Endogenous Prostaglandin E2 in Human and Experimental Duodenal Ulceration. By Stirling Pugh MRCP A thesis submitted to University College, London for the degree of Ph. D. in Clinical Science, From The Department of Surgery and Gastroenterology, Rayne Institute, Faculty of Clinical Science, University College, London. ProQuest Number: 10630942 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10630942 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 Abstract The aetiology of human peptic ulcer disease is multifactorial. Its association with anti-prostaglandin drugs has lead to the suggestion that mucosal deficiency of prostaglandins, which are known to be protective to the gastrointestinal tract, may be an aetiological factor. The thesis documents a short history of duodenal ulceration and previous work into its pathogenesis and epidemiology. The history of prostaglandins in the gastrointestinal tract, the phenomena of mucoprotection and the evidence for the protective role of these substances are explored. A method for using endoscopic biopsies to assess the endogenous ability to synthesise prostaglandin E 2 (PGE2 ) is described and validated. Synthesis of iPGE 2 (immunoreactive PGE 2 ) in the upper gastrointestinal tract in control subjects is detailed and the effect of demographic factors explored. Synthesis of iPGE2 in patients with duodenal ulceration is estimated and related to all stages of the disease and to histological and histochemical analyses. The human work suggested a deficiency in iPGE 2 in patients with duodenal ulceration, and the timing of this deficiency in relation to ulcer formation is explored in the cysteamine rat model of duodenal ulceration. 2. Dedication This work is dedicated to my wife Susan, to my parents and to Mike. 3. Table of contents Title page 1 Abstract 2 Dedication 3 Table of contents 4 Index to tables 11 Index to figures 14 Index to photomicrographs 15 Acknowledgements 16 Statement of originality 18 Introduction 19 Hypothesis and plan of research 21 Chapter 1 History, epidemiology, and pathogenesis of duodenal ulceration. 23 Short history of duodenal ulceration 24 Epidemiology of duodenal ulcer 30 Pathogenesis of duodenal ulcer 35 Introduction 35 Abnormalities of acid secretion 37 Abnormalities of pepsin secretion 39 Abnormalities of pylorus function 39 Mucosal protection 41 Mucus and bicarbonate secretion 41 Other protective factors 43 Non-gastrointestinal factors which may contribute to ulcerogenesis 43 Possible infective causes of duodenal ulcer 46 Sum m ary 50 4. Chapter 2 History of PGs, their biochemistry and physiology and the mucosal protection phenomenon. 52 History of PGs 53 Biochemistry of PGs and other eicosanoids 55 Nomenclature 58 Metabolism 59 Dietary deficiency in essential fatty acids 59 Inhibition of PG synthesis 60 Physiological and pharmacological effects of PGs 60 PGE2 in the gastrointestinal tract. 60 Action on muscle 60 Gastrointestinal secretion 61 The physiological roles of PGs in the stomach and duodenum 62 Histamine 2 receptor stimulation and acid secretion 62 Motility 63 Duodenal bicarbonate secretion 64 Eating 64 Permissive action of corticosteroids 64 Diet 65 In summary 66 NSAIDs and peptic ulceration 67 Mucosal protection 71 Introduction 71 History of 'cytoprotection' (mucosal protection). 71 Adaptive mucosal protection 76 Mechanisms of mucosal protection 76 Mucosal bicarbonate secretion 77 Mucus secretion 81 Mucosal blood flow 82 Mucosal cell turnover 85 Preservation of tight junctions 86 In summary 86 Mucosal protection by other substances 86 Carbenoxolone 86 Bismuth compounds 87 Other metal salts 90 5. Sucralfate 90 Other substances 92 Alternative views on mucosal protection and the role of PGs 95 Effect of PGs on isolated cells 98 PGs and mucosal protection in man. 100 Physiological actions 100 Mucosal protection in man 101 Alternative views on mucosal protection and the roles of PGs in man 104 In summary 106 The therapeutic use of PGs and their synthetic analogues 106 Chapter 3 Historical review of the methodology for assessing endogenous PG synthesis 109 Introduction 110 Earliest published method 111 Subsequent attempts 111 Using gastric juice 112 Using venous blood 114 Using mucosal samples 117 PG 'content' of mucosal tissue samples. 117 In vitro synthesis using mucosal tissue samples 119 In vitro synthesis following homogenisation 119 In vitro synthesis in whole mucosal tissue samples 122 In vitro synthesis using vortexed whole tissue samples 126 Chapter 4 Description of method 128 Preparations 129 Obtaining samples 129 H um ans 129 Animals 131 The samples 132 Transfer 132 Weighing 132 6. PG synthesis 133 Radioimmunoassay 134 Potassium phosphate buffer 134 Radioimmunoassay buffer 134 Tritiated PGE 2 135 A ntiserum 135 Standard PGE 2 135 Charcoal mixture 136 Standard curve 136 Validation of the radioimmunoassay 141 Validation of the method of in vitro PG synthesis 142 Chapter 5 Normal values for iPGE 2 in the rat and man 149 Rats 150 Introduction 150 M ethod 150 Results 151 Discussion 154 M an 155 Introduction 155 Subjects and Methods 155 Subjects 155 Biopsies 156 Collection, stratification and statistical analysis of data 156 Results 157 Demography 157 Discussion 166 Chapter 6 In vitro mucosal iPGE 2 synthesis in subjects with duodenal ulcer disease 173 Introduction 174 Methods 174 Subjects 174 Biopsies 175 Treatment 175 Results 176 Demography 176 7. Subjects with duodenal ulcer compared with control subjects 178 Quantitative demographic data of control subjects and the duodenal ulcer sub-groups. 179 In vitro synthesis of 1PGE2 analysed according to biopsy site and clinical group 182 Discussion 185 Early attempts to measure gastroduodenal PGs 189 Plasma PGs in peptic ulcer disease 190 Gastric juice PGs 192 PGs in mucosal samples 194 PG 'content' in mucosal samples 194 In vitro synthesis using mucosal samples 198 Homogenates 198 Culture of mucosal samples 203 Vortex stimulation of mucosal samples 206 In summary 208 Chapter 7 PG synthesis compared to histology and histochemistry 210 Introduction 211 Methods 211 Patients 211 Biopsies 212 iPGE2 synthesis 212 Histology and histochemical examination 212 Statistical analysis 214 Results 215 Histological and histochemical appearances 215 iPGE2 synthesis values 218 Association between histology and iPGE 2 synthesis 222 Discussion 225 Chapter 8 iPGE2 synthesis and histological changes in the cysteamine model of duodenal ulceration in the rat. 229 Introduction 230 M ethods 233 A nim als 233 8. Treatments 234 Experiment design 234 After sacrifice 234 Mucosal samples and IPGE 2 synthesis 235 Histological samples 235 Animal and sample coding 237 Results 237 Postmortem 237 Histology results 238 iPGE2 synthesis 241 Correlation between histological findings and PG synthesis 245 Discussion 248 Chapter 9 Summary and critical review of thesis 256 Summary of thesis 257 iPGE2 synthesis in health and duodenal ulceration 257 Relationship to histological findings 259 Deficiency of mucosal prostaglandins; cause or effect of duodenal ulceration? 260 Importance of the findings in this thesis to the understanding of pathogenesis and healing of duodenal ulceration 261 Therapeutic implications 262 Future research 263 Investigations into the cause of the demonstrated PG deficiency 263 Eicosanoid synthesis in other peptic ulcerative conditions 264 Further clinical studies into the roles of endogenous eicosanoids in duodenal ulcer disease 264 Critical review 265 Measurement of PGE 2 and validation of the assay 266 Patient study design 267 Histological analysis of samples 268 Cysteamine duodenal ulcer model in the rat 269 Hypothesis proved? 274 9. Appendix Table of abbreviations 273 Consent form 274 Patient data entry form 275 Appendices in inside back cover pocket 276 Human raw data - patient demography and iPGE 2 277 Histological data 294 Animal raw data 296 References 297 10. Index to tables Chapter 4 Table 4.1 iPGE2 content of pre- and post-vortex solutions. 144 Table 4.2 Reproducibility and coefficients of variation in duplicate results (pg iPGE 2 mg'1) from control subjects. 145 Table 4.3 Weights (mg) of biopsies from inflamed and non­ inflamed areas pre- and post-freeze drying, and calculated percentage water content. 147 Chapter 5 Table 5.1 Normal iPGE2 values for duodenal samples in female Wistar rats. 152 Table 5.2 Normal iPGE2 values for antral samples in female Wistar rats. 153 Table 5.3 iPGE2 synthesis (pg iPGE2 mg-1) in control subjects. 158 Table 5.4 iPGE2 synthesis (pg iPGE2 mg-1) analysed according to sex. 159 Table 5.5 Duodenal iPGE 2 synthesis (pg iPGE2 mg'1) analysed according to social habits. 159 Table 5.6 iPGE2 (pg iPGE2 mg'1) synthesis analysed according to site of biopsy and age grouping. 160 Table 5.7 iPGE2 (pg iPGE2 mg"1) synthesis analysed according to recruitment centre. 163 Table 5.8 iPGE2 synthesis (pg iPGE2 mg'1) in control subjects from the beginning and end of the study period. 163 Table 5.9 iPGE2 synthesis (pg iPGE2 mg"l) in subjects taking NSAIDs compared to control subjects. 164 Chapter 6 Table 6.1 Patient Groups. 176 Table 6.2 Demographic details of duodenal ulcer subjects. Numbers of subjects in each description within each group. 177 11. Statistical analysis of data in Table 6.2 using y 2 analysis. 178 Quantitative demographic data in control subjects and duodenal ulcer sub-groups. 180 In vitro synthesis of iPGE 2 analysed according to site and clinical group. 183 Statistical analysis of Table 6.5 using Student's t test (two-tailed) for unpaired data. 184 Patient groups 212 Histological and histochemical scoring of biopsies.