AN INVESTIGATION OF POTATO ALKALOID LEVELS IN THE NORMAL POPULATION AND THEIR RELATIONSHIP TO FOETAL NEURAL TUBE DEFECTS Submitted in fulfilment of the requirements for the degree: Doctor of Philosophy of the University of Surrey by: MICHAEL HUGH HARVEY October 1988 ProQuest Number: 10798521 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 10798521 Published by ProQuest LLC(2018). 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 CONTENTS Page SECTION 1 INTRODUCTION 1.1 Introduction 1 1.2 Chemistry of the Solanum Alkaloids 3 1.3 Biochemistry of the Solanum Alkaloids 10 1.4 Distribution of Glycoalkaloids Within the Potato 14 1.5 Factors Affecting the Formation of Glycoalkaloids 17 1.5.1 Light 17 1.5.2 Temperature and Storage 18 1.5.3 Mechanical Injury 20 1.5.4 Climatic Conditions 21 1.5.5 Cooking and Potato Processing 21 1.5.6 Genetic Control 23 1.5.7 Control of Tuber Glycoalkaloid Content 25 1.6 Physiological Functions of Potato Alkaloids ' , 28 1.7 Analysis of Potato Glycoalkaloids 30 1.7.1 Extraction of Glycoalkaloids from the Potato 30 1.7.2 Colorimetric Quantification 32 1.7.3 Thin-Layer Chromatography 33 1.7.4 Gas Chromatography 34 1.7.5 High Performance Liquid Chromatography 35 1.7.6 Immunoassay Methods 38 1.7.7 Other Methods of Glycoalkaloid Analysis 40 1.8 Pharmacology and Toxicology of Potato 41 Glycoalkaloids 1.8.1 Glycoalkaloid Poisoning in Man and Domestic 41 Anim als 1.8.2 Pharmacology of Potato Glycoalkaloids 44 1.8.3 Pharmacokinetics of Potato Glycoalkaloids 47 1.8.4 Toxicity of Glycoalkaloids in Experimental 49 Anim als 1.8.5 Teratogenic Effects of Potato Glycoalkaloids 52 1.8.6 Aims of the Present Study 52 Page SECTION 2 RADIOIMMUNOASSAY OF POTATO ALKALOIDS 2.1 Introduction 54 2.2 Materials and Methods 64 2.2.1 M aterials 64 2.2.1.1 Assay Buffer 64 2.2.1.2 Immunogen 64 2.2.1.3 Antiserum 64 2.2.1.4 Label 65 2.2.1.3 Standard 65 2.2.1.6 Alkaloid-Free Serum/Urine/Saliva 66 2.2.1.7 Dextran-Coated Charcoal 66 2.2.1.8 Scintillation Vials 66 2.2.1.9 Scintillation Fluid 67 2.2.2 Methods 67 2.2.2.1 Original Radioimmunoassay Method for Serum 67 Solanidine 2.2.2.2 Modified Radioimmunoassay Method for Serum 69 Solanidine 2.2.2.3 Radioimmunoassay for Serum Total Alkaloid 71 71 2.2.2.4 Radioimmunoassay Method for Salivary Solanidine and Total Alkaloid 2.2.2.3 Radioimmunoassay Method for Urinary Solanidine 72 and Total Alkaloid 2.2.2.6 Summary of Potato Alkaloid Radioimmunoassay 72 2.2.2.7 Quality Control 78 2.3 Results 81 2.3.1 Development and Validation of the Radioimmunoassay 81 for Potato Alkaloids 2.3.1.1 Radioactive 8-Counting 81 2.3.1.2 Label 82 2.3.1.3 Antiserum 85 2.3.1.4 Establishment of Total Alkaloid Assay 96 2.3.1.5 Optimisation of Sample Volume 96 2.3.1.6 Preparation of the Standard Curve 101 2.3.1.7 Assay Incubation Times 104 2.3.1.8 Detection Limit 106 2.3.1.9 Precision 108 2.3.1.10 Label Immunoreactivity 108 2.3.1.11 Assay Recovery 108 2.3.1.12 Non-Specific Binding 113 2.3.1.13 Zero Binding 113 2.3.1.14 50% Intercept 113 Page SECTION 3 NEURAL TUBE DEFECTS 3.1 Introduction 114 3.2 Potato Alkaloids and Neural Tube Defects 124 3.2.1 Introduction 124 3.2.2 M aterials and Methods 128 3.2.2.1 Collection of Specimens 128 3.2.2.2 Analytical Methods 129 3.2.3 Results 129 3.2.4 Discussion 140 3.3 Zinc and Neural Tube Defects 146 3.3.1 Introduction 146 3.3.2 Materials and Methods 149 3.3.2.1 Collection of Specimens 149 3.3.2.2 Analytical Methods 149 3.3.3 Results 151 3.3.4 Discussion 158 3.4 Folate and Neural Tube Defects 161 3.4.1 Introduction 161 3.4.2 Analytical Method 161 3.4.3 Results 162 3.4.4 Discussion 163 SECTION 4 ALKALOID CONCENTRATIONS IN SELECTED POPULATIONS 4.1 Alkaloid Concentrations in the Normal Population 165 4.1.1 Introduction 165 4.1.2 Materials and Methods 165 4.1.2.1 Collection of Samples 165 4.1.2.2 Analytical Methods 166 4.1.3 Results 167 4.1.4 Discussion 167 4.2 Toxicological Effects of Excessive Intake of 178 Potato Glycoalkaloids in Man 4.2.1 Introduction 178 4.2.2 Materials and Methods 178 4.2.2.1 Case History 178 4.2.2.2 Glycoalkaloid Analysis 179 4.2.3 Results 179 4.2.4 Discussion 180 Page SECTION 5 POTATO ALKALOIDS IN HUMAN SALIVA 5 J Introduction 186 5.2 Materials and Methods 193 5.2.1 Collection of Specimens 193 5.2.2 Analytical Methods 193 5.3 Results 195 5.4 Discussion 204 SECTION 6 AFFINITY CHROMATOGRAPHY 6.1 Introduction 206 6.2 Materials and Methods 213 6.2.1 M aterials 213 6.2.2 Analytical Methods 214 6.2.2.1 Preparation of Anti-Solanum Alkaloid Immunosorbent 214 Using Cyanogen Bromide Activated Sepharose 4B 6.2.2.2 Preparation of Solanidine Free Serum, Urine and 216 Saliva 6.2.2.3 Preparation of Porous Glass and Controlled Pore 218 Ceramic Immunosorbents 6.2.2.4 A ffinity Extraction of Serum Samples and Removal 218 of Alkaloid Cross-Reactants 6.2.2.5 Scale Up of Controlled Pore Ceramic Column for 219 A ffinity Chromatography for 24 Hour Urine Samples 6.3 Results 221 6.4 Discussion 225 SECTION 7 HPLC AND TLC OF POTATO ALKALOIDS 7.1 Introduction 226 7.2 Materials and Methods 235 7.2.1 M aterials 235 7.2.2 Analytical Methods 235 7.2.2.1 Hydrolysis and TLC Separation of Potato Alkaloids 235 7.2.2.2 Spectral Analysis of Potato Alkaloids 237 7.2.2.3 HPLC of Potato Alkaloids 237 7.2.2.4 Development of HPLC Method 238 7.3 Results 243 7.3.1 Spectral Analysis 243 7.3.2 TLC of Potato Alkaloids in Serum and Urine 243 7.3.3 HPLC Analysis of Potato Alkaloids in Serum and Urine 243 7.4 Discussion 254 Page SECTION 8 PHARMACOKINETICS OF POTATO ALKALOIDS 8.1 Introduction 256 259 8.2 Materials and Methods 259 8.2.1 M aterials 25q 8.2.2 Methods 25q 8.2.2.1 Preparation of the Potato Meal 25g 8.2.2.2 Pharmacokinetic Studies 26o 8.2.2.3 Analysis of Total Glycoalkaloid Content of Potatoes 8.2.2.4 Analysis of Alkaloid Content of Serum and Urine 261 8.3 Results 262 8.3.1 Total Glycoalkaloid Content of Potato Samples 262 8.3.2 Analysis of Serum and Urine from Pharmacokinetic 262 Study 8.4 Discussion 278 SECTION 9 GENERAL DISCUSSION AND CONCLUSIONS 9.1 Methodology 281 9.2 Alkaloid Concentrations in Normal Populations 285 9.3 Neural Tube Defects 287 9.4 Further Studies 289 ACKNOWLEDGEMENTS 291 REFERENCES 292 APPENDIX 307 SUMMARY Radioimmunoassay methods were developed for the measurement of solanidine and total alkaloid in serum, urine and saliva. Solanidine and total alkaloid concentrations were measured in a group of normal pregnant women and a group of women pregnant with a foetus subsequently shown to be affected by a neural tube defect (NTD). Serum alkaloid concentrations were, contrary to expectations, lower in the NTD group, casting doubt on the theory suggesting a link between potato alkaloids and NTD serum zinc and folate concentrations were also measured in the two groups and no significant differences were noted. Solanidine and total alkaloid concentrations were measured in the serum, urine and saliva of healthy volunteers and reference ranges established. Total alkaloid concentrations were higher than solanidine in serum and urine but similar in saliva, indicating solanidine as the predominant salivary alkaloid. Alkaloid concentrations measured in a case of potato poisoning and in subjects on high alkaloid intake had serum, urine and salivary solanidine and total alkaloid concentrations much greater than the reference ranges. A pharmacokinetic study was carried out to obtain body fluid alkaloid concentrations following a potato load. The glycosides were found to be rapidly absorbed before metabolism to solanidine;urinary excretion accounted for a very small percentage of the to ta l dose. A ffinity chromatography allowed concentration of potato alkaloids in urine and serum for qualitative analysis of metabolites. A high performance liquid chromatography (HPLC) method was developed and used in conjunction with thin layer chromatography (TLC) to separate the principle alkaloids found in urine and serum. As well as the glycosides, a- solanine and a-chaconine, the presence of the aglycone solanidine was established. Additionally, several of the lower glycosides were found in serum and urine. ABBREVIATIONS AFP Alpha Fetoprotein ASB Anencephaly and Spina B ifida Cystica BSA Bovine Serum Albumin B/Bo Percentage counts bound in zero tube c.p.m.