Chi Keung Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 219-251). / Chapter CHAPTER I --- LITERATURE REVIEW / Chapter 1 --- The structure of bone --- p.2 / Chapter 1.1. --- The cortical bone --- p.3 / Chapter 1.2. --- The cancellous bone --- p.3 / Chapter 2 --- The composition of bone --- p.3 / Chapter 2.1. --- Bone minerals --- p.4 / Chapter 2.2. --- The organic matrix --- p.4 / Chapter 2.3. --- The bone cells --- p.9 / Chapter 2.3.1. --- The osteoblast and the osteocyte --- p.9 / Chapter 2.3.2. --- The osteoclast --- p.11 / Chapter 3 --- Bone turnover - modelling and remodelling of bone --- p.13 / Chapter 3.1. --- Postulated sequence of bone remodelling --- p.14 / Chapter 4 --- Regulation of bone resorption --- p.16 / Chapter 4.1. --- Role of osteoblast and the lining cell on bone resorption --- p.17 / Chapter 5 --- Regulation of bone formation --- p.19 / Chapter 6 --- Effects of systemic hormones and local factors on bone metabolism --- p.20 / Chapter 6.1. --- Parathyroid hormone --- p.20 / Chapter 6.2. --- "1,25-dihydroxyvitamin D3" --- p.22 / Chapter 6.3. --- Calcitonin --- p.23 / Chapter 6.4. --- Prostaglandins --- p.23 / Chapter 6.5. --- Sex hormones --- p.24 / Chapter 6.6. --- Glucocorticoid --- p.26 / Chapter 6.7. --- Growth hormone --- p.27 / Chapter 6.8. --- Insulin --- p.28 / Chapter 6.9. --- Thyroid hormones --- p.29 / Chapter 6.10. --- Other systemic and local factors --- p.30 / Chapter 7 --- Indices of bone turnover --- p.34 / Chapter 8 --- Non-biochemical indices of bone metabolism --- p.34 / Chapter 8.1. --- Radionuclide bone scan --- p.34 / Chapter 8.2. --- Radiokinetic assessment --- p.35 / Chapter 8.3. --- Bone biopsy --- p.35 / Chapter 8.4. --- Bone densitometry --- p.36 / Chapter 9 --- Biochemical indices of bone metabolism --- p.37 / Chapter 10 --- Biochemical markers of bone formation --- p.38 / Chapter 10.1. --- Alkaline phosphatase --- p.38 / Chapter 10.1.1. --- Role and origin of bone alkaline phosphatase isoenzyme --- p.39 / Chapter 10.1.2. --- Measurement of bone alkaline phosphatase --- p.41 / Chapter 10.1.2.1. --- Heat inactivation --- p.42 / Chapter 10.1.2.2. --- Chemical inactivation --- p.43 / Chapter 10.1.2.3. --- Immunological methods --- p.44 / Chapter 10.1.2.4. --- High performance liquid chromatography --- p.45 / Chapter 10.1.2.5. --- Gel electrophoresis --- p.45 / Chapter 10.1.2.6. --- Isoelectric focusing --- p.47 / Chapter 10.2. --- Osteocalcin --- p.48 / Chapter 10.3. --- Osteonectin --- p.51 / Chapter 10.4. --- Matrix Gla-protein --- p.51 / Chapter 10.5. --- Other non-collagenous proteins --- p.52 / Chapter 10.6. --- Urinary Gla concentration --- p.52 / Chapter 10.7. --- Collagen peptides and extension peptides --- p.54 / Chapter 11 --- Biochemical markers of bone resorption --- p.55 / Chapter 11.1. --- Urine hydroxyproline --- p.55 / Chapter 11.2. --- Pyridinium cross-links --- p.58 / Chapter 11.3. --- Acid phosphatase --- p.60 / Chapter 11.3.1. --- Acid phosphatase isoenzymes --- p.60 / Chapter 11.3.2. --- The band 5 acid phosphatase isoenzyme genetics and characteristics --- p.62 / Chapter 11.3.3. --- Band 5 acid phosphatase as marker of osteoclastic function --- p.64 / Chapter 11.3.4. --- Measurement of osteoclastic acid phosphatase --- p.67 / Chapter 11.3.4.1. --- Specific chemical inhibitor --- p.67 / Chapter 11.3.4.2. --- Electrophoresis --- p.67 / Chapter 11.3.4.3. --- Immunological methods --- p.68 / Chapter 12 --- Problems with current biochemical markers of bone metabolism --- p.68 / Chapter 13 --- Aims of this study --- p.70 / Chapter CHAPTER II --- PURIFICATION OF TARTRATE-RESISTANT ACID PHOSPHATASE AND THE DEVELOPMENT OF AN IMMUNOASSAY FOR IT'S MEASUREMENT / Chapter 1 --- Introduction --- p.72 / Chapter 2 --- Materials and methods --- p.75 / Chapter 2.1. --- Chemicals and reagents --- p.75 / Chapter 2.1.1. --- Apparatus --- p.76 / Chapter 2.2. --- Methods --- p.77 / Chapter 2.2.1. --- Cord serum --- p.77 / Chapter 2.2.2. --- Measurement of tartrate-resistant acid phosphatase activity --- p.77 / Chapter 2.2.3. --- Measurement of protein concentration --- p.80 / Chapter 2.2.4. --- Purification of TRACP from cord plasma --- p.82 / Chapter 2.2.4.1. --- Cation-exchange column chromatography --- p.83 / Chapter 2.2.4.2. --- Gel filtration column chromatography --- p.84 / Chapter 2.2.4.3. --- Concanavalin A-affinity column chromatography --- p.85 / Chapter 2.2.4.4. --- Preparative isoelectric focusing (IEF) --- p.86 / Chapter 2.3. --- Characterisation of purified TRACP --- p.90 / Chapter 2.3.1. --- Polyacrylamide gel electrophoresis (PAGE) --- p.91 / Chapter 2.3.2. --- "Optimum pH, substrate specificity and the effects of potential activators and inhibitors on TRACP activity" --- p.99 / Chapter 2.3.3. --- Amino acid composition of purified TRACP --- p.101 / Chapter 2.4. --- Methods for raising anti-human TRACP antibody and characterisation of the antiserum --- p.102 / Chapter 2.4.1. --- Production of rabbit anti-human TRACP antibody --- p.102 / Chapter 2.4.2. --- Determination of the titre of rabbit anti-human TRACP antibody --- p.103 / Chapter 2.4.3. --- Immunoblotting analyses for cross reactivity study --- p.103 / Chapter 2.4.4. --- Immunohistochemical study for antibody specificity --- p.105 / Chapter 2.4.5. --- Cross reactivity study of the rabbit anti-human TRACP antibody to some tissue preparations --- p.107 / Chapter 2.5. --- Enzyme linked immunosorbent assay for TRACP --- p.109 / Chapter 2.5.1. --- Optimisation and evaluation of the new ELISA method for TRACP --- p.111 / Chapter 3 --- RESULTS --- p.113 / Chapter 3.1. --- "Precision of methods for the determination of protein, TRACP and phosphate." --- p.113 / Chapter 3.2. --- Isolation and purification of TRACP --- p.113 / Chapter 3.2.1. --- Concanavalin A affinity chromatography --- p.120 / Chapter 3.2.2. --- Isoelectric focusing (IEF) --- p.120 / Chapter 3.3. --- Characterisation and homogeneity of purified TRACP --- p.128 / Chapter 3.3.1. --- Characterisation of purified TRACP --- p.128 / Chapter 3.3.2. --- Homogeneity of purified TRACP --- p.132 / Chapter 3.3.3. --- Amino acid composition --- p.136 / Chapter 3.4. --- Characterisation of the rabbit anti-human TRACP antibody --- p.136 / Chapter 3.4.1. --- Antibody specificity - immunoblotting study --- p.139 / Chapter 3.4.2. --- Antibody specificity - cross reactivity with partially purified non-cord plasma TRACP --- p.142 / Chapter 3.4.3. --- Antibody specificity - immunohistochemical study --- p.145 / Chapter 3.5. --- Enzyme linked immunosorbent assay for TRACP --- p.145 / Chapter 3.5.1. --- Optimal concentration of antigen for coating of microtitre plate --- p.145 / Chapter 3.5.2. --- Kinetics of reaction with the primary rabbit anti-human TRACP antibody --- p.149 / Chapter 3.5.3. --- "Precision, recovery and assay range" --- p.149 / Chapter 4 --- DISCUSSION --- p.155 / Chapter 4.1. --- Purification of cord plasma TRACP --- p.155 / Chapter 4.2. --- Characterisation of cord plasma TRACP --- p.158 / Chapter 4.3. --- Characterisation of rabbit anti-human TRACP antibody --- p.163 / Chapter 4.4. --- Enzyme immunoassay for TRACP --- p.165 / Chapter CHAPTER III --- STUDY OF SERUM TRACP IN HEALTHY SUBJECTS AND IN PATIENTS WITH BONE RELATED DISEASES / Chapter 1 --- Introduction --- p.168 / Chapter 2 --- Materials and methods --- p.171 / Chapter 2.1. --- Subjects --- p.171 / Chapter 2.1.1. --- Healthy subjects --- p.171 / Chapter 2.1.2. --- Patients --- p.172 / Chapter 2.1.2.1. --- Post-menopausal women on hormone replacement therapy --- p.172 / Chapter 2.1.2.2. --- Hip fracture patients --- p.173 / Chapter 2.1.2.3. --- Other patients --- p.174 / Chapter 2.3. --- Measurement of other biochemical parameters --- p.175 / Chapter 2.3.1. --- Bone alkaline phosphatase --- p.175 / Chapter 2.3.2. --- "Measurement of urine hydroxyproline, creatinine, calcium, osteocalcin, thyroid hormones and parathyroid hormone" --- p.176 / Chapter 2.4. --- Statistics --- p.178 / Chapter 3 --- RESULTS --- p.179 / Chapter 3.1. --- Healthy subjects --- p.179 / Chapter 3.2. --- Serum TRACP concentration in post-menopausal women before and after hormone replacement therapy --- p.185 / Chapter 3.3. --- TRACP concentration in elderly subjects with hip fractures --- p.189 / Chapter 3.4. --- Serum TRACP concentrations in patients with other bone related diseases --- p.190 / Chapter 3.4.1. --- Hyperthyroidism --- p.194 / Chapter 3.4.2. --- Hyperparathyroidism --- p.198 / Chapter 3.4.3. --- Haemodialysis --- p.201 / Chapter 4 --- DISCUSSION --- p.204 / GENERAL DISCUSSION --- p.216 / REFERENCES --- p.219

by Chak Chi Wai. / Thesis (M.Phil)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 142-155). / ACKNOWLEDGMENTS --- p.i / TABLE OF CONTENT --- p.ii / LIST OF ABBREVIATION --- p.viii / ABSTRACT --- p.x / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- INTRODUCTION TO ALKALINE PHOSPHATASE --- p.2 / Chapter 1.1.1 --- The Alkaline Phosphatase Isoenzymes --- p.2 / Chapter 1.1.2 --- The Properties of Alkaline Phosphatases --- p.5 / Chapter 1.1.3 --- Serum Alkaline Phosphatases --- p.7 / Chapter 1.1.3.1 --- Intestinal Alkaline Phosphatase --- p.8 / Chapter 1.1.3.2 --- Placental Alkaline Phosphatase --- p.8 / Chapter 1.1.3.3 --- Renal Alkaline Phosphatase --- p.9 / Chapter 1.1.3.4 --- Skeletal Alkaline Phosphatase --- p.9 / Chapter 1.1.3.5 --- Hepatic Alkaline Phosphatase --- p.10 / Chapter 1.1.3.6 --- Miscellaneous Alkaline Phosphatases --- p.10 / Chapter 1.1.4 --- Problems in Discriminating the Skeletal and Hepatic Alkaline Phosphatase in Serum --- p.12 / Chapter 1.1.5 --- Wheat Germ Lectin Precipitation of the Bone- Specific Alkaline Phosphatase --- p.13 / Chapter 1.2 --- STRUCTURE OF BONE AND MECHANISMS OF CALCIFICATION --- p.15 / Chapter 1.2.1 --- Gross Structure of Bone --- p.15 / Chapter 1.2.2 --- The Elements of Bone --- p.17 / Chapter 1.2.2.1 --- Bone Cells --- p.17 / Chapter 1.2.2.2 --- Organic Substances of Bone --- p.19 / Chapter 1.2.2.3 --- Inorganic Substances of Bone --- p.21 / Chapter 1.2.3 --- Mechanisms of Calcification --- p.22 / Chapter 1.3 --- BONE FRACTURE HEALING --- p.24 / Chapter 1.3.1 --- Types of Fracture --- p.24 / Chapter 1.3.2 --- The Process of Bone Fracture Healing --- p.26 / Chapter 1.3.2.1 --- Stage of Hematoma --- p.26 / Chapter 1.3.2.2 --- Stage of Subperiosteal and Endosteal Cellular Proliferation --- p.28 / Chapter 1.3.2.3 --- Stage of Fibrocartilaginous Callus --- p.28 / Chapter 1.3.2.4 --- Stage of Bony Callus --- p.30 / Chapter 1.3.2.5 --- Stage of Remodeling --- p.31 / Chapter 1.4 --- THE OSTEOBLASTIC CHARACTERS OF UMR-106 OSTEOSARCOMA CELL LINE --- p.32 / Chapter 1.4.1 --- Classification of Osteosarcoma --- p.32 / Chapter 1.4.2 --- Derivation of UMR-106 Osteosarcoma Cell Line --- p.33 / Chapter 1.4.3 --- Osteoblastic Characters of UMR-106 --- p.34 / Chapter 1.4.3.1 --- ALP Expression --- p.34 / Chapter 1.4.3.2 --- Hormone Responsive Adenylate Cyclase System --- p.35 / Chapter 1.4.3.3 --- "Cytosolic Receptors for 1,25-Dihydroxy- cholecalciferol" --- p.35 / Chapter 1.5 --- IN VITRO CULTURE OF FETAL RAT CALVARIAL OSTEOBLASTS --- p.37 / Chapter 1.6 --- AIM AND SCOPE OF THIS DISSERTATION --- p.39 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- BONE FRACTURE OPERATION --- p.42 / Chapter 2.1.1 --- Animals --- p.42 / Chapter 2.1.2 --- Blood Sampling and Preparation of Plasma Samples --- p.42 / Chapter 2.1.3 --- Bone Fracture Operation --- p.43 / Chapter 2.1.3.1 --- Reagents and Apparatus --- p.43 / Chapter 2.1.3.2 --- Procedures --- p.44 / Chapter 2.1.4 --- Radiography --- p.50 / Chapter 2.1.5 --- Removal of Tibiae --- p.51 / Chapter 2.1.6 --- Extraction of Callus ALP --- p.51 / Chapter 2.1.6.1 --- Reagent --- p.51 / Chapter 2.1.6.2 --- Homogenization of the Callus --- p.51 / Chapter 2.1.6.3 --- Extraction of ALP --- p.52 / Chapter 2.1.7 --- Assay for Bone-Specific ALP --- p.53 / Chapter 2.1.7.1 --- Reagents --- p.53 / Chapter 2.1.7.2 --- Procedures --- p.54 / Chapter 2.1.8 --- Normal Curve for Plasma Bone-Specific ALP in Rabbits --- p.56 / Chapter 2.1.9 --- The Effects of Tibial Fracture on the Plasma Level of Bone-Specific ALP in Rabbits --- p.56 / Chapter 2.1.10 --- Profile of Plasma Bone-Specific ALP upon a Fracture Healing --- p.57 / Chapter 2.1.11 --- Profile of Callus Bone-Specific ALP at Different Stages of Fracture Healing --- p.57 / Chapter 2.2 --- CLINICAL STUDIES OF PLASMA BONE-SPECIFIC ALP --- p.58 / Chapter 2.2.1 --- Patient Groups --- p.58 / Chapter 2.2.1.1 --- Normal Adults --- p.58 / Chapter 2.2.1.2 --- Fracture Group --- p.58 / Chapter 2.2.1.3 --- Tumor Group --- p.59 / Chapter 2.2.2 --- Assays for Plasma Bone-Specific ALP --- p.59 / Chapter 2.3 --- "IN VITRO CULTURES OF FETAL, RAT OSTEOBLASTS AND UMR-106 OSTEOSARCOMA cell line" --- p.60 / Chapter 2.3.1 --- Animals --- p.60 / Chapter 2.3.2 --- UMR-106 Cell Line --- p.60 / Chapter 2.3.3 --- General Reagents Used for Cell Culture --- p.60 / Chapter 2.3.4 --- Isolation of Calvarial Osteoblasts --- p.64 / Chapter 2.3.4.1 --- Tools and Reagents --- p.64 / Chapter 2.3.4.2 --- Procedures --- p.65 / Chapter 2.3.5 --- Storage of UMR-106 Cell Line --- p.67 / Chapter 2.3.6 --- Subculture of Confluent Monolayer --- p.68 / Chapter 2.3.6.1 --- Reagents --- p.68 / Chapter 2.3.6.2 --- Procedures --- p.69 / Chapter 2.3.7 --- Staining for Calcium Deposits --- p.69 / Chapter 2.3.7.1 --- Reagents --- p.70 / Chapter 2.3.7.2 --- Procedures --- p.70 / Chapter 2.3.8 --- Protein Determination --- p.71 / Chapter 2.3.8.1 --- Reagents --- p.71 / Chapter 2.3.8.2 --- Procedures --- p.71 / Chapter 2.3.9 --- Microdetermination of Inorganic Phosphate --- p.72 / Chapter 2.3.9.1 --- Reagents --- p.72 / Chapter 2.3.9.2 --- Procedures --- p.73 / Chapter 2.3.10 --- Determination of Calcium --- p.73 / Chapter 2.3.10.1 --- Reagent --- p.73 / Chapter 2.3.10.2 --- Procedures --- p.73 / Chapter 2.3.11 --- Extraction and Assay for Cellular ALP --- p.74 / Chapter 2.3.11.1 --- Reagents --- p.74 / Chapter 2.3.11.2 --- Procedures --- p.75 / Chapter 2.3.12 --- Cell Surface ALP Assay --- p.75 / Chapter 2.3.12.1 --- Reagents --- p.75 / Chapter 2.3.12.2 --- Procedures --- p.76 / Chapter 2.3.13 --- Extraction of Calcium Phosphate Deposits --- p.76 / Chapter 2.3.13.1 --- Reagent --- p.76 / Chapter 2.3.13.2 --- Procedures --- p.76 / Chapter 2.3.14 --- Collagen Synthesis Assay --- p.77 / Chapter 2.3.14.1 --- Reagents --- p.77 / Chapter 2.3.14.2 --- Procedures --- p.78 / Chapter CHAPTER THREE: --- EFFECTS OF TIBIAL FRACTURE ON THE LEVEL OF BONE-SPECIFIC ALKALINE PHOSPHATASE IN RABBITS / INTRODUCTION --- p.81 / results: / Chapter 3.1 --- normal curve for plasma bone-specific alp in rabbits --- p.82 / Chapter 3.2 --- THE EFFECTS OF TIBIAL FRACTURE ON THE PLASMA LEVEL OF BONE-SPECIFIC ALP IN RABBITS --- p.84 / Chapter 3.3 --- PROFILE OF THE PLASMA ALP LEVEL UPON HEALING OF TIBIAL FRACTURE --- p.86 / Chapter 3.4 --- RADIOGRAPHY --- p.89 / Chapter 3.5 --- PROFILE OF CALLUS BONE-SPECIFIC ALP ACTIVITY UPON HEALING OF TIBIAL FRACTURE --- p.93 / DISCUSSION --- p.95 / Chapter CHAPTER FOUR: --- CLINICAL STUDIES OF PLASMA BONE-SPECIFIC ALKALINE PHOSPHATASE / INTRODUCTION --- p.100 / RESULTS: / Chapter 4.1 --- NORMAL VALUES --- p.100 / Chapter 4.2 --- FRACTURE GROUP --- p.101 / Chapter 4.3 --- BONE TUMOR GROUP --- p.102 / DISCUSSION --- p.102 / Chapter CHAPTER FIVE: --- IN VITRO CULTURE OF FETAL RAT OSTEOBLASTS AND UMR-106 CELL LINE / INTRODUCTION --- p.105 / RESULTS: / Chapter 5.1 --- IN VITRO MINERALIZATION OF UMR-106 CELLS AND PRIMARY RC CELLS --- p.107 / Chapter 5.2 --- STUDY OF BONE-SPECIFIC ALP RELEASED INTO MEDIUM BY UMR-106 CELLS AND PRIMARY RC CELLS --- p.113 / Chapter 5.3 --- STUDY OF CELLULAR ALP ACTIVITIES AND CALCIUM PHOSPHATE DEPOSITS --- p.116 / Chapter 5.4 --- STUDIES OF CELLULAR ALP ACTIVITIES AND RELATIVE RATES OF COLLAGEN SYNTHESIS --- p.125 / DISCUSSION --- p.128 / Chapter CHAPTER SIX: --- GENERAL DISCUSSION --- p.136 / BIBLIOGRAPHY --- p.142 / APPENDIX --- p.156

Several hundred inherited disorders of the skeleton have been delineated. Individually these conditions are rare, but as a group they cause much crippling and hardship. Several factors, including the rarity and complexity of the manifestations of these conditions, as well as semantic overlap, impede the accurate diagnosis which is essential for effective treatment. In this regard, the adoption of microcomputers warrants evaluation as a high technology aid. Microcomputers have developed tremendous capabilities during recent years. The state of the art has become such that a diagnostic aid facility on such a device has been demonstrated in various disciplines of medicine and may also be feasible in the area of inherited skeletal disorders. The study which forms the basis of this thesis, concerns the investigation of this feasibility and has led to the development of an effective working model which sets the basis for microcomputer-aided diagnosis. The design features followed in this project are similar to those conventionally employed for "Expert systems" on mainframe computers. A comprehensive knowledge base consisting of over 200 skeletal disorders and 700 radiographic and clinical manifestations, has resulted. Furthermore, the application is capable of "learning", although inference as employed by the inference engines of real expert systems, is not employed. In this context learning implies that the knowledge base, with the passage of time, improves considerably when used by experts. Serendipitous findings in this regard are: • 1) Considerable improvement of existing profile descriptions can occur without any increased demands on computer memory and storage space; • 2) Growth of the knowledge base in the form of additional disease profiles can be effected with very modest inroads on memory and storage resources. The computerized diagnostic aid which resulted from this thesis, has been demonstrated to be successful in both the Department of Human Genetics of the University of Cape Town and the Department of Paediatrics of the Johannes Gutenberg University in Mainz. Evaluated both in terms of efficiency and utility, the system provides an enhancement to the specialist genetic diagnostician. These achievements have been effected by means of a unique newly developed application of compressed bit-mapping, attained by writing the applicable programs in Turbo Pascal and 8086- assembler languages. Calculations indicate that much larger data bases may possibly be implemented on present-day microcomputers by means of the methods developed in this project.