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 , lipoxygenase and cytochrome P450 enzymes to a multitude of biologically active metabolites including (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 PGDH Prostaglandin dehydrogenase RIA Radioimmunoassay SRS Slow reacting substance TEAF Triethylamine formate

fcR Retention time Tx

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 of prostaglandins and hydroxy - eicosatetraenoic acids * 142 c) Separation of hydroxyeicosatetraenoic acids by normal-phase high performance liquid chromatograohy 150

2) Growth of Cells in Culture 153 a) Placental cells 153 b) Chorion laeve cells 153 c) Amnion cells 153

3) Metabolism of Arachidonic Acid by Cell Cultures 159

a) Uptake of labelled arachidonic acid 159 b) Analysis of oxygenative metabolism 165 i) Reproducibility 165 ii) Control experiments 165 iii) Qualitative and quantitative analysis of metabolism of exogenously added and endogenously incorporated arachidonic acid by cell cultures 169

First trimester placental cells 169 Placental cells obtained following spontaneous labour 177 Placental cells obtained following elective caesarean section 182

11 Chorion laeve cells obtained following spontaneous labour 186 Chorion laeve cells obtained following elective caesarean section 191 Amnion cells obtained following spontaneous labour 195 Amnion cells obtained following elective caesarean section 200

iv) Identification of monohydroxyeicosa- tetraenoic acids by normal-phase high performance liquid chromatography 206 Extraction of mono-HETEs 206 Normal-phase HPLC separation of HETEs 210

Arachidonic acid control 210 Placental cells 210 Chorion laeve cells 210 Amnion cells 212 v) Identification of mono-hydroxyeicosa- tetraenoic acids by gas-chromatography mass-spectrometry 212 vi) Quantitative analysis of total radioactivity 216

CHAPTER 4. DISCUSSION 221 1) High Performance Liquid Chromatography 222 a) Sample extraction 222 b) Separation of arachidonic acid metabolites 225

2) Cell Culture 230 a) Placenta 230 b) Chorion laeve 231 c) Amnion 233

12 3) Metabolism of Arachidonic Acid by Cell Cultures 234 a) Incorporation of -^H-arachidonic acid 234 b) Conversion to oxygenated metabolites 239 i) Autooxidation of arachidonic acid 240 ii) Placental and chorion laeve cells 241

iii) Amnion cells 247 c) Differences in metabolism of arachidonic acid between tissues and with gestational age 253

4) Regulation of Arachidonic Acid Metabolism 258

5) Biological Functions of Arachidonic Acid Metabolites in Pregnancy 263 a) Implantantion and development of utero­ placental circulation 263

b) Immunology ot pregnancy 265 c) Maintenance of bloodflow 265 d) Parturition 266 e) Regulation of the endocrine function of the placenta 267

6) Conclusions 271

7) Further Studies 272

13 LIST OF FIGURES, HPLC PROFILES, TABLES, CHARTS AND PLATES.

Figures. 1) Structures of phospholipids. 21 2) Specificity of phospholipases and mechanisms of arachidonic acid release. 24

3) Synthesis of endoperoxides. 26 4) :Further transformations of PGH2. 30 5) Catabolic pathways of primary prostaglandins. 34

6) Mechanism of action of lipoxygenases. 37 7) Possible products of lipoxygenase attack following abstraction of a single H-atom. 39

8) Transformation of arachidonic acid following attack by 5-lipoxygenase. 40 9) The further metabolism of LTC^. 44

10) Oxyqenation of arachidonic acid by cytochrome P450. 46 11) Mechanism of autooxidation of polyunsaturated fatty acids. 47 12) Release of arachidonic acid in fetal membranes. 58 13) Development and implantation of blastocyst. 77 14) Structure of fetal villus. 81 15) Relationship ‘between maternal and fetal circulations. 83 16) Structure of human amnion and chorion. 87 17) Extraction of arachidonic acid metabolites from culture supernatants. 113 18) Separation of arachidonic acid metabolites by reverse-phase HPLC. 117 19) Separation of hydroxyeicosatetraenoic acids by normal-phase HPLC. 119 20) Determination of peak height and area. 122

14 21) Determination of peak area and baseline correction. 124 22) Determination of incompletely separated peaks. 12 23) Protocol used for the analysis of metabolism of arachidonic acid by cultures of cells derived from human fetal membranes. 132 24) Hypothetical scheme for the assimilation of arachidonic acid from maternal blood by the human placenta. 238 25) Metabolism of arachidonic acid by placental cells. 248

26) Metabolism of arachidonic acid by chorion laeve cells. • 250 27) Metabolism of arachidonic acid by amnion cells. 252 28) Sites of regulation of arachidonic acid metabolism in human fetal membranes. 257

29) Possible functions of arachidonic acid metabolites produced by amnion. 268

30) Hypothetical scheme for roles of arachidonic acid metabolites in the placenta. 270

HPLC Profiles. 1) Separation of a mixture of radioactively labelled arachidonic acid metabolites by reverse-phase high performance liquidchromatography. 143 2) Ultra-violet detection of prostaglandins (e.g. 6-keto-PGE-^ ). 144 3) Ultra-violet detection of PGB2. 145 4) Ultra-violet detection of hydroxyeicosa- tetraenoic acids. 146 5) Separation of mono-hydroxyeicosatetraenoic acids by normal-phase high performance liquid chromatography. 152

6) Duplicate reverse-phase chromatograms of metabolites of ^H-arachidonic acid produced by cultures of placental cells obtained after spontaneous labour. 167

15 7) Reverse-phase chromatograms of compounds produced during incubation of ^H-arachidonic acid in the absence of cells. 168 8 ) Reverse-phase chromatograms of compounds produced from exogenous substrate by cultures of placental cells obtained during the 1st trimester. 170 9) Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of placental cells obtained during the 1st trimester. 175 10) Reverse-phase chromatograms of compounds produced from exogenous substrate by cultures of placental cells obtained after spontaneous labour. 178 11) Reverse-phase chromatograms * of metabolites produced from endogenous substrate by cultures of placental cells obtained after spontaneous labour. 180 12) Reverse-phase chromatograms of compounds produced from exogenous substrate by cultures of placental cells obtained after elective caesarean section. 183 13) Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of placental cells obtained after elective caesarean section. 185 14) Reverse-phase chromatograms of compounds produced from exogenous substrate by cultures of chorion laeve cells obtained after spontaneous labour. 187

15) Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of chorion laeve cells obtained after spontaneous labour. 189 16 ) Reverse-phase chromatograms of compounds produced from exogenous substrate by cultures of chorion laeve cells obtained after elective caesarean section. 192 17) Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of chorion laeve cells obtained after elective caesarean section. 194 18 ) Reverse-phase chromatograms of compounds produced from exogenous substrate by cultures of amnion cells obtained after spontaneous labour. 196

16 19 ) Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of amnion cells obtained after spontaneous labour. 198

20) Reverse-phase chromatograms of compounds produced from exogenous substrate by cultures of amnion cells obtained after elective caesarean section. 201

21 ) Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of amnion cells obtained after elective caesarean section. 203

22 ) Reverse-phase chromatograms of products of exogenous ^H-arachidonic acid produced by cultures of placental cells obtained after spontaneous labour, before and after partitioning of HETEs with chlorobutane. 207

23 ) Reverse-phase chromatograms of fractions before and after extraction of mono-HETEs from culture supernatants of amnion cells obtained after spontaneous labour. 209

24) Normal-phase separation of mono-HETEs. 211 25 ) Normal-phase separation of mono-HETEs following extraction with chJLorobutane after incubation of amnion cells with JH-arachidonic acid. 213

Tables.

1) Recovery of arachidonic acid metabolites following extraction with C 18 Sep-Paks. 138

2 ) The effect of serum and ethanol on the extraction of arachidonic acid and prostaglandins from tissue culture medium. 139 3) Extraction of arachidonic acid metabolites from tissue culture medium supplemented with 10% horse serum using C 18 Sep-Paks. 141 4) Retention data for arachidonic acid metabolites on reverse-phase high performance liquid chromatography. 149 5) Retention data for mono-HETEs on normal-phase high performance liquid chromatography. 151 6 ) Time course of uptake of 3H-arachidonic acid by placental cells in monolayer culture. 160

17 Charts. 1) Effect of arachidonic acid concentration on uptake of ^H-arachidonic acid by cultures of placental cells obtained after spontaneous labour. 161 2) Radioactivity incorporated by cell cultures after 24 hours incubation with ^H-arachidonic acid. 163 3) Distribution of radioactivity between metabolites produced from exogenous substrate by cultures of placental cells obtained during the 1st trimester. 173 4) Distribution of radioactivity between metabolites produced from endogenous substrate by cultures of 1st trimester placental cells. 176 5) Distribution of radioactivity between metabolites produced from exogenous substrate by cultures of placental cells obtained after spontaneous labour. 179 6) Distribution of radioactivity between metabolites produced from endogenous substrate by cultures of placental cells obtained after spontaneous labour. 181 7) Distribution radioactivity between metabolites produced from exogenous substrate by cultures of placental cells obtained after elective caesarean section. 184 8) Distribution of radioactivity between metabolites produced from exogenous substrate by cultures of chorion laeve cells obtained after spontaneous labour. 188 9) Distribution of radioactivity between metabolites produced from endogenous substrate by cultures of chorion laeve ceils obtained after spontaneous labour. 190 10) Distribution of radioactivity between metabolites produced from exogenous substrate by cultures of chorion laeve cells obtained after elective caesarean section. 193 11) Distribution of radioactivity between metabolites produced from exogenous substrate by cultures of amnion cells obtained after spontaneous labour. 197

18 12) Distribution of radioactivity between metabolites produced from endogenous substrate by cultures of amnion cells obtained after spontaneous labour. 199 13) Distribution of radioactivity between metabolites produced from exogenous substrate by cultures of amnion cells obtained after elective caesarean section. 202 14) Distribution of radioactivity between metabolites produced from endogenous substrate by cultures of amnion cells obtained after elective caesarean section. 204

15) Determination of 12-HETE by gas-chromatography mass-spectrometry. 215 16) Quantitation of radioactivity recovered from reverse-phase HPLC analysis of exogenously added •^H-arachidonic acid, '217

17) Quantitation of radioactivity recovered from Reverse-phase HPLC analysis of metabolites of endogenous ^H-arachidonic acid. 219

Plates.

1) Monolayer of term placental cells, showing syncytial like structure (48 hrs, haemotoxylin and eosin xllO). 155 2) Monolayer of term placental cells, showing syncytial like structure (72 hrs, haemotoxylin and eosin xllO). 155

3) Immunofluorescent staining of 1st trimester cells with antibody against hCG. Positive cells fluoresce light green xllO. 156 4) Monolayer of term chorion laeve cells showing fibrobalst and epithelial like cells (24 hrs, haemotoxylin and eosin xllO). 156

5) Monolayer of term chorion laeve cells showing syncytial structure and single epithelial cells (48 hrs, haemotoxylin and eosin xllO). 157

6) Monolayer of term chorion laeve cells showing syncytial structure (72 hrs, haemotoxylin and eosin xllO). 157 7) Monolayer of term amnion cells (48 hrs, unstained xllO). 158

19 CHAPTER 1.

INTRODUCTION.

1) Metabolism of Arachidonic Acid.

a ) Incorporation into complex lipids.

In mammalian cells most arachidonic acid is found esterified into complex lipids including phosphoglycerides, cholesterol esters and

triacylglycerols. The amount of .free arachidonic acid is low, for example in whole platelets it is less than 0.5%

_of the total free fatty acid (Marcus et a 1. 1969).

Phosphoglycerides, which are characteristic components of

cell membranes, are phosphoric esters in which one of the alcohol groups of glycerol is esterified to phosphoric acid (figure 1(a)), the other alcohol groups being esterified to fatty acids (figure 1(b)). Phosphoglycerides also contain a polar head group whose

hydroxyl group is esterified to the phosphoric acid moiety (figure 1(c)). The most abundant phosphoglycerides are phosphatidylcholine and phosphatidylethanolamine. Two other important phosphoglycerides are phosphatidylserine and phosphatidylinositol. In the tissues of higher animals the 3-hydroxy group of cholesterol is esterified to long chain fatty acids.

However the cholesteryl ester content of the placenta is low (Simpson & Burkhart 1980), perhaps to preserve cholesterol for progesterone synthesis (Winkel et al. 1981).

20 Figure 1. Structures of Phospholipids

a) Parent compound; sn-GlyceroL-3-phosphoric acid

ch2oh

HO— CH OH

H 2 C-- 0 -- P---OH !i

b) L-Phosphatidic acid

= Saturated fatty CH2 O — C R acid. R„ = Unsaturated fatty aci d . R — C— O — CH 2 ii i OH nO I1 I CH2 _ ° _ P OB

c ) Polar head groups

O — CH 2---CH2--- NH^ Ethanolamine

O — CH2— CH2 N(CH3)3 Choline

O — CH- CH— COO Serine 2 i n h 3 +

Inositol

R = phosphatidic acid

21 Triacylglycerides are the most abundant family of lipids and are major components of storage or depot fats in animal cells. Fatty acids are esterified at all 3 alcohol groups of glycerol and their content of arachidonic acid is usually low. Arachidonic acid is principally found esterified at the sn-2 position of phosphoglycerides (Lands & Samuelsson

1968, Irvine 1982). Relatively little is found in triacylglycerols or cholesterol-esters although the latter may be selectively metabolised in some tissues such as adrenocortical cells (Vahouny et al. 1978). Arachidonic acid is rapidly and specifically incorporated into cells, appearing covalently bound to phospholipids, whereas other fatty acids compete with each other for acylation (Chern &

Kinsella 1983) and may accumulate as free fatty acids within membranes (Spector et al. 1970). The first step in its incorporation into phosphoglycerides is the formation of an acyl-CoA ester from fatty acid, CoA and ATP - catalysed by an acyl-CoA synthetase. In platelets an acyl-CoA synthetase which exhibits specificity for arachidonic acid and 5,8,14-eicosatrienoic acid has been demonstrated (Wilson et al. 1982). In contrast to other acyl-CoA synthetases , which exhibit broad substrate specificity, the arachidonic acid specific enzyme also exhibits a relatively fast rate of reaction. It was suggested that in prostaglandin— producing cells, an arachidonic-acid— specific acyl-CoA synthetase might regulate the level of free arachidonic acid by diverting

22 arachidonic acid accumulated from the environment towards incorporation into complex lipids. Following conversion to acyl-CoA, acyl transferases esterify arachidonic acid into complex lipids (Lands et al. 1982). In the liver, unsaturated fatty acids are preferentially incorporated but there is no distinction between fatty acids of the n-6 and n-3 series. Arachidonate, eicosatrienoate and eicosapentaenoate are handled preferentially to other fatty acids. b ) Release from complex lipids.

Arachidonic acid may be metabolised by enzymes which insert oxygen atoms into the fatty acid chain.

Oxygenative metabolism has a requirement for free or unesterified arachidonic acid (Lands & Samuelsson 1968,

Kunze 1970 ). The level of free fatty acid appears to be regulated by its release from intracellular lipid stores, although the potential for esterification and de­ esterification would also be important.

The release of fatty acids from complex lipids is catalysed by acyl-hydrolases. Phosphoglyceride-specific acyl hydrolases (phospholipases) release arachidonic acid from the quantitatively important store of phosphoglycerides in a variety of ways depending upon substrate specificity.

Phospholipase A enzymes cleave fatty acids from the 1 or 2 positions producing lysophosphoglycerides and free fatty acids (figure 2). Phospholipase C produces a

23 Figure 2 a) Specificity of Phospholipases

ll + , HCOa”P —— 0CHoCH N (CH_) - A| A 2 2 3 3 PLC PLD

PLA = Phospholipase A PLC = Phospholipase C PLD = Phospholipase D

= Saturated fatty acid

R 2 = Unsaturated fatty acid (usually arachidonic acid 4o)6)

b ) Mechanisms of arachidonic acid release. --R,

R,

PLA, PX PLC (X=Inositol)

----- OH — Ri • > PLA„ 2 — 2 HO — R 2 r __ PX __ PX .OH

Lysopho spholipase Lipases 4 A,A. 4

*0H R.

HO HO

.PX .OH 24 diacylglycerol and phospholipase D produces phospatidic acid and free base by cleavage of the phosphate group and base respectively (figure 2). Diacylglycerols and lysophosphoglycerides are substrates for diacylglycerol- and lysophospholipases which release free fatty acids.

Arachidonic acid may also be released from cholesterol esters by cholesterol-esterases and from neutral lipids by lipases. c ) Oxygenative metabolism. i) Cyclooxygenase pathway.

Endoperoxide synthesis: Arachidonic acid, derived from intracellular lipids is converted via the cyclooxygenase pathway to prostaglandins (PG) and (Tx). The first step is the oxygenative cyclization of arachidonic acid to produce the endoperoxide, PGG2 (figure 3), via an enzyme whose activity is termed fatty acid cyclooxygenase (Hamberg et al. 1974). The same protein also catalyses the second step in which PGG2 is reduced at the 15-hydroperoxide to form the 15-hydroxyl of PGH2* This activity is termed hydroperoxidase (figure 3). Thus, one protein has been demonstrated to have activities jointly referred to as prostaglandin endoperoxide synthetase (Miyamoto et al.

1976), or PGH synthetase (Titus et al. 1982). The first step is a bis-dioxygenation (Samuelsson 1965) in which one oxygen molecule forms a peroxide bridge

25 Figure 3. Synthesis of Endoperoxides.

Arachidonic Acid

Cyclooxygenase

The proposed mechanism of action of PG Endoperoxide Synthase.

26 linking carbons 9 and 11, while the other is incorporated into a hydroperoxide at carbon 15 (figure 3). Although the actual mechanism is unknown, that outlined in figure 3 has been proposed by several workers (Samuelsson 1965, Nugteren et al. 1966, Hamberg & Samuelsson 1967).

Purification from a particulate fraction of ovine (Nugteren et al. 1966, Samuelsson 1967) or bovine (Yoshimoto et al. 1970, Takeguchi et al. 1971) vesicular gland by centrifugation at 100,000 xg suggests a microsomal site. Monoclonal antibodies have been used to demonstrate it on endoplasmic reticulum and nuclear membrane, but not mitochondrial or plasma membranes of

Swiss mouse 3T3 fibroblasts (Rollins & Smith 1980). Microsomes of many other tissues have been shown to contain fatty acid cyclooxygenase (Christ & Van Dorp 1972, Yoshimoto et al. 1977, Bhat et al. 1978, Shimizu et al. 1979, Ho et al. 1980). The bovine vesicular gland enzyme has been purified to apparent homogeneity (Miyamoto et al. 1976) and has no absorption in the visible region, suggesting no haem or flavin content (Ogino et al. 1979).

The ovine vesicular gland enzyme may contain a significant amount of non-haem iron (Hemler et al. 1976) and may be a glycoprotein (van der Ouderaa et al. 1977). The haem free form of the enzyme is essentially inactive (van der

Ouderaa et al. 19 7 7) and it has been shown that haemoglobin is the major activating factor (Yoshimoto et al. 1970, Miyamoto et al. 1974). Non-purified protein extracts have no requirement for added haem (Rome & Lands

27 1975a) which suggests that haem may be a native cofactor in vivo, which may be lost during purification.

Paradoxically, the haem cofactor also causes inactivation of both activities of the enzyme, the rate of loss of activity being dependent upon the haem to enzyme ratio (Ogino et al 1979). Protection from inactivation may be provided by tryptophan, adrenaline and hydroquinone (Hemler et al 1976 ), phenol (Hemler & Lands 1980) and other compounds which are cofactors for the hydroperoxidase activity.

The enzyme also undergoes an autocata1ytic destruction (Smith & Lands 1972, Egan et al. 1976,

Miyamoto et al. 1976, Ogino et al. 1979). In the absence of a cofactor, and despite the availability of substrates, the reaction will slow down and cease. Further addition of fresh enzyme causes the reaction to proceed further. The addition of an oxygen radical scavenger such as methional increases the half life, suggesting that the deactivation may be due to oxidative destruction by a free oxygen radical. Lipid peroxides are also required as activators. The most important is PGG2 which exerts a feed-back stimulation of its own synthesis (Hemler et al. 1979). Peroxidase co-substrates such as phenolic compounds, also increase synthesis of prostaglandins in vitro (Hemler & Lands 1980). These compounds may act by reducing oxidised haem forms of the enzyme so that they may participate further in the cyclooxygenase reaction.

28 The enzymic activity is not specific forarachidonic acid, since e icosadienoic acid is also oxygenated at C-18

(Hemler et al. 1978). The endoperoxide may be further converted to the primary prostaglandins and thromboxane by a number of specific enzymes. These enzymes are mainly isomerases which attack the 9,11-endoperoxide (figure 4).

Thromboxane A Synthetase: Thromboxane (Tx) A2 synthesis can be detected in a variety of tissues (Pace-

Asciak & Rangaraj 1977, Sun et al. 1977, Moncada & Vane 1979). The enzyme responsible has been demonstrated in the microsomal fraction (Sun et al. 1977). The immediate product of the reaction, TXA2, is extremely unstable and rapidly hydrolyses to TxB2- The reaction also produces hydroxyheptadecatrienoic acid (HHT) and malondialdehyde (MDA). Recently Ullrich and Graf (1984) demonstrated that Tx synthetase is a cytochrome P450 enzyme.

Prostaglandin D Synthetase: The isomerisation of the

9,11-endoperoxide to 9-alpha-hydroxy-ll-keto produces PGD2. This activity has been demonstrated in a variety of tissues including brain and placenta (Christ-Hazelhof &

Nugteren 1979, Shimizu et al. 1979, Mitchell et al. 1982) and is predominantly cytosolic (Christ-Hazelhof & Nugteren

1979). The brain enzyme has no requirement for glutathione (Shimizu et al. 1979) but other sources are stimulated by added glutathione (e.g. spleen, Christ- Hazelhof & Nugteren 1979).

29 Figure 4. Further transformations of PGH^»

30

f Prostaglandin E Synthetase: Prostaglandin E synthetase catalyses isomerisation of the 9,11- endoperoxide to 9-keto-ll-alpha-hydroxyl. The enzyme is microsomal in a variety of tissues (Nugteren & Hazelhof 1973, Bhat et al. 1978) and is unstable when solubilised. It can be protected by various thiol compounds and requires glutathione as a specific coenzyme.

Prostaglandin F Synthetase: Prostaglandin F may be synthesised in three ways: Firstly by the direct cleavage of the 9,11-endoperoxide (Hamberg & Samuelsson 1967). An enzyme with this activity has been located in the microsomes of cow and guinea-pig uterus (Wlodawer et al. 1976) which has a requirement for a reducing agent. Prostaglandin F may also be formed non-enzymatically from PGH2 (Christ-Hazelhof 1976). Secondly, the reduction of the 9-keto group of E type prostaglandins to the 9-hydroxy group of PGF by a 9-keto reductase has been shown to occur in a variety of tissues (Hamberg & Israelsson 1970, Leslie

& Levine 1973). Finally, the reduction of the 11-keto group of PGD2 to the 11-alpha-hydroxy of PGF has also been demonstrated (Reingold et al. 1981, Wong 1981).

Prostaglandin I2 Synthetase: Prostaglandin I2 (, Epoprostenol) is produced by the conversion of the 9,11-endoperoxide of PGH2 to a 6,9- epoxide and an 11-alpha-hydroxyl group (figure 4). This structure is unstable, especially at acid pH, and

31 hydrolyses to form 6-keto-PGF-^. The enzyme activity has been demonstrated in a variety of tissues (Moncada & Vane 1979, Peterson & Gerrard 1979) located in the microsomes (Moncada et al. 1976a, Anderson et al. 1978) and is

susceptible to inhibition by 15-HPETE (Gryglewski et al. 1976). Prostaglandin I2 synthetase has also been shown to be a cytochrome P450 enzyme (Ullrich & Graf 1984).

Prostaglandin interconversions: Both PGE2 and PGD2

may be converted enzymatically to PGF2^f. Prostaglandin E2 may also be dehydrated to PGA2 with subsequent

isomerisation of delta-10 to delta-11 yielding PGC2r and delta-11 to delta-8 yielding PGB2. These reactions may be catalysed by enzymes which have been demonstrated mainly

in serum or plasma of several species including man (Jones

et al. 1972, Polet & Levine 1975). Prostaglandin A2^ PGB2 or PGC2 may be created artificially. A 9-hydroxy prostaglandin dehydrogenase enzyme which

transforms PGI2 or 6-keto-PGF^ to 6-keto-PGEj has been demonstrated in the cytoplasm of platelets (Wong et al. 1980) and may be physiologically important, as 6-keto

PGE-l shares equal potency in many biological actions with PGI2 and is more stable (Moore & Griffiths 1983). Prostaglandin 9-keto-reductase activity has also been demonstrated (Lee et al. 1975)

32 ii) Catabolism of prostaglandins and thromboxane.

The potent biological activities of prostaglandins renders their catabolism an important factor in the regulation of their physiological effects. Most prostaglandins are rapidly inactivated enzymatically, the principal sites in the circulation being the lungs, liver, kidney and placenta (Anggard & Samuelsson 1964, Pace- Asciak 1977). The initial reaction is the conversion of the 15- hydroxyl group to a keto group (figure 5). This considerably reduces the biological activity and is generally followed by an enzymatic reduction of the delta-

13 double bond. The resulting 15-keto-13,14-dihydro- prostaglandins are essentially inactive.

15-hydroxy-prostaglandin dehydrogenases The first catabolic reaction is catalysed by a 15-hydroxy- prostaglandin dehydrogenase, 15-PGDH. The enzyme is particularly abundant in lung, kidney, spleen and placenta (Anggard et al. 1971, Jarabak 1972, Schlegel & Greep 1975, Sun et al. 1976 ), but also occurs in smaller amounts in many other tissues (Anggard et al. 1971, Jarabak 1972, Sun et al. 1976). Five types of 15-PGDH have been demonstrated.

Type 1, NAD—linked 15-hydroxy-prostaglandin dehydrogenase: The NAD linked enzyme has been purified from lung, kidney and human placenta (Marrazzi &

33 Figure 5. Catabolic Pathways of Primary Prostaglandins

P.G.

15-Hydroxy-Prostaglandin

Dehydrogenase

15'

A ^ Reductase P.G.s Tetranor 13,14-dihydro-15-keto-P,G.

JJ-Oxidation \ 19- hydroxy 13,14-dihydro-15-keto-P.G. Dinor 20- hydroxy Tetranor

1 Dicarboxylic acid

\ | i Oxidation

Dinor Dicarboxylic acid Tetranor

13 A Reductase refers to the enzyme which saturates the 13,14 carbon-carbon double bond

34 Matschinsky 1972, Braithwaite & Jarabak 1975, Schlegel & Greep 1975, Sun et al. 1976). It appears to be cytosolic

and shows a degree of substrate specificity.

Prostaglandins E, F, A, I2 and 6-keto-PGF^ are good substrates whereas PGs B, D and TXB2 are poor substrates.

Type 2, NADP- linked 15-hydroxy-prostaglandin dehydrogenase: This enzyme catalyses the same reaction as

the type 1 enzyme but utilises NADP preferentially as an hydrogen acceptor. The enzyme has also been purified from human placenta (Westbrook et al. 1977) as well as other

tissues (Lee & Levine 1975, Lee et al. 1975), shows specificity for PGB rather than PGE or F, and also shows 9-keto-prostaglandin reductase activity.

Other types of 15-hydroxy-prostag1andin dehydrogenase: Three other types of 15-hydroxy- prostaglandin dehydrogenase have been described. They are PGA specific, PGD specific and PGI specific (Oien et al. 1976, Watanabe et al. 1980, Korff & Jarabak 1981).

The placental type 1 and type 2 enzymes have not been separated (Lin & Jarabak 1978 ) and both are sensitive to inhibition by ethacrynic acid, furosemide and indomethacin.

Delta-13-prostaglandin reductase: Fifteen-keto- prostaglandins are substrates for an enzyme which saturates the delta-13 double bond. The activity has been isolated from a variety of tissues (Anggard et al. 1971,

35 Lee & Levine 1974) including a highly purified form from human placenta (Westbrook & Jarabak 1978). The reaction is irreversible and leads to the formation of 13,14- dihydro-15-keto-prostaglandins.

Beta and omega oxidation: In common with other long chain fatty acids, prostaglandins may undergo both beta and omega oxidation (figure 5). The beta oxidation system of rat liver mitochondria can shorten the alpha side chain of various prostaglandins into carbon 18 and carbon 16 homologues (Hamberg 1968). Omega oxidation proceeds via omega or omega minus 1 hydroxylation, catalysed by hepatic microsomal cytochrome P450 systems (Kupfer et al. 1978). The main urinary metabolites of prostaglandins are di- and tetra- nor prostaglandins but may contain 19- or 20- hydroxy or carboxylic acid groups.

iii) Formation of hydroxyeicosatetraenoic acids and leukotrienes via lipoxygenase enzymes.

Lipoxygenase enzymes catalyse the incorporation of an oxygen molecule into the chain of a polyunsaturated fatty acid producing a hydroperoxide (figure 6). The reaction is accompanied by a shift in double bond position and a change in configuration from cis to trans. Mammalian lipoxygenases have a requirement for a cis, cis-1,4- pentadiene system and the products contain a 5- hydroperoxy-l-cis-3 trans-pentadiene (Gibian & Galaway 1977).

36 Figure 6. Mechanism of action of Lipoxygenases.

ROOC-(CH2)n ' R 3

cis,cis conjugated pentadiene

Insertion of molecular oxygen

1 2

ROOC-(CH2)n

0-0H

5-Hydroperoxy 1-cis,-3-trans-conjugated diene

37 Arachidonic acid contains 3 overlapping cis,cis-l,4- pentadiene systems with labile hydrogens at carbon atoms

7, 10 and 13 (Smith 1981). Abstraction of one hydrogen atom allows insertion of oxygen at two carbon atoms producing a pair of lipoxygenase products (hydroperoxides, figure 7). In the mammalian cascade, lipoxygenase action may be followed by a reductive step to produce a hydroxyeicosatetraenoic acid (HETE). Three pairs of HETEs may be produced from precursor hydroperoxyeicosatetraenoic acids (HPETEs) and for each pair of HPETEs formed one of the pair may be metabolised to products other than the corresponding HETE. For example, abstraction of hydrogen from carbon atom 10, may give rise to 8-HPETE or 12-HPETE. Eight-HPETE is further reduced to 8-HETE, whereas 12-HPETE may either be reduced to 12-HETE or converted to an epoxide which in turn can be converted to a tri-HETE. Other tranformations may occur following abstraction of hydrogen from carbon atom 7. Firstly, 9-HPETE or 5- HPETE may be formed. Nine-HPETE is reduced to 9-HETE and 5-HPETE may be reduced to 5-HETE or to 5,6-oxido-7,9- trans-ll,14-cis-eicosatetraenoic acid (leukotriene A 4, LTA4).

Leukotriene A 4 is the precursor of the biologically important leukotrienes B4, C 4, D4 and E 4 (figure 8). Leukotriene B4 is formed by the action of an epoxide hydrolase/and non-enzymatic hydrolysis may give rise to

38 Figure 7. Possible Products of Lipoxygenase attack

following abstraction of a single Hydrogen

atom .

Removal of one hydrogen atom from a single carbon atom

allows the insertion of molecular oxygen into the fatty

acid chain at one of two carbon atoms. In the case

below hydrogen is removed from CIO prior to insertion

of oxygen at either C8 or C12. The immediate products

of lipoxygenase action can theh be further converted to

a variety of compounds.

Arachidonic acid

Insertion of

8-HPETE 12-HPETE Epoxidation ------► 12 ,13 -E p o x id e

Reduction Reduction Oxidation ▼ ▼ 8-HETE 12-HETE TriHETE

39 Figure 8. Transformation of Arachidonic Acid following

attack by 5-Lipoxygenase.

Arachidonic Acid

5-lipoxygenase V

5S-Hydroperoxy-6-1 rans

-8,11,14-cis-Eicosatetraenoic Acid (ETE)

5S-Hydroxy-6R-s 5S,12R-Dihydroxy-6,14- -Glutathione- cis-8,10-trans-ETE 7f9-trans-llf14-cis-ETE (LTB^)

(l t c 4)

40 stereoisomers (Borgeat & Samuelsson 1979 a, b, c, d). The addition of glutathione to carbon atom 6 of LTA4 by glutathione-S-transferase gives rise to LTC4 which is a component of slow reacting substance of anaphylaxis (SRS, Murphy et al. 1979, figure 8). There is also evidence for formation of analogous leukotrienes following oxygenation at carbons 12 and 15 (Jubiz et al. 1981, Lundberg et al. 1981, Lindgren et al. 1982). Further hydroxylation of 5-HETE via the action of 12- lipoxygenase may form 5S,12S-dihydroxy-6,10-trans-8,14- cis-eicosatetraenoic acid (Borgeat et al. 1981, Lindgren et al. 1981, figure 8). A 5,15-leukotriene may be formed in a similar way (Maas et al. 1981).

Properties of lipoxygenase enzymes: Various lipoxygenase enzymes have been purified and some of their properties are known. Five-lipoxygenase has been found in the 100,000 xg supernatant of human polymorphonuclear leucocytes (Goetzl 1980a) and is stimulated by calcium ions (Goetzl 1980b). The production of 5-HETE by rat neutrophils has a requirement for calcium ions (Siegel et al. 1981).

Twelve-lipoxygenase has been demonstrated in the particulate and cytosolic fractions of human platelets. The cytosol activity can be separated into 2 components; one fraction has little peroxidase activity and produces mainly 12-HPETE, whereas the other fraction also contains peroxidase activity and produces 12-HETE (Butler et al.

41 1979). Twelve-lipoxygenase, present in the cytosol of porcine polymorphonuclear leukocytes, utilises 5-HETE to produce 5S,12S-di-HETE (Yoshimoto et al. 1982). A 15-lipoxygenase has been identified in rabbit polymorphonuclear leucocytes. The enzyme catalyses the production of 15-HPETE, which subsequently undergoes non- enzymatic degradation to 15-hydroxy-, 13-hydroxy-14,15- epoxy- and 11,14,15-trihydroxy-fatty acids (Fridovich &

Porter 1981). The formation of 5-HETE (Borgeat et al. 1976 ), 5,12- diHETE (Borgeat & Samuelsson 19 7 9d) and 5,6-diHETE

(Borgeat & Samuelsson 1979c) have been demonstrated in human polymorphonuclear leucocytes. iv) Further metabolism of lipoxygenase products.

Re-esterification of mono-HETEs: Re-esterification of mono-HETEs into membrane lipids has been demonstrated in macrophages and granulocytes (Bonser et al. 1981, Stenson et al. 1983). The incorporation is rapid and specific for monohydroxy-fatty acids and there appears to be a degree of specificity for lipid pools (Stenson et al. 1983).

Omega oxidation of fatty acids: In human polymorphonuclear leucocytes, LTB^ is metabolised by omega oxidation to 20-hydroxy-LTB4 and, to a lesser extent, 19- hydroxy-LTB^. 20-hydroxy-LTB4 may be further metabolised to a dicarboxylic acid. The dicarboxylic acid is less active than LTB^ in causing leucocyte adherence

42 but is equally active in contracting guinea-pig lung parenchyma. Isomers of LTB4 such as 5S-I2S-LTB4 have also been shown to be metabolised by omega oxidation. A biologically active leukotriene may be formed in human leucocytes by omega oxidation (Hansson et al. 1981).

Further conversion of LTC^: LTC4 is enzymatically converted to LTD4 by -glutamyltranspeptidase (Orning et

al. 1980, Parker et al. 1980, figure 9). Leukotriene D4 may then be transformed to LTE^ by removal of a glycine residue (Parker et al. 1980 ). It-trans isomers of

LTC4 (Clarke et al. 1980 ), LTD4 and LTE4 (Lewis et al. 1980) have also been described, although their routes of

synthesis are unknown. Leukotrienes C4, D4 and E4 all have biological activities but their relative potencies

vary amongst different tissues. It is possible that these conversions may represent modulation of leukotriene activity rather than inactivation. Conversion of LTE4 to

LTF4 via X-glutamyltranspeptidase in the presence of glutathione reduces biological activity (Anderson et al. 1982) and may therefore be a mechanism for inactivation. Leukotriene C4 can also be converted to compounds containing sulphoxide groups.

v) Metabolism of arachidonic acid via cytochrome P450.

Apart from its roles in the synthesis of PGI2 and TXA2, cytochrome P450 may metabolise arachidonic acid in two other ways: omega 1 and omega 2 oxidation may produce

43 --Figure2------9. The--- further------4 metabolism of LTC.

LTC, COOH

0 ii

LTD;

LTE;

44 monohydroxyfa tty acids (Capdevila et al. 1982 ) and epoxidation may produce a variety of epoxides between carbon atoms 5 and 6, 8 and 9, 11 and 12 and 14 and 15 (Chacos et al 1982, figure 10). These epoxides may be rapidly converted to vicinal diols by the action of microsomal or soluble epoxide hydrolases (Oliw & Oates 1981a, Oliw et al. 1982a, figure 10). In the absence of hydrolases the epoxides may be isolated (Chacos et al.

1982, Oliw et al 1982b). Vicinal diols are substrates for omega 1 and omega 2 oxidation, producing a variety of trihydroxy fatty acids, which may have polarities similar to prostaglandins (Oliw & Oates 1981b). Epoxidation and hydroxylation may be catalysed by different types of enzymes (Oliw et al. 1982b, Oliw & Moldeus 1982). Unlike omega 1 and omega 2 hydroxylations, the'formation of epoxides has not been demonstrated in vivo, although isolated rat renal cells and hepatocytes have been found to metabolise arachidonic acid to 11,12-dihydroxy and 14,15-dihydroxy fatty acids (Oliw & Moldeus 1982). vi) Autooxidation of arachidonic acid.

Polyunsaturated fatty acids can be non-enzymatically transformed to hydroperoxides and other products (figure 11). The reaction can be catalysed by haematin and haematin-containing compounds. The major products of autooxidation are 6-cis,trans conjugated diene hydroperoxides. The first step is the abstraction of hydrogen from carbon atoms 7,10 and 13 by a free radical

45 Figure 10. Oxygenation of Arachidonic Acid by Cytchrome P450

\ — / \ /V/ \/ Arachidonic Acid Epoxidation 14,15-Epoxide 11,12-Epoxide 8,9-Epoxide 5,6-Epoxide

Epoxide Hydrolase ] i i I HO OH HO OH /=V=W /=S/=W /~V=W /“W WV/W \_/WW WWW WWW HO OH HO OH

Oxidation

Tri-hydroxy-fatty acids HO OH HO OH

HO OH OH HO OH

46 Figure 11, Mechanism of Autooxidation of Polyunsaturated Fatty Acids.

Ha /H R 'M'Conformation radical

00H

Hydroperoxide mechanism to produce a radical with W conformation. The next step is formation of an hydroperoxide group at carbons 5, 8, 9, 11, 12 or 15 to produce a cis,trans conjugated hydroperoxide. The cis, trans isomers are major products but trans,trans isomers may also be formed in very small amounts due to the loss of #00H as oxygen addition to pentadienyl radicals is reversible. Five- and

15-HPETE are the major products of arachidonic acid autooxidation.

48 2) Regulation of Arachidonic Acid Metabolism.

Cyclooxygenase and lipoxygenase enzymes can only convert unesterified arachidonic acid and there is a requirement for a free carboxyl group (Vonkman & Van Dorp 1968). Since most arachidonic acid is found esterified

into complex lipids, the incorporation being specific and rapid, its release by acylhydrolases may be important in regulating the availability of substrate for oxygenative metabolism. a ) Regulation of phospholipases. Membrane-bound phospholipases A have a requirement for calcium ions (Kunze et a 1. 1974) and are therefore affected by compounds which regulate intracellular calcium levels.

In the porcine pancreas the enzyme may also be activated by proteolysis (de Haas et al. 1968). Non enzymatic activating factors may also bind to the enzyme, perhaps causing a conformational change and thereby stimulating activity. One such factor has been derived from platelets (Duchesne et al. 1972). De novo synthesis of an inhibitory peptide induced by anti-inflammatory steroids inhibits phospholipases (Flower

& Blackwell 1979). Inhibitory peptides have been demonstrated in macrophages and leucocytes (Irvine 1982) and kidney (Cloix et al. 1983 ) and have been termed, macrocortin, lipomodulin and renocortin respectively. Target-tissue-specific hormones may also regulate

49 phospholipases. An androgen-controlled enzyme has been demonstrated in rat epididymis (Bjerve & Reitan 1978). Regulation by thyroid stimulating hormone has been shown

in the thyroid (Haye et al. 1973 ) and stimulation of activity has been demonstrated in rat uterus by steroids (Dey et al. 1982). Stimulation of activity by 17-beta- oestradiol was modulated by progesterone and inhibited by dexamethasone. Peptide hormones may also stimulate activity.

Prolactin has been shown to stimulate lipases in mammary gland (Rillema & Wild 1977) as has angiotensin II in

kidney (Schwartzman & Raz 1979), renomedu11ary interstitial cells (Zusman & Brown 1980), vascular tissue (Nolan et al. 1981), uterus (Campos et al. 1983) and in placenta (Glance et al. in preparation). Bradykinin stimulates lipases in heart (Hsueh et al 1977), kidney (Schwartzman & Raz 1979), lung (Zusman & Reiser 1977) and skin (Flower 1978).

b) Regulation of oxygenative pathways.

i) Cyclooxygenase pathway.

Cyclooxygenase has a requirement for haem and peroxide activators as cofactors. The formation of lipid peroxides may be limited by superoxide dismutase, catalase

and radical scavengers (Chance et al. 1979). Antioxidants such as tocopherol may also exert their effect in this way (Cohen 1975). Other compounds such as hydroxyl

50 scavengers, mannitol and benzoate (Fong et al 1973), beta carotene or furan derivatives (Kellog & Fridovich 1975) may also function by limiting formation of hydroperoxides. Reduction to hydroxy acids following lipoxygenase attack or removal by glutathione peroxidase may also limit the level of peroxides in the cell (Flohe et al. 1976).

Cyclooxygenase may be inhibited in a competitive manner by other fatty acids binding at the active site.

Carbon 18 and carbon 20 acetylenic fatty acids can act in this way causing reversible inhibition (Vanderhoek & Lands

1973). The tetrayne analogue of arachidonic acid causes irreversible inhibition (Ahern & Downing 1970). Fatty acids with n-3 double bonds are good substrates but are inhibitors (Lands et al. 1972). The fatty acid constitution of cellular lipids may therefore be a mechanism by which PG synthesis may be controlled. Aromatic acids may also cause selective competitive

interaction at the binding site. Arylacetic and 2- phenylpropanoic acids such as and indomethacin are members of a group of non-steroidal anti-inflammatory drugs (NSAIDs) which are thought to exert at least part of their pharmacological action through the inhibition of cyclooxygenase (Lands et al. 1974). Since binding is competitive, cyclooxygenase activity can be protected by competition with drugs which bind in a similar fashion

(Lands et al. 1974). Addition of substrate and inhibitors together would prevent inhibition, since arachidonic acid binds more tightly than either aspirin or indomethacin.

51 Inhibition following pre-incubation with inhibitor cannot be overcome by the addition of substrate since a time- dependent irreversible inhibition also occurs (Rome & Lands 1975b) which may involve acetylation of the active site. Phenolic components which inhibit the reaction act via radical-trapping mechanisms. This can give rise to paradoxical effects (Hemmler & Lands 1980). At concentrations of peroxides which cause time-dependent inactivation of the enzyme phenolic compounds stimulate activity. At lower peroxide concentrations which are required to initiate the reaction phenolic compounds will cause inhibition. Compounds which exhibit these effects include dihydroxynaphthalenes and phenolic antioxidants (Dewhirst 1980). ii) Prostaglandin synthetases.

The synthesis of prostaglandins may be further modulated by compounds which affect the activity of prostaglandin synthetases.

Prostaglandin I2 synthetase: PGI2 synthetase appears to be sensitive to fatty acid hydroperoxides (Salmon et al. 1978). Fifteen-HPETE was the first inhibitor of this type to be demonstrated (Gryglewski et al. 1976) and is commonly used as a specific antagonist of PGI2 production. The decrease in vascular synthesis caused by vitamin E deficiency may be due to the resultant increase in hydroperoxides (Carpenter 1981). Other antioxidants such

52 as high concentrations of catecholamines or propylgallate may increase PGI2 synthesis by regulating the level of inhibitory hydroperoxides (Pace-Asciak 1972, Carpenter 1981). Fatty acid hydroperoxides may be inactive when administered in vivo due to reduction to lipid hydroxides (Pace-Asciak & Gryglewski 1984). The monoamine oxidase inhibitor tranylcypromine has been found to be an inhibitor of PGI2 in vitro (Gryglewski et al. 1976). However, Jogee (1983) found it to be a very weak inhibitor which was cytotoxic at high concentrations in tissue culture. It has also been shown that its effects in vivo may not be mediated by inhibition of PGI2 (Clark & Harrington 1982).

In placental cell cultures Jogee et al. (1983) showed stimulation of 6-keto-PGF-^ by oestrogen and progesterone. It was not determined whether this was due to increased lipase activity, to increased activity of PGI2 synthetase or to decreased catabolism. Prostaglandin I2 biosynthetic activity is stimulated by oestradiol in rat aorta smooth muscle cells (Chang et al. 1980).

Thromboxane synthetase: Thromboxane A synthetase is inhibited by imidazole (Needleman et al. 1977), imidazole derivatives (Moncada et al. 1977) and endoperoxide analogues (Gorman et al. 1977).

Prostaglandin E synthetase: Prostaglandin E synthetase has a requirement for glutathione (Lands et al.

53 1971) and it has been postulated that the addition of glutathione to prostaglandin synthesising systems may divert arachidonic acid preferentially to PGE, thus distorting the spectrum of products (Lands et al. 1971).

Prostaglandin D synthetase: Prostaglandin D synthetase appears to have isomeric forms distributed amongst the tissues in which it has been isolated. These

isomers seem to have some differential requirements for activity. The addition of glutathione stimulates actvity in the spleen (Christ-Hazelhof & Nugteren 1979) and mast cells (Steinhoff et al. 1980) whereas the brain enzyme has no requirement for glutathione (Shimizu et al. 1979).

iii) Regulation of lipoxygenase enzymes.

Lipoxygenases also have a requirement for free arachidonic acid and are therefore dependent upon the flux of arachidonic acid controlled by release from phospholipids. Therefore, inhibition of cyclooxygenase pathways may cause an increased flux through the lipoxygenase pathways.

Most data on regulation of specific lipoxygenases have been obtained for platelet 12-lipoxygenase which has a requirement for Fe^+ (Greenwald et al. 1980) and is therefore inhibited by metal ion chelators.

Calcium ions stimulate 5-HETE and LTB^ production by rat neutrophils, whereas production of 11-HETE and 15-HETE does not require calcium ions (Siegel et al. 1981). The

54 release of chemokinetic and aggregatory factors by polymorphonuclear leucocytes produced via the lipoxygenase pathway is also stimulated by calcium (Bray et al. 1981). The general antioxidant nordihydroguaiaretic acid (NDGA) also inhibits lipoxygenases (Tappel et al. 1953). Antioxidants such as vitamin E may inhibit platelet 12-

lipoxygenases (Rao 1978) whereas other workers have demonstrated no effect (Butler et al. 1979).

Platelet lipoxygenase is insensitive to NSAIDs and cyanide (Hamberg & Samuelsson 1974); however, peroxidase activity may be inhibited by aspirin and indomethacin

(Siegel et al. 1979) causing a build up of hydroperoxides. Some compounds may directly affect both cyclooxygenase and lipoxygenase pathways or may indirectly affect 1 pathway by a direct affect on the other.

Interaction of lipoxygenase products has been shown, for example 15-HETE inhibits production of 12-HETE and 5-HETE (Van der Hoek et al. 1980). The novel antithrombotic

compound Nafazatrom (2,4-dihydro-5-methyl-2-[2-(2- napthyloxy)ethyl]-3H-pyrazol-3-one) has been shown to stimulate in vitro vascular synthesis of PGI2 (Vermylen et al. 1979 ) possibly by acting as a cofactor for PGI synthetase. It may also act as a reducing cofactor for

peroxidases thus protecting cyclooxygenase from peroxide

inactivation. Alternatively, it may inhibit synthesis of HETEs or HPETEs which themselves inhibit PGI2 synthesis.

Nafazatrom has also been shown to inhibit the synthesis of 5-HETE and 12-HETE (Busse et al. 1982, Honn & Dunn 1982);

55 however, it stimulates 15-HETE production (Mardin Busse 1983 ) .

56 3) Metabolism of Arachidonic Acid in Intrauterine Tissues. a ) Release of substrate from complex lipids. Okazaki et al. ( 19 78 , 19 81 ) and Di Renzo et al.

(1981) demonstrated the presence of a calcium—sensitive phospholipase A2 in amnion and chorion laeve which specifically releases arachidonic acid from the sn-2 position of phosphatidylethanolamine (figure 12) and the presence of a calcium-sensitive phospholipase C which specifically cleaves arachidonic acid from phosph&tidylinositol (figure 12) in conjunction with 2 other enzymes. A diacylglycerol lipase was also demonstrated in decidua vera as was a monoacylglycerol lipase which had specificity for arachidonic acid in the sn-2 position. These 2 enzymes may act sequentially to liberate arachidonic acid and the combined activities were found to be greater in decidua vera than in amnion or chorion laeve. The activity of diacylglycerol kinase, which prevents release of arachidonic acid by converting diacylglycerol to phosphatidic acid, is inhibited by calcium, figure 12. The activity of phospholipase A2 in placental villi was shown to increase with the onset of labour (Dimette 1980). b) Synthesis of oxygenated arachidonic acid metabolites by

intrauterine tissues.

Despite extensive investigation of arachidonic acid metabolism by intrauterine tissues the precise sites and regulatory mechanisms of cyclooxygenase and lipoxygenase

57 Figure 12. Release of Arachidonic Acid in Fetal

Membranes.

1) Release from Phosphatidylinositol by a succesion

of Lipases.

Phosphatidyl inositol

PLC + Ca^ Diacylglycerol lipases ------b- Diacylglycerol + Monoacylglycerol

++ Kinase - Ca I Lipases

Diacylglycerolphosphate Arachidonic Acid

2) Release from Phosphtiylethanolamine by PLA,

Phosphatidyl ethanol amine

PLA- Ca

Arachidonic acid + Lysophosphatidylethanolamine

Pathway 1 is greater in decidua vera than amnion or chorion.

Pathway 2 has been demonstrated in amnion and chorion.

Diacylglycerol kinase is greater in amnion than chorion or decidua.

58 enzymes in the pregnant uterus have not been established. The production of prostaglandins from exogenous and endogenous sources has been demonstrated in most

intrauterine tissues. These investigations have not provided consistent results which may be due to the experimental methods employed. Sykes et al. (1975) demonstrated that homogenates of decidua and myometrium converted added arachidonic acid to

PGE2 and PGF2<^ and that homogenates of chorion laeve and amnion produced mainly PGE2 and some PGF2^• However Kinoshita et al. (1977) detected only PGE2 production in homogenates of term decidua, amnion, chorion laeve and villi and thought that the decidua might contain an inhibitory factor of prostaglandin synthesis.

The use of homogenates has some disadvantages. Trauma and disruption of intracellular control mechanisms may affect release of arachidonic acid and its subsequent metabolism. Added arachidonic acid may not be converted to the same products as endogenous arachidonic acid. Christensen and Green (1983) followed the metabolic fate

of exogenous ^C-arachidonic acid in homogenates of human amnion, chorion laeve, placental artery, myometrium and placenta. Placenta and amnion produced mainly PGE2, whereas placental arteries and myometrium produced 6-keto-

PGF^. In a subsequent study Dimov et al. (1983) observed that homogenisation induced a substantial release of arachidonic acid into the medium thus reducing the specific activity of the added label and the concentration

59 of endogenous substrate. No qualitative correlation between conversion of endogenous and exogenous substrate was observed and it was suggested that the two sources of substrate did not mix freely. Similar results have been obtained in other tissues (Coene et al. 1982, Ehrman et

al. 1982). Superfusion techniques have been used to wash out products induced by trauma (Mitchell & Flint 1978 ) and to

demonstrate production of PGE2 and PGF2^by maternal and fetal cotyledons and myometrium and of TXB2 (Mitchell et al. 1978a) and PGD2 (Mitchell et al. 1982) by the placenta. In fetal tissues 6-keto-PGFj^ was found to be

the major metabolite with lesser amounts of PGF2o( and PGE2 (Strickland & Mitchell 1983). However this technique may not take into account the decrease in endogenous substrate

induced by trauma (Dimov et al. 1983). The use of whole cells may reduce some of the factors

which affect prostaglandin production by homogenates since the cells remain intact. Myatt and Elder (1977) observed that placental tissue released an anti-platelet

aggregatory activity in vitro and Jogee et al. (1983) demonstrated accumulation of 6-keto-PGF-^ in placental, cell cultures. Olson et al. (1983a) detected release

of PGE2f pGF2d, anc* smaller quantities of 6-keto-PGF^ by amnion cells in suspension. These systems may have been

affected by culture conditions. Jogee et al. (1983) showed that release of 6-keto-PGF^ decreased after 24 hours in culture during which time the cells adhered and

60 may have recovered from the trauma caused by dispersion.

In studying a cell suspension Olson et al. (1983a) did not allow cells to recover from trauma and thus production of

PGF2o( may have resulted from non-enzymatic conversion of endoperoxides synthesised from excess arachidonic acid.

Other workers (Casey et al. 1984, Kinoshita & Green 1980) have not observed P G F ^ production by amnion cells. The method of detection may also affect measurement of prostaglandins. Radioimmunoassay (RIA) is limited to compounds for which assays are available and chromatographic techniques are limited by their resolving power. Harper et al. (1983) determined conversion of radiolabelled arachidonic acid in human placenta and found that the major metabolites were 15-keto-PGE2 and 13,14- dihydro-15-keto-PGE2. Prostaglandin D2 and smaller amounts of PGF2A, its metabolites and PGE2, TxB2 and 6- keto-PGF^ were also formed. It was postulated that an endogenous inhibitor of prostaglandin synthesis was present. Kinoshita and Green (1980) showed formation of several hydroxy-fatty acids in homogenates of amniotic membranes but in a subsequent study (Christensen & Green 1983) hydroxy-fatty acids were not detected. Saeed and Mitchell (1982a) have, however, demonstrated production of 12-HETE in human amnion, decidua vera and placenta and 12-

and 5-HETEs in human myometrium and cervical tissue using thin layer chromatography. This method lacks the resolution necessary for these compounds which can be obtained with high performance liquid chromatography

61 (HPLC) and gas-chromatography mass-spectrometry (GC-MS). It was also postulated by Saeed and Mitchell (1982 & 1983) that the immediate precursor of 12-HETE, 12-HPETE, could inhibit PGI2 formation. Elliot et al. (1984) have demonstrated production of both prostaglandins and HETEs in rabbit fetal membranes and found that in mid pregnancy HETE synthesis predominated whereas prostaglandin synthesis increased towards term. Although the various methods employed may effect the metabolism of arachidonic acid, it appears that PGE2 is the major product of amnion, whereas myometrium and fetal vessels may sythesise mainly PGI2 (Terragno et al.

1978, Hamberg et al. 1979, Bamford et al. 1980). A variety of workers have shown in vitro production of PGD2, PGE2/

PGF2&' PGI2 an<^ TxB2 the placenta. However further metabolites of prostaglandins could also be produced since the placenta has an extensive catabolic capacity

(Braithwaite & Jarabak 1975, Westbrook et al. 1977). The production of HETEs has been demonstrated but has not been extensively investigated in vitro. c ) Regulation of arachidonic acid metabolism in intrauterine tissues.

The regulation of phospholipase activity by sex steroids in target tissues may influence prostaglandin production (Dey et al. 1982). The presence of sex steroid receptors in the placenta might allow regulation of prostaglandin production by a receptor-mediated mechanism

62 similar to that postulated for the uterus (Pakrasi et al. 1983). Ogle (1982) demonstrated progesterone receptors in rat trophoblast. McCormick et al. (1981) could not detect high affinity low capacity binding of progesterone in human term placentae in contrast to the findings of Younes and Besch (1981). Placental oestrogens may act as autocrine hormones stimulating prostaglandin synthesis in the same organ in which they are synthesised. The mechanism could be similar to that in which ovarian steroids stimulate granulosa cell hyperplasia (Richards 1979). Coulam and Spelsberg (1983) suggested that the placenta could be a target for glucocorticoids and androgens, but not oestrogen and progesterone on the basis of receptor determinations. Jogee et al. (1983) found that oestradiol and progesterone, both singly and combined, stimulated 6-ke to-PGF^ production in cell cultures of 1st trimester placentae. The effects were • _ c only significant at 10 M which is not indicative of a receptor- mediated mechanism. Combinations of 10“*^M oestradiol with increasing concentrations of progesterone had synergistic effects, significant at a concentration of

10“^M progesterone suggesting that the synergystic action of progesterone may have been receptor-mediated. In term placental cells oestradiol stimulated 6-keto-PGF^oC production in a dose-dependent fashion whereas progesterone had a slight inhibitory effect at 10“^M. In

63 combination, progesterone was found to antagonise the oestrogen effect at high concentrations (10~^M) but was synergystic at low concentrations. The effect of low concentration may be receptor-mediated but the effect at high concentration raises the possibility of an alternative mechanism such as membrane stabilisation and destabilisation. Further results suggested that the levels of 6-keto-PGFreleased were controlled at the level of catabolism via prostaglandin dehydrogenase.

It has also been suggested that steroid hormones regulate the levels of oxytocin receptors in the myometrium (McCracken et al. 1978). Oestrogen causes an increase in receptor concentration while progesterone opposes this effect. Since prostaglandin synthesis in the uterus is stimulated by oxytocin acting via its receptor, sex steroids may indirectly control prostaglandin synthesis through regulation of oxytocin receptor levels.

Stimulation of synthesis can also be obtained by removal of inhibition. The presence of an endogenous inhibitor of prostaglandin synthesis has been reported in the pregnant uterus (Harper et al. 1983, Kinoshita et al. 1977). Lipoxygenase products have been postulated to be inhibitors of PGI2 synthesis (Saeed & Mitchell 1983) and an inhibitor of PGI2 synthesis produced by blood elements trapped in the decidua may be a lipoxygenase product (El

Tahir & Williams 1981). Uterine prostaglandin synthesis may be inhibited by compounds of fetal origin. Willman and Collins (1976) observed that high levels of

64 prostaglandins found in the non-pregnant endometrium progressively declined with the course of pregnancy. Itoh et al. (1980) suggested that human chorionic gonadotrophin

(hCG) may maintain pregnancy by inhibition of PGF20C synthesis. Synthesis of prostaglandins by amnion has been found to be dependent upon calcium ions (Olson et al. 1983a) suggesting regulation at the level of phospholipases.

Synthesis was also stimulated by factors in fetal and adult urine and by isoproterenol, a beta-adrenergic agonist (Di Renzo et al. 1984a). The latter observation led to the suggestion that catecholamines present in amniotic fluid might regulate prostaglandin synthesis by fetal membranes. It is not known if any of the pathways outlined in figure 12 are sensitive to catecholamines but catecholamine receptors have been characterised in the amnion (Di Renzo et al. 1984b) and placenta (Moore & Whitsett 1981).

Prostaglandin production may also be regulated at the level of conversion of endoperoxide to primary prostaglandins. Olson et al. (1983d) found differential effects of 17-beta-oestradiol and 2-hydroxyoestradiol on amnion and decidual cells. These effects were only observed in cells obtained following spontaneous labour.

Synthesis of PGI2 by umbilical vessels was stimulated by 17-beta-oestradiol, this effect being inhibited by progesterone. Thromboxane B2 synthesis was not affected by either hormone (Makila et al. 1982).

65 4) Biological Roles of Arachidonic Acid Metabolites in

Pregnancy. a ) Prostaglandins. Prostaglandins have a wide range of biological activities in a variety of tissues. There is considerable

evidence that they act via specific receptors at the cellular level to modulate hormonal, neurohormonal or other stimuli and to exert a variety of pharmacological effects (Samuelsson et al. 1978, Kennedy et al. 1982).

In pregnancy prostaglandins have been implicated in ovulation, implantation, maintenance of blood flow and

placental perfusion and in parturition (Goldberg & Ramwell

1975). They may also be involved in the immunology of pregnancy.

i) Implantation.

During implantation a local inflammatory reaction occurs at the attachment site (Psychoyos 1967). An increase in uterine stromal capillary permeability has been detected which is thought to be necessary for successful implantation. Prostaglandins and histamine have been implicated in the mechanism based on their function in altering vascular permeability. Support for

this hypothesis is provided by the findings of Kennedy and

Zamecnik (1978) and Pakrasi and Dey (1982) who respectively determined elevated levels of 6-keto-PGF-j^.

and PGF2^ and PGE2 at the implantation site in the rabbit.

66 The levels were raised above those at non-implantation

sites and removal o£ the blastocyst lowered them. It was not determined whether the site of synthesis was the endometrium or the blastocyst. Prostaglandin I2 may be involved in the increase in permeability but also affects vascular tone and platelet

aggregation. Kuehl et al. (1977) suggested that PGG2 or a non-prostaglandin product might mediate the increase in

permeability. Williams (1983) showed that a LTD^-induced

increase in vasopermeability may be augmented by PGE^ and

p g e 2 . Further support for a role for prostaglandins in implantation was given by Lau et al. (1973) who showed that treatment with indomethacin in pregnancy prevented implantation in mice, an effect which was overcome by

administration of PGE2 or PGF2^.

ii) Maintenace of blood flow and placental perfusion.

The maintenance of blood flow and placental perfusion is a key role proposed for prostaglandins during pregnancy (Wallenberg 1981). Vasodilator prostaglandins,

especially PG^* may be responsible for reduced peripheral resisitance, vasodilation of uterine arteries and

refactoriness to the pressor action of angiotensin II in

the maternal circulation (Gant et al. 1973). Support for this role was provided by McLaughlin et al. (1978) who found that treatment with indomethacin causes enhanced

constriction of the uterine vasculature by angiotensin II

67 in sheep. Prostaglandin I2 may be important in preventing platelet aggregation and formation of infarcts in the spiral arteries and on the surface of the placenta and may play a role in the maintenance of the fetal circulation. Vessels of the umbilical cord have a considerable capacity to produce PGI2 (Hamberg et al. 1979) which is the most potent dilating substance for the umbilical artery. The rapid closure of the vessels of the umbilical cord at birth may be a result of switching from PGI2 to TXA2 synthesis (Tuvemo 1980), since TXA2 is the most potent contracting substance for the umbilical artery and the vessels have a large synthetic capacity for this compound. The thromboxane mimetic U46619 is also a powerful constrictor of the perfused human fetal placental vasculature (Glance et al. in preparation) whereas other primary / have little or no effect on their own. A possible role for vasodilator prostaglandins may be the protection of the placental circulation from the pressor effects of angiotensin II (Glance et al. in preparation) a situation analagous to that in the kidney but of importance in the placenta which may be a major site of activation of the renin-angiotensin system in the fetal circulation (Glance et al. 1984).

It has previously been suggested that prostaglandins may play dual roles on opposite sides of the placenta, having vasoconstrictor effects on one side and vasodilator

68 effects on the other, thereby overcoming the effects of perfusion inequalities (Rankin 1978). However in view of the considerable capacity for prostaglandin catabolism in the placenta (Braithwaite & Jarabak 1975, Westbrook et al. 1977) and lack of evidence for transplacental passage of prostaglandins (Glance et al. in preparation), this is unlikely. Further support for the importance of prostaglandins in the maintenance of pregnancy comes from studies of pregnancies complicated by fetal growth retardation or hypertension. It is frequently found that there is a decreased synthesis or concentration of vasodilator prostaglandins in these complicated pregnancies. Reduced PGI2 production has been found in umbilical and placental vasculature (Remuzzi et al. 1980, Lewis et al. 1981, Stewart et al. 1981) and in maternal subcutaneous and uterine vessels (Bussolino et al. 1980). Increased synthesis of vasoconstrictory TXA2 has been observed in platelets (Wallenberg & Rotmans 1982 ) and placentae (Makila et al. 1984, Walsh & Fenner 1984), from pregnancies complicated by hypertension. The latter observation could be accounted for by increased numbers of platelet aggregates within the placenta and it was not determined which side of the placenta was involved.

Stuart et al. (1981) found that although the uptake of ^C-arachidonic acid by umbilical cords from normal, pre-eclamptic and other pathological pregnancies did not vary, the pre-eclamptic samples had a diminished capacity

69 to produce PGI2. Ylikorkala et al. (1983) could find no correlation between intervillous bloodflow and 6-keto-PGF^ or TXA2 levels, but suggested that alterations in feto­ placental production may be aetiological factors in fetal growth retardation.

iii) Parturition.

One of the major functions of prostaglandins in pregnancy may be their involvement in parturition via their effect on myometrial activity. A role for prostaglandins in parturition is implied by the large release of prostaglandins observed during

labour (Bygdeman et al. 1970, Green et al. 1974, Granstrom & Kindahl 1976). The importance of prostaglandins in the process of labour was shown by the prolongation of labour

induced by prostaglandin synthesis inhibitors (Fuchs et al. 1982). The mechanism of action of prostaglandins is

unclear although both PGE2 and PGF2

1979 ) and the use of prostaglandins in induction of labour, ripening of the cervix and abortion is now well established (Bygdeman 1980).

Mitchell et al. (1978b) suggested a role for PGI2 in parturition based on their findings that fetal membranes

70 including amnion, synthesised 6-keto-PGF^^ and that synthesis increased during pregnancy.

Prostaglandin I2 has been shown to cause a biphasic response in non-pregnant myometrium in vitro consisting of initial contractions followed by a longer period of relaxation, with absence of spontaneous tone. The mechanism was thought to be mediated via an increase in intracellular cAMP (Omini et al. 1979). Further evidence showing inhibition of spontaneous contractility of non­ pregnant human myometrium by PGI2 was given by Karim and Adaikan (1979 ) and Wilhelmsson et al. (1981). In the pregnant uterus a similar effect was observed although the inhibition was transitory (Wikland et al. 1983). It was suggested that the uterine vessels are considerably more sensitive to the action of PGI2 than the myometrium and, by implication, that the site of action is in fact the uterine vasculature. However it has been shown that PGI2 synthetase is more abundant in uterine musculature than vasculature (Kierse et al. 1984). Further evidence against a role for PGI2 in parturition was suggested by the observation that although there appeared to be increased synthesis with labour, there was no continuing rise which correlated with physiological changes such as cervical dilation (Mitchell et al. 1979). The same was true for Tx B2 (Mitchell et al. 1978c). In contrast, measured values of PGE, PGF2(?^ and 13,14-dihydro-15-keto-

PGF2^ continued to rise with the progression of labour (Kierse 1979).

71 iv) Roles as second messengers: interaction with peptide

hormones.

The major role of prostaglandins in pregnancy may be their action as mediators or modulators of the effects of peptide hormones.

There is evidence to suggest that prostaglandins may participate in the maintenance of low peripheral resistance of maternal and fetal circulation by attenuating the pressor effects of angiotensin II (Gant et al. 1973, Glance et al. in preparation). Angiotensin II may also stimulate production of PGI2 in the myometrium and PGF2^in the endometrium of the pregnant rat (Campos et al. 1983 ) and production of vaso-dilator PGI2 and PGE in the human fetal-placental vasculature (Glance et al. in preparation).

Prostaglandins have also been suggested to mediate the contractile effects of vasopressin and oxytocin.

Laudanski et al. (1984) found that although the contractile effect of vasopressin in myometrium is not mediated by prostaglandins the effect in uterine artery may be attenuated by prostaglandins. In the endometrium oxytocin and vasopressin stimulate prostaglandin synthesis (Fuchs et al. 1981, Stromberg et al. 1983) and since prostaglandins potentiate the effect of vasopressin in the pregnant uterus (Laudanski & Akerlund 1980) it may be possible that prostaglandins synthesised at one site could exert their effect at another.

72 Towards the end of pregnancy there is an increased sensitivity of the uterus to the contractile effect of oxytocin. In the rat this increase in sensitivity is correlated with uterine PGF2C* production (Chan 1983). The increase in sensitivity has also been related to an increase in the amount of receptors (Alexandrova & Soloff 1980). McCracken et al. (1978) showed that oestrogens increased, the amount of receptor whereas progesterone had an inhibitory ef'fect. It can therefore be postulated that falling progesterone levels towards the end of pregnancy cause an increase in oxytocin receptors, thus increasing the sensitivity of the uterus to oxytocin.

This would lead to an increased synthesis of PGF2q( and possibly PGE2, which may mediate the contractile effect of oxytocin. Prostaglandin synthetic activity may also be modulated by steroid hormones, progesterone having an inhibitory effect.

Thus, prostaglandins may exert a combined effect with peptide hormones or may mediate or modulate the effect depending upon the site of action of the hormone and endocrine status of the tissue. -The effect of prostaglandins may be at the site of synthesis e.g. in the vasculature, or at different sites e.g. synthesis in the endometrium and action in the myometrium.

Further roles for prostaglandins in pregnancy may be the mediation of changes in cervical connective tissue (Liggins 1979) or modulation of the immune response via inhibition of lymphocyte function (Gemsa et al. 1981).

73 The evidence outlined above suggests that PGEz and

PGF2

The biological roles of leukotrienes (LT) and HETEs have not been investigated to the same extent as prostaglandins and most work has been centred on their role in the immune system.

Leukotrienes have been shown to be regulators and mediators of the immune response. Morris et al. (1980) have shown that LTC 4 , LTD^ and LTE 4 are the major components of slow reacting substance of anaphylaxis (SRS). Leukotrienes also have potent effects on the cardiovascular system. Leukotriene C4 and LTD4 are potent contractants of human pulmonary veins (Schellenberg &

Foster 1984), but have minimal activity on pulmonary arteries. In vivo 5-lipoxygenase products may cause an increase or a reduction in blood flow (Letts et al. 1984) and intrajugular injections cause a dose-dependent

74 increase in mean arterial pressure (Sirois et al. 1981).

Synthetic LTC4 and LTD4 can cause concentration-dependent contractions of the guinea-pig uterus (Weichman & Tucker 1982) which may be partially mediated by the stimulation of prostaglandin synthesis.

Leukotriene has a variety of functions. It may act as a potent chemoattractant for polymorphonuclear leucocytes (Ford-Hutchinson et al. 1980) and down regulates the immune response by inducing suppressor T cells (Rola-Plezsczynski & Sirois 1983). Similar effects have been demonstrated for LTD4 and LTE4 (Webb et al.

1982) and for 15-HETE in mice (Bailey et al. 1982). Leukotriene B4 also acts synergistically with PGE2 to increase vascular permeability (Williams 1983). Dihydroxy-fatty acids have been shown to cause contraction of guinea-pig lung parenchymal strips (Sirois et al. 1982). Leukotriene B4 is extremely potent and also causes release of prostaglandins and thromboxane. Five- HETE, 12-HETE and 15-HETE also have some effect on smooth muscle preparations.

Biological roles of mono-HETEs have not been studied in detail but it has been shown that 15-HETE (Vericell &

Lagarde 1980) and 12-HETE (Croset et al. 1983) have anti- platelet-aggregatory effects. An anti-aggregatory activity released from leucocytes in vitro may be due to a lipoxygenase product (Villa et al. 1983). The action may be mediated through antagonism of thromboxane receptors.

75 5) Fetal Membranes: development, structure and function. a ) Development of the membranes.

The cells from which the membranes derive differentiate from embryonic cells at an early stage in the development of the fertilised ovum. The ovum is fertilised in the ampulla of the fallopian tube and enters a period of rapid cellular division (figure 13). During this growth phase it is transported along the fallopian tube entering the uterus as a solid mass of cells called a morula. The formation of a central cavity within the morula leads to development of a blastocyst, and loss of the zona pellucida leaves the outermost cells of the blastocyst in direct contact with the endometrial epithelium. The outer layer of cells forms the trophoblast and the inner layer of cells the embryoblast.

The trophoblast differentiates into two layers: an inner layer of large mononuclear cells or cytotrophoblast and an outer layer of multinucleated syncytiotrophoblast, in which no cellular boundaries are observed.

The trophoblast proliferates rapidly into the endometrium causing degeneration of the endometrial epithelium, this process is termed implantation. Concomitantly, the endometrium undergoes a decidualisation reaction, which begins at the implantation site and spreads throughout the endometrium. During this process stromal connective tissue cells become enlarged and fill with glycogen and lipid/and endometrial arterioles

76 Figure 13, Development and implantation of blastocyst.

Embryoblast Uterine Epithelium

;* • I Cytotrophoblast Syncytium Amnion

Maternal vessels

Cytotrophoblast

Syncytium

Extra embyonic stroma Maternal lacunae

Cytotrophoblast shell

77 proliferate beneath the implanting blastocyst. The transformation into decidual tissue may fulfill a nutritional role and act as a mechanism which regulates

the depth to which the trophoblast invades. Following degeneration of the endometrial epithelium the rapidly proliferating trophoblast continues to invade

the decidua and begins to erode maternal sinusoids by digestion of their epithelium. Further erosion of

sinusoids leads to formation of interconnecting lacunae. Continued division of the cytotrophoblast leads to a

multilayered structure. During implantation the trophoblast forms a flat layer of cells on its inner surface. This layer of cells

attaches itself around the margin of the ectodermal disc and forms the amnion. The cavity enclosed by the amnion and the ectoderm is the amniotic sac, and folding of the amnion around the blastoderm and apposition and fusion of the folds gives rise to the amnionic membrane (figure 13). The formation of the amnion is succeeded by delamination of the extra-embryonic mesoderm from the inner surface of the trophoblast.

Further invasion by the trophoblast (figure 13) leads to engulfment and erosion of maternal blood vessels and the enlarging lacunae fill with blood. Cytotrophoblast

division within the trophoblast layer continues and cell columns form, further growth of which leads to primary

villus formation. Further development of the mesoderm within the cell columns results in the formation of

78 secondary villi. Distal extension of cytotrophoblast cell columns produces anchoring trophoblast columns which attach to the decidua. Lateral spreading of the columns leads to the formation of a cytotrophoblast shell which splits the syncytium into two layers. The definitive syncytium remains on the fetal side whereas the peripheral syncytium degenerates and is replaced by the fibrous layer of Nitabuch. This is the region where separation occurs at birth and is the limit of t'he depth to which the trophoblast invades, although radial expansion still occurs. Lateral spreading of trophoblast occurs within the

lacunae and begins with the formation of syncytial sprouts behind which cytotrophoblast columns form. Further growth of these columns and development of mesoderm leads to

secondary villi, which eventually provide the exchange surface within the intervillous space. Cytotrophoblast cells emigrate from the shell into the myometrium and may either fuse to form syncytial-like giant cells which colonize the placental bed or invade and

replace the endothelium of maternal spiral arteries (Brosens et al. 1967).

Endovascular trophoblast causes considerable disruption of the arterial wall with replacement of most

of the muscular tissue by fibrinoid. This has the effect of progressively distending the vessels. This adaptation accomodates the increase in blood flow which occurs with

79 the course of pregnancy. An absence of this secondary invasion is a pathological feature of pregnancies complicated by fetal growth retardation or hypertension

(Robertson 1976, Shepherd & Bonner 1976). b) Structure and function. i ) Placenta.

The development and functions of the placenta are well documented (Boyd & Hamilton 1970). The main function of the placenta is supply of oxygen and nutrients to, and removal of waste from, the fetus. The exchange region has a villous structure which brings fetal blood into close contact with maternal blood over a large surface area (figure 14). As pregnancy progresses there is a gradual decrease in the cytotrophoblast layer and maternal and fetal blood become separated by the syncytium, the basal membrane and the capillary endothelium. The syncytium is the area of exchange between the fetus and the mother and the cytotrophoblasts appear to be stem cells for syncytial formation. The stroma derives from the extra-embryonic mesoderm and has a cellular composition of fibroblasts and Hofbauer cells. The latter may represent differentiated monocytes to which a number of functions including a role as tissue macrophages have been ascribed. Fetal capillaries are continuous with the stroma and are lined with capillary endothelium, which provides part of the barrier between fetal and maternal blood.

80 Figure 14. Structure of a Fetal Villus

Cross sections of chorionic villi at different stages of gestation. (A) 4 week embryo; (B) 6 j week embryo; (C) I4th. week placenta; (D) Term placenta.

(taken from Llewellyn-Jones,D. , 1982, 'Fundamentals of Obstetrics and Gynaecology1, 3rd. edition, Faber & Faber, London, page 25.)

81 The relationship between the villi and the maternal circulation is shown in figure 15. Blood from the spiral arteries is driven into the intervillous space under the pressure of the maternal circulation. Its force is dissipated by the network of arteries diminishing in size from uterine to radial to basal to spiral, the shape of the latter causing a further reduction of force, and by the resistance of the villi. Blood spreads outwards and flows over the surface of the villi towards the basal plate where it drains out via outlets in the endometrial venous network. The draining is aided by the pressure of more blood entering the intervillous space from the spiral artery. The draining veins run parallel to the uterine wall (Ramsey & Davies 1963) and constriction by contractions of the uterus may prevent emptying of the intervillous space. Contractions may also constrict the spiral arteries thus affecting blood flow into the intervillous space, this mechanism may be important in preventing haemorrhage at parturition.

The placenta also fulfills a role as an endocrine organ (Diczfalusy & Troen 1961). It synthesises a variety of peptides (Saxena 1971) including hCG, human placental lactogen (hPL), corticotrophin and a variety of pregnancy specific proteins. Human chorionic gonadotrophin is similar to human luteinising hormone in structure and may maintain early pregnancy by stimulating ovarian oestrogen and progesterone production (Ross et al. 1970, Van de

82 Figure 15. Relationship between Maternal and Fetal circulations.

CHORIONIC PLATE DECIDUAL PLATE <------‘------v

FETAL ARTERIES MYOMETRIUM AND VEIN

MATERNAL VEIN

VILLI OF FETAL COTYLEDON

SEPTUM MATERNAL SPIRAL ARTERY

Diagram of an intervillous space showing flow of maternal blood over the villi of a fetal cotyledon. (taken from Llewellyn-Jones,D., 1982, 'Fundamentals of Obstetrics and Gynaecology', 3rd. edition, Faber & Faber, London, page 24.)

83 Wiele 19 7 0 ). It may also have immunosuppressive properties (Canfield et al. 1976). Human placental lactogen has lactogenic activity and may have an indirect growth-promoting effect via its antagonism of insulin action and the mobilization of free fatty acids thus maintaining the nutritional supply to the fetus. The functions of other placental peptides have not been elucidated.

The feto-placental unit synthesises a variety of steroid hormones. The major steroids released are oestrogens and progesterone. Oestrogens are synthesised from 3-beta-hydroxy-steroids supplied by the fetus. The major urinary metabolite of both pregnant and non-pregnant women is oestriol. In the latter it appears to be a catabolite of more active oestradiol whereas in the pregnant woman it is synthesised de novo by the feto­ placental unit. The pathway is via 16-hydroxylation of carbon-19 androgens and not through synthesis of oestrone or oestradiol. In addition to stimulating hypertrophy and hyperplasia of uterine muscle oestrogens may also exert other effects including stimulation of PGI2 synthesis by vascular tissue (Chang et al. 1980, Makila et al. 1982). A specific role for oestriol in pregnancy has not been demonstrated.

Progesterone is the other main steroid of the feto­ placental unit and is probably produced in situ from cholesterol via pregnenolone. Progesterone may have several functions in pregnancy. It may be involved in

8 4 reducing myometrial contractility, and in renal function may affect sodium balance. Progesterone also exerts catabolic effects such as increased protein breakdown and may balance the anabolic effects of oestrogens. The aromatization of 18—carbon and 19-carbon androgens involves microsomal cytochrome P450 enzymes which are specific for androgens. Placental mitochondria contain a cytochrome P450 enzyme which may catalyse cleavage of cholesterol to progesterone. Drug metabolism studies indicate that there is a degree of specificity of cytochrome P450 activity, which is similar to rodent hepatic P448 forms of cytochrome P450. A placental epoxide hydrase activity has also been demonstrated. The placenta may serve to preserve the fetus from immunological rejection. Maternal immunoglobulin G (IgG)

is transmitted to the fetus via Fc receptors on the syncytial membrane, which may be a source of IgG. This may protect the fetus against foreign antigens since the fetal immune system is not fully developed until some time post partum. If IgG is directed against fetal antigens, they may be absorbed within the placenta before entering the fetus (Johnson et al. 1977). The trophoblast layer in direct contact with the maternal circulation does not appear to initiate an immune reaction leading to rejection. This may be due to lack of histocompatibility (HLA) antigens (Page-Faulk 1981), binding of maternal proteins such as transferrin to the outer membrane (Faulk

85 & Galbraith 1979), or production of immunosuppressive hormones such as hCG (Beling & Weksler 1974) or progesterone (Kobayashi et a 1 . 1979). The immunosuppressive effect of hCG in crude preparations may be due, however, to contaminating factors (Morse 1976). ii ) Chorion laeve.

The chorion laeve is derived from the primary trophoblast of the blastocyst. Instead of developing a villous structure and a vascular system as the definitive placenta does, it degenerates to form an opaque membrane which contains ghost villi in its structure (Bourne 1960). The chorion laeve is composed of 4 layers: the cellular layer, the reticular layer, the pseudobasement membrane and the trophoblast layer (figure 16). The cellular layer is formed from a loose network of interlacing fibroblasts, which may be imperfect or absent from term chorionic tissue. The reticular layer is the thickest part of the chorion and consists of a reticular network which contains fibroblasts and Hofbauer cells. The pseudobasement membrane of the trophoblast is composed of dense argyrophilic (silver staining) connective tissue firmly attached to the reticular layer. Anchoring, branching fibres of basement membrane are sent down into the trophoblast layer. The trophoblast layer consists of

2 to 10 layers of trophoblast cells which are in direct contact with maternal decidua. Wang and Schneider (1982) have demonstrated myofibroblasts in the structure of the

86 Figure 16. Structure of human amnion and chorion.

( after Bourne,G.L. Am. J .Obstet.Cynec. 79 ,1070-1073 1960)

87 chorion, which, they suggested prevented the membrane from becoming over distended. The functions of the chorion laeve are not well known. It may provide an immunological barrier to protect the fetus by way of its trophoblast structure. There may be an extraplacental route for transfer of proteins via intercellular spaces and the discontinuity of the basement membrane (Wang & Maas 1983, Wang & Schneider 1983). The chorion also posseses extensive steroid- metabolising activity including sulphatase (Gant et al. 1975), 3-beta-hydroxy-delta-5-steroid dehydrogenase

(Jirasek et al. 1969) and aromatase (Gibb & Lavoie 1981), the latter being less active than that of the placenta.

Chorion laeve is also a rich source of arachidonic acid (Foster & Das 1984), the metabolism of which has been described and the chorio-decidua has been found to be a site of angiotensin I generation (Craven et al. 1983). The function of steroid metabolism by the chorion may be to supply oestrogens (Gibb & Lavoie 1981), cortisol (Murphy 1977) and 4-ene-3-keto-steroids (Gibb et al. 1978) to the amniotic fliud. iii) Amnion.

The structure of the amnion has also been described by Bourne (1960). It consists of 5 layers: an epithelium, a basement membrane, a compact layer, a fibroblast layer and a spongy layer (figure 16). The innermost layer is the epithelium and is in

88 direct contact with the amniotic fluid. It consists of a single layer of largely cuboidal cells which may be columnar over the placenta, or flattened on the reflected amnion. They are strongly adherent to the underlying basement membrane, but on their free surface have a brush border of microvilli, suggesting an exchange function. The cell membrane is well defined but is broken by complex intercellular canals. Viewed from above the epithelial cells appear as a mosaic of irregular polygonal shapes. The underlying basement membrane is composed of a network of reticular fibres. Interdigitations form a complex relationship with the epithelium. The compact layer is relatively dense, is almost devoid of cells, and consists of a complex network of reticular fibres. A loose network of fibroblasts embedded in a mass of reticulin forms the fibroblast layer. It is the thickest layer of the amnion and cells within it may have phagocytic activity. Between the amnion and the chorion is a compressed layer of extra-embryonic coelom which forms a spongy layer. it is composed of wavy bundles of reticulin bathed in mucin and may contain a few isolated fibroblasts. The chorion is firmly attached to the decidua and the spongy layer permits sliding of the amnion over the chorion.

The amnion may also play a role in the extraplacental transfer of proteins including alpha-feto-protein and albumin (Wang & Schneider 1982). More detailed

89 examination by light and scanning and transmission electron microscopy (Van Herendael et al. 1978) lead to the suggestion that its complicated structure permits it to perform other specialised functions. These include a covering and secretory epithelium as well as providing transcellular and intracellular routes of extraplacental transport. It also has a range of steroid-metabolising activities (Sulcova et al. 1974, 1976, 1977 & 1980) including 17-beta-hydroxy-steroid dehydrogenase and 3- beta-hydroxy-steroid dehydrogenase, but not sulphatase (Gant et al. 1975). A variety of other metabolic processes have been demonstrated: in vitro assessment has shown that it is able to perform glycolysis (Brame & Overly 1970), synthesise hexosamines (Mazurkiewicz- Kikzewska 1971) and that it can incorporate fatty acids into phospholipids (Schwartz et al. 1977), triglygerides and cholesterol esters (Pritchard et al. 1968). Goto and

Wiebel (1980) demonstrated a cytochrome P448 monooxygenase activity which metabolises benzo[A]pyrene via oxygenation of the benzo ring to an epoxide diol. In vitro the metabolic activity of amnion has been found to be high and maintenance in minimal media may lead to ultrastructural alterations suggestive of impaired function.

The incorporation of fatty acids, particularly arachidonic acid, into intracellular lipids, which may act as a pool for prostaglandin synthesis and the steroid-

90 metabolising capacity of both amnion and chorion laeve suggest a specialised function of these membranes which may be distinct from, or in addition to, the function of the definitive placenta.

91 6) Methods used to Assess Arachidonic Acid Metabolism. a ) Determination of arachidonic acid metabolites.

Arachidonic acid metabolites may be determined either by bioassays or by chemical measurement. i) Bioassays.

A bioassay may be used if a compound causes a dose dependent measurable biological response, such as muscle contraction, chemotaxis or platelet aggregation. Bioassays are sensitive and require small volumes with little or no sample preparation. The specificity of a response may not be absolute but use of a series of tissues produces a profile of responses. Such a profile enables the determination of a single, or, in a mixture, the major, active metabolite, preferably in the presence of antagonists of other bioactive compounds. Bioassay also enables determination of novel activities, especially short-lived ones, although it may not determine the composition of a mixture of compounds. This method is one of the most commonly used methods and has been instrumental in the discovery of prostaglandins (Von Euler 1936) and in elucidation of novel short-lived activities such as those of T x A 2 (Piper & Vane 1969) and PGI2 (Moncada et al. 1976a). ii) Radioimmunoassay (RIA).

Perhaps the' most common method of quantitation of

92 arachidonic acid metabolites is RIA. The method is based upon the competition between labelled and unlabelled ligand for the binding site of an antibody directed against that ligand. The technique may be sensitive , accurate and specific although these parameters may be affected by a variety of factors. Since its first application to the measurement of prostaglandins (Levine. & Van Vanakis 1970 ) RIAs have been developed for many metabolites of arachidonic acid. The range of compounds which can be measured is dependent upon the antigenicity of the ligand and the availability of a pure labelled ligand. The specificity of the assay is dependent upon the specificity of the antibody-ligand binding which is affected by various factors such as pH, protein, concentration of other lipophilic compounds and cross­ reactivity. The sensitivity of the assay is also affected by these factors and by the affinity of the antibody for the ligand. The accuracy of a RIA should be assessed by comparison of data with those of an independent method, the method of choice being mass spectrometry. The effects of cross-reactivity and non-specific factors on both bioassay and RIA can be minimised if an initial purification step is included. However such a step may limit the effectiveness of a bioassay in measuring activities of short-lived compounds such as PGI2 and T x A 2 and may introduce artifacts in RIAs. The development and use of RIAs for measuring prostaglandins

93 was described by Granstrom & Kindahl (1978). iii) Mass-spectrometry.

The most accurate method used to measure arachidonic acid metabolites is mass-spectrometry (MS). However, the accuracy is offset by the requirement for a suitable separation procedure, usually gas-chromatography (GC), a lengthy sample preparation step involving derivatization and the high cost of the apparatus. The technique minimises the effects of cross-reactivity and may be used to determine novel structures. Gas-chromatography may also be used in conjunction with other detectors such as flame ionization or electron capture. These methods are not as sensitive as mass spectrometry and may also require lengthy derivatization. However mass-spectrometry may be the only method of measuring certain metabolites of prostaglandins such as 2,3-dinor-TxB2 (Roberts & Oates 1984). iv) High performance liquid chromatography (HPLC).

Successful separation of most arachidonic acid metabolites can also be achieved using HPLC. The method is useful because its resolving power is high, recoveries are good and separation of mixtures can usually be achieved within 1 hour. Considering the multitude of arachidonic acid metabolites which have been identified, this method is suitable for the assessment of the

94 metabolic fate of arachidonic acid without the expense and time involved with GC-MS, with a greater range than RIA and more specificity than a bioassay. Oxygenated metabolites of arachidonic acid may be classified into the following groups; prostaglandins and thromboxanes, hydroxy-fatty acids an<^ hydroperoxy-fatty acids; and leukotrienes. These groups have different chemical properties which will affect the choice of mode of separation, the solvent system and the method of detection. Prostaglandins have a common absorbance spectrum with a maximum absorbance at 192.5 nm, with further maxima at

217nm, 228nm and 278nm for PGA2/ 15-keto-PGE2 and PGB2 respectively. They are soluble in both organic and aqueous-organic solvent mixtures rendering them suitable for both normal- and reverse-phase separations. Hydroperoxyeicosatetraenoic acids and their reduction products, HETEs, have hydroperoxy and hydroxy groups allylic to a conjugated diene. This structural component in the HETEs has an absorbance maximum at 235nm. The other non—conjugated diene function gives rise to another absorption band at 190nm. Hydroxy-fatty acids, including HHT, can be separated by either reverse- or normal-phase HPLC. These compounds are less polar than prostaglandins

but more polar than arachidonic acid.

Leukotrienes contain 4 carbon-carbon double bonds, including a triene system. The triene system has an absorbance maximum between 270nm and 280nm with prominent

95 shoulders at shorter and longer wavelengths. Leukotrienes are easily separated using reverse-phase HPLC and their relatively high extinction coefficients makes sub-nanomole detection by ultra-violet absorption more practical than for prostaglandins.

Sample preparation: although HPLC may be applied to underivatized compounds the nature of the columns used requires some preparation of 'the sample prior to separation. High performance liquid chromatography columns have comparatively high efficiency but low capacity. Therefore the sample load should be minimised to prevent overloading, it should be concentrated to a small volume to prevent peak-spreading and be applied to the column in an appropriate solvent with interferences from larger or insoluble compounds removed. There are four commonly used methods for chemical clean up: liquid/liquid partition gives good recovery but has a low specificity and is time-consuming; gel filtration may be used to remove large molecules but it is slow and lacks specificity; separation by thin layer chromatography is time-consuming as it requires a desorption step and its reproducibility is poor; liquid/solid chromatography has a higher specificity than

liquid/liquid partition and the recent development of commercially available octa-decylsilica (ODS) reverse- phase or silica normal-phase columns has led to a technique which is rapid, reproducible and relatively

96 specific (Powell 1980, Metz et al. 1982, Luderer et al.

1983 , Morris et al. 1984 ).

Filtration: after extracting the sample using one of the above methods, it can be redissolved in a solvent of similar composition to that used for HPLC. The volume should be minimised and large particles removed by filtration through membranes of pore size no larger than

0.45 )lm to prevent blocking of the column.

Separation: HPLC has been used successfully to separate prostaglandins (Carr et al. 1976, Russell & Deykin 1979, Wharton et al. 1979, Terragno et al. 1981),

HPETEs . HETEs and HHT (Goetzl & Sun 1979, Porter et al. 1979, Boeynams et al. 1980, Eling et al. 1980, Van Rollins et al. 1980) and leukotrienes (Mathews et al.

1981, Metz et al. 1982, Osbourne et al. 1983 ). The solubility of most arachidonic acid metabolites renders them suitable for separation on either reverse- phase or normal-phase HPLC. Since polar compounds such as prostaglandins have shorter retention times on reverse-phase, the choice of mode of separation is therefore determined by the polarity of the compounds of interest.

Detection: the absorption properties of leukotrienes and HETEs make their detection by ultra-violet absorbance the method of choice. Prostaglandins have also been detected by ultra-violet absorption (Desiderio et al.

97 1981, Terragno et al. 1981) but this requires sensitive spectrophotometers^ and solvents with good ultra-violet properties at wavelengths below 200nm. Prostaglandins may also be detected by RIA following separation by HPLC (Alam et al. 1979). The separation may improve the specificity of the assay by removing interfering factors but the combined technique is limited by the range of assays available. If insufficient quantities of a compound are available for Ultra-violet defection the sensitivity may be increased by using a radioactive tracer. Radioactivity may be detected by scintillation counting either directly with an on-line monitor or following fractionation of the eluting solvent and static counting in a liquid scintillation counter. b ) Experimental approaches to the determination of arachidonic acid metabolism.

Methods used to determine arachidonic acid metabolism may be divided into 2 categories: firstly the measurement of selected primary products or their metabolites and, secondly, following the fate of exogenously added arachidonic acid and determination of product profiles. These methods may be applied in vivo or in vitro. The determination of the roles of arachidonic acid metabolites has largely been inferred from observations of changes in the levels of metabolites concomitant with changes in physiology. In such studies, the results obtained may be

98 affected by the methods used. Several approaches have been taken to determine synthesis of arachidonic acid metabolites and their circulating levels. In vivo techniques using humans are

largely limited to analysis of body fluids and observing

changes under a variety of conditions. In vitro techniques may therefore be preferable in determination of

synthetic capacity.

i) Body fluids.

The direct determination of arachidonic acid

metabolites in body fluids such as serum, plasma or urine has been used frequently to demonstrate alterations associated with physiological or pathological observations.

The measurement of primary prostaglandins does not account for their rapid inactivation in the lungs and the

measurement of primary metabolites may give a more accurate representation (Granstrom & Kindahl 1984). However, a multitude of metabolites have been suggested to give a good reflection of prostaglandin levels

(Samuelsson et al. 1975, Granstrom et al. 1982, Frolich & Rosenkrantz 1984, Granstrom & Kindahl 1984). Measurements

made in body fluids may be affected by the mode of collection or circadian variationyand may not represent the concentration at the site of action since prostaglandins may act as local hormones. This has been demonstrated by

99 the observation that although the synthetic capacity of

PGI2 by various tissues from pathological pregnancies including umbilical cord (Stewart et al. 1981) and placenta (Jogee et al. 1983) may be reduced in vitro, maternal 6-keto-PGF^ levels in plasma do not correlate with pathological pregnancies (Ylikorkala et al.1981). ii) Whole organ studies.

In vivo, the capacity of the uterus to release prostaglandins has been implicated by measuring arterio­ venous differences (Barcikowski et al. 1974). Metabolism and extraction of various prostaglandins, such as PGI2/ by liver and kidney has also been shown (Dusting et al. 1978, Gerben et al. 1978). These methods are limited to use in animals and measurements may be affected by factors such as anaesthesia and operative trauma. Whole organs may be removed from animals and maintained in vitro using perfusion techniques. This method has been used to demonstrate conversion of arachidonic acid to prostaglandins such as PGI2 by various organs including kidney and heart (Needleman et al. 1978) and to investigate uptake and metabolism by organs including the human placenta (Glance et al. in preparation). The determination of arachidonic acid metabolism may be affected by trauma (Piper & Vane 1971) and blood contamination and is usually limited to short­ term investigations.

100 iii) Chopped or minced tissue.

Release of prostaglandins from chopped or minced tissue may be useful in determining the capacity of an organ to metabolise arachidonic acid. However, the results obtained may be complicated by the effects of trauma, blood contamination and alterations in substrate concentration (Dimov et al. 1983). Mitchell & Flint

(1978) developed a superfusion technique to reduce these effects. Their results suggested a decline in prostaglandin release over a 90-minute period before basal levels were reached. iv) Homogenates and microsomal preparations.

The use of fractions obtained following lysis of cells may provide useful information on the capacity of a tissue to metabolise arachidonic acid. Microsomal preparations may provide a source of enzyme with a high specific activity suitable for kinetics of inhibitor binding studies. However, these types of studies give no information about the control of arachidonic acid metabolism within intact cells or tissues. Lysis of cells disrupts intracellular regulatory mechanisms (Dimov et al. 1983) and also increases the requirement for cofactors, which may affect the product profile. The addition of exogenous arachidonic acid may not give rise to the same products as endogenous arachidonic acid (Coene et al. 1982) and may alter the relative proportion of metabolites

101 (Sun & McGuire 1978). v) Cell culture.

The deter ruination of release of prostaglandins by whole cells maintained in culture is a technique which provides advantages over those discussed above. The use of whole cells maintains intracellular control mechanisms and minimises the requirement for additional cofactors.

Whole cells may be cultured for prolonged periods allowing long-term assessment of factors affecting arachidonic acid metabolism and the use of a single cell type circumvents the problem of cellular diversity found within intact organs. The production of prostaglandins by cell cultures may be influenced by many factors, for example blood contamination, and trauma and cellular activity during processes such as adherence (Bockman 1981). These effects may be minimised by allowing establishment of a culture, and removal of blood elements prior to the start of an experiment. Many cultures contain antibiotics and serum, both of which have been shown to influence prostaglandin production (Hong & Levine 1976, Seid et al. 1983). Serum- free culture systems are now being developed in which cells can be grown in a well-defined system, which may reduce artifactual effects. Cell culture systems have been used to investigate arachidonic acid metabolism in a variety of tissues and

102 cell types. The growth of fetal membranes in culture provides a technique which allows determination of the contributions of amnion, chorion and placenta grown under similar conditions, which may not be possible using other methods. c ) Tissue culture of fetal membranes. i) Placenta.

There is a wealth of literature on the primary culture of placental cells in monolayer (Thiede 1960, Soma et al. 1961) or rocker cultures (Chung et al. 1969).

Placental cells grown in monolayer culture produce a mixture of 4 morphologically distinct cell types (Thiede 1960, Fox & Kharkongor 1970, Jogee et al. 1983); epithelial-like cells, multinucleated giant cells, amoeboid cells and spindle-shaped cells. Enzyme histochemical techniques have been used to relate these cell types to cells found in the intact villus (Fox & Kharkongor 1970). Epithelial cells were identified as cytotrophoblasts, the multinucleated giant cells as syncytium, the amoeboid cells as Hofbauer or macrophage cells and the spindle cells as fibroblasts. Jogee (1983) repeated these experiments with similar results.

In placental monolayers grown for extended periods it is often observed that fibroblasts outgrow trophoblast cells (Jogee 1983). Various methods have been used to obtain pure trophoblast growth including growth under

103 hypoxic conditions (Aladjem & Lueck 1981) and separation of cell types on the basis of differential densities (Cotte et al. 1980). Purified cytotrophoblasts obtained by these methods were observed to form multi nucleated syncytial masses in monolayer culture. Short-term cultures of less than 1 week of unpurified cells were found to contain predominantly trophoblast cells (Jogee

1983). The identification of cell types purely on a morphological basis may be unreliable and therefore various parameters have been measured in placental cell cultures as a marker for trophoblast function. The synthesis and release of hormones such as hCG (Chung et al. 1969, Stromberg et al. 1978, Cotte et al. 1980), steroids (Hall et al. 1977, Winkel et al. 1980) and hPL (Hall et al. 1977, Jogee 1983 ) have been measured. Human chorionic gonadotrophin has also been demonstrated using immunofluorescent techniques (Midgley & Pierce 1962) and similar methods using antibodies against cell-specific intermediate filaments (Contractor et al. 1984), or trophoblast antigens (Page-Faulk & McIntyre 1983) have also been described. Electron microscopy has been used to identify trophoblast cells on the basis of ultrastructural characteristics (Nehemiah et al. 1981).

ii) Chorion laeve.

Cells derived from chorion laeve have not been cultured as extensively as those of placenta.

104 Enzymatically dispersed cells have been grown in culture and some morphological and functional studies have been made. Acker et al. (1982) observed that chorion laeve cells grew rapidly to form a monolayer and that they could synthesise renin from labelled precursors. Zitcer et al.

(1955) reported that cultures of chorion laeve consisted of a mixture of epithelial and fibroblastic cells. iii ) Amnion.

Cells obtained from amnion have been grown in prolonged cultures and subcultured (Chang 1968). During their first and second subcultures they passed through proliferatory, stationary, degenerative and recovery phases. The addition of hydrocortisone was necessary to bring the culture into a recovery phase. Schwartz et al. (1977) found that the amnion was metabolically active and that it could be maintained in vitro provided that a suitable medium, that provides sufficient substrates, was used. The growth of enzymatically dispersed amnion cells has been described. Acker et al. (1982) described cultures of amnion cells which grew rapidlyy and Goto and Wiebel (1980) observed that the cells attached loosely during the first day of culture. They subsequently formed small colonies which grew as large sheets over 7 to 10 days. Although dispersion by trypsin required exposure to the enzyme for 3 hours few fibroblast—like cells were observed. Okita et al. (1983) have further characterised

105 amnion cells in primary culture by comparison with amnion tissue using morphological and biochemical criteria. In amnion cell cultures the main cell type observed is epithelial, which forms a monolayer having the typical "pavement" appearance of epithelial cells. Their biochemistry is similar to that of term membranes/and few other contaminating cell types have been observed.

106 Summary.

Arachidonic acid can be oxygenatively metabolised by several enzymes to produce a multitude of biologically active compounds including prostaglandins, thromboxane, hydroxy-, hydroperoxy-, and epoxy-fatty acids and leukotrienes. Prostaglandins may have many functions in the physiology and pathophysiology of pregnancy including implantation, maintenance of feto-placental and utero­ placental blood flows and low peripheral resistance, and in parturition. Deficient synthesis of vasodilator-, or increased concentrations of vasoconstrictor-prostaglandins may be aetiological factors in the pathology of pregnancies complicated by hypertension or fetal growth- retardation. The uterus may be a major source of prostaglandins during pregnancy with a substantial contribution from the fetal membranes.

The investigation of arachidonic acid metabolism is complicated by the effects of methods employed. Tlx vivo techniques with humans are largely limited to analysis of body fluids and may not allow the determination of regulatory mechanisms. In vitro techniques may be affected by trauma and blood contamination and are limited by the range of assays available. Intact cells grown in monolayer culture for extended periods allow observation of the basal metabolism of arachidonic acid and its regulation^since complicating

107 factors may be minimised. Separation of major classes of arachidonic acidmetabolites can be achieved using high performance liquid chromatography. Inclusion of a radioactively labelled tracer increases sensitivity and allows simultaneous detection of all compounds produced.

These methods were used to determine the basal metabolism of arachidonic acid by fetal membranes in culture. Placental, chorion laeve and amnion cells, obtained at different stages of pregnancy, were incubated with exogenous arachidonic acid which was either metabolised to oxygenated compounds or incorporated into the cells. Products of metabolism of exogenous and endogenous substrate were separated using high performance liquid chromatography and detected by liquid scintillation counting. Differences in product profiles were observed between exogenous and endogenous substrate, between different tissue types and before and after labour.

The major metabolites appeared to be produced by lipoxygenase or cytochrome P450 enzymes. Prostaglandin synthesis seemed to be related to the onset of labour, although exogenous substrate was converted via cyclooxygenase when endogenous substrate was not. The physiological implications of these findings are discussed.

108 CHAPTER 2.

MATERIALS AND METHODS.

The experiments described in this chapter were designed to determine the metabolic fate of arachidonic acid in human fetal membranes. Methods were developed for the extraction and separation of arachidonic acid metabolites using C18 Sep-Paks and high performance liquid chromatography. Cell cultures of human placentae, chorion laeve and amnion, obtained at various stages of pregnancy, were established in order to follow the metabolism of °H-•5 arachidonic acid. Experiments were performed to determine the uptake of added H-arachidonic acid into the cell cultures. Oxygenated metabolites of both exogenously added and endogenously incorporated JH-arachidonic acid were determined following extraction and separation.

1) Extraction of Arachidonic Acid Metabolites from Tissue Culture Medium using C18 Sep-Paks. a ) Materials.

C 18 Sep-Paks Waters, Northwich, UK. Laboratory distilled water. Petroleum spirit, Pronalys grade, May & Baker, Dagenham, UK.

Methyl formate, GPR, BDH, Poole, UK. Formic acid, GPR, _

Laboratory grade ethanol. Acetonitrile, HPLC grade, Rathburn Chemicals, Walkerburn, Scotland.

109 Triethylamine, Sequanal grade, Pierce & Warriner, Cheshire, UK. 0.45 |Jm SR filters, Millipore, Harrow, UK.

Glass syringe, 5ml. Sigmacote, Sigma Chemical Co., Poole, UK. Oxygen-free nitrogen, British Oxygen Corporation, London, UK.

Scintillation vials, Packard United Technologies, Caversham, UK.

Scintillation fluid, Unisolve 1, Koch Light, Haverhill, UK. Intertechnique SL 3000 liquid scintillation counter, Kontron, St. Albans, UK.

Radioactively labelled arachidonic acid metabolites:

6-keto- [5,8,9,11,12-, 14,15-3H(N)] -prostaglandin F-j^ , 120 Ci mMol-1, New England Nuclear, (Du Pont UK.), Stevenage, UK. [5,6,8,9,11,12,14,15-3H(N)]-thromboxane B2, 155 Ci mMol-1, New England Nuclear, (Du Pont UK.), Stevenage, UK.

[5,6,8,11,12,14,153H(N)]-prostaglandin E 2f 160 Ci mMol-1, Amersham International, Amersham, UK.

13,14-dihydro-15-keto-[5,6,8,11,12,14-3H(N)]- prostaglandin E2, 80-100 Ci mMol-1, Amersham International, Amersham, UK.

[5,6,8,11,12,14,15-3H(N)] -prostaglandin F20<, > 100 Ci mMol-1, Amersham International, Amersham, UK.

[5,6,8,9,11,12,14,15-3H(N)]-leukotriene B4, > 100 Ci mMol-1, Amersham International, Amersham, UK.

[5,6,8,9,11,12,14,15-3H(N)]-arachidonic acid, 135 Ci mMol 1, Amersham International, Amersham, UK. 1-stearoy1-2-[1-1^C]-arachidonyl-L-3- phosphatidylcholine, > 50 mCi mMol-1, Amersham International, Amersham, UK.

110 ^ C - 5 ,8,9,11,12- and 15-hydroxyeicosatetraenoic acids (HETEs) were gifts from Dr. P.W. Woolard, Institute of Dermatology, University of London. b ) Methods used for the extraction of arachidonic acid metabolites from tissue culture medium. i) Determination of extraction efficiencies.

"Waters" C 18 Sep-Paks were activated with acetonitrile (10 mis) and washed with distilled water (10 mis), petroleum ether (10 mis), methyl formate (10 mis), 80% ethanol (10 mis) and finally distilled water (10 mis).

Triplicate 20 ml samples of tissue culture medium 199

(TC-M 199) supplemented with 10% horse serum and containing either radioactively labelled 6-keto-PGF^ ,

Txl^, PGE2^ p g f 2

Sep-Paks with a glass syringe. The Sep-Pak was washed with 15% ethanol (10 mis), petroleum ether (10 mis), methyl formate (10 mis) and finally 80% ethanol (10 mis). Each solvent was collected into a scintillation vial, to which scintillation fluid (Koch Light Unisolve 1, 10 mis) was added. Recoveries were determined by comparing the radioactivity in each solvent with the total radioactivity added to the medium, counted in the same volume of each solvent since significant and different quenching occurred in each solvent.

Ill ii) Determination of the effects of horse serum and

ethanol on the efficiency of extraction.

Since recovery of arachidonic acid was low using the method described above, a comparison was made between extraction from TC-M199 both containing and not containing horse serum (10%) - in order to determine the effect of serum and between loading the sample onto Sep-Paks with increasing concentrations of ethanol (0-30% v/v) which precipitates protein but also changes the polarity of the solvent. iii) Extraction of culture supernatants.

Sep-Paks and tissue culture medium were prepared as before. After loading the sample,Sep-Paks were washed with distilled water (10 mis) Sc petroleum ether (10 mis), and the arachidonic acid metabolites eluted with methyl formate (5 mis). The Sep-Pak was washed with 80% ethanol prior to re-use.

The methyl formate fraction was reduced under oxygen- free nitrogen and the residue dissolved in HPLC solvent 1

(acetonitrile : triethylamine formate (TEAF) [4xlO”^M],

30:70 v/v, 1 ml) and filtered through Millipore Millex SR filters ( 0.45 Jjl m ) pr ior to separation by reverse-phase HPLC.

The method of extraction of arachidonic acid metabolites from culture supernatants is outlined in figure 17.

112 Figure 17. Extraction of arachidonic acid metabolites from culture supernatants.

i) Preparation of C18 Sep-Pak.

C18 Sep-Pak i Activate with iacetonitrile Wash with waterr petroleum spirit, methyl formate and 80% ethanol i Remove organic phase with water

ii) Extraction of arachidonic acid metabolites

Culture supernatant containing ^H-arachidonic acid metabolites

i Acidify with formic acid (8.6M, 5/4 1 ml-1)

Load onto prepared4 Sep-Pak I Wash Sep-Pak with distilled water (10 mis) i Remove water with petroleum spirit (10 mis)

Elute arachidonic4 acid metabolites with methyl formate (5 mis) I Wash Sep-Pak with 80% ethanol (10 mis) and reuse

113 2) Separation of Arachidonic Acid Metabolites using

High Performance Liquid Chromatography, a ) Materials. SP 8700 Solvent delivery system, Spectra Physics, St. Albans, UK. Fatty Acid Analysis column, Waters, Northwich, UK.

Silica 50-5 Nucleosil column, HPLC Technology, Macclesfield, UK.

Gilford 252 spectrophotometer, Gilford Instruments, Middlesex, UK. Glass syringe 1ml, Hamilton, Bonaduz, Switzerland. 0.45pm SR filters, Millipore, Harrow, UK.

Triethylamine, Pierce & Warriner, Cheshire, UK. Acetonitrile, HPLC grade, Hexane, Rathburn Chemicals, Isopropanol, Walkerburn, Scotland Methanol, Acetic acid, BDH, Poole, UK. Formic acid, Helium, Air Products, Bracknell, UK. Scintillation vials, Packard United Technologies, Caversham UK. Scintillation fluid, Unisolve 1, Koch Light, Haverhill, UK. Intertechnique SL 3000 liquid scintillation counter, Kontron, St. Albans, UK.

Frac 100 fraction collecter, Pharmacia GB, Central Milton Keynes, UK.

Radioactively labelled arachidonic acid metabolites were those used for the extraction method described in section (1).

114 LJnlabelled arachidonic acid metabolites were obtained from Sigma Chemical Co., Poole, UK., Prostaglandin D2

15-keto-prostaglandin E2 15-keto-prostaglandin F2p(

13,14-dihydro-15-keto-prostaglandin F2q( Prostaglandin A2 Prostaglandin B2 b ) Method used to separate arachidonic acid metabolites

by reverse-phase high-performance liquid chromatography A reverse-phase high performance liquid chromatography (HPLC) system was developed to separate prostaglandins, their further metabolites, di- and mono-

HETEs and free arachidonic acid using 3 isocratic solvent mixtures. The solvent composition and flow rate were controlled by a Spectra-Physics SP 8700 solvent programmer connected to a pump. Low pressure mixing of solvents was achieved by a ternary valve. Samples in 1ml of elution solvent 1 were injected onto the column (Waters "Fatty

Acid" column) through a rheodyne injector valve after filtration through 0.45 (Jim solvent-res is tant filters. Solvent 1 consisted of acetonitrile : TEAF (pH 3.15)

(30:70 v/v) for 25 minutes, solvent 2 consisted of

acetonitrile : TEAF (50 :50 v/v ) for 25 minutes and solvent 3 acetonitrile for 8 minutes. Flow was 1 ml per minute. Transition between solvents was over 1 minute and the separation was complete within 1 hour.

115 Eluting solvent was collected into fractions (0.5 ml. ) and radioactivity determined by liquid scintillation counting. Results are shown as radioactivity per fraction against retention time. The reverse-phase HPLC method is outlined in figure 18. The separation of arachidonic acid metabolites was calibrated using radioactively labelled and non-labelled standard metabolites. Retention times of labelled compounds were determined from peak maxima obtained following liquid scintillation counting of 0.5 ml fractions. Those of unlabelled standards were determined by continuous monitoring of ultra violet absorbance at 200 nm (for prostaglandins, except for PGB2 which was detected at 280 nm) and 234 nm (for HETEs). c ) Normal-phase separation of hydroxyeicosatetraenoic acids (HETEs). i) Extraction of HETEs with chlorobutane.

Following extraction of arachidonic acid metabolites from culture supernatants with methyl formate on C 18 Sep- Paks the residue was redissolved in alkaline phosphate buffered saline (pH 8.5,. 0.2M, 1ml) and HETEs extracted by shaking with chlorobutane (3xlml). The chlorobutane fraction was evaporated under oxygen-free nitrogen and redissolved in normal-phase HPLC solvent (hexane : isopropanol : methanol [methanol contains 5% acetic acid], 95.2 : 2.1 : 2.7 v/v).

116 Figure 18. Separation of arachidonic acid metabolites by reverse-phase high performance liquid chromatography.

Culture supernatant I . Extraction with C18 Sep-Paks

1 Resuspend in HPLC solvent 1 (1 ml, 30% acetonitrile / 70% TEAF) i Filter (0.45 |Jm SR filter) I Inject onto Fatty Acid Analysis column

1 Elute prostaglandins (30% acetonitrile)

1 Elute Di- and mono-HETEs (50% acetonitrile) i Elute arachidonic acid (100% acetonitrile)

117 ii) Separation of HETEs by normal-phase HPLC.

The HETEs were separated on a silica 50-5 column with normal-phase HPLC solvent, as described above, at a flow rate of 1 ml per minute. Eluting solvent was collected into 0.5 ml fractions using a "Frac 100" fraction collector. Radioactivity was determined by liquid scintillation counting. Separation of HETEs by normal-phase HPLC is outlined in figure 19. Separation of arachidonic acid and HETEs by hormal-phase HPLC was calibrated using radioactively labelled standards, as described above for reverse-phase HPLC .

118 Figure 19. Separation of Hydroxyeicosatetraenoic acids by normal-phase high performance liquid chromatography.

Culture supernatant

1 Extract with C18 Sep-Pak

1 Resuspend in alkaline phosphate (pH 8.5, 1 ml) I Partition with chlorobutane (3x1 ml) I Reduce chlorobutane under oxygen-free nitrogen

1 Resuspend in hexane : isopropanol : methanol (95.2 : 2.1 : 2.7 v/v, 1 ml)

1 Filter through solvent^resistant filter (0.45 JJL m)

1 Separate on Silica 50-5 column

119 d ) Analysis of reverse-phase chromatograms. High performance liquid chromatography profiles may be analysed in 2 principal ways. Firstly, the nature of the compounds giving rise to peaks on the chromatogram can be analysed qualitatively. This qualitative analysis of products is based upon the retention times of peaks eluting from the column. In the method employed the accuracy of tR determination is limited by the size of fraction collected (in this case 0.5ml.). Retention times may be determined at peak maxima, peak means or half heights on the leading side. Pauls & Rogers (1977) compared all 3 methods and found that different retention times were obtained with each method and that they were all affected by background noise. However the values

obtained from calculating differences in retention times between peaks were not statistically different using each method. Since all 3 methods give equivalent results peak maxima

were used in this study since they were more readily determined. Secondly the quantitative analysis aims to give a numerical value to the amount of compound present as indicated by the size of the peak it produces. This may

be determined by measurement of peak height or peak area.

i ) Peak height.

The determination of peak height is quicker than

areas although a plot of height against amount has a smaller linear portion than a plot of area against amount

120 (Komers & Kresci 1979). Peak height is determined by

measuring the perpendicular distance from the zero line to the peak maximum regardless of zero line drift (figure

20(i ) ) .

ii) Peak area.

There are a variety of methods for the determination

of peak areas. Peak areas are less sensitive to operating conditions than heights (Komers & Kresci 1979) although deficiencies of the pumping system may effect area

measurement (Bristow 1976). Methods commonly used for

determination of peak areas include planimetry, cutting and weighing, squaring of the curve, triangulation, graphical integration and use of mechanical or electronic integrators.

Manual measurement: direct evaluation of areas by planimetry or cutting and weighing have advantages in that

the shape of the peak does not effect the results. However both methods are lengthy and are of doubtful accuracy.

Curve squaring: curve squaring or determination of peak height and width at half height (figure 20(ii)) is rapid and easy, but is dependent upon peak shape.

Asymmetric curves or peaks with low height and long elution time are unsuitable.

121 Figure 20. Determination of peak height and area.

i) Measurement of retention peak height.

H.j and H2 are measured heights and B is the baseline.

i i) Measurement of areas as the product of the height and width at

half height.

H is the measured hight and W the width. The hatched area is

proportional to the area under the peak.

122 Triangulation: approximation with a triangle, or triangulation makes use of a triangle constructed by drawing two intersecting tangents to the inflexion points of the curve (figure 21(i)). The base of the triangle is delimited by intersection of tangents with the baseline.

This method is simple and the height is easily measured. The drawbacks are uncertainty in finding inflexion points and the unsuitability of narrow and high or asymmetrical peaks.

Graphical integration: graphical integration is based upon the division of the peak into vertical strips of equal width. The heights of the strips are then summed. This method is convenient for analysis of curves obtained following fractionation of the eluting solvent by collecting fractions at equal time intervals, and was therefore the method employed in this study.

Electronic integration: electronic integrators or computers with suitable hardware and software provide a quick and convenient method for determining areas and peak heights. The accuracy is greater than other methods described but the initial high cost of equipment may offset the accuracy.

Baseline correction; to obtain a meaningful result peaks must be integrated with respect to the baseline (Marson 1969). The rate of deviation of a baseline is dependent upon the type and size of the sample. Baseline

123 Figure 21. Determination of peak area and baseline correction.

i ) Measurement of area by triangulation. H=height, B=base, Area=0.5B x H

ii) Automatic baseline correction. a) No baseline correction. b) Integration with respect to last known baseline. c) Prediction of baseline by previous behaviour. d) Retrospective correction according to measurements before and after peak. 124 correction may be achieved in a variety of ways as shown in f igure 21(ii). In the case of incompletely separated components, peaks 0 and P in figure 22, separate peak areas may be determined by several methods including peak height or triangulation. The use of triangulation is shown in figure 22(i). Tangents are constructed through inflexion points for curves 0 and P. If the tangent to P crosses the baseline after the maximum of peak 0 and the tangent to 0 crosses the baseline prior to the maximum of curve P, peak height or triangulation may be used. If the overlap is so large that this is not the case then a perpendicular is dropped from the minimum of the curves, and areas are measured (figure 22(ii)). Comparison should then be made with standard compounds 0 and P. If one component is in great excess then the curve may be completed by inspection and the area calculated or measured (figure 22(iii).

Similar-sized peaks should be separated by drawing a perpendicular from the trough, or minimum, and an area correction factor applied to each peak, dependent on the other.

125 Figure 22. Determination of incompletely separated peaks.

126 3) Tissue Culture. a) Materials Sterile universal containers, 30 and 150 ml, Tissue culture dishes, Sterilin, 5.0cm dia., Teddington, UK. Sterile disposable pipettes, 5 and 1ml, "Linbro" tissue culture multiwell plates (12 well), Flow Labs, Irvine, Scotland.

Sterile 200 ml conical glass flasks, glass funnels, dissecting scissors, forceps and surgical gauze.

Phosphate buffered saline, Dulbecco's A Oxoid Ltd, Basingstoke, UK. Trypsin, Difco Bacto 1/250, Difco laboratories, East Mosely, UK. EDTA, Analar grade, BDH, Poole, UK. Dispase grade II, Boehringer, Mannheim, Germany. Tissue culture medium 199, Gibco, Uxbridge, UK. Horse serum (Mycoplasma free),

Protosol tissue solubiliser, New England Nuclear (Du Pont UK.), Southampton, UK.

[5,6,8,9,11,12,14,15-^H(N)-arachidonic acid 135 Ci mMol- , Amersham International, Amersham, UK. Magnetic stirrer,

Oven, 37°C, Gallenkamp, London, UK. Controlled CO2 incubator, Laminar flow cabinet, Envair UK. Ltd., Rossendale, UK. BTL Intermediate Centrifuge Baird & Tatlock, London, UK.

127 b ) Preparation of cells. Normal human placentae were obtained at delivery by elective caesarean section or following spontaneous labour. Placental tissue was also obtained following termination of 1st trimester pregnancies.

Samples of chorion laeve and amnion (10x10cm) were cut out away from term placentae, separated and placed in sterile phosphate buffered saline (PBS). The placenta was placed maternal-side up and portions of the maternal surface which showed no gross pathology were cut away exposing fetal villi. Samples of fetal villi were dissected out and placed in sterile PBS. Term and 1st trimester placental and chorion laeve samples were minced with scissors and washed with PBS until no further blood contamination could be removed. The tissue was then incubated in trypsin solution (0.5%, 100 mis, 37°C, 1 hr) with continuous, gentle stirring. Amnion was cut into strips (lcmxlOcms), washed thoroughly with PBS, incubated in EDTA solution (5xlO“^M, 10 mins, room temp.) and washed again with PBS (3x). The strips were then incubated with Dispase (0.25%, 100 mis, 45 mins, 37°C).

Placental and chorion laeve cells were collected by filtration through 2 layers of sterile gauze followed by centrifugation (150xg, 10 mins). Amnion cells were collected by transferring the strips to culture medium (TC-M199 containing 10% horse serum, 100 mis) and shaking vertically (lOOx). The strips were removed and cells

128 pelleted by centrifugation (150xg, 10 mins).

Cell pellets were resuspended in culture medium (100 mis), repelleted and resuspended in culture medium

(20 mis). An aliquot (0.3 mis) of each suspension was incubated with trypan blue (0.5%, 0.1 ml, 2 mins) and cell numbers determined by counting in a Neubauer chamber.

Cell suspensions were diluted to 2x1 0-^ per ml for experiments determining the uptake of arachidonic acid, and 5x10^ per ml for those determining the metabolism of arachidonic acid. c ) Metabolism of arachidonic acid by cultures of cells derived from human fetal membranes. i ) Uptake of arachidonic acid.

Cell suspensions• of 1 o t " trimester i and term placentae (2.5 mis) were plated out in tissue culture dishes (3.5 cm) and incubated (24 hrs, 37°C, 5% CO2 in air) in a humidified atmosphere. Culture medium was removed and adherent cells washed with PBS (3x). Culture medium containing ^H-arachidonic acid (0.2|UlCi ml”^ [ 1.48xlO“^M], 2.5mls) was added and the cells reincubated. Culture medium was removed from separate dishes after 1, 2, 4, 6 and 24 hours and cells harvested by trypsinisation (0.5%, lml, 37°C, 12 hrs). These cells were then incubated with

Protosol tissue solubiliser (2mls, 37°C, 12 hrs) and radioactivity determined by liquid scintillation counting. Unlabelled arachidonic acid (sodium salt) was

129 dissolved in ethanol (1x 10"3M) and further diluted in tissue culture medium containing 3H-arachidonic acid (1.48 x 10“^M) to concentrations of 10“3 molar to 10“^molar freshly for each experiment. The concentration of radioactively labelled arachidonic acid was adjusted to 10"-1-0 and 10”^ molar in 2 dishes per sample. The effect

of arachidonic acid concentration on uptake of 3 H- arachidonic acid was determined after 24 hours incubation.

ii) Conversion of arachidonic' acid to oxygenated metabolites.

Cell suspensions (lOmls, 5xlOJ ml x) of all 3 tissues were plated out into tissue culture dishes (5cm),

incubated in a humidified atmosphere (37°C, 5% CO2 in air, 24 hrs) and the supernatant removed. Non-adherent cells and blood elements were removed by washing with PBS (3x). Medium (lOmls) containing 3H-arachidonic acid (0.2 JJl Ciml"1)

was added to the monolayer. The cells were reincubated (24 hrs) and tissue culture medium removed and stored at

-2 0°C prior to analysis. To determine the total radioactivity in the cells, monolayers from 3 samples were washed and cells collected by trypsinisation and treated as previously described. To determine the metabolites released following conversion of this endogenously incorporated label, monolayers from 3 further samples were washed and medium (lOmls) was added. After further incubation (24 hrs) medium was collected and stored at -20°C. The protocol for the investigation of conversion

130 of arachidonic acid to oxygenated metabolites is shown in figure 23. Continuing viability of the monolayers was determined by the trypan blue exclusion test. iii) Control experiments.

To control for autooxidation of arachidonic acid culture medium (10 mis) containing -^H-arachidonic acid

(0.2 [4 Ci ml“^) was incubated in the absence of cells under identical conditions to those of cell cultures, and treated in the same manner as culture supernatants. iv) Reproducibility.

In order to determine the reproducibility of the analysis of arachidonic acid metabolism by cell cultures, 4 Petri dishes were set up as in section (ii). Medium was collected according to the protocol in figure 23 and duplicate 20 ml samples were then analysed by reverse- phase HPLC. v) Normal-phase high performance liquid chromatographic separation of hydroxyeicosatetraenoic acids.

Hydroxyeicosatetraenoic acids were extracted from culture supernatants and separated as in the protocol in figure 19. Following chlorobutane extraction the aqueous phase was acidified, re-extracted on Sep-Paks and separated on reverse-phase HPLC in order to determine non-extracted compounds. Organic phases from some experiments were also separated by reverse-phase HPLC.

131 Figure 23. Protocol used for the analysis of metabolism of arachidonic acid by cultures of cells derived from human fetal membranes.

Cell suspensions (5xl05 ml-1)

i Add 10 ml to each of 2 Petri dishes

i Incubate for 24 hours (37°C, 5% C02 in air)

Wash monolayers with sterile PBS (3 x 10 mis)

Add medium containing• ^ 3JH-arachidonic acid (10 mis, 0.2^Ci ml--*-) I Incubate for 24 hours (37°C, 5% C0o ) 1 Collect medium and store at -20°C i Wash cells with sterile PBS (3 x 10 mis) i Add fresh medium (no 3H-arachidonic acid) I Incubate for 24 hours (37°C, 5% C09) I Collect medium and store at -20°C i Test viability of monolayers by the trypan blue exclusion test

132 vi) Analysis of hydroxyeicosatetraenoic acids by gas- chromatography mass-spectrometry.

Cultures of amnion, chorion and placental cells were incubated with medium containing unlabelled arachidonic acid to the same concentration (1.48 x 10“^ M) as that of

labelled arachidonic acid used in previously described experiments. The supernatants were extracted on C18 Sep-

Paks as shown in figure 18. The compounds present in the methylformate fractions were separated by HPLC, and HETEs analysed by gas-chromatography mass-spectrometry (GC-MS) using the method of Woolard & Mallet (1984) at the

Institute of Dermatology, University of London. Since the culture medium contained lipid compounds which interfered with the analysis, and therefore gave rise to high background levels, the results are expressed as a percentage of the amount measured in culture medium only.

vii) Analysis of reverse-phase chromatograms.

Reverse-phase chromatograms of culture supernatants were analysed in 2 ways. Firstly, a qualitative method

was used to identify the peaks observed. Peaks were identified by comparing their retention times (t R),

measured at peak maxima, with those of known standards.

These results are shown in the form of chromatograms, or product profiles, with peak identification adjacent to the peak. Compounds which did not co-chromatograph with known standards were tentatively identified by comparison of

133 their chemical and chromatographic properties with literature reports of other compounds. Abbreviations used are as follows:

PGIm Compounds which eluted in the same volume as standard metabolites of PGI2.

Tx B2 Thromboxane B2.

p g f 2d< Prostaglandin F2(^ . p g e 2 Prostaglandin E2.

p g d 2 Prostaglandin D2. 15-K-PGE2 15-keto-metabolites of primary 15-K-PGF2^ prostaglandins. PGEm 13,14-dihydro-15-keto-prostaglandin PGFm metabolites.

l t b 4 Leukotriene B^. Di-HETE Dihydroxyeicosatetraenoic acid. HETE Hydroxyeicosatetraenoic acid.

A.A. Arachidonic acid.

Secondly, the major compounds identified on the product profiles were quantified using a graphical integration method to sum the radioactivity within the peak. Values were corrected for losses if a labelled standard was available. For HETEs the mean value for recovery was used, since all superantants appeared to contain a mixture of HETEs. Baseline corrections were made retrospectively and incompletely separated peaks were determined by drawing a perpendicular from the minimum between the 2 peaks. In the case of 1 compound being in excess, the curve was completed by eye to determine the lesser compound. The results are shown as bar charts,

134 with compounds which were not produced by at least 3 samples being shown without standard deviation bars. The total radioactivity from each sample was determined by summation of all the fractions. The proportion of the total radioactivity added to, or incorporated by, cell cultures which was converted to metabolites, was also calculated. Statistical comparisons were made using a 2 sample t test on log-transformed data in ‘the Royal Postgraduate Medical School Computer Centre (University of London).

135 CHAPTER 3 -

RESULTS.

1) High Performance Liquid Chromatography. a ) Extraction of arachidonic acid metabolites from tissue culture medium using C 18 Sep-Pak cartridges.

The objective of these experiments was to determine the optimum method for extraction of arachidonic acid metabolites from tissue culture medium 199 (TC-M199) and to quantitate their recoveries.

Octadecyl silica (ODS) Sep-Paks may provide a solid phase for the extraction of arachidonic acid metabolites by solid/liquid partition. They have been used for the extraction of arachidonic acid metabolites from a variety of media and their effectiveness for extraction from tissue culture medium was investigated in the present study.

The method of Powell (1980 ) was used to extract radiolabelled S-keto-PGF^ t TxB2/ 2

Sep-Paks which were then washed with 15% ethanol (to remove very polar compounds), distilled water (to remove the organic solvent), petroleum ether (to remove the aqueous phase), methyl formate (to elute arachidonic acid

136 metabolites) and 80% ethanol (to remove non polar compounds). Total radioactivity recovered in each fraction was determined by liquid scintillation counting.

The percentage recoveries are shown in table 1. The recoveries of prostaglandins in the methyl formate fraction were high (79%-94%) and that of phosphatidylcholine low (6.3%). The recovery of arachidonic acid was only 23.8% and it was thought that because most of the radioactivity eluted in the void volume, and had not therefore bound to the column, it may have bound with greater affinity to serum proteins which were carried through with the aqueous phase. This possibility was examined by comparing recoveries from serum-free and serum-supplemented medium and also by determining the effect of differing ethanol concentrations on extraction. The results of these experiments are shown in table 2. The recovery of arachidonic acid was greater from serum-free medium than serum-supplemented medium (p < 0.01), but no effect was observed on the recovery of prostaglandins. This suggested that the addition of serum reduced the recovery of arachidonic acid but not prostaglandins, perhaps because the binding of prostaglandins to serum proteins was weaker. Precipitation of protein by the addition of ethanol increased the recovery of arachidonic acid from serum(_ supplemented medium (p < 0.005), but also caused a decrease in the recovery of polar prostaglandins such as 6-keto-PGFl0( (p < 0.001).

137

choline 7.8 ± 1.2 0.6 ± 0.4 6.3 ± 1.3 Phosphatidyl­ 0.6 ± 0.0 48.9 ±4.7 4.3 ± 0.4 3.4 ± 4.9 acid 11.8 11.8 ± 1.7 1.9 ± 0.4 0.6 ± 0.0 Arachidonic 2 ± 0 .4 ± ± 2.1 ±5.3 58.8 ±0.3 ± ± 1.6 23.8 ± 1.7 ± 0.1 p g e 0.5 5.3 4.9 ± 4 .2 0.0 0.4 7.0 4.0 0.1 0.1 7.2 7 79. 0.1 0.1 ± 5.2 ± 3.0 ± 2.0 ± 0.2 ± 0.0 P G E 1 89.6 ± 1.9 ± 0.0 1.2 ± 0.1 0.1 0.1 ± 0 .0 P G F 2 * 2.5 ± 2 .4 94.1 94.1 ± 2.3 B 2 x T with C18 Sep-Paks. 0.2 ± 0.0 0.4 ± 0.4 0.4 ± 0 .3 0.4 ± 0.1 4.3 ± 2.6 K 3.8 ± 3.4 1.1 ± 0 .0 0.1 0.1 ± 0 .0 0.9 ± 0.7 88.7 ± 5.3 90.6 ± 2.9 following extraction Table 1. Recovery of arachidonic acid, prostanoids and phosphatidylcholine 8 Sep-Paks. 15% 15% Ethanol 80% 80% Ethanol 6.4 ± 5.2 Fraction 6-keto-PGF,. Petroleum Ether 0.1 ±0.0 Legend. Tissue culture medium-199 containing horse 10% serum and radioactively labelled prostaglandins, arachidonic acid Water wash culture medium (n = 3). Methylformate or 2-arachidonyI-phosphatidylcholine was extracted on C l8 Sep-Paks with the solvent shown. Results are expressed as the percentage recovery (to 1 decimal place ± 1 standard deviation) of the total radioactivity added to 20 mis of Aqueous

138 Table 2. The effect of serum and ethanol on the extraction of arachidonic acid and prostaglandins from tissue culture medium.

Extraction from tissue culture medium-199

% Recovery.

Compound 0% ethanol 10% ethanol 20% ethanol 30% ethanol

6-keto-PG F . 94 ± 1 92 ± 1 85 ± 1 58 ± 3 Ice T x B 2 92 ± 2 91 ± 3 88 ± 2 76 ± 3 72 + 2 70 ± 1 67 ± 2 60 ± 3 P G F 2« p g e 2 8 2 + 2 84 ± 4 81 ± 3 69 ± 4 A.A. 79 ± 4 78 ± 4 81 ± 4 86 ± 3

ii) Extraction from tissue culture medium-199 supplemented with horse serum (10%). % Recovery.

Compound 0% ethanol 10% ethanol 20% ethanol 30% ethanol

6-keto-PGF- 92 ± 1 91 ± 1 86 ± 2 61 ± 5 1 oc T x B 2 97 ± 2 98 ± 3 92 + 2 83 ± 3 59 ± 3 PGF~2°c 83 ± 2 81 ± 1 79 ± 2 p g e 2 86 ± 2 88 ± 4 83 ± 3 74 ± 4 A.A. 23 + 1 29 ± 3 43 ± 1 62 ± 6

Legend. Radioactively labelled arachidonic acid and prostaglandins were extracted from tissue culture medium-199 in the presence and absence of serum and with increasing concentrations of ethanol. Samples extracted in the absence of ethanol were not given an ethanol wash. Results are expressed as the percentage of total radioactivity added to 20 ml of tissue culture medium, to the nearest whole number ± 1 standard deviation. ( n = 3 ).

139 Since ethanol was observed to cause a decreased recovery of polar prostaglandins and may also cause elution of HETEs into the petroleum ether fraction (Powell 1980) it was omitted from the extraction of experimental culture supernatants. The recoveries of prostaglandins and HETEs from serum —supplemented medium using this modified method are shown in table 3.

The method used gave optimal recoveries of prostaglandins which were greater than those of HETEs. The recoveries of HETEs are similar to those reported by

Powell (1980). 5 -HETE, 12-HETE and LTB^ may form lactones which may alter their polarity, thus affecting their retentivity on reverse-phase columns. Since HETEs are less polar than prostaglandins they are not as soluble in aqueous medium and may therefore bind to proteins more avidly thus reducing their recovery.

140 Table 3. Extraction of arachidonic acid metabolites from tissue culture medium supplemented with 10% horse serum using C18 Sep-Paks.

Metabolite % Recovery in methylformate fraction 6-keto-PGF, 89 ± 1 1 T x B 2 91 ± 1 p g f 2cc 94 ± 1 p g e 2 80 ± 1 5-H ETE 32 ± 16 8 -H E T E 42 ± 5 9-H ETE .44 ± 11 11-H ETE 62 ± 5 12-H ETE 82 ± 20 15-HETE 60± 5 L T B ^ 59 ± 2 Arachidonic acid 24± 4 Phosphatidylcholine 6 ± 1

L e ge n d . Radioactively labelled arachidonic metabolites were extracted from TCM-199 containing horse serum (10%) using methylformate following washes with water and petroleum spirit on C l8 Sep-Paks. Recoveries are expressed as the percentage recovery of the total radioactivity added to 20 ml. of medium,to nearest whole figure ± 1 standard deviations=3.

141 b) Calibration of reverse-phase high performance liquid

chromatographic separation of prostaglandins and hydroxyeicosatetraenoic acids. S e p aration of a m i x t u r e of r a d iolabelled prostaglandins and HETEs on a "Fatty Acid Analysis" column was possible using 3 isocratic solvent mixtures at a flow rate of 1 ml per min, as shown in profile 1. Solvent 1 consisted of 30% acetonitrile in triethylamine formate pH 3.15 (TEAF) for 25 minutes which separated primary prostaglandins and their further metabolites. Solvent 2 consisted of 50% acetonitrile in TEAF for 25 minutes and eluted LTB^ and mono-HETEs. Two peaks were observed for

5-HETE and 12-HETE, which may be due to lactone formation. This solvent composition separated di- and mono-HETEs but did not give baseline separation of mono-HETEs. Solvent 3 consisted of 100% acetonitrile for 8 minutes and eluted free arachidonic acid.

Accurate and reproducible solvent mixtures are possible using a solvent programmer and a ternary valve, allowing mixing of solvents before entering the column. Retention times were determined from peak maxima,and peak widths were determined to aid quantitation.

Unlabelled prostaglandins were determined by ultra­ violet absorbance at 200 nm. Profile 2 shows the detection of 6-keto-PGE-^ as an example. Prostaglandin B2 was determined at 280 nm (profile 3). Unlabelled HETEs were detected at 234 nm (profile 4).

142 Profile 1. Separation of a mixture of radioactively labelled arachidonic acid metabolites by reverse-phase high performance liquid chromatography.

in co X c l

20

10i n 0 u

o \o

Legend. Radioactively labelled arachidonic acid metabolites were eluted from a "Fatty acid analysis" column using 3 isocratic solvent mixtures, at a flow riate of 1 ml min . Solvent 1 consisted of acetonitrile : TEAF (30:70), solvent 2 consisted of acetonitrile : TEAF (50:50) and solvent 3 consisted of acetonitrile only. Radioactively labelled compounds were determined in 0.5 ml fractions by liquid scintillation counting. Retention times were determined from peak maxima and peak widths between points of inflexion. The profile shown is representative of 3 separate determinations. PGEm= 1 3,1 4-dihydro-15-keto-PGE2

143 Profile 2. Ultra-violet detection of prostaglandins. Example shown 6-keto-PGE^.

0.05 a) 6-keto-PGE.j Ipg.

0. 05t

b) 6-keto-PGE^ 10 pg.

0 1 234 567 89 10 11 t R (m ins.)

Legend. Unlabelled prostaglandins were determined using ultra-violet absorbance at 200 nm. Increasing amounts of prostaglandin in 1 ml. of elution solvent were injected onto a "Fatty acid analysis column and eluted with a solvent of composition acetonitrile:TEAF 30:70 v/v at a flow rate of 1 ml. min . Absorbance of the eluting solvent was continuously monitored with a Gilford U.V.spectro­ photometer and recorded with a chart recorder. 144 Profile 3. Ultra-violet detection of PGB2

Legend. Unlabelled PGB2 was determined using ultra-violet absorbance at 280 nm. Increasing amounts of prostaglandin in 1 ml. of elution solvent were injected onto a "Fatty acid analysis " column through a rheodyne valve and eluted with a solvent consisting of acetonitrile TEAF 50:50 v/v at a flow rate of 1 ml. min . Absorbance of the eluting solvent was continuously monitored with a Gilford U.V. spectrophotometer and recorded with a chart recorder. 145 Profile 4. Ultra-violet detection of hydroxyeicosatetraenoic acids.

a) 5-HETE (200ng)

0.1

E c rocf rs

■Mto 'E D o

0

t^ (m in s.)

L e g e n d . Unlabelled HETEs were determined using ultra-violet absorbance at 234 nm. Two hundred ng of each HETE were injected in 1 ml. of elution solvent through a rheodyne valve onto a "Fatty acid analysis column" and eluted with a solvent of^composition acetonitrile TEAF 50:50 v/v, at a flow rate of 1 ml. min. Absorbance of the eluting solvent was monitored continuously with a Gilford U.V. spectrophotometer and recorded with a chart recorder. 146 Absorbance units (234 nm.) Absorbance units (234 nm.) Absorbance units (234 nm. Profile 4 (continued) U.V. Detection of HETEs ^ mi s.) in (m t^ 147 The sensitivity of ultra-violet absorbance at short wavelengths is low and requires use of non-absorbing solvents and large amounts of prostaglandins. The maximum absorbance of prostaglandins is at 192.4 nm (Terragno et al. 1982) which was below the minimum wavelength of the available equipment. This caused a further reduction in sensitivity and increased the minimum amount of prostaglandin required for detection, the minimum detectable amount at 200 nm was lOjulg (profile 2). This method was therefore unsuitable for the detection of prostaglandins produced by placental cells in culture, which have been shown to produce only about 2 ng per 10^ cells over 24 hours (Jogee et al. 1984). Prostaglandin B2 was more easily detected since it has a maximum absorbance peak at 280 nm (profile 3). Mono-hydroxyeicosatetraenoic acids were easily detected at 234 nm (profile 4), but since other compounds cannot be detected simultaneously this separation was also calibrated with radioactively labelled compounds (profile 1). The retention times and peak widths of all compounds investigated are shown in table 4. It was observed that prostaglandin metabolites were retained for a longer time than the parent compound. The method used separated prostaglandins, prostaglandin metabolites and di- and mono-HETEs but did not separate individual mono-HETEs sufficiently to identify the position of hydroxylation. It can be used to show the major classes of compounds produced but due to the complexity of metabolites produced

148 Table 4. Retention data for arachidonic acid metabolites on reverse-phase high performance liquid chromatography.

* represents unlabelled compounds.

Compound Peak width ( mins) (m ins)

8.0 7.0 _ 9.0 6-keto-PGF.1* 2,3-dinor-6-keto-PGF.' J oc. * 8.2 not determined 13.14- dihydro-6,15-di-keto-PGFl0c* not detected 2, 3-dinor-13,14-dihydro-6, 15-diketo-PGF ' 7.7 not determined 6-keto-PGE1 * 9.8 not determined

T x B 2 11.0 10.5 - 12.0 13.0 12.5- 14.5 P G F2oc 0 15-keto-PGF02oc * 18.0 not determined 13.14- dihydro-1 5-keto-PGF,^* 22.0 not determined

p g e 2 16.5 15.5 - 18.0 15-keto-PGE, 20.5 not determined 13.14- dihydro-15-keto-PGF2o<* 27.0 26.0 - 28.5

p g d 2 * 19.0 not determined PGA, * 29.0 not determined P G B , * 31.8 not determined LTB4 34.0 34.0 - 35.0 5 -H E T E [35.5 [34.5 - 36.5 [43.0 [42.0 - 44.0 8 - H ET E 42.5 41.5 - 43.0 9 - H E T E 44.0 43.0 - 44.5 11- H E T E 42.5 43.0 - 44.0 12- H E T E [35. 5 [35.0 - 36.0 [42.5 [41.5 - 43.0 15 -H E T E 42.0 41.5- 43.0 Arachidonic acid 58.0 57.5 - 58.5

Legend. Radioactively labelled and unlabelled arachidonic acid metabolites were eluted from a "Fatty acid analysis" column using 3 isocratic solvent mixtures, at a flow rate of 1 ml per minute. Solvent 1 consisted of acetonitrile : TEAF (30:70), solvent 2 consisted of acetonitrile : TEAF (50:50) and solvent 3 consisted of acetonitrile only. Radioactively labelled compounds were determined in 0.5 ml fractions by liquid scintillation counting. Unlabelled compounds were determined by continuous monitoring of absorbance of the e lu tin g solve n t at 200 nm for p ro sta g la n d in s, 280 nm for PG B and 234 nm for H E T E s. Retention times were determined from peak maxima, and peak Widths for labelled compounds only, between points of inflexion. Results are expressed as the average of 3 separate determinations. 149 from arachidonic acid it is difficult to analyse every compound. c ) Separation of hydroxyeicosatetraenoic acids by normal- phase high performance liquid chromatography.

Since baseline separation of mono-HETEs was not achieved by reverse-phase HPLC a normal-phase system was developed. The relative polarity of normal-phase HPLC means that mono-HETEs are eluted more quickly than following separation on reverse-phase. The retention times of mono-HETEs on normal-phase HPLC are given in table 5 and the separation is shown in profile 5. *r -

HETE showed 2 peaks which may again be due to delta- lactone formation. Mono-HETEs could therefore be separated within 30 minutes but a further extraction step was required to bring them into a suitable solvent. The method used was extraction from alkaline phosphate buffered saline using chlorobutane. The extraction was not quantitated as insufficient standards were available.

The HPLC separation was therefore qualitative only, although the individual HETEs may be extracted with high efficiencies (Barr et al. 1984).

150 Table 5. Retention data for mono-hydroxyeicosatetraenoic acids on normal-phase high performance liquid chromatography.

Compound Retention time (mins )

A.A. 7.5 5 -H E T E 12.5 5 -H E T E 27.5 8 -H ET E 22.0 9-H ETE 19.5 11-H ETE 18.5 12-H ETE 14.5 15 -H E T E 16.5

Legend. Radioactively labelled HETEs were injected in 1 ml of elution solvent through a rheodyne injector onto a silica 50-5 column and eluted with hexane : isopropanol : methanol (containing 5% acetjc acid) (95.2 : 2.1 : 2.7 v/v) at a flow rate of 1 ml min . Eluting solvent was collected into 0.5 ml fractions and radioactivity determined by liquid scintillation counting. Radioactivity per fraction was plotted against time and retention times determined from peak maxima. Results shown are the average of 2 determinations.

151 Profile 5. Separation of mono-hydroxyeicosatetraenoic acids by normal-phase high performance liquid chromatography.

Arachidonic acid

t^ (m in s.)

Legend ^ 3 Mixtures of 4C-HETEs and ^H-arachidonic acid were separated on a silica 50-5 column(hplc Technology) using hexane:isopropanol: methanol(con^aining 5% acetic acid) 95.2: 2.1 : 2.7 v/v at a flow rate of 1 ml. min. Radioactivity was determined in 0.5 ml. fractions and was plotted against time. The profile shown is representative of duplicate experiments. The numbers at the top of each peak repesent the position of hydroxylation for each of the HETE isomers.

152 2) Growth of cells in culture. a ) Placental cells. Placental cells formed a monolayer composed of epithelial, fibroblast and syncytial-like cells. The formation of large multinucleated syncytial-like masses was observed at 48 and 72 hours ( plates 1 and 2).

Suspensions of 1st trimester cells were stained for hCG by an immunofluorescent method ‘in the Department of Histochemistry at the Royal Postgraduate Medical School,

London. Approximately 30% of the cells showed positive staining (plate 3). b ) Chorion laeve cells. Chorion laeve cells showed a similar pattern of growth to placental cells. Following 24 hours in culture a mixture of cell types was seen which included epithelial and fibroblast-like cells (plate 4). The formation of syncytial-like structures was also observed (plates 5 and

6 ) . c ) Amnion cells.

Amnion cells formed a confluent monolayer within 24 hours with a typical "pavement" appearance characteristic of epithelial cells (plate 7). No other cell types were observed and no gross morphological changes occurred over the 3 days of culture. Amnion has previously been

reported to have a high metabolic rate in vitro (Schwartz et al. 1977). Tissue culture medium 199 is a rich growth

153 medium and when supplemented with serum should be adequate to maintain amnion cells in monolayer culture.

At the end of the culture period cells were tested for viability by the trypan blue exclusion testy and cultures which were less than 90% viable were discarded.

154 Plate 1. Monolayer of term placental cells, showing syncytial-like structure, (48 hours, haematoxylin s eosin xllO)

Plate 2, Monolayer of term placental cells, showing syncytial-like structure. (72 hours, haematoxylin & eosin xllO)

155 Plate 5. Imrounofluorescent staining of 1 st trimester cells with antibody against hCG. Positive cells fluoresce light green. xllO

Plate 4, Monolayer of term chorion Iaeve ceils showing fibroblast and epithelial—like cells. (24 hours, haemqtoxylin 8 eosin xllO)

156 Plate 5. lv!onolayer of term chorion laeve cells showing syncytial structure and single epithelial cells (48 hours, haematoxylin & eosin x llO ) ,

Plate 6. Monolayer of term chorion laeve cells showing syncytial structure (72 hours haematoxylin & eosin xllO ).

157 Plate 7. Monolayer of term amnion cells (48 hours, unstained xllO)

158 3) Metabolism of Arachidonic Acid by Cell Cultures. a ) Uptake of labelled arachidonic acid. Placental cells from 1st trimester and term placentae

were found to incorporate ^H-arachidonic acid in a time- dependent fashion (table 6). In ls^ trimester placental

cells there was an initial increase over 4 hours, no further incorporation from 4-6 hours and then a further

increase up to 24 hours of culture. Term placental cells showed increased incorporation over the initial 2 hours of culture, no further increase from 2-6 hours and then a

further increase up to 24 hours. This pattern of uptake was similar to that in other cell types (Pong et al. 1977)

and could represent incorporation into different classes of intracellular lipids.

In order to determine whether the level of free arachidonic acid in culture medium could affect the uptake of -^H-arachidonic acid, placental cell cultures were incubated with increasing concentrations of added arachidonic acid. The concentration of added ^H-

arachidonic acid was increased from 10”^^ to 10“^ molar and unlabelled arachidonic acid was added to give

concentrations of 10* " ® to 10 “ ^ molar. The increase in concentration was found to cause an

increase in uptake of arachidonic acid (chart 1). The

addition of unlabelled arachidonic acid over the range lO” -*-^- to 10“6 molar caused a linear increase in the uptake

of arachidonic acid as shown by the isotope dilution

experiment. There was no evidence of saturation at 10***^ molar. 159 3 Table 6. Time-course of uptake of H-arachidonic acid by placental cells in monolayer culture.

st ) 1 trimester placental cells.

Time (hours) CPM /well n % Total CPM. o +i 1 3451 + 1407 5 o 0.29

2 5316 + 2280 5 1.20± 0.46

3 6325 + 2218 5 1.40± 0.45

+ cr> l+ 4 7341 1588 5 o 0.32 ID +l 6 7120 + 1502 4 o 0.30 in + l 24 20620 + 7341 *4 O 1.49

ii) Term placental cells.

Time (hours) CPM /w ell n % Total CPM

+ o CO l+ 1 3874 327 3 o 0.07

+ OJ l+ 2 6414 464 3 o 0.09 3 6781 + 305 3 1 .50± 0.06 4 7273 + 1505 3 0. 90± 0.31 6 4747 + 1281 3 0. 90± 0.26

+ + 4= 0 24 2173Q 2711 3 GO 1 0.55

Legend. 6 Placental cells (0.5 x^O ) were plated out and grown for 24 hours and then incubated with JH-arachidonic acid (0.5/^Ci). The cells were harvested by trypsinization at various times, as shown, solubilised with an equal volume of NCS tissue solubiliser, at room temperature overnight. Radioactivity was determined by liquid scintillation counting. Total radioactivity wa^ determined in an equal volume of aqueous phase. Incorporation of H-arachidonic acid at each time point was assessed in duplicate for each sample.and results are expressed as the mean counts per minute ± 1 standard deviation.

160 log arachidonic acid incorporated (moles) uptake on concentration acid arachidonic of effect The 1. art h C n ulble aahdnc cd p o 10 to up acid arachidonic unlabelled and n slblzd ih ise slblzr t om eprtr over g t igh rn e v o temperature room trypsinization at by solubilizer harvested tissue were with cells ours, solubilized h 24 and r e rth fu a r fte A Legend. h mnlyr fre wr icbtd ih aahdnc cd at acid -arachidonic H with incubated were formed monolayers The te el ws acltd Thi vle a cnetd o amount to converted was value is h T calculated. was cells the y b rs. u o h 24 for n grow and out plated were ) 10 x .5 (0 cells Placental h pooto o add rcioi ai wih a be tkn up taken been had which acid arachidonic added of proportion the aiatvt ws eemie b lqi sitlain onig and counting scintillation liquid by ined determ was Radioactivity h eprmn ws eetd n ulct wt 3 ifrn smples sam different 3 with duplicate in repeated was experiment The 10 hc ae ersne b te y os - O d n a A , 0 bols sym the by represented are which mls ad lte agai t ocnrto o add rcioi acid. arachidonic added of concentration st in a g a plotted and (moles) ", ", 11—10-9 1 -1 0 1 r 10 * 0 oa or ih aahdnc cd t 0 molar 10 at acid -arachidonic H with r o molar 10 ** f rcioi ai b clue o paetl cells placental of cultures by acid arachidonic of band fe sotnos labour. spontaneous after obtained 11 o aahdnc cd ocnrto add (molar) added concentration acid arachidonic Log " 10 i ------

1 . 9 - „«7 *„-6 „ «-7 „„-8 9 - . -10 _ g 3 g 0 1 1 ------" 161 0 1 1 ------" >-8

0 ad 0 ® molar. 10 and 7 10 3 0 1 1— ' ------10 1

arachidonic acid was compensated for by an increase in uptake. Concentrations of arachidonic acid greater than 10“4 molar were found to be cytotoxic as judged by the failure of the monolayers to exclude trypan blue. Cell monolayers of all tissues were found to have

incorporated radioactivity after 24 hours incubation

(chart 2). First-trimester placental cells incorporated significantly more -^H-arachidonic acid during a 24-hour incubation period than all other cell types except chorion

laeve cells obtained after spontaneous labour (p < 0.001, chart 2). This could reflect the nutritional requirement of the early placenta which grows rapidly and may require arachidonic acid for the synthesis of cell membranes as well as providing a substrate for prostaglandin synthesis. Placental and amnion cells obtained following caesarean section incorporated less ^H-arachidonic acid than cells obtained following spontaneous labour (p < 0.05, chart 2). The mean amount of ^H-arachidonic acid incorporated by chorion laeve cells obtained after

spontaneous labour was more than that incorporated by cells obtained following elective caesarean section, but

the difference was not statistically significant possibly because of the large standard deviation for the former cells. This may represent a change in activity of the

metabolic pathways of arachidonic acid metabolism in fetal membranes at parturition. j The small sample number and large variation preclude any comment on the significance of these observations. 162 Chart 2. Radioactivity incorporated by cell cultures after 24 hours 3 incubation with H-arachidonic acid.

ED 1st trimester ua Spontaneous labour □ Elective caesarean section.

+ ■kit

Placenta Chorion Amnion

Legend. ^ Placental, chorion laeve and amnion cells (5 xlO°) obtained at various stages of pregnancy, were plated out and grown for 2^ hours. The monolayers formed were incubated with medium containing H-arachidonic acid ( 2pCi) for a further 24 hours. Cells were then harvested by trypsinization and solubilized with tissue solubilizer at room temperature overnight. Radio­ activity was determined by liquid scintillation counting and is shown as mean radioactivity ± 1 standard deviation, per dish, (n = 3) Statistical comparisons were by an unpaired t-test. st * 1 trimester placenta significantly greater than other cell types except chorion laeve obtained after spontaneous labour p<0.001

** Cells obtained after spontaneous labour significantly greater than cells obtained after caesarean section p<0.05

+ Chorion and amnion cells obtained after spontaneous labour significantly greater than the corresponding placental cells p<0.05.

++ Chorion and amnion cells obtained after elective caesarean section significantly greater than the corresponding placental cells p<0.001. 163 Placental cells obtained after both caesarean section and spontaneous labour incorporated less arachidonic acid than either their equivalent amnion or chorion laeve cells (p < 0.05 for samples obtained after spontaneous labour, p < 0.001 for samples obtained after elective caesarean section). This could reflect the different

roles of the definitive placenta and other membranes with respect to transport of fatty acids and prostaglandin synthesis.

164 b ) Analysis of Oxygenative Metabolism. i) Reproducibility.

In order to determine the reproducibility of chromatographic traces, duplicate samples (20 mis) of supernatants from placental cell cultures were extracted, separated and counted separately. The profiles shown in profile 6 are duplicate profiles for 1 sample. The same major peaks were observed on the basis of retention times

(table 4) and the peaks were superimposable.

Experiments with other samples demonstrated that for individual samples the profiles were similar and therefore the only differences were those between samples.

ii) Control experiments.

Four duplicate 10ml samples of tissue culture medium,

supplemented with 10% horse serum, containing3H-arachidonic acid (0.2 JJ Ci ml“^), were incubated at 37°C for 24 hours

in a humidified atmosphere of 5% CC>2 in air. The chromatograms obtained following extraction and reverse-

phase HPLC are shown in profile 7. A major peak which eluted in the void volume (tR at peak maximum = 5.0 min) was observed. This peak may have been due to polar products of autooxidation such as short chain, C18, C16

and C14 fatty acids. Some compounds were also observed

which eluted in the same fractions as prostaglandins,& di- and mono-HETEs. The recovery of free arachidonic acid was variable and since this is affected by protein binding

165 could be accounted for by variations in binding capacity between batches of horse serum.

166 Profile 6. Duplicate reverse-phase chromatograms of metabolites of 3 H-arachidonic acid produced by cultures of placental cells obtained after spontaneous labour.

o »“* X

Legend. In order to determine the reproducibility witfjin experiments, placental cells obtained after spontaneous labour (5x10°) were plated out in tissue culture medium (10 mis) and grown for 24 hours. The monolayers were washed andgincubated for a further 24 hours with medium (10 mis) containing n-arachidonic acid (2 pCi). The medium was collected and arachidonic acid metabolites extracted on C l8 Sep-Paks using methyl- formate. Separation of metabolites was by reverse-phase HPLC. The solvent was collected in 0.5 ml fractions and radioactivity was determined in each fraction by liquid scintillation counting. The two profiles shown above are duplicates from one sample, showing the reproducibility within individual experiments. 167 Profile 7. Reverse-phase chromatograms of compounds produced during 3 incubation of H-arachidonic acid in the absence of cells.

Sample 1.

Sample 2.

Sample 3.

Sample 4.

Legend. 2 In order to determine the extent of autooxidation, H-arachidonic acid (2 pCi) was incubated in tissue culture medium (10 mis) for 24 hours at 37°C in an humidified atmosphere of 5% C 0 2 in air. The medium was acidified and the products of autooxidation were then extracted by solid/ liquid partition with methylformate, using C18 Sep-Paks as the solid phase. Polar products of autooxidation were separated using reverse- phase HPLC. Three isocratic elution solvents were used which separate prostaglandins (solvent 1, acetonitrile : TEAF 30 : 70), mono- and di- hydroxyfatty acids (solvent 2, acetonitrile : TEAF 50 : 50) and free arachidonic acid (acetonitrile 100%). Eluting solvent was collected into 0.5 ml fractions and radioactivity determined in each fraction by liquid scintillation counting and plotted against time. 168 iii) Qualitative and quantitative analysis of metabolism of exogenously added and endogenously incorporated arachidonic acid by cell cultures.

After incubation of cultures with exogenous arachidonic acid the products formed were identified by comparing retention times of major peaks on reverse-phase chromatograms with those of control experiments and known

standards.

Following removal of medium containing exogenously added arachidonic acid and washing of cultures, the products of metabolism of endogenous substrate released

into the medium were also analysed by reverse-phase HPLC.

First-trimester placental cells: the profile of compounds produced after incubation with exogenous substrate are shown in profile 8. The radioactivity eluting in the void volume was a consistent feature of profiles of incubations with

exogenous arachidonic acid and as it also appeared in the control experiments was probably a result of autooxidation

of arachidonic acid. Metabolites of PGI2 were poorly separated from this peak and since there appeared to be relatively less radioactivity in this fraction they were observed as shoulders on the major peak. The profile of prostaglandin products varied between samples but peaks corresponding to TxE^f PGD2 and PGF and PGE metabolites were observed.

169 Profile 8. Reverse-phase chromatograms of metabolites produced from exogenous substrate by cultures of placental cells obtained st during the 1 trimester.

Legend. st Monolayer cultures of placental cells obtained during the 1 trimester, which had been cyown for 24 hours, were incubated in tissue culture medium (10 mis) containing H-arachidonic acid (2 pCi) for a further 24 hours at 37°C, in a humidified 5% CO. in air atmosphere. Supernatant was collected, acidified and oxygenated products of L metabolism were extracted by solid/liquid partition using methylformate and C18 Sep- Paks. The extracted compounds were then separated by reverse-phase HPLC using 3 isocratic solvent mixtures on a "Fatty acid analysis" column. Solvent 1 consisted of acetonitrile : TEAF (30:70), solvent 2 consisted of acetonitrile : TEAF ( 50:50) and solvent 3 acetonitrile (100%). Eluting solvent was collected into 0.5 ml fractions and radioactivity determined by liquid scintillation counting and plotted against elution time. The resulting profiles of 4 samples are shown. Metabolites were identified by comparison of retention times of peaks observed on the chromatograms with those of authentic standards or literature values of relative retention times on reverse-phase HPLC. 170 rfl 8 continued. 8, Profile

Radioactivity (cpm x10 20 16 171 ape 4. Sample ape 3. Sample First —trimester placental cells also produced compounds which co-chromatographed with LTB4 and mono- HETEs, as well as a compound which did not co­ chromatograph with any of the available standards. A tentative identification of this compound was made on the basis of its relative polarity and stability in acid conditions. It is possible that the peak was due to an acid-stable epoxide similar to that synthesised by liver microsomes (Chacos et al. 1982). Both trophoblast and amnion contain cytochrome P450 enzymes which could form epoxides although the presence of a placental epoxide hydrase could lead to formation of vicinal diols which would have increased polarity on reverse-phase HPLC. Peaks which co-chromatagraphed with LTB4 and mono-

HETEs, including isomers of 5-HETE and 12-HETE, were also identified. Other peaks which eluted between LTB4 and mono-HETEs could have been due to products of enzymatic hydrolysis of epoxides, further hydroxylations, epoxy­ hydroxy compounds or non-enzymatic conversions. since standards of di-HETE isomers were not available it was not determined whether they were separated adequately enough to distinguish them from each other. Compounds which eluted in the same fraction as LTB4 are therefore termed di-HETEs.

Chart 3 shows that 1st trimester cells produced significantly greater amounts of di- and mono-HETEs compared to controls. The major prostaglandin appeared to

172 CO Radioactivity (cpm x10 100 0 - 60 0 . 80 0 - 40 oxidation hat . srbuto f aiatvt bten eaoie produced metabolites between radioactivity of tion u istrib D 3. art Ch

ulue o paetl el otie durng h 1lrmester 15ltrim the g rin u d obtained cells placental of ltures cu : d n e g e L opud wih lt bten 0 n 4 mins. 45 and 40 between elute which compounds xei ns Res t ae hwn s en ons e minute per counts mean as n show are lts by su e R d roduce p acid ents. experim arachidonic added f o metabolites major The ttsia cmprsn bten oto ad utr spenaa ts atan ern sup culture and control between parisons com Statistical e 2 ihs 1 tndad dvain oe bas ersn culture represent ars b open deviation, ard d stan 1 ± dishes 2 per ee dniid n h bass rtnin ek osre i control in observed peaks retention f o sis a b the on identified were hc wr sg fcnty geaer n utr spraat are pounds Com supernatants culture in data. r ed ate gre log-transform tly on ifican st sign t-te were two-sample which a y b were uentns n6 ad oi br rpeet otos (n=4). controls represent bars solid and (n=6) supernatants niae b p aus bv te bar. the above values p by indicated EEa: opud wih lt bten 5 n 4 mn. HETElb): mins. 40 and 35 between elute which compounds HETEjal: o- ^ E x T - to u A p<0. 05 p<0. band ng te ti ester. trim 1 the g in r u d obtained rm xgnu s tat y utrs f lcna cells placental of cultures by te stra b su exogenous from

PE - HEE Epoxide ETE H i- D PGEM 9 D G P 1 TE a HE A. . .A A E ET H (a) E ET H p<0. 005 r p<0. st 173 p<0.05 b) (b

be PGD2 although it was not produced by all samples. The production of T x B 2 / autooxidation products and PGI metabolites was not significantly greater than that in controls. Profiles of products released following the metabolism of endogenous substrate are shown in profile 9.

Only 1 sample of 1st trimester placenta cells produced cyclooxygenase products, PGI2 metabolites, TXB2 anc^ PGE2* A peak which may have been due to autooxidation products was also observed. Compounds which co-chromatographed with di- and mono-HETEs and the putative epoxide were observed in all samples. The mean radioactivity incorporated into each metabolite is shown in chart 4^

174 Profile 9. Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of placental cells obtained st during the 1 trimester.

Sample 1.

HETE Epoxide D i- H E T E , ^ Sample 2. PGEm A.A.

m o

E ua >S ■M Sample 3. > ’•M u roo *■5 to OZ

Sample 4,

Legend. In order to determine the products of metabolism of endogenously incorporated H-aracfn^lonic acid, monolayer cultures of placental cells ^btained during the i trimester were incubated with medium containing H-arachidonic acid for 24 hcyrs. The medium was then replaced with fresh medium not containing H-arachidonic acid and incubated for a further 24 hours. Metabolites released into the medium during the second incubation were analysed by reverse-phase HPLC. Culture conditions, extraction and separation methods were as outlined in profile 8. 175 Chart 4. Distribution of radioactivity between metabolites rmetr ee dniid n h bss f eeto pas observed b l peaks the retention in of basis obtained H-arachkjJonic the cells on placental identified of incorporated ltures were cu ester by trim endogenously of released acid metabolites major The e 2 ihs 1 t d dvain ( = 6) = n ( deviation. rd a d n sta 1 ± dishes 2 per n rfl 9 Rdociiy e mtblt ws nertd n the and integrated was metabolite per Radioactivity 9. profile in eed 3 metabolite per minute per counts mean the as shown are results Legend. Radioactivity (cpm x 10 ) 16 r 18 14 10 12 - Eoie .A A Epoxide s E T E H E T E i-H D o f l b L t r i m e s t e r p l a c e n t a l c e l l s . produced from endogenous substrate by cultures 176

Placental cells obtained following spontaneous labour: placental cells obtained following spontaneous labour produced a similar profile of products to 1st trimester cells (profile 9), when incubated with medium containing ^H-arachidonic acid .(profile 10). Thromboxane

B2, PGE2/ PGD2/ PGE metabolites, di-HETEs, mono-HETEs and the putative epoxide were identified. The range of prostaglandins varied between samples and the HETE peak appeared to consist of more than 1 component.

In chart 5 it is shown that only the production of mono-HETEs and the postulated epoxide were significantly above controls. The production of prostaglandins was not significantly different from the controls. Profiles of metabolites of endogenous substrate are shown in profile 11. One sample produced a compound which eluted with a PGE2 metabolite. Small peaks eluting with mono-HETEs and the putative epoxide were observed in all 3 samples. The total radioactivity incorporated into each of these metabolites is shown in chart 6.

177 Profile 10. Reverse-phase chromatograms of metabolites produced from exogenous substrate by cultures of placental cells obtained after spontaneous labour.

Sample 1

Sample 2,

Sample 3.

Legend. In order to determine the products of metabolism of added H-arachidonic acid in cultures of placental cells obtained after spontaneous labour, the procedure followed was that described in profile 8.

178 s no Radioactivity (cpm xlO Chart5. Distribution of radioactivitybetween metabolites produced f lcna el otie atr pnaeu lbu wr ietfe on identified were labour spontaneous after obtained cells placenta of h mjr eaoie o add H-rcioi ai poue b cultures by produced acid -arachidonic ^H added of metabolites major The ons e mnt pr ds ± sa ad eito. pn ars b Open deviation. dard stan 1 ± s e dish 2 per minute per counts Legend. Legend. eaoie a itgae ad a cmae t te oa radioactivity total the to compared was and integrated was metabolite ttsia cmprsn bten oto ad utr spraat wr by were supernatants culture and control between parisons com Statistical h bai o rtnin ek os v i poie 0 Rdociiy per Radioactivity 10. profile in d rve obse peaks retention of asis b the e pa osre i cnrl xei ns Reut ae hw a mean as shown are esults R ents. experim control in observed peak per bv te bar. the above tosml tts o lgtasome dt. o ons hc were which pounds Com data. ed log-transform on t-test two-sample a ersn clue uper tnt ( - ) n sld s ersn controls represent rs a b solid and 3) - (n ts atan rn e p su culture represent b) cmpud wih lt bten 0 n 4 minutes. 45 and 40 between elute which pounds com : ) (b E T E H iniia l getr n utr spr tnt ae niae b p values p by indicated are ts atan supern culture in greater tly ifican sign (n (n 100 0 . 60 0 - 80 0 - 40 =4). oxidation

o TxB x T to- u A S. .S N a) cmpud wih lt bten 5 n 4 minutes. 40 and 35 between elute which pounds :com ) (a E T E H obtained after spontaneous labour. from exogenous substrate by cultures of placenta cells

PGD9 Di HE HE Epoxide E ET H E ET H i- D 7 A G P 9 D G P - E G P 2 179 TE () b) A.A. ) (b (a) E ET H p<0.05

Profile 11. Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of placental cells obtained after spontaneous labour.

Sample 1.

CO

Sample 2.

Sample 3.

Legend. In order to determine the products of metabolism of endogenously incorporated ^H-arachidonic acid in cultures of placental cells obtained after spontaneous labour, the procedure was that described in profile 9.

180 Chart 6. Distribution of radioactivity between metabolites produced from endogenous substrate by cultures of placental cells obtained after spontaneous labour.

10 r.

8 -

ro o X as u

4-<>S > •Mu oro ’■5OJ O'

D i-H E T E HETEs Epoxide A.A

L e ge n d . 2 The major metabolites of endogenously incorporated JH-arachidonic acid released by cultures of placental cells obtained after spontaneous labour were identified on the basis of retention peaks observed in profile 11. Radioactivity per metabolite was integrated and the results are shown as mean counts per minutes per metabolite per 2 dishes ± 1 standard deviation. ( n = 3 ).

181 Placental cells obtained following elective caesarean section: cells obtained following elective caesarean section showed low activity of all pathways when incubated with medium containing ^H- arachidonic acid (profile 12).

Two samples produced compounds which eluted with PGI2

metabolites and 1 sample produced metabolites of PGF2

significantly above the control experiments (chart 7). Placental cells obtained following elective caesarean section showed little metabolism of endogenous label

(profile 13). One sample produced compounds eluting with

PGF2o^ and PGE2J and a peak corresponding to HETEs was observed in another sample. There was not a consistent pattern of metabolites produced. The mean value for radioactivity per metabolite was not, therefore, calculated. The total radioactivity for the metabolites

produced by each sample was low in contrast to those produced by 1st trimester placental cells which produced a consistent pattern of metabolites.

182 Profile 12. Reverse-phase chromatograms of metabolites produced from exogenous substrate by cultures of placental cells obtained after elective caesarean section.

Sample 1

A.A.

CO o r— X E a u Sample 2. ■M ’> COo o ’•5(0 O'

Sample 3.

L e ge n d . 2 In order to determine the products of metabolism of added H-arachidonic acid in cultures of placental cells obtained after elective caesarean section, the procedure followed was that described in profile 8.

183 Radioactivity (cpm x10 Chart 7. Distribution of radioactivity between metabolites produced n h bass f eeto pek osre i poie 2 Rdociiy per Radioactivity 12. identified profile in were section observed caesarean eaks p elective r retention fte a of sis a b obtained the cells on placental of ek bevd n oto eprmet. ut ae hw a ma counts mean as shown are sults e R ents. experim control in observed peak utr spraat ( = ) n sld r rpeet otos n 4). = (n controls represent ars b solid and 3) = (n supernatants culture h mjr eaoie o add H-rcioi ai poue by c t es re ltu cu y b produced acid -arachidonic °H added of metabolites major The e mnt pr dihes 1 t d dvain Oe bar represent rs a b Open per deviation. rd a d n radioactivity sta 1 total ± the s e to ish d 2 compared per was minute and per integrated was metabolite eed ^ Legend. xdto HETE H oxidation o PI TXB X T PGIm to- u A o b t a i n e d a f t e r e l e c t i v e c a e s a r e a ns e c t i o n . from exogenous substrate by cultures of placental cells 2 Gm Gm - TE pxd A. . .A A Epoxide E ET H i- D PGEm PGFm 184

Profile 13. Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of placental cells obtained after elective caesarean section.

4- A.A,

d i- /mono- - Sample 1 2 H E T E s ro PGEm o 0 X E Q. U 4 1 >S D i- A.A, > 2 - Sample 2, u oro 0 s pf:H^ TE i '"O ro O'

Sample 3.

Legend. In order to determine the products of metabolism of endogenously incorporated H-arachidonic acid in cultures of placental cells obtained after elective caesarean section, the procedure was that described in profile 9.

185 Chorion laeve cells obtained following spontaneous l a b o u r : chorion laeve cells obtained following spontaneous labour produced similar products to their equivalent placental cells when incubated with medium

containing ^H-arachidonic acid (profile 14). Peaks

corresponding to Txl^, PGE2, PGD2f PGE metabolites, di- HETEs, mono-HETEs and the putative epoxide were observed.

The range of prostaglandins was again variable.

Although the mean values for di- and mono-HETEs were

above those in control experiments only the HETEs which

eluted between LTB^ and HETE standards were significantly

greater. Prostaglandin synthesis was not significantly

greater than that in controls (chart 8).

Chorion laeve cells obtained following spontaneous labour also showed very little metabolism of endogenous substrate, profile 15. One sample produced metabolites eluting with di- and mono-HETEs and the putative epoxide, whereas the other samples were less active. Four peaks were consistently observed in each sample and were identified as di-HETE(s), HETE, putative epoxide and

arachidonic acid. The mean values of the total radioactivity per peak for these metabolites are shown in chart 9.

186 Profile 14. Reverse-phase chromatograms of metabolites produced from exogenous substrate by cultures of chorion laeve cells obtained after spontaneous labour.

o

t R (m in s.)

Legend. 2 In order to determine the products of metabolism of added ^H-arachidonic acid in cultures of chorion laeve cells obtained after spontaneous labour, the procedure followed was that described in profile 8.

187 Radioactivity (cpm x!0 Chart 8. Distribution of radioactivity between metabolites produced n h bai o rtnin ek osre i poie 4 Radioactivity 14. identified profile were in labour observed peaks spontaneous r afte retention of obtained asis b cells the laeve on chorion of ons e mnt pr dihes 1 tnad eito. pn rs a b Open deviation. standard 1 ± s e ish d 2 per minute per counts e pa osre i cnrl xei ns Rsls r son s mean as shown are Results ents. experim control in cultures by observed peak produced per acid ^H-arachidonic added of metabolites major The b) cmpud wih lt bten 0 n 4 minutes. 45 and 40 between elute which pounds com : ) (b E T E H e mtblt ws nertd n ws oprd o h ttl radioactivity total the to compared was and integrated was metabolite per ersn clue uper tnt ( 3 ad oi br rpeet controls represent bars solid and 3) = (n ts atan rn e p su culture represent Legend. Legend. ttscl o aios ewe cnrl n cluespraat ee by were supernatants culture and control between parisons com Statistcal bv te bar. the above n 4. a) cmpud wih lt bten 5 n 4 minutes. 40 and 35 between elute which pounds com : ) (a E T E H 4). = (n tosml tts o lgtasome dt. o ons hc were which pounds Com data. ed log-transform on t-test two-sample a iniia l getr n ulue uenaa s r idctd values p y b indicated are ts atan supern lture cu in greater tly ifican sign xdto m TE a) b) (b ) (a E ET H m oxidation A uto- uto- A obtained after spontaneous labour. fromexogenous substrate by cultures of chorion laeve cells TxB0 P C D - PG F P G A - D i- H ETE H ET E Epoxide Epoxide E ET H ETE H i- D - A G P F PG - D C P 2 188

A.A.

Profile 15. Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of chorion laeve cells obtained after spontaneous labour.

>N Sample 2. > U O03 ’•o 03 CC 4-,

2- D i-H E T E Sample 3. PGEm ^ E^xid^V. A.

o-n ~ T “i------1------1------1----- 1 0 10 20 30 40 50 60 t R (m ins)

Legend. In order to determine the products of metabolism of endogenously incorporated JH-arachidonic acid in cultures of chorion laeve cells obtained after spontaneous labour, the procedure was that described in profile 9.

189 Chart 9. Distribution of radioactivity between metabolites produced from endogenous substrate by cultures of chorion laeve cells obtained after spontaneous labour

16 r

14 -

12 CO o X 10 . S ua 8 -

‘> +J u 6 * ora ,J5co CL 4 -

2 -

0

Di-HETE HETEs Epoxide A.A.

Legend. 3 The major metabolites of endogenously incorporated ^H-arachidonic acid released by cultures of chorion laeve cells obtained after spontaneous labour were identified on the basis of retention peaks observed in profile 15. Radioactivity per metabolite was integrated and the results are shown as mean counts per minute per metabolite per 2 dishes ± 1 standard deviation. ( n = 3 )

190 Chorion laeve cells obtained following elective caesarean section: chorion laeve cells obtained following elective caesarean section had little metabolic activity when incubated with medium containing -^H-arachidonic acid (profile 16). Two samples produced PGE2 metabolites and di- and mono-HETEs. One sample had no activity. None of the metabolite peaks were significantly greater than those in control experiments (chart 10). Metabolism of endogenous substrate by chorion laeve cells obtained following elective caesarean section was relatively low (profile 17). Only 1 sample produced metabolites which eluted with di- and mono-HETEs and the putative epoxide. The production of metabolites from endogenous substrate by one sample did not allow a statistical analysis of the quantitative incorporation of radioactivity into oxygenated metabolites.

191 Profile 16. Reverse-phase chromatograms of metabolites produced from exogenous substrate by cultures of chorion laeve cells obtained after elective caesarean section.

Sample 2.

4

2 _ Sample 3. | A. A. ___ _ 1 0 i | | | i 0 10 20 30 HQ 50 60 t R (m ins)

Legend. ^ In order to determine the products of metabolism of added H-arachidonic acid in cultures of chorion laeve cells obtained after elective caesarean section, the procedure was that described in profile 8.

192 Chart 10. Distribution of radioactivity between metabolites produced from exogenous substrate by cultures of chorion laeve cells obtained after elective caesarean section.

m

Legend. 3 The major metabolites of added H-arachidonic acid produced by cultures of chorion laeve cells obtained after elective caesarean section were identified on the basis of retention peaks observed in profile 16. Radioactivity per metabolite was integrated and was compared to the total radioactivity per peak observed in control experiments. Results are shown as mean counts per minute per 2 dishes ± 1 standard deviation. Open bars represent culture supernatants (n = 3) and solid bars represent controls (n = 4).

193 Profile 17. Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of chorion laeve cells obtained after elective caesarean section.

HETE Epoxide 2 P i- H ET E ^ J ^

0.

o X

0 10 20 30 40 50 60 t^ (m in s.)

Legend. In order to determine the products of metabolism of endogenously incorporated H-arachidonic acid in cultures of chorion laeve cells obtained after elective caesarean section, the procedure was that described in profile 9.

194 Amnion cells obtained following spontaneous labour: the major prostaglandin produced by amnion cells obtained following spontaneous labour was PGE2 (profile 18), although some T x B 2 and PGE metabolites were also observed. Peaks corresponding to di- and mono-HETEs and the putative epoxide were also seen. Amnion cells produced significantly more PGE2, di-

HETEs, mono-HETEs and epoxide than controls. The synthesis of TxB2 and autooxidation and PGI metabolites was not significantly greater than in controls (chart 11). Amnion cells obtained following spontaneous labour produced PGE2, di- and mono-HETEs and the putative epoxide from endogenous label (profile 19). The mean value of radioactivity incorporated into these metabolites is shown in chart 12.

195 Profile 18. Reverse-phase chromatograms of compounds produced during incubation of amnion cells obtained following 3 spontaneous labour with medium containing H- arachidonic acid.

A u to-oxid .

(m in s.)

Legend. 2 In order to determine the products of metabolism of added H-arachidonic acid in cultures of amnion cells obtained after spontaneous labour, the procedure was that described in profile 8. 196 Radioactivity (cpm xlO Chart 11. Distribution of radioactivity between metabolites produced Legend. Legend. f min el otie atr pnaeu lbu wr ietfe o the on identified were labour spontaneous after obtained cells amnion of i o rtnin ek os v i poie 8 Rdociiy per Radioactivity 18. profile in d rve obse peaks retention of sis a b utr spenaa s n 3 ad oi bas ersn cnrl ( 4). = (n controls represent ars b solid and 3) = (n ts atan ern sup culture ek bevd n oto eprmet. eut ae hw a ma counts mean as shown per are Results radioactivity total ents. the to experim compared per control was in minute and per observed integrated peak was metabolite h mjr eaoie of de H-rc dni ai poue by cultures y b produced acid ic idon -arach H added f o metabolites major The a) cmpud whc eue ewe 3 ad 0 minutes. 40 and 35 between elute hich w pounds com : ) (a E T E H b) cmpud wih lt bten 0 n 4 minutes. 45 and 40 between elute which pounds com : ) (b E T E H bv te bar. the above y tosml tts o lgtasome dt. o ud wih were which punds Com were data. ed supernatants culture log-transform and on control t-test between two-sample a parisons com by Statistical iniia l rae i clue u r tnt ae niae by values p y b indicated are ts atan ern sup culture in greater tly ifican sign obtained after spontaneous labour. from exogenous substrate by cultures of amnion cells 2 s ± s e ish d 1 t d dvain Oe bar represent rs a b Open deviation. rd a d n sta 2 197

Profile 19. Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of amnion cells obtained after spontaneous labour.

Sample 1.

Sample 2.

Sample 3.

Legend. In order to determine the products of metabolism of endogenously incorporated H-arachidonic acid in cultures of amnion cells obtained after spontaneous labour, the procedure was that described in profile 8.

198 cd eesd yc t es f min el otie atr spontaneous idonic after -arach H obtained cells amnion of incorporated s re ltu by*cu endogenously f o released acid metabolites major The Radioactivity (cpm x 10 ) rfl 1. doatvt pr eaoie a itgae ad the and integrated was metabolite per activity adio R 19. profile per per Chart 12. Distribution of radioactivity between metabolites ee . ^ d. Legen eut ae hw a a te en ons e mnt pr metabolite per minute per counts mean the as as shown are results aor ee dniid n h bss rtnin ek osre in observed peaks retention f o basis the on identified were labour 12 16 20 0 L 2 ihs ± dishes PGE o f a m n i o n c e l l s o b t a i n e d a f t e r s p o n t a n e o u s l a b o u r . produced from endogenous substrate by cultures 2 1 tndad dvain (n = n ( deviation. ard d stan - Eoie A, .A A Epoxide s E T E H E T E i-H D 199 3 )

Amnion cells obtained following elective caesarean section: amnion cells obtained following elective caesarean section converted exogenous arachidonic acid to the same products as amnion cells obtained following

spontaneous labour (profile 20). Prostaglandin E2 was the major prostaglandin and other peaks were identified as

PGI 2 metabolites, Tx B2 r di- and mono-HETEs and the

putative epoxide. Synthesis of HETEs was significantly greater than in control experiments (chart 13). Amnion cells obtained following elective caesarean section did not produce any prostaglandins from endogenous substrate but produced di- and mono-HETEs and the putative epoxide (profile 21). The mean values of radioactivity incorporated into these metabolites is shown in chart 14.

200 Profile 20. Reverse-phase chromatograms of metabolites produced from exogenous substrate by cultures of amnion cells obtained after elective caesarean section.

Sample 1.

Sample 2

t R (mins)

Legend. 2 In order to determine the products of metabolism of added ^H-arachidonic acid in cultures of amnion cells obtained after elective caesarean section, the procedure was that described in profile 8.

201 ro Chart 13. Distribution of radioactivity between metabolites produced n h bass f eeto pek os v i poie 0 Radioactivity 20. per profile minute in d per rve obse counts eaks p retention of sis a b the on fano cls band fer lcie asra scin ee identified were section caesarean elective r afte obtained cells amnion of e pa osre i cnrl xei ns Res t ae hw a mean as shown are lts su e R radioactivity total the ents. to experim compared control was in and observed integrated peak was per metabolite per h mjr eaoie o add aahioni ai poue b c t es re ltu cu by produced acid ic n ido -arach H added of metabolites major The Legend. Legend. n 4. a) cmpud wih lt bten 5 n 4 minutes. m 40 minutes. 45 and 35 controls and 40 between represent elute between ars b elute which solid which pounds and com : 3) = compounds : ) (n (a E T ) E ts H (b E atan T E rn e H p su 4). = (n culture represent ttsia cmprsn bten oto ad utr spr nt wr by culture were in ts an greater supern were which culture and pounds Com control between t-test.* parisons two-sample com a Statistical u naa s r idctd y vle aoe h bar. the above values p by indicated are ts atan rn e sup Radioactivity (cpm x10 60 20 40 xdto PGE G P oxidation A u to - - to u A , J S. .S N o b t a i n e da f t e r e l e c t i v e c a e s a r e a ns e c t i o n . from exogenous substrate by cultures of amnion cells S. .S N T x B 9 E G P 2 s ± sa ad eito. pn rs a b Open deviation. dard stan 1 ± s e ish d 9 5- Di HETE HEE pxd A.A A Epoxide ETE H E T E H i- D - -k 15 202 3 2 TE () (b) (a) E ET H S. .S N .05 0 < p

i

Profile 21. Reverse-phase chromatograms of metabolites produced from endogenous substrate by cultures of amnion cells obtained after elective caesarean section.

Sample 1.

o 4-»>> ’> o O03 (0 * 6

Sample 2.

L ege n d . In order to determine the products of metabolism of endogenously incorporated H-arachidonic acid in cultures of amnion cells obtained after elective caesarean section, the procedure was that described in profile 9.

203 Chart 14. Distribution of radioactivity between metabolites produced from endogenous substrate by cultures of amnion cells obtained after elective caearean se ctio n .

24 r

20

o 16 X S a 12 u x > u(0 O t(05 C*

Di-HETE HETEs Epoxide A.A.

Legend. . ^ The major metabolites of endogenously incorporated JH-arachidonic acid released by cultures of amnion cells obtained after elective caesarean section were identified on the basis of retention peaks observed in profile 21. Radioactivity per metabolite was integrated and the results are shown as mean counts per minute per metabolite per 2 dishes ± 1 standard deviation, (n = 3 )

204 In order to determine whether there was a switch of arachidonic acid metabolism from lipoxygenase to cyclooxygenase pathways associated with parturition the ratios of PGE2 to the other major metabolites were determined. Amnion cells obtained following spontaneous

labour produced significantly more PGE2 from exogenously added arachidonic acid than cells obtained following elective caesarean section (p < 0.05). The production of

HETEs and the putative epoxide from exogenously added arachidonic acid did not change significantly with the onset of parturition. The production of di-HETEs appeared

to increase with labour since in samples obtained following labour it was significantly greater than controls (chart 11), whereas in samples obtained following

elective caesarean section, it was not (chart 13). Since no PGE2 was detected from endogenous substrate by cells obtained following elective caesarean section, the amount

of radioactivity incorporated into PGE2 by cells obtained following spontaneous labour was significantly greater (p < 0.005). Th$ release of HETEs and the putative epoxide did not significantly change following spontaneous labour, although the mean amount of the putative epoxide appeared to decrease with spontaneous labour. This may have been due to the small sample number (n = 3). The ratio of PGE2 : HETEs produced from exogenous substrate appeared to increase following spontaneous labour (spontaneous labour 0.49+0.22; elective caesarean section 0.25+0.22) as did the ratio of PGE2 + di-HETE : HETEs (spontaneous labour 0.98+0.33; elective caesarean

205 section 0.87+0.20). However, these changes were not statistically significant. The ratios of PGE2 to the putative epoxide were very variable and although the mean values increased from elective ceasarean section

(0.29+0.22) to spontaneous labour (2.10+2.10) the increase was not statistically significant, possibly due to the large variations. Therefore although the synthesis of PGE2 was associated with labour, there did not seem to be a diversion of radioactivity from lipoxygenase to cyclooxygenase products. Use of non-parametric statistics was precluded because of the small sample number. iv) Identification of mono-hydroxyeicosatetraenoic acids by normal-phase high performance liquid chromatography.

The reverse-phase HPLC separation did not give baseline separation of mono-HETEs and therefore it was not possible to determine their identities with this method. Further identification was attempted using 2 other methods

- normal-phase HPLC and GC-MS.

Extraction .of mono-HETEs: following Sep-Pak extraction of arachidonic acid metabolites from tissue culture medium, mono-HETEs were extracted using liquid/liquid partition between phosphate buffered saline

(pH 8.4) and chlorobutane. By comparing reverse-phase HPLC profiles of placental culture supernatants before and after phosphate/ chloro­ butane partition (profile 2 2 ) it was found that

chlorobutane extracted compounds which eluted after LTB4.

206 Profile 22. Reverse-phase chromatograms of metabolites of 3 exogenously added H-arachidomc acid produced by cultures of placental cells obtained after spontaneous labour, before and after partitioning of HETEs with chloro-butane.

i) Before extraction

ii) Chloro-butane

fraction. .

iii) Phosphate fraction.

t R (m in s.) Legend. Cultures of placental cells obtained after spontaneous labqur were incubated with tissue culture medium (10 mis) containing ^H-arachidonic acid (2 pCi). Culture supernatants from 8 plates were pooled and acid­ ified . Oxygenated metabolites of arachidonic acid were extracted using solid/liquid partition with methylformate and C l 8 Sep-Paks. The methyl- formate extract was divided into 2 aliquots and the solvent evaporated under nitrogen. The residue from 1 aliquot was redissolved in reverse- phase HPLC solvent 1 (acetonitrile : TEAF, 30:70) and metabolites separated by reverse-phase as previously described in profile 8. The other residue was redissolved in 1 ml alkaline phosphate buffered saline (pH 8.4) and extracted with chloro-butane (3x1 ml). The phosphate fraction was re-acidified with formic acid, re-extracted on C18 Sep-Paks and arachidonic acid metabolites separated by reverse-phase HPLC. The chloro-butane fraction was dried down under nitrogen, the residue redissolved in reverse-phase HPLC solvent 1 and arachidonic acid metabolites separated by reverse-phase HPLC. 207 This indicated that these compounds were mono-HETEs on the basis of their polarity, although compounds which are less polar than mono-HETEs, such as arachidonic acid epoxides, would also have been extracted. Similar results were obtained with amnion supernatants (profile 23). The HETE fraction which eluted between 35.0 and 38.5 minutes was recovered with 60.9% efficiency and the HETE fraction which eluted between 40.0 and 44.0 minutes was recovered with 50.9% efficiency. The putative epoxide was recovered with only 14.6% efficiency. The low recovery of epoxide could be due to its relative polarity which may make it less soluble than the HETEs in aqueous solution. Chlorobutane was therefore useful for selectively extracting compounds which were less polar than LTB^. The efficiency of recovery for HETEs was quite high but less—polar metabolites of arachidonic acid may not be recovered as efficiently.

208 Profile 23. Reverse-phase chromatograms of fractions before and after extraction of mono-HETEs from culture supernatants of amnion cells obtained after spontaneous labour.

Before extraction.

Phosphate fraction.

Legend. Amnion cells obtained after spontaneous labour were cultured for 24 hours in tissue culture medium (10 mis) containing H-arachidonic acid (2 pCi) Supernatants from 8 plates were pooled and acidified. Oxygenated metabolites of arachidonic acid were extracted using solid/liquid partition with methylformate and C l 8 Sep-Paks. The methylformate fraction was treated as described in profile 2 2.

209 Normal-phase HPLC separation of HETEs: the chlorobutane-extracted compounds were separated on normal-

phase HPLC. Representative profiles of arachidonic acid control, term placenta, chorion laeve and amnion are shown

' in profiles 24 and 25.

Arachidonic acid control: the major compound eluted between 11- and 9-HETE and a smaller peak eluted with 5-

HETE (profile 2 4(i)). Autooxidation of arachidonic acid results in formation of 5- and 15-HPETEs but the stereochemistry of reduced autooxidation products may be

different to that of enzymatic products. Therefore their retention times may be altered. Arachidonic acid autooxidation may be catalysed by haemoglobin, the products of which may be different from spontaneous

autooxidation products.

Placental cells: term placental cells appeared to produce mainly 15- and 5-HETE from exogenous arachidonic

acid (profile 24(iii)). This is in agreement with the findings of Kinoshita and Green (1980). Minor peaks

rcorresponding to 9- and 12-HETE were also observed.

Chorion laeve: chorion laeve cells produced 5- and 12-HETE* peaks corresponding to other HETEs were not observed in the sample shown, and did not appear to be

major metabolites (profile 24(iv)). Following separation of the products of metabolism of endogenous arachidonic acid there was too little activity

210 rfl 2. oma-hs sprto o mono-hydroxyeicosatetraenoic m of separation al-phase Norm 24. Profile

Radioactivity (cpm xIO acids. smers. isom E T E H h nmbr r er o h psto o hy ox aton of h varous u rio a v the f o n tio la xy ro yd h of position the to r fe re bers num The is ulur eim 1 mi. cnann ^ aahdnc acid -arachidonic ^H containing is.) m (10 medium re ltu cu e tissu n 5- slc clmn i hi slet t fo rt of ml. 1 f o rate aflow at solvent is th g sin u n colum silica ^50-5 a on t es cls i rmpaet, mno, n chorion and nion, am placenta, from d e riv e d cells f o s re ltu u C i . utng s vent a cletd no . m. rcin and fractions ml. 0.5 into collected was t n e lv so g tin lu E . min a r sov n m. hsht bfee sln (H 8.4). (pH saline buffered phosphate 1ml. in d issolve d re was n oxg t tblts aahdnc cd ee extracted were acid arachidonic f o etabolites m d ate n xyge o and Legend. n potd ns euin ie HETEs ee dniid n the on identified were s E T E H time. elution st in a g a d plotted issolve d re and was ue sid re The . n ge itro n r e d n u evaporated then e tyfr t wa eaoae udr tog n te esdue sid re the and n ge itro n under evaporated as w ate ethylform m he T y oi lq d parii t mehlomt a Sep- s. k a -P p e S 8 l C d an ethylformate m ith w n rtitio a p id liqu solid by av otie afer p a lbu wr icbtd ith w incubated were labour s u o e tan n spo r fte a obtained laeve i o rtnin i s peas bevd n o aio with w parison com in s. rd observed a d n aks e ta p s f o ntic es e th tim au f o separated retention were those anol p of rp s E T :isop E sis e a H b xan e h and f o 2.7) g : sistin n 2.1 o c : t n e lv (95.2 so C L P H ethanol se a h al-p rm o isopropanol:m n ml. 1 in ooutn ( x ml) a ue t etat ad was and s E T E H extract to used was l.) 1m x (3 tane lorobu h C adi ciiy a dtr nd y iud cnilto cuntng tin n cou scintillation liquid by ined determ was activity io d ra 2. .0 (2 ) oto . i Amnion. ii) . Control i) jj ) o 2 hr. h s naa s r cletd acidified collected, ere w ts atan rn e p su The rs. h 24 for i) C 211

in culture supernatants from placenta and chorion laeve cells to detect peaks.

Amnion cells: amnion cells produced mainly 5- and 15- HETE and smaller amounts of 12-, 8- and 11-/9-HETES.

Eleven-HETE appeared to result from autooxidation in all samples (profile 24(i i )). Amnion cells appeared to release mainly 12- and 15- HETE from endogenous arachidonic acid although some 5-HETE was also observed (profile 25(iii)).

Although the resolving power of this method is high, . . *3 products of autooxidation of exogenous JH-arachidonic acid confused the analysis and the low level of HETEs released from endogenous label made their further identification possible only in amnion cell culture supernatants. v) Identification of mono-hydroxyeicosatetraenoic acids by gas-chromatography mass-spectrometry.

Gas-chromatography mass-spectrometry analysis was performed on Sep-Pak extracts of supernatants from cultures incubated with unlabelled arachidonic acid at the same concentration as labelled arachidonic acid in other experiments by Dr. P.Woollard at the Institute of Dermatology, University of London. The method of Woollard and Mallet (1984) was employed.

Gas-chromatography mass-spectrometry is the most sensitive method for the determination of arachidonic acid

212 Profile 25. Normal-phase separation of mono-hydroxyeicosatetraenoic acids following extraction with chloro-butane after incubation of 3 amnion cells with medium containing H-arachidonic acid.

12 11 8

3 H-arachidonic acid only.

Amnion exogenous substrate

Amnion endogenous substrate.

Legend. In order to determine whether the HETEs produced from exogenous and endogenous substrate were different, cultures of amnion cells obtained after spontaneous labour wer^incubated with medium containing JH-arachidonic acid for 24hrs. The medium was removed, the cultures washed and incubated for a further 24 h r| . with fresh medium not containing °H-arachidonic acid. Supernatants from both incubations were treated as described in profile 24. The numbers refer to the position of tR (m in s.) hydroxylation of the various HETE isomers.

213 metabolites^ however the high level of interference from lipid compounds originating from the horse serum permitted

analysis of 12-HETE only. The results obtained from mass-spectrometry are shown

in chart 15. The level of 12-HETE measured by mass- spectrometry is represented as the percentage peak area of

that obtained for culture medium blank, since there was a detectable level in the blank. The results showed that only amnion cells produced an increased level of 12-HETE

above that of the medium blank. Although chorion laeve cells appeared to produce 12-HETE from labelled substrate the amount of unlabelled substrate converted may not have been high enough to detect an increase above the level in the medium blank. It also appeared that placenta and chorion laeve cells may have incorporated 12-HETE from the medium in the absence of added exogenous arachidonic acid, since the percentage peak area was less than that of the

medium blank. Because of the high levels in culture medium alone determination of HETEs by GC-MS was not

continued. In the case of 12-HETE the medium blank contained 0.53yng per ml. The analysis was performed on culture supernatant from only one sample of each tissue.

214 Chart 15. Determination of 12-HETE by gas chromatography-

mass spectrometry.

23 Endogenously incorporated arachidonic acid

□ Exogenously added arachidonic acid.

Legend. Cultures of placenta, chorion laeve and amnion cells obtained after spontaneous labour were incubated with tissue culture medium (10 mis.) containing unlabelled aracidonic acid to the same concentration as n-arachidonic acid used in previously described experiments. After. 24 hrs. the supernatants were collected and the cells were incubated for a further 24 hrs. in tissue culture medium containing no added arachidonic acid. The supernatants from both incubations were extracted on C 1 8 Sep-Paks with methylformate. The methylformate was evaporated under nitrogen and the residue redissolved in phosphate buffered saline ( pH 8. 4) HETEs were extracted with chlorobutane and determined by GC- MS following normal-phase HPLC by Dr. Woolard (Institute of Dermatology, University of London.) using the method of Woolard and Mallet (1 984).

215 vi) Quantitative analysis of total radioactivity.

The total radioactivity recovered following reverse- phase HPLC was quantified by summation of radioactivity per fraction for the whole chromatogram. The proportion of this value which was in the peaks identified as major metabolites was also determined (to control for variation in baseline) and the proportion of total added radioactivity converted to metabolites was calculated for each tissue for exogenous and endogenous substrate (charts

16 and 17). After incubation with exogenously added arachidonic acid significantly less radioactivity was recovered from cultures of placental cells obtained following elective caesarean section than from those obtained in the ls^ trimester (p < 0.005) and also less from chorion laeve cells obtained after elective caesarean section compared with spontaneous labour(p < 0.05). There were no significant differences between placental cells obtained after spontaneous labour compared with 1 st trimester or elective caesarean section, or between amnion cells obtained before and after labour (chart 16 (i)). Although some of the differences were not significant the mean values for radioactivity recovered from cells obtained after elective caesarean section were always lower than those for cells obtained following labour. This could be caused by a greater uptake from the medium and/or subsequent conversion to oxygenated metabolites Cultures

of cells obtained following elective caesarean section incorporated less ^H-arachidonic acid than those obtained

2.1 6 > 4

Chart 16. Quantitation of Radioactivity recovered from Reverse-phase HPLC analysis of Exogenously 3 added H-Arachidonic Acid.

i ) Total radioactivity recovered ii) Proportion of total radioactivity iii) Proportion of total radioactivity in metabolite fraction. added to culture converted to C31S Trimester. 100 □ Caesarean section. metabolites. E2 Spontaneous labour. 90 * 80 15 p 70 / / 60 /. to 50 7 ■ k-k-k-k H 10 - vJ / / / ~ 40 / u o / / ro / no / o o 30 . ’■5 / ra 5 / ‘*ra 5 / /• / / 20 / (0 / o I o 10 / H h- o\o o\o / / / 0 1 Placenta Chorion Amnion Placenta Chorion Amnion Placenta Chorion Amnion Legend.

Cultures o f cells derived from placenta, chorion laeve and amnion, obtained after delivery by spontaneous labour or elective caesarean section or during the 1i>ltrimester (placenta only), were incubated with culture medium containing 3H-arachidonic acid for 24 hours as described in methods. Supernatants were collected and treated as in profile 8. The total radioactivity recovered for each tissue type was calculated (i) as was the proportion of this total radioactivity which was accounted for by the major metabolites (ii). The proportion of the total radioactivity added to the medium which was converted to metabolites ct was also calculated (iii). * 1 t r im e s t e r > term placentae p < 0.005. ** Caesarean section < spontaneous labour p < 0.005. *** Caesarean section < spontaneous labour p < 0.01. **** Caesarean section < spontaneous labour p <0.05. following spontaneous labour. The lower recovery of radioactivity from elective caesarean section samples must have been due to a decreased conversion to oxygenated . o metabolites, since recovery of JH-arachidonic acid was ; less than that of oxygenated metabolites (table 3 ). The proportion of total radioactivity which was

accounted for by major metabolites previously identified was not different between the cells obtained at different

stages of pregnancy (chart 16 (ii)). This indicates that the decrease in recovery of radioactivity was not due to

relative changes in baseline on the chromatograms. Chart 16 (iii) shows that cells obtained following elective caesarean section converted less of the added arachidonic acid to oxygenated metabolites than the cells obtained at other stages of pregnancy (p < 0.01 for

placental cells, p < 0.05 for chorion laeve cells and p < 0.05 for amnion cells). Analysis of endogenous radioactivity released into the medium (chart 17) showed that the amount recovered

from 1st trimester placental cells was greater than that from cells obtained following elective caesarean section

(p < 0.005 ) or spontaneous labour (p < 0.025). There was

no difference between the last 2 groups for placental, chorion laeve and amnion cells (chart 17(i)). The proportion of total radioactivity incorporated

into metabolites did not vary (chart 17 (ii) suggesting no

relative alteration in baseline. The proportion of total incorporated radioactivity which was re-released appeared to increase with gestational age (p < 0.05) in the placenta but did not change before or after labour in any

218 Chart 17. Quantitation of Radioactivity recovered from Reverse-Phase HPLC analysis of 3 Metabolites of Endogenous H-Arachidonic Acid.

i) Total radioactivity recovered. ii) Proportion of total radioactivity iii) Proportion of total incorporated in metabolite fraction. substrate released into medium. G3 lSt Trimester. □ Caesarean section. £2 Spontaneous Labour.

to 1—* v£>

Legend 3 After incubation with medium containing ^H-arachidonic acid, cultures of cells derived from placenta, amn^n and chorion laeve obtained after delivery by spontaneous labour gr elective caesarean section or during the i Trimester (placenta only), were incubated with medium not containing JH-arachidonic acid for a further 24 hours. Supernatants were treated as previously described (profile 9). The total radioactivity recovered for each tissue type was calculated (chart 17 (i)) as well as the proportion of this total which was accounted for by major metabolites (chart 17 (ii)). T h e proportion of total incorporated radioactivity which was released by each cell type was also calculated (chart 17{iii))» Statistical comparisons were by an unpaired t-test. * l s *trimester > term placentae p<0.025 **spontaneous labour > caesarean section p<0.05 *** term placentae > 1s*trimester p<0.05 of the tissues studied (chart 17 (iii)). The differences in the metabolites of arachidonic acid observed could reflect the relative activity of arachidonic acid metabolic pathways at different stages of gestation. Although the total amount of radioactivity recovered from the column was less for samples obtained following elective caesarean section than that for samples, obtained following spontaneous labour, the proportion of this radioactivity which had been incorporated into major metabolites did not change. This indicates that the conversion of arachidonic acid to oxygenated metabolites

by fetal membranes increases with parturition for both exogenously added and endogenously incorporated substrate.

This observation suggests that there may be an increase in the activity of enzymes in the metabolic pathways as well as an increase in the activity of enzymes which jregulate substrate availability. The observation that the proportion of incorporated substrate which was released did not increase with labour, but that there was an increase in prostaglandin synthesis

from endogenous substrate (particularly in amnion), also supports the idea that the rate—limiting steps of arachidonic acid metabolism may not be confined solely to

the release of arachidonic acid from phospholipids.

220 CHAPTER 4. DISCUSSION.

The aim of the studies reported in this thesis was to determine the metabolic fate of arachidonic acid in human fetal membranes and placenta. The production of prostaglandins is well documented but the observations reported may be due to different methodologies previously employed. In this thesis monolayer cell culture was used for studying arachidonic acid metabolism. This provides a system where the biological mechanisms which regulate arachidonic acid metabolism may be maintained in vitro.

Other factors such as blood contamination and trauma may also be minimised thus allowing the study of changes which may occur in vivo and which may otherwise be obscured by these non-specific factors.

Reverse-phase HPLC coupled with a radioactively labelled tracer was chosen as a method for separating and detecting the products of oxygenative metabolism. The major advantage of this technique is that it allows the simultaneous detection of products of the known pathways of arachidonic acid metabolism with high sensitivity. The technique also allows detection of novel metabolites.

Cell culture combined with reverse-phase HPLC was used to follow the metabolism of arachidonic acid in human fetal membranes at various stages of gestation in order to determine the major metabolic pathways and any changes which may occur during pregnancy and at parturition.

221 1 ) High Performance Liquid Chromatography,

High performance liquid chromatography requires a preparatitive step which concentrates and purifies the sample and leaves it in a solvent which is miscible with, and has the same polarity as, the eluting solvent. a ) Sample extraction. Octadecylsilica (ODS) Sep-Paks were used as a reverse-phase solid support for the extraction of arachidonic acid metabolites from tissue culture medium using solid/liquid partition. Solid/liquid partition has several advantages over other commonly used methods including 1 iquid/liquid partition - it is less time consuming and gives greater and more reproducible recoveries. The recoveries of prostaglandins, HETEs and LTB 4 were similar to those of Powell (1980). The extraction made use of the partition between an aqueous phase and ODS, following acidification to prevent ionization of the carboxyl group, and resulted in the

binding of arachidonic acid metabolites to the column. After removal of the aqueous phase from the column by

washing with petroleum ether the arachidonic acid metabolites were eluted with methyl formate, a strong volatile solvent. The method used, based on that of

Powell (1980), was rapid and gave good recoveries of prostaglandins and 12 HETE ( > 80%). The recoveries of 5- 8-, 9-, 11- and 15-HETE and LTB^ were less than those of prostaglandins (40-60%) and the recovery of arachidonic

222 acid and phosphatidylcholine (23.8% and 6.4% respectively) were low. The low recovery of arachidonic acid was thought to be due to binding to serum proteins. The method described by Powell (1980) included loading of the sample in an aqueous/ethanol mixture. This decrease in polarity makes the solid phase miscible with petroleum ether which causes elution of HETEs, reduces the recovery of polar prostaglandins such as 6 -keto-PGF-j^. (table 2) and also precipitates proteins in the medium. The addition of ethanol to tissue culture medium gave an increased recovery of arachidonic acid (table 2) possibly because of its effects on proteins but also caused a reduced recovery of 6-keto-PGF-^. It was suggested by the data of Powell (1980) that subsequent removal of the aqueous phase by petroleum ether may cause elution of HETEs also. The addition of ethanol to the sample was therefore omitted in

this study since optimal recoveries of polar compounds

such as G-keto-PGF^oC were required because of the need to understand their potential roles in pregnancy. Arachidonic acid recovery was also increased following extraction from tissue culture medium which did not contain horse serum compared to extraction from that which did. The role of serum in tissue culture techniques

is the provision of growth factors and nutrients to the cells and although culture of amnion cells in serum-free media has been described (Del'Aquila & Gaffney 1982 ) the

223 establishment of serum-free culture techniques which allows growth of cells from amnion, placenta and chorion laeve under identical conditions was not within the objectives of the present study. The lower recovery of HETEs in comparison with prostaglandins may also be due to protein binding since they are less polar than prostaglandins and would therefore be less soluble in aqueous media. The recovery of peptide- containing leukotrienes has been found to be reduced by serum and whole blood (Morris et al. 1984) and this phenomenon raises the possibility of systemic as well as local effects by these compounds since they may be bound to blood proteins. Acidification protonates the weakly acidic carboxylic acid groups thus reducing ionic interactions but would further reduce the solubility of relatively non-polar compounds, thereby reducing their recovery. The chemical structure of the HETEs may also affect their extraction since 5-HETE and 12-HETE appear to form lactones between their hydroxyl and carboxyl groups which changes th$ir polarity. Recovery of LTB^ and its isomers, which contain both 5- and 12- hydroxyl groups, may also be affected in a similar fashion, since it may also form lactones (Borgeat & Samuelsson 1979d).

Separation of HETEs- with normal-phase HPLC required a further extraction step. Following elution from Sep-Paks with methyl formate the sample was redissolved in PBS (pH 8.4) and HETEs were extracted by liquid/liquid partition with chlorobutane. This method has been reported to be

224 specific for mono-HETEs and gives a 90% recovery of 15-HETE (Barr et al. 1984). Reverse-phase HPLC before and after extraction indicated that all compounds which were

less polar than LTB4 were extracted with equal efficiency. After elution of arachidonic acid metabolites from

Sep-Paks with methyl formate the solvent may be easily evaporated by blowing nitrogen gently over the surface.

Methyl formate is quite volatile and evaporates quickly. However, the presence of a small quantity of water slows evaporation and therefore the Sep-Pak was washed with petroleum ether, which is an immiscible solvent, to remove

the aqueous phase. Prior to reverse-phase HPLC samples were redissolved in 1ml of the initial eluting solvent and filtered (0.45 jAm) prior to loading to prevent particulate matter blocking the inlet of the column.

b ) Separation of arachidonic acid metabolites. The choice of mode of separation depends upon the

nature of the compounds of interest. Prostaglandins are relatively polar compounds and are more quickly eluted on

reverse-phase than normal-phase HPLC whereas HETEs are relatively non-polar and are therefore eluted more quickly on normal-phase HPLC. Prostaglandins synthesised by fetal membranes have been implicated in playing many roles

during pregnancy and a reverse-phase HPLC system was developed to separate primary prostaglandins and their metabolites using an isocratic solvent mixture consisting of acetonitrile (30%) and TEAF(pH 3.15, 70%). Increasing

225 the organic solvent proportion eluted and separated di- HETEs and HETEs but did not give baseline separation of HETEs. Acetonitrile was then used to elute non-polar arachidonic acid. This step-wise isocratic solvent profile was controlled by a solvent programmer connected directly to a HPLC pump via a ternary valve allowing low pressure solvent mixing, which is accurate and reproducible. The high resolving power of HPLC made it possible to separate compounds differing from each other by one carbon-carbon double bond e.g. PGE^ and PGE2. The technique has been used to separate compounds labelled with different isotopes (e.g. 3H- and 14C- labelled compounds, Powell 1981) and stereoisomers of LTB^ (Masters

& McMillan 1983) by changing elution parameters such as solvent composition and flow rate. Borgeat (1984) has recently developed a method for separating upwards of 16 different lipoxygenase products but this degree of resolution increases the separation time and therefore reduces the throughput. Since lipoxygenase products had not been previously described as major products of arachidonic acid metabolism in fetal membranes, the technique was optimised to give baseline separation of primary prostaglandins and separation of di- and mono-

HETEs. . Stereo-and structural-isomers of LTB^ were not available for this study and it was therefore not possible to determine whether the method was able to resolve them into individual compounds. Compounds which co­

226 chromatographed with LTB4 were therefore identified as di-

HETEs. Following the observation that arachidonic acid may be largely metabolised via lipoxygenases to HETEs,as shown

by the reverse-phase HPLC separation^ normal-phase system was developed in an attempt to give a rapid separation of

HETEs following specific extraction with chlorobutane. A simple isocratic solvent mixture consisting of isopropanol

(2.1%), hexane (95.2%) and methanol (2.7%) was found to separate the HETEs on a silica column within 30 minutes.

This was considered preferable to extending the separation

on reverse-phase HPLC.

On both reverse-phase and normal-phase HPLC the ^ c - 5-HETE standard produced 2 peaks. Carbon-14 labelled 12-

HETE also produced 2 peaks on reverse-phase but only 1 peak on normal-phase. The production of 2 peaks may have been due to lactone formation, as previously discussed on

page 224. A similar observation might also be expected for LTB^, although its lactone-containing isomers may be relatively non-polar. Providing that a suitable method of detection is available HPLC offers a rapid method for determining arachidonic acid metabolites. The high resolving power enables very selective separation of individual compounds

from a mixture which may contain very similar isomers. In order to obtain separation of some metabolites the separation may have to be repeated with different solvents and in a different phase (Masters & McMillan 1983).

227 In the present study metabolites of radioactively labelled arachidonic acid were detected by liquid scintillation counting following fractionation of the eluting solvent. Bioassay, radioimmunoassay and ultra­ violet absorbance are other commonly used methods of detection. Bioassay may be rapid and is useful for detecting short-lived activities but was not suitable following HPLC since the solvents could interfere with the assay and detection of unstable compounds would not be possible. Radioimmunoassay is suitable for quantitation but is time-consuming and is restricted to compounds for which assays have been developed. This technique has been used in conjunction with HPLC (Alam et al. 1979) but requires the use of volatile buffers or buffers which do not affect the binding of the ligand with the antibody.

Radioimmunoassay was not employed in the present study following separation by HPLC since it was the purpose of the study to determine the major metabolites, and assays were not available for the whole range of known metabolites of arachidonic acid.

Ultra-violet absorbance may be used for the immediate detection of many compounds during HPLC. It is frequently used for the detection of leukotrienes and HETEs since they absorb maximally at wavelengths (234 nm); at which the solvents used for HPLC do not absorb strongly, and they have high coefficients of absorbance. Prostaglandins have also been detected by ultra-violet absorbance (Desiderio

228 et al. 1981, Terragno et al. 1981) but they absorb maximally at much shorter wavelengths (192 nm) and are therefore detected with less sensitivity, the detection requiring non-absorbing solvents. Hydroxyeicosatetraenoic acids were easily detected at

234 nm (profile 4) but the low sensitivity of prostaglandin detection would require the application of considerably larger amounts of prostaglandins to the column (1 yw g) than are released into culture medium by cell cultures. Detection with ultra-violet absorbance also requires monitoring at several wavelengths for the simultaneous detection of many metabolites which have different absorbance maxima. The ultra-violet detection of prostaglandins requires sensitive detection at low wavelengths. The solvents used in the present study have optimum absorbance properties compared with other solvents commonly used for HPLC. Triethylamineformate is non- ultra-violet absorbing (Desiderio et al. 1981) and acetonitrile has slightly better ultra-violet absorption properties than methanol at lower wavelengths (DMS Ultra­ violet Atlas of Organic Compounds, vol. 1, Butterworths). However the background absorbance was too high to allow detection of the low levels of prostaglandins released into culture supernatants.

Since insufficient quantities of prostaglandins for ultra-violet detection may be present in culture supernatant sf and simultaneous detection of several compounds was required, a radioactive tracer was used.

229 This increased the sensitivity, reduced interference from endogenous compounds and enabled simultaneous detection of all the products of metabolism. The choice of tracer was determined by experimental requirements. Carbon-14 labelled arachidonic acid is of lower specific activity than tritiated arachidonic acid (e.g. for compounds obtained from Amersham International, the specific activity for -^C-arachidonic acid is 50 mCi mmol-^ and that for ^H-archidonic acid is 120 Ci mmol"-*-). Although -**^C is detected more efficiently than higher concentrations of arachidonic acid may be required to detect conversion at low rates. These higher concentrations may artifactually stimulate metabolism.

The single atom may also be susceptible to loss following chain shortening by beta oxidation. Tritiated arachidonic acid was therefore thought to be preferable since conversion may still be detected after addition of low concentrations of substrate, and short chain metabolites may still be detected.

2) Cell Culture, a ) Placenta.

Placental cells derived before and after spontaneous labour and from 1st trimester pregnancy were cultured using a monolayer culture method previously described

(Jogee et al. 1983). After more than 1 day of culture cells were observed to form syncytial-like masses (plates 1 & 2). This characteristic has been previously reported

230 for purified cytotrophoblast cells (Cotte et al. 1980, Aladjem & Lueck 1981). Cotte et al. (1980) also reported that syncytial elements present in the cell suspension at the time of plating did not persist in culture after 24 hours and may be removed by washing. This was also observed in the present study. The formation of syncytial structures from adherent cells in vitro may therefore reflect the in vivo behavior of cytotrophoblast cells which are thought to give rise to the syncytiotrophoblast (Midgeley & Pierce 1962). First- trimester cell suspensions were found to stain positively for hCG, although this does not necessarily demonstrate synthesis in culture. Human placental lactogen synthesis has been

4- ( demonstrated m 1 trimester cell cultures prepared in the same way (Jogee 1983). During culture of placental cells over 8-10 days biochemical functions such as release of acid-stable phosphatase and beta-hCG may decline (Cotte et al. 1980, Morgan & Toothill 1984). This could be associated with a change in the populations of different cell types (Contractor et al. 1984). It therefore appears that in long- term cultures of placental cells a progressive change in cellular function may take place, which suggests that short term primary culture may be a better model for studying placental cell function. b) Chorion laeve.

The growth of cells derived from chorion laeve in culture has not been as extensively investigated as that

231 of placental cells. In the present study cells were dispersed from chorion laeve and grown in culture using identical conditions to those used for placental cells. Chorion laeve cells were observed to grow in a similar fashion to placental cells and the formation of syncytial- like structures was also observed (plates 5 & 6). In vivo the villi of chorion laeve degenerate to form an opaque membrane, the trophoblast layer of which consists of several layers of cytotrophoblast cells. This degeneration may be a result of nutritional deficiency, and growth of cells in a medium which corrects this deficiency could restore their capacity to develop. Chorion laeve cells were grown since the membrane may be an important site of prostaglandin synthesis, particularly during labour, as it contains a considerable amount of arachidonic acid (Foster & Das 1984). Since the chorion laeve is an avascular tissue and its structure is not as complicated as that of the definitive placenta it may provide a source of cytotrophoblast cells, with less contamination from other cell types. The chorion laeve also contains Hofbauer cells (Bourne 1960) and fibroblast cells. Although the fibrous layer appeared to be resistant to trypsinization these cells may contaminate cultures. However, only a small proportion of' fibroblast­ like cells were observed (plate 4). The observation that placental and chorion laeve cells grew in a similar fashion indicated that the chorion laeve would be a source of cytotrophoblast cells similar to those of the

232 definitive placenta. The appearance of syncytial-like elements in chorion laeve cell cultures supports the hypothesis that villous degeneration may result from a nutritional deficiency and that villous formation can be restored by supplying a nutritionally rich growth medium. Cytotrophoblast cells are of particular interest since they are the dividing cell types whose growth determines the development of the placenta. They also invade the

spiral arteries possibly increasing the vessel s' capacity to synthesise vasodilating and platelet anti-aggregating

substances to maintain bloodflow through them. Deficient growth of, or invasion by, cytotrophoblasts may be factors associated with the pathology of complicated pregnancies which are characterised by decreased utero-placental bloodflow (Shepherd & Bonnar 1976).

c ) Amnion. Cells dispersed from amnion were readily grown in culture and, when plated at a density of 5x10^ cells per

dish, formed a confluent monolayer by 24 hours of culture. The "pavement-like" morphology was similar to that reported by Okita et al. (1983) and is characteristic of epithelial cells. Okita et al. (1983) investigated some

biochemical properties of cells grown in monolayer culture and reported that they were similar to those of cells dispersed from term membranes, apart from a lower activity of lipolytic enzymes associated with arachidonic acid release. This was suggested to be due to either cofactor

233 deficiency, presence of inhibitor or absence of stimuli. Since trauma is a maior stimulus for arachidonic acid lower lipolytic activity release could also reflect the relative lack of trauma of cells grown in culture compared to cells immediately dispersed from membranes. It has been shown that prostaglandin release may also be stimulated by trauma (Piper & Vane 1969) and mechanical manipulation and that cells may release prostaglandins during adherence to culture plates (Bockman 1981). In a previous study (Jogee et al. 1983) 6-oxo-PGF-^ production by placental cells was observed to increase and reach a plateau over the ls^ 24 hours of culture? a similar observation has been made on TXB2 production by placental cells (Rose, unpublished observations). In the present study cells were therefore cultured for 24 hours to overcome the effect of trauma and adherence, before the addition of ^H-arachidonic acid, thus allowing the study of the basal metabolism of arachidonic acid.

Over the period of culture no gross morphological changes were observed in cultures, and their continuing viability ( > 90%) was demonstrated by the trypan blue exclusion test.

3) Metabolism of Arachidonic Acid. a ) Incorporation of -^H-arachidonic acid. In order to determine the possible optimum time for incorporation of ^H-arachidonic acid#uptake of

234 radioactivity from the culture medium was investigated in cultures of ls^ trimester and term placental cells. Cells obtained from 1st trimester placentae incorporated ^H- arachidonic acid from the medium in a time— dependent pattern similar to that for other cell types reported in the literature. -Pong et al. (1977) reported that transformed mouse fibroblasts incorporated -^H-arachidonic acid into phosphatidylcholine and that a steady state was reached in 5 hours. Incorporation into phosphatidylethanolamine and phosphatidylserine was slower but increased up to 20 hours. Incorporation into triglycerides resembled that into phosphatidylcholine until 5 hours when a decrease was observed. The time course of incorporation observed in this study was similar to that of Pong et a 1. (1977) and could therefore represent incorporation into different complex lipids which occurs at different rates.

In order to determine whether uptake of arachidonic acid was saturable and whether there was an optimum concentration for maximum uptake, the total amount of arachidonic acid in the culture medium was increased by adding increasing amounts of unlabelled arachidonic acid to a constant amount of labelled arachidonic acid.

Results showed that there was no saturation of uptake over the range of concentrations tested. The amount of radioactivity incorporated remained constant with the increasing total arachidonic acid concentration. It was concluded that the uptake was not saturable over the range of concentrations tested and that the level 235 of arachidonic acid in serum-supplemented tissue medium itself would not affect uptake of radioactively labelled arachidonic acid. The results also suggest that the placenta may extract free arachidonic acid from blood. It can also synthesise arachidonic acid from linoleic acid and may therefore be a source of arachidonic acid in late pregnancy (Zimmerman et al. 1979 ). The situation in culture may not accurately reflect the uptake of arachidonic acid in vivo since there is little free arachidonic acid in the blood. Ramwell et al. (1977) reported that the level of free arachidonate in plasma is approximately 3 g per ml. which amounts to only 1-2% of the total free fatty acid. This free arachidonate is, however, bound electrostatically and hydrophobically to albumin (Spector et al. 1969). Most arachidonic acid is covalently bound in phospholipids and cholesterol esters of lipoproteins (Kuksis 1978). The release from lipoproteins depends upon lipoprotein lipase activityy and the removal of free arachidonic acid from the medium may bypass rate-regulating steps in its further metabolism, if the release from lipoproteins is coupled to acylation of phospholipids in cell membranes. The placenta has lipase activity (Mallov & Alousi 1965) and it has been found that other fatty acids are transported as free fatty acids following release from triglycerides (C. Thomas, personal communication). They appear to be bound non-covalently to fatty acid binding proteins which may act as a mechanism

236 for transport of farcy acids. A possible pathway for the uptake, incorporation and transport of arachidonic acid by the placenta is shown in figure 24. The total amount of radioactivity incorporated into placenta, amnion and chorion laeve cells obtained at various stages of pregnancy were determined at the end of a 24 hour incubation with exogenous ^H-arachidonic acid. Investigation of the time course of uptake of ^ H- arachidonic acid revealed no significant difference between 1st trimester placental cells and placental cells obtained following spontaneous labour, when plated out at

0.5 x 106 cells per well. However, significant differences were observed between the various cell types obtained at different stages of gestation after plating

fZ out at 5 x 10° cells per well.

Cells from 1 s t trimester • placentae incorporated more radioactivity than other cell types, apart from chorion laeve cells obtained after spontaneous labour. During early pregnancy the placenta is a rapidly growing organ and arachidonic acid may be required for a structural function in cell membranes as well as a substrate for prostaglandin synthesis. The synthesis of vasodilating and platelet anti-aggregating eicosanoids may be vital at this stage of pregnancy when trophoblast cells are invading the spiral arteries. Arachidonic acid may also be required for the development of the fetus and the placenta may need to extract sufficient arachidonic acid to meet all these requirements.

237 Figure 2*J. Hypothetical scheme for the assimilation of arachidonic acid from maternal blood by the human placenta.

Cell membrane

Arachidonic acid is released from maternal lipoproteins by the action of lipases. It is either rapidly incorporated into membrane phospho­ lipids (P.L.) or transported , bound to fatty acid binding proteins, across the placenta, following esterification to CoA. Placental arachidonic acid may also be derived from linoleic acid by chain elongation and desaturation. Arachidonic acid incorporated into membrane phospho­ lipids may serve as a substrate for cyclooxygenase or lipoxygenase enzymes or may perform a structural role.

238 There was a greater uptake of arachidonic acid by cells derived from all tissues obtained after spontaneous labour than those obtained after elective caesarean section. The difference was significant for placental and amnion cells but not for chorion laeve cells, possibly because of the large variation in the total radioactivity recovered. The increased uptake following labour may reflect an increase in the activities of arachidonic acid metabolic pathways associated with parturition. The lower incorporation by samples obtained before labour could also reflect transportation of arachidonic acid. After uptake into the cell arachidonic acid could be released, either as free acid or covalently bound to lipoproteins rather than being incorporated into complex lipids (figure 24). Greater uptake following labour could therefore be due to the replenishment of complex lipids from which arachidonic acid has been released during parturition, probably in order to provide substrate for prostaglandin synthesis. b) Conversion to oxygenated metabolites.

The products of oxygenative metabolism of exogenously added and endogenously incorporated ^H-arachidonic acid were analysed by reverse-phase HPLC, aftera24 hour incubation. Metabolites were identified on the basis of their retention times and were compared with those produced in control experiments in which ^H-arachidonic acid was incubated in the absence of cells under identical conditions to those of cell cultures.

239 i) Autooxidation of arachidonic acid.

Polyunsaturated fatty acids may undergo autooxidation which may lead to the production of a variety of compounds including hydroxyfatty acids (Porter et al. 1980, 1981), short chain fatty acids which contain aldehyde and ketone groups (Esterbauer 1982) and compounds with polarities similar to prostaglandins (Nugteren et al. 1967). In order to determine the degree to which autooxidation may occur during the incubation of cell cultures ^H- arachidonic acid (0.2 ^Ci ml"-*-, lOmls) was incubated in tissue culture medium in the absence of cells under identical conditions to those of cell cultures. The reverse-phase chromatograms of these experiments indicated that some autooxidation may occur during the incubation period. The main products either eluted in the void volume or co-chromatographed with LTB4 or HETE standards. Some peaks which co-chromatographed with prostacyclin metabolites and TXB2 were also observed. Peaks with the same retention times which were identified following reverse-phase HPLC of culture supernatants were compared with those observed in control experiments to determine whether they were significantly different. The mean value of total radioactivity incorporated into polar compounds following autooxidation ( 3.60% + 0.87% ) was less than the mean value of total radioactivity incorporated into major peaks in supernatants of 1st trimester placental cells (13%+4%), placental cells obtained following spontaneous

240 labour (ll%+4%), chorion laeve cells obtained following spontaneous labour (11.0% + 4.4%) and amnion cells obtained following spontaneous labour (12.8%+2.8%) and caesarean section (8.8% + 1.0%). The peaks identified in culture supernatants of placental and chorion laeve cells obtained after elective caesarean section were not greater than in control experiments. Autooxidation of polyunsaturated fatty acids occurs by a free radical mechanism which jnay be catalysed by haem- containing compounds and could therefore be stimulated by proteins present in serum. The products of autooxidation may have detrimental effects on biological systems including damage to cell membranes (Halliwell & Gutteridge 1984). The reduced capacity to synthesise prostacyclin, characteristic of complicated pregnancies, may be associated with the increased level of lipid peroxide observed in the serum of affected individuals (Wickens et al. 1981 ). ii) Placental and chorion laeve cells.

Supernatants from placental cell cultures incubated with medium containing ^H-arachidonic acid contained compounds which co-chromatographed with polar compounds produced by autooxidation, primary prostaglandins, prostaglandin metabolites, dihydroxyfatty acids, monohydroxyfatty acids and also a compound whose retention time was greater than that of the HETEs and was therefore less polar than the HETEs.

241 The relative polarity and acid-stabi1ity of this compound was similar to that of acid-stable epoxides which may be synthesised by cytochrome P450 enzymes. Compounds with an epoxide structure have been isolated from liver microsomes following incubation with tritiated arachidonic acid in the presence of epoxide hydrolase inhibitors (Oliw et al. 1982b). Hepatic monooxygenases may metabolise arachidonic acid to 4 epoxides which are homoallylic and stable at physiological pH. Chacos et al. (1982) have identified 5f6-r 8,9-, 11,12- and 14,15- epoxyeicosatrienoic acids produced by liver cytochrome P450. Both placenta (Meigs & Ryan 1968) and amnion (Goto

& Weibel 1980) contain cytochrome P450 enzymes which appear to have specific drug-metabolising activities and are also involved in steroid metabolism. Metabolism of arachidonic acid by cytochrome P450 has not previously been described either in fetal membranes or by whole cells but the formation of epoxides by cytochrome P450 may result in production of a compound whose polarity is less than that of HETEs. The presence of placental epoxide hydrolase could cause conversion to di-hydroxy compounds which may have polarities similar to LTB^. Successive actions of lipoxygenases and epoxygenases could lead to the synthesis of epoxy-hydroxy compounds (Pace-Asciak et al. 1983) whose polarities would be intermediate between those of di-HETEs and HETEs (Woolard, personal communication). Hydrolysis of the epoxide may lead to formation of trihydroxy compounds. Pace-Asciak et al.

(1983) demonstrated conversion of 12-hydroperoxy-eicosa-

5,8, 10,14-tetraenoic acid by a 0-30% ammonium sulphate fraction of rat lung parenchyma into 2 hydroxyepoxides which were further converted by a 30-50% ammonium sulphate fraction into 2 trihydroxy compounds suggesting the

successive actions of epoxygenases and epoxide hydrolases. Cholesterol esters of fatty acids are also less polar

than HETEs but may also be less'polar than arachidonic acid on reverse-phase HPLC judging by their behavior on thin-layer chromatography (Woolard, personal communication). Although the placenta may synthesise cholesterol esters (Simpson & Burkhart 1980), cholesterol may be used preferentially for steroid biosynthesis (Winkel et al. 1981). Studies on other cell types suggest that relatively little arachidonic acid is esterified to cholesterol in comparison with phospholipids (reviewed by Kuksis 1978) and that a relatively small amount of cholesterol esters contain arachidonic acid. Therefore it is unlikely that the non-polar compounds released by placental cells are cholesterol esters of arachidonic acid. Thus phospholipids may be the predominant site of arachidonic acid esterification within the cell in a storage capacity.

Metabolites of H-arachidonic acid with the same retention time as HETEs were also observed. The formation of hydroxyfatty acids by fetal membranes has been previously reported. Saeed and Mitchell (1982b) reported

243 production of 5- and 12-HETE and postulated that compounds

produced by lipoxygenase enzymes could inhibit PGI2 production. The source of HETEs in placental cell cultures may be lipoxygenase or cytochrome P450 enzymes.

Lipoxygenases lead to production of 5-, 8-, 9-, 11-, 12-

and 15-HETEs via hydroperoxide intermediates whereas cytochrome P450 enzymes may produce omega and omega-1

hydroxylated metabolites. Chlorobutane was found to extract all of the compounds eluting after LTB4 indicating that they were HETEs, since chlorobutane is specific for HETEs and compounds of similar polarity (Barr et al. 1984). Compounds eluting between LTB^ and HETEs may

therefore be HETE isomers, since an isomer of 12-HETE was found to elute just after LTB 4. The polarity of

intermediate compounds is also indicative of hydroxyepoxy compounds. Normal-phase analysis of chlorobutane- extracted compounds suggested that the major product was 15-HETE, in contradiction to the results of Saeed and Mitchell (1982b). Gas-chromatography mass-spectrometry analysis was restricted to 12-HETE, due to interference from lipids present in serum which gave a background level above which cell products could not be detected. The

amount of compounds in the medium blank measured in the

12-HETE channel was 0.53 jAg per ml. Twelve-HETE was detected in culture supernatants from amnion but not chorion laeve or placental cell cultures.

A compound which co-chromatographed with LTB4 was also observed. Dihydroxyfatty acids may arise from enzymatic and non-enzymatic hydrolysis of epoxides such as

LTA^. Double hydroxylations may give rise to 5,12- and ,14,15-di-HETEs. Since standards of all the many known di-

HETEs were not available it cannot be concluded if they were separated or whether, therefore, the observed peak was composed of several components which were not resolved. It is unlikely that di-HETE isomers were resolved from each other since following chlorobutane extraction only 1 peak remained in the hydroxyfatty acid portion of the chromatogram.

A variety of compounds were observed to elute with standard prostaglandins and their further metabolites. The detection of PGI2 as determined by stable compounds eluting between 7 and 12 minutes was complicated by the presence of the large peak presumed to contain polar products of autooxidation. Prostaglandin I2 metabolites appeared to be present in relatively small amounts and could only be identified as shoulders on the main peak. The total radioactivity within this region of the chromatogram was not significantly different from that in control experiments. 6 -keto-PGF-j^synthesis has been demonstrated in placental cell cultures (Jogee et al. 19 83 ) although its synthesis was not detected in homogenates of placental cells using both RIA and GC-MS

(Dembele-Duchesne et al. 1982). Prostaglandin I2 synthesis may be inhibited by products of lipoxygenases such as 15-HPETE, which may explain the lack of synthesis

245 observed in the present study. A peak corresponding to

Tx E*2 was also observed but the total radioactivity was not significantly above that observed in control experiments.

Time course studies with TXB2 antibodies have shown that production declined after the ls^ 24 hours of culture

(Rose, unpublished observations). Thromboxane B2 production has also been demonstrated by Mitchell et al.

(1978a) in the placenta but its synthesis may be associated with trauma or ischaemia. Compounds which co­ chromatographed with PGD2r PGF20£ and PGE2 were also identified. Production of PGD2 (Mitchell et al. 1982),

PGF2

(Braithwaite & Jarabak 1975, Westbrook et al. 1977) explains the presence of peaks which elute with prostaglandin metabolites and the apparently small peaks of primary prostaglandins. The metabolism of prostaglandins by 15-hydroxy-prostaglandin dehydrogenase is not altered by the onset, or during the process of, spontaneous labour (Kierse et al. 1976). Since metabolism does not change, alterations in prostaglandin profiles must be due to changes in synthesis. In vivo, prostaglandins may also be metabolised by 15-hydroxy- prostaglandin dehydrogenase and delta-13-prostaglandin reductase in the myometrium and endometrium (Abel & Kelly 1983), and in the chorion laeve (Kierse & Turnbull 1976).

246 The major pathways of metabolism of both exogenously added and endogenously incorporated arachidonic acid by placental cells observed in this study are summarised in figure 25. The metabolism of arachidonic acid by chorion laeve cells was investigated to determine whether the pattern of products was similar to that of placental cells. Chorion laeve cells also produced an unidentified compound which was postulated to be a fatty,acid epoxide. Peaks corresponding to HETEs were also observed and although only 5- and 12-HETE could be detected on normal-phase

HPLC, GC-MS did not detect levels of 12-HETE above those found in the medium. A peak corresponding to LTB^ and a similar pattern of prostaglandins and their further metabolites were also observed. It therefore appeared that the profiles of metabolites produced by both placenta and chorion laeve cells were similar. The major pathways of metabolism of exogenously added and endogenously incorporated arachidonic acid are summarised in figure 26. iii) Amnion cells.

The profile of hydroxyfatty acids and the putative epoxy fatty acid on reverse-phase HPLC was similar to that of placental cells. Normal-phase HPLC showed that 15-HETE was the major product although GC-MS data suggested that 12-HETE was also synthesised. Prostaglandin E2 was clearly the major cyclooxygenase product of metabolism and although the amnion has not been

247 Figure 25 (a) Metabolism of arachidonic acid in 1 st. trimester

placental cells.

Exogenously added

Minor pathways PL= Phospholipid

In c re a sin g CE= Cholesterol ester

jr activity TG= Triglyceride

I---->M ajor pathways

248 figure 25 (b) Metabolism of arachidonic acid in term placental cells.

Exogenously added

Elective caesarean section

Spontaneous labour

> Minor pathways PL= Phospholipid. in creasin g activity CE= Cholesterol ester. ▼ Major pathways TG= Triglyceride.

249 Figure 26. Metabolism of arachidonic acid in chorion laeve cells.

Exogenously added

A.A. A.A.

Spontaneous labour A.A. A.A.

Epoxide

Minor pathways PL= Phospholipid

I Increasing CE= Cholesterol ester ^ a c tiv ity TG= Triglyceride.

Major pathways

250 demonstrated to contain a high prostaglandin catabolic activity, further metabolites were observed. These may possibly be 13,14-dihydro-15-keto-PGE2 or PGA2' which may arise non-enzymatically from PGE2 and chromatographs with

the PGE2 metabolite, 13,14-dihydro-15-keto-PGE2. Prostaglandin E2 production by amnion cells has been previously demonstrated (Mitchell & Flint 1978, Olson et al. 1983c). The latter group also observed 6-keto-PGF-^ and PGF2(* production. Thromboxane B2 synthesis has also been reported. Peaks corresponding to thromboxane B2 and 6-keto-PGF^ were observed in the present study as well as

PGE2 but not P G F 2 P r o s t a g l a n d i n F2 may be produced non-enzymatically from endoperoxides (Christ-Hazelhof &

Nugteren 1979) and stimulation of arachidonic acid release without a corresponding increase in prostaglandin synthetase activity could result in its formation artifactually via the hydrolysis of prostaglandin endoperoxides. In the study by Olson et al. (1983c), p g f 2o( was measure<^ i-n supernatants following short-term incubations of cell suspensions which could result in the stimulation of arachidonic acid release following trauma due to the dispersion of cells from the tissue. The major pathways of metabolism of exogenously added and endogenously incorporated arachidonic acid in amnion cells observed in this study are outlined in figure 27.

251 Figure 27. Metabolism of arachidonic acid in amnion cells.

Exogenously added

Spontaneous labour

A.A.

PL= Phospholipid I Increasing X a c tiv ity CE= Cholesterol ester

TG= Triglyceride

|--^ >major pathway,

252 c) Differences in the metabolism of arachidonic acid

between tissues and with gestational age. First-trimester placental cells were found to produce

PGD2 and PGE metabolites via the cyclooxygenase pathway, di-HETEs, HETEs and the putative epoxide via lipoxygenase

or cytochrome P450 enzymes. The only reproducible products of placental cells obtained following spontaneous

labour were the putative epoxide, HETEs and compounds which eluted between HETEs and di-HETEs, whereas placental cells obtained following elective caesarean section did not synthesise arachidonic acid metabolites.

These changes may reflect the relative functional

state of the placenta at different stages of pregnancy, in a manner similar to that previously discussed for the uptake of arachidonic acid by cell cultures. The major

difference between 1 trimester and term placental cells was the apparent synthesis of di-HETEs. The roles of

hydroxyfatty acids in pregnancy are unknown but LTB^ has a variety of effects including chemotaxis for

polymorphonuclear leucocytes (Ford-Hutchinson et al. 1980 ) and the induction of suppressor T cells (Rola- Pleszczynski & Sirois 1983). The latter role may be important in preventing rejection of the conceptus by the maternal immune system. During implantantion and in early pregnancy the invasion of maternal arteries may be aided by the synergistic action of LTB^ and PGE2 in increasing

vascular permeability (Williams 1983). When the placenta has reached the limit of its development, production of

253

V LTB^ may not be necessary. During parturition increased vasopermeability could lead to excessive fluid loss, which may explain the apparent change in synthesis. A similar increase in activity following parturition appeared to take place with chorion laeve cells, although only the compounds eluting between di-HETEs and HETEs were significantly above the control levels.

The major qualitative change occurring in the metabolism of exogenous arachidonic acid by amnion cells was the apparent increase in synthesis of PGE2 and di- HETEs with labour. The ratios of PGE2 : HETEs and PGE2 + di-HETEs : HETEs appeared to increase minimally (PGE2 : HETEs from 0.25 + 0.22 to 0.40 + 0.22, PGE2+di- HETE : HETE from 0.87 + 0.20 to 0.98 + 0.20). This is in contrast to the finding in placental tissue and suggests that the major source of prostaglandins during parturition is the amnion. During parturition there could therefore be a switch from the synthesis of HETEs and the putative epoxide to PGE2 and, possibly, di-HETEs. Prostaglandin E2 may be involved in myometrial contractions but the role of di-HETEs in parturition is unknown. D i - hydroxyeicosatetraenoic acid(s) may also therefore play a role in the process of parturition.

The metabolism of endogenous arachidonic acid also showed some differences. Only 1st trimester cells released significant amounts of metabolites, whereas cells obtained following caesarean section and spontaneous

254 labour were not as active. A similar pattern was observed for chorion laeve cells, although one sample obtained following spontaneous labour released HETEs and the putative epoxide. Amnion cells obtained both before and after labour released HETEs and the putative epoxide but, perhaps more importantly, only cells obtained after spontaneous labour released PGE2. In common with other experimental models there was low conversion of both exogenously added and endogenously incorporated arachidonic acid to oxygenated metabolites. First trimester placental cells and term amnion cells appeared to be the most active. This may be explained by the fact that during the 1 trimester implantation and the development of the utero-placental circulation take place. Concomitantly the trophoblast tissue surrounding the embryo develops rapidly and arachidonic acid may be an important constituent of its cell membranes. Oxygenated metabolites of arachidonic acid may be important in preventing the rejection of the conceptus and in the maintenance of utero-placental bloodflow at this time in gestation. Towards term the amnion appears to be the major source of arachidonic acid metabolites in fetal membranes rather than the placenta or chorion laeve.

These observations may explain the relative activities of these cell types.

The overall conclusion of these observations is that although cells of all 3 membranes studied were able to metabolise exogenous free arachidonic acid, the metabolism

255 of endogenous substrate may be different. The metabolism of arachidonic acid may be regulated at several levels

(figure 28). The release of substrate from complex lipids is a prerequisite for metabolism by lipoxygenase or

cyclooxygenase pathways. Phospholipase A2 activity has been found to increase in placental villi at term (Dimette

1980 ) which may be an explanation for the relatively low release of metabolites from placental and chorion laeve

cells before labour. However the•observations that amnion cells only synthesise PGE2 from endogenous substrate following labour but that synthesis of HETEs and the putative epoxide appears to be constitutive suggest that there may be regulation at the level of cyclooxygenase and

lipoxygenase enzymes also. Since the amount of free arachidonic acid is low it is suggested that addition of

free arachidonic acid may not provide a suitable model for the study of arachidonic acid metabolism. Placental and chorion laeve cells appeared to have similar metabolic profiles but amnion cells produced PGE2 specifically and also synthesised 12-HETE, which was not

detected in placental cell cultures by GC-MS. This difference suggests a separate role for amnion distinct from

that of chorion or the definitive placenta. Prostaglandin E2 is thought to be involved in parturition due to its

contractile effects on uterine musculature and its synthesis was associated with spontaneous labour. The function of 12-HETE is unknown but it has contractile

256 Figure 28. Sites of regulation of arachidonic acid metabolism

Prostaglandins HETEs Epoxides.

(1) Following formation of Acyl-CoA. incorporation into phospholipids is rapid.

(2) Release from phospholipids may be rate-limiting but different phospholipases or phospholipid pools may provide substrate for different oxygenative pathways.

(3) There may also be regulation at the level of oxygenative metabolism.

(4) Addition of free arachidonic acid may bypass the rate-limiting de-esterification of arachidonic from phospholipids.

25 7 effects on smooth muscle preparations (Carraher et al. 1983) and has also been shown to be a platelet anti­ aggregatory substance (Croset et al. 1983 ).

4) Regulation of Arachidonic Acid Metabolism.

Arachidonic acid metabolism may be regulated by a multitude of factors in vitro and in vivo. The rate- limiting step is generally considered to be the deacylation of phospholipids which is catalysed by phospholipases (Levine 1978). The activity of phospholipases determines the amount of free fatty acid available for metabolism via cyclooxygenase and lipoxygenase pathways. The metabolite profile obtained following reverse-phase HPLC of cell culture supernatants showed that fetal membranes may metabolise arachidonic acid via cyclooxygenase, lipoxygenase andi cytochrome P450 pathways and that the relative abundance of metabolites was in favour of those produced by lipoxygenases or cytochrome P450. This evidence suggests that there may also be mechanisms for the regulation of arachidonic acid metabolism at steps following its release from phospholipids. Alternatively it is possible that release of arachidonic acid from different classes of phospholipids may be coupled to different metabolic pathways. Ehrman et al. (1982) observed that in rabbit kidney medulla arachidonic acid release from different pools occurred by different mechanisms; one mechanism was calcium— sensitive and another calcium-insensitive. Only

2 52 the calcium-sensitive pool was efficiently coupled to prostaglandin endoperoxide synthetase. The rate-limiting effect of phospholipases may be

bypassed by the addition of exogenous arachidonic acid (figure 28). The addition of serum to media, which is one of the major stimuli of prostaglandin synthesis in cell culture systems/may act partly bjsupplying free arachidonic acid. In the present study the use of a radioactively labelled tracer showed that this exogenous substrate was not metabolised in the same fashion as endogenous substrate, an observation previously made in other systems (Coene et al. 1982, Ehrman et al. 1982). However, these experiments allowed the metabolism of the 2 pools to be differentiated. The activity of phospholipases may be affected by non-specific factors such as trauma and mechanical manipulation. These events may disturb the membrane thereby activating phospholipases causing release of arachidonic acid. The release of prostaglandins during adherence of cells to culture dishes (Bockman 1981) may be mediated in a similar way. In the present study, these non-specific factors were minimised by preincubating the cells for 24 hours prior to the addition of arachidonic acid. Blood contaminants such as platelets did not persist over the 24 hour preincubation period and were removed by washing, thus reducing their effects. In vivo phospholipases may be regulated by hormonal factors and the activity of a variety of tissue

259 phospholipases may be modulated by target-tissue-specific hormones. In the vasculature of the fetal placenta angiotensin has been shown to stimulate PGI2 and PGE release (Glance et al. 1985), and oestrogen and progesterone have been shown to stimulate placental 6- keto-PGF^ synthesis (Jogee et al. 1983). In amnion cell suspensions, beta-adrenergic agonists were shown to stimulate PGE production (Di Renzo et al. 1984a).

However, lipoxygenase products were not analysed and the non-specific effects of trauma were not taken into account in these studies. The apparent discrepancy between the results of the present study and previously reported experiments,, wherein prostaglandins did not appear to be released < constitutively I although lipoxygenase- and cytochrome P450-products were released, may reflect the activity of different phospholipases coupled to the different pathways. Addition of exogenous substrate bypasses the physiological regulation of arachidonic acid metabolism. Release of PGE2 by amnion cells obtained following spontaneous labour may be a result of irreversible activation of the cyclooxygenase pathway following a change in their hormonal status. In the case of the amnion this may result from some change in amniotic fluid, since both amniotic fluid (Mitchell et al. 1982) and adult and fetal urine (Casey et al. 1983) contain factors which stimulate PGE2 synthesis in the amnion. In the case of trophoblast cells which are in direct contact with maternal tissue, the regulating factors may be of

260 maternal rather than of fetal origin. Prostaglandin synthesis by placental tissue may therefore only occur following addition of a specific stimulus which was absent

in culture. Beta adrenergic receptors on the placenta have been characterised (Moore & Whitsett 1981) and could be coupled to prostaglandin synthesis in a similar fashion to that in amnion cells (Di Renzo et al. 1984a).

Serum may also provide factors such as peptide and steroid hormones which may affect arachidonic acid metabolism. Since differences in the metabolism of endogenous substrate were observed at different stages of pregnancy it was unlikely that hormonal influences of serum did not affect the metabolic pathways in a way that may have produced misleading results. The metabolism of arachidonic acid in the tissues

investigated may therefore be dependent upon factors of either fetal or maternal origin. These may not only regulate the availability of substrate but also the activity of the various pathways, the presence of which is related to the' stage of pregnancy. The previous discussion indicates that the addition of exogenous substrate may obscure physiological changes in arachidonic acid metabolism. The metabolism of endogenously released

substrate may therefore be a better reflection of the in vivo situation. Considering only this, placental cells

showed changes in their metabolic profile throughout pregnancy and amnion cells showed a change associated with

261 labour. This agrees with the observations of Olson et al.

(1983c) who detected increased synthesis of prostaglandins in amnion and chorion laeve associated with labour.

Increased synthesis in chorion laeve cells was not observed in the present study. Trophoblast cells may require a specific stimulus for prostaglandin synthesis in the absence of non-specific effects of trauma due to preparation of the cells. First-trimester placental cells released hydroxy-and the postulated epoxy-fatty acids, whereas cells at term both before and after labour showed relatively little activity. Amnion cells synthesised hydroxys and epoxy-fatty acids before and after labour but only synthesised PGE2 after labour. Similar studies with rabbit membranes (Elliott et al. 1984) have shown a change in activity of enzymes of both pathways during gestation, lipoxygenase enzymes being more active in mid-gestation and cyclooxygenase more active towards term. However, the use of homogenates may only reflect alterations in activity at the level of these enzymes. The use of whole cells allows the manifestation of specific regulatory mechanisms at the cellular level. Both pathways in human placental cells were relatively quiescent before labour for both exogenous and endogenous substrate whereas cells after labour could metabolise exogenous substrate. The placenta may therefore have a requirement for free arachidonic acid, a possible source being the amnion or amniotic fluid during labour, although a control mechanism at the level of cyclooxygenase

262 may exist to prevent metabolism of free arachidonic acid before labour. The release of arachidonic acid and its conversion to prostaglandins within trophoblast cells may require continual stimulation from external sources.

However a change in the biochemistry of amnion cells may

lead to constitutive synthesis of PGE2 during and after labour.

The change in relative activities of lipoxygenase and

cyclooxygenase activities may be a secondary effect which is dependent upon their mutual interaction. Hydroperoxy- fatty acids may inhibit PGI2 synthesis and production of PGI2 may therefore not only require activation of cyclooxygenase but also inhibition of 15-lipoxygenase.

5) Biological Functions of Arachidonic Acid Metabolites in Pregnancy. a ) Implantation and development of utero-placental

circulation.

During implantation in most species studied the first macroscopically-demonstrable event is a localised increase in endometrial vascular permeability (Psychoyos 1973). There is considerable evidence that prostaglandins have an obligatory role in these changes (Kennedy 1983). However, little is known of their sites of synthesis or their sites and modes of action. It is also probable that they interact with other compounds. The development of placentation following implantation could be mediated in a

263 similar fashion. It is possible that the blastocyst and/or invading trophoblast tissue could be the source of prostaglandins during the 1st trimester of pregnancy. In this study human placental cells in culture utilised free exogenously added arachidonic acid to synthesise prostaglandins. However endogenously derived arachidonic acid was not converted to prostaglandins. Unless the endometrium or blood provides a source of free arachidonic acid the trophoblast of the implanting blastocyst may not therefore be a source of prostaglandins during implantation. The blastocyst or trophoblast may therefore provide a signal which may stimulate the endometrium to produce prostaglandins which could then mediate the vascular changes associated with placentation. The observation that human placental cells may metabolise arachidonic acid largely through the lipoxygenase pathway to produce compounds which co-chromatograph with di-HETEs and HETEs could provide a mechanism whereby the invading trophoblast stimulates prostaglandin synthesis in the endometrium. It has been shown that leukotrienes may stimulate prostaglandin biosynthesis in macrophages (Feuerstein et al. 1981) and in guinea-pig lung parenchyma (Sirois et al. 1980). The trophoblast could also be a source of LTB^ which has been shown to act synergistically with prostaglandins to increase vascular permeability

(Williams 1984). The observation that ls^ trimester placental cells produced relatively more di-HETEs than term placental cells supports this idea.

264 b) Immunology of pregnancy. Despite the fact that the conceptus is allogeneic it does not appear to provoke a maternal immune response resulting in its rejection. There is some evidence to suggest that the placenta synthesises compounds which suppress the maternal immune system, and hCG (Beling & Weksler 1974) and progesterone (Kobayashi et al. 1979) have been implicated as immunosuppressive agents.

Lipoxygenase products have been shown to be involved in the immune response and the observation that the fetal membranes may metabolise arachidonic acid via lipoxygenase pathways could provide an alternative mechanism for interaction with the maternal immune system. Active suppressor cells are induced by LTB^ (Rola-Pleszczynski &

Sirois 1983). These cells could prevent rejection of the conceptus by the down-regulation of the maternal immune response and may be induced by compounds present in culture supernatants which co-chromatograph with LTB^ on reverse-phase HPLC. Suppressor T cells appear early in pregnancy and have been demonstrated in the decidua (Daya et al. 1985). Mono-HETEs may also be involved in the immunology of pregnancy since they play a part in the regulation of human neutrophil migration (Goetzl 1984a) and in human eosinophil function (Goetzl et al. 1980). c ) Maintenance of bloodflow.

The development of the fetus is dependent upon an adequate supply of maternal blood to the placenta. This

265 may be ensured by dilation of the spiral arteries and inhibition of platelet aggregation within the arteries in the inter-villous space. The placenta has been suggested to be a source of platelet anti-aggregatory activity which could be due to PGI2 production (Myatt & Elder 1977),

ADPase (Duchesne et al. 1972) or inhibition of TxA2« In this study 6-keto-PGF^^ was not a major product of placental arachidonic acid metabolism. The presence of HETEs could be an additional source of inhibition of platelet aggregation since 12-HETE (Croset et al. 1983) and 15-HETE (Vericell & Lagarde 1980) have been shown to inhibit platelet aggregation. d) Parturition,

The role of prostaglandins in parturition is well documented. Although the sources of prostaglandins during labour have not been completely described it is generally thought that the major source is the amnion which produces PGE2. In this study amnion cells in culture specifically produced PGE2 via the cyclooxygenase pathway. Exogenously added arachidonic acid was metabolised by cells obtained both prior to and following spontaneous labour. However only samples obtained after spontaneous labour produced

PGE2 from endogenous substrate. The stimulation of PGE2 production by amnion cells may be initiated by substances produced by the fetus and released into the amniotic fluid by the fetal urine (Casey et al. 1983). The stimulant appeared to cause a change in the PGE2 synthetic capacity

266 of amnion cells which is maintained in vitro and could therefore involve protein synthesis. The possible roles of arachidonic acid metabolites produced by amnion cells are outlined in figure 29. Little evidence was obtained for either placenta or chorion laeve being a major source of prostaglandins during parturition. Lipoxygenase products of arachidonic acid metabolism may also be involved in parturition. . tZ r-HETE causes contractions of the myometrium (Carraher et al. 1983) and closure of the ductus arteriosus may, in part, depend on inhibition of PGI2 synthesis by an endogenous hydroperoxy fatty acid (Needleman et al. 1981). e ) Regulation of the Endocrine Function of the Placenta.

The placenta is a source of peptide hormones with structural and functional analogies with pituitary peptide hormones (Krieger 1982). Lipoxygenase products have been implicated in the regulation of hCG release in chorion (Ilekis & Benveniste 1984) and fatty acid epoxides have been implicated as intermediates in the release of a variety of pituitary peptides including luteinising hormone (Snyder et al. 1983). The synthesis of steroid hormones by the placenta, chorion laeve and amnion could also be regulated by endogenous lipoxygenase products in a similar fashion to that in the Leydig cell (Dix et al. 1984). The products of lipoxygenases or cytochrome P450 enzymes could therefore be endogenous mediators of hormone

267 Figure 29. Possible functions of arachidonic acid metabolites produced by the amnion.

A. A.

▼ Epoxide------h C G ^

(1) & (2) predominate before labour and play a role in the maintenance of pregnancy.

(3) & (4) increase with the onset of parturition.

HPETEs may inhibit myometrial prostaglandin synthesis thus inhibiting myometrial contractions.

268 release in the fetal membranes. Prostaglandins did not appear to be major metabolites of arachidonic acid in placenta and chorion laeve and may only be produced in association with parturition in the amnion. The synthesis of prostaglandins by fetal membranes previously described may have been affected by trauma and other non-specific effects. It is possible that prostaglandin synthesis may require a specific stimulus and elucidation of the roles of prostaglandins may require the identification of such stimuli. It is unlikely that prostaglandins of the E and F type are produced in large amounts by the placenta and chorion laeve since this could lead to myometrial contractions. Similarly production of PGI2 could play a role in the onset or progression of labour since it may potentiate the effects of oxytocin (Willens et al. 1979 ) and may cause myometrial contractions (Omini et al. 1979). However Wikland et al. (1983) observed a transitory inhibition of myometrial contractions. Prostaglandin I2 could, however, play a role as an inhibitor of platelet aggregation and also as a vaso-dilator in the spiral arteries and umbilical cord. Prostaglandin D2 is also an inhibitor of platelet aggregation. The production of TXB2 in these vessels could be a mechanism whereby blood loss is reduced I during parturition. However its synthesis may only be associated with trauma or ischaemia.

A summary of the hypothetical roles of arachidonic acid metabolites in the placenta is outlined in figure 30.

269 Figure 30. Hypothetical scheme for roles of arachidonic acid metabolites in the placenta.

270! 6) Conclusions. The results of the present study showed that cells derived from fetal membranes were able to metabolise arachidonic acid via cyclooxygenase and, largely, lipoxygenase and (possibly) cytochrome P450 enzymes. High performance liquid chromatography allowed the resolution of the major classes of arachidonic acid metabolites produced by these enzymes.

The synthesis of prostaglandins was dependent upon substrate source, stage of pregnancy and tissue type. Placental cells produced prostaglandins from exogenous substrate during early pregnancy and at term following spontaneous labour. Both cell types produced a variety of prostaglandins and their further metabolites. Amnion cells,however, only produced PGE2 and were able to synthesise it from exogenous substrate both before and after labour. Endogenous substrate was only converted to PGE following spontaneous labour. These results suggest that the addition of free arachidonic acid to the medium may override physiological regulatory mechanisms. This may therefore obscure physiological changes in regulatory mechanisms associated with parturition. The use of intact cells in monolayer culture provides a useful model in which non-specific effects can be minimised and cellular alterations associated with physiological changes may be maintained in vitro. Products of lipoxygenase enzymes have been previously described in fetal membranes but the relative activities of the various pathways have not been

271 investigated. Cytochrome P450 enzymes have not been shown to metabolise arachidonic acid in fetal membranes, although the observation has been made in liver. These results suggest that further studies are required to

investigate the regulation of these alternative pathways and the biological roles of their products.

7) Further Studies.

Further studies are required to determine the precise chemical nature and biological roles of lipoxygenase and cytochrome P450 products and how the products of all pathways may interact with each other. The elucidation of the chemical structure of unidentified compounds, such as the putative epoxide and di-HETE compound(s)^ could be determined by GC-MS. The investigation of the regulatory mechanisms governing arachidonic acid metabolism should be confined to the fate of endogenous substrate rather than free exogenous substrate. This is due to the possibility that the addition of free arachidonic acid may bypass key roles of phospholipases in the regulation of the level of free arachidonic acid in the cell. Further studies should be carried out to determine whether deacylation and metabolism are coupled or whether some released substrate

is free to be metabolised indiscriminately through the various pathways. If the products of metabolism of arachidonic acid are dependent upon the endocrine environment, which changes throughout pregnancy, it is

272 necessary to determine how the product profile may change within the course of normal pregnancy before determining how it may be altered in complicated pregnancies or how it may be modulated by pharmacological agents.

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