THE EFFECT OF a-SOLANINE ON ACETYLCHOLINESTERASE ACTMTY IN WO:AN EXAMINATION OF UNDOCUMENTED BELIEFS Airdrie M. Waker A Thesis Submitted to the Faculty of Graduate Studies in partial fiifdiment of the requirements for the degree of Master of Science University of Manitoba Winnipeg, Manitoba (c) Airdrie M. Waker, 1997. National Libraiy Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services senrices bibliographiques The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Li- of Canada to Bibliothèque nationale du Canada de reproduce, loan, distniute or sell reproduire, prêter, distn'buer ou copies of this thesis in microform, vendre des copies de cette hese sous paper or electroaic fonnats. la forme de mic~che~nim,de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. COPYRIGET PERMISSION PAGE THE EFFECT OF a-SOLANINE ON ACTEYLCROLINESTERASE ACTIVITY --IN VIVO: ANEXAKDUTIONOF UNDOCüMENTED BELIEFS A Thcrir/Pmcticum sabmittd to the Faculty of Graduate Studics of The Univenity of Manitoba in partial falfillmet of the reqaimmenti of the dm of MASTER OF SCIENCE Airdrie M. WaLker 1997 (c) Pennissiom hm ken granted to the Librnry of The University of Minitoba to Itnd or seiî copies of thu thesis/prptcticum, to the Natiooai Library of CanPàa to micronlm this thesis and to lend or sel1 copies of the mm, and to Dissertations Abstracb IaternatiooaI to pablisb an abstnct of this thesidprpcticam. The author racmotk publication rigbts, and neither thir thesislprrcticum nor esternive extncts from it may bc printd or othede rtproductd without the author's written permission. I hereby declare that I am the sole author of thïs thesis. 1 authorize the University of Manitoba to lemi this thesis to other institutions or individuals for the purpose of scholarly reswch. Airdne M. Walker I further authorize the University of Manitoba to reproduce this thesis by photocopying or by other means, in toial or in part, at the request of other institutions or indivuals for the purpose of scholarly research. Airdrie M. Walker The University of Manitoba requises the signatures of ail pesons using or photocopying this thesis. Please sign klow, and give addrcss md date. Dedication Dedicated to the memory of my loving and much beloved parents. Solanaceous plants, such as potato, contain glycosidic steroidal alkaloids. The potato glycoalkaioids, principdiy a-solanine and a-chaconine, have been implicated in numerous cases of toxicity to humans and animais. The mecbaaism of action for the toxic efieci is poorly definecl but toxicity has bew attributed to their anti-cholinesterase property. Much of the support for this notion has derived from in vitro studies, limired animal studies in which potato peels were fed, and rewspective case studies of humans. The evidence for glycoalkaloid toxicity is weak and anecdotal in nature. The present study examined the effect of a-solanine in vivo under controlled experimental conditions. Shce dietary protein can alter the rate of biotransfonnation lipophilic xenobiotics to hydrophilic metabolites and the excretion of xenobiotics, the study examiwd the effect of a-sol- on cholinesterase enzyme activity at two levels of dietary protein. Eighty-sut (86) weanling male Sprague-Dawley rats were fed a low (7.5%) or standard (15%) protein diet ad libitum for four weeks. At the end of the dietary ueamient period they were injected intraperitoneally with or-solanine suspended in 8% ethyl alcolhol at a dose level of 1 mgMg B.W. Enzyme activity was meawred in erythrocytes, plasma, liver and brain at three tuw periods: 3, 8 and 12 hours post- injection. The study showed that a-solanine at this dose level inhibiteci the cholinesterase activity in the liver but only at 12 houn pst-injection in the anirnals fed the standard protein diet. In the other tissues the enzyme activity was reduced due to the glycoalkaloid v treafment but the difference was not statistically significaot. For example. Ïn the erythrocytes at 8 hours pst-injection the eiityme activity was 2.3 * 1.2 I.U.B. in the connol group compared to 1.5 10.6 I.U.B. in the treated group but this difference was not significant staûsticaily. The study examined the relationship between die- factors and the ami- chohesterase effect which has not been snidied previously . The effect of low dietary protein was evident in al1 tissues but was only significantly diffent in the liver. In the Iiver, a signifiant difference in enzyme activity was observed in both the low and standard protein groups at al1 three the points. In otber tissues there was a reductioa in activity due to the low protein diet but it was not statistically significant. For example, in brain at 3 houn pst-injection the activity was 12.014.4 in the standard protein control group compared to 9.3 k2.7 in the low protein control group. This rnay be explained by differences in unique tissue response to dietary protein. This study suggests that the anti-choünesterase effect of a-solaniw is a factor in the toxicity of animals resulting fiom the ingestion of potato glyoalkaloids and toxicity may be greater in animals with inadequate nutritional status. Most importantly, I wish to gratefuily achowledge the conaiion of Dr. Demis Fitzpatnck in the completion of this work and for the academic oppominity afforded to me. I would also like to convey my appreciation to other members of the faculiy of the Depamnent of Foods anâ Nutrition who have shown interest and offered encouragement to me during the course of my studies. 1 am indebted to Ms. Marilyn Latta who wiiiîngly provided invaluabie assistance in the Iaboratory and to Dr. Paz-AIiaga, Arequipa, Peru for his participation in this project . My shcerest thanks are exteaded to feiiow graduate midents whose friendship and support has been abundant. To my family, Duncan. Jeanne and Graeme, who are exceptionally generous in their enthusiasm and support of my endeavoun, 1 am truly grateful. vii TABLE OF CONTENTS ABSTRACT ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS CHAPTERS PAGE 1.O INTRODUCTION 1.1 Thesis 1.2 Background 1.3 Relevance of Research 1.4 Objectives and Seope 1.5 MethodoIopical Considerations 2.0 REVIEW OF LITERATURE 2.1 Toxicology 2.2 Nutritional Todcology 2.2.1 Dose Response Relationship viii 2.2.4 Factors Affecthg Biotransformation 14 2.2.5 Efféct of DKt Biotransfomiaton 17 23 2.3.1 Structure and Identification 2.3.2.1 Distibution 2.3.23 Physiological Function 2.3.2.3 Tolerances 2.3.2.4 Factors Mecting Levels 28 2.3.2.5 Wild Species 31 2.3.2.6 Effects of Processing and Cooking 32 2.3.2.7 Bioavaiiability 2.3.2.8 Toxicity a) Dose Response 39 b) Structural Features Governing Toxicity 39 c) Adverse Effects 41 2.3.2.9 Phazmacokinetics 2.3.2.10 Acceptable Daiiy Intake 2-4.3 Acety lcholinesterase and Butyry lcholinesterase 56 3.0 MATERiALS AND METHODS 3.1 Animais 3.2 Experimental Protocol 3.3 Experimental Design 3.4 Diet Formulation 3.4.1 Methods Erythrocytes Plasma Liver and Brah 3.5 statistical Anaiysis 4.0 RESULTS 4.1 Animai Weight 4.2 Food C~llsumption 4.3 Feed Efficiency 4.4 Enzyme Assays 4.5 Rotein Effect 5.0 DISCUSSION 5.1 Animals 5.2 Food Consumption 5.3 Feed Efficiency 5.4 Enzyme AJseys 5.5 Rotein Effect 6.0 CONCLUSIONS REF'ERENCES APPENDICES A. LIST OF MAJOR EQUIPMENT B. NUTRIENT SUPPLIERS ACh AChE BBB BuCh BuChE butyrylcho~sterase B.W. body weight CHO carbohydrate CNS central newous system cyt-P450 cytochrome P-450 DMSO dimethyl suifoxide DTNB ECm concentration cawing malformation in 50 % of the of the surviving embryos FA0 Food and Agriculture Organization FETAX fiog embryo teratogenisis assay-Xenopus FWB fresh weight bais GA glycoakaioid(s) g-i. gastrointestinal i.m. i.p. i.v. LDH lactate de hydrogenase xii LCm median concentration causing 50% embryo lethaliv MAS metabolic activation system MF0 mixed fiuiction oxidase NOAEL no observed adverse effect ievel NTD murai tube defect ODC ornithine decarboxylase PEM protein energy malnutrition SGOT aspartate transaminase SGPT gamma glutarnyl trarisminase TGA total glycoalkaloid TI teratogenic index xiii LIST OF TABLES -Table Pane 1* Food Supply Per Caput Per Day .. .. -. - - .-. .- . 8 2. Distribution of or-solanine in the Potato Plant .. .. ... .. ... 26 3. Glyco~oidContent in Various Potato Products ................. 26 4. GlycoaWoid Content of Some Common North American and European Commercial Potato Varieties .. .. - .. .. .- . 29-30 5. Toxic and LEM Doses of Glycoalicaloids in Man ...... ................ 37 6. Effect of a-solanine admuiistered to different species .. 38 7. Experimental Design .. -. .... .. -. .. .. .. .. ... .. .- -. ...--...... 63 8. Diet Composition (% weight) ........................ .... ....... ..-....--65 9. Effect of Dietary Protein on Body Weight .. - - 74 10. Effect of Dietary Protein on Food Intake ............................. 75 11. Feed Efficiency .. .. .- .. .. .. - .. .. - ... .. - .. .. 75 12- Effect of a-solme on Acetylcholinesterase Activity in Erythrocytes 3 hours Post-injection .. .. .. .. .. .... ... 76 13. Effect of a-solaniw on Acetylcholinesterase Activity in Erythrocytes 8 hours Post-injection .. .- .. 76 14. Effect of a-solanine on Acetylcholiwsterase Activity in Erythrocytes 12 houn Post-injection .. .... .. .. .. 76 15. Effect of a-solanine on Butyrylcholinesterase Activity in Plasma 3 hours Post-injection .. .. .. 77 xiv Effect of a-solanine on Butyrylcholinesterase Activity in Plasma 8 hours Post-injection ...................................... 77 Effect of U-solanine on Bu~lcholinesteraseActivity in Plasma 12 hours Post-injection ....................................