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Arachidonic Acid Metabolism by Human Fetal Membranes

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Arachidonic Acid Metabolism by Human Fetal Membranes Arachidonic Acid Metabolism by Human Fetal Membranes in Tissue Culture. Matthew P. Rose BSc. Institute of Obstetrics & Gynaecology, Hammersmith Hospital, Du Cane Road, London, W12. U.K. Submitted for the degree of PhD. in the Faculty of Science, London University. 1985 1 ABSTRACT. Arachidonic acid (A.A.) may be oxygenated by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes to a multitude of biologically active metabolites including prostaglandins (PGs), leukotrienes (LTs), hydroxy (HETEs) and epoxy (EETs) fatty acids. Prostaglandins synthesised by intrauterine tissues have been implicated in the development and maintenance of pregnancy and in parturition. The synthesis of other oxygenated metabolites by intrauterine tissues has not been studied and their physiological roles are unknown. Cells obtained from human amnion, chorion laeve and placenta have been grown in monolayer culture and incubated with tritiated A.A. to determine its metabolic fate. Products of oxygenative metabolism of both exogenously added and endogenously incorporated A.A. have been extracted from tissue culture medium and separated using high p e r f o r m a n c e liquid chromatography* Chromatographic profiles have been evaluated by comparison of retention times of metabolites with those of known standards and summation of radioactivity in major peaks. The profiles of- products were dependent upon tissue type, gestational age and source of substrate. Prostaglandins were produced by all tissues. Amnion produced mainly PGE^r whereas trophoblast produced a variety of PGs and their further metabolites. Cells obtained in the Is trimester of pregnancy or following labour were metabolically more active than those obtained from elective caesarean section. The latter cells only produced PGs from exogenous substrate whereas cells obtained following labour produced PGs from endogenous substrate as well. These observations lend further support for a role for PGs in parturition, but also suggest that the addition of exogenous substrate may bypass key regulatory steps in the A.A. cascade. The relative proportions of free and esterified A.A. at various stages of gestation may therefore be physiologically important. All tissues also synthesised products which co­ chromatographed with mono- and di-HETEs. A putative product of cytochrome P450 systems was also observed. Despite differences between tissues and changes with gestational age these products appeared to be the major metabolites of A.A. in intrauterine tissues. The physiological significance of these findings is not understood since very little is known about synthesis of these compounds by intrauterine tissues and their roles in pregnancy. 2 ACKNOWLEDGEMENTS. I am indebted to the many people who have helped and encouraged me during the course of these studies. I would particularly like to thank Professor M.G. Elder for his continued support throughout my studies and Les Myatt for his neverending attentive supervision of both my experimental and written work. I would also like to acknowledge other members of staff at the Institute of Obstetrics and Gynaecology, especially John White, whose encouragement, comments and criticisms were of great help in seeing me through some minor crises, and David Glance •who provided a continual source of thought-provoking ideas. I am also grateful to Kathryn Philipson, Tim Scane, Girish Parmar, Mohammed Jogee, Julian Guest and David Cukier for their helpful discussions. I acknowledge the financial support of the Institute of Obstetrics and Gynaecology Appeal Fund, and all those who contributed to it, and also the Spastics Society. Finally, I am indebted to Jane Mundin who patiently and tirelessly corrected and typed the manuscript and who was a perpetual source of encouragement and inspiration. 3 DEDICATION. To my family, past, present and future. 4 LIST OF ABBREVIATIONS. ATP Adenosine triphosphate CAMP Cyclic adenosine monophosphate CoA Coenzyme A GC-MS Gas chromatography mass spectrometry hCG Human chorionic gonadotrophin HETE Hydroxyeicosatetraenoic acid HHT Hydroxyheptadecatrienoic acid HPETE Hydroperoxyeicosatetraenoic acid hPL Human placental lactogen HPLC High performance liquid chromatography IgG Immunoglobulin G LT Leukotriene MDA Malondialdehyde NAD(P) Nicotinamide adenine dinucleotide (phosphate) NDGA Nordihydroguaiaretic acid NSAIDs Non-steroidal anti-inflammatory drugs ODS Octadecyl silica PBS Phosphate buffered saline PG Prostaglandin PGDH Prostaglandin dehydrogenase RIA Radioimmunoassay SRS Slow reacting substance TEAF Triethylamine formate fcR Retention time Tx Thromboxane 5 CONTENTS. Title page 1 Abstract 2 Acknowledgements 3 Dedication 4 List of abbreviations 5 List of contents 6 List of figures, HPLC profiles, charts and plates 14 References , 274 CHAPTER 1. INTRODUCTION. 20 1) Metabolism of Arachidonic Acid. 20 a) Incorporation into complex lipids 20 b) Release from complex lipids 23 c) Oxygenative metabolism 25 i) Cyclooxygenase Pathway 25 Endoperoxide synthesis 25 Thromboxane A synthetase 29 Prostaglandin D synthetase 29 Prostaglandin E synthetase 31 Prostaglandin F synthetase 31 Prostaglandin I2 synthetase 31 Prostaglandin interconversions 32 ii) Catabolism of prostaglandins and thromboxane 33 15-hydroxy-prostaglandin dehydrogenase 33 Type 1, NAD-linked 15-hydroxy- prostaglandin dehydrogenase 33 Type 2, NADP^linked 15-hydroxy- prostaglandin dehydrogenase 35 6 Other types of 15-hydroxy- prostaglandin dehydrogenase 35 Delta-13-prostaglandin reductase 35 Beta and Omega oxidation 36 iii) Formation of hydroxyeicosatetraenoic acids and leukotrienes via lipoxygenase enzymes 36 Properties of lipoxygenase enzymes 41 iv) Further metabolism of lipoxygenase products 42 Re-esterification of mono-HETEs 42 Omega oxidation of fatty acids 42 Further conversion of LTC^ 43 v) Metabolism of arachidonic acid via Cytochrome P450 43 vi) Autooxidation of arachidonic Acid 45 2) Regulation of Arachidonic Acid Metabolism 49 a) Regulation of phospholipases 49 b) Regulation of oxygenatiye pathways 50 i) Cyclooxygenase pathway ^ 50 ii) Prostaglandin synthetases 52 Prostaglandin 12 synthetase 52 Thromboxane synthetase 53 Prostaglandin E synthetase 53 Prostaglandin D synthetase 54 iii) Regulation of lipoxygenase enzymes 54 3) Metabolism of Arachidonic Acid in Intrauterine Tissues 57 a) Release of substrate from complex lipids 57 7 b) Synthesis of oxygenated arachidonic acid metabolites by intrauterine tissues 57 c) Regulation of arachidonic acid metabolism in intrauterine tissues 62 4) Biological Roles of Arachidonic Acid Metabolites in Pregnancy 66 a) Prostaglandins 66 i ) Implantation 66 ii) Maintenance of blood flow and placental perfusion 67 iii ) Parturition * 70 iv) Roles as second messengers: interaction with peptide hormones 72 b) Lipoxygenase products 74 5) Fetal Membranes: Development, Structure and Function 76 a) Development of the membranes 76 b) Structure and function 80 i ) Placenta 81 ii) Chorion laeve 86 iii) Amnion 88 6) Methods Used to Assess Arachidonic Acid Metabolism. 92 a) Determination of arachidonic acid metabolites 92 i) Bioassays 92 ii) Radioimmunoassay (RIA) 92 iii) Mass spectrometry 94 iv) High performance liquid chromatography (HPLC) 94 Sample preparation 96 Filtration 97 8 Separation 97 Detection 97 b) Experimental approaches to the determination of arachidonic acid metabolites 98 i ) Body fluids 99 ii) Whole organ studies 100 iii) Chopped or minced tissue 101 iv) Homogenates and microsomal preparations 101 v) Cell culture 102 c) Tissue culture of fetal membranes 103 i) Placenta 103 ii) Chorion laeve 104 iii) Amnion 105 Summary 107 CHAPTER 2. MATERIALS AND METHODS 109 1) Extraction of Arachidonic Acid Metabolites from Tissue Culture Medium using C18 Sep-Paks 109 a) Materials 109 b) Methods used for the extraction of arachidonic acid metabolites from tissue culture medium 111 i) Determination of extractionefficiencies 111 ii) Determination of the effects of horse serum and ethanol on the efficiency of extraction 112 iii) Extraction of culture supernatants 112 2) Separation of Arachidonic Acid Metabolites using High Performance Liquid Chromatography 114 a) Materials 114 9 b) Method used to separate arachidonic acid metabolites by reverse-phase high performance liquid chromatography 115 c) Normal-phase separation of hydroxyeicosa- tetraenoic acids 116 i) Extraction of hydroxyeicosatetraenoic acids with chlorobutane 116 ii) Separation of hydroxyeicosatetraenoic acids by normal-phase high performance liquid chromatography 118 d) Analysis of reverse-phase chromatograms 120 i) Peak height 120 ii) Peak area 121 Manual measurement 121 Curve squaring 121 Triangulation 123 Graphical integration 123 Electronic integration 123 Baseline correction 123 3) Tissue Culture 127 a) Materials 127 b) Preparation of cells 128 c) Metabolism of arachidonic acid by cultures of cells derived from human fetal membranes 129 i ) Uptake of arachidonic acid 129 ii ) Conversion of arachidonic acid to oxygenated metabolites 130 iii) Control experiments 131 iv) Reproducibility 131 v) Normal-phase high performance liquid chromatographic separation of hydroxy­ eicosatetraenoic acids 131 10 vi) Analysis of hydroxyeicosatetraenoic acids by gas chromatography-mass spectrometry 133 vii) Analysis of reverse-phase chromatograms 133 CHAPTER 3. RESULTS 136 1) High Performance Liquid Chromatography 136 a) Extraction of arachidonic acid metabolites from tissue culture medium using C 18 Sep- Pak cartridges 136 b) Calibration of reverse-phase high performance liquid chromatography separation
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