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Investigations on hypervitaminosis E in rats Macdonald, Ian Bruce 1979

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INVESTIGATIONS ON HYPERVITAMINOSIS E IN RATS by IAN BRUCE MAGDONALD B.H.E., Un i v e r s i t y of B r i t i s h Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES D i v i s i o n of HUMAN NUTRITION SCHOOL OF HOME ECONOMICS We accept t h i s t h e s i s as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1979 © Ian Bruce Macdonald, 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t n f Human N u t r i t i o n The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e A p r i l 2k, 1979 i ABSTRACT In view of the f a c t t h a t some f a t soluble vitamins are t o x i c i n large doses to experimental animals and man, t h i s study was i n i t i a t e d to investigate the long-term e f f e c t s of low, moderate and high l e v e l s of d i e t a r y vitamin E on various metabolic parameters i n the r a t . Six groups of female Wistar r a t s (50 g) were fed f o r as long as 16 months the basal vitamin E-free d i e t with supplements ranging from 0 to 25»000 IU vitamin E (DL-a-tocopheryl acetate) per kilogram d i e t . The l e v e l s of vitamin E chosen were 0 , 2 5 , 250, 2 , 5 0 0 , 10,000 and 25,000 ITj/kg d i e t ; 0 representing vitamin E - f r e e , 25 representing moderate l e v e l and 250 to 25»000 representing large doses. A l l n u t r i e n t s i n the basal d i e t except vitamin E were adequate. The focus of t h i s study was on the e f f e c t s of large doses of d i e t a r y vitamin E on : ( l ) the hematological indices such as hematocrit and hemoglobin l e v e l s , prothrombin time and erythrocyte hemolysis at 9 , 12 and 16 months of treatment; (2) urinary creatine and c r e a t i n i n e l e v e l s at 11 months of treatment; (3) body weight and various organ weights at 8 and 16 months of treatment; (4) femoral parameters such as ash content, and calcium and phosphate concentrations of bone at 8 and 16 months of treatment; and (5) the l e v e l s of a-tocopherol, vitamin A, t o t a l l i p i d s , and cholesterol i n l i v e r and plasma at 8 and 16 months of treatment. Rats fed 10,000 and 25.000 IU vitamin E/kg d i e t f o r 8 and 16 months had s i g n i f i c a n t l y reduced body weights i n comparison to those r e c e i v i n g the moderate l e v e l of vitamin E. The depressing e f f e c t of excess dietary vitamin E on body weight was not as marked as that of vitamin E d e f i c i e n c y . There was l i t t l e difference between the moderate and high vitamin E supplemented groups with respect to the weights of l i v e r , uterus and kidney. However, high l e v e l s of d i e t a r y vitamin E increased the r e l a t i v e heart weights a f t e r 8 months and the spleen weights a f t e r 16 months. Hemoglobin and hematocrit values were not influenced by excessive amounts of vitamin E a f t e r 9 or 12 months of treatment. At 16 months however, the hematocrit values of r a t s fed 10,000 and 25,000 IU vitamin E/kg d i e t were increased s i g n i f i c a n t l y over those of r a t s fed 25 Iu"/kg d i e t . The prothrombin time was reduced i n r a t s treated with excess dietary vitamin E f o r 12 and 16 months. Only vitamin E d e f i c i e n c y , but not excess vitamin E, was associated with increased membrane f r a g i l i t y of erythrocytes. In r a t s subjected to excess vitamin E f o r 16 months the ash content of bone was decreased. High l e v e l s of d i e t a r y vitamin E increased the plasma a l k a l i n e phosphatase a c t i v i t y a f t e r 16 months of treatment. These r e s u l t s i n d i c a t e that there may be increased mineral turnover i n bones of r a t s f e d high l e v e l s of vitamin E f o r prolonged periods. Urinary l e v e l s of creatine and creatinine were not a f f e c t e d by high l e v e l s of d i e t a r y vitamin E. However, i n the vitamin E d e f i c i e n t r a t s , the creatine excretion increased while the creatinine excretion decreased, r e s u l t i n g i n a very high r a t i o of creatine/creatinine i n urine. The ot-tocopherol stored i n l i v e r rose s i g n i f i c a n t l y with increas- ing d i etary vitamin E. A logarithmic r e l a t i o n was demonstrated between l i v e r ot-tocopherol concentration and d i e t a r y l e v e l s of vitamin E. The t o t a l a-tocopherol i n whole l i v e r of r a t s f e d the d i f f e r e n t l e v e l s of vitamin E f o r 16 months was approximately double that i n r a t s treated f o r 8 months. A c u r v i l i n e a r r e l a t i o n s h i p between plasma tocopherol and the logarithm of d i e t a r y vitamin E was found i n r a t s treated f o r 8 and 16 months. T o t a l l i p i d s i n l i v e r increased s i g n i f i c a n t l y with increasing dietary vitamin E i n r a t s treated f o r 8 months, but not i n r a t s treated f o r 16 months. There was l i t t l e difference i n l i v e r c h o l e s t e r o l concen- t r a t i o n between the moderately supplemented and highly supplemented groups. Increasing d i e t a r y vitamin E s i g n i f i c a n t l y lowered plasma t o t a l l i p i d s and cholesterol i n r a t s treated f o r 16 months. A quantita- t i v e examination of the data showed that the reduction i n plasma t o t a l l i p i d s was not simply a r e f l e c t i o n of the cholesterol l e v e l s , and suggests that a high dietary l e v e l of vitamin E a f f e c t e d one or more of the constituents of the t o t a l l i p i d s (phospholipids and/or t r i g l y c e r - ides) other than c h o l e s t e r o l . From the f i n d i n g s of t h i s long-term study, i t appears that high l e v e l s of dietary vitamin E r e s u l t i n biochemical changes i n some aspects of metabolism i n r a t s . Some of the changes worth recognition are the depression i n body weight, increase i n r e l a t i v e spleen and heart weights, decrease i n ash content of bones with concurrent increase i n plasma a l k a l i n e phosphatase a c t i v i t y , increased hematocrit value and f a t t y l i v e r i n r a t s treated f o r 8 months. A logarithmic r e l a t i o n s h i p was observed between dietary l e v e l s of vitamin E and the concentrations of t h i s vitamin i n l i v e r and plasma. The r e s u l t s of t h i s study suggest that excess vitamin E over prolonged periods of time have some harm- f u l e f f e c t s i n r a t s . ACKNOWLEDGEMENT To my family, f r i e n d s and professional colleagues who gave much encouragement throughout my Master's Program, I extend my sincere thanks. Special thanks i s extended to Dr. N.Y. Jack Yang f o r h i s knowledge and assistance throughout the course of t h i s study. I am als o g r a t e f u l f o r the valuable discussion and moral support of Dr. J.F. Angel. Acknowledgement i s expressed to my advisor, Dr. I.D. Desai f o r h i s cooperation and assistance i n t h i s study; to Dr. J . Lei c h t e r and to Professor B.E. March f o r serving on my committee; and to V i r g i n i a Green f o r computer programming and s t a t i s t i c a l a n a l y s i s of the r e s u l t s . This study was supported i n part by Grant No. 67-^686 to Dr. I.D. Desai from the National Research Council of Canada and a Graduate.Student Fellowship (1975-1976) from the O f f i c e of the President, U n i v e r s i t y of B r i t i s h Columbia. TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENT i v LIST OF TABLES v i i i LIST OF FIGURES i x CHAPTER I. INTRODUCTION 1 I I . REVIEW OF THE LITERATURE 3 A. History of Vitamin E 3 B. Vitamin E Deficiency - Occurrence 5 1. Human Infants 5 2. Human Adults 6 3< Animals 7 C. C e l l u l a r Function of Vitamin E 8 D. Pharmacological E f f e c t of Vitamin E i n Human Subjects 10 E. Pharmacological E f f e c t of Vitamin E i n Animals 13 1. Growth 13 2. Hematology 13 3« Endocrine Function 15 4 . Bone C a l c i f i c a t i o n 15 5» Tissue Storage of Vitamin E 17 6. Tissue Storage of Vitamin A 18 7. L i v e r L i p i d Levels 20 8. Blood L i p i d Levels 21 v i III. MATERIALS AND METHODS 22 A. Animal Care 22 B. Experimental Diets 22 C. Experimental Groups 22 D. Experimental Procedure 224 E. Biochemical Determinations 25 1. Hemoglobin and Hematocrit 25 2. Prothrombin Time 25 3« Erythrocyte Hemolysis 27 4. Urinary Creatine and Creatinine 27 5- Plasma Vitamin A 28 6. Plasma Vitamin E 28 7. Plasma Cholesterol 30 8. Plasma Total Lipids 32 9• Plasma Alkaline Phosphatase 32 10. Liver Lipid Extraction 34 11. Liver Vitamin A 35 12. Eiver Vitamin E ' 36 13• Liver Total Lipids 39 14. Liver Cholesterol 39 15« Femoral Ash 40 16. Femoral Calcium 40 17. Femoral Inorganic Phosphate 42 F. Sta t i s t i c a l Analysis 4c; IV. RESULTS 46 A. Body and Organ Weights 46 B. Hematological Parameters 4Q v i i C. Femoral Parameters 54 D. Urinary Creatine and Creatinine 57 E. Fat Soluble Vitamins 57 1. L i v e r and Plasma Vitamin E 57 2. L i v e r and Plasma Vitamin A 59 F. L i p i d s 65 1. L i v e r T o t a l L i p i d s and Cholesterol 65 2. Plasma To t a l L i p i d s and Cholesterol 68 V. DISCUSSION 71 A. Body and Organ Weights 71 B. Hematological Parameters 73 C. Femoral Parameters 74 D. Urinary Creatine and Creatinine 75 E. Fat Soluble Vitamins 76 1. L i v e r and Plasma Vitamin E 76 2. L i v e r and Plasma Vitamin A 77 F. L i p i d s 78 1. L i v e r T o t a l L i p i d s and Cholesterol 78 2. Plasma Total L i p i d s and Cholesterol 79 VI. SUMMARY 80 LITERATURE CITED 82 v i i i LIST OF TABLES Table No. Page 1 Syndromes Resulting From Vitamin E Deficiency 8 2 Composition of the Basal Diet 23 3 Body and Organ Weights of Rats on D i f f e r e n t Levels of Dietary Vitamin E f o r 8 Months 47 4 Body and Organ Weights of Rats on D i f f e r e n t Levels of Dietary Vitamin E f o r 16 Months 48 5 Hemoglobin Values of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E 50 6 Hematocrit Values of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E 51 7 Erythrocyte Hemolysis of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E 52 8 Prothrombin Times of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E 53 9 Femoral Parameters of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E f o r 8 Months 55 10 Femoral Parameters of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E f o r 16 Months 56 11 Urinary Creatine and Creatinine of Rats on D i f f e r e n t Levels of Dietary Vitamin E f o r 11 Months 58 12 The Concentrations of ot-Tocopherol i n L i v e r s of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E f o r 8 and 16 Months 60 i x Standard Curve f o r Hemoglobin Standard Curve f o r Plasma Vitamin A 29 Standard Curve f o r Plasma Vitamin E 31 Standard Curve f o r Plasma Cholesterol 33 Standard Curve f o r L i v e r Vitamin A 37 Standard Curve f o r L i v e r Vitamin E 38 Standard Curve f o r L i v e r Cholesterol 41 Standard Curve f o r Calcium 43 Standard Curve f o r Phosphate 44 LIST OF FIGURES Figure No. Page 1 Structure and Nomenclature of the Tocopherols 4 2 3 4. 5 6 7 8 9 10 11 P l o t of the Logarithm of L i v e r a-Tocopherol Concen- t r a t i o n versus the Logarithm of Dietary Vitamin E, i n Rats Treated f o r 8 and 16 Months 6 l 12 Plasma a-Tocopherol Concentration of Rats Fed D i f f - erent Dietary Levels of Vitamin E f o r 8 and 16 Months 62 13 L i v e r Vitamin A Concentration of Rats Fed D i f f e r e n t D ietary Levels of Vitamin E f o r 8 and 16 Months 63 14 Plasma Vitamin A Concentration of Rats Fed D i f f e r - ent Dietary Levels of Vitamin E f o r 16 Months 64 15 T o t a l L i p i d s i n L i v e r of Rats Fed D i f f e r e n t D i e t - ary Levels of Vitamin E f o r 8 and 16 Months 66 16 L i v e r Cholesterol Concentration of Rats Fed D i f f - erent Dietary Levels of Vitamin E f o r 8 and 16 Months 67 17 T o t a l L i p i d s i n Plasma of Rats Fed D i f f e r e n t D i e t - ary Levels of Vitamin E f o r 8 and 16 Months 69 18 Cholesterol Concentration i n Plasma of Rats Fed D i f f e r e n t D i e t a r y Levels of Vitamin E f o r 8 and 16 Months 70 1 CHAPTER 1 INTRODUCTION Vitamin E has generally been considered to be non-toxic. In recent years there has been considerable i n t e r e s t among the l a y public regarding the possible pharmacological r o l e of vitamin E when taken i n large d i e t a r y supplements ("megavitamin E therapy"). At the present time, there i s no s a t i s f a c t o r y s c i e n t i f i c or c l i n i c a l evidence to prove that vitamin E supplementation i s b e n e f i c i a l f o r health. In i s o l a t e d cases, amounts greatly exceeding the normal d i e t a r y intake have been administered to human subjects with no s i g n i f i c a n t , adverse c l i n i c a l e f f e c t s ( F a r r e l l and B i e r i , 1975)* Nevertheless, i t i s f a r from c e r t a i n that chronic ingestion of vitamin E i n megadoses i s e n t i r e l y safe. In human beings, s i d e - e f f e c t s of excess vitamin E have been reported as fatigue (Briggs et a l . , 1974), c r e a t i n u r i a (Briggs et a l . , 1974; Hillman, 1957) and lengthened prothrombin time when taken i n excess along with warfarin and c l o f i b r a t e treatment (Corrigan and Marcus, 1974). There have been reports of metabolic abnormalities induced i n experimental animals by excess vitamin E. March et a l . (1973) report- ed that hypervitaminosis E induced r e t i c u l o c y t o s i s , lowered hematocrit value, reduced thy r o i d a c t i v i t y and increased requirements f o r vitamin D and vitamin K i n chicks. Hypervitaminosis E has al s o been found to depress the a c t i v i t y of glutathione peroxidase i n l i v e r and plasma of r a t s (Yang et a l . , 1976). E a r l y studies reported that excess vitamin E caused t e s t i c u l a r degeneration and reduced f e r t i l i t y 2 in male rats (Bscudero and Herraiz, 1942), and affected the length of estrus cycle and ovarian activity i n female rats (Reiss, 1941). In view of the reports of hypervitaminosis E in experimental animals the purpose of this study was to investigate further the long-term effects of high intakes of dietary vitamin E on rats treated with levels ranging from 0 to 25,000 iu/kg diet. The focus was on the effect of excess intake of vitamin E on the following metabolic parameters: (l) hematocrit and hemoglobin levels, prothrombin time and erythrocyte hemolysis; (2) urinary creatine and creatinine levels; (3) body weight and various organ weights; (4) bone ash content, and calcium and phosphate concentration of bone; and (5) the levels of Ot-tocopherol, vitamin A, total l i p i d s and cholesterol in l i v e r and plasma. These parameters were compared s t a t i s t i c a l l y with the same parameters in rats receiving a moderate or normal level of dietary vitamin E. 3 CHAPTER II REVIEW OF LITERATURE A. History of Vitamin Evans and Bishop (1922) discovered a f a t soluble a n t i s t e r i l i t y f a c t o r f o r the r a t , which was designated vitamin E by Sure ( 1924) . Evans proposed to name the substance tocopherol, from the Greek words "tocos" meaning c h i l d b i r t h , "phero" meaning to bring f o r t h and the s u f f i x " o l " , i t being an a l c o h o l . Much of the pioneer h i s t o r y of vitamin E was reviewed by Evans 419-62) and by Mason (1977)- The multiple nature of the vitamin began to unfold i n 1936, when Evans et a l . (1936) succeeded i n i s o l a t i n g from wheat germ o i l two compounds with vitamin E a c t i v i t y , a-tocopherol and B-tocopherol. Since that time, studies of vitamin E have been conducted by numerous inv e s t i g a t o r s (Pennock et a l . , 1964; Stern et a l . , 1947). To date, eight s t r u c t u r a l l y s i m i l a r forms, a l l d e r i v a t i v e s of chroman-6-ol, have been discovered to have v a r i e d amounts of vitamin E a c t i v i t y . The tocopherols belong to two d i s t i n c t s e r i e s of compounds, the tocopherols and the t o c o t r i e n o l s . The basic structure and the c l a s s - i f i c a t i o n of these compounds accepted by the IUPAC-IUB Commission on Biochemical Nomenclature (1979) are shown i n Figure 1. The e l u c i d a - tion' of the structure and synthesis of the tocopherols has been reviewed by S e b r e l l and Harris ( 1972) . The d i f f e r e n c e s i n number and p o s i t i o n of the methyl groups a f f e c t the b i o l o g i c a l a c t i v i t y of the various forms of tocopherols. The evaluation of the r e l a t i v e potency of the many compounds which have D e f i n i t i o n of terms: The accepted names are vitamin E or tocopherols. FIGURE 1 STRUCTURE AND NOMENCLATURE OF THE TOCOPHEROLS1 R^= CH 2(CH 2CH=CCH 2) 3H Tocol S t r u c t u r e T o c o t r i e n o l S t r u c t u r e Methyl P o s i t i o n s a - tocopherol 3 - t o c o p h e r o l y-tocopherol 6 - t o c o p h e r o l a - t o c o t r i e n o l 3 - t o c o t r i e n o l y - t o c o t r i e n o l 6 - t o c o t r i e n o l 5,7, 5,8 7,8 8 ^UPAC-IUB Commission on Biochemical Nomenclature (1979) Generic D e s c r i p t o r s and T r i v i a l Names f o r Vitamins and Related Compounds. J . Nutr. 109, 8-15. 5 vitamin E a c t i v i t y has been c a r r i e d out using i n v i t r o t e s t s (Bunyan et a l . , I960; Rose and Gyorgy, 1952) and bioassays (Bunyan et a l . , I 9 6 0 ; Dicks and Matterson, 1962; Friedman et a l . , 1958» Rose and Gyorgy, 1952). Investigation on the r e l a t i o n s h i p between dosage and response f o r vitamin E i n the f e t a l resorption t e s t ( B l i s s and Gyorgy, 1951) l e d to the acceptance by the National Formulary of the American Pharmaceutical Association ( i 9 6 0 ) of the conversion f a c t o r s f o r the various forms of vitamin E as shown below.1 1 mg dl-ot-tocopherol acetate = 1.0 International Unit 1 mg dl-ot-tocopherol = 1.1 International Unit 1 mg d-ot-tocopherol acetate = I . 3 6 International Unit 1 mg d-ot-tocopherol = 1.4-9 International Unit B. Vitamin E D e f i c i e n c y - Occurrence 1. Human Infants The c l i n i c a l evidence of vitamin E d e f i c i e n c y has been seen i n the earlypphase of l i f e , u s u ally with small premature i n f a n t s . This r e s u l t s from the poor t r a n s f e r of vitamin E across the placenta, sotithe i n f a n t s have low l e v e l s of vitamin E i n both t i s s u e s and blood. The anemia i n premature i n f a n t s i s hemolytic i n nature and associated with an abnorm- a l l y elevated erythrocyte f r a g i l i t y by hydrogen peroxide (Bunyan et a l . , I960; Rose and Gyorgy, 1952). In t r e a t i n g the anemia, Gross and Melhorn (1972) have found that the absorption of o r a l l y administered ot-tocopherol acetate i s i n e f f i c i e n t i n g e s t a t i o n a l l y immature in f a n t s and i s followed by a favorable hematologic response only when the chronologic equivalent of g e s t a t i o n a l maturity i s reached. A state of vitamin E d e f i c i e n c y occurs i n i n d i v i d u a l s who have 6 a defect i n t h e i r a b i l i t y to absorb f a t (Binder and Shapiro, 196?; Machon and Neals, 1970; Muller and- H a r r i s , 1969)- A large number of these cases are i n c h i l d r e n and young adults with c y s t i c f i b r o s i s ( B i e r i and F a r r e l l , 1976). Lower than usual blood tocopherol l e v e l s are observ- ed i n diseases where i n t e s t i n a l absorption i s a f f e c t e d , but no symptom- atology which responds to vitamin E has been observed. A thorough review of the information a v a i l a b l e on vitamin E status i n other malabsorptive states has been conducted by B i e r i and F a r r e l l (1976). 2. Human Adults There are no reported c l i n i c a l evidences of a d e f i c i e n c y of vitamin E i n normal human adults because of the considerable t i s s u e storage of the vitamin and the consequent extended period required f o r depletion ( B i e r i , 1975)* I t has been suggested that serum tocopherol l e v e l s below 0.5 mg/lOO ml could be c l a s s i f i e d as d e f i c i e n t ( B i e r i and F a r r e l l , 1976). Some i n v e s t i g a t o r s have shown that there i s a tendency f o r serum ot-tocopherol to r i s e and f a l l i n proportion to the amounts of c h o l e s t e r o l , phospholipid and t r i g l y c e r i d e s present i n the blood (Davies et a l . , 1969; Horwitt et a l . , 1972). Hence, i n t e r p r e t a t i o n of the status of vitamin E n u t r i t u r e from blood data may not accurately r e f l e c t e i t h e r the l e v e l of intake or t i s s u e storage. Furthermore, since blood tocopher- o l i s only about 1 per cent of the t o t a l body tocopherol pool, i t i s sometimes d i f f i c u l t to r e l a t e hlood tocopherol to vitamin E n u t r i t u r e . A long-term study by the Food and N u t r i t i o n Board of the National Research Council (Horwitt, I962) was c a r r i e d out by feeding a p a r t i a l l y d e f i c i e n t vitamin E d i e t to men f o r 5 years. There were no obvious c l i n i c a l signs of vitamin E d e f i c i e n c y i n these subjects even though the 7 blood tocopherol l e v e l s f e l l up to 0 .3 mg/lOO ml. The h a l f - l i f e of the erythrocytes was decreased, but there were no obvious manifestations of anemia. 3 • Animals Vitamin E d e f i c i e n c y can be demonstrated i n animals f e d d i e t s low i n vitamin E. There are a number of vitamin E d e f i c i e n c y states i n d i f f e r e n t species of animals, but s k e l e t a l muscle i s the most u n i v e r s a l l y a f f e c t e d t i s s u e . Some of the signs of vitamin E d e f i c i e n c y i n d i f f e r e n t species of animals are shown i n Table 1. A thorough documentation of the vitamin E d e f i c i e n c y states i n animals are reviewed by Green (1972a) and Scott ( 1970) . C. C e l l u l a r Function of Vitamin E A f u l l understanding of the mode of action of vitamin E at the molecular l e v e l has not yet been reached. With the several d i f f e r e n t , apparently unrelated disease states i n d i f f e r e n t animal species a r i s i n g from vitamin E d e f i c i e n c y i t has been d i f f i c u l t to determine a basic r o l e f o r the vitamin i n c e l l u l a r metabolism. There are two major i n t e r p r e t a t i o n s put f o r t h by i n v e s t i g a t o r s to explain the mechanism of a c t i o n of vitamin E, the b i o l o g i c a l antioxidant theory and the s p e c i f i c metabolic f u n c t i o n theory. The b i o l o g i c a l f u n c t i o n of vitamin E as a l i p i d antioxidant has been investigated f o r nearly f o r t y years, since O l c o t t and Mattel (19^1) discovered the antioxidant a c t i v i t y of vitamin E. The b i o l o g i c a l a n t i - oxidant theory suggests that t i s s u e unsaturated l i p i d s are constantly under attack by free r a d i c a l s and that i n the presence of oxygen they become peroxidized. I f s u f f i c i e n t vitamin E i s not present, the TABLE 1 Syndromes Resulting Prom Vitamin E Deficiency' Animal Species Syndrome Rat (male) (female) (both sexes) Rabbit Dog and guinea pig GhickenB Primate s t e r i l i t y f e t a l resorption l i v e r necrosis muscular dystrophy myocardial degeneration myocardial degeneration encephalomalacia exudative d i a t h e s i s macrocytic anemia muscular dystrophy From Nair, 1972. 9 peroxidation of l i p i d s becomes extensive and uncontrolled, leading to widespread damage to i n t r a c e l l u l a r membranes, enzymes and c e r t a i n metabolites such as vitamin A and phospholipids. A l l the diverse e f f e c t s of vitamin E d e f i c i e n c y i n animals are considered to be secondary, stemm- ing from one primary process, l i p i d peroxidation. S c i e n t i f i c evidence attempting to show that vitamin E functions as a l i p i d antioxidant has been presented i n numerous reviews (Tappel, 1962; Tappel, 1972; Witting, 1970). In recent years there has been considerable research which has exposed major weaknesses i n the antioxidant theory of vitamin E (Bunyan et a l . , 1968; Green and Bunyan, 1969> Green, 1972b). Controversy has developed as to whether or not l i p i d peroxidation occurs during vitamin E d e f i c i e n c y . There i s no doubt that vitamin E has antioxidant properties which can i n h i b i t t i s s u e l i p i d peroxidation i n v i t r o . Several i n v e s t - i g a t o r s have argued that l i p i d peroxidation does not occur i n vivo and therefore the b i o l o g i c a l f unction of the vitamin must be unrelated to i t s antioxidant a c t i v i t y . However, a recent experiment by Hafeman and Hoekstra (1977) i n d i c a t e d that l i p i d peroxidation occurs i n vivo i n r a t s as a r e s u l t of vitamin E d e f i c i e n c y and the peroxidation process i s g r e a t l y accelerated during the terminal phase of the f a t a l condition. T h i s report opposes the b a s i c argument of the c r i t i c s of the antioxidant theory. Much more research, however, i s needed to strengthen the antioxidant hypothesis. Several i n v e s t i g a t o r s have r e c e n t l y postulated that vitamin E may act as a c a t a l y s t or regulatory agent i n intermediary metabolism, at a s p e c i f i c s i t e which i s of fundamental importance i n metabolism (Green, 1972b{ Nair, 1972; Schwarz, 1972). In spite of the huge amount of data 10 on vitamin E published i n the l i t e r a t u r e , an unequivocal, d i r e c t involve- ment of vitamin E i n s p e c i f i c metabolic functions has yet to be i d e n t i f i e d . I t i s known that the a c t i v i t y of many enzyme systems are a l t e r e d i n vitamin E d e f i c i e n t animals (Green, 1972a; Mason and Horwitt, 1972a). Whether the a l t e r a t i o n of enzymatic a c t i v i t y i s primary or secondary to the breakdown of other t i s s u e components i s s t i l l a controversy. Research has been stimulated at the molecular l e v e l f o r a d i r e c t involvement of vitamin E i n many enzyme functions. Muscle creatine kinase (Olson, 1974), l i v e r microsomal enzyme drug hydroxylating complex (Carpenter and Howard, 1974), l i v e r xanthine oxidase (Catignani et a l . , 1974), bone marrow y -aminolevulonic a c i d synthetase and l i v e r Y ~ a m i n o ~ l e v u l o n i c a c i d dehydratase (Caasi et a l . , 1972; Nair, 1972) are some of the enzymes that have received most atten t i o n . These studies on rates of enzyme synthesis suggest a r o l e f o r vitamin E i n the r e g u l a t i o n of p r o t e i n synthesis. Just how vitamin E may possibly p a r t i c i p a t e i n t h i s sequence of events i s not known. This area has been thoroughly reviewed by Molenaar et a l . (1972) and B i e r i and F a r r e l l ( 1976) . At t h i s time there i s no d e f i n i t i v e evidence to explain many of the biochemical derangements evoked by a d e f i c i e n c y of vitamin E i n animals. Investigators at t h i s time must consider that both the b i o l o g i c a l antioxidant theory and the s p e c i f i c metabolic f u n c t i o n theory of vitamin E a c t i o n are v i a b l e and that they may be neither inconsistent nor mutually exclusive. D. Pharmacological E f f e c t s of Vitamin E i n Human Subjects Even though i t seems u n l i k e l y that a natural d e f i c i e n c y of vitamin E occurs i n man there i s good reason to believe that a large \ 11 segment of the North American population i s consuming supplementary doses of vitamin E (Farrell and B i e r i , 1975)- Much of the popular interest in vitamin E stems from articles in magazines, hooks and newspapers deal- ing with the therapeutic efficacy of vitamin E for disorders ranging from cardiovascular disease to muscular dystrophy. A c r i t i c a l appraisal of the therapeutic value of vitamin E has been made by Marks ( I962) and Bie r i and F a r r e l l (1976) . Tocopherol supplements either self-administered or prescribed by physicians vary widely in dosage, but average to about 400 IU vitamin E/day. Vitamin E has been presumed to be nontoxic to human and animals (Briggs and Briggs, 1974; F a r r e l l and Bi e r i , 1975» Horwitt and Mason, 1972). While the undesirable side effects have been rarely reported, i t i s d i f f i c u l t to evaluate what possible pharmacological action results from the ingestion of vitamin E at many times the generally recognized nutritional requirement. Very few c r i t i c a l studies of megavitamin E supplementation in man have ever been carried out. In the only systematic investigation of megavitamin E supplementation i n human, Fa r r e l l and Bi e r i (1975)> gave 100 to 800 IU vitamin E/day for 3 years to 28 adults. Laboratory screening for toxic side effects of vitamin E supplementation by c l i n i c a l blood tests f a i l e d to reveal any disturbance i n l i v e r , kidney, muscle, thyroid gland, erythrocytes, leucocytes, coagulation parameters or blood glucose. It was concluded that megavitamin E supplements in this group produced no toxic side effects. Beckman (1955) has reported that vitamin E was given to patients for months, both orally and parenterally at a dosage level of 300 IU vitamin E:/ day without any adverse c l i n i c a l effects. Greenblatt (1957) supplemented 12 the diet of six men with a massive dose, 40 g d-a-tocopherol acetate/day for one month. There were no adverse c l i n i c a l signs reported. However, Hillman (195?) reported that ingestion of 2 to 4 g vitamin E/day by an individual for 3 months produced creatinuria, cheilosis, angular stomatitis, gastrointestinal disturbance and muscular weakness. These toxic side effects ceased within two weeks after the vitamin E supplemen- tation was discontinued. Vogelsang et a l . (1947) reported that vitamin E supplementation resulted in hypoglycemia and depressed prothrombin levels, the lat t e r suggesting a relative vitamin K deficiency. The involvement of vitamin E i n potentiating some anti-coagulation activity has been reported i n two studies. Korsan-Bengtsen et a l . (1974) reported prolonged plasma clotting time in 9 subjects receiving 300 IU a-tocopherol/day. Corrigan and Marcus (1974) observed a prolonged prothrombin time in a patient ingesting 800 IU vitamin E/day, plus warfarin and clofibrate. A reduction of the level of vitamin K-dependent coagulation factors was noted during the period of vitamin E ingestion, which returned to base-line levels after the patient stopped taking the vitamin E. An examination of the mechanism by which vitamin E might be antagonistic to vitamin K-dependent clotting activity has led to an evaluation of the biological metabolites of the tocopherols. Woolley (1945) reported that a-tocopherylquinone was an antimetabolite of vitamin K^. This structural analog of vitamin was reported to cause hemorrhages in the reproductive systems of pregnant mice. The action of the a-tocopheryl- quinone was prevented by small amounts of vitamin K^. Subsequent research by March et a l . (1973) with chickens, and Rao and Mason (1975) with rats, offers further evidence that metabolites of the tocopherols may serve as 13 competitive i n h i b i t o r s of vitamin . E. Pharmacological E f f e c t s of Vitamin E i n Animals 1. Growth The f i n d i n g s on the e f f e c t of excess d i e t a r y vitamin E on the growth rate i n animals vary widely. March et a l . (1973) found a depressed growth rate i n chicks f e d a 2,200 IU vitamin E/kg d i e t from hatching to 50 days. Growth rate was not seen to be affe c t e d by supplementation of 1,000 IU vitamin E/kg d i e t . Nockels et a l . (1975) reported that feeding chicks 2,000 or 4,000 IU vitamin E/kg d i e t f o r 5 weeks had no s i g n i f i c a n t e f f e c t on body weight. However, higher l e v e l s of vitamin E supplementation, such as 8,000 and 64,000 iu/kg d i e t , were reported to reduce the chick body weight s i g n i f i c a n t l y . McGuaig and Motzok (1970) f e d a 10,000 IU vitamin E/kg d i e t to chicks and found the growth rate was unaffected by the supplementary vitamin E treatment. Similar r e s u l t s have also been reported i n the ra b b i t (Awad and Gilbreath, 1975) and the r a t ( A l f i n - S l a t e r et a l . , 1972) f e d excess vitamin E. However, Jenkins and M i t c h e l l (1975) found that the growth rate was increased when r a t s were f e d e i t h e r a 600 or 6,000 IU vitamin E/kg d i e t f o r 2 months. 2. Hematology March et a l . (1973) examined r e t i c u l o c y t o s i s i n response to various dietary-antioxidants i n chicks. They found that supplementation of e i t h e r 120 or 220 IU vitamin E./kg d i e t induced r e t i c u l o c y t o s i s . At these l e v e l s of vitamin E; supplementation hematocrit l e v e l s were not aff e c t e d . In a l a t e r study, treatment of chicks with l a r g e r doses of vitamin E 14 (2,200 iu/kg d i e t ) was noted to induce both r e t i c u l o c y t o s i s and a reduction i n hematocrit values (March et a l . , 1973)- Jenkins and M i t c h e l l (1975) f e d 600 or 6,000 IU vitamin E/kg d i e t to r a t s and found no s i g n i f i c a n t e f f e c t on the hemoglobin l e v e l s . A s i g n i f i c a n t lengthening of the prothrombin time was observed by March et a l . (1973) when a 2,200 IU vitamin E/kg d i e t was f e d to chicks. The lengthened prothrombin time was r a p i d l y normalized by i n j e c t i o n with menaquinone. An e a r l i e r study by Melette and Leone (i960) was the f i r s t to observe that vitamin E supplementation may prolong prothrombin time i n r a t s f e d nonirradiated as well as i r r a d i a t e d beef i n the d i e t . The mechanism by which hypervitaminosis E a f f e c t s prothrombin time has not yet been f u l l y elucidated. The observation by March et a l . (1973) that an i n j e c t i o n of vitamin K reversed the lengthened prothrombin time l e d them to speculate that a metabolite of vitamin E may be a s t r u c t u r a l analogue of vitamin K. One such compound has been i d e n t i f i e d i n the l i v e r , ot-tocopherol-p -quinone (Csallany e t a l . , I962). An e a r l i e r study by Woolley (1945) found that administration of ot-tocopherylquinone to pregnant mice caused hemorrhage i n the reproductive system. The a c t i o n of quinone was prevented by small amounts of vitamin , but not by large doses of dl-ot-tocopheryl acetate. S u f f i c i e n t amounts of Ot-tocopherylquinone may be produced f o l l o w i n g excessive intake of vitamin E to increase the d i e t a r y requirement f o r vitamin K^. It has been well established that vitamin E d e f i c i e n c y i s character- iz e d by spontaneous hemolysis of the erythrocyte or extensive i n v i t r o hemolysis induced by hydrogen peroxide or d i a l u r i c a c i d . Low erythrocyte hemolysis i n v i t r o though does not c l e a r l y i n d i c a t e adequacy of t i s s u e vitamin E stores ( B i e r i and Poukka, 1970). The existence of v a r i a b l e s 15 other than vitamin E intake that can a f f e c t i n v i t r o hemolysis has added to the uncertainty of t h i s t e s t (Macdougall, 1972; Melhorn et a l . , 19715 Stocks and Dormandy, 1971a; Stocks et a l . , 1 9 7 i b ) . Stocks and Dormandy (1971a) i l l u s t r a t e d that peroxide induced erythrocyte autoxidation was influenced by a number of substances such as albumin, plasma and ascorbic a c i d . Melhorn et a l . (1971) have shown that hydrogen peroxide hemolysis of greater than 20 per cent can occur i n a wide v a r i e t y of hematological disorders i n which vitamin E concentration i s normal. Some of these hematological disorders are: hereditary and acquired anemias, i r o n d e f i c i e n c y anemia, and hemoglobinopathies. Hypervitaminosis E has been found to change the f a t t y a c i d pattern of erythrocytes ( A l f i n - S l a t e r et a l . , 1972). Whether excessively high doses of vitamin E w i l l a l t e r the s t a b i l i t y of the erythrocyte membrane has not yet been determined. 3• Bone C a l c i f i c a t i o n Excess amounts of vitamin E were found to depress bone c a l c i f i c a t i o n i n chicks f e d e i t h e r calcium-deficient or vitamin D-deficient d i e t s . March et a l . (1973) found that the adverse e f f e c t of hypervitaminosis E on bone c a l c i f i c a t i o n was overcome when vitamin D was f e d at over 300 iu/kg d i e t . The mechanism by which the excess d i e t a r y vitamin E increased the requirement of vitamin D f o r maximum bone c a l c i f i c a t i o n i s not presently known. 4. Endocrine Function Several studies have shown a l t e r e d endocrine function i n experimental animals due to excessive d i e t a r y vitamin E intake. The endocrine organs 16 reported to be a f f e c t e d are the sexual organs (Czyba, 1966a; Escudero and Herraiz, 194-2; Masson, 1941; Reiss, 1941), adrenal gland ( H i l l and Hamed, 1979; F o r n i et a l . , 1955. Jenkins and M i t c h e l l , 1975), thymus (Forni et a l . , 1955); and the thyro i d gland (Czyba et a l . , 1966b; Huter, 1947; March et a l . , 1973 5 V a l e n t i and B o t t a r e l l i , I 9 6 5 ) . While there i s l i t t l e conclusive evidence of any adverse e f f e c t of vitamin E on the former three organs, the e f f e c t on the t h y r o i d gland i s f a i r l y well established. An e a r l y i n v e s t i g a t o r of hypervitaminosis E i n female r a t s observed a hypertrophy of the ovary and a l t e r a t i o n i n the length of estrus cycle (Reiss, 1 9 4 l ) . Other studies found that excess dietary vitamin E reduced male f e r t i l i t y i n r a t s (Escudero and Herraiz, 1942) and hamsters (Czyba, 1966a). However, Masson (1941) reported that feeding excessive amounts of vitamin E to hens had no e f f e c t on the b i r d s ' f e r t i l i t y . The evidence of adverse e f f e c t s of hypervitaminosis E on adrenal function i s contradictory. F o r n i et a l . (1955) found that excess vitamin E caused an increase i n adrenal weight i n r a t s . In contrast, Jenkins and M i t c h e l l (1975) f e d a d i e t containing up to 6 ,000 IU vitamin E/k, f o r eight weeks and reported a s i g n i f i c a n t decrease i n adrenal weight. H i l l e t a l . ( i 9 6 0 ) observed that hypervitaminosis E caused adrenal degeneration. O The only reported e f f e c t of hypervitaminosis E on the thymus was made by F o r n i et a l . (1955)> who observed a decrease i n thymus weight. Hypervitaminosis E has been reported to have an adverse e f f e c t on the thyroid gland. Huter (1947) was the f i r s t to report an i n j u r y to the thyroid gland i n r a b b i t s caused by excess d i e t a r y vitamin E. V a l e n t i and B o t t a r e l l i (1965) found that hypervitaminosis E reduced t h y r o i d a c t i v i t y i n the. r a t . Czyba et a l . (1966) reported that the administration of 17 vitamin E caused a t r a n s i t o r y stimulation of t h y r o i d a c t i v i t y , which was followed by a depression of t h y r o i d function. March et a l . (1973) f e d a 220 IU vitamin E/kg d i e t to chicks and assessed the t h y r o i d a c t i v i t y by 131 measuring the rate of uptake and release of I by the t h y r o i d gland. They found that the a c t i v i t y of the t h y r o i d was s i g n i f i c a n t l y suppressed i n response to excess vitamin E. I t would be expected that a decrease i n thyr o i d a c t i v i t y would be accompanied by some decrease i n growth r a t e . This was not seen though at t h i s l e v e l of vitamin E supplementation, but feeding a t e n - f o l d greater amount of vitamin E (2,200 iu/kg) caused a decreased growth r a t e . 5» Tissue Storage of Vitamin E The major pathway of vitamin E absorption from the i n t e s t i n e p a r a l l e l s f a t absorption (Pomeranze and l i u c a r e l l o , 1953)• Following absorption, the tocopherol i s transported, v i a the lymphatics, i n the chylomicrons (Blomstrand and Forsgren, 1968). Gloor et a l . (1966) have shown that y -tocopherol was absorbed from the i n t e s t i n e almost as e f f i c i e n t l y as was a-tocopherol. Since y ^tocopherol i s the predominant tocopherol i n the North American d i e t , c a l c u l a t i o n s based on only a-tocopherol s i g n i f i c a n t l y underestimate vitamin E intakes ( B i e r i and Poukka Evarts, 1973). There have been numerous studies attempting to determine the quantitative r e l a t i o n s h i p of i n c r e a s i n g l y higher l e v e l s of vitamin E intake versus plasma and l i v e r tocopherol l e v e l s . Losowsky et a l . (1972) have examined the e f f i c i e n c y of absorption of d i e t a r y tocopherol i n both man and animals. Over a narrow d i e t a r y range of intake the percentage absorp- t i o n f a l l s o f f as the dose i s increased. Excretion measurements with r a t s 18 i n d i c a t e d a marked decrease i n tocopherol absorption e f f i c i e n c y as the dose was increased from the microgram to milligram range (Losowsky et a l . , 1972). B o l l i g e r and Bolliger-Quaife (1956) i n experiments with r a t s have reported that the r e l a t i o n s h i p between the dose of tocopherol and i t s storage i n l i v e r i s l i n e a r when both are expressed as logarithms. They also suggested a l i n e a r r e l a t i o n s h i p between plasma tocopherol l e v e l and the l o g of the dose of vitamin E intake. In experiments with the chick, Wiss et a l . (1962) reported s i m i l a r r e s u l t s as the former study. B i e r i (1972) a l s o reported a l i n e a r r e l a t i o n s h i p between plasma tocopherol concentration and the l o g of the di e t a r y vitamin E i n experiments with r a t s . Gray (i960) disagreed that such a r e l a t i o n s h i p existed. Inaa 28 week study of high vitamin E intake i n r a t s , A l f i n - S l a t e r et a l . (1972) found that the plasma tocopherol l e v e l s r e f l e c t e d the d i e t a r y vitamin E intake. The plasma l e v e l s were not proportional to the dose administered. Also tocopherol l e v e l s i n females were almost two-times greater than i n male r a t s . Awad et a l . (1975) i n a 4 week study with r a b b i t s reported that supplementation with 5i000 IU vitamin E/kg d i e t increased the plasma and l i v e r tocopherol l e v e l s , but only the l a t t e r was s i g n i f i c a n t l y increased. These r e s u l t s suggest that the r e l a t i o n s h i p between the d i e t a r y l e v e l of vitamin E and tiss u e storage may be va r i a b l e , depending on the animal species, growth rate of the animal, length of the t e s t period, dosage l e v e l and the t i s s u e being examined. 6. Tissue Storage of Vitamin A I t has been recognized f o r many years that there i s a n u t r i t i o n a l r e l a t i o n s h i p between vitamin E and vitamin A. Many i n v e s t i g a t o r s have 19 found a "sparing" e f f e c t of vitamin E on vitamin A. Moore et a l . (1940) o r i g i n a l l y reported that vitamin E increased l i v e r storage of vitamin A i n r a t s over a period of 8 to 12 months. Hichman et a l . (1944) confirmed t h i s f i n d i n g . Other e a r l y workers though found no "sparing" e f f e c t of vitamin E i n experiments l i m i t e d to 4 weeks (Lemley, 1947; Herbert and Morgan, 1953) ' The contradictory evidence demonstrated that there was not a simple r e l a t i o n s h i p between the two vitamins, but instead a complex e f f e c t , dependent on d i e t , the dosage regimen of the two vitamins and the length of the experiment. In spite of the contradictory r e s u l t s reported i n the early studies, more recent i n v e s t i g a t i o n s i n d i c a t e d that d i e t a r y vitamin E increases tissue l e v e l s of vitamin A (Cawthorne et a l . , 1968; Jenkins and M i t c h e l l , 1975i Prodouz and Navari, 19755 Roels et a l . , 1964). Prodouz and Navari (1975) chose dietary l e v e l s of vitamin E ranging from 0 .00 iu/week to 3«5 iu/week and examined the e f f e c t of vitamin E on vitamin A storage i n r a t s . They found a much l a r g e r increase i n l i v e r vitamin A per IU vitamin E fed than per IU of vitamin A i n the d i e t . In examining the depletion of l i v e r stores of vitamin A Cawthorne and colleagues (1968) reported that supplementary vitamin E s i g n i f i c a n t l y decreased the rate of depletion of vitamin A reserves i n the r a t , thus confirming the r e s u l t s of Moore et a l . (1940). This vitamin E e f f e c t was shown at remarkable low intakes; even 1 mg was s u f f i c i e n t to produce a t h r e e - f o l d d i f f e r e n c e on vitamin A storage within 6 weeks i n r a t s . This e f f e c t was demonstrable though only when the i n i t i a l reserves were high, about 3 0 , 0 0 0 IU vitamin A per l i v e r . The same e f f e c t was not observed when the i n i t i a l l i v e r reserves of vitamin A were only 3 ,000 IU, which suggests a r o l e f o r vitamin E i n a l t e r i n g the capacity of the l i v e r to bind vitamin A. 20 The e f f e c t of supplementation with high l e v e l s of vitamin E on ti s s u e vitamin A storage has been examined by two groups. Roels et a l . (1964) reported that a t e n - f o l d increase i n dietary vitamin E intake (50 to 500 iu/kg d i e t ) r e s u l t e d i n a 11 percent increase i n l i v e r vitamin A storage. In examining the e f f e c t of supplementation with 600 or 6,000 IU vitamin E:/kg d i e t Jenkins and M i t c h e l l (19750 confirmed that vitamin E increased the storage of vitamin A i n the l i v e r . They also found that the plasma vitamin A was s i g n i f i c a n t l y increased when high l e v e l s of vitamin E were fed. Green and Bunyan (1969) suggested that vitamin E may "spare" vitamin A by protection from oxidation i n the gut, by increasing vitamin A absorption, by increasing vitamin A effeciency, and/or by increasing the storage of vitamin A. They noted that the antioxidant properties of vitamin E may or may not be s i g n i f i c a n t i n the mechanism. Roel et a l . (1964) and Jenkins and M i t c h e l l ' s (!1975) f i n d i n g s cannot be explained by the antioxidant e f f e c t of vitamin E. Even the supplementary ~̂~) J 6 ' l e v e l i n the experiment of Roel et a l . (1964), 50 IU vitamin E/kg d i e t X was more than adequate f o r the r a t s needs, yet excessively l a r g e r doses of vitamin E accentuated the "sparing"effect of vitamin E on vitamin A. This supports the proposal of Tappel (1973). DiLuzio (1973) and Green (1972b) that vitamin E may have a more s p e c i f i c i n vivo biochemical r o l e i n a d d i t i o n to i t s suggested i n vivo and/or i n v i t r o antioxidant properties. 7« L i v e r L i p i d Levels According to A l f i n - S l a t e r et a l . (1972) l i v e r c h o l e s t e r o l and t o t a l l i p i d l e v e l s increased progressively as the dietary vitamin E intake was 21 increased. This e f f e c t was observed i n r a t s fed high l e v e l s of vitamin E f o r a 28-week period. Other workers have examined the simultaneous e f f e c t s of various l e v e l s of vitamin E and vitamin A ( H a r r i l l et a l . , 19655 Jenkins and M i t c h e l l , 19755 Prodouz and Navari, 1975). or vitamin E and arginine or methionine ( H a r r i l l and G i f f o r d , 1966) on l e v e l s of l i v e r c h o l e s t e r o l and t o t a l l i p i d s . Contrary to the r e s u l t s of A l f i n - S l a t e r et a l . (1972), H a r r i l l and G i f f o r d (1966) found that increasing the dietary l e v e l of vitamin E decreased the l e v e l of c h o l e s t e r o l and t o t a l l i p i d s i n r a t l i v e r . These f i n d i n g s are not cons i s t e n t l y seen though when examining the simultaneous e f f e c t s of vitamin E and A on t i s s u e l i p i d l e v e l s . Prodouz and Navari (1975) and H a r r i l l et a l . (1965) reported that increasing d i e t a r y vitamin E s i g n i f i c a n t l y decreased l i v e r t o t a l l i p i d s and increased l i v e r c h o l e s t e r o l . However, Jenkins and M i t c h e l l (1975) reported that increasing d i e t a r y vitamin E s i g n f i c a n t l y increased t o t a l l i p i d s and decreased c h o l e s t e r o l i n r a t l i v e r . The reason f o r the discrepancy i n r e s u l t s i n t h i s area remains obscure. I t might well be that the r a t i o of vitamin E to vitamin A i s the decisive f a c t o r determining the e f f e c t of these vitamins on t i s s u e l i p i d l e v e l s i n these experiments. 8. Blood L i p i d Levels The r e l a t i o n s h i p between high d i e t a r y vitamin E and plasma l i p i d l e v e l i s not yet c<hear. Most i n v e s t i g a t i o n s i n t h i s area have examined the a b i l i t y of supplemental vitamin E to a l t e r plasma ch o l e s t e r o l l e v e l s . Some studies have reported a decrease i n serum c h o l e s t e r o l i n r a t s f e d vitamin E supplemented d i e t s (Chen e t ' a l . , 1972; H a r r i l l et a l . , I965; 21a Prodouz and Navari, 1975)- Chen et a l . (1972) showed that r a i s i n g the d i e t a r y vitamin E intake r e s u l t e d i n lower serum c h o l e s t e r o l l e v e l s , proportional to the amount supplemented. The regression curves of c h o l e s t e r o l l e v e l to vitamin E intake (up to 50 iu/kg d i e t ) were not l i n e a r though. However, several workers have reported that high dietary vitamin E intakes had no e f f e c t on serum c h o l e s t e r o l l e v e l s i n r a b b i t s (Awad and Gi l b r e a t h , 1975; Horn et a l . , 1962), chicks (Koyangi et a l . , 1966), and r a t s (Jenkins and M i t c h e l l , 1975). Awad and Gilbreath (1975) found that d i e t s formulated to contain 5»000 IU vitamin E/kg d i e t had no e f f e c t on serum ch o l e s t e r o l i n r a b b i t s . Jenkins and M i t c h e l l (1975) also f e d high l e v e l s of vitamin E (6,000 iu/kg d i e t ) to r a t s and observed that plasma cho l e s t e r o l l e v e l s were not s i g n i f i c a n t l y a f f e c t e d . Some i n v e s t i g a t o r s have reported that high doses of vitamin E a c t u a l l y caused hypercholesterolemia (Bruger, 194-5; Campbell, 1952). L i t t l e evidence of the e f f e c t of large doses of vitamin E on blood l i p i d l e v e l s can be gained though from e i t h e r of these two studies because of the unnatural experimental conditions employed. Both studies f e d a t h e r o s c l e r o t i c d i e t s to r a b b i t s and vitamin E was i n j e c t e d intramuscularly. The l e v e l of vitamin E supplementation, the length of treatment and the d i e t a r y ingredients vary widely i n the experiments reported above. Whether one or more of these conditions can account f o r the wide d i v e r s i t y of f i n d i n g s reported i n the l i t e r a t u r e i s not yet known. 22 CHAPTER I I I MATERIALS AND METHODS A. Animal Care Ninety female weanling Wistar r a t s , 45 - 55 g i n weight, were obtained from Biobreeding Laboratories, Ottawa, Ontario. Upon a r r i v a l they were randomly divided into s i x groups of f i f t e e n animals each. For the i n i t i a l two-week period they were housed i n p a i r s , a f t e r which they were housed s i n g l y i n screen-bottomed s t a i n l e s s s t e e l cages kept i n an air - c o n d i t i o n e d room maintained at 23-25°C. L i g h t i n g was regulated automatically to provide alternate 12-hour periods of l i g h t and darkness ( l i g h t on from 6:00 a.m. to 6:00 p.m.). Food and water were given ad l i b i t u m throughout the experimental period of sixteen months (December 1973 t o A p r i l 1975)• B. Experimental D i e t s Six experimental d i e t s were used: a tocopherol-free d i e t , and the same d i e t supplemented with e i t h e r 25, 250, 2,500, 10,000 or 25,000 IU vitamin E (dl-a-tocopherol acetate) per kg d i e t . These were based on a modified Draper's (1964) Standard Vitamin E-Free d i e t . The composition of the d i e t s and that of the mineral and vitamin mixes used are shown i n Table 2. Die t a r y ingredients were obtained from Texlab M i l l s , Madison, Wisconsin, U.S.A. C. Experimental Groups The s i x experimental groups were designated as shown below. Group A : Vitamin E-free d i e t - Basal Group B : Basal d i e t plus 25 IU vitamin E/kg Group C : Basal d i e t plus 250 IU vitamin E/kg Group D : Basal d i e t plus 2,500 IU vitamin E/kg TABLE 2 Composition of the Basal Diet Ingredient f0 D-Dextrose 64.9 Vitamin-free casein 20.0 Corn o i l , tocopherol stripped 10.0 Salt mix (no. 4l64)2 4.0 Vitamin mix 0.6 Choline chloride 0.5 Modified from Draper, H.H., et a l , (1964) J. Nutr. 84, 395-400. Trovided the following as g/kg diet: CaCO^, 6-54; CaHPO^ s 2H20, 14.2; NaCl, 4.3; K2HP0^, 3.09; K3(C6R"50;p -H20, 9-46; MgCO^, 1.64; FetCgH^) • 3H20, 0.64; MnS0^-H20, 0.055; ZnCO^, 0.018; CuSO^ • 5H 20, 0.007i KI, 0.0018. 'Provided the following amount per kg diet (in III): 25.000 vitamin A as retinyl palmitate; 2,000 ergocalciferol; (inmg): menadione, 1; biotin, 0.1; vitamin B^2, 0.1; calcium pantothenate, 10; f o l i c acid, 1; niacin, 25» pyridoxine HC1, 5*0; riboflavin, 5-0; thiamine HC1, 10. 2k Group E : Basal diet plus 10,000 IU vitamin E/kg Group F : Basal diet plus 25i000 IU vitamin E/kg D. Experimental Procedures The experiment was continued over a sixteen month period during which animals were randomly chosen from each group and the following protocol was carried out at predetermined times. The hematological indices were measured at 9,12 and 16 months of treatment. Blood samples were taken by t a i l cutting after anesthetizing the rats with anhydrous diethyl ether (Fisher Scientific) for the deter- mination of hemoglobin, hematocrit and erythrocyte hemolysis. Blood was directly drawn from the t a i l into a sodium oxalate coated Miale prothrombin pipet for estimation of prothrombin time. At 11 months, a 24-hour urine sample was collected for the deter- mination of urinary creatine and creatinine. The samples were stored i n plastic bottles without preservative at -20°G unt i l analysis. Four rats from each group were k i l l e d after 8 months and the others were k i l l e d at the end of 16 months of dietary treatment. The animals were f i r s t weighed and then l i g h t l y anesthetized with anhydrous diethyl ether. Blood was drawn from the inferior vena cava using a heparinized syringe. Plasma was obtained by centrifugation and placed into small plastic tubes and frozen at -20°G u n t i l further processing. The storage of the individual plasma aliquots permitted avoidance of repeated thawing and re-freezing. Plasma samples were analyzed for vitamin E, cholesterol and total l i p i d s . Plasma alkaline phosphatase activity and vitamin A (retinol) were also measured in rats treated for 16 months. Immediately after exsanguination, the l i v e r , spleen, heart, kidney and uterus were rapidly removed. They were trimmed for extraneous 25 t i s s u e s , washed i n cold p h y s i o l o g i c a l saline s o l u t i o n and then weighed. The l i v e r was frozen at -20°C f o r a n a l y s i s of vitamin A, vitamin E, t o t a l l i p i d s and c h o l e s t e r o l . The l e f t femur was removed and stripped of s o f t t i s s u e . I t was then frozen at -20°C f o r a n a l y s i s of hone ash, calcium and inorganic phosphate. E.. Biochemical Determinations 1. Hemoglobin and Hematocrit Hemoglobin was determined by the spectrophotometric method described by E i l e r s (1967). A 0.02 ml aliq u o t of blood was d i l u t e d with 5 ml of cyanmethemoglobin reagent (Hyland D i v i s i o n , Travenol Laboratories Inc., Casta Mesa, C a l i f . , U.S.A.) and then read at 5̂0 nm using the Beckman DU-2 spectrophotometer. Hemoglobin concentration was calculated by m u l t i p l y i n g o p t i c a l density (0D) at 5̂ 0 nm with a f a c t o r determined on the hemoglobin c a l i b r a t i o n curve as shown i n Figure 2. Hematocrit was read from a heparinized micro-hematocrit tube (Fisher S c i e n t i f i c ) a f t e r c e n t r i f u g a t i o n at 11,500 x G f o r 5 minutes ( E i l e r s , 1967). 2. Prothrombin Time The prothrombin time was determined by a micromethod of the standard one-stage prothrombin time method described by Miale and Winningham (1967). T h i s procedure used a s i l i c o n i z e d Miale Prothrombin Pipet (Dade, Miami, F l . ) f o r the c o l l e c t i o n of c a p i l l a r y blood. I t was mixed with a measured amount of sodium oxalate s o l u t i o n (100 mM) and centrifuged to obtain oxalated plasma. The t e s t was then performed by blowing the oxalated plasma i n t o a t e s t tube of thromboplastin-CaCl 2 mixture (Dade) at 37°C and the c l o t t i n g time was noted. FIGURE 2 Standard Curve For Hemoglobin g Hb/lOO ml 27 3• Erythrocyte Hemolysis The hemolysis procedure was that described by Draper and Gsallany (1966). It i s based on the degree of spontaneous hemolysis of erythrocytes in a buffered isotonic saline solution. Following incubation of the erythrocyte aliquots, the absorbance of the supernatants were read at 415 nm on the Beckman DU-2 spectrophotometer. Therper cent hemolysis was calculated from the formula shown below. Ab % hemolysis = x 100 c where = absorbance of buffer solution at 415 nm A = absorbance of Ĥ O solution at 415 nm c 2 4 . Urinary Creatine and Creatinine The urinary creatine and creatinine levels were determined by a method based on the Jaffe reaction as described by Henry et a l . (1974) . Creatinine was determined by quantitating the red pigment, alkaline creatinine picrate. The optical density was measured with a Beckman DU-2 spectrophotometer at 500 nm. The urinary creatinine level was calculated by the following formula. A x mg creatinine/ml urine = — s where A x = absorbance of unknown at 500 nm A x = ahsorbance of standard at 500 nm The urinary creatinine of the rats was then expressed as follows, mg creatinine/kg body weight/24 hours 28 The urinary creatine l e v e l was determined by the d i f f e r e n c e i n creatinine before and a f t e r the dehydration of creatine to c r e a t i n i n e . The urinary creatine l e v e l was then calculated as follows. t o t a l creatinine (mg reformed creatinine x plus mg creatine as creatinine/ml urine) ^s mg creatine as creatinine/ml urine = t o t a l creatinine - preformed creatinine The urinary creatine of the r a t s was then expressed as follows, mg creatine/kg body weight/24 hours 5. Plasma Vitamin A Plasma vitamin A l e v e l s were determined according to the method described by Neeld and Pearson (1963)> which i s a modification of the c l a s s i c Carr-Price technique. The blue chromophore produced by the i n t e r a c t i o n of t r i f l u o r o a c e t i c a c i d and vitamin A i n chloroform was measured at 620 nm on a Beckman DU-2 spectrophotometer and gave an i n d i c a t i o n of the amount of vitamin A present i n the plasma. Standard curves f o r mg vitamin A/lOO ml plasma were established using a l l trans r e t i n y l acetate (Hoffmann-La Roche Inc., Nutley, N.J., U.S.A.). The average slope of the curve at 620 nm was found to be 7«53• An i l l u s t r a t i o n of t h i s curve i s shown i n Figure 3- Plasma vitamin A l e v e l s were calculated from the standard curve ( F i g . 3) and expressed as u,g per 100 ml of plasma. 6. Plasma Vitamin E Plasma vitamin E l e v e l s were determined according to the method described by Fabianek et a l . (1968), which i s a modification of the c l a s s i c FIGURE 3 STANDARD CURVE FOR PLASMA VITAMIN A 0.5 Llg Retinol/tube (2.0 ml chloroform) 30 Emmerie-Engel technique. The a n a l y s i s i s based on a reduction of f e r r i c ion to the ferrous form by tocopherols, with the resultant formation of a pink complex of ferrous ions with 4,7-diphenyl-10,10-phenanthroline. The use of phosphoric a c i d prevents the photochemical reduction of f e r r i c chloride and also reduces interference of carotene to a minimum. The complex was measured with a Beckman DU-2 spectrophotometer at 536 nm. Standard curves f o r mg dl-a-tocopherol/lOO ml plasma were established using dl-a-tocopherol (Hoffmann-La Roche Inc., Nutley, N.J., U.S.A.). The average slope of the curve at 536 nm was found to be 2.54 ( F i g . 4). The plasma tocopherol concentration was then calculated from the standard curve ( F i g . 4) and expressed as mg tocopherol per 100 ml of plasma• 7. Plasma Cholesterol Plasma cholesterol was assayed by an enzymatic color procedure described by Roschlau et a l . (1974). A 0.02 ml a l i q u o t of plasma was mixed with 5 m l of c h o l e s t e r o l reagent mixture (1.7 M methanol; 0.57 M ammonium phosphate buffer, pH 7> 0.02 M acetylacetone5 0.1% hydroxypolyethoxydodecane; catalase > 670 u/ml; cholesterol*- ., esterase > 26 mU/ml). The contents of the t e s t tubes were mixed well using a Vortex mixer and 0.02 ml of cholesteroloxydase (4 u/ml) was added. The samples were incubated at 37°C f o r 60 minutes and the o p t i c a l density was read at 410 nm against a sample blank on a Beckman DU-2 spectrophotometer. Standard curves f o r mg cholesterol/lOO ml plasma were determined using pure ch o l e s t e r o l ( P r e c i s e t Cholesterol ). The average slope of the Boehringer Mannheim GmbH, Mannheim, W. Germany FIGURE 4 STANDARD CURVE FOR PLASMA VITAMIN E 0.40 0.80 1.20 1.60 2.00 mg dl-a-tocopherol/lOO ml ethanol 32 curve at 4 1 0 nm was found to be 8 1 8 . 1 ( i l l u s t r a t e d i n Figure 5)- The plasma cho l e s t e r o l l e v e l s were calculated as shown below, mg cholesterol/lOO ml plasma = 0 D ^ ^ Q ^ x 8 1 8 . 1 8. Plasma T o t a l L i p i d s Total l i p i d s i n plasma were measured by the method of Amenta ( 1 9 7 0 ) . L i p i d s were extracted from the plasma i n t o a chloroform-methanol sol u t i o n 1.5*1 ( v / v ) and n o n - l i p i d impurities and methanol were removed by a wash with an aqueous GaClg s o l u t i o n (0.5%)• An a l i q u o t of the l i p i d - c o n t a i n i n g chloroform phase was evaporated and the t o t a l l i p i d measured by reacting with an a c i d dichromate reagent (0.5%)' The amount of dichromate reduced was determined by the change i n absorption measured at 430 nm on a Beckman DU=2 spectrophotometer which was d i r e c t l y proportional to the l i p i d present. The standard f o r t o t a l l i p i d s was l e c i t h i n (0.1%) and pa l m i t i c a c i d (0.15%), d i s s o l v e d i n chloroform. To t a l l i p i d s i n plasma were then determined according to the formula shown below. A:-x mg t o t a l l i p i d s / l O O ml plasma = x Z A s where A = 0 D , , O A method blank — 0 D , , o „ sample x 430 nm 430 nm * A = 0 D , l O O reagent blank — 0 D , . . o A standard s 4 3 0 nm ^° (00 nm Z = concentration of the standard x d i l u t i o n f a c t o r 9• Plasma Alk a l i n e Phosphatase Plasma a l k a l i n e phosphatase was assayed by a procedure described by Henry et a l . (1974). A 0.1 ml a l i q u o t of plasma was mixed with 1 ml of 0.02 M phenol 33 FIGURE 5 STANDARD CURVE FOR PLASMA CHOLESTEROL O.50 4- 34 phosphate. The hydrolysis product, phenol, was condensed with 4-aminoanti- pyrine and then oxidized with a l k a l i n e f e r r i c y a n i d e to give a red complex which was measured at 5°0 nm on a Beckman DU-2 spectrophotometer. 0 One u n i t of a l k a l i n e phosphatase a c t i v i t y was defined as the amount of enzyme i n 100 ml of plasma which l i b e r a t e d 1 mg phenol i n 15 minutes at 37°C. The amount of a l k a l i n e phosphatase i n the plasma was then calculated as follows. A — A x c un i t s a l k a l i n e phosphatase/lOO ml plasma = x Z A s where A x = absorbance of unknown at 500 nm A c = absorbance of control at 500 nm A = absorbance of standard at 500 nm s Z. = concentration of the standard x d i l u t i o n f a c t o r 10. L i v e r L i p i d E x t r a c t i o n The concentrations of vitamin A, cholesterol and t o t a l l i p i d s i n Mvermofsrats were measured i n the chloroform-extract of l i v e r , prepared by a modification of the methods of Folch et a l . (1957) and Amenta (1970). The l i p i d e x t raction procedure was c a r r i e d out as follows. One h a l f g of l i v e r was minced and then homogenized i n 1 ml d i s t i l l e d water, f i r s t with a S o r v a l l micro-homogenizer attatched to a So r v a l l omni-mixer and then with a Potter-Elvehjem glass and t e f l o n plunger type of homogenizer. One h a l f m i l l i l i t e r of crude homogenate was extracted with 3 ml of chloroform-methanol 1.521 ( v / v ) i n a glass stoppered centrifuge tube by a g i t a t i n g vigorously f o r 3 minutes with a 3 5 Vortex mixer. The tubes were then centrifuged at 1 , 2 0 0 x G f o r 5 minutes. The upper chloroform phase was pipe t t e d o f f and retained. The supernatant phase was extracted with 3 ml of chloroform-methanol 1 . 5 * 1 ( v / v ) as before and recentrifuged. The l i q u i d phase was combined with the chloroform phase from the f i r s t e x t r a c t i o n . The mixture was then washed with 3 ml of aqueous CaClg s o l u t i o n ( 6 7 - 5 mM) by shaking vigorously f o r 3 minutes and then centrifuged at 1 , 2 0 0 x G. Aliquots of the l i p i d - c o n t a i n i n g chloroform phase were then ready f o r the vitamin A, ch o l e s t e r o l and t o t a l l i p i d analyses. 1 1. L i v e r Vitamin A The l e v e l of vitamin A i n l i v e r was determined according to the method of Neeld and Pearson ( 1 9 7 3 ) ' An aliq u o t of the l i p i d - c o n t a i n i n g chloroform phase was d i l u t e d 1 : 3 with chloroform, from which 0 . 2 ml was used f o r the vitamin A a n a l y s i s . The blue chromophore produced by the i n t e r a c t i o n of t r i f l u o r o a c e t i c a c i d and vitamin A i n chloroform extract was measured at 6 2 0 nm on a Beckman DU - 2 spectrophotometer. Standard curves f o r r e t i n o l equivalents per tube were established using a l l t r a n s r e t i n y l acetate (Hoffmann-La Roche Inc.). The average slope of the curve at 6 2 0 nm was found to be 7 ' 1 9 . An i l l u s t r a t i o n of t h i s c a l i b r a t i o n curve i s shown i n Figure 6 . In the preliminary laboratory work, known amounts of a l l trans r e t i n y l acetate were added to l i v e r before the l i p i d e x t r a c t i o n procedure. Analysis was c a r r i e d out according to the method discussed above and the per cent recovery was cal c u l a t e d . I t was found that recovery of 1 0 3 per cent was attained. The vitamin A concentration i n l i v e r was then calculated from the 36 standard curve ( F i g . 6 ) and expressed as fig per g of l i v e r . 1 2 . L i v e r Vitamin K The l e v e l of a-tocopherol i n l i v e r was determined according to the t h i n - l a y e r chromatography (TLC) method of B i e r i (1969). Two-dimensional a n a l y s i s was c a r r i e d out on precoated s i l i c a g e l G TLC p l a t e s (Redi/Plate, F i s h e r S c i e n t i f i c ) using benzene-elthanol ( 9 9 J l ) and hexane-ethanol ( 9 ! l ) mixtures as solvents. A f t e r the solvent had evaporated from the second dimension run the chromatograms were sprayed with a 0 . 0 0 2 5 % s o l u t i o n of sodium f l u o r e s c e i n i n methanol. This aided i n v i s u a l i z a t i o n and i d e n t i f i c a t i o n of the a-tocopherol spot. Following e l u t i o n , a c o l o r i m e t r i c determination of the a-tocopherol i n the ethanol eluate was c a r r i e d out. The method e s s e n t i a l l y consisted of extracting the ethanol eluate with xylene, followed by the ad d i t i o n of 0 . 4% 4 , 7-diphenyl - 1 0 , 1 0-phenanthroline, 0 . 6 % f e r r i c chloride and 8 5 % orthophosphoric a c i d . Standard curves f o r fig a-tocopherol per tube were established using dl - a-tocopherol (Hoffmann-La Roche Inc.).^ The average slope of the curve at 5 3 6 nm was found to be 1 0 . 2 ( i l l u s t r a t e d i n F i g . 7 ) ' In the preliminary laboratory work, known amounts of dl - a-tocopherol were added to l i v e r from vitamin E-free treated r a t s p r i o r to s a p o n i f i c a - t i o n . Analysis was c a r r i e d out and the per cent recovery was determined. I t was found that up to 8 4 . 6 per cent recovery could be obtained. Consequently a correction f a c t o r of 1.18 was employed to compensate f o r t h i s l o s s . The a-tocopherol concentration i n l i v e r was then c a l c u l a t e d from the standard curve ( F i g . 7 ) and expressed as fig per g of l i v e r and also as fig per whole l i v e r . FIGURE 6 STANDARD CURVE FOR LIVER VITAMIN A 0 . 9 4 |lg Retinol/tube (2.0 ml chloroform) FIGURE 7 STANDARD CURVE FOR LIVER VITAMIN E 3 9 1 3• L i v e r T o t a l L i p i d s Total l i p i d s i n l i v e r were determined by the method described by Amenta ( 1 9 7 0 ) . One h a l f m i l l i l i t e r of the l i p i d - c o n t a i n i n g chloroform phase was mixed with 1 . 5 ml chloroform, from which 0 . 4 ml was evaporated and the t o t a l l i p i d measured by r e a c t i n g with an a c i d dichromate reagent. The amount of dichromate reduced was determined by the change i n absorption when measured at 4 3 0 nm on a Beckman DU - 2 spectrophotometer and was d i r e c t l y proportional to the amount of l i p i d present. In the preliminary laboratory work, known amounts of l i p i d were added to l i v e r before the l i p i d e x t r a c t i o n procedure. Analysis was c a r r i e d out according to the method discussed above and the per cent recovery was calculated. I t was found that the recovery of 1 0 1 per cent was a t t a i n a b l e . T o t a l l i p i d s i n l i v e r were calculated by the same formula used f o r determining plasma t o t a l l i p i d s (See Section 8.) and were expressed as follows. mg t o t a l l i p i d / g l i v e r 14. L i v e r Cholesterol T o t a l c h o l e s t e r o l i n l i v e r was determined by the enzymatic color t e s t of Roschlau et a l . ( 1 9 7 4 ) . A 1 . 5 nil a l i q u o t of the l i p i d - c o n t a i n i n g chloroform phase was evaporated to dryness by f l u s h i n g with nitrogen i n a t e s t tube placed i n a heating block set at 5 0 ° C The c h o l e s t e r o l - residue was disso l v e d i n 5 ml of c h o l e s t e r o l reagent mixture (See Section 7> Plasma Cholesterol f o r a d e s c r i p t i o n of the reagent mixture) by sonic treatment at O^C. The contents of the t e s t tube were mixed well using a Vortex mixer and 0 . 0 2 ml of cholesteroloxydase ( 4 u/ml) was added. The samples were incubated at 3 7°C f o r 6 0 minutes and the o p t i c a l density was 40 read at 410 nm against a sample blank on a Beckman DU-2 spectrophotometer. Calibration curves of p,g cholesterol per tube were established using pure cholesterol (Preciset Cholesterol, Boehringer Mannheim GmbH) as standard. The calibration factor of the curve at 410 nm was found to be 368.1 (illustrated i n Figure 8). In the preliminary laboratory work, known amounts of cholesterol were added to l i v e r before the l i p i d extraction procedure. Analysis was carried out according to the method discussed above and per cent recovery was calculated. It was found that recovery of 103 per cent was attained. The cholesterol content i n the l i v e r was calculated from the standard curve (Fig. 8) and expressed as mg per g of l i v e r . IS' Femoral Ash The femoral bone was ashed by a modification of the procedure of gipken et a l . (1959)- The femur was f i r s t weighed, then defatted with a mixture of chloroform-methanol 2:1 (V:V) for 24 hours. The defatted bone was dried i n an isothermal oven at 105°C for 24 hours, weighed and then ashed i n a furnace at 650°C for 18 hours. The bone ash was weighed and then dissolved i n 4 ml of 3 N HC1. The per cent ash of the femur was calculated as shown below. 16. Femoral Calcium The calcium in bone was determined by atomic absorption spectrophotom- etry using a method described by W i l l i s (i960). The whole dried femoral bone dissolved in 3 N HC1 was diluted with lanthanum chloride solution per cent ash = weight of ash (g) x 100 weight of defatted dry bone (g) so that the calcium concentration lay between 5 and 20 mg/l. 41 FIGURE 8 STANDARD CURVE FOR LIVER CHOLESTEROL 42 Analysis was carried out using a Unicam SP90 Atomic Absorption Spectrophotom- eter. Calibration curves for mg per cent calcium were determined using AnalaR calcium carbonate (Canadina Lab. Supplies Ltd.) i n AnalaR hydrochloric acid. The calibration factor of the curve at 422.7 mu. was found to be 6.67- An i l l u s t r a t i o n of this calibration curve i s shown in Figure 9' The total calcium in the femur was calculated from the calibration curve (Fig. 9) and expressed as g calcium per femur. The per cent calcium in the dry and defatted femur was calculated as shown below. per cent weight of calcium i n femur (g) calcium = v—rz— T; 7—v~ x 100 „ weight of defatted dry femur (g) in femur ^ 17- Femoral Inorganic Phosphate Inorganic phosphate in the femur was determined by a modified method of Fiske and Subbarrow (1970). The inorganic phosphate analysis of the diluted bone-HCl solution involved photometric determination of the molybdenum blue formed by reduction of the molybdenum diphosphate, using aminonaphtholsulfonic acid as the reducing agent. Calibration curves for Llg phosphate per tube were determined using a phosphorus standard (5 |ig/ml). The calibration factor of the curve at 660 nm was found to be 35*8 (illustrated in Fig. 10). The total inorganic phosphate in the femur was calculated from the calibration curve (Fig. 10) and expressed as g inorganic phosphate per femur. The per cent phosphate in the dry defatted femur was calculated as shown below. FIGURE 9 STANDARD CURVE FOR CALCIUM mg % Calcium FIGURE 10 STANDARD CURVE FOR PHOSPHATE 0 .6 + fig Phosphate/tube (4.0 ml H»6) 45 per cent phosphate = i n femur weight of phosphate i n femur (g) weight of defatted dry femur (g) x 100 F. S t a t i s t i c a l Analysis of the Data The raw data were analyzed s t a t i s t i c a l l y by computer at the Computing Centre of the U n i v e r s i t y of B r i t i s h Columbia. The SPSS computer program package ( K i t a , 1976) was employed to draw up a program f o r the desired analyses. Experimental data were tested by applying one-way a n a l y s i s of variance. The homogeneity of variances was tested by Cochrans C t e s t . A l o g transformation was used f o r data with heterogeneous variance. S t a t i s t i c a l comparisons were made using regression analysis and Duncan's new multiple-range t e s t f o r data containing equal number of samples among groups or by Scheffe's range t e s t f o r data containing unequal number of samples on a p r o b a b i l i t y l e v e l of at l e a s t 95 per cent f o r a l l measurements. 46 CHAPTER IV RESULTS A. Body and Organ Weights Results of the e f f e c t of d i f f e r e n t l e v e l s of dietary vitamin E on growth i n r a t s treated f o r 8 and 16 months are presented i n Tables 3 and 4 r e s p e c t i v e l y . As can be seen i n Table 3 , high l e v e l s of vitamin E had a s i g n i f i c a n t depressing e f f e c t (P< 0 . 0 5 ) on growth of r a t s treated f o r 8 months. Vitamin E d e f i c i e n c y r e s u l t e d i n a greater growth depression than excess d i e t a r y vitamin E supplementation. The r e s u l t s i n Table 4 a l s o show that growth rate was s i g n i f i c a n t l y reduced i n r a t s treated with high l e v e l s of dietary vitamin E f o r 16 months. The organ weights (expressed i n mg/lOO g body weight) of r a t s treated with d i f f e r e n t l e v e l s of vitamin E f o r 8 and 16 months are a l s o shown i n Tables 3 and 4 . Treatment f o r 8 months at a l l l e v e l s of vitamin E supplementation had no s i g n i f i c a n t e f f e c t on l i v e r , uterus, spleen or kidney weight. However, increasing the dietary l e v e l of vitamin E was found to s i g n i f i c a n t l y increase (P<-0.05) the r e l a t i v e heart weights i n r a t s treated f o r 8 months. The l i n e a r r e l a t i o n s h i p between r e l a t i v e heart weights (Y) and l o g dietary vitamin E (x) was X= 232.316 + 11.347X, the c o r r e l a t i o n coeffecient (R) being 0.465 (P< 0 . 0 5 ) - Results f o r the r e l a t i v e organ weights of the vitamin E d e f i c i e n t r a t s show that a l l organs, except uterus, were s i g n i f i c a n t l y l a r g e r ( P < 0 . 0 l ) than those r e c e i v i n g vitamin E treatment. Table 4 shows that treatment f o r 16 months at a l l l e v e l s of vitamin E supplementation had no s i g n i f i c a n t e f f e c t on the r e l a t i v e weights of l i v e r , uterus or kidney. However, a s i g n i f i c a n t increase 47 TABLE 3 Body and Organ Weights of Rats on D i f f e r e n t 1 Levels of Dietary Vitamin E For 8 Months Dietary vitamin E Body weight L i v e r Uterus Heart Spleen Kidney iu/kg d i e t g mg/lOOg ; body weight 0 I66a 5,215a 183 416a 313a l,159a (13) (292) (27) (24) (26) (32) 25 366M 2,790b 189 248b2 I60b 536b (3D (123) (20) (16) (4) (42) 250 332cd 2,470b 204 269b2 I6lb 560b (12) (70) (25) (12) (8) (30) 2,500 356M 2,469b 198 2 5 l b 2 I64b 496b (16) (204) (13) (10) (5) (32) 10,000 3l3cd 2,540b 176 282b2 159b 576b (8) (102) (19) (13) (14) (29) 25,000 301c 2,451b 197 289b2 I67b 547b (16) (157) (16) (10) (6) (19) Values are means of four r a t s with t h e i r SEM given i n parentheses. Values within each column not sharing a common superscript l e t t e r are s i g n i f i c a n t l y d i f f e r e n t (P<0.05) using Duncan's new multiple-range t e s t . 2 L i n e a r response s i g n i f i c a n t (P<0.05) against dietary vitamin E using regression a n a l y s i s . The f u n c t i o n a l r e l a t i o n s h i p between r e l a t i v e heart weights (Y) and l o g dietary vitamin E (X) was Y= 232.316 + 11.347X, the c o r r e l a t i o n c o e f f i c i e n t (R) being 0.465 (P<0.05). 48 TABLE 4 Body and Organ Weights of Rats on D i f f e r e n t Levels of Dietary Vitamin E For 16 Months Dietary Number Body L i v e r Uterus Heart Spleen-' Kidney vitamin E r a t s weight iu/kg d i e t g mg/lOOg body weight 0 25 4 408 a 1,957 165 268 167 5 9 9 a b (12) (109) (25) (8) (14) (25) 250 9 4 5 7 a 2,702 217 229 168 5 0 1 a (20) (166) (20) (9) (6) (20) 2,500 6 4 l 4 a 3,088 214 263 184 5 8 6 a b (17) (175) (14) (7) (11) (41) 10,000 7 3 9 8 a b 3,065 202 268 218 5 8 3 a b (21) (-1'07) (19) (6) (14) ,(21) 25,000 5 3 5 8 b 2,873 ' 248 294 240 6 2 7 b (10) (135) (22) (6) (34) (21) xValues are means with t h e i r SEM given i n parentheses. Values within each column not sharing a common superscript l e t t e r are s i g n i f - i c a n t l y d i f f e r e n t (P<0 . 0 5 ) using Scheffe's range t e s t . Quadratic response s i g n i f i c a n t (POO.Ol) against d i e t a r y vitamin E. The fu n c t i o n a l r e l a t i o n s h i p between r e l a t i v e heart weight (Y) and l o g dietary vitamin E (x) was Y = 355-603-85.825 X + 16.219 X 2, the multiple c o r r e l a t i o n c o e f f i c i e n t (R) being 0.64-9 (P<0.0l). Linear response s i g n i f i c a n t (P<0.008) against dietary vitamin E using regression a n a l y s i s . The f u n c t i o n a l r e l a t i o n s h i p between r e l a t i v e spleen weight (Y) and l o g d i e t a r y vitamin E (x) was Y= 2.127 + 0.051 X, where R=0-580 (P< 0.008). 4 9 (P<O.Ol) i n relative heart weight was shown in rats fed high levels of vitamin E for 1 6 months. The functional relationship between relative heart weights (Y) and log dietary vitamin E (x) was Y = 3 5 5 - 6 0 3 - 8 5 . 8 2 5 X + 16.219 X2, the multiple correlation coefficient (R) being 0.649 (P<0.0l). The relative spleen weights were also significantly increased (P< 0.008) in rats fed high levels of vitamin E for 16 months. The linear relationship between relative spleen weight (Y) and log diet- ary vitamin E (X) was Y = 2 . 1 2 7 + 0 . 0 5 1 X, where R = 0-580 (P< 0.008). B. Hematological Parameters The influence of treatment with different levels of dietary vitamin E for 9 » 12 and 16 months on hemoglobin and hematocrit values, erythrocyte hemolysis and prothrombin time are presented i n Tables 5 » 6 , 7 and 8 respectively. There was no significant difference in hemoglobin levels (Table 5 ) when the rats were fed different dietary levels of vitamin E for 9 , 12 and 1 6 months. The hematocrit values of rats treated with different levels of vitamin E are shown in Table 6 . There were no significant differences in the hematocrit values of rats treated for 9 or 12 months. However, the hematocrit values were significantly increased (P<0.04) by treatment with excess vitamin E for 1 6 months. The functional relationship between hematocrit value (Y) and log dietary vitamin E (x) was Y = 41.018 + 1.242X, where R= 0.482 (P<0.04). Results of the effect of different levels of dietary vitamin E on the spontaneous hemolysis of the erythrocytes i n a buffered isotonic saline solution in rats treated for 9, 12 and 1 6 months are presented in TABLE 5 Hemoglobin Values 1 of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E Dietary Feeding period (months) vitamin E 9 1 2 1 6 iu/kg d i e t (4) (3) (4) 0 1 5 . 9 ± 0.4 2 5 1 5 . 3 ± 0 . 1 1 5 . 2 ± 0 . 3 14.8± 0 . 5 2 5 0 1 5 . 9 ± 0 . 1 15.5± 0 . 7 1 5 . 5 ± 0.4 2 , 5 0 0 15.6+ 0 . 2 1 5 . 0 ± 0.4 14.8± 0 . 3 1 0 , 0 0 0 1 5 . 6 ± 0 . 1 14.5± 1 . 2 1 5 . 1 ± 0 . 5 2 5 , 0 0 0 14.6± 0 . 3 1 5 . 7 + 0.4 12.4± 2 . 2 Each value represents mean i SFJM f o r the number of r a t s given i n parentheses above each column. 51 TABLE 6 1 Hematocrit Values of Rats Fed Different Levels of Dietary Vitamin E Dietary Feeding I period (months) vitamin E 9 12 1 6 2 iu/kg diet Hematocrit (%) (4) (3) (4) 0 45.6± 0 .9 25 45.5± 0 . 5 44 .7± 1.2 42.61 0 . 8 250 4 5 . 1 ± 0 . 9 44.3± 0.6 45.41 0 . 7 2,500 45.2± 0 . ? 42.9± 1.1 42.6+ 0 . 8 10,000 44.2± 0 . 6 43.4± 3-1 46.61 1.0 25,000 44 .1± 1.1 46.21 0.7 47.2+ 2 . 0 Each value represents mean 1 SEM for the number of rats given in parentheses above each column. Linear response significant (P<0.04) against dietary vitamin E using regression analysis. The functional relationship between hematocrit value (Y) and log dietary vitamin E was Y= 41.018 + 1.242X, where R= 0.482 (P<0.04). 52 TABLE 7 Erythrocyte Hemolysis of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E Dietary Feeding I period (months) vitamin E 9 12 16 iu/kg d i e t Hemolysis (%) (4) (3) (4) 0 86.8± 2 . 4 a 25 2.4± 0 . 5 b 2.2± 0 . 3 3-7± 0 . 6 a 250 1 .6± 0 . 2 b 2.3± 0 . 4 2.6± 0 . 2 a b 2,500 1.8± 0 . 4 b 2.7± 0 . 7 3.4± 0 . 6 a b 10,000 2.0± 0 . 1 b 2.0± 0 .1 2 . 1 ± 0 . 4 b 25,000 1 . 7 ± 0 . 2 b 1.4± 0 . 3 3-7± 0 . 3 a Each value i s the mean ± SEM f o r the number of r a t s given i n parentheses above each column. Values within each column not sharing a common superscript l e t t e r are s i g n i f i c a n t l y d i f f e r e n t (P< 0 . 0 5 ) using Duncan's new multiple-range t e s t . TABLE 8 Prothrombin Times of Rats Fed Different Levels of Dietary Vitamin E Dietary Feeding period (months) vitamin E 9 1 2 2 16" iu/kg diet Prothrombin Time (sec) (4) ( 3 ) (4) 0 1 3 . 1 ± 0 . 2 2 5 14.9± 0 . 8 14.6± 0 . 1 13.0± 0.4 2 5 0 12.9± 0.4 1 5 . 6 ± 0 . 5 14.5± 0 . 2 2 . 5 0 0 1 3 . 8 ± 0 . 5 14.3± 0 . 7 1 3 .6± 0 . 6 1 0 , 0 0 0 1 3 . 9 ± 0 . 5 1 1 . 3 ± 0 . 3 1 2 . 8 ± 0 . 5 2 5 , 0 0 0 14 .2+ 0 . 6 1 1 . 3 ± 0.4 12.7± 0 . 3 1 Each value i s the mean ± SEM for the number of rats give in parentheses above each column. Quadratic response significant (P<0.000l) against dietary vitamin E. The functional relationship between prothrombin time (Y) and log dietary vitamin E (x) was Y= 10.409 + 4 . 4 3 9 - 0 . 9 9 1 X , where R= 0 . 8 9 4 (P<0.000l). Quadratic response significant (P<0.02) against dietary vitamin E using regression analysis. The functional relationship between prothrombin time (Yl and log dietary vitamin E (X) was Y = 9.819 + 3.208X- 0 . 5 9 7 X , where R= 0 . 6 0 9 (P<0.02). 54 Table 7» Vitamin E supplementation d i d not s i g n i f i c a n t l y a f f e c t the s t a b i l i t y of the erythrocyte at any period of treatment. Vitamin E de f i c i e n c y f o r 9 months s i g n i f i c a n t l y increased the f r a g i l i t y of the erythrocyte membrane. The prothrombin time values of r a t s treated with d i f f e r e n t l e v e l s of vitamin E are shown i n Table 8 . Treatment f o r 9 months at a l l l e v e l s of vitamin E supplementation had no s i g n i f i c a n t e f f e c t on prothrombin time. In r a t s treated with high d i e t a r y l e v e l s of vitamin E f o r 12 and 16 months the prothrombin time was s i g n i f i c a n t l y shorter. In r a t s treated f o r 12 months the f u n c t i o n a l r e l a t i o n s h i p between prothrombin time (Y) and l o g dietary vitamin E (x) was Y= 10.409 + 4 . 4 3 9 X - 0-991 X 2, where R= 0.894 (P<0.0001). In r a t s treated f o r 16 months the f u n c t i o n a l r e l a t i o n s h i p between prothrombin time (Y) and l o g d i e t a r y vitamin E (x) was Y = 9.819 + 3-208X--0 . 5 9 7 X 2 , where R= 0 .609 (P<0.02). C. Femoral Parameters The influence of excess vitamin E administration f o r 8 and 16 months on ash content and calcium and phosphate concentration of bone are shown i n Tables 9 and 10. Included i n Table 10 i s the plasma a l k a l i n e phosphatase a c t i v i t y which was measured at 16 months as an a d d i t i o n a l parameter of bone c a l c i f i c a t i o n . Treatment with d i f f e r e n t l e v e l s of vitamin E f o r 8 months d i d not s i g n i f i c a n t l y a f f e c t the ash content of the femur.(Table 9). However, a f t e r 16 months of high d i e t a r y vitamin E supplementation (Table 10) the ash content of bone decreased s i g n i f i c a n t l y (P<0 . 0 0 0 5 ) . The f u n c t i o n a l r e l a t i o n s h i p between the percentage ash content of bones (Y) and l o g di e t a r y vitamin E (x) was Y= 68.970 - i.20?X, where R = 0.703 (P< 0 . 0 0 0 5 ) . 5 5 TABLE 9 Femoral Parameters of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E f o r 8 Months Dietary Feeding period : 8 months vitamin E Ash Calcium Phosphate iu/kg d i e t % % % 0 6 5 . 3 ± 0 . 7 2 3-41 0 . 3 A B 1 2 . 6 1 0.4 a 2 5 6 5 . 5 ± 0 . 2 ah 2 2 . 9 1 0 . 3 1 2 . 0 1 0 . 3 A B 2 5 0 6 6 . 2 + 0 . 3 2 3 . 6 1 0 . 3 A B 11.81 0 . 1 A B 2 , 5 0 0 6 5 . 7 1 0 . 7 2 2.81 0 . 5 A 11.31 0 . 2 B 1 0 , 0 0 0 6 6 . 0 1 0 . 9 2 3 . 9 1 0.4 b 1 1 . 9 1 0 . 2 A B 2 5 , 0 0 0 6 6.31 0 . 5 ah 2 3 . 0 1 0 . 2 1 1 . 5 1 o.i b 'Each value i s the mean 1 SEM f o r 4 r a t s . Values within each column not sharing a common superscript l e t t e r are s i g n i f i c a n t l y d i f f e r e n t ( P < 0 . 0 5 ) using Duncan's new multiple-range t e s t . TABLE 1 0 Femoral Parameters of Rats Fed Different Levels of Dietary Vitamin E For 1 6 Months Dietary Feeding period : 1 6 months 2 vitamin E Ash Calcium Phosphate Plasma alkaline phosphatase3 »4 iu/kg diet % % 0 25 67.41 0.5 23-1± 250 65.9± 0.4 21.9+ 2,500 65.0± 0.6 23.6± 10,000 64.21 1.1 22.5± 25,000 63.6± 1.0 21.0± % units/100 ml 0 . 5 11.9± o . i a b 17-9± 1.1 0 . 8 1 1 . 6 ± 0 . 6 A B 14.11 1 . 8 1 . 1 1 2 . 4 ± 0 . 6 B 15.71 0 . 1 0 . 5 10.9± 0 . 3 A 2 2 . 1 1 4 . 6 0 - 3 1 1 . 4 ± 0 . 3 A B 2 4 . 6 1 4.3 """Each value i s the mean 1 SEM for four rats. Values within each column not sharing a common superscript letter are significantly different (P<0 . 0 5 ) using Duncan's new multiple-range test. ^Linear response significant (P<0 . 0 0 0 5 ) against dietary vitamin E. The functional relationship between the percentage ash content of bones (Y) and log dietary vitamin E (x) was Y= 68.970 - 1.207X, where R= 0 . 7 0 3 ( P < 0 . 0 0 0 5 ) . Quadratic response significant (P<0.04) against dietary vitamin E using regression analysis. The functional relationship between plasma alkaline phosphatase^activity (Y) and log dietary vitamin E (x) was Y = 13-789 + 0.468X , where R= 0 . 4 6 3 (P<0.04). 4 One unit of alkaline phosphatase activity was defined as the amount of enzyme in 100 ml of plasma which liberated 1 mg phenol in 1 5 minutes at 3 7 C. 5 7 Femoral calcium content was not significantly affected by excess vitamin E supplementation for either 8 or 16 months. There were some slight, but significant differences (P<0 . 0 5 ) in femoral phosphate concentration in rats treated with different levels of vitamin E for 8 and 16 months. Regression analysis though was unable to show any significant relationship between the phosphate concentration and the dietary level of vitamin E supplemented. As can be seen in Table 10, excess vitamin E supplementation, from 2 5 0 to 25,000 Ill/kg diet increased plasma alkaline phosphatase activity after 16 months treatment. The functional relationship between plasma alkaline phosphatase activity (Y) and log dietary vitamin E (x) was Y= 13 - 7 8 9 + 0.469 X 2, where R = 0.463 (P<0.04). D. Urinary Creatine and Creatinine Data from the analysis of urinary creatine and creatinine of rats treated with different levels of vitamin E for 11 months are presented in Table 11. Vitamin E supplementation at a l l levels did not influence t urinary levels of either creatine or creatinine. Vitamin E deficiency significantly increased (P<0.0l) the urinary creatine excretion, while the urinary creatinine excretion decreased significantly (P<0.0l). E. Fat Soluble Vitamins 1. b Liver and Plasma Vitamin E The influence of high levels of dietary vitamin E on l i v e r storage of a-tocopherol after 8 and 16 months of treatment i s shown in Table 12. The results are reported as both, the a-tocopherol concentration of the l i v e r (p.g/g l i v e r ) and the total a-tocopherol content of the l i v e r (|lg/whole l i v e r ). 5 8 TABLE 11 Urinary creatine and creatinine of rats on different levels of dietary vitamin E for 11 months1 Dietary vitamin E Creatine Creatinine Creatine/Creatinine iu/kg diet mg/kg/24 hr mg/kg/24 hr ratio 0 5 4 . 0 7 ± 1 9 . 9 0 A 14 . 5 0 1 2 . 3 1 A 5.041 2 . 8 6 A 2 5 3-141 0 . 6 2 B 2 7 - 5 0 1 1 . 9 5 B o . l l l o . 0 3 B 2 5 0 1.441 0 . 2 0 B 2 7 - 1 2 1 l - 3 2 B 0 . 0 5 1 0 . 0 1 B 2 , 5 0 0 i.92± 0 . 3 9 B 2 7 - 8 3 1 1.28b 0 . 0 7 1 0 . 0 2 B 10,000 3 . 2 0 ± 0.46 b 2 5 . 7 2 ± 0 . 6 0 B 0 . 1 2 1 0 . 0 2 B 2 5 , 0 0 0 1.941 0 . 3 8 B 27-ll± 1 . 4 9 B 0 . 0 7 1 0 . 0 2 B 'Each value i s the mean 1 SEM for 4 rats. Values within each column not sharing a common superscript letter are significantly different (P<0.0l) using Duncan's new multiple-range test. 59 As can be seen i n Table 12, increasing the l e v e l of d i e t a r y vitamin E up to 10 , 0 0 0 iu/kg d i e t f o r 8 and 1 6 months s i g n i f i c a n t l y increased (P< 0.000l) the vitamin E concentration of the l i v e r . A d d i t i o n a l vitamin E supplementation, above 10 , 0 0 0 Ill/kg d i e t , d i d not s i g n i f i c a n t l y increase the l i v e r vitamin E concentration any f u r t h e r . Analysis of the data revealed a l i n e a r r e l a t i o n s h i p between the d i e t a r y l e v e l s of vitamin E and the vitamin E concentration i n l i v e r when both were expressed as logarithms ( F i g . 1 1 ) . The logarithmic r e l a t i o n s h i p between l i v e r vitamin E and dietary vitamin E was 9 4 % l i n e a r f o r r a t s treated f o r 8 months and 98% l i n e a r f o r r a t s treated f o r 1 6 months. The f u n c t i o n a l r e l a t i o n s h i p between l o g l i v e r vitamin E (Y) and l o g di e t a r y vitamin E (x) i n r a t s treated f o r 8 months was Y= - 0 . 1 0 6 + 1 . 2 5 2 X - 0.10?X2, where R =0.980 (P<0.0001); and i n r a t s treated f o r 1 6 months was Y = 0 - 7 6 2 * 0 . 6 5 2 X , where R= 0-988 (P< 0.000l). Shown i n Figure 12 are the concentrations of vitamin E i n plasma of r a t s f e d d i f f e r e n t d i etary l e v e l s of vitamin E f o r 8 and 1 6 months. In r a t s treated f o r 8 months plasma l e v e l s of vitamin E were s i g n i f i c a n t l y increased ( P < 0 . 0 0 5 ) with increasing l e v e l s of dietary vitamin E intake. The f u n c t i o n a l r e l a t i o n s h i p between l o g plasma vitamin E (Y) and l o g dietary vitamin E (x) i n r a t s treated f o r 8 months was Y = — 1 . 4 1 9 * 0.949X- 0.132X2, where R= 0.84 ( P < 0 . 0 0 5 ) . The plasma vitamin E l e v e l s were over 2 - f o l d higher i n r a t s f e d various l e v e l s of vitamin E f o r 1 6 months than those f e d f o r 8 months. The f u n c t i o n a l r e l a t i o n s h i p bet- ween l o g plasma vitamin E (Y) and l o g dietary vitamin E (x) i n r a t s treated f o r 1 6 months was Y= - 0 . 6 2 2 + 0-595X - 0 . 0 6 4 X 2 , where R = 0 - 9 6 (P<0.0000l). 2 . L i v e r and Plasma Vitamin A The e f f e c t of d i f f e r e n t l e v e l s of dietary vitamin E on the TABLE 1 2 The Concentrations of ot-Tocopherol i n Li v e r s of Rats Fed Di f f e r e n t Levels of Dietary Vitamin E For 8 and 1 6 Months Dietary L i v e r a-tocopherol vitamin E 8 months 16 months iu/kg d i e t / T 2 fj.g/g l x v e r fj.g/whole l i v e r (ig/g l i v e r |lg/whole l i v e r 0 0.5± 0.1 4.61 1 . 1 2 5 27.1± 1.8 2 7 7 . 5 1 33.5 43.71 1-4 577-61 29.7 2 5 0 204.9+ 15-5 1,691.91 182.0 214.2+ 1 7 . 1 2,535-71 296.4 2 , 5 0 0 7 0 3 . o ± 8 8 . 7 6,104.41 882.3 1,172.61181.7 14,323.8+2,150.2 10,000 1 , 9 5 2.3± 2 0 3.1 15,403 .511,456.7 2'?822.61283-1 30,994.715,826.5 25,000 2,214.7±587.8 1 7 , 1 3 7 . 3 1 6 , 5 7 2.0 3,411.11513.1 37,236.616,268.2 y Each value represents mean 1 SEM f o r four r a t s . Quadratic response s i g n i f i c a n t ( P < 0 . 0 0 0 1 ) against dietary vitamin E i n supplemented r a t s . Linear response s i g n i f i c a n t ( P < 0 . 0 0 0 1 ) against dietary vitamin E. FIGURE 11 Plot of the logarithm of l i v e r a-tocopherol concentration versus the logarithm of d i e t a r y vitamin E, i n r a t s treated f o r 8 and 16 months.  FIGURE 12 Plasma a-tocopherol concentration of r a t s fed d i f f e r e n t d i e t a r y l e v e l s of vitamin E f o r 8 months (open bars) and 16 months (closed bars). Each point represents mean ± SEM of four r a t s . In r a t s treated f o r 8 months plasma tocopherol i n group A i s s i g n i f i c a n t l y lower than other groups (P< 0 . 0 1 ) using Duncan's multiple-range t e s t . In r a t s treated f o r 8 months, quadratic response i s s i g n i f i c a n t (P< 0 . 0 0 5 ) against dietary vitamin E. In r a t s treated f o r 16 months,, quadratic response i s s i g n i f i c a n t ( P< 0 . 0 0 0 l ) against dietary vitamin E. P l a s m a oc-Tocopherol ( mg /100 m l ) as 3 ca a 5' to 01 to en o 2° o o o b o o to b o o N b CO b b o O N FIGURE 1 3 L i v e r vitamin A concentration of r a t s fed d i f f e r e n t d i e t a r y l e v e l s of vitamin E f o r 8 months (open bars) and 16 months (closed bars). Each point represents mean ± SEM of four r a t s . In r a t s treated f o r 8 months group A i s s i g n i f i c a n t l y lower than a l l other groups (P<0.0l). In r a t s treated f o r 16 months group G i s s i g n i f i c a n t l y higher than groups D, E and F (P<0.05)« V i t a m i n E ( l U / k g D i e t ) FIGURE 14 Plasma vitamin A concentration of r a t s fed d i f f e r e n t d i e t a r y l e v e l s of vitamin E f o r 16 months. Each point represents mean ± SEM of four r a t s .  6 5 concentration of vitamin A in l i v e r i s shown in Figure 13• Supplementation with high levels of vitamin E for 8 and 16 months had no significant influence on l i v e r vitamin A storage. However, after 8 months, vitamin E deficiency was found to significantly decrease (P<0.01) the l i v e r vitamin A storage. The influence of high levels of vitamin E on plasma vitamin A levels was examined only after 16 months treatment (Fig. 14). Long-term treatment with excess vitamin E had no significant effect on plasma vitamin A levels. F. Lipids 1. Liver Total Lipids and Cholesterol" The influence of high levels of vitamin E on l i v e r concentrationso of total l i p i d s and cholesterol are presented in Figures 1 5 and 16 respectively. The l i v e r total l i p i d s were significantly increased (P<0.000l) after 8 months treatment with high levels of dietary vitamin E (Fig. 1 5 ) - The l i v e r total l i p i d s were increased from 4 . 5 % of the l i v e r weight i n rats fed 2 5 IU vitamin E/kg diet to 14.0% in rats fed 25,000 IU vitamin E/kg diet. The linear relationship between log l i v e r l i p i d s (Y) and log diet- ary vitamin E (X) was Y= 1.497 + 0.144X, where R = 0.86 (P<0.000l). With a longer experimental period - 1 6 months, i t was interesting to observe that there was no significant difference in l i v e r total l i p i d s at different levels of vitamin E supplementation. A l l groups treated for 16 months, except those fed 2 5 IU vitamin E/kg diet, had lower l i p i d levels than those groups treated for 8 months at comparable levels of vitamin E supplementation. FIGURE 15 T o t a l l i p i d s i n l i v e r of r a t s fed d i f f e r e n t d i e t a r y l e v e l s of vitamin E f o r 8 months (open bars) and 16 months (closed bars). Each point represents mean + SEM of four r a t s . Group A i s s i g n i f i c a n t l y lower than other groups (P< 0 . 0 1 ) using Duncan's new multiple-range t e s t i n r a t s fed f o r 8 months. In r a t s supplemented f o r 8 months, l i n e a r response s i g n i f i c a n t (P< 0 . 0 0 0 1 ) against dietary vitamin E. V i t a m i n E ( l U / k g Diet ) FIGURE 16 L i v e r cholesterol concentration of r a t s f e d d i f f e r e n t d i e t a r y l e v e l s of vitamin E f o r 8 months (open bars) and 16 months (closed bars). Each point represents mean ± SEM of four r a t s . Group G i s s i g n i f i c a n t l y higher than other groups (P< 0 . 0 5 ) except group F r a t s fed f o r 8 months.  6 8 As shown in Figure 1 6 , l i v e r cholesterol was unaffected by high levels of vitamin E after 8 and 1 6 months of treatment. In contrast to the results of l i v e r total l i p i d s , rats treated for 1 6 months had generally higher l i v e r cholesterol levels than those treated for 8 months. 2. Plasma Total Lipids and Cholesterol Results of the effect of different levels of dietary vitamin E on plasma total l i p i d s and cholesterol are shown in Figure 1 7 and 18 respectively. Treatment with high levels of dietary vitamin E for 8 months had no significant effect on plasma total l i p i d s . However, the plasma total l i p i d s of rats fed 2 5 or 2 5 0 IU vitamin E/kg diet for 8 months were higher than in rats fed more than 2 , 5 0 0 iu/kg diet. This was also observed in the rats treated for 16 months. Regression analysis revealed that increasing the dietary level of vitamin E significantly decreased (P<0.024) the plasma total l i p i d s in rats treated for 1 6 months. The linear relationship between log plasma l i p i d s (Y) and log dietary vitamin E (x) i n rats treated for 1 6 months was Y = 3 - 0 3 0 6 - 0.00814X, where R = 0.4l (P<0.024). Vitamin E supplementation for 8 months had no significant effect on plasma cholesterol (Fig. 18). The plasma cholesterol levels of vitamin E deficient rats was significantly lower (P<0 . 0 5 ) than those supplemented with 2 , 5 0 0 IU vitamin E/kg diet for 8 months. Treatment with excess levels of dietary vitamin E, 2 , 5 0 0 iu/kg diet or higher for 1 6 months significantly lowered (P<0 . 0 5 ) the plasma cholesterol level (Fig. 18). FIGURE 17 Total l i p i d s in plasma of rats fed different dietary levels of vitamin E for 8 months (open bars) and 16 months (closed bars). Each point represents mean ± SEM of number of rats shown in parentheses. In rats treated for 16 months, linear response i s significant (P<0.024) against dietary vitamin E. 6 9 ; lOOOf 800+ 6004 4 0 0 200 25 250 2,500 10,000 V i t a m i n E (iu/kg D i e t ) 25,000 FIGURE 18 Cholesterol concentration in plasma of rats fed different dietary levels of vitamin E for 8 months (open bars) and 1 6 months (closed bars). Each point represents mean ± SEM of four rats. In rats treated for 8 months, group D i s significantly higher ( P < 0 . 0 5 ) than group A. In rats treated for 1 6 months, groups B and C are significantly higher ( P < 0 . 0 5 ) than groups D, E and F. E O 25 250 2.500 10.000 25,000 V i t a m i n E (iu/kg D i e t ) 7 1 CHAPTER V DISCUSSION A. Body and Organ Weights The body weights of r a t s treated with high l e v e l s of vitamin E ( 1 0 , 0 0 0 and 2 5 , 0 0 0 iu/kg d i e t ) f o r 8 months were s i g n i f i c a n t l y depressed ( P < 0 . 0 5 ) . Body weights were also s i g n i f i c a n t l y reduced i n r a t s f e d 2 5 , 0 0 0 IU vitamin E/kg d i e t f o r 1 6 months compared to those r e c e i v i n g 2 5 to 2 , 5 0 0 iu/kg d i e t . From these r e s u l t s i t appears that excess d i e t a r y vitamin E f e d to r a t s over an extended period of time depressed t h e i r body weights. The r e s u l t s of research on the e f f e c t of excess d i e t a r y vitamin E on the growth rate i n animals vary widely. March et a l . ( 1 9 7 3 ) concluded that growth rate i n chicks appeared to be r e l a t i v e l y i n s e n s i t i v e to excess dietary vitamin E ( 1 , 0 0 0 iu/kg d i e t ) , although a depression occurred with 2 , 2 0 0 IU vitamin E/kg d i e t i n t h e i r short term study. Nockels et a l . ( 1 9 7 5 ) also reported that high l e v e l s of vitamin E supplementation ( 8 , 0 0 0 and 64 , 0 0 0 iu/kg d i e t ) s i g n i f i c a n t l y reduced the chick body weight. However, McCuaig and Motzok ( 1 9 7 0 ) f e d a 1 0 , 0 0 0 IU vitamin E/kg d i e t to chicks and reported that growth rate was unaffected. S i m i l a r r e s u l t s have also been reported i n the r a b b i t (Awad and G i l b r e a t h , 1 9 7 5 ) * E f f e c t s of excess vitamin E on growth rate of r a t s have also been studied. In a 28 week study, A l f i n - S l a t e r et a l . ( 1 9 7 2 ) found there were no d i f f e r e n c e s i n weight gains of r a t s f e d 1 0 0 mg vitamin E/day compared to those fed 3 0 mg vitamin E/day. However, Jenkins and M i t c h e l l ( 1 9 7 5 ) reported an increase i n body weight of r a t s f e d 6 0 0 and 6 , 0 0 0 IU vitamin E/kg d i e t f o r 2 months. Treatment with d i e t a r y vitamin E at 6 , 0 0 0 iu/kg d i e t (Jenkins and M i t c h e l l , 1 9 7 5 ) i s a comparable l e v e l to an o r a l supplement 72 of 100 mg vitamin E/day ( A l f i n - S l a t e r et a l . , 1972) i f i t s assumed that the r a t consumes 15 g diet/day. The reason f o r the wide discrepancy i n r e s u l t s i n t h i s area remains to be investigated. The organ weights (expressed as mg/lOO g body weight) f o r r a t s f e d d i f f e r e n t l e v e l s of vitamin E f o r 8 and 16 months are shown i n Tables 3 and k r e s p e c t i v e l y . For r a t s given dietary vitamin E from 25 to 25»000 iu/kg d i e t f o r 8 months, there were no s i g n i f i c a n t d i f f e r e n c e s among groups with respect to weights of l i v e r , uterus, kidney and spleen. However, high l e v e l s of vitamin E s i g n i f i c a n t l y increased the groups r e l a t i v e heart weight a f t e r 8 months treatment. The regression a n a l y s i s also revealed that a f t e r extending the treatment to 16 months, the groups fed excess vitamin E continued to have r e l a t i v e heartt weights l a r g e r than those f e d moderate l e v e l s of vitamin E. Also at t h i s time the r e l a t i v e spleen weights were s i g n i f i c a n t l y increased. There were no s i g n i f i c a n t d i f f e r e n c e s among the r a t s fed d i f f e r e n t l e v e l s of vitamin E with respect to weights of l i v e r , kidney and uterus. Hypervitaminosis E i n r a t s has been reported to increase r e l a t i v e adrenal weight, but not e f f e c t r e l a t i v e weight of l i v e r , kidney or spleen (Jenkins and M i t c h e l l , 1975). With the exception of the uterus, the r e l a t i v e organ weights i n the vitamin E-free r a t s were s i g n i f i c a n t l y l a r g e r than those groups r e c e i v i n g vitamin E supplements f o r 8 months. This may be due to the depression i n body weight a f t e r 3 to k months on the vitamin E-free d i e t . The weight l o s s represents massive muscle atrophy i n vitamin E-free r a t s , with the organs not being a f f e c t e d as much during the same period of time. As a consequence, the r e l a t i v e s i z e s of the organs appear to be l a r g e r i n vitamin E-free r a t s with the exception of the uterus. 73 B. Hematological Parameters There was no evidence i n t h i s study to suggest that excess vitamin E would lengthen the prothrombin time of r a t s . Instead, at the l a t t e r two te s t periods, 12 and 16 months, excess vitamin E a c t u a l l y r e s u l t e d i n decreased prothrombin times. These f i n d i n g s indicateathat r a t s r e c e i v i n g adequate dietary vitamin K do not develop prolonged prothrombin time even when they are fed a very high l e v e l of vitamin E. According to March et a l . (1973)> prothrombin timeswas lengthened i n chicks f e d excess amount of vitamin E. The prothrombin time was r a p i d l y reversed by i n j e c t i o n of vitamin K, which indicated an increase i n the dietary requirement f o r vitamin K i n the presense of excess d i e t a r y vitamin E. One previous study also reported that i n some s t r a i n s of r a t s the prothrombin l e v e l declined as higher doses of vitamin E were administered (Mellette and Leone, i960). I t i s d i f f i c u l t to compare the f i n d i n g s of March et a l . (D973) with those of t h i s study, however, because of the differences i n the d i e t a r y requirement of vitamin K i n r a t s and chickens. The induction of vitamin K d e f i c i e n c y i s also a f f e c t e d by other p h y s i o l o g i c a l f a c t o r s , such as the s t r a i n , age and sex of. the experimental animal. Mellette and Leone (i960) have shown clear-cut d i f f e r e n c e s between s t r a i n s of r a t s and s u s c e p t i b i l i t y to prolonged prothrombin time, as an i n d i c a t i o n of vitamin K d e f i c i e n c y . Also the female r a t , as used i n t h i s study, i s more r e s i s t a n t to vitamin K def i c i e n c y than the male r a t (Johnson et a l . , i960). The r e s u l t s of t h i s study, shown i n Table 5» i n d i c a t e that high l e v e l s of vitamin E f o r prolonged periods d i d not a f f e c t hemoglobin l e v e l s s i g n i f i c a n t l y . These f i n d i n g s are i n agreement with the observation of Jenkins and M i t c h e l l s ' (1975) In a short term experiment with r a t s . 7 4 Hematocrit values were not influenced by high d i e t a r y l e v e l s of vitamin E a f t e r 9 and 1 2 months of treatment (Table 6 ) . However, a f t e r 1 6 months of treatment hematocrit values were s i g n i f i c a n t l y increased when vitamin E was f e d at a l e v e l of 1 0 , 0 0 0 iu/kg d i e t or higher. March et a l . ( 1 9 7 3 ) have reported that hematocrit values were reduced i n chicks f e d 2 , 2 0 0 IU vitamin E/kg d i e t f o r 5 0 days. They observed that the reduction was more severe when the chicks were younger. At present i t i s not possible to a s c e r t a i n whether the hemotoe p o i e t i c system i s influenced by an excess of vitamin E. Only r e c e n t l y has a t t e n t i o n been centered on a possible role of vitamin E i n heme and hemeprotein synthesis (Murty et a l . , 1 9 7 0 ; Gaasi et a l . , 1 9 7 2 ; Nair, 1 9 7 2 ) . Other i n v e s t i g a t o r s , however, have not been able to show any involvement of vitamin E i n heme synthesis (Carpenter, 1 9 7 2 ; Diplock, 1 9 7 4 ) . The r e s u l t s of the spontaneous hemolysis of erythrocyte i n a buffered i s o t o n i c slaine s o l u t i o n showed that only red blood c e l l s of vitamin E d e f i c i e n t r a t s were susceptible to hemolysis. Excess d i e t a r y vitamin E d i d not a l t e r the s t a b i l i t y of the erythrocyte membrane to i n v i t r o hemolysis. C. Femoral Parameters Bone composition of the r a t s i n t h i s experimet were not a f f e c t e d by high l e v e l s of vitamin E fed f o r 8 months. However, the presence of excess vitamin E i n the d i e t f o r 1 6 months s i g n i f i c a n t l y (Table 1 0 ) reduced ( P < 0 . 0 0 0 5 ) the ash content of bones. Treatment with increasing l e v e l s of dietary vitamin E, ranging from 2 5 0 to 2 5 , 0 0 0 iu/kg d i e t , increased the plasma a l k a l i n e phosphatase a c t i v i t y s i g n i f i c a n t l y . The al t e r e d a l k a l i n e phosphatase a c t i v i t i e s and ash content a f t e r 1 6 months may indi c a t e increased turnover i n bones of r a t s fed high l e v e l s of 7 5 vitamin E f o r prolonged periods. March et a l . ( 1 9 7 3 ) reported that hone c a l c i f i c a t i o n was depressed when excess vitamin E ( 2 , 2 0 0 iu/kg d i e t ) was administered to chicks f e d e i t h e r calcium-deficient or vitamin D - d e f i c i e n t d i e t s . They concluded that excess vitamin E increased the requirement f o r vitamin D. There i s considerable species v a r i a b i l i t y i n the d i e t a r y requirements of vitamin D, calcium and phosphorus. Animal performance depends on the absolute amounts of each nutr i e n t as well as the r e l a t i v e amounts. Chickens require a higher calcium:phosphorus r a t i o than r a t s do f o r optimal growth. In r a t s , there i s no extensive evidence to i n d i c a t e that vitamin E i s required f o r normal c a l c i f i c a t i o n when the d i e t a r y calcium and phosphorus are balanced and adequate (National Research Council, 1 9 7 2 ) . The National Research Council ( 1 9 7 2 ) has recommended that approximately 1 , 0 0 0 IU vitamin D/kg d i e t be f e d to growing r a t s . The vitamin D content i n the experimental d i e t was 2 , 0 0 0 iu/kg d i e t . Thus, even i f excess vitamin E has increased the requirement f o r vitamin D as March et a l . ( 1 9 7 3 ) have suggested i n t h e i r work with chickens, t h i s would not have been observed i n t h i s experiment since the animals received adequate l e v e l s of vitamin D with balanced l e v e l s of calcium and phosphorus. D. Urinary Creatine and Creatinine Urinary excretion of creatine and creatinine were apparently normal i n a l l r a t s r e c e i v i n g high l e v e l s of dietary vitamin E f o r 1 1 months. Vitamin E - d e f i c i e n t r a t s had s i g n i f i c a n t l y higher creatine and lower creatinine l e v e l s i n urine©.. C r e a t i n u r i a i s a recognized symptom associated with vitamin E d e f i c i e n c y . Hillman ( 1 9 5 7 ) and Briggs ( 1 9 7 4 ) have described c r e a t i n u r i a i n three human subjects r e c e i v i n g large doses of vitamin E. 76 Briggs reported that an elevated serum creatine kinase accompanied the creatinuria. There was no indication in this study that excess vitamin E induces damage to skeletal muscle. E. Fat Soluble Vitamins 1. Liver and Plasma Vitamin E The relationship between dietary levels of vitamin E and the storage of this vitamin in l i v e r after 8 and 16 months treatment was linear when both values were expressed as logarithms (Figure 11). There was a significant deviation from the relationship between logarithm of vitamin E intake and log l i v e r tocopherol concentration when the dietary level increased beyond 10,000 IU vitamin E/kg diet. As shown in Figure 11, further increases i n dietary vitamin E had no significant effect on increasing the storage of this vitamin in l i v e r . The total vitamin E content in the l i v e r (Table 12) was approximately two-fold greater i n rats_treated for 16 months than those treated for 8 months. This accumulation of vitamin E was a result of both an increase i n concentration of a-tocopherol and enlargement of l i v e r size as the experimental period was extended. The findings of a linear relationship between increasing levels of vitamin E intake and l i v e r levels when both were expressed i n logarithmic units i s supported by the work of Bolliger and Bolliger-Quaife (1956) and Wiss et a l . (1962). In this study the plasma tocopherol level increased significantly as the dietary vitamin E level was raised (Figure 12). The plasma tocopherol levels were not proportional to the vitamin E intake at a l l dietary levels, though. In addition, the plasma tocopherol levels were at least two-fold higher i n the rats fed for 16 months, than those treated for 8 months. 77 According to B o l l i g e r and Bolliger-Quaife ( 1 9 5 6 ) , Wiss et a l . ( 1 9 6 2 ) and B i e r i ( 1 9 7 2 ) , there i s a l i n e a r r e l a t i o n s h i p between plasma tocopherol and the logarithm of the dose fed . The former two studies were short term experiments, while the report by B i e r i ( 1 9 7 2 ) was 2 5 weeks long and examined the e f f e c t of feeding a low l e v e l of vitamin E, 3 2 iu/kg d i e t . In the only long term study examining the e f f e c t of large doses of vitamin E on plasma tocopherol l e v e l s i n r a t s , A l f i n - S l a t e r et a l . ( 1 9 7 2 ) reported that plasma tocopherol l e v e l s r e f l e c t e d the tocopherol l e v e l supplemented, but were not proportional to the dose. 2 . L i v e r and Plasma Vitamin A In t h i s study, a f t e r 8 months treatment, the l i v e r vitamin A storage of a l l vitamin E supplemented groups was s i g n i f i c a n t l y higher than of. vitamin E-free groups. However, i n the r a t s treated f o r 8 and 1 6 months, the change i n dietary vitamin E ranging from 2 5 to 2 5 , 0 0 0 Ill/kg d i e t showed no s i g n i f i c a n t e f f e c t on a l t e r i n g l i v e r vitamin A storage. The plasma vitamin A content of r a t s f e d various dietary l e v e l s of vitamin E f o r 8 months was not tested, but those f o r 1 6 months were measured and no s i g n i f i c a n t d i f f e r e n c e s were observed between the groups. Therefore, i t : may be concluded that there was no i n t e r a c t i o n between high l e v e l s of dietary vitamin E and vitamin A i n l i v e r or plasma i n t h i s study. Workers have confirmed that increased intakes of vitamin E increase the storage of vitamin A i n the l i v e r (Cawthorne et a l . , I 9 6 8 ; Prodouz and Navari, 1 9 7 5 ) * This vitamin E "sparing" e f f e c t on vitamin A has been shown at widely varying l e v e l s of vitamin E intake, f o r example, from 1 Ill/week (Cawthorne et a l . , 1 9 6 8 ) up to 6 , 0 0 0 Ill/kg d i e t (Jenkins and M i t c h e l l , 1 9 7 5 ) have been reported to increase the l i v e r vitamin A storage 7 8 i n r a t s . Jenkins and M i t c h e l l ( 1 9 7 5 ) also reported that there was a s i g n i f i c a n t increase i n plasma vitamin A with increasing l e v e l s of vitamin E i n the d i e t . The mechanism of a c t i o n between these two vitamins i s s t i l l unknown, but according to Cawthorne et a l . , ( 1 9 6 8 ) the r e l a t i o n s h i p between vitamin E and vitamin A i n vivo cannot be regarded as that between an antioxidant and a peroxidizable substrate. F. L i p i d s 1. L i v e r T o t a l L i p i d s and Cholesterol T o t a l l i p i d s i n l i v e r were s i g n i f i c a n t l y increased by excess vitamin E supplementation (from 2 5 0 to 2 5 , 0 0 0 iu/kg d i e t ) i n r a t s treated f o r 8 months. Contrary to the r e s u l t s found a f t e r 8 months treatment, l i v e r t o t a l l i p i d l e v e l s were not s i g n i f i c a n t l y a l t e r e d among the r a t s treated f o r 1 6 months with d i f f e r e n t l e v e l s of vitamin E. No mechanism has been proposed to explain why excess vitamin E should increase l i v e r t o t a l l i p i d s only i n younger r a t s . This cannot be accounted f o r by an increase i n the l e v e l of l i v e r c h o l e s t e r o l , because at both 8 and 1 6 months excess vitamin E d i d not s i g n i f i c a n t l y a f f e c t l i v e r c h o l e s t e r o l concentration. High dietary l e v e l s of vitamin E have been reported to increase the l e v e l of t o t a l l i p i d s i n l i v e r ( A l f i n - S l a t e r et a l . , 1 9 7 2 ; Jenkins and M i t c h e l l , 1 9 7 5 ) ' Increasing d i e t a r y vitamin E intake a l s o has been shown to enhance the development of alcohol induced f a t t y l i v e r (Levander et a l . , 1 9 7 3)• Contrary to the above f i n d i n g s , other workers have report- ed that increasing d i e t a r y l e v e l s of vitamin E w i l l decrease the l e v e l of t o t a l l i p i d s i n r a t l i v e r s ( H a r r i l l et a l . , 1 9 6 5 ; H a r r i l l and G i f f o r d , 1 9 6 6 ; Prodouz and Navari, 1 9 7 5 ) - The l e v e l s of vitamin E used were much lower and the length of treatment was much shorter i n these l a t t e r 7 9 i n v e s t i g a t i o n s compared to the reports showing increases i n the l e v e l of t o t a l l i p i d s i n l i v e r . 2 . Plasma Total L i p i d s and Cholesterol The r e s u l t s of t h i s study, shown i n Figures 1 7 and 18, i n d i c a t e that the plasma t o t a l l i p i d s and c h o l e s t e r o l were not s i g n i f i c a n t l y a l t e r e d following 8 months treatment with thigh l e v e l s of vitamin E. However, those r a t s treated f o r 1 6 months with high l e v e l s of d i e t a r y vitamin E (over 2 , 5 0 0 Ill/kg d i e t ) had s i g n i f i c a n t l y lower plasma t o t a l l i p i d s and c h o l e s t e r o l . The regression curves of plasma t o t a l l i p i d s on vitamin E were l i n e a r , while those on plasma cholesterol were not l i n e a r . Further- more, the decrease i n plasma t o t a l l i p i d s was greater than that of plasma cholesterol i n r a t s f e d high l e v e l s of vitamin E suggesting that other components, such as t r i g l y c e r i d e s or phospholipids might also he a f f e c t e d . I t i s d i f f i c u l t to compare the r e s u l t s of plasma t o t a l l l i p i d s and c h o l e s t e r o l i n t h i s study with those of other workers, since the l e v e l of vitamin E supplementation, the length of treatment and the d i e t a r y ingredients vary widely i n the experiments. Vitamin E may play a r o l e i n a l t e r i n g plasma t o t a l l i p i d and c h o l e s t e r o l , hut the r e s u l t s reported i n the l i t e r a t u r e are inconsistent. I t has been reported i n numerous short term studies that high l e v e l s of d i e t a r y vitamin E w i l l lower plasma ch o l e s t e r o l i n r a t s (Chen et a l . , 1 9 7 2 ; H a r r i l l et a l . , 1 9 6 5 ; Prodouz and Navari, 1 9 7 5 ) - However, other workers have reported that high dietary vitamin E had no e f f e c t on serum c h o l e s t e r o l i n r a t s (Jenkins and M i t c h e l l , 1 9 7 5 ) , r a b b i t s (Awad and G i l b r e a t h , 1 9 7 5 ) and chicks (Koyangi et a l . , 1 9 6 6 ) . 80 CHAPTER VI SUMMARY The purpose of t h i s study was to investigate the long-term e f f e c t of high l e v e l s of dietary vitamin E on various metabolic parameters i n the r a t . Six groups of female r a t s were f e d f o r as long as 1 6 months the basal vitamin E-free d i e t with supplements ranging from 0 to 2 5 , 0 0 0 IU vitamin E (dl-a-tocopheryl acetate) per kilogram d i e t . The l e v e l s of vitamin E chosen were 0 , 2 5 , 2 5 0 , 2 , 5 0 0 , 1 0 , 0 0 0 and 2 5 , 0 0 0 iu/kg d i e t . A l l n u t r i e n t s i n the basal d i e t except vitamin E were adequate. The metabolic parameters studied i n the r a t s fed excess vitamin E were compared s t a t i s t i c a l l y with the same parameters i n r a t s r e c e i v i n g a moderate or normal l e v e l of d i e t a r y vitamin E. Theefindings of t h i s study on the long-term e f f e c t of excess intake of vitamin E i n the r a t were as follows: ( 1 ) Body weights were depressed i n the groups f e d 1 0 , 0 0 0 and 2 5 , 0 0 0 IU vitamin E/kg d i e t f o r 8 and 1 6 months. ( 2 ) High l e v e l s of d i e t a r y vitamin E increased the r e l a t i v e heart weight a f t e r 8 months and r e l a t i v e spleen weight a f t e r 1 6 months. ( 3 ) Hemoglobin values and spontaneous erythrocyte hemolysis were not influenced by excessive amounts of vitamin E. The prothrombin time was reduced a f t e r 1 2 months, while elevated hematocrit value was observed a f t e r 1 6 months.of treatment. ( 4 ) The ash content of bone decreased with concurrent increase i n plasma a l k a l i n e phosphatase a c t i v i t y a f t e r 1 6 months of treatment. ( 5 ) Urinary l e v e l s of creatine and creatinine were not af f e c t e d by high l e v e l s of d i e t a r y vitamin E. (6) A logarithmic r e l a t i o n s h i p was observed between dietary l e v e l s of vitamin E and the concentrations of t h i s vitamin i n l i v e r and plasma. (7) The concentrations of vitamin A i n l i v e r and plasma were not affected by high l e v e l s of dietary vitamin E. (8) To t a l l i p i d s i n l i v e r were s i g n i f i c a n t l y increased by excess vitamin E supplementation i n r a t s fed f o r 8 months, but not i n r a t s f e d f o r 16 months. 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