids conserved between these two peptides.

activity. There are considerable differences in the sequences of the carboxy-terminal two thirds of these molecules. In several different biological assays, the order of potency of the calcitonins is teleost > artiodactyl > human, although it is important to note that the absolute efficacies vary greatly between CTRs of different species and of receptor isoforms within species. For example, hCT is 3- to 50-fold less potent than sCT at the human CTR (hCTR) 58-6~ and more than 1000-fold less potent at sheep brain membrane receptors. 61 Studies of substituted, deleted, and otherwise modified calcitonins have provided considerable information regarding structure/activity relationships of the calcitonin molecule. For example, it was deduced that the ring structure serves to protect and stabilize the molecule so that replacement of the disulfide bridge with an N - N bond in aminosuberic 1 - 7 eel calcitonin was found to provide increased biological stability and potency. 62 Despite this, linear analogs of sCT may retain full hypocalcemic activity and adenylate cyclase activation. 63 Circular dichroism studies indicate that sCT exhibits considerable secondary structure in the presence of lipid, consistent with the generation of an amphipathic oL-helix between residues 8 and 2 2 . 64,65 However, the less potent hCT has reduced propensity to form this secondary structure. Modifications of residues in the 8 - 2 2 sequence that alter the ability of sCT to form secondary structure have yielded conflicting results, in terms of the hypocalcemic potency of the analogs. 64-69 In some cases analogs with less secondary structure had correspondingly lower hypocalcemic activity, 65'67 whereas other analogs are fully active, 64'66'68 suggesting that conformational flexibility may also contribute to activity 67'69 and that factors other than secondary structure are also critical for ligand binding and receptor activation. Likely reasons for the conflicting results are first, the relative stability in vivo, compared with in vitro, of the various peptides; second, the use of different receptor types to assess the efficacy of the peptides; and third, a divergence between the relative potency of the peptides for

binding and activation of adenylate cyclase. These latter two points are illustrated in the following examples from our own work. The use of sCT analogs with varying capacities to form oL-helices revealed divergence in the responses of different receptors. 7~ This was most apparent for the stimulation of cyclic adenosine monophosphate (cAMP) production by the rat receptor isoforms C la and C l b (see Section VI.B). In cells expressing the C la receptor, helical analogs were equipotent with both sCT and analogs that have reduced or absent helical structure, consistent with their potencies in the rat hypocalcemic assay, and suggest that this latter function is mediated via interaction with C la receptors. In contrast, the nonhelical analogs were 100- to 1000-fold less potent than, for example, sCT at the C lb receptor. In addition, the general finding across receptors of several species was that reduction in the ability of sCT analogs to form helix structures had a greater impact on the potency of the analogs in competition for 125I-sCT binding than in cAMP accumulation. This disparity between the relative potencies of the peptides in studies of binding competition and cAMP accumulation was also seen in experiments to examine the basis of the high potency of sCT, relative to hCT, using chimeric sCT/hCT molecules. 7~These studies also highlighted the importance of the type of receptor used to study relative calcitonin peptide potencies. It was found that residues present in the carboxy-terminal half of sCT are more important for binding competition with the rat C la, rat C lb, and human CT receptors, whereas residues in the amino-terminal half of sCT are more important for binding competition with the porcine CTR.

III. BIOSYNTHESIS A. C a l c i t o n i n a n d Its P r e c u r s o r s The medullary carcinoma of the thyroid provided the source for studies of the amino acid sequence of hCT

100

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON The complete sequences of the cDNA for human, v4 rat, 75'76 chicken, 77 and sheep 78 calcitonins and the DNA sequence of the full human calcitonin gene, 79 have been determined. These show that the hormone is flanked in the precursor by N- and C-terminal peptides (Fig. 4 - 4 ) , but the biological significance of these peptides is unknown. The human calcitonin gene has been located in the p 14-qter region of chromosome 11.8~

and has allowed detailed analysis of the calcitonin gene in rat and humans. Thus calcitonin is synthesized as a large molecular-weight precursor (136 amino acids) with a leader sequence at the amino terminus, which is cleaved during transport of the molecule into the endoplasmic reticulum. The location of dibasic amino acid residues at either end of the (1-32) calcitonin sequence in the precursor molecule ensures cleavage of the prohormone to yield mature calcitonin (Fig. 4 - 4 ) . In addition to cleavage, however, it is essential, as discussed earlier, that the proline residue at position 32 is amidated. The residue immediately following the proline in the precursor is glycine, which has been suggested 71 to provide the amide group for the preceding amino acids. A potentially important posttranslational modification of calcitonin is that of glycosylation. 72 It had been noted that the tripeptide sequence, Asn-Leu-Ser, found within the amino-terminal ring structure of calcitonin (Fig. 4 3) is invariate among the calcitonins of different species. This sequence is an acceptor site for N-linked glycosylation. This, together with evidence for glycosylation of tumor calcitonin, led to detailed studies showing that the calcitonin precursor is indeed a glycoprotein 73 and that the only N-linked glycosylation site in the entire precursor was within the calcitonin portion itself. The biological significance of calcitonin glycosylation has yet to be determined.

B. C a l c i t o n i n G e n e - R e l a t e d P e p t i d e a n d A l t e r n a t i v e S p l i c i n g o f the P r i m a r y C a l c i t o n i n Gene mRNA Transcript It has been found that the calcitonin gene transcript actually encodes two distinct peptides, which arise by tissue-specific alternative splicing of the mRNA. The original observation was that serially transplanted rat medullary thyroid cancers changed from states of high to low or absent calcitonin production. 81 The low producers were found to produce different mRNA transcripts, which encoded a peptide termed "calcitonin gene-related peptide" (CGRP). The mature CGRP and calcitonin mRNAs predict proteins which share sequence identity in the amino terminal regions (Fig. 4 - 4 ) , but in the carboxy-terminal regions the nucleotide sequences

GENE 5'

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FIGURE 4--4 Organizationof rat CT/CGRP gene illustrating alternative patterns of processing of the primary transcript and subsequent protein processing. Exons are denoted E, introns are represented by a single line. Coding exons are labeled CT or CGRP. A putative intron enhancer85 is shown between exons 4 and 5.

CHAPTER 4 C

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are entirely different. The mature, secreted 32- and 37amino-acid CT and CGRP peptides, respectively, result from cleavage of both amino- and carboxy-terminal flanking sequences, at cleavage sites depicted in Figure 4 - 4 . 8z The pattem of expression of calcitonin and CGRP mRNA transcripts corresponds to the immunochemical analysis of the peptides. 83 Calcitonin mRNA is found almost exclusively in the thyroid and CGRP mRNA is found primarily in the nervous system. However, aberrant expression of CGRP may be seen in medullary thyroid carcinoma, as suggested above. Two different calcitonin/CGRP genes, oL and [3, have been identified in man and rat. 84 The calcitonin/CGRP gene was one of the first examples of a cellular gene exhibiting alternative, tissuespecific processing of its primary mRNA transcript and has served as an important paradigm to study the molecular mechanisms of RNA splicing. Processing of the pre-mRNA to the calcitonin mRNA transcript involves usage of exon 4 as a 3'-terminal exon with concomitant polyadenylation at the end of exon 4 (Fig. 4 - 4 ) . Processing to produce the CGRP mRNA involves the exclusion of exon 4 and direct ligation of exon 3 to exon 5, with polyadenylation at the end of exon 6. Much work has been done to illuminate the mechanisms by which this differential splicing is achieved, 85 although it remains to be fully elucidated. Briefly, however, the hCT/ CGRP exon 4 can be characterized as having weak processing signals, like many differential exons. Weak differential exons are frequently associated with special enhancer sequences that facilitate exon recognition in the presence of accessory factors that bind to the enhancer. 86 Indeed, such an enhancer, located in the intron downstream of exon 4, has been described for the calcitonin/ CGRP gene. In addition, sequences within exon 4 are necessary for the inclusion of exon 4. 85 Another hormone of the calcitonin family is amylin (Fig. 4 - 3 ) , a 37-amino-acid peptide produced by the pancreatic [3-cells which modulates glycogen synthesis and glucose uptake in skeletal muscle and can induce insulin resistance in this tissue in v i t r o . 87"88 Amylin has about 40% sequence homology with CGRP (Fig. 4 - 3 ) , and its gene has been localized to chromosome 12. Although amylin and CGRP have biological effects distinct from those of calcitonin, at high concentrations they may mediate calcitonin-like effects, such as inhibition of bone resorption. 89 A receptor distinct from calcitonin or CGRP receptors with high affinity for amylin has recently been identified. 39 However, it is important to note that sCT can also compete for binding with high affinity at amylin binding sites. 39 Since sCT has been the ligand primarily used for calcitonin binding and functional analysis of calcitonin action, it is possible that this has introduced a degree of ambiguity into interpretation of these studies.

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IV. SECRETION AND METABOLISM Calcitonin secretion from the C cells is clearly dependent on the prevailing serum calcium level. Any tendency toward lowering of the calcium level results in storage of calcitonin within the granules of the C cells; these stores are readily discharged as the serum calcium is elevated. Although there is little doubt that calcium is an important secretagogue for calcitonin in normal or tumor C cells, the exact mechanisms by which calcium provokes exocytosis of calcitonin have not been fully elucidated. It has recently been shown, however, that the same extracellular calcium-sensing receptor that is found in parathyroid cells, 9~ where its activation leads to decreased parathyroid hormone secretion, is also found in C cells and that its presence correlates with the extracellular calcium sensing function. 91 This calcium receptor is likely to represent the primary molecular entity through which C cells detect changes in extracellular calcium and control calcitonin release, suggesting that activation of the same receptor can either stimulate or inhibit hormone secretion in different cell types. Reduction in numbers of granules and vesicles occurred during induced calcitonin secretion in v i v o 17 and from thyroid s l i c e s , 92 but alteration of calcium levels did not influence the release of calcitonin from isolated pig thyroid granules, 93 consistent with the view that calcitonin release may be mediated at the cell membrane, and intracellular stores repleted from the storage granules as the intracellular concentration falls. Agents that elevate C cell cAMP may stimulate calcitonin secretion, since cAMP analogs have been shown to have this effect in v i v o 94 and in vitro. 95 Probably the most important calcitonin secretagogues apart from calcium, however, are the gastrointestinal hormones. In the pig, gastrin appears to be an effective physiological secretagogue, 96 providing part of the evidence that has led to a view of calcitonin's physiological role as a hormone important postprandially, capable of counteracting the effect of a calcium meal and of parathyroid hormone action by preventing the efflux of calcium from bone into blood. 97 Although there is some evidence in favor of this role in the pig and r a t , 97 it is difficult to envisage it as being important in adult humans. However, this awaits further study. Other gastrointestinal hormones, including glucagon, cholecystokinin, and secretin, are also capable of promoting calcitonin secretion. 97 The gastrin analog pentagastrin has been used clinically as a provocative test for calcitonin secretion in patients with medullary carcinoma of the thyroid. Other hormones that influence calcium homeostasis may also directly or indirectly influence calcitonin secretion. 1,25-dihydroxyvitamin D3 [1,25(OH)zD3] adminis-

102

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

tration has been reported to increase plasma calcitonin levels; this was suggested to occur via specific thyroid C cell receptors for 1,25(OH)2D3, which modify secretion of calcitonin. 98 Both calcitonin and 1,25(OH)2D3 levels are raised in pregnancy and lactation, 99 and it has been suggested that calcitonin may act to protect the skeleton in the face of increased calcium demand by the fetus. Most of this information on the regulation of calcitonin secretion comes from observations made during acute experiments with assays of calcitonin released into plasma or culture medium. The development of cDNA and oligonucleotide probes has allowed studies of the factors influencing calcitonin mRNA production. The serum and thyroid concentrations of calcitonin increase markedly with age in the rat, and this is associated with substantial increases in thyroid content of calcitonin mRNA. TM The mechanisms of this are undetermined. In the same study, TM increasing calcium concentrations did not alter hybridizable calcitonin mRNA in response to calcium. T M Thus, in normal rats subjected to acute calcium stimulation in vivo, thyroid calcitonin mRNA was increased as measured in hybridization experiments and by translatable preprocalcitonin. TM Furthermore, in a medullary thyroid carcinoma cell line, phorbol esters selectively increased calcitonin transcription while inhibiting cellular proliferation. 1~176 On current evidence it seems that calcium can stimulate both synthesis and secretion of calcitonin by thyroid C cells. Delineation of the molecular mechanisms by which translatable calcitonin is increased by calcium will be of considerable interest. Calcitonin is degraded by liver and kidney to inactive fragments, the half-life of the peptide in blood being only a few minutes. Teleost calcitonins are considerably

60

,.-d

more resistant to breakdown by tissue and serum enzymes than are the mammalian calcitonins. Injected salmon calcitonin, for example, has a much longer half life than either pig or human calcitonin. 1~ Although this might contribute to the greater biological potency in vivo of sCT, the more important factor is the greater affinity of sCT for receptors. Using the most specific and sensitive radioimmunoassays, the level of calcitonin in human blood appears to be less than 10 pg/ml in normal subjects.

V. ACTIONS OF CALCITONIN A. Bone Resorption Addition of calcitonin to resorbing bone in vitro inhibited bone resorption, 1~176 an effect that appeared to be explained by a direct action on osteoclasts, inhibiting their production and activity. Calcitonin treatment of resorbing bone in vitro resulted in rapid loss of osteoclast ruffled borders and decreased release of lysosomal enzymes. In vivo evidence was also consistent with an inhibitory action upon bone resorption. Thus, calcitonin infused into rats resulted in an immediate reduction in the rate of excretion of hydroxyproline, consistent with the action of the hormone inhibiting the breakdown of bone collagen. 1~ Furthermore, kinetic studies in rats led to similar conclusions, with no evidence to suggest any increase in the active uptake of calcium by bone. 1~176 For example, when calcitonin was infused into rats that had been injected 12 hours previously with 45Ca, hormone treatment lowered plasma calcium without affecting plasma 45Ca levels (Fig. 4 - 5 ) . Under these experi-

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CHAPTER 4

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mental conditions, disappearance of radioactivity from the plasma reflected uptake of 45Ca by the skeleton. The failure of calcitonin to influence this reflects an action of the hormone to prevent calcium efflux from bone and is not consistent with active stimulation of calcium uptake by bone. Studies of the actions of hormones on isolated bone cell populations have established that calcitonin acts directly on osteoclasts. Autoradiographic experiments using biologically active iodinated sCT as receptor ligand have pointed to osteoclasts as the only discernible bone cell targets. ~~ Consistent with this are the observations of its actions in organ culture, especially the demonstration that calcitonin-treated osteoclasts in cultured mouse calvaria rapidly lose their ruffled borders. TM A similar in vivo observation of loss of ruffled border in osteoclasts has been made in patients with Paget's disease, in w h o m bone biopsies were taken before and 30 minutes after an injection of calcitonin. 1'2 In the same clinical study, calcitonin was noted to decrease the number of osteoclasts in addition to altering their ultrastructure. Studies using isolated osteoclast preparations 1~3'114 point to a direct effect of calcitonin upon the osteoclast, in which the hormone rapidly inhibits the activity of osteoclasts. In further experiments 115 it was also noted that, while isolated osteoclasts remained quiescent in calcitonin as long as the hormone was present, they regained activity when osteoblasts were added to the culture. This escape of osteoclasts from inhibition by calcitonin took place at a rate proportional to the number of osteoblasts with which they were in contact. In other studies, Chambers showed that calcitonin reduced the cytoplasmic spreading of isolated osteoclasts in a dose-dependent manner. 115 Parathyroid hormone had no effect unless osteoblasts were co-cultivated with the osteoclasts, in which case addition of parathyroid hormone resulted in a marked increase in cytoplasmic spreading of osteoclasts. It cannot be assumed that these phenomena reflect the responses of cells in bone, but this work provided for the first time some useful direct observations of actions of hormones on isolated bone cell preparations containing osteoclasts. These observations are consistent with the view that the osteoblasts (or " l i n i n g " cells) might mediate the actions of bone-resorbing hormones by producing factors that stimulate the osteoclast 1~6 and also with the view that calcitonin acts directly upon the osteoclast. The molecular mechanisms by which calcitonin decreases osteoclast function have yet to be fully defined. The rapid effects of the hormone may be brought about through actions on a cytoskeletal function of osteoclasts, after initial events involving generation of several intracellular second messengers. Early events in calcitonin signal transduction have been studied in a variety of cell

n

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types and are described later (see Section VI.C). With the development of improved methods of studying isolated osteoclasts, it has been possible to establish clearly that mammalian osteoclasts possess abundant, specific, high-affinity receptors for calcitonin and that calcitonin stimulates cAMP formation in a sensitive and dose-dependent manner 117 as well as increases in intracellular Ca 2+ levels. 115 The other means by which calcitonin could inhibit resorption is through inhibition of osteoclast formation. In vivo data and results from calcitonin inhibition of resorption in organ culture are consistent with this. The development of methods of studying osteoclast formation in vitro from hemopoietic precursor cells 118 has allowed this question to be addressed directly. There were several reports of calcitonin inhibiting osteoclast-like cell formation in bone marrow cultures of human, 119 baboon, 12~ and mouse 118 origin. However, these experiments were all conducted at relatively high calcitonin concentrations. In recent studies using lower concentrations of calcitonin, which nevertheless reduced calcitonin receptor m R N A expression in developing mouse osteoclasts 12~ (Fig. 4 - 6 ) , there was no reduction in osteoclast formation. The multinucleated osteoclasts formed under calcitonin treatment, however, had fewer nuclei, and, notably in this and in another study, 122 evi-

FIGURE 4--6 Effectof continuous treatment of mouse bone marrow cultures with CT. Cultures were maintained for 8 days in the presence of 1,25(OH)2D3 with (+) or without (-) 10-1~ sCT. Reverse transcription/PCR of mRNA extracted at the times indicated, was amplified using primers specific for the mouse CTR sequence or murine GAPDH. In the presence of CT, osteoclast-like cells developed, which were deficient in CTR and CTR mRNA. (Data from the authors' group.)

104 dence was obtained for the generation of osteoclasts which are deficient in calcitonin receptor m R N A and protein, but nevertheless capable of resorbing bone. This observation may be relevant to the mechanism of "esc a p e " from calcitonin action, which is considered in Section VI.D of this chapter. It should be stressed that the failure of calcitonin to inhibit osteoclast formation in such osteoclast-generating cell culture systems, except perhaps at very high calcitonin concentrations, does not exclude the possibility that inhibition of osteoclastogenesis contributes to calcitonin action in vivo. The emergence in vitro of osteoclasts deficient in calcitonin receptors complicates such experiments. Although it is interesting to consider that such a phenomenon might take place also in vivo, and even contribute to calcitonin resistance, it seems unlikely to be a consistent and major feature of in vivo responses. Although there has been no demonstration of calcitonin receptors in osteoblast-like cells, or of a direct biochemical effect of the hormone upon such cells, the administration of calcitonin has been observed to produce rapid changes in osteocytes, in which osteocyte shrinkage took place and was followed by the formation of hydroxyapatite crystals in the perilacunae and pericanaliculae. ~23 The rapid changes in the bone lining cells produced by calcitonin were enhanced by phosphate, ~24 and could be related to the possible involvement of phosphate ions in the action of calcitonin, which has been argued by Talmage. 97 Most important, however, the proposal that calcitonin acts directly upon lining cells to reduce calcium efflux from bone (in direct opposition to the action of parathyroid hormone) implies that these cells should possess receptors for calcitonin, but there is no direct evidence for this. Parathyroid hormone and the other major bone-resorbing hormones have been shown to increase plasminogen activator production by osteoblast-like cells, both normal and malignant, ~25 raising the possibility of a neutral protease derived from osteoblasts contributing to the process of matrix degradation, either directly or indirectly. This increase is not influenced by calcitonin. At present there is no convincing evidence for the existence of calcitonin receptors in cells of the osteoblast lineage, but the possibility cannot be excluded that a calcitonin-responsive subpopulation exists. It is of interest to note that calcitonin receptors and c A M P response have been found to occur in late passage cultures of a parathyroid h o r m o n e - r e s p o n s i v e osteogenic sarcoma cell line that is phenotypically osteoblast. Subclones have been developed that respond to both parathyroid hormone and calcitonin. ~26 Although it is possible that such cells reflect the existence within the osteoblast series of cells capable of a calcitonin response, it is also likely that this is a property of these malignant cells, with no relevance to normal osteoblasts.

T.J. MARTIN, D. M. FINDLAY,J. M. MOSELE¥, AND E M. SEXTON B. B o n e F o r m a t i o n Although there is no good evidence for a stimulatory effect of calcitonin upon bone formation, some early evidence was obtained for a stimulatory effect on osteoblasts. In rats treated chronically with calcitonin, an increase in the number of osteoblasts was observed in the b o n e s . 127 Furthermore, calcitonin treatment in vitro of cultures of mouse radius rudiments led to an increased net amount of bone tissue that was associated with an increased number of osteoblasts, 128 leading to the suggestion that calcitonin might have a stimulatory effect on bone formation in addition to its inhibition of resorption. It is difficult to explain such observations in the light of current views of the coupling of bone resorption to formation. It is considered that any change in bone resorption is rapidly followed by a change in formation rate in the same direction. Thus, inhibition of bone resorption by calcitonin would be expected to be accompanied by inhibition of bone formation. Indeed, this is the experience, for example, in the use of calcitonin in the treatment of Paget's disease. In in vivo experiments, no effect of calcitonin was detected on the incorporation of labeled proline into bone hydroxyproline in rats chronically treated with calcitonin. 129 To the present time, therefore, it cannot be concluded that calcitonin has an anabolic effect on bone, and indeed it seems more likely that it would be "antianabolic." In normal humans, calcitonin was shown to inhibit the excretion of hydroxyproline-containing peptides, consistent with its effect of inhibiting bone resorption, but treatment also inhibited the excretion of nondialyzable hydroxyproline, which reflects bone collagen synthesis. ~3° This can be interpreted as an inhibitory effect of calcitonin upon bone collagen synthesis, accompanying the decrease in breakdown in bone. It is interesting to note that in those acute experiments in humans, ~3° calcitonin decreased urinary hydroxyproline excretion acutely after injection, but several hours later the rates of excretion returned to pretreatment levels. With continued treatment, however, there was a gradual fall in hydroxyproline excretion, such as is seen in Paget's disease patients treated chronically with calcitonin. This is interpreted as a dual action of calcitonin, on the one hand acutely inhibiting the function of osteoclasts, and on the other a chronic effect of inhibiting the generation of new osteoclasts. Calcitonin treatment of rats led to increased matrixinduced bone formation TM which appeared to be associated with a stimulated proliferation of cartilage and bone precursor cells. The effect was seen provided the calcitonin treatment was begun before cartilage formation. After that time no effect was observed. It is not clear that this can be regarded as evidence for an ana-

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bolic effect of calcitonin upon bone formation. The conclusion at present must be that there is no direct effect of calcitonin upon bone cells resulting in increased anabolic function. Rather, it seems likely that the reverse is the case, as an indirect consequence of inhibition of bone resorption by calcitonin.

C. Calcitonin, Bone, and Calcium Homeostasis It is worth considering how the discovery of calcitonin and its mechanism of action have influenced modern views of the regulation of the extracellular fluid calcium, and the contribution to this of bone. Older concepts of calcium homeostasis that considered only parathyroid hormone and bone were questioned with the suggestion that if bone were the only means of regulating the serum calcium level in conjunction with parathyroid hormone, control would be inadequate. Hence the suggestion that the parathyroid hormone action on the kidney might contribute. The arrival of a new calcium-lowering hormone seemed to solve the problem. However, events proved otherwise. Concepts of the role of bone in maintaining extracellular fluid calcium had relied upon observations made in the young, growing rat. Hence it was calculated that for the tibia in a young rat there was an accretion rate of 6.2% of the bone calcium per day, a resorption rate of 4.7% of the bone calcium per day, and an exchangeable fraction equivalent to 3.0% of the total calcium in the bone. 132 Thus it was clear that if accretion continued at the same rate and resorption was inhibited, the result would be a lowering of plasma calcium. The younger the animal, the more rapid the bone resorption rate. It would therefore be expected that the calciumlowering effect of calcitonin should be greater in younger than in older animals. This was indeed the case in the r a t 133 (Fig. 4 - 7 ) , in which it was noted that in the biological assay of calcitonin, which depends on the calcium-lowering effect of the hormone, the response became less marked with increasing age of the animals. It should be noted, however, that the ability of calcitonin to counteract the effect of a calcium load was not impaired in older animals, at least in the r a t , TM a n observation that has not been explained and that has not been extended to other species. Dependence of the hypocalcemic action of calcitonin upon the prevailing rate of bone resorption was also noted in other species, and it soon became clear that in normal adult humans even quite large doses of calcitonin had little effect on plasma calcium levels. In those subjects in whom bone turnover was increased (e.g., in thyrotoxicosis, Paget's disease), calcitonin treatment acutely inhibited bone resorption and resulted in a lowering of the plasma calcium. ~35

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It has been proposed that the hypocalcemic action of calcitonin is related to the availability of circulating inorganic phosphate. 9v In r a t s , 136 and in patients with chronic renal failure, 137 the degree of calcium lowering in response to calcitonin was found to be proportional to the initial blood phosphorus concentration. It has been claimed that no hypocalcemia followed calcitonin injection in rats maintained for several weeks on a phosphorus-deficient diet. 97 In contrast, Robinson et al. showed that in parathyroidectomized rats fed a low-phosphorus, high-calcium diet for 17 days, calcitonin lowered plasma calcium by a mechanism consistent with inhibition of bone resorption. 1~ It seems that the acute effect of calcitonin on serum calcium is related to the prevailing rate of bone resorption. If that is accepted, lack of a calcium-lowering effect of the hormone in the mature animal or human is not surprising, since the process of bone resorption is a slow one in maturity. It may be that the role of calcitonin in its effect on bone throughout life is that of a regulator of the bone resorptive process, whatever the overall rate of the latter. In the young, or in pathological states of increased bone resorption in maturity (e.g., Paget's disease, thyrotoxicosis), calcitonin inhibition of bone resorption can lower the serum calcium level, and there

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T.J. MARTIN, D. M.

may even be a calcium homeostatic role for endogenous calcitonin in those circumstances. In a normal adult animal, however, when bone turnover is slow, no effect on serum calcium is obtained with calcitonin. The physiological function of calcitonin in maturity may nevertheless be to regulate the bone resorptive process, in either a continuous or intermittent manner. It follows that calcitonin should not necessarily be regarded as a "calcium-regulating hormone" in maturity, but may yet be shown to be such in stages of rapid growth (e.g., in the young or in states of increased bone turnover). It is nevertheless important that bone resorption be regulated, and calcitonin is the only hormone known to be capable of carrying out this function by a direct action on bone. Such a role might become more important in circumstances in which skeletal loss particularly needs to be prevented (e.g., in pregnancy and lactation). 138 Evidence in support of such an important physiological role for endogenous calcitonin in protecting against bone loss is provided by the experiments of Yamomoto e t al., 139 who showed that cancellous bone loss in thyroparathyroidectomized rats treated with parathyroid hormone was greater than that in similarly treated shamoperated controls.

D. Renal Effects of Calcitonin Although calcitonin lowered plasma calcium in the absence of the kidneys, it was noted that in parathyroidectomized rats with very low levels of plasma calcium, calcitonin had no effect on calcium but lowered phosphorus. 137 When this phosphate-lowering effect was found to be prevented by nephrectomy 1~ it was considered that in some circumstances the kidneys might be involved in the phosphate-lowering effect of calcitonin. Indeed, a phosphaturic effect of thyroid extract had been shown in intact r a t s , 14~ and infusion of calcitonin in parathyroidectomized rats led to a dose-dependent phosphat u r i a . 141 The effect on phosphate excretion was only a minor one in comparison with the phosphaturic effect of parathyroid hormone, and although it was demonstrated in human subjects also, 135 in several species calcitonin failed to have any effect on phosphate excretion. Thus, it has seemed unlikely that the phosphaturic effect is of any major physiological significance. The hormone was also noted to promote excretion of inorganic sulfate in r a t s 142 and humans, ~45 probably reflecting a shared renal tubular transport system between sulfate and phosphate. A number of other renal effects of calcitonin have been noted, including a transient increase in calcium excretion,135,143-145 due probably to inhibition of renal tubular calcium reabsorption. Although this has not usually been regarded as an important effect of calcitonin,

FINDLAY,

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MOSELEY, AND

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recent observations link it to the calcium-lowering effect of calcitonin in patients with metastatic bone disease. The use of calcitonin in the treatment of hypercalcemia due to cancer has been based exclusively on the inhibition of osteolysis by calcitonin. Some evidence has been produced that failure of the kidneys to excrete the calcium load derived from bone breakdown is a major contributor to the hypercalcemia. 146 This prompted careful studies of the relative contributions to the hypocalcemic effect of calcitonin of its renal and skeletal components. 147 It was concluded that inhibition of renal tubular reabsorption by calcitonin can induce a rapid fall in serum calcium, and that the magnitude of this effect depends upon the correction of volume depletion, which inevitably accompanies hypercalcemia. Thus, the calciuretic action of calcitonin may assume greater importance than hitherto suspected. Calcitonin was found to produce a natriuretic effect in human subjects, 135''48 and study of the renal effects of calcitonin in rats pointed to striking increases in sodium excretion. 149 The effects were more marked with sCT than with calcitonins of mammalian origin, 149 raising the possibility that the natriuretic property of calcitonin from lower vertebrates might be of functional significance in those species, or that these effects were mediated by C3 receptors, which display high affinity for both sCT and amylin (see Section VI.B). Calcitonin receptors have been demonstrated clearly in rat kidney, 15~ and a further action on the kidney is to enhance 1-hydroxylation of 25-hydroxyvitamin D in the proximal straight tubule of the kidney. 15! Since autoradiographic studies ~52 and polymerase chain reaction (PCR) 152 analysis of calcitonin receptor mRNA expression have failed to localize calcitonin receptors in the proximal tubule, it seems unlikely that these actions are mediated by direct actions on calcitonin receptors. The action of calcitonin upon adenylate cyclase activity has been localized in the human nephron predominantly to the medullary and cortical portions of the thick ascending limb and to the early portion of the distal convoluted tubule. 154 The co-localization of the calcitonin receptor mRNA expression (Fig. 4 - 8 ) and cell surface receptors 152 with G-protein-sensitive adenylate cyclase is consistent with cAMP being an important mediator of calcitonin action in this organ (Fig. 4 - 8 ) . A further renal effect of calcitonin was noted as a result of studies of calcitonin effects upon a pig kidney cell line. 155 The hormone was found to stimulate greatly the production of the neutral protease, plasminogen activator. 156 This effect appeared to be related to calcitonin's actions on cAMP formation in the cells, and hormone treatment also was associated with marked inhibition of cell replication. In the same cells, calcitonin treatment enhanced the transcription of plasminogen ac-

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activation of C3 receptors, which have high affinity for amylin and sCT, but not hCT or rCT. These include inhibition of gastric acid secretion, inhibition of pancreatic enzyme secretion, and impairment of glucose tolerance.

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FIGURE 4--8 Distributionof Cla-receptor mRNAs along rat nephron. Each point represents mean value obtained from one animal (a total of 5 to 13 samples were analyzed for each structure). Dashed line indicates detection threshold of assay (50 molecules sample). (Redrawn from Firsov D, Bellanger A-C, Marsy S, et al: Quantitative RT-PCR analysis of calcitonin receptor mRNAs in the rat nephron. Am J Physiol 38:F702-F709, 1995.)

tivator mRNA, the data indicating that calcitonin was able to activate the plasminogen activator gene in those cells. 157 Subsequent studies in humans showed that calcitonin treatment resulted in increased urinary plasminogen activator activity. 158 The plasminogen activator/ plasmin system is an important local regulator of many functions, the nature of which depends on the local tissue environment. Its involvement in the local actions of calcitonin is an intriguing possibility that merits further study. The tissue effects of plasminogen activator depend on its location--thus, they are likely to be very different between kidney and bone. It is worth noting that the bone-resorbing hormones increased plasminogen activator production in osteoblast-like cells, 125 and that this effect was not influenced by calcitonin.

E. Calcitonin and the Gastrointestinal Tract It has been suggested that the function of calcitonin is to prevent rises in plasma calcium taking place after ingestion of calcium-containing m e a l s . 97'159 This is based on the observation made in pigs that calcium meals do not alter the plasma calcium, but that during the feeding period there is a rise in plasma calcitonin levels. This is a possible role for the hormone in humans, particularly in stages of growth, in which intermittent secretion in response to oral calcium loads could inhibit bone resorption and decrease the movement of calcium from bone to blood at a time when calcium was being absorbed from the intestine. However, much more study is needed in humans to define the relationship of calcitonin secretion to feeding and to gastrointestinal hormones. Other gastrointestinal effects of calcitonin are probably not physiological and, as discussed above, may relate to

F. Calcitonin in the Central Nervous System The origin of calcitonin-producing cells in the neural crest raised the possibility of calcitonin involvement in neural function. Both immunoreactive calcitonin and calcitonin receptors have been demonstrated in the brain and nervous system of rats, humans, and other species. 21'23'33 Immunoreactive calcitonin related antigenically to human calcitonin has been demonstrated in the nervous systems of protochordates, lizards, and pigeons. 21 Recent data, again obtained with radioimmunoassay, point to the presence of low levels of sCT-like peptide material in the human thyroid and in the paraventricular mesencephalic region of the brain. 16~ It has been suggested that this might be a vestige of a highly conserved gene for calcitonin. 16~ In addition to the well-characterized calcitonin receptors of bone and kidney, calcitonin receptors are also abundant in the central nervous system (CNS), 23'161-163 where central injection of calcitonin induces potent effects that include analgesia and inhibition of appetite and gastric acid secretion (reviewed in Sexton15~ Autoradiographic mapping of rat CT-binding sites revealed high densities associated with parts of the ventral striatum and amygdala, the hypothalamic and preoptic areas, as well as most of the circumventricular organs. High-density binding also occurs in parts of the periaqueductal gray, the reticular formation, most of the midline raphe nuclei, parabrachial nuclei, locus coeruleus, and nucleus of the solitary tract. 164-169 The central actions of calcitonin correlate well with the location of calcitonin-binding sites. The periaqueductal gray is important in central regulation of pain, and calcitonin binding within this region is likely to be involved in calcitonin-induced analgesia, 165 ' 170 ' 171 while the hypothalamic binding parallels the multiple hypothalamic actions of calcitonin, which include modulation of hormone r e l e a s e , 172'173 as well as decreased a p p e t i t e , 174-176 gastric acid secretion, 177'178 and intestinal motility. 179 Recently, and as also discussed in Section VI.B, cloning studies demonstrated that calcitonin receptors in rat brain are heterogeneous and exist as two distinct isof O r l T I S . 37'180 Calcitonin-specific binding sites in brain have been termed C1 sites 15~ and the two receptor isoforms are termed C la and C lb receptors. The two receptors are identical, except that the C lb sequence encodes a 37-amino-acid insert in the second extracellular domain, which confers altered ligand recognition. Both receptors

108

demonstrate high apparent affinity for sCT, but differ greatly in their affinity for hCT. C la receptors bind hCT with an apparent affinity two to three orders of magnitude less potent than sCT, whereas C lb receptors have negligible affinity for hCT. 37'38'18~PCR-based studies on the distribution of calcitonin receptor mRNA confirmed that message for both receptor isoforms is present in rat brain. 37,18~ Studies with helical and nonhelical analogs of sCT had previously suggested the existence of two potential subtypes of calcitonin-binding sites in rat brain membranes. 66 A calcitonin-linear (CT-L) type interacted with nonhelical sCT analogs with high affinity, and a calcitonin-helical (CT-H) type interacted with these analogs with a low affinity. While both types of receptors bind the helical sCT with high affinity, only the CT-L receptor binds with high affinity to hCT, which also has a reduced helical structure. The partial competition of 125I-sCT binding by hCT in the rat forebrain lsz supported the existence of multiple binding sites. These receptors are analogous to the C l a and C lb calcitonin receptor isoforms, with the specificity of interaction of the cloned receptors with helical and nonhelical sCT analogs equivalent to the CT-L and CT-H receptors, respectively. 7~ Receptor localization studies have predominantly used the fish-type calcitonins to identify receptors. 164-168'183Salmon CT, however, does not differentiate between rat C 1a and C lb receptors. Moreover, sCT also binds with high affinity to C3-amylin receptors, 39'4~ complicating interpretation of the binding distribution and its potential physiological significance. By utilizing the differences in ligand-specificity of the Cla, Clb, and C3-amylin receptors, Hilton and colleagues 51 demonstrated that distribution of C la and C lb receptors in brain was predominantly parallel. Brain regions containing C la but little or no C lb binding sites were restricted to the nucleus of the solitary tract, area postrema, and the intermediate lobe of the pituitary. In contrast, no nuclei containing exclusively C lb receptors were found, although parts of the mesencephalic and pontine reticular formation and the thalamic paraventricular nucleus were enriched in C lb receptors, relative to the density of C 1a receptors in other brain regions. The C3-amylin receptors are almost ubiquitously expressed in regions also expressing the C la receptor isoform but occur in only a subset of the nuclei labeled also with the C la-specific ligand 125IhCT. 4~ However, in restricted parts of the accumbens nucleus and fundus striati, the binding sites appear to be predominantly of the C3-receptor phenotype. 51 Only very limited structure/activity studies have been carried out in regard to the central actions of calcitonin. Indeed, in many experiments, sCT has been used to characterize either receptor localization or pharmacological action of calcitonin in the CNS. Consequently, in many

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

cases it is unclear whether the reported actions of calcitonin are mediated through the classic C 1-type calcitonin receptors or via C3-type amylin receptors of equivalent distribution. For example, although the analgesic action of calcitonin can be delineated as acting independently of C3-amylin receptors based both on receptor distribution studies and lack of an amylin-induced analgesic action, n~ the anorexic action of calcitonin may be primarily due to interaction with the C3 receptors. In support of the later assertion, the actions of amylin and calcitonin are paralleled (both are nonaversive agents) 184'185 and the mammalian calcitonins are relatively weaker at inhibiting appetite than lowering plasma calcium (a Cla-mediated action), 186'187 consistent with the specificity of C3-amylin receptors. 39'4~ Clearly, further studies need to be done to resolve the roles that the different receptors play in the central actions of calcitonin and related peptides. CGRP has been identified by immunocytochemistry in the central and peripheral nervous systems 188'189 and its receptors have been mapped in the brain, w~ It is uncertain whether CGRP has any significant effect on calcium metabolism, but its wide distribution and potent biological effects could indicate an important local function in several organ systems.

VI. CALCITONIN

RECEPTOR

Because preparation of osteoclasts suitable for biochemical study has only recently been possible, information on calcitonin receptor interactions was initially obtained from studies in other cell types. In mammals, calcitonin receptors were identified by direct binding studies in many cell types (e.g., in rat kidney, 192 human placenta, 193 human 23 and r a t 26'162'163 brain, human lymphoid cells, 194 cultured pig kidney cells, 156 human cancer cell lines derived from lung 195 and breast, 196 and a subclone from rat osteogenic sarcoma cells126). Calcitonin receptors have also been demonstrated in trout gill. w7

A. R e c e p t o r C l o n i n g Understanding of calcitonin receptor biology has undergone a dramatic change with the recent cDNA cloning of the calcitonin receptor of pig, 198 human, 58 rat, 37'18~ mouse, 199 and rabbit 2~176 origin. Based on amino acid homology, these receptors belong to a sub-family of the very large 7-transmembrane domain, G-protein-linked receptor class (Fig. 4 - 9 ) . This subfamily includes receptors for parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP), 2~ secretin, 2~ growth hormone releasing hormoneY 3 vasoactive intestinal

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polypeptide, TM glucagon-like peptide-I, 2~ pituitary adenylate cyclase activating peptide, 2~ gastric inhibitory polypeptide, 2~ and corticotropin releasing factor. 2~176 Nucleotide sequence analysis of the cloned calcitonin receptors indicated open reading frames that translate to polypeptides of around 500 amino acids, depending on the pattern of mRNA splicing. 37'58'198 Hydropathy plots indicate the presence of 7-transmembrane domains and a signal peptide at the far N-terminus. 37 There are four potential N-linked glycosylation sites in the hCTR and rCTR sequences, three of which are conserved in the porcine (p)CTR receptor. The presence of glycosylation was suggested by previous biochemical studies which indicated that the hCTR is associated with glycosyl moieties, the major contributors of which are N-acetyl-Dglucosamine residues. 21~ Posttranslational modification of the calcitonin receptor was confirmed by comparison of the predicted receptor molecular masses of--~50 kDa with those estimated from cross-linking 21~ or western blot 212 analysis, which suggest molecular masses of ---80 kDa.

B. R e c e p t o r I s o f o r m s Receptor cloning led to the discovery of calcitonin receptor isoforms (Fig. 4 - 9 ) , which arise from alternative splicing of the primary mRNA transcript and result in receptor heterogeneity. In the case of the rCTR, two isoforms were identified by cDNA cloning from a hypothalamic library. 37 These two forms, termed C la and C lb, differ structurally in that C lb contains a 37-aminoacid sequence in the second extracellular loop, which is not present in the C la form. The nomenclature here relates to that of Sexton et al., 181 who reported binding sites in rat brain, designated C1 (which bind calcitonin with high affinity), C2 (corresponding to high-affinity CGRP sites), and C3 (which interact with both peptides and which were later characterized as being high-affinity

n

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amylin receptors). 39 Functional studies of these isoforms have revealed interesting differences between C la and C lb. These isoforms displayed different ligand recognition, with the C lb form essentially not recognizing hCT o r r f T . 37'38'70 The kinetics of ligand binding were also very different; sCT binds to C la receptors essentially irreversibly, while binding to C lb receptors is rapidly and completely reversible. 38 Salmon CT activates intracellular signal transduction similarly via either receptor type (see below). Although the cloning data provided the first direct evidence for heterogeneity of the rCTR, there were some earlier data indicating that this might be the case. In studying calcitonin-binding sites in the brain, Nakamuta et a l . 66 found these to be heterogeneous with respect to recognition of analogs of calcitonin with different propensity for helix formation in solution. As discussed above (see Sections II.A and V.F), cells stably transfected with C la receptors have been used to show that helical calcitonin analogs are equipotent with analogs that have reduced or absent helical structure. In contrast, at C lb receptors, the nonhelical analogs were several orders of magnitude less potent than sCT.70 Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of rat tissues and cells, the distribution of mRNA encoding C l a and C l b receptor isoforms was determined. 37'18~ Both C l a and C l b mRNA are abundant in the hypothalamus, nucleus accumbens, cerebral cortex, and brainstem. The predominant mRNA species outside the central nervous system (e.g., in the kidney) is Cla. UMR106-06 osteogenic sarcoma cells express essentially C 1a receptor mRNA only. Osteoclasts express predominantly C l a receptor, 2~3 whereas no calcitonin receptor could be detected by RTPCR in rat calvarial osteoblasts. In general, these studies showed an unexpectedly wide tissue distribution of calcitonin receptor mRNA, and this was the case also with studies in human tissues. 214'215 The functional signifi-

FIGURE 4--9 Schematiclinear diagram illustrating known splice variations within the CTR. At least four different insertions or deletions into the coding region of the receptors have been described. (1) a 71-bp insertion into the 5' end of the receptor, which provides an upstream, in-frame, potential translation start site,2~8(2) a deletion of 125-bp located in the N-terminus of the receptor coding region, which is predicted to generate an N-terminal deletion of 47 amino acids in the mature protein,TM (3) a 48-bp insertion into the predicted first intracellular domain of the r e c e p t o r , 58'214'216 and (4) a lll-bp insertion into the predicted second extracellular domain of the r e c e p t o r . 37 e, extracellular domain; i, intracellular domain; TM, transmembrane domain.

110 cance of the calcitonin receptor in most tissues remains to be explored. It is likely that the structural variations of the rCTR species are the result of alternative splicing of the primary RNA transcript, although no classic splice consensus sequences have been found in the nucleotide sequences surrounding the insert sequence in C lb. However, Southern blot analysis results were consistent with a single rCTR gene. 37 Although the rCTR gene has not been isolated, the insert sequence of C lb does occur at an equivalent position to the junction of exons 8 and 9 in the pCTR gene. 216 The hCTR gene is located on chromosome band 7q21.2-q21.3 217 or band q22. 2~8 The mouse (m)CTR gene has been localized to the proximal region of chromosome 6,199 which is homologous to the 7q region of the human chromosome 7. The structural heterogeneity within the rCTR is also present in the mCTR, which possesses virtually the same insert in the second extracellular loop. 199 T h e pCTR gene was mapped to chromosome band 9ql 1-q12. 216 In the case of the hCTR, there is no compelling evidence for a receptor isoform corresponding to the rat and mouse C lb form, although its presence in some tissues was suggested by a minor PCR fragment in one study. 214 However, several forms of the hCTR have been identified (Fig. 4 - 9 ) . The human isoforms expressed most abundantly are, first, the form originally cloned an ovarian cancer cell line, possessing a 16-amino-acid insert in the intracellular domain58; and second, an isoform cloned from the T47D breast cancer cell line, in which the insert sequence is absent. 214 The insert-negative receptor appears to be much more abundant in most tissues than the insert-positive receptor; however, transcripts encoding the latter are well represented in ovary and placenta, 214 suggesting tissue-specific regulation of this receptor isoform. Our own studies, 219 and those by other groups, 217'218'22~have now investigated the functional significance of the receptor isoforms. We found that the binding affinity and kinetics were identical for the two forms. In contrast, the presence of the insert greatly reduced the ability of the receptor to couple to adenylate cyclase, with EDs0 values 100-fold higher than those of the insert-negative form. Calcitonin-induced stimulation of a transient intracellular Ca 2+ response, via activation of phospholipase C, was abolished for the insert-positive receptor isoform. 219 The rate of ligand-induced internalization of the insert-positive form of the receptor was significantly impaired relative to the insert-negative form, suggesting that this process may be dependent on intracellular signaling. 219 Unlike the pig calcitonin receptor gene, where the 16-amino-acid insert sequence was contiguous with exon 8, 216 nucleotide sequence encoding the 16-amino-acid insert in the hCTR has been located in a separate exon of the hCTR gene. 219'22~ An additional

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

hCTR isoform has been described in which the first 47 amino acids of the amino-terminal extracellular domain are truncated. 222 This transcript represents a minority mRNA species in many of the tissues expressing the fulllength calcitonin receptor mRNA. Comparison of the truncated and full-length molecules by transfection revealed similar binding constants as well as calcitoninmediated cAMP production. 222 Sequencing of cDNA clones from a giant cell tumor revealed the presence or absence of a 71-bp insert in the 5'-untranslated region of the mRNA. 218 The functional significance of this sequence remains unclear. In addition to intraspecies variants of the calcitonin receptor, there are primary structural differences between species, which are likely to influence tertiary structure and hence ligand recognition or signal transduction. Indeed, and as discussed above, studies to date have revealed several dramatic differences in the ligand recognition of the rCTR hCTR and pCTRs. 7~ For example, sCT and hCT were almost equipotent in activating the hCTR, while hCT was essentially inactive at the pCTR. Analysis of the predicted amino acid sequences of the pig, human, and rat receptors revealed 78% identity between the human receptor cloned from BIN 67 ovarian cancer cells 58 and the rat C l a receptor, 37 and 67% identity between the pig receptor cloned from LLC-PK1 cells 198 and the rat C la receptor. The availability of a large number of calcitonins and analogs continues to facilitate detailed investigation of functional differences among calcitonin receptors, now that it is possible to investigate properties of expressed recombinant receptors. Already there are a number of studies from which it is possible to derive a working model of calcitonin/calcitonin receptor interactions. Studies using receptor chimeras of hCTR and glucagon receptor provided evidence for a two-site hormonereceptor interaction model, the data suggesting that both the N-terminal extracellular extension and the extracellular loops cooperate to bind calcitonin, while activation of signaling is primarily via interaction with the extracellular loops. 212 Supportive of this model are the results of experiments using CTR/PTHR receptor chimeras together with CT/PTH peptide chimeras. This work suggested that the N-terminus of the calcitonin molecule interacts with the extracellular loops and/or the transmembrane domains to activate the receptor, while binding is stabilized by interactions between C-terminal portions of the calcitonin molecule and the extracellular extension of the receptor. 223 To date there are few studies to elucidate the structure/function of intracellular domains of the calcitonin receptor. As discussed above, the naturally occurring insert in the first intracellular loop of the hCTR blocks calcitonin-induced activation of phospholipase C and subsequent increases in intracellular Ca 2§ levels. 219 Deletion of the C-terminal intracellular

CHAPTER4 Calcitonin tail of the pCTR interfered with calcitonin-induced internalization of this receptor in transfected cells. TM

C. Signal Transduction Early experiments showed that osteoclast-rich cultures derived from mouse calvariae 2z5 show a rise in cAMP formation in response to calcitonin. Confirmation that the osteoclast is a direct target for calcitonin, and that calcitonin acts in these cells to increase cAMP levels, came from experiments using highly enriched rat osteoclasts 226 or human osteoclastoma cells. 227 Moreover, both dibutyryl cAMP 228 and forskolin, which directly activate adenylate cyclase, 229 inhibit bone resorption. Calcitonin was shown to increase adenylate cyclase activity in the medullary and cortical portions of the thick ascending limb of Henle's loop, in the early portion of the distal convoluted tubule, and to some extent in the collecting tubule. 154 Interestingly, recent quantitative RTPCR confirmed the selective expression of calcitonin receptor mRNA in the same regions of the nephron previously shown to be calcitonin responsive, ~53 which was consistent with our autoradiographic localization of renal calcitonin receptor. 152 Calcitonin induction of cAMP has now been documented in a large number of calcitonin receptor-beating cell lines in culture including LLC-PK~ pig kidney cells 23~ and cancers of lung, ~95 breast, 196 and bone, 126 the evidence suggesting that coupling of the calcitonin receptor to adenylate cyclase activation occurs via receptor interaction with the G proteins of the G~s family. It is now clear that many of the G-protein-coupled receptors interact with multiple G proteins. In the case of the calcitonin receptor, there is ample evidence that calcitonin may also induce increases in cytoplasmic calcium concentrations ([Ca2+]i). For example, it is apparent that in the osteoclast, signaling through both cAMP and changes in [Ca2+]i are important in calcitonin action. TM Although controversial and potentially species specific, retraction of osteoclasts by calcitonin, and calcitonin-induced cytoskeletal changes, at least in the mouse, appear to be mainly mediated by the protein kinase A pathway. 232 On the other hand, inhibition of osteoclastic bone resorption by calcitonin can be mimicked by both dibutyryl cAMP and phorbol esters or blocked by protein kinase C inhibitors. 233 These results suggest that alternative G protein coupling can mediate calcitonin activation of either adenylate cyclase or phospholipase C, leading in the latter case to raised intracellular inositol triphosphate levels and thence increased [Ca2+], which together with the co-liberated diacylglycerol, activate protein kinase C. Calcitonin apparently causes either no change, or an inhibition of adenylate cyclase activity, in

111 brain tissue. 234'e35 Despite this, calcitonin receptors cloned from brain are capable of coupling to G~s protein in other cell types. 37'18~ In hepatocytes, calcitonin-induced activation of adenylate cyclase has not been shown, but calcitonin, even at very low concentrations, is capable of increasing [Ca2+]i .236 There is a recent report that calcitonin prevented CCl4-induced oxyradical formation and cellular damage in hepatocytes by a C lb receptor-mediated activation of protein kinase C . 237 In LLC-PK~ pig kidney cells, calcitonin can, in a cell cycle-dependent manner, induce changes mediated by either cAMP or [cae+]i .e38 Finally, expression of cloned receptors in a variety of cell types has conclusively shown that calcitonin receptors of human, 2~4 rat, 28 and porcine 239'e4~origin are capable of signaling through both cAMP- and CaZ+-activated second-messenger systems. It is important to note that comparison of the calcium response in cell lines expressing different calcitonin receptor levels has suggested that the magnitude of the response is proportional to the receptor density. 241 This relationship has been clearly shown for the TSH 242 and PTH/PTHrP receptors 243 and it is possible that relative receptor density in target tissues may influence the signaling pathway(s) activated. Calcitonin-induced [cae+]i fluxes are rapid and sustained in the presence of extracellular calcium. TM In the absence of extracellular calcium, the sustained phase is not seen. These results suggest that the initial response is mediated by inositol 1,4,5-triphosphate (IP3), which stimulates the release of calcium from intracellular stores. Calcitonin stimulation of IP3, probably generated by Gq-mediated activation of phospholipase C, has been shown in the case of cloned pCTR 239'24~ and hCTRs. 214 The sustained phase of the Ca 2+ response is dependent on extracellular calcium, and current evidence, as discussed below, suggests that this is mediated by calcium inflow through plasma membrane calcium channels. An interesting "calcium-sensing" function of the hCTR was recently reported, whereby calcitonin receptor-bearing cells were rendered sensitive to extracellular calcium in terms of increased [Ca2+]i .241 Although initially reported to be independent of calcitonin, subsequent work in cells transfected with the hCTR, 245 rCTR, and pig CTRs TM showed that sensitivity to extracellular calcium is in fact dependent upon preexposure of cells to calcitonin. Thus calcitonin treatment of calcitonin receptor-bearing cells, in the presence of extracellular calcium, causes a sustained rise in [Ca2+]i, the extent of which is dependent on the concentration of the extracellular calcium. Since osteoclasts, which express high levels of calcitonin receptor, ~7 are reportedly exposed to calcium concentrations as high as 26 mM during bone resorption, 246 this phenomenon may have par-

1 12 ticular relevance for this cell type. In isolated osteoclasts, calcitonin and extracellular C a 2+ both produce intracellular C a 2+ transients. 247 Interestingly, calcitonin and [CaZ+]e greatly potentiate the signal produced by either agent alone. 248 The mechanisms underlying this phenomenon are not yet known but two possibilities are: (1) that this is analogous to the receptor-activated calcium entry seen for a number of other receptor systems where calcium inflow apparently results from emptying of intracellular calcium stores 249 and (2) that this involves a mechanism independent of these events, since there is now evidence that calcium "sensing" results from lower concentrations of calcitonin than those required to produce intracellular calcium transients. 245 It is becoming recognized that signals generated by cell surface receptors of various classes are subject to modulation by other receptors. An intriguing example of this "cross-talk" is a finding in osteoclasts that calcitonin can down-regulate the cell signals induced by a fragment of the bone extracellular matrix molecule, bone sialoprotein (BSP). 25~ BSP and osteopontin, a related molecule also found in bone extracellular matrix, can both bind to osteoclasts, via the OLv[33integrin receptor, and induce intracellular C a 2+ mobilization. TM Calcitonin was also shown to inhibit osteopontin mRNA in isolated rabbit osteoclasts. 252 Given that BSP and/or osteopontin can act as attachment molecules for osteoclasts, 253 reduction of osteopontin formation by calcitonin could be one means by which calcitonin inhibits osteoclast activity and thus bone resorption. Calcitonin can potently modulate the growth of some calcitonin receptor-beating cells. Calcitonin was shown to stimulate the growth of human prostate cancer cells, in which calcitonin increases intracellular C a 2+ and cAMP levels. TM On the other hand, calcitonin treatment inhibited the growth of T47D human breast cancer cells, an action believed to be mediated by the specific activation of the type II isoenzyme of the cAMP-dependent protein kinase. 255 Recently, several points of intersection of cAMP-mediated pathways and those of growth factor-stimulated proliferation pathways have been identified, 256'257 although none of these intracellular mechanisms have yet been explored with respect to calcitonin. Recent reports also support a role for Ca2+-ac tivated pathways in growth modulation, with depletion of intracellular C a 2+ stores by thapsigargin being implicated in inhibition of cell growth 258 while mitogenesis by thrombin and bradykinin appeared to involve Gq-mediated increases in [Ca2+]i .259 Again, the role of these pathways in calcitonin-mediated growth modulation remains to be determined, but the above considerations are of potential importance, given the demonstrated inhibition of growth, by calcitonin, of human breast cancer cells. 255

Y.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

D. Receptor Regulation: The "Escape" Phenomenon The molecular and cellular biology of the calcitonin receptor in osteoclasts has been amenable to study following the development of a means to derive osteoclasts in vitro, 118 in addition to the cloning of the calcitonin receptor. In osteoclasts generated in mouse bone marrow cultures treated with 1,25(OH)2D3, as well as in freshly isolated osteoclasts from newborn rat, the calcitonin receptor and calcitonin receptor mRNA are abundantly expressed. 213 In this cell type the C la receptor was found to be the isoform most abundantly expressed, with C lb detectable in lesser amounts. 213 In mouse marrow cultures, calcitonin receptor mRNA could be detected by RT-PCR very early, before receptor detection by autoradiography. Furthermore, at a stage when only very few calcitonin receptor-positive cells were seen against a background of mainly stromal cells in these cultures, it was even possible to detect calcitonin receptor m R N A by the less sensitive method of Northern blot hybridization. This observation implies that the developing osteoclasts in the marrow cultures are very rich in calcitonin receptor mRNA. The calcitonin receptor has been shown to be subject to both homologous and heterologous regulation, the latter by a number of factors including glucoc o r t i c o i d s , 26~ activators of protein kinase C, 262 and transforming growth factor ~.263 Calcitonin-induced down-regulation of the calcitonin receptor was first demonstrated in various transformed cell l i n e s , 264'266 and later in primary cultures of kidney cells. 267 Receptor loss from the cell surface was shown to be due to an energydependent cellular internalization of the ligand-receptor complex. 268 The molecular mechanisms involved in these events remain to be fully elucidated, but appear to be cell type dependent. As noted for the PTH/PTHrP receptor, 269 ligand-induced phosphorylation of the hCTR has recently been demonstrated 27~ and may be important in receptor regulation. Preincubation of calcitonin receptor-beating cell lines with calcitonin resulted also in persistent activation of adenylate cyclase, which was shown to be due to persistent occupancy of cell-surface receptors by poorly dissociable sCY. 271'272 Calcitonin treatment also resulted in desensitization of the cells to a subsequent challenge with calcitonin, which was thought to be due to receptor loss from the cell surface and possibly to an uncoupling of the remaining receptors from signal t r a n s d u c t i o n . 271'272 Although calcitonin inhibits bone resorption, it has been found in vitro and in vivo that calcitonin inhibition is followed by " e s c a p e . ''273'274 " E s c a p e " is defined as an increase in resorption in bones stimulated to resorb

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by a resorptive agent, despite the continued presence of concentrations of calcitonin that were maximally inhibitory. Furthermore, rats treated chronically with calcitonin become refractory to the hypocalcemic action of the peptide) 75 Data from the in vitro experiments suggested that "escape" was due to a change in responsiveness to calcitonin rather than a loss of activity of the hormone. An interesting feature of the phenomenon is that the development of escape in vitro can be prevented by concomitant treatment of the bones w i t h glucocorticoid, 276 which more recent experiments indicate might be due to antagonism by glucocorticoid of calcitonin-induced calcitonin receptor down-regulation in osteoclasts. 261 The phenomenon of "escape" is such an integral part of calcitonin action that it has to be borne in mind whenever the hormone is being used therapeutically. It helps to explain why calcitonin is less effective than might be expected in the treatment of such states of excessive bone resorption as hyperparathyroidism and malignant hypercalcemia. The biochemical and cellular mechanisms by which calcitonin induces refractoriness to its own action have been the subject of intense study in recent years. Studies of osteoclasts in c u l t u r e 276-278 have shown that calcitonin treatment results in down-regulation of the calcitonin receptor, which is nevertheless different from that seen in other cell types in that down-regulation is considerably prolonged. 276 These results were consistent with the hypothesis that the resistance to calcitonin that typifies the clinical phenomenon of "escape" might be due to reduced calcitonin sensitivity of osteoclasts. Evidence for this had previously been obtained by elegant experiments in bone organ culture. TM We have shown that either s h o r t 276'279 o r continuous 121 treatment of osteoclasts with calcitonin results in a rapid and prolonged downregulation of calcitonin mRNA, whereas in the nonosteoclastic human breast cancer T47D cells, there was no decrease in calcitonin receptor mRNA levels, z76 Significantly, calcitonin-treated osteoclasts regained the ability to resorb bone, 122 again suggesting that functional, calcitonin-resistant osteoclasts result from exposure to pharmacological doses of calcitonin. It was also found that in the continuous presence of calcitonin, resorptive osteoclast-like cells, that had very low levels of calcitonin receptor mRNA, formed in mouse bone marrow cultures TM (Fig. 4-6). These results, therefore, indicate that calcitonin-induced decreased responsiveness of osteoclasts to calcitonin is at least partly due to suppression of calcitonin receptor and its mRNA in existing osteoclasts and to the development of newly formed osteoclasts that have very low levels of calcitonin receptor. It is interesting to note that in bone organ culture, "escape" was prevented by irradiation, which would inhibit proliferation of osteoclast precursors, 28~ and that "es-

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cape" was accompanied by the formation of small, newly formed active osteoclasts. TM Down-regulation of calcitonin receptor and of calcitonin receptor mRNA in mature mouse osteoclasts, but not in nonosteoclastic cells, appears to be mediated by activation of cAMPdependent protein kinase. 282

VII. CALCITONIN IN CLINICAL MEDICINE The discussions in this chapter of the mechanism of action of calcitonin provide background to the use of calcitonin in therapy. Its specific uses are considered in detail elsewhere in this volume, but one of the most important unanswered questions concerning the role of calcitonin in physiology, pathology, and therapeutics is whether its role as an inhibitor of bone resorption is such that calcitonin deficiency can lead to the development of osteoporosis. The corollary would be that calcitonin could be used in the treatment of osteoporosis. Initial reports that basal calcitonin levels are lower in women than in men, 283'284 fall with age in both, 285 and are reduced in osteoporotic women 286 gave rise to considerable interest in the possible use of calcitonin in the treatment of postmenopausal osteoporosis. However, enthusiasm has been tempered by subsequent data showing no difference in basal calcitonin levels between postmenopausal osteoporotic and age-matched normal w o m e n . 287'288 Calcitonin has an established place in the treatment of Paget's disease of bone, and is increasingly used in osteoporosis, where its effectiveness in nasal spray form has been established. 289

VIII. SUMMARY Calcitonin is a polypeptide hormone whose major recognized effect in mammals, including humans, is to inhibit bone resorption. This it does by acutely inhibiting osteoclast activity, and perhaps also by inhibiting the generation of osteoclasts, although the in vitro evidence for the latter is conflicting. Because bone resorption is rapid enough to contribute to the maintenance of extracellular fluid calcium only in stages of growth or in certain disease states in maturity, in which bone resorption is increased, it follows that only in those circumstances does calcitonin injection result in a significant fall in plasma calcium. Thus, although calcitonin was discovered as a calcium-lowering hormone in experiments that were carried out in young animals, it may be that its function throughout life is that of a regulator of the bone resorption process. This might not necessarily contribute

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T . J . MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

to acute maintenance of plasma calcium in maturity. Consideration of the "physiological" role of calcitonin often fails to take into account the need to regulate the bone resorptive process, however slowly that may be proceeding, and whether or not it contributes to maintenance of the plasma calcium level. Thus, the general role of calcitonin in skeletal metabolism may indeed be that of the most important inhibitor of the bone resorptive process, acting to prevent excessive skeletal loss throughout life, and especially at times at which skeletal loss becomes a risk, for example, in rapid growth, pregnancy and lactation, immobilization, and certain pathological states (Paget's disease of bone, thyrotoxicosis, hyperparathyroidism). This working model of calcitonin action and function can be applied to any discussion of the hormone's role in therapy or in pathogenesis of bone disease. The last few years have opened fascinating new aspects of calcitonin physiology. This includes particularly the cloning of the calcitonin receptor and the finding of a number of functionally different calcitonin receptor isoforms, the discovery of calcitonin and of calcitonin receptor sites in the brain, leading to the possible role of calcitonin as a neuropeptide. The most interesting areas we have to address now are the physiological significance of the receptor isoforms, the details of calcitonininduced intracellular signaling, and the potential role of calcitonin in cell growth and differentiation.

Acknowledgments These authors acknowledge the support of The National Health and Medical Research Council of Australia and The Anti-Cancer Council of Victoria in funding their work. Their thanks are also extended to Mrs. A. Carruthers for her help in the preparation of the manuscript.

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199. Yamin M, Gom AH, Flannery MR, et al: Cloning and characterization of a mouse brain calcitonin receptor complementary deoxyribonucleic acid and mapping of the calcitonin receptor gene. Endocrinology 135:2635-2643, 1994. 200. Shyu J-F, Baron R, Home WC: Rabbit osteoclasts express two isoforms of the calcitonin receptor, one of which is novel. J Bone Miner Res 10:$486, 1995. 201. Jtippner H, Abou Samra AB, Freeman M, et al: A G proteinlinked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 254:1024-1026, 1991. 202. Ishihara T, Nakamura S, Kaziro Y, et al: Molecular cloning and expression of a cDNA encoding the secretin receptor. EMBO J 7:1635-1641, 1991. 203. Mayo KE: Molecular cloning and expression of a pituitaryspecific receptor for growth hormone-releasing hormone. Mol Endocrinol 6:1734-1744, 1992. 204. Sreedharan SP, Robichon A, Peterson KE, et al: Cloning and expression of the human vasoactive intestinal peptide receptor. Proc Natl Acad Sci USA 88:4986-4990, 1991. 205. Wheeler MB, Lu M, Dillon JS, et al: Functional expression of the rat glucagon-like peptide-I receptor, evidence for coupling to both adenylyl cyclase and phospholipase-C. Endocrinology 133:57-62, 1993. 206. Spengler D, Waeber C, Pantaloni C, et al: Differential signal transduction by five splice variants of the PACAP receptor. Nature 365:170-175, 1993. 207. Usdin TB, Mezey E, Button DC, et al: Gastric inhibitory polypeptide receptor, a member of the secretin-vasoactive intestinal peptide receptor family, is widely distributed in peripheral organs and brain. Endocrinology 133:2861-2870, 1993. 208. Chen R, Lewis KA, Perrin MH, et al: Expression cloning of a human corticotropin-releasing factor receptor. Proc Natl Acad Sci USA 90:8967-8971, 1993. 209. Chang C, Pearse RVI, O'Connell S, et al: Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 11:11871195, 1993. 210. Moseley JM, Findlay DM, Gorman JJ, et al: The calcitonin receptor on T47D breast cancer cells. Evidence for glycosylation. Biochem J 212:609-616, 1983. 211. Moseley JM, Findlay DM, Martin TJ, et al: Covalent crosslinking of a photoactive derivative of calcitonin to human breast cancer cell receptors. J Biol Chem 257:5846-5851, 1982. 212. Stroop SD, Kuestner RE, Serwold TF, et al: Chimeric human calcitonin and glucagon receptors reveal two dissociable calcitonin interaction sites. Biochemistry 34:1050-1057, 1995. 213. Ikegame M, Rakopoulos M, Zhou H, et al: Calcitonin receptor isoforms in mouse and rat osteoclasts. J Bone Miner Res 10: 5 9 - 6 5 , 1994. 214. Kuestner RE, Elrod RD, Grant FJ, et al: Cloning and characterization of an abundant subtype of the human calcitonin receptor. Mol Pharmacol 46:246-255, 1994. 215. Kenny MA, Moore CX, Pittner R, et al: Salmon calcitonin binding and stimulation of cyclic AMP generation in rat skeletal muscle. Biochem Biophys Res Commun 197:8-14, 1993. 216. Zolneirowicz S, Cron P, Solinas-Tolda S, et al: Isolation, characterization and chromosomal localization of the porcine calcitonin receptor gene. J Biol Chem 269:19530-19538, 1994. 217. Nussenzveig DR, Mathew S, Gershengom MC: Altemative splicing of a 48-nucleotide exon generates two isoforms of the human calcitonin receptor. Endocrinology 136:2047-2051, 1995. 218. Gom AH, Rudolph SM, Flannery MR, et al: Expression of two skeletal calcitonin receptor isoforms cloned from a giant

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120 234. Koida M, Yamamoto Y, Nakamuta H, et al: A novel effect of salmon calcitonin on in vitro Ca-uptake by rat brain hypothalamus: The regional and hormonal specificities. Jpn J Pharmacol 32:981-986, 1982. 235. Shah GV, Kennedy D, Dockter ME, et al: Calcitonin inhibits thyrotropin-releasing hormone-induced increases in cytosolic Ca 2§ in isolated rat anterior pituitary cells. Endocrinology 127: 613-620, 1990. 236. Yamaguchi M: Stimulatory effect of calcitonin on Ca 2§ inflow in isolated rat hepatocytes. Mol Cell Endocrinol 75:65-70, 1991. 237. Chen S, Morimoto S, Tamatari M, et al: Calcitonin prevents CCl4-induced hydroperoxide generation and cytotoxicity possibly through C l b receptor in rat hepatocytes. Biochem Biophys Res Commun 218:865-871, 1996. 238. Chakraborty M, Chatterjee D, Kellokumpe S, et al: Cell cycledependent coupling of the calcitonin receptor to different G proteins. Science 251:1078-1082, 1991. 239. Force T, Bonventre JV, Flannery MR, et al: A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C. Am J Physiol 2 6 2 : F l l 1 0 - F l l 1 5 , 1992. 240. Chambre O, Conklin BR, Lin HY, et al: A recombinant calcitonin receptor independently stimulates 3'-5' cyclic adenosine monophosphate and CaZ§ phosphate signaling pathways. Mol Endocrinol 6:551-556, 1992. 241. Stroop SD, Thompson DL, Kuestner RE, et al: A recombinant human calcitonin receptor functions as an extracellular calcium receptor. J Biol Chem 268:19927-19930, 1993. 242. Gershengorn MC, Heinflink M, Nussenzveig DR, et al: Thyrotropin releasing hormone (TRH) receptor number determines the size of the TRH-responsive phosphoinositide pool: Demonstration using controlled expression of TRH receptors by adenovirus mediated gene transfer. J Biol Chem 269:6779-6783, 1994. 243. Bringhurst FR, Jtippner H, Guo J, et al: Cloned, stably expressed parathyroid hormone (PTH)/PTHrP-related peptide receptors activate multiple messenger signals and biological responses in LLC-PK1 kidney cells. Endocrinology 132:2090-2098, 1993. 244. Findlay DM, Houssami S, Sexton PM, et al: Calcium inflow in cells transfected with cloned rat and porcine receptors. Biochem Biophys Acta 1265:213-219, 1995. 245. Stroop SD, Moore EE: Intracellular calcium increases mediated by a recombinant human calcitonin receptor. J Bone Miner Res 10:524-532, 1995. 246. Silver IA, Murrils RJ, Etherington DJ: Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp Cell Res 175:266-276, 1988. 247. Zaidi M, Shankar VS, Bax CMR, et al: Linkage of extracellular and intracellular control of cytosolic Ca 2§ in rat osteoclasts in the presence of thapsigargin. J Bone Miner Res 8:961-967, 1993. 248. Malgaroli A, Meldolesi J, Zambonin Zallone A, et al: Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin. J Biol Chem 264: 14342-14347, 1989. 249. Putney JW: A capacitative model for receptor-activated calcium entry. Adv Pharmacol 22:251-269, 1991. 250. Paniccia R, Riccioni T, Zani BM, et al: Calcitonin downregulates immediate cell signals induced in human osteoclastlike cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism. Endocrinology 136:1177-1186, 1995. 251. Miyauchi A, Alvarez J, Greenfield EM, et al: Recognition of osteopontin and related peptides by an ctv[33 integrin stimulates immediate cell signal in osteoclasts. J Biol Chem 266:2036920374, 1991.

T . J . MARTIN, D. M. FINDLAu J. M. MOSm.Eu AND P. M. SEXTON 252. Kaji H, Sugimoto T, Miyauchi A, et al: Calcitonin inhibits osteopontin mRNA expression in isolated rabbit osteoclasts. Endocrinology 135:484-487, 1994. 253. Flores ME, Norgard M, Heinegard D, et al: RGD-directed attachment of isolated rat osteoclasts to osteopontin, bone sialoprotein, and fibronectin. Exp Cell Res 201:526-530, 1991. 254. Shah GV, Rayford W, Noble MJ, et al: Calcitonin stimulates growth of human prostate cancer cells through receptor-mediated increase in cyclic adenosine 3',5'-monophosphate and cytoplasmic Ca z+ transients. Endocrinology 134:596-602, 1994. 255. Ng KW, Livesey SA, Larkins RG, et al: Calcitonin effects on growth and on selective activation of type II isoenzyme of cyclic adenosine 3':5'-monophosphate-dependent protein kinase in T47D human breast cancer cells. Cancer Res 43:794-800, 1983. 256. Wu J, Dent P, Jelinek T, et al: Inhibition of EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science 262:1065-1069, 1993. 257. Cook SJ, McCormick F: Inhibition by cAMP of Ras-dependent activation of Raf. Science 262:1069-1072, 1993. 258. Short AD, Bian J, Ghosh TK, et al: Intracellular Ca z§ pool content is linked to control of cell growth. Proc Natl Acad Sci USA 90:4986-4990, 1993. 259. LaMorte VJ, Harootunian AT, Spiegel AM, et al: Mediation of growth factor induced DNA synthesis and calcium mobilization by Gi and Gi2. J Cell Biol 121:91-99, 1993. 260. Kurokawa M, Michelangeli VP, Findlay DM: Induction of calcitonin receptor expression by glucocorticoids in T47D human breast cancer cells. J Endocrinol 130:321-326, 1991. 261. Wada S, Akatsu T, Tamura T, et al: Glucocorticoid regulation of calcitonin receptor in mouse osteoclast-like multinucleated cells. J Bone Miner Res 9:1705-1712, 1994. 262. Findlay DM, Michelangeli VP, Robinson PJ: Protein kinase-Cinduced down regulation of calcitonin receptors and calcitoninactivated adenylate cyclase in T47D and BEN cells. Endocrinology 125:2656-2663, 1989. 263. Schneider H-G, Michelangeli VP, Frampton RJ, et al: Transforming growth factor-J3 modulates receptor binding of calciotropic hormones and G protein-mediated adenylate cyclase responses in osteoblast-like cells. Endocrinology 131:1383-1389, 1992. 264. Findlay DM, deLuise M, Michelangeli VP, et al: Independent down-regulation of insulin and calcitonin receptors on a human tumour cell line. J Endocrinol 88:271-281, 1981. 265. Findlay DM, Martin TJ: Relationship between internalization and calcitonin-induced receptor loss in T47D cells. Endocrinology 115:78-83, 1984. 266. Findlay DM, Martin TJ: Kinetics of calcitonin receptor internalization in lung cancer (BEN) and osteogenic sarcoma (UMR 106-06) cells. J Bone Miner Res 1:277-283, 1986. 267. Schneider H-G, Raue F, Bollenbach N, et al: Internalization of calcitonin receptors in primary rat kidney cell cultures. Acta Endocrinol (Copenh) 122:255-262, 1990. 268. Findlay DM, Ng KW, Niall M, et al: Processing of calcitonin and epidermal growth factor after binding to receptors in human breast cancer cells (T47D). Biochem J 206:343-350, 1982. 269. Blind E, Bambino T, Nissenson RA: Agonist-stimulated phosphorylation of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein. Endocrinology 136: 4271-4277, 1995. 270. Nygaard SC, Kuestner RE, Moore EE, et al: Phosphorylation of the calcitonin receptor localized at the C-terminus. J Bone Miner Res 10:S141, 1995. 271. Lamp SJ, Findlay DM, Moseley JM, et al: Calcitonin induction of a persistent activated state of adenylate cyclase in human

CHAPTER 4

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breast cancer cells (T47D). J Biol Chem 256:12269-12274, 1981. Michelangeli VP, Findlay DM, Moseley JM, et al: Mechanisms of calcitonin induction of prolonged activation of adenylate cyclase in human cancer cells. J Cyclic Nucleotide Prot Phos Res 9:129-142, 1983. Raisz LG, Wener JA, Trummel CL, et al: Induction, inhibition and escape as phenomena in bone resorption. Excerpta Medica Int Congr Series No. 243:446-453, 1972. Tashjian AH Jr, Wright DR, Ivey JL, et al: Calcitonin binding sites in bone: Relationship to biological response and "escape." Recent Prog Horm Res 34:285-334, 1978. Messer HH, Copp DH: Changes in response to calcitonin following prolonged administration to intact rats. Proc Soc Exp Biol Med 146:643-647, 1974. Wada S, Martin TJ, Findlay DM: Homologous regulation of the calcitonin receptor in mouse osteoclast-like cells and human breast cancer T47D cells. Endocrinology 136:2611-2621, 1995. Takahashi S, Goldring S, Katz M, et al: Downregulation of calcitonin receptor mRNA expression by calcitonin during human osteoclast-like cell differentiation. J Clin Invest 95:167-171, 1995. Lee SK, Goldring SR, Lorenzo JA: Expression of the calcitonin receptor in bone marrow cell cultures and in bone: A specific marker of the differentiated osteoclast that is regulated by calcitonin. Endocrinology 136:4572-4581, 1995. Rakopoulos M, Ikegame M, Findlay DM, et al: Short treatment of osteoclasts in bone marrow culture with calcitonin causes prolonged suppression of calcitonin receptor mRNA. Bone 17: 4 47- 453, 1995.

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280. Kreiger NS, Feldman RS, Tashjian AH: Parathyroid hormone and calcitonin interactions in bone: Irradiation-induced inhibition of escape in utero. Calcif Tissue Int 34:197-203, 1982. 281. Klaushofer K, Horandner H, Hoffman O, et al: Interferon ~/and calcitonin induce differential changes in cellular kinetics and morphology of osteoclasts in cultured neonatal calvaria. J Bone Miner Res 4:585-606, 1989. 282. Wada S, Udagawa N, Martin TJ, et al: Physiological levels of calcitonin regulate the mouse osteoclast calcitonin receptor by a protein kinase A-mediated mechanism. Endocrinology 137: 3 1 2 - 320, 1996. 283. Heath H, Sizemore GW: Plasma calcitonin in normal man. Differences between men and women. J Clin Invest 60:1135-1140, 1977. 284. Hillyard CJ, Stevenson JC, Maclntyre I: Relative deficiency of plasma-calcitonin in normal women. Lancet 1:961-962, 1978. 285. Deftos LJ, Weisman MH, Williams GW, et al: Influence of age and sex on plasma calcitonin in human beings. N Engl J Med 302:1351-1353, 1980. 286. Milhaud G, Benezech-Le Fevre M, Moukhtar M: Deficiency of calcitonin in age-related osteoporosis. Biomedicine 29:272-276, 1978. 287. Chestnut CH, Baylink DJ, Sisom K, et al: Basal plasma immunoreactive calcitonin in postmenopausal osteoporosis. Metabolism 29:559-562, 1980. 288. Tiegs RD, Body J-J, Rolfe J, Heath H: Do calcitonin levels decrease with age? Reassessment with a new technique. Calcif Tissue Int 36:479 (abstract), 1984. 289. Azaria M, Copp DH, Zanelli JM: 25 Years of salmon calcitonin: From synthesis to therapeutic use. Calcif Tissue Int 57:405-408, 1995.

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~HAPTER

.

Vitamin D Metabolism and Biological Function MICHAEL

F. H O L I C K

J O H N S. A D A M S

Boston University Medical Center, Boston, Massachusetts 02118 University of California, Los Angeles, Los Angeles, California 90048

I. II. III. IV. V.

History of Vitamin D Photobiology of Vitamin D3 Intestinal Absorption of Vitamin D Metabolism of Vitamin D to 25-Hydroxyvitamin D Metabolism of 25-Hydroxyvitamin D to 1,25-Hydroxyvitamin D VI. Alternative Metabolism of 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D VII. Metabolism of Vitamin D2

VIII. Biological Actions of 1,25(OH)2D IX. Biological Actions of 1,25(OH)2D in Tissues Regulating Calcium Balance X. Actions of Vitamin D Metabolites and Analogs in Nonclassical Target Tissues XI. Assays for Vitamin D and Its Metabolites XII. Conclusion References

The secosterol vitamin D has its origin dating back at least 0.5 billion years ago, when it was produced in ocean dwelling phytoplankton while they were being exposed to sunlight, possibly to act as a sunscreen. 1 With the evolution of the terrestrial vertebrates, vitamin D became important for the development and maintenance of the ossified skeleton. One of the principal physiological functions of vitamin D is to maintain normal circulating concentrations of calcium and phosphorus for support of neuromuscular function and bone ossification. Vitamin D is able to accomplish this by enhancing the efficiency of the small intestine to absorb dietary calcium and phosphorus and increasing the mobilization of calcium stores from the bone. During the past two decades, intense research has revealed that vitamin D is not a vitamin but a hormone. Once vitamin D is made in the skin or ingested in the diet it undergoes successive hydroxylations in the liver METABOLIC BONE DISEASE

and kidney to be transformed to 1,25-dihydroxyvitamin D [1,25(OH)2D]. 2 1,25(OH)2D is the hormonally active form of vitamin D that leaves the kidney and travels in the circulation to its various target tissues and cells to carry out its physiological functions. This knowledge has led to the development of assays for vitamin D metabolites, which have been invaluable in obtaining solid clinical evidence that acquired and inherited disorders of vitamin D metabolism are the cause of several hypo- and hypercalcemic disorders. Although the major target tissues for 1,25(OH)2D are the small intestine, bone, and kidney, recently it has been revealed that such diverse tissues and cells as the skin, pancreas, parathyroid gland, stomach, gonads, brain, monocytes, and activated T and B lymphocytes possess low-capacity, high-affinity nuclear receptors for this hormone. In a variety of experimental systems, both in vivo and in vitro, 1,25(OH)2D has been shown to inhibit tu123

Copyright 9 1998 by Academic Press. All rights of reproduction in any form reserved.

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MICHAEL E HOLICK AND JOHN S. ADAMS

mor cell proliferation and promote cellular differentiation; to stimulate insulin, thyroid-stimulating hormone, and interleukin-1 synthesis or release; to inhibit parathyroid hormone (PTH) and interleukin-2 synthesis and secretion; and to induce peripheral monocytes to mature into osteoclast-like cells. Although the physiological relevance of these observations is unclear at present, these revelations have given rise to speculation that 1,25(OH)2D is important for the recruitment of bone marrow stem cells to form new osteoblasts and osteoclast-like cells and to the novel use of 1,25(OH)2D for the treatment of lymphomas and psoriasis. The purpose of this chapter is to review the recent advances in the photobiology, biochemistry, and physiology of vitamin D and to recount how this fundamental knowledge has been useful for the diagnosis and treatment of a variety of disturbances in calcium and bone metabolism as well as diseases as diverse as psoriasis and cancer.

I. HISTORY OF VITAMIN D A. Rickets and the Environment There is firm evidence that most plants and animals that live on the Earth today have the capacity to produce vitamin D during exposure to sunlight. 1'3 In terms of human history, however, historians state that the disease rickets was reported to occur in humans as early as the 2nd century AD, but the disease was not considered a significant health problem until people began to congregate into the cities in northern Europe just prior to its industrialization. 4 In the mid-17th century, Whistler, Glisson, and DeBoot each independently recognized that many of the children who lived in the sunless alleyways in the industrialized cities developed a severe disease of the bones. They noted that this disease was associated with deformities of the skeleton, particularly enlargement of the epiphyses of the joints of the long bones and the rib cage (commonly referred to as the rachitic rosary), bending of the spine, enlargement of the head, curvature of the thighs, and weak and toneless muscles, especially of the extremities (Fig. 5-1).4'5 The incidence of this debilitating bone disease increase dramatically during the Industrial Revolution, especially in northern Europe and North America, and by the latter part of the 19th century, autopsy studies done in Leiden, the Netherlands, suggested that approximately 80% to 90% of children raised in the crowded cities of these areas had the disease. 6 As early as 1822, the Polish physician Sniadecki realized the importance of sun exposure for the prevention and cure of rickets. 7 He realized that children living in the inner city of Warsaw had a very high incidence of

FIGURE 5 - - 1 Child with rickets showing rachitic rosary of the rib cage, bowed legs, deformity of the long bones, and muscle weakness. (From Fraser D, Scriver CR: Hereditary disorders associated with vitamin-D resistance or defective phosphate metabolism. In DeGroot L, et al (eds): Endocrinology, vol 2. New York, Grune & Stratton, 1979, pp 797-807.)

this bone disease, whereas children living in the rural areas outside of Warsaw were essentially free of this disorder. He advocated if the parents' financial status permits, it is best to take the children out into the country and keep them as much as possible in the dry, open, and pure air. If not, at least they should be carried out in the open air, especially in the sun, direct action of which on our bodies must be regarded as one of the most efficient methods for the prevention and cure of this disease. 7 However, little attention was focused on the environment as a cause for this disorder until 1889 when an investigative committee of the British Medical Association reported that rickets was unknown in the rural districts of the British highlands but that it was prevalent in the large industrial towns. 8 A year later, Palm 9 reported his epidemiological survey that included clinical observations from a number of physicians throughout the British Empire and the Orient. His information revealed that

CI-IAPTER 5 Vitamin D Metabolism and Biological Function rickets was rare in children living in impoverished cities in China, Japan, and India where the people received poor nutrition and lived in squalor, whereas the children of the middle-class and poor who lived in the industrialized cities in the British Isles had a high incidence of rickets. Based on this survey he urged the following: the establishment of a sunshine recorder in the heart of the city to record the chemical activity of the sun's rays rather than its heat; the removal of rachitic children, as early as possible, from the large towns to a locality where sunshine abounds and the air is dry and bracing; the systematic use of sunbaths as a preventative and therapeutic measure in rickets and other diseases; and the education of the public to the appreciation of sunshine as a means of health. However, it was difficult at the time for people to believe that such a simple remedy as exposure to sunlight could have any significant effect on curing this crippling bone disease. At the turn of the 20th century, many theories had surfaced as to the cause of the debilitating disease, including infection, poor nutrition, lack of activity, and an inherited disorder. In 1905, Buchholz 6 exposed 16 rachitic children to a carbon-arc light source and suggested that there was a favorable response. However, in 1919 Huldschinsky demonstrated unequivocally for the first time that phototherapy alone was curative. He reported four patients with severe tickets who were cured (based on x-ray examination) after being treated with exposure to a mercury-vapor quartz lamp. ~~ He speculated that the radiation responsible for this cure was the same radiation that causes melanization of the skin. He also showed that the effect of phototherapy was not local, inasmuch as exposure of one arm had equal and dramatic curative effects on both arms. Two years later, Hess and Unger ~ exposed seven rachitic children on the roof of a New York City hospital to varying periods of sunshine and reported that x-ray examinations showed marked improvement of the tickets of each child as evidenced by calcification of the epiphyses.

B. V i t a m i n D" T h e N u t r i e n t During the 19th century, cod-liver oil was frequently used for the prevention and cure of rickets. This knowledge prompted an intense investigation to determine what nutrient was responsible for preventing rickets. In 1919, Mellanby ~2 reported that he could produce rickets in dogs by feeding them oatmeal and could cure the disease by adding cod-liver oil to their diet. In 1921, McCollum and colleagues 13 reported that profound influence of dietary phosphorus on the calcification of cartilage and ossification of bone in growing rats. They demonstrated that rats given a diet deficient in phosphorus and the antirachitic factor developed rickets, whereas

125 rats given diets adequate in calcium and phosphorus but deficient in the antirachitic factor developed osteoporosis but not tickets. With this experimental model they examined the question of whether the antirachitic factor in cod-liver oil was identical to or distinct from vitamin A. In 1922, these workers oxidized cod-liver oil, destroying all vitamin A activity, and showed that the oxidized oil retained its antirachitic properties. Thus, it became clear that the antirachitic factor present in cod-liver oil was not vitamin A but a new fat-soluble vitamin that was to be called vitamin D. The fact that the antirachitic factor could be generated in the skin after exposure to sunlight or ultraviolet radiation or could be obtained from codliver oil caused some confusion as to whether there was more than one antirachitic factor. This issue was resolved when Powers et al. TM reported that radiation from a mercury-vapor quartz lamp had similar if not identical healing effects on rachitic rats when compared with those brought about by the administration of cod-liver oil. Once it was known that exposure to sunlight could prevent and cure the disease, Steenbock and Black 15 and Hess and Weinstock 16 independently demonstrated that exposure of food and a variety of other substances to the vitamin D-producing radiation from the mercury-vapor quartz lamp could impart antirachitic properties to these substances. These investigators reported that exposure of rat liver, human serum, olive, cotton and linseed oils, lettuce, growing wheat, rat chow, and a variety of other substances endowed each with antirachitic properties. This concept was used to make milk antirachitic by adding the precursor of vitamin D to milk and exposing it to radiation from a mercury-arc lamp. Today, 400 IU (10 txg) of vitamin D2 or vitamin D3 is directly added to milk and other foods, resulting in almost complete elimination of rickets in the United States and in countries that use this practice. Once it was established that the antirachitic factor could be produced in vitro by the ultraviolet (UV) irradiation of plant and animal tissues, several investigators began the quest to isolate and structurally identify the antirachitic factor. Originally, it was thought that the substance that was activated by UV radiation was cholesterol in animals and phytosterol in vegetable foods. 15'17 However, cholesterol lost the property of becoming antirachitic when it was purified by chemical means. It was concluded that the precursor of vitamin D (called provitamin D) was not cholesterol itself, but a substance associated with it. TM The findings that ergosterol, a yeast sterol, had an intense ultraviolet absorption band at 280 nm (which was similar to the UV absorption maximum for the cholesterol-like sterol that had antirachitic properties) and that this yeast sterol could be rendered antirachitic by exposure to ultraviolet radiation suggested that the parent substance of vitamin D was either ergos-

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terol or a highly unsaturated sterol similar to ergosterol. These observations prompted the quest of several laboratories to isolate and structurally identify the antirachitic factor. Reerink and van Wijk m and Askew et al. 2~ purified vitamin D from irradiated ergosterol and reported almost identical UV absorption spectra with X max at 265 and 262 nm, respectively. Initial characterization of vitamin D established that the structure retained the secondary hydroxyl group of the parent ergosterol, that ring B was opened between carbon 9 and 10, and that there were three double bonds in conjunction with each other (Fig. 5 - 2 ) . Finally, in 1948 x-ray crystallographic analysis of a heavy atom derivative of vitamin D yielded the spacial orientation of vitamin D as a molecule that is quite different from its parent sterol (Fig. 5 - 2 ) . The first vitamin D that was isolated from the irradiation of ergosterol was designated vitamin D1. However, the product was found to be an impure mixture of vitamin D and lumisterol and the term was dropped. Vitamin D was finally purified from its irradiation products and was named ergocalciferol (vitamin D2) (Fig. 5 - 2 ) . Initially, it was thought that vitamin D2 was iden-

tical to the vitamin D that was present in fish-liver oils and was produced in the skin by exposure to sunlight. However, in the 1930s it was reported that vitamin D obtained from the irradiation of ergosterol had little antirachitic activity in chickens, whereas the vitamin D that was isolated from the irradiation of cholesterol-like sterol yielded a very potent antirachitic substance. 21-23 The confusion as to whether vitamin D2 w a s identical to the substance produced in skin was resolved when Windaus et al. 24 reported the synthesis of a new provitamin D analogue that was similar to ergosterol with the exception that the side chain was that of cholesterol (Fig. 5 - 2 ) . This provitamin D was called provitamin D3 or 7dehydrocholesterol and upon irradiation gave rise to cholecalciferol (vitamin D3) (Fig. 5 - 2 ) . This new vitamin D had equal antirachitic activity in chick and rat to the vitamin D that was isolated from the cholesterol-like sterol and was identical to the vitamin D found in fishliver oils and mammalian skin. 25 Therefore, it was concluded that 7-dehydrocholesterol rather than ergosterol was the parent compound present in the skin and that the resulting photoproduct was vitamin D3.

22

21

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Structure of vitamins D3 and D2 and their respective precursors, 7-dehydrocholesterol and ergosterol. The only structural difference between vitamins D2 and D3 is their side chains; the side chain for vitamin D2 contains a double bond between carbon-22 and carbon-23 and a carbon-24 methyl group. (From MacLaughlin JA, Holick MF: Photobiology of vitamin D3 in the skin. In Goldsmith LA (ed): Biochemistry and Physiology of the Skin, vol 2. London, Oxford University Press, 1983, pp 734-754.)

CHAPTER 5

127

Vitamin D Metabolism and Biological Function

II. P H O T O B I O L O G Y

OF VITAMIN

imately 10% is reflected and the other 90% is absorbed or scattered. 26 During exposure to sunlight, UV-B photons are transmitted into the epidermis and dermis where cytoplasmic stores of provitamin D3 are located. The 5,7diene of provitamin D3 absorbs this radiation, causing cleavage of ring B between carbons 9 and 10 and the formation of a 6,7-cis-conjugated triene to form a 9,10seco (seco from the Greek term split) sterol known as previtamin D3 (Fig. 5--3). 27 In adult skin, approximately 50% of the entire cutaneous stores of provitamin D3 are

D3

A. P h o t o s y n t h e s i s of P r e v i t a m i n D3 in H u m a n Skin The sun emits a broad spectrum of radiation. The high-energy photons that are most damaging to life on Earth (below 290 nm) are absorbed by the thin layer of ozone that envelops the Earth. When radiation between 290 and 315 nm (UV-B radiation) hits the skin, approx-

SUN

~

SUN

SUN

PREVITAMINDs ~

.

CHs

TACHYSTEROL3

s~(~_

HOr

[ so,

VITAMIN Ds

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I OH

I

"

SUPRASTEROL I

HO

SUPRASTEROL II 5,6-TRANSVITAMIN D3

FIGURE 5--3 Photochemical events that lead to the production of vitamin D 3 and the regulation of vitamin D 3 in the skin. (From Holick MF: McCollum Award Lecture, 1994: Vitamin D: New horizons for the 21st century. Am J Clin Nutr 60:619-630, 1994.)

128

MICHAELE HOLICKAND JOHN S. ADAMS

found in the epidermis while the other 50% reside in the dermis. 27 When adult Caucasians and blacks are exposed to sunlight, approximately 70% to 80% and 95% to 98% of the UV-B photons are absorbed in the epidermis, respectively. Therefore, approximately 80% to 90% of the previtamin D3 that is formed occurs in the actively growing layers of the epidermis (stratum basale and stratum spinosum) while less than 20% occurs in the dermis. 27 In neonates, about 50% of the provitamin D3 stores in the skin are present in the dermis, and because the epidermis transmits more UV-B photons into the dermis, the dermis is also a major site for previtamin D3 synthesis. Once previtamin D3 is made in the skin, it immediately begins to thermally equilibrate to vitamin D3 by a temperature-dependent process (Fig. 5--3). 27 This thermal equilibration takes approximately 10 hours to reach completion at body temperature (37 ~ C) in humans. 27'28 Because most of the cutaneous previtamin D3 synthesis in adults occurs in the actively growing layers of the epidermis, which is in close proximity to the dermal capillary bed, changes in the temperature of the surface of the skin resulting from exposure to very warm or cold climates do not significantly alter the rate of conversion of previtamin D3 to vitamin D3 in humans. 27 Although the exact mechanism for how vitamin D3 exits the epidermal cells and journey to the dermal capillary bed is unknown, it is now appreciated that previtamin D3 is made in the plasma membrane and as a result is entrapped within the membrane due to its conformation. 29 Once it isomerizes to vitamin D3, the change in conformation results in it being jettisoned into the extracellular space where it is drawn into the dermal capillary bed by the vitamin D-binding protein.

B. Regulation of Previtamin D3 Synthesis in Human Skin In 1967, Loomis 3~ popularized a theory that skin pigmentation evolved for the purpose of regulating vitamin D3 synthesis in the skin. He suggested that peoples living at or near the equator would have died of vitamin D intoxication as a result of daily exposure to intense solar radiation were it not for the evolution of more melanin pigmentation in the skin. Melanin is a natural sunscreen that is produced by the epidermis and acts as a neutral density filter absorbing wavelengths of sunlight that are responsible for producing previtamin D3 in human skin. 31 Although it is clear that melanin can compete with 7dehydrocholesterol for UV-B photons and therefore limit previtamin D3 synthesis in the skin, this cannot be the only explanation for why Caucasians exposed to prolonged sunlight do not become vitamin D intoxicated.

It is now appreciated that there is a more fundamental process that regulates the photosynthesis of previtamin D3 in human skin. Previtamin D3 is sensitive to both thermal energy and ultraviolet radiation. Once previtamin D3 is formed in the epidermis and dermis, it can either thermally isomerize to vitamin D3 or during exposure to sunlight absorb a photon of UV-B radiation and isomerize to biologically inert isomers lumisterol and tachysterol (Fig. 5-3). 32 Thus, if a Caucasian is exposed to sunlight at the equator, during the initial few minutes of exposure provitamin D3 is rapidly converted to previtamin D3 (Fig. 5-4). Prolonged exposure to sunlight, however, does not increase previtamin D3 production, but rather previtamin D3 absorbs UV-B radiation and undergoes isomerization to form lumisterol and, to a small extent, tachysterol (Fig. 5-4). 32 Thus, prolonged exposure to sunlight does not necessarily increase previtamin D3 synthesis. In addition to the photolysis of previtamin D3 in human skin that limits the amount of previtamin D3 that is ultimately formed during a single exposure to sunlight, it is now appreciated that vitamin D3 is exquisitely sensitive to exposure to sunlight. On a sunny day in Boston in June, vitamin D3 is efficiently converted to 5,6-trans-vitamin D3 and suprasterols 1 and 2 (Fig. 5--3). 33 The physiological roles of the photoisomers of previtamin D3 and vitamin D3 are unknown at present.

C. Other Factors That Regulate the Cutaneous Production of Vitamin D3 The photoproduction of previtamin D3 in any layer of skin is dependent on the concentration of provitamin D3, the presence of chromophores that compete with provitamin D3 for UV-B photons, and the quantum of UV-B photons that are able to penetrate the skin and are absorbed by the provitamin D3 chromophore. The average concentration of provitamin D3 in a 6.25 cm 2 area of young adult human skin is approximately 5 Ixg for the epidermis and 1 to 3 Ixg for the dermis. 34 There is an inverse relation between the concentrations of provitamin D3 in the epidermis with age. 34 Compared with elderly adults, young adults can make two to three times more previtamin D3 in their skin when they are exposed to the same amount of sunlight (Fig. 5--5). 34-36 Sunscreens, which are effective in preventing the damaging effects of sunlight, also prevent the beneficial effects of sunlight, the photosynthesis of previtamin D3 in human skin. 31 The topical application of a sunscreen with a sun protection factor of 8 can completely block the photosynthesis of previtamin D3 in human skin and prevent the elevation of the concentration of vitamin D3 in the circulation after a whole-body exposure to a dose of ul-

CHAPTER 5

129

Vitamin D Metabolism and Biological Function

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absorbing chromophores that are present in the skin, such as melanin, urocanic acid, proteins, and RNA and DNA. An increase in the zenith angle either by the daily rotation of the Earth or by an increase in the distance north or south from the equator shifts the spectral distribution of sunlight toward longer wavelengths because of greater subtraction of shorter wavelengths (UV-B) by atmospheric absorption and scattering. 5 It is well known that in northern latitudes children are more prone to develop rickets during and immediately after the winter when compared with spring, summer, and fall. Originally it was thought that the principal cause for this was that children wore more clothing and were outdoors less. However, there is a more fundamental reason for this phenomenon. There is now evidence that exposure to sunlight between the months of November and March, in Boston (42 ~ N), does not result in any cutaneous production of previtamin D3. 33 However, in Los Angeles

8

(hours)

8O

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FIGURE 5 - 4 An analysis of the photolysis of 7-dehydrocholestrol (7-DHC) in the basal cells and the appearance of the photoproducts previtamin D3 (preD3), lumisterol (L), and tachysterol (T) with increasing time of exposure to equatorial simulated sunlight. (From Holick MF, MacLaughlin JA, Doppelt SH: Science 211:590, 1981. Copyright 1981 by the American Association for the Advancement of Science.)

.=_ E >

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traviolet radiation that is equivalent to a minimal erythemal dose (Fig. 5--6). 31'36 Clothing-like sunscreens absorb solar ultraviolet radiation and therefore also prevent the cutaneous synthesis of vitamin D3 .37 The percentage conversion of cutaneous provitamin D3 to previtamin D3 is also influenced by the solar zenith angle, which is inversely related to the amount of ultraviolet B photons in the solar spectrum, and by U V - B -

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FIGURE 5 - - 6 Circulating concentrations of vitamin D in young adults who applied either a sunscreen with sun protection factor of 8 or a topical placebo cream followed by a single whole-body exposure to 1 MED of simulated sunlight. (From Holick MF: McCollum Award Lecture, 1994: Vitamin D: New horizons for the 21st century. Am J Clin Nutr 60:619-630, 1994.)

130

MICHAEL E HOLICK AND JOHN S. ADAMS rj "1-

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Photosynthesis of previtamin D3 after exposure of 7-dehydrocholesterol (7-DHC) to sunlight in Boston (42 ~ N) for 1 hour (-o-) and 3 hours (-e-) Edmonton, Canada (52 ~ N) for 1 hour (-A-) each month for 1 year, Los Angeles (34 ~ N) (A) and Puerto Rico (18 ~ N) in January (~). (From Holick MF: McCollum Award Lecture, 1994: Vitamin D: New horizons for the 21st century. Am J Clin Nutr 60:619-630, 1994.)

(34 ~ N) and Puerto Rico (18 ~ N), sunlight produces previtamin D3 in the skin throughout the year (Fig. 5 - - 7 ) . 36,38

D. Effect o f W h o l e - B o d y U l t r a v i o l e t I r r a d i a t i o n on C i r c u l a t i n g C o n c e n t r a t i o n s o f V i t a m i n D

""

20

40

60

TIME (HOURS)

Month FIGURE 5 - 7

O

; % . A L o,ooo ,u

FIGURE 5 - - 8 Serum concentrations of vitamin D from healthy young adults who received a single oral dose of either 10,000 or 25,000 IU of vitamin D2 or a whole-body exposure to 1 MED of simulated solar ultraviolet B radiation. (From Holick MF: Sunlight, vitamin D and human health. In Holick MF, Jung EG (eds): Proceedings, Symposium on the Biological Effects of Light. Berlin, Water De Gruyter & Co, 1994, pp 3 - 1 5 . )

after exposure, whereas exposure to the same amount of radiation had no effect on serum vitamin D concentrations in the black volunteers (Fig. 5 - 9 ) . 41 The black subjects were then exposed to a dose of radiation that was equivalent to 6 times the MED for the Caucasian subjects (exposure of the Caucasian subjects to this amount of radiation would have caused severe second-degree

a n d Its M e t a b o l i t e s When young, healthy adult volunteers were exposed to a single whole-body dose of ultraviolet radiation that caused minimal erythema (1 minimal erythemal dose [MED]), the circulating vitamin D concentrations increased from a baseline value of 2 ng/ml to 24 ng/ml within 24 hours and retumed to a baseline value within 1 week after the exposure (Fig. 5 - - 8 ) . 39 This is equivalent to taking between 10,000 and 25,000 IU of vitamin D2 orally (Fig. 5-8). 40 The apparent half-life of vitamin D in the serum was determined to be about 48 h o u r s . 39 Based on the assumption that the plasma volume is about 5% of the mean body weight, it was estimated that at least 30 Ixg of vitamin D3 w a s released from each square meter of body surface area after exposure to 1 MED of ultraviolet radiation. 39 The role of skin pigment and ethnic background on limiting the cutaneous formation of vitamin D3 w a s examined by exposing slightly pigmented Caucasians, moderately pigmented immigrants of Indian and Pakistani extraction, and heavily pigmented black volunteers to a single dose of ultraviolet radiation. Whole-body exposure of Caucasian subjects to 1.5 times their MED greatly increased the serum vitamin D concentrations by up to 60-fold 24 to 48 hours

A

t

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oJ

r

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I

,

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FIGURE 5--9

Serum vitamin D concentration in two lightly pigmented Caucasian (A) and three heavily pigmented black subjects (B) after total-body exposure to 0.054 J/cm 2 of UVR. C, Serial change in circulating vitamin D after reexposure of one black subject (e in panel B) to a 0.32 J/cm 2 dose of UVR. (From Clemens TL, Adams JS, Henderson SL, Holick MF: Increased skin pigment reduces the capacity of the skin to synthesize vitamin D. Lancet 1:74, 1982.)

CHAPTER 5 Vitamin D Metabolism and Biological Function sunburn), and circulating concentrations of vitamin D increased approximately 30-fold during the 24 hours after exposure (Fig. 5--9). 41 It is well recognized that Asians of Indian and Pakistani extraction living in Great Britain are more prone to develop rickets and osteomalacia. Although this may be due to the high phytate content in their diet, there has been some question as to whether these individuals have the same capacity as that of Caucasians and blacks to produce vitamin D3. Indians and Pakistanis have the same capacity as that of Caucasians and blacks to produce vitamin D3 in their skin as evidenced by increasing circulating concentrations of vitamin D that are comparable to those of Caucasian subjects exposed to 1 MED of ultraviolet radiation. 42 Hence, blacks and Asians of Indian and Pakistani extraction have the same capacity as that of Caucasians to produce vitamin D3 but require a much larger dose of ultraviolet radiation to do so because of the pigmentation that is present in their skin. Although a single whole-body exposure to ultraviolet radiation has a dramatic effect on elevating the circulating concentration of vitamin D in a dose-dependent fashion, the effect on the circulating concentration of 25(OH)D and 1,25(OH)2D is minimal. The serum 25(OH)D concentration increased only gradually, reaching a 50% increase in 7 to 14 days after exposure. 39 There was no significant increase in circulating concentrations of 1,25(OH)2D in the Caucasian subjects exposed to 1 and 3 MEDs of ultraviolet radiation. 39 Vitamin D deficiency does not appear to alter provitamin D concentrations in the skin. 39 When vitamin D-deficient patients were exposed to 1 MED of whole-body ultraviolet radiation, their circulating concentrations of vitamin D and 25(OH)D increased in a fashion almost identical to that seen for the vitamin D-sufficient volunteers. 39 However, the serum 1,25(OH)2D concentrations in the vitamin D-deficient patients increased by three- to fourfold within 7 days after the exposure and persisted to this concentration throughout the next 2 weeks. 39 In these patients, the increase in circulating concentrations of 1,25(OH)2D was most likely due to the fact that the high circulating concentrations of PTH enhanced the renal metabolism of 25(OH)D to 1,25(OH)2D (see separate section on vitamin D metabolism and regulation for details). 39

131 angle of the sun does not permit sufficient vitamin D3 production in skin. 38'4~ V e r y few foods contain vitamin D naturally; these include liver, egg yolks, and fish-liver oils. Several countries practice the fortification of some foods with vitamin D. In the United States, milk is the principal dietary component that is subject to vitamin D fortification with either vitamin D2 or vitamin D3. However, milk cannot always be depended on as the sole source of vitamin D, since it is now recognized that the vitamin D content in milk can be highly variable. Three separate studies have revealed that about 80% of milk samples tested did not contain more than 80% of the vitamin D that was supposed to be in the milk and about 10% of skim milk had no detectable vitamin D. 43-45 In other countries, some cereals, margarine, and breads also have small quantities of vitamin D added to them. When vitamin D is ingested, this fat-soluble compound is incorporated into the chylomicron fraction and absorbed through the lymphatic system. Approximately 80% of the vitamin D enters the body via this mechanism. After the ingestion of a single dose of 50,000 IU of vitamin D2, circulating concentrations of vitamin D are increased by as early as 4 hours, peak at 12 hours, and gradually decline to near baseline by 72 hours (Fig. 5 - 1 0 ) . 46 Although aging significantly decreases the capacity of the skin to produce vitamin D3, aging does not appear to significantly affect the intestinal absorption of vitamin D (Fig. 5 - 1 0 ) . 47 On the other hand, intestinal malabsorption syndromes such as Crohn's disease, cystic fibrosis, and Whipple's disease can effectively prevent

9O 80 E

70

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For most of the population of the world cutaneous synthesis of vitamin D3 is the principal source of this prohormone. The exceptions are probably peoples living in very northern and southern latitudes where the zenith

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TIME (HOURS) FIGURE 5--10 Vitamin D2 absorption in young (e) and elderly (o) adults. Each subject received an oral dose of 50,000 IU of vitamin D2, and at various times blood determinations were made for circulating concentrations of vitamin D. (From Holick MF: Vitamin D requirements of the elderly. Clin Nutr 5:121, 1986.)

132

MICHAEL E HOLICK AND JOHN S. ADAMS 8O

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FIGURE 5--11 Serum vitamin D concentrations in seven patients with intestinal fat malabsorption syndromes after a single oral dose

of 50,000 IU (1.25 mg) of vitamin D2. For comparison, the means and standard errors of vitamin D concentrations measured in seven normal control subjects after a similar dose are indicated by the closed circles and dashed lines (-o-). Note that two patients, one with Crohn' s ileocolitis (patient F) and one with ulcerative colitis (patient G), had essentially normal absorption curves. Five patients, however, showed a dramatic lack of response, with no values shown above 10 ng/ml. (From Lo CW, Paris PW, Clemens TL, et al: Vitamin D absorption in healthy subjects and in patients with intestinal malabsorption syndromes. Am J Clin Nutr 42:644, 1985. Reproduced with permission from the American Society for Clinical Nutrition and The American Journal of Clinical Nutrition.)

the intestinal absorption of vitamin D, whereas diseases that affect the more distal small intestine and large intestine appear to have little effect on the intestinal absorption of this fat-soluble vitamin (Fig. 5-11).46 A simple method to determine whether an individual is capable of absorbing vitamin D is to conduct a provocative vitamin D absorption test. A blood sample is obtained before and 12 hours after a single oral administration of 50,000 IU of vitamin D2 (Fig. 5 - 1 1 ) . 46 If no elevation in the circulating concentration of vitamin D is observed, complete malabsorption of vitamin D should be suspected; however, an increase in the circulating concentration of vitamin D is reflective of vitamin D absorption, and the patient's dose of vitamin D can therefore be tailored accordingly. 46 Thus, patients who suffer from chronic liver disease or who have a disease of the small intestine are more prone to develop vitamin D deficiency owing to the inability to absorb this fatsoluble vitamin.

IV. METABOLISM OF VITAMIN D TO 25-HYDROXYVITAMIN D A. Hepatic Metabolism Vitamin D3 made in the skin or ingested in the diet along with dietary vitamin D2 enters the circulation and

is bound to a vitamin D - b i n d i n g protein. 2 Vitamin D (the use of the term vitamin D without a subscript refers to either or both vitamin D2 and vitamin D3) is transported to the liver where it is hydroxylated on carbon 25 to generate the major circulating form of vitamin D, 25-hydroxyvitamin D [25(OH)D] (Fig. 5 - 1 2 ) . 2,48 The vitamin D 25-hydroxylases are located in the mitochondria and microsomes of the parenchymal cells. The enzymatic reaction is supported by reduced N A D P and molecular oxygen. 48 Although the mammalian liver has a large reserve of the vitamin D-25-hydroxylase, it is not the sole site for vitamin D 25-hydroxylation. In avian species, the kidney and intestine are capable of metabolizing vitamin D3 to 25(OH)D3, 49 and hepatectomized rats metabolize vitamin D3 to 25(OH)D3 to a small degree. 5~ Vitamin D2 and vitamin D3 as well as vitamin D analogues, such as dihydrotachysterol and l c~hydroxyvitamin D3, are metabolized in the liver to their 25-hydroxy counterparts. 2 There is some evidence, however, that because the vitamin D - b i n d i n g protein does not bind to vitamin D2 as tightly as it does to vitamin D3, vitamin D2 is more available as a substrate for the hepatic enzyme and, therefore, is more efficiently converted to 25(OH)D2. 51 The half-life of 25(OH)D in the human circulation is about 2 to 3 weeks, and its concentration is a good reflection of the cumulative effects of dietary intake of vitamin D and exposure to sunlight. 2 It should be recognized, however, that the liver vitamin D-25-hydroxylase is relatively well regulated, inasmuch as the relative increase in circulating concentration of 25(OH)D3 in comparison to the cumulative intake of vitamin D3 is relatively small (Fig. 5 - 1 3 ) . 52 This may, in part, be due to negative feedback regulation of the hepatic vitamin D-25-hydroxylase by vitamin D, 25(OH)D, and/or 1,25(OH)2D3 .53

B. Clinical Disorders Patients with severe parenchymal and cholestatic liver disease often have low circulating concentrations of 25(OH)D (Table 5 - 1 ) . 54 This is partly due to the associated intestinal malabsorption of vitamin D as well as a decrease in the reservoir of the vitamin D-25-hydroxylase in the liver. Originally, it was believed that the low circulating concentration of 25(OH)D was responsible, in part, for the debilitating bone disease associated with severe liver failure. However, there is no correlation between the severity of the bone disease and circulating concentrations of 25(OH)D, and treatment of these patients with 25(OH)D or its metabolites provides no benefit. 54'55 However, because these patients are more prone to vitamin D deficiency due to associated fat malabsorption, it is wise to increase the vitamin D intake and

CHAPTER 5

Vitamin D Metabolism and Biological Function

133 ,4CE TA TE

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~ .0~

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FIGURE 5--12 The photochemical, thermal, and metabolic pathways for vitamin D 3. Circled letters and numbers denote specific enzymes" (Z)7-dehydrocholesterol reductase; Q vitamin D-25-hydroxylase; (~ 25(OH)D- 1c~-hydroxylase; (~ 25(OH)D-24R-hydroxylase; (~)25(OH)D-26-hydroxylase. (From Holick MF, Potts JT Jr: Vitamin D. In Isselbacher KJ, et al (eds): Harrison's Principle of Internal Medicine, 10th ed. New York, McGraw-Hill, 1983, pp. 1944-1949.)

monitor circulating concentrations of 25(OH)D. The 25(OH)D concentrations in these patients should be maintained in the mid-normal range of approximately 25 to 45 ng/ml. Some patients will benefit by increasing the dose or frequency of oral administration of vitamin D once it has been determined, using the provocative oral

vitamin D absorption test, 46 that the patient can absorb vitamin D. Otherwise, intravenous or intramuscular injections of vitamin D or increased exposure to sunlight will often provide adequate vitamin D nutrition for these patients. In disease states in which there is an increase in the metabolism of 25(OH)D to 1,25(OH)2D, such as

134

MICHAEL E HOLICK AND JOHN S. ADAMS 160-

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FIGURE 5-- 13 Serum levels of 25(OH)D3 observed in response to various oral doses of vitamin D3, given to vitamin D-deficient rats. There is a linear correlation that extends well into the pharmacological range for both parameters. (Courtesy of the Nichols Institute.)

vitamin D - d e p e n d e n t rickets type II, sarcoidosis and other chronic granulomatous disorders, primary hyperparathyroidism, and hyperphosphatemic tumoral calcinosis, it has been reported that the circulating concentrations of 25(OH)D are often decreased, but usually not below the normal values. 53 Patients with nephrotic syndrome who have marked proteinuria (>4 g/24 hr) may have decreased concentrations of 25(OH)D in the circulation as a result of a loss in the urine of the vitamin D - b i n d i n g protein (which is similar in molecular weight to albumin) with its tightly bound 25(OH)D. Thus, the turnover of 25(OH)D is markedly increased, and these patients will often benefit from additional vitamin D sup-

TABLE 5--1 Serum Concentrations of 25(OH)D in Disorders of Calcium, Phosphorus, and Bone Metabolism Serum

Disease State

25(OH)D

Vitamin D deficiency Intestinal malabsorption syndromes Liver disorders Nephrotic syndrome Osteopenia in the aged Vitamin D intoxication

$ $ $ $ N or $ 1"

plementation. 56 There is no need to treat these patients with either 25(OH)D3 or 1,25(OH)2D3. It is well documented that there is an association between osteomalacia or rickets and anticonvulsant drug therapy in epileptic patients. 57 Initially, it was thought that this resistance was because phenytoin and phenobarbital induced liver microsomal enzymes that rapidly metabolized and inactivated vitamin D and its metabolites. However, it now appears that these drugs also disrupt calcium homeostasis by additional mechanisms. Phenytoin inhibits vitamin D - d e p e n d e n t and vitamin D-independent intestinal calcium transport. Phenobarbital increases bile secretion (thereby increasing the turnover of vitamin D) and has a negative effect on the kinetics of the vitamin D-25-hydroxylase. 58 This problem appears to be more severe in children and adults who are institutionalized and who are taking several antiseizure medications. There does not appear to be a disruption in calcium or bone metabolism in otherwise healthy individuals who are taking a single anticonvulsant drug for prolonged periods of time. It has been reported that free-living cardiac patients free of known disorders of calcium metabolism have normal circulating concentrations of 25(OH)D despite 2 years of treatment with conventional doses of phenytoin. 59 The hallmark of this disorder is low circulating 25(OH)D. The associated disorders in calcium and bone metabolism are reversed

CHAPTER 5 Vitamin D Metabolism and Biological Function

135 of this hormone with skin, bone cells, and/or peripheral monocytes being touted as the most likely tissues with the enzymatic machinery 65-68 and capacity 69'7~to produce enough 1,25(OH)2D to be detectable in blood; patients who have had a bilateral nephrectomy or who have severe renal failure have very low but detectable levels of the hormone. 71 In the absence of disease, whether other tissues besides the kidney can produce enough 1,25(OH)2D to maintain normal serum levels is unlikely. The renal 25(OH)D-l-hydroxylase (1-hydroxylase) is a mitochondrial cytochrome P-450, mixed-function oxidase that requires reduced NADP and molecular oxygen for its activity (Fig. 5-14). 48 The enzyme is located in the epithelial cells of the proximal convoluted tubule of the normal mammalian kidney. 72 One group of investigators recently claimed to have isolated the enzyme in pure f o r l T l , 73 while another group TM reported the isolation of a cDNA for the 1-hydroxylase mRNA from a vitamin D-deficient rat kidney library; the latter group employed as probe the cDNA for the rat 24-hydroxylase mRNA. Confirmation of the successful isolation and cloning of the 1-hydroxylase will require documentation of its fulllength amino acid sequence and the expression of a vitamin D-metabolizing P-450, which has a high affinity for substrate 25(OH)D in cells that normally lack the 1hydroxylase.

when the oral intake of vitamin D is increased to raise 25(OH)D into the normal range. 60

V. METABOLISM OF 25-HYDROXYVITAMIN D TO 1,25-HYDROXYVITAMIN D A. R e n a l 2 5 ( O H ) D - l o L - H y d r o x y l a s e Once 25(OH)D is formed in the liver, it is transported on the serum vitamin D - b i n d i n g protein to other tissue sites for further metabolism, primarily for hydroxylation on either C-1 of the A-ring or C-24 in the molecular side chain of 25(OH)D (Fig. 5-12). 2,48,60 In most mammalian species, including humans, the principal site for the metabolism of 25(OH)D to 1,25(OH)2D is the kidney. Normal circulating concentrations of 1,25(OH)2D are between 15 and 65 pg/ml, with the circulating half-life of the hormone between 4 and 6 hours. 6] During the third trimester of pregnancy the placenta plays a significant part in maintaining relatively high circulating concentrations of 1,25(OH)2D by metabolizing 25(OH)D to 1 , 2 5 ( O H ) 2 D . 62-64 There have also been reports that there are other " n o r m a l " extrarenal sites for the production

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FIGURE 5--14 Mechanismof production of 1,25(OH)2D3 by chick kidney mitochondria, illustrating the energydependent transhydrogenation reaction involved in electron supply for the hydroxylation reaction. (From DeLuca HF: The metabolism, physiology, and function of vitamin D. In Kumar R (ed): Vitamin D, Basic and Clinical Aspects. Boston, Martinus Nijhoff Publishing, 1984, pp 1-68.)

136

MICHAEL E HOLICK AND JOHN S. ADAMS

of the ferredoxin which contributes electrons to the 1hydroxylase enzyme, s3'84 Whether messengers in these same transduction pathways also regulate expression of the gene encoding the 1-hydroxylase has not been determined since the gene for the enzyme has not been cloned (see below). A change in the serum phosphate concentration is the other major regulator of 1,25(OH)zD production; in adult humans dietary phosphorus restriction causes an increase in circulating concentrations of 1,25(OH)zD to 80% above control values, an increase not due to accelerated metabolic clearance of this hormone. 79 Dietary phosphorous supplementation will have the opposite effect. 79 Human volunteers who received phosphorus supplements to their diet showed an abrupt decrease in the serum concentration of 1,25(OH)zD reaching a nadir within 2 to 4 days; after 10 days of supplementation the mean concentration of 1,25(OH)2D was 29% lower than the value measured when the phosphorus was normal and the production rate of hormone decreased significantly without any change in the metabolic clearance rate (Fig. 5-15). Although the mechanism by which a drop in the serum phosphate level will increase renal 1,25(OH)zD production remains uncertain, there is no doubt that there exists a concerted, cooperative attempt of the calciumphosphorus-PTH axis in man to regulate the conversion of 25(OH)D to 1,25(OH)zD in the kidney (Fig. 5-16). It is well known that the body adapts to the increased need for calcium by enhancing the efficiency of the intestine to absorb dietary calcium during pregnancy, 62-64

B. R e g u l a t i o n o f 2 5 ( O H ) D M e t a b o l i s m The synthesis of 1,25(OH)2D by the renal 1-hydroxylase is normally strictly regulated, with levels of the hormone product 1,25(OH)2D being some 1000-fold less plentiful in the circulation than that of the principal substrate for the enzyme 25(OH)D. 61 Hormone synthesis in the kidney is stimulated by an increase in the serum PTH concentration, a decrease in the serum phosphate concentration, and a decrease in the activity of the competing vitamin D-24-hydroxylase. 3'8'6~ In vivo, hypocalcemia also enhances renal 1-hydroxylase activity, but the majority of this activity is indirectly mediated through an increase in the secretion of parathyroid hormone. 48'77A drop in the serum calcium concentration will be immediately registered by the parathyroid cell calcium-sensing receptor, which will release its inhibition on PTH production and secretion. An increase in the circulating PTH will directly stimulate the renal 1hydroxylase, while a PTH-mediated phosphaturic response and a subsequent decrement in the serum phosphate level will indirectly promote 1,25(OH)zD production. 48'6~176 The mechanism by which parathyroid hormone exerts direct influence on the renal metabolism of 25(OH)D has not been firmly established, but recent information suggests that interaction of PTH with its receptor on cells in the proximal convoluted tubule transduces a stimulatory signal through both the protein kinase A and protein kinase C pathways, 81'82 effecting transient changes in the phosphorylation status

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TIME (days) FIGURE 5-- 15 Effect of changes in the oral intake of phosphorus on the fasting serum concentrations of 1,25(OH)2D and phosphorus in six healthy men. The bracketed points depict mean values + SEM. (From Portale AA, Halloran BE Murphy MM, Morris RC Jr: Oral intake of phosphorus can determine the serum concentration of 1,25-dihydroxyvitamin D by determining its production rate in humans. J Clin Invest 77:7-12, 1986.)

CI-IAr'TEg5 Vitamin D Metabolism and Biological Function

FIGURE 5-- 16 Photosynthesis of vitamin D3 and the metabolism of vitamin D3 to 25-OH-D3 and 1,25(OH)2D3. Once formed, 1,25(OH)2D3 carries out the biological functions of vitamin D3 on the intestine and bone. Parathyroid hormone (PTH) promotes the synthesis of 1,25(OH)2D3, which, in tum, stimulates intestinal calcium transport and bone calcium mobilization and regulates the synthesis of PTH by negative feedback. (From Holick MF: Vitamin D: Photobiology, metabolism, mechanism of action, and clinical application. In Favus MJ (ed): Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, 3rd ed. Philadelphia, Lippincott-Raven, 1996, pp 7 4 - 8 1 . )

lactation, and periods of skeletal growth. 75 Hence, it is not surprising that there is evidence for estrogen, 85'86prolactin, 87 and growth hormone, 88-9~ either directly or indirectly through insulin-like growth factor 1 (IGF-1), 91 enhancing the renal production of 1,25(OH)2D in various in vitro and in vivo animal models. However, patients with hyperprolactinemia and acromegaly, who have high circulating concentrations of prolactin and growth hormone, respectively, do not exhibit increased circulating concentrations 1,25(OH)2D. 6~ The role of estrogen and progesterone on production of 1,25(OH)2D in the

137 human kidney also remains unclear. For example, although mean serum 1,25(OH)2D levels are known to be higher in growing children than in a d u l t s , 91'92 the circulating concentrations of the vitamin D hormone are not significantly altered in adolescent women with estrogen deficiency caused by anorexia nervosa. 93 Concentrations of testosterone increase dramatically at the onset of sexual maturation in boys, but these changes are not temporally associated with an increase in serum concentrations of 1,25(OH)2D. 92 At the opposite end of the reproductive scale it has been shown that menopausal females have lower 1,25(OH)2D levels than menstruating women. 86 However, this association appears not to be due more to an age-related diminishment of responsiveness of the 1-hydroxylase to PTH (Fig. 5 - 1 7 ) 94 than to any effect of gonadal steroids on the renal metabolism of 25(OH)D3 to 1,25(OH)2D3. The other major contributor to the circulating 1,25(OH)2D level is the activity of the vitamin D-24hydroxylase. Like the 1-hydroxylase, 24-hydroxylase is a magnesium-dependent, heme-binding mitochondrial enzyme requiring NADPH and molecular oxygen. 83 The cDNA and part of the gene for the human, rat, and chicken enzyme, now referred to as P450cc24, have been cloned. 95-98 Expression of P450cc24 is stimulated in kidney cells by 1,25(OH)2D, especially if the protein kinase C (PKC) pathway is also up-regulated. 99'1~176 PTH appears to exert an opposite, inhibitory effect on P450cc24 gene transcription 98 and 24,25(OH)2D synthesis. 1~176 There are two modes by which this mitochondrial, cytochrome P-450-1inked enzyme system regulates vitamin D balance in adult animals including man. Because it is co-expressed in the kidney along with the vitamin D-1-hydroxylase, the first mode of regulation is on substrate availability to the 1-hydroxylase. Like the 1-hydroxylase, the 24-hydroxylase exhibits a preference for 25-hydroxylated secosterol substrates. 84 Although its affinity for 25(OH)D is somewhat less than that of renal 1-hydroxylase, its capacity for substrate is substantially greater. 83 Hence, when up-regulated under the influence of circulating 1,25(OH)2D or diminished serum PTH levels, the 24-hydroxylase has the capacity to compete with the 1-hydroxylase for substrate 25(OH)D. Under physiological circumstances, this state of competitive substrate deprivation for the 1-hydroxylase will persist until the serum calcium and PTH concentration are normalized. The second mode of regulation imparted on the circulating 1,25(OH)2D concentration by the 24hydroxylase is at the level of catabolism of the 1,25(OH)2D hormone. The affinity of the 24-hydroxylase for 1,25(OH)2D is as great as it is for 25(OH)2D. 84 Considering the fact that the 24-hydroxylase is the initial step in the conversion of 1,25(OH)2D to non-biologically active, water-soluble, excretable metabolites of the hor-

138

MICHAEL F. HOLICK AND JOHN S. ADAMS

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HOURS FIGURE 5 - - 17 Effect of synthetic parathyroid hormone [hPTH-(1 - 34)] on concentrations of 1,25(OH)2D in normal subjects (solid circles). All values are expressed as the mean + SEM. The single asterisk denotes significant differences at p < 0.01, and the double asterisk at p < 0.05, between the level in the patients and that in the controls at corresponding time points. Asterisks also refer to significant differences between the preinfusion baseline levels and levels at particular time points. To convert values for 1,25(OH)zD to picomoles per liter, multiply by 2.36. (From Slovik DM, Adams JS, Neer RM, et al: Deficient production of 1,25-dihydroxyvitamin D in elderly osteoporotic patients. N Engl J Med 305: 372-374, 1981.)

mone, 76 up-regulation of this enzyme will contribute to the lowering of 1,25(OH)2D hormone levels.

C. Extrarenal Metabolism of 25(OH)D As early as 1940, Henneman et al. 1~ suggested that the intestine was hypersensitive to vitamin D in patients with sarcoidosis. The fact that an active metabolite of 25(OH)D could be made outside of the kidney was first confirmed by Barbour and colleagues in 1981.1~ These investigators reported high concentrations of a vitamin D metabolite detected as 1,25(OH)2D in the circulation of a hypercalcemic, anephric patient with the granulomaforming disease sarcoidosis (Fig. 5-18). Two years later, Adams et al. ~~ determined the macrophage to be the extrarenal source of this active vitamin D metabolite with unequivocal structural characterization of the metabolite as 1,25(OH)2D obtained by these same investigators in 1985.1~ It is now widely accepted that the overproduction of 1,25(OH)2D or a 1,25(OH)2D-like metabolite can cause hypercalciuria and frank hypercalcemia in many different granuloma-forming diseases ~~ of both infectious and noninfectious origin (Table 5 - 2 ) . By the 1980s data were accumulating to suggest that a vitamin D - m e d i a t e d disturbance in calcium metabolism was not confined to patients with granuloma-

forming diseases and could also be observed in patients with lymphoproliferative neoplasms. 1~ Most recent reports 114'115 indicate that the extrarenal overproduction of 1,25(OH)2D is the most common cause of hypercalciuria and hypercalcemia in patients with non-Hodgkin's and Hodgkin's lymphoma, especially in patients with Bcell neoplasms and whether or not the tumor is associated with the acquired immunodeficiency syndrome (AIDS). 1~ There are now at least four clear lines of clinical evidence to indicate that endogenous 1,25(OH)2D production in hypercalcemic/hypercalciuric patients with granuloma-forming diseases and lymphoma is dysregulated and not bound by the same set of endocrine factors known to regulate 1,25(OH)2D synthesis in the kidney. First, hypercalcemic patients with sarcoidosis possess a frankly high or inappropriately elevated serum 1,25(OH)2D concentration, although their serum PTH level is suppressed and their serum calcium and phosphate concentration is relatively elevated. 116'117 If 1,25(OH)2D synthesis were under the regulation of PTH, phosphate, and 1,25(OH)zD itself, then 1,25(OH)zD concentrations in such patients should be low. Second, the serum 1,25(OH)zD concentration in patients with active sarcoidosis is very sensitive to an increase in available substrate, ~ls while the serum 1,25(OH)zD level in normal individuals is not. Clinically, this aspect of dysregulation

CHAPTER 5 Vitamin D Metabolism and Biological Function Mediastinal Lymph Node Bx Bilateral (granuloma) Nephrectomy a ~t

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FIGURE 5-- 18 Relation of changes in serum levels of 1,25(OH)2D, iPTH, and calcium to doses of prednisone administered to control hypercalcemia. The solid portion of the bar representing the administration of prednisone indicates daily therapy, and the open portion indicates alternate-day therapy. Bx denotes biopsy. To convert values for 1,25(OH)2D to picomoles per liter, multiply by 2.36. To convert values for calcium to millimoles per liter, multiply by 0.25. (From Barbour GL, Coburn JW, Slatopolsky E, et al: Hypercalcemia in an anephric patient with sarcoidosis, evidence for extrarenal generation of 1,25-dihydroxyvitamin D. N Engl J Med 305:440-443, 1981.)

is manifest by the long-recognized association of the appearance of hypercalciuria and/or hypercalcemia in sarcoidosis patients in the summer months 119 or following beach-going holidays to geographic locations at lower latitudes than those at which the patient normally resides. 12~ Third, the rate of endogenous 1,25(OH)2D production, which is significantly increased in patients with sarcoidosis, is unusually sensitive to inhibition by factors (i.e., drugs) that do not influence the renal vitamin D-l-hydroxylase at the same doses. For example, antiinflammatory concentrations of glucocorticoids have long been recognized as effective combatants of sarcoidTM

TABLE 5--2 Human Granuloma-Forming Diseases Associated with the Extrarenal Overproduction of an Active Vitamin D Metabolite Sarcoidosis Tuberculosis Leprosy Disseminated candidiasis Histoplasmosis Coccidiodomycosis Crytococcosis Berylliosis Silicone-induced granulomatosis Eosinophilic granuloma Wegener' s granulomatosis Massive infantile fat necrosis

139 osis-associated hypercalcemia 1~ and shown to effectively lower elevated 1,25(OH)2D levels. 117 On the other hand, giving the same amount of glucocorticoid to patients without sarcoidosis is not associated with a clinically relevant reduction in the serum 1,25(OH)2D or calcium concentration. Chloroquine and its analog, hydroxychloroquine, are other examples of agents that appear to act preferentially on the extrarenal vitamin D-1hydroxylation reaction that is active in patients with sarcoidosis. 124-126 And fourth, in patients with either granuloma-forming disease or lymphoma the serum calcium and 1,25(OH)2D concentrations are positively correlated to indices of disease activity. ]~ Investigators 1~ have now generated a substantial body of experimental data from cells, including inflammatory cells harvested directly from patients with sarcoidosis, to indicate that the dysregulated vitamin D hormone synthesis in sarcoidosis is due to expression of a 1hydroxylase that is not different from the renal 1-hydroxylase, but rather to expression of the authentic 1hydroxylase in a macrophage, not a kidney cell. In fact, each of the above mentioned pieces of clinical evidence for dysregulated vitamin D hormone production in this disease can be borne out in vitro in cells from patients with this disease. 13~

VI. ALTERNATIVE METABOLISM OF 25-HYDROXYVITAMIN D AND 1,25-DIHYDROXYVITAMIN D

A. Metabolism of 25-Hydroxyvitamin D to 24,25-Dihydroxyvitamin D The body possesses a variety of enzymes that have the capacity to transform 25(OH)D into innumerable dihydroxy, trihydroxy, and tetrahydroxy metabolites (Fig. 5--19). 48'60'76'131-133 In the early 1970s, it was appreciated that vitamin D - d e f i c i e n t rats, when provided radioactive vitamin D3, efficiently converted the vitamin first to 25(OH)D3 and then to 1,25(OH)2D3 .48']33 However, when these animals were given a diet that was high in calcium and contained vitamin D, [3H]25(OH)D3 was converted to a metabolite that was more polar and was structurally identified as 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] 135 (Fig. 5 - 1 2 ) . Despite the fact that 25(OH)D2 has a methyl group in the S configuration on carbon-24, it, too, is metabolized to 24,25(OH)2D2. 24,25(OH)2D is the major circulating metabolite of 25(OH)D, and its concentration, which is usually 2 to 4 ng/ml, is a reflection of the 25(OH)D concentration. 48'6~ Although the kidney is the primary site for its production, most other tissues that possess nuclear TM

140

MICHAELE HOLICKAND JOHN S. ADAMS

25 (,OH)D3

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23S,25(OH)2D3

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FIGURE 5--19 Pathwayof 25(OH)D3 metabolism to 25(OH)D326,23-1actone. (From Napoli JL, Horst RL: Vitamin D metabolism. In Kumar R (ed): Vitamin D, Basic and Clinical Aspects. Boston, Martinus Nijhoff Publishing, 1984.)

receptors for 1,25(OH)2D also have the enzymatic machinery to produce this metabolite (Fig. 5--20). 48'6~ The renal 25(OH)D-24-hydroxylase is a mitochondrial enzyme requiring NADPH +, molecular oxygen, and magnesium ions. 48'6~ Originally, it was believed that parathyroid hormone, which enhanced the renal production of 1,25(OH)2D, was also responsible for decreasing the synthesis of 24,25(OH)zD. However, there is now firm evidence that parathyroid hormone does not influence the renal metabolism of 25(OH)D to 24,25(OH)zD. Parathyroid hormone stimulates the production of 1,25(OH)2D. This hormone, in turn, shuts off its own renal production and enhances the kidney's production of 24,25(OH)zD. 84'101'102'136'137 The physiological role of the 24-hydroxylation of 25(OH)D is unsettled at the present time. There are reports that 24,25(OH)2D3 is capable of (1) promoting hatchability of chicken eggs, ~38 (2) directly stimulating bone formation in vitro only in the presence of parathyroid hormone and 1,25(OH)2D3,139 (3) increasing the synthesis of proteoglycans in cultured chondrocytes, 14~ (4) improving calcium retention in anephric patients, TM and (5) more effectively promoting bone mineralization in humans when combined with 1,25(OH)2D3 than 1,25(OH)2D3 could by itself. ~42However, it is known that

24,25(OH)2D3 at physiological concentrations is biologically inert in anephric rats in inducing either intestinal transport or bone calcium mobilization. ~43 Its biological activity is restored when it is hydroxylated on carbon-1 to act as an analogue of 1,25(OH)2D3. This trihydroxy metabolite, 1,24,25-trihydroxyvitamin D3, is less active than 1,25(OH)2D3 in the stimulation of intestinal calcium transport and bone calcium mobilization. 143Furthermore, this 1-hydroxylated metabolite does not have any specific function in enhancing bone mineralization. To further evaluate the role of the carbon-24 hydroxylation of 25(OH)D3, an analogue of 25(OH)D3 that had its hydrogens at carbon-24 replaced with flourines (thus preventing this analogue from being hydroxylated on carbon-24), was synthesized and its biological activity evaluated. It was found that the 24,24'-difluoro-25hydroxyvitamin D3 has the same biological activity as 25(OH)D3 in (1) enhancing intestinal calcium transport, (2) mobilizing calcium from bones, and (3) healing rachitic lesions in rats. ~44 These results suggest that the carbon-24 hydroxylation of 25(OH)D3 is not essential for the physiological actions of vitamin D3 in the rat. An alternative explanation for the presence of the 25(OH)D24-hydroxylase in the kidney as well as other tissues that possess receptors for 1,25(OH)2D3 is that this enzyme acts as the initiator for the side-chain catabolism of 25(OH)D and 1,25(OH)2D. 145

B. Side-Chain and A-Ring Metabolism of 25(OH)D3

and 1,25(OH)zD3

Under physiological conditions, 25(OH)D is metabolized on carbon-26 to form 25S,26-dihydroxyvitamin D3 [25,26(OH)zD3] (Fig. 5--12). 146 The circulating concentrations of 25,26(OH)zD are also reflective of the vitamin D nutritional status of humans. Although 25,26(OH)zD3 does not have any special biological functions, it can mimic the biological actions of 1,25(OH)zD3 once it is hydroxylated in the kidney, 1,25S,26trihydroxyvitamin D3 (Fig. 5-12). 132'147Although little is known about the regulation of this metabolic step, it appears that the kidney is the principal site for this metabolism. The metabolism of 25(OH)D3 on carbon-23 yields a 23S,25-dihydroxyvitamin D3 (Fig. 5--19). 132'147 This metabolite is apparently a precursor for an additional metabolic step that gives rise to a rather unusual metabolite that has been identified as 25-hydroxyvitamin D323,26-1actone [25(OH)D3-1actone] (Fig. 5-19). 132 This lactone is found in the circulation of humans and other animals that have received pharmacological doses of vitamin D. Although there is no evidence that this lactone has vitamin D - l i k e activity on either the intestine or

CHAPTER5 Vitamin D Metabolism and Biological Function

141

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FIGURE 5--20 Proposed mechanism of action of 1,25(OH)2D3 in target cells resulting in a variety of biological responses. The free form of 1,25(OH)2D3 (D3) enters the target cell and interacts with its nuclear vitamin D receptor (VDR), which is phosphorylated (P). The 1,25(OH)2D3-VDR complex combines with the retinoic acid X receptor (RXR) to form a heterodimer, which, in turn, interacts with the vitamin D-responsive element (VDRE), causing an enhancement or inhibition of transcription of vitamin D-responsive genes such as the 25-OH-D-24-hydroxylase (24OHase). (From Holick MF: Vitamin D: Photobiology, metabolism, mechanism of action, and clinical application. In Favus MJ (ed): Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, 3rd ed. Philadelphia, Lippincott-Raven, 1996, pp 74-81.)

bone, this compound is made in the kidney. It is curious that the vitamin D - b i n d i n g protein recognizes this metabolite with a binding affinity that is about five to seven times greater than that for 2 5 ( O H ) D 3 .48'132 In addition to the multiple hydroxylations that can occur in the side chain, the hydroxyls can also be oxidized. 24,25(OH)2D3 and 23,25(OH)2D3 are oxidized to 24-keto-25(OH)D3 and 23-keto-25(OH)D3, respectively. 48'132 The multitude of hydroxylations and oxidations that 25(OH)D3 can undergo are also seen for 1,25(OH)2D3. Therefore, the side chain of 1,25(OH)2D3 is recognized as a 25(OH)D3 substrate and can undergo further oxidation and hydroxylation at carbon23, carbon-24, and carbon-26 to form a number of metabolites including 1,24,25(OH)D3; 1,25,26(OH)D3; 24-keto-l,23,25(OH)3D3; 1,23,25(OH)3D3; and 1,25(OH)2D3-23,26-1actone. 48'6~ It is believed that the metabolic importance of the side-chain modifications is for the deactivation and rapid clearance of 1,25(OH)2D3. This is particularly true for the carbon-23 oxidation, which ultimately gives rise to a biologically inactive

water-soluble acid derivative known as let(OH)24, 25,26,27-tetranor-23(COOH) vitamin D3 (calcitroic a c i d ) . 48'6~ To date, more than 22 metabolites of vitamin D have been structurally identified. All of these metabolites are less biologically active on a weight basis than 1,25(OH)2D. In addition to the multiple hydroxylations in the side chain, 25(OH)D3 can have its A-ring oxidized whereby the carbon-19 is replaced with a keto group. This reaction occurs in vivo in ruminants and in vitro in chick kidney homogenates, resulting in the conversion of 25(OH)D3 to the cis and trans isomers of 10-keto-19-

nor-25-hydroxyvitamin D3.48'60'132

VII. METABOLISM

OF VITAMIN

D2

In the 1930s it was first appreciated that the vitamin D isolated from the irradiation of the sterol ergosterol yielded a vitamin D (vitamin D2) that had minimum biological activity in the chicken when compared with the

142 vitamin D (vitamin D3) that was isolated from the irradiation of 7-dehydrocholesterol. 2~-23 It is now recognized that vitamin D2 is about 10 to 20 times less active than vitamin D3 in the chicken. It has been assumed in mammals, including humans, that vitamin D2 and vitamin D3 have equal biological potency and are metabolized in identical fashion. However, it is known that in New World monkeys, as in chickens, the activities of vitamin D2 and vitamin D3 differ. 6~ Originally, it was believed that vitamin D: is metabolized in a fashion identical to that of vitamin D3. However, there are some exceptions. It is now recognized that the vitamin D-binding protein binds vitamin D2 1.5 to 2 times less efficiently than vitamin D3. This difference may be important because higher concentrations of the free form of vitamin D2 can enter into the liver parenchyma and be more efficiently metabolized to 25(OH)D2. 6~ Indeed, when a rat liver is perfused with an equal amount of vitamin D2 and vitamin D3, vitamin D2 is preferentially metabolized to 25(OH)D2 when compared with vitamin D3.51 Similar to vitamin D3, vitamin D: is metabolized to 25(OH)D2, which, in turn, is metabolized to 1,25-dihydroxyvitamin D2, 24R,25dihydroxyvitamin D2, and 25S,26-dihydroxyvitamin D2.149 1,25(OH)2D2 is further metabolized to 1,24,25trihydroxyvitamin D 2 u a process believed to be important for the deactivation of 1,25(OH)zD2. 6~176 Because vitamin D2 has a methyl group at carbon-28, unlike vitamin D3, this carbon is a target for a further hydroxylation to give rise to a variety of 28-hydroxylated metabolites, including 24,25,28-trihydroxyvitamin D~ and 1,24,25,28-tetrahydroxyvitamin D2.152 The physiological role of these metabolites remains uncertain; however, it is interesting to consider the possibility that 24,25, 28(OH)3D2 and 1,24,25,28(OH)4D2 could be metabolized so that the carbon-28 methyl is oxidized and removed to yield a side chain that looks more like the side chain for vitamin D3 .6~

VIII. BIOLOGICAL ACTIONS OF 1,25(OH)2D A. Cellular Mechanism of Action: Genomic There is agreement that most of the actions of 1,25(OH)2D to alter gene transcription directly are initiated by interaction of the hormone with the vitamin D receptor (VDR) (Fig. 5--20). 153-158 Similar to other steroid hormones, 159 the mammalian VDR is a rare intracellular protein. Depending on the species from which it is derived, the VDR has a molecular weight in the range of 48,000 to 60,000 daltons; the human VDR is the smallest VDR yet characterized. 48'16~It selectively binds the active metabolite, 1,25(OH)2D, with high affinity

MICHAEL E HOLICK AND JOHN S. ADAMS

(equilibrium dissociation "~10 -1~ M). 155 The classic dogma 159 for steroid hormones holds that the hormone traverses the cell membrane and binds to a receptor in the cytosol of the cell. Binding of the steroid to its specific receptor "activates" the receptor and promotes binding of the receptor to acceptor sites (regulatory sequences) on nuclear DNA. Binding of the hormonereceptor complex in the nucleus regulates the transcription of hormone-specific mRNAs which, in turn, govern the translation of protein. As has been recently suggested for other steroid hormones, 161-163 such a simple scenario may not be the case with 1,25(OH)zD3. Autoradiographic studies with radiolabeled 1,25 (OH)2D3164 as well as immunoperoxidase staining with anti-VDR monoclonal antibody 165 demonstrate predominantly nuclear localization of both the 1,25(OH)zD hormone and the VDR in target cells. Nuclear localization of the VDR and 1,25(OH)zD most likely represents specific binding of the VDR to the "downstream half site" of the vitamin D response element (VDRE) on genomic DNA and specific binding of the hormone to the receptor, respectively (Fig. 5-21B). 166-168 However, this interaction of the 1,25(OH)zD-VDR complex requires at least one other accessory protein, the retinoid X receptor (RXR). 169 Like the VDR, the RXR is a member of the steroid receptor superfamily and when given the opportunity will specifically bind 9-cis retinoic acid. ~7~When paired with the unliganded RXR, the VDR assumes a conformation that promotes 1,25(OH)zD binding and stable RXR-VDR dimer formation. These events, in turn, enhance the ability of the DNA binding domains of the RXR-VDR heterodimer to interact with the VDRE. When all critical elements of the receptor-DNA binding reaction are in place, the RXR, the VDR, 1,25(OH)zD and VDRE, then it is proposed that this complex promotes transcription of the vitamin D-responsive gene by interacting with other key elements (protein complexes) of the cell' s transcription machinery. ~7~A specific nucleotide sequence for a VDRE has now been identified in the promoter of six different genes recognized to be important in mammalian and avian mineral homeostasis (Fig. 5-21B). With the exception of the calbindin-28K gene VDRE in the mouse whose composite half sites are separated by four instead of three nucleotides, ~6~ all VDREs so far characterized are made up of an imperfect direct repeat of a hexnucleotide sequence separated by a three-nucleotide "spacer." Although the VDR's hormone-binding domain is distinct and at some intramolecular distance from the DNAbinding domain, ~56 as just discussed, it is apparent that these two separate regions of the protein are functionally linked168; hormone binding induces a covalent modification (phosphorylation) of the receptor, promotes heterodimerization with RXR, increases the receptor's af-

CHAPTER 5

143

Vitamin D Metabolism and Biological Function

C m Genes Regulated by a Vitamin D Response Element Gcne

~

,

Tissue

_

osteocalcin

bone

ostepontin

bone

1~3 integrin

bone

cal bind in- 28K

intestine

parathyroid hormone

parathyroid gland

vitamin D-24-hydroxylase

diverse

FIGURE 5--21 Panel A provides a schematic view of the functional domains of the vitamin D receptor. With the possible exception of a hormone-independent transcriptional activation domain in the amino terminal region of the receptor molecule, all residues (shown as dark lines) known to be important in hormone binding, receptor dimerization, and transcriptional activation reside in the carboxy terminal region of the receptor; this includes the phosphorylation site on the serine residue at position 208 which is considered important for the initiation of ligand-directed dimerization and transactivation. Panel B shows the preferred association of the hormone-occupied vitamin D receptor (VDR) with the unliganded retinoid X receptor (RXR) and interaction of the dimer partners with the consensus vitamin D response element (VDRE). Panel C summarizes those genes whose promoters are known to harbor a functional VDRE which acts to enhance gene transcription.

finity for binding to DNA, and activates (initiates) transcription. The domains of the V D R responsible for each of these functions is pictured schematically in Figure 5 - 2 1 A . Most noticeable is the spacial separation of the DNA-binding domain in the amino-terminus and the hormone-binding domain in the carboxy-terminus of the VDR. These two domains are separated by the so-called " h i n g e " region of the molecule. The " h i n g e " can vary substantially in size, and it is this part of the V D R that generally accounts for the variability in mass of the receptor from species to species. Also of interest is the fact that, aside from D N A binding, all major functions of the V D R reside within the hormone-binding domain. 16~ In fact, the functions of h o r m o n e binding, di-

merization, and transcriptional activation are reliant upon amino acid residues at the extremes of the hormone-binding domain. 168 The " p o l a r i t y " of these functional residues suggests that the central portion of the hormone-binding d o m a i n may be looped out in three-dimensional space, allowing relatively distant residues to c o m e in close contact with one another; confirmation of this hypothesis awaits x-ray crytallographic characterization of the VDR. In conclusion, the trancription of genes (Fig. 5 - 2 1 C ) under the influence of 1,25(OH)2D is subject to control at several different levels. This includes (1) the level of 1,25(OH)2D to which the target cell is exposed, (2) access to and binding of 1,25(OH)zD3 to the VDR, (3) the

144 appropriate phosphorylafion of the VDR, at least at the serine residue in position 208,16~ (4) the state of dimerization with the RXR or other potential dimer partners, 172'173 and (5) the access and interaction of the receptor complex with the VDRE; the latter may be determined by both the specific sequence of the response element as well as the presence of other proteins in the nucleus of the cell that may be competitive for the same D N A sequence 174-176 or competitive for the receptor complex itself. 173

B. Cellular Mechanism of Action: Nongenomic Binding of the 1,25(OH)2D3 to the VDR, binding of receptor-hormone to specific DNA sequences, gene transcription, and new protein synthesis may not be a prerequisite for all of the cellular actions of 1,25(OH)2D3. TM In fact, of the more than fifty documented, specific biological actions of the vitamin D hormone, a minority have been confirmed to take place at the level of gene transcription. 16~ In the intestinal epithelial cell, for instance, there is evidence that receptor-bound 1,25(OH)2D3 activates transcription of the calbindin 28K gene, 176 which codes for a calcium-binding protein postulated to be important in the transcellular transport of calcium in the intestine. 177'178However, administration to animals of inhibitors of either transcription or translation, which terminate 1,25(OH)2D3-directed CaBP production, will not block the 1,25(OH)2D3-mediated uptake of calcium across the luminal membrane of the cell. 179'18~ Furthermore, time-course experiments indicate that calcium uptake by the enterocyte precedes or coincides with the synthesis of hormone-specific CaB P, additional evidence that this 1,25(OH)2D3 effect is not dependent on new protein synthesis. Investigators have also demonstrated a 1,25(OH)zD3-specific change in the lipid composition of the brush border membrance that (1) either preceded or occurred simultaneously with the change in calcium transport rate across the membrane and (2) was also not affected by the administration of inhibitors of protein synthesis. These kinds of experimental results led to investigation of a direct effect of 1,25(OH)2D3 on the cell membrane. 181'182 In fact, currently available evidence suggests that the interaction of 1,25(OH)2D3 with a target cell may result in hormonespecific alterations in the cell at two levels, one of which is dependent on nuclear localization of the h o r m o n e VDR complex and another that is independent of traditional transcriptional events. TM The best characterized nongenomic action of 1,25(OH)zD3 is in fact the rapid transmembrane transport of calcium or "transcaltachia," 183 which is initiated by the opening of voltage-gated calcium (Ca 2+) channels in the

MICHAEL E HOLICK AND JOHN S. ADAMS

membrane. Vitamin D hormone-driven transcaltachia has been demonstrated to occur in the plasma membranes of intestinal epithelial cells, osteoblast like cells, hepatocytes, muscle, and parathyroid cells. TM According to Nemere et al., 184 transcaltachia is initiated by the interaction of the vitamin D compound with a plasma membrane receptor protein. Although not yet purified or extensively characterized from a functional standpoint, 131'185on the basis of its preference to bind vitamin Ds with a 6-s-cis rather than a 6-s-trans conformation, this plasma membrane receptor is proposed to be distinct from the nuclear VDR protein. It is proposed that when "activated" with ligand the putative membrane receptor opens a voltage-dependent Ca 2+ channel in the membrane that in tum activates phospholipases coupled to a variety of intracellular signaling pathways. 186

IX. BIOLOGICAL ACTIONS OF 1,25(OH)2D IN TISSUES REGULATING CALCIUM BALANCE A. Actions of 1,25(OH)2D on the Intestine The intestine was one of the first target tissues in which the actions of vitamin D and its active metabolite 1,25(OH)2D were carefully studied187; 50% or more of active intestinal calcium transport is vitamin D dependent. Vitamin D facilitates (1) the entry of calcium into the enterocyte, (2) the transit of calcium through the cell, and (3) the exit of the cation into the extracellular space at the basolateral membrane of the enterocyte. There are a number of vitamin D-dependent proteins in the intestine that appear to be temporally related to the transcellular transport of calcium. 188 At the brush border (luminal) membrane of the intestinal epithelial cell there is the aforementioned voltage-gated calcium channel that is functionally linked to a locally stimulated plasma membrane receptor for vitamin D and provides access of luminal calcium ions to the interior of the enterocyte. Once inside the cell, calcium binds to the high-affinity intestinal calcium binding protein, calbindin-28K. The mRNA for calbindin-28K gene is regulated by 1,25(OH)2D at both the level of transcription and message stability. 169 The calbindin-28K protein is proposed to be critical in buffeting the normally low cytoplasmic calcium concentration and in the transcellular movement of calcium. 178'187-189 Finally, there is a vitamin D responsive Ca-ATPase present in the basolateral membrane of the enterocyte. This protein is proposed to be the pump in the antiluminal membrane responsible for the movement of calcium out of the cell against a strong electrochemical gradient187; the mode of its regulation

145

CHAPTER5 Vitamin D Metabolism and Biological Function under the influence of 1,25(OH)2D, whether direct via the VDR or plasma membrane vitamin D receptor or indirect, is not known. 16~

these direct actions are controlled by the plasma membrane vitamin D receptor instead of the nuclear VDR is unknown.

B. A c t i o n s of 1,25(OH)2D in B o n e

C. A c t i o n s of 1,25(OH)2D in K i d n e y

Although vitamin D-deprived states in animals and man have long been associated with failure to normally mineralize bone, the prevailing view is that neither vitamin D nor any of its metabolites are directly responsible for the process of skeletal mineralization. Rather, 1,25(OH)2D, through its ability to stimulate the intestinal absorption of mineral ions, is critically important in the supply of calcium and phosphate to bone for adequate hydroxyapatite formation. 94 There is little convincing evidence that the hormone influences mature bone formation in vivo. In fact, more convincing are data that support a key role for the vitamin D hormone in regulation of the mobilization of calcium from bone. 19~ However, if 1,25(OH)2D does have a direct effect on bone and that effect is mediated at the genomic level, it is most likely controlled through the bone-forming cell, the osteoblast; expression of the VDR gene can only be demonstrated in mature osteoblasts, ~9~ not osteoclasts. For example, 1,25(OH)zD-mediated recruitment and differentiation of osteoclast progenitors into osteoclast-like cells appears to be dependent upon the physical presence of osteoblasts (or preosteoblasts), m Furthermore, it has been shown that exposure of osteoblasts to 1,25(OH)zD results in release of osteoclast-activating activity (OAF) that acts on neighboring bone-resorbing cells to mobilize calcium from the skeleton. ~5~ In vitro 1,25(OH)zD increases calcium uptake, IGF-1 receptor expression, and synthesis of specific collagenous and noncollagenous proteins in mature osteoblast-like c e l l s . 19~ More recently, the hormone has also been shown to downregulate expression of the id gene in osteoblast-like cells. 197This gene product is a member of the helix-loophelix family of proteins, which are suppressors of cell differentiation; 1,25(OH)zD-mediated suppression of this suppressor of differentiation is in accord with the acknowledged tenet that vitamin D hormones, like the retinoids, promote the progression of a cell to a more differentiated phenotype 198 (see section below). However, there may be some exceptions to the tenet that 1,25(OH)zD acts only indirectly on the osteoclast. 1,25(OH)zD appears to be vitally important in the promotion of osteoclast progenitor cell fusion into functionally competent, multinucleated osteoclasts ~9~ and may directly promote an increased [33 integrin expression in the osteoclast's bone-resorbing ruffled border199; integrin molecules appear to be key in localizing and anchoring osteoclasts to the resorbing surface of bone. Whether

Unlike PTH for which there is a clear role in the renal handling of phosphate and calcium and in stimulating 1,25(OH)2D production, 189 there is little evidence that 1,25(OH)2D3, or any other vitamin D metabolite or analog for that matter, has a direct physiological effect on the management of filtered calcium or phosphate) ~176 Although the VDR as well as vitamin D-responsive calcium-binding proteins (calbindins) are found in the distal nephron segments, their participation in regulating transcellular calcium transport in this part of the kidney is not well defined. 16~ Compared to some other tissues, the kidney is relatively enriched in the 1,25(OH)2Dinducible vitamin D 24-hydroxylase) ~ Interaction of 1,25(OH)2D with the VDR in renal tubule preparations results in enhanced expression of the 24-hydroxylase gene 2~ and increased local catabolism of 25(OH)D and 1,25(OH)2D.

D. A c t i o n s o f 1,25(OH)2D on P T H P r o d u c t i o n In comparison to the effects of 1,25(OH)2D to increase transcription of genes whose promoters contain the "classical" VDRE (Fig. 5-21C), 1,25(OH)2D will inhibit the synthesis and secretion of PTH by inhibition of PTH gene transcription. 2'2~176 In fact, 1,25(OH)2D administration has become the therapy of choice for patients with chronic renal failure and secondary hyperparathyroidism. T M Repression of transcription of the human PTH gene under the influence of 1,25(OH)2D is apparently mediated through a single, seven-nucleotide VDRE (AGGTTCA) rather than the two six-nucleotide half-site motifs that legislate up-regulation of transcription. 16~

X. A C T I O N S METABOLITES NONCLASSICAL

OF VITAMIN D AND ANALOGS TARGET

IN

TISSUES

A major impetus for investigators to search for a function of active vitamin D metabolites in tissues other than intestine, bone, kidney, and the parathyroid gland were studies documenting the localization of the VDR and radiolabeled 1,25(OH)2D in a variety of tissues not previously thought to be a site for the action of the hormone (Table 5-3). It should be pointed out that many of these

146

MICHAEL E HOLICK AND JOHN S. ADAMS

TABLE 5--3 1,25(OH)2D3 Receptor Distribution Among Mammalian Tissues Intestine Kidney Bone Parathyroid Brain Pituitary Parotid Pancreas Stomach Skin

Thymus Lymphocytes Monocytes/macrophages Testes Ovary Uterus Placenta Breast Embryonic liver Embryonic muscle

less of progress in analog development, it is of considerable interest to note that the naturally occurring hormone 1,25(OH)2D still appears to be the most effective vitamin D metabolite in "side-by-side" testing with synthetic analogs irrespective of the cell, tissue, or disease process being investigated. 131'2~ The apparent superiority of the naturally occurring hormone begs the question whether 1,25(OH)2D is operating through genomic, nongenomic, or both pathways in cellular targets responsive to synthetic analogs.

B. D i f f e r e n t i a t i n g and A n t i p r o l i f e r a t i v e Effects o f 1,25(OH)2D nonclassical target tissues and proposed "alternative" functions of 1,25(OH)2D were initially discovered in experiments performed in vitro, so the physiological significance of some of these effects remains to be definitively elucidated in vivo in humans. What follows is a discussion of the effects of vitamin D metabolites and analogs in tissues and cells where these "alternative" actions have been best characterized. There is no doubt that our knowledge of these and of a number of less well-characterized functions of the family of vitamin D molecules in other tissues will be expanding in the future. However, what we do know currently is that many of these functions of 1,25(OH)2D are not directly related to maintenance of mineral ion homeostasis. Furthermore, we know that some of these "alternative" actions may be affected by analogs of vitamin D that do not have the same calcium regulating potential of the naturally occurring hormone, 1,25(OH)2D; implicit in this argument is the suggestion that actions of these vitamin D analogs are not transduced through the VDR. 2~176

A. N o n h y p e r c a l c e m i c A n a l o g s of V i t a m i n D 1,25(OH)2D is attractive as a therapeutic agent. The hormone is active at relatively low circulating concentrations, it has an easily manageable and manipulatable biological half-life in the human host, it can be measured in the circulation with relative ease, and it can be taken orally. The problem is its narrow therapeutic index; when employed in concentrations great enough to transduce the desired effect in the target cell or tissue of interest, the vitamin D hormone is also likely to induce hypercalciuria and/or hypercalcemia. This has prompted investigators and pharmaceutical concerns to design and develop vitamin D analogs capable of transducing a specific effect without putting the patient at risk for hypercalciuria, renal stone disease, possible renal insufficiency, and eventually chronic hypercalcemia. 198 Many such compounds have now been developed. TM Regard-

Because of the identification in the VDR in cells of the myeloid series, the first evidence that 1,25(OH)2D possessed potent differentiating activity was provided by workers examining the effects of the hormone on mouse and human myeloblastic leukemia cell lines. Under the influence of 1,25(OH)2D these cells could be induced to differentiate into monocyte/macrophages (Table 5-3). The differentiating effect of 1,25(OH)2D3 has been extended to human cells; investigators showed that 1,25(OH)2D would promote the maturation of mononuclear cells from human bone marrow into monocytes or multinucleated macrophages. 2~176 In conjunction with its ability to promote the differentiated phenotype, 1,25(OH)2D has also been shown to exert an antiproliferative effect on almost every animal or human cultured cell population, malignant or nonmalignant, in which it has been examined. 21~ In fact, inhibition of cell proliferation by 1,25(OH)2D has been used as a bioassay of the hormone-receptor interaction in cultured human cells. 166 The universality of the prodifferentiating and antiproliferative effect of 1,25(OH)2D suggests that this action of the hormone is not cell specific. In fact, investigators have recently discovered that the endogenous cell cycle-blocking protein p21, a cyclin-dependent kinase inhibitor, is under the transcriptional control of 1,25(OH)2D. 217 When activated, the VDRE in the p21 promoter in the poorly differentiated myelomonocytic cell line U937 up-regulates transcription of p21, driving the cell to a more differentiated macrophage-like phenotype. These observations suggested early on that 1,25(OH)2D may be useful as adjuvant or even primary therapy for human neoplastic disorders. Experimental results with animals showed promise that the antiproliferative effect of the hormone might also be observed in vivo. The survival time of mice inoculated with myeloid leukemia cells can be prolonged, 213 while growth of human VDR-bearing tumor xenografts in immunoincompetent mice can be slowed by parenteral administration of

CHAPTER5 Vitamin D Metabolism and Biological Function 1 , 2 5 ( O H ) 2 D . 214 Unfortunately, the results of the first clinical trials of vitamin D metabolites and analogs in the management of myelodysplastic disorders have been disappointing. 218"219Nonetheless, investigation in this direction continues. 198 The differentiating effects of 1,25(OH)2D on two other cell types has received considerable attention in the recent past. As just discussed, it is now known that 1,25(OH)2D (1) can act as an important switch to direct marrow stem cells away from macrophage development and toward osteoclastogenesis 19~ and (2) can induce the fusion of monocytes into multinucleated giant cells with bone-resorbing potential in vitro. 2~ Given the fact that mature osteoclasts lack the V D R , 216 it is tempting to speculate that the action of 1,25(OH)2D at the level of bone is to amplify the pathway of maturation of stem cells in bone into multinucleated osteoclasts. The differentiation program of keratinocytes, skin cells that give rise to the epidermis, can also be altered by 1,25(OH)2D and related analogues. 22~As will be discussed below, this action of vitamin D metabolites and derivatives has been successfully transformed into therapy of patients with hyperproliferative, dedifferentiating epidermal diseases like psoriasis.

147 TABLE 5--4 Immunological Actions of Active Vitamin D Metabolites and Analogs Target Cell

Action

Macrophage or Antigen-presenting cell

q" Differentiation 1" p21 expression $ Apoptosis $ Proliferation J, c-myc expression 1" chemotaxis 1" [32 integrin expression 1" cell adhesion 1" giant cell formation 1" phagocytosis 1" reactive oxygen metabolite production 1" cidal activity 1" Fc receptor expression 1" IL-1 expression and secretion 1" hsp synthesis ,1, IL-12 expression and production $ HLA-DR, -DE -DQ expression $ TH cell profileration 1" TS cell proliferation $ IL-2 ,l, IFN-gamma ,l, IL-4 $ IL-5 ,l, IL-6 ,l, IL-7 $ TH cell-directed IgG2A production ,], delayed-type hypersensitivity ,[, IL-12 ,], cytotoxic capacity

Activated T-lymphocyte

C. Immunoregulatory Effects of 1,25(OH)2D The expression of the VDR in mitogen- or antigenactivated human lymphocytes and peripheral blood monocytes 221-226 as well as the ability of stimulated macrophage-like cells to make 1,25(OH)2 D227 suggests that production and interaction of active vitamin D metabolites with cells in the lymphocyte and monocyte/ macrophage lineage may modulate the immune response in animals, including man. A comprehensive compilation of the various effects of 1,25(OH)zD on immune cells is provided in Table 5 - 4 . It is important to point out that the immunomodulatory effects of endogenously synthesized 1,25(OH)zD have not been detected in v i v o in the peripheral blood of human subjects. Therefore, it is likely that the circulating concentration of 1,25(OH)zD is quantitatively inadequate to modulate the peripheral immune response. More likely is the possibility that the hormone is designed to act in an intracrine, autocrine, or paracrine mode at tissue sites of inflammation and not as an endocrine modifier of the human immune response. 224'228 Looking carefully at Table 5 - 4 , one can discern that the actions of 1,25(OH)ED and related analogs are generally directed toward stimulation of lymphokine-driven macrophage f u n c t i o n 228-231 o n the one hand and inhibition of lymphocyte responsiveness on the o t h e r . 229-233 The former includes promotion of macrophage phago-

Activated B-lymphocyte Natural killer cell

cytosis, killing, antigen processing, antigen presentation, and adjuvant monokine production. By contrast, the latter generally includes inhibition of the proliferation and action of the T-helper lymphocyte, type 1 (TH1) population, a33 including inhibition of (1) T-cell-directed immunoglobulin ( I g G 2 A ) p r o d u c t i o n , (2) lymphokine synthesis, and (3) generation of the delayed-type hypersensitivity r e a c t i o n . 224-226'229"23~ While there is abundant information on the effects of active vitamin D compounds on stimulated T-helper cell function, relatively little is known regarding the direct effects of active vitamin D metabolites and analogs on T-suppressor cell, natural killer (NK) cell, and B-lymphocyte function. Preliminary data suggest that 1,25(OH)2D may inhibit Tsuppressor cell proliferation, NK-cell activity, and selectively block IL-12 secretion by B-lymphocytes. 233

D. Potential Endocrine Effects of 1,25(OH)2D on Immune Cells When given parenterally, 1,25(OH)2D3 and related analogs have been shown to inhibit the development of

148

MICHAEL E HOLICKAND JOHN S. ADAMS

TH1 lymphocyte-directed autoimmune disease in mice, such as diabetes, systemic lupus erythematosis, and autoimmune encephalitis, and transplantation rejection in experimental animals. 131'23~ The potential utility of vitamin D derivatives in the treatment of human autoimmune disorders or in the prevention of tissue rejection in human organ transplant recipients has not yet been assessed. Furthermore, increased circulating concentrations of 1,25(OH)2D from either endogenous overproduction of the hormone (as can occur in some patients with granuloma-forming diseases or lymphoma) or from exogenously administered 1,25(OH)2D have not been shown to alter the function of either circulating monocytes or lymphocytes in man.

E. E f f e c t s o f 1,25(OH)zD3 on S k i n C e l l s In addition to being the source of vitamin D3 synthesis, 27 mammalian skin also appears to be a target for the active form of the hormone. Receptors for 1,25(OH)zD3 have been identified in rodent skin 236 and in cultured dermal fibroblasts and keratinocytes 212 from human hosts. Among the reported effects of 1,25(OH)zD3 on cultured human cells are inhibition of proliferation of fibroblasts and keratinocytes, 212 stimulation of 7dehydrocholesterol (provitamin D3)synthesis, 237'23s cornification of keratinocytes, 239 and increased melanogenesis in melanoma cells. 24~ The lack of bioeffective receptors for 1,25(OH)2D3 in the developing skin of patients with vitamin D - d e p e n d e n t rickets type II has been suggested as a cause for the alopecia observed in some of these patients242; however, administration of large doses of 1,25(OH)2D3 in such patients does not stimulate hair growth. 243 Oral (Fig. 5 - 2 2 ) 80,244 as well as topical

administration of 1,25(OH)2D3245 to patients with psoriasis, a hyperproliferative disorder of the epidermis, has been shown to have remarkable therapeutic effects. Several other analogs of 1,25(OH)zD3 have been topically administered for the treatment of p s o r i a s i s . 243'246-25~ One of the most widely studied 1,25(OH)zD3 analogs for the treatment of psoriasis is calcipotriol (calcipotriene). 185'246'251 This analog is rapidly metabolized, and therefore, has minimal calcemic activity. This medication is now considered the treatment of choice for psoriasis.

XI. ASSAYS FOR VITAMIN D AND I T S METABOLITES The ability to measure with precision vitamin D2 and vitamin D3 (collectively termed vitamin D) and their metabolites in the serum or plasma of human subjects has improved dramatically in the last 10 to 15 years. Before 1971, when the first competitive protein-binding assays for 25(OH)D were reported, 252'253 an estimate of the circulating concentration of active vitamin D metabolites was obtained solely by bioassay techniques. The "antirachitic" activity of human serum was determined by feeding a lipid extract of human serum to rats that were maintained on a high-calcium, low-phosphorus, vitamin D-deficient diet. One week later, the radii and ulnae were obtained, split, and stained with silver nitrate. The thickness of the line of new mineralization at the epiphyses "line test" was a measure of the effectiveness of the extract. 254 The ability of extracts of human serum to augment 45Ca transport in the duodenum of vitamin D deficient animals was another technique that was employed to measure vitamin D activity. 255'256 The advent

FIGURE 5--22 Viewsof the back of the legs of a female patient with erythrodermal psoriasis prior to treatment (A) and after 3 months of treatment with a daily oral dose of 2.0 lxg of 1,25(OH)2D3 (B). (From Holick MF: Vitamin D and the kidney. Kidney Int 32:912, 1987.)

CHAPTER 5 Vitamin D Metabolism and Biological Function

149

of competitive protein-binding assays and the discovery that vitamin D was metabolized to more active compounds 257'258 ushered in the modem era of vitamin D metabolite essay technology. The introduction of highperformance liquid chromatography (HPLC) for sample purification and the synthesis of radioligands of high specific activity are the two most important technological advances that have allowed the measurement of the less plentiful metabolites of 25(OH)D. For instance, it is now possible to detect 1,25(OH)2D, the active metabolite of the hormone, as low as 4.5 pg (10 fM) in a single milliliter of serum. 259 Although the progress in assay technology has been rapid in terms of the vitamin D metabolites that have been measured, only measurements of the serum levels of 25(OH)D and 1,25(OH)2D have been shown to be of clinical utility. For that reason, particular attention will be paid to a description of the methods and significance of assaying these two metabolites and only brief mention will be made of the measurement of vitamin D and some of its other metabolites.

A. Assay Techniques 1. MEASUREMENT OF VITAMIN D2 AND VITAMIN D3 Quantitation of vitamin D2 and vitamin D3 in the serum is an arduous task and, at the present time, provides little useful information to the clinician. Because vitamin DE and vitamin D3 are less polar than their hydroxylated metabolites, they cannot be purified or adequately resolved from one another on normal-phase HPLC. Therefore, if direct quantitation of vitamin D2 and vitamin D3 is desired, at least 2 ml of serum must be extracted and the lipid extract subjected to preparative open-column chromatography prior to purification on reverse-phase HPLC and quantitation on straight-phase HPLC (Table 5 - - 5 ) . 260-262 Although serial determinations of the vitamin D3 concentrations may provide an index of the

TABLE 5--5 25(OH)D

1,25(OH)2D

amount of vitamin D3 synthesized in the skin after exposure to UV-B radiation (Fig. 5 - - 7 ) , 39 it is not an adequate measure of the vitamin D status of the individual. This is simplified in Figure 5 - 2 3 , which shows the serum vitamin D concentrations in a group of normal subjects, all of whom had a normal serum concentration of 25(OH)D, who were sampled once during the summer and again in the winter. Because of the rapid disappearance of vitamin D from the serum, 39 the serum vitamin D concentration is variable and unpredictable. It may even be low in the summer if the person was not recently exposed to a significant amount of sunlight. However, there is now evidence suggesting that the serum vitamin D2 level may be useful in judging the adequacy of absorption of an oral dose of 50,000 IU of vitamin D2 (see Section III for details), thus providing a sensitive index of intestinal fat absorption and chylomicron formation. 46,263 2. MEASUREMENT OF 25-HYDROXYVITAMIN D2 AND 25-HYDROXYVITAMIN D3 As shown in Table 5 - 5 , the approach to determination of the 25(OH)D concentration may be varied, depending on the clinical situation and whether it is important to know the relative contributions of 25(OH)D3 and 25(OH)D2 to the overall circulating concentration of 25(OH)D. In assays not employing HPLC with direct UV absorbance for detection, a 25(OH)D concentration can be obtained easily from a few hundred microliters of serum. In all routine 25(OH)D assays, regardless of the method of detection, lipid extraction of the serum is necessary to dislodge the compound from the protein constituents of the serum that bind 25(OH)D. Lipid extraction for the 25(OH)D assay can be accomplished with a variety of solvents including chloroform and methanol, 264 methylene chloride and methanol, 265 diethyl ether, 216 acetonitrile, 267 or simply absolute ethanol. 268 The crude lipid extract may then be placed directly into a competitive protein-binding assay with some form of

Indications for Assay

Vitamin D deficiency Intoxication with exogenously administered vitamin D or 25(OH)D3 Following therapy with exogenously administered vitamin D or 25(OH)D3 Vitamin D-dependent rickets, type I Vitamin D-dependent rickets, type II Hypercalcemia of sarcoidosis and other granulomatous diseases Humoral hypercalcemia associated with lymphoma Intoxication with exogenously administered let(OH)D3 and 1,25(OH)zD3 Absorptive hypercalciuria Idiopathic hypercalcemia of infancy (Williams syndrome) Renal failure

150

MICHAEL F. HOLICK AND JOHN S. ADAMS 24-~

20,

-I00

50o o

16

12"

40-

{}

o o o

30

60

o

81,

20

8-

40

!

o

le

o

20

I0o oe

VITAMIN D (ng/ml)

80

A AA

25 (,OH)D (ng/ml)

1,25 (OH)2 D (pg/ml)

FIGURE 5--23 Distributionof circulating concentrations of vitamin D, 25(OH)D, and 1,25(OH)2D in healthy subjects determined in the authors' laboratory. Vitamin D and 25(OH)D values determined in subjects during summer months (open symbols) were higher than those determined during winter months (closed symbols), whereas no seasonal variation was apparent for 1,25(OH)2D concentration. Elderly adults (triangles) (aged 50 to 80 years) had significantly lower 25(OH)D concentrations than those in younger adults (circles). (From Clemens TL, Holick MF, Anast CS, Gray TK (eds): Perinatal Calcium and Phosphorus Metabolism. New York, Elsevier, 1983, pp 1-23.)

the serum vitamin D - b i n d i n g protein (DBP) and [3H]25(OH)D3 as the displaceable ligand. Most 25(OH)D assays employ whole serum from vitamin D - d e f i c i e n t rats as a source of binding protein. 25z'269 However, since only about 1% of the total binding sites on DBP are occupied by vitamin D or its metabolites, even in the vitamin D-sufficient state, serum from a vitamin D replete animal can be used without significantly altering the sensitivity of the assay. Obviously, whole lipid extracts will contain the total lipid complement of serum in addition to vitamin D, 25(OH)D, and other vitamin D metabolites. Therefore, it is not surprising that crude extracts containing 24,25(OH)zD, 25,26(OH)zD, and 25(OH)D3-23,26lactone, all of which are bound with high affinity by DBP, 27~ as well as other n o n - v i t a m i n D lipids that can bind to DBP yield values that are proportionately higher than values obtained after chromatography of the lipid extract, z7~ Hence, a clearly low or undetectable value for 25(OH)D in a nonchromatographic assay is indicative of vitamin D deficiency; however, a value in the lownormal range in such an assay does not exclude vitamin D deficiency. Preparative chromatography of the lipid extract on silicic acid 253 or LH-20 Sephadex 272 to isolate a 25(OH)D-containing fraction of the extract prior to assay provides a more accurate estimate of the serum concentration of 25(OH)D. Further chromatography on normal-phase HPLC can be employed to separate

25(OH)D2 and 25(OH)D3 so that the two metabolites can be assayed independently. 273 It should be emphasized that the mammalian DBP does not significantly discriminate between 25(OH)D3 and 25(OH)D2, so [3H]25(OH)D3 may be the radioligand used in assaying either of the 25(OH)D metabolites. The truest possible estimate of the total 25(OH)Dz and 25(OH)D3 concentration is obtained by direct quantitation of the metabolites by a sensitive UV detector (coupled to an H P L C ) . 26~ Inasmuch as the lower limits of detection by such an assay are in the range of 2 to 5 ng, direct quantitation of 25(OH)D2 and 25(OH)D3 requires the extraction of a larger volume of serum (2 to 5 ml), especially if the patient is thought to have vitamin D deficiency. Measurement of the 25(OH)D2 is of some clinical importance in the patient taking a multi-vitamin preparation containing vitamin D2. Since the hepatic microsomal vitamin D-25-hydroxylase does not distinguish between vitamin D2 and vitamin D3 as substrates,275 a low circulating concentration of 25(OH)D3 in such an individual may indicate that endogenous vitamin D3 synthesis is deficient and that the patient is dependent on dietary or therapeutic supplements of vitamin D2 to achieve an adequate vitamin D nutritional status. It should be noted, however, that in the United States foods are supplemented with either vitamin D2 or vitamin D3, and measurement of 25(OH)DE and 25(OH)D3 cannot be used as a method for determining the source of vitamin D.

CHAPTER5 VitaminD Metabolism and Biological Function

151

3. MEASUREMENT OF 1,25-DIHYDROXYVITAMIN D

receptor, many antibodies raised against 1,25(OH)2D3 analogues bind 1,25(OH)zD2 less avidly than 1,25(OH)aD3; therefore, the radioimmunoassays suffer from an inability to determine accurately the 1,25(OH)aDE contribution to the overall 1,25(OH)2D concentration. Second, antibodies raised against 1,25(OH)zD3 exhibit cross-reactivity with other vitamin D metabolites. Both of these deficiencies are related to the immunogen, which is usually a derivative of 1,25(OH)2D3 conjugated to bovine serum albumin through the side chain of the molecular or in the A-ring at the carbon-3 position. The lack of good immunogen is undoubtedly the explanation for the failure of workers to develop a good antibody against the hormone, a86-29~ Stern et al. 291'e92 have developed a very sensitive bioassay for 1,25(OH)2D that measures the release of 45Ca from prelabeled rat ulna and tibia maintained in organ culture. Although relatively labor-intensive by current standards, the bioassay does allow one to quantitate the biological effectiveness of a vitamin D metabolite or analogue. The cytoreceptor assay of Manolagas et al. 293'294 employs intact, receptor-possessing renal osteogenic sarcoma (ROS) cells and has the advantage of obviating the need for extensive chromatographic purification of the sample prior to assay. During the cytoreceptor assay, the ROS cells are incubated in an extracellular medium containing DBP, which binds 1,25(OH)2D with less affinity than many of the contaminating vitamin D metabolites; this leaves relatively more 1,25(OH)2D free to enter the ROS cells and to bind its own high-affinity, intracellular receptor.

The first serum assay for 1,25(OH)zD was reported in 1974276 just 3 years after the structural identification of 1,25(OH)D3 as the active metabolite of vitamin D3.277-279 The key to development of the assay was the detection of a high-affinity, low-capacity binding protein (receptor) in chick intestinal epithelium that could be employed as a specific binding protein in a radioligand binding assay. 28~ The original assay required extraction of a large volume of serum (20 ml) and three separate chromatographic purification steps before assay and was sensitive to only 50 pg/tube. In recent years the availability of tracers of high specific activity, the application of sophisticated chromatography for sample purification, and the use of techniques to promote receptor stability have combined to greatly improve sample preparation and the analysis. The 1,25(OH)2D assay is now available in most university hospitals and through several commercial laboratories. Although the assay for 1,25(OH)2D in patients' serum or plasma is obtained with relative ease, there exists significant variability in its performance and there are caveats in its application to human health or disease (see Section IX.B). A purified lipid extract of human serum or plasma can be analyzed for its content of 1,25(OH)2D in a variety of ways (Table 5-5). The classic binding protein in the competitive protein-binding assay for 1,25(OH)2D is the hormone's own receptor protein isolated from intestinal epithelium of vitamin D-deficient chicks. Vitamin D deficient birds are employed in order to improve the extraction of receptor that is not occupied by endogenously synthesized 1,25(OH)2D. A disadvantage in the use of the chick intestinal receptor in the binding assay is that it discriminates against 1,25(OH)2D2 binding in favor of 1,25(OH)2D3. Therefore, these two metabolites, which are biologically equipotent in humans, cannot be measured with comparable efficiency. This may account for the reports that the chick receptor assay underestimates the 1,25(OH)2D2 concentration by 23% to 6 0 % . 281-283 This failure is a significant liability when determining the serum concentration of 1,25(OH)2D in patients relying on ingested vitamin D2 for their vitamin D nutrition. These problems have been resolved with the increased use of the receptor derived from calf thymus, which binds 1,25(OH)2D3 and 1,25(OH)2D2 with equivalent affinity and capacity. T M In fact, this assay can now be employed for the measurement of 1,25(OH)2D in lipid extracts of small quantities of human serum (0.5 to 1.0 ml) after purification on a single silica cartridge. 285 Several radioimmunoassays have been developed for detection of 1,25(OH)2D in the s e r u m . 286-29~ Although potentially advantageous, radioimmunoassays suffer two major drawbacks. First, similar to the chick intestinal

4. MEASUREMENT OF 24,25-DIHYDROXYVITAMIN D

Owing to the high affinity of DBP for 24,25(OH)2DY 5 this metabolite can be readily detected with ease in a competitive protein-binding assay (Table 5-5). However, 24,25(OH)2D2 is bound two or three times less avidly than is 24,25(OH)zD3, 296'297 making an estimation of the overall 24,25(OH)2D concentration misleading in a person ingesting significant amounts of vitamin DE. Accurate estimation of the total 24,25(OH)zD concentration must, therefore, employ chromatographic techniques that resolve 24,25(OH)zD2 and 24,25(OH)zD3 and binding assays that use the appropriate radioinert metabolite in a standard curve. Recently, the use of a methylene chloride-isopropanol solvent system on a cyano-bonded ~-silica HPLC column has been shown to be capable of separating 24,25(OH)zD2 and 25(OH)D3-23,26-1actone, 298 two metabolites of 25(OH)D3 that co-migrate with 24,25(OH)2D on normal-phase HPLC ~-silica columns developed in hexane-isopropanol. It might be questioned whether determination of the circulating 24,25(OH)zD concentration warrants the effort required. Although suggested by a number of investigators, ~42'299a

152

MICHAEL F. HOLICK AND JOHN S. ADAMS

physiological role for 24,25(OH)2D in human mineral metabolism has not been unequivocally demonstrated (see Section VI.A). Until more convincing data are available, routine assay of 24,25(OH)2D cannot be advocated. 5. OTHER VITAMIN D METABOLITES

In recent years the measurement of two additional metabolites, 25,26(OH)2D and the 25(OH)D-23,26lactone, has been extensively investigated. For both, HPLC purification of the serum lipid extract is required prior to detection in a DBP-based competitive proteinbinding assay (Table 5-5). Neither metabolite has been shown to be of physiological significance in humans. In fact, the 25(OH)D3-23,26-1actone, a renal metabolite of 23,25(OH)2D3, 3~176 is detected in human serum only in states of vitamin D excess. The significance of the extraordinarily high affinity of DBP for the lactone is not currently known. High circulating concentrations of this metabolite can theoretically displace 25(OH)D and 1,25(OH)2D from the DBP, thereby facilitating the binding of these metabolites to the cellular receptor for 1,25(OH)2D in 12i1,'O.301

B. Clinical Utility of the 25(OH)D and 1,25(OH)2D Assays (Table 5-5) 1. 25(OH)D ASSAY The 25(OH)D concentration is the test of choice for determining the adequacy of vitamin D nutrition from either endogenous or exogenous sources. 2'6~ A very low or undetectable value in the competitive protein binding assay is indicative of (1) deficient substrate for the hepatic vitamin D-25-hydroxylase; (2) disease or hormonerelated alteration in the activity of the hepatic vitamin D-25-hydroxylase; (3) failure to synthesize DBP or loss of DBP-bound 25(OH)D; or (4) accelerated catabolism of 25(OH)D. The first may result from diminished cutaneous sunlight exposure of the patient or from a combination of deficient sunlight exposure and intestinal malabsorption of vitamin D in persons consuming a diet supplemented with vitamin D. In either case, it is important to point out that deficient endogenous synthesis of vitamin D is required for vitamin D deficiency to occur. Clements et al. 3~ recently reported that after oral or intravenous administration, neither vitamin D nor 25(OH)D undergoes significant enterohepatic circulation, precluding the possibility that one can become vitamin D - or 25(OH)D-deficient in the presence of adequate production of vitamin D3 in the skin. A number of hepatocellular diseases can contribute to 25(OH)D deficiency in patients in whom the availability of substrate, vitamin DE, or vitamin D3 is compromised. However, it

is unusual to encounter 25(OH)D deficiency in patients who are vitamin D-sufficient except for patients with severe liver disease, 54 because the " f r e e " 25(OH)D concentration in such individuals is usually not altered. 3~ Because human DBP is an alpha globulin with a relatively low molecular weight (58,000 daltons; albumin MW = 69,000), it is lost through the glomerular basement membrane in patients with nephrotic syndrome. The total serum concentration of 25(OH)D in such patients is directly correlated with the degree of proteinuria, and is expected to be low in patients with greater than 4 g of proteinuria per day. 56 Although patients with protein-losing enteropathies or severe burns may be subject to loss of DBP, 25(OH)D deficiency in such settings is rarely due to only a loss of DBP through the gastrointestinal tract or skin. Finally, in principle at least, the potential exists for accelerated 25(OH)D clearance from the serum as a cause for diminished circulating concentrations of the metabolite. It is known that administration of pharmacological doses of 1,25(OH)2D3 will accelerate 25(OH)D3 clearance in experimental animals 3~ and ameliorate the increase in the serum 25(OH)D concentration in humans 53 given large daily doses of vitamin D2 orally. An assessment of the adequacy of therapy and patient compliance with therapy with vitamin D3, vitamin D2, or 25(OH)D3 in patients requiting vitamin D supplementation (i.e., hypoparathyroidism) can be made by following the serum concentrations of 25(OH)D. Therapy in most cases is aimed at maintaining the 25(OH)D level in the high-normal range, usually between 25 and 45 ng/ ml. 2 Because 25(OH)D is bound with high affinity by the serum DBP, it has a long serum half-life, with estimates ranging from 12 to 60 days. 2 Its prolonged halflife makes infrequent measurements of the serum 25(OH)D concentration (every 2 to 3 months) adequate for monitoring therapy in the noncompliant patient. Measurement of the 25(OH)D concentration is also the test of choice in establishing the diagnosis of vitamin D intoxication from ingestion or parenteral administration of vitamin D or 25(OH)D3. Ingestion of large quantities of vitamin D may result in 25-hydroxylation of vitamin D by hepatic mitochondria, 3~176 which possess a vitamin D-25-hydroxylase of lower affinity for vitamin D (Km = 10 -6 M) than hepatic microsomes (Km = 10 -8 M) 3~176 but a greater capacity for conversion of vitamin D to 25(OH)D. 3~ The low-affinity, high-capacity mitochondral system 31~ does not appear to be productinhibitable as is its microsomal counterpart 31~ and is, therefore, capable of prolific 25(OH)D generation given enough substrate. Interestingly, in normal human subjects, excessive exposure to sunlight will not result in vitamin D3 intoxication, because only a limited amount of vitamin D3 will be formed in the skin (see Section II.B); with continued photon bombardment of the skin,

CHAPTER5 Vitamin D Metabolism and Biological Function

153

previtamin D3 will be preferentially converted to two nonbiologically active photoisomers, tachysterol and lumisterol (Fig. 5 - 3 ) and vitamin D3 is degraded to 5,6trans-vitamin D3 and suprasterols 1 and 2 (Fig. 5-5).

2. 1,25(OH)2D ASSAY In the authors' view there are few clinical circumstances in which a single determination of the serum concentration of 1,25(OH)2D, a metabolite with a circulating half-life of only 4 to 6 hours, will be of diagnostic value (Table 5 - 6 ) . The finding of a very low or undetectable serum 1,25(OH)2D concentration in a rachitic child with a normal or elevated serum concentration of 25(OH)D is indicative of vitamin D-dependent rickets type I, an inheritable defect, or absence of the renal 25(OH)D-loL-hydroxylase. TM A very high circulating concentration of 1,25(OH)2D in a rachitic child on vitamin D therapy is indicative of end-organ unresponsiveness to 1,25(OH)2D and the diagnosis of vitamin D dependent tickets type 11. 312 A high value for the 1,25(OH)2D assay may occur in patients intoxicated with either dihydrotachysterol (DHT), lc~-hydroxyvitamin D3 [ loL(OH)D3], o r 1,25(OH)2D3 .6~ Intoxication with the former compounds is dependent to a great extent on hepatic 25-hydroxylation of DHT and of l c~(OH)D3 to 25-hydroxydihydrotachysterol and 1,25(OH)2D3, respectively, which cross-react with the 1,25(OH)2D receptor. A frankly elevated or high-normal serum 1,25(OH)2D concentration in a hypercalcemic patient with sarcoidosis, 119'313 tuberculosis, 314 siliconeinduced granulomatous disease, 315 disseminated candidiasis, 316 o r l y m p h o m a 1~ represents inappropriate

TABLE 5 - - 6

endogenous production of the hormone in the presence of hypercalcemia (also see Section V.C). The autonomous, extrarenal synthesis of 1,25(OH)2D in sarcoidosis is almost always associated with extensive disease and high-intensity alveolitis. 317'318 The finding of an inappropriately high circulating 1,25(OH)2D concentration in such a patient with granulomatous disease or lymphoma portends an excellent calcium-lowering effect with glucocorticoid therapy. In an older individual with suspected granulomatous disease or lymphoma, the failure of glucocorticoids to lower the serum 1,25(OH)2D and calcium concentration within a few days strongly suggests the presence of a coexistent hypercalcemia-causing disease (i.e., primary hyperparathyroidism). Extrarenal 1,25(OH)2D synthesis by the placenta will elevate the total and " f r e e " concentration (that fraction of the total serum concentration not bound by a circulating binding protein) of 1,25(OH)2D in the serum during the third trimester of pregnancy. 319'32~ A meaningful interpretation of the serum 1,25(OH)25(OH)2D concentration in states of vitamin D deficiency is difficult. It may be increased, decreased, or normal, depending on the circumstances in which the sample is obtained. Obtaining serial values for 1,25(OH)2D in a single individual after a provocative stimulus to endogenous 1,25(OH)2D synthesis is currently being investigated in various clinical settings (e.g., postmenopausal osteoporosis). 94'321'322 However, at present, these techniques require frequent sampling and often prolonged stimulation of the renal 25(OH)D-lot-hydroxylase, such as a 6- to 12-hour infusion of hPTH(1-34), in order to measure a

Assay of Vitamin D and Vitamin D Metabolites Purification

Sterol

Detection

LPLC

HPLC

UV

X X

X X

X

X X X

X X

X X X X

X X X

24,25(OH)2D

X

X

25,26(OH)zD

X

X

25(OH)D-23,26-1actone

X

X

Vitamin D

CPBA

RIA

BIO

25(OH)D

1,25(OH)2D

LPLC, open column (low pressure) liquid chromatography; HPLC, high-performance liquid chromatography; UV, assay by ultraviolet light absorbance on HPLC; CPBA, competitive proteinbinding assay; RIA, radioimmunoassay; BIO, bioassay.

154

MICHAEL E HOLICK AND JOHN S. ADAMS

significant effect on the serum 1,25(OH)2D concentration (Fig. 5-17). For the sake of practicality, the provocative tests are not yet a clinically useful tool, but they may become so in the future.

C. The Use of Vitamin D Assays in the Evaluation of the Hypocalcemic and Hypercalcemic/Hypercalciuric Patient When confronted with a hypocalcemic patient, it is useful to consider whether the reduction in the serum ionized calcium concentration is due to deficiency in one or both of the calcemic hormones, 1,25(OH)zD and PTH. In the case of the former hormone, measurement of the serum 25(OH)D concentration provides the most valuable information in the majority of cases. Owing to its long serum half-life, a 25(OH)D measurement provides an excellent index of the amount of vitamin D that is metabolized in the liver and the amount of substrate [25(OH)D] available for synthesis of the active metabolite, 1,25(OH)zD. In the hypocalcemic patient, a frankly low 25(OH)D level almost always indicates deficient cutaneous synthesis and dietary intake of vitamin D. Patients with severe hepatocellular disease or nephrotic syndrome may have a low total serum calcium and 25(OH)D concentration due to either a decrease in hepatic synthesis or urinary loss of proteins that bind calcium (albumin, prealbumin) and 25(OH)D (DBP, albumin, lipoproteins) in the circulation. In such cases, a low serum concentration of ionized calcium and a compensatory elevation in the circulating PTH concentration may aid in establishing the presence of true ( " f r e e " ) 25(OH)D deficiency. Hypocalcemia with a normal or elevated 25(OH)D level points either to deficiency or decreased bioeffectiveness of PTH, an acquired abnormality in the metabolism of 25(OH)D to 1,25(OH)zD (i.e., renal failure), an inherited defect in 1,25(OH)2D synthesis, or a defect in the action of 1,25(OH)zD at its target tissues. The last two situations, both rare, can usually be distinguished on biochemical grounds by the circulating 1,25(OH)zD concentration, which will be low or nondetectable in patients with vitamin D-dependent rickets type I, and often dramatically increased in patients with vitamin D-dependent tickets type II. 2'6~ In patients with hypocalcemia and diminished synthesis, release, or endorgan effectiveness of PTH (i.e., patients with hypoparathyroidism, magnesium deficiency, or pseudohypoparathyroidism), the 1,25(OH)zD concentration will be inappropriately low. This is due to the lack of a stimulatory effect of PTH on the renal 25(OH)D-loL-hydroxylase and the inhibitory effect of hyperphosphatemia, a usual consequence of hypoparathyroidism, on the same

enzyme. However, because of overlap of the serum 1,25(OH)zD concentration into the normal range in such patients, measurement of this metabolite is not particularly helpful in establishing the diagnosis of hypoparathyroidism. Whereas the 25(OH)D assay has a central role in the evaluation of the hypocalcemic patient, the utility of this assay in the workup of a hypercalcemic patient is limited. Only if 25(OH)D is present in high concentrations in the circulation and it (or its metabolites) binds to the high-affinity receptor for 1,25(OH)zD is it capable of inducing hypercalcemia. Intoxication from an exogenous source may arise after ingestion of large amounts of vitamin D or 25(OH)D3, whereas intoxication with endogenously synthesized 25(OH)D may be the etiological explanation for the idiopathic hypercalcemia of infancy. The latter, referred to as the Williams syndrome when accompanied by the phenotypic markers of supravalvular aortic stenosis, elfin facies, and mental retardation, is a rare disorder that has been associated with an exaggerated increase in the serum concentration of 25(OH)D in response to a challenge with orally administered vitamin D2 .322 These children have also been shown to possess inappropriately elevated serum concentrations of 1,25(OH)zD. 323 In 25(OH)D intoxication, from either an exogenous or endogenous source, the serum 1,25(OH)zD concentration may be normal or even reduced unless there is some additional abnormality in the regulation of the l oL-hydroxylation of 25(OH)D. As alluded to previously, normal individuals do not become vitamin D [25(OH)D] intoxicated from endogenously synthesized vitamin D. Lifeguards, for instance, may develop circulating 25(OH)D concentrations that are well above the normal range (>100 mg/ml) but do not develop hypercalcemia or hypercalciuria. 3~ The usefulness of the 1,25(OH)zD assay in evaluating a patient with hypercalcemia and/or hypercalciuria is, for the most part, restricted to patients with suppressed PTH secretion; an elevation in the serum 1,25(OH)zD concentration is an expected accompaniment of primary hyperparathyroidism (unless renal failure is present), and there is little need for it to be measured when that diagnosis is certain or highly likely. Therefore, an inappropriate elevation in the serum 1,25(OH)zD concentration and a suppressed plasma PTH concentration in a hypercalciuric/hypercalcemic patient is highly suggestive of (1) exogenous intoxication with lc~(OH)D3, DHT, or 1,25(OH)zD3; (2) endogenous intoxication with 1,25(OH)zD as may occur in sarcoidosis, other granulomatous diseases, and lymphoma; or (3) absorptive hypercalciuria. 324 Determination of the serum 1,25(OH)zD concentration may be of help in discerning the presence of primary hyperparathyroidism in patients who are suspected to

CHAPTER 5 Vitamin D Metabolism and Biological Function harbor the disease but in whom confirmatory laboratory data are lacking. It should also be recognized that a frankly elevated serum 1,25(OH)2D concentration may not be present in a hypercalcemic/hypercalciuric patient with concomitant hepatocellular disease or nephrotic proteinuria resulting in diminished production or loss of serum proteins that bind 1,25(OH)2D. Although the "total" concentration of the hormone may be relatively low in such cases, the " f r e e " concentration of 1,25(OH)2D, which can adequately be determined from a knowledge of the serum albumin and DBP levels, 325 will be higher than normal.

XII. CONCLUSION As recently as 20 years ago, vitamin D was considered to be "just another one of those fat-soluble vitamins." The revelation that vitamin D must be hydroxylated in the liver and kidney to 1,25(OH)zD before it is biologically active opened a new chapter for vitamin D research, and clinical medicine. The finding that the kidney was responsible for the final activation step for vitamin D metabolism provided a new insight into the cause of vitamin D resistance that was often associated with patients who suffered from chronic renal failure. The availability of reliable assays for the measurement of the circulating concentration of 25(OH)D and 1,25(OH)zD has led to the identification of inbom and acquired disorders in 25(OH)D metabolism. These assays have provided new insights into the cause of a variety of hypo- and hypercalcemic disorders including vitamin D-dependent rickets types I and II, 326 primarily hyperparathyroidism, and tumor-induced hypercalcemia associated with certain lymphomas and chronic granulomatous disorders. The knowledge of the crucial role of the kidney's metabolic conversion of 25(OH)D to 1,25(OH)zD prompted the chemical synthesis of this kidney hormone and its 25-deoxy analogue, l oL-hydroxyvitamin D3. These drugs have provided the clinician with an effective means for the treatment of a variety of hypocalcemic disorders that are caused by acquired and inbom errors in the metabolism of 25(OH)D to 1,25(OH)zD. The discovery of a rare intracellular protein (receptor) that binds 1,25(OH)zD with high affinity and specificity has greatly advanced our understanding of the cellular mode of action of the hormone. In the 1970s the observation that a variety of tissues and cells (that are not classic target tissues for vitamin D) possessed nuclear receptors for 1,25(OH)zD3 opened up a new and exciting chapter in the evolving vitamin D story. It was found that such diverse tissues as the brain, parathyroid gland, gonad, thymus, pancreas, and skin had high-affinity,

155 low-capacity nuclear receptors for 1,25(OH)2D (Table 5 - 3 ) . In addition, several circulating mononuclear cell populations including monocytes and activated B and T lymphocytes possessed receptors for this hormone. The initial reaction to these observations was that this was an epiphenomenon with little biological importance. However, the observation that 1,25(OH)zD could induce differentiation of 1,25(OH)zD3 receptor-positive promyelocytic leukemic cells generated an enormous amount of interest regarding the potential biological effects of 1,25(OH)zD on "nonclassical" target tissues. In vivo and in vitro studies have revealed that 1,25(OH)zD can inhibit the proliferation and induce cellular differentiation of 1,25(OH)zD3 receptor-positive cells and enhance or inhibit the synthesis and secretion of a variety of hormones, growth factors, and immunoglobulins. The physiological importance of 1,25(OH)zD on (1) regulating hormone secretion, (2) inducing maturation of a variety of tissues including the skin, (3) altering immune function, and (4) affecting myocardial activity is unknown. However, it is known that patients with vitamin D-dependent tickets type II or simple vitamin D deficiency do not have overt alterations in their immune systems, cardiac function, skin appearance, or hormone responsiveness to provocative stimulation. Thus, although it is unlikely that 1,25(OH)zD is essential for the normal functioning of these tissues, the observation that 1,25(OH)zD can alter the biochemistry and physiology of receptor-positive tumor and normal cells has wide-ranging pharmacological and physiological applications. For example, the findings that parathyroid glands possess nuclear receptors for 1,25(OH)zD3 and that in vitro 1,25(OH)zD3 inhibits the expression of the parathyroid hormone gene 327 led to the intravenous use of 1,25(OH)zD3 as a method to suppress parathyroid hormone secretion in chronic renal failure patients with marked secondary hyperparathyroidism. 328 The observation that circulating monocytes possess receptors for 1,25(OH)zD3 and differentiate into osteoclast-like cells when exposed to this hormone has prompted the concept that 1,25(OH)2 may be important for the mobilization of stem cells for the bone remodeling process. Initially it was thought that 1,25(OH)zD would have great potential as an antitumor agent. However, it is now known that antimitogenic activity of 1,25(OH)zD3 is reversible 329 and that tumor clones that have a decreased number of receptors for this hormone are resistant to its antimitogenic activity. Furthermore, when the hormone is given in pharmacological doses, it causes severe hypercalcemia. 33~ Despite these problems, 1,25(OH)zD3 may be of great pharmacological value, especially for dermatology. Unlike tumor cells that can dedifferentiate when 1,25(OH)zD3 is removed from their environment, when 1,25(OH)zD3 induces human keratinocytes to dif-

156

MICHAEL E HOLICK AND JOHN S. ADAMS

ferentiate, it does so in an irreversible manner. One practical application is the effective use of 1,25(OH)2D3 for the treatment of hyperproliferative skin disorders such as psoriasis. It is well documented that 1,25(OH)2D3 causes many if not most of its biological effects by a nuclear-mediated mechanism. However, there is intriguing evidence that in some cells 1,25(OH)2D may also increase intracellular calcium concentrations and alter phosphoinositol metabolism in both receptor-positive and -negative c e l l s . 331'332 Thus, it may be possible to develop analogues of 1,25(OH)2D3 that have selective effects on the plasma membrane and cytosolic calcium concentrations, while others could be developed that would interact with the nucleus to induce antimitogenic activity. The next decade holds great promise for the field of vitamin D research. 1,25(OH)2D and its analogues offer great promise in areas of immunology, dermatology, cardiology, oncology, nephrology, and endocrinology. Thus, 1,25(OH)2D3 is a hormone whose clinical pharmacological potential is yet to be realized.

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163 294. Manolagas SC, Culler FL, Howard JE, et al: The cytoreceptor assay for 1,25-dihydroxyvitamin D and its application to clinical studies. J Clin Endocrinol Metab 56:751-760, 1983. 295. Haddad JG, Min C, Mendelsohn M, et al: Competitive proteinbinding radioassay of 24,25-dihydroxyvitamin D 3 in sera from normal and anephric subjects. Arch Biochem Biophys 182:390 -3 9 5 , 1978. 296. Horst RL, Littledike ET, Gray RW, Napoli JL: Impaired 24,25dihydroxyvitamin D production in anephric man and pig. J Clin Invest 67:274-280, 1981. 297. Hay AWM, Jones G: The elution profits of vitamin DE metabolites from Sephadex LH20 columns. Clin Chem 25:473-475, 1979. 298. Jones G: Chromatographic separation of 24(R),25-dihydroxyvitamin D3 and 25-hydroxyvitamin Da-26,23-1actone using a cyano-bonded phase packing. J Chromatogr 276:69-74, 1983. 299. Hodsman AB, Wong EGC, Sherrard DJ, et al: Preliminary trials with 24,25-dihydroxyvitamin D3 in dialysis osteomalacia. Am J Med 74:407-414, 1983. 300. Napoli JL, Pramanik BC, Partridge JJ, et al: 23S,25-Dihydroxyvitamin D3 as a circulating metabolite of vitamin D3: Its role in 25-hydroxyvitamin Da-26,23-1actone biosynthesis. J Biol Chem 257:9634-9639, 1982. 301. Horst RL, Reinhardt TA, Napoli JL: 23-keto-25-hydroxyvitamin D3 and 23-keto-1,25-dihydroxyvitamin D3: Two new metabolites with high affinity for the 1,25-dihydroxyvitamin D3 receptor. Biochem Biophys Res Commun 107:1319-1322, 1982. 302. Clements MR, Chalmers TM, Fraser DM: Enterohepatic circulation of vitamin D: A reappraisal of the hypothesis. Lancet 1: 1376-1379, 1984. 303. Halloran BE Bikle DD, Levens MJ, et al: Chronic 1,25-dihydroxyvitamin D 3 administration reduces the serum concentration of 25-dihydroxyvitamin D 3 by increasing the metabolic clearance rate. J Bone Miner Res 1:18(abstract), 1986. 304. Bell NH, Shaw S, Turner RT: Evidence that 1,25-dihydroxyvitamin D3 inhibits the hepatic production of 25-hydroxyvitamin D in man. J Clin Invest 74:1540-1544, 1984. 305. Bjorkhem I, Holmberg I: Assay and properties of a mitochondrial 25-hydroxylase active on vitamin D3. J Biol Chem 253: 8 4 2 - 8 4 9 , 1978. 306. Bjorkhem I, Holmberg, I, Oftebro H, Pedersen JI: Properties of a reconstituted vitamin Da-hydroxylase from rat liver mitochondria. J Biol Chem 255:5244-5249, 1980. 307. Yoon PS, DeLuca HF: Resolution and reconstruction of soluble components of rat liver microsomal vitamin Da 25-hydroxylase. Arch Biochem Biophys 203:529-540, 1980. 308. Andersson S, Holmberg I, Wikvall K: 25-Hydroxylation of C27steroids and vitamin D3 by a constitutive cytochrome P-450 from liver microsomes. J Biol Chem 258:6777-6781, 1983. 309. Pedersen JI, Bjorkhem I, Gustafsson J: 25-Hydroxylation of C27steroids by soluble liver mitochondrial cytochrome P-450. J Biol Chem 254:6464-6469, 1979. 310. Bhattacharyya MH, DeLuca HF: The regulation of rat liver calciferol-25-hydroxylase. J Biol Chem 248:2969-2973, 1973. 311. Fraser D, Kooh SW, Kind HE et al: Pathogenesis of hereditary vitamin-D-dependent tickets: An inborn error of vitamin D metabolism involving defective conversion of 25-hydroxyvitamin D to lc~,25-dihydroxyvitamin D. N Engl J Med 289:817-822, 1973. 312. Brooks MH, Bell NH, Love L, et al: Vitamin-D-dependent tickets type II: Resistance of target organs to 1,25-dihydroxyvitamin D. N Engl J Med 298:996-999, 1978. 313. Bell NH, Stem PH, Pantzer E, et al: Evidence that increased circulating l oL,25-dihydroxyvitamin D is the probable cause for

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abnormal calcium metabolism in sarcoidosis. J Clin Invest 64: 2 1 8 - 225, 1979. Gkonos PJ, London R, Hendler ED: Hypercalcemia and elevated 1,25-dihydroxyvitamin D levels in a patient with end-stage renal disease and active tuberculosis. N Engl J Med 311:1683-1685, 1984. Kozeny GA, Barbato AL, Bansal VK, et al: Hypercalcemia associated with silicon-induced granulomas. N Engl J Med 74: 1103-1105, 1984. Kantarjian HM, Saad MF, Estey EH, et al: Hypercalcemia in disseminated candidiasis. Am J Med 74:721-723, 1983. Sandier LM, Winearls GC, Fraher LJ, et al: Studies of the hypercalcemia of sarcoidosis: Effect of steroids and exogenous vitamin D3 on the circulating concentrations of 1,25-dihydroxyvitamin D 3. Q J Med 210:615-630, 1984. Adams JS, Gacad MA, Endres DB, Sharma OP: Biochemical indicators of disordered vitamin D and calcium homeostasis in sarcoidosis. Sarcoidosis 3:1-6, 1986. Bouillon R, Van Assche FA, Van Baelen H, et al: Influence of the vitamin D-binding aprotein on the serum concentration of 1,25-dihydroxyvitamin D3. J Clin Invest 67:589-596, 1981. Bikle DD, Gee E, Halloran B, Haddad JG: Free 1,25-dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease. J Clin Invest 74:1966 - 1977, 1984. Prince RL, Wark JD, Omond S, et al: A test of 1,25-dihydroxyvitamin D3 secretory capacity in normal subjects and application in metabolic bone diseases. Clin Endocrinol 18:127133, 1983. Taylor AB, Stern PH, Bell NH: Abnormal regulation of circulating 25-hydroxyvitamin D in the Williams syndrome. N Engl J Med 306:972-975, 1982. Garabedian M, Jaeqz E, Guillozo H, et al: Elevated plasma 1,25dihydroxyvitamin D concentrations in infants with hypercalcemia and elfin facies. N Engl J Med 312:948-952, 1985.

324. Broadus AE, Insogna KL, Lang R, et al: A consideration of the hormonal basis and phosphate leak hypothesis of absorptive hypercalciuria. J Clin Endocrinol Metab 58:161-169, 1984. 325. Bikle DD, Siiteri PK, Ryzen E: Serum protein binding of 1,25dihydroxyvitamin D: A reevaluation by direct measurement of free metabolite levels. J Clin Endocrinol Metab 61:969-975, 1985. 326. Fraser D, Scriver CR: Hereditary disorders associated with vitamin-D resistance or defective phosphate metabolism. In DeGroot L, et al (eds): Endocrinology, vol 2. New York, Grune & Stratton, 1979, pp 797-807. 327. Silver J, Russell J, Sherwood LM: Regulation of preproparathyroid hormone mRNA in bovine parathyroid cells in culture by vitamin D metabolites. In Norman AW, Schaefer K, Grigoleit H-G, von Herrath D (eds): Vitamin D, Chemical, Biochemical and Clinical Update. Proc 6th Workshop, Vitamin D, Merano, Italy, 1985. Berlin, de Gruyter, 1985, pp 24-33. 328. Slatopolsky E, Weerts C, Thielan HR, et al: Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxycholecalciferol in uremic patients. J Clin Invest 74:2136-2143, 1984. 329. Bar-Shavit Z, Kahn AJ, Stone KR, et al: Reversibility of vitamin D-induced human leukemia cell-line maturation. Endocrinology 118:679-686, 1986. 330. Koeffler HP, Hirjik S, Itri L, and the Southern California Leukemia Group: 1,25-Dihydroxyvitamin D3. In vivo and in vitro effects on human preleukemic and leukemic cells. Cancer Treat Rep 69:1399-1407, 1985. 331. Baran DT, Moira ML: 1,25-Dihydroxyvitamin D increases hepatocyte cytosolic calcium levels: A potential regulator of vitamin D-hydroxylase. J Clin Invest 77:1622-1626, 1986. 332. Smith E, Holick MF: The skin: The site of vitamin D3 synthesis and a target tissue for its metabolite 1,25-dihydroxyvitamin D3. Steroids 49:103-131, 1987.

2 H A P T E R (~

Pathophysiology of Calcium, Phosphate, and Magnesium Absorption ROBERTO CIVITELLI, KONSTANTINOS ZIAMBARAS, AND RATTANA LEELAWATTANA Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri 63110

I. Calcium A. Physiology of Calcium Absorption B. Increased Calcium Absorptive States C. Decreased Calcium Absorptive States II. Phosphate A. Physiology of Phosphate Absorption B. Clinical Manifestations of Phosphate Disorders

C. Increased Phosphate Absorptive States D. Decreased Phosphate Absorptive States III. Magnesium A. Physiology of Magnesium Absorption B. Clinical Manifestations of Magnesium Disorders C. Increased Magnesium Absorptive States D. Decreased Magnesium Absorptive States References

I. C A L C I U M

A. P h y s i o l o g y o f C a l c i u m A b s o r p t i o n 1. CALCIUM BALANCE

Calcium is an essential element for survival. It not only serves as the principal component of the skeleton but also plays a vital role in a variety of physiological and biochemical processes, such as regulation of nerve excitability, muscle contraction, and blood coagulation. Calcium balance is a tightly controlled homeostatic mechanism and active processes are critical to the absorption of calcium through the intestine. Accordingly, pathological conditions that affect the major regulators of calcium absorption--primarily the vitamin D systemmresult in clinically relevant syndromes conditioned and maintained by an abnormal intestinal calcium absorption. METABOLIC BONE DISEASE

The relationship between calcium absorption and dietary calcium is curvilinear, reflecting the two major absorption mechanisms, passive diffusion and active, saturable absorption (Fig. 6-1). Up to a daily calcium intake of 1000 mg, net absorption increases linearly with calcium intake. Above that limit, calcium absorption tends to plateau, so that normally no more than 300 mg/ day is absorbed on average even if the dietary intake is raised. This threshold increases up to 500 mg/day at around puberty (discussed in Section I.A.5), but within these genetic limits, there is a wide individual variability in the efficiency of calcium absorption, which is believed 165

Copyright 9 1998by AcademicPress. All rightsof reproductionin any formreserved.

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to be accounted for by wide variations of active calcium transport among healthy individuals. On the other hand, fractional calcium absorption is inversely correlated to dietary calcium intake, thus providing a partial compensation for conditions in which calcium dietary is insufficient (discussed in Section I.C.1). Unlike sodium and potassium, calcium is only partially absorbed from the intestinal lumen. Approximately 15% of the total intestinal calcium secretion is assumed to be nonabsorbable, even under conditions in which dietary calcium is maximally absorbed. Net calcium absorption represents the difference of two unidirectional processes: the total transfer of calcium from intestinal lumen to plasma, minus the transfer of calcium from plasma to lumen, or endogenously secreted calcium. Fecal calcium consists of the unabsorbed dietary calcium, the calcium present in the intestinal secretions, and that lost with desquamating intestinal cells, --~140 to 150 mg/ day in adult women. 1'2 Considering other obligatory calcium losses from the skin (--~ 60 to 80 mg/day through sweat, skin, and hair desquamation) 2'3 and in the urine (the kidney cannot produce calcium-free urine), a minimal dietary intake of at least 400 to 500 mg of calcium is required to maintain zero calcium balance. Diets providing less than 400 mg/day of the element result in a negative balance (Fig. 6 - 1 ) , with dire consequences for growth in children and skeletal health in adults. 2. CALCIUM INTAKE The definition of a correct dietary intake of calcium has concerned physicians and investigators alike for sev-

eral years, and although guidelines have been established in this regard, controversy still exists on a number of related issues. It is a common assumption that the current average diet in the United States, and also in many European and developing countries, contains an insufficient amount of calcium. The most recent, 1989 Nationwide Food Consumption Survey confirmed that a large proportion of the U.S. population consumes diets providing an average daily calcium intake of about 900 mg/day, substantially less than the current recommended dietary allowance (RDA) of 1200 mg 4 (Fig. 6 - 2 ) . However, this limit was suggested in 1989 by the Food and Nutritional Board of the National Academy of Sciences as a minimal allowance to prevent negative calcium balance, and it is now felt to be inadequate to meet the requirements for all age ranges of a healthy population. A National Institutes of Health Consensus Conference was called in 1994 to review the current status of knowledge on dietary calcium intake, and to provide recommendations as to the appropriate daily intake for different strata of the general population. After reviewing the available data, the panel recommended that the optimal daily calcium intake in young adults be between 1200 and 1500 mg/ day, 5 whereas the current R D A for infants (400 to 600 mg) and young children up to age 5 (800 mg) was felt to be adequate. A daily intake of above 800 mg of calcium was recommended for children between ages 6 and 10. For men and women in the fertile period, an intake of 1000 mg/day was considered satisfactory to maintain bone mass after peak is achieved. After the menopause, a daily intake of at least 1500 mg is required, unless estrogen replacement therapy is instituted. Finally, individuals of both sexes of 65 years or older should consume 1500 mg of calcium daily. 5 These recommendations set daily intakes of calcium at above the R D A for most age ranges, thus it is conceivable that a revision of the current R D A limits will be forthcoming (Table

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CHAPTER6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption TABLE 6--1 Optimal Calcium Requirements Recommended by the National Institutes of Health Consensus Panel a Age group Infant B i r t h - 6 months 6 months- 1 year Children 1 - 5 years 6 - 1 0 years Adolescents/young adults 1 1 - 2 4 years Men 2 5 - 6 5 years Over 65 years Women 2 5 - 50 years Over 50 years (postmenopausal) On estrogen Not on estrogen Over 65 years Pregnant and nursing

Optimal calcium intake (mg/day)

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aReproduced from NIH Consensus Development Panel on Optimal Calcium Intake: Optimal Calcium Intake, JAMA 272:1942-1948; 1994.

6-1). Athletes, and especially males, may also require additional dietary calcium to prevent the bone loss and calcium loss through sweat, that can be as high as 400 mg per training session. 6 3. C A L C I U M SOURCES Most of the calcium is ingested from food, and in particular, dairy products, grains, bony fish, and spinach. Other common food constituents such as fruits, vegetables, pasta, breads, and beverages contain much smaller amounts of calcium. Although food fortification with calcium has been aggressively pursued in Western countries, the fear of higher fat intakes limits somewhat the possibility of effectively correcting a low calcium intake by dietary adjustments. This consideration and the relatively low cost of supplements explain why calciumcontaining preparations currently represent the preferred way of attaining optimal calcium intake, especially in elderly individuals. To date, there are more than a dozen commonly prescribed calcium supplements and hundreds of different preparations available over-thecounter. Numerous factors that can affect the bioavailability of calcium have to be considered when prescribing a calcium supplement, including solubility, interference from co-ingested medications or food, dosage, and timing of ingestion. The most common calcium

167

supplement currently used is calcium carbonate, the salt with the highest concentration of calcium by weight (40%). However, calcium carbonate is relatively insoluble, especially at neutral pH. Preparations containing calcium citrate have a lower calcium content (21% by weight) but they are much more soluble than calcium carbonate, an important factor in achlorhydric patients (see below). Citrate is also considered helpful in increasing calcium solubility in the urine, thus reducing the risk of renal stones in hypercalciuric patients. The most soluble calcium salt available as supplement is calcium citrate maleate. This salt is reportedly six times more soluble than the citrate salt, and perhaps more absorbable than calcium carbonate. In general, there seems to be a correlation between solubility and bioavailability of calcium preparations. 4 However, the solubility of the calcium salt may be of limited practical importance, because of the minor differences in net absorption among the different preparations at intakes at or above the RDA. Ultimately, co-existing morbid conditions and other factors will determine the choice of the most appropriate calcium supplement in each individual patient. 4. M E C H A N I S M OF INTESTINAL C A L C I U M ABSORPTION

Calcium absorption by the intestine occurs through the combined action of two pathways, a paracellular and a transcellular pathway. The transcellular pathway is a carrier-mediated (and thus, saturable), energy-dependent, active transport mechanism, which carries calcium from the intestinal lumen across the brush border of the luminal membrane, through the cytoplasm, and extrudes the ion into the vascular side of the epithelium. This mechanism operates in the duodenum and is regulated by vitamin D. The paracellular pathway is a nonsaturable, voltage-dependent, vitamin D-independent, passive transport mechanism, its driving force being the electrochemical gradient of calcium across the intestinal mucosa. This is the mechanism operating in most segments of the intestine, resulting in calcium diffusion through the tight intercellular space between enterocytes. Active transport accounts for most of the calcium absorption when luminal calcium is low (--~3 mM), whereas the passive route takes over at higher luminal calcium concentrations (which usually saturates the active mechanism), v-9 Active calcium transport across the intestinal epithelium occurs in three steps: entry into the epithelial cell across the brush border membrane, transport across the cytoplasm to the basolateral membrane, and finally transport across the basolateral membrane. All these steps are under the control of 1,25-dehydroxyvitamin D2 [1,25(OH)zD3] (see below). Calcium uptake by the mucosal surface requires no metabolic energy, as calcium moves along a downhill electrochemical gradient. 1~The

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ROBERTO CIVITELLI, KONSTANTINOS ZIAMBARAS, AND RATTANA LEELAWATTANA

involvement of a calcium channel or a carrier has been suggested to facilitate luminal calcium entry into epithelial cells. 1~ Alkaline phosphatase, present in the brush border region of intestinal epithelial cells, was thought to be involved in controlling calcium entry into the intestinal cells, because its activity had been shown to coincide with enhanced calcium entry shortly after 1,25(OH)zD3 administration, ll'12 However, inhibition of alkaline phosphatase activity by either L-phenylalanine or theophylline does not decrease calcium uptake, 13 suggesting that the role of this enzyme in calcium entry is minor, if any. Once inside the cell, calcium is carried across the cytoplasm to the basal membrane complexed with a specific calcium-binding protein, calbindin D (CaBP), which mediates interactions between microtubules and membrane-bound vesicles. 14 Once calcium has reached the basal membrane, it is extruded in the extracellular space through a high-affinity CaZ+-ATPase pump. 15 Calcium is also extruded via the Na+/Ca 2+ exchanger, which is driven by the sodium electrochemical gradient. This process also requires energy to maintain the sodium gradient across the plasma membrane, a function operated by the Na+/K+-ATPase pump. 16 5. MEASUREMENT OF CALCIUM ABSORPTION

Quantization of intestinal absorption of calcium is difficult, and despite the many approaches and techniques developed over the years, assessment of calcium absorption remains primarily a research application. True calcium absorption can only be reliably measured by metabolic balance methods. These techniques allow the assessment of various minerals, and they are also used to calculate phosphate and magnesium absorption. Typically, after a 7- to 10-day adaptation period of controlled mineral intake, a balance period follows, during which subjects consume diets containing determined amounts of calcium, phosphorus, and magnesium for approximately 6 days. During this period, the content of each element is measured in urine and feces, as well as in equal amounts of food consumed. Net intestinal absorption is calculated by subtracting the average daily urinary and fecal excretion from the dietary intake. This cumbersome, time-consuming, and expensive method is not suitable outside experimental settings, and it has been replaced by methods based on calcium isotopes. The early techniques developed were based on a single calcium isotope. 17-19 After oral administration of the tracer~generally the radioactive 47Ca i s o t o p e ~ a s a fixed carrier salt (usually CaCI2), the circulating fraction of the absorbed dose is calculated from appearance of radioactivity in the circulation, using mathematical deconvolution models. ~7Thus, these methods measure fractional calcium absorption, rather than the net input of the element. Because passive absorption contributes as

a constant to the fraction of dietary calcium that is absorbed, differences in fractional calcium absorption reflect primarily differences in active transport. To avoid the potential accumulation of a radioactive isotope into the bone, strontium has been used as an alternative to 47Ca or 45Ca, based on the assumption that strontium is absorbed through the same mechanism as calcium, and has a similar volume distribution. 2~ Because the clearance rate of calcium and the distribution volume cannot be precisely assessed using single isotopes, doubleisotope methods have been developed. By following the radioactivity in the forearm, or in the urine, or feces after oral and intravenous administration of 47Ca and 45Ca, one can more precisely estimate fractional calcium absorption. 18'21 The major limitation of these methods stems from the use of a fixed calcium carrier rather than food, which precludes an estimation of the effect of dietary calcium on calcium absorption, leaving the requirement of balance studies to determine true fractional calcium absorption. In the attempts to overcome these problems, an alternative, relatively simple method has been recently proposed based on stable calcium isotopes. One of them (44Ca) is mixed with food, and the other (42Ca) is given intravenously. 22 True fractional absorption can thus be calculated by measuring each isotope in the urine after 24 hours. This method has the advantage of tracing calcium in a habitual diet, rather than in a fixed carrier, and of using stable, rather than radioactive isotopes. True fractional absorption obtained by double stable isotopes is highly correlated with that calculated from balance studies. 22 The cost of these isotopes and, more importantly, the need of facilities and equipment to measure stable isotopes still limit a more widespread applicability of this potentially useful technique. An alternative, simple, though less precise method has been used since the 1970s to estimate the increased absorption rate in patients with absorptive hypercalciuria. The test consists of the measurement of calcium excretion following an oral load of calcium, after an overnight fast. The increase of urine calcium corrected for creatinine in 2-hour urine collections after the load is an index of absorption efficiency. 23 Obviously, the wide individual variability, the difficulty in establishing exactly the total calcium intake, and its strict dependence on the glomerular filtration rate are just some of the most important factors that limit the precision of this method. Nevertheless, the calcium load test may still provide a quick, inexpensive method for screening subjects with hyperabsorptive states. 6. REGULATION OF CALCIUM ABSORPTION

a. Hormonal Regulators VITAMIN D. Vitamin D is the single most important factor regulating the intestinal absorption of calcium in

CHAPTER 6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption humans and animals. 1~ As noted above, 1,25(OH)zD3, the active metabolite of vitamin D, has been shown to affect several steps of the calcium transport process. Early reports demonstrating a significant alteration in membrane lipid composition of the epithelial cells as an early response to 1,25(OH)2D3 administration led to the "liponomic" theory. 25 According to this hypothesis, alterations in the brush border lipid membrane structure and its fluidity state would lead to increased permeability of the membrane to calcium. Membrane fluidity was shown to be diminished in vitamin D - d e p l e t e rats, with rapid restoration of normal fluidity after 1,25(OH)2D3 administration. 26 These changes of membrane fluidity, which are correlated with alterations in membrane phospholipid fatty composition, occur within 1 to 2 hours after administration of 1,25(OH)2D3 and precede the increase in calcium absorption. Therefore, changes in fatty composition and fluidity of the brush border membrane cannot be the sole mechanism by which vitamin D modulates calcium absorption. 26 Vitamin D metabolites directly regulate the expression of the carrier proteins that translocate calcium from the luminal to the basal membrane. A number of CaBPs have been found in the intestinal epithelial cell membrane and cytosol. The most important, calbindin-D, is a high-affinity CaBP. Its dependence on vitamin D is demonstrated by its absence in vitamin D - d e p l e t e chicks, 27 and by its increased synthesis in association with enhanced enterocyte calcium transport after administration of 1,25(OH)2D3. 28 Calcium entry into the cell and transcellular transport are two strictly linked but distinct processes. Although 1,25(OH)zD3 rapidly enhances cellular calcium entry, there is a considerable time lag between entry and subsequent transport of the ion through the cytoplasm. 1~ Furthermore, neither translocation of calcium across the luminal membrane nor binding to the microvilli requires 1,25(OH)2D3, whereas the mobilization of microvillarbound calcium into the cytosol d o e s . z9 In fact, the luminal cell membrane does not represent a substantial barrier to calcium entry into the cell. 3~ Microvilli-bound calcium is initially sequestered in the apical region of the cell. Under the influence of 1,25(OH)2D3, the calcium that has entered the cell can now move, presumably bound to calbindin-D, 31 through the cytoplasm to the basolateral pole where it is actively extruded by the calcium pump. Thus, although vitamin D metabolites may enhance the calcium entry rate, their primary action is to provide the necessary transport elements. Large amounts of parathyroid hormone-related protein (PTHrP) are secreted in human, bovine, and rat milk. 32 The milk concentration of PTHrP is three orders of magnitude higher than that present in the serum of patients with humoral hypercalcemia of malignancy. 32'33 The function of PTHrP in maternal and/or neonatal phys-

169

iology remains speculative. Because PTHrP modulates transplacental calcium gradients during gestation, 34 it is reasonable to believe that the hormone may somehow be involved in the control of calcium transport from blood to milk during lactation. Furthermore, if PTHrP is absorbed intact, or as small active peptides, it could regulate mineral metabolism in the infant in a similar manner as PTH by interacting with PTH/PTHrP receptors. Alternatively, milk PTHrP could directly activate PTH/ PTHrP receptors in the intestine without requiring absorption. The hormone could then directly increase calcium absorption, or stimulate intestinal cell proliferation and differentiation. However, a recent study performed in neonatal pigs failed to detect any effect of synthetic PTHrP-(1-86) added to a soy formula on calcium absorption, renal handling of calcium, or bone mineralization. 35 The only significant effect was an attenuation of the transient increase of serum and urine phosphorus during the first 2 weeks of life, suggesting that some effects of PTHrP may occur before the development of full response to PTH. A direct effect of PTH on intestinal calcium transport has been described in perfused duodena, 36 thus implying the presence of PTH/PTHrP receptors in the intestine. These observations do not exclude the possibility that more distal, non-PTH-like regions of PTHrP may in fact be physiologically active in the neonate, perhaps acting through different, PTHrP-specific receptors. ESTROGEN. A role of estrogen in the physiological regulation of calcium absorption is indirectly demonstrated by the decreased absorption efficiency in postmenopausal women with osteoporosis, 37'38 which can be corrected by estrogen replacement, 39 and by observations in animals suggesting that calcium absorption varies during the normal menstrual cycle in a direct relationship with estrogen levels. 4~ Two non-mutually exclusive hypotheses have been entertained to explain the mechanisms of this estrogen action on intestinal function, including a direct effect of female hormones on the intestinal epithelium, and an indirect effect via the synthesis of 1,25(OH)zD3. In support of the first hypothesis are observations that intestinal cells express functional estrogen receptors, 42"43 and that 17[3-estradiol enhances the uptake of calcium by the intestine in 1;ivo 44 and in v i t r o . 43 Furthermore, vitamin D receptors are decreased in intestinal cells of ovariectomized r a t s , 45 and estrogen replacement can restore a normal intestinal response to 1,25(OH)zD3 in women after surgical menopause, 46 thus suggesting a potential direct regulatory action of estrogen on intestinal vitamin D receptors. On the other hand, the findings that estrogen administration is followed by an increase of 1,25(OH)zD3 blood levels in postm e n o p a u s a l w o m e n 47'48 and by an enhancement of renal l et-hydroxylase activity in lower species, 49 would be

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ROBERTO CIVITELLI, KONSTANTINOS ZIAMBARAS, AND RATTANA LEELAWATTANA

consistent with an indirect, vitamin D-mediated mechanism. However, vitamin D-binding globulins are also increased by estrogen, and whether the free fraction of 1,25(OH)2D3--the hormonally active f o r m - - i s also increased is still debated. 5~ Ultimately, both mechanisms may contribute to the regulatory action of estrogen on intestinal calcium absorption. Several lines of evidence demonstrate that prolactin may enhance intestinal calcium transport. Prolactin has been shown to stimulate renal l oL-hydroxylase activity, 53 thus raising serum 1,25(OH)2D3 levels. 54'55 However, other mechanisms may be involved, since animal studies indicate that prolactin increases calcium absorption even in vitamin D-deficient conditions. 56'57Moreover, prolactin action on active calcium absorption is rapid, reaching maximal effect within 5 to 10 minutes of treatment in sexually mature rats, 58 and an acute (within 2 minutes) increase in apical uptake of 47Ca in mucosa cells also occurs after administration of prolactin. 58 The finding of prolactin receptor messenger ribonucleic acid (mRNA) transcripts in the lamina propria of the intestinal mucosa raises the possibility that this hormone may act directly on the enterocytes. 59 Other actions of prolactin can indirectly contribute to its stimulatory effect on calcium absorption. Thus, prolactin is able to enhance both gastric emptying rate, 6~ resulting in a faster delivery of calcium to the proximal small intestine, and sodium and water transport across the jejunum, probably by stimulating the activity of enterocyte basolateral Na§ § ATPase. 61 Because prolactin stimulation of calcium absorption is partly sodium dependent, an increase in sodium absorption could create a transepithelial osmotic gradient, which in tum would induce water flow from the lumen to circulation, dragging calcium and other ions with it (solvent drag). 61 b. Gastrointestinal Function GASTRIC ACIDITY. With the increasing age of populations in developed countries and the high prevalence of reduced gastric acid secretion in elderly people, 62 the role of gastric acidity in calcium bioavailability may in theory be of importance in elderly people. A low pH is required to solubilize mineral-containing salts, and since only soluble calcium can be absorbed by the intestine, gastric acidity may condition intestinal calcium absorption. As detailed above, the solubility of calcium salts is somewhat correlated with ambient pH, which presumably affects their absorption (see also Section I.A.3). Early studies of the effect of gastric acid alone on intestinal calcium absorption gave conflicting results. 63-65 Whereas a decreased calcium absorption from calcium carbonate was observed in achlorhydric subjects compared with normal individuals in the fasting state, 63'66 this difference disappeared when calcium carbonate was

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given with a meal 64 (Fig. 6-3). Thus, gastric acid may be necessary to solubilize the insoluble calcium-containing salts in the fasting state, but this requirement does not appear to be clinically relevant when calcium is taken with meals, perhaps because of the longer time the element remains available at absorption sites. 67 As a corollary, patients requiring calcium supplements should be recommended to take their calcium tablets with meals. INTESTINAL MOTILITY. By controlling the delivery of calcium to the small intestine, gastric and intestinal motility can affect the rate of calcium absorption. Calcium absorption is inversely related to gastric emptying. 68'69 Although the maximal rate of calcium absorption occurs in the duodenum, 7'7~ the transit time through this short section of the small intestine is normally very rapid. This is the major reason why the largest fraction of the total calcium is absorbed at sites distal to the duodenum. 7 Accordingly, any condition that slows down the rate of gastric emptying and exposes the duodenal epithelium to calcium for longer time increases calcium absorption, whereas disorders and conditions that increase transit time will reduce calcium absorption. 68 Importantly, the rate of gastric emptying depends heavily on the caloric content of food; the higher the caloric content, the slower the gastric emptying, vl This may explain the better absorption of calcium supplements when they are taken with meals of high caloric content, rather than on empty stomach.67 Likewise, the beneficial effect of certain sugars on calcium absorption is probably in part related to

CHAPTER 6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption the slow gastric emptying caused by their high caloric content. 72 BILE SALTS. The main role of bile salts is to solubilize lipids for absorption by forming micelles. This increases the absorption of liposoluble vitamins, including vitamin D. In addition, bile salts facilitate the luminalvascular transport of minerals directly, and in fact, calcium absorption is enhanced by bile salts even in vitamin D-deficient conditions. 73 The negatively charged surface of micelles strongly binds calcium and sodium ions, which are thus carried into the bloodstream. Binding of calcium to micelles may contribute to the fact that the bulk of calcium absorption occurs in the ileum, even though the physicochemical environment of the ileum favors calcium precipitation. TM Through a similar mechanism, bile salts can maintain a very high calcium concentration in the gallbladder, and facilitate its absorption back into the bloodstream. 75 Studies in vitro also seem to suggest that bile salts may directly regulate intestinal calcium transport either by increasing transepithelial electric conductance, TM thus facilitating paracellular ionic flow, or by acting as calcium ionophores. 77

c. Age and Reproductive Function The efficiency of calcium absorption varies during a lifetime in response to a variety of exogenous and endogenous factors. The ability to extract calcium from the diet is of particular importance in children during growth, when the requirements of mineral for skeletal build-up are maximal. It has been estimated that infants can retain up to 44% of calcium from cow m i l k - b a s e d formulas, and up to 60% from breast feeding. When the rate of growth in children slows down, there is a pro78 portional decline in calcium absorption. Peak periods of calcium retention occur at around the time of highest calcium requirements coinciding with the increased bone remodeling for skeletal consolidation. Fractional calcium absorption is approximately 35% in premenarcheal girls. 79'8~ This is roughly 45% higher than in adult women consuming equivalent amounts of calcium, 81'82 and significantly higher than prepubertal or late pubertal (postmenarcheal) girls, whose fractional absorption is consistently below 3 0 % . 79 Accordingly, calcium deposition into bone decreases substantially in the first 5 years following menarche. 83 '84 Because the nonlinear relationship between calcium intake and absorption frac81 tion is qualitatively similar in girls and adult women, it has been proposed that the adaptive response to dietary calcium (see below) in girls is operating around a higher set point compared to adults (Fig. 6 - 4 ) . Interestingly, a daily intake of 1200 mg seems to be sufficient to provide maximal calcium retention in adolescent girls, and any further increase in calcium intake results in little additional increase in calcium retention. 85 Thus, when the

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absorbed calcium reaches 500 mg/day, it probably saturates the ability of skeleton to retain it, and further increases may actually lead indirectly to suppression of calcium absorption. 85 Sufficient data are available to indicate that calcium absorption efficiency declines after puberty. This decline starts soon after initiation of menarche in girls and continues during adulthood, v9'82 It is estimated that after the age of 40, calcium absorption decreases at a rate of 0.21% per year. 81 Because in women this age-related decline in absorption occurs well before the menopause, it cannot be attributed entirely to the loss of sex hormones. As discussed in more detail under Section I.C.3, menopause is accompanied by an additional --~2% decrease of fractional calcium absorption. 8] Thus, the combined effect of menopause and age leads to a 20% to 25% decrease in absorption efficiency in women between ages 40 and 60. 86

d. Gender and Ethnicity African-Americans have greater bone density and a lower risk of osteoporotic fractures compared to Caucasians in the United States, 87 despite a lower calcium intake and a higher frequency of lactose intolerance. 88 Little and inconsistent information is available on racial differences in calcium absorption. 84 '89 Although no difference in fractional calcium absorption between white and black adolescents was reported earlier, s9 a recent

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ROBERTO CIVITELLI, KONSTANTINOS ZIAMBARAS, AND RATTANA LEELAWATTANA

study using a dual-isotope technique demonstrated a markedly greater true calcium absorption in black girls compared to white peers. 84 These racial differences were partly dependent on pubertal status, with the largest difference occurring after menarche. The recent controversy about the vitamin D receptor (VDR) haplotype as a possible genetic determinant of osteoporosis adds to the uncertainty. Indeed, different VDR allelic frequencies have been observed in different ethnic groups, 9~ but the physiological correlates of the different genetic make-ups remain to be established. e. Dietary Factors A number of food products or additives may alter calcium absorption. The most important dietary factor is calcium itself. At intakes above 300 mg/day, which saturate the active transport mechanisms, increasing calcium in the diet results in higher net calcium absorption via increased passive transport (Fig. 6-1). At lower intakes, the intestine is able to adapt to the dietary calcium deficiency with an increased efficiency of absorption. 37'92'93 Thus, fractional absorption increases when the available calcium in the intestinal lumen is low (Fig. 6-5). The relationship between fractional absorption and dietary calcium is not linear, so that at low intakes the absorption fraction is very sensitive to small dietary changes, whereas above 300 to 400 mg/day absorption efficiency virtually does not change with increased dietary content (Fig. 6-5). Although in the initial stages of adaptation a PTH-mediated increase of 1,25(OH)2D3 correlates well with the increased calcium a b s o r p t i o n , 37'93 for prolonged calcium deprivation such an association is lost, 94 implying that other mechanisms may mediate the

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adaptive response. A similar adaptive mechanism has been invoked to explain the higher absorption efficiency at puberty (see above). A high sodium intake increases natriuresis, which is accompanied by calciuresis. As a response to the sustained renal leak of calcium, serum 1,25(OH)2D3 increases to maintain normal serum calcium levels by increasing intestinal calcium absorption. 95 This compensatory mechanism requires the presence of intact parathyroid glands, because it does not occur in hypoparathyroid patients, 95 and a rise in serum PTH and urinary cyclic adenosine monophosphate (cAMP) precede the enhanced calcium absorption. 96 The presence of high concentrations of glucose in the intestinal lumen results in a marked enhancement of intestinal calcium absorption. There are at least three possible mechanisms for this effect. First, glucose might directly stimulate active calcium absorption by providing a readily available energy source. Second, calcium may cross the mucosa barrier by solvent drag, entrapped in the transepithelial water flow, which is driven by the increased glucose transport. Third, glucose-stimulated water absorption may create a calcium concentration gradient along the intestinal lumen, which allows calcium ions to passively flow from the lumen to extracellular fluid. 97 Certain amino acids, such as L-lysine have been reported to affect intestinal calcium transport in early animal studies. 98"99 The notion that lysine may somehow be involved in skeletal homeostasis is underlined by the observation that individuals with lysinuric protein intolerance present with childhood osteopenia, ~~176 which can be corrected only by normalizing plasma lysine. 1~176 Short-term dietary supplementation with L-lysine enhances intestinal absorption of calcium in postmenopausal osteoporotic women with deficient intestinal calcium a b s o r p t i o n . 1~ This action seems to be specific for L-lysine, although early data suggested that L-arginine, another cationic amino acid, could also be effective. 99 The mechanisms underlying this effect of L-lysine are mostly unknown.

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FIGURE 6--5 The relationship between fractional calcium absorption and dietary intake of calcium in unselected women between ages 30 and 60. (From Heaney RP, Recker RR, Stegman MR, Moy AI: Calcium absorption in women: Relationships to calcium intake, estrogen status, and age. J Bone Miner Res 4:469-475, 1989.)

a. Milk-Alkali Syndrome In 1915 a new diet was introduced for patients suffering from peptic ulcer. The diet consisted of large amounts of calcium and alkali in the form of milk and absorbable alkaline salts of calcium and magnesium. Before being adequately appreciated what later came to be known as the "milk-alkali syndrome," it was a significant cause of irreversible renal

CHAPTER6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption TABLE 6 - 2

Disorders Associated with Increased Calcium Absorption

Increased calcium bioavailability Milk-alkali syndrome Increased intestinal saccharases (lactose) activity Vitamin D excess Vitamin D intoxication Sarcoidosis, other granulomatous diseases, and lymphomas Hyperparathyroidism Growth hormone excess Hyperprostaglandin E syndrome Renal hypercalciuria Vitamin D-independent calcium hyperabsorption Absorptive hypercalciuria Pregnancy Lactation

failure. 1~ The syndrome classically is characterized by hypercalcemia, metabolic alkalosis, and renal impairment with normal or low urine calcium excretion. The increased intestinal absorption of calcium is due to the tremendous calcium load that the intestine is being exposed to, although an intrinsic inability to reduce fractional absorption of ingested calcium cannot be completely excluded. Intact PTH is typically suppressed by the hypercalcemia, 1~176 and it is likely that occasional case reports of increased PTH levels were due to measurement of the carboxy-terminal portion of the PTH molecule in patients with compromised renal function. TM Serum 1,25(OH)2D3 level is also low normal or low. This syndrome is seldom seen nowadays, because of the drastic changes to the therapy of ulcers. Nonetheless, such a condition should be kept in mind, considering that many currently available over-the-counter antacids contain absorbable calcium salts which, when abused, may cause a similar syndrome. b. Increased Intestinal Saccharase Activity Lactose is a disaccharide that is hydrolyzed by lactase to glucose and galactose in distal parts of the small intestine where these monosaccharides are absorbed. The remaining lactose is metabolized in the large intestine to volatile fatty acids (propionic, butyric, and acetic acids), thus lowering the pH of the fluid within the cecum and colon. 1~ Lactulose, an analogue of lactose that is resistant to endogenous saccharases in the small intestine, 1~ can be metabolized by the bacterial flora to volatile fatty acids in the large intestine. Animal studies have shown that both saccharides increase fractional intestinal calcium absorption when present in food at high concentrations. 107 ,~08 This effect is not specific for lactose and lactulose, since other carbohydrates such as xylitol, sorbitol, arabinose, and gluconate are able to increase calcium absorption in

173

a similar manner and extent. 1~ Furthermore, oral lactose increases calcium absorption in vitamin D-deficient rats, and lactulose action is additive to that of 1,25(OH)2D3 .1~ Therefore, these saccharides appear to stimulate the passive, vitamin D-independent component of calcium absorption. In fact, long-term lactose feeding can even reduce duodenal (active) calcium absorption and CaBP content, possibly as an adaptive reaction to compensate the saccharide-induced increase of passive calcium absorption from the distal intestine. 1~ There are few potential mechanisms by which high luminal carbohydrate concentrations affect calcium absorption. First, the hydrolytic products of lactose (i.e., glucose and galactose) may induce a solvent drag as a consequence of an increased water absorption, 11~ as discussed in Section I.A.e. Second, the presence of osmotically active sugars in the small intestine (i.e., hydrolyzed lactose or lactulose) increases the volume of fluids within the lumen, which might increase the distention and permeability of the intercellular junctions between enterocytes, thereby increasing the passive absorption of calcium. 1~ This phenomenon can be reproduced by increasing the fluid content of jejunal loops in situ with hypertonic solutions. 112 Finally, the reduced pH from saccharide hydrolysis in the large intestine may increase the concentration of soluble calcium available for absorption. 113

2. VITAMIN D-DEPENDENT HYPERABSORPTION a. Vitamin D Intoxication Excess administration of vitamin D metabolites, although less common than in the past, still presents a potentially serious clinical problem. In most cases, the source of the excess vitamin D is calciferol, the most widely used form of vitamin D supplement. Preparations containing "megadoses" (i.e., 50,000 IU per tablet) are sometimes prescribed on a weekly schedule to elderly patients with presumptive signs of vitamin D insufficiency. While the incidence of vitamin D deficiency, most commonly in a subclinical form, is still relatively high even in the United States, TM the doses contained in these preparations greatly exceed the daily requirements and the metabolic clearance capacity of the liver, thus leading to accumulation of calciferol in adipose tissue stores. Because the 25hydroxylation in the liver is not as tightly regulated as the l oL-hydroxylation reaction, the massive bulk of substrate ingested results in increased production of 25(OH)D. Although this metabolite is certainly far less potent than 1,25(OH)2D3, it appears to contribute to regulate calcium absorption even at physiological concentrations. 69'115 Thus, when its circulating levels rise significantly as it occurs in vitamin D intoxication, 25(OH)D becomes the major factor that conditions the increased calcium absorption. In fact, not only are serum

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ROBEI~TO CIVITELLI, KONSTANTINOSZIAMBARAS, AND RATTANA LEELAWATTANA

levels of 25(OH)D better correlated with calcium absorption than circulating 1,25(OH)zD3 in healthy individuals, 69 but it is 25(OH)D, rather than 1,25(OH)zD3 that invariably increases in vitamin D intoxication. However, more recent data indicate that although serum 1,25(OH)zD3 is usually normal in vitamin D intoxication, probably because of the hypercalcemia, the free fraction of 1,25(OH)zD3 is indeed increased, thus providing a possible contribution to the pathogenesis of this condition. 116 The ensuing hypercalciuria, hypercalcemia, and hyperphosphatemia are sustained by the increased active calcium absorption, and can lead to renal stones. Like calciferol, 25(OH)D has a half-life of approximately 3 weeks, because it can accumulate in adipose tissue and muscles. Consequently, following the withdrawal of vitamin D, several weeks or months may be required before hypercalcemia and the enhanced absorption of calcium are corrected. b. Sarcoidosis, Other Granulomatous Diseases, and Lymphomas Sarcoidosis is a disease of unknown origin, characterized by a granulomatous infiltrate of multiple tissues. It is associated with mild to severe hypercalcemia in 10% of the patients. Calcium balance studies have repeatedly demonstrated the association of hypercalcemia with increased intestinal calcium absorption, increased urinary calcium excretion, and decreased fecal calcium excretion. 117'118 The pathogenetic mechanism of calcium hyperabsorption is an increased synthesis of 1,25(OH)zD3 by macrophages in the sarcoid tissue. 119-121 Cells of the monocyte/macrophage lineage express a 1oLhydroxylase, which is very similar to but distinct from the renal enzyme, and whose physiological function is still unknown. Patients with active sarcoidosis will benefit from dietary restriction of calcium and vitamin D and from limited exposure to sunlight. Glucocorticoid treatment is very effective in correcting the hypercalcemia, because it inhibits l et-hydroxylase activity, thus reducing the increased calcium absorption. 122 The same mechanism can account for the hypercalcemia associated with other granulomatous diseases, such as coccidioidomycosis, 123 histoplasmosis, 124 berylliosis, 125 cryptocCOCOSiS,126 tuberculosis, 127'128 and rare cases of chronic infections from Candida albicans 129 and Pneumocystis carinii. ~3~Ectopic production of 1,25(OH)zD3 by tumorinfiltrating macrophages is the pathogenetic mechanism of the increased calcium absorption in non-Hodgkin's lymphoma as well. TM Finally, T-lymphocytes transformed by the human T-cell lymphotropic virus (HTLV1), can also synthesize 1,25(OH)zD3 resulting in hypercalcemia in patients with HTLV-1 leukemia. 132 c. Primary Hyperparathyroidism Direct stimulation of 1,25(OH)zD3 production by abnormal secretion of

PTH in parathyroid adenomas or hyperplastic glands is one of the physiological paradigms of vitamin D dependent calcium hyperabsorption. The excessive calcium absorbed via increased active transport is the primary mechanism that maintains the hypercalciuria in primary hyperparathyroidism. In spite of the enhanced distal tubular reabsorption of calcium that is stimulated by PTH, the large bulk flow of filtered calcium generated by the increased absorption results in a very high total urine calcium output. d. Growth Hormone Excess Human and animal studies have demonstrated an elevation in calcium and phosphate intestinal absorption after growth hormone (GH) administration. ~33'~34Furthermore, hypercalciuria is a well-known feature in acromegalic patients, who also demonstrate a tendency toward high-normal, if not frankly elevated serum calcium. A positive correlation between calcium absorption and serum or urine calcium levels exists, 133 suggesting that the main cause of hypercalciuria in acromegaly is excessive absorption from the gut resulting in increased filtered load of calcium. ~33 Some reports have demonstrated a correlation between GH-induced calcium absorption and 1,25(OH)zD3 levels and jejunal concentration of calbindin (CaBP9k), 135 although such association is still controversial. TM In vitro studies indicate that this effect of GH is, at least in part, mediated by insulin-like growth factor 1 (IGF-1), which can either directly stimulate absorption of calcium and phosphate, or increase 1,25(OH)zD3 synthesis. 134'136 A marked mucosa atrophy is seen in hypophysectomized rats, in association with decreased calcium absorption, changes that are reversed upon GH repletion. 137 Although the anabolic effect of GH on the intestinal epithelium may theoretically enhance intestinal absorption efficiency, it is unlikely that it represents the primary mechanism for GH action on calcium intestinal absorption. e. Hyperprostaglandin E Syndrome Increased urinary excretion of prostaglandins of the E series (PGE) is observed in children with elevated aldosterone and plasma renin activity, electrolyte abnormalities, and hypercalciuria. In many aspects this condition resembles Barrter's syndrome, although variant forms have been reported in the literature. 138 Children affected by this disorder can be in positive calcium balance, 139 suggesting that the hypercalciuria is probably the result of both a primary defect in renal calcium reabsorption, and an excessive enteric calcium absorption. 138'14~A positive correlation has been found between serum 1,25(OH)2D3 (usually elevated in these patients), calcium absorption, and urinary PGE2 .141 Because PGE2 is able to stimulate l oL-hydroxylase activity in v i v o 142 and in v i t r o , 143 a vi-

CHAPTER 6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption tamin D-dependent hyperabsorption is considered the pathogenetic mechanism of this syndrome. Although calcium absorption, as well as serum 1,25(OH)2D3 and PGE2 tend to normalize after treatment with indomethacin or aspirin, 138"143some degree of hypercalciuria may persist, probably as a result of the PGE2-induced bone resorption, that may lead to osteopenia in these children. TM

f Renal Hypercalciuria The term "idiopathic hypercalciuria" was used to identify the association of renal stones with hypercalciuria, defined as a urinary calcium excretion of greater than 250 mg/day in females, greater than 300 mg/day in males, or greater than 4 mg/ kg/day in either sex, while on random diet. Hypercalciuric syndromes are now recognized as a group of pathophysiologically heterogenous disorders, 144'145 whose major entities are represented by absorptive hypercalciuria and renal hypercalciuria. Clinically, these two forms differ in their response to therapy. A reduction in dietary calcium or an overnight fast resolves the hypercalciuria in the absorptive form but not in the renal variant. 23'146 On the other hand, therapy with thiazides persistently reduces urine calcium in renal hypercalciuria, whereas in patients with absorptive hypercalciuria the decrease of urinary calcium is transient, and hypercalciuria recurs in about 50% of the patients after 3 months of therapy. ~47 Renal hypercalciuria represents one end of the spectrum, in which the primary abnormality is an impairment in the renal tubular reabsorption of calcium. 148 The renal calcium loss causes a mild decrease in serum calcium, which in turn induces a secondary increase of PTH as homeostatic response. The attendant increase of 1,25(OH)2D3 enhances intestinal calcium absorption which, along with mobilization of skeletal calcium by PTH, maintains normal serum calcium levels. Thus in renal hypercalciuria, the increased calcium absorption is vitamin D dependent and secondary to a primary renal defect. Unlike in the absorptive form, thiazides induce a significant inhibition of intestinal calcium absorption in renal hypercalciuria, 147 the consequence of a correction of the secondary hyperparathyroidism and of improved renal calcium conservation. 149 3. VITAMIN D-INDEPENDENT CALCIUM HYPERABSORPTION

a. Absorptive Hypercalciuria The primary abnormality in the absorptive form of hypercalciuria is an increased intestinal calcium absorption. This results in a slight rise in serum calcium, which in turn increases the filtered calcium load with consequent hypercalciuria and suppressed parathyroid function. The enhanced excretion of calcium contributes to keep serum calcium within the

175

normal range. TM Thus, although calcium absorption is increased in both absorptive and renal hypercalciuric syndromes, the pathogenesis and biochemical abnormalities are substantially different. Controversy persists regarding the cause of the intestinal calcium hyperabsorption of absorptive hypercalciuria. Although by strict definition, absorptive hypercalciuria should be vitamin D-independent, elevated levels of 1,25(OH)2D3 have been reported in 30% to 80% of patients with absorptive hypercalciuria, defined using rigorous criteria. T M These observations may reflect a difficulty in distinguishing between the two forms in many patients, who may present overlapping features of absorptive and renal hypercalciuria. Nonetheless, serum PTH and phosphate are generally normal in patients with the absorptive form. TM An increased sensitivity to 1,25(OH)zD3, perhaps reflecting the ability of 1,25(OH)zD3 to up-regulate its own receptor, 152 has been considered as a possible pathogenetic mechanism. ~53 Experimental animals with absorptive hypercalciuria have increased 1,25(OH)2D3 receptors in their intestines and kidneys, ~54 but similar findings are still unavailable for humans. Nevertheless, a subset of patients with absorptive hypercalciuria do exhibit a genuine vitamin D-independent increase in intestinal calcium absorption. 15~ In these cases, high calcium absorption occurs almost exclusively in the jejunum, pointing to a primary defect of the active transport mechanism. ~55

b. Pregnancy Pregnancy is a state of increased intestinal calcium absorption, associated with an increased calcium excretion. 156'157 Fractional calcium absorption, which is --~40% during the first trimester of pregnancy, may increase up to 60% in the second and third trimesters. 157 The factors that induce this rise are still not well defined. Maternal serum concentrations of 1,25(OH)2D3 rise progressively since the first trimester of pregnancy, 157'~58in parallel with the changes in intestinal calcium absorption. 157 However, the concentration of serum vitamin D-binding protein also increases during pregnancy because of elevated estrogen level, and the free fraction of 1,25(OH)2D3 does not increase until the last month of gestation. 159'16~Similarly, PTH rises only during the last few weeks of pregnancy, ~56'~57indicating that the adaptive reaction of the PTH/vitamin D axis is not entirely responsible for the hyperabsorptive state of pregnancy. Since calcium absorption increases in vitamin D-deficient rats during pregnancy as well, 161 additional mechanisms might be operating. A direct effect of estrogen on intestinal VDR expression remains a possibility (see Section I.B.5). Alternatively, the elevated prolactin and/or human placental lactogen may also facilitate calcium absorption through mechanisms that are not yet totally clarified.

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ROBERTO CIVITELLI, KONSTANTINOS ZIAMBARAS, AND RATTANA LEELAWATTANA

c. Lactation Breast-feeding is a highly demanding period with respect to daily calcium needs, if one considers that lactating women secrete --~210 mg/day of calcium in breast milk. 162 This additional output of calcium may result in a transient and reversible bone lOSS, 163'164 to which adolescents are especially exposed. 165 Despite the widely held belief that the extra calcium requirements of lactating women are met by increased intestinal calcium absorption, accumulating data seem to disprove this contention. A number of human studies have failed to show significant changes in intestinal calcium absorption during lactation. 156'157'166 However, lactating women who resume menses do exhibit a higher fractional calcium absorption compared to postpartum amenorrheic counterparts, or nonlactating control peers. It may appear paradoxical that calcium absorption does not increase during lactation, a time of significant calcium loss, yet it increases after weaning when the calcium drain has ceased. Changes in hormonal status and bone remodeling rates help explain these puzzling findings. Serum levels of tartrate-resistant acid phosphates and urine deoxypyridinoline, markers of osteoclast activity and bone resorption, increase during late pregnancy and lactation. 157 The increased extraction of calcium from bone maintains serum calcium at normal levels, without changes in calcium absorption. Thus, during lactation calcium balance is maintained by increased bone resorption, rather than increased calcium absorption. The relative hypoestrogenism during breast-feeding may be responsible for the increased bone resorption, and the restored estrogen levels after weaning may reequilibrate bone remodeling. Therefore, in the recovery period, intestinal calcium absorption increases to meet the calcium requirements for bone accretion consequent to the transient lOSS.157'167 Although estrogen deficiency may be sufficient to explain these homeostatic changes, the increased secretion of other hormones during lactation, in particular prolactin and PTHrP, 168'169 may contribute to the increased bone resorption of breast-feeding.

C. Decreased Calcium Absorptive States Conditions characterized by a decreased intestinal calcium absorption are listed in Table 6 - 3 . 1. DECREASED CALCIUM BIOAVAILABILITY a. Dietary Deficiency Despite the growing evidence that an adequate calcium intake is critical not only to achieve an optimal peak bone mass but also to minimize the age-dependent bone loss, a large number of Americans still fail to meet the currently recommended guidelines for calcium intake. 5 The issues concerning the op-

TABLE 6--3

Disorders Associated with Decreased Calcium Absorption

Decreased calcium bioavailability Dietary calcium deficiency Lactose intolerance Reduced absorptive surface Vitamin D deficiency Dietary vitamin D deficiency Hepatobiliary diseases with malabsorption Anticonvulsant medications Chronic renal failure Nephrotic syndrome Hyperthyroidism Immobilization Diabetes mellitus Preeclampsia Intrinsic defects of intestinal calcium transport Aging Menopause Glucocorticoids Hypomagnesemia Alcohol abuse Excessive caffeine intake Extrinsic factors that inhibit calcium absorption Drugs: phenytoin, neomycin, colchicine, chemotherapeutic agents Dietary components/additives: phosphate, phytate, oxalate

timal calcium intake at the different ages in relation to the current RDA are discussed under Section I.A.2. The inadequacy of standard diets to provide sufficient calcium remains a problem of national relevance in most Western countries, food fortification notwithstanding. Thus, despite the ability of the intestine to adapt to low calcium in the diet with increased absorption efficiency, inadequate dietary calcium intake remains one of the leading risk factors for osteoporosis in the 1990s. b. Lactose Intolerance Abdominal symptoms such as bloating, abdominal pain, flatulence, and diarrhea after ingestion of milk or dairy products are usually attributed to an inefficient lactose metabolism, with consequent bacterial fermentation of the nonabsorbed lactose in the intestine. This syndrome, often referred to as lateonset, primary lactase deficiency, is generally ascribed to an age-dependent decrease of brush border disaccharidases, including sucrase, maltase, and lactase. These abnormalities have been invoked to explain lactose malabsorption in elderly subjects, a condition that may lead to the onset of abdominal symptoms and consequently induce the avoidance of milk and dairy products from the diet. Since dairy products provide most of the daily dietary requirement of calcium, lactose malabsorption is considered a risk factor for osteoporosis, 17~even though a direct link between lactase deficiency and low bone mass has never been consistently found. 171'172 However,

CHAPTER 6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption subjects with lactose intolerance and positive abdominal symptoms tend to have a lower bone density compared to those without symptoms, and an inverse correlation exists between severity of symptoms and calcium intake. 172 Thus, calcium intake and bone mass are not influenced directly by lactose malabsorption, but the appearance of symptoms of intolerance seems to negatively affect bone mass through a reduced calcium intake. 172 It is worth noting that subjects with primary lactase deficiency can absorb lactose more efficiently from yogurt than from milk, because of the lactase activity present in yogurt bacteria. 173 Few data are currently available on secondary lactose malabsorption, a condition frequently observed in subjects with decreased absorptive surface, such as in the short-bowel syndrome, or as a consequence of chemical or toxic injury to intestinal cells, as occurs after chemotherapy. In these conditions, lactose malabsorption can be greatly improved by consuming yogurt rather than m i l k . 174A75 Obviously, adequate calcium intake in these cases can be easily obtained by calcium supplements or by the addition of exogenous lactase to milk, if dairy intake is required for other reasons.

c. Reduced Absorptive Surface Reduction of the absorptive surface area is responsible for calcium malabsorption in gastrointestinal diseases, such as Crohn' s disease, celiac disease, ~76 as well as in subjects with short bowel syndrome. 177 The malabsorption in these conditions is obviously not limited to calcium, and it involves phosphate, magnesium, and other minerals. Malabsorption of vitamin D and loss of bile salts add to the inadequate mineral input. At the skeletal level, this results in osteopenia sometimes associated with osteomalacia, depending on whether the vitamin D or the calcium deficit prevails. Celiac disease represents a good paradigm for the complex calcium/vitamin D malabsorption in intestinal disorders. In celiac disease, serum levels of 25(OH)D are lower than normal, whereas 1,25(OH)2D3 is either normal or even increased. 178 Thus, the increase of 1,25(OH)2D3, driven by a secondary rise of PTH, is not able to compensate for the absorption inefficiency. 178 Although these changes may be interpreted on the basis of a primary intestinal defect which alters the responsiveness to vitamin D, one could speculate that the subnormal 25(OH)D levels may be the cause of the calcium malabsorption. In fact, correction of the malabsorption with gluten-free diet restores normal levels of the vitamin D metabolite, and prevents the high bone turnover osteopenia characteristic of untreated celiac disease. 178'~79 Furthermore, long-term supplement with vitamin D (1000 IU/day) prevents bone mineral loss in patients with Crohn's diseases, provided that serum 25(OH)D3 is normal. 180

177

2. VITAMIN D DEFICIENCY a. Dietary Vitamin D Deficiency As noted repeatedly in this chapter, the widespread use of calcium supplements containing vitamin D and fortification of food products have only partially altered the prevalence of relative vitamin D deficiency in the Western hemisphere. Although overt clinical syndromes of nutritional tickets or osteomalacia are now very rarely seen, a subclinical vitamin D deficiency, defined biochemically as low 25(OH)D with low-normal serum calcium and phosphate, increased alkaline phosphatase, and mild secondary hyperparathyroidism, was found in approximately 10% of ambulatory women living in the midwestern United States, referred for evaluation of osteoporosis. TM A more recent study estimated that almost 50% of homebound, community-dwelling, elderly persons may suffer from vitamin D deficiency. TM Thus, the possibility of subclinical deficiency of vitamin D and calcium malabsorption should be considered in elderly subjects with poor nutrition, and who spend most of their time indoors. Particular mention should be made to patients with anorexia nervosa. Calcium balance studies suggest that fractional absorption is blunted in these patients, compared to normal individuals. 182 Although the mechanism is still hypothetical, nutritional deficiency of vitamin D 183 and estrogen, as well as a glucocorticoid hyperproduction 183 may all contribute to the defective calcium absorption efficiency in anorexia nervosa.

b. Hepatobiliary Diseases The important physiological role of bile salts in intestinal absorption of calcium is underlined by the observation that patients with distal gut resection are calcium deficient. Loss of bile salts and ensuing steatorrhea contribute to the calcium malabsorption occurring in gastrointestinal conditions characterized by decreased absorptive surface (see above). Excess intraluminal unabsorbed fat not only binds calcium and limits its accessibility to the transport site but also impairs the absorption of vitamin D. B iliary cirrhosis and other chronic cholestatic syndromes with impairment of bile salt secretion result in marked reduction of serum 25(OH)D levels and calcium absorption. 184 On the other hand, patients with cirrhosis and loss of parenchymal cells, but without significant cholestasis, have only modestly reduced serum 25(OH)D levels. Thus, the enzymatic reserve for 25-hydroxylation of vitamin D is sufficient to sustain the requirement of vitamin D metabolism, even in the presence of marked hepatocellular losses. Accordingly, calcium absorption is impaired in patients with chronic parenchymal nonbiliary liver disease and cholestasis, whereas anicteric patients conserve a normal calcium absorption. Therefore, the defec-

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ROBERTO CIVITELLI, KONSTANTINOS ZIAMBARAS, AND RATTANA LEELAWATTANA

tive calcium absorption of hepatobiliary disease is primarily related to vitamin D deficiency. c. Anticonvulsant Medications Anticonvulsant medications, especially phenobarbital and phenytoin, have long been recognized as potential causes of disordered mineral homeostasis. Long-term anticonvulsant therapy can lead to hypocalcemia with biochemical evidence of secondary hyperparathyroidism. 185'186 The most consistent abnormality is a decreased serum 25(OH)D, 185'186 whereas the concentration of circulating 1,25(OH)2D3 is usually normal or even elevated in individuals treated with anticonvulsant agents. 187 Reduced calcium absorption and increased fecal calcium excretion have been demonstrated in epileptic patients after treatment with either phenobarbital or phenytoin. 188'189The selective reduction of serum 25(OH)D points to a hepatic defect in these subjects. Barbiturates induce the hepatic P-450 microsomal enzyme activity, resulting in an increased catabolic hydroxylation of calciferol and 25(OH)D to biologically inactive, more polar metabolites that are eliminated in the bile and u r i n e , 187 thus reducing the availability of 25(OH)D. 188 Because 1,25(OH)2D3 is not significantly decreased, the pathophysiological changes would imply that decreased 25(OH)D accounts for the blunted intestinal calcium absorption associated with anticonvulsant therapy. Higher frequency of osteomalacia was in fact reported in early studies. 19~ Nevertheless, some individuals on long-term therapy with antiepileptic agents have normal serum 25(OH)D, and in fact the incidence of histologically proven osteomalacia is quite rare in patients who are ambulatory and supplemented with vitamin D. 189'191 The discrepant findings may be related to the different ages and general clinical status of the different study populations, vitamin D deficiency being more frequent in nonambulatory or institutionalized subjects. Furthermore, there is enough evidence to suggest that phenytoin may have a direct effect on intestinal calcium a b s o r p t i o n , 192'193 which certainly contributes to the hypocalcemia in long-term users. In osteoblasts, phenytoin decreases calcium influx and increases cytoplasmic calcium efflux, 191 which may represent a mechanism by which phenytoin interferes with intestinal calcium transport. d. Chronic Renal Failure This is associated with a marked impairment of intestinal absorption of calcium and phosphate, and poor adaptation to alterations in dietary calcium. 194 Abnormal calcium absorption is usually detectable when serum creatinine rises above 2.5 mg/ dl, 195 and an inverse correlation between blood urea nitrogen (BUN) and calcium absorption has been reported for patients with BUN levels between 25 and 250 mg/

dl. 196 With glomerular filtration rate of 50 ml/min or

greater, intestinal calcium absorption is normal, 197 whereas fractional absorption of calcium and phosphate is markedly impaired in dialysis patients. 194 In renal failure, the calcium malabsorption is directly related to the defective production of 1,25(OH)2D3, and thus, it is the active calcium transport that fails, although in theory, uremic toxins may also directly alter enterocyte metabolism and proliferation rate. 198 A significant decrease of CaB P content in uremia has been reported in some studies but not o t h e r s . 199'2~176 Since passive calcium absorption is essentially intact, uremic patients can absorb sufficient calcium when their dietary intake is augmented to 4 to 10 g/day. Although these large amounts of dietary calcium may overcome the malabsorption by forcing the diffusion of calcium through the intestinal mucosa, such doses are usually not well accepted by patients. Oral administration of 1,25(OH)zD3 increases fractional calcium absorption up to 45% to 50% 2~ and is the mainstay of therapy in chronic renal failure. As expected, successful renal transplantation normalizes calcium and phosphate absorption. The success of the transplant in terms of improved calcium absorption depends on both the glomerular filtration rate of the transplanted kidneys, and the steroid dosage for immunosuppression. Doses of prednisone lower than 30 mg/day and glomerular filtration rates above 50 ml/min are usually required to avoid the development of clinically relevant calcium malabsorption. 197

e. The Nephrotic Syndrome The nephrotic syndrome is commonly associated with vitamin D deficiency, hypocalcemia, and reduced intestinal calcium absorption. 2~176 Although hypocalcemia was initially attributed to hypoalbuminemia, subsequent studies showed that ionized calcium is also reduced. 2~ Typically, the concentration of 25(OH)D is reduced because of the urinary loss of vitamin D metabolites bound to the vitamin D-binding protein (DBP). 2~ In fact, urinary excretion of DBP increases in nephrotic syndrome, proportionally to the degree of proteinuria, 2~176 whereas absorption of vitamin D is normal. 2~ 1,25(OH)2D3 is also lost in the urine bound to DBE 2~ although l oLhydroxylase may also be defective in the nephrotic syndrome. 2~ As a result of the global vitamin D deficiency, calcium absorption declines, contributing to lower serum calcium, and to the secondary hyperparathyroidism. The chronic hypotonicity of circulating fluids that characterizes the nephrotic syndrome may further contribute to calcium malabsorption. Conceivably, the net plasma-tolumen water movement generated by the hypotonic plasma may obstruct the flow of calcium from the lumen to the plasma.

CHAPTER 6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption

f Hyperthyroidism Intestinal absorption of calcium is reduced in hyperthyroidism. 2~176 On the contrary, urinary and fecal calcium excretion are frequently increased. Low serum levels of 1,25(OH)zD3 have been demonstrated in patients with active hyperthyroidi s m , 2~176 probably the result of suppressed 1,25(OH)zD3 synthesis, secondary to the increased bone turnover. Both active and passive intestinal calcium absorption are decreased, 2~ the latter being the result of multiple factors, such as increased volume of intestinal secretions and increased gastrointestinal motility caused by hyperthyroidism. These changes are entirely reversible, since normalization of plasma thyroid hormones restores normal jejunal calcium absorption and normalizes serum 1,25(OH)2D3. z~ g. Immobilization Acute immobilization is complicated by rapid bone loss and severe hypercalciuria. A similar process occurs during space flight, and it is thought to result from a concurrent elevation of bone resorption and depression of bone formation. Balance studies have demonstrated a negative calcium balance with elevated urinary and fecal excretion during longterm bed rest. 2~~ These changes are accompanied by a decrease in true calcium absorption and a parallel decrease of 1,25(OH)2D3 .211 Calcium absorption returns to normal within 5 to 6 weeks of rehabilitation. h. Diabetes Mellitus Calcium metabolism is markedly altered in conditions of insulin deficiency. While the impact of diabetes on calcium metabolism has been extensively studied in animals, some caveats must be borne in mind in interpreting these results. Most importantly, a distinction must be made between short-term and long-term adaptation of mineral metabolism in response to insulin deficiency. Short-term diabetic rats (2 to 4 weeks) develop fasting hypocalcemia, hypercalciuria, increased fecal calcium excretion, and diminished duodenal calcium absorption 212 leading to a negative calcium balance. 2~3 Despite the ensuing secondary hyperparathyroidism, s e r u m 1,25(OH)2D3214'215 and CaBP9K215 are depressed in diabetic rats. The low 1,25(OH)2D3 levels are probably secondary to decreased conversion of 25(OH)D to 1,25(OH)2D3 in an insulin-deficient environment. 2~6 On the contrary, chronic (>6 to 7 weeks) diabetic rats develop a markedly increased intestinal calcium absorption, despite persistently low 1,25(OH)2D3 levels) ~7 At this stage of chronic diabetes, urinary losses of calcium are substantial, 217 and this may drive an enhancement of intestinal calcium absorption as a compensatory mechanism. Furthermore, chronic insulin deficiency causes intestinal hypertrophy, increased mucosa cell proliferation, and stimulation of several membrane transport systems. An insulin-dependent increase in in-

179

testinal absorption of calcium along with marked hypercalciuria has also been reported in humans with poorly controlled diabetes. 218

i. Preeclampsia Preeclampsia is associated with several abnormalities of calcium metabolism, such as marked hypocalciuria, 2~9 decreased 1,25(OH)zD3, 22~ elevated parathyroid hormone, T M and reduced level of ionized calcium. 222 In some patients, although not all, intestinal calcium absorption may be l o w . 219'221 3. INTRINSIC DEFECTS OF INTESTINAL C A L C I U M TRANSPORT

a. Aging A decreased intestinal calcium absorption 37'86'223 and a decline in the intestinal ability to adapt to a low-calcium diet have been consistently noted in elderly individuals. 22'81 As mentioned under Section I.A.5, two major hypotheses have been entertained to explain the declining calcium absorption, i.e., a decrease of 1,25(OH)2D3 and an intestinal resistance to the vitamin D metabolite. Although age-dependent declines in serum 1,25(OH)2D3 levels have been observed, 37'224 others have reported either unchanged 47'225 or even increased circulating 1,25(OH)2D3 .226'227 Furthermore, whereas a decreased conversion of 25(OH)D to 1,25(OH)2D3 has been found, kinetic studies in rats indicate that synthesis of 1,25(OH)2D3 actually increases with aging. 192 The latter finding would suggest that an intestinal resistance to 1,25(OH)2D3 develops with aging. Accordingly, a selective resistance to treatment with 25(OH)D in elderly women with fractures compared to subjects without fractures has been observed, despite equal increments in 1,25(OH)2D3 levels. 228 Furthermore, an age-related decrease of intestinal VDR was demonstrated in duodenal biopsies of normal w o m e n , 226 a s well as in r a t s . 223 However, other observations have not been consistent with the hypothesis of an intestinal resistance to vitamin D metabolites. Prolonged administration of 1,25(OH)2D3 could consistently improve calcium absorption in elderly women with vertebral fractures, 229 pointing to the existence of a reduced ability to synthesize 1,25(OH)2D3 in the elderly, rather than to an end-organ resistance. The two mechanisms might not be mutually exclusive, and the apparent discrepancies could be reconciled by observing that serum 1,25(OH)zD3 increases until age 65, starting to decline only thereafter 227'230 (Fig. 6 - 6 ) . These observations could be interpreted to suggest that vitamin D resistance develops earlier, representing the major factor responsible for the declining calcium absorption until the seventh decade; whereas a defect in 1,25(OH)zD3 production occurs in a later phase of life. Recent studies have shed a different light on this issue. Using a doubleisotope method to assess true fractional absorption, Ebel-

180

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ing et al. TM were able to induce increases of intestinal calcium absorption and serum 1,25(OH)2D3 of similar magnitude in elderly and young women after a 4-day low-calcium diet. TM To further complicate this matter, the same group did not find the expected correlation between age and true fractional calcium absorption in their patients, although the adaptation to low dietary calcium content was confirmed. 227'231 These most recent data appear to dampen the pathophysiological relevance of a decline of intestinal calcium absorption in elderly people. b. Menopause A decreased intestinal calcium absorption in postmenopausal women with fractures has been recognized since the early 1 9 6 0 S , 223'232'233 and a reduced ability of estrogen-deficient women to absorb calcium is considered one of the pathophysiological mechanisms of postmenopausal (type I) osteoporosis. 234 The incidence of calcium malabsorption in osteoporotic women can be as high as 60% to 70%, and fecal calcium exceeds calcium intake in a third of these women. Several lines of evidence support the notion that the reduced calcium absorption consequent to estrogen depletion is independent of aging. First, ovariectomy is followed by a rapid decline in net intestinal calcium absorption. 38'235'236 Second, in the transition between the preand postmenopausal period, fractional calcium absorption declines an additional 2%, independent of age. 81 Finally, among postmenopausal women, those who are osteoporotic have lower calcium absorption compared to " n o r m a l " peers. 38 Despite this rather convincing evidence, the pathogenesis of the menopause-related

decline of calcium absorption efficiency remains very controversial. Although estrogen deficiency acts independent of aging, similar pathophysiological mechanisms have been invoked to explain these phenomena. As in the case of aging, reduced serum levels 1,25(OH)zD3 have been reported in postmenopausal osteoporotic patients by some investigators, 47'224'237'23s but not by o t h e r s . 239-241 The increased bone resorption produced by estrogen deficiency, and the attendant reduction of parathyroid activity, could in theory explain a diminished 1,25(OH)zD3 synthesis, and in turn the decreased calcium absorption and negative calcium balance. Nevertheless, a number of women with calcium malabsorption have normal plasma 1,25(OH)zD3 levels, suggesting that these individuals may lose responsiveness to the action of 1,25(OH)2D3. In support of this hypothesis is the observation that stimulation of calcium absorption by 1,25(OH)2D3 is blunted in ovariectomized women, compared to age-matched controls, and that estrogen supplementation preserves the intestinal response to the vitamin D metabolite 46 (Fig. 6 - 7 ) . On the other hand, an earlier study demonstrated that it is possible to correct the calcium malabsorption in elderly postmenopausal women with 1,25(OH)2D3 therapy, provided that the treatment is continued long enough, 229 perhaps allowing for up-regulation of VDR. 152 Although the clinical evidence of a menopause-induced intestinal resistance cannot be considered conclusive, this hypothesis has been borne out of in vitro studies demonstrating a reduced number of intestinal vitamin D receptors after ovariectomy, 45'242 in addition to a decreased production of calbindin-D, and a reduced capacity of the basolateral membranes to actively accumulate c a l c i u m . 243'244 As in the case of aging, the hypotheses of an end-organ resistance and a deficient production of 1,25(OH)2D3 may coexist in postmenopausal women. The recent description of an association between osteoporosis and a certain VDR haplotype 9~in theory would provide a pathogenetic basis for a decreased endogenous responsiveness to vitamin D in postmenopausal women with osteoporosis. If different VDR alleles produce receptors with different functional activities, then a heterogeneity in calcium absorption efficiency could be expected in women, according to their VDR haplotype. A recent study seems to partially support this hypothesis, since fractional calcium absorption was found to be lower in women with a VDR genotype associated with low bone density. 245 However, such a difference was evident only at very low calcium intakes, 245 and the association between VDR allele distribution and osteoporosis is far from being universally accepted. c. Corticosteroid Excess Glucocorticoids are among the most commonly prescribed drugs, and unfortunately

CHAPTER 6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption

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they also have a profound impact on calcium homeostasis and bone mass. Either exogenously administered (i.e., dexamethasone, prednisone) or endogenously secreted by pituitary or adrenal tumors (cortisone), glucocorticoids reduce intestinal calcium absorption 246-249 and renal calcium conservation. 25~ The ensuing negative calcium balance, coupled with the steroids' direct actions on bone turnover, leads to marked reduction in bone mineralization with consequent osteoporosis. 251'252 Glucocorticoids are known to inhibit the production of active vitamin D metabolites, 253 but this may not be the sole nor the major mechanism by which they alter intestinal calcium absorption, for the negative effect on intestinal calcium transport seems unrelated to 25(OH)D and 1,25(OH)2D3 levels. T M Glucocorticoids may directly affect intestinal cells, since they markedly alter villus height and crypt depth of the intestinal mucosa. 255 Nonetheless, vitamin D metabolites can correct the corticosteroid-induced calcium malabsorption, as patients with Cushing's syndrome with severe calcium malabsorption respond to short-term treatment with 25(OH)D with a significant improvement of calcium metabolism. In fact, additional vitamin D, or one of its metabolites, is usually prescribed to patients under chronic steroid therapy. In vitro, dexamethasone decreases the steady-state mRNA and protein levels of VDR in osteosarcoma cells, 256 an effect that would lead to reduced cellular response to vitamin D. Although glucocorticoids alter phosphate absorption in a similar way (see Section II.D.5), only calcium absorption fully recovers within 1 month after

withdrawal of the drug, whereas only a partial restoration of phosphate absorption occurs. 6~ d. Hypomagnesemia Magnesium deficiency has diverse and critical effects in calcium homeostasis (see Section III.B). Although in normal subjects an acute fall in serum magnesium stimulates PTH secretion, chronic and severe magnesium deficiency as well as hypermagnesemia inhibit PTH secretion. 257 Patients with hypomagnesemia develop peripheral resistance to PTH, in addition to a blunted PTH secretion in response to low calcium. These changes are conducive to decreased intestinal calcium absorption, which is one of the pathophysiological mechanisms of the hypocalcemic syndrome that characterize magnesium deficiency. Unfortunately, these patients are resistant not only to PTH but also to vitamin D, which complicates the treatment of the calcium malabsorption of magnesium deficiency. e. Alcohol Abuse The actions of ethanol on intestinal calcium transport are various and complex. Besides its direct toxic effect on the intestinal epithelium, 258 ethanol can indirectly affect calcium transport through its negative effects on other organs, such as the pancreas and the liver. Malnutrition and malabsorption secondary to alcohol-induced liver and pancreatic disease (see Section I.C.2) contribute to calcium malabsorption in alcoholism. Ethanol also interferes directly with calcium transport in the intestine. Acute exposure of the duodenum to high concentrations of ethanol causes a re-

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ROBERTO CIVITELLI,KONSTANTINOSZIAMBARAS, AND RATTANA LEELAWATTANA

versible decrease in its ability to transport c a l c i u m , 258'259 whereas it enhances plasma-to-lumen calcium flux in the ileum) 59 The combined actions result in net calcium secretion and increased fecal excretion. Histologically, there is necrosis of the villus epithelium and inflammation of the remaining crypts with infiltration of lymphocytes and plasma cells, leading to decreased absorptive surfaces. 26~ Despite the absence of clear histological changes of the enteric epithelium, chronic alcohol ingestion does cause a defect in intestinal absorption, independent of pancreatic or liver disease. 258 Lower calcium accumulation rates have been observed in isolated duodenal brush border membrane vesicles in chronic ethanol u s e r s , 262 and a change in lipid composition of enterocyte plasma membrane may alter calcium transport by changing the cell membrane fluidity. 262

f Excessive Caffeine Intake Caffeine is consumed daily by a large majority of Western populations in the form of beverages, foods, and medications. There is an inverse correlation between caffeine intake and calcium balance, 263'264 such that for every 6-fl-oz serving of caffeine-containing coffee, calcium balance is decreased by 4.3 mg/day. 265 Part of this effect is related to decreased intestinal calcium absorption, 266 in addition to increased intestinal calcium secretion. 265 Caffeine inhibition of renal l oL-hydroxylase activity 267 and intestinal calcium transport is similar to the effect of other methylxanthines, and seems to parallel their inhibition of phosphodiesterase activity. 264 Nonetheless, most of the detrimental effects of caffeine are the consequence of altered renal calcium handling. Caffeine, acting as mild diuretic, transiently increases the urine output of sodium, 268 which drives the calcium loss. However, these changes are in most part compensated by homeostatic responses, and an adequate calcium intake protects against any harmful effects that caffeine may have on calcium metabolism and bone density. In fact, no permanent deleterious effects of caffeine occur when daily calcium intake is at least 600 to 800 mg. 266'269'270 Caffeine may also indirectly affect calcium bioavailability, because individuals who drink coffee tend to avoid consuming milk and dairy products. 265 4. EXTRINSIC FACTORS THAT INHIBIT CALCIUM ABSORPTION

a. Drugs Several drugs can interfere with calcium bioavailability via different mechanisms. As discussed earlier in this chapter, the most commonly used antiepileptic medications, barbiturates and phenytoin, negatively affect calcium homeostasis through different mechanisms, including a direct inhibition of calcium absorption (see Section I.C.2). Neomycin sulfate when ad-

ministered orally increases fecal fat content and fecal calcium excretion. This results in a decreased calcium absorption and urinary excretion. 271 Colchicine produces a dose-dependent inhibition of calcium transport with no effect on water transport. At the concentration of 0.5 mM, colchicine causes a significant decrease of calcium uptake and accumulation by duodenal cells, leading to inhibition of the unidirectional influx of calcium across the small intestine. Cytotoxic chemotherapeutic agents can induce profound malabsorption of calcium by damaging the intestinal epithelium, which is extremely sensitive to these agents owing to the rapid turnover of the intestinal mucosa epithelium. Occasionally, tetany may occur with cis-platinum, although hypocalcemia in this case is caused primarily by the renal tubular toxicity and hypomagnesemia produced by this potent drug. Calcium channel-blocking agents are widely used in the treatment of hypertension and myocardial ischemia. In theory, blockage of calcium may interfere with transcellular calcium transport. However, at therapeutic doses these drugs do not inhibit intestinal calcium transport, 272 and they may even enhance absorption through increased production of 1,25(OH)2D3. 273

b. Dietary Phosphate Phosphate present in the diet is commonly believed to interfere with calcium absorption by forming poorly soluble calcium phosphate salts. 274 However, this "common wisdom" assumption has been challenged by some cross-sectional human studies that have related high-phosphorus, low-calcium diet to positive rather than negative effects on bone mineral content and density. 275'276 Likewise, increasing dietary phosphorus up to 2000 to 2700 mg/day results in no changes in intestinal absorption of calcium. Although intestinal calcium secretion does increase with highphosphate diets, this is balanced by a significant decrease in urinary calcium lOSS.265 Therefore, calcium balance remains essentially unchanged within a wide range of dietary phosphate content. 264 However, this premise may not hold in all circumstances. For example, a negative correlation between phosphorus intake and calcium absorption has been reported in teenage girls, indicating that at a time of maximal calcium absorption efficiency, dietary phosphorus may produce substantial effects. 277 Furthermore, phosphate contained in carbonated soft drinks can decrease ionized serum calcium with modest increase of PTH, presumably because the phosphoric and citric acid contained in the carbonated soft drinks may acidify the urine, thereby increasing the secretion of urinary c a l c i u m . 278'279 Finally, some undigestible organic phosphates, such as phytates, present in wheat bran, unpolished rice, and other cereals (see below), can indeed form insoluble calcium salts, thus preventing intestinal calcium absorption.

CHAPTER6 Pathophysiologyof Calcium, Phosphate, and Magnesium Absorption

c. Dietary Fibers In recent years, the level of fiber in the diet of many Americans has increased, mostly because of their putative therapeutic role in the control of serum lipids or in glucose absorption in diabetes. The decreased intestinal calcium absorption and the ensuing negative calcium balance produced by diets high in fiber represent a potential drawback for this approach. T M This consideration is particularly important in elderly people as they are often encouraged to increase their dietary fiber intake to enhance bowel motility, at a time when their calcium intake may be inadequate. There are various mechanisms by which some components of dietary fibers alter calcium bioavailability. Calcium may bind directly to uronic acid components of fibers, 282 or to phytic acid, the storage form of phosphorus present in the outer husks of cereal grains, 282'283 thus forming insoluble calcium salts which reduce the availability of the mineral for absorption by the small intestine. Phytate is the major factor that hampers the absorption of calcium from whole-wheat products and other cereals. Conversely, because of their lower (almost 1/20) content of phytate, wheat flour-based products, such as wholewheat bread, provide more bioavailable calcium than wheat bran-based products. T M Presumably, leavening reduces the phytic acid content during bread-making, thus improving the bioavailability of calcium in leavened bread. On the contrary, high-phytate soybean based meals result in --~25% lower calcium absorption compared to low-phytate soybean meal. 285 The presence of phytase, an enzyme that cleaves phytate to soluble phosphate, is also a factor conditioning mineral absorption, by making more phosphate as well as calcium available for absorption in wheat and rye. 286 The bioavailability of spinach calcium is also less than optimal, presumably because of the relatively high content of oxalic acid in these vegetables. 91'287 Although earlier calcium balance studies in humans yielded conflicting results, 288 recent data conclusively indicate that spinach calcium is poorly absorbed compared to dairy calcium, probably because of the formation of insoluble complexes with oxalic acid. 91'289 The presence of oxalic acid also greatly limits the bioavailability of calcium in other vegetables, such as beet greens, rhubarb, peanuts, and green beans. Other fibers, such as psyllium-based charged fibers, a popular remedy for constipation, has little effect on the bioavailability of co-ingested calcium when used at typical therapeutic doses. 29~ Likewise, alginate, a food additive used as a thickening agent in ice creams, jams, and baking products, does not seem to interfere with calcium, magnesium, phosphorus, or zinc absorption, although it may inhibit iron absorption. TM When the diet is high in fiber, the colon becomes an important site for calcium absorption because colonic bacterial enzymes can break down complex fibers and

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release calcium for a b s o r p t i o n . 282'292'293 I n addition, calcium-fiber binding is pH dependent, with increased binding at a higher pH. T M B e c a u s e fibers can partially buffer gastric acidity, they help create an environment conducive to the formation of calcium-fiber complexes, and therefore, less suitable for calcium absorption. For instance, calcium-phytate complexes begin to precipitate at pH 4 to 5, whereas at pH 6, 70% of calcium is bound to phytate, and at neutral pH only less than 10% of calcium is in a soluble state suitable for absorption by the small intestine. T M

II. PHOSPHATE Phosphorus is not only a major constituent of the skeleton as a component of apatite crystals but is also a mediator of energy transfer and participates in a wide variety of intracellular metabolic processes. In body fluids, phosphorus exists in both organic form, consisting essentially of phospholipids and energy storage molecules (i.e., adenosine triphosphate [ATP]), and inorganic phosphate salts. A sensibly large amount of phosphorus is present in the erythrocytes as 2,3-diphosphoglyceric acid, a critical regulator of oxygen binding to hemoglobin. The concentration of total phosphorus in the circulation is --~14 mg/dl, of which --~8 mg/dl is in the organic form, and ---4 mg/dl in the inorganic form. Of the latter, --~15% is protein bound; --~85% is present either as the undissociated ions, HPO 2-, and H2PO4; or complexed with Ca 2+, Na 2+, and Mg 2+. It is the inorganic orthophosphate moiety (PO43-) that is measured in clinical medicine. Inorganic phosphate is also the form that is absorbed by the intestine. It is important to recognize that although the terms "phosphate concentration" and "phosphorus concentration" are used interchangeably, it is the phosphate ions that are regulated by the homeostatic mechanisms that maintain phosphate concentration in the plasma. The term "phosphorus concentration" should then be used to indicate the content of the element in body fluids, tissues, or foodstuffs.

A. P h y s i o l o g y o f P h o s p h a t e A b s o r p t i o n 1. PHOSPHATE SOURCES AND INTAKE In adults, a dietary intake of approximately 800 mg of phosphate per day is required to maintain phosphate balance. During pregnancy and lactation this requirement is increased by 50%. Fortunately, phosphate is relatively abundant in a wide variety of foods and it is more efficiently absorbed than calcium, so that nutritional phosphate deficiency is infrequent. Approximately four

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fifths of the dietary phosphorus is contained in milk products, grains, and meat, and there is also a significant amount in food additives. Dairy products constitute a major source of dietary phosphorus, with 1 cup of skim milk containing 247 mg and 1 oz of processed American cheese containing 210 mg. Phosphorus-containing food additives are used primarily in baked goods, cheeses, and other dietary products, and phosphoric acid is also contained in colas and carbonated beverages. In fact, food additives may contribute as much as 30% of the phosphorus of an average adult diet. 297 Dietary supplements of phosphorus are also readily available as mixed sodium and potassium mono- and dibasic salts. The laxative Fleet Phospho-Soda contains 129 mg/ml, so that 1 tsp three times daily can provide over 1900 mg of phosphorus per day. Capsules and tablets are also available containing 250 or 500 mg of phosphorus as the sodium or potassium salt. Consumption of phosphate in excess of 1800 mg will often be complicated by diarrhea.

3. MECHANISMS OF PHOSPHATE ABSORPTION

Phosphate, like calcium, can diffuse through intestinal mucosa by two mechanisms, active transcellular, sodium-dependent transport, and passive paracellular sodium-independent diffusion. 3~ Because of the abundance of phosphate in standard diets, active absorption becomes physiologically relevant only when the intraluminal phosphate concentration is low (<500 mg/day). Phosphate ions are transported through the enterocyte brush border plasma membrane via a Na+/Pi transporter protein, driven by the Na § electrochemical gradient. This process requires energy and is partly vitamin D dependent. 8'3~ In ruminants, phosphate is exchanged with protons, instead of sodium, through a different transport system. 3~ Active intestinal phosphate transport can be stimulated by vitamin D metabolites, although the overall impact of hormonal regulators on phosphate absorption is much lower than it is for calcium. Passive absorption is the major route of phosphate input. This mechanism depends on the electrochemical gradient between the luminal and vascular sides of the intestinal 2. PHOSPHATE BALANCE mucosa. Because of the negative intracellular electric potential, the intestinal epithelium constitutes a powerful The dietary content of phosphate is rarely below 600 barrier to the transepithelial transport of anions. Accordmg/day, and the usual phosphate intake is usually beingly, phosphate is better passively absorbed in the form tween 700 and 2000 mg/day. Within this range, 60% to of H2PO4 than HPO 2-, and an acidic environment pro80% of dietary phosphate is absorbed. Thus, in most motes H2PO4 formation. 3~ Thus, the duodenum is the circumstances the active absorption mechanisms are major site of passive phosphate absorption because of its fully saturated, and the amount of phosphate absorbed is relatively acid milieu, whereas the jejunum is the privirtually a direct function of dietary intake. Only at very mary site of active phosphate absorption. The less neglow intakes (<300 mg/day) do net phosphate losses ocatively charged H2PO4 ions that can accumulate in more cur, the consequence of active secretion through intesproximal segments of the intestine are more readily tinal juices and cell death. Experimental phosphate deptransported through paracellular pathways when the lurivation using low-phosphate-containing synthetic diets minal phosphate concentration is high, whereas the more rapidly leads to hypomagnesemia, hypermagnesuria, and charged HPO 2species is absorbed primarily via active hypercalciuria in addition to hypophosphatemia. 298-3~176 transport. 3~ Despite the high efficiency of absorption of These changes are more frequent in females than in the duodenum, most of the dietary phosphate is absorbed males, and they occur early after phosphate deprivation. in the jejunum because of the longer jejunal transit time, For short periods of dietary phosphate restriction, fasting which allows a higher net absorption than in the duoserum phosphate levels do not appreciably change, but denum. The ileum does not absorb phosphate as effimean 24-hour serum phosphate decreases and the paciently as the proximal intestine, and the colon absorbs tients develop negative phosphate balance through ina negligible amount of phosphate. However, significant testinal lOSS.3~ The homeostatic response includes amounts of phosphate can be absorbed by the colon with increased 1,25(OH)2D3 production and consequent inphosphate-containing enema solutions. 3~ This condition creased urinary calcium excretion, while serum calcium can be the cause of fatal hyperphosphatemia in pediatric is maintained at normal levels. 3~176 The hypercalciuria is patients. 3~ reversible upon correction of phosphate depletion. Longer periods of phosphate deprivation (2 weeks of 4. MEASUREMENT OF PHOSPHATE ABSORPTION dietary phosphorus intake of about 600 mg/70 kg/day) lead to clinically apparent hypophosphatemia assoA number of approaches to assess intestinal phosphate ciated with hypomagnesemia, hypercalciuria, and inabsorption have been pursued. The most reliable has creased urinary magnesium. The low serum magnesium been the classic balance technique, which measures the and the higher 1,25(OH)2D3 levels decrease PTH net retention or loss of dietary phosphate over a period secretion. of 1 to 2 weeks (see Section I.A.6). Not only is the

CHAPTER6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption technique tedious and impractical, it also does not measure intestinal phosphate transport specifically. A more simplified approach is the measurement of the rise in serum phosphate after an oral load of phosphate. 3~ Although admittedly a crude estimation of the phosphate absorptive capacity of the intestine, this simple method is less affected by the rate of phosphate utilization for cell metabolism or bone deposition, or by intestinal secretion of phosphate, than balance studies. Unfortunately, the high concentration of phosphate essential for studies of phosphate absorption limits the ability to assess active intestine transport of phosphate against a more physiological concentration gradient. Isotopic techniques based on measurement of radioactivity after oral administration of 32p offer a reliable estimate of fractional phosphate absorption. The theoretical bases are the same as for single calcium isotope methods, and the same limitations apply (see Section I.A.6), including exposure to radioactivity from 32p. In addition, because of the large amounts of filtered phosphate and its sensitivity to glomerular filtration rate, blood levels of 32p must be corrected for renal clearance of 32p.194,247 Measurement of calcium and phosphate absorption following oral administration of 45Ca and 32p has been successfully applied to healthy individuals 247 and patients with renal failure. TM A significant reduction of the absorbed fraction of both elements was observed in hemodialysis patients, 3~ confirming the potential applicability of these methods to clinical settings. Peroral jejunal biopsy after ingestion of 32p has been proposed as an alternative approach to measure directly the intestinal transport of phosphate. The rate of 32p accumulation in the mucosal tissue is determined as an index of absorption. 3~ Obviously, this technique measures the tissue accumulation of the isotope, rather than the actual transport of phosphate across the mucosa. Thus, the parameter obtained is an indirect estimation of phosphate absorption. A problem common to all techniques is constituted by the rapid organification of the phosphate upon entering the cytoplasm. This limits the ability to ascertain the effective concentration gradient of phosphate across the brush border. 5. REGULATION OF PHOSPHATE ABSORPTION

Phosphate absorption is altered by the concomitant presence of other dietary factors, which interfere with passive absorption. Certain hormones can regulate active phosphate transport, although this aspect of phosphate homeostasis is of little physiological relevance when dietary intake is normal. Obviously, to allow efficient phosphate absorption the intestinal mucosa should be intact and the transit time normal. Mucosal integrity and intestinal motility are important factors considering that phosphate is absorbed primarily via passive diffusion.

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On the other hand, gastric acid secretion appears to be a less important factor than it is for calcium absorption. Although a low pH is required for the solubilization of calcium-phosphate salts, treatment with inhibitors of gastric acid secretion does not prevent the postprandial increase of serum phosphate, thus suggesting that intestinal absorption of this anion is not significantly dependent on gastric pH. 31~ The active metabolite of vitamin D, 1,25(OH)2D3 is the major regulator of active phosphate absorption, although the precise mechanisms of this action are not yet completely understood. In vitro, 1,25(OH)2D3 stimulates the synthesis of the Na+/Pi transporter, 3~ and promotes phosphate transport at both brush border and basolateral membranes. 3~2 However, the manner in which phosphate ions flow from one pole to the other of the membrane is not clear, and a specific phosphate cartier protein, analogous to CaBP, has not been identified. Thus, the hypothesis that the stimulatory action of vitamin D on phosphate absorption could be mediated, at least in part, by nongenomic effects on plasma membrane fluidity, as described in proximal tubule brush border membranes, 313 cannot be entirely ruled out. Parathyroid hormone, the most important regulator of mineral homeostasis, promotes urinary phosphate excretion by inhibiting renal tubular reabsorption. Its effect on intestinal absorption of phosphate is the result of two opposing actions. While PTH can indirectly increase absorption via stimulation of 1,25(OH)2D3 synthesis, in vitro studies suggest that PTH can also directly affect the enterocyte, resulting in inhibition of intestinal phosphate transport. 314'315 However, the latter finding remains controversial. 316 Calcitonin shares some of the effects of PTH on phosphate homeostasis. Calcitonin promotes renal phosphate excretion by inhibiting renal tubular reabsorption, but the evidence for an action on intestinal absorption remains controversial. Although some studies would suggest that intestinal absorption of phosphate is decreased by calcitonin in the jejunum, the site of active transport, passive phosphate transport is unaffected. 317 Furthermore, direct effects of calcitonin on enterocyte phosphate transport and cAMP accumulation have never been demonstrated in v i t r o . 315'316 While estrogen has a physiological role in the control of calcium absorption, its action on phosphate absorption is minor at best. Not only are data in humans unavailable, but administration of 17[3-estradiol to young fertile female rats seems to increase phosphate absorption only marginally. 44 A role of intestinal alkaline phosphates on phosphate absorption had been advocated on the basis of the hydrolytic activity of this ectoenzyme, which theoretically would make inorganic phosphate ions available 66 70 at sites of absorption.' Neutralizing antibodies and alkaline phosphates inhibitors, such as theophylline and

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Z n 2+, can inhibit brush border membrane phosphate uptake, 66 and L-phenylalanine and beryllium, which also in-

hibit alkaline phosphate activity, decrease intestinal phosphate absorption in mice. 7~ Nonetheless, there is no evidence that any of these enzyme inhibitors can produce clinically relevant phosphate hypoabsorption syndromes.

B. Clinical M a n i f e s t a t i o n s of Phosphate Disorders The clinical manifestations of hypophosphatemia can be attributed to two metabolic consequences of cellular phosphate depletion: (1) the reduction of ATP synthesis and thus the cell's source of energy; and (2) a shift in the oxyhemoglobin dissociation curve of the red blood cell. 318 Because anaerobic glycolysis is directly dependent on serum phosphate concentration, the production of ATP is impaired when serum phosphate levels fall. As a consequence of the reduced cellular ATE the integrity and deformability of the erythrocyte membrane is reduced, resulting in severe hemolytic anemia in patients with phosphate levels below 0.3 mg/dl. 319 Neurological and muscular dysfunction associated with hypophosphatemia may also be attributed to the depletion of intracellular ATE Symptoms of weakness, malaise, joint stiffness, and intention tremors occur in patients with serum phosphorus levels of 2 mg/dl. 32~ More profound hypophosphatemia results in heart failure, rhabdomyolysis, hemolysis, platelet dysfunction, paralysis, seizures, coma, and death. 321'322 Low serum phosphate is particularly dangerous for patients with chronic obstructive pulmonary disease. In these patients, diaphragmatic muscle contracts poorly, leading to hypoventilation and further contributing to the impaired gas exchange. This life-threatening outcome is reported in an anorexia nervosa patient, in whom severe hypophosphatemia may result in bulbar palsy. 323 A n additional factor contributing to these neuromuscular symptoms may be tissue hypoxia resulting from the impaired delivery of oxygen. 3~8 Red cell phosphate depletion causes a decrease in the glycolyric intermediate 2,3-diphosphoglycerate, leading to a leftward shift of the oxyhemoglobin dissociation curve and consequent decreased delivery of oxygen to tissues. Lack of phosphate has serious consequences for the structural integrity of the skeleton. Phosphate deficiency is one of the leading causes of calcification defects, which result in clinical syndromes of tickets in children and osteomalacia in adults. In hypophosphatemic tickets or osteomalacia, the mineralization defect and bone mineral loss appear to be independent of vitamin D and parathyroid hormone, underlining the critical role of phosphate itself in the production of a normal, mature bone tissue. However, the role of a defective intestinal

phosphate absorption in the pathogenesis of phosphate depletion syndromes is probably minor, as compared to the heavy phosphate losses that can occur via the kidney. Nonetheless, it is difficult to establish the real contribution of phosphate malabsorption in pathological conditions because of the difficulties inherent in the assessment of intestinal phosphate absorption (see Section II.A.4). Hyperphosphatemia is very rare when renal function is normal. Modestly elevated serum phosphate levels can occasionally occur in severe vitamin D intoxication, especially when large amounts of phosphate are ingested. Although hyperphosphatemia can cause acute renal failure in subjects with normal renal function, such an event is very infrequent. On the contrary, hyperphosphatemia is very common in end-stage renal failure, where it contributes to further deterioration of renal function and perpetuation of the renal osteodystrophy and secondary hyperparathyroidism. Therefore, it is important to control hyperphosphatemia by reducing intestinal phosphate absorption in these patients.

C. I n c r e a s e d P h o s p h a t e A b s o r p t i v e States Conditions characterized by increased intestinal absorption of phosphate are listed in Table 6 - 4 . 1. VITAMIN D-DEPENDENT PHOSPHATE HYPERABSORPTION a. Vitamin D I n t o x i c a t i o n Although administration of vitamin D metabolites, especially 1,25(OH)2D3 to vitamin D-deficient individuals, stimulates jejunal phosphate absorption, elevations of serum 1,25(OH)zD3 in healthy subjects only slightly increases fractional phos-

TABLE 6--4

Disorders Associated with Increased Phosphate Absorption

Vitamin D-dependent phosphate hyperabsorption Vitamin D intoxication Primary hyperparathyroidism Growth hormone excess Metabolic acidosis Dietary phosphate deficiency Chronic dietary calcium deficiency Vitamin D-independent active phosphate hyperabsorption Hyperthyroidism Acute glucocorticoid administration Chronic dietary phosphate deficiency Increased phosphate bioavailability High dietary lactose High meat content in diet High dietary fat

CHAPTER6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption phate absorption. TM This finding is consistent with the notion that dietary sources are the major component of phosphate absorption in normal individuals. Such premise has been borne out of animal studies indicating that while 1,25(OH)2D3 repletion in young vitamin D deficient rats does restore a normal jejunal phosphate absorption, such an effect is not observed in n o n vitamin D-deficient animals. 8 In fact, 1,25(OH)2D3 administered intraperitoneally does not increase phosphate absorption in normal adult rats. TM Nonetheless, an increased intestinal absorption of phosphate may certainly contribute to the hyperphosphatemia that seldom develops with overdosage of 1,25(OH)ED3, or in vitamin D intoxication. On the other hand, therapeutic doses of 1,25(OH)2D3 enhance phosphate absorption in patients with hypoparathyroidism and X-linked hypophosphatemic rickets, resulting in improved phosphate balance. High serum phosphate levels are not usually seen in these conditions even after 1,25(OH)2D3 therapy at high doses, for hypercalciuria and/or hypercalcemia may develop sooner. b. Primary Hyperparathyroidism In patients with primary hyperparathyroidism, surgical removal of parathyroid adenomas is followed by a decrease in intestinal fractional phosphate absorption. 247 Although this observation implies that phosphate absorption is increased in primary hyperparathyroidism, the phosphaturic action of PTH overrides the higher phosphate bioavailability in these patients, whose serum phosphate is lower than normal. c. Growth Hormone Excess In acromegaly or after administration of growth hormone, intestinal absorption and renal tubular reabsorption of phosphate are increased. 247 As discussed under Section I, growth hormone can stimulate renal 1oL-hydroxylase either directly, or via IGF-I. 135 Although other mechanisms may be involved, increased production of 1,25(OH)zD3 appears to mediate the stimulatory action of growth hormone on calcium and phosphate absorption. d. Metabolic Acidosis Metabolic acidosis enhances the action of 1,25(OH)zD3 on intestinal phosphate absorption. Adult vitamin D-replete rats exhibit an enhanced phosphate absorption in response to 1,25(OH)zD3 if they are rendered acidotic by ingestion of NH4C1, relative to control nonacidotic rats at comparable levels of circulating 1,25(OH)zD3. 325 Although hyperphosphatemia occurs in acute metabolic acidosis, there is no evidence of increased intestinal phosphate absorption in diabetic ketoacidosis. In this condition, phosphate balance is negative owing to urinary phosphate loss, resulting in hypophosphatemia after correction of acidosis.

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2. VITAMIN D-INDEPENDENT ACTIVE PHOSPHATE HYPERABSORPTION

a. Triiodothyronine Triiodothyronine (T3) synergizes with the action of 1,25(OH)2D3 on jejunal enterocytes in stimulating phosphate absorption. Enterocytes cultured in medium totally lacking T3 exhibit a defect in phosphate transport, 3~ thus suggesting that T3 may play a physiologically relevant role in phosphate homeostasis. Accordingly, an increased phosphate absorption may contribute to the hyperphosphaturia often seen in patients with hyperthyroidism, in the presence of normal serum phosphate levels. 3~ b. Glucocorticoids Glucocorticoids have complex and contrasting effects on the different pathways of phosphate absorption. They increase passive phosphate absorption but decrease active transport. The acute effect of corticosteroids is to enhance passive phosphate flux, especially in the jejunum. 326 This effect is independent of 1,25(OH)zD3, and it is an indirect consequence of the increased absorption of water and sodium, which glucocorticoids stimulate. The resulting increased phosphate concentration in the lumen creates a higher phosphate chemical gradient between the lumen and the vascular side of the mucosa, which drives the phosphate into the circulation. 32v Such an effect can be seen only when dietary phosphate is high enough to produce an adequate concentration gradient. However, this phenomenon has very little pathophysiological relevance, since a single administration of pharmacological doses of glucocorticoids can actually cause hypophosphatemia from renal phosphate

l o s s . 322

c. Chronic Dietary Phosphate Deprivation This enhances active intestinal absorption via vitamin D dependent a n d - i n d e p e n d e n t mechanisms. Phosphate deficiency stimulates 1,25(OH)zD3 production as a homeostatic adaptation that attempts to correct the phosphate lack by increasing intestinal absorption and reducing urinary loss. However, the efficiency of phosphate absorption improves after phosphate deprivation even in conditions of vitamin D deficiency, implying that vitamin D-independent adaptive mechanisms also exist. TM VDR-binding capacity is not increased in animals after prolonged dietary phosphate deprivation, 328 whereas the Vmax of the Na+/Pi transporter system seems to increase after dietary phosphate deprivation, independently of PTH, 1,25(OH)2D3, or other hormones. TM Adaptation is apparently reduced in older animals, TM although aging does not seem to affect the action of 1,25(OH)2D3 in elderly rats. d. Prolonged Dietary Calcium Restriction This also induces higher fractional phosphate absorption. 329 This

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effect persists in older rats, and it appears to be only partially mediated by increased 1,25(OH)zD3, since the increased fractional absorption of phosphate after chronic calcium and/or phosphate deprivation does not correlate with the age-dependent decline of 1,25(OH)zD3 le ve l s . 247,329,33~

3. INCREASED PHOSPHATE BIOAVAILABILITY Dietary lactose promotes phosphate solubility, thus making more phosphate available for passive absorption. TM When lactose is high in the diet, more lactic acid is produced from lactose by the intestinal lactase, and the luminal pH decreases, a condition permissive for passive phosphate absorption. High meat content in the diet, up to 20% of total energy intake, is associated with increased fractional phosphate absorption, as compared to diets containing 10% or less of meat as total energy intake. 332 The mechanism of this phenomenon is not clear, although the widespread presence of large amounts of phosphorus in meat may provide additional substrate for absorption. Finally, high dietary fat, either animal or vegetable, provides higher phosphate than standard diets, thus leading to higher net phosphate absorption, as demonstrated in lactating cows. 333 The increased absorption may be related to the phospholipid content in fat. Obviously, the increased dietary phosphate brought about by these food products rarely, if ever, constitutes a single cause of significant abnormalities of phosphate homeostasis.

D. D e c r e a s e d P h o s p h a t e A b s o r p t i v e States A list of the major conditions and disorders associated with decreased phosphate absorption is given in Table 6-5. 1. NUTRITIONAL DEFICIENCY

Severe nutritional deficiency can cause negative phosphate balance because of the obligatory phosphate losses. Likewise, malnutrition secondary to any intestinal or nonintestinal diseases, as well as anorexia nervosa, can lead to negative phosphate balance and hypophosphatemia. It is important that parenteral nutrition is mentioned, for inadequate phosphate supplements have been reported to cause hypophosphatemia. 322'334 2. INTESTINAL DISEASES WITH MALABSORPTION

Gastrointestinal disorders that result in steatorrhea such as pancreatic insufficiency and bile salt deficiency, intrinsic bowel disease (Crohn's disease and sprue), and short bowel syndromes (intestinal fistula, ileal bypass) can be associated with decreased intestinal absorption of phosphate and hypophosphatemia. Although the pre-

TABLE 6--5

Disorders Associated with Decreased Phosphate Absorption

Nutritional phosphate deficiency Intestinal diseases with malabsorption Intrinsic bowel diseases Short bowel syndromes Disorders of vitamin D metabolism Nutritional vitamin D deficiency Hepatobiliary disease Chronic renal failure Hypoparathyroidism Familial X-linked hypophosphatemic rickets Oncogenic osteomalacia Vitamin D-dependent rickets (types 1 and 2) Intrinsic intestinal defects of phosphate absorption Aging Magnesium deficiency Chronic glucocorticoid therapy Extrinsic factors inhibiting phosphate absorption Cations: A13§ Mg 2§ Ca 2+ Dietary factors: fibers, phytate

dominant mechanism leading to hypophosphatemia in these disorders is the renal wasting of phosphate caused by the secondary hyperparathyroidism reactive to the malabsorption of calcium and vitamin D, 335 the reduced phosphate absorption caused by the increased intestinal transit and poor food intake contributes to the negative phosphate balance. Malabsorption of vitamin D occurs in patients with steatorrhea, as one of the consequences of the reduced absorption of fat-soluble vitamins, which requires bile salts. Their defective synthesis in patients with severe liver disease and biliary cirrhosis accounts for the osteomalacia seen in these disorders. In patients with primary intestinal diseases, such as Crohn's disease, Whipple's disease, or in subjects with postileal resections or jejunal bypass, a reduced absorptive surface is the predominant mechanism of phosphate malabsorption, along with the vitamin D deficiency. 3. DISORDERS OF VITAMIN D METABOLISM Any factors interfering with vitamin D metabolism result in decreased active phosphate absorption. Thus, dietary vitamin D deficit, hypoparathyroidism, malabsorption syndromes, chronic liver and kidney diseases, and vitamin D-resistant states are associated with phosphate malabsorption secondary to abnormal vitamin D metabolism. While in most of these cases the low phosphate absorption conditions and maintains a low serum phosphate level, in patients with chronic renal failure and in hypoparathyroidism hyperphosphatemia occurs despite the lower phosphate absorption brought about by the failure to convert 25(OH)D3 to 1,25(OH)zD3. This

CHAPTER6 Pathophysiology of Calcium, Phosphate, and Magnesium Absorption pathophysiological paradigm exemplifies the preponderant role of renal handling over intestinal absorption in the control of circulating phosphate levels. Patients with X-linked hypophosphatemic rickets have a defective tubular reabsorption of phosphate with hypophosphatemia. Although the pathogenesis of this inborn disorder has not been completely elucidated, recent data seem to suggest that a phosphaturic factor, also called "phosphatonin," is present in the circulation of these patients. 336 This factor is believed to inhibit both tubule phosphate reabsorption, thus causing phosphate loss, and the synthesis of 1,25(OH)2D3. Patients with Xlinked hypophosphatemic rickets have in fact "inappropriately normal" 1,25(OH)2D3 levels, which may contribute to the hypophosphatemia because of the inefficient response of the vitamin D system to the low phosphate. Consequently, active intestinal phosphate absorption is also impaired in this c o n d i t i o n . 3~176 However, phosphate absorption is widely variable in these subjects, probably because the defect is compounded with other factors that may also affect phosphate absorption efficiency. For example, the phosphate absorption defect, detectable in young patients, has been reported to resolve as the affected individuals reach a d u l t h o o d . 3~ A hypersecretion of phosphatonin or an equivalent phosphaturic factor by tumoral fibrous tissue is most probably the cause of the hypophosphatemia of oncogenic osteomalacia. 336'339-341 Although a defective intestinal phosphate absorption has never been reported, it is very likely that the depressed 1,25(OH)2D3 levels condition a low phosphate absorption, thus contributing to the hypophosphatemia. Finally, the autosomal recessive disorders of vitamin D-dependent rickets, in which the renal 1oL-hydroxylase

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Intestinal absorption of phosphate has not been carefully studied as a function of age in humans. A crosssectional study in which fractional absorption of calcium and phosphate were measured using isotopic methods demonstrated that both calcium and phosphate absorption efficiency decline with advancing age in healthy individuals 247 (Fig. 6-8). As in the case of calcium absorption, the few data available are not consistent with a single, clear mechanism by which the malabsorption may occur. Although a reduced intestinal response to vitamin D may develop with aging (see Section I.C.3), phosphate absorption appears to decline in parallel with serum 1,25(OH)2D3 in elderly people. 343 Obviously, the clinical relevance of a decreased phosphate absorption efficiency in aging is minor as compared to the potential consequences of calcium malabsorption. An interesting but complex disorder of phosphate absorption occurs in magnesium deficiency (see Section III). Severe magnesium deficiency in humans is associated with hypocalcemia consequent to impaired parathyroid hormone secretion and peripheral resistance to the hormone. This disorder is further complicated by an ap-

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is either defective (type I) or the vitamin D receptor is absent, resulting in a functional resistance to 1,25(OH)2D3 (type II), provide proof of the importance of vitamin D control of phosphate absorption. 342 By the first year of life these children develop hypophosphatemia and tickets resistant to vitamin D replacement but correct completely by treatment with 1,25(OH)2D3 with restoration of normal growth and return of biochemical abnormalities to normal.

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parent resistance to vitamin D. Despite the functional hypoparathyroidism, serum phosphate is often below normal. The hypophosphatemia can be attributed in part to an impairment in the intestinal absorption, associated with a reduced renal tubular reabsorption of phosphate. Magnesium supplementation corrects the defect of phosphate balance. It is postulated that magnesium deficiency commonly associated with excessive ethanol ingestion may be one of the principal factors contributing to the hypophosphatemia of chronic alcoholism. 344 Finally, it is worth mentioning that hypophosphatemia develops in the spontaneous hypertensive rat (SHE) strain. The onset of hypertension is usually in adulthood, when hypophosphatemia also develops. In vitro studies demonstrated an impaired sodium-dependent phosphate transport in jejunal epithelial cells of adult SHE, a defect that can be corrected by 1,25(OH)zD3 administration only in vitamin D-deficiency conditions. 345 This animal strain may thus prove to be a useful experimental tool to analyze some aspects of phosphate transport in the intestine. 5. EXTRINSIC FACTORS INHIBITING PHOSPHATE ABSORPTION

A number of factors contained in common food products, dietary supplements, or over-the-counter medications influence the intestinal absorption of phosphate. Aluminum and magnesium ions, the principal elements of widely used antacids, form insoluble salts with phosphate and are not absorbed. 346-348 Because of this action, these agents have been successfully used as phosphate binders in chronic renal failure patients. 347'348In animals, relatively low doses of aluminum hydroxide administered either with drinking water (2000 ppm), or added to the food (80 mg/kg/day), can significantly decrease duodenal p h o s p h a t e . 346'349 Accordingly, inappropriate and continued use of aluminum-containing phosphatebinding antacids results in chronic hypophosphatemia, hypercalcemia, increased propensity toward renal calculi formation, and osteomalacia. 32~In addition, the excess of antacids can complex the endogenously secreted phosphate, thereby depleting the body of inorganic phosphate. When present at sufficiently high concentrations in the intestinal lumen, zinc can also form insoluble phosphate salts, thus impairing phosphate absorption. Much less tenable is the hypothesis that the inhibitory effect of zinc is mediated by inhibition of alkaline phosphatase activity, considering the poor evidence of a physiological role of this enzyme in regulating mineral absorption. Phosphate in the form of inositol hexaphosphoric acid or phytate, the major component of organic phosphate in cereals, forms insoluble salts with calcium and is, therefore, not available for absorption. Although phytate is not absorbable, the presence of phytase, an enzyme

that cleaves phytate in certain foodstuff such as wheat and rye, increases the phosphate available for absorption. 286 On the contrary, other phytate-containing vegetables (i.e., corn) have little phytase and provide less bioavailable phosphate. Food processing can also change the bioavailability of phosphate in dietary products. For example, the process of extrusion, to which some cereals are subjected, destroys phytase and results in more residual phytate than the original vegetable. 35~ Thus, there are several mechanisms that condition different phosphate bioavailability for different types of starch present in aliments. When dietary phosphate is low and active transport plays a more important role, the inhibitory effect of glucocorticoids on active transport results in lower intestinal phosphate absorption. A single high dose of methylprednisolone (100 mg intraperitoneal) causes a significant decline of sodium-dependent phosphate transport in animals. 89 This effect is dependent on both the dose and the type of steroid. For example, betamethasone has been shown to be more potent than prednisone in inhibiting phosphate absorption when used at equip o t e n t doses. 247 Pharmacological doses of glucocorticoids inhibit intestinal phosphate absorption, independently of 1,25(OH)2D3 levels. 247'352 In fact, serum 1,25(OH)2D3 may be elevated owing to the slightly low serum calcium typically observed in steroid-treated patients. Glucocorticoids exert varied actions on the intestinal epithelium. In rapidly growing rabbits, these agents can decrease the synthesis of the Na+/Pi transporter protein and retard the increased rate of enterocyte turnover that is associated with neonatal development by interfeting with enterocyte migration from the bottom to the top of the villi. 352 Finally, chronic high-dose glucocorticoid therapy also decreases intestinal villi height, resulting in shallowing of the crypt, and consequent decease of the absorptive surface. 247 Thus, the mechanisms by which glucocorticoids affect phosphate absorption are substantially different than the mechanism that leads to the reduced calcium absorption. In spite of an equal impairment of calcium and phosphate absorption, the absorptive capacity for calcium rapidly recovers after discontinuation of steroid treatment, whereas recovery from phosphate malabsorption is slower. 247

III. M A G N E S I U M Magnesium is the most abundant intracellular cation. It is an essential element involved in many cellular functions, including production and utilization of energy, regulation of enzymatic activities, and control of intracellular and extracellular electrolyte concentration. A 70-kg adult contains 25 to 28 g of magnesium, of which 60%

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to 65% is present in bone, about 30% in soft tissues (mostly muscle), and only less than 2% in the extracellular space, with serum accounting for 0.3% of total body magnesium.

sium absorbed. Magnesium from dietary sources is as bioavailable as soluble magnesium acetate and is better absorbed than commercially available enteric-coated magnesium chloride. 356 2. MECHANISMS OF MAGNESIUM ABSORPTION

A. P h y s i o l o g y of M a g n e s i u m A b s o r p t i o n 1. MAGNESIUM BALANCE AND INTAKE

Magnesium is a basic constituent of cells, both animal and vegetable, in which the element is present as a porphyrin complex in chlorophyll. Therefore, green leaf vegetables are a good source of magnesium, as is any diet containing food of cellular origin. Unlike calcium, magnesium absorption is directly proportional to dietary magnesium intake, so that diets with sufficient caloric intake ensure adequate intestinal absorption of magnesium. On an average diet of approximately 200 to 700 mg of magnesium, about 40% to 50% is a b s o r b e d . 353'354 Because vitamin D and other regulators affect only minimally intestinal magnesium absorption, individual variations are related to both the amount of dietary magnesium and the presence of other dietary food components that interfere with the absorption of the element (see below). Although severe dietary deficiency of magnesium is rare, one must consider that intestinal magnesium loss occurs at intakes lower than 30 to 50 mg/day, as a consequence of the magnesium lost via secretion in intestinal juices and with intestinal cell death. The established RDA for magnesium in healthy adults is 280 to 350 mg/day, or 5 mg/kg/day. Although this intake is marginally adequate for maintaining magnesium balance, most diets in developed countries contain more magnesium than the RDA would require. However, not only is the recommended magnesium requirement for lactating and pregnant women higher than the current RDA, but this limit may not be adequate in certain conditions, such as diabetes mellitus, or after chronic use of loop diuretics, or extensive bums, in which magnesium losses may require far higher intakes. Because of the high intracellular content of magnesium, shifts to the extracellular milieu may occur before the extracellular concentration of magnesium declines in magnesium-deficient states. Therefore, the circulating levels of magnesium do not accurately reflect magnesium deficiency. Nonetheless, total serum magnesium is still most commonly used to assess magnesium state. A more appropriate assessment of magnesium status can be provided by urinary magnesium excretion after magnesium loading. 355 Since most of the magnesium is passively absorbed, treatment of hypomagnesemia in general consists of increasing the dietary intake of the element, in order to increase the net amount of magne-

Dietary magnesium is absorbed by three mechanisms: active absorption, passive peffusion, and solvent drag. 357'358 Active absorption is a saturable, transcellular transport process, and is regulated by 1,25(OH)2D3. Similar to phosphate, active absorption becomes physiologically important for magnesium input only when intraluminal magnesium concentration is low. For standard dietary intakes, the absolute amount of actively absorbed magnesium is relatively small compared to the passive mechanisms. Consequently, the role of vitamin D in the regulation of magnesium absorption is marginal. Passive peffusion occurs by paracellular ion diffusion, and depends on luminal magnesium concentration. Passive absorption accounts for most of the magnesium absorbed in the jejunum and ileum. 358 Whereas in the proximal jejunum absorption increases linearly with luminal magnesium concentration, in the ileum the magnesium absorptive process is fully saturated at luminal concentrations above 10 mM 359 (Fig. 6-9). Because magnesium movement across the intestine follows the water flux across the intestinal cells, 36~ solvent drag represents an additional mechanism of magnesium absorption. In general, the whole intestine can absorb magnesium. Therefore, the net input depends on both the efficiency of absorption and the transit time at each single intestinal A V) k,,,

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Net magnesium absorption (solid line), assessed from metabolic balance method, as a function of intake in healthy individuals. The equation for net absorption, e 1 " 2 8 6 - 2 " 8 8 9 / ( x + 1) at - 0.0710X, represents the combination of a hyperbolic function (dotted line) plus a linear function (dashed line). (From Fine KD, Santa Ana CA, Porter J, Fordtran JS: Intestinal absorption of magnesium from food and supplements. J Clin Invest 88:396-402, 1991. Copyright 1991, The American Society for Clinical Investigation.)

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segment. Accordingly, although the highest rate of magnesium absorption occurs in the duodenum, most of the dietary magnesium is absorbed in the distal intestine because of the longer transit time. 357 Species differences exist as to the role of the colon in this function. In avians and lower mammals the colon is the major site of magnesium absorption, whereas in humans most of the element is absorbed before entering the colon. 357'358 However, one should consider that colonic absorption may occur when enemas containing magnesium are administered. 361 3. MEASUREMENT OF MAGNESIUM ABSORPTION The limitations of the current knowledge about magnesium absorption are reflected by the technical problems and limitations related to its measurement. Balance studies have been employed in a similar manner as for calcium balance, but this approach is time consuming and impractical for clinical use. 357 Fractional magnesium absorption can be assessed using isotopic retention techniques after oral administration of a radioactive tracer by applying the same mathematical models used for calcium (see Section I.A.6). The major limitations of these methods are the short half-life and high cost of 28Mgm the radioactive isotope most commonly u s e d ~ a n d patients' exposure to radioactivity. An alternative method is based on the assessment of urinary magnesium after an oral magnesium load. 355 This method is based on the same assumptions applied to the oral calcium load test, and as such, it bears the same limitations of sensitivity and reproducibility (see Section I.A.5). Nonetheless, this simple method could be used in clinical settings, and its results have been shown to correlate reasonably well with 28Mg-based fractional absorption. 36z 4. REGULATION OF MAGNESIUM ABSORPTION As noted above, because the major mechanism of magnesium absorption is passive transport, hormonal control is not a crucial factor that regulates the net input of magnesium. No direct correlation exists between intestinal magnesium absorption or serum magnesium and serum levels of 1,25(OH)2D3 .363'364 Furthermore, substantial magnesium absorption occurs in individuals with no detectable blood levels of 1,25(OH)2D3 .363 This observation is in apparent contrast with findings of enhanced magnesium absorption after vitamin D administration to vitamin D-depleted animals 359'365 and humans. 365 Since interactions between magnesium and phosphatemand perhaps calciummabsorption occur (see below), it is reasonable to assume that the reported regulatory action of 1,25(OH)2D3 on intestinal magnesium transport may be in part the result of indirect changes in phosphate and/ or calcium absorption in response to the vitamin D me-

tabolite. Unlike calcium, magnesium absorption does not seem to be altered in aging. 357

B. Clinical M a n i f e s t a t i o n s o f Magnesium Disorders Because of its role in many fundamental physiological functions, magnesium deficiency leads to a variety of cellular and organ dysfunctions (Table 6-6). Hypomagnesemia is associated with other electrolyte imbalances, in particular hypokalemia and hypocalcemia. The latter is of concern because it responds only to magnesium repletion and is caused by an end-organ resistance to PTH and 1,25(OH)2D3, associated with a functional hypoparathyroidism. 366 In hypomagnesemic conditions, infusion of PTH does not raise either serum calcium or 1,25(OH)2D3 levels. Urinary cAMP production is also blunted in most patients with severe hypomagnesemia. 367 Because magnesium is required for adenylate cyclase activity, 368 is it likely that a reduced synthesis of cAMP may be responsible for the PTH resistance of hypomagnesemia, although other aspects of PTH signal transduction may be involved. Magnesium is involved in the regulation of phospholipase C activity 369 and inhibits inositol-trisphosphate binding to its receptor, 37~a signaling pathway that is stimulated by P T H . 371'372 As a consequence of the peripheral resistance to PTH, serum 1,25(OH)2D3 also declines in spite of the hypocalcemia. Thus, a reduced intestinal absorption of calcium is one of the pathogenetic factors of hypocalcemia in hypomagnesemia (see Section I.C.3). Magnesium deficiency also reduces skeletal responsiveness to 1,25(OH)2D3, although the intestinal response to 1,25(OH)2D3 is maintained, because administration of 1,25(OH)2D3 and calcium can maintain normal serum calcium level in hypomagnesemic animals. 373 The parathyroid gland needs adequate magnesium to maintain an appropriate

TABLE 6 - 6

Disorders Associated with Decreased Magnesium Absorption

Dietary deficiency Intestinal diseases with decreased transit time Vitamin D deficiency Chronic renal failure Hypoparathyroidism Intrinsic defects of intestinal magnesium transport Primary hypomagnesemia Diabetes mellitus Extrinsic factors inhibiting magnesium absorption Cations: A13+, Zn 2+, K + Anions: phosphate, oxalate, phytate

CHAPTER6 Pathophysiologyof Calcium, Phosphate, and Magnesium Absorption secretory function. At normal serum calcium and magnesium concentrations, both cations can equally regulate PTH secretion. 374'375When serum magnesium is low, the ability of parathyroid cells to respond to hypocalcemia with increased hormone secretion is impaired, thus causing a condition of hypoparathyroidism. 367 Therefore, normal PTH levels in hypocalcemia of hypomagnesemia should be considered inappropriate for the hypocalcemia. Magnesium deficiency also results in increased urinary potassium excretion and hypokalemia, which corrects only upon restoring normal magnesium level. It also contributes to insulin resistance, hyperlipidemia, and atherosclerosis in diabetes mellitus. Unfortunately, magnesium is lost through the urine in poorly controlled diabetic patients, whose metabolic control may thus worsen. Magnesium repletion reduces platelet aggregability, and increases lipoprotein lipase activity and lecithin-cholesterol transferase activity. 376 More severe and protracted hypomagnesemia can lead to cardiac arrhythmias and congestive heart failure.

C. I n c r e a s e d M a g n e s i u m A b s o r p t i v e States Conditions characterized by increased magnesium absorption occur, but rarely do they result in metabolic imbalances of clinical relevance. In most cases, the increased absorption is a secondary response to mineral losses through the kidney. Excess dietary intake of magnesium (>800 mg/day) is seldom seen as a spontaneous occurrence. Additional magnesium may be provided by laxatives containing magnesium hydroxide or citrate salts, although hypermagnesemia may occur only when significant impairment of kidney function is associated. Accordingly, magnesium-based laxatives should not be given to renal failure patients. Patients with primary hyperparathyroidism have elevated serum magnesium and 1,25(OH)2D and increased urinary magnesium excretion after oral load compared to normal individuals. 362 Because fasting serum magnesium is not altered in these patients while their urinary magnesium is high, it seems reasonable to assume that the hypermagnesuria in primary hyperparathyroidism is in part related to an increased intestinal magnesium absorption. Although the mechanism of this increased absorption is not totally clear, the increased 1,25(OH)2D3 may provide a pathogenetic explanation. On the other hand, magnesium absorption is normal in patients with absorptive hypercalciuria, whose primary pathophysiological abnormality is an inappropriately high intestinal calcium absorption (see Section I.B.3). This observation further underlines the notion that calcium and magnesium absorption are mediated and regulated by different physiological pathways.

193

An increased intestinal absorption has also been advocated to explain the increased urinary excretion of magnesium occurring in normal subjects during very low dietary calcium (200 mg/day) regimens, as compared to individuals who consume high-calcium diets (1900 mg/ day). The increased 1,25(OH)2D3 driven by the secondary hyperparathyroidism of calcium deficiency may account for this phenomenon. 362 Dairy products are a very good source of calcium, phosphate, and magnesium, not only because of the substantial amount of these elements contained in milkderived food products but also because of the presence of lactose. When lactose or other nonabsorbable carbohydrates, such as fructo-oligosaccharides, are fermented by either intestinal lactase or the bacteria of the colonic flora, luminal pH becomes acidic, which favors magnesium solubilization. Addition of lactose to a highmagnesium diet increases apparent absorption by 17%, thus promoting more magnesium retention. 377 Likewise, fractional magnesium absorption can increase up to almost 80% in rats fed with fructo-oligosaccharides. 378Because water absorption does not change while colonic pH decreases as magnesium absorption increases, coIonic acidification is responsible for the magnesium hyperabsorption, rather than solvent drag. 379 A similar effect can be obtained with other saccharides resistant to digestion. However, this property is lost with food processing through heating and cooling, the so-called "retrograde process." It is believed that retrograded food is nonfermentable, so it cannot contribute to colonic acidification, and consequently it does not provide the additional beneficial effect on mineral absorption. 38~The monosaccharide fructose does not share the same positive action on magnesium absorption with disaccharides. Rats fed with fructose absorb more magnesium than rats fed with glucose. However, because those in the high-fructose group also excrete higher amounts of magnesium by the kidney, the result is actually a negative magnesium balance, despite the enhanced intestinal absorption. TM

D. D e c r e a s e d M a g n e s i u m A b s o r p t i v e States 1. DIETARY DEFICIENCY Because magnesium absorption is strictly dependent on the luminal concentration of the element, any chronic dietary restriction or deficiency results in reduced absorption. However, given the widespread presence of magnesium in most food products, such an occurrence is rare. Hypomagnesemia is not uncommon in hospitalized patients, 376 in whom many factors may contribute to magnesium loss, including acidosis, continuous use of diuretics, intravenous administration of sodium-

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containing fluid, and drugs that increase magnesuria. Whether nutritional deficiency and magnesium hypoabsorption participate in the mechanism leading to hypomagnesemia remains to be determined. 2. INTESTINAL DISEASES

Primary intestinal diseases associated with decreased transit time (stomach resection, chronic diarrhea), or loss of absorptive surface (short bowel syndrome), or with insufficient bile secretion (biliary fistula or atresia) can cause a magnesium deficit. It is questionable whether the vitamin D deficiency that develops in hepatobiliary disorders as a consequence of fat malabsorption has any contribution to the decreased magnesium absorption, which is largely a vitamin D-independent process. Abuse of laxatives may also cause hypomagnesemia secondary to decreased transit time. This possibility should be considered in lactating women. As a consequence of the mother's magnesium deficiency, the milk is poor in magnesium content and hypomagnesemia may develop in the infant, with deleterious consequences. 382 3. VITAMIN D DEFICIENCY a. Chronic Renal Failure

Patients with chronic renal failure often have high serum magnesium levels because of the inability to eliminate the element through the kidney. However, intestinal magnesium absorption in these subjects is reduced to less than 20% of dietary magnesium, as compared to the normal 40% to 50% fractional absorption. Although the precise mechanism that causes this relative magnesium hypoabsorption is still unknown, this phenomenon may represent an adaptive reaction to the severe hypermagnesemia, which may be related to the lower serum 1,25(OH)2D3 levels. b. Hypoparathyroidism and Pseudohypoparathyroidism A similar mechanism can be considered to explain

the hypomagnesemia that complicates hypoparathyroidism and pseudohypoparathyroidism. Intestinal magnesium absorption is reduced in these patients, whose serum 1,25(OH)2D3 levels are also lower. 362 Typically, the absorptive disorder of magnesium corrects after therapy with 1,25(OH)2D3 or other vitamin D metabolites. 362 4. INTRINSIC DEFECTS OF INTESTINAL MAGNESIUM ABSORPTION a. Primary Hypomagnesemia Primary hypomagnesemia is a rare genetic disease transmitted by autosomal recessive trait that causes hypomagnesemia in infants. The affected babies present with convulsions, hypocalcemia, and hypomagnesemia between 9 days and 4 months of age. Males are affected more frequently than

females. 382 The primary defect is an intestinal magnesium hypoabsorption caused by a defective active transport. If dietary magnesium is increased early, and serum magnesium levels are maintained within an acceptable range, the prognosis for these patients is relatively good. 383 b. Diabetes Mellitus In patients with diabetes mellitus, magnesium deficiency may decrease peripheral sensitivity to insulin, and thus worsen diabetic vasculopathy. 384 The mechanism for hypomagnesemia in diabetes is probably related to increased urinary magnesium loss, which occurs in poorly controlled disease. 384-386 Experiments in untreated insulin-dependent diabetic rats suggest that total magnesium absorption is not significantly decreased. However, because bowel weight is increased, the efficiency of magnesium absorption per gram of intestine is in fact decreased. 38v The mechanism of this phenomenon is still unknown, but it occurs early after rats develop insulin-dependent diabetes mellitus. Owing to the relative hypoabsorption and the dietary restrictions of grains and nuts, patients with diabetes should be considered at high risk of developing magnesium deficiency, especially when their metabolic abnormality is poorly controlled. 385 5. EXTRINSIC FACTORS THAT INHIBIT MAGNESIUM ABSORPTION

A number of dietary constituents may interfere with magnesium and decrease its absorption by reducing its bioavailability at sites of absorption. a. Cations Some cations can decrease the bioavailability of dietary magnesium via mechanisms that are not totally clear. Zinc interferes with magnesium absorption, as well as with phosphate absorption. In normal adult males, diets with high zinc content (142 mg/day) can decrease magnesium absorption by more than 10%, resulting in negative body magnesium balance, if dietary magnesium is low. 388 The mechanism of this inhibitory effect is uncertain. Competition of zinc and magnesium for the same absorptive sites along the intestine has been considered. Studies in animals also suggest that potassium may interfere with magnesium absorption. Lambs fed high-potassium diets (4.8% total potassium content in the diet) is associated with a 20% decrease of magnesium absorption. 389 However, there is no evidence of such a finding in humans. The evidence that dietary calcium can interfere with magnesium absorption is tenuous at best. Although rats fed high-calcium diet (five times the standard diet content) absorb significantly less magnesium than rats fed regular calcium, 39~such observation has not been reproduced in humans. Furthermore, increasing dietary calcium to 2000 mg/day does not affect

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m a g n e s i u m absorption in either normal individuals, 361'362 or in patients with chronic renal failure. 361'391 Aluminum can slightly diminish m a g n e s i u m absorption. The presence of 2000 p p m a l u m i n u m in drinking water decreases m a g n e s i u m absorption by almost 10% in w e t h e r s . 346 This interference with m a g n e s i u m is evident only in early phases of a l u m i n u m administration (5 days), and after prolonged exposure of a l u m i n u m this effect is lost. The m e c h a n i s m by which a l u m i n u m alters m a g n e s i u m absorption is unknown. A l u m i n u m does not decrease magnesium bioavailability, but it can interfere with the absorption of fat-soluble vitamins. 392 Thus, a transient vitamin D deficiency can theoretically account for the decrease in m a g n e s i u m absorption.

b. Anions Of more concern for the possible development of m a g n e s i u m deficiency is the interference of m a g n e s i u m with anions, and in particular phosphate and oxalate. As it occurs for calcium, insoluble m a g n e s i u m phosphate or oxalate salts can form in the intestinal lumen, thus preventing the absorption of either ion. This possibility is exemplified by the special milk formulas that are used to feed very-low-birth-weight babies. These formulas contain high amounts of phosphate, and are given under the assumption that the high phosphate content may facilitate the skeletal d e v e l o p m e n t of these babies. 393 Unfortunately, when phosphate is increased to a calcium/phosphate ratio lower than 2:1, fractional intestinal m a g n e s i u m absorption decreases. 393 Thus, the possibility of h y p o m a g n e s e m i a should be considered in small babies when using formulas with high phosphate content. Although green leaf vegetables are an important source of magnesium, its absorbability is reduced by the presence of oxalic acid in some vegetables, such as spinach. Food processing can also modify the bioavailability of magnesium. For example, raw spinach has a poorer m a g n e s i u m bioavailability c o m p a r e d to boiled or fried spinach; however, raw spinach can still efficiently correct m a g n e s i u m deficiency in r a t s . T M Notably, the same considerations do not apply to calcium, for boiling or frying does not improve spinach calcium bioavailability. T M Phytate contained in m a n y vegetables negatively affects m a g n e s i u m absorption, as it does with calcium. Studies in rats would suggest that phytate can also increase intestinal loss of endogenous magnesium. 395 As noted for calcium, soybeans contain large amounts of phytate, which inhibits the absorption of cations, thus making diets based exclusively on soybeans inadequate to provide the daily requirements of minerals. A similar caution can be extended to infants fed with extruded food formula, in which phytase is destroyed by the extrusion process, and consequently m a g n e s i u m and calcium absorption is decreased by interaction with the remaining intact phytate. 35~

c. Other Other food products or additives that can theoretically interfere with mineral absorption have proven not to alter m a g n e s i u m homeostasis. A m o n g these, alginate, which interferes with iron absorption, does not inhibit either calcium or m a g n e s i u m absorption. T M Likewise, a high dietary meat content does not alter m a g n e s i u m absorption, despite the high amount of zinc contained in m e a t . 332 Although subjects who abuse alcohol are prone to h y p o m a g n e s e m i a and m a g n e s i u m deficiency, the cause of the deficiency is a combination of low m a g n e s i u m intake, pancreatic diseases, vitamin D deficit, vomiting, and urinary losses. Intestinal magnesium absorption is not impaired in these individuals. 396

References 1. Heaney RP, Skillman TG: Secretion and excretion of calcium by the human gastrointestinal tract. J Lab Clin Med 64:29-41, 1964. 2. Heaney RP: Calcium in the prevention and treatment of osteoporosis. J Intern Med 231:169-180, 1992. 3. Jensen FT, Charles P, Mosekilde L, Hansen HH: Calcium metabolism evaluated by 47-calcium-kinetics: A physiological model with correction for fecal lag time and estimation of dermal calcium loss. Clin Physiol 3:187- 204, 1983. 4. Levenson DI, Bockman RS: A review of calcium preparations. Nutr Rev 52:221-232, 1994. 5. Consensus Development Conference Panel: Optimal calcium intake. JAMA 272:1942-1948, 1994. 6. Klesges RC, Ward KD, Shelton ML, et al: Changes in bone mineral content in male athletes. Mechanisms of action and intervention effects. JAMA 276:226-230, 1996. 7. Civitelli R, Avioli LV: Calcium, phosphate, and magnesium absorption. In Johnson LR (ed): Physiology of the Gastrointestinal Tract. New York, Raven Press, 1994, pp 2173- 2181. 8. Lee DBN, Hardwick LL, Hu M, Jamgotchian N: Vitamin Dindependent regulation of calcium and phosphate absorption. Miner Electrolyte Metab 16:167-173, 1990. 9. Sheikh MS, Ramirez A, Emmett M, et al: Role of vitamin Ddependent and vitamin D-independent mechanisms in absorption of food calcium. J Clin Invest 81:126-132, 1988. 10. Fullmer CS: Intestinal calcium absorption: Calcium entry. J Nutr 122:644-650, 1992. 11. Bachelet M, Lacour B, Ulmann A: Early effects of loL,25-dihydroxyvitamin D3 o n phosphate absorption. A role for alkaline phosphatases. Miner Electrolyte Metab 8:261-266, 1982. 12. Nasr LB, Monet J, Lucas PA: Rapid (10-minute) stimulation of rat duodenal alkaline phosphatase activity by 1,25 dihydroxyvitamin D 3. Endocrinology 123:1788-1782, 1988. 13. Pansu D, Bellaton C, Roche C, Bronner F: Theophylline inhibits transcellular Ca transport in intestine and Ca binding by CaBP. Am J Physiol 257:G497-G943, 1989. 14. Nemere I, Norman AW: Transcaltachia, vesicular calcium transport and microtubule-associated calbindin-D28k: Energizing views of 1,25-dihydroxyvitamin D3-mediated intestinal calcium absorption. Miner Electrolyte Metab 16:109-114, 1990. 15. Ghijsen WE, DeJong MD, Van Os CA: ATP-dependent calcium transport and its correlations with Ca-ATPase activity in basolateral membranes of rat duodenum. Biochim Biophys Acta 689: 327-336, 1982.

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346. Allen VG, Fontenot JP, Rahmena SH: Influence of aluminum citrate and citric acid on mineral metabolism in wether sheep. J Anim Sci 68:2496-2505, 1990. 347. O'Donovan R, Baldwin D, Hammer M, et al: Substitution of aluminum salts by magnesium salts in control of dialysis hyperphosphatemia. Lancet 1:880-881, 1986. 348. Parsons V, Baldwin D, Moniz C, et al: Successful control of hyperparathyroidism in patients on continuous ambulatory peritoneal dialysis using magnesium carbonate and calcium carbonate as phosphate binders. Nephron 63:379-383, 1993. 349. Schwarz KB, Zimmerman DC, Alpers DH, Avioli LV: Gender differences in antacid-induced phosphate deprivation in rats. Gastroenterology 89:313- 320, 1985. 350. Kivisto B, Andersson H, Cederblad G, et al: Extrusion cooking of a high-fiber cereal product 2. Effects on apparent absorption of zinc, iron, calcium, magnesium, and phosphorus in humans. Br J Nutr 55:255-260, 1986. 351. Roubaty C, Portmann P: Relation between intestinal alkaline phosphatase activity and brush border membrane transport of inorganic phosphate, D-glucose, and D-glucose-6-phosphate. Pflugers Arch 412:482-490, 1988. 352. Borowitz SM, Granrud GS: Glucocorticoids inhibit intestinal phosphate absorption in developing rabbits. J Nutr 122:12731279, 1992. 353. Schwartz R, Spencer H, Welsh JJ: Magnesium absorption in human subjects from leafy vegetables intrinsically labeled with stable 26Mg. Am J Clin Nutr 39:571-576, 1984. 354. Schwartz R, Spencer H, Wentworth RA: Measurement of magnesium absorption in man using stable 26Mg as a tracer. Clin Chim Acta 87:265-273, 1978. 355. Harris I, Wilkinson AW: Magnesium depletion in children. Lancet 2:735-736, 1971. 356. Fine KD, Santa Ana CA, Porter J, Fordtran JS: Intestinal absorption of magnesium from food and supplements. J Clin Invest 88:396-402, 1991. 357. Kayne LH, Lee DBN: Intestinal magnesium absorption. Miner Electrolyte Metab 19:210- 217, 1993. 358. Hardwick LL, Jones MR, Brautbar N, Lee DBN: Site and mechanism of intestinal magnesium absorption. Miner Electrolyte Metab 16:174-180, 1990. 359. Hardwick LL, Jones MR, Brautbar N, Lee DBN: Magnesium absorption: Mechanisms and the influence of vitamin D, calcium and phosphate. J Nutr 121:13- 23, 1991. 360. Behar J: Magnesium absorption by the rat ileum and colon. Am J Physiol 227:334-340, 1974. 361. Brannan PG, Vergne-Marini P, Pak CYC: Magnesium absorption in the human small intestine. Results in normal subjects, patients with chronic renal disease and patients with absorptive hypercalciuria. J Clin Invest 57:1412-1418, 1976. 362. Nicar MJ, Pak CYC: Oral magnesium load test for the assessment of intestinal magnesium absorption. Miner Electrolyte Metab 8:44-51, 1982. 363. Hodgkinson A, Marshall DH, Nordin BEC: Vitamin D and magnesium absorption in man. Clin Sci 57:121-123, 1979. 364. Wilz DR, Gary RW, Dominguez JH: Plasma 1,25(OH)2-vitamin D concentrations and net intestinal calcium, phosphate, and magnesium absorption in humans. Am J Clin Nutr 32:20522060, 1979. 365. Pointillart A, Denis I, Colin C: Effects of dietary vitamin D on magnesium absorption and bone mineral contents in pigs on normal magnesium intakes. Magnes Res 8:19-25, 1995. 366. Leicht E, Biro G: Mechanisms of hypocalcaemia in the clinical form of severe magnesium deficit in the human. Magnes Res 5: 37-44, 1992.

367. Rude RK, Oldham SB, Singer FR: Functional hypoparathyroidism and parathyroid hormone end-organ resistance in human magnesium deficiency. Clin Endocrinol 5:209-224, 1976. 368. Northup JK, Smigel MD, Gilman AG: The guanine nucleotide activating site of the regulatory component of adenylate cyclase: Identification by ligand binding. J Biol Chem 257:1141611423, 1982. 369. Litosh I: G protein regulation of phospholipase C activity in a membrane-solubilized system occurs through a Mg 2+- and timedependent mechanism. J Biol Chem 266:4764-4771, 1991. 370. Volpe P, Alderson-Lang BH, Niklols GA: Regulation of inositol 1,4,5-triphosphate-induced Ca 2§ release. I. Effect of magnesium. Am J Physiol 258:C1077-C1085, 1990. 371. Fujimori A, Cheng S, Avioli LV, Civitelli R: Dissociation of second messenger activation by parathyroid hormone fragments in osteosarcoma cells. Endocrinology 128:3032-3039, 1991. 372. Fujimori A, Cheng S, Avioli LV, Civitelli R: Structure-function relationship of parathyroid hormone: Activation of phospholipase C, protein kinase A and C in osteosarcoma cells. Endocrinology 130:29- 36, 1992. 373. Risco F, Traba ML, De La Piedra C: Possible alterations of the in vivo 1,25(OH)ED3 synthesis and its tissue distribution in magnesium-deficient rats. Magnes Res 8:27-35, 1995. 374. Ferment O, Gamier PE, Touitou Y: Comparison of the feed-back effect of magnesium and calcium on parathyroid hormone secretion in man. J Endocrinol 113:117-122, 1987. 375. Wallace J, Scarpa A: Regulation of parathyroid hormone secretion in vitro by divalent cations and cellular metabolism. J Biol Chem 257:10613-10616, 1982. 376. Abbot LG, Rude RK: Clinical manifestations of magnesium deficiency. Miner Electrolyte Metab 19:314-322, 1993. 377. Greger JL, Gutkowski CM, Khazen RR: Interactions of lactose with calcium, magnesium and zinc in rats. J Nutr 119:16911997, 1989. 378. Ohta A, Ohtsuki M, Baba S, et al: Effects of fructooligosaccharides on the absorption of iron, calcium, and magnesium in iron-deficient anemic rats. J Nutr Sci Vitaminol 41:281-291, 1995. 379. Ohta A, Ohtsuki M, Baba S, et al: Calcium and magnesium absorption from the colon and rectum are increased in rats fed fructooligosaccharides. J Nutr 125:2417-2424, 1995. 380. Schulz AGM, Van Amelsvoort JMM, Beynen AC: Dietary native resistant starch but not retrograded resistant starch raises magnesium and calcium absorption in rats. J Nutr 123:17241731, 1993. 381. Van der Heijden A, Van den Berg GJ, Lemmens AG, Beynen AC: Dietary fructose v. glucose in rats raises urinary excretion, true absorption and ileal solubility of magnesium but decreases magnesium retention. Br J Nutr 72:567-577, 1994. 382. Geven WB, Monnens LAH, Willems JL: Magnesium metabolism in childhood. Miner Electrolyte Metab 19:308-313, 1993. 383. Milla PJ, Agget PJ, Wolff OH, et al: Studies in primary hypomagnesaemia: Evidence for defective carrier-mediated small intestinal transport of magnesium. Gut 20:1028-1033, 1979. 384. White JR, Campbell RK: Magnesium and diabetes. Ann Pharmacother 27:775-780, 1993. 385. Campbell RK, Nadler JL: Implications of Magnesium Deficiency in Diabetes. Bimark, New Jersey, 1993, pp 2 - 2 5 . 386. Whang R, Hampton EM, Whang DD: Magnesium homeostasis and clinical disorders of magnesium deficiency. Ann Pharmacother 28:220-226, 1994. 387. Miller LD, Schedl HP: Effects of diabetes on intestinal magnesium absorption in the rat. Am J Physiol 231:1039-1042, 1976.

CHAPTER 6

Pathophysiology of Calcium, Phosphate, and Magnesium Absorption

388. Spencer H, Norris C, Williams D: Inhibitory effects of zinc on magnesium balance and magnesium absorption in man. J Am Coil Nutr 13:479-484, 1994. 389. Green LW, Fontenot JP, Webb KE: Effect of dietary potassium on absorption of magnesium and other macroelements in sheep fed different levels of magnesium. J Anim Sci 56:1208-1213, 1983. 390. Kaup SM, Behling AR, Choquette L, Greger JL: Calcium and magnesium utilization in rats: Effect of dietary butter fat and calcium and of age. J Nutr 120:266-273, 1990. 391. Spencer H, Lesniak M, Gatza CA, et al: Magnesium absorption and metabolism in patients with chronic renal failure and in patients with normal renal function. Gastroenterology 79:2634, 1980. 392. Harvey SC: Gastric antacids, miscellaneous drugs for treatment of peptic ulcer, digestants and bile acids. In Goodman Gilman A, Goodman LS, Rail TW (eds): The Pharmacological Basis of

393.

394.

395.

396.

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Therapeutics. New York, Macmillan Publishing, 1985, pp 980993. Rodder SG, Mize CE, Forman LP, Uauy R: Effects of increased dietary phosphorus on magnesium balance in very low birthweight babies. Magnes Res 5:273-275, 1992. Kikunaga S, Ishii H, Takahashi M: The bioavailability of magnesium in spinach and the effect of oxalic acid on magnesium utilization examined in diets of magnesium-deficient rats. J Nutr Sci Vitaminol 41:671-685, 1995. Brink EJ, Van den Berg J, Van der Meer JM, et al: Inhibitory effect of soybean protein vs. casein on apparent absorption of magnesium in rats is due to greater excretion of endogenous magnesium. J Nutr 122:1910-1916, 1992. Rivlin RS: Magnesium deficiency and alcohol intake: Mechanisms, clinical significance and possible relation to cancer development. J Am Coil Nutr 13:416-423, 1994.

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~HAPTER

,

Disorders of Phosphate Homeostasis KEITH HRUSKA A N D

ANANDARUP

GUPTA

Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110

C. Differential Diagnosis of Hypophosphatemia D. Treatment of Hypophosphatemia III. Hyperphosphatemia A. Causes of Hyperphosphatemia B. Clinical Manifestations of Hyperphosphatemia C. Treatment of Hyperphosphatemia References

I. Phosphate Homeostasis A. Gastrointestinal Absorption of Phosphorus B. Renal Reabsorption of Phosphorus C. Bone Remodeling and Phosphate Transport II. Hypophosphatemia A. Causes B. Clinical and Biochemical Manifestations of Hypophosphatemia

many cellular reactions including biosynthesis derives from hydrolysis of adenosine triphosphate (ATP). Organic phosphate is an important component of phospholipids in cell membranes. Its concentration influences the activity of several metabolic pathways such as ammoniagenesis, glycolysis, gluconeogenesis, and the formation of 1,25-dihydroxyvitamin D3 [1,25(OH)zD3] from 25-hydroxyvitamin D3 [25(OH)D3]. Changes in serum phosphorus may also influence the dissociation of oxygen from hemoglobin through its regulation of the concentrations of 2,3-diphosphoglycerate (2,3-DPG).

I. PHOSPHATE HOMEOSTASIS The physiological concentration of serum phosphorus ranges from 2.8 to 4.5 mg/dl (0.9 to 1.45 mM) in adults. 1 There is a diurnal variation in serum phosphorus of 0.6 to 1.0 mg/dl the nadir occurring between 8 AM and 11 AM. Ingestion of meals rich in carbohydrate decreases serum phosphorus concentrations as a result of movement of phosphorus from the extracellular to the intracellular space. In the extracellular fluid, phosphorus is present predominantly in the inorganic form. In serum, phosphorus exists mainly as the free ion, and only a small fraction (<15%) is protein bound. 2'3 Approximately 80% to 85% of the phosphorus is present in the skeleton. Phosphorus is a critical, basic component of hydroxyapatite, the mineral phase of bone. The rest of the phosphorus is widely distributed throughout the body in the form of organic phosphate compounds. Phosphorus plays a fundamental role in several aspects of cellular metabolism. The energy required for METABOLIC BONE DISEASE

A. G a s t r o i n t e s t i n a l A b s o r p t i o n o f P h o s p h o r u s Approximately 1 g of phosphorus is ingested daily in an average diet in the United States. About 300 mg is excreted in the stool, and 700 mg is absorbed (Fig. 7-1). Most of the phosphorus is absorbed in the duodenum and jejunum, with minimal absorption occurring 207

Copyright 9 1998by AcademicPress. All rightsof reproductionin any formreserved.

208

KEITH HRUSKA AND ANANDARUP GUPTA

FIGURE 7-- 1 Overviewof phosphorus (PI) metabolism: Pathways involved in Pi balance and turnover rates in major tissue compartments. (From Feldman D, Glorieux F, Pike JW (eds): Vitamin D. New York, Academic Press, 1997.)

in the ileum. 4 Phosphorus transport in proximal segments of the small intestine appears to involve both passive and active components and to be under the influence of vitamin D. The movement of phosphorus from the intestinal lumen to the blood requires: (1) transport across the luminal brush border membrane of the intestine, (2) transport through the cytoplasm, and (3) transport across the basolateral plasma membrane of the epithelium. 1. LUMINAL BRUSH BORDER TRANSPORT

The mechanism of transport across the intestinal brush border epithelial membrane involves a sodiumphosphate co-transport system as suggested by Berner et al. 5 The role of 1,25(OH)2D3 and 25(OH)D3 in this transport system has been studied by several investigators. 6-8 Murer and Hildman have shown that in vivo administration of 1,25(OH)2D to rabbits affects in vitro uptake of phosphorus by intestinal brush border membrane vesicles. 7 Reduction of endogenous 1,25(OH)2D3 decreases uptake of phosphorus by approximately 65% in brush border membrane vesicles. This uptake increased threefold after injection of high doses of

1,25(OH)2D3. These studies are in agreement with those of Fuchs and Peterlik, who found similar results utilizing chick intestinal epithelial brush border membrane vesicles. 4 The events that lead to stimulation of membrane uptake of phosphorus by vitamin D are of major interest but have not been completely elucidated. Studies of phosphorus accumulation by rat intestinal brush border vesicles have demonstrated that at physiological pH phosphorus uptake is dependent on luminal sodium but is unaffected by the transmembrane potential, a finding that is consistent with an electroneutral co-transport of sodium and monovalent phosphate (H2PO4); at lower pH values (6.0) the increased uptake is independent of intraluminal sodium concentration and appears to involve transfer of net negative charge. Vitamin D seems to stimulate phosphorus absorption by two m e c h a n i s m s ~ a calcium-dependent duodenal process and a calciumindependent jejunal system. In the rat the major effect of 1,25(OH)2D3 on phosphorus absorption appears to involve increased active transport. Parathyroid hormone (PTH) may influence phosphorus absorption in the gut

CHAPTER7 Disorders of Phosphate Homeostasis but presumably does so indirectly by stimulating the renal synthesis of 1,25(OH)2D3. Low-phosphorus diets increase the absorption of phosphorus from the intestine but may do so by stimulating the formation of 1,25(OH)ED3. 8 In animals fed a high-phosphorus diet, suppression of 1,25(OH)ED3 may result in decreased intestinal absorption of phosphorus. 2. TRANSCELLULAR MOVEMENT OF PHOSPHORUS The second component of transcellular intestinal phosphorus transport involves the movement of phosphorus from the luminal to the basolateral membrane. Although little is known about the cellular events that mediate this transcellular process, evidence suggests a role for the microtubule system of intestinal cells. 4 Microtubules in the cell may be important in conveying phosphorus from the brush border membrane to the basolateral membrane and may be involved in the extrusion of phosphorus at the basolateral membrane from the epithelial cell. 3. PHOSPHATE EXIT AT BASOLATERAL MEMBRANE Little is known about the mechanisms of phosphorus extrusion at the basolateral membrane of intestinal epithelial cells. The electrochemical gradient for phosphorus favors movement from the intracellular to the extracellular compartment because the interior of the cell is electrically negative compared with the basolateral external surface. 7 Phosphate may exit the cell via an anion exchange mechanism, 7 although there have been reports of an ATP and Na+-dependent transport process. 9

B. Renal Reabsorption of Phosphorus Most of the inorganic phosphorus in serum (90% to 95%) is ultrafilterable at the level of the glomerulus. At physiological levels of serum phosphorus, approximately 7 g of phosphorus is filtered daily by the kidney, of which 80% to 90% is reabsorbed by the renal tubules and the remainder excreted in the urine (~700 mg on a 1-g phosphorus diet) (Fig. 7-1).1~ Micropuncture studies have demonstrated that 60% to 70% of the filtered phosphorus is reabsorbed in the proximal tubule (Fig. 7-2). However, there is also evidence that a significant amount of filtered phosphorus is reabsorbed in distal segments of the nephron. 1~ When serum phosphorus levels increase and the filtered load of phosphorus increases, the capacity to reabsorb phosphorus also increases. However, a maximum rate of transport (Tm) for phosphorus reabsorption is obtained usually at serum phosphorus concentrations of 6 mg/dl. There is a direct correlation between Tm phosphorus values and glomerular filtration rate (GFR) even when the latter is varied over a broad

209 Loop

Distal

Urine

100

:i7 o 50

25

FIGURE 7--2

Profile of PO43 reabsorption along the nephron derived from micropuncture data. (From Feldman D, Glorieux F, Pike JW (eds): Vitamin D. New York, Academic Press, 1997.)

range. Micropuncture studies suggest two different mechanisms responsible for phosphorus reabsorption in the proximal tubule. In the first third of the proximal tubule, in which only 10% to 15% of the filtered sodium and fluid is absorbed, the ratio of tubular fluid (TF) phosphorus to plasma ultrafilterable phosphorus (UF) falls to values of approximately 0.6. This indicates that the first third of the proximal tubule accounts for approximately 50% of the total amount of phosphorus reabsorbed in this segment of the nephron. In the last two thirds of the proximal tubule the reabsorption of phosphorus parallels the movement of salt and water. In the remaining 70% of the pars convoluta, the TF/UF phosphorus ratio remains at a value of 0.6 to 0.7, whereas fluid reabsorption increases to approximately 60% to 70% of the filtered load. Thus, in the last two thirds of the proximal tubule, the TF/UF phosphorus reabsorption is directly proportional to sodium and fluid reabsorption. A significant amount of phosphorus, perhaps on the order of 20% to 30%, is reabsorbed beyond the portion of the proximal tubule that is accessible to micropuncture. There is little phosphorus transport within the loop of Henle, 12 most distal transport occurring in the distal convoluted tubule, in this location, Pastoriza-Munoz et al. 1~ found that approximately 15% of filtered phosphorus is reabsorbed under baseline conditions in animals subjected to parathyroidectomy, but the value falls to about 6% after administration of large doses of PTH. The collecting duct is a potential site for distal nephron reabsorption of phosphorus. 13-15 Transport in this nephron segment may explain the discrepancy between the amount of phosphorus delivered to the late distal tubule in micropuncture studies, and the considerably smaller amount of

210

KEITH HRUSKAAND ANANDARUPGUFTA

phosphorus that appears in the final urine of the same kidney. Phosphorus transport in the cortical collecting tubule is independent of regulation by PTH. This is in agreement with the absence of PTH-dependent adenylate cyclase in the cortical collecting tubule. 15 1. COMPARISON OF SUPERFICIAL AND DEEP NEPHRON TRANSPORT The contribution of superficial and deep nephrons to phosphorus homeostasis differs. Nephron heterogeneity in phosphorus handling has been evaluated in a number of conditions by puncture of the papillary tip and of the superficial early distal tubule, the recorded fractional delivery representing deep and superficial nephron function, respectively. Using this technique, Haramati et al. 16'17 have demonstrated that in thyroparathyroidectomized (TPTX) rats fed a normal phosphorus diet, phosphorus reabsorption is greater in deep than in superficial nephrons, and this heterogeneous handling of phosphorus can be mitigated by both PTH infusion 17 and a lowphosphorus diet. 16 Studies in TPTX rats fed a normal phosphorus diet have presented in vivo measurements of maximum tubular reabsorptive capacities for phosphorus of deep and superficial nephrons. Deep nephron values (5.05 pM/ml/min) were significantly greater than the values obtained in superficial nephrons (3.38 pM/ml/min). These results suggest that the juxtamedullary nephrons are more responsive to body phosphorus requirements than are the superficial nephrons. Also, microinjection of phosphorus tracer into thin ascending and descending limbs of Henle's loop reveal that only 80% of phosphorus was recovered in the urine, whereas 88% to 100%

FIGURE 7--3

of phosphorus was recovered when the tracer was injected into the late superficial distal tubule. It was concluded that a significant amount of phosphorus must be reabsorbed by juxtamedullary distal tubules and/or by segments connecting the juxtamedullary distal tubules to the collecting ducts to account for the discrepancy between the results of superficial nephron injection and injection into the juxtamedullary ascending limb of Henle's loop. These data seem to support an increased reabsorptive capacity for phosphorus in deep as opposed to superficial nephrons. In summary, phosphorus transport occurs in the distal nephron, particularly in the distal convoluted tubule and cortical collecting tubular system. This transport may be considerable under certain experimental conditions, but the importance of the terminal nephron system in dayto-day phosphorus homeostasis remains to be defined. It is also evident from data obtained from various micropuncture and microinjection studies that juxtamedullary and superficial nephrons have different capacities for phosphorus transport. The increased responsiveness of the deep nephrons to phosphorus intake suggests a key regulatory role for this system in phosphorus homeostasis. 2. CELLULAR MECHANISMS OF PHOSPHATE REABSORPTION IN THE KIDNEY The apical membrane of renal tubular cells is the initial barrier across which phosphorus and other solutes present in the tubular fluid must pass to be transported into the peritubular capillary network (Fig. 7-3). Because the electrical charge of the cell interior is negative to the exterior and phosphorus concentrations are higher

Phosphate transport in the proximal convoluted tubule. (From Feldman D, Glorieux F, Pike JW (eds): Vitamin D. New York, Academic Press, 1997.)

CHAPTER7 Disorders of Phosphate Homeostasis in the cytosol, phosphorus must move against an electrochemical gradient into the cell interior, whereas, at the antiluminal membrane, the transport of phosphorus into the peritubular capillary is favored by the high intracellular phosphorus concentration and the electronegafivity of the cell interior. Studies with apical membrane vesicles have demonstrated co-transport of Na + with phosphate across the brush border membrane, whereas the transport of phosphorus across the basolateral membrane is independent of that of Na+. TM The apical membrane Na+-phosphate co-transport protein (NaPi) energizes the uphill transport of phosphate across the brush border membrane by the movement of Na + down its electrochemical gradient. The latter gradient is established and maintained by active extrusion of Na + across the basolateral cell membrane into the peritubular capillary through the action of Na+-K+-ATPase (Fig. 7-3). 19 Two families of NaPi co-transport proteins of the proximal tubule (NaPi-1, NaPi-2) have been cloned using Xenopus oocyte expression strategies. 2~ The DNA clones encode 80- to 90-kDa proteins that reconstitute Na+-dependent concentrative or "uphill" transport of phosphate upon chromosomal RNA injection in oocytes or transfection of sf9 cells. 2~ The NaPi-2 proteins are similar between several species including human (Fig. 7-4). 2~ Nephron localization of NaPi-2 proteins have been limited to the proximal tubule of superficial and deep nephrons (greatest in the latter, concordant with physiological studies). 24 The nature of the apical phosphate co-transport protein of the distal nephron, the enterocyte co-transporter, and the osteoblast phosphate cotransporter proteins remains to be described at this writing. Immunolocalization studies in renal epithelial cells demonstrated apical membrane and subapical membrane vesicle staining, 24 suggesting that a functional pool

FIGURE 7--4 Structure of the type II Na-dependent phosphate cotransporters (NaPi 2/3/4/6). (From Feldman D, Glorieux F, Pike JW (eds): Vitamin D. New York, Academic Press, 1997.)

211 of transporters is available for insertion or retrieval from the brush border membrane itself. This has been shown to be a major mechanism of inorganic phosphate (Pi) transport regulation 21'25'26 in response to acute changes in Pi and PTH. The role of the NaPi-1 family of co-transporters in physiological regulation has not been elucidated. The NaPi-1 transporter family may be constitutive and represent a basic "housekeeping" function. The NaPi-2 family is up-regulated by chronic feeding of low Pi diets. 27'28 Both the levels of messenger ribonucleic acid (mRNA) message and the amount of transport protein in the apical membrane are increased chronically, suggesting a transcriptional event (Fig. 7-5). Acutely, there appears to be an early stimulation of protein insertion into the apical membrane which precedes increased transcription (Fig. 7-5). The NaPi-2 family is also regulated by PTH, mainly through removal of transport protein from the apical membrane; and chronically there is also a decrease in the m R N A . 26 Studies of phosphorus exit across the basolateral membrane suggest that it is accompanied by the net transfer of a negative charge and occurs down a favorable electrochemical gradient via sodium-independent mechanisms. 29 3. FACTORS THAT AFFECT THE URINARY EXCRETION OF PHOSPHORUS

Several factors are known to affect the urinary excretion of phosphorus. Of the multiplicity of factors that regulate phosphate transport in the kidney (Table 7-1), the most important are phosphate delivery and PTH.

a. Effects of PTH on Phosphorus Reabsorption By the Kidney Parathyroidectomy decreases urinary phosphorus excretion, whereas administration of PTH rapidly increases phosphorus excretion. 3~ Micropuncture studies indicate that PTH inhibits phosphorus transport in the proximal t u b u l e 32'33 and probably in segments of the nephron located beyond the proximal tubule. 11 TF/ UF phosphorus reaches a value of 0.6 by the $2 segment of the proximal tubule, and once achieved, this equilibrium ratio is maintained along the accessible portion of the proximal tubule. Within 6 to 24 hours of parathyroidectomy, the proximal TF/UF phosphorus falls to a value of 0.2 to 0.4, indicating an increase in phosphorus reabsorption. 33-35 Tubular fluid phosphorus falls progressively with continuous fluid absorption along the length of the tubule, so that by the end of the proximal tubule the reabsorption of phosphorus is 70% to 85% of the filtered load, resulting in decreased phosphorus delivery to distal segments of the nephron. Since decreased delivery of phosphorus out of the proximal tubule complicates the evaluation of any distal effects of PTH on

212

KEITH HRUSKA AND ANANDARUP GUFTA

FIGURE 7--5

Biphasic response of proximal tubular phosphate transport to reduced delivery of PO43. (From Feldman D, Glorieux F, Pike JW (eds): Vitamin D. New York, Academic Press, 1997.)

phosphorus excretion, maneuvers have been designed to increase phosphorus delivery to the distal nephron to study distal effects of parathyroidectomy on phosphorus reabsorption (e.g., phosphorus loading by intravenous infusion). 36'37 In the non-phosphorus-loaded, acutely parathyroidectomized animal, virtually all the distal load of phosphorus is reabsorbed by the distal nephron, reducing urinary phosphorus excretion to very low levels. In the phosphorus-loaded animal, the distal reabsorption of phosphorus increases until saturation is approached and urinary phosphorus excretion begins to r i s e . 38'39 Acute administration of PTH to phosphorus-loaded, parathy-

TABLE 7--1

Factors Regulating Na/Pi Co-transport

Parathyroid hormone Calcitonin Atrial natriuretic factor cAMP/cAMP analogues Nicotinamide Phosphate loading Metabolic acidosis Fasting Glucocorticoids Growth hormone Insulin IGFs Thyroid hormone lct-25(OH)2D3 Phosphate deprivation Stanniocalcin

Renal

Osteogenic

$ ,I, $ $ $ $ $ $ $ 1" 1" 1" 1" 1" 1" 1"

1" 1" ? 1" ? $ $ ? ? 1" 1" 1" 1" 1" 1" 1"

roidectomized dogs sharply lowers the distal reabsorption. These experiments indicate that PTH inhibits reabsorption of phosphorus in the distal as well as in the proximal nephron. Administration of PTH in vivo results in decreased rates of Na+-dependent phosphorus transport in brush border membrane vesicles isolated from the kidneys of treated rats. 4~ The uptakes of D-glucose and Na § were not affected by administration of PTH. Intravenous infusion of dibutyryl cyclic adenosine monophosphate (cAMP) also decreased Na+-dependent phosphorus uptake in isolated brush border vesicles, but neither PTH nor dibutyryl cAMP decreased phosphate transport when added directly to membrane vesicles. 4~ These observations suggested that the effects of PTH on renal phosphate transport were mediated through altered functional characteristics (decreased Vmax) of the renal brush border membrane Na+-dependent phosphate transporter. 41 Measurements of renal reabsorption of phosphorus, in vivo, and calculations of kinetic parameters of Na +dependent phosphorus transport in membrane vesicles isolated from the renal brush border membranes of normal dogs, parathyroidectomized dogs, dogs fed a lowphosphorus diet, and dogs receiving human growth hormone were performed (Table 7--2). 41'42 The latter three groups of dogs had greater baseline values for absolute tubular reabsorption of phosphorus compared with normal dogs. Na+-dependent phosphate transport in brush border membrane vesicles isolated from kidneys of these dogs were significantly increased compared with transport in brush border vesicles from kidneys of normal

CHAPTER 7 Disorders of Phosphate Homeostasis

213

TABLE 7--2 The Effect of Parathyroid Hormone on Kinetic Parameters of Phosphate Uptake in Brush Border Membrane Vesicles Vmax

Animals Normal Normal + PTH Thyroparathyroidectomized Thyroparathyroidectomized + PTH Phosphate-depleted Phosphate-depleted + PTH GH-treated GH-treated + PTH

Km (IxM) 51 45 43 55 39 36 52 48

• • • • • _ _ _

2 3 4 8 6 2 1 4

(pMPi/20sec/ mg protein) 1585 • 892 • 2490_ 1259 • 3364 • 2680 • 2124 • 1388 •

20 68 139 68 75 69 99 104

Adapted from Klahr S, et al: Renal effects of parathyroid hormone and calcitonin. In Dunn M (ed): Renal Endocrinology.Baltimore, Williams & Wilkins, 1983, p 269.

dogs. Administration of PTH decreased significantly the apparent Vmax for Na§ phosphorus transport in brush border membrane vesicles isolated from kidneys of each of the four groups of dogs (Table 7-2). The apparent Km (intrinsic binding affinity) for Na+-dependent phosphorus transport was not significantly changed by experimental maneuvers. Absolute tubular reabsorption of phosphorus measured in vivo was decreased by administration of PTH in each group of dogs with the exception of the dogs fed a low-phosphorus diet. 41'42 Thus alterations in phosphorus reabsorption measured in vivo were paralleled by alterations in Na+-dependent phosphorus transport in isolated membrane vesicles, and the administration of PTH in vivo resulted in altered transport characteristics of the isolated brush border membranes. The cloning of the NaPi co-transport proteins has further elucidated the mechanisms of PTH action on phosphate transport as yet. Because the phosphaturic effect of PTH can be reproduced by analogues of cAMP, the intracellular mechanism of phosphate transport regulation is thought to involve the cAMP/protein kinase A signal pathway. However, the NaPi transport proteins are not characterized by a protein kinase A - m e d i a t e d phosphorylation site. 43 Phosphorylation of brush border membrane proteins in vitro occurs in parallel with inhibition with NaPi co-transport, 41 and these may be involved in endocytic traffic of NaPi-2 transporters. Parathyroidectomy of rats causes a two- to threefold increase in the NaPi-2 protein content of brush border membrane vesicles. 44 Immunocytochemistry reveals the increase in protein exclusively in apical brush border membranes of proximal tubules. PTH treatment of parathyroidectomized rats for 2 hours decreased protein levels in the brush border membrane and decreased the abundance of

NaPi-2-specific mRNA by 31%.44 Parathyroidectomy did not affect NaPi-2 mRNA levels. The effects of PTH were apparent within 2 hours of administration and indicate that PTH regulation of NaPi is determined in large part by changes in the expression of NaPi-2 protein in the renal brush border membranes by a mechanism that results in endocytic withdrawal of NaPi-2 into a cytoplasmic pool. 44 b. Effects o f Changes in Acid-Base Balance on Phosphate Excretion The effect of acid-base status on the renal excretion and transport of phosphate is complex. Acute respiratory acidosis increases and acute respiratory alkalosis decreases phosphate excretion. 45 These effects occur independent of PTH, and plasma or luminal bicarbonate levels, but they may be mediated by changes in plasma phosphate. 45 Acute metabolic acidosis has minimal effects on phosphate excretion; however, the phosphaturic effect of PTH is blunted. Acute metabolic alkalosis causes an increase in phosphate excretion independent of P T H . 46-5~ This effect is due, in part, to volume expansion produced by the infusion of bicarbonate. 47'48 Chronic acidosis increases phosphate excretion, again independent of PTH or changes in ionized C a 2 + . 5~ The effect appears to be directly on the sodium-dependent phosphate transport mechanism. 54 Chronic alkalosis decreases phosphate excretion, probably by the same mechanism as acidosis, operating in the opposite direction. 46"55 The effects of acid-base perturbations are complex and depend on antecedent dietary intake, the chronicity of the change, and whether the change affects luminal or intracellular pH, or both. c. Adrenal Hormones Administration of pharmacological amounts of cortisol leads to phosphaturia. Acute adrenalectomy diminishes GFR and increases the reabsorption of phosphorus in the proximal tubule. Frick and Durasene 56 concluded that glucocorticoid hormones could play an important role in the regulation of fractional reabsorption of phosphorus. The current notion of an important role for cellular metabolism in regulating the proximal tubular reabsorption of phosphorus is relevant to these observations. An effect of glucocorticoid hormones in altering carbohydrate metabolism within the proximal tubular cells could underlie the effects described. d. Vitamin D Controversy still surrounds the regulatory role of vitamin D in renal phosphorus handling. Several past studies have demonstrated that the chronic administration of vitamin D to parathyroidectomized animals is phosphaturic. 57-59 Conversely, other investigators reported that vitamin D acutely stimulates proxi-

214 mal tubular phosphorus transport in both parathyroidectomized and vitamin D-depleted rats. 6~ A unifying interpretation of these studies was hampered by the fact that the dosages of vitamin D administered and the status of the serum calcium, phosphorus, and PTH varied considerably from study to study. Liang and co-workers 61 administered 1,25(OH)2D3 to vitamin D-deficient chicks and subsequently examined the transport characteristics of isolated renal tubule cells. Three hours following the in vivo administration of vitamin D, phosphorus uptake by the cells was significantly increased, whereas 17 hours after the administration of vitamin D, phosphorus uptake was reduced. The serum phosphorus concentration, however, was significantly increased at the 17-hour time point, and administration of phosphorus to vitamin D-depleted animals so that their serum phosphorus reached a level comparable to that of the 17-hour vitamin D-replete group resulted in a similar decrease in phosphorus uptake. 61 In response to in vitro preincubation with as little as 0.01 pM of 1,25(OH)2D3, renal cells isolated from vitamin D-deficient chicks demonstrated a specific increase in sodium-dependent phosphorus uptake, which was blocked by pretreatment with actinomycin D. The stimulatory effect was relatively specific for 1,25(OH)2D3, and kinetic analysis indicated that the Vmax of the phosphorus transport system was increased, whereas the affinity of the system for phosphorus was unaffected. 61 Kumik and H r u s k a 62 also examined the relationship between vitamin D and renal phosphorus excretion in a normocalcemic, normophosphatemic weanling rat model fed a vitamin D-deficient diet. The animals were mildly vitamin D deficient [92 pg/ml of 1,25(OH)zD3 vs. 169 pg/ml in controls] but had no evidence of secondary hyperparathyroidism. Clearance studies performed in the basal, partially vitamin D-deficient state showed an increase in both absolute and fractional phosphorus excretion compared to controls. Animals replete with 1,25(OH)zD3 and maintained on diets designed to protect against the development of hyperphosphatemia demonstrated a significant decrease in urinary phosphorus excretion. Other animals were similarly repleted with vitamin D but did not receive dietary adjustment, and in this group, both the serum phosphorus and the urinary phosphorus excretion levels increased significantly. A third group was fed a normal diet and received smaller doses of 1,25(OH)zD3 (15 pM/g body weight) for shorter periods of time, and although this dose had no effect on the serum phosphorus concentration, the phosphaturia was completely resolved. Studies on brush border membrane vesicles prepared from these animals revealed that in the partially vitamin D-deficient state, sodium-dependent phosphorus uptake was significantly reduced compared with control ani-

KEITH HRUSKA AND ANANDARUP GUFTA

mals. Animals repleted with vitamin D and fed a controlled diet had a greater sodium-dependent phosphorus uptake than both vitamin D-depleted animals and vitamin D-repleted animals not maintained on controlled diets. The results of this series of studies suggest that the primary action of 1,25(OH)zD3 is to increase tubular phosphorus reabsorption. Long-term administration of vitamin D, however, represents a more complex situation, and here, phosphaturia may occur secondary to changes in the filtered load of phosphorus, in the body distribution of phosphorus, or in intracellular phosphorus activity. e. Growth Hormone An increase in serum phosphorus and a rise in renal phosphorus transport are characteristics of growth hormone excess either during the period of rapid growth in the child, during acromegaly, or during exogenous growth hormone administration to experimental animals. Hammerman et al. reexamined this phenomenon in the brush border membrane vesicle preparation in the dog 63 and demonstrated that growth hormone treatment resulted in an increased sodiumdependent phosphorus transport. These data reassert the importance of brush border membrane phosphorus uptake in regulating overall renal phosphorus reabsorptive capacity. f Alterations in Dietary Phosphorus Intake The mechanism by which the kidney conserves phosphorus when dietary phosphorus is reduced or increased continues to be intriguing. Early micropuncture studies suggested that the most striking adaptive increase in phosphorus transport occurs in the proximal tubule, but later studies 16 suggested that the entire nephron participates in the reduction of phosphorus excretion during dietary phosphorus deprivation. It has been shown that isolated perfused tubules obtained from rabbits fed a normal or low-phosphorus diet differ in their capacity to reabsorb phosphate. In normal animals the proximal convoluted tubule is capable of reabsorbing 7.2 _+ 0.8 pM/ml/min, whereas tubules obtained from phosphorus-deprived animals reabsorb 11.1 _+ 1.3 pM/ml/min. Conversely, animals fed a high dietary phosphorus intake show reduced phosphorus reabsorption when the proximal tubules are perfused in vitro (2.7 + 2.6 pM/ml/min). Based on renal brush border membrane preparations, it has been suggested that the effect of reduced dietary phosphorus to stimulate renal phosphorus transport is intrinsic to the renal tubular epithelium and occurs specifically at the brush border membrane Na+-phosphate cotransporter. Considerable evidence has accrued from studies performed in cell lines isolated from mammalian kidneys, indicating that the adaptation to phosphate

CHAPTER7 Disorders of Phosphate Homeostasis supply by the sodium phosphate co-transporter is biphasic. 64-66 These studies demonstrated that incubation of cells in low-phosphate medium result in a twofold increase in Na+-independent phosphate co-transport. The first phase of adaptation is observed rapidly (within 10 minutes) and is characterized by an increase in the Vmax of the transporter. This initial phase is independent of new protein synthesis. A slower phase resulting in a doubling of the phosphate transport rate, again through an increase in V .... occurs over several hours (max --~15) and is inhibited by blocking new protein synthesis. These studies have been interpreted to indicate that the immediate response to reduced phosphate availability is the insertion of new transport units into the brush border membrane from an intracellular store (Fig. 7 - 5 ) . Second, through gene transcription and increased NaPi protein synthesis, additional units are produced and inserted into the brush border (Fig. 7-5). The reverse of these processes is produced by high phosphorus availability and by PTH. The cloning of the NaPi-2 phosphate co-transport proteins has enabled further confirmation of results of the above-mentioned Pi transport studies. As discussed above, chronic feeding of low-Pi diet increases steadystate mRNA levels of NaPi-6 in rabbits and NaPi-2 in rats. 2v'28Acute Pi deprivation does not affect NaPi-2 family mRNA levels, compatible with a protein synthesisindependent action related to insertion of new transport proteins from subapical vesicles into the brush border membrane. 25,67 g. Stanniocalcin Stanniocalcin is a new calciumregulating hormone recently found in serum and the kidney. 68 Its name derives from its synthesis by the corpuscles of stannous, endocrine glands found in the kidneys of bony fishes. In mammals, stanniocalcin lowers calcium transport and increases phosphate reabsorption. The bony fishes use this action to increase phosphate deposition into bone and scales. The role of stanniocalcin in human physiology requires elucidation, as does the regulation of its secretion.

C. B o n e R e m o d e l i n g a n d P h o s p h a t e T r a n s p o r t The main physiological function of bone is to act as a mechanical support. This function is achieved by mineralization of an extracellular matrix formed by specialized cells. Inorganic phosphate is an essential element for the physiological functioning of bone cells, not only because it is an integral component of appatite crystals but also because it can affect the production rate of bone matrix and regulate bone resorption. 69'7~ Until recently, little was known about the transport of phosphorus in

215 bone cells. Recent advances have led to the description of phosphate transport as an exciting component of bone cell physiology which offers opportunity for regulation, leading to modulation of bone remodeling. 1. OSTEOCLAST FUNCTION AND PHOSPHATE TRANSPORT

Osteoclasts are polarized cells involved in bone resorption. They are exposed to high ambient concentrations of inorganic phosphate during the active process of bone resorption. Osteoclasts possess specific phosphate transport systems including a sodium-dependent Pi co-transporter whose activity is dependent upon ATP production and Na-K ATPase activity to maintain the inwardly directed Na gradient driving force for concentrative Pi co-transport. TM When osteoclasts are isolated in vitro and allowed to attach to bone particles, there is stimulation of Pi transport, without an increment of transport protein synthesis. TM The stimulatory effect of bone particles on Pi transport was inhibitable by peptides containing the Arg-Gly-Asp cell adhesion motif, an effect that implicates integrins and cell matrix interaction in the regulation of Pi transport. Western blot analysis of osteoclast lysates utilizing antibodies to the NaPi-2 family proteins demonstrate the detection of proteins of 100 and 105 kDa. Immunofluorescent studies of osteoclasts cultured on glass coverslips reveals the proteins to be present in discrete cytosolic vesicles. Following attachment to bone, these vesicles fuse with the basolateral membrane of osteoclasts coordinate with the stimulation of phosphate transport. Immunofluorescence-based confocal microscopy reveals co-localization of the osteoclast NaPi co-transporter with the Nail antiporter on the basolateral membrane. The osteoclast isoform of the NaPi-2 protein family exhibits an apparent molecular mass greater than the 80 to 90 kDa of the renal tubule. Analogous to the renal tubule, vesicular traffic regulates membrane transporter protein levels in acute regulatory responses of phosphate transport. TM In addition, agents that specifically inhibit the renal tubule phosphate transporter also inhibit osteoclast phosphate transport and bone resorption, vl Phosphate transport is essential to osteoclast function providing the substrate for ATP synthesis, which is required to support the high rates of proton transport involved in bone resorption. 2. PHOSPHATE TRANSPORT AND OSTEOGENIC

CELL FUNCTION The osteogenic cells, osteoblasts, and chondrocytes do not exhibit a phosphate transporter with sequence homology to the NaPi-1 or NaPi-2 protein family. 69 Recently, a new NaPi co-transport system, which is expressed in several tissues including bone marrow, has been identified. 72 This transport system also serves as a

216 membrane receptor for some retroviruses. It is a candidate protein for the Na-dependent Pi co-transporter of osteogenic cells that has not as yet been identified at the molecular level. Analysis of Na-dependent Pi transport in osteoblasts reveals that phosphate transport in these cells differs significantly from that of renal epithelial cells. Affinity constants for Pi are significantly higher (300 to 500 IxM compared to 50 to 100 IxM for renal epithelial cells). The affinity of the transporter for Na is similar to that of epithelial cells (40 mM). Pi transport activity of osteoblasts is stimulated by an acidic extracellular pH in contradiction to the renal epithelial transporter. Regulation of osteoblast phosphate transport also differs from that of renal epithelial cells. Parathyroid hormone stimulates phosphate transport in osteoblast-like cells through the cAMP signal transduction pathway. 73 This is directionally opposite to the effect of PTH in the renal epithelium. Insulin-like growth factor 1 (IGF-1) is also a selective stimulator of Pi transport in osteoblasts and chondrocytes. 74 Fluoride, another agent which, at low doses, stimulates bone formation, also increases Pi transport in osteoblasts. 75 The effect of fluoride may be mediated by the formation of fluoroaluminate. 69 a. M a t r i x Vesicles Biological mineralization is a complex process involving different types of tissues. Primary mineralization is regulated by chondrocytes, osteoblasts, and odontoblasts, and is observed, respectively, in epiphyseal cartilage, embryonic bone, postfetal ossification, and the development of predentin. The mineralization process mediated by osteoblasts in bone formation is not fully understood. Two general but not mutually exclusive theories exist on how calcification is initiated in skeletal tissues: (1) collagen nucleation and (2) matrix vesicles. 76 The first theory holds that the collagen fibril is the major site of crystal nucleation such that Ca phosphate crystals are deposited in the whole region of these fibrils. The alternative theory involves matrix vesicle production by osteogenic cells. When matrix vesicles are calcified in the extracellular matrix, they serve as nucleation sites for mineralization. Matrix vesicles are small organelles with a diameter of 100 to 200 nm that have been observed in mineralizing tissues in cultures of osteogenic cells often in contact with initial mineral crystals. Matrix vesicles arise from boneforming cells by a process of budding from elongated tubular extensions that project from the plasma membrane of these cells. 77 Matrix vesicles may participate in the uptake of mineral ions dependent upon the presence of labile forms of mineral within the vesicles that serve as a trap for newly entering mineral ions. 78 Calcium enters matrix vesicles by a protease-sensitive carrier that may be related to the annexins. 79 Matrix vesicles have a

KEITH HRUSKAAND ANANDARUPGUPTA

similar Na-dependent Pi transport system to osteoblasts and chondrocytes. 8~ Formation of phosphate-calcium phospholipid complexes in the vesicular space may maintain a favorable Pi gradient, allowing the continuation of phosphate transfer into matrix vesicles. In this system, sodium only facilitates translocation of Pi across the membrane of matrix vesicles, since a mechanism of maintaining the sodium gradient is not present as in the plasma membrane of cells. Studies 81 have recently shown that Pi transport activity is low in matrix vesicles released during the proliferative phase of osteoblast differentiation, but is significantly increased during osteoblast differentiation, peaking at the time of bone matrix formation. This observation suggests the existence of specific mechanisms for enhanced phosphate transport activity in matrix vesicles released during the formation of the collagenous matrix. It may be explained by enrichment of matrix vesicles with phosphate carriers during the differentiation process. Since the activity of Pi transport in osteogenic cells is regulated by calcitropic hormones and growth factors, regulation of transport may represent a mechanism by which osteogenic cells regulate the calcification of their extracellular matrix.

II. HYPOPHOSPHATEMIA Hypophosphatemia refers to serum phosphorus concentrations of less than 2.5 mg/dl. Hypophosphatemia usually results from one or a combination of the following factors82'83: (1) increased excretion of phosphorus in the urine, (2) decreased gastrointestinal absorption of phosphorus, or (3) translocation of phosphorus from the extracellular to the intracellular space. The major causes of hypophosphatemia are listed in Table 7 - 3 .

A. Causes 1. INCREASED EXCRETION OF PHOSPHORUS IN THE URINE

Several pathophysiological conditions may increase excretion of phosphorus in the urine. Some of these conditions are characterized by elevated levels of circulating PTH. Since PTH decreases phosphorus reabsorption by the kidney, modest to marked elevations of this hormone may increase urinary excretion of this anion. Decreased tubular reabsorption of phosphorus may also occur without increased levels of PTH and may be due to changes in the reabsorption of salt and water or to renal tubular defects specific for the reabsorption of certain solutes and/or phosphorus. Hypophosphatemia may also occur in the diuretic phase of acute tubular necrosis or in post-

CHAPTER7 Disorders of Phosphate Homeostasis TABLE 7 - 3

Causes of Hypophosphatemia

I. Increased excretion of phosphorus in the urine A. Primary hyperparathyroidism B. Secondary hyperparathyroidism C. Renal tubular defects D. Diuretic phase of acute tubular necrosis E. Postobstrucfive diuresis F. After renal transplantation G. ECF volume expansion H. Familial 1. X-linked hypophosphatemia 2. McCune-Albright syndrome II. Decrease in gastrointestinal absorption of phosphorus A. Malabsorpfion B. Malnutrition-starvation C. Administration of phosphate binders D. Abnormalities of vitamin D metabolism 1. Vitamin D deficiency m rickets 2. Familial a. Vitamin D-resistant rickets b. X-linked hypophosphatemia III. Miscellaneous causes/translocation of phosphorus A. Diabetes mellitus: during treatment for ketoacidosis B. Severe respiratory alkalosis C. Recovery phase of malnutrition D. Alcohol withdrawal E. Toxic shock syndrome F. Leukemia, lymphoma G. Severe bums

obstructive diuresis, presumably owing to a combination of high levels of PTH and decreased tubular reabsorption of salt and water. 2. PRIMARY HYPERPARATHYROIDISM This is a common entity in clinical medicine. 84 PTH is secreted in excess of the physiological needs for mineral homeostasis owing to either adenomas or hyperplasia of the parathyroid glands. 85 This results in decreased phosphorus reabsorption by the kidney. The losses of phosphorus in the urine result in hypophosphatemia. The degree of hypophosphatemia may vary considerably among patients because mobilization of phosphorus from bone will in part mitigate the hypophosphatemia. Moreover, if the patient ingests large amounts of dietary phosphorus, the degree of hypophosphatemia observed may be mild. Since these patients also have elevated levels of serum calcium, the diagnosis is made relatively easy in the majority of cases by the finding of elevated levels of immunoreactive PTH. 3. SECONDARY HYPERPARATHYROIDISM Although secondary hyperparathyroidism is present in most patients with chronic renal disease, hyperphosphatemia rather than hypophosphatemia occurs in such patients owing to decreased phosphorus excretion in the

217 urine as a consequence of the fall in GFR. However, certain conditions characterized by malabsorption of calcium from the gastrointestinal tract may produce hypocalcemia, leading to development of secondary hyperparathyroidism. 86 The elevated levels of PTH will decrease phosphorus reabsorption by the kidney, resulting in hypophosphatemia. Thus patients with gastrointestinal abnormalities resulting in calcium malabsorption and secondary hyperparathyroidism will have low levels of both serum calcium and phosphorus. In these patients, the hypocalcemia is responsible for the increased release of PTH. In addition, decreased intestinal absorption of phosphorus as a result of the primary gastrointestinal disease may also contribute to the decrement in the levels of serum phosphorus. In general, these patients have urinary losses of phosphorus that are out of proportion to the hypophosphatemia in contrast to patients with predominant phosphorus malabsorption and no secondary hyperparathyroidism in whom urinary excretion of phosphorus is low. 4. RENAL TUBULAR DEFECTS

Several conditions characterized by either single or multiple tubular defects have been described in which phosphorus reabsorption is decreased. 87 In the Fanconi syndrome, 87 patients excrete not only increased amounts of phosphorus in the urine but also increased quantities of amino acids, uric acid, and glucose, resulting in hypouricemia and hypophosphatemia. There are other conditions in which an isolated defect in the renal tubular transport of phosphorus has been found as, for example, in fructose intolerance, an autosomal recessive disorder. 88 Following renal transplantation, an acquired renal tubular defect may be responsible for the persistence of hypophosphatemia in some patients. 89 5. DIURETIC PHASE OF ACUTE TUBULAR NECROSIS

Most patients with acute renal failure develop secondary hyperparathyroidism and hyperphosphatemia during the oliguric phase. During the recovery phase of acute renal failure, the combined occurrence of a profound diuresis, secondary hyperparathyroidism, and continued use of phosphate binders may lead to development of severe hypophosphatemia. This hypophosphatemia is usually short lived, and serum phosphorus levels return to normal as the diuretic phase of acute tubular necrosis (ATN) subsides. 6. POSTOBSTRUCTIVE DIURESIS A marked phosphaturia may develop in some patients after relief of urinary tract obstruction. This phosphaturia may be severe enough in a few patients to lead to development of hypophosphatemia. 9~

218 7. POSTRENAL TRANSPLANTATION

Hypophosphatemia, sometimes severe, is common in patients after renal transplantation. 91 Patients coming to renal transplantation usually have severe secondary hyperparathyroidism. 91 As GFR is restored after placement of the graft, the high levels of PTH acting on the transplanted kidney markedly increase phosphorus excretion in the urine. However, in some patients after renal transplantation, even after the levels of PTH have returned to normal, a persistent phosphaturia that is out of proportion to the levels of serum phosphorus has been observed. In these patients it is presumed that a tubular defect for phosphorus reabsorption has developed. Thus hypophosphatemia in the first few days after renal transplantation may result from a combination of factors: (1) ingestion of phosphate binders that has continued even after the transplant has adequate function, (2) secondary hyperparathyroidism in the presence of near normal GFR, and (3) a renal tubular defect for phosphorus reabsorpfion. When phosphate binders are decreased or discontinued and hyperparathyroidism subsides, phosphorus levels usually retum to normal in most patients with a renal graft. However, in some patients, as mentioned above, hypophosphatemia does not subside, possibly owing to a tubular defect in phosphorus reabsorption. 89 Prolonged periods of severe hypophosphatemia may cause bone disease characterized by severe osteomalacia in some of these patients. 8. EXTRACELLULAR FLUID VOLUME EXPANSION

Expansion of the extracellular fluid (ECF) volume by the administration of solutions containing sodium increases the urinary excretion of phosphorus. An important mechanism by which ECF volume expansion produces phosphaturia consists of a fall in ionized calcium and subsequent release of PTH. 35 This condition is probably of minor importance in clinical medicine, and restoration of ECF volume to normal results in return of phosphorus reabsorption to physiological levels. 9. X-LINKED HYPOPHOSPHATEMIC RICKETS

X-linked hypophosphatemic rickets (XLH) is an X-linked dominant disorder characterized by hypophosphatemia, decreased reabsorption of phosphorus by the renal tubule, decreased absorption of calcium and phosphorus from the gastrointestinal tract, and varying degrees of tickets or osteomalacia. The roles of alterations in vitamin D sensitivity and/or synthesis in the defective transport of phosphorus observed both in the gut and the kidney have been investigated in the murine homologue of X-linked hypophosphatemia. 92 Tenenhouse and Scriver found that administration of small doses of 1,25(OH)2D3 to normal animals significantly increased

KEITH HRUSKAAND ANANDARUPGUFTA plasma calcium, plasma phosphorus, and fractional calcium excretion without a change in fractional phosphorus excretion or phosphorus transport as measured in proximal tubular brush border membrane vesicles. 93 However, there was no response to this dose of 1,25(OH)2D3 in the Hyp mice. At a fivefold higher dose of vitamin D the familial hypophosphatemic mice did show increased plasma calcium and fractional calcium excretion as well as plasma phosphorus levels, but again there was no change in fractional phosphorus excretion or in sodium-dependent phosphorus transport in brush border membrane vesicles. Although 1,25(OH)2D3 increased phosphorus transport in the intestine, there was no defect of phosphorus transport in the intestine of untreated hypophosphatemic mice. The authors concluded that vitamin D treatment influenced phosphorus homeostasis in Hyp mice by pharmacological stimulation of phosphorus absorption from the gastrointestinal tract. The defect in renal phosphorus reabsorption remained unchanged despite high levels of vitamin D. These observations are consistent with studies performed in humans with familial hypophosphatemia in whom the renal reabsorptive defect persists despite correction of growth by administration of vitamin D and oral phosphate supplements. 94'95 There is, however, evidence of defective or altered metabolism of vitamin D3 in Hyp mice. Meyer et al. found that on a normal diet plasma 1,25(OH)2D3 levels were the same in Hyp and normal mice, 96 although 25(OH)D3 levels were reduced in Hyp mice. Because hypophosphatemia increases plasma 1,25(OH)2D3 levels, hypophosphatemic mice were resistant to the stimulatory effect of hypophosphatemia. To test this possibility, these authors fed the animals a low-phosphorus diet and found a paradoxical reduction in 1,25(OH)2D3 levels in hypophosphatemic mice. They concluded that Hyp mice have a defective control system for plasma 1,25(OH)2D3 that is unresponsive to a low-phosphate diet stimulus. The issue is further complicated by studies by Beamer et al., 97 who studied the effect of various preparations of vitamin D3 on intestinal transport of phosphorus. The Hyp mice responded to ltx-dihydroxyvitamin D3 but not to 1,25(OH)2D3, suggesting that intestinal phosphorus transport is not genetically absent in this model but rather does not respond to normal endogenous levels of 1,25(OH)2D3. Thus mice with familial hypophosphatemia appear to have impaired metabolism of vitamin D, but this impairment does not cause the renal phosphate transport defect. There is also evidence suggesting that the decreased tubular reabsorption of phosphorus is not due to increased PTH levels. 97 It seems that a component of renal phosphorus transport, which may be PTH independent, may be abnormal and responsible for the increased phosphaturia. Tenenhouse et al. 98 have shown that tissue phosphate levels were normal in familial hy-

219

CHAPTER7 Disorders of Phosphate Homeostasis pophosphatemic mice, whereas these levels tended to be low in animals that have hypophosphatemia secondary to reduction in dietary phosphorus. It would seem, therefore, that XLH is a selective disorder of the transepithelial transport of phosphate. However, there is no correlation between the degree of hypophosphatemia and the severity of bone disease. Moreover, alterations in vitamin D metabolism cannot be explained solely by the presence of hypophosphatemia. Therefore, some undefined pathological mechanism may involve both abnormal phosphate transport and renal 1-hydroxylase function. Tenenhouse et al. 99 have extended their studies in Hyp mice to demonstrate that the message levels for the NaPi-2 co-transport protein are reduced by 50% in the renal cortex, similar to the reduction in apical membrane vesicle NaPi-2 protein levels. This demonstrates a significant underexpression of the proximal tubule Nadependent phosphate transport protein in Hyp mice. However, the mapping of NaPi-2 and NaPi-1 to chromosomes 5 and 6, respectively, ~~176176 indicates that these genes are not candidates for the genetic defects leading

FIGURE 7--6

to X-linked hypophosphatemia or Hyp. One possibility is that X-linked hypophosphatemia and Hyp are produced by defective transcription of the NaPi-2 and NaPi3 genes, respectively. 1~ Another possibility is that the pathogenesis of X-linked hypophosphatemia and in Hyp mice is due to abnormal levels of a circulating hormonal factor. Results of the cross-perfusion studies of Meyer et al. 1~ and the cross-transplantation studies of Nesbitt et al. TM have been interpreted to indicate the presence of a circulating factor. Furthermore, Nesbitt et al. TM were unable to demonstrate transmission of the Hyp renal defect by transplanting a Hyp kidney into a normal recipient. Likewise, transplantation of a normal kidney into a Hyp recipient failed to correct the Hyp phenotypic abnormalities. These studies, in addition to the recent studies by Cai et al., ~~ in oncogenic osteomalacia, suggest that the presence of a circulating humoral factor could be capable of producing the Hyp and X-linked hypophosphatemia phenotype. The circulating humoral factor hypothesis (Fig. 7 - 6 ) for the etiology of X-linked hypophosphatemia has

Conceptual diagram of a new phosphate regulatory system. The discovery that the genetic defect in X-linked hypophosphatemia is in a gene (PEX) encoding an endopeptidase gives even greater interest to findings in oncogenic osteomalacia.., wherein a circulating substance that inhibits PO42 transport has been described. The concept is that PEX normally inactivates phosphatonin unless it is overproduced as in oncogenic osteomalacia. (From Feldman D, Glorieux F, Pike JW (eds): Vitamin D. New York, Academic Press, 1997.)

220 gained considerable support from the recent discovery of the defective gene, PEX, through positional cloning. 1~ The PEX gene product encodes a neutral endopeptidase of the same family as endothelin-converting enzyme, and it is characterized by zinc regulation and a single transmembrane-spanning domain. The model proposed by this result suggests that a circulating phosphaturic factor is normally catabolized by the PEX gene product. When PEX is mutated, the phosphaturic factor levels are increased and hypophosphatemia results. An interesting aspect of the Hyp phenotype is the presence of a normal Na-dependent phosphate co-transport in osteoblasts. 1~ However, the osteoblasts of Hyp have been shown to be defective in models of endochondral bone formation. 1~176 Defective mineralization is an important aspect of osteomalacia, and mineralization is controlled by calcium, phosphate, and bone matrix proteins. We recently demonstrated defective phosphorylation of a key bone matrix protein, osteopontin, in Hyp mice and abnormal low activity of a protein kinase (casein kinase-II-like activity) responsible for osteopontin phosphorylation. 1~176 Thus, multiple proteins appear to be defective in the Hyp phenotype. This is compatible with multiple targets of the PEX gene such as phosphatonin and an inhibitor of casein kinase-II, which achieve greater activity when PEX is defective. 1~176 From a therapeutic point of view, the combination of neutral phosphate and 1,25(OH)2D3 has led to an improvement in the bone disease of some patients with X-linked hypophosphatemia and in the Hyp m i c e . 111'112 The administration of phosphorus in X-linked hypophosphatemia is usually divided into four doses, with the total amount ranging between 1 and 4 g/day. Pharmacological doses of calcitriol on the order of 1 to 3 Ixg/ day may be necessary to correct the skeletal alterations. 1,25(OH)2D3 does not correct the increased fractional excretion of phosphate. The enthusiasm for this regimen is tempered by a high incidence of nephrocalcinosis and occasional renal failure. 111-113 10. MCCUNE-ALBRIGHT SYNDROME

This syndrome is characterized by the clinical triad of polyostotic fibrous dysplasia, caf6 au lait skin pigmentation, and endocrine/metabolic disorders. The endocrine disorders include autonomous secretion of various hormones such as growth hormone, thyroid hormone, cortisol, estradiol, and testosterone. Rickets and osteomalacia due to hyperphosphaturic hypophosphatemia are prominent components of the syndrome. The disorders of the syndrome share in common excessive function of cells whose actions are normally regulated by hormones that induce cAMP generation. The molecular basis for the phenotype is an activating mutation of the Gs~ protein (the oL component of the stim-

KEITH HRUSKA AND ANANDARUP GUFTA

ulatory heterotrimeric guanosine triphosphate [GTP] -binding protein) in cells from affected tissues from patients with the syndrome. 114 Kidney tissue, presumably proximal tubule, from patients has been reported to contain cells with the mutation. 114 11. ABNORMALITIES OF VITAMIN D METABOLISM Vitamin D and its metabolites play an important role in phosphorus homeostasis. 115 Vitamin D promotes the intestinal absorption of calcium and phosphorus and is necessary to maintain the normal mineralization of bone. Dietary deficiencies of vitamin D increase the amount of osteoid tissue in the skeleton and decrease normal mineralization. Bone mineralization is a complex process that is not completely understood. Normally, the osteoblast is responsible for laying down normal collagen that is well organized and distributed in a lamellar fashion. Between the recently deposited collagen and the old bone, there is an area called the mineralization front. Initially, amorphous calcium-phosphate is deposited in the mineralization front and eventually matures into hydroxyapatite [Ca10 (PO4)6(OH)2]. Thus, the osteoid tissue changes into bone. Optimal mineralization requires the following: (1) normal bone cell activity, (2) normal supply of minerals, (3) the appropriate pH (7.4 to 7.6), (4) normal synthesis and composition of the matrix, and (5) control of inhibitors of calcification. The appositional growth rate in normal bone is about 1 Ixm/day and complete mineralization of the osteoid requires 13 to 21 days. Thus, the thickness of the osteoid usually does not exceed 20 )xm. Less than 20% of the surface of the bone is normally covered by osteoid. When a biopsy is performed in a normal subject who has previously ingested two doses of tetracycline separately and 3 weeks apart, one normally detects two fluorescent tings or bands, indicating the locations of the mineralization front. In a patient with osteomalacia, usually a single band, no band, or an irregular and spotty uptake of tetracycline is seen. In tickets or osteomalacia, there is a quantitative and qualitative defect in bone mineralization. 12. VITAMIN D-DEFICIENT RICKETS

Diets deficient in vitamin D lead to the metabolic disorder known as rickets when it occurs in children or osteomalacia when it appears in adults. 116 Deficiency of vitamin D in childhood results in severe deformities of bone because of rapid growth. These deformities are characterized by soft, loose areas in the skull known as "craniotabes" and costochondral swelling or bending (known as "rachitic rosary"). The chest usually becomes flattened, and the sternum may be pushed forward to form the so-called pigeon chest. Thoracic expansion may be greatly reduced, with impairment of respiratory

CHAPTER7 Disorders of Phosphate Homeostasis function. Kyphosis is a common finding. There is remarkable swelling of the joints, particularly the wrists and ankles, with characteristic anterior bowing of the legs, and fractures of the "greenstick" variety may also be seen. In adults, the symptoms are not as striking and are usually characterized by bone pain, weakness, radiolucent areas, and pseudofractures. Pseudofractures represent stretch fractures in which the normal process of healing is impaired owing to a mineralization defect. Mild hypocalcemia may be present; however, hypophosphatemia is the most frequent biochemical alteration. This metabolic abnormality responds well to administration of small amounts of vitamin D.

221 outgrow the disease. Human vitamin D-resistant rickets as a genetic defect in the vitamin D receptor varies significantly from other genetic diseases of steroid hormone receptors caused by resistance to thyroid hormone, androgens, and estrogens. 124-126 For instance, individuals heterozygous for vitamin D receptor mutations are apparently completely normal. Second, no dominant negative mutations, which are prominent in thyroid hormone resistance, have been identified as a cause of human vitamin D-resistant rickets. Thus, much remains to be learned from the genetic analysis of this disease. The treatment of this condition requires large pharmacological doses of calcium that overcome the receptor defects and maintain bone remodeling. 122

13. VITAMIN D-RESISTANT RICKETS

This is a recessively inherited form of vitamin D-refractory rickets. This condition is also characterized by hypophosphatemia, hypocalcemia, elevated levels of serum alkaline phosphatase, and, sometimes, generalized aminoaciduria and severe bone lesions. Currently, two main forms of vitamin D-resistant rickets have been characterized. The serum concentrations of 1,25(OH)2D3 serve to differentiate the two types of vitamin D resistant rickets. Type I is an inborn error in conversion of 25-(OH)D to 1,25(OH)zD3 due to deficiency of the renal 1-hydroxylase enzyme. 117 This condition responds to very large doses of vitamin D2 and D3 (100 to 300 times the normal requirement of physiological doses); however, 0.5 to 1.0 txg/day of 1,25(OH)ED3. Type II is characterized by an end-organ resistance to 1,25(OH)ED3. Plasma levels of 1,25(OH)zD3 are elevated. This finding, in association with radiographic and biochemical signs of tickets, implies resistance of the target tissue to calcitriol. Cellular defects found in patients with vitamin D-resistant tickets type II are heterogenous, providing in part an explanation for the different clinical manifestations of this disorder. Among the cellular defects are: (1) decreased number of cytosolic receptors, (2) deficient maximal hormonal binding, (3) deficient hormone-binding affinity, (4) normal hormonal binding but undetectable nuclear localization, and (5) abnormal DNA-binding domain for the 1,25(OH)zD3 receptor. 118 Numerous studies 119-126 demonstrated that hereditary vitamin D-resistant tickets is a genetic disease affecting the vitamin D receptor. Defects in the hormone-binding domain 119'12~ and the DNA-binding domain 121'122 have been defined. In addition, several cases of human vitamin D-resistant tickets have been studied and no abnormality in the coding region of the vitamin D receptor has been found, 123 suggesting a defect elsewhere in the hormone action pathway. An unexplained feature of this disease in adolescents is the tendency for calcium levels to normalize and for the radiographic abnormalities of tickets to improve, thus giving the appearance that they

14. ONCOGENIC OSTEOMALACIA

This rare syndrome is characterized by hypophosphatemia associated with tumors. It was described initially in association with mesenchymal tumors; however, recent reports emphasized the association of this syndrome with malignant tumors. 127"128 The other characteristics of this syndrome are increased phosphate excretion, low plasma 1,25-dihydroxyvitamin D concentrations, and osteomalacia. All of the biochemical and pathological abnormalities disappear when the tumor is resected. The tumors associated with this syndrome are thought to secrete a substance that inhibits the renal tubular reabsorption of phosphate and suppresses 25-hydroxycholecalciferol 1 oL-hydroxylase activity. Whether this factor interacts directly with renal tubular cells is unknown. Recent studies by Cai et al. 1~ have investigated the ability of conditioned media from sclerosing hemangioma cell cultures to affect Na-dependent phosphate transport. The tumor was removed from a patient with osteogenic osteomalacia. They found that the medium inhibited sodiumdependent phosphate transport, without increasing cellular concentrations of cAME The medium has PTH-like immunoreactivity but no PTH-related protein (PTHrP) -like immunoreactivity, and the action of the tumor medium was not blocked by a PTH antagonist. The plasma 1,25(OH)2D3 concentrations are low in patients with oncogenic osteomalacia despite the presence of hypophosphatemia, 129-131 which usually increases plasma 1,25(OH)2D3 concentrations by stimulating the renal 25(OH)D3 l oL-hydroxylase in a PTH-independent manner. 132 Besides the hypophosphatemia, deficient production of 1,25(OH)2D3 is a factor contributing to the pathogenesis of the osteomalacia in these patients. Miyauchi et al. 133 have reported that 25(OH)D3 l e~-hydroxylase activity of cultured renal tubular cells was decreased by incubating the cells with tumor extracts. This supports the concept that the tumor extracts contain a substance that inhibits the formation of 1,25(OH)2D3 in the proximal tubule.

222 The varied studies of oncogenic osteomalacia and some studies associated with X-linked hypophosphatemic rickets support the possibility that a hormone primarily responsible for the regulation of renal phosphate reabsorption is abnormally produced in these conditions. Econs and Drezner TM have termed the substance "phosphatonin." The similarity between oncogenic osteomalacia and X-linked hypophosphatemia (XLH) raises the possibility that phosphatonin is the factor normally degraded by the PEX gene product that causes abnormal phosphate transport in XLH and Hyp. 15. MALABSORPTION

Since most of the absorption of phosphorus from the gastrointestinal tract occurs in the duodenum and jejunum, gastrointestinal disorders such as celiac disease, tropical and nontropical sprue, and regional enteritis may decrease the absorption of phosphorus. 86 Phosphorus malabsorption has also been described in patients who have undergone surgical bypass procedures for morbid obesity. The degree of hypophosphatemia varies among patients, with intestinal malabsorption being extremely mild in some and severe in others. 16. MALNUTRITION Most of the phosphorus ingested in the diet is present in protein, particularly meat, cheese, milk, and eggs. In many parts of the world where protein consumption is extremely low, hypophosphatemia occurs predominantly in children. Overall growth is retarded, and a series of metabolic abnormalities are present. 135 17. ADMINISTRATION OF PHOSPHATE BINDERS Certain compounds, mainly aluminum salts (aluminum hydroxide, aluminum carbonate gel) and calcium carbonate, are used in the treatment of hyperphosphatemia. 136 However, when these compounds are given in excess, they may produce profound hypophosphatemia. These gels trap phosphorus in the small intestine and increase the amount of phosphorus in the stool. Patients ingesting large amounts of phosphate binders and not followed closely may develop phosphate depletion. Over a period of time such individuals may develop severe weakness, bone pain, and osteomalacia. 18. OTHER CAUSES OF HYPOPHOSPHATEMIA

Recent reviews of the causes of hypophosphatemia in hospitalized patients 137'138 attributed the majority of instances to intravenous administration of carbohydrate. However, a wide variety of other causes were found, including diuretic usage, hyperalimentation, alcoholism, respiratory alkalosis, and use of phosphate binders. 139 A 31% incidence of hypophosphatemia was seen in patients admitted to a general medical ward, and a further

KEITH HRUSKA AND ANANDARUP GUPTA

fall in serum concentrations occurred in all patients with acute alcoholism between days 2 and 5 after admission to a medical ward. 14~Hypophosphatemia is also seen frequently during treatment for diabetic ketoacidosis. ~41 When diabetic patients develop ketoacidosis they usually have an increase in phosphate excretion in the urine; however, serum phosphate may be slightly elevated due to acidosis. During the administration of insulin there is a rapid decrease in the levels of glucose with translocation of phosphate from the extracellular to the intracellular space, resulting in hypophosphatemia. Acute respiratory alkalosis decreases urinary phosphate excretion but produces marked hypophosphatemia. 142 In contrast, patients who receive sodium bicarbonate excrete large amounts of phosphate in the urine; however, the hypophosphatemia that may develop is only moderate in nature. It has been postulated that in respiratory alkalosis there is an increase in the intracellular pH with activation of glycolysis and increased formation of phosphate-containing sugars leading to a precipitous fall in the concentration of serum phosphorus. The mild hypophosphatemia that may be seen during administration of sodium bicarbonate is probably secondary to increased renal phosphate excretion due to a decrease in ionized calcium and release of PTH as well as to the consequences of ECF volume expansion. 48 In addition, new clinical disorders have been identified in which hypophosphatemia is an important aspect of the pathological condition. Marked hypophosphatemia has been associated with acute leukemia or with lymphomas in the leukemic phase. 143-145 These individuals typically present with hypophosphatemia, normocalcemia, and no evidence of excess PTH activity. Urinary phosphate is typically extremely low. Although kinetic studies have not been performed in this setting, the fact that serum phosphate concentration correlated with a growth phase of the tumors and that hyperphosphatemia was seen when cells were destroyed by chemotherapy or radiotherapy strongly suggested that serum phosphorus was initially utilized in the rapid growth of new cells. Because these patients are often severely ill and under treatment with glucose infusions as well as antacids and other drugs known to induce hypophosphatemia, they may be at great risk of developing severe acute phosphorus depletion. Another clinical condition in which hypophosphatemia has been a prominent feature is the toxic shock syndrome. Chesney et al. 146 described 22 women with this disorder who showed hypocalcemia and hypophosphatemia as prominent manifestations. Whether respiratory alkalosis or staphylococcal sepsis induced release of substances that were responsible for acute phosphorus shifts into cells is unknown. Lindquist et a l . 147 studied in a prospective fashion the importance of hypophospha-

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temia in patients with severe burns. In 33 patients studied for 2 weeks after injury, transient hypophosphatemia was seen in the second to tenth day in all these individuals. Five of seven patients who died from complications of the terminal injury had severe hypophosphatemia. Because urinary phosphorus excretion was not increased, tissue uptake seems to be the predominant mechanism responsible for the hypophosphatemia. Levy reported the occurrence of severe hypophosphatemia during the rewarming phase in a profoundly hypothermic patient. 148 In this individual urinary excretion of phosphorus was minimal, suggesting that a shift of phosphate into the cells occurred as a consequence of rewarming. Finally, the development of hypophosphatemia as a consequence of refeeding clinically starved patients has been emphasized. Silvis et al. showed that the classic phosphorus depletion syndrome, consisting of paresthesias, weakness, seizures, and hypophosphatemia, can occur in individuals who receive oral caloric supplements following a prolonged period of starvation. 149 In order to further evaluate this issue, they performed studies in normal dogs who had been starved or had received normal diets and found that the infusion of calories through an intragastric catheter to previously starved animals resulted

TABLE 7 - 4

in a fall in serum phosphorus from an average of 4.8 mg/dl to 1.6 mg/dl. Nearly 50% of starved animals developed clinical signs of phosphate depletion following oral refeeding. Weinsier and Krumdiek reported two patients who developed the phosphorus depletion syndrome in association with cardiopulmonary decompensation following overzealous hyperalimentation after prolonged caloric deprivation, is~

B. Clinical and B i o c h e m i c a l M a n i f e s t a t i o n s of Hypophosphatemia The manifestations of hypophosphatemia are presented in Table 7 - 4 . It has been suggested that the clinical manifestations of hypophosphatemia and severe phosphorus depletion are related to disturbances in cellular energy and metabolism. Studies have examined the effects of phosphate depletion on cellular energetics and other components of cell function. A study of glycolytic intermediates and adenine nucleotides during insulin treatment of patients with diabetic ketoacidosis emphasized the important effects of insulin-induced cellular phosphate depletion on cell metabolism. TM These results

Clinical and Biochemical Manifestations of Marked Hypophosphatemia

I. Cardiovascular and skeletal muscle A. Decreased cardiac output B. Muscle weakness C. Decreased transmembrane resting potential D. Rhabdomyolysis II. Carbohydrate metabolism A. Hyperinsulinemia B. Decreased glucose metabolism III. Hematological alterations A. Red blood cells 1. Decreased ATP content 2. Decreased 2,3-DPG 3. Decreased Ps0 4. Increased oxygen affinity 5. Decreased lifespan 6. Hemolysis 7. Spherocytosis B. Leukocytes 1. Decreased phagocytosis 2. Decreased chemotaxis 3. Decreased bactericidal activity C. Platelets 1. Impaired clot retraction 2. Thrombocytopenia 3. Decreased ATP content 4. Megakaryocytosis 5. Decreased lifespan

IV. Neurological manifestations A. Anorexia B. Irritability C. Confusion D. Paresthesias E. Dysarthria F. Ataxia G. Seizures H. Coma V. Skeletal abnormalities A. Bone pain B. Radiolucent areas (x-ray) C. Pseudofractures D. Rickets or osteomalacia VI. Biochemical and renal manifestations A. Low PTH levels B. Increased 1,25(OH)2D3 C. Hypercalciuria D. Hypomagnesemia E. Hypermagnesuria E Hypophosphaturia G. Decreased GFR H. Decreased T m for bicarbonate I. Decreased renal gluconeogenesis J. Decreased titratable acid excretion K. Increased creatinine phosphokinase L. Increased aldolase

Adapted from Slatopolsky E: Pathophysiology of calcium, magnesium, and phosphorus. In Klahr S (ed): The Kidney and Body Fluids in Health and Disease. New York, Plenum Press, 1983, p 269.

224

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demonstrated that the reduced level of 2,3-DPG seen during insulin treatment of diabetes is due to intracellular phosphorus depletion producing a decrease in glyceraldehyde-3-phosphate dehydrogenase (GAPD) activity rather than inhibition of the phosphofructokinase enzyme system. Ditze1152 has suggested that repeated transient decreases in red cell oxygen delivery due to reduced 2,3-DPG with insulin-induced hypophosphatemia could contribute over many years to the microvascular disease seen in diabetics. It should be pointed out that patients with mild degrees of hypophosphatemia are usually asymptomatic. However, if hypophosphatemia is severe m that is, if serum phosphorus levels are below 1.5 m g / d l m a series of hematological, neurological, and metabolic disorders may develop. In general, the patient becomes anorectic and weak, and mild bone pain may be present if the hypophosphatemia persists for several months (see Table 7-4).

phosphate depletion. DeFronzo and Lange 159 have used the glucose and insulin clamp technique to study the kinetics of glucose metabolism in patients with a variety of chronic hypophosphatemic conditions including vitamin D-resistant rickets. When glucose was infused to maintain constant glycemia at 125 mg/dl, hypophosphatemic individuals required 36% less glucose to maintain these glycemic levels than did controls. Also, when euglycemia was achieved by combined insulin and glucose infusion, the hypophosphatemia individuals required 40% less glucose to maintain euglycemia than did controis. Insulin catabolism was apparently unaffected in these hypophosphatemia individuals. These data indicate that hypophosphatemia is associated with impaired glucose metabolism in both hyperglycemic and euglycemic patients.

1. CARDIOVASCULAR AND SKELETAL MUSCLE MANIFESTATIONS

Hematological abnormalities of hypophosphatemia are a major manifestation of this syndrome. 16~ In addition to defects in affinity of oxyhemoglobin leading to generalized tissue hypoxia, there may be increased hemolysis. 163'164 Quantitative and functional defects have also been described in platelets and leukocytes. 165 These defects lead to diminished platelet aggregation as well as abnormalities in chemotaxis and phagocytosis of white blood cells. The latter may contribute to the increased risk of gram-negative sepsis reported in hypophosphatemic patients. 166 This is of particular concern in immunosuppressed patients receiving phosphate-poor alimentation through a central venous line.

Severe cardiomyopathy with decreased cardiac output has been described in patients and animals with severe hypophosphatemia. ~53'~54Studies revealed that the resting muscle membrane potential fell, sodium chloride and water content of the tissue increased, and potassium content decreased in severe hypophosphatemia. 155These values returned to normal after phosphate was administered. Skeletal muscle weakness and electromyographic abnormalities are associated with chronic hypophosphatemia and phosphate depletion. Dogs fed low-phosphate diets for several months developed changes in muscle, rhabdomyolysis, and characteristic increases in their levels of creatine phosphokinase (CPK) and aldolase in blood. 156 The syndrome of rhabdomyolysis has been observed in alcoholic patients with hypophosphatemia. 157 Knochel et al. 156 showed that myopathy associated with phosphate depletion in dogs did lead to changes in cell water content, sodium concentration, and transmembrane potential difference. Kretz et al. 158 examined the possibility that changes in calcium transport in the sarcoplasmic reticulum of muscle were responsible for the clinical myopathy seen in acute phosphate depletion. Despite significant hypophosphatemia and a reduction in muscle phosphorus concentration they found no significant changes in the rate of calcium uptake of calcium-concentrating ability in vesicles prepared from muscle sarcoplasmic reticulum of phosphate-depleted rats. Thus the role of altered transcellular calcium movements in phosphate-depleted tissues is yet to be completely resolved. 2. EFFECTS ON CARBOHYDRATE METABOLISM Hyperinsulinemia and abnormal glucose metabolism suggesting insulin resistance have been described in

3. HEMATOLOGICAL MANIFESTATIONS

4. NEUROLOGICAL MANIFESTATIONS

Manifestations at the level of the central nervous system resulting in generalized anorexia and malaise or more severe disturbances such as ataxia, seizures, and coma have been described in hypophosphatemia. 167-169 Neuromuscular abnormalities include paresthesias and weakness, the result of both myopathic changes and diminished nerve conduction. ~7~ 5. SKELETAL ABNORMALITIES

The skeletal abnormalities associated with hypophosphatemia, particularly in vitamin D-resistant rickets, may be quite marked. In addition, bony abnormalities, including osteomalacia and pathological fractures, have been described in antacid-induced phosphate depletion 171'172 as well as in hypophosphatemic patients undergoing hemodialysis who did not receive phosphatebinding gels. 173 A rheumatic syndrome resembling ankylosing spondylitis also has been reported in hypophosphatemic patients. 174

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Disorders of Phosphate Homeostasis

6. GASTROINTESTINAL DISTURBANCES These manifestations include anorexia, nausea, and vomiting. 168 It has been speculated that hypophosphatemia in the alcoholic patient may further impair hepatic function through hypoxic insult. 7. RENAL MANIFESTATIONS

There is a decreased phosphorus excretion and decreased tubular reabsorption of calcium, magnesium, bicarbonate, and glucose. 175-18~ The renal conservation of phosphorus occurs early in the syndrome and is the result of a primary increase in the tubular reabsorption of the anion as well as a decrease in GFR and consequently in the filtered load of phosphorus. 18~ This mechanism results in complete renal conservation of phosphorus, with net losses representing only a small fraction of total body phosphorus stores. 168 The increase in phosphorus reabsorption seen with phosphorus depletion is independent of several hormones known to influence phosphorus transport under other circumstances, including PTH, vitamin D, calcitonin, and thyroxine. 182The possibility that serum phosphorus concentration p e r s e (or intracellular phosphorus) may in some manner regulate its absorption along the nephron seems plausible. Hypercalciuria of enough magnitude to produce negative calcium balance is seen commonly in hypophosphatemic patients. Several factors contribute to this increase in calcium excretion including increased calcium mobilization from bone, enhanced gastrointestinal calcium absorption, and inhibition of renal tubular calcium reabsorption. 175'18~These effects appear to be independent of PTH activity and may be the result of a direct effect of phosphate on these transport processes. 8. ACID-BASE DISTURBANCES

Renal bicarbonate wasting, diminished titratable acid excretion, and decreased ammoniagenesis have been reported in hypophosphatemia. T M However, these defects are counterbalanced to some extent by the mobilization of alkali from bone. Thus, steady-state pH may be near normal at the expense of skeletal buffers. 177

C. Differential D i a g n o s i s of H y p o p h o s p h a t e m i a In general, the cause of hypophosphatemia can be determined from the medical history or from the clinical setting in which it occurs. When the cause is in doubt, measurement of the urinary phosphorus excretion level may be of help. If the urinary phosphorus concentration is less than 4 mg/dl when the serum phosphorus level is less than 2 mg/dl, then renal losses may be excluded. TM Of the three major extrarenal causes including dimin-

ished phosphorus intake, increased extrarenal losses (gastrointestinal tract), and translocation into the intracellular space, the last is the most common, especially in the hospitalized patient. 137'185When urine phosphorus excretion is high, the differential diagnosis includes hyperparathyroidism, a primary renal tubular abnormality, or vitamin D-dependent or -resistant renal tickets. Measurements of serum calcium, PTH, and vitamin D and its metabolites as well as urinary excretion of other solutes (glucose, amino acids, bicarbonate) will usually elucidate the underlying disturbance that is responsible for the hypophosphatemia.

D. T r e a t m e n t of H y p o p h o s p h a t e m i a There are several general principles that apply to the treatment of patients with hypophosphatemia. As with any predominantly intracellular ion (e.g., potassium), the state of total body phosphorus stores, as well as the magnitude of phosphorus losses, cannot be readily assessed by measurement of the concentrations in serum. In fact, under conditions in which a rapid shift of phosphorus has resulted as a consequence of glucose infusion or hyperalimentation, total body stores of phosphorus may be normal, although with diminished intake and renal losses there may be severe phosphorus depletion. Furthermore, the volume of distribution of phosphorus may vary widely, reflecting in part the intensity and duration of the underlying cause. 186 In clinical situations in which hypophosphatemia is to be expected (i.e., glucose infusion or hyperalimentation in the alcoholic or nutritionally compromised patient during treatment of diabetic ketoacidosis), careful monitoring of the concentration of serum phosphorus is crucial. In these situations, addition of phosphorus supplementation to prevent the development of severe hypophosphatemia may prove to be very helpful. Certainly, other contributing causes of hypophosphatemia in this setting should be identified and treated. This is especially true of the use of phosphate-binding antacids (aluminum and magnesium hydroxide) for peptic ulcer disease, which may be replaced by aluminum phosphate antacids (Phosphagel) or cimetidine (Tagamet). It is now generally recommended that hyperalimentation solutions contain a phosphorus concentration of 12 to 15 mM (37 to 46.5 mg/dl) to provide an appropriate amount of phosphorus in the patient in whom renal impairment is absent. 186 Phosphorus supplementation during glucose infusion or during the treatment of ketoacidosis is usually withheld until the serum phosphorus levels decrease below 1 mg/dl. Phosphorus may be given orally to these patients and others with mild asymptomatic hypophosphatemia in the form of skim milk, which contains 0.9

226 mg/ml, Nutraphos (3.3 mg/ml), or phosphorus soda (129 mg/ml). However, intestinal absorption is quite variable, and diarrhea often complicates the oral administration of phosphate-containing compounds. For these reasons, parenteral administration is usually recommended in the hospitalized patient. If oral therapy is permissible, Fleet Phospho-Soda may be given at a dose of 60 mM/day in three doses (21 mM/5 ml or 643 mg/5 ml). A convenient method is to provide the phosphorus together with potassium replacement in these patients. Addition of 5 ml of potassium phosphate (K phosphate) into 1 liter of intravenous fluid provides 22 mEq of potassium and 15 mM (466 mg) of phosphorus. 186 However, since potassium losses may greatly exceed the phosphorus deficit, the repletion of potassium should not be totally linked to phosphorus therapy. In patients with severe phosphate depletion it is difficult to determine the magnitude of the total deficit of phosphorus and to calculate a precise initial dose. It is usually prudent to proceed with caution and repair the deficit slowly. The most frequently recommended regimen is 0.08 mM/kg body weight (2.5 mg/kg body weight) given over 6 hours for severe but uncomplicated hypophosphatemia and 0.016 mM/kg body weight (5 mg/kg body weight) in symptomatic patients. 186 Parenteral administration should be discontinued when the serum phosphorus concentration is greater than 2 mg/dl. Calcium administration may be needed during phosphate repletion to prevent severe hypocalcemia. Calcium must not be added to bicarbonate or phosphatecontaining solutions because of the potential precipitation of calcium salts. Intravenous infusion of calcium gluconate or calcium chloride may be given until tetany abates. In addition to hypocalcemia, metastatic calcification, hypotension, hyperkalemia, and hypernatremia are potential side effects of parenteral infusion of phosphorus. These problems can be prevented by judicious use of therapy and frequent monitoring of serum electrolyte concentrations.

III. HYPERPHOSPHATEMIA Hyperphosphatemia is said to occur when serum phosphorus concentration exceeds 5 mg/dl in adults. It should be remembered that in children and adolescents serum levels of phosphorus of up to 6 mg/dl may be physiological. The most frequent cause of hyperphosphatemia is decreased excretion of phosphorus in the urine as a consequence of a fall in GFR. However, increases in serum phosphorus can also occur as a result of increased entry into the extracellular fluid due to excessive intake of phosphorus or increased release of

KEITH HRUSKA AND ANANDARUP GUFTA

phosphorus from tissue breakdown. The major causes of hyperphosphatemia are listed in Table 7 - 4 .

A. C a u s e s of H y p e r p h o s p h a t e m i a 1. DECREASED EXCRETION OF PHOSPHORUS IN URINE

a. D e c r e a s e d R e n a l Fu n ct i o n In progressive renal failure, phosphorus homeostasis is maintained by a progressive increase in phosphorus excretion per nephr o n . 187'188 As a consequence of this increased phosphorus excretion, it is unusual to see marked hyperphosphatemia until GFR values fall below 25 ml/min. 189 Under physiological conditions with a GFR of 120 ml/min, a fractional excretion of 5% to 15% of the filtered load of phosphorus is adequate to maintain phosphorus homeostasis. However, as renal insufficiency progresses and the number of nephrons decreases, fractional excretion of phosphorus may increase to as high as 60% to 80% of the filtered load. This progressive phosphaturia per nephron, as renal disease progresses, serves to maintain the concentration of phosphorus within normal limits in plasma (see Fig. 7-3). However, when the number of nephrons is greatly diminished, if the dietary intake of phosphorus remains constant, phosphorus homeostasis can no longer be maintained and hyperphosphatemia develops. This usually occurs when the GFR falls below 25 ml/min. As hyperphosphatemia develops, the filtered load of phosphorus per nephron increases, phosphorus excretion rises, and phosphorus balance is reestablished but at higher concentrations of serum phosphorus. Hyperphosphatemia is a usual finding in patients with faradvanced renal insufficiency unless phosphorus intake in the diet has been decreased through dietary manipulations or the patient is receiving phosphate binders such as calcium carbonate or aluminum-containing salts that will decrease the absorption of phosphate from the gastrointestinal tract. 19~In patients with acute renal failure, hyperphosphatemia is a usual finding. TM The degree of hyperphosphatemia in patients with acute renal failure varies considerably. It is quite marked in patients with renal insufficiency secondary to severe trauma or known traumatic rhabdomyolysis, as frequently occurs in patients ingesting large amounts of alcohol or in heroin addicts. 192 The degree of hyperphosphatemia depends on the amount of phosphorus released from damaged tissue, since phosphorus intake and decreased GFR, to less than 2 ml/min, is constant across different forms of oliguric acute renal failure. In most patients with acute renal failure, hyperphosphatemia is transitory, and serum phosphorus values return toward normal as renal function improves. However, in some of these patients infection

CHAPTER7 Disorders of Phosphate Homeostasis or tissue destruction due to many causes may maintain relatively high serum phosphorus values even during the recovery phase of renal function. 2. DECREASED OR ABSENT LEVELS OF CIRCULATING PTH

Hypoparathyroidism is characterized by low or absent levels of PTH, low levels of serum calcium, and hyperphosphatemia. 193 The most common cause of hypoparathyroidism results from injury to the parathyroid glands or their blood supply during thyroid, parathyroid, or radical neck surgery. Idiopathic hypoparathyroidism is a rare disease. Since PTH normally inhibits the renal reabsorption of phosphorus, its absence leads to an elevation in the Tm for phosphorus and decreased excretion of the anion in the urine. Balance is reestablished when the serum phosphorus rises to concentrations of 6 to 8 mg/dl. At this concentration of serum phosphorus the filtered load of phosphate is increased exceeding the Tm for phosphorus reabsorption, and a new steady state is reestablished. Patients with hypoparathyroidism are easily diagnosed by the findings of a low level of serum calcium, hyperphosphatemia, and undetectable levels of circulating immunoreactive PTH. After several years of hypoparathyroidism other signs may become manifest such as cataracts and bilateral symmetrical calcification of the basal ganglia on x-rays of the skull. The most striking symptoms in patients presenting with hypoparathyroidism are related to an increase in neuromuscular excitability resulting from a decrease in the levels of ionized calcium in serum. Some patients may not develop hypocalcemia and severe tetany, but increased neuromuscular excitability may be demonstrated by contraction of facial muscles in response to stimulus over the facial nerve (Chvostek's sign) or by carpal spasm (Trousseau's sign) occurring 2 or 3 minutes after inflating a blood pressure cuff around the arm above systolic blood pressure. In other patients psychiatric disturbances, paresthesias, numbness, muscle cramps, and dysphagia may be presenting symptoms. 3. PSEUDOHYPOPARATHYROIDISM

This is a relatively rare condition characterized by end-organ resistance to the action of PTH. TM Characteristically, the kidney and skeleton do not respond appropriately to the action of PTH. Some patients with pseudohypoparathyroidism may have specific somatic characteristics such as short stature, round face, short metacarpal bones and phalanges, and some degree of mental retardation. Biochemically, these patients, like those with hypoparathyroidism, have low concentrations of serum calcium and hyperphosphatemia. However, there are two important points in the differential diagnosis. In the majority of patients with pseudohypopara-

227 thyroidism the circulating levels of immunoreactive PTH are elevated, whereas in patients with true hypoparathyroidism PTH levels are low or absent. Second, patients with pseudohypoparathyroidism do not respond to the administration of exogenous PTH with phosphaturia. Patients with true hypoparathyroidism demonstrate a heightened phosphaturic response to administration of exogenous PTH. Two major types of pseudohypoparathyroidism have been described. In type I, patients fail to increase the excretion of cAMP or phosphate in the urine in response to the administration of exogenous PTH. This abnormal response seems to be related, at least in some of these patients, to a defect in the GTPbinding protein, Gs, protein of the adenylate cyclase complex. 195'196 In other patients there is an increase in cAMP in response to the administration of exogenous PTH but no phosphaturic response. This condition has been termed "pseudohypoparathyroidism type II." 197 4. ABNORMAL CIRCULATING PTH

This syndrome is characterized by hyperphosphatemia, hypocalcemia, chronic tetany, and cataracts. These manifestations, as described above, are those observed in patients with hypoparathyroidism, but these patients have normal or high serum levels of PTH. However, in contrast to patients with pseudohypoparathyroidism, they do respond to the exogenous administration of PTH with an increase in the excretion of cAMP and phosphaturia. It has been postulated that the defect in these patients relates to an abnormal form of endogenous PTH that is devoid of physiological effects. 198 However, this postulate has not been substantiated by characterization and analysis of the circulating PTH in these patients. 5. ACROMEGALY

Growth hormone decreases the urinary excretion of phosphorus and increases the Tm for phosphorus. 199 Hypersecretion of growth hormone may lead to development of gigantism if the increased secretion occurs prior to the closure of the epiphysis or to acromegaly if the excessive secretion occurs after puberty. Hyperphosphatemia has been described in patients with acromegaly. It is known that serum phosphorus concentrations are higher in children (5 to 8 mg/dl) than in adults. This may be related in part to increased levels of circulating growth hormone in children. 6. TUMORAL CALCINOSIS

Although the etiology of this entity is not completely characterized, its pathogenesis is probably related to a primary increase in phosphorus reabsorption by the kidhey. 2~176 This condition, which is seen more frequently in young blacks, is characterized by hyperphosphatemia,

228 ectopic calcification around large joints, normal levels of circulating immunoreactive PTH, and a normal response to administration of exogenous PTH. 2~176 The extensive calcification of soft tissues observed in patients with this condition is most likely due to an elevated phosphoruscalcium product in blood. Despite the development of hyperphosphatemia, patients with tumoral calcinosis do not develop secondary hyperparathyroidism. This may be due to the fact that circulating levels of 1,25(OH)2D3 remain normal in these patients despite hyperphosphatemia. These normal levels of 1,25(OH)2D3 maintain a normal gastrointestinal absorption of calcium. This, combined with the decreased urinary calcium observed in these patients, may serve to maintain normal serum calcium values and prevent the development of secondary hyperparathyroidism. 7. ADMINISTRATION OF BISPHOSPHONATES Administration of bisphosphonates, which are used in the treatment of Paget's disease, may result in the development of hyperphosphatemia and osteoporosis. 2~ The mechanisms by which bisphosphonates increase serum phosphorus are not completely clear at present but may involve an alteration in phosphate distribution between different cellular compartments and a decrease in renal phosphorus excretion. It appears that both the levels of circulating PTH and the urinary excretion of cAMP after administration of exogenous PTH are normal in patients receiving bisphosphonates. 8. REDISTRIBUTION OF PHOSPHORUS BETWEEN INTRACELLULAR AND EXTRACELLULAR POOLS (TUMOR LYSIS SYNDROME)

Various syndromes of tissue breakdown may result in the development of hyperphosphatemia and subsequent hypocalcemia. Hyperphosphatemia has been described in patients with several types of lymphomas. Patients receiving treatment for lymphoblastic leukemia may develop hyperphosphatemia with a concomitant decrease in serum calcium. 2~ The phosphorus load originates primarily from the destruction of lymphoblasts, which have about four times the concentration of organic and inorganic phosphorus present in mature lymphocytes. Similar findings have been described during treatment of Burkitt's lymphoma. Cohen et al. 2~ reviewed the acute tumor lysis syndrome associated with the treatment of Burkitt's lymphoma. In 37 patients with American Burkitt's lymphoma, azotemia occurred in 14 patients and preceded chemotherapy in 8. Pretreatment azotemia was associated with elevated levels of lactate dehydrogenase (LDH) and uric acid and sometimes extrinsic ureteral obstruction by the tumor. Following chemotherapy, major metabolic complications related to tumor lysis were associated with large tumors and high LDH levels

KEITH HRUSKA AND ANANDARUPGUPTA

and were manifest by hyperkalemia, hyperphosphatemia, and hyperuricemia. Elevated phosphorus levels were seen in 31% of nonazotemic patients and in all azotemic patients. Hemodialysis was required in three patients for control of azotemia, hyperuricemia, hyperphosphatemia, and/or hyperkalemia. Tsokos et al. 2~ studied the renal metabolic complications of other undifferentiated lymphomas and lymphoblastic lymphomas. These workers found that serum LDH prior to chemotherapy correlated well with the stage of disease and predicted the serum levels of creatinine, uric acid, and phosphorus in the posttreatment period. Patients with LDH values greater than 2000 IU were likely to develop severe hyperphosphatemia. When azotemia developed in the postchemotherapy period it was attributed to hyperuricemia and/or hyperphosphatemia. Some of these patients had elevated serum phosphorus levels in the range of 20 to 30 mg/dl, which may contribute to the development of renal insufficiency due to calcium deposition in the kidney and other tissues. Thus there is a great risk of hyperphosphatemia in patients undergoing chemotherapy for rapidly growing malignant lymphomas. The best method of prevention of this complication as well as the best therapeutic intervention has not been well defined. Initially it appears useful to attempt to increase the renal excretion of phosphate during the induction of remission by chemotherapy in these patients. This requires infusion of large amounts of saline and possibly bicarbonate, which has been shown to increase renal phosphorus excretion above and beyond the mere effects of volume expansion. Acetazolamide, a potent phosphaturic agent, might also be beneficial in these individuals. The general recommendation of hemodialysis as the prime therapeutic modality for hyperphosphatemia and acute renal insufficiency resulting from tumor lysis is not based on experimental data. Although hemodialysis no doubt rapidly lowers serum phosphorus levels, the mass of phosphorus continually presented to the extracellular space from ongoing tissue breakdown is not continuously treated by this modality. Thus it is possible that combined hemodialysis and peritoneal dialysis, or even peritoneal dialysis alone, might be as, if not more, beneficial and safer in individuals undergoing tumor lysis syndrome. 9. INCREASED CATABOLISM

Conditions characterized by increased protein breakdown (severe tissue muscle damage, severe infections) may sometimes be accompanied by hyperphosphatemia. Although the hyperphosphatemia may be related simply to translocation of phosphorus into the extracellular space, other factors seem to play a role. Hyperphosphatemia has been described in patients with ketoacidosis prior to treatment. After administration of intravenous

CHAPTER7 Disorders of Phosphate Homeostasis fluids and insulin therapy the entrance of glucose into the cells is usually followed by movement of phosphorus back into the intracellular space, and some patients now may develop hypophosphatemia. Thus the combination of dehydration, acidosis, and tissue breakdown in different catabolic states may lead to hyperphosphatemia. 10. RESPIRATORY ACIDOSIS

Acute respiratory acidosis may lead to a marked increase in serum phosphorus. 2~ By contrast, chronic respiratory acidosis is usually not manifested by sustained elevated levels of serum phosphorous. Acute rises in Pc02 in experimental animals have been shown to lead to increased serum phosphorus levels. The modest degree of hyperphosphatemia seen in chronic respiratory acidosis is probably related to renal compensation and increased phosphorus excretion via the kidney to maintain phosphorus homeostasis. 11. ADMINISTRATION OF PHOSPHATE SALTS OR VITAMIN D OR ITS METABOLITES Administration of vitamin D3 or its metabolites, particularly 1,25(OH)2D3, may result in increases in serum phosphorus, particularly in uremic patients. These compounds very likely may result in hyperphosphatemia in uremic individuals by increasing phosphorus absorption from the gut and perhaps by potentiating the effect of PTH on the skeleton with increased release of phosphorus from bone. Decreased renal function limits the compensatory mechanism of the kidney to excrete the increased load of phosphate entering the extracellular space. In addition to elevating serum phosphorus, vitamin D metabolites may result in hypercalcemia. An increase in the phosphorus-calcium product may result in tissue deposition of calcium, particularly in the kidney, leading to further renal functional deterioration. 12. INGESTION AND/OR ADMINISTRATION OF SALTS CONTAINING PHOSPHATE

Hyperphosphatemia has been observed in adults ingesting laxative-containing phosphate salts or after administration of enemas containing large amounts of phosphate. 2~176 Intravenous phosphate administration has been used in the treatment of hypercalcemia of malignancy. The administration of 1 to 2 g of phosphate intravenously decreases the concentration of serum calcium. Unfortunately, the severe hyperphosphatemia induced by administration of large amounts of phosphorus intravenously may lead to calcium precipitation in important organs such as the heart and kidney, and several deaths have been reported as a consequence of this form of therapy. Hyperphosphatemia may develop in newborn infants fed cow's milk, which is higher in phosphorus

229 content than human milk. This may be an important factor in the genesis of neonatal tetany.

B. Clinical Manifestations

of Hyperphosphatemia Most of the clinical effects of hyperphosphatemia are related to secondary changes of calcium metabolism. Hyperphosphatemia produces hypocalcemia by several mechanisms (Fig. 7-4), including decreased production of 1,25(OH)2D3,115 precipitation of calcium, 21~ and decreased absorption of calcium from the gastrointestinal tract, presumably due to a direct effect of phosphorus on calcium absorption. TM In addition to the manifestations by hypocalcemia, which are described elsewhere in this chapter, ectopic calcification is one of the important manifestations of hyperphosphatemia. The association of hyperphosphatemia and ectopic calcification has been observed in several clinical settings including patients with chronic renal failure, hypoparathyroidism, and tumoral calcinosis. It appears that when the calciumphosphorus product exceeds 70, the likelihood for calcium precipitation is greatly increased. In addition to the calcium-phosphorus product, local tissue factors may play an important role in calcium deposition. For example, regional changes in pH (local alkalosis) may favor calcification in tissue such as cornea and lungs. In patients with severe calcification (calciphylaxis), it appears that high levels of circulating PTH may also aggravate this condition. Hyperphosphatemia plays a key role in the development of secondary hyperparathyroidism in patients with renal insufficiency. It has been observed that when phosphate ingestion is decreased and hyperphosphatemia is prevented in experimental animals with induced renal insufficiency, hyperparathyroidism can be prevented. 212 The mechanisms presumably relate to maintenance of serum calcium levels with prevention of hyperphosphatemia and, at the same time, continued synthesis of 1,25(OH)2D3, the circulating levels of which may directly influence the secretion of PTH. 213'214 In patients on chronic hemodialysis, the degree of hyperparathyroidism correlates well with the concentration of serum phosphorus. Patients who do not adhere to their therapeutic prescriptions requiting ingestion of phosphate binders seem to develop more severe and persistent hyperphosphatemia with marked secondary hyperparathyroidism and bone disease than do patients who adhere carefully to dietary and therapeutic prescriptions. Vascular calcification has been observed in some patients with chronic renal insufficiency and severe calcification, hyperphosphatemia, and hyperparathyroidism leading to necrosis and gangrene of extremities. Slit lamp examination may show ocular calcification, and some patients

230

KEITH HRUSKA AND ANANDARUP GUPTA

may develop acute conjunctivitis, the so-called red eye syndrome of uremia. Precipitation of calcium in the skin may be in part responsible for pruritus, a symptom that is usually seen in patients with far-advanced uremia. It has been reported that parathyroidectomy in such patients may alleviate the symptoms. From the therapeutic point of view, the most efficacious way of controlling hyperphosphatemia is through the use of phosphate binders that decrease the absorption of phosphorus from the gastrointestinal tract. In patients with adequate renal function, expansion of the ECF with saline will greatly increase phosphorus excretion in the urine and contribute to correction of the hyperphosphatemia.

C. Treatment of Hyperphosphatemia Decreased absorption of phosphate from the gastrointestinal tract is a cornerstone of treatment of hyperphosphatemia. Phosphate absorption from the gastrointestinal tract can be markedly decreased either by decreasing the amount of phosphorus in the diet or by administering phosphate-binding agents capable of decreasing absorption of phosphorus. Since protein requirements limit the amount of phosphorus restriction that can be achieved through dietary manipulation, from a practical point of view administration of agents capable of decreasing phosphorus absorption from the gastrointestinal tract is the mainstay of treatment. Administration of calcium salts has replaced aluminum salts as the traditional treatment to control hyperphosphatemia. Most of these preparations require the administration of two to four tablets or capsules three or four times daily. If the patient develops constipation, one of the complications of such medications, magnesium salts may be incorporated into these preparations. However, if the patient has hyperphosphatemia secondary to severe renal insufficiency, magnesium should not be given because of the likelihood of producing severe hypermagnesemia, which may lead to magnesium intoxication, muscle paralysis, and death. The elucidation of aluminum toxicity, which results from prolonged administration of aluminum-containing salts as phosphate binders in patients with chronic renal insufficiency, has led to diminished use of these agents or their elimination. 215'216 Several studies indicated that calcium carbonate 217-219 is an effective agent for control of hyperphosphatemia in chronic renal failure. Although decreased gastrointestinal absorption of phosphorus is an effective way to control hyperphosphatemia in patients with renal insufficiency, excretion of phosphorus through the kidney is also an important mechanism. Thus expansion of the extracellular fluid volume may markedly increase phosphorus excretion by the kidney. This result

is presumably related both to direct effects of volume expansion on the kidney, which decreases salt and water reabsorption and hence phosphorus reabsorption, and to increased PTH release, particularly as a consequence of decreased ionized calcium during volume expansion. In patients with marked renal insufficiency or with marked degrees of hyperphosphatemia due to tumor lysis or chemotherapy, peritoneal dialysis or hemodialysis may be used to remove large quantities of phosphorus from the extracellular space. Redistribution of phosphorus from the intracellular to the extracellular space can sometimes be rapidly corrected by the administration of glucose and insulin. In general, mild degrees of hyperphosphatemia can be tolerated, particularly if calcium levels are not markedly elevated. The goal in patients with chronic renal insufficiency is to keep phosphorus levels below 4.5 mg/dl to avoid falls in serum ionized calcium and marked development of severe hyperparathyroidism. Although PTH increases the excretion of phosphorus, administration of PTH is not a practical or effective way to treat patients with chronic hyperphosphatemia. It should be remembered that in some patients prolonged administration of phosphorus-binding gels may result in development of phosphate depletion and hence hypophosphatemia.

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In Bouillon R, Norman AW, Thomasset M (eds): Vitamin D, A Pluripotent Steroid Hormone: Structural Studies, Molecular Endocrinology and Clinical Applications. New York, de Gruyter, 1994, p 704. Glorieux FH, Made PJ, Pettifor JM, Delvin EE: Bone response to phosphate salts, ergocalciferol, and calcitriol in hypophosphatemic vitamin D-resistant rickets. N Engl J Med 303:10231031, 1980. Verge CF, Lam A, Simpson JM, et al: Effect of therapy in X-linked hypophosphatemic rickets. N Engl J Med 325:18751877, 1991. Friedman NE, Lobaugh B, Drezner MK: Effects of calcitriol and phosphorus therapy on the growth of patients with X-linked hypophosphatemia. J Clin Endocrinol Metab 76:839-844, 1993. Weinstein LS, Shenker A, Gejman PV, et al: Activation mutations of the stimulatory G protein in the McCune-Albright syndrome. N Engl J Med 325:1688-1695, 1991. Gray RW, Wilz DR, Caldas AE, Lemann J Jr: The importance of phosphate in regulating plasma 1,25(OH)2 vitamin D levels in humans: Studies in healthy subjects, in calcium-stone formers and in patients with primary hyperparathyroidism. J Clin Endocrinol Metab 45:299-306, 1977. Frame B, Parfitt AM: Osteomalacia current concepts. Ann Intern Med 89:966-982, 1978. Fraser D, Kooh SW, Kind HP, et al: Pathogenesis of hereditary vitamin-D-dependent tickets. An inborn error of vitamin D metabolism involving defective conversion of 25-hydroxyvitamin D to l alpha,25-dihydroxyvitamin D. N Engl J Med 289:817822, 1973. Liberman UA, Eil C, Marx SJ: Resistance of 1,25 dihydroxyvitamin D. Associated with heterogeneous defects in cultured skin fibroblasts. J Clin Invest 71:192- 200, 1983. Feldman D, Chen T, Cone C, et al: Vitamin D resistant tickets with alopecia: Cultured skin fibroblasts exhibit defective cytoplasmic receptors and unresponsiveness to 1,25(OH)2D3. J Clin Endocrinol Metab 55:1020-1022, 1982. Chen TL, Hirst MA, Cone CM, et al: 1,25-dihydroxyvitamin D resistance, tickets and alopecia: Analysis of receptors and bioresponse in cultured fibroblasts from patients and parents. J Clin Endocrinol Metab 59:383- 388, 1984. Malloy PJ, Hochberg Z, Pike JW, Feldman D: Abnormal binding of vitamin D receptors to deoxyribonucleic acid in a kindred with vitamin D-dependent tickets, type II. J Clin Endocrinol Metab 68:263-269, 1989. Hochberg Z, Weisman Y: Calcitriol-resistant tickets due to vitamin D receptor defects. Trends Endocrinol Metab 6:216-220, 1995. Hewison M, Rut AR, Kristjansson K, et al: Tissue resistance to 1,25-dihydroxyvitamin D without a mutation of the vitamin D receptor gene. Clin Endocrinol 39:663-670, 1993. Refetoff S, Weiss RE, Usala SJ: The syndromes of resistance to thyroid hormone. Endocr Rev 14:348-399, 1993. McPhaul MJ, Marcelli M, Zoppi S, et al: Genetic basis of endocrine disease. 4. The spectrum of mutations in the androgen receptor gene that causes androgen resistance. J Clin Endocrinol Metab 76:17-23, 1993. Smith EP, Boyd J, Frank GR, et al: Estrogen resistance caused by a mutation in the estrogen receptor gene in a man. N Engl J Med 331:1088-1089, 1994. Parker MS, Klein I, Haussler MR, Mintz DH: Tumor-induced osteomalacia: Evidence of a surgically correctable alteration in vitamin D metabolism. JAMA 245:492-493, 1981. Rowe PSN, Ong ACM, Cockerill FJ, et al: Candidate 56 and 58 kDa protein(s) responsible for mediating the renal defects in

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KEITH HRUSKA AND ANANDARUP GUPTA oncogenic hypophosphatemic osteomalacia. Bone 18:159-169, 1996. Sweet RA, Males JL, Hamstra AJ, DeLuca HF: Vitamin D metabolite-levels in oncogenic osteomalacia. Ann Intern Med 93: 279, 1980. Weidner N: Review and update: Oncogenic osteomalaciarickets. Ultrastruct Pathol 15:317-333, 1991. Drezner MK, Feinglos MN: Osteomalacia due to l alpha,25dihydroxycholecalciferol deficiency. Association with a giant cell tumor of bone. J Clin Invest 60:1046-1053, 1977. Ribovich ML, DeLuca HF: Effect of dietary calcium and phosphorus on intestinal calcium absorption and vitamin D metabolism. Arch Biochem Biophys 188:145-156, 1978. Miyauchi A, Fukase M, Tsutsumi M, Fujita T: Hemangiopericytoma-induced osteomalacia: Tumor transplantation in nude mice causes hypophosphatemia and tumor extracts inhibit renal 25-hydroxyvitamin D 1-hydroxylase activity. J Clin Endocrinol Metab 67:46-53, 1988. Econs MJ, Drezner MK: Tumor-induced osteomalacia: Unveiling a new hormone. N Engl J Med 330:1645-1649, 1994. Klahr S, Davis TA: Changes in renal function with chronic protein-calorie malnutrition. In Mitch WE, Klahr S (eds): Nutrition and the Kidney. Boston, Little, Brown & Co, 1988, pp 59-79. Shields HM: Rapid fall of serum phosphorus secondary to antacid therapy. Gastroenterology 75:1137-1141, 1978. Juan D, Elrazak MA: Hypophosphatemia in hospitalized patients. JAMA 242:163-164, 1979. Larsson L, Rebel K, Sorbo B: Severe hypophosphatemia--a hospital survey. Acta Med Scand 214:221 - 223, 1983. Betro MG, Pain RW: Hypophosphatemia and hyperphosphatemia in a hospital population. Br Med J 1:273-276, 1972. Ryback RS, Eckardt MJ, Pautler CP: Clinical relationships between serum phosphorus and other blood chemistry values in alcoholics. Arch Intern Med 140:673-677, 1980. Seldin DW, Tarail R: The metabolism of glucose and electrolytes in diabetic acidosis. J Clin Invest 29:552-565, 1950. Mostellar ME, Tuttle EP Jr: Effects of alkalosis on plasma concentration and urinary excretion of urinary phosphate in man. J Clin Invest 43:138-149, 1964. Aderka D, Shoenfeld Y, Santo M, et al: Life-threatening hypophosphatemia in a patient with acute myelogenous leukemia. Acta Haematol 64:117-119, 1980. Matzner Y, Prococimer M, Polliack A, et al: Hypophosphatemia in a patient with lymphoma in leukemic phase. Arch Intern Med 141:805-806, 1981. Zamkoff KW, Kirshner JJ: Marked hypophosphatemia associated with acute myelomonocytic leukemia: Indirect evidence of phosphorus uptake by leukemic cells. Arch Intern Med 140: 1523-1524, 1980. Chesney PJ, Davis JP, Purdy WK, et al: Clinical manifestations of toxic shock syndrome. JAMA 246:741-748, 1981. Lennquist S, Lindell B, Nordstrom H, Sjoberg HE: Hypophosphatemia in severe burns: A prospective study. Acta Chir Scand 145:1-6, 1979. Levy LA: Severe hypophosphatemia as a complication of the treatment of hypothermia. Arch Intern Med 140:128-129, 1980. Silvis SE, DiBartolomeo AG, Aaker HM: Hypophosphatemia and neurologic changes secondary to oral caloric intake: A variant of hyperalimentation syndrome. Am J Gastroenterol 73: 215-222, 1980. Weinsier RL, Krumdiek CL: Death resulting from overzealous total parenteral nutrition: The refeeding syndrome revisited. Am J Clin Nutr 34:393-399, 1981.

151. Kono N, Kuwajima M, Tarui S: Alteration of glycolytic intermediary metabolism in erythrocytes during diabetic ketoacidosis and its recovery phase. Diabetes 30:346-353, 1981. 152. Ditzel J: Changes in red cell oxygen release capacity in diabetes mellitus. Fed Proc 38:2484-2488, 1979. 153. Darsee JR, Nutter DO: Reversible severe congestive cardiomyopathy in three cases of hypophosphatemia [retracted by Nutter DO, Glenn JF. In Ann Intem Med 99:275-276, 1983]. Ann Intern Med 89:867-870, 1978. 154. Zazzo JF, Troche G, Ruel P, Maintenant J: High incidence of hypophosphatemia in surgical intensive care patients: Efficacy of phosphorus therapy on myocardial function. Intensive Care Med 21:826-831, 1995. 155. Fuller TJ, Carter NW, Barcenas C, Knochel JP: Reversible changes of the muscle cell in experimental phosphorus deficiency. J Clin Invest 57:1019-1024, 1976. 156. Knochel JP, Barcenas C, Cotton JR, et al: Hypophosphatemia and rhabdomyolysis. J Clin Invest 62:1240-1246, 1978. 157. Knochel JP, Bilbrey GL, Fuller TJ: The muscle cell in chronic alcoholism: The possible role of phosphate depletion in alcoholic myopathy. Ann NY Acad Sci 252:274-286, 1975. 158. Kretz J, Sommer G, Boland R, et al: Lack of involvement of sarcoplasmic reticulum in myopathy of acute phosphorus depletion. Klin Wochenschr 58:833-837, 1980. 159. DeFronzo RA, Lang R: Hypophosphatemia and glucose intolerance: Evidence for tissue insensitivity to insulin. N Engl J Med 303:1259-1263, 1980. 160. Bellingham AJ, Detter JC, Lenfant C: The role of hemoglobin affinity for oxygen and red cell 2,3-diphosphoglycerate in the management of diabetic ketoacidosis. Trans Assoc Am Physicians 83:113-120, 1970. 161. Lichtman MA, Miller DR, Cohen J, Waterhouse C: Reduced red cell glycolysis, 2,3-diphosphoglycerate and adenosine triphosphate concentration and increased hemoglobin-oxygen affinity caused by hypophosphatemia. Ann Intern Med 74:562-568, 1971. 162. Travis SF, Sugarman HJ, Ruberg RL, et al: Alterations of redcell glycolytic intermediates and oxygen transport as a consequence of hypophosphatemia in patients receiving intravenous hyperalimentation. N Engl J Med 285:763-768, 1971. 163. Jacob HS, Amsden T: Acute hemolytic anemia and rigid red cells in hypophosphatemia. N Engl J Med 285:1446-1450, 1971. 164. Klock JC, Williams HE, Mentzer WC: Hemolytic anemia and somatic cell dysfunction in severe hypophosphatemia. Arch Intern Med 134:360-364, 1974. 165. Craddock PR, Yawata Y, Van Santen L, et al: Acquired phagocyte dysfunction: A complication of the hypophosphatemia of parenteral hyperalimentation. N Engl J Med 290:1403-1407, 1974. 166. Riedler GF, Scheitlin WA: Hypophosphatemia in septicemia: Higher incidence in gram-negative than in gram-positive infections. Br Med J 1:753-756, 1969. 167. Lotz M, Ney R, Bartter FC: Osteomalacia and debility resulting from phosphorus depletion. Trans Assoc Am Physicians 77: 281-295, 1964. 168. Lotz M, Zisman E, Bartter FC: Evidence for a phosphorusdepletion syndrome in man. N Engl J Med 278:409-415, 1968. 169. Prins JG, Schrijver H, Staghouwer JM: Hyperalimentation, hypophosphatemia and coma. Lancet 1:1253-1254, 1973. 170. Boelens PA, Norwood W, Kjellstrand C, Brown DM: Hypophosphatemia with muscle weakness due to antacids and hemodialysis. Am J Dis Child 120:350-353, 1970. 171. Baker LRI, Ackrill P, Cattell WR, et al: Iatrogenic osteomalacia and myopathy due to phosphate depletion. Br Med J 3:150152, 1974.

CHAPTER 7

Disorders of Phosphate Homeostasis

172. Cooke N, Teitelbaum S, Avioli LV: Antacid-induced osteomalacia and nephrolithiasis. Arch Intern Med 138:1007-1009, 1978. 173. Ahmed KY, Varghese Z, Willis MR, et al: Persistent hypophosphatemia and osteomalacia in dialysis patients not on oral phosphate-binders: Response to dihydrotachysterol therapy. Lancet 1:439-442, 1976. 174. Moser CR, Fessel WJ: Rheumatic manifestations of hypophosphatemia. Arch Intem Med 134:674-678, 1974. 175. Cobum JW, Massry SG: Changes in serum and urinary calcium during phosphate depletion: Studies on mechanisms. J Clin Invest 49:1073-1087, 1970. 176. Dominguez 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 25-OHvitamin D. J Clin Endocrinol Metab 43:1056-1068, 1976. 177. Emmett M, Goldfarb S, Agus ZS, Narins RG: The pathophysiology of acid-base changes in chronically phosphate-depleted rats: Bone-kidney interactions, J Clin Invest 59:291 - 298, 1977. 178. Gold LW, Massry SG, Arieff AI, Cobum JW: Renal bicarbonate wasting during phosphate depletion: A possible cause of altered acid-base homeostasis in hyperparathyroidism. J Clin Invest 52: 2556-2561, 1973. 179. Gold LW, Massry SG, Friedler RM: Effect of phosphate depletion on renal tubular reabsorption of glucose. J Lab Clin Med 89:554-559, 1977. 180. Goldfarb S, Westby GR, Goldberg M, Agus ZS: Renal tubular effects of chronic phosphate depletion. J Clin Invest 59:770779, 1977. 181. Muhlbauer RC, Bonjour JP, Fleisch H: Tubular localization to dietary phosphate in rats. Am J Physiol 234:E294, 1978. 182. Steele TH, Stromberg BA, Underwood JL, Larmore CA: Renal resistance to parathyroid hormone during phosphorus deprivation. J Clin Invest 58:1461-1464, 1976. 183. O'Donovan DJ, Lotspeich WD: Activation of kidney mitochondrial glutaminase by inorganic phosphate and organic acids. Nature 212:930-932, 1966. 184. Agus ZS, Goldfarb S, Wasserstein A: Disorders of calcium and phosphate balance. In Brenner BM, Rector FC Jr (eds): The Kidney. Philadelphia, WB Saunders Co, 1981, p 940. 185. Harris CA, Bauer PG, Chirito E, Dirks JH: Composition of mammalian glomerular filtrate. Am J Physiol 227:972-976, 1974. 186. Lentz RD, Brown DM, Kjellstrand CM: Treatment of severe hypophosphatemia. Ann Intern Med 89:941-944, 1978. 187. Slatopolsky E, Gradowska L, Kashemsant C, et al: The control of phosphate excretion in uremia. J Clin Invest 45:672-677, 1966. 188. Slatopolsky E, Robson AM, Elkan I, Bricker NS: Control of phosphate excretion in uremic man. J Clin Invest 47:18651874, 1968. 189. Goldman R, Bassett SH: Phosphorus excretion in renal failure. J Clin Invest 33:1623, 1954. 190. Rutherford E, Mercado A, Hruska K, et al: An evaluation of a new and effective phosphate binding agent. Trans Am Soc Artif Intem Organs 19:446-449, 1973. 191. Massry SG, Arieff AI, Cobum JW, et al: Divalent ion metabolism in patients with acute renal failure: Studies on the mechanism of hypocalcemia. Kidney Int 5:437-445, 1974. 192. Koffler A, Friedler RM, Massry SG: Acute renal failure due to nontraumatic rhabdomyolysis. Ann Intem Med 85:23-28, 1976. 193. Parfitt AM: The spectrum of hypoparathyroidism. J Clin Endocrinol Metab 34:152-158, 1972.

235 194. Albright F, Bumett CH, Smith PH, Parson W: Pseudohypoparathyroidism - - a n example of "Seabright-Bantam syndrome." Endocrinology 30:922-932, 1942. 195. Boume HR, Kaslow HR, Brickman AS, Farfel Z: Fibroblast defect in pseudohypoparathyroidism, type I: Reduced activity of receptor-cyclase coupling protein. J Clin Endocrinol Metab 53: 636-640, 1981. 196. Farfel Z, Brickman AS, Kaslow HR, et al: Defect of receptorcyclase coupling protein in pseudohypoparathyroidism. N Engl J Med 303:237-242, 1980. 197. Drezner M, Neelon FA, Lebovitz HE: Pseudohypoparathyroidism type II: A possible defect in the reception of the cyclic AMP signal. N Engl J Med 289:1056-1060, 1973. 198. Connors MH, Irias JJ, Golabi M: Hypo-hyperparathyroidism: Evidence for a defective parathyroid hormone. Pediatrics 60: 343-348, 1977. 199. Lambert PP, Corvilan J: Site of action of parathyroid hormone and role of growth hormone in phosphate excretion. In Williams PC (ed): Hormones and the Kidney (Memoirs of the Society of Endocrinology, No. 13). New York, Academic Press, 1963, p 139. 200. Mitnick PD, Goldbarb S, Slatopolsky E, et al: Calcium and phosphate metabolism in tumoral calcinosis. Ann Intem Med 92: 482-487, 1980. 201. Lufkin EG, Wilson DM, Smith LH, et al: Phosphorus excretion in tumoral calcinosis: Response to parathyroid hormone and acetazolamide. J Clin Endocrinol Metab 50:648-653, 1980. 202. Zerwekh JE, Sanders LA, Townsend J, Pak CY: Tumoral calcinosis: Evidence for concurrent defects in renal tubular phosphorus transport and in l alpha,25-dihydroxycholecalciferol synthesis. Calcif Tissue Int 32:1-6, 1980. 203. Walton RJ, Russell RG, Smith R: Changes in the renal and extrarenal handling of phosphate induced by disodium etidronate (EHDP) in man. Clin Sci Mol Med 49:45-56, 1975. 204. Zusman J, Brown DM, Nesbit ME: Hyperphosphatemia, hyperphosphaturia and hypocalcemia in acute lymphoblastic leukemia. N Engl J Med 289:1335-1340, 1973. 205. Cohen LF, Balow JE, Magrath IT, et al: Acute tumor lysis syndrome. A review of 37 patients with Burkitt's lymphoma. Am J Med 68:486-491, 1980. 206. Tsokos GC, Balow JE, Spiegel RJ, Magrath IT: Renal and metabolic complications of undifferentiated and lymphoblastic lymphomas. Medicine 60:218- 229, 1981. 207. Giebisch G, Berger L, Pitts RF: The extra-renal response to acute acid-base disturbances of respiratory origin. J Clin Invest 34:231-245, 1955. 208. Honig PJ, Holtzapple PG: Hypocalcemic tetany following hypertonic phosphate enemas. Cllin Pediatr 14:678-679, 1975. 209. McConnell TH: Fatal hypocalcemia from phosphate absorption from laxative preparation. JAMA 216:147-148, 1971. 210. Payne JW, Walser M: Ion association. II. The effect of multivalent ions on the concentration of free calcium ions as measured by the frog heart method. Bull Johns Hopkins Hosp 105: 298-310, 1959. 211. Morgan DB: Calcium and phosphorus transport across the intestine. In Girdwood RM, Smith AW (eds): Malabsorption. Baltimore, Williams, & Wilkins, 1969. 212. Slatopolsky E, Caglar S, Gradowska L, et al: On the prevention of secondary hyperparathyroidism in experimental chronic renal disease using "proportional reduction" of dietary phosphorus intake. Kidney Int 2:147-151, 1972. 213. Golden P, Mazey R, Greenwalt A, et al: Vitamin D: A direct effect on the parathyroid gland. Miner Electrolyte Metab 2:1, 1979. 214. Slatopolsky E, Weerts C, Thielan J, et al: Marked suppression of secondary hyperparathyroidism by intravenous administration

236 of 1,25-dihydroxy-cholecalciferol in uremic patients. J Clin Invest 74:2136-2143, 1984. 215. Berlyne GM, Ben-Ari J, Pest D, et al: Hyperaluminaemia from aluminum resins in renal failure. Lancet 2:494-496, 1970. 216. Ward MK, Feest TG, Ellis HA, et al: Osteomalacic dialysis osteodystrophy: Evidence for a water-borne aetiological agent, probably aluminum. Lancet 1:841-845, 1978. 217. Moriniere PH, Roussel A, Tahira Y, et al: Substitution of aluminum hydroxide by high doses of calcium carbonate in patients

KEITH HRUSKA AND ANANDARUP GUPTA on chronic hemodialysis: Disappearance of hyperaluminaemia and equal control of hyperparathyroidism. Proc Eur Dial Transplant Assoc 19:784-787, 1982. 218. Slatopolsky E, Weerts C, Lopez-Hilker S, et al: Calcium carbonate as a phosphate binder in patients with chronic renal failure undergoing dialysis. N Engl J Med 315:157-161, 1986. 219. Slatopolsky E, Weerts C, Norwood K, et al: Long-term effects of calcium carbonate and 2.5 mEq/liter calcium dialysate on mineral metabolism. Kidney Int 36:897-903, 1989.

7HAPTER t

Bone Biopsies: A Modern Approach MARIE=CLAUDE MONIER=FAUGERE, M.

CHRIS LANGUB, AND

HARTMUT H. MALLUCHE Division of Nephrology, Bone and Mineral Metabolism, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky 40536

I. II. III. IV. V.

Function and Structure of the Skeleton Bone Biopsies Mineralized Bone Histology Techniques Molecular Bone Histology Evaluation of Bone

VI. Indications For and Information Derived from Bone Biopsies VII. Information Derived from Molecular Histology References

During the last two decades, bone biopsies, mineralized bone histology, and bone morphometry have advanced our understanding of normal bone physiology and the pathological mechanisms leading to various metabolic bone diseases. To date, bone biopsies represent the most powerful and informative diagnostic tool and a significant research method in the area of metabolic bone diseases. Bone biopsies have become the "gold standard" against which the noninvasive "diagnostic" markers of bone metabolism are tested. The ongoing search for such markers has provided clinicians with useful new diagnostic tools, such as serum and urinary concentrations of bone collagen degradation products. However, none of the currently measurable serum or urinary indices, alone or in combination, can predict the various underlying bone diseases with absolute certainty. Bone biopsies still occupy an important place among the diagnostic tools of metabolic bone diseases. As a research tool, bone biopsies have most often been used to test the effects on bone of newly available therapeutic modalities. However, new methods to assess bone architecture and the application of molecular biology to the field of METABOLIC BONE DISEASE

bone histopathology have recently opened a new and exciting area of research. With the isolation and genomic coding of numerous systemic and local biomolecules that play a role in bone metabolism, their localization, intensity of expression, and synthesis in bone and its microenvironment can now be studied through in situ hybridization histochemistry and immunohistochemistry. This chapter presents (1) a short review of the functional and structural organization of bone, (2) the conditions under which bone biopsies should optimally be performed, (3) established and novel techniques in bone morphology, (4) bone morphometric methods, and (5) indications for and information derived from bone biopsies.

I. FUNCTION AND STRUCTURE OF THE SKELETON The skeleton has a dual mechanical and metabolic function. The rigidity of the skeleton is responsible for maintenance of the human body configuration, protec-

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tion of soft organs, and mobility through transmission of forces originated by muscle contraction. It is also the main reservoir for calcium, phosphate, and bicarbonate; thus, the skeleton contributes to the minute-to-minute regulation of extracellular fluid. This osseous framework comprises 206 distinct, differently shaped pieces, the organ bones. Each bone consists of bone tissue, hematopoietic marrow, and vasculature. Bones from the axial skeleton include the skull, spine, thorax, and pelvis, whereas bones from the extremities comprise the appendicular skeleton. Pathological processes and therapies can affect these skeletal sites in different ways. This is due to the relative amount of the two main structural types of bone tissues, that is, cortical and cancellous bone.

A. C o r t i c a l a n d C a n c e l l o u s B o n e Cortical or compact bone can be distinguished macroscopically from cancellous or trabecular bone. Cortical bone is a dense tissue that contains less than 10% soft tissue. Cancellous or spongy bone is made up of trabecules shaped as plates or rods interspersed between bone marrow that represents more than 75% of the cancellous bone volume. Cortical bone forms the external layer of all bones but is found predominantly in the appendicular skeleton, particularly in diaphysis of long bones. Cancellous bone is found mainly in the axial skeleton, located between the cortices of smaller flat and short bones such as scapulae, vertebrae, and pelvis. It is also present in limited amounts in the juxtaarticular extremities of appendicular skeleton. Cortical bone represents 80% of the skeletal mass and therefore supports most of the mechanical function. 1 Cancellous bone is only 20% of the skeletal mass but is metabolically four times more active per unit volume than cortical bone. Thus, the metabolic function is equally distributed between cortical and cancellous bones. 1

FIGURE 8 - 1 Bone envelopes. Periosteal surface (P), haversian surface (H), and endosteal surfaces (E). On one side of the longitudinal cut of bone (left), the three-dimensional orientation of trabeculae is illustrated; on the other side (fight), the histological appearance of a bone section, that is, its two-dimensional image, is shown. (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.)

modeling activities take place on bone surfaces. However, the levels of activity vary from one surface to another and can be differently affected by physiological or pathological events as well as by therapeutic agents.

C. T h e B o n e S t r u c t u r a l U n i t Between the periosteal and endosteal envelopes, bone is further organized in several structural elements, the bone structural units (BSUs). The spatial arrangement of these smallest, individual units of bone and their cohesion are responsible for bone strength. In cortical bone, BSUs are represented by the osteon or haversian system (Fig. 8 - 2 ) . Each osteon consists of

B. B o n e E n v e l o p e s a n d B o n e S u r f a c e s At the microscopic level, each bone exhibits two envelopes. The periosteal envelope represents the outer layer of connective tissue that encloses both hard and soft tissue and separates bone from other organs (Fig. 8 - 1 ) . The inner or endosteal envelope surrounds all soft tissue within the bone (except osteocytes) and is the boundary between soft tissue, mainly bone marrow, and bone tissue. Within the endosteal envelope, three distinct but continuous surfaces are observed: the intracortical, including the haversian and Volkmann's canals, the endocortical, and the trabecular surfaces. Modeling and re-

FIGURE 8--2 Osteons--smallest functioning units of bone. Concentric layers of osseous lamellae surrounding central canals. Interstitial lamellae interspersed between intact osteons. Undecalcified, unstained, 7 I~m thick section of cortical human bone. Polarized light with red filter (X31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

CHAPTER8 Bone Biopsies: A Modem Approach

239

FIGURE 8 - 3

Trabecular osteon. The difference in orientation of lamellae allows identification of the boundaries of the osteon for measurement of mean wall thickness. Undecalcified, unstained, 7 ~m thick section of human iliac bone. Phase-contrast microscopy (x80). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8 - 4 Lamellar osteoid and lamellar bone. Viewing under polarized light allows recognition of the orderly lamellar pattern of osteoid. Undecalcifed, 3 txm thick section of human iliac bone (modified Masson-Goldner stain; X125). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

a 200- to 250-1~m-wide cylinder running parallel to the long axis of cortical bone. The osteon center is occupied by a 40- to 50-txm haversian canal containing blood vessels, nerves, and connective tissue. The haversian canals of adjacent osteons are linked transversally by Volkmann's canals, creating an intracortical network, which is also connected with the periosteum and bone marrow. The central haversian canal is surrounded by concentric layers of 20 to 30 osseous lamellae making the osteon wall approximately 70 to 100 Ixm thick. Osteons are densely packed together and separated only by interstitial lamellae, which are the remains of incompletely resorbed osteons (Fig. 8-2). In cancellous bone, BSUs are flat and can be envisioned as longitudinally cut and unfolded osteons (Fig. 8-3). They appear as semilunar packets roughly parallel to the central axis of the trabecule. The trabecular surface corresponding to the open haversian canal follows the shape of the trabecule and is in contact with bone marrow. With trabecules, the BSUs are separated from each other by interstitial bone, which, as in cortical bone, represents the remainder of older incompletely resorbed packet.

exhibits an orderly lamellar pattern, as circular layers alternate with longitudinal ones. The change in collagen fiber direction from layer to layer is responsible for the birefringence of bone under polarized light microscopy (Fig. 8-4). Contrasting with the regularity of lamellar bone, woven bone is composed of loose and randomly arranged collagen bundles. Woven bone is formed by irregular and unpolarized extrusion of protocollagen by osteoblasts. This matrix consists of an unordered, crisscross texture that lacks the birefringence typical of lamellar bone under polarized light (Fig. 8-5). Woven bone is present in embryonic skeleton and in both cortical and cancellous bones during stages of rapid growth. After completion of bone growth, woven bone is replaced by lamellar bone in the normal skeleton. However, woven bone is also observed in certain pathological conditions,

D. L a m e l l a r and W o v e n B o n e Each BSU consists of a specialized connective tissue, the osseous tissue made of a mineralized protein matrix. At the microscopic level, two different types of bone can be identified by the arrangement of collagen bundles. In normal mature skeleton, bone is of the lamellar type. In this bone, the orientation of collagen fibers alternates regularly from layer to layer. Each layer is approximately 3 txm thick with all collagen fibers deposited in the same direction. The deposited collagen

FIGURE 8 - - 5 Woven osteoid and woven bone in predominant hyperparathyroid bone disease. Abundance of irregular woven osteoid in the center and presence of lamellar osteoid at the periphery of the trabecular bone. Undecalcified, 3 I~m thick section of human iliac bone. Polarized light microscopy (modified Masson-Goldner stain; x50). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

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such as Paget's disease of bone, fracture healing, osteogenesis imperfecta, and in primary or secondary hyperparathyroidism. In adults, woven bone is indicative of rapid, uncontrolled bone formation and high bone turnover that is attributable to either local or systemic factors.

E. B o n e M o d e l i n g a n d R e m o d e l i n g During life, the skeleton is not static but undergoes numerous transformations. First, there is growth, also called bone modeling, when the overall shape and size of bones change. The two types of bone growth are longitudinal and appositional modeling. Longitudinal growth occurs by enchondral ossification, a process that takes place at the growth plate (Fig. 8-6), when cartilage proliferates and progressively calcifies creating new trabeculae until the epiphyseal growth plate fuses. Appositional modeling (i.e., the growth of bone in width) proceeds by periosteal apposition of new bone and endosteal resorption of old bone. After epiphyseal growth plates close, the adult skeleton continues to renew itself without noticeable changes in macroscopic shape. This is bone remodeling. The primary function of bone remodeling is to replace old bone with new bone. Old bone contains high mineral density and microfractures that decrease its mechanical properties. Approximately 3% of cortical bone and 25% of cancellous bone are renewed per year in the mature human skeleton. 2 Bone remodeling occurs in distinct locations on bone surfaces, the bone remodeling units (BRUs). 1 BRUs require the involvement of a team of different cells, the bone multicellular units (BMUs). The remodeling of a "packet" or "quantum" of bone entails sequential events known as the remodeling cycle. This re-

FIGURE 8--6

Growth plate. Longitudinal growth through enchondral ossification. Layers of proliferating and hypertrophic columnar cartilage followed by primary and secondary spongiosa. Undecalcifled, 3 Ixm thick section of growing dog bone (modified MassonGoldner stain; • (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

modeling cycle includes activation, resorption, reversal, formation, and quiescence. The signaling factors responsible for the initiation of the remodeling cycle are not well understood. Structural and/or biomechanical characteristics of old bone packets may play a role in this early phase, and signals may be relayed through the osteocyte-lining cell system to osteoblasts and bone marrow cells. 1 Numerous factors known to stimulate bone resorption are probably involved in the activation of bone remodeling that also requires interaction between hormones, cytokines, and growth factors and their receptors in bone cells and bone marrow cells (see Chapter 1). Activation of bone surface is accompanied by mobilization of mononucleated osteoclast precursors, retraction of the lining cell layer, and exposure of bone matrix chemotactic substances such as osteocalcin, transforming growth factor [3 (TGF-[3), and type I collagen. 3-7 The progressive fusion of mononucleated osteoclast precursors results in a team of one to four mature and active osteoclasts at each remodeling site. The team of osteoclasts then adheres to bone surface along a ring (i.e., the sealing zone), leaving an extracellular bone resorbing compartment, and then bone resorption begins. Osteoclasts dissolve bone mineral through acidification of the subosteoclastic compartment. This process involves proton pumps and carbonic anhydrase. Bone matrix is hydrolyzed by proteolytic enzymes such as collagenase. 8 Osteoclasts are motile cells and move along the erosion cavity. They are responsible for the rapid resorption (---7 days) of two thirds of the final eroded cavity. Mononucleated cells resorb the other one third at a slower rate (---36 days). 9 These mononucleated resorbing cells consist of either unfused original osteoclast precursors or segmented osteoclasts. The rapid osteoclastic resorption leaves behind a rather rough surface that the mononuclear cell erosion transforms into a smoother bone surface. In cortical bone, the resorption process takes place along the long axis of bone, and osteoclasts are observed in the cutting cone (Fig. 8 - 7 ) with depths reaching 100 txm. 1 In normal cancellous bone, osteoclasts erode bone parallel to the bone surface, forming a shallow cavity with a depth of 40 to 60 Ixm (Fig. 8-8). After resorption ceases, there is a transition period before bone formation occurs (i.e., the reversal phase). During this 1- to 2-week phase, a layer of material with particular optical characteristics is deposited at the bottom of the resorption lacunae. On histological section this "line" is positive for acid phosphatase staining and presents with a different birefringence under polarized or phase contrast light microscopy. This line (surface) serves as a "cement" or " g l u e " between old and new bone to be apposed and is referred to as the reversal or cement line. Only mononucleated cells are seen in front

CHAPTER 8 Bone Biopsies: A Modem Approach

FIGURE 8 - 7

241

Bone remodeling. Cutting cone with several osteoclasts at its tip followed by peripheral deposition of new bone by osteoblasts. Between osteoblasts and osteoclasts, undifferentiated mesenchymal cells are seen. The sequential character of bone modeling is illustrated in this haversian remodeling site. Undecalcified, 3 Ixm thick section of human cortical bone (modified Masson-Goldner stain; X50). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

Osteoblasts. Active osteoblastsIepithelium-like layer of osteoblasts depositing newly formed bone. Regular arrangement of the mononucleated osteoblasts with one to three nucleoli. Undecalcified, 3 Ixm thick section of human iliac bone (modified Masson-Goldner stain; X 125). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

of the resorption lacunae during this phase and probably are responsible for the deposition of cement line. The exact origin of these cells is not known. Concurrent with the reversal phase, other events take place that are responsible for the coupling between resorption and formation. In the adult skeleton, bone is formed only on a preexisting eroded surface. This implies that signals are emitted to promote osteoblast proliferation and to direct osteoblast precursors to a precise location on the bone surface. The complex mechanisms responsible for this coupling phenomenon are not fully understood; however, several local agents such as chemotactic substances, growth factors, and cytokines are probably involved in this event. After differentiation, osteoblasts form a layer in front of the reversal lacunae and deposit matrix protein (i.e., osteoid) (Fig. 8-9). Mineralization of osteoid seam starts

5 to 15 days later when the osteoid seam is approximately 20 txm thick. Apposition of osteoid is rapid at first, and then progressively slows down. The mineral apposition rate follows the same pattern. First, osteoid is mineralized at a rate of 1 to 2 txm/day and subsequently slows down but is still faster than matrix apposition at that time. When matrix apposition ceases, the remaining layers of osteoid are slowly mineralized. The first hydroxyapatite crystals are small and immature, and therefore calcium ions can chelate other substances present at high concentration in the bone microenvironment. These substances may include aluminum, fluoride, and others, in particular tetracycline hydrochloride. Under fluorescent light microscopy, deposits of tetracycline in new bone are spontaneously visible on unstained sections and appear as bright yellow bands. The technique of tetracycline double-labeling (see below) uses this phenomenon to determine the rate of mineralization. However, when tetracycline is administered, it chelates reversibly to all exposed bone surfaces (i.e., resorptive cavities and at the mineralization front). Therefore, it is necessary to perform a bone biopsy within a few days after the last administration of the antibiotic, usually 4 days. This ensures that the tetracycline chelated at the mineralization front is protected from leaching out by a thin layer of newly mineralized bone. During bone formation, osteoblasts are first cuboidal (Fig. 8 - 9 ) and very active; thereafter they flatten (Fig. 8 - 1 0 ) and become lining cells when the osteoid seam is completely mineralized. Moreover, some osteoblasts are embedded in bone matrix and become osteocytes. Also, some osteoblasts may locally undergo apoptosis (programmed cell death). ~~ The total duration of the bone formative phase in normal skeleton is approximately 3 months.

FIGURE 8 - - 8 Osteoclastic bone resorption. Shallow resorption of bone by an osteoclast with two nuclei and prominent nucleoli. Undecalcified, 3 txm thick section of human iliac bone (modified Masson-Goldner stain; x125). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8--9

242

MARIE-CLAUDEMONIER-FAUGERE,M. CHRIS LANGUB,AND HARTMUTH. MALLUCHE bone turnover represents the rate at which the skeleton is renewed. This depends on the activation rate and the extent and distribution of the remodeling sites among the various bone envelopes. If bone turnover is high, the minute changes observed at the bone remodeling sites are amplified. For example, in case of negative bone balance, bone loss is greater in individuals with high bone turnover.

G. B o n e Cells FIGURE 8 - 1 0 Osteoblasts. Resting osteoblasts- - flat cells over a thin layer of osteoid, forming a barrier between the osteoid surface and the bone marrow space. Undecalcified, 3 txm thick section of human iliac bone (modified Masson-Goldner stain; X125). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

The detailed descriptions of the cellular components of bone are detailed in Chapter 1. In the following section, only cellular characteristics pertaining to histological observation are described.

1. THE OSTEOCLASTS A phase of quiescence follows. During the beginning of this phase (3 to 6 months), the newly formed " y o u n g " bone packet will mature by increasing its mineral density. New bone is separated from bone marrow by the layer of lining cells and a thin collagenous membrane. In normal adult bone, the majority of bone surface is in a quiescent stage (80%). Three types of bone remodeling have been described: random, selective, and redundant. With random remodeling, any packet of bone has the same probability to be remodeled, regardless of its age. Selective remodeling involves the remodeling of the older, more fragile bone due to fatigue fracture, local trauma, or osteocyte death. Redundant remodeling renews the youngest bone packets. In normal adult bone, remodeling is mainly random, and the overall age of bone has been calculated to be 20 years in cortical and 4 years in cancellous bone. 1 The age of bone and its mechanical properties can be influenced by pathological processes that can preferentially induce a specific mode of remodeling. 1

Under light microscopy, osteoclasts are large multinucleated cells located in resorption lacunae in the vicinity of mineralized bone (Fig. 8-8). Osteoclasts represent the main cells in the breakdown of bone matrix and bone mineral. They vary in size from 20 to 100 txm in diameter and usually display projections and lobes that give them an irregular appearance. Osteoclasts are highly mobile and go through cycles of resorption and rest. Thus, it is not surprising that these cells vary in histological appearance, depending on the stage of the cycle at which they are observed. In normal skeleton, osteoclasts are somewhat larger than macrophages and may have from two to five nuclei. In pathological states, osteoclasts are large, with up to 100 nuclei (Fig. 8-11). The nuclei are found in the center of the cell, they are characteristically round or oval, and they usually contain one or two prominent nucleoli. Osteoclasts may appear mononucleated depending on the plane of the sections;

F. B o n e B a l a n c e a n d B o n e T u r n o v e r In normal young adults, coupling between the amount of bone resorbed by osteoclasts and the amount of bone formed by osteoblasts results in bone balance. Uncoupling between formation and resorption will result either in negative or positive bone balance at the remodeling site. In states of negative bone balance, the amount of bone resorbed is disproportionately higher than bone formed; conversely, more bone is deposited than resorbed when bone balance is positive. If bone remodeling represents the cellular-based events that occur at a specific site of the bone surface,

FIGURE 8--11 Osteoclasticbone resorption. Lacunar resorption. Osteoclasts resorbing mineralized bone in individual resorption zones forming a deep lacuna. Undecalcified, 3 txm thick section of human iliac bone (modified Masson-Goldner stain; x 125). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

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however, serial sections show other nuclei. These cells exhibit a characteristic pink staining with the modified Masson-Goldner trichrome stain due to abundance of mitochondria, lysosomes, and ribosomes in their cytoplasm. Their foamy appearance reflects the presence of numerous endocytic and lysosomal vacuoles. The ruffled border, the ultrastructural trademark of osteoclasts, consists of numerous foldings of the apical membrane and is occasionally identified at high magnification on thin histological bone sections. However, in the majority of bone samples, the ruffled border is not recognizable. 2. THE OSTEOBLAST LINEAGE CELLS Osteoblasts are mononucleated cells responsible for production of bone matrix and are involved in its mineralization. Under light microscopy, mature osteoblasts in normal bone form a monolayer of cells in front of the bone formation site (Fig. 8-9). They are cuboidal, polarized cells measuring between 15 and 25 ~ m in diameter. Their round nuclei are located at the basal pole of the cells (away from bone) and contain one or more nucleoli. The cytoplasm is strongly basophilic due to the large amount of endoplasmic reticulum and Golgi apparatus responsible for active production of type I collagen and other substances found in the bone matrix. Osteoblasts at a bone formation site give the appearance of an epithelium-like organization. They exhibit gap junctions that ensure their cohesion and provide cell-tocell communication. During active bone formation, osteoblast cells are plump. Towards completion of the new bone packet, the osteoblast shape flattens and its cytoplasm loses its basophilic characteristics, and its nuclei become elongated (Fig. 8-10). The majority of these resting osteoblasts become bone lining cells observed above the quiescent surfaces. Also, approximately 10% of osteoblasts become embedded in the bone matrix before its mineralization and become stellar cellular complexes with a central nucleus and numerous long cellular processes, the osteocytes (Figs. 8 - 1 2 and 8-13). Gap junctions have also been described between the peripheral processes of osteocytes, osteoblasts, and bone lining cells from the same bone area. When matrix mineralizes, osteocytes are found in osteocytic lacunae, the border of which has been found to be calcified by osteocytes. Long cytoplasmic processes are seen in canaliculae. Newly embedded osteocytes conserve the cellular characteristics of osteoblasts, and the oldest ones in deeper bone areas lose signs of protein synthesis and accumulate glycogen. The periosteocytic space between osteocytes and mineralized bone contains the bone extracellular fluid (1 to 1.5 liters) and the bone lining cell-osteocyte network plays an important role in the minute-to-minute calcium homeostasis, as the osteocytes are rapidly exposed to changes in

FIGURE 8 - 1 2 Osteocytes. Osteocytes situated in lacunae with processes traversing the matrix in canaliculi and connecting the osteocytes with each other. (From Malluche HH, Faugere MC: Arias of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

circulating factors. Due to their deep location in mineralized bone, osteocytes are also considered sensors for fatigue damage and microfractures and thus may represent an important contributor to the regulation of bone remodeling and bone turnover.

3. BONE MARROW CELLS Besides bone cells, other cells are present in the bone microenvironment. There is ample evidence that the bone marrow cells play a role in the local regulation of bone remodeling and bone turnover. Cells from the monocyte/macrophage and lymphoid lineages produce various substances such as cytokines and growth factors that directly or indirectly act on bone cell recruitment and activity." Moreover, macrophages have the capability to produce calcitriol. 12''3 Mast cells, which produce heparin, a proven stimulator of bone resorption, may also be involved in the local regulation of bone. 14'15

FIGURE 8--13 Osteocytes surrounded by osteoid, indicating either new bone formation by osteocytes or removal of mineral from the bone matrix. Undecalcified, 3 Ixm thick section of human iliac bone (modified Masson-Goldner stain; x198). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

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In bone biopsies, it is common to observe that states of high bone turnover are often associated with various degrees of bone marrow cell hyperplasia, whereas low turnover states of bone are accompanied by variable hypoplasia of hematopoietic cells. 16

II. BONE BIOPSIES A. Prerequisites for Bone Biopsies The major prerequisite for any biopsy technique is that the operator must obtain a representative sample that will yield sufficient information on the tissue under investigation. This implies that the biopsy specimen has to contain an adequate amount of cortical and deep cancellous bone. The bone sample should also be obtained without artifacts; that is, without breakage or fractures of cortex and trabecules, compression of trabecular bone that leads to overlapping of bone components, hemorrhage in bone marrow, and cellular damage generated by heat during the drilling process. Achievement of optimal quantity and quality depends upon both the bone biopsy instrument employed and the skill and experience of the operator (see below). Another prerequisite to further enhance the informative value of bone biopsies is labeling with bone markers. This adds a dynamic dimension to the regular static evaluation of bone structure, formation, and resorption. Labeling with specific, nontoxic, in vivo markers before bone biopsy allows the operator to determine the level of bone turnover and rate of bone formation, and to identify possible mineralization abnormalities. 17 Labeling entails administration of time-spaced bone markers (i.e., antibiotics from the tetracycline family) prior to biopsy. These compounds form calcium chelate complexes that have spontaneous fluorescence (Fig. 8 - 1 4 ) and bind to actively forming bone surfaces. 8-2~ A double-labeling technique is the best approach, since it provides information on mineralization rate and bone formation. 21 It offers more specific information than either (1) staining of bone slides with toluidine blue 22 at an acidic pH, which indicates the degree of bone remodeling, or (2) using one prolonged single tetracycline administration, which can simplify the labeling schedule for patients, but does not allow differentiation between a mineralization defect and active mineralization. 23 In most cases, administration of the first label for 2 days is followed by a free interval period which varies from 8 to 15 days. An interval that is too short can lessen the distinction between labels, particularly in the case of a mineralization defect. If the interval is too long, it may artificially increase the number of single labels because the probability exists that the number of forming sites

FIGURE 8-- 14 Labeling of bone. Tetracycline double-labeling at the bone-osteoid interface. Two regular distinct labels. The outer golden yellowish label represents administration of democlocycline; the inner greenish yellow label represents administration of tetracycline. Undecalcified, unstained, 7 txm thick section of human cancellous bone. Fluorescent light microscopy (X 126). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

being completed or started will increase. During the 2 to 4 days following the free interval period, a second course of antibiotics is given. Bone biopsy is then performed 4 to 6 days after the last administration of tetracycline. This recommended delay allows the tetracycline to be slightly buried within mineralized osteoid, thus preventing the antibiotic from leaching out. However, if a bone biopsy is performed more than 10 days after the last label, the chance is increased that labels will not correspond to active formative sites, because there is the probability that the first label has undergone resorption. In case of emergency, labeling of bone can be shortened to a 1-day-on, 6-days-off, and 1-day-on schedule. This schedule was recently advocated for clinical purposes 24 with a single dose of oral tetracycline per day (1.0 to 1.5 g of tetracycline or 600 to 900 mg of Declomycin). However, it is not known whether gastrointestinal side effects are greater with this approach. Accurate assessment of the mineralization rate is ensured by the use of two labels with different colors. Tetracycline hydrochloride has a light yellow and demeclocycline hydrochloride (Declomycin) a yellow-orange fluorescence (Fig. 8-14). If only one type of tetracycline is used, the two labels can appear as one when they merge in the presence of a profound decrease in mineralization rate and thus are unrecognizable as a double label. Dosages of tetracycline hydrochloride and Declomycin are usually 500 mg and 300 mg t.i.d., respectively, for patients with normal renal function. Dosages should be reduced to 500 mg and 300 mg b.i.d, in patients with impaired kidney function. While patients generally tolerate tetracycline doublelabeling, some side effects, such as allergic reactions, vomiting, and photosensitivity, might be observed. The

CHAPTER 8 Bone Biopsies: A Modem Approach pathologist can encounter problems such as a lack of fluorescent labels or evidence of only one label on bone slides, even though tetracycline double-labeling was prescribed. This usually occurs when patients did not want to take additional pills or forgot one or two doses. Thus, physicians need to inform patients carefully about the importance of labeling. Recognition of tetracycline in bone slides can be confounded if tetracycline is ingested with meals or with antacids, which decrease the intestinal absorption of tetracycline and therefore its uptake by bone. The pathologist may also run into difficulty if the patient has previously been taking tetracycline antibiotics.

B. Perceived Constraints of Bone Biopsies In the past, bone biopsies have been confined to research conducted in a few specialized centers, except in the case of severely symptomatic patients. At present, bone biopsies are more widely used for both diagnosis and research. The U.S. Food and Drug Administration (FDA), for instance, has included histological data as a prerequisite for therapeutic trials in metabolic bone diseases. However, traditional constraints continue to be perceived because of the: (1) invasiveness of the procedure; (2) lack of technical training; (3) potential for pain suffered by patients; (4) costs associated with the procedures; (5) lack of specialized centers with expertise to interpret bone samples; (6) time delays between biopsy and pathological reports; and (7) limited understanding of the nature of information provided by histological results. These perceived limitations have prompted an ongoing search to find or develop noninvasive serum or bone markers that predict bone turnover, mineralization status, cellular abnormalities, and bone aluminum accumulation. This research has resulted in improved determinations of serum levels of various calciotropic hormones, isolation of proteins and enzymes from bone, and development of commercially available assays. However, these alternatives have not proven to be specific or sensitive enough to effectively determine the potential value of a specific therapeutic regimen. Therefore, efforts have been made to minimize the constraints associated with bone biopsies. For example, instrument design and biopsy techniques have improved (see below). More intensive and detailed training of clinicians and pathologists has been implemented. Advances in bone sample processing have resulted in faster turnaround time between bone biopsy and availability of histological results. This has enhanced the value of bone biopsy in routine patient care. Also, bone morphometrists have adopted a uniform and simple nomenclature

245 for bone histomorphometric parameters25; thus, nonmorphometrists can now better understand bone biopsy resuits. Moreover, the number of individuals involved in bone morphometry has steadily increased, resulting in the establishment of the International Society for Bone Morphometry.

C. Bone Biopsy Sites, Instruments, and Techniques Metabolic bone diseases encompass systemic alterations of the skeleton. Therefore, bone samples of adequate size and containing both cortical and cancellous bone should provide useful information necessary to establish a diagnosis. Over the years, the iliac crest has been the site of choice for compelling reasons. This bone site is easily accessible, does not require extensive surgery (as needed for rib biopsies), and is associated with fewer postoperative complications. Local forces known to affect regional bone remodeling, such as direct weight-beating or tension and forces exerted by muscle pull, are minimized at the anterior iliac crest. Moreover, normal values have been easier to collect and comparisons between iliac crest values indicate good correlation with vertebrae, 26 tibiae, and femora. 27'28 Iliac crest bone biopsies can be obtained in a vertical or horizontal direction. The vertical approach allows assessment of subcortical cancellous bone and deep cancellous bone without size restrictions. The horizontal direction provides information on the outer and inner cortices, but sample size is limited by the thickness of the iliac bone. Coefficients of variance have been established for routinely used histomorphometric parameters in bone samples located at the iliac crest. 29'3~These include the (1) left or fight, (2) anterior or posterior, (3) horizontal or vertical, and (4) superior or inferior iliac crest bone samples. When considering variances, there is no difference in cancellous bone volume between sections of vertical and horizontal biopsies. However, all other histomorphometric parameters are significantly higher in vertical biopsies with the exception of lower trabecular thickness. 31 This finding can be explained by inclusion of more and deeper cancellous bone in the specimen. Available bone biopsy instruments are classified as manual trochars 32-38 or electric drills (Fig. 8 - - 1 5 ) . 39-41 A bone sample of the desired size should be obtained with minimal surgical invasiveness. While overzealous use of physical force with manual instruments can produce artifacts, undue pressure exerted with an electric drill can result in "bone powder" accumulation in the periphery of spongy bone. Drilling speeds that are too high can result in heat artifacts of bone cells.

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FIGURE 8 - 1 5 Bone biopsy instruments. Electric drill (right) for transiliac and vertical samples of 0.5 cm inner diameter and 3 to 4 cm length. Drilling bits of smaller diameter can be used for children. Manual trochar (center) for transiliac bone samples of 0.8 cm diameter. The 8-gauge Jamshidi needle (left) vertical bone samples of 0.3 cm diameter and 2 to 3 cm length. (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.)

Generally, bone samples of 0.4 to 0.5 cm diameter and 2.5 to 3.5 cm length taken vertically (Fig. 8 - 1 6 ) or 0.8 cm diameter obtained horizontally (Fig. 8 - 1 7 ) from the anterior iliac crest are sufficient for qualitative and quantitative bone histology. 16 We conducted a comparative study of established electrical drill techniques and the manual Jamshidi needle while performing vertical anterior iliac crest b o n e biopsies (with 3 and 5 m m diameter, respectively) on 14 patients suffering from various metabolic b o n e diseases. 42 The anterior and posterior samples obtained f r o m alternately using both instruments s h o w e d that even though there was relatively little difference in terms of quality b e t w e e n sampies, results of the osteoclast surface/bone surface did not correlate and the mineral apposition rate was different. This indicates that b o n e samples of 3 m m inner diameter provided by the Jamshidi technique do not pro-

FIGURE 8 - 1 6 Bone slide of 3 ~m thickness cut from samples of 5 mm diameter. Undecalcified, 3 Ixm thick section of human iliac bone (modified Masson-Goldner stain). Photograph taken from original slide. (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.)

FIGURE 8 - 1 7 Bone slide of 3 Ixm thickness cut from a sample obtained horizontally using a trocar of 8 mm inner diameter. Inner and outer cortices are included in the sample. See Color Plate.

vide the same useful information as that derived from 5 - m m samples. Based on this study, we cannot support the use of the relatively noninvasive and inexpensive Jamshidi technique for routine diagnostic bone biopsies. A recently introduced electric drill (Straumann, Cambridge, M A ) performs like the established electric drill with the addition of a one-step drilling and extraction procedure, which shortens the surgical time for performing bone biopsies (Fig. 8 - 1 8 ) . This improves patients' acceptance of the procedure. Also, the use of disposable drill bits decreases infection risks, avoids p r o b l e m s with dull drill bits, and eliminates the risk of breakage of drill bits that accompanies frequent resharpening.

D. Potential Complications

of Bone Biopsies

Complications as a result of bone biopsies can include pain, h e m a t o m a , w o u n d infection, and rarely neuropathy. However, studies show that horizontal or transiliac and vertical or superior biopsies of the anterior iliac crest

FIGURE 8--18 Electrical drill with the addition of one-step drilling and extraction procedure for vertical samples of 5 mm inner diameter and 3 to 4 cm in length.

CHAPTER 8 Bone Biopsies: A Modern Approach result in very small morbidity and no mortality as a result of the procedure. The operator's experience is an important factor in keeping morbidity to a minimum and in maintaining adequacy of the specimen. Use of recently developed techniques should further decrease any complications. Data by Duncan et al., 43 gathered from a questionnaire sent to 18 different hospitals performing routine biopsies, showed an overall 0.52% frequency of complications based on a total sampling of 14,810 biopsies. For the horizontal approach, complications were found in 0.63% and for the vertical approach in 0.36% of biopsies. Also, instruments allowing smaller biopsy samples resulted in fewer complications. A pain score assessed discomfort experienced by patients during and subsequent to bone biopsies. Of the 37 subjects assessed, 21% reported no pain, 46% reported acceptable pain, and 30% experienced moderate pain. One patient (3%) said the pain was severe. Similar observations were made at the Mayo Clinic, where a power-driven transiliac trephine was used after application of local anesthesia to the external and internal surfaces of the ilium. 41

III. MINERALIZED BONE HISTOLOGY TECHNIQUES A. Fixation and Dehydration of Bone The fixation process should inhibit postmortem changes without removing the mineral from bone, thus allowing preservation of bone tissue constituents and bone cells in a condition as lifelike as possible. 44 A prerequisite to any good fixation is rapid inactivation of acidic lysosomal enzymes by immersion of the biopsy sample in the fixative as soon as possible after the biopsy. There are two major groups of fixative: (1) precipitant fixatives, which transform cytosolic proteins into a coagulum and (2) nonprecipitant fixatives, which denature proteins. Several fixatives have been used for bone biopsies: absolute or 70% ethanol, buffered 10% formalin and methanol, and osmium tetroxide. The choice of fixative and temperature at which the biopsy sample should be kept depends upon the planned staining techniques to be later applied to bone slides (see below). For routine mineralized bone histology, ethanol at room temperature is the fixative of choice. This relatively weak fixative does not remove calcium from bone within several weeks of exposure. Formalin is more potent than ethanol but, like some strong fixatives, has a tendency to leach out calcium, aluminum, and tetracycline from bone. Although osmium tetroxide is a good fixative, it tends to react with and adds onto many cellular struc-

247 tures. This promotes contrasts and, in a sense, "stains" tissue. The portion of the bone sample placed in fixative should be small enough to allow rapid diffusion of ethanol into the innermost part of the bone tissue. The total volume of the fixative should be at least 10 to 20 times that of the tissue. Duration of fixation depends on sample size, and usually 24 to 48 hours are sufficient for regular samples. It is essential to dehydrate bone sample very thoroughly before embedding, since all plastic monomers commonly used are not miscible with water. To obtain optimal dehydration, bone samples should be passed at least five times (24 hours each) through fresh absolute ethanol. The use of microwaves dramatically reduces time requirements for fixation, dehydration, and penetration. Use of a heat sink (unpublished observation) reduces the risk of microwave irradiation. This principle has recently been embodied in a tissue processor allowing rapid fixation, dehydration, and penetration of bone for mineralized bone histology and processing of soft tissue (Straumann Medical, Waldenburg, Switzerland).

B. Embedding of Bone Finding the appropriate embedding medium is important for good section quality. After testing numerous substances, we found that methyl methacrylate introduced by Amold and Jee 45 is among the most useful. We used the following criteria: (1) the final degree of hardness should approximate the hardness of bone; (2) the substance should readily penetrate bone specimen without causing artifacts such as bubbles; (3) it should be nonflammable, nontoxic, and adjustable in hardness for final cutting; and (4) solvents should be available for its dissolution after cutting. Plastic monomers such as bioplastic and glycol methacrylate can also be employed; however, they have certain drawbacks. Progressive and full penetration of the monomer within the bone sample may be facilitated by vacuum application. After complete penetration of the liquid monomer, polymerization (hardening) is achieved by heat and/or addition of benzoyl peroxide. The temperature has to be kept at 48 ~ C, because higher temperatures promote rapid hardening and gas bubbles accumulate within the block.

C. Sectioning of Bone Cutting mineralized bone requires special microtomes equipped with carbide edges or diamond knives. The

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most popular microtomes for larger sectioning are the older Jung microtome model K (Nussloch, Germany) and the newer Reichert-Jung polycut. The Universal microtome series (Model 1130-1150, Nussloch, Germany) and the newer Reichert-Jung Universal rotary microtome Model 2050 (Cambridge Instruments, Buffalo, NY) or the Microm Universal microtome (Zeiss, Thornwood, NY) are preferred for smaller bone samples. It should be noted, however, that microtome quality cannot replace the need for a skilled technician. Various cutting fluids (water, ethanol) are used to keep the block and the section moist. The number of sections to be cut depends on the number of different stains that will be used. Moreover, for bone morphometry, the number of sections are decided not only by the amount of cancellous or cortical bone available in the bone sample (the smaller the bone sample, the higher the number of histological sections) but also by the reproducibility of the quantitative method employed (see below). Ideally, several sets of serial sections taken at different depths within the block should be obtained to cover the variation within the block. Within one set, serial sections make it possible to quantitate dynamic parameters of bone on the closest section (3 txm) to the one used to measure static parameters.

D. S t a i n i n g o f B o n e S e c t i o n s Staining of sections may be performed by flotation or following initial mounting on slides. The flotation technique requires thick plasticized sections of bone and therefore provides less cellular definition under the microscope. Also, uneven staining could result from incomplete exposure of the sections to the various staining fluids. When initially sealed on the slides, sections can be very thin, and plastic can be removed prior to staining. Removal of plastic provides excellent cellular details for bone cells and especially bone marrow cells. However, the process of plastic removal has to be gentle, since application of harsh solvents may cause artifacts. There are several staining techniques for discriminating calcified bone from uncalcified matrix. The modified Masson-Goldner trichrome stain, 46 the solochrome cyanine stain, 47 and von Kossa's stain 48 are the most valuable and most frequently used techniques. Special stains are also available for differentiation or for further classification. For instance, osteoblasts can be differentiated from mononucleated osteoclasts by the pyronine green stain. Active osteoclasts can be identified by histochemical staining of the lysosomal enzyme acid phosphat a s e . 49'5~ Villanueva has described a special method for thick sections that stains the entire bone specimen. 51 Fur-

ther documentation will determine whether staining the entire specimen affects special staining for aluminum, iron, etc. Several independent techniques can be used to document accumulation of aluminum in bone: (1) energydispersive x-ray analysis (EDAX), (2) atomic absorption spectrophotometry (i.e., measurement of aluminum content of bone), and (3) histochemical staining for identification of aluminum at the mineralization front or at other sites. EDAX involves processing small pieces of bone for transmission electron microscopy. The electron microscope sample is then exposed to an energy-active x-ray source and finally analyzed by an energy-dispersive x-ray detector. Aluminum presents with a characteristic peak (K = 1.485 eV). This technique is very sensitive and specific for aluminum. However, EDAX does not allow assessment of the exact extent and severity of aluminum deposition in bone. Determination of bone aluminum content provides an accurate estimate of the magnitude of the aluminum load in bone but cannot discriminate between aluminum embedded within mineralized bone or at the mineralization front or in bone marrow. There are two available histological staining techniques to detect aluminum on bone slides: the aurintricarboxylic acid 52 and the solochrome azurine methods. 53 Classically, both methods are used with counterstaining; however, in our experience the counterstain may cover, at various degrees, the aluminum deposits. It is therefore recommended not to counterstain and to view the bone section under fluorescent light or phase contrast microscopy to precisely locate the aluminum deposits. The aurin-tricarboxylic acid method stains aluminum deposits as red bands (Fig. 8-19),

FIGURE 8--19 Aluminum deposits in bone. Stainable bone aluminum at the osteoid-mineralized bone interface (i.e., the mineralization front). Undecalcified, 3 Ixm thick section of human iliac bone (aurin-tricarboxylic acid stain; x31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

CHAPTER 8 Bone Biopsies: A Modem Approach whereas aluminum appears dark purple-blue with the solochrome azurine stain. Both techniques have advantages and disadvantages and are complementary. The aurintricarboxylic acid stain is very specific; however, deposits of light intensity are sometimes difficult to visualize, since the human eye is less sensitive to red than other colors. Examination of bone slides with green filter and under fluorescent light helps in recognizing those light aluminum deposits. With the solochrome azurine stain, aluminum deposits are easily recognizable at the mineralization front and also within bone, since this technique involves partial decalcification of the trabecules. However, this stain is less specific than the aurin-tricarboxylic acid stain and nonspecific staining can be seen at the edge of bone sections and when artifactual disruption of the trabecules occurs. Both staining techniques have been validated in our laboratory by energy-dispersive x-ray microanalysis. 54 A characteristic peak for aluminum was invariably observed at the mineralization front whenever a positive histochemical staining for aluminum was found. Importantly, those bone slides lacking stainable bone aluminum at the mineralization front did not exhibit the typical peak for aluminum by energy-dispersive x-ray analysis. Histopathological changes associated with aluminum accumulation in bone correlate better with the extent of stainable bone aluminum at the mineralization front than with results of direct measurements of total aluminum content in bone. 55 Therefore, stainable bone aluminum represents a more useful clinical tool than the measurement of bone aluminum content for assessing a patient's aluminum-related bone disease. Abnormal amounts of iron may be found in bone. Of the four available staining methods for demonstration of iron, 56-59 we found that the modified Gomori method 59 supplied the best results, primarily because of its high specificity for iron (Fig. 8-20). Amyloid deposits can be detected by using the Congo red stain, viewed under polarized light microscopy. Other stains used in surgical pathology may also be of use when abnormalities in bone marrow are found.

249

FIGURE 8--20 Histochemical staining of iron in bone. Iron can be demonstrated as blue linear deposits at the marrow-bone interface and in the bone marrow. Undecalcified, 7 lxm thick section of human iliac bone (modified Gomori stain; • (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

A. In Situ H y b r i d i z a t i o n H i s t o c h e m i s t r y When applied to bone sections, in situ hybridization histochemistry (ISHH) is an attractive technique that reveals cellular sources and differential gene expressions of specific biomolecules including matrix proteins, cytokines, and receptor species involved in bone metabolism. ISHH detects cellular nucleic acids based on the formation of double-stranded hybrids between a nucleic acid fragment (the probe) and a DNA or RNA sequence present within bone cells. The probe is labeled with a reporter molecule and is subsequently detected. When required, ISHH can become a quantitative method, with the aid of computer-assisted image analysis and stereological applications for the evaluation of the actual content of specific nucleic acids in the volume of a single cell. 6~ Since bone is a nonhomogenous tissue, ISHH, with its precise cellular localization, has the advantage over methods such as Northern blot or nuclease protection assay that analyze total RNA extracts. ISHH also allows analysis of the relationship between gene expression and cell size and activity directly in the bone microenvironment. 1. BONE PROCESSING

IV. MOLECULAR

BONE

HISTOLOGY

Among the powerful methodologies emerging in the investigation of bone cell metabolism are in situ hybridization histochemistry and immunohistochemistry. Obviously, however, the application of these techniques as routine morphological tools in the study of bone is still in its infancy. This section examines the basic principles of these methods and aims to serve as a comprehensive guide to molecular bone histology.

Bone samples to undergo ISHH may be processed frozen or after fixation. Sectioning of frozen specimens requires the use of heavy-duty cryostats. To date, these microtomes do not provide sections thin enough to allow precise cellular detection. Some techniques involve decalcification and embedding in paraffin. Even though processing of decalcified bone is easy, it does not allow the study of the relationship between cell gene expression and dynamics of bone. Alternatively, bone samples may be fixed with fresh 4% buffered formalin kept at

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4 ~ C for 24 hours, dehydrated, embedded in methyl methacrylate, and polymerized at cold temperature. 61 Plastic-embedded bones can be stored at 4 ~ C until use. Blocks can then be sectioned with regular microtomes equipped with carbine-edged knives (see above). Preparation of bone sections for hybridization after plastic removal includes (1) proteinase K permeabilization/digestion to allow probe penetration, (2) 0.2% buffered glycine rinses to prevent residual digestion by proteinase K, and (3) incubation in 0.25% acetic anhydride/ 0.1 M triethanolamine to eliminate electrostatic binding of probe to tissue sections. 2. PROBE PREPARATION Originally, in the late 1970s and early 1980s, DNA probes of incomplete homology (i.e., R N A - D N A hybrids) were used to analyze DNA gene sequences in tissue sections and cytological specimens. 62-64 By the early 1980s, the introduction of tritiated complementary DNA (cDNA) probes allowed the quantitation of RNA cellular content. 65 Initially, double-stranded cDNA probes were used. Because both sense (identical) and antisense (complementary) DNA strands are present in the hybridization mixture, a suboptimal denaturation can lead to selfreannealing of the cDNA strands, hence reduced formation of c D N A - m R N A hybrids. Even though single-stranded cDNA probes were preferred, their use resulted in rather unstable c D N A - m R N A hybrids. In the last decade, the development of cRNA probes has offered a convenient alternative to single-stranded cDNA probes in I S H H . 66 Single-stranded RNA probes are the most sensitive but require experience in handling of nucleic acids and access to cDNA templates. These synthetic oligonucleotide probes are short (25 to 45 bases), single strands of DNA complementary to a designated sequence of target mRNA, which can be chosen on the basis of published cDNA maps. These probes can be synthesized and labeled by run-off transcription of cDNA templates subcloned into expression vectors containing specific RNA polymerase promoters. 67 Antisense RNA probes display several features that make them attractive when studying mRNAs expressed at low levels. Ribonucleotides are efficiently incorporated and produce RNA probes with a high degree of specificity. From the same template, sense probes can be synthesized with sequences identical to those of target RNA, providing suitable controls for the evaluation of nonspecific binding and background. In addition, nonspecifically bound probes can be removed by RNase A digestion, because R N A - R N A hybrids are not degraded by this enzyme. This results in a favorable signal/background ratio for the detection of low-abundance RNA copies. To date, bone laboratories perform ISHH with

single-strand antisense RNA probes complementary to m R N A s . 68-73

Nucleic acid probes must be labeled with reporter molecules that allow their detection. The labeling must be stable enough to survive denaturing conditions, yield a good cellular resolution, and provide sensitivity for the detection of low mRNA copies. Some procedures for probe labeling employ isotopically labeled nucleotides (33p and 35S) that can be detected by autoradiographic liquid emulsion or by contact autoradiographic film. The level of sensitivity of ISHH employing isotopically labeled probes depends mainly on the specific activity of the probe (i.e., the amount of radioactivity incorporated per microgram of probe). Probes of high specific activity give high levels of autoradiographic signal per copy of target mRNA. However, this signal will be recognized only if the background is low. Thus, equally critical is a good signal/background ratio. The inconvenience of handling radioactive material has prompted the development of nonisotopic labeling protocols for nucleic acid probes. Most nonradioactive probes are produced by coupling of nucleotides with reporter molecules that can be detected by histochemical procedures such as immunohistochemistry or immunofluorescence. Biotin is one of the first molecules to be used for nonradioactive probe labeling. Once incorporated either as biotinylated-nucleotide or direct chemical biotinylation of the nucleic acid, biotin can be revealed by conventional histochemical technique. Most protocols use avidin/strepavidin systems conjugated with alkaline phosphatase or horseradish peroxidase. Alternatively, the biotinylated probe can be detected by binding of enzyme-linked antibiotin antibodies and subsequent immunohistological steps. Depending on the detection system used, the specific signal appears as deposition of either a colored substrate or a fluorescent emission on target cells. B iotinylated probes are very stable and can be prepared in large batches. With tissues rich in endogenous biotin, adequate blockade of endogenous biotin and negative control slides should always be included in each experiment. Digoxigenin-labeled nucleotides have recently been developed for labeling cDNA, cRNA, and oligonucleotide probes. Digoxigenin is detected by the immunohistochemical approach with specific antidigoxigenin antibodies. The procedure's specificity is due to the lack of an endogenous tissue source for digoxigenin in animal tissues. The controversy of sensitivity of nonradioactive versus radioactive labeling techniques is still open for discussion, but in the experience of the authors, antisense 33p-labeled cRNA probes should be used when high sensitivity is needed for the detection of low-copy-number mRNAs.

CHAPTER8 Bone Biopsies: A Modem Approach 3. PROBE-TISSUE HYBRIDIZATION

During the hybridization protocol, 1 million cpm of synthesized probe is applied per slide in 50 ml of hybridization buffer containing 50% formamide. The slides are then placed in a chamber humidified with 50% formamide and incubated overnight at 50 ~ C. The following day, coverslips are rinsed off in 2X SSC and unbound probe digested in 100 mg/ml RNase A. The slides are washed twice in 2X SSC (10 min/wash), incubated in 2X SSC at 65 ~ C for 1 hour, then transferred into fresh 2X SSC. Dehydration through graded ethanol and air drying follow. The slides are subsequently prepared for x-ray and/or emulsion autoradiography. Validation of the ISHH studies are ensured by the concomitant inclusion of control slides (1) pretreated with RNase A and (2) hybridized with sense-strand probes. Despite the invaluable information it provides, ISHH also has several limits that must be kept in mind when interpreting results. The sensitivity of ISHH is difficult to assess in different laboratories, and low levels of RNA gene expression may escape detection if the corresponding autoradiographic signal is dispersed over a large surface of the section or is masked by background. Therefore, caution must be used before important biological conclusions are drawn from negative results. Finally, specificity relies on the choice of suitable sequences of probe and stringency adopted for the hybridization procedure. Moreover, ISHH reveals only intracellular RNA transcripts and does not provide information about the subsequent steps of protein synthesis and extracellular secretion unless associated with immunohistochemistry.

B. I m m u n o h i s t o c h e m i s t r y In synergy with ISHH, application of immunohistochemistry (IHC) to bone tissue clearly provides an accurate assessment of the metabolic state of bone cells by visualizing protein production. Although the technique of IHC is well established in soft tissue, hard tissue such as bone provides an exceptional challenge. Thus, this highly complementary procedure to ISHH remains relatively unexplored as evidenced by a limited amount of literature describing the use of this technique in bone. Establishment of this technique in bone tissue will afford researchers the link between transcription and translation mechanisms in cells. Because the well-established technique of IHC is rarely being applied to bone tissue, care and caution must be exercised to ensure a reliable and reproducible outcome. Clearly, some of the factors that must be considered are type of fixative, tissue embedding procedure,

251 antibody compatibility and stability, and relative abundance of target protein in tissue. Immunohistochemistry studies of bone tissue have been performed on frozen tissue, TM paraffin, 75 or plastic76embedded sections. Application of this technique in frozen bone sections has it own limitations. Frozen sections provide poor cytological preservation, but antigen survival is better preserved. In contrast to frozen sectioning, morphological and cytological details are well preserved in paraffin sections, but the antigenicity is much reduced. Cold polymerization technique of plastic embedding allows for a good compromise between preservation of morphological features and antigenic stability. 76 Application of IHC to bone sections allows detection of antibody-antigen complex in bone. In general, whether it is a paraffin-embedded or plastic-impregnated bone sample, initiation of IHC requires removal of the embedding medium to uncover antigenic sites on tissue. This is achieved either by deparaffinization with xylene or plastic removal by use of methoxyethylacetate. In both cases, rehydration in phosphate buffer is required prior to initiation of the IHC protocol. Once the tissue is ready, slides are pretreated to remove endogenous peroxidases, preincubated in a blocking serum at room temperature, and then incubated in the selected primary antibody at 4 ~ C overnight. The following day, the slides are incubated in secondary antibody and processed through chromogenic reaction. This visualization of immunolabeled product is achieved by using either the basic immunoperoxidase method or commercially available diaminobenzidene (DAB) substrate kits. Once the reaction is stopped by excess phosphate buffer, the slides are taken through graded ethanol and xylene, affixed with mountant to coverslips, and examined under bright field light microscopy. Control slides are processed similarly with the exception of primary antibody omission resulting in absence of positive immunolabeling. Another control procedure requires preabsorption of the primary antibody prior to application on bone tissue sections. One or both controls should be performed in each IHC experimental applications.

V. E V A L U A T I O N

OF BONE

Optimal evaluation of bone at the tissue, osteon, cellular, or molecular levels relies on the observer's expertise in both qualitative and quantitative assessment of bone.

A. Q u a l i t a t i v e E v a l u a t i o n The end-point in clinical bone laboratories is the diagnosis based on microscopic examination of mounted

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sections of bone and bone marrow. Diagnosis represents a judgment call that is the pathologist's responsibility. The final decision may be based on qualitative or quantitative assessment of bone sections. Qualitative evaluation relies entirely on the experience, the process of continual reassessment, and the education of the pathologist. The principle of anatomic pathology, "The eye sees what the brain is prepared to see," 77 is particularly correct for bone pathologists. Although clinical diagnosis of metabolic bone diseases does not generally require quantification (such as overt osteopenia, osteomalacia, Paget's disease of bone, aluminum accumulation, etc.), it is highly desirable that a qualitative diagnosis be made by individuals with bone training and longstanding experience in bone morphometry. In other cases, quantification and appropriate controls should serve as the basis of a qualitative diagnosis. However, before quantitation of bone, the observer must decide whether a bone biopsy is suitable for morphometric analysis, where histological structures should be measured, how much area of the bone sample is required, and what elements must be evaluated. These decisions require training and regular evaluation.

B. Q u a n t i t a t i v e E v a l u a t i o n The goal of bone morphometry is to assign numerical values to the various elements constituting bone in order to statistically evaluate potential differences between groups of patients, normal individuals, or changes occurring after treatment modalities. Through the microscope, the bone section appears as a two-dimensional image consisting of profiles of cortical osteons and trabeculae, bone cells, and bone marrow. The primary measurements that can be performed are length, width, area, and profile counts. The principles of stereology have been applied to relate the two-dimensional profiles to the three-dimensional structure of bone78; therefore, length, width, and area become surface, thickness, and volume, respectively. Validity of the calculations involved in the transformation from two to three dimensions of bone profiles has been verified for all measurements except for profile count such as trabecular and cellular number, which is expressed in two dimensions. 78 Initially, measurements were performed by the manual or point-counting technique. This method involves the use of a grid placed in the microscope eyepiece. Several different grids can be employed. They consist of points linked by straight or curved lines that are projected over the bone section. The fraction of points overlying a structure (bone, osteoid, mineralized bone, or bone marrow) represents the area, or volume, of the

studied structure. Surfaces are similarly derived from the number of intersections between the grid lines and the boundaries of a structure. Distances between points or lines are measured with a micrometer mounted in the eyepiece. This cumbersome, time-consuming method requires measurements of a great number of optical fields, and the operator must calculate the histomorphometric parameters. The semiautomatic computerized method was introduced by Malluche et al. in the late 1970s. 79 It involves the use of a microscope equipped with a drawing tube overlying a digitalized tablet. The histological field is projected on the tablet, which is linked to a cursor with a central light and, optimally, several buttons. On the tablet a square is delineated representing an optical field. This square is bordered by a menu consisting of small squares that represent different types of measurements, such as bone volume, osteoid, erosion, osteoblast and osteoclast interface, cell count, fibrosis, and tetracycline labeling. When measuring an optical field, the operator activates the desired square on the menu. In each field, the operator measures area and length by tracing around structures or along interfaces. Profile counts are made by clicking the cursor button over each individual element. Using a cursor with several buttons allows, in addition to measurements of trabeculae, osteoid and erosion surfaces, and bone cells, to separately evaluate (1) lamellar or woven osteoid, (2)erosion surface with or without osteoclasts (reversal surfaces), and (3) fibrosis in front of erosion or formation surface. These various measurements are represented by different colors on a monitor to allow verification of the measurements. When an individual field has been measured, the bone slide is moved to the adjacent field. When the predetermined number of fields is reached (see below), the software automatically computes the histomorphometric parameters applying principles of stereology. Several software programs with various degrees of sophistication have been developed for use with the semiautomatic method. All programs are reliable for calculations of areas and counts; however, there are some differences in computation of lengths (surface). An entire trabecule is often analyzed over several optical fields. Within one field computer programs record the area accurately, whereas the boundary of the trabecule (surface) may also include the intercepts of the trabeculum with the borders of the field. This overestimates all surface parameters, which also affects all derived parameters. Therefore, a software must be chosen that automatically avoids this problem. Computerized-assisted histomorphometric analysis of bone also has the advantage of providing many means of data verification. Images of individual fields and the entire area of bone measured are stored, as well as all

CHAPTER 8 Bone Biopsies: A Modem Approach raw data and calculated parameters per field and for the entire bone area. Another advantage is that the data obtained can be automatically transferred to any statistical software, thus avoiding entry error. The semiautomatic method is rapid, reliable, and precise. The fully automated image analyzers include a video camera (color or black-and-white) that projects the image of an optical field onto a monitor. The computer is programmed to automatically separate components of bone according to their color or gray value. Modem automated analyzers are equipped with autofocus and an automatic scanning stage. They also represent the best means to measure parameters of bone microarchitecture such as star volume (see below). Automatic assessment of bone structure drastically reduces the time required for evaluation of bone slides. However, at present these analyzers are limited to assessment of parameters of bone structure. Discrimination of cellular details, detection of woven versus lamellar bone, and recognition of erosion surface by the currently available computers are not yet completely reliable. Complete automatic analysis of bone may be possible in the future with improved video cameras, other staining techniques, and enhanced computerized image-analysis capabilities. Regardless of the method employed, several standards have to be established before undertaking bone morphometry. The magnification at which bone fields may be evaluated should represent the best compromise between visual recognition of details and the greatest possible area in order to guarantee accuracy and save time. The degree of magnification also depends on the parameters to be measured. For instance, determination of bone architecture should be done at the lowest magnification possible in order to include the greatest possible number of trabecules, whereas counting bone cells should be performed using high magnification. The number of slides to be analyzed in a bone sample needs to be determined. For transiliac crest biopsies, several nonconsecutive sections are needed because of the small area of bone sample obtained and the greater variance in histomorphometric parameters between superior and inferior portions of the iliac crest. The number of sections needed is also much higher when the manual technique is employed. Bone samples obtained vertically do not present this problem. Because of the lower variance in parameters between anterior and posterior iliac bone, the number of sections needed is dramatically reduced. Moreover, the total area of bone (i.e., the number of fields to be measured) has to be determined statistically using classical methods of assessing the variance of histomorphometric parameters with increasing number of fields in order to obtain the smallest variance possible.

253 This total number of fields to be measured needs to be determined for each different disease entity under investigation and/or site of the skeleton to be assessed. When dynamic parameters of bone or aluminum deposition are analyzed, optimally the bone sections should be serial to the slides analyzed for static parameters of bone structure, formation, and resorption. This allows a more precise evaluation of derived parameters of bone dynamics and aluminum deposition, which include static parameters in their calculations.

C. H i s t o m o r p h o m e t r i c P a r a m e t e r s o f B o n e The nomenclature of histomorphometric parameters has been recently standardized by a committee of histomorphometrists assembled by the American Society of Bone and Mineral Research. The symbols and units adopted are detailed in the manuscript published by this committee. 25 Briefly, the method to report most of the data is as follows: S o u r c e - - Measurement~eferent The source indicates the structure that was analyzed. These structures include total biopsy core (Tt), cortical bone tissue (Ct), cancellous bone (Cn), periosteal surface (Ps), transitional zone (Tr.Z), diaphyseal bone (Dp), etc. The measurements are primary, consisting of area, perimeter, distance, and counts; or they are derived by computing parameters from several primary measurements. These measurements are reported in relation to a referent, for instance, bone surface (BS), bone volume (BV), tissue volume (TV), core volume (CV), osteoid surface (OS), erosion surface (ES), etc. 1. PARAMETERS OF BONE STRUCTURE

Bone volume/tissue volume and cortical thickness give information on bone mass in cancellous and cortical bone. However, it has been shown that bone volume relates only partially to bone mechanical strength and that microarchitecture and the degree of mineralization of bone matrix represent other determinants of bone strength. 8~ Architectural quality of cancellous bone relies on the number of trabecules, their shape, orientation, thickness, and connectivity. The number of trabecules may be assessed either through calculation 8~ or directly by counting the number of trabecular profiles observed on bone sections. However, a more accurate representation of trabecular number can be provided by other methods with three-dimensional reconstruction from two-dimensional images captured from serial bone sections. 81-84 These methods use computed tomography or ultrasound technology and

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MARIE-CLAUDE MONIER-FAUGERE, M. CHRIS LANGUB, AND HARTMUT H. MALLUCHE

either directly measure the number of trabecules or apply mathematical models. 85-87 Trabecules may appear as plates, especially in young adults, or as rods in older individuals. 8~ Moreover, trabecules are oriented vertically and horizontally to mechanical load. Measurements of their thicknesses can also be calculated or directly assessed. Formulae for calculations and methods of measurements differ according to the trabecule's shape and orientation. Three-dimensional reconstruction helps in determining trabecular shape and orientation and allows an accurate estimate of trabecular thickness. Connectivity can be directly assessed by determination of marrow star volume. 88 In this method, random points are taken in bone marrow and a number of lines are drawn radiating around the chosen points. The mean distances between these points and their intersection with bone represent the marrow star volume. The same technique can also be used to determine bone star volume. A bone sample with numerous perforations of trabecules and loss of connectivity presents with high marrow and low bone star volume. Another method to assess connectivity is strut analysis, which includes the measurements of the number of nodes and free ends (termini). 89'9~ Nodes represent the site in the trabecular network where trabecules are branched together. The loss of connectivity is associated with low number of nodes and high free ends. The ratio between nodes and termini and the strut length between node-to-node, terminus-to-terminus, node-to-loop, and node-to-terminus are also indices of connectivity. The degree of mineral density of the bone trabecules at the microscopic level also plays an important role in the mechanical ability of the skeleton to sustain load and deformation. Bone biopsies may be analyzed for their mineral content and quality. Several methods with various levels of sophistication are available to determine mineral content. Ash weight has been supplemented by ultrastructural biochemistry 91 or backscattered electron image analysis. 92'93 Newly formed bone presents with immature crystals, low mineral density, and high elasticity, whereas old bone packets are made up of mature crystals and denser bone, which is more brittle. The relative distribution between young and old bone packets plays a role in mechanical strength of bone. 2. PARAMETERS OF BONE FORMATION AND MINERALIZATION

Bone formation and mineralization are assessed by measurements of static and dynamic parameters. Static parameters include osteoid volume/bone volume (OV/ BV), osteoid surface/bone surface (OS/BS), osteoid thickness (O.Th), osteoblast surface/bone surface (Ob.S/ BS), and number of osteoblasts/bone perimeter (NOb/

BPm). In states associated with rapid and irregular formation of woven osteoid such as Paget's disease of bone, renal osteodystrophy, primary hyperparathyroid bone disease, and other conditions, it is essential to separately measure woven (Wo) or lamellar (Lm) osteoid and osteoblast parameters. Assessment of total osteoid and osteoblast parameters (including lamellar and woven parameters) may be misleading in studies with crosssectional and especially longitudinal designs, since osteoid parameters may be similar; whereas the relative distribution between the two osteoid types may be very different and denotes different pathological processes. Tetracycline double-labeling of bone allows determination of mineralization status, bone formation rates, and remodeling times. Osteoid and bone surfaces undergoing active mineralization show either double or single labels. The mineralizing surface is calculated as the sum of doubly labeled plus half of singly labeled surfaces per bone surface (MS/BS). Mineral apposition rate (MAR) represents the mean speed at which individual osteoid seams are mineralized. This entails the measurement of the mean distance between the centers of the two tetracycline labels (corrected for obliquity) divided by the number of days separating the administration of the two labels. This is expressed in txrn/day. This parameter indicates the mean rate at which individual active osteoid seams are being mineralized at their remodeling sites. At the bone level, adjusted apposition rate (Aj.AR) gives a more precise representation of the rate at which all osteoid seams are mineralized and is calculated as MAR * MS/OS. Similarly, parameters that assess the timing of mineralization, that is, the mean interval between deposition of osteoid and mineralization, may be calculated at individual remodeling sites (osteoid maturation time, Omt = O.Th/MAR) or over all the osteoid seams involved in active mineralization (mineralization lag time, Mlt = O.Th/Aj.AR). Estimates of these parameters are essential for the diagnosis of osteomalacia or mineralization defect. In the absence of osteomalacia, bone formation rates (BFRs) may be calculated; they represent the mean amount of mineralized bone formed per unit of time. 25 They can be expressed over different referents. Bone formation rate per tissue volume (BFR/TV) is a useful parameter when studying relationships between bone metabolism and systemic markers of bone. BFR at the bone level (BFR/BV) gives information on bone age. BFR expressed per bone surface (BFR/BS) represents the parameter of choice when assessing the effects of a drug on bone remodeling. BFR at the osteoid level (BFR/OS) is similar to Aj.AR. The activity of individual osteoblasts can also be determined by calculating BFR per osteoblast (BFR/Ob). This parameter indicates whether a

CHAPTER 8 Bone Biopsies: A Modem Approach given rate of bone formation depends on the number or activity of individual osteoblasts or both. 3. PARAMETERS OF BONE RESORPTION

Unlike bone formation, bone resorption can be assessed only by static parameters because of the lack of a safe in vivo dynamic marker of bone erosion. Static parameters are essentially represented by erosion surfaces, number of osteoclasts, and erosion depth. Erosion surface per bone surface (ES/BS) represents the total percentage of bone surface that shows signs of past or present resorption. This comprises active resorption lacunae with osteoclasts, lacunae with mononuclear cells, and empty resorption spaces. The last represents reversal surface (RS). Optimally, these different erosion surfaces are measured separately and indicate the intensity of bone remodeling. The number of osteoclasts and their interface with bone surface are also good indicators of the intensity of bone resorption. Erosion depth (E.De) is the sole parameter that reflects, even though statically, osteoclastic bone-resorbing activity. Several methods m direct and indirectmhave been employed to measure e r o s i o n d e p t h . 94-98 Depths of erosion lacunae have been determined either by the number of lamellae eroded from the trabecular surface multiplied by lamellar t h i c k n e s s 95'98 o r by computerized reconstruction of the resorptive s p a c e . 96-98 Lamellar count is time consuming, whereas computerized reconstruction is rapid and less cumbersome. The characteristics of the resorptive space under study have also varied among investigators. Some measure erosion surface containing no or mononucleated c e l l s , 95'96 whereas others reconstruct the erosion space from sites of newly formed bone. 97 Comparison of these various methods of E.De measurements in the same bone samples showed that all results were highly correlated but were significantly different and had different levels of variance. 98 On histological bone slides, the number of fully completed resorption sites is low, and this is responsible for a high variance. Moreover, it is impossible for the observer to know whether the mononucleated cells are still engaged in resorption or represent preosteoblasts. On the other hand, reconstruction of resorptive spaces from formative sites allows clear visualization of the cement line, which marks the time between cessation of resorption and onset of formation. This allows more accurate assessment of E.De and compares better with wall thickness in the assessment of bone balance (see below). Moreover, resorption proceeds much faster than formation, and therefore the number of formative sites is much higher than completely resorbed lacunae, thus reducing the variance of E.De measurements. More recently, techniques employing scanning electron and confocal microscopy to obtain three-dimen-

255 sional estimates of erosion depth have been investigated. 99 Even though these techniques can provide more accurate assessments of E.De, scanning of an entire bone biopsy by these techniques may be time consuming and limit bone analysis to bone resorption. 4. PARAMETERS OF BONE REMODELING, BONE TURNOVER, AND BONE BALANCE

The time needed for completion of a remodeling cycle that achieves the complete renewal of a bone packet is called the remodeling period (Rm.P). It includes the resorption (Rs.P), reversal (Rv.P), and formation (FP) periods. In addition, the duration between two consecutive activations of the same bone surface includes the quiescent period (QP) and is represented by the total period (Tt.P = Rm.P + QP). The formation period represents the time needed to complete a new bone packet, with a measurable width, that is, wall thickness (W.Th). FP is calculated by dividing W.Th by adjusted mineralization rate (Aj.AR). All other periods are derived from FP, and calculations of Rv.P, Rs.P, and QP assume steady state between every phase of the remodeling cycle, when percentage of space (bone surface) equates percentage of time (period). TM These periods are calculated as FP multiplied by the ratio of the appropriate surface (Rv.S, Rs.S, and QS) by osteoid surface (OS). The histomorphometric assessment of bone turnover is done by activation frequency (Ac.f), which indicates the probability for a new remodeling cycle to be initiated at any point of the trabecular surface. It is calculated, therefore, as the inverse of total period (Tt.P) and this probability is expressed per year. A simplification of the formula shows that Ac.f = BFR/BS + W.Th. Therefore, assessing bone turnover by use of BFR/BS alone may sometimes be inaccurate when great variations in wall thickness occur. Bone balance represents the net effect of the remodeling cycle on the amount of bone gained or lost. Bone balance on a bone section can be assessed by calculating the difference between mean values of W.Th and E.De (i.e., the average of bone formed minus the average amount of bone resorbed). 5. MOLECULAR PARAMETERS OF BONE

The same general principles of morphometry (see above) apply to molecular morphometry. In particular, it is critical when counting radiolabeled grains per cell to determine variability between various runs of the techniques, to assess the number of cells to be measured, and to correct for background nonspecific grains. Quantitation may be done manually or semiautomatically with specially designed software (e.g., the National Institutes of Health [NIH] imaging software program). For each individual molecule tested, the relative number of oste-

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oblasts, osteoclasts, lining cells, osteocytes, or bone marrow cells containing grains may be measured, as well as the number of grains per individual cell. These data can also be directly correlated to activity of osteoblasts measured by bone formation rate per cell or erosion depth. The total number of grains expressed may also be related to parameters of bone remodeling and bone turnover.

and the laboratory environment. The external component includes proficiency testing, continual education, and audit or inspection by certified agencies. Quality assurance activities usually follow several steps of monitoring and evaluation. First, a set of quality indicators is determined. Thresholds or levels of tolerance at which corrective actions should be taken are then established for each step.

D. N o r m a l Values Since metabolic bone diseases are mainly characterized as a departure from normal in quantitative terms, the establishment of normal ranges for the various histomorphometric and molecular parameters is essential for diagnosis and research purposes. Indeed, great efforts have been made to collect bone from normal subjects. 44'1~176176 However, most normative data were obtained from cadaveric bone; thus cellular components are of borderline quality and no information on dynamic parameters can be obtained. Several investigators have succeeded in gathering bone samples from normal living subjects after tetracycline labeling, ~~176176 and their results have been used extensively. However, there is still a need to increase the pool of normative values. In each study, normative data should be obtained from individuals with the same gender, age, geographical area, socioeconomic background, lifestyle, and nutritional habits. Moreover, processing methods and histomorphometric techniques should be similar to those applied to assess bone parameters in patients or the study population.

E. Q u a l i t y A s s u r a n c e a n d Q u a l i t y C o n t r o l Bone laboratories involved in diagnosis and/or research may benefit greatly from adopting the principles and implementing the measures of quality assurance and quality control as used in laboratories of other disciplines. This issue was addressed during the Vlth International Congress of Bone Morphometry, 1992, held in Lexington, KY, 1~ and the adopted recommendations are summarized in the proceedings of this meeting. 1~ Briefly, implementation of quality assurance helps to identify and prevent mistakes and contributes to precise and accurate results. Precision is defined as the closeness with which replicate analysis is made. Internal controls are employed to quantify and minimize imprecision. Accuracy, on the other hand, is the closeness to true value. Usually, external controls are employed in order to estimate accuracy. The internal component of quality assurance includes organization of laboratory and personnel, specimen handling, reports, performance evaluation,

VI. INDICATIONS FOR AND INFORMATION DERIVED FROM BONE BIOPSIES Over the years, indications for bone biopsies have continuously evolved depending upon clinicians' attitudes; changes in pattern of individual metabolic bone diseases, such as in renal osteodystrophy; the development of new noninvasive measures of bone mass or bone turnover; and the institution of new, efficient therapies. Regardless of the numerous advances made in the diagnosis and management of patients suffering from metabolic bone diseases, bone biopsy represents the most informative and only tool that allows assessment of the mechanisms underlying the various bone diseases and the effects of therapies at the cell, osteon, and tissue levels.

A. O s t e o p o r o s i s Osteoporosis is the most frequent metabolic bone disease, and its economic cost creates an enormous burden on society, especially because of the aging baby boomers. During the past several decades, intense research has been conducted to better understand, prevent, and treat this crippling condition. Information derived from bone biopsies has been instrumental in comprehending the cellular mechanisms that lead to osteoporosis and in evaluating the various available preventive and therapeutic approaches. The role of bone biopsy in the diagnosis of osteoporosis has changed over the years, however. Early on, osteoporosis was defined as low bone mass (compared to young adults) and was associated with fractures with minimal or no trauma. The diagnosis was made after fractures occurred. However, before the fractures occurred, measurement of bone volume/tissue volume on bone biopsy was the only available means to establish the diagnosis of low bone mass (i.e., osteopenia) (Fig. 8-21). This index was rather crude m especially when transiliac bone biopsies were u s e d - - b e c a u s e of the large variance in bone volume between superior and inferior

CHAPTER 8 Bone Biopsies: A Modem Approach

257

FIGURE 8 - 2 1 Osteopenia. Trabeculae cut perpendicularly to their long axis. Reduced connectivity between trabeculae. Undecalcified, 3 txm thick section of human iliac bone (modified MassonGoldner trichrome stain; X7.5). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8 - 2 2 Osteopenia with osteomalacia. Reduced cancellous bone mass and increase in osteoid seam thickness. Undecalcified, 3 Ixm thick section from tibia of an oophorectomized and vitamin Ddeficient rat (modified Masson-Goldner stain; x20). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

parts of the iliac bone. Bone volume/tissue volume was found to be lower in patients with fractures than agematched controls; however, there was a large overlap in bone volume between these two populations. 1~ With the growing emphasis not only on bone mass but also on architecture and mechanical competence of bone, osteoporosis is now defined as a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture (definition adopted by the World Health Organization). Noninvasive and sensitive techniques to measure bone mineral density have been refined and have supplanted bone biopsies as a means to assess bone mass. Thus, the role of bone biopsy to diagnose and manage osteoporosis has changed and reflects the great progress made in the field. Briefly, there are three main reasons that bone biopsies can be indicated to diagnose and manage patients at risk of developing osteopenia or with established fractures: (1) to establish bone quality, in particular the degree of mineralization and microarchitectural derangements; (2) to assess bone turnover and bone balance; and (3) to analyze the effects of treatment on bone structure and bone turnover. Mineralized bone histology after tetracycline doublelabeling is the only means that can establish whether bone tissue undergoes a normal pattern of mineralization and ensure that the patient does not suffer from a mineralization defect of frank osteomalacia with or without concomitant osteopenia (Figs. 8 - 2 2 and 8 - 2 3 ) . Confusion between these two diseases or their association is not uncommon. Vertebral crush fractures have been described in patients with osteomalacia. '~ Also, biochemical profiles indicative of osteomalacia have been inconclusive or normal in patients who underwent bone

biopsies for other reasons and who revealed histologically a mineralization defect. 16'1~ The clinical significance of the diagnosis of mineralization defect or osteomalacia is obvious, since appropriate measures can be undertaken to correct this abnormality, whereas available antiosteoporotic treatments would not have any effect or might even be harmful. Indeed, defective mineralization has been discovered in patients who underwent bone biopsies to find an explanation for antiosteoporotic treatment failure. Bone microarchitecture, another indicator of bone quality, is also a predictor for successful therapy. When bone mass decreases in cancellous bone, it is essential to determine whether this phenomenon is due to thinning of trabecules and/or disruption of the trabecular network because of trabecular perforation. Patients presenting mainly with thin trabecules are more amenable to treatments designed to increase trabecular thickness, whereas there is no current proven therapy that can restore tra-

FIGURE 8--23 Osteopeniawith mineralization defect. Presence of single tetracycline label with wide osteoid seam. Undecalcified, unstained, 7 lxm thick section of human iliac bone (X31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

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becular connectivity (i.e., the network). In cortical bone, thinning of the cortices and increased porosity are indices of loss of cortical integrity. Both envelopes or one envelope only may or may not be altered concomitantly. It has been shown that patients with vertebral crush fractures present predominantly with alterations in cancellous bone structure and architecture, whereas those suffering from hip fractures have thinner cortices. It remains to be shown whether these differences are due to sequential bone loss in the two bone compartments or to constant compartmental differences. The exact degree of bone turnover and bone balance (i.e., the mechanisms underlying the ongoing bone loss) can also only be determined through the use of mineralized histology. Even though bone loss is the overall result of more bone resorption than formation, osteopenia is not a homogeneous disease and is characterized by a high degree of heterogeneity with time in the same patient and between patients. With time, for instance, men and w o m e n lose bone through two different patterns. As men age, they lose bone slowly and continuously. Women, on the other hand, start to lose bone slowly before menopause but experience a rapid bone loss after menopause, which lasts a few years and then slows down. We have shown in an experimental model of cessation of ovarian function that the early rapid bone loss (seen in the first 4 months) was accompanied by a dramatic increase in osteoclastic activity as evidenced by deeper erosion cavities. 1~ This was followed later by an increase in bone turnover, normalization of erosion depth, and decrease in osteoblastic activity. 1~ The late decrease in bone formation was confirmed by the observation of thinner walls with age. ll2 Therefore, the nature of the imbalance between bone formation and resorption varies with time, but also any increase in bone turnover worsens the effects of this imbalance, thus accelerating bone loss. Bone turnover also varies from patient to patient. Noninvasive serum and urinary parameters have a limited use because they are sensitive to very high bone turnover. 113 In the majority of patients, available noninvasive parameters do not indicate bone turnover. Approximately 30% of osteoporotic patients present with high bone turnover; the others show low or normal turnover. High-turnover osteopenia (Figs. 8 - 2 4 to 8 - 2 7 ) is characterized by an increase in osteoid surface and volume with normal osteoid thickness. There is a highnormal or increased number of osteoclasts and osteoblasts. Mineralizing surface/bone surface and activation frequency are increased, whereas mineral apposition rate and mineralization lag time are normal (Fig. 8 - 2 7 ) . Low-normal turnover osteopenia (Figs. 8 - 2 8 and 8 29) is characterized by few remodeling foci and a re-

FIGURE 8 - 2 4 High turnover osteopenia. High fraction of trabecular surface covered by osteoid and, on the opposite side of the trabecular, extended resorption zones. Undecalcified, 3 Ixm thick section of human iliac crest bone (modified Masson-Goldner stain; x31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8 - 2 5 High turnover osteopenia. High fraction of trabecular surface covered by osteoid and presence of numerous osteoblasts covering the osteoid seam. Undecalcified, 7 Ixm thick section of human iliac bone (modified Masson-Goldner stain; x40). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8--26 High-turnoverosteopenia. Extended resorption lacunae with and without multinucleated osteoclasts. Undecalcified, 3 pLm thick section of human iliac bone (modified Masson-Goldner stain; X50). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

CHAPTER 8

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Bone Biopsies: A Modem Approach

FIGURE 8 - 2 7 High-turnover osteopenia. High fraction of trabecular surface exhibiting tetracycline double labels. However, distance between labels varies from normal to low with appearance of merging of labels. Undecalcified, unstained, 7 txm thick section of human iliac bone. Fluorescent light microscopy (x25). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8--29

Low-turnover osteopenia. Reduced tetracycline uptake. No double labels, only single labels of low intensity. Undecalcifed, 7 txm thick section of human iliac bone. Fluorescent light microscopy (X31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

B. O s t e o m a l a c i a duction in osteoblast and osteoclast number. Accordingly, osteoid, erosion, and mineralizing surfaces are usually lower than normal (Fig. 8-28), and activation frequency is low (Fig. 8-29). Bone biopsies and histomorphometry have become an FDA-required outcome of any experimental or clinical trials assessing the effects of antiosteoporotics on bone. With the recent availability of antiresorptive agents and bone formation stimulators, bone biopsies performed before and after long-term therapy provide information on the effects of a drug on: (1) bone microarchitecture; (2) bone turnover; and thus (3) bone quality including bone age determined by direct assessment of bone mineral density, quality of apatite crystals, and microbiomechanics of trabecules. Moreover, analysis of pre- and posttreatment biopsies may help in delineating characteristics separating responders from nonresponders to the drug under investigation.

FIGURE 8 - 2 8

Low-turnover osteopenia. Absence of osteoclasts and resorption zones. Absence of osteoid and osteoblasts. Undecalcified, 3 ~m thick section of human iliac (modified Masson-Goldner stain; X31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

Osteomalacia was first described by PomlTler 114 as an abnormality in which newly formed bone matrix fails to mineralize in a normal or timely manner. The consequence of this condition is an accumulation of unmineralized matrix (i.e., osteoid), which leads to bone fragility. Osteomalacia in its fully developed stage or its milder form (called mineralization defect) develops in a large variety of pathological conditions that include renal failure (see below), nutritional deficiency, vitamin D resistance, gastrointestinal and liver diseases, status following bypass surgeries, medications inducing the cytochrome P-450 such as anticonvulsants, and the Fanconi syndrome. Florid nutritional tickets and osteomalacia with clinical, biochemical, and roentgenologic manifestations are rarely seen in Western countries, and few biopsies are performed to establish the diagnosis of osteomalacia. Histologically, these severe forms exhibit "active" osteomalacia because of the combination of vitamin D deficiency with secondary hyperparathyroidism. Osteoid volume and surface are increased, but, most importantly, osteoid seams are wide (Fig. 8-30). However, to establish the diagnosis of abnormal mineralization, wide osteoid seams must be accompanied by increased mineralization lag time. Mineralizing surfaces may be high, double labels are rare, and tetracycline uptake is seen as broad, diffuse, single labels. Mineral apposition rate is very low or cannot be measured (Fig. 8-31). Active osteomalacia is also characterized by the numbers of osteoblasts and osteoclasts varying from low-normal to high-normal (Fig. 8-32). Woven bone, woven osteoid (Fig. 8-33), and peritrabecular or marrow fibrosis may be seen. Besides these now rare and dramatic histological changes, a mineralization defect can be diagnosed in occult forms of nutritional deficiency, especially in the el-

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FIGURE 8 - 3 0 Osteomalacia. Excessive accumulation of osteoid and increased width of osteoid seams. Undecalcified, 3 Ixm thick section of human iliac bone (modified Masson-Goldner stain; x20). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8 - 3 1

Osteomalacia. Diffuse broad single labels of intense fluorescence. Undecalcified, unstained, 7 Ixm thick section of human iliac bone. Fluorescent light microscopy (x126). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8 - 3 3 Osteomalacia. Lamellar osteoid interspersed with woven osteoid and woven bone. Surface osteoid is mainly of the lamellar type. Undecalcified, 3 Ixm thick section of human iliac bone. Polarized light microscopy (modified Masson-Goldner stain; x 126). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate. derly, alone or associated with osteopenia. In these cases, bone biopsy is the only means to diagnose the mineralization defect, which might be found unexpectedly. The histological characteristics are similar to florid osteomalacia but less pronounced. Osteoid thickness is above normal but not as severely increased as in florid osteomalacia and is accompanied with normal or increased osteoid surface. In these cases, tetracycline doublelabeling is clearly needed to establish the uncoupling between osteoid apposition and its mineralization as evidenced by an increase in mineralization lag time. Bone cell numbers may be increased, normal, or low. When they are low, tetracycline labels usually appear as thin single labels (Figs. 8 - 3 4 and 8-35). C. P r i m a r y H y p e r p a r a t h y r o i d i s m Over the years, the need for bone biopsy to establish the diagnosis of primary hyperparathyroidism has dra-

FIGURE 8--32

Osteomalacia. Increase in fraction of trabecular surface covered by osteoid. Osteoid seam thickness high normal to increased. Trabecular surface without osteoid coating is being resorbed. Resorption cavities within mineralized trabecules under osteoid. Undecalcified, 3 Ixm thick section of human iliac bone (modified Masson-Goldner stain; x50). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8 - - 3 4 Mineralization defect. Increased fraction of trabecular surface covered by osteoid. Osteoid seam thickness increased. Undecalcified, 3 Ixm thick section of human iliac bone (modified MassonGoldner stain; x31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

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CHAPTER 8 Bone Biopsies: A Modem Approach

FIGURE 8 - 3 5 Mineralization defect. Absence of tetracycline double labels, only a few thin or broad labels of low intensity. Undecalcified, unstained, 7 lxm thick section of human iliac bone. Fluorescent light microscopy (X31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

matically decreased. A routine serum chemistry profile detects mild hypercalcemia. Sensitive radioimmunoassays or immunoradiometric assay for determination of serum parathyroid hormone (PTH) 115 confirm the diagnosis of primary hyperparathyroidism. In developed countries, advanced cases of primary hyperparathyroidism with clinical and radiological skeletal abnormalities and histological signs of severe "osteitis fibrosa cystica" have virtually disappeared. However, in approximately 10% of c u r r e n t c a s e s , 116 normocalcemic patients with borderline or inconsistent elevations in serum PTH levels or hypercalcemic patients with serum PTH levels in the normal range might benefit from a bone biopsy. The reason is that bone reflects the integral of PTH activity over time, whereas serum PTH levels fluctuate throughout the day; thus, interpretation of a single blood test may be inconclusive. Also, bone biopsies performed for other medical reasons, such as osteoporosis or nephrolithiasis, may reveal signs of excessive PTH activity on bone. In most cases (i.e., mild to moderate primary hyperparathyroidism), mineralized bone histology shows various degrees of an increase in bone turnover with signs of abnormal bone formation. Quantitative evaluation is sometimes needed for borderline cases. The increase in osteoid volume and surface may be accompanied by normal or slightly elevated osteoid thickness. Typically, some osteoid seams exhibit a woven texture (Fig. 8-36), and some peritrabecular fibrosis (Figs. 8 - 3 7 and 8 - 3 8 ) may be seen. Extent of erosion surface and erosion depth are also increased (Fig. 8-38). The number and size of all bone cells, including osteoclasts, osteoblasts, and osteocytes are increased (Figs. 8 - 3 6 to 8 38). Fluorescent light microscopy reveals that the number of osteoid seams undergoing active mineralization is increased (Fig. 8-39). The mineralization apposition

FIGURE 8--36

Primary hyperparathyroid bone disease. Appearance of woven osteoid. Undecalcified, 3 Ixm thick section of human iliac bone. Polarized light microscopy (modified Masson-Goldner stain; • (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

rate is normal or elevated (Fig. 8-39), and there is typically no delay in mineralization. Woven osteoid seams may show double labels or diffuse, broad, single uptake of tetracycline (Fig. 8-40). Bone structure may also be altered. Typically, there is an increase in porosity of cortical bone, which can be pronounced and undergo "cancellization." However, in cancellous bone, bone volume/tissue volume and trabecular connectivity may vary depending on the prevailing bone balance. When bone balance is normal or positive, PTH has been shown to exert an anabolic effect on b o n e 117 and therefore bone volume may be normal or increased. If primary hyperparathyroidism occurs in postmenopausal women and patients with malnutrition or prolonged immobilization, osteopenia may ensue. D. R e n a l O s t e o d y s t r o p h y Renal failure induces a wide array of metabolic abnormalities, and those affecting mineral and bone me-

FIGURE 8--37

Primary hyperparathyroid bone disease. Increased number of osteoblasts. Peritrabecular fibrosis. Undecalcified, 3 p,m thick section of human iliac bone (modified Masson-Goldner stain; x 126). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

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FIGURE 8--40

FIGURE 8--38

Primary hyperparathyroid bone disease. Increase in the number of osteoclasts and increased extent of trabecular surface exhibiting resorption lacunae. Increased density of osteocytes. Marrow fibrosis. Undecalcified, 3 txm thick section of human iliac bone (modified Masson-Goldner stain; • (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

Primary hyperparathyroid bone disease. Appearance of bright and widespread labels reflecting tetracycline uptake in woven bone. Undecalcified, 7 Ixm thick section of human iliac bone. Fluorescent light microscopy (x126). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

tabolism have been well established. 11s-~22 Bone metabolism is profoundly altered to compensate for the systemic mineral abnormalities. As a result, renal osteodystrophy develops in the early stages of loss of excretory kidney function. 123-125 More than 50% of patients exhibit abnormal bone histology when their glomerular filtration rate falls to less than 50% of normal, x26 When end-stage renal failure (ESRD) ensues, requiring chronic maintenance dialytic therapy, nearly all patients have abnormal bone histology. Despite a common pathogenic pathway, renal osteodystrophy is not a uniform bone disease. Classically, the various forms of renal osteodystrophy encompass (1) predominant hyperparathyroid bone disease, (2) low-turnover bone disease including osteomalacia and adynamic bone disease, and (3) mixed uremic osteodystrophy. 16'127-129 Transformation from one form to another can occur.

BONE DISEASE

1. PREDOMINANT HYPERPARATHYROID

FIGURE 8--39

Primary hyperparathyroid bone disease. High fraction of trabecular surface exhibiting tetracycline uptake. Undecalcifled, unstained, 7 txm thick section of human iliac bone. Fluorescent light microscopy (• (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

The main abnormality in predominant hyperparathyroid bone disease (PHBD) is a marked increase in bone turnover. Trabecules are irregular in shape and display numerous abnormal remodeling sites (Fig. 8-41). An unusually high number of bone cells are irregular in shape and arrangement. Numerous enlarged osteoclasts contain multiple nuclei with prominent nucleoli. Deep, irregular resorption cavities frequently dissect or tunnel the trabecules. The normal cuboidal shape of the osteoblasts becomes polygonal or spindle shaped. Nuclei containing several nucleoli lose their polarity and occupy random positions within the cytoplasm of osteoblasts. Atypical multilayered arrangements of cells with variable often-

FIGURE 8--41 Renal osteodystrophy--predominant hyperparathyroid bone disease. High fraction of trabecular surface covered by osteoid seams. High osteoid-osteoblast interface. High boneosteoclast interface with appearance of tunneling resorption. Marrow fibrosis. Undecalcified, 3 lxm thick section of human iliac bone (modified Masson-Goldner stain; x31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

CHAPTER 8 Bone Biopsies: A Modem Approach tation toward the bone surface (from parallel to perpendicular) can replace the usual palisade-like monolayer of osteoblasts. These morphological abnormalities of osteoblasts are accompanied by profound perturbations of their function. There is an increase in osteoid surface and volume, and sometimes an osteoid seam thickens because of an overproduction of collagen. The resulting osteoid is primarily of the woven, irregular type (Fig. 8 - 4 2 ) . Because collagen fibers accumulate towards the bone surfaces and are deposited between osteoblasts and towards the bone marrow, peritrabecular and bone marrow fibrosis ensues. In advanced cases, bone marrow can be entirely replaced by fibrosis. This reflects the irregular activity of disorganized groups of osteoblasts in which the regular activity of individual cells is decreased. It is conceivable that the cellular hyperplasia might represent a compensatory p h e n o m e n o n to the cellular insufficiency. Numerous and irregular osteocytic lacunae within woven osteoid and mineralized bone result from the increased number of osteoblasts in bone, while the extent and number of mineralizing sites and the mineral apposition rate are notably increased (Fig. 8 - 4 3 ) . The deposition of calcium in woven osteoid proceeds irregularly, incompletely, and diffusely. Findings of thick osteoid seams and irregular, diffuse tetracycline uptake in woven bone are sometimes erroneously diagnosed as osteomalacia (Fig. 8 - 4 4 ) . A marked increase in bone turnover often leads to cancellization of the cortical bone, which causes a net decrease in cortical bone volume most evident in the appendicular skeleton. P H B D usually presents with high bone volume. However, malnutrition, immobilization, or other causes can also markedly reduce bone volume. Ad-

263

FIGURE 8 - 4 3 Mineralization in predominant hyperparathyroid bone disease. Increased fraction of trabecular surface exhibiting tetracycline uptake. The majority of the tetracycline labels exhibit regular double labels. Undecalcified, unstained, 7 Ixm thick section of human iliac bone. Fluorescent light microscopy (X22.5). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

ditionally, areas of high bone mass may be adjacent to pseudocysts, which primarily consist of fibrotic or hyperplastic bone marrow. In any case, bone strength cannot be equated with high bone volume in these patients because the irregular trabeculae may lose their proper three-dimensional architecture and their connectivity. Moreover, the trabeculae are poorly mineralized and consist mainly of mechanically deficient woven bone with a propensity to fracture. Since characteristics of this bone disease create a particularly fragile bone prone to fractures, the use of the term "osteosclerosis" is inappropriate.

2. Low-TURNOVER BONE DISEASE The second histological type of renal osteodystrophy is low-turnover uremic osteodystrophy, which has two subgroups: osteomalacia (LTOM) and adynamic renal

FIGURE 8--42 Woven osteoid and woven bone in predominant hyperparathyroid bone disease. Appearance of irregular crisscross pattern of mineralized bone (blueish gray) and osteoid (golden yelloworange) surrounding an island of previously deposited normal lamellar bone exhibiting the typical birefringence (rust-brown). Undecalcified 3 Ixm thick section of human iliac bone. Polarized light microscopy (modified Masson-Goldner stain; x50). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8--44 Mineralization in predominant hyperparathyroid bone disease. Presence of diffuse intense single labels in woven bone adjacent to double labels. Undecalcified, unstained, 7 Ixm thick section of human iliac bone. Fluorescent light microscopy (x 31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

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FIGURE 8 - 4 5 Renal osteodystrophy--low-turnover osteomalacia. Increase in osteoid volume. Absence of osteoblasts and surface resorption. Undecalcified, 3 Ixm thick section of human iliac bone (modified Masson-Goldner stain; • (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8 - 4 7 Mineralization defect in low-turnover osteomalacia. Absence of double labels. Thin single labels at the osteoid-bone interface of low intensity. Undecalcified, unstained, 7 Ixm thick section of human iliac bone. Fluorescent light microscopy (• (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

b o n e disease (ABD). This type is characterized by a dramatic reduction in the n u m b e r of b o n e - f o r m i n g and bone-resorbing cells and is associated with a significant decrease in bone formation and mineralization. L o w - t u r n o v e r uremic osteodystrophy represents the other end of the spectrum from P H B D . The histological hallmark of this group is a profound decrease in the n u m b e r of active r e m o d e l i n g sites (Figs. 8 - 4 5 and 8 - 4 6 ) . Certain features reflect a m a r k e d decrease in osteoblastic activity. The majority of the trabecular bone is c o v e r e d by lining cells, and there are few osteoclasts and osteoblasts. B o n e structure is predominantly lamellar, sites of active b o n e formation are rarely seen, and the extent of mineralizing surfaces is markedly reduced. Usually only a few thin single labels of tetracycline are observed (Figs. 8 - 4 7 and 8 - 4 8 ) . The two histological subgroups within this type can be identified depending on the sequence of events lead-

ing to a decline in the n u m b e r and/or activity of the osteoblasts. LTOM is characterized by an accumulation of unmineralized matrix in which a diminution in mineralization precedes or is m o r e p r o n o u n c e d than the inhibition of collagen deposition. B o n e volume/tissue volu m e m a y vary; however, mineralized bone v o l u m e is always low. In the case of LTOM, unmineralized bone represents a sizable fraction of trabecular bone volume. The increased lamellar osteoid v o l u m e is due to the presence of wise osteoid seams that cover a large portion of the trabecular surface. The occasional presence of w o v e n bone buried within the trabecules indicates past high bone turnover. W h e n osteoclasts are present, they are usually seen within trabecular bone (Fig. 8 - 4 9 ) or at the small fraction of trabecular surface left without osteoid coating.

FIGURE 8 - 4 6 Adynamic uremic bone disease. No accumulation of osteoid. Absence of bone formation of resorption. Undecalcified, 3 txm thick section of human iliac bone (modified Masson-Goldner stain; • (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8--48 Adynamic uremic bone disease. Absence of tetracycline uptake documenting no mineralizing activity. Undecalcified, 7 Ixm thick section of human iliac bone. Fluorescent light microscopy (X31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

CHAPTER 8 Bone Biopsies: A Modem Approach

FIGURE 8 - 4 9

Renal osteodystrophy--low-turnover osteomalacia. Osteoclasts resorbing bone within trabecular bone and underneath osteoid. Fibers filling the resorption lacunae and thin layer of peritrabecular fibrosis (modified Masson-Goldner stain; • 126). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

With ABD, bone volume is frequently reduced. A reduction in mineralization is coupled with a concomitant and parallel decrease in bone formation. ABD is characterized by few osteoid seams. 3. MIXED UREMIC OSTEODYSTROPHY

Mixed uremic osteodystrophy (MUO) is caused primarily by increased parathyroid hormone activity on bone and defective mineralization with or without increased bone formation. These features may coexist in varying degrees in different patients. Bone volume/tissue volume is extremely variable and depends on a dominant pathogenic cause. Increased numbers of heterogeneous remodeling sites can be seen (Fig. 8-50). The number of osteoclasts is usually increased. Because active foci with numerous cells, woven osteoid seams, and peritra-

FIGURE 8 - - 5 0 Mixed uremic osteodystrophy. Increased fraction of trabecular surface covered by osteoid. Mean thickness of osteoid seams is increased, resulting in high osteoid volume. Increased extent of trabecular surface exhibiting resorption lacunae filled with or void of osteoclast. Undecalcified, 3 Ixm thick section of human iliac bone (modified Masson-Goldner stain; x7.5). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

265

FIGURE 8 - 5 1 Mixed uremic osteodystrophy. Increased width of osteoid seams and resorption lacunae with multinucleated osteoclasts. Elevated osteoid-osteoblast interface. Mild peritrabecular fibrosis. Undecalcified, 3 lxm thick section of human iliac bone (modified Masson-Goldner stain; x31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

becular fibrosis coexist with adjacent lamellar sites with a more reduced activity, greater production of lamellar or woven osteoid causes the accumulation of osteoid with normal or increased thickness of osteoid seams (Fig. 8-51). While active mineralizing surfaces are frequently found in woven bone with higher mineralization rate and diffuse labeling, mineralization surfaces may be low in lamellar bone with a low mineral apposition rate (Fig. 8-52). 4. EVOLUTION OF RENAL OSTEODYSTROPHY

In the 1970s, the heterogeneity in bone lesions was attributed, at least in part, to coexistence of aluminum In patients with ESRD, the matoxicity in b o n e . 16'13~ jor sources of contamination are high aluminum content of water and dialysate and aluminum-containing phosphate binders. Severe aluminum intoxication is accom-

FIGURE 8 - - 5 2 Mixed uremic osteodystrophy. Regular double uptake of tetracycline with increased distance between labels, single uptake, and irregular diffuse uptake. Undecalcified, unstained, 7 lxm thick section of human iliac bone. Fluorescent light microscopy (x20). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

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MARIE-CLAUDE MONIER-FAUGERE, M. CHRIS LANCUB,AND HARTMUT H. MALLUCHE

panied by high morbidity and mortality, and its treatment through deferoxamine (DFO) chelation, though efficient, is not without risks. Therefore, every effort was made to develop diagnostic tests capable of recognizing this disease. Determining serum levels of aluminum before and after DFO challenge, alone or in association with serum PTH, was found useful, but only in a minority of patients (i.e., in patients with high baseline serum aluminum levels who showed a marked increase after DFO challenge and had low serum PTH l e v e l s ) . 133 Therefore, bone biopsies represented the only unequivocal means to establish aluminum accumulation in bone and determine the type of renal osteodystrophy. Today, control of aluminum content in dialysate and replacement of aluminum-containing phosphate binders with calcium salts are widely implemented. To overcome parathyroid gland overactivity, therapeutic regimens attempting to ensure better control of serum calcium and phosphorus levels and correction of calcitriol deficiency are also widely u s e d . 134-137 In a recent study, we analyzed the changing pattern of renal osteodystrophy in 2248 patients over 13 years (from 1983 to 1995). 138 Distribution of the four histological forms of renal osteodystrophy was as follows: PHBD, 24.5%; MUO, 52.9%; LTOM, 6.2%; and ABD, 16.4%. Over the 13 years of the survey, however, this distribution varied. MUO was found in the majority of patients over the years, but a slight decrease in the number of patients with this diagnosis was seen in 1990 and 1995. PHBD was observed in a large number of patients in 1983. The frequency of this diagnosis decreased constantly until 1988 but then increased gradually to reach a level in 1995 similar to the one found in 1983. The incidence of LTOM decreased progressively during the last 13 years. ABD was first diagnosed in 1984, and the number of patients with this histological bone disease increased until the late 1980s. Thereafter, the number of patients with ABD stayed approximately the same. When patients on hemodialysis and peritoneal dialysis were compared, the trends were similar for the four histological groups. However, patients on peritoneal dialysis consistently exhibited more histological signs of ABD and fewer signs of PHBD than patients on hemodialysis. Overall, 58.5% of the patients exhibited positive stainable aluminum at greater than 30% of the trabecular surface, while 41.5% displayed negative aluminum staining (<30%). In 1983, approximately 40% of the biopsied patients were positive for aluminum. The proportion of aluminum-positive patients increased to approximately 60% in 1984 and remained stable until 1992, when a trend towards a decrease was observed. However, in 1995 approximately one fourth of the biopsies analyzed still showed aluminum deposition in

bone. The extent of aluminum deposited in bone was higher in patients with low bone turnover, either LTOM or ABD, than in patients with other forms of renal osteodystrophy, and this did not change with time. There were more patients with stainable aluminum greater than 30% in the LTOM, ABD, and MUO groups than in the PHBD group. From 1984 to 1995, the proportion of patients with bone aluminum accumulation decreased within each histological group. In 1995 aluminum accumulation was mostly seen in patients with MUO and ABD. In summary, despite recent rational strategies, aluminum accumulation has not completely disappeared, and dialyzed patients still present with PHBD, MUO, and ABD. Recent evidence shows that low bone turnover is not only a histological entity but has clinical relevance. Patients with this abnormality were found to have abnormal calcium homeostasis, 139 more bone pain, higher incidence of fractures, delayed healing, and higher morbidity and mortality rates than patients exhibiting other histological abnormalities. 14~ The present challenge to nephrologists is to determine a patient's level of bone turnover in order to apply the correct therapeutic regimen. Serum PTH levels measured with the radioimmunometric assay are commonly used to assess bone turnover in dialyzed patients because these levels have been found to be more sensitive than the previously employed radioimmunoassays. 141 However, there is no consensus at the present time regarding the serum level of PTH that reflects normal bone turnover in patients with ESRD. In a recent study, 142 our laboratory found that serum PTH levels between 65 and 450 pg/ml, seen in the majority of dialysis patients, are unpredictive of the underlying bone disease. Because bone biopsies provide a sensitive measurement of bone changes, they more accurately determine the type of renal osteodystrophy and can indicate potential aluminum accumulation in dialyzed patients. When ESRD patients undergo kidney transplantation, renal function is only partially restored, and signs of moderate renal osteodystrophy may still be observed. Persistence of severe secondary hyperparathyroidism is not uncommon. Aluminum accumulation may also persist up to 2 years after renal grafting. However, the major bone disease associated with transplantation is osteoporosis because of aggressive steroid and immunosuppressive therapy.

E. B o n e A b n o r m a l i t i e s in R e n a l S t o n e F o r m e r s Tubular defects such as the adult Fanconi syndrome may cause mineral losses and associated severe bone abnormalities including osteomalacia (see above). Pa-

CHAPTER 8 Bone Biopsies: A Modem Approach tients suffering from renal stone disease may have a bone abnormality contributing to their hypercalciuria and proclivity to form stones. In fact, the bone abnormality may prove to be the primary cause, as in the cases of patients with resorptive hypercalciuria143; the bone abnormality may be a secondary phenomenon resulting from renal calcium losses144; or the bone abnormality may represent an unexplained finding deserving further studies before we speculate on cause and effect. 145 This form is characterized by signs of secondary hyperparathyroidism and local decrease in bone remodeling and/or defective mineralization. Active foci with numerous osteoblasts, woven osteoid, peritrabecular fibrosis, and increased tetracycline uptake coexist with adjacent lamellar sites with lower cellularity and tetracycline uptake. The ratio between high- and low-turnover remodeling sites may vary from patient to patient. The number of osteoclasts is usually increased.

267

FIGURE 8 - 5 3 Paget's disease of bone. Increased trabecular surface covered by osteoid. Seam width increased. Lamellar osteoid and woven osteoid coexist. Undecalcified, 3 Ixm thick section of human iliac crest bone under polarized light (modified Masson-Goldner stain; x31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

F. P e d i a t r i c D i s e a s e s Indications for bone biopsies in children are similar to those listed for adults. However, bone biopsies for mineralized bone histology are particularly helpful in the diagnosis and management of tickets, osteogenesis imperfecta, osteopetrosis, and other rare pediatric bone diseases. Note that the dosage required for diagnostic tetracycline labeling of bone neither discolors teeth nor affects skeletal growth. Also, growth is not affected by the biopsy even though the continuity of the growth plate may be temporarily disrupted by the biopsy.

G. M i s c e l l a n e o u s

FIGURE 8 - 5 4 Bone abnormalities associated with malignancies. Metastatic epidermoid carcinoma. Presence of malignant cells in the bone marrow and stimulation of bone formation evidenced by high number of osteoblasts producing woven osteoid, woven bone, and fibers surrounding the trabecules and filling the marrow space. Undecalcified, 3 Ixm thick section of human iliac bone (modified Masson-Goldner stain; X50). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

Bone biopsies are indicated in patients with unexplained bone pain, unexplained elevation of serum alkaline phosphatase, and unexplained deviations from normal levels in serum calcium and phosphorus. They are also indicated in patients with delayed fracture healing; pseudofractures; fractures after minimal trauma; and prolonged healing after bone and joint surgery, bone implants, or other orthopedic interventions. Bone biopsies may also be useful in documenting the diagnosis of Paget's disease of bone (Fig. 8 - 5 3 ) in borderline cases. Also, in sporadic cases, bone biopsies might reveal an unexpected carcinoma (Fig. 8 - 5 4 ) , myeloma (Figs. 8 - 5 5 and 8 - 5 6 ) , oxalosis (Fig. 8 - 5 7 ) , or sarcoidosis (Figs. 8 - 5 8 and 8 - 5 9 ) . The bone biopsy may also be helpful in understanding the radiological finding of osteosclerosis through histological findings consistent with fluorosis or Albers-SchOnberg disease.

FIGURE 8--55 Bone abnormalities associated with malignancies. Multiple myeloma. Diffuse infiltration of the bone marrow by plasma cells without invasion of calcified bone. Undecalcified, 3 txm thick section of human iliac bone (modified Masson-Goldner stain; x31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

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MARIE=CLAUDE MONIER=FAUGERE,M. CHRIS LANGUB, AND HARTMUT H. MALLUCHE

FIGURE 8--56 Bone abnormalities associated with malignancies. Multiple myeloma. High-power detail of plasma cells and Russell bodies. Undecalcified, 3 Ixm thick section of human iliac bone (modified Masson-Goldner stain; x198). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8 - 5 9

Sarcoidosis.A characteristic asteroid body within a giant cell. Close-up view of bone marrow from the same patient shown in Figure 8-45 (hematoxylin-eosin stain; x 126). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

VII. INFORMATION DERIVED FROM MOLECULAR HISTOLOGY

FIGURE 8--57 Oxalosis.Invasive crystal growth within trabecular bone and osteoid. Undecalcified, 3 p~m thick section of human iliac bone. Partially polarized light microscopy (modified MassonGoldner stain; x50). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

FIGURE 8--58 Sarcoidosis. Sarcoid noncaseating granuloma in bone marrow consisting of giant cells and histiocytic cells. Undecalcified, 3 Ixm thick section of human iliac bone (von Kossa's stain, counterstained with methylene blue; X31). (From Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986.) See Color Plate.

At present, there are few reports on histological demonstration of local factors involved in bone cell activity. Molecular histology techniques have been used in studies on growing or mature normal bone, ovariectomized rats, Paget's disease of bone and, renal osteodystrophy. Expressions of bone matrix proteins including osteocalcin, osteopontin, and type I collagen associated with cell-to-cell or cell-to-mineralized matrix adhesion have been documented by ISHH. 68-7~ For example, m R N A expression for osteocalcin and osteopontin in bone tissue from newborn and adult rats processed by I S H H was reported by Ikeda et al. in 1992. 68 The same group of researchers reported age-related diminutions in expression levels of osteopontin m R N A that may be indicative of decreased biological activity of bone cells. 69 More recently, I S H H technique was e m p l o y e d to document changes in biological activity as m e a s u r e d by osteopontin and type I collagen m R N A expressions in bone cells from ovariectomized rats. 7~ Both osteopontin and type I collagen m R N A levels were increased in individual cells from 7 to 10 days postoperation and were sustained for 60 days. Moreover, the n u m b e r of osteocytes that expressed osteopontin m R N A was increased only in bones that were preselected to be resorbed after ovariectomy, suggesting that some osteocytes may be stimulated to express osteopontin m R N A by estrogen deficiency. Other factors believed to play important roles in regulation of bone turnover have been demonstrated by I S H H methodology to be expressed in bone cells. For example, the transducer of vitamin D effects in m a n y tissue systems sensitive to the hormone, the vitamin D receptor (VDR) is expressed in bone osteoblast and os-

CHAPTER 8

269

Bone Biopsies: A M o d e m Approach

FIGURE 8 - - 6 0 Brightfield photomicrographs of bone tissue processed for IL-6 receptor (IL-6R) mRNA in situ hybridization histochemistry (5 Ixm thickness). A, IL-6R mRNA localized to multinucleated osteoclasts in a renal failure patient. B, Serial section processed through RNase A pretreatment before hybridization with antisense IL-6R probe. No labeling was observed in osteoclasts.

teoclast c e l l s . 72'73 Using riboprobe for VDR mRNA, Mee et al. 72 detected VDR transcripts in osteoblast cells within sections taken from normal bone as well as actively remodeling bone tissues including Paget's disease, renal osteodystrophy and healing fracture callus. Osteoclasts contained low levels of VDR mRNA. Osteoblasts, osteocytes, and dental epithelial cells in the orofacial mineralized bone tissue have also been demonstrated to express VDR n l R N A . 73 These reports suggest that in addition to vitamin D-mediated effects upon osteoprogenitor cells, the hormone could also exert direct effects on active bone-resorbing cells. The cytokine interleukin-6 (IL-6) is postulated to play a significant role in bone remodeling by its ubiquitous presence in bone microenvironment and its stimulation of differentiation and proliferation of the bone-resorbing osteoclast-progenitor c e l l s . 147 In the developing bone and cartilage, the emergence of cytokines as key factors in the control of modeling and remodeling of tissue has been explored. ~48 Specifically, expression of interleukin- 1[3 (IL- 1[3), IL-6, and transforming growth factor [31 (TGF-[31) were assessed during endochondral and intramembraneous ossifications. IL-l[3 mRNA localized in osteoblasts during intramembranous but not during endochondral ossification. IL-6 mRNA expression was noted in preosteoblasts and active osteoblasts, but only a weak signal was detected in inactive osteoblasts, chondroblasts, and chondrocytes. In osteoblasts next to bone or cartilage matrix, the intensity of expression correlated with matrix secretion and this was also observed in chondroblasts and chondrocytes in relatively lower levels. Altogether, the three cytokines were differentially expressed temporally and spatially in developing bone and cartilage, suggesting different roles for each in cellular function. Recently, combining histomorphometric and ISHH analyses, our laboratory documented mRNA expressions of IL-6 and its receptor, IL-6R, in patients with renal osteodystrophy. 7~ We determined that IL-6 mRNA is expressed in osteoblasts, osteocytes, osteoclasts, and bone

marrow cells. Similar observations were noted for cellular expression of IL-6R mRNA. Semiquantitative analysis of hybridization signal strengths indicated that activity of osteoclasts assessed by erosion depth of resorbing lacunae is paralleled by IL-6R mRNA expression (Fig. 8-60). This finding suggests that IL-6 and particularly IL-6R are associated intricately in osteoclastic bone resorption. Clearly, the value of molecular histology in bone research is obvious. As limitations to its applicability rapidly diminish with the development of more efficient methods that can be economically performed (i.e., without undue time restrictions), this methodology will find its way into many basic and clinical bone research programs.

References 1. Parfitt AM: The physiologic and clinical significance of bone histomorphometric data. In Recker RR (ed): Bone Histomorphometry: Techniques and Interpretation. Boca Raton, FL, CRC Press, 1983, pp 132-223. 2. Dempster DW: Bone remodeling. In Riggs BL, Melton LJ III (eds): Osteoporosis. Philadelphia, Lippincott-Raven, 1995, pp 67-91. 3. Malone JD, Teitelbaum S, Griffin GL, et al: Recruitment of osteoclast precursors by purified bone matrix constituents. J Cell Biol 92:227, 1982. 4. Mundy GR, Power JW: Chemitactic activity of the ~/-carboxyglutamic acid containing protein in bone. Calcif Tissue Int 35: 164, 1983. 5. Mundy GR, Varani J, Orr W, et al: Resorbing bone in chemotactic for monocytes. Nature 35:164, 1983. 6. Chambers TJ: Regulation of osteoclast development and function. In Rifkin BR, Gay CV (eds): Biology and Physiology of the Osteoclast. Boca Raton, FL, CRC Press, 1992, pp 105-128. 7. Glowacki J: The influence of matrix components on osteoclasts. In Rifkin BR, Gay CV (eds): Biology and Physiology of the Osteoclast. Boca Raton, FL, CRC Press, 1992, pp 187-206. 8. Delaisse JM, Gilbert V: Mechanism of mineral solubilization and matrix degradation in osteoclastic bone resorption. In Rifkin BR, Gay CV (eds): Biology and Physiology of the Osteoclast. Boca Raton, FL, CRC Press, 1992, pp 2 8 9 - 314.

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9. Eriksen EE Melsen F, Mosekilde L: Reconstruction of the resorptive site in the trabecular bone: A kinetic model for bone resorption in 20 normal individuals. Metab Bone Dis Rel Res 5: 235-242, 1984. 10. Jilka RL, Weinstein RS, Bellido T, et al: Demonstration of programmed cell death (apoptosis) in osteoblasts: Evidence for modulation of this process by growth factors and cytokines. J Bone Miner Res ll(Suppl 1):S144, 1996. 11. Canalis E: Regulation of bone remodeling. In Favus MJ (ed): Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. New York, Raven Press, 1993, pp 33-37. 12. Cohen MS, Gray TK: Phagocytic cells metabolize 25-hydroxyvitamin D3 in vitro. Proc Natl Acad Sci USA 81:931-934, 1984. 13. Koeffler HE Reichel H, Bishop JE, Norman AW: ~/-Interferon stimulates production of 1,25-dihydroxyvitamin D3 by normal human macrophages. Biochem Biophys Res Commun 127: 596-603, 1985. 14. Goldhaber P: Heparin enhancement of factors stimulating bone resorption in tissue culture. Science 147:407-408, 1965. 15. Sakamoto S, Sakamoto M, Goldberg L, et al: Mineralization induced by beta-glycerophosphate in cultures leads to a marked increase in collagenase synthesis by mouse osteogenic MC3T31 cells under subsequent stimulation with hepadn. Biochem Biohys Res Commun 162:773-780, 1989. 16. Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. Basel, Karger, 1986. 17. Malluche HH, Faugere M-C: The role of bone biopsy in the management of patients with renal osteodystrophy. J Am Soc Nephrol 4(9):1641-1642, 1994. 18. Milch R, Rall D, Tobie J: Fluorescence of tetracycline antibiotics in bone. J Bone Joint Surg 40A:897-910, 1958. 19. Harris WH: A microscopic method of determining rates of bone growth. Nature 188:1038-1039, 1960. 20. Frost HM, Villanueva AR: Tetracycline staining of newly formed bone and mineralized cartilage in vivo. Stain Technol 35:135-138, 1960. 21. Frost HM: Tetracycline-based histological analysis of bone remodeling. Calcif Tissue Res 3:211 - 237, 1969. 22. Bordier PJ, Tun Shot S: Clinics in endocrinology and metabolism. In MacIntyre I (ed): Quantitative Histology of Metabolic Bone Disease, vol 1. Philadelphia, WB Saunders Co, 1972, pp 197-215. 23. Teitelbaum SL, Hruska KA, Shieber W, et al: Tetracycline fluorescence in uremic and primary hyperparathyroid bone. Kidney Int 12:366-372, 1977. 24. Sherrard DJ, Maloney NA: Single-dose tetracycline labeling for bone histomorphometry. Am J Clin Pathol 91:682-687, 1989. 25. Parfitt AM, Drezner MK, Glorieux FH, et al: Bone histomorphometry: Standardization of nomenclature, symbols and units. J Bone Miner Res 6:595-610, 1987. 26. Meunier PJ, Courpron P, Edouard CM: Physiological and comparative histological data. Clin Endocrinol Metab 2:239-259, 1973. 27. Faugere MC, Dorr LD, Malluche HH: Static and dynamic bone histology in primary and revision total knee arthroplasty. Orthop Trans 7:299- 300, 1983. 28. Dorr L, Malluche HH, Faugere MC, et al: Bone structural and cellular characteristics in patients with total hip arthroplasty. 31 st Annual Meeting, Orthopaedic Research Society, 1985. 29. Faugere MC, Malluche HH: Comparison of different bone biopsy techniques for qualitative and quantitative diagnosis of metabolic bone diseases. J Bone Joint Surg 65A:1314-1319, 1983. 30. Goldhaber P: Bone resorption factors, cofactors and giant vacuole osteoclasts in tissue culture. In Gaillard PJ, Talmage RV,

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Budy AM (eds): The Parathyroid Glands. Chicago, University of Chicago Press, 1965, pp 153-171. Fornassier VL, Vilaghy MI: The results of bone biopsy with a new instrument. Am J Clin Pathol 60:570-573, 1973. Bordier P, Matrajt H, Miravet L, Hioco D: Mesure histologique de la masse et da la r6sorption des trav6es osseuses. Path Biol Paris 12:1238-1243, 1964. Hocking DR: Bone marrow biopsy: A routine including marrow trephine. Med J Aust ii:915-917, 1964. Ellis LD, Jensen WN, Westerman MP: Needle biopsy of bone and marrow. An experience with 1,445 biopsies. Arch Intern Med 114:213-221, 1964. Duursma SA, Visser WJ, van Zoeren M, Korver MF: A bone biopsy procedure. Calcif Tissue Res 4:269-273, 1969. Jamshidi K, Swaim WR: Bone marrow biopsy with unaltered architecture: A new biopsy device. J Lab Clin Med 77:335-342, 1971. Courpron P, Giroud JM, Bringuier JP, Meunier P: Histomorphometrie de l'os spongieux iliaque. Influence des techniques de pr6paration et de lecture des coupes histologiques sur la d6termination du volume trab6culaire osseux iliaque Lyon Med 232: 515-522, 1974. Chappard D, Alexandre C, Bousquet G, Riffat G: Nouvelles modifications du trocart de Bordier pour la biopsie osseuse quantitative. Revue Rhum Mal Osteoartic 50:307-308, 1983. Burkhardt R: Technische Verbesserungen und Anwendungsbereich der Histo-Biopsie von Knochenmark und Knochen. Klin Wochenschr 44:326-334, 1966. Malluche HH: The value of bone biopsies for diagnosis of renal bone disease. In Moorehead J, Baillod R, Mion C (ed): Dialysis, Transplantation, Nephrology. London, Pitman Medical, 1973, pp 111-115. Johnson KA, Kelly PJ, Jowsey J: Percutaneous biopsy of the iliac crest. Clin Orthop 123:34-36, 1977. Malluche HH, Sherman D, Meyer W, Massry SG: A new semiautomatic method for quantitative static and dynamic bone histology. Calcif Tissue Int 34:439-448, 1982. Duncan H, Rao SD, Parfitt AM: Complication of bone biopsy. In Jee W, Parfitt A (ed): Bone Histomorphometry. Pads, Soci6t6 Nouvelle de Publications M6dicales et Dentaires, 1981. Malluche HH, Meyer W, Sherman D, Massry SG: Quantitative bone histology in 84 normal American subjects. Micromorphometric analysis and evaluation of variance of iliac bone. Calcif Tissue Int 34:449-455, 1982. Amold JS, Jee WSS: Embedding and sectioning undecalcifed bone and its application to radioautography. Stain Technol 29: 225-239, 1954. Goldner J: A modification of the Masson trichrome technique for routine laboratory purposes. Am J Pathol 14:237-243, 1938. Matrajt M, Hioco D: Solochrome cyanin R as an indicator dye of bone morphology. Stain Technol 41:97-100, 1966. Von Kossa G: Ueber die im Organismus kuenstlich erzeugbaren Verkalkungen. Beitr Pathol Anat 29:163- 202, 1901. Burstone MS: Histochemical demonstration of acid phosphatase activity in osteoclasts. J Histochem Cytochem 7:39-41, 1959. Evans RA, Dunstan CR, Baylink DJ: Histochemical identification of osteoclasts in undecalcifed sections of human bone. Miner Electrolyte Metab 2:179-185, 1979. Villanueva AR: An improved stain for fresh mineralized bone sections. Am J Clin Pathol 47:78-84, 1967. Lillie PD, Fullmer HM: Histopathologic Technique and Practical Histochemistry, 4th ed. New York, McGraw-Hill, 1976, p 534.

CHAPTER 8

Bone Biopsies: A M o d e m Approach

53. Denton J, Freemont AJ, Ball J: Detection of distribution of aluminum in bone. J Clin Pathol 37:136-142, 1984. 54. Smith AJ, Faugere MC, Abreo K, et al: Aluminum related bone disease in mild and advanced renal failure. Am J Nephrol 6: 275-283, 1986. 55. Faugere MC, Malluche HH: Stainable aluminum and no aluminum content reflect histologic changes in bone of dialyzed patients. Kidney Int 30:717-722, 1986. 56. Perls M: Nachweis von Eisenoxyd in gewissen Pigmenten. Virchows Arch Pathol Anat 39:42-48, 1867. 57. Quincke H: Ueber directe Fe-Reaction in thierischen Geweben. Arch Exp Pathol Pharmkol 37:183-190, 1896. 58. Schmeltzer W: Der mikrochemische Nachweis von Eisen in Gewebselementen mittels Rhodan-Wasserstoffsaure und die Konservierung der Reaktion in Paroffinol. Z Wiss Mikrosk 50: 99-102, 1933. 59. Gomori G: Microtechnical demonstration: A criticism of its methods. Am J Pathol 12:655-663, 1936. 60. McCabe JT, Bolender RP: Estimation of tissue mRNAs by in situ hybridization. J Histochem Cytochem 12:1777-1783, 1993. 61. Chappard D, Palle S, Alexandre C, et al: Bone embedding in pure methyl methacrylate at low temperature preserves enzyme activities. Acta Histochem 81:183-190, 1987. 62. Gall G, Pardue ML: Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci USA 63:378-381, 1969. 63. Buongiorno-Nardelli M, Amaldi F: Autoradiographic detection of molecular hybrids between rRNA and DNA in tissue sections. Nature 225:946- 948, 1970. 64. John JL, Birnstiel ML, Jones KW: RNA-DNA hybrids at the cytological level. Nature 223:582-591, 1969. 65. Brahic M, Haase AT, Cash E: Simultaneous in situ detection of viral RNA and antigens. Proc Natl Acad Sci USA 81:54455448, 1984. 66. Stahl WL, Eakin TJ, Baskin DG: Selection of oligonucleotide probes for detection of mRNA isoforms. J Histochem Cytochem 12:1735-1740, 1993. 67. Melton DA, Krieg PA, Rebagliati MR: Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acid Res 12:7035-7056, 1984. 68. Ikeda T, Nomura S, Yamaguchi A, et al: In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections. J Histochem Cytochem 40(8):1079-1088, 1992. 69. Ikeda T, Nagai Y, Yamaguchi A, et al: Age-related reduction in bone matrix protein mRNA expression in rat tissues: Application of histomorphometry to in situ hybridization. Bone 16(1): 1 7 23, 1995. 70. Ikeda T, Yamaguchi A, Yokose S, et al: Changes in biological activity of bone cells in ovariectomized rats revealed by in situ hybridization. J Bone Miner Res 11(6):780-788, 1996. 71. Langub MC, Koszewski NJ, Turner HV, et al: Bone resorption and mRNA expression of IL-6 and IL-6 receptor in patients with renal osteodystrophy. Kidney Int 50(2):515-520, 1996. 72. Mee AP, Hoyland JA, Braidman IP, et al: Demonstration of vitamin D receptor transcripts in actively resorbing osteoclasts in bone sections. Bone 18(4):295-299, 1996. 73. Davideau J-L, Papagerakis P, Hotton D, et al: In situ investigation of vitamin D receptor, alkaline phosphatase, and osteocalcin gene expression in oro-facial mineralized tissue. Endocrinology 137(8):3577-3585, 1996. 74. Braidman IP, Davenport LK, Carter DH, et al: Preliminary in situ identification of estrogen target cells in bone. J Bone Miner Res 10(1):74-80, 1995.

271 75. Johnson JA, Grande JP, Roche PC, Kumar R: Ontogeny of the 1,25-dihydroxyvitamin D3 receptor in fetal rat bone. J Bone Miner Res 11(1):56-61, 1996. 76. Lebeau A, Muthman H, Sendelhofert A, et al: Histochemistry and immunohistochemistry on bone marrow biopsies: A rapid procedure for methyl methacrylate embedding. Pathol Res Pract 191:121-129, 1995. 77. Derman H, Chandler FW, Hicklin MD, Penner DW: Anatomic Pathology in Quality Assurance Practices for Health Laboratories. Washington, DC, American Public Health Association, 1978, pp 381-409. 78. Parfitt AM: Stereologic basis of bone histomorphometry; theory of quantitative microscopy and reconstruction of the third dimension. In Recker RR (ed). Bone Histomorphometry: Techniques and Interpretation. Boca Raton, FL, CRC Press, 1983, pp 53-87. 79. Malluche HH, Sherman D, Manaka R, Massry SG: Comparison between different histomorphometric methods. In Jee WSS, Parfitt AM (ed): Bone Histomorphometry. Paris, Socirt6 Nouvelle de Publications Mrdicales et Dentaires, 1981, pp 449-451. 80. Parfitt AM: Implications of architecture for the pathogenesis and prevention of vertebral fracture. Bone 13:541-547, 1992. 81. Feldkamp LA, Goldstein SA, Parfitt AM, et al: The direct examination of three-dimensional bone architecture in vitro by computed tomography. J Bone Miner Res 4:3-11, 1989. 82. Garrahan NJ, Boyde A, Croucher PI, et al: Method for the validation of structural analysis of two-dimensional histological sections using computerised three-dimensional reconstruction. Bone 13:A12, 1992. 83. Hahn M, Vogel M, Pompesius-Kempa M, Delling G: Trabecular bone pattern factor m a new parameter for simple quantification of bone microarchitecture. Bone 13:327-330, 1992. 84. Odgaard A, Andersen K, Ullerup R, et al: Three-dimensional reconstruction of entire vertebral bodies. Bone 15(3):335-342, 1994. 85. Gundersen HJG, Boyce RW, Nyengaard JR, Odgaard A: The conneulor: Unbiased estimation of connectivity using physical disectors under projection. Bone 14:217- 222, 1993. 86. van Rietbergen B, Weinans H, Huiskes R, Odgaard A: A new method to determine trabecular bone elastic properties and loading using micromechanical finite-element models. J Biomech 28(1):69-81, 1995. 87. Gluer CC, Wu CY, Jergas M, et al: Three quantitative ultrasound parameters reflect bone structure. Calcif Tissue Int 55(1):45-52, 1994. 88. Vesterby A: Star volume of marrow space and trabeculae in iliac crest: Sampling procedure and correlation to star volume of first lumbar vertebra. Bone 11:149-155, 1990. 89. Garrahan NJ, Croucher PI, Compston JE: A computerised technique for the quantitative assessment of resorption cavities in trabecular bone. Bone 11:241-245, 1990. 90. Compston JE, Garrahan NJ, Croucher PI, et al: Quantitative analysis of trabecular bone structure. Bone 14:187-192, 1993. 91. Boskey Boskey AL, Pleshko N, Doty SB, Mendelsohn R: Applications of FT-IR microscopy to the study of mineralization in bone and cartilage. Cells Materials 2:209-220, 1992. 92. Boyde A, Elliott JC, Jones SJ: Stereology and histogram analysis of backscattered electron images: Age changes in bone. Bone 14:205-210, 1993. 93. Boyde A, Jones SJ, Aerssens J, Dequeker J: Mineral density quantitation of the human cortical iliac crest by backscattered electron image analysis: Variations with age, sex, and degree of osteoarthritis. Bone 16(6):619-627, 1995. 94. Courpron P: Bone tissue mechanisms underlying osteoporoses. Orthop Clin North Am 12:513-543, 1981.

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95. Eriksen EF: Assessment of erosion depth by lamellar counting. Bone 14:443-447, 1993. 96. Croucher PI, Mellish RWE, Vedi S, et al: The relationship between resorption depth and mean interstitial bone thickness: Age-related changes in man. Calcif Tissue Int 45:15-19, 1989. 97. Cohen Solal M, Shih M-S, Lundy MW, Parfitt AM: A new method for measuring cancellous bone erosion depth. Application to the cellular mechanisms of bone loss in postmenopausal osteoporosis. J Bone Miner Res 6:1331-1338, 1991. 98. Juvin R, Faugere MC, Malluche HH: Comparison between different methods of measurement of bone resorption. Bone 13(5): A17, 1992. 99. Jones SJ, Boyde A: Histomorphometry of Howship's lacunae formed in vivo and in vitro: Depths and volumes measured by scanning electron and confocal microscopy. Bone 14:455-460, 1993. 100. Kimmell DB: An evaluation of existing normal iliac trabecular bone volume (BV/TV) data in bone morphometry. Niigata, Japan, Nishimura Co, 1990, pp 2 0 8 - 215. 101. Kimmel DB, Recker RR, Lappe JM: Histomorphometry of normal pre and postmenopausal women. Bone 13(5):A18, 1992. 102. Glorieux FH, Travers R, Taylor A, Norman M: Normative data for iliac crest bone histomorphometry in growing children. Bone 13(5):A12, 1992. 103. Parisien M, Morgan D, Shen V, et al: Bone histomorphometry reveals subtle differences in bone formation between black and white premenopausal women. J Bone Miner Res 10(Suppl 1): $444, 1995. 104. Proceedings of the Sixth International Congress of Bone Morphometry, Lexington, KY, October 4 - 9 , 1992, Bone 14(3), 1993. 105. Monier-Faugere M-C: Quality assurance-quality control in bone histopathology and bone histomorphometry. Bone 14:559-572, 1993. 106. Meunier PJ, Sellami S, Brianqon D, Edouard C: Histological heterogeneity of apparently idiopathic osteoporosis. In De Luca HF, Frost, H, Jee W, et al (eds): Osteoporosis: Recent in Pathogenesis and Treatment. Baltimore, University Park Press, 1981, pp 2 9 3 - 301. 107. Meunier PJ: Bone histomorphometry. In Riggs BL, Melton LJ III (eds): Osteoporosis: Etiology, Diagnosis, and Management. 2nd ed. Philadelphia. Lippincott-Raven, 1995, pp 299-318. 108. Faugere MC, Friedler RM, Fanti P, Malluche HH: Bone changes occurring early after cessation of ovarian function in beagle dogs: A histomorphometric study employing sequential biopsies. J Bone Miner Res 5(3):263-272, 1990. 109. Malluche HH, Faugere MC, Rush M, Friedler RM: Osteoblastic insufficiency is responsible for maintenance of osteopenia after loss of ovarian function. Endocrinology 119:2643-2654, 1986. 110. Monier-Faugere M-C, Friedler RM, Bauss F, Malluche HH: A new bisphosphonate, BM21.0955, prevents bone loss associated with cessation of ovarian function in experimental dogs. J Bone Miner Res 8(11):1345-1355, 1993. 111. Lips P, Courpron P, Meunier PJ: Mean wall thickness of trabecular bone packets in the human iliac crest: Changes with age. Calcif Tissue Int 26:13-17, 1978. 112. Geng ZP, Faugere MC, Qi QL, Malluche HH: Predictive value of serum osteocalcin for bone turnover in patients with osteoporosis. J Bone Miner Res 9:$402, 1994. 113. Pommer G: Rachitis and Osteomalacia. Leipsiz, Vogel, 1885. 114. Ritz E, Krempien B, Bommer J, et al: Skeletal x-ray findings and bone histology in patients on hemodialysis. Kidney Int 13: 316-326, 1978. 115. Perry HM, Weinstein RS, Teitelbaum SL, et al: Pseudofractures in the absence of osteomalacia. Skeletal Radiol 8:17-19, 1982.

116. Nussbaum SR, Zahradnik RJ, Lavigne JR, et al: Highly sensitive two-site immunoradiometric assay of parathyrin and its clinical utility in evaluating patients with hypercalcemia. Clin Chem 33: 1364-1367, 1987. 117. Bilezikian JP: Primary hyperparathyroidism. In Favus MJ (ed): Primer on the Metabolic Bone Diseases and Disorders or Mineral Metabolism, 2nd ed. New York, Raven Press, 1993, pp 155-159. 118. Eriksen EF, Melsen F, Mosekilde L: Drug therapy: Formationstimulating regimens. In Riggs BL, Melton LJ III (ed): Osteoporosis: Etiology, Diagnosis, and Management, 2nd ed. Philadelphia, Lippincott-Raven, 1995, pp 403-434. 119. Brown EM, Gamba G, Riccardi D, et al: Cloning and characterization of an extracellular Ca-sensing receptor from bovine parathyroid. Nature 366:575-579, 1993. 120. Moallem E, Silver J, Naveh-Many T: Post-transcriptional regulation of PTH gene expression by hypocalcemia due to protein binding to the PTH mRNA 3UTR. J Bone Miner Res 10(Suppl 1):S142, 1995. 121. Slatopolsky E, Finch J, Ritter C, et al: High phosphate (PO4) directly stimulates PTH secretion in tissue culture, and PO4 restriction suppress parathyroid cell growth in chronic renal failure (CRF). J Bone Miner Res 10(Suppl 1):$480, 1995. 122. Silver J, Russell J, Sherwood LM: Regulation by vitamin D metabolites of messenger ribonucleic acid for pre-proparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA 82:4270-4273, 1985. 123. Russell J, Lettieri D, Sherwood LM: Suppression by 1,25(OH)2D3 of transcription of the pre-proparathyroid hormone gene. Endocrinology 119:2864- 2866, 1986. 124. Malluche H, Ritz E, Hodgson M, et al: Skeletal lesions and calcium metabolism in early renal failure. Proc EDTA 11:443450, 1974. 125. Llach F, Massry S, Singer F, et al: Skeletal resistance to endogenous parathyroid hormone in patients with early renal failure. A possible cause of secondary hyperparathyroidism. J Clin Endocrinol Metab 41:339-345, 1975. 126. Massry S, Llach F, Singer F, et al: Homeostasis and action of parathyroid hormone in normal man and patients with mild renal failure. In Moorhead JS, Mion C, Baillod R (ed): Dialysis Transplantation Nephrology. London, Pitman Medical, 1975, pp 451-456. 127. Malluche HH, Ritz E, Kutschera J, et al: Calcium metabolism and impaired mineralization in various stages of renal insufficiency. In Norman AW, Schaefer K, Grigoleit HG, et al (eds): Vitamin D and Problems Related to Uremia Bone Disease. Berlin and New York, Walter de Gruyer & Co, 1975, pp 5 1 3 522. 128. Teitelbaum SL: Renal osteodystrophy. Hum Pathol 15:306-323, 1984. 129. Sherrard DJ, Baylink DJ, Wergedal JE, Maloney NA: Quantitative histological studies on the pathogenesis of uremic bone disease. J Clin Endocrinol Metab 39:119-135, 1974. 130. Hruska KA, Teitelbaum SL: Renal osteodystrophy. N Engl J Med 333:166-174, 1995. 131. Malluche HH, Faugere MC: Renal bone disease 1990: An unmet challenge for the nephrologist. Kidney Int 38:193-211, 1990. 132. Ott SM, Maloney NA, Klein GL, et al: Aluminum is associated with low bone formation in patients receiving chronic parenteral nutrition. Ann Intern Med 98:912-914, 1983. 133. Hodsman AB, Sherrard DJ, Alfrey AC, et al: Bone aluminum and histomorphometric features of renal osteodystrophy. J Clin Endocrinol Metab 54:539-546, 1982. 134. Malluche HH, Faugere MC: The value of desferal infusion test for diagnosis of aluminum bone disease in patients with end stage

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renal failure. In DeBroe ME (ed): Aluminum and Iron Overload in Hemodialysis. Toronto, Lewiston, NY, Bern, Gottingen, Stuttgart, Hogrefe & Huber Publishers, 1989, pp 11-15. Massry SG, Goldstein DA, Malluche HH: Current status of the use of 1,25(OH)2D3 in the management of renal osteodystrophy. Kidney Int 19:409-418, 1980. Voights AL, Felsenfeld AJ, Llach F: The effects of calciferol and its metabolites on patients with chronic renal failure. Arch Intern Med 143:1205-1211, 1983. Malluche HH, Faugere M-C: Effects of 1,25(OH)2D3 administration on bone in patients with renal failure. Kidney Int 38(Suppl 29):$48-$53, 1990. Baker LRI, Abrams SML, Roe CJ, et al: 1,25(OH)2D3 administration in moderate renal failure: A prospective double-blind trial. Kidney Int 35:661-669, 1989. Faugere MC, Malluche HH: Trends in renal osteodystrophy: A survey from 1983 to 1995 in a total of 2,248 patients. Nephrol Dial Transplant ll(Suppl 3):111-120, 1996. Kurz P, Monier-Faugere MC, Bognar B, et al: Evidence for abnormal calcium homeostasis in patients with adynamic bone disease. Kidney Int 46:855-861, 1994. Hercz G, Sherrard DJ, Chan W, Pei Y: Aplastic osteodystrophy: Follow-up after 5 years. J Am Soc Nephrol 5(3):851, 1994. Cohen Solal ME, Sebert JL, Boudailliez B, et al: Comparison of intact, midregion, and carboxy terminal assays of parathyroid

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_~HAPTER .

Noninvasive Assessment of Bone MARTIN UFFMANN, THOMAS P. FUERST, MICHAEL JERGAS,* AND HARRY K. GENANT Osteoporosis & Arthritis Research Group, Department of Radiology, University of California, San Francisco, Califomia 94143; and *Department of Radiology, St. Josef Hospital, Ruhr-University of Bochum, Bochum, Germany

I. II. III. IV.

V. Clinical Applications Acknowledgments References

Introduction Radiation-Based Assessment of Bone Assessment of Bone without Radiation Standardization and Quality Assurance in Dual X-Ray Absorptiometry

rameters. Therefore, established methods for estimation of those nondensitometric properties of bone have been improved, and recently developed techniques will be available for clinical use in the near future. Osteoporosis is a ubiquitous disorder causing considerable socioeconomic burden on a global scale. Postmenopausal women represent the most important and fastest growing group that suffers from osteoporosisrelated symptoms. Finally, osteoporosis represents the final common pathway of a variety of underlying disordersm besides deprivation of estrogen after the menopause.

I. INTRODUCTION Methods for bone assessment play an increasingly important role in the diagnosis of metabolic bone diseases. Osteoporosismas the most common metabolic bone disordermis characterized by low bone mass and microarchitectural deterioration, leading to bone fragility and subsequently increased fracture risk. In the past decade, considerable effort has been expended in the development of methods for assessing the skeleton noninvasively in order to provide early detection and precise monitoring of this disease. Since the definition of osteoporosis includes bone mass as a parameter, several mineral density measures have been employed. These radiation-based methods provide imaging capabilities that are unique in diagnostic medicine in terms of their physical nature. On the other hand, osteoporosis is characterized by changes in geometrical and structural paMETABOLIC BONE DISEASE

A. Chapter Objectives This chapter describes bone measurements from different perspectives, beginning with a brief presentation of different techniques and their underlying principles.

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This is followed by a description of currently used applications for assessment of bone properties. Recent promising developments that may be useful in the near future have been included. Some critical remarks for interpretation of measurement results are provided. The chapter is concluded by a discussion of clinical applications for bone assessment.

body sites like the hand (radiographic absorptiometry)or the heel (single x-ray absorptiometry, quantitative ultrasound), locations that are unlikely sites of osteoporotic fractures. Since osteoporosis is a generalized disorder and since density measurements show correlations across different sites, this implies that examinations at one site may be predictive for fractures at another site, albeit at varying levels of reliability.

B. Anatomical Considerations D. Acronyms in Bone Densitometry Bone is the tissue that provides the support and stability of the human body. The skeleton is made up of about 80% cortical or compact bone and 20% trabecular or cancellous bone. 1 The appendicular skeleton predominantly consists of cortical bone (80% to 95%), whereas the axial skeleton is composed of a combination of trabecular (in the center of the vertebral body) and cortical bone (in the end plates and posterior elements). Since metabolic bone turnover is about eight times higher in trabecular than in compact bone 2 (partly as a result of the different surface/volume ratios), measurements of trabecular bone usually detect loss of mineralization more effectively than those of compact bone. Osteoporotic fractures generally occur earliest in the vertebra or the distal radius, sites with a relatively high proportion of trabecular bone. The ultradistal region of the radius adjacent to the radiocarpal joint contains 60% trabecular bone, whereas the radial shaft contains mainly cortical bone (95%). 3 Both regions are typical measurement sites for forearmdensitometry (often referred as "ultradistal" and "1/3 radius" measurement sites). The two anatomical parts of the vertebramposterior processes and b o d y - - e a c h comprise about 50% of the entire vertebral bone mass. 4 The proportion of trabecular bone is about 50% in the vertebral body, whereas the posterior part contains substantially less. The proportion of cortical bone in the spine is greater than usually assumed (> 50%), and contributes significantly to spinal bone mass measurements. 5

C. Comparison of Common Fracture Locations and Bone Measurement Sites There are several clinically established methods used for noninvasive bone assessment. Some of them are performed at typical fracture sites like the forearm [single x-ray absorptiometry and peripheral quantitative computed tomography (CT)], the spine (dual x-ray absorptiometry and quantitative CT), and the hip (dual x-ray absorptiometry). Other techniques are applied at remote

Bone densitometry has gained wide acceptance. Since different techniques have been developed and are clinically available, a variety of abbreviations exist. For example, the acronym DXA has been proposed and widely accepted for dual x-ray absorptiometry. 6 Still, different abbreviations (DEXA, DER, DRA, QDR, and DPX) are being used and therefore contribute to some confusion. There is general agreement that standard abbreviations for bone densitometry are necessary. Manufacturers and users in clinic and research have joined efforts in an International Standard Committee. 7 The following proposal for terminology reflects the development in bone densitometry and will be understood by both clinicians and researchers: DXA for dual x-ray absorptiometry, DPA for dual-photon absorptiometry, SXA for single xray absorptiometry, SPA for single photon absorptiometry, QCT for quantitative computed tomography, and pQCT for peripheral quantitative computed tomography. For newer techniques like ultrasound attenuation, ultrasound velocity, and magnetic resonance measurements appropriate acronyms still have to be agreed upon. QUS for quantitative ultrasound and QMR for quantitative magnetic resonance would be consistent with QCT and will be used here. However, these abbreviations need consensus development. 8

E. Principal Bone Measurement Techniques and Their History Numerous methods for the assessment of bone mineral density (BMD) have been developed during the last three decades. Some of their preceding techniques and principles reach back even to the 1930s. Radiation-based techniques like single photon absorptiometry, dual x-ray absorptiometry, and quantitative computed tomography have become standard methods with widespread use and acceptance in both clinical and research application. During the last few years, several new non-radiationbased methods have been applied for assessment of both bone density and structure. Promising results have been

CHAPTER9 NoninvasiveAssessment of Bone obtained, especially for ultrasound and magnetic resonance techniques. QUS, which in principle was introduced for bone assessment in the 1960s, has emerged as a promising option for the assessment of bone properties in recent years. New developments in hardware and software have considerably enhanced precision and improved clinical feasibility. However, the suitability of ultrasound methods for routine application has not yet been established. The newest modality for noninvasive bone assessment--quantitative magnetic resonance--is still in an early state of development. Similar to ultrasound, it combines the advantages of being a nonionizing-radiation technique and potentially providing structural information about bone. A brief survey of the various major techniques and their history is listed in Figure 9 - 1 .

II. RADIATION-BASED ASSESSMENT OF BONE

A. Appearance of Bone Loss in Conventional Radiographs Since the first days of roentgenology, osteoporotic fractures have been depicted; therefore conventional radiography is the oldest approach for assessing bone mineralization. As early as 1936 Lachmann and Whelan stated that only under very favorable radiographic conditions could decalcification of less than 20% be recognized. 9 These and other authors assumed that the loss in bone density must be as much as 20% to 40% before it can be detected on lateral radiographs of the thoracic and lumbar spine. 1~ Criteria such as increased radiolucency, reduction of cortical thickness, accentuation of vertebral end plates, and loss of horizontal trabeculae combined with the prominence of vertical trabeculae are generally regarded as signs of spinal osteopenia. However, Doyle et al. found none of these criteria to be reliable in assessing longitudinal changes in bone density in the course of osteoporosis, ll Besides its subjective character and its dependence on the reader, this method is influenced by many technical factors such as x-ray equipment, exposure settings, soft tissue thickness, film/ screen combination and film processing. In an attempt to classify radiographic manifestations of spinal osteopenia without the presence of fractures, Saville 12 introduced a score from 0 to 4. This index, however, has never gained widespread acceptance due to subjectivity and dependence on the experience of the observer. Therefore, efforts to detect bone mineral loss radiographically have been replaced by absorptiometric density measurements.

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B. Radiographic Morphometry 1. VERTEBRAL BODY DEFORMITY Since the morbid event in osteoporosis is fracture, and the most common ones are vertebral fractures, they have been described as hallmarks of osteoporosis (e.g., one classical definition requires the presence of nontraumatic spinal compression fractures before the diagnosis can be made13). The assessment of vertebral fractures from spinal radiographs plays an important role in clinical practice. The same applies to radiography in the context of epidemiological studies or pharmaceutical trials, where osteoporotic fractures of the spine are a primary end point. Various efforts have been made to define objective, reliable, and reproducible methods to assess vertebral deformity as a measure of osteoporosis. The numerous methods can basically be divided into quantitative and semiquantitative assessment, although some of them have features from both approaches.

a. Quantitative Approach. One of the first studies using measurements of vertebral dimensions on lateral spine radiographs was described by Barnett and Nordin. TM They introduced an index that compares the anterior and midvertebral height in order to quantify vertebral biconcavity. Several quantitative morphometric approaches based on those vertebral height measurements have been developed, increasingly sophisticated with up to ten marker points placed on a vertebral body. 15-23 The methods employed have often been compared within the same population to examine the impact of methodology on assessment of vertebral fracture in both individual patients and individual vertebrae. Considerable differences in fracture sensitivity and specificity have been found by different a u t h o r s . 17'24-26 The intermethodology correlation has been only modest with two- to four-fold differences in the estimation of prevalent fractures. Unfortunately, there is no true "gold standard" for vertebral fractures and therefore the different methods or cutoff criteria cannot be judged independently. Despite having well-defined, objective tools, quantitative morphometry brings up several problems that may explain the difficulties in reliably distinguishing between osteoporotic fractures and other nonfracture deformities. For example, poor positioning, obesity, and spinal abnormalities like scoliosis and osteoarthritis result in magnified or distorted vertebral projection, which lowers the precision of measurement. Another problem is to define normal vertebral dimensions, since osteoporosis-related deformities in the spine are accompanied by degenera-

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FIGURE 9-- 1

Noninvasive methods for assessment of bone and preceding techniques.

tive changes with a broad overlap in vertebral measurement parameters. Nevertheless quantitative morphometry plays an important role in epidemiological studies and in clinical drug studies, particularly for long-term changes in follow-up examinations.

b. Semiquantitative Approach. Semiquantitative assessment of vertebral deformities means grading of vertebrae into a few categories based on visual determination only (Fig. 9-2). The reading includes alterations of vertebral heights as well as its shape relative to adjacent vertebrae and expected normal appearance. These criteria add a strong qualitative aspect to the interpretation. Therefore, an experienced reader is able to compensate for conditions that would lead to misclassification in quantitative analysis. In several studies semiquantitative assessment of vertebral fractures has rendered excellent intra- and interobserver reproducibility. 27'28

2. TRABECULAR PATTERN OF THE FEMUR Another approach to semiquantitative grading of osteopenic bone on radiographs has been described by Singh et al. 29 They developed a femoral trabecular index based on previous findings about altered trabecular patterns of the proximal femur in women with hip fracture. 3~ The radiographic appearance of the hip can be classified into six grades according to the distribution, thickness, and spacing of the compressive and tensile trabeculae assuming that during the process of bone loss the deterioration of trabeculae follows a distinct sequence (Fig. 9-3). The Singh index has been applied in a number of studies revealing varying associations with bone mass and fracture occurrence. A recent publication refused the hypothesis of organized loss of femoral trabeculae and instead indicated a generalized loss of bone. 31 The quantitative methods such as SPA, SXA, DPA, and DXA have changed the status of conventional ra-

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FIGURE 9 - 3 The Singh index is based on the assumption that the trabeculae in the proximal femur disappear in a predictable sequence depending on their original thickness. The classification ranges from grade VI (normal, all trabecular groups visible) to grade I (marked reduction of even the principal compressive trabeculae) according to the degree of bone loss. (Modified from Singh YM, Nagrath AR, Maini PS: Changes in trabecular pattern of the upper end of the femur as an index of osteoporosis. J Bone Joint Surg 52-A: 457-467, 1970.)

FIGURE 9--2 Grading scheme for a semiquantitative evaluation of vertebral deformities. A, Grade 0, normal. B, Grade 1, mild deformity, 20% to 25% reduction of any vertebral height. C, Grade 2, moderate deformity, 25% to 40% reduction of any vertebral height. D, Grade 3, severe deformity, >40% reduction of any vertebral height. The drawings illustrate reductions of the anterior height that correspond to the grade of the deformity. Reductions of the middle or posterior vertebral height or combinations thereof can be evaluated using the same grading scheme. (Courtesy of Dr. C. Y. Wu.)

diography for the assessment of osteoporosis. Nevertheless, quantitative radiography remains useful for the detection of specific alterations in certain clinical cases (e.g., subperiosteal resorption in hyperparathyroidism). Although D X A equipment already offers some imaging capabilities, radiography is still the primary modality for skeletal imaging, having unequaled spatial resolution. For detection of complications of osteoporosis such as fractures as well as differential diagnosis of other metabolic bone diseases or bone tumors, conventional radiography remains indispensable.

tours cortical indices as well as cortical area can be derived, assuming a circular shape of the bone cross-section (Fig. 9 - 4 ) . Virtually every long bone can be assessed, although the most c o m m o n l y used sites are the metacarpals. Initially, the precision was relatively poor due to operator interaction. Technical modifications like semiautomation and electronic calipers have improved this technique considerably, with reported shortterm precision errors between 0.4% and 2.0% for the cortical index in 12ivo. 33 Even in the older studies, a remarkable age-related loss of cortical indices has been found. In postmenopausal w o m e n with estrogen substitution after o o p h o r e c t o m y the annual decrease of combined cortical thickness (outer minus inner cortical diameter) was considerably smaller compared to the 34 control group (0.5% vs. 1.45%, respectively). In several studies, r a d i o g r a m m e t r y has been found to be a good discriminator b e t w e e n postmenopausal w o m e n with and without vertebral 35'36 or hip fractures, 37 even though the relatively simple m e a s u r e m e n t of cortical bone d o e s n ' t estimate intracortical resorption and porosity. The information provided by this technique is more useful in clinical research than for individual patient m a n a g e m e n t .

C. Radiogrammetry

D. Radiographic Absorptiometry

R a d i o g r a m m e t r y was the first approach for assessing 14 32 cortical bone quantitatively. ' With simple measurements of the inner and outer diameter of the bone con-

Since the early days of radiography it has been known that the photographic density on a film is roughly proportional to the mass of bone the x-ray b e a m passes

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MARTIN UFFMANN, THOMAS P. FUERST, MICHAEL JERGAS, AND HARRY K. GENANT

FIGURE 9 - 4 Schematicrepresentation of a cross section of a tubular bone, showing several parameters determined by radiogrammetry. [From Steiner E, Jergas J, Genant HK: Radiology of osteoporosis. In Marcus R, Feldman D, Kelsey J (eds): Osteoporosis. San Diego, Academic Press, 1996, pp 1019-1054.]

through. Based on this observation radiographic absorptiometry (RA), or photodensitometry, was developed by different investigators. 38-4~ The underlying principle had already been described in 1939. 41 This technique makes possible bone mass measurements from radiographs of the peripheral skeleton, most commonly the metacarpal and phalangeal bones. The measurement procedure can be performed by any standard radiology department. It simply requires a reference wedge on the film and an appropriate exposure technique (Fig. 9 - 5 ) . The developed radiographs are sent to a central reading facility where the reference and relevant bone locations are evaluated using a photodensitometer. RA became relatively widely used as a research technique in the 1960s, although it has subsequently been superseded by photon absorptiometry and x-ray absorptiometry. More recently, with the advent of computerized image processing, RA has become more precise 42 and gained new respect for use in the diagnosis of osteoporosis. In addition, recent studies in which radiographic absorptiometry was used have demonstrated that the detection of accelerated bone loss in the early menopause with this technique is comparable to other densitometry techniques. 43-45 Being cost-effective, easy to perform, and universally available, all make radiographic absorptiometry an increasingly attractive option for noninvasive bone assessment in both research and clinical practice.

The decay of this radionuclide can be measured with a whole-body counter and the total body calcium can be derived. The radiation dose required is relatively high (< 2.5 mSv). Although having good precision (1.5% to 2%) and accuracy (5%), 48'49 this technique reflects primarily

E. C o m p t o n S c a t t e r i n g T e c h n i q u e / N e u t r o n Activation Analysis With neutron activation analysis the total body calcium can be estimated. This technique was developed in the 1960S. 46'47 The patient is exposed to a uniform beam of neutrons designed to irradiate the whole body. A small fraction (about 0.2%) of the total c a l c i u m m t h e isotope 4 8 C a n i s converted into 49Ca with a half-life of 8.8 min.

FIGURE 9--5 Radiographicabsorptiometry. The simultaneous exposure of an aluminum wedge on a hand radiograph allows for a reproducible determination of bone density with an appropriate exposure technique. [FromJergas M, Genant HK: Quantitativebone mineral analysis. In Resnick D (ed): Diagnosis of Bone and Joint Disorders, 3rd ed. Philadelphia, WB Saunders Co, 1995, pp 1854-1884.]

CHAPTER 9 NoninvasiveAssessment of Bone cortical bone and does not reveal a change confined to a small part of the skeleton. Also, it cannot distinguish between normal bone and heterotopic calcium. Nevertheless, neutron activation analysis was once considered the gold standard of bone mass measurements and may have some implications as a reference method for calibration of absorptiometric bone mass measurement systems. For instance, the manufacturer of DXA instruments relies on cross-calibration for updating new instruments and analysis software. This may be appropriate for longitudinal precision, but also can propagate accuracy errors into newer instrument generations. 49'5~ Compton scattering techniques have been employed for bone mass measurements since the early 1970s. 51'52 They provide density estimation of selected volumes of bone and utilize the quantitative information extracted from the scattered radiation, not from the primary beam transmitted through the bone target. For inducing Compton scattering a relatively high photon energy is necessary, usually generated from a monoenergetic isotope source. Details about the effective dose of Compton scattering have not been published yet. However, when applied to the peripheral skeleton for a small volume, the radiation dose should be small. The bone target to be measured is defined by the position of the highly collimated gamma ray source and a highly collimated detector. Their projected pathways intersect perpendicularly to each other within the object and therefore define the volume 56 (Fig. 9 - 6 ) . Measurements obtained with this technique reflect not only the density of mineral substance within the bone

281 but also include a composite of all medullary components. The detected scatter is proportional to the electron density, independent of the atomic number. For this reason one must be aware of marrow fat effects. The precision of the technique has been established as 2% to 5%. Because of their extreme technical requirements and relatively high radiation dose, both neutron activation analysis and Compton scattering technique are regarded as investigational techniques limited to research. For clinical practice they have been superseded by absorptiometric devices for bone assessment, which are much more applicable, cost-effective, and easy to perform.

F. Photon and X-ray Absorptiometry with One or Two Spectrum Technique 1. SINGLE PHOTON AND X-RAY ABSORPTIOMETRY

Absorptiometric bone measurement techniques were developed in the 1960s and have been modified and improved since then. In 1963 Cameron and Sorenson introduced SPA; they even mentioned the dual energy approach in their original report. 54 A highly collimated photon beam from a radionuclide source is used to measure radiation attenuation at the measurement site. If the attenuation coefficients of bone and soft tissue are known, then the measured attenuation can be converted to BMD or bone mineral content (BMC) by using a

FIGURE 9--6 Schematicrepresentation of the source and the scatter for Compton scatter densitometry. [From Jergas M, Genant HK: Quantitative bone mineral analysis. In Resnick D (ed): Diagnosis of Bone and Joint Disorders, 3rd ed. Philadelphia, WB Saunders Co, 1995, pp 1854-1884.]

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FIGURE 9--7

Soft tissue contributes critically to the measured absorption in SPA. The introduction of a water bath in which the measured body part is immersed corrects for the inconstant path length that is caused by the soft tissue, as illustrated by the intensity profiles at the bottom line of the figure. [From Jergas M, Genant HK: Quantitative bone mineral analysis. In Resnick D (ed): Diagnosis of Bone and Joint Disorders, 3rd ed. Philadelphia, WB Saunders Co, 1995, pp 1854-1884.]

known standard (Fig. 9-7). For optimal tissue and bone separation the photon energy should be under 70 keV; mostly 1251is used. For correct measurement the bone must be surrounded by soft tissue of constant thickness. This is normally achieved by immersing the limb in a water bath, surrounding the scanning site with a water bag or other tissue equivalent material, or by compressing the limb to constant thickness. Therefore, SPA is confined to the appendicular skeleton. Clinical instruments based on this principle were available during the 1970s and 1980s. The precision obtained with early devices was 3% to 5% and the accuracy was about 3%. 55 Replacing the radionuclide source by an x-ray tube in more recently developed devices (SXA) has improved precision considerably. Other advantages are reduced examination time and cost-effectiveness for these systems.

2. DUAL PHOTON AND X-RAY ABSORPTIOMETRY Single energy measurements are not possible at sites with variable soft tissue thickness and composition (i.e., the axial skeleton, hip, or whole body). For these purposes dual energy techniques are employed to correct for unknown path length in the body. The initial approach to dual energy absorptiometry, dual photon absorptiometry (DPA), uses a radionuclide source at two effective discrete energy levels. Radiation of distinct energies is attenuated by tissues to different extents. From the attenuation profiles of both a high- and a low-energy beam, an attenuation profile ideally reflecting the bony components may be calculated (Fig. 9-8). The radionuclide sources for early DPA densitometers consisted of radionuclide combinations to generate two distinct energy levels. The dual emitter 153Gd (44 and 100 keV)

CHAPTER 9 NoninvasiveAssessment of Bone

283

FIGURE 9--8

Principle of DPA and DXA. Radiation of distinct energies is attenuated by tissues to different extents. In both soft tissue and bone, a low-energy beam is attenuated to a greater degree than a high-energy beam. In bone, this attenuation occurs to a much greater extent than in soft tissue. By entering the attenuation profiles for a low- and high-energy beam into a mathematical equation system, an attenuation profile of the bony components may be calculated. [From Jergas M, Genant HK: Quantitative bone mineral analysis. In Resnick D (ed): Diagnosis of Bone and Joint Disorders, 3rd ed. Philadelphia, WB Saunders Co, 1995, pp 1854-1884.]

was introduced in the 1970s and became the standard radionuclide source for DPA. 56 A dual energy technique involving x-rays was first introduced by Krokowski and Schlungbaum, who took lateral x-rays of the lumbar spine at 62 and 250 kV and then calculated bone density from photometric measurements of the lumbar vertebrae and the adjacent soft tissue. 57 Dual x-ray absorptiometry (DXA), based on this principle and the method of x-ray spectrophotometry, was introduced commercially as the direct successor to DPA in 1987. 58-61 In DXA, the radionuclide source is replaced by an x-ray tube. Depending on the manufac-

turer, two distinct energy level beams are either generated by the x-ray generator or filtered from an x-ray spectrum. The main advantages of an x-ray system over a DPA radionuclide system are shortened examination time due to an increased photon flux of the x-ray tube and greater accuracy and precision due to higher resolution and the lack of radionuclide decay. 62 DXA has taken the place of DPA, thereby reaching great acceptance in clinical practice and research. The usual anatomical sites for DXA measurement of bone mineral include the lumbar spine, proximal femur, and whole body, but peripheral sites can also be scanned. The digital im-

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age resulting from the measurement allows a gross survey of the region examined (Fig. 9 - 9 ) . The software of all DXA devices allows one to identify regions of interest with distinct compositions of trabecular and cortical bone such as the femoral neck and Ward's triangle, and the ultradistal or the distal third of the radius. Fractured vertebrae may be excluded from analysis. The examination procedure at the lumbar spine with the initial devices took 6 to 15 minutes. Newly developed devices using enhanced generators or a fan beam instead of a pencil beam x-ray source make the process even faster, shortening the examination time to 2 minutes o r l e s s . 63 The in vivo precision of the posteroanterior (PA) DXA examination of the lumbar spine is 0.5% to 1.5% with

an accuracy error of 1% to 10%. 6 4 - 6 9 Due to osteophytes, aortic calcifications, degenerative facet hypertrophy, and intervertebral disk space narrowing in degenerative disk disease, the measured bone mineral density may be increased artificially in the PA measurement of the lumbar spine (an important drawback of this method, especially in elderly patients); furthermore, the area projectional measurement includes substantial portions of compact bone, thereby reducing discrimination between osteoporotic and nonosteoporotic s u b j e c t s . 7~ A lateral examination of the lumbar spine makes possible an evaluation of the vertebral b o d y - - w i t h almost exclusive measurement of the trabecular bone. Therefore, the correlation between lateral DXA and QCT, which measures

FIGURE 9--9 Lumbarspine (upper left), hip (upper right), whole body (lower left), and forearm (lower right) as they are depicted by DXA. These are the typical anatomical sites that are used for the application of DXA.

CHAPTER 9 NoninvasiveAssessment of Bone the vertebral body, has been found to be stronger than that of PA DXA and Q C T . TM This method can reduce the errors intrinsic in the PA examination of the lumbar spine. However, overlap of the iliac crest may substantially increase the measured bone density primarily at the level L4, and L2 is overlapped by ribs in almost all patients. Nevertheless, the inclusion of all vertebral levels L2 through L4 usually yields the best precision and diagnostic sensitivity. 75"76Beyond that, the reproducibility of the lateral DXA measurement is poorer due to greater thickness and nonuniformity of the soft tissue in the lateral projection. 75'77-8~ The adverse effect on reproducibility of measurements of the spine in lateral decubitus has been addressed with newer densitometers using a tube-detector system that can be rotated. This G arm allows for lateral spine scanning with the patient in the supine position, thereby reducing obliquity with pelvis and rib overlap and improving the in vivo reproducibility to a b o u t 2 % . 76,82 Several studies indicate that an age decrease is more pronounced in lateral B MD. Formica et al. recently reported that this greater decrease with age may be explained by the increased fat content in the soft tissue baseline region in elder women. 83 Lateral supine DXA is also more strongly associated with prevalent vertebral fractures than is standard PA B MD, indicating a potentially superior diagnostic sensitivity of this m e t h o d . 76,84-86

Dual x-ray absorptiometry is also employed for measurements at the appendicular skeleton. Most standard DXA densitometers allow for the highly precise measurement of the radius using regions of interest like those derived from SPA and SXA measurements and also userdefined subregions. 87'88 Recently, specially designed DXA densitometers exclusively for the forearm have been introduced that may provide these measurements at a lower cost. Measurements of the calcaneus using standard DXA scanners have been performed recently by several researchers, demonstrating the ability of DXA to measure bone density reliably at this site. 89-9~ Low radiation dose, availability, and ease of use have made DXA the most widely used technique for measurements of bone density in clinical trials and epidemiological s t u d i e s . 92'93 Different DXA densitometers of one manufacturer usually yield comparable results, which, depending on the scan mode and region scanned, are often within the precision error of the densitometer. 94-98 For densitometers of different manufacturers, however, the results may vary substantially due to differences in bone standards, edge-detection algorithms, and regions of interest. Under the auspices of an international DXA standardization committee including all leading manufacturers of DXA equipment, a standardized BMD (sBMD, given in milligrams per square centimeter) for measurements at the lumbar spine, based on

285 the excellent in vivo correlation among all densitometers at this site, has been proposed. 99 The standardized B MD provides compatibility of DXA results at the lumbar spine obtained on different scanners. To provide similar standardization at other sites, changes of the analysis software are required due to substantial differences in the regions of interest between the different manufacturers. Being a projectional technique, the measured bone density in DXA does not reflect a true volumetric density but rather an area density, calculated as the quotient of the B MC and the area. This normalization by the projected area partly reduces the effect of body size. However, it does not take the true volume (e.g., of a vertebra) into account. For a constant volumetric bone density, a larger vertebra would typically yield higher area BMD results than a smaller one. Several volumetric estimates of bone density derived from either PA DXA or both PA and lateral DXA of the lumbar spine have been proposed. 84'1~176176 Volumetric estimates of bone density from paired PA and lateral measurements may be more strongly associated with prevalent vertebral fractures than the standard DXA measurements. 1~ A volumetric estimate of femoral neck bone density, bone mineral apparent density (BMAD), did not improve the predictive value of standard BMD measurements for future hip fractures in the context of a large epidemiological study. 1~ Further studies are required to confirm these early results and to establish the role of volumetric estimates of projectional bone density. Due to the high resolution, anatomical details of the examined region are depicted clearly with the resulting digital images. Using DXA for obtaining lateral images of the lumbar spine offers the advantage that the scanning b e a m m i n contrast to conventional cone beam radiographymis always parallel to the vertebral end plates. This allows a better definition of vertebral dimensions for a morphometric analysis. This method has been called morphometric x-ray absorptiometry, a name referring to the DXA approach. ~~176 Overlying structures like ribs or iliac crest may have an adverse effect on the morphometric analysis. To enhance the accuracy of this method, technical modifications of the x-ray tube and the detector system may provide images with higher resolution and thus enhance the analysis of vertebral deformities. The disadvantage of a higher resolution for MXA devices may be the required higher radiation dose to the patient compared to regular DXA. Beyond that, the same limitations that apply to the morphological analysis of conventional radiographs apply to MXA. These techniques are in a developmental stage, and image quality throughout the spine is still a major concern among researchers. However, if proven suitable for a diagnosis of vertebral fractures, for certain indications (e.g., clinical drug trials) MXA may be able to take the

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place of conventional spine radiography, offering low radiation exposure and simultaneous measurements of bone density. Architectural properties derived from conventional pelvic radiographs, such as thickness of the femoral cortex or width of the trochanteric region, were found to be associated with future hip fractures. 1~ However, one disadvantage of conventional radiography is that positioning is not as strictly standardized as it is with DXA. When using pencil beam technique with DXA there is no substantial magnification of the object studied. Researchers have studied geometric properties of the femur on DXA scans and found that the hip axis length was significantly associated with future hip fractures independent of age and bone mineral density. 1~ Measurement of the hip axis length has been automated, allowing for an uncomplicated and reproducible assessment of an individual's hip axis length. 1~ Similarly, geometric variables derived from DXA scans of the radius predicted the fracture load in vitro. 11~These studies primarily document the importance of architectural bone properties for the biomechanics of fracture and may potentially account for differences in the fracture risk between ethnic groups. ~1'112Further studies in this field are required, and the assessment of simple geometric variables may be an interesting asset to dual x-ray absorptiometry.

G. Q u a n t i t a t i v e C o m p u t e d T o m o g r a p h y 1. INTRODUCTION

Based on fundamental methods and technical evaluation described by numerous investigators in the 1970s, 113-12~clinical QCT of the spine was introduced in the early 1 9 8 0 S . 34"121 The usefulness of this technique, which has been widely investigated in recent years, lies in its ability to precisely determine true volumetric density (in milligrams per cubic centimeter) in three dimensions at any skeletal site. For measurements in the spine, the potential advantages of quantitative CT over DPA or DXA are its capability for providing both a direct density measurement and spatial separation of highly responsive trabecular bone from less responsive cortical bone. Therefore, QCT has been principally employed to determine trabecular bone density in the vertebral centrum. In this application, QCT has been used for assessment of vertebral fracture risk, 121-125 measurement of age-related bone loss, 85"126'127and follow-up of osteoporosis and other metabolic bone diseases. 128 The validity of this technique for measurement of vertebral cancellous bone is widely accepted and it is used at over 4000 centers worldwide. Generally, spinal QCT is performed on standard clinical

CT scanners. It employs an external bone mineral reference phantom to calibrate the CT number measurements to bone-equivalent values as well as special software to place regions of interest inside the vertebral body. 2. ACQUISITION TECHNIQUE

a. How Does QCT Measure Bone Density? For the measurement of vertebral bone density, a calibration phantom, containing several cylindrical channels of known densities of hydroxyapatite (HA) or KzHPO4, is placed under the patient's back and scanned simultaneously (Fig. 9-10). Normally, a lateral scout view is first taken to determine the mid levels of the lumbar vertebrae being scanned (usually L1 through L3), and each vertebra is subsequently imaged using a 1-cm slice thickness, with the gantry angled appropriately. Following data acquisition, the reconstructed image is analyzed with software that places a ROI in the central trabecular bone or cortical rim of the vertebral body. The average attenuation observed in the region of interest is converted to HA- or KzHPO4-equivalent densities using the equation of a regression line determined between the known densities and measured attenuation values of the calibration phantom channels. The calibrated BMD values measured for the individual vertebrae are subsequently averaged and the patient's average value is compared to a population-specific normative database adjusted for age and the type of CT scanner. In order to improve precision and reduce acquisition and analysis time, the sagittal location of mid vertebral slices and the axial placement of regions of interest can be highly automated. 129'13~The software automatically locates the vertebral body, maps its outer edges, and employs anatomical landmarks such as the spinous process and spinal canal to calculate size and location of the region of interest. The systems can place trabecular, cortical, or integral regions of interest. Typical automatic analysis time for a vertebral body is about 5 seconds, and total scanning time is several minutes. b. Single and Dual Energy Approach for QCT. QCT can be performed in single energy (SEQCT) or dual energy (DEQCT) modes, which differ in accuracy, precision, and radiation. The accuracy of SEQCT for spinal bone mineral determination depends on variable marrow fat composition in the vertebrae, the accuracy of the calibration standard, and beam hardening errors, and scatter, among other factors. 131-133 The principal source of error is marrow fat, which causes SEQCT measurements to underestimate BMD and overestimate BMD loss. However, vertebral marrow-fat content increases with age, and a simple correction procedure that takes this

CHAPTER 9

Noninvasive Assessment of Bone

FIGURE 9--10 Quantitative CT. The lateral scout view (A) provides a rapid and simple method to define the midplane of four vertebral bodies (A), and a single 8- to 10-mm-thick section is obtained at each level (B). The classic oval region of interest is placed anteriorly in the middle of the vertebral body of three to four consecutive lumbar vertebrae and contains purely trabecular bone. For reference, regions of interest also are placed in the compartments of the calibration standards that contain distinct solutions of K2HPO4.

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into account can reduce the BMD accuracy errors to levels that are small compared with the biological variation. T M Additionally, marrow-fat errors can be further reduced by using a kVp setting that minimizes the fat sensitivity for the given scanner. Although it is possible to improve accuracy by employing DEQCT, this approach incurs reduced in vivo precision and higher dose, and thus is recommended only for research studies that require higher accuracy. 135'136 3. CLINICAL APPLICATIONS OF SPINAL

QCT

a. Fracture Discrimination. The in vivo precision errors of 2% to 4% and the accuracy errors of 5% to 15% reported for spinal QCT are generally higher than those observed for posteroanterior DXA of the spine and comparable with those of lateral DXA. However, QCT's ability to selectively assess the metabolically active and structurally important trabecular bone in the vertebral c e n t r u m 85'137-139 results in excellent ability to discriminate vertebral fracture and to measure bone loss, generally with better sensitivity than projectional methods such as DXA or DPA. Ross et al. employed prospective data to assess the predictive power of various BMD measurements for vertebral fracture and found that a spinal QCT measurement two standard deviations (2 SD) below the normative value was 40% more predictive of future vertebral fracture than was the corresponding spinal DPA measurement. Interestingly, they also found that both spinal DPA and QCT had statistically significant associations with fracture even when they were combined in the fracture prediction model, indicating that these two techniques may provide independent information about vertebral fracture risk. Other studies have examined BMD decrements between normal subjects and those with vertebral fractures. These studies reported that the decrement as measured by spinal QCT is significantly higher than that observed by posteroanterior DXA and that vertebral fracture discrimination is generally superior with QCT. 85'91'124'14~ b. Age- and Menopause-Related Bone Changes. Because the metabolic rate in the vertebral trabecular bone is substantially greater than that of the surrounding cortical bone, the ability of QCT to selectively measure trabecular bone gives it comparatively good sensitivity for measurement of bone loss following the menopause. In a cross-sectional study of 108 post-menopausal women, Gulgielmi et al. 85 measured overall bone loss rates of 1.96% per year with QCT compared with 0.97% per year and 0.45% per year, respectively, for lateral DXA and posteroanterior DXA. Generally, it has been found that the cross-sectional bone loss rate in females is typically 1.2% per year when measured with QCT and

a little over one half that value when measured with DXA or DPA. 128Block et al. carried out a comprehensive QCT study of the patterns of age-related bone loss rate and found that bone loss in women was best described by a two-phase (linear-exponential) regression, with a linear bone loss of 0.45 mg/cc/year up to the menopause, followed by a 25 mg/cc decrement during the early menopause, and an exponential pattern of bone loss of 1.99 mg/cc/year following the menopause. 126 4. PERSPECTIVES ON QCT a. Volumetric QCT at the Spine. While use of QCT has centered on two-dimensional characterization of vertebral trabecular bone, there is interest in developing three-dimensional, or volumetric, computed tomography (vQCT) techniques both to improve spinal measurements as well as to extend QCT assessments to the proximal femur (Fig. 9-11). These three-dimensional techniques encompass the entire object of interest either with stacked-slice or spiral CT scans and can employ anatomical landmarks to automatically define coordinate systems for reformatting of the CT data into anatomically relevant projections. In the spine, three-dimensional methods have been investigated to improve both longitudinal performance and discriminatory capability. Volumetric methods would be expected to improve the in vivo precision of QCT, first by employing image alignment techniques to reproducibly quantify the same volume of tissue in longitudinal studies, 142'143 and second by assessing the trabecular bone from the entire vertebral centrum, a volume roughly nine to ten times larger than the standard elliptical region of interest. However, assessment of a larger volume of interest coveting the trabecular bone in the centrum does not necessarily improve the identification of vertebral fracture over standard two-dimensional QCT methods. Thus, volumetric studies of regional B MD, which examine subregions of the centrum that may vary in their contribution to vertebral strength 144 ' 145 and studies of the cortical shell, 146-149the condition of which may be important for vertebral strength in osteoporotic individuals, 15~ are of interest for future investigation. b. Volumetric QCT at the Hip. Because of the proximal femur's complex architecture and dramatic threedimensional variation in its density, the two-dimensional QCT methods widely used in the spine cannot be used to assess the proximal femur. Thus, there is no clinically accepted QCT technique for the hip and virtually all densitometric assessment of the proximal femur is performed with DXA, which provides an integral measurement of trabecular and cortical bone. Early attempts to apply QCT methods to the proximal femur measured

CHAPTER 9 Noninvasive Assessment of Bone

289

FIGURE 9-- 11 A, Three-dimensional representation of excised lumbar vertebral body. The vertebral body, mounted in a water-filled cylinder, was encompassed with 3-mm contiguous slices; segmentation was obtained by mapping the bone surface using a contour tracking algorithm. B, Three-dimensional representation of proximal femur of patient with osteoporosis secondary to paraplegia. Proximal femur was encompassed with 3-mm contiguous slices, and segmentation was obtained by mapping the bone surface using a contour-tracking algorithm. (From Genant HK, Engelke K, Fuerst T, et al: Noninvasive assessment of bone mineral and structure: State of the art. J Bone Miner Res 11:707-730, 1996.)

purely trabecular bone, 151'152 because trabecular bone shows the earliest loss and will most effectively identify individuals at risk for fracture. However, the contributions of trabecular and cortical bone to proximal femur strength vary with the proximal femur site. 153 Thus, welldefined volumes of interest selectively measuring trabecular and cortical bone, as provided by QCT, may be important for assessment of bone strength at various sites in the proximal femur. Additionally, the crucial role of geometry in determining proximal femur strength has been well documented. 1~ QCT, with the inherent ability to resample data along any axis of interest, yields geometric information not obtainable with projectional techniques. For example, Lotz et al. resampled CT data along an axis defined by the peaks of the greater and lesser trochanters and found that the product of average intertrochanteric CT number and intertrochanteric area correlated extremely well (R2= 0.90) with in vitro fracture load in a configuration simulating a fall to the side. ~Sv In addition to assessment of proximal femur strength, vQCT could play a useful role in monitoring differential trabecular or cortical bone response to pharmacological interventions.

c. High-Resolution and Micro-Computed Tomography (HRCT/txCT). While the average BMD measured within a relatively large region of interest is a valuable

tool for the assessment of osteoporosis, an improved assessment of bone strength and fracture risk prediction may also require microstructural analysis. Apart from trabecular BMD, two main factors that affect bone strength are the architecture of the trabecular network and the thickness of the cortical shell. While the spatial resolution of clinical CT scanners (typically > 0.5 mm) is inadequate for highly accurate cortical measurements and for an analysis of discrete trabecular morphological parameters, newer CT developments try to address these issues. Two main approaches can be distinguished: (1) the development of new image acquisition and analysis protocols using existing clinical CT scanners; and (2) the development of new CT scanners for in vivo investigations of peripheral bones or for in vitro two- or threedimensional IxCT for structural analysis of very small bone samples (typically < 1 cm3). These efforts to develop new imaging and analysis protocols for existing scanners with limited spatial resolution have often focused on a regional analysis of BMD. In studies on the spine, Sandor et al. 146'158 divided the trabecular area into several regions of interest in the form of a spider net. The BMD was distributed in a Wshaped pattem with maximum B MD in the lateral and anterior portions of the vertebral body. Regions with highest BMD showed the highest loss with age. Hangartner and Gilsanz 159 and Sumner et al. 16~ addressed

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techniques to determine the peak appendicular cortical density and vertebral cortical thickness, respectively. Flynn et al. 161 used CT to determine regional bone density in 18 small cylindrical regions of interest in the lower lumbar spine. Pattern classification methods identified vertebral architectural density patterns that potentially provide enhanced fracture discrimination. Instead of the usual trabecular BMD analysis, BrailIon and colleagues 162 suggested analysis of the standard deviation of the BMD values as a parameter that partially reflects structural variations in the cross-sections of the lumbar vertebrae. A high BMD standard deviation indicates a high degree of gray level variations in the image and thus a highly networked bone architecture. Engelke et al., using a very-low-dose technique, applied this idea to a data set of 214 w o m e n . 163 However, this study did not confirm a significant potential of the BMD standard deviation as measured in trabecular spinal QCT to improve the capability of B MD to separate osteoporotic from nonosteoporotic s u b j e c t s . 163 Nevertheless, this technique could possibly be useful at higher radiation, which provides better depiction of structure. Another direction is the development of in vivo, highresolution, thin-slice CT (slice thickness, 1 to 1.5 mm). A high-resolution image of a vertebral body that clearly displays structural information in a higher dose CT image is shown in Figure 9 - 1 2 . However, the quantitative extraction of this information is difficult and the results often vary substantially according to which image processing technique is used. Some investigational work using thin-slice tomography has been published recently by Chevalier et al. 164 They measured a feature termed the trabecular fragmentation index (length of the trabec-

ular network divided by the number of discontinuities) to separate osteoporotic subjects from normal subjects. However, this index did not readily separate postmenopausal osteoporotic women with vertebral fractures from normal or osteopenic subjects. 164 A similar trabecular texture analysis approach was also reported by Ito. 165 Ultra-high-resolution CT scanners for peripheral skeletal in vivo measurements have been developed by Rtiegsegger et al. 114'166-168 The images, with a spatial resolution of 100 to 200 Ixm, show trabecular structure in the radius and the tibia. These state-of-the-art scanners probably approach the limits of spatial resolution achievable in vivo when administering acceptable exposure rates. The images can be used for quantitative trabecular structural analysis and also for a separate assessment of cortical BMD. Feldkamp et al. 169'170 constructed a IxCT system for in vitro three-dimensional analysis of small bone samples. The spatial resolution of 60 to 100 Ixm clearly separates individual trabeculae and thus allows for a three-dimensional analysis of a trabecular network. Based on data sets from this CT scanner, Engelke et al. 171'172 developed a three-dimensional digital model of trabecular bone that can be used to compare two- and three-dimensional structural analysis methods and to investigate the effect of decreasing spatial resolution and image processing technique on the extraction of structural parameters. Three-dimensional data sets can be used not only for calculating classic histomorphometric parameters like trabecular thickness a n d separation 173'174 but also for determining topological measurements like the Euler number, which is a measure of three-dimensional connectivity. 175 Another in vitro CT scanner with a spatial

FIGURE 9--12 High resolution (500 X 500 Ixm) CT image of a 1.0-mm slice of a vertebral body imaged in vivo. Trabecular structure is well delineated in gray-scale (A) and skeletonized (B) images. (From Genant HK, Engelke K, Fuerst T, et al: Noninvasive assessment of bone mineral and structure: State of the art. J Bone Miner Res 11:707-730, 1996.)

CHAPTER 9 Noninvasive Assessment of Bone resolution of 20 Ixm has recently been developed by Rtieggsegger et al. 176'177 Whereas the CT scanners described above use an x-ray tube as their radiation source, other investigators 178-18~ have explored the potential of high-intensity, tight-collimation synchrotron radiation, which allows for either faster scanning or higher spatial resolution for imaging bone specimens. 5. QCT AT PERIPHERAL SITES Special purpose peripheral QCT (pQCT) scanners have been employed for measurements of BMC and BMD of the peripheral skeleton. Initially, a radionuclide source (usually 125I) was used; however state-of-the-art scanners employ x-ray s o u r c e s . 168'181-186 Peripheral QCT allows for a true volumetric density measurement of appendicular bone without superimposition of other tissues and provides exact three-dimensional localization of the target volume. Ease of use and the ability to separately assess cortical and trabecular bone, and to measure BMD, BMC, and axial cross-sectional area, make the method an interesting alternative to SPA or SXA. There are about 700 pQCT systems in use, mostly in Europe. The great majority of these systems are clinical pQCT scanners; about 20 are ultra-high-resolution, highprecision pQCT systems for research applications. With the commonly used clinical pQCT scanner, measurements in the distal radius are performed at only one site with a single axial slice of 2.5 m m thickness located at the level that represents 4% of the ulnar length from the distal radial cortical end plate (Fig. 9-13). The short-term in vivo precision of the clinical pQCT has been measured using groups of healthy young vol-

291 unteers. Butz et a l . 187 found relative precision errors (CV) of 1.7% for trabecular, 0.8% for total, and 0.9% for cortical BMD measurements. Lehmann et al. 188 (pQCT with an x-ray source) and Schneider et al. 183 (pQCT with a radionuclide source) calculated absolute precision errors for trabecular regions of interest between 2.6 and 3.1 mg/cm 3, which resulted in CVs of under 1%. In a study by Grampp et al. 189 of pre- and postmenopausal women, the average absolute precision errors for the trabecular and total region were of the same order as in the previous studies (1.8 to 3.4 mg/cm 3, 3.8 to 8.5 mg/ cm 3, respectively), but the resulting CVs of the postmenopausal population were higher (0.9 to 2.1%, 1.1 to 2.6%, respectively), because of lower average BMD in their groups. Long-term in vitro precision with phantom measurements was calculated by Wapniarz et al. to be about 0 . 9 % . 19~ In vitro, the accuracy of the method was calculated to be about 2%. TM In a cadaver study in which radii were measured with pQCT and then ashed, Takada et al. found high correlations between total pQCT BMC and ash weight (r - 0.90) and between pQCT total BMD and ash weight (r = 0 . 8 2 ) . 191 The relationship between pQCT parameters and aging in healthy subjects was evaluated in several studies. Using a high-resolution scanner, Rtiegsegger et al. found that in contrast to trabecular BMD, which declined with age, cortical density (but not cortical B MC or area) remained constant between the ages of 20 and 70 years. TM Similar observations with a clinical pQCT scanner were made by Grampp et al., 192 who found only relatively small annual BMD changes in healthy volunteers of - 0 . 3 0 % in total, - 0 . 2 5 % in trabecular, and - 0 . 1 9 % in

FIGURE 9--13 pQCT cross-sectional image of forearm (left) showing declination of cortical bone and central trabecular bone (center). The cross-section is located at the level that represents 4% of the ulnar length from the distal radial end plate (right).

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MARTIN UFFMANN, THOMAS P. FUERST, MICHAEL JERGAS, AND HARRY K. GENANT

cortical BMD. In this study, the highest age-related changes in pQCT parameters measured at the radius occurred in the cortical thickness measures, with an average annual decrease of - 0 . 6 9 % in cortical BMC and - 0 . 5 2 % in cortical area indicating principally a thinning of the cortex by endosteal resorption. 192 Other studies found higher annual changes in BMD but did not consider BMC or cortical area. Schneider et al. 183 found annual decreases of 0.5% in the trabecular BMD of healthy women and 1.9% in osteoporotic women, and Butz et al. 187 found changes of 0.9% in the trabecular and 1.1% in the total BMD. The differences between the studies are not entirely clear but may be related to different criteria in the definition of the study subjects. The influence of B MD in trabecular and in cortical bone on the total BMD measured by pQCT was evaluated i n a study by Rico et al. with healthy young male and female volunteers. 193 Here, the cortical BMD proved to be more closely related to the total BMD than was trabecular BMD. This was indicated by the higher correlation coefficients for comparisons of pQCT total versus cortical BMD (r = 0.95), as compared to total versus trabecular (r = 0.62), and of trabecular versus cortical BMD (r = 0.43). In some studies, pQCT measurements of B MD at the radius were found to be successful in distinguishing between osteoporotic and nonosteoporotic patients, and in monitoring subjects during clinical studies. 168'181 However, other authors have reported conflicting results, especially for peripheral trabecular B M D . 194-196 The importance of the measurement of cortical bone p e r s e was suggested by Sparado et al., who found in a biomechanical study that the cortical shell contributes substantially to the mechanical strength of the distal radius. 197 The thinning of the cortical rim at the radius was a potential mechanism contributing to osteoporosis, 168'~81 and it identified this compartment as a promising location for B MC and thickness measurements. These findings were supported in a study by Grampp et al. w2 that examined the ability of BMD, BMC, and cross-sectional area to detect osteoporotic changes. Only the cortical area and BMC significantly distinguished between women with nontraumatic vertebral fractures and healthy postmenopausal women; these two parameters also showed the highest age-adjusted odds ratio for fracture risk. These data suggest that pQCT measurement of cortical rather than trabecular bone at the radius may have greater diagnostic sensitivity in terms of appendicular measurements. Modem pQCT scanners also incorporate a multislice data acquisition capability coveting a larger volume of bone as compared with the commonly used single-slice technique. ~98'199The measurement of several slices is potentially more representative of changes in the distal ra-

dius and may therefore reflect the bone status of an individual more accurately. If studies employing this multislice pQCT technique are successful, they may contribute to more extensive use (currently about 1000 systems worldwide) of this already promising technique.

III. ASSESSMENT OF BONE WITHOUT RADIATION A. Quantitative Ultrasound 1. BONE DENSITY AND QUALITY MEASUREMENT USING ULTRASOUND

The use of QUS for the assessment of skeletal status has seen continued and accelerating interest in recent years, z~176 The attractiveness of QUS lies in its low cost, portability, ease of use, and freedom from ionizing radiation. These benefits, combined with preliminary clinical results showing good diagnostic sensitivity and the ability to predict fracture risk, have encouraged further basic investigation and commercial development. Currently there are more than 12 commercially available QUS devices, although none has been approved for clinical use in the United States by the Food and Drug Administration (FDA). For this reason, in the United States QUS devices for bone assessment are found primarily at research centers. Nevertheless, QUS devices have a very wide distribution in Europe and Asia and are routinely used for clinical assessment of osteoporosis. By the end of 1996 more than 3000 clinical ultrasound units will be in use worldwide. 2. QUANTITATIVE ULTRASOUND PARAMETERS

These devices are used to measure both the velocity and the attenuation of ultrasound as it travels through bone. The choice of sites for the measurement of ultrasound properties is driven by the need for easy access to the bone (minimal intervening soft tissue) and the type of bone one wishes to examine (trabecular or cortical). Currently, devices are available to interrogate the calcaneus, phalanges, tibia, and patella. In each case the devices are of the transmission type, using two ultrasound transducers (a transmitter and receiver) positioned on each side of the bone to be measured (Fig. 9 - 1 4 ) . These devices measure ultrasound parameters in primarily trabecular bone at the calcaneus and patella, cortical bone at the tibia, and integral bone at the phalanges. The velocity parameter measured is often called the ultrasound transmission velocity (UTV) or more commonly the speed of sound (SOS). The ultrasound attenuation parameter is called broadband ultrasound attenuation (BUA).

CHAPTER 9

Noninvasive Assessment of Bone

293

FIGURE 9--14 Schematic diagrams showing method of acquisition for two quantitative ultrasound measures. A, Calcaneal ultrasound measurement using a water bath for acoustic coupling. Paired transmitters and receivers measure both attenuation and velocity of ultrasound through the heel. Dry systems are available that use ultrasound gel for coupling. B, Ultrasound velocity measurement at the tibia with the SoundScan 2000. Probe measures speed of ultrasound wave propagation longitudinally through the tibial cortex. The fixed distance between the receivers and the difference in signal arrival time determine velocity.

a. Speed of Sound. The velocity of ultrasound wave propagation, or speed of sound, through bone is determined by dividing the distance traversed (e.g., bone diameter or length) by the transit time. The resulting velocity is quoted in meters per second (m/sec). Measurements of SOS are c o m m o n l y performed at the calcaneus, tibia, and phalanges. Velocity has also been measured in the patella, but a c o m m e r c i a l system is not currently available. Thus skeletal sites with both predominantly cortical or trabecular bone are assessable with ultrasound, although different devices are required for the various measurements. Table 9 - 1 shows typical values of SOS measured at these sites. The velocity of ultrasound in cortical bone is two to three times higher than at trabecular sites.

SOS at the heel is well established and several manufacturers (Achilles by Lunar Corporation, USA, Sahara by Hologic, Inc., USA, and CUBAclinical by M c C u e Ultrasonics, Ltd., UK) are producing devices to measure the heel in the mediolateral direction. While similar in concept, these devices differ in transducer design, analysis algorithms, and whether water or ultrasound gel are used to acoustically couple the transducers to the heel. Typical values range from 1400 to 1900 m/sec and precision errors are reported to be between 0.2% and 1 . 5 % . 201-203 Heel thickness and water temperature are parameters k n o w n to affect the velocity and are potential error sources for this measurement. 2~176 Clinical assessment of SOS at the phalanges was evaluated and first described by Jergas et al. 2~ M e a s u r e m e n t s

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TABLE 9--1 Different Approaches for Assessment of Bone with Quantitative Ultrasound Anatomical Site

Biological

Precision

Range

(CV) a

Parameter

4 0 - 1 2 0 b 1.3-6.0%

Examination Time

Os calcis

BUA (dB/MHz)

Os calcis

SOS (m/s)

1400-1900

0.2-1.5%

10 min

Mid tibia

SOS (m/s)

3400-4200

0.2-1.0%

15 min

Phalanges

SOS (m/s)

1800-2000

0.5-1.0%

8 min

40

N -r" 30.

:

10 min

t'- 20~ O

.

r" aCV = coefficient of variation. bDependent on manufacturer.

performed at this site revealed significant differences in SOS between healthy and osteoporotic postmenopausal women. 2~ Independently, a group in Italy used a similar approach and recently developed a commercially available device (DBM Sonic 1200 by Igea s.r.1, Italy). 2~ Unlike other SOS evaluations, the measurement of SOS in the phalanges is amplitude dependent and is often referred to as AD-SOS. This in some sense combines both velocity and attenuation properties into a single measure. AD-SOS values (1800 to 2000 m/sec) are generally higher than those found in the calcaneus. The coefficient of variation for AD-SOS is reported to be 0 . 5 % . 209'210 The AD-SOS measurement has greater dependence on the operator than the heel measurements, and operator skill will determine precision. Another recently developed instrument measures SOS at the mid tibia in the longitudinal direction (SoundScan 2000 by Myriad Ultrasound Systems, Ltd., Israel) (see Fig. 9-14). At this site the device measures cortical bone and records higher velocities (3600 to 4200 m/sec). Precision of this measurement (0.2% to 1%) is as good as or better than other QUS techniques 21~-2~3 and, like the phalangeal measurement, is influenced by the operator. b. Broadband Ultrasound Attenuation. Broadband ultrasound attenuation is the second parameter measured by QUS. Ultrasound attenuation results from scattering and absorption of the ultrasound signals in bone, bone marrow, and surrounding soft tissue. Langton et al. were the first to show that the attenuation of ultrasound in bone is linear in the frequency range of 200 to 600 kHz. 214 Hence the slope of this relationship defines the attenuation parameter BUA, which has units of dB/MHz. Figure 9 - 1 5 shows examples of attenuation data collected over this range of frequencies. Currently, BUA is measured only in the heel. The short-term in vivo precision errors for BUA range from 1.3% to 6 % . 202'203'215 The relatively poor precision can be explained in part by variation in foot position and consequently the region of interest measured. One study of

<

200

.

.

.

.

I

300

.

.

.

.

I

400

.

.

.

.

I

500

Frequency (kHz)

.

.

.

.

600

FIGURE 9-- 15

Examples of attenuation data collected from measurements in the calcaneus in two postmenopausal women. Signal is quite linear of the range of frequencies from 200 to 600 kHz. Regression slope is used to determine broadband ultrasound attenuation (BUA). The lower curve suggests that this woman is at increased risk of fracture.

the factors influencing precision has shown that rotation of the foot about the axis of the leg and translation in the heel-toe direction have the largest impact on the B UA measurement. 215 Foot positioning is considered the primary source of error in the B UA measurement and is probably due to the inhomogeneity of the calcaneus. Two new devices have recently been introduced that allow imaging of the BUA properties of the calcaneus (UBIS 3000 by Diagnostic Medical Systems, France, and DTU-1 by Osteometer, Denmark). 216 Like the previous generation of devices, these measure the heel in the mediolateral direction. To allow imaging of the heel, focused transducers are used to produce an ultrasound beam with an effective diameter of approximately 5 mm. This beam is scanned across the heel in 1-mm steps to image the entire calcaneus. The advantages of the BUA image are (1) that it would allow the evaluation of standardized regions of interest in all patients; (2) larger regions of interest can be used, potentially improving precision; and (3) measurement artifacts can be clearly identified and avoided. The Lunar Achilles system also reports a calculated variable named "stiffness" (which should not be confused with the biomechanical term), which is a combination of BUA and SOS having the best correlation with bone density. From the point of view of clinical interpretation, a single parameter like "stiffness" which combines attenuation and velocity can simplify interpretation when one is high and the other low.

CHAPTER 9 Noninvasive Assessment of Bone c. Standardization o f Q US Parameters. Depending on the manufacturer, velocity at the calcaneus is defined as the mean velocity between transducers (called timeof-flight velocity), through the heel (heel velocity), or through the calcaneus alone (bone velocity). 2~ Different velocity results are obtained with these various definitions. In addition, BUA will vary systematically between the various manufacturers. Thus, velocity as well as BUA differ substantially between the various devices, and results from different equipment types are not comparable. For a broader acceptance of QUS a standardization of the technique must be achieved in the future. 3. WHAT DOES ULTRASOUND MEASURE?

Recent investigations in vivo have demonstrated the utility of QUS measurements to provide information about bone that is relevant to the management of osteoporosis. While these results show the promise of QUS, the exact mechanisms of ultrasound interaction with bone and the physical properties measured remain undetermined. It is currently believed that QUS parameters are influenced by the mechanical properties of bone, which in turn are determined by bone's material and structural properties. Thus both bone mineral density and bone architecture could be expected to play a role in determining QUS properties. a. QUS and BMD. Early investigations of QUS showed a dependence of attenuation and velocity parameters on bone mineral density. Correlation coefficients have been reported from approximately 0.3 to 0.9 for both BUA and S O S . 202'213'217-220 Site-matched comparisons of BMD and QUS measurements have produced correlations of about 0 . 7 . 217'218 Thus only about 50% of the variability of QUS measurements can be explained by B MD. The remainder may be determined by other properties of bone related to bone strength, but this remains to be proven. An interesting application of QUS is as a surrogate for BMD measurement by DXA or other radiation-based technique. However, it has been demonstrated that the error in predicting lumbar spine or femoral BMD from calcaneal QUS is large (15% to 30%) and consequently unreliable. 2~ Thus estimation of bone mass is an inappropriate use of QUS. However, this does not mean QUS is not useful for fracture risk estimation. b. Q US and Bone Structure. The relationship between ultrasound parameters and bone structure has been demonstrated in several in vitro studies. First, it has been shown that B UA measured in trabecular bone cubes is dependent on trabecular orientation, being 50% higher along the axis of compressive trabeculae. 221-224 Other investigations have demonstrated the relationship of QUS

295 with stereological measures of bone structure. 225'226 In one of these studies B UA and SOS were related to trabecular spacing and connectivity, and this association was independent of B M D . 225 c. Q US and Bone Strength. These relationships between ultrasound and the material and structural properties of bone have been confirmed by tests of biomechanical strength. 223'224'227-229 The mechanical properties (elastic modulus and ultimate strength) of trabecular bone specimens measured in vitro are well correlated with both B UA and SOS. The empirical data fit reasonably with established theoretical relationships. Moreover, the anisotropy of bone structure, which is detectable with ultrasound but not absorptiometry, is similarly reflected in the elastic modulus. In studies more applicable to the clinical use of ultrasound, Bouxsein et al. 227 have shown that B UA and SOS in the calcaneus are strong predictors of femoral failure loads (r 2 = 0.5 1 and 0.40, respectively) compared to calcaneus BMD (r 2 = 0.63) and femoral neck BMD (r 2 = 0.79). These studies confirm the potential utility of QUS for bone assessment and fracture risk prediction. 4. CLINICAL ASSESSMENT WITH ULTRASOUND

The relationships of QUS with bone mass, structure, and strength suggest that ultrasound is a useful tool in the clinical assessment of osteoporosis. Measurements in vivo have established normative data and the patterns of changes in BUA and SOS with aging. Studies have also been conducted to investigate the ability of ultrasound to discriminate patients with osteoporotic fracture from age-matched controls. a. Age-Related Changes in Women. Numerous cross-sectional studies of ultrasound normative data have been reported. 2~176 They all show that QUS parameters are inversely correlated with age later in life, especially after the menopause. Before this age both BUA and SOS are relatively stable, although two studies have shown steady declines from age 20 years. 2~176 Typical rates of change are 0.5 to 1.0 dB/MHz (0.5% to 1.0%) per year for BUA and 1 to 5 m/sec (0.1% to 0.3%) per year for SOS at the heel, patella, and tibia. Only one longitudinal study has been performed to date and the results are similar to cross-sectional data ( - 0 . 5 % per year for BUA and - 0 . 4 % per year for SOS). Some studies have reported accelerated changes immediately after the menopause with slower declines later in life. b. QUS and Fracture Risk. Cross-sectional 219'220'231'234-240 studies have demonstrated that QUS can be used to discriminate normal from osteoporotic subject groups as well as traditional bone densitometry ap-

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proaches. Moreover, prospective s t u d i e s 241-244 of osteoporotic fracture have shown the utility of calcaneal QUS to predict fracture risk with the same power as, and in some cases independent from, measurements of bone mass. The majority of investigations have been made with QUS at the calcaneus. Associations have been found with both vertebral and hip fractures as well as fractures of the forearm. Logistic regression analyses have found that risk of fracture (measured with odds ratios) increases approximately 1.5- to 2.5-fold for each standard deviation decrease in BUA or SOS. This makes QUS comparable to but slightly lower than x-ray absorptiometry techniques, which typically have odds ratios of 1.5 to 3.0 depending on the measurement site and the population studied. Two large recent prospective studies 243'244 in Europe and the United States have confirmed the usefulness of BUA and SOS to predict hip fractures in elderly women that was first reported by Porter et al. in 1990. 241 Interestingly, the European study found this association to be independent of femoral neck B MD, while this was not true in the U.S. study. This may be due to the older population studied in Europe. In two cross-sectional studies of hip fractures, the association of BUA with the type of fracture (trochanteric vs. cervical) was investigated. 234'24~ Both studies performed measurements within 2 weeks of the fracture event. In one study, significantly lower BUA was found in cases of trochanteric fracture while the other showed no difference. As the calcaneus is composed primarily of trabecular bone, a better association with trochanteric fractures may be expected. However, this question requires further investigation. Recently, reports have begun to appear showing similar capabilities for ultrasound measurements at the patella, tibia, and p h a l a n g e s . 231'232'242"245-248 These crosssectional studies are not as extensive as those done for calcaneal ultrasound and have had positive and negative findings. Association with prevalent spine fracture has been demonstrated for all three techniques. However, in some studies the association did not reach statistical significance. 213'245Tibial SOS has also been associated with incident nonspine fractures. 245 In one report of 11 women with hip fracture, tibial SOS was significantly lower in cases than in age-matched controls. 23z Larger prospective studies are needed to evaluate these methods more thoroughly.

c. Q US and Longitudinal Monitoring. Reports of QUS being used to evaluate longitudinal changes due to aging or intervention are also appearing. Three studies with 2-year follow-up measurements have shown positive changes in QUS parameters between controls and women treated with calcitonin (+4.2% for BUA and

+0.8% for SOS compared to c o n t r o l s ) , 249 hormone replacement therapy (HRT) (+ 3.6% for BUA and +0.7% for SOS), 25~ and bisphosphonates (estimated + 18% for BUA). 251 These changes were statistically significant for treatment with calcitonin and HRT but not with the bisphosphonate. The power of the bisphosphonate study was limited by very small size (n = 12). These results suggest some utility for QUS to monitor patients longitudinally. However, the imprecision of the measurement will make evaluation of changes in the individual more difficult.

B. Q u a n t i t a t i v e M a g n e t i c R e s o n a n c e a n d M a g n e t i c R e s o n a n c e M i c r o s c o p y for Bone Assessment 1. QUANTITATIVEMAGNETIC RESONANCE Magnetic resonance (MR) is a complex technology that has evolved rapidly since its introduction to medical science in the early 1970s. Based upon the application of high magnetic fields, transmission of radiofrequency (RF) waves, and detection of RF signals from excited hydrogen protons, this technique has revolutionized medical imaging in general. Recently, the ability of QMR and magnetic resonance microscopy (t.LMR) to assess osteoporosis has been explored. To date, most MR imaging techniques have been limited to the study of soft tissue or of gross skeletal structure because compact bone does not generate any detectable MR signal. However, newly developed QMR techniques have been used to study trabecular bone, specifically. The presence of the trabecular bone matrix affects the signal intensity of bone marrow, an effect that is particularly enhanced in specific imaging sequences. The magnetic properties of trabecular bone and bone marrow are significantly different. These differences produce distortions of the magnetic lines of force, which make the local magnetic field within the tissue inhomogeneous and alter the relaxation properties of tissue, such as the apparent transverse relaxation time T*, in gradient-echo images. From theoretical considerations, such changes in T* should directly relate to the density of the surrounding trabecular network and its spatial geometry. The resultant shortening of relaxation time becomes greater with an increase in the concentration of trabecular bone in the surrounding homogeneous marrow tissue. Thus, in a normal dense trabecular network, T* shortening should be more pronounced than in rarefied osteoporotic trabeculae. Experimental studies have confirmed the theoretical predictions, suggesting QMR as a promising tool for studying trabecular bone architecture and assessing os-

CHAr'TEI~ 9 Noninvasive Assessment of Bone teoporosis. Davis et a l . 252 have shown a reduction in the in vitro T* of both water and cottonseed oil in the presence of bone powder at a magnetic field strength of 5.9 tesla. Rosenthal et al. 253 have measured a reduction in the T* of water present in the trabecular spaces compared with extratrabecular water, using specimens of excised human vertebrae at 0.6 tesla. Majumdar et al., TM using specimens of dried human vertebral bodies of various bone densities, have examined susceptibilitymediated relaxation effects. The mean trabecular bone density for each specimen, measured by QCT, was significantly related to the overall relaxation rate (l/T*) of intratrabecular saline. Similar relations in vivo have also been established in the forearm, distal femur, and proximal tibia sites at which the trabecular bone network shows significant variations as a function of the distance from the joint line, with the bone density and relaxation rate, l/T*, being greatest in the epiphysis and progressively decreasing towards the metaphysis and diaphysis. 255 In studies on the distal radius, precision errors in measured T* times were found to range from 1.3 to 2.9 msec, corresponding to 3.8% to 9.5% C V o 256 In a similar fashion, Ford and Wehrli 257 have used a method called MR interferometry to assess variations in T* between osteoporotics and normal subjects, and found reasonable discriminatory capability. Theoretically and from computer simulations 254'258and phantom studies, 258a the relaxation time T* of bone marrow is affected not only by the density of the trabecular matrix but also by its spatial architecture. In early experiments with specimens, Majumdar et a l . 259 have shown correlation between T* and the elastic modulus, which reflects the biomechanical properties of trabecular bone. Chung et a l . 26~ found a strong correlation (r = 0.9 1) between the Young's modulus of elasticity and l/T* in trabecular bone specimens from human lumbar spine, while Jergas et a l . 261 have shown similar relationships using specimens of tibial bone. Thus, this measured QMR parameter T* may enhance the capability for fracture discrimination and fracture risk prediction. This technique has the advantage that it can be performed at medium to low resolution, resulting in decreased acquisition times, and permitting the use of scanners at fields of 1.5 tesla or less. 2. MAGNETIC RESONANCE MICROSCOPY lxMR may prove to be an additional valuable MRbased technique for the quantitative study of trabecular microarchitecture both in vitro and in vivo. In vitro, using small RF surface coils in high-field scanners, MR microscopy can be performed at resolutions high enough to discriminate individual bone t r a b e c u l a e . 262'263 In vitro images have been obtained at in-plane resolutions as low as 33 Ixm, while in vivo images range from resolutions

297 of 78 X 78 X 300 Ixm through the phalanges TM to images at a resolution of 156 X 156 X 700 Ixm in the distal radius 265 and 200 x 234 x 1000 Ixm in the calcaneus. 266 Typically, the image is first segmented into bone and marrow phases, and histomorphometric analysis is then performed on the resulting binary image. Stereological parameters such as the trabecular bone area and volume fraction, mean intercept length, mean trabecular width, and mean trabecular spacing and trabecular number can thus be calculated. Wehrli et al. have compared the standard measures with stereological measures of bone volume fraction derived from MR images obtained at 9.4 tesla and found good correlations, 263 while Antich et al. have conducted similar experiments and found changes that accord with histomorphometry measures. 262 Kapadia et al. have extended the in vitro techniques to obtain images in an ovariectomized rat model and have shown the ability to measure changes in trabecular structure following ovariectomy. 267 Because of limitations of the signal-to-noise ratio and total imaging time, smaller individual trabeculae usually cannot be resolved with the resolution achievable in vivo at clinical field strengths, but the images show the larger trabeculae and the texture of the trabecular network. However, using standard techniques of stereology as well as texture analysis tools such as fractal analysis, the trabecular structure can still be quantified. In an early study establishing the feasibility of using such images to quantify trabecular structure, MR images of the distal radius have been obtained using a modified gradient echo sequence on a 1.5-tesla imager, at a spatial resolution of 156 txm and slice thickness of 0.7 mm. 265 It is well known that the amount of trabecular bone is greatest at distal sites of the radius and decreases proximally and this is readily seen in the MR images. Similar images obtained in the calcaneus of a normal subject are shown in Figure 9 - 1 6 . The orientation of the trabeculae is significantly different in the subtalar region as compared, for example, with the posterior region. The ellipses, representing the mean intercept length, show a preferred orientation and hence map the anisotropy of trabecular structure. In preliminary in vivo studies in the calcaneus, gray-scale reference values from fat, muscle, and tendon were used to calculate a reproducible threshold value. This approach gave a midterm in vivo precision of ---3% to 5% CV for trabecular width and spacing. 268 In MR the appearance of the image depends on several factors other than image resolution. The pulse sequence used to obtain the image, whether it is a spinecho or gradient echo, the echo time, and the magnetic field strength are all important factors that may modify the trabecular dimensions depicted in an MR image. 269 Furthermore, when the image resolution is comparable

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MARTIN UFFMANN, THOMAS P. FUERST, MICHAEL JERGAS, AND HARRY K. GENANT

IV. STANDARDIZATION AND QUALITY ASSURANCE IN DUAL X-RAY ABSORPTIOMETRY

A. Quality Assurance in DXA The validity of quantitative assessments of bone is dependent on the accuracy and precision with which the measurements are made. Principally two factors can influence accuracy and precision: (1) performance of the operators who acquire and analyze the scans and (2) the performance of the instrument used to make the measurements. Both need to be carefully monitored and controlled to achieve reliable information on skeletal health. These topics will be discussed in the context of measurements of bone mineral density with DXA. However, the specific details to be described are easily generalized to any other method of quantitative bone assessment. 1. O P E R A T O R P E R F O R M A N C E

FIGURE 9-- 16

High-resolution magnetic resonance image through the sagittal plane in the calcaneus. The anisotropy of trabeculae (dark striations in the bright marrow) is clearly seen. The mean intercept length, a measure of the mean trabecular width as a function of angle, is shown. The major axis of the ellipse defines the preferred trabecular orientation. The anisotropy of trabeculae is most pronounced in the subtalar region as is also demonstrated by the elliptical plot of the mean intercept length. The ellipses are scaled identically, thus it can also be seen that the thickest trabeculae are found in the subtalar region. (From Genant HK, Engelke K, Fuerst T, et al: Noninvasive assessment of bone mineral and structure: State of the art. J Bone Miner Res 11:707-730, 1996.)

to the trabecular dimensions, a small error is manifested as a large relative error, and hence the stereological measures from MR images are likely to be subject to these effects. In the analysis of such images the threshold is also shown to have a significant effect on the estimated parameters. 265 Technical challenges brought about by standardization of image acquisition and analysis and improved understanding of those processes underlying image formation could allow MR to become a potential tool for assessment of trabecular bone structure in vivo. As a noninvasive, nonionizing technique, MR can provide three-dimensional images in arbitrary orientations, and can also depict trabecular structure. Although it is a relatively expensive, time-consuming technique to use for primary screening for osteoporosis, it provides a potential platform for identifying particularly high-risk patients after initial bone densitometry and perhaps for assigning these patients to more aggressive therapies.

Efforts to ensure the correct acquisition and analysis of quantitative data begins with proper training and certification of the operators. Operator training should be provided by the manufacturer of the instrument at the time of installation. Typically, the training involves several days of hands-on exercises in acquisition and analysis as well as a review of radiation safety issues. For already-installed systems, training of new staff is often handled in-house with experienced operators training the new personnel. This yields varying degrees of success and depends on the expertise of the mentor and the time they have to devote to the training. Peer-to-peer training opens the possibility of bad habits being passed on to the next generation of trainees. Retraining by the manufacturer will generally be more reliable. A second important step in operator quality assurance is peer review of measurements. This can be done by an experienced operator or by the physician responsible for interpretation of the exams. This procedure will be effective in catching most mistakes that result from carelessness or haste. Moreover, the interpreting physician should have the knowledge necessary to evaluate the quality of the exam and be familiar with those factors that can lead to measurement errors (e.g., patient positioning, image artifacts, incorrect region-of-interest placement). 2. INSTRUMENT PERFORMANCE As with any device used for quantitative measurements, proper calibration of DXA scanners is vital. Correct and consistent calibration ensures accurate and precise measurements. Each manufacturer has developed methods for setting the calibration at the time of man-

CHAPTER 9 NoninvasiveAssessment of Bone ufacture. This can be done using an absolute standard whose true values are known or by comparison to a "gold standard" instrument that has been calibrated to an absolute standard. This calibration ensures that all scanners measure patients similarly. Standardization to within _ 1% can be expected. 95'27~ After installation, stable performance is monitored by daily quality control checks, which vary between manufacturers. These tests check basic electrical and mechanical performance as well as instrument calibration. Calibration is checked by scanning a test object and comparing measured bone density to historical values. Deviation from an established reference value is determined visually or with statistical tests and the operator is alerted when a value falls out of bounds. In that case, the quality control test is usually repeated; a second failure warrants a call to the manufacturer's service department. If the test is passed the second time, the alarm is ignored. The choice of test object for longitudinal monitoring of scanner performance is difficult. Ideally, the phantom would simulate the in vivo measurements as closely as possible, being constructed with appropriate materials and designed to represent the soft tissue and bone compartments of the body. In principle, a separate phantom should be available for each of the anatomical sites measured, at least for the spine and femur. However, practical concerns regarding cost and ease of production prevent the construction of such a phantom. Moreover, the desire to evaluate accuracy requires strict definition of the size and composition of the phantom, allowing the calculation of true values. This requirement limits the degree to which the phantom can simulate the patient.

299 The most commonly used test object is the Hologic spine phantom. It is provided with all Hologic densitometers and has been the standard of choice for multicenter clinical trials. It is semianthropomorphic in design, being composed of four molded hydroxyapatite vertebrae embedded in epoxy resin. The volumetric density of the hydroxyapatite is uniform, allowing measurement at only a single BMD value. Another recently introduced phantom is the European Spine Phantom (ESP). 272 This phantom was designed under the auspices of the Committee d'Actions Concert6s-Biomedical Engineering and has several advantages over the Hologic phantom. It is constructed to strict geometric criteria and includes components to model both trabecular and cortical bone. It also has different volumetric and areal density that allow the evaluation of scanner linearity and stability across the clinically relevant range of densities. Whether such elaborate phantoms offer significant advantages over simpler tools is not proven. However, it is clear that some procedure for monitoring scanner performance over time is required.

B. C o m p a r i s o n a n d C r o s s - C a l i b r a t i o n o f Different Scanners The differences in calibration among 105 machines is demonstrated in Figure 9 - 1 7 . The calibration techniques employed by the manufacturers generally result in interscanner differences less than 1% as measured with the Hologic spine phantom. This close agreement indicates that the normative data provided by each manufacturer is appropriate for use on all machines of that type with-

FIGURE 9--17 Comparisonof calibration of 105 densitomers (22 Lunar and 83 Hologic) measured with a single Hologic spine phantom scanned on each machine. Difference from mean was calculated for the two scanner types separately. About 50% of the densitomers were within 0.5% of the mean and 75% were within 1%.

300

MARTIN UFFMANN, THOMAS P. FUERST, MICHAEL JERGAS, AND HARRY K. GENANT

out concern for differences in calibration. However, with regard to longitudinal monitoring of disease progression or response to therapy, changing scanners between visits is not recommended. Rates of bone loss or gain are typically only 2% or less per year. Using a different scanner for follow-up assessment can mask the true changes in bone mass. It is important to use a single, stable scanner for follow-up measurements. Eventually equipment wears out and needs to be replaced, or changing to a new scanner with hardware improvements may be deemed necessary. In this case i