Prostaglandins and related substances a practical approach

Transcription

1 O Prostaglandins and related substances a practical approach Edited by c Benedetto Institute of Obstetrics and Gynecology, Universita di Torino, Via Ventimiglia 3, Torino 10126, Italy R C McDonald-Gibson Department of Biochemistry, Brunei University, Uxbridge, Middlesex UB8 3PH, UK S Nigam Department of Gynecological Endocrinology, Prostaglandin Research Laboratories, Klinikum Steglitz, Freie Universitat, D-1000 Berlin 45, FRG T F Slater Department of Biochemistry, Brunei University, Uxbridge, Middlesex UB8 3PH, UK OIRL PRESS OXFORD WASHINGTON DC

2 Contents SECTION I SECTION II SECTION III SECTION IV SECTION V SECTION VI APPENDIX INTRODUCTION AND METABOLISM 1. Introduction to the Eicosanoids 1 2. Metabolism of Prostaglandins and Lipoxygenase Products: Relevance for Eicosanoid Assay 5 SAMPLING AND EXTRACTION 3. Tissue Sampling and Preparation Extraction of Eicosanoids from Biological Samples 45 PHYSICO-CHEMICAL METHODS 5. Thin-Layer Chromatography (including Radio Thin- Layer Chromatography and Autoradiography) of Prostaglandins and Related Compounds High-Pressure Liquid Chromatography in the Analysis of Arachidonic Acid Metabolites Gas Chromatography and Mass Spectrometry of Eicosanoids 99 PHARMACOLOGICAL AND IMMUNOASSAYS 8. Measurement of Prostaglandins, Thromboxanes and Leukotrienes by Smooth Muscle Bioassay Aggregometry Techniques for Prostanoid Study and Evaluation Radioimmunoassay of Eicosanoids Enzyme Immunoassay 197 ENZYMOLOGY AND LIPID PEROXIDATION 12. Cyclo-Oxygenase: Measurement, Purification and Properties Lipoxygenases: Measurement, Characterization and Properties Lipid Peroxidation 243 RELATED TECHNIQUES 15. Quantitative Measurement of Arachidonic Acid in Tissues or Fluids Methods in Prostanoid Receptor Classification Paf-Acether (Platelet-Activating Factor) 305 EICOSANOIDS: STRUCTURE, AVAILABILITY, STORAGE AND STABILITY 313 IX

3 ABBREVIATIONS xix 1. INTRODUCTION TO THE EICOSANOIDS 1 T.F.Slater and R.G.McDonald-Gibson General Background 1 Nomenclature, Structure and Biosynthesis 1 References 4 2. METABOLISM OF PROSTAGLANDINS AND LIPOXY- GENASE PRODUCTS: RELEVANCE FOR EICOSANOID ASSAY 5 E.Granstrom and M.Kumlin Introduction: The 'What, When, and Where' Problem 5 Metabolism of Prostaglandins 7 General comments 7 Individual metabolic fates of the prostaglandins 9 Metabolism of Thromboxanes 16 General comments 16 Metabolism of thromboxane B 2 16 Interconversions of Prostaglandins and Related Compounds 18 Metabolism of Lipoxygenase Products 20 General comments 20 Metabolism of hydroperoxy acids and monohydroxy acids 20 Metabolism of the epoxy intermediates, LTA4 and 14,15-LTA 4 22 Metabolism of dihydroxy acids including LTB 4 23 Metabolism of the peptido-leukotrienes,- LTC 4, LTD 4 and LTE 4 23 Conclusions 25 References TISSUE SAMPLING AND PREPARATION 29 C.Benedetto and T.F.Slater Introduction 29 Sampling Blood and Urine 29 Blood 29 Urine 32 Sampling Solid Tissues and Intracellular Fractions 33 Endogenous levels in solid tissue in situ 33 Sampling cells and intracellular suspensions 37 Tissue Preparations 38 Perfused organs 39 Tissue slices and rings 39 Isolated cells 39 Homogenizing Tissue Samples 40

4 Use of inhibitors Rapidity and cooling Homogenate complexity Separation of intracellular organelles Acknowledgements References 4. EXTRACTION OF EICOSANOIDS FROM BIOLOGICAL SAMPLES S.Nigam Introduction Methods of Extraction Organic solvent extraction and open column chromatography Powell method for extraction of eicosanoids other than peptidoleukotrienes from biological samples using octadecylsilyl (ODS)-silica Extraction of peptido-leukotrienes Recoveries of Standard Eicosanoids from Biological Samples General Considerations on the Extraction of Eicosanoids Materials required Extraction device and dry-down apparatus Preparation of the sample Influence of ph on the extraction Precautions required for assaying eicosanoids in biological samples Acknowledgements References THIN-LAYER CHROMATOGRAPHY (INCLUDING RADIO THIN-LAYER CHROMATOGRAPHY AND \ AUTORADIOGRAPHY) OF PROSTAGLANDINS AND RELATED COMPOUNDS 53 J.S.Hurst, S.Flatman and R.G.McDonald-Gibson Introduction 53 Equipment 53 Thin-layer plates 53 Applicators 54 Solvents 54 Development chambers (tanks) 54 Reagent-sprayers 55 UV-cabinets 55 Procedures 55 Concentration and preparation of the sample 55 Preparation of the plate 55 xi

5 Marking of the plate 56 Application of standards and sample 57 Development 58 Detection 59 Solvent Systems 62 A9 system 62 Al system 63 Systems for lipoxygenase metabolites 63 Other solvent systems 63 Elution and Recovery 64 Other Thin-Layer Chromatography Techniques 65 Two dimensional t.l.c. 65 High performance thin-layer chromatography 65 Radio-TLC 66 Experimental details 66 Instrumentation 66 Quantitation 68 Limitations 69 Autoradiography 72 References HIGH-PRESSURE LIQUID CHROMATOGRAPHY IN THE ANALYSIS OF ARACHIDONIC ACID METABOLITES 75 W.S.Powell Introduction 75 Chromatographic Behaviour of Arachidonic Acid Metabolites 76 Preparation of Samples 76 Eicosanoids other than peptido-leukotrienes 76 Precolumn extraction of samples containing peptido-leukotrienes 77 Detection of Eicosanoids ; 79 UV-absorbance 79 Radioactivity 79 Normal-Phase High-Pressure Liquid Chromatography 80 Separation of cyclo-oxygenase products by NP-h.p.l.c. 80 Separation of lipoxygenase products by NP-h.p.l.c. 81 Effects of injection medium on chromatographic behaviour 81 NP-h.p.l.c. of methyl esters 83 Argentation High-Pressure Liquid Chromatography 83 Preparation of stationary phase 83 Mechanism of Ag-h.p.l.c. 83 Applications of Ag-h.p.l.c. 85 Reversed-phase High-Pressure Liquid Chromatography 87 xn

6 Separation of mixtures of eicosanoids not containing peptidoleukotrienes 88 High-pressure liquid chromatography of peptido-leukotrienes 89 Gradients for reversed-phase-h.p.l.c. of arachidonic acid metabolites 92 Quantitation of Eicosanoids by High-Pressure Liquid Chromatography 95 Quantitation by u.v.-absorbance or fluorescence 95 Other methods of quantitation by h.p.l.c. 97 Acknowledgements 97 References GAS CHROMATOGRAPHY AND MASS SPECTROMETRY OF EICOSANOIDS 99 S.E.Barrow and G.W.Taylor Introduction 99 Principles of Gas Chromatography 99 General Principles of Mass Spectrometry 101 Magnetic sector instruments 101 Quadrupole mass spectrometers 103 Ionization 103 Sample Preparation 105 General precautions 105 Sample extraction 106 Sample purification 107 Use of stable isotope internal standards 109 Estimation of efficiency of extraction and purification 111 Derivatization 114 Gas Chromatography of Eicosanoids 117 Identification by comparison of retention times 118 Mass Spectrometry of Eicosanoids 118 Preliminary structural studies 118 Electron impact mass spectrometry 120 Chemical ionization mass spectrometry 127 Electron capture ionization 128 Soft ionization 133 Future Trends 137 Liquid chromatography-mass spectrometry 139 Ion trap g.c. detector 139 Acknowledgements 140 References 140 General Bibliography 141 xm

7 8. MEASUREMENT OF PROSTAGLANDINS, THROM- BOXANES AND LEUKOTRIENES BY SMOOTH MUSCLE BIOASSAY 143 P. J. Piper Introduction 143 Superfusion 144 Prostaglandins 145 Thromboxane A Leukotrienes 146 Cysteinyl-containing LTs 146 Leukotriene B Conclusion 149 References AGGREGOMETRY TECHNIQUES FOR PROSTANOID STUDY AND EVALUATION 151 B.J.R.Whittle Introduction 151 Basic Principles of Aggregometry 151 Optical aggregometry 151 Measurements of aggregation in whole blood 155 Washed platelets 156 Data analysis 158 Materials 158 Inhibition of Platelet Aggregation by Prostanoids 159 Species sensitivity 159 Studies in whole-blood 160 Washed platelets 161 Bioassay of Inhibitory Prostanoids 161 Formation of prostacyclin 162 Characterization of prostacyclin-like activity 162 Platelet Prostanoid Receptor Classification 163 Prostanoid receptor antagonists 163 Prostanoid receptor agonists 164 Thromboxane A 2 mimetics and antagonists 164 Lipoxygenase Products 164 Conclusions 165 References RADIOIMMUNOASSAY OF EICOSANOIDS 167 E.Granstrom, M.Kumlin and H.Kindahl xiv Introduction 167 Development of an Eicosanoid Radioimmunoassay 168

8 Preparation of the antigen 168 Preparation of the antibody 169 Preparation of the tracer (labelled ligand) 173 Performance of the assay 175 Counting of radioactivity 176 Mathematical handling of data 177 Validation of the Eicosanoid Radioimmunoassay 178 Common Problems in Eicosanoid Radioimmunoassay 180 Instability of the target substance 180 Problems related to the preparation of the immunogen 182 Problems related to the preparation of tracer 185 Deterioration of assay performance with time 187 Major Sources of Error in Eicosanoid Radioimmunoassay 190 Acknowledgements 194 References ENZYME IMMUNOASSAY 197 S.Yamamoto, K.Yokota, T.Tonai, F.Shono and Y.Hayashi Introduction 197 Enzyme Immunoassay of 6-keto-PGF la 197 Outline of the assay 197 Preparation of anti-6-keto-pgf la antiserum 198 Conjugation of 6-keto-PGF la and /3-galactosidase 200 Reagents 201 Standard assay conditions 201 Calibration curve and cross-reactivity 202 Validity of the assay 203 Application to human serum 203 Enzyme Immunoassay of TXB Outline of the assay 204 Preparation of anti-txb 2 IgG 204 Conjugation of TXB 2 and /3-galactosidase 205 Reagents 205 Standard assay conditions 205 Calibration curve and cross-reactivity 206 Validity of the assay 207 Application to human blood and urine 207 References CYCLO-OXYGENASE: MEASUREMENT, PURIFICATION AND PROPERTIES 209 R.J.Kulmacz and W.E.M.Lands Introduction 209 Measurement of Cyclo-oxygenase Activity 209 xv

9 General features of the assay 210 Assay of cyclo-oxygenase with the polarographic oxygen electrode 210 Assay of cyclo-oxygenase with radioisotope 214 Assay of cyclo-oxygenase with spectrophotometry 215 Purification of Cyclo-oxygenase 215 Properties of Cyclo-oxygenase 217 Size of the synthase and its subunit composition 217 Haem requirement 218 Absorbance spectrum of the synthase 218 Protease sensitivity 220 Requirement for hydroperoxide initiator 220 Substrate requirements 224 Interaction with cyclo-oxygenase inhibitors 224 Acknowledgements 226 References LIPOXYGENASES: MEASUREMENT, CHARACTERIZATION AND PROPERTIES 229 T.Schewe, H.Kiihn and S.M.Rapoport Introduction 229 Detection and Assay 229 Methods to measure lipoxygenase activity in purified systems 230 Strategy to detect lipoxygenase activities in cells and tissues 234 Detection of lipoxygenases by activity staining 237 Immunological and molecular-biological detection of lipoxygenases 238 Principles of Isolation and Purification of Lipoxygenases 239 Characterization of Lipoxygenases 239 Positional and steric specificity 240 Special lipoxygenase-catalysed reactions 240 Acknowledgements 242 References LDPID PEROXIDATION 243 T.F.Slater and K.H.Cheeseman xvi Introduction 243 Methods for Measuring Lipid Peroxidation 244 Models for Studying Lipid Peroxidation 245 Homogeneous reactions 245 Purified enzymes and lipid micelles 246 Isolated organelles 246 Isolated cells 248 Whole tissue and whole organ studies 248 Whole animal 249

10 Practical Aspects 249 Protocol for NADPH-dependent lipid peroxidation 249 Protocol for CCl 4 -stimulated lipid peroxidation 251 Protocol for NADPH-ADP iron stimulated lipid peroxidation 252 Protocol for peroxidation stimulated by cumene hydroperoxide 253 Protocol for 7-irradiation 253 Malonaldehyde estimation 254 Diene conjugation measurement 255 Lipid hydroperoxide measurement 256 Procedures for measuring alkane production 256 Measurements of polyunsaturated fatty acids 256 Measurements of 4-hydroxy-alkenals 256 Concluding Remarks 256 References QUANTITATIVE MEASUREMENT OF ARACHIDONIC ACID IN TISSUES OR FLUIDS 259 R. G. McDonald-Gibson Introduction 259 General Theory 259 Internal Standards 260 Total Lipid Extraction 260 Thin-Layer Chromatographic Separation of Lipid Classes 261 Preparation of Methyl Esters for Gas Chromatography 262 Equipment 262 Reagents 262 Method 262 Gas Liquid Chromatography 263 General equipment 263 Packed column separations 263 Capillary column separations 264 Calculations 264 References METHODS IN PROSTANOID RECEPTOR CLASSIFICATION 267 R.A.Coleman Introduction 267 The Classification and Nomenclature 268 Pharmacologically Active Agents in Prostanoid Receptor Classification 269 Use of the standard agonists 269 Synthetic agonists 271 Antagonists 275 Determination of Potency of Pharmacologically Active Agents 278 xvii

11 Determination of agonist potency 278 Antagonists 282 Useful Preparations in Prostanoid Receptor Classification 285 Criteria 285 Smooth muscle 286 Blood platelets 301 Concluding Remarks 302 Acknowledgements 303 References PAF-ACETHER (PLATELET-ACTIVATING FACTOR) 305 J.Benveniste Introduction 305 Synthesis of Paf-Acether 305 The formation and release of paf-acether 305 Assay and characterization of paf-acether 307 Sources and Metabolism of Paf-Acether 308 Effects of Paf-Acether 309 Specific Inhibition of Paf-Acether-Induced Platelet Activation 310 References 310 APPENDIX Eicosanoids: Structure, Availability, Storage and Stability 313 S.Nigam INDEX 321 xvm