CHROMIUM, VANADIUM AND ASCORBATE EFFECTS ON LIPIDS, CORTISOL, GLUCOSE AND TISSUE ASCORBATE OF GUINEA PIGS by WOLE KOLA OLADUT, B.S., M.S. A DISSERTATION IN HOME ECONOMICS Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved áxigust. tq^ 11;^ ACKNOWLEDGMENTS - <"-^ f/K^ 1 would like to express my sincere gratitude and respect to Dr. Barbara J. Stoecker for her patience, encouragment and direction throughout my graduate program and research project. I am also grateful to other members of my committee, Professors Elizabeth Fox, Donald Oberleas, Marvin Shetlar and Shiang P. Yang. My appreciation is extended to my colleagues, Dr. Reza Zolfaghari for his technical advice, Ms. Gay Riggan, my typist and my coworkers at Methodist Hospital Laboratory. Special thanks are extended to my mother, Mrs. Comfort Oladutemu, my brother, Jide Oladutemu, Dr. Bayode Lasekan and all other family members and friends for their love, help and encouragement during the completion of my graduate education. 11 CONTENTS Page ACKNOWLEDGMENTS il LIST OF TABLES v LIST OF FIGURES viii LIST OF ABBREVIATIONS Ix I. INTRODUCTION 1 II. REVIEW OF LITERATURE 4 Ascorbate and Carbohydrate Metabolism 4 Ascorbate and Lipid Metabolism 6 Chromium and Carbohydrate Metabolism 12 Chromium and Lipid Metabolism 15 Vanadium and Carbohydrate Metabolism 19 Vanadium and Lipid Metabolism 20 Cholesterol-fed Guinea Pigs 22 Conclusion 27 III. ADRENAL ASCORBATE AND CORTISOL CONCENTRATIONS OF GUINEA PIGS SUPPLEMENTED WITH CHROMIUM AND/OR VANADIUM .... 29 Abstract 29 Introduction 30 Materials and Methods 32 Results and Discussion 36 IV. TISSUE ASCORBATE, ADRENAL 4-'^^C-CH0LESTER0L AND PLASMA CORTISOL OF GUINEA PIGS SUPPLEMENTED WITH CHROMIUM AND/OR ASCORBATE 46 Abstract 46 Introduction 47 Materials and Methods 49 Results and Discussion 52 V. PLASMA AND TISSUE ASCORBATE CONCENTRATIONS OF CHOLESTEROL-FED GUINEA PIGS SUPPLEMENTED WITH VANADIUM AND/OR ASCORBATE 65 Abstract 65 Introduction 66 Materials and Methods 67 Results and Discussion 70 111 Page VI. LIPIDS, GLUCOSE AND HEMATOLOGICAL EVALUATION OF CHOLESTEROL-FED GUINEA PIGS SUPPLEMENTED WITH VANADIUM AND/OR ASCORBATE 77 Abstract 77 Introduction 78 Materials and Methods 81 Results and Discussion 83 VII. EPIDIDYMAL FAT PAD LIPOPROTEIN LIPASE AND TISSUE ASCORBATES OF CHOLESTEROL-FED GUINEA PIGS SUPPLEMENTED WITH CHROMIUM AND/OR ASCORBATE 92 Abstract 92 Introduction 92 Materials and Methods 93 Results and Discussion 96 LIST OF REFERENCES 102 APPENDIX 112 IV LIST OF TABLES Page 3.1 Basal diet composition for guinea pigs supplemented with chromium and/or vanadium 33 3.2 Initial and final body weights of guinea pigs supplemented with chromium and/or vanadium 36 3.3 Organ weights of guinea pigs supplemented with chromium and/or vanadium 39 3.4 Tissue ascorbate of guinea pigs supplemented with chromium and/or vanadium 41 3.5 Plasma and adrenal cortisol of guinea pigs supplemented with chromium and/or vanadium 42 3.6 Epididymal fat-pad lipoprotein lipase of guinea pigs supplemented with chromium and/or vanadium 44 14 3.7 4- C-cholesterol activity of guinea pig tissues after supplementation with chromium and/or vanadium 45 4.1 Basal diet composition for guinea pigs supplemented with chromium and/or ascorbate 50 4.2 Initial and final body weight of guinea pigs supplemented with chromium and/or ascorbate 53 4.3 Organ weights of guinea pigs supplemented with chromium and/or ascorbate 57 4.4 Tissue ascorbate of guinea pigs supplemented with chromium and/or ascorbate 58 4.5 Plasma and adrenal cortisol of guinea pigs supplemented with chromium and/or ascorbate 60 4.6 Epididymal fat pad lipoprotein lipase of guinea pigs supplemented with chromium and/or ascorbate 63 14 4.7 4- C-cholesterol of guinea pigs supplemented with chromium and/or ascorbate 64 5.1 Basal diet composition for cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 68 5.2 Initial and final body weights of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 71 v Page 5.3 Tissue weights of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 74 5.4 Tissue ascorbate of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 75 6.1 Plasma cholesterol of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 84 6.2 Plasma triglyceride of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 85 6.3 Biliary cholesterol of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 86 6.4 Hematocrit and zlnc protoporphyrin of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate. • . 87 6.5 Plasma glucose at fasting and 2 hours after 2 g/kg body weight glucose loading of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 88 6.6 Morphological evaluation of blood smears of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate. 89 6.7 Epididjnnal fat pad lipoprotein lipase activity of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 90 7.1 Basal diet composition for cholesterol-fed guinea pigs supplemented with chromium and/or ascorbate 94 7.2 Initial and final body weights of cholesterol-fed guinea pigs supplemented with chromium and/or ascorbate 97 7.3 Plasma and tissue ascorbates of cholesterol-fed guinea pigs supplemented with chromium and/or ascorbate 100 7.4 Epididymal fat pad lipoprotein lipase of cholesterol-fed guinea pigs supplemented with chromium and/or ascorbate. 101 A.l Furnace program for dietary chromium analysis 113 A.2 Furnace program for dietary vanadium analysis 114 A.3 Complete blood counts of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate 115 VI Page A.4 Differential analysis of blood smears of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate. 116 A.5 Morphological evaluation of blood smears of cholesterol-fed guinea pigs supplemented with vanadium and/or ascorbate. 117 A.6 Lipoprotein lipase activity of frozen tissue and acetone powder of epididymal fat pad of guinea pigs 118 vii LIST OF FIGURES Page 2.1 Metabolic pathway for cortisol formation 10 3.1 Body weights of guinea pigs supplemented with chromium and/or vanadium 37 4.1 Body weights of guinea pigs supplemented with chromium and/or ascorbate 54 5.1 Body weights of guinea pigs supplemented with vanadium and/or ascorbate 72 7.1 Body weights of cholesterol-fed guinea pigs supplemented with chromium and/or ascorbate 98 Vlll LIST OF ABBREVIATIONS AA Ascorbic acid ACTH Adrenal corticotrophic h( ADCC Antibody-dependent phagoi cytotoxicity Aniso Anisocytosis Baso Basophils BS Basophilic stipplings +C Ascorbate adequate -C Ascorbate deprived CE Cholesterol esters co^ Carbon dioxide CrCl^ Chromium chloride -Cr Chromium deprived +Cr Chromium supplemented CPM Counts per minute D Decreased DHAA Dehydroascorbic acid dL deciliter Eosin Eosinophils Fe Iron 8 gram GRR Glucose removal rate GTF Glucose tolerance factor HCl Hydrochloric acid IX Mono Monocytes N Normal NADH Nicotinamide adenine dinucleotide NaK-ATPase Sodium-Potassium adenine triphosphatase NH^VOg Ammonium vanadate NIDDM Non-insulin dependent diabetes mellitus OGTT Oral glucose tolerance test Pest Platelet estimate PLT Platelet count ppb Parts per billion ppm Parts per million poik Poikilocytosis Poly Polychromasia PUFA Polyunsaturated fatty acids RBC Red blood cells RCR Relative chromium response RDW Red cell distribution rpm revolution per minute RQ Respiratory quotient SAS Statistical analysis system Seg Segmented neutrophils SEM Standard error of the mean TCA Tricholoroacetic acid TGL Triglyceride TPN Total parenteral nutrition XI UC Unesterified cholesterol ug microgram umol micromoles -V Vanadium deprived +V Vanadium supplemented V02+ Vanadayl ion VO3 Vanadate VLDL Very low density lipoproteins WBC White blood cells Wk Week w/v weight per volume ZPP Zinc protoporphyrin Xll CHAPTER I INTRODUCTION Ascorbate is essential for guinea pig and primates. The role of ascorbate in a variety of stressful situations including burns, severe injuries, surgery and infection, was elucidated in the 1940's and 1950's. The involvement of ascorbate in key regulatory enzjnnes such as cortisol formation in the adrenal and bile acid formation in the liver has been of major interest to scientists in recent years. Studies by Ginter and coworkers (37-41) have shown the importance of ascorbate. In ascorbate deprivation, there was elevation of serum cholesterol (41). Holloway and Rivers (48) have also studied long-term effects of inadequate and excessive dietary ascorbate on bile acid metabolism in guinea pigs. Chromium may have a function in maintaining normal serum lipids. Abraham and colleagues (1-3) have associated chromium deprivation with cholesterol-induced atherosclerosis. Hyperlipidemia and impaired glucose tolerance in adult men were reversed by chromium supplementation in a 12 week double blind study by Riales and Albrink (92). Mertz had earlier hypothesized that chromium may function as a cofactor for insulin. An in vitro study by Mertz and colleagues (76) showed a significant glucose uptake into the epididymal fat tissue of rats in the presence of insulin when trivalent chromium chloride was supplemented. Studies conducted by Schroeder and coworkers (101, 102) found no significant changes in total lipid content or the incidence of aortic 1 plaques in rats, but they observed a decreased serum cholesterol in animals receiving 5 ppm chromium in drinking water. However, a short term study by Preston and colleagues (89) demonstrated that 0.125 ppm chromium had no effect on weight gain and serum cholesterol in guinea pigs. Dietary experiments in animals have indicated an effect of vanadium on glucose, lipid metabolism and body growth. Addition of vanadate has been shown to stimulate the activity of phospho- glucomutase and glucose-6-phosphate dehydrogenase. The rate limiting enzyme in cholesterol synthesis and the HMG-CoA inactivating enzyme have been reported by Menon and coworkers (71) to be inhibited by vanadate.