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
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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
vii
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
in
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-
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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
|
7
5
FIGURE 3--5
6
7
8
7
28
9
4n
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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Io I |
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r
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-I {3" ILl |
.~i
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)
.
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.
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.
:~
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--
/ / ....~.... )days 10 - 2
!
:~
,
6
,
, l'0hr
H
i
,, 2:3days
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
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rat mouse chicken
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110
120
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K
150
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E
.
N
.
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.
R
.
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.
T
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.
H
H
M
160
Q
N
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P
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V
170
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130 T B T ~
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T T
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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
CHAPTER 3 ParathyroidHormone and Parathyroid Hormone-Related Peptide
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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
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90
120
150
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FIGURE 4 - 1
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
N N N N N
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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.
n
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0
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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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Effect of CT infusion on plasma calcium, radioactivity, and specific activity in control (o) and in CTtreated rats (o). 45Ca was injected 12 hours before beginning the 4-hour infusion of CT or control solution. (From Robinson CJ, Martin TJ, Matthews EW, et al: Mode of action of thyrocalcitonin. J Endocrinol 39:71-77, 1967.)
CHAPTER 4
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t
o
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
i
n
1
0
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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-
CHAPTER 4
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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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FIGURE 4--7 Decreasing hypocalcemic response to calcitonin with increasing age of the rat. (From Cooper CW, Hirsch PF, Toverud SV, et al: An improved method for the biological assay of thyrocalcitonin. Endocrinology 81"610-617, 1967.)
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
106
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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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
CHAPTER 4 C
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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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tumor of bone. The first intracellular domain modulates ligand binding and signal transduction. J Clin Invest 95:2680-2691, 1995. Moore EE, Kuestner RE, Stroop SD, et al: Functionally different isoforms of the human calcitonin receptor result from altemative splicing of the gene transcripts. Mol Endocrinol 9:959-968, 1995. Egerton M, Needham M, Evans S, et al: Identification of multiple human calcitonin receptor isoforms; heterologous expression and pharmacological characterization. J Mol Endocrinol 14: 179-189, 1995. Nakamura M, Hashimoto T, Nakajima T, et al: A new type of human calcitonin receptor isoform generated by altemafive splicing. Biochem Biophys Res Commun 209:744-751, 1995. Albrandt K, Brady EMG, Moore CX, et al: Molecular cloning and functional expression of a third isoform of the human calcitonin receptor and partial characterization of the calcitonin receptor gene. Endocrinology 136:5377-5384, 1995. Bergwitz C, Gardella TJ, Flannery MR, et al: Full activation of chimeric receptor by hybrids between parathyroid hormone and calcitonin: Evidence for a common pattern of ligand-receptor interaction. J Biol Chem 271:26469-26472, 1996. Findlay DM, Houssami S, Lin HY, et al: Truncation of the porcine calcitonin receptor cytoplasmic tail inhibits intemalization and signal transduction but increases receptor affinity. Mol Endocrinol 8:1691-1700, 1994. Luben RA, Wong GL, Cohn DV: Biochemical characterization with parathormone and calcitonin of isolated bone cells: Provisional identification of osteoclasts and osteoblasts. Endocrinology 99:526-534, 1976. Nicholson GC, Moseley JM, Sexton P, et al: Characterization of calcitonin receptors and cyclic AMP responses in osolated osteoclasts. In Cohn DV, Martin TJ, Meunier PJ (eds): Calcium Regulation and Bone Metabolism: Basic and Clinical Aspects, Vol 9. Amsterdam, Elsevier Science Publishers, 1987, pp. 3 4 3 348. Nicholson GC, Horton MA, Sexton PM, et al: Calcitonin receptors of human osteoclastoma. Horm Metab Res 19:582-586, 1987. Murrills RJ, Dempster DW: The effects of stimulators of intracellular cyclic AMP on rat and chick osteoclasts in vitro: Validation of a simplified light microscope assay of bone resorption. Bone 11:333-344, 1990. Lemer UH, Fredholm BB, Ransj6 M: Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro. Biochem J 240:529-539, 1986. Jans DA, Gajdas EL, Dierks-Ventling C, et al: Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin. Biochim Biophys Acta 930:392-400, 1987. Alam ASMT, Bax CMR, Shankar VS, et al: Further studies on the mode of action of calcitonin on isolated rat osteoclasts: Pharmacological evidence for a second site mediating intracellular Ca 2+ mobilization and cell retraction. J Endocrinol 136:7-15, 1993. Suzuki H, Takahashi N, Nakamura I, et al: Calcitonin-induced alteration in the cytoskeleton is mediated by the signal pathway associated with protein kinase A in osteoclasts. Bone 16:171S, 1995. Zaidi M, Datta HK, Moonga BS, et al: Evidence that the action of calcitonin on rat osteoclasts is mediated by two G-proteins acting via separate post-receptor pathways. J Endocrinol 126: 4 7 3 - 4 8 1 , 1990.
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
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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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MICHAEL E HOLICK AND JOHN S. ADAMS
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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~8
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CH5 CH3
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VITAMIN D2
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
SUN
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
c~ lOO] .c_ E 80 ._ >
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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 -
Without Sunscreen
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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
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60
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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
,
.... t
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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
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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
.o~t~
~ .0~
7- Dehydrocholesterol
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_ _
SKIN]TEMPERATURE
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~
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OH
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O~,25S,26(OH)303] o
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HO
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I~, 24R, ZS(OH)3%]"
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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23,25,26(OH)3D 3
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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.
166
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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)
400 600 800 800-1200 1200-1500 1000 1500 1000 1000 1500 1500 1200-1500
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
170
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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Fractional calcium absorption as a function of calcium intake in adult women (solid line) and in girls (dotted line). The latter correlation was extrapolated from the curve for adult women multiplied by a factor of 1.45 (the degree by which fractional absorption in girls appears to be increased relative to adult women), and the open circles represent the average absorption fraction of 11 girls during two different dietary calcium regimens. (From O'Brien KO, Abrams SA, Liahg LK, et al: Increased efficiency of calcium absorption during short periods of inadequate calcium intake in girls. Am J Clin Nutr 63:579-583, 1996. Copyright 1996, American Society for Clinical Nutrition.)
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
172
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
174
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-
178
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
ROBERTO CIVITELLI, KONSTANTINOS ZIAMBARAS, AND RATTANA LEELAWATTANA
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FIGURE 6--6 Serumlevels of 1,25(OH)2D3 as a function of age in healthy individuals. (From Eastell R, Yergey AL, Vieira NE, et al: Interrelationship among vitamin D metabolism, true calcium absorption, parathyroid function, and age in women: Evidence of an agerelated intestinal resistance to 1,25-dihydroxyvitaminD action. J Bone Miner Res 6:125-132, 1991.)
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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Effect of a 5-day course of 1,25(OH)2D3 on fractional calcium absorption in ovariectomized women treated with either conjugated equine estrogens (0.625 mg/day) or placebo. (From Gennari C, Agnusdei D, Nardi P, Civitelli R: Estrogen preserves a normal intestinal responsiveness to 1,25(OH)2D3 in oophorectomized women. J Clin Endocrinol Metab 71:1288-1293, 1990.)
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-
182
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
183
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.
185
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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ROBERTO CIVITELLI, KONSTANTINOSZIAMBARAS, AND RATTANA LEELAWATTANA
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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ROBERTO CIVITELLI, KONSTANTINOS ZIAMBARAS, AND RATTANA LEELAWATTANA
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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Pathophysiology of Calcium, Phosphate, and Magnesium Absorption
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
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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
KEITH HRUSKA AND ANANDARUPGUFTA
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.
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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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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
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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.
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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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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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Copyright 9 1998 by Academic Press. All fights of reproduction in any form reserved.
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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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Noninvasive Assessment of Bone
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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)
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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
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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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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
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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%.
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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 it is recommended that the new system be calibrated to match the one being replaced. 95 The phantoms discussed above can be used for this purpose. Scanner crosscalibration based on measurements of a phantom will generally ensure that the two scanners are matched to within 1%. This should be satisfactory for routine clinical use. However the validity of phantom measurements for cross-calibration has been questioned. 273 Thus for scanners used in longitudinal research it is recommended that a series of measurements in vivo be used for comparing and matching calibrations. It has been recommended that 30 subjects be scanned on both systems and the calibration adjusted based on the results of these measurements. 271 This procedure represents a substantial investment of time and resources that may not be practical. However, the limitations of phantom-based measurements must be acknowledged and weighed against the effort required for in vivo cross-calibration. Changing the brand of densitometer has much larger implications and can lead to much larger differences as described below. For this reason it is highly recommended to avoid changing manufacturer, especially in longitudinal studies.
From the clinical perspective these differences lead to confusion in the interpretation of the BMD values reported. Unlike blood pressure or cholesterol readings, significant differences in results can be attributed to the instrumentation used for the measurement. As discussed above, the implication for research is the inability to accurately compare values measured across systems. To address these issues steps have been taken for the universal standardization of DXA measurements. In coordination with the International DXA Standardization Committee, a comparison of Lunar, Norland, and Hologic scanners was undertaken with the aim of developing cross-calibration equations between these systems. 99 The result was the definition of a new variable called "standardized BMD," (sBMD) and has units of milligrams per square centimeter rather than grams per square centimeter to distinguish it from uncorrected measurements. For measurements of spine BMD, the following equations were derived to convert from manufacturerspecific values to the standardized values: sBMD = 1.0761 9BMDNorland sBMD = 0.9522 ~ BMDLunar sBMD = 1.0755 9BMDHologic For any patient the sBMD of the spine reported from any densitometer should be comparable. Similar measurements were made at the femur, but differences in the definitions of the various regions of interest precluded the derivation of comparable equations. To circumvent this problem the Lunar and Norland companies have developed new software that defines a total femur ROI matched to that already in existence for Hologic. Using this new ROI, standardization equations will be developed for the femur.
C. S t a n d a r d i z a t i o n o f D X A M e a s u r e m e n t s V. C L I N I C A L In contrast to the close agreement between scanners from the same manufacturer, measurements made with equipment from different manufacturers are likely to be very different. Systematic differences up to 17% have been reported. 274-277 This results from several factors. One is differences in the calibration of the scanners. This results from the use of different absolute standards for the initial calibration of the systems. Second is the use of different edge-detection algorithms to segment the image into bone and soft tissue. Third is differences in the definitions of the various regions of interest in which B MD is measured. This is especially true for the femur scan, where marked differences exist in all of the regions studied (femoral neck, trochanter, Ward's region).
APPLICATIONS
A. C l a s s i f i c a t i o n o f O s t e o p o r o s i s w i t h Bone Assessment In 1993, a new classification of osteoporosis was proposed by an expert panel of the World Health Organization, in which a bone mass measurement result of more than 2.5 SD below peak bone mass (i.e., T-score below - 2 . 5 ) classifies for osteoporosis. This cutoff value for diagnosing osteoporosis follows the "fracture threshold concept." It has been proposed before based on the findings of large epidemiological studies that showed that the prevalence of fractures increases sharply below a certain value for mineral density. 278'279While the classic definition of osteoporosis requires the presence of frac-
CHAPTER 9 NoninvasiveAssessment of Bone tures, the new approach of the WHO reflects effort to diagnose the disease before any fractures occur. Although this concept offers significant guidance for diagnostic and therapeutic procedures from a clinical perspective, the term "fracture threshold" in its literal sense may be misleading. For any bone measurement technique there is a substantial overlap in bone mineral density in patients with and without fractures. An absolute discrimination between these groups is not possible for the following reasons. 1. DIFFERENCES IN NORMATIVE DATA T-scores based on the manufacturer's young adult normative database may not be representative of normative data in another geographic area. When BMD measurements are compared with two different reference populations, this will likely result in differences in classification. T M Furthermore, discrepancies between the normative databases of various DXA manufacturers have been reported. 282 2. IMPACT OF MEASUREMENT SITES
Classification of osteoporosis based on densitometry may vary considerably depending on the site used. In a group of 120 healthy women 65 years of age and older, bone density measurements at various sites have been performed. Using the WHO criteria, lateral spine BMD determinations classified two thirds of the women as being osteoporotic in contrast to less than one third using the PA projection. Osteoporosis was diagnosed in 55% of women using measurements of the femoral neck, compared to approximately 20% of women when the total femur, ultradistal radius, or total body was examined. 283 The rates of bone loss differ at different sites with increasing age. But even for a younger population of perimenopausal women, a similar discordance of bone mass measured at various sites has been reported. T M Although being valid from an epidemiological perspective, the WHO classification may be deficient when applied to an individual patient and when applied for a treatment threshold. A low bone mineral density does not account entirely for an increased risk of fracture. Other variables, such as an increased risk of falling, restricted protective responses during fall, and bone properties other than density, also are significant. Furthermore, the discrimination between healthy and osteoporotic individuals is not the reason why bone mass or other bone properties are measured. Rather, such measurements should be used as an estimate of the risk of future fractures. Osteoporosis should be understood as the lower part of a continuum of bone density, assuming a continuous gradient of increasing fracture risk with decreasing bone mass. 285'286
301
B. Interpretation of Measurement Results Changes in densitometric and ultrasonic parameters are observed with aging, and differences exist between sexes and ethnic groups. Therefore, bone measurement results must be compared with those of age-, sex-, and race-matched controls. With most of the techniques currently in use, patient results are reported on a graph showing the normal age-related mean regression with 95% confidence intervals. The results are also expressed as T- and Z-scores, which have become an important concept for measurement interpretation. The Z-score gives the patient's results as the deviation from the mean of age-matched controls divided by the standard deviation of this mean, which is an indication of biological variability. The T-score is referenced to the peak bone mass of young normal adults and calculated similarly to the Z-score.
C. Which Site to Measure? Several studies that compared different measurement sites in the peripheral and axial skeleton have found only moderate correlations between the sites. 287-29~Although being statistically significant, those correlations are not strong enough to permit the prediction of bone mineral density values for one site from measurements from another. TM This supports the widespread opinion that for diagnosing spinal osteoporosis a site-matched bone mass measurement would be required. However, other investigations have shown that bone density derived from the axial and peripheral skeleton may have comparable results in discriminating osteoporofic from nonosteoporotic women, and that differences between the techniques are marginal. 292-295 In the assessment of the aging spine a relative insensitivity for discrimination of osteoporotic fractures has been observed for projectional techniques. For DXA in the PA direction degenerative changes and inclusion of cortical bone of the posterior elements may mask agerelated bone loss. For this particular patient population QCT proved to be superior in its discriminatory ability to identify patients with osteoporotic fractures. Lateral spine DXA by excluding most of the degenerative changes from analysis has been shown promising, 76'1~ but further study is required. In light of these results, the choice of an appropriate site for bone mass measurements remains controversial. It appears that the established methods are complementary rather than competitive. Besides the measurement site, the composition of bone measured and the technique used play a major role in the assessment and monitoring of bone density. 53
302 D. D i f f e r e n t T e c h n i q u e s ~ W h a t
MARTIN UFFMANN, THOMAS P. FUERST, MICHAEL JERGAS, AND HARRY K. GENANT
Is I m p o r t a n t ?
Numerous techniques are available for quantitative assessment of the skeleton in osteoporosis. Whenever a method is applied on a single patient (in a clinical setring) or a population group (in an epidemiological setting), it is important to assess the following abilities and compare them with other techniques: 1. Accurately discriminating the high-risk patient or group from a general population at risk for osteoporofic fracture. 2. Detecting early postmenopausal bone loss. 3. Evaluating the efficacy of therapeutic intervention. These discriminatory abilities of a given method depend on its precision, accuracy, and sensitivity. Precision is the deviation of measurements, often represented on a percentage basis as coefficient of variation. Accuracy means the reliability of a method; that is, assurance that the measured value reflects the true properties (e.g., mineral content or ultrasound velocity). Sensitivity is the capacity to readily separate an abnormal state from the normal in a population or to readily detect changes over time in a patient or in a population. Whereas the precision can be assessed relatively easily with multiple measurements, the determination of accuracy requires a precise reference technique. For example, bone ash techniques are used for determination of true bone mineral content. The sensitivity of a given method depends on several parameters, such as the composition of the normative population used for comparison and the statistical methods applied for separation of an abnormal state from normal. Also, the sensitivity depends on the method's precision. In general, when used as a diagnostic procedure to identify a patient with osteoporosis, accuracy and sensitivity are important. For serial determination in a given patient or group, the ability to monitor changes in skeletal status is crucial. This ability, here referred to as longitudinal sensitivity, is typically based on an assessment of the technique's precision (i.e., reproducibility). However, a technique that shows poorer precision but demonstrates larger changes over time may be more sensitive than a competing method with high precision. For example, the precision errors reported for spinal DXA (PA) are generally lower than those observed for QCT of the lumbar spine. But in DXA aortic and osteophytic calcification can produce falsely elevated BMD values, thus obscuring serial bone loss. QCT, being much less dependent on degenerative changes and selectively assessing trabecular bone with high metabolic activity may be more responsive to long-term changes. Taking the re-
TABLE 9--2 Comparison of Approximate Worldwide Distribution, and of Precision Error, Accuracy Error, and Radiation Dose of Techniques for Noninvasive Bone Assessment a Technique (Worldwide Distribution)
Precision Error (%)
Accuracy Error (%)
Effective Dose Equivalent (IxSv)
RA (500) Phalanx/metacarpal
1- 2
5
---5
1- 2
4- 6
<1
D X A (6000) PA spine Lateral spine Proximal femur Forearm Whole arm
1-1.5 2- 3 1.5- 3 --- 1 ---1
4-10 5-15 6 5 3
-~ 1 ---3 --- 1 <1 ---3
Q C T (4000) Spine trabecular Spine integral
2-4 2-4
5-15 4-8
---50 ---50
p Q C T (1000) Radius trabecular Radius total
1- 2 1- 2
9 2- 8
--- 1 --- 1
SXA/DXA (3000) Radius/calcaneus
QUS (2000) SOS calcaneus/tibia B U A calcaneus
0.3-1.2 1.3-3.8
?
0
?
0
aDose for annual background ---2000 txSv, for abdominal radiograph ---500 ~Sv, and for abdominal CT ---4000 ixSv. 3~ The numbers given for precision errors and accuracy errors are from various publications. Since these numbers were obtained using different methods and sometimes distinct statistical approaches, they have to be perceived as a guideline for clinical practice.
sponsiveness into account, QCT provides better longitudinal sensitivity despite its inferior precision compared to PA DXA. Precision, accuracy, radiation dose, and general availability of different techniques are listed in Table 9 - 2 .
E. A b o u t M e t h o d C o m p a r i s o n The T-score and Z-score concept is widely used by manufacturers of both bone densitometers and ultrasound devices. Since these scores are easy to calculate and interpret and they do not have different measurement units, one may be inclined to use them for comparison of two or more different techniques with respect to their discriminatory ability, assuming that a bigger mean Zscore is an indication of better discrimination. However, conclusions of method comparison based on these scores are limited. The statistical tests based on these measures are valid only under certain assumptions (e.g., equal variances, large sample size). Violation of these assumptions will result in misclassification. There-
CHAPTER 9 Noninvasive Assessment of Bone fore, T- and Z-scores should not be used for comparing the discriminative ability of different techniques. More appropriate approaches for this purpose are statistical techniques such as logistic regression and discriminant analysis. They measure fracture risk (odds ratio) for a specific technique and thus can be used to decide which of the several methods are associated with uncovering a higher risk compared to the c o n t r o l s . 296
F. A p p r o p r i a t e C l i n i c a l I n d i c a t i o n s The greater efficiency provided by the recent innovations in bone densitometry, aside from improving technical performance, has allowed reductions in the cost of examinations. Given the current impetus to disseminate information about osteoporosis, to make new instrumentation more readily available, and to limit its costs, these methods of bone densitometry are becoming widely used in routine medical practice. A consensus is forming about the clinical indications for the appropriate use of densitometry, and four indications have emerged. 297 1. EVALUATION OF PERIMENOPAUSAL WOMEN FOR INITIATION OF PROTECTIVE THERAPY
Decisions about the initiation of estrogen therapy may be contingent on a number of factors, including the current level of bone density, the severity of menopausal symptoms, patient or physician preferences, laboratory evidence of rapid bone loss, and the long-term risk of cardiovascular disease. 298 The absolute level of bone density at the menopause and the magnitude of subsequent bone loss are important considerations in assessing the risk for fracture. The decision to begin prophylaxis against osteoporosis, therefore, can be facilitated by knowledge of a woman's bone density. Furthermore, compliance with estrogen therapy may be enhanced by quantitative information concerning fracture risk and efficacy of treatment. 2. DETECTION OF OSTEOPOROSIS AND ASSESSMENT OF ITS SEVERITY
Quantitative evaluation of the skeleton may be indicated in individuals in whom osteoporosis is suspected or in whom atraumatic fracture is suspected based on radiographic findings. Recent evidence, as reviewed here, supports the concept that the absolute level of bone density is predictive of fracture risk; for example, most of the variance in bone strength can be attributed to bone density, and a gradient of increasing fracture risk is associated with declining levels of bone density. 299-3~ Thus, bone density p e r s e provides the primary standard of osteoporosis risk and provides an important basis for therapeutic decisions.
303 3. EVALUATION OF PATIENTS WITH METABOLIC DISEASES THAT AFFECT THE SKELETON
Many metabolic disorders, such as hyperparathyroidism, Cushing's syndrome, and chronic corticosteroid therapy, have profound influences on calcium metabolism and may adversely affect the skeleton. Additional relatively common disorders known to influence bone include testerone deficiency, amenorrhea, eating disorders, treatment with suppressive doses of thyroid hormone, alcoholism, disuse, treatment with multiple anticonvulsants, treatment with heparin, and renal osteodystrophy. In these secondary forms of osteoporosis, bone density measurements may be important because they carry prognostic as well as therapeutic implications. 4. MONITORING OF TREATMENT AND EVALUATION OF DISEASE COURSE
The importance of bone density measurements would be diminished were there no clinically useful treatments capable of affecting bone mass and the consequent risk of fracture. There is an extensive literature surrounding the effect of various treatments upon bone mass, but a much smaller literature surrounding fracture risk. Prospective, randomized, controlled trials have demonstrated both a beneficial effect upon bone density and a decrease in spinal fracture rate for etidronate, 3~176 alendronate, T M and transdermal estrogen treatment. 3~ In the past, bone density measurements were associated with large precision errors relative to estimated rates of change, and could not reliably monitor changes in bone density in individual patients. Given the recent improvements in measurement precision and speed of performance and the large skeletal effects observed in certain metabolic disorders and with certain medical regimens, monitoring of individual patients may be considered when important therapeutic decisions are to be made. In general, such serial measurements are usually obtained at the spine using DXA or QCT because of their favorable longitudinal sensitivity. However, specific guidelines regarding the appropriate techniques, site, and frequency of measurement are yet to be established.
Acknowledgments Sections of this chapter are adapted in part from Genant HK, Engelke K, Fuerst T, et al: Noninvasive assessment of bone mineral and structure: State of the art. J Bone Min Res 11:707-730, 1996.
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2. Frost HM: Dynamics of bone remodeling. In Frost HM (ed): Bone biodynamics. Little, Brown, Boston, 1964, pp 315-334. 3. Schlenker RA, Von Seggen WW: The distribution of cortical and trabecular bone mass along the lengths of the radius and ulna and the implications for in vivo bone mass measurements. Calcif Tissue Res 20:41 - 52, 1976. 4. Seeman E, Formica C, Mosekilde L: Equivalent deficits in bone mass of the vertebral body and posterior processes in women with vertebral fractures: Implications regarding the pathogenesis of spinal osteoporosis. J Bone Miner Res 10:2005-2010, 1995. 5. Nottestad SY, Baumel JJ, Kimmel DB, et al: The proportion of trabecular bone in human vertebrae. J Bone Miner Res 2: 221 - 229, 1987. 6. Wilson CR, Collier BD, Carrera GF, Jacobson DR: Acronym for dual-energy x-ray absorptiometry. Radiology 176:875, 1990. 7. Nord RH, Stein JA, Mazess RB, Pommet RP: Letter. Bone Miner 13:85, 1991. 8. Genant HK, Gltier CC, Faulkner KG, et al: Acronyms in bone densitometry. Radiology 184:878, 1992. 9. Lachmann E, Whelan M: The roentgen diagnosis of osteoporosis and its limitations. Radiology 26:165-177, 1936. 10. Virtama P: Uneven distribution of bone mineral and covering effect of nonmineralized tissue as reasons for impaired detectability of bone density from roentgenograms. Ann Med Intern Fenn 49:57-65, 1960. 11. Doyle FH, Gutteridge DH, Joplin GF, Fraser R: An assessment of radiological criteria used in the study of spinal osteoporosis. Br J Radiol 40:241-250, 1967. 12. Saville PD: A quantitative approach to simple radiographic diagnosis of osteoporosis:: Its application to the osteoporosis of rheumatoid arthritis. Arth Rheum 10:416-422, 1967. 13. Schapira D, Schapira C: Osteoporosis: The evolution of a scientific term. Osteoporosis Int 2:164-167, 1992. 14. Barnett E, Nordin BEC: The radiological diagnosis of osteoporosis: A new approach. Clin Radiol 11:166-174, 1960. 15. Hedlund LR, Gallagher JC: Vertebral morphometry in diagnosis of spinal fractures. Bone Miner 5:59-67, 1988. 16. Davies KM, Recker RR, Heaney RP: Revisable criteria for vertebral deformity. Osteoporosis Int 3:265-270, 1993. 17. Smith-Bindman R, Cummings SR, Steiger P, Genant HK: A comparison of morphometric definitions of vertebral fracture. J Bone Miner Res 6:25- 34, 1991. 18. Melton LJ III, Kan SH, Frye MA, et al: Epidemiology of vertebral fractures in women. Am J Epidemiol 129:1000-1011, 1989. 19. Eastell R, Cedel SL, Wahner HW, et al: Classification of vertebral fractures. J Bone Miner Res 6:207-215, 1991. 20. Minne HW, Leidig G, Wfister C, et al: A newly developed spine deformity index (SDI) to quantitate vertebral crush fractures in patients with osteoporosis. Bone Miner 3:335-349, 1988. 21. Black DM, Cummings SR, Stone K, et al: A new approach to defining normal vertebral dimensions. J Bone Miner Res 6: 883-892, 1991. 22. McCloskey EV, Spector TD, Eyres KS, et al: The assessment of vertebral deformity: A method for use in population studies and clinical trials. Osteoporosis Int 3:138-147, 1993. 23. Ross PD, Yhee YK, He Y-F, et al: A new method for vertebral fracture diagnosis. J Bone Miner Res 8:167-174, 1993. 24. Sauer P, Leidig G, Minne HW, et al: Spine deformity index (SDI) versus other objective procedures of vertebral fracture identification in patients with osteoporosis. J Bone Miner Res 6:227- 238, 1991.
25. Hansen M, Overgaard K, Nielsen V, et al: No secular increase in the prevalence of vertebral fractures due to postmenopausal osteoporosis. Osteoporosis Int 2:241-246, 1992. 26. Adami S, Gatti D, Rossini M, et al: The radiological assessment of vertebral osteoporosis. Bone 13(Suppl):S33-S36, 1992. 27. Genant HK, Wu CY, vanKuijk C, Nevitt M: Vertebral fracture assessment using a semi-quantitative technique. J Bone Miner Res 8:1137-1148, 1993. 28. Wu CY, Li J, Jergas M, Genant HK: Comparison of semiquantitative and quantitative techniques for the assessment of prevalent and incident vertebral fractures. 5:354-370, 1995. 29. 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. 30. Urist MR: Observations bearing on the problem of osteoporosis. In Rodahl K, Nicholson JT, Brown EM Jr (eds): Bone As a Tissue. New York, McGraw-Hill, 1960, pp 18-45. 31. Saitoh S, Nakatsuchi Y, Latta L, Milne E: An absence of structural changes in the proximal femur with osteoporosis. Skeletal Radiol 22:425-431, 1993. 32. Meema HE, Meema S: Measurable roentgenologic changes in some peripheral bones in senile osteoporosis. Am J Geriatr Soc 11:1170-1182, 1963. 33. Matsumoto C, Kushida K, Yamazaki K, et al: Metacarpal bone mass in normal and osteoporotic Japanese women using computed x-ray densitometry. Calcif Tissue Int 54:324-329, 1994. 34. Genant HK, Cann CE, Ettinger B, Gordan GS: Quantitative computed tomography of vertebral spongiosa: A sensitive method for detecting early bone loss after oophorectomy. Ann Intern Med 97:699-705, 1982. 35. Meema HE, Meema S: Postmenopausal osteoporosis: Simple screening method for diagnosis before structural failure. Radiology 164:405-410, 1987. 36. Meema HE: Improved vertebral fracture threshold in postmenopausal osteoporosis by radiographic measurements: Its usefulness in selection for preventative therapy. J Bone Miner Res 6:9-14, 1991. 37. Jergas M, San Valentin R, Black D, et al: Radiogrammetry of the metacarpals predicts future hip fracture. A prospective study, J Bone Miner Res 10:$371, 1995. 38. Heuck F, Schmidt E: Die quantitative Bestimmung des Mineralgehaltes des Knochens aus dem R6ntgenbild. Fortschr Rontgenstr 93:523-554, 1960. 39. Steinbach HL: The roentgen appearance of osteoporosis. Radiol Clin North Am 2:191, 1964. 40. Mack PB, Vose GP, Nelson JD: New developments in equipment for the roentgenographic measurement of bone density. Am J Roentgenol 82:303- 310, 1959. 41. Mack PB, O'Brian AT, Smith JM, Bauman AW: A method for estimating degree of mineralization of bones from tracings of roentgenograms. Science 89:467, 1939. 42. Yang S-O, Hagiwara S, Engelke K, et al: Radiographic absorptiometry for bone mineral measurement of the phalanges: Precision and accurate study. Radiology 192:857-859, 1994. 43. Trouerbach WT, Vecht-Hart CM, Collette HJA, et al: Crosssectional and longitudinal study of age-related phalangeal bone loss in adult females. J Bone Miner Res 8:685-691, 1993. 44. Trouerbach WT, Birkenh~iger JC, Collette BJA, et al: A study on the phalanx bone mineral content in 273 normal pre- and post-menopausal females (transverse study of age-dependent bone loss). Bone Miner 3:53-62, 1987. 45. Yates AJ, Ross PD, Lydick E, Epstein RS: Radiographic absorptiometry in the diagnosis of osteoporosis. Am J Med 98: 41S-47S, 1995.
CHAPTER 9
Noninvasive Assessment of Bone
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Noninvasive Assessment of Bone
205. Hans D, Schott AM, Arlot ME, et al: Influence of anthropometric parameters on ultrasound measurements of the os calcis. Osteoporosis Int 5:371-376, 1995. 206. Barkmann R, Heller M, GlUer CC: The influence of soft tissue and waterbath temperature on quantitative ultrasound transmission parameters: An in vivo study. Osteoporosis Int 6:181, 1996. 207. Jergas M, Uffmann M, Wittenberg R, et al: Ultraschallgeschwindigkeitsmessungen an belastungstragenden und nichtbelastungstragenden Stellen des peripheren Skeletts. Der Einfluf5 k6rpedicher Aktivit~it bei Ful3ballspielern. Fortschr Rontgenstr 157:420-424, 1992. 208. Jergas M, Uffmann M, MUller P, K6ster O: Ultraschallgeschwindigkeitsmessungen zur Diagnose der postmenopausalen Osteoporose. Fortschr Rontgenstr 158:207-213, 1993. 209. Lusenti T, Cadossi R, Franco V, et al: Bone quality assessment by ultrasound at proximal phalanges of the hand. J Bone Miner Res 10(Suppl 1):$471, 1995. 210. Duboeuf F, Hans D, Schott AM, et al: Ultrasound velocity measured on proximal-phalanxes. Osteoporosis Int 6:189, 1996. 211. Fan B, Zucconi F, Fuerst T, et al: Precision assessment: Ultrasonic velocity measurement of the mid-tibia versus other techniques. J Bone Miner Res 10(Suppl 1):$368, 1995. 212. Foldes AJ, Rimon A, Keinan DD, Popovtzer MM: Quantitative ultrasound of the tibia: A novel approach for assessment of bone status. Bone 17:363-367, 1995. 213. Rosenthall L, Tenenhouse A, Caminis J: A correlative study of ultrasound calcaneal and dual-energy X-ray absorptiometry bone measurements of the lumbar spine and femur in 1000 women. Eur J Nucl Med 22:402-406, 1995. 214. Langton CM, Palmer SB, Porter RW: The measurement of broadband ultrasound attenuation in cancellous bone. Eng Med 13:89-91, 1984. 215. Evans WD, Jones EA, Owen GM: Factors affecting the in vivo precision of broad-band ultrasonic attenuation. Phys Med Biol 40:137-151, 1995. 216. Laugier P, Fournier B, Berger G: Ultrasound parametric imaging of the calcaneus: In vivo result with a new device. Calcif Tissue Int 58:326-331, 1996. 217. Gltier CC, Vahlensieck M, Faulkner KG: Site-matched calcaneal measurements of broadband ultrasound attenuation and single x-ray absorptiometry: Do they measure different skeletal properties? J Bone Miner Res 7:1071-1079, 1992. 218. Salamone LM, Krall EA, Harris S, Dawson-Hughes B: Comparison of broadband ultrasound attenuation to single x-ray absorptiometry measurements at the calcaneus in postmenopausal women. Calcif Tissue Int 54:87-90, 1994. 219. Ross P, Huang C, Davis J, et al: Predicting vertebral deformity using bone densitometry at various skeletal sites and calcaneus ultrasound. Bone 16:325-332, 1995. 220. Funke M, Kopka L, Vosshenrich R, et al: Broadband ultrasound attenuation in the diagnosis of osteoporosis: Correlation with osteodensitometry and fracture. Radiology 194:77-81, 1995. 221. Nicholson PHF, Haddaway MJ, Davie MWJ: The dependence of ultrasonic properties on orientation in human vertebral bone. Phys Med Biol 39:1013-1024, 1994. 222. Gltier CC, Wu CY, Genant HK: Broadband ultrasound attenuation signals depend on trabecular orientation: An in-vitro study. Osteoporosis Int 3:185-191, 1993. 223. Njeh CF, Hodgskinson R, Currey JD, Langton CM: Orthogonal relationships between ultrasonic velocity and material properties of bovine cancellous bone. Med Eng Phys 18:373-381, 1996.
309 224. Bouxsein ML, Radloff SE, Hayes WC: Quantitative ultrasound of the calcaneus reflects trabecular bone strength, modulus, and morphology. J Bone Miner Res 10(Suppl 1):S175, 1995. 225. GlUer CC, Wu CY, Jergas M, et al: Three quantitative ultrasound parameters reflect bone structure. Calcif Tissue Int 55: 4 6 - 52, 1994. 226. Hans D, Arlot ME, Schott AM, et al: Do ultrasound measurements on the os calsis reflect more the bone microarchitecture than the bone mass?: A two dimensional histomorphometric study. Bone 16:295- 300, 1995. 227. Bouxsein ML, Courtney AC, Hayes WC: Ultrasound and densitometry of the calcaneus correlate with the failure loads of cadaveric femurs. Calcif Tissue Int 56:99-103, 1995. 228. Rho JY, Ashman RB, Turner CH: Young's modulus of trabecular and cortical bone material: Ultrasonic and microtensile measurements. J Biomech 26:111-119, 1993. 229. Langton CM, Njeh CF, Hodgskinson R, Currey JD: Prediction of mechanical properties of the human calcaneus by broadband ultrasonic attenuation. Bone 18:495-503, 1996. 230. Schott AM, Hans D, Sornay-Rendu E, et al: Ultrasound measurements on os calcis: Precision and age-related changes in a normal female population. Osteoporosis Int 3:249-254, 1993. 231. Heaney RP, Avioli LV, Chestnut CH, et al: Osteoporotic bone fragility: Detection by ultrasound transmission velocity. JAMA 261:2986- 2990, 1989. 232. Funck C, Wtister C, Alenfeld FE, et al: Ultrasound velocity of the tibia in normal German women and hip fracture patients. Calcif Tissue Int 58:390-394, 1996. 233. van Daele PLA, Burger H, Algra D, et al: Age-associated change in ultrasound measurements of the calcaneus in men and women: The Rotterdam Study. J Bone Miner Res 9:1751 - 1757, 1994. 234. Schott AM, Weill-Engerer S, Hans D, et al: Ultrasound discriminates patients with hip fracture equally well as dual energy X-ray absorptiometry and independently of bone mineral density. J Bone Miner Res 10:243-249, 1995. 235. Bauer DC, GlUer CC, Genant HK, Stone K: Quantitative ultrasound and vertebral fracture in post menopausal women. J Bone Miner Res 10:353-358, 1995. 236. Turner CH, Peacock M, Timmerman L, et al: Calcaneal ultrasonic measurements discriminate hip fracture independently of bone mass. Osteoporosis Int 5:130-135, 1995. 237. Stewart A, Reid DM, Porter RW: Broadband ultrasound attenuation and dual energy x-ray absorptiometry in patients with hip fractures: Which technique discriminates fracture risk. Calcif Tissue Int 54:466-469, 1994. 238. Baran DT, Kelly AM, Karellas A, et al: Ultrasound attenuation of the os calcis in women with osteoporosis and hip fractures. Calcif Tissue Int. 43:138-142, 1988. 239. Gltier CC, Cummings SR, Bauer DC, et al: Associations between quantitative ultrasound and recent fractures. Radiology 199:725-732, 1996. 240. Dretakis EK, Kontakis GM, Steriopoulos K, et al: Broadband ultrasound attenuation of the os calcis in female postmenopausal patients with cervical and trochanteric fracture. Calcif Tissue Int 57:419-421, 1995. 241. Porter R, Miller C, Grainger D, Palmer S: Prediction of hip fracture in elderly women: A prospective study. BMJ 301: 6 3 8 - 6 4 1 , 1990. 242. Heaney RP, Avioli LV, Chestnut CH III, et al: Ultrasound velocity through bone predicts incident vertebral deformity. J Bone Miner Res 10:341-345, 1995. 243. Bauer DC, GlUer CC, Pressman AR, et al: Broadband ultrasonic attenuation (BUA) and the risk of fracture: A prospective study. J Bone Miner Res 10:S175, 1995.
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244. Hans D, Dargent P, Schott AM, et al: Ultrasound parameters predict hip fracture independently of hip bone density: The EPIDOS prospective study. J Bone Miner Res 10:S 169, 1995. 245. Stegman MR, Heaney RP, Travers-Gustafson D, Leist J: Cortical ultrasound velocity as an indicator of bone status. Osteoporosis Int 5:349-353, 1995. 246. Uffmann M, Fan B, Fuerst TP, et al: Quantitative ultrasound of the tibia: A risk assessment of vertebral fractures. Osteoporosis Int 6:155, 1996. 247. McCloskey E, Orgee J, Bernard J, et al: Vertebral fracture risk. A comparison of ultrasonic and absorptiometric techniques. Osteoporosis Int 6:217, 1996. 248. Alenfeld FE, Wiister C, Beck C, et al: Quantitative ultrasound of the phalanges: Separation of osteoporotic and non-osteoporotic fractures. J Bone Miner Res 10(Suppl 1):$272, 1995. 249. Cepollaro C, Gonnelli S, Martini S, et al: Ultrasound parameters in the follow up of osteoporotic women treated with calcitonin. Osteoporosis Int 6:180, 1996. 250. Giorgino R, Lorusso D, Paparella P: Ultrasound bone densitometry and 2-year hormonal replacement therapy efficacy in the prevention of early postmenopausal bone loss. Osteoporosis Int 6:226, 1996. 251. Ryan P, Herd R, Blake GC, Fogelman I: Calcaneal BUA changes in a 2 year placebo controlled study of pamidronate in post menopausal osteoporosis. Osteoporosis Int 6:212, 1996. 252. Davis CA, Genant HK, Dunham JS: The effects of bone on proton NMR relaxation times of surrounding liquids. Invest Radiol 21:472-477, 1986. 253. Rosenthal H, Thulborn KR, Rosenthal DI, Rosen BR: Magnetic susceptibility effects of trabecular bone on magnetic resonance bone marrow imaging. Invest Radiol 25:173-178, 1990. 254. Majumdar S: Magnetic field in homogeneity effects induced by inherent tissue susceptibility differences in gradient echo magnetic resonance imaging: Computer simulations. Magn Reson Med 22:101-110, 1991. 255. Majumdar S, Genant H: In vivo relationship between marrow T2* and trabecular bone density determined with a chemical shift-selective asymmetric spin-echo sequence. J Magn Reson Imaging 2:209-219, 1992. 256. Grampp S, Majumdar S, Jergas M, et al: MRI of bone marrow in the distal radius: In vivo precision of effective transverse relaxation times. Eur Radiol 5:43-48, 1995. 257. Ford JC, Wehrli FW: In vivo quantitative characterization of trabecular bone by NMR interferometry and localized proton spectroscopy. Magn Res Med 17:543-551, 1991. 258. Ford JC, Wehrli FW, Chung H: Magnetic field distribution in models of trabecular bone. Magn Res Med 30:373-379, 1993. 258a. Engelke K, Majumdar S, Genant HK: Impact of trabecular structure on marrow relaxation time, T2*: Phantom studies. Mag Res Med 31:380-387, 1994. 259. Majumdar S, Keyak J, Lee I, et al: Relationship between marrow relaxation time T* and elastic modulus. Society of Magnetic Resonance, Berlin, Germany, 1992. 260. Chung H, Wehrli FW, Williams JL, Kugelmass SD: Relationship between NMR transverse relaxation, trabecular bone architecture, and strength. Proc Natl Acad Sci USA 90:1025010254, 1993. 261. Jergas MD, Majumdar S, Keyak JH, et al: Relationships between young modulus of elasticity, ash density, and MRI derived effective transverse relaxation T* in tibial specimens. J Comput Assist Tomogr 19:472-479, 1995. 262. Antich P, Mason R, McColl R, et al: Trabecular architecture studies by 3D MRI microscopy in bone biopsies. J Bone Miner Res 9:327, 1994.
263. Chung HW, Wehrli FW, Williams JL, et al: Quantitative analysis of trabecular microstructure by 400 MHz nuclear magnetic resonance imaging. J Bone Miner Res 10:803-811, 1995. 264. Jara H, Wehrli FW, Chung H, Ford JC: High-resolution variable flip angle 3D MR imaging of trabecular microstructure in vivo. Magn Reson Med 29:528-539, 1993. 265. Majumdar S, Genant H, Grampp S, et al: Analysis of trabecular bone structure in the distal radius using high resolution MRI. Eur Radiol 4:517-524, 1994. 266. Majumdar S, Genant H, Gies A, Gugliemi G: Regional variations in trabecular structure in the calcaneus assessed using high resolution magnetic resonance images and quantitative image analysis. J Bone Miner Res 8:351, 1993. 267. Kapadia RD, High W, Bertolini D, Sarkar SK: MR microscopy: A novel diagnostic tool in osteoporosis research. In Christiansen C (ed): 4th International Symposium on Osteoporosis & Consensus Development Conference, Hong Kong, 1993, p 38. 268. Ouyang X, Lang P, Selby K, et al: Analysis of high resolution MRI images of Calcaneus: Gray-level thresholding and trabecular quantification. Society of Magnetic Resonance Third Scientific Meeting, Nice, France, 1995. 269. Majumdar S, Newitt D, Jergas M, et al: Evaluation of technical factors affecting the quantification of trabecular bone structure using magnetic resonance imaging. Bone 17:417-430, 1995. 270. Blake GM, Tong CM, Fogelman I: Intersite comparison of the Hologic QDR-1000 dual energy x-ray bone densitometer. Br J Radiol 64:440-446, 1991. 271. Finkelstein JS, Butler JP, Cleary RL, Neer RM: Comparison of four methods for cross-calibrating dual-energy x-ray absorptiometers to eliminate systematic errors when upgrading equipment. J Bone Miner Res 9:1945-1952, 1994. 272. Kalender WA, Felsenberg D, Genant HK, et al: The European Spine P h a n t o m - - a tool for standardization and quality control in spinal bone mineral measurements by DXA and QCT. Eur J Radiol 20:83-92, 1995. 273. Blake GM: Replacing DXA scanners: Cross-calibration with phantoms may be misleading. Calcif Tissue Int 59:1-5, 1996. 274. Laskey MA, Flaxman ME, Barber RW, et al: Comparative performance in vitro and in vivo of Lunar DPX and Hologic QDR-1000 dual energy x-ray absorptiometers. Br J Radiol 64: 1023-1029, 1991. 275. Pocock NA, Sambrook PN, Nguyen T, et al: Assessment of spinal and femoral bone density by dual x-ray absorptiometry: Comparison of Lunar and Hologic instruments. J Bone Miner Res 7:1081 - 1084, 1992. 276. Khai CL, Goodsitt MM, Murano R, Chestnut CH: A comparison of two dual-energy x-ray absorptiometry systems for spinal bone mineral measurement. Calcif Tissue Int 50:203-208, 1992. 277. Tothill P, Fenner JAK, Reid DM: Comparisons between three dual-energy x-ray absorptiometers used for measuring spine and femur. Br J Radiol 68:621-629, 1995. 278. Wahner HW: Single- and dual-photon absorptiometry in osteoporosis and osteomalacia. Semin Nucl Med 17:305-315, 1987. 279. Nordin BEC: The definition and diagnosis of osteoporosis. Calcif Tissue Int 40:57-58, 1987. 280. Looker AC, Johnston CC Jr, Wahner HW, et al: Prevalence of low femoral bone density in older U.S. women from NHANES III. J Bone Miner Res 10:796-802, 1995. 281. Ryan PJ, Spector TP, Blake GM, et al: A comparison of reference bone mineral density measurements derived from two sources: Referred and population based. Br J Radiol 66:11381141, 1993.
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282. Faulkner KG, Roberts LA, McClung MR: Discrepancies in normative data between Hologic and Lunar systems. J Bone Miner Res 10:S 146, 1995. 283. Greenspan SL, Maitland-Ramsey L, Myers E: Classification of osteoporosis in the elderly is dependent on site-specific analysis. Calcif Tissue Int 58:409-414, 1996. 284. Pouilles JM, Tremollieres F, Ribot C: Spine and femur densitometry at the menopause: Are both sites necessary in the assessment of the risk of osteoporosis? Calcif Tissue Int 52: 3 4 4 - 347, 1993. 285. Genant HK, Block JE, Steiger P, et al: Appropriate use of bone densitometry. Radiology 170:817-822, 1989. 286. Wasnich R: Fracture prediction with bone mass measurements. In Genant HK (ed): Osteoporosis Update 1987. San Francisco, Radiology Research and Education Foundation, 1987, pp 9 5 101. 287. Riis B J, Christiansen C: Measurement of spinal or peripheral bone mass to estimate early postmenopausal bone loss? Am J Med 84:646-653, 1988. 288. Grubb SA, Jacobson PC, Awbrey BJ: et al: Comparison of single and dual photon absorptiometry in postmenopausal bone mineral loss. J Nucl Med 26:1257-1262, 1984. 289. Nilas L, Gotriedsen A, Hadberg A, Christiansen C: Age-related bone loss in women evaluated by the single and dual photon technique. Bone Miner 4:95-103, 1988. 290. Aloia JF, Vaswani A, Ross P, Cohn S: Aging bone loss from the femur, spine, radius, and total skeleton. Metabolism 39: 1144-1150, 1990. 291. WHO: Assessment of fracture risk and its application to screening for postmenopausal osteoporosis: A report of a WHO study group. Geneva, Switzerland, World Health Organization, 1994. 292. Nordin BEC: Osteoporosis, osteomalacia and calcium deficiency. Clin Orthop 17:253-258, 1960. 293. Van Berkum FNR, Birkenh~iger JC, Van Veen LCP, et al: Noninvasive axial and peripheral assessment of bone mineral content: A comparison between osteoporotic women and normal subjects. J Bone Miner Res 5:679-685, 1989. 294. Reinbold WD, Genant HK, Reiser UJ, et al: Bone mineral content in early-postmenopausal osteoporotic women and post-
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2HAPTEI;
Biochemical Markers of Bone Turnover CLAUS CHRISTIANSEN, CHRISTIAN HASSAGER, AND BENTE JUEL RIIS Center for Clinical and Basic Research, Ballerup, Denmark
I. II. III. IV. V.
VI. VII. VIII. IX.
Introduction Bone Matrix, Minerals, and Cells Bone Turnover: Modeling and Remodeling Biochemistry of Bone Turnover Markers of Bone Formation
five diagnosis may often mean lifelong intervention, with not only the desired effects but also undesired adverse effects. This encourages attempts to optimize the diagnostic procedures. The understanding of the pathophysiology of the postmenopausal bone loss has improved significantly during the last decade and has placed the role of bone turnover in focus as an independent risk factor for osteoporosis. Increased bone turnover results in bone loss and perhaps deteriorated bone structure. The bone turnover is reflected by the concentration in serum and urine of either enzymatic activities of the bone cells involved in the turnover processes or by-products, which are released during bone formation or bone degradation. These so-called "biochemical markers of bone turnover" have been rapidly improved in specificity and sensitivity within the last 5 years. Furthermore, the understanding of the indication for use and interpretation of results are evolving. The potential roles of these markers will be discussed in this chapter.
I. I N T R O D U C T I O N Osteoporosis is a major health problem that is becoming increasingly important in our aging society. In many Western countries, patients with hip fracture occupy more hospital beds than any other disease. Measures to combat osteoporosis are urgently needed, but the best way of doing so is uncertain. Osteoporosis prevention may be targeted at subgroups of the population at high risk of disease. Alternatively, the intervention may be aimed at the entire population. Both preventive strategies have been proposed. Some researchers thus advocate population programs to increase weight-beating exercise and calcium intake or to reduce smoking, but there is little direct evidence of their effectiveness. Others advocate for population screening or screening of subgroups to identify individuals at risk that would benefit from pharmacological intervention. Such screening programs may, for example, include a bone mass measurement, which is widely believed to be the most accurate diagnostic predictor of osteoporosis risk. A posiMETABOLIC BONE DISEASE
Markers of Bone Resorption 24-Hour Variation in Markers of Bone Turnover Potential Use of Biochemical Markers Conclusions References
313
Copyright 9 1998 by Academic Press. All rights of reproduction in any form reserved.
314
CLAUS CHRISTIANSEN, CHRISTIAN HASSAGER, AND BENTE JUEL RIIS
TABLE 10-- 1 Mineral --~65% of dry weight Matrix --~35% of dry weight
Cells
Composition of Bone Hydroxyapatite Collagen --~90% Other proteins Lipids + carbohydrates? Osteoblasts Lining cells Osteocytes Osteoclasts
Water
II. BONE MATRIX, MINERALS, AND CELLS Bone is essentially made up of organic matrix, inorganic mineral, cells, and water (Table 10-1).
performed in a closed, sealed-off microenvironment located between the cell and the bone surface. This boneresorbing zone is called the clear zone and is closed on one side by the cell membrane and on the other side by the bone surface. The cell membrane, which covers the bone, is called the ruffled border. From this ruffled border two types of products are secreted, both leading to bone destruction. The first category is the H + ions, which dissolve the bone mineral. It originates from H2CO3 as a result of the action of carbonic anhydrase and is secreted by means of an ATPase. The second category includes various proteolytic enzymes, especially cathepsins and collagenases, which digest the matrix. Bone resorption can be modulated by altering two basic processes, namely, the recruitment of new osteoclasts or the activity of mature osteoclasts. Both are influenced by a series of cytokines and hormones.
D. Osteoblasts
A. Organic Matrix The organic matrix constitutes about one third of the dry weight of bone. It consists of 90% collagen, which is thus by far the most abundant bone protein. This collagen is almost exclusively type I collagen. The remainder of the bone matrix is made of various noncollagenous proteins, the role of which is not yet well understood. The most abundant ones are osteonectin; osteocalcin, formerly called bone gla-protein (BGP); osteopontin; and bone sialoprotein.
B. Inorganic Mineral The mineral amounts to about two thirds of the total dry weight of bone. It is made of small crystals in the shape of needles, plates, and rods located within and between the collagen fibrils. Chemically it is basically hydroxyapatite, Calo(POa)6(OH)2; however, it is not pure and contains many other constituents, among others carbonate, citrate, magnesium, sodium, fluoride, and strontium.
C. Osteoclasts The osteoclasts are usually large, multinucleated cells. They originate from a different lineage than that of the other bone cells, namely, from the hemopoietic compartment, more precisely from the granulocytemacrophage colony-forming unit (GM-CFU). The role of osteoclasts is to resorb bone. The bone resorption is
The osteoblasts, which derive from mesenchymal stem cells located in the bone marrow, are the cells that synthesize the bone matrix. They form an epithelial-like structure at the surface of the bone where they secrete the osseous organic matrix. In a second step, this matrix then calcifies extracellularly. As a consequence of the time lag between the formation of the matrix and its calcification, there is often a layer of unmineralized matrix under osteoblasts, called osteoid. The modulation of bone formation is still poorly understood. It may occur at the level of the recruitment of new osteoblasts as well as through the alteration of osteoblast function. Although many hormones and cytokines influence osteoblasts in vitro, among them the insulin-like growth factors (IGFs), transforming growth factor [3 (TGF-[3), platelet-derived growth factor (PDGF), bone morphogenetic proteins (BMPs) and prostaglandins, their role in vivo is not yet clear.
III. BONE TURNOVER: MODELING AND REMODELING Bone is continuously being turned over by the two processes of modeling and remodeling. In the former, which takes place principally in the child, new bone is formed at a location different from the one destroyed. This results in a change in the shape of the skeleton. In remodeling, which is the main process in the adult, resorption and formation occur in the same place, so that no change occurs in the shape of the bone. Both modeling and remodeling, however, result in the replacement
CHAPTER 10 Biochemical Markers of Bone Turnover of old bone by new bone. This allows the maintenance of the mechanical integrity of the skeleton. The turnover also allows the bone to play its role as an ion bank. As mentioned, bone remodeling consists of two processes: bone resorption and bone formation and the sequence is always the same. The process is performed by small, geographically defined assemblies of cells, the basic multicellular units (BMUs). In response to stimulation by unknown factors, a number of osteoclasts gather and start to resorb bone, thus producing minute resorption lacunae (Howship's lacunae). This resorption phase is followed by a formation phase, in which a group of osteoblasts lay down organic bone matrix (osteoid) in the resorption lacunae. Eventually the osteoid in mineralized. Resorption is always followed by formation, a phenomenon called "coupling." There are several ways in which the remodeling system can change or fail. The number of (active) BMUs throughout the skeleton may vary, the speed at which each BMU works may vary, and an imbalance between the resorption and formation processes may occur. The remodeling rate is between 2% and 10% of the skeletal mass per year. It is increased by parathyroid hormone, thyroxin, growth hormone, and 1,25-dihydroxyvitamin D [1,25(OH)2D], and it is decreased by calcitonin, estrogen, and glucocorticosteroids. It is also modulated by mechanical conditions. The cancellous bone, which represents about 20% of the skeletal mass, makes up 80% of the turnover, while the cortex, which represents 80% of the bone, makes up only 20% of the turnover. For some years after cessation of growth, bone mass probably increases to consolidate the skeleton. At skeletal maturity men have 50% more skeletal mass than women. It is not quite certain at what age peak bone mass is achieved, and it may vary from one society to another and with many different factors. At a certain point thereafter, the skeletal mass will start to decrease; that is, bone density decreases, whereas bone size and shape stay more or less the same (with a few exceptions). The bone loss in men seems to follow a linear function. This age-related loss of bone has been compared to the loss of other functions with age (e.g., that of muscle mass). The bone loss in men is low, probably on the order of 3% to 5% per decade, which explains the rather low incidence of osteoporotic fractures in men. In women, the process is more complicated. Bone loss before the menopause is small and probably parallels that in men; however, significant premenopausal bone loss may occur in women with conditions that affect estrogen production. It is not known how large a proportion of premenopausal women have relative estrogen deficiency, and whether this proportion increases/changes with changing lifestyle, habits, stress, birth rate, etc.
315 Around the menopause, however, bone loss accelerates, averaging 2% per year. This bone loss continues for many years after menopause and is still present in elderly women. 1 The accelerated postmenopausal bone loss and the relatively lower peak bone mass explains why osteoporosis is much more common in women than in men. The rate of loss in postmenopausal years varies from one women to another, ranging from less than 1% to more than 5% per year. The bone turnover in postmenopausal women is characterized by increased bone resorption and bone formation. The postmenopausal bone loss implies that bone resorption is more increased than bone formation (Fig. 10-1). Women have increased bone turnover many years after m e n o p a u s e u p r o b a b l y the rest of their lives - - a n d therefore also increased bone loss. The higher the bone turnover, the bigger the difference between bone resorption and formation, and the bigger the resulting rate of bone loss.
IV. BIOCHEMISTRY OF BONE TURNOVER The rate of formation and degradation of the bone tissue can be assessed in blood and urine by measuring enzymatic activity or bone matrix components released into the circulation during formation or resorption. The measured parameters have been separated into markers of formation and resorption, but in disease states where both events are coupled and change in the same direction, either of these markers will reflect the overall rate of bone turnover. Bone markers cannot discriminate between turnover changes of a specific skeletal envelope (i.e., trabecular vs. cortical), but reflect the overall net changes of resorption and formation. These markers are of unequal specificity and sensitivity, which implies that it is not yet possible to estimate the net excess of resorption over formation, by comparing the relative increase of resorption marker over a formation marker. Circulating levels of these markers can be influenced by factors other than bone turnover such as their metabolic clearance (liver uptake, renal excretion, and/or trapping on bone hydroxyapatite), and by the pattern of immunoreactive moieties recognized by a given antibody. thus, each of these assays needs to be carefully validated in a specific clinical situation before conclusions about their clinical utility can be drawn. In contrast to metabolic bone diseases such as Paget's disease of bone or renal osteodystrophy, both of which are characterized by dramatic changes of bone turnover, the postmenopausal bone turnover is in a condition where moderate modifications of the bone remodeling activity can lead to a substantial loss of bone mass after
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CLAUS CHRISTIANSEN, CHRISTIAN HASSAGER, AND BENTE JUEL RIIS
9 Resorption
>
Resorption
0 (-. k,.
C 9 r~
Formation
o, 2'0 FIGURE 10--1
9 Age (yrs.)
80
2'0
Age (yrs.)
|
Ib
8O
Changes in bone mineral content (BMC) and bone turnover with age.
a long period of time. Consequently, there have been efforts to develop specific and sensitive biochemical markers of bone turnover, which are able to pick-up such moderate changes in bone remodeling.
V. MARKERS OF BONE FORMATION A. Alkaline Phosphatases The concentration of serum total alkaline phosphatase (T-AP) has been used widely as a marker of bone tumover, and in controlled settings it is a valuable marker. 2'3 T-AP consists of tissue nonspecific (liver, bone, kidney), intestinal, and placental AP, 4 and any disease that affects one or more of these extraskeletal sources of AP will decrease the value of T-AP as a marker of bone turnover. Bone specific alkaline phosphatase (B-AP) is localized in the plasma membrane of the osteoblast and is released into the circulation as a dimer. The exact role of B-AP is unknown, but in vivo it may be involved in bone formation and skeletal mineralization. 4 Using a new immunoassay, it has been demonstrated 5'6 that the cross-reactivity to the liver contribution of total AP is 11% to 16%. Based on a 16% cross-reactivity and the assumption that in nonbone disease, such as liver disease, the increase in T-AP is only accounted for by an increase in L-AP, it has been calculated that the B-AP assay can be used as long as T-AP does not exceed 2.6-fold of the upper limit of the normal range. 5 Accordingly, it was shown that determi-
nations of B-AP fall within the normal range in patients with liver diseases with less than 4.5 ixkat/liter of T-AP activity. 5 However, serum samples from patients with more severe liver disease and much higher elevated T-AP levels may of course show elevated results also in B-AP. T-AP and B-AP are increased in women both fight after and 20 years after menopause (Table 10-2). It has previously been suggested that bone turnover is considerably higher just after menopause than 20 years after menopause, but recent data contradict such speculations and support that bone turnover stays high many years after menopause. T-AP and B-AP decrease gradually in response to hormone therapy. As a marker of bone formation, a relatively slow decrease is expected, since the response is secondary to the response in bone resorption (Fig. 10-2). B-AP responds to hormone and bisphosphonate therapy more or less to the same extent as osteocalcin, but more pronounced than T-AP. B-AP correlates relatively strongly to both T-AP and osteocalcin, The advantage of B-AP over T-AP is not striking. However, B-AP has the advantage of being relatively independent of diseases affecting other sources of AP. Compared to osteocalcin, B-AP performs virtually similar, but the two molecules may reflect somewhat different aspects of the bone remodeling.
B. Osteocalcin Osteocalcin (formerly called bone Gla protein or the vitamin K-dependent protein of bone) was discovered
CHAPTER 10 Biochemical Markers of Bone Turnover TABLE 10--2
317
Serum T-AR B-AR Osteocalcin, and Bone Mineral Density (BMD) in Three Groups of Women (Mean _+ 1 SD) Premenopausal (n = 71)
B-AP (ixg/liter) T-AP (U/liter) Osteocalcin (ng/ml) BMCarm (g) BMDspine (g/cm1)
9.5 121 5.8 3.097 1.064
__ 2.8 _+ 32 +_ 2.4 ___0.341 +_ 0.120
during the study of various tissues for proteins containing the vitamin K - d e p e n d e n t amino acid gammacarboxyglutamic acid (Gla). 7 Osteocalcin is found at high levels only in the extracellular matrix of bone and tooth dentine in all vertebrates tested to date. 8 In most species it is one of the most abundant noncollagenous bone proteins; that is, the osteocalcin content of rat, bovine, chicken, and swordfish bone is typically 1 to 3 mg/g of dry nonmineralized bone tissue, whereas the osteocalcin content of human adult bone is much lower, with a level of 0.05 to 0.1 mg/g of dry nonmineralized tissue. 7,8 Osteocalcin has been detected in the blood of all vertebrates examined. The amount of osteocalcin in rat and calf plasma is typically 100 to 300 ng/ml. In human plasma the concentration is only 5 to 19 ng/ml. The physiological function of osteocalcin is not k n o w n . 9
FIGURE 10--2 Changesin bone mineral content (BMC) and bone turnover during treatment with an antiresorptive agent.
50-year-old (n = 122) 13.6 174 12.2 3.118 0.968
_ 5.5 __ 49 _ 5.2 __ 0.446 __ 0.122
70-year-old (n = 182) 13.3 __ 3.9 184 __ 42 12.0 _+ 5.1 2.205 __+0.308 0.802 __ 0.138
The primary structure of osteocalcin has been determined in more than ten different species. 1~The molecule is small, typically 49 or 50 residues (molecular weight of 5200 to 6100, 5879 in humans), and has within the molecule three residues of Gla. A comparison of the structure of osteocalcin in these different species reveals a remarkable degree of conservation over evolutionary time. Osteocalcin is synthesized by osteoblasts and odontoblasts as a 10-kDa precursor. The Gla residues in osteocalcin are formed by posttranscriptional carboxylation of glutamic acid by a vitamin K - and carbon dioxide-dependent carboxylase enzyme complex. Vitamin D is also involved in the biosynthesis of osteocalcin, but the synthesis is stimulated by Vitamin D rather than dependent on vitamin D. The metabolism of osteocalcin is only partially understood. After synthesis, osteocalcin, in a constant ratio, is either incorporated into bone matrix or diffuses into blood, where it can be measured by immunoassay, a Osteocalcin is thus believed to reflect bone formation, and studies have shown that in normal subjects, osteocalcin reflects changes in bone turnover as a function of age, sex, and hormonal status. 11'12 Osteocalcin is elevated in diseases characterized by high bone turnover such as hyperparathyroidism and Paget's disease of bone, and decreased in diseases characterized by low bone turnover such as myxedema. Osteocalcin increases after menopause as a marker of the increased bone turnover, and decreases in response to hormone replacement, bisphosphonate, and other antiresorptive therapy. Finally, osteocalcin, perhaps in combination with other markers of bone turnover, may be helpful in the diagnosis of osteoporosis risk. There are several different in-house as well as commercial osteocalcin kits available. The different antibodies measure different parts of the osteocalcin molecule. Since osteocalcin is fragile and present in serum in various fragments, these assays give different absolute results, but after proper normalization the assays are comparable. 13 Osteocalcin is also labile frozen. However, one fragment of osteocalcin, which can be mea-
318
CLAUS CHRISTIANSEN, CHRISTIAN HASSAGER, AND BENTE JUEL RIIS
sured by a commercial two-site assay (N-mid, Osteometer), seems to be relatively stable at both room temperature and in the freezer. 14
C. F r e e B o n e G l a P r o t e i n Gla is an amino acid formed by posttranslational carboxylation of glutamic acid. The process requires the presence of vitamin K. Gla binds calcium and hydroxyapatite, and the largest reservoir of Gla in humans is bone, where Gla occurs in two major noncollagenous proteins, osteocalcin and matrix Gla protein (MGP) which contain three and five residues of Gla, respectively. ~5 Gla is also present in several vitamin K dependent proteins that circulate in blood. These include plasma proteins C, S, and Z and the coagulation factors II, VII, IX, and X. Gla is also found in small amounts in various tissues such as the kidneys and atherosclerotic plaques. 16 The urinary excretion of Gla has been proposed as a marker of bone tumover, because it was found to be increased in patients with osteoporosis, ~7 primary hyperparathyroidism, Paget's disease, osteolytic multiple myeloma, and malignant hypercalcemia. ~8 To further elucidate the usefulness of free Gla, a sensitive and reproducible method to determine serum concentrations of free Gla was developed. ~9 Studies with this method were, however, disappointing: menopause and hormone replacement therapy (HRT) did not change serum freeGla concentrations, and serum-free Gla did not correlate with bone loss or with other biochemical markers of bone turnover. It may thus be suggested that serum-free Gla might be sensitive enough to detect the large increases of bone turnover occurring in metabolic bone diseases, but not sensitive enough to detect the moderate increase in bone tumover following the menopause. One open question is what are the relative contributions of bone metabolism and metabolism of the vitamin K dependent clotting factors to the serum levels of free Gla. Presently, serum-free Gla cannot be used as a marker of bone turnover changes induced by menopause.
D. P r o c o l l a g e n P e p t i d e s During the conversion of type I procollagen to type I collagen (the formation of new collagen), relatively large parts of the molecule are split off from both the carboxyl-terminal and the amino-terminal ends of the molecule and are released into the extracellular fluid. 2~ The extension that is cleaved off from the carboxylterminus, known as the carboxyl-terminal propeptide of
type I procollagen (PICP), is a glycoprotein with a molecular weight of about 1 0 0 , 0 0 0 . 21 PICP splits o f f f r o m the procollagen in a 1:1 molar ratio to newly formed collagen and is probably not incorporated into bone, but is released into the extracellular fluid. Theoretically, therefore, the concentration of PICP in serum may be a marker of osteoblast function or of bone formation without being affected by bone resorption. Serum PICP correlates weakly but significantly with histomorphometric bone formation rates in osteoporotic populations. 22'23The menopause induces a significant but marginal (20%) increase in serum PICE and serum PICP is similarly decreased by estrogen replacement therapy. Serum PICP is not correlated to the subsequent rate of bone loss measured by densitometry. 24 Contrasting with a significant increase in bone alkaline phosphatase and osteocalcin in patients treated with fluoride, serum PICP decreased with fluoride therapy. 25 The reasons for this relative lack of sensitivity are not clear. Type I collagen is not specific for bone and it is not known how much of the PICP in serum originates from bone. The metabolism of this peptide in humans is unknown, but it has been shown in the rat that PICP is cleared rapidly from the circulation mainly by the mannose receptor in liver endothelial cells. The peptides that are split off from the amino-terminal end of the procollagen, called PINE has been reported to circulate in concentrations that are 100 times higher than for PICE 25 The concentration was not increased in metabolic bone diseases characterized by increased bone turnover such as Paget's disease. There are too many open questions concerning PINP to recommend it as a marker of bone turnover at the present time.
VI. MARKERS OF BONE RESORPTION A. C a l c i u m and H y d r o x y p r o l i n e The urinary excretion of calcium and hydroxyproline (Hpr) are the traditional markers of bone resorption. As all urinary markers, they have to be corrected for the excretion of creatinine. Although not specific for bone turnover these parameters have been of appreciable value for both calcium metabolism research and in the clinical situation. The measurement of fasting urinary calcium is performed by routine laboratory methods, whereas fasting urinary hydroxyproline usually is measured by a spectrophotometric analysis. The method is cumbersome and time consuming and it requires quite some expertise to run the assay with low accuracy and precision errors. Hpr, furthermore, has some inherent problems when
CHAPTER 10 Biochemical Markers of Bone Turnover used as a marker of bone resorption. First of all, Hpr is not bone specific since it is found in other connective tissues than bone, in C 1q of the complement system and also in N-terminal propeptides from type I collagen. Secondly, 80% to 90% of all Hpr in the circulation is metabolized by the liver in normal individuals, 26 and thirdly the urinary excretion of Hpr is influenced by diets rich in Hpr, requiring diet restrictions before determination. 27 The urinary excretion of calcium is also influenced by other factors than changes in bone resorption (e.g., changes in calcium intake, intestinal calcium absorption rate, and renal function).
B. Hydroxylysine Hydroxylysine is another amino acid unique to collagen and proteins containing collagen-like sequences. Like hydroxyproline, hydroxylysine is not re-utilized for collagen biosynthesis, and although it is much less abundant than hydroxyproline, it is a potential marker of collagen degradation. 28 Hydroxylysine is present in part as galactosyl-hydroxylysine and in part as glucosylgalactosyl-hydroxylysine. The relative proportion and total content of galactosyl-hydroxylysine and glucosylgalactosyl-hydroxylysine varies in bone and soft tissues, which suggests that their urinary excretion might be a more sensitive marker of bone resorption than urinary hydroxyproline. Urinary galactosyl-hydroxylysine increases with aging. 29 This marker deserves further evaluation in osteoporosis but its development is limited by the current high-pressure liquid chromatography (HPLC) technique.
C. Plasma Tartrate-Resistant Acid Phosphatase Acid phosphatase is a lysosomal enzyme that is present primarily in bone, prostate, platelets, erythrocytes, and spleen. These different isoenzymes can be separated by electrophoretic methods, which lack sensitivity and specificity. The bone acid phosphatase is resistant to L(+)-tartrate, whereas the prostatic isoenzyme is inhibited. 3~ Acid phosphatase circulates in blood and shows higher activity in serum than in plasma because of the release of platelet phosphatase activity during the clotting process. In normal plasma, tartrate-resistant acid phosphatase (TRAP) corresponds to plasma isoenzyme 5, which originates partly from bone, as osteoclasts contain a tartrate-resistant acid phosphatase that is released into the circulation. 31 Plasma TRAP is increased in a variety of metabolic bone disorders with increased bone turnover, 32 and is elevated after oophorectomy 33 and in vertebral osteoporosis, 34 but it is not clear whether this
3 19 marker is actually more sensitive than urinary hydroxyproline. 33 In children with osteopetrosis, a condition characterized by a marked decrease of bone resorption despite numerous but inactive osteoclasts, plasma TRAP was elevated, was correlated with osteoclast counting on bone biopsy, and decreased after marrow transplantation that induced an increase of bone matrix resorption. 35 Perhaps serum TRAP is more related to osteoclast number than to osteoclast activity. These data illustrate the fact that different markers reflect different events of the sequence of bone remodeling. The lack of specificity of plasma TRAP activity for the osteoclast, its instability in frozen samples, and the presence of enzyme inhibitors in serum are potential drawbacks that will limit the development of enzymatic assays of TRAP in osteoporosis. Conversely, the development of new immunoassay using monoclonal antibodies specifically directed against the bone isoenzyme of TRAP 36 should be valuable to assess the ability of this marker to predict bone resorption in osteoporosis.
D. Pyridinoline and Deoxypyridinoline Studies of a new class of potential markers of bone resorption led to the discovery of the intermolecular cross-linking amino acids of the collagen molecule. The extracellular matrix is stabilized by the formation of covalent cross-links between adjacent collagen chains, and the reducible aldimine cross-links initially formed are converted to mature nonreducible compounds. 37 Two mature cross-linking amino acids of the 3-hydroxypyridinium family have been identified by their natural fluorescent properties. Pyridinoline (Pyr) has been identified in very low concentrations in many connective tissues, such as bone, cartilage, and to a lesser extent in other connective tissues. Deoxypyridinoline (D-Pyr) is present in bone tissues where it accounts for about 21% of the total mature cross-links, and in dentine, tendons, and aorta. 38 Neither of the markers have been found in skin, which is a rich source of type I collagen. The crosslinks are released during collagen breakdown and excreted unmetabolized in the urine. Because bone is the most abundant source of collagen matrix and because its rate of turnover is higher than some other connective tissues such as cartilage, Pyr and D-Pyr in biological fluids are likely to be primarily derived from bone. Pyr and especially D-Pyr are thus potential markers of bone resorption. Diet does not seem to influence these two markers .27 Pyr and D-Pyr concentrations are determined in hydrolyzed urine by HPLC, which is a complicated and time-consuming procedure. The results reflect the total concentration of cross-links (i.e., both the free and the
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CLAUS CHRISTIANSEN, CHRISTIAN HASSAGER, AND BENTE JUEL RIIS
peptide-bound fractions). The results that have been obtained have been of significant importance for the osteoporosis research and for the understanding of the biochemical markers of bone turnover. Urinary excretion of Pyr and D-Pyr are sensitive to the changes in bone turnover that occur at the menopause and reflect the decreases after antiresorptive therapies such as HRT, calcitonin, and bisphosphonates. Because of the complicated HPLC procedure involved in the determination of total Pyr and D-Pyr, much effort has been invested in the development of immunoassays that could reflect bone resorption as good or better than the above-mentioned HPLC determination of Pyr and D-Pyr.
E. Free Pyr and D-Pyr Pyr and D-Pyr when measured with HPLC includes the total urinary excretion of the two molecules (i.e., both the peptide-bound and the free fractions). Some years ago, it was reported that about 40% of urinary Pyr and D-Pyr is present in the free form, and measurements of the free form correlate with total Pyr and D-Pyr in normal pre- and postmenopausal subjects and in those with levels elevated due to metabolic bone disease. Since it was demonstrated that the peptide-bound forms of Pyr and D-Pyr are mixtures of several molecular species and that free Pyr is a major contributor to total Pyr in urine, an immunoassay was developed that uses antibodies that preferentially recognize free Pyr. 39 Later, an assay to determine free D-Pyr was introduced. 4~ Urine concentrations of free Pyr and D-Pyr have been shown to be elevated in women after menopause and in patients with metabolic bone disease. The markers also decrease during treatment with estrogens. However, to much surprise, free Pyr and D-Pyr did not show the expected decrease during treatment with some of the second- and third-generation bisphosphonates. 41 The exact explanation for this phenomenon is not yet clear, but it points in the direction of a changed free to peptidebound ratio of Pyr and D-Pyr under different conditions.
F. Cross-Links Bound to the N-Terminal Part of the Collagen Molecule (Osteomark) Another group of researchers came to the conclusion that because the majority of the pyridinoline cross-links were known to be excreted in peptide-bound form (60%) it was a good idea to design an immunoassay that could detect the cross-links that were bound to peptide fragments of the collagen molecule. They set out to identify
a specific pyridinoline-containing fragment of type I collagen in urine that could provide a quantitative index of the total pyridinoline pool. Their research revealed that the best candidate was a fragment from the aminoterminal end of the collagen molecule which contained 60% of the D-Pyr found in human bone collagen. 42 The target peptides behaved as a digestion product resulting from the endogenous proteolytic degradation of bone collagen fibrils. The cross-linked N-telopeptide (NTx) presumably resists further proteolysis (in bone, liver, and kidney) because they present compact structures. Measuring the urinary excretion rate of the NTx antigen appears to provide a reproducible measure of bone resorption rate. The results correlate well with urinary hydroxyproline concentrations in the same samples as well as with total Pyr and D-Pyr determined with HPLC. 42 The marker (now known under the trade name "Osteomark") reflects the changes in bone turnover across the menopausal transition 43 and responds as expected to treatment with antiresorptive drugs. 44'45 Recent observations reveal that changes in bone metabolism caused by estrogen deficiency are reflected in urinary NTx, the latter apparently more sensitive and specific than pyridinoline or hydroxyproline markers. 46
G. Degradation Products of Type I Collagen (CrossLaps) This enzyme-linked immunoabsorbent assay (ELISA) assay is based on an immobilized synthetic peptide having an amino acid sequence specific for a part of the Cterminal telopeptide of the alfa-1 chain of the collagen molecule (Glu-Lys-Ala-His-Asp-Gly-Gly-Arg). 47 The reasons for selecting this part of the molecule were several: The sequence contains an important region for intermolecular cross-links, and the proximity of crosslinking residues and the compact structure of the molecules may protect such peptides from further renal degradation. Urinary CrossLaps excretion for creatinine correlates highly significantly to Pyr and D-Pyr excretion, which supports that CrossLaps is a marker of bone resorption. Urinary CrossLaps is significantly increased in patients with metabolic bone diseases such as hyperparathyroidism and Paget's disease. The increase at menopause in CrossLaps is pronounced (more than 70%), indicating that it is a sensitive marker of the changes in bone turnover taking place at the menopause. Accordingly, CrossLaps decreases substantially in response to hormone replacement therapy, which opens the possibility that it might be a useful marker to monitor treatment efficacy. Furthermore, the correlation between CrossLaps and the rate of loss mea-
CI-IAPTEn 10 Biochemical Markers of Bone Turnover
321
sured by single-photon absorptiometry markers was found to be higher than what have previously been found using other markers of bone turnover.48 Urine CrossLaps concentrations might thus be used to predict the rate of loss.
H. I C T P The carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP) in serum has also been proposed as a possible new serum marker of bone resorption. 49 ICTP is a 9-kDa fragment of type I collagen, which is released into the extracellular fluid during the resorption of mature bone collagen, and its concentration in serum has recently been reported to correlate with histomorphometric measurements of bone resorption in a group of patients with various metabolic bone diseases. 5~ However, early postmenopausal women only had slightly higher serum ICTP values than premenopausal women, and the effect of hormone replacement therapy on serum ICTP is very modest. 51 Furthermore, treatment with the anabolic steroid nandrolone decanoate resulted in a pronounced increase in serum ICTP in postmenopausal women, even though such treatment is not believed to increase bone resorption. 52 These data indicate lack of sensitivity and specificity of serum ICTP as a marker of modest changes in bone resorption. Part of the reason could be that the assay was not optimal for measuring serum ICTP in normal and osteoporotic women, as the lower range in normal subjects was near the detection limit. With improved sensitivity of the assay it may be possible to detect changes brought about by hormone replacement therapy in osteoporotic women.
VII. 24-HOUR VARIATION IN MARKERS OF BONE TURNOVER It has long been known that some of the biochemical markers of bone turnover show a quite substantial variation over a 24-hour period. 53 The variation is of a magnitude that demands a strict control and standardization of the sample collection conditions. The most pronounced diurnal variation is seen in the urinary markers of bone resorption exemplified here (Fig. 10-3) as deoxypyridinoline excretion. 54 Both formation (e.g., OC and PICP) and resorption (e.g., Pyr, ICTP, NTx, and CrossLaps) markers are higher at night than during the daytime. The biological half-life of circulating skeletal alkaline phosphatase is on the order of 1 to 2 days. Thus a circadian variation in the release of AP from the osteoblasts will be buffered by a large circulation pool and therefore difficult to detect.
FIGURE 10--3 Circadianvariation in urinary excretion of deoxypyridinoline/creatinine (D-Pyr/Cr) in 24 healthy postmenopausal women (mean _ SD).
As the excretion level of pyridinium cross-links reflects the breakdown product from bone, 55 variations in urinary pyridinium cross-links may be connected to an intrinsic rhythmic activity occurring in bone. Diurnally regulated skeletal growth was first observed by Okada and Mimura in 1940. 56 These researchers reported that bone mineralization was most active during the dark environmental period. This observation was further documented in rats and mice. 57'58 The etiology of the circadian rhythms in the various biochemical markers of bone turnover are not known. It seems to be relatively unaffected by age, menopause, gender, and physical factors such as movements. 59 It could be related to systemic or local hormones, growth factors, or cytokines, but it does not seem to be caused by obvious candidates such as cortisol, parathyroid hormone, or growth hormone. 6~ Even though the etiology of the circadian rhythms is unknown, the magnitude of the variation demands a strict control and standardization of the sample collection timing.
VIII. POTENTIAL U S E O F BIOCHEMICAL MARKERS Below is listed a number of diagnostic potentials for the biochemical markers of bone remodeling: 1. What effect does the menopause and therapy have on bone turnover? 2. What is the rate of bone loss? 3. Does an instituted therapy have the expected effect?
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CLAUS CHRISTIANSEN, CHRISTIAN HASSAGER, AND BENTE JUEL RIIS
% 160
Effect of H RT
Effect of menopause
TT T
% I00
t_ t.)
C3
80
140 60 120 -
L (.9
r~
r-
II -U I
40
U
100 -
FIGURE 10--4
The effect of menopause and hormone replacement therapy on biochemical markers of bone resorption (ICTP, serum carboxy-terminal telopeptide of type I collagen; fU Hpr/cr and fU DPyr/cr, fasting urinary excretion of hydroxyproline and deoxypyridinoline corrected for creatinine) and bone formation (Oc, serum osteocalcin; Alk. Ph., serum total alkaline phosphatase). Left, Mean _ SEM of measurements in 44 postmenopausal women given as a percentage of mean premenopausal values. Right, Changes after 1 year of hormone replacement therapy in 20 postmenopausal women (mean _ SEM, percentage of initial values). Data from Hassager et al (1994).
A. The Effect of Menopause and Therapy Figure 1 0 - 4 A shows the effect of menopause on several parameters of bone formation and resorption. The menopause has a substantial effect on most of the markers. There are differences in terms of percentage increase and variation including the biological variation (the scatter around the mean). Often, the markers increase to a level reaching the upper end of the normal premenopausal range (defined as mean -+- 2 SD; shown by hatched area). The parameters of bone degradation, generally speaking, increase most as a response to menopause, when compared to the other markers. However, these markers also showed a relatively large scatter around the mean. Figure 1 0 - 4 B shows the effect of HRT on the different biochemical markers. Again, generally speaking all markers decrease in response to t h e r a p y m s o m e more pronounced than others.
B. Rate of Bone Loss Decreased bone mass is the main risk factor for sustaining a fracture as a result of minor trauma or even no trauma 9 This evidence is supported by several retrospective and longitudinal s t u d i e s . 63'64 Bone loss, by simply decreasing the bone mass, is probably an important determinant of fracture risk, but other factors associated with bone loss such as devel-
opment of microarchitectural abnormalities and microdamage could be contributing as well. 65 Some individuals lose bone at a more rapid rate than others and this rapid rate may persist in some of these individuals over a period of years. 48'66'67 Such rapid loss could result in trabecular damage and additional risk of fracture above that contributed by the loss of bone alone. It is also possible that even a relatively short period (2 to 3 years) of rapid bone loss might contribute to perforation of trabecular and thus to increased fracture incidence. Long-term prospective studies of bone loss from the spine and hip have yet to be published due primarily to the more recent availability of techniques to measure these sites. However, the same group of women was followed from menopause (range, 6 months to 3 years after the last menstruation) and prospectively for 15 years. 67 At baseline, the women were stratified according to the rate of bone lOSS. 48 Those women with an estimated rate of loss above 3% per year in the first 2 following years were called fast bone losers and those with a bone loss below were called normal bone losers. The bone mineral density was similar in the two groups at baseline. At 15 years, determination of biochemical markers of bone resorption (CrossLaps) and formation (osteocalcin) revealed that the fast bone losers had significantly higher bone turnover than the normal losers (Table 10-3). Furthermore, the fast bone losers had lower bone mineral density at all sites of the skeleton (forearm, spine, hip) when compared to the normal bone losers. The differ-
CHAPTER 10 Biochemical Markers of Bone Turnover TABLE 10--3 Biochemical Markers of Bone Turnover at 15 Years in the Groups of Fast and Normal Bone Losers As They Were Defined 15 Years Ago Rate of Bone Loss Fast (n = 49) Height (cm) Weight (kg) CrossLaps/Cr (nM/M) Osteocalcin (ng/ml)
161.6 _ 5.8 *62.9_ 12.5 **0.43 ___0.17 **11.0 _ 4.8
323 TABLE 10--4 The Correlation Between a Baseline Value of a Biochemical Marker of Bone Turnover and the Bone Loss in the Following 2 Years in Early Postmenopausal Women Rate of Bone Loss
Normal (n = 133) 160.2 +__5.6 67.3 _ 12.7 0.33 + 0.14 9.0 _ 4.8
*p < 0.05. **p < 0.01.
ence was approximately 10%, which is equivalent to approximately 1 SD. The role of baseline bone mass and rate of loss on fracture risk was evaluated. The data showed that a low baseline bone mass (below - 1 SD) and a fast rate of loss predisposed to fracture to the same extent (Odds ratio 1.9 and 2.0, respectively; both p < 0.05). A combination of a low baseline bone mass and a fast rate of loss increased the Odds ratio of getting a fracture to 3.0. Another more recent prospective study of risk factors for hip fractures conducted in a large cohort of elderly healthy women in France supports this hypothesis. 68 In those women who had a hip fracture within a 2-year follow-up, baseline measurements of urinary CrossLaps and Pyrilinks-D (an immunoassay of D-pyr) were higher than in nonfractured controls; increased CrossLaps above the normal range of premenopausal w o m e n was associated with an 80% increase of the risk of hip fracture. The biochemical markers of bone turnover reflect bone loss; the higher the markers the higher the rate of loss. A r e c e n t s t u d y 69"7~ showed that CrossLaps correlates to the rate of bone loss with an r value of 0.75 (forearm) and 0.79 (spine) (Table 1 0 - 4 ) . If a bone loss of more than 3.0% per cent per year is defined to be a risk factor, the use of a CrossLaps cut-off limit of 600 ixg/mM creatinine gave a diagnostic sensitivity of 73% and a specificity of 79%. Another g r o u p 71 found that hydroxyproline, Pyr, and D-Pyr correlated to the spinal rate of loss with r values of 0.66, 0.63, and 0.66, respectively (Table 10-4). What should the clinician use now as criteria for preventive intervention against osteoporosis? A practical approach may be suggested. A measurement of bone mass should be made only when intervention to prevent bone loss can be prescribed and will be accepted. Most commonly such a measurement would take place at menopause to consider intervention with HRT. But other
Marker
Forearm r Value
Spine r Value
References
U-CrossLaps/Cr (n = 9)
0.75
0.79
69, 70
U-Calcium/Cr (n = 19) U-Hpr/Cr (n = 35) U-Pyr/Cr (n = 35)
0.33 m m
0.48 0.66 0.63
69, 70 71 71
U-D-Pyr/Cr (n = 35) S-Osteocalcin (n = 19)
~ 0.52
0.66 0.48
71 69, 70
S-AP (n = 19)
0.28
0.25
69, 70
forms of therapy are under development for which similar criteria could be applied. If the measurement was greater than 1 SD above the mean for young normals, no further measurement would be needed and no intervention undertaken. If the measurement was lower than - 1 SD below the mean for young normals, intervention would be r e c o m m e n d e d (provided no contraindications were present). For those within _ 1 SD, the bone mass measurement could be repeated in 1 to 5 years. A measurement at 1 year would probably pick up the extremely fast bone losers, but a clinically relevant bone loss of 3% per year could often be within the error of the method used to measure bone density. 72 Waiting for 5 years could thus result in a 15% to 30% loss of bone mass before treatment was instituted. Alternatively, a determination of one of two biochemical markers of bone turnover could reveal the rate of loss.
C. D o e s t h e I n t e r v e n t i o n H a v e the Expected Effect? There have been some indications that the biochemical markers may aid in choosing the treatment that will yield the best result. In a study of Civitelli et al., 73 elderly w o m e n with osteoporotic fractures were treated with salmon calcitonin. A retrospective analysis of the data showed that the w o m e n with baseline high bone turnover had the highest response to treatment in bone mineral density. Corresponding results have been reported by Overgaard et al., TM but the area needs further investigation. It is a c o m m o n request from physicians and patients that an instituted therapy should be proved to be effective. The efficacy of prevention and treatment of bone
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CLAUS CHRISTIANSEN, CHRISTIAN HASSAGER, AND BENTE JUEL RIIS
loss and osteoporosis may be followed-up by serial bone mass measurements. Because of the relatively small changes in bone mass and the precision of the bone mass measurement methods, which is of a magnitude comparable to the changes in bone mass, this procedure may take an unacceptably long time. It is possible, however, that the biochemical markers may be useful to predict the effect of therapy. The advantage of using the biochemical markers is that they change 50% to 100% in response to HRT within a few months, whereas the precision of the methods are about 5%. This gives a beneficial ratio between change and precision when compared to bone mass measurements. Preliminary data show that the change in biochemical markers from baseline until after 6 months' treatment correlates with the change in bone mass with an r value of 0.67 (data not shown). The data indicate that there is a large potential here for the biochemical markers, but further investigations are needed to increase our understanding of this area.
IX. CONCLUSIONS The present survey of the clinical use of the current available biochemical marker of bone turnover shows that most of them have a role to play in the diagnostic procedures associated with osteoporosis. Combination of three to four biochemical markers determined at baseline in early postmenopausal women can predict the rate of bone loss. It appears that the new and more specific markers are even more closely related to the rate of bone loss. For example, urinary excretion of CrossLaps at menopause predicts postmenopausal bone loss as well as a combination of four of the classical markers of bone turnover combined. Determination of the rate of bone loss may add significantly to the prediction of fracture risk if combined with a bone mass measurement. Furthermore, the rate of loss may also be a determinant for bone quality, because rapid bone loss may deteriorate the bone structure of the cancellous network. One final indication for the use of biochemical markers of bone turnover is an evaluation of the effect on bone mass of a given therapy. Although this area is promising, more research is required before definitive statements can be made at this time.
References 1. Ensrud KE, Palermo L, Black DM, et al, for the Study of Osteoporotic Fractures Research Group: Hip bone loss increases with advancing age: Longitudinal results from the study of osteoporotic fractures. J Bone Miner Res 9(Suppl 1): S130, 1994.
2. Meunier PJ, Salson C, Matheiu L: Skeletal distribution and biochemical parameters of Paget's disease. Clin Orthop 217:37-44, 1987. 3. Riis BJ, Thomsen K, StrCm V, Christiansen C: The effect of percutaneous estradiol and natural progesterone on postmeneopausal bone loss. Am J Obstet Gynecol 156:61-65, 1987. 4. Fishman WH: Alkaline phosphatase isoenzymes: Recent progress. Clin Biochem 23:99-104, 1990. 5. Garnero P, Delmas PD: Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease. J Clin Endocrinol Metab 77: 1046-1053, 1993. 6. Panigrahi K, Delmas PD, Singer F, et al: Characteristics of a twosite immunoradiometric assay for human skeletal alkaline phosphatase in serum. Clin Chem 40:822-828, 1994. 7. Hauschka PV, Lian JB, Gallop PM: Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue. Proc Natl Acad Sci USA 72:3925-3929, 1975. 8. Price PA, Willamson MK, Lothringer JW: Origin of the vitamin K-dependent bone protein found in plasma and its clearance by kidney and bone. J Biol Chem 256:12760-12766, 1981. 9. Price PA, Lothringer JW, Nishimoto SK: Absence of the vitamin K-dependent bone protein in fetal rat mineral. Evidence for another gamma-carboxyglutamic acid-containing component in bone. J Biol Chem 255:2938-2942, 1980. 10. Hauschka PV: Osteocalcin: The vitamin K-dependent Ca 2§ binding protein of bone matrix. Haemostasis 16:258- 272, 1986. 11. Delmas PD, Stenner D, Wahner HW, et al: Increase in serum bone gamma-carboxyglutamic acid protein with aging in women. Implications for the mechanism of age-related bone loss. J Clin Invest 71:1316-1321, 1983. 12. Delmas PD, Wahner HW, Mann KG, Riggs BL: Assessment of bone turnover in postmenopausal osteoporosis by measurement of serum bone Gla-protein. J Lab Clin Med 102:470-476, 1983. 13. Delmas PD, Christiansen C, Mann KG, Price PA: Bone Gla protein (osteocalcin) assay standardization report. J Bone Miner Res 5 : 5 - 1 1 , 1990. 14. Rosenquist C, Bonde M, Qvist P: A competitive immunoassay (ELISA) for the detection of N-terminal-related osteocalcin; improved stability of osteocalcin in serum. J Bone Miner Res l(Suppl 1):S140 (abstract 89), 1994. 15. Price PA, Urist MR, Otawara Y: Matrix Gla protein, a new gamma-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone. Biochem Biophys Res Commun 117:765-771, 1983. 16. Petersen TB, ThCgersen HC, Magnusson S: In Sottrup-Jensen K, Suttie JW (eds): Vitamin K-dependent metabolism and vitamin K-dependent proteins. Baltimore, University Park Press, 1979, pp 171-174. 17. Gundberg CM, Lian JB, Gallop PM, Steinberg JJ: Urinary gamma-carboxyglutamic acid and serum osteocalcin as bone markers. Studies in osteoporosis and Paget's disease. J Clin Endocrinol Metab 57:1221-1225, 1983. 18. Founier B, Gineyts E, Delmas PD: Measurement of free gammacarboxyglutamic acid in serum: A new marker of bone turnover. J Bone Miner Res 3(abstract 402), 1988. 19. Johansen JS, Delmas PD, Riis BJ, et al: Serum-free gamma carboxyglumatic acid (free Gla) is a poor biochemical marker of bone resorption in early postmenopausal women. Bone 12:257160, 1991. 20. Prockop DJ, Kivirikko KI, Tuderman L, et al: The biosynthesis of collagen and its disorders. N Engl J Med 301:12-23, 1979. 21. Olsen BR, Guzman NA, Engel J, et al: Purification and characterization of a peptide from the carboxy-terminal region of chick tendon procollagen type I. Biochemistry 16:3030-3036, 1977.
CHAPTER 10
Biochemical Markers of Bone Turnover
22. Parfitt AM, Simon LS, Villanueva AR, et al: Procollagen type I carboxy-terminal extension peptide in serum as a marker of collagen biosynthesis in bone. Correlation with iliac bone formation rates and comparison with total alkaline phosphatase. J Bone Miner Res 2:427-436, 1987. 23. Hassager C, Jensen LT, Johansen JS, et al: The carboxy-terminal propeptide of type I procollagen in serum as a marker of bone formation: The effect of nandrolone decanoate and female sex hormones. Metabolism 40:205-208, 1991. 24. Hassager C, Fabbri-Mabelli G, Christiansen C: The effect of the menopause and hormone replacement therapy on serum carboxyterminal propeptide of type I Collagen. Osteoporosis Int 3 : 5 0 52, 1993. 25. Ebeling PR, Peterson JM, Riggs BL: Utility of type I procollagen propeptide assays for assessing abnormalities in metabolic bone diseases. J Bone Miner Res 7:1243-1250, 1992. 26. Kivirikko KI: Urinary excretion of hydroxyproline in health and disease. Int Rev Connect Tissue Res 5:93-163, 1970. 27. Colwell A, Eastell R, Assiri AMA, Russell RGG: Effect of diet on deoxypyridinoline excretion. In Christiansen C, Overgaard K (eds): Osteoporosis. Copenhagen, Osteopress Aps 1990, pp 5 9 0 591. 28. Krane SM, Kantrowitz FG, Byrne M, et al: Urinary excretion of hydroxylysine and its glycosides as an index of collagen degradation. J Clin Invest 59:819-827, 1977. 29. Moro L, Mucelli RSP, Gazzarrini C, et al: Urinary [3-1-galactosylO-hydroxylysine (GH) as a marker of collagen turnover in bone. Calcif Tissue Int 42:87-90, 1988. 30. Li CY, Chuda RA, Lam WKW, et al: Acid phosphatase in human plasma. J Lab Clin Med 82:446-460, 1973. 31. Minkin C: Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int 34: 2 8 5 - 290, 1982. 32. Stepan JJ, Silinkova-Malkova E, Havrenek T, et al: Relationship of plasma tartrate-resistant acid phosphatase to the bone isoenzyme of serum alkaline phosphatase in hyperparathyroidism. Clin Chim Acta 133:189-200, 1983. 33. Stepan JJ, Pospichal J, Presl J, et al: Bone loss and biochemical indices of bone remodeling in surgically induced postmenopausal women. Bone 8:279-284, 1987. 34. Piedra C, Torres R, Rapado A, et al: Serum tartrate resistant acid phosphatase and bone mineral content in postmenopausal osteoporosis. Calcif Tissue Int 45:58-60, 1989. 35. Glorieux FH, Travers R, Lanoue G, et al: Tartrate-resistant acid phosphatase levels are an index of the number but not of the activity of osteoclasts. Bone 16(Suppl 1):1015 (abstract Bone ICCRH), 1993. 36. Kraenzlin M, Lau KHW, Liang L: Development of an immunoassay for human serum osteoclastic tartrate-resistant acid phosphatase. J Clin Endocrinol Metab 71:442-451, 1990. 37. Eyre DR, Dickson IR, Van Ness KP: Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues. Biochem J 252: 4 9 5 - 500, 1988. 38. Eyre DR, Koob TJ, Van Ness KP: Quantification of hydroxypyridinium crosslinks in collagen by high-performance liquid chromatography. Anal Biochem 137:380-388, 1984. 39. Robins SP, Woitge H, Hesley R, et al: Direct, enzyme-linked immunoassay for urinary deoxypyridinolin as a specific marker for measuring bone resorption. J Bone Miner Res 9:1643-1649, 1994. 40. Seyedin S, Zuk R, Kung V, et al: An immunoassay to urinary pyridinoline: The new marker of bone resorption. J Bone Miner Res 8:635-642, 1993.
325 41. Garnero P, Gineyts E, Arbault P, et al: Different effects of bisphoshonate and estrogen therapy on free and peptide-bound crosslinks excretion. J Bone Miner Res 10:641-649, 1995. 42. Hanson DA, Weiss MALE, Bollen AM, et al: A specific immunoassay for monitoring human bone resorption: Quantitation of type I collagen cross-linked N-telopeptides in urine. J Bone Miner Res 7:1251-1258, 1992. 43. Ebeling PR, Atley LM, Guthrie JR, et al: Bone turnover markers and bone density across the menopausal transition. J Clin Endocrinol Metab 81:3366-3371, 1996. 44. Rosen HN, Dresner-Pollak R, Moses AC, et al: Specificity of urinary excretion of cross-linked N-telopeptides of type I collagen as a marker of bone turnover. Calcif Tissue Int 54:26-29, 1994. 45. Garnero P, Shih WJ, Gineyts E, et al: Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to alendronate treatment. J Clin Endocrinol Metab 79:1693-1700, 1994. 46. Gorai I, Taguchi Y, Chaki O, et al: Specific changes of urinary excretion of cross-linked N-telopeptides of type I collagen in preand postmenopausal women: Correlation with other markers of bone turnover. Calcif Tissue Int 60:317-322, 1997. 47. Bonde H, Quist P, Fledelius C, et al: Immunoassay for quantifying type I collagen degradation products in urine evaluated. Clin Chem 40:2022- 2025, 1994. 48. Christiansen C, Riis BJ, RCbro P: Screening procedure for women at risk of developing postmenopausal osteoporosis. Osteoporosis Int 1:35-40, 1990. 49. Risteli J, Elomaa I, Niemi S, et al: Radioimmunoassay for the pyridinoline cross-linked carboxyterminal telopeptide of type I collagen. A new serum marker of bone collagen degradation. Clin Chem 39:635-640, 1993. 50. Eriksen EF, Charles P, Melsen F, et al: Serum markers of type I collagen formation and degradation in metabolic bone disease: Correlation with bone histomorphometry. J Bone Miner Res 8: 127-132, 1993. 51. Hassager C, Risteli J, Risteli L, Christiansen C: Effect of the menopause and hormone replacement therapy on the carboxyterminal pyridinoline cross-linked telopeptide of type I collagen. Osteoporosis Int 4:349-352, 1994. 52. Hassager C, Jensen LT, PCdenphant J, et al: The carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen in serum as a marker of bone resorption: The effect of nandrolone decanoate and hormone replacement therapy. Calcif Tissue Int 54: 3 0 - 33, 1994. 53. Bollen AM, Martin MD, Leroux BG, et al: Circadian variation in urinary excretion of bone collagen cross-links. J Bone Miner Res 10:1885-1890, 1995. 54. Schlemmer A, Hassager C, Jensen SB, Christiansen C: Marked diumal variation in urinary excretion of pyridinium cross-links in premenopausal women. J Clin Endocrinol Metab 74:476-480, 1992. 55. Robins SP, Stewart P, Astbury C, Bird HA: Measurement of the cross linking compound, pyridinoline, in urine as an index of collagen degradation in joint disease. Ann Rheum Dis 45:969973, 1986. 56. Okada M, Minura T: Zur physiologie und pharmakologie der hartgewebe. J Med Sci Pharmacol 13:95- 96, 1940. 57. Simmons DJ: Daily rhythm of 5-32 incorporation into epiphyseal cartilage in mice. Experientia 24:363-364, 1968. 58. Simmons DJ, Nichols G: Diumal periodicity in the metabolic activity of bone tissue. Am J Physiol 210(Suppl 2):411-418, 1966. 59. Schlemmer A, Hassager C, Pedersen BJ, Christiansen C: Posture, age, menopause and osteopenia do not influence the circadian
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63.
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variation in the urinary excretion of pyridinium cross-links. J Bone Miner Res 9:1883-1888, 1994. Schlemmer A, Hassager C, Alexandersen P, et al: The circadian variation in bone resorption is not related to serum cortisol. J Bone Miner Res 10(Suppl 1):$357, 1995. Eveling PR, Butler PC, Eastell R, et al: The noctumal increase in growth hormone is not the cause of the noctumal increase in serum osteocalcin. J Clin Endocrinol Metab 73:368-372, 1991. Ledger GA, Bumitt M E Kao PC, et al: Acute suppression of parathyroid hormone by a 24 hour calcium infusion does not prevent the noctumal increase in bone resorption in normal women. J Bone Miner Res 10(Suppl 1):B378, 1994. G/irdsell P, Johnell O, Nilsson BE: The predictive value of bone loss for fragility fractures in women: A longitudinal study over 15 years. Calcif Tissue Int 49:90-94, 1991. Melton JL, Atkinson EJ, O'Fallon WM, et al: Long-term fracture prediction by bone mineral assessed at different skeletal sites. J Bone Miner Res 8:1227-1233, 1993. Parfitt AM, Kleerekoper M, Villaneuva AR: Increased bone age: Mechanisms and consequences. In Christiansen C, et al (eds): Osteoporosis 1987. Proceedings of Intemational Symposium on Osteoporosis, Aalborg, 1987, pp 301 - 308. Christiansen C, Riss BJ, RCdbro P: Prediction of rapid bone loss in postmenopausal women. Lancet 1:1105-1108, 1987.
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67. Hansen MA, Overgaard K, Riis B J, Christiansen C: Role of osteoporosis; 12 year study. Br Med J 303:961-964, 1991. 68. Gamero P, Hauscher E, Chapuy MC, et al: Markers of bone resorption predict hip fracture in elderly women: The EPIDOS prospective study. J Bone Miner Res 11:1531-1538, 1996. 69. Qvist P, Bonde M, Rosenqvist C, Christiansen C: Use of a new biochemical marker (CrossLapSTM) for the estimation of rate of postmenopausal bone loss. J Bone Miner Res l(Suppl 1):$334, 1994. 70. Bonde M, Qvist P, Fledelius C, et al: Applications of an enzyme immunoassay for a new marker of bone resorption (CrossLapSTM)--follow up on hormone replacement therapy and osteoporosis risk assessment. J Clin Endocrinol Metab 80:864-868, 1995. 71. Kraenzlin ME, Kraenzlin CA, Haas HG: Comparison of the utility of various biochemical markers to predict bone loss in clinical practice. J Bone Miner Res l(Suppl 1):S157, 1994. 72. Heaney RP: En recherche de la diff6rence (p < 0.05). Bone and Miner 1:99-114, 1986. 73. Civitelli R, Gonnelli S, Zacchei F, et al: Bone turnover in postmenopausal osteoporosis. Effect of calcitonin treatment. J Clin Invest 82:1268-1274, 1988. 74. Overgaard K, Hansen MA, Herss Nielsen V-A, et al: Discontinuous calcitonin treatment of established osteoporosisDeffect of withdrawal of treatment. Am J Med 89:1-6, 1990.
0 s te o rn al ac i a an d Related Disorders A. M.
PARFITT
Division of Endocrinology and Center for Osteoporosis and Metabolic Bone Disease, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
VI. Osteomalacia Resulting from Abnormal Phosphate Metabolism VII. Osteomalacia with Normal Vitamin D and Phosphate Metabolism VIII. Therapeutic Intervention in Osteomalacia Acknowledgments References
I. Bone Mineralization and the Mechanisms of Osteoid Accumulation II. Manifestations of Osteomalacia III. Etiological Classification and Pathogenesis of Osteomalacia IV. Osteomalacia Resulting from Abnormal Vitamin D Metabolism V. Vitamin D and Age-Related Osteoporosis
combination of reduced dietary intake and reduced production in skin, and intrinsic, due to some combination of impaired intestinal absorption and increased catabolism, frequently initiated by malabsorption of calcium. 5 Osteomalacia defined in this way has characteristic clinical, biochemical, radiographic, and histological features collectively referred to as the osteomalacic syndrome 1 that have been described and illustrated many times. However, the deformed skeleton of advanced osteomalacia, with potentially catastrophic complications such as obstructed labor, is almost never seen except when and where extrinsic vitamin D deficiency is endemic, as in prerevolutionary China 6 and in some parts of present day India 4 and the Middle East. 7 Since it became feasible to examine bone microscopically during life, osteomalacia has usually been defined in histological terms as the accumulation of osteoid tissue because of defective bone mineralization. 3 Unfortunately, because of erroneous notions of how defective mineralization should be identified histologically, some definitions of osteomalacia obscure the differences from
The term " o s t e o m a l a c i a " originally referred to generalized softening of bone leading to crippling deformities. 1'2 Histological differentiation of osteomalacia from osteoporosis and osteitis fibrosa was first made by the German pathologist Pommer in the late 19th century, 3 but at that time bone was examined only in cadavers. A restatement of his observations in terms of current concepts of bone remodeling is that resorbed bone is replaced by the same volume of normal lamellar bone in young adults, by a lesser volume of normal lamellar bone in age-related bone loss and osteoporosis, by a complex mixture of woven bone and fibrous tissue in osteitis fibrosa, and by unmineralized bone matrix, or osteoid tissue, usually lamellar but occasionally woven, in osteomalacia. Soon after the discovery of vitamin D it became apparent that almost all cases of bone softening were the result of vitamin D deficiency, and osteomalacia came to be equated with the disease that could be cured by vitamin D administration. 1'2 Vitamin D deficiency can conveniently be classified 4 as extrinsic, due to some METABOLIC BONE DISEASE
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328 other metabolic bone diseases to the extent that the term osteomalacia ceases to serve any useful purpose, thus effectively negating Pommer's pioneering work. One of the principal themes of this chapter is the formulation of a more rigorous histomorphometric definition of osteomalacia that restores many of its traditional connotations, allows more accurate evaluation of other diagnostic tests, clarifies the relationships to other forms of metabolic bone diseases, and elucidates pathogenesis. Even with a more precise histological definition, osteomalacia can result from a wide variety of causes other than vitamin D deficiency. 1'2'4'5'8-1~In children, in whom disordered mineralization is expressed as tickets, these other conditions, although individually rare, have in the developed countries become collectively more common than vitamin D deficiency. Diseases presenting in childhood include virtually all of the genetically determined forms of tickets and osteomalacia2; they are considered here only to the extent that they raise issues of pathogenesis, diagnosis, or treatment that are relevant to adult medicine. One disadvantage of a histological rather than a clinical definition of osteomalacia is that many of its rarer forms differ in one or more respects from the standard textbook description. Statements based on observations in one form of osteomalacia must never be assumed without evidence to apply to another. Even in vitamin D - r e l a t e d osteomalacia, with more widespread use of bone biopsy and consequent earlier diagnosis, some classic features such as Looser's zones are now quite rare, and complete absence of symptoms, or the presence only of musculoskeletal symptoms unrelated to vitamin D, is much more common than in the past. Although there are many nonhereditary causes of osteomalacia, intestinal malabsorption of calcium and vitamin D (intrinsic deficiency) still accounts for more than 75% of all current cases in U.S. adults without renal failure. This is so despite a lower prevalence than in the past of osteomalacia in some malabsorptive states, such as adult celiac disease, which are now earlier recognized and more effectively treated. An important difference from extrinsic vitamin D deficiency is that malabsorption of calcium is more severe, since it can occur also as a result of the primary intestinal disease, unrelated to lack of vitamin D. Patients with intestinal malabsorption are also at risk for nonosteomalacic osteopenia, associated sometimes with high bone turnover due to secondary hyperparathyroidism and sometimes with low bone turnover; the latter is likely related to malabsorption of other nutrients, some known and some unknown. A detailed characterization of the different forms of intestinal bone disease and their interrelationships is another major theme of this chapter. Apart from problems resulting from incorrect histological criteria for diagnosis, several other misconcep-
A.M. PARFITT tions about osteomalacia can be found in recent writings on the subject. Some authors have claimed that osteomalacia can frequently be present even when all the noninvasive diagnostic tests usually performed are normal, 4 but in the author's experience this is a rare occurrence. In some series, previously unsuspected osteomalacia has been found in up to 10% of patients with apparent agerelated osteoporosis and compression fractures. 11 This probably reflects the referral bias that inevitably occurs at centers of tertiary care, since the prevalence of osteomalacia in unselected series of patients with postmenopausal osteoporosis in Detroit is less than 1%, although it may be higher in European countries that do not practice fortification of dairy products with vitamin D . 4 Nevertheless, patients with low bone density due to osteomalacia usually remain for many years misdiagnosed as having only osteoporosis. But the most important misconception about osteomalacia is that all of its manifestations are curable. Although symptoms can readily be relieved, and biochemical and histological abnormalities corrected, there has usually been irreversible damage to the skeleton by the time the diagnosis is made. 12 Symptomatic relief is gratifying to physicians, but its overemphasis can be counterproductive, because many patients have unrelated symptoms that are not relieved. This leads to poor compliance with treatment, the need for which is lifelong, regardless of symptoms. In most patients, osteomalacia is preceded for many years by clinically silent secondary hyperparathyroidism that accelerates the irreversible age-related loss of cortical, and to a lesser extent, cancellous bone. 13 Exposition of this concept is aided by using the term "hypovitaminosis D osteopathy" (HVO) to encompass the totality of osseous complications of deficiency or altered metabolism of vitamin D . 14'15 The possible role of HVO in the pathogenesis of age-related fractures, especially of the upper femur, represents a confusing but important borderland between osteoporosis and osteomalacia that is in need of clarification.
I. BONE MINERALIZATION AND THE MECHANISMS OF OSTEOID ACCUMULATION
A. The Remodeling Context for Mineralization Bone remodeling is a replacement mechanism. For reasons that are poorly understood, once bone has reached a certain age, it becomes less able to carry out its functions, whether mechanical or metabolic, and must be renewed. 16 The instrument of bone remodeling is the
CHAPTER 11 Osteomalacia and Related Disorders basic multicellular unit (BMU), a unique temporary anatomical structure consisting of a team of osteoclasts in front; a team of osteoblasts behind; and associated blood vessels, nerves, and connective tissue. 17 Each BMU originates at a particular place at a particular time and travels towards its target, either through the bone (osteonal remodeling) or across the surface of bone (hemiosteonal remodeling), continues beyond its target for a variable distance, and terminates when its supply of precursor cells is interrupted. During its progression, each BMU creates and leaves behind successive cycles of resorption followed by formation, each cycle slightly out of step with the one before and each cycle representing an individual remodeling transaction. The number of new cycles initiated per unit time (activation frequency) is the product of the number of new BMUs originated, equivalent to the number of new remodeling projects, and the average distance traveled by each BMU. ~6The end result is the formation of a new bone structural unit (BSU), either an osteon in cortical bone or a hemiosteon in cancellous bone. The sequence of events within a representative cycle can be reconstructed from standard histological sections, which are usually oriented at an angle to the direction of advance of the BMU, but the relationship between successive cycles can be observed only in sections that are parallel to the direction of advance. Such longitudinal sections are difficult to prepare in cortical bone TM and occur rarely and only by chance in cancellous bone because of its geometrical complexity. 17 Nevertheless, two-dimensional sections can be correctly interpreted only as interceptions of the remodeling events that occur in time and s p a c e . 17'19
B. I n t e r p r e t a t i o n o f T e t r a c y c l i n e D a t a The use of in vivo double tetracycline labeling as a diagnostic and investigative tool is described elsewhere in this volume, but some additional details are important for the understanding of osteomalacia. Tetracycline chelates calcium, and when the blood level is raised, it binds reversibly to every bone surface accessible to the circulation, but is permanently incorporated into bone only at the mineralization fronts, which are the sites of currently active mineralization. 5'2~ A few days after it has disappeared from the blood, tetracycline fluorescence is seen only in relation to these sites, which can also be identified by a band of granular structure and somewhat blurred outline that stains deeply with toluidine blue, and by a wide variety of other histochemical features. 3'4 Tetracycline binds preferentially to the most recent formed mineral which is of small crystal size and high surface area2~ the existence of amorphous mineral with special affinity for tetracycline has been challenged, but its pres-
329 ence as a very small fraction has not been excluded. 21 Because of the relatively low density and high water content of recently formed bone, tetracycline diffuses into it, away from the surface, to form the zone of instantaneous labeling, usually 2 to 3 Ixm in thickness.* As will be discussed later, in osteomalacia there can be an increase in the width of this band as well as a decrease in the distance between bands. In all studies from the author's laboratory that form the basis of the conclusions to be presented later, by scheduling the biopsy before labeling is begun, the time interval between the end of the second labeling period and the biopsy has been kept constant at 4 days. Permanent tetracycline fixation occurs because during this time interval a layer of new mineral is deposited of sufficient thickness to prevent the escape of tetracycline when its blood level falls to zero, as occurs from all bone surfaces at which there is no mineral deposition. Depending on the particular tetracycline used and on the physical and chemical characteristics of the new mineral, there is some outward diffusion, so that the outermost border of the second fluorescent band may be quite close to the current location of the mineralization front, which has moved 2 to 3 txm away from its location at the time of administration of the second label. There are several potential pitfalls in the interpretation of tetracycline measurements. 19 A double band of fluorescence establishes unequivocally that bone formation occurred during the relevant time period, but only a single label can be deposited if formation begins or ends between the periods of label administration. Most single labels are evidence of bone formation, especially if they are in continuity with double labels, but in osteomalacia there are several mechanisms (described later) for the retention of a single label in the absence of any recent mineralization. Conversely, some mineralization can occur in the absence of tetracycline uptake, because if mineral apposition is very slow, too few tetracycline molecules may be retained to exceed the threshold for visible fluorescence. 22 As a result, in some cases a higher proportion of the osteoid surface is unlabeled than can be accounted for by the delay in onset of mineralization, and the double tetracycline method underestimates the true bone formation rate by about 5% to 10%. Based on the study of cortical bone, it has been suggested that some unlabeled osteoid results from a temporary cessation of mineralization to form a so-called resting seam, 23 but no evidence for this phenomenon was found in cancellous bone. 24 *In the ilium, thickness (in three-dimensional space) = width (measured in two-dimensional sections) x ~/4, because of section obliquity.
3
3
0
A
C. L i f e H i s t o r y o f I n d i v i d u a l O s t e o i d S e a m s The formation of each new BSU begins at the cement surface, a thin layer of lowly mineralized collagen-poor but glycoprotein-rich connective tissue 25 that is laid down on the floor of the resorption cavity at the end of the reversal phase of each remodeling cycle. In twodimensional histological sections stained with toluidine blue or gallocyanine, the three-dimensional cement surface is represented by the cement line, forming the boundary in the section of the n e w B S U . 26 The cement line, which remains in the same location, separating new bone from old, must be distinguished from the boundary between mineralized and unmineralized bone, referred to as the o s t e o i d - b o n e interface, which normally moves away from the cement line during bone formation (Fig. 11-1). The mineralization front is always located at the o s t e o i d - b o n e interface, but if mineralization fails to begin, or ceases temporarily or permanently, the mineralization front either never appears or disappears, but the interface remains. During each cross-sectional remodeling cycle, soon after deposition of the cement surface, a team of osteoblasts assembles and begins to deposit a layer of bone matrix, referred to as an osteoid seam. Because of the local geometry, osteoid seams in standard histological sections appear in cortical bone as rings, and in cancellous bone as crescents tapering at each end, but in three dimensions each seam extends much farther along the BMU. 17 Although their individuality is less apparent than in cortical bone and is obscured by the usual methods of histomorphometry, osteoid seams in cancellous bone have a predictable range of dimensions and a measurable lifespan during which characteristic changes occur in the
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M. PARFITT
morphological features and function of the osteoblasts. 19 A provisional description of the cross-sectional life history of a typical osteoid seam in adult human cancellous bone, albeit simplified and incomplete, can be derived from tetracycline-based kinetics (Figs. 1 1 - 1 and 1 1 - 2 ) , and the principle that distance from the cement line is an index of the passage of time. 19 Matrix apposition is most rapid (2.0 to 3.0 txm/day) at the outset and the seam reaches a maximum thickness of approximately 15 to 20 Ixm after about 10 to 15 days, just before mineralization begins. Mineral apposition is also most rapid at the outset (1.0 to 1.5 Ixrn/day), and both matrix and mineral apposition progressively slow down with time, as the osteoblasts become flatter and more extended in shape and less basophilic in staining. About 50 days after the onset of mineralization when the bone surface has retumed to its previous location about 40 Ixm from the cement line, matrix synthesis stops. The osteoid seam thickness has by now fallen to about 6 Ixm, and mineral apposition continues at a progressively slower rate for a further 30 days, until the osteoid seam disappears, because all the new matrix has become mineralized. The osteoblasts have now completed their histological transformation into lining cells, and construction of the new BSU at that cross-sectional location is finished. According to this model, the durations of matrix synthesis and of mineralization overlap, but are neither coextensive nor necessarily identical. Three separate stages can be recognized (Fig. 11-1). In the first stage, matrix synthesis occurs alone without mineralization. In the second stage, matrix synthesis and mineralization occur together, and in the third stage mineralization occurs alone without matrix synthesis. In an alternative model, 27 the second and third stages are merged, matrix synthesis and
FIGURE 1 1 - 1 Stagesin the completion of a new bone structural unit at one cross-sectional location. O.Th., osteoid thickness; Md.B.Th, mineralized bone thickness; W.Th, wall thickness of completed structural unit. Note that bone interface (BI) moves away from the cement line during mineralization and that osteoblasts change progressively from cuboidal to flat.
CHAPTER 11
Osteomalacia and Related Disorders
331
FIGURE 11--2 Model of bone formation, with growth curves for matrix apposition above and mineral apposition below, showing distances from cement line as functions of time at a single cross-sectional location of a representative BMU. At any distance from the cement line, the horizontal distance between the lines is the instantaneous mineralization lag time at that distance. At any time, the vertical distance between the lines is the instantaneous osteoid thickness at that time. At any point the slopes of the lines (tangents) represent instantaneous apposition rates. For example, at t = 30 days, the instantaneous values are 20 pum for mineralized bone thickness, 16 lxm for osteoid thickness, 0.5 txm/day for matrix osteoid apposition rate, and 0.8 Ixm/day for mineral apposition rate; the matrix deposited at that time will have a mineralization lag time of 26 days. Formation period (FP) is counted from the onset of matrix synthesis to the completion of mineralization, and in this example is 120 days, at which time completed wall thickness (W.Th) equals 50 txm. It is evident that the total area between the curves is given by FP X O.Th and by W.Th X Mlt, so that these expressions are equal. Furthermore, it follows that O.Th = Mlt x Mx.AR. [Modified from Parfitt AM: The physiologic and pathogenetic significance of bone histomorphometric data. In Coe FL, Favus MJ (eds): Disorders of Bone and Mineral Metabolism. New York, Raven Press, 1992, pp 475-489.]
mineralization terminating simultaneously. In either case, the third stage is w h e n mineral apposition may often be too slow for tetracycline fixation to o c c u r . 22 In considering the relationship between this model and the results of bone h i s t o m o r p h o m e t r y as ordinarily performed, it is important to distinguish between the m e a n values of various measured and derived quantities found in one individual and the instantaneous values that occur at different stages of the osteoid seam life history in that individual (Fig. 1 1 - 2 ) ; where the context makes it necessary, a symbol that refers to such m e a n values will be identified by an upper horizontal bar. For the m e a n value to be an accurate representation of the whole sequence of events, it is even more important that sampling is unbiased with respect to time than with respect to space. This is possible only if bone remodeling is in a steady state during the period of labeling and biopsy.
of osteomalacia is the mineralization lag time, which is defined as the time interval between the deposition of an individual moiety of bone matrix and its mineralization. 2s During this time interval new matrix will be added, so that the osteoid seam thickness (O.Th) is determined by the mineralization lag time (Mlt) and the osteoid apposition rate (OAR):
D. Mineralization Lag Time and Osteoid Maturation Time
Mlt - O . T h / M A R .
A key quantity in understanding the m e c h a n i s m s of osteoid accumulation and the pathogenesis and diagnosis
O.Th = M l t . O A R , which on rearrangement gives Mlt = O . T h / O A R .
(1)
This concept was derived from studies of periosteal bone formation in rat tibia and cannot be applied without modification to adult h u m a n bone remodeling. In the growing rat, formation occurs over the entire periosteal surface and all the osteoid labels with tetracycline. Consequently, the osteoid apposition rate is virtually identical with the mineral apposition rate (MAR), so that: (2)
In h u m a n bone, the fraction of osteoid surface that labels with tetracycline (MS/OS), whether based on the m e a n of the separately m e a s u r e d lengths of first and second label or on the length of the second label alone, is sig-
3
3
2
A
nificantly less than unity, probably because too little tetracycline fixation occurs during terminal mineralization. 19'22 Since the total volumes of matrix and of mineralized bone formed are normally the same, the average rates of matrix and mineral apposition must also be the same. Consequently, the best estimate of the osteoid apposition rate is the mineral apposition rate averaged over the entire osteoid surface, referred to as the adjusted apposition rate (Aj.AR):
3.0 Mx.AR 2.0 (p.m/d)
1.0 0 3.0
MAR
(l~m/d)
2.0 1.0 0
Aj.AR = M A R . MS/OS
30
which in combination with equation 1 gives: Mlt = O . T h / M A R . MS/OS = O.Th/Aj.AR.
M. PAm~ITT
.
(3)
In the rat, bone formation is continuous, and the distinction between mean and instantaneous values is unimportant, but in humans, bone formation is cyclical. The instantaneous Mlt in young normal subjects increases progressively from about 10 days at the beginning of an osteoid seam lifespan (initial Mlt) to about 30 days at the end of the lifespan (terminal Mlt) with a mean value of about 16 days (Fig. 11-3). The onset of mineralization is delayed because of a progressive increase in collagen cross linking and other biochemical changes in the matrix that prepare it for mineralization8'19'28; the time needed for these changes to occur is the osteoid maturation time (Omt). Because Aj.AR has a lower limit of zero, 29 Mlt and Omt have upper limits of infinity. This drawback can be circumvented by using osteoid mineralization rate (OMR) as the reciprocal of Mlt and osteoid maturation rate (OmR) as the reciprocal of Omt, quantities that express the fraction of osteoid thickness that mineralizes or matures in 1 day. If mineralization occurs as soon as maturation is complete, as is likely in the growing rat, then Mlt is the same as Omt. This probably also holds in man at the onset (initial Mlt = Omt), but the cause of the subsequent increase in Mlt, which has no counterpart in the rat, is unknown. One possibility is that the time required for matrix maturation increases with the age of the osteoblast, and changes in matrix apposition and lag time together determine the progress of mineralization (Fig. 11-4). In this case, lag time would be an independent variable that remained identical with maturation time, and the changes in mineral apposition rate would follow automatically. Alternatively, the rates of matrix and mineral apposition could be separately and independently regulated. For example, there could be a decline in the supply of mineral, since the net inward calcium flux characteristic of osteoblasts must at some point change to the outward calcium gradient without net flux characteristic of lining cells. 21 In this case, lag time would exceed maturation time, and it would not be a genuine
O.Th. (pro)
20 10 0
30 MII (d)
20 10 0
0
I
I
I
I
I
I
I
120 20 40 60 80 100 TIME FROM ONSET OF MATRIX SYNTHESIS (DAYS)
FIGURE 1 1 - 3 Changesin various indices of bone formation during the life history of an individual osteoid seam. Values correspond to the time course depicted in Figure 11-2. Mx.AR, matrix (osteoid) apposition rate; MAR, mineral apposition rate; O.Th, osteoid seam thickness; Mlt, mineralization lag time. [Modified from Parfitt AM: Osteomalacia and related disorders. In Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990, pp 329-396.]
independent variable but would automatically follow changes in the other variables (Fig. 11-4). It is also possible that each of these mutually exclusive relationships could be true in different circumstances. Fortunately, the uncertainty in the pathophysiological significance of the lag time does not detract from its usefulness in the understanding of histomorphometric data and of the mechanisms of osteoid accumulation. 19'2~
E. Mineral Accumulation and Fluorescent Label Width The rate at which the bone interface advances ("horizontal" mineralization in Fig. 1 1 - 5 ) must be distinguished from the rate at which mineralization proceeds after it is initiated. 19 In an individual moiety of bone matrix, mineral accumulation ( " v e r t i c a l " mineralization in Fig. 1 1 - 5 ) as a function of time is a continuous process that is conveniently subdivided into two stages.
CH~"rE~ 11
333
Osteomalacia and Related Disorders INDEPENDENT
Ma.AR;MIt
DEPENDENT
MiAR
Ma.AR;Mi.AR MIt
a.
b.
s
DISTANCE (pm)
t
/
L," l--
l
I
0
I
I
l
40
I
.
80
I
120 TIME
I
I
0
|
l
40
l
80
--.
120
(DAYS)
FIGURE l 1 - 4 Two models of the relationship between matrix apposition, mineral apposition and mineralization lag time. a, Matrix (Ma) apposition rate and mineralization lag time are separately and independently regulated as functions of osteoblast age, and the rate of mineral apposition changes as an automatic consequence, b, Rates of matrix and mineral apposition are separately and independently regulated as functions of time and the lag time changes as an automatic consequence. In both cases, the genuine independent variables are depicted by solid lines and the automatically determined variables by interrupted lines. [Modified from Parfitt AM: The physiologic and pathogenetic significance of bone histomorphometric data. In Coe FL, Favus MJ (eds): Disorders of Bone and Mineral Metabolism. New York, Raven Press, 1992, 475-489.]
M.Fr 5
M,Fr o
I
I
I I I I s I / I / I / I / I r I I I I ! I ! I! II
I t
i Advance of M, Fr I 80"
Mineral Oensity (M.Dn)
('/,,Max.)
60" . 40i,
20" 0"
Distance (/~ m)
I
I
I
I
I
!
I
I
|
|
'a
5
4
3
2
1
0
-1
-2
-3
-4
-5
I
Time (days)
FIGURE 11--5
6
,,
|
|
l
!
I
|
|
|
l
l
|
5
4
3
2
1
0
-1
-2
-3
-4
-5
!
Distinction between two aspects of mineralization. In a plane perpendicular to a bone-forming surface, mineral density (M.Dn) as percentage of maximum is plotted as a function of distance from the location of the mineralization front (M.Fr) at time zero (M.Fr0) and of time; osteoid is to the left and mineralized bone to the fight. The correspondence between distance and time scales depends on movement of the M.Fr from fight to left at a constant rate, assumed here to be 0.8 Ixm/day. The curved solid line shows M.Dn at different locations within mineralized bone at time zero. The curved interrupted line shows M.Dn at different locations 5 days later, during which the M.Fr has advanced 4 lxm to M.Fr5 and mineral density at M.Fr0 has increased to 75% of maximum. The distance from M.Fr0 at which M.Dn has increased to the level that prevents diffusion of tetracycline molecules determines the width of the fluorescent band. The rate of advance of M.Fr or mineral apposition rate ("horizontal mineralization") and the rate of mineral accumulation ("vertical mineralization") can vary independently, although they usually change in the same direction. [Modified from Parfitt AM: The Physiologic and Clinical Significance of Bone Histomorphometric Data. In Reeker R (ed): Bone Histomorphometry. Techniques and Interpretations. Boca Raton, CRC Press, 1983. Copyright CRC Press, Inc.]
3
3
4
A
.
There is an early rapid increase to about 75% to 80% of m a x i m u m within the first few days, referred to as primary mineralization. It involves multiplication in the number of crystals, occurs close to the osteoblast, and may be influenced by its function. A much slower increase to about 95% of m a x i m u m or more over many months is referred to as secondary mineralization. It involves slow growth in the size of crystals (with displacement of water), occurs remote both in time and space from the osteoblast, and is presumably governed entirely by physicochemical factors. 19 As mineral density increases, a point is reached at which the bone is no longer permeable to tetracycline. For a given rate of mineral apposition, the time taken to reach this point determines the width of the fluorescent band. 3~ In the rat, this relationship permits calculation of the rate of initial mineral accumulation, which is closely correlated with the rate of osteoid maturation. 28'3~ In human subjects, this calculation cannot yet be formalized, but increased label width, presumably due to slower primary mineralization, is frequently observed in osteomalacia.
M. PARFITT
Rate oforemodel ing~ Acti vati n
.-._OS/BS~
Rateoftiomatrix, apposi n J~' Mi neralizationf ~ Lag time
O~V/BV, ~ o.
#
FIGURE 11--6 Relationshipbetween kinetic indices of bone formation and static indices of osteoid accumulation. The bars indicate an absence of effect of matrix apposition rate on OV/BV, since OS/ BS and O.Th are affected reciprocally by changes in matrix apposition rate in the steady state.
OS/BS(%) =
Ac.f(/y).FP(y)
x 100.
Osteoid volume is determined entirely by the fractional rate of bone turnover, which is the same as the volumebased bone formation rate (BFR/BV), and by the mean lifespan of an individual moiety of osteoid, which is the same as the mineralization lag time: OV/BV(%) = BFR/BV(%/y) 9Mlt(y).
F. Osteoid Indices in Relation to Tetracycline-Based Kinetics It is customary to ignore individual osteoid seam profiles and determine length, thickness, and volume for the entire section. Thickness (width x -rr/4) should be measured directly rather than calculated indirectly from volume and surface, both because of its intrinsic importance and because the frequency distribution of individual measurements sometimes gives more useful information than the mean value. The three osteoid indices are related as follows: 31
(5)
(6)
Since in the steady state bone turnover is determined entirely by the frequency of remodeling activation and the surface/volume ratio and since FP is inversely proportional to the osteoid apposition rate, 19 the relationships between the static indices of osteoid accumulation and the kinetic indices can be summarized as in Figure 11-6. Note that each osteoid index is determined by different aspects of bone remodeling and bone cell function 31 and, in particular, that osteoid volume is independent of matrix (or mineral) apposition rate, which in the steady state affects surface and thickness in opposite directions.
OV/BV(%) = O.Th(mm)
*OS/BS(%)*
BS/BV
(mm2/mm3).
(4)
A fall in trabecular thickness, as occurs to a modest extent in aging and in osteoporosis, will increase BS/BV and so increase OV/BV even if surface and width are unchanged, and for the most accurate interpretation, OV/ BV should be corrected to the expected trabecular thickness for age and s e x . 32 The relationship between osteoid thickness and its kinetic determinants is given by rearrangement of equations 1 and 3. Osteoid surface per unit of bone surface is determined entirely by the mean osteoid seam lifespan or formation period (FP) and by the average frequency with which new osteoid appears at any point on the bone surface, which in the steady state is the same as the frequency of remodeling activation (Ac.f):
G. Problems in the Definition of Impaired Mineralization Although a reduction in mineral apposition rate is frequently taken to indicate defective mineralization, it is evident from Figure 1 1 - 2 that matrix and mineral apposition are closely coupled and that the mean mineral apposition rate can never exceed the mean matrix apposition rate. Consequently, a reduction in the mean rate of matrix apposition inevitably leads to, and is much the most common cause of, a reduction in the mean rate of mineral apposition. In postmenopausal osteoporosis, the adjusted apposition rate as previously defined averages about 65% of normal, with some values as low as 10%, reflecting different degrees of impairment of matrix synthesis by teams of osteoblasts. 33 Both in normal subjects
CHAPTER 11
Osteomalacia and Related Disorders
335
and in every metabolic bone disease except osteomalacia, there is a significant positive correlation between mean osteoid thickness and mean adjusted apposition rate, with broadly similar slopes (b) and intercepts (a) of the regression lines. 31 Although such a relationship is to be expected, it has an unanticipated consequence for the interpretation of the mineralization lag time, since we can write: O.Th = b(OAR) + a.
(7)
If this is combined with equation 3, we obtain: Mlt = b + a/OAR.
(8)
Because of this relationship, which defines a rectangular hyperbola, when the osteoid apposition rate falls, the mineralization lag time increases (Fig. 11-7). This occurs in old normal subjects and in osteoporotic patients 33 and also in hypothyroidism. 34 Since prolongation of the lag time is an inevitable consequence of a fall in adjusted osteoid rate it does not by itself indicate defective mineralization. 31 Another way of arriving at the same conclusion is to consider the effect of prolongation of FP, which is also an inevitable consequence of a reduction in osteoid apposition rate. From Figure 1 1 - 2 it is clear that: F P . O.Th = W . T h . Mlt.
(9)
Since O.Th has a minimum value (the intercept in equation 7) and W.Th (the mean thickness of a completed BSU) is effectively constant in the short term, an increase in FP must be accompanied by an increase in Mlt. An increase in surface extent but decrease in thickness of osteoid, a reduction in the proportion of labeled osteoid (or mineralization front), a fall in either mineral 200 ~>,
a
E
li'/", ',
tOM
(/)
160
I--
120
_J co
80
I
\\
PMN"
MIt = 6.3/AJ.AR + 11.9
P M o P M,t = 5 0 / A J / A R +
~
OM:
MIt = 27.5/AJ.AR + 100
. .N_
~s o) i.c-
40 -
PM OP
~
'
.........
,,O. 1 i ~ 0.2 0.3 0 14 Adjusted Apposition Rate (pm/d)
or adjusted apposition rate, and a prolongation of mineralization lag time can all be nonspecific consequences of impaired matrix synthesis by osteoblasts. Using any of these, alone or in combination, as a definition of osteomalacia serves only to obscure its distinction from osteoporosis. Although mineralization is a kinetic process, it tums out to be impossible to frame a rigorous and consistent definition of impaired mineralization in vivo using kinetic data alone without reference to osteoid thickness. To explain further why this is so, it is necessary to examine the evolution of hypovitaminosis D osteopathy as earlier defined.
0 '.5
FIGURE 11--7 Hyperbolic relationships (equation 8) between mineralization lag time (Mlt) and adjusted apposition rate (Aj.AR; an estimate of matrix or osteoid apposition rate). PMN, postmenopausal normal; PMOP, postmenopausal osteoporosis; OM, osteomalacia due to hypovitaminosis D. Equations of the three lines given in inset based on published regression equations.3~ See also Table 11-2.
H. Histological Evolution of HVO and the Kinetic Definition of Osteomalacia There is no relationship between osteoid thickness and osteoid surface either in normal subjects or in patients with postmenopausal osteoporosis, but in HVO, whether due to dietary deficiency or to intestinal malabsorption of vitamin D, there is a hyperbolic relationship between these variables 31'32 (Fig. l l - 8 A ) . This indicates that osteoid surface increases first and that osteoid thickness increases only slightly until OS/BS exceeds 70%, after which further increases in osteoid volume are due mainly to increasing thickness. 14'15 In the same patients, osteoid thickness shows a more complex relationship to Aj.AR (Fig. 11-8B).When this is above 0.1 Ixm/day, there is the usual positive relationship between these variables; below 0.1 p~m/day further decrements in Aj.AR are associated not with a fall in osteoid thickness as in all other situations, but with a progressive increase limited only by the normal thickness of new matrix or W.Th. 35 This reversal is the cardinal kinetic characteristic of defective mineralization; a similar hyperbolic relationship is found between osteoid thickness and the fraction of osteoid surface undergoing mineralization. 1~ The concept is further illustrated in Figure 11-7, which shows that the mineralization lag time in osteoporosis, although prolonged in absolute value compared to normal subjects, is shortened relative to Aj.AR, whereas in osteomalacia the lag time is prolonged relatively, as well as absolutely; other differences are listed in Table 11-1. Based on these relationships, the author defines osteomalacia by a combination of mean mineralization lag time more than 100 days and mean osteoid thickness above the upper 95% confidence limit predicted by the regression of osteoid thickness on Aj.AR in normal and osteoporotic subjects (Fig. 11-8), or more simply, above an absolute value of 12.5 Ixm (corrected for obliquity). Patients with HVO who do not meet these criteria have increased volume and surface but not thickness of os-
336
A 60
50
E
.
A Static
t!-"
HPT
(D-) (D+)
I
/
/
30
/
/
/
MIt
100d
/ /
,~
10 a
o 9 l~Ip~, e 9
\ /
I
20
i
I
40
i
/
I .\ ,
\
9 9
-+/+,
~ --.
a
/
\
9 ~_~
./i.
* * a'a
/
\
\
/
\
9
__.
~
/
\
9
20
oc'-
/ /
0 9
/
///
\ \
9
/
// /
t~ el) CO O tl) .,i-, o)
9
HPT
E
-g
I I
2ry
(I) r
.o
9
I I
40
B Kinetic
I
9 OM a 2ry ,~
M. PARFITT
'.
;C -4--
I,
60
0
/
~o oAC
,y+ ~.. B~.
..o * ~ " qL
/
a
--
/
\ \ --'~'--
/
/ "boa
"-~--
I i
I
80
i
!
100
Osteoid Surface (% Bone Surface)
0
--
.B
- . , - -A-o-o
*_--___9 0.1
0.2
0.3
0,
a
.
0.4
.
.
0.5
.
0.6
Adjusted Apposition Rate (pm/d)
FIGURE 1 1--8 Osteoid thickness relationships in vitamin D deficiency, extrinsic or intrinsic. On left (A, static measurements), horizontal lines define mean and range for osteoid thickness and surface in normal subjects. The interrupted horizontal lines indicate the range for these variables in patients with secondary hyperparathyroidism with (D-; D) or without (D+; A) vitamin deficiency. The latter values represent patients with intestinal malabsorption of calcium alone. The curved solid line depicts the hyperbolic relationship found in osteomalacia (O.Th = 7.2/([1-0.0075) OS/BS]) and the interrupted lines depict the 95% confidence limits. On the fight (B, kinetic measurements), the straight solid line is the regression line of osteoid thickness on adjusted apposition rate (Aj.AR) in normal and hyperparathyroid subjects (O.Th = 7.2 Aj.AR + 7.8) and the 95% confidence limits for individual values. The oblique interrupted line represents mineralization lag time (Mlt) of 100 days. Note that in osteomalacia the osteoid thickness increases as adjusted apposition rate declines instead of decreasing as in all other situations. Patients with anticonvulsant bone disease (AC) and biliary cirrhosis (BC) individually identified because of the rarity of these causes of osteomalacia.
teoid, increased bone formation rate, the normal positive relationship b e t w e e n O.Th and Aj.AR, and increased osteoclast indices 14'15 (Fig. 1 1 - 9 ) , resembling in every respect the histological features of primary hyperparathyroidism. 4 This analysis identifies the earliest stage of H V O (HVOi), when osteoid accumulation is due mainly to increased frequency of remodeling activation and b o n e turnover, before the e m e r g e n c e of a significant mineralization defect, as due to secondary hyperparathyroidism. For some purposes it is useful to refer to such patients as having preosteomalacia. Patients with H V O who m e e t the criteria for defective mineralization are further subdivided into those who retain some tetracycline double labels (HVOii) and those with no double labels (HVOiii). A different perspective on the fundamental nature of osteomalacia can be gained f r o m the m o d e l of osteoid seam lifespan (Fig. 1 1 - 1 0 ) . In every other condition, all matrix f o r m e d eventually mineralizes; the slopes of the curves representing matrix and mineral apposition, al-
though initially divergent, eventually converge, and the loop f o r m e d by these curves u l t i m a t e l y closes. B y contrast, in untreated osteomalacia some matrix remains permanently unmineralized, the slopes of matrix and mineral apposition remain divergent, and the loop n e v e r closes. The m o d e l also illuminates the difference between HVOii, in which the earliest f o r m e d matrix bec o m e s mineralized but the later f o r m e d matrix does not, and HVOiii, in which none of the matrix f o r m e d bec o m e s mineralized. Since all patients with HVOiii at the time of biopsy have likely been through the stage of HVOii, they show a mixture of the two types of osteoid seam depicted in Figure 1 1 - 1 0 . Both thickness and volu m e of osteoid are significantly greater in H V O i i i than in H V O i i 14 (Fig. 1 1 - 9 ) , but even in the m o s t severe cases individual values for m e a n osteoid thickness fall within the reference range for m e a n wall thickness. 35 A final point about the kinetic definition of osteomalacia is the identification of two variant forms. The definition does not explicitly refer to osteoid volume,
CHAPTER 11
337
Osteomalacia and Related Disorders TABLE 11--1
Kinetic Differences Between Osteoporosis and Osteomalacia
Feature
Osteoporosis
Osteomalacia
Mineralization lag time (Mlt)
Low for Aj.AR
High for Aj.AR
Osteoid maturation time
Normal
Increased
Adjusted apposition rate (Aj.AR)
Relative fall more than relative rise in Mlt Never zero
Relative fall less than relative rise in Mlt Often zero
Osteoid thickness (O.Th)
Low
High
O.Th correlation with OS/BS
None
Positive
O.Th correlation with Aj.AR
Positive
Negative
Osteoblast defect
Matrix
Mineral
OS/BS, osteoid surface/bone surface.
which is the best index of the overall severity of osteoid accumulation. In cases that meet the kinetic criteria OV/ BV is almost always greater than 10% (twice the upper limit of normal), but occasionally it is normal. In these cases, contrary to the general rule, osteoid thickness increases earlier or to a relatively greater extent than osteoid surface, so that osteoid thickness is above the upper 95% confidence limit depicted in Figure l l - 8 B . This feature defines focal osteomalacia 36 as occurs in some
Apposition Rate (iJm/d)
BFR/BS (IJm3/IJm2/d)
drug-induced defects in mineralization, in which the initial increase in activation frequency that characterizes H V O is absent for one or another reason. In mild cases, focal osteomalacia, although qualitatively evident on inspection, m a y be shown by m e a s u r e m e n t only as an abnormal frequency distribution of individual osteoid seam thickness. A second and even rarer variant is the presence of substantial osteoid accumulation ( O V / B V > 10%) in the absence of any increase in osteoid thickness,
Oc.S/Md.S (%)
O.Th (pm) .
N
i
ii
iii
N
i
ii
N
iii
i
ii
.
MIt (d)
_
Ill
N
0.7
35
21
35
0.6
30
18
30
0.5
25
15
25
0.4
20
12
20
0.3
15
15
0.2
10
10
0.1
5
5
0
0
i
ii
iii
N
i
ii
iii OO
1000
100
10
0
FIGURE 11--9 Histological evolution of HVO. Progression from normal through the successive stages is accompanied by declines in apposition rates and fractional osteoid labeling and increases in osteoclast surface related to mineralized surface, osteoid thickness, and mineralization lag time, but the changes in bone formation rate are biphasic, increasing in HVOi and then progressively falling to zero.
338
A
.
M. PARFITT
FIGURE 11-- 10 Kineticsof matrix and mineral apposition in osteomalacia. Evolution of bone formation at a single cross-sectional location. Each panel is constructed in a manner similar to Figure 11- 2, but with altered time scale. Mlt, mineralization lag time; O.Th, osteoid thickness; W.Th, wall thickness. Note that in mild osteomalacia (HVOii) mineralization is delayed in onset, retarded in rate, and premature in termination, whereas in severe osteomalacia (HVOiii) no mineralization occurs at all [From Parfitt AM: Bone forming cells in clinical conditions. In Hall BK (ed): Bone: A Treatise, vol 1. The Osteoblast and Osteocyte. New Jersey, Telford Press, Inc, 1990.]
even when the frequency distribution of individual measurements is considered. This is referred to as atypical osteomalacia and may reflect a more severe impairment of matrix synthesis by osteoblasts than ordinarily occurs in osteomalacia. 32 The definitions of generalized, focal, and atypical osteomalacia and of H V O and its stages are summarized in Table 1 1 - 2 ; this classification will be used as a basis for describing the clinical, radiographic, and biochemical features of H V O and for comparing it with other forms of osteomalacia.
II. MANIFESTATIONS OF OSTEOMALACIA Osteomalacia is accompanied by clinical, histopathological, radiographic, biochemical, and radiokinetic features, the nature and frequency of which vary to some extent with the etiological factor. The typical effects of vitamin D - r e l a t e d osteomalacia are described and the main exceptions, which will be covered in more detail in relation to specific types, are noted briefly. An important general principle is that the effects can differ markedly according to the age of onset. W h e n defective mineralization begins in childhood, as well as tickets
there is usually osteomalacia. If this persists untreated into adulthood, as characteristically occurs in endemic vitamin D deficiency, severe deformity and gross radiographic abnormalities are common. W h e n osteomalacia begins later in life without previous rickets, as is now the rule in the indigenous populations of developed countries, its manifestations are more subtle. The later the onset, the more the clinical features can resemble those of age-related osteoporosis and the more easily overlooked are the clues to osteomalacia, since its symptoms can easily be attributed to the locomotor or neurological effects of aging. Except for Asian immigrants to Great Britain, advanced endemic type osteomalacia is found only in an occasional patient with celiac disease or vitamin D dependency. 2'4
A. C l i n i c a l F e a t u r e s The classic symptoms are bone pain and tenderness, muscle weakness, and difficulty in walking, often unremitting but lessening at the end of summer in mild cases. In the genetic forms of osteomalacia and in adult celiac disease there may be residual deformities of rickets, 2 usually associated with short stature. Deformities due to
CHAPTER 11 Osteomalaciaand Related Disorders
339
TABLE 1 1--2 Criteria for Different Morphological Forms of Osteomalacia and Different Stages of the Evolution of Hypovitaminosis D Osteopathy (HVO) Form
Stage
O.Th a (p~m)
Mlt (d)
Preo steomalacia
HV Oi
< 12.5 c
< 100
>5
Osteomalacia: generalized Generalized
HVOii HVOiii
> 12.5 > 12.5
> 100 od
> 10 > 10
---
> 12.5 <12.5
> 100 >50
Focal Atypical
OV/BV b (%)
<5e >5r
aCorrected for regression on adjusted apposition rate in borderline cases. bCorrected for trabecular thickness in borderline cases. CUpper limit higher in renal osteodystrophy. dNo double label, so apposition rate zero. eCases with OV/BV between 5% and 10% are transitional between focal and generalized. fin cases with OV/BV between 5% and 10% or with Mlt between 50 and 100, will need BFR to discriminate from HVOi or other forms of high-turnover osteoporosis. O.Th, mean osteoid thickness (corrected for section obliquity); Mlt, mineralization lag time; OV/BV, osteoid volume per unit of bone volume.
softening of the adult skeleton include kyphosis, coxa vara, and pigeon breast. 1'2'4 Less obvious is subclinical basilar impression. 37 Protusio acetabuli can ultimately compress the pelvic inlet to a triradiate shape and narrow the pubic arch, so that childbirth is only possible by cesarian section. 6 But in most patients there is no deformity other than the normal effects of aging on the spine. Less usual manners of presentation include algodystrophy, probably as a result of increased load bearing by the remaining mineralized b o n e 38'39 and plantar fasciitis. 4~ The various types of fracture that occur in osteomalacia will be described after the radiographic changes. A few patients present with the effects of hypocalcemia, which are described in detail elsewhere in relation to hypoparathyroidism. Some rarer symptoms, possibly due to hypocalcemia, that have been reported in osteomalacia but not in hypoparathyroidism, are impaired function of the posterolateral columns of the spinal cord in the absence of vitamin B12 deficiency, and cochlear deafness. 4 1.
B O N E P A I N AND T E N D E R N E S S
Like bone pain in general, pain in osteomalacia is dull and poorly localized but clearly felt in the bones rather than in the j o i n t s . 2'4'41'42 I t is often persistent, made worse by weight-bearing and contraction of locally attached muscles, and rarely relieved completely by rest but sometimes by the adoption of a particular posture. The pain is usually symmetrical, beginning in the low back, later spreading to the pelvis and hips, upper thighs, upper back, and fibs. It is never of sciatic radiation and in the absence of fracture is rarely felt below the knees. The distribution of tenderness to percussion is similar, but usually includes the shins. Lateral compression of the
ribs and posterior compression of the sternum are useful maneuvers to elicit pain. The anatomical localization to the axial rather than the appendicular skeleton has been attributed to its higher proportion of cancellous bone, 2 which accumulates relatively more osteoid than cortical bone and probably undergoes deformation more easily. Although pain conforming to this description in every particular is easily recognized, usually it is less characteristic and is an uncertain basis for differential diagnosis. Some patients carry for many years one or more diagnoses that cover a broad spectrum of rheumatological and orthopedic practice. Often these diagnoses are erroneous, but the patients commonly have other reasons for pain that do not respond to treatment of osteomalacia. A significant minority of patients are completely free of pain, especially those with severe hypocalcemia. 6'43 Others suffer excruciating pain with the least movement or the slightest touch. In X-linked hypophosphatemia (XLH) there is often no bone pain until middle age despite lifelong osteomalacia in the absence of treatment, possibly because the cortices of weight-bearing bones are thicker, so that even softened cancellous bone is less subject to strain. Although the general characteristics of bone pain as a form of deep somatic pain can be accounted for by the type and distribution of nerve fibers in bone, 42 its specific characteristics in different clinical circumstances are unexplained. 2. M U S C L E W E A K N E S S
Muscles of the proximal limb girdles, especially the lower, are often weak in osteomalacia, the severity varying from a slight abnormality detectable only on careful examination to severe disability verging on complete paralysis. In the only quantitative study, in 12 vitamin D -
340 depleted patients, the force exerted during maximum voluntary isometric contraction of the quadriceps ranged from 14% to 67% of normal, with a mean of 37%. 44 Atrophy is mild in relation to the severity of weakness, tone is reduced, and fasciculation is absent, but deep tendon reflexes are increased. 4 In mild cases, true weakness must be distinguished from unwillingness to tense muscles because of pain. Specific symptoms include difficulty in rising from a chair 1~ or walking up or down stairs without using the arms, and a characteristic gait, described later. It is essential for the physician to observe the movements that the patient reports to be difficult. 5 Electromyography usually shows motor unit potentials that are of short duration and reduced amplitude and often polyphasic. 45'46 Muscle biopsy shows no unequivocal features of primary muscle disease but mean fiber cross-sectional area is reduced to about the same extent as muscle strength,44 with preferential loss of type II fibers. 46 The syndrome is commonly referred to as a myopathy, but the site of the lesion is unknown. There is variable evidence of a neurogenic component to the weakness, 4 but this could result from associated deficiencies of nutrients other than vitamin D. 45 The syndrome can occur in every form of osteomalacia except XLH, but it is more common in vitamin D-related cases. Among these, the frequency is only 10% to 30% when the diagnosis is made early by biopsy, but approaches 100% in endemic nutritional osteomalacia. 45 In gastrointestinal disorders with a high frequency of neurological abnormality, " m y o p a t h y " is found only in patients with vitamin D malabsorption and osteomalacia, 4 and further evidence for specificity is induction of the muscle histological changes by experimental vitamin D deficiency in the rat. 46 The similarity in clinical and laboratory findings between patients with " m y o p a t h y " associated with hypophosphatemia of various causes but variable severity suggests that a common mechanism is depletion of phosphate at some critical intracellular location; this could be the result of severe phosphate deficiency, a specific transport defect in muscle under genetic control, or lack of some direct action of one or more vitamin D metabolites on muscle. 47 In patients with vitamin D depletion, muscle cell adenosine triphosphate (ATP) and phosphoryl creatinine are reduced but do not correlate with the degree of weakness, 44 and preliminary nuclear magnetic resonance (NMR) studies in one patient with hypophosphatemic osteomalacia were inconclusive. 48 Experimental phosphate depletion reduces muscle cell ATP and actomyosin content in the rat, 4 but several other mechanisms are possible. Severe proximal " m y o p a t h y " in the aluminum-related osteomalacia that occurs during hemodialysis may be an exception to this generalization, reflecting instead the neurotoxicity of aluminum. 4
A . M . PARFITT
3. DIFFICULTY IN W A L K I N G Abnormal gait is the most frequent clinical manifestation of osteomalacia regardless of etiology49; it can be the result of either pain or weakness, but usually both contribute. A change in gait discriminates more reliably between young adults with and without nutritional osteomalacia than any other symptom. 5~Many patients feel pain only when walking, which they consequently undertake sparingly and gingerly. Because of weakness the legs may feel heavy and the patient tires easily, walks more slowly with a flat-footed, springless gait, and is more likely to stumble. 1 If the hip muscles can no longer keep the pelvis horizontal with asymmetrical support, the upper body bends laterally away from the outwardly swinging trailing leg, keeping the center of gravity over the leading leg. The combination of trunk oscillation, short steps, and wide track constitutes the classic penguin or duck-like waddling gait of advanced osteomalacia, ~'2'4'5 which is still common where vitamin D deficiency is endemic, 7'45 but is otherwise rare. A waddling gait must be differentiated from a broad-based gait resuiting from outward bowing of the femora in the absence of muscle weakness.
B. H i s t o p a t h o l o g y o f H V O The diagnostic criteria have already been discussed, but some additional histological features are important for understanding the structural abnormalities in the skeleton and for the interpretation of noninvasive indices of bone remodeling. Osteoid accumulation increases about 15-fold, with a range of 5- to 30-fold, but the absolute increase in OV/ B V is much greater in cancellous bone (10% to 60%) than in cortical bone (1% to 10%) because of the difference in their normal rate of turnover. 12'51 The corresponding decreases in mineralized bone volume are also much greater in cancellous bone, with values similar to those of patients with osteoporotic vertebral compression fractures. 12 Total cancellous bone volume, including osteoid, is usually normal or increased in extrinsic vitamin D deficiency but often reduced in intrinsic deficiency to levels intermediate between normality and established vertebral osteoporosis. 32 Because of increased bone turnover in HVOi, cortical bone porosity is increased about twofold, with little further change in HVOii and iii. Osteoid accumulation and porosity are reversible, but as a result of prolonged secondary hyperparathyroidism there is thinning of cortical bone due to increased net endosteal resorption, which is irreversible and forms the largest component of the total body bone mineral deficit. 13'5~With the increase in cancellous tissue space at the expense of the inner third of the cortex, the absolute amount of total
CHAPTER 11 Osteomalaciaand Related Disorders cancellous bone may be normal even if its relative amount (analogous to concentration) is reduced. 51 There are also qualitative changes in the bone. If completion of secondary mineralization is delayed, scattered areas of bone of subnormal mineral density may be revealed by quantitative microradiography or by permeability to basic fuschin. 4 Because of its high water content, such bone is more accessible than normal bone to exchange of mineral ions with the extracellular fluid. Low-density bone is seen especially around osteocyte lacunae, which may also be lined by thin osteoid-like seams. 4 The asymmetrical perilacunar abnormality observed in XLH appears to be specific for that condition. 52 As well as being wider, tetracycline fluorescent bands are frequently blurred and indistinct. 3'8 The second label may still be visible at the zone of demarcation, even when mineralization is completely arrested, either because outward diffusion is retarded by thicker osteoid seams or because of binding to some constituent of the cement surface. 4 Finally, scattered small foci of particulate mineral may be seen within the osteoid in toluidine-bluestained sections 3 and by electron microscopy. 53 The interpretation of osteoclast indices, whether expressed as number of cells or nuclei or as extent of surface in contact with osteoclasts, rests on the observation that osteoclasts normally resorb only mineralized bone and avoid osteoid, 4'54 probably because their mechanism of attachment to the bone surface is impaired. 55 In HVOi the surface extent of osteoclasts is increased, whether related to the total surface or the mineralized surface. As osteoid increases in extent, the osteoclast surface per unit of mineralized surface increases substantially, but in advanced osteomalacia the osteoclast surface per unit of total surface may remain unchanged. For example, if the relative osteoid surface is 95% of bone surface and mineralized surface 5%, an osteoclast surface of 1% of bone surface is within the normal reference range for postmenopausal females but represents 20% of the mineralized surface. With the combination of reduced surface available for normal resorption and severe hyperparathyroidism, osteoclasts in typical Howship's lacunae are observed on osteoid, which appears to be undergoing resorption that is morphologically indistinguishable from the resorption of mineralized bone. Although the relative extent is small, osteoid resorption can account for more than half of the total osteoclast surface. 54 For a full description of bone remodeling in osteomalacia it is necessary to consider the formation, resorption, and balance of unmineralized bone and mineralized bone separately. In a few patients with severe osteomalacia there are the typical findings of osteitis fibrosamdissecting or tunneling intratrabecular resorption, giant subendocortical resorption cavities, increased fibrous tissue deposition adjacent to the cancellous and endocortical surfaces,
341 within resorption cavities and within the marrow spaces, and formation of w o v e n b o n e . 42'56'57 In contrast to the osteitis fibrosa of hyperparathyroidism alone without a significant mineralization defect, in osteomalacia the osteoblasts cover a smaller than normal fraction of the osteoid surface 58 and when mineralization ceases altogether, the osteoid eventually becomes covered entirely by flat lining cells. 35 In vitamin D-related osteomalacia, the morphological effects of hyperparathyroidism become more evident as mineralization becomes more defective, in contrast to the inverse relationship observed in the aluminum-related osteomalacia of hemodialysis. 4 Osteoclast indices are also less increased in non-vitamin D-related hypophosphatemic osteomalacia, osteitis fibrosa is rare, and osteoid resorption does not occur.
C. S k e l e t a l R a d i o l o g y o f H V O Structural changes in bone detectable on x-ray result either from increased parathyroid hormone (PTH) secretion or from impaired mineralization. Secondary hyperparathyroidism accelerates net loss of bone from endocortical surfaces, leading to generalized thinning of cortical bone detected either by radiogrammetry or radial photon absorptiometry. 13'51'59 If the loss is rapid, the endosteal surfaces may appear more irregularly scalloped than usual. 42 Increased bone turnover increases cortical porosity, recognizable as cortical striation in the metacarpals and phalanges on high-resolution films of the hands. 6~For a particular level of increase in turnover, the extent of cortical striation remains stable, but if mineralization becomes defective with the transition from HVOi to HVOii, cortical striations accumulate because refilling of resorption tunnels by osteoid does not alter their radiographic appearance. 4 By contrast, cortical thinning is probably retarded by osteoid insulation of the endocortical surface. Phalangeal subperiosteal erosion, the most specific sign of hyperparathyroidism, is not as common as in renal osteodystrophy 4 but in the absence of Looser's zones (see later) may be the most tangible radiographic evidence of osteomalacia, since it is not seen in the usually less severe hyperparathyroidism of HVOi. Very rarely, patients with longstanding intestinal malabsorption develop generalized osteitis fibrosa cystica. 4 In no case has adequate bone histology been performed, so these patients could have undergone progression of HVOi without transition via osteomalacia, as occurs in renal osteodystrophy. The most common radiographic manifestation of impaired mineralization in cancellous bone is a nonspecific reduction in density, 42 usually referred to in the older literature as "demineralization" and in current literature as "osteopenia." Distinctive qualitative abnormalities in
342 trabecular pattern occur only when severe osteomalacia began in childhood, as in celiac disease, or in endemic vitamin D deficiency. 4 Many trabeculae were never formed and those present are more widely and irregularly spaced, and may become thicker and more clearly visible despite their lower mineral concentration. Horizontal lines of increased density in the metaphyses (Harris lines) resulting from resumption of growth after temporary arrest are also frequently seen in such patients. 4 Minor degrees of coarsening, blurring, and loss of detail have often been described but have not been validated by adequate bone histology and are of questionable value in differential diagnosis. 42 In most patients the appearances are indistinguishable from those of moderately severe age-related bone loss. Of greater specificity are changes in bone shape. Minor degrees of protrusio acetabuli, detected by measuring the relationship between the acetabular and ilioischial lines, are much more common in osteomalacia than in osteoporosis. 61 More familiar, although rare in current practice, are changes in the spine, with biconcavity that is symmetrical about the horizontal axis of each vertebra and of similar degree in adjacent vertebrae, contrasting with the irregular distribution of altered vertebral shape in osteoporosis. 2'42 If anterior vertebral height is preserved there is no deformity, but a regular kyphosis with moderate height loss may result from generalized anterior wedging. In some patients with associated osteitis fibrosa, the abnormal vertebral shape is accompanied by irregular end-plate sclerosis as in renal osteodystrophy, but osteosclerosis commonly occurs in osteomalacia only in X L H . 42'62 Bizarre spinal deformities can be seen in advanced endemic osteomalacia, 4 but substantial loss of trunk height with generalized vertebral compression is otherwise rare except in sporadic nonfamilial hypophosphatemia of adult onset, which in contrast to XLH is associated with severe trabecular osteopenia. 62 Osteomalacia in general has been claimed both to increase the risk of the osteoporotic type of vertebral compression f r a c t u r e 63 and to protect against such fractures2; this issue will be discussed in Section II.D. The best known radiographic feature of osteomalacia is the Looser's zone, a lucent band adjacent to the periosteum that represents an unhealed insufficiency type stress fracture. 2'8'42'64 Stress fractures are incomplete fissures without displacement that occur as a result of repeated nonviolent subthreshold trauma; they are commonly divided into fatigue fractures in normal bone subjected to abnormal stress and insufficiency fractures in abnormal bone subjected to normal s t r e s s . 42 In the osteomalacia of cadmium poisoning, the lucent band consists mainly of unmineralized woven bone; during healing, sometimes spontaneous, woven osteoid mineralizes and is replaced by lamellar bone. 65 Looser's zones
A . M . PARFITT
TABLE 1 1--3
Comparison of Classic Looser's Zones and Typical Stress Fractures a Looser's Zone
Stress Fractures
Direction
Perpendicular
Can be oblique
Lucent band
Present
Usually absent
Margins
Parallel
Can diverge
Adjacent bone
Normal
Can be abnormal b
Number
Multiple
Usually single
Symmetry
Present
Absent
Sclerosis
Absent
Present
Visible callus
Absent
Present
Healing
Slow or absent
Rapid
aModified from Parfitt AM: Bone fragility in osteomalacia: Mechanisms and consequences. In Uhthoff H (ed): Current Concepts of Bone Fragility. New York, Springer-Verlag, 1986, pp 2 6 5 -270. bFor example, in Paget's disease.
occur most commonly in ribs, pubic rami, and outer borders of scapulae and less commonly in femoral necks, metatarsals, and shafts of long bones. 1'2 At some sites of predilection, localization may be determined by proximity to arterial pulsation. 4'9 Although occasionally painless, they are more commonly associated with local tenderness and pain on activity. If the lesions are multiple, symmetrical, and perpendicular to the periosteum with parallel margins and, in the absence of treatment, persist without callus or adjacent sclerosis, the diagnosis of osteomalacia is certain, 4 but exact conformity to this complete description (to which the term Looser's zone should perhaps be restricted) is observed in fewer than 5% of patients with osteomalacia in current practice. More commonly the appearances are intermediate in one or more respects between Looser's zones and typical stress fractures 66 (Table 1 1-3). Such lesions can occur in the absence of osteomalacia and so lack diagnostic specificity 8'64 (Fig. 1 1 - 1 1). Indeed, in our recent experience, atypical lucent stress fractures are more often the result of osteoporosis than of osteomalacia, 64 although the relative frequency remains much lower in the more common condition. Looser's zones, like other types of stress fracture, show increased focal uptake on bone scan, which can lead to a mistaken diagnosis of metastatic disease 67 and to a fruitless and sometimes fatal search for a primary tumor. 4
D. Fractures Several types of fracture occur in patients with osteomalacia. 4 Looser's zones, like other types of stress frac-
CHAPTER 11 Osteomalaciaand Related Disorders
343
FIGURE 11-11
Atypical fractures in osteomalacia and osteoporosis. Upper panel, left: metatarsal fracture with central lucency and abundant callus formation in a patient with histologically verified osteomalacia; fight: metatarsal fractures with more organized callus but persistent central lucency in a patient with histologically verified absence of osteomalacia. Resemblance to a classic Looser's zone is somewhat greater in the latter case. Lower panel, left: incomplete fracture on medial aspect of upper femur in a patient with histologically verified osteomalacia; fight: similar fracture in patient with histologically verified absence of osteomalacia. Although subtrochantefic location is more suggestive of osteomalacia, the other characteristics of the fractures do not differ significantly.
ture, can extend to produce a complete fracture with separation or displacement. This is common in the metatarsals and is probably also the mechanism for subtrochanteric fractures in the upper femoral shaft. Multiple rib fractures in osteomalacia have caused death from hemothorax. Occasionally, a greenstick-type fracture can occur in severe osteomalacia, as in rickets. 42 When osteomalacia begins in childhood, the adult bones tend to be soft rather than brittle and fractures are rare; con-
versely, when osteomalacia begins in adult life, fractures are more common, occurring mainly in the extremities. They differ from fractures in a normal skeleton only in needing a lesser degree of trauma, and from fractures in an osteoporotic skeleton only in a more varied anatomical distribution. An exception to this generalization is spontaneous fracture of the sternum, which is virtually confined to adult-onset osteomalacia and myelomatosis (Fig. 11-12).
344
A.M. PARFITT
FIGURE 1 1 - 1 2 Combinationof angulation of sternum due to spontaneous fracture and vertebral biconcavity in a patient with aluminum-related dialysis osteomalacia.
The major factor contributing to increased fracture risk is accelerated loss of cortical bone due to secondary hyperparathyroidism, so that repeated fractures are a common mode of presentation in HVOi. ~3'15 The biomechanical significance of osteoid accumulation and cortical porosity is less clear. In an elderly population the ash content of a vertebral body is highly correlated with its compressive strength, which is equally reduced whether mineralized bone tissue is lost or replaced by unmineralized osteoid. 68 This effect is unlikely to be quantitatively important unless OV/BV exceeds 20%, but could increase vertebral fracture risk in those who have already lost cancellous bone. 63 Conversely, if bone matrix volume is normal, osteoid accumulation could decrease fracture risk by increasing elasticity. 1 Appendicular fractures are less common in HVOii and iii than in HVOi. ~5 This is partly because activity is limited by pain and weakness--indeed, fracture risk increases when these symptoms are relieved by treatment. But the main reason is that loss of cortical bone is less severe. In HVOi the deficit in forearm bone density is about 25%, ~3'15 whereas in HVOii and iii, it is about 17%. 51 This can be explained by more rapid progression and earlier diagnosis, which would reduce the durations of both accelerated bone loss and exposure to increased fracture risk. The role of deficiency and altered metabolism of vitamin D in the pathogenesis of hip and vertebral fractures will be discussed in more detail in Section V.
E. N o n i n v a s i v e I n d i c e s o f B o n e R e m o d e l i n g A bone biopsy gives detailed information about cellular events in a small region but is subject to appreciable sampling variation. 19 By contrast, radiokinetic and biochemical indices reflect the integrated activity of the entire skeleton, but are governed mainly by the frequency of remodeling activation and give no information on individual cell function. In osteomalacia there are increases both in urinary total hydroxyproline, an index of whole-body bone resorption, 69-71 and serum total alkaline phosphatase, an index of whole-body bone formation. 1'2'4'9 Newer indices of bone formation, such as osteocalcin 71 and C terminal collagen propeptide (PICP), 72 are also increased but to a relatively smaller extent. In addition, radiocalcium kinetics often show an increase in accretion, an estimate of bone formation, 4'9 and there are also increases in skeletal uptake of various bone-seeking isotopes such as radiostrontium 73 and technetium-labeled bisphosphonates 74 and in bone blood flow. 75 It is commonly inferred that bone turnover is high in osteomalacia, 9'~~ but this interpretation is at variance with the histomorphometric evidence. One explanation for the discrepancy is failure to differentiate between the different stages in the evolution of HVO. In HVOi, all methods of study are in agreement in showing increases in resorption, formation, and turnover, and it is likely that some patients included in previous reports did not have osteomalacia as rigorously
CHAPTER 11
Osteomalacia and Related Disorders
345 though the distribution of h y d r o x y p r o l i n e - c o n t a i n i n g peptides b e t w e e n different c h r o m a t o g r a p h i c fractions is not altered. 69 M o r e difficult to explain are the increase in serum alkaline phosphatase, osteocalcin, and PICP. There is a high and significant correlation (r > 0.8) b e t w e e n log serum bone-specific alkaline phosphatase and log urinary hydroxyproline/creatinine in normal subjects and in several generalized disorders of bone. The regression slopes were similar, but in osteomalacia, alkaline phosphatase was higher relative to urinary h y d r o x y p r o l i n e than in any other condition. 78 Alkaline phosphatase also correlates (r > 0.7) with radiostrontium uptake in osteomalacia due to vitamin D depletion 79 but not with bone blood flow. 75 Matrix apposition rate in osteomalacia is reduced, but total matrix synthesis could be increased, balancing the increase in unmineralized matrix resorption; this could account for the significant correlations b e t w e e n several indices of osteoid accumulation and both serum alkaline phosphatase 8~ and osteocalcin. 71 Since osteocalcin is incorporated into b o n e matrix after mineralization begins, failure of mineralization w o u l d divert m o r e osteocalcin into the circulation, vl Inappropriate release of alkaline phosphatase might also reflect a disorder of osteoblast function induced by excess PTH, since aluminum-related osteomalacia in dialysis patients is usually a c c o m p a n i e d by relatively low levels of both P T H and alkaline phosphatase. 81
defined, but rather p r e o s t e o m a l a c i a with the histological, biochemical, and kinetic c o n s e q u e n c e s of secondary hyperparathyroidism. 58 But other patients in the cited studies had u n d o u b t e d osteomalacia despite lack of adequate histology. A n o t h e r possibility is the local increase in cellular activity in relation to healing fractures, as mentioned in the previous section. There m a y also have been inadvertent inclusion of patients in the early stages of healing when histologically determined formation rates are very high, but this is unlikely in patients needing a pharmacological rather than a physiological dose of vitamin D. Furthermore, in patients studied by double tetracycline labeling, the level of serum alkaline phosphatase increases with the severity of the mineralization defect, and is highest w h e n active mineralization has ceased 15 (Table 1 1 - 4 ) . Apparently increased kinetic accretion can probably be accounted for by the presence of significant amounts of bone of low mineral density and increased permeability, since this will enhance short- and m e d i u m - t e r m e x c h a n g e of radioactive calcium or strontium that m a y be impossible to differentiate from accretion without extended observation. 21 Increased uptake of labeled bisphosphonate correlates with serum alkaline phosphatase but not with radiocalcium k i n e t i c s . 74 It has been proposed that the bisphosphonate binds to some constituent of bone matrix and so is an index of osteoid accumulation with or without mineralization, 76 but in aluminumrelated osteomalacia, bisphosphonate uptake is low despite abundant osteoid. 7v Total h y d r o x y p r o l i n e excretion is highest in patients with subperiosteal erosion, 69 and could reflect P T H - m e d i a t e d resorption of unmineralized osteoid 54 even if the absolute level of resorption of mineralized bone was normal or even reduced. Utilization of n e w l y synthesized collagen might be defective, 4 al-
TABLE 11--4
F. A b n o r m a l i t i e s
of Bone Mineral Metabolism in H V O
The cardinal metabolic abnormality is reduced net intestinal absorption of c a l c i u m . 1'2'4-6'1~ Fecal calcium ex-
B i o c h e m i c a l Evolution of H V O
Normala (n = 23)
___2.1 ___0.5 _ 4.7(11) _ 0.11t
50.5 6.8 39.1 7.95
_ 6.3 _ 0.9(10) _ 3.7(8) ___0.39*
HVOiii (n = 28)
60.3 27.3 40.8 9.64
Plasma phosphate (mg/dl)
3.47 _ 0.08
3.36 +__0.11
2.91 _ 0.23*
2.64 _ 0.13
Plasma Ca • P (mg/dl)2
33.5 ___0.7
30.7 _ 1.1"
23.3 _ 1.6'
21.2 _ 1.2
Alkaline phosphatase (IU)
82.8 _ 3.9
132 _ 7.2t
201 +__31.1
284 _ 24.2*
4.01 _ 0.34
6.62 _ 0.79
5.94 _ 0.61
2.0 _ 0.26
57.2 6.0 46.0 9.12
HVOii (n = 11)
Age (years) Plasma calcidiol (ng/ml) Plasma calcitriol (pg/ml) Plasma calciums (mg/dl)
NcAMP (nM/dlGF)
+__ 1.3 ___3.0 +__6.9 _ 0.08
HVOi (n = 26)
58.1 4.1 21.7 8.02
___2.4 +__0.6(18)* _ 3.2(10) t _ 0.17
aVolunteers for bone biopsy. bCorrected for albumin. Stages defined as in Table 11-2. Number of analyses shown in parentheses when less than number of subjects. Data shown as mean _ SE. Significance levels shown for difference in mean values from column immediately to the left. *p < 0.05, *p < 0.01, *p < 0.001.
346 cretion is close to and can even exceed dietary intake, but urinary calcium is low and calcium balance rarely more negative than - 1 0 0 mg/day. 82 There is an equimolar deficit in net absorption of inorganic phosphate, but the relative change is much smaller. ~ According to the usual interpretation, calcium malabsorption leads in sequence to a fall in plasma calcium, secondary hyperparathyroidism, reduced renal tubular reabsorption of phosphate, hypophosphatemia, and reduction in calcium • phosphate product, which falls even further with the advent of more severe hypocalcemia. Eventually, deposition of mineral in osteoid is impaired because the supply of the relevant ions is reduced, and the alkaline phosphatase then rises. This traditional scheme requires considerable modification with regard to the order in which the changes occur, their pathophysiology, and their diagnostic significance. In HVOi, the mean plasma calcium is slightly reduced, but the individual values are almost always normal (Table 1 1-4). There is no adult counterpart to the early hypocalcemia of infantile nutritional rickets, 83 which reflects difficulty in releasing calcium from the rapidly growing skeleton, and is only rarely observed in older children. 84 PTH secretion is increased as shown both by radioimmunoassay 85-88 and by excretion of nephrogenous cyclic adenosine monophosphate (cAMP) ~5 (Table 11-4). Although mean TmP/GFR and plasma phosphate are both slightly reduced, individual values are usually normal. Twenty-four-hour urinary calcium excretion and fasting urinary calcium/creatinine are often but not invariably reduced. 85 As already mentioned (see Section II.E), a moderate elevation of alkaline phosphatase is the most consistent abnormality, but can be absent in subjects without osteopenia. 86 Unlike the various types of osteomalacia with hypophosphatemia due to n o n PTH-dependent defects in tubular phosphate reabsorption (referred to for convenience as primary), Albright and Reifenstein's first stage of "chemical osteomalacia with normal phosphatase ''89 does not occur during the evolution of HVO. The vitamin D metabolite levels depend on the etiology. In extrinsic vitamin D depletion the plasma calcidiol level at which abnormal mineral metabolism can first be detected in an individual is usually below 5 ng/ ml, 5'1~ but in subjects with values between 5 and 10 ng/ml there is a slight but statistically significant depression of mean plasma calcium and phosphate and urinary calcium and elevation of PTH, 85'87and most patients with histologically verified HVOi have calcidiol values in this range 15 (Table 11-4). With calcidiol values between 10 and 20 ng/ml, some subjects have a state of vitamin D insufficiency, in which calcidiol administration is followed by increases in serum calcitriol and calcium absorption, responses which do not occur in pa-
A.M. PARNTT tients with vitamin D sufficiency. 1~ In patients with intrinsic vitamin D depletion, the complete biochemical, histological, and bone densitometric syndrome of HVOi can occur at plasma calcidiol level between 10 and 20 ng/ml, 15 presumably because there is an independent mechanism for calcium malabsorption and consequent secondary hyperparathyroidism that is unrelated to vitamin D. Increased PTH secretion accounts for the normal mean level of plasma calcitriol. Although not evident from Table 11-4, which reports only data from patients who had bone biopsy, it is likely that in the earlier stages of intrinsic HVOi, plasma calcitriol levels are increased, 9~ as can also occur in anticonvulsanttreated patients. 9~ Further discussion of this will be postponed until Section III. Normal calcitriol levels in patients with extrinsic deficiency 7~'85 are even more puzzling, since in such patients the malabsorption of calcium that is thought to be the initial event is without obvious explanation. These paradoxes will be discussed later in relation to pathogenesis. With progression to HVO stages ii and iii, in general all the biochemical abnormalities become more severe. Plasma and urinary calcium, TmP/GFR, and plasma phosphate levels become lower, and PTH, NcAMP, and alkaline phosphatase levels become higher ~5 (Table 1 1 4), but there are many individual exceptions. PTH hypersecretion and parathyroid gland hyperplasia occur in response not only to hypocalcemia but to the independent stimulus of calcitriol deficiency. 92 Patients with severe hyperparathyroidism may get impaired tubular reabsorption of bicarbonate and amino acids as well as phosphate, resembling proximal renal tubular acidosis or the Fanconi syndrome, 4'~~ except for increased rather than decreased tubular reabsorption of calcium. Hypophosphatemia is adequately explained by increased PTH secretion without the need to postulate an additional effect of vitamin D metabolite deficiency. 47 Indeed, for the same increase in NcAMP, TmP/GFR is higher in secondary than in primary hyperparathyroidism because of the independent effect of plasma calcium on phosphate reabsorption. 43'93 Hypophosphatemia is of significantly lesser degree of HVO than in primary impairment of phosphate reabsorption, for both mean values and the frequency of individual low values. 1'41'94 Separation between the two groups is even clearer when the inverse relationship between TmP/GFR and NcAMP is considered. 43'47 Conversely, both individual and mean values for plasma calcium are almost always normal in patients with primary hypophosphatemia. The mean calcidiol level is not significantly lower in HVOii than in HVOi, but does fall further in HVOiii (Table 11-5). By contrast, the calcitriol levels can be normal in stage ii and do not become consistently subnormal until stage iii. Others have also found both normal and subnormal cal-
CHAPTER 11 Osteomalacia and Related Disorders
347
TABLE 11--5 Contrasting Features of Two Main Etiological Categories of Osteomalacia Primary Disorder Vitamin D Plasma calcium Plasma phosphate PTH secretion Alkaline phosphatase Osteoclast surfaceb Osteitis fibrosa Cortical thickness Cancellous volume
N or ,l, N or %a 1" or 1"1" 1"1" 1"1" Frequent $$ N or $
Phosphate N %%
N or 1" 1" 1" Rare $ 1" or $c
aOccasionally increased. bDifference clearer if referent is bone surface rather than mineralized surface. OVaries with type. N, normal: 1", $ change mild or slight; 1"1",$$ change severe or marked.
citriol levels in osteomalacia, although not classifying their cases in the same manner. 71'8~ In either HVOii or HVOiii, a minority of patients present with hypocalcemic tetany, often with absence of bone pain and radiographically less severe bone d i s e a s e . 4-6'43'79'95 Whether this is a pathogenetically distinct syndrome or simply one end of a spectrum is unclear, although probit analysis of plasma calcium values indicates two populations, 1 and in adolescent patients the values are bimodally distributed. 5 Because of lesser depression of phosphate reabsorption, higher than expected plasma phosphate, lower alkaline phosphatase, and lower incidence of phalangeal subperiosteal resorption, absence of the expected increase in PTH secretion was postulated. 4'79"89This has been refuted, although PTH assays (greater increase in normocalcemic patients) 95 and NcAMP excretion (greater increase in hypocalcemic patients) 43 have given different results. Patients with severe hypocalcemia are resistant to the effect of PTH, on both calcium release from bone and on tubular phosphate reabsorption but maintain a normal cAMP response to endogenous and sometimes exogenous P T H . 43'95-97 The biochemical resemblance to pseudohypoparathyroidism type II is even closer in patients with absolute rather than relative hyperphosphatemia, 95-97 especially if the cAMP response to exogenous PTH is impaired, 97 as may result from increased receptor occupancy. 98 The syndrome has been attributed to magnesium depletion, 1~ but this is only rarely the explanation, a3 The effect of PTH on phosphate reabsorption is blunted by
hypocalcemia, and is restored by treatment that raises the plasma calcium, 4'43 so that plasma calcium and phosphate levels change in opposite directions (instead of in the same direction, as in the usual response). 5 The role of a relative failure to increase tubular reabsorption of calcium in response to PTH 47 is difficult to assess. In vitamin D-deficient monkeys, the syndrome can be reproduced by reducing dietary calcium intake to about 5% of normal, '~176 but in osteomalacic patients no such clear relationship to dietary calcium intake is evident. 4'6 Neither insulation of bone surfaces by osteoid nor defective osteoclastic bone resorption accounts for severe hypocalcemia, since no histological difference was found between osteomalacic patients with low or with normal plasma calcium levels, 43 and some patients with the syndrome have radiographic as well as histological evidence of osteitis f i b r o s a . 95'96 Furthermore, PTHmediated resorption of cultured bone is unaffected by vitamin D deficiency, l~ There appears rather to be a failure of the calcium homeostatic system in bone, a system based not on osteoclastic bone resorption but on equilibration at quiescent bone surfaces. 21 The failure is unexplained, but its frequency during adolescence may be related to rapid skeletal growth. 5 The same mechanism operating with varying severity is probably the most important factor in all degrees of hypocalcemia in H V O . 1'47 The rarest and least well understood biochemical abnormality in HVO is hypercalcemia. 4 '95 .,02 Affected patients invariably have substantial parathyroid enlargement and high PTH levels and usually have radiographic osteitis fibrosa. Hypocalcemia is the primary stimulus to parathyroid cell proliferation as well as hormone secretion102; progression to hypercalcemia indicates that excessive cell proliferation has continued beyond the point needed to restore normocalcemia, in response to a different stimulus. The nature of the stimulus has not been established in vitamin D deficiency, but study of the analogous situation in chronic renal failure has suggested several possible mechanisms. These include calcitriol deficiency, loss of calcitriol receptors in parathyroid cells, and an increase in PTH secretory setpoint, '~ each of which could account for hypercalcemic secondary hyperparathyroidism. Because increased cell proliferation increases the probability of a mutation, 1~ some patients develop parathyroid adenomas. 102 ' 104 It is to such cases, that combine the hyperplasia of secondary hyperparathyroidism with the monoclonality of primary hyperparathyroidism, that the term "tertiary hyperparathyroidism" should be restricted. '~ In regions where vitamin D deficiency is endemic, it may be impossible in individual patients to distinguish between tertiary hyperparathyroidism as defined and coincidental primary hyperparathyroidism, '~ which may intensify vitamin D deficiency 106 by increasing the hepatic catabolism of calcidiol. In
348
A.M. PARFITT
either case, vitamin D deficiency may blunt the severity of the hypercalcemia, which may become evident only after vitamin D administration. 4'85
G. Summary of Temporal Evolution of HVO In HVOi or preosteomalacia, there are characteristically no symptoms until a fracture occurs, which is the main reason why the existence of this intermediary stage was unrecognized for so long. The only biochemical abnormality that would be revealed by routine screening is a raised plasma alkaline phosphatase. Both fasting and 24-hour urinary calcium excretion are usually reduced. Skeletal radiographs are either normal or show only nonspecific osteopenia, but bone densitometry reveals that age-related loss of bone is accelerated, especially appendicular cortical bone but also axial trabecular bone, with a corresponding increase in fracture risk. Plasma calcidiol is usually but not invariably low. There is both biochemical and histological evidence of secondary hyperparathyroidism and of increased bone turnover. Defective mineralization is either absent or no more severe than in primary hyperparathyroidism. Despite the lack of symptoms, lifelong treatment with some form of vitamin D is necessary to reduce PTH secretion to normal and prevent further irreversible bone loss. As mentioned earlier, the deficit in forearm bone density is greater in HVOi than in HVOii and iii despite less severe hyperparathyroidism (Table 11-5). This can only be explained by slower progression and consequent longer duration of accelerated cortical bone loss and increased fracture risk. It is likely that some patients remain arrested at this stage; a few progress to more severe hyperparathyroidism with radiographic osteitis fibrosa; but others eventually develop the complete clinical, biochemical, radiographic, and histological syndrome of osteomalacia. However, it may reasonably be assumed that all patients in stages ii or iii at the time of diagnosis traveled earlier through stage i.
H. Diagnostic Considerations As in all fields of medicine, the most important step in diagnosis is to keep the condition in mind in the appropriate clinical settings, which will be described in Section IV. In each setting the diagnostic value of a measurement must be independently validated and cannot be inferred from its importance in pathophysiology. But some aspects of diagnosis are logically considered here, because the existence of HVOi or preosteomalacia has
an important beating on the diagnostic process. Although crucial to understanding the disease, distinction between the different stages in the evolution of HVO is not essential for diagnosis, since it should not usually influence the decision to treat an individual patient with vitamin D. Consequently, the aim of diagnosis should be to identify HVO as early as possible in its evolution. Unfortunately, most diagnostic algorithms 49'50'94'107'108have been based on the erroneous assumption that only osteomalacia, defined rather strictly, needs treatment and hence recognition. The aim of preventing both occult bone loss and overt disease is best served by applying tests of high sensitivity (the probability of an abnormal result in a patient with disease) to the screening of populations at increased risk, 8'1~ even if such tests are of low specificity (the probability of a normal result in a patient without disease). Current commercial assays for intact PTH can easily detect modest increases due to mild vitamin D deftciency. 87'88 Other good screening tests are the plasma levels of total alkaline phosphatase and of calcidiol. For many tests, but especially alkaline phosphatase and urinary calcium, sensitivity is reduced by a large coefficient of variation, so that a biologically significant change could have occurred in an individual even though the result is still within the wide reference range. Sensitivity for alkaline phosphatase can be improved by more careful attention to sex and age differences, and both sensitivity and specificity can be improved by measurement of the bone-specific component. A low plasma calcidiol level is a poor predictor of bone histology,4'1~176just as a low vitamin B12 level is a poor predictor of bone marrow histology. Nevertheless, it is the best available index of suboptimal vitamin D nutriture and consequent increased risk of HVO. For the diagnosis of established osteomalacia without resort to an invasive procedure, test specificity is more important than sensitivity. 8 From this standpoint no test is very efficient when considered alone, especially in the very old in whom other causes for abnormal results are more prevalent. 11~ Nevertheless, if the plasma calcium and phosphate levels (or calcium • phosphate product) are low and the alkaline phosphatase is high, osteomalacia is likely, 1~ and if all three values are normal it is unlikely. ~1~Between these extremes, the diagnostic value can be improved by various discriminant functions based on biochemical values alone 94'1~ or combined with clinical and radiographic features. 1~ But the validity of any particular discriminant function depends on the prevalence of the condition of interest in a specific community and will not be the same in a young adult with suspected migrant osteomalacia, a nursing home resident with a hip fracture, or a patient with some form of intestinal malabsorption.
CHAPTER 11 Osteomalacia and Related Disorders
349
III. ETIOLOGICAL CLASSIFICATION AND PATHOGENESIS OF OSTEOMALACIA Osteomalacia can occur in diverse clinical settings, but in most types there is either a primary disorder of vitamin D metabolism or a primary ( n o n - P T H dependent) defect in the renal tubular reabsorption of phosphate. 5'8-1~ In the former, hypocalcemia and secondary hyperparathyroidism are usual and hypophosphatemia mild, whereas in the latter normocalcemia is the rule, secondary hyperparathyroidism is slight or absent, and indices of bone turnover are less increased but hypophosphatemia more severe (Table 11-5). In some forms of primary hypophosphatemia there are separate but interrelated disorders of both vitamin D and phosphate metabolism. Less common etiological categories are the presence of a mineralization inhibitor, a primary abnormality of bone matrix, or a defect in alkaline phosphatase.8-10
A. Overview of Defects in Vitamin D Metabolism Vitamin D metabolism can be affected at one of six levels (Table 11-6). Identification of the level is important in planning treatment, although the summation of independent factors at several levels may be needed to produce clinical effects, and some diseases affect more than one level. Each level is associated with a characteristic profile of vitamin D metabolite concentrations in blood (Table 11-6), but these must be interpreted with caution, since changes in vitamin D - b i n d i n g protein (DBP) can alter total concentrations without altering free
TABLE 11--6
concentrations. 111'112 Body stores of vitamin D, located mainly in fat and to a lesser extent in muscle, are derived either from the photochemical production in skin of cholecalciferol or from dietary intake and intestinal absorption of either chole- o r e r g o c a l c i f e r o l . 47"113'114 Although the former source is more physiological, 115 with current lifestyles the latter is equally important. 116 The distinction was made earlier between extrinsic vitamin D depletion, due to some combination of reduced skin synthesis and reduced intake, and intrinsic depletion, due to intestinal malabsorption of vitamin D, augmented by an additional mechanism for increased fecal loss of vitamin D . 117 Two infrequently emphasized features of normal vitamin D metabolism are relevant to the pathogenesis of osteomalacia: first, its wastefulness in normal circumstances, 5 and second, its susceptibility to disruption by calcium deficiency. ~8 Metabolic pathways leading from calciferol to calcitriol have been extensively investigated but are preferentially followed only when body stores are greatly depleted. Normally, about 70% of the daily supply of both calciferol and calcidiol is converted to more polar metabolites of low or absent biological activity that undergo biliary and eventually fecal excretion, 5 so that only about 10% of available calciferol is used for calcitriol production. The proportion following the alternate pathways can decrease to very low levels when necessary but increases to 90% for calciferol and 99% for calcidiol in vitamin D - t r e a t e d hypoparathyroidism. ~9 Despite their quantitative importance in overall vitamin D economy, little is known about either the metabolites formed or their mechanism of production, and even less about how distribution between different pathways is regulated. Because of the usual wide margin of safety, malabsorption of dietary vitamin D is rarely of sufficient se-
Possible Levels of Disturbances of Vitamin D Metabolism Plasma Metabolite Concentration
Level
Calciferola
Calcidiol
Calcitriolb
Extrinsic depletion Intrinsic depletion Impaired 25-hydroxylation Increased catabolism Impaired 1-hydroxylation Receptor defect
$ $ N ($)c N N
$ $ $ $ N N
N or $ N or $ N or $ N or $ $ 1"
alnferential, few measurements available. bMay be increased if there is a vitamin D-independent mechanism for reduced intestinal calcium absorption and secondary hyperparathyroidism.5 CDepends on compound affected.
35
0
A
verity to be the sole mechanism responsible for vitamin D depletion. The first additional mechanism to be proposed was interruption of a conservative enterohepatic circulation of calcidiol. 12~ This proposal accounted for depletion of vitamin D of dermal as well as dietary origin, but the magnitude of this pathway in human subjects, if it occurs at all, is much too small to fulfill its postulated role. 5'121 It now seems much more likely that the additional mechanism is accelerated catabolism of calcidiol in the liver initiated by calcium deprivation, 122 whether due to dietary deficiency or intestinal malabsorption. This effect is mediated by secondary hyperparathyroidism, either as a direct effect of increased circulating PTH 118'123'124or as an indirect effect of increased serum levels of calcitriol. 118'125 This mechanism explains the occurrence of vitamin D deficiency in geographic regions with high sun exposure but low calcium intake, 118 and contributes to vitamin D deficiency in a variety of gastrointestinal disorders. 5 The hepatic 25-hydroxylation of calciferol to calcidiol provides the principal transport form of vitamin D and an additional component of body stores, located mainly in m u s c l e . 113'114 This process is impaired in cirrhosis of the liver, 126 but rarely to a level that causes osteomalacia (unless there is also malabsorption, as in biliary cirrhosis) because the liver has such a large reserve capacity. 114 Significant calcidiol deficiency that is not due to depletion of its precursor is most commonly the result of increased catabolism to biologically inactive metabolites from drug-induced enzyme induction, 127 or from stimulation of existing enzymes by calcitriol o r P T H . 122'125 Loss of calcidiol bound to protein (both DBP and albumin) occurs in the nephrotic syndrome and leads to secondary hyperparathyroidism and osteoid accumulation in the absence of impaired renal f u n c t i o n . 128'129 A similar mechanism operates during continuous ambulatory peritoneal dialysis (CAPD), and urinary loss of calcidiol is also increased in patients with biliary cirrhosis.
114
Calcitriol deficiency with normal body stores of vitamin D is most commonly the result of chronic renal failure, but can also be due to a genetic defect in renal lt~-hydroxylation, referred to as hereditary hypocalcemia or vitamin D dependency type I. Plasma calcitriol levels are reduced by magnesium depletion, 114'13~ but osteomalacia as a consequence has not been demonstrated. Calcitriol synthesis is impaired by deficiency of PTH, but it is doubtful whether this causes osteomalacia, possibly because bone turnover is so low. 85 In a case be131 lieved initially to exemplify this relationship, the patient was subsequently found to have concurrent X L H . 132 However, there is one adequately documented case of osteomalacia due to pseudohypoparathyroidism with secondary hyperparathyroidism. 133 Very low plasma cal-
.
M. PARFITT
citriol levels are found during prolonged total parenteral nutrition, but have not been clearly related to the presence or type of metabolic bone disease. TM Finally, calcitriol may be ineffective because of one of several kinds of defect in its receptors, referred to as vitamin D dependency type II. Hereditary defects in calcitriol synthesis or receptor binding will not be considered further as causes of osteomalacia in this chapter. The role of such defects in the pathogenesis of age-related bone loss and fractures is discussed briefly in Section V.
B. O v e r v i e w o f D e f e c t s in Phosphate Metabolism The plasma phosphate level is regulated mainly by the tubular reabsorption of phosphate, best expressed as the mean renal threshold (TmP/GFR). 135 Intestinal absorption is much more efficient for phosphate than for calcium, net intestinal phosphate absorption remaining positive even with severe intestinal mucosal disease or with a large reduction in dietary intake. 47 Only if net absorption falls below 50 mg/day and the tubular transport mechanism is operating on the splay portion of the curve between the appearance and mean thresholds 135 can there be a sustained fall in plasma phosphate level below 2.5 mg/dl. Consequently, whole-body phosphate depletion sufficient to cause osteomalacia occurs only when net intestinal phosphate absorption becomes negative as a result of prolonged ingestion of large doses of phosphate-binding aluminum salts used as antacids. 136 With this exception, chronic hypophosphatemia causing osteomalacia is invariably the result of a low renal phosphate threshold. When not due to hyperparathyroidism, primary or secondary, reduced phosphate reabsorption can be either a solitary or principal defect, with or without glucosuria and/or glycinuria, or one component of the Fanconi syndrome 47 (Table 1 1-7). This term refers to a global disorder of proximal tubular function with glucosuria, generalized aminoaciduria, and impaired reabsorption of bicarbonate, urate, and less commonly, potassium, calcium, and sodium. Either type can be hereditary, usually presenting in infancy or early childhood, or nonhereditary, occurring at any age but most commonly during adolescence or adulthood. This simple approach to classification breaks down in some causes of the Fanconi syndrome, such as galactosemia or cystinosis, in which proximal tubule function is affected, not directly but as an indirect consequence of a nephrotoxic substance that accumulates in abnormal amounts because of a genetically determined enzyme defect that is unrelated to renal tubular transport. The classic form of hereditary hypophosphatemia is familial hypophosphatemic vitamin D-refractory tickets
CHAPTER 11 Osteomalacia and Related Disorders TABLE 1 1 - 7
351
Classification of Primary (non-PTH-Dependent) Defects in Tubular Reabsorption of Phosphate Causing Osteomalacia Plasma Calcitriol
Type Phosphate alone -L-_glucose
Hereditary Nonhereditary
Multiple (Fanconi syndrome)
Hereditary Nonhereditary
Classic Hypercalciuric Tumor-induced b Idiopathic
Normal a High Low Low c
Primary d Secondary e Internals External g Idiopathic
Low c Low c Variable Unknown Low c
relative to plasma phosphate. bProbably includes fibrous dysplasia and neurofibromatosis. CData inconclusive. dReduced reabsorption as a direct consequence of the genetic defect. eReduced as an indirect consequence of the genetic defect, via accumulation of a nephrotoxic agent such as cystine or galactose. SResulting from extrarenal disease, such as light-chain nephropathy. gResulting from the harmful effect of a drug or environmental toxin; except for cadmium, such agents usually do not cause osteomalacia. aLow
(or osteomalacia), often referred to as XLH because of the most common mode of inheritance. 62 Plasma calcitriol levels in the untreated state are normal for the rate of growth, but are lower than expected for the degree of hypophosphatemia 137 (Table 1 1-7). In a clinically similar disorder, hereditary hypercalciuric hypophosphatemia, the expected increase in plasma calcitriol levels occurs, leading to increased intestinal absorption of calcium. 138 In nonhereditary hypophosphatemia, most commonly due to a mesenchymal tumor, both symptoms and bone disease are more severe, and there is an absolute, not just a relative, deficiency of calcitriol. 139 There are numerous causes of the Fanconi syndrome, but most cases in adults leading to osteomalacia are either idiopathic or the result of light-chain nephropathy. 14~Limited data suggest that in the Fanconi syndrome, whether hereditary or acquired, the plasma calcitriol level is usually low, either relatively or absolutely. 47'~41 Renal tubular acidosis can be either a component of the Fanconi syndrome or unaccompanied by other primary defects in tubular reabsorption. 47 In the latter condition, the frequency and severity of hypophosphatemia are slightly greater than in vitamin D depletion, but less than in other types of hypophosphatemic osteomalacia. 1 There is no evidence for either absolute or relative calcitriol deftciency, 142 and it is possible that metabolic acidosis impairs mineralization directly, as well as by reducing phosphate reabsorption. Finally, severe hypophosphatemia probably accounts for the rare occurrence of osteomalacia in primary hyperparathyroidism without vitamin D deficiency, and after renal transplantation. 4
C. P a t h o g e n e s i s o f D e f e c t i v e M i n e r a l i z a t i o n Despite much progress 143 we still lack a complete understanding of how and why biological mineralization normally occurs only in some types of connective tissue and under close temporal and spatial control. Consequently, the pathophysiology of osteomalacia at the physicochemical, molecular, and cellular levels can be discussed only in a provisional and somewhat speculative manner. But several important general principles are known with reasonable certainty. 3 "8 58 ' 135 .143First, calcium, phosphate, and carbonate ions must be supplied and hydrogen ions removed for mineralization to occur. Second, the thermodynamic activities of the relevant ions in the fluid phase at sites of mineralization are influenced by, but are not the same as, those in the systemic extracellular fluid. Third, concentration gradients for the relevant ions between mineralizing and nonmineralizing sites can be maintained by cells (osteoblasts in bone or chondroblasts in cartilage), by structures derived from the cells, such as matrix vesicles, and by the ion-binding properties of macromolecules synthesized by the cells. Fourth, as well as chelating calcium, diverse proteins can have many other effects on mineralization, some positive and some negative. Fifth, the exact chemical composition and three-dimensional structure of connective tissue matrices are the most important determinants of where and when mineralization can occur. Although collagen is the most important matrix constituent, other structural proteins such as fibronectin and sialoprotein are also essential. Finally, there are differences as well as similar-
35
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A
ities between the mineralization of growth plate cartilage and bone, TM which explain why rickets and osteomalacia can, under some circumstances, vary independently in severity and in response to treatment. 145 The role of vitamin D in sustaining normal mineralization has given rise to two related controversies. 146'147 First, is the action of vitamin D mediated solely by changes in the calcium and phosphate concentrations in E C F , 4'148 o r does it influence mineralization more directly? 4'149 Second, is calcitriol the only metabolite of physiological importance, other than as a precursor,85'148'15~ or must some other metabolite also be considered? 4'~49'151 In both cases the contestants have often failed to recognize the difference between an essential function that confers an absolute requirement and a contributory function that confers only a relative requirement. There is evidence that the morphological effects of vitamin D deficiency can be corrected by giving enough calcium and phosphate intravenously both in man 152 and in the rat, and that defects 153 in calcitriol receptor binding can also be bypassed by providing sufficient mineral substrate. ~54'~55 In the clinical studies the evidence is inconclusive 156 but if taken at face value then vitamin D is clearly not essential for mineralization. Nevertheless, it could have a direct action on bone cells that contributes 4 135 149 to the process in normal circumstances.' ' Similarly, calcitriol alone can completely prevent or correct all the effects of vitamin D deficiency both in humans 4'157'158and in the r a t , 159'16~ claims to t h e c o n t r a r y 149'151 reflecting the inability of intermittent oral administration to sustain an adequate blood level. 159 Clearly, other metabolites are not essential, but it remains possible that one or more contributes to the evolution and reversal of defective mineralization. Alternatively, locally produced as well as systemic calcitriol could be involved. 1.
E V I D E N C E FOR D I R E C T AS W E L L AS I N D I R E C T
E F F E C T S OF V I T A M I N D
A plasma total Ca • P product, in (mg/dl) 2, of less than 30 is a useful guide to the presence of infantile nutritional rickets, 4'~~ but in older children and adults there is rarely such a consistent relationship between the plasma composition and the state of mineralization. 1'2'4'5'8'161 There are significant correlations between plasma phosphate and adjusted apposition rate and between plasma calcium and mean osteoid thickness, 162but their magnitude is too low for useful prediction in individual patients. Calculation of an ion product more clearly related to the physical chemistry of bone mineral may remove some discrepancies,4 but many remain. A more serious flaw in this line of reasoning is that single measurements in the fasting state, as in normal clinical practice, do not adequately represent body fluid composition because of the substantial circadian varia-
.
M. PARFITT
tion. 21'163 Nevertheless, it seems unlikely that such variation could account for the absence of osteomalacia in some patients with a degree of persistent hypophosphatemia that in other patients would be regarded as a sufficient explanation for their osteomalacia. 162"164 Even in the rat, a species in which mineralization probably depends more closely on plasma composition than in humans, healing of rickets can be detected radiographically in response to vitamin D administration while the Ca • P product is still subnormal. 16~ The persistence in early osteomalacia of some doubly-labeled surfaces with normal or only moderately reduced rates of mineral apposition (see Section I.H) indicates that mineralization can proceed at the beginning of the osteoid seam lifespan, although it ceases prematurely. Mineralizing and nonmineralizing osteoid seams are often close together, sometimes even in direct continuity, and are exposed to the same microcirculation, so that the difference between them cannot be explained in terms of chemical changes alone. But at doubly-labeled seams a higher proportion of the surface is lined by osteoblasts, 35'165 suggesting that these cells, possibly in conjunction with the osteocytes derived from them lying within the osteoid, 166 are able to promote mineralization in the face of a moderate reduction in plasma ion product, but do so for a shorter period of time than normal in vitamin D depletion. When this function is lost, mineralization ceases even though matrix apposition continues slowly and the osteoid seam gets progressively thicker. In more severe osteomalacia, osteoblasts are fewer or absent altogether, 57 mineralization never begins, and double labels are not found. A similar relationship is observed during t r e a t m e n t u t h e recovery of mineralization in response to calcitriol administration, indicated by double labeling, occurs preferentially at surfaces where new osteoblasts have appeared. 167"168 The bone histological data in patients with osteomalacia strongly suggest that deficiency of calcitriol (and possibly also other metabolites) impairs some function of the osteoblast that favors mineralization. This proposal is consistent with the presence in osteoblasts of calcitriol receptors, 169 the autoradiographic localization of labeled calcitriol in osteoblast nuclei, 17~ the stimulation by calcitriol of the in vitro production by osteoblasts of alkaline phosphatase 171 and osteocalcin, 172 the in vivo enhancement by calcitriol of mineral apposition rate in young m i c e , 173 and the morphological changes induced by calcitriol in the cells lining quiescent bone surfaces 174 that are of osteoblast lineage. 19 It is also consistent with the abnormalities in collagen cross-linking and other changes in bone matrix maturation and composition that 28 146 have been found in vitamin D deficiency, ' although it is less clear that these are the result of a direct rather than an indirect effect of vitamin D on osteoblast func-
CHAPTER 11 Osteomalacia and Related Disorders tion. The proposal is also not in conflict with the evidence that unphysiologically high doses of calcitriol given to growing rats inhibit several aspects of osteoblast function and in some circumstances may even impair mineralization and lead to osteoid accumulation, 4'5'175 accounting for the paradoxical osteomalacia of prolonged hypervitaminosis D . 176 The usual approach to mineralization has been to study the physical chemistry of the solution in which the ions originate, and the events taking place in bone, and to largely ignore what happens in between. But the circulating ions have to traverse a rather complex pathway before they arrive at the site of mineralization (Fig. 11-13). Having left the capillary and diffused through marrow connective tissue, they must pass through a layer of osteoblasts and a layer of osteoid before they can reach the site of mineral deposition. Osteoblasts on the surface, osteocytes within the osteoid, and osteocytes within mineralized bone are joined by a communicating network of cellular processes within the canaliculi. Very little is known about how mineral ions actually travel through this complex structure, but it would be surprising if cellular transport mechanisms of some kind were not involved in the movement of ions from the extracellular fluid to the site of mineral deposition. Indeed, it
353 seems likely that calcitriol could stimulate the inward transport of calcium and/or phosphate ions through or between cells at sites of mineralization, 47'85 consistent with its known effects on the cells of the intestinal mucosa and possibly the renal tubule. The osteoblast thus influences mineralization in two ways, by its effects on matrix maturation, and by its effects on mineral transport. 5'9 Furthermore, calcium and phosphate ions must be regarded not just as substrates for apatite formation but as part of the environment of the cell which is involved in their transport, since osteoblast function is influenced by the circulating and presumably also local levels of calcium 3~ and phosphate. 5'162 The concept that mineralization normally depends both on the availability of substrate ions via the circulation and on the activity of osteoblasts, although by no means rigorously established, enables all the apparently conflicting data, laboratory and clinical, to be reconciled. The concept has the additional merit of providing a basis for unifying the pathogenesis of all major forms of osteomalacia, since hereditary or acquired defects in phosphate transport across the renal tubular epithelium that covers all bone s u r f a c e s . 47'177 2. EVIDENCE THAT THE EFFECTS OF VITAMIN D ARE NOT MEDIATED SOLELY BY CIRCULATING C A L C I T R I O L
FIGURE 1 1--13
Biochemical and morphological approaches to mineralization. On the left are shown the directional movements of ions between a blood vessel above and the bone below without reference to intervening structures. These structures are depicted diagrammatically on right. 25HCC, 25-hydroxycholecalciferol (calcidiol); 1,25DHCC, 1,25-dihydroxycholecalciferol (calcitriol); M., mineralization; interface, boundary between osteoid and mineralized bone. The osteoblasts and osteocytes can be influenced by circulating levels of calcium, phosphate, and calcitriol, and also by locally produced calcitriol, either autocrine or paracrine. [From Parfitt AM: Human bone mineralization studied by in vivo tetracycline labeling: Application to the pathophysiology of osteomalacia. Excerpta Medica International Congress, 4th International Symposium on Chemistry and Biology of Mineralized Tissues 1992, pp 465-474.]
In patients with histologically verified osteomalacia or with radiographically unambiguous rickets, plasma calcitriol concentrations can be within the appropriate reference ranges 4'1~176 (Table 11-5). The levels are indeed inappropriately low for the degrees of PTH hypersecretion and hypophosphatemia, 179 as is indicated by the very high levels attained during the early stages of treatment with vitamin D , 157'158'181 but the lack of target cell responses to a concentration of calcitriol that is normally adequate requires explanation. In adults with osteomalacia, both biochemical and histological indices of vitamin D depletion appear to correlate better with either the sum of calcidiol (in ng/ml) and calcitriol (in pg/ml) concentrations, or with calcidiol alone, than with calcitriol alone. 1~ This suggests that calcidiol, or some other metabolite for which calcidiol is a precursor, such as 24-hydroxycalcidiol, might function as an agonist for calcitriol. In infants with untreated tickets, the plasma Ca • P product correlated with the plasma concentration of calcitriol and not calcidiol, although a higher than normal calcitriol level was needed to maintain a normal product. TM This suggests that some other metabolite functions in permissive manner, so that a fall in its concentration below a critical level would increase the need for calcitriol, but without dose-related effects above the critical level. 4
35
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Higher than normal calcitriol levels could be needed to correct hypocalcemia and to restore normal mineralization when the calcitriol-responsive cells are separated from the mineralized bone by a much wider than normal layer of uncalcified osteoid through which the mineral ions must travel 5 (Fig. 1 1-13). But no similar reason is evident for the failure of intestinal mucosal cells to accomplish normal calcium transport, at least in patients who do not have an independent cause for impaired calcium absorption, such as intestinal disease or anticonvulsant administration. 182 Theories that ascribe all manifestations of osteomalacia to deficiency of circulating calcitriol alone may be able to account for its persistence, but have much greater difficulty accounting for its initiation. At the onset of HVO, what sustains calcium malabsorption and a small but significant fall in plasma calcium when plasma calcitriol is maintained at a normal (or even increased) level by secondary hyperparathyroidism? A similar argument applies to the increased vitamin D requirement of primary hyperparathyroidism, due to accelerated calcidiol catabolism in the presence of increased plasma calcitriol levels. 1~ During the evolution of HVO there is an early fall in plasma concentrations of both calcidio115'41'8~ (Table 11-5) and 24-hydroxycalcidiol. 149'17sCalcium absorption and retention in bone are increased in man by pharmacological doses of 24-hydroxycalcidiol, 151 but there is no evidence for such effects at physiological blood levels. Calcidiol binds to intestinal receptors for calcitriol, but with approximately 500- to 1000-fold lower affinity, although calcidiol is only 100 times less effective than calcitriol in promoting bone resorption in vitro. 183 Seemingly, these differences in activity could be offset by the much higher total plasma concentration of calcidiol, but there is only a tenfold difference in free concentrations, based on recent estimates of the association constants for binding to the same circulating protein. 1~1 Consequently, a fall in plasma calcidiol level below normal could not significantly modify total receptor occupancy in the target cells that respond to circulating calcitriol, although some more complex effect on receptor function remains possible. 184 A more promising approach to the clinical paradox is the possibility that one or more dihydroxylated metabolites are produced locally in target tissues, as is strongly suggested by studies with isolated bone and intestinal cells 185'186and by the in vivo intestinal response to a pharmacological oral dose of calcidiol. ~87 If bone cell and intestinal cell l ot-hydroxylases were less influenced by PTH and phosphate than is the renal l et-hydroxylase, as is generally the case for extrarenal calcitriol production, 1~3 local production of calcitriol would be more substrate dependent than circulating calcitriol and would be impaired by a fall in plasma calcidiol concentration be-
.
M. PARFITT
low normal. Similar considerations would apply to local production of 24-hydroxycalcidiol, if this compound could be shown to have a physiologically important function. But it is more consistent with the evidence that only calcitriol is essential 5'159 to postulate that circulating calcitriol is most important for the regulation of calcium homeostasis, locally produced calcitriol is most important for the regulation of bone remodeling and mineralization, and both are important for the regulation of calcium absorption.
IV. OSTEOMALACIA
RESULTING
FROM ABNORMAL VITAMIN D METABOLISM A. E x t r i n s i c V i t a m i n D D e p l e t i o n Synthesis of vitamin D in the skin is reduced by residence at latitude distant from the equator, atmospheric pollution, and increased skin pigmentation. 5'8'~3'188 But the most important determinants are the duration of direct exposure to sunlight and the type and extent of protective clothing, which reflect cultural and social influences 5'7'8'189'19~ as well as individual choice. Seasonal fluctuation is important in the U.K.; vitamin D status in the winter is largely dependent on the extent of body stores accumulated during the previous summer and in many persons is only marginally adequate, with a high prevalence of subclinical deficiency. 5'~13'~9~ The same probably holds in many European countries. 116 Any chronic disease that impairs independence and mobility will inevitably reduce sun exposure or eliminate it altogether. 192 Differences in individual behavior, for whatever reason, can override the effect of latitude. ~9~ Foods naturally abundant in Vitamin D, such as swordfish, are consumed rarely if at all by most persons, so that dietary intake of vitamin D depends mainly on regional and national policy concerning fortification of food, for example, milk in the U.S. and margarine in the U.K. Differences in dietary intake of vitamin D contributed more than differences in latitude to variation in mean plasma calcidiol level between countries, although both effects were significant. ~6'~88 Loss of dietary vitamin D, especially the contribution from less exotic fish and from eggs, is most likely to be important in those with an aversion to fatty foods or in strict vegetarians. 1 9 3 - 1 9 5 Vitamin D in multiple nutritional supplements is an unreliable source because most of the other constituents promote its chemical decomposition. 196 The dietary requirement of vitamin D is increased in the elderly, ~3'1~6 who are more likely to be housebound or confined to nursing homes and are subject to age-related declines in the efficiency of vitamin D absorption, in the
CHAPTER 11 Osteomalacia and Related Disorders photochemical activation of 7-dehydrocholesterol in the skin, and in the ability of the kidney to make c a l c i t r i o l . 197 If the total supply of vitamin D, dermal and alimentary, is borderline, the occurrence of osteomalacia (and also of rickets) will be determined by other factors. Osteomalacia can occur locally in pagetic lesions 198because bone of high turnover either needs more vitamin D or permits the more rapid development of the histological expression of impaired mineralization; the opposite probably applies to bone of low turnover. 85 Pregnancy depletes vitamin D stores because calcidiol is transferred preferentially to the f e t u s , 199 which produces biochemical deterioration 2~176 and can precipitate overt osteomalacia. 6'2~ The sacrifice of maternal vitamin D stores ceases at part u r i t i o n , 196 but the calcium drain of lactation further weakens the bone particularly if the dietary intake of calcium is l o w . 6 Apart from its contribution to vitamin D deficiency, 118 calcium deficiency alone does not cause osteomalacia in adults but can do so in growing children. 2~ The risk of osteomalacia is probably greater in populations that use breads made from whole meal or high extraction flour as a staple source of c a l o r i e s . 4'2~ Such breads are rich in phytate, which binds dietary calcium and reduces its absorption, but the high fiber content is probably more important. Wheat fiber increases fecal excretion of bile acids 4 which could impair the absorption of vitamin D. A high-fiber diet also reduces the plasma half-life of labeled calcidiol with increased fecal loss, T M an effect probably initiated by calcium malabsorption, as previously explained. 122 In current practice in the developed countries, osteomalacia due to extrinsic (or privational) vitamin D depletion occurs mainly in two groups of p e r s o n s ~ i n young adults who have migrated from India or Pakistan, directly or via East Africa, to the U.K. and other European countries, ~~ and in the elderly of every country. 113'197 Migrant osteomalacia has received the most attention. Although the roles of genetic susceptibility, skin pigmentation, and type of cereal consumption have been debated for many years, it is now clear that the major risk factor for the migrant population as a whole is a drastic reduction in solar exposure together with persistence of a very low dietary vitamin D i n t a k e . 41'193'2~176 Although earlier studies were inconclusive, 2~ it now seems clear that a higher intake of chapatti and a low intake of meat, fish, and egg are additional risk factors205-207; the histological severity increases with the strictness of vegetarian practice. 2~ In one northem U.K. city there was a substantial improvement in various indices of vitamin D nutriture in the children of migrants over a 10-year period, probably reflecting adaptation to a Western lifestyle, but no improvement in the adults, 2~ a public health problem that is only partially s o l v e d . 2~176176
355 It is generally agreed that osteomalacia in the elderly is much more common in the U.K. than in the U.S., although prevalence in a random community sample has not been determined in a n y c o u n t r y . 2~ Reasons for increasing susceptibility with age were given earlier, why women remain more at risk than men long after cessation of childbearing is unknown. In the U.K., mean plasma calcidiol falls almost linearly with age in women from 30 to 90 years, 5'1~ subclinical vitamin D depletion is especially common in t h e elderly, 116'19~ and is the most important reason for impaired calcium absorption, 21~ but similar data are not available for men. According to reasonable histological criteria, osteomalacia is found in about 4% of unselected geriatric admissions in the U.K., a proportion that remained stable for almost 20 years. 21~ The prevalence is higher in nursing home residents and higher still in hip fracture patients113'214; whether the latter represents an etiological relationship will be discussed later. No comparable histological studies have been carried out in the U.S., although there are strong reasons for believing that the prevalence there would be lower in each of the four populations mentioned, but with the same rank order. 113'197 Even some healthy free-living persons in the U.S. have subclinical vitamin D depletion. 197'215 Contrary to earlier belief, privational osteomalacia does occur, especially with multiple risk f a c t o r s , 197'216 and being less common is more likely to be overlooked than in the U.K. 217
B. I n t r i n s i c V i t a m i n D D e p l e t i o n a n d Gastrointestinal Bone Disease Although much less prevalent worldwide than extrinsic vitamin D depletion, in the U.S. and other countries that practice fortification of dairy products with vitamin D, intrinsic depletion is the commonest cause of osteomalacia. In countries where it is common, extrinsic vitamin D depletion is a major determinant of the effect of intestinal disease on the skeleton218; this interaction is less evident in the U.S., although in Rochester (Minnesota) plasma calcidiol concentration in patients with adult celiac disease was significantly correlated with the duration of sun exposure. 219 Diverse other factors can influence the expression of vitamin D depletion in patients with gastrointestinal and hepatobiliary disease. Some have calcium malabsorption and consequent secondary hyperparathyroidism for reasons other than, or in addition to, vitamin D depletion. 15 Protein deficiency is common and could affect bone in many different ways. Finally, other nutrients not normally thought of in the context of metabolic bone disease may turn out to have important influences on the function of bone cells. 32 Probably for this reason nonosteomalacic osteopenia
356
A
.
with low bone turnover is especially common in patients with intestinal malabsorption, whether or not they have vitamin D depletion and secondary hyperparathyroidism. Vitamin D needs intraluminal bile salts for absorption by a similar mechanism to other lipid-soluble substances, and enters the circulation via chylomicra in mesenteric lymph. In patients with both intestinal malabsorption and vitamin D depletion, a causal relationship is commonly assumed but difficult to prove. It is necessary to measure fecal excretion of radioactively labeled vitamin D after oral administration, since the rise in plasma vitamin D reflects also the rates of tissue uptake for storage or metabolism. 22~ Calcidiol absorption is less dependent on bile and occurs directly into the portal circulation. 187'221 The increase in plasma calcidiol or the peak level after an oral load is even less specific than the rise in plasma vitamin D, correlating neither with measured absorption nor with the degree of steatorrhea. 222 But a low value, although mainly indicative of reduced body stores of vitamin D, sometimes provides more information than the basal plasma level alone 222'223 and so may help to establish the existence of vitamin D depletion, although not necessarily establishing the mechanism. Absorption of vitamin D is generally more depressed than absorption of calcidiol, but even with gross steatorrhea rarely falls below 40% of intake, 22~ a level that would rarely cause vitamin D depletion in an adult ingesting the U.S. RDA of 10 I~g/day. Extrinsic factors that need to be considered even in relatively sunny regions are self-imposed avoidance of fatty foods to reduce fecal bulk, and limited sun exposure because of chronic illness or concern with body image. 32 But in many gastrointestinal disorders, the frequency and severity of vitamin D depletion are difficult to explain by these factors alone. For example, osteomalacia can occur after intestinal bypass surgery despite prescription of 1.25 mg/day of vitamin D , 224 from which absorption of only 1% would prevent depletion. As previously mentioned, the most likely explanation is that biliary excretion of vitamin D metabolites is increased by calcium malabsorption and secondary hyperparathyroidism, either directly or via an increase in serum calcitriol. 125 Several authors have indicated the frequency of different absorptive and digestive disorders as causes of 165 223 osteomalacia ' (Table 11-8). Such lists reflect variation in surgical practice and individual physician interest as well as geographical differences in disease prevalence, and serve only to demonstrate the range of possibilities rather than to reveal epidemiological truth. An increase in serum total alkaline phosphatase can result from hepatobiliary disease or reflect the intestinal isoenzyme even in the absence of bone disease and so may be less useful in studies of prevalence than in extrinsic vitamin D depletion. But despite these possible
M. PARFITT TABLE 1 1--8 Frequency of Different Types of Intestinal Malabsorption as Causes of Osteomalacia at Henry Ford Hospital, 1976-1985 Type of Malabsorption
n
(%)
Postgastrectomy Adult celiac disease Bypass surgery Chronic pancreatitis Other short bowel Biliary Cirrhosis Total
14 8 7 4 2 1 36
(39) (22) (19) (11) (6) (3) (100)
drawbacks, total alkaline phosphatase is a useful predictor of HVO in adult celiac disease. 225 Not surprisingly, there is much more information on the frequency and severity of vitamin D depletion (indicated by low plasma calcidiol levels) and of abnormal bone mineral metabolism than of histologically verified HVO. Currently unexplained is the high frequency of osteomalacia in the absence of vitamin D depletion after biliopancreatic bypass for obesity. 226 Evidence for another form of bone disease in addition to HVO comes from several sources. Vertebral deformity and fractures are more common in patients who have had gastrectomy than in control subjects 227 and 5% of patients referred for evaluation of vertebral fracture gave a history of gastrectomy compared with only 1% in control subjects. 228 Significant cortical and/or trabecular osteopenia without osteoid accumulation and in the absence of other etiological factors such as corticosteroid therapy has been found after gastrectomy, 229 intestinal bypass surgery for obesity 32 and small bowel resection for Crohn's disease, 23~in primary biliary cirrhosis, TM and in longstanding ethanol a b u s e . 232 In all these disorders a common bone histomorphometric profile can be discerned with thinner but more extensive osteoid seams, a low adjusted apposition rate indicating reduced collagen synthesis by teams of osteoblasts, and a low bone formation rate indicating reduced remodeling activation. Reduced trabecular thickness is mainly the result of reduced wall thickness. 233 A very similar disorder is also found in beagles with intestinal malabsorption. 234 The state of decreased bone remodeling at the time of biopsy was probably preceded in many patients by high bone turnover typical of HVOi. Many of the patients still have vitamin D depletion and/or secondary hyperparathyroidism, to which their bone is not responding in the usual manner. A few patients show atypical osteomalacia as previously defined, with similar kinetic defects to low-turnover osteopenia but with increased
CHAPTER 11
Osteomalaciaand Related Disorders
surface and volume but not thickness of osteoid. 32 This most likely represents a transition from HVOi, in which the usual morphological expression of osteomalacia is blunted by an unusually severe defect in bone matrix synthesis by osteoblasts224; the same defect developing earlier in the course of the disease and accompanied by depression rather than stimulation of remodeling activation could prevent any osteoid accumulation in the patients with nonosteomalacic osteopenia (Fig. 11-14). Since these defects are similar to those found in patients with postmenopausal osteoporosis, 14'33 it is likely that in patients who have undergone accelerated bone loss whether from secondary hyperparathyroidism or from menopausal estrogen deficiency, impaired recruitment and activity of osteoblasts limit bone repair and predispose to fracture. 32'33 The previous discussion has emphasized the similarities between the various causes of intrinsic vitamin D deficiency, but there are also important differences. In postgastrectomy osteomalacia, extrinsic factors have been emphasized in the U . K . , 115'218'235 but hyperosteoidosis is common in countries where extrinsic vitamin D depletion is r a r e . 236'237 In most patients, steatorrhea is mild or absent and the most likely mechanism of vitamin D depletion is enhanced catabolism of 25-hydroxy D initiated by calcium malabsorption as previously described. 122'124 In representative asymptomatic patients studied on average 9 years after operation, plasma calcidiol was reduced and calcitriol increased, but there was moderate impairment of calcium absorption and appendicular osteopenia. 238 In cystic fibrosis, mild vitamin D
.:~,O~.~O NORMAL
HVOi
= GENERALIZED OM (ii) ~ .
. ~
GENERALIZED OM (iii)
-%--,~"LTO"
~-.,~ f = ATYPICAL OM
DEFECTIVE MATRIX APPOSITION DOMINANT
FIGURE 1 1 - 1 4
Possible deviations from normal development of HVO in intestinal bone disease. Transition from HVOi to atypical osteomalacia (OM) rather than to stage ii generalized OM reflects unusually severe depression of matrix synthesis, but if this continues very slowly, osteoid thickness will increase with eventual progression to stage iii generalized OM. If the initial increase in remodeling activation is blocked, the same defect in matrix synthesis with impaired mineralization will prolong formation period and lead directly to atypical osteomalacia. If remodeling activation is depressed by some additional agent, there will be no osteoid accumulation, the disorder of cell function resembling low turnover osteoporosis (LTO) but with normal instead of reduced osteoid seam thickness. If the mineralization defect persists, there will be gradual transition to atypical osteomalacia.
357 depletion and moderate osteopenia a r e c o m l T l O n , 239 but rickets is very rare despite the severity of steatorrhea 24~ and hyperosteoidosis has been verified histologically only in two adults, both with focal biliary cirrhosis and severe secondary hyperparathyroidism. 241'242 Osteomalacia is also rare in chronic pancreatitis and probably requires alcoholic liver disease as well as pancreatic enzyme deficiency. Vitamin D depletion is common in Crohn's disease, but is severe enough to cause osteomalacia only in patients who have undergone small intestinal resection, 243 especially if treated with cholestyramine for bile acidinduced diarrhea. T M After intestinal shunt surgery for obesity, serum calcidiol falls rapidly in the first 6 months and more slowly thereafter, and appendicular bone mass begins to fall after about 1 year despite little change in serum calcitriol. 245 Osteomalacia develops in about 12% of patients in Europe and in about 4% in the U.S., but for unknown reasons is less likely if hypomagnesemia is more severe. 32 Spontaneous improvement has been observed, 246 but progressive worsening of symptoms in the absence of diagnosis is more common. The potential risk of osteomalacia is greatest in adult celiac d i s e a s e 223'247'24s because the mucosal defect impairs absorption of vitamin D and calcium directly 73 and may also reduce local calcitriol synthesis. 187 Patients with mild subclinical celiac disease may manifest all the features of HVOi, which improve with a gluten-free diet. 249 In patients untreated for many years, osteomalacia develops in more than half, but can be forestalled by timely diagnosis. 247 Osteomalacia can occur even without steatorrhea and may be the presenting manifestation. 247'24sThe distinctive features conferred by onset in childhood (even if subclinical) were emphasized previously. 4 Unlike postgastrectomy osteomalacia, there is no response to ultraviolet irradiation 4 or to moderate doses of vitamin D in the absence of a gluten-free diet. 25~
C. Impaired 25-Hydroxylation and Hepatobiliary Bone Disease Despite the physiological necessity of 25-hydroxylation, its impairment is of only minor importance in clinical medicine, unless the supply of vitamin D is low. If liver destruction is so extensive that too little calcidiol can be produced despite adequate substrate, the patient will usually die before developing HVO. Furthermore, no genetic defect in the 25-hydroxylase has yet been identified. To understand what has been termed hepatic osteodystrophy, TM the separate effects of alcohol excess, loss of liver parenchyma, and cholestasis must be distinguished. Alcohol can cause osteopenia by a variety of
358 adverse effects on bone mineral metabolism unrelated to vitamin D . 232'252 Alcoholics are also especially prone to extrinsic vitamin D depletion, 253 which probably accounts for the coexistence of alcoholism and osteomalacia in a few patients with aseptic osteonecrosis. 254 Patients with cirrhosis of the liver, whether or not due to alcohol, often have mild steatorrhea 255 and impairment of vitamin D absorption. 4 Moderate reductions in plasma calcidiol in cirrhotic patients correlate with plasma albumin and other indices of liver function 254 probably because 25-hydroxylation is mildly impaired, ~26but plasma calcidiol can be raised above normal by oral vitamin D in the dose range 180 to 625 ixg/day. 4 In hemochromatosis venesection therapy increases plasma calcidiol, suggesting that iron accumulation can impair 25-hydroxylation independent of other effects on hepatocellular function. 256 In advanced cirrhosis, PTH secretion is slightly increased, but osteomalacia does not occur unless there is either cholestasis or extrinsic vitamin D depletion. 257 In chronic cholestasis, whether in primary biliary cirrhosis or due to hepatitis or to extrahepatic biliary tract disease, intraluminal bile salt deficiency causes steatorrhea and impaired absorption of both vitamin D and calcidiol. 258 Also, some metabolite of vitamin D other than calcidiol is lost in the urine in proportion to the rise in serum bilirubin. 5'114 Consequently, plasma calcidiol levels are low and there is malabsorption of calcium that can be reversed by treatment with vitamin D or its metabolites, 259 but the frequency of osteomalacia due only to these abnormalities has been greatly exaggerated. 251 Based on the kinetic criteria given earlier, or the osteoid measurements characteristic of these criteria, or on unequivocal clinical findings, the only acceptable published cases are from the north of England, 257'258or the far north of Sweden, 26~ where extrinsic vitamin D deficiency is common. Other patients were being treated for pruritus with cholestyramine, a drug that independently reduces vitamin D absorption. 261 In many other cases osteomalacia has been misdiagnosed because of one or more of the errors described in Section I, failure to exclude HVOi, or applying an unvalidated method to decalcified bone. 4 Data from the only definite case in the U.S. are shown in Figure 1 1-8. By far the most important component of hepatic osteodystrophy is severe osteoporosis, 25~'262made worse in a few cases by glucocorticoid therapy. 263 Bone pain and tenderness are frequently mentioned in clinical descriptions but they are the result of the multiple fractures to which these patients are especially prone, not of osteomalacia. Muscle weakness is also common, but can occur in primary biliary cirrhosis for several reasons unrelated to HVO. Even in asymptomatic patients there is significant vertebral osteopenia. 262 Bone turnover is usu-
A.M. PARFITT ally reduced, often markedly so, with low values for surface extent and volume of osteoid and extent and separation of tetracycline l a b e l i n g , 262'263 indicating both depressed remodeling activation and impaired osteoblast function. TM In other series bone tumover is increased in some patients. T M The indices of bone structure in transilial biopsies in primary biliary cirrhosis are very similar to those found in the nonosteomalacic osteopenia after intestinal shunt surgery, with cortical thinning and reduced thickness rather than density of trabecular plates. TM
D. Enzyme Induction and Anticonvulsant Bone Disease The quantitative importance of inactive vitamin D metabolites made in the liver was mentioned earlier. By analogy with other steroid hormones, their production in some cases depends on hepatic microsomal mixed function oxidases. 127 These enzymes are inducible, with increased microsomal content of one of several types of cytochrome P-450, by a wide variety of drugs, including barbiturates, phenytoin and several other anticonvulsants, and the antituberculous drug rifampicin. 267 The degree of enzyme induction is roughly indicated by the increase in metabolic clearance of antipyrine or in urinary excretion of D-glucaric acid or 6[3-hydroxycortisol, each marker showing a wide variation between different subjects taking the same drug, but correlating weakly with dose and blood level. 26v Some patients on long-term anticonvulsant therapy develop a syndrome of low plasma calcidiol, intestinal malabsorption of calcium, slight fall in plasma calcium, secondary hyperparathyroidism, and cortical osteopenia. 127'268-27~Although commonly referred to as anticonvulsant osteomalacia, this term is misleading on several counts. Radiographic evidence of mild tickets has been found in 8% of children, 4 but systematic surveys of bone histology in adults have invariably failed to disclose osteomalacia, 27~-273 except for a few doubtful cases in Scotland where privational vitamin D depletion is common, 4 and one case in an elderly psychiatric population. 274 With double tetracycline labeling, the bone formation rate is increased without defective mineralization, 57'271'273 and in this and every other respect the syndrome conforms exactly to the description of HVOi given earlier, with the exception of a higher incidence of acquired resistance to the phosphaturic effect of PTH 268 and disproportionate elevation of serum osteocalcin. 275 Osteomalacia, according to adequate clinical and radiographic and/or histological criteria, has been found in a few cases, but is largely confined to patients with only marginally adequate vitamin D supply, pro-
CHAPTER 11 Osteomalacia and Related Disorders longed treatment with multiple drugs, or other risk factors 4'5'127'276'277 (Fig. 11-8). The term "anticonvulsant osteomalacia" is also misleading because the implication of uniform pathogenesis takes no account of significant differences between drugs. 278 Phenobarbital is a more potent enzyme inducer than phenytoin and has been convincingly shown to enhance the catabolism of calciferol and calcidiol in the liver 4'127 but does not usually by itself reduce plasma calcidiol levels 279 or cause rickets, 4 probably because the formation of calcidiol is increased as well as its destruction. 4'127'278 Phenytoin has not been shown to have any direct effect on vitamin D catabolism, 114'275 but is more commonly associated with abnormal bone mineral metabolism than phenobarbitone because it can lead to hypocalcemia and secondary hyperparathyroidism by mechanisms unrelated to vitamin D , 269'273 such as impaired calcium release from bone and reduced calcium absorption. 4'5'~82'275 This would be expected to enhance calcidiol catabolism indirectly by the mechanism previously described. 5 A further complexity is that in the only prospective study, the early effect of phenytoin in newly diagnosed epileptics was to increase plasma calcitriol at the expense of calcidiol, with a corresponding increase in calcium absorption but without change in parathyroid function, 28~ consistent with the experimental demonstration of increased 1ot-hydroxylase activity. 4"278Multiple effects of phenytoin on vitamin D metabolism could account for differences in vitamin D m e t a b o l i t e profile 4'91'127'273'28~but make it more difficult to explain the production of osteomalacia, convincingly shown experimentally in the rat. TM Paradoxically, the best evidence for the clinical importance of enzyme induction and enhanced vitamin D catabolism comes from studies not with anticonvulsants, but with rifampicin and isoniazid. 282'283 Whatever the mechanism, anticonvulsant administration appears to increase vitamin D requirement by 10 to 15 txg/day in children 4'5 and possibly more in a d u l t s , 127'277'284 but whether this is clinically significant depends on the total dermal and dietary supply of vitamin D . 4'5'127'271 In otherwise healthy persons in sunny climates the effect is trivial, although non-vitamin D related effects of phenytoin may cause osteopenia. 273 By contrast, in mentally retarded institutional residents there is a substantial risk of clinically significant vitamin D depletion and its consequences. 277 At any level of supply, the amount by which requirement is increased depends on the dose and especially the number of drugs used, but also reflects individual susceptibility to enzyme induction. Consequently, both the frequency and the severity of the syndrome vary considerably between different populations. In some institutions, fractures due in part to anticonvulsant-induced osteopenia are a major
359 health problem and overt rickets is common, 285 but in other institutions prolonged immobility appears a more important determinant of bone density than anticonvulsant therapy. 4 As already emphasized, symptomatic bone disease is rare in noninstitutional settings, but the long-term effects of asymptomatic osteopenia are unknown. Although the clinical, biochemical, and histological abnormalities respond well to treatment, 127'286cortical bone loss is largely irreversible. 127 There is currently no consensus on whether all or only some anticonvulsant-treated patients should be given prophylactic vitamin D, if the latter, how they should be selected, and in either case whether ergoor cholecalciferol is more effective. ~27'27~
E. I m p a i r e d l o L - H y d r o x y l a t i o n a n d Renal Bone Disease The belief that chronic renal failure can impair the mineralization of bone has a long and controversial history. The term renal rickets was coined many years ago, 287 but its prevalence has been greatly overestimated because the radiographic appearances of metaphyseal osteitis fibrosa have so often been attributed mistakenly to rickets. 288 Quantitative bone histology frequently reveals substantial osteoid accumulation, 289 but the relative contributions of increased activation frequency and prolonged mineralization lag time (Fig. 1 1 - 6 ) could not be determined without double tetracycline labeling. In severe uremic hyperparathyroidism, osteoid seams are not only more extensive but thicker than normal because initial matrix apposition is more rapid, 19 so that all the static indices of osteoid accumulation can be reproduced, even though mineralization is normal. 31'57 The extent to which osteoid accumulation was excessive in relation to the histological severity of osteitis fibrosa was inversely related to the serum Ca • P product, 289 but in retrospect this relationship was most likely accounted for by the high prevalence of subclinical vitamin D deficiency in northern England that was not evident until measurement of serum 25(OH)D became possible. 5 Using tetracycline-based bone histomorphometry, the prevalence of osteomalacia in undialyzed patients with uncomplicated chronic renal failure was found to be very l o w . 29~ The risk of osteomalacia is increased by vitamin D deficiency, both in populations as already mentioned, and in individuals. TM Although not formally demonstrated, the adverse effects of vitamin D deficiency are probably potentiated by concomitant renal failure. Prolonged administration of aluminum-containing antacids to undialyzed patients can also predispose to osteomalacia because of either relative hypophosphatemia, 292 discussed in Section VI.A, or aluminum intoxication, 293 dis-
360 cussed in Section VII.A. Severe metabolic acidosis also increases the risk of osteomalacia. 294 The rarity of osteomalacia in chronic renal failure in the absence of one of these complications despite severe calcitriol deficiency is due partly to the protective effect of hyperphosphatemia, 292 and partly to the maintenance or calcitriol production in bone, provided the supply of its precursor calcidiol is maintained (Fig. 1 1-13).
V. VITAMIN D AND AGE-RELATED OSTEOPOROSIS Despite their obvious differences (Table 1 1-1), osteomalacia and osteoporosis have much in common. In both there is malabsorption of calcium, negative calcium balance, osteopenia, and increased fracture risk. In both, an initial state of high bone turnover, increased resorption, and accelerated bone loss is often followed by a state of low bone turnover and impaired osteoblast function. These similarities suggest a possible role for vitamin D in the pathogenesis, differential diagnosis, and treatment of osteoporosis. 4'295-297
A. Pathogenesis of Bone Loss and Fracture In type I (postmenopausal) osteoporosis, low-normal plasma calcitriol levels and impaired calcium absorption are secondary consequences of increased estrogendependent bone loss, and parathyroid function is normal or slightly depressed, but in type II (senile) osteoporosis, a primary abnormality in vitamin D metabolism probably contributes to bone loss. Functioning renal tissue mass declines with age, which reduces the ability to synthesize calcitriol in response to stimulation by PTH; aging itself may delay the response but does not alter its magnitude. 298 Because of this defect, augmented probably by other consequences of impaired renal function and possibly by an age-related decline in calcitriol receptor number or binding affinity, 299 PTH secretion increases with age to an extent that depends on the prevailing level of vitamin D nutriture. 21~176176 Since all forms of secondary hyperparathyroidism that have been adequately studied accelerate the age-related loss of cortical and to a lesser extent trabecular bone, ~3 it would be surprising if the same did not apply to the secondary hyperparathyroidism of aging. In France, where food is not fortified with vitamin D, HVOi may be an important cause of high bone turnover in patients with vertebral compression fractures, 63 but in late postmenopausal women without fracture, HVOi made only a small contribution to increased turnover. 3~176
A.M. PARFITT In the U.K., subclinical vitamin D deficiency is also common, and both lumbar spine and hip bone density correlate inversely with the severity of HVOi. 3~ In the U.S., high bone turnover is less common than in Europe in patients with compression fracture 11'33and reflects unusually prolonged effects of estrogen deficiency 295 rather than secondary hyperparathyroidism, except among those who have had a gastrectomy or other cause of intrinsic vitamin D deficiency. 228 But even in the U.S., serum 25(OH)D levels are reduced in some compression fracture patients. 3~ HVOi due to impaired 1 et-hydroxylation could account for the mild increase in OV/BV (3.5% to 8%), reversible by administration of alfacalcidol, in patients with vertebral compression fractures, who because of their advanced age would be classified as "type II" rather than "type I" osteoporosis. 3~ Muscle weakness and abnormal muscle histochemistry may also respond to alfacalcidol administration in these patients. 3~ As mentioned earlier, the prevalence of subclinical extrinsic vitamin D depletion, secondary hyperparathyroidism, and excess osteoid is frequently 1~3'3~176 but not invariably ~13'3~176 increased in patients with hip fracture. The available data are consistent with HVOi as an etiological factor, but it remains possible that hypovitaminosis D is a nonspecific marker of ill health and inactivity. In a controlled trial, vitamin D supplements failed to increase the ability of elderly hospitalized patients with low plasma calcidiol levels to carry out activities of daily living. 311 But hip fracture patients with normal osteoid indices and low plasma calcidiol levels have increased surface extent of osteoclastic resorption and more severe cortical osteopenia than those with normal levels. 12Also, increased osteoid accumulation and occasionally frank osteomalacia have been found in hip fracture patients with normal plasma levels of calcidiol but slightly reduced levels of calcitriol3~2; such an abnormality is less likely to be a nonspecific marker of ill health. These data increase the likelihood that HVOi, whether due to depletion or impaired metabolism of vitamin D, is a risk factor for hip fractures, but it is probably of etiological importance in only a small proportion of patients. 3~176
B. Differential Diagnosis and Treatment of Osteoporosis In all patients presenting with osteoporotic fractures of the spine or hip, especially after the age of 70, the physician should consider HVOi, which is both more common and easier to overlook than osteomalacia. If noninvasive indices of bone remodeling are increased, hyperparathyroidism (primary or secondary) and hyperthyroidism (endogenous or exogenous) must be excluded before resorting to estrogen replacement therapy. Serum
CHAPTER 11 Osteomalaciaand Related Disorders
361 larly nursing home residents. Indeed, 800 IU of vitamin D, together with 1200 mg of calcium, significantly reduced the risk of hip fracture in such s u b j e c t s . 319 Since the protective effect was evident within 6 months, 32~ it could have reflected increased muscle strength and decreased propensity to fall rather than any change in bone strength. A protective effect of vitamin D against hip fracture was not observed in healthy free living elderly people 321 and may be confined to the frail elderly with low body mass. 322
osteocalcin correlates better with bone histology than alkaline phosphatase or urinary hydroxyproline 313 but has not yet been shown to be a better guide to the selection of treatment. Single-photon absorptiometry of the radius, although an inaccurate predictor of vertebral bone status, is a good index of PTH-dependent bone loss. 51 Finally, although iliac bone histomorphometry after double tetracycline labeling is primarily a research tool, measurement of osteoid in a Jamshidi needle specimen is a simple and nontraumatic procedure with the potential for wide application. 314 Vitamin D in a pharmacological dose is commonly used in the management of spinal osteoporosis, on the grounds that calcium alone would be inadequately absorbed and that subclinical osteomalacia would be corrected without the need for accurate diagnosis. It is doubtful whether in any other branch of medicine a treatment is so popular that combines such complete absence of supporting evidence with such potential for serious h a r m . 315'316 In addition to its well-known toxic effects on the kidney, a high dose of vitamin D has an adverse effect on bone 317 because one or more of its metabolites stimulates bone resorption. 175 Nevertheless, because subclinical vitamin D deficiency is common, small physiological doses in the range of 5 to 20 Ixg/day significantly reduce the rate of bone loss in healthy postmenopausal women, 318 and it is reasonable to give such doses to those at increased risk of vitamin D depletion, particu60-
VI. OSTEOMALACIA ABNORMAL
FROM
RESULTING
PHOSPHATE
METABOLISM Many of the similarities and differences between vitamin D-related and phosphate-related osteomalacia (Table 11-5) have already been referred to and can be readily accounted for, but one major question remains: What corresponds to HVOi in hypophosphatemic osteomalacia? In established cases, the relationships between osteoid seam thickness and both osteoid surface and effective apposition rate are the same as in HVO (Fig. 11-15), although seam thickness is greater and a higher proportion are in stage iii rather than stage ii. But there are almost no data on the mode of evolution at an earlier
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0.3
Aj.AR (pm/d)
Osteoid thickness relationships in hypophosphatemic osteomalacia. Layout as in Figure 11-8 with static measurements on left (A) and kinetic measurements on right (B). Data are from 18 patients accumulated over 15 years, including 6 adults with XLH, 7 with nonhereditary hypophosphatemia (5 apparently without and 2 with a tumor, 1 cured by excision, with pre- and postoperative values joined), 2 with antacid-induced phosphate depletion (F-I), 1 with renal tubular acidosis secondary to Sj6gren's syndrome (A), 1 adult with hereditary hypercalciuric hypophosphatemia (O), and 1 with atypical osteomalacia following renal transplantation (A).
362
A.M. PARFITT
stage. The absence of focal osteomalacia indicates that, as in HVO, osteoid surface increases before osteoid thickness, but without a stimulus to increased remodeling activation analogous to secondary hyperparathyroidism, osteoid surface can increase only as a result of prolongation of the formation period, long known to be characteristic of hypophosphatemic osteomalacia. 323 It can be inferred that all patients with impaired mineralization due to hypophosphatemia evolve through a stage of atypical osteomalacia, in which a reduction in the rate of mineral apposition is accompanied by a parallel reduction in the rate of matrix apposition or, looked at from a different viewpoint, by an inversely proportional prolongation of mineralization lag time. As osteoid surface increases, the surface available for initiation or progression of remodeling would decrease, with a corresponding fall in bone formation rate, which would fall even further as mineralization became more defective. Except for one instance of atypical osteomalacia after renal transplantation (Fig. 11-15), data confirming this inference are lacking, most likely because the patients are not yet symptomatic and therefore not subjected to biopsy. Patients with hypophosphatemia, impaired osteoblast function, and reduced bone turnover but without osteomalacia 162'164 are presumably at an earlier stage at which disease progression is arrested because the fall in remodeling activation occurred sooner than would be dictated by the increase in osteoid surface, presumably from an independent cause. The situation is very similar to that in the nonosteomalacic low turnover form of intestinal bone disease (Fig. 11-14), with the difference that in hypophosphatemia this is the usual manner of evolution, whereas in HVO it is an uncommon variant.
dl), but the most consistent and diagnostically reliable abnormality is that urine phosphorus excretion is always less than 50 mg/24 hours and often much lower. By contrast, urinary calcium excretion is increased, sometimes to very high levels326; in one case nephrolithiasis resulted and led to unnecessary parathyroid s u r g e r y . TM Hypercalciuria was absent in the solitary case of tickets, possibly because the mineral content of bone was subnormal. 328 The clinical and biochemical syndrome of phosphorus depletion, except for osteomalacia, can be induced experimentally within a few weeks in normal subjects by high-dose antacid administration. 329'33~ The osteomalacia is histologically typical, without evidence of aluminum deposition, and with increased osteoclast extent, less than in HVO but more than in other forms of primary h y p o p h o s p h a t e m i a . 136'324'327'331 Consequently, the hypercalciuria probably results from increased net bone resorption as well as from increased calcium a b s o r p t i o n . 47"33~ Since plasma calcidiol and PTH levels are normal, both sources of urinary calcium probably depend on an increased plasma calcitriol concentration, known to be induced by phosphate depletion 329 and demonstrated in the most recent c a s e s . 136'324'326-328 In retrospect, this abnormality was first observed more than 30 years ago in a patient with an increased plasma level of vitamin D activity, measurable at that time only by bioassay. 333 All manifestations are quickly reversed with cessation of antacid administration combined, if necessary, with supplemental phosphate. Looser's zones have been observed to heal, and in one case there was histological verification of c u r e . 327 Other causes of osteomalacia with an increase in plasma calcitriol and/or hypercalciuria are shown in Table 11-9. In some of these conditions, calcitriol excess may paradoxically impair mineralization, as it does in the rat. 175
A. P h o s p h o r u s D e p l e t i o n Osteomalacia, according to reasonable criteria, has resulted from chronic phosphorus depletion in 16 c a s e s , 136'324-327 including two shown in Figure 11-15, and rickets in one case. 328 The patients with osteomalacia have ranged in age from 26 to 75 years and included 14 women and 2 men, in keeping with the greater susceptibility of women to hypophosphatemia on a lowphosphate diet. 329 They had taken phosphate-binding antacids--aluminum hydroxide with or without magnesium hydroxide--in large quantities usually for at least 2 years. The clinical and radiographic features do not differ from osteomalacia in general, although symptoms of general debility may be more frequent and severe. 33~ The plasma alkaline phosphatase is usually raised, but the abnormalities of bone mineral metabolism are unique. Plasma calcium is always normal and plasma phosphate usually but not invariably low (0.9 to 3.2 mg/
B. H e r e d i t a r y H y p o p h o s p h a t e m i a The gene responsible for this condition has been localized, both in a murine model and in XLH, the most common fOrlTl. TM In adults, the five separate but related components - - hypophosphatemia, impaired mineralization, retarded growth, osteosclerosis, and ligamentous ossificationmare of different relative importance and manner of expression than in c h i l d r e n . 62'335'336 Impaired tubular reabsorption of phosphate is lifelong, but in other family members may be accompanied only by relative shortness of stature. After epiphyseal closure, treatment is often withdrawn, but bone biopsy invariably shows osteomalacia and persistence of the asymmetrical perilacunar hypomineralization even in the absence of symptoms337'338; it is not known whether the same applies to hypophosphatemic relatives who never had rickets.
CHAPTER 11
363
Osteomalacia and Related Disorders TABLE 1 1 - 9
Unusual Causes of Osteomalacia with Increase in P l a s m a Calcitriol Concentration or Urinary Calcium Excretion or Both a Plasma Calcitriol
Phosphate depletion Hypercalciuric hypophosphatemiab Wilson's diseasec Cadmium poisoningc Hypercalcemic HPTd with D Myeloma + LCN (c.e)
Urinary Calcium Mixed
1" (Osseous)
$
1" 1" ? 9 $ $ or N
Cystinosisc
$
Oculocerebrorenal syndromec
9
Renal tubular acidosis Bartter's syndromeI
N ?
Vitamin D dependency type IF Calcium deficiencyh
1" 1"
aModified from Parfitt AM: Bone as a source of urinary calcium-osseus hypercalciuria. In Coe F (ed): Hypercalciuric States--Pathogenesis, Consequences and Treatment. New York, Grune & Stratton, 1984, pp 313-378, where additional references are given for conditions not discussed in present text. bReference 138; previously referred to as Gentil-Dent syndrome.332 CMultiple renal tubular defects of Fanconi type. dHyperparathyroidism (primary or tertiary) and vitamin D deficiency.1~ eLight-chain nephropathy. IRadiographic evidence of rickets; osteomalacia not verified. ~Defect in calcitriol receptor. hReference 202. Mixed hypercalciuriamincrease in net intestinal absorption as well as net bone resorption. Osseous hypercalciuriamincrease in net bone resorption a l o n e . 332
Some patients experience recurrence of bone pain and difficulty in walking, appearance or reappearance of L o o s e r ' s zones, and progression of deformity after cessation of treatment. Others develop s y m p t o m s for the first time in late adult life, and adults are especially prone to dental abscesses and degenerative joint disease. 336 These differences cannot be related to the apparent histological severity of the mineralization defect. In a unique family with X-linked recessive inheritance, the clinical features, mainly progressive lateral bowing of the femora, did not begin in any subject until adult life. 4 The coarsened trabecular pattern often present in children may progress to a generalized increase in trabecular bone density, especially in the axial skeleton, producing a radiographic resemblance to osteopetrosis or renal osteodystrophy. 62'335 Osteosclerosis may be especially severe in families with autosomal recessive rather than the more usual X-linked dominant inheritance. 339 Measurement of bone mineral density by a variety of methods 4'34~ indicates reduced thickness of n o n weight-bearing bone in the extremities and normal or increased cancellous mineralized bone volume, consistent with the histological findings. 338'34~ The radiographic
appearances reflect both the excess mineralized bone and the increased radiodensity of osteoid relative to other soft tissues because of its high sulphur content. Cortical thickness is often increased in weight-bearing bones because of compensatory buttressing during growth. 62'34~ Osteosclerosis is asymptomatic, but ossification of interosseous m e m b r a n e s , ligaments, tendons, and joint capsules at their sites of attachment to bone, collectively known as entheses, is an important cause of disability in adults with hereditary hypophosphatemia. 335'344'345 The process begins as a roughening and irregularity of the periosteal surfaces, but progresses by extension of ossification beyond the original confines of the bone. Occasionally, ossicles due to ectopic ossification not continuous with the bone appear in the extremities and around the joints of the pelvis. There is usually pain, stiffness, and limitation of motion in relation to affected sites, and both x-ray appearances and clinical disability increase with age without relation to sex or treatment. Probably because of retarded growth and consequent shortness of the pedicles, the lumbar spinal canal is often narrow with increased susceptibility to cord compression requiting surgical intervention. 62'346 Mild sensorineural
36
4
A
.
hearing loss probably due to involvement of the cochlea by ligamentous ossification and bony expansion is also quite c o m m o n . 347 The occurrence of similar lesions in a patient with cadmium-induced osteomalacia 335 suggests that they arise in response to prolonged tension on the surface of softened bones rather than representing an independent component of the genetic syndrome as previously suggested. 62
C. Nonhereditary Hypophosphatemia, Idiopathic and Oncogenous Hypophosphatemic osteomalacia sometimes appears for the first time in adolescence or adult life in the absence of tickets or retarded growth during childhood and with a negative family history. 62'348'349 In many cases the clinical, biochemical, and histological abnormalities have been cured by removal of a mesenchymal tumor~139'350 and several lines of evidence suggest that such a tumor is present in all cases. First, the characteristics that differ from hereditary hypophosphatemia (Table 11-10) are essentially identical in cases with and apparently without a tumor, including the frequency distribution of age of onset by decade. Second, with more
TABLE 11--10 Differences Between Two Forms of Hypophosphatemic Osteomalacia a Feature
Hereditary
Nonhereditary ~
Muscle weakness
No
Yes
Bone pain
No C
Yes
Increased glycinuria
No
Yes
Osteopenia
No
Yes
Vertebral collapse
No
Yes
Loss of trunk height
No
Yes
Fractures a
No
Yes
Perilacunar low-density bone
Yes
No e
Osteosclerosis
Yes
No
Enthesopathy
Yes
No i
Deafness
Yes
No
Phosphate depletion
No
Yes
aModified from Parfitt AM, Kleerekoper M: Clinical disorders of calcium, phosphorus and magnesium metabolism. In Maxwell M, Kleeman CR (eds): Clinical Disorders of Fluid and Electrolyte Metabolism, 3rd ed. New York, McGraw Hill, 1980, pp 9 4 7 - 1 1 5 2 . ~vVith or without a tumor. CMay occur in adults but rarely disabling. dLooser's zones occur in both. eEvidence inconclusive. I M a y depend on duration without treatment. Differences dependent only on age of onset are excluded.
M. PARFn'T
widespread recognition of the need to search for a tumor in adult-onset osteomalacia, reports of idiopathic cases have become notably less frequent, 35~ whereas twice as many tumorous cases were reported from 1980 through 1989 than in the preceding decade. 35~ Third, in many cases the tumors are so small that in an unfavorable location they could escape even the most sophisticated current methods of detection. TM Finally, in at least one case initially reported as idiopathic, a tumor has been discovered during more extended observation. 355 But in one case encountered by the author, a tumor has still not been found nearly 50 years after the onset of symptoms, so that an occult tumor must not only be very small but very slowly growing. The issue will likely not be settled until the pathophysiology is better understood. The interval between onset of symptoms and diagnosis in published cases has averaged about 5 years, with a range of 4 months to 19 years, and has not declined significantly in recent years. 35~ The onset can be at any age but in most cases is between 20 and 50 years. In less than 10% of cases the symptoms begin before age 10 or after age 70, the former presenting as rickets rather than as osteomalacia. 356 Unlike adults with hereditary hypophosphatemia, most patients have bone pain, muscle weakness, and difficulty in walking and many have Looser's zones. In contrast to hereditary hypophosphatemia, ligamentous ossification does not occur, and instead of axial osteosclerosis, there is often severe vertebral osteopenia with multiple compressions, loss of trunk height, and kyphosis, usually beginning 1 to 2 years after the other symptoms and progressing r a p i d l y . 348'349 Osteopenia is often generalized, with pronounced cortical thinning; multiple fractures, especially of the femoral necks, are common. Due to the severity of the disease and the frequent delay in diagnosis, many of these patients in the past developed bizarre and crippling deformities including multiple angulations of the long bones, scoliosis as well as kyphosis, and pigeon-chest due to fracture of the sternum. 4'6a Close to half of the patients, with or without tumor, have been bedridden before effective treatment w a s begun. 62'139 The biochemical features are similar to those of hereditary hypophosphatemia, but malabsorption of calcium is more severe and the plasma phosphate level is lower. The mean value was 1.64 mg/dl in 27 nontumorous cases tabulated by Fanconi 349 and 1.52 mg/dl in 41 tumorous cases tabulated by Ryan and Reiss, 139 whereas in adults with hereditary hypophosphatemia, the plasma phosphate is usually above 2.0 mg/dl. Renal tubular responsiveness to exogenous PTH was blunted in one tumoral c a s e 357 but exaggerated in one nontumoral c a s e , 353 in keeping with the reported effect of parathyroidectomy, 358 but the data are too fragmentary to conclude that there is a difference in pathogenesis. Plasma
CHARTER 11 Osteomalacia and Related Disorders calcitriol levels are uniformly low in the tumoral c a s e s , 139'35~ but have not been studied systematically in the nontumoral cases; in neither group does calcitriol administration consistently correct the defect in tubular reabsorption o f p h o s p h a t e , 139'35~ probably because this requires maintenance of a supraphysiological plasma level. Urinary calcium excretion is either normal or sometimes increased, presumably owing to increased net bone resorption as a result of phosphate depletion. 348 Urinary excretion of glycine is often increased but other amino acids are normal, although a few cases manifest the Fanconi syndrome with generalized aminoaciduria and glucosuria with or without hyperchloremic acidosis. 139'359 Histologically, the bone is similar to severe vitamin D deficiency but, as in hereditary hypophosphatemia, osteoclastic resorption is usually less p r o m i n e n t 4'351'357'36~ (Fig. 11-15). But in one case, resorption of osteoid was observed, indicating profound stimulation of osteoclast recruitment and activity. 361 Resection of a tumor when present is followed within a few days or weeks by a rise in low levels of plasma phosphate and calcitriol to normal, and by rapid improvement and eventual disappearance of symptoms and healing of Looser's zones. Return of normal mineralization and healing of the osteomalacia has been confirmed histologically in a few c a s e s 36~ (Fig. 11-15). The tumors presumably secrete one or more humoral agents that impair both phosphate reabsorption and l oLhydroxylation; in vitro confirmation of this presumption has been accomplished in different w a y s , 334'362"363 but the identity of the agent is still unknown. The tumors are mesenchymal but of variable origin in soft tissue or bone and with variable histological features, often diagnosed as hemangiopericytoma. Almost all have the two main features of extreme vascularity and large numbers of spindle cells and multinucleated giant ce11s139'364'365; about 10% of cases are malignant. 366 Osteomalacia (or tickets) that is clinically, biochemically, radiographically, and histologically very similar (except for earlier age of onset) occurs in some patients with fibrous dysplasia of bone (13 c a s e s ) , 367'368 neurofibromatosis (12 cases)369'37~ and linear sebaceous nevus syndrome (4 c a s e s ) . TM In these disorders also, a humoral origin is likely, but is in most cases impossible to prove, since the extent and multiplicity of lesions preclude surgical cure. However, partial excision of fibrous dysplasia in one case led to significant clinical and biochemical improvement. 367 If a tumor is not found, treatment with some form of vitamin D in pharmacological dose, together with supplemental phosphate and calcium, will lead to symptomatic relief and biochemical and radiographic improvement. 348 By analogy, with hereditary hypophosphatemia calcitriol should be superior to calciferol and has been very effective in tumoral c a s e s . 139'372 Only a moderate
365 response was noted initially with short-term administration in nontumoral c a s e s , 351'352 but an excellent long-term response was recently observed. 373 As in XLH, hypercalcemic hyperparathyroidism develops with unusual frequency during treatment, 374'375 and is probably analogous to tertiary hyperparathyroidism with longstanding vitamin D depletion. These issues are discussed more fully in Section VIII. A final peculiarity of idiopathic nonhereditary hypophosphatemia is that in at least two cases, spontaneous complete recovery has occurred, allowing treatment to be w i t h d r a w n . 36~ Osteomalacia has also been reported in a few patients with carcinoma of the prostate. 35~176 The plasma calcitriol level has been low in the three cases measured, 378'379 and the biochemical syndrome has been induced in nude mice transplanted with tumor tissue from affected patients, TM but there are several differences from the usual form of oncogenous osteomalacia just described. First, with one exception 377 osteomalacia has occurred in patients already known to have widespread osteoblastic metastases, with a total tumor burden that is much larger than in patients with a benign mesenchymal tumor. Second, hyperosteoidosis is due at least in part to stimulation of new woven bone by tumor c e l l s 38~ and is found also in patients with osteosclerosis for other reasons such as thorotrast toxicity 383 or myeloid metaplasia. T M Third, affected patients are usually hypocalcemic as well as hypophosphatemic, 382 the abnormalities being of similar severity to those in patients who have osteoblastic metastases without osteomalacia. 47 Finally, many patients with this form of osteomalacia lack clinical effects from it, with no proximal muscle weakness, bone pain no worse than can be accounted for by the osseous metastases, and no Looser's zones or other radiographic features of osteomalacia. However, two patients treated with vitamin D 377 o r calcitrio1379 derived substantial relief of bone pain.
D. F a n c o n i S y n d r o m e For the most part this is a childhood disorder, 1'2'141 although Wilson's disease occasionally presents with osteomalacia without preceding rickets. 385 Onset in adult life does not rule out a genetic basis, probably always autosomal dominant, 386'387 but most cases are sporadic. Some are due to a known cause of renal tubular damage such as industrial or environmental cadmium intoxication 4'5'65'335'388'389 o r light-chain proteinuria. 14~ Fanconi syndrome due to external toxicity other than cadmium rarely causes osteomalacia, presumably because the effects are usually reversible, 39~ but can cause rickets. TM Some cases are tumor induced, as mentioned earlier, but many are diagnosed by exclusion as idiopathic. 39e'393
366
A.M. PARFITT
There is usually a close clinical resemblance to nonhereditary hypophosphatemia without the Fanconi syndrome, although osteopenia is less prominent and the manifestations are less severe if bone biopsy is used to facilitate early diagnosis. 14~ Calcitriol levels when measured have usually been lOW, 141'394'395 except in one case of light-chain nephropathy, 14~probably because the defect in the proximal tubule occurs at a site of calcitriol synthesisS; experimental induction of the Fanconi syndrome by administration of maleic acid also impairs l oL-hydroxylation. 396 Consistent with calcitriol deficiency, calcium absorption is impaired, 397 except in Wilson's disease 398 and cadmium poisoning, 335 in which for unknown reasons it can be increased. Urinary calcium excretion is often high (Table 1 1-9); this sometimes results from associated proximal renal tubular acidosis and is correctable by alkali administration, 397 sometimes from phosphate depletion, 399'4~176 and sometimes from a separate defect in tubular reabsorption of calcium. Osteomalacia in the adult Fanconi syndrome responds well to treatment with oral phosphate either a l o n e , 399'4~176 o r combined with calcitriol if the plasma level is l o w . 14~ In the autosomal dominant form there may be a greater need for alkali as well, 2'387 which also helps to preserve renal function. 388
E. Renal Tubular Acidosis and Ureteral Diversion These two conditions have in common chronic metabolic acidosis in which the low plasma bicarbonate is balanced by an increase in chloride rather than in normally unmeasured anions such as lactate. Hyperchloremic acidosis also occurs with carbonic anhydrase administration and with congenital absence of carbonic anhydrase, 4~ but these conditions have not been shown to cause osteomalacia. Renal tubular acidosis (RTA) is conveniently classified as proximal, in which bicarbonate reabsorption is reduced and distal, in which the urine cannot be maximally acidified. 4~ Proximal RTA is usually a component of the Fanconi syndrome (see Section VII.D). Lone proximal RTA with rare exceptions occur only in infants, with spontaneous recovery after a few years, and does not cause rickets. 4 The concurrence of lone proximal RTA and osteomalacia 4~ is usually a consequence of vitamin D depletion and secondary hyperparathyroidism. 99 In a possible exception to this rule, 4~ the Fanconi syndrome was not adequately excluded. Adult-onset distal RTA in most cases is a complication of Sjrgren's syndrome or other cause of hyperglobulinemia. 4~176176 The major effects of RTA are potassium depletion, nephrolithiasis, and nephrocalcin o s i s , 1'2'141'4~ their severity varying with the dietary acid
load. 20steomalacia was frequent in the past, 4~ although rarely verified histologically. 332'4~176176 Overt osteomalacia is n o w uncommon, 4~176 most likely because treatment is started earlier as a result of routine biochemical screening 4~ and is more likely to require bone biopsy for diagnosis 4~ (Fig. 1 1-15). It closely resembles vitamin D-related osteomalacia 1'2 including the presence of proximal muscle weakness, 411 and secondary hyperparathyroidism. 412'413 In one case the latter caused bone disease from osteitis fibrosa rather than from osteomalacia, 414 as sometimes happens in gluten enteropathy (see Section II.C), but hyperparathyroid effects usually are less severe than in HVO. 1 In the related condition of ureteral diversion, hyperchloremic acidosis is the result of preferential reabsorption of chloride and/or hydrogen ions from urine in contact with colonic or ileal epithelium. After ureterosigmoidostomy, histologically verified osteomalacia has developed in 5 to 18 years, 4~ but with ileal replacement of ureters, osteomalacia has been manifest clinically in 2 years and histologically (in the absence of symptoms) in 6 months. 419 This form of osteomalacia is more common in the U.K. than in the U.S., and in several cases there has been marginal vitamin D deficiency. 417,418 In both forms of hyperchloremic acidosis, the pathogenesis of osteomalacia is obscure. Despite the combined effects of hypokalemia, acidemia, and hyperparathyroidism, which all independently reduce tubular reabsorption of phosphate, mean plasma phosphate is only slightly lower than in vitamin D depletion I and plasma calcium is normal, so that the relevant formation product is probably not as low. Hypophosphatemia is significant only in an occasional patient with osteomalacia due to ureteral diversion. 416 Ammonium chloride administration increases urinary calcium excretion with little change in plasma calcium by simultaneously increasing net bone resorption and decreasing tubular reabsorption of calcium, but PTH secretion is unaffected so that this is an imperfect model for R T A . 42~ Furthermore, absolute hypercalciuria is uncommon in RTA. 332 Contrary to the predictions from animal experiments, metabolic acidemia does not impair calcitriol synthesis in humans 42~ unless they have renal failure, 422 and calcitriol levels are normal in both RTA 141 and ureteral di404 418 version ' unless there is significant renal insufficiency. 417 The usual malabsorption of calcium in RTA is not explained by calcitriol deficiency, but may be another effect of acidemia or an indirect consequence of retarded growth. 5 Acidemia probably impairs minerali5 404 zation directly,' either by lowering trivalent p 043- disproportionately, 423 or by compromising the removal of hydrogen ions from sites of mineral deposition. But several paradoxes remain; nonhyperchloremic acidosis, as in glycogen storage disease, does not impair bone min-
CHAPTER 11 Osteomalacia and Related Disorders eralization, and in one family with isolated proximal RTA, hyperchloremic acidosis of similar severity to distal RTA was unaccompanied by any disorder of bone mineral metabolism. 424 Whatever the explanation, administration of alkali 1.5 to 2.5 mEq/kg body weight per day will usually raise plasma phosphate and heal the osteomalacia both in R T A 425-427 and in ureteral divers i o n 416'418'419 s o that vitamin D is only needed in severe cases, or when delay in diagnosis has resulted in significant renal failure from nephrocalcinosis. 2
VII. OSTEOMALACIA NORMAL
VITAMIN
WITH
D AND
PHOSPHATE M E T A B O L I S M A. I n h i b i t o r s o f M i n e r a l i z a t i o n Impaired mineralization and osteomalacia can be produced by sodium etidronate used in the treatment of Paget's disease, sodium fluoride used in the treatment of osteoporosis, and aluminum from a variety of sources in patients on maintenance hemodialysis. Aluminum accumulation also contributes to bone disease in patients receiving total parenteral nutrition, 428 and a similar mechanism could be involved in thorium-related osteomalacia resulting from thorotrast exposure. 383 In all varieties of toxic osteomalacia there is a high prevalence of both atypical and focal forms as defined in Section I.G. Indeed, with the exceptions of pseudohypoparathyroidism 133 and hypophosphatasia, 429 focal osteomalacia is observed only with inhibitors of mineralization. Sodium etidronate blocks mineralization in vitro and probably binds to the crystal surface of newly deposited mineral in 1;ivo. 43~ Osteoid accumulation and reduced tetracycline uptake were first observed with the use of etidronate in the treatment of osteoporosis, 431 but were regularly seen in the treatment of Paget's disease in a dose of 10 mg/kg/day or more, associated with increasing bone pain and increased fracture r i s k . 432'433 The mineralization defect develops earlier and is more severe in pagetic than in normal bone, 9 presumably because of the difference in t u r n o v e r . 58'433 In the largest series with individual data reported, osteomalacia was generalized in 1 1, focal in 5, and atypical in 4. 432 Although a dose of 5 mg/kg/day is generally considered to be s a f e , 433'434 focal osteomalacia is quite common after treatment with this dose for 6 months or longer 36 (Fig. 1 1-16), and in one case there was generalized osteomalacia presenting with a pathological fracture. 435 Even infrequent cyclical administration has led to atypical osteomalacia. 436 The high frequency of focal osteomalacia presumably results from the depression of remodeling activation by etidron-
367 ate leading to reduced bone turnover before the emergence of a significant mineralization defect, but the occurrence of atypical osteomalacia is less easy to explain. Etidronate-induced osteomalacia is reversible, but the excess osteoid may not begin to mineralize for 3 to 6 months after the drug is discontinued. 58 Newer bisphosphonates are much less potent inhibitors of mineralization and do not cause osteomalacia, but may induce secondary hyperparathyroidism. 437 In patients with osteoporosis treated with sodium fluoride, significant increases in the surface extent, thickness, and volume of unmineralized osteoid have usually been observed, but initially they result from stimulation of matrix synthesis rather than from impaired mineralization. 438'439 Histological evidence of osteomalacia was found in 8 of 14 patients after 2 years of treatment but was clinically manifest only in o n e . 438 Impaired mineralization can occur as early as 6 months but may improve spontaneously during continued administration. 439 Symptomatic osteomalacia attributed to fluoride treatment has been reported only in a very few individual cases, 44~ probably because osteoid is usually added to mineralized bone (appositional osteomalacia) rather than replacing it (substitutional osteomalacia), so that structural failure is less likely. 58 Osteomalacia is also found in some patients after prolonged treatment with niflumic acid, a fluorine containing antiinflammatory agent. 442 We have observed osteomalacia in ten cases, of which six were generalized, three focal and one atypical (Fig. 11-16). Symptoms were present only in one patient with focal osteomalacia who had a painful stress fracture of a pubic ramus resembling a Looser's zone. The available data suggest that sodium fluoride in high doses significantly impairs mineralization in some patients. The defect is not prevented by physiological levels of vitamin D or related to abnormal vitamin D metabolism; the value of pharmacological doses of vitamin D 443 o r its metabolites 439 is unclear. Lack of substrate for mineralization may contribute to the defect, which may or may not be associated with secondary hyperparathyroidism. 439 Probably, there is both a physicochemical effect of fluoride at the mineralization front and a direct toxic effect on osteoblasts. Since trabecular thickness increases more in patients who develop osteomalacia than in those who do not (Kleerekoper and Parfitt, unpublished data), osteomalacia could be a stage in the evolution of a successful therapeutic response rather than a harmful side effect, 439 but may contribute to an increased fracture rate in the first year of fluoride treatment, especially if the dose is too high. Disabling osteomalacia with multiple spontaneous fractures, sometimes including the sternum (Fig. 11-12), was frequent during the 1980s among patients on maintenance hemodialysis, 81'444 especially after parathyroid-
3
6
8
A !I !
50
E
I I
v::3.
. MIt
100 d I
I
to 40 to
M. PARFITT
I ~
./
/
/
t-o t--
30-
/
E
/
I
I
\
\
\
."
/
."
.I
/
/
9
-o 20-
.,
0
10-
] -',7, ._•.,_.., 0
' 2'0 ' 4'0 ' 6 ' 0 ' g 0 ' 1 6 0 OS/BS (%)
6
B
0'.1
0'.2
0'.3
Aj .AR (pm/d)
0:4
0'.5
FIGURE 1 1--16
Osteoid thickness relationships in drug-induced osteomalacia. Layout as in Figures 1 1 - 8 and 1 1 - 1 5 with static measurements on left (A) and kinetic measurements on right (B). Data are from three patients with Paget's disease treated with etidronate 5 mg/kg body weight for 12 to 20 months giving rise to focal osteomalacia (single closed triangles), and 10 patients with osteoporosis treated with sodium fluoride in various regimens, with pretreatment values (open symbols) and posttreatment values (closed symbols) joined. Osteomalacia was focal in three cases (triangles), atypical in one case (squares), and generalized in six cases (circles). Note that complete arrest of mineralization was observed in all three etidronate-treated patients but in only 1 of 10 fluoride-treated patients.
ectomy. 445 The prevalence declined with better control of aluminum exposure, but too much aluminum in municipal water can cause osteomalacia even in undialyzed patients. 446 Both atypical osteomalacia, referred to in this context as aplastic 81 or type 11,444 and focal osteomalacia, 447 are common. Some patients have low-turnover nonosteomalacic osteopenia, 448 which occurs also with relative hypoparathyroidism in the absence of aluminum. 449 The different histological patterns reflect varying severity of prior hyperparathyroid bone disease, and of independent effects of aluminum and other factors to inhibit bone matrix synthesis and mineralization 9450 In patients with generalized osteomalacia, the bone formation rate is no lower than in the osteomalacia of vitamin D depletion, but there is a closer resemblance to hypophosphatemic osteomalacia. The morphological expression of hyperparathyroidism is less apparent and the mineralization defect is more severe, with osteoid seams that are relatively thicker (Fig. 1 1-17), and tetracycline uptake more often completely absent. 451 Aluminum inhibits mineralization both in vitro 452 and in v i v o 453 and dialysis osteomalacia is undoubtedly associated with aluminum deposition at the bone interface, 444-45~ and can develop in less than 1 year after an abrupt increase in water aluminum concentration. 453 But aluminum in the cement line, which is the initial location of the bone interface, frequently does not block mineralization in patients with osteitis fibrosa, 45~ and mineralization resumes after renal transplantation 456 or after deferoxamine treatment 57 despite persistence of stainable aluminum at the cement line. Furthermore,
nonuremic vitamin D-deficient animals given aluminum accumulate it in bone without impairing the healing response to vitamin D. 457'458 Clearly other factors must be involved in the pathogenesis of aluminum-related osteomalacia. One such factor could be the plasma level of citrate, which complexes aluminum to form a more potent inhibitor of mineralization than any other aluminum salt. 459 Another could be direct effects of aluminum on the function of osteoblasts, in which cells aluminum can be demonstrated within mitochondria. 45~
B. Inability of Matrix to Mineralize Although abnormal matrix maturation may contribute to osteomalacia in vitamin D depletion, osteomalacia due to defective bone matrix alone in the absence of any disorder of bone mineral or vitamin D metabolism has been established only in fibrogenesis imperfecta ossium. 46~ In this rare condition, normal lamellar bone is replaced by matrix with no birefringence in polarized light and no fiber pattem detectable by light microscopy, but which by electron microscopy consists of a tangled mass of very thin, short, and irregularly curved fibers. 461 Mineralization of the abnormal matrix is delayed and retarded, but because the protein content of the matrix is reduced by as much as 75%, mineral density ultimately becomes higher than normal. 46~ Nevertheless, bone surfaces are covered by thick and extensive layers of unmineralized matrix, the ultrastructural appearance of the mineralization front is abnormal, 462 and conform-
CHAPTER 11 Osteomalaciaand Related Disorders
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FIGURE 1 1 - - 1 7 Osteoid thickness relationships in aluminum-related osteomalacia occurring during maintenance hemodialysis. Layout as in Figures 1 1 - 8 , 11-15, and 11-16, with static measurements on left (A) and kinetic measurements on right (B). Data are from 46 patients, 15 with osteitis fibrosa (C)), 25 with generalized osteomalacia (O), 1 with focal osteomalacia (A), and 5 with aplastic bone disease (11); these represent an early stage of atypical osteomalacia with OV/BV between 5% and 10%. Note that there is considerable overlap in patients with and without osteomalacia by the static measurements, but clear demarcation by the kinetic measurements.
ity to the kinetic criteria for generalized osteomalacia has been demonstrated by double tetracycline labeling. 463 The condition most often presents in the fifth or sixth decade with intractable pain and multiple fractures, the patients often becoming unable to walk and dying within a few y e a r s . 464 The radiographic appearances are characteristic but can be confused with Paget's d i s e a s e . 463'465 Since there is always some normal bone, the matrix defect must begin in adult life, probably only a few years before the onset of symptoms. 46~In one case, histological and biochemical indices of bone resorption were increased, but there was no therapeutic response to calcitonin. 462 There is a high frequency of monoclonal gammopathy, and in one case with kappa light chains in serum and urine, complete clinical remission and resumption of normal bone formation and mineralization were achieved by combined treatment with cyclical melphalan and prednisolone. 466 A possible explanation for this extraordinary observation, which has far-reaching implications for the cell biology of bone, is that cytotoxic therapy eliminated a population of abnormal osteoblast precursors.
C. Disorders of Alkaline Phosphatase The role of alkaline phosphatase in mineralization has been discussed for many years and is still unclear, but osteomalacia occurs in hypophosphatasia, a genetic defect in the synthesis of alkaline phosphatase 467 and in so-
called axial osteomalacia, in which morphologically inactive osteoblasts stain with paradoxical intensity for alkaline phosphatase. 468 Hypophosphatasia is usually an autosomal recessive disorder that presents in infancy with rickets, raised intracranial pressure, hypercalcemia, nephrocalcinosis, and early death; in mild cases the onset is in later childhood, and spontaneous radiographic improvement c a n o c c u r . 467 There is partial and variable deficiency (10% to 30% of normal) of the bone/liver/kidney isoenzyme in cultured skin fibroblasts, 469 similar reductions of the bone-specific component of serum alkaline phosphatase, 47~and normal intestinal and placental isoenzymes. The plasma level and urinary excretion of phosphorylethanolamine are increased, most likely as a consequence of altered hepatic metabolism. But more diagnostically specific is an increased plasma level of pyridoxal 5'-phosphate. 467 The plasma level and urinary excretion of inorganic pyrophosphate are also increased; alkaline phosphatase functions as a pyrophosphatase in bone, and failure to hydrolyze pyrophosphate in matrix vesicles could account for the mineralization defect, which persists when epiphyseal cartilage from affected subjects is incubated in vitro in solutions that calcify normal cartilage. 467 There is no evidence for any abnormality in vitamin D metabolism. 471 Adult hypophosphatasia is much commoner than previously suspected and is characterized by early loss of permanent teeth; calcium pyrophosphate arthropathy; osteopenia and increased risk of stress fractures; and os467
370 teomalacia of variable severity, usually atypical, less commonly generalized, and occasionally f o c a l . 57'472 Alkaline phosphatase staining in osteoblasts is of markedly reduced intensity, and its surface extent is inversely correlated with the amount of osteoid. 472 Most patients with abnormal bone histology are asymptomatic. The same biochemical abnormalities are found in blood and urine as in the childhood forms. In some cases there is a history of tickets in childhood and the presence of deformities common in the childhood form such as prominent sternum and loss of normal curvature of the thoracic spine, 473 but in several large kindreds with autosomal dominant transmission the onset of clinical effects was clearly in adult l i f e . 467'472 The clinical expression of the biochemical defect varies considerably both within and between families. The term "axial osteomalacia" is applied to a disorder in which trabecular bone of the axial skeleton is of increased radiodensity with an irregular, coarsened, and sponge-like appearance. 474 In the most recent case, very high spinal bone density was confirmed by dual energy x-ray absorptiometry (DEXA). 475 Pain in affected bone is mild or absent, and fractures do not occur. Histologically, the trabecular plates are irregularly thickened and closely packed, with increased surface extent and width of osteoid and reduced extent and separation of tetracycline uptake, but both mineralized and unmineralized bone is of lamellar structure with normal birefringence. 1~ The mineralization defect may be f o c a l . 475 The appendicular skeleton is radiographically normal; in one case osteoid was abundant in the ilium and absent in a tibial malleolus, providing the first direct evidence that the mineralization defect is confined to the axial skeleton. 476 The disorder is sometimes classified with fibrogenesis imperfecta ossium as a matrix defect, but supporting evidence is lacking. The serum alkaline phosphatase has been raised only in 3 of 13 published cases, two from one family, all other cases being sporadic. 468 The only clue to pathogenesis is the increased alkaline phosphatase content of osteoblasts, which is disproportionate to the increase in serum level and inconsistent with other indices of osteoblast activity. A defect either in the release of alkaline phosphatase at sites of bone formation or in its biological effectiveness could account for impaired mineralization, but would not explain the osteosclerosis.
VIII. THERAPEUTIC INTERVENTION IN O S T E o M A L A C I A There is an important difference between the two main etiological categories. Vitamin D-related osteomalacia occur for the most part only in persons already
A.M. PARFITT known to be at risk and so in principle is largely preventable. By contrast, phosphate-related osteomalacia is usually less predictable, so that prevention is less often possible. Occasional measurement of urinary phosphate in patients on long-term antacid therapy would prevent phosphate depletion, and appropriate family surveillance might forestall symptoms in some genetic disorders. It is reasonable to continue treatment in familial hypophosphatemia after cessation of growth to minimize adult complications. 336 Routine multichannel biochemical screening probably leads to treatment of renal tubular acidosis before the onset of osteomalacia in most cases, but accidentally discovered hypophosphatemia is usually either transient or mild and of uncertain significance. 4'478 With these few exceptions, prevention can be usefully discussed only in relation to vitamin D.
A. Prevention of HVO The prevention of intrinsic vitamin D depletion is primarily an issue of public health rather than of individual patient care, but many studies carried out with the object of influencing public policy have been poorly designed. 2~ Attempts to eradicate migrant osteomalacia in the U.K. have included provision of free vitamin D tablets and various types of education, but neither measure has been convincingly demonstrated to improve health. 2~ Any policy that requires ostensibly healthy Asian adults to permanently change their habits with respect to diet or sun exposure or to take daily medication indefinitely will inevitably fail. More realistic is to give a single oral dose of 2.5 mg of calciferol annually just before winter, an effective and safe method of maintaining adequate plasma calcidiol levels in Asians throughout the y e a r . 479 Compliance with medical advice is likely to be better during pregnancy and 25 Ixg of calciferol daily during the last trimester would improve fetal growth and ossification and minimize the occurrence of neonatal hypocalcemia as well as protecting the mother. 48~ The same principles apply to prevention in the elderly. Very few will take a weekly serving of sardines or an annual vacation by the Mediterranean because of medical advice. Therapeutic ultraviolet irradiation is both physiological and effective, but it is also cumbersome, expensive, and potentially hazardous. ~13'481An oral vitamin D supplement of 20 Ixg/day given to old people at varying levels of risk in Ireland maintained satisfactory plasma levels of calcidiol, corrected any biochemical abnormalities indicative of subclinical HVO, and appeared to be entirely safe. 482 Unfortunately, the preparation used also supplied 60,000 IU/day of vitamin A, the long-term safety of which has not been rigorously established. Nevertheless, the observation supports the
CHAPTER 11 Osteomalaciaand Related Disorders proposal that the RDA for vitamin D in the U.S. should be increased to 15 to 20 Ixg/day in the elderly ~3 and should be heeded by those responsible for their nutritional needs. It should also encourage the governments of Northern European countries to adopt vitamin D fortification, at least to the level practiced in the U.S. and Canada, and preferably extended to a wider range of foods. There is still no satisfactory pharmaceutical preparation for giving a physiological daily dose of vitamin D to adults, w6 Prevention of HVO in patients with gastrointestinal or hepatobiliary disease or receiving anticonvulsant therapy depends on medical awareness of the risk in these populations. The best policy has not yet been determined, but to be successful, it should be applied before serum alkaline phosphatase has increased or cortical osteopenia has developed. The degree of vitamin D depletion varies widely between patients, and no single oral dose is uniformly safe and effective in prophylaxis. Absorption from intramuscular injections of vitamin D dissolved in oil is unpredictable, 483 and a water-soluble preparation that can be given intravenously is not generally available, w6 Individual monitoring is unavoidable and a reasonable policy is to give a capsule (not tablet) of 1.25 mg of ergocalciferol at a frequency that maintains plasma calcidiol between 20 and 40 ng/ml, although higher plasma levels will be needed in some patients with intestinal resection or refractory sprue. For most patients the requisite frequency will lie somewhere between once a month and once a day. An amount of supplemental calcium should be added, such that the combination maintains urinary calcium excretion between 100 and 250 mg/24 hours (or calcium/creatinine ratio between 0.1 and 0.2 mg/mg) and plasma calcium, alkaline phosphatase, and the best available index of parathyroid function within normal limits. With these guidelines, some patients will be treated unnecessarily, in the sense that they would never have developed symptoms even if nothing was done, but many patients will benefit and none will be harmed.
B. T r e a t m e n t o f H V O The aims of treatment are to relieve symptoms, restore bone strength by promoting mineralization of osteoid, and preserve mineralized bone by correcting secondary hyperparathyroidism. It is important that all three aims are explained to the patient and that the first is not permitted to overshadow the second and third. In extrinsic vitamin D depletion these aims are best served by giving a modest dose of vitamin D (25 to 50 Ixg/day), together with adequate supplemental calcium. Urgent treatment of hypocalcemia is occasionally required in
371 patients with osteomalacia. The responses to treatment and time course of healing will be described first, followed by some practical aspects of management and modifications of the regimen needed for other causes of HVO. 1. RESPONSE TO VITAMIN D
Giving a physiological dose of vitamin D to a patient with extrinsic vitamin D depletion produces a characteristic series of changes. 1'2'6'1~ The plasma calcitriol level begins to rise immediately and quickly reaches supranormal levels, intestinal calcium and phosphate absorption promptly increase, plasma calcium (if low) returns to normal in 1 to 4 weeks, PTH secretion falls rapidly but not to normal, and plasma phosphate rises to normal in 4 to 8 days and then to above normal for several months. Osteoclast function improves, and there is a rapid increase in urinary hydroxyproline. 7~Osteoblast function improves and mineralization resumes rapidly, probably within a week, plasma alkaline phosphatase increases further, calcium and phosphate are rapidly deposited in bone and external balance becomes positive usually by the second 4-day period, with low fecal as well as urinary calcium excretion. 6'41 Symptoms improve soon after and a previously bedridden patient may become able to walk without pain in a few weeks, but restoration of normal muscle power 44 and normal parathyroid function 85 can take up to 2 years or even longer, and it is important that the asymptomatic patient not default from follow-up. Osteoid indices and cortical porosity eventually return to normal, accompanied by large increases in vertebral and hip bone mineral density,10 but loss of cortical thickness is permanent. 57 Some of these changes will now be described in more detail. 2. EFFECT ON BONE MINERAL METABOLISM
When their substrate is depleted, both 25- and l cthydroxylase enzymes are maximally active and vitamin D is rapidly and almost quantitatively converted to calcitriol, so that the doses of calciferol and calcitriol needed for most rapid healing are almost the same, despite the 1000-fold difference in doses in other circumstances, such as the treatment of hypoparathyroidism. 484 Plasma calcidiol may not begin to rise for several weeks, but plasma calcitriol reaches three to five times the nor157 158 mal level and can remain elevated for many months, ' sustained by continued hypersecretion of PTH despite restoration of normocalcemia 85'86 and probably also by continued low blood levels of calciferol and calcidiol. Plasma phosphate rises by about 1.0 mg/dl to reach a normal value in the first week with a transient fall in urinary phosphate indicating increased tubular reabsorption, ~'4 and continues to rise to a peak value about 1.0 mg/dl higher than normal by 3 to 4 weeks, and then falls
3
7
2
A
slowly for the next 6 m o n t h s . 41 Although both the rise in plasma calcium and the initial fall in PTH may contribute to the early rise in plasma phosphate, the later increase in tubular reabsorption of phosphate above normal is most likely the result of continued elevation of plasma calcitriol acting directly on the kidney. 484 3. EFFECT ON BONE MINERALIZATION AND REMODELING
Tetracycline-based histomorphometry during the healing of osteomalacia is limited, but serial measurement of the extent of toluidine blue stainable mineralization front supports the inference from balance studies that substantial mineral deposition begins within a week of starting treatment. 166'485'486 However, it is unlikely that normal mineralization is immediately restored over the entire osteoid surface, since this would require calcium retention of 2.0 to 3.0 g/day rather than the 0.5 to 1.0 g/day usually found. Furthermore, even the thickest osteoid seam should mineralize completely within 3 months, but mean seam thickness can take much longer to return completely to normal. The most likely explanation for the delay is that mineralization can begin immediately only where osteoblasts are still present. 35'165-167 Elsewhere, the flat cells coveting the osteoid surface must reacquire osteoblast function and morphological features, and additional osteoblasts must be recruited to maintain coverage of the surface, processes that probably continue for several months and are responsible for the further increase in serum alkaline phosphatase that begins soon after starting treatment. At some of the thickest seams mineralization may resume closer to the surface than the bone interface, leaving a layer of osteoid that remains permanently unmineralized as a residual "scar."35 In biopsies taken at varying times between 3 and 12 months after treatment was begun, mean values for mineral apposition rate were about 30% above normal and mean values for bone formation rate about five times greater than normal (Teotia and Parfitt, unpublished data). It is likely that osteomalacia heals by reversing its sequence of development, so that a patient in stage iii at the onset of treatment would pass successively through stage ii (return of double labels, but seam thickness still increased) and stage i (seam thickness normal, but formation rates still high) before returning completely to normal. This histological evolution is accompanied by a gradual fall in alkaline phosphatase and, when remineralization is almost complete, by a rise in urinary calcium excretion. With the increase in mineralized bone surface and continued hyperparathyroidism there is an increase in absolute surface extent and number of osteoclasts, and a decline in total bone matrix volume, even though mineralized bone volume is increasing. Bone turnover prob-
.
M. PARFITT
ably remains elevated until the completion of parathy102 roid involution, which is a very slow process. 4. PRACTICAL ASPECTS OF MANAGEMENT The ability of 1et-hydroxylated metabolites to cure the osteomalacia of extrinsic vitamin D depletion 4'5'1~is important to the understanding of physiology and pathogenesis, but it is simpler and safer to give the precursor and rely on the regulated endogenous production of calcitriol which cannot lead to overtreatment and vitamin D intoxication. 484 Even in patients with age-related decline in renal function and l oL-hydroxylase activity, no advantage over calciferol has been demonstrated. 481 The dose of vitamin D is not critical, healing can be initiated with only 2 to 3 Ixg/day but will proceed more rapidly with a larger d o s e . 196'485 Calcium conservation is very efficient during the early stages of recovery,6 but supplemental calcium 1.0 to 2.0 g/day will restore mineralized bone and suppress parathyroid hypersecretion more quickly 488 and make it less likely that replenishment of the axial skeleton will occur at the expense of the appendicular skeleton. 13 The most impressive calcium retention has been achieved with microcrystalline hydroxyapatite, which provides all the required minerals in the correct proportion, 41'484 but this preparation is not currently available in the U.S. and any form of calcium is satisfactory. Cortical bone that was lost during the development of the disease cannot be replaced, 51 and with the resumption of normal physical activity the patient should be advised how to minimize the chance of falling. The treatment of osteomalacia due to intrinsic vitamin D depletion follows the same general principles, but differs in several points of detail. First, as for prevention, the dose of vitamin D varies over a wide range and is unpredictable. Second, although vitamin D itself is the cheapest and safest compound for prevention, and for the correction of extrinsic depletion, calcidiol has several potential advantages in patients with intestinal or hepatobiliary disease. It is more precisely formulated, 119 better absorbed, 22~bypasses any defect in 25-hydroxylation, leads to more predictable blood levels that can be monitored directly, 4'5'316 and is more rapid in onset and offset of its effects, so that the response to a change in dose can be detected more rapidly, 119'484but retains physiological regulation of 1-hydroxylation. Third, whatever compound is used, the dose requirement will be greatly affected by the response of the underlying disease to treatment, 4'5's and will (for example) show a larger and more rapid decline during the successful use of a glutenfree diet in celiac disease than during the attempted correction of pancreatic enzyme deficiency. Finally, because the dosage is both higher and varies more with time, more frequent and careful monitoring of the therapeutic response is required to avoid vitamin D intoxication. 47'196
CHAPTER 11 Osteomalaciaand Related Disorders This should be on the same lines as previously recommended for prevention, but with particular attention to the approach of plasma alkaline phosphatase to normal and a rise in urinary calcium excretion, which may both give warning of a need to reduce the dose. A moderate increase in plasma creatinine during healing may reflect a change in muscle metabolism rather than a decline in renal function, and so is not by itself an indication for any change in treatment. 489
C. Treatment of Hypophosphatemic Osteomalacia XLH, nonhereditary hypophosphatemia, and the Fanconi syndrome differ in many ways, but they have in common a primary (non-PTH-dependent) defect in tubular phosphate reabsorption and either relative or absolute impairment of calcitriol synthesis. In the absence of a tumor or of some reversible cause of renal tubular damage, 386 optimum treatment of all three conditions is based on some combination of supplemental phosphate and calcitriol. 8- ~o 24-hydroxy calcidiol may contribute to the control of phosphate-induced secondary hyperparathyroidism, 49~ and nonhypercalcemic analogues of calcitriol may prove to be advantageous. 49~ Otherwise the only medications that may be needed are supplemental calcium in severe nonhereditary hypophosphatemia, and alkali in renal tubular acidosis, primary or secondary, as was previously indicated.
373 The principal benefit of supplemental phosphate is direct enhancement of bone mineral deposition, but there can be additional benefits unrelated to a rise in plasma level. In patients with defective urinary acidification as well as impaired phosphate reabsorption, metabolic acidosis is improved by provision both of alkali and of additional urinary buffer to facilitate hydrogen ion excretion. 495 Also, phosphate supplements increase tubular reabsorption of calcium and reduce calcium excretion, even in the absence of metabolic acidosis. 47 When used in the treatment of hypercalcemia, supplemental phosphate may lead to hypocalcemia, soft tissue calcification, and impaired renal function47; these effects are not usually observed during the treatment of hypophosphatemic osteomalacia, although in patients treated with calcitriol as well, the severity of nephrocalcinosis is related to the dose of phosphate. 496 Diarrhea is troublesome in some patients, but the most serious complication of long-term treatment with phosphate is the emergence of hypercalcemic hyperparathyroidism needing surgical intervention. This has been reported after 2 to 18 years of treatment in 16 cases, 1 1 with XLH and 5 with nonfamilial hypophosphatemia, 3 with and 2 without a tumor. 374'375'497 The parathyroid pathology has been variable but with a predominance of multiple gland involvement. The pathogenesis is probably similar to the tertiary hyperparathyroidism of longstanding vitamin D depletion (see Section II.F), but a parathyroid adenoma was found in one patient with XLH who had never received phosphate. 374 2. CALCITRIOL
1. PHOSPHATE SUPPLEMENTATION Since phosphate is reasonably safe and sometimes effective by itself, 14~176176 it is logical to use it initially as the only treatment. In the hypercalciuric form of hereditary hypophosphatemia, only phosphate is needed and calcitriol is contraindicated. 492 With normal renal function, even a large phosphate supplement can produce only a modest increase in plasma phosphate, which depends much more on the renal threshold than on the phosphate load to be excreted. Assuming 70% to 75% absorption and a glomerular filtration rate (GFR) of 100 ml/min, the mean plasma phosphate will rise by about 0.5 mg/dl for each 1.0 g/day increment in elemental phosphorus intake, with a smaller rise in fasting plasma phosphate. Even this theoretical maximum will be attained only if the phosphate is administered as the potassium rather than as the sodium salt, since expansion of the extracellular fluid volume by sodium may reduce tubular phosphate reabsorption and partly offset the rise in plasma phosphate. 493 Conversely, the effect of the supplement can be augmented by dietary salt restriction 47 or by diuretic therapy. 494
The effect of vitamin D in a pharmacological dose in a nondepleted subject is quite different from its effect in physiological dose in a depleted subject. ~96 First, the 25and loL-hydroxylase enzymes are relatively inactive and are suppressed further by excess of the substrate so that the response is much slower in onset. Second, even when the maximum response has been achieved, less of the absorbed calcium and phosphate is retained in bone and much more is excreted in the urine. Finally, when treatment is stopped the effect may persist for many months because of the large capacity of body storage sites for calciferol and calcidiol. There may be only a modest or even no increase in plasma calcitriol, but a substantial rise in plasma calcidiol, 338 probably sufficient to increase the occupancy of calcitriol receptors despite its much lower affinity. Although with careful attention to detail, satisfactory results can be achieved with calciferol, 62'196'348 and early results in nonhereditary hypophosphatemia have shown little advantage for l ct-hydroxylated compounds, 351'352 it is likely that with increasing experience and better definition of optimum dose requirements calcitriol (or alfacalcidol, which produces
374
A.M. PARFIrr
calcitriol after hepatic 25-hydroxylation) will be the vitamin D metabolite of choice in all forms of hypophosphatemic osteomalacia. 1~ In vitamin D-replete patients, calcitriol increases intestinal absorption of calcium and phosphate more reliably than calciferol, but its most important therapeutic effect is to increase the renal tubular reabsorption of phosphate with a consequent rise in plasma phosphate. 338 Although commonly attributed to suppression of PTH secretion, 498 it is more likely a direct renal tubular effect of supraphysiological plasma levels of calcitrio1338'499 as occurs during the treatment of extrinsic vitamin D depletion. 484 In children, the doses needed to achieve this effect are in the range of 50 to 80 ng/kg/day, which corresponds to 2.5 to 5 Ixg/day (or 4 to 8 Ixg/dl of alfacalcidol) for most adults, preferably administered at frequent short intervals because of the short half-life. Even though the hypercalciuric and hypercalcemic effects are partly neutralized by concurrent administration of phosphate, that is very close to the intoxicating dose, particularly as the osteomalacia heals, and frequent and meticulous supervision is essential. Even so, it is difficult to avoid the development of nephrocalcinosis. 496 Once healing has been achieved, the dose can be reduced to a safer and more manageable level. 5'1~ The need for such large doses with their attendant hazards should be considered only after smaller doses have proved ineffective, since healing and symptomatic relief can often be accomplished by a dose that increases calcium absorption without increasing phosphate reabsorption, together with an adequate phosphate supplement. 14~ Concurrent calciferol does not prevent phosphate-induced tertiary hyperparathyroidism; the likely greater effectiveness of calcitriol, 497 24-hydroxy calcidiol, 49~ or nonhypercalcemic analogues of calcitrio1491 in this respect remains to be demonstrated.
Acknowledgments Many colleagues, present and former, contributed to the data and concepts presented in this chapter, but two were indispensable. D.S. Rao performed almost all of the transiliac bone biopsies, demonstrating that in expert hands it can be one of the safest and least unpleasant of all invasive procedures. A.R. Villanueva performed almost all the bone histomorphometry, with results that are a testament to his insight, skill, and experience.
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383 375. Knudtzon J, Halse J, Monn E, et al: Autonomous hyperparathyroidism in X-linked hypophosphataemia. Clin Endocrinol 42: 199- 203, 1995. 376. Dent CE, Friedman M: Hypophosphataemic osteomalacia with complete recovery. Br Med J 1:1676-1679, 1964. 377. Hosking DJ, Chamberlain MJ, Shortland-Webb WR: Osteomalacia and carcinoma of prostate with major redistribution of skeletal calcium. Br J Radiol 48:451-456, 1975. 378. Lyles KW, Berry WR, Haussler M, et al: Hypophosphatemic osteomalacia: Association with prostatic carcinoma. Ann Intern Med 93:275-278, 1980. 379. Kabadi UM: Osteomalacia associated with prostatic cancer and osteoblastic metastases. Urology 21:65-67, 1983. 380. Charhon SA, Chapuy MC, Delvin EE, et al: Histomorphometric analysis of sclerotic bone metastases from prostatic carcinoma with special reference to osteomalacia. Cancer 51:918-924, 1983. 381. Brazy PC, Lobaugh B, Lyles KW, Drezner MK: The pathogenesis of tumor-induced osteomalacia: A new perspective. In Frame B, Potts JT Jr (eds): Clinical Disorders of Bone and Mineral Metabolism. Amsterdam, Excerpta Medica, 1983, pp 242-246. 382. Coindre JM, Mage P, Bui BN, et al: Mrtastases ostrocondensantes prostatiques et ostromalacie. La Presse Med 14:18231827, 1985. 383. Murphy WA, Seligman PA, Tillack T, et al: Osteosclerosis, osteomalacia, and bone marrow aplasia: A combined late complication of thorotrast administration. Skeletal Radiol 3:234-238, 1979. 384. Coindre JM, Reiffers J, Goussot JF, et al: Histomorphometric analysis of sclerotic bone from idiopathic myeloid metaplasia (nine cases). J Pathol 144: 163-169, 1984. 385. Monro P: Effect of treatment on renal function in severe osteomalacia due to Wilson's disease. J Clin Pathol 23:487-491, 1970. 386. Brenton DP, Isenberg DA, Cusworth DC, et al: The adult presenting idiopathic Fanconi syndrome. J Inherit Metab Dis 4: 211-215, 1981. 387. Harrison NA, Bateman JM, Ledingham JGG, Smith R: Renal failure in adult onset hypophosphatemic osteomalacia with Fanconi Syndrome: A family study and review of the literature. Clin Nephrol 35:148-150, 1991. 388. Adams RG, Harrison JF, Scott P: The development of cadmiuminduced proteinuria, impaired renal function, and osteomalacia in alkaline battery workers. Q J Med 38:425-443, 1969. 389. Kasuya M, Teranishi H, Aoshima K, et al: Renal toxicology with special reference to cadmium. In Abudulla M, Dashti H, Sarkar B, et al (eds): Metabolism of Mineral and Trace Elements in Human Disease. London, Smith-Gordon, 1989, pp 111-121. 390. Ghozlan R, Dupuis M, Baviera E: Osteomalacie revelatrice d'un syndrome de Toni-Debre-Fanconi secondaire a la prise de methyl-3-chromone. Rev Rhum 52:61-62, 1985. 391. Skinner R, Pearson ADJ, Price L, et al: Hypophosphataemic rickets after ifosfamide treatment in children. Br Med J 298: 1560-1561, 1989. 392. Mallette LE, Patten BM: Neurogenic muscle atrophy and osteomalacia in adult Fanconi syndrome. Ann Neurol 1:131-137, 1977. 393. Glassock RJ, Malluche HR, Tate M, et al: Recurrent fractures, hypophosphatemia, and renal insufficiency in an elderly woman. Am J Nephrol 4:329-335, 1984. 394. Baran DT, Marcy TW: Evidence for a defect in vitamin D metabolism in a patient with incomplete Fanconi syndrome. J Clin Endocrinol Metab 59:998-1001, 1984. 395. Colussi G, De Ferrari ME, Pontoriero C, et al: Vitamin D metabolites in osteomalacia associated with the adult Fanconi syn-
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437. Rossini M, Gatti D, Zamberlan N, et al: Long-term effects of a treatment course with oral alendronate of postmenopausal osteoporosis. J Bone Miner Res 9:1833-1837, 1994. 438. Briancon D, Meunier PJ: Treatment of osteoporosis with fluoride, calcium, and vitamin D. Orthop Clin North Am 12:629648, 1981. 439. Lundy MW, Stauffer M, Wergedal JE, et al: Histomorphometric analysis of iliac crest bone biopsies in placebo-treated versus fluoride-treated subjects. Osteoporos Int 5:115-129, 1995. 440. Grennan DM, Palmer DG, Mathus RS, et al: Iatrogenic fluorosis. Aust NZ J Med 8:528-531, 1978. 441. Compston JE, Chadha S, Merrett AL: Osteomalacia developing during treatment of osteoporosis with sodium fluoride and vitamin D. Br Med J 281:910-911, 1980. 442. Bonvoisin B, Bouvier M, Meunier PJ, Lejeune E: Osteomalacie histologique induite par l'administration prolongee d'acide niflumique. Nouv Presse Med 11:1636, 1982. 443. Kleerekoper M, Frame B, Villanueva AR, et al: Treatment of osteoporosis with sodium fluoride altemating with calcium and vitamin D. In DeLuca HF, et al (eds): Osteoporosis: Recent Advances in Pathogenesis and Treatment. Baltimore, University Park Press, 1981. 444. Dunstan CR, Hills E, Norman AW, et al: The pathogenesis of renal osteodystrophy: Role of vitamin D, aluminum, parathyroid hormone, calcium and phosphorus. Q J Med 55:127-144, 1985. 445. Andress DL, Ott SM, Maloney NA, Sherrard D J: Effect of parathyroidectomy on bone aluminum accumulation in chronic renal failure. N Engl J Med 312:468-473, 1985. 446. O'Brien AAJ, Moore DP, Keogh JAB: Aluminum osteomalacia in chronic renal failure patients neither on dialysis nor taking aluminum containing phosphate binders. Ir J Med Sci 159:7476, 1990. 447. Boyce BF, Elder HY, Elliot HL, et al: Hypercalcaemic osteomalacia due to aluminum toxicity. Lancet 2:1009-1013, 1982. 448. Charhon SA, Chavassieux PM, Chapuy MC, et al: Low rate of bone formation with or without histologic appearance of osteomalacia in patients with aluminum intoxication. J Lab Clin Med 106:123-131, 1985. 449. Moriniere P, Cohen-Solal M, Belbrik S, et al: Disappearance of aluminic bone disease in a long term asymptomatic dialysis population restricting AI(OH)3 intake: Emergence of an idiopathic adynamic bone disease not related to aluminum. Nephron 53: 9 3 - 1 0 1 , 1989. 450. Parfitt AM: The localization of aluminum in bone: Implications for the mechanism of fixation and for the pathogenesis of aluminum-related bone disease (editorial). Int J Artif Organs 11: 7 9 - 9 0 , 1988. 451. Parfitt AM, Rao D, Stanciu J, Villanueva AR: Comparison of aluminum related with vitamin D related osteomalacia by tetracycline based bone histomorphometry. In Massry SG, Olmer M, Ritz E (eds): Phosphate and Mineral Homeostasis. Adv Exp Biol Med 208:283-287, 1986. 452. Blumenthal NC, Posner AS: In vitro model of aluminuminduced osteomalacia: Inhibition of hydroxyapatite formation and growth. Calcif Tissue Int 36:439-441, 1984. 453. Goodman WG, Henry DA, Horst R, et al: Parenteral aluminum administration in the dog: II. Induction of osteomalacia and effect on vitamin D metabolism. Kidney Int 25:370-375, 1984. 454. O'Brien AAJ, Moore DP, Keogh JAB: Acute epidemic aluminum osteomalacia secondary to water supply contamination. Ir J Med Sci 159:71-73, 1990. 455. Coumot-Witmer G, Zingraff J, Plachot JJ, et al: Aluminum localization in bone from hemodialyzed patients: Relationship to matrix mineralization. Kidney Int 20:375-385, 1981.
385 456. Podenphant J, Salem N, Sypitkowski C, et al: Reversal of aluminum related dialysis osteomalacia after transplantation. Proceedings, Fourth International Workshop on Bone Histomorphometry. Bone 6:405, 1985. 457. Hodsman AB, Anderson C, Leung FY: Accelerated accumulation of aluminum by osteoid matrix in vitamin D deficiency. Miner Electrolyte Metab 10:309-315, 1984. 458. Quarles DL, Dennis VW, Gitelman HJ, et al: Aluminum deposition at the osteoid-bone interface. An epiphenomenon of the osteomalacic state in vitamin D deficient dogs. J Clin Invest 75: 1441-1447, 1985. 459. Thomas WC, Meyer JL: Aluminum-induced osteomalacia: An explanation. Am J Nephrol 4:201 - 203, 1984. 460. Baker SL, Dent CE, Friedman N, Watson L: Fibrogenesis imperfecta ossium. J Bone Joint Surg 48B:804-825, 1966. 461. Swan CHJ, Shah K, Brewer DB, Cooke WT: Fibrogenesis imperfecta ossium. Q J Med 45:233-253, 1976. 462. Lang R, Vignery AMC, Jensen PS: Fibrogenesis imperfecta ossium with early onset: Observations after 20 years of illness. Bone 7:237-246, 1986. 463. Frame B, Frost HM, Pak CYC, et al: Fibrogenesis imperfecta ossium. A collagen defect causing osteomalacia. N Engl J Med 285:769-772, 1971. 464. Stoddart PGP, Wickremaratchi T, Hollingworth P, Watt I: Fibrogenesis imperfecta ossium. Br J Radiol 57:744-751, 1984. 465. Byers PD, Stamp TCB, Stoker D J: Case report 296. Skeletal Radiol 13:72-76, 1985. 466. Ralphs JR, Stamp TCB, Doppi~-,g-Hepenstal PJC, Ali SY: U1trastructural features of the osteoid of patients with fibrogenesis imperfecta ossium. Bone 10:243-249, 1989. 467. Whyte MP: Hypophosphatasia. In Scriver CR, et al (eds): The Metabolic and Molecular Bases of Inherited Disease, 7th ed, vol III. New York, McGraw-Hill, 1995. 468. Whyte MP, Fallon MD, Murphy WA, Teitelbaum SL: Axial osteomalacia. Clinical, laboratory and genetic investigation of an affected mother and son. Am J Med 71:1041-1049, 1981. 469. Whyte MP, Vrabel LA, Schwartz TD: Alkaline phosphatase deficiency in cultured skin fibroblasts from patients with hypophosphatasia: Comparison of the infantile, childhood, and adult forms. J Clin Endocrinol Metab 57:831-837, 1983. 470. Whyte MP, Walkenhorst DA, Fedde KN, et al: Hypophosphatasia: Levels of bone alkaline phosphatase immunoreactivity in serum reflect disease severity. J Clin Endocrinol Metab 81: 2142-2148, 1996. 471. Whyte MP, Seino Y: Circulating vitamin D metabolite levels in hypophosphatasia. J Clin Endocrinol Metab 55:178-180, 1982. 472. Fallon MD, Teitelbaum SL, Weinstein RS, et al: Hypophosphatasia: Clinicopathologic comparison of the infantile, childhood, and adult forms. Medicine 63:12-24, 1984. 473. Bethune JE, Dent CE: Hypophosphatasia in the adult. Am J Med 28:615-622, 1960. 474. Frame B, Frost HM, Ormond RB, Hunter RB: Atypical osteomalacia involving the axial skeleton. Ann Intem Med 55:632639, 1961. 475. Demiaux-Domenech B, Bonjour JP, Rizzoli R: Axial Osteomalacia: Report of a new case with selective increase in axial bone mineral density. Bone 18:633-637, 1996. 476. Whyte MP: Sclerosing bone dysplasias. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, 2nd ed. New York, Raven Press, 1993, pp 327-344. 477. VanErpecum KJ, Kroon HM, VanGroningen K, Harinck HIJ: Central and peripheral bone biopsy in a patient with axial osteomalacia. Neth J Med 28:505-508, 1985.
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_]HAPTER 1~
Osteoporosis Pathogenesis and Therapy MICHAEL
KLEEREKOPER
LOUIS V. AVIOLI
Department of Medicine, Wayne State University, Detroit, Michigan 48201 Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri 63110
E. Biochemical Markers of Bone Remodeling F. Clinical Characteristics IV. Classification V. Management A. Nonpharmacological Intervention B. Pharmacological Intervention C. On the Horizon References
I. Definition A. Epidemiology II. Physiological Osteoporosis III. Diagnostic Aids A. Radiology B. Densitometry C. Ultrasound D. Histomorphometry
having BMD greater than 2.5 SD below peak adult bone mass, whether or not a fragility fracture has occurred. 4 In order to distinguish this asymptomatic condition of having low bone mass without fractures from what has traditionally been termed osteoporosis, the WHO has suggested that the term severe osteoporosis be applied to subjects with low bone mass and fragility fractures. The Food and Drug Administration (FDA) in the United States has recently approved therapies for the treatment of osteoporosis, defining the disease as being present when BMD is greater than 2.0 SD below peak bone mass. Whether one chooses to define osteoporosis in an individual patient using the WHO or the FDA criteria is immaterial in most clinical circumstances. These arbitrarily defined cut-points should really be applied to an individual patient not so much as defining or diagnostic cut-points, but rather as intervention decision-making cut-points. The situation is entirely analogous to the diagnosis of hypertension or hypercholesterolemia. Diagnostic levels for both of these asymptomatic states have been defined by various authoritative groups but individual practitioners have developed their own intervention criteria, which vary with the clinical circumstance and are not strictly based on numerical criteria. With an
I. D E F I N I T I O N Osteoporosis, which has actually been described in prehistoric populations, 1 is a brittle bone disease manifest clinically by fragility fractures defined as fractures occurring in the absence of trauma or in response to only trivial trauma (force equal to or less than a fall from a standing height). In almost all patients, these fragility fractures are preceded by a long, silent period during which the bones become progressively more brittle without fracture occurring. While there are a number of putative risk factors for progressive bone loss and fracture risk, these are only applicable in population studies. In an individual subject the only means of detecting osteoporosis during this silent period is by direct, noninvasive measurement of bone mineral density (BMD). Reference intervals for peak adult bone mass have been established for each method of measuring BMD, and epidemiological studies have indicated that an individual's risk of sustaining an osteoporotic fracture doubles for each standard deviation (SD) by which the measurement is below the mean value for peak adult bone mass. 2'3 From this information, the World Health Organization (WHO) has defined osteoporosis as the state of METABOLIC BONE DISEASE
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MICHAEL KLEEREKOPER AND LOUIS V. AVIOLI
elevated blood pressure or serum cholesterol, or a low bone mass, clearly the further from the reference value is the observation in the individual patient, the more aggressive the intervention and monitoring. In fact, great caution should be used when considering putting a diagnostic label (hypertension, hypercholesterolemia, osteoporosis) on an asymptomatic patient simply on the basis of a physiological measurement. In addition to the anxiety such a label may cause in the patient, the diagnosis may have profound adverse implications for the patient's life and health insurance. Furthermore, it has been suggested that absenteeism from the work force is significantly greater in patients with a diagnosis of hypertension than their nonhypertensive co-workers, even when the stated cause for absenteeism is unrelated to the elevated blood pressure or its management. Given the frequency with which backache is the stated cause for absenteeism from the workplace, there is considerable risk that patients with osteoporosis without fracture would be absent even more frequently if they were additionally concerned about fracture risk, or erroneously related their backache to the low bone mass.
A. E p i d e m i o l o g y Peak adult bone mass is achieved sometime during the fourth and fifth decades of life, with a gradual, progressive decline in BMD beginning at about age 40 to 45 in all personsd Peak adult bone mass is greater in men than in women, and greater in African-Americans than in non-Hispanic whites. These differences in peak bone mass are reflected in gender and ethnic differences in the prevalence and incidence of osteoporosis and osteoporotic fractures later in life. At the menopause women experience a 7 to 10-year period of accelerated bone loss as a result of estrogen deficiency, widening this gender gap in the prevalence and incidence of osteoporosis and osteoporotic fractures. Based on a relatively small sample of women in Rochester, Minnesota, Melton has estimated the prevalence of osteoporosis (defined by the WHO criteria detailed above) to be in excess of 20 million women in the United States 6 (Table 12-1 and Fig. 12-1). The WHO has predicted the future occurrence of osteoporosis throughout the world (Fig. 12-2). The most startling aspect of these projections is that all of the persons projected to have a hip fracture in the year 2050 are already alive. There is a definite pattern to the development of osteoporotic fractures, with distal forearm (Colles') fractures being extremely uncommon in men at any age but rising sharply in incidence in women within 5 to 10 years of the menopause. 7 It has been estimated that at least 90% of all hip and spine fractures among elderly
TABLE 12-1 WHO Diagnostic Criteria for Osteoporosis a'b Normal
BMD within 1 SD of peak adult bone mass (PABM)
Low bone mass
BMD --> 1.0 but < 2.5 SD below PABM
Osteoporosis
BMD --> 2.5 SD below PABM
Severe osteoporosis
BMD --> 2.5 SD below PABM, with fractures
aFrom Melton LJ III: How many women have osteoporosis now? J Bone Miner Res 10(2):176, 1995. bproportion of women with osteoporosis (BMB > 2.5 SD below peak bone mass).
white women should be attributed to osteoporosis. 8 Fractures of the vertebral body are the most prevalent of the osteoporotic fractures occurring, with a peak age incidence between 65 and 70 in women. The incidence in men is about one third that in women and occurs some 5 to 10 years later. Fractures of the proximal femur (hip) are the most devastating of the osteoporotic fractures, occurring with increasing frequency after age 75 in women and age 80 in men. There are approximately 300,000 hip fractures each year in the United States in persons over age 65, with the incidence being twice as high in women. 9 Estimates of short-term (1 year or less) mortality following these hip fractures vary between 5% and 20% in different series, with mortality being twice as high in men than in women. 1~It should be emphasized that these mortality statistics reflect excess mortality after adjustment for age and comorbidities. Osteoporosis and its dreaded sequelae of progressive disability with associated neurological deficits, poor rehabilitation profile, 11'12excessive deaths, and escalating medical care expenditures is becoming a worldwide problem. In 1990 North America and Europe accounted for the majority of the 1.66 million fractures that occurred internationally. Since the elderly component of the population is increasing most rapidly in Africa, Asia, Latin America, and the Middle East, it has been projected that these geographical areas will account for over 70% of the 6.3 million fractures expected in the year 2050.13 More than 95% of all osteoporotic hip fractures occur after a fall, usually a fall to the side, but only a small fraction of falls result in hip fractures. 14 Fall prevention and attention to fall mechanics are assuming increasing importance in attempts to lessen the societal burden of osteoporotic hip fractures, which in 1995 exceeded $10 billion per annum in the United States. 15 While most attention is focused on fractures of the wrist, spine, and hip, fractures of the proximal humerus, distal femur, distal tibia and fibula, and ribs are increasingly common in the elderly and must be considered as clinical manifestations of osteoporosis. In fact, the contribution of nonhip fractures to
CHAPTER 12
Osteoporosis Pathogenesis and Therapy
389
FIGURE 12--1 Estimated skeletal status of United States white women in 1990, by age group. Osteopenia is bone density of the hip, spine, or distal forearm more than 1.0 but less than 2.5 SD below the young normal mean. Osteoporosis is bone density at one or more of these sites more than 2.5 SD below the mean and, when linked with a history of fracture, is deemed established osteoporosis. (From Melton LJ III: How many women have osteoporosis now? J Bone Miner Res 10:176, 1995.)
the substantial m o r b i d i t y and e x p e n d i t u r e s associated with o s t e o p o r o s i s has b e e n u n d e r e s t i m a t e d b y many. 15
II.
PHYSIOLOGICAL
OSTEOPOROSIS
T h e p h e n o m e n o n o f a p r o g r e s s i v e d e c l i n e in b o n e m a s s with age, after age 40 to 45, has a l r e a d y b e e n m e n -
tioned, as has the a c c e l e r a t e d b o n e loss of the m e n o p a u s e that occurs in all w o m e n w h o live l o n g e n o u g h . S i n c e t h e s e are u n i v e r s a l p h e n o m e n a in h u m a n s , this m u s t to s o m e e x t e n t be r e g a r d e d as part o f the n o r m a l p h y s i o l o g y o f a g i n g i n a s m u c h as e v e r y o n e w h o lives l o n g e n o u g h will e v e n t u a l l y d e v e l o p o s t e o p o r o s i s as defined in t e r m s o f a r e d u c t i o n f r o m p e a k b o n e mass. T h e r e is also an a p p a r e n t p h y s i o l o g i c a l o s t e o p o r o s i s o f p u b e r t y
FIGURE 12--2 Estimated number of fractures for men and women in different regions of the world in 1990, 2025 and 2050. (From Kanis JA, and WHO Study Group: Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. WHO Technical Report Series 843:13, 1994.)
390 with a peak incidence of distal forearm (greenstick) fractures occurring in boys and gifts between ages 9 and 16. This observation may be explained by the fact that linear skeletal growth precedes the accumulation of skeletal mass during the growth spurt, in much the same manner that accumulation of muscle bulk occurs after the growth spurt of puberty. In boys, this peak pubertal incidence of distal forearm fractures returns to low levels after age 16 with little fluctuation thereafter throughout life. The same pattern is seen in gifts immediately following puberty, only to rise sharply again in women a few years after the menopause. The precise pathogenesis of the accelerated bone loss of the menopause is unknown. Although previous studies have revealed that peak bone mass is under genetic influence the contribution of genetic factors to postmenopausal bone turnover and bone loss is relatively insignificant. 16 That the bone loss is related to the cessation of gonadal steroid production is clearly established such that whenever there is a decline in gonadal steroids (estrogen in women, androgen in men) as a result of disease, therapy, or surgery, there is a transient (approximately 5- to 7-year) period of increased bone remodeling and accelerated bone loss reverting to basal, physiological rates of bone loss thereafter. Estrogen receptors have been identified in bone but at concentrations that are at least one order of magnitude lower than in more classic estrogen-dependent tissues such as the endometrium or breast. Therefore, it is unlikely that estrogen deficiency has direct effects on the skeleton. There is compelling evidence that estrogen modulates the local production of cytokines, interleukin-1 (IL-1) and interleukin-6 (IL-6), which in turn are responsible for increased bone resorption. 17-22 Considerable controversy surrounds the relative roles of IL-1 and IL-6 in this regard, 18'19 and quite possibly both are significantly involved. It is not yet known whether these, and possibly other cytokines, act directly on the skeleton or mature bone cells, or are indirectly involved in the recruitment and maturation of osteoprogenitor cells. 2~ Very little is known about possible cellular mechanisms underlying age-related bone loss, although most would agree that estrogen status and heredity are major determinants of premenopausal bone loss. 23 It seems well established that bone loss begins before there is any decline in ovarian estrogen production in women and commences well before any age-related decline in androgen production in men. A putative role for an agerelated decline in renal production of calcitriol (1,25dihydroxyvitamin D) with resultant decrease in intestinal absorption of calcium, elevation in parathyroid hormone, and negative skeletal balance has been postulated by several investigators. There is some observational and experimental support for this hypothesis, but in general
MICHAEL KLEEREKOPER AND LOUIS V. AVIOLI
the process of age-related bone loss is so slow, and the physiological changes so subtle, that it has proven extremely difficult to formulate and substantiate any single hypothesis to account for the universal phenomenon of age-related bone loss. A most tenable hypothesis is that late consequences of estrogen deficiency rather than agerelated processes p e r s e a r e the principal causes of the secondary hyperparathyroidism and increased bone resorption in elderly women. 24 We should also recognize the observations that high bone turnover is associated with low bone mass and vertebral fractures in postmenopausal women, z5 and although bone turnover normally declines progressively after the menopause, it does not in patients with vertebral fractures and osteoporosis. 26
III. DIAGNOSTIC AIDS A. Radiology Plain radiographs are usually required for confirmation of fracture occurrence but have little additional role in most patients with osteoporosis. A decrease in total skeletal mass can be seen in advanced stages of bone loss, but it has been estimated that fully 30% of skeletal mass must be lost before it can be detected radiographically. Newer densitometric methods have supplanted plain radiographs for this purpose. Most long-bone fractures (proximal femur, distal radius, proximal humerus, distal tibia and fibula) can be detected clinically and confirmed radiographically. Fractures of the vertebral bodies pose a particular problem for several reasons. There is considerable variability in vertebral body size and shape, and several developmental and acquired abnormalities, such as Schuermann's disease, can mimic vertebral fractures. Additionally, a proportion of vertebral body fractures have either occurred spontaneously without symptoms or the proximate cause of the fracture has been forgotten by many patients. Several attempts have been made to develop population-specific reference data for vertebral body dimensions from the fourth thoracic (dorsal) through the fifth lumbar vertebra. Vertebral body dimensions can then be measured on radiographs of the thoracic and lumbar spine taken in the lateral projection, and deviations from the reference data can be used to define the presence or absence of a vertebral body fracture. These morphometfic approaches to detecting vertebral fractures probably result in some overestimate of fracture occurrence, and visual inspection of the radiograph by a trained and skilled musculoskeletal radiologist remains the "gold standard" for fracture detection in individual cases. Morphometfic methods have greater relevance in population studies and in clinical trials. 27 Since pelvic
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CHAPTER 12 Osteoporosis Pathogenesis and Therapy radiographs present sufficient spatial resolution and contrast to evaluate the anatomical structures of the proximal femur, prediction of hip fractures can often be made using a combination of measurements 28 (Fig. 12-3). One important function of plain radiography in the evaluation of individual patients suspected of having an osteoporotic fracture is the detection of other diseases that might mimic osteoporosis. Fragility fractures can complicate metabolic diseases such as osteomalacia, hyperparathyroidism, and systemic mastocytosis, but these diseases generally have radiographic features that distinguish them from osteoporosis. The same is usually true also for genetic disorders such as osteogenesis imperfecta, and malignant diseases such as skeletal metastases and multiple myeloma, each of which is associated with fragility fractures. Occasionally, fractures prove difficult to verify with plain radiographs due to the site (fibs) or impaction without obvious skeletal deformity. In such cases radionuclide skeletal scintigraphy may be of benefit with new fractures associated with intense increased isotope uptake. Scintigraphy may also be of benefit in assessing the age of a vertebral fracture, with increased isotope
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\ i
i
36
FIGURE 12--3 Femoral and pelvic bones and placement of selected radiographic measurements: the thickness of the medial shaft cortex (SC) 3 cm below the lesser trochanter, the thickness of the medial cortex at the center of the femoral neck (NC), the width of the femoral head (HW), the width of the intertrochanteric region (TW), and acetabular bone width (AW). The region of the principle tensile group of trabeculae (PT) is marked. (From Gluer CC, Cummings SR, Pressman A, et al, for The Study of Osteoporotic Features Research Group: Prediction of hip fracture from pelvic radiographs: The study of osteoporotic fractures. J Bone Miner Res 9: 671, 1994.)
uptake persisting from 6 months to 2 years after the fracture. The pattern of fractures seen scintigraphically may also aid in the diagnosis of metastatic disease, particularly if there is discordance between the plain radiograph and the scintigraph (i.e., increased isotope uptake at a site without obvious radiographic abnormality).
B. Densitometry Dual energy x-ray absorptiometry (DEXA) has become the gold standard for the noninvasive measurement of B MD. The method is accurate (> 95% to 98%), precise (0.5% to 2.5% error for repeat measurements), painless, and rapid (approximately 5 minutes per measurement site) and exposes the patient to very little more than background radiation. Densitometry (or other method for B MD measurement) remains the only method for diagnosing osteoporosis prior to fracture as described in detail in Section I of this chapter. Current equipment commercially available can measure BMD at the lumbar spine, the forearm, and the proximal femur, as well as the total body (where it can also provide accurate and precise information about body composition). As with any other clinical test, densitometry should only be obtained if a clinical decision is going to be made on the basis of the result obtained. For example, perimenopausal women who have already elected to begin estrogen replacement therapy do not require a bone mass measurement prior to initiating therapy, since more than 95% of these women will derive skeletal benefit from the therapy. Some women are reluctant to take estrogen unless a specific skeletal need can be demonstrated in their case. This can only be done with direct BMD measurement. In this regard, it should be emphasized that both lumbar spine and proximal femur measurements should be made when women are using bone density measurements as a means to effect the decision of whether or not to use hormonal replacement therapy. 29"3~Another indication is the patient in whom spine radiographs have demonstrated a vertebral deformity and there is uncertainty as to whether this resulted from low bone mass or from trauma. Patients on corticosteroid therapy and at increased risk for secondary osteoporosis should have BMD measured, as should patients with mild, asymptomatic primary hyperparathyroidism where the documentation of a low BMD is an indication for parathyroidectomy. There is no current consensus concerning the role of BMD measurement in patients who clearly have severe osteoporosis with fragility fractures. Some clinicians feel that a baseline measurement is important in order to properly monitor the response to therapy. Others feel that the therapeutic options are still so
392
MICHAEL KLEEREKOPER AND LOUIS V. AVIOLI
limited that knowledge of BMD will not alter management. This dilemma will probably be solved as the cost of BMD testing decreases m currently $100 to $250 (U.S.) per measurement s i t e m a n d as more therapeutic options become available. Even greater controversy surrounds the timing of serial measurements of B MD. While the precision error is quite low, it is nonetheless extremely close to the anticipated annual rate of change in BMD, either progressive bone loss or in response to therapy. Unless there is reason to suspect a very rapid loss of bone (e.g., during the early phase of corticosteroid therapy) there is little clinical justification in obtaining repeat BMD measurements more frequently than once a year, and in many circumstances biannual measurement will suffice.
D. Histomorphometry
C. Ultrasound The definition of osteoporosis includes a statement conceming bone quantity ( " a reduction in bone mass") and quality ("microarchitectural deterioration"). The former can be measured by densitometry, but this method provides no measure of quality. Ultrasonography is gaining increasing acceptance as a measure of bone quality, although precisely what quality of bone is measured remains unclear. Ultrasound has several important potential advantages over densitometry in that it does not expose the patient to ionizing radiation, and is available using portable, small equipment that can be purchased generally at far lower cost than densitometry equipment. Ultrasound equipment has been developed to measure speed-of-sound ( S O S ) - - a l s o termed apparent velocity of ultrasound (AVU) and ultrasound transmission velocity ( U T V ) m , broadband ultrasound attenuation (BUA), and stiffness. Studies have been performed with measurements obtained predominantly at the calcaneus or patella, but other sites are accessible and have been studied. When osteoporosis has been defined on the basis of low BMD, all ultrasound parameters have been significantly lower in those with osteoporosis compared to appropriate control groups. More importantly, recent studies have demonstrated that ultrasound predicts incident vertebral deformity 31 and can also provide estimates of relative risk of hip fracture that are independent of B MD. 32 The potential of ultrasound is still emerging and will not be fully realized until there is a more clear identification of just where this technology fits in the intervention decision analysis of patients at risk for osteoporosis, and whether it is a surrogate for BMD, adjunctive to BMD, or perhaps preferred over BMD.
Most of what is now known about skeletal metabolism has been learned from microscopic examination of the skeleton, particularly dynamic histomorphometry with in vivo double tetracycline labeling. With this technique one can assess the extent of surface activity of osteoclasts and osteoblasts, the rate at which bone is being formed with deposition of new bone matrix, and the extent to which this newly formed matrix is being properly mineralized. Diagnostic information can be gleaned from skeletal histomorphometry, static or dynamic, which cannot be obtained with measurements of bone mineral density or biomarkers of bone tumover particularly if there is clinical suspicion of osteomalacia and serum or plasma levels of the vitamin D metabolites have provided equivocal results. A second example might be a rare case of systemic mastocytosis where it would be important to document excess mast cells in the biopsy specimen. Some authorities consider that all patients, particularly males, with unexplained or unexpected osteoporosis should have a bone biopsy.
E. Biochemical Markers of Bone Remodeling The mature adult skeleton is subject to continuous turnover, or remodeling, with removal of old bone and replacement at that site with new bone. Remodeling always takes place on skeletal surfaces, and always in discrete packets termed basic multicellular units (BMUs). Under normal physiological circumstances, 80% of skeletal surfaces are quiescent and 20% are actively involved in remodeling. What factor(s) determines the site and extent of remodeling is unknown. The process begins with recruitment of preosteoclasts that mature into osteoclasts which resorb a cavity to a depth of 50 /zm at a rate of 5 Ixrn/day. During this phase of bone resorption breakdown products of the fully mineralized skeleton (matrix protein and mineral, predominantly calcium) are removed from the skeleton, and transiently enter the circulation from which they are rapidly cleared by the kidney. Monitoring the urinary excretion of these breakdown products of bone resorption has recently become clinically available with the development of highly specific and sensitive assays for the pyridinium crosslinks of type I collagen of bone, and the amino- and carboxyl-terminal telopeptides of these cross-links (Table 12-2). Either a specific product of the osteoclast, or something released from the skeleton during the phase of osteoclast resorption, is a stimulus to the recruitment and/ or activation of osteoblasts. These cells line the newly
CHAPTER 12 OsteoporosisPathogenesis and Therapy
393
TABLE 12--2 Biochemical Markers of Bone Remodeling
disease in which red cell survival is not decreased but erythropoiesis is impaired. Assessing rates of bone remodeling are of potential importance in the evaluation of patients with osteoporosis. These biochemical studies have not yet been demonstrated to independently predict fracture risk (except in small studies of elderly women), nor can they reliably estimate current B MD, although attempts have been made to use the biochemistry as surrogates for densitometry. 33 However, biochemical assessment of the imbalance between resorption and formation can be used to predict, and monitor, changes in B MD, and this remains the current major role of these measurements in clinical medicine. The precision error for repeat measurements is 10% to 15% for serum-based assays (formation) and 25% to 30% for urine-based assays (resorption), which initially appears unacceptable in relation to the 0.5% to 2.5% precision of DEXA. However, anticipated rates of change in biochemical markers in response to therapy for osteoporosis is greatly in excess of these precision errors, exactly the opposite of the situation with DEXA. More importantly, these changes in biochemistry occur rapidly, 34 with significant decrements in markers of resorption detectable within 4 weeks of initiating antiresorptive therapy. Because of the sequence of resorption preceding formation, significant changes in markers of formation are not seen until 2 to 3 months after initiation of such therapy. Finally, and perhaps most importantly, these early biochemical responses to therapy predict changes in BMD detected 24 months later. The current best use of biochemical markers of bone remodeling is limited because of the limited therapeutic options for osteoporosis. Most markers of bone remodeling are increased in elderly subjects with vitamin D insufficiency and vary with its correction. 35 Ideally, one should also be able to tailor the dose of antiresorptive therapy to the magnitude of the abnormality in rates of resorption, adjusting the dose in relation to the skeletal response as reflected in the biomarkers of bone remodeling. Unfortunately, of the FDA-approved therapies available in the United States, dose adjustment is only possible with estrogen and alendronate, but not yet with calcitonin nasal spray. Estrogen and calcitonin are usually most effective in patients in whom the primary abnormality is an increase in bone resorption (high turnover), whereas alendronate appears to work equally well in patients with normal, low, and high turnover. Patients with low or normal turnover would also be expected to respond more favorably to agents that directly stimulate bone formation such as fluoride or parathyroid hormone. Since currently both of these approaches must still be regarded as experimental, it will be some time before the concept of tailoring therapy (drug type and dose) can be fully implemented into clinical practice.
Resorption Serum tartrate resistant acid phosphatase Urine hydroxyproline Pyridinoline: free and peptide-bound Deoxypyridinoline: free and peptide-bound Teleopeptides of collagen cross-links: amino-terminal, carboxylterminal Formation Serum bone specific alkaline phosphatase Serum procollagen extension peptides: amino-terminal carboxylterminal Turnover (both resorption and formation) Serum osteocalcin
formed resorption cavity and begin the repair (formation) process by filling in the cavity, from the base towards the surface, with matrix proteins, predominantly type I collagen. As newly synthesized bone matrix is deposited it becomes progressively more mineralized, again from the base towards the surface, until after approximately 90 days, a new BMU has refilled the resorption cavity. This phase of the remodeling cycle can be monitored by measuring circulating levels of proteins (or their breakdown products) synthesized and secreted by the osteoblast (Table 12-2). While the skeleton is in balance for that short period of time after peak adult bone mass is attained (see Section I), the amount of bone removed during resorption is normally completely replaced. Subsequently in later years, with each remodeling cycle there is always a slight excess of resorption over formation. This is a mechanistic way of describing the progressive negative balance of age-related bone loss. Any perturbation that increases the rate at which the skeleton is being remodeled will accelerate the rate of bone loss even though loss per remodeling cycle is unchanged. This has been termed "high-turnover" bone loss and is analogous to the rapid turnover of erythrocytes in hemolytic anemias, which are characterized by decreased red cell survival ("resorption") and an increased but inadequate reticulocytosis ("formation"). Most of the identifiable secondary causes of osteoporosis (see Section IV) result in high-turnover bone loss. Under other circumstances, most often with advanced age, there is an apparent defect in the coupling or signaling between resorption and formation. When this occurs, skeletal loss per remodeling cycle is increased so that accelerated bone loss can occur with normal or even low turnover. To continue the anemia analogy, this would be akin to anemia of chronic
394
MICHAEL KLEEREKOPERAND LOUIS V. AVIOLI F. C l i n i c a l C h a r a c t e r i s t i c s
Osteoporosis diagnosed only on the basis of a low BMD is entirely without symptoms. Once osteoporosis is complicated by fractures the clinical features relate to the site of fracture. The goal of orthopedic intervention for osteoporotic long-bone fractures is complete restoration of function to prefracture levels, and this can be achieved in most patients. A small proportion of patients sustaining a Colles' fracture are left with residual pain, deformity, and restricted mobility of the wrist. This also happens, but to a lesser extent, with distal tibia and fibula fractures. In contrast, restoration of prefracture functional state is achieved in less than half of the patients who sustain an osteoporotic hip fracture. Osteoporotic vertebral fractures require separate consideration. 27 An acute fracture episode will result in severe spinal pain, tenderness, and limitation of motion for about 6 weeks after which the tenderness should subside, the pain should be more paraspinal than spinal, and there should be improved range of motion. If the acute symptoms, particularly direct spinal tenderness, persist for longer than 8 weeks one must consider diagnoses other than osteoporosis such as metastases or myeloma. Only rarely does an acute osteoporotic vertebral fracture impinge on the spinal cord with resultant long tract symptoms and signs. 36 If this is present, diagnoses other than osteoporosis must again be considered, even if the fracture can be defined as a fragility fracture. As noted, some vertebral fractures can occur without an identifiable acute episode. Here the clinical presentation is progressive loss of stature, progressive spinal deformity, chronic back pain, protuberant abdomen (occasionally with gastrointestinal complaints such as early satiety), and altered body image. 1 Of particular importance is the progressive loss of stature. We and others have documented that women (and presumably men) who sustain vertebral fractures lose stature two to three times more rapidly than those who do n o t . 37 Progressive vertebral deformity is associated with an increased risk of general disability and the use of devices to assist ambulation in both men and women, with severe age-related progression of this syndrome observed primarily in women. 38 It is worthwhile to accurately record stature at each clinic visit, just as weight is recorded. Patients with acute back pain or exacerbation of existing pain who have not sustained 0.5 to 1.0 inch or more of height loss are unlikely to have sustained a new vertebral fracture as a cause of the back pain. Similarly, well-documented stature loss of 1.0 cm or more between clinic visits should alert the clinician to the possibility of an asymptomatic vertebral fracture. As mentioned, the presence of a fracture is a reliable, independent (of BMD) predictor of future fractures so
that detection of asymptomatic fractures is clinically important. 39 The clinical evaluation of any patient with osteoporosis, or being evaluated for osteoporosis risk, must include a careful history and physical examination with the intention of identifying potential secondary causes of osteoporosis (see Table 12-3), since these may be amenable to specific therapy. The initial investigations should also be geared towards screening for these secondary causes even in the absence of clinical clues from the history and physical exam. Since the yield will be low, these investigations should not be exhaustive and can be accomplished with a biochemical profile (hyperor hypocalcemia, renal and hepatic dysfunction, hypoalbuminemia, hyperglobulinemia, marked elevation of alkaline phosphatase), a complete blood count (CBC) (nutritional deficiency, marrow infiltrative diseases),
TABLE 1 2 - 3
A Pathogenetic Classification of Osteoporosis
Primary Juvenile Senile Idiopathic Young/middle-aged males Pregnancy Regional migratory II. Secondary Hormone excess Parathyroid (1~ or 2~ Thyroid Cortisol Hormone deficiency Estrogen Premenopausal Disease Drugs Postmenopausal Testosterone Diseases Postgastrectomy without vitamin D deficiency Systemic mastocytosis Immobilization Following organ transplantation Drugs Corticosteroids Thyroxine Alcohol Anticonvulsants Barbiturates Phenytoin Anticoagulants Heparin Coumadin Antimetabolites Methotrexate Cyclosporine
395
CHAPTER 12 Osteoporosis Pathogenesis and Therapy thyroid-stimulating hormone (TSH) (hyperthyroidism), and free testosterone in males. Although there is no indication for measuring circulating parathyroid hormone, calcitonin, or the vitamin D metabolites routinely, blood levels of 25(OH)D are often appropriate in the elderly (particularly if housebound or institutionalized) because of their tendency to become vitamin D deficient. 35 A more rigorous search for secondary osteoporosis should be undertaken in premenopausal women and in men who are neither hypogonadal nor alcoholic. A similar rigorous search should be undertaken in patients whose measured BMD is not only > 2.5 SD below peak adult bone mass but is also > 2.0 SD below mean values after adjustment for age, gender, and ethnicity. Occult Cushing's syndrome, 4~ late presentations of osteogenesis imperfecta, and systemic mastocytosis may manifest for the first time with fragility fractures or marked spinal deformities (Fig. 12-4). These evaluations will be dictated by the clinical presentation, but from extensive clinical experience it should be noted that in occasional patients osteoporosis with an increased urine free cortisol may be the only manifestation of hypercortisolism. Similarly, patients may present with "osteoporotic x-rays" as the manifestation of nontropical sprue. A typical history in these patients who turn out to have osteomalacia and not osteoporosis, is one of frequent but regular bowel movements for 5, 10, or more years. It is not peculiar for the patient to have denied any change in bowel habits during this time when questioned by physicians. Finally, it cannot be overemphasized that multiple myeloma and skeletal metastases and primary hyperparathyroidism can present clinically in a manner indistinguishable from osteoporosis. 42 The clinician must maintain a high index of suspicion for these disorders when entertaining a diagnosis of osteoporosis. Osteoporosis in males deserves special mention. 43-46 Males do not generally report gonadal dysfunction to their physicians and physicians in turn infrequently enquire about gonadal function in their male patients. Yet 30% to 50% of males with osteoporosis have had unrecognized hypogonadism for a decade or more before presenting with an osteoporotic f r a c t u r e . 46 Far less common, almost rare in most practices, is "idiopathic" osteoporosis in young to middle-aged males. 47 This disorder, of unknown etiology but often associated with hypercalciuria, presents with acute back pain resulting from a fragility fracture of a vertebral body during the course of daily work or leisure activity in otherwise healthy males aged 35 to 55. The radiographs have no distinguishing characteristics other than the fracture and the initial, appropriate evaluation is to seek an occult malignancy as the cause of the fracture. Unless idiopathic osteoporosis is considered early and intervention started, several more vertebral fractures complicate this
seemingly self-limited disease (4 to 5 years) while the fruitless search for identifiable cause continues. It must be remembered that nothing about the evaluation or management of idiopathic osteoporosis should interfere with the remainder of the workup for malignancy. Whether or not stress fractures related to athletic activity represent osteoporosis or simply mechanical overload of an otherwise healthy, normal skeleton remains unresolved. There is no doubt that these are overload fractures and the patient should be counseled about reducing her or his physical activity until the fracture heals, restarting the activity at a less intense level. In women with stress fractures from physical activity (which frequently occurs in those serving with the armed forces) it is essential to obtain a complete menstrual history, as many of these women will have oligomenorrhea or even amenorrhea and can be assumed to have estrogen deficiency bone loss. In that event a direct measurement of BMD will most likely reveal low bone mass, but it is questionable whether this would be sufficient to convince the patient to cut back on her exercise program. Most such women are aware that altered menses results from their physical activity, but pay scant attention to this. Measurement of BMD in women with stress fractures who have demonstrated no change in menses is probably not indicated, since it is unlikely to lead to specific intervention other than advice to decrease the amount of physical activity. In men with stress fractures from physical activity it is probably appropriate to measure serum free testosterone. B MD measurement is probably not justified unless the circulating testosterone is low.
IV. CLASSIFICATION Of the many ways to classify osteoporosis we have elected to focus on the etiological classification provided in Table 12-3. In this system the only primary forms of osteoporosis are those in which there is no understanding of the pathogenesis. All other forms of osteoporosis, including the most common postmenopausal osteoporosis, are classified as secondary. Amongst the secondary osteoporotic syndromes there are many conditions where the proximate cause of bone loss is known (e.g., postgastrectomy) even though there is little known about the basic mechanism(s) underlying the bone loss. Finally, there is a group of diseases or conditions manifest by low bone mass and fragility fractures that mimic osteoporosis, which are best not labeled or classified as osteoporosis. The majority of these disorders are characterized by both a localized and/or generalized effect on the skeleton, while we consider osteoporosis a generalized phenomenon in most instances.
FIGURE 12--4
Severe vertebral kyphosis in a young female with Cushing's syndrome before (A and B) and following surgical intervention (C and D).
CHAPTER 12 Osteoporosis Pathogenesis and Therapy V. M A N A G E M E N T A. N o n p h a r m a c o l o g i c a l i n t e r v e n t i o n 1. LIFESTYLE Osteoporosis can affect anyone, including those individuals who pay very careful attention to their lifestyle in that they eat properly; participate in regular load-bearing exercise; do not smoke; consume alcohol in only moderate amounts; and have no diseases, conditions, or medication use that might predispose to osteoporosis. Patients with osteoporosis who are not quite so particular about their lifestyle must be counseled about all the activities they can change in their daily living that might slow down the progression of bone loss. Patients who have had vertebral fractures need very specific instructions concerning changes in their activities of daily living such that they learn to bend, lift, stoop, and so forth in a manner that does not place undue stress and strain on the spine. Similar advice should also be given to those with very low bone mass who have not yet fractured. This advice needs to be very specific and include not only the use of postural exercises and the use of posture training supports 48 but also such mundane items as instructing check-out personnel at the supermarket to place fewer items in each shopping bag, not lifting up the comers of heavy mattresses in order to neaten the sheets and blankets, and careful attention to bending over a crib to pick up a grandchild. Patients must be counseled that an osteoporotic fracture is a very good predictor of the next fracture, and while encouraged to remain as active as possible, the activity must be done properly. Patients at risk for a hip fracture need additional advice about fall prevention, particularly "fall-proofing" the home environment. Poor lighting, electric and telephone cords, loose scatter rugs, and highly polished floors can all contribute to an increased risk of falling with resultant fracture. Those with failing vision must wear proper corrective spectacles and make sure to change them when appropriate if moving from indoors to the outside. Travel outdoors should be with some form of ambulatory support in the frail elderly (less frail spouse, child or companion, cane, walker), particularly in wet or icy conditions. 2. NUTRITION While osteoporosis is not a calcium deficiency disease, there is ample evidence that an adequate calcium supply throughout life minimizes osteoporotic fracture risk later in l i f e . 49 In studies that reported cross-sectional data on BMD and hip fracture prevalence from two communities in Yugoslavia, the farming community (and one
397 with greater exercise) that consumed more calcium had higher BMD and lower fracture prevalence than the urban community that consumed less calcium. 5~While presumptive evidence that lifetime dietary calcium intake has an influence on osteoporosis risk, it is not clear whether other differences between these communities (e.g., daily physical activity) might have accounted for the observed differences. Calcium supplements have been provided to monozygotic twin boys with one from each pair receiving calcium for 3 years while the other received placebo. 51 The twins on calcium supplements, particularly those who were studied before puberty, had a higher BMD than their brothers on placebo. This benefit in BMD was not maintained after the calcium supplements were discontinued, again suggesting that lifetime calcium intake is important. These and other d a t a 52'53 resulted in a National Institutes of Health (NIH) Consensus Development panel deriving new figures for suggested optimum daily calcium intake throughout life (Table 12-4). 54 These recommendations do ensure that less than 5% of those consuming these recommended amounts will have dietary calcium deficiency. Since calcium is available in many foods and calcium supplements are inexpensive and generally free of side-effects, this is a reasonable community prescription. In contrast to the benefits of calcium in optimizing B MD and minimizing osteoporosis risk, the situation concerning calcium therapy for established osteoporosis is unclear. 52'53 The accelerated bone loss of the menopause can be retarded to some extent by large doses of
TABLE 1 2 - 4
Optimal Calcium Requirements a
Group Infants Birth-6 months 6 months- 1 year Children 1- 5 years 6-10 years Adolescents/Young Adults 11-24 years Men 25-65 years Over 65 years Women 25- 50 years Over 50 years (postmenopausal) On estrogens Not on estrogens Over 65 years Pregnant and nursing
Optimal Daily Intake (mg) 400 600 800 800-1200 1200-1500 1000 1500 1000 1000 1500 1500 1200-1500
aFrom NIH Consensus Conference: Optimal calcium intake. JAMA 272:1942, 1994.
398 supplemental calcium, but not to the same extent as is seen with estrogen replacement. Not surprisingly, greatest benefit is obtained in those women with the lowest habitual intakes (< 400 mg/day) and in those studied 5 or more years following menopause where the overwhelming effects of estrogen deficiency are dissipating. 52 There is inadequate data on other nutrients and osteoporosis. The renal handling of calcium is controlled to some extent by the renal handling of sodium such that a high sodium intake promotes hypercalciuria with mild acceleration of bone loss. 55 For most persons this does not pose a significant problem, but occasional patients with hypercalciuria also have increased urine sodium excretion. The hypercalciuria will resolve with a reduced sodium intake. This is particularly worth considering in elderly folk who either eat frequently at restaurants or who consume many prepared foods characterized by preservation with high salt contents. A high-acid-ash diet (protein rich) has been implicated as a potential aggravating factor for osteoporosis, but clearly an adequate protein intake must be consumed during growth and development of the skeleton. Excess caffeine is alleged to be a risk factor for the development of osteoporosis, but on the basis of very limited data. 56 Similarly, alcohol when taken to excess is a risk factor for osteoporosis (e.g., direct toxic effects on the skeleton, liver disease, poor general nutrition, hypercortisolism, excess falls). Since excess alcohol is a risk factor for poor health and life expectancy in general, it is really not necessary to focus on the osteoporosis risk when counseling patients to avoid excess alcohol. It should be acknowledged, however, that women who drink alcohol and use oral estrogens for estrogen replacement may have significantly higher circulating estradiol levels than those reported in studies advocating the use of estrogen replacement therapy. 57 Vitamin D is a co-factor in normal skeletal growth and development, but surprisingly little is known about lifetime requirements for this vitamin; 400 IU/day (from sunlight exposure and/or oral intake) is sufficient to prevent tickets in children, but it is not known that higher intakes result in a higher BMD. Adult requirements for vitamin D are unknown but, while undoubtedly less than in children, it seems prudent to maintain an intake of 400 IU throughout life. Unlike calcium, where excess intake is harmless for the most part, excess vitamin D intake (> 5000 IU/day) should be avoided to minimize the risk of vitamin D toxicity, especially in the elderly, since body vitamin D content decreases with age, primarily as a result of restricted sunlight exposure, reduced capacity of the sun to produce vitamin D, and reduced vitamin D intake. An important prospective trial was recently completed in France wherein elderly (70 years and older) institu-
MICHAEL KLEEREKOPER AND LOUIS V. AVIOL!
tionalized women have been randomized to receive either a placebo or 1000 mg calcium plus 800 IU of vitamin D daily. 58 During 3 years of prospective follow-up, those on the supplements have sustained significantly fewer osteoporotic hip and other nonvertebral fractures. It is not certain whether this observation would be valid in the United States, where the food chain is fortified with vitamin D. It is also unclear whether the benefit seen in this French study results from calcium, vitamin D, or both. Vitamin D deficiency is associated with a mild proximal myopathy and increased risk of falling, and the observed effects could be explained by an effect on muscle rather than bone. Whatever the mechanism, it must be recognized that many elderly, particularly if institutionalized, do not have adequate sunlight exposure and are at risk for vitamin D deficiency. It would seem prudent, safe, and cost-effective to recommend a daily supplement of 1000 mg of calcium and 800 IU of vitamin D to all persons over age 75 in order to minimize the occurrence of secondary hyperparathyroidism and high-bone-turnover osteoporotic syndromes. 59 3. EXERCISE
Immobilization results in accelerated bone loss, while vigorous load-beating exercise during growth and development results in increased BMD. Between these extremes the skeletal benefits of exercise are unclear. Since there are many nonskeletal benefits of a regular aerobic and load-bearing exercise program, it is appropriate for all persons to participate in such activities. At times of accelerated bone loss (menopause, disease, or medication) even vigorous exercise is of minimal help in retarding bone loss, and certainly cannot substitute for estrogen replacement, for example. However, there are reports testifying to the additive effects of weightbeating exercises and estrogens on bone mineral density in older women. 6~ Persons who have sustained osteoporotic fractures, especially of the vertebral body or proximal femur and to a lesser extent the wrist, must be encouraged to enter an active physical therapy/rehabilitation program. Even non-load-bearing activities such as swimming will be of overall benefit to these patients despite the limited likelihood of a direct benefit to BMD. Such a program will markedly diminish the chronic back pain of vertebral fractures, improve posture, and probably reduce the risk of further fracture occurrence, although this is by no means well documented. Likewise, rehabilitation is essential following a hip fracture to reduce morbidity and mortality. The improved balance achieved by these activities will also reduce the risk of subsequent falls and fractures. The importance of balance, coordination, and muscle strength and tone in minimizing risk of fall and fracture cannot be overemphasized. 61'62
CHAPTER 12 Osteoporosis Pathogenesis and Therapy
399
B. Pharmacological Intervention
femur. Whenever ERT is started, bone loss will be prevented, no matter how many years have passed since the menopause. However, gains in bone mass with ERT are quite small (1% to 2% per year for 1 to 2 years) so that it is essential to commence ERT as soon after the menopause as possible to minimize irreversible bone loss. This therapy prevents bone loss as long as it is administered, but bone loss resumes as soon as therapy is discontinued. Epidemiological studies suggest that a minimum of 10 years of ERT/HRT is required to obtain maximal prophylactic benefit. 67 In established osteoporosis (low BMD with or without fracture) ERT/HRT prevents further bone loss and is associated with slight gains in BMD on the order of 1.5% to 2.5% per year for 1 to 2 years, with maintenance of B MD thereafter. There are recent data suggesting that response of the skeleton to estrogen is enhanced in osteoporosis with larger gains in bone mass seen in older women compared to women in the first few years after the menopause. In one study conducted in women aged 65, 1 year of transdermal estradiol was sufficient to significantly reduce prospectively measured vertebral fracture occurrence. 68 There is a prevailing opinion that ERT/HRT should be administered and continued indefinitely in order to prevent fracture. 69 It is also worth emphasizing that ERT/HRT initiated after 60 years of age and continued thereafter offers almost comparable protection as would be anticipated when treating younger postmenopausal women. 7~ Estrogen suppresses bone remodeling with a reduction in biochemical markers of resorption and formation seen within 3 months of therapy. 72 Discontinuation of therapy also results in a rapid loss of bone and a return of these markers to pretreatment levels within 3 months of interrupting therapy. Given the poor patient acceptance of and compliance with ERT/HRT, and that the concerns linking therapy to an increased occurrence of breast cancer 73 are duration dependent, a case could be made for intermittent therapy. However, to our knowledge, to date no formal prospective studies of this approach have been recorded.
1. ESTROGEN For the prevention of rapid bone loss at the menopause there is little doubt that estrogen replacement therapy (ERT) is the most effective and cost-effective intervention. In women with an intact uterus, progesterone should be prescribed together with estrogen [hormone replacement (HRT)] to minimize and possibly eliminate the potential for ERT-induced carcinoma of the endometrium. There are many potential compounds, doses, and combinations of ERT and HRT as well as many nonskeletal benefits, side effects, and other considerations, but a full discussion of these is beyond the scope of this chapter. There have been few formal dose-finding studies, and even fewer studies comparing one therapeutic option with another. However, there is considerable evidence that 0.625 mg/day of oral conjugated equine estrogen (CEE), or equivalent (Table 12-5), is sufficient to prevent early postmenopausal bone loss in 90% to 95% of all women. 63 While relatively large doses of progesterone alone (e.g., 20 mg/day of medroxyprogesterone acetate) will retard bone loss without estrogen, there is no evidence that HRT imparts a skeletal benefit over ERT. 63 Combining 0.3 mg/day of CEE with 1500 mg/ day of a calcium supplement has been shown to be as effective as 0.625 mg of CEE alone. 64 There is little cost advantage to this approach, and the potential compliance benefit of lesser estrogen dose must be weighed against the observations that compliance, which is a characteristic problem for women who consider estrogen therapy, 65 generally falls after the first year of estrogen treatment. 66 At the menopause, ERT/HRT is effective at the spine and proximal femur, although some studies have suggested that a higher dose (> 0.625 mg CEE) may be required to fully prevent bone loss from the proximal
TABLE 1 2 - 5 Hormone Replacement Therapies Approved for the Prevention and Treatment of Osteoporosis in the United States Drug
Dosage
Premarin a
0.625 mg/day
PremPro
0.625/2.5 mg/day
Premphase
0.625/5 mg/day
Ogen a
0.625 mg/day
Estrace a
0.5 mg/day
Estraderm a
0.05 mg twice a week
aEstrogen-only preparations, usually prescribed with progesterone in women with intact uterus.
2. CALCITONIN Although the physiological role of calcitonin in humans has not been established, it is well documented that this hormone can profoundly inhibit osteoclast function and retard bone loss, particularly when due to increased resorption (i.e., high-turnover bone lOSS). 72-81 Clinical trials have been conducted with synthetic salmon, eel, and human calcitonin (hCT), with salmon calcitonin (sCT) being the most potent of these. Until recently, sCT was only available as an injectable preparation requiting self-administered subcutaneous injections, severely limiting patient acceptance of this therapy. However, sCT is
400
MICHAEL KLEEREKOPER AND LOUIS V. AVIOLI
now available as a nasal spray, with each metered dose providing 200 IU of the drug. The altered bioavailability of the nasal spray preparation makes this 200-unit dose approximately equivalent to 100 IU of the injectable preparations of sCT, which has also been approved by the FDA for osteoporosis therapy. The short- and long-term safety and efficacy of sCT in the treatment of osteoporosis has been well established during two decades of experience with the injectable preparation. Compared to patients treated with calcium alone, sCT increases B MD by approximately 1.5% to 2.0% over a 2-year period. 79 Placebo-controlled prospective studies are also consistent with reduction in fracture frequency. 79'8~Although intermittent therapy using 200 IU of nasal spray has proven effective in some postmenopausal patients, 82'83 this is not always the case 81 (Fig. 12-5). The response to calcitonin is similar to that obtained with ERT/HRT. 77 While there is a dosedependent response to sCT, side effects (nausea, flushing) limited the use of doses greater than 100 IU subcutaneously daily. Nasal spray sCT is not available in doses less than 200 IU/day, an effective dose for 75% of patients, and there is only minimal incremental benefit from 400 1-U/day.84 Short-term side effects of nausea and flushing are of minimal concern with this preparation. The major side effects relate to mild rhinitis and nasal stuffiness. Although sCT is currently considerably more expensive than ERT/HRT, it is certainly a viable alternative approach for those women who cannot or will not take estrogen therapy, and can also be considered appropriate therapy for nonhypogonadal males with osteoporosis. A few well-controlled short-term prospective studies in patients with vertebral fractures have indicated that VERTEBRAL BMD
3. BISPHOSPHONATES
1 0
SCT 200 iu daily
(n=29)
%
change
-1 SCT 200
2
iu M/W/F (n=29)
3
PLACEBO (n--39) 0
FIGURE 12--5
sCT has significant analgesic properties, 85'86presumed to relate to calcitonin-mediated release of endorphins. In these trials, which were conducted in patients where therapy was initiated in the immediate postvertebral fracture period, sCT (injectable and nasal spray) reduced overall morbidity, self-assessment of pain, and consumption of analgesics. 85'86 Since many clinicians report substantial albeit anecdotal experience of the analgesic benefits of sCT in the acute postfracture stage, this might be the best indication to commence sCT therapy. Without intervention, the acute pain following a vertebral fracture should subside within 6 weeks, but the patient is often left with chronic back pain. Since there are no data to suggest that sCT has any analgesic benefit for this chronic pain, it appears that the early benefit derives from local actions at the skeleton and not central, endorphin-mediated effects. An analgesic effect in patients with Paget' s disease of bone or skeletal metastases would support this local action. Estrogen, an equipotent inhibitor of bone resorption, has no apparent analgesic effects. While the mechanisms are unclear, as mentioned, relief of acute postfracture pain remains a good indication for initiating sCT therapy in osteoporotic patients who present with painful fracture syndromes. As with estrogen, sCT therapy can be shown to decrease rates of bone turnover and at least one study has indicated that intermittent (discontinuous) sCT therapy is effective. 8~ Certainly the major effect of sCT (and estrogen) to increase BMD is complete after 2 to 3 years, and it seems appropriate to reevaluate the therapeutic response at that time. At the end of therapy biochemical markers of remodeling should be in the normal range. These could be monitored at 3- to 6-month intervals, with therapy reinstituted as the markers begin to increase as a result of progressive bone loss.
6
12
18
24
(months)
Percentage change in vertebral BMD in all patients treated with either intranasal salmon calcitonin (sCT) 200 IU daily, sCT 200 IU twice weekly (MWF), or placebo. Vertical bars represent SEM (*P <-- 0.02). *Baseline mean +_ SD mg/cm 3 and mean % change. (From Ellerington MC, Hillard TC, Whitcroft SIJ, et al: Intranasal salmon calcitonin for the prevention and treatment of postmenopausal osteoporosis. Calcif Tissue Int 59:6, 1996.)
The bisphosphonate class of drugs are analogs of pyrophosphate in which the central oxygen atom has been replaced by a carbon atom, allowing many structural variations in the side chains (Fig. 12-6). These side-chain differences impart different properties and potencies to the several bisphosphonates available for the treatment of osteoporosis. The precise mechanism(s) of action of the bisphosphonates is uncertain, although direct actions on osteoclasts and osteoblasts have been implicated. 88 These agents have a high affinity for bone crystals and, once in the circulation (from an intravenous or oral route), accumulate in the skeleton where bone resorption is inhibited. Bisphosphonates have a very long half-life in the skeleton, 10 years or longer, but do not accumulate in any other organs. This organ specificity of bisphosphonates provides a wide margin of safety for therapeutic use, provided any potential for skeletal toxicity can
CHAPTER 12 Osteoporosis Pathogenesis and Therapy
O" CH3 I
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Astra; Boehringer Mannheim; tiludronate Gentili; Leiras; Rh6ne-Poulenc Rorer Sanofi
Boehringer Mannheim
I S I
bis-phosphonate
FIGURE 1 2 - 6 Chemicalstructure of available bisphosphonates commercially available at the time for therapy of osteoporosis. (From Faranci RM: Oral facies in the treatment of osteoporosis: a review. Curr Ther Res 56:831-851, 1995.)
be minimized. In addition to inhibiting bone resorption, bisphosphonates may also inhibit mineralization of bone, and it is the therapeutic range between these two opposing actions of this class of drugs (with respect to skeletal integrity) that governs clinical use of the several bisphosphonates currently available. 88 The dose of etidronate sodium, the first bisphosphonate clinically available, that impairs mineralization is similar to the dose required to inhibit resorption so that this drug can only be administered in an intermittent fashion. 89'9~With alendronate sodium, the most recent bisphosphonate to be approved for therapeutic use in the United States, there is approximately a 500-fold difference between the dose that inhibits resorption and the dose that impairs mineralization, making it a safe and effective-to-administer therapy. Other bisphosphonates in use outside the United States or in clinical trials at present (clodronate, ibandronate, pamidronate, risedronate, tiludronate) have a therapeutic profile similar to that of alendronate.
B isphosphonates are poorly absorbed from the intestinal tract (< 1% of an orally administered dose) so that they must be taken in the postabsorptive state, generally after an overnight fast. 88 Even this low absorption is abolished if there is food or other medication in the stomach so that after each morning dose it is essential to avoid any food, drink (other than water, which is advised), calcium supplements, or other medications for at least 30 minutes and preferably 2 hours after ingestion of the bisphosphonate. This class of drugs is remarkably free of side effects because of the very high affinity for the skeleton. The major side effects are seen in the digestive tract, with some nausea and abdominal discomfort. More worrying, but unexplained, is the potential for erosive esophagifis with the more potent bisphosphonates, particularly pamidronate 91 and alendronate. 92 With alendronate, esophagifis (which can occur in over 20% of patients on therapy 92) is minimized if the patients avoid a recumbent position for the first 30 minutes after taking the drug.
402 The longest clinical experience with bisphosphonates in the treatment of osteoporosis is with cyclical etidronate (400 mg/day for 14 days followed by a 12-week drug-free interval during which 1500 mg/day of calcium should be prescribed as a supplement). This regimen increases spinal BMD by approximately 2.5% to 4.0% over 2 years, with BMD remaining stable thereafter for up to 4 years. 9~ There is only a gradual decrease in B MD during the first 2 years following discontinuation of therapy and BMD will again increase, albeit to a lesser extent, when therapy is reinstituted. Hip BMD also increases with cyclical etidronate therapy, but to a slightly lesser extent than the increase in spine BMD. The two long-term placebo-controlled clinical trials of etidronate were not initially designed to monitor antivertebral fracture efficacy as a defined outcome variable of the studies. Nonetheless, a significant reduction in prospectively measured vertebral fracture occurrence was reported in individuals with more advanced osteoporosis. 93'94 However, while this drug is available by prescription in the United States, it is not approved by the FDA with an osteoporosis indication, presumably because of inadequate fracture data and some concerns about the longterm skeletal safety. In a recent study designed to compare the effects of estrogen to etidronate in osteoporotic individuals, 33% of patients treated with etidronate in the routine cyclical manner developed osteomalacia. These untoward effects were reversed when estrogen was added to the treatment regimen. 89 Alendronate sodium in a dose of 10 mg/day was approved for oral use in the treatment of postmenopausal osteoporosis in the United States in the fall of 1995. The indication was for postmenopausal women with established osteoporosis with fractures, and/or those in whom measured BMD at any site is more than 2.0 SD below peak adult bone mass. This is a seemingly new definition of osteoporosis by the FDA (compared to the WHO definition of osteoporosis as BMD more than 2.5 SD below peak bone mass) for unclear reasons. Parathetically it should be noted that nasal spray calcitonin, also approved by the FDA in the fall of 1995, is also indicated when BMD is more than 2.0 SD below peak bone mass. In the reported clinical trials with alendronate, almost 90% of subjects on therapy had an increase in spine BMD of at least 3% over 2 years, with the average increase being 6% to 8%, clearly greater than with ERT/ HRT, calcitonin, or etidronate. 95'96 In common with these other therapies, all of which inhibit bone resorption, the most rapid increase in spine BMD was seen during the first 2 years of alendronate therapy (Fig. 12-7). However, there was a further increase in spine BMD, at a much slower rate, during the third year of these studies. This has led to speculation that alendronate may have properties to increase bone formation (directly or indi-
MICHAEL KLEEREKOPER AND LOUIS V. AVIOLI
rectly) in addition to its documented inhibitory effect on bone resorption. 97 Only limited data are available concerning the preservation of spine BMD following cessation of alendronate therapy, but it appears that there is a very slow, insignificant decline in BMD during the first year off therapy. Femoral neck BMD also increased with alendronate (Fig. 12-7). Vertebral and hip fractures were a defined outcome variable of the alendronate trials, and these studies did demonstrate a significant 48% reduction in prospectively measured vertebral fracture rate and a 49% decrease in hip fractures. 98 It must be emphasized, however, that most of the subjects in these trials did not have any vertebral fractures at the start of the trials and that the fracture incidence on those on placebo was only 6.2%. Perhaps more important than the fracture data itself is the observation that stature was more preserved on the alendronate than on placebo, even in patients on alendronate who sustained a vertebral fracture. Thus it would appear that alendronate not only reduced fracture occurrence but also limited the severity of the fractures. Clodronate 99 and pamidronate 1~176 are also currently commercially available in a variety of international locales. Clodronate does not inhibit bone mineralization in doses recommended for osteoporosis therapy. Although there are reports of decrease in fracture frequency in patients with malignant skeletal disease treated with clodronate, extrapolation of these data to potential therapeutic effects of clodronate in postmenopausal osteoporotic populations should be considered conjectural at best. Moreover, enthusiasm for this bisphosphonate is dampened by the fact that it is a " w e a k " bisphosphonate and that some patients developed leukemia during clodronate therapy, although a causal relationship between the disease and the drug has yet to be established. Pamidronate is an aminobisphosphonate that is about 100 times more potent than etidronate in inhibiting bone resorption in experimental animals and, like clodronate, can inhibit bone resorption in doses that are not detrimental to bone mineralization and formation. This bisphosphonate has approval only for the treatment of hypercalcemia and Paget's disease in the United States. In open nonrandomized studies using a limited number of osteoporotic individuals, pamidronate has been administered either in a continuous (150 mg/day) or cyclical intermittent 250 to 300 mg/day (2 months o n - 2 months off) fashion. Continuous administration of the drug resulted in an increase in vertebral bone mineral density of 3% per annum for 5 to 6 years, whereas annual increments of 2.5% were recorded for the first 2 years of intermittent therapy with no further changes during the subsequent 3 years. More recent observations albeit in a limited population of postmenopausal osteoporotic women subjected to pamidronate in an oral dose of 150 mg/day revealed a 7.0% increase in vertebral and 5%
403
CHArteR 12 Osteoporosis Pathogenesis and Therapy
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increase in femoral trochanteric bone mineral density after 2 years. The results of administering tiludronate to postmenopausal osteoporotic populations are still limited and require more extensive follow-up and documentation. In doses of 100 mg/day for a 6-month period the drug has been shown to inhibit bone loss for at least 1 year using appropriate biochemical markers. 4. SODIUM FLUORIDE Each of the classes of therapies described in the previous three sections exert their effects on the skeleton primarily by inhibition of bone resorption. Any effect on bone formation, as has been suggested for alendronate, for example, is only a minor component of the therapeutic effectiveness. This can be seen in serial measurements of B M D where the most rapid increase is noted in the first year of therapy as the remodeling (resorption)
space is closed in. In sharp contrast, sodium fluoride (NaF) stimulates bone formation directly and gains in B M D are linear over time for at least 6 years a~ and probably for as long as 10 years. 1~ Although this response should be the ideal therapy for osteopenia and osteoporosis, more than 30 years of clinical research has failed to fully document the therapeutic efficacy and safety of this drug. All of the many studies confirm the linear increase in B MD in an apparent dose-dependent manner, with increases of approximately 6% to 8% per year with doses of 50 to 75 rag/day. The problems arise when this increase in spine B M D is not always translated 101,102 into a reduction in vertebral fracture occurrence. Most of the uncontrolled clinical trials have reported antifracture efficacy, 1~ while most of the controlled clinical trials have not been able to confirm this. Pak et al. recently demonstrated beneficial results with a daily dose
404 of 50 mg of slow-release (SR) NaF. The benefit was only apparent in patients whose BMD was 65% of peak bone mass or higher. 1~176 A second potential problem with fluoride therapy is the suggestion from some of the studies that the increase in spine BMD occurs at the expense of bone mass in the proximal femur, with a resultant increased occurrence of hip fractures. Obviously, if this is indeed the case, fluoride would be a totally inappropriate therapy for osteoporosis. While there are no doubt a few patients in whom fluoride-induced hip fractures are well documented, this has usually been in patients where very large doses have been employed. There are no data to support an increased occurrence of complete hip fractures when doses 75 mg/day or lower are used. What has been well documented are instances of the "painful lower extremity" syndrome complicating therapy at doses of 75 mg/day. ~~ The pathology in this syndrome is unclear, but it appears to be an exaggerated bone formation response to therapy with local accumulation of excess osteoid, not progressing to true fracture. Whatever the etiology of this syndrome, it does limit the potential effectiveness of NaF therapy. Fluoride therapy in the form of monofluorophosphate (MFP) has been recently investigated. While few long-term studies have been completed, preliminary data indicate that this is a viable alternative to NaF and may in fact be slightly more potent (per milligram fluoride). As with SR sodium fluoride, and most of the enteric-coated formulations, MFP does not appear to be associated with any of the gastrointestinal side effects that have been reported with non-enteric-coated NaF. Finally, recent reports of "calcium-depleted" states in individuals on NaF therapy, with associated increments in circulating parathyroid hormone, must also be acknowledged. ~~ In summary, although fluoride remains the most potent drug available with respect to increasing spinal BMD, there remain several concerns about its clinical safety and antifracture efficacy. The recent studies with SR NaF were favorably reviewed by an FDA advisory panel in the fall of 1995, but final approval by the FDA is still pending at this time. 5. IPRIFLAVONE
Ipriflavone is a flavinoid derivative that has recently been demonstrated to have both direct and indirect effects on the skeleton. ~~ Not only does this drug inhibit the differentiation of precursors to osteoblasts and inhibit bone resorption, it also appears to stimulate osteoblast production and activity. Limited short-term studies reveal therapeutic effectiveness in preventing bone lOSS.1~ A recent controlled clinical trial in 70 early postmenopausal women demonstrated that ipriflavone 200 mg t.i.d, orally increased BMD in the distal radius an average of 5% per year during 2 years of therapy. 113
MICHAEL KLEEREKOPERAND LOUIS V. AVIOLI
The greatest increase was seen in those women who had the lowest initial BMD. That same study also evaluated the effect of the therapy on spinal BMD in an additional 35 women and, while those on placebo lost bone, there was preservation (but no gain) of spinal BMD during the 2 years. 113 The dominant effect of treatment, which was well tolerated, was inhibition of bone resorption with a significant reduction in urinary hydroxyproline excretion. This drug, which is currently being used in a prospective fashion to determine its influence on vertebral fractures, clearly has potential as another antiresorptive therapy for osteoporosis. 6. CALCIDIOL~ CALCITRIOL~ AND ALFACALCIDOL Only a few formal studies of these polar metabolites of vitamin D have been completed. Calcidiol, or 25hydroxyvitamin D [25(OH)D], is the major storage form of the vitamin and probably will only be of benefit in that small proportion of patients with vitamin D deficiency secondary to intestinal malabsorption, such as patients with nontropical sprue. Calcitriol, or 1,25-dihydroxyvitamin D [1,25(OH)2D], may have a more central role in the treatment of osteoporosis, particularly in the elderly, but this is by no means well established. Calcitriol production by the senescent kidney appears to decline with age to a greater extent than can be accounted for by age-related declines in glomerular filtration rate. This may in part explain the mild increases in parathyroid hormone (PTH) seen with aging and contribute to bone loss via this mechanism. Controlled clinical trials with calcitriol in osteoporosis have yielded conflicting results. One multicenter study carried out in New Zealand demonstrated a significant difference in prospectively measured vertebral fracture rate between those on calcitriol plus calcium compared to those on calcium alone. However, most of this difference could be accounted for by a sharp increase in the number of fractures in the calcium-only group. ~4 A similar study conducted in the United States failed to demonstrate any differences in fracture rates between the two groups. 115 The biggest difference between these two studies was in the incidence of adverse biochemical events (hypercalciuria and hypercalcemia), which were of concern in the U.S. study but far less frequent in the New Zealand study. The reason for this marked difference remains unclear. Nonetheless, there is concern about the safety of calcitriol, since both hypercalciuria and hypercalcemia occur without symptoms 115'~16 and have potential for serious clinical consequences. Thus, frequent biochemical monitoring would be essential in patients receiving this therapy, adding to the expense and inconvenience of use. Currently, several pharmaceutical companies are exploring a number of analogs of calcitriol that may have more selective effects on bone and
CHAPTER 12 Osteoporosis Pathogenesis and Therapy on calcium homeostasis such that a skeletal benefit might be possible without concern for hypercalcemia, for example. 115'116 One of these, alfacalcidol [lce(OH)D3], has been utilized as a specific therapeutic modality primarily in Japan for the past 6 to 7 y e a r s . 117 Although placebo-controlled, double-blind, prospective studies are essential to substantiate the recorded effects of this agent, preliminary results indicate that treatment with alfacalcidol can decrease the incidence of new fractures in Japanese women, even in the absence of beneficial effects on B MD. 7. THIAZIDE DIURETICS The renal tubules generally handle sodium and calcium in concert such that factors that promote a natriuresis also promote calciuresis. One exception to this is the class of thiazide diuretics which, while promoting natriuresis, inhibit renal excretion of calcium. This has made these drugs useful in the treatment of hypercalciuric states, particularly when complicated by nephrolithiasis. On the assumption that at least some of the progressive skeletal loss that characterizes osteoporosis can be explained by excess renal losses of calcium, several groups have evaluated the potential of thiazides as therapy for osteoporosis. Most of this work has been retrospective or cross-sectional epidemiological studies of fragility fracture rates on thiazide users versus nonusers, with several studies demonstrating a protective effect. ~18 Prospectively, thiazides have also been shown to minimize bone loss, but these studies were largely adjunctive to studies where therapy was provided for nonskeletal indications. 119 More recent double-blind, placebo-controlled studies do support a causal relationship between thiazide-like diuretics and reduced bone l o s s . 12~ In the absence of firm data it is difficult to recommend the use of thiazides specifically for prevention and/or treatment of osteoporosis. However, current use of thiazides remains an important variable to consider when evaluating osteoporosis risk and therapy in an individual patient. When diuretics are being considered for a nonskeletal indication, it would seem prudent in the elderly at greatest risk for osteoporosis to favor the use of thiazides, recognizing the subtle side effects of chronic therapy such as hypomagnesemia and cardiac arrhythmias. Similarly, use of diuretics such as furosemide that promote calciuresis should be avoided as much as possible in those with osteoporosis.
C. On the Horizon 1. PARATHYROID HORMONE PTH is one of the most intriguing emerging therapies for osteoporosis. 121-124 It has long been known that
405 chronic excess PTH leads to increased bone resorption and progressive bone loss. Less well known, although the initial observations were made many years ago, is that the initial skeletal response to a single pulse of PTH is hypocalcemia and stimulation of osteoblasts. As the structure of human PTH became known and supplies of synthetic human PTH (hPTH) became available, investigators began studying this early response to pulses of PTH and noted that this hormone, when administered in a pulsatile fashion rather than chronically, was in fact trophic to the skeleton, resulting in gains in bone mass rather than the intuitively more obvious loss of bone. 123-125 Studies with intermittent hPTH have progressed over the last decade or so, indicating that this is a very safe, effective way to stimulate bone formation, with increases in BMD similar to those seen with NaF. Dose-finding studies are nearing completion as are studies using hPTH in combination with antiresorptive therapies such as estrogen. Shortterm studies also reveal that hPTH can prevent the bone loss that is usually observed in women following suppression of ovarian function. 26 Parathyroid hormone-related protein (PTHrP), a major factor in the pathogenesis of humoral hypercalcemia of malignancy, has homology with hPTH in its first 13 amino-terminal amino acids and binds avidly to PTH receptors. Given the interest and favorable response to hPTH it was a logical extension of studies with PTHrP (and analogs) to evaluate the potential use in osteoporosis. These studies have not progressed to human protocols to date but, by direct comparison with PTH in the same experimental models, there is very real promise for this approach to treatment of osteoporosis in the future. 127,128 2. SELECTIVE ESTROGEN RECEPTOR MODULATORS Several analogs of estrogen, with target-organ selective affinity for estrogen receptors, have potential to be either agonist or antagonist to the actions of estrogen. The first of these in widespread clinical use was tamoxifen as adjunctive therapy for breast cancer because of documented antagonism of this drug to the effects of estrogen on the breast. It has now been demonstrated that tamoxifen, like estrogen, is an agonist for skeletal tissue and affects lipid profiles in a favorable fashion. 129'13~ Women with breast cancer who experienced menopause as a consequence of cancer therapy sustain BMD on tamoxifen despite postmenopausal estrogen levels. 129 Node-positive breast cancer remains the major indication for tamoxifen therapy, but it may ultimately be considered a viable alternative for prevention of osteoporosis in women with breast cancer for whom estrogen is contraindicated and tamoxifen not otherwise indicated. As noted in patients treated with estrogens, tamoxifen therapy also results in endometrial hyperplasia
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and endometrial carcinoma. 131'132 As a consequence, other selective estrogen receptor modulators (SERMs) have been developed specifically as potential therapy for osteoporosis without this serious side e f f e c t . 133 Since drugs in this category suppress bone loss without inducing marked uterine effects, it is most likely that one or more of this class of drugs will become available for prevention and/or treatment of osteoporosis within the next decade. Currently, one of these SERMS, raloxifene, is in the final stages of clinical testing as an osteoporosis preventative in an international placebo-controlled study. 134,135
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120. Wasnich RD, Davis JW, He Y-F, et al: A randomized, doublemasked, placebo-controlled trial of chlorthalidone and bone loss in elderly women. Osteoporosis Int 5:247-251, 1995. 121. Lane NE, Kimmel DB, Nilsson MHL, et al: Bone-selective analogs of human PTH(1-34) increase bone formation in an ovariectomized rat model. J Bone Miner Res 11:614-625, 1996. 122. Boyce RW, Paddock CL, Franks AF, et al: Effects of intermittent hPTH(1-34) alone and in combination with 1,25(OH)2D3 or risedronate on endosteal bone remodeling in canine cancellous and cortical bone. J Bone Miner Res 11:600-613, 1996. 123. Tam CS, Heersche JNM, Murray TM, Parsons JA: Parathyroid hormone stimulates the bone apposition rate independent of its resorptive action: Differential effects of intermittent and continuous administration. Endocrinology 110:506-512, 1982. 124. Ma YF, Jee WSS, Ke HZ, et al: Human parathyroid hormone(1-38) restores cancellous bone to the immobilized, osteopenic proximal tibial metaphysics in rats. J Bone Miner Res 10:496, 1995. 125. Reeve J, Davies UM, Hesp R, et al: Human parathyroid peptide treatment of osteoporosis substantially increases spinal trabecular bone (with observations on the effects of sodium fluoride therapy). Br Med J 301:314-318, 477, 1990. 126. Finkelstein JS, Klibanski A, Schaefer EH, et al: Parathyroid hormone for the prevention of bone loss induced by estrogen deficiency. N Engl J Med 331:1618, 1994.
409 127. Weir EC, Terwilliger G, Sartori L, Insogna KL: Synthetic parathyroid hormone-like protein (1-74) is anabolic for bone in vivo. Calcif Tissue Int 51: 3 0 - 34, 1992. 128. Stewart AF: PTHrP(1-36) as a skeletal anabolic agent for the treatment of osteoporosis. Bone 19:303- 306, 1996. 129. Love RR, Mazess RB, Barden HS, et al: Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N Engl J Med 326:852-856, 1992. 130. Love RR, Barden HS, Mazess RB, et al: Effect of tamoxifen on lumbar spine bone mineral density in postmenopausal women after 5 years. Arch Intern Med 154:2585-2588, 1994 131. Seoud MAF, Johnson J, Weed JC Jr: Gynecologic tumors in tamoxifen-treated women with breast cancer. Obstet Gyneco182: 165-169, 1993. 132. Lahti E, Blanco G, Kauppila A, et al: Endometrial changes in postmenopausal breast cancer patients receiving tamoxifen. Obstet Gynecol 81:660-664,1993. 133. Sata M, Rippy MK, Bryant HU: Raloxifene, tamoxifen, nafoxidine or estrogen effects on reproductive and nonreproductive tissues in ovariectomized rats. FASEB J 10:905-912, 1996. 134. Tanouje E: New pill may offer safer alternative to estrogenreplacement therapies. The Wall Street Journal, August 2, 1995. 135. Brody JE: Drug researchers working to design customized estrogen. New York Times, March 4, 1997.
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7HAPTER 1S
Primary Hyp erp arathyro i di s rn JOHN T. POTTS, JR.
I. II. III. IV.
Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
Introduction Etiology and Pathology Clinical Features: Changing Clinical Presentation Diagnosis and Differential Diagnosis
V. Medical Management of Hypercalcemia and Hyperparathyroidism VI. Summary References
I. INTRODUCTION
calcemic parathyroid crisis. Patients may present with multiple signs and symptoms, including recurrent nephrolithiasis, peptic ulcers, mental changes, and, less frequently, extensive bone resorption. However, with greater awareness of the disease and wider use of multiphasic screening tests, including blood calcium determinations, the diagnosis is frequently made in patients who have no symptoms and minimal, if any, signs of the disease other than hypercalcemia and elevated levels of parathyroid hormone. The annual incidence of the disease is estimated to be as high as 0.2% in patients over age 60, with an estimated prevalence due to undiscovered asymptomatic patients of 1% or higher. 11-16 Prevalence (defined as the proportion of a defined population affected at a given point in time when screening tests are applied) is higher than true incidence rates (the number of new cases found on a yearly basis when a defined population is repeatedly screened), as is true in any disease that has many patients with few or no symptoms. Incidence figures, however, may be overestimated by ascertainment bias. When screening tests are systematically applied, it has been noted that incidence figures
Primary hyperparathyroidism is a generalized disorder of calcium, phosphate, and bone metabolism that results from an increased secretion of parathyroid hormone. 1'2 Hyperparathyroidism was first recognized as a clinical entity over 50 years ago, when it was discovered that surgical removal of parathyroid adenomas led to cure of the malady. 3-7 Albright and associates contributed considerably toward the understanding of the pathophysiology of this disease and described many of its protean manifestations. 8'9 An account of one of the first patients studied in depth was reported by Bauer and Federman. 1~ The excessive concentration of circulating hormone causes hypercalcemia and hypophosphatemia. There is great variation in the clinical presentation. The clinical manifestations of hyperparathyroidism may be subtle, and the disease may have a benign course for many years or a full lifetime. Rarely, the disease seems to appear abruptly, and patients may exhibit severe complications, such as marked dehydration and coma, so-called hyperMETABOLIC BONE DISEASE
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Copyright 9 1998 by Academic Press. All fights of reproduction in any form reserved.
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decline when repeated over a number of consecutive years ll. Prevalence figures for hyperparathyroidism, on the other hand, make the assumption that the majority of the patients in whom hypercalcemia is detected have hyperparathyroidism; careful tests, with clinical followup and/or the application of the specific parathyroid hormone (PTH) radioimmunoassay (RIA) to confirm the diagnosis, are not part of the data in the reported series.
II. E T I O L O G Y
AND PATHOLOGY
Normally, four parathyroid glands are present in the neck in tissue adjacent to the thyroid and/or thymus (Fig. 13-1). In a study of the distribution of normal glands in 160 autopsies of patients dying from causes unrelated to parathyroid disease, 156 subjects had four glands, three had five glands, and one had six glands. 7a (See Chapter 15 for further details of the anatomy of the parathyroids.) Enlargement of only a single parathyroid gland is sufficient to cause excessive hormone secretion (Fig. 132). The single enlarged gland is usually classified as a benign adenoma; only rarely is it a malignant carcinoma. Enlargement and hypercellularity of all four glands may also be seen (Fig. 13-3). Hyperplasia involving clear
FIGURE 13--1 Gross appearance of four normal parathyroid glands removed at autopsy to illustrate relatively small size and variation in appearance. (Courtesy of the Department of Pathology, Massachusetts General Hospital.)
FIGURE 13--2 A 0.2 g parathyroid adenoma arising from a parathyroid gland to illustrate greater size than normal glands. Adenoma is at top and is compressing remaining normal parathyroid tissue below. (Courtesy of the Department of Pathology, Massachusetts General Hospital.)
cells as a cause of primary hyperparathyroidism was described many years ago. 2 Later, appropriate emphasis was directed to the frequency of chief cell hyperplasia as a cause. 17 As many as 80% to 90% of patients with primary hyperparathyroidism have a single abnormal gland or adenoma as the only abnormality; usually an effective cure can be obtained by surgical removal of the single abnormal gland. 1'2'17-2s Although an adenoma of a single gland is regarded by most groups l'w-zs as the most common cause of primary hyperparathyroidism, there has been some controversy regarding cause and management that should be acknowledged. Several pathologists and surgeons have advanced a minority viewpoint, namely that hyperplasia in multiple glands is the most frequent cause of primary hyperparathyroidism, or at least much more common than generally thought. 29-33 The changes involved either diffuse hyperplasia and/or nodular hyperplasia. Focal hyperplasia within several parathyroid glands also has been proposed as the principal pathological entity in patients with primary hyperparathyroidism by one group, who use a special set of criteria for defining gland pathology in arriving at their classification. 32 One reason for the continuing dispute about the pathology is that it is often essentially impossible by either
CHAPTER 13 Primary Hyperparathyroidism
FIGURE 1 3 - 3 Gross appearance of primary chief cell hyperplasia of the parathyroids removed at surgery. Glands are irregular in outline and enlargement is variable. The fourth, a smaller gland, was left in the neck. (Courtesy of the Department of Pathology, Massachusetts General Hospital.)
gross or histological examination of a given gland to distinguish between adenoma and hyperplasia, and whether abnormalities are present in only one or all four parathyroid glands although, sometimes, as shown in Figure 13-4, the histology is helpful. We know now (see below) that both hyperplastic glands and solitary adenomas can be monoclonal cellular outgrowths. The most practical approach to resolution of the uncertainty would seem to be information regarding recurrence of hyperparathyroidism following resection of a presumed solitary hypercellular gland. Available studies have shown that recurrence of disease is rare after surgical removal of only one macroscopically enlarged parathyroid g l a n d . 25"26'34-36 It will be important, however, to measure both blood calcium and PTH levels at intervals in patients who have had a single gland removed to detect occult recurrence, if any, of hyperparathyroidism which if it occured could lead to symptomatic patients years later. On the other hand, careful follow-up studies seem necessary in those patients who have had extensive resection to detect borderline or definite hypoparathyroidism, the principal clinical concern with extensive resection. 36 Parathyroid carcinoma has been estimated to occur in approximately 3% of patients with hyperparathyroidism, 1'19-24'37-39 but more recent experience suggests that its incidence is much lower, perhaps 0.1% to 10~.27'38-40
4 13 This lower incidence reflects both the increasing recognition of the disease as asymptomatic or mild, and the application of strict criteria in classifying a tumor as carcinoma. Carcinoma of the parathyroid is often characterized by the tenacious adherence of glandular tissue to surrounding structures. Classic histological criteria of malignancy 41 include the presence of nuclear mitotic figures (normally none is seen in an entire section), fibrous bands, and capsular or blood vessel invasion. However, the criteria necessary for diagnosing parathyroid carcinoma are controversial because of overlap with apparently benign but atypical adenomas. 42-47 The diagnosis can be made unequivocally when extraparathyroid invasion or metastases are present; few would contest the diagnosis in the setting of locally invasive recurrent disease with positive histological criteria. Also (again see below), we now know that the cellular genetic defects are distinctive in carcinoma. Parathyroid carcinoma is usually not aggressive in character. 37-43 Long-term survival without recurrence is common if at initial operation the entire gland is removed without rupture of the capsule. 37-43 E v e n recurrent parathyroid carcinoma is usually slow-growing with local spread in the neck, and surgical correction of recurrent disease may be feasible. Occasionally, however, parathyroid carcinoma is more aggressive in character, with distant metastases (lung, liver, and bone) found at the time of initial operation. It may be difficult to appreciate initially that a primary tumor is carcinoma; increased numbers of mitotic figures and increased fibrosis of the gland stroma may precede invasive features. The diagnosis of carcinoma is often made in retrospect. Hyperparathyroidism from a parathyroid carcinoma may be clinically indistinguishable from other forms of primary hyperparathyroidism; a potential clue to the diagnosis of carcinoma, however, is provided by the degree of calcium elevation. Calcium values of 14 to 15 mg/dl are frequent with carcinoma; this finding may alert the surgeon to remove the abnormal gland with care to avoid capsular rupture. A specific genetic defect involving the retinoblastoma gene has been identified in a number of patients with parathyroid carcinoma (see below). Two major syndromes of multiple hereditary endocrinopathy are recognized and are genetically distinct. The first, termed multiple endocrine neoplasia type I (MEN-I), consists primarily of tumors of the parathyroid, pituitary, and pancreas. The second hereditary syndrome, termed multiple endocrine neoplasia type IIA (MENIIA), consist of medullary carcinoma of the thyroid, adrenal tumors (pheochromocytoma), and, again, tumors of the parathyroids. An alternate form of this syndrome called MEN-IIB is more aggressive with respect to the malignant character of the medullary carcinoma, lacks hyperparathyroidism, and shows additional features (Ta-
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FIGURE 1 3--4 Microscopic appearance of a chief cell adenoma of the parathyroid gland. Adenoma is in lower left half of the field. Normal parathyroid tissue with characteristic fatty stroma and islands of chief cells and oxyphil cells is seen in upper right field. (Courtesy of the Department of Pathology, Massachusetts General Hospital.)
ble 1 3 - 1 ) . Benign and malignant forms of familial hyperparathyroidism without associated manifestations of M E N have also been described, 48-58 including one synd r o m e in which cystic parathyroid adenomas are found together with ossifying fibromas of the jaw; this syndrome is clearly distinct from either M E N syndrome. 55'56 The other hereditary hyperparathyroid-like condition is familial benign hypocalciuric hypercalcemia (FBHH). F B H H is an autosomal dominant condition characterized by a cellular insensitivity to calcium, an altered parathyroid gland set point, and low urinary calcium excretion. 51'57'58 This condition is now understood also in terms of genetic defect (see below). W e r m e r 59 was the first to define the genetic aspects of the earliest recognized multiple endocrine syndrome, describing a pattern that featured pituitary, pancreatic, and parathyroid tumors (what is now termed MEN-I).
TABLE 13--1
Multiple Endocrine Disorders
MEN-I (autosomal dominant): 1. Parathyroid hyperplasia 2. Pituitary tumors A. Acromegaly B. Chromophobe 3. Islet cell tumors A. Gastrin B. Insulin C. Glucagon D. Others 4. Multiple lipomata 5. Positive carcinoid, adrenal adenoma, others MEN-IIA: Hyperparathyroidism, medullary thyroid carcinoma, pheochromocytoma MEN-IIB: Medullary thyroid carcinoma, pheochromocytoma, multiple neuromata, marfanoid habitus
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CHAPTER 13 Primary Hyperparathyroidism Wermer correctly deduced that the high frequency with which one or more of these tumors were seen in family members from affected kindreds was compatible with an autosomal dominant inheritance. The alternate syndrome, that of multiple endocrine neoplasia type II, was first reported, as a suspected separate clinical entity, by Sipple. 6~ Schimke and Hartmann, 61Schimke et al., 62 and Steiner et a l . 63 all noted the linkage between pheochromocytoma and medullary carcinoma of the thyroid as originally described by Sipple, plus the additional features of parathyroid tumors and mucosal neuromas. Steiner et a l . 63 deduced an autosomal dominant pattern of inheritance in a total of 29 families who exhibited pheochromocytoma, medullary carcinoma of the thyroid, and hyperparathyroidism, and suggested the term multiple endocrine neoplasia type II (MEN-II) for the disorder involving the thyroid and adrenal medulla inasmuch as tumors of the pituitary and pancreas, common in the alternate endocrinopathy (which was then renamed multiple endocrine neoplasia type I syndrome, or MEN-I), were absent from the kindreds with thyroid and adrenal tumors. Several studies suggested that the Zollinger-Ellison syndrome, 64'65 familial medullary thyroid c a r c i n o m a , 66'67 and familial pheochromocytoma, 63 may occur as well-established hereditary syndromes in certain families without any evidence, despite careful screening, of any other endocrine disorder. 68 Significant progress has been made recently in elucidating the molecular genetic mechanisms underlying
FIGURE 13--5
parathyroid tumor development. Analysis of kindreds with MEN-I and MEN-IIA and cytogenetic studies of tumor tissue from patients with solitary adenomas have documented that at least two molecular defects are involved in the genesis of the hyperparathyroid state, namely, overactivity of a protooncogene, or growthpromoting gene, and loss of function of growth-retarding genes or antioncogenes. 69-74 A gene locus on chromosome 1 1 appears to be responsible for MEN-I, and the normal allele of this gene is likely to be an antioncogene74'vs; the gene has recently been cloned, but has not yet been characterized functionally. TM The available data based on analysis of genetic events in neoplasia generally is as follows. In MEN-I, the loss of one allele is inherited as an autosomal trait, and loss of the other allele via somatic cell mutation leads to monoclonal expansion and tumor development in the parathyroids. In approximately 25% of sporadic parathyroid adenomas, the same locus on chromosome 1 1 appears to be lost, implying that the same defect responsible for MEN-I can also cause the common disease. 71'74 In MEN-I patients there is one defective allele in all cells at birth (often traceable from the affected parent), and in patients with sporadic adenomas one copy is lost in a parathyroid cell as a silent somatic mutation. In both disorders, the second allele becomes mutated or deleted in a parathyroid cell; this second somatic mutation results in a monoclonal outgrowth (Fig. 13-5). These findings also explain why hyperparathyroidism
Schematic illustration of the potential mechanisms of loss of growth control genes in parathyroid neoplasia. [From Arnold A: Genetic basis of endocrine disease. Molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab 77(5):1108-1121, 1993.]
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TABLE 1 3 - 2
Molecular Genetic Mechanisms In Parathyroid Adenomas a
9 Rearrangement and overexpression of PRAD1 (cyclin D1) 9 Inactivation of putative tumor suppressor genes Frequent allelic loss of DNA markers on: lp (most common, 40% of adenomas) 6q 1 lq (MEN-I region) 15q aclonal DNA defects currently demonstrable in 70% of parathyroid adenomas. (Based on data from Cryns VL, Yi SM, Tahara H, et al., 1995.)
develops earlier in the M E N syndromes (starts with a germ line defect) than with parathyroid adenomas (two somatic mutations required). Additional genetic abnormalities (loss of another antioncogene) seem likely to be involved in parathyroid adenomas because allelic loss (of heterozygosity) involving several specific genetic loci in addition to the MEN-I region is found in these tumors 7~ (Table 1 3 - 2 ) (loss of one allele at these additional chromosomal sites implies that, as in MEN-I, this event is a marker for loss/ inactivation of both gene copies, the mutation at the second allelic site being invisible to chromosomal analysis). Stimuli that cause excessive proliferation of parathyroid cells may favor such somatic mutations; more than 60% of patients operated on for secondary hyperparathyroidism (initially simple polycellular proliferation) (see Chapter 3) due to renal failure appear to have monoclonal proliferation associated with loss of heterozygosity that includes an X c h r o m o s o m e locus (Table 13--3). 69 Figure 1 3 - 6 schematically illustrates some of the cel-
TABLE 13--3
Monoclonal Parathyroid Neoplasms in Chronic Renal Failure a
9 63% of resected glands from hemodialysis patients with severe secondary/tertiary hyperparathyroidism were monoclonal neoplasma 9 64% of patients harbored at least one monoclonal parathyroid tumor 9 Independent clonal origins of discrete tumors in same patient aValues may underestimate true frequencies. (Based on data from Arnold A, Kim HG, 1995.)
lular proliferative effects that may occur in these parathyroid gland disorders. In secondary hyperparathyroidism, various stimuli (such as renal failure, phosphate retention, and low 1,25-dihydroxyvitamin D3[1,25(OH)2D3] lead to chronic hypocalcemia which in turn results in a generalized parathyroid cell hyperplasia that is not growth autonomous in that it reverses with correction of the hypocalcemic stimulus. 77'78 Frequently repeated cell divisions is a setting in which genetic mutations can occur; these in turn lead to clonal emergence and then clonal expansion of a growth autonomous parathyroid tumor, no longer reversible (Fig. 1 3 - 6 ) . In the hyperplasia of the MEN-I syndrome there may be a proliferative phase also before a clonal outgrowth occurs; data reported years ago suggested a circulating factor in these patients that is mitogenic for the parathyroids. 79 In some parathyroid adenomas, a second mechanism of abnormal growth is operative, namely, activation of a protooncogene. The synthesis of parathyroid hormone is under the control of a promoter that, in the presence of a reciprocal translocation involving c h r o m o s o m e 11, can also drive the expression of a gene product termed PRAD-1, later shown to be a D-cyclin protein that plays
FIGURE 13--6 Schematicillustration of sequential changes in the cells of the parathyroid gland during pathophysiological alterations. Initial stimulus is for generalized gland enlargementmall cells are involved in a generalized hyperplasia. One cell (dark) undergoes sufficient genetic alteration to become growth autonomous and begins a rapid expansion resulting in a large tumor with many identical cells (clonal expansion)m many dark cells. (Courtesy of Dr. Andrew Arnold.)
CHAPTER 13 Primary Hyperparathyroidism
41'/
a critical role in normal cell division. 73 If overexpressed (or mutated), cyclins contribute to abnormal cell replication. This translocation is found in 5% or more of parathyroid adenomas, usually in larger glands 73 (Fig. 13-7). Still different mechanisms appear to be operative in parathyroid carcinoma, namely, loss of both copies of the retinoblastoma gene. Hence, unlike the situation with some cancers in which there appears to be progression from benign to malignant neoplasms as cellular genetic defects accumulate, parathyroid adenomas do not seem to be precursors of parathyroid carcinoma 71 (Fig. 13-5). There are several familial forms of heritable medullary carcinoma and the MEN-II syndrome with specific phenotypes (Table 13-4). The two most common associated findings are pheochromocytomas, usually bilateral in distribution, and hyperparathyroidism, usually due to parathyroid hyperplasia (Table 13-1). In MEN-HA the frequency of hyperparathyroidism varies within families and between families, is less common than in MEN-I, and is usually mild with surgical management much more effective than in MEN-I (see Chapter 15). 8~ In MEN-IIB, in addition to the bilateral pheochromocytomas, there are other neuroectodermal features leading to a striking and distinctive facial appearance. These features, often termed the mucocutaneous syndrome or ganglioneuromatosis phenotype, include small polypoid neuromas of the eyelids, lips, and tongue, usually with profuse thickening of the lips (Table 13-4). Previously, the most reliable method for establishing the diagnosis of medullary thyroid carcinoma was the immunoassay for calcitonin; plasma calcitonin concentration may be increased before there is any clinical evidence of the presence of the tumor. There have been some limitations with the use of the calcitonin assay, however, especially in the aggressive MEN-IIB syndrome, which attacks at an early age 72 (Table 13-4). Some patients have been found to have metastatic disease even with early exploration. 72 Fortunately, recent genetic findings have established that mutations in the gene that encodes another
TABLE 13--4
FIGURE 13--7 Schematic illustration of the nature and consequences of gene rearrangement and overexpression of the PRADI protooncogene. [From Habener JF, Arnold A, Potts JT: Hyperparathyroidism. In DeGroot LJ, Besser M, Burger HG (eds): Endocrinology, 3rd ed, vol 2. Philadelphia, WB Saunders Co, 1995, pp 1044-1060.]
protooncogene, a tyrosine kinase, termed the RET protooncogene, accounts for most cases of familial medullary carcinoma, MEN-IIA and MEN-IIB. The locus of the mutation correlates with the phenotype (Fig. 13-8). In this disease, it is now possible to use formal genetic testing to direct patient care, including a firm recommendation for prophylactic thyroidectomy, especially
Types of Syndromes Associated with Medullary Thyroid Carcinoma Medullary Thyroid Carcinoma
Syndrome
Patient Age at Onset ( Y r s )
Familial MEN-IIA MEN-IIB
>50 <20 < 10
Multicentric 100% 100% 100%
Bilateral 100% 100% 100%
Pheochromocytoma 0% 10- 50% 10- 50%
HPT
GN
0% 10- 20% Rare
0% 0% 100%
HPT, hyperparathyroidism; GN, ganglioneuromatosis phenotype. (Modified from Ledger GA, et al: Genetic testing in the diagnosis and management of multiple endocrine neoplasia type II. Ann Int Med 122:118, 1995.)
418
FIGURE 13--8
The R E T protooncogene showing the different domains of the transmembrane by tyrosine kinase receptor protein and site of mutations in medullary carcinoma syndromes. (From Ledger GA, et al: Genetic testing in the diagnosis and management of multiple endocrine neoplasia type II. Ann Int Med 122:118, 1995.)
family members with the MEN-II syndrome who are found to have the mutated R E T protooncogene. 72 Since the R E T mutation is carried in all cells as a germ line mutation, this underlying mutation is necessary but not sufficient for the occurrence of a monoclonal outgrowth of a medullary carcinoma; other factors must cooperate but are as yet unknown. The principal practical utility of this assay is that it makes it possible to make the diagnosis definitely and operate early to prevent metastatic spread of medullary carcinoma. 72
III. CLINICAL FEATURES: C H A N G I N G C L I N I C A L PRESENTATION Primary hyperparathyroidism is diagnosed most commonly between the fifth and seventh decades. 1'15'19'2~As many as 50% to 80% of patients seen at major medical referral centers present with no clearly identifiable symptoms attributable to hyperparathyroidism. 82'83 Patients who have no symptoms traditionally attributable to hyperparathyroidism, however, can have occult signs attributable to the disease, such as nephrolithiasis or osteopenia, or may have intercurrent medical conditions that may or may not be causally related to the hyper-
JOHN T. POTTS, JR. parathyroidism, such as hypertension, joint complaints, or depression. Patients with asymptomatic hyperparathyroidism are defined as those with presumptive hyperparathyroidism who have neither symptoms nor signs commonly attributable to the disease. 83 How to optimally manage such patients led to the convening of a National Institutes of Health (NIH) Consensus Conference to discuss the issues thoroughly as reviewed in several subsequent sections. 83 Hyperparathyroidism is primarily a disease that concerns morbidity and not mortality. The death rate attributable to hyperparathyroidism in the United States in 1986 was only 57 for the entire population. 84 However, the data on death certificates are usually incomplete for endocrinological disorders, 85 so that the numbers of deaths due at least indirectly to hyperparathyroidism and/or hypercalcemia may be considerably higher than these figures indicate. On the other hand, some recent reports suggest that hyperparathyroidism may be a risk factor for an earlier mortality than would be expected in an elderly population in whom hyperparathyroid patients were compared with an age-matched group that had untreated primary hyperparathyroidism. These reports (which have not been prospective nor randomized but largely retrospective in some cases or small series in which hyperparathyroid patients were compared to agematched controls) have stressed cardiovascular causes of death or potentially reversible cardiac functional abnormalities. 86-94 NIH Consensus Conference participants concluded that the issue of whether increased mortality is associated with the disease should be properly investigated by a prospective study of sufficient size and design to provide a definitive answer. 83 For the present, it was not accepted that hyperparathyroidism causes increased mortality but, rather, that it was possible to recommend that patients over the age of 50 be placed under medical surveillance if criteria regarding bone mass and renal function were met in truly asymptomatic patients (see Section V. B). The manifestations of renal and skeletal disease in patients presenting with primary hyperparathyroidism have changed dramatically over the last two decades. The classic manifestations seen earlier--high frequency of kidney stones and severe bone disease with accompanying severe morbidity and m o r t a l i t y - - h a v e been replaced by more subtle changes, such as mild renal insufficiency and diffuse osteopenia with minimal morbidity and essentially no mortality. The reason for this change in disease manifestations is almost certainly the improved diagnostic approaches that have uncovered a tenfold greater prevalence of primary hyperparathyroidism in the population, much of it milder disease, and have led to detection of the disease at early stages of development. 83 It is unusual to encounter osteitis fibrosa
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CHAPTER 13 Primary Hyperparathyroidism cystica or severe hypercalcemia in patients with hyperparathyroidism; even nephrolithiasis is much less frequent. The absolute numbers of patients with hyperparathyroidism who have severe renal and bone disease may be little different from that seen earlier, but because the overall pool of patients with the disease has expanded by as much as tenfold, 80% of the patients have the mild form of the disease.
A. R e n a l M a n i f e s t a t i o n s In earlier decades, kidney involvement (particularly, recurrent nephrolithiasis) was reported in 60% to 70% of patients. 95-1~ In recent studies, nephrolithiasis was found in only 5% to 10% of patients. 15'~~ A review of clinical features and factors that infuence the occurrence of renal disease in hyperparathyroidism is helpful because some patients still exhibit organ dysfunction even if the incidence is greatly reduced. The observations suggest that it is merely the relative incidence of the complications that is declining, and continuing study of potential causative factors may be useful to protect asymptomatic patients who elect medical surveillance. A variety of renal function abnormalities occur in hyperparathyroidism, even in the absence of detectable nephrocalcinosis and/or nephrolithiasis. 1~176 These include elevation of blood urea nitrogen and serum creatinine, reflecting modest to marked reduction in glomerular filtration rate, and numerous renal tubular defects, particularly impairment of proximal tubular function. Parathyroid hormone has multiple direct effects on renal tubular function. In severe hyperparathyroidism these effects cause a reduction in net acid secretion--renal tubular acidosis (RTA) of the proximal (or type II) category, proximal RTAI~ well as aminoaciduria and glycosuria. Increased phosphate clearance with readily detectable abnormalities in measured parameters of renal tubular phosphate reabsorption leads to fasting hypophosphatemia. These overall defects occur in a continuous spectrum of severity and are influenced by anatomical changes in the kidney, such as diffuse calcium deposition (nephrocalcinosis) and the inflammation due to bacterial infection. With careful evaluation of renal function, it is found that (proximal) RTA is a rather common finding in primary hyperparathyroidism. This situation frequently results in mild hyperchloremic acidosis and reduction in serum bicarbonate. On the other hand, 1~176 hypercalcemia p e r se produced experimentally 1~ or in disease states associated with hypercalcemia not due to hyperparathyroidism, such as carcinoma and acute vitamin D intoxication, is usually associated with mild alkalosis rather than a c i d o s i s . 1~176 Hyperchloremia and/or mild
reduction in serum bicarbonate helps to distinguish hyperparathyroidism from other causes of hypercalcemia, 111'112 although the distinction is not always seen. 113 Partial or complete reversibility of some or all of the defects in glomerular function and tubular function including acidification mechanisms and renal concentrating ability has been documented after surgical correction of hyperparathyroidism. 6'1~ (See Chapter 25 for a discussion of the nature and causes of nephrolithiasis and the previous edition 1~9 for a tabular analysis of the changing pattern of renal involvement of hyperparathyroidism over recent decades.)
B. S k e l e t a l I n v o l v e m e n t There is clearly also a changing pattern of the manifestations of skeletal disease in hyperparathyroidism. Osteitis fibrosa cystica, the classic pathognomonic form of skeletal disease in hyperparathyroidism, 12~ is declining sharply in relative frequency. In its place, a subtle, often asymptomatic form of skeletal disorder is being detected that resembles the diffuse osteopenia characteristic of osteoporosis. Recent studies 123 have confirmed earlier reports 102.124that skeletal symptoms are infrequent and bone density, especially in trabecular bone, is spared. In an analysis of 138 cases of primary hyperparathyroidism, 96 it was noted that, during the decades between 1930 and 1949, 53% of patients had symptomatic generalized osteitis fibrosa cystica confirmed by radiological examination. In the decade between 1949 and 1960, only 21% were so afflicted, 96 and between 1965 and 1973 only 9% of 57 patients seen had skeletal pain or evidence of osteitis fibrosa cystica. TM No skeletal symptoms at all were detected in the 58 patients with primary hyperparathyroidism seen at the Henry Ford Hospital from 1980 to 1983.1~ On the other hand, all patients with hyperparathyroidism show skeletal changes consistent with high turnover. In one careful study, 125 it was shown that all patients, when examined histomorphometrically, had evidence for increased turnover relative to age-matched controls; elevated values of osteoid surface and volume, as well as mineralized surfaces, were increased. Recent reports have again emphasized that cortical bone thickness is significantly lower than normal, whereas cancellous bone volume was significantly greater. 123'126 Current techniques of noninvasive measurements of bone mass include single- and dual-photon absorptiometry, quantitative computed tomography (CT), and dual energy x-ray absorptiometry (DEXA). 127 The precision of these techniques is very high. 125'127Measurements that reflect high content of trabecular bone, such as spinal vertebrae and the trabecular portion of the iliac crest, were
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FIGURE 13--9
A, Magnification of skull x-ray film from a patient with primary hyperparathyroidism (top) and of a normal subject (bottom) to emphasize the appearance of localized resorption. [Courtesy of the Department of Radiology, Massachusetts General Hospital; from Potts JT Jr, Deftos LJ: Parathyroid hormone, calcitonin, vitamin D, bone and bone mineral metabolism. In Bondy PK, Rosenberg LE (eds): Duncan's Diseases of Metabolism. Philadelphia, WB Saunders Co, 1974.] B, Radii and ulnae of a patient with primary hyperparathyroidism. Severe osteopenia, increased trabeculation, and large areas of cystic degeneration, are evident. (Courtesy of the Department of Radiology, Massachusetts General Hospital.)
CHAPTER 13
Primary Hyperparathyroidism
FIGURE 13--10
Hand films of patient with hyperparathyroidism and severe osteitis fibrosa cystica before (A) and after (B) removal of parathyroid adenoma. Cystic destruction of bone, subperiosteal resorption, and a pathological fracture of the second proximal phalanx are evident. Some remineralization has occurred since the parathyroidectomy. (Courtesy of the Department of Radiology, Massachusetts General Hospital.) C, Magnification of x-ray film of normal finger (left) and finger from two patients with primary hyperparathyroidism (middle and right) to emphasize typical features. The abnormal fingers show decreased demineralization in the phalangeal tuft (middle) and subperiosteal bone resorption (middle and right). [Modified from Potts JT Jr, Deftos LJ: Parathyroid hormone, calcitonin, vitamin D, bone and bone mineral metabolism. In Bondy PK, Rosenberg LE (eds): Duncan's Diseases of Metabolism. Philadelphia, WB Saunders Co, 1974.]
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FIGURE 1 3 - 1 1 Microscopic appearance of osteitis fibrosa cystica in bone from an iliac crest bone. A, Extensive replacement of marrow with fibrovascular tissue; cystic cavities are evident. Osseous trabeculae showed evidence of both osteoclastic resorption and areas of osteoblastic bone formation. B, Classic appearance of dissecting osteitis within lamellar bone of trabeculae; several areas of fibrous replacement of marrow are evident. (Courtesy of Department of Pathology, Massachusetts General Hospital.)
CHAPTER 13 Primary Hyperparathyroidism often found to be normal or slightly increased, whereas areas where cortical bone is more dominant, such as the appendicular skeleton, are lower than expected. It has been observed that although bone density may be lower at the time of initial examination, there was no further loss over an average of 3 years. ~23'128It was speculated that in many patients there may be an initial period of rapid development of manifestations of the hyperparathyroidism, with consequent loss in bone mass, followed by a period of more stable disease with little progression. ~29 Clinically, however, a number of features of bone disease, such as the presence of osteitis fibrosa cystica, are of importance in providing diagnostic clues to the presence of the disease or in helping to assess the need for surgical treatment as well as the response of the patient to surgical correction. When severe osteitis fibrosa cystica develops, radiological examinations or bone biopsy can directly establish the diagnosis of hyperparathyroidism (Fig. 13-9). The most important roentgenograms for the diagnosis of generalized osteitis fibrosa cystica are those of the hands in the posteroanterior view ~3~ (Fig. 1 3 - 1 0). In the majority of patients with roentgenographically demonstrable bone lesions, subperiosteal erosion of the radial aspects of the middle phalanges is seen. In advanced stages of the disease, there is evidence of erosion of the outer cortical surfaces of bone through the skeleton, generalized demineralization of bones, and localized destructive lesions, often of cystic character. Histological examination of bone specimens from patients with severe osteitis fibrosa cystica reveals a number of changes that collectively define the presence of parathyroid overactivity (Fig. 13-1 1). There is a reduction in the number of trabeculae, an increase in multinucleated osteoclasts seen in scalloped areas on the surface of the bone (Howship's lacunae), and a marked replacement of normal cellular and marrow elements by fibrovascular tissue. In general as discussed below in Section on V.B., the relative stability of bone mineral density especially in trabecular bone in patients with hyperparathyroidism has been crucial to a rationale for nonsurgical treatment but rather medical surveillance. However, the natural history of the skeletal response to parathyroidectomy has not been established. Recent reports have confirmed the relative stability of bone mineral density for up to 6 years in a group of carefully observed hyperparathyroid patients. 123 Confusing, however, is the report from the same group that in a subset of patients who were operated upon there was a progressive increase in bone mineral density of up to 12% over 4 years at lumbar spine and femoral neck, less at the radius. TM This degree of change could be significant for bone strength and fracture prevention. Since this subgroup triggered guidelines for surgery, their disease may have been more severe than the
423 majority of the study group, whose bone density remained stable. The results, however, reveal the continuing uncertainty about the natural history of the disease and the potential value of a prospective study of a larger group of patients. ~26
C. Neuromuscular and Neuropsychiatric Manifestations Profound muscle weakness and atrophy were recognized in several of the earliest described cases of severe hyperparathyroidism. 2'132 The majority of patients seen today lack clear-cut evidence of neuromuscular syndromes, although some observers point to a high frequency of mild weakness that improves with surgical treatment. 133-137 The pathophysiological features of neuromuscular involvement seen in some patients with hyperparathyroidism have been studied in some detail. 124'133 Symptoms can include extreme weakness and fatigue, particularly involving proximal musculature (the lower more frequently than the upper extremities); often the symptoms disappear dramatically with corrective surgery. There is, however, considerable disagreement concerning the frequency of these symptoms. Although most reports, including the majority opinion of the NIH Consensus Conference on the topic of asymptomatic hyperparathyroidism 83 stress that many patients have no neuromuscular symptoms at all, 15'1~ some studies have continued to emphasize the frequency of such symptoms. 2~ The higher incidence in a few centers may reflect ascertainment bias as a result of referral patterns or the severity of hyperparathyroidism at the time of diagnosis. In the specific syndrome that reverses with parathyroidectomy, the muscle weakness may be so profound that an initial diagnosis of amyotrophic lateral sclerosis, muscular dystrophy, or other serious, irreversible neuromuscular disorder is entertained. 133 Gross atrophy may be detectable in involved muscle groups. Striking reversal of muscle weakness and atrophy is noted in these patients after successful correction of the hyperparathyroidism. Patten et a1133 emphasized that the overall findings, including microscopic examination of the affected tissue, are consistent with a neuropathic rather than a myopathic origin of the neuromuscular disease. In addition to the neuromyopathic manifestations of hyperparathyroidism, mental disturbances and impairment of higher central nervous system (CNS) functions in some patients are reported. 96 ' 135 , 31 6 - 1 4 5 Depression, personality changes, psychomotor retardation, memory impairment, and, occasionally, overt psychosis may occur. What is not established, as was evident in lengthy discussions at the 1990 NIH Consensus Conference, 83 is a clear cause-and-effect, as distinct from an associative
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linkage. In severe hyperparathyroidism, mental obtundation and coma may be observedl4~ the debate is whether psychiatric symptoms are seen commonly in mild hyperparathyroidism, are due to the disease, and reverse after parathyroidectomy. 83 In some reports, a striking improvement in a patient's sense of well-being, including relief of fatigue and depression that may not have been noticeable prior to surgery, is r e p o r t e d . 96'146 In one s u c h study, 96 59 patients with primary hyperparathyroidism were evaluated using a comprehensive psychopathological rating scale both before and after the successful parathyroid surgery. Most of the patients had symptoms of depression before surgery that were greatly ameliorated after the surgery. Symptoms that were elicited and that improved after surgery included fatigability, lassitude, concentration difficulties, sadness, indecision, inner tension, and aggressiveness. It should be emphasized, however, that mild depression, fatigue, and vague constitutional complaints alone are difficult to accept as an indication for surgery because these symptoms are so common in the population in the absence of hyperparathyroidism, especially among elderly people. What is lacking in these otherwise interesting clinical s t u d i e s 96'134-142'146 is an appropriate control population and study design to eliminate placebo effect (e.g., positive reinforcement from physician and hospital attention), and controlled, long-term follow-up of the patients' clinical course. 83
D. G a s t r o i n t e s t i n a l I n v o l v e m e n t Peptic ulcer disease was seen in earlier decades in patients with primary hyperparathyroidism with such high frequency that many investigators concluded that ulcer disease is causally related to the hyperparathyroidism. As with neuropsychiatric symptoms, the incidence of the two diseases in the population must be considered if a causal relation is to be established. Even if it is true, as seems likely, that the incidence of ulcer disease in asymptomatic hyperparathyroidism is much l o w e r 15'19'96 than that found in earlier reports in symptomatic disease, 147 an increased incidence in symptomatic patients greater than that found in the control population would suggest a causal relationship, particularly given the known effects of hyperparathyroidism to increase gastric acid production, as discussed below. Some patients with peptic ulcer disease, particularly those with severe symptoms and multiple ulcers, are found to have Zollinger-Ellison syndrome 148 or MEN-I with gastrin-producing tumors or diffuse hyperplasia of the pancreatic islet cells. These patients, however, constitute a small minority of the patients with hyperparathyroidism and peptic ulcers; most patients have no recognized abnormalities of the endocrine pancreas.
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The etiological basis for the increased incidence of ulcer disease in hyperparathyroidism may be increased gastrin secretion and resultant increased gastric acid production, induced by the hypercalcemia. 149 Levels of gastrin are elevated, although lower than in ZollingerEllison syndrome. In hyperparathyroidism, both basal acid secretion 15~ and serum immunoreactive gastrin 15~ are increased. Following parathyroidectomy, the excessive acid production, hypergastrinemia, and peptic ulcer symptoms usually return to n o r m a l . 149'15~ Evidence indicates that the increased gastrin secretion and hyperacidity in hyperparathyroidism are due to the hypercalcemia and not to direct effects of the PTH on gastrin production or on acid production p e r s e . Even in Zollinger-Ellison syndrome, the hyperacidity, elevated gastrin levels, and peptic ulcer symptoms may be reversed acutely by parathyroidectomy. 15~ Long-term follow-up of some of these patients has revealed ultimate recurrence of the Zollinger-Ellison syndrome, however, despite the persistence of normal parathyroid function as would be expected given the benign or malignant pancreatic tumors that overproduce gastrin in the ZollingerEllison syndrome. 155 Thus, although most patients with hyperparathyroidism diagnosed today do not have ulcer symptoms, specific management issues do arise when the two diseases coexist. Pancreatitis was believed to be another gastrointestinal manifestation of hyperparathyroidism. 156'157If an association of pancreatitis and hyperparathyroidism exists, the basis for it is u n k n o w n . 158-162 Later studies of patients with surgically proven hyperparathyroidism, however, have failed to show a high frequency of coexisting pancreatitis. 163 Although a cause-and-effect relation between pancreatitis and hyperparathyroidism is uncertain, awareness of this association does have several practical consequences. One should still be aware of the possible development of pancreatitis as a complication of severe hyperparathyroidism, particularly in acute parathyroid crisis, a condition in which an incidence of 25% has been reported. 164 It is also well-documented that both acute and chronic pancreatitis (the latter due to malabsorption) may cause a decrease in serum calcium concentration to deceptively normal or even low levels in hyperparathyroidism. Thus, the findings of even normal levels of serum calcium in the face of pancreatitis may be a clue to the concomitant existence of primary hyperparathyroidism. 159
E. A r t i c u l a r M a n i f e s t a t i o n s A number of articular and periarticular disorders have been recognized in association with primary hyperparathyroidism, such as chondrocalcinosis with or without
CHAPTER 13 Primary Hyperparathyroidism acute attacks of pseudogout; juxtaarticular erosions; subchondral fractures; and traumatic synovitis, calcific periarthritis, and u r a t e g o u t . 165-167 The frequency of chondrocalcinosis in primary hyperparathyroidism was reported to be from 7.5% to 18% in the 1 9 6 0 ' S . 168'169 As with the other disease manifestations, the incidence of articular diseases in hyperparathyroidism seems much lower at present. It may still be cost-effective to screen patients with chondrocalcinosis for hyperparathyroidism, however.
F. H y p e r t e n s i o n There have been numerous reports that hypertension occurs more frequently in hyperparathyroidism, particularly with elderly patients, than in the general population. 136'17~ This thesis has been challenged recently, however, as many patients with milder forms of hyperparathyroidism are evaluated. 83'185Earlier estimates of the prevalence of hypertension in patients with primary hyperparathyroidism ranged from 30% to more than 6 0 % . 171'173'178'179'186 As the prevalence of hypertension in the general population is estimated to be from 20% to 30%, depending upon age and other factors, 187 it is necessary to question seriously whether a true association exists between hypertension and hyperparathyroidism based on statistical analysis of prevalence below data and the ascertainment bias in hospitalized, surgically treated patients. At the present time, no single mechanism explains hypertension as a correlate of hyperparathyroidism; there is little agreement also on the reversibility of the hypertension with parathyroidectomy. 1'177'184 Recent studies 185 have emphasized that patients with asymptomatic hyperparathyroidism do not have either systolic or diastolic blood pressure that differs from euparathyroid age-matched individuals. With regard to reversibility of hypertension following parathyroidectomy, despite one report, ~84 the weight of the evidence indicates that there is little benefit. 1'177'188 Thus, even if hyperparathyroidism contributes to hypertension in some patients, reports that it remains essentially unabated following successful parathyroidectomy and cure of hyperparathyroidism 1'177'188led the NIH Consensus group to reject the thesis that hypertension is an absolute indication for surgery in hyperparathyroidism. 83
G. O t h e r Clinical M a n i f e s t a t i o n s Acute primary hyperparathyroidism (parathyroid poisoning, parathyroid crisis, parathyroid intoxication) represents an acute exacerbation of primary hyperparathyroidism. 189 The syndrome features acute or subacute
425 onset of life-threatening hypercalcemia (> 15 mg/dl) with anorexia, nausea, polyuria, polydipsia, dehydration, and lethargy, rapidly progressing to confusion and, in a number of cases, coma. Parathyroid hormone levels average 20 times above normal. 189 Medical therapy is useful but sometimes ineffective in severe cases. Rapid diagnosis by PTH assay and emergency parathyroidectomy is the recommended management. The parathyroid pathology is usually a large benign adenoma. Precipitating causes are unknown; there is a sudden stimulus to excessive PTH secretion, then a cycle of worsening hypercalcemia, dehydration, and finally deteriorating renal function. Typically, patients have evidence of bone and renal disease, so a severe form of hyperparathyroidism is underlying. Parathyroid carcinoma can present similarly and is occasionally seen at surgery in some typical cases of parathyroid c r i s i s . 189 Primary hyperparathyroidism in patients under 16 years of age is rare. 19~ Two types of primary hyperparathyroidism are recognized, one occurring in neonates and the other during childhood. In neonates, serum calcium levels may be above 15 mg/dl and were reported to be as high as 30.2 mg/dl in one case. ~92 Neonatal hyperparathyroidism is often genetically transmitted as an autosomal dominant trait and is fatal unless recognized and treated early. Near-total parathyroidectomy is required to control the disease. The prognosis following surgery is good, provided that a delay in diagnosis and treatment has not resulted in irreversible changes. This condition may result from a double dose of the gene believed responsible for the otherwise benign syndrome known as familial hypocalciuric hypercalcemia 193'~94(see below and Chapter 16). In children, as distinct from neonates, hyperparathyroidism usually does not have known genetic inheritance, is caused by parathyroid adenomas, and should be considered merely an early onset of adult-acquired primary hyperparathyroidism. 195-198 Rickets can occasionally occur in children with primary hyperparathyroidism. 199 Rickets is usually mild but may be severe in some. The proposed pathogenesis of the rickets is that PTH enhances bone resorption and turnover, there is renal wasting of phosphate, and dietary calcium intake is limited due to anorexia. The combination of these factors leads to a deficiency of bone mineralization in the rapidly growing skeleton of the child. Primary hyperparathyroidism is a rare disease during pregnancy. 2~176176 If the diagnosis is overlooked, however, the subsequent hypercalcemia often has deleterious effect on both mother and fetus. The serious complic a t i o n s ~ o f which neonatal tetany, stillbirth, and abortion are the most f r e q u e n t ~ c a n usually be avoided if surgical treatment is instituted in time. The diagnosis of hyperparathyroidism during pregnancy depends on the recognition of the usual symptoms and the finding of
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hypercalcemia. One should be aware, however, that the total serum calcium levels in pregnant women are often reduced because of a decrease in the protein-bound fraction of calcium. Consequently, corrected and/or ionized serum calcium concentration should be measured if hyperparathyroidism is suspected in a pregnant woman.
H. S u m m a r y o f Clinical F e a t u r e s The increasing frequency of detection of hyperparathyroidism, particularly in mild form, raises questions about the natural history of the disease, especially the indications for surgical treatment versus simple nonoperative medical surveillance. Frequency of the various signs and symptoms is difficult to accurately describe, as discussed above, in view of the different rates of detection of the disease in surveys published several decades ago, 2~ versus frequency of detection in the present era. The diagnosis is now relatively easy to establish but management remains uncertain. Hyperparathyroidism, even if benign, could be a potential risk factor for osteopenia, renal failure, or even a slight increase in mortality associated with cardiovascular disease when compared with euparathyroid patients (as has been suggested 86-94'2~ but not generally accepted because of the limitations in the type of data). 83 Prospective longterm clinical studies would provide a more accurate picture of the true incidence of signs and symptoms in the disease and would help to clarify certain unresolved issues regarding hyperparathyroidism.
IV. D I A G N O S I S A N D DIAGNOSIS
DIFFERENTIAL
A. I n t r o d u c t i o n Whenever hypercalcemia is confirmed, a definitive diagnosis must be established. Although hyperparathyroidism, a frequent cause of asymptomatic hypercalcemia, is a chronic disorder in which manifestations, if any, may be expressed only over months or years, hypercalcemia also can be the earliest clue to the presence of malignancy, the second most common case of hypercalcemia in the adult. Hyperparathyroidism and cancer account for 90% of cases. Diagnosis can usually be established. 2~ However, the optimal management of asymptomatic hyperparathyroidism is still unsettled, 2~ and treatment of the hypercalcemia of cancer is difficult (see Chapter 22). Before undertaking an evaluation of hypercalcemia, it is essential to be sure that true hypercalcemia, not a false-positive laboratory test, is present. 2~ Hypercal-
cemia is a chronic problem, and it is cost-effective to obtain several serum calcium measurements; these tests need not be in the fasting state. False-positive calcium tests are usually the result of inadvertent hemoconcentration during blood collection of elevation in serum proteins, particularly albumin. Measurement of ionized calcium is not quite technically feasible, but the potential advantages in measurement of ionized rather than total calcium have not been clearly established, although research on this issue continues. 2~ Clinical features there alone are helpful in differential diagnosis. Hypercalcemia in an adult who is asymptomatic is usually due to primary hyperparathyroidism. 2~ In most cases of malignancy-associated hypercalcemia the disease is not occult; rather, symptoms of the underlying malignancy bring the patient to the physician, and hypercalcemia is discovered during the workup. In patients with malignancy, the interval between detection of hypercalcemia and death is often less than 6 months. Accordingly, if an asymptomatic individual has had hypercalcemia or some manifestation of hypercalcemia, such as kidney stones, for more than 1 or 2 years, it is unlikely that malignancy is the cause. As discussed below, however, differentiating primary hyperparathyroidism from occult malignancy can occasionally be a problem, and careful evaluation of patients is required, particularly when the duration of the hypercalcemia is unknown. A variety of diseases cause hypercalcemia in the remainder of cases (< 10%) not due to hyperparathyroidism and malignancy. Table 13-5 groups the other causes into three categories: those associated with excessive vitamin D action; those associated with high bone turnover from any of several causes; and, finally, renal failurerelated hypercalcemia, occurring as a complication of the management of patients with renal failure or in which renal failure occurs as part of a hypercalcemia syndrome, as after an excessive ingestion of milk and alkali. (See Chapter 22 for a discussion of hypercalcemia and malignancy; Chapters 5, 11, 14, and 20 for review of vitamin D-related bone and mineral disorders; and Chapter 16 for details of disorders associated with abnormalities in the calcium sensor.)
B. O t h e r P a r a t h y r o i d - R e l a t e d C a u s e s of Hypercalcemia
1. JANSEN'SDISEASE Jansen's disease is a rare disorder characterized by a severe osseochondrodysplastic deformity, short-limbed dwarfism, and severe hypercalcemia with features suggestive of hyperparathyroidism but in fact recently dis-
CHAPTER 13 Primary Hyperparathyroidism TABLE 13--5 Classification of Causes of Hypercalcemia a Parathyroid related Hyperparathyroidism Jansen's disease (constitutive activation of the PTH receptor) Familial benign hypocalciuric hypercalcemia Lithium therapy Cancer related Hematological malignancies Bone metastases Humoral syndrome Vitamin D related Intoxication (dietary) Hereditary and acquired disorders of vitamin D metabolism High bone turnover states Hyperthyroidism Paget's disease Immobilization Thiazides Vitamin A intoxication Renal failure related Severe secondary (tertiary) hyperparathyroidism Milk-alkali syndrome Aluminum intoxication aEtiology > 90%.
covered to be due to constitutive activation of the receptor, a PTH-independent activation of the PTH receptor. 2~ For a thorough analysis of skeletal and other phenotypic features see recent reviews. 2~ 209 Severe hypercalcemia develops after a few months and persists lifelong (range, 1 1 to 17 mg/dl). Other laboratory findings are consistent with hyperparathyroidism but both PTH and PTHrP are undetectable. 2~ The deduction that this disease was due to constitutive activation of the PTH receptor was confirmed by analysis of the coding exons of the PTH receptor by use of the polymerase chain reaction (PCR) of genomic DNA from blood leukocytes. 2~ Two sites of nucleotide point mutation were found resulting in a histidine to arginine mutation at residue 223 (in patients from several different families) and threonine to proline (one patient) at residue 410. Both mutations were shown to cause constitutive activation (see Chapter 3 for details). 2. FAMILIAL BENIGN HYPOCALCIURIC HYPERCALCEMIA
There are multiple reports of syndromes of familial hypercalcemia 2 1 1 - 2 2 0 that do not involve other endocrine abnormalities and thus differ from the MEN syndromes. Some have very unusual features in the kindreds, such as multiple ossifying adenomas of the jaw and evidence for a unique responsible genetic lOCUS.216'221'222 The most
427 frequently reported is the syndrome that is now termed in most reviews familial benign hypercalciuric hypercalcemia (FBHH), (sometimes called familial benign hypercalcemia or familial hypercalciuric hypercalcemia 211"217'218"22~ which closely resembles but is distinct from primary hyperparathyroidism. The pathophysiological defect has now been defined as due to loss-offunction mutations in the receptor that regulates PTH secretion. 211'223-226Recognition of the disorder is important because members of affected kindreds do present themselves, particularly to specialists in referral centers, for management of their hypercalcemia; surgical exploration of the parathyroids is essentially never indicated because it will not cure the disorder. 224-226 The calcium-sensing receptor cloned by Brown and colleagues 2 2 7 - 2 3 0 (see Chapter 16) has seven transmembrane-spanning segments typical of G-protein-coupled receptors; the large extracellular domain is involved in calcium binding. The receptor is present in kidney and several other sites, as well as the parathyroid cell, and is a member of a distinctive evolutionary group of Gprotein-linked receptors that includes the metabotropic glutamate receptor. TM Point mutations have been identified that interfere with function in many patients with FBHH (a single allele is either null or acts as a dominant negative). A more severe phenotype results when both alleles are defective (in one case due to consanguinous marriage of two related, affected individuals). 228"237The diagnosis can now be made definitively by demonstration of the mutations (see Chapter 16 for a recent review and details). 232 3. LITHIUM THERAPY
Lithium therapy, employed in the management of bipolar and other psychiatric disorders, causes hypercalcemia in patients treated with customary doses for extended periods of t i m e . 233-236 Several reports, in which large series of patients on lithium were surveyed, confirm hypercalcemia, often with elevated PTH levels, in about 10% o f patients. 233'234'237 The hypercalcemia was clearly lithium-dependent, as it remitted and recurred with stopping and restarting. 237 The pathophysiology of the disorder is confusing, however, because some patients had adenomas when they were explored. 235'238Lithium has been demonstrated in vitro to cause increased PTH secretion through a shift in the dose-response curve that relates PTH secretion to ambient calcium concentration. 239 This finding and the reversibility in the majority of patients when lithium was stopped could explain the hypercalcemia and increased PTH concentration as merely a functional change in the glands. As outlined in Chapter 16 that reviews findings conceming the calcium sensor in the parathyroids, the locus
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of the lithium effect is undoubtedly an alteration of the sensitivity of this calcium sensor. Why a few patients are reported to have adenomas is unclear 235'238 except perhaps by analogy with secondary hyperparathyroidism where persistent hyperplasia may lead to a monoclonal outgrowth 69 (see Fig. 13-6).
C. Vitamin D-Related Hypercalcemia Hypercalcemia related to excessive vitamin D action is due to either excessive ingestion or abnormal metabolism of the vitamin (see Chapter 5 for details of vitamin D metabolism and action). 24~ Abnormal metabolism of the vitamin is usually acquired in association with some widespread granulomatous disorder, such as sarcoidosis (see Chapter 20). 2~ One rare hereditary form of vitamin D sensitivity is seen in infants in association with a variety of other developmental anomalies. 247 These inherited and acquired defects are due to defective regulation of the production of 1,25(OH)ED. 243 Normally there is tight regulation of the renal 1-hydroxylase by several controlling factors, but this normal feedback suppression is less effective in infants than in adults and operates poorly, if at all, on the 1-hydroxylase enzyme activity present in nonrenal ectopic sites, as in sarcoidosis (see Chapter 20). 243
D. High Bone Turnover States 1. HYPERTHYROIDISM
Severe and symptomatic hypercalcemia is a rare complication of thyrotoxicosis (see Chapter 18 for details). Minimal elevations of serum calcium have been reported, however, as in patients with hyperthyroidism. 249'25~Hypercalcemia in hyperthyroidism is secondary to increased bone resorption, probably due to direct effects of thyroxine and triiodothyronine on the skeleton. 251-256 Patients with thyrotoxicosis are more sensitive than normal persons to the hypercalcemic effects of l Y r n . 257-259 It has been emphasized 26~ that patients with thyrotoxicosis who develop with severe hypercalcemia may have concomitant hyperparathyroidism, inasmuch as t h e hypercalcemia of uncomplicated thyrotoxicosis is usually mild. One thyrotoxic patient with a serum calcium of 15 mg/dl later proved to have a parathyroid adenoma. TM Another group 262 described 17 patients with thyrotoxicosis and concomitant parathyroid adenomas. The diagnosis of hyperthyroidism usually offers no difficulties because, in most cases, it is the thyrotoxicosis rather than the hypercalcemia that brings the patients to
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the physician. Particularly in the elderly, however, the signs of thyrotoxicosis may be minimal. During the treatment of thyrotoxicosis and hypercalcemia, repeated determinations of serum calcium should be made. If the serum calcium does not return toward normal as the thyrotoxicosis is brought under control, another cause for the hypercalcemia, such as hyperparathyroidism, should be suspected. 2. IMMOBILIZATION
Immobilization is rarely associated with hypercalcemia in the absence of an associated disease in adults but may be associated with hypercalcemia in children and young adolescents, particularly after spinal cord injury and paraplegia or quadriplegia. 263 If significant ambulation again becomes possible, the hypercalcemia in young patients usually returns to normal spontaneously. The mechanism involves a relative change in bone turnover rates with sudden loss of weight bearing with resorption favored over formation. 3. THIAZIDES
Administration of benzothiadiazides (thiazides) 264-268 can cause hypercalcemia in patients with high rates of bone turnover, such as in patients with hyperparathyroidism, z69,z7~ hypoparathyroidism treated with high doses of vitamin D, 264 and juvenile, high-turnover osteoporosis. 27~Thiazides have been used as a provocative test to bring out borderline hypercalcemia in patients suspected of hyperparathyroidism. 271 Thiazide administration to normal individuals causes a transient increase in blood calcium, usually within the normal range, which then reverts to preexisting levels after a week or more of continued thiazide administration. 266 The pharmacology of the drug action is complex; the overall result seems to be a challenge to calcium homeostasis by actions at multiple levels and by effects on renal calcium excretion, bone calcium turnover, and intestinal calcium absorption. The efficacy of parathyroid action p e r s e is increased and may explain in some patients the observed e f f e c t s . 264-267
Several aspects of the mode of action of the thiazides, however, particularly in normal subjects and in patients with hypoparathyroidism, remain unclear. Chronic thiazide administration lowers urinary calcium excretion in all normal subjects and persists for the duration of the treatment. This hypocalciuric effect was not seen in several initial studies in hypoparathyroid patients. 264'266'267 In subsequent clinical and experimental animal studies, however, significant hypocalciuria has been demonstrated in hypoparathyroidism. 272'273 The results indicate an action of thiazides on the renal tubule despite deficiency of PTH; the actions of the drug are not merely
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CHAPTER 13 Primary Hyperparathyroidism an augmentation of the biological actions of PTH. TM In fact, the substantial hypocalciuric effect that can be elicited with concomitant dietary manipulations in hypoparathyroid patients on high-dose vitamin D and oral calcium replacement has been used successfully as an adjunct to therapy by achieving a satisfactory elevation of serum calcium without severe hypercalciuria. 273 The overall results suggest that if normal hormonal function and calcium and bone metabolism are present, homeostatic controls can be reset to counteract the calcium-elevating effect of thiazides. In the presence of hyperparathyroidism or increased bone turnover from another cause, such as with high-dose vitamin D therapy, homeostatic mechanism cannot be reset. 264'266 The presence of hypercalcemia in association with thiazide administration points to an underlying disorder in calcium metabolism or bone turnover, not a manifestation of iatrogenic disease, in a normal subject. The severity of hypercalcemia in hyperparathyroidism may be exaggerated if thiazides are being given. Treatment with thiazides should be stopped in hypercalcemic patients because it exaggerates the apparent degree of hypercalcemia and can confuse judgments about the need for surgery. The abnormal effects of thiazides on calcium metabolism disappear within days of cessation of treatment with the drug.
E. Vitamin A Intoxication Hypercalcemia due to vitamin A intoxication is rare. Most examples of vitamin A intoxication result from accidental overdose from nutritional supplements. 274'275 Calcium levels have been reported to be elevated into the 12 to 14 mg/dl range in patients who have taken 50,000 to 100,000 IU of vitamin A daily, 10 to 20 times the minimum daily requirement. The patients have typical features of severe hypercalcemia, including fatigue, anorexia, severe muscle pain, and sometimes diffuse bone pain. 274"275 The mechanism of action of the excess vitamin A intake is presumed to be increased bone resorption. Diagnosis can be established by history and by confirmatory measurements of vitamin A levels in serum, which are often increased severalfold above normal. Occasionally, characteristic skeletal radiographs reveal very distinctive periosteal calcifications, particularly in radiographs of the hands. 276 Treatment consists of withdrawal of the vitamin, which is usually associated with the prompt disappearance of the hypercalcemia and reversal of the skeletal changes; if needed, administration of 100 mg of cortisone or its equivalent per day leads to rapid normalization of the calcium. 274-276
F. Hypercalcemia Associated with Renal Failure 1. SEVERE SECONDARY HYPERPARATHYROIDISM The spectrum of bone disease in patients with endstage renal failure has evolved under the impact of changing therapy in recent decades. Attempts to control secondary hyperparathyroidism and to treat renal osteodystrophy led to the extensive use of calcitriol and supraphysiological dialysate calcium. Awareness of aluminum toxicity in chronic renal failure patients also led to increased use of calcium compounds, rather than aluminum salts, to control serum phosphate. (See Chapter 14 for details of these clinical problems, especially those related to vitamin D and aluminum intoxication and mineral metabolism, and see also Chapters 5, 6, and 7.) 2. MILK-ALKALI SYNDROME
The milk-alkali syndrome has been described in several distinctive clinical presentations m acute, subacute, and chronic--all of which feature the triad of hypercalcemia, alkalosis, and renal failure. 277-279 The syndrome is caused by excessive ingestion of calcium and absorbable antacids such as milk or calcium carbona t e . 277'279'280'282 The disorder has become much less frequent since its widespread recognition and the availability of nonabsorbable antacids and Hz-receptor antagonists, such as cimetidine and ranitidine, for the treatment of gastric and duodenal ulcers. 3. ALUMINUM INTOXICATION
Aluminum intoxication occurs in patients on chronic dialysis because of the accumulation of aluminum in the body, especially bone, due to aluminum present in dialysis fluids and/or aluminum-containing antacids. Discase manifestations can include an acute dementia and a peculiar form of unresponsive severe osteomalacia. 129"281 The patients often develop severe bone pain, multiple nonhealing fractures, particularly of the ribs and pelvis, and a proximal myopathy. 129'282Hypercalcemia is encountered in these patients when attempts are made to treat them as one would other patients with renal osteodystrophy and renal failure m namely, by administering calcium and vitamin D or 1,25(OH)2D. 129'282 Details of this unusual entity are reviewed in Chapter 14.
G. Differential Diagnosis: Summary of General Approach Several repeat calcium measurements are indicated before beginning an evaluation of hypercalcemia. 283 Diagnosis in hypercalcemic disorders is best approached,
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JOHN T. PoTrs, JR.
as in most diseases, by weighing clinical evidence carefully. Hyperparathyroidism and cancer account for 90% of the cases of hypercalcemia. The clinical points that deserve greatest emphasis are (1) the presence or absence of clinical symptoms or signs and (2) evidence of chronicity. If one discounts reports of fatigue or depression, unfortunately common in the population without relating to specific illness, then most patients with asymptomatic hypercalcemia will prove to have primary hyperparathyroidism; symptoms of malignancy are usually present when hypercalcemia due to cancer is detected. Hypercalcemia, however, can be an early manifestation of an occult malignancy in the absence of symptoms; hence, the importance of careful confirmatory laboratory testing with the PTH assay, as reviewed below. In the other 10% of patients with hypercalcemia not due to primary hyperparathyroidism or cancer, the signs of specific disease manifestations, such as renal failure, if not the symptoms, may be evident on an initial routine laboratory test screening. Chronicity is the second most important clinical point. If evidence indicates that hypercalcemia has been present for more than a year (record of laboratory values or history of kidney stones), even occult malignancy as the cause of the hypercalcemia can usually be excluded on clinical grounds alone. A striking feature of malignancyassociated hypercalcemia is the rapidity of the disease course, whereby signs and symptoms related to the underlying malignancy are evident within months of the
first detection of hypercalcemia. T M Therefore, hyperparathyroidism rather than malignancy is overwhelmingly likely in patients with chronic hypercalcemia. Diseases other than hyperparathyroidism, such as sarcoidosis or vitamin D intoxication, are rare altemative causes of chronic hypercalcemia, however. A careful history of dietary supplements and drug use may reveal intoxication with vitamin D or A or the use of thiazides, often in proprietary combinations given for blood pressure maintenance. Sometimes, however, there are no clinical clues to the diagnosis, either because the patient is entirely asymptomatic or because chronic illness obscures symptoms or signs that might provide a clue to the presence of malignancy. The new double-antibody, immunometric assays for PTH have become the principal diagnostic test in laboratory evaluation of hypercalcemic patients (Fig. 13-12). Patients with hyperparathyroidism have elevated levels of PTH (or persistent normal values despite hypercalcemia); patients with malignancy and the other causes of hypercalcemia (other than those related to parathyroid abnormalities, such as lithium-induced hypercalcemia and FBHH) have PTH levels below normal or undetectable (Fig. 13-13). A cost-effective approach to the differential diagnosis of hypercalcemia can be focused on combining clinical probabilities with the PTH assay. Other tests used in the past such as phosphate clearance and urinary cyclic adenosine monophosphate (cAMP) measurements are I000._I
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A, Dose-response plot for IRMA of PTH. Inset, Linear plot for lower concentrations of PTH. Rest of plot is log-log. B, Intact PTH 1 - 8 4 measured by IRMA in sera from 72 normal individuals, 37 patients with surgically proven hyperparathyroidism (1 ~ HPT), and 24 patients with hypercalcemia associated with malignancy. The normal reference interval is 12 to 65 pg/ml. Mean PTH in patients with hyperparathyroidism was 206 (+_ 43 SEM) pg/ ml and 3.35 ( _ 0.8 SEM) pg/ml in individuals with hypercalcemia associated with malignancy. (A, and B from 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.)
FIGURE 13-- 12
CHAPTER 13 Primary Hyperparathyroidism
431
9 Hyperparathyroidism 9Hypercalcemia of Malignancy
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tailed screening for the possibility of MEN should be undertaken in the patient and family. If in a patient with no clear-cut symptoms and only a short or no prior history of hypercalcemia PTH levels are suppressed or undetectable, occult malignancy must be considered as a possible diagnosis clinically. A careful screening for occult malignancy is then a high priority by standard techniques such as chest radiography, CT of chest and abdomen, and bone scan. Careful attention should also be paid to clues of underlying hematological disorders, such as anemia, increased globulin, and abnormal serum immunoelectrophoresis; bone scans can be helpful but have been reported to be negative in patients with multiple myeloma. If no sign of malignancy is found after such a workup for occult malignant disease or if an asymptomatic patient has history or other evidence for chronic hypercalcemia yet PTH values that are suppressed, it is then useful to search for the other illnesses that cause hypercalcemia (Table 13-5).
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V. MEDICAL MANAGEMENT OF HYPERCALCEMIA AND HYPERPARATHYROIDISM
A. Treatment of Hypercalcemia
FIGURE 13-- 13
Results of PTH 1-84 immunometric assay in 101 consecutive patients with surgically confirmed hyperparathyroidism and 79 patients with hypercalcemia of malignancy studied at the Massachusetts General Hospital. The area within the box represents normal serum calcium and serum PTH concentrations. [From Nussbaum SR, Potts JT Jr: Immunoassays for parathyroid hormone 1-84 in the diagnosis of hyperparathyroidism. J Bone Miner Res 6(Suppl 2): $ 4 3 - $ 5 0 , 1991.]
unnecessary for clinical u s e . 285 There is high sensitivity and specificity with the double antibody or immunometry assay methods that have replaced the older PTH immunoassay methods (see reference 285 for extensive review of diagnostic methods). If the patient is asymptomatic and has evidence by history of some chronicity to the hypercalcemia, hyperparathyroidism is almost certainly the cause; if PTH levels in the newer assays are elevated, the presumptive diagnosis is established. Strictly speaking, hyperparathyroidism is never confirmed until abnormal parathyroid tissue is surgically removed, correcting the hypercalcemia, but patients with asymptomatic hypercalcemia who have elevated concentrations of PTH to support the diagnosis can be followed (with high confidence in the diagnosis), according to the general principles of medical surveillance discussed below or recommended for surgery with reasonable confidence of cure by neck exploration. If the family history is suggestive of other endocrine abnormality, more de-
One perspective on medical management of hypercalcemia is reviewed in Chapter 22. What follows is a brief review of the most efficacious therapies, listing general principles and advantages and disadvantages of the different therapies (Table 13-6), as well as review of issues encountered and management options in nonsurgical treatment of asymptomatic hyperparathyroidi s m . 296
Patients with severe hypercalcemia are markedly dehydrated and require intravascular volume repletion. If, however, treatment focuses exclusively on fluid therapy for prolonged intervals, complications of volume overload and electrolyte disturbance can occur. Since the underlying mechanism of severe hypercalcemia in most cases, especially cancer, is predominantly increased bone resorption, antiresorptive agents such as the potent aminobisphosphonates should be administered at the initiation of saline rehydration and diuresis. Early use of antiresorptive agents may prevent complications due to infusion of large amounts of normal saline for prolonged times. For life-threatening hypercalcemia (corrected serum calcium > 16 mg/dl), treatment with calcitonin also should be initiated. Calcitonin's antiosteoclast action will have an onset within hours, contrasted with the 24to 36-hour onset of action of the bisphosphonates or gallium. Unfortunately, tachyphylaxis to calcitonin occurs
TABLE13-6 Treatment Most useful therapies Hydration with saline Forced diuresis; saline loop diuretic
Onset of Action
Therapies for Severe Hypercalcemia
Duration of Action
Advantages
Disadvantages
Hours Hours
During infusion During treatment
Rehydration invariably needed Rapid action
1-2 days
5-7 days in doses used
Hyperphosphatemia; 3-day infusion
1-2 days
10- 14 days after high dose
Hours
2-3 days
First available bisphosphonate; intermediate onset of action High potency; intermediate onset; prolonged duration of action Rapid onset of action; useful as adjunct in severe hypercalcemia
7-10 days
High potency
Plicamycin
Day after 5-day administration 3-4 days
Days
Potent antiresorptive
Length of IV administration; cannot be used with renal failure Liver, kidney, and marrow toxicity; bleeding
Phosphate Oral
24 hours
During use
Low toxicity if P < 4 mgldl
Intravenous
Hours
Rapid action, highly potent
Glucocorticoids
Days
During use and 24-48 hours afterward Days, weeks
Dialysis
Hours
During use and 24-48 hours afterward
Useful in renal failure; onset of effect in hours; can immediately reverse lifethreatening hypercalcemia
Bisphosphonates 1st generation: etidronate 2d generation: pamidronate Calcitonin Other therapies Gallium nitrate
+
Oral therapy, antitumor agent
Cardiac decompensation, intensive monitoring electrolyte disturbance, hypokalemia, hypomagnesemia
Fever in 20%; hypophosphatemia, hypocalcemia, hypomagnesemia Often limited calcium lowering, rapid tachyphylaxis
Limited use except as adjuvant or chronic therapy Ectopic calcification; severe hypocalcemia Active only in certain malignancies; glucocorticoid side effects Complex procedure, reserved for extreme or special circumstances
CHAPTER
13 Primary Hyperparathyroidism
within several days, and even the limited efficacy of this therapy, usually lowering calcium by 2 to 3 mg/dl, is no longer seen. The long-term management of hypercalcemia has been more problematic, and most therapies have been associated with refractoriness and cumulative toxicities; the most effective long-term treatment for hypercalcemia is to treat the underlying disease whenever possible.
433
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1. M O S T USEFUL THERAPIES
a. Hydration and Mild Diuresis. Severe hypercalcemia is associated with a cycle of decreasing renal concentrating ability followed by dehydration, decreased renal blood flow, decreased glomerular filtration rate (GFR), and enhanced reabsorption of calcium along with sodium. Because the underlying hypercalcemia most often results from marked osteoclastic bone resorption, there is continued efflux of calcium from the skeleton into the extracellular fluid (ECF). Rehydration is the essential first step in the treatment of severe hypercalcemia. 286'287It often takes 4 to 6 liters of saline to restore volume status during the initial 24 hours. b. Forced Diuresis. In certain disease states, particularly malignancies with widespread bone destruction, the elevation of serum calcium can become immediately life threatening. Under such circumstances, diuretic therapy can be pursued much more aggressively by giving 6 liters of isotonic saline (900 mEq sodium) daily plus potent loop diuretics such as furosemide, bumetanide (Bumex), or ethacrynic acid; furosemide, for example, in doses of 80 to 120 mg every 3 hours, increases urinary calcium excretion to levels of 1000 mg/day or more. 288 Serum calcium may decrease by as much as 4 mg/dl within 24 hours in such patients. Severe potassium and magnesium depletion are inevitable unless replacements are given; pulmonary edema can obviously be precipitated. The potential complications can be averted by intensive monitoring of central venous pressure and plasma or urine electrolytes, but the demands on the patient and medical staff are significant. A bladder catheter is usually necessary to allow the patient to sleep. With the effectiveness of potent antiresorptive agents now available that can be given at the same time as fluid therapy is initiated, forced diuresis should be seen as a short-term therapy, reserved for patients with very severe hypercalcemia. It is then to be used for 1 to 3 days at a maximum, until the antiresorptives, such as aminobisphosphonates, take effect. c. Bisphosphonates (Diphosphonates). The bisphosphonates are analogues of pyrophosphate. These drugs have high affinity for hydroxyapatite of bone, especially
pamidronate
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I
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FIGURE 13--14
Structural formulas of commonly used bisphosphonates and the generic formulas. Alendronate: R1 = ( C H 2 ) 3 - NH2; R2 - OH. Clodronate: R~ - C1; R2 = C1. [From Nussbaum SR, Neer RM, Potts JT: Medical management of hyperparathyroidism and hypercalcemia. In DeGroot LJ (ed): Endocrinology, 3rd ed, vol 2. Philadelphia, WB Saunders Co, 1995, pp 1094-1105.]
in areas of increased bone turnover such as those that occur at metastatic lesions or those involved with Paget's disease. These bone-seeking bisphosphonates are stable in the body because phosphatase enzymes cannot hydrolyze their central carbon-phosphorus-carbon bond (Fig. 13-14). Concentrated in areas of high bone turnover, the bisphosphonates are taken up by osteoclasts and inhibit osteoclast action289; the detailed mechanism of action of the bisphosphonates, however, is not well understood. 3~176 Etidronate, a first-generation bisphosphonate, is poorly absorbed and, like more potent later-generation bisphosphonates, generally must be given intravenously to be effective in hypercalcemia. With further study of structure/activity relations, a newer class of bisphosphonates with more favorable properties in efficacy and duration of action has emerged, although poor oral absorption remains a problem for all currently used bisphosphonates. 3~176 The newer bisphosphonates have a more favorable bone action, blocking bone resorption with less effect on decreasing bone mineralization. 3~176 Pamidronate is one of the new generation of aminobisphosphonates that are extremely potent in their inhibition of osteoclast-mediated skeletal resorption yet do not cause mineralization defects at doses used clinically. 29~ Between 60 and 90 mg of pamidronate, given as a single intravenous dose, has been shown to normalize serum calcium in the majority of patients for weeks or e v e n months21~ refractoriness, however, is reported with continued use. Alendronate under the trade name Fosamax has been introduced for therapy of osteoporosis, but has not been evaluated/reported for use in hypercalcemia. 3~ d. Calcitonin. Calcitonin acts within minutes of its administration, through receptors on osteoclasts, de-
434 creasing the release of skeletal calcium, phosphorus, and hydroxyproline, z98'299 One group 3~ reported a mean decline of serum calcium of 2 mg/dl with a maximal response in 6 hours; doses of 2 to 8 U/kg body weight intravenously, subcutaneously, or intramuscularly every 6 to 12 hours have been reported with variable responses. 297'298'3~176 Escape from the hypocalcemic effects of calcitonin is seen in vitro, where it can be prevented by glucocorticoids. In vivo in humans, administration of glucocorticoids in combination with calcitonin may augment or prolong the action of calcitonin, 3~ although this effect has not been widely studied (see Chapter 22). 2. OTHER THERAPIES
Gallium nitrate exerts its hypocalcemic action by inhibiting bone resorption. 3~ The reductions seen in levels of biochemical markers of bone resorption and the overall clinical efficacy indicate that it is a potent inhibitor of b o n e resorption. 3~176 Plicamycin is a tumoricidal antibiotic that was used initially in the treatment of embryonal cell carcinoma of the testes. 3~176 Plicamycin inhibits RNA synthesis, and thus may interfere with the differentiation of osteoclast precursors into mature osteoclasts. Although quite effective in many patients after intravenous use, because of its toxicity, its use has been supplanted by the intravenous bisphosphonates. Pharmacological doses of glucocorticoids (e.g., 40 to 100 mg of prednisone daily in divided doses) reverse the hypercalcemia of vitamin D intoxication, 31~ sarcoidosis, tuberculosis, and other granulomatous diseases, 311'312 as well as that caused by certain malignancies, typically, hematological malignancies such as multiple myeloma, leukemias, Hodgkin's disease, and other lymphomas where an antitumor effect occurs also. 313 One of the most dramatically effective therapies for severe hypercalcemia, although one fraught with potential hazards, is intravenous phosphate. 312"314-317 Calcium kinetic studies show a rapid disappearance of calcium from the circulation within minutes of beginning a phosphate infusion. Calcium phosphate precipitates in the skeleton; however, it also precipitates in kidney, soft tissues, and the heart. Intravenous phosphate is now largely superseded by other therapies such as bisphosphonates. Oral phosphate may be useful in certain patients with chronic hypercalcemia and may lower calcium without exceeding the calcium-phosphate solubility product. Phosphate therapy should not be used if renal insufficiency and/or normophosphatemia or hyperphosphatemia is present. Hypercalcemia complicated by severe renal failure is difficult to manage; if treatment is appropriate in this clinical setting, dialysis is the first choice. Peritoneal di-
JOHN T. POTTS, JR.
alysis can remove 500 to 2000 mg of calcium in 24 to 48 hours and lower the serum calcium concentration by 3 to 12 mg/dl if calcium-free dialysis fluid is used. 318'319
B. M e d i c a l T r e a t m e n t o f H y p e r p a r a t h y r o i d i s m Asymptomatic hyperparathyroidism is defined as the clinical profile of patients with documented (presumptive) hyperparathyroidism without signs or symptoms commonly attributable to the disease. The importance of the topic, coupled with the limitations of published data, led the NIH to hold a Consensus Conference on Management of Asymptomatic Hyperparathyroidism in 1990. 83 Recent reviews have summarized the views presented, including controversies about natural history and treatment, directions for future clinical and basic research, and an interim practical set of recommendations regarding surgery for hyperparathyroidism. 83'125'185'2~ In summary, patients over age 50 who wish to avoid surgery, who do not show the obvious symptoms of hyperparathyroidism, and have a bone density that is normal for age (or at least not lower than 2 SD below agematched normals), may be considered for medical surveillance. Table 1 3 - 7 lists the principal guidelines for recommending surgery. If these guidelines do not apply, it was felt reasonable to avoid surgery and undertake careful medical monitoring, which includes particularly monitoring bone mass and renal function. 83'126 The conference panel did not make a specific recommendation about a method of monitoring bone mass despite many improvements in techniques 232 in view of the uncertainties about the type of bone loss in hyperparathyroidism and its implications for f r a c t u r e . 123'126'131 In the opinion of the author, it is reasonable in the United States at present to use only single-photon absorptiometry to follow cortical bone. This test is relatively easy and inexpensive; the body of data at present suggests that, in the United States at least, increased loss of cancellous bone is not seen in most patients, and higher risk of vertebral fractures in hyperparathyroidism may not be a major issue. 83'123'126'131 The more complex issue of monitoring spi-
TABLE 13--7 Asymptomatic Hyperparathyroidism: Suggested Guidelines for Surgery 1. 2. 3. 4. 5. 6.
Serum calcium-- 1.0-1.6 mg/dl above normal; 11.4-12.0 mg/dl History of life-threatening hypercalcemia Reduced creatinine clearance--<30% of age-matched normal Kidney stones documented 24-hour urinary calcium excretion of >400 mg/24 hours Bone mass-->2 SD below age-matched normal
CHAPTER 13
435
Primary Hyperparathyroidism
nal bone mass by DEXA can be reserved as a research issue for controlled clinical studies. The point at which changes in the patient's condition, either objective decline in bone mass or changes in renal function without clear-cut symptoms, should indicate the need for surgery will remain a highly individual decision involving the patient's overall circumstances and attitudes. Uncertainty persists about changes in skeletal mass, especially after surgery as discussed above under skeletal signs and symptoms. 126 Cost-benefit calculations for surgery versus medical monitoring are unclear at this time, but obviously, the younger the patient, the more surgical treatment seems indicated from the point of view of both medical safety and overall cost and convenience. Surgery should always be appreciated as the standard therapy, with its high success rate and low morbidity. A clearer knowledge of long-term clinical outcomes based on prospective, randomized studies is needed to develop more firm guidelines about methods of monitoring and definition of predictive indices that would permit an early decision for surgical therapy on a fully rational basis for those patients who will ultimately require it.
VI. SUMMARY Primary hyperparathyroidism can now be diagnosed with confidence using the highly specific and sensitive double-antibody radioimmunoassays for detection of elevated PTH levels. The genetic basis of parathyroid gland hyperfunction has been clarified; loss of antioncogenes and activation of protooncogenes have both been defined as contributory to gland pathology in various syndromes including adenomas, (severe) secondary hyperparathyroidism, and primary chief cell hyperplasia, the latter often part of associated heritable multiple endocrine disorders (MEN-I and MEN-II). The advances in understanding the genetic pathogenesis have proven helpful in clinical management in some situations. Definitive diagnosis is possible in the MEN-II syndrome (detection of mutated R E T protooncogene), but not yet in the MEN-I syndrome (although the suspected antioncogene has just been cloned). TM This ability to determine which family member carries the abnormal gene in the germ line greatly aids management of MEN-II kindreds. Knowledge that poorly treated (but still reversible) parathyroid hyperplasia may progress though clonal emergence to a growth-autonomous tumor should spur efforts to optimally control secondary hyperparathyroidism. The clinical spectrum of hyperparathyroidism exhibits typically a much milder phenotype than was seen several decades ago, primarily due to the increased incidence of
the disease, as much as tenfold higher, with early diagnosis in minimally affected or asymptomatic patients. Surgical management by removal of the hyperfunctioning parathyroid tissue is still the standard and usually effective therapy, but in older patients with no symptoms and lack of renal or osseous deterioration, medical surveillance may be justified without surgery. We still lack a clear knowledge of the natural history of the disease, particularly with regard to progression and/or reversibility of osteopenia, however, and uncertainty therefore persists about optimal medical management of hyperparathyroidism.
References 1. Browder W, Rakinic J, Schlecter R, Krementz ET: Primary hyperparathyroidism in the seventies: A decade of change. Am J Surg 146:360-365, 1983. 2. Albright F, Reifenstein EC Jr: The parathyroid glands and metabolic bone disease: Selected studies. Baltimore, Williams & Wilkins, 1948. 3. Mandl F: Klinisches und experimentelles zur frage der lokalisierten ostitis fibrosa: B. Die generalisierte form der ostitis fibrosa. Arch Klin Chir 143:1, 1926. 4. Gold E: Mitt Grenzgeb Med Chir 41:63, 1928. 5. Barr DP, Bulger HA: The clinical syndrome of hyperparathyroidism. Am J Med Sci 179:449-476, 1930. 6. Wilder RM: Hyperparathyroidism: Tumor of the parathyroid glands associated with ostitis fibrosa. Endocrinology 13:231, 1929. 7. Wang CA: Ann Surg 183:271, 1976. 7a. Akerstrrm G, Malmaeus J, Bergstrom R: Surgical anatomy of human parathyroid glands. Surgery 95:14, 1983. 8. Albright F, Aub JC, Bauer W: Hyperparathyroidism. A common and polymorphic condition as illustrated by seventeen proved cases from one clinic. JAMA 102:1276-1287, 1934. 9. Albright F, Reifenstein EC Jr: The Parathyroid Glands and Metabolic Bone Disease: Selected Studies. Baltimore, Williams & Wilkins, 1948. 10. Bauer W, Federman DD: Metab Clin Exp 11:21, 1962. 11. Melton LJ III: Epidemiology of primary hyperparathyroidism. J Bone Miner Res 6(Suppl 6):$25-$29, 1991. 12. Palmer M, Jakobsson S, Akerstrom G, Ljunghall S: Prevalence of hypercalcaemia in a health survey. A 14-year follow-up study of serum calcium values. Eur J Clin Invest 18:39-46, 1988. 13. Stenstrom G, Heedman P-A: Clinical findings in patients with hypercalcaemia. Acta Med Scand 195:473-477, 1974. 14. Mundy GR, Cove DH, Fisken R: Primary hyperparathyroidism: Changes in the pattern of clinical presentation. Lancet 1: 1317-1320, 1980. 15. Heath H III, Hodgson SF, Kennedy MA: Primary hyperparathyroidism: Incidence, morbidity, and potential economic impact in a community. N Engl J Med 302:189-193, 1980. 16. Christensson T, Hellstrom K, Wengle B, et al: Prevalence of hypercalcaemia in a health screening in Stockholm. Acta Med Scand 200:131-137, 1976. 17. Cope O, Barnes BA, Casfleman B, et al: Vicissitudes of parathyroid surgery: Trail of diagnosis and management in 51 pa-
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41. Schantz A, Castleman B: Parathyroid carcinoma: A study of 70 cases. Cancer 31:600-605, 1973. 42. Holmes EC, Morton DL, Ketcham AS: Parathyroid carcinoma: A collective review. Ann Surg 169:631-640, 1969. 43. Trigonis C, Cedermark B, Willems J, et al: Parathyroid carcinoma problems in diagnosis and treatment. Clin Oncol 10:1119, 1984. 44. Smith JF, Coombs RRH: Histological diagnosis of carcinoma of the parathyroid gland. J Clin Pathol 37:1370-1378, 1984. 45. Levin KE, Galante M, Clark OH: Parathyroid carcinoma versus parathyroid adenoma in patients with profound hypercalcemia. Surgery 101:649-660, 1987. 46. McKeown PP, McGarity WC, Sewell CW: Carcinoma of the parathyroid gland: Is it overdiagnosed? Am J Surg 147:292298, 1984. 47. Anderson BJ, Samaan NA, Vassilopoulou-Sellin R, et al: Parathyroid carcinoma: Features and difficulties in diagnosis and management. Surgery 94:906-915, 1983. 48. Streeten EA, Weinstein LS, Norton JA, et al: Studies in a kindred with parathyroid carcinoma. J Clin Endocrinol Metab 75: 363- 366, 1992. 49. Sandler LM, Moncrieff MW: Familial hyperparathyroidism. Arch Dis Child 55:146-157, 1980. 50. Allo M, Thompson NW: Familial hyperparathyroidism caused by solitary adenomas. Surgery 92:486-490, 1982. 51. Law WMJ, Heath HI: Familial benign hypercalcemia (hypocalciuric hypercalcemia): Clinical and pathogenic studies in 21 families. Ann Intern Med 102:511-519, 1985. 52. Cutler RE, Reiss E, Ackerman LV: Familial hyperparathyroidism: A kindred involving eleven cases, with a discussion of primary chief-cell hyperplasia. N Engl J Med 270:859-865, 1964. 53. Christensson T: Familial hyperparathyroidism. Ann Intern Med 85:614-615, 1976. 54. Marsden P, Anderson J, Doyle D, et al: Familial hyperparathyroidism. Br Med J 3:87-90, 1971. 55. Mallette LE, Malini S, Rappaport MP, Kirkland JL: Familial cystic parathyroid adenomatosis. Ann Intern Med 107:54-60, 1987. 56. Jackson CE, Norum RA, Boyd SB, et al: Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: A clinically and genetically distinct syndrome. Surgery 108:1006-1013, 1990. 57. Foley TP, Harrison HC, Arnaud CD, Harrison HE: Familial benign hypercalcemia. J Pediatr 81:1060-1067, 1972. 58. Marx SJ, Attie MF, Levine MA, et al: The hypocalciuric or benign variant of familial hypercalcemia: Clinical and biochemical features in fifteen kindreds. Medicine 60:397-412, 1981. 59. Wermer P: Genetic aspects of adenomatosis of endocrine glands. Am J Med 16:363- 371, 1954 60. Sipple JH: The association of pheochromocytoma with carcinoma of the thyroid gland. Am J Med 31:163-166, 1961. 61. Schirnke RN, Hartmann WH: Familial amyloid-producing medullary thyroid carcinoma and pheochromocytoma: A distinct genetic entity. Ann Intern Med 63:1027-1039, 1965. 62. Schimke RN, Hartmann WH, Prout TE, et al: Syndrome of bilateral pheochromocytoma, medullary thyroid carcinoma and multiple neuromas: A possible regulatory defect in the differentiation of chromaffin tissue. N Engl J Med 279:1-8, 1968. 63. Steiner AL, Goodman AD, Powers SR: Study of a kindred with pheochromocytoma, medullary thyroid carcinoma, hyperparathyroidism and Cushing's disease. Multiple endocrine neoplasia, Type 2. Medicine 47:371-409, 1968.
CHAPTER 13
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A Chemical, Biochemical and Clinical Update. Berlin, Walter de Gruyter, 1985, pp 5 7 - 5 8 . Bell NH, Stern PH, Pantzer E, et al: Evidence that increased circulating 1,25-dihydroxyvitamin D is the probable cause for abnormal calcium metabolism in sarcoidosis. J Clin Invest 64: 2 1 8 - 225, 1979. Bell NH: Vitamin D-endocrine system. J Clin Invest 7 6 : 1 - 6 , 1985. 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: 4 4 0 - 4 4 3 , 1981. Adams JS, Singer FR, Gacad MA, et al: Isolation and structural identification of 1,25-dihydroxyvitamin D3 produced by cultured alveolar macrophages in sarcoidosis. J Clin Endocrinol Metab 60:960-966, 1985. Lambert PW, Stern PH, Avioli RC, et al: Evidence for extrarenal production of 1,25-dihydroxyvitamin D in man. J Clin Invest 69:722-725, 1982. Garabedian M, Jacqz E, Guillozo H: Elevated plasma 1,25dihydroxyvitamin D concentrations in infants with hypercalcemia and an elfin facies. N Engl J Med 31:948-952, 1985. Newman LS, Cecile SR, Maier LA: Sarcoidosis. N Engl J Med 336:1224-1234, 1997. Rose E, Boles RS Jr: Hypercalcemia in thyrotoxicosis. Med Clin North Am 37:1715, 1953. Bryant LR, Wulsin JH, Altemeier WA: Hyperparathyroidism and hyperthyroidism. Ann Surg 159:411, 1964. Aub JC, Bauer W, Heath C, et al: Studies of calcium and phosphorus metabolism. III. Effects of thyroid hormone and thyroid disease. J Clin Invest 7:97, 1929. Laake H: Osteoporosis in association with thyrotoxicosis. Acta Med Scand 151:229, 1955. Krane SM, Brownell GL, Stanbury JB, et al: The effect of thyroid disease on calcium metabolism in man. J Clin Invest 35:874, 1956. Adams PH, Jowsey J, Kelly PJ, et al: The effect of thyroid disease on calcium metabolism in man. J Clin Invest 35:874, 1956. Adams PH, Jowsey J: Bone and mineral metabolism in hyperparathyroidism: An experimental study. Endocrinology 81:735, 1967. Krane SM, Goldring SR: Hyperthyroidism: Skeletal system; neuromuscular system; emotions and mentation. In Werner SC, Ingbar SH (eds): The Thyroid: A Fundamental and Clinical Test. New York, Harper & Row, 1976, pp 7 2 7 - 7 5 6 . Harrison MT, Harden RMcG, Alexander WD: Some effects of parathyroid hormone in thyrotoxicosis. J Clin Endocrinol Metab 24:214, 1964. Bouillon R, DeMoor P: Parathyroid function in patients with hyper- or hypothyroidism. J Clin Endocrinol Metab 38:9991004, 1974. Epstein FH, Freedman LR, Levitin H: Hypercalcemia, nephrocalcinosis and reversible renal insufficiency associated with hyperparathyroidism. N Engl J Med 258:782, 1958. Epstein FH: Bone and mineral metabolism in hyperthyroidism. Ann Intern Med 68:490, 1968. Baxter JD, Bondy PK: Hypercalcemia of thyrotoxicosis. Ann Intern Med 65:429, 1966. Breuer RI, McPherson HT: Hypercalcemia in concurrent hyperthyroidism and hyperparathyroidism. Arch Intern Med 118: 310, 1966. Winters JL, Kleinschmidt AG Jr, Frensilli JJ, et al: Hypercalcemia complicating immobilization in the treatment of fractures: A case report. J Bone Joint Surg 48A:1182, 1966.
441 264. Parfitt AM: The interactions of thiazide diuretics with parathyroid hormone and vitamin D: Studies in patients with hypoparathyroidism. J Clin Invest 51:1879-1888, 1972. 265. Koppel MH, Massry SG, Shinaberger JH, et al: Thiazideinduced rise in serum calcium and magnesium in patients on maintenance hemodialysis. Ann Intern Med 72:895-901, 1970. 266. Brickman AS, Massry SG, Coburn JW: Changes in serum and urinary calcium during treatment with hydrochlorothiazide: Studies on mechanisms. J Clin Invest 51:945-954, 1972. 267. Popovtzer MM, Subryan VL, Alfrey AC, et al: The acute effect of chlorothiazide on serum-ionized calcium: Evidence for a parathyroid hormone-dependent mechanism. J Clin Invest 55: 1295-1302, 1975. 268. Goodman LS, Gilman A: The Pharmacological Basis of Therapeutics, 4th ed. New York, Macmillan, 1970. 269. Duarte DG, Winnacker JL, Becker KL, et al: Thiazide-induced hypercalcemia. N Engl J Med 284:828, 1971. 270. Parfitt AM: Chlorothiazide-induced hypercalcemia in juvenile osteoporosis and hyperparathyroidism. N Engl J Med 281:55, 1969. 271. van der Sluys VJ, Birkenhager JC, Smeenk D: De invloed van oraal toegediende diuretica op de c a l c i u m - - e n fosfaatstofwisseling. Ned Tijdschr Geneeskd 109:1795, 1965. 272. Rizzoli R, Hugi K, Fleisch H, Bonjour JP: Effect of hydrochlorothiazide on 1,25-dihydroxyvitamin D3 induced changes in calcium metabolism in experimental hypoparathyroidism in rats. Clin Sci 60:101 - 107, 1981. 273. Porter RH, Cox BG, Heaney P, et al: Treatment of hypoparathyroid patients with chlorthalidone. N Engl J Med 298:577581, 1978. 274. Katz CM, Tzagourounis M: Chronic adult hypervitaminosis A with hypercalcemia. Metabolism 21:1171, 1972. 275. Frame B, Jackson CE, Reynolds WA, et al: Hypercalcemia and skeletal effects in chronic hypervitaminosis A. Ann Intern Med 80:44, 1974. 276. Caffey J: Chronic poisoning due to excess of vitamin A: Description of clinical and roentgen manifestations in seven infants and young children. Am J Rad Ther Nucl Med 65:12, 1951. 277. Burnett CH, Commons RR, Albright F, et al: Hypercalcemia without hypercalciuria or hypophosphatemia, calcinosis and renal insufficiency: A syndrome following prolonged intake of milk and alkali. N Engl J Med 240:787, 1949. 278. McMillan DE, Freeman RB: The milk alkali syndrome: A study of the acute disorder with comments on the development of chronic condition. Medicine 44:485, 1965. 279. Henneman PH, Benedict PH, Forbes AP, et al: Idiopathic hypercalciuria. N Engl J Med 259:802-807, 1958. 280. Vincent PC, Radcliff FJ: The effect of large doses of calcium carbonate on serum and urinary calcium. Am J Dig Dis 2: 2 8 6 - 2 9 5 , 1966. 281. 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. 282. Orwoll ES: The mill alkali syndrome: Current concepts. Ann Intern Med 97:242-248, 1982. 283. Ladenson JH: Calcium determination in primary hyperparathyroidism. J Bone Miner Res 6(Suppl 2):$33-$41, 1991. 284. Ralston SH, Gallacher SJ, Patel U, et al: Cancer-associated hypercalcemia: Morbidity and mortality. Ann Intern Med 12: 4 9 9 - 504, 1990. 285. Segre GV, Potts JT: Endocrinology, 3rd ed. Philadelphia, WB Saunders, 1995, pp 1094-1105. 286. Hosking DJ, Cowley A, Bucknall CA: Rehydration in the treatment of severe hypercalcemia. QJ Med 200:473-481, 1981.
442 287. Walser M: Treatment of hypercalcemia. Mod Treat 7:662, 1970. 288. Suki "VVN, Yium JJ, Von Minden M, et al: Acute treatment of hypercalcemia with furosemide. N Engl J Med 283:836-840, 1970. 289. Sato M, Grasser W, Endo N, et al: Bisphosphonate action: Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 88:2095-2105, 1991. 290. Carano A, Teitelbaum SL, Konsek JD: Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro. J Clin Invest 85:456-561, 1990. 291. Fleisch H: Bisphosphonates: Pharmacology and use in the treatment of tumor-induced hypercalcemia and metastatic bone disease. Drugs 42:919-944, 1991. 292. Reitsma PH, Teitelbaum SL, Bijoet OLM, Kahn AJ: Differential action of the bisphosphonates (3-amino-l-hydroxypropylidene)-l, 1-bisphosphonate (APD) and disodium dichloromethylidene bisphosphonate (C12MDP) on rat macrophage-mediated bone resorption in vitro. J Clin Invest 70:927-933, 1982. 293. Body JJ, Borkowski A, Cleeren A, Bijvoet OLM: Treatment of malignancy-associated hypercalcemia with intravenous aminohydroxypropylidene diphosphonate. J Clin Oncol 4 : 1 1 7 7 1183, 1986. 294. Body JJ, Pot M, Borkowski A, et al: Dose/response study of aminohydroxypropylidene bisphosphonate in tumor-associated hypercalcemia. Am J Med 82:957-963, 1987. 295. Cantwell BMJ, Harris AL: Effect of single high dose infusions of aminohydroxypropylidene diphosphonate on hypercalcemia caused by cancer. Br Med J 294:467-469, 1987. 296. Potts JT, Nussbaum SR, Neer RM: Medical management of hyperparathyroidism and hypercalcemia. In DeGroot L, et al (eds): Endocrinology, 3d ed. Philadelphia, WB Saunders Co, 1995, pp 1094-1106. 297. Thiebaud D, Jaeger P, Jacquet AF, Burckhardt P: A single-day treatment of tumor-induced hypercalcemia by intravenous aminohydroxypropylidene bisphosphonate. J Bone Miner Res 1:555-562, 1986. 298. Deftos LJ, First BP: Calcitonin as a drug. Ann Intern Med 95: 192-197, 1981. 299. Hosking DJ, Gilson D: Comparison of the renal and skeletal actions of calcitonin in the treatment of severe hypercalcemia of malignancy. Q J Med 53:359-368, 1984. 300. Lin JH: Bisphosphonates: A review of their pharmacokinetic properties. Bone 18:75-85, 1996. 301. Kammerman S, Canfield R: Effect of porcine calcitonin on hypercalcemia in man. J Clin Endocrinol Metab 31:70-75, 1970. 302. Attie MF: Treatment of hypercalcemia. Endocrinol Metab Clin North Am 18:807-828, 1989. 303. Bilezikian JP: Etiologies and therapy of hypercalcemia. Endocrinol Metab Clin North Am 18:389-413, 1989. 304. Bilezikian JP: Management of acute hypercalcemia. N Engl J Med 326:1196-1203, 1992.
JOHN T. POTTS, JR.
305. Binstock ML, Mundy GR: Effect of calcitonin and glucocorticoids in combination on the hypercalcemia of malignancy. Ann Intern Med 93:269-272, 1980. 306. Warrell RP Jr, Bockman RS: Gallium in the treatment of hypercalcemia and bone metastasis. Important Adv Oncol 1: 2 0 5 - 220, 1989. 307. Liberman Uri A, Weiss Stuart R, Broil Johann, et al: Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. N Engl J Med 333(22): 1437-1443, 1995. 307a. Warrell RP Jr, Israel R, Frisone M: Gallium nitrate for acute treatment of cancer-related hypercalcemia. Ann Intern Med 108:669-674, 1988. 308. Cortes EP, Holland JF, Muskowitz R, Depoli E: Effects of mithramycin on bone resorption in vitro. Cancer Res 32:7476, 1972. 309. Elias E, Reynoso G, Mittelman A: Control of hypercalcemia with mithramycin. Ann Surg 175:431-435, 1972. 310. Verner JV Jr, Engel FL, McPherson HT: Vitamin D intoxication: Report of two cases treated with cortisone. Ann Intern Med 48:765-773, 1958. 311. Henneman PH, Dempsey EF, Carroll EL, Albright F: The cause of hypercalciuria in sarcoid and its treatment with cortisone and sodium phytate. J Clin Invest 35:1229-1242, 1956. 312. Fulmer DH, Dimish AB, Rothschild ED, Myers WP: Treatment of hypercalcemia: Comparison of intravenously administered phosphate, sulfate and hydrocortisone. Arch Intern Med 129: 9 2 3 - 930, 1972. 313. Mundy GR, Rick ME, Turcotte R, et al: Pathogenesis of hypercalcemia in lymphosarcoma cell leukemia: Role of an osteoclast activating factor-like substance and mechanism of action for glucocorticoid therapy. Am J Med 65:600-606, 1978. 314. Ayala G, Chertow B, Shah J, et al: Acute hyperphosphatemia and acute persistent renal insufficiency induced by oral phosphate therapy. Ann Intern Med 83:520, 1975. 315. Baylink D, Wergedal J, Stauffer M: Formation, mineralization, and resorption of bone in hypophosphatemic rats. J Clin Invest 50:2519-2530, 1971. 316. Goldsmith R, Ingbar S: Inorganic phosphate treatment of hypercalcemia of diverse etiologies. N Engl J Med 274:1-7, 1966. 317. Hebert L, Lemann J, Petersen J, Lennon E: Studies of the mechanism by which phosphate infusion lowers serum calcium concentration. J Clin Invest 45:1886-1894, 1966. 318. Nolph KD, Stoltz M, Maher JF: Calcium free peritoneal dialysis: Treatment of vitamin D intoxication. Arch Intern Med 128:809-814, 1971. 319. Stoltz ML, Nolph KD, Maher JF: Factors affecting calcium removal with calcium-free peritoneal dialysis. J Lab Clin Med 78:389-398, 1971.
Renal Osteodystrophy EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ The Renal Division and Chromalloy American Kidney Center, Washington University School of Medicine, St. Louis, Missouri 63110
I. II. III. IV. V.
VI. Radiographic Features of Renal Osteodystrophy VII. Extraskeletal Calcifications VIII. Therapeutic Approach to Renal Osteodystrophy Acknowledgments References
Introduction Secondary Hyperparathyroidism and Osteitis Fibrosa Osteomalacia Bone Histology Clinical and Biochemical Features of Altered Divalent-Ion Metabolism
that the lives of patients with chronic renal failure can be prolonged through better conservative therapy, readily available dialysis, and successful renal transplantation, the morbidity associated with disordered divalent ion metabolism and bone disease has assumed greater clinical significance. Currently, the clinical features of renal osteodystrophy are better defined, several abnormal mechanisms have been clarified, and a rational approach to the prevention and treatment of renal osteodystrophy is possible.
I. INTRODUCTION A dramatic improvement in our understanding of the factors involved in the pathogenesis of "renal osteodystrophy" has occurred over the past 25 years. The term renal osteodystrophy is used in a genetic sense to include all skeletal disorders that occur in patients with renal failure, including osteitis fibrosa, osteomalacia, osteosclerosis, and growth retardation. The association of renal failure with hyperplasia of the parathyroid glands, abnormal vitamin D metabolism, and skeletal abnormalities has been known for many years. 1-4 It is not surprising that metabolic bone diseases develop in patients with chronic renal disease because the kidney plays a major role in mineral homeostasis. It maintains external balance for phosphorus, calcium, and magnesium; synthesizes 1,25-dihydroxyvitamin D [ 1,25(OH)2D] (calcitriol); degrades and removes parathyroid hormone (PTH) from the circulation; and is the main organ responsible for the excretion of aluminum. Under normal conditions small amounts of aluminum that may be ingested are rapidly eliminated in the urine. In renal failure, if the ingestion or exposure to aluminum is increased, the inability of the diseased kidney to excrete the metal imposes a serious risk in dialysis patients. Now METABOLIC BONE DISEASE
II. SECONDARY HYPERPARATHYROIDISM AND OSTEITIS FIBROSA Hyperplasia of the parathyroid glands and high levels of serum immunoreactive PTH (I-PTH) are among the most consistent pathogenetic factors affecting divalent ion metabolism in patients with chronic renal failure. Increased levels of I-PTH have been reported in patients with moderately abnormal renal function 5.6 as shown in Figure 14-1. 443
Copyright 9 1998by AcademicPress. All rightsof reproductionin any formreserved.
444
EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ
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Calcitriol and PTH values in 150 patients with various levels of chronic renal failure. [Adapted from Martinez I, Saracho R, Montenegro J, Llach F: A deficit of calcitriol synthesis may not be the initial factor in the pathogenesis of hyperparathyroidism. Nephrol Dialysis Transplant $3:22-28, 1996.]
Ionized calcium (ICa) and 1,25(OH)2D (1,25D) are the two major regulators of PTH hemostasis in man. The suppressant action of ICa on PTH secretion is very fast (< 3 minutes) and that of 1,25D is slow (> 6 hours). Hence, 1,25D plays a key role in the day-to-day maintenance of calcium balance. PTH stimulates the production of 1,25D by activating the renal l o~-hydroxylase enzyme 7, and 1,25D suppresses the synthesis and secretion of PTH. 8-~~ Thus, both PTH and 1,25D directly affect calcium homeostasis, and each exercises important regulatory effects on the other.
A. Alterations in Vitamin D Metabolism in Renal Failure" The Receptor Control of gene transcription by 1,25D is thought to be mediated by a receptor protein in target cells that has a high affinity and high specificity for the vitamin D metabolite. Although the mechanisms by which the receptor carries out the nuclear action of 1,25D is not fully understood, it is clear that the receptor can determine the response of the target cell to 1,25D. Korkor ~1 demonstrated that parathyroid glands (PTG) from patients with chronic renal failure (CRF) contained one third the number of receptors compared to parathyroid adenomas. Merke et al. 12 found in rats, 6 days after subtotal nephrectomy, that the PTG contained only half the number of receptors compared to PTG of sham-operated controis. Similar results were found by Brown et al. 13 in dogs. Although it has not been rigorously proven that vitamin D receptors (VDR) play a role in suppressing PTH synthesis and determining the sensitivity of the parathyroid gland to calcium, it is possible that the reduced
VDR numbers in PTG of uremic patients render the glands less responsive to the inhibitory action of 1,25D. Whether the levels of serum 1,25D determine the content of VDR in PTG is unclear. Navey-Many et al. 14 demonstrated that the administration of 1,25D led to a dosedependent increase in the mRNA for the VDR in the PTG of normal rats. Recently, Denda et al. demonstrated that the administration of 1,25D or its analog 22-oxacalcitriol (OCT) increases the number of VDR in the parathyroid glands of uremic rats. 15 These data are consistent with the view that 1,25D regulates its own receptor in parathyroid cells. An additional effect of 1,25D on PTG comes from the studies of Kremer et al. 16 who showed that exposure of quiescent bovine parathyroid cells in culture to serum resulted in increased 3H t h y midine incorporation, followed by an increase in the cell number. These changes were preceded by an increase in c-myc and c-fos protooncogene mRNA levels. However, 1,25D, when added to serum, blocked the increase in cmyc mRNA, and there was no increase in the number of parathyroid cells. These results indicated that 1,25D may directly modulate parathyroid cell proliferation by altering the expression of replication associated with specific oncogenes. Thus, 1,25D may be an important inhibitory growth factor of parathyroid cells, and low levels of 1,25D may allow parathyroid cells to proliferate. In addition, studies by Szabo et al. 17 in experimental animals with renal failure suggest that 1,25D administration suppresses parathyroid hyperplasia independent of changes in serum calcium. Once established, hyperplasia was not reversed by short-term 1,25D treatment. Fukuda et al. 18 have provided evidence for a decreased 1,25D receptor density in patients who have parathyroid hyperplasia. The investigators studied the VDR distribution of surgically excised PTG obtained from dialysis patients. They classified the parathyroids as exhibiting nodular or diffuse hyperplasia. They found a lower density of the VDR in PTG showing nodular hyperplasia than in those showing diffuse hyperplasia. A significant negative correlation was found between VDR density and the weight of the PTG (Fig. 14-2). In other words, the higher the levels of PTH in serum, the greater the degree of PTG hyperplasia and the lower the density of the VDR. These studies provide rational basis for understanding the difficulties in suppressing secondary hyperparathyroidism (SH) when the PTH levels are extremely high (> 1500 pg/ml). In addition, it is important to emphasize that in the PTG there is a component of the secretory mechanism that cannot be suppressed by either high ICA or pharmacological doses of 1,25D. Although this component of the secretory mechanism represents a small percentage of the total amount of PTH secreted by the PTG in normal individuals, it becomes extremely important in those patients in whom the PTG
CHAPTER 14 Renal Osteodystrophy
FIGURE 14--2 The relationship between parathyroid gland weight and percentage positive VDR staining. (Adapted from Fukuda N, Tanaka H, Tominaga Y, et al: Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest 92: 1436-1443, 1993.)
are 50 to 100 times normal size. Many years ago, Gittes et al. 19 clearly demonstrated this p h e n o m e n o n in rats by implanting a large number of PTG in rats that had undergone a previous parathyroidectomy (PTX). The serum calcium decreased following PTX. However, after the implantation of 20 or 80 PTG, the rats developed severe hypercalcemia. One would expect that the hypercalcemia should suppress the release of PTH. However, it was clear from these experiments that the hypercalcemia continued due to the fact that a nonsuppressible component was present, and the large amount of tissue was responsible for the maintenance of hypercalcemia. Furthermore, when Mayer et al. 2~ measured the A-V difference of PTH across the PTG in cows, they found that despite induction of severe hypercalcemia (serum calcium up to 18 to 20 mg/dl), there was still a small component of PTH secretion from the PTG (Fig. 1 4 - 3 ) . Thus, the development of hyperplasia with a consequent increase of a nonsuppressible component and the low number of 1,25D receptors makes the PTG more resistant to the use of 1,25D in the treatment of SH.
B. Decreased Sensitivity to Calcium Evidence exists for an intrinsic abnormality of the PTG in uremia that leads to disordered calciumregulated PTH secretion. An insensitivity to the suppressive effect of calcium on PTH secretion has been shown in glands obtained from patients with CRF. 21'22 These observations suggest that one mechanism for the increased PTH levels in CRF may be a shift in the set-
445
FIGURE 14--3 Levels of plasma PTH before and after a calcium infusion. (Adapted from Mayer GP, Habener JF, Potts JT Jr: Parathyroid hormone secretion in vivo. Demonstration of a calcium-independent, nonsuppressible component of secretion. J Clin Invest 57:678683, 1976.)
point (defined as the ICa level that causes a 50% suppression of maximally stimulated PTH) for calciumregulated PTH secretion in addition to the increase in the mass of parathyroid tissue. The set-point for calcium in normal PTG was --~1.0 m M of calcium, whereas in patients with SH, the set-point was found to be increased to 1.26 m M of calcium. These abnormalities also are manifested by an increase in the calcium concentration required for the inhibition of the adenylate cyclase activity in membranes prepared from hyperplastic PTG obtained from patients with C R E 23 It is possible, therefore, that normal concentrations of ICa in serum may not be sufficient to suppress hyperplastic PTG. Thus, the serum calcium levels may have to be increased to the upper limits of normal to control the rise of PTH in patients with SH. Although the precise mechanism responsible for the decreased sensitivity to calcium in hyperplastic PTG is unknown, alterations in vitamin D metabolism (i.e., low levels of 1,25D and decreased number of V D R in the PTG) may account, in part, for the abnormal secretion of PTH in renal failure. Brown et al. 24 cloned a calcium receptor (Ca-R) localized in bovine parathyroid cell membranes. Although the molecular nature of such receptors(s) is unknown, parathyroid cells possess an extracellular calciumsensing mechanism that recognizes trivalent and polyvalent cations (such as neomycin) and regulates PTH secretion by changes in phosphoinositide turnover and
446 cytosolic calcium levels. This receptor features a large, extracellular domain containing clusters of acidic amino acids that are possibly involved in calcium binding. The extracellular domain is coupled to a cellular membranespanning domain similar to the G-protein-coupled receptor superfamily. 24 Pollack et al. 25 demonstrated that mutations in the human calcium-sensing receptor cause familiar hypocalciuric hypercalcemia and severe neonatal hyperparathyroidism. Recently, Kifor et al. 26 using immunohistochemistry techniques, demonstrated a 59% decrease in Ca-R in parathyroid glands of uremic patients. Similar results were demonstrated in adenomas of parathyroid glands obtained from patients with primary hyperparathyroidism. These investigators concluded that the degree of Ca-R reduction would be sufficient to account, at least in part, for the altered sensitivity to Ca observed in secondary hyperparathyroidism. Arnold et al. 27 examined the abnormality in primary and secondary hyperparathyroidism using chromosome inactivation analysis with the M2713 DNA polymorphism and by searching for monoclonal allelic losses at M2713 and at loci on chromosome l lq13. Seven of 11 (64%) uremic patients demonstrated at least one monoclonal parathyroid tumor, as did 38% of patients with primary chief cell hyperplasia. These findings suggest that monoclonal transformation of previous hyperplastic parathyroid tissue may be another factor responsible for the development of autonomous hyperparathyroidism. Thus, the combination of an enlarged parathyroid cell (hypertrophy), increased numbers of parathyroid cells (hyperplasia), abnormal calcium-regulated secretion of PTH, nodular appearance with loss of vitamin D receptors, decreased number of calcium receptors, and monoclonal changes may all participate in the development of nonsuppressible secondary hyperparathyroidism (Fig. 14-4). To further characterize the potential role of 1,25D on the abnormal set-point for the suppression of PTH by calcium, Delmez et al. 28 studied the suppression of PTH by calcium before and after 2 weeks of intravenous (IV) 1,25D in a group of hemodialysis patients. During hypercalcemic suppression, the calcium set-point for PTH fell from 5.24 mg/dl to 5.06 mg/dl after the administration of 1,25D. During hypocalcemic stimulation, the parathyroid response was attenuated by 1,25D. Thus, the suppression of PTH secretion during treatment with 1,25D appears to be due, in part, to an increase in the sensitivity of the PTG to ambient calcium levels (Fig. 14-5). Dunlay 29 found similar inhibitory effects with IV 1,25D in dialysis patients. After 10 weeks of IV 1,25D, there was a significant decrease in the levels of serum PTH. These investigators also found that the relation of ionized calcium-PTH sigmoidal curve was shifted to the
EDUARDO SLATOPOLSKYAND JAMES A. DELMEZ
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left, suggesting an increase in the suppressive effect of calcium. Others have been unable to confirm these results. Several years ago 3~ we showed that the IV administration of 1,25D was very effective in the suppression of Ca
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The relationship between ionized calcium and serum PTH before ( e - - e ) and after treatment with 1,25(OH)zD3 (o--o). (Adapted from Delmez JA, Tindira C, Grooms P, et al: Parathyroid hormone suppression by intravenous 1,25-dihydroxyvitamin D: A role for increased sensitivity to calcium. J Clin Invest 83: 1349-1355, 1989.)
CHAgrER 14 Renal Osteodystrophy
447
FIGURE 1 4 - 6 Mean 1,25(OH)2D3 serum concentrations at various times after (o--o) bolus injection of 35 pM 1,25(OH)2D3 and at various times during (omo) continuous infusion of 70 pM 1,25(OH)2D3. (Adapted from Reichel H, Szabo A, Uhl J, et al: Intermittent versus continuous administration of 1,25-dihydroxyvitamin D3 in experimental renal hyperparathyroidism. Kidney Int 44:1259-1265, 1993.)
SH. Studies performed in 20 patients maintained on chronic hemodialysis given IV 1,25D demonstrated a marked suppression of PTH (70.1 _ 3.2%). On the other hand, studies performed in a group of patients treated with oral daily doses (0.5 txg) of 1,25D showed less suppression. We also demonstrated 31 in primary culture of bovine parathyroid cells that the suppression of PTH by 1,25D was dose dependent. Tsukamoto et al. 32 showed that 1,25D, when given as an oral pulse of 3 to 4 txg twice weekly, achieved a greater suppression than 0.5 Ixg daily. Reichel et al. 33 compared the effect of intermittent versus continuous administration of 1,25D in uremic rats with SH. One group of animals received 35 pM of 1,25D via intraperitoneal (IP) bolus on days 0 and 4. A second experimental group received a continuous infusion of 70 pM of 1,25D over 6 days via an osmotic minipump. Thus, over the same period of time, the two groups received the same amount of 1,25D. Peak 1,25D concentrations were significantly higher and PTH values were significantly lower in the group that received the two IP boluses compared to the group that received the continuous infusion (Fig. 14-6). The calculated timeaveraged increase in 1,25D serum concentrations during the 6 days was 254 pg/ml (42.3 pg/ml/24 hr) in continuous infusion-treated animals and 156 pg/ml (26.0 pg/ ml/24 hr) in bolus-treated animals. The degree of suppression of the prepro PTH/13-actin m R N A was also greater in the group that received the IP bolus compared to those animals treated with a constant infusion (Fig.
14-7). Moreover, the growth of the parathyroid glands was prevented only with the bolus administration (Fig. 14-8). Thus, these investigators conclude that the concentration of 1,25D achieved in serum is an important determinant of the response of PTG to 1,25D. Although 1,25D and calcium are negative regulatory elements in the upstream region of the PTH gene, they act on different locations. Both are of great importance and simultaneously may participate in the suppression of PTH secretion. The calcium responsive element was found around - 3 . 5 Kb upstream from the human PTH
FIGURE 14--7 Effectof bolus versus continuous 1,25(OH)2D3administration on prepro PTH mRNA. (Adapted from Reichel H, Szabo A, Uhl J, et al: Intermittent versus continuous administration of 1,25dihydroxyvitamin D3 in experimental renal hyperparathyroidism. Kidney Int 44:1259-1265, 1993.)
448
EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ
FIGURE 1 4 - - 8 Effect of bolus versus continuous 1,25(OH)2D3 administration on parathyroid gland weight. (Adapted from Reichel H, Szabo A, Uhl J, et al: Intermittent versus continuous administration of 1,25-dihydroxyvitamin D 3 in experimental renal hyperparathyroidism. Kidney Int 44:1259-1265, 1993.)
gene, and its sequence consists of 12 palindromic bases with a gap of three bases. 34 In contrast, the negative vitamin D-responsive element was found in a 25-bp oligonucleotide spanning from - 1 2 5 to - 1 0 1 . 35 In order to determine the relationship of these two elements (ICa and 1,25D) in the suppression of the secretion of PTH in uremic rats, we induced hypocalcemia by feeding uremic rats a low-calcium diet. 36 We clearly demonstrate that pharmacological doses of 1,25D, that usually suppress SH, failed to block the synthesis of prepro PTH mRNA and the secretion of PTH. Thus, correction of both calcium and 1,25D are crucial in the treatment of SH.
C. P h o s p h a t e R e t e n t i o n Phosphate retention plays a key role in the pathogenesis of secondary hyperparathyroidism. 37-39 Reiss et al. 4~ reported that an oral load of phosphorus, providing 1.0 g of elemental phosphorus, led to an increase in serum phosphorus, a fall in ICa levels, and an increase in serum I-PTH in normal subjects. Several investigators have shown that long-term feeding of animals with a diet high in phosphate can produce parathyroid hyperplasia, increased levels of I-PTH, and a mild reduction in serum calcium l e v e l s . 41'42 Studies in experimental renal failure have shown that the restriction of dietary phosphate in proportion to the decrease in glomerular filtration rate (GFR) can prevent the development of secondary hyperparathyroidism in azotemic dogs followed for 2 months. 38 Subsequent studies 43 showed a substantial reduction of serum I-PTH levels in animals with renal failure treated with a phosphate-restricted diet for 2 years. Llach et al. 44 indicated that a reduction in dietary phos-
phate intake in proportion to the decrease in GFR over a 2-month period in humans with creatinine clearances of 50 to 90 ml/min was associated with a decrease in serum I-PTH to normal levels. When phosphate intake was reduced in patients with more advanced renal insufficiency, serum I-PTH fell substantially but remained above normal levels. 45 Other studies have shown that serum I-PTH levels correlate positively with the degree of hyperphosphatemia in patients undergoing dialysis, 46 an observation providing support for a role of hyperphosphatemia in causing parathyroid hypersecretion, particularly in patients with advanced uremia. Thus, phosphate loading could cause a small fall in the level of ionized calcium, or it may decrease the renal production of 1,25D 47 which may permit more secretion of PTH at any given levels of serum calcium. As renal disease advances and GFR falls below 25 ml/min, hyperphosphatemia is usual, 48 and hypocalcemia is more directly related to a markedly increased level of serum phosphorus. Although high concentrations of serum phosphate (7 to 9 mg/dl) precipitate calcium in soft tissues, the mechanism by which mild hyperphosphatemia affects the concentration of ionized calcium in serum is not known. It may decrease the release of calcium from bone 49 or affect the activity of the renal enzyme l oL-hydroxylase responsible for the conversion of 25(OH)D to 1,25(OH)2D. 47 Portale et al. 5~ demonstrated that, in patients with moderate renal insufficiency, phosphate restriction increased plasma 1,25D levels with a concomitant normalization of plasma PTH. This occurred despite no changes in serum phosphorus levels. Thus, the effect of phosphate on the l et-hydroxylase enzyme in early renal insufficiency may not be responsible for the development of hypocalcemia that may be seen in patients with advanced renal failure and severe hyperphosphatemia. Since reduced renal mass may limit the production of 1,25D in advanced renal insufficiency, we performed further studies in uremic dogs 52 to clarify the mechanism by which dietary phosphate restriction improved SH. We examined how dietary phosphate restriction could ameliorate SH without increasing the levels of 1,25D (Fig. 14-9). Our results confirmed an important effect of phosphorus restriction on suppressing SH. However, in contrast to previous findings in patients with moderate renal insufficiency, progressive reduction of dietary phosphorus did not increase plasma 1,25D levels (Fig. 14-10). Thus, in severe chronic renal failure phosphorus appears to regulate PTH secretion by a mechanism that is independent of calcitriol. In agreement with our results, Lucas 53 found that the administration of a low-phosphorus diet to patients with advanced renal insufficiency did not increase 1,25D levels. Plasma PTH levels significantly decreased, while serum calcium levels did not change. Schaefer 54 obtained similar results in
CHAPTER 14 Renal Osteodystrophy
FIGURE 14--9 Effects of dietary phosphorus and calcium on plasma ionized calcium (top), plasma phosphorus (middle), and PTH (bottom) in five uremic dogs. (Adapted from Lopez-Hilker S, Dusso A, Rapp N, et al: Phosphorus restriction reverses secondary hyperparathyroidism in chronic renal failure independent of changes in calcium and 1,25 dihydroxycholecalciferol. Am J Physiol 259:F432F437, 1990.)
a group of 17 patients with advanced renal insufficiency (plasma creatinine 8.7 mg/dl) in whom ketoacids were added to the diet. After 8 weeks of treatment, they found a significant decrease in the levels of plasma phosphorus and IPTH. There were no changes in plasma calcium or
FIGURE 14--10
Effects of dietary phosphorus and calcium on plasma 1,25(OH)2D3 in five uremic dogs. (Adapted from Lopez-Hilker S, Dusso A, Rapp N, et al: Phosphorus restriction reverses secondary hyperparathyroidism in chronic renal failure independent of changes in calcium and 1,25 dihydroxycholecalciferol. Am J Physiol 259: F432-F437, 1990.)
449 1,25D values. Tessitore et al. 55 also showed that dietary phosphorus restriction did not have an effect on 1,25D concentration when the GRF was less than 20 ml/min. In vitamin D-deficient rats, Dabbagh et al. 56 observed that in the presence of a normal plasma calcium concentration, the administration of a phosphorus-restricted diet prevented the development of SH. Recently we have shown that dietary phosphate restriction prevented parathyroid cell growth in uremic rats. In addition, high phosphate concentrations in the media post-transcriptionally stimulated PTH secretion in tissue culture. 57 Studies were performed in normal and uremic rats fed a low-phosphorus (0.2%) or highphosphorous (0.8%) diet for a period of 2 months. Parathyroid gland weight and serum PTH were similar in both groups of normal rats and uremic rats fed the 0.2% phosphorus diet. On the other hand, in uremic rats fed the 0.8% phosphorus diet, the parathyroid gland weight increased by approximately 120% compared to the normal animals fed the same diet. In the uremic rats fed a high-phosphorus diet PTH also increased from 29.1 • 6.1 Pg/ml to 130 • 25 pg/ml. There were no changes in ICa or 1,25D. In vitro studies with parathyroid glands of normal rats demonstrated that, when the phosphorus in the culture medium was increased from 0.2 mM to 2.8 mM, the amount of PTH secreted into the medium increased from 810 • 155 to 1492 • 182 pg/Ixg DNA per 5 hours. Since it took a minimum of 3 hours for phosphorus to increase the amount of PTH in the medium, the effect of phosphorus was mainly on PTH synthesis and eventually on secretion (Fig. 14-11). The addition of cycloheximide blocked the effect of phosphorus, suggesting that protein synthesis is necessary for the increment in PTH secretion. In conclusion, dietary phosphorus restriction improves secondary hyperparathyroidism in animals and subjects with advanced renal failure. This effect is not mediated by an increase in the levels of 1,25D or plasma ICa. In addition, high-phosphorus diets induce chief cell hyperplasia of parathyroid glands in uremic rats. Moreover, studies in vitro demonstrate that high phosphorus levels increase PTH synthesis and secretion. Thus, in addition to a well-known effect of phosphorus in the regulation of 1,25D synthesis, a high-phosphorus diet may have direct effect on the secretion of PTH. Although the mechanism of this effect is not yet known, phosphorus may potentially affect the phospholipid composition of the parathyroid cell membrane, calcium fluxes, and VDR, and perhaps have an effect on the calcium receptor in the parathyroid cell membrane. From the clinical point of view, significant data have accumulated indicating that, in the present of hyperphosphatemia, the parathyroid glands are resistant to the action of 1,25D. The new information of a direct action of
450
EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ 2000
*
1800
2.8 P mM
~- 1600 Z s
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,
9
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;
-
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FIGURE 14-11
Time course for PTH secretion by normal intact rat parathyroid glands, incubated in a low (0.2 mM) (o) (n - 8) or high (2.8 mM) (o) (n = 8) phosphorus in the media (P < 0.05). The effects of phosphorus were not evident until 3 hours (From Slatopolsky E, Finch J, Denda M, et ah Phosphate restriction prevents parathyroid cell growth in uremic rats and high phosphate directly stimulates PTH secretion in tissue culture. J Clin Invest 97:2534-2540, 1996.)
phosphorus on PTH synthesis and chief cell hyperplasia emphasizes the importance of controlling serum phosphorus in chronic renal failure. Further studies are necessary to determine the precise mechanism at the molecular level by which phosphorus contributes to the regulation of PTH secretion in chronic renal failure.
The concept of skeletal resistance to PTH as an important factor in the development of secondary hyperparathyroidism has been challenged. Kaplan et al. 6~ fed dogs with chronic renal failure a low-phosphate diet and prevented the development of SH. Nonetheless, these animals developed skeletal resistance to the calcemic action of PTH. Thus, despite the presence of skeletal resistance to PTH, the dogs did not develop secondary hyperparathyroidism. However, Llach et al. 44 found that dietary phosphate restriction improved the calcemic response to an infusion of PTH in patients with mild renal insufficiency. It is possible that alterations in vitamin D metabolism could be responsible for the resistance to the calcemic action of PTH seen in uremia. In dogs with acute renal failure, 6~ treatment with 1,25(OH)2D3 resuited in partial correction of the blunted calcemic response to parathyroid extract. Other observations suggest that hyperphosphatemia accounts for the skeletal resistance to parathyroid extract in rats with acute renal failure. 62 Galceran et al. 63 studied the calcemic effects of PTH in dogs before and after the induction of chronic renal failure. The administration of calcitriol did not improve the calcemic response to PTH (Fig. 14-12). However, when the uremic dogs underwent a parathyroidectomy 24 hours before the infusion of PTH, the calcemic response returned to normal. Therefore, despite low levels of 1,25(OH)2D3 and maintenance of a uremic environment, removal of PTH from the circulation restored the calcemic response to exogenous PTH to normal. These 1.5
i D. Skeletal Resistance to the Calcemic Action of Parathyroid Hormone Skeletal resistance to the calcemic action of PTH may be an important cause of hypocalcemia in patients with renal insufficiency. The calcemic response to the infusion of parathyroid extract is blunted in hypocalcemic patients with renal failure compared to normal subjects or patients with hypoparathyroidism. 58 The finding of a delayed recovery from ethylenediaminotetraacetic acid (EDTA)-induced hypocalcemia in patients with mild renal insufficiency, despite a greater augmentation in serum I-PTH levels compared to normal subjects, indicates that the skeletal resistance to endogenous PTH appears early in the course of renal insufficiency. 59 Such data suggest that higher levels of PTH may be needed to maintain normal serum calcium levels in patients with renal failure.
1.0
oo 0.5
7 days NS
FIGURE 14--12
90 days
180 days 1,25(OH)2D 3 1,25(OH)2D 3 +TPTX T-PTX
P<0.05 P<0.05 P<0.02
NS.
NS
Calcemic response to the administration of PTH in a group of dogs before and after the induction of renal failure. After 90 days renal insufficiency the dogs had a blunted response to PTH. The administration of 1,25(OH)2D3 did not improve the calcemic response. A T-PTX 24 hours before the administration of exogenous PTH returned the calcemic response to normal. (Adapted from Galceran T, Martin KJ, Morrissey J, Slatopolsky E: Role of 1,25 dihydroxyvitamin D on the skeletal resistance to parathyroid hormone. Kidney Int 32:801-807, 1987.)
CHAPTER 14 Renal Osteodystrophy results clearly demonstrated that low levels of circulating calcitriol are not responsible for the skeletal resistance to PTH in uremia. They suggest that desensitization to high levels of endogenous PTH prevents a normal calcemic response to the administration of exogenous PTH in uremia.
E. R o l e o f R e d u c e d D e g r a d a t i o n o f P T H Increased secretion of PTH is the major factor responsible for high plasma levels of I-PTH in patients with renal insufficiency. Since the kidney plays an important role in the degradation of PTH, this could be a factor contributing to the high levels of PTH in uremia. Because the kidney may be the only organ removing the carboxyl-terminal fragments of PTH from the circulation, 64 the levels of these fragments are greatly increased in renal failure. Measurements of I-PTH specific for the carboxyl fractions reveal values that are much higher in uremic patients than in patients with primary hyperparathyroidism. 65 However, the kidney also plays a role in the degradation of the intact PTH(1-84) molecule and studies of the metabolic clearance of bovine PTH(1-84) suggest that it is decreased with renal failure. 66
III. O S T E O M A L A C I A Osteomalacia is another important feature of the skeletal disease in some patients with advanced renal failure. Bone biopsies show an increase in the osteoid seam width and impaired mineralization activity. The pathogenesis of decreased skeletal mineralization in patients with chronic renal failure is not clear. It is conceivable that altered vitamin D metabolism leads to impaired mineralization of bone. Whether 1,25(OH)2D3 directly stimulates bone mineralization or leads to mineral deposition by increasing the levels of calcium and phosphate in the extracellular fluid surrounding bone is controversial. Although the plasma levels of 1,25(OH)2D3 are reduced in patients with advanced renal insufficiency, overt osteomalacia is found in only a small fraction of patients. 67 Moreover, osteomalacia may be absent even in anephric patients. 68 Thus, other factors appear to participate in the pathogenesis of osteomalacia in uremic patients. One factor that may influence the development of osteomalacia is the plasma phosphorus levels. Hypophosphatemia p e r s e can produce severe osteomalacia even in patients with normal renal function. Alterations in collagen synthesis and maturation may also contribute to the development of osteomalacia. Experimental studies in uremic rats have shown a preponderance of immature soluble collagen that fails to mineralize normally. Treat-
451 ment of such uremic animals with 25(OH)D can normalize collagen maturation. 69 Bone crystal formation also is affected in patients with renal insufficiency. Immature bone contains large quantities of amorphous calcium phosphate. Other abnormalities in the bone of uremic patients include an increase in magnesium and pyrophosphate content and decreased amounts of carbonate. A combination of these factors may play a role in bone maturation and contribute to the development of uremic osteomalacia. Acidosis also may contribute to the pathogenesis of renal osteodystrophy. Administration of bicarbonate and correction of the acidosis in azotemic patients can reduce fecal calcium and improve calcium balance, TM and therefore may be of therapeutic benefit. It is clear that the retention of aluminum plays an important role in the development of osteomalacia. In patients with advanced renal failure treated with dialysis it was believed that the major source of aluminum was in the dialysate. In geographical areas with a high aluminum content in the tap water used for dialysate, the incidence of osteomalacia decreased substantially with subsequent deionization treatment. 71 However, currently it is universally accepted that the main source of aluminum accumulation is the ingestion of phosphate binders containing aluminum. Finally, evidence suggests that a lack of PTH, as has been seen in uremic patients after parathyroidectomy, can precipitate the development of osteomalacia. 72,73
IV. BONE HISTOLOGY A. S e c o n d a r y H y p e r p a r a t h y r o i d i s m (High-Turnover Bone Disease) Examination of bone is often important for the diagnosis and management of patients with renal osteodystrophy (Figs. 14-13 and 14-14). The most common histological finding is osteitis fibrosa secondary to high levels of PTH. In these patients, the histological findings are osteoclastosis and increased bone resorption surfaces. Osteoblastic activity is also substantially increased. The total number of osteoblasts is usually increased, and a large portion of the surface of bone is involved in bone formation. Bone biopsies characteristically show increased bone turnover and increased quantifies of woven osteoid. This differs from normal lamellar osteoid in that there is a haphazard arrangement of collagen fibers. Woven osteoid can be mineralized, but the calcium may be deposited as amorphous calcium phosphate rather than as hydroxyapatite. The excess deposition of calcium phosphate in woven osteoid may explain the finding of osteosclerosis in some uremic patients.
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EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ
FIGURE 1 4 - - 1 3 Baseline bone biopsy from a patient on chronic maintenance dialysis exhibiting signs of marked secondary hyperparathyroid bone disease. Features include irregular trabecular structure, increased osteoid surface and volume, abundance of osteoblasts and osteoclasts, and marked bone marrow fibrosis. (Mineralized bone histology. Under calcified 3-1xm-thick section. Modified Masson-Goldner trichrome; original magnification • (Courtesy of Dr. Malluche.)
B. Osteomalacia and Adynamic Bone (Low-Turnover Bone Disease) Osteomalacia is characterized by a reduction in the number of bone-forming and bone-resorbing cells along with an accumulation of lamellar osteoid or unmineralized bone collagen in trabecular bone. Osteoidosis, however, does not indicate the presence of osteomalacia or defective mineralization. The amount of osteoid present in bone depends on the rate and extent of its formation by osteoblasts versus the rate and extent of calcification. Thus, in order to make the histological diagnosis of osteomalacia, an abnormal rate of mineralization must be present. The use of double tetracycline labeling in conjunction with quantitative histomorphometric techniques provides the best method for identification of defective mineralization. The bone turnover rate is normal or increased in patients with osteitis fibrosa, whereas it is decreased in patients with osteomalacia. Dialysis patients with pure osteomalacia usually show little or no evidence of secondary hyperparathyroidism. 74 Osteomalacia in patients with chronic renal disease can result from vitamin D deficiency or from hypocalcemia and/or hypophosphatemia. However, in patients treated with chronic dialysis, aluminum accumulation currently accounts for most of the osteomalacic lesions. In these patients, stainable aluminum deposits at the bone-osteoid surface are usually seen.
A disorder termed "aplastic" or " a d y n a m i c " refers to bone histology exhibiting normal amounts of osteoid, no tissue fibrosis, decreased number of osteoblasts and osteoclasts, and low rates of bone formation. Patients with this lesion may have had a high exposure to aluminum. However, this lesion has also been observed in the absence of aluminum in patients with relatively low levels of circulating PTH (PTH < 200 pg/ml). 75 The long-term clinical consequences of this lesion are unknown. However, it has been reported that patients with adynamic bone have less bone fractures and symptoms of bone pain and myopathy than those with osteitis fibrosa and aluminum-related bone disease. 76
C. Mixed Lesion Some patients demonstrate histological evidence of both osteitis fibrosa and osteomalacia on bone biopsy. This pattern is termed a "mixed lesion" of renal osteodystrophy. 77 Such patients frequently have biochemical findings characteristic of high PTH in addition to features that are associated with impaired bone formation and mineralization. Tetracycline studies reveal a decrease in the double-labeled lamellar osteoid seams. Usually there is an increased number of osteoblasts and osteoclasts and moderate peritrabecular fibrosis. In the past, mixed renal osteodystrophy was seen in patients
CHAPTER 14 Renal Osteodystrophy
453
FIGURE 14--14 Bone biopsy in the same patient as in Figure 14-13, 1 year after therapy with oral 1,25(OH)2D3 therapy. There was a decrease in the severity of hyperparathyroidism with decreased osteoid volume and surface, reduction in the number of osteoblasts and osteoclast, and less marrow fibrosis. (Mineralized bone histology. Undecalcified 3-1xm-thicksection. Modified Masson-Goldner trichome; original magnification x20.) (Courtesy of Dr. Malluche.)
with previously established osteitis fibrosa who were developing aluminum-related bone disease. A large number of patients with mixed lesions have significant stainable aluminum at the mineralization front.
V. CLINICAL AND BIOCHEMICAL FEATURES OF ALTERED D I V A L E N T - I O N METABOLISM Alterations in mineral homeostasis may appear early in the course of renal insufficiency, and their identification should assist the physician in initiating prompt treatment to prevent renal osteodystrophy. Symptoms from altered divalent-ion metabolism, however, usually appear only in patients with advanced renal failure. Bone pain can develop and progress to the point where the patient becomes incapacitated. This can occur if the skeletal abnormality is osteitis fibrosa or osteomalacia. The bone pain is often vague and commonly is located in the lower back, hips, knees, and legs. Low back pain may arise from collapse of a vertebral body, and sharp chest pain may result from spontaneous rib fractures. However, physical findings frequently are lacking. Muscular weakness, when present, is usually proximal. It appears slowly and often progresses. Plasma levels of the muscle enzyme, creatinine phosphokinase, usually are normal,
and the electromyographic changes are nonspecific. The pathogenesis of the muscle weakness is uncertain. In patients with such myopathy, electron micrographic studies have revealed localized disorganization of the myofibrils and dispersion of Z bands. These changes have been shown to revert to normal following treatment with 25(OH)D. 78 Others have attributed the muscular weakness to secondary hyperparathyroidism on a neuropathic basis. 79 Finally, we have seen the resolution of muscle weakness following treatment of aluminum excess with chelation therapy. Pruritus, an especially common symptom in uremic patients with severe secondary hyperparathyroidism, has been attributed to an increased amount of calcium in the skin. Moreover, pruritus can develop in uremic patients with hypercalcemia or severe hyperphosphatemia. In patients with chronic renal failure, peripheral ischemic necrosis and vascular calcification have been reported. The lesions may involve the tips of the toes and fingers, where the skin becomes violaceous. Ulceration and scar formation may occur, with clear demarcation of the lesions from the surrounding skin. Most patients with such problems have severe secondary hyperparathyroidism. Some have responded to a subtotal parathyroidectomy (Fig. 14-15). Acute pain and swelling around one or more joints may develop in uremic patients. This syndrome of calcified periarthritis may be caused by the deposition of hydroxyapatite crystals and is associated with marked
454
EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ
FIGURE 14-15 A, Necrosis of the toes, 5 months after renal transplantation, in a patient with a long history of chronic renal failure. B, Complete healing of lesions in same patient 3 months after subtotal parathyroidectomy. (From Massey SG, et al: Secondary hyperthyroidism. Arch Intem Med 124:431-439, 1969. Copyright 1969, American Medical Association.)
FIGURE 14--16 hyperphosphatemia. 8~Abnormal collagen metabolism, as is believed to exist in uremic bone, may also occur in tendons and may predispose to spontaneous tendon rupture. Patients maintained on chronic dialysis for more than 5 years often develop amyloid deposition in bone and soft tissues. This type of amyloid is comprised of [32microglobulin. 8~ Since [32-microglobulin, a polypeptide, is degraded by the kidney, the serum levels increase substantially in renal failure. Amyloid deposition in the musculoskeletal system may cause symptoms that could be confused with those of renal osteodystrophy. A common manifestation is the occurrence of the carpal tunnel syndrome with entrapment of the median nerve by fibrous and amyloid tissue. In addition, erosive arthritis of the shoulders, interphalangeal joints, and knees is frequently seen. X-rays may demonstrate the presence of cysts in the humerus (Fig. 14-16), femoral head, acetabulum carpal, and tarsal bones. Fractures can develop in bones affected and weakened by these cystic changes. 82 Currently, it is not known why all dialysis patients are not affected by amyloidosis nor if certain methods of dialysis will influence the natural history of this disorder. Unfortunately, the management of this pathological condition is unsatisfactory. It is likely, however, that early transplantation would prevent its development. Once established, the radiographic changes of amyloid do not regress with transplantation. The symptoms, however, often improve.
Two large cysts in the head of the humerus (From Alfred A: American Kidney Foundation. Nephrol Lett 6:(4), 1989.)
Skeletal deformities are common in growing azotemic children. Bowing of the tibiae and femurs and deformities due to slipped epiphyses are not uncommon. Children with renal tickets sometimes exhibit typical radiographic findings of vitamin D deficiency. Growth retardation is usually seen in young children both before and during maintenance hemodialysis. Several factors, such as malnutrition, chronic acidosis, and severe osteomalacia, may contribute to the retarded growth. Caloric supplementation, correction of acidosis, and treatment with 1,25(OH)2D3 and growth hormone may improve the growth rate. In adults with renal failure, particularly those with osteomalacia, marked skeletal deformities, with lumbar scoliosis, thoracic kyphosis, and deformities of the thoracic cage, may be observed. The latter two conditions may cause respiratory compromise. From the biochemical point of view, one of the early changes in patients with renal insufficiency (GFR between 60 and 80 ml/min) is the presence of elevated levels of circulating I-PTH. As the disease progresses (GFR < 30 ml/min), hypocalcemia may be present. Alterations in circulating ionized calcium levels are seen earlier than in total serum calcium. This is because of
CHAPTER 14 Renal Osteodystrophy an increase in the complexed fraction of blood calcium. However, the serum calcium levels usually remain close to normal in patients with advanced renal insufficiency with values below 7.5 mg/dl noted infrequently. Usually, hypocalcemia is most marked in patients with osteomalacia, profound metabolic acidosis, or severe hyperphosphatemia. Occasionally, hypercalcemia may be observed in uremic patients, particularly in those undergoing long-term dialysis. This can arise from severe secondary hyperparathyroidism, high levels of calcium in the dialysate, ingestion of large amounts of calcium or vitamin D, unrelated diseases such as sarcoidosis or malignancy, or from low bone turnover due to either osteomalacia or adynamic bone disease. Hyperphosphatemia is common in patients with a GFR less than 25 ml/min. The degree of hyperphosphatemia depends on the amount of phosphate ingested in the diet, the fraction absorbed by the intestine, and the quantity excreted into the urine. With judicious use of phosphate binders and dietary phosphorus restriction, the serum phosphorus may remain normal despite advanced renal insufficiency. PTH can also influence the concentration of serum phosphorus. Although PTH decreases the reabsorption of phosphorus by the renal tubule, it also mobilizes phosphorus from bone. Thus, patients with severe hyperparathyroidism and advanced renal insufficiency usually have high serum concentrations of phosphate. Hypermagnesemia may occur in patients when the GFR falls below 15 ml/min. Intake of magnesium-containing antacids by patients with severe renal failure can lead to an abrupt and marked hypermagnesemia. Increased serum magnesium is usually associated with an increased content of magnesium in bone, which may in turn decrease mineralization. In patients receiving chronic hemodialysis, with a dialysate magnesium concentration of 1.5 mEq/liter, hypermagnesemia with blood levels of 2.5 to 3.0 mEq/liter, are common. Serum alkaline phosphatase activity is often increased in uremic patients with osteitis fibrosa, osteomalacia, or mixed lesions. Although serum alkaline phosphatase activity is composed of isozymes, activity arising from intestine, liver, kidney, and bone, routine alkaline phosphatase measurements, when increased in uremic patients, usually suggest increased osteoblastic activity. In dialysis patients, serum alkaline phosphatase activity correlates with the extent of bone osteoid surface covered with osteoblasts, resorbing surfaces, the number of osteoclasts, and the percentage of osteoid seams that take up tetracycline. 83'84 Coexistent liver disease, however, should be excluded as a cause of elevated alkaline phosphatase in uremic patients. Bone GLA-protein, also known as osteocalcin, synthesized by osteoblasts is a marker of bone formation. However, plasma levels are
455 increased when renal function is impaired. In dialysis patients there is a good correlation between the levels of osteocalcin, PTH, and alkaline phosphatase. Patients with high bone turnover and severe secondary hyperparathyroidism usually have high levels of circulating osteocalcin.
VI. RADIOGRAPHIC FEATURES OF RENAL OSTEODYSTROPHY Standard radiographic methods, as applied in clinical practice, often are insensitive in identifying progression of renal osteodystrophy. Techniques to increase the sensitivity of x-ray studies (which are particularly applicable for views of the hands) include the use of fine-grain film (i.e., Kodak M industrial film or mammography film). The main radiographic feature of secondary hyperparathyroidism is increased bone resorption, most commonly seen on the subperiosteal surfaces of bone. Erosions that occur in conjunction with formation of new bone may appear as cysts or osteoclastomas (brown tumors). The presence of subperiosteal erosions correlates with serum I-PTH and histomorphometric features of osteitis fibrosa on biopsy. Subperiosteal erosions of the phalanges (detected by fine-grain hand radiographs) may be the most sensitive radiographic sign of secondary hyperparathyroidism (Fig. 14-17). The tuft of the terminal phalanx of the second or third digit commonly shows resorption, and there may be collapse of the overlying soft tissue, so that the finger appears clubbed. Bone erosions also may occur at the proximal end of the tibia, the neck of the femur or humerus, and the inferior surface of the distal end of the clavicle. In the skull, there is a mottled and granular appearance, with areas of resorption commonly associated with areas of osteosclerosis. The lamina dura of the teeth often shows erosions in primary hyperparathyroidism, but is uncommonly affected by secondary hyperparathyroidism. Osteosclerosis, another feature of osteitis fibrosa, is due to an increase in the thickness and number of trabeculae in spongy bone and accounts for the typical "rugger jersey" appearance of the spine. Although the Looser's zone or pseudofracture is considered pathognomonic of tickets in children or of osteomalacia in the adult, the x-ray features of osteomalacia often are less distinctive than those of secondary hyperparathyroidism. The typical x-ray feature of tickets (i.e., widening of the epiphyseal growth plate) cannot develop after epiphyseal closure, and hence this radiographic sign of defective bone mineralization is limited to children. With mechanical stress on the skeleton affected by tickets or osteomalacia, a Looser's zone may
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EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ
VII. EXTRASKELETAL CALCIFICATIONS The factors that predispose the appearance of soft tissue calcification include an increase in the circulating calcium-phosphate product in plasma, the degree of secondary hyperparathyroidism, the magnitude of alkalosis, and local tissue i n j u r y . 86 Three major categories include (1) calcification of medium-size arteries; (2) articular or tumoral calcification; and (3) visceral calcification affecting the heart, lung, and kidney. Arterial calcification often involves the media of the vessel. These calcifications are diffuse and continuous along the vessels, and their appearance contrasts to the regular discrete appearance of calcified intimal plaques. Such medial calcification of the vessels may be seen first in the dorsalis pedis artery, where it appears as a ring or tube as it descends between the first and second metatarsals. Other sites commonly involved are the ankles, abdominal aorta, feet, pelvis, hands and wrists (Fig. 14-18).
VIII. THERAPEUTIC APPROACH TO RENAL OSTEODYSTROPHY A. Phosphate Retention FIGURE 14--17
Serial radiographs of the right index of a 43year-old man with chronic renal failure under treatment with peritoneal dialysis. Three views illustrate the value of fine-grain film and the use of simple magnification techniques to detect changes in periosteal resorption. The upper panel are magnified x 3, the lower • 12. A, The periosteal resorption at the midshaft is poorly identified, owing to the large-grain film (Kodak dental film). B, The radiolucent areas in the cortex are more clearly visualized, but only the higher magnification shows convincingly that there is an intact periosteal layer of bone covering these juxtaperiosteal resorption cavities. C, Six months later, juxtaperiosteal resorption had progressed to periosteal resorption. (B and C, Kodak M-film.) (From Meema HE, Meema S: Clin Orthop 130:297-310, 1978.)
extend across a bone and produce a true fracture. 85 Features such as increased haziness or indistinctiveness of the trabeculae, biconcavity of the vertebral bodies (particularly in association with normal bone density), and deformities of long bones are said to be typical of osteomalacia, but these findings are uncommon and may not be easily recognized. Furthermore, uremic patients with osteomalacia may have secondary hyperparathyroidism with concomitant x-ray features of the latter. Thus, the definitive diagnosis of osteomalacia requires a bone biopsy.
Hyperphosphatemia is a major factor accounting for the development and maintenance of secondary hyperparathyroidism in uremia. High serum phosphorus levels also contribute to the development of soft tissue calcification in patients with advanced uremia. Thus, restriction of dietary phosphorus and prevention of hyperphosphatemia are critical in the management of renal bone disease. The intake of phosphorus depends primarily upon meat and dairy products in the diet. The usual phosphorus intake by normal adults in the United States is 1.0 to 1.4 g/day. Dietary intake of phosphorus can be lowered by restricting the intake of dairy products and by rigid adherence to a low-protein diet. However, there is great difficulty in lowering phosphorus intake in proportion to the reduced GFR in patients with advanced renal failure by use of dietary manipulation alone. Lowphosphate diets are generally unpalatable to the tastes of most people living in the United States, and aluminumcontaining compounds that reduce the intestinal absorption of phosphorus used to be prescribed frequently. 87 These compounds include aluminum hydroxide and aluminum carbonate gels, which are available in liquid, tablet, and capsule form. The goal of such therapy in dialysis patients is to reduce serum phosphorus to near normal in patients treated conservatively. Predialysis se-
CHAPTER 14
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Renal Osteodystrophy TABLE 14--1. Guidelines for M a n a g e m e n t of Renal O s t e o d y s t r o p h y
Early Treatment Begin treatment early, (i.e., when the GFR is 30-40 ml/min) for the control of serum phosphate and calcium malabsorption. Control of Serum Phosphate (3.5-4.5 mg/dl)
Restrict phosphorus intake in diet to 600-800 mg/day Predialysis phosphorus: 4.5- 5.5 mg/dl Calcium carbonate: 1-4 g with each meal or Calcium acetate 0.5-3 g with each meal If serum phosphorus is greater than 8 mg/dl and Ca • P product is greater than 80 mg/dl, phosphate binders containing aluminum could be added in small amounts and for short periods of time (Basajel, Dialume, Alucap, one to four capsules with each meal) Adequate Calcium Intake
Oral calcium supplements at night providing 1-2 g/day when serum P is controlled: Os-Cal, Titralac, Tums Dialysate Ca, 5.0-5.5 mg/dl (2.5-2.75 mEq/liter) Use of Vitamin D Sterols
FIGURE 14--1 8 Severe metastatic calcification of the left thumb in a patient maintained on chronic dialysis with a high Ca • P product.
25(OH)D3 (calcifediol), 20-100 Ixg/day (Calderol) 1,25(OH)2D3 (calcitriol), 0.5-1.0 I~g/day orally (Rocaltrol) 1,25(OH)2D3 (calcitriol), 2.0-4.0 Ixg orally twice per week (Rocaltrol) 1,25(OH)2D3 (calcitriol), 1.0-3.0 Ixg intravenously three times per week (Calcijex) Parathyroid Hormone Levels: Although, the upper limit for normal
r u m phosphorus levels are ideally maintained at 5.0 to 5.5 mg/dl. In patients with creatinine clearances below 20 ml/min and in those undergoing treatment with dialysis, dietary phosphorus should be restricted to 800 to 900 mg/day. One to three capsules of a l u m i n u m hydroxide or a l u m i n u m carbonate were often taken with each meal (Table 1 4 - 1 ) . It was generally a s s u m e d that a l u m i n u m hydroxide and a l u m i n u m carbonate were nonabsorbable and safe. However, data suggest that orally administered alumin u m m a y be absorbed in significant amounts in m a n and in experimental animals, and that such treatment can lead to increased plasma and tissue a l u m i n u m content. 88'89 It is clear that dialysis dementia, myopathy, microcytic anemia, 89 and l o w - b o n e - t u r n o v e r osteodystrophy 9~ are related to the tissue accumulation of aluminum. Therefore, alternative binders of phosphorus in the gut have been sought. Clarkson et al. 91 showed that a large dietary intake of calcium carbonate in patients with chronic renal failure decreased the gastrointestinal absorption of phosphorus. Subsequently, m a n y investigators have shown that calcium carbonate is an effective phosphate binder 92'93 (Fig. 1 4 - 1 9 ) . However, the administration of calcium carbonate is not completely free of side effects. Transient episodes of h y p e r c a l c e m i a have been observed frequently by several investigators. 92-94 In the United States most patients undergo hemodialysis with dialysate containing 3.0 to 3.5 mEq/liter of calcium.
serum intact PTH is 60 pg/ml, to prevent the development of adynamic bone disease, the serum PTH should be kept between 150 and 250 pg/ml Parathyroidectomy: Severe secondary hyperparathyroidism (bone
erosions and increased IPTH) plus any of the following: Persistent hypercalcemia (serum Ca > 11.5-12.0 mg/dl) Progressive or symptomatic extraskeletal calcification Persistently elevated serum Ca x P products Pruritus not responsive to medical treatment Calciphylaxis (ischemic ulcers and necrosis)
Since a positive calcium balance is induced during dialysis with dialysate calcium levels above 3.5 mEq/liter, the administration of large amounts of calcium carbonate could increase the risk of h y p e r c a l c e m i a and extraskeletal calcifications. Several investigators 95-97 have att e m p t e d to decrease the incidence of hypercalcemic episodes by diminishing the a m o u n t of calcium in the dialysate. Slatopolsky et al. 97 evaluated the long-term effect of administration of calcium carbonate (mean dose, 10.5 g/day, range 2.5 to 28 g/day) and lowering the dialysate calcium to 2.5 mEq/liter on h y p e r p h o s p h a t e m i a in hemodialysis patients. They confirmed the effectiveness of calcium carbonate as a phosphate binder. Moreover, the d e v e l o p m e n t of h y p e r c a l c e m i a was greatly m i n i m i z e d by the reduction of calcium concentration in the dialysate. It is importaot to e m p h a s i z e that patients maintained on a dialysate concentration of 2.5 mEq/liter
458
EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ Control
]DC Aluminum J
CalciumCarbonate
9Calcium O Phosphorus
8m "O
_
6
-~~x:~,
MONTH
FIGURE 14--19
Temporal relationship between serum calcium and phosphorus in a group of 20 patients maintained on chronic dialysis. During the control part of the studies, the patients ingested phosphate binders containing aluminum. In the second part the aluminum binders were discontinued. In the third part of the study, the patients received calcium carbonate as a phosphate binder. (From Slatopolsky E, Weerts C, Lopez-Hilker S, et al: Calcium carbonate is an effective phosphate binder in dialysis patients. N Engl J Med 315: 157-161, 1986. Copyright 1986, Massachusetts Medical Society.)
shown that half the amount of elemental calcium can be given to bind phosphorus in dialysis patients when given in the form of calcium acetate versus calcium carbonate. Unfortunately, this treatment did not decrease the incidence of hypercalcemia. It has been shown ~~ that calcium citrate increases aluminum absorption. Thus, this salt should not be given to patients with renal failure. If the serum calcium reaches a concentration of 11.0 mg/dl, the prescribed amount of calcium carbonate should not increase further. In the patients who remained hyperphosphatemic, phosphate binders containing aluminum may be added to the regimen. Recently, Delmez et al. 1~ demonstrated that magnesium carbonate in conjunction with calcium carbonate is an effective phosphate binder. To prevent the development of hypermagnesemia the investigators reduced amounts of magnesium in the dialysate from 1.8 mg/dl to 0.6 mg/dl. The patients tolerated doses of elemental magnesium as high as 400 to 600 mg/day without any complications and maintained good control of serum phosphorus levels.
B. C a l c i u m S u p p l e m e n t s may develop severe secondary hyperparathyroidism with the use of small amounts of calcium carbonate. Therefore, the prescription of a dialysate formulated in this manner should be restricted to those patients who demonstrate compliance with the ingestion of the necessary doses of calcium carbonate. When calcium carbonate is prescribed as a phosphate binder, it must be ingested with meals, both to increase the efficiency of phosphate binding and to minimize the absorption of calcium and the consequent risk of hypercalcemia. 98 We recommend that dietitians determine the total amount of phosphorus ingested over 24 hours and during each meal. There is both patient-to-patient variability and meal-to-meal variability in the same patient. Thus, to prevent the development of hypercalcemia, it is important to prescribe calcium carbonate according to the amount of phosphorus ingested with each meal. The dose of calcium carbonate ingested with each meal should be adjusted to match the phosphorus intake at that time. In general, 1.0 to 2.0 g of calcium carbonate with each meal would be a reasonable initial dose. Sheik et al. 99 in acute studies, demonstrated that calcium acetate may bind phosphorus more efficiently than calcium carbonate or calcium citrate. These investigators found that calcium acetate bound 1.0 _ 0.1 mg of phosphorus per milligram of calcium absorbed. This was better than both calcium carbonate (0.57 _ 0.5 mg of phosphorus per milligram of calcium absorbed) and calcium citrate (0.47 _ 0.5 mg of phosphorus per milligram of calcium absorbed). In long-term studies, we 1~176 and others 1~ have
Impaired calcium absorption exists in patients with advanced renal failure and in those undergoing dialysis. Since their diets generally contain suboptimal quantities of calcium, oral calcium supplements are usually necessary. Studies of net intestinal calcium absorption suggest that a neutral or positive calcium balance can be achieved in uremic patients when supplemental calcium increases the total elemental calcium intake above 1.5 g m / d a y . 1~
Long-term treatment with large doses of oral calcium supplements has been reported to reduce the incidence of bone resorption lesions, fractures, and episodes of pseudogout and extraskeletal calcification. 91 Others have shown lower plasma levels of alkaline phosphatase and I-PTH in uremic patients receiving calcium supplements compared to controls. 1~ Treatment with oral calcium supplements is not without risk. Calcium supplements should not be given to a patient with marked hyperphosphatemia because of the risk of increasing the Ca • P product and the consequent predisposition for the development of extraskeletal calcification. Hypercalcemia can develop in uremic patients during oral therapy with calcium salts. Thus, careful monitoring of the calcium and phosphorus levels is essential. Calcium carbonate is currently the first choice as a source of supplemental calcium, because it contains a high fraction of calcium and is inexpensive, tasteless, and relatively well tolerated. Calcium carbonate contains 40% elemental calcium. Calcium carbonate is available
CHAPTER 14 Renal Osteodystrophy in several proprietary preparations, including Os-cal, Titralac, and Tums. Calcium acetate contains 23% elemental calcium, and is commercially available by the name of Phos-Lo.
C. Concentration of Calcium in the Hemodialysis Dialysate A number of studies have observed an effect of different dialysate calcium levels on the progression of renal bone disease. With the use of dialysate calcium levels lower than 6.0 mg/dl (3 mEq/liter), evidence of progressive bone disease has been suggested by increases in plasma alkaline phosphatase 1~ and evidence of bone deterioration on radiographs. 1~176 This, in part, was due to the flux of diffusable calcium from the serum to the dialysate. From such information, it was generally recommended that the dialysate calcium concentration be 3.0 to 3.5 mEq/liter. However, such data were obtained in patients ingesting aluminum-containing phosphate binders. In patients ingesting large doses of calcium carbonate as a phosphate binder, there is considerable risk of hypercalcemia with the use of such dialysate. Under these circumstances, lowering the dialysate calcium to 2.5 mEq/liter may confer an additional level of safety. 97
D. Use of Vitamin D and Metabolites Despite dietary control of phosphate, the use of phosphate binders, and adequate dietary calcium, and appropriate levels of calcium in the dialysate, uremic patients may still develop skeletal disease. Thus, vitamin D and its metabolites are important and effective agents in the treatment of renal osteodystrophy. Calcitriol, the most active metabolite of vitamin D, is the drug of choice in the treatment of hypocalcemia and secondary hyperparathyroidism. The usual dose is 0.5 to 1 txg/day given orally. Recent evidence suggests that "oral pulse" (2 to 4 Ixg twice or thrice weekly) is more effective than the usual small dose (0.5 to 1.0 txg) given daily. 1~ Calcitriol, given IV, or orally, at the dose of 1 to 3 p~g three times per week, is the best approach to suppress secondary hyperparathyroidism. With the intravenous preparation of calcitriol, high concentrations of 1,25(OH)2D3 can be obtained in serum. 11~ This is important, because the low number of receptors in the parathyroid gland make the hyperplastic tissue more resistant to the action of calcitriol. Moreover, calcitriol can up-regulate its own number of receptors. ~5 If osteomalacia without evidence of aluminum accumulation predominates on bone bi-
459 opsy, excellent results have been obtained with the use of 25(OH)D3 (20 to 100 Ixg/day) in addition to calcitrio1.113 With the use of vitamin D or its metabolites, hypercalcemia and hyperphosphatemia may occur as side effects.
E. Parathyroidectomy The types of treatment previously discussed often lead to improvement of calcium and phosphorus homeostasis, reversal of symptoms of bone disease, and suppression of parathyroid secretion. However, such measures may be unsuccessful, and certain features of secondary hyperparathyroidism may necessitate parathyroid surgery. When this problem becomes a consideration, there should be ample evidence for the presence of severe secondary hyperparathyroidism (e.g., very high levels of serum I-PTH with bone erosions or the presence of osteitis fibrosa on bone biopsy). Also, it is important that aluminum-related bone disease be excluded. In addition to very high levels of I-PTH, the clinical features that may indicate the need for parathyroid surgery include (1) persistent hypercalcemia, particularly when symptomatic; (2) intractable pruritus that does not respond to dialysis or other medical treatment; (3) progressive extraskeletal calcifications that occur in conjunction with a Ca • P product that is consistently greater than 75 to 80, despite appropriate phosphate restriction; (4) severe and progressive skeletal pain or fractures; and (5) the appearance of calciphylaxis (ischemic lesions of soft tissue and skin and vascular calcification). 114-116 Persistent and significant hypercalcemia (serum calcium level > 12.0 mg/dl) that occurs in association with symptoms of nausea or vomiting or evidence of ulcer disease may indicate a need for subtotal parathyroidectomy. Hypercalcemia can also develop in uremic patients who lack evidence of secondary hyperparathyroidism, and parathyroid surgery should not be undertaken unless bone erosions and elevated serum I-PTH levels are documented. The presence of marked hyperphosphatemia and overt secondary hyperparathyroidectomy in a noncompliant uremic patient presents a difficult therapeutic dilemma for the physician. Parathyroid surgery may lead to a transient lowering of blood phosphorus and even to the resolution of ectopic calcifications, but secondary hyperparathyroidism may recur, with all its manifestations, unless the patient follows the prescribed treatment with dietary phosphorus restriction and phosphate binders. After parathyroid surgery, hypocalcemia may pose a problem, although this may be less serious when some parathyroid tissue is left in place. In general, the worse
460 the secondary hyperparathyroidism, the more severe the hypocalcemia. If hypocalcemia does not occur, either the patient did not have severe osteitis fibrosa or excess parathyroid tissue remains. The postoperative treatment of hypocalcemic patients with 1,25D, intravenous infusions of calcium gluconate, and oral calcium supplements may be needed. Tetany and even seizures can occur during the postoperative period. Serum levels of phosphorus and magnesium may also decrease after parathyroid surgery, and aluminum-containing phosphate binders should be withheld if the serum phosphorus falls below 2.5 mg/dl. However, serum phosphorus should not be allowed to increase above 4.5 mg/dl because of the risk of aggravating hypocalcemia. Rapid remineralization of the skeleton is usually occurring during this period, and blood calcium will usually begin to rise after the " h u n g r y " bones have been repleted with calcium. A fall in the elevated plasma alkaline phosphatase toward normal may be a clue that rapid skeletal remineralization is nearly completed and indicates that calcium supplements and vitamin D dosage may be reduced or discontinued. At the time of parathyroidectomy, four parathyroid glands should be identified, with the selection of one gland that is to be partially resected. A part of this gland is removed, leaving 60 to 80 mg of viable tissue in place. After the frozen sections are available from the first gland, each of the other three glands may be removed. The remnant parathyroid tissue left in situ may undergo hyperplasia leading to the reappearance of overt secondary hyperparathyroidism. The management of such a situation may be difficult. A second surgical procedure may be associated with greater technical difficulties and significant complications compared with the initial operation. Because of the difficulty and risk of a second surgical procedure, total parathyroidectomy with autotransplantation of some parathyroid tissue to the forearm has been recommended. 117 Such tissue transplanted to the forearm may be more accessible for subsequent surgical removal. To prevent recurrence of hyperparathyroidism, some advocate using tissue that is histologically hyperplastic as opposed to nodular. Another risk of parathyroid surgery is the development of hypoparathyroidism. To avoid this, parathyroid tissue should be frozen and stored to be implanted later if hypocalcemia persists. Although controversial, we feel that a total parathyroidectomy has little place in the management of renal bone disease, since it may predispose uremic patients to the development of an isolated mineralizing defect. There are new analogues of calcitriol that have the potential to cause less hypercalcemia. Whether they may be effective in leading to long-term suppression of the parathyroid glands must await clinical evaluation.
EDUARDO SLATOPOLSKY AND JAMES A. DELMEZ
F. A l u m i n u m A c c u m u l a t i o n The presence of excessive quantities of trace metals, particularly aluminum, in the water used to prepare dialysate can cause bone disease in uremic patients. Therefore, the purity of the water supply should be routinely tested and the methods of water treatment periodically reevaluated. The amount of aluminum in the dialysate should not exceed 10 txg/liter. However, the main source of aluminum responsible for the development of bone disease is the ingestion of phosphate binders containing aluminum. Several reports have shown that deferrioxamine (DFO), a chelating agent, is useful as a diagnostic tool as well as therapeutic agent for patients with aluminum accumulation. 118'119When DFO is given IV in the dose of 10 to 20 mg/kG, an increase in the levels of serum aluminum is produced. If the increment in serum aluminum observed after the administration of DFO is greater than 200 ixg/liter, it is likely that the patient is at risk for the development of bone disease secondary to aluminum accumulation. Aluminum can be deposited in the mineralization front at the interface between bone and osteoid and may impair the normal mineralization of bone. Most of these patients also have low levels of circulating I-PTH. Although the " D F O test" may be a useful noninvasive tool in the diagnosis of aluminum accumulation, bone histomorphometry with special staining for aluminum is the definitive test in the diagnosis of aluminum-induced bone disease. 12~ In this situation, its use is strongly encouraged. If a patient requires DFO treatment for aluminum-induced osteomalacia, the usual dose of DFO is 0.5 to 1.0 g/week for 3 to 6 months. A second bone biopsy after 1 year can conclusively determine the effectiveness of the treatment. The use of dialyzers utilizing polysulfone membranes or cartridges containing activated colloidin-coated charcoal greatly increases the removal of aluminum during hemodialysis. 122
Acknowledgments The authors wish to express their appreciation to Sue Viviano for her assistance in the preparation of this manuscript. This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases grants DK-09976, DK-30178, and DK-07126.
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CHAPTER 14
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77.
78.
failure: A possible cause for secondary hyperparathyroidism. J Clin Endocrinol Metab 41:339-345, 1975. Kaplan MA, Canterbury JM, Gavellas GA, Jaffe D: The calcemic and phosphaturic effects of parathyroid hormone in the normal and uremic dog. Metabolism 27:1785-1792, 1978. Massry SG, Stein R, Garty J, et al: Skeletal resistance to the calcemic action of parathyroid hormone in uremia: Role of 1,25(OH)2D3. Kidney Int 1:467-474, 1975. Rodriguez M, Martin-Malo A, Martinez ME, et al: Calcemic response to parathyroid hormone in renal failure: Role of phosphorus and its effect on calcitriol. Kidney Int 40:1053-1067, 1991. Galceran T, Martin KJ, Morrissey J, Slatopolsky E: Role of 1,25 dihydroxyvitamin D on the skeletal resistance to parathyroid hormone. Kidney Int 32:801-807, 1987. Martin KJ, Hruska KA, Lewis J, et al: The renal handling of parathyroid hormone. Role of peritubular uptake and glomerular filtration. J Clin Invest 60:808-814, 1977. Hruska KA, Kopelman R, Rutherford WE, et al: Metabolism of immunoreactive parathyroid hormone in the dog. The role of the kidney and the effects of chronic renal disease. J Clin Invest 56: 39-46, 1975. Hruska K, Korkor A, Martin K, Slatopolsky E: The peripheral metabolism of parathyroid hormone: Role of the liver and kidney and the effect of chronic renal failure. J Clin Invest 67: 885-892, 1981. Sherrard DJ, Baylink DJ, Wergedal JE, Maloney N: Quantitative histological studies on the pathogenesis of uremic bone disease. J Clin Endocrinol 39:119-135, 1974. Bordier PJ, Tun-chot S, Eastwood JB, Fournier A: Lack of histological evidence of vitamin D abnormality in the bones of anephric patients. Clin Sci 44:33-41, 1973. Russell JE, Avioli LV: 25-hydroxycholecalciferol-enhanced bone maturation in the parathyroprivic state. J Clin Invest 56:792798, 1975. Litzow JR, Lemann J Jr, Lennon EJ: The effect of treatment of acidosis on calcium balance in patients with chronic azotemic renal disease. J Clin Invest 46:280-288, 1967. Ward MK, Feest TG, Ellis HA, et al: Osteomalacic dialysis osteodystrophy: Evidence for a water borne etiological agent, probably aluminum. Lancet 1:841-845, 1978. Teitelbaum SL, Bergfeld MA, Freitag J, et al: Do parathyroid hormone and 1,25-dihydroxyvitamin D modulate bone formation in uremia? J Clin Endocrinol Metab 51:247-251, 1980. Felsenfeld AJ, Harrelson JM, Gutman RA, Wells SA Jr: Osteomalacia after parathyroidectomy in patients with uremia. Ann Intern Med 96:34- 39, 1982. Cobum JW, Brickman AS, Sherrard D J, et al: Defective skeletal mineralization in uremia without relation to vitamin D, serum Ca or P. Kidney Int 12:455-459, 1977. Sherrard DJ, Hercz G, Pei Y, et al: The spectrum of bone disease in end-stage renal failure. An evolving disorder. Kidney Int 43: 436-442, 1993. Pei Y, Hercz G, Greenwood C, et al: Risk factors for renal osteodystrophy: A multivariant analysis. J Bone Miner Res 10: 149-156, 1995. 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. Schoenfeld PJ, Martin JA, Barnes B, Teitelbaum SL: Amelioration of myopathy with 25-dihydroxyvitamin D3 therapy (25(OH)D3) in patients on chronic hemodialysis. Abstract Book. Third Workshop on Vitamin D. Asilomar, 1977, p 160.
CHAPTER 14
Renal Osteodystrophy
79. Mallette LE, Patten BM, Engel WK: Neuromuscular disease in secondary hyperparathyroidism. Ann Intern Med 82:474-479, 1975. 80. Mirahmadi KS, Coburn JW, Bluestone R: Calcific periarthritis and hemodialysis. JAMA 223:548-552, 1973. 81. Kleinman KS, Coburn JW: Amyloid syndromes associated with hemodialysis. Kidney Int 34:567-575, 1989. 82. DiRaimondo CR, Cassey TT, DiRaimondo CV, Stone WJ: Pathologic fractures associated with idiopathic amyloidosis of bone in chronic hemodialysis patients. Nephron 43:22-27, 1986. 83. Hruska KA, Teitelbaum SL, Kopelman R, et al: The predictability of the histologic features of uremic bone disease by noninvasive techniques. Metab Bone Dis Rel Res 1:39-44, 1978. 84. Ritz E, Malluche HH, Bommer J, et al: Metabolic bone disease in patients on maintenance hemodialysis. Nephron 12:393-404, 1974. 85. Parfitt AM: Clinical and radiographic manifestations of renal osteodystrophy. In David DS(ed): Calcium Metabolism in Renal Failure and Nephrolithiasis, New York, John Wiley & Sons, 1977, pp 150-169. 86. Parfitt AM: Soft tissue calcification in uremia. Arch Intern Med 124:544-552, 1969. 87. Fournier AE, Johnson WJ, Taves DR, et al: Etiology of hyperparathyroidism and bone disease during chronic hemodialysis. I. Association of bone disease with potentially etiologic factors. J Clin Invest 50:592-598, 1971. 88. Berlyne GM, Ben-Ari J, Pest D, et al: Hyperaluminaemia from aluminum resins in renal failure. Lancet 2:494-499, 1970. 89. Alfrey AC, LeGendre GR, Kaehny WD: Dialysis encephalopathy syndrome: Possible aluminum intoxication. N Engl J Med 294:184-187, 1976. 90. 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. 91. Clarkson EM, Eastwood JB, Koutsaimanis KG, DeWarner HE: Net intestinal absorption of calcium in patients with chronic renal failure. Kidney Int 3:258-263, 1973. 92. Morniere PH, Roussel A, Tahiri Y, et al: Substitution of aluminum hydroxide by high doses of calcium carbonate in patients on chronic hemodialysis: Disappearance of hyperaluminemia and equal control of hyperparathyroidism. Proc Eur Dial Transplant Assoc 19:784-787, 1982. 93. Slatopolsky E, Weerts C, Lopez-Hilker S, et al: Calcium carbonate is an effective phosphate binder in dialysis patients. N Engl J Med 315:157-161, 1986. 94. Salusky IB, Coburn JW, Foley, et al: Effects of oral calcium carbonate on control of serum phosphorus and changes in plasma aluminum levels after discontinuation of aluminumcontaining gels in children receiving dialysis. J Pediatr 108: 767-770, 1986. 95. Mactier RA, VanStone J, Cox A, et al: Calcium carbonate is an effective phosphate-binder when dialysate calcium concentration is adjusted to control hypercalcemia. Clin Nephrol 28:222-226, 1987. 96. Sawyer N, Noonan K, Altmann P, et al: High-dose calcium carbonate with stepwise reduction in dialysate calcium concentration: Effective phosphate control and aluminum avoidance in haemodialysis patients. Nephrol Dial Transplant 3:1-5, 1988. 97. 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.
463 98. Schiller LR, Santa Ana CA, Sheikh MS, et al: Effect of the time of administration of calcium acetate on phosphorus binding. N Engl J Med 320:1110-1103, 1989. 99. Sheikh MS, Maguire JA, Emmett M, et al: Reduction of dietary phosphorus absorption by phosphorus binders. A theoretical, in vitro and in vivo study. J Clin Invest 83:66-73, 1989. 100. Delmez J, Tindeira C, Windus D, et al: Calcium acetate as a phosphorate binder in hemodialysis patients. J Am Soc Nephrol 3:96-102, 1992. 101. Mai ML, Emmett M, Sheikh MS: Calcium acetate, an effective phosphorus binder in patients with renal failure. Kidney Int 36: 690-695, 1989. 102. Slanina P, Frech W, Ekstrom LG, Loof L, et al: Dietary citric acid enhances absorption of aluminum in antacids. Clin Chem 32:534-541, 1986. 103. Delmez JA, Keller S, Norwood K, et al: Magnesium carbonate as a phosphorus binder: A prospective controlled crossover study. Kidney Int 49:63-167, 1996. 104. Kopple JD, Coburn JW: Metabolic studies of low protein diets in uremia. II. Calcium, phosphorus and magnesium. Medicine 52:597-607, 1973. 105. Meyrier A, Marsac J, Richet G: The influence of a high calcium carbonate intake on bone disease in patients undergoing hemodialysis. Kidney Int 4:146-153, 1973. 106. Curtis JR, DeWardener HE, Gower PE, Eastwood JB: The use of calcium carbonate and calcium phosphate without vitamin D in the management of renal osteodystrophy. Proc Eur Dial Transplant Assoc 7:141-144, 1970. 107. Mirahmadi KS, Duffy BS, Shinaberger JH, et al: A controlled evaluation of clinical and metabolic effects of dialysate calcium levels during regular hemodialysis. Trans Am Soc Artif Intern Organs 17:118-124, 1971. 108. Goldsmith RS, Johnson WJ: Role of phosphate depletion and high dialysate calcium in controlling dialytic renal osteodystrophy. Kidney Int 4:154-160, 1973. 109. Tsukamoto Y, Nomura M, Takahashi Y: The "oral 1,25(OH)2D3 pulse therapy" in hemodialysis patients with severe secondary hyperparathyroidism. Nephrology 57:23-28, 1991. 110. Slatopolsky E, Weerts C, Thielan J, et al: Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25(OH)2D3 in uremic patients. J Clin Invest 75:2136-2143, 1984. 111. Dunlay R, Rodriguez M, Felsenfeld AJ, Leach F: Direct inhibitory effects of calcitriol on parathyroid function (sigmoidal curve) in dialysis. Kidney Int 36:1093-1098, 1989. 112. Canella G, Bonucci E, Rolla D, et al: Evidence of healing of secondary hyperparathyroidism in chronically hemodialyzed uremic patients treated with long-term intravenous calcitriol. Kidney Int 46:1124-1132, 1994. 113. Eastwood JB, Stamp TCB, DeWardener HE, et al: The effect of 25-hydroxyvitamin D3 in osteomalacia of chronic renal failure. Clin Sci Mol Med 52:499-508, 1977. 114. Wilson RE, Hampers CL, Bernstein DS, et al: Subtotal parathyroidectomy in chronic renal failure. Ann Surg 174:640-649, 1971. 115. Katz AD, Kaplan L: Parathyroidectomy for hyperplasia in renal disease. Arch Surg 107:51-59, 1973. 116. David SD. Calcium metabolism in renal failure. Am J Med 58: 48-56, 1975. 117. Wells SA Jr, Gunnells JC, Shelburne JD, et al: Transplantation of the parathyroid glands in man: Clinical indications and resuits. Surgery 78:34-41, 1975. 118. Ackrill P, Day JP, Gargstang FM, et al: Treatment of fracturing renal osteodystrophy by desferrioxamine. Proc Eur Dial Transplant Assoc 19:203-207, 1982.
464 119. Milliner DS, Nebeker HG, Ott SA, et al: Use of the desferrioxamine infusion test in the diagnosis of aluminum-related osteodystrophy. Ann Intern Med 101:775-780, 1984. 120. Malluche HH, Smith AJ, Abreo K, Faugere MC: The use of desferrioxamine in the management of aluminum accumulation in bone in patients with renal failure. N Engl J Med 311:140144, 1984. 121. Molitoris BA, Alfrey AC, Alfrey PS, Miller NL: Rapid removal of DFO-chelated aluminum during hemodialysis using polysul-
EDUARDO SLATOPOLSKYAND JAMES A. DELMEZ fone dialyzers. Kidney Int 34:98-101, 1988. 122. Delmez J, Weerts C, Lewis-Finch J, et al: Accelerated removal of deferoxamine mesylate-chelated aluminum by charcoal hemoperfusion in hemodialysis patients. Am J Kidney Dis 13:308311, 1989. 123. Martinez I, Saracho R, Montenegro J, Llach F: A deficit of calcitriol synthesis may not be the initial factor in the pathogenesis of secondary hyperparathyroidism. Nephrol Dialysis Transplant $3:22-28, 1996.
~HAPTE~
Surgical Treatment for Hyperparathyroidism GERARD M. DOHERTY AND SAMUEL A. WELLS, JR.* Section of Endocrine and Oncologic Surgery and *Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110
V. Operative Management of Patients with Parathyroid Carcinoma VI. Operative Management of Patients with Renal Osteodystrophy VII. Postoperative Management References
I. History of Surgery for Hyperparathyroidism II. Primary Hyperparathyroidism: Preoperative Evaluation III. Primary Hyperparathyroidism: Conduct of the Operation IV. Primary Hyperparathyroidism: Management of Patients with Persistent or Recurrent Hyperparathyroidism
I. HISTORY OF SURGERY FOR HYPERPARATHYROIDISM
formed by the Viennese surgeon Felix Mandl in 1925. 5 The patient was a Viennese streetcar conductor who was admitted to the hospital clinic with hypercalcemia, hypercalciuria, roentgenographic changes of osteitis fibrosa cystica generalisata, and a broken leg. After surgical therapy, the calcium in the blood and urine decreased, the bones became more dense and pain-free, and the patient became ambulatory. Within 6 years, however, his disease recurred and became progressively worse, ultimately causing death. The patient had a repeat neck exploration, but no enlarged parathyroid gland was found then or at necropsy. Following the report of Mandl's case, there were a series of reports of neck exploration for hyperparathyroidism. Perhaps the most famous was Charles Martel, a sea captain, who underwent five unsuccessful neck explorations for hyperparathyroidism at the Massachusetts General Hospital, before an enlarged mediastinal parathyroid gland was found at the sixth operation. 6 He subsequently died in tetany during a ureterolithotomy. 7
The operative treatment of patients with hyperparathyroidism is a relatively recent intervention strategy. This is mainly because the parathyroid glands were not discovered until 1880.1 Moreover, this great anatomical discovery was not appreciated until 11 years later, when Gley "rediscovered" the glands and showed that their removal led to tetany. 2 Erdheim was the first to appreciate the relationship of parathyroid gland enlargement to bone disease, and he proposed that the parathyroid changes were secondary to abnormalities in the skeleton) Schlagenhaufer also noted the association of bone disease and enlarged parathyroid glands, but he proposed that single parathyroid gland enlargement might be the primary rather than the secondary abnormality, relative to the bone disease, and that resection of the single enlarged gland might be curative. 4 Based on this reasoning, the first parathyroidectomy was perMETABOLIC BONE DISEASE
465
Copyright 9 1998by AcademicPress. All rightsof reproductionin any formreserved.
466
GERARD M. DOHERTY AND SAMUEL A. WELLS, JR.
~
The first American patient to have successful diagnosis and treatment of hyperparathyroidism by operation was an Illinois farm woman. She was treated at Barnes Hospital in 1928. 8 She first presented in 1927 because of progressive muscular hypotonia, hyperflexibility, weakness, urinary symptoms, and a bony tumor of the fight index finger. 9 Further evaluation demonstrated extensive bilateral nephrolithiasis. Her fight index finger was amputated for an apparently benign "giant-cell tumor of the epulis type." She was discharged with little improvement, but returned 1 year later with a humerus fracture. Her serum calcium concentration was 12.4 to 16.6 mg/dl, and extensive metabolic studies documented a negative calcium balance. At operation, a 3-cm left lower parathyroid gland was resected. Postoperatively she developed severe hypocalcemia, and required substantial calcium replacement, initially intravenously, and subsequently orally. Postoperatively she maintained a positive calcium balance, and had return of muscle strength, regression of her bone tumors, and partial recalcification of her skeleton. However, she suffered spinal cord compression and lower extremity paralysis, and died of progressive uremia 14 months after operation. 1~ Thus in the early days of the surgical treatment of hyperparathyroidism the patients presented with advanced disease. Even if the operations were technically successful, patients often died of the complications of the disease, particularly renal insufficiency. A great deal more is known about primary hyperparathyroidism today, compared to 70 years ago when Mandl performed the first parathyroidectomy. As a result, most patients are diagnosed when their disease is in an early stage and treatment can be planned with a high likelihood of cure. In this chapter, we will primarily review the preoperative, operative, and postoperative management of patients with primary or secondary hyperparathyroidism.
varied; most are diagnosed because an elevated serum concentration of calcium is detected on a routine laboratory test, often performed without suspicion of parathyroid disease. Currently, approximately 20% of patients have or have had renal stone disease, and less than 5% have evidence of osteitis fibrosa cystica generalisata. Most patients have minimal symptoms, and many are described as "asymptomatic." On careful questioning, patients often complain of weakness, tiredness, loss of energy, loss of memory, poor sleep habits, constipation, and polyuria. 11 Perhaps because the onset of hyperparathyroidism is almost always insidious, many patients do not notice that they are feeling poorly, and attribute their symptoms to "aging." 1. PHYSICAL FINDINGS
Except in patients with the classic deformities of advanced bone disease, physical examination is seldom helpful in making the diagnosis. Diseased parathyroids are rarely palpable, except in patients with parathyroid carcinoma, where approximately half of the patients have a palpable mass. A mass in the anterior neck in a patient with primary hyperparathyroidism is usually a thyroid nodule. 2. CALCIUM Hypercalcemia is the single most important diagnostic finding; however, particularly in early or mild cases, serial analyses may show fluctuations in and out of the normal range. Coexistent hypoalbuminemia and acidosis may result in an apparently normal total serun; calcium concentration, even though the ionized fraction is actually elevated. Although cumbersome, methodology is available for determining the ionized serum calcium concentration. This value can be helpful in the evaluation of some patients. 3. PARATHYROID HORMONE
II. PRIMARY HYPERPARATHYROIDISM: PREOPERATIVE EVALUATION Careful preoperative evaluation includes a history and physical examination as well as clinical and biochemical confirmation of the diagnosis of hyperparathyroidism. It is also important to assess for the presence of familial hyperparathyroidism and prior x-ray treatment to the head and neck area.
A. D i a g n o s i s Primary hyperparathyroidism is mainly a disease of postmenopausal women. The presentation of patients is
In the United States, the measurement of plasma parathyroid hormone (PTH) has become an important method for establishing the diagnosis of hyperparathyroidism. Because of the heterogeneity of the various circulating forms of PTH, the initial clinical experience with radioimmunoassays gave conflicting and often confusing results. The methodology continues to be refined, and most current assays are sufficiently sensitive, specific, and reliable to recommend wide clinical use. Middle-region and two-site assays, as opposed to aminoterminal, carboxyl-terminal, and intact hormone assays, appear to be the most dependable. The demonstration of an elevated plasma PTH concentration alone does not establish the diagnosis of hyperparathyroidism. In the setting of an inappropriately elevated serum calcium concentration, however, this finding is virtually diagnos-
CHAPTER 15
Surgical Treatment for Hyperparathyroidism
467
tic of the disease. Familial hypercalcemic hypocalciuric hyperparathyroidism (FHHH) can present with mildly elevated serum concentrations of calcium and PTH. It is important to diagnose this disease because operation is of no benefit and should be avoided. In patients for w h o m this etiology is considered, it can be confirmed or excluded by m e a s u r e m e n t of the 24-hour urinary excretion of calcium, which is nearly always elevated in patients with primary hyperparathyroidism, but is normal or low in people with FHHH. 4.
PHOSPHATE
Parathyroid h o r m o n e stimulates renal excretion of phosphate and, in about half of patients, hypophosphatemia is present. In the presence of renal disease, however, the serum phosphate levels may be normal or significantly elevated.
5. BICARBONATE Parathyroid h o r m o n e also increases bicarbonate excretion such that patients may develop a hyperchloremic metabolic acidosis. It has been suggested that the finding of an elevation of the serum chloride/phosphate ratio may be helpful in the differential diagnosis of hypercalcemia. A ratio greater than 30 in the presence of hypercalcemia is highly suggestive of hyperparathyroidism.
6. ALKALINE PHOSPHATASE Patients with elevated serum concentrations of calcium and alkaline phosphatase have bone disease, which
TABLE 15--1 Author/Site Satava et al./Mayo Clinic 12
Years
may not be evident clinically or on routine radiographs. However, they are at risk for a m a r k e d and prolonged decrease in the serum calcium concentration postoperatively because of " b o n e h u n g e r " for calcium during remineralization.
7. INDICATIONS FOR OPERATION Once the diagnosis of primary hyperparathyroidism is established, the issue of appropriate treatment must be addressed. Very few clinicians would argue that neck exploration is indicated in patients who have renal stones, classic bone disease, or b o t h e r s o m e symptoms. Otherwise, m a n y physicians consider mild hyperparathyroidism well tolerated, and do not advise neck exploration unless there is demonstrable demineralization on bone density studies or a serum calcium concentration above 12 mg/dl. Most endocrine surgeons consider the diagnosis of primary hyperparathyroidism an indication for neck exploration, if the patient is otherwise healthy. The reasons for this position are: (1) neck exploration for hyperparathyroidism in experienced hands is curative in greater than 95% of patients (Table 1 5 - 1 ) ; (2) the operative and convalescent times are short; (3) skeletal calcium loss is a serious problem for postmenopausal women, and there is clear evidence that bone remineralization occurs after correction of the hyperparathyroidism24-26; and (4) there are convincing data that hypercalcemic hyperparathyroidism is associated with increased mortality, although
Results of Operation for Primary Hyperparathyroidism Follow-up
N
Patients with Normocalcemia
Patients w i t h Hypocalcemia
Patients with Hypercalcemia
Permanent RLN Injury
1970-72
2-4 yr
327
94.2%
1.2%
4.5%
0.3%
1956-79 1956-79
289 441
92.4% 84.1%
0% 3.4%
7.6% 12.5%
NR NR
1961-83 1963-84 1980-88 1953-82 NR
Mean 5 yr 4-27 yr NR (7.5% lost to follow-up) Mean 5.2 yr 1-8 yr Mean 12.9 yr Mean 13 yr
311 180 59 896 274
71.0% 96.6% 93.2% 92.7% 94.2%
10.6% 1.1% NR 4.2% 1.1%
10.9% 2.2% 6.8% 3.0% 4.7%
2.9% NR NR NR NR
Ud6n et al./San Francisco11
1980-90
NR
250
98.8%
0
1.2%
0
Kairaluoma et al./Finland2~
1982-89
Mean 2.3 yr
92
85.9%
5.4%
8.7%
2.2%
Doherty et al./St. Louis21
1992-93
< 2 yr
39
0
0
0
Oertli et al./Basel, Switzerland22 Chigot et al./Paris 23'a
1977-92 1978-92
Mean 25 mo Mean 3 yr
92.3%
4.1%
3.5%
0.6%
94.8%
0
1.3%
0
Ronni-Sivula and Sivula/ Helsinki 13 Rudberg et al./SwedenTM Kristoffersson et al./ Sweden15 Roka et al./Vienna16 Wendt and Geipel/Berlin17 Hedback et al./SwedenTM Bonjer et al./Rotterdam19
170 78
100%
aOnly patients > 75 yr of age; operative mortality 3.8% all prior to 1984; major morbidity 4%. NR, not reported; RLN, recurrent laryngeal nerve.
468 TABLE 15--2 NIH Consensus Conference Indications for Operation in Asymptomatic Primary Hyperparathyroidism a On initial evaluation: Markedly elevated serum calcium History of an episode of life-threatening hypercalcemia Reduced creatinine clearance Presence of kidney stone(s) detected by abdominal radiograph Markedly elevated 24-hour urinary calcium excretion Substantially reduced bone mass as determined by direct measurement During monitoring of an asymptomatic patient, these developments: Typical symptoms of the skeletal, renal, or GI systems Sustained serum calcium > 1.0 to 1.6 mg/dl above normal Significant decline in renal function (> 30% decline in creatinine clearance) Nephrolithiasis or worsening calciuria Significant decline in bone mass (to < 2 SD below age/gender/racematched mean) Significant neuromuscular or psychological symptoms Inability or unwillingness of patient to continue medical surveillance aModified from Potts JT Jr, Ackerman IP, Barker CF, et al: Diagnosis and management of asymptomatic primary hyperparathyroidism: Consensus development conference statement. Ann Intem Med 114:593-597, 1991. GI, gastrointestinal; SD, standard deviations.
proof that parathyroidectomy can prevent this is lacking. 18'27-29 A National Institutes of Health Consensus Development Conference reviewed this subject in 1990. 30 The panel confirmed that operation is indicated for all patients with symptoms of bone disease or renal stone disease. The indications for operation in asymptomatic patients (Table 15-2) were outlined, and the panel suggested semiannual follow-up for patients managed nonoperatively. In addition, operation was recommended for patients in whom medical surveillance was neither desirable nor suitable, as when the patient requests an operation, consistent follow-up is unlikely, coexistent illness complicates management, or the patient is less than 50 years of age. While the initial cost of a parathyroid operation is higher than medical monitoring, with time the cost of follow-up, including bone densitometry studies, laboratory work, and other tests, exceeds the cost of operative intervention; the operative cost can be minimized if the patient is treated by an experienced endocrine surgeon. 2~ Definitive resolution of this question regarding the best management of asymptomatic patients with hyperparathyroidism will require a randomized, controlled trial. 8. LOCALIZATION
That there is no need for parathyroid gland localization studies prior to cervical exploration in a previously
GERARD M. DOHERTY AND SAMUEL A. WELLS, JR.
unoperated patient with hyperparathyroidism is an area of some controversy. This view is reflected in the statement of the recent National Institutes of Health Consensus Conference statement: "Preoperative localization in patients without prior neck operation is rarely indicated and not proven to be cost-effective."3~ The fact that the surgeon must examine all four parathyroid glands before making a decision as to which should be resected obviates the routine use of localization procedures.
B. S p e c i a l E t i o l o g i c a l C o n s i d e r a t i o n s 1. FAMILIAL DISEASE Familial hyperparathyroidism can present as an isolated, autosomal dominant inherited disease, or more commonly as a part of multiple endocrine neoplasia (MEN) types I or IIA. A thorough family history may reveal the presence of a familial endocrinopathy. This is clinically important because the parathyroid disease in these patients is multiglandular. This demands an operative strategy designed for multiglandular disease, even if there is asymmetrical hypertrophy and one or more of the parathyroid glands appears grossly normal. 31 In addition, patients with MEN-I are also at risk for developing tumors of the pancreatic islet cells (especially functional gastrinomas or insulinomas), the pituitary gland (particularly prolactinomas), and the adrenal cortex. All patients with MEN-IIA develop medullary thyroid carcinoma, and about half of them develop pheochromocytomas. Management of these nonparathyroid tumors requires specific strategies designed around their natural histories. FHHH and neonatal severe hyperparathyroidism are rare conditions caused by a defect in the gene coding for the calcium-sensing receptor. Homozygote infants are often bom with hypotonia, poor feeding, constipation, respiratory distress, and neonatal severe hyperparathyroidism. 32 The 1-year survival in symptomatic untreated patients is less than 50%, and total parathyroidectomy with autotransplantation appears to be the treatment of choice. 33 A more conservative approach may be justified if the infant is asymptomatic. People who have one normal gene copy have FHHH. This benign condition represents an elevation in the calcium set-point. The patients have elevated serum calcium concentrations and mildly elevated serum PTH concentrations, but no complications of primary hyperparathyroidism. This disease can be distinguished from primary hyperparathyroidism by the normal 24-hour urine calcium. The genetic abnormalities associated with MEN-I, MEN-IIA, and FHHH have been identified, and it is now possible to identify affected family members by direct
CHAPTER 15 SurgicalTreatment for Hyperparathyroidism
genetic testing. 34-36'34a This simplifies the diagnosis and management of these patients.
469 asymptomatic patient, the altemative of nonoperative or medical management should be presented.
2. RADIATION EXPOSURE
Exposure to external beam radiation, particularly during childhood, carries an increased risk of later primary hyperparathyroidism. 37'38 This risk factor is important to elucidate, not because of differences in the parathyroid pathology, which is similar to patients without a history of radiation exposure, but because of the increased frequency of associated tumors in the thyroid or salivary glands, which might require attention at operation. 39 Careful examination of the neck preoperatively and postoperatively is important in patients with this history. The patient should be informed of the possible need for resection of a thyroid or salivary gland tumor if found at operation.
C. General Preoperative Assessment Patients undergoing neck exploration for hyperparathyroidism must be prepared for general anesthesia. The preoperative assessment should include a thorough history and physical examination to evaluate the risk of anesthesia for the particular patient. Neck exploration is typically not a physiologically stressful procedure, as there are only minor fluid shifts, and rarely significant blood loss. Severe hypercalcemia should be corrected before operation by saline diuresis and pharmacological means. Patients who have had previous cervical operations should have documentation of bilateral vocal cord function.
D. Informed Consent A thorough preoperative discussion can help the patient by giving them appropriate expectations about the operation. The patient should be made fully aware of the complications associated with the neck exploration, including potential damage to the superior and recurrent laryngeal nerves and the development of transient or permanent hypocalcemia. The possibility of an unsuccessful parathyroid search should be mentioned, and the patient should be made aware that repeat operation, including a mediastinal exploration, may be required if the planned operation is unsuccessful. Although the likelihood of these complications is small, it is best to discuss them prior to the initial neck exploration. In addition, the complications associated with general anesthetic, including allergic reactions to medications, and the rare occurrence of wound complications, such as postoperative bleeding or infection, should be presented. In the rare, truly
III. PRIMARY HYPERPARATHYROIDISM: CONDUCT OF THE OPERATION There are five cardinal rules which should be followed when operating on patients with primary hyperparathyroidism for the first time: (1) explore both sides of the neck and identify all four parathyroid glands; (2) determine which of the parathyroid glands is (are) enlarged; (3) resect the enlarged parathyroid glands and leave the normal size parathyroid gland(s) in situ; (4) when all four parathyroid glands are enlarged, perform either a subtotal (31/2-gland) parathyroidectomy, or a total parathyroidectomy with a heterotopic parathyroid autotransplantation; and (5) if no enlarged parathyroid glands are identified, do not resect a normal parathyroid gland. There is no special preoperative preparation for the patient. Most are admitted to the hospital early on the day of the operation; however, there have been reports of patients having parathyroidectomy on an outpatient basis, with discharge on the same day as the operation.
A. Exploration Most parathyroid glands are found in the usual anatomical positions; some variations in gland location are so frequent as to be considered "normal," and should be investigated in patients in whom a gland is not identified in a more usual site (Fig. 15-1). It is essential that the surgeon have a clear understanding of parathyroid gland embryology. The upper glands are derived from the fourth pharyngeal pouch, while the lower glands are derived from the third pharyngeal pouch. The thymus gland co-migrates with the lower gland; in the lower neck these structures separate so that the lower parathyroid lies close to the lower pole of the thyroid gland, while the thymus resides in the mediastinum. A remnant of the thymus gland can be identified protruding from the thoracic inlet adjacent to the inferior pole of the thyroid gland. The search for the lower parathyroid should begin in the thyrothymic ligament, since the parathyroid gland is usually close to, or within, it. The thyrothymic ligament is usually well formed, and appears as a cylindrical grayish structure below the thyroid lobe. In some people, the thymus gland may descend only a short distance, or not at all, during embryogenesis. If the associated parathyroid gland also fails to migrate, it will reside high in the neck as an "undescended para-
470
GERARD M. DOHERTY AND SAMUEL A. WELLS, JR.
FIGURE 15-- 1 Illustration of frequent ectopic locations of parathyroid tissue. (From Wells SA Jr, Leight GS, Ross AJ: Primary hyperparathyroidism. Curr Prob Surg 17:400-463, 1980.)
thymus." There can be varying stages of maldescent, and the surgeons must be aware of this, if the lower parathyroid gland cannot be found in its expected position. The most common sites of an ectopic lower parathyroid gland are either high in the neck, in the lateral neck associated with thymus tissue, or in the anterior mediastinum in association with the thymus gland. Also, as lower parathyroid glands increase in size, they may be pulled into the upper anterior mediastinum by negative intrathoracic pressure. Parathyroid glands within the mediastinum can frequently be removed by mobilizing the thymus through the cervical incision. Embryologically, the upper glands migrate with the lateral thyroid complex for only a short distance. Upper glands are more constant and posterior in location than the lower glands but can still present anywhere from the retropharyngeal area, to the carotid sheath or the posterior mediastinum. One common "acquired ectopic" position that occurs with enlarged upper parathyroid glands is along the esophagus reaching inferiorly into the mediastinum. These glands usually are tethered at the upper end, near the crossing of the inferior thyroid artery and the recurrent laryngeal nerve. With dissection of the local structures, the parathyroid can be separated from its usual location, slide inferiorly, and be missed. If exploration of these areas is unsuccessful in identifying a missing parathyroid gland, the thyroid lobe on the side
of the missing gland should be mobilized and palpated. Intraoperative ultrasound examination may be useful to identify an intrathyroidal parathyroid gland, or a gland in another ectopic location within the soft tissues of the neck. As a last resort, blind excision of the ipsilateral thyroid lobe is advocated by some surgeons. In patients with multinodular goiter, the upper parathyroid is prone to become enfolded within the thyroid parenchyma, where it may be difficult to identify. These anatomical landmarks are utilized to carefully explore each side of the neck, with the goal of identifying all four parathyroid glands in every patient. Although in the past some surgeons have advocated unilateral exploration if a single enlarged parathyroid gland and a normal parathyroid gland are identified on the first side of the neck explored, most surgeons now agree that all four parathyroid glands need to be identified at the initial exploration because of the possibility of multiplegland disease. 4~ Abnormal parathyroid glands on the contralateral side of the neck will be missed at exploration in about 10% of patients if a unilateral approach is utilized. Although frozen section has not been helpful in differentiating diseased and normal glands, many endocrine surgeons consider it essential for confirming the presence or absence of parathyroid tissue. Small, thin biopsy specimens are sharply incised from each gland with extreme care taken to avoid damaging their delicate
CHAPTER 15 Surgical Treatment for Hyperparathyroidism
471
blood supply. Some surgeons, alternatively, utilize frozen section selectively, to document only the abnormal parathyroid gland. 22 When an enlarged gland is identified, it is removed, trimmed of fat, and weighed. Parathyroid gland weight is the single most valuable datum obtained during the operation. If, after meticulous exploration, three or four parathyroid glands have been identified, none of which is enlarged, the operation should be terminated. Supernumerary glands may be present and should be sought at the initial procedure. This is particularly important in those patients with familial hyperparathyroidism or renal osteodystrophy, since the incidence of supernumery glands is higher. For this reason, the cervically accessible thymic tissue should be removed in these patients.
B. E x t e n t o f R e s e c t i o n The operative procedure performed is based on the number of enlarged glands identified (Table 15-3). In many instances, the pathologist cannot reliably distinguish a parathyroid adenoma from parathyroid hyperplasia on frozen section. If the surgeon determines that a parathyroid gland is enlarged, then it should be resected. Typically, single-gland disease has been treated by simple excision, whereas any combination of two- or threegland enlargement is treated by resection of the diseased tissue with the normal glands left in place. The question of whether two- or three-gland enlargement implies the presence of disease in all glands (hyperplasia) has not
TABLE 15--3 Author/Site Satava et al./Mayo Cliniclz Ronni-Sivula and Sivula/ H e l s i n k i 13
Rudberg et a l . / S w e d e n 14 Kristoffersson et al./Swedenis Roka et al./Vienna16 Wendt and Geipel/Berlin17 Hedback et al./SwedenTM Ud6n et al./San Francisco~ Kairaluoma et al./Finland2~ Doherty et al./St. L o u i s 21 Oertli et al./Basel, Switzerland22 Chigot et a l . / P a r i s 23
been resolved. If one gland is large and the remaining three are normal in size, resection of the single parathyroid cures virtually all patients. Patients with two- or three-gland disease should be managed by excising only the large glands. The incidence of persistent or recurrent hypercalcemia in these patients is 1 0 . 5 % . 41 The management of patients with four-gland disease is more difficult. In many of these patients, the disease occurs as a component of one of the familial syndromes, particularly MEN-I. Patients with four-gland parathyroid hyperplasia can be managed by subtotal parathyroidectomy (removing 31/2 glands) or by total parathyroidectomy with autotransplantation of some parathyroid tissue into the nondominant forearm. Both operations depend on meticulous identification of all parathyroid tissue. The putative advantage of the subtotal parathyroidectomy is that it leaves the remaining parathyroid tissue with its native blood supply. Total parathyroidectomy has the advantage of removing all abnormal parathyroid tissue from the neck and placing it in a site where reoperation for recurrent hyperparathyroidism would be simpler. In either operation, parathyroid tissue should be viably cryopreserved to allow later autografting if the patient has persistent hypoparathyroidism postoperatively. Subtotal parathyroidectomy for nonfamilial parathyroid hyperplasia has a reported incidence of recurrent hypercalcemia of 0% to 16%; the incidence of permanent hypoparathyroidism is 4% to 5%. Patients with MEN-I, however, have a recurrence rate of 26% to 36% with long term follow-up after subtotal parathyroidectomy (average time to recurrence > 5 years). Total parathyroidectomy has a similar risk of permanent hypo-
Pathology at Operation for Primary Hyperthyroidism Two- or Three-Gland Diseasea
Hyperplasia or Four-Gland Disease
Years
N
Adenoma
1970-72
327
84.7%
NR
9.2%
1956-79 1956-79 1961-83 1963-84 1980-88 1953-82 1980-90 1982-89 1992-93 1977-92 1978-92
289 441 311 186 59 896 250 92 39 170 78
84.1% 77% 80% 82.8% 74.5% 84.0% 78.4% 63% 76.9% 82.1% 94.9%
NR NR NR 11.3% 15.3% NR 6% 2.2% 2.6% NR 3.8%
15.2% 18% 15% 2.2% 1.7% 15.7% 14% 31.5% 20.5% 14.5% 1.3%
aSome authors report two- or three-gland enlargement separately from four-gland disease; this is noted where recorded. NR, not reported.
Carcinoma 0% 0.7% 0 0.3% 3.2% 1.7% 0.2% O% O% O% 3.5% O%
472
GERARDM. DOHERTYAND SAMUELA. WELLS,JR.
parathyroidism (5%) and a higher reported risk of recurrent hypercalcemia (familial, 64%; nonfamilial, 20%). Reoperation for recurrent hypercalcemia is greatly simplified by total parathyroidectomy with autotransplantation approach. Thus, given the currently available data, sporadic parathyroid hyperplasia can be acceptably treated by either operation. However, the substantial risk of recurrent hypercalcemia following either operation makes total parathyroidectomy with autotransplantation an attractive option for patients with familial disease. To attempt to definitively answer this question for patients with familial hyperparathyroidism, our group has recently begun a randomized prospective clinical trial of subtotal parathyroidectomy versus total parathyroidectomy with autograft. n
IV. PRIMARY HYPERPARATHYROIDISM: MANAGEMENT OF PATIENTS WITH PERSISTENT OR RECURRENT HYPERPARATHYROIDISM Patients with unsuccessful neck explorations almost always have either persistent or recurrent hypercalcemia. Persistent hyperparathyroidism pertains to patients who either have continued unabated hypercalcemia in the immediate postoperative period, or develop it within 6 months of the neck exploration. Recurrent hypercalcemia describes patients who are normocalcemic for 6 months after neck exploration before they develop an elevated blood calcium level. More often, persistent rather than recurrent hypercalcemia is the mode of presentation in patients who have unsuccessful neck explorations for hyperparathyroidism. Inadequately excised hyperplastic tissue is a common cause of both persistent and recurrent disease. Less common is the late local recurrence of adenoma or carcinoma as a result of tissue spilled from a broken gland that implanted at the time of initial operation. In most cases, review of the original operative notes and pathology report provides clues as to the position of residual tissue and the nature of the recurrence. 42 The location of parathyroids responsible for failed operations due to missed single adenoma found on reexploration in one large series is shown in Figure 15-2. Patients should have a clear indication for surgical management of their disease before reexploration. These can include serum level of calcium greater than 11.5 mg/dl, or complications of hypercalcemia, such as renal stones or osteopenia. Reexploration of asymptomatic patients with mild hypercalcemia is generally considered
%
1. Tracheo-esophageal groove
n=59 (27%)
2. Anterior mediastinum/thymus
n=38 (18%)
3. Normal upper
n=28 (13%)
4. Normal lower
-n=26 (12%)
5. Intrathyroid
n=22 (10%)
6. Undescended
n=18 (8.4%)
7. Carotid sheath
n=8
(3.7%)
8. Retroesophageal
n=7
(3.3%)
9. Other mediastinal
n=3
(1.4%)
10. Strap muscles
n=3
(1.4%)
11. Other
n=3
(1.4%)
FIGURE 1 5 - - 2 Sites of parathyroid resection in patients with persistent hyperparathyroidism due to missed parathyroid adenoma. [From Jaskowiak N, Fraker DL, Alexander HR, et al: A prospective trial evaluating a standard approach to reoperation for missed parathyroid adenoma. Ann Surg 224:308-320, 1996.]
unwise because the increased risk associated with the reoperation is thought to outweigh the potential benefits.
A. Localization In contrast to patients having their first operation for hyperparathyroidism, most surgeons agree that localization studies should be performed prior to reexploration. A combination of noninvasive studies should be utilized initially. If these are unsuccessful in demonstrating an abnormality, invasive studies (selective angiography and venous sampling for parathyroid hormone) are indicated. There are several noninvasive techniques for localizing hyperfunctioning parathyroid tissue. Barium swallow, cineesophagography, and thyroid scanning are
CHAPTER 15
473
Surgical Treatment for Hyperparathyroidism
rarely helpful, whereas c o m p u t e d tomographic (CT) scans, magnetic resonance imaging (MRI), technetiumthallium subtraction scanning, 99mTc-sestamibi scanning, and high-resolution real-time ultrasonography are variably useful in identifying enlarged parathyroid glands in patients studied (Table 1 5 - 4 ) . Sestamibi scanning is the newest of these techniques, and utilizes a radionuclide imaging agent originally d e v e l o p e d for cardiac imaging (Fig. 1 5 - 3 ) . During these studies, this agent was observed to image parathyroid tissue on delayed scans. Recently, sestamibi has been used for parathyroid imaging. The single-nuclide nature, short half-life, and highenergy profile of this technique provides advantages in lateral, oblique, and three-dimensional imaging compared to technetium-thallium scanning. Sestamibi scans and C T scans are most useful for identifying lesions in the neck and mediastinum. Ultrasound and C T examinations can be coupled with needle aspiration of an identified mass, followed by assay of the aspirate for PTH. Aspirate parathyroid h o r m o n e values that are twice the m e a n b a c k g r o u n d (peripheral venous) levels of parathyroid h o r m o n e indicate the presence of parathyroid tissue. 43 Specific testing algorithms depend on the available facilities and expertise, as the utility of each examination
TABLE 15--4
Ultrasound Sensitivity False-positive Scintigraphy Sensitivity False-positive CTscan Sensitivity False-positive MRIscan Sensitivity False-positive Angiography Sensitivity False-positive Parathyroid venous sampling Sensitivity False-positive Intraoperative ultrasound Sensitivity False-positive
will vary with the skill and e q u i p m e n t of the invasive radiologist. A typical strategy w o u l d be to obtain two initial c o m p l e m e n t a r y studies, such as ultrasound and sestamibi scanning. If these each demonstrate the same abnormality consistent with parathyroid tissue, no other studies are p e r f o r m e d and the patient is explored. If either study is negative or if one is contradictory of the other, additional tests are p e r f o r m e d to evaluate the area of concern. If no two noninvasive imaging tests provide reliable, confirmatory results, then selective arteriography and venous sampling for parathyroid h o r m o n e are employed. 44,4s Selective arteriography (Fig. 1 5 - 4 ) identifies hyperfunctioning parathyroid tissue in m a n y cases (Table 1 5 - 4 ) . Venous sampling may also be helpful in some patients, although interpretation is often complicated by the d e v e l o p m e n t of collateral venous drainage postoperatively. B e c a u s e selective venous sampling with P T H determination provides no direct image, but only lateralizes the side of the neck where the hyperfunctioning tissue is located, it m a y only help to direct the exploration to one or the other side of the neck. In a series from the National Institutes of Health, invasive studies identified the abnormal parathyroid gland in 41 of 43 patients w h o had negative or equivocal noninvasive
Parathyroid Localization Studies for Persistent or Recurrent H y p e r p a r a t h y r o i d i s m Miller et al. (1987) NIH44'57
Cheung et al. (1989) University of Michigan s8
Levin et al. (1987) UCSF45
n = 50 36.0% 14.0% n 34a 26.5% 5.95 % n=51 47.1% 2.0% n = 16 50% NR n = 43 60.4% 4.7% n = 30 80.0% 0 n = 18 77.7% 0
n = 17 23.5% 5.9% n 9a 55.6% 11.1% n = 15 26.7% 20.0% m
n = 41 58% 13% n = 39a 49% 23 % n=41 46% 15% n = 17 65% 18%
=
aTechnetium-thallium subtraction scanning. b 99 mTc-sestamibi-123 I imaging. NR, not reported.
=
n=9 22.2% 11.1% n = 23 39.1% 26.1% --
n = 28 71% 0
Weber et al. (1993) Emory University 59
n = 14~ 85.7% NR
Thompson et al. (1994) Mayo Clinic 6~ n = 39 43.6% 10.3% n = 45b/15a 62%/60% 2.2%/6.7% n=10 20% 0 n=3 0 0
474
GERARD M. DOHERTYAND SAMUELA. WELLS, JR. abnormal parathyroid tissue localized preoperatively. A direct approach to the localized disease should be used. If the disease is identified on one side of the neck, the opposite side is not typically explored, to avoid injury to the remaining parathyroids or the recurrent laryngeal nerve. The side of the neck that is explored should be fully investigated, to avoid any need for a third dissection in that area in the future. Intraoperative ultrasound is a useful adjunct during reoperative neck exploration (Fig. 1 5 - 5). 46,47
Surgical reexploration can be difficult. The goal of the reoperation should be to identify and remove the
If the disease has been localized in the anterior superior mediastinum, the neck should almost always be reexplored first. If the thymic remnant has not already been removed, it should be excised at this time. If the parathyroid tissue is not included in this tissue, or if scar tissue makes the exposure difficult, successful transcervical mediastinal exploration is sometimes possible using the Cooper thymectomy retractor, which permits more extensive mediastinal exploration and thymectomy through a cervical incision. 48 Any remaining thymic tissue is first isolated and examined; other adjacent tissues in the anterior mediastinum can be dissected as well. Median stemotomy and exploration is necessary in only 1% to 2% of patients with primary hyperparathyroidism. Usually, a vertical incision is made from the center of the cervical incision to the xiphoid. With this incision, there is excellent exposure of the mediastinum. Functional tests to prospectively detect the removal of abnormal parathyroid tissue while still in the operating room have been used on a limited basis. Urinary levels of cyclic adenosine monophosphate (cAMP) can be measured, and reflect circulating parathyroid hormone
FIGURE 15--4 Angiogramdemonstrating a recurrent parathyroid adenoma (arrow) in the aortopulmonary window supplied by a branch of the internal thoracic artery. Note the surgical clips from previous resection of adenomatous parathyroid tissue in this area.
FIGURE 15--5 Intraoperativeultrasound demonstrating a hypoechoic intrathyroidal parathyroid adenoma (arrow).
FIGURE 1 5 - 3 99mTC-sestamibi scan identifying parathyroid tissue in the right lower position in a patient with hyperparathyroidism (arrow). Symmetrical uptake higher in the neck represents submandibular glands.
studies. 44 Importantly, false-positive results, which could lead to fruitless exploration, were uncommon. These studies, while very accurate in centers with demonstrated expertise, also have potentially significant risks, such as transverse myelitis, cerebrovascular accident, and death.
B. Reoperation
CHAPTER 15 SurgicalTreatment for Hyperparathyroidism levels. This test has been used in the reoperative parathyroidectomy setting, but does not appear to add substantially to the intraoperative judgment of the experienced surgeon. 49'5~ More recently, some investigators have begun using rapid determinations of serum levels of parathyroid hormone intraoperatively in an attempt to detect when all of the abnormal parathyroid tissue has been removed. The utility of the approach remains to be demonstrated. Surgical reexploration is successful in experienced hands in more than 80% of cases; however, there is an increased incidence of postoperative complications. Unilateral recurrent laryngeal nerve injury occurs in 5% to 10% of patients and permanent hypoparathyroidism occurs in 10% to 20% of patients. Cryopreservation of excised parathyroid tissue is a mandatory component of the management of these patients, as it allows later autotransplantation if the patient becomes hypoparathyroid postoperatively. The risks of these complications are clearly outweighed by the clinical improvement in patients with advanced disease. Rarely, patients with symptomatic persistent hyperparathyroidism are not operative candidates because of concurrent illnesses; these patients may be considered for nonoperative angiographic parathyroid ablation if an enlarged parathyroid gland is identified. 51
V. O P E R A T I V E M A N A G E M E N T OF PATIENTS WITH PARATHYROID CARCINOMA Parathyroid carcinoma is a rare condition, accounting for less than 1% of all cases of hyperparathyroidism (Table 15-3). This diagnosis should be suspected when (1) a palpable neck mass is present; (2) the serum calcium level exceeds 14 mg/dL; (3) the serum PTH level is markedly elevated; and (4) a previously unoperated patient is hoarse from a recurrent laryngeal nerve invasion. 52 Compared with patients with benign disease, these patients tend to be younger and to have more symptoms. In contrast with the marked female predominance in benign disease, the male/female ratio in carcinoma is equal. Patients may have manifestations of both renal disease and bone disease. The affected gland is palpable in almost half of patients. At operation, the parathyroid carcinoma appears white and very firm, unlike adenomas, which are reddish brown and soft. It may be difficult to distinguish early parathyroid carcinoma from atypical parathyroid adenoma by histological and clinical criteria: the diagnosis is securely made only on the basis of local invasion or distant metastases. In these cases, DNA cytometry can be helpful as an aneuploid
475 pattern and higher nuclear DNA content are typical of carcinomas compared to adenomas. 53 The initial operation must be aggressive, yet meticulous, with e n b l o c resection of the parathyroid tumor and all adjacent invaded tissues, including the ipsilateral thyroid lobe. It is very important not to rupture the capsule and spill the tumor. Radical neck dissection is reserved for patients with clinically overt cervical node metastases. Neither chemotherapy nor radiation therapy have been shown to be of any benefit to patients with parathyroid carcinoma. Tumor recurrence is generally apparent within 6 months to 3 years of operation and may denote an incurable process. If the disease recurs, an attempt should be made to resect it because, if untreated, these patients usually succumb to uncontrolled hypercalcemia. The long-term prognosis is poor, and the opportunity for survival depends on complete initial resection. 54,55
VI. O P E R A T I V E M A N A G E M E N T OF PATIENTS WITH RENAL OSTEODYSTROPHY The indications for parathyroidectomy in patients with renal osteodystrophy (secondary hyperparathyroidism) are: (1) persistent and symptomatic hypercalcemia in prospective renal transplant patients; (2) bone pain or pathological fractures; (3) ectopic calcification; and (4) intractable itching. These patients can be managed by subtotal (31/2-gland) parathyroidectomy or total parathyroidectomy with heterotopic (forearm) autotransplantation. As in other forms of hyperplasia, reexploration for recurrent hyperparathyroidism is simplified by autotransplantation to the nondominant forearm. A randomized, prospective trial has been reported for patients with secondary hyperparathyroidism, comparing subtotal parathyroidectomy to total parathyroidectomy with autotransplantation (Fig. 15-6). 56 Of patients randomized to subtotal parathyroidectomy, 20% developed hypercalcemia and indications for reoperation within 40 months after the initial procedure. Because of improved clinical parameters (less pruritus and muscle weakness), improved radiological parameters, and the need for no reoperations, the total parathyroidectomy with autotransplantation is the procedure of choice.
VII. POSTOPERATIVE
MANAGEMENT
The postoperative recovery of patients after parathyroidectomy is generally uncomplicated, typically requiring a one- or two-night stay in the hospital. Cervical
476
GERARD M. DOHERTYAND SAMUELA. WELLS, JR. Patients with Chronic Renal Failure and Renal Osteodystrophy
.11
PANIX~tT'E
Subtotal Parathyroidectomy (n=20)
~
Total Parathyroidectomy + autotransplantation (n=20)
wo,, I
2 patients hypercalcemic but not yet reoperated
3 Dead
I
17 Well
3 1/2 year follow-up
FIGURE 15--6 Diagramof a randomized trial of parathyroidectomytechniques in patients with renal osteodystrophy (From Rothmund M, Wagner PK, Schark C: Subtotal parathyroidectomy versus total parathyroidectomy and autotransplantation in secondary hyperparathyroidism: A randomized trial. World J Surg 15:745-750, 1991.) Patients with conventional indications for parathyroidectomy in renal failure were randomized to total or subtotal resection. After 3.5 years, patients in the total parathyroidectomy/autotransplantationgroup were more likely to have normal serum calcium and alkaline phosphatase concentrations (p < 0.05), to have radiological improvement, and to have clinical improvement (pruritis and weakness, p < 0.05), than patients in the subtotal parathyroidectomy group. In addition, there were no patients who required, or who had indications for, reoperation in the total parathyroidectomy/autotransplantationgroup.
incisions heal well, and infectious complications are rare. However, the postoperative management of patients after parathyroidectomy requires consideration of some specific issues.
A. Airway Protection Any patient who has had operation on both sides of the neck, with exposure of the recurrent laryngeal nerves, is at risk for temporary or permanent, unilateral or bilateral, vocal cord paresis. This rare complication must nevertheless be considered at the completion of the operative procedure, as the patient emerges from general anesthesia and the endotracheal tube is removed. Unilateral vocal cord paresis affects phonation, but does not usually endanger ventilation or oxygenation. Patients with bilateral cord paresis may initially ventilate adequately through a patent anterior glottic chink, and may have nearly normal phonation, but may subsequently have decreased ventilatory capacity due to further edema or fatigue. Careful attention to the early postoperative airway is vital to avoid devastating complications.
B. Calcium Replacement After removal of hyperfunctioning parathyroid tissue with normal, vascularized parathyroid tissue left in situ, most patients become transiently hypocalcemic, and many require temporary oral calcium replacement be-
cause of serum calcium levels below 7.0 mg/dl, or symptoms of hypocalcemia. Typically, oral calcium is delivered as calcium gluconate 500 mg, two to four doses daily. Occasional patients will require oral vitamin D as well. The need for calcium and/or vitamin D replacement appears to correlate with the extent of preoperative bone disease. The nadir of the serum calcium concentration is typically reached within 48 to 72 hours after operation. Patients who become severely hypocalcemic with marked symptoms, and those who are unable to take the medications orally because of concomitant medical illness, will require intravenous calcium supplementation. Calcium gluconate (10% solution available as ampules containing 90 mg elemental calcium/10 ml) can be administered intravenously emergently over 10 minutes. Intravenous infusion supplementation is then begun as 60 ml (6 ampules) of 10% calcium gluconate in 500 ml of D5W (1 mg/ml elemental calcium) at 0.5 to 2.0 mg/kg/hr. The serum calcium concentration should be monitored periodically and the infusion adjusted appropriately. Patients who have total parathyroidectomy and autotransplantation will develop significant hypocalcemia, and should be started on oral calcium and vitamin D replacement immediately after operation in order to avoid severe hypocalcemia. Replacement should be adjusted to maintain the serum calcium concentration at the lower limit of normal. The parathyroid autografts typically perform adequately within 8 to 12 weeks. Autograft function is documented by comparing the parathyroid hormone levels in serum samples drawn from
CHAPTER 15
477
Surgical Treatment for Hyperparathyroidism
each antecubital fossa, above the parathyroid autograft. If there is a gradient between the arms, but the patient remains hypocalcemic, supplementation should be continued a n d m o r e t i m e a l l o w e d f o r t h e a u t o g r a f t to b e s t i m u l a t e d to p r o d u c e a d e q u a t e h o r m o n e . I f t h e r e is n o g r a d i e n t , t h e n it is l i k e l y t h a t t h e p a r a t h y r o i d g r a f t v e n o u s e f f l u e n t is d r a i n e d t h r o u g h a r o u t e o t h e r t h a n t h e a n t e c u b i t a l vein. Patients with renal osteodystrophy, and those rare patients w i t h v i t a m i n D r e s i s t a n t t i c k e t s , m a y h a v e v e r y marked
hypocalcemia
after
total
parathyroidectomy,
probably because of the severity of the b o n e disease. T h i s is c o m p o u n d e d
in t h e p a t i e n t s w i t h v i t a m i n D re-
s i s t a n c e b y t h e difficulty in m a i n t a i n i n g
a d e q u a t e re-
p l a c e m e n t u s i n g t h e g a s t r O i n t e s t i n a l tract. T h e s e difficulties
should
be
anticipated
and
treated
by
more
v i g o r o u s , a n d early, r e p l a c e m e n t o f c a l c i u m .
References 1. Sandstrom, I: On a new gland in man and several mammals (gladulae parathyroideae). Upsala Lak Foren Forh 15:441, 1879. 2. Gley E: Sur les fonctions du corps thyroide. C R Soc Biol 43: 841, 1891. 3. Erdheim J: Uber epithelkorperchenbefunde bei osteomalacie. S B Akad Wiss Math Naturw C1 116:311, 1907. 4. Schlagenhaufer F: Zwei failer von parathyreoideatumoren. Wien Klin Wochenschr 28:1362, 1915. 5. Mandl F: Therapeutischer Versuch bei einem Falle von Ostitis fibrosa generalisata mittels exstirpation eines epithelk orperchen Tumors. Zentrabl Chir 5:260, 1926. 6. Richardson EP, Aub JC, Bauer W: Parathyroidectomy is oseomalacia. Ann Surg 90:730, 1929. 7. Albright F: A page out of the history of hyperparathyroidism. J Clin Endocrinol Metab 8:637-657, 1948. 8. Barr DP, Bulger HA, Dixon HH: Hyperparathyroidism. JAMA 92:951-952, 1929. 9. Barr DP, Bulger MA: The clinical syndrome of hyperparathyroidism. Ann J Med Sci 179:449, 1930. 10. Thomas CG Jr: Presidential address: The glands of O w e n - - a perspective on the history of hyperparathyroidism. Surgery 108: 939-950, 1990. 11. Udrn P, Chan A, Duh Q-Y, et al: Primary hyperparathyroidism in younger and older patients: Symptoms and outcome of surgery. World J Surg 16:791-798, 1992. 12. Satava RMJ, Beahrs OH, Scholz DA: Success rate of cervical exploration for hyperparathyroidism. Arch Surg 110:625-628, 1975. 13. Ronni-Sivula H, Sivula A: Long-term effect of surgical treatment on the symptoms of primary hyperparathyroidism. Ann Clin Res 17:141-147, 1985. 14. Rudberg C, Akerstrrm G, Palmer M, et al: Late results of operation for primary hyperparathyroidism in 441 patients. Surgery 99:643-651, 1986. 15. Kristoffersson A, Dahlgren K, Granstrand B, Jarhult J: Primary hyperparathyroidism in Northern Sweden. Surg Gynecol Obstet 164:119-123, 1987.
16. Roka R, Niederle B, Kovarik J, et al: Clinical long-term results after parathyroidectomy for primary hyperparathyroidism. Acta Chir Scand 153:513-520, 1987. 17. Wendt F, Geipel D: Clinical aspects of surgery in hyperparathyroidism. Exp Clin Endocrinol 94:163-170, 1989. 18. Hedback G, Tisell L-E, Bengtsson B-A, et al: Premature death in patients operated on for primary hyperparathyroidism. World J Surg 14:829-836, 1990. 19. Bonjer HJ, Bruining HA, Birkenhager JC, et al: Single and multigland disease in primary hyperparathyroidism: Clinical followup, histopathology, and flow cytometric DNA analysis. World J Surg 16:737-744, 1992. 20. Kairaluoma MV, Makarainen H, Kellosalo J, et al: Results of surgery in primary hyperparathyroidism. Ann Chir Gynaecol 81: 309-315, 1992. 21. Doherty GM, Weber B, Norton J: Cost of unsuccessful surgery for primary hyperparathyroidism. Surgery 116:954- 958, 1994. 22. Oertli D, Richter M, Kraenzlin M, et al: Parathyroidectomy in primary hyperparathyroidism: Preoperative localization and routine biopsy of unaltered glands are not necessary. Surgery 117: 392-396, 1995. 23. Chigot J-P, Menegaux F, Achrafi H: Should primary hyperparathyroidism be treated surgically in elderly patients older than 75 years? Surgery 117:397-401, 1995. 24. Wells SA Jr: Surgical therapy of patients with primary hyperparathyroidism: Long-term benefits. J Bone Miner Res 6:S143S149, 1991. 25. Kenny AM, MacGillivray DC, Pilbeam CC, et al: Fracture incidence in postmenopausal women with primary hyperparathyroidism. Surgery 118:109-114, 1995. 26. Kochersberger G, Buckley NJ, Leight GS, et al: What is the clinical significance of bone loss in primary hyperparathyroidism? Arch Intern Med 147:1951, 1987. 27. Hedback G, Oden A, Tisell L-E: The influence of surgery on the risk of death in patients with primary hyperparathyroidism. World J Surg 15:399-407, 1991. 28. Palmer M, Bergstrom R, Akerstrom G, et al: Survival and renal function in untreated hypercalcaemia. Lancet 1:59-62, 1987. 29. Palmer M, Adami H-O, Bergstrom R, et al: Mortality after surgery for primary hyperparathyroidism: A follow-up of 441 patients operated on from 1956 to 1979. Surgery 102:1-7, 1987. 30. Potts JT Jr, Ackerman IP, Barker CF, et al: Diagnosis and management of asymptomatic primary hyperparathyroidism: Consensus development conference statement. Ann Intern Med 114: 593-597, 1991. 31. Marx SJ, Menczel J, Campbell G, et al: Heterogeneous size of the parathyroid glands in familial multiple endocrine neoplasia type 1. Clin Endocrinol 35:521-526, 1991. 32. Pollak MR, Brown EM, Chou Y-HW, et al: Mutations in the human Ca-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75:1297-1303, 1993. 33. Key LL, Thorne M, Pitzer B, et al: Management of neonatal hyperparathyroidism with parathyroidectomy and autotransplantation. J Pediatr 116:923- 926, 1990. 34. Pollak MR, Chou Y-HW, Marx SJ, et al: Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism: Effects of mutant gene dosage on phenotype. J Clin Invest 93:11081112, 1994. 34a. Chandrasekharappa SC, Guru SC, Manickam P, et al: Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 276:404-408, 1997. 35. Mulligan LM, Kwok JBJ, Healey CS, et al: Germ-line mutation of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 363:458-460, 1993.
478 36. Donis-Keller H, Dou S, Chi D, et al: Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet 2(7):851-856, 1993. 37. Cohen J, Gierlowski TC, Schneider AB: A prospective study of hyperparathyroidism in individuals exposed to radiation in childhood. JAMA 264:581-584, 1990. 38. Christensson T: Hyperparathyroidism and radiation therapy. Ann Intern Med 89:216, 1978. 39. Tezelman S, Rodriguez JM, Shen W, et al: Primary hyperparathyroidism in patients who have received radiation therapy and in patients who have not received radiation therapy. J Am Coil Surg 180:81-87, 1995. 40. Duh Q-Y, Uden P, Clark OH: Unilateral neck exploration for primary hyperparathyroidism: Analysis of a controversy using a mathematical model. World J Surg 16:654-662, 1992. 41. Wells SA, Leight GS, Hensley M, Dilley WG: Hyperparathyroidism associated with the enlargement of two or three parathyroid glands. Ann Surg 202:533-538, 1985. 42. Levin KE, Clark OH: The reasons for failure in parathyroid operations. Arch Surg 124:911 - 915, 1989. 43. Macfarlane MP, Fraker DL, Shawker TH, et al: Use of preoperative fine-needle aspiration in patients undergoing reoperation for primary hyperparathyroidism. Surgery 116:959-965, 1994. 44. Miller DL, Doppman JL, Krudy AG, et al: Localization of parathyroid adenomas in patients who have undergone surgery. Part II. Invasive procedures. Radiology 162:138-141, 1987. 45. Levin KE, Gooding GAW, Okerlund M, et al: Localizing studies in patients with persistent or recurrent hyperparathyroidism. Surgery 102:917-925, 1987. 46. Kern KA, Shawker TH, Doppman JL, et al: The use of highresolution ultrasound to locate parathyroid tumors during reoperations for primary hyperparathyroidism. World J Surg 11: 579-585, 1987. 47. Norton JA, Shawker TH, Jones BL, et al: Intraoperative ultrasound and reoperative parathyroid surgery: An initial evaluation. World J Surg 10:631-639, 1986. 48. Wells SA, Cooper JD: Closed mediastinal exploration in patients with persistent hyperparathyroidism. Ann Surg 214:555-561, 1991.
GERARD M. DOHERTY AND SAMUEL A. WELLS, JR. 49. Darling GE, Marx SJ, Spiegel AM, et al: Prospective analysis of intraoperative and postoperative urinary cyclic adenosine 3',5'-monophosphate levels to predict outcome of patients undergoing reoperations for primary hyperparathyroidism. Surgery 104:1128-1136, 1988. 50. Norton JA, Brennan MF, Saxe AW, et al: Intraoperative urinary cyclic adenosine monophosphate as a guide to successful reoperative parathyroidectomy. Ann Surg 200:389-395, 1984. 51. Doherty GM, Doppman JL, Miller DL, et al: Results of a multidisciplinary strategy for management of mediastinal parathyroid adenoma as a cause of persistent primary hyperparathyroidism. Ann Surg 215:101 - 106, 1992. 52. Fujimoto Y, Obara T: How to recognize and treat parathyroid carcinoma. Surg Clin North Am 67:343, 1987. 53. Levin KE, Chew KL, Ljung B-M, et al: Deoxyribonucleic acid cytometry helps identify parathyroid carcinomas. J Clin Endocrinol Metab 67:779-784, 1994. 54. Wang C, Gaz RD: Natural history of parathyroid carcinoma. Diagnosis, treatment, and results. Am J Surg 149:522-527, 1985. 55. Fraker DL, Travis WD, Merendino JJJ, et al: Locally recurrent parathyroid neoplasms as a cause for recurrent and persistent primary hyperparathyroidism. Ann Surg 213:58-65, 1991. 56. Rothmund M, Wagner PK, Schark C: Subtotal parathyroidectomy versus total parathyroidectomy and autotransplantation in secondary hyperparathyroidism: A randomized trial. World J Surg 15:745-750, 1991. 57. Miller DL, Doppman JL, Shawker TH, et al: Localization of parathyroid adenomas in patients who have undergone surgery. Part I. Noninvasive imaging methods. Radiology 162:133-137, 1987. 58. Cheung PSY, Borgstrom A, Thompson NW: Strategy in reoperative surgery for hyperparathyroidism. Arch Surg 124:676-680, 1989. 59. Weber CJ, Vansant J, Alazraki N, et al: Value of technetium 99m sestamibi iodine 123 imaging in reoperative parathyroid surgery. Surgery 114:1011-1018, 1993. 60. Thompson GB, Mullan BE Grant CS, et al: Parathyroid imaging with technetium-99m-setsamibi: An initial institutional experience. Surgery 116:966-973, 1994.
7HAPTER 1 (
Familial Benign Hypocalciuric Hypercalcemia and Other Syndromes of Altered Responsiveness to Extracellular Calcium EDWARD M. BROWN,*
MEI BAI,* AND MARTIN POLLAKt
*Endocrine-Hypertension and #Renal Divisions, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115
I. Introduction II. Syndromes of Extracellular Calcium Resistance III. Autosomal Dominant Hypocalcemia--A Syndrome of Increased Responsiveness of Target Tissues to Cao2+
IV. Summary and Conclusions Acknowledgments References
the disorders but also the normal functions of the genes in question. Pioneering clinical investigators, in some cases working many years before the advent of molecular techniques, were remarkably astute in outlining a conceptual framework for understanding abnormalities in the interactions of hormones with their respective receptors. For example, Fuller Albright more than 50 years ago recognized that certain endocrine disorders result from target tissue resistance to hormones, such as the
I. I N T R O D U C T I O N The application of molecular techniques has greatly expanded our understanding of how cells communicate with one another. Endocrine diseases have proven to be a rich source of abnormalities in the mechanisms governing intercellular communication. Elucidation of the molecular details of these diseases, in turn, has frequently clarified not only the pathophysiology of METABOLIC BONE DISEASE
479
Copyright 9 1998 by Academic Press. All fights of reproduction in any form reserved.
480 resistance to the actions of parathyroid hormone (PTH) in pseudohypoparathyroidism. ~ Subsequent work showed Albright's prescience by documenting that pseudohypoparathyroidism is associated with inactivating mutations in the guanine nucleotide binding (G) protein (Gs) through which the PTH receptor activates one of its principal intracellular signaling pathways, the adenylate cyclase-cyclic adenosine monophosphate (cAMP) system. 2 Conversely, activating mutations of Gs cause the peculiar skeletal and skin lesions of another syndrome described by Albright, polyostotic fibrous dysplasia. 3 Additional disorders have been described subsequently that arise from alterations in the biological activities of the cell surface receptors that couple to their intracellular signaling systems via G proteins. These receptors are members of the superfamily of G proteincoupled receptors (GPCRs) that share a common overall structure comprising an extracellular amino-terminus of widely varying size, seven hydrophobic membranespanning helices, and a cytoplasmic carboxyl-terminus. This superfamily includes several hundred or more members and encompasses receptors for a wide variety of extracellular first messengers, including numerous peptide hormones (e.g., PTH and calcitonin), neurotransmitters (e.g., biogenic amines), and prostaglandins as well as environmental stimuli such as photons (i.e., rhodopsin) and even odorants. 4-6 Inactivating mutations of GPCRs cause syndromes of hormonal resistance, while activating mutations produce inappropriate stimulation of the second messenger and other effector pathway(s) regulated by that receptor. 7'8 For example, inactivating mutations in the adrenocorticotropic hormone (ACTH) receptor produce adrenal insufficiency, 9 while activating mutations in the luteinizing hormone (LH) or PTH receptors cause precocious puberty 1~ and Jansen's syndrome, ~ respectively. The latter is a hypercalcemic disorder with biochemical abnormalities resembling primary hyperparathyroidism despite normal or suppressed PTH levels because of constitutive activation of the common receptor for PTH and the PTH-related peptide (PTHrP). Recent studies on the hormonal control of mineral ion homeostasis have considerably expanded our understanding of the mechanisms through which the extracellular calcium concentration (Ca 2+) is regulated. 12 This body of knowledge, in turn, has enabled elucidation of the molecular basis for several previously poorly understood hyper- and hypocalcemic syndromes whose clinical features had suggested reduced or increased responsiveness, respectively, to Ca 2+. This chapter will describe three such disorders: familial benign hypocalciuric hypercalcemia (FBHH), ~3 neonatal severe hyperparathyroidism (NSHPT), 14 and a form of autosomal dominant hypocalcemia (ADH). 15 In many but not all
EDWARD M. BROWN, MEI BAI, AND MARTIN POLLACK
cases, these disorders are caused by mutations in a recently cloned extracellular Ca 2+-sensing receptor (CAR), 16 which is a central mediator of the direct actions of Ca 2+ on parathyroid and C-cells as well as on a variety of additional cell types within the kidneys, brain, and other tissues. ~2 Thus, as with other endocrine disorders, the evidence gleaned from careful clinical investigation of genetic diseases of Ca2+-sensing provided critical clues that ultimately led to a greater molecular understanding of the normal physiology of this process.
II. SYNDROMES OF E X T R A C E L L U L A R CALCIUM RESISTANCE A. Familial Benign
Hypocalciuric Hypercalcemia 1. CLINICAL AND BIOCHEMICAL FEATURES OF FBHH In 1972, Foley et al. called attention to the remarkably benign clinical features of a hypercalcemic syndrome that they called familial benign hypercalcemia (FBH). ~7 Although several forms of familial hypercalcemia had been described previously (including a family that was later classified as having FBH18), this report clearly outlined the unique characteristics of patients with this syndrome. Subsequent detailed clinical studies of families with this disorder were carried out by Marx and coworkers 13 as well as by Heath et al. in the late 1970s and 1980S. 19'z~ Marx et al. termed this syndrome "familial hypocalciuric hypercalcemia" because of the characteristic abnormality in renal calcium handling found in affected family members. 21 Although there has never been a consensus as to which of these two names should be used to describe this inherited abnormality in calcium metabolism, we have chosen to call it FBHH in this chapter. FBHH is a rare genetic disorder of mineral ion metabolism that is inherited in an autosomal dominant fashion and is characterized by lifelong, generally asymptomatic hypercalcemia of mild to moderate severity (usually < 12 mg/dl). 2~ Despite their hypercalcemia, affected patients are generally remarkably free of the characteristic symptoms and complications that can afflict other hypercalcemic patients. The latter include most commonly mental disturbances; gastrointestinal abnormalities, particularly anorexia, nausea, and constipation; and renal complications, such as reductions in renal function, defective urinary concentrating ability, and nephrolithiasis or nephrocalcinosis. 22'23 Occasional families exhibit a higher calcium concentration (e.g., 12 to 13 to as high as 14.6 mg/dl in one case13), but affected mem-
CHazrE~ 16 Familial Benign Hypocalciuric Hypercalcemia bers of even these kindreds are generally remarkably free of symptoms. Although nonspecific symptoms encountered in other forms of hypercalcemia, such as fatigue, were reported in some early studies of FBHH, 13 these have not been corroborated in later studies, 2~ perhaps because ascertainment bias attributed symptoms to the disease in probands that were not confirmed in more detailed analyses of entire kindreds. Patients from some kindreds with FBHH have also had pancreatitis and chondrocalcinosis, 13 raising the possibility that these might be true complications of this condition. Pancreatitis, however, does not appear to be present more commonly in affected than in unaffected family members or the general population. Moreover, most patients with FBHH who developed pancreatitis had other known factors predisposing to this complication, such as alcoholism or gallstones. 24 Subsequent studies also have not shown an increased incidence of chondrocalcinosis in FBHH when large numbers of kindreds were examined. 2~ Law and Heath 2~ have described an apparently increased incidence of gallstones in FBHH, but this association has not been emphasized in other clinical series. As far as is known there is no increased incidence of hypertension in FBHH [a complication which does appear to be present with increased frequency in patients with primary hyperparathyroidism (PHPT)22'23], nor is there any documented reduction in the life expectancy of affected members of FBHH families. The degree of hypercalcemia found in patients with FBHH is similar to that seen in those with PHPT of mild to moderate severity. Affected individuals generally have some reduction in serum phosphate concentration, although to a lesser extent than those with PHPT. 13'2~Serum magnesium levels are often in the upper part of the normal range, and mild hypermagnesemia may be present in some cases in contrast to patients with PHPT, whose magnesium concentrations tend to be in the lower half of the normal range. ~]'25 In addition, most patients with FBHH exhibit inappropriately "normal" circulating levels of PTH despite their hypercalcemia, suggesting abnormal regulation of parathyroid function by Ca2+o . 2 4 With the recently developed immunoradiometric assays, which measure predominantly the intact, biologically active form of the hormone, PTH levels in affected 26 27 family members are often midnormal ' and may in some cases be in the lower part of the normal range, although occasional patients may have elevated PTH levels. 2s While most patients with PHPT have frankly elevated circulating levels of intact PTH, it is sometimes difficult to differentiate patients with FBHH from those with mild PHPT, particularly in the 5% to 10% of hyperparathyroid individuals with levels of PTH within the upper part of the normal rangeY Patients with FBHH
48 1 also exhibit abnormal PTH dynamics when administered agents lowering or raising the serum ionized calcium concentration, exhibiting an increase in "set-point" (the level of Ca2o+ half-maximally lowering circulating PTH levels). 28"29 That is, to suppress the PTH level to any given extent, these individuals require a serum calcium concentration slightly higher than that necessary to achieve a comparable degree of reduction in normal subjects. These findings further supported the notion that the parathyroid glands of patients with FBHH have a defect in Cao2+-sensing, 9 exhibiting mild to moderate resistance to the inhibitory effects of extracellular calcium on PTH secretion. A similar defect is present in many patients with PHPT, with only occasional patients having true autonomy of PTH secretion (e.g., PTH secretion that is totally nonsuppressible even with large increases in Cao2+). 3~ Patients with PHPT do tend to have additional defects in secretory control for PTH, however, showing increases in both maximal secretory rates at low levels of Ca 2+o and in the minimal, nonsuppressible secretory rate at high Ca2o+. 29'3~ In FBHH, however, resected parathyroid glands have most commonly exhibited only very mild parathyroid hyperplasia or have been classified as normal. 31'32 Serum levels of 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] are generally within the normal range in FBHH, 33-35 and intestinal ab-
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Bone mineral densities of the lumber spine ( L 1 - L 4 ) , mid radius, and distal radius in 16 women (shown as triangles) and 15 men (shown as circles) from 14 families with FBHH. The pooled results are shown by the brackets as mean ___ SEM and are shown in comparison to their standard deviations above or below age- and sex-specific mean values. Note that values of - 2 and + 2 show the 95% confidence limits of normal bone mineral density. [From Law WJ, Heath H III: Familial benign hypercalcemia (hypocalciuric hypercalcemia). Clinical and pathogenic studies in 21 families. Ann Intern Med 105:511-519, 1985.]
482
EDWARD M. BROWN, MEI BAI, AND MARTIN POLLACK
sorption of calcium is normal or slightly reduced. Interestingly, some individuals with FBHH reported by Heath et al. exhibited normal gastrointestinal Ca 2§ absorption and 1,25(OH)2D levels despite being on a low-calcium diet, suggesting a blunted homeostatic response to reduced dietary calcium. 3s The normal levels of 1,25(OH)2D and of intestinal Ca 2§ absorption in FBHH contrast with the frequently elevated levels of these two parameters in PHPT. 23 Although indices of bone turnover, such as urinary hydroxyproline excretion, may be slightly elevated in patients with F B H H , 36'37 bone mineral density is normal (Fig. 16-1) 20,36,38 and there does not appear to be any increase in the risk of fractures or other complications of bone disease. In a recently described kindred from Oklahoma, several family members had radiological evidence of osteomalacia, but this form of bone disease is very uncommon in other FBHH
though occasional families have been described in which some family members have hypercalciuria, 4~ it is currently unclear whether such individuals represent a variant of FBHH or have a coexistent abnormality in renal calcium handling that overrides the hypocalciuric effect of the FBHH gene(s). Of interest, the enhanced renal tubular reabsorption of calcium (but not that of magnesium) persists following induction of hypoparathyroidism by total parathyroidectomy (Fig. 1 6 - 2 B ) . 33'41 This result implies that the abnormal renal handling of calcium is not dependent upon PTH and represents an independent defect in the renal sensing/handling of Ca 2§ One careful study localized the abnormal renal calcium handling to the thick ascending limb of the nephron, 41 while another suggested that it took place in the proximal tubule. 42 Several additional aspects of renal function in individuals with FBHH are also suggestive of altered renal responsiveness to Cao2§ Renal blood flow and glomerular filtration rate, which can each be reduced in hypercalcemic patients, are both normal in FBHH. 13Moreover, patients with this disorder can concentrate their urine normally despite their hypercalcemia, 43 which produces defective maximal urinary concentrating ability in some hypercalcemic patients and overt nephrogenic diabetes insipidus (DI) in a few. 44 Therefore, both the clinical and biochemical features of FBHH suggested that it repre-
k i n d r e d s . 39
A very characteristic feature of FBHH is abnormally avid renal tubular reabsorption of calcium and magnesium despite the hypercalcemia present in these patients (Fig. 16-2A). 19'21 The renal calciurn/creatinine clearance ratio is less than 0.01 in about 80% of patients with FBHH, while it is higher than this in the vast majority of patients with PHPT and markedly so in other hypercalcemic disorders, where PTH is suppressed thereby further reducing renal tubular Ca 2+ reabsorption. A1-
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FIGURE 16--2 Altered renal handling of calcium in patients with FBHH compared to other conditions. A, The urinary calcium to creatinine clearance ratio in patients with FBHH expressed as a function of creatinine clearance relative to the values for patients with typical primary hyperparathyroidism. Note that approximately 80% of patients with FBHH show a clearance ratio of less than 0.01, while only one patient with primary hyperparathyroidism had a value this low. [From Marx SJ, Attie MF, Levine MA, et al: The hypocalciuric or benign variant of familial hypercalcemia: Clinical and biochemical features in fifteen kindreds. Medicine (Baltimore) 60:397-412, 1981.] B, The relationship between serum calcium concentration and urinary calcium excretion in patients with FBHH rendered surgically aparathyroid (closed symbols) compared to those with hypoparathyroidism alone (open symbols). (From Attie M, Gill JJ, Stock J, et al: Urinary calcium excretion in familial hypocalciuric hypercalcemia. J Clin Invest 72:667-676, 1983.)
CHAPTER 16 Familial Benign Hypocalciuric Hypercalcemia sented an inherited defect in the sensing and/or handling of Ca 2§ by parathyroid, kidney, and perhaps other tissues (e.g., there is a lack of the gastrointestinal or mental symptomatology usually associated with hypercalcemia). Prior to the recognition of FBHH as a distinct clinical entity, a number of patients were subjected to partial or total parathyroidectomy in the belief that they had a form of primary parathyroid hyperplasia. Indeed, even following the clinical differentiation of the disorder from primary hyperparathyroidism, patients with FBHH were sometimes confused with those having PHPT. 25 The clinical course of FBHH following attempts at parathyroidectomy was unusual and provided an additional hint that this disorder differed from typical hyperparathyroidism. In those patients undergoing parathyroid surgery, hypercalcemia recurred rapidly (within a few days or weeks) in 21 of 27 patients who underwent from one to four neck explorations; only two were rendered permanently normocalcemic. 24 In contrast, recurrence of hypercalcemia in various forms of primary parathyroid hyperplasia, if it occurs at all, does not take place for several years. Only patients rendered totally aparathyroid avoided recurrence of hypercalcemia (5 of the 27 reported patients described above). Principally because of the benign clinical course of nearly all cases of FBHH, but also because of the difficulty of obtaining a biochemical "cure" (which accomplishes nothing in an asymptomatic patient with FBHH), a consensus has emerged that surgical intervention should be avoided in this condition. 24 2. GENETIC HETEROGENEITY OF FBHH FBHH is inherited as an autosomal dominant disorder, with greater than 90% penetrance. 13'2~ Genetic analysis of four families with FBHH mapped the disease gene to band 3q21-2426 and formally proved that individuals with FBHH represented the heterozygous form of the disorder (e.g., they have one copy of the disease gene). 26 Subsequent studies have confirmed that the great majority of families with FBHH exhibit this same genetic linkage. 45'46 In one family, however, the disorder mapped to chromosome 19, (band 19p13.3),45 confirming the genetic heterogeneity of the clinical syndrome of FBHH. Another kindred from Oklahoma, not linked to either chromosome 3 or 19, has certain atypical features, including osteomalacia in some affected family members as well as a tendency of patients to show progressive increases in serum PTH as they age. 39'47 A severe variety of hyperparathyroidism (NSHPT), which is encountered rarely in newborns from FBHH families, has been shown to represent in a few cases the homozygous form of the disease that is linked to chromosome 3 and will be discussed in detail later (see Section II.B). 46'48
483 3. CLONING AND CHARACTERIZATION OF AN EXTRACELLULAR Ca2+-SENSING RECEPTOR (CAR) An additional line of research directed at understanding the molecular basis of Ca 2§ " o-sensing was being actively pursued during the time that the clinical and biochemical features of FBHH were elucidated. It was recognized that parathyroid cells, calcitonin (CT)secreting C-cells of the thyroid gland, various renal cells, and, indeed, a variety of other cell types respond directly to physiologically relevant changes in Cao2+.49 The mechanism(s) underlying the capacity of various 2+ cells to sense Cao , however, was obscure. The parathyroid cell represents a particularly striking example of 2+ Cao -sensing, responding to even minute (e.g., 2% to 3%) changes in serum ionized calcium concentration with reciprocal, severalfold changes in PTH secretion that restore Ca o2+ to normal by actions on effector tissues (e.g., kidney and bone). 49 A series of studies in the 1980s showed that altering Cao> modulated intracellular second messengers in parathyroid cells in ways that were very similar to those brought about in target tissues by the actions of so-called Cai+-mobilizing hormones on their cell surface, G protein-coupled receptors (e.g., angiotensin II) (for review, see Brown49). For example, elevating Cao2§ produced a transient followed by a sustained increase in the cytosolic Ca 2§ concentration (Ca~)5~ in association with activation of phospholipase C (PLC) 51 and increased turnover of polyphosphoinositides. These data provided indirect evidence for the presence of a CaR on the surface of the parathyroid cell that was coupled to stimulation of PLC. High Cao2+ also evoked a pertussis toxin-sensitive inhibition of adenylate cyclase in bovine parathyroid cells, suggesting that the putative CaR might also be linked to adenylate cyclase in an inhibitory fashion via the inhibitory Gprotein, G~, similar to a number of other GPCRs. 52 Additional tissues respond to Ca2o§ in ways that imply that they too express a similar Cao2+-sensing mechanism. In the kidney, for example, elevating Cao2§ directly inhibits the 1-hydroxylation of 25(OH)D in the proximal tubule 53 in addition to acting indirectly in this regard by reducing PTH secretion (e.g., PTH directly stimulates 1hydroxylation). In addition, in the thick ascending limb (TAL) of Henle's loop, Ca2+o exerts several actions on tubular function of potential relevance to calcium, fluid, and electrolyte metabolism. Elevated levels of C a o2+ inhibit the reabsorption of sodium chloride, 54 which may contribute to the impaired urinary concentrating capacity of some hypercalcemic subjects by reducing the generation of the countercurrent gradient in the renal medulla. 44 Raising the peritubular but not the luminal level of Ca 2+ (or Mg2o+) in perfused segments of the TAL also inhibits the reabsorption of both Ca 2+ and Mg2+, 55 poten-
484 tially providing a mechanism for autoregulating the tubular handling of these ions. There is likewise a pertussis toxin-sensitive inhibition of vasopressin-stimulated cAMP accumulation by high Ca 2+oin the medullary TAL (MTAL) 56"57that is reminiscent of the effect of Ca 2+o on cAMP metabolism in the parathyroid cell. 52 Finally, the reduced vasopressin-mediated urinary concentrating capacity in some hypercalcemic individuals appears to involve an action of Ca 2+ on the collecting duct. 58 These effects of Ca o2+ on renal function as well as others on a variety of other cell types [i.e., osteoclasts, osteoblasts, intestinal cells, and numerous other epithelial cells (for review, see Brown 49) suggested that specific mechanisms for sensing Ca 2+ might be a more widespread property than generally recognized. It proved difficult, however, to obtain more direct evidence for the presence of a CaR. One approach that had proven useful for cloning G protein-coupled receptors that activated PLC was the technique of expression cloning in Xenopus laevis oocytes. This method takes advantage of the fact that injection of mRNA from a cell normally expressing such a receptor enables the oocyte to synthesize the relevant receptor protein and express it on the cell surface. The receptor can then couple to the endogenous, G protein-activated PLC of the oocyte, thereby stimulating PI turnover and elevating Caa when exposed to a receptor agonist. 59 The increase in Cai, in turn, stimulates a Ca2+-activated chloride channel, which provides a readily measurable bioassay that can be used to screen for expression of the receptor by oocytes during the expression cloning process. Both Nemeth and coworkers 6~ and Shoback et al. 61 showed that X. laevis oocytes became responsive to agents thought to activate the putative parathyroid Cao2 + -sensing receptor following injection of the oocytes with mRNA extracted from bovine parathyroid glands. Thus it was likely that the expression cloning approach would be a feasible one for isolating the CaR and characterizing its molecular properties. Brown et al. 16 subsequently used this approach successfully to isolate a single full-length clone of the bovine parathyroid CaR. The 5.3-Kb cDNA, called BoPCaR (bovine parathyroid Ca2+-sensing receptor), encodes a predicted protein of 1085 amino acids similar to that shown in Figure 1 6 - 3 (which is the human homolog of the CaR) with three principal structural domains. The first is a putative, extracellular amino-terminal domain (ECD) of 613-amino-acid residues that contains nine predicted N-linked glycosylation sites and begins with an --~20-residue signal peptide. Recent studies have suggested that polycationic agonists bind, at least in part, to this portion of the CaR 62 as described in more detail later. The second domain of the receptor is a central core with seven membrane-spanning helices indicating that
EDWARD M. BROWN, MEI B AI, AND MARTIN POLLACK
the receptor is a member of the superfamily of G proteincoupled receptors. The last part is a 222-amino-acid, carboxyl-terminal tail that is predicted to be cytoplasmic. Within the C-terminal tail and other intracellular domains of the receptor (e.g., intracellular loops between the membrane-spanning segments) are four predicted protein kinase C (PKC) phosphorylation sites 16 that may contribute to the inhibition of the activation of PLC by high Ca 2§ following treatment of parathyroid cells with activators of P K C . 63-66 Since the cloning of BoPCaR, additional CaRs have been isolated using hybridizationbased strategies from human parathyroid 67 and kidney 68 as well as from rat kidney, 69 brain, 7~ and C-cells. 71 All show greater than 90% identity in their amino acid sequences to BoPCaR and represent the various species homologs of the same ancestral gene. To date no other isoforms of the receptor have been isolated. A structurally unrelated protein with putative Ca o2+-sensing " properties has been cloned independently by two research groups. 72'73 This protein, called megalin by one group in view of its very large size (--~500 kDa), 73 is a member of the LDL receptor superfamily and is heavily expressed in the parathyroid gland, proximal tubule of the kidney, and placenta. Additional studies of the expressed protein will be needed to assess fully whether it actually senses Ca 2+ o a physiologically relevant manner. The CaR activates PLC in a pertussis toxin-insensitive fashion, 74 most likely through a member of the Gq family of G proteins. 75 The most abundant member of this family in bovine parathyroid gland is Gll with lesser amounts of Gq.75 As noted previously, high Ca 2+ also produces a pertussis toxin-sensitive inhibition of cAMP accumulation in parathyroid cells. 52 Rogers et al. have reported in preliminary form that human embryonic kidney (HEK293) cells stably transfected with the CaR also exhibit inhibition of agonist-stimulated cAMP accumulation, suggesting that the CaR also couples to inhibition of adenylate cyclase via Gi .76 Structure/function studies of the CaR are in an early stage. Hammerland et al. have taken advantage of the modest degree of homology between the CaR and metabotropic glutamate receptors (mGluRs) (GPCRs for glutamate, the principal excitatory neurotransmitter in the brain) to construct C a R - m G l u R chimeras in order to determine where agonists interact with these receptors. 62 If the CaR ECD is replaced by that of an mGluR, the chimeric receptor now responds to glutamate as its ligand. Conversely, the chimeric receptor with the CaR ECD responds to Ca2+o as its ligand, albeit with a somewhat reduced apparent affinity. Therefore, the principal determinants of the binding of polycationic ligands to the CaR appear to reside within the large ECD. It has been suggested that agonists interact with regions within the CaR ECD that have a relatively high density of
CHAPTER 16 Familial Benign Hypocalciuric Hypercalcemia
485
FIGURE 16--3 Schematic representation of the proposed topological structure of the extracellular Ca2+-sensing receptor cloned from human parathyroid gland. SP, signal peptide; HS, hyrophobic segment. Also shown are missense and nonsense mutations causing either familial benign hypocalciuric hypercalcemia (FBHH) or autosomal dominant hypocalcemia (ADH), which are indicated using the three-letter amino acid code, with the normal amino acid indicated before and the FBHH or ADH mutation shown after the number of the relevant codon. [Modified from Brown EM, Harris HW Jr, Vassilev PM, Hebert SC: The Biology of the Extracellular Ca2+-Sensing Receptor. In Principles of Bone Biology. Raisz LG, Rodan G, Bilezikian JP (eds): New York, Academic Press, pp 243-262, 1996.]
acidic residues (e.g., glutamates and/or aspartates) reminiscent of those thought to bind Ca 2+ in low-affinity Ca2+-binding proteins, such as calsequestrin. 16 The CaR is expressed in a variety of tissues, including parathyroid gland, C-cells, intestine, lung, a variety of cells within the brain (including the subfornical organ, which is involved in angiotensin I I - m e d i a t e d thirst), and several types of kidney c e l l s . 16'69'7~ Within the kidney, the receptor is expressed most heavily in the CTAL with lower levels of expression in the MTAL, distal convoluted tubule (DCT), and collecting d u c t . 77 The use of anti-CaR antibodies has made it possible to show that the receptor protein is expressed on the basolateral surface of the epithelial cells of the CTAL and on the apical surface of those of the inner medullary collecting duct (IMCD). v7 As noted above, all of these nephron segments are capable of responding directly to Cao2 + . The physiological functions of the receptor in the CTAL and IMCD as well as in the MTAL will be discussed later (see Section I.B.4). Using sensitive techniques [e.g., re-
verse transcriptase polymerase chain reaction (RTPCR)], even lower levels of expression of CaR transcripts can be detected in the glomeruli and proximal convoluted and straight tubules, but the physiological relevance of the receptor in these sites remains to be determined. 77 4. CAR MUTATIONS IN FBHH3q: F B H H AS A SYNDROME OF Ca 2+ "Resistance" o
Since FBHH is a disorder in which there is abnormal Ca2+-sensing by parathyroid, kidney, and probably other tissues, there was an obvious rationale for searching for mutations in the newly cloned CaR. Pollak et al. initially mapped the human homolog of the CaR gene to the long arm of chromosome 3. 48 They then used a ribonuclease A (RNAse A) protection assay to screen for mutations in the human CaR gene in affected members of three unrelated families with FHH linked to the locus on chromosome 3. They identified a single, unique missense mutation in each family (i.e., mutations in which a
486 change in a single nucleotide base substitutes a new amino acid for the one normally coded for) (Fig. 16-3). Moreover, the observed mutations were not found in the genomic DNA of 50 normocalcemic individuals. All three mutations occurred in amino acid residues conserved between the human and bovine receptor genes. Two of the three families exhibited a mutation in the ECD of the CaR [R185Q (inadvertently described as R186E in the original publicationmnote that the numbering for the bovine CaR 48 is one higher than that for the human CaR 78 because of the presence of an extra amino acid in the ECD of the former) and E297K] and might, therefore, interfere with the binding of polycationic ligands to the receptor. The third family harbored a mutation (R795W) within the third intracellular loop, which could be important for binding G protein(s) and initiating signal transduction, as in other GPCRs. 6 Xenopus laevis oocytes injected with synthetic mRNA encoding a CaR engineered to contain the R795W missense mutation exhibited a markedly blunted response to 2+ 3+ Cao , Gdo , and neomycin, providing convincing evidence that this mutation impaired Ca 2+o-sensing." 48 Subsequent studies by several groups have uncovered a variety of additional mutations in the CaR in families with FBHH linked to chromosome 3 (Fig. 1 6 - - 3 ) . 46,79-82 In most cases each family has its own unique mutation. Most are missense mutations that cluster within several discrete regions of the CAR--(1) the first half of the ECD; (2) a region proximal to TM1 (the first membranespanning domain); and (3) the transmembrane domains, extra- and/or intracellular loops of the receptor (Fig. 16-3). In addition, several apparently benign polymorphisms have been described in the C-terminal tail of the CaR that are not associated with any change in Ca 2+ in individuals harboring them and are present in a substantial proportion of the population (--~10% to 30%). 81 There are, however, several additional types of mutations that have been discovered recently. One family has a nonsense mutation in codon 607 (e.g., a point mutation that substitutes a stop codon for the amino acid normally coded for) just proximal to T M 1 . 46 Such a mutation should result in synthesis of a presumptively inactive, truncated receptor that might well be secreted into the extracellular fluid. Another mutation includes both a one-nucleotide deletion and a transversion of an adjacent nucleotide (change from one nucleotide to another) within codon 747, which would be predicted to modify the downstream reading frame leading to premature termination of the protein at a stop codon following residue 776 within the transmembrane domains. 46 One additional type of mutation in a Nova Scotian family is the insertion of a 383-bp Alu repetitive sequence at codon 876. 82 This sequence is in the opposite orientation to the CaR gene and contains an exceptionally
EDWARD M. BROWN, MEI BAI, AND MARTIN POLLACK
long poly-A tract. Stop signals are present in all three reading frames within the Alu sequence, leading to a predicted truncation of the CaR protein following a long stretch of repeated phenylalanines (encoded by the triplet AAA). Thus this mutation would likely result in production of a nonfunctional protein that might well have compromised biological activity and/or fail to reach the
100.
A ----O---- wildtype -" R62M ----42b--- R66C T138M R185Q -* R795W
80. r~
60
'~N 40 .m
N 20
0
10
20
30
40
50
Ca~o (mM) lOO
80 ~
t
60
.---o---
wild type e R62M .----o--- R66C
~
t
I~ ~ / ~ ' ` ` ~ - ~ . ' - - . &
~1
o
z
~ -*i
G143E T138M R185Q E297K
20
0
100
FIGURE 16--4
200 300 Gd3+ o (gM)
400
500
Expression of CaRs bearing FBHH mutations in HEK293 cells. Results indicate the effects of varying levels of CaZo§ (top panel) or the trivalent cation agonist, gadolinium (Gd3o§ (bottom panel), on the cytosolic calcium (Cai) level in HEK293 cells transiently transfected with the wild-type CaR or mutant CaRs bearing the indicated FBHH mutation. Note than CaRs containing the FBHH mutations show varying degrees of increase in ECs0 (the level of Caoz§ or Gd3o§ required to produce a half-maximal increase in Cai) and/or reduction in the maximum level of Ca~ achieved, thereby producing resistance of cells expressing the mutant CaRs in vivo to Ca oz§ and resultant hypercalcemia. [From Bai M, Quinn S, Trivedi S, et al: Expression and characterization of inactivating and activating mutations of the human Ca o2§-sensing " receptor. J Biol Chem 271" 19537-19545, 1996.]
CHAPTER 16 Familial Benign Hypocalciuric Hypercalcemia cell surface. Of interest, the size of the Alu element was found to approximately double in size in a subsequent generation of this family. 83 Despite the multiplicity of mutations identified to date in families with FBHH, only about two thirds of families with the form of the disorder linked to chromosome 3 have identifiable mutations within the coding sequence of the CaR gene or in regions close to the splice junctions of the seven exons of the CaR gene. In the remaining families, there are presumably mutations within introns or within upstream and/or downstream regulatory regions of the gene that interfere with its normal expression. Recent studies have undertaken expression of CaRs engineered to contain these various mutations in mammalian expression systems. 84 Examples of the effects of several of the point mutations on high CaoZ+-evoked increases in the Ca~ in transiently transfected human embryonic kidney (HEK293) cells are illustrated in Figure 16-4. Some of the mutations, such as R185Q and R795W, markedly reduce the apparent affinity and/or maximal activity of the mutated receptor. In other cases, however, such as T138M or R62M, the mutation only modestly reduces apparent affinity, without decreasing the maximal response to Cao2 + .84 Many of the mutant CaRs with the most severe reductions in biological activity show reduced quantities of the putatively mature, glycosylated form of the receptor on Western blot analysis. It is of interest that the elevation in serum calcium concentration in such families is generally mild. Indeed, the family with the nonsense mutation at codon 607 has very mild hypercalcemia 46 and, as described in greater detail below, mice with targeted deletion of one allele of the CaR also have mild hypercalcemia. 85 In contrast, mutations that severely impair the biological function of the receptor despite showing an essentially normal pattern on Western blot analysis can be associated with more severe hypercalcemia. This latter circumstance is exemplified by the families with the R795W or R185Q mutations. 84 In these two families, serum calcium concentrations average over 2 mg/dl and 3 mg/dl higher than the levels in unaffected family members, respectively. It seems likely that these two mutated receptors interfere in some way with the function of the normal receptor. This could conceivably occur through: (1) a reduction in the quantity of normal CaR reaching the cell surface; (2) formation of an inactive complex of mutant CaR with G protein(s), thereby decreasing the concentration of Gprotein available to the wild type receptor; and/or (3) formation of inactive complexes of normal and mutant receptors on the cell surface. Expression by transient transfection has also been performed of two mutant receptors containing the deletion and transversion in codon 74785a as well as the insertion of the Alu element in the region of the CaR gene encoding the carboxyl-terminal
487 tail of the CaR. 85b Both were totally nonfunctional and encoded truncated proteins on Western blot analysis that corresponded closely in their sizes to those predicted from the premature stop codons introduced by the mutations. Although the level of expression of the normal CaR in parathyroid and kidney has not been determined in FBHH, the development of mice with targeted deletion of the CaR (see Section II.B.5) has made it possible to carry out similar studies in an animal model of FBHH. In mice heterozygous for knockout of the CaR, the level of receptor protein in the parathyroid and kidney as assessed by immunocytochemistry and Western blot analysis, respectively, is approximately one half of that in the corresponding tissues from the wild-type mice. 85 It appears, therefore, that there is little up-regulation of expression of the CaR protein produced from the remaining normal allele and that a 50% reduction in receptor protein translates into a 10% to 20% increase in the setpoint for Ca2+-regulated PTH release in vivo. A further reduction in the level and/or function of the normal receptor on the cell surface produces a greater shift in setpoint, as in the cases of the families with the R185Q and R795W mutations. A related situation may exist in PHPT, where no point mutations could be found in the CaR gene, 86 but the intensity of CaR immunoreactivity was decreased in parathyroid adenomas relative to normal glands from the same patients. 87 The reduced immunostaining suggested a reduced level of expression of an otherwise normal CaR protein (at least in terms of its deduced amino acid sequence), thereby causing an increase in set-point for CaoZ+-regulated secretion. Of interest, the decrease in the intensity of CaR immunostaining averaged 60%, which might predict a slightly greater increase in set-point in patients with a parathyroid adenoma than in persons with FBHH, which is, in fact, the case. While much additional work is required to elucidate the structure/function relationships for the CaR, the following tentative conclusions can be drawn from the studies just described. Some FBHH mutations within the ECD produce a spectrum of alterations in the apparent affinity of the CaR for Ca 2+ (as well as for Gd3o+) without apparent changes in the level of expression of the mature protein, at least as assessed by Western blot analysis. 84 Therefore, the altered amino acid might be either directly or indirectly involved in determining the binding properties of the CaR for polycationic ligands. It seems unlikely, however, that the residues in question actually interact directly with the ligands, as in many cases the mutations are often associated with a net loss of positive charge (e.g., R62M). They might, on the other hand, determine more general features of the conformation of the ECD that, in turn, affect affinity for ligands. Even-
488 tually solution of the three-dimensional structure of the ECD will be needed to determine the precise nature of the interactions of the protein with its polycationic ligands. It seems likely that there is more than one binding site for Ca 2§ on the CaR. The Hill coefficient for activation of the transiently expressed CaR by Cao2§ is around 3, implying the presence of at least three interacting binding sites. 84 Conversely, the Hill coefficient for the activation of the receptor by Gd3o§ is about 1, raising the possibility that trivalent cations interact with the CaR in a manner that differs from that for the physiological ligand, Cao2+. Of interest in this regard, Hammerland et al., 62 in addition to studying a chimeric, C a R - m G l u R construct, also investigated a receptor engineered to lack essentially the entire ECD. This deletion mutant, while no longer activated by Ca o2+, still showed a relatively robust response to Gd3o§ albeit with a somewhat reduced apparent affinity. This result raises the possibility that there could be a site that binds Gdo3+, but perhaps not Cao2+ , within the extracellular loops or even TMDs of the CaR or that binding of Cao2§ to this site alone is not sufficient to initiate signal transduction. These observations with the CaR deletion mutant may also explain why CaRs harboring some FBHH mutations show no response to Ca 2§ but are activated reasonably well by Gd3o+84; perhaps the structure of the ECD in these mutated receptors has been severely compromised but a functional Gd3+o finding site remains intact. The mutations within the transmembrane domains of the CaR presumably disrupt conformational changes of the TMDs attendant upon the binding of ligand to the ECD that are required to transduce signals intracellularly to the site(s) of interactions of the receptor with G protein(s), most likely the intracellular loops (ICL). As noted previously, the one FBHH mutation described to date within an ICL p e r s e (R795W, within the third ICL), markedly impairs activation of PLC, presumably by interfering with this coupling to and/or activation of Gq or Gl1.48 While the third ICL plays a critical loop in determining the specificity of the coupling of many GPCRs to their G proteins, 4'5 recent studies have shown that for the homologous mGluRs, which couple to G proteins similar to those utilized by the CaR, the second loop may confer specificity for activation of Gi or Gq.88 Nevertheless, available data suggest that G proteins interact with multiple (probably all three) ICLs in GPCRs and likely with the carboxyl-terminal tail as well. 4-6 Therefore, it is not surprising that the substitution of a bulky hydrophobic residue for the basic arginine normally present at residue 795 of the CaR will interfere with the coupling of the receptor to its intracellular signal transduction system. Thus the spectrum of mutations encountered to date in FBHH has already provided useful clues
EDWARD M. BROWN, MEI BAI, AND MARTIN POLLACK
into structure/function relationships for the receptor. Moreover, it provides the basis for a targeted approach to elucidating these relationships further using sitedirected mutagenesis. Based on these results, we conclude the following: (1) FBHH is genotypically and phenotypically heterogeneous, but more than 90% of families likely have the form of the disorder linked to chromosome 3. (2) About two thirds of all individuals with FBHH linked to chromosome 3q show inactivating mutations within the coding region of the recently cloned CaR, and each family generally has its own unique mutation. The majority of mutations occur within the ECD and may reduce the affinity of the receptor for extracellular Ca 2§ or interfere with its biosynthesis or expression on the cell surface. Some occur in transmembrane or cytoplasmic domains and may disrupt processes involved in signal transduction. All likely impair the responsiveness of the CaR to Cao2+ , rendering patients with FBHH mildly to moderately "resistant" to the extracellular Ca 2§ signal. (3) Of the remaining one third of individuals with FBHH linked to the CaR gene, there are no detectable mutations in the coding region. These individuals may have defects in promoter or enhancer sequences of the CaR gene, which have not yet been characterized. (4) In occasional families the defect maps to other, as yet undefined genes either on chromosome 19p or elsewhere. (5) Because patients with FBHH have a generally benign clinical course, parathyroidectomy should not be performed except in very unusual clinical circumstances.
B. N e o n a t a l S e v e r e H y p e r p a r a t h y r o i d i s m 1. CLINICAL AND B I O C H E M I C A L FEATURES OF N S H P T
Neonatal primary hyperparathyroidism often presents dramatically as severe, symptomatic hypercalcemia with hyperparathyroid bone disease in infants under the age of 6 months. 24 This syndrome was actually described well before FBHH was recognized as an entity distinct from other forms of PTH-dependent hypercalcemia. 89'9~ A recent review of 49 cases of NSHPT found that most presented at birth or shortly thereafter, generally within the first week of life. 24 Common presenting symptoms include failure to thrive, anorexia, constipation, hypotonia, and respiratory distress. Additional manifestations comprise deformity of the chest wall and, less commonly, craniotabes, dysmorphic facies, and anovaginal or rectovaginal fistulas. 14"91-98 Respiratory complications may arise owing to thoracic deformity, including an actual flail chest syndrome because of multiple rib fractures, and are a major cause of morbidity. 24'91
CHAPTER 16 Familial Benign Hypocalciuric Hypercalcemia The hypercalcemia in NSHPT is typically severe, ranging from 14 to 20 mg/dl, and levels as high as 30.8 mg/dl have been described. 93 Interestingly, relative hypocalciuria has been documented in some cases, even in the absence of a family history of FBHH. 99 Although serum magnesium concentrations were not reported in many of the cases, when measured they have sometimes been elevated well above the upper limit of normal even when renal function was not compromised. 79 Serum PTH levels have been high in the majority of cases in which they were measured, often being increased five- to tenfold, although the degree of elevation can be mild in s o m e c a s e s . 99-1~ Skeletal radiographs show marked undermineralization, with fractures of the ribs and long bones, subperiosteal erosions, metaphyseal widening, and, occasionally, rickets. 91"1~ Histological examination of bone may show typical osteitis fibrosa cystica. TM At the time of parathyroidectomy, all four parathyroid glands are usually enlarged, sometimes being many times the mass of normal parathyroid glands in infants of this age. Histological examination shows chief cell or water-clear cell hyperplasia, although in cases where the glandular enlargement is less marked, the normal lack of fat in the parathyroid glands of children complicates interpretation of the histological findings. 14'24'1~ There have been no cases described in which a parathyroid adenoma caused NSHPT. There is, however, a relatively broad range of clinical severity encountered in NSHPT. In some cases, the hypercalcemia is less marked, in the range of 11 to 12 mg/dl, and cases have been reported where the disease ran a self-limited course, reverting to milder hypercalcemia by the age of 6 to 7 months with conservative treatment. 91'1~ The recent advent of techniques for identifying cases of NSHPT due to mutations in the CaR will doubtless widen the spectrum of the disease further. A particularly instructive case was recently identified of a homozygous female offspring of a consanguineous marriage of two individuals with heterozygous FHH whose serum calcium concentrations were in the upper part of the normal range. 79 This homozygous child did not present as NSHPT and was not diagnosed until age 32, when she was identified by mutational analysis. She was essentially asymptomatic, although she was mildly retarded, despite serum calcium concentrations of 15 to 17 mg/dl, accompanied by a serum magnesium concentration elevated to a similar extent (--~50% above the upper limit of the normal range) and a serum intact PTH level at the upper limit of normal. In spite of the degree of elevation of her serum calcium concentration, her renal function was normal, including her renal concentrating ability. Prior to 1982, 24 NSHPT was often fatal in severe cases if aggressive medical and surgical management
489 was delayed. As just noted, however, this is not invariably true in more recent cases, and wider recognition of the full clinical spectrum of the disorder as well as improvements in the medical therapy of severe hypercalcemia have permitted successful expectant medical management of NSHPT during the past 15 years. In symptomatic cases, initial management should include hydration and aggressive respiratory support. If the clinical condition is very severe or deteriorates during treatment, total parathyroidectomy with autotransplantation of part of one of the glands is recommended during the first month of l i f e . 24'1~176176Some authors recommend total parathyroidectomy with lifelong medical management of the resultant hypoparathyroidism. ~4'~~ The latter includes oral calcium supplementation and sufficient vitamin D replacement, generally with 1,25(OH)2D, to maintain low normal serum calcium concentrations. Because patients with CaR mutations would be expected to have relative hypocalciuria, the dose of vitamin D required for replacement is generally lower than needed for individuals with congenital or acquired primary hypoparathyroidism. There is dramatic clinical improvement following parathyroidectomy, with rapid healing of the skeletal abnormalities, even though the hypercalcemia usually recurs rapidly with less than total parathyroidectomy or following autotransplantation. ~4'24Indeed, during the past 15 years, similar clinical improvement has also been observed in infants with NSHPT managed medically. 24
2. G e n e t i c s o f N S H P T Since the first descriptions of FBHH, the presence of children with NSHPT in FBHH families has been noted by several investigators. 13'14'94'1~ In 1981, Marx et al. 13 reported their studies of 15 kindreds with FBHH, in which they found three patients from two families with neonatal severe hyperparathyroidism. One explanation for this association that they suggested was that NSHPT can in some cases be the homozygous form of FBHH. These investigators subsequently studied a family with two children affected by NSHPT. Their parents, who were related, had mild increases in serum ionized calcium concentration (total calcium levels were normal) as well as hypocalciuria. Additional family members had borderline and, in some cases, intermittent hypercalcemia. 1~ These clinical findings supported the hypothesis that NSHPT can be the homozygous form of FBHH and that apparently sporadic occurrences of NSHPT can result from the failure to recognize very mild hypercalcemia in family members who are heterozygous for the abnormal gene. (Indeed, the alteration in Cao2 + -sensing by the mutant CaR might in some cases be so mild that
490 overt hypercalcemia is not present, as in the recently reported Japanese family, 79 and that there is simply a subtle increase in serum calcium concentration to a level that is higher than in unaffected family members but remains within the normal range.) After localization of the FBHH gene to chromosome 3q in several families, 26 Pollak and co-workers investigated 11 FBHH families and showed that the abnormal gene mapped to chromosome 3q2 in all of them. 1~ Four families had consanguineous marriages of affected individuals resulting in children with NSHPT. The pattern of inheritance of genetic markers closely linked to the FBHH gene in these families was consistent with NSHPT being the homozygous form of the same disorder. Subsequent studies of the CaR gene in additional families in which both FBHH and NSHPT coexisted confirmed that inheriting two abnormal copies of the CaR gene can cause NSHPT. 48'8~ These patients do not have any normal CaRs, and as a result exhibit much more severe hypercalcemia than do patients with FBHH, with greater elevations in PTH, substantial parathyroid glandular enlargement, and markedly abnormal set-points for calcium-regulated PTH secretion. All of these findings indicate a state of severe parathyroid resistance to Ca 2+ in NSHPT, although if the defect in the FBHH gene is sufficiently mild, as in the Japanese family described above, NSHPT can escape detection in the neonatal period and be compatible with life. 3. MUTATIONS IN THE C A R IN N S H P T : HOMOZYGOUS VERSUS DE NOVO HETEROZYGOUS MUTATIONS
It is now established, as described in more detail below, that not all cases of NSHPT represent homozygous FBHH. Indeed, most cases of NSHPT have been reported to occur sporadically or in FBHH families with only one affected parent. 97"1~176 T h e latter situation might conceivably arise from the child with NSHPT being a compound heterozygote, harboring two CaR alleles, each with a with distinct mutation, one producing obvious hypercalcemia in one parent, but the other being sufficiently mild so as not to be biochemically detectable in the other parent. Alternatively, there might be a mutation in one allele of the CaR gene as well as in one allele of one of the other genes causing an FBHH-like clinical picture, including the one on chromosome 19p 45 as well as that present on neither chromosome 3 nor 19. 47 While the latter two circumstances have not been documented to date, the sizable proportion of FHH3q families without detectable mutations in the coding region of the CaR gene, as well as our ignorance concerning other genes causing FBHH, would make it difficult to exclude these scenarios at present.
EDWARD M. BROWN, MEI BAI, AND MARTIN POLLACK
Recent studies have documented the occurrence of clinically severe, neonatal hyperparathyroidism resulting from heterozygous d e n o v o CaR mutations in two sporadic cases 46 (i.e., caused by a single d e n o v o CaR mutation in the child of normal parents). Both infants had evidence of hyperparathyroid bone disease, although they had less severe hypercalcemia than is seen in NSHPT due to homozygous FHH. This may provide an explanation for the observed clinical spectrum in NSHPT, which ranges from the typically severe, lifethreatening manifestations (in patients homozygous for mutated CaR) to a more benign form of the disorder (i.e., in patients with d e n o v o heterozygous CaR mutations). Another possible explanation for the presence of a child with NSHPT whose father has FBHH but whose mother is unaffected would be the impact of the normal maternal calcium concentration on the abnormal Cao-2+ sensing of the parathyroid glands of the affected fetus in u t e r o . 1~ Calcium is actively transported across the placenta from mother to fetus, resulting in a higher fetal than maternal concentration of calcium. 1~ Therefore, a normal mother would expose fetal parathyroid glands with even mildly abnormal Ca2+-sensingo due to FBHH to a level of Ca 2+o that would be recognized as relatively hypocalcemic compared to that experienced by normal fetal parathyroid glands. This relative hypocalcemia would be expected to "overstimulate" the fetal parathyroids leading to the superimposition of " s e c o n d a r y " fetal/neonatal hyperparathyroidism upon the abnormal Ca2+-sensing already present due to FBHH. This hypothesis is supported by the observation that cases of NSHPT with autosomal dominant inheritance have been reported in cases where the father had FBHH while the mother was apparently n o r m a l . 14'24'25 With time postnatally, the secondary component of hyperparathyroidism would subside, causing eventual reversion to the clinical and biochemical features expected of FBHH. In most cases, however, it has not been apparent that children with FBHH who were born of a normal mother had more severe hypercalcemia than those born of an affected mother. Furthermore, there are no apparent phenotypic or biochemical differences between mice heterozygous for knockout of the CaR that are born to a normal mother or one who is heterozygous for CaR knockout. 85 Of interest, we recently documented another case of d e n o v o heterozygous NSHPT in a child harboring the same R185Q mutation described above that is associated with an elevation in serum calcium that is greater than observed in most cases of FBHH. 1~ In this case, the relatively large disparity between the set-points of the maternal and fetal parathyroid glands may have contributed to a greater degree of prenatal hyperparathyroidism and resultant hyperparathyroid bone disease in the fetus.
CHAPTER 16 Familial Benign Hypocalciuric Hypercalcemia These studies on NSHPT, which in the homozygous cases is a knockout of the human CaR, suggest the following perspectives on the function of the receptor in humans: (1) They highlight its importance in fetal and neonatal calcium metabolism; and (2) they also suggest a possible role for the CaR in the tonic inhibition of parathyroid cellular proliferation, since there is substantial parathyroid hyperplasia in NSHPT. 4. DEVELOPMENT OF MICE WITH TARGETED
DELETION OF THE CAR GENE Recently, Ho et al. have used the genetic technique of targeted disruption of the CaR gene to produce mice heterozygous or homozygous for deletion or knockout of the CaR gene that represent animal models of FBHH and NSHPT, respectively. These investigators introduced DNA coding for the neomycin resistance gene into the third exon of the CaR gene, with resultant absence of detectable CaR protein in the parathyroid and kidney of mice homozygous for CaR knockout and levels of the protein that were reduced by ---50% in the heterozygous mice. 8s Phenotypically, the heterozygous mice cannot be readily distinguished from their normal littermates, are fertile, and appear to have a normal lifespan. Their serum calcium concentrations (averaging 10.4 mg/dl) are about 10% higher than normal and they had magnesium levels that were likewise modestly but significantly higher than those of normal mice. Serum levels of PTH were about 50% higher than in the normal mice, and the calcium concentration in bladder urine was slightly lower than in the normal mice. Skeletal x-rays were not detectably different from those of the normal littermates. Thus the mice heterozygous for knockout of the CaR gene mimic in many ways the phenotypic and biochemical features of FBHH. The homozygous CaR knockout mice, in contrast, while of nearly normal size at birth, grow much more slowly than their normal or heterozygous littermates, 85 perhaps in part because they compete poorly for maternal milk with their more vigorous littermates. They are severely hypercalcemic, averaging 14.8 mg/dl with magnesium levels that were slightly but not significantly higher than those of the heterozygous mice. Serum PTH levels were nearly tenfold higher than the mean value of the normal mice, and the calcium concentration in bladder urine was lower than that of the normal mice despite the severe hypercalcemia. 85 Skeletal x-rays showed substantially reduced mineral density, kyphoscoliosis, and bowing of the long bones. Most of the homozygous mice died during the first 2 weeks of life, with only occasional ones surviving up to 3 or 4 weeks of age. Again, therefore, the clinical and biochemical abnormalities in mice homozygous for CaR knockout show many similarities to NSHPT in humans. While relatively little work has
491 been done using this animal model to study abnormalities in Ca2+-sensing in tissues normally expressing the CaR, these animal models of FBHH and NSHPT will likely be very useful for performing in vitro studies that are impossible in the human diseases. It is of interest, as noted before, that despite the complete lack of CaR production from one allele encoding the gene, there appears to be little, if any, up-regulation of production of the CaR protein from the remaining normal gene, as the levels of expression of the protein in parathyroid and kidney are approximately half of their normal levels. 85 In addition, this ---50% reduction in expression of the CaR protein in parathyroid is associated with a mild, --~10% increase in the apparent set-point of the parathyroid gland, consistent with the somewhat greater reduction in CaR immunoreactivity in parathyroid adenomas, which exhibit a somewhat greater increase in set-point in vitro. 87 Finally, most families have a degree of hypercalcemia that is comparable to that of mice heterozygous for knockout of the CaR, suggesting that the abnormal CaR allele in many cases acts as a null mutation (i.e., as if it were totally nonfunctional). 13'2~ In occasional families (e.g., those with R185Q or R795W), however, the abnormal protein appears to interfere in some fashion with the normal CaR, resulting in greater degree of elevation of Ca 2+. 5. IMPLICATIONS OF C A R MUTATIONS IN FBHH FOR UNDERSTANDING THE ROLE OF THE C A R IN THE
REGULATION OF PARATHYROID AND RENAL FUNCTION BY Ca2o§
FBHH3q and most cases of NSHPT represent, in effect, experiments in nature where there are reductions in the biological activity of the CaR. Therefore, examination of the alterations in the effects of Ca o2+ on various aspects of parathyroid and renal function in these patients provide useful clues into the normal role of the receptor in regulating these two tissues. Coupled with the recent development of mice with targeted deletion of the CaR gene, the tools are now in hand to elucidate which of the previously poorly characterized effects of Cao2§ on the function of various tissues are CaR mediated. a. The CaR a n d Ca o2 + -Regulated P a r a t h y r o i d Function.
As described previously, persons with FBHH have a modest (10% to 20%) increase in their set-point for 2+ Cao-regulated PTH secretion, 28'29 while parathyroid glands from two individuals with NSHPT that were studied in vitro showed considerably more severe resistance to Ca 2+o (the set-point was elevated by two- to fourfold or more). 1~176 Similarly, mice that are heterozygous or homozygous for knockout of the CaR gene exhibit mild and marked elevations, respectively, in both serum cal-
492 cium concentration and PTH. 85 Thus, the abnormalities in Ca2+-regulated PTH secretion in FBHH and NSHPT as well as in mice with deletion of one or both alleles of the CaR gene offer strong support for the CaR as a key mediator of the inhibitory effect of Ca 2+ on PTH secretion. Recent studies also suggest that the receptor is responsible for the high CaZ+-evoked suppression of PTH gene expression. ~1~Finally, the marked parathyroid cellular hyperplasia in NSHPT as well as in mice homozygous for targeted deletion of the CaR gene suggests that the CaR could also act to suppress parathyroid cellular proliferation. It remains to be determined whether the CaR is also responsible for additional effects of Ca 2+ on parathyroid function (for review, see Brown49), such as changes in cellular respiration, the activity of the hexose monophosphate shunt, and the intracellular degradation of PTH.
b. The CaR and CaZ+-Regulation of the Function of the TAL. As discussed above, FBHH is characterized by an inappropriate degree of reabsorption of calcium ions by the kidney given the ambient hypercalcemia, which persists even after parathyroidectomy. 33'4~ Studies by Attic and co-workers suggested that this abnormality in renal calcium handling takes place in the TAL, since individuals with FBHH showed an exaggerated calciuric response to administration of the loop diuretic, ethacrynic acid. 4~ The CaR is present in cells of the TAL based on studies using in situ hybridization, immunohistochemistry and RT-PCR. 77 Moreover, increases in the peritubular, but not luminal levels of Ca 2+ to which the TAL is exposed have been shown to inhibit reabsorption of calcium ions. 55 Recent studies suggest that Ca 2+ acts, at least in part, by inhibiting the activity of a potassium channel in the luminal membrane, whose activity is necessary to "recycle" potassium ions transported intracellularly by the apical Na+-K+-2C1 - co-transporter. 1~ The activity of the co-transporter, coupled with K + recycling, generates a lumen-positive potential gradient that drives passive, paracellular transport of Na +, Ca 2+, and Mg2+. 112 PTH stimulates this lumen-positive potential by a cAMP-dependent activation of the co-transporter. Following inhibition of the K + channel, there is an attendant reduction in net co-transporter activity because of the decrease in luminal K + concentration, thereby reducing the lumen-positive potential and divalent cation reabsorption. In effect, high Ca 2+ acts as a loop diuretic through its action on the overall activity of the co-transporter. Therefore, the alterations in the tubular handling of calcium in FBHH 4~ provide additional evidence that 2+ the CaR is responsible for high Cao -mediated modulation of Ca 2+ reabsorption by this segment of the nephron. In effect, the TAL, like the parathyroid cell, is "resistant" to Cao2+ in persons with FBHH, limiting
EDWARD M. BROWN, MEI BAI, AND MARTIN POLLACK
their ability to up-regulate urinary excretion of calcium in the presence of an elevated level of Cao2+, which, in turn, contributes to the maintenance of their hypercalcemia.
c. The CaR and the Control of Mg z+o Homeostasis. Individuals with FBHH can exhibit mild hypermagnesemia due, in large part, to excessive magnesium reabsorption in the distal nephron. 13 Moreover, in some individuals with NSHPT, Mg 2+o levels have been elevated roughly in proportion to the concomitant elevation in serum calcium concentration, raising the possibility that the CaR contributes to the "setting" of Mg2+. 79 Additional support for this hypothesis has come from the elevated serum levels of Mgo2+ in mice that are heterozygous or homozygous for knockout of the CaR gene, although the Mgo2+ level in the homozygotes was only slightly and not statistically significantly higher than in the heterozygotes. 85 What are the mechanisms through which the CaR could contribute to regulation of the serum Mg 2+ concentration? Previous in vitro studies had shown that elevated peritubular levels of not only Ca2+o but also Mg 2+o inhibit the reabsorption of both of these divalent cations in the TAL. 55 Moreover, both the cloned parathyroid 16 and renal 69 CaRs respond to Mg 2+ when expressed in X. laevis oocytes, although the potency of Mg o2+ is about two- to threefold less than that of Cao2+. The concentration of Mgoz+ in the TAL, however, is higher than in the initial glomerular filtrate, since relatively little Mg 2+ is reabsorbed prior to this segment of the nephron. 55 Perhaps the local Mg2o+ concentrations achieved in the TAL are sufficient to be sensed by the CaR, which could thereby play an important, although as yet unproven, role in the regulation of renal Mg 2+ reabsorption. In FBHH, the presence of abnormal CaRs with reduced activity leads to excessive Mg 2+ reabsorption and consequent hypermagnesemia. 13'2~However, unlike Ca 2+ reabsorption, the presence of PTH may be necessary for this inappropriate Mg 2+ reabsorption in FBHH, as this abnormality does not persist after parathyroidectomy. 4~ d. The CaR and Urinary Concentrating Ability. A long-recognized but poorly understood clinical consequence of hypercalcemia is a reduction in maximal uri' nary concentrating capacity, resulting in hyposthenuria and, in some cases, frank nephrogenic diabetes insipidus (NDI). 44 Studies in vivo have suggested that two principal factors contribute to this defect in urinary concentration: (1) Elevations in Caoz+ reduce NaC1 absorption in the thick ascending limb, 54 a site where the CaR is located on the basolateral side of the tubular epithelium. 77
CHAPTER 16 Familial Benign Hypocalciuric Hypercalcemia Reabsorption of NaC1 in the TAL contributes to generation of the countercurrent gradient that is required for vasopressin-elicited water flow. (2) Hypercalcemia also inhibits vasopressin action on water reabsorption in the collecting duct. 58 We recently demonstrated that the CaR is located on the apical surface of the tubular epithelium of the collecting ducts, particularly in the IMCD and papillary collecting ducts, where urinary concentration normally increases markedly during dehydration owing to the accompanying increase in circulating levels of vasopressin. 113 Individuals with FBHH show an alteration in maximal urinary concentration that provides additional indirect support for a role of the CaR in regulating water handing by the kidney. Unlike most hypercalcemic patients, persons with FBHH concentrate their urine normally despite their hypercalcemia. 43 This might occur because of deranged Ca2+-sensing in either MTAL or collecting duct as a result of reduced activity of mutant CaRs. Impaired Ca2+-sensingo in the MTAL could diminish the inhibitory action of hypercalcemia on NaC1 reabsorption and consequent generation of the medullary countercurrent gradient. Moreover, the presence of CaRs with reduced activity in the collecting duct could block the inhibitory action of hypercalcemia on vasopressin action in this nephron segment, likewise mitigating the development of NDI. In normal individuals, excreting a dilute urine during hypercalciuria could be a protective mechanism that reduces the risk of developing kidney stones and/or nephrocalcinosis when a concentrated urine is being elaborated because of dehydration. 112
C. Diagnostic and Therapeutic Implications of CaR Mutations Detection of mutations in the CaR in patients with sporadic asymptomatic hypercalcemia could clearly be helpful in diagnosing FBHH, although the size of the CaR coding sequence makes this a substantial undertaking if direct sequencing were performed. More rapid screening procedures, such as denaturing gradient gel electrophoresis or the use of RNase protection, could facilitate the process of screening for point mutations. 46'48 A negative screen for mutations is not of much value, however, as not all FBHH patients show CaR mutations, even when the disorder is linked to the chromosome 3 locus. Therefore, the diagnosis of FBHH will likely continue to be established by the traditional approach of documenting an autosomal dominant inheritance of asymptomatic hypercalcemia in family members other than the proband that is accompanied by relative hypocalciuria (calciurn/creatinine clearance ratio of < 0.01). In our experience, it is not uncommon for patients with
493 mild hyperparathyroidism to restrict their calcium intake voluntarily to the point where separation of their clearance ratios from those encountered in FBHH patients can be problematic. In this situation, particularly when firstdegree relatives are not readily available for family screening, dietary supplementation to a total of 1000 mg of elemental calcium daily can be very helpful, as it will usually increase urinary calcium excretion in patients with true primary hyperparathyroidism well above the levels seen with FBHH. Other causes of apparent hypocalciuria in otherwise typical primary hyperparathyroidism are vitamin D deficiency, the use of hypocalciuric agents such as lithium or thiazides and, occasionally, hypothyroidism. Screening of as many family members as possible of patients with a provisional diagnosis of FBHH for hypercalcemia is important, since even borderline hypercalcemic patients may harbor mutations. Mutational screening of the spouse of a hypercalcemic patient could potentially be of value in predicting risk for NSHPT in the offspring. In conjunction with family screening for hypercalcemia, mutational analysis could also be useful for the diagnosis of cases presenting with hyperparathyroidism in the neonatal period, especially in the absence of a family history of hypercalcemia. Finally, it will be of great interest to determine the nature of the additional gene defects that can give a clinical picture similar to the seen with FBHH3q. It is not currently known whether these represent additional components within the signal transductional pathway(s) utilized by the CaR, additional forms of CaR, or additional mechanisms through which parathyroid and kidney cells sense Cao2 + .
III. AUTOSOMAL DOMINANT HYPOCALCEMIA---A SYNDROME OF INCREASED RESPONSIVENESS OF TARGET TISSUES TO Ca2o§
A. The Spectrum of Inherited Hypocalcemias Familial isolated hypoparathyroidism is a rare disorder that occurs in autosomal dominant, autosomal recessive, or X-linked forms. TM The molecular basis for the autosomal recessive and X-linked forms of the disorder have not been defined. In a recent study of eight families with a diagnosis of autosomal dominant hypoparathyroidism, two were found to be linked to the gene for parathyroid hormone. 115 One was subsequently found to result from a mutation in the signal peptide-encoding region of the preproPTH gene. 116 Another family has subsequently been identified with a mutation in a splice
494
EDWARD M. BROWN, MEI BAI, AND MARTIN POLLACK
junction within the preproPTH gene. 117 The molecular basis for the disorder in other families with inherited forms of hypocalcemia, however, have been, until recently, obscure. The cloning of the CaR and the subsequent identification of inactivating mutations in the CaR gene in the autosomal dominant hypercalcemic disorder, FBHH, raised the possibility that an analogous, autosomal dominant hypocalcemic syndrome might result from activating mutations in the receptor. Subsequent studies described below established that this is indeed the case.
B. Clinical and Biochemical Features of Autosomal Dominant Hypocalcemia ADH has only been recognized as a distinct clinical entity for approximately 2 years, and approximately a dozen families have been described in which the syndrome has been proven to be present on the basis of the mutational analysis described in more detail later. 78"118-12~Nevertheless, the disorder appears to have certain characteristic clinical features that are in many ways what one might have predicted from the effects of activating CaR mutations that, in effect, " r e s e t " downwards the set-points of both the parathyroid and kidney for regulation by Ca 2+. That is, the disorder is the expression of a mild to moderate increase in responsiveness of target tissues to Cao2+ in contrast to the resistance to Ca 2+ present in FBHH. 12 ADH can be viewed, therefore, as "familial benign hypercalciuric hypocalcemia." As additional families are described, we will likely obtain a more detailed picture of the presentation and clinical course of this syndrome. Affected family members with ADH have hypocalcemia of a mild to moderate degree (---6 to 8 mg/dl) with an autosomal dominant pattern of i n h e r i t a n c e . 78'118-121 Individuals with ADH have relatively few symptoms, however, despite their hypocalcemia. Seizures are not uncommon, particularly in younger patients, although in many cases the seizures occur during febrile episodes and are not difficult to control. Other symptoms generally ascribed to hypocalcemia, such as paresthesias, tetany, and laryngospasm appear to be uncommon. Individuals with ADH, like those with classical primary hypoparathyroidism, tend to have hyperphosphatemia. In some kindreds, however, the serum phosphate may be normal in many affected family members, 12~perhaps because of the coexistent, normal levels of PTH (see below). Serum magnesium concentrations are not uncommonly in the lower part of the normal range or even subnormal in the untreated s t a t e . 121 Intact PTH levels are generally in the lower half of the normal range. In one case, further reduction in serum calcium concentration
in a patient with ADH caused a brisk increase in PTH level, suggesting a leftward shift in the set-point for Ca2+-regulated PTH release. ~5 The level of 1,25(OH)2D has been measured in relatively few cases and was generally normal. Urinary calcium excretion has been reported to be significantly higher in the untreated state than in patients with classical hypoparathyroidism, despite the fact that PTH levels are often well below the lower limit of normal in the latter and, therefore, lower than in ADH. 78'121 Careful studies will be needed, how ever, to define the relationship between serum and urinary calcium concentrations in ADH in the same way that this parameter was defined in FBHH and was instrumental in elucidating its pathophysiology. On the basis of the available data, it appears that patients with ADH respond to treatment with vitamin D and its metabolites in a characteristic manner that differs from the response of patients with true hypoparathyroidism. ADH patients seem to be unusually prone to marked hypercalciuria and to renal complications of "overtreatm e n t " with vitamin D, even in the absence of overt, treatment-related hypercalcemia. 121 These untoward effects of vitamin D therapy have included renal stones, nephrocalcinosis, and reversible (and even, in some cases, irreversible) reductions in renal function as well as polyuria and polydipsia, presumably as a result of nephrogenic diabetes insipidus. 121 That is, individuals with ADH appear to suffer from "hypercalcemic" complications even in the presence of normocalcemia, presumably as a result of resetting of their mineral ion homeostatic system as a result of enhanced responsiveness 2+ of target tissues to Cao .
C. Linkage of ADH to Chromosome 3 and Identification of CaR Mutations Finegold and c o - w o r k e r s 122 initially demonstrated that ADH was linked to a locus on chromosome 3 in the region where the gene for the CaR is located. Pollak and co-workers then identified a heterozygous missense mutation at codon 127 of the CaR (glu127ala). 12~ Additional missense mutations in the CaR have subsequently been identified in approximately ten families with A D H 78'118-12~ a s well as in a case of apparently sporadic hypocalcemia. 1~8 Most reside within the ECD of the receptor, providing further evidence for the key role of this part of the receptor in the mechanisms of its activation by polycationic agonists. Several mutations have recently been reported within the transmembrane helices of the receptor, 118 similar to the location of naturally occurring activating mutations that have been described in several other GPCRs. 7'8 In all cases this disorder has re-
CHAPTER 16 Familial Benign Hypocalciuric Hypercalcemia suited from heterozygous mutations, and, to date, no cases of homozygous A D H have been described. Pollak and co-workers expressed a mutant CaR engineered to contain the glu127ala mutation in X. l a e v i s oocytes and showed that the levels of IP3 were elevated relative to those in oocytes expressing the wild-type receptor at both low (0.5 mM) and high (5.0 mM) Uao'-2"+ . 120 We have recently expressed the same mutation in HEK293 cells using the same methodologies described above that were used to express the F B H H mutations. The mutated CaR shows a clear leftward shift for the CaZ+-evoked increase in Ca 2+ (Fig. 1 6 - 5 ) . The mechanism(s) by which these mutations could activate the CaR is presently unclear. Activating point mutations in other G-protein-coupled receptors, like the T S H and LH receptors, are present in transmembrane domains and presumably enhance the processes of signal transduction or mimic the active state of the receptor following ligand binding if the activating mutation is truly ligand independent. 1~ In the CaR, it is likely that mutations within the ECD enhance the affinity of the CaR for Ca 2+ or mimic the ligand-bound state of the extracellular domain, thereby initiating subsequent events in signal transduction at inappropriately low levels of extracellular calcium or even when C a o2+ is close to zero. Available
8O
o! r~
Wild type, EC5o=3.9a + 0.1 mM E127A/wt, EC5o=3.4b + 0.1 mM
~ " _
- " q : } - - E127A, EC5o=3.3b + 0.1 mM
7/
g~, 60
40,
495 data do not support an increased level of expression of the mutant CaR as a contributory factor, as the pattern observed on Western blot analysis for the glu127ala mutation was similar to that for the wild-type receptor. 84 Ultimately, it will be of considerable interest to determine from more detailed structural studies (e.g., x-ray diffraction) the mechanisms through which these activating mutations alter the binding properties of the CaR.
D. Clinical Features of ADH that Provide Clues to the Normal Physiological Role of the CaR Several clinical features of A D H provide additional indirect evidence for the importance of the CaR in setting the responsiveness of parathyroid and kidney to changes in Ca 2+. The parathyroid glands and kidneys of individuals with A D H appear overly responsive to extracellular calcium. Normalization of Ca 2+ in these individuals leads to hypercalciuria that may be excessive compared to that encountered in patients with primary hypoparathyroidism, as individuals with A D H seem unusually prone to the complications of hypercalciuria and hypercalcemia even at low-normal levels of Cao2+ . 121 These observations complement the ones described earlier in individuals with inactivating CaR mutations (who have inappropriate hypocalciuria despite their hypercalcemia) and confirm a major role for the CaR in regulating tubular reabsorption of calcium. Moreover, the apparently reciprocal abnormalities in water metabolism in A D H and F B H H provide further indirect evidence that the CaR is intimately involved in coordinating renal handling of C a 2+ and water.
..u
E
E. Diagnostic and Therapeutic Implications of Activating CaR Mutations
20.
0
1
2
3
4
5
Ca2+o (mM) FIGURE 16--5 Expressionof a mutant CaR bearing an ADH mutation (glu127ala) in HEK293 cells. Results indicate the effects of varying levels of Ca 2+ on the cytosolic calcium level in HEK293 cells transiently transfected with the wild-type CaR, a mutant CaR bearing the ADH mutation, glu127ala, or both the wild-type and mutant receptors. Note that the mutant receptor has an increase in its apparent affinity for C a o2+ and that in the co-transfected cells the Cao2 + -evoked increases in Cai take place at lower concentrations of Ca 2+ than in those transfected with the wild-type receptor alone. Thus in vivo the receptor is activated by lower than normal levels of Ca 2+ producing stable hypocalcemia. [From Bai M, Quinn S, Trivedi S, et ah Expression and characterization of inactivating and activating mutations of the human Cao2 + -sensing receptor. J Biol Chem 271"19537-19545, 1996.]
These studies suggest the existence of A D H as an entity distinct from typical hypoparathyroidism and could lead to recognition of a larger number of cases with ADH, many of which may previously have been classified as mild cases of familial isolated or sporadic hypoparathyroidism. This distinction is of great clinical importance, since patients with A D H may end up with irreversible renal damage if they are treated with calcitriol to normalize their serum calcium concentration. The recognition of cases with autosomal dominant hypocalcemia due to activating mutations of the CaR is important, since many of these individuals could be "overtreated" with calcium/vitamin D with deleterious, sometimes irreversible renal consequences if the disorder is not identified. TM The documentation of this condition
496
EDWARD M. BROWN, MEI BAI, AND MARTIN POLLACK
requires careful clinical and genetic characterization. The clinician should carefully consider this diagnosis in individuals with the p r e s u m e d diagnosis of familial hypoparathyroidism, particularly those who develop m a r k e d hypercalciuria and/or renal impairment when treated with vitamin D, so that complications such as nephrocalcinosis and renal failure can be prevented.
IV.
SUMMARY
AND
CONCLUSIONS
The recent cloning of a C a R from diverse tissues in various species demonstrates directly that cells can sense (i.e., recognize and r e s p o n d to) small changes in their ambient level of Ca 2+ through a G protein-coupled, cell surface receptor. Thus Ca 2+ acts as an extracellular first m e s s e n g e r in addition to carrying its better k n o w n role as an intracellular second messenger. Several of the tissues that express the C a R are key elements in the calcium homeostatic system that have been k n o w n for m a n y years to be able to sense Ca 2+, such as parathyroid and thyroidal C-cells. The presence of the C a R in the kidney, however, provides strong evidence that several of the well-recognized but poorly understood actions of Ca 2+o on renal function m i g h t be mediated by the CaR. These actions include the up-regulation of renal calcium and m a g n e s i u m excretion in response to h y p e r c a l c e m i a that c o m p l e m e n t s the indirect inhibition of renal reabsorption of Ca 2+ in this setting which results from high 2+ C a o - m e d i a t e d reduction for P T H secretion. The impaired renal concentrating capacity observed in some hypercalcemic patients is probably a manifestation of a homeostatically important integration of the regulation of renal calcium and water handling that decreases the risk of pathological deposition of calcium in the kidney w h e n there is a need to get rid of excessive calcium in the urine. In this regard, the h u m a n syndromes of Ca2o+ " r e s i s t a n c e " or " o v e r r e s p o n s i v e n s s " due to loss- or gain-of-function C a R mutations, respectively, have p r o vided useful experiments-in-nature that have helped to elucidate the importance of the C a R in normal physiology as well as in pathophysiology. M u c h remains to be learned, however, about the role of the C a R in places w h e r e it likely responds to changes in local rather than systemic levels of Ca 2+o, such as in the brain. In these locations, it m a y be an important modulator of the functions of neurons, responding to Ca 2+ as a n e u r o m o d u lator or even neurotransmitter. D e v e l o p m e n t of CaRbased therapeutics that either activate 124,125or inhibit the receptor m a y be very useful for treating a variety of disorders where the C a R is either under- or overactive, respectively. Finally, it w o u l d not be surprising if there were additional receptors for Ca 2+ or for other ions (the C a R may, in fact, function as a M g 2+ sensor), which
could malfunction in disease states and be a m e n a b l e to pharmacological manipulation with appropriate receptorbased drugs.
Acknowledgments The authors gratefully acknowledge the generous grant support of the USPHS (DK41415, 44588, 46422, and 48330 to E.M.B.; DK02138 to M.P.; and DK09436 to M.B.) as well as NPS Pharmaceuticals, Inc., and the St. Giles Foundation (to E.M.B.).
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2HAPTER
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Hypoparathyroidism and P s eudohyp op arathyroi di s in MICHAEL A.
I. II. III. IV. V.
LEVINE
Division of Endocrinology and Metabolism, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Introduction Pathophysiology of Hypocalcemia Signs and Symptoms of Hypocalcemia Specific Causes of Functional Hypoparathyroidism Pseudohypoparathyroidism
VI. Diagnosis VII. Treatment VIII. Conclusion Acknowledgments References
only 1% of the total body calcium within extracellular fluids and soft tissues. Calcium exists within three fractions in the serum: ---50% of serum calcium is ionized at normal plasma protein concentrations, ---10% is complexed to citrate and phosphate ions, and ---40% is protein bound. 1 Although conventional measurement of serum calcium implies determination of the total serum calcium concentration, more physiologically relevant information is obtained by measurement of the ionized calcium concentration. From a practical point of view, measurement of total serum calcium concentration provides a reasonable estimate of the ionized calcium concentration, but several caveats are worth noting. For example, decreased concentration of serum albumin, the major calcium-binding protein in the circulation, rather than a decrease in the concentration of ionized calcium, accounts for most cases of low total serum calcium in hospitalized patients. Sudden changes in the distribution of calcium between ionized and bound fractions may cause symptoms of hypocalcemia, even in patients who have normal hormonal mechanisms for the regulation of the ionized calcium concentration. Increases in the extracellular fluid
I. I N T R O D U C T I O N The term "functional hypoparathyroidism" refers to a group of metabolic syndromes in which hypocalcemia and hyperphosphatemia occur either from a failure of the parathyroid glands to secrete adequate amounts of biologically active parathyroid hormone (PTH) or, less commonly, from an inability of PTH to elicit appropriate biological responses in its target tissues. Plasma levels of PTH are typically reduced or absent in patients with true hypoparathyroidism. By contrast, patients with pseudohypoparathyroidism (PHP) have target tissue resistance to PTH that results in characteristically elevated plasma levels of PTH. Thus true hypoparathyroidism differs fundamentally and biochemically from PHP. Hypocalcemia is the biochemical hallmark of functional hypoparathyroidism. The serum concentration of calcium is normally maintained within narrow limits despite wide variations in dietary intake, the demands of the skeleton during growth, and losses during pregnancy and lactation. Approximately 99% of total body calcium is in the skeleton in the form of hydroxyapatite, leaving
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502 (ECF) concentration of anions, such as phosphate citrate, bicarbonate, or edetic acid, will increase the proportion of bound calcium and decrease ionized calcium until intact regulatory mechanisms normalize ionized calcium. Extracellular fluid pH also affects the distribution of calcium between ionized and bound fractions. Acidosis increases the ionized calcium, whereas alkalosis decreases it. Therefore, measurement of ionized calcium is preferred when evaluating symptoms of hypocalcemia in patients who have abnormal circulating proteins or normal levels of total serum calcium. 2 Plasma levels of ionized calcium can be measured in most clinical chemistry laboratories using now-standardized techniques. 3-5 However, when it is not possible, or practical, to determine the ionized calcium concentration directly, a "corrected" total calcium concentration can be derived using one of several proposed algorithms that are based on albumin or total protein concentrations. 2'6 None of these correction factors is absolutely accurate, but they often provide useful estimates of the true concentration of calcium in serum. 7 One widely used algorithm estimates that total serum calcium declines by approximately 0.8 mg/dl for each 1 g/dl decrease in albumin concentration, without a change in ionized calcium.
II. P A T H O P H Y S I O L O G Y OF HYPOCALCEMIA The concentration of extracellular ionized calcium is tightly regulated by PTH and 1,25-dihydroxyvitamin D [1,25(OH)zD; calcitriol]. PTH is synthesized in the four parathyroid glands as a preprohormone (115 amino acids), converted to a prohormone (90 amino acids) as it is transported across the rough endoplasmic reticulum, and stored in secretory granules as the mature 84-aminoacid hormone. PTH is secreted at a rate inversely proportional to the ambient serum ionized calcium concentration. Hormone secretion is tightly regulated through the interaction of extracellular calcium (and to a lesser extent other divalent cations) with specific calciumsensing receptors 3-1~ that are present on the surface of the parathyroid cell. Extracellular calcium stimulates a receptor-dependent signaling pathway that leads to the rapid but transient increase in intracellular calcium; the increase in cytosolic calcium inhibits release of PTH from the parathyroid cell. By contrast, PTH synthesis and secretion are increased when the extracellular calcium concentration is low. Over time, protracted hypocalcemia leads not only to increased PTH secretion but also to increased parathyroid gland mass. Fluctuations in the serum calcium concentration provoke rapid changes in PTH secretion that within minutes affect distal tubular calcium reabsorption and osteoclas-
MICHAEL A. LEVINE
tic bone resorption. In contrast to this short-loop feedback system, adjustments in the rate of gastrointestinal absorption of calcium via the P T H - v i t a m i n D axis occur over 1 to 2 days and constitute a long-loop feedback system. The integrated actions of PTH on these target tissues provides a precise system of control and maintains the serum ionized calcium concentration within a narrow range. PTH has direct effects on bone to regulate calcium exchange at osteocytic sites and to enhance osteoclastmediated bone resorption. In the kidney, PTH directly enhances distal tubular reabsorption of calcium, decreases the proximal tubular reabsorption of phosphate, and stimulates the metabolic conversion of 25-hydroxyvitamin D [25(OH)D] to 1,25(OH)2D, the active vitamin D metabolite. 1,25(OH)2D acts on bone to enhance bone resorption and on the gastrointestinal mucosa to increase absorption of dietary calcium. PTH exerts its effects on the target cells in bone and kidney by binding to specific membrane receptors that are coupled via guanine-nucleotide-binding proteins (G proteins) to the signal-generating enzymes adenylyl cyclase and phospholipase C (Fig. 17-1). The initial event in the expression of PTH action is binding of the hormone to specific receptors located on the plasma membrane of target cells. Molecular cloning of cDNAs encoding PTH receptors from several species ~1-~4 has indicated that bone and kidney cells express an identical receptor that binds PTH and parathyroid hormone-
Ri
PTH/PTHrPRc
G
PIP2~~ cAMP
1
PKA FIGURE 17-- 1
.
IP3
§
DAG PKC
Cell surface receptors for PTH are coupled to two classes of G proteins. Gs mediates stimulation of adenylyl cyclase (AC) and the production of cAME which in turn activates protein kinase A (PKA). Gq stimulates phospholipase C (PLC) to form the second messengers inositol-(1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG) from membrane-bound phosphatidylinositol-(4,5)bisphosphate. IP3 increases intracellular calcium (Ca 2+) and DAG stimulates protein kinase C (PKC) activity. Each G protein consists of a unique oL chain and a [3~/dimer.
503
CHAPTER 17 Hypoparathyroidism and Pseudohypoparathyroidism related protein (PTHrP) with equivalent affinity. The PTH/PTHrP receptor is a member of a superfamily of receptor proteins that span the membrane in a serpentine fashion, threading themselves across the lipid bylayer through a series of seven o~-helical domains. These receptors are coupled by heterotrimeric (c~[3~/) G proteins 15 to signal effector molecules localized to the inner surface of the plasma membrane (Fig. 17-1). Highly specific associations among at least 20 G~, five G~, and 12 Gv chains generate a diversity of heterotrimeric G proteins that have the ability to discriminate amongst a multitude of receptor and effector molecules. Hormone binding to a receptor facilitates activation of the G protein, a process in which the oL chain exchanges bound guanosine diphosphate (GDP) for guanosine triphosphate (GTP) and dissociates from the [3~/dimer and the receptor. The free, GTP-bound form of the oL chain is the primary modulator of relevant effector molecules, although [3~/ dimers can also influence activity of many effectors (e.g., some forms of adenylyl cyclase and phospholipase C). An intrinsic GTPase associated with the oL chain acts as a molecular timing mechanism, and after a predetermined interval GTP is hydrolyzed to GDE The inactive GDP-bound oL chain reassociates with a [3~/ dimer, and the heterotrimeric G protein is ready for another cycle of hormone activation. Hormone binding to the PTH/PTHrP receptor is followed rapidly by the generation of a variety of second messengers, 16'17 including c A M E 18'19 inositol 1,4,5trisphosphate and diacylglycerol, 2~ and cytosolic calcium, 23'24 indicating that a single PTH/PTHrP receptor can couple not only to Gs to stimulate adenylyl cyclase, but also to Gq and GI~ to stimulate phospholipase C (Fig. 17-1). The best characterized mediator of PTH action is cyclic adenosine monophosphate (cAMP), which rapidly activates protein ldnase A. 25 The relevant target proteins that are phosphorylated by protein kinase A and precise mode(s) of action of these proteins remain uncharacterized, though metabolic enzymes, transcription factors, and ion channel proteins are strong candidates. The intracellular accumulation of cAMP triggers a biochemical chain reaction that begins with phosphorylation of specific protein substrates by cAMP-activated protein kinase A, and ultimately concludes with the physiological response of the cell to agonist recognition. In contrast to the well-recognized biological effects of cAMP in PTH target tissues, the physiological importance of metabolites of phosphotidylinositol and intracellular calcium as PTH-induced second messengers has not yet been established. Clinical disorders causing hypocalcemia occur if the production of biologically active PTH or 1,25(OH)2D is impaired or if target organ responses to these hormones are abnormal, either because of a specific biochemical
defect or because of generalized target organ damage (Table 17-1). In the hypoparathyroid states, in which PTH secretion or action is deficient, the normal effects of PTH on bone and kidney are absent. Bone resorption, and release of calcium from skeletal stores, is diminished. Renal tubular reabsorption of calcium is decreased, but because of hypocalcemia and low filtered load, urinary calcium excretion is low. In the absence of PTH action, urinary clearance of phosphate is decreased, and hyperphosphatemia is common. The deficiency of PTH action and the hyperphosphatemia impair renal production of
TABLE 17-- 1 Clinical Disorders Causing Hypocalcemia Hypoproteinemia (factitious or "pseudohypocalcemia") Hypoparathyroidism (inadequate secretion of PTH) Postsurgical Toxic agents (alcohol, irradiation) Developmental disorders of the parathyroid gland Magnesium excess or deficiency Idiopathic Autoimmune Genetic defects in the PTH gene Genetic defects in the calcium-sensing receptor gene Infiltration (iron, copper, tumor) Transient neonatal hypoparathyroidism Resistance to PTH action Pseudohypoparathyroidism Hypomagnesemia Disorders of vitamin D metabolism Decreased precursors Dietary deficiency Malabsorption Nephrotic syndrome or peritoneal dialysis Liver disease Disrupted enterohepatic circulation Decreased metabolic activation Renal disease 1oL-hydroxylasedeficiency (vitamin D-dependent rickets, type I) Resistance to vitamin D action Genetic defects in the vitamin D receptor (vitamin D-dependent rickets, type II) Decreased bone resorption Antiresorptive drug treatment Increased bone formation Hungry bones Osteoblastic tumor metastasis Miscellaneous Hyperphosphatemia Rhabdomyolysis Tumor lysis Phosphate infusion, ingestion, enema Citrate administration Respiratory alkalosis Acute severe illness (pancreatitis, burns, sepsis)
504
MICHAEL A. LEVINE
1,25(OH)2D. The low circulating levels of 1,25(OH)2D result in reduced intestinal calcium absorption as well as decreased bone resorption. Functional hypoparathyroidism, therefore, is characterized by a decreased entry of calcium into the extracellular fluid compartment from bone, kidney, and intestine and is associated with hypocalcemia and hyperphosphatemia. In states of vitamin D deficiency or vitamin D insensitivity, absorption of calcium from the intestine is markedly impaired. 1,25(OH)2D is also a potent stimulator of bone resorption, and its absence may also decrease the availability of calcium from bone. Because the parathyroid glands are intact in the vitamin D-deficient states, hypocalcemia induces secondary hyperparathyroidism and renal phosphate clearance is enhanced. Thus, hypocalcemia in vitamin D deficiency results from decreased intestinal absorption of calcium and a limited availability of calcium from bone despite secondary hyperparathyroidism; characteristically, it is accompanied by hypophosphatemia.
III. SIGNS
serum calcium levels27; thus, this sign cannot be considered diagnostic of hypocalcemia unless it is known that it was previously absent. Trousseau's sign is present if carpal spasm occurs after compression of the nerves in the upper arm. A typical protocol consists of inflation
AND SYMPTOMS
OF HYPOCALCEMIA Ionized calcium, rather than total calcium, is the primary determinant of symptoms in patients with hypocalcemia. A low extracellular fluid ionized calcium concentration enhances neuromuscular excitability, an effect that is potentiated by hypomagnesemia. 26 There is substantial variation among patients in the severity of symptoms, and there does not appear to be an absolute level of serum calcium at which symptoms can be expected. Patients with chronic hypocalcemia sometimes have few, if any, symptoms of neuromuscular irritability despite markedly depressed serum calcium concentrations. By contrast, patients with acute hypocalcemia frequently manifest many symptoms of tetany. Most patients with hypocalcemia will have some mild features of tetany, including circumoral numbness, paresthesias of the distal extremities, or muscle cramps. Symptoms of fatigue, hyperirritability, anxiety, and depression are also common. Clinical manifestations of marked hypocalcemia consist of carpopedal spasm, laryngospasm, and focal or sometimes life-threatening generalized seizures (which must be distinguished from the generalized tonic muscle contractions that occur in severe tetany). Clinical signs of the neuromuscular irritability associated with latent tetany include Chvostek's sign and Trousseau's sign. Chvostek's sign is elicited by tapping the facial nerve just anterior to the ear to produce ipsilateral contraction of the facial muscles. Slightly positive reactions occur in 10% to 30% of adults with normal
FIGURE 17--2 Electrocardiogram (ECG) of a patient with severe hypocalcemia. This ECG demonstrates the characteristic prolongation of the (corrected) QT interval, measured from the beginning of the QRS complex to the end of the T wave (arrows in bottom panel). The QT interval corresponds to the duration of ventricular depolarization and repolarization. The interval increases with decreasing heart rate and may be corrected (QTc) by measuring the RR interval and employing the following formula: QTc = QT/~v/(R - R). The second or plateau phase (ST segment) of the action potential is influenced by the serum calcium, and is of greater duration in hypocalcemic subjects. As the exact end of the T wave may be difficult to determine, some clinicians utilize the Q,Tc interval, which comprises the onset of the QRS complex to the apex of the T wave. [From Becker KL (ed): Principles and Practice of Endocrinology and Metabolism, 2nd ed. Philadelphia, JB Lippincott, 1995, with permission.]
CHAPTER 17 Hypoparathyroidism and Pseudohypoparathyroidism of a cuff on the upper arm to above systolic blood pressure for 3 to 5 minutes. Both of these signs can be absent even in patients with definite hypocalcemia. Hypocalcemia is also associated with nonspecific electroencephalographic changes, increases in intracranial pressure, and papilledema. A markedly depressed serum calcium concentration can have profound affects on the heart. The corrected QT (QTc) interval may be prolonged on the electrocardiogram (Fig. 17-2), and may be associated with arrhythmias or cardiac dysfunction that is generally reversible with treatment of the hypocalcemia. 28'29 Cardiac dysfunction may range from mild abnormality that is noted only with exercise 3~ to life-threatening heart failure. 31 The somatosensoryevoked potential recovery period may provide a useful tool for assessing the effects and recovery from hypocalcemia. 32 Additional signs may be associated with longstanding hypocalcemia. Ectodermal findings such as dry skin, coarse hair, and brittle nails are common, but they are frequently overlooked. Dental and enamel hypoplasia and absence of adult teeth indicate that hypocalcemia has been present since childhood. 33-35 The pattern of dental abnormality can be used to determine the age at which the patient first developed hypocalcemia. Calcification of the basal ganglia, and to a lesser extent the cerebral cortex, occurs in all forms of hypoparathyroidism and can be detected by computed tomographic (CT) scanning even when routine skull radiographs do not demonstrate intracerebral calcification. 36'37 Rarely, calcification of the basal ganglia is associated with neurological signs or symptoms that resemble Parkinson's disease or chorea. 38 An unusual neurological manifestation of hypocalcemia is an increased susceptibility to dystonic reactions induced by phenothiazines. 39 Subcapsular cataracts are common in untreated hypoparathyroidism and are most readily detected by slit lamp examination. Treatment may reverse or decrease the progression of the cataracts. Rickets and osteomalacia, although not characteristic, do occur occasionally in hypoparathyroidism after prolonged hypocalcemia. 4~ Patients with longstanding hypoparathyroidism have been reported to have significantly increased bone mineral density 42 whether they are treated 43 or not. 44
IV. SPECIFIC FUNCTIONAL
CAUSES
OF
HYPOPARATHYROIDISM
A. S u r g e r y a n d T o x i c A g e n t s Surgery is the most common cause of acquired hypoparathyroidism. Hypoparathyroidism may occur after parathyroid or thyroid surgery or after radical surgery
505 for laryngeal or esophageal carcinoma. 45 The resulting hypoparathyroidism can be transient or permanent, and sometimes may not develop for many years. 46 A chronic state of "decreased parathyroid reserve ''46 may exist in some patients who manifest hypocalcemia only when mineral homeostasis is stressed further by other factors such as pregnancy, lactation, or illness. Hypocalcemia occurs soon after removal of a hyperfunctioning parathyroid adenoma because the remaining parathyroid tissue is "suppressed" by previous hypercalcemia and is unable to secrete adequate amount of PTH. Hypoparathyroidism is usually transient because the normal parathyroid glands recover function quickly (generally within 1 week), even after long-term suppression. Transient postoperative hypocalcemia may be exaggerated or prolonged in those patients who have significant preexisting hyperparathyroid bone disease. In these patients the acute reduction of previously elevated serum levels of PTH results in an increased movement of serum calcium (and phosphorus) into remineralizing "hungry bones. ''47 Treatment with calcium and a short-acting vitamin D metabolite may be required until the bones heal. Permanent hypoparathyroidism is unusual after an initial neck exploration for primary hyperparathyroidism and develops in fewer than 1% to 2% of patients. The incidence is greatly increased with repeated neck surgery for recurrent or persistent hyperparathyroidism, after subtotal parathyroidectomy for parathyroid hyperplasia, or when surgery is performed by an inexperienced operator. The incidence of permanent hypoparathyroidism after thyroid surgery varies widely, and reflects the underlying thyroid lesion, the extent of surgery, and the experience of the surgeon. Hypoparathyroidism may occur as a result of direct injury or inadvertent removal or devascularization of the parathyroid glands. Permanent hypoparathyroidism is unlikely to occur after a hemithyroidectomy and should be relatively uncommon even after total thyroidectomy. 48'49 By contrast, transient hypoparathyroidism may occur in up to 33% of patients who undergo a total thyroidectomy for thyroid cancer 5~ or thyrotoxicosis. The fall in plasma calcium level generally occurs within 24 to 48 hours after surgery and often is sufficient to provoke symptoms of tetany. The basis for this acute hypocalcemia is not well understood. One mechanism that has been proposed for patients with thyrotoxicosis is similar to the "hungry bones" phenomenon that occurs after parathyroid surgery in patients with marked primary hyperparathyroidism. 51 Patients with severe hyperthyroidism often have increased bone resorption, elevated plasma ionized calcium levels, and suppressed parathyroid function. Although it has been proposed that hypocalcemia occurs as calcium moves rapidly into remineralizing bones after surgical correc-
506 tion of thyrotoxicosis, 52 the early development of hypocalcemia after surgery often precedes significant reduction of serum levels of thyroid hormones. A more likely explanation is that unappreciated damage has occurred to the parathyroid glands during surgery. Whatever the initiating cause, the development of hypocalcemia must indicate that the secretory response of the parathyroid glands is inadequate to maintain a normal serum calcium concentration.53'54 1. RADIATION AND DRUGS
In contrast to many other endocrine tissues, the parathyroid glands are particularly resistant to damage by a great many toxic agents. The administration of radioactive iodine for the treatment of benign or malignant thyroid disease or for the deliberate induction of hypothyroidism has only rarely caused permanent, symptomatic hypoparathyroidism. Similarly, external beam radiation appears to have little or no effect on parathyroid gland function. Parathyroid function is altered only occasionally by most chemotherapeutic or cytotoxic agents. Notable exceptions include asparaginase, which causes parathyroid necrosis in rabbits, and ethiofos, a radio- and chemoprotector that causes a dose-dependent and reversible inhibition of PTH secretion. 55'56 Along with its effects on the parathyroid gland, ethiofos also inhibits osteoclast activity. Thus, a significant component of its calcium-reducing effect derives from the ability of this agent to inhibit osteoclast-directed bone resorption and calcium release from skeletal stores. The most common toxic agent to affect parathyroid function is alcohol. 57 Transient hypoparathyroidism has been associated with ingestion of large quantities of alcohol. 58'59 2. INFILTRATIVE DISEASE OF THE PARATHYROIDS Infiltrative processes that affect the parathyroid gland may impair gland function and inhibit release of PTH. Idiopathic hemochromatosis and chronic transfusion therapy are often associated with significant deposition of iron in the parathyroid glands. 6~ Patients occasionally develop clinical hypoparathyroidism, but more commonly manifest decreased "parathyroid reserve" as a consequence of iron infiltration. 61 A similar pathophysiological process has been described in one patient with Wilson's disease and increased copper storage who developed symptomatic hypoparathyroidism. 62 Pathological involvement of the parathyroid glands can also occur in metastatic neoplasia, miliary tuberculosis, amyloidosis, and sarcoid, but clinical hypoparathyroidism rarely occurs in these conditions. 3. MAGNESIUM DEFICIENCY AND EXCESS
Although calcium is the major regulator of PTH secretion, magnesium can modulate PTH secretion in a
MICHAEL A. LEVINE
similar manner. Recent studies show that magnesium has approximately 30% to 50% of the effect of calcium on either stimulating or suppressing PTH secretion. 63 Hypermagnesemia may cause reversible hypocalcemia. This situation is most commonly encountered in obstetrical practice when high-dose magnesium infusions are used for the treatment of toxemia or premature labor. 64'65 Because the hypocalcemia is accompanied by significant hypermagnesemia, neuromuscular irritability should be less than that expected when similar calcium concentrations occur with normal magnesium; clinical tetany usually does not o c c u r . 26 While elevations in extracellular magnesium can suppress PTH secretion and lower serum calcium levels, hypocalcemia is more often a manifestation of magnesium depletion, 65 particularly in patients with chronic alcoholism. 55 As the serum magnesium level falls, the parathyroid gland responds by increasing secretion of PTH. However, as intracellular magnesium depletion develops, the ability of the parathyroid gland to secrete PTH is impaired, and hypocalcemia may ensue. 67 Magnesium depletion must become severe before symptomatic hypocalcemia occurs, but even mild degrees of magnesium depletion may result in a significant reduction in the serum calcium level. The majority of patients with magnesium depletion have low levels of PTH, or "normal" levels of PTH that are in fact inappropriate for the degree of hypocalcemia. 68 Therefore, a state of relative or functional hypoparathyroidism exists in these patients. By contrast, serum levels of PTH are elevated in hypocalcemic patients who have more severe magnesium depletion. 67-69 These patients show an impaired response to exogenous PTH, suggesting that refractoriness to PTH may develop with increasing degrees of magnesium depletion. Hypocalcemia can be readily reversed by magnesium therapy alone, but is not corrected by administration of calcium or vitamin D.
B. I d i o p a t h i c H y p o p a r a t h y r o i d i s m The term "idiopathic hypoparathyroidism" describes a heterogeneous group of rare disorders that share in common deficient secretion of PTH. The use of the term "idiopathic" to describe this group of disorders has greater historical than scientific relevance at present, as recent molecular and biochemical studies have revealed the cause of hypoparathyroidism in many of these disorders. Accordingly, the term "idiopathic" is a misnomer for many of these conditions. Although most cases are sporadic, the familial occurrence of idiopathic hypoparathyroidism has been reported. Within these families hypoparathyroidism may occur as part of a complex autoimmune disorder associated with multiple endocrine deficiencies (i.e., type 1 polyglandular syndrome) or in
CHAPTER 17 Hypoparathyroidism and Pseudohypoparathyroidism association with diverse developmental abnormalities (e.g., nephropathy, lymphedema, nerve deafness, or tetralogy of Fallot). The pleiotropic nature of many of these various syndromes has suggested that the genetic basis of PTH deficiency is not related to a specific defect intrinsic to the parathyroid gland.
C. Autoimmune Hypoparathyroidism Autoimmune hypoparathyroidism may occur alone or as a component of the type 1 polyglandular syndrome. TM The type 1 polyglandular syndrome may be sporadic or familial with an autosomal recessive inheritance pattern. vl The classic triad of this syndrome is hypoparathyroidism, adrenal insufficiency, and mucocutaneous candidiasis (HAM). The recognition that affected patients may have additional components has led to the suggestion that a more inclusive term be used to describe the syndrome: autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (APECED). vl'v2 The syndrome is generally first recognized in early childhood, although a few individuals have developed the condition after the first decade of life. The clinical onset of the three principal components of the syndrome typically follows a predictable pattern, in which mucocutaneous candidiasis first appears at a mean age of 5 years, followed by hypoparathyroidism at a mean age of 9 years and adrenal insufficiency at a mean age of 14 years, v~ Patients may not manifest all three components of the clinical triad. Additional features occur in some patients, such as alopecia, keratoconjunctivitis, malabsorption and steatorrhea, gonadal failure, pernicious anemia, chronic active hepatitis, thyroid disease, and insulin-requiting diabetes mellitus. Antibodies directed against the parathyroid, thyroid, and adrenal glands are present in many patients v3 and a T-cell abnormality has been described. TM The presence of antibodies may not correlate well with the clinical findings. In those cases that have been examined pathologically, complete parathyroid atrophy or destruction has been demonstrated. In some patients, treatment of hypoparathyroidism has been complicated by apparent vitamin D "resistance," possibly related to coexistent hepatic disease or steatorrhea, or both. Despite increasing appreciation of the clinical and genetic features of this syndrome, the molecular basis for APECED remains unknown.
D. Isolated Hypoparathyroidism Isolated hypoparathyroidism, in which PTH deficiency is not associated with other endocrine disorders or developmental defects, is usually sporadic, but it may
507 occur on a familial basis. The age of onset is generally within the first decade, although hypocalcemia may not be first discovered until later in adult life. There is a high incidence of parathyroid antibodies in patients with isolated idiopathic hypoparathyroidism, and some cases may be examples of incomplete expression of the polyglandular type 1 syndrome (above). Some patients may possess antibodies that inhibit the secretion of PTH v5 rather than cause parathyroid gland destruction. 76 In other cases that have been examined pathologically, fatty replacement vv or atrophy with fatty infiltration and fibrosis TM has been described. Isolated hypoparathyroidism may be sporadic or familial, with inheritance of PTH deficiency by autosomal dominant, autosomal recessive, or X-linked modes of transmission, v9 The age at onset covers a broad range (1 month to 30 years), and the condition is often recognized first in the child rather than in the parent. Parathyroid antibodies are absent. As the location of the preproPTH gene has been mapped to l lp15, molecular genetic studies of familial isolated hypoparathyroidism have focused on kindreds in which inheritance of hypoparathyroidism is consistent with an autosomal mode of transmission. Genetic analysis of one pedigree in which hypoparathyroidism was inherited in an autosomal dominant manner showed linkage of hypoparathyroidism to the preproPTH gene locus. 8~ Subsequent DNA sequence analysis of the preproPTH gene in this kindred revealed that affected members had a heterozygous mutation consisting of a single base substitution (T ~ C) in exon 2. 81 This mutation results in the substitution of arginine (CGT) for cysteine (TGT) in the leader sequence of preproPTH. The substitution of a charged amino acid in the midst of the hydrophobic core of the leader sequence inhibits processing of the mutant preproPTH molecule to proPTH by signal peptidase 81 and is presumed to impair translocation of not only the mutant hormone but also the wild-type protein across the plasma membrane of the endoplasmic reticulum. Thus, this heterozygous mutation results in a dominant inhibitor phenotype that prevents processing of the wild-type preproPTH molecule. Familial hypoparathyroidism can also be transmitted as an autosomal recessive trait. An abnormality of the preproPTH gene has been found in one consanguineous family with autosomal recessive hypoparathyroidism. 82 Affected members of this family are homozygous for a single base transversion (G ~ C) at the exon 2-intron 2 boundary. This mutation alters the invariant gt dinucleotide of the 5' donor splice site that presumably affects annealing of the U 1-snRNP recognition component of the nuclear RNA splicing enzyme. Previous studies of similar donor splice site mutations in other genes have demonstrated that such mutations impair normal process-
508
ing of the nascent in mRNA. As PTH gene expression is generally confined to the parathyroid cell, analysis of preproPTH mRNA processing in these patients required reverse transcriptase polymerase chain reaction (RTPCR) to detect "illegitimate" or "ectopic" transcription of the PTH gene in cultured lymphoblasts. 82 Using this approach, a PTH cDNA that was 90 bp shorter than the corresponding wild-type form was amplified from cultured lymphoblasts of affected subjects. Nucleotide sequence analysis of the shortened cDNA revealed that exon 1 had been spliced to exon 3 in the mutant PTH mRNA, a process that resulted in the deletion of exon 2 from the mature transcript (i.e., exon skipping). 82 The loss of exon 2 eliminates both the initiation codon and the signal peptide sequence from the aberrant preproPTH mRNA, and explains the molecular basis for autosomal recessive hypoparathyroidism in this family. Although the molecular pathophysiology of hypoparathyroidism has been defined in these two families, both linkage analysis and gene sequencing have failed to disclose defects in the preproPTH gene in affected members of other autosomal kindreds. 8~ New insights into the molecular pathology of hypoparathyroidism have come from the recent cloning and characterization of the cDNA 9 and gene 85 encoding the calcium-sensing receptor, the cell surface protein that determines the calcium "set-point" of the parathyroid cell and thereby controls calcium-sensitive secretion of PTH. Initial studies demonstrated that heterozygous mutations that result in the loss of function of the calcium-sensing receptor are present in most patients with familial (benign) hypocalciuric hypercalcemia (FHH), an autosomal dominant disorder associated with decreased ability of calcium to suppress PTH secretion. 85-88 In several families some affected members have homozygous mutations that cause severe neonatal hyperparathyroidism, 89 a life-threatening hypercalcemic disorder in which parathyroid hyperplasia occurs. By contrast, mutations that lead to gain of function have been identified in several kindreds with autosomal dominant hypocalcemia, a syndrome associated with low serum levels of PTH and relative hypercalciuria. 9~ In other cases, linkage of hypocalcemia to the chromosomal locus for the calciumsensing receptor (3q21-24) has provided indirect evidence for the involvement of this gene with familial hypoparathyroidism. 92 Preliminary studies have identified similar activating mutations of the calcium-sensing receptor gene in many patients with sporadic hypoparathyroidism. In both familial and sporadic cases, each affected propositus has demonstrated a unique mutation, suggesting that new mutations must sustain this disorder in the population. These remarkable results suggest that mutation of calcium-sensing receptor gene may be the most common cause of genetic hypoparathyroidism.
MICHAEL A. LEVINE
The calcium-sensing receptor is expressed not only in the parathyroid gland but also in the kidney, where it appears to play an important role in regulating calcium reabsorption. 8'93'94Thus, loss of function mutations of the calcium-sensing receptor in patients with FHH is associated with decreased calcium clearance and hypocalciuria. By contrast, gain of function mutations in the calcium-sensing receptor are likely to account for the increased calcium clearance and relative hypercalciuria noted in patients with autosomal dominant hypocalcemia. Familial hypoparathyroidism can also be inherited as an X-linked disorder that is of course unrelated to specific defects in the preproPTH gene on chromosome 11. 95 Using a battery of X-chromosome gene markers, linkage studies of two large multigenerational families with X-linked hypoparathyroidism have localized a candidate gene to the region Xq26-27. 96 These results imply that the defective gene or genes in this syndrome may be important for parathyroid cell development or function. The early onset of hypocalcemia in affected individuals, and the apparent inability to identify parathyroid tissue in a single patient with this disorder at autopsy, 79 are consistent with a role for this genetic locus in the embryological development of the parathyroid glands (see below).
E. D e v e l o p m e n t a l D i s o r d e r s o f the Parathyroid Gland Hypoparathyroidism may result from agenesis or dysgenesis of the parathyroid glands. The most welldescribed example of parathyroid gland dysembryogenesis is the DiGeorge syndrome, in which maldevelopment of the third and fourth branchial pouches is frequently associated with congenital absence of not only the parathyroids but also the thymus. Because of thymic aplasia, T-cell-mediated immunity is impaired, and affected infants have an increased susceptibility to recurrent viral and fungal infections. Maldevelopment of the first and fifth branchial pouches occurs frequently as well, producing characteristic facial anomalies (Fig. 17-3), including hypertelorism; antimongoloid slant of the eyes; low-set and notched ears; short philtrum of the lip; and micrognathia (first branchial pouch); or aortic arch abnormalities, such as fight-sided arch, truncus arteriosus, or tetralogy of Fallot. Most cases of branchial pouch dysembryogenesis are sporadic, but familial occurrence with apparent autosomal dominant inheritance has been described. 97'98 Molecular mapping studies have demonstrated an association between the syndrome and deletions involving 22qll 99-1~ or 10p 1~176 in some patients, although it is important to note that most patients with
CHAPTER 17 Hypoparathyroidismand Pseudohypoparathyroidism
509
FIGURE 17--3
Photograph of an 18-month-old boy with DiGeorge syndrome showing characteristic features including mild hypertelorism; increased antimongoloid slant of the eyes; low-set, asymmetrical, malformed ears; and a short philtrum. Other features depicted are a broad nose, cupid bow mouth, and mandibular hypoplasia. Chest radiograph revealed a right-sided aortic arch and an absent thymic shadow. [From Kretschmer R, Say B, Brown D, Rosen F: Congenital aplasia of the thymus gland (DiGeorge's syndrome). N Engl J Med 279:1295, 1968.]
DiGeorge syndrome have normal karyotypes. A candidate gene (rnex 40) which encodes a putative DNAbinding protein that shares homology with the androgen receptor has been identified in this region. 1~ The loss of genetic material at 22qll results in hemizygosity of genes located in this region, and is associated with contiguous gene deletion syndromes that include not only the DiGeorge syndrome but also the overlapping conotruncal anomaly and velocardiofacial syndromes. 1~ Although most affected children with DiGeorge syndrome die of infections or cardiac failure by the age of 6, survival into adolescence or adulthood is possible when the syndrome is only partially expressed. Hypoparathyroidism occurs in more than 50% of patients who have the Kenney-Caffey syndrome, an unusual syndrome characterized by short stature, osteosclerosis, basal ganglion calcifications, and ophthalmic d e f e c t s . 79'1~ Hypoparathyroidism also occurs as a component of several other less well-characterized developmental syndromes. These include the Barakat syndrome, associated with familial nephrosis and sensorineural deafness~~ a novel syndrome described in a single kindred in which two affected brothers had lymphedema, prolapsing mitral valve, brachytelephalangy, and nephropathy~~ and a recently reported syndrome in one consanguineous family in which affected members showed severe growth failure plus dysmorphic features that include microcephaly, beaked nose, and crognathia. 1~~
F. Neonatal Hypocalcemia Shortly after birth there is a physiological fall in the serum calcium concentration, and during the first 3 days of life many normal infants will have serum calcium levels that are less than 8 mg/dl. Hypocalcemia in the neonate can be divided into early hypocalcemia, starting within the first 24 to 72 hours of life before feedings have been given, and late hypocalcemia, usually appearing after several days to weeks of feeding. 111'112 1. EARLY NEONATAL HYPOCALCEMIA Early neonatal hypocalcemia represents an exaggeration of the normal fall in serum calcium concentration and theoretically is due to deficient release of PTH by immature parathyroid glands. 1~3 Prematurity, low birth weight, hypoglycemia, maternal diabetes, difficult delivery, and respiratory distress syndrome are frequently associated findings. Hypocalcemia may be asymptomatic, but can manifest as irritability, muscular twitching, or convulsive seizures. Although the course is self-limited, symptomatic infants should be treated with oral or intravenous calcium. A more severe form of transient neonatal hypoparathyroidism and tetany can occur in children born to mothers with primary hyperparathyroidism or other causes of hypercalcemia. In these infants, exposure in utero to maternal hypercalcemia suppresses
510
MICHAEL A. LEVINE
parathyroid activity and apparently leads to impaired responsiveness of the parathyroid glands to hypocalcemia after birth. 1~4'115 2. LATE NEONATAL HYPOCALCEMIA Transient hypocalcemia that occurs 4 to 6 days (or later) after birth is considered to be a manifestation of relative immaturity of renal phosphorus handling or of the renal adenylyl cyclase system. Hypocalcemia may be precipitated by a high-phosphate diet and appears to occur particularly in those infants who are fed with artificial foods such as cow's m i l k - b a s e d formulas. ~16 In these infants, the renal response to PTH is inadequate and hypocalcemia ensues. The reduction of serum calcium ion concentration is probably secondary to elevated serum phosphate levels and should result in increased parathyroid gland activity. This form of hypocalcemia is the most common cause of seizures in the newborn period. Spontaneous recovery of normal mineral homeostasis typically occurs after a few weeks, but the serum calcium levels of symptomatic infants can be increased within 1 to 2 days by feeding a supplemented milk mixture with a high calcium/phosphorus (3:1 to 4:1) ratio.
due to an inability of the target organs, bone and kidney, to respond to PTH. In addition to the clinical and biochemical features of hypoparathyroidism, the patients described by Albright exhibited a distinctive constellation of developmental and skeletal defects, subsequently referred to as A1bright's hereditary osteodystrophy (AHO), and including a round face; short, stocky physique; brachydactyly; heterotopic ossification; and mental retardation. The relationship between the biochemical abnormalities (hypocalcemia and hyperphosphatemia) and A H O could not be explained by Albright, and yet remains unclarified. Indeed, in certain families some affected members may show both AHO and PTH resistance, whereas other family members may have A H O without evidence of any endocrine dysfunction, a disorder Albright termed "pseudopseudohypoparathyroidism" (pseudoPHP) to emphasize the physical similarities but biochemical differences between these patients and patients with PHE ~8 The diagnostic classification of PHP is further extended by the existence of additional variants in which patients manifest PTH resistance and biochemical hypoparathyroidism but lack any of the features of A H O . 119'12~ A classification of the many different forms of PHP is presented in Table 17-2.
V. P S E U D O H Y P O P A R A T H Y R O I D I S M In their original report of PHR Fuller Albright and his associates described the failure of patients with this syndrome to show a phosphaturic response to injected parathyroid extract, ll7 These observations led to the speculation that biochemical hypoparathyroidism in PHP was
TABLE 17--2
Characterization of the molecular basis for PHP commenced with the observations by Chase and Aurbach that cAMP mediates many of the actions of PTH on
Classification and Characteristic Features of the Various Forms of Pseudohypoparathyroidism PHP type Ia
Physical appearance
A. G e n e r a l P a t h o p h y s i o l o g y
PseudoPHP
PHP type Ib
PHP type Ic
PHP type II
Albright hereditary osteodystrophy, may be subtle or (rarely) absent
Normal
Albright hereditary osteodystrophy
Normal
Normal Normal Normal
Defective Defective Low
Defective Defective Low
Normal Defective Low
Hormone resistance
Defective Defective Low or (rarely) normal Generalized
Absent
Generalized
Gs~ activity Inheritance
Reduced Reduced Autosomal dominant
Normal Unknown
Limited to PTH target tissues Normal Unknown
Molecular d e f e c t
Heterozygous mutations in the GNAS1 gene
Limited to PTH target tissues Normal Autosomal dominant (most cases) Unknown (see t e x t )
Unknown
Unknown
Response to PTH Urine cAMP Urine phosphorous Serum calcium level
CHAPTER 17 Hypoparathyroidism and Pseudohypoparathyroidism kidney and bone, and that administration of biologically active PTH to normal subjects leads to a significant increase in the urinary excretion of nephrogenous cAMP and phosphate. 121 The PTH infusion test remains the most reliable test available for the diagnosis of PHP, and enables distinction between several variants of the syndrome (Fig. 17-4). Patients with PHP type I fail to show an appropriate increase in urinary excretion of both nephrogenous cAMP and phosphate, 121 suggesting that an abnormality in the renal PTH receptor-adenylyl cyclase complex that produces cAMP is the basis for impaired PTH responsiveness. Subsequent studies by Bell et al., in which administration of dibutyryl cAMP to patients with PHP type I produced a phosphaturic response, provided additional support for this theory, and demonstrated that the renal response mechanism to cAMP was intact. ~22 These studies have led to the conclusion that proximal renal tubule cells are unresponsive to PTH. By contrast, cells in other regions of the kidney appear responsive to PTH, as evidenced by the observation that urinary calcium excretion in patients with PHP type I is less than in patients with hormonopenic hypoparathyroidism after normalization of blood calcium levels in each group by vitamin D and oral calcium supplements. 123'124 Moreover, renal handling of calcium (and
FIGURE 17--4 Urinary cAMP excretion in response to an infusion of bovine parathyroid extract (300 USP units). The peak response in normal subjects (A) as well as those with pseudoPHP (not shown) is 50- to 100-fold times basal. Subjects with PHP type Ia (e) or PHP type Ib (o) show only a 2- to 5-fold increase. Urinary cAMP is expressed as nanomoles per deciliter (nM/dl) of GF, UcAMP(nM/dl GF) ~-~ UcAMP (nM/dl) • Scr e (mg/dl) UCr e (mg/dl). (From Levine MA, Jap TS, Mauseth RS, et al: Activity of the stimulatory guanine nucleotidebinding protein is reduced in erythrocytes from patients with pseudohypoparathyroidism and pseudopseudohypoparathyroidism: Biochemical, endocrine, and genetic analysis of Albright's hereditary osteodystrophy in six kindreds. J Clin Endocrinol Metab 62:497-502, 1986.)
511 sodium) in response to exogenous PTH also appears to be normal in patients with PHP type 1.125 These results indicate that calcium reabsorption in the distal tubule is responsive to circulating PTH in subjects with PHP type I, and imply that adequate amounts of cAMP are produced in these cells or that other second messengers (e.g., cytosolic calcium or diacylglycerol) may be responsible for PTH action (Fig. 17-1). Administration of PTH to subjects with the less common form of the disorder, PHP type II, produces a normal increase in urinary cAMP but fails to elicit an appropriate phosphaturic response. 119 These observations have suggested that PTH resistance in PHP type II resuits from a biochemical defect that is either unrelated or distal to the PTH-stimulated generation of cAMP. It has been generally assumed that bone cells in patients with PHP type I are innately resistant to PTH, but this remains unproved. In fact, cultured bone cells from a patient with PHP type I have been shown to increase intracellular cAMP normally in response to PTH treatment in v i t r o . 126 Evidence that bone cells are unresponsive to PTH is largely inferred from the observation that patients with PHP type I are hypocalcemic and that administration of PTH does not increase the plasma calcium level. However, clinical, roentgenographic, or histological evidence of increased bone turnover and demineralization (Fig. 1 7 - 5 ) is common in patients with PHP type I. There is a spectrum of bone disease in PHP: some subjects have apparently normal appearing bone, while others have radiological or histological evidence of significant bone resorption. 127 Patients with PHP type Ia typically have little or no evidence of diffuse skeletal involvement, while patients with PHP type Ib often demonstrate evidence of osteopenia or hyperparathyroid bone disease, including osteifis fibrosa cystica (Fig. 17-5). Cultured bone cells from one patient with PHP type Ib and osteitis fibrosa cystica were shown to have normal adenylyl cyclase responsiveness to PTH in vitro. 126 Patients with PHP may develop additional abnormalities in bone metabolism, including osteomalacia, 127 rickets, 128 renal osteodystrophy, 129 and osteopenia. 13~ These skeletal abnormalities result from excessive PTH or deficient 1,25(OH)2D3. One possible explanation for the variable bone responsiveness to PTH is the existence of two distinct cellular systems in bone upon which PTH exerts action: the remodeling system and the mineral mobilization or homeostatic system. The bone remodeling system appears to be more responsive to PTH in patients with PHP type I than the homeostatic system. This variability may reflect the lesser dependence of the remodeling system upon normal plasma levels of 1,25(OH)2D. Plasma levels of 1,25(OH)2D are reduced in hypocalcemic patients with PHP type I, 40 and could explain the concurrence of
512
FIGURE 17-5 Photograph and radiograph of hands of a patient with marked hyperparathyroid bone disease. Marked periosteal bone erosion in terminal phalanges has resulted in "pseudoclubbing." (From Levine MA, Parfrey NA, Feinstein RS: Pseudohypoparathyroidism. Johns Hopkins Med J 151:137-146, 1982.)
hypocalcemia and increased skeletal remodeling in many of these patients. Hypocalcemia leads to a compensatory overproduction of PTH, which could eventually overcome the 1,25(OH)2D dependency for remodeling but not for PTH-stimulated calcium mobilization. A role for 1,25(OH)2D in modulating the responsiveness of the calcium homeostatic system to PTH is suggested by several observations. First, the calcemic response to PTH is deficient not only in patients with PHP type I, but also in patients with other hypocalcemic disorders in which plasma levels of 1,25(OH)2D are low. Moreover, normalization of the plasma calcium level in patients with PHP type I by administration of physiological amounts of 1,25(OH)2D or pharmacological amounts of vitamin D restores calcemic responsiveness. TM Second, patients with PHP type I who have normal serum levels of calcium and 1,25(OH)2D3 without vitamin D treatment (so-called normocalcemic PHP) show a normal calcemic response to administered PTH. TM These findings suggest that 1,25(OH)2D deficiency is the basis for
MICHAEL A. LEVINE
the lack of a calcemic response to PTH in hypocalcemic patients with PHP type I, and challenge the premise that bone cells are intrinsically resistant to the actions of PTH. Subjects with PHP type I have increased serum levels of phosphate owing to an inability of PTH to decrease phosphate reabsorption in the kidney. Hypocalcemia p e r s e may also contribute to the development of hyperphosphatemia, as renal phosphate clearance is impaired by very low levels of intracellular calcium. Accordingly, restoration of plasma calcium levels to normal by chronic treatment with calcium and vitamin D can reduce elevated levels of serum phosphorus. Similar therapy has been shown to reverse the defective phosphaturic response to administered PTH in certain patients with PHP type I, although the urinary cAMP response remains markedly deficient. 132 Therefore, persistence of a blunted urinary cAMP response to PTH in PHP type I patients in whom chronic vitamin D therapy has led to normalization of plasma calcium levels and restoration of a phosphaturic response need not imply, as has been at least suggested, 132that there is no relationship between cAMP production and phosphate clearance. The overall evidence suggests that the disturbances in calcium, phosphorous, and vitamin D metabolism in most patients with PHP type I result directly or indirectly from reduced responsiveness of both bone and kidney to PTH. Hypocalcemia results from impaired mobilization of calcium from bone, reduced intestinal absorption of calcium [via deficient generation of 1,25(OH)2D], and urinary calcium loss. Of these defects, the diminished movement of calcium out of bone stores into the extracellular fluid probably has the greatest role in producing hypocalcemia. Intensive treatment with calcitriol [1,25(OH)2D] or other vitamin D analogs improves intestinal calcium absorption and bone calcium mobilization, restores plasma calcium to normal, and reduces circulating PTH levels. Thus, although PTH resistance appears to be the proximate biochemical defect, the major abnormalities in mineral metabolism found in patients with PHP type I can be largely explained on the basis of deficiency of circulating 1,25(OH)iD.
B. M o l e c u l a r Classification of P s e u d o h y p o p a r a t h y r o i d i s m Hormone action may be divided conceptually into prereceptor, receptor, and postreceptor events; defects in each of these steps have been proposed as the basis of hormone resistance in PHP (Fig. 17-1). For example, a circulating inhibitor of PTH action has been proposed as a cause of PTH resistance on the basis of studies showing an apparent dissociation between plasma levels of
CHAPTER 17 Hypoparathyroidism and Pseudohypoparathyroidism endogenous immunoreactive and bioactive PTH in subjects with PHP type I. Despite high circulating levels of immunoreactive PTH, the levels of bioactive PTH in many patients with PHP type I have been found to be within the normal range when measured with highly sensitive renal 133 and metatarsal TM cytochemical bioassay systems. Furthermore, plasma from many of these patients has been shown to diminish the biological activity of exogenous PTH in these in vitro bioassays. 135 Currently, the nature of this putative inhibitor or antagonist remains unknown. The observation that prolonged hypercalcemia can remove or reduce significantly the level of inhibitory activity in the plasma of patients with PHP has suggested that the parathyroid gland may be the source of the inhibitor. In addition, analysis of circulating PTH immunoactivity after fractionation of patient plasma by reversed-phase high-performance liquid chromatography has disclosed the presence of aberrant forms of immunoreactive PTH in many of these patients. 136 Although it is conceivable that a PTH inhibitor may cause PTH resistance in some patients with PHP, it is more likely that circulating antagonists of PTH action arise as a consequence of the sustained secondary hyperparathyroidism that results from the primary biochemical defect. By contrast, molecular studies have provided confirmation that defects in the PTH/PTHrP receptorG-protein-coupled signaling pathway are responsible for PTH resistance in many patients with PHP type I. 1.
PSEUDOHYPOPARATHYROIDISM
TYPE IA
AND PSEUDOPSEUDOHYPOPARATHYROIDISM
Cell membranes from patients with PHP type Ia show an approximately 50% reduction in expression or activity of Gs~ protein 137 (Fig. 17-6). This generalized deficiency of Gs~ may impair the ability of PTH, as well as many other hormones and neurotransmitters, to activate
1O 0 ._
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FIGURE 17--6 Gs. activity of erythrocyte membranes. Gs~ is quantified in complementation assays with $49 cyc membranes,which genetically lack Gs. but retain all other components necessary for hormone-response adenylyl cyclase activity. Activity is reduced approximately 50% in patients with AHO subjects with either PHP type Ia or pseudoPHP, but is normal in patients with PHP type Ib.
5 13 adenylyl cyclase and thereby may account for multihormone resistance. In addition to hormone resistance, patients with PHP type Ia also manifest the peculiar constellation of developmental and somatic defects that are collectively termed AHO 117 (Fig. 17-7). Early studies of PHP type Ia led to the identification of families in which some individuals had signs of AHO but lacked apparent hormone resistance (i.e., pseudoPHP). The observation that PHP type Ia and pseudoPHP can occur in the same family first suggested that these two disorders might reflect variability in expression of a single genetic lesion. Further support for this view derives from recent studies indicating that within a given kindred, subjects with either pseudoPHP or PHP type Ia have equivalent functional Gs~ deficiency (Fig. 17--6), 137'138 and that a transition from hormone responsiveness to hormone resistance may O c c u r . 139 It therefore seems reasonable to use the term AHO to simplify description of these two variants of the same syndrome, and to acknowledge the common clinical and biochemical characteristics that patients with PHP type Ia and pseudoPHP share. a. Molecular Defect The recent discovery that Gs~ deficiency results from heterozygous mutations in the gene encoding Gs~ (GNAS1) resolved the controversy surrounding the pattern of inheritance of AHO. Xlinked, 14~ autosomal dominant, 141 and autosomal recessive 142 inheritance of AHO had been proposed. However, the observation of father-to-son transmission of AHO with Gs~ deficiency excluded an X-linked mode of inheritance, 143 and the mapping of the GNAS1 gene to chromosome 20q13.2 --) 13.3144 provided final confirmation that AHO, including both PHP type Ia and pseudoPHP, is inherited in an autosomal dominant fashion. GNAS1 is a complex 20-Kb gene 145 comprised of 15 exons, including 2 alternative first exons. Alternative splicing of a nascent transcript accounts for the production of four mRNAs that encode Gs~. Deletion of exon 3 results in the loss of 15 codons from the mRNA, while use of an alternative splice site in exon 4 results in the insertion of a single additional codon into the mRNA. This generates two Gs~ proteins with apparent molecular weights of 45 kDa and two isoforms of apparent molecular weights of 52 k D a . 145 The alternatively spliced forms of Gs~ show a tissue-specific distribution. ~46 Biochemical characterization of the short and long forms of Gs~ has revealed subtle differences in the binding constant for GDP, the rate at which the forms are activated by agonist binding, efficiency of adenylyl cyclase stimulation, or the rate of GTP hydrolysis. None of these differences appear to be physiologically relevant, h o w e v e r . 147-149 Both long and short forms of Gs~ can stimulate adenylyl cyclase and open calcium channels. ~48
51
4
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I
C
H
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FIGURE 17--7
Typical features of Albright's hereditary osteodystrophy. A, A young woman with characteristic features of AHO; note the short stature, disproportionate shortening of the limbs, obesity, and round face. B, Radiograph of patient's hands showing marked shortening of fourth and fifth metacarpals. C, Archibald sign, the replacement of "knuckles" with "dimples" due to the marked shortening of the metacarpal bones. D, Brachydactyly of the hand, note thumb sign ("murderer's thumb" or "potter's thumb") and shortening of the fourth and fifth digits. [From Levine MA, Schwindinger WE Downs RW, Jr, Moses AM: Pseudohypoparathyroidism. In Bilezikian JP, Marcus R, Levine MA (eds): The Parathyroids: Basic and Clinical Concepts. New York, Raven Press, 1994.]
Additional complexity in the processing of the GNAS1 gene may derive from the use of at least two alternative first exons that generate novel Gs~ transcripts. In one case, a transcript is produced with an exon 1' that lacks an initiator ATG; thus, a truncated, nonfunctional Gs~ protein is translated from an inframe ATG in exon 2.15~ In the other case, a transcript is generated that encodes a larger Gs~ isoform (XL~s) in which a novel 51-kDa protein is spliced to exons 2 through 13.15~Because these two alternative forms of Gs~ lack amino acid sequences encoded by exon 1, which are required for interaction with G~v and attachment to the plasma membrane, it is
likely that neither of these proteins can function as a transmembrane signal transducer. Molecular studies of DNA from subjects with AHO have disclosed a variety of GNAS1 gene mutations 152-16~ that lead to decreased expression or function of Gs~ protein (Fig. 17-8). Although most gene mutations impair expression of Gs~ m R N A , 132'161 in some subjects levels of Gs~ mRNA a r e n o r m a l 138'161'162 and encode dysfunctional Gs~ proteins. 153'154'159 A large variety of mutations in the GNAS1 gene have been identified, including missense mutations, 153'155'157'159point mutations in sequences required for efficient splicing, 156 and small dele-
CHAPTER 17 Hypoparathyroidism and Pseudohypoparathyroidism
I bp deletion 43 bp deletion
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220 240 280 324
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$250R R258W(A) E259V
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Mutations in the GNAS1 gene. The upper panel (A) depicts the human GNAS1 gene, which spans over 20 Kb and contains at least 13 exons and 12 introns. Unique mutations that result in loss of Gs, function are depicted; missense mutations are denoted by the symbol *. The lower panel (B) indicates the position of missense mutations above the protein structure. Two polymorphisms are denoted by the symbol +, and the position of the unchanged amino acid is denoted beneath the predicted Gs~ protein (B). The site of two missense mutations that result in gain of function (replacement of either Arg 2~ or Gln 227) in patients with MAS 214'215'268-271 or in sporadic tumors 271,zvz are depicted in italics. The mutation in exon 1 eliminates the initiator methionine codon and prevents synthesis of a normal Gs~ protein. ~57 The 4-bp deletions in exon 7 163'164 and exon 8,155 and the 1-bp deletion and insertion in exon 10 all shift the normal reading frame and prevent normal m R N A and/or protein synthesis. Mutations in intron 3 and at the donor splice junction between exon 10 and intron 10 cause splicing abnormalities that prevent normal m R N A synthesis. ~56 The mutations indicated with an asterisk represent missense mutations154'~55'159; the resultant amino acid substitutions are indicated in the schematic diagram of the Gs~ protein at the bottom of the figure. Some of these mutations may prevent
FIGURE 17--8
normal protein synthesis by altering protein secondary structure; the R231H substitution in exon 9 prevents normal interaction of the oLchain with the 13~/dimer153; the R385H substitution in exon 13 appears to encode an altered protein that cannot couple normally to receptorsTM and the A366S mutation encodes an activated Gs~ protein that is unstable at 37~
Although novel mutations have been found in nearly all of the kindreds studied, a 4-base deletion in exon 7 has been detected in five families, 163'164 and an unusual missense mutation in exon 13 (A366S; see below) has been identified in two unrelated young boys, ~59 suggesting that these two regions may be genetic " h o t spots." t i o n s . 152'155'156'162
b. Albright's Hereditary Osteodystrophy Subjects with PHP type Ia or pseudoPHP typically manifest a characteristic constellation of developmental defects, termed Albright's hereditary osteodystrophy, that includes short stature, obesity, a round face, shortening of
the digits (brachydactyly), subcutaneous ossification, and dental hypoplasia (Fig. 17-7). 117'118 Considerable variability occurs in the clinical expression of these features even among affected members of a single family, and all of these features may not be present in every case. 165 On rare occasion, it may be impossible to detect any features of AHO in an individual with Gs~ deficiency. ~55'~56 Although patients with AHO may be of normal height and weight, approximately 66% of children and 80% of adults are below the 10th percentile for height. This reflects a disproportionate shortening of the limbs, as arm span is less than height in the majority of patients. Obesity is a common feature of AHO and about one third
5
1
6
M
I
C
of all patients with AHO are above the 90th percentile of weight for their age, despite their short stature ~65 (Fig. 17-7A). Patients with AHO typically have a round face, a short neck, and a flattened bridge of the nose. Numerous other abnormalities of the head and neck have also been noted. Ocular findings include hypertelorism, strabismus, nystagmus, unequal pupils, diplopia, microphthalmia, and a variety of abnormal findings on funduscopic exam that range from irregular pigmentation to optic atrophy and macular degeneration. Head circumference is above the 90th percentile in a significant minority of children. ~66 Dental abnormalities are common in subjects with PHP type Ia and include dentin and enamel hypoplasia, short and blunted roots, and delayed or absent tooth eruption. 167 Brachydactyly is the most reliable sign for the diagnosis of AHO, and may be symmetrical or asymmetrical and involve one or both hands or feet (Fig. 17-7). Shortening of the distal phalanx of the thumb is the most common abnormality; this is apparent on physical exam as a thumb in which the ratio of the width of the nail to its length is increased (so called "Murderer's thumb" or "potter's thumb"; Fig. 17-7D). Shortening of the metacarpals causes shortening of the digits, particularly the fourth and fifth. Shortening of the metacarpals may also be recognized on physical exam as dimpling over the knuckles of a clenched fist (Archibald's sign; Fig. 17-7C). Often, a definitive diagnosis requires careful examination of radiographs of the hands and feet (Fig. 17-7B). A specific pattern of shortening of the bones in the hand has been identified, in which the distal phalanx of the thumb and third through fifth metacarpals are the most severely shortened. 168'169This may be useful in distinguishing AHO from other unrelated syndromes in which brachydactyly occurs, such as familial brachydactyly, Turner's syndrome, and Klinefelter's syndrome. ~69 In addition to brachydactyly, several other skeletal abnormalities are present in AHO. Numerous deformities of the long bones have been reported, including a short ulna, bowed radius, deformed elbow or cubitus valgus, coxa vara, coxa valga, genu varum, and genu valgus deformities. 165 The most common abnormalities of the skull are hyperostosis frontalis interna and a thickened calvarium. The skeletal abnormalities of AHO may not be apparent until a child is 5 years old. 17~ Bone age is advanced 2 to 3 years in the majority of patients. 166 Spinal cord compression has been reported in several patients with AHO. 171 Patients with AHO develop heterotopic ossifications of the soft tissues or skin (osteoma cutis) that are unrelated to abnormalities in serum calcium or phosphorus levels. Osteoma cutis is present in 25% to 50% of cases of AHO, and is usually first noted in infancy or early childhood. Ossification of the skin and subcutaneous tissues may be the presenting cardinal feature of AHO in
H
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infancy or childhood in the absence of hypocalcemia or other features of A H O . 172'173 Blue-tinged, stony hard papular or nodular lesions that range in size from pinpoint up to 5 cm in diameter often occur at sites of minor trauma and may appear to be migratory on repeated exams. 173 Biopsy of these lesions reveals heterotopic ossification with spicules of mineralizing osteoid and calcified cartilage. The presence of these developmental and skeletal abnormalities does not necessarily indicate that the patient has AHO and Gs~ deficiency. Features of AHO, particularly shortened metacarpals or metatarsals, may occur in normal subjects, as well as in patients with hormone deficient hypoparathyroidism, 174-178 renal hypercalciu r i a , 179 and primary hyperparathyroidism. 18~ Moreover, several features of AHO, for example obesity, round face, brachydactyly, and mental retardation, are common to other disorders (e.g., Prader-Willi syndrome, acrodysostosis, Ullrich-Turner syndrome, Gardner's syndrome), many of which are associated with chromosomal defects. In some instances overlapping clinical features between AHO and other syndromes may lead to confusion. For example, AHO in a mother and her daughter has been associated with a proximal 15q chromosomal deletion resembling that found in Prader-Willi syndrome. 181 A growing number of reports have described small terminal deletions of chromosome 2q in patients with variable AHO-like phenotypes. Terminal deletion of 2q37 [del(2)(q37.3)] is the first consistent karyotypic abnormality that has been documented in patients with an AHO-like syndrome. 182'183These patients have normal endocrine function and normal Gso activity, however. 182 Thus, high-resolution chromosome analysis, biochemical/molecular analysis, and careful physical and radiological examination are essential in discriminating between these phenocopies and AHO. c. Multiple Hormone Resistance Although biochemical hypoparathyroidism is the most commonly recognized endocrine deficiency in PHP type Ia, early clinical studies described additional hormonal abnormalities, such as hypothyroidism 184'185 and hypogonadism. 186 Because available evidence suggests that Gs~ is present in all tissues, generalized deficiency of this protein could be the basis for not only PTH resistance, the hallmark of PHP type Ia, but could also explain the decreased responsiveness of diverse tissues (e.g., kidney, thyroid gland, gonads, and liver) to hormones that act via activation of adenylyl cyclase [e.g., PTH, thyroid-stimulating hormone (TSH), gonadotropins, and glucagon]. 123'18v'188 Primary hypothyroidism occurs in most patients with PHP type Ia. ~88 Typically, patients lack a goiter or antithyroid antibodies and have an elevated serum TSH with an exaggerated response to TRH. Serum levels of T4 may be low or low normal. Hypothyroidism may occur early
CHAPTER 17 Hypoparathyroidism and Pseudohypoparathyroidism in life prior to the development of hypocalcemia, and elevated serum levels of TSH are not uncommonly detected during neonatal screening. 189-191 Unfortunately, early institution of thyroid hormone replacement does not seem to prevent the development of mental retardation. 19~ Hypogonadism is common in subjects with PHP type Ia. Women may have delayed puberty, oligomenorrhea, and infertility. ~88 Plasma gonadotropins may be elevated, but are more commonly normal. Some patients show an exaggerated serum gonadotropin response to gonadotropin-releasing hormone ( G n - R H ) . 186'192 Features of hypogonadism may be less obvious in men. Serum testosterone may be normal or reduced. Testes may show evidence of a maturation arrest or may fail to descend normally. Fertility appears to be decreased in men with PHP type Ia. Deficiency of prolactin secretion (basal and in response to secretagogues such as TRH) had been reported in some patients with PHP type I, 193 but later studies have not confirmed these early findings. 188 Abnormal hormone responsiveness may occur in some tissues without obvious clinical sequelae. For example, the hepatic glucose response to glucagon is normal, although plasma cAMP concentrations fail to increase normally. 188'194 In other tissues significant hormone resistance does not occur despite the apparent reduction in Gs~. Diabetes insipidus is not a feature of AHO, and urine is concentrated normally in response to vasopressin in patients with PHP type Ia. 195 Although there is a report of adrenal insufficiency in a single individual with PHP type Ia, 196 hypoadrenalism is not a typical feature of PHP type Ia and adrenocortical responsiveness to ACTH is normal. 188
d. Neurosensory Abnormalities Patients with PHP type Ia frequently manifest distinctive o l f a c t o r y , 197 g u s tatory, 198 and auditory 199 abnormalities that are apparently unrelated to endocrine dysfunction. The molecular basis of these neurosensory deficits has become more obscure with the discovery of unique G proteins that regulate signal transduction pathways related to vision, 2~176176 olfaction, 2~ and taste. 2~ Mild to moderate mental retardation is common in patients with PHP type Ia. Farfel and Friedman assessed intelligence in 25 patients with PHP type I whose Gs~ activity had been determined. 204 The authors found an association between mental deficiency and Gs~ deficiency, and speculated that reduced cAMP levels in cortical tissue may lead to mental retardation. Other factors that might contribute to mental retardation in patients with PHP type Ia include hypothyroidism and hypocalcemia; however, efforts to control these have not prevented cognitive dysfunction in all patients, suggesting that Gs~ deficiency may cause a primary abnormality of neurotransmitter signaling.
517 e. Phenotypic Variability Molecular studies have provided a basis for Gs~ deficiency, but they do not explain the striking variability in biochemical and clinical phenotype. Why do some Gs~-Coupled pathways show reduced hormone responsiveness (e.g., PTH, TSH, gonadotropins), whereas other pathways are clinically unaffected [adrenocorticotropic hormone (ACTH) in the adrenal and vasopressin in the renal medulla]? Perhaps even more intriguing is the paradox that Gs~ deficiency can be associated with hormone resistance and AHO (PHP type Ia), AHO only (pseudoPHP), or no apparent consequences at all. 155 These observations, when considered in the context of studies showing that the number of Gs molecules in cell membranes greatly exceeds the number of either receptor or adenylyl cyclase molecules, 2~ raise issue with the hypothesis that a 50% deficiency of Gs~ can impair hormone responsiveness. Indeed, in vitro studies of the hormone responsiveness of tissues and cells from subjects with PHP type Ia have frequently provided conflicting results. 2~ Although the basis for the variable expression of Gs~ deficiency remains unknown, several observations argue against a simple genetic mechanism. First, clinical genetic studies have documented that PHP type Ia and pseudoPHP frequently occur in the same family, but are not present in the same generation. These findings are inconsistent with models in which chance determines phenotype or in which a second gene is interactive with the defective GNAS1 gene, as both PHP type Ia and pseudoPHP would be expected to occur with equal frequency and in the same sibship. Second, phenotypic expression of Gs~ deficiency appears to become more severe with each generation. 2~ This pattern had originally been attributed to genomic imprinting, 2~176 as reviews of published reports of AHO kindreds indicated that maternal transmission of Gs~ deficiency led to PHP type Ia, whereas paternal transmission of the defect led to pseudoPHP. 2~176176 Several lines of evidence argue against a model of simple genomic imprinting of GNAS1 as the basis for variable phenotype, however. First, analyses of available cells from patients with PHP type Ia and pseudoPHP have shown that these cells contain equivalent amounts of Gs~ protein. 2~ Second, the recent description of both maternal and paternal transmission of PHP type Ia 21~ in a single multiplex family with Gs, deficiency 211 has suggested that parental origin of the GNAS1 mutation may be unrelated to phenotype. And third, both GNAS1 gene alleles are actively transcribed in many fetal 212 and adult ~55'213human tissues, indicating than both GNAS1 alleles are expressed in a wide variety of tissues. These observations suggest that complex mechanisms, such as tissue-specific genomic imprinting, may be responsible for the variable expression of GNAS1 gene defects within members of a kindred. Further modification of the phenotype may derive from variability in
5 18 other components of the signal transduction pathway (e.g., there are multiple forms of adenylyl cyclase and phosphodiesterase that exhibit tissue-specific expression). Regardless of the mechanism, understanding how identical defects in GNAS1 can have such variable consequences in different tissues or in different individuals will reveal much about the biology of the GNAS1 gene. In AHO, inherited GNAS1 gene mutations reduce expression or function of Gs~ protein. By contrast, in the McCune-Albright syndrome, postzygotic somatic mutations in the GNAS1 gene (Fig. 17-8) enhance activity of the protein. 214'215 These mutations lead to constitutive activation of adenylyl cyclase, and result in proliferation and autonomous hyperfunction of hormonally responsive cells. The clinical significance of Gs~ activity as a determinant of hormone action is emphasized by the recent description by Iiri et al. 159 of two males with both precocious puberty and PHP type l a. These two unrelated boys had identical GNAS1 gene mutations in exon 13 (A366S) (Fig. 1 7 - 8 ) that resulted in a temperaturesensitive form of Gs~. This Gs~ is constitutively active in the cooler environment of the testis, while being rapidly degraded in other tissues at normal body temperature. Thus, different tissues in these two individuals could show hormone resistance (to PTH and TSH), hormone responsiveness (to ACTH), or hormone-independent activation (to LH). 2. PSEUDOHYPOPARATHYROIDISMTYPE IB
Subjects with PHP type I who lack features of AHO typically manifest hormone resistance that is limited to PTH target organs (Fig. 17-1) and have normal Gs~ activity (Fig. 17-6). 188 This variant is termed PHP type Ib. 216 Although patients with PHP type Ib fail to show a nephrogenous cAMP response to PTH, they often manifest skeletal lesions similar to those that occur in patients with hyperparathyroidism (Fig. 1 7 - - 5 ) . 217 These observations have suggested that at least one intracellular signaling pathway coupled to the PTH receptor may be intact in patients with PHP type Ib. The molecular basis for PTH resistance in PHP type Ib has not been identified. Specific resistance of target tissues to PTH, and normal activity of Gs~, has implicated decreased expression or function of the PTH/ PTHrP receptor as the cause for hormone resistance, however. Evidence in favor of this hypothesis comes from studies of cultured skin fibroblasts from patients with PHP type Ib. These studies suggest that the mechanisms responsible for PTH resistance are likely to be heterogeneous. Fibroblasts from some, but not all, PHP type Ib patients accumulate less cAMP in response to PTH 216'21s and contain decreased levels of mRNA encoding the PTH/PTHrP receptor. 219 Furthermore, in most cases, pretreatment of the cultured fibroblasts with dex-
MICHAEL A. LEVINE
amethasone normalizes the PTH-induced cAMP response and increases expression of PTH/PTHrP receptor mRNA. 219 Taken in the context of recent molecular studies that have failed to disclose mutations in the coding exons 22~ and promoter regions 221 of the PTH/PTHrP receptor gene or in the c D N A , 222 it is likely that the molecular defect in PHP type Ib resides in another gene(s) that regulates expression of the PTH/PTHrP receptor. Although most cases of PHP type l b appear to be sporadic, familial cases have been described in which transmission of the defect is most consistent with an autosomal dominant pattern. 12~ 3. PSEUDOHYPOPARATHYROIDISMTYPE IC
Resistance to multiple hormones has been described in several patients with AHO who do not have a demonstrable defect in Gs or Gi .172'188'224 This disorder is termed PHP type Ic. The nature of the lesion in such patients is unclear, but it could be related to some other general component of the receptor-adenylyl cyclase system, such as the catalytic unit. 225 Alternatively, these patients could have functional defects of Gs (or Gi) that do not become apparent in the assays presently available. 4. PSEUDOHYPOPARATHYROIDISMTYPE II
PHP type II is the least common form of PHE This variant of PHP is typically a sporadic disorder, although one case of familial PHP type II has been reported. 226 Patients do not have features of AHO. Renal resistance to PTH in PHP type II patients is manifested by a reduced phosphaturic response to administration of PTH, despite a normal increase in urinary cAMP excretion. 119 These observations suggest that the PTH receptoradenylyl cyclase complex functions normally to increase cAMP in response to PTH, and are consistent with a model in which PTH resistance arises from an inability of intracellular cAMP to initiate the chain of metabolic events that result in the ultimate expression of PTH action. Although supportive data are not yet available, a defect in cAMP-dependent protein kinase A has been proposed as the basis for this disorder. 119 Alternatively, the defect in PHP type II may not reside in an inability to generate a physiological response to intracellular cAMP: a defect in another PTH-sensitive signal transduction pathway may explain the lack of a phosphaturic response. One candidate is the PTH-sensitive phospholipase C pathway that leads to increased concentrations of the intracellular second messengers inositol 1,4,5trisphosphate and diacylglycero121'22 and cytosolic calcium 23'24'227'228 (Fig. 17 - 1 ) . In some patients with PHP type II the phosphaturic response to PTH has been restored to normal after serum levels of calcium have been normalized by treatment
CHAPTER 17 Hypoparathyroidismand Pseudohypoparathyroidism with calcium infusion or vitamin D . 229 These results point to the importance of Ca 2§ as an intracellular second messenger. Finally, a similar dissociation between the effects of PTH on generation of cAMP and tubular reabsorption of phosphate has been observed in patients with profound hypocalcemia due to vitamin D deficiency, 23~ suggesting that some cases of PHP type II may in fact represent vitamin D deficiency.
C. Natural History of Pseudohypoparathyroidism The natural history of PHP is quite variable. Although PHP is congenital, hypocalcemia is not present from birth, and the biochemical defects arise gradually during childhood. The initial manifestations of tetany typically occur between 3 and 8 years of age, but the significance of these findings may not be appreciated and the diagnosis of hypocalcemia may be delayed for months or even years. A progressive decline in serum calcium, preceded by increasing levels of serum phosphate, PTH, and 1,25(OH)2D3, has been documented in one child as he advanced from 3 to 31/2years of age. TM In a second report serial PTH infusions were used to evaluate hormone responsiveness. This child was shown to have a normal cAMP response at age 3 months when serum levels of calcium, phosphorous, and PTH were normal, but was found to have an abnormal cAMP response when retested at age 2.6 years after he had developed tetany and was found to be hypocalcemic. 139 At the time of his second PTH infusion, the child had markedly elevated serum concentrations of phosphorous and PTH and was receiving thyroxine for recently diagnosed hypothyroidi s m . 139 Some affected children show few symptoms of tetany and the diagnosis of PHP is recognized only later in life after hypocalcemia is inadvertently discovered or when features of AHO become obvious. Hypocalcemia may not always provide a clue to the clinical diagnosis of PHP, however, as some PHP patients are able to maintain a normal serum calcium level without treatment (i.e., normocalcemic PHP). 132
VI. DIAGNOSIS The diagnosis of hypoparathyroidism should be considered in any patient who has hypocalcemia and hyperphosphatemia. A low serum calcium level may be found during an evaluation of unexplained paresthesias or seizures, or may be discovered after multichannel analysis of a blood specimen obtained as part of a rou-
5 19 tine examination. PHP should be strongly suspected if the serum concentration of PTH is elevated, although occasionally serum levels of PTH are "inappropriately" normal in subjects with PHP owing to confounding hypomagnesemia 223 or other factors. 232 Hypocalcemia may be precipitated or worsened during times of "stress" on calcium homeostasis, such as during early pregnancy, lactation, or during an episode of acute pancreatitis. Although hypocalcemia is present in most patients with hypoparathyroidism by the end of the first decade of life, this biochemical finding may go undetected for many years. Cataracts and intracranial calcification, particularly of the basal ganglion, occur commonly in patients with all forms of chronic hypoparathyroidism. Thus the presence of these ectopic or metastatic calcifications does not help to discriminate among the various causes of hypocalcemia and hyperphosphatemia. 233 Intracranial calcifications are readily detected when CT scanning is employed, 234'235 and may occasionally be associated with symptoms such as Parkinson's disease. 236 Unusual presenting manifestations of PHP include neonatal hypothyroidism, 19~ unexplained cardiac failure, 237 Parkinson's disease, 236 and spinal cord compression. 238 A diagnosis of hypoparathyroidism can be made with reasonable certainty when hypocalcemia is accompanied by normal serum levels of phosphorous and magnesium and the plasma PTH concentration is low or inappropriately normal. By contrast, an elevated level of PTH should suggest the diagnosis of PHP, particularly when clinical features of AHO are present. Further corroboration of the diagnosis of PHP requires demonstration of normal renal function and normal serum levels of magnesium and 25(OH)D. The presence of AHO and/or manifestations of multihormone resistance, such as hypothyroidism or hypogonadism, favors a diagnosis of PHP type Ia. 188 When most or all of these features are present, more sophisticated tests may not be necessary to confirm the clinical diagnosis. Serum calcium levels can fluctuate in patients with PHP, and may spontaneously change from low to normal and vice versa, thus contributing to the confusion regarding the distinction between PHP and pseudoPHp.131'239 However, the abnormal cAMP response to administered PTH (below) does not become normal in PHP patients who become normocalcemic with or without treatment. Thus, the PTH infusion remains the most reliable test to distinguish between these two variants (Fig. 17-4).
A. Specialized Tests The biochemical hallmark of PHP is the failure of the target organs, bone and kidney, to respond normally to
520 PTH. Additional tests have been developed to identify subjects with PHP type Ia; these research tests, which are based on analysis of Gs~ protein or the GNAS1 gene, are only rarely indicated under typical clinical circumstances. The classical tests of Ellsworth and Howard, and of Chase, Melson, and Aurbach, involved the intravenous infusion of 200 to 300 USP units of bovine parathyroid extract (parathyroid injection, USP; Lilly) and subsequent measurement of urinary excretion of nephrogenous (or total) cAMP (Fig. 17-4) and phosphate. This relatively crude PTH preparation is no longer available, and has been replaced by synthetic peptides corresponding to the amino-terminal region of human PTH [e.g., Parathar; teriparatide acetate; hPTH(1-34); Rhone-Poulanc Rorer]. After infusion of synthetic hPTH(1-34), normal subjects and patients with hormonopenic hypoparathyroidism usually display a 10- to 20fold increase in urinary cAMP excretion, whereas patients with PHP type I (type Ia and type Ib) show a markedly blunted response regardless of their serum calcium concentration. The urinary cAMP response to infusion of synthetic hPTH fragments in patients with PHP type I is unrelated to serum calcium levels, but may be related to endogenous serum PTH levels. The maximal urinary cAMP response to PTH increases after suppression of endogenous PTH in patients with PHP type I, but nevertheless does not reach that of the normal range. 125 Thus, this test can distinguish patients with socalled normocalcemic PHP (i.e., patients with PTH resistance who are able to maintain normal serum calcium levels without treatment) from subjects with pseudoPHP (who will have a normal urinary cAMP response to PTH 12~'137) (Fig. 17-4). Tests that measure the calcemic response to PTH are no longer used as a means of diagnosing PHP. The definitive diagnosis of PHP at the present time depends on the demonstration of a deficient cAMP, 1,25(OH)2D3, or phosphate response to an active preparation of PTH. Several diagnostic protocols have been described in which the response to an infusion of synthetic hPTH(1-34) peptide is used to differentiate among disorders of PTH responsiveness. 24~ A standard protocol involves the infusion of this PTH fragment, 200 units in an adult and 3 units/kg body weight (200 units maximum) in children over the age of 3 years, intravenously over 10 m i n u t e s . 24~ Test subjects should be in a fasting state and active urine output should be initiated and maintained by the ingestion of 200 ml of water per hour, 2 hours prior to the infusion of PTH and continuing through the study. A baseline urine collection should be made in a 60-minute period preceding the PTH infusion. Starting at time 0 urine should be collected in separate collections at the 0- to 30-minute, 30to 60-minute and 60- to 120-minute time periods. Blood
MICHAEL A. LEVINE
samples should be obtained at time 0 and at 2 hours after the start of PTH infusion for measurement of serum creatinine and phosphorus concentrations. Urine samples should be analyzed for cAMP, phosphorus, and creatinine concentrations. The preferred unit for expression of urinary cAMP is nM/100 ml (or per liter) of glomerular filtrate (nM/dl GF). The cAMP response during the first 30 minutes after the start of PTH infusion best differentiates patients with PHP type I from those with hypoparathyroidism and from normal subjects than other parameters of cAMP metabolism. 243 The change in urinary cAMP per milligram creatinine during the same 30-minute period also discriminates well between patients with PHP and normal subjects or patients with hypoparathyroidism. 243 Several metabolic abnormalities such as hypo- and hypermagnesemia and metabolic acidosis may interfere with the renal generation and excretion of cAMP in response to P T H . 68'244-246 These abnormalities should be corrected if possible, but probably do not interfere with the interpretation of the test. Calculation of the phosphaturic response to PTH as the percentage decrease in tubular maximum for phosphate reabsorption (percentage fall in TmP/GF) during the first hour after PTH infusion yields the best separation between normal subjects and patients with PHP or hypoparathyroidism. 243 However, distinction between groups is also possible when the results are expressed as the fall in percentage tubular reabsorption of phosphorus (decrease in % TRP). A nomogram has been developed that facilitates calculation of TmP/GFR. 247 TmP/GFR is elevated in patients with PHP and hypoparathyroidism. Patients with hormone-deficient hypoparathyroidism have a steep fall in TmP/GFR during the first hour after beginning the infusion of PTH. This fall does not occur in patients with PHP (for further details see references by Mallette et al.24~ Although a normal phosphate response may occur in PHP type I patients with serum calcium or PTH levels in the normal range, 125 in patients with PHP type II the phosphaturic response to PTH is not changed despite at least a tenfold increase in cAMP excretion. Unfortunately, interpretation of the phosphaturic response to PTH is often complicated by random variations in phosphate clearance, and it is sometimes not possible to classify a phosphaturic response as normal or subnormal regardless of the criteria employed. More perplexing yet is the observation that biochemical findings that resemble PHP type II have been found in patients with various forms of vitamin D deficiency. 23~ In these patients, marked hypocalcemia is accompanied by hyperphosphatemia due presumably to an acquired dissociation between the amount of cAMP generated in the renal tubule and its effect on phosphate clearance.
CHAPTER 17 Hypoparathyroidism and Pseudohypoparathyroidism The plasma cAMP response to PTH can also be used to differentiate patients with PHP type I from normal subjects and from patients with hypoparathyroidi s m . 242'248'249 Patients with PHP type II can be expected to have normal responsiveness, however. This test offers few advantages over protocols that assess the urinary excretion of cAMP, as changes in plasma cAMP in normal subjects and patients with hypoparathyroidism are much less dramatic than changes in urinary cAMP, and urine must still be collected if one wishes to assess the phosphaturic response to PTH. One reasonable indication for measuring the plasma cAMP response to PTH is the evaluation of patients in whom proper collection of urine is not possible, such as young children. 248 The plasma 1,25(OH)2D3 response to PTH has been used to differentiate between hormone-deficient and hormone-resistant hypoparathyroidism. 241'25~ In contrast to normal subjects and patients with hypoparathyroidism, patients with PHP had no significant increase in circulating levels of 1,25(OH)2D3. This proposed test readily demonstrates the difference in the pathophysiology between hypoparathyroidism and PHP. Its clinical relevance is probably limited to distinguishing type I from type II PHP where the expected increase in the latter form of PHP might be a more reliable parameter than the phosphaturic response to PTH.
VII. TREATMENT Urgent treatment of acute or severe symptomatic hypocalcemia in patients with hypoparathyroidism is best accomplished by the intravenous infusion of calcium. Vitamin D is not required. The goal is alleviation of symptoms and prevention of laryngeal spasm and seizures. Hyperphosphatemia, alkalosis, and hypomagnesemia should be corrected. The serum calcium should be increased to the mid-normal range. The desired serum calcium levels can usually be obtained by injecting 1 to 3 g of calcium gluconate (93 to 279 mg of elemental calcium, 10 to 30 ml of 10% calcium gluconate) over a 10-minute period followed by continuous infusion of calcium (up to 100 mg/hr) using a solution of D5W containing 100 ml of 10% calcium gluconate (930 mg of elemental calcium) per liter. A 10% solution of calcium chloride is available for intravenous use but it is very irritating to the veins. The serum calcium level should be measured at frequent intervals, and the amount of intravenous calcium should be adjusted accordingly. Electrocardiographic monitoring is advisable when patients are receiving digitalis-like drugs because increasing serum calcium levels can predispose to digitalis toxicity. Oral calcium and vitamin D therapy should be
521 started as soon as possible and gradually adjusted to replace the need for intravenous calcium. TM The long-term treatment of hypocalcemia in patients with hypoparathyroidism involves the administration of oral calcium and vitamin D or analogues. Patients with PHP may require less intensive therapy than patients with PTH deficiency. The goals of therapy are to maintain serum ionized calcium levels in the normal range, to avoid hypercalciuria, and, in patients with PHP, to suppress PTH levels. Patients with hypoparathyroidism have increased urinary calcium excretion in relation to serum calcium and are therefore prone to hypercalciuria. 252 By contrast, patients with PHP have significantly lower urinary calcium in relation to s e r u m c a l c i u m 252'253 and can tolerate serum calcium levels that are within the normal range without developing hypercalciuria. TM Once normocalcemia has been attained, attention should be directed towards suppression of PTH levels to normal. This is important because elevated PTH levels in patients with PHP are frequently associated with increased bone remodeling. Hyperparathyroid bone disease, including osteitis fibrosa cystica 17~ and cortical osteopenia (Fig. 17-5), ~3~can occur in patients with PHP type Ib. These subjects may have elevated serum levels of alkaline phosphatase T M and urine hydroxyproline. 13~ In this regard calcitriol has an advantage over other vitamin D preparations, since it may inhibit PTH release directly 255 in addition to the indirect inhibition caused by elevating the serum calcium. Oral calcium is usually administered in amounts from 1 to 3 g of elemental calcium per day in divided doses. To assure optimal absorption, oral calcium supplements should be taken with water or other fluids, and with food in the stomach. 256 Many considerations are involved in the selection of a calcium supplement, and none are unique to the treatment of hypoparathyroidism. Calcium carbonate is an inexpensive form of calcium that is very convenient owing to its high content of elemental calcium (40%). When taken with food, absorption of calcium from calcium carbonate is adequate even in achlorhydric patients. Due to the low content of elemental calcium in calcium lactate (13%) and calcium gluconate (9%), patients must take many tablets to obtain adequate amounts of calcium. Thus, these salts are inconvenient and are often not acceptable to the patients. Calcium citrate is 21% calcium and is well absorbed even in the absence of gastric acid) 57 Although more expensive than many other forms of calcium, calcium citrate has the advantage of causing fewer gastrointestinal side effects. For those who prefer a liquid calcium supplement, calcium glubionate is very palatable and contains 252 mg calcium/10 ml. Ten to 30 ml of a 10% calcium chloride solution (360 to 1080 mg calcium) every 8 hours may
522 be very effective in patients with achlorhydria. 258 Hyperchloremic acidosis may occur, which can be prevented by giving half of the calcium as chloride and half as carbonate simultaneously. 258 Calcium phosphate salts should be avoided. All patients with hypoparathyroidism who are hypocalcemic will require vitamin D or analogues in addition to calcium. Calcitriol, the active form of vitamin D, is the most physiological treatment choice. Patients with PHP require about 75% as much calcitriol to maintain normocalcemia as do patients with hypoparathyroidi s m . 259 Almost all patients with hypoparathyroidism or PHP can be effectively treated with calcitriol in the amount of 0.25 Ixg twice a day to 0.5 Ixg four times a day. Because of the expense of calcitriol and the need to administer the drug several times per day, other vitamin D preparations may be preferred. Patients with all forms of hypoparathyroidism and PHP will respond to pharmacological doses of ergocalciferol and calcifidiol. Ergocalciferol is the least expensive choice for vitamin D therapy, and provides a long duration of action (with corresponding prolonged potential toxicity). Patients with PHP require lower doses of vitamin D than patients with hypoparathyroidism, 259 an observation that reflects the response of bone and renal distal tubular cells to endogenous P T H . 26~ Treatment with calcium and vitamin D usually decreases the elevated serum phosphate to a high normal level because of a favorable balance between increased urinary phosphate excretion and decreased intestinal phosphate absorption. In general, phosphate-binding gels such as aluminum hydroxide are not necessary. Attention should be directed to a number of special situations. Because thiazide diuretics can increase renal calcium reabsorption in patients with hypoparathyroidism, 261-263 the inadvertent institution or discontinuation of these drugs may respectively increase or decrease plasma calcium levels. 264 By contrast, furosamide and other loop diuretics can increase renal clearance of calcium and depress serum calcium levels. The administration of glucocorticoids antagonizes the action of vitamin D (and analogues) and may also precipitate hypocalcemia. The development of hypomagnesemia may also interfere with the effectiveness of treatment with calcium and vitamin D . 265 Experimental treatments for hypoparathyroidism include transplantation of cultured human parathyroid c e l l s 266 and daily injection of synthetic human P T H ( 1 3 4 ) . 267 P T H ( 1 - 3 4 ) has been shown to effectively normalize serum concentrations of calcium and phosphorous while causing less hypercalciuria than treatment with comparable doses of calcitriol. 267 Unfortunately, uncertainties regarding the future availability of human PTH(1-34), and the need to administer the drug by in-
MICHAEL A. LEVINE
jection, lessen overall enthusiasm for this form of treatment. Patients with AHO may require specific treatment for unusual problems related to their developmental and skeletal abnormalities. Patients with PHP type Ia should be treated for their associated hypogonadism and hypothyroidism. Ectopic calcification occurs in about 30% of patients with A H O , 166 but rarely causes a problem. However, at times large extraskeletal osteomas may o c c u r . 173 These may require surgical removal to relieve pressure symptoms.
VIII.
CONCLUSION
The identification, and molecular characterization, of the signal transduction pathways that regulate PTH secretion and action have facilitated development of new approaches to the investigation of the hypoparathyroidism and PHP. Advanced immunoassays now make possible the accurate and precise measurement of circulating concentrations of biologically active PTH, and innovative genetic techniques now offer the promise of future molecular diagnosis of these disorders. Ultimately, the insights gained from studies of these unusual patients will provide new information concerning the physiological regulation of PTH responsiveness in classical target tissues, such as bone and kidney, as well as in nonclassical targets. As with many other human disorders for which the disease gene has been identified, it is predicted that ability to diagnose these disorders on a molecular level will extend the clinical spectrum of disease. This prediction has already been fulfilled through our understanding of defects in genes encoding Gs~ and the calcium-sensing receptor. Future work will be directed towards identification of the molecular basis for other forms of hypoparathyroidism and PTH resistance so that all disorders of PTH action can be described on the basis of their pathophysiology.
Acknowledgments This work has been supported in part by grants from the National Institutes of Health (DK-34281 and DK-46720).
References 1. Marshall RW: Plasma fractions. In Nordin BEC (ed): Calcium, Phosphate and Magnesium Metabolism. London, Churchill Livingstone, 1976, p 162.
CHAPTER 17
Hypoparathyroidism and Pseudohypoparathyroidism
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CHAPTER 17
Hypoparathyroidism and Pseudohypoparathyroidism
162. Mallet E, Carayon P, Amr S, et al: Coupling defect of thyrotropin receptor and adenylate cyclase in a pseudohypoparathyroid patient. J Clin Endocrinol Metab 54:1028-1032, 1982. 163. Weinstein LS, Gejman PV, de Mazancourt P, et al: A heterozygous 4-bp deletion mutation in the Gsa gene (GNAS 1) in a patient with Albright hereditary osteodystrophy. Genomics 13: 1319-1321, 1992. 164. Yu S, Yu D, Hainline BE, et al: A deletion hot-spot in exon 7 of the Gs alpha gene (GNAS1) in patients with Albright hereditary osteodystrophy. Hum Mol Genet 4:2001 - 2002, 1995. 165. Faull CM, Welbury RR, Paul B, Kendall Taylor P: Pseudohypoparathyroidism: Its phenotypic variability and associated disorders in a large family. Q J Med 78:251-264, 1991. 166. Fitch N: Albright's hereditary osteodystrophy: A review. Am J Med Genet 11:11 - 29, 1982. 167. Croft LK, Witkop CJ, Glas J: Pseudohypoparathyroidism. Oral Surg Oral Med Oral Pathol 20:758-770, 1965. 168. Graudal N, Galloe A, Christensen H, Olesen K: The pattem of shortened hand and foot bones in D- and E-brachydactyly can pseudohypoparathyroidism / pseudopseudohypoparathyroidism. ROFO Fortschr Geb Rontgenstr Nuklearmed 148:460-462, 1988. 169. Poznanski AK, Werder EA, Giedion A: The pattem of shortening of the bones of the hand in PHP and P P H P - - a comparison with brachydactyly E, Tumer syndrome, and acrodysostosis. Radiol 123:707-718, 1977. 170. Steinbach HL, Rudhe U, Jonsson M, et al: Evolution of skeletal lesions in pseudohypoparathyroidism. Radiol 85:670-676, 1965. 171. Alam SM, Kelly W: Spinal cord compression associated with pseudohypoparathyroidism. J R Soc Med 83:50-51, 1990. 172. Izraeli S, Metzker A, Horev G, et al: Albright hereditary osteodystrophy with hypothyroidism, normocalcemia, and normal Gs protein activity. Am J Med 43:764-767, 1992. 173. Prendiville JS, Lucky AW, Mallory SB, et al: Osteoma curls as a presenting sign of pseudohypoparathyroidism. Pediatr Dermatol 9:11 - 18, 1992. 174. Le Roith D, Burshell AC, Ilia R, Glick SM: Short metacarpal in a patient with idiopathic hypoparathyroidism. Isr J Med Sci 15:460-461, 1979. 175. Izraeli S, Metzker A, Horev G, et al: Albright hereditary osteodystrophy with hypothyroidism, normocalcemia, and normal Gs protein activity: A family presenting with congenital osteoma cutis. Am J Med Genet 43:764-767, 1992. 176. Shapira H, Friedman E, Mouallem M, Farfel Z: Familial A1bright's hereditary osteodystrophy with hypoparathyroidism: Normal structural Gsalpha gene. J Clin Endocrinol Metab 81: 1660-1662, 1996. 177. Isozaki O, Sato K, Tsushima T, et al: A patient of short stature with idiopathic hypoparathyroidism, round face and metacarpal signs. Endocrinol Jpn 31:363-367, 1984. 178. Moses AM, Rao KJ, Coulson R, Miller R: Parathyroid hormone deficiency with Albright's hereditary osteodystrophy. J Clin Endocrinol Metab 39:496-500, 1974. 179. Moses AM, Notman DD: Albright's osteodystrophy in a patient with renal hypercalciuria. J Clin Endocrinol Metab 49:794-797, 1979. 180. Sasaki H, Zsutsu N, Asano T, et al: Co-existing primary hyperparathyroidism and Albright's hereditary osteodystrophy--an unusual association. Postgrad Med J 61:153 - 155, 1985. 181. Hedeland H, Bemtorp K, Arheden K, Kristoffersson U: Pseudohypoparathyroidism type I and Albright's hereditary osteodystrophy with a proximal 15q chromosomal deletion in mother and daughter. Clin Genet 42:129-134, 1992.
527 182. Phelan MC, Rogers RC, Clarkson KB, et al: Albright hereditary osteodystrophy and del(2)(q37.3) in four unrelated individuals. Am J Med Genet 58:1-7, 1995. 183. Wilson LC, Leverton K, Oude Luttikhuis ME, et al: Brachydactyly and mental retardation: An Albright hereditary osteodystrophy-like syndrome localized to 2q37. Am J Hum Genet 56: 400-407, 1995. 184. Werder EA, Illig R, Bernsasconi S, et al: Excessive thyrotropinreleasing hormone in pseudohypoparathyroidism. Pediatr Res 9: 12-16, 1975. 185. Marx SJ, Hershman JM, Aurbach GD: Thyroid dysfunction in pseudohypoparathyroidism. J Clin Endocrinol Metab 33:822828, 1971. 186. Wolfsdorf JI, Rosenfield RL, Fang VS, et al: Partial gonadotrophin-resistance in pseudohypoparathyroidism. Acta Endocrinol 88:321-328, 1978. 187. Tsai KS, Chang CC, Wu DJ, et al: Deficient erythrocyte membrane Gs alpha activity and resistance to trophic hormones of multiple endocrine organs in two cases of pseudohypoparathyroidism. Taiwan I Hsueh Hui Tsa Chih 88:450-455, 1989. 188. Levine MA, Downs RW Jr, Moses AM, et al: Resistance to multiple hormones in patients with pseudohypoparathyroidism. Association with deficient activity of guanine nucleotide regulatory protein. Am J Med 74:545-556, 1983. 189. Yokoro S, Matsuo M, Ohtsuka T, Ohzeki T: Hyperthyrotropinemia in a neonate with normal thyroid hormone levels: The earliest diagnostic clue for pseudohypoparathyroidism. Biol Neonate 58:69-72, 1990. 190. Weisman Y, Golander A, Spirer Z, Farfel Z: Pseudohypoparathyroidism type Ia presenting as congenital hypothyroidism. J Pediatr 107:413-415, 1985. 191. Levine MA, Jap TS, Hung W: Infantile hypothyroidism in two sibs: An unusual presentation of pseudohypoparathyroidism type Ia. J Pediatr 107:919-922, 1985. 192. Downs RW Jr, Levine MA, Drezner MK, et al: Deficient adenylate cyclase regulatory protein in renal membranes from a patient with pseudohypoparathyroidism. J Clin Invest 71:231235, 1983. 193. Carlson HE, Brickman AS, Bottazzo CF: Prolactin deficiency in pseudohypoparathyroidism. N Engl J Med 296:140-144, 1977. 194. Brickman AS, Carlson HE, Levin SR: Responses to glucagon infusion in pseudohypoparathyroidism. J Clin Endocrinol Metab 63:1354-1360, 1986. 195. Moses AM, Weinstock RS, Levine MA, Breslau NA: Evidence for normal antidiuretic response to endogenous and exogenous arginine vasopressin in patients with guanine nucleotide-binding stimulatory protein-deficient pseudohypoparathyroidism. J Clin Endocrinol Metab 62:221-224, 1986. 196. Ridderskamp I, Schlaghecke R: Klin Wochenschr 68:927-931, 1990. 197. Weinstock RS, Wright HN, Speigel AM, et al: Olfactory dysfunction in humans with deficient guanine nucleotide-binding protein. Nature 322:635-636, 1986. 198. Henkin RI: Impairment of olfaction and of the tastes of sour and bitter in pseudohypoparathyroidism. J Clin Endocrinol Metab 28:624, 1968. 199. Koch T, Lehnhardt E, Bottinger H, et al: Sensorineural heating loss owing to deficient G proteins in patients with pseudohypoparathyroidism: Results of a multicentre study. Eur J Clin Invest 20:416-421, 1990. 200. Lochrie MA, Hurley JB, Simon MI: Sequence of the alpha subunit of photoreceptor G protein: Homologies between transducin, ras, and elongation factors. Science 228:96-99, 1985.
528 201. Lerea CL, Somers DE, Hurley JB, et al: Identification of specific transducin alpha subunits in retinal rod and cone photoreceptors. Science 234:77-80, 1986. 202. Jones DT, Reed RR: Golf: An olfactory neuron specific-G protein involved in odorant signal transduction. Science 244:790795, 1989. 203. McLaughlin SK, McKinnon PJ, Margolskee RF: Gustducin is a taste-cell specific G protein closely related to the transducins. Nature 357:563-568, 1992. 204. Farfel Z, Friedman E: Mental deficiency in pseudohypoparathyroidism type I is associated with Ns-protein deficiency. Ann Intern Med 105:197-199, 1986. 205. Levis MJ, Bourne HR: Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity. J Cell Biol 119:1297-1307, 1992. 206. Levine MA: Pseudohypoparathyroidism. In Bilezikian JP, Raisz LG, Rodan GA (eds): Principles of Bone Biology. San Diego, Academic Press, 1996, pp 853-876. 207. Wilson LC, Trembath RC: Albright's hereditary osteodystrophy. J Med Genet 31:779-784, 1994. 208. Davies SJ, Hughes HE: Imprinting in Albright's hereditary osteodystrophy. J Med Genet 30:101 - 103, 1993. 209. Wilson LC, Oude Luttikhuis ME, Clayton PT, et al: Parental origin of Gs alpha gene mutations in Albright's hereditary osteodystrophy. J Med Genet 31:835-839, 1994. 210. Schuster V, Kress W, Kruse K: Paternal and maternal transmission of pseudohypoparathyroidism type Ia in a family with A1bright hereditary osteodystrophy: No evidence of genomic imprinting [letter]. J Med Genet 31:84, 1994. 211. Schuster V, Eschenhagen T, Kruse K, et al: Endocrine and molecular biological studies in a German family with Albright hereditary osteodystrophy. Eur J Pediatr 152:185-189, 1993. 212. Campbell R, Gosden CM, Bonthron DT: Parental origin of transcription from the human GNAS 1 gene. J Med Genet 31:607614, 1994. 213. Namnoum AB, Merriam GR, Moses AM: Ovarian dysfunction in Albright's hereditary osteodystrophy. J Clin Endocrinol Metab 1997 (in press). 214. Weinstein LS, Shenker A, Gejman PV, et al: Activating mutations of the stimulatory G protein in the McCune-Albright syndrome. N Engl J Med 325:1688-1695, 1996. 215. Schwindinger WE Francomano CA, Levine MA: Identification of a mutation in the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase in McCune-Albright syndrome. Proc Natl Acad Sci USA 89:5152-5156, 1992. 216. Silve C, Santora A, Breslau N, et al: Selective resistance to parathyroid hormone in cultured skin fibroblasts from patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 62:640-644, 1986. 217. Kidd GS, Schaaf M, Adler RA, et al: Skeletal responsiveness in pseudohypoparathyroidism: A spectrum of clinical disease. Am J Med 68:772-781, 1980. 218. Silve C, Suarez F, el Hessni A, et al: The resistance to parathyroid hormone of fibroblasts from some patients with type Ib pseudohypoparathyroidism is reversible with dexamethasone. J Clin Endocrinol Metab 71:631-638, 1990. 219. Suarez F, Lebrun JJ, Lecossier D, et al: Expression and modulation of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in skin fibroblasts from patients with type Ib pseudohypoparathyroidism. J Clin Endocrinol Metab 80:965-970, 1995. 220. Schipani E, Weinstein LS, Bergwitz C, et al: Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related
MICHAEL A. LEVINE
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238. Cavallo A, Meyer WJ III, Bodensteiner JB, Chesson AL: Spinal cord compression: An unusual manifestation of pseudohypoparathyroidism. Am J Dis Child 134:706-707, 1980. 239. Breslau NA, Notman D, Canterbury JM, Moses AM: Studies on the attainment of normocalcemia in patients with pseudohypoparathyroidism. Am J Med 68:856-860, 1980. 240. Mallette LE: Synthetic human parathyroid hormone 1-34 fragment for diagnostic testing. Ann Intern Med 109:800-804, 1988. 241. McElduff A, Lissner D, Wilkinson M, et al: A 6-hour human parathyroid hormone (1-34) infusion protocol: Studies in normal and hypoparathyroid subjects. Calcif Tissue Int 41:267-273, 1987. 242. Furlong TJ, Seshadri MS, Wilkinson MR, et al: Clinical experiences with human parathyroid hormone 1-34. Aust N Z J Med 16:794-798, 1986. 243. Mallette LE, Kirkland JL, Gagel RF, et al: Synthetic human parathyroid hormone-(1-34) for the study of pseudohypoparathyroidism. J Clin Endocrinol Metab 67:964-972, 1988. 244. Beck N, Kim HP, Kim KS: Effect of metabolic acidosis on renal action of parathyroid hormone. Am J Physiol 228:1483-1488, 1975. 245. Beck N, Davis BB: Impaired renal response to parathyroid hormone in potassium depletion. Am J Physio1228:179-183, 1975. 246. Slatopolsky E, Mercado A, Morrison A, et al: Inhibitory effects of hypomagnesemia on the renal action of parathyroid hormone. J Clin Invest 58:1273-1279, 1976. 247. Walton RJ, Bijvoet OLM: Nomogram for derivation of renal threshold phosphate concentration. Lancet 309:310, 1975. 248. Stirling HF, Darling JA, Barr DG: Plasma cyclic AMP response to intravenous parathyroid hormone in pseudohypoparathyroidism. Acta Paediatr Scand 80:333-338, 1991. 249. Sohn HE, Furukawa Y, Yumita S, et al: Effect of synthetic 1-34 fragment of human parathyroid hormone on plasma adenosine 3', 5'-monophosphate (cAMP) concentrations and the diagnostic criteria based on the plasma cAMP response in EllsworthHoward test. Endocrinol Jpn 31:33-40, 1984. 250. Mirua R, Yumita S, Yoshinaga K, Furukawa Y: Response of plasma 1,25-dihydroxyvitamin D in the human PTH(1-34) infusion test: An improved index for the diagnosis of idiopathic hypoparathyroidism and pseudohypoparathyroidism. Calcif Tissue Int 46:309-313, 1990. 251. Lebowitz MR, Moses AM: Hypocalcemia. Semin Nephrol 12: 146-158, 1992. 252. Litvak J, Moldawer MP, Forbes AP, Henneman PH: Hypocalcemic hypercalciuria during vitamin D and dihydrotachysterol therapy of hypoparathyroidism. J Clin Endocrinol Metab 18: 246-252, 1958. 253. Yamamoto M, Takuwa Y, Masuko S, Ogata E: Effects of endogenous and exogenous parathyroid hormone on tubular reabsorption of calcium in pseudohypoparathyroidism. J Clin Endocrinol Metab 66:618-625, 1988. 254. Kolb FO, Steinbach HL: Pseudohypoparathyroidism with secondary hyperparathyroidism and osteitis fibrosa. J Clin Endocrinol Metab 22:59-64, 1962. 255. Slatopolsky E, Weerts C, Thielan J, et al: Marked suppression of secondary hyperparathyroidism by intravenous administration
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SHAPTER
1t
Bone Disease In 9 H y p er t h y r o l"d i s m DOUGLAS S. Ross
I. II. III. IV.
Thyroid Unit, Massachusetts General Hospital, Boston, Massachusetts 02114
Grades of Hyperthyroidism Overt Hyperthyroidism Endogenous Subclinical Hyperthyroidism Exogenous Subclinical Hyperthyroidism: Thyroid Hormone Suppressive Therapy
V. Thyroid Hormone Replacement Therapy VI. Treatment and Prevention of Thyroid Hormone-Mediated Bone Loss VII. Conclusions References
Severe osteopenia and fractures were among the primary clinical consequences of thyrotoxicosis prior to the availability of antithyroid drugs. In 1891 von Recklinghausen first described the " w o r m eaten" appearance of the long bones of a young woman who died from hyperthyroidism. 1 Plummer in 1928 described hyperthyroid patients with multiple fractures; at autopsy bones were friable, "easily crushed between the fingers," and "almost translucent when held up to the light." 2 With the introduction of the thionamide antithyroid drugs and radioiodine in the 1940s, severe manifestations of hyperthyroid bone disease became rare. Even today, however, thyrotoxic patients may still suffer from significant long-term adverse effects on skeletal integrity. In the last decade, the effects of even minimal hyperthyroidism on bone, and the potential detrimental effects from the use of thyroid hormone supplements, have been the major focus of clinical investigations.
TSH release, in turn, is regulated by the negative feedback of thyroid hormones on the pituitary thyrotrope. Because the negative feedback relationship between thyroxine and TSH is a linear-log relationship, small changes in thyroxine concentrations, even within the normal range, result in large changes in serum TSH concentrations. 3 With the advent of sensitive TSH assays in the late 1980s, serum TSH concentrations have replaced thyroxine levels as the most sensitive indicator of thyroid function. 4 Overt thyrotoxicosis refers to patients with elevated serum thyroxine (T4) and/or triiodothyronine (T3) concentrations and subnormal TSH concentrations. Subclinical hyperthyroidism is defined as a subnormal serum TSH level with normal serum levels of T4 and T3. Such patients have minimal hyperthyroidism, but are not necessarily asymptomatic. 5 If the hyperthyroidism originates from thyroidal overactivity or release of hormone from an inflamed thyroid gland, then the patient has endogenous hyperthyroidism. Exogenous hyperthyroidism occurs when patients are taking thyroid hormone supplements. Such patients may have overt hyperthyroidism if they are purposely taking thyroxine inappropriately (e.g., for weight reduction). The majority of patients with exogenous hyperthyroidism, however, have subclinical hy-
I. GRADES OF HYPERTHYROIDISM The thyroid gland is regulated by pituitary thyroidstimulating hormone (TSH; thyrotropin) production. METABOLIC BONE DISEASE
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perthyroidism. If the purpose of thyroid hormone therapy in hypothyroidism is to restore hormone levels to normal, replacement therapy with levothyroxine is administered. Overzealous replacement therapy may result in subclinical hyperthyroidism. Many patients are given thyroid hormone to suppress goitrous tissue or prevent regrowth of thyroid remnants or neoplasms. The goal of such therapy is to suppress serum TSH concentrations. Suppressive therapy by definition results in subclinical hyperthyroidism. This chapter is divided into separate sections. First, the effects of overt hyperthyroidism on bone and mineral metabolism are discussed, followed by consideration of endogenous subclinical hyperthyroidism, exogenous subclinical hyperthyroidism (suppressive therapy), and thyroid hormone replacement therapy.
II. OVERT HYPERTHYROIDISM Thyroid hormones have a direct resorptive effect upon bone. As a result hyperthyroid patients have loss of bone density, and a significant negative calcium balance. Basic studies will be reviewed with respect to cellular mechanisms by which thyroid hormone regulates bone cell metabolism, followed by consideration of clinical effects of thyroid hormone on bone and mineral metabolism, clinical thyrotoxic bone disease, and the effects of hyperthyroidism on bone mineral density.
A. C e l l u l a r E f f e c t s o f T h y r o i d H o r m o n e on Bone 1. CELL CULTURE
Thyroid hormone action at the cellular level requires T3 to bind a nuclear thyroid hormone receptor, which in turn interacts with thyroid response elements on thyroid hormone-responsive genes. 6 T4, the principal hormone secreted by the thyroid gland, is deiodinated to form T3 by one of two specific 5'-monodeiodinases present in most peripheral organs. 7 Nuclear T3 receptors have been characterized in several osteoblast cell lines including rat osteosarcoma ROS 17/2.8 cells 8'9 and UMR 106 cells, m mouse MC3T3-E1 cells, 1~ and human osteoblast cell lines. 12 Receptors have also been demonstrated in primary cultures of rat 13'14 and mouse ~5 osteoblasts. Although at high doses T3 usually inhibits cell replication in rat and mouse osteoblast cell lines, 9-~1'13'15 there is one report describing proliferation and differentiation of human osteoblast-like cells after T3 was added to culture medium. 16 T3 may also have direct nongenomic effects on bone via stimulation of the inositol phosphate secondmessenger pathway. ~7
G
L
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S. ROSS
T3 stimulates production of the bone proteins alkaline phosphatase 8'9'11'13 and osteocalcin (bone Gla protein) 8'9'13 in cultured rat and mouse osteoblast cell lines. Regulation of the levels of these mRNAs requires the presence of retinoids. TM T3 also increases IGF-1 and IGF-1 binding protein-2 production in cultured rat osteoblast cells 19'2~ and IGF-1 mRNA levels in cultured MC3T3-E1 osteoblast cells, 2~ although the significance of this finding with respect to osteoblast differentiation is not fully elucidated. T3 receptors have not been described in osteoclasts. Isolated osteoclasts do not respond directly to T3,22'23 although T3 increases bone resorption when rat osteoclastoma cells are co-incubated with rat osteoblast UMR cells in a bone slice resorption assay. 23 This effect occurs even when osteoblast cells are pretreated with T3, and the osteoblast cells are subsequently incubated with osteoclasts in the absence of T3. Thus osteoblasts appear to mediate thyroid hormone stimulation of osteoclast bone resorption, presumably via paracrine factors. 2. ORGAN CULTURE
Mundy et al. first demonstrated the direct effect of thyroid hormone on bone resorption in 1976. Using prelabeled rat femurs in organ culture, both T4 and T3 increased release of 45Ca into the medium. 24 Thyroid hormone also increases the release of both alkaline phosphatase and IGF-I. 25 Organ cultures of both long bones and calvaria have been utilized to study the effects of various paracrine factors on osteoclastic-mediated bone resorption as assessed by calcium release. Prostaglandin (PG) E2 and PGFI~ are increased after the addition of T3 to cultures or neonatal rat calvaria. 26 Nevertheless, indomethacin may inhibit 14'26'27 or have no effect 24"28'29 on calcium release from cultured bone. Interferon-~/26 and cyclosporin A 3~ inhibit thyroid hormone-mediated calcium release. Transforming growth factor 13 (TGF-[3) enhances thyroid hormone bone resorption 3~ and monoclonal antibody to TGF-[3 inhibits thyroid hormone-stimulated calcium release into media. ~5 Recent studies suggest that interleukin-6 (IL-6) is produced by osteoblasts and may stimulate osteoclast formarion and osteoclast-mediated bone resorption. 31 T3 potentiates the effects of IL-113 upon IL-6 release and bone resorption in cultured rat long bones. 27
B. B o n e a n d M i n e r a l M e t a b o l i s m The direct effects of thyroid hormone on bone resorption and the consequences thereof, as well as the additional effects of hyperthyroidism on calcium absorption, result in a significant negative calcium balance in
CHAPTER 18 Bone Disease in Hyperthyroidism
533 ~~.~I,"
1" T4, T3
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URINE
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I,9gut absorption of Ca and PO 4
FIGURE 18--1 Bone mineral metabolism in hyperthyroidism. (From Wartofsky L: Does replacement thyroxine therapy cause osteoporosis? Adv Endocrinol Metab 4:157-175, 1993.)
thyrotoxic patients 32-34 (Fig. 18-1). Hypercalcemia due to resorption of bone occurs in only 5% to 8% of patients. 35 Serum albumin levels are lower in hyperthyroid patients than euthyroid c o n t r o l s , 36 and increased ionized calcium concentrations are found in up to 47% of patients. 37'38 Attempts to measure parathyroid hormone (PTH) levels in hyperthyroid patients were initially confounded by imprecise assays and occasional patients with coexistent primary hyperparathyroidism. Using intact PTH immunoassays, it is now clear that the elevated serum ionized calcium concentration suppresses PTH secretion. 39 This in turn results in an increased renal tubular reabsorption of phosphorus, and reduced conversion of 25-hydroxyvitamin D [25(OH)D] to 1,25-dihydroxyvitamin D [1,25(OH)2D]. 4~ 1,25(OH)2D levels are also subnormal in hyperthyroidism due to an increase in its metabolic clearance rate. 41 The reduced 1,25(OH)2D levels result in poor absorption of calcium and phosphorous from the gut. Calcium malabsorption in hyperthyroidism has been demonstrated in humans with tracer quantities of 45Ca administered orally. 42 This malabsorption is further aggravated by steatorrhea and increased gut motility seen in thyrotoxic patients, resuiting in further significant fecal calcium lOSS. 43 Calcium is also lost in the urine; parathyroid hormone suppression results in reduced renal tubular reabsorption of calcium, resulting in significant hypercalciuria. 44 The overall result is that hyperthyroid patients have a significant negative calcium balance.
C. Thyrotoxic Bone Disease Meunier and colleagues have reviewed the clinically apparent consequences of hyperthyroidism on the skel-
eton. 45 Fifteen of 187 patients (8%) with hyperthyroidism had symptoms. All were women, 80% were over the age of 50, two thirds had a fracture or severe bone pain, and three quarters had been hyperthyroid for less than a year. Radiological studies revealed generalized osteopenia. Vertebral compression fractures were common and in the absence of fracture, there was increased radiolucency of the spine. The phalanges showed a latticelike appearance to the trabecular bone, and a flaky appearance of cortical bone with striations on magnified views. Histomorphometric studies demonstrate more significant effects on cortical compared to trabecular bone. Trabecular bone volume was assessed by placing a grid over bone and determining the percentage of the grid occupied by calcified bone. Patients with overt hyperthyroidism had a 2.7% reduction in trabecular bone volume. In contrast, cortical bone demonstrated a 40% increase in osteoclast resorption surfaces and a 324% increase in cortical bone porosity. Although earlier studies indicate increased osteoid volume in hyperthyroidism, in this study there was no evidence of osteomalacia. Both cortical and trabecular bone undergo constant remodeling. Osteoclasts initiate the remodeling sequence by resorbing bone. Osteoblasts lay down new osteoid matrix which is subsequently remineralized. During the normal remodeling cycle, the amount of mineralized bone at the end of the cycle is equal to the amount of bone resorbed by osteoclasts, thus maintaining skeletal integrity. In overt hyperthyroidism, histodynamic studies using double tetracycline labeling demonstrate that the rate of remodeling and new remineralization fronts are increased to twice normal. Bone histomorphometry has been used by Eriksen to create three-dimensional reconstructions of the remod-
534
DOUGLASS. ROSS
eling sequence. 46 These reconstructions show that phases of resorption, laying down new matrix, and remineralization are all shortened in overt hyperthyroidism, but they are not shortened proportionately. Consequently, the normal remodeling cycle of approximately 200 days is halved, and each cycle is associated with a 9.6% loss of mineralized bone. The integrity of trabecular bone may also be undermined by the random accidental occurrence of osteoclast cutting cones perforating individual trabeculae, eliminating their ability to bear external stress. Since the rate of remodeling is increased twofold in overt hyperthyroidism, trabecular perforations also occur twice as frequently. Biochemical markers of bone turnover are increased in hyperthyroid patients. Alkaline phosphate is invariably increased in overt hyperthyroidism, and may remain elevated for months following treatment of hyperthyroidism; levels may increase during initial correction of hyperthyroidism, presumably reflecting increased osteoblastic activity as hyperthyroidism resolves. 47 Serum osteocalcin (bone Gla protein) is similarly increased in hyperthyroidism. 48'49 Urinary concentrations of bone collagen-derived pyridinium cross-links are increased in overt hyperthyroidism, and normalize shortly after treatment. 5~
D. B o n e D e n s i t y Bone density is reduced in overt hyperthyroidism. Controversy remains as to whether bone density can be normalized after treatment of the hyperthyroidism. In one of the earliest reports in 1971, Fraser and colleagues, using a simple x-ray assessment of bone density of the middle metacarpal, reported in a cross-sectional study that hyperthyroid patients had a 14% reduction in bone density, and that treated, previously hyperthyroid patients had a 5% reduction in bone density. 51 A recent study using dual energy x-ray absorptiometry (DEXA) of the lumbar spine demonstrated a 7.4% reduction in bone density in overt hyperthyroidism. 52 In several studies bone density has been measured before and after treatment of hyperthyroidism. Nielson and colleagues, using single-photon absorptiometry (SPA) of the forearm, found a 12% reduction in bone density in hyperthyroid patients that normalized after treatment. 53 Lind and Friis demonstrated a 28% reduction in SPA of the calcaneus that normalized after treatment. 54 In contrast, Krolner and colleagues, using dual-photon absorptiometry measurements of the lumbar spine, found a 13% reduction in bone density in hyperthyroidism, which was increased 1 year after treatment, but by only 3.7%. 55 Diamond and colleagues found hyperthyroid patients had a
12% reduction in bone density of the lumbar spine and a 13% reduction in bone density of the femoral trochanter that increased by only 6.6% and 3.2%, respectively, after 1 year of treatment. 56 Toh and colleagues found a 20% reduction in forearm SPA in hyperthyroid men that was only minimally improved 3 years after treatment. 57 In one other study an 11% increase in the DEXA of the lumbar spine was seen in treated hyperthyroid patients after 3 years. 58 There are two potentially confounding variables that need to be considered when assessing these data. First, the age of the patient, specifically the patient's menstrual status may be important when assessing the ability of bone to recover from thyrotoxicosis. Second, since thyroid hormone therapy may itself be a cause of reduced bone density (see below), it is important to note whether patients are receiving hormone replacement following ablative treatment for hyperthyroidism. Two recent studies help to address these issues. 59'6~ Franklyn and colleagues found reduced bone density of the femoral trochanter and lumbar spine in postmenopausal women with a history of thyrotoxicosis treated with radioiodine whether or not they were presently taking levothyroxine. 59 There was no reduction in bone density in premenopausal patients. Grant and colleagues also found postmenopausal women whose thyrotoxicosis had been treated with radioiodine 15 to 20 years earlier had reduced bone density (SPA distal forearm) whether or not they were taking levothyroxine. 6~Women treated by surgery 20 to 25 years earlier (i.e., patients treated with surgery were treated at a younger age) who were not receiving levothyroxine had normal bone density, while those receiving levothyroxine had reduced bone density. Overall, these data suggest that a prior history of hyperthyroidism is a risk for reduced bone density. Consistent with this are two recent reports which document that a prior history of hyperthyroidism results in an increased risk for hip fracture later in life. 61'62
III. ENDOGENOUS
SUBCLINICAL
HYPERTHYROIDISM Patients with endogenous subclinical hyperthyroidism have the mildest form of hyperthyroidism with normal serum concentrations of free T4 and T3, but subnormal serum TSH concentrations. 5 These patients are frequently elderly women with autonomy that has developed within a multinodular goiter; however, any form of hyperthyroidism can be subclinical. Mudde and colleagues measured the SPA of the forearm in patients with nodular goiter and subclinical hyperthyroidism, and compared their age-specific Z-scores to those of patients with nodular goiter who were euthyroid. 63 The patients
CHAPTER 18 Bone Disease in Hyperthyroidism
535
with subclinical hyperthyroidism had a significant reduction in mean Z-score to - 0 . 6 9 ; Z-scores were normal in the euthyroid patients. In addition, Z-scores were negatively correlated with serum free T 4 concentrations. F61des and colleagues also found that postmenopausal (but not premenopausal) women with nodular goiter and subclinical hyperthyroidism had reduced bone density at the radius and femoral neck, but not lumbar spine. 64 Treatment of postmenopausal women with endogenous subclinical hyperthyroidism with antithyroid drugs prevented further bone loss and resulted in significantly higher bone density after 2 years when compared to untreated controls with subclinical hyperthyroidism. 65 Parameters of bone metabolism have also been measured in endogenous subclinical hyperthyroidism. Faber and colleagues have shown elevations in serum osteocalcin concentrations in patients with nodular goiter and subclinical hyperthyroidism. 66 Serum osteocalcin levels were inversely correlated to serum TSH concentrations. These studies argue that endogenous subclinical hyperthyroidism has an adverse effect upon bone, especially in postmenopausal women, and that such patients may benefit from therapy aimed at normalizing serum TSH concentrations.
IV. EXOGENOUS SUBCLINICAL HYPERTHYROIDISM: THYROID HORMONE SUPPRESSIVE THERAPY Excessive use of exogenous thyroid hormone is a well-known cause of o v e r t hyperthyroidism. In 1982 Ettinger and Wingerd described reduced bone density in patients taking 3 to 4 grains of thyroid extract, 67 and in 1983 Fallon and colleagues described severe osteoporosis and fractures in three women taking excessive doses of thyroid hormones. 68 After sensitive TSH assays were introduced in the late 1980s, it became clear that in over half of the patients
TABLE 18-1
who were given levothyroxine in commonly recommended and prescribed doses, subclinical hyperthyroidism resulted. 69 A large number of clinical studies have been published since 1987. 7~ Overall, they support the conclusion that subclinical hyperthyroidism from levothyroxine therapy reduces cortical bone density more than trabecular bone density, and that significant reductions in bone density are seen primarily in postmenopausal women. Selected clinical studies are discussed below and recent selected studies are summarized in Tables 18-1 and 18-2.
A. B o n e D e n s i t y 1.
INITIAL
CROSS-SECTIONAL
STUDIES
Ross and colleagues first reported in 1987 that 28 premenopausal women who were taking levothyroxine for suppressive therapy of goitrous tissue had a 5% reduction in the SPA of the wrist after 5 years and a 9% reduction in bone density after 10 years of treatment. 72 None of these women had a prior history of hyperthyroidism. The average administered dose of levothyroxine was 0.171 mg/day, an accepted and recommended dose at the time, 73 but one that presently would be considered excessive even for suppressive therapy. Some of these patients were found to have elevated free T 4 concentrations and therefore would not precisely be considered " s u b c l i n i c a l " - - a concept that was in evolution at the time these data were published. TM Nonetheless, these data suggested that commonly prescribed doses of levothyroxine may be deleterious to bone. These results were confirmed by Paul and colleagues in a similar study of 31 premenopausal patients taking an average dose of 0.175 mg of levothyroxine. 75 There was a 12.8% reduction in the bone density of the femoral neck and a 10.1% reduction in the bone density of the femoral trochanter, although lumbar spine density, trabecular-rich bone, was unchanged. Concordant with
Thyroid Hormone Suppression and Bone Density: Cross-Sectional Studies in Premenopausal Women
Study
N
Wrist
D i a m o n d et al. 87
14
--
L e h m k e et al. 88
25
--
G o n z a l e z et al. 89
16
~
S t e p a n a n d L i m a n o v a 9~
20
Calcaneus
Hip $ 1 1%
--
F r a n k l y n et al. 93
18
M a r c o c c i et al. 94
47
~ --
G a r t o n et al. 95
20
--
Lumbar spine
536
DOUGLAS S. Ross
TABLE 1 8 - 2
Thyroid Hormone Suppression and Bone Density: Cross-Sectional Studies in Postmenopausal Women
Study
N
Wrist
Diamond et al. 87
10
$ 11%
Lehmke et al. 88
16
$ 15%
Gonzalez et al. 89
34
$
Stepan & Limanova 9~
25
Kung et al. 91
34
Franklin et al. 93
26
Schneider et al. 84
120
Giannini et al. 92
13
Hawkins et al. 82
84
Fujiyama et al. 83
12
Calcaneus
Hip $ 23%
$ 22%
Lumbar spine $ 16% __a
0.6 SD
+ lSD
$
7%
,l, 13%
$ 18%
$
,l, 5%
8%
$ 7%
a--indicates no change.
the histomorphometric studies in patients with overt hyperthyroidism (see above), this study suggested a more pronounced effect of thyroid hormone on cortical than trabecular bone. The study by Taelman and colleagues was one of the first to compare premenopausal women to postmenopausal women. 76 The reduction in bone density at the wrist was 5% in the premenopausal and 20% in the postmenopausal women; however, thyroid hormone dose was poorly controlled and many of the older women were likely on excessive doses of liothyronine. Two earlier studies highlighted the importance of excluding patients with a prior history of thyrotoxicosis. Adlin and colleagues reported that femoral neck bone density was reduced 9% and lumbar spine density 11% in 19 patients taking levothyroxine, although these charges no longer reached statistical significance if those patients with a prior history of hyperthyroidism were excluded. 77 Greenspan and colleagues also found that after excluding patients with a prior history of hyperthyroidism, changes in bone density were minimal. TM 2.
SUPPRESSIVE V E R S U S R E P L A C E M E N T T H E R A P Y
In several studies suppressive versus replacement doses of levothyroxine were compared; no significant difference in the two doses was found. 79-83 Ribot and colleagues reported that 21 patients taking 0.135 mg of levothyroxine daily with normal serum TSH concentrations had similar lumbar bone densities as 28 patients taking 0.154 mg/day with suppressed serum TSH concentrations. 79 Grant and colleagues reported that 34 patients taking 0.132 mg of levothyroxine with normal serum TSH concentrations had forearm bone density similar to those of 44 patients taking on average 0.155 mg of levothyroxine. In this study forearm density was
reduced 5% in the patients with suppressed serum TSH concentrations, but this did not reach statistical significance. 8~ Gam and colleagues 81 and Hawkins and colleagues 82 failed to show a difference in lumbar bone density between patients with normal and suppressed serum TSH. And in a recent study by Fujiyama and colleagues, the bone density was similar in patients taking on average 0.152 mg/day of levothyroxine with serum TSH levels less than 0.1 mU/liter compared to those taking 0.096 mg of levothyroxine with detectable serum TSH concentrations. 83 It is critical to note that the doses of levothyroxine used in these later studies were 54% to 90% of those in the initial reports, suggesting that clinician behavior had already been changed as a result of the first reports. Additionally, only the study by Grant and colleagues 8~had measured bone density at a cortical bone-rich site and comparisons made to controls who were not taking levothyroxine. Most of these studies are limited by small numbers of patients. Schneider and colleagues published a larger population-based study of bone density in 991 postmenopausal women that included 196 women taking thyroid hormone preparations. 84 Those women taking more than 1.6 ~g/kg body weight of levothyroxine daily ("suppressive therapy") had a 7% reduction in bone density at the distal radius, an 8% reduction at the hip, and a 5% reduction at the spine. In contrast, those women taking less than 1.6 ~g/kg body weight of levothyroxine ("replacement therapy") had no reduction in bone density (Fig. 18-2). 3.
SUPPRESSION F O R T H Y R O I D C A N C E R V E R S U S
SUPPRESSION F O R B E N I G N G O I T E R
In two studies bone losses in patients on suppressive therapy for benign goiter versus suppressive therapy for
CHAPTER 18 Bone Disease in Hyperthyroidism
537
FIGURE 1 8 - 2
Bone mineral densities in postmenopausal women living in Rancho Bemardo, CA, by current thyroid hormone dose level adjusted for age, body mass index, smoking, and use of thiazide diuretics, oral corticosteroids, and estrogen. (From Schneider DL, Barrett-Connor EL, Morton DJ: Thyroid hormone use and bone mineral density in elderly women: Effect of estrogen. JAMA 271:1245-1249, 1994.)
thyroid cancer were compared. The assumption is that the cancer groups were treated more aggressively with levothyroxine suppression than the patients with benign goiter. Mtiller and colleagues found that patients on suppressive therapy for thyroid cancer, but not patients with goiter, had a 5% reduction in bone density at extremity cortical-rich sites when compared to controls. Additionally, the patients with cancer had a 12% reduction in calcium bone index, a measure of total calcium in the central third of the skeleton, when compared to patients with goiter, s5 McDermott and colleagues found significant reductions in bone density in treatment of thyroid cancer or benign goiter in both cortical- and trabecularrich bone compared to controls. 86 In patients with thyroid cancer, bone density at cortical-rich sites (radius and femoral neck), but not trabecular-rich sites (lumbar spine), was lower than that in those receiving suppressive therapy for benign goiter. 4. R O L E OF MENSTRUAL STATUS Finally, most recent studies of suppressive therapy in thyroid cancer patients have noted the importance of menstrual status (Tables 18-1 and 18-2). Diamond and colleagues documented a 11% reduction in bone density of the hip in premenopausal women and a 23 % reduction in hip density in postmenopausal women; bone density was reduced 11% at the wrist and 16% at the lumbar spine but only in postmenopausal patients. 87 Lehmke and colleagues also reported significant reductions in density
at the radius and calcaneus, but not lumbar spine, that were significant only for postmenopausal women. 88 Gonzalez and colleagues found significant reductions at the wrist but not lumbar spine in postmenopausal women only. 89 Stepan and Limanova found reductions in lumbar spine but only in postmenopausal women. 9~ Kung and colleagues found an 18% reduction in lumbar spine and 12% to 13% reduction in femoral neck and trochanter in postmenopausal women 91 and Giannini and colleagues found a 7% reduction in bone density of the lumbar spine only in postmenopausal women. 92 In contrast, Franklyn and colleagues, in a well-designed study in which average levothyroxine dose was 0.19 1 mg/day reported no change in either femoral or vertebral bone density 93 in postmenopausal women. Nevertheless, Marcocci et al. 94 and Garton et al. 95 found no significant change in either femoral or vertebral bone density in premenopausal women on levothyroxine suppressive therapy. Later, however, a significant correlation of levothyroxine dose with annualized bone loss at the femoral neck, was observed. 5. LONGITUDINAL STUDIES
All of the studies discussed above are cross-sectional and therefore have limitations. In two small studies patients have been followed longitudinally. Stall and colleagues described ten patients who were inadvertently included in a large study following bone density in women but were retrospectively identified as having sub-
5
3
8
D
O
U
clinical hyperthyroidism due to levothyroxine therapy. 96 Their annualized rate of loss of density of the femoral neck was increased four- to fivefold, while bone loss of the spine was increased two- to threefold; only the loss at the spine was statistically significant. Pioli and colleagues prospectively followed 14 premenopausal women taking on average 0.147 mg of levothyroxine following thyroidectomy for goiter or thyroid cancer, and compared them to 24 age-matched controls over 3 years. 97 Spinal density fell at a rate of 2.6% per year in the levothyroxine-treated patients, and 0.2% per year in the controls. Curiously, there was no change in radial bone density. a. Meta-analysis A recent meta-analysis of the literature regarding bone density in patients with subclinical hyperthyroidism due to levothyroxine therapy found that there was a significant reduction in bone density for postmenopausal women only. 98 The analysis was based upon a theoretical bone containing 30% distal forearm, 29% femoral neck, and 41% lumbar spine.
G
L
A
S
S. ROSS
have failed to demonstrate an adverse effect of carefully monitored levothyroxine suppressive therapy in premenopausal women. Nevertheless, annualized bone loss in premenopausal women is correlated with levothyroxine dose. This suggests that the reduced doses of levothyroxine prescribed both as a result of the early reports in patients taking higher doses, and the use of sensitive TSH assays to closely monitor therapy, have significantly ameliorated the problem for premenopausal women.
B. Biochemical Markers of Bone Mineral Metabolism
Several parameters of bone metabolism other than bone density have been measured in addition in patients with subclinical hyperthyroidism taking thyroid hormone supplements. Variable abnormalities have been reported. Harvey and colleagues found that postmenopausal women with subclinical hyperthyroidism taking levothyroxine had increased urinary concentrations of the bone b. Does Calcitonin Deficiency Result in Loss of collagen-derived pyridinium cross-links pyridinoline Bone? It is unclear what role if any calcitonin defiand deoxypyridinoline. 1~ Ross and colleagues demonciency might have upon bone. In one prospective study strated an inverse negative correlation between serum by Pioli and colleagues, 97 the thyroidectomized patients osteocalcin and TSH concentrations in patients taking would have been calcitonin deficient compared to the levothyroxine. 1~ Krakauer and Kleerekoper found incontrol group. In most of the cross-sectional studies creased urinary hydroxyproline concentrations in pathere is the potential for significant differences in calcitients with subclinical hyperthyroidism taking an avertonin concentrations between patients and controls, since age dose of only 0.130 mg of levothyroxine daily. 1~ hypothyroidism from both surgery and radioiodine, 99'1~176 Giannini and colleagues found increased serum alkaline or chronic thyroiditis TM all adversely affect C-cell funcphosphatase and urine hydroxyproline concentrations in tion. Several studies have attempted to address this quespostmenopausal women taking on average 144 Ixg of tion. Prior to the appreciation that levothyroxine therapy levothyroxine daily, but not in premenopausal women might reduce bone density, McDermott and colleagues receiving on average 152 txg of levothyroxine daily. 92 In attributed reductions in bone density in thyroidectomized contrast, in three other studies, serum osteocalcin levels patients to calcitonin deficiency. ~~ Hurley and coland urinary excretion pyridinium cross-links were not leagues failed to show reduced bone density in a small increased in premenopausa195 or postmenopausa183 heterogeneous group of patients. ~~ Unfortunately, it is women taking levothyroxine, and serum osteocalcin levnow clear that as yet no experimental design has satisels were not increased in postmenopausal women taking factorily separated the variable of calcitonin deficiency levothyroxine. 82 from concurrent levothyroxine therapy, and therefore the potential role of calcitonin deficiency in these patients remains undefined. 89'1~176 6. CONCLUSIONS The data reviewed in this section suggest that subclinical hyperthyroidism from levothyroxine suppressive therapy adversely affects bone density in postmenopausal women at cortical-rich sites such as femoral neck. Significant reduction in bone at trabecular-rich sites such as lumbar spine have been variable. In contrast, despite initial reports that premenopausal women had reduced bone density at cortical-rich sites, most recent studies
C. Fractures Presently there is uncertainty as to whether patients taking levothyroxine have an increased rate of fractures despite almost a decade of clinical research on the effects of thyroid hormone on bone density. One preliminary study suggested a twofold increased risk of hip fracture in women taking thyroid supplements although serum TSH concentrations were not measured. 1~ Leese and colleagues reported on 1180 patients taking levothyrox-
539
CHAPTER 18 Bone Disease in Hyperthyroidism ine: 59% had suppressed serum TSH concentrations and 38% had normal serum TSH levels. 11~Over a 5-year period the overall fracture rate in women over age 65 was 0.9% for patients with a normal TSH and 2.5% for women with a subnormal TSH. These differences were not statistically significant. Solomon and colleagues interviewed 330 women taking levothyroxine but did not find an increased fracture rate. TM In contrast, as noted previously, women with a prior history of hyperthyroidism and bone density values similar to those noted in patients taking suppressive doses of levothyroxine do have an increased fracture r i s k . 61'62 One would anticipate that the reported reductions in bone densities in patients with subclinical hyperthyroidism would be sufficient to result in an increased fracture rate112; perhaps larger studies are required.
V. THYROID HORMONE REPLACEMENT THERAPY Overtly hypothyroid women who are started on levothyroxine for replacement therapy have a reduction in bone density. Krolner and colleagues prospectively followed eight women whose lumbar spine density fell by 13% after 1 year of levothyroxine replacement therapy. 55 Ribot and colleagues followed ten women for 1 year after starting levothyroxine and found a 5.4% and 7% reduction in the bone density of the lumbar spine and femoral neck, respectively. 79 In contrast Toh and Brown failed to show a reduced bone density in eight hypothyroid men who were treated with levothyroxine for 3 years. 113 The reason for this is suggested by Ericksen's analysis of the bone remodeling cycle in overt hypothyroidism. 46 The cycle is increased from about 200 to almost 700 days in overt hypothyroidism, and each cycle is associated with a 17% increase in mineralized bone. The histomorphometric study of Coindre and colleagues TM demonstrates the likely cause of this apparent loss in bone following institution of thyroid hormone replacement. Ten untreated hypothyroid patients were compared to 15 patients treated with levothyroxine and euthyroid controls. The untreated hypothyroid patients had a mean cortical width that was actually higher than that of euthyroid controls, and levothyroxine treatment was associated with a significant increase in resorption surfaces and cortical bone porosity, and a decrease in mean cortical width to levels similar to those seen in euthyroid controls. TM Thus it is likely that replacement therapy reduces bone density from elevated to normal values. Ross studied postmenopausal women with subclinical hypothyroidism in a randomized trial of levothyroxine therapy to determine if replacement therapy p e r s e might
be detrimental to bone. 115 If the loss in bone density seen during the early treatment of overt hypothyroidism were due to increased remodeling and osteoclast resorption preceding the return to normal steady-state conditions, then one would not expect a similar reduction in bone density when levothyroxine was administered to patients with subclinical hypothyroidism, since the initial abnormality in bone remodeling would be trivial in these patients. There was no reduction in bone density at either the wrist or lumbar spine after 14 months of levothyroxine replacement therapy in postmenopausal women with subclinical hypothyroidism. 115 Thus the apparent loss in bone during the initial treatment of hypothyroidism partly reflects the delay in reestablishing normal steadystate conditions. Nystrom and colleagues demonstrated increased serum concentrations of procollagen III peptide 6 months after the initiation of levothyroxine therapy, but procollagen III peptide levels were no different from untreated controls after 10 years of levothyroxine replacement. 116 These data also support the hypothesis that transient abnormalities in bone metabolism shortly after initiating levothyroxine replacement therapy reflect a delay in achieving normal steady-state dynamics. Many of the cross-sectional studies noted above comparing suppressive with replacement therapy demonstrated normal bone mineral density in women receiving replacement doses of levothyroxine. 79-83 Nevertheless, there is one cross-sectional study that did demonstrate reduced bone density in patients on replacement therapy. Kung and Pun reported on 26 premenopausal hypothyroid women with chronic lymphocytic thyroiditis who were treated with an average dose of 0.111 mg/day of levothyroxine for an average of 7.5 years. 117 TSH levels were carefully monitored and were normal throughout the study. Bone density of the femoral trochanter was reduced 7%, while there was no change in the density of the lumbar spine. While a review of present data suggest that this study is an outlier, it is possible that even replacement doses of levothyroxine could be detrimental to bone, since they fail to perfectly mimic normal physiology. Euthyroid patients taking levothyroxine have serum T4 concentrations that are on average 1 to 2 Ixg/dl higher than euthyroid untreated controls at a time when both s e r u m T3 and TSH concentrations are normal. ~18
VI. TREATMENT AND PREVENTION OF THYROID HORMONE-MEDIATED BONE LOSS Overt hyperthyroid requires rapid treatment to avoid skeletal as well as other complications of thyrotoxicosis. While treatment of endogenous subclinical hyperthyroid-
540
DOUGLAS
ism remains controversial, recent data demonstrate the benefit of antithyroid drugs in minimizing further loss in bone density. 65 Based on current information, one should strongly consider antithyroid therapy in estrogendeficient postmenopausal women with even minimal subclinical hyperthyroidism. For patients with exogenous subclinical hyperthyroidism, the major focus is on prevention of bone loss. Careful titration of levothyroxine replacement therapy should avoid subclinical hyperthyroidism altogether. Patients who are intentionally given suppressive doses of levothyroxine by definition have subclinical hyperthyroidism. It is still uncertain as to the degree of TSH suppression necessary in specific clinical settings. For example, many authors have recommended that patients with thyroid cancer maintain fully suppressed TSH concentrations (<0.01 mU/liter in a third-generation TSH assay). A recent report suggests, however, that serum thyroglobulin concentrations do not fall further when TSH is suppressed below 0.1 mU/liter. 119 Since serum osteocalcin concentrations are inversely proportional to serum TSH concentrations, 1~ and inversely proportional to bone density in overt hyperthyroidism, 12~it is possible that bone loss in subclinical hyperthyroidism can be minimized by carefully titrating the TSH to minimally subnormal values. Several recent reports have tested interventions that might prevent thyroid hormone-mediated bone loss. In the population-based study of Schneider and colleagues
S. Ross
which included 120 postmenopausal women taking suppressive doses of thyroid hormone (defined not by TSH measurement, but by doses in excess of 1.6 Ixg/kg body weight daily), the reduction in bone density in patients taking thyroid hormones was completely ameliorated if estrogens had been co-administered 84 (Fig. 18-3). These data support the conclusion that subclinical hyperthyroidism reduces bone density predominantly in postmenopausal women, and suggests that bone loss in such patients can be prevented by estrogen replacement therapy. A recent smaller study confirms the benefit of estrogen therapy for thyroid hormone-mediated bone loss: estrogen abolished the reduction in bone density seen in postmenopausal women with a prior history of thyrotoxicosis currently on levothyroxine therapy. TM Presently there is little information regarding the use of alternatives for postmenopausal women who cannot take estrogens. Calcitonin has been shown to reduce urinary hydroxyproline and calcium concentrations in overt hyperthyroidism. 122 Although in rats, calcitonin reduced thyroid hormone-mediated increases in serum osteocalcin, it did not prevent the loss in bone density. 123'124Pamidronate 125 and etidronate 124'126prevent thyroid hormonemediated bone loss in the rat, and pamidronate infusions reduce thyroid hormone-mediated increases in indices of bone t u m o v e r . 127 Patients pretreated with pamidronate before liothyronine administration had lower concentrations of serum osteocalcin and urine hydroxyproline, calcium, and pyridinium cross-links compared to patients
FIGURE 18--3 Bonemineral densities in postmenopausal women living in Rancho Bernardo, CA, by current thyroid hormone use and estrogen use adjusted for age, body mass index, smoking, and use of thiazide diuretics and oral corticosteroids. (From Schneider DL, Barrett-Connor EL, Morton DJ: Thyroid hormone use and bone mineral density in elderly women: Effect of estrogen. JAMA 271:1245-1249, 1994.)
CHAPTER 18
541
Bone Disease in Hyperthyroidism
receiving liothyronine a l o n e , a27 While no data are currently available, it is therefore possible that agents such as alidronate may be useful to ameliorate the adverse effects o f s u p p r e s s i v e d o s e s o f t h y r o i d h o r m o n e o n b o n e d e n s i t y in p o s t m e n o p a u s a l w o m e n w h o c a n n o t t a k e estrogens.
2. 3.
4.
VII. CONCLUSIONS Thyroid hormone has a direct resorptive effect on bone. Thyroid hormone receptors are present on osteoblasts. Osteoblasts mediate osteoclastic bone resorption presumably by the release of paracrine factors. Elevated serum ionized calcium levels in overt hyperthyroidism suppresses PTH production, resulting in lower 1,25(OH)2D levels, reduced calcium absorption from the gut, and increased fecal and urinary calcium losses. Hyperthyroid patients have a significant negative calcium balance. Overt hyperthyroidism and iatrogenic hyperthyroidism result in significant osteoporosis and fractures. Postmenopausal women with nodular goiter and subclinical h y p e r t h y r o i d i s m h a v e r e d u c e d b o n e density, a n d f u r t h e r b o n e loss c a n b e p r e v e n t e d b y a n t i t h y r o i d t h e r a p y. S u b c lin i c a l h y p e r t h y r o i d i s m d u e to o v e r z e a l o u s r e p l a c e m e n t or i n t e n t i o n a l s u p p r e s s i v e d o s e s o f l e v o t h y r o x i n e r e s u l t in r e d u c e d c o r t i c a l b o n e d e n s i t y in e s t r o g e n - d e f i c i e n t p o s t m e n o p a u s a l w o m e n . P r e m e n o p a u s a l w o m e n are n o t e f f e c t e d w h e n o n e u s e s the m i n i m a l d o s e o f l e v o t h y r o x ine n e e d e d to a c h i e v e t h e r a p e u t i c g o a l s , b u t h i g h e r d o s e s m a y r e s u l t in b o n e loss. C o n c u r r e n t t r e a t m e n t w i t h est r o g e n r e p l a c e m e n t t h e r a p y or d i p h o s p h o n a t e s m a y b e u s e f u l to p r e v e n t t h y r o i d h o r m o n e - m e d i a t e d b o n e loss. H y p o t h y r o i d i s m is a s s o c i a t e d w i t h an i n c r e a s e d cor-
5. 6. 7.
8. 9.
10.
11.
12. 13.
14.
15.
tical w i d t h o n h i s t o m o r p h o m e t r i c s t u d i e s o f iliac c r e s t b i o p s i e s d u e to a p r o l o n g a t i o n o f t h e r e m o d e l i n g c y c l e to n e a r l y 7 0 0 days. W i t h i n 6 to 12 m o n t h s o f i n i t i a t i n g l e v o t h y r o x i n e t h e r a p y in o v e r t l y h y p o t h y r o i d p a t i e n t s , t h e r e is a r e d u c t i o n in b o n e d e n s i t y to v a l u e s s i m i l a r to that s e e n in u n t r e a t e d e u t h y r o i d c o n t r o l s . R e d u c t i o n s in b o n e d e n s i t y are n o t n o t e d w h e n l e v o t h y r o x i n e t h e r a p y is initiated in p o s t m e n o p a u s a l w o m e n w i t h s u b c l i n i c a l h y p o t h y r o i d i s m . C h a n g e s in b o n e d e n s i t y s h o r t l y after starting l e v o t h y r o x i n e l i k e l y d o n o t reflect s t e a d y - s t a t e c o n d i t i o n s . M o s t s t u d i e s d e m o n s t r a t e n o b o n e loss w i t h
16.
17.
18.
19.
l o n g - t e r m l e v o t h y r o x i n e r e p l a c e m e n t therapy.
20.
References 21. 1. von Recklinghausen FD: Die Fibr6se oder deformierende Ostitis, die Osteomalazie und die osteoplastische Carzinose in ihren
gegenseitigen Beziehungen. Festchrift Rudolf Virchow. Berlin, George Reimer, 1891, pp 1-89. Plummer WA: Cases showing osteoporosis due to decalcification in exophthalmic goiter. Staff Meet Mayo Clin 3:119-121, 1928. Spencer CA, LoPresti JS, Patel A, et al: Applications of a new chemiluminometric thyrotropin assay to subnormal measurement. J Clin Endocrinol Metab 70:453-460, 1990. Ross DS, Daniels GH, Gouveia D: The use and limitations of a chemiluminescent thyrotropin assay as a single thyroid function test in an out-patient endocrine clinic. J Clin Endocrinol Metab 71:764-769, 1990. Ross DS: Subclinical thyrotoxicosis. Adv Endocrinol Metab 2: 89-105, 1991. Evans RM: The steroid and thyroid hormone receptor superfamily. Science 240:889-895, 1988. Larsen PR, Silva JE, Kaplan MM: Relationships between circulating and intracellular thyroid hormones: Review of recent findings and their clinical implications. Endocr Rev 2:87-102, 1981. Rizzoli R, Poserj, Btirgi U: Nuclear thyroid hormone receptors in cultured bone cells. Metabolism 35:71-74, 1986. Sato K, Han DC, Fujii Y, et al: Thyroid hormone stimulates alkaline phosphatase activity in cultured rat osteoblastic cells (ROS 17/2.8) through 3,5,3'-triiodo-L-thyronine nuclear receptors. Endocrinology 120:1873-1881, 1987. LeBron BA, Pekary AE, Mirell C, et al: Thyroid hormone 5' deiodinase activity, nuclear binding, and effects of mitogenesis in UMR-106 osteoblastic osteosarcoma cells. J Bone Miner Res 4:173-178, 1989. Kasono K, Sato K, Han DC, et al: Stimulation of alkaline phosphatase activity by thyroid hormone in mouse osteoblast-like cells (MCT3T): A possible mechanism of hyperalkaline phosphatasia in hyperthyroidism. Bone Miner 4:355-363, 1988. Allain TJ, McGregor AM: Thyroid hormones and bone. J Endocrinol 139:9-18, 1993. Egrise D, Martin D, Neve P, et al: Effects and interactions of 17[3-estradiol, T 3 and 1,25(OH)2D3 on cultured osteoblasts from mature rats. Bone Miner 11:273-283, 1990. Kreiger NS, Stappenbeck TS, Stern PH: Characterization of specific thyroid hormone receptors in bone. J Bone Miner Res 3: 473-478, 1988. Klaushofer K, Varga F, Glantschnig N, et al: The regulatory role of thyroid hormones in bone cell growth and differentiation. J Nutr 125:1966S- 2003S, 1995. Kassem M, Mosekilde L, Eriksen EF: Effects of triiodothyronine on DNA synthesis and differentiation markers of normal human osteoblast-like cells. Biochem Mol Biol Int 30:779-788, 1993. Lakotos P, Stern PH: Evidence for direct non-genomic effects of triiodothyronine on bone rudiments in rats: Stimulation of the inositol phosphate second messenger system. Acta Endocrinol (Copenh) 125:603-608, 1991. Williams GR, Bland R, Sheppard MC: Retinoids modify regulation of endogenous gene expression by vitamin D3 and thyroid hormone in three osteosarcoma cell lines. Endocrinology 136: 4304-4314, 1995. Schmid C, Schlapfer I, Futo E, et al: Triiodothyronine (T3) stimulates insulin-like growth factor (IGF)- 1 and IGF binding protein (IGFBP)-2 production by rat osteoblasts in vitro. Acta Endocrinol (Copenh) 126:467-473, 1992. Lakatos P, Caplice MD, Khanna V, Stern PH: Thyroid hormones increase insulin-like growth factor I content in the medium of rat bone tissue. J Bone Miner Res 8:1475-1481, 1993. Varga F, Rumpler M, Klaushofer K: Thyroid hormones increase insulin-like growth factor mRNA levels in the clonal osteoblastic cell line MC3T3-E1. FEBS Lett 345:67-70, 1994.
542 22. Allain TJ, Chambers TJ, Flanagan AM, McGergor AM: rift_ iodothyronine stimulates rat osteoclastic bone resorption by an indirect effect. J Endocrinol 133:327-331, 1992. 23. Britto JM, Fenton AJ, Holloway WR, Nicholson GC: Osteoblasts mediate thyroid hormone stimulation of osteoclastic bone resorption. Endocrinology 134:169-176, 1994. 24. Mundy GR, Shapiro JL, Bandelin JG, et al: Direct stimulation of bone resorption by thyroid hormones. J Clin Invest 58:529534, 1976. 25. Stracke H, Rossol S, Schatz H: Alkaline phosphatase and insulin-like growth factor in fetal rat bone under the influence of thyroid hormones. Horm Metabol Res 18:794, 1986. 26. Klaushoffer K, Hoffman O, Gleispach H, et al: Bone-resorbing activity of thyroid hormones is related to prostaglandin production in cultured neonatal mouse calvaria. J Bone Miner Res 4: 3 0 5 - 312, 1989. 27. Tarjan G, Stern PH: Triiodothyronine potentiates the stimulatory effects of interleukin-l[3 on bone resorption and medium interleukin-6 content in fetal rat limb bone cultures. J Bone Miner Res 10:1321 - 1326, 1995. 28. Hoffman O, Klaushofer K, Koller K, et al: Indomethacin inhibits thrombin-, but not thyroxin-, stimulated resorption of fetal rat limb bones. Prostaglandins 31:601-608, 1986. 29. Kawaguchi H, Pilbeam CC, Woodiel FN, Raisz LG: Comparison of the effects of 3,5,3'-triiodothyroacetic acid and triiodothyronine on bone resorption in cultured fetal rat long bones and neonatal mouse calcariae. J Bone Miner Res 9:247-253, 1994. 30. Lakatos P, Stern PH: Effects of cyclosporins and transforming growth factor beta 1 on thyroid hormone action in cultured fetal rat limb bones. Calcif Tissue Int 50:123-128, 1992. 31. Ishimi Y, Miyaura C, Jin CH, et al: IL-6 is produced by osteoblasts and induces bone resorption. J Immunol 145:3296-3303, 1990. 32. Auwerx J, Bouillon R: Mineral and bone metabolism in thyroid disease: A review. Q J Med 60:737-752, 1986. 33. Benker G, Breuer N, Windeck R, Reinwein D: Calcium metabolism in thyroid disease. J Endocrinol Invest 11:61-69, 1988. 34. Mosekilde L, Eriksen EF, Charles P: Effects of thyroid hormones on bone and mineral metabolism. Endocrinol Metab Clin North Am 19:35-63, 1990. 35. Parfitt AM, Dent CE: Hyperthyroidism and hypercalcemia. Q J Med 39:171-187, 1970. 36. Daly JG, Greenwood RM, Himsworth RL: Serum calcium concentration in hyperthyroidism at diagnosis and after treatment. Clin Endocrinol (Oxf) 19:397-404, 1983. 37. Frizel D, Malleson A, Marks V: Plasma levels of ionized calcium and magnesium in thyroid disease. Lancet i:1360-1361, 1967. 38. Burman KD, Monchik JM, Earll JM, Wartofsky L: Ionized and total serum calcium and parathyroid hormone in hyperthyroidism. Ann Intern Med 84:668-671, 1976. 39. Ross DS, Nussbaum SR: Reciprocal changes in parathyroid hormone and thyroid function after radioiodine treatment of hyperthyroidism. J Clin Endocrinol Metab 68:1216-1219, 1989. 40. Jastrup B, Mosekilde L, Melsen F, et al: Serum levels of vitamin D metabolites and bone remodeling in hyperthyroidism. Metabolism 31:126-132, 1982. 41. Karsenty G, Bouchard P, Ulmann A, Schaison G: Elevated metabolic clearance rate of 1ct,25-dihydroxyvitamin D3 in hyperthyroidism. Acta Endocrinol 110:70-74, 1985. 42. Shafer RB, Gregory DH: Calcium malabsorption in hyperthyroidism. Gastroenterology 63:235-239, 1972. 43. Thomas FB, Caldwell JH, Greenberger NJ: Steatorrhea in thyrotoxicosis. Relation to hypermotility and excessive dietary fat. Ann Intern Med 78:669-675, 1973.
DOUGLAS S. R o s s 44. Lalau JD, Sebert JL, Marie A, et al: Effect of thyrotoxicosis and its treatment on mineral and bone metabolism. J Endocrinol Invest 9:491-496, 1986. 46. Meunier PJ, S-Bianchi GG, Edouard CM, et al: Bony manifestations of thyrotoxicosis. Orthop Clin North Am 3:745-774, 1972. 46. Eriksen EF: Normal and pathological remodeling of human trabecular bone: Three dimensional reconstruction of the remodeling sequence in normals and in metabolic bone disease. Endocr Rev 7:379-408, 1986. 47. Cooper DS, Kaplan MM, Ridgway EC, et al: Alkaline phosphatase isoenzyme patterns in hyperthyroidism. Ann Intern Med 90: 164-168, 1979. 48. Garrel DR, Delmas PD, Malaval L, Tourniaire J: Serum bone Gla protein: A marker of bone turnover in hyperthyroidism. J Clin Endocr Metab 62:1052-1055, 1986. 49. Luckert BP, Higgins JC, Stoskopf MM: Serum osteocalcin is increased in patients with hyperthyroidism and decreased in patients receiving glucocorticoids. J Clin Endocr Metab 62:10561058, 1986. 50. MacLeod JM, McHardy KC, Harvey RD, et al: The early effects of radioiodine therapy for hyperthyroidism on biochemical indices of bone turnover. Clin Endocrinol 38:49-53, 1993. 51. Fraser SA, Anderson JB, Smith DA, Wilson GM: Osteoporosis and fractures following thyrotoxicosis. Lancet i:981 - 9 8 3 , 1971. 52. Wakasugi M, Wakao R, Tawata M, et al: Bone mineral density in patients with hyperthyroidism measured by dual energy x-ray absorptiometry. Clin Endocrinol 38:283-286, 1993. 53. Nielsen HE, Mosekilde L, Charles P: Bone mineral content in hyperthyroid patients after combined medical and surgical treatment. Acta Radiol Oncol 18:122-128, 1979. 54. Linde J, Friis TH: Osteoporosis in hyperthyroidism estimated by photon absorptiometry. Acta Endocrinol 91:437-448, 1979. 55. Krolner B, Jorgensen JV, Nielsen SP: Spinal bone mineral content in myxoedema and thyrotoxicosis. Effects of thyroid hormone(s) and antithyroid treatment. Clin Endocrinol 18:439-446, 1983. 56. Diamond T, Vine J, Smart R, Butler P: Thyrotoxic bone disease in women: A potentially reversible disorder. Ann Intern Med 120:8-11, 1994. 57. Toh SH, Claunch BC, Brown PH: Effect of hyperthyroidism and its treatment on bone mineral content. Arch Intern Med 145: 833-836, 1985. 58. Rosen CJ, Adler RA: Longitudinal changes in lumbar bone density among thyrotoxic patients after attainment of euthyroidism. J Clin Endocrinol Metab 75:1531 - 1534, 1992. 59. Franklyn J, Betteridge J, Holder R, et al: Bone mineral density in thyroxine treated females with or without a previous history of thyrotoxicosis. Clin Endocrinol 41:425-432, 1994. 60. Grant DJ, McMurdo MET, Mole PA, Parerson CR: Is previous hyperthyroidism still a risk factor for osteoporosis in postmenopausal women? Clin Endocrinol 43:339-345, 1995. 61. Cummings SR, Nevitt MC, Browner WS, et al: Risk factors for hip fracture in white women. N Engl J Med 332:767-773, 1995. 62. Wejda B, Hintze G, Katschinski B, et al: Hip fractures and the thyroid: A case control study. J Intern Med 237:241-247, 1995. 63. Mudde AH, Reijnders FJL, Nieuwenhuijzen Kruseman AC: Peripheral bone density in women with untreated multinodular goitre. Clin Endocrinol 37:35-39, 1992. 64. Frldes J, Tarjan G, Szathmari M, et al: Bone mineral density in patients with endogenous subclinical hyperthyroidism: Is this thyroid status a risk factor for osteoporosis? Clin Endocrinol 39: 5 2 1 - 5 2 7 , 1993. 65. Mudde AH, Houben AJH, Nieuwenhuijzen Kruseman AC: Bone metabolism during anti-thyroid drug treatment of endogenous
CHAPTER 18
66.
67. 68.
69.
70. 71. 72.
73.
74.
75.
76.
77.
78.
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80.
81.
82.
83.
84.
85.
Bone Disease in Hyperthyroidism
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543 86. McDermott MT, Perloff JJ, Kidd GS: A longitudinal assessment of bone loss in women with levothyroxine-suppressed benign thyroid disease and thyroid cancer. Calcif Tissue Int 56:521525, 1995. 87. Diamond T, Nery L, Hales I: A therapeutic dilemma: Suppressive doses of thyroxine significantly reduce bone density measurements in both premenopausal and postmenopausal women with thyroid carcinoma. J Clin Endocrinol Metab 72:11841188, 1991. 88. Lehmke J, Bogner U, Felsenberg D, et al: Determination of bone mineral density by qualitative computed tomography and single photon absorptiometry in subclinical hyperthyroidism: A risk of early osteopaenia in post-menopausal women. Clin Endocrinol 36:511-517, 1992. 89. Gonzalez DC, Mautalen CA, Correa PH, et al: Bone mass in totally thyroidectomized patients. Role of calcitonin deficiency and exogenous thyroid treatment. Acta Endocrinol 134:521525, 1991. 90. Stepan JJ, Limanova Z: Biochemical assessment of bone loss in patients on long-term thyroid hormone treatment. Bone Miner 17:377-388, 1992. 91. Kung AW, Lorentz T, Tam SCF: Thyroxine suppressive therapy decreases bone mineral density in post-menopausal women. Clin Endocrinol 39:535-540, 1993. 92. Giannini S, Nobile M, Sartori L, et al: Bone density and mineral metabolism in thyroidectomized patients treated with long-term L-thyroxine. Clin Sci 87:593-597, 1994. 93. Franklyn JA, Betteridge J, Daykin J, et al: Long-term thyroxine treatment and bone mineral density. Lancet 340:9-13, 1992. 94. Marcocci C, Golia F, Bruno-Bossio G, et al: Carefully monitored levothyroxine suppressive therapy is not associated with bone loss in premenopausal women. J Clin Endocrinol Metab 78: 818-823, 1994. 95. Garton M, Reid I, Loveridge N, et al: Bone mineral density and metabolism in premenopausal women taking L-thyroxine replacement therapy. Clin Endocrinol 41:747-755, 1994. 96. Stall GM, Harris S, Sokoll LJ, Dawson-Hughes B: Accelerated bone loss in hypothyroid patients overtreated with L-thyroxine. Ann Intern Med 113:265- 269, 1992. 97. Pioli G, Pedrazzoni M, Palummeri E, et al: Longitudinal study of bone loss after thyroidectomy and suppressive thyroxine therapy in premenopausal women. Acta Endocrinol 126:238-242, 1992. 98. Faber J, Galloe AM: Changes in bone mass during prolonged subclinical hyperthyroidism due to L-thyroxine treatment: A meta analysis. Eur J Endocrinol 130:350, 1994. 99. Body JJ, Demeester-Mirkine N, Corvillian J: Calcitonin deficiency after radioactive iodine treatment. Ann Intern Med 109: 590-591, 1988. 100. Tzanela M, Thalassinos NC, Nikou A, et al: Effect of 1311 treatm e n t on the calcitonin response to calcium infusion in hyperthyroid patients. Clin Endocrinol 38:25-28, 1993. 101. Body JJ, Demeester-Mirkine N, Borkowski A, et al: Calcitonin deficiency in primary hypothyroidism. J Clin Endocrinol Metab 62:700-703, 1986. 102. McDermott M, Kidd G, Blue P, et al: Reduced bone mineral content in totally thyroidectomized patients: Possible effect of calcitonin deficiency. J Clin Endocrinol Metab 56:936-939, 1983. 103. Hurley D, Tiegs R, Wahner H, Heath H III: Axial and appendicular bone mineral density in patients with long term deficiency of excess of calcitonin. N Engl J Med 317:537-541, 1987. 104. Lowry W, Thomas C, Awbrey B, et al: The late effect of subtotal thyroidectomy and radioiodine therapy on calcitonin secretion
544
105.
106.
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113.
114.
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116.
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117.
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chronic L-thyroxine treatment. Clin Endocrinol 31:143-150, 1989. Kung AWC, Pun KK: Bone mineral density in premenopausal women receiving long-term physiological doses of levothyroxine. JAMA 265:2688-2681, 1991. Fish LH, Schwartz HL, Cavanaugh J, et al: Replacement dose, metabolism, and bioavailability of levothyroxine in the treatment of hypothyroidism. Role of triiodothyronine in pituitary feedback in humans. N Engl J Med 316:764-770, 1987. Burmeister LA, Goumaz MO, Mariash CN, Oppenheimer JH: Levothyroxine dose requirements for thyrotropin suppression in the treatment of differentiated thyroid cancer. J Clin Endocrinol Metab 75:344-350, 1992. Lee MS, Kim SY, Lee MC, et al: Negative correlation between the change in bone mineral density and serum osteocalcin in patients with hyperthyroidism. J Clin Endocrinol Metab 70: 766-770, 1990. Franklyn JA, Betteridge J, Holder R, Sheppard MC: Effect of estrogen replacement therapy upon bone mineral density in thyroxine-treated postmenopausal women with a past history of thyrotoxicosis. Thyroid 5:359-363, 1995. Hendriks JThAM, Smeenk D: Investigation of bone and mineral metabolism in hyperthyroidism before and after treatment using calcitonin, 47Ca and balance studies. Acta Endocrino191:77 - 88, 1979. Ongphiphadhanakul B, Alex S, Braverman L, Baran DT: Excessive L-thyroxine therapy decreases femoral bone mineral densities in the male rat: Effect of hypogonadism and calcitonin. J Bone Miner Res 7:1227-1231, 1992. Kung AWC, Ng F: A rat model of thyroid hormone-induced bone loss: Effect of antiresponsive agents on regional bone density and osteocalcin gene expression. Thyroid 4:93-98, 1994. Rosen HN, Sullivan EK, Middlebrooks L, et al: Parental pamidronate prevents thyroid hormone-induced bone loss in rats. J Bone Miner Res 8:1255-1261, 1993. Ongphiphadhanakul B, Janis LG, Braverman LE, et al: Etidronate inhibits the thyroid hormone-induced bone loss in rats assessed by bone mineral density and messenger ribonucleic acid markers of osteoblast and osteoclast function. Endocrinology 133:2502-2507, 1993. Rosen HN, Moses AC, Gundberg C, et al: Therapy with parental pamidronate prevents thyroid hormone-induced bone turnover in humans. J Clin Endocrinol Metab 77:664-669, 1993.
2HAPTEI~
Paget's Disease of Bone FREDERICK R. STEPHEN M.
I. II. III. IV. V. VI.
S I N G E R John Wayne Cancer Institute, Santa Monica, California 90404 KRANE
Department of Medicine, Harvard Medical School and Arthritis Unit, Massachusetts General Hospital, Boston, Massachusetts 02114
Historical Aspects Incidence and Epidemiology Histopathology Focal Manifestations Local Complications Metabolic Aspects of Paget's Disease
VII. VIII. IX. X.
Paget's disease of bone is a focal disorder of unknown etiology characterized initially by excessive resorption and subsequently by excessive formation of bone, culminating in a " m o s a i c " pattern of lamellar bone associated with extensive local vascularity and increased fibrous tissue in adjacent marrow. In the strict sense this is not a metabolic bone disease, since some portion of the skeleton is spared, no matter how widespread the disease process may be. It is the extraordinary extent of the abnormal remodeling seen in some cases and its metabolic consequences that make necessary a detailed consideration of the disorder in this volume.
Descriptions of the disorder had been published earlier: Rullier, 4 Wrany, 5 Wilks, 6 and Czerny 7 all reported single cases that resembled those of Paget. Nagant de Deuxchaisnes and Krane 8 have summarized evidence that the disease may have existed in prehistoric times. A detailed historical review of Paget's disease can be found in Paget's Disease of Bone by B a r r y . 9
II. INCIDENCE AND E P I D E M I O L O G Y The incidence of Paget's disease in a given population is difficult to estimate, since the great majority of affected individuals are asymptomatic. 1~The types of studies that have been undertaken to obtain data are autopsies of unselected persons, review of roentgenograms, and review of hospital admissions. The first major study was that of Schmor111 in Dresden, who found at autopsy that 3% of 4614 patients over 40 years of age had Paget's disease. Collins ~2 in England found an incidence of 3.7% in 650 autopsies. These figures were confirmed by the radiological survey of Pygott, ~3 who found an incidence of 3.5% in London in 9775 patients over 45 years of age. The incidence in hospitalized patients, however,
I. HISTORICAL ASPECTS Sir James Paget in 1876 described the clinical and pathological aspects of a disease resulting in focal enlargement and deformity of the skeleton I (Fig. 19-1). His subsequent papers provided further details. 2'3 It was his opinion that the pathological basis for the disease was a chronic inflammation of bone, hence the term "osteitis deformans." METABOLIC BONE DISEASE
Systemic Complications and Associated Diseases Drug Treatment Surgery Etiology References
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Copyright 9 1998by AcademicPress. All rightsof reproductionin any formreserved.
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FIGURE 19--1 A drawing of the original patient of Sir James Paget showing marked skeletal deformities including enlargement of the skull, represented here by change in hat size.
is less than 1%. Newman 14 reported 82 cases out of 127,000 admissions at the Hospital of the University of Pennsylvania. Rosenkrantz and colleagues 15 found 111 cases out of 94,112 patients admitted to the New York Veterans Administration Hospital. BalTy 9 reviewed admissions to the main teaching hospitals of Australia and found 2630 cases out of 1,769,664 admissions (1 in 673). The most striking aspect of the epidemiology of Paget' s disease is the great variation in prevalence throughout the world. 16 By roentgenographic survey it occurs most frequently in England, 17 but it is also commonly found in Australia, 18 New Zealand, 19 North America, 2~ and France. 22 Although the autopsy study of Schmorl indicated a common occurrence in Dresden, 11 a roentgenographic survey comparing Europe to Great Britain indicated a low prevalence in Essen, West Germany. 22
FREDERICK R. SINGER AND STEPHEN M. KRANE
Great variability of prevalence may be found even within one country as evidenced by the strikingly high prevalence of Paget's disease in Lancashire 17 compared with the rest of England and by the much lower prevalence in the southern compared with the northern United States. 2~ Surprisingly, in Ireland few affected individuals have been identified. 23 The disease is very rare in Scandinavia, 22 Japan, 24 and China. In one epidemiological study past dog ownership was more common in pagetic patients than in control subjects with diabetes mellitus. 25 A second study examining exposure to pets in patients with Paget's disease found no overall risk in dog owners, but there was increased risk with ownership of mongrel dogs, unvaccinated dogs, cats, and birds. 26 In one study in England 27 and another in the United States 28 no relationship of Paget's disease to dog ownership was found. Paget's disease is usually first identified in individuals over the age of 40 years, although the disorder has been detected in younger persons, almost none of whom have been under 20 years of age. The incidence then rises with increasing age so that by the ninth decade it has been reported to range from 5% to 11%. In the large series that have been reported, men are affected somewhat more frequently than are women. In Barry's series of 2630 patients, 9 1420 were men, a ratio of seven men to six women. There is a rare syndrome in children of congenital hyperphosphatasia characterized by fragile and deformed bones, which has also been called juvenile Paget's disease. The involved bones do not have the morphological characteristics of Paget's disease and this disorder should therefore be considered as a separate entity. 29 In two large studies that analyzed questionnaires, a positive family history was found in 14% 3o and 12.3% 31 of patients with Paget's disease. Only 2.1% of spouse controls had a positive family history in the latter study. 31 Since mild Paget's disease may go undiagnosed, Morales-Piga and colleagues studied in detail the families of 35 patients using bone scans to detect subtle disease and found that 40% of the patients had at least one other first-degree relative with Paget's disease. 32 Patients with "familial" Paget's disease were likely to be younger at time of diagnosis and to have more bones affected. Typical Paget's disease has been observed in as many as seven members of one kindred with probable disease in two other members. 33 At least 40 members of a kindred in northern Ireland have been reported to inherit an autosomal dominant trait that resembles Paget's disease but has unusual features. 34 In this variant of Paget's disease, the diagnosis is usually apparent in the early adult years and although focal expansible lesions are characteristic, unlike typical Paget's disease there are more generalized radiological alterations in trabecular bone. Other features not typical of Paget's disease are
CI-IAVrER 19 Paget's Disease of Bone the absence of sclerotic lesions, relative lack of osteoblastic response in histopathological sections, the high prevalence (95%) of conductive type deafness with onset often before age 20, and loosening and fracture of the teeth, common at an early age. 35 This dysplasia has been termed familial expansile osteolysis. In most families with typical Paget's disease the pattern of distribution is consistent with autosomal dominant inheritance. Inexplicably, Martin and Melick were able to find only five recorded cases of Paget's disease in identical twins. 36 In an attempt to identify possible genetic markers for the disease, the frequencies of HLA antigens in patients with Paget's disease were determined by several investigators. 37-41 The phenotypic frequencies of the HLA-A and HLA-B antigens were found not to differ consistently from those of control populations. In five kindreds with a pattern of inheritance consistent with autosomal dominant transmission, however, typing of the HLA-A, B, C (class I) antigens and linkage analysis revealed an association between the haplotype and clinical demonstration of the d i s e a s e . 4~ Studies of class II HLA antigens have revealed an increased frequency of HLA-DR2 and HLA-DQW1 antigens in Paget's disease. 44'45 In the only study of HLA-DR and DQ alleles in Paget' s disease, HLA-DRB 1" 1104 gene frequency was found to be significantly increased in Ashkenazi Jews with Paget's disease. 46 These findings suggest that an immunoregulatory abnormality could be linked to the class II histocompatibility genes in patients with Paget's disease.
III. HISTOPATHOLOGY (see also Chapter 8) There is a general agreement that the pathological process of Paget's disease of bone may be divided into t h r e e phases. 11'47-49 In the first, the osteolytic or " h o t " phase, there is intense resorption of existing bone. This is soon accompanied by accelerated deposition of spicules of lamellar bone in a disorganized fashion (the mixed phase). In the final phase, the osteoblastic or " c o l d " phase, bone formation is dominant, and the irregularly shaped trabeculae are characterized by the " m o s a i c " pattern of cement lines, which appear haphazardly between fragments of lamellar bone. Any single bone may have separate foci, each of a different phase of the disease process, or various bones may be in different phases. It is possible for all three phases of the disorder to be found in the same focus in a single bone. The diagnostic feature of Paget's disease is the abnormal architecture of lamellar bone. The osteons in normal compact bone are cylindrical, whereas they form regular arches or packets in spongy bone. The lamellar
547 arrangement of normal bone results from the parallel arrangement of collagen fibers in a regular orientation and is best visualized by polarized light. There are relatively few osteocytes per unit area of matrix, and there is uniformity in size and shape of the osteocytes, which generally appear as small cells with dark nuclei. There are a number of types of lamellar bone in the normal cortex. The outer and inner circumferential lamellae are those on the periosteal and endosteal surfaces, respectively. These surround the osteons or haversian systems, which are composed of concentric lamellae. The interstitial lamellae found between osteons are the remnants of previously formed and partially resorbed circumferential lamellae. The trabeculae of the medulla are also normally composed of lamellar bone but do not contain osteons. There are few, if any, complete osteons composed of lamellar bone in Piaget's disease. Woven bone, which is almost never found under normal conditions after the epiphyses close except in areas of rapid remodeling, is the type of bone most frequently seen in response to a stress, for example, fracture or tumor. In woven bone there is a seemingly chaotic, unorganized pattem of deposition of collagen fibers, large numbers of osteocytes per unit area of matrix, and large variations in size and shape of the osteocytes. This type of bone is frequently seen in "tissue patches" of Paget' s disease, which have scalloped contours and prominent cement lines where there is interaction with other patches. 11 This pattern of deposition of woven bone should be distinguished from the diagnostic " m o s a i c " pattern of pagetic lamellar bone. Although woven bone may occasionally be the predominant finding in an active phase of Paget's disease, it is not a diagnostic feature, and the percentages of woven and lamellar bone vary from patient to patient and lesion to lesion. 47 The major changes seen in the osteolytic phase of the disease are due to the activity of osteoclasts. These large multinucleated cells are often found in great profusion adjacent to any bone surface and are presumably derived from similar hematopoietic precursor cells as are normal osteoclasts (Fig. 19-2). In Paget's disease, however, the osteoclasts may assume bizarre shapes, and often contain more than 8 to 12 or as many as 100 nuclei. 48'5~ Osteoclasts of this type are rarely seen in other forms of nonneoplastic bone disorders characterized by increased remodeling, such as hyperparathyroidism. The osteoclastic resorption occurs prior to the fibrous tissue replacement of adjacent normal fatty or hematopoietic bone marrow by a fibrous stroma. Noteworthy at this stage of the disease is the early development of vascular hypertrophy, hyperplasia, and venous ectasia in the marrow spaces and the influx of undifferentiated mesenchymal cells adjacent to the osteoclastic foci. As resorption continues, there is an increase in the deposition
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FREDERICK R. SINGERAND STEPHENM. KRONE
FIGURE 19--2 n large multi-nucleated pagetic osteoclast in a resorption lacuna in a spicule of lamellar bone. This cell contains at least 30 nuclei (H and E; •
of this fibrous stroma, and the vascularity becomes more prominent. There is evidence consistent with increased vascular perfusion in this bone but not for the existence of arteriovenous shunts, as will be discussed subsequently. In the cortex, the osteoclasts widen the osteons centrifugally until they become confluent with other osteons or Volkmann's canals. This process may continue until resorption extends to the endosteal and periosteal surfaces (Fig. 1 9 - 3 ) . In some cases the outer circumferential lamellae will remain intact. If there is penetration of the outer circumferential lamellae, formation of periosteal new bone takes place; often woven bone is deposited in these areas in a manner similar to that observed in fracture callus. If the disease continues, this new bone will also be resorbed and eventually replaced by pagetic lamellar bone with its characteristic " m o saic" pattern. Despite the fact that osteoclasts are the principal cells of interest in the lytic phase, foci of intense osteoblastic activity may also be seen at this stage. These osteoblasts are responsible for the deposition of lamellar bone, often adjacent to areas of irregular resorption (Fig. 19-4), although woven bone is also occasionally seen in these areas. The newly formed woven bone may be resorbed soon after deposition based on the pattern of tetracycline labeling. 53 Similar changes are seen in the cancellous bone of the medulla, where the trabecular bone may be rapidly resorbed by numerous osteoclasts. Thin bands of new bone of either lamellar or woven type may also be found in the marrow cavity adjacent to areas of resorption. Oc-
casionally, hemorrhagic cysts surrounded by fibrous marrow containing hemosiderin-laden macrophages are found in the medulla. Such cysts may persist into the third phase of the disease. These defects develop from microinfarcts of trabecular bone and marrow secondary to rupture of numerous thin-walled dilated vessels. 47 In most patients with Paget's disease there is abundant evidence for abnormal bone resorption. The large osteoclasts (which contain the inclusions consistent with viral nucleocapsids, as will be discussed subsequently) are deeply embedded in the resorption lacunae. Meunier et al. 5~ have observed that a few normal sized osteoclasts may be found adjacent to the giant osteoclasts, which they interpret as evidence for the survival of the normal bone remodeling process adjacent to the pagetic lesion. In their series of 72 patients, Meunier et al. 5~ have attempted to quantitate the abnormal resorption in Paget's disease. The mean value for the total resorption surface as compared with total surface was found to be 23.1% +__ 8.3%, approximately sixfold that of the mean in the 130 normal controls (3.6% -+- 1.1%). The number of osteoclasts was found to be 3.18 ___ 2.3/mm 2 in pagetic bone compared with 0.33 ___ 0.23/mm 2 in nonpagetic bone. Weinstein has observed that pagetic osteoclasts can bore rapidly through cancellous bone doubly-labeled with tetracycline and that this is markedly reduced by treatment. 54 The " m i x e d " phase of Paget's disease occurs at that stage when osteoclasts become less numerous and their activity begins to wane, and osteoblasts become more numerous. New lamellar bone formation occurs with
CHAPTER 19 Paget's Disease of Bone
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FIGURE 19--3 Osteoclastic resorption of the cortex at the endosteal surface and within an osteon in pagetic bone. The outer circumferential lamellae are still intact (H and E; X 190).
lines of easily recognized plump osteoblasts closely applied to bone. The appearance of classic pagetic bone with the " m o s a i c " arrangement of the c e m e n t lines then follows. This diagnostic histological feature of P a g e t ' s disease is characterized by irregular, jigsaw-shaped pieces of lamellar bone with an erratic arrangement of c e m e n t lines (Fig. 1 9 - 5 ) . The scalloped appearance of
these c e m e n t lines represents sites of prior osteoclastic resorption. As mentioned earlier, it is unusual to find completely intact osteons and/or to be able to trace a single unit of lamellar bone for any distance (Fig. 1 9 6). Bone lamellae haphazardly faceted together are readily observed using polarized light microscopy (Fig. 1 9 7). The interfaces between these units or patches of pa-
FIGURE 19--4 Resorption of the cortex is almost complete, although a thin rim of outer circumferential lamellae is still intact. Note the prominent bone formation surfaces lined by osteoblasts. Much of the new bone in this section is woven bone (H and E; X190).
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FREDERICK R. SINGER AND STEPHEN M. KRANE
FIGURE 1 9 - 5 The mixed phase of Paget's disease with prominent rows of osteoclasts lining lamellar bone, which is arranged in the typical "mosaic" pattern. Note the replacement of fatty marrow by loose fibrovascular stroma. The crack (arrow), which is an artifact, separates two units of lamellar bone and is usually seen as a blue cement line (H and E; x 190).
getic b o n e are r e p r e s e n t e d by the c e m e n t lines o b s e r v e d using light microscopy. E a c h n e w w a v e of osteolysis will leave a resorption line, w h i c h subsequently r e m a i n s as a c e m e n t line w h e n n e w lamellar b o n e is deposited. Thus, pagetic b o n e reflects the repeated pattern of resorption and deposition of lamellar b o n e w a x i n g and
w a n i n g in the same area. T h e m a r r o w c o n t i g u o u s with areas of osteoblastic and osteoclastic activity is replaced by the fibrovascular stroma (Fig. 1 9 - 8 ) . A l t h o u g h m o s t attention has b e e n directed t o w a r d the a b n o r m a l osteoclasts and their role in initiating the increased b o n e resorption that characterizes P a g e t ' s dis-
FIGURE 19--6 An incomplete osteon in a focus of Paget's disease. This subtle change is very common and often overlooked (H and E; X 190).
CHAPTER 19 Paget' s Disease of Bone
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FIGURE 1 9 - 7 Polarized views of lamellar bone. A, Normal cortex with circumferential and concentric lamellar bone (H and E; X150). B, Lamellar bone in Paget's disease with the typical "mosaic" pattern (H and E; X190).
ease, osteoblastic function is also abnormal. Numerous, plump osteoblasts can readily be found lining formation surfaces, which are considerably increased in pagetic bone. It is the increased number and activity of the osteoblasts that accounts for elevations of serum alkaline phosphatase and, to a lessor extent, osteocalcin levels.
FIGURE 19--8
There is no conclusive evidence, however, whether the abnormalities in osteoblastic number and function originate in the osteoblasts themselves or are induced by factors produced by osteoclasts or the proliferating stromal cells that replace the normal hematopoietic bone marrow within or adjacent to the pagetic lesions. The
A cross-sectional view of a rib showing the mixed phase of Paget' s disease. Most of the cortex has been replaced by irregular spicules of pagetic bone and loose fibrovascular stroma instead of normal marrow. The remnants of medulla and hematopoietic marrow are seen in the lower center of the photomicrograph (H and E; x44).
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"hyperosteoblastosis" is associated with marked changes in the amount and distribution of osteoid tissue. Meunier et al. 5~ found osteoid volume to be increased in pagetic bone (2.5-fold in males; 4.2-fold in females) as well as osteoid surfaces (3.4-fold in males; 5.0-fold in females). On the other hand, they found that the thickness index of pagetic osteoid borders was slightly decreased compared with controls. These results were interpreted as indicating that the calcification rate is slightly faster than the osteoblastic appositional rate in Paget's disease, the reverse of what is found in osteomalacia. The calcification rate was calculated to be 1.36 _ 0.27 ixm/day in pagetic subjects compared with 0.72 _ 0.12 in controls. Meunier et al. 5~ considered that the nonpagetic bone in the patients with Paget's disease was also abnormal with significant increases in trabecular resorption and osteoid surfaces. In later stages that constitute the so-called osteoblastic or sclerotic phase of the disease, dense, irregular masses of pagetic bone are found with relatively little cellular activity (Fig. 19-9). The normal marrow is almost entirely replaced with fibrous tissue containing scattered telangiectatic vessels, and osteoblasts line bone surfaces. There may be scattered chronic inflammatory cells present as well. Periodic a c i d - S c h i f f - p o s i t i v e droplets are found in the marrow adjacent to some osteoblasts. The pagetic bone in the sclerotic phase has no tendency to orient around vascular canals or to form osteons and is therefore relatively avascular and grossly hard to cut. Belanger, 52 using microradiography, found that the thin metachromatic cement lines of the early phases formed
into high-density bands of calcification in the late sclerotic phase. It was proposed by Belanger et al. 55 that the first morphological event in Paget's disease is the resorption of surrounding lamellar bone by osteocytes (osteocytic osteolysis). Microradiographs of lamellar bone in a focus of Paget's disease close to the junction of normal bone do show widened osteocyte lacunae with irregularly bosselated or etched borders. Belanger et al. 52 suggested that these lacunae are continuously enlarged by osteocytes until they communicate with the haversian system or Volkmann's canal. Occasional osteocyte lacunae also may appear scalloped and enlarged even by random light microscopy (Fig. 19-10). Evidence has been presented indicating, however, that the increased size of the periosteocytic lacunae does not represent "osteolysis" but is a feature of the woven bone p e r se, which characteristically contains relatively large osteoblasts (osteocytes).50'56 The cement lines, seen with hematoxylin and eosin staining as blue lines at the interfaces between circumferential lamellae and osteons, between interstitial lamellae and osteons, or in trabecular bone, appear when a new unit of bone is added to preexisting bone. Cement lines, therefore, are at the interface between units of bone, even between woven and lamellar bone. The later accumulation of these cement lines in lamellar bone creates the diagnostic " m o s a i c " pattern seen in Paget's disease. Although there may be more than normal bone per unit volume in Paget's disease, it is architecturally un-
FIGURE 19--9 The osteoblastic phase of Paget's disease with thickened irregular spicules of pagetic bone and a loose fibrous stroma. There is very little cellular activity in this section even at higher magnification (H and E; •
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FIGURE 19--10 Enlarged osteocytic lacunae in a section of bone. A, An enlarged scalloped osteocyte lacuna that may represent osteocytic osteolysis (H and E; X790). B, Electron micrograph of demineralized bone from a different patient with Paget's disease illustrating an osteocyte surrounded by a large zone in which mineralized bone is absent. The osteocyte has a large lobulated nucleus and clumped chromatin. Note the large number of peripherally located dense bodies in the cytoplasm with the appearance of lysosomes. There are also vacuoles and dilated cisternae of the rough endoplasmic reticulum. The edge of the cell has numerous filapodia extending into the lacunar space, which contains amorphous material and loose collagen fibrils. The perilacunar wall is frayed and the osmiophilic lamina is lost. The collagenous structure of the bone is disorganized and there are no distinct lamellae (x6840). (Courtesy of Dr. Barbara Mills.)
sound. Grossly, the bone in the osteolytic phase may be soft enough to be cut with a knife without prior demineralization if resorption and the decrease in the mass of mineralized bone are severe enough. In the osteoblastic phase, the macerated bone often crumbles like a p u m i c e stone. This is presumably due to the improper alignment along stress lines of the collagen fibers in each spicule or plate of bone. Although each spicule of bone is arranged according to stress lines, each is w e a k e r than a comparable normal spicule of lamellar bone and will readily break. The characteristic biological abnormalities of P a g e t ' s disease are obvious in all phases of bone remodeling even though it is likely that the initial event is focal osteoclastic bone resorption. Although it has been generally assumed that the formation of pagetic bone is a coupled response to the increased resorption, there is evidence that all of the pagetic bone is abnormal: osteoblast number, osteoblastic surface, and osteoblastic activity are all increased. These altered remodeling events can be interpreted as consistent with an increased " b i r t h r a t e " of all of the bone cell populations. Meunier et al. 5~ have also pointed out that their quantitative data also indicate that there is an increased occurrence of new " b o n e cell differentiation f o c i " and a decrease in the ratio of appositional to resorption surfaces. These findings are consistent with a shortened phase of formation or a prolonged phase of resorption or both.
A. Pertinent Aspects of Histological Differential Diagnosis F r o m the point of view of diagnostic histopathology, there are a few entities that may mimic the pattern of pagetic bone, although none has the striking " m o s a i c " pattern of P a g e t ' s disease. In hyperparathyroidism, irregular fragments of lamellar bone surrounded by a fibrous m a r r o w adjacent to brown tumors or cysts are seen. However, there is no great profusion of c e m e n t lines, and dissecting osteitis, not seen in P a g e t ' s disease, is frequently present. Similarly, periosteal reactions in syphilis or fracture callus may include spicules of lamellar bone with m a n y c e m e n t lines, but complete osteons can still be identified, and the irregular c e m e n t lines are rarely as evident as in Paget's disease. Slowly f o r m e d reactive lamellar bone, such as that about a Schmorl's nodule in the spine, a Brodie's abscess in a long bone, or endosteal callus, may also be confused with P a g e t ' s disease. These lesions often contain large, irregularly shaped trabeculae of lamellar bone, but cem e n t lines are not very numerous. Accessory scaphoid bones, especially those subjected to constant pressure, also have a confusing histological picture, but again cem e n t lines are not numerous. 47 Osteoblastic metastases may stimulate formation of lamellar bone spicules, which are e m b e d d e d in a sea of fibrous tissue. However, numerous c e m e n t lines are lacking in this new bone, and
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FREDERICK R. SINGER AND STEPHEN M. KRANE
metastatic tumor cells should be identified in adjacent marrow.
In fibrous dysplasia, the trabeculae are irregularly shaped and consist of woven bone with numerous cement lines surrounded by dense fibrovascular connective tissue. However, prominent osteoblasts on the surface of bone trabeculae, a characteristic feature of Paget's disease, are not seen in fibrous dysplasia. Moreover, abnormal lamellar bone, diagnostic of Paget's disease, is absent in fibrous dysplasia. In the few reported cases of so-called congenital hyperphosphatasia or hyperphosphatasemia studied microscopically, 57-61 the bone contains irregular trabeculae of woven bone instead of compact normal cortex with osteon formation. This rare, autosomal recessive disease, which occurs in children and has also been called juvenile Paget's disease, osteoclasia, osteochalasia, and hyperostosis corticalis deformans juvenilis, is characterized by rapid tumover of subperiosteal bone and may share certain clinical and biochemical features of Paget's disease, for example, multiple bone involvement, skull deformities, elevated total urinary hydroxyproline excretion and serum alkaline phosphatase activity, and even the presence of angioid streaks. Histologically, however, the skeletal lesions can be readily differentiated from those of Paget's disease, since hyperphosphatasemia represents a lack of normal cortical remodeling; hence, the classic "mosaic" pattern of faceted units of lamellar bone is absent. We have had the opportunity to review the tumorous left radius in one of the three cases reported by Thompson et al. 58 The mass had numerous misshapen spicules of mineralized woven bone surrounded by a dense fibrovascular marrow, similar to a focus of fibrous dysplasia. The pattern of Paget's disease was not present, and we feel that the term "juvenile Paget's disease" is not warranted.
IV. FOCAL
MANIFESTATIONS
The clinical presentation of patients with Paget's disease depends on the extent and site of skeletal involvement. 6z The disease may be monostotic or polyostotic, with or without symptoms. When polyostotic, the disease may lead to crippling deformities. Symptoms arise from fractures, enlargement, and excessive vascularity of bone as well as from compression of neural structures. Joints may undergo so-called degenerative changes due to abnormal mechanical stress on the articular cartilage resulting from structurally altered, deformed bones. It is our impression that some patients with very severe, extensive disease may be withdrawn or somnolent, or complain of fatigue and weakness. This is especially true in those with severe skull involvement. On the basis of an-
giographic studies it has been postulated that in some of these individuals there may be a shunting of blood through the extemal carotid system to the detriment of the brain, a so-called pagetic steal (vol pagetique). 63 A. P a i n Although the majority of pagetic lesions encountered are not painful, 64 pain may be prominent in patients with Paget's disease. Pain may result from the primary pagetic process or from complications that arise because of the abnormal bone. The pain associated with an uncomplicated pagetic lesion is usually dull and boring but may occasionally be sharp and radiating. Weight-bearing may increase the severity of pain in lesions in the vertebrae, pelvis, and lower extremities, but nocturnal pain is also common in these areas. Khairi et al. found that pagetic lesions detected by Na18F bone scan but not seen on roentgenograms are generally painful. 65 An evaluation by Vellenga and colleagues of 45 roentgenographically normal lesions detected by 99mTc-EHDP scanning, however, indicated that only 11% were associated with typical pain of Paget's disease. 66 Fogelman and Carr also found that symptoms were not associated with any of 33 roentgenographically normal lesions in 23 patients. 67 The pathogenesis of bone pain in Paget's disease in the absence of a demonstrable fracture is not established. Steindler postulated that stretching of the periosteum as the bone enlarges and associated hyperemia of the marrow cavity stimulate somatic sensory endings in these areas. 68 Sensory nerve endings may also be stimulated by high local concentrations of proinflammatory cytokines in the lesions. Interleukins and tumor necrosis factor are candidates for inducing this response, 69 and, as will be discussed, interleukin-6 can be found in elevated concentrations in bone marrow aspirated from pelvic bones affected by Paget's disease, v~ In weight-bearing areas microfractures may be a significant cause of pain. The overgrowth of bone encroaching on nervous tissue may also result in severe pain syndromes; sensory findings, however, are usually absent. Frequently pain may be due to joint involvement by other disease processes distinct from Paget's disease, such as degenerative arthritis, gouty arthritis, calcific periarthritis, or rheumatoid arthritis and its variants. 71 It is of great importance to appreciate the variety of factors that can account for such pain in order to be able to institute the appropriate mode of therapy. B. T h e S k u l l Paget's disease involves the skull in two major patterns, although both may be present at the same time or
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FIGURE 19 - 1 1 Progressionof osteoporosis circumscripta in Paget's disease. A, The skull of a 52-year-old man, which shows a focal irregular osteoporotic area. B, The same patient, 1 year later, now with almost total skull involvement.
one may develop into the other. Presumably the earliest lesion and one of the pattems seen in the skull that is usually asymptomatic has been termed osteoporosis circumscripta 72'73 (Fig. 19-11). Histologically the inner and outer tables are resorbed and replaced by fibrovascular t i s s u e . 47 Therefore, the involved area is grossly red-violet because of the vascular marrow unobscured by the bone tables. These circumscribed radiolucent areas in the calvarium may persist for years before new bone deposited in a patchy fashion is appreciated radiologically (Fig. 19-12). A second pattem, which probably develops decades after the stage of osteoporosis circumscripta (or possibly independently), is enlargement of the cranium,
usually greatest in the occipital and frontal regions. In extreme instances, the circumference of the skull may increase by as much as 15 cm. This increase results from cortical thickening, primarily the result of deposition of pagetic bone on the outer table. As the process continues, however, the diploe is also replaced, resulting in a calvarium that is coarsely thickened yet still vascular. Roentgenograms at this stage may show the classic "cotton w o o l " appearance (Fig. 19-13). The patient with an enlarged cranium may be asymptomatic or have a variety of problems. Rarely, the increased weight of the skull can make it difficult for the patient to hold the head erect, leading to spasm in the
FIGURE 19--12 An 83-year-old woman with a "honeycomb" skull produced by patch deposition of new bone in a focus of osteoporosis circumscripta.
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FIGURE 19--13 An enlarged skull with the "cotton wool" pattern of diffuse cortical thickening and platybasia. Chamberlain's line (black arrow), which extends from the posterior end of the hard palate to the dorsal rim of the foramen magnum, is well below the tip of the dens. McGregor's line (white arrow), which extends from the posterior surface of the hard palate to the most caudal point of the occipital curve, is below the dens by more than 4 mm. Both of these measurements aid in defining platybasia.
muscles of the neck. A potentially more serious complication caused by pagetic remodeling of the base of the skull is protrusion of bone through the foramen m a g n u m . The degree of such basilar invagination or platybasia may be m e a s u r e d radiographically as an increase in the angle b e t w e e n the basisphenoid and the basilar portion of the occiput (Fig. 1 9 - 1 3 ) . Although basilar invagination is not u n c o m m o n in Paget's disease, neurological abnormalities resulting from compression of structures in the posterior f o s s a 74 o r less c o m m o n l y from cerebellar tonsillar herniation 75 are rare. W h e n there is such neural compression, l a m i n e c t o m y of the upper cervical vertebrae and suboccipital craniectomy have been successfully performed to relieve pressure on the structures of the posterior fossa. 74 Hydrocephalus may also develop in patients with P a g e t ' s disease, which produces disturbances in gait, urinary incontinence, and varying degrees of dementia 76 (Fig. 1 9 - 1 4 ) . Ventricular shunts may reverse these abnormalities. 76 Paget's disease of the temporal bone is c o m m o n l y associated with hearing loss and less c o m m o n l y with tinnitus or difficulty in maintaining balance. Conductive hearing loss has been ascribed to direct involvement of the ossicles or the external auditory canal by the pagetic process. 77-79 Sensorineural hearing loss could be a consequence of cochlear i n v o l v e m e n t or compression of the
FIGURE 19--14 Magnetic resonance imaging of the skull and spine of a 74-year-old woman with polyostotic Paget's disease who developed weakness of the lower extremities and loss of bladder control. Note the markedly thickened calvaria, basilar invagination, low lying cerebellar tonsils, and dilated ventricles. Anterior offset of T2 on T3 secondary to pagetic deformity and thickening of facial bones are also present. (Courtesy of Dr. Patrick Colletti.)
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FIGURE 1 9 - 1 5 Computed tomographic scan of the head of a 79year-old man with extensive Paget's disease who presented with 6 months of blurring of vision in his right temporal field. There is narrowing of the structures at the orbital apex and optic foramina bilaterally.
eighth cranial nerve in the auditory foramen. Although mixed sensorineural and conductive hearing loss is common in Paget's disease of the temporal bone, examination of 26 affected temporal bones in one study failed to define any definitive explanation for the hearing loss. 8~ Recent studies utilizing computed tomography (CT), suggest that the most common reason for hearing loss in Paget's disease is cochlear dysfunction. 81'82 In contrast, patients with familial expansile osteolysis, a bone dysplasia resembling Paget's disease, develop conductive heating loss due to osteoclastic resorption of the ossicles. 83 Although disturbances of function of other cranial nerves are unusual, optic atrophy has been reported as a complication of Paget's disease and may be more common than is generally thought. 84 Direct bony compression of the optic nerve appears to account for only a minority of the cases of optic atrophy, however. An example of pagetic bone overgrowth causing compression of the optic nerve is shown in Figure 19-15.
FIGURE 1 9 - 1 6 Severe displacement of the maxillary teeth in a patient with Paget's disease.
Paget's disease involving the bone of the alveolar socket may produce serious dental problems. 85 The bone adjacent to the teeth can be involved in all phases of the disease, but the enamel and dentin are spared. Particularly prominent is hyperplasia of the cementum surrounding the teeth, partial loss of the lamina dura, and displhcement of the teeth (Figs. 19-16 and 19-17).
C. T h e J a w s The jaws and bones of the face are uncommonly involved with Paget's disease. The maxilla is most often affected. 85 Leontiasis ossea, which occurs when all the facial bones are involved, and is more typically a feature of fibrous dysplasia, is a rare complication of Paget's disease.
FIGURE 1 9 - 1 7 A Panovex view of the mouth showing a marked hypercementosis appearing as a bosselated radiodense mass. Such a lesion could easily be confused with a dental tumor were it not for the associated pagetic changes in the adjacent bone. (Courtesy of Dr. W. Guralnick.)
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The spine, particularly in the lumbar and sacral regions, is one of the commonest sites in which Paget's disease is found. Although Paget's disease of the spine is most often asymptomatic, pain may be ascribable to the pagetic lesions. The early vertebral lesion may resemble osteoporosis, especially when the entire body is involved. More commonly the vertebral bodies appear enlarged radiologically with thickening at the margins and central coarse vertical striations producing a characteristic framed pattern (Fig. 19-18). Although multiple adjacent vertebrae may be affected, the disease process frequently skips vertebrae. The vertical height of a vertebral body may be decreased owing to inadequate mechanical strength and the development of micro- and/ or macrocompression fractures. In extreme instances a vertebral body may nearly completely resorb, take on the appearance of a thin transverse osseous rod, and perhaps be mistakenly identified as a calcified intervertebral disk.
Compression of the spinal cord or nerve roots may result from enlarging vertebral bodies, pedicles, and laminae involved by Paget's disease. 86-88 Neurological compression of this sort is most common in the thoracic area. The symptoms resulting from these lesions include back pain, numbness and paresthesias of the feet, difficulty in walking, and progressive paresis of the legs. If untreated, the patient may develop spastic paraparesis with abnormal bladder and bowel function and an upper thoracic sensory loss. Myelography performed in the lumbar and suboccipital regions reveals the inferior and superior limits of the compression, which often extends over multiple adjacent thoracic segments. 89 The noninvasive techniques of CT (Fig. 19-19) and magnetic resonance imaging (MRI) are excellent methods of determining anatomical abnormalities of the spine, particularly in the neural arch, where routine roentgenograms are least helpful. In the presence of neural compression of this sort, the treatment of choice is decompressive laminectomy if recovery cannot be induced by medical therapy (to be dis-
FIGURE 19--1 8 Paget's disease of the spine in a 60-year-old man. A, Enlarged vertebral bodies with thickened margins and coarse central vertical striations producing the appearance of the so-called framed vertebrae. B, A higher power view.
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FIGURE 1 9 - 1 9 Computed tomographic scan of the third lumbar vertebra of a 71-year-old man with Paget's disease. Typical changes of Paget's disease are seen in the vertebral body.
cussed). An uncommon feature found in some patients is the development of a discrete paraspinal mass, which consists of partially calcified osteoid tissue that infiltrates the epidural region by direct extension from the vertebral periosteum and paraosseous connective t i s s u e . 9~ We observed such a lesion in a 61-year-old man with extremely severe, generalized Paget's disease (urinary hydroxyproline 1200 mg/24 hours), which appeared, over a period of months, on roentgenograms of the chest as a rapidly enlarging mass (Fig. 19-20). An exploratory thoracotomy performed to rule out a pleural tumor or malignant transformation revealed a large oval mass arising from several thoracic vertebrae, which was composed of a central marrow cavity and a peripheral capsule of pagetic bone. Spinal cord compression may be a complication of this phenomenon. Rarely, an acute cauda equina syndrome may occur in which a large epidural mass originating in pagetic bone can produce acute neurological dysfunction. 93 Although severe clinical problems that are the consequence of neural compression are most often seen with thoracic involvement, lumbar spine pain associated with lumbosacral involvement is more common. For example, in the series of Altman and Collins, 94 significant lumbar spine pain was present in 106 patients compared with only six patients with dorsal spine pain. In the latter, compression of vertebral bodies due to associated osteoporosis was thought to be the underlying pathological process. Indeed, it is important to consider that intervertebral disk disease or arthritis unrelated to Paget's disease may be the cause of lumbar pain as well. An evaluation of 70 patients with Paget's disease of the spine revealed that spinal stenosis was present in 24 patients, but not all were symptomatic. 95 Pain was more
FIGURE 19--20
A tomogram of a 61-year-old man with severe generalized Paget's disease and a rapidly growing paraspinal mass arising from several thoracic vertebrae.
likely in the presence of stenosis and zygapophyseal arthropathy in the lumbar region. In another study of Paget's disease of the spine, 9 of 84 patients were observed to have intradiskal invasion from adjacent vertebrae affected by Paget's disease. 96 Spinal pain was present in 67% of patients with this finding. An association of ankylosing spondylitis and Paget's disease was reported by Bitar 97 and Layani and colleagues 98 in a total of six patients. We evaluated an additional six patients with Paget's disease who also had physical findings that suggested ankylosing spondylitis. 71 Limitation of chest expansion was present in each. Spinal flexion was impaired in four, and four had peripheral joint disease. Apparent ossification of spinal ligaments was present in two patients, and extensive osteophytosis in the other four. Sacroiliac joint obliteration was found in four patients, and squaring of the vertebral bodies in two. Syndesmophytes were not seen, however. Paget's disease was present in the pelvis of five patients and in the lumbosacral spine of four. Fatigue, stiffness, and back pain were common complaints. Although the clinical findings in this group of patients were strongly suggestive of ankylosing spondylitis, it is conceivable that
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FIGURE 19-21 An osteolytic focus of Paget's disease in the cortex of the tibia. Note the coarsely trabeculated pattern of bone spicules and the rich vascular marrow within this focus. (From Taylor GW, Castleman B: Clinicopathological conferences of the Massachusetts General Hospital. N Engl J Med 269:808-813, 1963.)
pagetic involvement adjacent to certain joints might mimic spondylitis. Our approach to understanding the nature of spinal symptoms in Paget's disease is to consider the results of HLA typing. From 88% to 96% of patients with ankylosing spondylitis have the HLA antigen B27 as compared with approximately 8% of control Caucasian populations. 99'~~176 In four of our six patients with Paget's disease associated with the features of spondylitis, none had the B27 HLA antigen. It is thus likely that small joints of the spine and the costovertebral joints are affected secondarily by Paget's disease in adjacent bone, and that the resultant clinical syndrome mimics that produced by ankylosing spondylitis. In the two cases described by Altman and Collins, 94 syndesmophytes were present consistent with true MarieStrtimpell's spondylitis coexisting with Paget's disease.
so-called pagetic coxopathy, a form of degenerative joint disease of the hip, which may cause considerable morbidity from pain and decreased mobility. 1~ Although Barry 9 felt that the incidence of this problem was low, w e 71 and Altman and C o l l i n s 94 have found pagetic coxopathy to be a common source of discomfort in Paget's disease. Involvement of the hip joint could result from pagetic changes in the subchondral bone or from altered mechanics at the hip due to deformity of the entire bone. Roper ~~ suggested that if the narrowing of the hip joint space in patients with Paget's disease is predominantly
E. T h e P e l v i s a n d E x t r e m i t i e s In the extremities, the femur and tibia are most commonly involved with Paget's disease. It is not unusual to find a monostotic focus in either of these bones (Fig. 19-21). When these bones are involved, clinical manifestations such as pain, deformity, and increase in skin temperature are frequently present. The deformity associated with Paget's disease of the femur is an outward bowing combined with a coxa vara deformity causing external rotation of the lower leg. The tibia is usually bowed anteriorly and laterally (Fig. 19-22); on the other hand, the fibula is almost never affected. The upper extremities are less frequently affected by Paget's disease and are often asymptomatic (Figs. 19-23 and 19-24). However, involvement of the humerus and ulna does occur. Rarely the disease appears in the hands and feet; it is usually in a single bone and is almost always asymptomatic (Figs. 19-25 and 19-26). The major clinical problem that may develop after years of involvement of the pelvis and upper femur is
FIGURE 19--22 A transillumination of a macerated hemisected tibia with Paget's disease showing the thickened cortex and densely packed, coarsely thickened trabeculae, as well as typical bowing deformity.
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FIGURE 19-23 An osteolytic monostotic focus of Paget's disease in the radius of a patient who presented with wrist pain. Note the flame shape or sawtooth pattern produced by the advancing edge of osteoclastic activity which distinguishes this lesion from a malignant tumor.
superior in the zone of maximal weight-bearing, coincidental, unrelated degenerative joint disease is probably the cause. He concluded that if the joint space narrowing is medial and accompanied by protrusio acetabuli, the Paget's disease is the cause. In our study of 41 patients with disease involving 76 hips, we have found that pagetic involvement of the proximal femur in the absence of acetabular involvement is rare, whereas acetabular disease without femoral involvement is common 71 (Fig. 19-27). The latter is associated with narrowing of the superior joint margin, usually without protrusio acetabuli, and relatively few symptoms. The most severe disease occurs in patients with both acetabular and proximal femoral disease (Fig. 19-28). Protrusio acetabuli is usually associated with both superior and medial joint space narrowing (Fig. 19-29). In these patients the abnormal remodeling of pagetic bone permits the forces of weight-bearing to drive the femoral head in a superior and medial direction. Some symptomatic relief from the pain of hip disease is obtained by the use of orthopedic
FIGURE 19--24 Paget's disease of the humerus. Note the cyst in the humoral head representing cystic degeneration, which may occur in a focus of Paget's disease. The cortices are markedly thickened by coarse trabeculae.
devices, salicylates, or indomethacin (or related antiinflammatory drugs), but in others total hip replacement is required for adequate relief of pain and definitive treatment. 103 Knee pain is common in patients with Paget's disease of the distal femur and proximal tibia 71 and is more likely to be present if the disease is adjacent to the joint. Severe joint space narrowing is observed in these patients and is almost always accompanied by pain, al-
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FIGURE 19--26
Paget's disease of the third metatarsal. Note the increased trabeculation and dimensions of the bone.
the distorted anatomy of the distal lower extremity. ~~ Knee replacement is occasionally required for pain relief. 1~ Paget's disease may also involve the patella, which may become enlarged and warm to the touch. The focal nature of the disease is illustrated by the fact that a pagetic patella may be present in the absence of any other involvement of the particular extremity and is not necessarily associated with Paget's disease of the femur or tibia (Fig. 19-30).
V. L O C A L
COMPLICATIONS A. F r a c t u r e s
FIGURE 19--25
Involvement of two phalanges in a 78-year-old man with severe generalized Paget's disease. Note the thickened cortex of the proximal phalanx and the coarsely thickened trabeculae of the middle phalanx. These lesions were not symptomatic.
though the severity of the pain and impaired mobility are usually less than in patients with pagetic hip disease. Osteophytes may also be found. When synovial effusions occur, they usually have the characteristics of noninflammatory (type I) f l u i d s . 94 Excellent resolution of the pain and impaired mobility in patients with tibial disease can be achieved by tibial osteotomy and realignment of
Fractures are a common complication of Paget's disThey are characteristically transverse whether complete or incomplete, and lie perpendicular to the cortex. The incomplete fissure fracture, or infraction, seen in the long bones is frequently a precursor to complete transverse fractures 1~ and need not result from external trauma. Femoral fractures are twice as common as fractures of the tibia4V; the main femoral site is just below the lesser trochanter, whereas the main tibial site is at the upper third of the shaft. 1~ The small cortical incomplete fractures often occur at multiple sites on a single long bone, primarily along the periosteal edge of the convex surface of the curved bone, and penetrate to e a s e . 1~
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FIGURE 19--27 Early involvement of the right half of the pelvis characterized by a dense rim of pagetic bone surrounding the acetabulum, the "brim" sign. This is a frequent early finding in the pelvis.
various depths through the cortex. These lesions are seen on r o e n t g e n o g r a m s as narrow slitlike radiolucent transverse lines (Fig. 1 9 - 31). Repeated occurrence and remodeling of incomplete fractures is partly responsible for the increase in total degree of curvature in diseased tubular bone. The majority of incomplete fractures
occur during the osteolytic phase, whereas complete fractures tend to be more c o m m o n in the osteoblastic phase. This would be anticipated, since the energy required for the propagation of the fracture is dissipated by the numerous separate trabeculae of the bone of the lytic phase.
More severe involvement of the left pelvis and femur by Paget's disease. Note that the left portion FIGURE 1 9 - 2 8 of the sacrum is also involved.
564
FIGURE 1 9 - 2 9
Protrusio acetabuli in Paget's disease involving the proximal femur and acetabulum.
Fractures in pagetic bone can heal as efficiently as in normal bone while the callus underlying the break itself undergoes the bony changes of Paget's disease. In the largest reported series of 182 complete femoral fractures, however, the incidence of nonunion was high, that is, 4 0 % . l~ In complete fractures, which are usually transverse as if the bone were snapped like a piece of chalk, the periosteum will be torn with a wide separation of fragments (Fig. 19-31D). There is a bias (over 30%) for the occurrence of complete shaft fractures at a site just below the lesser trochanter, with resultant flexion abduction deformity of the proximal fragment. The frequency of this subtrochanteric site of fracture in Paget's disease
FIGURE 19--30
Paget's disease of the patella. There is marked exaggeration of the cortical and medullary bone produced by the pagetic bone, following normal stress lines.
FREDERICK R. SINGER AND STEPHEN M. KRANE
should be contrasted with the frequency of femoral neck site of fracture in older persons without Paget's disease. Since incomplete fractures are most common at the subtrochanteric level, this area would be expected to be the site of the most extensive femoral remodeling and proliferation of abnormal bone in Paget's disease. Accordingly, a combination of internal expansion and remote compression trauma might be sufficient to elicit complete fracture. Tibial fractures follow the femoral pattern. At times the fibula, which is almost never involved with Paget's disease, may nevertheless be fractured as a consequence of the tibial fracture. Pelvic fracture is usually through one or both pubic or ischial rami. It is often difficult to determine whether vertebral body deformity results from pagetic resorption p e r s e or superimposed authentic fracture. It has been noted that spontaneous fracture may occur through an osteolytic lesion of Paget's disease and result in rapid local spread of the pagetic osteolytic process. 9'11~ Osteolysis may also be accelerated by total hip replacement in the presence of an osteolytic focus in the femoral shaft. 111 These events may be confused radiographically with malignant transformation.
B. A s s o c i a t e d N e o p l a s i a The precise incidence of malignant change in Paget's disease is unknown. Although there have been estimates as high as 10% in generalized, severe disease 47 an incidence of approximately 1% probably reflects a more accurate picture if all cases are taken into a c c o u n t . 9'1~ Rarely, osteosarcomas may develop in multiple members of families with Paget's d i s e a s e . 113'114 In all patients with bone sarcoma over the age of 60 years, over half will have Paget's disease. 115 In Barry's series 9 of 116 sarcomas arising in Paget's disease, men were affected twice as often as were women. Malignant change is rare below the age of 50 years. The most common symptoms are pain and swelling, which in some instances predate radiographic findings by a period of months. Pain may be absent in malignancies involving the skull. In some cases the neoplasm itself, as a soft tissue tumor, may be the presenting sign. A postulated correlation of fractures in pagetic bone and the site of sarcomatous change has never been substantiated, since histological demonstration of neoplasms arising at a fracture site has rarely been reported even though Paget's disease and neoplasms may be found in the same bone. 9 Furthermore, in one survey no predisposition to malignancy was found following fracture. 116 Therefore, local injury or excessive trauma, so often invoked as causally related to malignant transformation, is more
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FIGURE 19-31 Fractures in Paget's disease. A, Two fissure fractures of a femur involved by Paget's disease. B, One month later, a complete "chalk stick" fracture occurred at the site of the distal fissure fracture. Note that the proximal fissure fracture is still present. C, A month after the fracture was pinned, it began to heal. D, Two years later, a complete "chalk stick" fracture occurred at the site of the proximal fissure fracture.
likely to be the event that draws attention to the previously undetected tumor. Sarcomas in Paget's disease are found most frequently in the pelvis, femur, humerus, skull, and facial bones, but are uncommon in v e r t e b r a e . 9'112'117-119 The distribution of sarcomas can be contrasted with the distribution of Paget's disease itself, which affects the vertebrae, skull, pelvis, femur, tibia, and humerus in that order. The reasons why sarcomatous change favors the humerus are obscure. 9 The malignant changes always
take place within a focus of Paget's disease. Therefore, only in cases of advanced and extensive polyostotic disease are multifocal s a r c o m a s f o u n d , 47'117 and when they do occur, the skull is the most common site. 9 Jaffe 4v described cases in which sarcoma developed in the only bone involved with Paget's disease. In the studies of Collins, Paget's disease was polyostotic in two thirds of the patients with sarcoma. 1~ Autopsy data suggest that when multiple sarcomas are present, each is of independent origin and not metastatic. Multiple scalp tumors
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FREDERICK R. SINGER AND STEPHEN M. KRANE
were seen in 24 cases in Barry's series of 116 sarcomas. 9 Such lesions were often associated with lysis of the underlying calvarium and invasion directly into the brain. Death ensued within 6 months after diagnosis. The sarcomas vary widely in cell composition,9,47,112,115-119 reflecting the pluripotentiality of the mesenchymal elements of the bone marrow. Sarcomatous tumors have a spectrum of histological patterns, which include the following: (1)cell-poor, collagen-rich fibrosarcomas (Fig. 19-32); (2)poorly differentiated fibrosarcomas with abundant giant cells; (3)anaplastic sarcomas with giant cells, spindle cells with plump nuclei, and osteoclast-like giant cells resembling so-called malignant giant cell tumors of bone; (4)rare chondrosarcomas, nine of which were reviewed by Barry9; (5) conventional giant cell tumors found mostly in the skull, but with occasional spinal, innominate, or femoral involvement; (6) sarcomatous stroma that may contain osteoid and may be classified as osteosarcoma; (7)reticulum cell sarcoma12~ and (8)multiple myeloma. 121 This histological spectrum appears to be a continuum from benign to malignant, since benign but bizarre cellular changes in fibrous bone marrow remote from the tumor site may be seen. 47'122 Thus, there is a danger in interpreting a biopsy taken from one portion of a tumor, since different histological patterns may be found in different parts of the neoplasm, and for this reason we prefer the broadly descriptive term " s a r c o m a . " As Jaffe 47 concluded: "It would seem that the connective tissue from which they [sarcomas] arise may reveal, even within the same tumor, manifold potentialities for differentiation and dedifferentiation along mesenchymal lines." Radiographically, sarcomas usually appear as small, irregular radiolucent foci with mottled or speckled areas of calcification super-imposed on the background of Pa-
get's disease (Fig. 19-33). The dense areas of tumor bone and the sunburst pattern (periosteal reaction) characteristic of osteosarcoma of the young are not usually seen in sarcomas associated with Paget's disease. 123 In addition, the tumors associated with Paget's disease are not confined to the ends of the bones but occur anywhere along the shaft. For example, in Barry's series 9 of 24 tumors involving the femur, 10 were in the proximal third of the shaft, 5 in the mid third, and 9 in the distal third. Most tibial tumors were in the proximal portion of the shaft. Sarcomas appear to originate in the medulla, 9'47 as manifested by the radiographic signs of early subcortical medullary bone lysis. This progresses to cortical bone loss and the eventual development of a soft tissue mass. Computed tomography is now essential in delineating the soft tissue extension. A pathological fracture may be seen as a terminal event of this biological sequence. 9 Although many patients with Paget's disease who develop osteosarcomas have increased levels of serum alkaline phosphatase activity compared with previous levels, so-called explosive rises of serum enzyme activity are not seen. The prognosis for sarcomas arising in Paget's disease is d i s m a l . 9'47'112'115-119 The 5-year survival in pagetic patients with sarcomas in one study was 7.5% as compared with a survival of 37% in elderly patients in whom the tumor arose d e n o v o . 115 Most patients die within 2 years after the diagnosis is established. In Barry' s series of 116 tumors, 9 no patient survived longer than 5 years, with the average duration of survival being 12 months from diagnosis. Those patients with multiple sarcomas died within 6 months with pulmonary metastases. 47 At present, radiotherapy is only palliative, and the treatment of choice appears to be amputation if the tumor is painful or disfiguring. In contrast to the remarkable increase in efficacy
FIGURE 19--32 A spindle cell sarcoma. A, Abnormal fibroblasts mainly compose the tumor, which surrounds fragments of pagetic bone (H and E; • 190). B, Atypical mitoses are seen in several of the spindle sarcoma cells (H and E; X750).
CHAPTER 19 Paget's Disease of Bone
FIGURE 19--33 An 82-year-old man with osteosarcoma arising in a tibia involved with Paget's disease. The tumor has extended into the soft tissue and has multiple radiodense areas, some of which have the trabecular pattern of bone formation. Note the underlying changes of Paget's disease in the tibia.
567 of several chemotherapeutic programs used to treat osteosarcomas in children, TM adjuvant chemotherapy has not yet proved effective in prolonging survival when the osteosarcomas arise in patients with Paget's disease. 115 A peculiar neoplasm deserving special mention here is the giant cell tumor associated with Paget's disease. 125 These are uncommon tumors that most often involve some portion of the calvarium or facial bones. They have rarely been reported to occur in familial Paget's disease. 126 In Barry's review 9 of 15 such tumors (five in the calvarium; two each in the maxilla, mandible, and tibia; and one each in the humerus, ilium, sacrum and ethmoid), a spectrum ranging from classic benign giant cell tumors to malignant sarcomas with giant cells was seen. Similar cases have been reported under the broad term of giant cell tumor. Although the prognosis varies with each case, survival rates appear to be more favorable than for other neoplasms associated with Paget's disease. Jaffe 47 has described an astounding case in which, over an 8-year period, 12 such tumors appeared successively in the frontal, parietal, temporal, occipital, and facial bones. We have also observed these tumors to occur in multiple bones (all pagetic) in the same individual. 127 We had under our care a man with extensive polyostotic Paget's disease known since age 35 (serum alkaline phosphatase levels as high as 114 Bodansky units) who at age 46 was discovered to have a tumor containing giant cells in the mandible. He was treated with curettage and radiation but the tumor recurred on several occasions over several years. Eleven years after the mandibular lesion was identified, he developed a fight iliac bone tumor with iliac vein obstruction, which was identical histologically to the lesion in the jaw (Fig. 19-34). The pelvic lesion did not respond to radiation therapy but became smaller with the administration of calcitonin. At the time of this patient's death from coronary heart disease, 19 years after the jaw lesion was discovered, there was no evidence of metastases. Microscopically these tumors consist of benign spindle cells with plump fusiform nuclei and clumped chromatin or nucleoli (Fig. 19-34). Mitoses are rare. Scattered among these cells are osteoclast-type giant cells not randomly dispersed, but congregated about foci of hemorrhage or scar and often associated with hemosiderin-laden macrophages. Some of the giant cells have phagocytized erythrocytes. Small scattered fragments of pagetic bone were found adjacent to some tumor cells in addition to reactive woven bone spicules, which are generally peripherally disposed. Fibrous marrow adjacent to these tumors has similar gradations of cellular atypicality such that the tumors merge subtly with the nearby fibrous marrow. We feel that some of these lesions represent an atypical proliferative process similar to the giant cell repar-
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FREDERICK R. SINGER AND STEPHEN M. KRANE
FIGURE 19--34
A giant cell reparative granuloma in the right iliac bone of a 57-year-old man with extensive Paget's disease of most bones. A, The lesion is seen as a large cystic defect in the ilium. There is severe Paget' s disease of the pelvis and femur. This lesion was histologically indistinguishable from a giant cell reparative granuloma of the
jaw in the same individual. B, The jaw and iliac lesions were composed of benign-appearing spindle cells with plump fusiform nuclei and prominent nucleoli. The many osteoclast-type giant cells were generally arranged about areas of hemorrhage (H and E; X270). C, Such giant cells often contained large aggregates of erythrocytes (H and E; X700).
ative granulomas of the jaw. This conclusion was reached after review of four additional cases at the Massachusetts General Hospital as well as 35 others in the literature. 127 The reasons for considering the lesions distinct from true giant cell tumors are as follows: Histologically the giant cell lesions of Paget's disease sometimes differ from true giant cell tumors in the number and distribution of giant cells, the benign appearance of the stromal cells, and the increased production of collagenous matrix. Clinically, the true giant cell tumors occur predominantly in the ends of the long bones, whereas the pagetic tumors are usually found in facial bones. A small percentage of true giant cell tumors may be malignant as documented by occurrence of distant metastases, whereas reparative granulomas do not metastasize. The cases reported by Jacobs et al. 126 also re-
semble ours clinically and histologically. What is of particular interest in that s e r i e s 126 is t h a t three of five patients were related and all patients traced their ancestral roots to Avellino, Italy (three of the five patients in our series were also of Italian ancestry, at least one of whom had relatives from Avellino). It is possible that all or most of the previously reported cases are reparative granulomas, although not all pathologists agree with the interpretation that these lesions are not true giant cell tumors. 128 Whether or not the giant cell lesions found in association with Paget's disease are neoplasms, they are frequently very aggressive and locally destructive and attain large dimensions. The soft tissue mass in one of our patients extended at least 28 to 30 cm, estimated by computed tomography (Fig. 19-35). Although the lesions may respond to radiation therapy, they do recur,
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FIGURE 19--35 Computedtomographic scan of the lumbar spine of a 71-year-old woman with extensive Paget's disease and a paraspinal mass consistent with giant cell reparative granuloma. This scan was similar to that previously reported as Patient 1.126
and chemotherapy with agents such as doxorubicin may be required. 128 Dexamethasone may be of limited value as reported in the series of Jacobs et al. 126 Multiple extraskeletal osteoclastomas, resembling giant cell tumors of bone, have been reported in a Korean man with polyostotic familial Paget's disease. 129 These soft tissue lesions diminished in size with dexamethasone treatment. Attempts have been made to correlate the skeletal distribution of Paget's disease with that of metastatic cancer, but the correlation would hold only for the axial skeleton, which is among the most favored sites for metastases of many types of cancer. The ribs are a preferential site for metastases, but rib involvement in Paget's disease is unusual. On the other hand, the tibia, which is among the most favored sites of Paget's disease, is an infrequent location for metastatic cancer. 9 A correlation would seem to depend on the degree of abnormal bone vascularization. The few examples of the coexistence of multiple myeloma and Paget's disease, two diseases that commonly affect the axial skeleton of the elderly, appear to be coincidental. 121 Paget's disease is also occasionally the site of skeletal metastases of carcinomas. We have encountered a few such instances that included a metastasis from prostatic carcinoma and lung carcinoma. In view of the high vascularity of pagetic bone, it is surprising that metastases to such bone are not encountered more frequently. By 1991 only 29 cases had been reported. 13~
VI. METABOLIC ASPECTS OF PAGET'S DISEASE A. M i n e r a l M e t a b o l i s m Clinical and histopathological evidence suggests that the primary defect in Paget's disease is excessive resorption of bone in a focal area. Characteristically this excessive resorption is soon accompanied by an increase in bone formation, which may continue even after the resorption has slowed. Despite the focal nature of the disorder and its limited extent in some patients, the increased remodeling is readily detected by overall assessment of the turnover of the mineral and organic phases of bone. As bone is resorbed, mineral ions are released into the extracellular fluid. If excessive resorption occurs in one part of the skeleton, the increased concentration of mineral ions in the extracellular fluid would, because of the resultant hypercalcemia, decrease secretion of parathyroid hormone and increase renal clearance of calcium. In patients with Paget' s disease, however, concentrations of calcium and phosphorus in plasma are usually normal, concentrations of parathyroid hormone measured by radioimmunoassay are usually normal, 131'132 and urinary calcium excretion is not characteristically increased in the absence of fractures of bone or immobilization of patients in bed. 8 These observations imply that the in-
570 creased release of mineral ions that results from increased bone resorption must be accompanied by closely geared local reutilization of such ions for the formation of the new mineral phase. Therefore, the external calcium balance in Paget's disease, which reflects differences in bone formation and resorption, does not usually give any indication of the r a t e s of these processes. It should be pointed out, however, that calcium balances in pagetic subjects are rarely more than 200 mg/day positive or negative. Analysis of the disappearance from the plasma of tracer doses of mineral ions does indicate how greatly accelerated the turnover of these ions may be, even though the process may be confined to a limited region of the skeleton. The results of studies of several authors utilizing kinetic analysis of plasma disappearance rates and/or skeletal uptake of 47Ca, 45Ca, 855r,133'134 and Mg 135 or 99mTcdiphosphonate, 136'137 although not strictly comparable quantitatively, have all shown that the rates of both bone formation and resorption can be greatly increased in patients with Paget's disease. From data of this sort it is possible to calculate the size of the exchangeable calcium pool and rates of entrance into (Vo+) and exit from (Vo_) this pool. 8 Although there is disagreement as to the significance of such calculations and their physiological meaning and what corrections need to be applied to take into account factors such as long-term isotope exchange, it is possible to use the numbers derived to compare different subjects with each other and with normals. The increased values for Vo+ and Vo_ calculated in patients with Paget's disease are mean rates for the whole skeleton. It should be emphasized that some areas of bone would be remodeling at a rate manyfold that of others. Calculations of mineral ion kinetic parameters probably overestimate rates of calcium resorption, Vo- (as well as calcium deposition, Vo+). Nevertheless, in comparison with the rates calculated for normal subjects, both formation and resorption may be increased occasionally more than 20-fold. Fluoride ions can be incorporated into the hydroxyapatite crystal lattice, and evidence of increased retention of absorbed fluoride has also been found in patients with Paget's disease. 8 Although the whole-body turnover of mineral ions is increased in pagetic subjects, results obtained by external scanning following administration of the radioactive isotopes 47Ca, 855r, 18F, and bisphosphonate coupled to 99mTC136-14~indicate that the tracer is localized mainly to diseased areas of the skeleton. This phenomenon has led to the use of bone-seeking isotopes in defining the extent and activity of the disease in clinical practice. The pattern of uptake of 99mTc-labeled bisphosphonate in monostotic and polyostotic Paget's disease is illustrated in Figure 19- 36.
FREDERICK R. SINGER AND STEPHEN M. KRANE
It was found earlier that pagetic bone initially takes up more 47Ca than normal bone and retains the isotope for longer times. 8 This increased uptake is not due entirely to rapid exchange processes, since the high level of activity persists in the involved bones even when the specific activity of 47Ca in plasma has fallen to low levels. Whether such retention is the result of the formation of a new mineral phase or some long-term exchange process 141'142is not yet known. On the basis of the uptake of 47Ca and the subsequent decay of this isotope to its daughter isotope 47Sc, we concluded that the calcium that enters pagetic bone becomes "fixed" and no longer available for short-term exchange processes. 8 Thus a model can be proposed in which there is rapid formation of new bone with the calcium-phosphate mineral phase of the older bone "insulated" by newly deposited mineral phase. Skeletal uptake of agents such as 99mTcdiphosphonate would be proportional to the surface of newly formed calcium-phosphate mineral phase as well as to the magnitude of the blood flow to the pagetic bone. 137 As discussed previously, although there is increased blood flow through dilated superficial vessels overlying a pagetic bone, there is convincing evidence using 18F clearance techniques that blood flow is increased to pagetic bone. The intensity of the remodeling in diseased bone may also result in the rapid clearance of tracer ions from the blood, leaving little tracer available for uptake by normal bone. Thus, uptake over an uninvolved tibia at an arbitrary interval following injection of 47Ca is less in patients with Paget's disease than in normal subjects. 8 The kinetic data obtained in patients with Paget's disease imply that despite enormous increases in both bone resorption and formation, the rates of the two processes are very similar. We have speculated 8 that the increase in bone formation is the homeostatic response of the organism to maintain the concentration of calcium in the plasma constant despite the great egress of calcium from bone to extracellular fluid. It is possible that it is this homeostatic requirement for bone formation that accounts for the irregular deposition of new bone that characterizes Paget's disease. The delicate equilibrium between formation and resorption is easily upset by such events as fractures or immobilization of the patients and would be reflected in alterations in calcium balance or in elevation of serum calcium levels. Serum magnesium concentrations were found to be low in some pagetic patients. 143 Metabolic balance studies indicated that the hypomagnesemic individuals had a significantly more positive balance and a higher net absorption of magnesium than normomagnesemic individuals. It was suggested that the hypomagnesemia probably resulted from increased uptake by bone. In a study assessing the level of vitamin D metabolites in the circulation, serum 25(OH)D and 1,25(OH)2D3 lev-
CHAPTER 19 Paget' s Disease of Bone
571
FIGURE 1 9 - - 3 6 Bone scans in two patients with Paget's disease. A, An example of monostotic disease in which nearly the entire right femur shows increased uptake of 99mTc-bisphosphonate. B, Polyostotic Paget's disease involving the skull, multiple vertebrae, the right hemipelvis, and multiple long bones.
els were within the normal range, but serum 24,25(OH)2D3 levels were more than 50% lower than those of an age-matched control group. 144 The degree of disease activity was higher in the patients with the lower levels of 24,25(OH)2D3. The significance of these findings remains to be established.
B. M e t a b o l i s m of B o n e M a t r i x 1. PAGETIC BONE MATRIX
When bone is resorbed, not only is there release of mineral ions from the inorganic phase, but there is resorption of the organic phase as well. The organic phase of bone consists mainly of collagen in addition to several noncollagenous proteins. The major, if not the exclusive, type of collagen in normal bone is type I collagen in addition to small amounts (--~3%) of type V collagen and type XI collagen. 145 Type III collagen is also found in the walls of blood vessels. The amino acid composition of pagetic bone analyzed after demineralization in ethylenedia-
minetetracetic acid (EDTA) is indistinguishable from normal bone. Furthermore, the few samples of pagetic bone that have been examined by cyanogen bromide digestion and chromatography showed a pattern also indistinguishable from that of normal bone and do not show the presence of detectable amounts of type III collagen peptides. 146 Moreover, pagetic bone fragments in organ culture synthesize only type I collagen, whereas skin fibroblasts synthesize both types I and III collagens. 147 It has not been proved by microdissection techniques that all of the collagen in lamellar as well as woven pagetic bone is type I. Histological examination of specimens of pagetic bone, however, characteristically shows a loose, fibrous stroma replacing normal marrow elements. This fibrous stroma probably includes types III and V collagens. It is possible that the inability to detect type III collagen chemically in bone is accounted for by the relatively low percentage of stroma in the samples exampled. 148 Normal bone and skin both contain the same heterotrimeric type I collagen. A single gene (COL1A1) encodes both OLl(I) chains and another single gene
572 (COL1A2) encodes the oL2(I) chain. Unless there is some alternative pathway of processing the primary gene transcript of these type I collagen chains, then the amino acid sequence of the type I collagen chains in skin and bone should be identical. Skin and bone collagens are not identical from the point of view of posttranslational modifications, however. For example, the pattern of glycosylation of the hydroxylysine residues is clearly different in human skin and bone. 149-151 The ratio of glucosyl-galactosyl hydroxylysine [Hyl(Glc-Gal)] to galactosyl-hydroxylysine [Hyl(Gal)] is 0.47 + 0.01 in normal human bone and 2.06 _+ 0.47 in normal human skin, whereas the total glycosylated hydroxylysine is similar in the collagens from these tissues. 15~In five pagetic bone samples the ratio of Hyl(Glc-Gal)/Hyl(Gal) ranged from 0.40 to 0.74, not significantly different from normal. ~52 The reducible crosslinks of normal bone can also be distinguished from those of normal skin. 145'153 In normal bone the predominant compound is hydroxylysino-hydroxynorleucine, the reduced Schiff base of hydroxylysine and hydroxylysine aldehydes. The major crosslinks in bone are the mature hydroxypyridinoline (HP) and lysylpyridinoline (LP) compounds. 145'153'154 Of the two pyridinoline-forming sites in bone type I collagen fragments, those bridging the N-telopeptide to the helix are the most abundant. The N-telopeptide interactions are predominantly a l(I) and OL2(I) to OL2(I), a feature that distinguishes bone type I collagen from other tissue type I collagens. Measurement of these peptide-bound pyridinoline compounds as well as total and free HP and LP (155) are thus useful markers of bone collagen resorption. It is probable that there is no major abnormality of the type, primary structure, and/or distribution of collagen in pagetic bone proper. There may be minor differences in posttranslational modifications of bone collagens that could reflect the "maturity" of the bone sampled and the turnover, that is, the rates of formation and resorption. If the sample is from the more loosely structured cancellous bone, then there might be a greater relative contribution of stromal collagens that would include types III and V collagens. A systematic study of all of the noncollagenous proteins in pagetic bone compared with normal bone has yet to be reported. The concentrations of O[ 2 HS-glycoprotein, albumin, and sialic acid (for bone sialoprotein) have been measured in a small number of samples and found to be significantly increased in pagetic bones. 156 This has been attributed to the increased rate of bone turnover. There is also evidence that the distribution of the noncollagenous proteins in the pagetic bone matrix is abnormal. ~57 Differences have been found in the intensity of immunostaining in pagetic bone sections using antibodies to osteonectin, osteocalcin, and decorin, whereas staining for
FREDERICK R. SINGER AND STEPHEN M. KRANE
other noncollagenous proteins such as biglycan is unchanged. These findings probably reflect the abnormal deposition of bone, particularly incomplete haversian systems, that results from the intensity and rapidity of the remodeling process. 2. COLLAGENOLYSIS
In order for bone to be resorbed, it is essential to remove the mineral phase so that the matrix can be attacked by proteolytic enzymes. Current evidence suggests that these processes occur in the uniquely acidic environment of the extracellular compartment adjacent to the ruffled border of the osteoclast. ~58-161 At the low pH in this compartment, dissolution of the calciumphosphate of bone is presumably the initial event. It was first proposed by Vaes 162 and subsequently elaborated upon further 163 that acid hydrolases, which would function optimally at the low pH in this compartment, would be involved in the resorption of the organic matrix. Candidate acid cysteine proteinases include cathepsins B, L, and K. Recently, it has been shown that cathepsin K, highly expressed in osteoclasts, 164'~65is the target of mutations in pycnodysostosis, 166 a disorder characterized by osteosclerosis. It is likely that cathepsin K would also be expressed in pagetic osteoclasts. Collagenase, a member of the matrix metalloproteinase (MMP) family, is expressed predominantly in osteoblasts in the mouse rather than in osteoclasts. 167-169 Collagenase-3 (MMP13), the human homologue of murine collagenase, is also expressed in skeletal cells and is probably important in several aspects of bone remodeling. Since the rate of bone resorption in Paget's disease is characteristically increased, it follows that the rate of collagenolysis is also increased, which is reflected in the urinary excretion of peptides derived from collagen. Whether this enhanced collagenolysis is due to increased numbers of cells (and which cells) or enhanced collagenolytic activity per cell is not known. 3. HYDROXYPROLINE AND HYDROXYLYSINE EXCRETION In normal animals and humans, the free hydroxylysine and hydroxyproline released from collagen-derived peptides are not reutilized for collagen synthesis and are degraded to small carbon fragments. 17~ Oligopeptidebound hydroxyproline and hydroxylysine 8 and glycosylated hydroxylysine, however, are excreted in the urine, the rate of excretion roughly reflecting the rate of collagen degradation. 146'149-152'172-175 The presence of increased urinary excretion of peptides containing hydroxyproline and other collagen markers in patients with Paget's disease provides a useful tool for the study of collagen turnover in humans and for assessing responses to therapy.
CHAPTER 19 Paget' s Disease of Bone The excretion of hydroxyproline-containing peptides is greater than normal in almost all patients with Paget's disease, even in those with monostotic involvem e n t . 146'171'176 In general, the amount of hydroxyproline excreted is directly correlated with the extent of the Paget's disease as well as with the degree of disease activity as determined, for example, by 47Ca kinetics. 8 The fact that the concentrations of oligopeptide hydroxyproline in the plasma are also increased, 177"178 despite rapid clearance by the kidney, 179 indicates that the increased urinary excretion is not due simply to increased renal clearance. Although the evidence is indirect, it is likely that the excessive urinary excretion of hydroxyproline and hydroxylysine and its glycosides in pagetic subjects has its source in bone. The evidence is as follows: (1)The level of excretion of hydroxyproline is directly related to the extent and the degree of the activity of the Paget's disease. (2) Treatment of Paget's disease with agents that have their major effects on bone (e.g., calcitonin) results in decreased hydroxyproline excretion. (3)There is no histopathological evidence that collagens in tissues other than bone, such as the dermis, are involved to a degree sufficient to explain the increased collagen degradation, although minor morphological changes in appearance of the dermis in pagetic subjects have been observed. (4)The total urinary excretion of hydroxylysine as well as hydroxyproline is increased in Paget's disease, and the pattern of glycosylated hydroxylysines excreted in the urine of several patients studied resembles that of bone collagen. The ratio of Hyl(Glc-Gal)/Hyl(Gal) in patients with extensive Paget's disease ranged from 0.40 to 0.74, closer to that of bone collagen (0.47) than to that of skin (2.06) or other collagens (>2.0). 149 The urinary ratios are distinctly lower than the mean in nonpagetic subjects of 1.49.172 It is possible that the measurement of pyridinoline crosslinks in urine will eventually prove to be a better discriminant to assess pagetic activity than measurement of total hydroxyproline. 153'155'18~ Nevertheless, in the relatively small series published so far, urinary excretion of the C-telopeptide crosslink compound (CrossLaps), the N-telopeptide or free pyridinoline and deoxypyridinoline correlates well with that of hydroxyproline but none is a much better discriminant than hydroxyproline. In patients with mild Paget's disease, however, measurement of total pyridinoline or either of telopeptide crosslinks could turn out to be a better and more readily measured marker than total hydroxyproline. It has been proposed that the superiority of measurements of peptide-bound crosslinks (e.g., NTX) to those of free pyridinolines in Paget's disease could be explained by a rate-limiting step in this disorder in further metabolism of the peptide-bound pyridinolines.183
573 4. NONDIALYZABLE URINARY HYDROXYPROLINE
The urinary excretion of nondialyzable urinary peptides containing 4-hydroxyproline has provided some information about skeletal matrix remodeling in Paget's disease. These heterogeneous peptides have an apparent molecular weight of 5000 kD, or less, contain Gly-XY repeats, are substrates for bacterial collagenase, have structural characteristics of gelatins, and are rapidly labeled in vivo after administration of [14C]L-proline.184-186 There is evidence indicating that at least some of these peptides are by-products of collagen synthesis. The peptide whose composition was reported by Szymanowicz 187 could comprise --~5 Gly-Pro-Hyp triplets and could therefore originate from the minor helix in type I procollagen and be cleaved in the course of its processing. Measurements of total nondialyzable 4-hydroxyproline peptides show that their excretion increases relative to total hydroxyproline (including oligopeptides) when bone resorption is decreased acutely with calcitonin. 188 We have interpreted the acute fall in excretion of the nondialyzable peptides after calcitonin administration to indicate that there are acute adjustments of bone formation in response to the decreases in bone resorption as additional homeostatic responses in Paget's disease. Chronic treatment with bisphosphonates that results in a decrease in urinary hydroxyproline excretion also results in increases in the ratio of nondialyzable to dialyzable hydroxyproline with a corresponding decrease in the excretion of the total nondialyzable peptides. These findings indicate a change in the balance of bone collagen synthesis relative to resorption in favor of increased formation. The pattern of alteration in the metabolism of mineral ions and the matrix components considered up to this point in patients with Paget's disease is thus consistent with the high rate of skeletal turnover inferred from radiological and histological observations of the course of the disorder. We noted that the mineral ion kinetics probably overestimate the mean rate of bone calcium deposition and resorption and that the total urinary hydroxyproline excretion reflects both (bone) collagen synthesis and degradation. The urinary hydroxyproline contained in low-molecular-weight fractions largely reflects collagen breakdown. An unknown portion of these peptides, however, is cleaved to yield free hydroxyproline, which in tum is further degraded in the liver. 171 Therefore, the calculation of collagen degradation based on the excretion of hydroxyproline peptides alone gives too low an estimate. Determination of the glycosylated hydroxylysines provides a more accurate index of collagen degradation, since their further metabolism is relatively limited. Despite these caveats, we have calculated the amount of bone resorbed in a number of pagetic patients
574 based on 47Ca kinetics compared with that based on total urinary hydroxyproline excretion. We have estimated that --~50 g of bone per day may be resorbed in individuals with the most extensive, active, Paget's disease. 5. SERUM PROCOLLAGEN PEPTIDES
The potential value of assays of procollagen peptides as markers of collagen synthesis was first explored by Goldberg et al. 189'19~and Taubman et al. 189 who developed antibodies to carboxyl-terminal fragments of type I collagen purified from human fibroblast-conditioned medium. Since the procollagen peptide extensions of type I collagen are cleaved prior to fibril formation, measurements of these peptides would theoretically be of considerable value if they were not extensively degraded by proteases before release into the circulation. The carboxyl-terminal propeptide of type I procollagen is a heterotrimer of approximately 1 10 kDa that is stabilized by interchain disulfide bonds. Taubman et al. 191 described elevated levels of these peptides (which we termed pColl-I-C 125) in the serum of patients with Paget's disease compared with normal subjects, and the levels of these peptides correlated with the extent and activity of the disease, determined by the activity of serum alkaline phosphatase. We subsequently corroborated these findings and others PICP in which we also observed a decrease in the levels of pColl-I-C in pagetic subjects who received dichloromethylene bisphosphonate for 4 to 7 months. 148 Furthermore, we observed an acute fall in the levels of pColl-I-C in patients given single subcutaneous injections of salmon calcitonin. There are several explanations for these results, including the possibilities that these fragments are released when bone is resorbed or that these therapies alter the metabolism of the procollagen peptides. We interpreted these results, however, as indicating that the fall in pColl-I-C following administration of calcitonin or bisphosphonates reflects a decrease in bone formation coupled to the decrease in resorption, similar to that observed using measurements of nondialyzable urinary hydroxyproline. 188 Levels of the aminoterminal propeptide of type III procollagen (pColl-III-N), determined by radioimmunoassay, also are elevated in serum from patients with Paget's disease and the levels correlate in most individuals with the extent and/or activity of the disease. 148'192 We presume that the elevated serum levels of pColl-III-N in Paget's disease result from synthesis of type III procollagen by the stromal fibroblasts in close proximity to the pagetic bone spicules. Elevations of serum levels of PICP in Paget's disease have been found in other series, although the increases are not nearly as striking as those of total or bonespecific alkaline phosphatase. 18~-~82 This may be due to
FREDERICK R. SINGER AND STEPHEN M. KRANE
problems intrinsic to the immunoassay or reflect metabolism of the type I collagen C-propeptides. There is no evidence that the type I collagen C-propeptides in bone are trapped in the extracellular matrix as it is deposited. Resorption of the fibrous stroma that is a characteristic feature of the pagetic lesion could result in release of amino-terminal peptides of type III procollagen. It should also be pointed out that the amino-terminal peptides of both types I and III procollagens have a potential functional role in bone remodeling. The aminoterminal propeptides have been proposed as negative feedback regulators of collagen synthesis that inhibit translation of procollagen m R N A s . 193'194 6. METABOLISM OF NONCOLLAGENOUS BONE PROTEINS IN PAGET'S DISEASE OF BONE Circulating levels of oL2HS-glycoprotein have been measured in patients with Paget's disease of bone and found on the average to be lower than those in normal adults. 195 The levels are inversely related to the activity of plasma alkaline phosphatase. These decreases of the concentration of tx2HS-glycoprotein in pagetic s e r u m a r e to be contrasted with increases in pagetic b o n e , 156 and are consistent with the idea that tx2HS-glycoprotein is synthesized in the liver, circulates in plasma, and is taken up into newly synthesized bone m a t r i x . 196 When the pagetic subjects were treated either with a bisphosphonate or calcitonin, the serum levels of tx2HS-glycoprotein tended to increase toward normal. 195 The changes observed after calcitonin therapy were considered to be too rapid and too large to be accounted for on the basis of decrease in bone formation alone, and the possibility of increased hepatic synthesis was raised. Levels of osteocalcin (bone Gla-protein or BGP) have also been found to be increased on the average in individuals with Paget's disease compared with normal subjects. 182'197-2~ Although in some series very high levels of alkaline phosphatase activity have been associated with only slight increases in osteocalcin levels, there has been on the average a significant positive correlation between serum levels of osteocalcin and activity of alkaline phosphatase in untreated patients with Paget's disease. 2~176 The serum levels of bone Gla-protein, however, have not always fallen when the Paget's disease has been treated with bisphosphonates to the extent predicted based on changes in total urinary hydroxyproline excretion and serum activity of alkaline phosphatase. Thus, in Paget's disease, levels of osteocalcin are not sensitive markers of pagetic bone turnover and may not reflect the same aspects of bone metabolism or even the same cellular source (e.g., osteoblasts vs. bone stromal fibroblasts). There are observations that serum and urine from patients with Paget's disease contain immunoreactive fragments of osteocalcin not identified in normals or subjects
CHAPTER 19 Paget' s Disease of Bone with other diseases except uremia. 2~ The presence of these fragments, possibly the result of aberrant metabolism, might account for the lack of correlation of levels of osteocalcin with other markers of skeletal turnover in Paget's disease. The significance of altered levels of osteocalcin must now be interpreted with respect to the recent findings in osteocalcin-deficient mice. 2~ Introduction of a homozygous null mutation in the osteocalcin gene results in an increase in bone formation without an impairment of bone resorption. Thus osteocalcin can be considered a negative biological regulator of bone formation rather than simply a structural component of the extracellular matrix and a "passive" marker of bone formation. Observations that urinary excretion of total Gla (not bone Gla-protein) is not different in pagetic subjects compared with normals 2~ simply reflect the fact that there are many sources of Gla (such as several clotting factors) other than bone Gla-proteins. Total urinary Gla is therefore not a useful discriminant in Paget's disease. Measurements have also been made of the serum levels of several other noncollagenous proteins that are relatively abundant in bone. Levels of osteonectin were found to be increased in patients with Paget's disease but not to the extent observed even using immunoassays for osteocalcin. 2~ Indeed, no correlation was found between serum levels of osteonectin and those of osteocalcin in Paget's disease or other forms of metabolic bone disease. Levels of osteonectin, osteopontin, and bone sialoprotein in serum reflect in large part the high content of these proteins in blood platelets. 2~ In some disorders such as hyperparathyroidism, hyperthyroidism, and postmenopausal osteoporosis, there is a correlation between serum levels of [32-microglobulin and bone remodeling. No such correlation, however, was observed in patients with Paget's disease. 212 It was proposed that in Paget's disease [32-microglobulin is reutilized in new bone formation rather than being released into the circulation. Although there is a report indicating that measurement of serum bone sialoprotein is useful as a marker of bone turnover in patients with multiple myeloma and metastatic malignancy, the levels in Paget's disease are not significantly elevated and do not approach the magnitude predicted from the levels of alkaline phosphatase. 213
C. A c u t e E f f e c t s o f C a l c i t o n i n The use of the calcitonins in treatment of Paget's disease will be discussed subsequently. The acute effects of calcitonin are so striking in pagetic subjects, however, that it is appropriate to discuss them as they relate to bone turnover. Bijvoet et a l . 177 w e r e the first to demonstrate that a decrease in serum calcium and phosphorus
575 concentrations was readily produced by injection of porcine calcitonin in pagetic patients, whereas no such response was detected in normal subjects. The fall in serum calcium was accompanied by a marked decrease in plasma levels and urinary excretion of hydroxyproline peptides. These results were confirmed and extended to include the effects of salmon calcitonin. 188 The major decrease in hydroxyproline excretion occurs in the oligopeptide fraction. Observations that in response to calcitonin administration, the ratio of Hyl(Glc-Gal)/ Hyl(Gal) rises (from the low values of bone) as the total hydroxylysine and hydroxyproline fall are consistent with a calcitonin-induced decrease in degradation of bone c01lagen rather than collagen from some other source. 152 The acute decrease in bone resorption induced by calcitonin appears to be due to an inhibition of osteoclastic activity. Within 30 minutes after administration of calcitonin to pagetic patients, the osteoclasts become less adherent to bone, lose their ruffled borders, and begin to fragment (Fig. 19-37). It thus seems reasonable to conclude, since the calcitonins have been shown to have their major action on decreasing bone resorption, that the pagetic subjects in whom bone turnover is markedly accelerated would show the greatest serum calcium response to a decrease in bone resorption. This conclusion is based in part on the demonstration that normal subjects respond to calcitonin by decreasing total urinary hydroxyproline excretion without significantly changing serum calcium levels. 152 The fall in total urinary hydroxyproline excretion in normal subjects is similar in percentage to that seen in patients with Paget's disease, although the absolute decrease is orders of magnitude less. In the normal subjects, as in the patients with Paget's disease, the decrease in excretion of polypeptide hydroxyproline and the increase in the ratio of polypeptide/oligopeptide excreted occur at the nadir of the calcitonin response. As an explanation of these observations, we proposed that the decrease in plasma and urinary hydroxyproline is greater than the decrease in plasma calcium because the size of the exchangeable calcium pool is large relative to rates of entry into this pool, even in Paget's disease in which the amount of calcium entering and leaving the pool is much greater than normal. In contrast, the size of the pool of small peptides containing hydroxyproline is small relative to rates of entry into this pool from degradation of collagen. Therefore, a given decrease in resorption would more profoundly affect the size of the oligopeptide hydroxyproline pool than that of the calcium pool. The results of this study further suggest that an acute decrease in bone matrix formation (as shown by decrease in excretion of polypeptide hydroxyproline) accompanies the fall in bone resorption. Other evidence for a decrease in bone matrix formation is provided by observations that there
576
FREDERICK R. SINGERAND STEPHENM. I~ANE
FIGURE 19--37 Electronmicrographs of iliac crest biopsies taken from a 60-year-old woman with Paget's disease prior to A and 30 minutes following 20 txg salmon calcitonin IV B. A, Edge of active osteoclast showing wide ruffled border containing collagen fibrils and bone mineral. Note numerous as well as large vacuoles immediately adjacent to the ruffled border (• B, Osteoclast displaying reduction in activity shown by the loss of the ruffled border and relatively smooth appearance of the cytoplasmic membrane bordering bone. Note the reduction in numbers of vacuoles but an increase in autophagic vacuoles compared with the initial biopsy (• (Courtesy of Dr. Barbara G. Mills.)
is a corresponding acute fall in circulating levels of the carboxyl-terminal procollagen peptide within hours after administration of calcitonin. 148 Whether this response is mediated by parathyroid hormone, which can acutely decrease collagen synthesis, or by a fall in the level of plasma phosphorus is not known.
D. Serum Alkaline Phosphatase Alkaline phosphatases in humans are encoded by four specific genes. One of these, localized to human chromosome 1, encodes the tissue-nonspecific isoenzyme. The latter is enriched in liver, kidney, and skeletal tissues, but the enzymes from these tissues have different properties, due to different posttranslational modifications. 214'215 The tissue nonspecific alkaline phosphatases are covalently bound to cell surfaces through a glycosylphosphatidylinositol (GPI) anchor. 216 The bone-specific enzyme is cleaved from osteoblasts at the GPI anchor where it is particular enriched, and released into the circulation. Although the functions of alkaline phosphatase in bone have not been established, recent experiments still support its postulated role in mineralization. The GPI-linked enzyme is thought to be the important biological form. Elevation of the level of alkaline phosphatase in serum is characteristically seen in patients with Paget's disease, an observation made first by Kay. 217 Indeed, the highest recorded values for alkaline phospha-
tase are found in patients with Paget's disease. The level correlates with the extent as well as the activity of the disease, as does the level of urinary hydroxyproline excretion. Serum alkaline phosphatase and urinary hydroxyproline excretion also correlate with each other 171 (Fig. 19-38). In almost all studies, the circulating alkaline phosphatase in Paget's disease is indistinguishable from that normally present in bone or in plasma in other bone diseases. Alkaline phosphatase purified from pagetic serum was found to behave differently from the enzymes purified to homogeneity from several other human tissues, that is, liver, kidney, intestine, and placenta. 218 When the native pagetic enzyme was desialylated, however, it behaved identically with respect to electrophoretic and isoelectric focusing properties with the desialylated forms from liver and kidney, but could be distinguished from the desialylated enzyme from placenta or intestine. In two pagetic subjects who were plasmaphoresed and the plasma replaced with albumin, the alkaline phosphatase activity that fell immediately to one third the initial levels returned to baseline within 10 to 15 days. The biological half-life of the enzyme in these subjects was therefore approximately 1 to 2 days. 219 Several commercially available immunoassays have recently been used to measure bone-specific serum alkaline phosphatase levels in Paget's disease. These assays appear to be more sensitive than assays of total alkaline phosphatase in discrimination of patients with mild disease from normal controls. 182 Increases in levels
CHAPTER 19
Paget's Disease of Bone
577 ,(208)
:~ 160
(214)
120
eo
.% 9, 4
40
Q
O0
0 %
i ,r 0
1
9
L
200
1
1
400
L
1
600
1
1
800
1
|
1000
TO TAL URINARY HYDROXYPROLINE (rag/24hr)
1
1
1200
FIGURE 19--38
The relationship between serum alkaline phosphatase and urinary hydroxyproline excretion in 42 patients with Paget's disease. (Data calculated from Franck WA, Bress NM, Singer FR, Krane SM: Rheumatic manifestations of Paget's disease of bone. Am J Med 56:592-603, 1974.)
are proportionally greater in patients with more extensive Paget's disease and it is likely that the bone-specific assays will be more widely applied to follow responses during therapy. It is distinctly unusual to see markedly elevated levels of alkaline phosphatase in subjects with Paget's disease who have isolated involvement of a small bone such as a vertebral body, and, indeed, the levels may be normal in 15% of patients with only one or two lesions determined by 99mTc-diphosphonate imaging. Isolated involvement of larger bones, however, is associated with a broad range of phosphatase levels. In our experience, involvement of the skull alone is often associated with levels of phosphatase higher in proportion to the percentage of skeleton involved; the converse is true for pelvic involvement. In general, with increasing numbers of pagetic lesions there is an approximately linear correlation of number with total or bone-specific alkaline phosphatase levels. 22~ When the disorder enters the sclerotic or osteoblastic phase and cellular activity decreases, even extensive involvement may be associated with limited elevations. In patients followed over years, the levels of alkaline phosphatase tend to show a long-term upward trend with a tendency to plateau in a range characteristic for that individual. TM Some patients show cyclic fluctuations about this more or less constant baseline. Although "explosive" elevations in serum alkaline phosphatase have been observed in individuals in whom osteosarcoma de-
velops on the background of Paget's disease, this is an unusual occurrence and is not discussed at all in most large s e r i e s . 222'223
E. Other Phosphatases In patients with Paget's disease and moderately elevated alkaline phosphatase levels, the activity of 5'nucleotidase is usually normal. Modest elevations in 5'nucleotidase may be seen, however, in serum from patients with very high alkaline phosphatase levels. 224 The level of total acid phosphatase is usually normal in patients with Paget's disease, although in subjects with widespread, advanced lesions and very high alkaline phosphatase levels, mild elevations of acid phosphatase levels may also be encountered. 225'226 Tartrate-resistant acid phosphatase (TRAP) is highly enriched in osteoclasts and measurements of TRAP are frequently utilized as an osteoclast marker in cell culture and histological analysis. Levels of TRAP are, on average, increased in serum of patients with Paget's d i s e a s e . 181'182'227 The increases are small, however, and clear-cut elevations are seen only in patients with extensive active disease and marked elevations in alkaline phosphatase levels. The superiority of the serum alkaline phosphatase assays probably reflects the unique nature of alkaline phosphatase as a GPI-linked ectoenzyme.
578
FREDERICK R. SINGER AND STEPHEN M. KRANE
VII. SYSTEMIC COMPLICATIONS AND ASSOCIATED DISEASES
A. Hypercalciuria, Renal Calculi, and Hypercalcemia Urinary calcium excretion in patients with Paget's disease may be high, low, or normal, since the amount excreted appears to be dependent on the rates of bone resorption and formation relative to each other, 8 as well as on homeostatic influences such as variations in the secretion of parathyroid hormone in response to changes in plasma calcium concentrations. In the majority of patients with Paget's disease, however, rates of resorption and formation are similar and therefore urinary calcium excretion is usually normal. In those patients in whom bone resorption is greater than formation, hypercalciuria is more likely to occur. Hypercalciuria may also occur when this equilibrium is disturbed, for example, following fractures or in association with extended immobilization. 228 In an individual patient, hypercalciuria may predispose to the formation of renal calculi. This is not a common complication of Paget's disease. Nagant de Deuxchaisnes and Krane, 8 after reviewing the literature on this subject, found an overall incidence of 5% among 1382 pagetic patients. Two factors other than the Paget's disease probably account for such an association. First, since patients with urinary calculi frequently have roentgenograms of the abdomen and pelvis, asymptomatic and presumably incidental Paget's disease in these areas would be detected more readily. Second, prostatic hypertrophy, which is common in elderly males, often leads to urinary tract obstruction, infection, and calculi. RidI o n 229 found that more than half of 22 pagetic patients with urinary calculi had prostatic hypertrophy. Hypercalcemia is distinctly uncommon in patients with Paget's disease. Nagant de Deuxchaisnes and Krane 8 were able to find in the literature only 17 patients with a serum calcium greater than 12 mg/dl after excluding those patients with coincidental hyperparathyroidism and multiple myeloma. In several of these reported instances it was uncertain that analysis of serum calcium levels was performed with sufficient accuracy to be confident about its interpretation. Reifenstein and Albright 228 reported originally that hypercalcemia developed in immobilized patients with Paget's disease, and it is difficult to separate the general effects of immobilization from the local effects of fractures. Hypercalcemia in patients with Paget's disease may also develop as a result of coincidental hyperparathyroidism 23~ or malignancy. Rarely hyperparathyroidism may be misdiagnosed as Paget's disease because of ra-
diological similarities in pelvic lesions. A complete skeletal survey and, if necessary, measurement of serum parathyroid hormone concentration should distinguish the two disorders. Secondary hyperparathyroidism with or without mild hypocalcemia has been reported in some patients with Paget's disease and normal renal function. 232'233 This has been attributed to an increased rate of bone formation compared with that of resorption that may occasionally be observed. It was pointed out earlier that histomorphometric findings consistent with secondary hyperparathyroidism have been found in nonpagetic bone from patients with Paget's disease. 48
B. Hyperuricemia and Gout The association of Paget's disease and gout is commented upon in some of the early descriptions of Paget's disease. Nevertheless, there had been little evidence to indicate that patients with Paget's disease have an increased incidence of hyperuricemia or gout until we reported our studies of the concentration of serum uric acid and the incidence of gout in 47 patients with Paget's disease. 71 The results suggested that the pagetic process could be an etiological factor in the development of gout. In this study of 28 men and 18 women, 19 patients (40.4%), 16 of whom were men, were found to be hyperuricemic. Serum uric acid concentrations in the group correlated with both the extent and the activity of the bone disease as defined by the percentage of bone involved radiologically, the serum alkaline phosphatase, and the total urinary hydroxyproline excretion. Seven of the males had clinical episodes of gouty arthritis. Urinary uric acid excretion was measured in six patients. Three were hyperuricosuric and three were hypouricosuric. The one patient who had a family history of gout had the most severe form of the disease (Fig. 19-39). It seemed reasonable to speculate that the turnover of nucleic acids in the active cells of pagetic bone might increase the urate pool and result in secondary hyperuricemia. 234 In a second large series, a history of gouty arthritis was present in only 11 of 149 patients and hyperuricemia was present in 20% of 118 patients not on allopurinol. 94 These findings were interpreted as indicating no increase in the levels of serum urate or the incidence of gout above that expected in the general population. It is probable that our series 71 included individuals with more severe disease and greater skeletal turnover, which may explain the different results. In a study of radionuclide imaging in patients with gout, 23% of the patients were found to have Paget's disease compared with 2.1% of a reference population who had had bone scans ordered for other reasons. 235
CHAPTER 19 Paget's Disease of Bone
579
FIGURE 19--39 A 59-year-old man with gout and generalized Paget's disease who has both diseases involving the left first metatarsal. The pagetic bone is radiodense and widened. The associated scalloped destruction of the metatarsal head is due to gouty arthritis. (From Franck WA, Bress NM, Singer FR, Krane SM: Rheumatic manifestations of Paget' s disease of bone. Am J Med 56:592-603, 1974.)
C. C a l c i f i c P e r i a r t h r i t i s a n d C h o n d r o c a l c i n o s i s Franck et a l . 71 reviewed the roentgenograms of multiple joints from 55 patients with Paget's disease and found a 36% incidence of periarticular calcifications. The association, although striking, could still be coincidental. The shoulders were most commonly involved but the elbow, wrist, hip, knee, metacarpophalangeal, and interphalangeal joints were also affected. Acute inflammatory episodes occurred in relation to these deposits. In general the calcifications were not adjacent to areas of Paget's disease and there was no correlation between the extent of the Paget's disease and the calcifications. Radi et a l . 236 have reported that 6 of 14 patients with Paget's disease had chondrocalcinosis on roentgenograms of wrists and knees. In contrast, Franck et a l . 71 found only two instances of chondrocalcinosis in 54 patients who had wrist and knee films. The main difference between the two series was that the patients without chondrocalcinosis had a mean serum alkaline phosphatase level seven times greater than the mean in the patients with chondrocalcinosis. It is conceivable that the high concentrations of alkaline phosphatase, which acts as an inorganic pyrophosphate at neutral pH, by decreasing ambient concentrations of inorganic pyrophosphate would protect against the deposition of cal-
cium pyrophosphate dihydrate crystals that characterize chondrocalcinosis.
D. C a r d i o v a s c u l a r C o m p l i c a t i o n s In the osteolytic and mixed phases of Paget's disease there is a marked increase in blood flow through involved extremities, manifested often by elevated skin temperature over the affected bone. 237 That this increased blood flow is not due to anatomical arteriovenous anastomoses but rather to increased perfusion of pagetic bone was shown by Rutishauser et al. 238 who injected the vessels of such bone at autopsy, and by Rhodes et a l . 239 who showed, using injections of microspheres, that there are no such channels greater than 15 Ixm in diameter. Others have concluded that there is associated vascular hyperplasia and ectasia in pagetic bone. 24~ It has been found that most, if not all, of the increased blood flow to pagetic extremities can be accounted for by increased cutaneous flow, as measured with water plethysmographs and by demonstrating that epinephrine iontophoresis reduces blood flow of an affected extremity almost to normal. 241 In addition, local heating of the pagetic extremity fails to increase blood flow in the pagetic limb as it does in the normal limb. Thus, reflex cutaneous dilation is the likely explanation for the cu-
FREDERICKR. SINGERAND STEPHENM. KI~NE
580
taneous hyperthermia and the elevated P02 in the venous blood of an involved limb. The extent to which increased vascularity of the bone proper contributes has been demonstrated using methods such as 18F clearance, 242 which show increased flow in pagetic bone and reduction following treatment. 243'244 Increased cardiac output secondary to increased blood flow to skeletal and nonskeletal tissue may be found in patients with generalized disease of at least 15% of the skeleton i n v o l v e d , 237'245-247 although we have observed increased cardiac output in patients with Paget's disease limited to the skull. This increased cardiac output in elderly individuals with Paget's disease may contribute to congestive heart failure, but particularly in the presence of other coexistent heart disease. It is generally responsive to the usual modes of treatment. Intracardiac calcification has been reported in association with Paget's disease. Harrison and Lennox 248 concluded that such calcification of the cardiac tissues was five times more common in patients with generalized Paget's disease than in control subjects. Since the calcification may involve the interventricular septum and the aortic and mitral valves, heart block may develop as a result of septal calcifications. Usually, however, the valvular calcifications are not of clinical significance, although in one report a patient with Paget's disease and calcification of the interventricular septum and aortic and mitral valves developed complete heart block and required a permanent transvenous pacemaker. 249 Recently Strickberger et al. 25~reported a correlation of calcific aortic valve disease with the severity of Paget's disease. Arterial calcifications are also common in patients with Paget's disease. For example, O'Reilly and Race TM found an incidence of 43% in their series. The calcifications are particularly common in limb arteries and may be detected by radiographic examination in as many as 50% of patients. 252 Such calcifications are generally of the Mofickeberg type (medial involvement and preservation of the lumen) and are usually of no clinical importance.
cluded by linkage analysis. 256 It is likely that this disorder is due to a defect in one of the elastic fiber microfibril-associated glycoproteins. The relationship between the abnormalities in pseudoxanthoma elasticum and Paget's disease remains obscure. The vast majority of patients with Paget's disease have grossly normal skin, even those with angioid streaks. We have examined skin biopsies from six patients with Paget's disease. Abnormal patterns of elastic fibers seen in four of these biopsies included a decrease in the number of the superficial fine elastic fibers with fragmentation and clumping of fibers as seen with elastic tissue stains. In the reticular layer, the coarse fibers were sparse and the few remaining ones greatly fragmented so that few interconnecting bundles were present. Such changes were also noted by P a t o n , 256 but he felt that after treatment with various connective tissue stains and digestion with beta-glucuronidase there was no evidence of any abnormality in the collagen or elastic fibers. Pat o n 256 explained his findings on the basis of the great variation of the cutaneous elastic fibers found normally in various portions of the body. We have not been able to refute this valid point, since the normal variations of cutaneous elastic fibers have not been well documented. Therefore, although the changes in the skin may be purely specious, we have recorded our findings so that future investigators may deal with this problem. Although angioid streaks are found in 8% to 15% of advanced cases of Paget's disease, 255'256 comparatively little has been written about such lesions in Paget's disease (Fig. 19-40). The changes described for the angioid streaks seen in Paget's disease are histologically identical to those of pseudoxanthoma elasticum and include
E. P s e u d o x a n t h o m a E l a s t i c u m , A n g i o i d S t r e a k s , and Skin Changes The association of pseudoxanthoma elasticum and Paget's disease 253-255 has provoked speculation that Paget's disease may be a widespread disorder of connective tissue rather than a disease limited to the skeleton. Characteristics common to these two diseases include angioid streaks, vascular calcification, and skin calcification. 256'257 The molecular defect in pseudoxanthoma elasticum has not yet been identified, although the genes for elastin, fibrillin-1, fibrillin-2, and lysyl oxidase have been ex-
FIGURE 19--40 Multipleangioid streaks in Paget's disease (arrow indicates most extensive one).
CHAPTER 19 Paget' s Disease of Bone basophilia and cracking of Bruch's membrane and proliferation of fibrovascular tissue through the defects. It was suggested that since calcification may occur at the site of elastic fibers in the skin of patients with Paget's disease, the same event may occur in Bruch's membrane. 255 Bruch's membrane is located between the retinal pigment epithelium and the choriocapillaris. This membrane comprises a central elastic layer; inner and outer collagenous layers; and other layers containing type IV collagen, laminin, and heparan sulfate proteoglycan. 259 Thus, Bruch's membrane has features of basement membranes as well as the extracellular matrices of vascular walls. Fibrillins and other elastic fiber microfibril-associated proteins must also be present and abnormalities in their structures are probably responsible for the pathology. Recently it has been shown that deposits in Bruch's membrane that are found in Sorsby's fundus dystrophy consist of the product of a mutant gene for tissue inhibitor of matrix metalloproteinases-3 ( T I M P - 3 ) . 26~ Eventually the molecular pathology of these interesting fundus and vascular lesions will be elucidated in Paget's disease. Some evidence that supports the concept of a generalized connective tissue abnormality in Paget's disease was reported by Francis and Smith, 262 who examined the nature of skin collagen in 1 1 patients with Paget's disease and control subjects. They found a decreased amount and stability of the polymeric collagen fraction in the pagetic patients and speculated that a progressive failure of glycoprotein and collagen crosslinking reactions could account for the observed abnormalities. As previously discussed, however, there are no other indications of a major structural abnormality of bone collagen specific for Paget's disease.
F. Malabsorption Syndrome Somayaji 263 described eight patients with Paget's disease who had a variable pattern of gastrointestinal malabsorption. Diarrhea, steatorrhea, xylose malabsorption, and folate deficiency were prominent features. It was postulated that secondary folate deficiency or relative ischemia of the bowel secondary to increased bone blood flow might be responsible for the observations. No confirmatory reports have been published.
VIII. DRUG TREATMENT A. Indications Many patients with Paget's disease require no treatment, since they are asymptomatic and the localization
581 of their disease makes it unlikely that even in the future there would be significant morbidity from complications. On the other hand, there are patients who can receive great benefits from the administration of effective agents that have become available during the past quarter of a century. In Table 19-1 some reasons for treating Paget's disease are summarized. Probably the most common reasons to treat Paget's disease are to relieve bone pain and to attempt to prevent complications of the disease. Because of the development of potent agents that can be used intermittently, the latter indication may become far more common in clinical practice.
B. Calcitonin Calcitonin inhibits osteoclast activity by first binding to a well-defined receptor on the cell membrane of osteoclasts TM and progenitor cells. 265 The clinical experience in Paget's disease with all forms of calcitonin has, in general, been similar. Calcitonins are usually administered by subcutaneous or intramuscular injection, usually on a daily basis. With few exceptions, affected individuals have responded with a fall in urinary hydroxyproline excretion within days and a fall in serum alkaline phosphatase levels within weeks. Calcium balance usually becomes more positive during the initial 1 to 4 months of treatment, 266-268 and continued treatment (9 to 19 months) is not associated with any change in calcium balance, e69 In one series of 38 patients treated up to 5 years, mean total body calcium as assessed by neutron activation actually fell by 4 % . 270 Histological examination of sequential bone biopsies in patients treated with calcitonin shows a reduction in osteoclast number as well as a reduction in woven bone and the extent of fibrosis of the bone m a r r o w . 268'27a The reported clinical benefits associated with chronic calcitonin therapy include relief of bone p a i n , 272'273 red u c t i o n of increased cardiac output, 274 reversal of central and peripheral neurological deficits, 275 stabilization of heating deficits, 276 healing of osteolytic lesions, 277'278 reduction of skeletal blood f l o w , 279 and prevention of excessive hemorrhage associated with orthopedic surgery. TM Of particular interest is the radiological improvement of osteolytic lesions, which is readily demonstrable during long-term therapy. An example of the efficacy of salmon calcitonin on healing of bone lesions is shown in Figure 19-41. In some patients the formation of a more normal cortex in long bones, the restoration of the corticomedullary junction, and a reduction in the total diameter of the shaft of expanded bones is observed. Beneficial effects on the skeletal lesions observed radiologically may, however, require larger doses
582
FREDERICK R. SINGER AND STEPHEN M. KRANE
TABLE 19--1
Reasons to Treat Paget's Disease with Drugs Outcome
Problem Bone pain attributable to Paget's disease
Usually relieved but bisphosphonates may temporarily increase pain
Increased cardiac output
Decreases concomitantly with reduced bone turnover; may be of considerable benefit to patients with underlying heart disease but many patients have no cardiac symptoms
Recurrent urinary calculi associated with hypercalciuria
Hypercalciuria is usually decreased by all therapies and should reduce the incidence of stone formation
Hypercalcemia due to Paget's disease
This is unusual and usually associated with immobilization in patients with extensive disease; good response to therapy of Paget's disease
Neural compression syndromes
Dramatic clinical improvement occasionally reported but difficult to predict response
Fracture of pagetic lesion
Treatment with newer bisphosphonates should prevent worsening of osteolytic process and promote healing; etidronate probably predisposes to fracture and may induce osteomalacia
Elective surgery on pagetic bone or if postoperative state will require prolonged immobilization
Treatment with calcitonin or bisphosphonates (preferably not etidronate) is likely to reduce bleeding in pagetic lesions and prevent immobilization, hypercalcemia, and worsening of osteolytic lesions, although these effects are hard to prove
Disabling weakness or fatigue or uncertain cause in patients with extensive disease
Benefits are not well documented
Early onset of Paget's disease in sites where significant complications would be expected (i.e., temporal bone, spine, lower extremity)
Intermittent use of more potent bisphosphonates would likely be effective in preventing deformation; less expensive than older therapeutic agents
of calcitonin than are needed to produce maximal biochemical suppression. 277 As would be expected, improvement in bone scans during long-term calcitonin therapy is also observed. 28~ Gallium scans appear to be a more sensitive means of detecting responses to calcitonin therapy, perhaps because gallium uptake is a better index of bone cell activity. 281 This concept is supported by the finding that gallium localizes in the nuclei of osteoclasts in Paget's disease. 282 Several patterns of response to calcitonin are apparent during long-term treatment of Paget's disease. In one pattern, there are complete biochemical and clinical remissions 268 (Fig. 19-42). A more common pattern (the "plateau response") is that in which there is an initial decrease in indices of bone remodeling, but normalization of bone remodeling does not take place with continued treatment (Fig. 19-43). Patients who show this pattern usually maintain clinical improvement associated with biochemical evidence of decreased bone turnover. On the average, the biochemical indices in these patients are stabilized at approximately 50% of pretreatment levels. A third pattern is characterized by an initial response comparable to that in the other groups, but as treatment is continued, biochemical and clinical abnormalities recur 283-287 (Fig. 19-44).
The plateau response has been observed in patients treated with each type of calcitonin. In an attempt to exclude inadequate dosage of hormone as a factor in the plateau response, Singer and colleagues 2ss increased the dose of salmon calcitonin up to tenfold in four patients but found no significant further decreases in the biochemical indices of disease. The third pattern of acquired resistance to the effects of calcitonin has been found in some patients treated with porcine, 287 s a l m o n , 283,284,zs6,zs9-291 human calcitonin. 286 In the great majority of patients treated with the nonhuman calcitonins who become resistant to metabolic effects, high titers of antibodies to these species of calcitonin have been demonstrated in the circulation. Resistance to porcine, 287 s a l m o n , 286,zs7,zs9 and human calcitonin, 285 however, may also rarely develop in the absence of detectable circulating antibodies. With the exception of a single individual treated with human calcitonin who developed clinically insignificant antibody titers, 292 antibodies to human calcitonin in treated patients have not been detected. It is not agreed upon whether antibodyassociated calcitonin resistance is a rare or common event. Martin a n d colleagues 289'293'294 found that only 1 individual out of 36 who were treated with salmon calcitonin became resistant owing to high antibody titers.
CHAPTER 19 Paget's Disease of Bone
583
FIGURE 19--4 1 Osteolytic lesion in right proximal femur. A, Before treatment. B, Two years after treatment with salmon calcitonin. (Courtesy of Dr. Jane Mahaffey.)
Results of our own experience 283'286 suggest that resistance due to the formation of salmon calcitonin antibodies is a common event in the United States. Resistance to salmon calcitonin developed during long-term treatment in 22 of our 85 patients (26%). 286 Antibodies to salmon calcitonin were detected in 56 of the 85 patients (66%), and in 19 of the 19 patients whose antibody titer was 1:1500 or greater, calcitonin resistance was evident. Antibodies, in titer less than 1:1500, did not appear to interfere with the efficacy of long-term therapy. In the three patients who became resistant to calcitonin in the absence of antibodies, no other explanation could be established for the mechanism of resistance. Further evidence for the clinical significance of high antibody titers is established by the observation that 15 of 15 resistant patients (including five unpublished cases) have responded to treatment with human calcitonin. 286"295These results are consistent with those studies in vitro in which it has been demonstrated that antibodies to salmon calcitonin do not bind human calcitonin. An example of successful response to human calcitonin of a patient who was resistant to salmon calcitonin is illustrated in Figures 19-45 and 19-46. Another factor that might alter the clinical response of patients treated with calcitonin is the stimulation of parathyroid hormone secretion secondary to calcitonin-
induced hypocalcemia. 288 Dube and colleagues 296 observed a slight elevation of the levels of serum immunoreactive parathyroid hormone in five patients treated with porcine calcitonin and proposed that the development of secondary hyperparathyroidism could account for resistance to calcitonin. Burckhardt and colleagues TM studied the parathyroid hormone response to calcitonininduced hypocalcemia in 12 patients with Paget's disease before and during treatment with salmon calcitonin and also observed increases in serum levels of parathyroid hormone. Hypersecretion after prolonged treatment, however, was not observed. Other investigators also found no evidence of hyperparathyroidism in their patients. 269'287'297-299 The possibility remains, however, that the persistent stimulation of parathyroid hormone secretion may interfere with the clinical response. Conclusive evidence for this response would be provided only if the histological changes of hyperparathyroidism were to be documented in bone uninvolved with Paget's disease. There is suggestive evidence of hyperparathyroidism in nonpagetic bone of individuals treated with human calcitonin, 297 although a similar appearance has also been described in untreated patients. 5~ Attempts have also been made to suppress the activity of Paget's disease by stimulating endogenous calcitonin secretion by means of oral calcium supplementation in
584
FREDERICK R. SINGER AND STEPHEN M. KRANE
FIGURE 19--42
Result of a 45-week period of daily administration of salmon calcitonin to a 78-year-old man with Paget's disease. Data are expressed as mean _ SE of multiple determinations made during a 1-week period before and during treatment. [From Singer FR, Keutmann HT, Neer RM, et al: Pharmacological effects of salmon calcitonin in man. In Talmage RV, Munson PL (eds): Calcium Parathyroid Hormone and the Calcitonins. Amsterdam, Excerpta Medica, 1972.]
combination with the diuretic chlorthalidone. 3~176 This regimen has been shown to significantly lower serum alkaline phosphatase activity and urinary hydroxyproline excretion as well as improve symptoms in most patients, although no direct evidence of increased calcitonin secretion has been presented. It is unlikely that this program is as effective as treatment with exogenous calcitonin, since the abnormal biochemical indices are not suppressed to the same degree and healing of osteolytic lesions has not been reported. Nevertheless, a trial of 1500 mg of supplemental calcium with 25 mg of chlorthalidone or equivalent of another thiazide diuretic may be worth considering in selected patients who have bone pain and mainly sclerotic lesions who are unable (e.g., for financial reasons) to embark on a course of calcitonin therapy. On the basis of more than 20 years of successful clinical use of the calcitonins in Paget's disease it can be concluded that these hormones can effectively and safely suppress abnormal bone remodeling as well as induce a variety of beneficial clinical responses. Although side effects of nausea and facial flushing may be troublesome in some individuals, rarely has significant toxicity been reported. 272 It should be stressed that joint pain, a corn-
mon complication, will probably not respond to calcitonin therapy. The optimum dose of the salmon calcitonin is in the range of 50 to 100 MRC units administered daily or three times weekly by subcutaneous self-injection. If treatment is continued for at least 1 year and then discontinued, there is considerable variation in the time period when manifestations of the disease reappear. 3~ It may therefore be worthwhile to wait until symptoms reappear or the alkaline phosphatase increases. In those individuals in whom neurological deficits are improved, the hormone may have to be administered indefinitely. The elucidation of the mechanism of calcitonin resistance in those individuals who do not develop significant titers of neutralizing antibodies will probably require studies of calcitonin receptors in the plasma membrane of the osteoclasts. It is possible that clones of osteoclasts without calcitonin receptors could develop during chronic treatment. The pathogenesis of nausea and facial flushing after calcitonin injection is not understood. The mode of administration of calcitonin by selfinjection or even by a nurse or relative is not accepted by some patients. Preliminary results obtained with nasal spray and suppository formulations of salmon calcitonin
CHAPTER 19 Paget's Disease of Bone 2000
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Result of a 72-month period of daily subcutaneous administration of human calcitonin to a 68year-old man with Paget's disease. The upper limit of normal for urinary hydroxyproline is 40 mg/24 hr and for serum alkaline phosphatase activity is 3 Bessey-Lowrey-Brock units.
have produced modest results. 3~176 In our own experience, only a minority of patients treated with salmon calcitonin by nasal spray had a response equivalent to that produced by parenteral administration. It should also be noted that human calcitonin is no longer commercially available. Since the advent of potent bisphosphonate therapy the use of parenteral salmon calcitonin therapy has decreased. In selected patients with mild to moderate disease activity it is still a reasonable option if bisphosphonate therapy proves ineffective or not tolerable.
C. Bisphosphonates Bisphosphonates (formerly diphosphonates) are synthetic analogues of inorganic pyrophosphate in which -P-C-P- bonds are substituted f o r - P - O - P - bonds. The bisphosphonates bind to the surface of calcium phosphate mineral. This property is responsible in part for localization of the bisphosphonates coupled to a radionuclide, in regions of active bone formation. In vitro, the bisphosphonates retard precipitation of calcium phosphate from solution and slow the growth and dissolution of hydroxyapatite crystals. Extensive studies in experimental animals and humans indicate that they inhibit
bone resorption and formation. The mode of action of bisphosphonates is complex and likely to involve direct effects on osteoclasts and osteoclast precursors as well as indirect effects on these cells through possible interactions with osteoblasts and macrophages. 3~ The mode of action also appears to be dependent on the structure of the side chains. The first bisphosphonate used to treat Paget's disease was disodium etidronate, which is usually administered at a dose of 5 mg/kg body weight daily for 6-month periods, e78'3~ Treatment with this dose results in a slow decrease in serum alkaline phosphatase activity and urinary hydroxyproline excretion, which reach a nadir between 3 and 6 months after beginning treatment. The decrease in urinary hydroxyproline excretion usually occurs prior to the decrease in serum alkaline phosphatase activity. After a 6-month period of treatment, their biochemical abnormalities may remain suppressed for 1 year or more 3~ in many patients. As has been found with calcitonin therapy, there are several patterns of biochemical response to disodium etidronate. 3~ Approximately 40% of patients have a prolonged response after a single course of therapy. These individuals are often those with modest initial elevation of biochemical indices of disease, although occasionally patients with markedly elevated alkaline phosphatase levels may also have a pro-
586
FREDERICK R. SINGER AND STEPHEN M. KRANE
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FIGURE 1 9 - 4 4 The control and treatment levels of urinary hydroxyproline, plasma alkaline phosphatase and salmon calcitonin-125I binding titer during an 11.5-month period when a 5 I-year-old male received 1200 MRC units of salmon calcitonin daily. The hydroxyproline data points represent the mean SE of at least five 24-hour urine collections during a 1-week period. The upper limit of normal is 40 mg/24 hours. The alkaline phosphatase data points represent the mean SE of at least three blood samples taken during the week. The upper limit of normal is 2.6 Bessey-Lowrey-Brock units. The binding titer is plotted on a log scale. (From Singer FR, Aldred JP, Neer RM, et al: J Clin Invest 51:2331, 1972.)
longed remission. A second group of patients 3~ (---45%) experience a good response to the initial course of therapy but require retreatment with disodium etidronate 3 to 63 months after the initial therapy, and then respond similarly to 20 mg/kg/day but not as well to 5 mg/kg/ day. The final group of patients 3~ (---15%) do not respond to retreatment during an average follow-up of 6 years. Resistance is best predicted by an initial urinary hydroxyproline level of more than ten times normal. Following high-dose disodium etidronate therapy, a reduction in cardiac output is observed coincident with reduction of biochemical activity. 31~The mean reduction of---27% is probably explained by a proportionate reduction in skeletal blood flow. 242 Other features of the clinical response to disodium etidronate are not identical to those produced by calcitonin, however. Bone pain may be relieved in 50% or more of patients 3~1 over a period of several months, but not infrequently a dramatic increase in bone pain develops in the site of pagetic lesions. 3~ This is more likely to occur with 20 mg/kg/ day, but we have noted a paradoxical increase in pain
even in patients treated with 5 mg/kg/day. After treatment is stopped, the pain usually resolves within several weeks to months. The pathogenesis of disodium etidronate-induced bone pain is not known but is likely related to inhibition of bone mineralization, which will be discussed subsequently. Another puzzling aspect of the response to disodium etidronate is that despite considerable suppression of biochemical indices of disease, the extent of osteolytic lesions may increase during treatment. 278'312-314 An example is illustrated in Figure 19-47. The increase in radiolucent lesions associated with disodium etidronate therapy is most likely to occur with a dose of 10 or 20 mg/kg/day. We have also observed worsening of osteolytic lesions in several patients treated with 5 mg/kg/day, although this is observed only in a minority of treated patients. Little evidence for healing of osteolytic lesions has been presented. In one report, two of three lesions healed during multiple courses of therapy. 315 In another report, radiological healing was evident in three patients with multiple osteolytic lesions but the results were not
CHAPTER 19 Paget's Disease of Bone
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consistent. 278 A minority of the lesions improved, but the majority deteriorated. After treatment is stopped, healing may occur spontaneously 3~6 and may be accelerated if calcitonin therapy is instituted. 278 The explanation for the paradoxical increase in bone pain experienced by some patients and the worsening of osteolytic lesions may be found in an examination of bone biopsies from treated patients. A characteristic find-
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588
FREDERICK R. SINGER AND STEPHEN M. KRANE
FIGURE 19--47 The evolution of osteolytic skull lesions in a 60-year-old man treated with disodium etidronate, 5 mg/kg/day, for 7 months. A, Osteolytic lesion of the skull at the time treatment was started. B, New osteolytic lesions have appeared 6 months after treatment was discontinued despite the observation that serum alkaline phosphatase activity had decreased by 66% at this time.
5 to 8 mg/kg/day. An unusual feature observed in this study was that thickened osteoid seams with absent mineralization were found in a focal distribution along the trabecular surfaces. This finding contrasts with the diffuse distribution of the thickened osteoid seams of vitamin D deficiency. It has also been reported that 1 month of treatment with 20 mg/kg/day of disodium etidronate results in a clinical response superior to that of 5 mg/kg/day for 6 m o n t h s . 319 Although bone histomorphometry revealed transient impaired mineralization in these subjects, a sustained decrease in bone resorption was produced. These observations led to the proposal that short-term high-dose therapy may maximize suppression of disease activity but decrease the likelihood of clinically significant osteomalacia. These conclusions were disputed as a result of similar studies by another group of investigators who nevertheless reported similar histological changes. 32~ Impairment of mineralization in disodium etidronate-treated patients is probably not ascribable to inhibition of vitamin D metabolism, since in one study of this problem, intestinal calcium absorption actually increased and serum 1,25(OH)2D levels re321 mained within the normal range. A desirable therapeutic effect observed histologically in disodium etidronate-treated patients is a reduction in osteoclastic bone resorption, which is more profound in patients treated with 20 mgflKg/day. 311'312'317 Ultrastructural analysis of osteoclasts reveals considerable evidence of cell degeneration, although the characteristic nuclear and cytoplasmic inclusions are unaltered. 322 Bone marrow fibrosis is reduced, and areas of woven bone may be replaced by lamellar bone. Side effects of disodium etidronate therapy, in addition to increased bone pain, include nausea and diarrhea,
both of which are uncommon except at higher dosages. Hyperphosphatemia is a common occurrence at high dosages and is a consequence of increased renal tubular reabsorption of phosphate. Mild hyperphosphatemia may develop even following treatment with low dosages 3~8 and may indicate that greater than the average amount of drug has been absorbed. There is some controversy whether the incidence of fractures is increased by disodium etidronate therapy. After a multicenter review of 737 patients treated for varying periods with 2.5 to 20 mg/kg/day, it was concluded that treatment was not associated with an increased fracture incidence. 319a Nevertheless, it is likely that high-dose therapy, and, in some instances, low-dose therapy, can contribute to the development of fissure fractures 318 and completed pathological f r a c t u r e s . 3~ This is likely to result from the decreased strength of active pagetic bone when the resorptive phase is dominant and when the new bone formed is inadequately mineralized. Because of this, we feel that disodium etidronate therapy should be avoided, if possible, in patients with osteolytic lesions in weightbearing regions of the skeleton. It seems likely that few will use disodium etidronate as physicians become more acquainted with the growing number of more potent bisphosphonates, all of which are more effective in Paget's disease at doses that rarely induce a mineralization defect. The next two bisphosphonates, clodronate and pamidronate, introduced into clinical trials in the 1970s, do not produce a mineralization defect in the doses generally used to treat Paget's disease. Clodronate (dichloromethylene bisphosphonate) has been successfully used both orally 324 and intravenously 325 to treat Paget's disease. Unlike etidronate, treatment with clodronate can
CHAPTER 19 Paget's Disease of Bone cause transient hypocalcemia and secondary hyperparathyroidism, 324'326 but this does not interfere significantly with the therapeutic response. As bone turnover returns to the normal range, normocalcemia and a euparathyroid state follow as the coupling of bone formation and resorption is restored. In addition to the absence of a mineralization defect associated with the use of clodronate, 326 an increase in bone pain does not occur and hyperphosphatemia is not observed in treated patients. Clodronate is available for general use in many countries but not in the United States. This came about because clinical trials were stopped after several reports of leukemia in treated patients appeared. 327 In retrospect, leukemia appears to have been a coincidence or could be attributed to benzene exposure. Pamidronate [aminohydroxypropylidine bisphosphonate (APD)], the first aminobisphosphonate in clinical use, has proved to be a highly effective therapy of Paget's disease since its introduction in 1979. 328 Because of relatively poor oral tolerance it has mainly been given intermittently by intravenous infusion. In patients with mild Paget's disease (serum alkaline phosphatase activity about two times normal) a single infusion of 60 mg can induce a biochemical remission for 1 year or longer 329 (Fig. 19-48). This dose is equally effective and well tolerated when infused for 1, 4, 8, or 24 h o u r s . 33~ In the United States the drug has been approved to be given as an intravenous infusion of 30 mg over 4 hours on 3 consecutive days. This is a highly inefficient protocol for treatment, as this regimen is no more effective than a single 60-mg infusion and it is often inadequate
FIGURE 19--48 Plasmaalkaline phosphatase (o) and urinary hydroxyproline (o) excretion (mean _+ SEM) in 11 patients (except for days 270 and 360 where n = 6) with Paget's disease before and up to 1 year after a single intravenous infusion of 60 mg of AHPrBP. [From Thiebaud D, Jaeger P, Gobelet C, et al: A single infusion of the bisphosphonate AHPrBP (APD) as treatment of Paget's disease of bone. Am J Med 85:207-212, 1988.]
589 in restoring bone turnover to normal or near normal levels in patients with moderate to severe Paget's disease. A bewildering number of treatment regimens with pamidronate have been suggested for patients with alkaline phosphatase levels greater than twice normal. The regimens vary from relatively low-dose infusions (15 to 45 mg) given weekly for up to 12 w e e k s 331'332 to daily infusions for up to 10 c o n s e c u t i v e d a y s . 333 With each regimen generally good suppression of disease activity has been noted and biochemical parameters are often suppressed into the normal range. In patients with the most severe disease, however, normalization of biochemical parameters may not be achieved with a total treatment dose up to 2520 mg. 334'335 U s e of high doses of pamidronate (180 to 360 mg over 6 or 9 weeks) has been reported to produce osteomalacia (one of ten patients) or mineralization defects (three of ten patients), although adverse clinical effects were not observed in o n e s e r i e s . 336 Should patients show relative resistance to treatment with pamidronate, it may be advisable to consider use of another potent bisphosphonate. Pamidronate therapy has been shown to be far superior to etidronate with respect to the radiological progression of osteolytic lesions. 337 Significant healing was observed in 17 of 20 lesions, whereas deterioration was noted in 50% of lesions in etidronate-treated individuals. As would be expected, reduced uptake of bone scanning agents is commonly found after pamidronate treatment. 338 Patients who have been treated with intravenous pamidronate generally have few significant side effects. A temporary increase in bone pain has been noted. About one third of patients experience transient fever and myalgias that generally disappear after several d a y s . 339 This usually occurs only after the first infusion. There is also a transient leukopenia of no clinical consequence. As with clodronate there is also transient hypocalcemia and secondary hyperparathyroidism that reverses as bone turnover becomes normal. Rarely, ocular inflammation may o c c u r . 340 In one patient, multiple infusions appeared to produce heating loss, vertigo, and tinnitus. 341 A second aminobisphosphonate, alendronate (hydroxybutylidene bisphosphonate), is considerably more potent than pamidronate and has been approved as an oral agent for Paget's disease in the United States and other countries. Earlier studies with intravenous alendronate (2.5 to 25 mg/day for 4 days) demonstrated a 70% suppression of the levels of biochemical markers of bone turnover that was sustained for 2 to 18 months off treatment. 342 The highest doses produced the longest biochemical remissions. A comparative study of oral alendronate (40 mg/day) with etidronate (400 mg/day) for 6 months revealed the clear superiority of alendronate. 343 There were significantly greater decreases in both serum
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alkaline phosphatase activity (79% vs. 44%) (Fig. 1949) and urinary deoxypyridinoline excretion (75% vs. 51%). Normal serum alkaline phosphatase activity was achieved in 63% of the alendronate-treated patients but only in 17% of the etidronate-treated patients. In another study of alendronate therapy, similar suppression of biochemical activity as well as improvement in the appearance of osteolytic lesions in nearly half of the patients was found. 54 No evidence of a mineralization defect was observed in either study and no significant gastrointestinal side effects occurred. The most recently approved bisphosphonate for treatment of Paget's disease in the United States is tiludronate (chlorophenyl thiomethylene bisphosphonate). At a dose of 400 mg daily for 3 months serum alkaline phosphatase activity is reduced at 3 months by 45% and at 6 months by 49%. 344 The drug is well tolerated, and at relatively high doses in animals no mineralization defect is found. Other promising bisphosphonates are still undergoing research trials. Of particular interest are olbandronate, risedronate, and zoledronate. Olbandronate (dimethylaminohydroxypropylidene bisphosphonate) is a particularly interesting aminobisphosphonate. Although an aminobisphosphonate, it causes no gastrointestinal symptoms and is about fivefold more potent than oral pamidronate. 345'346It does produce a febrile response in about 30% of patients that is associated with a transient increase in circulating bioactive interleukin6. 347 A dose of 200 mg/day for 10 days is sufficient to induce a biochemical remission in the majority of cases.
Risedronate is another bisphosphonate with potency somewhat greater than alendronate. Preliminary studies indicate that a 2-month course of 30 mg daily can reduce biochemical markers of bone turnover to near normal levels in most patients with Paget's disease whose serum alkaline phosphatase activity is 1000 IU/ml or greater. 348 Zoledronate is a bisphosphonate containing an imidazole ring that is biologically active in microgram amounts in patients with Paget's disease. 349 As little as 216 Ixg intravenously lowers urinary calcium and hydroxyproline excretion over a 14-day period. This great potency may allow the development of a transdermal route of administration of the drug.
D. Mithracin This drug is being described for historical reasons, since it is seldom used. Mithracin (formerly mithramycin) is an antibiotic that has proved to be an effective antitumor drug in the treatment of embryonal tumors of the testis. The drug also has a potent cytotoxic effect on bone cells, particularly osteoclasts, possibly through inhibition of RNA synthesis. This has made it an effective agent in the acute control of severe hypercalcemia. Ryan and colleagues have pioneered the use of mithracin in the treatment of patients with Paget' s disease. 35~ 352 They and subsequent investigators have demonstrated occasionally dramatic effects of this drug in patients with high skeletal turnover. In a manner analogous to the effects of calcitonin or intravenous bisphosphonates, acute hypocalcemia and hypophosphatemia, a reduction in urinary hydroxyproline excretion, and a rise in serum parathyroid hormone levels result from a single infusion of mithracin. 353 In contrast to calcitonin, however, daily infusions produce persistent mild hypocalcemia, which resolves after treatment is discontinued. These effects are consistent with a potent inhibition of osteoclastic bone resorption. After ten daily infusions of mithracin at 10 to 25 Ixg/ kg body weight, urinary hydroxyproline excretion and serum alkaline phosphatase activity may decrease into the normal range in many patients. These decreases are maintained for a variable period ranging from several months to as long as 12 years (rare). Weekly infusions of 15 to 25 Ixg/kg body weight also have been reported to produce prolonged biochemical remissions. Bone scans may revert toward a normal pattern 354 and roentgenograms also, although the latter have not been extensively evaluated. A reduction in bone pain (frequently rapid) has been noted in the great majority of treated patients, although no double-blind trials have been undertaken. There is a good correlation between pain relief and biochemical re-
CHAPTER 19 Paget's Disease of Bone mission as well as recurrence of pain and worsening of biochemical parameters. There are significant problems encountered in using mithracin. The drug can be administered only by intravenous infusion and must be infused carefully, since extravasation can lead to painful soft tissue injury. Side effects may be troublesome. Nausea and vomiting may occur but are less likely to develop if the drug is infused over 4 hours or more rather than as a bolus. Toxic effects from mithracin therapy include abnormal bleeding ascribable to impaired platelet function and decreased production, hepatitis as manifested by increased levels of circulating hepatic enzymes, and renal failure. In most patients, the toxic effect is transient, although persistence of reduced glomerular filtration rate has been reported. 352 If smaller doses or weekly therapy is utilized, the incidence of toxic effects is considerably reduced. Although mithracin and another cytotoxic drug, actin o m y c i n D, 355 have been shown to suppress the activity of Paget's disease, these agents should not be considered first-line agents in therapy of the disease. The use of potentially lethal drugs in a benign disorder seems unwarranted in the majority of patients. Their primary use should be reserved for patients who have not responded to the other available agents or others who cannot tolerate such therapies. Careful monitoring of platelet number, liver enzymes, and renal function is critical during the treatment course if toxicity is to be minimized. Reduction of dosage is recommended in patients with liver or renal disease.
E. Gallium Nitrate Gallium nitrate, another compound originally developed as a cancer chemotherapeutic agent, is an effective inhibitor of osteoclast function. 357 Cyclical low-dose subcutaneous administration of gallium nitrate to patients with Paget's disease has been shown to produce modest suppression of bone turnover. 358 More extensive study of the optimum dose of this agent and its safety is necessary before it is likely to be acceptable for general use.
F. Miscellaneous Drugs Many other drugs have been used to treat Paget's disease, including arsenic, magnesium carbonate, aluminum acetate, anabolic steroids, folic acid, phenylbutazone, acetylsalicylic acid, adrenocorticoid-steroids or ACTH, sodium fluoride, phosphate, glucagon, and colchicine. 356 Some of these drugs were evaluated at a time when only subjective criteria were available to assess efficacy. Others, particularly acetylsalicylic acid and glucocorticoids, have
591 produced significant although transient suppression of the disease, although relatively large doses were required and their use was accompanied by intolerable side effects.
G. Combination Therapy Combinations of calcitonin and disodium endronate, 278'314'359 calcitonin and mithracin, 36~ mithracin and
glucagon, 36~ and mithracin and disodium etidronate 352 have been utilized in a relatively small number of patients to attempt to obtain additive suppressive effects on the disease and perhaps reduce toxicity. The results suggest that combination therapy may produce greater suppression of biochemical indices, but no compelling clinical evidence has been presented that patients will therefore benefit to greater extent than from single-agent therapy. On the contrary, the calcitonin-disodium etidronate combination is not as useful in inducing healing of osteolytic lesions as is calcitonin alone. 278'314
H. Radiological and Biochemical Monitoring
of Therapy In symptomatic patients with radiologically demonstrable osteolytic lesions, a radiograph 1 year after initiating treatment is helpful in demonstrating the efficacy of the therapeutic regimen. Thereafter, a biannual evaluation is probably adequate if evidence of resolution of the osteolytic focus has been detected. For most patients, serial measurements of serum total alkaline phosphatase activity are sufficient to determine the efficacy of the treatment. These could be obtained every 1 to 2 months until a plateau of response (preferably in the normal range) is noted. Then measurements could be obtained every 3 to 4 months to assess a recrudescence of disease activity. Measurement of bonespecific alkaline phosphatase may provide a slightly more sensitive marker of disease activity, 182 but in the absence of liver disease, there is no great advantage to its use. There also appears to be no need for routine measurement of urinary collagen crosslink determinations in most patients. The N-telopeptide assay does appear to give the greatest percentage decrease of the bone resorption markers during a treatment course. 183 It also has been used to help predict the adequacy of treatment with a 10-day course of potent bisphosphonates. 361 In patients whose urinary N-telopeptide excretion was suppressed by greater than 75%, serum alkaline phosphatase activity was found to be within the normal range 1 year after treatment. Therefore, an early assessment of N-telopep-
592
FREDERICK R. SINGER AND STEPHEN M. KRANE
tide excretion may aid in determining the adequate dose of a potent bisphosphonate for patients with severe Paget's disease.
IX. S U R G E R Y Surgery in patients with Paget's disease is often deferred or withheld for a variety of reasons. Some feel that the advanced age of many patients is a relative contraindication. The technical problems associated with operations on sclerotic bone, the fear of excessive hemorrhage from bone or adjacent soft tissue, the possibility of delayed union or nonunion, and the unproven speculation that surgery may predispose to induction of osteosarcomas have all contributed to a reluctance to operate on patients in whom these are otherwise appropriate indications for surgery. The problem of excessive hemorrhage has been considerably reduced by treatment with one of the available agents prior to surgery. In our experience, the benefits of appropriate surgery considerably outweigh any of the other potential problems. In patients with basilar impression, suboccipital craniectomy and laminectomy of upper cervical vertebrae may be necessary to decompress the posterior fossa and thereby relieve neurological impairment. 74 Ventricular shunts have been proposed as a means of preventing progressive dementia arising from progressive communicating hydrocephalus. 76 The more common problem of
hearing loss in patients with skull involvement has been treated with equivocal results utilizing stapes mobilization or stapedectomy. Spinal stenosis or nerve root compression as a consequence of intervertebral foraminal encroachment may require laminectomy or foraminotomy. Results of surgery on the spine are generally quite good. 86-88 The complication of arthropathy involving joints adjacent to pagetic bone disease provides the most frequent indication for surgery. Total hip replacement is highly effective in patients with intractable hip pain and impaired mobility ~~ (Fig. 19-50). Heterotopic ossification may develop postoperatively (Fig. 19-51) but fortunately does not often produce symptoms or disability. It is our impression that this complication may be more common in Paget's disease than in the general population of patients undergoing hip replacement. Involvement of the knee joint is a common problem in patients with distal femoral disease or severe bowing of the tibia. Correction of the varus deformity by tibial and fibular osteotomy may relieve knee pain and improve mobility ~~ (Fig. 19-52). It is occasionally necessary to carry out total knee replacement in these patients. 1~ Open reduction and fixation of fractures in Paget's disease is usually reserved for fractures of the f e m u r . 1~ Because of the high incidence of nonunion of femoral fractures, various forms of fixation are required to produce optimum healing. Fractures of the tibia and other long bones generally heal with immobilization by casting alone.
FIGURE 19--50 Totalhip joint replacement in a 68-year-old patient with severe pagetic coxopathy. Note the joint space narrowing of the left hip which may be an indication for a future second hip joint replacement.
CHAPTER 19 Paget's Disease of Bone
FIGURE 1 9--5 1 Heterotopic ossification surrounding a hip joint replacement in a 72-year-old patient 3 months postoperatively.
In patients in whom elective surgery is scheduled, suppression of disease activity should be accomplished over a 2- to 3-month period utilizing one of the available drugs. If serum alkaline phosphatase activity is reduced by approximately 50% prior to surgery, hemorrhage should be minimized. The optimum length of postoperative treatment has not been established, but it seems reasonable to continue treatment at least until healing is considered complete.
X. E T I O L O G Y Speculation as to the cause of Paget's disease began with the initial paper of Paget. 1 His concept of the disease as a chronic inflammatory disorder did not gain widespread acceptance, in large part because there are few inflammatory cells in pagetic lesions. Since Paget proposed an inflammatory etiology of Paget's disease, a variety of other theories of etiology and pathogenesis have been proposed. 362 These include considerations of Paget's disease as a disorder of secretion of growth hormone, parathyroid hormone of calcitonin, a benign neoplastic process, a primary vascular disorder, and an inborn error of connective tissue. No significant evidence
599 to support any of these hypotheses has been reported. The role of calcitonin perhaps deserves closest scrutiny, since this hormone has such a potent effect upon osteoclasts and does improve many manifestations of the disease. However, athyroidal subjects with low levels of circulating calcitonin do not develop pagetic lesions with increased frequency, and calcitonin levels in patients with Paget's disease are within the normal range. In 1974, the first study of the ultrastructure of bone cells in Paget's disease appeared. 364 Rebel and colleagues observed intranuclear inclusions that resembled nucleocapsids of the Paramyxoviridae virus family in the osteoclasts of four French patients (Fig. 19-53). These results led to the concept that Paget's disease might be considered a slow virus infection of bone, analogous to the post-measles virus infection of the brain, subacute sclerosing panencephalitis. 365 Subsequently, investigators in the United States, 366 Germany, 367 Italy, 368 Canada, 369 England, 37~ and J a p a n 371'372 f o u n d identical inclusions in the nuclei and, at times, the cytoplasm of pagetic osteoclasts in each patient from whom adequate bone specimens were obtained. These nucleocapsid-like structures have not been observed in any other bone or marrow cells and have not been found in osteoclasts from normal subjects or from patients with a variety of metabolic disorders such as primary and secondary hyperparathyroidism. Similar structures have, however, been described in a small percentage of giant cells in some patients with giant cell tumors of b o n e 373 and in the osteoclasts of several patients with pyknodysostosis, 374 osteopetrosis, 375 and primary o x a l o s i s . 376 In addition to the morphological evidence of virusrelated structures in pagetic osteoclasts, immunohistological studies have shown the presence of viral antigens. Initially, Rebel and colleagues reported the presence of measles virus antigens in pagetic osteoclasts, 377 whereas Mills and colleagues found respiratory syncytial virus antigens in osteoclasts (Fig. 19-54) and cultured mononuclear cells from pagetic bone specimens. 378 Subsequently, in a later study, measles virus and respiratory syncytial virus antigens were both identified in serial sections of the same osteoclasts. 379 Basle and colleagues have also described the presence of SV5 and parainfluenza 3 viral antigens in some pagetic specimens but initially could not detect respiratory syncytial virus antigens. 38~ Subsequently, they did confirm the presence of measles virus and respiratory syncytial virus antigens in the same osteoclasts (personal communication). Sensitive molecular techniques in virology were applied to specimens of Paget's disease beginning in 1986. Basle and colleagues in France utilized in situ hybridization to detect measles virus nucleocapsid mRNA not only in osteoclasts but also in osteoblasts, osteocytes, fibroblasts, and other marrow elements. TM Subsequently,
594
FREDERICK R. SINGER AND STEPHEN M. KRANE
The results of a right tibial osteotomy to correct bowing and recurrent knee effusions in a patient FIGURE 1 9 - 5 2 with Paget's disease. On the left is the tibia prior to surgery. (From Singer FR: Paget's Disease of Bone. New York, Plenum, 1977.)
Gordon and colleagues in England demonstrated that the nucleocapsid and fusion genes of canine distemper virus were present in 40% of bone samples from pagetic patients using antisense RNA probes for hybridization. 382 These investigators also found that hybridization was present not only in osteoclasts but also in osteoblasts, osteocytes, lymphocytes, and monocytes. Unlike the study in France, a measles virus mRNA probe failed to detect mRNA sequences in English patients. In the United States, a study utilizing measles-specific polymerase chain reaction (PCR) -amplified cDNA also failed to detect measles virus RNA by in situ hybridi-
z a t i o n . 383 Results of RNA extraction from pagetic bone have also been inconsistent. Gordon and colleagues, utilizing PCR to amplify for specific paramyxovirus sequences, found that 8 of 13 pagetic bone specimens had canine distemper virus nucleotide sequences whereas 1 of 10 had measles virus sequences. 384 Sequencing of the PCR products revealed 2% base pair (bp) changes in a 187-bp fragment from within position 1231 - 1464 of the canine distemper virus nucleocapsid gene, as compared to a strain of the wild type. Other studies in the United Kingdom, however, failed to find any evidence for either of these viruses, respiratory syncytial virus or parain-
595
CHArtER 19 Paget's Disease of Bone
FIGURE 1 9 - 5 3 Nucleus of an osteoclast from a patient with Paget's disease. The arrow indicates the characteristic nuclear inclusion in longitudinal section. Each microfilament measures approximately 1 nm in diameter (decalcified; • 250). [From Singer FR, et al: Acute effects of calcitonin on osteoclasts in man. Clin Endocrinol 5(Suppl): 333S-340S, 1976. Permission from Blackwell Scientific Publications, Ltd.)
fluenza 3 virus in pagetic bone 385 or cells obtained from cultured explants. 386 Another approach to pursuing the identity of the putative viral nucleocapsids in pagetic osteoclasts has been to culture marrow cells obtained from aspiration of iliac crests affected by Paget's disease. The osteoclast-like cells generated in these cultures by addition of calcitriol
FIGURE 19--54
as well as mononuclear cells in the cultures have been shown to harbor measles and respiratory syncytial virus antigens by immunohistological staining. 387 Using reverse transcriptase and PCR methodology, aspirated marrow mononuclear cells from these patients exhibited measles virus nucleocapsid transcripts. 388 Sequencing of the PCR fragments revealed several mutations within the position 1360-1371 base pairs of the nucleocapsid gene as compared to the wild-type virus. The mutations were analogous to those previously reported in the canine distemper virus nucleocapsid gene; these viruses have about 50% homology in nucleocapsid structure. Measles virus nucleocapsid transcripts were also found in peripheral blood mononuclear cells in patients with Paget's disease. 389 Since mononuclear osteoclast precursors are known to circulate in peripheral blood, it is possible that the transcripts are in such cells. In preliminary studies, measles virus nucleocapsid transcripts have been identified in early erythroid and more primitive multipotent myeloid progenitors as w e l l . 39~ Since no cells other than osteoclasts have been demonstrated to contain the nucleocapsid-like structure, it is possible that a primitive hematopoietic stem cell may act as a reservoir for one or more paramyxoviruses but only the mature osteoclast is capable of full expression of the viral nucleocapsid. Most of the preceding data point to a viral presence in pagetic bone, although it is premature to conclude that a slow virus infection is the cause of Paget's disease. Many questions remain concerning the definitive structure of the osteoclast inclusions and whether a variety of paramyxoviruses can initiate the pathology. What is the meaning of the observation of both measles and respiratory syncytial virus antigens in osteoclasts? Is there
A, A group of osteoclasts in a pagetic lesion. The multinucleated cells exhibit strong immunofluorescence when treated by an indirect immunofluorescent technique utilizing an antibody to respiratory syncytial virus. The cytoplasm but not the nuclei is stained. B, Serial section showing the same group of osteoclasts as in A. The cells exhibit a similar degree and localization of fluorescence when treated with an antibody to measles virus (original magnification • (From Mills BG, Singer FR, Weiner LP, Hoist PA: Immunohistological demonstration of respiratory syncytial virus antigens in Paget's disease of bone. Proc Natl Acad Sci USA 78:1209, 1981.)
596
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a dual infection, recombinant virus, or numerous mutations that occurred over a period of decades prior to diagnosis? In classical measles or respiratory syncytial virus infections, mature virions can be seen budding off from the plasma membrane of infected cells. This had not been seen in earlier studies in Paget's disease, but recently, convincing electron microscopic evidence of budding was found in two Japanese patients. 372 The rarity of this finding may explain why it has not been possible to detect an infectious virus by classical viral rescue techniques.391,392 If infection with a paramyxovirus is the initiating event of Paget's disease, what might be the molecular and cellular events responsible for the evolution of the disease? It has been shown that CFU-GM obtained from marrow aspirations of pagetic bone form osteoclast-like cells in culture with lower concentrations of calcitriol than do normal C F U - G M . 393 This may be a consequence of the increased concentration of interleukin-6 found in marrow plasma aspirated from pagetic lesions. 7~ Interleukin-6 may be generated in pagetic osteoclasts as well as in osteoblasts. 394 This may account for the observation that the c-fos protooncogene is up-regulated in pagetic osteoclasts. 395 The seminal role of measles virus or a related virus is suggested by the observation that interleukin-6 is induced by measles virus in glioma cell l i n e s . 396
The genetics of Paget's disease is now an emerging area of research. The gene for familial expansile osteolysis has been linked to chromosome 18q. 397 T h e same linkage has been reported in preliminary studies of families with classic Paget's d i s e a s e . 398'399 Identification of the gene and its function may aid in determining the relevance of the paramyxoviruses and/or the immune system in the pathogenesis of Paget's disease. Peyronie's disease (inflammatory fibrosis of the tunica albuginea corpora cavernosa of the penis) has been found in 31.4% of 61 men with Paget's disease and was reported to be present in 14.5% of 597 male patients who answered a questionnaire by mail. 4~176 In the general male population over 50 years, 1% of men have Peyronie's disease. The explanation for this association is not known.
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and of combined therapy (EHDP and calcitonin) in Paget' s disease of bone. In Maclntyre I, Szelke M (eds): Molecular Endocrinology. Amsterdam, Elsevier/North Holland, 1979, pp 405-433. Murphy WA, Whyte MP, Haddad JG Jr: Healing of lytic Paget bone disease with diphosphonate therapy. Radiology 134:635637, 1980. Krane SM: Etidronate disodium in the treatment of Paget's disease of bone. Ann Intern Med 96:619-625, 1982. Russell RGG, Smith R, Preston C, et al: Diphosphonates in Paget's disease. Lancet 1:894-898, 1974. Boyce BF, Smith L, Fogelman I, et al: Focal osteomalacia due to low-dose disphosphonate therapy in Paget's disease. Lancet 1:821-824, 1984. Preston CJ, Yates AJP, Beneton MNC, et al: Effective short term treatment of Paget's disease with oral etidronate. Br Med J 292:79-80, 1986. Johnston CC Jr, Altman RD, Canfield RE, et al: Review of fracture experience during Paget's disease of bone with etidronate disodium. Clin Orthop Rel Res 172:186-194, 1983. Gibbs CJ, Aaron JE, Peacock M: Osteomalacia in Paget's disease treated with short term, high dose sodium etidronate. Br Med J 292:1227-1229, 1986. Norman DA, Zerwekh JE, Pak CYC: An apparent 1,25dihydroxyvitamin D-independent stimulation of intestinal calcium absorption in patients with Paget's disease of bone during a short-term diphosphonate therapy. Metabolism 30:290-292, 1981. Basle MF, Rebel A, Renier JC, et al: Bone tissue in Paget's disease treated by ethane- 1,hydroxy- 1,1-diphosphonate (EHDP). Structure, ultrastructure, and immunocytology. Clin Orthop Rel Res 184:281-288, 1984. Evans RA, Hills E, Dunstan CR, Wong SYP: Pathologic fracture due to severe osteomalacia following low-dose diphosphonate treatment of Paget's disease of bone. Aust NZ J Med 13:277- 279, 1983. Meunier PJ, Chapuy MC, Alexandre C, et al: Effects of disodium dichloromethylene diphosphonate on Paget's disease of bone. Lancet 2:489-492, 1979. Yates AJP, Percival RC, Gray RES, et al: Intravenous clodronate in the treatment and retreatment of Paget's disease of bone. Lancet 1"1474-1477, 1985. Harris ST, Neer RM, Segre GV, et al: Secondary hyperparathyroidism associated with dichloromethylene diphosphonate treatment of Paget's disease. J Clin Endocrinol Metab 55" 11001107, 1982. Delmas PD, Chapuy MC, Vignon E, et al: Long-term effects of dichloromethylene diphosphonate in Paget's disease of bone. J Clin Endocrinol Metab 54:837-844, 1982. Frijlink WB, Bijvoet OLM, te Velde J, Heynen G: Treatment of Paget's disease with (3-amino-l-hydroxypropylidene)-l,1bisphosphonate (A.P.D.). Lancet 1:799-803, 1979. Thiebaud D, Jaeger P, Gobelet C et al: A single infusion of the bisphosphonate AHPrBP (APD) as treatment of Paget's disease of bone. Am J Med 85:207-212, 1988. Thiebaud D, Portmann L, Burckhardt P: Maladie de Paget mod6r6e, traitee par le pamidronate (APD)" Exp6rience chez 43 patients avec une perfusion unique de 60 mg d'une dure6 variable de 1 ~ 24 heures. Schweiz Med Wothenschr 122:18891894, 1992. Cantrill JA, Buckler HM, Anderson DC: Low dose intravenous 3-amino-l-hydroxypropylidene-l,l-bisphosphonate (APD) for the treatment of Paget's disease of bone. Ann Rheum Dis 45" 1012-1018, 1986.
604 332. Stone MD, Hawthorne AB, Kerr D, et al: Treatment of Paget's disease with intermittent low-dose infusions of disodium pamidronate (APD). J Bone Miner Res 5:1231-1235, 1990. 333. Harinck HIJ, Bijvoet OLM, Blanksma HJ, DahlinghausNienhuys PJ: Efficacious management with APD in Paget's disease of bone. Clin Orthop Rel Res 217:79-98, 1987. 334. Fenton AJ, Gutteftdge DH, Kent GN, et al: Intravenous aminobisphosphonate in Paget's disease: Clinical, biochemical, histomorphometric and radiological responses. Clin Endocrinol 34:197-204, 1991. 335. Cundy T, Wattie D, King AR: High-dose pamidronate in the management of resistant Paget's disease. Calcif Tissue Int 58: 6 - 8 , 1996. 336. Adamson BB, Gallacher SJ, Byars J, et al: Mineralisation defects with pamidronate therapy for Paget's disease. Lancet 342: 1459-1460, 1993. 337. Dodd GW, Ibbertson HK, Fraser TRC, et al: Radiological assessment of Paget's disease of bone after treatment with the bisphosphonates EHDP and APD. Br J Radiol 60:846-860, 1987. 338. Vellenga CJLR, Pauwels EKJ, Bijvoet OLM, et al: Quantitative bone scintigraphy in Paget's disease treated with APD. Br J Radiol 58:1165-1172, 1985. 339. Nagant de Deuxchaisnes C, Rombouts-Lindemans C, Huaux JP, et al: Treatment of Paget's disease with the diphosphonate APD. A biological and radiological study. In Donath A, Courvoisier B (eds): Symposium CEMO IV Diphosphonates and Bone. Brussels, Editions Medicine et Hygiene, 1982, pp 3 0 3 327. 340. Macorol V, Fraunfelder FT: Pamidronate disodium and possible ocular adverse drug reactions. Am J Ophthalmol 118:220-224, 1994. 341. Reid IR, Mills DAJ, Wattie DJ: Ototoxicity associated with intravenous bisphosphonate administration. Calcif Tissue Int 56:584-585, 1995. 342. Adami S, Salvagno G, Guarrera G, et al: Treatment of Paget's disease of bone with intravenous 4-amino-l-hydroxybutlidene1,1-bisphosphonate. Calcif Tissue Int 39:226-229, 1986. 343. Sifts E, Weinstein RS, Altman R, et al: Comparative study of alendronate versus etidronate for the treatment of Paget's disease of bone. J Clin Endocrinol Metab 81:961-967, 1996. 344. Reginster JY, Calson F, Morlock G, et al: Evaluation of the efficacy and safety of oral tiludronate in Paget's disease of bone. A double-blind, multiple-dosage, placebo-controlled study. Arthritis Rheum 35:967-974, 1992. 345. Papapoulos SE, Hoekman K, L6wik CWGM, et al: Application of an in vitro model and a clinical protocol in the assessment of the potency of a new bisphosphonate. J Bone Miner Res 4: 775- 781, 1989. 346. Schweitzer DH, Zwinderman AH, Vermij P, et al: Improved treatment of Paget's disease with dimethylaminohydroxypropylidene bisphosphonate. J Bone Miner Res 8:175-182, 1993. 347. Schweitzer DH, Oostendorp-Van de Ruit M, Van dr Pluijm G, et al: Interleukin-6 and the acute phase response during treatment of patients with Paget's disease with the nitrogencontaining bisphosphonate dimethylaminohydroxypropylidene b bisphosphonate. J Bone Miner Res 10:956-962, 1995. 348. Singer FR, Clemens TL: Risedronate and resistant Paget's disease of Bone. J Bone Miner Res (Supp) 7:$291, 1992. 349. Arden-Cordone M, Sifts ES, Lyles KW, et al: Antiresorptive effect of a single infusion of microgram quantities of zoledronate in Paget's disease of bone. Calcif Tissue Int 60:415-418, 1997. 350. Ryan WG, Schwartz TB, Perlia CP: Effects of mithramycin on Paget's disease of bone. Ann Intern Med 70:549-557, 1969.
FREDERICK R. SINGER AND STEPHEN M. KRAr~
351. Ryan WG, Schwartz TB, Fordham EW: Mithramycin and long remission of Paget's disease of bone. Ann Intern Med 92: 129-130, 1980. 352. Ryan WG, Schwartz TB: Mithramycin treatment of Paget's disease of bone. Exploration of combined mithramycin-EHDP therapy. Arthritis Rheum 23:1155-1161, 1980. 353. Bilezikian JP, Canfield RE, Jacobs TP, et al: Response of 1,25dihydroxyvitamin D3 to hypocalcemia in human subjects. N Engl J Med 299:437-441, 1978. 354. Pembrook RC, Chung CH, Carvallo AP: Effects of mithramycin and calcitonin in cardiovascular complications of Paget' s disease of bone. Conn Med 39:209-214, 1975. 355. Somerville PJ, Evans RA: Actinomycin D in the treatment of Paget's disease of bone. Med J Aust 2:13-16, 1975. 356. Singer FR, Schiller AI, Pyle EB, Krane SM: Paget's disease of bone. In Avioli LV, Krane SM (eds): Metabolic Bone Disease. New York, Academic Press, 1978, pp 558-559. 357. Hall RJ, Chambers TJ: Gallium inhibits bone resorption by a direct effect on osteoclasts. Bone Miner 8:211-216, 1990. 358. Bockman RS, Wilhelm F, Sifts E, et al: A multicenter trial of low dose gallium nitrate in patients with advanced Paget's disease of bone. J Clin Endocrinol Metab 80:595-602, 1995. 359. O'Donoghue DS, Hosking DS: Biochemical response to combination of disodium etidronate with calcitonin in Paget's disease. Bone 8:219-225, 1987. 360. Hadjipavlou AG, Tsoukas GM, Siller TN, et al: Combination drug therapy in treatment of Paget's disease of bone. J Bone Joint Surg 59A:1045-1051, 1977. 361. Papapoulos SE, Frohlich M: Prediction of the outcome for treatment of Paget's disease with bisphosphonates from short term changes in the rate of bone resorption. J Clin Endocrinol Metab 81:3993- 3997, 1996. 362. Singer FR, Mills BG: The etiology of Paget's disease of bone. Clin Orthop Rel Res 127:37-42, 1977. 363. Kanis JA, Heynen G, Walton RJ: Plasma calcitonin in Paget's disease of bone. Clin Sci Mol Med 52:329-332, 1977. 364. Rebel A, Malkani K, Basle M: Anomalies nucleaires de la maladie osseuse de Paget. Nouv Presse Med 3:1299-1301, 1974. 365. Dubois DM, Coblentz JM, Pleet AB: Subacute sclerosing panencephalitis. Unusual nuclear inclusions and lengthy clinical course. Arch Neurol 31:355-363, 1974. 366. Mills BG, Singer FR: Nuclear inclusions in Paget's disease of bone. Science 194:201-202, 1976. 367. Schulz A, Delling G, Ringe JD, Ziegler R: Morbus Paget des knochen Untersuchugen zur Ultrastruktur des Osteoclasten und ehrer Cytopathogenese. Virchows Arch A [Pathol Anat] 376: 309-328, 1977. 368. Gherardi G, LoCascio V, Bonucci E: Fine structure of nuclei and cytoplasm of osteoclasts in Paget's disease of bone. Histopathology 4:63-74, 1980. 369. Howatson AF, Fornasier VL: Microfilaments associated with Paget's disease of bone: Comparison with nucleocapsids of measles virus and respiratory syncytial virus. Intervirology 18: 150-159, 1982. 370. Harvey L, Gray T, Beneton MNC, et al: Ultrastructural features of the osteoclasts from Paget's disease of bone in relation to a viral etiology. J Clin Pathol 35:771-779, 1982. 371. Higo Y, Ohno T: An ultrastructural study of osteoclasts in Paget's disease of bone. J Clin Electron Microscopy 16:879-880, 1983. 372. Abe S, Ohno T, Park R et al: Viral behavior of paracrystalline inclusions in osteoclasts of Paget's disease of bone. Ultrastruct Pathol 19:455-461, 1995.
CHAPTER 19
Paget's Disease of Bone
373. Vacher-Lavenu M-C, Louvel A, Daudet-Monsac M, et al: Inclusions tubulofilamenteuses intranucleaires dans les cellules multinuclees des tumeurs a cellules geantes des os. Etude ultrastructurale d'une serie de 31 tumeurs. C R Acad Sci Paris 293: 639-644, 1981. 374. Beneton MNC, Harris SC, Kanis JA: Paramyxovirus-like inclusions in two cases of pycnodysostosis. Bone 8:211-217, 1987. 375. Mills BG, Yabe H, Singer FR: Osteoclasts in human osteopetrosis contain viral nucleocapsid-like nuclear inclusions. J Bone Miner Res 3:101-106, 1988. 376. Bianco P, Silvestrini G, Ballanti P, Bonucci E: Paramyxoviruslike nuclear inclusions identical to those of Paget's disease detected in giant cells of primary oxalosis. Virchows Arch A Pathol Anat Histopathol 421:427-433, 1992. 377. Rebel A, Basle M, Pouplard A, et al: Viral antigens in osteoclasts from Paget's disease of bone. Lancet 1:344-346, 1980. 378. Mills BG, Singer FR, Weiner LP, Hoist PA: Immunohistological demonstration of respiratory syncytial virus antigens in Paget's disease of bone. Proc Natl Acad Sci USA 78:1209-1213, 1981. 379. Mills BG, Singer FR, Weiner LP, et al: Evidence for both respiratory syncytial virus and measles virus antigens in the osteoclasts of patients with Paget's disease of bone. Clin Orthop Rel Res 183:303-311, 1984. 380. Basle MF, Russell WC, Goswami KKA, et al: Paramyxovirus antigens in osteoclasts from Paget's bone tissue detected by monoclonal antibodies. J Gen Virol 66:2103-2110, 1985. 381. Basle MF, Fournier JG, Rosenblatt S, et al: Measles virus RNA detected in Paget's disease bone tissue by in situ hybridization. J Gen Virol 67:907-913, 1986. 382. Gordon MT, Anderson DC, Sharpe PT: Canine distemper virus localized in bone cells of patients with Paget's disease. Bone 12:195-201, 1991. 383. Nuovo MA, Nuovo GJ, MacConnell P, et al: In situ analysis of Paget's disease of bone for measles-specific PCR-amplified cDNA. Diagn Mol Pathol 1:256- 264, 1992. 384. Gordon MT, Mee AP, Anderson DC, Sharpe PT: Canine distemper virus transcripts sequenced from pagetic bone. Bone Miner 19:159-174, 1992. 385. Ralston SH, Digiovine FS, Gallacher SJ, et al: Failure to detect paramyxovirus sequences in Paget's disease of bone using the polymerase chain reaction. J Bone Miner Res 6:1243-1248, 1991. 386. Birch MA, Taylor W, Fraser WD, et al: Absence of paramyxovirus RNA in cultures of pagetic bone cells and in pagetic bone. J Bone Miner Res 9:11-16, 1994.
605 387. Mills BG, Frausto A, Singer FR, et al: Multinucleated cells formed in vitro from Paget's bone marrow express viral antigens. Bone 15:443-448, 1994. 388. Reddy SV, Singer FR, Roodman GD: Bone marrow mononuclear cells from patients with Paget's disease contain measles virus nucleocapsid messenger ribonucleic acid that has a mutation in a specific region of the sequence. J Clin Endocrinol Metab 80:2108- 2111, 1995. 389. Reddy SV, Singer FR, Mallette L, Roodman GD: Detection of measles virus nucleocapsid transcripts in circulating blood cells from patients with Paget's disease. J Bone Miner Res 11: 1602-1607, 1996. 390. Reddy RV, Singer FR, Anderson JL, Roodman GD: Measles virus nucleocapsid transcript expression is not restricted to osteoclast lineage in patients with Paget's disease. J Bone Miner Res 1 l(Suppl 1):$370, 1996. 391. Mills BG, Singer FR, Weiner LP, Hoist PA: Long-term culture of cells from bone affected by Paget's disease. Calcif Tissue Int 29:79-87, 1979. 392. Mills BG, Hoist PA, Stabile EK, et al: A viral antigen-bearing cell line derived from culture of Paget's bone cells. Bone 6: 193-200, 1985. 393. Demulder A, Takahashi S, Singer FR, et al: Abnormalities in osteoclast precursors and marrow accessory cells in Paget's disease. Endocrinology 133:1978-1982, 1993. 394. Hoyland J, Freemont AJ, Sharpe PT: Interleukin-6, IL-6 receptor and IL-6 nuclear factor gene expression in Paget's disease. J Bone Miner Res 9:75-80, 1994. 395. Hoyland J, Sharpe PT: Upregulation of c-fos protooncogene expression in pagetic osteoclasts. J Bone Miner Res 9:11911194, 1994. 396. Schneider-Schaulies J, Schneider-Schaulies S, ter Meulen V: Differential induction of cytokines by primary and persistent measles virus infections in human glial cells. Virology 195: 218-228, 1993. 397. Hughes AE, Shearman AM, Weber JL, et al: Genetic linkage of familial expansile osteolysis to chromosome 18q. Hum Mol Genet 3:359-361, 1994. 398. Leach RJ, Singer FR, Lewis TB, et al: Evidence for a locus for Paget' s disease on human chromosome 18Q. J Bone Miner Res ll(Suppl 1):$99, 1996. 399. Haslam SI, Thompson JMG, Haites NE, Ralston SH: Genetic mapping in Paget's disease of bone: Evidence for a susceptibility locus on chromosome 18q. J Bone Miner Res 1 l(Suppl 1):$369, 1996. 400. Lyles KW, Gold DT, Newton RA, et al: Peyronie's disease is associated with Paget's disease of bone. J Bone Miner Res 12: 9 2 9 - 9 3 4 , 1997.
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7HAPTER 2(
S arcoldosls and Related Disorders NORMAN H.
BELL
Departments of Medicine and Pharmacology, Medical University of South Carolina and Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, South Carolina 29401
I. Granulomatous and Infectious Diseases A. Sarcoidosis B. Tuberculosis C. Other Granulomatous Diseases D. Rheumatoid Arthritis II. Lymphoma and Solid Tumors A. Lymphoma, Leukemia, and Hodgkin's Disease B. Metastatic Leioblastoma C. Seminoma D. Small Cell Carcinoma of the Lung
III. Miscellaneous Diseases A. Increased Serum 1,25(OH)2D of Unknown Cause B. Williams Syndrome C. Infants with Subcutaneous Fat Necrosis D. Vitamin D Intoxication E. 1,25(OH)2D3 Intoxication E Calcipotriol Intoxication References
Hypercalcemia and abnormal calcium metabolism can result from increased serum 1,25-dihydroxyvitamin D [ 1,25(OH)zD] in association with a number of diseases. Hypercalcemia and abnormal calcium metabolism also may be produced in response to treatment with vitamin D and its analogs. The diseases in which hypercalcemia caused by increases in serum 1,25(OH)zD and other metabolites or analogs of vitamin D are listed in Table 2 0 - 1 .
D metabolism has been obtained. Therefore, much of this chapter will be devoted to this subject.
A. Sarcoidosis Sarcoidosis is a systemic disease of unknown cause in which noncaseous granulomas are found in affected organs. Lymph nodes, lungs, eyes, and skin are the most frequent sites of involvement. 1'2 1. I M M U N O L O G Y AND P A T H O L O G Y
I. G R A N U L o M A T O U S AND INFECTIOUS DISEASES
Sarcoidosis can involve any tissue or organ. However, the finding that 90% of patients have lesions in the lungs, thoracic lymph nodes, or both provides evidence that the disease probably begins in the respiratory tract and associated lymphatic system. 2 Pulmonary sarcoidosis be-
Sarcoidosis is the disease in which most information about pathogenesis and treatment of abnormal vitamin METABOLIC BONE DISEASE
607
Copyright 9 1998 by Academic Press. All rights of reproduction in any form reserved.
NORMAN
608 TABLE 20" 1 Hypercalcemia Caused by Increased Circulating 1,25(OH)2D Granulomatous or infectious diseases Sarcoidosis Tuberculosis AIDS with Pneumocystis carinii pneumonia AIDS with cytomegalovirus infection AIDS with Cryptococcus neoformans infection Disseminated candidiasis Leprosy Rheumatoid arthritis Silicone-induced granulomas Wegener's granulomatosis Lymphoma and solid tumors Hodgkin's disease Lymphoma Histiocytic lymphoma T-cell leukemia Plasma cell granuloma Leiomyblastoma Seminoma Miscellaneous Calcitriol intoxication Calcipotriol intoxication Vitamin D intoxication Hypercalcemia of infancy Idiopathic Subcutaneous fat necrosis
gins as an alveolitis or an accumulation of inflammatory cells within the alveolus. 3'4 Large numbers of lymphocytes are present within the alveolar interstitium and on alveolar surfaces. 5'6 Most of the cells are T lymphocytes, many of which are activated. 5'7 The majority are T-helper cells] The increased number of T lymphocytes occurs because of increased replication. 8-11 Enhanced proliferation occurs in part because of synthesis and release by the activated T lymphocytes of interleukin-2, a Tlymphocyte growth factor. 9'~~In addition, antigen-driven, alveolar macrophage-modulated T-cell proliferation may also be a f a c t o r . 11 Thus, antigen-pulsed alveolar macrophages from sarcoid patients induce a greater increment in proliferation of T lymphocytes than do cells from normal subjects. On the other hand, patients with sarcoidosis have a deficiency of T lymphocytes in the peripheral circulation. 5'8 In active pulmonary sarcoidosis, activated T lymphocytes spontaneously produce a number of factors in addition to interleukin-2 that are important in the development and maintenance of the inflammatory process: macrophage chemotactic factor, ~2 leukocyte inhibitory factor, 5 ~/-interferon, 13 but not interleukin-1.14 The macrophages in granulomas are derived from monocytes that originate in bone marrow and reach the site of inflammation by way of the peripheral circulation. 12 By release
H. BELL
of monocyte chemotactic factor, the activated alveolar T lymphocytes recruit peripheral monocytes, thereby providing maintenance of granuloma formation. Neutrophils are seldom found, and their absence may be accounted for in part by leukocyte inhibitory factor. 5 Also, serum lysozyme, an enzyme that inhibits the migration of neutrophils, 15 is increased in sarcoidosis. 16 Enhanced antibody production by pulmonary B-cells in sarcoid lesions apparently accounts for hypergammaglobulinemia that commonly occurs in the disease. ~7 In this regard, it was proposed that the activated T lymphocytes stimulated B-cells in the lungs to differentiate into immunoglobulin-producing cells. In patients with active sarcoidosis, the number of pulmonary B lymphocytes that secrete immunoglobulins is markedly increased and there is a significant positive correlation between the percentage of T-cells and percentage of B-cells secreting IgG. ~8 These results account for the paradox that circulating lymphocytes of patients with sarcoidosis do not release increased amounts of immunoglobulins despite hypergammaglobulinemia in the peripheral circulation. 16 Alveolar macrophages from bronchoalveolar lavage of patients with sarcoidosis and hypercalcemia caused by increased circulating 1,25(OH)2D converted [3H]25(OH)D3 to [3H]-l,25(OH)zD3 in vitro. 19-22 Production of 1,25(OH)2D in this system is enhanced in a dosedependent fashion by ~/-interferon. 21'22 ~/-Interferon is produced spontaneously by activated T-cells and alveolar macrophages in sarcoidosis, 13 and the lymphokine evidently plays a key role in the pathogenesis of synthesis of 1,25(OH)2D in granulomatous disease. First, the concentration of ~/-interferon is increased in fluid from pleural effusions of patients with tuberculous pleuritis. Second, the capacity of the fluid to stimulate synthesis of 1,25(OH)zD by cultured pulmonary alveolar macrophages from patients with sarcoidosis correlates with the concentration of ~/-interferon in the fluid. Third, production of 1,25(OH)2D in response to pleural fluid is prevented by a monoclonal antibody to ~/-interferon. 23 Receptors for 1,25(OH)zD3 are present in activated peripheral T lymphocytes from normal human subjects, 24 and 1,25(OH)2D3 inhibits proliferation and suppresses interleukin-2 activity 25 and synthesis of ~/-interferon 26 by phytohemaglutin-stimulated human peripheral lymphocytes. 1,25(OH)zD3 inhibits activated T helper-inducer lymphocyte activity from normal human subjects in vitro. z7 If receptors for 1,25(OH)2D are present in activated pulmonary T lymphocytes and if 1,25(OH)zD3 inhibits proliferation of activated T-cells and their secretion of interleukin-2 and ~/-interferon in sarcoid granulomas, production of 1,25(OH)zD by alveolar macrophages could provide a compensatory mechanism to inhibit the inflammatory process. Regardless of the role of
CHAPTER20 Sarcoidosis and Related Disorders 1,25(OH)2D in modification of inflammation, it is evident that nonrenal production of 1,25(OH)2D is responsible for the abnormal vitamin D and mineral metabolism that occurs in sarcoidosis. 28 In sarcoidosis, granulomas consist of collections of inflammatory and immune effector cells. In developing granulomas, loosely arranged epithelioid cells, derived from macrophages, are surrounded by a few lymphocytes and initially the number of macrophages, monocytes, and lymphocytes is greater than the number of epithelioid cells. Later the number of epithelioid cells increases as the number of macrophages, monocytes, and lymphocytes declines. 4 When alveolitis predominates, few or no granulomas are present. In contrast, when extensive granulomas occur, alveolitis is minimal or absent. 5'29 Alveolitis, therefore, is thought to precede the development of granulomas in sarcoid. 4-17 After formation, granulomas either undergo resolution with little in the way of residual morphological alterations or progress to fibrosis. 3~ The fibrotic process begins as a deposition of collagen around the periphery of the granuloma. 3'29 It is assumed but not established that collagen is derived from fibroblasts that surround the granuloma. 29 As fibrosis develops, fibroblasts proliferate so that there is eventual destruction of granuloma, fibrosis, and destruction of the normal structures of the lung. 32'33 Thus, pulmonary sarcoidosis either resolves leaving little in the way of impairment of function, as occurs in some 80% of patients, or progresses to fibrosis with the development of pulmonary insufficiency and cor pulmonale, as occurs in the remaining 20% of them. 2. ABNORMAL CALCIUM METABOLISM
In sarcoidosis, the characteristic changes in calcium metabolism are increases in serum and urinary calcium and, as shown by balance studies and investigations with radiolabeled calcium, enhanced intestinal absorption of calcium. 34-37 Increases in turnover of 47Ca were found in patients with sarcoidosis and hypercalcemia that were similar to changes produced by pharmacological doses of vitamin D in normal subjects and patients with hypoparathyroidism. 37 Hypercalcemia and hypercalciuria in sarcoidosis occur spontaneously and are produced by small doses of vitamin D that are without effect in normal subjects 34-36 as well as by brief irradiation with ultraviolet light. 38 The importance of ultraviolet radiation in the pathogenesis of abnormal vitamin D and mineral metabolism in sarcoidosis is underscored by the seasonal incidence of hypercalcemia, which usually occurs during the summer months, 39 and by the demonstration that the abnormal calcium metabolism is reversed by omission of vitamin D from the diet and prevention of exposure to sunlight. 4~
609 Abnormal metabolism of calcium also occurs in patients with sarcoidosis who are norlnocalcemic. 41'42 Enhanced intestinal absorption of calcium, determined in studies with labeled calcium, was found and correlated with increased renal excretion of calcium. 41 Thus, hypercalciuria in sarcoid occurs in the absence of hypercalcemia and results from increased intestinal absorption of calcium. 41'42 Since increases in urinary hydroxyproline, an index of bone resorption, also occur in sarcoido s i s , 43 it is likely that excess urinary calcium also may be derived from the skeleton. In sarcoidosis, it is clear that hypercalciuria is present in greater frequency than hypercalcemia. 41'44The true incidence of hypercalciuria is difficult to establish, since there is known seasonal variation in the serum calcium 39 and considerable variation in urinary calcium in normal subjects. Further, urinary calcium varies with race and body habitus. Thus, compared to nonobese white men and women, normal nonobese black subjects and obese white individuals have secondary hyperparathyroidism with increased circulating 1,25(OH)2D and urinary cyclic adenosine 3',5'-monophosphate and decreased urinary calcium. 45,46 3. PATHOGENESIS
a. Normal Physiology of Vitamin D. Vitamin D3 is synthesized in the skin from 7-dehydrocholesterol, provitamin D3, with previtamin D3 as an intermediate. On exposure to ultraviolet light, 7-dehydrocholesterol absorbs a photon of light energy at the site of conjugated double bonds in the B-ring, As'7-diene, which produces rearrangement of the double bonds and cleavage of the double bond between carbons 9 and 10 to form provitamin D3. Since some 80% of the 7-dehydrocholesterol is in the stratum spinosum and stratum basale, the major site of photochemical conversion of provitamin D to previtamin D3 is at that site. 47 Previtamin D3 is thermally unstable and undergoes isomerization to vitamin D3 (cholecalciferol), a conversion that is temperature-dependent and takes place over a period of days. 47 Vitamin D3, which is thermally stable, is preferentially transported from the dermis bound to vitamin D-binding protein and is removed by the capillary bed that is adjacent to the deeper layer of skin. Vitamin D-binding globulin has a high affinity for vitamin D3 but little, if any, affinity for previtamin D3 so that previtamin D3 remains in the dermis for eventual conversion to vitamin D3. Vitamin D2 (ergocalciferol) and vitamin D3 in the diet are absorbed in the proximal small intestine, a step requiring bile acids, by the lymphatic system. They are transported to the liver in chylomicrons and are released into the systemic circulation bound to vitamin D -
61
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binding protein. Vitamins D2 and D3 are hydroxylated in the liver to their respective metabolites of 25(OH)D by action of the enzyme vitamin D-25-hydroxylase. 48 25(OH)D is transported to the kidney where it is hydroxylated further to 1,25(OH2)D, 49 the most biologically active metabolite of vitamin D that acts to enhance the intestinal absorption of calcium 5~ and the skeletal release of the ion. 51 1,25(OH)2D also acts to reduce the concentration of its precursor 25(OH)D 52 by enhancing its metabolic clearance. 53 Synthesis of 1,25(OH)2D by the kidney normally is tightly regulated by serum calcium 54 and parathyroid hormone. 55'56 Values are low in hypoparathyroidism, are elevated in primary hyperparathyroidism, and are increased by administration of parathyroid extract in normal subjects and in patients with hypoparathyroidism. 55'56 1,25(OH)2D is the major determinant of calcium absorption in humans, and adaptation to changes of calcium intake is mediated by changes in circulating 1,25(OH)2D. 57'58 Thus, serum 1,25(OH)2D increases when dietary calcium is restricted and decreases when dietary calcium is augmented. Recently, a calcium receptor was found in parathyroid tissue that mediates the inhibitory effects of calcium on secretion of the hormone. The gene for the receptor was cloned and sequenced. 59 The receptor is coupled to G proteins, apparently has seven membrane-spanning domains, a large extracellular region with nine glycosylation sites, and a limited homology with G-protein receptors. Mutations of the calcium receptor cause familial hypocalciuric hypercalcemia in heterozygotes and severe neonatal hypercalcemia in homozygotes. 6~ The receptor also is present in renal cortex. It is likely that the receptor mediates renal regulation of production of 1,25(OH)2D by calcium. 59 b. Abnormal Vitamin D Metabolism. Increased intestinal absorption of calcium, hypercalcemia, and hypercalciuria in sarcoidosis are caused by increased circulating 1,25(OH)2D. 28'61-64 Values are either increased or at the upper range of normal in hypercalcemic patients, and serum immunoreactive parathyroid hormone is either suppressed or in the low n o r m a l range. 28'57-61'65 In small doses, 10,000 units a day for 12 days, vitamin D increases serum 1,25(OH)zD and urinary calcium in normocalcemic patients with sarcoid and a history of hypercalcemia, but does not alter serum 1,25(OH)2D or urinary calcium in patients with normal calcium metabolism. 61 In larger doses, 100,000 units/day for 4 days, vitamin D increased serum 1,25(OH)zD and serum calcium in some patients with sarcoid who had normal calcium metabolism, but did not change the serum calcium in n o r m a l subjects. 66 This defect in the regulation of circulating 1,25(OH)zD provides the mechanism for in-
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creased sensitivity to vitamin D that is so characteristic of the disorder. Enhanced dermal production of vitamin D3 by sunlight during summer months produces seasonal variation in serum calcium in patients with sarcoidosis 39 and accounts for the fact that increases in serum 1,25(OH)2D and hypercalcemia usually occur in the summer in the northern hemisphere. 61-64 This occurs because the production of 1,25(OH)2D is not regulated in sarcoidosis so that the concentration of 1,25(OH)2D is more likely to be influenced by the concentration of the substrate 25(OH)D. In normal subjects as a result of differences in the time of exposure to sunlight, there is seasonal variation in serum 25(OH)D and 1,25(OH)2D. 67 Serum 1,25(OH)zD also may be increased in patients who do not have hypercalcemia. 42'58'66 In these individuals, increases in fractional intestinal absorption of calcium occur in association with hypercalciuria. 42 In sarcoidosis, hypercalcemia usually occurs in patients with some degree of renal insufficiency and limitation in the ability of the kidneys to excrete the excess calcium resulting from increased intestinal absorption and skeletal release of the ion. 61 Hypercalcemia is also related to calcium intake and can be corrected or prevented by restriction of dietary calcium. 35'36 Demonstration of increased circulating 1,25(OH)2D, hypercalcemia, and suppression of serum immunoreactive parathyroid hormone in an anephric patient with sarcoidosis was the first indication of extrarenal synthesis of 1,25(OH)2D. 28 As noted already, conversion of [3H]1,25(OH)2D3 by cultured pulmonary alveolar macrophages from patients with sarcoidosis was observed. 19-21 The structure of the putative 1,25(OH)D3 produced by the macrophages was confirmed by mass spectral analysis. 2~ Kinetic analysis of the synthesis of 1,25(OH)2D3 by pulmonary alveolar macrophages from five patients with sarcoidosis revealed a Km that varied from 52 to 210 nM. 21 This range is similar to values obtained with mammalian renal tissues including homogenates of mouse kidney, 68 mouse kidney cells in culture, 69 rat renal cortical cells, 69 and partially purified 25(OH)D-loLhydroxylase from rat kidney. 7~ As noted, increased production of 1,25(OH)2D by alveolar macrophages is attributed to ~/-interferon. 23 On the other hand, the 25(OH)D-let-hydroxylase activity in pulmonary alveolar macrophages from patients with sarcoidosis differs from the renal enzyme in a number of important respects. In contrast to the kidney, 68-7~ stimulation of 25(OH)D-24-hydroxylase activity in the macrophages occurs only at very high concentrations of 1,25(OH)2D3. 22 This means that there is little competition for the substrate 25(OH)D to be converted to 24,25(OH)2D, which has only modest biological activity, and that 1,25(OH)2D will not undergo further hydrol-
CHAPTER20 Sarcoidosis and Related Disorders
611
ysis to 1,24,25(OH)3D which has less biological activity than 1,25(OH)2D. 72'73 Also, unlike the mammalian kidney,69'74 there is only modest inhibition of 25(OH)D-loLhydroxylase activity by 1,25(OH)2D3 in the pulmonary alveolar macrophages. 22 Activity is inhibited by dexamethasone added directly to the culture system, and the inhibition is dose dependent. 21'22 These in vitro findings account in part for the clinical observations that production of 1,25(OH)2D in sarcoidosis is not regulated by the same factors that regulate renal synthesis and is inhibited by g l u c o c o r t i c o i d s . 28"61-64 As shown in Figure 20-1, recent studies showed that increases in daily calcium intake from 400 to 1000 mg lowered serum 1,25(OH)zD in 17 normal subjects but not in 11 patients with sarcoidosis who had never had hypercalcemia or abnormal vitamin D metabolism. 58 Serum angiotensin-converting enzyme was elevated in only three of the patients. In contrast, serum angiotensinconverting enzyme is usually increased in patients with sarcoidosis and hypercalcemia. 75 The results indicate that abnormal vitamin D metabolism occurs in normocalcemic patients with sarcoidosis and modulates calcium metabolism. The dietary calcium suppression test is the most sensitive means yet developed for determination of disease activity in sarcoidosis. It is likely that activated pulmonary alveolar macrophages possess a 25(OH)D-
NORMAL (17)
SARCOID (11)
7060-
9BLACK 0 WH ITE
50SERUM
1'25(OH)2D 40 pg/ml
30 20
10P <0.001
0I
-Ca
FIGURE 20--1
N.S. i
I
+Ca -Ca
i
+Ca
Effects of calcium intake on serum 1,25(OH)2D in normal subjects and normocalcemic patients with sarcoidosis. Daily calcium intake was 400 mg ( - C a ) and 1000 mg (+Ca). Note that serum 1,25(OH)2D was suppressed by increased calcium intake in the normal subjects but not in the patients and was within the normal range in all except three of the patients. (From Basile JN, Liel Y, Shary J, Bell NH: Increased calcium intake does not suppress circulating 1,25-dihydroxyvitamin D in normocalcemic patients with sarcoidosis. J Clin Invest 91:1396-1398, 1993.)
l c~-hydroxylase with an associated cytochrome P-450 system that is similar to the one in the kidney but is regulated by different mechanisms. Because of its low abundance, the enzyme has not been isolated, cloned, or sequenced from any tissue. A 25(OH)D-loL-hydroxylase with an associated cytochrome P-450 system is present in the chick myelomonocytic cell line HD-11. 7 6 - 7 8 Synthesis of 1,25(OH)2D by the cells is enhanced by ~/-interferon and insulin-like growth factor-I and is inhibited by dexamethasone, 1,25(OH)2D3, and inhibitors of cytochrome P - 4 5 0 . 77 Synthesis was not inhibited by calcium or phosphorus. Nitric oxide appears to be involved in synthesis of 1,25(OH)2D. TM Depletion of L-arginine, substrate for endogenous nitrous oxide production, diminished production of 1,25(OH)2D, and production was restored by addition of L-arginine. Further, synthesis was competitively inhibited by the synthetic analogs NW-nitro-Larginine methyl ester and NW-nitro-L-arginine. These studies indicate that the HD-11 chick cell line is a useful and novel model to study regulation of synthesis of 1,25(OH)2D by cells with a number of characteristics of the human macrophage. 4. CLINICAL MANIFESTATIONS
a. Calcium Metabolism. The true incidence of abnormal calcium metabolism, hyperabsorption of calcium, hypercalcemia, and hypercalciuria, is difficult to assess because of variation in urinary calcium in the general population such as occurs, for example, in obese subjects and in blacks. 45'46 Urinary calcium is lower in these individuals than in white nonobese men and women. Hypercalcemia probably occurs in less than 10% of patients with sarcoidosis, but this, too, is difficult to assess because of seasonal variation in the serum calcium. 39 Hypercalcemia of clinical importance probably occurs in less than 5% of patients. Symptoms related to abnormal calcium metabolism may be categorized relative to the gastrointestinal tract, kidney, and central nervous system. Anorexia, nausea, and vomiting may lead to dehydration and weight loss. Hypercalcemia and hypercalciuria may result in nephrogenic diabetes insipidus with polydipsia, polyuria, frequency, and nocturia. 79 The renal concentrating defect is reversible upon correction of the abnormal calcium metabolism. Renal stones may produce renal colic, hematuria, dysuria, urgency, and frequency. Hypercalcemia may be asymptomatic or produce generalized symptoms of muscle weakness, easy fatigue, and lethargy and, if severe and prolonged, may produce a variety of mental disturbances and even coma. In sarcoidosis, however, the hypercalcemia usually does not exceed 14 or 15 mg/dl and usually does not produce a hypercalcemic crisis.
612
NORMANH. BELL
Nephrolithiasis and nephrocalcinosis may produce pyelonephritis. Hypercalcemia may also produce impaired renal function. The author has seen two patients with sarcoid referred because of anemia who were found to have chronic renal failure as a result of hypercalcemia. The anemia in them was caused by renal insufficiency. Clinical findings in patients with sarcoid related to abnormal calcium metabolism include calcium deposits in the conjunctivae and costovertebral angle tenderness in patients with urinary tract infections and kidney stones. 5. TREATMENT
a. Glucocorticoids. In sarcoidosis, the abnormal metabolism of vitamin D and calcium consistently responds to treatment with glucocorticoids (Table 20-2). Serum and urinary calcium are decreased to normal and fecal calcium is increased. 35-37 Glucocorticoids thus inhibit the intestinal absorption and turnover of calcium as determined with labeled c a l c i u m . 34'37 Increased circulating 1,25(OH)2D is lowered to values that are within or even below the normal range, 28'6~-64 and serum immunoreactive parathyroid hormone increases as the serum calcium declines. 6~-64 Glucocorticoids also reduce serum 1,25(OH)2D and the intestinal absorption of calcium even in patients with sarcoidosis and a normal serum calcium. 42 In contrast, values in normal subjects do not change. 8~ Glucocorticoids act to reverse the abnormal calcium metabolism in sarcoid by correcting the altered metabolism of vitamin D. They may also act by inhibiting the peripheral actions of 1,25(OH)2D. 81 Glucocorticoids may be effective in reducing serum 1,25(OH)2D in sarcoidosis by suppressing the inflammatory response. They inhibit the proliferation of T lymphocytes, 82"83 the production of T-cell growth factor, 84 and the production of macrophage migration inhibitory factor. 85 Since sarcoidosis is a granulomatous disease that is perpetuated by activated T lymphocytes, glucocorticoids could act by inhibiting the growth, develop-
TABLE 20--2 Drugs Used for Treatment of Hypercalcemia Caused by Increased Serum 1,25(OH)2D Drugs that lower serum 1,25(OH)2D Glucocorticoids Chloroquine Hydroxychloroquine Ketoconazole Drugs that do not lower serum 1,25(OH)2D Flurbiprofen Mithramycin Phytate Saline
ment, and maintenance of granulomas. In this regard it is interesting to note that glucocorticoids lower serum angiotensin-converting enzyme in patients with sarcoidosis and hypercalcemia, 75 that serum angiotensinconverting enzyme is present in sarcoid granulomas, 86 and that it is produced by peripheral monocytes in this disease. 87
b. Other Therapy. Other modes of therapy that may be effective in treatment of abnormal calcium metabolism in sarcoidosis include reduction of calcium intake, 35 administration of sodium phytate or edetate disodium, which bind dietary calcium and prevent its absorption, 35'88 and omission of dietary vitamin D and exposure to ultraviolet light. 38 The prostaglandin synthesis inhibitor flurbiprofen was used to successfully treat hypercalcemia in one patient with sarcoidosis. 89 Unlike glucocorticoids, which reduce serum 1,25(OH)2D, flurbiprofen corrected the hypercalcemia but did not influence the increased circulating 1,25(OH)2D. Since prostaglandin E2 is produced by osteoblasts and produces bone resorption, flurbiprofen could act by inhibiting prostaglandin E2 synthesis and prostaglandin E2-mediated bone resorption. If the effectiveness of flurbiprofen is confirmed, it could be a useful alternative means to treat abnormal calcium metabolism in sarcoidosis. Glucocorticoids cause substantial loss of bone in patients with sarcoidosis. 9~ Chloroquine and hydroxychloroquine are effective in reducing serum 1,25(OH)2D and correcting abnormal calcium metabolism resulting from the disease. 9~'92 Loss of central visual acuity with granular pigmentation of the macula and retinal artery constriction are the toxic effects of greatest concern with the drugs. 91 These effects, however, correlate with total accumulated dose and can be prevented by periodic opthalmic examinations and stopping the drug in the event that alteration in visual acuity or retinal changes occur. Ketoconazole, an inhibitor of cytochrome P-450, reduces serum 1,25(OH)2D and corrects the abnormal calcium metabolism in s a r c o i d o s i s . 93'94
B. T u b e r c u l o s i s Hypercalcemia and hypercalciuria occur in pulmonary as well as miliary tuberculosis, 95-1~ and hypercalcemia is associated with suppression of serum immunoreactive parathyroid h o r m o n e . 96-98 Patients with tuberculosis are abnormally sensitive to vitamin D, and the hypercalcemia is reversed by g l u c o c o r t i c o i d s . 95'97'1~ Hypercalcemia in pulmonary tuberculosis is caused by increased circulating 1,25(OH)2D. 1~176 Further, regulation of serum 1,25(OH)2D is abnormal in normocalcemic
CHAPTER20 Sarcoidosis and Related Disorders patients with pulmonary tuberculosis. TM A modest but significant increase in mean serum 1,25(OH)2D occurred in a group of 11 patients given vitamin D, 100,000 units/ day for 4 days, but no change in mean serum 1,25(OH)2D in a group of normal subjects. Abnormalities in calcium metabolism in pulmonary tuberculosis are similar to those that occur in sarcoidosis. However, the clinical course of hypercalcemia in tuberculosis usually differs from that in sarcoidosis in several important respects. As noted already, hypercalcemia in sarcoidosis often develops following exposure to sunlight during the summer months and may be the cause for hospitalization. 61-63 The abnormal calcium metabolism frequently persists; usually requires treatment with glucocorticoids; and may be associated with nephrolithiasis, nephrocalcinosis, and recurrent urinary tract infections. As a consequence, renal failure may develop and even result in death. In contrast, hypercalcemia in tuberculosis may develop after several months of antituberculous therapy and is readily managed by hydration, and impairment of kidney function is short lived and does not require hospitalization, l~ Thus, the prognosis for the abnormal calcium metabolism in the two diseases is quite different. In pulmonary tuberculosis, it is likely that 1,25(OH)2D is synthesized by the granulomas. As noted, the concentrations of ~/-interferon and 1,25(OH)2D are increased in pleural fluid from patients with tuberculous pleural effusion and production of 1,25(OH)zD by alveolar macrophages from patients with sarcoidosis is stimulated by pleural fluid from patients with tuberculous pleural effusion. 23 The findings that the occurrence of hypercalcemia in tuberculosis varies with the extent of the disease 97 and that hypercalcemia and increased circulating 1,25(OH)zD occurred in a patient with tuberculosis and end-stage renal disease 1~ are consistent with this possibility. The delay in onset of hypercalcemia that usually develops in the course of treatment suggests that abnormal production of 1,25(OH)zD may be related in some way to antituberculous therapy. However, no changes in serum 25(OH)D or 1,25(OH)zD were found in a long-term study of eight patients with pulmonary tuberculosis during their treatment with rifampin and isoniazid. 1~ In two children with pulmonary tuberculosis and hypercalcemia caused by increased serum 1,25(OH)2D, ketoconazole lowered serum 1,25(OH)2D and corrected the abnormal calcium metabolism, 1~
C. Other Granulomatous Diseases Hypercalcemia was reported in histoplasmosis, 1~ disseminated candidiasis, 1~ disseminated coccidioidomy-
613 cosis, 1~ berylliosis, 11~ leprosy, T M silicone-induced granulomas, 112 Wegener's granulomatosis, 113"114 acquired immunodeficiency syndrome (AIDS) with Pneumocystis Carinii pneumonia, 115 and A I D S with Cryptococcus neoformans infection. 116 Increased serum 1,25(OH)2D is assumed to be the cause for the abnormal calcium metabolism in these granulomatous diseases. Hypercalcemia and elevated 1,25(OH)2D were corrected by treatment with salmon calcitonin, saline, furosemide, and mithramycin in the patient with candidiasis, by prednisone in the patients with leprosy and silicone-induced granulomas, and by chloroquine in a patient with Wegener's granulomatosis. The patient with AIDS and Pneumocystis carinii pneumonia was treated with prednisone, saline, and etidronate. 115 The patient with AIDS and cryptococcal infection had an elevated serum 1,25-(OH)2D, suppressed serum immunoreactive parathyroid hormone, and hypercalcemia, that responded to treatment of the infection. The cause for abnormal calcium metabolism in the other diseases was not determined. A low serum 1,25(OH)2D was demonstrated in a patient with hypercalcemia and disseminated coccidioidomycosis, 117 in a patient with hypercalcemia and leprosyll8; and in two patients with hypercalcemia, AIDS, and a cytomegalovirus infection. 119 This indicates that sometimes hypercalcemia may result from mechanisms not related to abnormal vitamin D metabolism in these disorders.
D. Rheumatoid Arthritis Hypercalcemia, hypercalciuria, and suppression of immunoreactive parathyroid hormone caused by increased circulating 1,25(OH)zD was found in a patient with rheumatoid arthritis. 12~ The patient showed lack of regulation of serum 1,25(OH)zD in response to small doses of vitamin D. The abnormal calcium metabolism and increased serum 1,25(OH)zD were retumed to normal by prednisone. It is not established whether abnormal metabolism of vitamin D in this patient is related to rheumatoid arthritis.
II. LYMPHOMA AND SOLID TUMORS A. Lymphoma, Leukemia, and Hodgkin' s Disease Hypercalcemia associated with increases in serum 1,25(OH)2D that responds to glucocorticoids occurs in patients with histiocytic tympnoma, " " 1 2 1'1 2 2 T-cell leukemialymphoma, 121 B cell lymphoma, 123 Hodgkin 's disease, 124-127 128 and plasma cell granuloma. The hypercalcemia is as-
614
NORMANH. BELL
sociated with suppression of serum immunoreactive parathyroid hormone. Studies carried out in one of the patients with histiocytic lymphoma indicated that the hypercalcemia was associated with hyperabsorption of calcium and hypercalciuria. 121 T-cell lymphoma resulting from human T-cell lymphotrophic virus (HTLV-1) is characterized by lymphocytosis, hepatosplenomegaly, hypercalcemia, and a rapidly progressive c o u r s e . 129 One patient with the disease, as noted, had hypercalcemia caused by increased circulating 1,25(OH)zD. 121 Other patients with T-cell leukemia were found who had hypercalcemia and suppressed serum 1,25(OH)zD. 13~Thus, the hypercalcemia in them was not caused by abnormal vitamin D metabolism. Human T lymphocytes from cord blood of normal infants infected with HTLV-1 by incubation of the lymphocytes with lymphoma cells carrying the virus secrete HTLV-1 proteins and exhibit morphological and functional properties of lymphoma cell. 131'132 Cord blood lymphocytes transformed by this means were shown to convert [3H]25(OH)D3 to [3H]l,25(OH)zD3.133 The putative 1,25(OH)zD3 caused release of 45Ca from long bones of fetal rats in tissue culture, displaced [3H]l,25(OH)zD3 from the receptors of rat osteosarcoma cells in the same manner as authentic 1,25(OH)zD3, and had the same retention times as authentic 1,25(OH)zD3 on high-pressure liquid chromatography in a number of different solvent systems. The identity of the metabolite was confirmed as 1,25(OH)zD3 by mass spectral analysis. 133 It remains to be demonstrated, however, that lymphocytes from patients with lymphoma produce 1,25(OH)zD. The question arises also whether activated lymphocytes synthesize 1,25(OH)zD in sarcoidosis.
B. Metastatic Leioblastoma Hypercalcemia with increased serum 1,25(OH)2D was reported in a patient with metastatic leiomyoblastoma of the jejunum. TM No skeletal lesions were detected. Moderate renal insufficiency developed, and the abnormal vitamin D and calcium metabolism persisted despite subtotal parathyroidectomy. The hypercalcemia and abnormally elevated 1,25(OH)zD were reversed by prednisone, phenytoin, and phenobarbital.
C. Seminoma Hypercalcemia with increased circulating 1,25(OH)2D was reported in a patient with seminoma. 135 These abnormal findings were reversed by partial resection of the tumor and chemotherapy.
D. Small Cell Carcinoma of the Lung A sterol tentatively identified as 1,24(OH)2D3was observed in extracts of serum and tumor in a patient with small cell carcinoma of the lung and hypercalcemia. 136 The sterol comigrated with authentic 1,25(OH)2D3 on high-pressure liquid chromatography and displaced [3H]l,25(OH)2D3 bound to the chick intestinal receptor in a manner similar to that of the authentic sterol. Further, the sterol and synthetic 1,24(OH)2D3 showed identical biological activity in producing release of 45Ca from mouse calvariae in tissue culture. The results provide evidence that the hypercalcemia in this patient resulted from increased production of 1,24(OH)2D3. This important investigation is the first indication that an endogenously produced sterol other than 1,25(OH)2D may be responsible for abnormal calcium metabolism in a clinical disorder.
III. MISCELLANEOUS DISEASES
A. Increased Serum 1,25(OH)2D of Unknown Cause Three patients were reported in whom hypercalcemia occurred in association with increased circulating 1,25(OH)zD, low normal or undetectable serum immunoreactive parathyroid hormone and urinary cyclic adenosine 3',5'-monophosphate (cAMP), hypercalciuria, nephrolithiasis, and no apparent systemic disease. 137-139 All were males. In two of them, hypercalcemia was corrected by glucocorticoids. The site of the production of 1,25(OH)zD is not known. The fact that serum 1,25(OH)zD is unregulated strongly suggests a nonrenal source for the metabolite.
B. Williams Syndrome Hypercalcemia, hypercalciuria, and increased intestinal absorption of calcium associated with anorexia, vomiting, impaired renal function, mental retardation, and failure to thrive occurs in newborn infants. 14~ 146 Patients show increased sensitivity to vitamin D, 141'142 and the hypercalcemia and abnormal calcium metabolism are corrected by g l u c o c o r t i c o i d s . 141'142'144 The illness usually occurs in the first 2 years of life and is self-limited. There is an association with the Williams syndrome in which supravalvular aortic stenosis, an elfin facies, and mental retardation are found in association with a number of other congenital d e f e c t s . 146-15~ White children with the syndrome show an exaggerated increase in se-
CHAPTER 20
Sarcoidosis and Related Disorders
rum 25(OH)2D after vitamin D challenge compared with normal children. 151 This is attributed to a lack of increase in serum 1,25(OH)zD in the patients and lack of feedback reduction of circulating 25(OH)D. 51'52 In four infants with elfin facies and hypercalcemia, increases in serum 1,25(OH)2D were observed. TM Values declined with time, and hypercalcemia was successfully treated with a low calcium diet. In another child with hypercalcemia, the serum 1,25(OH)zD was abnormally low. T M This indicates that hypercalcemia in the syndrome of hypercalcemia of infancy may result not only from elevation of circulating 1,25(OH)zD but from other causes. In infants with abnormal elevations of circulating 1,25(OH)zD, the source of the metabolite is not known. Restriction of calcium intake, hydration, and glucocorticoids are effective for treatment of children with hypercalcemia and abnormal vitamin D metabolism in this disorder. 143,145,151,152 Recent findings indicate that supravalvular aortic stenosis results from translocations in the elastin gene and that similar mutations occur in patients with the Williams syndrome in w h o m supravalvular aortic stenosis is common. 154-156 Whether mutations in one or more adjacent genes are responsible for other manifestations of the Williams syndrome remains to be determined.
C. I n f a n t s w i t h S u b c u t a n e o u s
Fat Necrosis
Subcutaneous fat necrosis occurs transiently and most often in newborns as a consequence of trauma or asphyxia at birth. Necrosis of fat is associated with granulomatous infiltration and foreign b o d y - t y p e cells. 157 Hypercalcemia, hypercalciuria, suppression of serum immunoreactive parathyroid hormone, and increased serum 1,25(OH)2D that were corrected by glucocorticoids occurred in two infants with the disorder. 157 The pathogenesis of abnormal vitamin D and calcium metabolism in the infants is not established but is attributed to granuloma.
D. V i t a m i n D I n t o x i c a t i o n In vitamin D intoxication, hypercalcemia and hypercalciuria that are corrected by corticosteroids and associated with suppression of serum immunoreactive parathyroid hormone and urinary c A M P occur. 158 In patients with vitamin D intoxication, abnormal calcium metabolism is caused primarily by excess circulating 25(OH)D. Serum 25(OH)D is markedly increased and serum 1,25(OH)2D is either normal or only modestly elevated. 159 However, free 1,25(OH)2D is increased and may contribute to production of abnormal calcium me-
615 tabolism. 16~ Hypercalcemia may be caused by overfortification of vitamin D in m i l k . 161'162 The diagnosis of vitamin D intoxication is made by the history of excessive intake of vitamin D and is confirmed by the finding of markedly elevated serum 25(OH)D, 200 ng/ml or higher.
E.
1,25(OH)zD3 Intoxication
Hypercalcemia may also occur in patients who are receiving 1,25(OH)zD3 .163 The hypercalcemia develops spontaneously even after a dose has been established that maintains the serum calcium in the normal range. Hypercalcemia is associated with abnormal increases in serum 1,25(OH)2D and suppression of serum immunoreactive parathyroid hormone. It has been reported to occur in a number of disorders including renal failure, osteoporosis, hypoparathyroidism, and pseudohypoparathyroidism. The diagnosis is made from the history and measurement of serum 1,25(OH)2D.
F. C a l c i p o t r i o l I n t o x i c a t i o n Hypercalcemic crisis occurred in a 17-year-old w o m a n with ichthyosis after excessive topical use of calcipotriol, an analog of vitamin D with much reduced activity on calcium metabolism. 164 She was successfully treated by stopping the calcipotriol and rehydration with intravenous fluids and electrolytes.
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Bone Disease in Rheumatological Disorders STEVEN R. GOLDRING AND RICHARD P. POLISSON 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; New England Baptist Bone and Joint Institute, Boston, Massachusetts 02215
I. Introduction II. Rheumatoid Arthritis III. Seronegative Spondyloarthropathies
IV. Systemic Lupus Erythematosus V. Juvenile Rheumatoid Arthritis References
matological disorder in which the pathological processes are restricted almost entirely to the joint structures, including the articular cartilage, synovium, and juxtaarticular (subchondral) bone. OA also may affect amphiarthroses such as the intervertebral disks. In this case, the disks themselves, and the adjacent bony tissues, are the principal sites of involvement. Many of the rheumatic diseases, particularly those associated with the various types of inflammatory arthritis, may affect extraarticular tissues such as the lung, central nervous system, kidney, cardiovascular system, and gastrointestinal system. These conditions are often accompanied by significant systemic symptoms such as fatigue, weight loss, and fever that may dominate the clinical picture. These illnesses, which include, for example, conditions such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE), are initiated by a dysregulated immune response that targets various organs and tissue sites, including the joints. In these illnesses, the widespread systemic inflammatory process may produce generalized effects on bone remodeling that affect the entire skeleton.
I. I N T R O D U C T I O N The rheumatological disorders include a diverse group of diseases that share in common their propensity to affect the anatomical components of articular and juxtaarticular tissues. Of the structurally distinct types of joints the amphiarthrodial and diarthrodial joints are the most commonly involved~ Amphiarthroses are characterized by a fibrocartilaginous union which joins the tWo opposing bone surfaces. These joints permit little motion, and are best represented by the intervertebral disks, which are a common site for certain inflammatory and degenerative joint disorders. Diarthrodial joints, the most common of the articular design patterns, consist of two articulating surfaces covered by hyaline cartilage. The joint cavity is lined by a highly specialized synovial membrane, which separates the joint from the surrounding connective tissues. This membrane is the site of production of synovial fluid, which provides the nutritional requirements for the articular cartilage and also "lubricates" the articulating surfaces of the opposing bones. Osteoarthritis (OA) is a prototypical example of a rheuMETABOLIC BONE DISEASE
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II. RHEUMATOID ARTHRITIS Among the rheumatological diseases, RA represents an excellent model for gaining insights into the effects of local as well as systemic consequences of inflammatory processes on skeletal tissue remodeling. RA is characterized by three types of bone loss: (1) local subchondral and joint margin bone erosion, (2) periarticular osteopenia adjacent to inflamed joints, and (3) generalized osteoporosis involving the axial and appendicular skeleton. 1
A. Focal Subchondral and Marginal Bone Erosions RA is a systemic autoimmune inflammatory disorder in which multiorgan system involvement is occasionally observed. The hallmark of RA is the presence of an intense immune-mediated inflammatory process that affects the synovial membrane of diarthrodial joints (Fig. 21-1). The synovial lesion is characterized by proliferation of the synovial lining cells and infiltration of the synovium with inflammatory cells, including lymphocytes, plasma cells, and activated macrophages. 2-4 As this lesion evolves, the synovial tissue extends over the articular surfaces forming a pannus or mantle-like c o v -
FIGURE 21-1
ering that invades the hyaline cartilage and results in progressive loss of the articular cartilage. At points where the synovial pannus contacts the joint margins, there is often erosion of the bone surfaces resulting in focal regions of osteolysis. These excavations of the bone surfaces give rise to the characteristic cystic bone erosions that can be detected radiographically (Fig. 2 1 2). The synovial tissue also may invade the marrow spaces, where it can produce additional focal areas of subchondral bone loss. There is still considerable debate concerning the specific cellular and biochemical mechanisms responsible for the progressive loss of articular cartilage, focal bony erosions, and subchondral osteolysis that characterizes the synovial lesion of RA. Results from studies employing histochemical and immunolocalization techniques support the concept that the production of collagenase and other proteinases at the cartilage-pannus junction play a prominent role in the pathogenesis of the cartilage destruction. 5-s The aggregation of mast cells to this site provides an additional mechanism for the cartilage destruction. 6'9 When activated, these cells produce a large repertoire of degradative proteinases and inflammatory mediators. Bromley and Woolley 9-11 have provided important insights into the cellular events associated with the marginal and subchondral bone erosions associated with the bone-pannus junction. They have noted the presence of
Photomicrograph demonstrating the histopathological changes associated with rheumatoid arthritis (RA). The rheumatoid synovium (shown at the top) invading the juxtaarticular bone. An arrow identifies a multinucleated osteoclast-like cell in a resorption lacuna.
CHAPTER 21 Bone Disease in Rheumatological Disorders
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FIGURE 21--2 Plain radiograph showing evolution of a bone erosion associated with the invading rheumatoid synovial lesion (pannus). A, Early RA--no erosion. B, Later RA- - erosion of the proximal interphalangeal joint identified by an arrow. C, Later RA--with progression of bone erosion demonstrated by an arrow.
increased numbers of multinucleated cells with phenotypic features of osteoclasts that are localized to osteolytic lacunae at the junction of the subchondral bone and adjacent marrow space. Many of these appeared to be in direct contact with the overlying cartilage matrix, and these multinucleated tartrate-resistant acid phosphatasepositive cells were characterized as "chondroclasts." lo These so-called chondroclasts were observed in 30% of large joint specimens where they appeared to contribute to the subchondral cartilage erosions. These cells were frequently observed in specimens from small joints, and the investigators speculated that the mechanisms associated with small joint pathology in RA might differ from that in the large joints. These observations are supported by the studies of Allard et al. 12 Additional analyses of the cell types at the b o n e pannus junction and characterization of cells isolated from rheumatoid synovium suggest that cells with phenotypic features and functional activities of authentic os-
teoclasts are responsible for at least a component of the focal bone resorption that characterizes the RA synovial l e s i o n . 13'14 The demonstration of osteoclasts at sites of active resorption does not preclude the possibility that macrophages or macrophage polykaryons (also present in the synovial lesions) also may have the capacity to resorb bone. These cell types have been shown to resorb bone, although their resorptive capacity is much less than osteoclasts.14'15 In our lab, we have used in situ hybridization techniques in which labeled cDNA probes derived from the recently cloned calcitonin receptor have been employed to characterize cells at the bone-pannus junction. 16-18 These studies confirm that the multinucleated cells (and some mononuclear cells) in resorption lacunae express abundant calcitonin receptor mRNA, adding further support to the concept that they are functionally indistinguishable from authentic osteoclasts (Fig. 21-1). Of interest, these cells do not express mRNA for the para-
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thyroid hormone receptor. This observation is consistent with the observations of others who speculate that PTH does not act directly on osteoclasts but rather mediates its effects indirectly via effects on osteoblast lineage and/or marrow stromal cells. 19-21 The origin of the osteoclast-like cells in the RA lesions remains speculative. There is some evidence that they may derive from mononuclear cell precursors present within the inflamed synovium, and that they are induced to differentiate into osteoclasts by the cytokines that are produced locally within the RA synovium. 1~
B. P e r i a r t i c u l a r B o n e L o s s Periarticular osteopenia is the radiographic hallmark of early RA; it typically appears in areas of active synovitis and usually precedes the appearance of focal erosions (Fig. 21-3). The etiology of this radiographic phenomenon is unknown; however, histological studies have suggested that increased bone resorption occurs in these areas. Shimizo et al. 22 observed increased turnover of
juxtaarticular bone in 12 patients with rheumatoid arthritis undergoing surgery. Bone histomorphometry revealed both increased bone formation as well as bone resorption. This finding contrasts with observations in the axial skeleton. Elaboration of proinflammatory mediators, hypervascularity of the synovium and surrounding tissue, and joint immobility, all have been implicated as possible mechanisms to explain this radiographic and histological finding. 3'z3-27 Because localized osteopenia is most noticeable in periarticular areas, it is intriguing to implicate a biological process in which increased bone resorption is caused by a gradient effect of proinflammatory mediators that are produced in the synovial tissue. 28 Prostaglandin E2 (PGE2) is abundantly produced by rheumatoid synovium, and it has been shown to stimulate osteoclastic bone resorption. 29 Inflammatory cells within the pannus produce many cytokines that have been implicated in pathological bone and cartilage matrix degradation, including interleukin-1 (IL-1), IL-6, and tumor necrosis factor-tx ( T N F - ~ ) . 23-26'3~ These cytokines likely play a role in the pathogenesis of the focal bone lesions of RA but may also contribute to the periarticular bone loss. Finally, immobilization of joints secondary to inflammation and pain may result in significant localized bone loss. 22
C. G e n e r a l i z e d B o n e L o s s
FIGURE 21--3
Plain radiograph of early RA revealing periarticular osteopenia affecting the metacarpalphalangeal joints.
Diffuse bone loss has been a well known clinical feature of RA since it was first described in 1865 by Barwell. The distribution and magnitude of bone loss in the axial spine and appendicular bone distant from synoviallined joints, however, has only recently become the focus of study. 28'32-37 S o m e observations suggest there is as much as a 10% reduction in bone mineral density (BMD) 33"38 in patients with RA compared to controls, while other studies have shown no significant differe n c e . 39 A compelling study of bone density as measured by dual energy x-ray absorptiometry (DEXA) in monozygotic twins (one with RA and one disease-free) showed significant reduction in BMD in the lumbar spine (4.6%), femoral neck (9.7%), and total body (5.7%) in the affected subjects. 4~ Histomorphometric analysis of bone biopsies from patients with RA indicate that the cellular basis of reduced bone mass is related to a decrease in bone formation rather than an increase in bone r e s o r p t i o n . 4x-43 More recently, biochemical markers of bone turnover have been used to study bone remodeling in patients with RA. Results from these studies indicate that there is an increase in the rate of bone resorption in these patients, 44'45 although these findings must be interpreted with caution, since the patients were receiving glucocorticoids.
CHAPTER21 Bone Disease in Rheumatological Disorders Analysis of BMD and bone histomorphometry data in patients with RA strongly suggests an independent effect of the disease itself on bone, perhaps related either to the systemic effects of the chronic inflammation or to the functional status and physical activity of the subjects. However, evaluating causation of this clinical phenomenon has been fraught with confounding factors, including patient sex, age, mobility, disease activity and duration, and the concomitant use of immunosuppressive therapies, all of which are known to have profound and independent effects on bone metabolism. Epidemiological studies have also been flawed by design problems, small numbers of subjects, and inconsistent methods of measurement. Some studies evaluate patients crosssectionally, but these studies do not provide dynamic information about evolution of the process through time. In addition, end-point determination of BMD have been highly variable, thereby complicating cross-study comparisons. Nevertheless, there is compelling evidence that patients with RA exhibit increased risk of fracture. 46-49 1. EFFECTS OF AGE
In healthy subjects, the effects of age on bone mass are well known. Bone mass peaks at approximately age 35 and then decreases progressively. In women, the estrogen deficiency that characterizes the perimenopausal period produces a rapid acceleration in the rate of bone lOSS.21 The same patterns of age-dependent and sex hormone-associated bone loss are observed in patients with RA. Sambrook et al. 33 observed no difference in age-related bone loss in patients with RA compared to normal controls. 2. DISEASE ACTIVITY]DURATION Disease activity and duration may be associated with accelerated reduction in bone mass. 5~ In a large longitudinal prospective study, Gough and co-workers 51 concluded that significant amounts of generalized skeletal bone was lost early in RA and that this loss was associated with disease activity. These findings support the previous observations of Als et al. 52 who also observed a significant decrease in bone mass during the early phases of RA. In a longitudinal study by Sambrook et al., 35 two clinical measures of disease activity, joint count and C-reactive protein (CRP), were correlated with trabecular bone loss in the lumbosacral spine. Many of these studies are flawed by confounding issues, such as level of physical activity and the concomitant use of drugs that affect bone remodeling. Although it is likely that an association exists between RA and BMD reduction, further studies are needed to accurately determine the natural history and factors that are predictive of generalized bone loss in patients with RA.
625 3. FUNCTIONAL STATUS Weight-bearing exercise affects bone mass in normal individuals. 53 The generalized osteoporosis observed in patients with RA has in part been ascribed to sedentary lifestyle related to chronic pain and joint inflammation. Patients with active synovitis experience significant stiffness of the joints after immobility, and pain is increased with motion or impact-loading of joints. As a result, the general level of physical activity in patients with RA is reduced. Functional status, as assessed by several different instruments, is now viewed as an important end point in most clinical studies of RA. Epidemiological studies have consistently shown the inverse relationship between physical activity and functional level and bone density in these patients. Using the Framingham Activity Index (a measure of functional status), Sambrook et al. observed that the level of activity correlated with femoral neck bone mineral density. 33 Gough et al., using the Health Assessment Questionnaire (HAQ), corroborated the association with disability level and bone loss in the femoral neck and axial spine. 51 In both of these studies, the effects of disease activity on BMD were not independently assessed. Magaro et al. 54 and Egglemeijer 55 studied RA patients not exposed to glucocorticoids and observed a significant correlation of functional class with bone loss. In a longitudinal study of RA patients, those with higher levels of functional disability, as measured by the HAQ for 18 months prior to bone mineral density measurement, had increased bone loss in the hip. 4. DRUG EFFECTS
Chronic administration of glucocorticoid therapy in high doses is associated with an adverse effect on skeletal mass. 56 The mechanisms responsible for the associated bone loss include decreased intestinal calcium absorption, reduction in the rate of bone formation, and the presence of a state of mild sustained secondary hyperparathyroidism. 57 On the other hand, low doses of glucocorticoids are known to improve symptoms of RA and functional capacity in the short term, which might be expected to have a beneficial effect on bone density, especially in the axial spine. 58 In a cross-sectional study of patients with RA, lower levels of BMD were observed in RA patients compared to normal controls, but no difference was noted in the low-dose glucocorticoid-treated RA group compared to RA patients not similarly exposed. 34 In a longitudinal study by the same group of investigators, 59 the effects of low doses of glucocorticoids were studied over a 24-month period. Baseline measurements of BMD in the glucocorticoid-treated group were lower than in the untreated cohort, suggesting that patients who received glucocorticoids had more disease activity and lower functional status. Interestingly,
626 after 24 months the rate of change across both groups was not significantly different. However, other crosssectional studies have suggested that even low doses of glucocorticoids have a detrimental effect on bone density. Laan et al. 6~ utilized quantitative computed tomography (QCT) to show that low doses of glucocorticoids (mean dose of 6.8 mg of prednisone per day) reduced bone density in the glucocorticoid-treated postmenopausal group. Similar effects were noted in other trials, utilizing other measurements of bone density. One of the more compelling studies involved a case-control analysis of 112 RA patients on long-term low-dose glucocorticoid therapy. Cases were matched to 112 control patients by sex, race, and disease duration. Glucocorticoid exposure was a significant predictor of adverse events, which included fractures, gastrointestinal events, and infections; however, the potential confounding effects of disease activity were not assessed. 61 Methotrexate is frequently used as treatment for patients with RA. Many individuals remain on this drug for prolonged periods of time, and concern exists that sustained exposure to moderate doses of this drug may adversely affect bone mass. Methotrexate has been shown to inhibit osteoblastic bone formation in animal models, 62 and in pediatric patients receiving high doses of methotrexate for treatment of malignancy, there is evidence suggesting accelerated bone loss. RA patients on low doses of methotrexate may be similarly affected, as indicated by a single study of a small number of methotrexate-treated RA patients in whom examination of bone histomorphometry revealed osteoporotic changes that reversed after discontinuation of the d r u g . 63 5. FRACTURE Although bone mass as assessed by B MD is an important predictive indicator of osteoporosis, fracture frequency remains the most significant end point for evaluating the effects of RA on bone. In 388 patients with RA analyzed by Hooyman e t al., 48 an increased risk of fracture was observed for the pelvis (relative risk, 2.56) and hip (relative risk, 1.51). Regression analysis suggested age, duration of disease, disability level, and glucocorticoid exposure were risk factors for fractures, whereas increased weight and estrogen exposure appeared to be protective. Similarly, a multivariate analysis of 390 RA patients identified glucocorticoid use and a prior diagnosis of osteoporosis as independent risk factors for fracture with an overall 5-year probability of fracture estimated at 34%. In this same cohort, men had no increased risk of fracture. 47 6. TREATMENT OF OSTEOPOROSIS
The results of treatment intervention to prevent or reduce the risk of osteoporosis in patients with RA is con-
STEVEN R. GOLDRING AND RICHARD P. POLISSON
troversial. Because epidemiological observations suggest that the biological process of inflammation, perhaps through elaboration of bone-active cytokines, may have an adverse effect on bone, control of the underlying disease hypothetically should reduce the risk of bone loss and fracture. 51'58 However, no good clinical studies exist that substantiate this hypothesis. Indeed, as noted above, methotrexate, which has been shown to ameliorate the level of disease activity in some patients with RA, may in fact have an adverse effect on bone mass by reducing the rate of bone formation. The use of other agents, particularly estrogens, has been shown in prospective studies to improve bone density modestly in postmenopausal women with R A . 64-67 Kroger et al. 68 examined the effects of calcitonin treatment on bone histomorphometry and biochemical markers of bone turnover. In a 3-month trial they were able to detect a small but significant increase in trabecular bone volume, although there were no detectable differences in the biochemical markers in the treatment group compared to the control group. In a double-blind, placebo-controlled study, Sileghem et al. detected a significant increase in BMD in both the spine and forearm in RA patients treated with intranasal calcitonin for 1 year. However, the increase in bone density in the calcitonin-treated subjects was not sustained over the following 12 months. In a recently completed prospective controlled 3-year trial with the bisphosphonate pamidronate, 105 patients with RA demonstrated a significant increased B MD in the lumbar spine and hip compared to the placebo group. Urinary hydroxyproline excretion (corrected for creatinine excretion) was significantly less in the pamidronate-treated group. 69
III. SERONEGATIVE SPONDYLOARTHROPATHIES The seronegative spondyloarthropathies encompass a broad range of multisystem inflammatory disorders in which the spine, peripheral joints, and periarticular structures are involved. This group of disorders includes ankylosing spondylitis, reactive arthritis, Reiter's syndrome, spondylitis and arthritis associated with psoriasis or inflammatory bowel disease, and juvenile-onset spondyloarthropathy. The poly- and oligoarthritis that accompany these disorders exhibit many of the same histopathological features that characterize the joint pathology in RA. Synovial hyperplasia, lymphoid infiltration, and pannus formation are frequently observed in affected joints. However, the spondylitic disorders lack several of the features commonly seen in RA, including proliferation of synovial villi, fibrin deposits, and localized regions of cell necrosis. Joint margin and subchon-
CHAPTER21 Bone Disease in Rheumatological Disorders dral erosions and cartilage destruction are often seen, although the inflammation may be restricted to only a few isolated joints. The pattern of distribution is typically asymmetrical, affecting proximal as well as distal joints including the distal interphalangeal joints that are not typically affected in RA. Inflammation of the enthesis (sites of tendinous or ligamentous attachment to bone), especially in the axial spine, is the pathological hallmark of the spondyloarthropathies, and this feature distinguishes this group of disorders from the other inflammatory forms of arthritis. Similar to those individuals with RA, patients with ankylosing spondylitis and the related spondyloarthropathies have both localized and generalized osteoporosis (Fig. 21-4). The enthesopathic erosion is a wellrecognized early radiographic feature of these disorders, whereas calcification and ossification of the enthesis is a latter manifestation. Few studies have addressed the pathophysiology of bone loss localized to tendon insertions or periarticular areas in these diseases. Recently, Braun et al. TM used immunohistological and in situ hybridization techniques to study the pathogenesis of inflammation and new bone formation in the sacroiliac
FIGURE 21--4
Plain radiograph of ankylosing spondylitis revealing osteopenia of the vertebral bodies and irregularity of the vertebral body end plates with syndesmophytes identified by the arrows.
627 joints of patients with spondylitis. Computer-assisted tomography was used to obtain biopsies of the sacroiliac joint in five patients with ankylosing spondylitis. Synovial tissue revealed dense infiltrates of T lymphocytes (CD4 + and CD8 +) and macrophages (CD 14 +). Localized nodules containing active foci of endochondral ossification were noted within the pannus. In situ hybridization identified abundant message for TNF-et (but not IL-l[3) within the inflammatory cells. In addition, message for transforming growth factor-[32 (TGF-[32) was noted in close proximity to the regions of new bone formation. The authors hypothesized that local production of growth factors by the inflammatory tissue may induce the new bone formation that gives rise to ossification and eventual bony ankylosis of the sacroiliac joints that occurs in some individuals. A similar process could account for the development of syndesmophytes that characterize the spinal pathology associated with the spondyloarthropathies. The initial lesion in the spine consists of inflammatory granulation tissue at the junction of the annulus fibrosus of the intervertebral disk and the margin of the vertebral bone. Replacement of the outer annular fibers of the intervertebral disks with bone, perhaps by mechanisms similar to those described by Braun and co-workers, TM would eventually lead to formation of the syndesmophyte with resultant ankylosis of the adjoining vertebral bodies. Despite the presence of localized regions of new bone formation associated with the formation of the syndesmophytes, many patients with spondylitis exhibit evidence of severe ostopenia of the spine. The generalized bone loss that occurs following bony ankylosis of the spine is a common feature, which is often ascribed to spinal immobility. 7~ This hypothesis, however, has been challenged. Will et al. 72 evaluated 25 patients with early ankylosing spondylitis using dual photon absorptiometry (DPA) and compared them to 25 age- and sex-matched controls. They observed a significant reduction in bone mineral density in the lumbosacral spine and hip in the spondylitis cohort, and they concluded that bone loss occurred early in disease before bony ankylosis and spinal immobility developed. They hypothesized that these observations were perhaps related to the biological effects of inflammation on bone metabolism. Ralston et al. 73 studied the issue of spontaneous insufficiency fractures in patients with ankylosing spondylitis, which hitherto had been considered a relatively rare occurrence. Of 111 consecutive patients with ankylosing spondylitis evaluated prospectively, 15 developed radiographic evidence of vertebral compression fractures. Interestingly, bone mineral density as assessed by single photon absorptiometry (SPA) of appendicular bone was normal, suggesting that osteoporosis in these patients is primarily localized to the axial spine. Devogelaer et al., TM utilizing
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radiographs, SPA, DPA, and QCT methods, observed that males with ankylosing spondylitis tended to develop significant bone loss in trabecular bone of the vertebral bodies. However, they observed an interesting but opposite trend in the appendicular skeleton. The latter observation lends credence to the hypothesis that spinal deformity with characteristic dorsal kyphosis might in fact be related to progressive anterior wedging of the vertebral bodies, secondary to trabecular osteoporosis. Both bone mineral density and insufficiency fractures in this group of patients are sometimes obscured by the bone accretion process associated with the formation of syndesmophytes.
IV. SYSTEMIC LUPUS ERYTHEMATOSUS SLE is characterized by an immunological dysregulation that results in the production of a diverse array of so-called autoantibodies. These antibodies are directed against a wide spectrum of self-molecules found in the nucleus, cytoplasm and on the surface of target cells. The pathological findings of lupus consist of inflammation of blood vessels that encompass both a bland vasculopathy and vasculitis, in part mediated by immune complex deposition. The pathological process may affect any organ system, including the articular tissues, periarticular tissues, and bone. Involvement of the skeletal tissues by this immune process may, in part, account for the relatively high frequency of osteonecrosis in individuals with SLE. Chronic use of high-dose corticosteroids is clearly a contributing factor in many patients, but the vasculopathy that accompanies this condition may also contribute to the occurrence of bone infarction. In contrast to patients with RA, the focal marginal articular erosion is not a feature of the arthropathy of SLE, despite similar histological appearance of the synovial membrane. Nevertheless, the arthritis of SLE can result in weakening of periarticular tissues leading to subluxation of joints and severe deformity (Fig. 21-5). Significant bone loss and vertebral compression fractures are well-recognized clinical complications typically observed in elderly females with SLE. Kalla et al. utilized DEXA to evaluate bone mineral density of the lumbosacral spine and hip in 46 premenopausal female patients with SLE and compared them with 108 controls. They observed a significant reduction in BMD of the lumbosacral spine and hip; however, no obvious differences could be discerned when the glucocorticoidtreated and untreated groups were compared. 75 These observations suggest that osteoporosis in SLE patients may be due to the disease itself, independent of other factors, such as menopausal status or glucocorticoid exposure.
FIGURE 21--5 Plainradiograph of systemic lupus erythematosus (Jaccoud's arthritis) with nonerosive, but severely deforming arthritis.
This is not surprising, since patients with SLE often experience severe systemic constitutional symptoms that lead to a marked reduction in the level of physical activity. Similar to patients with RA, the effects of chronic sustained systemic inflammation may also adversely affect bone remodeling, leading to reduced bone mass independent of the effects of glucocorticoids or other therapies. Further studies are needed to more firmly establish the relationship of SLE to reduced bone mass, since other investigators such as Dhillon et al. have failed to establish a linkage between SLE and reduced bone density. 76
V. JUVENILE RHEUMATOID ARTHRITIS Bone loss in children with inflammatory polyarthritis is a well-recognized though poorly understood clinical phenomenon (Fig. 21-6). It is likely due to multiple causes. Similar to adults with RA, certain "risk factors" for the development of reduced bone mass have been identified or proposed, including the effects of medications, reduced level of physical activity, dietary deficiencies, and the adverse systemic effects of inflammatory mediators and cytokines. 77
CHAPTER21 Bone Disease in Rheumatological Disorders
FIGURE 21--6 Plain radiograph of juvenile rheumatoid arthritis with severe osteopenia of the vertebral bodies.
Early description of the natural history of juvenile rheumatoid arthritis (JRA) defined the often dramatic effects of this condition on skeletal maturation, manifested both locally and generally, by retardation of linear skeletal g r o w t h . 78'79 Hillman et al. 8~provided direct evidence for disordered bone remodeling in children with JRA. They analyzed biochemical markers of bone remodeling and total body calcium in 44 children with active polyarticular or oligoarticular JRA. Their findings were consistent with a low bone formation rate with an overall reduction in bone turnover. Conclusive evidence for an increase in bone resorption rates was lacking. Hopp et al. 81 made similar observations. They studied spinal bone density in 20 children with active JRA using DPA and noted that the bone density was reduced in postpubertal girls compared with healthy controls. These observations, if confirmed, illustrate the impact of inflammation in preventing normal skeletal accretion during puberty. Adolescents with active disease may thus be particularly vulnerable to the impact of the inflammatory process on their capacity to achieve expected peak bone mass. Surprisingly, prepubertal girls did not differ from controls at any of the sites measured. In part, this could be related to the effects of treatment on the activity of
629 the disease in this group. The importance of disease activity on bone remodeling is further supported by the studies of Reed and co-workers, 82 who evaluated radial BMD repetitively over a 3-year period in children with JRA. They noted that improvement in the disease activity was associated with an increase to normal levels of serum osteocalcin. In contrast, in children with persistent joint inflammation serum osteocalcin and B MD remained below normal levels. Similar to the findings in patients with RA, children with JRA show evidence of multiple distinct patterns of bone loss, including focal marginal erosions, juxtaarticular osteopenia, and generalized osteoporosis. Although the cellular and pathological processes underlying these changes are similar in RA and JRA, the effects of the suppression of bone formation in children, especially during the pubertal growth period, may have a major adverse effect on the achievement of normal peak bone mass. This predisposes these individuals to a markedly increased fracture risk in adulthood. Studies, in fact, document the increased fracture risk in subjects with a history of J R A . 83'84 As in RA, there is evidence that several cytokines and their receptors may be implicated in the disturbance of local as well as generalized bone remodeling in children with JRA. In a prospective study of 40 patients with JRA, serum levels of soluble TNF receptor (sTNFR), soluble IL-2 receptor (slL-2R), and IL-6 correlated well with markers of inflammation (ESR, CRP) indicating an association of these cytokine receptors and IL-6 with disease activity. The highest levels of these mediators were observed in patients with active systemic disease or with polyarthritis.
A. R e f l e x S y m p a t h i c D y s t r o p h y The reflex sympathic dystrophy syndrome (RSD) is characterized by localized dependent edema, altered skin temperature (coldness or warmth), increased or decreased sweating, hyperesthesia, decreased range of motion in affected joints, and dystrophic skin changes. 85-88 RSD typically affects the distal extremities with involvement of the whole foot or hand85-89; however, occasionally, a single digit or more proximal sites such as the knee may be affected. It has been given different names such as causalgia, Sudeck's atrophy, and shoulder-hand syndrome, all of which are likely descriptors of the same general disorder. 9~ In part, the heterogeneity in the clinical picture can be explained by the tendency of this condition to progress through a series of stages. The stage at which the patient presents will therefore determine the descriptive clinical pattern.
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RSD most often occurs after injury or trauma to the affected site. 85-87 The syndrome has been divided into three stages. The first stage is characterized by burning pain, tenderness, swelling, and vasomotor changes that may last for 3 to 6 months. The second stage is characterized by persistent pain and burning, swelling, and induration with dystrophic skin changes. This stage may persist for an additional 3 to 6 months. The final stage evolves gradually and is characterized by skin and subcutaneous atrophy with the development of contractures in the affected extremity. A wide variety of precipitating factors have been associated with reflex sympathetic dystrophy. Although the mechanisms responsible for the initiation of this syndrome are not well defined, a common factor may be injury to either central or peripheral tissue, including peripheral nerve fibers. 86'9~The symptoms may begin gradually, or may manifest within a few hours of the injury. Sensitization of peripheral mechanoreceptors (and possibly pain receptors) has been implicated in the initiation of the symptoms, but additional factors must also be involved. The syndrome should be suspected when persistent and apparently disproportionately severe pain (allodynia or hyperpathia) occurs after an injury to an extremity. Radiographic changes occur in over 75% of patients. 87'92Plain radiographs initially show diffuse, patchy osteopenia that later progresses to a "ground-glass" appearance (Fig. 21-7). Fine-detail radiographs reveal
subperiosteal bone resorption, striation, and tunneling in the cortices, as well as excavations and tunneling of the endosteal surfaces. These changes have been observed in patients with hyperparathyroidism, thyrotoxicosis, and other conditions associated with increased bone turnover. These changes are not specific for reflex sympathetic dystrophy, but their presence after an injury should raise concern regarding this diagnosis. Immobilization and the decreased movement that accompanies RSD likely is responsible for these changes in bone remodeling, although other factors related to neovascular alterations may also contribute. 85 The three-phase bone scan may be more helpful in establishing the diagnosis. 93 The first phase of the scan (which reflects blood flow) and the second phase (which reflects the "blood pool") typically show asymmetrical blood flow and pooling, which probably reflects the neurovascular component of RSD. Diminished blood flow and uptake are seen in about 15% to 20% of patients. A majority of patients also have an abnormal third phase of the bone scan that reflects the " b o n e " component of the scan accompanied by increased periarticular uptake. Treatment of reflex sympathetic dystrophy is most effective when initiated in the early stages of the disorder, and the prognosis worsens following any delay. 85-88 The mainstays of therapy consist of analgesics and/or nonsteroidal antiinflammatory drugs accompanied by a physical therapy program that incorporates range-ofmotion exercises involving the affected extremity. Sys-
FIGURE 2 1--7 Plainradiograph of reflex sympathetic dystrophy (RSDS) revealing severe asymmetrical osteopenia of the left hand and wrist.
CHAPTER 21 Bone Disease in Rheumatological Disorders temic corticosteroids are useful in some patients, but should be used with caution, since their efficacy has not been firmly established. Several reports have also indicated that parenteral calcitonin may be of benefit in this condition, particularly when administered early in the course of the illness. However, these studies have not been well controlled and further evaluation is needed to establish its efficacy. 94'95 Paravertebral sympathetic blockade has been used successfully to treat many patients with RSD, and this approach is presently the most widely recommended treatment for patients who have not responded to the more conservative therapies. 87'88 Paravertebral sympathetic ganglionectomy is recommended for patients who respond to the initial transient blockade but fail to achieve successful sustained control of symptoms. Several series have established that the success rate with surgical parasympathectomy in selected patients may exceed 75%. The most important factor in predicting the response to treatment, however, is early recognition, since patients with longstanding disease are less likely to recover.
B. Regional Migratory Osteolysis Regional migratory osteolysis is an uncommon disorder that most often occurs in middle-aged m a l e s . 96-98 It is characterized by spontaneous attacks of periarticular pain and swelling, often accompanied by atrophy of adjacent muscles, affecting primarily the joints of the lower extremities. The ankle, foot, and knee joints are the most frequently affected joints. The characteristic radiographic findings are juxtaarticular osteoporosis with preservation of joint space. Bone scans reveal a striking increase in radionuclide uptake, 99 which in some cases may precede overt clinical symptoms. Synovial biopsies from affected joints demonstrate nonspecific synovitis. Laboratory tests most often are normal. Transient osteoporosis of the hip is a disorder that shares many of the clinical and radiographic features of transient migratory osteoporosis. ~~176 The condition was originally described in women with onset during the last trimester of pregnancy who presented with hip and thigh pain associated with demineralization of the femoral head without joint space narrowing. 1~ The major distinction between transient osteoporosis of the hip and regional migratory osteoporosis is the restriction of transient osteoporosis of the hip to a single joint. Both conditions tend to be self-limited and abate within less than a year without permanent joint changes. The etiology of migratory osteoporosis remains uncertain. Of interest, the radiological and scintigraphic features of this condition are similar to the findings in
631 reflex sympathetic dystrophy or so-called Sudeck's atrophy. 85-89 The absence of prior trauma and the lack of autonomic and neurovascular features, however, distinguish these disorders. Regional migratory osteolysis must be differentiated from other inflammatory and infectious disorders of the joints. Appropriate radiological, serological, and bacteriological data can be used to distinguish these conditions. Regional migratory osteoporosis must also be distinguished from so-called multicentric osteolysis. ~~176 This group of disorders consists of a complex of rare conditions that are characterized by resorption and destruction of involved bones. Five different types have been identified based on the characteristic clinical and radiological features. Of interest, two of the subtypes are associated with renal abnormalities, although the pathogenic mechanisms linking the renal and bone disease are not known. Each type has its onset in early childhood and may occur either as a hereditary or sporadic disorder. Typically, the syndrome begins as a painful swelling of the wrists and feet accompanied by progressive bone resorption affecting the carpal and tarsal bones. Although the osteolysis rarely progresses beyond adolescence, the children are often left with severe residual joint deformities. This group of disorders must be distinguished from other inflammatory forms of arthritis, especially juvenile rheumatoid arthritis. The absence of actual synovitis in the affected joints, as well as the lack of systemic features help to differentiate these disorders.
C. Osteoarthritis OA is the most common of the joint diseases. In contrast to RA and other forms of inflammatory arthritis, patients with OA lack systemic disease manifestations. The pathological changes are restricted to the joint structures, including the cartilage, subchondral bone, and synovium. This condition is characterized by progressive loss of articular cartilage, appositional new bone formation in the subchondral bony trabeculae, and formation of new bone and cartilage at the joint margins giving rise to osteophytes that are the radiological feature of OA (Fig. 21-8). Although low-grade synovitis occurs, it is most often restricted to localized regions adjacent to the joint margins and does not appear to play a primary role in the direct damage to the articular cartilage. Numerous studies have suggested that there is an inverse relationship between the risks for osteoarthritis and osteoporosis, the two most common disorders of bones and joints. 1~176 Others have failed to confirm this relationship. 1~176 The discrepancy in these results likely is related to the small number of patients studied and the absence of appropriately matched controls. In addition,
632
FIGURE 21--8 Plain radiograph of osteoarthritis (OA) revealing Heberden's and Bouchard's nodes/osteophytes.
particularly in the earlier studies, the techniques for assessment of bone mass were relatively imprecise. More recently, Hart et al. using DEXA examined the relationship between OA of the hand, hip, and spine and the bone density of the spine and femoral neck in a large population of middle-aged women. 1~ They found that there were small increases in B MD in women with early radiological evidence of OA of the hands, knees, and lumbar spine. Adjustments for confounders such as smoking, alcohol consumption, hormone replacement therapy, social class, and osteophytes did not alter the results. Nevitt et al. came to similar conclusions in a different population of women. 1~ In a cross-sectional study they examined the association between radiographic features of OA of the hip and BMD of the hip, spine, and appendicular skeleton in a population of Caucasian women 65 and older. Similar to the findings of the studies by Hart et al., they found that women with moderate to severe radiographic evidence of OA had higher BMD than women without OA. Higher BMD at both the appendicular and axial sites was associated with the presence and size of hip osteophytes; however, isolated narrowing of the hip joint without osteophytes was not associated with a higher BMD. These differences were not the result of potential confounding variables such as weight, physical activity, or the use of medica-
STF.VEN R. GOLDRING AND RICHARD P. POLISSON
tions that affect bone density. They speculated that thickening of the medial cortex and trabecular hypertrophy in the proximal femoral neck region in the women with OA might be related to a number of factors, including an altered distribution of mechanical stresses, reduced area and "shock-absorbing" capacity of cartilage, and stiffening and increased vascularization of periarticular bone. They suggested that the smaller increases in bone density in the trochanteric and intertrochanteric regions were related to a relatively smaller effect of the osteoarthritic changes on the local biomechanical factors and bone remodeling in this region of the femur. The findings of Nevitt et al. 1~ are consistent with the speculations of others that high bone mass creates a regional biomechanical environment that contributes to the pathogenesis of OA of the hip and other weight-beating joints. 1~176 According to this hypothesis, the higher bone density that is associated with a more dense, stiffer periarticular bone leads to the transfer of increased mechanical stress to the articular cartilage during joint loading (Fig. 21-9). This repeated "stress" leads to cartilage damage and eventually to an increased risk for the development and progression of OA. In the studies of Nevitt and co-workers, the local changes in bone remodeling secondary to OA of the hip did not account for the elevated BMD at sites that were remote from the hip. 1~ They were unable to account for the higher BMD in women with OA of the hip by adjusting for differences in anthropometric and other determinants of bone mass. They speculated that other confounding factors, includ-
FIGURE 21--9 Plain radiograph OA revealing bony sclerosis (white arrowhead) and joint space narrowing.
CHAPTER 21
Bone Disease in Rheumatological Disorders
ing genetic predisposition to both hip OA and higher bone mass, might be involved. An additional finding of the Nevitt study was the strong association between BMD and the size and extent of osteophytes of the hip joint. 1~ Hannan et al. 112 had noted a similar association between osteophytes of the knee joints and bone mineral density of the hip. These findings are consistent with the speculation that individuals who exhibit an increased frequency of OA have an inherent tendency to form bone at sites of tissue injury. 1~ Dequeker et al. have suggested that individuals with a propensity to OA produce increased levels of bone growth factors at skeletal sites 113 and that this may account for the association of increased generalized bone mineral density and OA. Of interest, several studies suggest that individuals with hip and knee OA have increased bone remodeling, particularly in the periarticular regions of affected joints. 114'115 It is not clear, however, whether these changes in bone turnover precede or occur in response to the abnormalities in cartilage metabolism. It is possible that local paracrine or autocrine factors produced at the sites affected by the OA process contribute to the disturbance in cartilage and bone remodeling. Identification of these "factors" could yield useful insights into the pathogenesis of OA.
References 1. Deodhar AA, Woolf AD: Bone mass measurement and bone metabolism in rheumatoid arthritis: A review. Br J Rheumatol 35:309-322, 1996. 2. Fassbender HG: Histomorphological basis of articular cartilage destruction in rheumatoid arthritis. Coil Rel Dis 3:141-155, 1983. 3. Harris EDJ: Rheumatoid arthritis. Pathphysiology and implications for therapy. N Engl J Med 322:1277-1289, 1990. 4. Krane SM, Conca W, Stephenson ML, et al: Mechanisms of matrix degradation in rheumatoid arthritis. Ann NY Acad Sci 580:340-354, 1990. 5. Woolley DE, Crossley MJ, Evanson JM: Collagenase at sites of cartilage erosion in the rheumatoid joint. Arthritis Rheum 20: 1231-1239, 1977. 6. Woolley DE, Bartholomew JS, Taylor DJ, et al: Mast cells and rheumatoid arthritis. In Galli S J, Austin KF (eds): Mast cell and basophil differentiation and function in health and disease. New York, Raven Press, 1989, pp 183-193. 7. Nguyen Q, Mort JS, Roughley PJ: Cartilage proteoglycan aggregate is degraded more extensively by cathepsin L than B. Biochem J 266:569-573, 1990. 8. Maciewicz RA, Wotton SF, Etherington DJ, et al: Susceptibility of the cartilage collagens type II, IX, and XI to degradation by the cysteine proteinases, cathepsin B and L. FEBS Lett 269: 189-193, 1990. 9. Bromley M, Fisher WD, Woolley DE: Mast cells at sites of cartilage erosion in the rheumatoid joint. Ann Rheum Dis 43: 7 6 - 7 9 , 1984.
633 10. Bromley M, Woolley DE: Chondroclasts and osteoclasts at subchondral sites of erosions in the rheumatoid joint. Arthritis Rheum 27:968- 975, 1984. 11. Bromley M, Woolley DE: Histopathology of the rheumatoid lesion; identification of cell types at sites of cartilage erosion. Arthritis Rheum 27:857-863, 1984. 12. Allard SA, Muirden KD, Maini RN: Correlation of histopathological features of pannus with patterns of damage in different joints in rheumatoid arthritis. Ann Rheum Dis 50:278-283, 1991. 13. Ashton BA, Ashton IK, Marshall MJ, et al: Localization of vitronectin receptor immunoreactivity and tartrate resistant acid phosphatase activity in synovium from patients with inflammatory and degenerative arthritis. Ann Rheum Dis 52:133-137, 1993. 14. Chang JS, Quinn JM, Demaziere A, et al: Bone resorption by cells isolated from rheumatoid synovium. Ann Rheum Dis 51: 1223-1229, 1992. 15. Chambers TJ, Thomson BM, Fuller K: Resorption of bone by isolated rabbit osteoclasts. J Cell Sci 66:383, 1984. 16. Harada Y, Wang JT, Gorn AH, et al: Identification of the cell types responsible for bone resorption in rheumatoid arthritis. Arthritis Rheum 37(suppl):S211, 1994. 17. Gorn AH, Lin HY, Yamin M, et al: The cloning, characterization and expression of a human calcitonin receptor from an ovarian carcinoma cell line. J Clin Invest 90:1726-1735, 1992. 18. Gorn AH, Rudolph SM, Flannery MR, et al: Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. J Clin Invest 95:2680-2691, 1995. 19. Rodan GA, Martin TJ: Role of osteoblasts in hormonal control of bone r e s o r p t i o n - - a hypothesis. Calcif Tissue Int 33:349351, 1981. 20. Suda T, Takahashi N, Martin TJ: Modulation of osteoclast differentiation. Endocr Rev 13:66-80, 1992. 21. Raisz LG: Local and systemic factors in the pathogenesis of osteoporosis. N Engl J Med 318:818-828, 1988. 22. Shimizo S, Shiozawa S, Shiozawa K, et al: Quantitative histological studies on the pathogenesis of periarticular osteoporosis in rheumatoid arthritis. Arthritis Rheum 28:25-31, 1985. 23. Deleuran BW, Chu CQ, Field M, et al: Localization of interleukin- let, type 1 interleukin- 1 receptor and interleukin- 1 receptor antagonist in the synovial membrane and cartilage/pannus junction in rheumatoid arthritis. Br J Rheumatol 31:801-809, 1992. 24. Firestein GS, Alcaro-Garcia JM, Maki R: Quantitative analysis of cytokine gene expression in rheumatoid arthritis. J Immunol 144:3347-3353, 1990. 25. Chu CQ, Field M, Feldman M, et al: Localization of tumour necrosis factor-a in synovial tissue and at the cartilage-pannus junction in patients with rheumatoid arthritis. Arthritis Rheum 34:1125-1132, 1991. 26. Chu CQ, Field M, Allard S, et al: Detection of cytokines at the cartilage/pannus junction in patients with rheumatoid arthritis; implications for the role of cytokines in cartilage destruction and repair. Br J Rheumatol 32:653-661, 1992. 27. Mazess RB, Whedon GD: Immobilization and bone. Calcif Tissue Int 32:265-267, 1983. 28. Joffe I, Epstein S: Osteoporosis associated with rheumatoid arthritis: Pathogenesis and management. Semin Arthritis Rheum 20:256- 272, 1991. 29. Robinson DR: Prostaglandin-stimulated bone resorption by rheumatoid synovium. J Clin Invest 56:1181, 1975. 30. Miyasaka N, Sato K, Goto M, et al: Augmented interleukin-1 production and HLA-DR expression in the synovium of rheumatoid arthritis patients. Arthritis Rheum 31:480, 1988.
634 31. Saxne T, Palladino MA, Heinegard D, et al: Detection of tumor necrosis factor-a but not tumor necrosis factor-[3 in rheumatoid arthritis synovial fluid and serum. Arthritis Rheum 31:1041, 1988. 32. Sambrook P, Nguyen T: Vertebral osteoporosis in rheumatoid arthritis. Br J Rheumatol 31:573-574, 1992. 33. Sambrook PN, Eisman A, Champion G, et al: Determinants of axial bone loss in rheumatoid arthritis. Arthritis Rheum 30: 721 - 728, 1987. 34. Sambrook P, Eisman J, Yeates M, et al: Osteoporosis in rheumatoid arthritis: Safety of low-dose corticosteroids. Ann Rheum Dis 45:950, 1986. 35. Sambrook P, Ansel B, Foster S, et al: Bone turnover in early rheumatoid arthritis; longitudinal bone density studies. Ann Rheum Dis 44:580, 1985. 36. Peel NF, Eastell R, Russell RGG: Osteoporosis in rheumatoid arthritis--the laboratory perspective. Br J Rheum 30:84-85, 1991. 37. Woolf AD: Osteoporosis in rheumatoid arthritis--the clinical viewpoint. Br J Rheumatol 30:82-84, 1991. 38. Compston J, Crawley E, Evans C, et al: Spinal trabecular bone mineral content in patients with non-steroid treated rheumatoid arthritis. Ann Rheum Dis 47:660, 1988. 39. Verstraeten A, Dequeker J: Vertebral and peripheral bone density content and fracture incidence in postmenopausal patients with rheumatoid arthritis; effects of low-dose corticosteroids. Ann Rheum Dis 45:852-857, 1986. 40. Sambrook P, Spector T, Seeman E, et al: Osteoporosis in rheumatoid arthritis: A monozygotic co-twin control study. Arthritis Rheum 38:806-809, 1995. 41. Kroger H, Arnala I, Alhava EM: Bone remodeling in osteoporosis associated with rheumatoid arthritis. Calcif Tissue Int 49: $90, 1991. 42. Mellish RWE, O'Sullivan MM, Garrahan NJ, et al: Iliac crest trabecular bone mass and structure in patients with non-steroid treated rheumatoid arthritis. Ann Rheum Dis 46:830-836, 1987. 43. Compston JE, Vedi S, Croucher PI, et al: Bone turnover in nonsteroid treated rheumatoid arthritis. Ann Rheum Dis 53:163166, 1994. 44. Hall GM, Spector TD, Delmas PD: Markers of bone metabolism in postmenopausal women with rheumatoid arthritis. Effects of corticosteroids and hormone replacement therapy. Arthritis Rheum 38:902-906, 1995. 45. Gough AK, Peel NF, Eastell R, et al: Excretion of pyridinium crosslinks correlates with disease activity and appendicular bone loss in early rheumatoid arthritis. Ann Rheum Dis 53:14-17, 1994. 46. Spector TD, Hall GM, McCloskey EV, et al: Risk of vertebral fracture in women with rheumatoid arthritis. BMJ 306:558, 1993. 47. Michel B, Block D, Fries J: Predictors of fracture in early rheumatoid arthritis. J Rheumatol 18:804-808, 1991. 48. Hooynman JR, Melton LJ, Nelson AM, et al: Fractures after rheumatoid arthritis; a population based study. Arthritis Rheum 27:1353-1361, 1984. 49. Peh WCG, Gough AKS, Sheeran T, et al: Pelvic insufficiency fractures in rheumatoid arthritis. Br J Rheumatol 32:319-324, 1993. 50. Laan RF, van-Riel PL, van-de-Putte LB: Bone mass in patients with rheumatoid arthritis. Ann Rheum Dis 51:826-832, 1992. 51. Gough AK, Lilley J, Eyre S, et al: Generalized bone loss in patients with rheumatoid arthritis. Lancet 344:23-27, 1994. 52. Als OS, Gotfredsen A, Riis BJ, et al: Are disease duration and degree of functional impairment determinants of bone loss in rheumatoid arthritis? Ann Rheum Dis 44:406-411, 1985.
STEVEN R. GOLDRING AND RICHARD P. POLISSON 53. Pocock N, Eisman J, Yeates M, et al: Physical activity is a major determinant of femoral neck and lumbar spine mineral density. J Clin Invest 78:618, 1986. 54. Magaro M, Tricerri A, Piane D, et al: Generalized osteoporosis in nonsteroid treated patients with rheumatoid arthritis. Rheumatol Int 11:73, 1991. 55. Egglemeijer F, Camps J, Valkema R, et al: Bone mineral density in ambulatory non-steroid treated female patients with rheumatoid arthritis. Clin Exp Rheumatol 11:1 - 38, 1993. 56. Fries JF, Williams CA, Ramsey DR, et al: The relative toxicity of disease-modifying antirheumatic drugs. Arthritis Rheum 44: 4 0 6 -4 1 1 , 1993. 57. Lukert BP, Raisz LG: Glucocorticoid-induced osteoporosis: Pathogenesis and management. Ann Intern Med 112:352-362, 1990. 58. Kirwan JR: The effects of glucocorticoids on joint destruction in rheumatoid arthritis. N Engl J Med 333:142-146, 1995. 59. Sambrook P, Cohen M, Eisman J, et al: Effects of low-dose corticosteroid on bone mass in rheumatoid arthritis: A longitudinal study. Ann Rheum Dis 48:535, 1989. 60. Laan R, van Riel P, van Erning L, et al: Vertebral osteoporosis in rheumatoid arthritis patients; effects of low-dose prednisone therapy. Br J Rheumatol 31:91 - 96, 1992. 61. Saag K, Rochelle K, Caldwell J, et al: Low dose longterm corticosteroid therapy in rheumatoid arthritis; an analysis of serious adverse events. Am J Med 96:115-123, 1994. 62. Friedlander GE, Tross RR, Doganis AC, et al: Effects of chemotherapeutic agents on bone. J Bone Joint Surg Am 60:602, 1984. 63. Preson SJ, Diamon T, Scott A, et al: Methotrexate osteopathy in rheumatic disease. Ann Rheum Dis 52:582, 1993. 64. MacDonald AG, Murphy EA, Capell HA, et al: Effects of hormone replacement therapy in rheumatoid arthritis: A double blind placebo-controlled study. Ann Rheum Dis 53:54-57, 1994. 65. van den Brink HR, Lems WF, van Everdingen AA, et al: Adjuvent oestrogen treatment increases bone mineral density in postmenopausal women with rheumatoid arthritis. Ann Rheum Dis 52:302-305, 1993. 66. Sambrook P, Birmingham J, Kelly P, et al: Postmenopausal bone loss in rheumatoid arthritis: Effects of estrogens and androgens. J Rheum 19:357-361, 1992. 67. Hall GM: Effect of hormone replacement therapy on bone mass in rheumatoid arthritis. Arthritis Rheum 37:1770-1773, 1994. 68. Kroger H, Arnala I, Alhava EM: Effect of calcitonin on bone histomorphometry and bone metabolism in rheumatoid arthritis. Calcif Tissue Int 50:11 - 13, 1992. 69. Egglemeiger F, Papapoulos S, van Paassen HC, et al: Increased bone mass with pamidronate treatment in rheumatoid arthritis. Arthritis Rheum 39:396-402, 1996. 70. Braun J, Bollow M, Neure L, et al: Use of immunohistologic and in situ hybridization techniques in the examination of sacroiliac joint biopsy specimens from patients with ankylosing spondylitis. Arthritis Rheum 4:499-505, 1995. 71. Spencer D, Park W, Dick H, et al: Radiologic manifestations in 200 patients with ankylosing spondylitis; correlations with clinical features and HLA-B27. J Rheumatol 6:305, 1979. 72. Will R, Bhalla A, Palmer R, et al: Osteoporosis in early ankylosing spondylitis; a primary pathological event? Lancet 23: 1483-1485, 1989. 73. Ralston SH, Urquhart GD, Brzeski M, et al: Prevalence of vertebral compression fractures due to osteoporosis in ankylosing spondylitis. Br Med J 300:563-565, 1990. 74. Devogelaer J-P, Maldague B, Malghem J, et al: Appendicular and vertebral bone mass in ankylosing spondylitis. A compari-
CHAPTER 21
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son of plain radiographs with single- and dual-photon absorptiometry and with quantitative computed tomography. Arthritis Rheum 35:1062-1067, 1992. Kalla AA, Fataar AB, Jessop SJ, et al: Loss of trabecular bone mineral density in systemic lupus erythematosus. Arthritis Rheum 36:1726-1734, 1993. Dhillon VB, Davies MC, Hall ML, et al: Assessment of the effect of oral corticosteroids on bone mineral density in systemic lupus erythematosus; a preliminary study with dual energy xray absorptiometry. Ann Rheum Dis 49:624-626, 1990. Cassidy JT, Langman CB, Allen S, et al: Bone mineral metabolism in children with juvenile rheumatoid arthritis. Pediatr Clin North Am 42:1017-1033, 1995. Ansell BM, Bywaters EGL: Growth in Still's disease. Ann Rheum Dis 15:295-318, 1956. Bernstein BH, Stobie D, Singsen BH, et al: Growth retardation in juvenile rheumatoid arthritis (JRA). Arthritis Rheum 29: 212-216, 1977. Hillman L, Cassidy JT, Johnson L, et al: Vitamin D metabolism and bone mineralization in children with juvenile rheumatoid arthritis. J Pediatr 124:910-916, 1994. Hopp R, Degan JA, Gallagher JC, et al: Estimation of bone mineral density in children with juvenile rheumatoid arthritis. J Rheumatol 18:1235-1239, 1991. Reed A, Haugen M, Pachman L, et al: Repair of osteopenia in children with juvenile rheumatoid arthritis. J Pediatr 122:693696, 1993. Badley BWD, Ansell BM: Fracture's in Still's disease. Ann Rheum Dis 19:135-138, 1960. Varonos S, Ansell BM, Reeve J: Vertebral collapse in juvenile chronic arthritis; its relationship with glucocorticoid therapy. Calcif Tissue Int 41:75-78, 1987. Kozin F, McCarty DJ, Sims J, et al: The reflex sympathetic dystrophy syndrome. I. Clinical and histological studies: Evidence for bilaterality, response to corticosteroids and articular involvement. Am J Med 60:321-331, 1976. Kozin F: Reflex sympathetic dystrophy syndrome. Bull Rheum Dis 36:1-7, 1986. Schwartzman RJ, McLellan TL: Reflex sympathetic dystrophy: A review. Arch Neurol 44:555-561, 1987. Schutzer SF, Gossling HR: Current concepts review: The treatment of reflex sympathetic dystrophy. J Bone Joint Surg 66A: 625-629, 1984. Silber TJ, Maid M: Reflex sympathetic dystrophy syndrome in children and adolescents. Am J Dis Child 142:1325-1330, 1988. Steinbrocker O, Argyros TG: The shoulder-hand syndrome: Present status as a diagnostic and therapeutic entity. Med Clin North Am 42:1533-1553, 1958. Stanton RP, Malcolm JR, Wesdock KA, et al: Reflex sympathetic dystrophy in children: An orthopedic perspective. Orthopedics 16:773-780, 1993. Goldsmith DP, Vivivo FB, Eichenfield AH, et al: Nuclear imaging and clinical features of childhood reflex neurovascular dystrophy: Comparison with adults. Arthritis Rheum 32:480485, 1989. Macinnon SE, Holder LE: The use of three-phase radionuclide scanning in the diagnosis of reflex sympathetic dystrophy syndrome. J Hand Surg 9A:556-563, 1984.
635 94. Bickerstaff DR, Kanis JA: The use of nasal calcitonin in the treatment of post-traumatic algodystrophy. Br J Rheumatol 30: 291-294, 1991. 95. Gobolet C, Meier JL, Schaffner W, et al: Calcitonin and reflex sympathetic dystrophy syndrome. Clin Rheumatol 5:382-388, 1986. 96. Naides S, Resnick D, Zvaifler N: Idiopathic regional osteoporosis. J Rheumatol 12:763-768, 1984. 97. Banas MP, Kaplan FS, Fallon MD, et al: Regional migratory osteoporosis. Clin Orthop Rel Res 250:303-309, 1988. 98. Gupta RC, Popovtzer MM, Huffer WE, et al: Regional migratory osteoporosis. Arthritis Rheum 16:363- 368, 1973. 99. Bray ST, Partain CL, Teates CD: The value of the bone scan in idiopathic regional migratory osteoporosis. J Nucl Med 20: 1268-1271, 1979. 100. Schapira D: Transient osteoporosis of the hip. Semin Arthritis Rheum 22:98-105, 1992. 101. Curtis PH, Kincaid WE: Transitory demineralization of the hip in pregnancy: A report of three cases. J Bone Joint Surg Am 4: 1327-1333, 1959. 102. Hardegger F, Simpson LA, Segmeller G: The syndrome of idiopathic osteolysis. J Bone Joint Surg 67B:89-93, 1985. 103. Warady BA, Haug SJ, Lindsley CB: Multicentric osteolysis: An infrequently recognized renal-rheumatologic syndrome. J Rheumatol 18:142-145, 1991. 104. Dequeker J: The relationship between osteoporosis and osteoarthritis. Clin Rheum Dis 11:271 - 296, 1985. 105. Hart DJ, Mootoosamy I, Doyle DV, et al: The relationship between osteoarthritis and osteoporosis in the general population: The Clingford Study. Ann Rheum Dis 53:158-162, 1994. 106. Nevitt MC, Lane NE, Scott JC, et al: Radiographic osteoarthritis of the hip and bone mineral density. Arthritis Rheum 38:560562, 1995. 107. Cooper C, Cook PL, Osmond C, et al: Osteoarthritis of the hip and osteoporosis of the proximal femur. Ann Rheum Dis 50: 540-542, 1991. 108. Reid DM, Kennedy NSJ, Smith MA, et al: Bone mass in nodal primary generalized osteoarthrosis. Ann Rheum Dis 43:240242, 1984. 109. Price T, Hesp R, Mitchell R: Bone density in generalized osteoarthritis. J Rheum 14:560-562, 1987. 110. Radin EL, Rose RM: Role of subchondral bone in the initiation and progression of cartilage damage. Clin Orthop 213:34-40, 1986. 111. Radin EL, I1 P, Rose RM: Role of mechanical factors in the pathogenesis of primary osteoarthritis. Lancet 1:519-521, 1972. 112. Hannan MT, Anderson JJ, Zhang Y, et al: Bone mineral density and knee osteoarthritis in elderly men and women: The Framingham Study. Arthritis Rheum 36:1671-1680, 1993. 113. Dequeker J, Mohan S, Finkelman RD, et al: Generalized osteoarthritis associated with increased insulin-like growth factor types I and II and transforming growth factor 13 in cortical bone from the iliac crest; possible mechanism of increased bone density and protection against osteoporosis. Arthritis Rheum 36: 1702-1708, 1993. 114. MacDonald AG, McHenry P, Robins SP, et al: Relationship of urinary pyridinium crosslinks to disease extent and activity in osteoarthritis. Br J Rheumatol 33:16-19, 1994. 115. Dieppe P, Cushnaghan J, Young P, et al: Prediction of the progression of joint space narrowing in osteoarthritis of the knee by bone scintigraphy. Ann Rheum Dis 52:557-563, 1993.
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2 H A P T E R 2~
Hypercalcemia of Malignancy GREGORY R.
MUNDY
University of Texas Health Science Center, San Antonio, Texas 78284
I. Pathophysiology A. Types of Malignancies Associated with Hypercalcemia B. Pathophysiology of Hypercalcemia C. Hematological Malignancies D. Solid Tumors with Metastasis E. Solid Tumors Associated with Hypercalcemia and without Metastasis
Hypercalcemia is a very frequent complication of malignant disease. It occurs in ---30% of patients with advanced breast cancer and possibly up to 20% of patients with lung cancer, the two most common forms of malignant disease seen in Western communities. This chapter reviews the pathophysiology, the types of malignant disease that are associated with hypercalcemia, the clinical features of hypercalcemia associated with malignant disease, and finally the management of patients with hypercalcemia associated with malignancy.
I. P A T H O P H Y S I O L O G Y
A. Types of Malignancies Associated with Hypercalcemia
II. Clinical Features III. Treatment A. Indications for Treatment B. Choice of Treatment Acknowledgments References
between the presence of bone metastases and the occurrence of hypercalcemia. For example, tumors of the gastrointestinal tract and female genitalia are frequently associated with metastatic bone disease but rarely are associated with hypercalcemia. The tumors most frequently associated with hypercalcemia are squamous cell carcinomas of the lung, head, and neck; breast cancer of all varieties; hematological malignancies including myeloma and T-cell lymphomas; renal cancer; and some unusual malignancies such as the VIPomas and cholangiocarcinomas. Unlike other ectopic hormone syndromes, hypercalcemia is rare in oat cell carcinoma of the lung. The relative frequencies of the types of malignant disease associated with hypercalcemia are listed in Table 2 2 - 1 .
B. Pathophysiology of Hypercalcemia
Although hypercalcemia is seen frequently in common malignancies such as lung cancer and breast cancer, there are other common forms of malignancies in which hypercalcemia is rare. 1 There is not a direct relationship METABOLIC BONE DISEASE
Hypercalcemia associated with malignant disease is due to a combination of complex pathophysiological
637
Copyright 9 1998 by Academic Press. All rights of reproduction in any form reserved.
638
GREGORY R. MUNDY
TABLE 22--1 Malignancies Associated with Hypercalcemia Types of Malignancy
Percentage of Total
Lung Breast Hematological (myeloma, lymphoma) Head and neck Renal Prostate Unknown primary Others
35 25 14 6 3 3 7
calcemia is due primarily to an increase in bone resorption, which is due to the production of factors that stimulate osteoclast activity. These factors vary with different types of tumors. The syndrome of hypercalcemia of malignancy can be divided arbitrarily into three clinical groups on the basis of the differing pathogenic mechanisms that are responsible for increasing the entry of calcium from bone into the extracellular fluid, although this classification is overly simplistic and there is overlap among the groups.
C. H e m a t o l o g i c a l M a l i g n a n c i e s events affecting fluxes of calcium across the skeleton, kidney, and gut. The primary cause is the release of calcium from bone into the extracellular fluid by increased osteoclastic bone resorption. However, it is also clear that renal factors are important. Under most circumstances, increased entry of calcium from bone will be counterbalanced by enhanced renal excretion so that homeostatic control of the extracellular fluid calcium concentration is maintained. However, in malignant disease, a number of events occur in the kidney that interfere with this homeostatic regulation of calcium. These include (1) volume depletion, with consequent impairment of glomerular filtration rate, which in turn leads to increased renal tubular sodium and calcium reabsorption in the proximal convoluted tubules; and (2) increased renal tubular calcium reabsorption independent of volume depletion. Many clinical studies have shown that there is increased renal tubular reabsorption of calcium for any level of the serum calcium in essentially all patients with hypercalcemia of malignancy, whether it is due to humoral mechanisms or to local osteolytic bone disease. 2 The mechanism for the increase in renal tubular calcium reabsorption is the only issue in doubt. In many patients, it may be related to increased production of the parathyroid hormone-related protein (PTHrP). However, some patients, most notably those with myeloma, hypercalcemia occurs without increased circulating PTHrP. They also have increased renal tubular calcium reabsorption 2 and, in these patients, the mechanism is unknown. The relative importance of these renal effects varies from tumor to tumor. In myeloma, hypercalcemia is an unusual occurrence in the absence of impaired glomerular filtration. However, in solid tumors associated with hypercalcemia, glomerular filtration is usually adequate, and increased renal tubular calcium reabsorption occurs, because of the effects of tumor-derived systemic factors such as PTHrP. 3'4 Although these renal effects may be important in enhancing hypercalcemia, it is unlikely that renal calcium reabsorption alone can produce hypercalcemia. Hyper-
1. MYELOMA In myeloma, osteolytic bone lesions are almost invariable, and hypercalcemia occurs in 20% to 40% of patients. As already indicated, hypercalcemia rarely occur unless the patient has impaired glomerular filtration. Patients with myeloma are predisposed to renal failure because of Bence Jones Proteinuria, urinary tract infection, and uric acid nephropathy. The bone destruction is due to local osteotropic cytokines released by the myeloma cells. 5'6 Release of these factors results in activation of adjacent osteoclasts, which in turn are responsible for resorbing the bone and causing the characteristic bone lesions. In myeloma, one of the major mediators responsible for increased osteoclast activity may be lymphotoxin. Myeloma cells express lymphotoxin messenger RNA and release lymphotoxin biological activity. 7 They also release bone resorbing activity, which can be partially inhibited by neutralizing antibodies to lymphotoxin. Lymphotoxin itself causes hypercalcemia when infused into normal intact m i c e . 7 Interleukin-113 (IL-1 [3) and IL6 have also been implicated in the bone destruction of myeloma based on in vitro studies, 8'9 and increases in PTHrP noted in clinical studies. 1~Both IL-1 ot and PTHrP have been implicated in the hypercalcemia that occurs in some T-cell lymphomas as well as solid tumors. 11'12 The majority of T-cell lymphomas express PTHrP and cause a syndrome identical to humoral hypercalcemia of malignancy (HHM) (see later). Since the observation that myeloma cells mediate bone destruction by the stimulation of osteoclasts, attention has been focused on the identification of the presumed soluble factors produced by myeloma cells that are responsible. However, the interactions between myeloma cells and bone cells may be much more complicated. Recent data have suggested that myeloma cells may grow so well in bone compared with other hematological malignancies because of products produced as a consequence of osteoclastic bone resorption, and in
CHAPTER 22 Hypercalcemia of Malignancy particular the cytokine IL-6, which is a major growth regulatory factor for myeloma cells. Thus, bone may not be simply a passive bystander in this disease, but rather an active participant as the source of the myeloma cell stimulant. If this concept is correct, then a vicious cycle may exist between myeloma cells and osteoclastic bone resorption whereby myeloma cells stimulate osteoclasts to resorb bone by the production of osteotropic cytokines such as lymphotoxin, IL-l[3 and IL-6, but the cytokine IL-6 is generated in prodigious amounts by cells involved in the resorption process and this enhances the growth of myeloma cells in bone. This vicious cycle could mean that the greater the bone destruction, the more aggressive the behavior of myeloma cells, which then may cause even greater bone destruction. This concept is based on the recent information that osteoclasts produce very large amounts of IL-6, certainly more than other types of bone cells. 13'14 The only cells that produce comparable amounts of IL-6 are endometrial cells. 2. LYMPHOMAS
Some types of lymphomas are also associated with bone lesions and hypercalcemia. 15 A syndrome of adult T-cell lymphoma has been reported in which bone lesions and hypercalcemia have been an almost universal finding. 16 This condition has occurred in clusters in Japan, the West Indies, and the southern United States. The disease is due to a human type C retrovirus that is carried by mature T. lymphocytes of the helper-inducer type. In many of these tumors, particularly those in Japan, it has been shown that the lymphoma cells have the capacity to secrete PTHrP and the patients have increased serum PTHrP concentrations and other features of the HHM syndrome. 17 It also appears that some tumors have the capacity to synthesize 1,25-dihydroxyvitamin D [1,25(OH)2D], a powerful bone-resorbing factor, which could also play a role in the osteolytic bone lesions seen in this condition. TM Some patients have increased serum 1,25(OH)2D. 19-22 It is unclear what the relationship is between the lymphokine produced by these cells and the factor produced by myeloma cells, which are the neoplastic progeny of a different subset of lymphocytes. Occasionally other types of lymphomas that are not associated with known viruses cause hypercalcemia. The mechanism in these conditions remains to be clarified.
D. Solid Tumors with Metastasis In many patients with sive bone destruction due Many tumors may cause with bone metastasis, but
hypercalcemia there is extento osteolytic bone metastasis. hypercalcemia in association the tumor most frequently as-
639 sociated with osteolytic bone destruction and hypercalcemia is breast cancer. Although it is possible that in some of these patients hypercalcemia is due to a humoral systemic factor produced by the tumor cell and the presence of the metastases is irrelevant, it is clear that in some cases the extent of bone metastasis is related to the hypercalcemia. This is most apparent in breast cancer, in which hypercalcemia almost never occurs early in the course of the disease or in the absence of extensive bone metastasis. 1 The cellular mechanism responsible for local destruction of bone by tumor cells has been a controversial issue for some years. Although definitive proof is still not available, it is likely that the predominant mechanism for bone destruction is an increase in osteoclast activity. The alternative possibility is that tumor cells destroy bone directly independent of osteoclasts. 23 The evidence in favor of osteoclastic bone resorption being the predominant mechanism is twofold. First, when looked for carefully using techniques such as scanning electron microscopy, distinctive osteoclast resorption lacunae are universally present. 24 Such studies show no evidence of smaller resorption lacunae corresponding to the size of tumor cells. Second, drugs that effectively inhibit osteoclast activity such as the bisphosphonates, plicamycin, and gallium nitrate work very effectively in the hypercalcemia of malignancy. Since these drugs work through the inhibition of osteoclast function, these data suggest that osteoclasts are the major (possibly sole) mediator of the bone destruction. An important issue then is the mechanism by which osteoclasts are stimulated in patients with cancer. Osteoclasts are stimulated by a variety of factors, some of which are produced directly by the tumor cells, and some by host cells (Table 2 2 - 2 ) . The mechanisms by which tumor cells metastasize to bone surfaces have not been clearly defined, although in vitro techniques have been developed to clarify some of the molecular mechanisms involved. 25-27 Tumor cells metastasize by being shed from the primary site, possi-
TABLE 22--2 Factors Associated with Osteoclast Stimulation in Malignancy PTHrP PTH Interleukin-1oL Interleukin-1 [3 Interleukin-6 Lymphotoxin Tumor necrosis factor Prostaglandins of E series Transforming growth factor-or Vitamin D metabolites
640 bly related to the production of proteolytic enzymes by the tumor tissue itself. Once shed from the primary site, tumor cells can enter the circulation and then spread to vascular organs such as the red bone marrow. Once within the marrow cavity, the tumor cells migrate through wide-channeled marrow sinusoids. They may be attracted out of the sinusoids toward endosteal bone surfaces to cause bone destruction by the release of local osteoclast-stimulating agents or by direct effect of the tumor cells themselves. We have found in vitro that tumor cell migration can be enhanced by the conditioned medium that is harvested from resorbing or remodeling bone. z5'26 In addition, tumor cell migration c~in be stimulated by fragments of type 1 collagen, which are chemotactic for tumor cells. 27 It is possible that these fragments of collagen could be released from bone by the action of tumor cells at endosteal bone surfaces and cause the attraction of the tumor cells toward bone, where they could then, in turn, stimulate resorption either by a direct effect of the tumor cells themselves as indicated previously or by releasing local factors that stimulate osteoclasts that lie along endosteal bone surfaces. There are many other molecular mechanisms that have recently been implicated in the metastasis of tumor cells to the skeleton. Many of these insights have come through the use of the technique first described by Arguello et al. 28 and modified by Nakai et al. 29 In this system, human tumor cells are inoculated directly into the left ventricle of the heart of nude mice. During the following 4 weeks, the mice develop characteristic osteolytic lesions adjacent to tumor cells with similar morphology to that seen in patients with bone metastases. Yoneda and colleagues 3~ have recently clarified some of the important molecular interactions that occur between tumor cells and the host microenvironment during this process of tumor metastasis and the housing of tumor cells in the skeleton. These include expression by the tumor cells of matrix metalloproteinases, the src protooncogene, and the cell attachment molecule Ecadherin. 3~Each of these represents a potential molecular target for the development of drugs to inhibit the metastatic process. It is clear that much work needs to be done to understand the cellular and molecular events by which tumor cells are selectively attracted toward bone in patients with metastatic disease and then how they are capable of destroying bone to produce the osteolytic deposit. Bone provides a very favorable niche for tumor cells and it is clear that in this microenvironment many tumors grow very well. In part, the reason may be that bone is a large repository for growth regulatory factors. 31 In particular, bone is rich in transforming growth factor[3 (TGF-[3), but also stores other growth regulatory fac-
GREGORY R. MUNDY
tors that may act as tumor growth factors including bone morphogenetic proteins, heparin-binding fibroblast growth factors, platelet-derived growth factors, and insulin-like growth factors I and II. These factors may be made available locally when bone is resorbed. This has been shown best in the case of TGF-[3, which is produced in active form when bone is resorbed. 32 TGF[3 may alter the behavior of many tumor cells, and in particular breast cancer cells, to enhance the production of PTHrP. 33 Recent data indicate that PTHrP may play a critical role in the process of metastasis of some solid tumors to bone, and particularly in osteoclast activation. In breast cancer, PTHrP is expressed much more frequently when the tumor is resident in bone than it is in the primary site or in soft tissue. 34 Recently, it has been found that in a model of human breast cancer-mediated osteolysis in bone, (1) neutralizing antibodies to PTHrP prevent and reverse bone osteolysis 35 and (2) tumor cells that are nonresponsive to TGF-[3 have reduced capacity to produce PTHrP and cause bone osteolysis. 36 Although it is not clear how common this phenomenon is, the data of Powell et a l . 34 suggest it may be very frequent in breast cancer. Overall, the results are consistent with the concept that active TGF-[3 produced in the bone microenvironment enhances PTHrP production by breast cancer cells, which in turn causes increased bone resorption, leading to further increased bone osteolysis and TGF-[3 production. The lack of suitable animal models has severely limited the study of bone metastasis. Although this has slowed the accumulation of knowledge in the pathophysiology of bone metastasis, it has been less of a problem in investigating other types of metastases and in particular lung metastases. 37 Arguello et al. 28 devised a technique where tumor cells are injected directly into the left ventricle of mice. This leads to the colonization of bone in regions containing hematopoietic bone marrow by appropriate tumor cells with potential to metastasize to bone. This leads to multiple lytic lesions resembling those seen in patients with cancer. In these models, bone metastasis occurs when tumor cells have ready access to the arterial circulation. As indicated earlier, we have modified this model by the use of human tumor cells in nude m i c e , 29 and concentrated particularly on human breast cancer cells. 38
E. Solid Tumors Associated with Hypercalcemia and without Metastasis Hypercalcemia occurs in some cases in the absence of bone metastasis or in the presence of minimal bone metastasis. In these situations it is clear that the tumor
CHAWrEg 22 Hypercalcemiaof Malignancy cells are producing a humoral factor that stimulates osteoclastic bone resorption to increase the entry of calcium into the extracellular fluid. This is the syndrome of HHM, characterized by increased PTHrP and increased generation of nephrogenous cyclic adenosine monophosphate (cAMP). The major factor responsible for this syndrome is PTHrP, although increased production of PTHrP cannot account for all of the features. There is a great amount of current research aimed at identifying and characterizing the humoral factors responsible for increasing osteoclastic bone resorption. A number of candidates have been suggested over the years. 1. PARATHYROID HORMONE-RELATED PROTEIN
Most solid tumors associated with hypercalcemia secrete a PTHrP. PTHrP has been purified, 39-41 molecularly cloned, 42'43 and tested extensively in vitro and in vivo. 44-47 This protein binds to and activates the PTH receptor 41 and seems to have all the biological effects of PTH. It causes hypercalcemia after single bolus injection, repeated injections, o r i n f u s i o n s . 44-47 It also stimulates bone resorption in vitro and in v i v o 44'47 and enhances renal tubular calcium reabsorption. 44'47 It has also been identified in keratinocytes, 48 in placentas, in sheep fetal parathyroid glands, 49 and in rat lactating breast. 5~ Neutralizing antibodies to this factor have lowered the plasma calcium concentration in some animal models of hypercalcemia of malignancy. 51 Despite its biological equivalency to PTH, it does not account for all of the features of humoral hypercalcemia of malignancy. For example, decreased bone formation, decreased 1,25(OH)2D production, and metabolic alkalosis are features of the HHM but not of primary hyperparathyroidism. Our notion is that those features of HHM that cannot be explained by PTHrP excess are due to other factors that are produced together with PTHrP. It appears likely that the complete syndrome of HHM is caused by multiple mediators working in concert. For example, transforming growth factor-cx (TGF-cx) is frequently produced by the same tumors that produce PTHrP and may be responsible for those features of the hypercalcemic syndrome that cannot be ascribed to PTHrP. 52 It is now apparent that some of these factors, such as TGF-cx, tumor necrosis factor (TNF), TGF, and IL-1, can modulate bone cell and renal tubular cell response to PTH and P T H E P . 53'54 PTHrP was first discovered using in vitro assays for PTH in tumors derived from lung, breast, and the kidney. 39-41 PTHrP is now known to be expressed by many squamous cell carcinomas and has also been described in T-cell lymphomas. 17 It is a 141-amino-acid peptide that bears some homology to parathyroid hormone in the first 13 amino acids. It binds to and activates the PTH receptor, and this is presumably the reason it mimics the biological effects of PTH on bone, kidney,
641 and the gut. It is now known that it is produced by about 50% of patients with breast cancer, and its production may be enhanced at the bone site by factors that have not yet been clearly determined. 34 Radioimmunoassays have been developed for PTHrP, although these assays have not shown a perfect relationship between the presence and severity of hypercalcemia and expression of the protein. 4~ PTHrP may have roles other than as a mediator of cancer hypercalcemia that have not yet been clearly determined. It has been found in lactating rat breast tissue as well as in human milk. 5~ It has been suggested that it may be involved in placental calcium transport. 49 Recently, PTHrP has been demonstrated in increased amounts in the amniotic fluid and produced by amniotic cells. 57 Its expression in amniotic cells decreases when the membranes rupture. It may have a role as a smooth muscle relaxant in this situation and be involved in labor and delivery. On the basis of these studies, it is possible that it may be an important hormone during embryonic life that is important for calcium transport in the fetus. Recently, in an attempt to study the normal physiological functions of PTHrP, a null mutation was introduced into the PTHrP gene by targeted disruption in embryonic stem cells followed by homologous recombination. 58 Resultant homozygous mice deficient in PTHrP expression did not survive embryonic life. These mice showed marked abnormalities at the growth plate. Thus, PTHrP expression may also be important for normal endochondral bone formation. 2. TRANSFORMING GROWTH FACTORS
Transforming growth factors are a family of polypeptide stimulators of cell growth and replication that have biological properties that overlap with those of epidermal growth factor (EGF). 59 These polypeptides are acid-soluble and acid-stable compounds that are probably produced by all tumors. They represent a family of factors and not all have the same biological properties. However, the one property that they all have in common is their capacity to maintain the transformed phenotype in cells that are normally nonneoplastic. This is assayed in vitro by their capacity to stimulate soft agar colony formation of fibroblasts, which do not grow otherwise in anchorage-independent conditions. 6~ We have studied purified preparations of the transforming growth factors and found that they are powerful stimulators of osteoclastic bone resorption. Transforming growth factors belong to two general classes, one that depends on occupancy of the EGF receptor for its biological effects (TGF-00, 61 and one that functions independently of the EGF receptor (TGF-~). 62 TGF-oL is produced by many solid tumors including breast carcinomas. It is frequently produced in associa-
642 tion with PTHrP. TGF-oL is a powerful stimulator of osteoclast formation and osteoclastic bone resorption. 63-66 Its effects on osteoclastic bone resorption may be exerted primarily on osteoclast progenitors. 67 TGF-oL also causes increased bone resorption and hypercalcemia in v i v o . 52 This has been shown in nude mice inoculated with Chinese hamster ovarian cells overexpressing TGF-oL. Its effects on hypercalcemia are enhanced by PTHrP. 52 3. PARATHYROID HORMONE
It is clear that PTH cannot account for the hypercalcemia of malignancy associated with the vast majority of nonparathyroid tumors. In most current immunoassays, immunoreactive parathyroid hormone concentrations are suppressed. 1'68 Moreover, a specific cDNA probe for parathyroid hormone that employs base pair hybridization of cloned PTH DNA to detect mRNA for parathyroid hormone shows that most tumors associated with the hypercalcemia of malignancy do not contain detectable PTH mRNA. 69 Nevertheless, there are now several well-documented reports of nonparathyroid tumors expressing PTH. 7~ These tumors were a small cell cancer, a neuroectodermal tumor and an ovarian carcinoma. One possible mechanism demonstrated in one of these tumors 7~ was a DNA arrangement in the region of the promoter. 4. PROSTAGLANDINS
Although prostaglandins may be responsible for increased bone resorption occurring in some patients with metastatic cancer, it is unlikely that they could be responsible for the humoral hypercalcemia associated with human malignancies. No circulating bone-resorbing prostaglandin has been described that is powerful enough to induce hypercalcemia. Moreover, prostaglandin production by tumors does not correlate well with calcium levels in those situations in which a humoral mechanism is responsible. Nevertheless, it is possible that prostaglandins of the E series can be responsible for the hypercalcemia that has been described in several animal m o d e l s . 73'74 Drugs that inhibit prostaglandin synthesis such as indomethacin are generally ineffective in the treatment of cancer associated with hypercalcemia. 75 5. COLONY-STIMULATINGACTIVITY Recently, some workers have shown that tumors associated with hypercalcemia also produce factors that have colony-stimulating activity for cells of the monocyte-macrophage family. 76-79 Nude mice carrying these tumors may develop leukocytosis, and it has been postulated that a colony-stimulating factor that could affect cells of the monocyte-phagocyte series (the pre-
GREGORYR. MUNDY sumed precursors of osteoclasts) could also be associated with leukocytosis as well as increased osteoclast activity and hypercalcemia. Yoneda and colleagues have recently shown the association between leukocytosis and hypercalcemia in a number of patients with squamous cell carcinomas. 8~ The mechanisms responsible for the leukocytosis are multiple and include production by the tumor cells of colony-stimulating factors of the granulocyte-macrophage, monocyte, and granulocyte varieties. These factors may each have been produced alone or in combination, and have now been demonstrated in a number of tumor m o d e l s . 79'83-86"
II. C L I N I C A L
FEATURES
The clinical features of hypercalcemic patients with malignant disease are similar to the clinical features associated with hypercalcemia due to other causes. These have been well described before on numerous occasions. The most outstanding clinical features of hypercalcemia are neurological and gastrointestinal. The neurological features include lethargy, confusion, nightmares, stupor, and in extreme cases coma followed by death. The most prominent gastrointestinal features are nausea and vomiting associated with anorexia and occasionally constipation. Constipation is a much rarer symptom in patients with malignancy than it is in patients with primary hyperparathyroidism. Other organ systems may also be affected, particularly the urinary tract. Hypercalcemia is always associated with loss of concentrating ability and insensitivity to antidiuretic hormone. In some patients there may also be impaired renal function, particularly when the serum phosphorus is high or when the patient has myeloma. Renal stones, which are common in patients with hyperparathyroidism, are rare in patients with the hypercalcemia of malignancy, presumably because hypercalcemia is not of longstanding enough duration for this complication to occur. Although the clinical features of hypercalcemia due to malignancy are similar to the features of hypercalcemia due to other causes, nevertheless there are some special features of hypercalcemia associated with malignancy. Hypercalcemia may develop very rapidly, and for this reason symptoms may be apparent at relatively lower serum calcium concentrations. For instance, patients with primary hyperparathyroidism of longstanding duration may tolerate a serum calcium that is in the range of 15 mg/dl without noticeable symptoms. In contrast, patients with malignant disease who have been normocalcemic but develop hypercalcemia very rapidly may become symptomatic with much lesser elevations in the serum calcium. Another point is that the symptoms of hypercalcemia that are so distressing for the patient,
CHAPTER22 Hypercalcemiaof Malignancy in particular gastrointestinal symptoms, may be confused with other causes in patients with malignant disease. For example, patients treated with cytotoxic drug therapy or irradiation therapy frequently develop nausea and vomiting, and the symptoms caused by hypercalcemia can be confused with those due to these therapies.
III. T R E A T M E N T A. Indications for Treatment Patients in whom the serum calcium is greater than 13 mg/dl or patients who are symptomatic should be treated urgently. Asymptomatic patients with corrected total serum calcium less than 12 mg/dl should be followed very carefully. In the hypercalcemia of malignancy, hypercalcemia is progressive and may develop very rapidly over a period of a few weeks. This contrasts with primary hyperparathyroidism, in which hypercalcemia may remain constant for many years. This observation influences our treatment decision. Even in asymptomatic patients with minor elevations in the serum calcium, we advise treatment, since it is likely that more severe and possibly life-threatening hypercalcemia will develop rapidly. Similar elevations in the serum calcium in primary hyperparathyroidism may not cause any symptoms if the serum calcium has been constant for years, and in that situation active treatment may not be necessary.
B. Choice of Treatment The treatment of hypercalcemia of malignancy may be considered under two categories: 1. Urgent treatment for those patients who are symptomatic or who have a serum calcium greater than 13 mg/dl. 2. Chronic treatment, for those patients who have mild hypercalcemia and are ambulant. Before therapy for hypercalcemia is commenced, treatment of the underlying malignancy should be considered. In many patients and particularly in patients with myeloma, chemotherapy that is effective against the underlying malignancy will relieve the hypercalcemia. In addition, any drugs that can potentiate or precipitate hypercalcemia, such as thiazide diuretics, estrogens, or antiestrogens (in the case of breast cancer), should be discontinued, and immobilization, which enhances bone loss, should be avoided if possible.
643 1. URGENT TREATMENT FOR HYPERCALCEMIA Intravenous saline is recommended because a sodium diuresis is accompanied by a calcium diuresis. Saline should be delivered with some care because some patients may become hypernatremic, particularly if they are obtunded and do not have an intact thirst mechanism. The pathophysiology of saline-induced hypernatremia is probably related to the loss of concentrating ability and relative resistance to antidiuretic hormone that occurs with hypercalcemia. This results in continued excretion of free water and subsequent increase in the serum sodium concentration when large volumes of isotonic saline are administered, particularly to the obtunded patient who does not have normal thirst sensation. Hypernatremia can be readily reversed by the administration of hypotonic fluids. One of the most rapid and effective ways of treating hypercalcemia urgently is to use a combination of calcitonin and pamidronate. 87 The maximal effects of pamidronate are usually delayed for 48 to 72 hours, whereas calcitonin acts within 12 hours. The combination is very effective, since calcitonin acts to rapidly lower the serum calcium although the effect is not sustained. By the time that escape is occurring from calcitonin therapy, pamidronate is effective. Calcitonin can be used in doses of 200 to 400 MRC units intramuscularly or subcutaneously every 12 hours, and pamidronate as a single infusion of 90 mg/kg body weight given over 4 hours. Calcitonin and glucocorticoids are relatively safe and effective in the acute treatment of hypercalcemia, particularly if the hypercalcemia is due to a hematological malignancy. 88 They have the advantage that they can be used at the same time as rehydration and they can be used in patients with poor renal function. They are effective in about 80% of patients and work within 4 to 6 hours. Salmon calcitonin should be administered in doses of 200 to 400 MRC units each 12 hours subcutaneously, and hydrocortisone 100 mg intravenously each 6 hours. In our experience, they are least effective in patients with primary hyperparathyroidism or parathyroid carcinoma, but there are notable exceptions recorded in the literature, 89 and their safety and relative efficacy make them worthy of trial. Although furosemide has been widely used, it is unlikely to be effective unless it is used in very large doses (100 mg every hour), 9~ and when used in this way it is not very convenient. The administration of furosemide in this manner requires the patient to be housed on an intensive care unit with careful monitoring of all fluids and electrolytes. In addition the patient must be fully rehydrated before these doses of furosemide are commenced, because this diuretic may promote extracellular fluid volume depletion, which will worsen hypercal-
644 cemia rather than relieve it. There is no evidence that furosemide in smaller doses has any beneficial effect on hypercalcemia, and it will worsen it if the patient is not fully rehydrated. The evidence that even in larger doses it has substantive effects beyond that of the fluid load is weak. Furosemide may be indicated for those unusual patients with hypercalcemia who are fluid overloaded, but here the indication for treatment with furosemide is for fluid overload and not because of its efficacy in lowering the serum calcium. Parenteral phosphate is very effective in lowering the s e r u m c a l c i u m . 91 However, this therapy is dangerous because it lowers the serum calcium by depositing calcium in soft tissues, including the lungs, heart, and kidney. 92 The mortality associated with this form of therapy may be high, and we believe it should be reserved for desperate situations in which no other therapy is possible or effective. Peritoneal or hemodialysis has been occasionally used for the treatment of hypercalcemia. 93 It may be transiently effective for lowering the serum calcium but has no effect on the increased bone resorption that is the primary cause of hypercalcemia. The mortality is usually high in these patients and this is probably due to the moribund state of patients who have severe hypercalcemia combined with renal failure. We recommend dialysis only for those patients who have severe renal failure combined with hypercalcemia, but it should only be used with the understanding that its beneficial effects will be temporary and that other forms of therapy will be necessary. 2. CHRONIC TREATMENT The agents available for the chronic treatment of malignant hypercalcemia include oral phosphate, corticosteroids, bisphosphonates, plicamycin (mithramycin), and indomethacin. An ideal form of therapy for the chronic management of hypercalcemia would be an orally administered agent without side effects that could maintain normocalcemia during the last few months of the patients' lives. Most of these patients have underlying widespread disease and are within the last 6 months of life. The aim of treatment is to keep the patient relatively symptom-free and ambulant as long as possible. a. Bisphosphonates The bisphosphonates are a family of synthetic compounds that are stable analogues of pyrophosphate. The basic structural difference between pyrophosphate and the bisphosphonates is the substitution of the P-O-P bond of pyrophosphate with a P-C-P bond of the bisphosphonates. Consequently, the bisphosphonates are resistant to enzymatic hydrolysis by pyrophosphatases that are present in the intestine and kidney, so they have a prolonged half-life. They were
GREGORY R. MUNDY
originally developed as additives to toothpaste to prevent calcification and plaque formation. This effect is related to their capacity to impair crystal growth by binding to hydroxyapatite, thereby inhibiting mineralization. 94'95 During these earlier studies on their mode of action on bone, it was found that they were also effective inhibitors of osteoclastic bone resorption. 96 This is the basis for their usefulness in the treatment of bone diseases, and appears to be a universal property of all the bisphosphonates that have so far been studied. These effects on bone occur at doses that seem to have few significant effects in other tissues. Newer generation agents were developed that were powerful inhibitors of bone resorption but had lesser effects on mineralization. These include clodronate, pamidronate, alendronate, risedronate, ibandronate, and tiludronate. 97'98 These are much more potent than etidronate, and include clodronate, pamidronate, alendronate, risedronate, tiludronate, and ibandronate. The only two of these latter bisphosphonates available at the present time in the United States are pamidronate and alendronate, although others may become available over the next several years. Clodronate is widely available in Europe, but the other new generation bisphosphonates are also likely to be much more widely used in Europe over the next few years. Pamidronate [amino-hydroxypropylidene bisphosphonate (APD)] is widely used in Europe and the United States and is extremely effective when administered intravenously for patients with hypercalcemia of malignancy or osteolytic bone disease. In the treatment of hypercalcemia, the bisphosphonates cause a lowering of the serum calcium by decreasing osteoclastic bone resorption. The mechanism by which they inhibit osteoclastic activity is not known. It has been suggested that they may interfere with mineralized bone surfaces to prevent mature osteoclasts from resorbing bone. However, in vitro studies now suggest that they have a direct cellular effect. Recently, we have shown that they promote osteoclast apoptosis. 99 Following a single intravenous infusion of pamidronate, the serum calcium should return to the normal range between 3 and 7 days (mean, 4 days). 1~176176 The duration of action of APD is variable in most patients; the serum calcium often stays within the normal range for 3 w e e k s . 1~176176 Pamidronate should be given as a single intravenous infusion of 15 to 90 mg, depending on the severity of the hypercalcemia, over 2 to 24 hours in 0.9% saline. In patients in whom a rapid response is required, calcitonin should be given concomitantly for the first 72 hours. 87 Pamidronate can be used again by intravenous infusion as required. Since it was first shown that the newer generation bisphosphonates will relieve bone pain and produce a
CHAPTER 22 Hypercalcemiaof Malignancy rapid, sustained and significant decrease in the urinary excretion of calcium and hydroxyproline, indicators of decreased bone t u r n o v e r , 1~176 the bisphosphonates have been proposed as potential therapies for patients with painful bone lesions without hypercalcemia. Studies in experimental models of human breast cancer metastasis have shown the beneficial effects of agents such as risedronate and ibandronate not only in decreasing the skeletal complications of cancer but also in decreasing tumor burden. 38 B isphosphonates have recently been approved by the U.S. Food and Drug Administration (FDA) for this indication in patients with solid tumors. In myeloma, pamidronate is also currently available and approved for use in patients with osteolytic bone disease without hypercalcemia. B isphosphonates may also be useful in the medical therapy of hypercalcemia due to hyperparathyroidism, at least in the short term. This has been known for a number of years. 1~~ Newer generation bisphosphonates such as pamidronate and risedronate will lower the serum calcium in patients with primary hyperparathyroidism during short-term treatment, TM but their beneficial effects on serum calcium over longer periods of treatment remain to be demonstrated. Other bisphosphonates that have been used in the treatment of hypercalcemia of malignancy in the United States include etidronate and alendronate. Etidronate has been approved by the FDA for this indication. Although it is available in both oral and intravenous preparations, it is ineffective orally. Even parenterally, its effectiveness is considerably less than that of the new generation bisphosphonates, which should be selected in preference. Etidronate is so rarely effective orally and should not be used in this manner. 75 However, it is effective in a variable number of patients when administered intravenously. It is difficult to know at the present time how frequently it will work intravenously, but current experience suggests that approximately 60% to 70% of patients will respond after a 48-hour period of treatment with intravenous infusions of etidronate. ~l: This agent is still being evaluated in investigative studies at the present time. On the other hand, clodronate is very effective whether used intravenously o r o r a l l y . 1~ However, because of unexpected side effects including the development of acute leukemia in several patients on longterm therapy, this drug is not being used at the present time. It is possible that the development of acute leukemia was unrelated to the drug, and should this become apparent by further observation of the many patients who received this drug, then it may be reintroduced. It is very effective in reversing hypercalcemia and relieving bone pain as well as in the treatment of Paget's disease. The other bisphosphonate that has received widespread use is pamidronate. 97'1~ This useful agent is almost uni-
645 formly effective when given intravenously. It is also frequently effective when used orally. 75 It has few side effects, although some patients have developed mouth ulcers with the oral preparation, fever, and leukocytosis. These have not been important complications. The availability of alendronate in oral form is a considerable advance. Although it is not FDA-approved for this use, it has a very similar spectrum of activity to that of pamidronate and can be very convenient for the ambulant patient, since it is orally administered. The bisphosphonates are the best drugs currently available for the treatment of hypercalcemia and increased bone resorption associated with malignant disease, and it is possible that over the next few years newer agents of this class will be developed that will be even more effective and freer of side effects than those that have been used to date. b. Oral Phosphate Oral phosphate is a relatively reliable form of therapy, since it usually lowers the serum calcium, particularly in patients who have a serum phosphorus of less than 3.7 mg/dl. However, its usefulness is limited because it cannot be used safely in patients with impaired renal function, and many patients cannot tolerate it because of diarrhea. Moreover, we have found that some patients treated with oral phosphate "escape" from the beneficial effects (lowering of serum calcium) with prolonged therapy. 75 This "escape" is not always related to the serum phosphorus concentration. In other words, this escape phenomenon cannot be explained simply by the increase in serum phosphorus that oral phosphate therapy causes. c. Corticosteroids Corticosteroids work in about 30% of all patients with hypercalcemia of malignance. 75 They are probably most effective in patients with hematological malignancies. In patients with solid tumors, corticosteroids may reduce PTHrP circulating concentrations in some patients by effects on PTHrP gene transcription. 115-119 They are very useful in most patients who respond, since many of these patients have only a short lifespan left to them. When needed for a period that is usually less than 6 months, the well-known side effects of corticosteroid therapy are not a problem. In addition, these agents are given orally and can be taken by patients who will respond before therapy is commenced. Some patients with breast and lung cancer respond, but some do not. Nevertheless, at the current time, we advise an early trial because if the patient is a responder, corticosteroid therapy is an extremely convenient form of treatment. d. Indomethacin Indomethacin is rarely effective and should not be used as routine treatment. 75 Despite
646 several earlier case reports suggesting efficacy, later experiences have been almost uniformly negative. Our o w n data suggest that less than 5% of patients with malignancy and h y p e r c a l c e m i a will respond to indomethacin. It appears unlikely that prostaglandins are responsible for the humoral h y p e r c a l c e m i a of malignancy, except in a few rare situations. One of these possible situations is estrogen-induced hypercalcemia. Indomethacin has not been used in this situation, but it m a y be effective for reasons indicated earlier in this review. 12~A l t h o u g h there have not been extensive studies of other prostaglandin synthesis inhibitors, it seems unlikely that these will prove to be m o r e effective than indomethacin.
e. Mithramycin (Plicamycin)
M i t h r a m y c i n is an effective agent, but it is also toxic. It is not convenient to use in ambulant patients because it must be given by intravenous infusion. Nevertheless, it is usually effective. In our experience, it works in m o r e than 80% of patients in w h o m it is a d m i n i s t e r e d ] 5 Its toxicity is probably greater than indicated in the literature and involves hepatocellular damage, bleeding unrelated to t h r o m b o c y topenia, nausea, anorexia and vomiting, and m a r r o w suppression. 121 One of the problems we have noticed with m i t h r a m y c i n is that it is difficult to space infusions of m i t h r a m y c i n appropriately. We have had the experience of a patient being treated with m i t h r a m y c i n successfully but then developing acute severe hypercalc e m i a again over a few hours, which proved to be fatal before the patient could be retreated. 75 M i t h r a m y c i n probably works because it is toxic to bone cells and inhibits their capacity to cause bone resorption. 122
f Other Agents Two other cytotoxic agents that have b e e n suggested as effective therapeutic agents for the treatment of h y p e r c a l c e m i a are gallium nitrate 123 and cis-platinum. 124 Both of these agents are drugs that are used as chemotherapeutic agents in patients with malignant disease and probably have a similar m o d e of action to mithramycin, that is, they are probably cytotoxic to osteoclasts and inhibit b o n e resorption by inhibiting osteoclast activity. It has been suggested that cis-platinum m a y also w o r k by lowering the serum m a g n e s i u m , which w o u l d predispose to hypocalcemia, but this seems unrelated to effectiveness in the h y p e r c a l c e m i a of cancer. These agents are still being evaluated at the present time, so their place in the treatment of h y p e r c a l c e m i a of malignancy cannot be assessed. They also both have the disadvantage that they are associated with severe side effects, particularly i m p a i r m e n t of renal function; renal function is often impaired prior to treatment in patients with hypercalcemia. The outstanding efficacy of the bisp h o s p h o n a t e s in the treatment of h y p e r c a l c e m i a has m e a n t that these agents are never likely to receive a
GREGORY R. MUNDY p r o m i n e n t place in therapeutic regimens. These cytotoxic drugs are probably not as effective, and certainly m u c h m o r e toxic.
Acknowledgments Some of the studies described in this paper were supported by grants AR28149, CA63628, and RR01346 from the National Institutes of Health. The author is grateful to Nancy Garrett for typing this manuscript.
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phate sulfate and hydrocortisone. Arch Intern Med 129:923930, 1972. Carey RW, Schmitt GW, Kopaid HH: Massive extraskeletal calcification during phosphate treatment of hypercalcemia. Arch Intern Med 122:150-155, 1968. Miach PJ, Dawborn JK, Martin TJ, et al: Management of the hypercalcemia of malignancy by peritoneal dialysis. Med J Aust 1:782-784, 1975. Fleisch H, Russell RGG, Francis MD: Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture and in vivo. Science 165:1262-1264, 1969. Francis MD, Russell RGG, Fleisch H: Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vivo. Science 165:1264-1266, 1969. Russell RGG, Muhlbauer RC, Bisaz S, et al: The influence of pyrophosphate, condensed phosphates phosphonates, and other phosphate compounds on the dissolution of hydroxyapatite in vitro and on bone resorption induced by parathyroid hormone in tissue culture and in thyroparathyroidectomized rats. Calcif Tissue Res 6:183-196, 1970. Frijlink WB, Bijvoet OLM, te Velde J, Heynen G: Treatment of Paget' s disease with (3'-amino- 1-hydroxypropylidene)- 1, 1-bisphosphonate (APD). Lancet i:799-803, 1979. Meunier PJ, Chapuy MC, Alexandre C: Effects of disodium dichloromethylene diphosphonate on Paget's disease of bone. Lancet ii:489-492, 1979. Hughes DE, Wright KR, Uy HL, et al: Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res 10:1478-1487, 1995. Sleeboom HP, Bijvoet OLM, van Oosterom AT, et al: Comparison of intravenous (3'-amino-l-hydroxypropylidene)-l, 1-bisphosphonate and volume repletion in tumour-induced hypercalcemia. Lancet ii:239-243, 1983. Yates AJP, Jerums GJ, Murray RML, et al: A comparison of single and multiple intravenous infusions of 3-amino-hydroxypropylidene-1, 1-bisphosphonate (APD) in the treatment of hypercalcemia of malignancy. Aust NZ J Med 17:387-391, 1987. Thiebaud D, Jaeger PH, Jacquet AF, et al: Dose response in the treatment of hypercalaemia of malignancy by a single infusion of the bisphosphonate AHPrBP (APD). J Clin Oncol 6:762-768, 1988. Portmann L, Haefliger JM, Bill G, et al: Un traitement simple de l'hypercalcemie tumorale: l'amino-hydroxypropylidene bisphosphonate (APD). Schweiz Med Wochenschr 51:1960-1963, 1983. Body JJ, Borkowski A, Cleeren A, et al: Treatment of malignancy-associated hypercalcemia with intravenous aminohydroxypropylidene disphosphonate. J Clin Oncol 8:1177-1183, 1986. Body JJ, Pot M, Borkowski A, et al: Dose-response study of aminohydroxypropylidene bisphosphonate in tumor-associated hypercalcemia. Am J Med 82:957-963, 1987. Cappelli R, Adami S, Bnalia AK, et al: Bisphosphonates and the acute phase response: In Chon DV, Martin TJ, Meunier PJ (eds): Calcium Regulation and Bone Metabolism. Basic and Clinical Aspects, vol 9, Amsterdam, Excerpta Medica, 1987, p 699. Coleman RE, Rubens RD: 3 (amino-l, 1-hydroxypropylidene) bisphosphonate (APD) for hypercalcemia of breast cancer. Br J Cancer 56:465-469, 1987.
649 108. Van Breukelen FJM, Bijvoet OLM, Van Oosterom AT: Inhibition of osteolytic bone lesions by (3-amino-l-hydroxypropylidene)1, 1-bisphosphonate (APD). Lancet 1:803-805, 1979. 109. Sifts ES, Sherman WH, Baquiran DC, et al: Effects of dichloromethylene diphosphonate on skeletal mobilization of multiple myeloma. N Engl J Med 302:310-315, 1980. 110. Shane E, Bacquirian DC, Bilezikian JP: Effects of dichloromethylene diphosphonate on serum and urinary calcium in primary hyperparathyroidism. Ann Intern Med 95:23-27, 1981. 111. Reasner CA, Stone MD, Hosking DH, Mundy GR: Acute changes in calcium homeostasis during treatment of primary hyperparathyroidism with risedronate. J Clin Endocrinol Metab 77: 1067-1071, 1993. 112. Ryzen E, Rude RK, Singer FR, et al: Intravenous didronel (etidronate disodium) in the treatment of hypercalcemia of malignancy. Calcif Tissue Int 36:483 (abstract), 1984. 113. Chapuy MC, Meunier PJ, Alexandre CM, et al: Effects of disodium dichloromethylene diphosphonate on the hypercalcemia produced by bone metastases. J Clin Invest 65:1243-1247, 1980. 114. Jacobs TP, Sifts ES, Bilezikian JP, et al: Hypercalcemia of malignancy. Treatment with intravenous dichloromethylene diphosphonate. Ann Intern Med 94:312-316, 1981. 115. Lu C, Ikeda K, Deftos LJ, et al: Glucocorticoid regulation of parathyroid hormone-related peptide gene transcription in a human neuroendocrine cell line. Mol Endocrinol 3:2034-2040, 1989. 116. Ikeda K, Lu C, Weir EC, et al: Transcriptional regulation of the parathyroid hormone-related peptide gene by glucocorticoids and vitamin D in a human C-cell line. J Biol Chem 264: 15743-15746, 1989. 117. Glatz JA, Heath JK, Southby J, et al: Dexamethasone regulation of parathyroid hormone-related protein (PTH-rP) expression in a squamous cancer cell line. Mol Cell Endocrinol 101:295-306, 1994. 118. Rizzoli R, Feyen JHM, Grau G, et al: Regulation of parathyroid hormone-related protein production in a human lung squamous cell carcinoma line. J Endocrinol 143:333-341, 1994. 119. Sebag M, Henderson J, Goltzman D, Kremer R: Regulation of parathyroid hormone-related peptide production in normal human mammary epithelial cells in vitro. Am J Physiol 267: C723-C730, 1994. 120. Valentin A, Eilon G, Saez S, Mundy GR: Estrogens and antiestrogens stimulate release of bone resorbing activity by cultured human breast cancer cells. J Clin Invest 75:726-731, 1985. 121. Mundy GR, Rodan SB, Majeska RJ, et al: Unidirectional migration of osteosarcoma cells with osteoblast characteristics in response to products of bone resorption. Calcif Tissue Int 34: 542-546, 1982. 122. Minkin C: Inhibition of parathyroid hormone stimulated bone resorption in vitro by the antibiotic mithramycin. Calcif Tissue Res 13:249-257, 1973. 123. Warrell RP, Brockman RS, Coonley CJ, et al: Gallium nitrate inhibits calcium resorption from bone and is effective treatment for cancer-related hypercalcemia. J Clin Invest 73:1487-1490, 1984. 124. Abramson EC, Kukla LJ, Bowser EN, et al: A model for human hypercalcemia of malignancy in athymic nude mouse. Calcif Tissue Int 35:A61, 1983.
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~ H A P T E R 2]
Osteogenesis lmperfecta DAVID W. ROWE JAY R. SHAPIRO
I. II. III. IV. V.
Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut 06032 Department of Medicine, Johns Hopkins Medical School, Baltimore, Maryland 21224
VI. Biochemical and Molecular Tools for the Identification of Mutations in Patients with OI VII. Molecular Pathophysiology of OI VIII. Therapy IX. Future Diagnostic and Therapeutic Directions References
Introduction Perspective: Clinical Introduction Clinical Features Pathophysiology Collagen Biochemistry and Bone Cell Biology as Related to OI
will be included that reflect studies that have appeared through 1996. Because OI is the best example of a single-gene defect that results in heritable bone disease, we wish to provide a basis for the clinical application of basic molecular research and the emerging field of somatic gene therapy, 1 and to suggest how these recent findings relate to other more c o m m o n inherited or acquired disorders of the skeleton.
I. I N T R O D U C T I O N With the introduction of molecular diagnostic techniques to the study of osteogenesis imperfecta (OI), this disease has served as the paradigm for understanding the molecular and genetic basis for all the heritable diseases of connective tissue. Most of this progress in molecular correlation with disease phenotype was discussed in the previous edition of this volume, so relative to the activity obtained in the chondrodystrophies, few major conceptual advances have occurred. However, this is because the field has encountered the same problems common to all heritable diseases, that is, the design and implementation of effective therapies utilizing either pharmacological or somatic genetic principles. As in the previous edition, the objectives of this chapter are to provide an overall view of this disease from a clinical perspective, to review specific aspects of connective tissue biology and biochemistry that apply to heritable disorders of bone, and to relate these with clinical examples so as to serve as a basis for understanding OI and related clinical syndromes. Modifications of these principles METABOLIC BONE DISEASE
II. PERSPECTIVE: CLINICAL I N T R O D U C T I O N A. Historical The history of OI can be traced to Egyptian times as evidenced by a skeleton preserved at the British Museum) The more modem history of the disease has been summarized by Weil, 3 and is recorded in the comprehensive text on OI by Smith et al. 4 The first recorded case of OI is attributed to Malebranche, 5 who reported
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DAVID W. ROWEAND JAY R. SHAPIRO
a subject who appeared "like a man broken on a wheel." The first detailed clinical description of OI is to be found in a thesis for the degree of Doctor of Medicine presented by Olaus Jacob Eckman, Chief Surgeon to the Swedish Royal Calvary Regiment, at the University of Upsala. 6 Describing a form of congenital osteomalacia, Eckman provided a comprehensive report of OI in three successive generations afflicted with fragilitas ossium and skeletal deformities. The association of fractures with joint laxity was noted by Velpeau, 7 with blue sclerae by Spurway 8 and Eddowes, 9 and with deafness by Adair-Deighton. l~ Bauer suggested that OI could result from a generalized mesenchymal disorder. 1~ Extensive clinical and genetic analyses of OI have been compiled by S e e d o r f 12 and Smars. ~3 Over the years a series of eponyms have been associated with the OI syndrome. The terms "OI congenita" and "OI tarda" were introduced by Looser in 1906 to indicate the age of onset of fractures, and by inference, the severity of the disease. 14 These terms are imprecise and should be discarded in view of recent research in OI that aims towards an integrated clinical and biochemical definition of the syndrome.
reliable because: (1) classification based on scleral color is inaccurate, since it is difficult to measure and may change with age; (2) ---10% of the cases defy classification because of overlapping clinical features; and (3) inheritance may be indeterminate. Thus, reliance on clinical criteria for recurrence risk counseling in a family without a prior history of the disease will be hazardous until precise biochemical diagnosis can differentiate between a new mutation and co-dominant or recessive inheritance. A currently popular classification of OI was proposed in 1979 by Sillence et al., 18'19 and has subsequently been expanded and modified by Levin 1~ and Shapiro 21 (Table 23-1). While a convenient tool in the absence of relevant biochemical and molecular data, this nosology may be inadequate in specific cases, since OI is sporadic in one third of cases and in two thirds of families. 22 In 1975 Bauze et al. classified OI into three groups based on the extent of skeletal deformity as mild, moderate, or severe, 23 while in 1992, Hanscom used similar criteria and five degrees of radiological severity. 24 Assignment of individual subjects may be open to dispute depending on observer, age, trauma, and so forth. Distribution of clinical findings between these broad categories is illustrated in Table 23-1.
B. Clinical Heterogeneity OI is genetically and clinically a heterogeneous disorder of bone and connective tissue characterized by osteoporosis, fragile bones, hyperextensible joints, dentinogenesis imperfecta (DI), bluish coloration of the sclerae, and adult-onset hearing loss. Genetic heterogeneity and variable expressivity are principles of clinical genetics that are illustrated in O1.15 The former implies that a limited number of clinical phenotypes will result from a larger number of gene defects, while variable expressivity refers to quantitative and qualitative variation within each phenotype. For example, the incidence of fractures or the extent of skeletal deformity will vary considerably among kindreds or among members of an affected family. Variable gene expression may result from tissue-specific modifier genes, the inherent genetic background of the host, somatic mosaicism, 16 or environmental factors. For many years, genetic heterogeneity and variable expressivity were obstacles to the precise definition of OI phenotypes. Nevertheless, one useful classification put forth by Ibsen in 1967 permits separation of most cases into subgroups according to the frequency of fractures; the extent of skeletal deformities; the presence or absence of DI, blue sclerae, and hearing loss; and the mode of inheritance. 17 This classification combines variable clinical signs of the disease with mendelian patterns of inheritance. However, these schema are not fully
C. Distinctive OI Phenotypes The following discussion will present clinical phenotypes in order of decreasing severity rather than the numerical order used in the Sillence classification. 1. NEONATAL LETHAL Ol (SILLENCE, TYPE II: BAUZE, SEVERE OI; HANSCOM, TYPE E Lethal OI represents the most extreme form of this disease. Skeletal mass is markedly decreased. Neonates suffer multiple gestational fractures causing shortened and deformed limbs, and may sustain cerebral trauma and even dismemberment during delivery. The common radiological appearance is of a large deformed cranium; broad, crumpled extremities (concertina deformity); a characteristic beading of the fibs; and fractured vertebrae (Fig. 23-1). However, a spectrum of radiological severity has been demonstrated. These children are hypotonic, feed poorly, and develop respiratory insufficiency shortly after birth. The majority succumb within days or weeks from pulmonary failure. Lethal OI affects ---10% of patients and --~20% of families. 22 The birth of multiple affected siblings to apparently normal parents is well documented and interpreted to indicate recessive inheritance in most cases. 25 New autosomal dominant mutations were first suggested by Langness and Beehnke in 1970 as an etiology in le-
CHAPTER 23
Osteogenesis Imperfecta TABLE 23-- 1
IIB IIC III
IV
Clinical Features of OI Phenotypes a Pattern of Inheritance
Phenotype
Type
IIA
653
Mild OI, relatively few fractures, nondeforming, mild scoliosis. Blue sclerae, adult hearing loss. Dentinogenesis imperfecta (type IB) in 25%. Lethal OI. Multiple intrauterine fractures, broad short femora, continuous beading of fibs, defective mineralization of the calvarium. As IIA, except that ribs are thin, with or without continuous beading, and the calvarium is better mineralized. Less frequent than type A or B. Femora and humeri are longer, undermodeled, and irregular. Calvarium is poorly mineralized. Severe OI with frequent fracture rate and marked deformity. Marked-growth retardation. Scoliosis severe and may progress to compromised pulmonary function. White or blue sclerae. Moderately severe disease. Heterogenous phenotype, white or blue sclerae. Fractures may be frequent and are associated with obvious skeletal deformity. Scoliosis is moderately severe. Denfinogenesis imperfecta (type IVB) in 25%, adult hearing loss.
AD AD: new mutations AR AR (data limited) AD (75%) AR (25%) AD
Key: AD: autosomal dominant; AR: autosomal recessive. aModified from Sillence DO: Osteogenesis imperfecta: Nosology and genetics. Ann NY Acad Sci 543:1-15, 1988.
thai O1. 26 This impression is supported by recent biochemical information demonstrating that new mutations may frequently underlie this form of O1. 27-29 In a comprehensive analysis of 71 affected infants, Byers noted that most cases occurred as sporadic events in which biochemical analysis of the parents strongly supported the abnormality in the affected infant as a new mutation. 3~ In the families with more than one affected infant, pedigree and biochemical data are most consistent with germinal mosaicism of one parent. 31 A recurrence risk of 6% to 8% was obtained when all cases were analyzed, reflecting the germinal mosaicism of a dominant trait rather than recessive or co-dominant inheritance. Because there is a finite risk of recurrence in a family with one affected infant, subsequent pregnancies should be examined by ultrasound at the 16th to 18th weeks of gestation to determine if the skeleton of the fetus is deformed or n o r m a l . 32'33
annulus juvenilis or senilis is common, and hearing loss may appear earlier than in other types, due to a greater degree of cranial deformity. Severe OI occurs in --~20% of affected patients, and in ---15% of affected families. 22 The vast majority occur as sporadic cases without a prior family history of bone disease. It is now well recognized that apparently normal parents may produce more than one affected offspring, compatible with autosomal recessive inheritance. 35 Type III OI has been reported in dizygotic twins. 36 However, most of these cases of recurrent OI can now be accounted for by somatic mosaicism. 32'33'37 Prenatal diagnosis of type III OI had been made in the second trimester and thus would be of value in families with a previously affected individual. 3s
2. SEVERE NONLETHAL OI (SILLENCE, TYPE III OR
This form of OI presents a spectrum of characteristics that range between the severely deforming and the mildly affected. As defined by Sillence, the distinguishing feature c o m m o n to these subjects is blue sclerae when young, which decrease to a lighter hue in childhood, and a normal color in adults. Individuals with white sclerae are the minority in moderate OI, and we have observed adult patients with moderately severe skeletal disease typical of this phenotype OI with blue sclerae. Thus, we believe that classification of individual cases based on scleral color may be arbitrary and may prove inconsistent with biochemical data.
PROGRESSIVE DEFORMING; BAUZE, SEVERE; HANSCOM, TYPE C, D) Fractures, deformity of long bones, molding of the calvarium, and elongated vertebrae pedicles are recognized at birth in this form of O1. 34 The natural history is one of marked growth retardation, frequent and deforming fractures of the long bones (Fig. 2 3 - 2 ) , and scoliosis with deformity of the chest wall that may ultimately lead to pulmonary insufficiency. These individuals are wheelchair-bound. Joint laxity is more marked than in other nonlethal types. The sclerae are typically white,
3. MODERATE AND DEFORMING Ol (SILLENCE, TYPE IV OI; BAUZE, MILD O1; HANSCOM, TYPE B, C)
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DAVID W. ROWE AND JAY R. SHAPIRO
4. M I L D NONDEFORMING O I (SILLENCE, TYPE I; BAUZE, M I L D OI; HANSCOM, TYPE A)
This type includes about 60% of patients. 19 Bone fragility and osteoporosis are mild compared to other types and usually produce no skeletal deformity (Fig. 2 3 - 4 ) . Fractures are rarely present at birth (--~10%), and usually occur when the child ambulates. Fracture incidence declines markedly after puberty. These patients demonstrate mild growth retardation and mild scoliosis. Triangular-shaped facies are common, all have blue sclerae, and 25% have DI. The latter finding has been used to subdivide this form of OI into group IA (with DI) and group IB (without DI). 2~ A limited degree of joint laxity is common. Heating impairment affects --~70%, only 10% of whom will require a hearing aid. Mild OI is inherited as an autosomal dominant trait. When it occurs as a new mutation, the phenotype is subsequently transmitted as a dominant. The disease appears to breed true (e.g., only rarely has there been marked variation in severity from the pattern established with a family). 41 5. RELATED SKELETAL/CONNECTIVE TISSUE SYNDROMES
FIGURE 23--1
Lethal OI (Sillence, type II; Bauze, severe). This radiograph illustrates the "broad-boned" type of lethal OI and the markedly defective mineralization of the calvarium and extremities. The accordion-pleated deformity of the femurs is present. Also characteristic is the beaded appearance of the fibs, a result of intrauterine fracture with callus formation. Slender long bones may also be seen in certain patients with lethal OI.
Despite the difficulties in defining scleral color, there exist other clinical characteristics of this form of OI. A moderate degree of growth retardation is usually present. Deformity of the extremities frequently necessitates bracing or a cane to assist ambulation (Fig. 23-3). Severe scoliosis may occur. Joint laxity is common. Hearing loss occurs, and --~20% require hearing aids. DI is most often found in this group, and such individuals have been identified as type IVB in contrast to type IVA, who lack dental changes, z~ Patterson et al. has observed that the distinguishing features of this phenotype, when compared to type I OI, have a greater frequency of fractures at birth (28.3% vs. 12.3%) but no difference in later years and a lesser tendency to bruising. 39 The authors' experience is that these patients develop more skeletal deformity than do individuals with type I OI. This group is also genetically heterogeneous. The majority demonstrate autosomal dominant inheritance, but the occurrence of sporadic cases suggests that new mutations may occur. Increased paternal age does not appear to contribute a new mutation. 4~
Several patients have been reported with multiple fractures suggestive of OI, associated with clinical features of the other heritable disorders of connective tissue. This may represent co-dominant inheritance of two gene defects. OI associated with Ehlers-Danlos syndrome (EDS) was reported in a child with blue sclerae, lax skin, bilateral hip dislocations, and fractures of vertebrae and both femurs. 42 This child's father also had lax skin. In another family, features of both OI and arthrochalasis multiplex (EDS, type VII) were present, and a biochemical explanation for these findings was demonstrated. 43 OI was observed in 23 members of an Irish kindred comprised of 102 individuals in four generations. 44 The propositus had multiple fractures, osteoporosis, blue sclerae, Marfan's habitus, and aortic and mitral regurgitation. He apparently inherited the OI gene from his father and the marfanoid complex from his mother. His siblings were affected with scoliosis, pectus excavatum, ligamentous laxity, arachnodactyly, osteoporosis, and the helmutshaped skull of OI. An "arthropathic form" of OI has been suggested in a 14-year-old girl who developed severe osteoporosis and generalized destructive joint disease resulting in ankyloses. 45 Similarity to OI was suspected because of a relative increase in type III collagen synthesized by cultured skin fibroblasts. 6. HERITABLE OSTEOPOROSIS The evaluation of large numbers of adult osteoporotic patients has focused attention on a subset in which os-
CHAPTER 23
655
Osteogenesis Imperfecta
FIGURE 23--2 Severe, nonlethal OI (Sillence, type III; Bauze, severe OI). This depicts the narrow diaphysis seen in severe nonlethal OI, which classically broadens to a bulbous metaphyseal/epiphyseal zone. The epiphyseal zones are severely hypomineralized and dysplastic. Whorls of partially calcified cartilage give the "popcorn appearance." Cystic changes may predominate in some patients.
FIGURE 23--3 Moderately severe disease (Sillence, type IV; Bauze, moderate OI). Both deformity and hypomineralization are apparent. The epiphyseal zones are dysplastic and show cystic irregularities. Scoliosis is present in these patients. Peripheral skeletal deformities frequently require bracing.
FIGURE 2 3 - 4 Mild and nondeforming OI (Sillence, type I; Bause, moderate OI). This radiograph illustrates the ability of these patients to heal a significant fracture without incurring a deformity. Hypomineralization is less marked than in other types, and the epiphyseal zones are normal.
teoporosis is apparently transmitted from generation to generation. Osteoporosis is m o r e c o m m o n in specific ethnic populations, 46 and m o t h e r / d a u g h t e r pairs are m o r e likely to have osteoporosis 47 or lower b o n e mass. 48 A n u m b e r of studies of individuals with mild connective tissue findings and heritable osteoporosis have d e m o n strated mutations consistent with mild OI. 49'5~ Once the mutation is identified within the pedigree, it is relatively easy to distinguish affected f r o m unaffected individuals, and, in retrospect, features of mild OI can be identified. Nevertheless, there is still the suspicion that subtle mutations could underlie certain pedigrees of heritable osteoporosis 51 even though attempts to identify t h e m have not yet been successful. 52'53 If one considers the m a r k e d effect of race and sex on b o n e density, it is possible that other genetic determinants affecting either collagen or another of the extracellular matrix c o m p o n e n t s could influence the d e v e l o p m e n t of osteoporosis in both m e n and women.
7. MURINE MODELS OF O l The m o u s e is b e c o m i n g the primary experimental m o d e l for heritable diseases because of its h o m o g e n e o u s
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DAVID W. ROWE AND JAY R. SHAPIRO
genetic background and the ability to introduce mutations into the genes that cause disease in humans. A variety of naturally occurring and experimentally induced mutations in type I collagen with a spectrum of severity that resembles human OI now exist. The first examples were based on mutation causing type II OI precluding the development of a stable mouse strain for repetitive analysis. 54 Lethal O155 and viable O156 have been produced in transgenic mice strains, although this approach has an exceptional and unexplained phenotypic variability. 57 A naturally occurring form (OIM mouse) resembling type III OI is now in wide use in a number of laboratories. 58 Mice closely resembling type I OI (Movl3 mouse) unexpectedly arose in an experimental setting and have been extensively characterized by Bonadio. 59 Other models are certain to follow that model specific human forms of O1. 60 Frequent reference to these murine strains will be made because of their contribution to understanding the pathophysiology of OI and as platforms to develop therapeutic strategies. 61
D. D i f f e r e n t i a l D i a g n o s i s 1. IN N E O N A T E S AND INFANTS
Several connective tissue syndromes that usually appear as sporadic or recessive disorders must be considered in the newborn period. The congenital lethal form of hypophosphatasia can present with severe bone disease, the result of a generalized failure of normal ossification. Osteopenia, micromelia, fractures, and a soft calvarium are present. 62 Death may occur due to respiratory failure. The level of alkaline phosphatase is diagnostically low in blood, bone, and viscera, while it is high in normal growing infants or in those with traumatic fractures. Serum calcium is high normal or elevated, and urinary phosphoethanolamine excretion is increased in this disorder, in contrast to OI. Radiologically, the infant develops rachitic changes with broadened epiphyses, bowing of the extremities, and prominent costochondral junctions. Mutations within the bone/kidney alkaline phosphatase gene underlie this disorder of bone mineralization. 63 The heritable chondrodystrophies can resemble lethal or severe OI due to their small size, facial features, short limbs, and hypotonia and are routinely detected by ultrasonography 64 and are differentiated by radiographic criteria. 65 In achondroplasia, typical skeletal manifestations should be apparent in the newborn period, and include characteristic craniofacial abnormalities with bossing and flattening of the bridge of the nose, extremely short extremities, and broad long bones having an abrupt widening at their metaphyseal ends. 62 A horizontal ace-
tabular roof and narrowing of the interpeduncular distance in the lumbar vertebrae are also found. In spite of severe micromelia, there is less marked reduction in height than in severe OI. Thanatophoric dwarfism is the prototype of the chondrodystrophies, which are incompatible with survival beyond the neonatal period. 66 Extreme micromelia is associated with increased head circumference. The thorax is narrow and the ribs are short with flaring. The epiphyses of long bones are absent at birth. Vertebral bodies are decreased in height with normally developed pedicles, which produces an H-shaped deformity. Activating mutations of the FGF3 receptor that primarily affect the proliferation/differentiation pathway of the chondrocyte are responsible for the chondrodystrophies. 67'68 Congenital spondyloepiphyseal dysplasia is characterized by short stature due to abnormal growth of the spine and extremities with normal appearing hands and f e e t . 69 Delayed bony maturation of the spine associated with a trapezoid appearance of vertebral bodies due to shortened length of their posterior borders is typical. Skeletal fragility and limb deformity are not features of this disorder, which is inherited either as an autosomal dominant or new mutation. Finally, lethal OI should be differentiated from achondrogenesis, a chondrodysplasia incompatible with survival. Dwarfism is severe, skull volume is increased, and there is micromelia. Vertebral ossification is markedly impaired. The ribs are short, with lateral spur formation and irregular ossification. A wide variety of mutations within type II collagen that interfere with the formation of a stable cartilage matrix account for the spectrum of clinical severity characteristic of the chondrodystrophies. 7~ In general, neonatal fractures are not a feature of the metaphyseal dysplasias, including diastrophic dwarfism, metatropic dwarfism, and punctate chondrodysplasia. These syndromes are characterized by the presence of severe micromelia, and result from mutations that affect the formation or function of the hypertrophic chondrocyte. 72'73 2. IN
CHILDHOOD
It is frequently necessary to differentiate OI from the , 74 75 "battered baby' syndrome. ' The presence of soft tissue trauma, metaphysial fractures, and periarticular calcifications, or evidence of psychosocial deprivation, should alert the physician to the possibility of abuse as a cause of recurrent fractures. 76 However, radiographic differentiation is not diagnostic. 77 Blue sclerae, wormian bones, and a parental history of fractures are clues to the presence of OI that are frequently overlooked when children are seen in an emergency setting. While biochemical studies can occasionally be helpful in distinguishing the two possibilities, 78 a careful clinical examina-
CHAPTER 23 OsteogenesisImperfecta tion should identify the characteristic features of O I . 79'80 In a family with an isolated case of mild OI, it may be difficult to convince well-meaning social agencies that child abuse is not present. A poorly described and understood clinical diagnosis of temporary brittle bone disease needs to be used with great caution. 81'82 Childhood hypophosphatasia can be confused with severe OI (type III). Affected children have radiological features of tickets including growth retardation, poor cranial ossification with bossing, early shedding of teeth, rachitic rosary of the chest cage, fractures, and widened epiphyses. The serum alkaline phosphatase remains low and urinary phosphoethanolamine excretion is elevated. There is autosomal recessive inheritance. Congenital hyperphosphatasia can present by the second year of life with painful deformities of the extremities and pathological fractures. 83 It is characterized by a 20- to 40-fold elevation of the serum alkaline phosphatase, and high urinary hydroxyproline excretion. Bones of the skull are irregularly thickened, and the long bones demonstrate evidence of markedly increased remodeling. Treatment with calcitonin may arrest the high rate of bone turnover and, in some children, prove life-saving. This rare disorder, which has been reported in siblings, is transmitted as an autosomal recessive trait. Homocystinuria is a heritable disorder of connective tissue characterized by a marfanoid appearance associated with the early onset of osteoporosis with fractures of the vertebrae and extremities. 84 Children typically develop inferior subluxation of the lens, chest wall deformities, kyphoscoliosis, slender extremities, tall stature, and mental retardation. For unexplained reasons, they are subject to lethal thromboses of major vessels. Several EDS variants have been recognized in children that do not conform to the currently recognized phenotypes. 85 These are usually mild sporadic cases, with excessive joint laxity, mitral valve prolapse, and occasionally, osteoporosis with fractures in adults. As noted above, OI and the EDS may coexist in the same kindred. Juvenile osteoporosis may be confused with OI when appearing at a young age. 86 Cases of this disorder have been noted as early as 4 or 5 years. However, in the majority there is a peripubertal onset of vertebral demineralization with fracture, pain, and loss of height. Pain in the extremities may result from metaphyseal fractures, which are uncommon in OI. To date, no biochemical markers of this disorder have been found. As in OI, the disease tends to remit following puberty. Malabsorption syndromes in children may be associated with osteomalacia, fractures, and growth retardation. Therefore, an evaluation of the osteopenic child should include consideration of occult gastrointestinal disease. Additional possibilities presenting as osteopenia include lymphoma or leukemia with fractures and en-
657 docrine disorders including hyperthyroidism, Cushing's disease, and hyperparathyroidism. 3. IN ADULTS
The presence of mild OI in the adult is frequently recognized after the diagnosis is made in an infant or child. For example, the occurrence of vertebral or extremity fractures in the premenopausal female may call attention to blue sclerae, or a family history consistent with the dominant transmission of skeletal fragility. The authors are increasingly aware of patients or kindreds in whom the initial diagnosis is osteoporosis, but where the presence of a systemic connective tissue lesion is suggested by scoliosis, hyperextensible joints, blue sclerae, and short stature. An understanding of these patients will depend on the elucidation of their biochemical lesion. Mild adult hypophosphatasia with growth retardation, tickets, hyperthyroidism, hyperparathyroidism and hypogonadism, chronic renal disease, and unrecognized malabsorption must be considered in this differential diagnosis. 87
E. Prevalence and Incidence OI has been reported in all ethnic groups and races without a geographic predilection. The incidence of OI is underestimated because many mildly affected subjects go undetected throughout childhood and adult life, while severely affected fetuses, stillborn or those dying shortly after birth, may be undiagnosed. Thus, the most reliable estimates of the frequency of OI are based on the recognition of fractures occurring in the newborn period. OI has a reported incidence of 1:20,000 to 1:60,000 live births, with a general prevalence in several reports from 1 to 5 X 10-5"19. 88-93
III. CLINICAL FEATURES
A. The Skeletal System 1. CLINICAL COURSE OF LONG BONE FRACTURES
Although OI is a systemic disorder of connective tissue, fragile bones are the dominant clinical manifestation. The morbidity and mortality associated with OI are directly related to the degree of skeletal fragility. 94 Connective tissue involvement in other organs carries relatively little morbidity. The most severe forms of OI suffer fractures in utero and may succumb shortly after birth, or they may survive with the handicap of severe skeletal deformity throughout their lives. Marked fragility carries the risk of death, particularly from head trauma. The less severely affected patients are always
658 osteoporotic, with variable amounts of deformity. We do not fully understand the relationship between the described gene defects and the diverse clinical phenotypes in OI, or why healing occurs without deformity in certain patients and with severe deformity in others. In the milder types of OI, type I, and the less severe cases of type IV disease, there may be minimal skeletal deformity (Fig. 2 3 - 4 ) . Probably most common is anterior bowing of the distal tibia. While fractures may be present at birth in --~10% of milder cases, they usually occur between the age of 6 months to 3 years as the child starts to stand and walk. Boys tend to experience more fractures than girls. 95 Occasionally, the first fracture occurs at an older age, and rarely in the teens. Metaphyseal and epiphyseal architecture are normal in mild O1. 96 Ashier noted small metaphyseal fractures in 7 out of 41 OI children with gross skeletal abnormalities. Most fractures in children involved the lower extremities, particularly in the proximal or distal femur. Older children may injure the distal forearm or fingers and toes. Skeletal deformities are more prominent in the type IV patient, where osteopenia and scoliosis are more severe (Fig. 23-3). As children first stand, we have watched the proximal femur change within a few months from no deformity to a "shepherd's crook" deformity, presumably the result of microfractures. As a consequence, unlike the type I patient, those with type IV are frequently dependent on crutches or canes to assist in walking. Children with severe OI (type III), are bom with obvious cranial deformities and fractures of the clavicles, extremities, or fibs. These may not be recognized for weeks or until callus has formed. Irritability or failure to thrive may be the only symptom of fractures in the nursery. Here, fractures occur with minimal stress and heal with residual deformity. Nonunion may occur but is uncommon. Repeated fractures and microfractures induce progressive deformity and may require immobilization of a limb or the trunk in a body spica cast, aggravating osteopenia and increasing the susceptibility to new fractures. In more severe cases one sees progressive angulation of the neck of the femur; a "sabre shin-like" anterior angulation of the lower tibia; and lateral bowing or rotation of the humerus, radius, and ulna. Once developed, these deformities represent areas highly susceptible to fracture with minimal trauma. In severe OI, the diaphysis of long bones frequently is very narrow, widening to an enlarged and dysplastic metaphysealepiphyseal a r e a . 97 The presence of clusters of small, scalloped, irregularly calcified radiolucencies at the ends of long bones have been termed " p o p c o m " calcifications. 98 The association of these lesions with disappearance of the normal horizontal growth plate suggests a posttraumatic disturbance in endochondral ossification (Fig. 23-2).
DAVID W. ROWE AND JAY R. SHAPIRO
Endochondral bone formation is markedly deficient in the neonatal lethal form of OI. The long bones typically appear broad and are severely osteopenic with multiple intrauterine fractures simulating an "accordian pleated" or "concertina bone" appearance. In addition to cranial fractures, rib fractures in utero produce a characteristic beaded appearance on x-ray (Fig. 23-1). Sillence et al. 25 have proposed that three variants of lethal OI may be recognized radiologically: group A, characterized by markedly shortened broad and rectangular crumpled limbs, strikingly impaired cranial ossification, and short thick ribs with prominent beading; group B, radiographs were similar to group A, but ribs showed only occasional beading; and group C, long bones were slender with multiple fractures and inadequate modeling, and beading of the ribs was less prominent than in group A. The spectrum of radiographic finding has been expanded further to five groups. Group 1 is more severe than group B with calvaria and long bone showing no modeling. Group 5 is less severe than group C with normal long bone modeling and metaphyseal flaring. The severity of the radiographic appearance correlated with the length of survival after birth. 99 In the nonlethal forms of OI there is marked variation in fracture rate even within individual families. Fractures are five- to tenfold more frequent before puberty. Moorfield and Miller found that an average of 91% of fractures occurred prior to puberty, while after puberty fractures apparently require more than trivial trauma. 1~176 The factors responsible for this increase in bone strength after puberty are unknown. Obviously, a change in hormonal milieu is crucial, since following the menopause the fracture rate starts to climb again in each of the phenotypes. Decreased calcium stores and the postmenopausal loss of estrogen may play a role. As reported by Paters o n , 95 the fracture rate in postmenopausal females with OI is about sevenfold that in the general population (e.g., 26 fractures per 100 patient years, for ages 50 to 70 years vs. 3.5 to 4.8 fractures per 100 patient years in comparably aged females). Males over age 50 also experience an increase in fracture rate, but less than that in the females. Fractures in OI are frequently transverse, at times subperiosteal, and usually in alignment, depending on the thickness of cortex and the nature of the trauma. Certain patients may suffer a fracture with merely a change in position: milder cases require substantial trauma to cause fracture. The authors have examined patients with obvious (mild) OI as children, who after puberty played contact sports with little injury. Contrary to a common perception, fractures in OI are painful, although minor fractures may resolve with minimal d i s c o m f o r t . 13
Three skeletal complications may occur following a fracture: (1) severe angulation, which may limit function
CHAPTER 23 OsteogenesisImperfecta if not properly corrected; (2) nonunion which, while uncommon, involves long bones and may prove disabling; and (3) hyperplastic callus, which occurs in less than 1% of OI fractures. T M This is a rapidly developing inflammatory overgrowth of new bone that occurs unexpectedly. Associated with an elevated erythrocyte sedimentation rate (ESR), local erythema, and warmth, hyperplastic callus presents as a tender mass at the site of a healing fracture. It appears more commonly in boys, and usually involves the femur.
2. SPINE Scoliosis is a frequent complication of OI. It may be present at an early age and is directly related to the severity of the disease and probably the degree of joint laxity. 1~ King and Bobechko found the incidence of scoliosis to vary from 28% in the milder forms to 43% in type III OI, while Falvo et al. TM found a 92% incidence in OI congenita. Weakness of the paraspinal musculature may promote asymmetrical growth of the spine, particularly in young children. Either thoracic scoliotic curves or a double primary curve may develop. Most curves accelerate during the pubertal growth spurt and many stabilize in adulthood. 1~ The majority, as reported by Renshaw et al., 1~ measure from 10 to 20 degrees; curves in excess of 20 degrees are found in type III patients. In the adult, scoliosis frequently increases in severity and becomes increasingly disabling as thoracic volume declines. 1~ Osteopenia and lax paraspinal ligaments lead to vertebral crush fractures ("codfish" deformity) or burst fractures, 1~ which are more common in adults than in children. 1~ Vertebral fractures in childhood are more likely to be a feature of juvenile osteoporosis than O1.11~ Neurological sequelae of scoliosis or vertebral collapse are usually not present. Upper cervical cord compression has been reported as a cause of death in lethal O1. TM 3. SKULL Deformity of the skull occurs frequently in OI. Typically, the circumference of the head is increased, although in some the circumference may only appear increased relative to the small size of the trunk. In mild cases the face may present a triangular appearance. Softening or early fractures of the mandible may produce a type III malocclusion with protrusion of the mandible. 112 In three families, 13 subjects with OI had radiolucent or radiopaque lesions of the maxilla or mandible. 113 In more severe cases, because ossification of the skull is deficient or delayed, softening leads to flattening in the anteroposterior diameter, particularly in the hypotonic, bedridden infant with little head control. Closure of the fontanels may be delayed for several years. Moulding may lead to prominence of the frontal and occipital bones,
659 simulating the bossing seen in rickets. Softening of the calvarium causes the " h e l m e t " or "tam-o'-shanter" deformity with a palpable occipital overhang. TM Prominence of the eyes and flattening of the forehead may combine to produce the characteristic "sunset sign" appearance of the severely affected child. The majority of severe cases of OI present with multiple wormian bones, and Cremin et al. have described these as detached portions of the primary ossification centers in adjacent membranous bones. 115 Significant persistence of wormian bones is defined as more than ten in number that are greater than 6 • 4 m m in size. They are frequently located in the occipital area and may persist for several years. Although wormian bones are an important diagnostic feature of OI, they are also found occasionally in normal growing skulls and in other heritable disorders of connective tissue, such as cleidocranial dysostosis, Menkes' syndrome, Prader-Willi syndrome, and progeria. Either basilar impression or platybasia may occur where there is extreme softening at the base of the skull. 116'117 These may be responsible for variable hydrocephalus with dilatation of the third and lateral ventricles, upper and lower motor neuron lesions, cranial nerve abnormalities, and cerebellar dysfunction. 118 Cortical atrophy in OI may be a consequence of macrocephaly. 1~9 Distortion of venous sinuses seen on computed tomography (CT) scans may be the cause of chronic headaches experienced by severely affected patients. Rarely, decompression of the posterior fossa may be required. ~2~ Trauma to the skull or face, while uncommon in OI, is a constant hazard after an accidental fall from a wheelchair or following an automobile accident.
4. OTHER SKELETAL ABNORMALITIES Additional skeletal abnormalities include protrusio acetabuli, coxa vara deformity, genu valgum, and piano valgus deformities of the feet. Various deformities of the chest wall (pectus excavatum and carinatum, asymmetry of the rib cage) accompany scoliosis of the spine. 122 Marked elongation of the pedicles of vertebrae and posterior rib angulation have been observed in type III O 1 . 34
B. O t h e r C o n n e c t i v e T i s s u e s
1. JOINT HYPERMOBILITY Clinical criteria related to joint hypermobility have been presented by Beighton and Horan. 123 To a variable extent, --~70% of OI patients may demonstrate significant joint hypermobility due to underdevelopment of the ligaments. Lax joints are more common in the more se-
660
DAVID W. ROWE AND JAY R. SHAPIRO
verely affected subjects. This is most evident in the younger patients and usually involves the distal rather than the larger proximal joints, which are affected in the Marfan's syndrome and EDS. There is no relationship of joint mobility to scleral color. The OI infant may appear loose jointed and floppy. While sprains and dislocations are not frequent in OI, with sufficient stress, troublesome dislocations may occur at the hip, knee, ankle, or shoulders. Muscle tone is poor in the more severely affected subjects, either secondary to disuse or to the underlying connective tissue abnormality also present within the tendons. Pes planus is a common finding in the OI patient. 2. SKIN
Diminished skin thickness is probably a consequence of diminished collagen production. 124 Skin tensile strength is decreased where type I collagen production is diminished. 125 Skin laxity is usually mild, and substantially less than the hyperelasticity characteristic of EDS. In contrast to the friability of skin in EDS, wound healing in OI is usually normal. Elastosis perforans serpiginosa, an unusual lesion common to other connective tissue disorders such as EDS and pseudoxanthoma elasticum, has been reported in O1.126 This lesion appears as a keratotic papule that grows to a serpiginous macule, usually on the arms or trunk. Patients may form thin broad scars, and occasionally keloids may be found. 127
3. TEETH
Dentin, the organic matrix of teeth, is composed of type I collagen and proteoglycans. The opalescent brown teeth, characteristic of DI, is the result of insufficient support of the overlying normal enamel. 128 With the continued trauma of eating, the enamel fractures, exposing the underlying dentin, and the tooth wears to the level of the gum (Fig. 23-5). Permanent teeth are usually less severely affected. Dentin, which contains the same genetic collagen as bone (type I), has increased amounts of hydroxylysine relative to lysine in OI patients without obvious DI, 129 a finding consistent with the overmodified collagen molecule found in the connective tissues in severe OI. When viewed by scanning electronmicroscopy, the dentin has an irregular tubular organization that contrasts with the highly ordered tubular pattern of normal dentin. 13~ Radiographic characteristics of DI include a normal density and thickness of enamel, an exaggerated constriction at the junction between crown and roots, and pulp chambers that may initially be larger than normal, but which become obliterated by abnormal dentin. 131 Approximately 25% in each clinical phenotype are also found to have DI. Smith et al. have found DI to be relatively uncommon in patients with mild disease and more common in those with severe bone disease. 132 However, careful histological examination of teeth from OI patients lacking obvious DI can reveal features of D I . 133'134 Genetic evaluation of families with dominantly
FIGURE 23--5 Dentinogenesisimperfecta of the secondary dentition. The teeth have a translucent appearance. There is eventual erosion and fracture of enamel exposing the dentin core, which has been worn to the gingiva.
CHAPTER 23 Osteogenesis Imperfecta inherited OI has revealed that when DI is present in a family, all affected individuals will have both skeletal and dental disease. TM However, there is no relationship between the severity of skeletal disease and the presence of DI in affected families. Rare families have been reported to have DI and blue sclerae, but no bone disease. 94
C. Other Organs Containing Type I Collagen 1. EYE
Although blue sclera has been recognized as a prominent clinical sign in OI since 1831, its pathogenesis still remains uncertain. 135 Type I collagen is found in the cornea, sclera, choroid, iris, and ciliary body. 136'137 A thin scleral coat permitting blue coloration has not been a consistent finding. Reudeman found a deficiency of scleral collagen and an increased density of PAS-fuscin stain, suggesting a relative increase in the mucoid component of the sclera. 138 Eicholtz and Muller examined blue sclerae by electron microscopy and found that while they were of normal thickness, the scleral cells had distended endoplasmic reticulum, and in one case there was deposition of dense material between scleral lamellae. 139 Corneal fiber diameter is diminished by about 25%, while the diameter of the scleral fibers is smaller by 50%. 14~ Bowman's membrane and Bruch's membrane are poorly developed. Clinical evidence for a structural defect in the scleral coat is suggested by diminished ocular rigidity as measured by tonometry. This finding correlated directly with the degree of bluishness and was normal in patients with white sclerae. 141 Blue sclerae have been observed in each of the clinical types of OI. 141'142 In type IV OI, sclerae tend to lighten with age, and in type III OI, white sclerae are commonly observed in the presence of severe bone disease. Our experience is that blue sclerae are not unique to any one phenotype, and thus should not be used to define clinical subgroups. Blue sclerae are also observed in other heritable disorders of connective tissue (e.g., EDS, certain patients with Marfan's syndrome, Menkes' syndrome) and may occur in otherwise normal subjects. Clinical evidence of diminished comeal thickness has been reported by Pederson and Bramsen143; this was not confirmed by Kaiser-Kupfer et al. TM Comeal collagen fibers have also been reported by Habera et al. TM to be thin and irregularly arranged. Several ocular abnormalities have been observed in a small number of patients with blue sclerae such as keratoconus, comeal perforations, megalocomea, and breaks in Descemet's membrane. 145 Most commonly observed is arcus juvenilis or senilis, which may be seen in an absence of blue sclerae. 13 Myopia, which is commonly observed in other
661 connective tissue disorders (e.g., Marfan's syndrome, homocystinuria) is not associated with OI. 2. EAR
The otological and maxillofacial features of OI have been summarized by Bergstrom. 146 Functional deafness requiring the use of a hearing aid appears during the second or third decade in --~10% of OI patients. However, the incidence of subtle defects in auditory function approaches 90% in older subjects. There has been a dispute as to the nature of this defect and the functional disturbance that leads to progressive loss of heating in some patients, but not in others. For many years the anatomical defect in OI was considered identical to otosclerosis; these are now recognized as distinct entities. 147 Biochemical differences in the ossicle bones between OI and otosclerosis have been reported. 148-15o Pederson et al. have demonstrated that in contrast to OI, diminished skeletal bone mineral content is not present in patients with otosclerosis. TM Furthermore, the histopathology of the temporal bone is qualitatively similar to that of the peripheral skeleton in OI. Deficient ossification occurs in the tympanic ring, ossicles, cochlea, and otic capsule, 152 and there may be intracochlear hemorrhage. 153 Microfractures occur in the incus and stapes, and the stapes may be reduced to thin fibrous threads. The stapes footplate may be embedded in vascular fibrous tissue, which has led to an association with otosclerosis. TM Recent studies suggest that sensorineural heating loss is more common than conductive loss. A characteristic audiological pattem of sensorineural heating loss, consisting of a high-pitched sensorineural loss starting at 6000 dB that increases at high frequencies, is found in the OI patient. With aging, the loss involves lower frequencies. 155 Testing of hearing and middle ear function in 55 patients with OI revealed sensorineural heating loss in 49% of patients less than 30 years of age and 94% of those over 30. In contrast, conductive loss was present in only 40% of patients and mixed hearing loss in 10%. Another characteristic feature was revealed by tympanometry. OI patients demonstrated increased compliance of the tympanic membrane suggestive of discontinuity or excessive mobility of the middle ear components. 155'156 However, Reidner et al. have described conductive hearing loss and a stiff middle ear system in O1.157 There are few data regarding the effectiveness of stapedectomy in OI. In an uncontrolled study, Cremess and Garretsen found that no technical problems were encountered during surgery, and that both short- and longterm gains in acuity occurred followed stapedectomy. 158'159However, while surgery may improve heating initially, the long-term value of the procedure remains to be proven. 160
662 3. HEART
The myocardium contains an extensive collagen network and the heart valves are largely collagen. Types I, III, and V collagens are found throughout these structures, although the last two are found primarily in vascular components of heart muscle, the valves, and perimyocytic supporting fibers. 161 Detailed biochemical analysis of heart valves or aortic connective tissue in OI has not been performed. There are several reports of mitral valve prolapse, aneurysms and dissection of the aortic r o o t 162 or cervical vessels, 163 and Ebstein's anomaly TM in OI; however, these occur much less frequently than in either EDS or Marfan's syndrome. 165'166 Only rarely are these lesions clinically important, and they do not appear related to either the severity of skeletal disease or the presence of blue sclerae. A patient with mitral and aortic insufficiency experienced mitral valve dehiscence due to extreme friability of the mitral annulus. 167 Three large series have utilized echocardiography to investigate these lesions in patients with OI and in their relatives. ~68-17~ The results were similar in each. Auscultatory findings of mitral valve prolapse occur in ---1% to 2% of OI patients, while echocardiographic evidence will be found in 10%, contrasted with 5% in the general population. Dilatation of the aortic root occurs in 10% to 12% of the OI population and is unrelated to the presence of mitral valve disease. The murmur of aortic regurgitation is heard in ---2% of patients. Unlike Marfan's syndrome, progression of aortic root dilatation has not been observed in OI. Aortic dilatation appears to occur within certain families: familial clustering of mitral valve prolapse has not been established. 17~ Successful surgical correction of the valvular defects has been reported. ~73 4. PULMONARY FUNCTION IN O l
Impaired pulmonary function occurs in the neonate with severe skeletal deformities, and in a gradually progressive manner in children or adults with severe scoliosis. In the lethal form of OI, death appears to be the result of pulmonary insufficiency: detailed pathological descriptions fail to indicate the cause of this lesion. The authors have observed pulmonary hypoplasia in a neonate with lethal O 1 . TM This child had evidence of a mutant oL 1(I) procollagen. Pulmonary function is variable in children and adults with type III OI; certain patients may develop fight ventricular failure in association with restrictive lung disease and hypoxemia. 175 D. E n d o c r i n e a n d M e t a b o l i c F u n c t i o n in O I 1. SHORT STATURE
Size at birth appears to reflect severity of the disGrowth retardation becomes apparent during
e a s e . 176
DAVID W. ROWE AND JAY R. SHAPIRO
the first 2 years. 177 Diminished growth of the skeleton is found in each OI phenotype, and the majority of patients fall below the third percentile for height. Normal height is attained only in rare patients with mild disease. While in part related to the severity of deformities, Albright and Grunt 177 and Wynne-Davies et al. 88 observed that diminished height occurred independent of the frequency of fractures. In the severely affected, growth may virtually cease after the age of 3 or 4; type III adults may be no taller than 3 ft. In less severely affected subjects, there may be a small growth spurt prior to puberty. At that age, scoliosis, the effects of long bone fractures, and deformities may magnify what is an intrinsic defect in the response of OI bone to normal growth stimuli. Mediators of somatic growth, growth hormone and somatomedin C, have been measured in different types of nonlethal O1.178 Provocative tests of growth hormone reserve, including overnight sampling of growth hormone secretion, have yielded normal values. Also, the meaning of decreased levels of baseline or stimulated somatomedin C in subjects with OI remains open to question, 179 since several investigators have failed to correlate either normal or low somatomedin C with rates of growth, or the growth response to growth hormone in normal or growth hormone-deficient children. 18~ Treatment trials with growth hormone have been carried out showing an initial stimulation of growth rate179'182 ; however, the long-term value of this approach is yet to be demonstrated. 2. THYROID FUNCTION Increased perspiration, frequently mentioned by the families of OI patients, has led to the determination of oxygen consumption rates and thyroid function. Cropp observed basal body temperature in OI subjects to be 37.2~ versus 36.6~ in normals. 183 Also, prepubertal OI patients were found to have significantly higher oxygen consumption and higher calculated metabolic rates than normal. These findings require confirmation, as do reports of increased thyroid function in OI. Distiller et al. found raised thyroid hormone levels that were attributed to a disturbance in serum thyroid-binding globulins in their subjects. TM Evaluation of thyroid function by Shapiro et al. demonstrated normal levels of thyroid hormone, and a normal response of these to the administration of thyrotropin releasing hormone (TRH). 185 Malignant hyperpyrexia has been associated with OI as a result of infrequent individual case reports of hyperpyrexia appearing with anesthesia. 186A metabolic link to OI is undetermined at this time. 187 Constipation with recurrent impaction and abdominal pain is not uncommon in type III OI. 188
CHAPTER 23 OsteogenesisImperfecta 3. MISCELLANEOUS LABORATORY TESTS There are no specific abnormalities in clinical tests that are characteristic of OI. Hypercalciuria is frequently found in children with OI that was unrelated to an endocrine or nutritional cause, but the degree did correlate with disease severity. 189 Despite the continued hypercalciuria, no evidence of renal impairment could be demonstrated, a9~ although nephrocalcinosis and renal stones were present in Vetter et al.'s series. TM The fraction of serum alkaline phosphatase derived from bone may be elevated following a fracture. Aminoaciduria with increased excretion of serine, threonine, valine, ethanolamine, and phosphoethanolamine has been observed in both individual patients and affected families. 192 Kinnett and Bullough observed increased excretion of phosphoethanolamine and cystine in several OI patients. However, urinary-free amino acid excretion was found to be normal, w3 Excretion of bone-derived collagen crosslinks are elevated after fractures land in the more severe forms of OI, reflecting increased basal bone turnover. TM The authors have found that serum parathyroid hormone levels are normal, as are serum determinations of vitamin D metabolites. Increased levels of serum copper and ceruloplasmin have been reported, but Matkovic et al. have found serum and urinary determinations of copper and zinc to be normal. 195 4. CLOTTING
Easy bruising is common in OI and in several of the heritable disorders of connective tissue, particularly in EDS. While several reports have documented abnormal blood platelets, 196 clinically significant bleeding is rarely a complication. ~97Abnormal platelet thromboplastin generation, macroplatelets, and abnormal platelet granulation have been observed. ~98 A study of hemostasis in 58 subjects with mild OI revealed that 35% had increased capillary fragility, 33% had decreased platelet retention, and 23% had reduced factor VII antigen. Reduced ristocetin cofactor, deficient platelet aggregation by collagen, and prolonged bleeding time were less common. 199 5. O l AND PREGNANCY
Most women with mild OI tolerate pregnancy without difficulty; however, severe skeletal deformities complicate pregnancy and delivery for both the mother and the fetus. 2~176176 The incidence of maternal fractures is not increased during pregnancy. Premature rupture of membranes, characteristic of EDS, does not occur in OI. Successful pregnancy and delivery have occurred in patients with type III OI, but these are likely to be complicated by cephalopelvic disproportion. The decision as to whether a patient should have a cesarian section should depend on maternal preference and the physician's judg-
663 ment. Previously unrecognized pelvic fractures, susceptibility to perineal lacerations, and a bleeding tendency may necessitate cesarean section. Uterine rupture has been observed complicating a second pregnancy following an uneventful first pregnancy. TM Prenatal diagnosis of type II OI has been accomplished with the use of ultrasonography performed at about the 18th week of gestation. 2~176 At that time one may visualize fractures, beading of fibs, foreshortening and bowing of long bones, or an enlarged head. Nonlethai but severe OI has been detected in the second trimester, and this is useful when another family member has the disease. 21~ Diagnosis may be not possible in mild, dominantly inherited OI, where severe deformity of the fetus is not likely to be observed. Molecular diagnosis from chorionic villus samples can be informative even prior to detection by ultrasonography provided that the mutation likely to be inherited from the parent is known.33,212,213
IV. PATHOPHYSIOLOGY Diseases such as OI that alter the dynamics of bone turnover need to be clarified at the histomorphological and metabolic levels before their genetic mutations can be integrated with specific phenotypes. Underlying this is the awareness that various gene defects will be expressed in a limited number of similar clinical phenotypes. Analysis of the bone in the murine models of OI may provide one approach with which to overcome the heterogeneity of findings in humans.
A. Skeletal Histopathology and Biochemistry in OI The interpretation of skeletal pathology in OI has been complicated by virtue of (1) clinical heterogeneity, which confuses clinicopathological correlation; (2) the fact that few bone biopsies have been obtained from areas not involved by fracture, or doubly labeled with tetracycline for histomorphometry; and (3) the differences in techniques used for processing samples of bone for histological analysis. The interpretation of a bone biopsy must include consideration of factors that may alter its histomorphometry such as age, level of activity, and exposure to androgenic and estrogenic hormones or other forms of therapy. 214 Furthermore, unapparent abnormalities of collagen in other connective tissues have not been sought. As a result, there are no histological markers that unequivocally distinguish the pathological changes in OI from other osteopenic syndromes, nor is
664 there a good correlation between the severity of OI and histological appearance. Nevertheless, there are several features at the tissue level that assist in the differentiation of the various OI phenotypes. 1. LETHAL (TYPE II) Ol
DAVID W. Rowe AND JAY R. SHAPIRO creased in diameter but have a normal banding pattern. 22~ Osteoblasts show large dilated rough endoplasmic reticulum, which probably represents retained mutant and overmodified type I c o l l a g e n . 221'222 This finding may be explained by the faulty secretion of type I collagen in this form of OI. 223
The striking feature in this group is the paucity of type I collagen in the extracellular matrix. As a result, lamellar bone formation is severely impaired. This can be best seen at the epiphysis where, in normal individuals, vertically oriented columns of chondrocytes differentiate into osteoblasts. In lethal disease there is an abrupt failure of normal ossification with persistence of hypercellular, cartilaginous metaphyseal tissue surrounded by immature woven bone (Fig. 2 3 - 6 ) . The maturation of lamellar bone is also markedly retarded in medullary and cortical zones as evidenced by the presence of multiple pregestational fractures showing partial healing. 2~5-217 Immunostaining demonstrates relative enrichment of type II and IV collagen reflecting the immaturity of the bone matrix, 21s while osteoblasts lie in a proteoglycan-filled lacuna that may adversely affect matrix calcification. 2~9 In contrast to other forms of OI, the osteoblasts appear diminished in number. At the electron microscopic level, there are a reduced number of extracellular type I collagen fibers, which are markedly de-
Severe osteopenia is present from birth. Bone cortices are markedly thinned. Variable amounts of immature or woven bone may be present depending on the severity of the disease. As a consequence of defective periosteal growth and remodeling, the shafts of long bones are slender, widening to a bulbous metaphyseal/epiphyseal zone that can fracture and further contribute to the growth failure. 224 The periosteum and perichondrium appear normal by conventional microscopy. The trabeculae are thin, disorganized, and separated by wide spaces of intervening marrow. Hyperosteocytosis is prominent in this phenotype (Fig. 2 3 - 7 ) , but a consistent abnormality of osteoblast, osteocyte, or osteoclast morphology has not been reported. Secondary centers of ossification are distorted and the epiphysis may contain several small whorls of partially calcified cartilaginous material surrounded by woven bone which disrupt the end plate. 225'226 Electron microscopy has revealed that while bone and
FIGURE 23--6 Bone biopsy of epiphyseal growth plate from a patient with lethal OI. The columns of maturing chondrocytes appear only slightly irregular. There is abrupt failure of endochondral bone formation proximal to the growth plate leaving poorly ossified irregular lamellae with a central cartilage core.
FIGURE 23--7 Toluidineblue stain of an iliac crest bone biopsy in type III OI. The characteristic findings are diminished trabecular volume, hyperosteocytosis, and an irregular pattern of lamellar staining.
2. SEVE~ NONLETHAL (TYPE IlI) Ol
CHAPTER 23 OsteogenesisImperfecta dermal collagen fiber diameter is normal, there are fibers that show fraying and breakage. Using scanning electron microscopy on bone samples from three patients with severe OI, Teitelbaum et al. found that the collagen fibers were diminished in size compared to normals and were arranged in thin sheets suggesting defective bundle formation. 227 Falvo et al. also found bone collagen fiber to have diminished diameter but normal periodicity. TM Fraying and breakage of type I collagen is not unique to OI, having been observed in other heritable disorders of collagen including type IV EDS. 228 3. MILD Ol WITH OR WITHOUT DEFORMITY (TYPES I AND IV)
The mild and moderately severe forms of OI share histopathological as well as phenotypic features. However, the type IV patients present skeletal findings of advanced osteoporosis with cortical thinning ranging from moderate to severe. Considering overlap in phenotype, individual cases range from nearly normal in type I patients, to severe cortical and medullary bone loss in type IV. Microscopically, one sees a decreased number of trabeculae of diminished width, while the depth of osteoid seams and osteoid volume are usually normal. Children with the " t a r d a " variety of OI were found to have low trabecular bone volume associated with an increased bone tumover rate. 229 A decrease in the function of individual osteoblasts was observed in spite of increased formation at the tissue level. An apparent increase in unmineralized osteoid in some patients may be a consequence of a marked decrease in trabecular bone volume. 23~ The staining of bone matrix with PAS or toluidine blue may be abnormal. The absolute number of osteocytes is increased, and an increased number of osteoclasts may be seen in some biopsies. Histomorphometric analyses have demonstrated a decreased fractional area of bone, an absolute increase in the number of osteocytes, and an increased number of resorption surfaces. 23~ In agreement with Baron et al., 229 calculation of static and dynamic parameters of bone function after tetracycline labeling indicated an increase in the rate of bone turnover despite the apparent decrease in the synthesis of bone matrix by individual osteoblasts. St. Marie et al. reported histomorphometric analysis of iliac crest bone following tetracycline labeling from 12 patients with type I or IV OI. TM In the adults, trabecular bone volume was considerably decreased, and the thickness index of osteoid seams was diminished. Trabecular osteoblastic surfaces were increased in four adults, while parameters of resorption were slightly increased. Jones et al. 232 described decreased type I collagen fiber diameter (0.04 to 0.06 txm vs. 0.06 to 0.08 txm in normals) from skin in OI, while Shapiro et al. 233 reported decreased fiber diameter and the presence of marked irreg-
665 ularity of individual fiber outline in a patient with coexistent mild OI and Paget's disease of bone. Biochemical characterization of bone matrix from the more severe forms of OI yields a stoichiometry that is similar to that made by fetal bone cells. T M Normal fetal and newborn bone cells have relatively low levels of type I collagen and decorin 235 and high levels of thrombospondin, fibronectin, hyaluronan, 236 and proteoglycans. 237 Similar changes have been observed in the OIM mouse. 238
B. Bone Mineral Content and Hydroxyapatite Quality in OI Methods available for the quantization of bone mineral content in vivo include: (1) radiogramometry based on the measurement of metacarpal cortical/medullary width239'24~ (2) single photon (125I) densitometry, which measures predominately cortical bone in the radius24~; (3) computerized axial tomographic (CAT) determination of trabecular bone mineral content of vertebral bodies T12 to L4242'243; (4) dual photon absorptiometry (DPA), which employ a 153Gd to measure both cortical and trabecular bone in the lumbar spine244; (5) digital radiography of the axial or vertebral bone245; and (6) dual-energy x-ray absorptiometry (DEXA). Quantitative computed tomography ( Q C T ) 246'247 and D E X A 248'249 appear to have sufficient accuracy and patient acceptance for quantitating bone mineral content. The studies consistently show low bone mineral density relative to agematched controls, which can be used as a diagnostic criterion in certain settings. The demineralization was roughly proportional to the severity of the OI. Type I OI patients appeared to lose bone mineral at a rate two to three times greater than nonaffected subjects. Demineralization was most severe in type III patients. Although the diminished bone density probably reflect reduced bone matrix, there is increasing evidence that the quality of the hydroxyapatite crystals 25~ and their interaction with the collagen fibrils 252'253 are disturbed in OI. Similar techniques have now been applied to murine models of OI and have found the same abnormalities in hydroxyapatite composition 254'255 and apposition. 256 This strengthens the value of the mice as model for human OI.
C. Bone Mechanics and Remodeling The murine models of OI have provided a method for evaluating the mechanical consequences of the abnormal matrix in OI bone. The initial transgenic mice yielded a lethal phenotype precluding the development of a strain for extensive laboratory analysis. However, bone ob-
666
DAVID W. ROWE AND JAY R. SHAPIRO
tained from the milder forms of murine OI (Mov 13) 257 or transgenic strain 258 show decreased resistance to strain and increased brittleness. Interestingly, the mechanical properties improve after the mouse enters puberty by increasing the cortical thickness and bone shaft diameter. This adaptive alteration is seen in human osteoporosis. 259'26~ However, when the mouse ages the remodeling changes are lost and mechanical properties diminish. Abnormal mechanical properties are even present in the heterozygous oim/+ mouse, which cannot be otherwise distinguished from the + / + littermate. 261 This underscores the reserve capacity of normal bone matrix for mechanical loading. There is evidence for increased bone turnover in humans with the more severe forms of OI, although there is great overlap in values depending on the pubertal status, concurrent fractures, and degree of ambulation. Bone-specific crosslinks appear to form normally in OI bone, 262 so the increased urinary excretion does reflect degradation of preformed bone matrix. A similar conclusion was reached utilizing an elegant 42Ca stable isotope dilution technique. 263 Markers of bone formation such as procollagen propeptides are distinctly low in type I OI, consistent with the underlying molecular defect of a null collagen allele. 264'265 However, there is greater variability in this marker in the more severe forms of O1. 266 Failure to see high level in certain individuals may reflect a mutation that causes much of the synthesized collagen to be destroyed prior to its secretion from the cell.
D. Summary and Speculation The above data indicate that there is insufficient type I collagen in bone and suggest that different factors underlie deficient matrix accumulation. There may be either underproduction or excessive turnover of collagen and other matrix components. The woven bone seen in more severely affected cases has not been chemically characterized, but suggests defective osseous maturation possibly due to improper collagen fiber formation. Failure to form a normal mature matrix probably accounts for the qualitative differences in hydroxyapatite interactions with the collagen fibril, further weakening the bone and contributing to rapid bone turnover. The hypercellularity frequently found within OI bone may reflect an effort by the osteoblast to compensate for the inadequate bone matrix, formation through some as yet undefined mechanism. 267 Since formation and turnover are coordinated through a bone coupling factor(s), 268 the increased number of osteoclasts and osteoblasts seen in association with measurements of enhanced bone turnover may be secondary to the production of a defective bone collagen
or faulty association of collagen and glycosaminoglycans in extracellular matrix. High bone turnover would be anticipated during periods of rapid somatic growth, limiting the time available to remodel woven bone to a more mature lamellar matrix. This may account for the high fracture frequency in growing children. With the cessation of somatic growth concurrent with the acquisition of full sexual maturity, fracture frequency slows. It is during this time that widening and strengthening of the cortical bone occur as well as remodeling of woven bone into lamellar bone. Because the turnover demands during sexual maturation are relatively low, the OI osteoblast does gradually accumulate a more stable matrix. However, when turnover again increases in association with loss of the sex hormones, the OI osteoblast cannot produce adequate amount of matrix and the fracture rate begins to increase.
V. C O L L A G E N BONE
CELL
BIOCHEMISTRY
BIOLOGY
AND
AS RELATED
TO OI It is now apparent that various mutations within the type I collagen genes underlie the pathophysiology of OI. The challenge to the clinician and molecular biologist in the next decade will be the integration of clinical and molecular disciplines to obtain more accurate diagnosis, improved genetic counseling, and effective therapy. As a starting point for the clinician, a review of the essential structural and regulatory elements of bone collagen pertinent to OI will be presented. A general discussion of type I collagen has been given elsewhere in this book. At least 15 genetically distinct steps must be successfully carried out between the first step of transcribing the gene and the final extracellular incorporation of the collagen molecule into a crosslinked microfibril. Many of these involve multiple sites within procollagen mRNA or the translated protein. This degree of complexity undoubtedly accounts for the genetic heterogeneity associated with the heritable disorders of connective tissue. In this section, the major steps in this process will be discussed with emphasis on how a regulatory or structural gene defect could result in altered collagen production or stability, and ultimately in the fragile skeleton of OI.
A. S t r u c t u r e o f C o l l a g e n a n d its G e n e 1. COLLAGEN: THE PROTEIN Although there are a minimum of 19 genetically distinct collagenous proteins, 5~ type I is the most prevalent
CHAPTER 23 OsteogenesisImperfecta and germane to diseases of bone. It is principally found in bone, tendons, skin, ligaments, dentin, and sclera, and to lesser extent in lung, blood vessels, and other visceral supporting tissues. Therefore, the heritable disorders involving type I collagen predominantly affect bone, ligaments, and skin; cause minimal involvement of visceral structures; and have no effect on cartilage (type II collagen) or basement membranes (type IV collagen). 269 Type I collagen molecules are organized in a threedimensional arrangement of crosslinks in fibers as interwoven fascicles surrounded by tissue-specific proteoglycans. Little is known of the precise organization of these macromolecular complexes. Therefore, attention has been focused on defining the biochemical nature of the individual fiber components in the absence of knowledge regarding those properties that normally facilitate their interaction. Thus in the following discussion, the reader should bear in mind that a thorough understanding of the heritable connective diseases will only be appreciated when these basic interactions are unraveled. The microfibril is the basic organizational unit of the collagen fiber. 27~ It consists of five collagen molecules arranged in a three-dimensional cylindrical structure and in which individual molecules are placed in quarter stagger relative to their neighbors. 272 The overlapping periodic arrangement of the collagen molecules is responsible for the cross-striations observed at 640 ,~ in collagen fibers by electron microscopy. 273 Each collagen molecule within the microfibril is a rod-shaped structure composed of three polypeptide chains (et chains) arranged in a tight triple-helical conformation. The type I collagen molecule is a heterotrimer composed of two identical oL1 chains and another structurally different e~2 chain, each containing 1000 amino acids. The triplehelical conformation is the result of the repeating triplet (gly-X-Y) that occurs approximately 330 times. The X position is filled with a proline, while the Y position may be proline or hydroxyproline. Hydroxyproline is a postranslational modification of the procollagen oL chains unique to collagen that adds conformational stability to the helix at physiological temperatures. 274 Certain lysine residues are hydroxylated and several of these are also mono- or diglycosylated with glucose and galactose. In the extracellular space lysine and hydroxylysine residues participate in the covalent crosslinkage of adjacent collagen molecules. The formation of these crosslinks ultimately determines the strength of collagenous tissues; however, the formation of normal crosslinks requires that the preceding intra- and extracellular steps of fiber formation were accomplished correctly. 275 Since the recurring (gly-X-Y) triplet is invariant in all collagens, it can be inferred that this sequence is required for normal triple-helical conformation. In fact, the presence of the glycine in the first position permits the chain
667 to maintain a tightly coiled state; a substitution of any other amino acid for a glycine will disrupt the highly ordered structure of the helix. 276 Under experimental conditions, when the temperature is elevated to 41.5~ the triple helix of normal collagen will begin to melt or uncoil. 277 However, if the collagen molecule contains a mutation that disrupts its tight helical configuration, the melting temperature may be reduced to less than physiological levels. 278'28~Occasionally, the disruption can be visualized by electron microscopy of procollagen molecules or in artificially formed fibrils in v i t r o . 281-283 Not only is the correct primary amino acid sequence of the e~ chain essential to the formation of the triple helix, it also contributes polar and nonpolar charges (domains) on the exterior of the triple helix that are essential for orderly fibril f o r m a t i o n . 271'284-286 Fibril aggregation occurs spontaneously in vitro without the aid of enzymes or other extracellular proteins, 287-289 and the process is slowed when an aggregate of normal and mutant collagen molecules are co-polymerized. 29~Ultimately, the stability of the microfibril is derived from enzyme-generated covalent crosslinks that form once the collagen molecules are arranged in register within m i c r o f i b r i l . 278 It is also assumed that noncollagenous proteins and glycosaminoglycans interact with the microfibril leading to a fiber composed of many microfibrils, 291-294 and further stabilized by covalent crosslinks. Thus, the formation of a collagen fiber with normal tensile strength represents the composite effect of many layers of organization that ultimately depend on the correct amino acid sequence of the collagen ct chains. Depending on the type, size, and location of mutation within an ct chain, there may be sufficient disruption of the triple helix to impair formation of the microfibril and c r o s s l i n k s . 28~ However, other mutations may not affect microfibril assembly but instead act to specifically reduce the extent of crosslinking. The location, size, and type of such molecular defects may determine whether the subsequent phenotype resembles either OI or Ehler-Danlos syndrome type V I I . 296
2. COLLAGEN: THE GENE The structure of the human collagen genes have been defined in great d e t a i l . 297-299 Like other genes, it is not continuous but has regions that code for mRNA (exons) and intervening regions that are not present in mature mRNA (introns; see Fig. 2 3 - 8 , step A). There is one copy of each collagen gene per haploid set of chromosomes; oLl(I) is located on chromosome 17, 3~176 while a2(I) is on chromosome 7. TM The collagen genes contain 50 to 51 exons spaced over a range of 18 to 49 Kb. The size of the exons range from 54 to 450 bp: most are less than 100 bp in size. Within the helical coding region, most of the exons code for amino acids in multiples of
DAVID W. ROWE AND JAY R. SHAPIRO
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18 that begin with glycine and end with the Y position codon. 3~176 The relative position and size of each exon is fairly constant between collagen genes, but the intron size varies widely. The gene also codes for the precursor procollagen propeptides (see below) and contains a transcription control region (promoter) at the 5' end of the gene and transcriptional termination signals at the 3' end of the g e n e . 297'3~176
B. Collagen Biosynthesis and Assembly 1. GENE TRANSCRIPTION AND MRNA PROCESSING
Similar to other eukaryotic genes, the collagen promotor contains a TATA sequence that directs the start of
transcription 30 bases "downstream" (Fig. 2 3 - 8 , step A). Sequences up to 150 bases 5' to the TATA region are highly conserved within mammalian species and appear to be crucial to the transcriptional activity of the gene. 3~176 Functional analysis of the promoter in transgenic mice has shown that domains at or beyond - 1 . 7 Kb of the gene control activity in specific types of type I collagen-producing cells. In particular, a "bone element" appears to be essential for activity of the COL1A1 gene in differential osteoblasts, while sequences elsewhere are sufficient for activity in preosteoblastic cells. 3~176 These upstream regions presumably contain hormone-binding sites that control the varied responses that are induced in bone collagen production by such hormones as parathyroid hormone (PTH), 1,25-
CHAPTER23 OsteogenesisImperfecta dihydroxyvitamin D [1,25(OH)2D], epidermal growth factor (EGF), and insulin. 311'312Mutations in any of highly conserved or bone-specific regions would be expected to diminish the activity of the gene, although those mRNAs transcribed from the gene would be structurally normal. Analyses of the proximal COL1A1 promoter domain for a mutation in association with type I OI have been performed without S u c c e s s , 313 but further study of selected individuals is still warranted. Recently, linkage of a polymorphism within the first intron of the COL1A1 gene to familial osteoporosis has been demonstrated, suggesting that a regulatory element in this region could influence transcriptional activity. 3~4 Although there is one copy for each type I collagen allele, twice as much etl(I) mRNA is present in the nucleus and cytoplasm as the oL2(I) mRNA (Fig. 2 3 - 8 , step B). At this point it is not known if this difference in oLchain ratio is due to more active transcription of the oLl(I) gene or a shortened half-life or altered processing of the oL2(I) mRNA. The primary mRNA transcript of the gene contains both introns and exons, that while within the nucleus is processed into mature mRNA (Fig. 2 3 - 8 , step C). For type I collagen, this requires that 49 to 50 splicing events must occur to remove all the introns. This process recognizes mRNA sequences that flank the boundaries of the intron (donor and acceptor sites) in addition to sequences within the body of the intron. 315 Mutations of these sequences can result in either exon skipping or intron inclusion depending on the site mutated and the size of the intron. Inactivation of a splice acceptor site always leads to exon skipping. 316 Skipping also occurs in donor site mutations when the intervening intron is large (> 200 bp) but results in intron inclusion when the intron is small. 317 These differences follow the rules of exon definition. 318 Most examples of exon skipping yield a truncated RNA that is translated normally into a truncated procollagen chain. In contrast, intron inclusion often includes a premature termination codon, which precludes the production of a protein product. Mutations that introduce a stop codon within an exon independent of splicing have a similar outcome. 319 Since polyadenylation must also be completed before a functional mRNA is transported to the cytoplasm, 32~mutation affecting this step will diminish the production of a functional RNA. TM The amount of normal protein produced by the affected allele is reduced or absent, resulting in a null allele. However, the protein made from the mRNA transcribed from the other allele is normal. 2. P R O C o L L A G E N ASSEMBLY AND SECRETION Fully processed nuclear procollagen mRNA is transported to the cytoplasm and translated into a procollagen oL chain in the rough endoplasmic reticulum 322 (Fig. 2 3 - 8 , step D). The initial size of the polypeptide chain
669 is approximately 1750 amino acids, comprising four specific domains: 1. a hydrophobic leader sequence that directs the nascent procollagen chain into the Golgi apparatus, during which time the leader sequence is cleaved. 323 It is within the Golgi apparatus that the posttranslational hydroxylation steps are initiated, which continue until helix formation is complete (Fig. 2 3 - 8 , step E). 2. The N-terminal propeptide, a 200-amino-acid region that may regulate procollagen mRNA translation 324 and may also affect the size of a collagen fiber. 325 The likelihood of translational regulation of procollagen mRNA is strongly suggested by a highly conserved nucleotide sequence within the 5' nontranslated region of procollagen m R N A . 326 3. The helical domain, described above. 4. The C-terminal domain, which contains the information that directs the initial alignment of the three prooL chains. 327 The completed pros chains spontaneously adopt a triple-helical conformation in a C--->N terminal direction after the procollagen chains have been aligned and stabilized by intrachain disulfide bonds present in the Cterminal domain (Fig. 2 3 - 8 , step F). The presence of glycine substitutions within the gly-X-Y triplet slow the rate of helix assembly. 328'329The presence of the proet2(I) chain facilitates helix assembly because homotrimeric molecules are slow to assemble. 33~ Until the triple helix is complete, specific proline and lysine residues can be posttranslationally modified by hydroxylation and, in the case of lysine, glycosylation with glucose and galactose. TM If the rate of helix formation is slowed, then the posttranslational modification process will continue, causing overhydroxylation and excessive glycosylation of lysine residues in the helical domain distal, or Nterminal to the point of helix stability. 332 This polarity of posttranslational modification provides an indirect localization of a mutation. The completed procollagen molecule is secreted from the cell by an exocytotic process that is poorly understood (Fig. 2 3 - 8 , step G). Mutant molecules with helical instability are inefficiently secreted from the cell and thus undergo intracellular degradation. 223 Chaperone proteins resident within the endoplasmic reticulum play a role in distinguishing certain types of mutation, particularly those within the C-terminal propeptide that interfere with procollagen chain assembly. 333-335 Specific extracellular procollagen peptidases cleave the N- and C-terminal propeptides, permitting the helical segment of the molecule to enter into the formation of the microfibril. 336 Lysyl oxidase, acting on specific lysine residues
670
DAVID W. ROWEAND JAY R. SHAPIRO
within the microfibril, initiates the formation of covalent crosslinks between procollagen molecules. 278 Mutations within procollagen that alter its susceptibility to enzymatic cleavage of collagen propeptides have been described. 337 In contrast to most enzymatic diseases that have a recessive inheritance, these structural mutations affect the procollagen substrate for these enzymes. The dominant inheritance pattems occur because one half of the molecules that accumulate in the tissue contain the abnormality. Mutations that primarily affect this aspect of collagen biosynthesis do not result in OI but are associated with EDS type VII. However, "overlap syndromes" with features of OI and EDS have been described that appear to result from mutations that destabilize the helix and affect the cleavage of the Nterminal propeptide. 43'338'339
C. C e l l u l a r R e g u l a t i o n o f B o n e C o l l a g e n Synthesis and Degradation Although bone collagen synthesis and degradation are highly regulated by the calcitropic hormones, 34~ cytokines, and growth f a c t o r s , 341 the influence of these factors on collagen synthesis in OI is unknown. In a manner yet to be defined, the osteoblast can increase collagen production when sensing mechanical stress while bone resorption occurs rapidly upon removal of gravitational f o r c e . 342 In cultured OI fibroblasts, mutant molecules that are initially incorporated into the matrix are gradually lost, providing a mechanism to improve the quality if not the quantity of m a t r i x . 343'344 However, OI bone cells do incorporate the mutated proteins into the matrix both in vitro and in intact bone but not skin. 345'346 This finding may be a partial explanation why the type I collagen mutations are expressed more dramatically in bone than in skin. The factor(s) mediating the increased bone turnover in certain forms of OI does not appear to be a primary or secondary alteration in calcitropic hormones. Clearly, areas for future research will be the mechanisms by which (1) cytokines, and locally acting growth factors each influence total collagen synthesis by the osteoblast, 347 and (2) how the cell recognizes the structurally compromised collagen molecule either before or after it is secreted into the matrix. The relative contribution of increased cell matrix production can result from a combination of increased output per osteoblast unit and an increase in the number of units per unit volume of bone tissue. Despite the fact that bone turnover in OI is high and frequently the bone tissue demonstrates hyperosteocytosis, there is a fundamental defect of matrix production and cell proliferation inherent to the OI bone cell. The inherent properties of the OI bone cells are revealed when grown in tissue
culture where they are removed from the local regulatory factors within the bone matrix. While the properties of OI bone cells indicate that they are fully differentiated osteoblasts, 348 the synthesis and accumulation of collagen and other proteins characteristic of mature bone matrix are signifcantly less than bone cells from an agematched individual. 349-352 The rate of proliferation and the density at confluence of OI-derived cells are significantly less than in cells from normal donors. The relative inefficiency of OI cells in cell culture is revealed when the two populations of cells derived from a mosaic individual are examined. 13 Although initially the cells that grow out in the dish contain the OI mutation, over time the cells containing the mutation are lost from the culture as the normal cells outgrow the OI cells. The synthetic and cell growth aspects appear to be more severe in OI bone cells than in fibroblasts. In many respects, cultured OI cells resemble cells from aged donors, as judged by their growth properties and the rates and even the relative proportion of the matrix protein produced. While this inherent property may be a primary consequence of the mutated collagen protein, it might reflect the chronic proliferative stress placed on the osteoprogenitor lineage pathway. In many respects, OI resembles a hemolytic anemia such as thalassemia or sickle cell disease. The hematopoietic lineage is dramatically stimulated in a futile attempt to produce adequate amounts of a functional globin. Because there appears to be a finite capacity of stem or early progenitor cells to maintain this replicatory activity, the long-term consequence can be an aplastic crisis. It would not be surprising that the same phenomenon could occur in OI and further complicate the pathogenic mechanisms for poor formation. The early stages of OI are characterized by a robust proliferative and synthetic output in the face of the inherent limited capacity. Over time, the lineage potential is gradually lost, which will contribute another major factor toward bone fragility.
VI. BIOCHEMICAL
AND MOLECULAR
TOOLS FOR THE IDENTIFICATION OF MUTATIONS
IN PATIENTS
WITH
OI
Cultured dermal fibroblasts have served as osteoblasts from affected individuals as useful tissues in which to study abnormalities in the synthesis or structure of type I collagen. Although the regulation of type I collagen in the fibroblast is not the same as that of the osteoblast, genetic abnormalities of bone collagen are presumed to be accurately reflected by the collagen synthesized by the fibroblast. This is not surprising, since there is only one copy of each type I collagen gene per haploid cell,
CHAPTER 23 Osteogenesis Imperfecta and therefore a mutation in the gene would be expressed in all cells that make type I collagen. Initial studies in OI characterized the abnormalities of type I collagen protein synthesis in OI, while newer techniques of polymerase chain reaction (PCR) and gene cloning have revealed the primary nucleotide alterations of collagen genes. Because these methods are becoming the basis for understanding the defects in all heritable connective tissue diseases, it is necessary that the clinician appreciate their underlying principles.
671 mutation can be localized to a specific cyanogen bromide fragment. 358 The consequences of a mutation that destabilizes the triple helix can be demonstrated by measuring the thermal stability of the molecule. 43'278 The intact collagen helix is resistant to proteolytic enzymes such as pepsin or trypsin. However, when the temperature is elevated toward 41.5~ the helix starts to unfold (denature) and becomes susceptible to the action of these enzymes. Thus, collagen molecules containing a mutation that destabilizes the helix will be degraded by these enzymes at or below physiological temperatures. 277'28~176
A. A n a l y s i s o f C o l l a g e n a n d P r o c o l l a g e n When dermal fibroblasts are cultured with radiolabeled proline, the radioactive collagen and procollagen bands can be identified by their electrophoretic mobility within polyacrylamide gels. From this analysis, amino acid deletions or insertions within the protein may be detected (see below). Quantization of the density of bands on the autoradiograph can be used to estimate the production rate of type I collagen and its oL chains. For example, diminished total synthesis can reveal a nonfunctional allele for one of the oL chains. 353 It can also indicate unbalanced production of oLchains; for example, if there is a population of molecules composed of three oL1(I) chains [oL1(1)3] rather than the normal heterotrimer of two oL1(I) chains and one oL2(I) chain. T M Evidence for impaired secretion of procollagen molecules can be obtained by separately analyzing the media and cell layer for their content of procollagen molecules. 223 Polyacrylamide gel electrophoresis of procollagen oL chains can demonstrate the presence of a gene deletion or insertion when it is greater than 30 amino acids. 28~ Since each collagen allele is diploid, the presence of such a mutation often results in a widened or duplicate oL chain band that represents both the normal and deleted allele. Evidence for even smaller mutations within the collagen helix can be obtained. 356 Since a mutation may delay the rate of helix formation, this results in excessive posttranslational hydroxylation and glycosylation of lysine residues which, in turn, results in a slowing of the migration rate within the acrylamide gel. A mutation of this type can be localized to a specific region of the helix if the collagen chains are cleaved into smaller fragments with cyanogen bromide and separated by electrophoresis. Since the collagen helix forms in a C--->N terminal direction, those cyanogen bromide fragments having delayed migration must have been positioned N-terminal to the mutation site, while those with a normal gel migration rate should be positioned C-terminal to the site of mutation. 357 Recent advances in protein microsequencing techniques also permit the identification of subtle mutations in type I collagen, particularly when the
B. M o l e c u l a r H y b r i d i z a t i o n Under proper experimental conditions, complementary strands of nucleic acid will anneal or hybridize in a very precise manner. 361 The minimum length of homology between two strands is 50 to 100 nucleotide bases, although hybridization between even shorter lengths is possible. A cloned segment of DNA is made radioactive and is used to detect the presence of a complementary strand of DNA or RNA within a mixture of many other base sequences. The hybridization probe can be either genomic DNA (containing introns and exons) or complementary DNA (cDNA produced by reverse transcription of mRNA) and used to detect a specific DNA or mRNA base sequence. Both forms of cloned DNA to type I collagen genes are now available from a number of research laboratories. They were constructed in a bacterial plasmid from RNA extracted from cultured fibroblasts or isolated from a genomic "library" constructed in lambda phage. Hybridization probes constructed with RNA have even greater sensitivity in detecting a complementary base sequence than do probes made from D N A . 362 However, it is the advent and application of PCR for amplifying segments of RNA or DNA for automated DNA sequencing that has led to an explosion of reported cases in OI in which the underlying mutation has been identified. 363'364A database of reported mutation has recently been established and is available through an intemet site (http://www.le.ac.uk/genetics/collagen/ collagen.html).365 At present the following methods can be undertaken with existing molecular hybridization probes. 1. SOUTHERN AND NORTHERN BLOT HYBRIDIZATION
A mixture of either DNA or RNA can be separated by molecular size within agarose gels and subsequently transferred and fixed to a nitrocellulose membrane. The membrane is incubated with a radiolabeled hybridization probe under defined conditions. After extensive washing,
672 a complementary strand of RNA or DNA is identified by exposing the membrane to an x-ray film. When DNA is identified by this procedure, it is referred to as a Southern blot (after the originator's name), while RNA blots are termed "Northern." In each case only complementary sequences will be hybridized from a complex mixture of unrelated sequences. This method is also used to indicate the size of a particular complementary RNA species or restriction fragment of DNA (see next). The limit of resolution by this method is 50 to 100 bases. 2. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS
Restriction fragments of DNA are generated by commercially available specific bacterial nucleases that cleave DNA through highly defined sequences 4 to 6 bases in length. Since these sites occur infrequently within genomic DNA, their presence can be used to map DNA in a highly reproducible manner. 366 The method of restriction fragment length polymorphism (RFLP) is based on the fact that certain restriction cleavage sites within genomic DNA are lost due to silent base changes within the cleavage sequence. These variations are polymorphic within a population; however, within a family these base changes are transmitted as mendelian dominants. The method does not itself indicate the site of the mutation, but it does demonstrate that the mutation is located within the gene adjacent to or surrounding the p o l y m o r p h i c site. 367 Thus the demonstration of a polymorphism linked to a specific OI phenotype would focus research effort to that ot chain gene. While the approach is no longer used for specific mutation identification, it still is of use in excluding mutation within a collagen gene in association with a bone disorder within a pedigree or subpopulation. 53'368 3. LOCALIZATION OF MUTATION WITHIN COLLAGEN M R N A OR D N A
Because of the size and the multiple exons of the collagen, many investigators have utilized techniques that point to a specific fragment of the gene likely to harbor a mutation prior to initiating DNA sequencing. Early success was obtained by directly assessing collagen RNA for subtle differences in hybridization to an RNA or DNA template that can be detected by chemical
DAVID W. ROWE AND JAY R. SHAPIRO
tension logarithmically amplify the original template such that sufficient DNA is available for analysis after 1 to 2 hours in an automated thermocycler. The template can be genomic DNA, in which case primers are selected to amplify clusters of exons and the included introns. Mature RNA lacking introns can be analyzed by first converting it to a cDNA with reverse transcriptase, giving rise to the acronym of RT-PCR. In either case, specific fragments are amplified throughout the length of the gene or RNA with primer sets derived from the known sequence of each COL1A1 or COL1A2 gene. A variety of methods have been developed to identify a single base pair mutation within the amplified fragment. They are based on a subtle difference of hybridization that occurs when DNA strands from the normal and mutant alleles are melted and reannealed. The resuiting mismatch can be detected by certain organic chemical r e a c t i o n s 373-376 or by subtle differences in migration within a denaturing gel electrophoresis. 377'378The third approach, which has attained the widest acceptance, is based on subtle differences in molecular conformation of single strands of DNA differing in a single base. Here, the PCR-amplified DNA is denatured and then separated by electrophoresis as single strands of DNA in a nondenaturing environment. The method is referred to as single-strand conformation polymorphism ( S S C P ) . 379 4. QUANTITATION OF A SPECIFIC M R N A
The most quantitative method is referred to as a dot blot hybridization. 38~ RNA extracted from tissue is diluted to different concentrations and fixed onto a nitrocellulose membrane. After the RNA is hybridized to a specific radiolabeled cDNA, the intensity of the radioactive spot on the x-ray film is compared to the signal from an RNA standard. However, if the probe crossreacts with other RNA species likely to be in the extract, then standard Northern blot hybridization is necessary. Although methods to quantitate using quantitative RT-PCR have been developed, 381 it is not always required because of the abundance of type I collagen mRNA in most tissue extracts. Any of these methods can be applied to RNA located within the cytoplasm or the nucleus of cultured cells and is valuable in studying regulatory mutations of collagen. 382
c l e a v a g e 369 or R N a s e s . 37~
All of the techniques now in common usage are based on PCR amplification of a specific fragment for subsequent analysis. Synthetic DNA primers that flank the region to be amplified and that are complementary to sequences on opposite strands of the DNA are used to hybridize its template. A thermostable DNA polymerase extends each primer making a copy of the template. Repeated cycles of denaturation, rehybridization, and ex-
C. D N A S e q u e n c i n g The advent of rapid and robotically automated DNA sequencing techniques now makes it possible to directly analyze for a mutation within a collagen gene by sequencing overlapping PCR-generated fragments. 383 While this approach is not routinely available, it has the
CHAPTER 23
673
Osteogenesis Imperfecta
advantage that all potential mutations within a fragment are identified, which is not the case for the method discussed above. However, direct sequencing to total cellular collagen RNA is unlikely to identify null mutation because the mutant template is less than 5% of the normal template. The most efficient approach combines the localization techniques with rapid sequencing.
VII. MOLECULAR PATHOPHYSIOLOGY
phenotypes will be presented in decreasing order of clinical severity.
A. L e t h a l ( T y p e II) O I The majority of research effort has been spent with cultured dermal fibroblasts derived from this type of OI. The best studied case, first identified by Penttinen, 386 has been shown to contain a 651-base-pair deletion within the midportion of one of the oLl(I) alleles (Fig. 2 3 - 9 , step A). This abnormality was first detected in the collagen oL1 chains by the presence of a doublet in the et 1(I) chain region by polyacrylamide gel electrophoresis. 223 Subsequently, a doublet was demonstrated in the etl(I) mRNA. When the etl(I) gene was analyzed it could be shown that one allele lacked a certain restriction site, while another restriction fragment was shortened by
OF OI
Because of the large number of cases of OI that have been studied at the protein and molecular level, the range of mutations of type I collagen that are associated with skeletal disease is well developed although still incompletely understood. 384'385 In the following discussion, the
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Type II OI. Deletion within one ctl(I) collagen allele. In this figure both the normal and abnormal allele of the diploid cell are depicted while only one of the two normal o~2(I) alleles are shown. A deletion within one of the ctl(I) alleles (step A) leads to an initial transcript (step B) and mature mRNA (step D) that is shortened relative to the product of the normal allele. The mRNAs are translated into proof chains that initiate assembly in a C---~N (step F). Any molecule that incorporated the translated product of the shortened et 1(I) mRNA will disrupt the stability of the helix distal to the location of the deletion. Since the procollagen molecules will incorporate either one or two of the abnormal chain, three quarters of the resulting procollagen population will be structurally unsound (step G).
674 about 500 bases. 387 The region of DNA containing the deletion has now been isolated and sequenced by two laboratories. 388'389 The analysis indicates that three exons and associated intervening sequences have been deleted, resulting in an mRNA lacking 252 bases which, in turn, code for a procollagen chain deficient in 84 amino acids (Fig. 2 3 - 9 , steps D and E). Mutations that interrupt the helix weaken the stability of all the procollagen molecules containing at least one of the abnormal procollagen oL chains. 28~ The concept is referred to as the "suicide model" and proposes that three out of four procollagen molecules will have incorporated one or two of the abnormal etl(I) alleles and will therefore be structurally unsound 39~(Fig. 2 3 - 9 , step G). The important findings that have been derived from this case are: (1) the destabilized collagen helix is more susceptible to thermal denaturation, which renders the molecule susceptible to tissue proteases43'278'28~ (2) interruption of the helix decreases the rate of secretion of abnormal molecules from the cell leading to dilatation of the rough endoplasmic reticulum, 223 thus a reduced amount of collagen accumulates in the extracellular space, and that which is secreted is structurally unsound and susceptible to extracellular proteolytic digestion391; and (3) interruption of the helix leads to posttranslational overmodification of the lysine residues in the helical domain, N-terminal to the point where the helix is disrupted. 392 This important point has been used to great advantage by Bonadio and Byers 393 to map the apparent mutations that interrupt the helix in other cases of lethal OI. Although the physiological significance of these changes is uncertain, it may affect the quality of fibril formation or the generation of intermolecular crosslinks ~ Despite the dramatic findings derived from the above lethal case, most cases of lethal OI do not have an oL1chain doublet, indicating that the mutation is below the resolution of this type of analysis. However, most cases do show a delay of oL-chain migration, suggesting posttranslational overmodification of lysine residues. 395 Byers has mapped the location of the putative mutation in some of these cases by determining which cyanogen bromide peptide has delayed migration on acrylamide gels. This analysis suggests that mutations in the Cterminal or midhelical domains of the oLl(I) chain are common to the lethal form of OI. These findings were confirmed by Bateman et a l . 396 and clearly shown to be the result of excessive hydroxylation and glycosylation of hydroxylysine residues consequent to the delay in helix formation. The data suggested that a mutation below the size of biochemical detectability was disrupting the formation of the helix. This possibility has been well demonstrated by two cases in which a cysteine substitution was found
DAVID W. ROWE AND JAY R. SHAPIRO
within type I collagen molecules. This mutation was detected because cysteine is not normally present in the helical domain of type I collagen, and when it is present within two chains of the same molecule, it causes the chain to run as a dimer under nonreducing conditions. Besides cysteine, the other possible substitutions of firstposition glycine include arginine, alanine, serine, aspartic and glutamic acid, tryptophan, and v a l i n e . 397-399 In the case described by Steinmann et a l . 278 the cysteine mutation was found in a patient with type II OI, while in the case of Nicholls et al., the child had a mild form of the disease. 4~176 Recent nucleotide sequencing of the cloned DNA obtained from the lethal case demonstrated that the cysteine was a substitution for a first-position glycine. 4~176 In the mild case, there was no evidence of thermal destabilization of the molecules containing the mutation, suggesting that the cysteine was probably in an X or Y position. 4~ Glycine substitution within the oL2(I) chain is found in association with perinatal lethal OI, although it is less c o m m o n . 4~176 Genetic compound has been suggested as another cause of OI, but the interpretation of these early molecular studies should be reexamined with the newer DNA diagnostic techniques. Other types of mutation have the same consequence of interrupting the integrity of the collagen helix. Errors of splicing that cause exon skipping can truncate a segment of helix, resulting in disease severity that is related to the domain that is deleted. 4~ Inclusion of a segment of nonhelical sequence due to an error of splicing 4~ or a partial gene deletion 41~has been reported. Finally, base substitutions that arise within the C-terminal propeptide and result in lethal OI indicate that chain assembly is initially determined by the interactions in this region of the m o l e c u l e . 411'412 In summary, the unifying abnormality in most forms of lethal OI appears to be the deletion of genetic material or a point mutation of a first-position glycine, which has a deleterious effect on the stability of the helix. As a rule, the severity of the disease may be related to the location of the mutation. The more C-terminal within the helix, the greater the length of unstable helix. 413 It should be anticipated that there will be a spectrum of clinical severity ranging from perinatal lethal to severe type III OI reflecting the locus of the mutation and the length of the destabilized collagen h e l i x . 414'415
B. S e v e r e ( T y p e III) O I Although this is the most severe of the nonlethal forms of OI, a clear biochemical basis for the disease has not yet emerged. In the best studied case (Fig. 2 3 10), the affected child failed to synthesize oL2(I) chains and instead produced a type I et collagen trimer. 354'416
CHAPTER 23
Osteogenesis Imperfecta
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Type III OI. Failure of e~2(I) production. Both e~2(I) alleles of the diploid cell contain the same mutation, which is a 4-bp deletion that places the remainder of the coding region out of phase. The process of transcription and mRNA processing is unaffected. Because the frameshift mutation changes the amino acids within the ot2(I) C-terminal propeptide that are required for chain assembly, this chain does not become incorporated into the procollagen molecule (step F). The result is an oL1(I) trimer that is not sufficient to replace the function of the normal heterotrimer. The unincorporated c~2(I) chains are degraded intracellularly. The parents of this infant are obligate heterozygotes for this mutation and do synthesize a mixture of normal and homotrimer molecules. They do not have OI but may have premature osteoporosis.
However, the cultured fibroblasts do contain oL2(I) m R N A and nascent procollagen e~2(I) chains can be demonstrated intracellularly. 417"418 An S1 nuclease analysis of the collagen m R N A from this case demonstrated a 4-bp deletion. 419 This type of deletion places the remainder of the m R N A codons out of the correct reading phase (frameshift mutation) so that all the amino acids distal to the mutation are nonsense. 42~ The location of this mutation is in the C-terminal propeptide that is essential for initial procollagen chain assembly. Since both alleles are affected (the child was the product of a consanguineous union), no oL2(I) chains are found within the procollagen molecule. The c~l(I) trimer is able to form a helix, although it is less than the heterotrimer, and it shows biochemical evidence of excessive posttranslational modification. 421 It is interesting to note that another case with deficient oL2(I)-chain synthesis has been described in a patient with EDS and no bone disease. 422 This finding points out that the type I collagen genes are not necessarily expressed in a similar manner in all tis-
sues and urges caution in extending results from cultured fibroblasts as necessarily reflective of events within bone. Except for the oim mouse, no other cases resembling this mutation have been observed, nor have deletions similar to those described under lethal OI been reported within either oL chain. However, a range of glycine substitutions and exon skips of the helical domain or base substitution within the C-terminal propeptide of either the oLl(I) or oL2(I) chain have now been amply documented. 423-42s While the impression that a gradient of severity can be correlated to the position of the mutation, many exceptions exist suggesting that there are specifc domains within the molecule that are more tolerant of disruption. 429 In certain cases the inheritance of severe nonlethal OI appears to be autosomal recessive within the black population in South Africa. 35 Molecular studies do not show linkage to type I collagen and no abnormalities of collagen production could be demonstrated. 43~ Another po-
676
DAVID W. ROWE AND JAY R. SHAPIRO
tential case of OI not linked to type I collagen was reported in an Irish pedigree. 43] Thus, mutation in genes other than type I collagen may produce an OI phenotype, but they are most unusual. Instead, most cases of apparent recessive inheritance of nonlethal OI are examples of germinal mosaicism of o n e p a r e n t . 432-434 However, undisclosed genetic compounds could be present.
C. Mild, Deforming OI (Type IV) Patients with this type of OI have been shown to synthesize two populations of type I collagen. One contains proet2 chains that migrate normally in polyacrylamide gels, the other containing slow migrating chains. The slow chain contains a mutation within the oL2(I) chain located in one case toward the N-terminal domain of the helix (Fig. 2 3 - 1 1). In this patient, a deletion of approx-
imately 30 amino acids was detected by the presence of a doublet of the ct2 chain on acrylamide gel electrophoresis, 352 which proved to result from an e x o n skip. 435 In other cases, thermal denaturation studies in association with a change in the oL2(I) chain 436 on delayed migration of the et2(I) chain in a patient with known linkage by RFLP to the tx2(I) gene 437 have been observed. A glycine substitution in the mid- or C-terminal region of the helix has been found in association with type IV O1, 438 although there are many exceptions to the correlations between disease severity and location of the helix interrupting mutation.
D. Mild Nondeforming OI (Type I) The abnormality common to type I OI leads to a decrease in the production of type I collagen rather than a
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CHAPTER 23
677
Osteogenesis Imperfecta
cated protein that is unstable and does not participate in helix formation, 443 a transcript that is subject to rapid d e g r a d a t i o n in either the n u c l e a r or c y t o p l a s m i c comp a r t m e n t of the cell, or a transcript that is retained within the nucleus. 444
structural m u t a t i o n of the protein (Fig. 2 3 - 1 2 ) . T h e mutation is e x p r e s s e d in fibroblasts in cell culture as an elevation in the ratio of type III to type I collagen. 439 U n d e r l y i n g the reduction in type I collagen synthesis in one of these variants is one silent allele for the oLl(I) chain that results in h a l f - n o r m a l oL1(I) m R N A . 44~ Since a stable collagen m o l e c u l e requires two oLl(I) chains, 442 the net p r o d u c t i o n of type I collagen is limited by the availability of the oLl(I) chains. 353 A h e t e r o g e n e o u s variety of m o l e c u l a r m e c h a n i s m s can inactivate the output o f a function p r o c o l l a g e n cx1 (I) chain. T h e m o s t c o m m o n appears to be a p r e m a t u r e stop c o d o n or 1- to 2-bp insertion/deletion that induces a stop c o d o n within the m a t u r e R N A transcript. T h e c o n s e q u e n c e is either a trun-
Alpha 1 (1) ........ A. ~ ......... Mutation within Intronl' ,L B.
c o n f i r m e d by m u t a t i o n a l analysis in specific patients. 45~ T h e recessive o i m / + m o u s e m o d e l of OI m a y be a m o d e l of this f o r m of OI. This subtype of m i l d OI m i g h t be e x p e c t e d to h a v e subtle clinical differences
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FIGURE 23--12 Type I OI. Functionally inactive c~l(I) allele. Both the normal and abnormal allele of the otl(I) gene present in the diploid cell are shown, while only one of the normal oL2(I) alleles is illustrated (step A). In this mutation, those mRNAs transcribed from the allele that contain a mutation that alters the steps of mRNA maturation do not readily enter the cytoplasm for translation (step E). This reduces the total amount of c~l(I) mRNA within the cytoplasm. Instead, the abnormal mRNA accumulates within the nucleus (steps C and D). Those abnormally processed mRNAs that reach the cytoplasm do not produce a normal otl(I) chain because the introns contain stop codons that prevent the completion of the chain (step E). The net effect is the synthesis of c~l(I) and oL2(I) procollagen chains in a 1:1 ratio instead of a 2:1 ratio. Since a stable type I collagen molecule requires two oLl(I) chains, the output of normal molecules is reduced by 50% (steps F and G). The ot2(I) chains present in excess are degraded intracellularly, since a molecule composed of two or three c~2(I) chains is unstable (steps F and G).
678
DAVID W. ROWE AND JAY R. SHAPIRO
from those due to a null allele because the pathophysiological basis of the disease is distinctly different. This is one example where correlating molecular and clinical phenotype may have practical significance. Dominant inheritance is usually present in most families with type I OI, although the variability in severity may be such that mildly affected individuals may go undetected. Since the biochemical findings are quite distinctive, once the abnormality can be demonstrated in affected individuals, it can be used to identify mildly affected family members. Family studies indicate that the disease can result as a new mutation that is then transmitted as a dominant trait.
E. Syndromes with Shared Features An interesting family with a dominantly inherited syndrome of joint and skin laxity, blue sclerae, and increased fracture history was reported by Sippola et al. 43 The molecular basis for the disorder was a small deletion at the extreme N-terminal portion of the oL2(I) chain. The effect of this mutation is an alteration in the rate of conversion of procollagen to collagen. This type of change has been observed in EDS type VII due to a mutation that affects the N-terminal procollagen peptidase cleavage site of the oL1(I) or oL2(I) chain. 28~ Apparently, the deletion present in this family places the cleavage site out of register with other determinants for the procollagen peptidase. 454 The abnormality also destabilizes a small portion of the collagen helix, which may account for the bone fragility.
F. Heritable Osteoporosis Pedigrees demonstrating familial transmission or clustering of osteopenia have been identified. 455 Subjects with this phenotype also display scoliosis and mild joint laxity. From the experience gained studying the milder forms of OI, there is reason to believe that abnormalities of type I collagen (or other matrix components) may be present in certain of these families. 456 A number of determined attempts to discern such patients from unselected osteoporotic patients 52 or pedigrees with familial osteoporosis have been unrewarding. 53 Nevertheless, it can be expected that variable degrees of osteopenia, reflecting the underlying defect of collagen or other matrix protein, do account for the transition in phenotype between type I OI and heritable osteoporosis. The problem is how to distinguish this potential subgroup clinically from other forms of osteoporosis.
VIII. THERAPY The physician involved in the treatment of OI assumes responsibility not only for acute events (e.g., fractures) but also for chronic care including physical and psychological support for both the patient and the family. This must be a team effort: excellent orthopedic care in the absence of comparable physical therapy will prove inadequate. A general pediatrician or internist should also be part of the team to evaluate the growth and development of the child with OI and care for the medical complications that appear in adulthood. Among the latter are pulmonary infection and respiratory insufficiency in severely affected patients of any age, dental care for those with DI, the evaluation and treatment of heating loss, and detection of secondary diseases which may aggravate the skeletal disorder such as diabetes mellitus or thyroid or parathyroid disease. Health professionals who treat this disease must appreciate the psychological needs of parents and the patient who is facing yet another crisis precipitated by a major fracture. 457 Families frequently relate stories of medical personnel who are insensitive to their pain or disability, or who do not appreciate the specific needs required by an individual with brittle bones. Parents are willing to teach the health team how they have learned to deal with these problems, but often find that the caregiver does not heed this advice. It is particularly important for the health professionals to listen to and learn from their patients with OI.
A. Medical Therapy A variety of hormones and mineral supplements considered effective in strengthening bone in postmenopausal osteoporosis and Paget's disease of bone have been administered to patients with OI. These have been summarized by Albright 458 and Gertner and R o o t , 459 who noted that 70% of the articles on medical treatment of OI claimed positive results for 20 different agents. It is not surprising that each ultimately proved ineffective, since none attack the primary defect in O I - - t h a t is, a failure to produce normal type I collagen. Modification of the collagen gene in vivo will be the ultimate curative therapy of OI. At this time, there is no effective hormonal, mineral, or vitamin therapy for any type of osteogenesis imperfecta. From the pathophysiology of OI as revealed by the histomorphological and molecular studies described above, it is now evident that the rationale for the previous therapeutic trials was not well founded. Salmon calcitonin was intended to suppress bone resorption and thereby favor bone f o r m a t i o n . 46~ Controlled studies of
CHAPTER 23 Osteogenesis Imperfecta calcitonin therapy have failed to confirm a significant effect on fracture rate or bone m o r p h o l o g y . 461'462 While it is true that certain forms of OI do have evidence of increased bone turnover, it is likely that defective bone collagen is the stimulus for this process. Inhibiting increased bone turnover would only lead to accumulation of more abnormal bone collagen and a compensatory fall in bone formation. However, a recent report of bisphosphonates in OI children suggest there is a significant reduction of bone pain and increase in ambulation. 463 If this observation is verified by controlled trials showing a long-term reduction in fracture frequency, then the role of inhibitors of resorption, and even the underlying pathophysiological concepts of OI bone, will require a major reevaluation. Several agents have been administered in an effort to increase bone formation. Clinical evidence suggests that anabolic steroids and estrogen therapy primarily decrease bone resorption; an effect on bone formation is probably minimal. 464 Ascorbic acid is a cofactor for prolyl hydroxylase, essential for the formation of stable collagen polymer. It promotes high collagen production by increasing gene transcription and stabilizing collagen m R N A , 465 and maintaining the osteoblast in a differentiated s t a t e . 466 However, there is no evidence that there is ascorbic acid resistance in OI or that supplemental ascorbic acid can increase collagen synthesis. 467 Fluoride therapy appears to increase bone mass in osteoporosis by stimulating proliferation of new osteoblasts. 468 Since a hypercellular bone matrix is frequently found in OI, it is unlikely that fluoride will be of value in most cases of OI. Indeed, two trials indicate that it is not effective in O I . 177'469 Growth hormone and insulin-like growth factor type 1 (IGF-1) are potent stimulators of bone formation and resorption in man 47~ and in rodent models. 472 A number of clinical trials of human growth hormone (hGH) are currently in progress. 179'182 While an anabolic and growth response characteristic of all individuals during the first treatment year was observed, there are no longterm data reported on efficacy or fracture frequency. Since the individuals treated had forms of OI resulting from the production of a mutant collagen molecule, the expectation would be that a fundamentally weak skeleton would be expected to carry a larger body mass. If any patient population might benefit from anabolic bone therapy, it would be a patient with a null COL1A1 gene within the category of type I OI. The experience with hGH or IGF-1 in osteoporotic humans (presumably not secondary to an underlying OI mutation) is still mixed and has not gained wide acceptance. The Mov-13 heterozygous mouse is an excellent example to test the hypothesis of GH utility in type I O1. 473,474 A particularly attractive strategy has been developed by King in which
679 the GH transgene driven by a globin promoter is expressed in erythroblasts. 475 This transgenic mouse strain has large bones and improved mechanical properties. However, when this transgene was bred into the oim/oin mouse, no improvement in mechanical properties was observed, as would be expected because the proportion of mutant c~1(I) trimeric molecules remains unchanged. 476 Unexpectedly, the mechanical properties of the oim/+ mouse did improve up to the level of the + / + mouse even though the oim/+ mouse is phenotypically normal. Clearly, the role of these growth factors, regardless of the way they are presented to bone, needs further evaluation of their effect on bone biology and mechanics before they receive widespread use in humans. Other agents have had an even less credible basis for their use. Calcium and vitamin D supplementation, 477 while not harmful when used in moderation, are inappropriate, since abnormalities in calcitropic hormones or calcium homeostasis have not been demonstrated. Magnesium supplements were intended to correct a postulated, but never confirmed, defect in pyrophosphate metabolism. 478 The flavinoids, ( + ) catechins, were intended to revert collagen and glycosaminoglycan synthesis towards " n o r m a l . " 479 Since these studies have not defined a specific abnormality, their use should be suspect. Flavinoids will decrease collagen production in vitro. 48~ Any future medical therapies must have a solid theoretical basis and be evaluated on a biochemically homogeneous group of patients. Furthermore, no longer can a change in fracture rate be used as the sole criterion of efficacy. Instead, accurate measurements of bone mineral content using dual photon absorptiometry or computer assisted tomography will be necessary to provide periodic quantitative and nonbiased assessments during therapy. Once positive changes are revealed by these techniques, then reliance on fracture rate or change in bone morphology will be possible.
B. S u r g i c a l T h e r a p y The primary goal in the surgical therapy of OI should be directed towards reducing deformity and promoting normal function. 482 This means that fractures must be carefully managed to diminish the possibility of deformity while limiting the period of immobilization. Aggressive surgical therapy should be considered so as to maintain limbs in as near a functional condition as possible. Muscle strength should be maintained between episodes of fracture. This requires the anticipation of deformity following fractures or with weight-beating, and the consideration of bone-straightening procedures even in severe, wheelchair-bound patients. Usually, realignment of a deformed limb requires the insertion of either
680
DAVID W. ROWE AND JAY R. SHAPIRO
a pin or rod once the deformity has been corrected by manual (osteoclasis) or surgical (osteotomy) techniques. Proper timing of the rodding procedure is important; early, rather than late correction of deformity is advisable. This may mean inserting rods by age 3 to 5, depending on the child's mobility and extent of deformity. 483 Internal fixation of the femur using an extensible intramedullary rod with separate bone fragments being threaded onto the rod after the femur has been realigned is utilized to reduce the need for re-rodding as the child grows. 484 Percutaneous rodding allowing more frequent changes during growth has also been used. 485 Complications of rodding procedures include restriction of joint motion (although most patients gain near full range of motion at large joints), migration of the ends of the rod requiting reoperation, bending of the rod, nonunion of the fractured fragments, and rarely, development of hyperplastic
c a l l u s . 486
Moorefield and Miller 1~176 have summarized the longrange results of corrective limb surgery in 31 OI adults studied an average of 19 years postoperatively. Preoperative complications of fracture included limb-length discrepancy, exuberant callus, and common peroneal and radial nerve palsy. Eighteen patients were nonambulatory prior to surgery, and 13 used braces or crutches. At follow-up, only 8 patients remained nonambulatory and wheelchair-bound, 18 were able to walk with braces or crutches, and 5 were independent. Of the 28 patients who had undergone a total of 174 operations (6.2 per patient), follow-up x-rays and clinical examination revealed improvement of the deformity in 17, and no improvement in 11. The overall complication rate was 18%. Once rodded, many children are able to ambulate, sometimes walking for the first time, albeit using crutches or a brace. Similar success with extensible rodding procedures has been reported. 487 The positive impact on the child's psychosocial development may easily justify the surgical trauma and the risk of additional surgery. Upper limb surgery also involves insertion of a pin or rod (Sofield rod, Rush pins) following multiple osteotomies or external fracture and realignment of the limb. Rodding of the radius or ulna is more difficult than with the humerus, but is required less frequently. 488 Limited experience with hip and knee joint replacement has been reported. 489 Leg straightening procedures based on the Ilizarov method of lengthening has been utilized in an adult with mild OI with s o m e s u c c e s s . 49~ The surgical management of deformities of the spine has been reviewed by Benson and Newman. 1~ Because mechanical support (Milwaukee brace, plaster bracing) is not generally useful in OI patients, these authors recommend the use of posterior correction, with fusion or the use of the Harrington instrument inserted at an early age if the scoliosis exceeds 50 degrees. However, the
complication rate is high. Young-Hing and MacEwen have reported that of 29 patients with posterior spinal fusion without bracing and 20 with bracing, there were 5 pseudoarthroses, 2 fractured rods, 1 case of rod protrusion through the skin, and 7 cases where hooks were displaced. 491 The general impression is that severe scoliosis resulting in pulmonary insufficiency cannot be reversed by surgical means.
C. Orthotic Therapy In addition to the primary bone abnormality in OI, immobilization and the absence of weight-bearing further impede an improvement in skeletal mass and muscle strength in these patients. Furthermore, OI children have lax ligaments, which contribute to the instability of large joints. During cycles of fracture and repair, time spent on weight-beating and exercise is lost. The prolonged use of body casts and a reluctance on the part of the family or physician to urge physical therapy cost dearly in the effort to maintain skeletal mass. For example, the stimulus for new bone formation provided by pubertal hormone secretion may be negated while an adolescent spends weeks or months recovering from a major fracture. Individualized orthotic care and rehabilitative therapy are key to gaining confidence and promoting independent activity once fractures have healed. As stressed by Binder, 492 positioning of the infant with OI is critical to maintaining the strength of respiratory, spinal, and neck muscles. Swimming in a tub or heated pool is thought to be one of the most useful procedures, and this can be started as early as age 6 months in many children. A variety of exercises can be employed to assist muscle strengthening prior to ambulation. Children should be encouraged to move in any manner and by any means as early as possible so that weight-beating may strengthen limb and trunk m u s c l e s . 493 However, ambulation is usually delayed depending on the extent of deformity and susceptibility to fracture. B leck has proposed the principle of compression of the incompressible fluid muscle as one way of increasing the stress o n b o n e . 494 Plastic orthoses that conform to limb contour have been designed to promote more effective weight-beating. The principle is to brace the child when ambulation is first anticipated. Binder et a l . 492 have designed ultralight polypropylene braces, extending to the pelvis and fitted for ischial weight-beating (Fig. 23-13). Initially cylindrical, knee joints are added after a year or so. The child's adaptability to these braces is quite remarkable. Trials indicate that children will readily adapt to mechanical support and will become fully active in braces: the positive psychological effect on both the child and family is remarkable. During a limited
CHAPTER 23 OsteogenesisImperfecta
681 healthy peers. Continuing efforts to maximize physical and intellectual development are required to ensure the achievement of independent living in spite of a significant physical handicap. 5~176176 There are a number of voluntary organizations that are concerned with many of these issues as well as promoting research of the diseases, including the following: Osteogenesis Imperfecta Foundation 804 W. Diamond Avenue Gaithersburg, MD 20878 Tel: (301) 947-0083 and Children's Brittle Bone Foundation P.O. Box 27 Highland Park, IL 60035 Tel: (847) 433-4981 The physician who cares for an OI individual should make contact with the local OI group to learn of the resources available for his patient.
FIGURE 23--13 This 3-year-old child is standing in lightweight polypropylene braces, using a walker for support. Creative application of orthotic principles is essential to promote early ambulation, peer acceptance, normal schooling, and normal social development in the child with severe OI.
study, no exercise-related fractures occurred in four children while they were in braces. 495 Weight-bearing walkers and A-flame orthoses have been designed to permit stress on the lower limbs without full weight-bearing. Children confined to a wheelchair require special support to minimize the risk of scoliosis. Additional work on the subject using controlled studies will be required before the effect of early weight-beating on long bones or the spine in OI can be adequately a s s e s s e d . 496'497
IX. FUTURE THERAPEUTIC
DIAGNOSTIC DIRECTIONS
AND
Because OI has such a severe and long-term impact on the OI patient and family, it is important that the clinician be aware of emerging diagnostic and therapeutic modalities. It is hard for the family to face their daily disappointments with OI if their physician is not enthusiastic about future developments that may affect the lives of their OI patients. The insights gained into the molecular basis of OI that have developed within the past 5 years are astounding. Recent advances in molecular technologies lead many investigators to feel that the tools to define any type of gene mutation are now available. When these methods are applied to the problem of OI, the following developments can be anticipated.
D. P s y c h o l o g i c a l S u p p o r t s A. D i a g n o s i s The stress of repeated hospitalizations and the need to adapt to prolonged immobility in the hospital or at home places an incredible strain on family life. Little attention has been devoted by the medical community toward an understanding of why some families succeed and others fail in this difficult t a s k . 498'499 Unlike many other chronic illnesses, these families must rear a child with an exceptional intellectual potential but whose disease will always limit their ability to compete with their
Commercial sites for obtaining a molecular diagnosis for OI are now available. 5~176 This approach will permit identification of subjects carrying (or free of) a specific mutation in the collagen gene that is of particular value to a family in which one parent is a somatic mosaic. Specific mutation identification may be predictive of the functional nature of the collagen abnormality and its clinical expression, which should promote development
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of the specific therapy aimed at the pathophysiological consequences of the mutation.
B. Animal Models for the Evaluation
of Therapy Until recently, every new therapeutic agent was evaluated in children, without much supportive animal data indicative that a positive outcome was possible. This approach is no longer acceptable, as there are many murine models now available to demonstrate potential efficacy. It is incumbent on the medical profession to demand animal data prior to human studies, because the young, impressionable, and desperate parents of an infant with severe OI are not in a position to be "informed consumers"; their infant, even less so. The potential risk and discomfort experienced by children treated with calcitonin and fluoride in the past pale against the use of growth factors, bisphosphonates, and bone marrow transplantation being considered in the future. Animal models have the added advantage that the effect of prolonged treatment or long-term consequences can be assessed in a relatively short period of time. An inherent danger of all medical trials has been apparent positive initial outcomes with little investigation of the long-term consequences. Fracture frequency in children is a notoriously bad indicator of bone strength, while this measurement can be made directly in a mouse. Finally, the murine models provide a stable genetic background to evaluate the effect of an intervention with the identical mutation in a large number of experimental subjects.
C. Somatic Gene Therapy Gene therapy for OI presents a radically different problem of therapy than most of the heritable disorders that are currently under investigation. These strategies are directed at diseases for which the underlying defect is a deficiency in the enzyme activity, receptor, or growth factor. OI more closely resembles the problems of gene therapy for the acquired immunodeficiency syndrome (AIDS), in which it is the presence of the abnormal gene product (the AIDS virus or mutant collagen molecules) that is causing the disease. In either case the essential problem is how to selectively remove the gene product while preserving the remaining cellular function. For OI it is even more complex because ideally only the mutant but not the normal gene product needs to be altered, yet the two products may differ by only a single base or amino acid. The clinical observation that suggests somatic gene therapy may have a role in OI comes from the obser-
vation of parents who are somatic mosaics for OI. Although they may have a degree of OI mosaicism reaching as high as 40%, rarely do they have evidence of bone disease, although other minor connective tissue features have been described. This suggests that the presence of the normal bone cells somehow inactivate the deleterious effect of the OI cells. Direct analysis of bone from the individuals with somatic mosaicism has never been performed, so the mechanism for the protective effect is unknown. It is possible that the OI bone cells have been completely replaced by normal cells due to the inefficiency of cell proliferation and production. Furthermore, the matrix synthetic capacity of the normal cells is so much greater that even if OI cells are present within bone, they may contribute little to the total matrix accumulation. A number of reports now suggest that bone marrow transplantation may achieve the desired mosaicism that might improve bone strength. Currently, very little is known about the ability of osteoprogenitor cells to be transplanted by a systemic route, even though this route is routinely used for hematopoietic engraftment. Studies of mice or humans who have had standard bone marrow transplantation suggest that marrow stromal and bone engraftment are controversial. 5~176 A number of research groups are exploring various ways in which the bone cell progenitors can be isolated, expanded, and then reinfused either systemically or directly within the bone marrow to achieve engraftment. 5~ This author feels that these very fundamental problems need to be investigated thoroughly in mice or other experimental animals before being applied to affected individuals. The second problem to overcome is the rejection of the transplant. Chronic use of immunosuppressives could be as damaging to bone as the underlying disease. The ideal strategy envisions harvesting stromal cell progenitors from the affected individual and genetically engineering them to reduce the production of the mutant gene product. It may be possible to specifically inactivate the mutant collagen gene with ribozymes that can discriminate a target that differs by a single base. 5~ This is a major advantage over standard antisense approaches, which inactivate transcripts from both collagen alleles, unless there is a major hybridization difference between the two transcripts. 51~ Trans-RNA splicing is another approach that requires evaluation. 512 While it does not discriminate at the nucleotide level, it has the advantage that an abnormal transcript is converted to a normal transcript. Finally, a technique to correct a mutation within genomic DNA of an intact cell population at an unexpected high frequency utilizing a chimeric RNA-DNA oligonucleotide has been reported. 513 If this approach proves to work in other laboratories, then the ideal tool for genetic engineering of a genomic mutation will be in hand.
CHAPTER 23
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683
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in a proband with osteogenesis imperfecta. J Biol Chem 258: 7721-7728, 1983. Gillerot Y, Druart JM, Koulischer L: Lethal perinatal osteogenesis imperfecta in a family with a dominantly inherited type I. Eur J Pediatr 141:119-122, 1983. Nicholls AC, Osse G, Schloon HG, et al: The clinical features of homozygous oL2(I) collagen deficient osteogenesis imperfecta. J Med Genet 21:257-262, 1984. Chu ML, Rowe DW, Nicholls AC, et al: Presence of translatable mRNA for pro et2(I) chains in fibroblasts from a patient with osteogenesis imperfecta whose type I collagen does not contain oL2(I) chains. Coil Relat Res 4:389-394, 1984. Deak SA, Nicholls FM, Pope FM, Prockop DJ: The molecular defect in a nonlethal variant of osteogenesis imperfecta. Synthesis of pro-a2(I) chains which are not incorporated into trimers of type I procollagen. J Biol Chem 258:15192-15197, 1983. Dickson LA, Pihlajaniemi T, Deak S, et al: Nuclease S1 mapping of a homozygous mutation in the carboxyl propeptide coding region of the pro ct2(I) collagen gene in a patient with osteogenesis imperfecta. Proc Natl Acad Sci USA 81:4524-4528, 1984. Pihlajaniemi T, Dickson LA, Pope FM, et al: Osteogenesis imperfecta: Cloning of a pro-ct2(I) collagen gene with a frameshift mutation. J Biol Chem 259:12941-12944, 1984. Deak SB, van der Rest M, Prockop DJ: Altered helical structure of a homotrimer of oL1(I) chains synthesized by fibroblasts from a variant of osteogenesis imperfecta. Coil Relat Res 5:305-313, 1985. Hata R-I, Kurata S-I, Shinkai H: Existence of malfunctioning proct2(I) collagen genes in a patient with a proet2(I)-chaindefective variant of Ehlers-Danlos syndrome. Eur J Biochem 174:231-237, 1988. Pack M, Constantinou CD, Kalia K, et al: Substitution of serine for alpha l(I)-glycine 844 in a severe variant of osteogenesis imperfecta minimally destabilizes the triple helix of type I procollagen. The effects of glycine substitutions on thermal stability are either position of amino acid specific. J Biol Chem 264: 19694-19699, 1989. Nicholls AC, Oliver J, Renouf DV, et al: Substitution of cysteine for glycine at residue 415 of one allele of the alpha 1(I) chain of type I procollagen in type III/IV osteogenesis imperfecta. J Med Genet 28:757-764, 1991. Forlino A, Zolezzi F, Valli M, Pignatti PF, et al: Severe (type III) osteogenesis imperfecta due to glycine substitutions in the central domain of the collagen triple helix. Hum Mol Genet 3: 2201 - 2206, 1994. Mackay K, De Paepe A, Nuytinck L, Dalgleish R: Substitution of glycine-172 by arginine in the alpha 1 chain of type I collagen in a patient with osteogenesis imperfecta, type III. Hum Mutat 3:324-326, 1994. Molyneux K, Starman BJ, Byers PH, Dalgleish R: A single amino acid deletion in the alpha 2(1) chain of type I collagen produces osteogenesis imperfecta type III. Hum Genet 90:621628, 1993. Oliver JE, Thompson EM, Pope FM, Nicholls AC: Mutation in the carboxy-terminal propeptide of the pro-alpha-l(1) chain of type I collagen in a child with severe osteogenesis imperfecta (OI type III)--possible implications for protein folding. Human Mutat 7:318-326, 1996. Wenstrup RJ, Shrago-Howe AW, Lever LW, et al: The effects of different cysteine for gylcine substitutions within alpha 2(1) chains. Evidence of distinct structural domains within the type I collagen triple helix. J Biol Chem 266:2590-2594, 1991. Wallis GA, Sykes B, Byers PH, et al: Osteogenesis imperfecta type III: Mutations in the type I collagen structural genes,
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COL1A1 and COL1A2, are not necessarily responsible. J Med Genet 30:492-496, 1993. Williams EM, Nicholls AC, Daw SC, et al: Phenotypical features of an unique Irish family with severe autosomal recessive osteogenesis imperfecta. Clin Genet 35:181-190, 1989. Mottes M, Gomez Lira MM, Valli M, et al: Patemal mosaicisim for a COL1A1 dominant mutation (alpha 1 Ser-415) causes recurrent osteogenesis imperfecta. Hum Mutat 2:196-204, 1993. Namikawa C, Suzumori K, Fukushima Y, et al: Recurrence of osteogenesis imperfecta because of paternal mosaicism: Gly862---~Ser substitution in a type I collagen gene (COL1A1). Hum Genet 95:666-670, 1995. Lund AM, Schwartz M, Raghunath M, et al: Gly802asp substitution in the proa2(I) collagen chain in a family with recurrent osteogenesis imperfecta due to paternal mosaicism. Eur J Hum Genet 4:39-45, 1996. Chipman SD, Shapiro JR, McKinstry MB, et al: Expression of mutant alpha (I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV. J Bone Miner Res 7:793-805, 1992. Wenstrup RJ, Hunter AGW, Byers PH: Osteogenesis imperfecta type IV: Evidence of abnormal triple helical structure of type I collagen. Hum Genet 74:47-53, 1986. Wenstrup RJ, Tsipouras P, Byers PH: Osteogenesis imperfecta type IV. Biochemical confirmation of genetic linkage to the pro e~2(I) gene of type I collagen. J Clin Invest 78:1449-1455, 1986. Superti-Furga A, Pistone F, Romano C, Steinmann B: Clinical variability of osteogenesis imperfecta linked to COL1A2 and associated with a structural defect in the type I collagen molecule. J Med Genet 26:358-362, 1989. Rowe DW, Shapiro JE, Poirier M, Schlesinger S: Diminished type I collagen synthesis and reduced et 1(I) collagen messenger RNA in cultured fibroblasts from patients with dominantly inherited (type I) osteogenesis imperfecta. J Clin Invest 76:604611, 1985. Willing MC, Pruchno CJ, Atkinson M, Byers PH: Osteogenesis imperfecta type I is commonly due to a COL1A1 null allele of type I collagen. Am J Hum Genet 51:508-515, 1992. Willing MC, Pruchno CJ, Byers PH: Molecular heterogeneity in osteogenesis imperfecta type I. Am J Med Genet 45:223-227, 1993. Tkocz C, Kuhn K: The formation of triple-helical collagen molecules from a l or oL2 polypeptide chains. Eur J Biochem 1: 454-462, 1969. Bateman JF, Lamande SR, Dahl HH, et al: A frameshift mutation results in a truncated nonfunctional carboxyl-terminal pro alpha 1(I) propeptide of type I collagen in osteogenesis imperfecta. J Biol Chem 264:10960-10964, 1989. Redford-Badwal DA, Stover ML, Valli M, et al: Nuclear retention of COL1A1 messenger RNA identifies null alleles causing mild osteogenesis imperfecta. J Clin Invest 97:1035-1040, 1996. Deak SB, Scholz PM, Amenta PS, et al: The substitution of arginine for glycine 85 of the alpha 1(I) procollagen chain resuits in mild osteogenesis imperfecta. The mutation provides direct evidence for three discrete domains of cooperative melting of intact type I collagen. J Biol Chem 266:21827-21832, 1991. Valli M, Zolezzi F, Mottes M, et al: Gly85 to Val substitution in pro alpha 1(I) chain causes mild osteogenesis imperfecta and introduces a susceptibility to protease digestion. Eur J Biochem 217:77-82, 1993.
694 447. Wallis G, Beighton P, Boyd C, Mathew CG: Mutations linked to the pro oL2(I) collagen gene are responsible for several cases of osteogenesis imperfecta type I. J Med Genet 23:411-416, 1986. 448. Sykes B, Ogilvie D, Wordsworth P, et al: Osteogenesis imperfecta is linked to both type I collagen structural genes. Lancet 2:69-72, 1986. 449. Grobler-Rabie AF, Wallis G, Brebner DK, et al: Detection of a high frequency Rsa I polymorphism in the human pro ot2(I) collagen gene which is linked to an autosomal dominant form of osteogenesis imperfecta. EMBO J 4:1745-1748, 1985. 450. Nuytinck LR, Dalgleish R, Spotila L, et al: Substitution of glycine-661 by serine in the alpha-1 (I) and alpha-2(I) chains of type I collagen results in different clinical and biochemical phenotypes. Hum Genet 97:324-329, 1996. 451. Nicholls AC, Oliver J, McCarron S, et al: Splice site mutation causing deletion of exon 21 sequences from the pro-alpha-2(I) chain of type I collagen in a patient with severe dentinogenesis imperfecta but very mild osteogenesis imperfecta. Hum Mutat 7:219-227, 1996. 452. Weil D, Bernard M, Combates N, et al: Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant. J Biol Chem 263:8561-8564, 1988. 453. Kuivaniemi H, Sabol C, Tromp G, et al: A 19-base pair deletion in the pro-oL2(I) gene of type I procollagen that causes in-frame RNA splicing from exon 10 to exon 12 in a proband with atypical osteogenesis imperfecta and in his asymptomatic mother. J Biol Chem 263:11407-11413, 1988. 454. Dombrowski KE, Vogel BE, Prockop DJ: Mutations that alter the primary structure of type I procollagen have long-range effects on its cleavage by procollagen N-proteinase. Biochemistry 28:7107-7112, 1989. 455. Shapiro JR, Rowe DW, Burn V: Familial osteoporosis pedigrees. J Bone Miner Res 2 (Suppl 1):344A, 1987. 456. Spotila LD, Constantinou CD, Sereda L, et al: Mutation in a gene for type I procollagen (COL1A2) in a woman with postmenopausal osteoporosis: Evidence for phenotypic and genotypic overlap with mild osteogenesis imperfecta. Proc Natl Acad Sci USA 88:5423-5427, 1991. 457. Shea-Landry GL, Cole DE: Psychosocial aspects of osteogenesis imperfecta. Can Med Assoc J 135:977-981, 1986. 458. Albright JA: Systemic treatment of osteogenesis imperfecta. Clin Orthop 159:88-96, 1981. 459. Gertner JM, Root L: Osteogenesis imperfecta. Orthop Clin North Am 21:151-162, 1990. 460. Rosenberg E, Lang R, Boisseau V, et al: Effect of long-term calcitonin therapy on the clinical course of osteogenesis imperfecta. J Clin Endocrinol Metab 44:346-355, 1977. 461. August GP, Shapiro JR, Hung W: Calcitonin therapy of children with osteogenesis imperfecta. J Pediatr 91:1001 - 1005, 1977. 462. Pedersen U, Charles P, Hansen HH, Elbrond O: Lack of effects of human calcitonin in osteogenesis imperfecta. Acta Orthop Scand 56:260-264, 1985. 463. Bishop NJ, Lanoue G, Chabot G, et al: Clinical and bone densitometric improvement after pamidronate infusion in children with severe osteogenesis imperfecta. J Bone Miner Res 11 :$645, 1996. 464. Cattell HS, Clayton B: Failure of anabolic steroids in the treatment of osteogenesis imperfecta. J Bone Joint Surg 50A: 123141, 1968. 465. Rowe LB, Schwarz RI: Role of procollagen mRNA levels in controlling the rate of procollagen synthesis. Mol Cell Biol 3: 241-249, 1983.
DAVID W. ROWE AND JAY R. SHAPIRO 466. Franceschi RT, Iyer BS, Cui Y: Effects of ascorbic acid on collagen matrix formation and osteoblast differentiation in murine MC3T3-E1 cells. J Bone Miner Res 9:843-854, 1994. 467. Kurz D, Eyring EJ: Effects of vitamin C on osteogenesis imperfecta. Pediatrics 54:56-61, 1974. 468. Farley JR, Wegedal JE, Baylink DJ: Fluoride directly stimulates proliferation and alkaline phosphatase activity of bone-forming cells. Science 222:330-332, 1983. 469. Shoenfeld Y, Fried A, Ehrenfeld NE: Osteogenesis imperfecta. Review of the literature and presentation of 29 cases. Am J Dis Child 129:679-687, 1975. 470. Johansson AG, Lindh E, Blum WF, et al: Effects of growth hormone and insulin-like growth factor I in men with idiopathic osteoporosis. J Clin Endocrinol Metab 81:44-48, 1996. 471. Brixen K, Kassem M, Nielsen HK, et al: Short-term treatment with growth hormone stimulates osteoblastic and osteoclastic activity in osteopenic postmenopausal women m a dose response study. J Bone Miner Res 10:1865-1874, 1995. 472. Andreassen TT, Jorgensen PH, Flyvbjerg A, et al: Growth hormone stimulates bone formation and strength of cortical bone in aged rats. J Bone Miner Res 10:1057-1067, 1995. 473. Bonadio JF, Stewart TA, Baker AR, Goldstein SA: Local expression of human growth hormone in bone results in impaired mechanical integrity in the skeletal tissue of transgenic mice. J Orthop Res 14:598-604, 1996. 474. Jepsen KJ, Goldstein SA, Kuhn JL, et al: Type I collagen mutation compromises the post-yield behavior of Mov 13 long bone. J Orthop Res 14:493-499, 1996. 475. Saban J, Schneider GB, Bolt D, King D: Erythroid-specific expression of human growth hormone affects bone morphology in transgenic mice. Bone 18:47-52, 1996. 476. King D, Saban J, Flay N, et al: Localized production of growth hormone improves bone quality in a mouse model of osteogenesis imperfecta. J Bone Miner Res 11:$78 (abstract), 1996. 477. Gertner J, Baron R, Vigery A, Lasng R: Cyclical therapy of osteogenesis imperfecta with fluroide and 25 hydroxyvitamin D. Calcif Tissue Int 31:62 (abstract), 1980. 478. Granada JL, Falvo KA, Bullough P: Pyrophosphate levels and magnesium oxide therapy in osteogenesis imperfecta. Clin Orthop 126:228-231, 1977. 479. Cetta G, Balduini C, Valli M, et al: Influence of a flavinoid on some abnormalities of connective tissues in osteogenesis imperfecta. Presp Inherit Metab Dis 3:181-183, 1979. 480. Becker Y, Stevely W, Hamaburger Y, et al: Effect of flavonoid (+) cyanidanol-3 on procollagen biosynthesis and transport in normal and ataxia telangiectasis cultured skin fibroblasts. Connect Tissue Res 8:77-84, 1981. 481. Blumenkrantz N, Asboe-Hansen G: Effect of (+) catechin on connective tissue. Scand J Rheumatol 7:55-60, 1978. 482. Cole WG: Early surgical management of severe forms of osteogenesis imperfecta. Am J Med Genet 45:270-274, 1993. 483. Engelbert RH, Helders PJ, Keessen W, et al: Intramedullary rodding in type III osteogenesis imperfecta. Effects on neuromotor development in 10 children. Acta Orthop Scand 66:361-364, 1995. 484. Stockley I, Bell MJ, Sharrard WJ: The role of expanding intramedullary rods in osteogenesis imperfecta. J Bone Joint Surg Br 71:422-427, 1989. 485. McHale KA, Tenuta JJ, Tosi LL, McKay DW: Percutaneous intramedullary fixation of long bone deformity in severe osteogenesis imperfecta. Clin Orthop 305:242-248, 1994. 486. Porat S, Heller E, Seidman DS, Meyer S: Functional results of operation in osteogenesis imperfecta: Elongating and nonelongating rods. J Pediatr Orthop 11:200- 203, 1991.
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487. Nicholas RW, James P: Telescoping intramedullary stabilization of the lower extremities for severe osteogenesis imperfecta. J Pediatr Orthop 10:219- 223, 1990. 488. Root L: Upper limb surgery in osteogenesis imperfecta. Clin Orthop 159:141-146, 1981. 489. Papagelopoulos PJ, Morrey BF: Hip and knee replacement in osteogenesis imperfecta. J Bone Joint Surg Am 75:572-580, 1993. 490. Ring D, Jupiter JB, Labropoulos PK, et al: Treatment of deformity of the lower limb in adults who have osteogenesis imperfecta. J Bone Joint Surg Am 78:220-225, 1996. 491. Yong-Hing K, MacEwen GD: Scoliosis associated with osteogenesis imperfecta: Results of treatment. J Bone Joint Surg 64B: 36-43, 1982. 492. Binder H, Hawks L, Graybill G, et al: Osteogenesis imperfecta: Rehabilitation approach with infants and young children. Arch Phys Med Rehabil 65:537-541, 1984. 493. Daly K, Wisbeach A, Sanpera I Jr, Fixsen JA: The prognosis for walking in osteogenesis imperfecta. J Bone Joint Surg Br 78:477-480, 1996. 494. Bleck EE: Nonoperative treatment of osteogenesis imperfecta: Orthotic and mobility management. Clin Orthop 159:111 - 122, 1981. 495. Gerber LH, Binder H, Weintrob J, et al: Rehabilitation of children and infants with osteogenesis imperfecta. A program for ambulation. Clin Orthop 251:254-262, 1990. 496. Binder H, Conway A, Gerber H: Rehabilitation approaches to children with osteogenesis imperfecta: A ten-year experience. Arch Phys Med Rehabil 74:386-390, 1993. 497. Binder H, Conway A, Hason S, et al: Comprehensive rehabilitation of the child with osteogenesis imperfecta. Am J Med Genet 45:265-269, 1993. 498. Brodin J: Children and adolescents with brittle bones--psychosocial aspects. Child Care Health Dev 19:341 - 347, 1993. 499. Cole DE: Psychosocial aspects of osteogenesis imperfecta: An update. Am J Med Genet 45:207-211, 1993. 500. Dubowski FM: Children with osteogenesis imperfecta. Nurs Clin North Am 11:709-715, 1976. 501. Werner P, Metz L, Dubowski F: Nursing care of an osteogenesis imperfecta infant and child. Clin Orthop 159:108-110, 1981. 502. Byers PH: Analysis is protein based with subsequent molecular studies. Contact Melanie Pepin at the University of Washington
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at 206-543-5464. Instructions for sample submission are provided at the web site: http://www.pathology.washington.edu/ byers.html. Prockop DJ: Analysis is genomic DNA based. Contact Maureen Kinnarney at Center for Gene Therapy, Alleghany University of Health Science at 215-762-7234. Instructions for sample submission are provided at the web site: http://healthlinks. washington.edu/helix/. Friedenstein AJ, Ivanov-Smolenski AA, Chajlakyan RK: Origin of bone marrow stromal mechanocytes in radiochimeras and heterotopic transplants. Exp Hematol 6:440-444, 1978. Hollings PE, Fitzgerald PH, Heaton DE, Beard MJ: Host origin of in vitro bone marrow fibroblasts after bone marrow transplantation in man. Int J Cell Cloning 2:348-351, 1984. Keating A, Singer JW, Killen PD, et al: Donor origin of the in vitro haemopoietic microenvironment after marrow transplantation in man. Nature 51:217- 305, 1982. Piersma AH, Ploemacher RE, Brockbank KM: Transplantation of bone marrow fibroblastoid stromal cells in mice via the intravenous route. Br J Haematol 54:285-292, 1983. Pereira RF, Halford KW, O'Hara MD, et al: Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci USA 92:4857-4861, 1995. Grassi G, Marini JC: Ribozymes - - structure, function and potential therapy for dominant genetic disorders. Ann Med 28: 499-510, 1996. Khillan JS, Li S-W, Prockop DJ: Partial rescue of a lethal phenotype of fragile bones in transgenic mice with a chimeric antisense gene directed against a mutated collagen gene. Proc Natl Acad Sci USA 91:6298-6302, 1994. Wang Q, Marini JC: Antisense oligodeoxynucleotides selectively suppress expression of the mutant alpha 2(1) collagen allele in type IV osteogenesis imperfecta fibroblasts. A molecular approach to therapeutics of dominant negative disorders. J Clin Invest 97:448-454, 1996. Mikheeva S, Jarrell KA: Use of engineered ribozymes to catalyze chimeric gene assembly. Proc Natl Acad Sci USA 93: 7486-7490, 1996. Colestrauss A, Yoon KG, Xiang YF, et al: Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide. Science 273:1386-1389, 1996.
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~ H A P T E R 2z
Skeletal Disorders Characterized By Osteosclerosis Or Hyperostosis MICHAEL P. WHYTE
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
I. II. III. IV. V. VI.
Introduction Osteopetrosis Carbonic Anhydrase II Deficiency Pycnodysostosis Osteomesopyknosis Progressive Diaphyseal Dysplasia (Camurati-Engelmann Disease) VII. Endosteal Hyperostosis VIII. Osteopoikilosis IX. Osteopathia Striata
X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII.
provide insight into mineral metabolism and skeletal biology. Hereditary, neoplastic, hematological, infectious, endocrinological, metabolic, and dietary disturbances can be at fault. 2'7 Cumulatively, the number of patients is significant. 1'2 This chapter reviews the principal dysplastic disorders that cause osteosclerosis and/or hyperostosis and dis-
I. I N T R O D U C T I O N Though most are rare, there are many disorders of bone formation or skeletal homeostasis that cause either focal or generalized increases in skeletal mass (Tables 2 4 - 1 and 2 4 - 2 ) . 1-8 Some are mere radiological curiosities; others are difficult clinical problems. Some METABOLIC BONE DISEASE
Melorheostosis Mixed Sclerosing Bone Dystrophy Fibrodysplasia Ossificans Progressiva Axial Osteomalacia Fibrogenesis Imperfecta Ossium Fluorosis Pachydermoperiostosis Hepatitis C-Associated Osteosclerosis Other Disorders References
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Copyright 9 1998by AcademicPress. All rights of reproductionin any formreserved.
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MICHAEL P. WHYTE
TABLE 24--1 Disorders that Cause Generalized Osteosclerosis a Dysplasias Craniodiaphyseal dysplasia Craniometaphyseal dysplasia Dysosteosclerosis Endosteal hyperostosis Van Buchem's disease Sclerosteosis Frontometaphyseal dysplasia Infantile cortical hyperostosis (Caffey's disease) Melorheostosis Metaphyseal dysplasia (Pyle's disease) Mixed sclerosing bone dystrophy Oculodento-osseous dysplasia Osteodysplasia of Melnick and Needles Osteoectasia with hyperphosphatasia (hyperostosis corticalis) Osteopathia striata Osteopetrosis Osteopoikilosis Progressive diaphyseal dysplasia (Engelmann's disease) Pycnodysostosis Metabolic Carbonic anhydrase II deficiency Fluorosis Heavy metal poisoning Hypervitaminosis A, D Hyper-, hypo-, and pseudohypoparathyroidism Hypophosphatemic osteomalacia Milk-alkali syndrome Renal osteodystrophy Other Axial osteomalacia Fibrogenesis imperfecta ossium Ionizing radiation Lymphoma Mastocytosis Multiple myeloma Myelofibrosis Osteomyelitis Osteonecrosis Paget's disease Sarcoidosis Skeletal metastases Tuberous sclerosis aFrom Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.
cusses briefly some of the secondary causes of increased skeletal mass. Osteosclerosis refers to thickening of trabecular (spongy) bone; hyperostosis refers to widening of cortical (compact) bone from apposition of osseous tissue at periosteal and/or endosteal surfaces)'* *Throughout the text, heritable disorders are referred to by the classification number(s) given by McKusick. 1 Recognition of the radiographic features of these conditions is essential for making a correct diagnosis; accordingly, I refer the reader to several excellent references that also illustrate the x-ray findings) -8
TABLE 24--2
Types of Osteosclerosis a
Cortical and Trabecular Bone (Both) Carbonic anhydrase II deficiency Dysosteosclerosis Osteopetrosis Pycnodysostosis Cortical Bone (Predominantly) Autosomal dominant osteosclerosis Diffuse idiopathic skeletal hyperostosis Endosteal hyperostosis (van Buchem's disease and sclerosteosis) Hypertrophic osteoarthropathy Pachydermoperiostosis Progressive diaphyseal dysplasia (Engelmann's disease) Trabecular Bone (Predominantly) Dysplastic Osteomesopyknosis Hematological Mastocytosis, myelofibrosis, polycythemia vera, sickle cell disease Metabolic Fluorosis, hyperparathyroidism, renal osteodystrophy, X-linked hypophosphatemic rickets, vitamin D toxicity Neoplastic Disorders Metastatic disease Myeloma, lymphoma, leukemia aFrom Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.
II. OSTEOPETROSIS Osteopetrosis (marble bone disease) was first described by Albers-Sch6nberg in 19049; more than 300 cases have been reported. As discussed below, we now understand that "osteopetrosis" is a generic term used for a group of disorders with a similar pathogenesis. ~~ Two principal clinical forms are generally recognized; an autosomal dominant benign type (McKusick 166600) with few or no symptoms, 12 and an autosomal recessive malignant type (McKusick 259700) that is fatal during infancy or early childhood if untreated. ~3 Nevertheless, there appear to be at least nine distinct forms of osteopetrosis in humans (Table 24-3). 1~ An especially rare "intermediate" form, inherited as an autosomal recessive trait (McKusick 259710), manifests during childhood with some of the clinical problems associated with the malignant type, but has an uncertain impact on life expectancy. 14'15Carbonic anhydrase II (CA II) isoenzyme deficiency is an autosomal recessive condition (McKusick 259730) that was formerly called the syndrome of osteopetrosis/renal tubular acidosis/cerebral calcification. 16 Because the etiology of CA II deficiency is now known, the following section describes this entity. Malignant osteopetrosis with neuronal storage disease has been delineated in several infants. 17 Still rarer "lethal,"
CHAPTER 24 Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis TABLE 24--3
Types of Osteopetrosis in Humans
Type
Inheritancea
Benign (adult) Type I Type II Malignant Carbonic anhydrase II deficiency Intermediate Lethal Malignant with neuronal storage disease Transient infantile Postinfectious
AD AR AR AR AR AR 9 m
aFrom Whyte MP: Osteopetrosis and the heritable forms of rickets. Royce PM, Steinmann B (eds): Connective Tissue and its Heritable Disorders. New York, Wiley-Liss, Inc, 1993, 563-589. AD: autosomal dominant; AR, autosomal recessive.
In
"postinfectious," and "transient" types of osteopetrosis have been reported. 1~ Although it is apparent that several genetic defects cause osteopetrosis in humans, the pathogenesis of all true forms is absent or greatly diminished osteoclast function. TM Consequently, there is accumulation of unresorbed skeletal tissue--including the primary spongiosa deposited at growth plates during endochondral bone formation. 1s-21 Though a generic term, "osteopetrosis" should no longer be used merely to refer to a generalized increase in skeletal mass. 10,11
A. Clinical Presentations Malignant forms of osteopetrosis cause symptoms beginning in infancy. 11'13 Nasal "stuffiness" is an early sign that has been attributed to malformation of the paranasal and mastoid sinuses. Subsequently, there is failure to thrive, and palsies of the optic, oculomotor, and facial nerves can result from compression by narrowed cranial foramina. Dentition is delayed and bones are fragile and often fracture. Short stature, a large head, frontal bossing, nystagmus, "adenoid appearance," hepatosplenomegaly, and genu valgum may be present. Some patients develop hydrocephalus or sleep apnea. 22 Retinal degeneration sometimes contributes to loss of vision. 23 Recurrent infection with spontaneous bruising and bleeding can occur from myelophthisis. Hypersplenism and hemolysis may exacerbate the anemia. Without successful treatment, death occurs during the first decade of life from pneumonia, sepsis, hemorrhage, or severe anemia. 13 Intermediate osteopetrosis causes short stature and there may be macrocephaly, cranial nerve deficits, ankylosed teeth (which predisposes to osteomyelitis of the
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jaw), recurrent fractures, and a mild or occasionally moderately severe anemia. 14'15 Benign osteopetrosis can manifest as a developmental disorder. Most affected individuals are asymptomatic. 12 The long bones are brittle, and pathological fractures may occur. Deafness, facial palsy, osteomyelitis of the jaw, visual or auditory impairment, psychomotor delay, carpal tunnel syndrome, 24 and osteoarthritis have also been reported. 12 Studies from Denmark propose that there are two types of benign osteopetrosis. 25'26However, the histological hallmark (unresorbed primary spongiosa) 1~ and presence of a biochemical marker in serum for osteoclast failure (creatine kinase, brain isoenzyme) 2v manifests only in the more common type II form (see below). 25'26
B. Laboratory Findings Serum acid phosphatase activity, apparently derived from defective osteoclasts, is often elevated. Recently, presence in serum of the brain isoenzyme of creatine kinase (BB-CK) has been found to distinguish the osteopetroses among disorders that increase skeletal m a s s .27 Standard biochemical parameters of mineral homeostasis are usually unremarkable. In malignant forms of osetopetrosis, however, serum calcium levels can be low with values reflecting the dietary calcium intake. 2s Furthermore, secondary hyperparathyroidism occurs with increased serum levels of calcitriol (1,25-dihydroxyvitamin D), 29 and hypocalcemia may lead to rachitic disease. There is a leukoerythroblastic anemia.
C. Radiological Features Generalized osteosclerosis with thick cortical bone is the principal radiological feature of osteopetrosis. 2-8 The apparent hyperostosis occurs because endosteal bone resorption fails to create a marrow cavity. Abnormal skeletal modeling and remodeling together cause the symmetrical increase in bone mass (Fig. 24-1). However, the skeleton may be uniformly dense, or the osteosclerosis may manifest as alternating dense and lucent bands suggesting a fluctuating disturbance in skeletal growth. Such banding is most commonly seen in the pelvis and near the ends of major long bones (Fig. 24-2). In the appendicular skeleton, metaphyses are typically widened because of the defective modeling. Erosion of the distal phalanges occurs rarely and is more characteristic of pycnodysostosis. Transverse pathological fracture of long bones is common (Fig. 24-3). Rachitic changes due to hypocalcemia have been described. 3~ In the axial
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FIGURE 2 4 - 1 Osteopetrosis (malignant form). Anteroposterior (AP) radiograph of the lower limbs of a neonate shows characteristic diffuse osteosclerosis, absence of medullary space, and poorly modeled metaphyses. Furthermore, growth plate widening and metaphyseal irregularity are consistent with rickets.
MICHAEL P. WHYTE
FIGURE 24--2
Osteopetrosis (intermediate form). AP radiograph of the distal femur of a 10-year-old boy shows a widened metadiaphyseal region with characteristic alternating dense and lucent bands. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co,
1990.] skeleton, the cranium is usually thick and dense, with the base having the greatest osteosclerosis (Fig. 2 4 - 4 ) , and there is underpneumatization of the paranasal and mastoid sinuses. Vertebrae may exhibit lucent central bands (Fig. 2 4 - 5 ) or show a "bone-in-bone" (endobone) configuration on lateral view. The two proposed types of benign osteopetrosis are characterized by progressive osteosclerosis with either pronounced diffusely increased radiodensity of the skull and other bones (type I) or more selective sclerosis of the base of the skull together with typical endobone formation in the spine (type II). 25'26 For patients with osteopetrosis, skeletal scintigraphy retains its utility to demonstrate fractures, osteomyelitis, and so on. 31 Magnetic resonance imaging (MRI) of a few affected individuals has revealed a weaker signal from marrow spaces in malignant compared to benign disease. 32 Accordingly, MRI may prove useful to monitor patients treated by bone marrow transplantation (see later). Cranial MRI and computed tomography (CT) findings in infants and children have been reported. 33
D. Histopathological Findings Although the radiological features of osteopetrosis are often diagnostic, failure of osteoclasts to resorb skeletal tissue provides a histological finding that can be considered pathognomonic (Fig. 2 4 - 6 , see Color Plate); that is, presence of "islands" or " b a r s " of calcified cartilage (unresorbed primary spongiosa) within bony trabeculae distant from sites of endochondral bone formation. Normally, such cartilage remnants are removed by functioning osteoclasts during skeletal remodeling. 19-21'34-36 Osteoclasts themselves have been reported to be increased, normal, or decreased in number in osteopetrosis. In malignant forms, typically there are many of these multinucleated cells appropriately positioned at bone s u r f a c e s . 37 Nevertheless, their nuclei are especially numerous and ultrastructural abnormalities may be present, including absence of "ruffled borders" and "clear
CHAPTER 24
Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis
FIGURE 24--3 Osteopetrosis(benign form). AP radiograph of the distal femur of a 22-year-old man shows homogeneous osteosclerosis with a transverse pathological fracture of the midshaft. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
zones," which are characteristics of osteoclasts that are actively resorbing bone. 34,3s'3s Marrow space is often crowded with fibrous tissue. 34'37 In benign osteopetrosis, osteoid may be increased, and osteoclasts can be scant and devoid of ruffled borders. 36
E. Etiology and Pathogenesis Although the more common forms of human osteopetrosis appear to be heritable (Table 2 4 - 3 ) , the defective gene loci of the types discussed thus far are unknown. Only CA II deficiency is understood at the molecular level (see below). 16 Theoretically, a defect in the microenvironment of the stem cell for the osteoclast, the stem cell itself, osteoclast
701
precursor cells, osteoclast heterokaryon formation, the mature osteoclast, or the skeletal matrix could account for osteopetrosis. ~8'2~ Animal models for osetopetrosis clearly illustrate various pathogenetic mechanisms. TM The osteopetrosis of CA II deficiency appears to result from failure of osteoclasts to secrete hydrogen ion for dissolution of o s s e o u s t i s s u e . 16 The etiology and precise pathogenesis are unknown for other human forms. 1~ Biosynthesis of an abnormal parathyroid hormone, 39 defective production of interleukin-2, 4~ and subnormal generation of superoxide necessary for bone resorption 41 are potential defects. The occurrence of osteopetrosis with neuronal storage disease (characterized by accumulation of ceroid lipofuscin) indicates that these patients may have a primary lysosomal defect. 17 Virus-like inclusions (nearly identical to those of the nucleocapsids of Paramyxoviridae) and antigens of respiratory syncytial virus and measles virus have been noted in some of the osteoclasts of several sporadic cases of benign osteopet r o s i s . 42 Their significance, however, is uncertain. Recently, retrovirus infection has been offered as an explanation for some types of osteopetrosis. 43 Primary immune defects may occur in osteopetrosis, but do not seem to be as common as believed previously. 2~ Detailed investigation of leukocyte function in malignant osteopetrosis has revealed abnormalities in circulating monocytes and granulocytes. 41'44 The myelophthisic disease in the malignant forms of osteopetrosis probably results from marrow crowding by bone, fibrous tissue, and numerous osteoclasts. 37 Skeletal fragility may be due to defective remodeling of woven bone to compact bone and/or to defective weaving of collagen fibrils necessary for osteons to become connected. 21 Presence of CK-BB in serum in true forms of osteopetrosis appears to reflect malfunctioning osteoclasts rather than a metabolic consequence of a hypoxic marrow. 27
F. Treatment Because the various forms of osteopetrosis can differ in outcome, a correct diagnosis (which may involve assessment of disease progression) and selection of an appropriate therapeutic approach a r e c r u c i a l . 1~ Transplantation of allogeneic bone marrow from a haploidentical donor has been followed by significant clinical improvement and reversal of neurological, hematological, radiological, and histopathological abnormalities in malignant osteopetrosis. 45-5~ Demonstration that osteoclasts, but not osteoblasts, were of donor origin after bone marrow transplantation has corroborated the hypothesis that osteoclast-mediated bone resorption is defective and that osteoclasts are normally derived from precursor cells in the marrow. 51 Transplantation of HLA-
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MICHAEL P. WHYTE
FIGURE 24--4 Osteopetrosis (intermediate form). Lateral radiograph of the skull of a 13-year-old boy shows osteosclerosis, especially apparent at the base. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
nonidentical marrow has considerably poorer outcome. 52 Since a variety of genetic defects appear to cause osteopetrosis, 1~ it is understandable that bone marrow transplantation may not be helpful in all patients. 18'21 Furthermore, older patients and those with especially severe sclerosis of the marrow cavity seem to fare less well with transplantation. 2~ Accordingly, bone biopsy may be helpful to support the rationale for and predict the outcome of bone marrow transplantation. It has been advised that rickets be treated before bone marrow transplantation so that donor osteoclasts will be able to resorb mineralized bone tissue. 54 Successful transplantation resuits in hypercalcemia in some patients. 55 Treatment with large oral doses of calcitriol together with a calcium-deficient diet has been reported to improve the malignant form of osteopetrosis as successfully as bone marrow transplantation. 28'53 Here, calcitriol appears to act therapeutically by stimulating osteoclast activity. However, a recent update indicates that some patients become resistant to this treatment. 56 A preliminary report of a long-term trial of this regimen in an adult with an apparently intermediate form of osteopetrosis describes improved hematopoietic function and decreased skeletal mass. 57 Early publications report some success with a calcium-deficient diet alone. 58 Ironically,
supplementation of dietary calcium may be necessary to correct rachitic disease from hypocalcemia. 54 Pharmacological doses of glucocorticoids appear to be beneficial for malignant osteopetrosis and are especially helpful in stabilizing patients with hepatomegaly and pancytopenia. 59'6~ Prednisone therapy together with a low-calcium, high-phosphate diet has been reported to be a potentially effective alternative treatment to marrow transplantation for malignant osteopetrosis. 61 Long-term infusion of parathormone seemed efficacious for one infant, 39 perhaps by increasing endogenous calcitriol levels. Recently, interferon g a m m a - l b treatment has been shown to diminish frequency of infection and improve hematological and skeletal manifestations in severe cases of osteopetrosis. 56'62 Osteomyelitis of the jaw can be treated with antibiotics and hyperbaric oxygenation. 63 Surgical decompression of the facial and optic nerves (evaluated best by CT rather than by radiological views of the optic foramena) may be SUCCessful. 22'23 Conventional radiographic study has failed to diagnose malignant osteopetrosis in utero at 20 weeks' gestation 64 but did detect the disease later in pregnancy. 65 Prenatal diagnosis of lethal disease at 18 weeks' gestation by ultrasound has recently been reported. 66
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703
f a m i l i e s . 16"74-76 Consanguinity is a common finding in
kindreds from the Arabian peninsula. 77 Perinatal history is generally unremarkable. Manifestations present in infancy or early childhood and include developmental delay, failure to thrive, or fracture. Investigation of short stature may also lead to the diagnosis. 16'76 Patients commonly, but not always, have mental subnormality. Dental malocclusion and compression of the optic nerves develop in some patients. The renal tubular acidosis is usually a " m i x e d " type (proximal and distal) and may account for apathy, muscle weakness, and hypotonia. Intermittent hypokalemic paralysis has been reported. Although most patients do not fracture, recurrent breaks in long bones can cause significant morbidity. TM The disorder, however, appears to be compatible with long life. 7°,75
B. L a b o r a t o r y F i n d i n g s
FIGURE 24--5 Osteopetrosis (intermediate form). Lateral radiograph of the lower thoracic spine of an 8-year-old boy shows osteosclerosis of ribs and vertebrae. Note the characteristic central lucent bands in each vertebra. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
Anemia, if present, is usually mild and apparently of nutritional origin. Bone marrow aspiration reveals normal findings. Metabolic acidosis has been noted as early as the neonatal p e r i o d , 77 and both proximal and distal renal tubular acidosis have been reported. TM Distal (type I) renal tubular acidosis seems established best; however, further studies are needed to define more precisely the defect in acid-base homeostasis. 78'79 Aminoaciduria and glycosuria are absent. TM
C. R a d i o l o g i c a l F e a t u r e s
III. CARBONIC ANHYDRASE II DEFICIENCY CA H deficiency is an inborn error of metabolism. 16 In 1972, three independent g r o u p s 67-69 described a new autosomal recessive syndrome characterized by radiological changes of osteopetrosis in patients with renal tubular acidosis. Cerebral calcification and histopathological confirmation of osteopetrosis were first reported in 1 9 8 0 . 7°'71 Three years later, deficiency of CA II isoenzyme was identified as the primary biochemical defect. 72 Mutations in the " c a n d i d a t e " CA II gene were first reported in 1992. 73
The radiological features of CA II deficiency are similar to those of other forms of osteopetrosis, except that the osteosclerosis can diminish spontaneously over years (Fig. 2 4 - 7 ) and basal ganglia calcification will typically appear. TM Radiological abnormalities of the skeleton were noted at the time of clinical presentation in all case reports, although one neonate had only very subtle findings. 77 CT has shown that the cerebral calcification is developmental (appearing between 2 and 5 years of age), increases during childhood, occurs in the gray matter of the cortex and basal ganglia, and is similar if not identical to that seen in idiopathic hypoparathyroidism or pseudohypoparathyroidism. 8°
D. H i s t o p a t h o l o g i c a l F i n d i n g s A. C l i n i c a l P r e s e n t a t i o n Descriptions of more than 20 patients with this rare inborn error of metabolism (McKusick 259730) reveal that there is clinical variability among affected
Autopsy studies have not been reported. Specimens of bone taken from four patients from two families have been examined histopathologically. 68'7° Changes characteristic of osteopetrosis TM were documented in each of
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MICHAEL P. WHYTE
FIGURE 24--7
Carbonic anhydrase II deficiency. A, AP radiograph of the proximal tibia and fibula of a 2-year-old girl shows features of osteopetrosis including widening of the metaphysis, diffuse osteosclerosis, and altemating radiodense and radiolucent bands. B, AP radiograph of proximal tibia and fibula of the same patient, at age 17, shows nearly complete resolution of features of osteopetrosis. A few very faint sclerotic lines remain in what appears to be otherwise normal bone. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
the three sisters first found to have CA II deficiency (Fig. 24-8). E. E t i o l o g y a n d P a t h o g e n e s i s CAs catalyze the initial step in the reaction C O 2 -Jv H + + H C O 3 and, therefore, function importantly in acid-base regulation. CA II has been identified in a variety of cell types and tissues including erythrocytes, eye, brain, kidney, cartilage, liver, lung, skeletal muscle, pancreas, and gastric mucosa 81's2 and is the most catalytically active isoenzyme of the CA family. 83 The other CA isoenzymes have a more restricted tissue distribution. Initially, studies of erythrocyte lysates indicated that deficiency of CA II accounted for this form of osteopetrosis; now a variety of mutations have been found in the CA II isoenzyme gene. 16 Red cell lysates reveal a deficiency of CA II but not of CA I. 72'74 Autosomal recessive inheritance for this disorder was supported by the observation that CA II levels are approximately halfnormal in heterozygous parents of patients. 16'72'74 H 2 0 ---) H2CO3 ~
Presence of osteopetrosis and renal tubular acidosis in CA II-deficient patients reveals a significant physiological role for CA II in the skeleton and kidney of humans. 16 Indeed, a variety of in vivo and in vitro studies indicate that CA is important for osteoclast function, likely by enabling pericellular pH to be lowered by the action of a H + pump. 18 Pharmacological inhibition of CA activity can block bone resorption in organ culture. 84 CA II is the CA isoenzyme present in osteoclasts. 85 The observation that CA II-deficient patients respond appropriately to an acetazolamide challenge with bicarbonaturia has provided evidence that a CA isoenzyme in addition to CA II functions importantly in the kidney. 78 Whether cerebral calcification is a direct or indirect effect of CA II deficiency is unclear. Mouse models for this disorder should help to define the role of CA II in mammal s. 16,86 The gene for CA II is located on chromosome 8 in humans. 87 Some of the clinical and biochemical heterogeneity observed among affected kindreds appears to be explained by different allelic defects in the CA II g e n e . 16,73
CHAPTER 24 Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis
705
FIGURE 24--8 Carbonic anhydrase II deficiency. In this iliac crest specimen, defective osteoclastic function and true osteopetrosis is revealed by the presence of cartilage "islands" (short arrows) that reflect unresorbed primary spongiosia in trabecular bone (TB). Osteoclasts (long arrows) are present (Goldner stain; x 50) [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
F. Treatment Prenatal diagnosis of CA II deficiency has not been reported. Renal tubular acidosis in CA II deficiency has been treated briefly with HCO3 supplementation, but the long-term outcome of this therapy has not been assessed fully. 77 Transfusion of CA II-replete erythrocytes to one patient failed to correct her systemic acidosis. This finding indicates that the renal acidification defect is not the result of CA II deficiency in erythrocytes, but intrinsic to the kidney. 88 Hence, bone marrow transplantation might affect the skeletal disease, but would not reverse the renal acidification defect. 16
IV. PYCNODYSOSTOSIS
Pycnodysostosis (McKusick 265800), first delineated in 1962, 89,90 is the disorder that is believed to have affected the French impressionist painter Henri de Toulouse-Lautrec (1864-1901).91 More than 100 cases from 50 kindreds have been described. 92 Pycnodysostosis is inherited as an autosomal recessive trait93-95; parental consanguinity has been described for about 30% of patients. Most reported cases have originated in Europe or the United States, but the condition has been
encountered in Israel and Indonesia, in Asian Indians, and in African blacks; it is especially common in the Japanese. 96
A. Clinical Presentation Pycnodysostosis is usually diagnosed during infancy or childhood because of disproportionate short stature together with additional dysmorphic features including a relatively large cranium with fronto-occipital prominence, small facies, obtuse mandibular angle, small chin, dental malocclusion with persistence of carious deciduous teeth, high-arched palate, proptosis, bluish sclerae, and a pointed and beaked nose. 5'93 The anterior fontanel is often palpably open, as are other major cranial sutures. The hands are small and square, the fingers are short and clubbed from acroosteolysis or aplasia of terminal phalanges, and the nails are hypoplastic. Recurrent deforming fractures (usually of the lower limbs), a narrowed thorax, pectus excavatum, kyphoscoliosis, increased lumbar lordosis, and genu valgum may o c c u r . 92 Mental retardation is present in about 10% of c a s e s . 93 Adult height ranges between 4'3" and 4'11". Atypical patients have been described, for example, with visceral manifestations and with rickets. 97
706
MICHAEL P. WHYTE B. L a b o r a t o r y F i n d i n g s
Patients are not anemic. Serum calcium and inorganic phosphate levels and alkaline phosphatase activity are generally normal.
and the orbital ridges are dense (Fig. 2 4 - 9 ) . There is hypoplasia of facial bones, sinuses, and terminal phalanges (Fig. 24-10). Vertebrae are sclerotic (transverse processes are uninvolved) and may have anterior and posterior concavities. Lumbosacral spondylolisthesis is not uncommon, and lack of segmentation of the atlas and axis can occur. Madelung's deformity may be present in the forearms. 92
C. R a d i o l o g i c a l F e a t u r e s Many of the radiological findings of pycnodysostosis are similar to those of osteopetrosis; for example, both conditions are characterized by generalized osteosclerosis and recurrent fractures. 98'99 However, pyknodysostosis differs from osteopetrosis by the following additional features; wormian bones; delayed closure of sutures and fontanels (prominently the anterior); obtuse mandibular angle; gracile clavicles that are hypoplastic at the lateral ends; partial absence of the hyoid bone; and hypoplasia or aplasia of the distal phalanges and ribs. 1~176Radiodense striations and endobones (bonewithin-bone) do not o c c u r . 2'5-7 In pycnodysostosis, the osteosclerosis is uniform, becomes apparent in childhood, and increases with age. However, it is not accompanied by the considerable modeling defects of osteopetrosis, although the long bones have thick cortices with narrow medullary cavities. In the skull, the calvarium and base are sclerotic
D. H i s t o p a t h o l o g i c a l F i n d i n g s Histopathological studies reveal normal cortical bone structure in pycnodysostosis, yet decreased osteoblastic and osteoclastic activity. 1~ A depressed rate of skeletal turnover has been reported. 1~ Electron microscopy of bone from two patients revealed findings consistent with defective degradation of skeletal collagen--perhaps due to an abnormality in the bone matrix itself, or in osteoclast function. 1~ In fact, osteoclasts in pycnodysostosis have been found to contain somewhat large cytoplasmic vacuoles that are usually filled with collagen fibrils. These observations suggest that there is defective extracellular or intracellular degradation of bone collagen. 1~ Ultrastructural study of cartilage, however, has also revealed abnormal inclusions in chondrocytes. 1~
FIGURE 24--9 Pycnodysostosis. Lateral radiograph of the skull of an infant shows that the cranial sutures are markedly widened. The base is sclerotic. (Courtesy of William H. McAlister, St. Louis, MO.) [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
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707 F. T r e a t m e n t
There is no effective medical therapy for pycnodysostosis. Fractures of the long bones are usually transverse and heal at a satisfactory rate; however, massive callus formation and delayed union have been reported, l~ Internal fixation of long bones is made difficult by their hardness. Patients are, however, generally able to walk independently. 92 Extraction of teeth is difficult and several mandibles have been broken. 93 Osteomyelitis of the jaw responds to combined surgical and antibiotic therapy. 1~
V. OSTEOMESOPYKNOSIS
FIGURE 24--10 Pycnodysostosis. Posteroanterior (PA) radiograph of the hand shows that the bones are sclerotic and the distal phalanges are hypoplastic. (Courtesy of William H. McAlister, St. Louis, MO.) [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
E. E t i o l o g y a n d P a t h o g e n e s i s In 1996, the molecular basis of pycnodysostosis was identified as cathepsin K gene defects. 1~ Cathepsin K is lysosomal cystine protease that is highly expressed in osteoclasts. A study of calcium homeostasis using 47Ca and 45Ca in one patient revealed a normal exchangeable calcium pool and unremarkable rate of bone accretion and calcium excretion. 1~ However, when the increased skeletal mass was considered, both the rate of bone accretion and the size of the exchangeable calcium pool appeared to be reduced. TM Thus, decreased bone resorption may account for the osteosclerosis. Similar kinetic studies, using SSSr, have been reported. 9~ Paramyxovirus-like inclusions, similar to those found in Paget's bone diseases, have been reported in the osteoclasts of two brothers with pycnodysostosis. 1~
Osteomesopyknosis (McKusick 166450) was first described in 1979 l~ and named in 1980.11~ This condition is inherited as an autosomal dominant trait. ~ Fewer than 20 cases have been reported. 1~ Patients are usually discovered incidentally during adolescence or early adulthood by radiographic study; lowback pain appears to be a common complaint. The youngest patient with characteristic lesions was 10 years old. 113 One affected woman was infertile from "ovarian sclerosis. ''1~1 Physical examination is generally unremarkable except for tenderness of the back. Routine biochemical studies were normal in all patients but one who had renal tubular acidosis. Osteomesopyknosis is characterized by patchy osteosclerosis localized to the spine, pelvis, and proximal parts of long bones. The osteosclerosis is especially prominent in the vertebral end plates (Fig. 2 4 - 1 1 ) . Lesions appear to be well demarcated by CT. 113 Bone scintigraphy has been reported to be normal. 113 Radiographic abnormalities may be unchanged for more than a decade. TM Histopathological studies of osteomesopyknotic bone have not been described.
VI. PROGRESSIVE DIAPHYSEAL DYSPLASIA (CAMURATI-ENGELMANN DISEASE) Progressive diaphyseal dysplasia (McKusick 131300) was first described in 1920 by Cockayne. TM Camurati noted in 1922 that the disorder could be inherited. 115 Engelmann characterized the severe form in 1929 and the condition is often called Engelmann's disease. 116 This is a developmental disorder that is inherited as an autosomal dominant trait, but with variable clinical and radiological penetrance. 1'117-119 More than 100 cases
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MICHAEL P. WHYTE
head, proptosis, and cranial nerve palsies when the skull is involved. ~23'124 Some patients have delayed puberty due to hypothalamic hypogonadotropic hypogonadism (personal observation). Increased intracranial pressure can occur. Physical examination may reveal palpable bony enlargement and hepatosplenomegaly and elicit diffuse skeletal tenderness on palpation. Some patients have Raynaud's phenomenon and evidence of vasculitis. 125
B. R a d i o l o g i c a l F e a t u r e s
FIGURE 24--11
Osteomesopyknosis. Lateral radiograph of the thoracic spine of this teenage girl reveals characteristic radiodense end plates.
have been reported. 119 All races appear to be affected. Hyperostosis occurs gradually along both the endosteal and periosteal surfaces of tubular bones. In especially severe cases, osteosclerosis is generalized and affects the skull and axial skeleton. Some adult carriers of the disorder have no radiographic abnormalities. Mild forms (believed to be transmitted as an autosomal recessive trait) were first reported by Ribbing. 117'12~
A. Clinical P r e s e n t a t i o n Leg pain, muscle wasting, decreased subcutaneous fat in the extremities, and a limping or broad-based and waddling gait during childhood are characteristic features of progressive diaphyseal dysplasia. Unless appropriate radiological studies are performed, the condition may be mistaken for a muscular dystrophy. 121'122Severely affected patients have a characteristic body habitus (Fig. 24-12) that includes an enlarged head, prominent fore-
Gradually spreading cortical hyperostosis of long bone diaphyses, secondary to proliferation of new bone on both the periosteal and the endosteal surfaces, is the primary radiographic feature of progressive diaphyseal dysplasia. 2-7 Hyperostosis is nearly symmetrical and occurs in the diaphyseal and metaphyseal regions of long bones. An important radiological feature is epiphyseal sparing by this process (Fig. 24-13). Diaphyseal width slowly increases. CT has shown that endosteal involvement is more extensive than periosteal hyperostosis. 126 Characteristically, the shafts of long bones have irregular surfaces (Fig. 24-14). The age of onset, rate of progression, and degree of diaphyseal sclerosis vary considerably among patients. Tibiae and femora are involved most frequently; less commonly the humeri, radii, and ulnae are affected. Occasionally, the short tubular bones show radiological changes. Progressive sclerosis of the vertebrae, skull, scapulae, clavicles, and pelvis may also occur. Cranial involvement, which appears as calvarial hyperostosis and sclerosis of the base of the skull (Fig. 24-15), can cause diagnostic confusion with mild forms of craniodiaphyseal dysplasia. With maturation of newly formed bone, the affected areas become more sclerotic. In severely affected children, osteopenia can accompany new bone formation. Focally increased radionuclide accumulation occurs during bone scintigraphy. In the milder forms of progressive diaphyseal dysplasia, which develop in adolescents or young adults, radiographic and scintigraphic studies may show skeletal abnormalities limited to the long bones of the lower limbs (Fig. 24-16). Comparison of the scintigraphic, radiographic, and clinical findings in four patients at different stages of the disease revealed generally concordant findings. In some patients, however, bone scans were rather unremarkable despite distinct abnormalities present radiographically; in these patients the disease process appeared to be quiescent. 127 Sequential radiological findings reveal a variable course for the disorder, but progressive bony involvement is the rule. Arrest of the disease process appears to
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Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis
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FIGURE 24-- 12 Progressivediaphyseal dysplasia. A and B, This prepubescent 17-year-old girl with severe disease involvement has the characteristic body habitus including a paucity of muscle and subcutaneous fat in the limbs, thickening of the long bones, and cranial enlargement. [From Whyte MP: Rare disorders of skeletal formation and homeostasis. In Becker KL (ed): Principles and Practice of Endocrinology and Metabolism, 2nd ed. Philadelphia, JB Lippincott, 1995, pp 594-606.]
occur in s o m e c a s e s . 126'128 The MRI features of multiple cranial neuropathies have been described. 123
C. Histopathological Findings New bone formation is present in areas affected by progressive diaphyseal dysplasia. Peripheral to healthy cortical bone, there appears to be centripetal maturation and "cancellous compaction" of disorganized (woven) hyperostotic bone. 1iv In muscle, electron microscopy reveals atrophy of isolated muscle fibers, accumulation of endomyseal collagen fibrils, and thickening of pefivascular basement m e m b r a n e - - c h a n g e s that are similar in sporadic and familial cases. ~2~ Type II muscle fiber atrophy was noted in one patient with clinical evidence of myopathy but without degenerative changes on electron microscopy. ~22
D. E t i o l o g y a n d P a t h o g e n e s i s The autosomal gene defect that causes progressive diaphyseal dysplasia has not been mapped in the human genome. Some especially mild cases may reflect a separate, autosomal recessive disorder, called " R i b b i n g ' s disease." 117,120,129 However, clinically mild forms of progressive diaphyseal dysplasia can be transmitted as an autosomal dominant trait (MP Whyte and WA Murphy, unpublished observation). Some investigators argue that the clinical and laboratory findings in the full-blown condition and their responsiveness to glucocorticoid therapy indicate that progressive diaphyseal dysplasia is a systemic disorder that should be included among the inflammatory connective tissue diseases. 125 Greatly increased blood flow to affected bone was reported in two of four patients. 13~Ab-
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MICHAEL P. WHYTE
FIGURE 24-- 13
Progressive diaphyseal dysplasia. AP radiograph of the left forearm of a 19-year-old woman with severe disease shows osteopenia with superimposed sclerosis of the diaphyses of the radius and ulna from proliferation of bone on the periosteal and endosteal surfaces. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
normal differentiation of monocytes-macrophages ultimately to osteoblasts has been described as a potential pathogenetic factor. TM
FIGURE 24-- 14
Progressive diaphyseal dysplasia. AP radiograph of the left forearm of a 41-year-old man (father of patient shown in Fig. 24-11) shows dense sclerosis resulting in thickening of the radial and ulnar cortices. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
toms in one patient in whom radiographic findings were progressing. 138 However, I have not observed symptomatic benefit in several patients treated with etidronate. In a few cases, my colleagues and I have noted relief of pain following a biopsy (cortical window) of an affected diaphysis. 117
E. Treatment Radiological studies show that the evolution of progressive diaphyseal dysplasia is slow and unpredictable. 127 Skeletal symptoms may remit during adolescence. Since 1967, glucocorticoid therapy (prednisone given in small doses on an alternate-day schedule) has been recognized to relieve bone pain. 132'133 Histological improvement of affected skeletal tissue has also been reported. 134-137 Intermittent courses of bisphosphonate (disodium etidronate) therapy seemed to improve symp-
VII. ENDOSTEAL HYPEROSTOSIS Endosteal hyperostosis is characterized by cortical bone thickening on the medullary (marrow) surface. Two principal forms have been describedmvan Buchem's disease and sclerosteosis. The form of endosteal hyperostosis first reported by van Buchem and colleagues in 1955139-141 is also called hyperostosis corticalis generalisata (McKusick 239100). This disorder is heritable,
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711
FIGURE 24--15 Progressivediaphyseal dysplasia. Lateral radiograph of the skull of a 19-year-old woman shows diffuse sclerosis of the cranial vault and base of the skull. Frontal bossing is present. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
either as an autosomal recessive type that is clinically severe (van B u c h e m ' s disease ) or as an autosomal dominant type that is more benign (Worth type). 142'143 H o w ever, these types of endosteal hyperostosis do not appear to be as common as the older literature suggests. In 1977, review of 41 reported cases showed that only 6 patients fit uniform diagnostic criteria; most had clinical and radiological features of sclerosteosis or other craniotubular disorders including craniodiaphyseal dysplasia. TM Sclerosteosis (cortical hyperostosis with syndactyly), like van B u c h e m ' s disease, is an autosomal recessive craniotubular hyperostosis; it is usually reported in the Afrikaner population of South Africa (McKusick 269500). Cases described from elsewhere often have Dutch ancestry as well. 143 Sclerosteosis was distinguished from van B u c h e m ' s disease in 1958 by some radiographic differences and by the presence of syndactyly. 145,146
A. Clinical Presentations Van Buchem's disease has been reported in children and adults. The gender distribution appears to be equal.
All adult patients have a markedly thickened jaw with wide angle, yet there is no prognathism, and dental malocclusion is uncommon. Progressive asymmetrical enlargement of the mandible occurs during puberty. Patients often live normally without complaints. However, recurrent facial paralysis, deafness, and optic atrophy due to narrowing of the cranial foramina are common and may begin during infancy. 147 There is no predisposition to fracture. Palpably thickened clavicles and ribs may also be present. Long bones can be painful to pressure, but range of motion appears to be normal. Sclerosteosis had until recently 14s been differentiated from van B u c h e m ' s disease because excessive height and syndactyly are present. Syndactyly may be the only clinical finding in sclerosteosis at birth. 149 During early childhood, however, there is overgrowth and sclerosis of the skeleton, especially the skull, with attendant facial disfiguration. 149 Patients are tall and heavy beginning in childhood. " G i g a n t i s m " has been used to describe their appearance. The jaw has a very square appearance. Deafness and facial palsy from cranial nerve entrapment may be among the presenting clinical manifestations. Some patients develop raised intracranial pressure and headache from a small cranial cavity. Brainstem compression may also occur. Syndactyly due to cutaneous or bony
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MICHAEL P. WHYTE
ized osteosclerosis occurs because of involvement of most other bones, including the base of the skull (Fig. 24-18), facial bones and mandible (Fig. 24-19), vertebrae, pelvis, and ribs (Fig. 24-20). 2-8 In sclerosteosis, the skeleton, except for possible syndactyly, is normal in early childhood. The major radiological feature is progressive osteosclerosis and widening of the skull and mandible (including prognathism). 152The fibs, pelvis, vertebral pedicles, and tubular bones are somewhat dense. Modeling defects occur in the long bones where the cortices are thickened. Syndactyly, usually of the index and long fingers, is common (Fig. 2 4 21). Cranial CT findings in sclerosteosis have recently been reported in detail and have revealed that fusion of the ossicles as well as narrowing of the internal auditory canals and cochlear aqueducts account for the deafness. ~53
D. Histopathological Findings Van Buchem and co-workers suggested that the disorder is due to excessive formation of normal bone tissue. 141
Progressive diaphyseal dysplasia. AP radiograph of the left leg of a 19-year-old man with a mild form of Engelmann's disease shows focal sclerosis and cortical thickening of the midtibia (arrows). [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
A recent multidisciplinary study of an American kindred with sclerosteosis included histomorphometric analysis of calvarium following in vivo tetracycline labeling. 151 Dense thickened trabeculae were associated with active appearing osteoblasts, increased total bone volume, elevated relative osteoid volume, and increased linear extent of bone formation and appositional rate. Osteoclastic bone resorption seemed to be depressed. 151
fusion of the index and middle fingers is a characteristic finding, but of variable severity. Fingernails are dysplastic. Intelligence is not affected. Life expectancy may be
E. Etiology and Pathogenesis
FIGURE 24-- 16
s h o r t e n e d . 15~
B. Laboratory Findings Alkaline phosphatase activity in serum is of bone origin, and may be increased in van Buchem's disease. Serum levels of calcium and inorganic phosphate are normal.
C. Radiological Features The principal radiographic feature of van Buchem's disease is endosteal hyperostosis producing dense homogeneous diaphyseal cortices that narrow the medullary canal (Fig. 24-17). 2-8 Long bones are properly shaped, but appear dense because of widening of the cortex from selective endosteal hyperostosis. General-
Osteoblast hyperactivity, with failure of osteoclasts to compensate, appears to account for the osteosclerosis of sclerosteosis. 151 No abnormality of pituitary function or of calcium homeostasis has been found. 154 A detailed assessment of the pathogenesis of the neurological defects is available. 151 Recent review of 50 patients with sclerosteosis in South Africa and 15 patients with van Buchem's disease in Holland revealed similar clinical and radiographic features that were more severe in sclerosteosis. Accordingly, Beighton and co-workers suggested that both disorders share the same faulty gene(s), but that the phenotypic variation is due to the epistatic effect of modifying genes. ~48
F. Treatment Surgical decompression of narrowed foramina may be helpful in some patients with cranial nerve palsy. 155 There is no effective medical treatment.
CHAPTER 24
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713
FIGURE 24--17 Endostealhyperostosis. A, AP radiograph of the left leg of a 9-year-old boy shows bones that are of normal width yet have very thick cortices that constrict the medullary canal. B, Similar changes are present in his left forearm and hand. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
In sclerosteosis, syndactyly usually requires surgical intervention, but is especially difficult to correct if there is bony fusion. Patients are not prone to fracture. Surgery to correct prognathism is complicated by dense mandibular bone. A thorough discussion of the m a n a g e m e n t of the neurological dysfunction was published in 1983.154
VIII. OSTEOPOIKILOSIS Osteopoikilosis (McKusick 166700), literally translated, means "spotted b o n e s . " This radiological curiosity is inherited as an autosomal dominant trait with a high degree of penetrance. 156 In some kindreds, affected subjects also have connective tissue nevi (dermatofibrosis lenticularis disseminata), in which case the con-
dition is then called the Buschke-Ollendorff syndrome, 157 but family members can have either manifestation alone.
A. Clinical Presentation Osteopoikilosis is often diagnosed when radiological study is performed for unrelated reasons. Musculoskeletal pain has been described in some cases; however, the bony lesions are considered asymptomatic. If the condition is not recognized as a benign skeletal dysplasia, affected individuals may be subjected to diagnostic studies for other important disorders including metastatic disease to the skeleton. 158 The cutaneous nevi of dermatofibrosis lenticularis disseminata are most often noted on the lower trunk or limbs and appear before puberty. They may be congen-
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FIGURE 24-- 18 Endosteal hyperostosis. Lateral radiograph of the skull of the boy depicted in Figure 2 4 - 1 7 shows dense sclerosis of the cranial vault. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
FIGURE 24--19
Endosteal hyperostosis. Lateral radiograph of the mandible and facial bones of the boy depicted in Figures 2 4 - 1 7 and 2 4 - 1 8 show dense sclerosis of all osseous structures. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
P. WHYTE
CHAPTER 24 Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis
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poikilosis revealed excessive amounts of unusually broad, interlacing elastin fibers in the dermis. 157 The epidermis was unremarkable. Electron microscopy showed markedly branched elastin fibers. After digestion with pancreatic elastase, the lesions were formed to contain large amounts of desmosine. 157 The bony abnormalities are thickened trabeculae that merge with surrounding normal bone, or islands of cortical bone with haversian systems. Remodeling of mature lesions is inactive. ~56
D. Treatment Osteopoikilosis does not require treatment.
IX. OSTEOPATHIA STRIATA
FIGURE 24--20 Endosteal hyperostosis. AP radiograph of the ribs of the boy depicted in Figures 24-17 to 24-19 shows homogeneous dense osteosclerosis of all ribs (note: the central portions of the vertebrae are also sclerotic). [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
ital. This dermatosis appears as small asymptomatic papules. Occasionally, the lesions are yellow or white disks or plaques or deep nodules, or streaks. 157'159
B. Radiological Features The principal radiologic feature of osteopoikilosis is focal bony sclerosis composed of numerous small overgrowths in the trabecular portions of the skeleton. Commonly affected sites are the metaepiphyseal regions of the long bones (Fig. 2 4 - 2 2 ) , the ends of the short tubular bones, and the carpal, tarsal, and pelvic bones (Fig. 2 4 - 2 3 ) . These foci are of variable shape, but tend to be round or oval. Once manifest, they are stable in size and shape for decades 2-s and do not avidly accumulate radionuclide on bone scintigraphy. 15s Metastatic lesions may mimic the radiographic appearance of osteopoiki1osis. 16~
C. Histopathological Studies Examination of dermatofibrosis lenticularis disseminata in 12 patients in two unrelated kindreds with osteo-
Osteopathia striata is a radiographic abnormality characterized by asymptomatic linear striations at the ends of long bones and in the ilium. 2-s This finding occurs alone or in a variety of syndromes such as (1) osteopathia striata with cranial sclerosis (McKusick 166500), inherited as an autosomal dominant trait ~61-~65" (2) osteopathia striata with focal dermal hypoplasia, also called Goltz's syndrome (McKusick 305600), 166'167 inherited as an X-linked recessive trait; or (3) osteopathia striata with pigmentary dermopathy (including white forelock), in which X-linked dominant inheritance seems likely (McKusick 311280) and the bony abnormalities have been shown to be developmental in early childhood. 168 Osteopathia striata also occurs as a component of a sclerosing bone disorder, even more radiologically complex, called mixed sclerosing bone dystrophy (see later).
A. Clinical Presentation When osteopathia striata occurs as an isolated finding, affected individuals are generally asymptomatic and the condition is a radiological curiosity. Musculoskeletal complaints of some sort may initiate the radiological studies that lead to the diagnosis. However, when there is cranial sclerosis, cranial nerve palsies and palatine malformations a r e c o m m o n . 164'169 In one such affected family, the diagnosis was reportedly made when a pregnant family member underwent ultrasound examination that disclosed an increased fetal biparietal diameter. ~7~ Osteopathia striata with focal dermal hypoplasia (Goltz's syndrome) is the most serious disorder featuring osteopathia striata. Affected boys have widespread linear lesions of dermal hypoplasia through which adipose tissue
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MICHAEL P. WHYTE
FIGURE 24--21 Sclerosteosis.A, PA radiograph of the hand of a 36-year-old man shows thickened, sclerotic tubular bones. B, PA radiograph of the hand of a 38-year-old man shows syndactyly of his second and third fingers in addition to osteosclerotic tubular bone. (Radiographs courtesy of Dr. Peter Beighton, Capetown, South Africa.) [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
can herniate and have a variety of skeletal defects affecting the l i m b s . 166'167 Osteopathia striata with dermopathy and white forelock 168 may be associated with developmental deafness (personal observation). B. R a d i o l o g i c a l F e a t u r e s
D. T r e a t m e n t There is no medical treatment for osteopathia striata.
X. MELORHEOSTOSIS
Gracile linear striation of bone is the principal radiologic feature of osteopathia striata) -s This abnormality develops in the cancellous portions of the skeleton, particularly the metaepiphyseal ends of the long bones (Fig. 2 4 - 2 4 ) and the periphery of the iliac bones (Fig. 2 4 - 2 5 ) . Involvement of the carpal, tarsal, and tubular bones of the hands and feet is less common and more subtle. Once manifest, the striations are stable in appearance for years. They do not show focal radionuclide accumulation on bone scintigraphy. 158
Melorheostosis (MuKusick 155950), translated from the Greek, means flowing hyperostosis of the limbs. The radiological appearance of affected long bones has been likened to melted wax dripping down the side of a candle. The disorder was first described in 1922.171 About 200 cases have been reported. 172 No mendelian basis for melorheostosis has been established. 173'174
C. H i s t o p a t h o l o g i c a l F i n d i n g s
Melorheostosis usually manifests during childhood. Monomelic involvement is most common. Bilateral disease is asymmetrical. Pain and stiffness are the predom-
Histopathological studies have not been reported.
A. C l i n i c a l P r e s e n t a t i o n
CHAPTER 24 Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis
7 17
B. Laboratory Findings Routine laboratory studies in melorheostosis (e.g., serum calcium and inorganic phosphate levels and alkaline phosphatase activity) are normal.
C. Radiological Features Dense, eccentric, irregular hyperostosis of the cortex and adjacent medullary canal of a single bone or of several adjacent bones is the principal radiological feature of melorheostosis. 2-8'~82 Any bone or anatomical region may be involved, but the condition is more common in the lower limbs (Figs. 2 4 - 2 6 and 24-27). Ectopic bone may develop in soft tissue adjacent to involved skeletal areas, particularly near joints. Melorheostotic bone accumulates radionuclide during bone scintigraphy. 158'183
D. Histological Findings
FIGURE 24--22 Osteopoikilosis.AP radiograph of the knee of a young woman shows multiple sclerotic foci of variable shape located in the metaepiphyseal regions of the distal femur and proximal tibia.
inant symptoms. Affected joints may become contracted and deformity also occurs. Cutaneous changes can overlie affected skeletal regions. Of 131 patients reported in one study, 17% had a dermatosis characterized by linear scleroderma-like areas, 175 sometimes with hypertric h o s i s . 176 Fibromas, fibrolipomas, lymphangiectasis, capillary hemangiomas, and arterial aneurysms may occur. 177 Vascular abnormalities have been documented in at least 5% of reported patients. ~78 The soft tissue abnormalities are often discovered earlier than the underlying hyperostosis. Therefore, it has been suggested that the linear scleroderma might represent the primary abnormality that extends deep into the skeleton. 179 In affected children, soft tissue contractures and premature fusion of epiphyses can cause leg length inequality as a principal clinical feature18~ however, their pain is less frequent than in adults. The skeletal changes appear to progress most rapidly during childhood; during the adult years they can stabilize or more gradually extend. ~8~
Unlike the skin lesions of true scleroderma, the sclerodermatous skin lesions of melorheostosis contain normal-appearing collagen. Therefore, the condition has been called "linear melorheostotic s c l e r o d e r m a . ''176'179'184 A melorheostotic skeletal lesion consists of endosteal thickening during infancy and periosteal bone formation during adulthood. 182 Affected bone is sclerotic and thickened with irregular lamellae that obliterate haversian systems. Fibrosis of intertrabecular spaces can occur. 182
E. Etiology and Pathogenesis The appearance of melorheostosis and soft tissue lesions in the distribution of sclerotomes, myotomes, and dermatomes suggests that a segmentary embryogenetic defect accounts for this sporadic mesodermal disorder. 172'182 An early postzygotic mutation has been suggested. 178
F. Treatment Surgical correction of contractures in children with melorheostosis is difficult; recurrent deformity is usual. The calcium channel blocking agent nifedipine has been reported to alleviate pain in one patient. ~85
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MICHAEL P. WHYTE
FIGURE 24--23 Osteopoikilosis.AP radiograph of left hemipelvis of this 39-year-old woman shows multiple small sclerotic foci throughout the innominate bone and the metaepiphyseal region of the left femur. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
XI. MIXED SCLEROSING BONE DYSTROPHY Mixed sclerosing bone dystrophy is the term offered by Walker in 1964 to describe two patients in w h o m radiological features of melorheostosis, osteopoikilosis, and osteopathia striata were present in combination. 186 Subsequently, cranial sclerosis and/or other skeletal defects were recognized to also occur (see Section IX) in some affected individuals. 187-19~ Although this is clearly a very heterogeneous disorder, review of the literature in 1981 resulted in a tentative classification that might help to identify possible subgroups. 19~
FIGURE 24--24 Osteopathia striata. AP radiograph of left knee of a 25-year-old woman shows vertical linear striations in the metaepiphyseal regions of the distal femur and proximal tibia. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
ically associated with each of these findings. The skull can be enlarged (Fig. 2 4 - 2 8 ) and cranial nerve entrapment may occur. Skeletal pain can trouble the patient.
B. Radiological Features Two or more patterns of osteosclerosis described thus f a r m t h a t is, osteopoikilosis, osteopathia striata, melorheostosis, focal osteosclerosis, cranial sclerosis, generalized cortical hyperostosis, or progressive diaphyseal d y s p l a s i a m a r e found in one patient (Figs. 2 4 - 2 9 and 2 4 - 3 0 ) . Often, only a portion of the skeleton is involved. 186'19~ Skeletal scintigraphy will reveal increased radionuclide uptake in areas of greatest osteosclerosis. 190,191
A. Clinical Presentation C. Histopathological Findings Patients with mixed sclerosing bone dystrophy who have components of cranial sclerosis and/or melorheostosis may be symptomatic from the complications typ-
Although some patients with mixed sclerosing bone dystrophy and generalized osteosclerosis have been re-
CHAPTER 24
Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis
FIGURE 2 4 - 2 5 Osteopathia striata. AP radiograph of the left hemipelvis of this 25-year-old woman shows linear striations that are distributed in a fan-like pattern in the iliac wing. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
ported to have " o s t e o p e t r o s i s , " detailed histopathological study of iliac crest bone from several affected individuals has failed to show remnants of primary spongiosa, thereby excluding absence of osteoclastmediated bone resorption. 19~
D. Etiology and Pathogenesis Delineation of m i x e d sclerosing bone dystrophy suggests a c o m m o n pathogenetic m e c h a n i s m for these forms of osteosclerosis or hyperostosis w h e n they occur separately. However, whereas osteopoikilosis and osteopathia striata have been shown clearly to be heritable, m i x e d sclerosing bone dystrophy, like melorheostosis, has been reported only as a sporadic disorder.
E. Treatment There is no effective medical treatment for mixed sclerosing bone dystrophy. Surgical correction of con-
719
FIGURE 24--26 Melorheostosis. AP radiograph of the left knee of a 42-year-old woman shows asymmetrical eccentric distribution of cortical thickening and dense medullary hyperostosis of the distal femur. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
tractures or of neurovascular c o m p r e s s i o n by osteosclerotic lesions m a y be necessary.
XII. FIBRODYSPLASIA OSSIFICANS PROGRESSIVA Fibrodysplasia ossificans progressiva (myositis ossificans progressiva) is characterized by a variety of congenital skeletal deformities together with recurrent, painful, progressive episodes of heterotopic formation of true bone in fascia, aponeuroses, ligaments, tendons, and connective tissue of voluntary muscles; smooth muscle is spared. Since the disorder was first described in 1692,192 more than 500 cases have been reported, w3'194 It appears to be transmitted as an autosomal dominant trait, but with very variable expressivity ( M c K u s i c k 135100). M o s t cases, however, are sporadic. 1 Caucasians are re-
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MICHAEL P. WHYTE
muscles of the mandible may lead to severe limitation of jaw movement and impair nutrition. Involvement of the musculature of the thorax can deform the chest (Fig. 2 4 - 3 2 ) and thereby cause restrictive lung disease and predispose the patient to pneumonia. Scoliosis occurs frequently. ~95 Although secondary amenorrhea is not unusual, successful reproduction has been reported. ~96 Deafness and alopecia occur with increased frequency.
B. Laboratory Findings Serum alkaline phosphatase activity in fibrodysplasia ossificans progressiva may be increased. Other routine biochemical studies are usually normal.
C. Radiological Features
FIGURE 24--27 Melorheostosis. AP radiograph of the left hemipelvis of a 42-year-old woman shows dense, eccentric, irregular hyperostosis of the acetabulum and proximal femur. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
ported most commonly; however, the disorder has been described in blacks. 192
A. Clinical Presentation Fibrodysplasia ossificans progressiva usually becomes manifest during the first decade of life, 194 but the age at presentation is highly variable. There are reports of involvement in u t e r o and beginning as late as early adulthood. Nevertheless, the diagnosis may be made at birth by the presence of a variety of congenital skeletal anomaliesmthe most characteristic being hallux valgus with microdactyly (Fig. 24-31). Microdactyly of the thumb may also be present. There can be synostosis and hypoplasia of the phalanges. Recurrent episodes of tender, rubbery, painful soft tissue swellings (that are sometimes associated with minor trauma) are followed by calcification and then heterotopic true bone formation. Early on, torticollis with involvement of the sternocleidomastoid muscle is noted, or the muscles of the shoulder girdle and dorsum of the trunk are affected. Fever may occur during these times and mimic an infectious process. Progressive episodes of heterotopic bone formation result in decreased range of motion, especially in the neck and shoulders. Involvement of the
A combination of anomalies of the digits and soft tissue ossification is the radiological hallmark of fibrodysplasia ossificans progressiva. ~97'198 There is ossification of fascia, tendons, aponeuroses, and other tissues in a characteristic pattern of progression. 199 The neck (Fig. 24-33A) and shoulders (Fig. 24-33B) tend to be involved earlier than the lower extremities (Fig. 24-34). Paraspinal muscles are especially prone to ossification. Ankylosis of the spine, rib cage, and joints limits mobility. During severe inflammation, portions of the skeleton are osteopenic. Otherwise, bones are generally well mineralized. Chief among the skeletal anomalies is disturbance of the formation of the great toe (Fig. 24-35). CT to detect soft tissue calcification appears to be the best radiological technique to diagnose an early lesion. 2~176 Skeletal scintigraphy with 99mTCmethylene diphosphonate will also be abnormal before ossification will be detected radiographically. New areas of disease activity can be detected early by this technique. 2~
D. Histopathological Findings Early lesions of fibrodysplasia ossificans progressiva are characterized by edema of fascial planes. The soft tissue mass is an edematous muscle or group of muscles. Multifocal interconnective nodules then form that are composed of fibroblasts. Later, osteoid, cartilage, and bone are present at the center of a fibrous connective tissue m a t r i x . 2~176 Fasciae, tendons, ligaments, and joint capsules may be involved. The pathological process has been identified as a form of endochondral bone formation. 2~ The ossification is characterized macroscopically by dense, flat, irregular areas of bone within the connective tissue of fascial planes, and this ossification ex-
CHAPTER 24
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FIGURE 24--28 Mixed sclerosing bone dystrophy. A and B, This 49-year-old man has a very large head with prominent jaw and forehead. (From Pacifici R, Murphy MA, Teitelbaum SL, et al: Mixed-sclerosing-bone-dystrophy: 42-year follow-up of a case reported as osteopetrosis. Calcif Tissue Int 38:175-185, 1986.)
tends partly or completely around a muscle. 2~ Cancellous bone can eventually form. As the lesions mature, hematopoietic tissue will appear within the areas of trabecular bone. 2~ Muscle fibers may undergo secondary degenerative and atrophic change.
damental abnormality in the skeletal muscle (myositis), because histological and electromyographic abnormalities may occur prior to connective tissue proliferation. 2~ Recently, evidence for a fundamental disturbance in the biosynthesis of a bone morphogenetic protein has been demonstrated. 2~
E. E t i o l o g y a n d P a t h o g e n e s i s F. T r e a t m e n t a n d P r o g n o s i s The autosomal gene defect that causes fibrodysplasia ossificans progressiva is unknown. Paternal age appears to contribute importantly to the incidence of new dominant mutations, ~94'2~ Most cases appear to be sporadic. 1'2~ The pathogenesis is incompletely understood. It is unclear whether the disorder is due to a connective tissue abnormality that secondarily affects muscle or a primary abnormality in muscle itself. The term "fibrodysplasia ossificans progressiva" is favored by those who believe that connective tissue is primarily involved and its proliferation within skeletal muscle leads to atrophy and heterotopic bone formation. In support of this hypothesis, CT has shown that characteristic swelling of muscular fascial planes occurs before development of ectopic ossification. Multifocal sites of new bone formation develop adjacent to and extend around muscles. 2~ Some scholars in the field, however, see a fun-
There is no satisfactory medical treatment for fibrodysplasia ossificans progressiva. Adrenocorticotropic hormone or corticosteroids, calcium binders in the diet, and ethylenediaminetetraacetic acid (EDTA) infusion have not been successful. 21~ Treatment with the bisphosphonate disodium etidronate has resulted in a variable response. 211 Use of warfarin to inhibit gamma-carboxylation of osteocalcin in an effort to prevent ectopic bone formation has been of no noticeable clinical benefit. 212 Surgical release procedures for joint contractures, neurovascular entrapment, or to increase mandibular range of motion often provoke additional ectopic bone formation as does intramuscular vaccination. CT is useful to guide critical surgical intervention by identifying soft tissue changes and ossification that is not yet apparent by conventional radiography. 2~ In some cases, a course
722
FIGURE 24--29 Mixed sclerosing bone dystrophy. AP radiograph of the man shown in Figure 24-28; the left knee shows osteopoikilosis of the distal femoral epiphysis, osteopathia striata of the distal femoral and proximal tibial metaepiphyses, and melorheostosis of the distal femoral diaphysis. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
of e t h a n e - l - h y d r o x y - l , l - d i p h o s p h o n a t e (EHDP) appeared to delay the mineralization of newly formed bone matrix following surgery. 211 Despite widespread ectopic ossification, some patients live into the fifth decade. Most patients, however, die earlier from respiratory complications secondary to restricted pulmonary ventilation due to chest wall involvement. 194
XlII. AXIAL OSTEOMALACIA Axial osteomalacia is a rare disorder characterized by coarsening of the trabecular pattern on radiographic examination of the axial but not appendicular skeleton. The disorder was first described in 1961 by Frame and coworkers; the axial skeletons of three patients were noted radiographically to have " a unique coarsening and
MICHAEL P. WHYTE
FIGURE 24--30 Mixed sclerosing bone dystrophy. AP radiograph of the man shown in Figure 24-28; the left hemipelvis shows focal osteosclerosis of the ilium and a melorheostosis pattem of the proximal femur. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
sponge-like appearance. ''213 Their skulls and limbs were unremarkable. Osteoidosis was found on examination of undecalcified sections of bone. Fewer than 20 cases have been described. No mendelian pattern of transmission has been established, but autosomal dominant inheritance seems possible (McKusick 109130).
A. Clinical Presentation Most patients with axial osteomalacia have been middle-aged or elderly men. A few affected middle-aged w o m e n have been described. Radiographic manifestations, however, are likely to be present much earlier in life. 214 The disorder may be discovered incidentally; more frequently there is dull, vague, and chronic axial skeletal pain (often in the cervical region) that prompts x-ray study. Family histories have generally been reported to be negative for skeletal disease, but radiographic surveys have usually not been performed. Axial osteomalacia has been noted to be familial o n c e m a f fecting a black mother and son in w h o m polycystic liver
CHAPTER 24 Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis
723
FIGURE 24--3 1 Fibrodysplasiaossificans progressiva. Typical hallux valgus deformity with microdactylyis present in this 14-year-old boy. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
and kidneys were also present. 214 Two other patients had features of ankylosing spondylitis. 215
cervical spine and ribs, and to a lesser extent in the lumbar spine. The appendicular skeleton is unaffected.
B. Laboratory Studies
D. H i s t o p a t h o l o g i c a l F i n d i n g s
Four cases of axial osteomalacia have been described in whom serum inorganic phosphate levels tended to be l o w . 215 In other patients, defective bone mineralization occurred despite normal serum levels of calcium and inorganic phosphate and increased alkaline phosphatase activity (bone isoenzyme). Serum levels of 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] were unremarkable in one patient. 214 One affected individual was found to be in strongly positive calcium and phosphorus balance. 216 Symptoms, elevated serum creatine kinase activity, and muscle biopsy features consistent with myopathy have been reported in a single patient. 214
C. Radiological Features The radiographic features of axial osteomalacia are essentially limited to the spine (Fig. 2 4 - 3 6 ) and pelvis (Fig. 24-37); the trabecular pattern is somewhat coarse and resembles that found in other types of osteomalacia. 5 Looser's zones, however, have not been reported. Radiographic changes appear to be most pronounced in the
In axial osteomalacia, osseous collagen has a normal lamellar pattern as shown by polarized light microscopy of rib specimens. Iliac crest specimens have distinct corticomedullary junctions, yet cortices may have both increased width and porosity. Trabeculae can have varied thickness, and total bone volume may be increased. Osteoidosis has been documented; that is, the width and extent of osteoid seams on trabecular bone surfaces and in cortical spaces may be increased. Biopsy of ribs and iliac crest following tetracycline labeling has confirmed the presence of osteomalacia, since most fluorescent "labels" were single, wide, and irregular. 214 Osteoblasts are flat and inactive-appearing ("lining") cells with reduced rough endoplasmic reticulum and Golgi zones and increased cytoplasmic glycogen. Nevertheless, they may stain intensely for alkaline phosphatase activity. Osteoclasts can be few, but histochemical studies show normal levels of acid phosphatase activity. Evidence of secondary hyperparathyroidism is absent. 214 Histological changes of osteomalacia following tetracycline labeling 215 help to distinguish axial osteomalacia from fibrogenesis imperfecta ossium 216 (see Section XIV).
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MICHAEL P. WHYTE
FIGURE 2 4 - 3 2
Fibrodysplasia ossificans progressiva. A and B, Marked narrowing of the thorax and frozen shoulders are present in this affected 14-year-old boy. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
FIGURE 24--33
Fibrodysplasia ossificans progressiva. A, Lateral radiograph of the neck of a 6-year-old boy shows ossifcation of dorsal soft tissues and ankylosis of all cervical apophyseal joints. B, AP radiograph of the shoulder of the same patient shows extensive soft tissue ossification. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
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FIGURE 24--34 Fibrodysplasia ossificans progressiva. AP radiograph of the pelvis of a 17-year-old boy shows extensive ossification about the right hip (note that the femoral head is dislocated and that the joint is ankylosed by the ossified periarticular tissue). [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
E. Etiology and Pathogenesis F r a m e and colleagues suggested that axial osteomalacia is due to " u n k n o w n defects of local cellular origin. ''213 Electron microscopic studies of iliac crest tissue
FIGURE 24--36 Axial osteomalacia. Lateral radiograph of the lumbar spine of a 35-year-old man shows generalized osteosclerosis due to thickening of the trabeculae.
from one patient 214 s h o w e d osteoblasts with an inactive appearance, yet the presence of intact matrix vesicles within unmineralized osteoid. The possibility that this is a heritable disorder deserves further study. 214
F. Treatment
FIGURE 24--35 Fibrodysplasia ossificans progressiva. AP radiograph of the toes of a 9-year-old boy shows anomalies of the phalanges of the great toe. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
There is no effective medical therapy for axial osteomalacia. In one patient treated with stilbestrol and methyltestosterone, no i m p r o v e m e n t in s y m p t o m s or radiological features was noted. 217 Similarly, vitamin D2 therapy (as m u c h as 20,000 units/day for 3 years) resulted in no change in s y m p t o m s or radiographic pattern. 217 In a study of four cases, calcium and vitamin D2 therapy resulted in some small i m p r o v e m e n t in skeletal histology, but there was no s y m p t o m a t i c benefit; it was concluded that this treatment was of no practical value. 218 Since the s y m p t o m s and radiological findings in one patient did not change during an 18-year period, 217 and another patient r e m a i n e d well during 5 years of observation, 216 a relatively benign natural history for axial
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MICHAEL P. WHYTE
FIGURE 24--37
Axial osteomalacia. AP radiograph of the pelvis of a 35-year-old man shows generalized osteosclerosis. (From Whyte MP, et al: Am J Med 71:1041 - 1049, 1981.)
osteomalacia seems possible. Treatment with vitamin D2 and mineral supplementation would seem risky, since the disorder appears to be due to a defect in the bone tissue itself, rather than in mineral homeostasis.
XIV. FIBROGENESIS IMPERFECTA OSSIUM Fibrogenesis imperfecta ossium, first described by Baker and Turnbull in 1950, 219 is a very rare condition. 219-228 In 1986, Lang and co-workers described one case and summarized the findings in six others. 228 Although radiological studies suggest a generalized decrease in bone mass, the coarse and dense appearance of trabeculae explain why it is discussed among the osteosclerotic disorders. Christman and colleagues, in 1981, reported three cases of axial osteomalacia and two cases of fibrogenesis imperfecta ossium and contrasted the clinical, biochemical, radiological, and histopathological features of both disorders. 216
life. Both men and women are affected. There is gradual onset of intractable skeletal pain and progressive immobility. The disease then runs a rapidly progressive and debilitating course, with spontaneous fractures being a prominent feature. Marked bony tenderness is usual. Patients generally become bedridden. One affected individual suffered acute agranulocytosis. 223 Another subject with radiological changes that simulated fibrogenesis imperfecta ossium had macroglobulinemia. 229
B. Laboratory Findings In fibrogenesis imperfecta ossium, serum levels of calcium and inorganic phosphate are normal; alkaline phosphatase activity is increased. Urinary levels of hydroxyproline may be normal or elevated. 228 There is generally no evidence of renal tubular dysfunction or aminoaciduria. Monoclonal gammopathy appears to be common. 226
C. Radiological Features A. Clinical Presentation Fibrogenesis imperfecta ossium is an acquired disorder that presents clinically in middle-age or late adult
The radiological features of fibrogenesis imperfecta ossium are found throughout the skeleton except in the skull. At first there may be only osteopenia and a slightly
CHAr~rER 24 Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis abnormal appearance of trabecular bone. 228 Subsequently, the changes are generally consistent with osteomalacia; that is, alteration of the cancellous bone pattern, irregular bone density, and cortical thinning. Despite a generalized decrease in skeletal density, remaining trabeculae appear coarse and dense ("fishnet" pattern). Corticomedullary junctions then become indistinct, and cortices appear to be replaced by an abnormal trabecular pattern. A mixed lytic and sclerotic pattern may be present. 228 Pseudofractures can occur. Fracture deformities may be noted, although the contour of bones is generally normal. A "rugger jersey" spine is present in some patients and should not be confused with similar radiographic findings in patients with renal osteodystrophy. Periosteal reaction may occur along the shafts of long bones. The radiographic changes of fibrogenesis imperfecta ossium closely resemble those of axial osteomalacia; however, the disorders are distinguishable by different distributions of radiographic abnormalities
727
(generalized vs. axial). Furthermore, there are different histopathological findings. 218
D. H i s t o p a t h o l o g i c a l F i n d i n g s The basic lesion of fibrogenesis imperfecta ossium appears to be similar throughout the skeleton, but the amount of affected bone varies widely from site to site. Cortical bone (e.g., in the diaphyses of the femora and tibiae) may show the least abnormality. Osteoblasts and osteoclasts can be abundant, and osteoid seams are thick. There is an abnormal mineralization pattern. Tetracycline labeling reveals the presence of osteomalacia. 228 Characteristically, abnormal collagen is found where lamellar bone should be present (collagen in other tissues is unremarkable). On polarized light microscopy, bone collagen fibrils are not birefringent. Electron microscopy reveals them to be thin and randomly organized in a
FIGURE 24--38 Fluorosis. A, Lateral radiograph of the lower thoracic spine of an 82-year-old woman with osteoporosis shows demineralized vertebral bodies. B, Lateral radiograph of lower thoracic spine of the same patient, now age 87, shows mild osteosclerosis of vertebral bodies after five years of sodium fluoride therapy. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
728
MICHAEL P. WHYTE
"tangled pattern," rather than in a lamellar distribution. 23~ Narrow and irregular bands of normal collagen fibrils may occasionally traverse regions of abnormal bone matrix. Only a few scattered areas of lamellar bone are noted. In some areas of abnormal matrix, unusual circular bodies of 300 to 500 nm diameter are present. 228 This abnormal bone matrix does not calcify properly, and wide osteoid seams are present. Unless biopsy specimens are viewed with polarized light or electron microscopy, this disorder may be mistaken for osteoporosis or the more common forms of osteomalacia. 228
F. T r e a t m e n t There is no effective medical therapy for fibrogenesis imperfecta ossium. Although the clinical course is generally one of deterioration, brief periods of clinical improvement c a n occur. 22s Vitamin D2 (or active metabolites) with calcium supplements has been tried without significant benefit. Calcification of soft tissues and of tendons and ligaments occurred in one patient who was treated with large doses of vitamin D2. Sodium fluoride, synthetic salmon calcitonin, and 24,25(OH)zD have also been used without apparent benefit. 228 Courses of melphalan and prednisone were followed by dramatic remission in one patient who had a monoclonal g a m m o p a t h y . 226,23~
E. E t i o l o g y a n d P a t h o g e n e s i s
XV. F L U O R O S I S The etiology of fibrogenesis imperfecta ossium is unknown. The disorder has been reported only sporadically and, therefore, genetic factors have not been implicated. Although any specific biochemical defect awaits discovery, the condition appears to be an acquired disorder of collagen synthesis in lamellar bone that, in some way, inhibits mineralization of the osseous matrix. Only the skeleton seems to be affected. There does not appear to be a defect in subperiosteal bone formation or in collagen in nonosseous tissues. In one patient who also had monoclonal gammopathy, a toxin produced by bone marrow was postulated. 226
FIGURE 24--39
Fluoride may cause osteosclerosis when ingested or inhaled chronically in various forlTlS. 231-234 Endemic fluorosis occurs in some regions (e.g., Punjab, India) from contaminated well water. 232 Men are more commonly affected than women, perhaps because their water consumption, as they perform manual labor, is greater. Leafy vegetables and tea grown in soil with a high content of fluoride, certain wines, and cooking salt represent other dietary sources of excessive amounts of fluoride. 234 However, fluorosis has also been reported as an occupational hazard, for example, in conjunction with alu-
Pachydermoperiostosis. Characteristic marked clubbing of the fingers of a 33-year-old man. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
CHAPTER 24 Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis minum or fertilizer production. TM Prolonged administration of the nonsteroidal antiinflammatory agent niflumic acid, T M or sodium fluoride in attempted therapy for postmenopausal osteoporosis, 233 represent other potential etiologies.
A. Clinical Presentation Diffuse bone pain, stiffness, decreased joint mobility, and brownish discoloration and mottling of the teeth are the principal signs and symptoms in f l u o r o s i s . T M A "plantar fasciitis syndrome" characterized by painful and swollen feet has been described, but stress fracture of the calcaneus has been shown to be a likely cause of this discomfort. 235 Cranial nerve palsies, radiculopathies, and plexopathies can result from ligamentous calcification, exostoses, and osteophytoses.
B. Laboratory Findings Serum alkaline phosphatase activity may be increased in fluorosis, which could reflect osteoblast stimulation
729
by secondary hyperparathyroidism. Mineral balance studies have shown calcium retention. 236 Urinary hydroxyproline levels may be increased. Assay of fluoride in blood or urine can provide biochemical support for the diagnosis.
C. Radiological Features Fluorosis can result in osteosclerosis and radiographic changes consistent with osteomalacia. 235 In general, it is the cancellous portions of the skeleton that become sclerotic (Figs. 24-38). Characteristically, however, various ligaments also calcify. Irregularity at the insertion of muscles at the iliac spine and calcification of ligaments in the pelvis, vertebrae, and interosseous membranes are early radiological signs. 5 Later, there may be generalized osteosclerosis. Osteomalacia can develop, because fluoride stimulates osteoid synthesis, yet mineral deposition may be impaired. 237 In this circumstance, stress fractures in the proximal femur or calcaneus have been reported during fluoride therapy for osteoporosis. 235
D. Etiology and Pathogenesis Fluoride appears to cause osteosclerosis by a combination of mechanisms including direct stimulation of osteoblast mitogenic activity, formation of fluoroapatite crystals that are relatively resistant to osteoclastic resorption, and inducement of secondary hyperparathyroidism. 234'236'237
E. Treatment Treatment of fluorosis includes elimination of excessive intake of fluoride and, if osteomalacia is present, calcium and vitamin D supplementation to mineralize the fluoride-induced osteoidosis and to reduce the secondary hyperparathyroidism. 235
XVI. PACHYDERMOPERIOSTOSIS
FIGURE 24--40 Pachydermoperiostosis. PA radiograph of the hand of the patient shown in Figure 24-39 shows clubbing of the fingertips with associated hypertrophy of the distal phalangeal tufts (note also the widened tubular bones, particularly the middle phalangeal bases). [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
Pachydermoperiostosis (hypertrophic osteoarthropathy: primary or idiopathic) was first described in 1868. 238 The condition is characterized by clubbing of the digits (Fig. 24-39), hyperhidrosis with thickening of the skin of especially the face and forehead, and periosteal new bone formation distally in the limbs. Autosomal dominant transmission with variable expression is established, 239 but autosomal recessive inheritance also appears to occur (McKusick 167100). 1
730
MICHAEL P. WHYTE A. C l i n i c a l P r e s e n t a t i o n
Men appear to be more severely affected by pachydermoperiostosis than are women, and blacks have it more often than others. 24~ Symptoms usually begin during adolescence, although earlier and later presentations have been reported. 239'24~ Some patients have all three major findings (pachyderma, cutis verticis gyrata, periostitis); others have one or two features. These abnormalities develop for about a decade and then become quiescent. 241 Gradual progressive enlargement of the hands and feet results in a paw-like appearance. Some patients appear to be acromegalic. 242 Often there are arthralgias of the ankles, knees, wrists, elbows, and occasionally, the small joints. Symptoms consistent with pseudogout may occur; chondrocalcinosis with calcium pyrophosphate crystals in synovial fluid has been reported in one patient. 243 Acroosteolysis has also been described. T M Stiffness and restricted motion of both appendicular and axial skeleton can occur. Cranial or spinal
nerves may be compressed. When there are cutaneous changes, they may include coarsening, thickening, furrowing, pitting, and oiliness of the skin of the face and scalp. Some patients fatigue easily. Myelophthisic anemia with extramedullary hematopoiesis may o c c u r . 245 Life expectancy appears to be n o r m a l . 24~
B. L a b o r a t o r y F i n d i n g s Synovial fluid in pachydermoperiostosis generally does not indicate inflammation.
C. R a d i o l o g i c a l F e a t u r e s Severe periostitis causing thickening of the distal aspect of tubular bones of the l i m b s - - e s p e c i a l l y the tibia, fibula, radius, and u l n a - - i s the major abnormality in pachydermoperiostosis. Clavicles, metacarpal, tarsal/
FIGURE 24--41 A, Pachydermoperiostosis. AP radiograph of the distal leg and ankle of the patient in Figure 24-39 shows extensive shaggy periosteal reaction along the interosseous membrane between the tibia and fibula (note also the extensive proliferative bone formation along the medial malleolus). B, Hypertrophicpulmonary osteoarthropathy. AP radiograph of the distal leg and ankle of a 30-year-old woman shows smooth, layered periosteal new bone formation along the medial aspect of the distal tibia [note that the medial malleolus (epiphysis) is not involved]. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
CHAPTER24 SkeletalDisorders Characterized By Osteosclerosis or Hyperostosis metatarsals, phalanges, pelvis, and base of the skull may also be affected. The spine is rarely involved. 5 Clubbing is apparent radiographically (Fig. 24-40). Sclerosis and expansion of the diaphyseal region of tubular bones resuits from periosteal thickening. These changes are widespread and symmetrical. In longstanding cases, ankylosis of joints--especially hands and f e e t - - m a y occur. 5 Acroosteolysis has also been reported with pachydermoperiostosis. 244,246 The major consideration in the differential diagnosis is secondary hypertrophic osteoarthropathy (pulmonary or otherwise). The radiological features are, however, somewhat different for primary and secondary disease. In pachydermoperiostosis, periosteal proliferation is more extreme, has an irregular appearance, and often extends to the epiphysis (Fig. 24-41A). In hypertrophic pulmonary osteoarthropathy, the periosteal reaction has a smoother, undulating appearance (Fig. 24-41B). 247 Skeletal scintigraphy in both conditions reveals regular and symmetrical diffuse uptake along the cortical mar-
731
gins of long bones, especially in the legs, which produces a "double-stripe" sign. 248
D. H i s t o p a t h o l o g i c a l F i n d i n g s Periosteal deposition of bone in pachydermoperiostosis results in a roughened cortical s u r f a c e . 249 The new bone undergoes cancellous compaction and may be difficult to distinguish from original cortex. 249 Synovial membrane shows mild cellular hyperplasia and thickening of subsynovial blood vessels. 25~ Electron microscopy shows a layered basement membrane.
E. E t i o l o g y and P a t h o g e n e s i s The genetic defect of pachydermoperiostosis is unknown. Blood flow is decreased to involved areas of bone in pachydermoperiostosis, but is increased in cases of secondary c l u b b i n g . 239'241'251'252 T h e arthralgias appear to stem from the associated periostitis. 253
XVII. HEPATITIS
C-ASSOCIATED
OSTEOSCLEROSIS In 1992, a new syndrome was characterized that features remarkably severe, developmental, generalized osteosclerosis and hyperostosis in hepatitis C-positive, former intravenous drug abusers. T M Periosteal, endosteal, and trabecular bone thickening occurs throughout the skeleton except in the cranium (Fig. 24-42). The forearms and legs are painful. Densitometric studies show bone mass that may be 200% to 300% above mean values for age and sex. Skeletal remodeling can be abnormally rapid and respond to calcitonin therapy. Gradual spontaneous remission in pain and normalization of bone remodeling may o c c u r . T M Tainted blood exposure is the common underlying history. 255 The etiology is unknown, but virus-induced stimulation of osteoblast function seems possible. T M
XVIII. OTHER DISORDERS
FIGURE 24--42 Hepatitis C-associated osteosclerosis. AP radiograph of the hip and proximal femur of this middle-aged man shows marked endosteal and periosteal bone formation and severe osteosclerosis characterized by thickened trabeculae.
As summarized in Tables 2 4 - 1 and 24-2, in addition to the primarily dysplastic osteosclerotic disorders discussed here, a relatively large number of other conditions result in either focal or generalized increases in skeletal mass. Although limitation of space does not permit a detailed discussion of these entities (the reader will find references 2 through 8 to be especially helpful
7
3
2
M
I
C
H
A
E
L
P. WHYTE
Acknowledgments This work was made possible by grant 15958 from Shriners Hospitals for Children and grant RR-00036 from the General Clinical Research Center Branch, Division of Research Facilities and Resources, National Institutes of Health.
References
FIGURE 2 4 - - 4 3 Disseminated prostatic carcinoma. Lateral radiograph of the lumbar spine of a 47-year-old man with widespread prostatic carcinoma shows characteristic radiodense vertebrae. Except for the margins of L3 (arrows), the osteosclerosis is quite uniform. [From Avioli LV, Krane SM (eds): Metabolic Bone Disease and Clinically Related Disorders, 2nd ed. Philadelphia, WB Saunders Co, 1990.]
sources
of information),
some
generalities
should be
noted. S a r c o i d o s i s o f the s k e l e t o n is t y p i c a l l y a s s o c i a t e d w i t h cystic and coarsely reticulated bone. Rarely, however, s c l e r o t i c l e s i o n s i n v o l v e t h e a x i a l s k e l e t o n o r l o n g tubular bones. T h e s e changes m a y occur well after the p u l m o n a r y d i s e a s e is a r r e s t e d . 256 A l t h o u g h m u l t i p l e m y e l o m a typically presents with generalized osteopenia or with osteolytic changes, widespread osteosclerosis can O c c u r . 257'258 L y m p h o m a , m y e l o s c l e r o s i s , a n d m a s t o c y t o sis a r e a d d i t i o n a l h e m a t o l o g i c a l c a u s e s o f i n c r e a s e d b o n e mass.
Metastatic
carcinomamprimarily
24-43)mcommonly
causes
prostate
osteosclerosis.
(Fig.
Osteoscle-
r o s i s is a t y p i c a l r a d i o l o g i c a l f e a t u r e o f P a g e t ' s b o n e
disease. 259 Diffuse osteosclerosis is also a relatively frequent radiographic finding in secondary hyperparathy-
roidism (as with renal disease), but can occur in primary hyperparathyroidism a s w e l l . 26~ Intoxication with either vitamin
A 261 o r D , 262
heavy metal poisoning, 263 milk-
alkali s y n d r o m e , 264 i o n i z i n g r a d i a t i o n , 265 o s t e o m y e l i t i s , 265 a n d o s t e o n e c r o s i s 2'5 are a d d i t i o n a l e t i o l o g i c a l f a c t o r s (Table 24-1
and 24-2).
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CHAPTER 24
Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis
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734 67. Sly WS, Lang R, Avioli L, et al: Recessive osteopetrosis: New clinical phenotype. Am J Hum Genet 24:34 (abstract), 1972. 68. Guibaud P, Larbre F, Freycon MT, et al: Osteopetrose et acidose renale tubulaire deux cas de association dans une fratrie. Arch Fr Pediatr 29:269-286, 1972. 69. Vainsel M, Fondu P, Cadranel S, et al: Osteopetrosis associated with proximal and distal tubular acidosis. Acta Paediatr Scand 61:429-434, 1972. 70. Whyte ME Murphy WA, Fallon MD, et al: Osteopetrosis, renal tubular acidosis and basal ganglia calcification in three sisters. Am J Med 69:64-74, 1980. 71. Ohlsson A, Stark G, Sakati N: Marble brain disease: Recessive osteopetrosis, renal tubular acidosis and cerebral calcification in three Saudi Arabian families. Dev Med Child Neurol 22:72-84, 1980. 72. Sly WS, Hewett-Emmett D, Whyte ME et al: Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc Natl Acad Sci USA 80: 2752-2756, 1983. 73. Roth DE, Venta PJ, Tashian RE, Sly WS: Molecular basis of human carbonic anhydrase II deficiency. Proc Natl Acad Sci USA 89:1804-1808, 1992. 74. Sly WS, Whyte ME Sundaram V, et al: Carbonic anhydrase II deficiency in 12 families with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. N Engl J Med 313:139-145, 1985. 75. Cochat P, Loras-Duclaux I, Guibaud P: Deficit en anhydrase carbonique II: Osteopetrose, acidose renale tubulaire et calcifications intracraniennes. Revue de la literature a partir des trois observations. Pediatrie 42:121-128, 1987. 76. Whyte MP: Carbonic anhydrase II deficiency. Clin Orthop 294: 5 2 - 6 3 , 1993. 77. Ohlsson A, Cumming WA, Paul A, et al: Carbonic anhydrase II deficiency syndrome: Recessive osteopetrosis with renal tubular acidosis and cerebral calcification. Pediatrics 77:371-381, 1986. 78. Sly WS, Whyte ME Krupin T, et al: Positive renal response to acetazolamide in carbonic anhydrase II deficient patients. Pediatr Res 19:1033-1036, 1985. 79. Bourke E, Delaney VB, Mosawi M, et al: Renal tubular acidosis and osteopetrosis in siblings. Nephron 28:268-272, 1981. 80. Cumming WA, Ohlsson A: Intracranial calcification in children with osteopetrosis caused by carbonic anhydrase II deficiency. Radiology 157:325-327, 1985. 81. Tashian RE, Hewett-Emmett D, Goodman M: On the evolution and genetics of carbonic anhydrase I, II, and III. In Rattazzi ME, Scandalios JG, Whitt GS (eds): Isoenzymes: Current Topics In Biological and Medical Research, vol 7. New York, Alan R Liss, 1983, pp 7 9 - 1 0 0 . 82. Tashian RE: Evolution and regulation of the carbonic anhydrase enzymes. In Rattazzi ME, Scandalios JG, Whitt GS (eds): Isoenzymes: Current Topics in Biological and Medical Research, vol 2. New York, Alan R Liss, 1977, pp 2 1 - 6 2 . 83. Sanyal G, Swenson ER, Pessah NI, et al: The carbon dioxide hydration activity of skeletal muscle carbonic anhydrase: Inhibition by sulfonamides and anions. Mol Pharmaco122:211-220, 1982. 84. Raisz LG, Simmons HA, Thompson WJ, et al: Effects of a potent carbonic anhydrase inhibitor in bone resorption in organ culture. Endocrinology 122:1083-1086, 1988. 85. Sundquist KT, Leppilampi M, Jarvelin K, et al: Carbonic anhydrase isoenzymes in isolated rat peripheral monocytes, tissue macrophages, and osteoclasts. Bone 8:33-38, 1987.
MICHAEL P. WHYTE 86. Lewis SE, Erickson RP, Barnett LB, et al: N-ethyl-N-nicrosourea-induced null mutation at the mouse Car-2 locus: An animal model for human carbonic anhydrase II deficiency syndrome. Proc Natl Acad Sci USA 85:1962-1966, 1988. 87. Lee BL, Venta PJ, Tashian RE: DNA polymorphism in the 5prime flanking region of the human carbonic anhydrase II gene on chromosome 8. Hum Genet 69:337-339, 1985. 88. Whyte ME Hamm LL III, Sly WS: Transfusion of carbonic anhydrase-replete erythrocytes fails to correct the acidification defect in the syndrome of osteopetrosis, renal tubular acidosis, and cerebral calcification (carbonic anhydrase II deficiency). J Bone Miner Res 3:385-388, 1988. 89. Maroteaux P, Lamy M: La pycnodysostose. Presse Med 70:999, 1962. 90. Andrrn L, Dymling J-F, Hogeman KE, et al: Osteopetrosis, acroosteolytica: A syndrome of osteopetrosis, acro-osteolysis and open sutures of the skull. Acta Chir Scand 124:496-507, 1962. 91. Maroteaux P, Lamy M: The malady of Toulouse-Lautrec. JAMA 191:715-717, 1965. 92. Edelson JG, Obad S, Geiger R, et al: Pycnodysostosis: Orthopedic aspects, with a description of 14 new cases. Clin Orthop 280:263-276, 1992. 93. Elmore SM: Pycnodysostosis: A review. J Bone Joint Surg 49A: 153-162, 1967. 94. Meneses de Almeida L: Contribution a l'etude genitique de la pycnodysostose. Ann Genet 15:99-101, 1972. 95. Meneses de Almeida L: A genetic study of pycnodysostosis. In Papadatos CJ, Bartsocas CS (eds): Skeletal Dysplasias. New York, Alan R Liss, 1982, pp 195-198. 96. Sugiura Y, Yamada Y, Koh J: Pycnodysostosis in Japan: Report of six cases and a review of Japanese literature. Birth Defects X : 7 8 - 9 8 , 1974. 97. Santhanakrishnan BR, Panneerselvam S, Ramesh S, et al: Pycnodysostosis with visceral manifestation and rickets. Clin Pediatr (Phila) 25:416-418, 1986. 98. Maroteaux P, Faure C: Pycnodysostosis. Prog Pediatr Radiol 4: 403, 1973. 99. Roth VG: Pycnodysostosis presenting with bilateral subtrochanteric fracture: Case report. Clin Orthop Rel Res 117:247-253, 1976. 100. Wolpowitz A, Matisson A: A comparative study of pycnodysostosis, cleidocranial dysostosis, osteopetrosis and acroosteolysis. S Afr Med J 48:1011, 1974. 101. Soto TJ, Mautalen CA, Hojman D, et al: Pycnodysostosis, metabolic and histologic studies. Birth Defects V:109-115, 1969. 102. Bressot C, Meunier PJ, Bard J, et al: La pycnodysostose. Analyse histomorphometrique et dynamique de l'os dans une nouvelle observation. Rev Rhum 47:425-430, 1980. 103. Everts V, Aronson DC, Beertsen W: Phagocytosis of bone collagen by osteoclasts in two cases of pycnodysostosis. Calcif Tissue Int 37:25- 31, 1985. 104. Stanescu R, Stanescu V, Maroteaux P: Ultrastructural abnormalities in chondrocytes in pycnodysostosis. Nouveautes Medicales 437:247, 1975. 105. Gelb BD, Shi GP, Chapman HA, et al: Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science 273: 1236-1238, 1996. 106. Cabrejas ML, Fromm GA, Roca JF: Pycnodysostosis. Some aspects concerning kinetics of calcium metabolism and bone pathology. Am J Med Sci 271:215, 1976. 107. Beneton MNC, Harris S, Kanis JA: Paramyxovirus-like inclusions in two cases of pycnodysostosis. Bone 8:211-217, 1987. 108. Van Merkesteyn JPR, Bras J, Vermeeren JIJF, et al: Osteomyelitis of the jaws in pycnodysostosis. Int J Oral Maxillofac Surg 16:615-619, 1987.
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132. Royer P, Vermeil G, Apostolides P, et al: Maladie d'Engelmann resultat du traitement par la prednisone. Arch Franc Pediatr 24: 693-702, 1967. 133. Bourantas K, Tsiara S, Drosos AA: Successful treatment with corticosteroid in a patient with progressive diaphyseal dysplasia (letter). Clin Rheumatol 14:485-486, 1995. 134. Minford AMB, Hardy GJ, Forsythe WI, et al: Englemann's disease and the effect of corticosteroids: A case report. J Bone Joint Surg 63B:597-600, 1981. 135. Lindstrom JA: Diaphyseal dysplasia (Engelmann) treated with corticosteroids. Birth Defects X:504-507, 1974. 136. Naveh Y, Alon U, Kaftori JK, et al: Progressive diaphyseal dysplasia: Evaluation of corticosteroid therapy. Pediatrics 75:321323, 1985. 137. Allen DT, Saunders AM, Northway WH Jr, et al: Corticosteroids in the treatment of Engelmann' s disease: Progressive diaphyseal dysplasia. Pediatrics 46:523-531, 1970. 138. Guan DW, Moinuddin M, Pitcock J, et al: Intermittent diphosphonate in Engelmann's disease; a 5 year follow-up. J Bone Miner Res 1:117 (abstract), 1986. 139. Van Buchem FSP, Hadders HN, Ubbens R: An uncommon familial systemic disease of the skeleton. Hyperostosis corticalis generalisata familiaris. Acta Radiol 44:109-116, 1955. 140. Van Buchem FSP, Hadders HN, Hansen JE et al: Hyperostosis corticalis generalisata: Report of seven cases. Am J Med 33: 387-397, 1962. 141. Van Buchem FSP, Prick JJG, Jaspar HHJ: Hyperostosis Corticalis Generalisata Familaris (Van Buchem's Disease). Amsterdam, Excerpta Medica, 1976. 142. Perez-Vicente JR Jr, Rodriquez de Castro E, Lafuente J, et al: Autosomal dominant endosteal hyperostosis. Report of a Spanish family with neurological involvement. Clin Genet 31:161169, 1987. 143. Nakamura T, Yamada N, Nonaka R, et al: Autosomal dominant form of endosteal hyperostosis with unusual manifestations of sclerosis of the jaw bones. Skeletal Radiol 16:48-51, 1987. 144. Eastman JR, Bixler D: Generalized cortical hyperostosis (Van Buchem diease): Nosologic considerations. Radiology 125: 2 9 7 - 304, 1977. 145. Truswell AS: Osteopetrosis with syndactyly. A morphologic variant of Albers-Schonberg's disease. J Bone Joint Surg 40B: 208-218, 1958. 146. Hansen HG: Sklerosteose. In Opitz H, Schmid F (eds): Handbuch der Kinderheilkunde, vol 6. Berlin, Springer, 1967, pp 351-355. 147. Fryns JP, Van den Berghe H: Facial paralysis at the age of 2 months as the first clinical sign of van Buchem disease (endosteal hyperostosis). Eur J Pediatr 147:99-100, 1988. 148. Beighton P, Barnard A, Hamersma H, et al: The syndromic status of sclerosteosis and van Buchem diesase. Clin Genet 25: 175-181, 1984. 149. Beighton P, Durr L, Hamersma H: The clinical features of sclerosteosis: A review of the manifestations in twenty-five affected individuals. Ann Intern Med 84:393-397, 1976. 150. Barnard AH, Hamersma H, Kretzmar JH, et al: Sclerosteosis in old age. S Aft Med J 58:401-403, 1980. 151. Stein SA, Witkop C, Hill S, et al: Sclerosteosis: Neurogenetic and pathophysiologic analysis of an American kinship. Neurology 33:267-277, 1983. 152. Beighton P, Cremin BJ, Hamersma H: The radiology of sclerosteosis. Br J Radiol 49:934-939, 1976. 153. Hill SC, Stein SA, Dwyer A, et al: Cranial CT findings in sclerosteosis. AJNR 7:505- 511, 1986. 154. Epstein S, Hamersma H, Beighton P: Endocrine function in sclerosteosis. S Afr Med J 55:1105-1110, 1979.
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155. Ruckert EW, Caudill RJ, McCready PJ: Surgical treatment of van Buchem disease. J Oral Maxillofac Surg 43:801-805, 1985. 156. Berlin R, Hedensio B, Lilja B, et al: Osteopoikilosisma clinical and genetic study. Acta Med Scand 18:305-314, 1967. 157. Uitto J, Starcher BC, Santa-Cruz DJ, et al: Biochemical and ultrastructural demonstration of elastin accumulation in the skin of the Buschke-Ollendorff syndrome. J Invest Dermatol 76: 284-287, 1981. 158. Whyte MP, Murphy WA, Seigel BA: 99m Tc-pyrophosphate bone imaging in osteopoikilosis, osteopathia striata, and melorheostosis. Radiology 127:439-443, 1978. 159. Verbor J, Graham R: Buschke-Ollendorf syndrome--disseminated dermatofibrosis with osteopoikilosis. Clin Exp Dermatol 11:17-26, 1986. 160. Ghandur-Mnaymneh L, Broder LE, Mnaymneh WA: Lobular carcinoma of the breast metastatic to bone with unusual clinical, radiologic, and pathologic features mimicking osteopoikilosis. Cancer 53:1801 - 1803, 1984. 161. Bass HN, Weiner JR, Goldman A, et al: Osteopathia striata syndrome: Clinical, genetic, and radiologic considerations. Clin Pediatr 19:369-373, 1980. 162. Rabinow M, Unger F: Syndrome of osteopathia striata, macrocephaly, and cranial sclerosis. Am J Dis Child 138:821-823, 1984. 163. Horan FT, Beighton PH: Osteopathia striata with cranial sclerosis: An autosomal dominant entity. Clin Genet 13:201-206, 1978. 164. Jones MD, Mulcahy ND: Osteopathia striata, osteopetrosis, and impaired heating. A case report. Arch Otolaryngol 87:116-118, 1968. 165. Paling MR, Hyde I, Dennis NR: Osteopathia striata with sclerosis and thickening of the skull. Br J Radio154:344-348, 1981. 166. Happle R, Lenz W: Striation of bones in focal dermal hypoplasia: Manifestation of functional mosaicism? Br J Dermatol 96: 133-138, 1977. 167. Knockaert D, Dequeker J: Osteopathia striata and focal dermal hypoplasia. Skeletal Radiol 4:223-227, 1979. 168. Whyte MP, Murphy WA: Osteopathia stftata associated with familial dermopathy and white forelock: Evidence for postnatal development of osteopathia striata. Am J Med Genet 5:227234, 1980. 169. Kornreich L, Grunebaum M, Ziv N, et al: Osteopathia striata, cranial sclerosis with cleft palate and facial nerve palsy. Eur J Pediatr 147:101-103, 1988. 170. Winter RM, Crawford MD, Meire HB, et al: Osteopathia striata with cranial sclerosis: Highly variable expression within a family including a cleft palate in two neonatal cases. Clin Genet 18: 462-474, 1980. 171. Left A, Joanny J: Une affection non decrite des os. Hyperostose "en coul6e" sur toute la longueur d'un membre ou "melorheostose." Bul Mem Soc Hop Pads 46:1141-1145, 1922. 172. Murray RO, McCredie J: Melorheostosis and sclerotomes: A radiological correlation. Skeletal Radiol 4:57-71, 1979. 173. Morris JM, Samilson RL, Corley CL: Melorheostosis: Review of the literature and report of an interesting case with a nineteenyear follow-up. J Bone Joint Surg 45A:1191-1206, 1963. 174. Beauvais P, Faure C, Montagne JP, et al: Left's melorheostosis: Three pediatric cases and a review of the literature. Pediatr Radiol 6:152-159, 1977. 175. Soffa DJ, Sire DJ, Dodson JH: Melorheostosis with linear sclerodermatous skin changes. Radiology 114:577-578, 1975. 176. Miyachi Y, Horio T, Yamada A, et al: Linear melorheostotic scleroderma with hypertrichosis. Arch Dermatol 115:12331234, 1979.
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177. Applebaum RE, Caniano DA, Sun C-C, et al: Synchronous left subclavian and axillary artery aneurysms associated with melorheostosis. Surgery 99:249-153, 1986. 178. Fryns J-P: Melorheostosis and somatic mosaicism (letter). Am J Med Genet 58:199, 1995. 179. Muller SA, Henderson ED: Melorheostosis with linear scleroderma. Arch Dermatol 88:142-145, 1963. 180. Younge D, Drummond D, Herring J, et al: Melorheostosis in children: Clinical features and natural history. J Bone Joint Surg 61B:415-418, 1979. 181. Colavita N, Nicolais S, Orazi C, Falappa PG: Melorheostosis: Presentation of a case followed up for 24 years. Arch Orthop Trauma Surg 106:123-125, 1987. 182. Campbell CJ, Papademetriou T, Bonfiglio M: Melorheostosis: A report of the clinical, roentgenographic, and pathological findings in fourteen cases. J Bone Joint Surg 50A: 1281-1304, 1968. 183. Janousek J, Preston DF, Martin NL, et al: Bone scan in melorheostosis. J Nucl Med 17:1106-1108, 1976. 184. Wagers LT, Young AW Jr, Ryan SF: Linear melorheostotic scleroderma. Br J Dermatol 86:297-301, 1972. 185. Semble EL, Poehling GG, Prough DS, et al: Successful symptomatic treatment of melorheostosis with nifedipine. Clin Exp Rheumatol 4:277-280, 1986. 186. Walker GF: Mixed sclerosing bone dystrophies. J Bone Joint Surg 46B:546-552, 1964. 187. Abrahamson MN: Disseminated asymptomatic osteosclerosis with features resembling osteopoikilosis and osteopathia striata. J Bone Joint Surg 50A:991-996, 1968. 188. Ewald FC: Unilateral mixed sclerosing bone dystrophy associated with unilateral lympangiectasis and capillary haemangioma. J Bone Joint Surg 54A:878-880, 1972. 189. Kanis JA, Thompson JG: Mixed sclerosing dystrophy with regression of melorheostosis. Br J Radiol 48:400-402, 1975. 190. Whyte MP, Murphy WA, Fallon MD, et al: Mixed-sclerosingbone dystrophy: Report of a case and review of the literature. Skeletal Radiol 6:95-102, 1981. 191. Pacifici R, Murphy MA, Teitelbaum SL, et al: Mixed-sclerosingbone-dystrophy: 42-year follow-up of a case reported as osteopetrosis. Calcif Tissue Int 38:175-185, 1986. 192. Connor JM, Beighton P: Fibrodysplasia ossificans progressiva in South Africa: Case reports. S Afr Med J 61:404-406, 1982. 193. Rogers JG, Geho WB: Fibrodysplasia ossificans progressiva: A survey of forty-two cases. J Bone Joint Surg 61A:909-914, 1979. 194. Connor JM, Evans DAP: Fibrodysplasia ossificans progressiva: The clinical features and natural history of 34 patients. J Bone Joint Surg 64B:76-83, 1982. 195. Shah PB, Zasloff MA, Drummond D, et al: Spinal deformity in patients who have fibrodysplasia ossificans progressiva. J Bone Joint Surg 76A:1442-1450, 1994. 196. Fox S, Khoury A, Mootabar H, Greenwald EF: Myositis ossificans progressiva and pregnancy. Obstet Gynecol 69:453-455, 1987. 197. Cremin B, Connor JM, Beighton P: The radiological spectrum of fibrodysplasia ossificans progressiva. Clin Radiol 33:499508, 1982. 198. Thickman D, Bonakdar A, Clancy M, et al: Fibrodysplasia ossificans progressiva. Am J Roentgenol 139:935-941, 1982. 199. Kaplan FS, Tabas JA, Zasloff MA: Fibrodysplasia ossificans progressiva: A clue from the fly? Calcif Tissue Int 47:117-125, 1990. 200. Voynow JA, Charney EB: Fibrodysplasia ossificans progressiva presenting as osteomyelitis-like syndrome. Clin Pediatr 25: 373-375, 1986.
CHAPTER 24
Skeletal Disorders Characterized By Osteosclerosis or Hyperostosis
201. Fang MA, Reinig JW, Hill SC, et al: Technetium-99m MDP demonstration of heterotopic ossification in fibrodysplasia ossificans progressiva. Clin Nucl Med 11:8-9, 1986. 202. Sumiyoshi K, Tsuneyoshi M, Enjoji M: Myositis ossificans. A clinicopathologic study of 21 cases. Acta Pathol Jpn 35:11091122, 1985. 203. Maxwell WA, Spicer SS, Miller RL, et al: Histochemical and ultrastructural studies in fibrodysplasia ossificans progressiva (myositis ossificans progressiva). Am J Pathol 87:483-498, 1977. 204. Kaplan FS, Tabas JA, Gannon FH, et al: The histopathology of fibrodysplasia ossificans progressiva: An endochondral process. J Bone Joint Surg 75A:220-230, 1993. 205. Reinig JW, Hill SC, Fang M, et al: Fibrodysplasia ossificans progressiva: CT appearance. Radiology 159:153-157, 1986. 206. Cramer SF, Ruehl A, Mandel MA: Fibrodysplasia ossificans progressiva. Cancer 48:1016-1021, 1981. 207. Rogers JG, Chase GA: Paternal age effect in fibrodysplasia ossificans progressiva. J Med Genet 16:147-148, 1979. 208. Lutwak L: Myositis ossificans progressiva: Mineral, metabolic, and radioactive calcium studies of the effects of hormones. Am J Med 37:269-293, 1964. 209. Shafritz AB, Shore EM, Gannon FH, et al: Overexpression of an osteogenic morphogen in fibrodysplasia ossificans progressiva. N Engl J Med 335:551-561, 1996. 210. Smith R: Myositis ossificans progressiva: A review of current problems. Semin Arthritis Rheum 4:369-380, 1975. 211. Smith R, Russell RGG, Woods CG: Myositis ossificans progressiva: Clinical features of eight patients and their response to treatment. J Bone Joint Surg 58B:48-57, 1976. 212. Moore SE, Jump AA, Smiley JD: Effect of warfarin sodium therapy on excretion of 4-carboxy-L-glutamic acid in scleroderma, dermatomyositis, and myositis ossificans progressiva. Arthritis Rheum 29:344-351, 1986. 213. Frame B, Frost HM, Ormond RS, et al: Atypical axial osteomalacia involving the axial skeleton. Ann Intern Med 55:632639, 1961. 214. Whyte MP, Fallon MD, Murphy WA, et al: Axial osteomalacia: Clinical, laboratory and genetic investigation of an affected mother and son. Am J Med 71:1041 - 1049, 1981. 215. Arnstein AR, Frame B, Frost HM: Recent progress in rickets and osteomalacia. Ann Intern Med 67:1296-1330, 1967. 216. Christman D, Wenger JJ, Dosch JC, et al: L'osteomalacie axiale analyse comparee avec la fibrogenese imparfaite. J Radiol 62: 37-41, 1981. 217. Condon JR, Nassim JR: Axial osteomalacia. Postgrad Med J 47: 817-820, 1971. 218. Nelson AM, Riggs BL, Jowsey JO: Atypical axial osteomalacia: Report of four cases with two having features of ankylosing spondylitis. Arthritis Rheum 21:715-722, 1978. 219. Baker SL, Turnbull HM: Two cases of hitherto undescribed disease characterized by a gross defect in the collagen of the bone matrix. J Pathol Bacteriol 62:132-134, 1950. 220. Baker SL, Dent CE, Friedman M, et al: Fibrogenesis imperfecta ossium. J Bone Joint Surg 48B:804-825, 1966. 221. Thomas WC Jr, Moore T: Fibrogenesis imperfecta ossium. Trans Am Clin Climatol Assoc 80:54-62, 1968 222. Goldring FC: Fibrogenesis imperfecta. J Bone Joint Surg 50B: 619-622, 1968. 223. Golde D, Greipp P, Sanzenbacher L, et al: Hematologic abnormalities in fibrogenesis imperfecta ossium. J Bone Joint Surg 53A:365, 1971. 224. Frame B, Frost HM, Pak CYC, et al: Fibrogenesis imperfecta ossium, a collagen defect causing osteomalacia. N Engl J Med 285:769-772, 1971.
737
225. Swam CHJ, Shah K, Brewer DB, et al: Fibrogenesis imperfecta ossium. Q J Med 45:233-253, 1976. 226. Stamp TCB, Byers PD, Ali SY, et al: Fibrogenesis imperfecta ossium: Remission with melphalan. Lancet 1:582-583, 1985. 227. Byers PD, Stamp TCB, Stoker D J: Fibrogenesis imperfecta (case report 296). Skeletal Radiol 13:72-76, 1985. 228. Lang R, Vignery AM, Jensen PS: Fibrogenesis imperfecta ossium with early onset: Observations after 20 years of illness. Bone 7:237-246, 1986. 229. Stanley P, Baker SL, Byers PD: Unusual bone trabeculation in a patient with macroglobulinaemia simulating fibrogenesis imperfecta ossium. Br J Radiol 44:305-313, 1971. 230. Ralphs JR, Stamp TCB, Dopping-Hepenstal PJC, Ali-SY: U1trastructural features of the osteoid of patients with fibrogenesis imperfecta ossium. Bone 10:243-249, 1989. 231. Roholm K: Fluorine Intoxication. London, HK Lewis, 1937. 232. Jolly SS, Singh BM, Mathur OC: Endemic fluorosis in Punjab (India). Am J Med 47:553-563, 1969. 233. Vischer TL (ed): Fluoride in Medicine. Bern, Hans Huber, 1970. 234. Krishnamachari KAVR: Skeletal fluorosis in humans: A review of recent progress in the understanding of the disease. Prog Food Nutr Sci 10:279- 314, 1986. 235. Schnitzler CM, Solomon L: Histomorphometric analysis of a calcaneal stress fracture: A possible complication of fluoride therapy for osteoporosis. Bone 7:193-198, 1986. 236. Srikantia SG, Siddiqu AH: Metabolic studies in skeletal fluorosis. Clin Sci 28:477-485, 1965. 237. Kanis JA, Meunier PJ: Should we use sodium fluoride to treat osteoporosis? A review. Q J Med 53:145-164, 1984. 238. Friedreich N: Hyperostose des gesammten Skelettes. Virchows Arch [Pathol Anat] 43:83-87, 1868. 239. Rimoin DL: Pachydermoperiostosis (idiopathic clubbing and periostosis). Genetic and physiologic consideration. N Engl J Med 272:923-931, 1965. 240. Matucci-Cerinic M, Lott T, Jajic IVO, et al: The clinical spectrum of pachydermoperiostosis (primary hypertrophic osteoarthropathy). Medicine 79:208- 214, 1991. 241. Herman MA, Massaro D, Katz S: Pachyderm~176176 ical spectrum. Arch Intern Med 116:919- 923, 1965. 242. Harbison JB, Nice CM Jr: Familial pachydermoperiostosis presenting as an acromegaly-like syndrome. Am J Roentgenol Radium Ther Nucl Med 112:532-536, 1971. 243. Appelboom T, Busscher H, Famaey JP: Chondrocalcinosis as a possible cause of arthritis in pachydermoperiostosis (letter). Arthritis Rheum 21:174, 1978. 244. Guyer PB, Brunton FJ, Wren MWG: Pachydermoperiostosis with acro-osteolysis: A report of five cases. J Bone Joint Surg 60B:219-223, 1978. 245. Neiman HL, Gompels BM, Martel W: Pachydermoperiostosis with bone marrow failure and gross extramedullary hematopoiesis. Report of a case. Radiology 110:553-554, 1974. 246. Hedayati H, Barmada R, and Skosey JL: Acrolysis in pachydermoperiostosis (primary or idiopathic hypertropic osteoarthropathy). Arch Intern Med 140:1087-1088, 1980. 247. Ali A, Tetalman M, Fordham EW: Distribution of hypertrophic pulmonary osteoarthropathy. Am J Roentgenol 134:771-780, 1980. 248. DeVries N, Datz FL, Manaster BJ: Case report 399: Pachydermoperiostosis (primary hypertrophic osteoarthropathy). Skeletal Radiol 15:658-662, 1986. 249. Vogl A, Goldfischer S: Pachydermoperiostosis: Primary or idiopathic hypertrophic osteoarthropathy. Am J Med 33:166-187, 1962. 250. Lauter SA, Vasey FB, Htittner I, et al: Pachydermoperiostosis: Studies on the synovium. J Rheumatol 5:85-95, 1978.
7
3
8
M
I
C
251. Fam AG, Chin-Sang H, Ramsay CA: Pachydermoperiostosis: Scintigraphic, thermographic, plethysmographic, and capillaroscopic observations. Ann Rheum Dis 42:98-102, 1983. 252. Kerber RE, Vogl A: Pachydermoperiostosis: Peripheral circulatory studies. Arch Intern Med 132:245-248, 1973. 253. Cooper RG, Freemont AJ, Riley M, et al: Bone abnormalities and severe arthritis in pachydermoperiostosis. Ann Rheum Dis 51:416-419, 1992. 254. Whyte MP, Teitelbaum SL, Reinus WR: Doubling skeletal mass during adult life: The syndrome of diffuse osteosclerosis after intravenous drug abuse. J Bone Miner Res 11:554-558, 1996. 255. Whyte MP, Reasner CA: Hepatitis C-associated osteosclerosis after blood transfusion. Am J Med 102:219-220, 1997. 256. Abdelwahab IF, Norman A: Osteosclerotic sarcoidosis. AJR Am J Roentgenol 150:161 - 162, 1988. 257. Shim MS, Mowry RW, Bodie FL: Osteosclerosis (punctate form) in multiple myeloma. South Med J 72:226-228, 1979. 258. Edelman RR, Kaufman H, Kolodny G: Case report 350. Skeletal Radiol 15:160-163, 1986.
H
A
E
L
P. WHYTE
259. Hamdy RC: Paget's Disease of Bone: Assessment and Management. East Sussex, UK, Praeger Publishers, 1981. 260. Van Holsbeeck M, Roex L, Favril A, et al: Osteosclerosis in primary hyperparathyroidism. Fortschr R/3ntgenstr 147:690691, 1987. 261. Frame B, Jackson CE, Reynolds WA, Umphrey JE: Hypercalcemia and skeletal effects in chronic hypervitaminosis A. Ann Intern Med 80:44-48, 1974. 262. Dewind LT: Hypervitaminosis D with osteosclerosis. Arch Dis Child 36:373-380, 1961. 263. Murphy WA, Seligman PA, Tillack T, et al: Osteosclerosis, osteomalacia, and bone marrow aplasia: A combined late complication of thorotrast administration. Skeletal Radiol 3:234-238, 1979. 264. Punsar S, Somer T: The milk-alkali syndrome. A report of three illustrative cases and a review of the literature. Acta Med Scand 173:435-449, 1963. 265. Jacobsson S, Hollender L, Lindberg S, Lansson A: Chronic sclerosing osteomyelitis of the mandible. Scintigraphic and radiographic findings. Oral Surg 45:167-174, 1978.
~ H A P T E R 2.
Kidney Stones: Pathogenesis, Diagnosis and Therapy CHARLES
I. II. III. IV. V.
Y. C . P A K
University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235
Introduction Hypercalciuria Hyperuricosuria Hyperoxaluria Hypocitratufia
VI. VII. VIII. IX.
patients with appropriate medical treatments. 5 Third, most renal stones are spontaneously passed and may not require intervention for their removal. However, spontaneous stone passage is often associated with severe colic; this morbidity could be averted by medical prevention of new stone formation. Fourth, medical treatment could potentially correct extrarenal manifestations of the stone disease, 5 whereas surgical approach concentrates on stone removal alone. This chapter reviews advances in the medical area. Each of the principal causes of stone disease is discussed separately, with consideration of pathophysiology, diagnostic criteria, and medical prevention.
I. I N T R O D U C T I O N A. Current Status of the Field The recent introduction of extracorporeal shock wave lithotripsy I and nephrostolithotomy 2 have revolutionized the treatment of nephrolithiasis. Most stones can now be removed with greater ease and less morbidity. Because of these improved methods of stone removal, there has been a tendency to disparage the need for medical diagnosis and treatment. 3 However, it is clear that the medical approach cannot be ignored if ultimate control of nephrolithiasis is to be achieved. First, there is no evidence that removal of stones, no matter how easily achieved, would prevent recurrence of stones. Following lithotripsy, urinary biochemical abnormalities persist and stone formation recurs. 4 Second, it is now possible to identify the cause of stone formation in the vast majority of patients and to inhibit new stone formation in most METABOLIC BONE DISEASE
Gouty Diathesis Cystinuria Infection with Urea-Splitting Organisms Conservative Management References
B. Epidemiology and Chemical Composition Nephrolithiasis is common, affecting 1% to 5% of the population in the industrialized countries, with an annual incidence of 0.1% to 0.3%. Stones originating in the
739
Copyright 9 1998 by Academic Press. All rights of reproduction in any form reserved.
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CHARLES Y. C. PAK
bladder are rare in industrialized countries except in association with a foreign body (e.g., indwelling catheter), although they were common in antiquity. In industrialized countries, most stones are calcareous stones, composed mainly of calcium oxalate occurring alone (35% of all stones) or in combination with hydroxyapatite (35%). The remaining calcareous stones (5%) are represented by those composed principally of hydroxyapatite or brushite (CaHPO4-2H20). Noncalcareous stones, comprising up to 25% of all stones, are composed of uric acid, magnesium ammonium phosphate, cystine, and rarely xanthine, sodium urate, 2,8dihydroxyadenine, or triamterene. Stones composed principally of calcium oxalate are generally more common in men than in women, with a particular susceptibility for middle-aged, white men. Struvite stones and predominantly calcium phosphate stones are more common in women. Chemical composition of the stone may sometimes provide the diagnosis (e.g., cystine stones for cystinuria, struvite stones for urinary tract infection with urea-splitting organisms, and uric acid stones for gouty diathesis). The finding of calcium phosphate as the predominant phase suggests the diagnosis of distal renal tubular acidosis or primary hyperparathyroidism. However, the identification of the most common, calcium oxalate stones, has a limited diagnostic value, since they could result from a wide variety of metabolic and environmental disturbances.
C. Diagnostic Separation Based on "Physiological" Derangements It is known that patients with renal calculi suffer from a wide variety of physiological disturbances, 6 which are believed to be pathogenetically important in stone formation. It is presumed that these physiological disturbances could result from environmental influences as well as from metabolic factors (of hormonal or genetic origin). These derangements have served as the basis of a refined classification of nephrolithiasis 7 and a more rational approach to medical treatment. 8 According to this classification scheme (Table 25-1), calcareous renal calculi are due to hypercalciuria, hyperuricosuria, hyperoxaluria, inhibitor deficiency (hypocitraturia), and gouty diathesis. Noncalcareous calculi result from gouty diathesis, cystinuria, and urinary tract infection (with urea-splitting organisms). Some of these derangements are heterogeneous in origin. Thus, hypercalciuria may be due to an excessive intestinal calcium absorption, renal calcium leak, or excessive bone resorption. In the category of inhibitor deficiency, hypocitraturia will be described in detail, because of increasing rec-
TABLE 2 5 - 1
Classification of Nephrolithiasis
Calcareous Renal Calculi
Hypercalciuria Absorptive Renal Resorptive Hyperuricosuria Primary urate overproduction Dietary purine overindulgence Hyperoxaluria Primary Secondary Inhibitor deficiency Hypocitraturia Other Gouty diathesis Noncalcareous Renal Calculi
Gouty diathesis Uric acid stones Cystinuria Cystine stones Infection with urea-splitting organisms Struvite stones
ognition of its importance in stone formation. Other simple (small molecular weight) and complex (macromolecular) inhibitors of the crystallization of calcium salts have been identified in urine, including pyrophosphate, 9 glycopeptides, 1~ nephrocalcin, 11 glycosaminoglycans, 12 ribonucleic acids, and uropontins. ~3 Each cause of nephrolithiasis will now be considered in toto from the perspective of pathophysiology, diagnosis, and treatment.
II. HYPERCALCIURIA A. Causal Role of Hypercalciuria in Calcium Stone Formation Hypercalciuria could cause or contribute to calcium stone formation by the following mechanisms. First, it increases the saturation of urine with respect to stoneforming calcium salts. There is a direct correlation between urinary saturation of calcium oxalate or brushite and urinary calcium concentration. 14 Although a rise in urinary calcium may reduce the ionic oxalate through increased complexation of oxalate, this effect is generally less prominent than the increase in ionic calcium. Second, hypercalciuria induced by a high calcium intake raises the urinary saturation of calcium salts. 15 Despite suggestions to the contrary, the opposing effect of the reduction in urinary oxalate from binding by calcium in the intestinal tract is modest. Third, hypercalciuria re-
CHAPTER25 Kidney Stones: Pathogenesis, Diagnosis, and Therapy duces the urinary inhibitor activity against the crystallization of calcium salts through binding or inactivation of negatively charged inhibitors. 16 Fourth, persistent hypercalciuria is one of the most important determinants of continued stone formation during therapy. 17 Finally, correction of hypercalciuria by administration of thiazide or sodium cellulose phosphate restores the normal urinary physicochemical environment TM and retards the formation of new stones. 19 The etiological role of high urinary calcium concentration should not be confused with that of a high calcium intake. In stone-forming patients with normal intestinal calcium transport, a high-calcium diet may produce only a modest and transient hypercalciuria, because of intestinal adaptation reflected by a fall in calcium absorption from suppressed synthesis of parathyroid hormone (PTH) and calcitriol. 2~ In patients with absorptive hypercalciuria, however, a sustained marked hypercalciuria may ensue from the loss of intestinal adaptation. 2~
B. P a t h o p h y s i o l o g y o f H y p e r c a l c i u r i a The association of hypercalciuria with calcium nephrolithiasis has long been recognized. The term "idiopathic hypercalciuria" has been used to denote this entity. 22 The recent progress in pathophysiological elucidation mandates that this term be discarded. Our own view is to consider hypercalciuria of nephrolithiasis to comprise several entities of heterogeneous origin. This approach permits incorporation of prevailing major theories of hypercalciuria.
1. ABSORPTIVE HYPERCALCIURIA (FIG. 25-1) The primary abnormality in absorptive hypercalciuria is intestinal hyperabsorption of calcium. The consequent increase in the circulating concentration of calcium augments the renal filtered load and suppresses parathyroid function. Hypercalciuria ensues from the increased filtered load and the reduced tubular reabsorption of calcium associated with suppression of PTH secretion. The excessive renal loss of calcium compensates for the high calcium absorption from the intestinal tract and helps to maintain serum calcium concentration in the normal range. Absorptive hypercalciuria is heterogeneous and may be broadly categorized into vitamin D-independent and vitamin D-dependent subvariants. The former may be a primary jejunal abnormality, 23'24 because the high calcium absorption has been observed only in the jejunum and the absorption of magnesium and phosphate is normal. Moreover, the restoration of normal serum 1,25dihydroxyvitamin D [1,25(OH)2D] by ketoconazole does
741 not correct the exaggerated intestinal absorption or renal excretion of calcium. 25 In vitamin D-dependent absorptive hypercalciuria, there may be calcitriol overproduction or hypersensitivity. Ketoconazole therapy completely or partially corrects the hyperabsorption of calcium and hypercalciuria. 25 Calcitriol overproduction is supported by the finding of high serum 1,25(OH)2D concentration 26'27 and of accelerated calcitriol synthesis in vivo in some patients. 28'29 This subvariant may include hypophosphatemic absorptive hypercalciuria. Hypophosphatemia ensuing from renal phosphate leak is thought to cause intestinal hyperabsorption of calcium by stimulating the renal synthesis of 1,25(OH)2D. 3~ However, hypophosphatemia is uncommonly found and the full spectrum of this entity is rarely encountered in stone-forming patients. 31 Hypersensitivity to vitamin D is supported by the state of intestinal hyperabsorption of calcium in patients with normal circulating concentration of calcitriol, where ketoconazole challenge restores normal calcium absorption. 25 It resembles the state of up-regulation of the vitamin D receptor, produced by long-term treatment with 1,25(OH)2D in normal subjects. 32 Fasting urinary calcium may be high in some patients with absorptive hypercalciuria. In most of them, it is probably due to the incomplete renal clearance of excessively absorbed calcium. 33 Suppressed parathyroid function from increased intestinal absorption of calcium may contribute to fasting hypercalciuria by impairing renal tubular reabsorption of calcium. In some of them, fasting hypercalciuria may reflect the resorptive action of bone of vitamin D excess or hypersensitivity. 34 Some patients with absorptive hypercalciuria may show evidence of excessive bone loss. Spinal bone density may be depressed, although radial shaft density is generally normal. 35 Calcium balance may be negative. 36 The exact cause for bone loss is not known. It may be related to vitamin D excess or hypersensitivity. However, the available evidence does not indicate that the abnormality in absorptive hypercalciuria is attributable to mutations of the vitamin D receptor or is linked to a common vitamin D receptor genotype. 37 2. RENAL HYPERCALCIURIA The primary abnormality in renal hypercalciuria is believed to be the impairment of the renal tubular reabsorption of calcium (Fig. 25-1). The consequent reduction in the circulating concentration of calcium stimulates parathyroid function. There may be an excessive mobilization of calcium from bone and an enhanced intestinal absorption of calcium because of the PTH excess and the ensuing stimulation of the renal synthesis of 1,25(OH)2D. These effects restore serum calcium to-
742
CHARLES Y. C. PAK
Absorptive Hypercalciuria
I'~ Ca Absorption ] "~
I "~Serum Ca I ~, I
I* H.I
I u ,~
]
Renal Hypercalciuria
I§
I --~ I +serumCa I # !
~ . ,
,
I§ Ca Absorption ]
Resorptive Hypercalciuria
I~'PTHI ~-~ I~'BoneResorptionl ~-~ I-~SerumCal ~ """-~
1~"1,25-(OH)2D I ~
I~ UrinaryCa !
[+ Ca Absorption I
FIGURE 25--l Schemesfor absorptive hypercalciuria type I and type II, renal hypercalciuria, and resorptive hypercalciuria. Dashed line indicates compensatory inhibition. [From Pak CYC: Kidney Stones. In Foster SW, Wilson J (eds): Williams Textbook of Endocrinology. Philadelphia, WB Saunders Co, 1985.]
ward normal. Unlike primary hyperparathyroidism, serum calcium is normal, and the state of hyperparathyroidism is secondary. Some patients with hypercalciuric nephrolithiasis, albeit a minority in most series, have been shown to have biochemical presentation supporting operation of this scheme. The occurrence of high serum PTH or urinary cyclic adenosine monophosphate (cAMP), and elevated serum 1,25(OH)zD and intestinal calcium absorption, have been shown in such patients in the setting of normal serum calcium and fasting hypercalciuria (indicative of renal calcium l e a k ) . 26 The correction of renal calcium leak by thiazide restores normal serum 1,25(OH)zD and fractional calcium absorption commensurate with the correction of hyperparathyroidism. 38 These findings support the contention that 1,25(OH)zD synthesis is enhanced from secondary parathyroid stimulation and that intestinal calcium absorption is high owing to 1,25(OH)zD excess. The occurrence of a primary renal calcium leak is supported by three lines of evidence. First, fasting hypercalciuria is associated with parathyroid stimulation and is poorly corrected by the inhibition of intestinal calcium absorption (and removing the effect of absorbed calcium) with sodium cellulose phosphate. 33 Second, there is a unique natriuretic response to thiazide. When thiazide is given to block reabsorption of calcium and
sodium in the distal renal tubule, impaired proximal tubular function would be manifested as an exaggerated renal excretion of these cations. ! n our study, the exaggerated natriuretic and calciuric response to hydrochlorothiazide was encountered only in patients with documented renal hypercalciuria with secondary hyperparathyroidism. 39 Third, an exaggerated calciuric response to 100 g of glucose was encountered in patients with renal hypercalciuria with secondary hyperparathyroidism, not in those with absorptive hypercalciuria. 4~ Ingestion of readily metabolizable carbohydrate (without calcium) normally augments renal calcium excretion, believed to be due to an alteration in renal proximal tubular function. 41 It has been suggested that renal calcium leak is secondary to an excessive dietary intake of sodium. 42 However, in one preliminary study, institution of a low sodium intake (9 mEq/day) did not eliminate fasting hypercalciuria in patients with renal hypercalciuria. That renal calcium leak (and secondary hyperparathyroidism) had been longstanding is shown by changes disclosed in bone density. Although clinical bone disease is rare, bone density as measured by photon absorptiometry in the distal third of the radius is reduced in the group with renal hypercalciuria (as compared with age- and 43 sex-matched controls). These results indicate that secondary hyperparathyroidism exerts deleterious effects on
CHAPTER 25
743
Kidney Stones: Pathogenesis, Diagnosis, and Therapy
urinary calcium in normocalciuric patients with nephrolithiasis but not in normal subjects.
the skeleton. The lack of a more serious involvement is probably due to the compensatory intestinal hyperabsorption of calcium that results from the PTH-induced renal synthesis of 1,25(OH)zD. This compensation is often inadequate, since urinary calcium usually exceeds absorbed calcium indicative of negative calcium balance.
C. D i a g n o s t i c Criteria (Table 2 5 - 2 )
3. RESORPTIVE HYPERCALCIURIA Resorptive hypercalciuria (Fig. 25-1) is characterized by primary hyperparathyroidism. The initial event is excessive resorption of bone resulting from hypersecretion of PTH. Intestinal absorption of calcium is frequently elevated because of PTH-dependent stimulation of renal synthesis of 1,25(OH)zD. 27 These effects increase the circulating concentration and the renal filtered load of calcium. The occurrence of hypercalciuria in primary hyperparathyroidism seems paradoxical, since the primary renal effect of PTH is to stimulate tubular reabsorption of calcium. However, hypercalciuria is often encountered in primary hyperparathyroidism because PTHdependent augmentation of renal tubular reabsorption of calcium is "overcome" by an increase in the renal filtered load and by a suppressive effect of hypercalcemia on calcium reabsorption. There are other causes of resorptive hypercalciuria associated with nephrolithiasis. Excessive bone resorption from oncogenic hypercalcemia, thyrotoxicosis, and sarcoidosis occasionally result in stone formation. Enhanced renal excretion of prostaglandin E2 has been reported in patients with hypercalciuric nephrolithiasis. 44 Treatment with inhibitors of prostaglandin synthesis corrected the hypercalciuria in such patients and reduced
TABLE 2 5 - 2
Absorptive hypercalciuria type 17 is characterized by normal serum calcium and phosphorus, slightly high or normal fasting urinary calcium [<0.03 mM/liter (< 0.11 mg/dl) glomerular filtrate], increased urinary calcium after an oral calcium load [> 5 mM/mg (> 0.2 mg/mg) creatinine], normal or suppressed parathyroid function (normal serum immunoreactive PTH) and high urinary calcium level for a restricted diet [10 mM (400 mg) calcium and 100 mM sodium/day] of more than 5 mM/day (200 mg/day). These values reflect increased intestinal calcium absorption, resulting parathyroid suppression, and hypercalciuria. Spinal bone density may be low in some patients, 35 although radial shaft bone density is generally normal. 43 Absorptive hypercalciuria type II 7 is characterized by the same biochemical features as type I except for normal urine calcium [< 5 mM/day (< 200 mg/day)] for a restricted diet of 10 mM (400 mg) calcium and 100 mM sodium/day. If these patients are placed on a diet of 25 mM/day (1000 mg/day) calcium and 100 mM/day sodium, urinary calcium exceeds 0.1 mM/kg body weight/ day (4 mg/kg/day) or 6 mM/day (250 mg/day). Renal hypercalciuria 7 has the following features: normal serum calcium, high fasting urinary calcium of greater than 0.03 mM/liter glomerular filtrate (> 0.11 mg/dl glomerular filtrate), and enhanced parathyroid ac-
Diagnostic Criteria a
Serum
AH-I AH-II RH HUCU EH Hypocit RTA Gouty diathesis Struvite stones
Urinary
Ca
P
PTH
1,25
Ca Fasting
Ca Load
Ca Restricted
UA
Ox
Cit
pH
oL
N N N N N/$ N N N N
N N N N N/$ N N N N
N N "]" N N/'I" N N/I" N N
N/l" N/q" 1" N N N N N N
N/q" N 1" N $ N 1" N N
1" 1" 1" N $ N N N N/q"
1" N 1" N $ N N/q" N N/l"
N N N 1" $ N N N/$ N
N N N N 1" N N N N
N N N N $ $ $ N $
N N N N N N N/q" $ 1"
1" "I'/N 1" N $ N $ N N
aFasting samples represent 2-hour collections obtained in morning following an overnight fast. Ca load samples were obtained over a 4-hour period subsequent to oral ingestion of 1 g Ca. Fractional Ca absorption (o0 was obtained from fecal recovery of radioactivity following oral administration of radiocalcium with 100 mg Ca. PTH, immunoreactive parathyroid hormone; 1", high; $, low; N, normal; 1,25, 1,25(OH)2D; UA, uric acid; Ox, oxalate; Cit, citrate; AH-I, absorptive hypercalciuria type I; AH-II, absorptive hypercalciuria type II; RH, renal hypercalciuria; HUCU, hyperuricosuric calcium nephrolithiasis; EH, enteric hyperoxaluria; Hypocit, idiopathic hypocitraturic calcium nephrolithiasis; RTA, incomplete renal tubular acidosis.
744
CHARLES Y. C. PAK
tivity (high serum immunoreactive PTH). These results indicate a renal leak of calcium with compensatory parathyroid stimulation. Evidence of parathyroid stimulation (high serum PTH) is critical for the diagnosis of renal hypercalciuria. Radial shaft bone density may be low in patients with renal hypercalciuria. 43 Primary hyperparathyroidism is characterized by hypercalcemia, hypophosphatemia, hypercalciuria, and increased or inappropriately high serum PTH. Bone density in the radial diaphysis is often low. 43 Hypercalcemic symptoms, peptic ulcer, or bone disease (osteitis, pathological fractures, osteoporosis) may be present. Reduced density of femoral neck and spine is encountered less commonly. 45
D. Treatment of Hypercalciuric Calcium Nephrolithiasis (Table 25- 3) 1. ABSORPTIVE HYPERCALCIURIA TYPE I No treatment program is capable of correcting the basic abnormality of absorptive hypercalciuria, although several drugs restore normal calcium excretion. Sodium cellulose phosphate administration is not an optimal therapy. 46 When given orally, this nonabsorbable ionexchange resin binds calcium and inhibits calcium absorption. However, decreased calcium absorption is due to limitation of the amount of intraluminal calcium available for absorption and not to correction of the disturbance in calcium transport. There are two potential complications of sodium cellulose phosphate therapy. 47 First, the agent may cause magnesium depletion by binding dietary magnesium. Second, sodium cellulose phosphate may produce secondary hyperoxaluria 48 by binding divalent cations in the intestinal tract, reducing formation of divalent cationoxalate complexes, and making more oxalate available for absorption. These complications may be prevented by oral magnesium supplementation (magnesium citrate, 10 mEq given twice a day separately from sodium cellulose phosphate) and moderate dietary restriction of oxalate. Under such circumstances, sodium cellulose phosphate (10 to 15 g/day, given with meals) lowers urinary calcium level, reduces urinary saturation of calcium salts, and retards new stone formation without significantly altering urinary oxalate or magnesium. 8 This drug is contraindicated in other forms of hypercalciuria because it may further stimulate parathyroid function and worsen negative calcium balance. Because of this potential complication, sodium cellulose phosphate should be given only in severe cases of absorptive hypercalciuria type I and in thiazide-resistant hypercalciuria, in whom there is normal bone density.
Thiazide does not decrease intestinal calcium absorption, 26 but it is widely used to treat this disorder because of its hypocalciuric action. However, thiazide may have limited long-term effectiveness in absorptive hypercalciuria type 1.49 It is usually effective in reducing the urinary calcium level during the first 2 years of treatment, but thereafter urinary calcium generally returns to the pretreatment range (Fig. 25-2). Intestinal calcium absorption persistently remains elevated throughout thiazide treatment. Thiazide may cause accretion of calcium in bone during the early years of therapy; eventually a low-turnover state of bone interferes with continued calcium accretion in the skeleton. 5~The "rejected" calcium would then be excreted in urine. In contrast, calcium retention does not occur in renal hypercalciuria because thiazide causes a decrease in intestinal calcium absorption commensurate following the reduction in urinary calcium. 38'49
The following guidelines for the use of these two agents are recommended until more selective therapies are found. Sodium cellulose phosphate is appropriate for patients with severe absorptive hypercalciuria type I [urinary calcium > 8.75 mM/day (> 350 mg/day)] and for patients resistant to or intolerant of thiazide therapy, without osteopenia. In patients at risk for bone disease (growing children, postmenopausal women, or elderly men), thiazide is the first choice. When thiazide becomes ineffective in lowering the urinary calcium, this treatment may be temporarily replaced by sodium cellulose phosphate or orthophosphate treatment (for --~6 months). Restoration of hypocalciuric response to thiazide may then ensue, permitting resumption of thiazide therapy. Potassium citrate (e.g., 15 to 20 mEq twice a day) should be given along with thiazide (e.g., trichlormethiazide 4 mg/day) to prevent hypokalemia and to augment citrate excretion. 51 Recent studies suggest that slow-release neutral potassium phosphate may be a promising treatment for absorptive hypercalciuria. 52 It reduces intestinal absorption of calcium by direct binding of calcium in the intestinal tract and by impairing renal calcitriol synthesis. A sustained reduction in urinary calcium is accompanied by a rise in urinary inhibitors (citrate and pyrophosphate). 2. ABSORPTIVE HYPERCALCIURIA TYPE I I
In absorptive hypercalciuria type II, a low-calcium diet [10 to 15 mM/day (400 to 600 mg/day)] and a high fluid intake (sufficient to maintain urine output greater than 2 liters/day) are appropriate 8 because normocalciuria can be restored by dietary calcium restriction alone and because increased urine volume reduces urinary saturation of calcium oxalate, brushite, and monosodium urate and inhibits spontaneous nucleation of calcium oxalate.
TABLE25-3
Optimal Treatment Programs for Nephrolithiasis
Treatment
Physiological Action
-
Indication
Sodium cellulose phosphate
.1intestinal calcium (Ca) absorption & urinary Ca
Thiazide
= intestinal Ca absorption urinary Ca (transient) urinary citrate
Absorptive hypercalciuria type I1
Low-Ca diet
Renal hypercalciuria
Thiazide
Hyperuricosuric Ca nephrolithiasis
Allopurinol Potassium citrate
& intestinal Ca absorption .1urinary Ca & urinary Ca (sustained) & intestinal Ca absorption & urinary uric acid
Absorptive hypercalciuria type I
Enteric hyperoxaluria
1' urinary
& oxalate intake Potassium citrate
.1urinary oxalate I' urinary citrate
Magnesium gluconate Calcium citrate
1' urinary citrate
Hypocitraturic Ca nephrolithiasis
Potassium citrate
Gouty diathesis
Potassium citrate
1'urinary pH
? urinary Mg
I' urinary I' urinary
Cystinuria
Penicillamine or tiopronin"
Infection stones
Acetohydroxamic acid
-1, decrease; 1',increase; =, no change.
pH
citrate
1'urinary pH k/=urinary Ca 1' urinary pH 1 undissociated uric acid I' urinary
"a-Mercaptopropionylglycine.
citrate
urinary saturation of Ca oxalate
& Ca phosphate saturation -1 urinary saturation of Ca salts
& urinary saturation of Ca oxalate and Ca phosphate urinary saturation of Ca salts
.1urate-induced crystallization of Ca salts & urinary saturation of Ca oxalate .1urate-induced crystallization of Ca salts .1urinary saturation of Ca oxalate & urinary saturation of Ca oxalate 1' inhibitor activity & urinary saturation of Ca oxalate
1' inhibitor activity
4 urinary saturation of Ca oxalate 1' inhibitor activity & urinary saturation of uric acid & Ca oxalate crystallization
citrate
Mixed disulfide with cysteine & urinary cystine urease activity
& NH: 1pH
-
Physicochernical Action
.1urinary saturation of cystine & urinary saturation of struvite
746
CH,~RLES Y.
C. PAK
ABSORPTIVE HYPERCALCIURIA 450
400
350 t'~ CB
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Years on Thiazide Therapy FIGURE 25--2
Effect of long-term therapy on urinary calcium and fractional (intestinal) calcium absorption in absorptive and renal hypercalciurias. (From Preminger GM, Pak CYC: Eventual attenuation of hypocalciuric response to hydrochlorothiazide in absorptive hypercalciuria. J Urol 137:1104-1109, 1987.)
3. RENAL HYPERCALCIURIA Thiazide is the treatment of choice for renal hypercalciuria. 26'38 This agent corrects the renal leak of calcium directly by augmenting calcium absorption in the distal tubule and by causing extracellular volume depletion, which stimulates proximal tubular reabsorption of calcium. The ensuing correction of secondary hyperparathyroidism restores normal serum 1,25(OH)2D and intestinal calcium absorption. Physicochemically, the urine becomes less saturated with calcium oxalate and brushite, largely because of the reduced calcium excretion. 53
Moreover, urinary inhibitor activity, as reflected by an increase in the limit of metastability, occurs by an unknown mechanism. These effects are shared by hydrochlorothiazide (50 mg twice a day), chlorthalidone (25 mg/day), and trichlormethiazide (4 mg/day). Potassium supplementation (15 to 20 mEq twice a day) may be required to prevent hypokalemia and attendant hypocitraturia. Concurrent use of triamterene, a potassiumsparing agent, should be undertaken with caution because of the possibility of triamterene stone formation. 54 Amiloride may be used with thiazide, because it may also exert a hypocalciuric action, exaggerate the hypo-
CHAPTER 25 Kidney Stones: Pathogenesis, Diagnosis, and Therapy calciuric action of thiazide, and prevent hypokalemia. 55 However, amiloride does not augment citrate excretion. Thus in patients with hypercalciuric nephrolithiasis and hypocitraturia, in whom the use of potassium citrate is contemplated, it is probably wise to use thiazide alone without a potassium-sparing diuretic. 4. PRIMARY HYPERPARATHYROIDISM
There is no established medical treatment for the nephrolithiasis of primary hyperparathyroidism. Although orthophosphates have been recommended for disease of mild to moderate severity, 56 their safety or efficacy has not yet been proven. They should be used only when parathyroid surgery cannot be undertaken. Thiazide is contraindicated in primary hyperparathyroidism because of potential aggravation of hypercalcemia. Estrogen is a reasonable alternative in postmenopausal women with primary hyperparathyroidism in whom surgery is refused or contraindicated. 57 This treatment (e.g., conjugated estrogen 0.625 mg/day, 25 days of each month) has been shown to reduce serum calcium concentration (by inhibiting PTH-induced bone resorption) and thereby reduce urinary calcium. Parathyroidectomy is the optimal treatment for the nephrolithiasis of primary hyperparathyroidism. Following removal of abnormal parathyroid tissue, urinary calcium is restored to normal commensurate with a decline in serum concentration of calcium and intestinal calcium absorption. 58 The urinary environment becomes less saturated with respect to calcium oxalate and brushite, and its limit of metastability (formation product ratio, a measure of inhibitor activity) for these calcium salts increases. 59 There is typically a reduced rate of new stone formation, unless urinary tract infection is present.
III. HYPERURICOSURIA The association of hyperuricosuria with calcium nephrolithiasis is well known, and has received the appellation of hyperuricosuric calcium nephrolithiasis.
A. C a u s a l R o l e o f H y p e r u r i c o s u r i a in C a l c i u m Stone Formation Recurrent calcium nephrolithiasis (stones of calcium oxalate and/or calcium phosphate) can occur in subjects with hyperuricosuria with no other discernible cause for nephrolithiasis, provided that the urinary pH is greater than the dissociation constant (pKa) of 5.47 for the first proton of uric acid. 6~ The association of hyperuricosuria with calcium nephrolithiasis has led to the suggestion
747 that hyperuricosuria is pathogenetically important in calcium stone formation. 61 The following scheme has been proposed: the urine may be supersaturated with respect to monosodium urate because it has a high content of uric acid and a pH (> 5.5) in which monosodium urate is stable. 62 Either a colloidal or crystalline monosodium urate can form in such a supersaturated environment 63 and initiate the formation of calcium stones by (1) direct induction of heterogeneous nucleation of calcium oxalate or (2) adsorption of glycosaminoglycans (which are inhibitors of crystal aggregation or spontaneous nucleation of calcium oxalate). 64 The scheme is supported by the demonstration of supersaturation of urine with monosodium urate, 62 by the ability of monosodium urate to induce heterogeneous nucleation of calcium oxalate, 64 and by the capacity of monosodium urate to attenuate the inhibitory activity of heparin (model mucopolysaccharide) 64 or naturally occurring urinary macromolecules. 65 Moreover, the induction of hyperuricosuria by oral purine loading facilitates spontaneous precipitation of calcium oxalate in urine, commensurate with a rise in urinary saturation of monosodium urate. 63 Unfortunately, the presence of crystalline monosodium urate in urine has not yet been documented.
B. P a t h o p h y s i o l o g y o f H y p e r u r i c o s u r i a Uric acid is an end product of purine metabolism. It cannot be degraded in humans because they lack uricase, which is present in other mammalian species. The major site of disposal of uric acid is the kidney. Hyperuricosuria may ensue when the serum concentration and the renal filtered load of uric acid are increased as a result of (1) the availability of an excessive amount of substrate (e.g., from a diet high in purine-rich foods 66) or from accelerated degradation and turnover of nucleic acids, or (2) a disturbance in purine biosynthesis that causes overproduction of purine substrates for uric acid synthesis. A high urinary uric acid level can occur transiently when renal tubular reabsorption of uric acid is impaired, as during early stages of extracellular volume expansion or after administration of uricosuric agents such as probenecid. In the steady state, however, normal urinary uric acid concentration is restored in latter conditions because of the secondary decline in the serum concentration and renal filtered load of uric acid, even though the renal tubular reabsorption of uric acid remains impaired. 67 Hyperuricosuria may be the only observed physiological abnormality in patients with calcium nephrolithiasis. Such a defect (hyperuricosuric calcium urolithiasis) exists alone in approximately 10% of patients with renal
748
CHARLES Y. C. PAK
Although hyperuricosuria may coexist with the various forms of hypercalciuria, only the pure disorder is considered in this section. The most common cause of hyperuricosuria in patients with hyperuricosuric calcium oxalate nephrolithiasis is probably dietary overindulgence in purine-rich f o o d s . 66 Such individuals have a history of a liberal intake of meat, poultry, and fish. 63'66 Hyperuricosuria in such patients can be ameliorated by dietary purine c a l c u l i . 68"69
d e p r i v a t i o n . 63,66
However, about 30% of patients with hyperuricosuric calcium oxalate nephrolithiasis have hyperuricosuria as the result of uric acid overproduction. Hyperuricosuria persists despite long-term purine deprivation. No further studies have been performed to elucidate the nature of this apparent urate overproduction. During long-term thiazide therapy, hyperuricosuria and hyperuricemia may be encountered. 7~ Besides its customary action of augmenting renal tubular reabsorption of urate, thiazide may stimulate urate production or decrease the extrarenal disposal of urate.
C. Diagnostic Criteria (Table 25-2) Hyperuricosuric calcium oxalate nephrolithiasis 71 is characterized by hyperuricosuria [urinary uric acid > 3.6 mM/day (> 600 mg/day) in at least two of three urine samples], normal serum calcium, normal fasting and calcium load response, normal urinary calcium, normal urinary oxalate [< 500 ixM/day (< 45 mg/day)], and calcium nephrolithiasis. Hyperuricosuria, defined functionally here by the upper normal limit of 3.6 mM/day (600 mg/day), correlates with the urinary supersaturation with monosodium urate that is associated with the increased propensity for calcium stone formation. 62 [Other laboratories employ a higher upper limit for urinary uric acid, e.g., 4.5 mM/day (750 mg/day) for women and 4.8 mM/ day (800 mg/day) for men.] Urinary pH is typically greater than 5.5. Hyperuricosuria may be the only abnormality in patients with calcium stones, or it may coexist with various forms of hypercalciuria.
D. Treatment of Hyperuricosuric Calcium Nephrolithiasis (Table 25- 3) Allopurinol (300 mg/day) is the drug of choice in hyperuricosuric calcium oxalate nephrolithiasis resulting from uric acid overproduction, because of its ability to reduce uric acid synthesis and to lower urinary uric acid concentration. 61 Its use in hyperuricosuria associated with dietary purine overindulgence is also reasonable,
because dietary purine restriction may be impractical. Physicochemical changes ensuing from restoration of a normal urinary uric acid include an increase in the urinary limit of metastability of calcium oxalate. 63 Thus the spontaneous nucleation of calcium oxalate is retarded by treatment, probably by the inhibition of monosodium urate-induced stimulation of calcium oxalate crystallization. 64 Because of the potential exaggeration of the latter process, a moderate sodium restriction (<150 mM/ day) is advisable. Potassium citrate is an effective alternative to allopurinol for treatment of this condition. 72 Citrate can inhibit the heterogeneous nucleation of calcium oxalate by monosodium urate. Not only does the induced hypercitraturia from potassium citrate therapy reduce the urinary saturation of calcium oxalate (by complexing calcium), it also inhibits the urate-induced crystallization of calcium oxalate.
IV. HYPEROXALURIA The main cause of moderate to severe hyperoxaluria causing calcium oxalate nephrolithiasis is ileal disease (enteric hyperoxaluria). This entity will therefore be the principal focus of this section.
A. Causal Role of Hyperoxaluria in Calcium Stone Formation A role for hyperoxaluria in stone formation is suggested by its identification as a risk factor for stone formation and by its frequent association with calcium nephrolithiasis. Hyperoxaluria facilitates stone formation by increasing urinary saturation of calcium oxalate.
B. Pathophysiology of Hyperoxaluria Oxalate is derived from both in vivo synthesis and intestinal absorption. Once synthesized or absorbed, it is not further degraded, and the principal route of excretion is the kidney. Data on renal handling of oxalate are limited and conflicting. Of the 30 mg of oxalate excreted normally, 80% is derived from in vivo synthesis and the remainder is derived from the diet. No primary derangement in renal handling of oxalate has been recognized. Rather, hyperoxaluria is due to increased serum concentrations and increased renal filtered loads resulting from (1) high substrate availability, as caused by administration of methoxyflurane or ascorbic acid; (2) enzymatic disturbances in the oxalate biosyn-
CHAPTER25 Kidney Stones: Pathogenesis, Diagnosis, and Therapy thetic pathway that cause overproduction, as in primary hyperoxaluria (rare); or (3) increased intestinal absorption of oxalate, as in the hyperoxaluria of ileal disease. 73,74 Two factors probably act in concert to cause intestinal hyperabsorption of oxalate: (1) intestinal transport of oxalate may be primarily increased because of the action of bile salts and fatty acids on the permeability of intestinal mucosa to oxalate, and (2) the total amount of oxalate absorbed may be increased because of an enlarged intraluminal pool of oxalate available for absorption. For example, the intestinal fat malabsorption characteristic of ileal disease may exaggerate soap formation with divalent cations, limit the amount of "free" divalent cations available to complex oxalate, and thereby enlarge the available oxalate pool. The formation of calcium oxalate stones has multifactorial causes. In addition to the disturbance in oxalate metabolism, the intestinal absorption and renal excretion of calcium are often decreased in enteric hyperoxaluria, probably because of loss of the intestinal site of calcium absorption as a result of disease or resection, intraluminal binding of calcium by nonabsorbed fatty acids, and vitamin D deficiency associated with fat malabsorption. Urine volume may be reduced because of fluid loss from the intestinal tract. Urinary citrate may be depressed because of hypokalemia and metabolic acidosis, 75 and urinary magnesium may be low because of impaired intestinal magnesium absorption. Saturation of urine with calcium oxalate may be increased because of the high urinary oxalate, even though urinary calcium level may be low. Low urine volume exaggerates urinary supersaturation. Urinary saturation of calcium oxalate is further increased, because the intestinal loss of electrolytes reduces urinary ionic strength, thereby increasing activity coefficient and the fraction of ionic calcium and oxalate. Moreover, the inhibitor activity against the crystallization of calcium salts is reduced because of low renal excretion of citrate and magnesium. Other stones may form as well in chronic diarrheal states. Urinary pH is low from intestinal alkali loss predisposing to uric acid stones. Ammonium urate stones have also been described, probably from reduced urinary sodium and high urinary ammonium.
C. Diagnostic Criteria (Table 25-2) Hyperoxaluria, defined as urinary oxalate greater than 44 mg/day, is associated with calcium oxalate stones. If urinary oxalate is greater than 80 mg/day, primary or enteric hyperoxaluria is probably present. In primary hyperoxaluria, urinary glycolate or glycerate may be increased in addition to oxalate. Moreover, oxalosis (tissue
749 deposition of calcium oxalate), anemia, and renal failure are common in primary hyperoxaluria. In enteric hyperoxaluria, there is a history of small bowel disease, ileal bypass, or resection. TM Urinary calcium is typically low (< 100 mg/day). Serum calcium and magnesium may be low or low normal, and parathyroid function may be stimulated. Serum bicarbonate and urinary citrate may be reduced. Even in the absence of intestinal disease, a mild to moderate hyperoxaluria (urinary oxalate 44 to 80 mg/day) may occur with vitamin C therapy, overindulgence in oxalate-rich foods (particularly spinach), or severe dietary calcium deprivation. Mild hyperoxaluria may also occur in patients with increased calcium absorption, such as those suffering from absorptive hypercalciuria.
D. Treatment of Hyperoxaluric Calcium Nephrolithiasis (Table 25- 3) Stone formation in enteric hyperoxaluria is multifactorial. The treatment should therefore be directed at correcting the various disturbances, including hyperoxaluria, hypocitraturia and low urinary pH, hypomagnesiuria, and low urine volume. Oral administration of large amounts of calcium (0.25 to 1.0 g four times a day) or magnesium has been recommended for the control of calcium nephrolithiasis of ileal disease. Although urinary oxalate may decrease (probably because of binding of oxalate by divalent cations), the concurrent rise in urinary calcium may obviate the beneficial effect of this therapy, at least in some patients. TM Cholestyramine does not cause a sustained reduction in oxalate excretion, but limitation of dietary oxalate intake and partial replacement of dietary fat with medium-chain fatty acids may be helpful in patients with malabsorption. Hypomagnesiuria, due to impaired intestinal absorption of magnesium, contributes to calcium oxalate stone formation by reducing the complexation of oxalate by magnesium and thus raising the urinary saturation of calcium oxalate. Oral magnesium supplementation can partially correct hypomagnesiuria, although it may worsen diarrhea. Magnesium citrate (10 mEq two to four times a day) is better tolerated and absorbed than magnesium oxide or hydroxide. Magnesium chloride is contraindicated because it exaggerates metabolic acidosis. A high fluid intake is recommended to ensure adequate urine volume. Control of excessive intestinal fluid loss with an antidiarrheal agent may be necessary before a sufficient urine output can be achieved. Calcium citrate may theoretically have a role in the management of enteric hyperoxaluria. This treatment may lower urinary oxalate by binding oxalate in the in-
750
CHARLES Y. C. PAK
testinal tract and increase urinary citrate and pH by providing an alkali load. 77 Finally, it may correct the malabsorption of calcium and avert bone loss by enhancing calcium absorption. If hypercalciuria develops, thiazide may be added.
V. HYPOCITRATURIA A. Causal Role of Hypocitraturia in Calcium Stone Formation Hypocitraturia may be a risk factor for the formation of calcium stones because citrate is known to inhibit stone formation. Citrate reduces urinary saturation of calcium oxalate or calcium phosphate by forming a soluble complex with calcium and thereby reducing calcium ion activity. 5~ Citrate is an effective inhibitor of calcium phosphate crystal growth, and a modest inhibitor of calcium oxalate crystal growth. 78 However, citrate directly inhibits spontaneous nucleation of calcium oxalate. 79 Citrate may also be a potent inhibitor of the agglomeration of preformed calcium oxalate crystals. 8~ Other simple (low-molecular-weight) and complex (macromolecular) inhibitors of the crystallization of calcium salts in urine include pyrophosphate, glycopeptides, l~ glycosaminoglycans, 12ribonucleic acids, and glycoproteins, ll Renal excretion of these substances may be disturbed in certain patients with nephrolithiasis, but this has never been fully substantiated. However, the finding that a specific urinary glycoprotein in patients with calcium nephrolithiasis may be abnormal structurally and functionally is intriguing and could be important pathogenetically. 11 Measurement of these inhibitors may have diagnostic or predictive utility if simple and reliable assays could be developed. Furthermore, therapeutic approaches to augmenting the activity or content of these inhibitors in urine may become feasible.
B. Pathophysiology of Hypocitraturia Urinary citrate excretion is a function of filtration, reabsorption, peritubular transport, and synthesis by the renal tubular cell. Approximately 80% to 90% of filtered citrate is normally reabsorbed, and citrate secretion is usually negligible. Although the exact physiology of renal handling of citrate has not been elucidated, several factors influence citrate excretion. Citrate excretion is enhanced by alkalosis, 81 PTH, vitamin D, growth hormone, and estrogen and decreased by acidosis, 82 hypokalemia, 75 androgen, and urinary tract infection (probably because of bacterial
degradation of citrate). Acid-base status probably plays the most important role in citrate excretion. For example, acidosis reduces the urinary citrate by both enhancing renal tubular reabsorption and impairing peritubular uptake and synthesis of citrate. This mechanism accounts for the occurrence of hypocitraturia in distal renal tubular acidosis (complete or incomplete), 83 chronic diarrheal states (resulting from intestinal alkali lOSS), 84'85 hypokalemia (from intracellular acidosis), thiazide therapy (from hypokalemia), 75 and a diet high in animal protein (from elevated acid-ash content). 86 Urinary citrate concentration may also be low after strenuous physical exercise (because of lactic acidosis) 87 and after high sodium intake (probably because of sodium-induced potassium loss). 88 Distal acidification defect (type I) is the only form of renal tubular acidosis associated with nephrolithiasis. The stone formation is the result of high urinary pH and calcium and low urinary citrate. Calcium phosphate crystallization is promoted by increased urine saturation (resulting from increased dissociation of phosphate and hypercalciuria) and reduced inhibitor activity (from hypocitraturia). Crystallization of calcium oxalate may also be enhanced because of increased saturation (resulting from hypercalciuria and reduced citrate complexation of calcium) and impaired inhibitor activity (from hypocitraturia). The predominant stone constituent is calcium phosphate (hydroxyapatite), but calcium oxalate is also typically present as a minor constituent. Nephrocalcinosis commonly coexists with nephrolithiasis. Nephrolithiasis is not associated with proximal renal tubular acidosis (type II) or hyporeninemic hypoaldosteronism (type IV). In these disorders, the urinary citrate and calcium may be low because of acidosis and bicarbonaturia in type II, and because of renal insufficiency in type IV. 89 Moreover, urinary pH is normal in type IV. Distal renal tubular acidosis should not be confused with calcium oxalate nephrolithiasis of the chronic diarrheal syndrome. In the latter disorder, metabolic acidosis is secondary to intestinal alkali loss in the stool. Stone (calcium) formation is due to low urinary citrate and low urine volume. Gastrointestinal disorders that cause this syndrome include the postgastrectomy state, inflammatory disease of the small bowel, bowel resection or bypass, and colitis. States of malabsorption of fat, hyperoxaluria, and low urinary magnesium level may contribute to calcium oxalate stone formation. Uric acid lithiasis may develop as a result of low urinary pH. In many patients with hypocitraturic calcium nephrolithiasis, the cause of low urinary citrate is unknown. It is not due to a primary defect in citrate absorption from the gastrointestinal tract. 9~ Dietary habit that leads to a low gastrointestinal absorption of net alkali may be an important factor. 91
CHAPTER25 Kidney Stones: Pathogenesis, Diagnosis, and Therapy
C. Diagnostic Criteria (Table 25-2) Hypocitraturic calcium nephrolithiasis in the pure presentation is the condition in which hypocitraturia [urinary citrate < 1.7 mM/day (< 320 mg/day)] is present alone without other physiological derangements (such as hypercalciuria or hyperuricosuria). Hypocitraturia may be caused by distal renal tubular acidosis, metabolic acidosis of chronic diarrheal states, or thiazide-induced hypokalemia. Complete distal renal tubular acidosis (high serum chloride and low serum potassium and carbon dioxide levels) and high urinary pH (> 6.8) in the absence of infection of the urinary tract is an uncommon occurrence in renal stone disease. A more common presentation of renal tubular acidosis in stone disease is the incomplete form, which is characterized by normal serum electrolyte levels but an impaired ability to acidify the urine after ammonium chloride load. Both complete and incomplete forms may be associated with hypercalciuria, hypocitraturia, calcium nephrolithiasis, and nephrocalcinosis. Stone analysis typically shows a preponderance of hydroxyapatite with calcium oxalate as a minor constituent. Chronic diarrheal states 84 capable of producing hypocitraturia and calcium nephrolithiasis include ileal disease or ileal resection, gastrectomy, ulcerative colitis, and colectomy. The degree of hypocitraturia is generally proportional to the severity of intestinal fluid loss. In severe diarrheal states, urinary citrate concentration may be very low [< 0.3 mM/day (< 50 mg/day)]; serum electrolyte abnormalities of acquired metabolic acidosis (caused by intestinal fluid loss) and low urinary pH may also be present. Some patients show full features of enteric hyperoxaluria. In thiazide-induced hypocitraturia, 75 serum potassium level is low or low normal and urinary citrate level is low [< 1.7 mM/day (< 320 mg/day)]. Thiazide action may also cause a high serum bicarbonate and a low serum chloride concentration.
D. Treatment of Hypocitraturic Calcium Nephrolithiasis (Table 25- 3) 1. RENAL TUBULAR ACIDOSIS (DISTAL)
Potassium citrate therapy is capable of correcting both metabolic acidosis and hypokalemia. 83 Moreover, it may restore normal urinary citrate, although large doses (up to 120 mEq/day) may be required in severe acidotic states. Urinary calcium concentration typically decreases with the correction of acidosis. The overall rise in urinary pH is small because the urinary pH is high to begin
751 with; urinary pH is generally below 7.5 during treatment unless a urinary tract infection is present. Thus, potassium citrate treatment produces a sustained decline in the urinary saturation of calcium oxalate (through a reduction in urinary calcium and rise in citrate complexation of calcium). The urinary saturation of calcium phosphate does not increase because the rise in phosphate dissociation is relatively small (as a result of the modest rise in pH) and is adequately compensated by a decrease in ionic calcium. Moreover, inhibitor activity against the crystallization of calcium oxalate and calcium phosphate is augmented by the direct action of citrate. The treatment with potassium citrate improves calcium balance by increasing intestinal calcium absorption (through an unknown mechanism), and by reducing urinary calcium. 92 If renal sodium leak is significant, alkali might be provided as a mixed sodium-potassium salt. However, renal sodium wasting is not prominent in most patients with renal tubular acidosis presenting with stones. 2. CHRONIC DIARRHEAL SYNDROME In patients with mild to moderate severity of intestinal fluid loss in whom hypocitraturia is not severe [urinary citrate in the range of 0.5 to 1.5 mM/day (100 to 300 mg/day)], potassium citrate (40 to 60 mEq in three or four divided doses in a liquid form) is generally effective in restoring normal urinary citrate and pH. 85 Urinary calcium generally remains low. In those with severe hypocitraturia [urinary citrate < 0.5 mM/day (< 100 mg/ day)], even high doses of potassium citrate (up to 120 mEq/day) may be ineffective in restoring a normal urinary citrate. A liquid preparation of potassium citrate is preferable to a slow-release tablet preparation in these states because some of the patients may have abnormal intestinal motility and may be more prone to obstruction caused by a tablet preparation. Furthermore, a slow-release tablet medication may be poorly absorbed because of rapid intestinal transit. A frequent dose schedule (three or four times a day) is recommended for the liquid preparation because of the relatively short duration of biological action. In other hypocitraturic conditions, the solid slow-release preparation is preferable or is better tolerated. A less frequent dose schedule (twice a day) is acceptable for the solid preparation because of its slow-release characteristic. 3. THIAZIDE-INDUCEDHYPOCITRATURIA Hypokalemia resulting from thiazide may cause hypocitraturia, probably causing intracellular acidosis, and may thereby attenuate the beneficial hypocalciuric effect of therapy on renal stone formation.
7
5
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H
A
It has been suggested that potassium citrate may be less effective than potassium chloride in correcting the thiazide-induced hypokalemia because of the poor reabsorbability of citrate from the renal tubules. 93 However, severe chloride depletion is uncommon in patients with thiazide-induced hypocitraturia. Potassium citrate (15 to 20 mEq twice a day) is usually effective in preventing hypokalemia and maintaining normal urinary citrate. 4. IDIOPATHIC HYPOCITRATURIC CALCIUM NEPHROLITHIASIS Idiopathic hypocitraturic calcium oxalate nephrolithiasis can be the result of isolated hypocitraturia with calcium stones or of hypocitraturia occurring in conjunction with absorptive and renal hypercalciurias or with hyperuricosuric calcium oxalate nephrolithiasis. 94 Stones formed are composed predominantly of calcium oxalate. Potassium citrate treatment (15 to 30 mEq twice a day) produces a sustained increase in urinary citrate excretion. 94 Urinary pH can be maintained at 6.5 to 7.0. Along with these changes, the urinary saturation of calcium oxalate declines to normal limits.
VI. GOUTY DIATHESIS A. Causal Role of Low Urinary pH in Formation of Calcium or Uric Acid Stones Persistent passage of unusually acid urine (pH < 5.5) may cause both uric acid and calcium stones. 95 Because urinary pH is close to or less than the dissociation constant of uric acid, the concentration of undissociated uric acid is high, leading to uric acid crystallization. The uric acid so formed may cause the crystallization of calcium oxalate by the same mechanism described for monosodium urate. Although uric acid is less efficient than monosodium urate in inducing heterogeneous nucleation of calcium oxalate (on a weight basis), its crystalline dimensions are more compatible with epitaxy. Moreover, uric acid has been shown to remove naturally occurring urinary macromolecular inhibitors, thereby attenuating their activity. 65
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ingly soluble. Some patients present with gouty arthritis or hyperuricemia. Stone analysis has disclosed uric acid alone or in combination with calcium oxalate and/or calcium phosphate. In some patients, we have found these features of gouty diathesis except for the lack of uric acid on stone analysis. The stone analysis has disclosed only the presence of calcium oxalate and/or calcium phosphate. No obvious cause has been detected for the unusually low urinary pH, such as excessive intestinal alkali loss or a consumption of an animal protein-rich diet. It is presumed that urate-induced crystallization of calcium salts accounts for calcium nephrolithiasis. 64 It is our contention that gouty diathesis represents varying phases of primary gout; the occurrence of hyperuricemia and gouty arthritis represents the full manifestation of the syndrome, whereas the picture of low urinary pH and uric acid/calcium nephrolithiasis without hyperuricemia or gouty arthritis reflects a forme fruste or an early phase of classic gout. Stone symptoms may precede articular symptoms in 40% of gouty patients, occasionally by more than 10 years. 2. PRIMARY GOUT
In primary gout, the principal abnormality, identifiable in all patients, is a persistently acid urinary pH. 97 In fasting morning specimens, the urinary pH is 5.0 or less in about half of these patients, in comparison with 15% of normal subjects. The abnormality is also manifest by a loss of the normal alkaline tide in the gouty population. In spite of a relatively large amount of literature on the subject, the basis of this tendency to a persistently acid urine remains unclear. 98-1~ The data indicate that patients with gout excrete relatively more titratable acid and relatively less ammonia than do normal subjects, 1~ but it is unclear whether the defect in ammonia production is primary, whether it is dependent on a primary abnormality in the excretion of titratable acid, or whether both abnormalities reflect an unknown fundamental defect in acid-base regulation. Many patients, but not all, present with hyperuricosuria as well due to urate overproduction. Such patients are at particular risk for uric acid nephrolithiasis. 3. OTHERS
B. Pathophysiology of Gouty Diathesis 1. GOUTY DIATHESIS
We have previously utilized the term gouty diathedescribe the overall clinical entity of uric acid lithiasis associated with or resulting from primary gout. The invariant feature is the persistent passage of unusually acid urine (pH < 5.5) in which uric acid is sparsis 95'96 t o
Other causes of uric acid nephrolithiasis include certain inborn errors of uric acid metabolism, secondary causes of urate overproduction, chronic diarrheal states, and use of uricosuric agents. Three well-studied enzymatic disorders of uric acid metabolism are hypoxanthine-guanine phosphoribosyl transferase deficiency (Lesch-Nyhan syndrome), phosphoribosyl pyrophosphate synthetase overactivity, and glucose-6-phosphatase deficiency (type I glycogen storage disease). Affected
CHAPTER 25 Kidney Stones: Pathogenesis, Diagnosis, and Therapy patients have extreme rates of uric acid overproduction. In myeloproliferative disorders, leukemia, neoplasia, or hemolytic anemia, hyperuricemia and hyperuricosuria occur because of an increased rate of nucleoprotein turnover. As many as 50% of patients with myeloproliferative disorders may form uric acid stones, which often is the initial clinical manifestation. Certain drugs such as probenecid and high-dose salicylates, as well as x-ray contrast agents, may produce an acute uricosuria by inhibiting net uric acid reabsorption and increasing the fractional excretion of uric acid. The chronic administration of these agents, however, resuits in a new steady state, in which the rate of uric acid excretion should be no greater than the pretreatment rate. Thus, the risk of stone formation occurs early, and not late, except in the setting of urate overproduction. Several gastrointestinal disorders have been associated with a high incidence of uric acid lithiasis. The three most common conditions are ulcerative colitis, regional enteritis, and the presence of ileostomy. The pathogenesis of uric acid stones in these disorders is related to variable degrees of dehydration and bicarbonate loss resulting in an unusually acid and concentrated urine. The excretion rate of uric acid is usually normal in these patients. In the preceding entities, calcium nephrolithiasis could also occur.
753 ration of calcium oxalate and retarding the crystallization of calcium oxalate. Sodium alkali is as effective as potassium citrate in preventing uric acid nephrolithiasis because it has a similar capacity for raising urinary pH. However, calcium stones may sometimes occur with sodium alkali therapy. 95 In patients with hyperuricemia or moderate to marked hyperuricosuria, allopurinol (e.g., 300 mg/day) is recommended as well.
VII. CYSTINURIA Cystinuria stones develop in patients with homozygous cystinuria. 1~ Homozygous cystinuria is characterized by urinary cystine excretion of more than 250 mg/g creatinine.
A. Causal Role of Cystine in Stone Formation Cystine is sparingly soluble in urine. Its solubility is greater at higher pH and is enhanced by electrolytes and macromoleculesl~ however, it rarely exceeds 1.7 mM/ liter (400 mg/liter). Cystine stones may form when urinary cystine concentration exceeds the solubility of cystine.
C. Diagnostic Criteria (Table 25-2) B. Pathophysiology of Cystinuria The invariant feature in gouty diathesis is the persistent passage of unusually acid urine (pH < 5.5). 95 The cause for low urinary pH cannot be readily ascertained. Some patients may give a positive personal or family history of gouty arthritis. Some patients may present with hyperuricemia or hypertriglyceridemia. Stones formed may be uric acid alone, calcium oxalate-phosphate alone, or the mixture of the two. Some patients may form uric acid on one occasion and calcium stones on another occasion. Uric acid stones are radiolucent.
D. Treatment of Gouty Diathesis (Table 25-3) Potassium citrate is the treatment of choice for this condition. A dosage of 30 to 60 mEq/day in divided doses increases the low urinary pH (< 5.5) to the desired range (> 6). 95 Because of the resulting enhanced dissociation of uric acid, the amount of undissociated uric acid decreases to levels found in normal controls [< 0.9 mM/day (< 150 mg/day)]. Thus potassium citrate therapy increases the solubility of uric acid, preventing uric acid stone formation. Moreover, this treatment inhibits formation of calcium stones by reducing urinary satu-
Normally, cystine is filtered and almost completely reabsorbed in the proximal nephron, so that less than 20 mg is excreted in urine each day. In cystinuria, the serum concentration and hence the renal filtered load of cystine are reduced. Exaggerated cystine excretion under this circumstance suggests a disturbance in renal handling of cystine. More than one defect can impair tubular reabsorption and back-diffusion of cystine. 1~ Similar defects in transport of other dibasic amino acids are present. However, exaggerated renal excretion of these amino acids and cystine may not be due to a single transport defect. 1~ Increasing the filtered load of one of these amino acids does not necessarily augment the excretion of others. T M The intestinal transport of dibasic amino acids may also be defective in cystinuria. The disorder has been classified into three types based on varying intestinal transport disturbances for these amino acids. 1~ The intestinal transport has been assessed by the in vitro uptake of radiolabeled amino acid by specimens of jejunal mucosa obtained by peroral biopsy and by studies of plasma cystine levels after oral cystine administration. In type I cystinuria, there is no uptake of cystine, lysine, or argi-
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nine by jejunal mucosa, and plasma cystine concentration is not elevated after an oral cystine load. Thus there is defective intestinal transport of all three dibasic amino acids. In types II and III, the intestinal transport of dibasic amino acids is disturbed but less severely than in the type I presentation. In type II, some cystine is taken up by jejunal mucosa but at a reduced rate, and oral cystine loading does not increase the plasma cystine level. In type III cystinuria, the uptake of cystine and lysine by jejunal mucosa is variably reduced and the increment in plasma cystine after oral cystine loading is blunted. In the homozygous state, all three types of cystinuria involve excessive renal excretion of all four dibasic amino acids. 1~ In the heterozygous state, type I cystinuria is characterized by normal cystine excretion, whereas types II and III have elevated cystine and lysine excretion (although not quite up to the level encountered in the homozygous state), probably because of a prevailing (although reduced) intestinal uptake of these amino acids. Recently, discrete mutations in the dibasic amino acid transporter gene have been found in certain cystinuric patients. ~~176
C. Diagnostic Criteria The urinary sediment (preferably in fresh first morning void) should be examined for the presence of typical hexagonal cystine crystals. The urine sample should also be screened for "qualitative" cystine by the cyanidenitroprusside test. A positive reaction suggests that cystine excretion exceeds 75 mg/liter. A false-positive test may be encountered in patients with homocystinuria and acetonuria. On roentgenological examination, cystine calculi are radiopaque (although not as much) like calcareous calculi, but are more rounded and homogeneous in appearance. They may attain a staghorn size. If these studies are suggestive of the presence of cystinuria, urinary cystine excretion should be quantitated. Urinary cystine exceeding 250 mg/g creatinine is usually diagnostic of homozygous cystinuria. Stones passed or removed should be analyzed. The presence of cystine provides a definitive diagnosis of cystinuria.
D. Treatment of Cystine Nephrolithiasis (Table 25 - 3) A low-methionine diet has often been recommended for the control of cystine nephrolithiasis because methionine is a precursor of cystine. Although such a dietary maneuver may reduce cystine excretion, rigid methionine restriction is impractical. Dietary sodium restriction
may also reduce cystine excretion, 11~ but this beneficial effect may be neutralized by reduced solubility of cysfine resulting from loss of the "solubilizing" action of sodium. 1~ In patients with cystine calculi and moderate cystinuria [ 1 to 2 mM/day (250 to 500 mg/day)], conservative measures of high fluid intake and alkali administration should be attempted. The aim of fluid therapy is to increase urine volume sufficiently to reduce the cystine concentration below the solubility limit. At least 3 liters of fluid should be provided, including two 8-oz glassfuls with each meal and at bedtime. Patients should be directed to wake up at night to urinate and drink water. Additional fluids should be consumed when excessive sweating or intestinal fluid loss is present. A minimum urine output of 2 liters/day on a consistent basis is attainable by most patients with proper and persistent instruction. In theory, alkali therapy would enhance cystine solubility by raising urinary pH. However, substantial increases in cystine solubility do not occur until the urinary pH exceeds 7.5. The provision of alkali, no matter how much, rarely raises urinary pH above 7.5. When urinary pH increases above 7.0 with alkali therapy, calcium phosphate nephrolithiasis may be enhanced because of the enhanced urinary supersaturation of hydroxyapatite in an alkaline environment. Excessive alkali therapy therefore is not indicated. 1~ Thus a modest amount of alkali is recommended to maintain urinary pH in a high normal range (6.5 to 7.0). Potassium citrate has the advantages over sodium citrate that it does not cause hypercalciuria, is less likely to promote development of calcium s t o n e s , 95'111 and does not induce increased cystine excretion, ll~ The object of treatment with penicillamine or riopronin (et-mercaptopropionylglycine) is to reduce total cystine excretion by complexing cysteine, the monomeric form of cystine. Penicillamine or tiopronin may be added to the conservative treatment program in patients with moderate cystinuria when the conservative treatment is ineffective in controlling stone formation. In patients with severe cystinuria [> 2 mM/day (> 500 mg! day)], in whom conservative management alone is not likely to be effective, penicillamine or tiopronin therapy (together with conservative measures) may be begun. Penicillamine and tiopronin share with cysteine a free sulfhydryl group. 112 Thus, they undergo thiol-disulfide exchange with cystine to form penicillamine-cysteine or tiopronin-cysteine disulfide, which is much more soluble than cystine. After oral administration, a sufficient amount of penicillamine or tiopronin can be excreted in urine to complex cysteine and thereby lower cystine excretion. Unfortunately, penicillamine therapy is associated with frequent and sometimes severe side effects,
CHAPTER 25 Kidney Stones: Pathogenesis, Diagnosis, and Therapy including nephrotic syndrome, dermatitis, and pancytopenia. 113 Tiopronin has biochemical and clinical actions similar to those of penicillamine. TM However, it has a lower toxicity profile than penicillamine.
VIII. INFECTION WITH UREA-SPLITTING ORGANISMS A. Causal Role of Infection and Pathophysiology of Struvite Stone Formation Infection of the urinary tract with urea-splitting organisms may be associated with renal stones of struvite (magnesium ammonium phosphate) and of calcium carbonate apatite. 115 The critical determinant is the formation of ammonia in urine due to enzymatic degradation of urea by bacterial urease. The ammonia undergoes hydrolysis to form ammonium and hydroxyl ions. The resulting alkalinity of urine augments dissociation of phosphate to form triphosphate ions, and reduces the solubility of struvite. Although struvite stones may form de novo from infection alone, they also occur as a complication of other causes of renal calculi, such as hypercalciuria.
B. Diagnostic Criteria (Table 25-2) Lithiasis due to infection is disclosed by the presence of magnesium ammonium phosphate on stone analysis. Such struvite stones are often associated with pyuria, positive urine culture for urea-splitting organisms (Proteus, certain species of Staphylococcus, Pseudomonas, and Klebsiella), and high urinary pH (> 7.5). 115 Struvite stones are radiopaque and sometimes may attain a large (staghorn) size; they usually occur as mixtures with calcium carbonate apatite and tricalcium phosphate or less commonly with calcium oxalate. Some patients with struvite stones may have hypercalciuria. Urinary citrate may be low owing to bacterial enzymatic degradation of citrate.
C. Treatment of Struvite Stones (Table 25-3) If longstanding control of infection with urea-splitting organisms can be achieved, new stone formation may be averted and some existing stones may be dissolved. Unfortunately, such control is difficult to obtain with antibiotic therapy. If a struvite stone is present, it is difficult to eradicate infection completely because the stone may harbor the organisms within its interstices. Even i f " s t e r -
755 ilization" of urine can be achieved by antibiotic therapy, reinfection by organisms harbored by the stones can occur. For this reason, surgical removal of the struvite stones is usually recommended. Acetohydroxamic acid, a urease inhibitor, reduces urinary saturation of struvite by preventing the formation of ammonium and hydroxyl ions. TM It may prevent stone growth and sometimes cause dissolution of existing stones. However, it may cause hemolytic anemia, thrombophlebitis, and nonspecific neurological symptoms (disorientation, tremulousness, and headache). 116
IX. CONSERVATIVE MANAGEMENT The conservative measures of high fluid intake and avoidance of dietary excess should be applied in all patients with nephrolithiasis. 117 They may be applied alone in patients with a single stone episode and inactive stone disease but should be instituted together with a specific medical treatment program in patients with recurrent stone disease, particularly if extrarenal manifestations are present. Some conservative programs are applicable to all forms of stone disease, whereas others are useful for particular causes. High fluid intake is the only nutritional modification that is useful in all forms of nephrolithiasis. 118 By increasing urine output, urinary concentration of constituent ions and saturation of stone-forming salts are lowered. Although dietary restriction of oxalate may be beneficial in all types of nephrolithiasis, it is particularly indicated when intestinal absorption of oxalate is increased, as in ileal disease and when calcium absorption is increased. Rigid calcium restriction [< 10 mM/day (< 400 mg/day)] is ill advised, even in patients who have high intestinal calcium absorption, because it is difficult to adhere to, may adversely affect general nutrition, and may cause negative calcium balance. However, moderate calcium restriction [10 to 15 mM/day (400 to 600 mg/ day)] may be useful in absorptive hypercalciuria because it alone may control the hypercalciuria in the less severe (type II) presentation and permit reduction of the dosage of medication necessary to restore normal urinary calcium concentration in the more severe (type I) presentation. Calcium restriction is neither necessary nor indicated in patients with nephrolithiasis with normal intestinal absorption of calcium. Moderate sodium restriction (100 mM/day) may be helpful in all forms of nephrolithiasis. Sodium excess raises urinary calcium and lowers urinary citrate. 88 Moreover, it attenuates the hypocalciuric action of thiazide and increases urinary saturation of monosodium urate. TM
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References 1. Chaussy C, Brendel W, Schmiedt E: Extracorporeally induced destruction of kidney stones by shock waves. Lancet 2:12651268, 1980. 2. Segura JW, Patterson DE, LeRoy AJ, et al: Percutaneous lithotripsy. J Urol 130:1051 - 1054, 1983. 3. Resnick MI, Pak CYC: Are metabolic studies of urolithiasis necessary? J Urol 137:960-961, 1987. 4. Fine JK, Pak CYC, Preminger GM: Effect of medical management and residual fragments on recurrent stone formation following shock wave lithotripsy. J Urol 153:27-33, 1995. 5. Pak CYC: Role of medical prevention. J Urol 141:798-801, 1989. 6. Pak CYC, Skurla C, Harvey J: Graphic display of urinary risk factors for renal stone formation. J Urol 134:867-870, 1985. 7. Pak CYC, Britton F, Peterson R: Ambulatory evaluation of nephrolithiasis: Classification, clinical presentation and diagnostic criteria. Am J Med 69:19- 30, 1980. 8. Pak CYC, Peters P, Hurt G, et al: Is selective therapy of recurrent nephrolithiasis possible? Am J Med 71:615-622, 1981. 9. Fleisch H, Bisaz S: Isolation from urine of pyrophosphate a calcification inhibitor. Am J Physiol 203:671-675, 1962. 10. Kitamura T, Zerwekh JE, Pak CYC: Partial biochemical and physicochemical characterization of organic macromolecules in urine from patients with renal stones and control subjects. Kidney Int 21:379- 386, 1981. 11. Nakagawa Y, Abram V, Parks JH, et al: Urine glycoprotein crystal growth inhibitors. J Clin Invest 76:1455-1462, 1985. 12. Bowyer RC, Brockis JG, McCulloch RK: Glycosaminoglycans as inhibitors of calcium oxalate crystal growth and aggregation. Clin Chim Acta 95:23-28, 1979. 13. Worcester EM, Blumenthal SS, Beshensky AM, Lewand DL: The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin. J Bone Miner Res 7:1029-1036, 1992. 14. Pak CYC, Holt K: Nucleation and growth of brushite and calcium oxalate in urine of stone-formers. Metabolism 25:665673, 1976. 15. Pak CYC: Idiopathic renal lithiasis: New developments in evaluation and treatment. In Fleisch H, Robertson WG, Smith LH, Vahlensieck W (eds): Urolithiasis Research. New York, Plenum Publishing Corp, 1976, pp 213-244. 16. Zerwekh JE, Hwang TIS, Poindexter J, et al: Modulation by calcium of the inhibitor activity of naturally occurring urinary inhibitors. Kidney Int 33:1005-1008, 1988. 17. Strauss AL, Coe FL, Deutsch L, Parks JH: Factors that predict relapse of calcium nephrolithiasis during treatment. Am J Med 72:17 -24, 1982. 18. Pak CYC, Galosy RA: Propensity for spontaneous nucleation of calcium oxalate. Quantitative assessment by urinary FPR-APR discriminant score. Am J Med 69:681-689, 1980. 19. Yendt ER, Cohanim M: Prevention of calcium stones with thiazides. Kidney Int 13:397-409, 1978. 20. Sakhaee K, Baker S, Zerwekh J, et al: Limited risk of kidney stone formation during long-term calcium citrate supplementation in non-stone forming subjects. J Urol 152:324-327, 1994. 21. Pak CYC: Calcium metabolism. J Am Coil Nut 8:46S-53S, 1989. 22. Henneman PH, Benedict PH, Forbes AP, Dudley RH: Idiopathic hypercalciuria. N Engl J Med 259:802-807, 1958. 23. Brannan PG, Morawski S, Pak CYC, Fordtran JS: Selective jejunal hyperabsorption of calcium in absorptive hypercalciuria. Am J Med 66:425-428, 1979.
24. Pak CYC, Nicar MJ, Krejs GJ: Intestinal absorption of calcium, magnesium, phosphate and oxalate: Deviation from normal in idiopathic urolithiasis. In Schwille PO, Smith LH, Robertson WG, Vahlensieck W (eds): Urolithiasis and Related Clinical Research. Germany, Plenum Publishing Garmisch-Partenkirchen, 1985, pp 127-133. 25. Breslau NA, Preminger GM, Adams BV, Pak CYC: Use of ketoconazole to probe the pathogenetic importance of 1,25-(OH)2D in absorptive hypercalciuria. J Clin Endocrinol Metab 75: 1446-1452, 1992. 26. Pak CYC: Physiological basis for absorptive and renal hypercalciurias. Am J Physiol 237:F415-F423, 1979. 27. Kaplan RA, Haussler MR, Deftos LJ, et al: The role of l et,25dihydroxyvitamin D in the mediation of intestinal hyperabsorption of calcium in primary hyperparathyroidism and absorptive hypercalciuria. J Clin Invest 59:756-760, 1977. 28. Insogna KL, Broadus AE, Dreyer BE, et al: Elevated production rate of 1,25-dihydroxyvitamin D in patients with absorptive hypercalciuria. J Clin Endocrinol Metab 61:490-495, 1985. 29. Broadus AE, Horst RL, Lang R, et al: The importance of circulating 1,25-dihydroxyvitamin D in the pathogenesis of hypercalciuria and renal-stone formation in primary hyperparathyroidism. N Engl J Med 302:421-426, 1980. 30. Gray RW, Wilz DR, Caldas AE, et al: 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. 31. Barilla DE, Zerwekh JE, Pak CYC: A critical evaluation of the role of phosphate in the pathogenesis of absorptive hypercalciuria. Miner Elecrolyte Metab 2:302-309, 1979. 32. Broadus AE, Erickson SB, Gertner JM, et al: An experimental human model of 1,25-dihydroxyvitamin D-mediated hypercalciuria. J Clin Endocrinol Metab 59:202-206, 1984. 33. Pak CYC, Galosy RA: Fasting urinary calcium and adenosine 3'-5'-monophosphonate: A discriminant analysis for the identification of renal and absorptive hypercalciuria. J Clin Endocrinol Metab 48:260-265, 1979. 34. Reynolds JJ, Holick MF, DeLuca HF: The role of vitamin D metabolites in bone resorption. Calcif Tissue Res 12:295-301, 1973. 35. Pietschmann F, Breslau NA, Pak CYC: Reduced vertebral bone density in hypercalciuric nephrolithiasis. J Bone Miner Res 7: 1383-1388, 1992. 36. Coe FL, Favus MJ, Crockett T, et al: Effects of low calcium diet on urine calcium excretion, parathyroid function and serum 1,25(OH)2D3 levels in patients with idiopathic hypercalciuria and in normal subjects. Am J Med 72:25-32, 1982. 37. Zerwekh JE, Hughes MR, Reed BY, et al: Evidence for normal vitamin D receptor messenger ribonucleic acid and genotype in absorptive hypercalciuria. J Clin Endocrinol Metab 80:29602965, 1995. 38. Zerwekh JE, Pak CYC: Selective effects of thiazide therapy on serum 1,25-dihydroxyvitamin D and intestinal calcium absorption in renal and absorptive hypercalciurias. Metabolism 29: 13-17, 1980. 39. Sakhaee K, Nicar MJ, Brater DC, Pak CYC: Exaggerated natriuretic and calciuric responses to hydrochlorothiazide in renal hypercalciuria but not in absorptive hypercalciuria. J Clin Endocrinol Metab 61:825-829, 1985. 40. Barilla DE, Townsend J, Pak CYC: An exaggerated augmentation of renal calcium excretion following oral glucose ingestion in patients with renal hypercalciuria. Invest Urol 15:486-488, 1978.
CHAPTER 25
Kidney Stones: Pathogenesis, Diagnosis, and Therapy
41. Lemann J, Piering WF, Lennon EJ: Possible role of carbohydrate-induced calciuria in calcium oxalate kidney stone formation. N Engl J Med 280:232-237, 1969. 42. Muldowney FP, Freaney R, Moloney MF: Importance of dietary sodium in the hypercalciuria syndrome. Kidney Int 22:292-296, 1982. 43. Lawoyin S, Sismilich S, Browne R, Pak CYC: Bone mineral content in patients with calcium urolithiasis. Metabolism 28: 1250-1254, 1979. 44. Buck AC, Lote CJ, Sampson WF: The influence of renal prostaglandins on urinary calcium excretion in idiopathic urolithiasis. J Urol 129:421-426, 1983. 45. Silverberg SJ, Shane E, Cruz L, et al: Skeletal disease in primary hyperparathyroidism. J Bone Miner Res 4:283- 291, 1989. 46. Pak CYC: Sodium cellulose phosphate: Mechanism of action and effect on mineral metabolism. J Clin Pharmacol 13:15-27, 1975. 47. Pak CYC: A cautious use of sodium cellulose phosphate in the management of calcium nephrolithiasis. Invest Urol 19:187190, 1981. 48. Hayashi Y, Kaplan RA, Pak CYC: Effect of sodium cellulose phosphate therapy on crystallization of calcium oxalate in urine. Metabolism 24:1273-1278, 1975. 49. Preminger GM, Pak CYC: Eventual attenuation of hypocalciuric response to hydrochlorothiazide in absorptive hypercalciuria. J Urol 137:1104-1109, 1987. 50. Pak CYC, Nicar MJ, Northcutt C: The definition of the mechanism of hypercalciuria is necessary for the treatment of recurrent stone formers. Contrib Nephrol 33:136-151, 1982. 51. Pak CYC, Peterson R, Sakhaee K, et al: Correction of hypocitraturia and prevention of stone formation by combined thiazide and potassium citrate therapy in thiazide-unresponsive hypercalciuric nephrolithiasis. Am J Med 79:284-288, 1985. 52. Breslau NA, Padalino P, Kok DJ, et al: Physicochemical effects of a new slow-release potassium phosphate preparation (UroPhos-K) in absorptive hypercalciuria. J Bone Miner Res 10: 394-400, 1993. 53. Woelfel A, Kaplan RA, Pak CYC: Effect of hydrochlorothiazide therapy on crystallization of calcium oxalate in urine. Metabolism 26:201-205, 1977. 54. Ettinger B, Oldroyd NO, Sorgel F: Triamterene nephrolithiasis. JAMA 244:2443-2445, 1980. 55. Leppla D, Browne R, Hill K, Pak CYC: Effect of amiloride with or without hydrochlorothiazide on urinary calcium and saturation of calcium salts. J Clin Endocrinol Metab 57:920-924, 1983. 56. Broadus AE, Magee JS, Mallette LE, et al: A detailed evaluation of oral phosphate therapy in selected patients with primary hyperparathyroidism. J Clin Endocrinol Metab 56:953-961, 1983. 57. Gallagher JC, Nordin BEC: Treatment with estrogens of primary hyperparathyroidism in postmenopausal women. Lancet i:503507, 1972. 58. Kaplan RA, Snyder WH, Stewart A, et al: Metabolic effect of parathyroidectomy on asymptomatic primary hyperparathyroidism. J Clin Endocrinol Metab 42:415-426, 1976. 59. Pak CYC: Effect of parathyroidectomy on crystallization of calcium salts in urine of patients with primary hyperparathyroidism. Invest Urol 17:146-148, 1979. 60. Finlayson B, Smith A: Stability of first dissociable proton of uric acid. J Chem Eng Data 19:94-97, 1974. 61. Coe FL: Hyperuricosuric calcium oxalate nephrolithiasis. Kidney Int 13:418-426, 1978. 62. Pak CYC, Waters O, Arnold L, et al: Mechanism for calcium nephrolithiasis among patients with hyperuricosuria: Supersaturation of urine with respect to monosodium urate. J Clin Invest 59:426-431, 1977.
7~7 63. Pak CYC, Barilla DE, Holt K, et al: Effect of oral purine load and allopurinol on the crystallization of calcium salts in urine of patients with hyperuricosuric calcium urolithiasis. Am J Med 65:593-599, 1978. 64. Pak CYC, Holt K, Zerwekh JE: Attenuation by monosodium urate of the inhibitory effect of glycosaminoglycans on calcium oxalate nucleation. Invest Urol 17:138-140, 1979. 65. Zerwekh JE, Holt K, Pak CYC: Natural urinary macromolecular inhibitors: Attenuation of inhibitory activity by urate salts. Kidney Int 23:838-841, 1983. 66. Coe FL, Kavalach AG: Hypercalciuria and hyperuricosuria in patients with calcium nephrolithiasis. N Engl J Med 291:13441350, 1974. 67. Breslau NA, Pak CYC: Lack of effect of salt intake on urinary uric acid excretions. J Urol 129:531-532, 1983. 68. Pak CYC: Medical management of nephrolithiasis in Dallas: Update 1987. J Urol 140:461-467, 1988. 69. Levy FL, Huet-Adams B, Pak CYC: Ambulatory evaluation of nephrolithiasis: An update from 1980. Am J Med 98:50-59, 1995. 70. Pak CYC, Tolentino R, Stewart A, et al: Enhancement of renal excretion of uric acid during long-term thiazide therapy. Invest Urol 3:191-193, 1978. 71. Coe FL, Raisen L: Allopurinol treatment of uric acid disorders in calcium stone formers. Lancet i:129-131, 1973. 72. Pak CYC, Peterson R: Successful treatment of hyperuricosuric calcium oxalate nephrolithiasis with potassium citrate. Arch Intern Med 146:863-868, 1986. 73. Earnest DL, Williams HE, Admirand WH: A physicochemical basis for treatment of enteric hyperoxaluria. Trans Assoc Am Physicians 88:224-234, 1975. 74. Smith LH, Fromm H, Hofmann AF: Acquired hyperoxaluric nephrolithiasis and intestinal disease. N Engl J Med 286:13711375, 1972. 75. Nicar MJ, Peterson R, Pak CYC: Use of potassium citrate as potassium supplement during thiazide therapy of calcium nephrolithiasis. J Urol 131:430-433, 1984. 76. Barilla DE, Notz C, Kennedy D, Pak CYC: Renal oxalate excretion following oral oxalate loads in patients with ileal disease and with renal and absorptive hypercalciurias. Am J Med 64: 579-585, 1978. 77. Harvey JA, Zobitz MM, Pak CYC: Calcium citrate: Reduced propensity for the crystallization of calcium oxalate in urine resulting from induced hypercalciuria of calcium supplementation. J Clin Endocrinol Metab 61:1223-1225, 1985. 78. Meyer JL, Smith LH: Growth of calcium oxalate crystals: Inhibition by natural urinary crystal growth inhibitors. Invest Urol 13:36-39, 1975. 79. Nicar MJ, Hill K, Pak CYC: Inhibition by citrate of spontaneous precipitation of calcium oxalate in vitro. J Bone Miner Res 2: 215-220, 1987. 80. Kok DJ, Bijvoet OLM, Papapoulos SE: Excessive crystal agglomeration with low citrate excretion in recurrent stone formers. Lancet i:1056-1058, 1986. 81. Simpson DP: Regulation of renal citrate metabolism by bicarbonate ion and pH: Observations in tissue slices and mitochondria. J Clin Invest 16:225-238, 1967. 82. Morrissey JF, Ochoa M, Lotspeich WD, et al: Citrate excretion in renal tubular acidosis. Ann Intern Med 58:159-166, 1963. 83. Preminger GM, Sakhaee K, Skurla C, Pak CYC: Prevention of recurrent calcium stone formation with potassium citrate therapy in patients with distal renal tubular acidosis. J Urol 134:20-23, 1985. 84. Rudman D, Dedonis JL, Fountain MT, et al: Hypocitraturia in patients with gastrointestinal malabsorption. N Engl J Med 303: 657-661, 1980.
7
5
8
C
H
A
85. Pak CYC, Fuller C, Sakhaee K, et al: Long-term treatment of calcium nephrolithiasis with potassium citrate. J Urol 134:1119, 1985. 86. Breslau NA, Brinkley L, Hill KD, Pak CYC: Relationship role of animal protein-rich diet to kidney stone formation and calcium metabolism. J Clin Endocrinol Metab 66:140-146, 1988. 87. Sakhaee K, Nigam S, Snell P, et al: Assessment of the pathogenetic role of physical exercise in renal stone formation. J Clin Endocrinol Metab 65:974-979, 1987. 88. Sakhaee K, Harvey JA, Padalino PK, et al: Potential role of salt abuse on the risk for kidney stone formation. J Urol 150:310312, 1991. 89. Uribarri J, Pak CYC: Renal stone risk factors in patients with type IV RTA. Kidney Int 23:784-787, 1994. 90. Fegan J, Khan R, Poindexter J, Pak CYC: Gastrointestinal citrate absorption in nephrolithiasis. J Urol 147:1212-1214, 1992. 91. Sakhaee K, Williams RH, Oh MS, et al: Alkali absorption and citrate excretion in calcium nephrolithiasis. J Bone Miner Res 8:787-792, 1993. 92. Preminger GM, Sakhaee K, Pak CYC: Hypercalciuria and altered intestinal calcium absorption occurring independently of vitamin D in incomplete distal renal tubular acidosis. Metabolism 36:176-179, 1987. 93. Kassirer JP, Berkman PM, Lawrenz DR, et al: The critical role of chloride in the correction of hypokalemic alkalosis in man. Am J Med 38:172-189, 1965. 94. Pak CYC, Fuller C: Idiopathic hypocitraturic calcium oxalate nephrolithiasis successfully treated with potassium citrate. Ann Intern Med 104:33-37, 1986. 95. Pak CYC, Sakhaee K, Fuller C: Successful management of uric acid nephrolithiasis with potassium citrate. Kidney Int 30:422428, 1986. 96. Khatchadourian J, Preminger GM, Whitson PM, et al: Clinical and biochemical presentation of gouty diathesis: Comparison of uric acid versus pure calcium stone formation. J Urol 154: 1665-1669, 1995. 97. Yu TF, Gutman AB: Uric acid nephrolithiasis in gout. Predisposing factors. Ann Intern Med 67:1133-1148, 1967. 98. Gutman AB, Yu TF: Uric acid nephrolithiasis. Am J Med 45: 7 5 6 - 779, 1968. 99. Henneman PH, Wallach S, Dempsey EF: Metabolic defect responsible for uric acid stone formation. J Clin Invest 4 1 : 5 3 7 542, 1962. 100. Gutman AB, Yu TF: Urinary ammonium excretion in primary gout. J Clin Invest 44:1474-1481, 1965. 101. Plante GE, Durivage J, Lemieux G: Renal excretion of hydrogen in primary gout. Metabolism 17:377- 385, 1968.
R
L
E
S
Y. C. PAK
102. Gutman AB, Yu TF: A three-component system for regulation of renal excretion of uric acid in man. Trans Assoc Am Physicians 74:353-365, 1961. 103. Thier SO, Segal S: Cystinuria. In Stanbury JB, Wyngaarden JB, Frederickson DS (eds): The Metabolic Basis of Inherited Disease. New York, McGraw-Hill, 1972, pp 1504-1519. 104. Pak CYC, Fuller CJ: Assessment of cystine solubility in urine and of heterogeneous nucleation between cystine and calcium salts. Invest Urol 129:1066-1070, 1983. 105. Broadus A, Thier S: Metabolic basis of renal stone disease. N Engl J Med 300:839-845, 1979. 106. Dent CE, Rose GA: Amino acid metabolism in cystinuria. Q J Med 20:205- 219, 1951. 107. Rosenberg LE, Downing S, Durant JL, et al: Cystinuria: Biochemical evidence for three genetically distinct diseases. J Clin Invest 45:365-371, 1966. 108. Calonge MJ, Gasparini P, Chillaron J, et al: Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine. Nat Genet 6:420-425, 1994. 109. Gitomer WL, Reed BY, Ruml LA, Pak CYC: A 335 base deletion in the mRNA coding for a dibasic amino acid transporterlike protein (SLC3A1) isolated from a patient with cystinuria. Genomics (in press). 110. Jaeger P, Portman L, Saunders A, et al: Anticystinuric effects of glutamine and of dietary sodium restriction. N Engl J Med 315: 1120-1123, 1986. 111. Sakhaee K, Nicar M, Hill K, Pak CYC: Contrasting effects of potassium citrate and sodium citrate therapies on urinary chemistries and crystallization of stone-forming salt. Kidney Int 24: 348-352, 1983. 112. Perrett D: The metabolism and pharmacology of D-penicillamine in man. J Rheumatol $8:41-50, 1981. 113. Halperin EC, Thier SO, Rosenberg LE: The use of D-penicillamine in cystinuria: Efficacy and untoward reactions. Yale J Biol Med 54:439-446, 1981. 114. Pak CYC, Fuller C, Sakhaee K, et al: Management of cystine nephrolithiasis with alpha-mercaptopropionylglycine (Thiola). J Urol 136:1003-1008, 1986. 115. Griffith DP: Struvite stones. Kidney Int 13:372-382, 1978. 116. Williams JJ, Rodman JS, Peterson CM: A randomized doubleblind study of acetohydroxamic acid in struvite nephrolithiasis. N Engl J Med 311:760-764, 1984. 117. Pak CYC, Smith LH, Resnick MI, Weinerth JL: Dietary management of idiopathic calcium urolithiasis. J Urol 131:850-852, 1984. 118. Pak CYC, Sakhaee K, Crowther C, Brinkley L: Evidence justifying a high fluid intake in treatment of nephrolithiasis. Ann Intern Med 93:36-39, 1980.
2 H A P T E R 2(
Metabolic Bone Disease in Children FRANCIS H.
GLORIEUX
Genetics Unit, Shriners Hospital for Children and Departments of Surgery and Pediatrics, McGill University, Montr6al, Qurbec, H3G 1A6 Canada
GERARD KARSENTY
Molecular Genetics, The University of Texas, MD Anderson Cancer Center, Houston, Texas 77030
R A J E S H W. T H A K K E R
MRC Molecular Endocrinology Group, Royal Postgraduate Medical School, Hammersmith Hospital, London, W12 0NN United Kingdom
III. Rickets and Osteomalacia References
I. Introduction II. Skeletal Development
lular matrix molecules. Although only a few of these anomalies have been presently ascribed to a metabolic bone disease in the strict sense of the term, it was felt that an overview of current knowledge on the genetic control of skeletal development would be a useful introduction to the present chapter.
I. I N T R O D U C T I O N As in adults, metabolic bone diseases in children are, for the most part, the clinical expression of alterations or dysfunctions of the various factors that control mineral homeostasis. The ensuing defective mineralization translates into tickets at the level of the epiphyseal growth plates, and osteomalacia on the endocortical and cancellous bone surfaces. The various forms of tickets and osteomalacia as they occur in the growing individual will be discussed in this chapter. It is important to keep in mind that these conditions will develop on the background of rapid skeletal growth and development. The clinical expression of rickets and osteomalacia will often include stunting of the growth rate and severe bone deformities. In recent years, the unraveling of the factors that control and influence skeletal development has grown at an exponential rate. Several human bone diseases have now been ascribed to functional anomalies of a growing list of factors, hormones and their receptors, and extracelMETABOLIC BONE DISEASE
II. S K E L E T A L
DEVELOPMENT A. O v e r v i e w
The vertebrate skeleton derives from several distinct embryological structures. 1-3 Most of the craniofacial skeleton originates primarily from neural crest cells (ectoderm), which emigrate from the neural tube (midbrain and rhombomeres) into predetermined branchial arches 4'5 and then migrate to the developing head. Only some pieces of the skull are of somitic origin (basi- and exooccipital) or derive of both ectodermal and mesodermal participation (sphenoid complex and otic capsule). 3 The 759
Copyright 9 1998 by Academic Press. All fights of reproduction in any form reserved.
760
FRANCISH. GLO~EUX, GERARDKARSENTY,AND RAJESHV. THAKKER
rest of the skeleton results exclusively from mesodermal contribution. The ribs and vertebrae are derived from the sclerotome, a differentiated area of the mature somites; each trunk vertebrae arises from the development and fusion of the posterior and anterior halves of two consecutive somites. 6 Lastly, the appendicular skeleton is derived from lateral mesoderm. At the cellular level there is a common event that occurs in every future bone piece during development regardless of its embryonic origin. This event is the aggregation of mesenchymal cells into a condensate that represents the outline of each of the future skeletal elements. Thereafter these mesenchymal condensations will evolve differently depending on the type of ossification they will undergo, endochondral or intramembranous. 7 In most of the bones, including the axial and appendicular skeleton, ossification will occur through endochondral ossification where condensations form a cartilaginous template that is later replaced by bone. At their earliest stage, cells present in the mesenchymal condensation transiently express type I and type III collagens, noncartilaginous proteoglycans, and fibronectin. 8 Shortly after these mesenchymal condensations are identifiable, overt chondrogenesis begins marked by the synthesis and secretion of an extracellular matrix rich in types II, IX, and XI collagens and proteoglycans. 7'8 This process forms the "anlage" or model of the future skeleton. Chondrocytes in the center of these future bones become hypertrophic and synthesize type X collagen and alkaline phosphatase. The hypertrophic cartilage is degraded by invading blood vessels coming from the perichondfium, the hypertrophic chondrocytes die, and osteoblasts brought in by the blood vessels deposit bone matrix in the space previously occupied by the hypertrophic cartilage. 8 The ossification process spreads centrifugally, much of the cartilage anlage is converted by this process, and chondrocytes only remain at the end of bone in the articular cartilage and as a structure located between epiphysis and dyaphysis called the growth plate cartilage or epiphyseal cartilage. The growth plate contains different populations of chondrocytes, such as resting, proliferating, and hypertrophic chondrocytes, organized in columns. This structure is responsible for the longitudinal growth of bones; the cartilaginous matrix is primarily ossified and, when calcified, is resorbed by osteoclasts and replaced by a bone matrix elaborated by osteoblasts. 9 During childhood the cartilaginous protein matrix of the growth plate diminishes, the cellular columns become disorganized, and the whole structure is replaced by bone by the end of puberty through an, as yet, unknown mechanism. The second mechanism of ossification, termed "intramembranous ossification," occurs mostly in the skull and clavicle, and also in the diaphyses of the long bones
where it promotes progressive thickening directly under the periosteum. In that case, epithelial-mesenchymal interactions promote proliferation of a subset of mesenchymal cells to form the preosteogenic condensations. 7 In contrast to what is seen in endochondral ossification, these condensations undergo direct osteogenesis, without any cartilaginous stages. The mesenchymal cells differentiate directly into osteoblasts synthesizing increasing amounts of type I collagen and alkaline phosphatase, but no cartilage-specific collagens or proteoglycans. Three mouse mutants characterized by mesenchymal condensation defects have been described. 7'1~The recessive lethal congenital hydrocephalus (ch) mutation leads to extensive skeletal abnormalities such as missing, fused or misshaped bones. These defects are ascribed to alterations of the size of both prechondrogenic and preosteogenic condensations. The second mutation, phocomelia (Pa), leads to a disproportionate dwarfism with missing or reduced bones and presence of ectopic cartilage. In this case formation of condensations is delayed by 24 hours, the size of some being subsequently reduced. No gene has yet been identified as responsible for any of these two mutations. The third mutant, brachipodism (bp) shows a decreased length of long bones in the limbs and changes in the number of phalanges in toes. This mutation has been demonstrated to be due to thin, malformed digit condensations and failure of cleavage of these condensations at the location of future joints structures. 1~ Recently, this phenotype has been ascribed to mutations in the gene encoding Gdf5, one of the members of the bone morphogenetic family of secreted signaling molecules (see below). 11 Mutations in the Gdf5 gene in humans causes the Hunter-Thompson chondrodysplasia.12
B. T r a n s c r i p t i o n a l R e g u l a t i o n of Skeletal P a t t e r n i n g and Cell D i f f e r e n t i a t i o n Skeletal development is marked by two major events. One is the patterning of various skeletal elements, for which we know of a large set of genes involved. Interestingly, these genes do not appear to control the second event that is the commitment of mesenchymal cells to chondrogenic and osteogenic lineages, followed by the terminal differentiation of precursor cells into three specialized cell types: the chondrocyte in cartilage, and the osteoblast and osteoclast in bone. Fewer genes have been implicated in this latter process. Several families of transcription factors have been shown by genetic means to control skeletal patterning (Table 26-1). One of the most extensively studied groups is the homeobox (Hox) family of proteins (see Sharpe 13 for review). The homeobox genes formed a
CHAPTER 26
761
Metabolic Bone Disease in Children TABLE 2 6 - 1
Gene
Origin of Mutation
Homeobox genes Hoxa-1 Hoxa-2
KO KOa
Hoxa-3
KO
Hoxa-10 Hoxa- 11 Hoxa- 13
KO KO mmc
T r a n s c r i p t i o n F a c t o r s I n v o l v e d in S k e l e t a l D e v e l o p m e n t Human Condition or Mouse Mutation
Phenotype Otic capsule defect HTb of 2nd branchial arch derivatives in 1st branchial arch derivatives Misshaped and shorter maxilla, mandible, and hyoid bone Misshaped femurs and knee joints HT of T13-L1 Short fingers and toes Abnormal wrist bone
52, 53 14 54
Hypodactyly (Hd) mutant Hand-foot-genital syndrome
Hm d
References
55 56 57 58 59 60
Hoxb-4 Hoxd-3
KO KO
Hoxd-ll Hoxd-13
KO Hm KO
Msxl Msx2 MHox
KO Hm KO
Cdxl
KO
Partial HT of C1-C2 Fusion of the basoccipital with C1 Partial HT of C2-C1 HT S1 in L7, misshaped radius and ulna Branched and misshaped metacarpal and metatarsal bones $4 fused and transformed in $3 Delayed ossification of digits Extra digits and digit fusions Multiple craniofacial defects Craniosynostosis Altered craniofacial skeleton Bowing and shortening of radius + ulna and tibia + fibula Anterior HT of vertebrae
Paired-box genes Paxl Pax3
mm Hm
Absence of intervertebral disks Craniofacial abnormalities
Undulated (Un) mutant Waardenburg' s syndrome
20 21
Other classes Sox9
Hm
Bowed bones, hypoplastic scapulae, and axial skeleton abnormalities Osteopetrosis, failure in osteoclast differentiation (Poly) syndactyly
Campomelic dysplasia
23, 24
c-Fos Gli3 Krox 20 Bmi-1 Atf2 Twist Kr
KO Hm min KO KO KO Hm KO mm
Mild endochondral ossification defect Anteroposterior HT of axial skeleton Uniform dwarfism, endochondral ossification defect Craniosynostosis, brachydactyly, facial dysmorphism Craniosynostosis Abnormal hyoid bone
Synpolydactyly type II
61 14 15
Boston type craniosynostosis
17 18 62
63
Greig's syndrome Extra toes (Xt) mutant
Saethre-Chotzen syndrome Kreisler (kr) mutant
25, 26 64 65 68 66 67 69, 70 71 72
knock out by gene targeting. bHT, homeotic transformation. Cmm, spontaneous mutation in mouse. dHm, human disease. aKO,
f a m i l y of g e n e s h i g h l y c o n s e r v e d across species, like for i n s t a n c e b e t w e e n Caenorhabditis elegans and h u m a n s . M o r e than 39 H o x g e n e s h a v e a l r e a d y b e e n identified in the m o u s e . In v e r t e b r a t e s , the H o x g e n e s are a r r a n g e d in four g e n e clusters, and w i t h i n a g i v e n c l u s t e r the l o c a t i o n of i n d i v i d u a l g e n e s c o r r e s p o n d to their a n t e r o p o s t e r i o r e x p r e s s i o n pattern. H o x g e n e s h a v e b e e n i m p l i c a t e d in the p a t t e r n i n g o f the axial and a p p e n d i c u l a r skeleton. I n d e e d , i n a c t i v a t i o n or o v e r e x p r e s s i o n of H o x g e n e s resuits in d e l e t i o n or a d d i t i o n of skeletal e l e m e n t s , or in
the t r a n s f o r m a t i o n o f the s h a p e o f certain skeletal elem e n t s such as v e r t e b r a e into the s h a p e of a n o t h e r vertebra. This p r o c e s s is c a l l e d h o m e o t i c t r a n s f o r m a t i o n . F o r instance, i n a c t i v a t i o n of the H o x a - 2 g e n e results in the d e l e t i o n of the c r a n i o f a c i a l e l e m e n t s arising f r o m the s e c o n d b r a n c h i a l arch, w h e r e H o x a - 2 is n o r m a l l y exp r e s s e d . T h e s e e l e m e n t s are r e p l a c e d b y skeletal elem e n t s r e s e m b l i n g t h o s e d e r i v e d f r o m the first b r a n c h i a l arch. TM N o t all H o x m u t a t i o n s l e a d to a loss of function, for i n s t a n c e the e x p a n s i o n o f a p o l y a l a n i n e stretch lo-
762
FRANCIS H. GLORIEUX, GERARD KARSENTY, AND RAJESH V. THAKKER
cated upstream of the DNA binding domain of Hoxd-13 causes synpolydactyly in heterozygous individuals. 15 This is characterized by the insertion of an extra digit between digits III and IV in association with variable syndactyly. In homozygotes, the mutation causes altered growth of the metacarpal and metatarsal anlagen so that they resemble those of carpal and tarsal bones instead of long bones. The expansion of the NH2-terminal alanine stretch does not affect DNA binding but is thought to affect the ability of Hoxd-13 to interact with other proteins. 14 Another argument suggesting that this is not a loss-of-function mutation stems from the analysis of the phenotype of Hoxd-13-deficient mice that have very different and more severe abnormalities. 15 Another subclass of homeobox genes, termed Msx genes, which does not belong to one of the clusters mentioned above, have been shown to contribute to skeletal development (Table 26-1). For instance, Msx 1 null mice exhibit multiple craniofacial defects, ~7 and a mutation in the Msx2 gene is at the origin of an autosomal dominant form of craniosynostosis (Boston type). ~8 A second highly conserved family of transcription factors involved in skeletal patterning are the paired-box (Pax) genes (Table 26-1).19 In Drosophila, as well as in vertebrates, members of the Pax gene family have important roles in segmentation and neurogenesis. Their expression patterns during mouse embryogenesis suggest that Pax l and Pax9 have a role in the development of the vertebral column, whereas Pax3 is probably involved in the patterning of several crest derivatives. Indeed, mutations in the mouse Pax l cause the undulated (Un) phenotype characterized by vertebral development defects and kinky tails. Several Un-alleles are known, displaying a spectrum of severity in their phenotype. Homozygotes of the most severe phenotype completely lack vertebral bodies in the lumbar region, indicating that Pax l has an important role in the differentiation of cells originating from the sclerotome. 2~ Mutations in Pax3 are at the origin of the Waardenburg's syndrome, a dominantly inherited condition characterized by hearing loss, and pigmentation and craniofacial abnormalities. 2~ The genes mentioned above control patterning (i.e., the number and shape of skeletal elements). However, in the mutant animals or the affected individuals, the unaffected skeletal pieces exhibit the three specific cell types: chondrocytes in cartilage, and osteoblasts and osteoclasts in bone. This observation indicates that other transcription factors must control cell differentiation of these lineages. To this date, the study of the transcriptional control of cell differentiation in the skeleton is still at an early stage, with the most significant progress being made in chondrocyte and osteoclast biology. Sox9 is an Sryrelated gene thought to play a role during chondrocyte
differentiation. Sox9 is expressed in the sclerotomal compartment of the somites, in the mesenchymal condensation of the forming long bones and stops to be expressed upon completion of the skeletal anlage. 22 Human campomelic dysplasia, characterized by bowing of long bones, small scapulae, and vertebral abnormalities, has been linked to heterozygosity for a mutation in S o x 9 . 23'24 Another transcription factor, c-Fos, has been shown through genetic analysis in mice to be important for osteoclast differentiation. 25'26 c-Fos is expressed in macrophages and chondrocytes, and in its absence the osteoclasts fail to differentiate. As a result the homozygous mutant mice develop an osteopetrotic phenotype. Interestingly, these animals have no skeletal patterning defect providing genetic evidence that different transcription factors are regulating skeletal patterning and cell differentiation in the skeleton. At present, our knowledge of the transcriptional control of osteoblast differentiation is much less advanced.
C. Growth Factor Involvement in Skeletal Development With the development of the homologous recombination technology that allows generation, virtually at will, of mouse mutants, several families of growth factors have been demonstrated to be involved at various steps during skeletal development. For historical reasons the group of growth factors that initially received the most attention were the bone morphogenetic proteins (BMPs) (Table 26-2). This generic name regroups more than 15 identified members termed BMPs or GDFs, for growth and differentiation factors. 27 All of these molecules, except B MP1, belong to the TGF-I3 superfamily and can induce ectopic bone formation when implanted subcutaneously or in muscle. 28 The most remarkable aspect of this de novo bone formation is that it recapitulates all the events that occur in endochondral bone formation during development. 29 There is recruitment of mesenchymal cells, mesenchymal condensation, chondrocyte differentiation, and then vascular invasion bringing in osteoblast progenitors and differentiated osteoblasts. Consistent with the putative role of the BMPs during patterning of the skeleton stage, two mouse mutants, short ear (se) and brachypodism (bp) have been shown to be due to the disruption of Bmp5 29 and GdfS, 11 respectively. Short ear mice have a variety of changes in the size and shape of many small bones where Bmp5 is expressed. Mutations in other Bmps generate much more severe phenotypes, generally leading to early embryonic (Bmp2, Bmp4) or perinatal lethality (Bmp7), and in agreement with their broad pattern of expression 27 af-
763
CHAPTER 26 Metabolic Bone Disease in Children TABLE 2 6 - 2 Gene Bmp5 Bmp7 GDF5
Growth Factors, Hormones, and Their Receptors Involved in Skeletal Development
Origin of Mutation mma KOb HmC
Human Condition or Mouse Mutation
Phenotype Reduced or misshaped bones Extra digit in hindlimbs Shortening of long bones, metatarsal, and metacarpal bones
m m
FGFR1 FGFR2 FGFR3
Hm Hm Hm
Craniosynostosis Craniosynostosis Dwarfism
PTHrP
KO KO
PTH/PTHrP Receptor
Hm KO
Endochondral bone overgrowth Dwarfism Accelerated chondrocyte differentiation Short-limbed dwarfism Accelerated chondrocyte differentiation
Short-ear (Se) mutant Hunter-Thompson chondrodysplasia
References 30 31, 32 12
Brachypodism (bp) mutant Pfeiffer' s syndrome Crouzon' s syndrome Hypochondroplasia Anchondroplasia Thanatophoric dysplasia
11 73 74 33 35 36 37 38
Jansen' s chondrodysplasia
39 40
,,
amm, spontaneous mutation in mouse. bKO, knock out by gene targeting. CHm, human disease.
fecting various internal organs. This suggests that there may be a functional hierarchy in the Bmp family. Factors, like Bmp2 and Bmp4, are primarily required during early embryogenesis for some critical function independent of skeletal development, while others appear to function mainly during skeletal patterning. To date the only other Bmp for which genetic evidence has shown that it is implicated in patterning of some skeletal elements is Bmp7, the inactivation of the gene leading to preaxial polydactyly of the hindlimbs. 31'32 Another family of growth factors involved in skeletal development are the fibroblast growth factors (FGFs) and their receptors (FGFRs). They regulate multiple cellular functions including cell proliferation. To date three members of this family have been implicated in human diseases (Table 2 6 - 2 ) and one of them, FGFR3, has been the subject of the most intense investigation. Several mutations in this gene have been reported in humans, affecting various domains of the molecule and resuiting in different forms and severity of achondroplasia (Table 26-2). For instance, a mutation in the kinase intracellular domain leads to hypochondroplasia 33 whereas point mutations in the region encoding the transmembrane domain of this receptor have been shown to result in Crouzon' s syndrome 34 or in achondroplasia, 35 the most frequent cause of dwarfism. These mutations are gainof-function mutations, since transfection of a cDNA with the "achondroplasia mutation" into cells causes the re-
ceptor to become constitutively active, 36 and that inactivation of the FGFR3 in mice results in a different phenotype, showing long bone overgrowth and vertebrae with enlargement of the hypertrophic zone of the growth plates. 37 Another group of molecules whose function in skeletal development has been extensively studied by genetic approaches is the signaling complex parathyroid hormone-related peptide (PTHrP), PTH/PTHrP receptor. PTHrP-deficient mice display severe chondrodysplasia at birth with shortening of the growth plate cartilage due to an abnormal hypertrophic zone. 38 Similarly, a mutation in the PTH/PTHrP receptor was found in a patient with Jansen's metaphyseal chondrodysplasia, a rare form of short-limbed dwarfism. 39 This defect of chondrocyte differentiation could be explained by recent studies suggesting that PTHrP lies downstream of Indian hedgehog, a signaling molecule involved in the regulation of the rate of hypertrophic differentiation. 4~ In the postnatal period, the rapid growth and development of a normally formed skeleton may be compromised by mutations in key factors regulating mineral homeostasis, and thus disturbing the normal mineralization of cartilage and bone. Recent advances in molecular genetics have led to the cloning of several of the genes involved in the etiology of the major heritable metabolic bone diseases. They will be extensively discussed in this chapter.
764
FRANCIS H. GLORIEUX,GERARDKARSENTY,AND RAJESH V. THAKKER
D. Role of Matrix Molecules in the Formation of the Skeleton Because of their structural importance, the first extracellular molecules shown to be implicated in skeleton formation were the two major collagens, type I collagen in bone and type II collagen in cartilage. Mutations in the genes encoding these two molecules have been found to generate various deficiencies, among them osteogenesis imperfecta (type I collagen) and Stickler's syndrome (type II collagen) (see references 42 through 45 for review). Other genes encoding minor collagens like the type IX, X, and XI collagens have also been implicated in some human diseases (Table 2 6 - 3 ) . 44 For example, mutations in the genes encoding the oL1 and oL2 chains of type IX collagen were shown to generate degenerative joint disease 46 and multiple epiphyseal dysplasia (MED), 47 respectively. It is interesting to note that this same MED syndrome was also ascribed to a mutation in the cartilage oligomeric matrix protein (COMP) gene, that encodes a glycoprotein of the cartilage matrix. 48 This same gene was also found mutated in cases of pseudoachondroplasia. 49 Lastly, two members of the Gla family of extracellular proteins, osteocalcin and matrix gla protein (MGP), were shown by gene inactivation in mice to affect bone formation 5~ and bone growth, 51 respectively. In this latter case, null mice become dwarfed, longitudinal bone growth being rapidly arrested by ectopic calcification of
TABLE 2 6 - 3
the growth plate cartilage leading to complete disorganization of the chondrocyte columns.
III. RICKETS AND OSTEOMALACIA A. Definition In the strict sense of the term, rickets is caused by any interference with the process of endochondral bone formation, that is, the sequence of events that takes place in the epiphyseal growth plates and results in lengthening of the long bones. The other processes that involve rapid matrix mineralization during growth include remodeling of cancellous bone (to accommodate for the making of the marrow cavity) and apposition of periosteal bone (to increase bone width and cortical thickness). Defective mineralization of osteoid tissue throughout the skeleton results in excessive accumulation of osteoid tissue, or osteomalacia. Thus tickets occurs during growth in children, while osteomalacia occurs in both children and adults.
B. History The first treatise on rickets was published in 1650 by Francis Glisson. 79 This detailed description of the clinical features of rickets and discussion of the pathophysiology of the disease remains in many aspects actual.
Extracellular Matrix Molecules Involved in Skeletal Development
Gene
Origin of Mutation
COL1A1, COL1A2 COL2A1
Hma Hm
Brittle bone ___skeletal deformities Mild to lethal achondroplasia
COL9A1 COL9A2 COL10A1 COL11A1 COL 11A2 COMP
KOb Hm Hm mmc Hm Hm
Osteoarthritis Osteoarthritis Chondrodysplasia Short limb, hunchback Osteochondrodysplasia Short stature, osteoarthrosis
Osteocalcin (BGP) MGP
KO
Increased bone formation
KO
Dwarfism, ectopic calcification of growth plate cartilage
aHm, human disease. bKO, knock out by gene targeting. Cmm, spontaneous mutation in mouse.
Phenotype
Human Condition or Mouse Mutation Osteogenesis imperfecta Hypochondrogenesis Achondrogenesis type II Spondyloepiphyseal dysplasia Kniest' s dysplasia Stickler' s syndrome Multiple epiphyseal dysplasia Schmid's metaphyseal dysplasia Chondrodysplasia (cho) mutant Stickler' s syndrome Multiple epiphyseal dysplasia Pseudoachondroplasia
References 42, 43 44, 45
46 47 75, 76 77 78 48 49 50 51
CHAPTER 26 Metabolic Bone Disease in Children The possibility that rickets could be a heritable condition was already mentioned by Glisson, who also suggested treatment with large amounts of dairy products. In the mid-19th century, the Industrial Revolution in Europe and North America was paralleled by a steep increase in the incidence of rickets that was attributed to air pollution by smoke-emitting industrial plants. Although the effectiveness of cod-liver oil in the treatment of rickets had been reported, it was ignored to a large extent during the latter half of the 19th century. In the 1920s vitamin D was identified, and its deficiency was established as the major cause of rickets in children and osteomalacia in adults. 8~ The use of vitamin D for prevention and treatment largely eliminated vitamin D deficiency tickets. Shortly thereafter it was recognized that a small number of patients did not respond to the usual doses of vitamin D. They were classified as vitamin D-refractory or vitamin D-resistant rickets, 8~ and it was later demonstrated that they formed a heterogeneous group of disorders of various pathogenesis. On the background of rapid unraveling of the biochemistry and physiology of vitamin D, important new insights have been provided, in the last 25 years, into the pathogenesis of several types of vitamin D-refractory rickets.
C. Pathophysiology Bone formation and skeletal growth require the controlled production of a matrix (cartilage in the epiphyseal plate, and osteoid in metaphyseal areas and at sites of intramembranous bone formation), that will rapidly calcify when adequate concentrations of extracellular calcium and phosphate are available. The mineral phase will first be amorphous and then become crystalline in the form of hydroxyapatite with the basic formula Calo(PO4)6(OH)2. Failure to mineralize because of inadequate supply of mineral will result in excessive accumulation of unmineralized matrix. This will cause the characteristic swelling around the growth plates particularly evident at the wrists and ankles, and at the costochondral junction of the ribs where they form the classic rosary. The alteration of the natural sequence of endochondral bone formation will also slow down the growth in length of the various pieces of the axial and appendicular skeleton, resulting in an overall decrease of the linear growth rate that, in the extreme cases, may be completely stopped. Unmineralized bone is weakened and will bend or twist under the weight of the body and the pull of muscles. Fractures may occur, but more frequently skeletal deformities will appear: genu varum, genu valgum, coxa vara, tibial and femoral torsion, pelvic deformities, chest deformations, scoliosis, and kyphosis. In the infant, poor
765 mineralization of the skull may result in craniotabes. Thoracic deformities include pigeon breast deformity and lower rib cage flaring due to softening of the ribs and depression at the sites of insertion of the diaphragm (Harrison's groove). In an attempt to correct the hypocalcemia characteristic of the calcipenic forms of tickets, secondary hyperparathyroidism develops leading to increased bone resorption. This will lead to progressive decrease in bone mass and severe osteopenia, further increasing bone brittleness. By contrast, osteopenia never occurs in the phosphopenic forms of tickets as normocalcemia does not induce parathyroid reaction.
D. C l a s s i f i c a t i o n A number of classification schemes have been employed to categorize the various forms of rickets. As bone mineral is mostly made of calcium and phosphate, rickets and osteomalacia may arise from either primary calcipenia or primary phosphopenia (Table 26-4). The intent is not to discuss all of the reported forms but, rather, to concentrate on those that most frequently present clinically as well as discuss the most recent insights into the genetics and the molecular mechanisms underlying some of the heritable forms of rickets.
TABLE 26--4
Classification of Rickets
Calcipenic forms Dietary calcium deficiency Simple vitamin D deficiency Vitamin D deficiency secondary to Fat malabsorption Liver disease (?) Renal insufficiency Heritable forms Pseudo-vitamin D deficiency (PDDR) 25(OH)D- 1ot-hydroxylase defect Hypocalcemic vitamin D-resistant rickets (HVDRR) Alterations of the vitamin D receptor (VDR) Phosphopenic forms Insufficient intake Prematurity Total parenteral nutrition Use of phosphate binders Increased renal loss Fanconi syndrome by tubular damage Tumor induced rickets and osteomalacia (TIO) Heritable Increased renal loss Familial hypophosphatemia Sex-linked dominant (XLH) Sporadic form (new mutations) Autosomal dominant Hypercalciuric form (HHRH)
766
FRANCIS H. GLORIEUX, GERARD KARSENTY, AND RAJESH V. THAKKER
E. Radiographic Findings The most marked changes occur at the cartilagemetaphysis junction just under the growth plates. The space is widened by the accumulation of uncalcified cartilage which will cause fraying, cupping, widening, and fuzziness of the zone of provisional calcification. These changes are better and earlier detected in the most active growth plates: distal ulna and femur, and proximal and distal tibia. Changes in the diaphyses may not be evident when metaphyseal changes are first detected. They will appear later as coarse trabeculation and, in case of increased resorption, as cortical thinning and subperiosteal erosion. Another sign of hyperparathyroidism is the disappearance of the lamina dura that normally surrounds tooth sockets.
F. Biochemical Findings In calcipenic rickets, the initial reaction to decreased intestinal calcium absorption is hypocalcemia which may cause tetany or convulsions. In a second stage secondary hyperparathyroidism corrects the calcemia at the expense of the skeleton. Increased resorption will translate into progressive osteopenia and restore normal serum calcium levels. It is at this stage that the radiological changes become evident. The renal response to increased PTH includes increased reabsorption of calcium and reduced reabsorption of phosphate, leading to increased phosphaturia which, if sustained, results in hypophosphatemia, further compromising the mineralization process. In a third stage, with further bone demineralization, the calcemic response to PTH decreases and hypocalcemia reappears. In phosphopenic rickets, since serum calcium remains normal, there are no clinical signs of nerve irritability or secondary hyperparathyroidism. Hence the absence of osteopenia. The hallmark of this form of rickets (except in the rare cases where there is insufficient phosphate intake) is an increased renal loss that may have different mechanisms (see below). It is usually more severe in the acquired forms [i.e., tumor-induced osteomalacia (TIO)] than in the familial forms of hypophosphatemia. Serum alkaline phosphatase activity is uniformly elevated in all forms of rickets, and the levels reflect the severity of the bone disease. Normal values are up to 300 IU/liter in growing individuals. Although alkaline phosphatase is not specific to bone, the bone isozyme represents over 80% of total activity in growing individuals, who have no liver dysfunction. There is thus no need to assess the bone specific isozyme activity in children, the simple testing of total alkaline phosphatase ac-
tivity is adequate to evaluate the severity and monitor treatment of rickets. Serum 25-hydroxyvitamin D [25(OH)D] concentrations are depressed in vitamin D-deficiency rickets and are usually below 14 ng/ml (34 nmol/liter). In all other forms, concentrations are normal (14 to 45 ng/ml or 34 to 91 nmol/liter) or may be very much elevated in case of prior treatment with large amounts of vitamin D at time of referral of a vitamin D-refractory form. Serum concentrations of 1,25-dihydroxyvitamin D [1,25(OH)2D] vary widely. Normal values are 27 to 56 pg/ml (65 to 134 pmol/liter). They are elevated in cases of calcium deficiency, target organ resistance by alterations of the vitamin D receptor (HVDRR), and the hypercalciuric form of familial hypophosphatemia (HHRH). In simple vitamin D deficiency they may be either low, normal, or elevated. The reason for the normal or elevated levels is uncertain but may be related to a maximal up-regulation of l oL-hydroxylase, due to the deficiency, and the blood sample obtained after brief exposure to sunlight or the ingestion of a small amount of vitamin D. In pseudodeficiency (PDDR), serum 1,25(OH)2D levels are markedly decreased, sometimes undetectable. They do not increase significantly after administration of large amounts of vitamin D, reflecting the defect in loLhydroxylase activity. Interestingly, they are similarly decreased in TIO. The ability of TIO patients to maintain normocalcemia despite depressed levels of 1,25(OH)2D has not been explained.
G. Vitamin D Therapy Vitamin D2 or D3 is used in the treatment of vitamin D deficiency rickets. Initial dose may vary from 2000 to 5000 IU/day. Such low dosages decrease the probability of toxic effects and do not mask non-nutrient forms of rickets. Healing requires 6 to 12 weeks of therapy. The most sensitive biochemical index of healing is a return to normal of alkaline phosphatase activity. When achieved, vitamin D supplementation may be reduced to the recommended daily allowance of 400 IU. An adequate calcium intake is necessary to avoid severe hypocalcemia following the inception of vitamin D therapy and to ensure healing. Dietary intake or supplementation should provide about 50 mg/kg/day of elemental calcium. The use of vitamin D metabolites or their analogs has no place in the treatment of vitamin D deficiency. They should be restricted to the treatment of the various heritable forms of rickets (see below).
CHAPTER 26 Metabolic Bone Disease in Children H. H e r i t a b l e C a l c i p e n i c R i c k e t s Patients whose tickets had similarity to vitamin D resistant (hypophosphatemic) tickets in requiting large therapeutic doses of vitamin D and yet differed markedly in its clinical and biochemical features, were recognized as suffering from a separate disorder. 82 The clinical course of this disorder, despite an adequate intake of vitamin D, was similar to that of ordinary rickets due to vitamin D deficiency, and the disorder was therefore named pseudovitamin D-deficiency rickets. 83 It was noted that therapy with very large doses of vitamin D resulted in remission of the disease, and the disease was also called vitamin D-dependent rickets. 84 Clarification of the abnormalities in vitamin D metabolism 82 led to the recognition that this condition was likely due to a defect in the renal l oL-hydroxylase enzyme. It is characterized by low serum 1,25(OH)zD concentration and rapid and complete therapeutic response to calcitriol (i.e., 1,25(OH)zD3). Subsequently, another condition was recognized that develops despite high circulating concentrations of calcitriol. It is due to end-organ resistance to 1,25(OH)zD, consecutive to a spectrum of mutations affecting the vitamin D receptor (VDR) in target tissues. 1. PSEUDOVITAMIN D DEFICIENCY
Almost all the clinical and biochemical features of PDDR are similar to those of vitamin D-deficient rickets. Clinically, the child is well at birth and within the first year of life develops hypotonia, muscle weakness, inability to stand or walk, growth retardation, convulsions, frontal bossing, and the signs of severe rickets-rachitic rosary, thickened wrists and ankles, bowed legs, and fractures. A history of adequate intake of vitamin D is usually obtained. Laboratory investigations (Table 2 6 - 5 ) reveal hypocalcemia with secondary hyperparathyroidism and associated increased urinary cyclic adenosine monophosphate (cAMP) and amino acid excretion, elevated serum alkaline phosphatase activity, either hypo- or normophosphatemia, phosphaturia, a low urinary calcium excretion, and decreased intestinal absorption of calcium. The condition is inherited as an autosomal recessive trait 84-86 and the incidence of parenteral consanguinity is high. It is particularly frequent in French-Canadians from the Saguenay region of Quebec, where the estimated gene frequency is 0.02. 87 A similar syndrome in a mutant strain of domestic pigs has been described. 88'89 This porcine model is morphologically and biochemically similar to the human condition, and is also inherited as an autosomal recessive trait. The pathogenesis of this disorder was elucidated by studying vitamin D metabolism in affected patients. It
767 was observed that massive doses of vitamin D3 and high doses of 25(OH)D3 but only small doses of 1,25(OH)zD3 were required to correct the clinical and biochemical abnormalities in PDDR patients. 82 This provided indirect evidence that the condition was due to an inborn error of vitamin D metabolism in which there was a defect in the renal l a-hydroxylase enzyme, the enzyme which converts 25(OH)D3 to 1,25(OH)2D3. Studies of circulating vitamin D metabolites in patients further supported this hypothesis. The serum 25(OH)D3 concentration was normal in untreated patients and high in patients treated with vitamin D, whereas the serum concentration of 1,25(OH)zD3 was low in untreated patients 9~ and remained low in patients treated with vitamin D3 .91 Absence of l e~-hydroxylase activity was recently documented in decidual cells of human placenta, 92 which thus represent another target for the PDDR mutation. The recommended treatment for PDDR patients is replacement therapy with calcitriol. An initial dose of 1 to 3 Ixg/day will induce healing of rickets within 7 to 9 weeks. The maintenance dose is about half the initial dose 9~ and will probably have to be continued throughout life. It is remarkably stable, with only temporary increase during pregnancy (unpublished data). Molecular genetic studies utilizing linkage studies in affected French-Canadian families have mapped the PDDR gene to chromosome 12q 14. 93 In addition, linkage disequilibrium data using three closely linked polymorphic loci from this region were consistent with the presence of a founder effect in the French-Canadian population. In order to clone this gene, which is likely to encode the l oL-hydroxylase, a rat renal cDNA library constructed from kidneys of vitamin D-deficient rats was screened at reduced stringency, using a 3' rat 24hydroxylase cDNA that encompasses a common hemebinding domain of the molecule. 94 A full-length 2.4 Kb cDNA that encodes a predicted 55-kDa protein with 78% amino acid sequence similarity to the 24-hydroxylase within the heme-binding domain was identified. The protein sequence showed reduced similarity outside this region, and transient expression of the 2.4 Kb cDNA in embryonal carcinoma cells resulted in the production of a vitamin D metabolite that had the assay characteristics of 1,25(OH)eD. 94 Further mapping studies of the human homologue of this novel rat 2.4-Kb cDNA, that encodes the putative 1oL-hydroxylase, to chromosome 12q14 and the detection of mutations in PDDR patients will help to establish the candidacy of this clone as the l oL-hydroxylase gene.
2. HEREDITARY 1,25(OH)2D-RESIsTANT RICKETS HVDRR is an autosomal recessive disorder in which there is end-organ resistance to 1,25(OH)zD3. 95-97 The clinical features are similar to those found in PDDR
TABLE26-5
Biochemical and Genetic Details of the Major Forms of Familial Ricketsa Serum
TYpe Renal tubular defect Hypophosphatemic rickets (XLH) Lowe's syndrome Dent's disease Vitamin D metabolism defect Pseudo-vitamin Ddeficient rickets (PDDR) Hypocalcemic vitamin Dresistant rickets (HVDRR)
Ca2+
PO:-
Urineb
1,25(OH),D,
PTH
N
1
LlN
N
N/& N
1 1
1lN
NIT
1
1lN
1
1lN
PO:-
Ca2+
Aminoaciduria
Glucose
pH <5.5
1‘
L/N
-
-
Yes
t
1 ‘ ~ t
+
+
2
2
Yes Nolyes
-
N
'r/N
1
"?
?
J
tl‘
7
t
1
t
+ +
Chromosomal Location
Gene
Xp22.1
PEX
Xq25 -q26 Xp11.22
OCRL CLCN5
Yes
12q14
(1aOHase)
Yes
12q12-q14
VDR
"In these disorders, the serum 25(OH)D, concentrations are normal and serum alkaline phosphatase activity is usually elevated. bUrine pH t5.5 refers to that found during a severe metabolic acidosis or an ammonium chloride loading test. PEX, phosphate-regulating gene with homologies to endopeptidases on the X chromosome; OCRL, oculocerebrorenal syndrome of Lowe gene, encoding an inositol polyphosphate phosphatase; CLCN5, voltage-gated chloride channel 5 gene, also mutated in X-linked recessive nephrolithiasis (XRN) and X-linked recessive hypophosphatemic rickets (XLRH); laOHase, the putative (denoted by parenthesis) renal la-hydroxylase gene; VDR, vitamin D receptor gene; N, normal; 1,decreased; ?, increased; +, present; -, absent.
CHAPTER26 Metabolic Bone Disease in Children (Table 26-5), with two major exceptions: in HVDRR, the circulating concentrations of 1,25(OH)zD3 are markedly elevated, and can be as high as 1000 pg/ml (2400 pmol/liter). Moreover, alopecia totalis may occur in some patients. There is an early onset of the disease with variable severity in its clinical and biochemical manifestations, which suggests heterogeneity in the underlying defects. 97 The resistance to 1,25(OH)zD3 therapy is variable, and some patients have improved following therapy with very large doses of vitamin D 98 or 1,25(OH)zD3 .99-1~ In patients who are refractory to vitamin D therapy, alternative treatments with oral calcium supplements have been tried with limited success. However, long-term nocturnal intravenous calcium infusions have successfully healed tickets and promoted mineralization in HVDRR patients, 1~ though there are considerable practical difficulties with this therapy. The elevated serum concentrations of 1,25(OH)zD3 in patients with HVDRR suggested an abnormality in the mode of action of the metabolite within the target tissues. Its functions are mediated by an intracellular receptor that binds DNA and concentrates the hormone in the nucleus, 1~ in a fashion analogous to the classical steroid hormones. 1~ The interactions between 1,25(OH)zD3 and its intracellular receptor have been studied using cultured skin fibroblasts from control subjects and HVDRR patients. ~~176 Several defects have been identified and include an absence of receptors, a decreased number of receptors with normal affinity, a normal receptor-hormone binding but a subsequent failure to translocate the hormone to the nucleus, and a postreceptor defect in which normal receptors are present but there is a deficiency in the induction of the 25(OH)D24 hydroxylase enzyme in response to 1,25(OH)zD. Thus, the heterogeneity suggested from clinical observations is demonstrated at the molecular level with various combinations of defective receptor-hormone binding and expression. The 1,25(OH)zD3 receptor (VDR), which is closely related to the thyroid hormone receptors and represents another member of the trans-acting transcriptional factors including the steroid and thyroid hormone receptors, is an intracellular protein that has a molecular weight of 60,000 daltons. The receptor site for the 1,25(OH)zD3 is in the C-terminal part of the protein, while the N-terminal part of the molecule possesses a region that binds specifically to DNA. ~~ Zinc and other divalent cations are important in maintaining the DNAbinding function of the receptor, possibly by determining the conformation of the protein and giving rise to processes that can interdigitate between the helices of DNA. This hormone-receptor complex binds to a region of DNA that is on the 5' side of the gene (i.e., upstream of the promoter) and initiates an increased expression of the calcium-binding protein gene.
769 Genetic abnormalities of the human VDR gene 1~ have been identified in HVDRR patients. 1~ The human VDR gene consists of nine exons; exons 2 and 3 encode the DNA-binding domain, while exons 5 to 9 encode the vitamin D-binding domain. The gene has been mapped to chromosome 12q12-q14 in man 11~ in close vicinity to the locus for the gene causing renal l oL-hydroxylase deficiency (PDDR). Mutational analysis of the VDR gene in HVDRR patients has demonstrated the presence of nonsense and missense mutations (Table 2 6 - 6 ) affecting different parts of the receptor. Expression of these mutations in COS-1 monkey kidney cells has demonstrated that they result in a reduction or a loss of VDR function similar to the heterogeneous effects observed in cultured fibroblasts from patients. Recently, null mutant (i.e., "knockout") mice for VDR have been produced by targeted gene disruption. TM The VDR mutant mice were found to have growth retardation, skeletal deformities, and an earlier mortality, and adult mice developed alopecia. In addition, biochemical investigations revealed that the VDR mutant mice were hypocalcemic and hypophosphatemic, with markedly elevated serum 1,25(OH)zD3 concentrations. Thus, the VDR null mutant mice have all the features consistent with the HVDRR phenotype. This mouse model will help to further elucidate the physiological role of VDR in the vitamin D signaling pathway.
I. H e r i t a b l e P h o s p h o p e n i c R i c k e t s
1. X-LINKED HYPOPHOSPHATEMICRICKETS (XLH) a. Clinical Features. The most frequent form of familial rickets was recognized by Albright et al. 81 The authors coined the term "vitamin D-resistant rickets" as the patients they described presented with changes in mineral metabolism that could only be overcome by very large daily doses of vitamin D. The classic triad is hypophosphatemia, rickets and osteomalacia causing limb deformities, and growth retardation. Although low serum phosphate is evident early after birth, it is only at the time of weight-bearing that progressive leg deformities (more often in varum than in valgum) and departure from normal growth rate become obvious. It is striking that, despite these symptoms, children are not sick. They never present with tetany or convulsion. They are normally active and never experience myopathy. Tooth eruption is often delayed, but teeth show with a normal enamel, in contrast with the enamel hypoplasia evident in the calcipenic forms of rickets. On x-ray, active rickets is present, reflecting the mineralization defect. There are no signs of subperiosteal erosion or excessive bone resorption. Bone density [by dual en-
770
FRANCIS H. GLORIEUX, GERARD KARSENTY, AND RAJESH V. THAKKER
TABLE 2 6 - 6 Some Vitamin D Receptor (VDR) Mutations in Hypocalcemic Vitamin D-Resistant Rickets (HVDRR) VDDR Domain/ Region
Base Change
Codon Change
References
DNA binding GGC-->GAC CAC---~CAG AAA---)GAA GCC---~GAC TTC~ATC CGA-->CAA CGA---)CAA CGA-->TGA CGA--~CAA CGG---~CAG
Gly 33Asp His35Gln Lys45Glu Gly46Asp Phe47Ile Arg507Gln Arg730Gln Arg73 Stop Arg73Gln Arg80Glu
CAG---~TAG
Gln152Stop
m
109 211 212 213 212 214 109 215 109 216 217
Hinge 215
218 Vitamin D binding
TGT~TGG CGC---~CTC TAC-->TAA
Cys190Trp Leu274Arg Tyr295 Stop
CAC---~CAG
His305Gln
_
m
m
218 219 215
m
ergy x-ray absorptiometry (DXA)] is normal to high normal. On histological sections of trabecular bone, osteomalacia is characterized by excessive accumulation of unmineralized osteoid tissue and very little resorption activity. There is not osteopenia in sharp contrast with what is observed in calcipenic rickets (with secondary hyperparathyroidism). In keeping with a sex-linked dominant mode of inheritance, the disease manifestations are more severe in males than in females, some of whom may be asymptomatic, have no skeletal involvement, and exhibit only hypophosphatemia. ~3 They represent the "carriers" for the trait and provide evidence that hypophosphatemia cannot solely explain the bone changes. b. Biochemical Findings. The major abnormalities are hypophosphatemia and hyperphosphaturia resulting from a low threshold (TmP/GFR) for renal phosphate. Renal function is otherwise normal and glycosuria, aminoaciduria, and acidification defects are notably absent. A normal serum calcium and an absence of secondary hyperparathyroidism in untreated patients are also important diagnostic features (Table 26-5). Urinary calcium excretion and intestinal absorption of calcium and phosphate are low. 114 Serum alkaline phosphatase activity, which varies with age and correlates with the rate of bone formation, ~15 is elevated in affected individuals with tickets, but is normal in hypophosphatemic individuals without active bone disease. The circulating con-
centration of 25(OH)D3 is normal in hypophosphatemic patients ~6 and that of 1,25(OH)3D3 is either norma1117-12~ or low. 91 It has been argued that 1,25(OH)zD levels were inappropriately low in the face of chronic hypophosphatemia. Whatever the cause, this relative decrease in 1,25(OH)zD synthesis is not severe enough to significantly impair intestinal calcium absorption. Normocalcemia is the rule and there is no secondary hyperparathyroidism. c. Treatment. XLH had traditionally been treated with large doses of vitamin D. 8~ Infants received 10,000 to 25,000 IU/day of vitamin D, and older children up to 300,000 IU/day. ~21 This resulted in significant healing of the rickets and reduced the need for osteotomies of the tibia and femora to correct the bone deformities. ~5 However, hypophosphatemia persisted, growth rate was not markedly improved, and vitamin D intoxication with hypercalcemia and renal damage sometimes occurred.lZ2 To offset the phosphate loss, oral phosphate supplements (1 to 4 g/day) were administered at frequent intervals, four to five times a day, and were effective in producing normophosphatemia. 123 However, the hypocalcemic effect of phosphate therapy often induced secondary hyperparathyroidism ~23'124 and large doses of vitamin D were added to prevent it, increasing the risk of vitamin D intoxication and renal damage. A combination of 1,25(OH)zD3 (0.5 to 1.0 Ixg/day) plus phosphate seems to be the most effective treatment for this disease. 125-128
CHAPTER 26 Metabolic Bone Disease in Children This combined therapy resulted in improved phosphatemia and growth velocity. It also healed rickets and osteomalacia as assessed by a fall in serum alkaline phosphatase activity, and normalized radiographs and bone histology. However, complications of treatment do still occur, with nephrocalcinosis developing in some patients. 129 Phosphate-induced secondary hyperparathyroidism is a constant concern, as in some patients it may become autonomous and require surgery. 13~ Particularly in boys, the growth-promoting effects of therapy are not always striking. For that reason, the adjunct of recombinant growth hormone, as a third therapeutic component, was recently advocated. Definite positive effects have been observed in young XLH patients. TM Larger scale studies will be necessary to definitely establish the long-term benefits of this therapeutic regimen. d. Molecular Genetics. The first familial occurrence of vitamin D-resistant rickets was described in a mother, her son, and her daughter. 132 Skeletal deformities were used to ascertain the affected phenotype, and an autosomal dominant mode of inheritance was proposed. However, it was observed that the severity of skeletal deformities varied and that some hypophosphatemic females had no evidence of rickets. When hypophosphatemia was used as the discriminant, an X-linked dominant mode of inheritance was established. 112'113'133 The males were uniformly more severely affected than the females, who sometimes had no evidence of rickets. This variability in female patients, which is expected in an X-linked disease, can be explained by the Lyon hypothesis of X-chromosome inactivation, which states that one of the X chromosomes in a pair is randomly inactivated in each cell of the early female e m b r y o . TM The subsequent daughter cells have inactivation of the same X chromosome as their mother cell. Thus a female hypophosphatemic patient is a mixture of cells some of which have an active normal X chromosome and some of which have an active "hypophosphatemic" X chromosome. The relative proportions of each cell type vary from female to female, due to the randomness of the inactivation process, and this would in turn determine the variable expression of the X-linked hypophosphatemic rickets gene in females. Further support for this comes from phosphate infusion studies in hypophosphatemic patients and normals. These demonstrated that hypophosphatemic males (hemizygotes) had decreased maximum tubular capacity for reabsorbing phosphate (TmP), while hypophosphatemic females (heterozygotes) had a TmP that was intermediate between that of normals and hypophosphatemic males. 123 About one third of the patients present with a negative family history but cannot otherwise be differentiated from the XLH form. We have observed in two instances, such
771 "sporadic" female subjects to give birth to affected babies (unpublished). This implies that sporadic cases carry, in fact, new XLH mutations. It also indicates that the mutation rate for the trait is rather high. Family segregation studies (Fig. 2 6 - 1 ) using restriction fragment length polymorphisms (RFLPs) and microsatellite polymorphisms mapped the HYP gene to Xp22.1 and defined an approximate 1 cM (equivalent to 1 Mb) map around the disease lOCUS. 136-142 A contig (i.e., a series of overlapping clones) of the region was constructed using yeast artificial chromosomes (YACs) 143'144 and the size of the region containing the HYP gene was defined to be 350 Kb. Cosmids were isolated from this region and were used to identify possible microdeletions in XLH patients. Four such microdeletions ranging in size from 1 Kb to 55 Kb were identified, and the cosmids detecting these were used to search for the HYP gene by two approaches. 145 In one approach the cosmids were used to screen cDNA libraries of fetal brain, fetal liver and adult muscle, and in the other approach the DNA sequences of the cosmids were determined and examined for possible encoded regions. Both approaches indicated the presence of a gene in an --~100-Kb region, and a partial 1.9-Kb cDNA was isolated. Sequence analysis revealed that the gene encoded a protein that had similarities to the family of endopeptidase genes such as neutral endopeptidase, endothelin-coverting enzyme-l, and the Kell-antigen, and the gene was therefore called PEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). These endopeptidases generally consist of a short cytoplasmic N-terminal domain, a single transmembrane domain, and a large extracellular C-terminal domain that contains a zincbinding motif and ten conserved cysteine residues. Analysis of the PEX partial sequence confirmed the presence of the putative transmembrane domain and part of the large extracellular domain that included the zincbinding motif and seven of the ten conserved cysteine residues. Mutational analysis of the PEX gene in XLH patients has identified a number of different genetic abnormalities which include nonsense mutations resulting from frame shifts due to base pair deletions and insertions; missense mutations; splice site mutations; and genomic deletions. 145-148 These mutations (i.e., the nonsense mutations) indicate that a loss of function of PEX is involved in the etiology of this form of hypophosphatemic tickets. However, the mechanisms whereby PEX mutations lead to the XLH phenotype remain to be elucidated by first determining the tissues in which PEX is expressed and second by defining its physiological role in these organs. An investigation for PEX expression by Northern blot analysis of human adult heart, brain, placenta, lung, liver, skeletal, muscle, kidney, and pancreas,
772
F ~ q c I s H. GLORIEUX, GERARD KARSENTY, AND RAJESH g. THAKKER
FIGURE 26-- 1 Segregation of DXS 1683 and HYP in family 5/95. Genomic DNA from the family members (upper panel) was used with [,y32p] adenosine triphosphate (ATP) for polymerase chain reaction (PCR) amplification of the polymorphic repetitive elements at the DXS 1683 locus. The PCR amplification products were detected by autoradiography on a polyacrylamide gel (lower panel). PCR products were detected from the DNA of each individual and were designated alleles as shown on the right. For example, individual 1.2, the affected mother, reveals two pairs of bands on autoradiography. The upper pair of bands is designated allele 2 and the lower pair of bands is designated allele 3. The upper band in the pair is the "true" allele, and the lower band in the pair is its associated "shadow" which results from slipped-strand mispairing during PCR. The segregation of these alleles together with the disease (HYP) can be studied in the family members, and the patemal allele is shown on the left. Thus, at DXS 1683, the disease is segregating with allele 3 and the probability that these two loci are linked can be expressed as a "LOD score," which is log10 of the odds ratio favoring linkage and is defined as the likelihood that two loci are linked at a specified recombination 19 versus the likelihood that the loci are not linked. In family 5/95, the disease and DXS1683 are co-segregating without recombination, and the likelihood of linkage at 19 = 0 is therefore 1. If the disease and DXS 1683 were not linked, then the disease would be associated with allele 2 in one half (1/2) of the children and with allele 3 in the remaining half (1/2) of the children. This is not observed in the seven children of generation II, in which all the affected children have inherited allele 3 and all the unaffected children have inherited allele 2. The likelihood that the two loci are not linked is (1/2). The odd ratio in favor of linkage between the disease and DXS 1683 loci at 19 = 0, in family 5/95, is therefore 1/(1/2) 7 (i.e., 128), and the LOD score is log10 128 = 2.10. LOD scores from different families can be combined and a LOD score >+3, establishes linkage between two loci. DXS1683 was shown by physical mapping studies and by construction of a YAC contig to be within less than 300 Kb of H Y P . 144 Individuals are represented as unaffected males (t3), affected male (I), unaffected female (o) and affected female (e).
a n d o f fetal h e a r t , b r a i n , liver, lung, a n d k i d n e y h a s n o t
e.
Tumor-Induced
Osteomalacia.
T u m o r - i n d u c e d or
a 6.6-Kb
o n c o g e n i c r i c k e t s a n d o s t e o m a l a c i a is a r a r e a c q u i r e d
m R N A t r a n s c r i p t h a s b e e n i d e n t i f i e d in m o u s e b o n e a n d c u l t u r e d o s t e o b l a s t s . 149 T h e g e n e r a l f u n c t i o n o f t h e n e u -
phaturia, low circulating 1,25(OH)zD levels, and rickets
identified PEX
mRNA
transcripts,
although
disorder characterized by hypophosphatemia, hyperphos-
and
a n d o s t e o m a l a c i a . It o c c u r s in p r e v i o u s l y u n a f f e c t e d in-
t h e r e b y alter t h e a c t i v i t y o f t h e s u b s t r a t e , w h i c h m a y b e
d i v i d u a l s . 14~ It is d i s c u s s e d h e r e b e c a u s e t h e r e are simi-
a h o r m o n e s u c h as o x y t o c i n , s u b s t a n c e P, a n g i o t e n s i n I, o r a n g i o t e n s i n II. 15~ T h e p u t a t i v e s u b s t r a t e t h a t P E X
phatemia
c l e a v e s h a s y e t to b e i d e n t i f i e d , a n d it is p o s t u l a t e d t h a t
d i f f e r e n c e s . T I O p a t i e n t s m a y h a v e u n d e t e c t a b l y l o w se-
this m a y p o s s i b l y b e a p h o s p h a t e - r e g u l a t i n g h o r m o n e , r e f e r r e d to as " p h o s p h a t o n i n , ''14~ that m a y a l s o b e se-
r u m concentrations of 1,25(OH)zD3 despite h y p o p h o s p h a t e m i a . T h e m e c h a n i s m s b y w h i c h T I O p a t i e n t s are
tral
endopeptidases
is
creted by some tumors
to
cleave
peptide
bonds
associated with hypophospha-
temic osteomalacia (see below).
larities b e t w e e n this t u m o r - i n d u c e d f o r m o f h y p o p h o s and
XLH.
There
are,
however,
important
a b l e to m a i n t a i n n o r m o c a l c e m i a d e s p i t e an e v i d e n t def e c t in 1 , 2 5 ( O H ) z D s y n t h e s i s h a v e n o t b e e n e l u c i d a t e d .
CHAPTER 26 Metabolic Bone Disease in Children Unlike XLH patients, TIO patients often suffer from a severe proximal myopathy. Aminoaciduria, most frequently glycinuria, and glycosuria are occasionally present. Typical rachitic changes are present on radiographs of bone metaphyses. There is also osteopenia, pseudofractures, and coarse trabeculation. On histological sections, almost all surfaces are covered by thick osteoid seams with no evidence of increased resorption. The tumors associated with TIO are usually of mesenchymal origin and include hemangiomas, angiosarcomas, giant cell tumors of bone, sclerosing angiomas, hemangiopericytomas, and nonossifying sarcomas. These tumors are often small, difficult to locate, and may involve any organ. Tumors of epithelial o r i g i n m f o r example, breast and prostatic carcinomas--have also been observed to be associated with this disorder. A complete surgical removal of the tumor rapidly corrects all the abnormalities, and it has been postulated that the tumor may secrete a factor, referred to as phosphatonin, 14~which inhibits the renal tubular reabsorption of phosphate. Recent studies of a sclerosing hemangioma from a patient with oncogenous osteomalacia have revealed that the medium in which the tumor cells were cultured was able to inhibit sodium-dependent phosphate transport in opossum kidney epithelial cells. TM This alteration in phosphate transport was not associated with an increase in cellular concentration of cAMP. In addition, the medium was found to have PTH-like immunoreactivity but no PTHrP immunoreactivity, and its action was not blocked by a PTH antagonist. The role of this possible humoral factor, which may represent a substrate for PEX in the control of phosphate homeostasis, remains to be elucidated. These functional studies are likely to be greatly facilitated by the investigation of two mouse models for human XLH (see below). The evident treatment of TIO is a complete resection of the involved tumor. When the tumor is not clearly identified or cannot be fully removed, medical therapeutic intervention is warranted. As in XLH, it is based on the association of 1,25(OH)2D3 (1 to 3 Ixg/day) and phosphate salts (2 to 4 g/day). Little information is available regarding the long-term effects of such treatment. It is likely that the same secondary effects in treated XLH patients (nephrocalcinosis and hyperparathyroidism) may also develop in treated TIO subjects. Thus careful monitoring of urinary calcium, and parathyroid and renal function is mandatory.
f Hypophosphatemic Mouse Models: hyp and gyro. The investigation of the etiology of X-linked hypophosphatemic rickets in man has been facilitated by the investigation of two hypophosphatemic mouse models. The first of them was reported when mutant male mice were observed to have a shortened trunk and limb
773 deformities in association with hypophosphatemia. 152 This mutant was allocated the gene symbol hyp. The second hypophosphatemic mouse model was called gyro, as the mutant mice exhibited circling behavior. ~53 The hyp mice showed close similarities to the human XLH. The hyp mice were characterized by dwarfism, diminished body weight, and bone deformities associated with rickets. Biochemical determinations demonstrated hypophosphatemia together with a high renal phosphate loss, elevated serum alkaline phosphatase, a marginally lower plasma calcium, a normal serum PTH concentration, and an absence of glycosuria and aminoaciduria. Serum 25(OH)D3 concentrations were either normal or low and 1,25(OH)2D3 concentrations were within the normal range. 122'154 Intestinal transport of phosphate was markedly reduced and that of calcium was mildly reduced. ~55 Vitamin D supplements had no effect on intestinal phosphate transport but increased intestinal calcium transport. Bone histology confirmed the presence of rickets and osteomalacia. 17~ The gyro mice exhibit circling behavior in addition to hypophosphatemic rickets. The original mutant was a circling female found among the offspring of a female that had received irradiation when a fetus of 17 days' gestation. 156 The gyro mice are similar to the hyp mice in morphological features, serum phosphate values, renal excretion of phosphate, and impairment of sodiumdependent phosphate co-transport by renal brush border membrane vesicles. ~53 Bone histology revealed osteomalacia. 153'157 The traits that differentiate the gyro from the hyp mice are a circling behavior, hyperactivity, deafness, lack of postural reflexes, oligospermia, male sterility, and a higher incidence of sudden death. Histopathological examination of the inner ear of male gyro mice showed three abnormalities in the organ of Corti: the acoustic ganglion was deficient in cell bodies, hair cells were degenerate, and the tectorial membrane was detached. The vestibular sense organs were also abnormal, the sacculus and utriculus were atrophic, and the semicircular canals were stenotic. These inner ear abnormalities were not found in male hyp mice. 153 Genetic studies of hyp and gyro mice mapped both disorders to the distal part of the mouse X chromosome, 152-153 and the location of the human HYP locus was found to be in the evolutionary conserved synthenic region. 136 The location of the murine hyp was refined by interspecific backcross studies, ~58 and pex, the murine homologue of the human PEX gene has been cloned. 149 The complete cDNA sequence of the mouse pex has been isolated and encodes a 749-amino-acid sequence that has 95% identity to the partial known human PEX sequence, which has homologies to the neutral endopeptidase family. Interestingly, Northern blot analysis of normal mouse bone and cultured osteoblasts revealed a
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FRANCIS H. GLORIEUX, GERARD KARSENTY, AND RAJESH V. THAKKER
6.6 Kb mRNA indicating the presence of large untranslated regions of the pex mRNA transcript. However, pex mRNA transcripts were not similarly detected by Northern blot analysis from hyp mouse osteoblasts. Initially mutational analyses could not detect mutations in the hyp mouse pex coding region, suggesting that in hyp mice, the pex mutations might be within the regulatory or untranslated regions that then result in reduced pex gene expression. 149 Recently, deletions of the 3' and 5' pex regions have been reported in hyp and gyro mice, respectively. 159 These findings establish that Hyp and Gy are allelic mutations and that both provide homologous mouse models for X-linked hypophosphatemia. The latter should facilitate functional studies to further understand the pathophysiology of the human condition.
g. Studies of Phosphate Transport. In vivo and in vitro studies of phosphate transport in the normal and hyp mouse have shown that renal phosphate transport is complex and involves two (or more) gene products. Renal cross-transplantation studies in the hyp and normal mice indicate that the kidney may not be the major organ involved in pex expression and thereby directly in the etiology of hyp. 160"161 Kidneys transplanted from hyp mice to normal nephrectomized mice resulted in a normal conservation of phosphate, and kidneys transplanted from normal mice to nephrectomized hyp mice resulted in phosphate wasting. Thus, the disease was neither transferred nor corrected by renal cross-transplantation, thereby indicating that the hyp kidney is unlikely to have a direct etiological role in this disease. However, in vitro studies have demonstrated a renal tubular defect of phosphate transport in the hyp mouse. The site of the defect has been localized by micropuncture studies to the cells of the proximal convoluted t u b u l e . 162'163 Further definition of the site of the phosphate transport defect was provided by observing separately the fluxes of phosphate across the basolateral and luminal brush border membranes from hyp and normal m i c e . 164 These studies indicated that the abnormality of phosphate transport in the hyp mouse was the result of a defect in renal sodiumphosphate co-transport in the brush border membranes and that it was associated with a decrease in the sodiumphosphate co-transporter NPT2 mRNA and protein. ~65 However, the human NPT2 gene has been mapped to c h r o m o s o m e 5q35,166 and the mechanisms whereby a defect in the X-linked PEX gene result in reduced expression of the autosomal NPT2 gene remain to be defined. This defect in phosphate transport demonstrated by the in vitro studies of the hyp mouse appears to be confined to the kidney, as phosphate transport has been demonstrated to be normal in the jejunum 167 and the 0 steoblast. 168,169
h. Studies of Osteoblast Function. Several observations indicate that low serum phosphate levels cannot fully account for the bone abnormalities in XLH and that an osteoblast dysfunction contributes to the bone disease. Indeed, the severity of the bone lesions does not correlate with the degree of hypophosphatemia. 113 Furthermore, phosphate supplementation in affected patients and mutant mice heals the rickets but fails to correct osteomalacia. 125'17~ An intrinsic osteoblast defect was first proposed by F r o s t 171 in view of the hypomineralized periosteocytic lesions present in XLH bone. These lesions also seen in Hyp mice 172 are not observed in other forms of chronic hypophosphatemia and never completely disappear in treated patients despite correction of the osteomalacia. 173 This was further investigated in the mouse model. Transplantation studies of hyp osteoblasts indicate that osteoblasts are likely to be a major site of pex expression, consistent with the results from Northern blot analysis. 149 Transplantation of hyp osteoblasts into normal mice resulted in the production of abnormal bone that had increased volume and osteoid thickness. 174 These effects were intrinsic to the osteoblasts and independent of extrinsic factors such as hypophosphatemia, as similarly transplanted osteoblasts from phosphatedepleted normal mice produced normal bone. In vitro studies have also demonstrated hyp osteoblasts to have an abnormal response to 1,25(OH)zD3. Normal mouse osteoblasts respond to 1,25(OH)zD3 by increasing alkaline phosphatase activity and by decreasing cell proliferation, but these responses are absent in the hyp osteoblasts. 175 However, increasing the phosphate concentrations rendered the hyp osteoblasts responsive, thereby indicating that extracellular phosphate may modulate the hyp osteoblastic response to 1,25(OH)D3. 2. HEREDITARY HYPOPHOSPHATEMIC RICKETS WITH HYPERCALCIURIA
In 1962176 and 1964,177 two groups reported on a small number of children presenting with hypophosphatemic rickets and hypercalciuria. No explanations were given at the time for this uncommon association. In 1985, Tieder et al. 178 reported on nine cases, all part of a single large pedigree in which they have subsequently identified a number of asymptomatic individuals with "idiopathic hypercalciuria." 179 The HHRH clinical picture resembles that of XLH: lower limb deformities and stunted growth. Muscle weakness (not part of XLH) has been reported in some patients. On x-ray, active rickets and osteopenia are evident. The mode of inheritance is most probably autosomal recessive. B iochemically, there is normocalcemia, increased alkaline phosphatase activity, and hypophosphatemia due
CHAPTER 26 Metabolic Bone Disease in Children to increased renal phosphate loss. Hypercalciuria is most evident after a meal or an oral calcium load. It disappears after a 15-hour fast. The salient abnormality is an elevated serum concentration of 1,25(OH)zD, which is the probable cause of increased intestinal calcium absorption. It has been suggested that these high concentrations of 1,25(OH)zD were the expected response to hypophosphatemia, in contrast to the "inappropriately low" levels of the metabolite in XLH serum. This hypothesis remains untested. The genetic defect in HHRH is unknown. There has been no report of attempts at locating the HHRH locus on the genome, to establish possible linkage with genes involved in the control of phosphate transport. Treatment is based on supplementation with high doses of phosphate salts (1 to 3 g/day in four or five divided doses). Within a few weeks, muscle strength and bone pain improve substantially, and growth rate increases with the healing of rachitic lesions. Unlike XLH, there is no need for calcitriol as an adjunct to phosphate therapy, as basal circulating levels are very high. There is no report on long-term effects of the treatment. Noticeably, the common complications found in XLH (nephrocalcinosis and hyperparathyroidism) are not encountered in HHRH patients. 3. FANCONI SYNDROME
Fanconi syndrome is characterized by multiple defects of the proximal renal tubule. There is renal wastage of amino acids, glucose, bicarbonate (leading to metabolic acidosis), phosphate (leading to hypophosphatemia), sodium, uric acid, proteins, and potassium (leading to hypokalemia). Serum calcium is normal, and calciuria varies. The clinical manifestations included rickets, lower limb deformities, and impaired linear growth. Clearly, hypophosphatemia is the major factor underlying rickets and osteomalacia. It is possible, however, that acidosis plays a contributing role, since bone serves as a buffer for excess of hydrogen ion. The excess acid is buffered by calcium carbonate leading to dissolution of bone mineral and may cause hypercalciuria. A possible impairment of 1,25(OH)zD synthesis, due to tubular dysfunction and/or acidosis was also documented in animals, ~8~ but not in human studies. 181 Circulating levels of 1,25(OH)2D are normal in Fanconi syndrome, 182 a finding similar to what is observed in XLH patients. Rickets is treated, as in XLH, with a combination of phosphate and 1,25(OH)2D3. Dosage and monitoring follow the same guidelines as in XLH. Alkali therapy may be used to correct the metabolic acidosis and potassium supplements to control hypokalemia. Fanconi syndrome is often idiopathic, but may also be caused by toxic agents including mercury, lead, cad-
775 mium, streptozotocin, and outdated tetracycline. It may also be found in association with inborn errors of metabolism that affect proximal renal tubule function. These include glycogen storage disease, galactosemia, cystinosis, tyrosinemia, and hereditary fructose intolerance. In recent years, advances have been made in the molecular understanding of two such forms of Fanconi syndrome: the syndrome of Lowe and Dent's disease. They are discussed below.
a. The Oculocerebrorenal Syndrome of Lowe. The oculocerebrorenal (OCRL) syndrome of Lowe is an Xlinked recessive disorder that is characterized by congenital cataracts; mental retardation; muscular hypotonia; rickets; and defective proximal tubular reabsorption of bicarbonate, phosphate, and amino acids. 183 The disease is nearly always confined to males, who develop renal dysfunction in the first year of life, have delayed bone age and reduced height, and may die in childhood. Female carriers who have normal neurological and renal function can be identified in 80% of cases by micropunctate cortical lens opacities. 184'185Family studies have confirmed the X-linked recessive inheritance. ~86,187 The Lowe's syndrome gene was mapped to the distal part of the long arm of the X chromosome at X q 2 5 q26, by using RFLPs in family linkage studies 188 and subsequent analysis of a patient with Lowe's syndrome who had an X-chromosome break point helped to further localize the gene. 189 The use of YACs, which spanned the break point, to screen cDNA libraries identified a candidate gene (designated OCRL), which was absent or of an abnormal size in male patients with Lowe's syndrome. The predicted protein encoded by this gene revealed a 71% similarity to the gene on chromosome l p for human inositol polyphosphate-5-phosphatase, INPP5B. 19~ This enzyme, which was originally derived from platelets, catalyses the 5-phosphate from 1,4,5inositol triphosphate and from 1,3,4,5-inositol tetraphosphate, thereby presumably inactivating them as second messengers in the phosphatidyl-inositol signaling pathway. Point mutations and deletions were demonstrated in affected individuals, providing conclusive evidence that an altered OCRL gene is the cause of Lowe's syndrome. 191 Further functional expression studies 192 of the OCRL cDNA in baculovirus-infected Sf9 insect cells have revealed that the OCRL protein catalyzes three different reactions in the inositol phosphate pathway. These are the conversions of: inositol 1,4,5-triphosphate to inositol 1,4-biphosphate; of inositol 1,3,4,5-tetrasphosphate to inositol 1,3,4-triphosphate; and of phosphatidyl inositol 4,5-bisphosphate to phosphatidyl inositol 4phosphate, which represents a marked difference from that of the platelet-derived INPP5B. These studies indi-
776
FRANCIS H. GLORIEUX, GERARD KARSENTY, AND RAJESH V. THAKKER
cate that O C R L is mainly a lipid phosphatase that m a y control the cellular levels of the critical metabolite, phosphosphatidyl inositol 4,5-biphosphate, and that a deficiency of this e n z y m e results in the protean manifestations of L o w e ' s syndrome.
b. Dent's Disease. D e n t ' s disease is a renal tubular disorder characterized by a low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and eventual renal failure. D e n t ' s disease is also associated with the other multiple proximal tubular defects of the Fanconi syndrome. ~93 However, the c o m m o n occurrences of hypercalciuria, nephrocalcinosis, and kidney stones in D e n t ' s disease and the unusual or rare association of these in other forms of the Fanconi synd r o m e are important differences. 194 H y p o p h o s p h a t e m i c rickets m a y develop as a c o n s e q u e n c e of h y p o p h o s p h a temia and cause severe limb deformities and shortness of stature. Treatment will be based on phosphate sup-
plements, but the adjunct of 1,25(OH)2D3 is very difficult to monitor in view of the underlying hypercalciuria (unpublished observation). An X-linked inheritance for D e n t ' s disease was postulated on the basis of a greater disease severity in males and an absence of m a l e - t o - m a l e transmission in the families. 193'195 Family linkage studies using X-linked polymorphic genetic markers localized the gene to X p l 1.22.195 In addition, a microdeletion involving the D N A probe M2713, which defines the locus D X S 2 5 5 , was identified in one family with D e n t ' s disease (Fig. 2 6 - 2 ) . This microdeletion was further characterized using YACs from a contig, and deletion m a p p i n g studies revealed that the microdeletion was approximately 515 Kb in size. A search for renal expressed genes from this region using the YACs as hybridization probes to screen a renal c D N A library identified a c D N A that e n c o d e d a protein of 746 amino acids with h o m o l o g i e s to the voltage-gated chloride channel (CLC) gene family, and
FIGURE 26--2 Segregation of Dent's disease with a microdeletion detected by M2713 in family 12/89 (upper panel). Probe M2713, which defines the locus DXS255 and has been localized to Xpll.22, hybridizes to EcoR1 fragments in the range 3 to 7 Kb in normal individuals, and heterozygosity in females exceeds 90%. Hybridization of the Southern blot (lower panel) from family 12/89 with probe M2713 demonstrated an absence of signals in all the affected males (11.3, 11.7, 111.3, 111.7, and IV.2) and only one fragment indicating heterozygosity was detected in the affected females (11.2, 11.6, 11.9, 11.12, and 111.2). A control hybridization of this Southern blot with the probe L1.28, which defines the locus DXS7, yielded signals from all the lanes and demonstrated the presence of DNA in each lane. Thus, a microdeletion involving M2713 is associated with Dent's disease in family 12/89, and this maps Dent's disease to Xpll.22. (From Pook MA, Wrong O, Wooding C, et al: Dent's disease, a renal Fanconi syndrome with nephrocalcinosis and kidney stones, is associated with a microdeletion involving DXS255 and maps to Xpll.22. Hum Mol Genet 2:2129-2134, 1993.)
CHAPTER 26 Metabolic Bone Disease in Children this novel channel is now referred to as CLC-5 and the gene as CLCN5. The CLC channels, which are structurally unrelated to other ion channels and form the only known large family of C1- channels, consist of about 12 transmembrane domains. 196 The first member, designated CLC-0, was cloned in 1990 from the electric organ of Torpedo marmorata and nine different CLCs (CLC-1 to CLC-7, and CLC-Ka and CLC-Kb) encoded, respectively, by genes CLCN1 to CLCN7, and CLCNKa and CLCNKb, have been identified in mammals. 196-198 These chloride channels are important for the control of membrane excitability, transepithelial transport, and possibly regulation of cell volume. 196 CLC channels are known to function as multimeric complexes, and recent studies have revealed that CLC-0 is a homodimer with two largely independent pores. 199-2~176 Some CLC genes are ubiquitously expressed (e.g., CLCN2), whereas others are tissue specific (e.g., CLCN1 is expressed in skeletal muscle and CLCN5 is expressed in kidney). To date, only CLCN1 and CLCN5 are known to have diseaseassociated mutations with the myotonia disorders of Thomsen and Becker, 2~176 and with the hereditary nephrolithiasis disorders (e.g., Dent's disease), respectively. T M The human CLCN5 gene consists of 12 exons that span 25 to 30 Kb of genomic DNA, 2~ and Northern blot hybridization analysis has identified a 9.5 Kb m R N A transcript that is predominantly expressed in the kidney and to a lesser extent in placenta and skeletal muscle. 196 The CLCN5 coding region, which consists of 2238 bp, is organized into 11 exons (exons 2 to 12) with 10 introns. Exon 2 and part of exon 3 encode the 57 amino acids of the N-terminal cytoplasmic domain; the 3' end of exon 3 and exons 4 to part of exon 10 encode the 491 amino acids of the transmembrane domains and loops; and the 3' end of exon 10, exon 11 and part of exon 12 encode the 198 amino acids of the C-terminal cytoplasmic domain. CLCN5 is highly conserved in primates, marsupials, rodents, reptiles, and birds. Heterologous expression of wild-type CLCN5 in Xenopus oocytes has revealed that the channel, CLC-5, conducts chloride currents that are outwardly rectifying and essentially time-independent. T M In addition, ion substitution experiments showed that there was a chloride---~iodide conductance sequence, which is consistent with that reported for the other chloride channels, CLC0, CLC-1, and CLC-2, of this family. Dent's disease is associated with different CLCN5 genetic abnormalities, and heterologous expression of such CLC-5 mutants in Xenopus oocytes has demonstrated an abolishment or a marked reduction in the chloride channels, thereby demonstrating their functional importance. T M Two other X-linked renal proximal tubular disorders associated with hypercalciuric nephrolithiasis and
777 similarities to Dent's disease have been reported. One of these disorders was reported in a North American family and referred to as X-linked recessive nephrolithiasis (XRN) 2~ and the other was reported in an Italian family and referred to as X-linked recessive hypophosphataemic rickets (XLRH). 2~ Family linkage studies of the XRN and XLRH kindreds localized both disease loci to X p l 1.22, 207,208 and genetic analysis of CLCN5 revealed mutations that resulted in a function loss of CLC-5. T M These results indicate that CLCN5 is a chloride channel whose functional loss results in a generalized proximal renal tubular defect (i.e., a Fanconi syndrome), which is associated with the hypercalciuria and nephrolithiasis of Dent's disease, XRN, and XLRH. However, the mechanisms whereby a loss of this renal chloride channel leads to hypercalciuria and the proximal tubular defects remains to be elucidated. The reabsorption of filtered protein occurs in the proximal tubule, whereas that of calcium occurs in the proximal tubule, thick ascending limb of Henle, and distal tubule; one possibility is that a loss of CLC-5 function in the proximal tubule may lead to a decrease in chloride reabsorption which in turn results in decreased calcium reabsorption. 2~ However, this does not explain the abnormal excretion of low-molecular-weight proteins, which are specifically absorbed in the proximal tubule by endocytosis and transported in an acidic vacuolar-lysomal system. 21~ A loss of chloride channel function in this system would prevent the dissipation of the charge that is generated by the electrogenic H+-ATPase pump for the provision of the acidic environment. However, these possibilities need to be explored, and the identification of the specific segments of the nephron that express CLCN5 will represent an important step in this pathway towards understanding further the role and function of CLC-5 in the etiology of hypercalciuria and renal stones.
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Index
A
Alkalosis, hypophosphatemia role, 222 Allopurinol, hyperuricosuric calcium nephrolithiasis treatment, 748 Aluminum hypercalcemia association, 429 hypophosphatemia role, 222 phosphate absorption effects, 190 renal osteodystrophy treatment, 460 Amylin, calcitonin biosynthesis, 101 Angioid streaks, Paget's disease association, 580-581 Ankylosing spondylitis Paget's disease association, 559-560 rheumatoid arthritis association, 626-628 Antibiotics, struvite stone treatment, 755 Anticonvulsant bone disease, enzyme induction, 358359 Antigens, Paget's disease diagnosis, 547, 560 Apatite, s e e Bone apatite Arthritis characteristics, 613 Paget's disease association, 579 rheumatoid arthritis, s e e Rheumatoid arthritis Autoimmune polyendocrinopathy- candidiasis- ectodermal dystrophy, characteristics, 507 Autosomal dominant hypocalcemia, 494-496 biochemical features, 494-495 chromosome 3 linkage, 495 clinical features, 494-495 diagnosis, 495-496 extracellular calcium ion-sensing receptor mutations, 495 therapy, 495-496 Axial osteomalacia clinical presentation, 722-723 etiology, 725 histopathological findings, 723-724
Absorptiometry, s e e s p e c i f i c t y p e s Acetohydroxamic acid, struvite stone treatment, 755 Acidosis hyperphosphatemia, 229 osteomalacia association, 366-367 phosphate absorption effects, 187 phosphate excretion effects, 213 Activating transcription factor 2, skeletal development role, 5 Adrenal hormones, phosphate excretion effects, 213 Age bone changes, computed tomography assessment, 288 calcium absorption effects, 171, 179-180 rheumatoid arthritis, 625 Albright's hereditary osteodystrophy, s e e a l s o Pseudohypoparathyroidism characteristics, 510, 515- 516 McCune-Albright syndrome, 220 Alcoholism hypophosphatemia role, 222 intestinal calcium transport effects, 181 - 182 Alendronate malignancy disease hypercalcemia treatment, 644645 osteoporosis management, 402 Paget's disease treatment, 589 Alfacalcidiol, osteoporosis management, 404-405 Alkaline phosphatase bone turnover marker, 316 characteristics, 7 hyperparathyroidism diagnosis, 467 mineralization defects, 369-370 Paget's disease association, 576-577, 591 785
786
Index
Axial osteomalacia (Continued) laboratory studies, 723 overview, 722 pathogenesis, 725 radiological features, 723 treatment, 725-726 Azotemia, hyperphosphatemia role, 228
B Benzothiadiazides, hypercalcemia diagnosis, 428-429 Bicarbonate bone apatite abnormalities, 41-45 hyperparathyroidism diagnosis, 467 Biglycan, characteristics, 7 Bile salts, calcium absorption role, 170-171, 177 Biochemical markers bone turnover assessment applications bone loss rate, 322-323 exogenous subclinical hyperthyroidism, 538 intervention assessment, 323-324 perimenopausal therapy, 322 biochemistry, 315-316 bone resorption markers calcium, 318-319 CrossLaps, 320-321 deoxypyridinoline, 319-320 free D-Pyr, 320 free Pyr, 320 hydroxylysine, 319 hydroxyproline, 318- 319 Osteomark N-terminal cross-links, 320 plasma tartrate-resistant acid phosphatase, 319 pyridinoline, 319-320 type I collagen carboxy-terminal pyridinoline cross-linked telopeptide, 321 type I collagen degradation products, 320-321 24-hour turnover marker variation, 321 marker formation alkaline phosphatases, 316 free bone gla protein, 318 procollagen peptides, 318 modeling, 314- 315 overview, 313- 314 remodeling, 314-315, 392-393 osteoporosis diagnosis, 392-393 Biopsies applications bone remodeling, 344-345 hyperparathyroidism, 260-261 low-turnover bone disease, 263-264 osteomalacia, 259-260, 344-345
osteoporosis, 256-259 pediatric diseases, 267 predominant hyperparathyroid bone disease, 262263 renal osteodystrophy, 261-266 renal stone formation, 266-267 constraints, 245 evaluation methods histomorphometric parameters, 253- 256 normal values, 256 qualitative evaluation, 251-252 quality control, 256 quantitative evaluation, 252-253 histology techniques applications, 267-269 dehydration, 247 embedding, 247 fixation, 247 immunohistochemistry, 251 sectioning, 247-248 in situ hybridization, 249-251,268-269 staining, 248-249 histomorphometric bone parameters balance, 255 bone formation, 254-255 bone structure, 253- 254 mineralization, 254- 255 molecular parameters, 255-256 remodeling, 255 resorption, 255 tumover, 255 pitfalls, 246-247 prerequisites, 244-245 techniques, 245-246 Bisphosphonates, see also specific types hypercalcemia management, 433 hyperphosphatemia role, 228 malignancy disease hypercalcemia treatment, 644645 osteoporosis management, 400-403, 626 Paget's disease treatment, 585-590 Blood clotting, osteogenesis imperfecta association, 663 hypophosphatemia manifestation, 224 Bone biopsies applications hyperparathyroidism, 260-261 low-turnover bone disease, 263-264 osteomalacia, 259-260, 344-345 osteoporosis, 256-259 pediatric diseases, 267 predominant hyperparathyroid bone disease, 262-263
Index Bone ( C o n t i n u e d ) renal osteodystrophy, 261-266 renal stone formation, 266-267 constraints, 245 pitfalls, 246-247 prerequisites, 244-245 techniques, 245-246 cell types, see specific types density measurement densitometry acronyms, 276 disease course evaluation, 303 ultrasound, 292, 392 development bone morphogenetic proteins, 3-4, 14, 762-763 cell-cell interaction, 12 cell lineage, 5-12 differentiation, 7-10 function, 10-12 origin, 9-10 phenotypes, 5-7 embryology, see Embryology fibroblast growth factor role, 3-5 histomorphometric parameters, 254- 255 limb development, 2-3 osteoblast cells, see Osteoblasts osteoclast cells, see Osteoclasts parathyroid hormone effects, 67 parathyroid hormone/parathyroid hormone-related peptide receptor effects, 77-78 parathyroid hormone-related peptide effects, 7273 pediatric disease role, 759-764 cell differentiation regulation, 760-762 growth factor involvement, 762-763 matrix molecules role, 764 transcriptional skeletal patterning regulation, 760-762 disease, see specific types evaluation methods, see also specific types histomorphometric parameters, 253- 256 noninvasive assessment techniques Compton scattering technique, 280-281 conventional radiographic bone images, 277 Cushing's syndrome, 303 densitometry acronyms, 276 disease course evaluation, 303 fracture locations, 276 historical perspectives, 276-277 magnetic resonance microscopy, 297-298 measurement sites, 276, 301 - 302 method comparison, 302 neutron activation analysis, 280-281 osteoporosis classification, 275, 301,303 perimenopausal evaluation, 303
787 quantitative computed tomography, 286-292 quantitative magnetic resonance, 296-297 quantitative ultrasound, 292-296 radiogrammetry, 279 radiographic absorptiometry, 279-280 radiographic morphometry, 277-279 relevance, 302 results interpretation, 301 single photon absorptiometry, 281-286 X-ray absorptiometry, 281-282, 298- 300, 391-392 normal values, 256 qualitative evaluation, 251-252 quality control, 256 quantitative evaluation, 252-253 fractures, see Fractures histology applications, 267-269 dehydration, 247 embedding, 247 fixation, 247 immunohistochemistry, 251 renal osteodystrophy adynamic bone, 452 divalent-ion metabolism alterations, 453-455 lesions, 452-453 osteomalacia, 452 secondary hyperparathyroidism, 451 sectioning, 247-248 in situ hybridization, 249-251,268-269 staining, 248-249 mineralization, see also Bone turnover biological function, 23-28 bone apatite phosphate abnormalities, 42-45 calcium-phosphorous deposition phase, 29-32 characteristics bone structure, 2 - 3, 28- 29, 314 crystal morphology, 32-36 histomorphometric parameters, 254-255 kinetic definition, 335-338 mineralization mechanisms, 328- 329 osteoid seam life history, 330-331 temporal changes, 28-29 defects bone apatite structure role, 36-41 calcitriol role, 353- 354 carbonate ion role, 41-45 hypovitaminosis D osteopathy histological evolution, 335-338 hypovitaminosis D osteopathy treatment, 371372 impaired mineralization, 334-335 magnesium ion role, 45-46 osteogenesis imperfecta, 665
788 Bone
Index (Continued)
Paget's disease, 569-571 phosphate ion role, 42-45 renal osteodystrophy, 453-455 vitamin D effects, 352-353 fluorescent label width, 332-334 octocalcium-phosphate deposition phase, 30-32 tetracycline-based kinetics, 334 tetracycline data interpretation, 329 modeling, s e e Bone turnover remodeling, s e e Bone turnover resorption calcitonin effects, 102-104, 113-114 1,25-dihydroxyvitamin D effects, 145 escape phenomenon, 112-113 histomorphometric parameters, 255 osteoclast cell role, s e e Osteoclasts skeletal structure balance, 242 bone marrow cells, 243-244 bone surfaces, 238 cancellous bone, 238 cortical bone, 238 histomorphometric parameters, 253- 254 inorganic minerals, 314 lamellar bone, 239-240 matrix, 314 modeling, 240-242 Paget's disease manifestations, 554-562 extremities, 560-562 jaw, 557 pelvis, 560-562 skull, 554-557 spine, 558-560 structural unit, 238-239 turnover, 242 woven bone, 239-240 Bone apatite abnormalities, 36-46 carbonate ions, 41-45 magnesium ions, 45-46 overview, 36-41 phosphate ions, 42-45 amorphous calcium-phosphorous deposition theory, 29-32 osteogenesis imperfecta pathophysiology, 665 temporal changes, 29 Bone density exogenous subclinical hyperthyroidism, 535-538 noninvasive assessment densitometry acronyms, 276 disease course evaluation, 303 ultrasound, 292, 392 overt hyperthyroidism, 534
Bone development, s e e Bone, development; Embryology Bone marrow cells characteristics, 243-244 transplantation calcium II deficiency syndrome treatment, 705 osteopetrosis treatment, 701-702 Bone matrix, s e e a l s o Collagen cell-matrix interactions, 16 characteristics, 314 metabolism, Paget's disease association, 571-575 collagenolysis, 572 hydroxylysine excretion, 572-573 hydroxyproline excretion, 572-573 noncollagenous bone protein metabolism, 574575 nondialyzable urinary hydroxyproline, 573-574 pagetic bone matrix, 571-572 serum procollagen peptides, 574 mineralization defects, 368-369 osteoid seam life history, 330-331 phosphate transport, 215 skeletal development role, 764 Bone morphogenetic proteins embryologic function, 3-4, 14 skeletal development role, 762-763 Bone sialoprotein, characteristics, 6 Bone turnover, s e e a l s o Bone, mineralization biochemical markers applications, 321-324 bone loss rate, 322-323 intervention assessment, 323-324 perimenopausal therapy, 322 biochemistry, 315- 316 bone resorption markers calcium, 318- 319 CrossLaps, 320-321 deoxypyridinoline, 319-320 free D-Pyr, 320 free Pyr, 320 hydroxylysine, 319 hydroxyproline, 318-319 Osteomark N-terminal cross-links, 320 plasma tartrate-resistant acid phosphatase, 319 pyridinoline, 319-320 type I collagen carboxy-terminal pyridinoline cross-linked telopeptide, 321 type I collagen degradation products, 320-321 exogenous subclinical hyperthyroidism, 538 24-hour turnover marker variation, 321 marker formation alkaline phosphatases, 316 free bone gla protein, 318
Index
789
Bone turnover ( C o n t i n u e d ) osteocalcin, 316- 318 procollagen peptides, 318 modeling, 314- 315 osteoporosis diagnosis, 392-393 overview, 313- 314 remodeling, 314-315, 392-393 characteristics, 242 histomorphometric parameters, 255 hyperthyroidism diagnosis, 428 low-turnover bone disease, 263-264 modeling overview, 314- 315 skeletal structure, 240-242 osteoclast cell role, see Osteoclasts remodeling bone mineralization, 328-329 cell-cell interaction, 12 function, 240-242 histomorphometric parameters, 255 hypovitaminosis D osteopathy treatment, 372 impaired mineralization, 367-369 minerals, see specific types noninvasive indices, 344-345 osteogenesis imperfecta pathophysiology, 665666 osteoid seam life history, 330-331 overview, 314- 315 Paget' s disease histopathology, 548- 553 phosphate transport role, 215-216 Brodie's abscess, Paget's disease histopathology, 553 Bums, hypophosphatemia association, 223
C Caffeine, intestinal calcium transport effects, 182 Calcidiol a-ring metabolism, 140-141 assay, 149-150 clinical disorders, 132-135 extrarenal regulation, 138-139 hepatic metabolism, 132 hepatobiliary disease association, 357-358 metabolic regulation, 136-138 osteoporosis management, 404-405 Paget's disease role, 570-571 renal bone disease association, 359-360 side-chain metabolism, 140-141 synthesis, 66 Calciferol, see Vitamin D Calcific periarthritis, Paget's disease association, 579 Calcinosis, see Chondrocalcinosis; Tumoral calcinosis Calcipotriol, intoxication, 615
Calcitonin actions, 102-108 bone formation, 104-105 bone resorption, 102-104, 113-114 calcium homeostasis, 105-106 gastrointestinal tract function, 107 neural function, 107-108 renal effects, 106-107 biosynthesis, 99-101 calcitonin gene mRNA transcript splicing, 100101 calcitonin gene-related peptide, 100-101 precursors, 99-100 calcitonin receptor, 108-113 cloning, 108-109 escape phenomenon, 112-113 isoforms, 109-111 regulation, 112-113 signal transduction, 111-112 characteristics, 96-98 calcitonin-producing cell origin, 96-97 chemistry, 98-99 vertebrate and nonvertebrates compared, 97-98 fibrogenesis imperfecta ossium treatment, 728 historical perspectives, 95-96 hypercalcemia management, 433, 643 malignancy disease hypercalcemia treatment, 643 metabolism, 101 - 102 osteogenesis imperfecta treatment, 678-679 osteoporosis management, 399-400, 626 Paget's disease association acute effects, 575-576 treatment, 581-585, 591 phosphate absorption effects, 185 secretion, 101 - 102 Calcitonin gene-related peptide calcitonin biosynthesis, 100-101 central nervous system distribution, 108 Calcitonin receptor cloning, 108-109 escape phenomenon, 112-113 isoforms, 109-111 regulation, 112-113 signal transduction, 111-112 Calcitriol antiproliferation effects, 146-147 a-ring metabolism, 140-141 assay, 151 biological actions, 142-144, 155-156 calcitonin secretion regulation, 101 - 102 calcium balance regulation, 144-145, 168-170, 173-174 differentiation effects, 146-147 endocrine effects, 147-148
790 Calcitriol ( C o n t i n u e d ) extrarenal 25-hydroxyvitamin D metabolism regulation, 138-139 25-hydroxyvitamin D metabolism regulation, 136138 hypercalcemic disorders calcipotriol intoxication, 615 Hodgkin' s disease, 613-614 leioblastoma, 614 leukemia, 613-614 lung carcinoma, 614 lymphomas, 613-614 rheumatoid arthritis, 613 sarcoidosis, 608-611 seminoma, 614 subcutaneous fat necrosis, 615 tuberculosis, 612-613 vitamin D intoxication, 615 William' s syndrome, 614-615 hyperphosphatemia manifestation, 229 hypoparathyroidism treatment, 522 hypophosphatemic osteomalacia treatment, 373-374 immunoregulatory effects, 147 intoxication, 615 luminal brush border phosphate transport, 208, 214 osteomalacia association deficiency effects, 350 treatment, 373-374 vitamin D regulation, 353-354 osteopetrosis treatment, 702 osteoporosis management, 404-405 Paget's disease role, 570-571 parathyroid hormone secretion regulation, 61 phosphate excretion regulation, 213- 214 pseudohypoparathyroidism pathophysiology, 511512, 520-521 renal 25-hydroxyvitamin D- lo~-hydroxylase, 135 renal osteodystrophy role, 444-445, 459 side-chain metabolism, 140-141 skin cell effects, 148 synthesis, 66 x-linked hypophosphatemic tickets role, 218-220 Calcium absorption pathophysiology, 165-183 decreased absorptive states, 176-183 bioavailability, 176-177 extrinsic inhibition, 182-183 intrinsic intestinal transport defects, 179-182 vitamin D deficiency, 177-179 increased absorptive states, 172-176 bioavailability, 173 vitamin D-dependent hyperabsorption, 173-175 vitamin D-independent hyperabsorption, 175176
Index physiology age function, 171 calcium balance, 165-166 dietary factors, 172 gastrointestinal function, 167-168, 170-171, 177 gender effects, 171 - 172 hormonal regulators, 168-170 intake, 166 intestinal absorption mechanisms, 167-168 measurement, 168 regulation, 168-172 reproductive function, 171 sources, 166-167 bone resorption marker, 318- 319 calcium II deficiency syndrome, 703-705 calcium-phosphorus bone mineral phase, 23-30 characteristics, 165 extracellular calcium response heightened response, s e e Autosomal dominant hypocalcemia overview, 479-480 resistance syndromes, s e e Familial benign hypocalciuric hypercalcemia; Neonatal severe primary hyperparathyroidism homeostasis regulation calcitonin role, s e e Calcitonin 1,25-dihydroxyvitamin D role, 52-53, 144-145 parathyroid hormone role, 52-53, 65-66, 105, 169, 445-448 hydroxyapatite crystals, s e e Bone, mineralization hyperoxaluric calcium nephrolithiasis treatment, 749 hyperparathyroidism treatment, 476-477 hypoparathyroidism treatment, 521 Paget's disease role, 570 parathyroid hormone secretion regulation, 60 renal osteodystrophy role parathyroid hormone secretion regulation, 445448 therapy, 458-459 sarcoidosis role, 609, 611 stone formation, s e e Nephrolithiasis Calcium citrate, hyperoxaluric calcium nephrolithiasis treatment, 749-750 Calcium gluconate, hypoparathyroidism treatment, 521 Calcium ion-sensing receptor altered response syndromes, s e e Autosomal dominant hypocalcemia; Familial benign hypocalciuric hypercalcemia; Neonatal severe primary hyperparathyroidism characterization, 483-485 cloning, 483-485, 496 mutations calcium ion resistance, 485-489 diagnostic effects, 493-496
Index Calcium ion-sensing receptor ( C o n t i n u e d ) homozygous versus d e n o v o heterozygous mutations, 490-491 identification, 493-494 parathyroid regulation, 492-493 renal function regulation, 482-483, 492-493, 496 targeted gene deletion mouse development, 491492 therapeutic effects, 493-496 overview, 479-480 physiology, 493-494 Calcium oxalate stones, s e e Nephrolithiasis cAMP calcitonin secretion regulation, 101, 104 pseudohypoparathyroidism pathophysiology, 510512, 517, 520-521 signal transduction role, 111-112 Camurati-Engelmann disease, s e e Progressive diaphyseal dysplasia Cancellous bone, characteristics, 238 Cancer, s e e Carcinomas; Sarcomas; Tumors Candidiasis-polyendocrinopathy- ectodermal dystrophy, characteristics, 507 Carbohydrates, hypophosphatemia manifestation, 224 Carbonate ions bone apatite abnormalities, 41-45 hyperparathyroidism diagnosis, 467 Carcinomas lungs, 614 parathyroid gland characteristics, 413-418 operative management, 475 Cardiovascular system hypophosphatemia manifestations, 224 osteogenesis imperfecta clinical features, 662 Paget's disease association, 579-580 Cartilage, s e e a l s o Bone, development bone articulation role, 27 skeletal development, 2 C cells calcitonin production, 96-97 calcitonin secretion, 101-102 Cell death, s e e Necrosis Central nervous system calcitonin role, 107-108 hypophosphatemia manifestations, 224 Chemotherapy, s e e s p e c i f i c t y p e s Children's diseases, s e e Pediatric diseases Chloroquine, sarcoidosis treatment, 612 Chondrocalcinosis familial benign hypocalciuric hypercalcemia association, 481 hyperparathyroidism association, 424 Paget's disease association, 579
791 Chondroclasts, s e e Osteoclasts Chondrodysplasia hypercalcemia association, 426-427 parathyroid hormone/parathyroid hormone-related peptide receptor mutation, 78-80 Chronic diarrheal syndrome, treatment, 751 Citrate, calcium stone formation, 750-752, 754 Clodronate osteoporosis management, 402 Paget's disease treatment, 588-589 Cloning calcitonin receptor, 108-109 calcium ion-sensing receptors, 483-485, 496 Clotting, osteogenesis imperfecta association, 663 Collagen, s e e a l s o Bone matrix bone three-dimensional structure, 24 characteristics, 5-6 markers bone resorption, 320-321 bone tumover, 318 osteogenesis imperfecta association biochemistry analysis, 671 assembly, 669-670 biosynthesis, 668-669 cellular regulation, 670 degradation, 670 gene structure, 667-668 protein structure, 666-667 secretion, 669-670 clinical features ear, 661 eye, 661 heart, 662 pulmonary function, 662 mRNA mutation localization, 672 Paget's disease association, bone matrix metabolism, collagenolysis, 572 procollagen peptides bone turnover marker, 318 osteogenesis imperfecta role, 671 Paget's disease association, bone matrix metabolism, 574 Colony-stimulating factors embryologic function, 13 malignant disease hypercalcemia pathophysiology, 642 Compton scattering technique, neutron activation analysis, 280-281 Computed tomography acquisition technique, 286-288 age-related bone changes, 288 fracture discrimination, 288 high-resolution tomography, 289-291
792
Index
Computed tomography ( C o n t i n u e d ) hip assessment, 288-289 menopause-related bone changes, 288 microcomputed tomography, 289-291 overview, 286 Paget's disease complications, 557-559 spine assessment, 288 volumetric tomography, 288-289 Cortical bone, characteristics, 238 Corticosteroids intestinal calcium transport effects, 180-181 malignancy disease hypercalcemia treatment, 645 Crohn' s disease calcium absorption effects, 177 phosphate absorption effects, 188 vitamin D depletion, 357 CrossLaps, bone resorption marker, 320-321 Crystallization, s e e Bone, mineralization Cystinuria, stone formation cystine role, 753 diagnostic criteria, 754 pathophysiology, 753-754 treatment, 754- 755
D Danlos-Ehlers syndrome, osteogenesis imperfecta association, 654, 660 Decorin, characteristics, 7 Demineralization, s e e Osteopenia Densitometry, s e e a l s o Ultrasound acronyms, 276 disease course evaluation, 303 exogenous subclinical hyperthyroidism, 535-538 osteoporosis diagnosis, 391-392 overt hyperthyroidism, 534 Dent's disease, characteristics, 776-777 Deoxypyridinoline, bone resorption marker, 319-320 Diabetes mellitus calcium absorption effects, 179 magnesium absorption effects, 193 Dialysis magnesium effects, 455 malignancy disease hypercalcemia treatment, 644 renal osteodystrophy therapy, 454-455, 459 Diarrheal syndrome, treatment, 751 Diet calcium absorption factors, 172 alcohol abuse, 181 - 182 caffeine effects, 182 fiber effects, 182-183 intake, 166, 176
lactose intolerance, 176-177 phosphate effects, 182 sources, 166-167 supplements, 458-459 vitamin D deficiency, 177 fiber intake, calcium absorption effects, 182-183 magnesium bioavailability, 194-195 deficiency, 193 intake, 191 osteoporosis management, 397-398 phosphate bioavailability, 188, 190 calcium effects, 187-188 deficiency, 188, 222 deprivation, 187 intake, 182-184 renal function, 214-215 sources, 183-184 Dietary fiber, calcium absorption effects, 182-183 DiGeorge syndrome, parathyroid gland development, 509 1,25-Dihydroxyvitamin D, s e e Calcitriol 24,25-Dihydroxyvitamin D assay, 151-152 metabolism, 139-140 Paget's disease role, 571 Diphosphonates, s e e Bisphosphonates Disodium etidronate fibrodysplasia ossificans progressiva treatment, 721 Paget's disease treatment, 586-588, 591 progressive diaphyseal dysplasia treatment, 710 Diuresis, hypercalcemia management, 217, 431-433, 644 DNA, sequencing, osteogenesis imperfecta analysis, 672-673 D-Pyr peptide, bone resorption marker, 320 Drugs, s e e Medications Dysplasia, s e e s p e c i f i c t y p e s
E Ear, osteogenesis imperfecta clinical features, 661 Ectodermal dystrophy, characteristics, 507 Edetate disodium, sarcoidosis treatment, 612 Ehlers-Danlos syndrome, osteogenesis imperfecta association, 654, 660 Electron microscopy bone apatite abnormality detection, 40-41 bone crystal structure analysis, 32-35 Ellison-Zollinger syndrome, peptic ulcer association, 424
Index Embryology bone development, 2-5, 104-105 bone morphogenetic proteins, 3-4, 14, 762-763 bone remodeling, cell-cell interaction, 12 cell-matrix interactions, 16 colony-stimulating factor role, 13 fibroblast growth factor role, 3-5, 14-15, 763 hepatocyte growth factor role, 15-16 limb development, 2-3 osteoblast cell lineage, 5-9 differentiation, 7-9 phenotypes, 5-7 osteoclasts, 9-12 differentiation, 9-10 function, 10-12 origin, 9-10 overview, 1 parathyroid gland development, 53, 469-471 parathyroid hormone-related peptide role, 4 pattern regulation, 2-3 platelet-derived growth factor role, 15 transforming growth factor [3 role, 13-14 vascular endothelial growth factor role, 15 Endocrine neoplasia, hyperparathyroidism role, 413-417 Endocrine system, s e e a l s o s p e c i f i c h o r m o n e s osteogenesis imperfecta role, 662-663 clotting, 663 pregnancy, 663 short stature, 662 thyroid function, 662 vitamin D metabolism, 147-148 Endosteal envelope, characteristics, 238 Endosteal hyperostosis characteristics, 710-712 treatment, 712-713 Engelmann-Camurati disease, s e e Progressive diaphyseal dysplasia Epidermal growth factor, malignant disease hypercalcemia pathophysiology, 641 Escape phenomenon, calcitonin receptor regulation, 112-113 Estrogen calcium absorption regulation, 169-170 hyperparathyroidism treatment, 747 osteoporosis management, 399, 405-406, 626 perimenopausal therapy evaluation, 303 phosphate absorption effects, 185 Estrogen receptor modulators, osteoporosis management, 405-406 Ethane- 1-hydroxy- 1,1-diphosphonate, fibrodysplasia ossificans progressiva treatment, 722 Ethnicity, calcium absorption effects, 171-172 Ethylenediaminetetraacetic acid, fibrodysplasia ossificans progressiva treatment, 721
793 Etidronate, s e e Disodium etidronate Exercise osteoporosis management, 398, 625 rheumatoid arthritis role, 625 Eye, osteogenesis imperfecta clinical features, 661
F Familial benign hypocalciuric hypercalcemia biochemical features, 480-483 clinical features, 480-483 differential diagnosis, 427 extracellular calcium ion-sensing receptor characterization, 483-485 cloning, 483-485 mutations, 485-489 resistance syndrome, 485-489 genetic heterogeneity, 483, 508 hypercalcemia association, 414, 427 parathyroid hormone secretion role, 59-61 renal function, 482-483, 492-493, 496 Familial hyperparathyroidism, s e e a l s o Hyperparathyroidism; Neonatal severe primary hyperparathyroidism etiology, 468-469 Familial hypoparathyroidism, s e e a l s o Hypoparathyroidism characteristics, 507-508 Familial hypophosphatemic vitamin D-refractory tickets, s e e Osteomalacia; Rickets Familial osteogenesis imperfecta, s e e Osteogenesis imperfecta Familial vitamin D-resistant rickets, characteristics, 771-772 Fanconi' s syndrome Dent's disease, 776-777 oculocerebrorenal syndrome of Lowe, 775-776 osteomalacia association, 365-366, 775 Fiber, calcium absorption effects, 182-183 Fibroblast growth factor limb development role, 3 skeletal development role, 4-5, 14-15, 763 Fibrogenesis imperfecta ossium characteristics, 726-728 treatment, 728 Fibrous dysplasia characteristics, 719-721 Paget's disease histopathology, 554 treatment, 721-722 Fluorosis characteristics, 728-729 treatment, 729 Flurbiprofen, sarcoidosis treatment, 612
794
Index
Fourier transform infrared spectroscopy amorphous calcium-phosphorus detection, 31-32 bone apatite abnormality detection, 36-40 Fractures exogenous subclinical hyperthyroidism complications, 538-539 noninvasive assessment computed tomography, 288 overview, 276 ultrasound, 296 osteogenesis imperfecta clinical features, 657-659 osteomalacia diagnosis, 342-344 Paget's disease complications, 562-564 rheumatoid arthritis complications, 626 Furosemide, malignancy disease hypercalcemia treatment, 643
progressive diaphyseal dysplasia treatment, 710 rheumatoid arthritis effects, 625-626 sarcoidosis treatment, 611-612 GNAS1 gene, pseudohypoparathyroidism pathophysiology, 513-515, 517-518, 520 Goiter, exogenous subclinical hyperthyroidism association, 536-537 Gout, Paget's disease association, 578 Gouty diathesis, calcium stone formation, 752-753 Granulomatous diseases, see specific types Growth factor, see specific types Growth hormone calcium absorption effects, 174 phosphate regulation, 187, 214
H G Gallium nitrate hypercalcemia management, 433 malignancy disease hypercalcemia treatment, 646 Paget's disease treatment, 591 Gastrointestinal tract calcium absorption, 167-168, 170-171, 177 hyperparathyroidism association, 424 magnesium absorption, 194 malignant disease hypercalcemia pathophysiology, 642 osteomalacia association, 355-357 phosphate absorption basolateral membrane phosphate exit, 209 hyperphosphatemia treatment, 230 hypophosphatemia, 222, 225 intestinal defects, 189-190 luminal brush transport, 208 malabsorption diseases, 188 transcellular phosphorus movement, 209 plasma calcium regulation calcitonin effects, 107 1,25-dihydroxyvitamin D effects, 144-145 vitamin D absorption, 131-132, 355-357 Gel electrophoresis, collagen analysis, osteogenesis imperfecta role, 671 Gender, calcium absorption effects, 171-172 Gene therapy, osteogenesis imperfecta management, 682 Genetic diseases, see specific types Gla protein, bone turnover marker, 318 Glucocorticoids hypercalcemia management, 434, 643 osteopetrosis treatment, 702 phosphate absorption effects, 187, 190
Heart, see Cardiovascular system Hedgehog proteins, skeletal development role, 4-5 Hemodialysis magnesium effects, 455 malignancy disease hypercalcemia treatment, 644 renal osteodystrophy therapy, 454-455, 459 Hemopoiesis, colony-stimulating factor role, 13 Hepatitis C-associated osteosclerosis, characteristics, 731 Hepatobiliary diseases calcium absorption effects, 177 25-hydroxylation impairment, 357-358 Hepatocyte growth factor, embryologic function, 1516 Hip computed tomography assessment, 288-289 walking difficulties, osteomalacia role, 340 Histology applications, 267-269 morphometric bone parameters balance, 255 bone formation, 254-255 bone structure, 253- 254 mineralization, 254-255 molecular parameters, 255-256 remodeling, 255 resorption, 255 turnover, 255 renal osteodystrophy, 451-453 adynamic bone, 452 high-turnover bone disease, 451 lesions, 452-453 low-turnover bone disease, 452 techniques dehydration, 247 embedding, 247
Index Histology ( C o n t i n u e d ) fixation, 247 immunohistochemistry, 251 sectioning, 247-248 in situ hybridization, 249-251,268-269 staining, 248-249 Histomorphometry, see also Radiographic morphometry bone biopsy parameters balance, 255 bone formation, 254-255 bone structure, 253-254 mineralization, 254- 255 molecular parameters, 255-256 remodeling, 255 resorption, 255 turnover, 255 osteoporosis diagnosis, 392 Hodgkin' s disease, characteristics, 613-614 H o x gene family, pattern regulation, 2-3 Human leukocyte antigen, Paget's disease diagnosis, 547, 560 Hydroxyapatite, see Bone, mineralization Hydroxychloroquine, sarcoidosis treatment, 612 Hydroxylysine bone resorption marker, 319 Paget's disease association, bone matrix metabolism, 572-573 Hydroxyproline bone resorption marker, 318-319 Paget's disease association, bone matrix metabolism, 572-574 25-Hydroxyvitamin D, see Calcidiol Hypercalcemia, see also Hypocalcemia calcitriol-induced disorders calcipotriol intoxication, 615 Hodgkin' s disease, 613-614 leioblastoma, 614 leukemia, 613-614 lung carcinoma, 614 lymphomas, 613-614 rheumatoid arthritis, 613 sarcoidosis, 608-611 seminoma, 614 subcutaneous fat necrosis, 615 tuberculosis, 612-613 vitamin D intoxication, 615 William' s syndrome, 614-615 familial benign hypocalciuric hypercalcemia biochemical features, 480-483 clinical features, 480-483 differential diagnosis, 427 extracellular calcium ion-sensing receptor characterization, 483-485
795 cloning, 483-485 mutations, 485-489 resistance syndrome, 485-489 genetic heterogeneity, 483 hyperparathyroidism diagnosis familial benign hypocalciuric hypercalcemia, 414, 427 Jansen's disease, 78-80, 426-427 lithium therapy, 427 preoperative evaluation, 466 malignant disease association clinical features, 642 pathophysiology colony-stimulating activity, 642 hematological malignancies, 638-639 lymphomas, 639 malignancy types, 637 metastasis solid tumors, 639-640 myeloma, 638-639 nonmetastasis solid tumors, 640-642 parathyroid hormone, 641-642 parathyroid hormone-related protein, 641 prostaglandins, 642 transforming growth factor, 641 treatment alendronate, 644-645 bisphosphonates, 644-645 calcitonin, 433, 643 corticosteroids, 645 furosemide, 643 gallium nitrate, 646 glucocorticoids, 643 hemodialysis, 644 indications, 642-643 indomethacin, 645 intravenous saline, 643 mithramycin, 645-646 oral phosphate, 645 pamidronate, 643-644 parenteral phosphate, 643 management bisphosphonates, 433, 644-645 calcitonin, 433, 643 diuresis, 217, 431-433, 644 gallium nitrate, 433, 646 glucocorticoids, 434, 643 mithramycin, 433-434, 645-646 phosphate, 434, 643-644 plicamycin, 433-434, 645-646 Paget's disease association, 578 Hypercalciuria calcium absorption effects, 175, 741 calcium stone formation causal role, 740-741
796 Hypercalciuria (Continued) diagnostic criteria, 743-744 pathophysiology, 741-743 treatment, 744-747 1,25-dihydroxyvitamin D therapy, 146 hereditary hypercalciuria hypophosphatemic tickets, 774-775 Paget's disease association, 578 sarcoidosis association, 609 vitamin D assay, 154-155 Hypermagnesemia, s e e a l s o Hypomagnesemia; Magnesium hypoparathyroidism, 194 Hyperostosis endosteal hyperostosis characteristics, 710- 712 treatment, 712- 713 melorheostosis, 716-717 overview, 697-698 Hyperoxaluria, calcium stone formation, 748-750 Hyperparathyroidism, s e e a l s o Hyperthyroidism; Hypoparathyroidism biopsy, 260-263 calcium absorption effects, 174, 743 clinical features articular manifestations, 424-425 gastrointestinal involvement, 424 hypertension, 425 neuromuscular manifestations, 423-424 parathyroid poisoning, 425 pediatric hyperparathyroidism, 425 pregnancy, 425-426 renal manifestations, 419, 743 skeletal involvement, 419-423 diagnosis, 426-431 aluminum intoxication, 429 differential diagnosis, 429-431 high bone turnover states hyperthyroidism, 428 immobilization, 428 thiazides, 428-429 hypercalcemia familial benign hypocalciuric hypercalcemia, 414, 427 Jansen's disease, 78-80, 426-427 lithium therapy, 427 milk-alkali syndrome, 173, 429 renal failure, 429 vitamin A intoxication, 429 vitamin D-related hypercalcemia, 428 etiology, 412-418 hypophosphatemia effects, 217 management, 434-435, 747 neonatal severe primary hyperparathyroidism
Index biochemical features, 489-490 clinical features, 489-490 extracellular calcium ion-sensing receptor mutations, 490-491 parathyroid regulation, 492-493 renal function, 492-493 targeted deletion mouse development, 491-492 genetics, 490 overview, 411-412 Paget' s disease histopathology, 553 pathology, 412-418 phosphate absorption effects, 187-188 renal osteodystrophy, 443-451 bone histology, 451 calcemic action resistance, 450-451 calcium sensitivity, 445-448 parathyroid hormone degradation, 451 parathyroid hormone resistance, 450-451 phosphate retention, 448-450 vitamin D metabolism alterations, 444-445 surgical treatment, 465-477 historical perspectives, 465-466 nephrolithiasis treatment, 747 operation conduct exploration, 469-471 resection extent, 471-472 parathyroid carcinoma, 475 postoperative management, 475-477 airway protection, 476 calcium replacement, 476-477 preoperative evaluation alkaline phosphatase measurement, 467 bicarbonate measurement, 467 calcium diagnosis, 466 familial disease etiology, 468-469 general assessment, 469 informed consent, 469 localization, 468 operation indicators, 467-468 parathyroid hormone measurement, 466-467 phosphate measurement, 467 physical findings, 465-466 radiation exposure, 469 recurrent hyperparathyroidism management, 472475 localization, 472-474 reoperation, 474-475 renal osteodystrophy, 459-460, 475 Hyperphosphatasemia, Paget's disease histopathology, 554 Hyperphosphatemia, s e e a l s o Hypophosphatemia causes acromegaly, 227 bisphosphonate administration, 228
Index Hyperphosphatemia (Continued) catabolism increase, 228-229 parathyroid hormone circulation, 227 phosphate salt administration, 229 pseudohypoparathyroidism, 227 respiratory acidosis, 229 tumoral calcinosis, 227-228 tumor lysis syndrome, 228 urinary excretion, 186, 226-227 vitamin D salt administration, 229 clinical manifestations, 229-230, 457-458 treatment, 230 Hyperprostaglandin E syndrome, calcium absorption effects, 174 Hyperthyroidism, s e e a l s o Hyperparathyroidism; Hypoparathyroidism calcium absorption effects, 178-179 diagnosis, 428 endogenous subclinical hyperthyroidism, 534-535 exogenous subclinical hyperthyroidism, 535-539 benign goiter suppression, 536-537 bone density, 535-538 bone mineral metabolism markers, 538 cross-sectional studies, 535- 536 fractures, 538-539 longitudinal studies, 537-538 menstrual status role, 537 suppressive versus replacement therapy, 536 thyroid cancer suppression, 536- 537 grades, 531 - 532 overt hyperthyroidism bone density, 534 mineral metabolism, 532-533 thyroid hormone effects, 532 thyrotoxic bone disease, 533- 534 Hypertrophic osteoarthropathy, characteristics, 729731 Hyperuricemia, Paget's disease association, 578 Hyperuricosuria, calcium stone formation, 747-748 Hypocalcemia, s e e a l s o Hypercalcemia inherited autosomal dominance, 494-496 biochemical features, 494-495 chromosome 3 linkage, 495 clinical features, 494-495 diagnosis, 495-496 extracellular calcium ion-sensing receptor mutations, 495 therapy, 495-496 magnesium deficiency effects, 189 neonatal hypocalcemia, 509- 510 pathophysiology, 502-504 symptoms, 501,504-505 vitamin D metabolism assays, 154-155 Hypocitraturia, calcium stone formation, 750- 752
797 Hypogonadism, pseudohypoparathyroidism association, 517 Hypomagnesemia, s e e a l s o Hypermagnesemia; Magnesium clinical manifestation, 192-193 intestinal calcium transport effects, 181 intestinal magnesium absorption defects, 194 treatment, 192 Hypoparathyroidism, s e e a l s o Hyperparathyroidism; Hyperthyroidism causes autoimmune hypoparathyroidism, 507 drugs, 506 idiopathic hypoparathyroidism, 506-507 isolated hypoparathyroidism, 507-508 magnesium imbalance, 506 neonatal hypocalcemia, 508-509 parathyroid gland development disorders, 508-509 parathyroid infiltrative disease, 506 radiation, 506 surgery, 505-506 toxic agents, 505-506 diagnosis, 519- 521 hypermagnesemia role, 194 hypocalcemia pathophysiology, 502-504 symptoms, 504-505 overview, 501-502 pseudohypoparathyroidism, 510- 519 molecular classification, 512-519 Albright's hereditary osteodystrophy, 510, 515516 multiple hormone resistance, 516-517 neurosensory abnormalities, 517 phenotypic variability, 517-518 type IA, 513-518 type IB, 518 type IC, 518 type II, 518-519 natural history, 519 pathophysiology, 510-512 treatment, 521 - 522 Hypophosphatemia, s e e a l s o Hyperphosphatemia biochemical manifestations, 223-225 causes acute leukemia, 222 acute respiratory alkalosis, 222 acute tubular necrosis diuretic phase, 217 alcoholism, 222 bums, 223 extracellular fluid volume expansion, 218 Fanconi's syndrome, 775-777 hereditary hypercalciuria hypophosphatemic tickets, 774-775
798
Index
Hypophosphatemia ( C o n t i n u e d ) hyperparathyroidism, 217 magnesium deficiency, 170, 218-220 malabsorption, 222 malnutrition, 222 McCune-Albright syndrome, 220 phosphate binder administration, 222 postobstructive diuresis, 217 post transplantation, 217- 218 renal tubular defects, 217 toxic shock syndrome, 222 urinary excretion, 217 vitamin D metabolism abnormalities, 188-189, 220 vitamin D-resistant tickets, 221 x-linked hypophosphatemic tickets, 188-189, 769-774 clinical manifestations, 186, 223-225 differential diagnosis, 225 osteomalacia association hereditary hypophosphatemia, 362-364 nonhereditary hypophosphatemia, 364-365 overview, 351 phosphorus depletion, 362 treatment, 225-226, 373-374 x-linked hypophosphatemic tickets biochemical findings, 770 clinical features, 188-189, 769-770 molecular genetics, 771-772 mouse models, 773-774 osteoblast function studies, 774 phosphate transport studies, 774 treatment, 770-771 tumor-induced osteomalacia, 772-773 Hypovitaminosis D osteopathy osteomalacia role bone mineral metabolism abnormalities, 345-348 diagnostic considerations, 348 histological evolution, 335-338 histopathology, 340-341 skeletal radiology, 341-342 temporal evolution, 348 prevention, 370-371 treatment bone mineral metabolism effects, 371-372 management aspects, 372-373 remodeling effects, 371-372 vitamin D response, 371
I Immunohistochemistry bone tissue applications, 251
1,25-dihydroxyvitamin D effects, 147-148 sarcoidosis, 607-609 Indomethacin, malignancy disease hypercalcemia treatment, 645 Infrared spectroscopy amorphous calcium-phosphorus detection, 31-32 bone apatite abnormality detection, 36-40 In situ hybridization applications, 268-269 techniques bone processing, 249-250 protocol, 251 Internet, osteogenesis imperfecta mutations, 671 Intestine, see Gastrointestinal tract Ipriflavone, osteoporosis management, 404
Jansen' s disease hypercalcemia association, 426-427 parathyroid hormone/parathyroid hormone-related peptide receptor mutation, 78-80 Jaw, Paget's disease manifestations, 557 Joints osteogenesis imperfecta clinical features, 659-660 Paget's disease manifestations, 561-562
K Ketoconazole, sarcoidosis treatment, 612 Kidneys 1,25-dihydroxyvitamin D metabolism, renal 25-hydroxyvitamin D-la-hydroxylase role, 135 25-hydroxyvitamin D metabolism, 138-139 kidney stones, see Nephrolithiasis renal function calcitonin effects, 106-107 calcium absorption, 178 calcium reabsorption, 65-66 chronic renal failure, 444-445 1,25-dihydroxyvitamin D synthesis, 66, 145 familial benign hypocalciuric hypercalcemia patients, 482-483, 492-493, 496 hypercalcemia association, 429, 482 hyperparathyroidism association, 418-419, 429 parathyroid hormone metabolism, 64-67 phosphorus reabsorption, 209-215 acid-base balance effects, 213 adrenal hormone effects, 213 cellular reabsorption mechanisms, 210-211 dietary phosphorus intake alterations, 214-215 growth hormone effects, 214 hyperphosphatemia, 186, 226-227
Index
799
Kidneys ( C o n t i n u e d ) hypophosphatemia, s e e Hypophosphatemia parathyroid hormone effects, 66, 211-213 stanniocalcin, 215 superficial-deep nephron transport compared, 210 vitamin D role, 213- 214 Kidney stones, s e e Nephrolithiasis Knee joint, Paget's disease manifestation, 561-562
L Lactate dehydrogenase, hyperphosphatemia role, 228 Lactation, calcium absorption effects, 175-176 Lactose intolerance, calcium absorption effects, 176-177 magnesium absorption effects, 193 Lamellar bone, characteristics, 239-240 Laxatives, magnesium absorption effects, 193 Leioblastoma, characteristics, 614 Leukemia characteristics, 613-614 hypophosphatemia role, 222 Leukocyte antigen, Paget's disease diagnosis, 547, 560 Levothyroxine, hyperthyroidism treatment, 537-538, 540-541 Lifestyle, osteoporosis management, 397 Limb development, embryology, 2-3 Lithium therapy, hypercalcemia association, 427 Liver calcium absorption effects, 177 hepatitis C-associated osteosclerosis, 731 25-hydroxylation impairment, 357-358 parathyroid hormone metabolism, 64-65 vitamin D-25-hydroxyvitamin D conversion, 132 Looser's zone, osteomalacia diagnosis, 342-344, 455456 Low-turnover bone disease, s e e a l s o Bone turnover biopsy, 263-264 Lungs osteogenesis imperfecta clinical features, 662 small cell carcinoma, 614 Lupus erythematosus, characteristics, 621,628 Lymphocytes, sarcoidosis pathology, 607-609 Lymphomas characteristics, 613-614 malignant disease hypercalcemia pathophysiology, 639
M Magnesium absorption pathophysiology, 191-194 decreased absorptive states, 193-195
disorders, clinical manifestations, 192-193 increased absorptive states, 193 physiology, 191-192 bone apatite abnormalities, 45-46 characteristics, 190-191 chronic hemodialysis effects, 455 hyperoxaluric calcium nephrolithiasis treatment, 749 hypomagnesemia, s e e Hypomagnesemia hypoparathyroidism induction, 506 intestinal calcium transport effects, 181 intestinal phosphate absorption effects, 189-190 Paget's disease role, 570 parathyroid hormone secretion regulation, 60-61 Magnetic resonance microscopy amorphous calcium-phosphorus detection, 31-32 bone apatite abnormality detection, 37, 40-41, 43 noninvasive bone assessment technique, 296-298 Markers, s e e Biochemical markers Marrow, s e e Bone marrow cells Matrix, s e e Bone matrix McCune-Albright syndrome, characteristics, 220 Medications, s e e a l s o s p e c i f i c t y p e s calcium absorption effects, 177-178, 182 hypoparathyroidism role, 506 osteogenesis imperfecta therapy, 678-679 Paget's disease treatment, 581-592 bisphosphonates, 585-590 calcitonin, 581-585 combination therapy, 591 gallium nitrate, 591 indications, 581 mithracin, 590-591 monitoring, 591-592 rheumatoid arthritis diagnosis effects, 625-626 rickets therapy, 134 Melorheostosis characteristics, 716-717 treatment, 717 Melphalan, fibrogenesis imperfecta ossium treatment, 728 Menopause, s e e a l s o Osteoporosis bone changes, computed tomography assessment, 288 bone turnover evaluation, 322 calcium absorption effects, 171 estrogen therapy evaluation, 303 intestinal calcium transport effects, 180 levothyroxine replacement therapy, 537-538, 540541 Microscopy, s e e s p e c i f i c t y p e s Migratory osteolysis, characteristics, 631 Milk-alkali syndrome calcium absorption effects, 173 characteristics, 429
800
Index
Milk production, calcium absorption effects, 175-176 Mineralization, s e e Bone, mineralization; Osteopenia Mithracin, Paget's disease treatment, 590-591 Mithramycin hypercalcemia management, 433-434 malignancy disease hypercalcemia treatment, 645646 Mixed sclerosing bone dystrophy, s e e a l s o Osteosclerosis characteristics, 718-719 treatment, 719 Mixed uremic osteodystrophy, s e e a l s o Renal osteodystrophy biopsy, 265 Morphometry, s e e Histomorphometry; Radiographic morphometry Mouse calcium ion-sensing receptor mutations, targeted gene deletion development, 491-492 hypophosphatemic tickets models, 773-774 osteogenesis imperfecta models, 655-656 Muscle, s e e Skeletal muscle Myeloma, malignant disease hypercalcemia pathophysiology, 638-639
N Necrosis acute tubular necrosis diuretic phase, 217 subcutaneous fat necrosis, 615 Neonatal hypocalcemia, characteristics, 509-510 Neonatal severe primary hyperparathyroidism biochemical features, 489-490 clinical features, 489-490 extracellular calcium ion-sensing receptor mutations, 490-491 parathyroid regulation, 492-493 renal function, 492-493 targeted deletion mouse development, 491-492 genetics, 490 Neoplasms, s e e Tumors Nephrolithiasis cystinuria, 753-755 formation, 266-267, 418 gouty diathesis, 752-753 hypercalciuria, 740-747 causal role, 740-741 diagnostic criteria, 743-744 pathophysiology, 741-743 treatment, 744-747 hyperoxaluria, 748-750 hyperuricosuria, 747-748 hypocitraturia, 750- 752
management, 755 overview chemical composition, 739-740 current field status, 739 diagnostic separation, 740 urea-splitting organism infections, 755 Nephrons, s e e Renal function Nephrotic syndrome, calcium absorption effects, 178 Nervous system calcitonin role, 107-108 hypophosphatemia manifestations, 224 Neural crest, development, 2 Neuromuscular system, hyperparathyroidism association, 423-424 Neurosensory abnormalities, pseudohypoparathyroidism association, 517 Neutron activation analysis, Compton scattering technique, 280-281 Nifedipine, melorheostosis treatment, 717 Northern blot, osteogenesis imperfecta mutation analysis, 671-672 Nuclear magnetic resonance microscopy amorphous calcium-phosphorus detection, 31-32 bone apatite abnormality detection, 37, 40-41, 43 noninvasive bone assessment technique, 296-298 Nutrition, s e e Diet
O Octocalcium phosphate, calcium-phosphorous mineral deposition, 30-32 Oculocerebrorenal syndrome of Lowe, characteristics, 775-776 Olbandronate, Paget's disease treatment, 590 Orthophosphates, hyperparathyroidism treatment, 747 Orthotics, osteogenesis imperfecta management, 680681 Osteitis fibrosa cyctica, hyperparathyroidism association bone histology, 451 calcemic action resistance, 450-451 calcium sensitivity, 445-448 parathyroid hormone degradation, 451 parathyroid hormone resistance, 450-451 phosphate retention, 448-450 skeletal changes, 419-423 vitamin D metabolism alterations, 444-445 Osteoarthritis, s e e a l s o Rheumatoid arthritis hypertrophic osteoarthropathy, 729-731 juvenile onset rheumatoid arthritis, 631-633 overview, 621
Index Osteoblasts cell lineage, 5 - 9 differentiation, 7 - 9 phenotypes, 5 - 7 characteristics, 243, 314 function studies, 774 mineralization influence, 353 Paget's disease histopathology, 5 4 8 - 5 5 1 , 5 9 3 - 5 9 4 parathyroid hormone effects, 67 phosphate transport, 215- 216 remodeling role, s e e Bone turnover Osteocalcin bone turnover marker, 316- 318 characteristics, 6 Paget's disease association, 574-575 Osteoclasts bone resorption regulation, 102-104, 113-114 characteristics, 9, 242-243, 314 differentiation, 9-10, 145 function, 10-12 malignancy stimulation factors, 639 origin, 9 - 1 0 Paget's disease histopathology, 547-548, 550-551, 593-595 parathyroid hormone effects, 67 phosphate transport, 215-216 signal transduction, 111-112 Osteodystrophy Albright' s hereditary osteodystrophy, 510, 515-516 biopsy, 261-266 renal osteodystrophy, s e e Renal osteodystrophy Osteogenesis imperfecta clinical features, 650-663 collagen-containing organs, 661-662 ear, 661 eye, 661 heart, 662 pulmonary function, 662 connective tissues, 659-661 joint hypermobility, 659-660 skin, 660 teeth, 660-661 differential diagnosis, 656-657 endocrine function, 662-663 clotting, 663 pregnancy, 663 short stature, 662 thyroid function, 662 heterogeneity, 652 historical perspectives, 651-652 phenotypes, 652-656 heritable osteoporosis, 654-655 mild deforming type IV, 653-654 mild nondeforming type I, 654
801 murine models, 655-656 neonatal lethal type II, 652-653 related skeletal-connective tissue syndromes, 654 prevalence, 657 skeletal system, 657-659 long bone fractures, 657-659 skull, 659 spine, 659 future research directions, 681-682 animal models, 682 diagnosis, 681-682 somatic gene therapy, 682 therapy evaluation, 682 mutation identification, 670-678 collagen analysis, 671 DNA sequencing, 672-673 molecular hybridization, 671-672 procollagen analysis, 671 overview, 651 pathophysiology bone mechanics, 665-666 bone mineral content, 665 collagen biochemistry, 666-670 assembly, 669-670 biosynthesis, 668-669 cellular regulation, 670 degradation, 670 gene structure, 667-668 protein structure, 666-667 hydroxyapatite quality, 665 molecular analysis, 673-678 heritable osteoporosis, 678 lethal type II, 673-674 mild deforming type IV, 676 mild nondeforming type I, 676-678 severe type III, 674-676 shared features, 678 skeletal histopathology, 663-665 lethal type II, 664 mild deforming type IV, 665 mild nondeforming type I, 665 severe nonlethal type III, 664-665 therapy, 678-681 medical therapy, 678-679 orthotic therapy, 680-681 psychological support, 680-681 surgical therapy, 679-680 Osteoids mineralization mechanisms maturation time, 331-332 osteoid seam life history, 330-331 tetracycline-based kinetics relationships, 334 Paget's disease histopathology, 552
802 Osteolysis, characteristics, 631 Osteomalacia, s e e a l s o Rickets axial osteomalacia clinical presentation, 722-723 etiology, 725 histopathological findings, 723-724 laboratory studies, 723 overview, 722 pathogenesis, 725 radiological features, 723 treatment, 725-726 biopsy, 259-260 bone mineralization mechanisms fluorescent label width, 332-334 hypovitaminosis D osteopathy histological evolution, 335-338 impaired mineralization, 334-335, 367-369 kinetic definition, 335-338 osteoid seam life history, 330-331 remodeling, 328-329 tetracycline-based kinetics, 334 tetracycline data interpretation, 329 juvenile onset, s e e Rickets manifestations bone remodeling noninvasive indices, 344-345 clinical features bone pain, 339 muscle weakness, 339-340 walking difficulty, 340 fractures, 342-344 hypovitaminosis D osteopathy bone mineral metabolism abnormalities, 345348 diagnostic considerations, 348 histopathology, 340- 341 skeletal radiology, 341-342 temporal evolution, 348 normal metabolic conditions alkaline phosphatase disorders, 369-370 matrix mineralization defects, 368-369 mineralization inhibitors, 367-368 oncogenic osteomalacia, 221-222 overview, 327-328 pathogenesis mineralization defects, 351 - 354 calcitriol role, 353-354 vitamin D effects, 352- 353 phosphate metabolism defects, 351 vitamin D metabolism defects, 349-350 phosphate metabolism defects, 361-367 depletion, 362 Fanconi's syndrome, 365-366, 775-777 hereditary hypophosphatemia, 362-364 nonhereditary hypophosphatemia, 364-365
Index renal tubular acidosis, 366-367 ureteral diversion, 366-367 renal osteodystrophy, 451-452, 455-456 therapeutic intervention hypophosphatemic osteomalacia treatment calcitriol, 373-374 phosphate supplementation, 373 hypovitaminosis D osteopathy treatment bone mineral metabolism effects, 371-372 management aspects, 372-373 prevention, 370-371 remodeling effects, 371-372 vitamin D response, 371-373 vitamin D metabolism defects anticonvulsant bone disease, 358-359 enzyme induction, 358-359 extrinsic depletion, 354-355 gastrointestinal bone disease, 355-357 hepatobiliary bone disease, 357-358 loL-hydroxylation impairment, 359-360 25-hydroxylation impairment, 357-358 intrinsic depletion, 355-357 pathogenesis, 349-350 renal bone disease, 359-360 Osteomark, bone resorption marker, 320 Osteomesopyknosis, characteristics, 707 Osteones, three-dimensional structure, 24 Osteopathia striata characteristics, 715- 716 treatment, 716 Osteopathy, s e e Hypovitaminosis D osteopathy Osteopenia bone turnover rate assessment, biochemical markers, 322-323 osteogenesis imperfecta association, 664-665 osteomalacia diagnosis, 342 osteoporosis pathogenesis, 360 rheumatoid arthritis diagnosis, 624-626 age effects, 625 disease activity, 625 drug effects, 625-626 fractures, 626 functional status, 625 osteoporosis treatment, 626 periarticular bone loss, 624 Osteopetrosis clinical presentations, 699 etiology, 701 histopathological findings, 700-701 laboratory findings, 699 overview, 698-699 pathogenesis, 701 radiological features, 699-700 treatment, 701-702
Index Osteopoikilosis characteristics, 713- 715 treatment, 715 Osteoporosis biopsy, 256-259 bone loss pathogenesis, 360 bone turnover assessment, s e e Bone turnover calcitonin role, 113 classification, 395- 396 definition, 387-388 diagnostic aids biochemical bone remodeling markers, 392393 clinical characteristics, 394-395 densitometry, 391- 392 histomorphometry, 392 radiology, 390- 391 ultrasound, 392 epidemiology, 388-389 fractures, s e e Fractures heritable forms, osteogenesis imperfecta association, 654-655, 678 management differential treatment, 360-361 future research directions parathyroid hormone, 405 selective estrogen receptor modulators, 405406 nonpharmacological intervention exercise, 398, 625 lifestyle, 397 nutrition, 397-398 pharmacological intervention alfacalcidiol, 404-405 bisphosphonates, 400-403, 626 calcidiol, 404-405 calcitonin, 399-400, 626 calcitriol, 404-405 estrogen, 399, 405-406, 626 ipriflavone, 404 pamidronate, 626 sodium fluoride, 403-404 thiazide diuretics, 405 rheumatoid arthritis patients, 626 noninvasive assessment, 275, 301,303 physiology, 389-390 Osteosclerosis, s e e a l s o Mixed sclerosing bone dystrophy associated disorders axial osteomalacia clinical presentation, 722-723 etiology, 725 histopathological findings, 723-724 laboratory studies, 723
803 overview, 722 pathogenesis, 725 radiological features, 723 treatment, 725-726 calcium II deficiency syndrome, 703-705 endosteal hyperostosis characteristics, 710-712 treatment, 712-713 fibrodysplasia ossificans progressiva characteristics, 719-721 treatment, 721-722 fibrogenesis imperfecta ossium, 726-728 fluorosis, 728-729 hepatitis C-associated osteosclerosis, 731 melorheostosis, 716-717 mixed sclerosing bone dystrophy, 718-719 osteomesopyknosis, 707 osteopathia striata, 715-716 osteopetrosis clinical presentations, 699 etiology, 701 histopathological findings, 700-701 laboratory findings, 699 overview, 698-699 pathogenesis, 701 radiological features, 699-700 treatment, 701-702 osteopoikilosis, 713- 715 pachydermoperiostosis, 729-731 progressive diaphyseal dysplasia, 707-710 clinical presentation, 708 etiology, 709-710 histopathological findings, 709 laboratory findings, 708-709 overview, 707-708 pathogenesis, 709-710 treatment, 710 pyknodysostosis, 705-707
P Pachydermoperiostosis, characteristics, 729-731 Paget' s disease differential diagnosis, 553- 554 drug treatment, 581-592 bisphosphonates, 585-590 calcitonin, 581-585 combination therapy, 591 gallium nitrate, 591 indications, 581 mithracin, 590-591 monitoring, 591-592 epidemiology, 545-547
804 Paget's disease ( C o n t i n u e d ) etiology, 593-596 focal manifestations, 554- 562 jaw complications, 557 knee pain, 561-562 pain, 554 pelvic deformities, 560-562 skull patterns, 554-557 spinal deformities, 558-560 histopathology, 547- 554 historical perspectives, 545, 593-596 incidence, 545-547 local complications, 562-569 fractures, 562-564 neoplasia, 564-569 metabolic aspects, 569-577 bone matrix metabolism, 571-577 calcitonin effects, 575-576 collagenolysis, 572 hydroxylysine excretion, 572-573 hydroxyproline involvement, 572-574 noncollagenous bone protein metabolism, 574575 pagetic bone matrix, 571-572 serum alkaline phosphatase, 576-577 serum procollagen peptides, 574 tartrate-resistant acid phosphatase, 577 mineral metabolism, 569-571 surgery, 592-593 systemic complications, 578-581 angioid streaks, 580-581 calcific periarthritis, 579 cardiovascular complications, 579-580 chondrocalcinosis, 579 gout, 578 hypercalcemia, 578 hypercalciuria, 578 hyperuricemia, 578 malabsorption syndrome, 581 pseudoxanthoma elasticum, 580-581 renal calculi, 578 skin changes, 580-581 Pain osteomalacia association, 339 Paget's disease association, 554, 561-562 Pamidronate malignancy disease hypercalcemia treatment, 643644 osteoporosis management, 402, 626 Paget's disease treatment, 588-589 Pancreatitis familial benign hypocalciuric hypercalcemia association, 481 hyperparathyroidism association, 424
Index Parathyroid gland carcinomas characteristics, 413-418 operative management, 475 circulating parathyroid hormone fragment origin, 63 -64 development, 53, 469-471 developmental disorders, 508-509 hyperparathyroidism role, s e e Hyperparathyroidism infiltrative disease, 506 parathyroidectomy, renal osteodystrophy therapy, 459-460, 475 parathyroid hormone biosynthesis, 58-59 renal failure role, 444-445 Parathyroid hormone action physiology bone, 67 kidneys calcium reabsorption, 65-66 phosphate reabsorption, 66, 211-213 renal effects, 67 vitamin D metabolite synthesis, 66 receptors, 73-82 biological role, 82-83 parathyroid hormone/parathyroid hormone-related peptide receptor, 74-80 parathyroid hormone-2 receptor, 80-81 structure-based design, 82 biosynthesis 1,25-dihydroxyvitamin D effects, 145 historical perspectives, 56-57 parathyroid hormone gene, 58, 507-508 precursor molecules, 57-58 regulation, 58-59 signal sequence function, 57-58 calcitonin production, 95 calcium homeostasis regulation, 52-53, 105, 169, 445 -448 chemistry, 53- 56 circulating forms, 61-65 biological significance, 61-63 fragment origin, 63-65 hyperparathyroidism role, s e e Hyperparathyroidism hyperphosphatemia, 227 hypocalcemia role, s e e Hypocalcemia hypophosphatemia, 217- 218 Jansen's disease role, 78-80, 426-427 magnesium absorption effects, 192-193 malignant disease hypercalcemia pathophysiology, 641-642 metabolism, 64-65 osteomalacia role, 345-348 osteoporosis management, 405 phosphate absorption effects, 185
Index Parathyroid hormone ( C o n t i n u e d ) phosphorus reabsorption role, 211-213 renal osteodystrophy role, s e e Renal osteodystrophy secretion, 59-61 x-linked hypophosphatemic tickets role, 218-220 Parathyroid hormone-related peptide action physiology, receptors, 73-82 biological role, 82-83 parathyroid hormone/parathyroid hormone-related peptide receptor, 74-80 parathyroid hormone-2 receptor, 80-81 structure-based design, 82 bone development role, 72-73 calcium homeostasis role, 69-72 embryologic function, 4 gene structure, 68-69 historical perspectives, 53, 67-68 hypocalcemia role, 503 malignant disease hypercalcemia pathophysiology, 638-639, 641 skeletal development role, 763 translated protein chemistry, 69-72 Parfollicular cells calcitonin production, 96-97 calcitonin secretion, 101-102 Pediatric diseases autoimmune polyendocrinopathy- candidiasis- ectodermal dystrophy, 507 bone biopsies, 267 DiGeorge syndrome, 509 hyperparathyroidism, 425 juvenile onset rheumatoid arthritis, 628-633 osteoarthritis, 631-633 reflex sympathic dystrophy, 629-631 regional migratory osteolysis, 631 neonatal hypocalcemia, 509- 510 neonatal severe primary hyperparathyroidism biochemical features, 489-490 clinical features, 489-490 extracellular calcium ion-sensing receptor mutations, 490-491 parathyroid regulation, 492-493 renal function, 492-493 targeted deletion mouse development, 491-492 genetics, 490 osteogenesis imperfecta, differential diagnosis, 656657 overview, 759 pseudohypoparathyroidism, 519 pyknodysostosis, 705-707 tickets, s e e Rickets skeletal deformities, 454 skeletal development, 759-764 cell differentiation regulation, 760-762
805 growth factor involvement, 762-763 matrix molecules role, 764 transcriptional skeletal patterning regulation, 760762 subcutaneous fat necrosis, 615 Pelvis, Paget's disease manifestations, 560-562 Penicillamine, cystine nephrolithiasis treatment, 754755 Peptic ulcers, hyperparathyroidism association, 424 Periarthritis, Paget's disease association, 579 Periosteal envelope, characteristics, 238 P E X gene, x-linked hypophosphatemic tickets role, 219 Phosphatase, s e e a l s o Alkaline phosphatase bone resorption marker, 319 Phosphate absorption pathophysiology clinical manifestations, 186, 351 decreased absorptive states, 188-190 extrinsic inhibiting factors, 190 intestinal malabsorption diseases, 188 intrinsic intestinal absorption defects, 189-190 nutritional deficiency, 188 vitamin D metabolism disorders, 188-189 parathyroid hormone effects, 66 calcium absorption effects, 182 characteristics, 183 homeostasis disorders hyperphosphatemia causes, 226-229 clinical manifestations, 229-230, 457-458 treatment, 230 hypophosphatemia, s e e Hypophosphatemia overview, 207- 216 bone remodeling, 215- 216 gastrointestinal absorption, 207-209, 222, 225, 230 renal reabsorption, 209-215 hydroxyapatite crystals, s e e Bone, mineralization hypercalcemia management, 434 hyperparathyroidism diagnosis, 467 hypophosphatemic osteomalacia treatment, 373 increased absorptive states, 186-190 bioavailability, 188, 190 vitamin D-dependent hyperabsorption, 186-187 vitamin D-independent hyperabsorption, 187-188 magnesium deficiency effects, 189-190 malignancy disease hypercalcemia treatment, 643, 645 physiology absorption mechanisms, 184 balance, 184 measurement, 184-185 regulation, 185
806 Phosphate ( C o n t i n u e d ) sources, 183-184 renal excretion, calcitonin effects, 106 renal osteodystrophy therapy, 457-458 transport studies, 774 Phosphorus amorphous calcium-phosphorus detection, 31-32 calcium-phosphorus bone mineral phase, 23-30 homeostasis regulation, vitamin D role excretion regulation, 213- 214 hyperabsorption, 186-187 uptake stimulation, 208, 220 renal reabsorption mechanisms, 209-215 acid-base balance effects, 213 adrenal hormone effects, 213 cellular reabsorption mechanisms, 210-211 dietary phosphorus intake alterations, 214-215 growth hormone effects, 214 hyperphosphatemia, 186, 226-227 hypophosphatemia, 217 parathyroid hormone effects, 66, 211-213 stanniocalcin, 215 superficial-deep nephron transport compared, 210 vitamin D role, 213-214 Photon absorptiometry, s e e a l s o X-ray absorptiometry noninvasive bone assessment dual photon absorptiometry, 282-286 single photon absorptiometry, 281-282 Platelet-derived growth factor, embryologic function, 15 Plicamycin hypercalcemia management, 433-434 malignancy disease hypercalcemia treatment, 645646 Polar activity zone, limb development, 3 Polyacrylamide gel electrophoresis, collagen analysis, osteogenesis imperfecta role, 671 Polyendocrinopathy- candidiasis- ectodermal dystrophy, characteristics, 507 Polyglandular syndrome, characteristics, 507 Polymerase chain reaction, osteogenesis imperfecta mutation analysis, 671-672 Potassium citrate cystine nephrolithiasis treatment, 754 gouty diathesis treatment, 753 hyperuricosuric calcium nephrolithiasis treatment, 748 hypocitraturic calcium nephrolithiasis treatment, 751-752 Prednisone fibrogenesis imperfecta ossium treatment, 728 progressive diaphyseal dysplasia treatment, 710 Predominant hyperparathyroid bone disease, biopsy, 262-263
Index Preeclampsia, calcium absorption effects, 179 Pregnancy calcium absorption effects, 175 hyperparathyroidism, 425-426 osteogenesis imperfecta complications, 663 Preosteomalacia, s e e Hypovitaminosis D osteopathy; Osteomalacia Primary hyperparathyroidism, s e e Hyperparathyroidism Procollagen peptides bone turnover marker, 318 osteogenesis imperfecta role, 671 Paget's disease association, bone matrix metabolism, 574 Progressive diaphyseal dysplasia, characteristics clinical presentation, 708 etiology, 709-710 histopathological findings, 709 laboratory findings, 708-709 overview, 707-708 pathogenesis, 709-710 treatment, 710 Prolactin, calcium absorption regulation, 169 Prostaglandins hyperprostaglandin E syndrome, 174 malignant disease hypercalcemia pathophysiology, 642 Pseudohypoparathyroidism, 510- 519, s e e a l s o Hypoparathyroidism hyperphosphatemia, 227 molecular classification, 512-519 Albright' s hereditary osteodystrophy, 510, 515516 multiple hormone resistance, 516- 517 neurosensory abnormalities, 517 phenotypic variability, 517-518 type IA, 513-518 type IB, 518 type IC, 518 type II, 518-519 natural history, 519 pathophysiology, 510-512 Pseudoxanthoma elasticum, Paget's disease association, 580-581 Psychological supports, osteogenesis imperfecta management, 680-681 Puberty, calcium absorption effects, 171 Pulmonary system osteogenesis imperfecta clinical features, 662 small cell carcinoma, 614 Pyknodysostosis characteristics, 705-707 treatment, 707 Pyridinoline, bone resorption marker, 319-321 Pyr peptide, bone resorption marker, 320
Index
807
R Race, calcium absorption effects, 171 - 172 Radiation, s e e a l s o Ultraviolet radiation exposure, hyperparathyroidism risk, 469 Radiographic morphometry, s e e a l s o Histomorphometry femur trabecular pattern assessment, 278-279 vertebral body deformity detection, 277-278 Radiography absorptiometry, 279-280 axial osteomalacia diagnosis, 723 calcium II deficiency syndrome diagnosis, 703 endosteal hyperostosis diagnosis, 712 fibrodysplasia ossificans progressiva diagnosis, 720 fibrogenesis imperfecta ossium diagnosis, 726-727 fluorosis diagnosis, 729 melorheostosis diagnosis, 717 mixed sclerosing bone dystrophy diagnosis, 718 osteopathia striata diagnosis, 716 osteopetrosis diagnosis, 699-700 osteopoikilosis diagnosis, 715 osteoporosis diagnosis, 390-391 pachydermoperiostosis diagnosis, 730-731 Paget's disease monitor, 555, 591-592 pyknodysostosis diagnosis, 706 radiogrammetry, 279 Reflex sympathic dystrophy, characteristics, 629-631 Regional migratory osteolysis, characteristics, 631 Remodeling, s e e Bone turnover Renal bone disease, l oL-hydroxylation impairment, 359-360 Renal calculi, Paget's disease association, 578 Renal function calcitonin effects, 106-107 calcium absorption, 178 calcium reabsorption, 65-66 chronic renal failure, 444-445 1,25-dihydroxyvitamin D synthesis, 66, 145 familial benign hypocalciuric hypercalcemia patients, 482-483, 492-493, 496 hypercalcemia association, 429, 482 hyperparathyroidism association, 418-419, 429 parathyroid hormone metabolism, 64-67 phosphorus reabsorption, 209-215 acid-base balance effects, 213 adrenal hormone effects, 213 cellular reabsorption mechanisms, 210-211 dietary phosphorus intake alterations, 214-215 growth hormone effects, 214 hyperphosphatemia, 186, 226-227 hypophosphatemia, s e e Hypophosphatemia parathyroid hormone effects, 66, 211-213 stanniocalcin, 215
superficial-deep nephron transport compared, 210 vitamin D role, 213- 214 stone formation, s e e Nephrolithiasis Renal osteodystrophy biopsy, 261-266 bone histology, 451-453 adynamic bone, 452 high-turnover bone disease, 451 lesions, 452-453 low-turnover bone disease, 452 divalent-ion metabolism alterations, 453-455 extraskeletal calcifications, 456-457 operative management, 475 osteomalacia, 451-452 overview, 443 radiographic features, 455-456 secondary hyperparathyroidism, 443-451 bone histology, 451 calcemic action resistance, 450-451 calcium sensitivity, 445-448 parathyroid hormone degradation, 451 parathyroid hormone resistance, 450-451 phosphate retention, 448-450 vitamin D metabolism alterations, 444-445 therapy, 457-460 aluminum accumulation, 460 calcium supplements, 458-459 hemodialysis dialysate calcium concentration, 459 parathyroidectomy, 459-460, 475 phosphate retention, 457-458 vitamin D, 459 Renal tubular acidosis hypophosphatemia association, 217 osteomalacia association, 366-367 Resorption, s e e Bone, resorption; Bone turnover Respiratory alkalosis, hypophosphatemia role, 222 Restriction fragment length polymorphisms, osteogenesis imperfecta mutation analysis, 672 Rheumatoid arthritis, s e e a l s o Osteoarthritis characteristics, 613 focal subchondral bone erosion, 622-624 generalized bone loss, 624-626 age effects, 625 disease activity, 625 drug effects, 625-626 fractures, 626 functional status, 625 osteoporosis treatment, 626 juvenile onset, 628-633 osteoarthritis, 631-633 reflex sympathic dystrophy, 629-631 regional migratory osteolysis, 631 marginal bone erosion, 622-624 periarticular bone loss, 624
808
Index
Rheumatological disorders, see specific t y p e s Rickets, see a l s o Osteomalacia biochemical findings, 766 classification, 765 definition, 764 drug therapy, 134 environmental association, 124-125 heritable calcipenic tickets, 767-769 heritable phosphopenic tickets, 769-777 Fanconi's syndrome, 775-777 hereditary hypercalciuria hypophosphatemic tickets, 774-775 x-linked hypophosphatemic rickets, 769-774 historical perspectives, 764-765 hypophosphatemia Fanconi's syndrome, 775-777 hereditary hypercalciuria hypophosphatemic tickets, 774-775 vitamin D-deficient rickets, 220-221 vitamin D-resistant tickets, 186, 221 x-linked hypophosphatemic tickets, 189, 218-220, 769-774 oncogenic osteomalacia, 221-222, 772-773 pathophysiology, 765 radiographic findings, 766 vitamin D therapy, 125-126, 766 Risedronate, Paget's disease treatment, 590 RNA calcitonin biosynthesis, calcitonin gene mRNA transcript splicing, 100-101 collagen mRNA mutation localization, osteogenesis imperfecta analysis, 672
S Saccharase, calcium absorption effects, 173 Saline, malignancy disease hypercalcemia treatment, 643 Sarcoidosis calcium absorption effects, 174 calcium metabolism abnormalities, 609 clinical manifestations, 611 immunology, 607-609 pathogenesis, 609-611,732 pathology, 607-609 treatment, 611-612 Sarcomas, Paget's disease complications, 564-569 Schmorl's nodule, Paget's disease histopathology, 553 Sclerosis, s e e Mixed sclerosing bone dystrophy; Osteosclerosis Sclerosteosis characteristics, 710-712 treatment, 712-713 Seminoma, characteristics, 614
Seronegative spondyloarthropathies, s e e s p e c i f i c t y p e s Sex type, calcium absorption effects, 171-172 Sialoprotein, characteristics, 6 Signal transduction, calcitonin role, 111-112 Skeletal development embryology, 2 fibroblast growth factor role, 3-5, 14-15, 763 pediatric disease role, 759-764 cell differentiation regulation, 760-762 growth factor involvement, 762-763 matrix molecules role, 764 transcriptional skeletal patterning regulation, 760762 Skeletal disorders, s e e s p e c i f i c t y p e s Skeletal muscle hyperparathyroidism association, 423-424 hypophosphatemia diagnosis, 224 osteomalacia effects, 339-340 Skeletal structure anatomy, 276 balance, 242 bone marrow cells, 243-244 bone surfaces, 238 cancellous bone, 238 cortical bone, 238 fractures, see Fractures lamellar bone, 239-240 modeling, 240-242 osteoblasts, s e e Osteoblasts osteoclasts, see Osteoclasts osteogenesis imperfecta clinical features, 657-659 structural unit, 238-239 turnover, 242 woven bone, 239-240 Skin osteogenesis imperfecta manifestations, 660 Paget's disease manifestations, 580-581 vitamin D metabolism 1,25-dihydroxyvitamin D effects, 148 photosynthesis, 127-128 Skull osteogenesis imperfecta clinical features, 659 Paget's disease manifestations, 554-557 Sodium alkali, gouty diathesis treatment, 753 Sodium cellulose phosphate, hypercalciuric calcium nephrolithiasis treatment, 744 Sodium citrate, cystine nephrolithiasis treatment, 754 Sodium fluoride fibrogenesis imperfecta ossium treatment, 728 osteoporosis management, 403-404 Sodium-phosphate co-transport proteins cellular phosphate reabsorption mechanisms, 210211,215 x-linked hypophosphatemic tickets role, 219
Index Sodium phytate, sarcoidosis treatment, 612 Somatic gene therapy, osteogenesis imperfecta management, 682 Southern blot, osteogenesis imperfecta mutation analysis, 671-672 SOX-9 transcription factor, skeletal development role, 5 Spine computed tomography assessment, 288, 558-559 osteogenesis imperfecta clinical features, 659 Paget's disease manifestations, 557-559 Spondylitis, s e e Ankylosing spondylitis Spondyloarthropathies, s e e s p e c i f i c t y p e s Stanniocalcin, phosphate excretion regulation, 215 Stress, osteogenesis imperfecta psychological management, 680-681 Struvite stones, formation, infection role, 755 Subcutaneous fat necrosis, characteristics, 615 Sudeck's atrophy, characteristics, 629-631 Surgery hyperparathyroidism treatment, 465-477 historical perspectives, 465-466 operation conduct exploration, 469-471 resection extent, 471-472 parathyroid carcinoma, 475 postoperative management, 475-477 airway protection, 476 calcium replacement, 476-477 preoperative evaluation alkaline phosphatase measurement, 467 bicarbonate measurement, 467 calcium diagnosis, 466 familial disease etiology, 468-469 general assessment, 469 informed consent, 469 localization, 468 operation indicators, 467-468 parathyroid hormone measurement, 466-467 phosphate measurement, 467 physical findings, 465-466 radiation exposure, 469 recurrent hyperparathyroidism management, 472475 localization, 472-474 reoperation, 474-475 renal osteodystrophy, 459-460, 475 hypoparathyroidism induction, 505-506 melorheostosis treatment, 717 osteogenesis imperfecta treatment, 679-680 Paget's disease treatment, 592-593 sclerosteosis treatment, 712-713 Syphilis, Paget's disease histopathology, 553 Systemic lupus erythematosus, characteristics, 621,628
809
T Tartrate-resistant acid phosphatase bone resorption markers, 319 Paget's disease association, 577 Teeth, osteogenesis imperfecta manifestations, 660661 N-Telopeptide bone resorption marker, 321 Paget's disease therapy monitor, 591-592 Tetracycline, bone mineralization mechanisms data interpretation, 329 kinetics, 334 Thiazides hypercalcemia diagnosis, 428-429 hypercalciuric calcium nephrolithiasis treatment, 744-746 hyperparathyroidism diagnosis, 428-429 osteoporosis management, 405 Thyrocalcitonin, s e e Calcitonin Thyroid gland calcitonin production, s e e Calcitonin osteogenesis imperfecta association, 662 Thyroid hormone, s e e a l s o Hyperthyroidism replacement therapy, 539-540 Thyroid-stimulating hormone, thyroid gland regulation, 531-532, 535-536, 538-540 Tiopronin, cystine nephrolithiasis treatment, 754-755 T lymphocytes, sarcoidosis pathology, 607-609 Tomography, s e e Computed tomography Toxic agents, hypoparathyroidism role, 505-506 Toxic shock syndrome, hypophosphatemia role, 222223 Toxins, hypoparathyroidism induction, 505-506 Transcription factors, skeletal development role, 5 Transforming growth factor embryologic function, 13-14 malignant disease hypercalcemia pathophysiology, 641 Transmission electron microscopy bone apatite abnormality detection, 40-41 bone crystal structure analysis, 32-35 Triiodothyronine, phosphate absorption effects, 187 Tuberculosis, characteristics, 612-613 Tumoral calcinosis, hyperphosphatemia, 227-228 Tumor lysis syndrome, hyperphosphatemia role, 228 Tumors calcitriol-induced hypercalcemia association, 613614 calcitriol therapy, 146-147 exogenous subclinical hyperthyroidism, thyroid cancer suppression, 536-537 malignant disease hypercalcemia association clinical features, 642
810
Index
Tumors ( C o n t i n u e d ) pathophysiology colony-stimulating activity, 642 hematological malignancies, 638-639 lymphomas, 613-614, 639 malignancy types, 637 metastasis solid tumors, 639-640 myeloma, 638-639 nonmetastasis solid tumors, 640-642 parathyroid hormone, 641-642 parathyroid hormone-related protein, 641 prostaglandins, 642 transforming growth factor, 641 treatment alendronate, 644-645 bisphosphonates, 644-645 calcitonin, 643 corticosteroids, 645 furosemide, 643 gallium nitrate, 646 glucocorticoids, 643 hemodialysis, 644 indications, 642-643 indomethacin, 645 intravenous saline, 643 mithramycin, 645-646 oral phosphate, 645 pamidronate, 643-644 parenteral phosphate, 643 osteomalacia induction, 772-773 Paget's disease complications, 564-569, 593 parathyroid carcinoma characteristics, 413-418 operative management, 475 pulmonary carcinoma, 614 seminoma, 614 Turnover, s e e Bone turnover
U Ultrasound, s e e a l s o Densitometry noninvasive bone assessment applications age-related changes, 295-296 bone strength measurement, 295 bone structure measurement, 295 fracture risk, 296 longitudinal monitoring, 296 osteoporosis, 295, 392 bone density measurement, 292 parameters, 292-295 broadband attenuation, 294-295 sound speed, 293-294
standardization, 295 Ultraviolet radiation, s e e a l s o Radiation vitamin D photobiology, 125-131 circulating concentrations, 130-131 cutaneous production, 128-130 human skin photosynthesis, 127-128 vitamin D production, 125-126 Uremic osteodystrophy, biopsy, 265 Ureteral diversion, osteomalacia association, 366-367 Uric acid stones, gouty diathesis, 752-753
V van Buchem's disease characteristics, 710-712 treatment, 712-713 Vascular endothelial growth factor, embryologic function, 15 Vascular system, s e e Cardiovascular system Vitamin A, hypercalcemia role, 429 Vitamin D hypercalcemia role, s e e Hypercalcemia hypermagnesemia role, 194 hypovitaminosis D osteopathy osteomalacia role bone mineral metabolism abnormalities, 345348 diagnostic considerations, 348 histological evolution, 335-338 histopathology, 340-341 skeletal radiology, 341-342 temporal evolution, 348 prevention, 370-371 treatment bone mineral metabolism effects, 371-372 management aspects, 372-373 remodeling effects, 371-372 vitamin D response, 371 intoxication, 615 metabolism abnormal physiology, 610-611 assays, 148-155 clinical utility, 152-154 1,25-dihydroxyvitamin Du measurement, 151 24,25-dihydroxyvitamin Du measurement, 151-152 25-hydroxyvitamin D measurement, 149-150 hypocalcemic evaluation, 154-155 vitamin Du measurement, 149 calcitriol effects, s e e Calcitriol defects extrinsic depletion, 354- 355 loL-hydroxylation impairment, 359-360
Index
811
Vitamin D ( C o n t i n u e d ) 25-hydroxylation impairment, 357-358 intrinsic depletion, 355-357 overview, 349- 350 saroidosis, s e e Sarcoidosis 1,25-dihydroxyvitamin D, s e e Calcitriol 24,25-dihydroxyvitamin Du conversion, 139140 historical perspectives, 124-126, 155 25-hydroxyvitamin D conversion, 132-135 a-ring metabolism, 140-141 clinical disorders, 132-135 hepatic metabolism, 132 side-chain metabolism, 140-141 intestinal absorption, 131 - 132 nonhypercalcemic analog actions, 146 normal physiology, 609-610 overview, 123-124 photobiology cutaneous production regulation, 128-130 photosynthesis in human skin, 127-128, 609 synthesis regulation, 128 ultraviolet irradiation effects, 130-131 renal osteodystrophy role secondary hyperparathyroidism, 444-445 therapy, 459 tickets, 124-125, 220-221,766 vitamin Du2, 141 - 142 osteomalacia role, s e e Osteomalacia osteoporosis association bone loss pathogenesis, 360 differential treatment, 360-361 phosphorus homeostasis regulation excretion regulation, 213- 214 hyperabsorption, 186-187 uptake stimulation, 208, 220 renal osteodystrophy therapy, 459 x-linked hypophosphatemic tickets treatment, 770771
W Warfarin, fibrodysplasia ossificans progressiva treatment, 721
William' s syndrome, characteristics, 614-615 Wilson's disease, osteomalacia association, 365-366 World wide web, osteogenesis imperfecta mutations, 671 Woven bone, characteristics, 239-240
X X-linked hypophosphatemic rickets, 769-774 biochemical fndings, 770 characteristics, 218-220 clinical features, 769-770 molecular genetics, 771-772 mouse models, 773-774 osteoblast function studies, 774 phosphate transport studies, 774 treatment, 770-771 tumor-induced osteomalacia, 772-773 vitamin D metabolism, 188-189 X-ray absorptiometry, s e e a l s o Photon absorptiometry noninvasive bone assessment dual X-ray absorptiometry cross-calibration, 299-300 methods, 282-286 osteoporosis diagnosis, 391-392 quality assurance, 298-299 standardization, 300 single X-ray absorptiometry, 281-282 X-ray diffractometry amorphous calcium-phosphorus detection, 31-32 bone apatite abnormality detection, 38-41 bone crystal structure analysis, 32-35 X rays hypovitaminosis D osteopathy detection, 341-342 renal osteodystrophy diagnosis, 455-456
Z Zoledronate, Paget's disease treatment, 590 Zollinger-Ellison syndrome, peptic ulcer association, 424 Zone of polar activity, limb development, 3
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FIGURE 24-6
Osteopetrosis. In this iliac crest specimen from a patient with the malignant form, defective osteoclastic activity is revealed by "islands" of lightly staining cartilage (white arrows) within dark staining mineralized bone (MB). Osteoclasts are numerous (black arrows) (Masson stain; ~150).
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