The Cytochemical Bioassay of Polypeptide Hormones

1. Nature of Cytochemistry.- A. Definitions and Origins.- I. Confusion Between Cytochemistry and Histochemistry.- II. Definition of Histochemistry.- III. Aims of Cytochemistry.- IV. Origin of Cytochemistry.- B. Relationship Between Cytochemistry and Biochemistry.- I. Material.- II. Reaction.- III. Precipitation.- 1. The Need for Precipitation.- 2. Methods for Obtaining an Insoluble Reaction Product.- a) Binding to an Insoluble Tissue Component.- b) Simultaneous Capture Methods.- c) Post-coupling Techniques.- d) Tetrazolium Salts.- IV. Measurement.- 2. Basic Concepts of Hormone Assays.- A. Two Ways of Assaying a Biologically Active Molecule.- I. General Considerations.- II. Assay of Enzymes.- B. Radio-immunoassay.- I. General.- II. Sensitivity of Radio-immunoassay.- C. Drawback of the Analytical Approach.- I. Problem of Fragments.- II. Problem of Different Forms of the Hormone.- III. Special Advantage of the Analytical Method.- D. The Need for Microbioassays.- 3. Introduction to Cytochemical Bioassay.- A. Criteria for Bioassays.- I. Basic Requirements.- 1. Nature of Bioassays.- 2. Parallelism.- 3. Precision of Bioassays.- 4. Accuracy.- 5. Specificity.- 6. Sensitivity.- 7. Variability.- II. Problems of Bioassays.- 1. Different Potencies Depending on Route of Administration.- 2. Problem of Desialation.- 3. Secondary Effects of Hormones.- B. Cytochemical Bioassays.- I. Basic Approach.- 1. Biochemical Effect of a Hormone.- 2. Hormonal Status of Tissues.- 3. Physiological State of the Target Cells.- 4. The Need for Essential Metabolites.- 5. Summary of the Basic Features of Cytochemical Bioassays.- II. Cytochemical Section Assays.- 1. Basic Procedures.- 2. Critical Importance of Colloid Stabilizers.- C. Special Features of Cytochemical Bioassays.- I. Details of the Assays.- 1. Dose-Response Calibration Graphs.- 2. Parallelism.- II. Deviations from Parallelism.- III. Use of Plasma.- 1. Loss of Biological Activity.- 2. Active Substances in Blood Cells.- 3. Configuration of Polypeptide Hormones.- IV. Physiological Studies.- 4. Maintenance Culture.- A. Importance of Organ Culture.- I. Changes Induced by Proliferative Culture.- II. Disadvantages of Isolated Cells.- B. Earlier Use of Organ Culture.- C. Maintenance Culture as Used in Cytochemical Bioassays.- I. Aims.- 1. Removal from the Influence of the Animal.- 2. Recovery from the Trauma of Excision.- 3. Restoration of Metabolites.- II. Procedure.- 1. Selection of the Animal Species.- 2. Segments.- 3. Culture Medium.- 4. Maintenance Culture.- III. Technical Considerations.- 5. Cytochemical Methods.- A. Basic Procedures.- I. Preparation of Tissue Slices.- 1. Histological Techniques.- 2. Chilling.- 3. Chilling Procedure Used in Cytochemical Bioassays.- 4. Mounting the Specimen.- 5. Sectioning.- 6. Flash Drying.- 7. Protection of Undenatured Sections.- B. Demonstration of Dehydrogenase Activity.- I. Nature of Reduced Tetrazolium Salt.- II. Terminology.- III. Use of an Intermediate Hydrogen Acceptor.- IV. Validation.- V. Function of Reducing Equivalents.- VI. 'NADPH-diaphorase' System.- C. Cytochemical Investigation of Lysosomal Function.- I. General.- II. Cytochemical Investigation of Lysosomal Function.- 6. Microdensitometry.- A. Development of Microspectrophotometry.- I. Background.- 1. Artefact of the 'Average' Nucleus.- 2. Ultraviolet Microspectrophotometry and Potential Errors.- 3. Non-specific Absorption.- 4. Diffraction.- B. Microdensitometry.- I. Basis of Measurement.- 1. Application of the Beer-Lambert Law.- 2. Optical Inhomogeneity.- II. Use of a Scanning and Integrating Microdensitometer.- 1. Procedure.- 2. Mean Integrated Extinction.- III. Validation of Microdensitometry.- 7. The Segment Assay of the Adrenocorticotrophic Hormone.- A. Rationale.- I. Background.- 1. The Hormone.- 2. Biological Action of ACTH on the Adrenal Gland.- II. Ascorbate Depletion and Hydroxylation.- III. Hypotheses Concerning the Mode of Action of ACTH.- B. Cytochemical Segment Assay of ACTH.- I. Introduction.- II. Procedure.- 1. Target Tissue.- 2. Apparatus.- 3. Culture Medium and Procedure.- 4. Function of the Maintenance Culture.- 5. Exposure to the Hormone.- 6. Cytochemical Reaction.- 7. Measurement.- III. Results and Validation.- 1. The 4 + 2 Assay: Precision.- 2. Sensitivity.- 3. Reproducibility.- 4. Accuracy and Specificity.- IV. Comparison with Other Assays.- V. Comparison with Radio-immunoassay.- 8. The Section Assay of Adrenocorticotrophin.- A. Background.- I. The Need for a More Routine Assay.- II. The Problems.- B. Cytochemical Section Assay of ACTH.- C. Validation.- 9. Application of Cytochemical Bioassays of Adrenocorticotrophin.- A. Physiological Studies.- I. Half-Time of Bioreactive ACTH.- II. Circadian Rhythm and Episodic Secretion of ACTH.- B. Advantages of a Very Sensitive Assay.- I. ACTH Levels in Neonates.- II. Hypopituitarism.- III. Studies on Small Animals.- C. Insulin-Hypoglycaemia Test of Hypothalamic-Pituitary-Adrenal Function.- I. Insulin-Hypoglycaemia Test.- II. Activity-Time Response in the Hypoglycaemia Test.- 10. The Assay of Thyroid Stimulators.- A. Background.- I. Thyroid-Stimulating Hormone.- 1. Nature of Thyrotrophin.- 2. Effect of TSH Acting on the Thyroid.- 3. Radio-immunoassay.- 4. Discrepancies Between Radio-immunoassay and Physiology.- II. Thyroid-Stimulating Immunoglobulins.- B. Cytochemical Segment Assay of Thyroid Stimulators.- I. Rationale.- II. Procedure.- III. Validation.- 1. Time Course of Response.- 2. Nature of the Response.- 3. Specificity and Accuracy.- 4. Sensitivity and Reproducibility.- 5. Inadvisability of Using Serum.- 6. Correlation with Radio-immunoassay.- 7. Circulating Levels of TSH and Discrepancies with Radio-immunoassay.- IV. Studies with Thyroid-Stimulating Immunoglobulins.- C. Cytochemical Section Assay of Thyroid Stimulators.- I. Background.- II. Procedure.- III. Validation.- D. A Possible Mechanism for the Action of Thyroid Stimulators.- E. Influence of Thyrotrophin on Oxidative Metabolism.- I. Significance of the Pentose Shunt.- II. NAD+-kinase.- III. Influence of Thyrotrophin.- 1. NADPH diaphorase.- 2. NAD+-kinase.- 11. Luteinizing Hormone.- A. Background Information.- I. Nature of the Hormone.- II. Methods of Assaying Luteinizing Hormone.- B. Cytochemical Bioassay of Luteinizing Hormone.- I. Original Procedure.- 1. Background.- 2. Original Cytochemical Segment Bioassay.- 3. Discrepancy Between Results with this Bioassay and with Radio-immunoassay.- II. Cytochemical Section Assay.- 1. Background.- 2. Procedure.- 3. Validation.- 12. Gastrin-like Activity.- A. Background.- I. Nature of Gastrin.- II. Biological Effects of Gastrin.- B. Cytochemical Segment Assay.- I. Carbonic Anhydrase.- 1. Background.- 2. Cytochemical Reaction.- 3. Rationale.- 4. Measurement.- II. Segment Assay.- 1. Background: pH of the Medium.- 2. Procedure.- 3. Precision.- C. Cytochemical Section Assay.- I. Problems.- II. Assay.- 1. Procedure.- 2. Precision.- D.Validation.- I. Effect of Cholecystokinin.- II. Effect of the Octapeptide of CCK.- III. Effect of Secretin.- IV. Effects of Other Mediators of Secretion.- V. Effect of Big Gastrin.- VI. Trasylol: Measurement of Little and Big Gastrin Activity.- VII. Comparison of the Cytochemical Bioassay and Radio-immunoassay.- E. Physiological Studies.- I. Effects of a Test Meal.- II. Studies on Pernicious Anaemia.- 13. Parathyroid Hormone.- A. Background Information.- I. The Hormone.- II. Bioassays.- III. In Vitro Assays.- B. Cytochemical Bioassay of Parathyroid Hormone.- I. Background.- 1. Possible Physiological Functions of the Hormone.- II. Cytochemical Studies on the Responses of the Renal Cortex to PTH.- 1. General Investigations.- 2. Method of Relating Activity to Histology.- 3. Effect of PTH on Different Regions of the Nephron.- III. Cytocherrfical Bioassay.- 1. Studies Required to Produce an Assay.- 2. Assay Procedure.- IV. Validation.- 14. Hypophysiotrophic Hormones: Two-Step Bioassays.- A. Background.- I. Hypothalamus as an Endocrine Organ.- II. Hypothalamic Hormones.- III. Problems in the Direct Bioassay of Hypophysiotrophic Hormones.- B. Thyrotrophin-Releasing Hormone.- I. Structure.- II. Distribution of Thyrotrophs in the Pituitary Gland.- III. Cytochemical Bioassay.- C. Corticotrophin-Releasing Factor.- I. Cytochemical Bioassay.- II. Results with the Bioassay.- 15. Prospects: Uses of the Cytochemical Bioassay System.- A. Bioassay of Hormones.- I. New Assays.- II. Assay of Similar Factors.- III. Influence of the 'Biologically Inactive' Part of the Molecule.- IV. Blocking or Stimulating 'Antibodies': The Importance of Parallelism.- B. Cytochemistry as Cellular Chemical Pathology.- C. Study of Biologically Active Molecules.- D. Conclusion.- Appendix 1. Maintenance Culture.- Appendix 2. Microdensitometry with the Viekers M85 Microdensitometer.- Appendix 3. Main Cytochemical Reactions Used in These Assays.- Appendix 4. Dilution of the Standard Reference Preparations.- References.