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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., U n i v e r s i t y  o f 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  in  THE FACULTY OF GRADUATE STUDIES Division of HUMAN NUTRITION SCHOOL OF HOME ECONOMICS  We a c c e p t t h i s t h e s i s a s conforming t o t h e required standard.  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1979 ©  I a n Bruce Macdonald, 1979  In p r e s e n t i n g t h i s  thesis in partial  an a d v a n c e d d e g r e e a t the  Library  I further for  shall  the U n i v e r s i t y  make i t  agree that  freely  of  extensive  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  this  written  thesis for  financial gain shall  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 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  A p r i l 2k,  1979  Columbia  the requirements I agree  r e f e r e n c e and copying of  this  that  not  copying or  for  that  study. thesis  by t h e Head o f my D e p a r t m e n t  i s understood  permission.  Department nf  Date  It  of  B r i t i s h Columbia,  available for  permission for  by h i s r e p r e s e n t a t i v e s . of  fulfilment  or  publication  be a l l o w e d w i t h o u t  my  i  ABSTRACT  I n view o f the f a c t t h a t some f a t s o l u b l e v i t a m i n s are t o x i c i n l a r g e doses t o e x p e r i m e n t a l  a n i m a l s and man,  t h i s study was  t o i n v e s t i g a t e the long-term e f f e c t s of low, o f d i e t a r y v i t a m i n E on v a r i o u s m e t a b o l i c  levels  g) were f e d f o r as l o n g  months the b a s a l v i t a m i n E - f r e e d i e t w i t h supplements r a n g i n g  0 t o 25»000 IU v i t a m i n E ( D L - a - t o c o p h e r y l The  moderate and h i g h  parameters i n the r a t .  S i x groups of female W i s t a r r a t s (50 16  initiated  l e v e l s of v i t a m i n E chosen were 0 ,  acetate) per kilogram  25,  250,  2,500,  as  from diet.  10,000 and  25,000  ITj/kg d i e t ; 0 r e p r e s e n t i n g v i t a m i n E - f r e e , 25 r e p r e s e n t i n g moderate l e v e l and  250  t o 25»000 r e p r e s e n t i n g l a r g e doses.  A l l nutrients i n  the b a s a l d i e t except v i t a m i n E were adequate. The  f o c u s of t h i s study  was  on the e f f e c t s of l a r g e doses o f  d i e t a r y v i t a m i n E on : ( l ) the h e m a t o l o g i c a l  i n d i c e s such as  and hemoglobin l e v e l s , prothrombin time and  erythrocyte hemolysis at  9,  12  and  16 months of treatment; (2)  l e v e l s a t 11  months of treatment; (3)  weights a t 8 and as ash 8 and  content,  body weight and (4)  femoral  v a r i o u s organ parameters such  c a l c i u m and phosphate c o n c e n t r a t i o n s of bone a t  16 months of treatment; and  v i t a m i n A, t o t a l l i p i d s , and 8 and  u r i n a r y c r e a t i n e and c r e a t i n i n e  16 months of treatment; and  hematocrit  (5)  the l e v e l s of  c h o l e s t e r o l i n l i v e r and  a-tocopherol, plasma a t  16 months o f treatment. Rats f e d 10,000 and  months had  2 5 . 0 0 0 IU v i t a m i n E/kg  d i e t f o r 8 and  s i g n i f i c a n t l y r e d u c e d body weights i n comparison t o  those  r e c e i v i n g the moderate l e v e l o f v i t a m i n E .  The  effect  of  e x c e s s d i e t a r y v i t a m i n E on body weight was  not as marked as t h a t  of  vitamin E deficiency.  There was  little  depressing  16  d i f f e r e n c e between the  moderate and h i g h v i t a m i n E supplemented groups w i t h r e s p e c t t o t h e weights o f l i v e r , u t e r u s and k i d n e y .  However, h i g h l e v e l s o f d i e t a r y  v i t a m i n E i n c r e a s e d the r e l a t i v e h e a r t weights a f t e r 8 months and the s p l e e n weights a f t e r 16 months. Hemoglobin and h e m a t o c r i t  v a l u e s were not i n f l u e n c e d by  e x c e s s i v e amounts o f v i t a m i n E a f t e r 9 o r 12 months o f At 16 months however, the h e m a t o c r i t  treatment.  v a l u e s o f r a t s f e d 10,000 and  2 5 , 0 0 0 IU v i t a m i n E/kg d i e t were i n c r e a s e d s i g n i f i c a n t l y over of r a t s f e d 25 Iu"/kg d i e t .  The prothrombin time was reduced  t r e a t e d with excess d i e t a r y v i t a m i n E f o r 12 and 16 months.  those in rats Only  v i t a m i n E d e f i c i e n c y , but n o t excess v i t a m i n E , was a s s o c i a t e d w i t h i n c r e a s e d membrane f r a g i l i t y  of erythrocytes.  In r a t s s u b j e c t e d t o excess v i t a m i n E f o r 16 months t h e a s h content  o f bone was d e c r e a s e d .  High l e v e l s o f d i e t a r y v i t a m i n E  i n c r e a s e d 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 o f treatment.  These r e s u l t s i n d i c a t e t h a t t h e r e may be i n c r e a s e d m i n e r a l  t u r n o v e r i n bones o f r a t s f e d h i g h l e v e l s o f v i t a m i n E f o r p r o l o n g e d periods. U r i n a r y l e v e l s o f c r e a t i n e and c r e a t i n i n e were n o t 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 v i t a m i n E d e f i c i e n t  r a t s , t h e c r e a t i n e e x c r e t i o n i n c r e a s e d w h i l e the c r e a t i n i n e e x c r e t i o n decreased,  r e s u l t i n g i n a very high r a t i o of c r e a t i n e / c r e a t i n i n e i n  urine. The ot-tocopherol s t o r e d i n l i v e r r o s e s i g n i f i c a n t l y with i n c r e a s ing d i e t a r y vitamin E.  A l o g a r i t h m i c r e l a t i o n was demonstrated between  l i v e r ot-tocopherol c o n c e n t r a t i o n and d i e t a r y l e v e l s o f v i t a m i n E . t o t a l a-tocopherol  The  i n whole l i v e r o f r a t s f e d t h e d i f f e r e n t l e v e l s o f  v i t a m i n E f o r 16 months was f o r 8 months.  approximately  double t h a t i n r a t s t r e a t e d  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 t o c o p h e r o l  the l o g a r i t h m of d i e t a r y v i t a m i n E was  found i n r a t s t r e a t e d f o r 8  and 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 d i e t a r y v i t a m i n E i n r a t s t r e a t e d f o r 8 months, but not f o r 16 months.  There was  little  i n rats treated  d i f f e r e n c e 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 h i g h l y supplemented groups.  I n c r e a s i n g d i e t a r y v i t a m i n E s i g n i f i c a n t l y l o w e r e d plasma  t o t a l l i p i d s and  c h o l e s t e r o l i n r a t s t r e a t e d f o r 16 months.  A quantita-  t i v e examination of the data showed t h a t the r e d u c t i o n 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 c h o l e s t e r o l l e v e l s ,  suggests t h a t a h i g h d i e t a r y l e v e l o f v i t a m i n E a f f e c t e d one  and or more  of the c o n s t i t u e n t s o f the t o t a l l i p i d s ( p h o s p h o l i p i d s and/or t r i g l y c e r i d e s ) o t h e r 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, l e v e l s of d i e t a r y vitamin E r e s u l t i n biochemical aspects  of metabolism i n r a t s .  are the d e p r e s s i o n  i t appears that h i g h changes i n some  Some of t h e changes worth r e c o g n i t i o n  i n body weight, i n c r e a s e i n r e l a t i v e s p l e e n  h e a r t weights, d e c r e a s e i n ash  content  of bones with c o n c u r r e n t  i n plasma a l k a l i n e phosphatase a c t i v i t y , i n c r e a s e d h e m a t o c r i t f a t t y l i v e r i n r a t s t r e a t e d f o r 8 months. was  t h a t e x c e s s v i t a m i n E over p r o l o n g e d ful effects in rats.  The  increase  value  and  A logarithmic relationship  observed between d i e t a r y l e v e l s o f v i t a m i n E and the  of t h i s v i t a m i n i n l i v e r and plasma.  and  concentrations  r e s u l t s of t h i s study  suggest  p e r i o d s o f time have some harm-  ACKNOWLEDGEMENT  To my f a m i l y , f r i e n d s and p r o f e s s i o n a l c o l l e a g u e s who gave much encouragement throughout my M a s t e r ' s Program, I extend my s i n c e r e thanks.  S p e c i a l thanks i s extended t o D r . N.Y. Jack Yang f o r h i s  knowledge and a s s i s t a n c e throughout t h e course o f t h i s study.  I am  a l s o g r a t e f u l f o r t h e v a l u a b l e d i s c u s s i o n and moral s u p p o r t o f Dr. J . F . A n g e l . Acknowledgement i s e x p r e s s e d t o my a d v i s o r , D r . I.D. D e s a i f o r h i s c o o p e r a t i o n and a s s i s t a n c e i n t h i s study; t o Dr. J . L e i c h t e r and t o P r o f e s s o r B.E. March  f o r s e r v i n g on my committee; and t o 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 o f t h e r e s u l t s . T h i s study was supported i n p a r t by Grant No. Dr. I.D. D e s a i from t h e N a t i o n a l Research Graduate.Student  67-^686  C o u n c i l o f Canada and a  F e l l o w s h i p (1975-1976) from t h e O f f i c e o f the  President, University of B r i t i s h  Columbia.  to  TABLE OF CONTENTS Page ABSTRACT  i  ACKNOWLEDGEMENT  i  LIST OF TABLES  v  viii  LIST OF FIGURES  ix  CHAPTER I.  INTRODUCTION  1  II.  REVIEW OF THE LITERATURE  3  A.  History of Vitamin E  3  B.  V i t a m i n E D e f i c i e n c y - Occurrence  5  1.  Human I n f a n t s  5  2.  Human A d u l t s  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  o f V i t a m i n E i n Human S u b j e c t s  10  E.  Pharmacological E f f e c t  o f V i t a m i n 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»  T i s s u e Storage  of Vitamin E  17  6.  T i s s u e Storage o f V i t a m i n A  18  7.  Liver L i p i d Levels  20  8.  Blood L i p i d L e v e l s  21  vi  III.  MATERIALS AND METHODS  22  A.  Animal Care  22  B.  Experimental Diets  22  C.  Experimental Groups  22  D.  Experimental Procedure  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 L i p i d s  32  9•  Plasma Alkaline Phosphatase  32  10.  L i v e r L i p i d Extraction  34  11.  Liver Vitamin A  35  12.  Eiver Vitamin E  13•  L i v e r Total L i p i d s  39  14.  L i v e r Cholesterol  39  15«  Femoral Ash  40  16.  Femoral Calcium  40  17.  Femoral Inorganic Phosphate  42  F. IV.  S t a t i s t i c a l Analysis  224  '  36  4c;  RESULTS  46  A.  Body and Organ Weights  46  B.  Hematological Parameters  4Q  vii  C.  Femoral Parameters  54  D.  U r i n a r y C r e a t i n e and C r e a t i n i n e  57  E.  Fat Soluble Vitamins  57  1.  L i v e r and Plasma V i t a m i n E  57  2.  L i v e r and Plasma V i t a m i n A  59  F. L i p i d s  V.  1.  L i v e r T o t a l L i p i d s and C h o l e s t e r o l  65  2.  Plasma T o t a l L i p i d s and C h o l e s t e r o l  68  DISCUSSION  71  A.  Body and Organ Weights  71  B.  H e m a t o l o g i c a l Parameters  73  C.  Femoral Parameters  74  D.  U r i n a r y C r e a t i n e and C r e a t i n i n e  75  E.  Fat Soluble Vitamins  76  1.  L i v e r and Plasma V i t a m i n E  76  2.  L i v e r and Plasma V i t a m i n A  77  F.  VI.  65  Lipids  78  1.  L i v e r T o t a l L i p i d s and C h o l e s t e r o l  78  2.  Plasma T o t a l L i p i d s and C h o l e s t e r o l  79  SUMMARY  LITERATURE CITED  80 82  viii  LIST OF TABLES  T a b l e No.  Page  1  Syndromes R e s u l t i n g From V i t a m i n E D e f i c i e n c y  2  Composition  3  Body and Organ Weights o f R a t s on D i f f e r e n t  23  o f the B a s a l D i e t Levels  of D i e t a r y V i t a m i n E f o r 8 Months 4  Body and Organ Weights o f R a t s on D i f f e r e n t  47 Levels  of D i e t a r y V i t a m i n E f o r 16 Months 5  6  7  8  9  10  11  12  8  48  Hemoglobin Values o f R a t s F e d D i f f e r e n t L e v e l s o f D i e t a r y Vitamin E  50  Hematocrit Values o f R a t s F e d D i f f e r e n t L e v e l s o f D i e t a r y Vitamin E  51  E r y t h r o c y t e Hemolysis o f Rats F e d D i f f e r e n t of D i e t a r y V i t a m i n E  52  Levels  Prothrombin Times o f R a t s F e d D i f f e r e n t L e v e l s o f D i e t a r y Vitamin E  53  Femoral Parameters of R a t s F e d D i f f e r e n t L e v e l s of D i e t a r y V i t a m i n E f o r 8 Months  55  Femoral Parameters o f R a t s Fed D i f f e r e n t L e v e l s o f D i e t a r y V i t a m i n E f o r 16 Months  56  U r i n a r y C r e a t i n e and C r e a t i n i n e o f R a t s on D i f f e r e n t L e v e l s o f D i e t a r y V i t a m i n E f o r 11 Months  58  The C o n c e n t r a t i o n s o f ot-Tocopherol i n L i v e r s o f R a t s Fed D i f f e r e n t L e v e l s o f D i e t a r y V i t a m i n E f o r 8 and 16 Months  60  ix  LIST OF FIGURES  F i g u r e No.  Page  1  S t r u c t u r e and Nomenclature o f the T o c o p h e r o l s  2  Standard  Curve f o r Hemoglobin  3  Standard  Curve f o r Plasma V i t a m i n A  29  4.  Standard  Curve f o r Plasma V i t a m i n E  31  5  Standard  Curve f o r Plasma C h o l e s t e r o l  33  6  Standard  Curve f o r L i v e r V i t a m i n A  37  7  Standard  Curve f o r L i v e r V i t a m i n E  38  8  Standard  Curve f o r L i v e r C h o l e s t e r o l  41  9  Standard  Curve f o r Calcium  43  10  Standard  Curve f o r Phosphate  44  11  P l o t o f the Logarithm o f L i v e r a - T o c o p h e r o l Concent r a t i o n v e r s u s the L o g a r i t h m o f D i e t a r y V i t a m i n E , i n Rats T r e a t e d f o r 8 and 16 Months  6l  Plasma a - T o c o p h e r o l C o n c e n t r a t i o n o f Rats 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 o f V i t a m i n E f o r 8 and 16 Months  62  L i v e r V i t a m i n A C o n c e n t r a t i o n of Rats 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 o f V i t a m i n E f o r 8 and 16 Months  63  12  13  14  15  16  17  18  4  Plasma V i t a m i n A C o n c e n t r a t i o n o f Rats 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 o f V i t a m i n E f o r 16 Months  64  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 a r y L e v e l s o f V i t a m i n E f o r 8 and 16 Months  66  L i v e r C h o l e s t e r o l C o n c e n t r a t i o n of Rats 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 o f V i t a m i n E f o r 8 and 16 Months  67  T o t a l L i p i d s i n Plasma o f Rats 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 o f V i t a m i n E f o r 8 and 16 Months  69  C h o l e s t e r o l C o n c e n t r a t i o n i n Plasma o f 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 and 16 Months  70  1  CHAPTER 1 INTRODUCTION  V i t a m i n E has g e n e r a l l y been c o n s i d e r e d t o be n o n - t o x i c .  In  r e c e n t y e a r s t h e r e has been c o n s i d e r a b l e i n t e r e s t among t h e l a y p u b l i c regarding the p o s s i b l e pharmacological  r o l e o f v i t a m i n E when t a k e n i n  l a r g e d i e t a r y supplements ("megavitamin E t h e r a p y " ) .  At the present  time, t h e r e 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 o r c l i n i c a l evidence t o prove t h a t v i t a m i n E supplementation i s b e n e f i c i a l f o r h e a l t h .  In  i s o l a t e d c a s e s , amounts g r e a t l y e x c e e d i n g t h e normal d i e t a r y i n t a k e have been a d m i n i s t e r e d  t o human s u b j e c t s 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 ti sfar  from c e r t a i n t h a t c h r o n i c i n g e s t i o n o f v i t a m i n E i n megadoses i s entirely safe.  I n human beings,  s i d e - e f f e c t s o f excess v i t a m i n E  have been r e p o r t e d a s f a t i g u e ( B r i g g s e t a l . , 1 9 7 4 ) , c r e a t i n u r i a ( B r i g g s e t a l . , 1974; H i l l m a n ,  1957) and lengthened  prothrombin time  when taken i n excess a l o n g with w a r f a r i n and c l o f i b r a t e ( C o r r i g a n and Marcus,  1974).  There have been r e p o r t s o f m e t a b o l i c experimental  treatment  animals by excess v i t a m i n E .  ed t h a t h y p e r v i t a m i n o s i s E induced  abnormalities induced i n March e t a l . (1973)  r e t i c u l o c y t o s i s , lowered  report-  hematocrit  v a l u e , reduced t h y r o i d a c t i v i t y and i n c r e a s e d r e q u i r e m e n t s f o r v i t a m i n D and v i t a m i n K i n c h i c k s .  Hypervitaminosis  E has a l s o been  found t o d e p r e s s the a c t i v i t y o f g l u t a t h i o n e p e r o x i d a s e plasma o f r a t s (Yang e t a l . , 1 9 7 6 ) .  i n l i v e r and  E a r l y studies reported  excess v i t a m i n E caused t e s t i c u l a r d e g e n e r a t i o n  and reduced  that fertility  2  i n male r a t s (Bscudero and Herraiz, 1942), and affected the length of estrus cycle and ovarian a c t i v i t y i n female r a t s (Reiss, 1941). In view of the reports of hypervitaminosis E i n experimental animals the purpose of t h i s study was to investigate f u r t h e r the long-term e f f e c t s of high intakes of dietary vitamin E on r a t s treated with l e v e l s ranging from 0 to 25,000 iu/kg d i e t .  The focus was  on  the e f f e c t of excess intake of vitamin E on the following metabolic parameters: ( l ) hematocrit and hemoglobin l e v e l s , prothrombin time and erythrocyte hemolysis;  (2) urinary creatine and creatinine l e v e l s ;  (3) body weight and various organ weights; (4) bone ash content, calcium and phosphate concentration of bone; and (5)  and  the l e v e l s of  Ot-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.  These parameters 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 dietary vitamin E .  3  CHAPTER I I REVIEW OF LITERATURE  A.  History of  Vitamin  Evans and Bishop  (1922) d i s c o v e r e d a f a t s o l u b l e 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  Evans proposed t o name the substance " t o c o s " meaning c h i l d b i r t h , suffix  reviewed  t o c o p h e r o l , from the Greek words  "phero" meaning t o b r i n g f o r t h and  " o l " , i t b e i n g an a l c o h o l .  v i t a m i n E was  v i t a m i n E by Sure ( 1 9 2 4 ) .  the  Much of the p i o n e e r h i s t o r y  of  by Evans 419-62) and by Mason ( 1 9 7 7 ) -  The m u l t i p l e nature  of the v i t a m i n began t o u n f o l d i n 1936,  when  Evans e t a l . (1936) succeeded i n i s o l a t i n g from wheat germ o i l two compounds with v i t a m i n E a c t i v i t y , a - t o c o p h e r o l and Since t h a t time,  B-tocopherol.  s t u d i e s of v i t a m i n E have been conducted by numerous  i n v e s t i g a t o r s (Pennock e t a l . ,  1964;  Stern et a l . ,  1947).  To  date,  e i g h t 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 d i s c o v e r e d t o have v a r i e d amounts of v i t a m i n E The  t o c o p h e r o l s belong  t o two  distinct  t o c o p h e r o l s and t h e t o c o t r i e n o l s . i f i c a t i o n o f these  s e r i e s of compounds, the  The b a s i c s t r u c t u r e and the  compounds a c c e p t e d  reviewed The  class-  by the IUPAC-IUB Commission  B i o c h e m i c a l Nomenclature (1979) a r e shown i n F i g u r e 1. t i o n ' o f the s t r u c t u r e and  activity.  on  The e l u c i d a -  s y n t h e s i s of the t o c o p h e r o l s has  been  by S e b r e l l and H a r r i s ( 1 9 7 2 ) . d i f f e r e n c e s i n number and p o s i t i o n o f 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 v a r i o u s forms o f t o c o p h e r o l s .  The  e v a l u a t i o n o f the r e l a t i v e potency o f the many compounds which have  D e f i n i t i o n of terms: The a c c e p t e d  names are v i t a m i n E or t o c o p h e r o l s .  FIGURE 1  STRUCTURE AND  NOMENCLATURE  OF THE TOCOPHEROLS  R^=  1  CH (CH CH=CCH ) H 2  2  Tocol Structure  Tocotrienol Structure  Methyl  a-tocopherol  a-tocotrienol  5,7,  3 - tocopherol  3 -tocotrienol  5,8  y-tocopherol  y-tocotrienol  7,8  6 - tocopherol  6-tocotrienol  8  2  3  Positions  ^ U P A C - I U B C o m m i s s i o n on B i o c h e m i c a l N o m e n c l a t u r e (1979) G e n e r i c D e s c r i p t o r s a n d T r i v i a l Names f o r V i t a m i n s and R e l a t e d Compounds. J . N u t r . 109, 8-15.  5  v i t a m i n E a c t i v i t y has been c a r r i e d out u s i n g i n v i t r o t e s t s (Bunyan e t a l . , I960; Rose and Gyorgy, 1952) D i c k s and M a t t e r s o n , 1962;  and b i o a s s a y s  (Bunyan e t a l . , I 9 6 0 ;  Friedman e t a l . , 1958» Rose and Gyorgy, 1 9 5 2 ) .  I n v e s t i g a t i o n on the r e l a t i o n s h i p between dosage and response f o r v i t a m i n E i n the f e t a l r e s o r p t i o n t e s t ( B l i s s and Gyorgy, 1951) the acceptance by the N a t i o n a l Formulary of the American  l e d to  Pharmaceutical  A s s o c i a t i o n ( i 9 6 0 ) o f the c o n v e r s i o n f a c t o r s f o r the v a r i o u s forms of v i t a m i n E as shown below.  1 mg  1  d l - o t - t o c o p h e r o l a c e t a t e = 1.0  International Unit  1 mg d l - o t - t o c o p h e r o l  = 1.1  1 mg d-ot-tocopherol a c e t a t e  = I.36  International Unit  1 mg  = 1.4-9  International Unit  d-ot-tocopherol  B.  Vitamin E D e f i c i e n c y - Occurrence  1.  Human I n f a n t s The  c l i n i c a l evidence  earlypphase  of l i f e ,  International Unit  of v i t a m i n E d e f i c i e n c y has been seen i n the  u s u a l l y w i t h s m a l l 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 v i t a m i n E a c r o s s the p l a c e n t a , sotithe i n f a n t s have low l e v e l s of v i t a m i n E i n both t i s s u e s and b l o o d . premature i n f a n t s i s h e m o l y t i c  i n nature  anemia i n  and a s s o c i a t e d w i t h an abnorm-  a l l y e l e v a t e d e r y t h r o c y t e f r a g i l i t y by hydrogen peroxide I 9 6 0 ; Rose and Gyorgy, 1 9 5 2 ) .  The  (Bunyan e t a l . ,  I n t r e a t i n g the anemia, G r o s s and  (1972) have found t h a t the a b s o r p t i o n of o r a l l y a d m i n i s t e r e d  Melhorn  ot-tocopherol  a c e t a t e 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 i n f a n t s and i s f o l l o w e d by a f a v o r a b l e h e m a t o l o g i c response o n l y when the c h r o n o l o g i c  equivalent  of g e s t a t i o n a l m a t u r i t y i s r e a c h e d . A s t a t e of v i t a m i n E d e f i c i e n c y o c c u r s i n i n d i v i d u a l s who  have  6  a d e f e c t i n t h e i r a b i l i t y t o a b s o r b f a t ( B i n d e r and S h a p i r o ,  1970;  Machon and N e a l s , these  M u l l e r and- H a r r i s ,  1969)-  196?;  A l a r g e number of  cases are i n c h i l d r e n and young a d u l t s w i t h c y s t i c f i b r o s i s  and F a r r e l l ,  1976).  Lower t h a n u s u a l b l o o d t o c o p h e r o l l e v e l s are  ed i n d i s e a s e s where i n t e s t i n a l a b s o r p t i o n i s a f f e c t e d , but no a t o l o g y which responds t o v i t a m i n E has been observed.  observ-  symptom-  A thorough r e v i e w  of the i n f o r m a t i o n a v a i l a b l e on v i t a m i n E s t a t u s i n o t h e r  malabsorptive  (1976).  s t a t e s has been conducted by B i e r i and F a r r e l l  2.  (Bieri  Human A d u l t s There are no r e p o r t e d c l i n i c a l e v i d e n c e s  of a d e f i c i e n c y o f  v i t a m i n E i n normal human a d u l t s because of the storage  o f the v i t a m i n and the consequent extended p e r i o d r e q u i r e d f o r  depletion (Bieri, l e v e l s below 0.5 Farrell,  considerable t i s s u e  1976).  1975)*  I t has been suggested t h a t serum  mg/lOO ml c o u l d be  c l a s s i f i e d as d e f i c i e n t  and  i n p r o p o r t i o n t o the amounts of  c h o l e s t e r o l , p h o s p h o l i p i d and t r i g l y c e r i d e s p r e s e n t  1969;  (Bieri  Some i n v e s t i g a t o r s have shown t h a t t h e r e i s a tendency  f o r serum ot-tocopherol t o r i s e and f a l l  et a l . ,  tocopherol  Horwitt  et a l . ,  1972).  i n the b l o o d  (Davies  Hence, i n t e r p r e t a t i o n of the  of v i t a m i n E n u t r i t u r e from b l o o d d a t a may the l e v e l o f i n t a k e o r t i s s u e s t o r a g e .  status  not a c c u r a t e l y r e f l e c t e i t h e r  Furthermore, s i n c e b l o o d  tocopher-  o l i s o n l y about 1 p e r cent of the t o t a l body t o c o p h e r o l p o o l , i t i s sometimes d i f f i c u l t t o r e l a t e h l o o d t o c o p h e r o l t o v i t a m i n E n u t r i t u r e . A l o n g - t e r m study by the Food and N u t r i t i o n Board o f the Research C o u n c i l ( H o r w i t t , I 9 6 2 ) was d e f i c i e n t v i t a m i n E d i e t t o men  c a r r i e d out by f e e d i n g a  f o r 5 years.  There were no  c l i n i c a l s i g n s of v i t a m i n E d e f i c i e n c y i n these  National partially  obvious  s u b j e c t s even though the  7  b l o o d t o c o p h e r o l l e v e l s f e l l up t o 0 . 3 e r y t h r o c y t e s was  mg/lOO ml.  The h a l f - l i f e  d e c r e a s e d , but t h e r e were no o b v i o u s  of the  manifestations  o f anemia.  3 •  Animals V i t a m i n E d e f i c i e n c y can be demonstrated i n a n i m a l s f e d d i e t s low  in  vitamin E.  There are a number of v i t a m i n E d e f i c i e n c y s t a t e s i n  d i f f e r e n t s p e c i e s of a n i m a l s , affected tissue.  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  Some of t h e s i g n s of v i t a m i n E d e f i c i e n c y i n d i f f e r e n t  s p e c i e s of a n i m a l s are shown i n T a b l e  1.  A thorough documentation of the  v i t a m i n E d e f i c i e n c y s t a t e s i n a n i m a l s are reviewed by Green (1972a) Scott  (1970).  C.  C e l l u l a r Function A full  molecular  of Vitamin  understanding  E  of the mode of a c t i o n of v i t a m i n E a t  l e v e l has not y e t been r e a c h e d .  apparently unrelated disease  and  With the  the  several d i f f e r e n t ,  s t a t e s i n d i f f e r e n t animal s p e c i e s  arising  from v i t a m i n E d e f i c i e n c y i t has been d i f f i c u l t t o determine a b a s i c r o l e f o r the v i t a m i n 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 t o e x p l a i n the mechanism of a c t i o n of v i t a m i n E , the b i o l o g i c a l a n t i o x i d a n t t h e o r y and the metabolic The  specific  f u n c t i o n theory. b i o l o g i c a l f u n c t i o n o f v i t a m i n E as a l i p i d a n t i o x i d a n t  has  been i n v e s t i g a t e d f o r n e a r l y f o r t y y e a r s , s i n c e O l c o t t and M a t t e l  (19^1)  discovered the antioxidant a c t i v i t y of vitamin E.  anti-  oxidant theory  suggests t h a t t i s s u e u n s a t u r a t e d  The b i o l o g i c a l  l i p i d s are  constantly  under a t t a c k by f r e e r a d i c a l s and t h a t i n the p r e s e n c e of oxygen they become p e r o x i d i z e d .  I f s u f f i c i e n t v i t a m i n E i s not p r e s e n t ,  the  TABLE 1  Syndromes R e s u l t i n g Prom V i t a m i n E D e f i c i e n c y '  Animal S p e c i e s  Syndrome  Rat  sterility f e t a l resorption l i v e r necrosis  (male) (female) (both sexes)  Rabbit  muscular d y s t r o p h y myocardial degeneration  Dog and guinea pig  myocardial  GhickenB  encephalomalacia exudative d i a t h e s i s  Primate  m a c r o c y t i c anemia muscular d y s t r o p h y  From N a i r ,  1972.  degeneration  9  p e r o x i d a t i o n of l i p i d s becomes e x t e n s i v e  and u n c o n t r o l l e d , l e a d i n g t o  widespread damage t o i n t r a c e l l u l a r membranes, enzymes and metabolites  such as v i t a m i n A and p h o s p h o l i p i d s .  o f v i t a m i n E d e f i c i e n c y i n animals a r e c o n s i d e r e d i n g from one attempting  primary p r o c e s s ,  l i p i d peroxidation.  certain  A l l the d i v e r s e  effects  t o be secondary, stemmScientific  evidence  t o show t h a t v i t a m i n E f u n c t i o n s as a l i p i d a n t i o x i d a n t  been p r e s e n t e d  i n numerous r e v i e w s ( T a p p e l , 1962;  Tappel,  1972;  has  Witting,  1970). I n r e c e n t years t h e r e has been c o n s i d e r a b l e r e s e a r c h which exposed major weaknesses i n the a n t i o x i d a n t t h e o r y et a l . , 1968;  Green and Bunyan, 1969> Green, 1 9 7 2 b ) .  developed as t o whether o r not l i p i d deficiency.  of vitamin E  has (Bunyan  Controversy  has  p e r o x i d a t i o n occurs d u r i n g v i t a m i n  There i s no doubt t h a t v i t a m i n E has a n t i o x i d a n t  which can i n h i b i t t i s s u e l i p i d p e r o x i d a t i o n i n v i t r o .  properties  Several invest-  i g a t o r s have argued t h a t l i p i d p e r o x i d a t i o n does not occur i n v i v o t h e r e f o r e the b i o l o g i c a l f u n c t i o n of the v i t a m i n must be u n r e l a t e d i t s antioxidant a c t i v i t y .  and to  However, a r e c e n t experiment by Hafeman and  H o e k s t r a (1977) i n d i c a t e d t h a t l i p i d p e r o x i d a t i o n o c c u r s i n v i v o i n r a t s as a r e s u l t o f v i t a m i n E d e f i c i e n c y and  the p e r o x i d a t i o n p r o c e s s  is  g r e a t l y a c c e l e r a t e d d u r i n g the t e r m i n a l phase of the f a t a l c o n d i t i o n . T h i s r e p o r t opposes the b a s i c argument of the c r i t i c s of the theory.  Much more r e s e a r c h , however, i s needed t o s t r e n g t h e n  antioxidant  antioxidant the  hypothesis.  S e v e r a l i n v e s t i g a t o r s have r e c e n t l y p o s t u l a t e d t h a t v i t a m i n E act  as a c a t a l y s t or r e g u l a t o r y agent i n i n t e r m e d i a r y  specific  may  metabolism, a t a  s i t e which i s o f fundamental importance i n metabolism (Green,  1972b{ N a i r , 1972;  Schwarz, 1 9 7 2 ) .  I n s p i t e of the huge amount of  data  E  10  on v i t a m i n E p u b l i s h e d i n the l i t e r a t u r e , an u n e q u i v o c a l , ment of v i t a m i n E i n s p e c i f i c m e t a b o l i c  direct involve-  f u n c t i o n s has y e t t o be  identified.  I t i s known t h a t the a c t i v i t y of many enzyme systems are a l t e r e d i n v i t a m i n E d e f i c i e n t a n i m a l s (Green, 1972a; Mason and H o r w i t t ,  1972a).  Whether the a l t e r a t i o n o f enzymatic a c t i v i t y i s p r i m a r y o r secondary t o the breakdown o f o t h e r t i s s u e components i s s t i l l  a  controversy.  Research has been s t i m u l a t e d a t the m o l e c u l a r  level for a  involvement of v i t a m i n E i n many enzyme f u n c t i o n s . kinase  Muscle c r e a t i n e  ( O l s o n , 1 9 7 4 ) , l i v e r microsomal enzyme d r u g h y d r o x y l a t i n g  ( C a r p e n t e r and Howard, 1 9 7 4 ) , l i v e r xanthine 1 9 7 4 ) , bone marrow y - a m i n o l e v u l o n i c  direct  oxidase  a c i d synthetase  complex  (Catignani et a l . , and l i v e r  Y~  a  m  i  n  l e v u l o n i c a c i d dehydratase ( C a a s i e t a l . , 1972;  N a i r , 1972)  t h e enzymes t h a t have r e c e i v e d most a t t e n t i o n .  These s t u d i e s on r a t e s  o  ~  a r e some o f  o f enzyme s y n t h e s i s suggest a r o l e f o r v i t a m i n E i n the r e g u l a t i o n of protein synthesis.  J u s t how  v i t a m i n E may  sequence of e v e n t s i s not known.  possibly participate i n this  T h i s a r e a has been t h o r o u g h l y  by Molenaar e t a l . (1972) and B i e r i and F a r r e l l  (1976).  At t h i s time t h e r e i s no d e f i n i t i v e evidence the b i o c h e m i c a l animals.  reviewed  t o e x p l a i n many of  derangements evoked by a d e f i c i e n c y of v i t a m i n E i n  I n v e s t i g a t o r s a t t h i s time must c o n s i d e r t h a t both the  a n t i o x i d a n t t h e o r y and t h e s p e c i f i c m e t a b o l i c a c t i o n are v i a b l e and  t h a t they may  f u n c t i o n theory  biological  of vitamin  be n e i t h e r i n c o n s i s t e n t n o r  mutually  exclusive.  D.  Pharmacological  E f f e c t s of V i t a m i n E i n Human S u b j e c t s  Even though i t seems u n l i k e l y t h a t a n a t u r a l d e f i c i e n c y of v i t a m i n E o c c u r s i n man  t h e r e i s good reason t o b e l i e v e t h a t a l a r g e  \  E  11  segment of the North American population i s consuming supplementary doses of vitamin E ( F a r r e l l and B i e r i , 1975)-  Much of the popular i n t e r e s t  i n vitamin E stems from a r t i c l e s i n magazines, hooks and newspapers dealing with the therapeutic e f f i c a c y of vitamin E f o r 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 ( I 9 6 2 ) and B i e r i and F a r r e l l ( 1 9 7 6 ) .  Tocopherol  supplements e i t h e r self-administered  or prescribed by physicians vary widely i n 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 B i e r i , 1975» Horwitt and Mason, 1 9 7 2 ) . While the undesirable side e f f e c t s have been r a r e l y reported, i t i s d i f f i c u l t t o evaluate what possible pharmacological  action r e s u l t s from  the ingestion of vitamin E a t many times the generally recognized n u t r i t i o n a l requirement.  Very few c r i t i c a l studies of megavitamin E  supplementation i n man have ever been carried out. In the only systematic i n v e s t i g a t i o n of megavitamin E supplementation i n human, F a r r e l l and B i e r i (1975)> gave 100 to 800 IU vitamin E/day f o r 3 years to 28 adults.  Laboratory screening f o r t o x i c side e f f e c t s of  vitamin E supplementation by c l i n i c a l blood t e s t s 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.  I t was concluded  that megavitamin E supplements i n t h i s group produced no t o x i c side effects. Beckman (1955) has reported that vitamin E was given to patients f o r months, both o r a l l y and parenterally a t a dosage l e v e l of 300 IU vitamin E:/ day without any adverse c l i n i c a l e f f e c t s .  Greenblatt (1957) supplemented  12  the diet of s i x men with a massive dose, 40 g d-a-tocopherol f o r one month.  acetate/day  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 i n d i v i d u a l f o r 3 months produced creatinuria, c h e i l o s i s , angular stomatitis, g a s t r o i n t e s t i n a l disturbance and muscular weakness.  These  t o x i c side e f f e c t s ceased within two weeks a f t e r the vitamin E supplement a t i o n was discontinued.  Vogelsang et a l . (1947) reported that vitamin E  supplementation resulted i n hypoglycemia and depressed prothrombin l e v e l s , the l a t t e r suggesting a r e l a t i v e vitamin K deficiency. The involvement of vitamin E i n potentiating some anti-coagulation a c t i v i t y has been reported i n two studies.  Korsan-Bengtsen et a l . (1974)  reported prolonged plasma c l o t t i n g time i n 9 subjects r e c e i v i n g 300 IU a-tocopherol/day.  Corrigan and Marcus (1974) observed a  prolonged  prothrombin time i n a patient ingesting 800 IU vitamin E/day, plus warfarin and c l o f i b r a t e .  A reduction of the l e v e l of vitamin K-dependent  coagulation f a c t o r s was noted during the period of vitamin E ingestion, which returned to base-line l e v e l s a f t e r the patient stopped taking the vitamin E. An examination of the mechanism by which vitamin E might be antagonistic to vitamin K-dependent c l o t t i n g a c t i v i t y has l e d to an evaluation of the b i o l o g i c a l metabolites of the tocopherols. reported that a-tocopherylquinone This structural analog of vitamin  Woolley (1945)  was an antimetabolite of vitamin  K^.  was reported to cause hemorrhages i n  the reproductive systems of pregnant mice.  The a c t i o n 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 r a t s , o f f e r s 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  1.  Growth  .  E f f e c t s o f V i t a m i n E i n Animals  The f i n d i n g s on t h e e f f e c t o f e x c e s s d i e t a r y v i t a m i n E on the growth r a t e i n animals v a r y w i d e l y .  March e t a l . (1973) found a d e p r e s s e d  growth r a t e i n c h i c k s f e d a 2,200 IU v i t a m i n E / k g d i e t from h a t c h i n g t o 50 days. 1,000  Growth r a t e was n o t seen t o be a f f e c t e d by supplementation o f  IU v i t a m i n E/kg d i e t .  Nockels e t a l . (1975) r e p o r t e d t h a t f e e d i n g  c h i c k s 2,000 o r 4,000 IU v i t a m i n E / k g 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, h i g h e r l e v e l s o f v i t a m i n E s u p p l e m e n t a t i o n ,  such a s 8 , 0 0 0 and 64,000 iu/kg d i e t , were r e p o r t e d t o reduce t h e c h i c k body weight  significantly.  McGuaig and Motzok (1970) f e d a 10,000 IU v i t a m i n E/kg d i e t t o c h i c k s and found t h e growth r a t e was u n a f f e c t e d by t h e supplementary vitamin E treatment. rabbit  S i m i l a r r e s u l t s have a l s o been r e p o r t e d i n t h e  (Awad and G i l b r e a t h , 1975) and the r a t ( A l f i n - S l a t e r e t a l . , 1972)  f e d excess vitamin E . However, J e n k i n s and M i t c h e l l (1975) found t h a t t h e growth r a t e was i n c r e a s e d when r a t s were f e d e i t h e r a 600 o r 6,000 IU v i t a m i n E / k g d i e t f o r 2 months.  2.  Hematology March e t a l . (1973) examined r e t i c u l o c y t o s i s i n response t o v a r i o u s  dietary-antioxidants i n chicks.  They f o u n d t h a t supplementation o f e i t h e r  120 o r 220 IU v i t a m i n E./kg d i e t induced  reticulocytosis.  of v i t a m i n E; supplementation h e m a t o c r i t  l e v e l s were n o t a f f e c t e d .  At these  l a t e r study, treatment o f c h i c k s with l a r g e r doses o f v i t a m i n E  levels In a  14  (2,200 iu/kg  d i e t ) was noted t o induce  i n hematocrit fed  values  600 o r 6,000 IU  (March e t a l . ,  b o t h r e t i c u l o c y t o s i s and a r e d u c t i o n  1973)-  J e n k i n s and M i t c h e l l  (1975)  v i t a m i n E / k g d i e t t o r a t s and f o u n d 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 March e t a l . The  lengthened  menaquinone. to  (1973)  when a  o f the prothrombin time was observed by  2,200 IU  v i t a m i n E/kg d i e t was f e d t o c h i c k s .  prothrombin time was r a p i d l y n o r m a l i z e d An e a r l i e r study by M e l e t t e  and Leone  by i n j e c t i o n  (i960) was  with  the f i r s t  observe t h a t v i t a m i n E supplementation may p r o l o n g prothrombin time i n  r a t s f e d n o n i r r a d i a t e d a s w e l l a s i r r a d i a t e d beef i n t h e d i e t . The  mechanism by which h y p e r v i t a m i n o s i s E a f f e c t s prothrombin time  has n o t y e t been f u l l y e l u c i d a t e d .  The o b s e r v a t i o n by March e t a l .  t h a t an i n j e c t i o n o f v i t a m i n K r e v e r s e d t h e lengthened led  them t o s p e c u l a t e t h a t a m e t a b o l i t e  analogue o f v i t a m i n K.  (1945)  One such compound has been i d e n t i f i e d i n t h e  I962).  An e a r l i e r  found t h a t a d m i n i s t r a t i o n o f ot-tocopherylquinone  pregnant mice caused hemorrhage i n the r e p r o d u c t i v e of quinone was p r e v e n t e d  by s m a l l amounts o f v i t a m i n  l a r g e doses o f d l - o t - t o c o p h e r y l a c e t a t e . Ot-tocopherylquinone  prothrombin time  o f v i t a m i n E may be a s t r u c t u r a l  l i v e r , o t - t o c o p h e r o l - p -quinone ( C s a l l a n y e t a l . , by Woolley  (1973)  system.  study to  The a c t i o n  , b u t n o t by  S u f f i c i e n t amounts o f  may be produced f o l l o w i n g e x c e s s i v e i n t a k e o f  v i t a m i n E t o i n c r e a s e the d i e t a r y requirement f o r v i t a m i n K^. I t has been w e l l e s t a b l i s h e d t h a t v i t a m i n E d e f i c i e n c y i s c h a r a c t e r i z e d by spontaneous h e m o l y s i s o f the e r y t h r o c y t e o r e x t e n s i v e h e m o l y s i s induced  by hydrogen peroxide  or d i a l u r i c acid.  i n vitro  Low e r y t h r o c y t e  h e m o l y s i s i n v i t r o though does n o t c l e a r l y i n d i c a t e adequacy o f t i s s u e v i t a m i n E s t o r e s ( B i e r i and Poukka,  1970).  The e x i s t e n c e o f v a r i a b l e s  15  o t h e r t h a n v i t a m i n E i n t a k e t h a t can a f f e c t i n v i t r o h e m o l y s i s has t o the u n c e r t a i n t y o f t h i s t e s t  (Macdougall,  1972;  Stocks and Dormandy, 1971a; S t o c k s e t a l . , 1 9 7 i b ) . (1971a) i l l u s t r a t e d t h a t p e r o x i d e  S t o c k s and Dormandy  induced e r y t h r o c y t e a u t o x i d a t i o n  Melhorn e t a l . (1971) have shown t h a t hydrogen p e r o x i d e  o f g r e a t e r than 20 p e r cent can o c c u r i n a wide v a r i e t y o f d i s o r d e r s i n which v i t a m i n E c o n c e n t r a t i o n i s normal. hematological  19715  Melhorn e t a l . ,  i n f l u e n c e d by a number o f s u b s t a n c e s such as albumin, plasma and acid.  added  was  ascorbic  hemolysis  hematological  Some of  these  d i s o r d e r s a r e : h e r e d i t a r y and a c q u i r e d anemias, i r o n  d e f i c i e n c y anemia, and Hypervitaminosis  hemoglobinopathies. E has been found t o change the f a t t y a c i d p a t t e r n  of e r y t h r o c y t e s ( A l f i n - S l a t e r e t a l . , 1 9 7 2 ) . doses o f v i t a m i n E w i l l a l t e r the  Whether e x c e s s i v e l y h i g h  s t a b i l i t y o f the e r y t h r o c y t e membrane  has not y e t been determined.  3•  Bone C a l c i f i c a t i o n E x c e s s amounts o f v i t a m i n E were f o u n d t o 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 c a l c i u m - d e f i c i e n t or vitamin D - d e f i c i e n t d i e t s . March e t a l . (1973) f o u n d t h a t the adverse e f f e c t of h y p e r v i t a m i n o s i s on bone c a l c i f i c a t i o n was 300  iu/kg d i e t .  The  overcome when v i t a m i n D was  E  f e d a t over  mechanism by which the e x c e s s d i e t a r y v i t a m i n E  i n c r e a s e d the requirement of v i t a m i n D f o r maximum bone c a l c i f i c a t i o n i s not p r e s e n t l y known.  4.  Endocrine  Function  S e v e r a l s t u d i e s have shown a l t e r e d e n d o c r i n e animals due  to excessive d i e t a r y vitamin E intake.  function i n The  experimental  endocrine  organs  16  r e p o r t e d t o be a f f e c t e d a r e t h e s e x u a l organs (Czyba,  1966a; Escudero and  H e r r a i z , 194-2; Masson, 1941; R e i s s , 1941), a d r e n a l g l a n d ( H i l l and Hamed, 1979; F o r n i e t a l . , 1955. J e n k i n s and M i t c h e l l , 1975), thymus ( F o r n i et  a l . , 1 9 5 5 ) ; and t h e t h y r o i d g l a n d (Czyba e t a l . , 1966b; Huter, 1947;  March e t 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 ) . c o n c l u s i v e evidence  o f any adverse  While t h e r e i s l i t t l e  e f f e c t o f v i t a m i n E on t h e former  t h r e e organs, t h e e f f e c t on t h e t h y r o i d g l a n d i s f a i r l y w e l l e s t a b l i s h e d . An e a r l y i n v e s t i g a t o r o f h y p e r v i t a m i n o s i s E i n female r a t s a hypertrophy  o f t h e ovary and a l t e r a t i o n i n t h e l e n g t h o f e s t r u s c y c l e  (Reiss, 1 9 4 l ) .  Other s t u d i e s f o u n d t h a t e x c e s s d i e t a r y v i t a m i n E  male f e r t i l i t y i n r a t s (Escudero 1966a). of  and H e r r a i z , 1942) and hamsters  reduced (Czyba,  However, Masson (1941) r e p o r t e d t h a t f e e d i n g e x c e s s i v e amounts  v i t a m i n E t o hens had no e f f e c t on t h e b i r d s ' The  evidence  o f adverse  function i s contradictory.  fertility.  e f f e c t s o f h y p e r v i t a m i n o s i s E on a d r e n a l  F o r n i e t a l . (1955) found t h a t  v i t a m i n E caused an i n c r e a s e i n a d r e n a l weight i n r a t s . J e n k i n s and M i t c h e l l for  observed  In contrast,  (1975) f e d a d i e t c o n t a i n i n g up t o 6 , 0 0 0 IU v i t a m i n E/k,  e i g h t weeks and r e p o r t e d a s i g n i f i c a n t decrease  H i l l e t a l . ( i 9 6 0 ) observed  i n a d r e n a l weight.  t h a t h y p e r v i t a m i n o s i s E caused a d r e n a l  O  degeneration. The  excess  o n l y r e p o r t e d e f f e c t o f h y p e r v i t a m i n o s i s E on t h e thymus was made  by F o r n i e t a l . (1955)> who observed Hypervitaminosis thyroid gland.  a decrease  i n thymus weight.  E has been r e p o r t e d t o have an adverse  Huter (1947) was t h e f i r s t t o r e p o r t an i n j u r y t o t h e  t h y r o i d g l a n d i n r a b b i t s caused by excess d i e t a r y v i t a m i n E . Bottarelli i n the. r a t .  e f f e c t on the  (1965) f o u n d t h a t h y p e r v i t a m i n o s i s E reduced  V a l e n t i and  thyroid activity  Czyba e t a l . (1966) r e p o r t e d t h a t the a d m i n i s t r a t i o n o f  17  v i t a m i n E caused a t r a n s i t o r y s t i m u l a t i o n of t h y r o i d a c t i v i t y , which f o l l o w e d by a d e p r e s s i o n o f t h y r o i d f u n c t i o n . 220  IU v i t a m i n E/kg  (1973)  March e t a l .  was fed a  d i e t t o c h i c k s and a s s e s s e d the t h y r o i d a c t i v i t y  by  131 measuring the r a t e o f uptake and r e l e a s e o f I They f o u n d t h a t t h e a c t i v i t y o f t h e t h y r o i d was i n response t o excess  vitamin E.  by the t h y r o i d g l a n d . significantly  I t would be expected  t h y r o i d a c t i v i t y would be accompanied by some decrease T h i s was  5»  in  i n growth r a t e .  (2,200 iu/kg)  but  caused a  growth r a t e .  T i s s u e Storage The  t h a t a decrease  not seen though a t t h i s l e v e l of v i t a m i n E supplementation,  f e e d i n g a t e n - f o l d g r e a t e r amount of v i t a m i n E decreased  suppressed  of V i t a m i n  E  major pathway of v i t a m i n E a b s o r p t i o n from the  p a r a l l e l s f a t a b s o r p t i o n (Pomeranze and l i u c a r e l l o ,  intestine  1953)•  Following  a b s o r p t i o n , the t o c o p h e r o l i s t r a n s p o r t e d , v i a t h e l y m p h a t i c s , i n t h e chylomicrons  (Blomstrand  and F o r s g r e n ,  shown t h a t y - t o c o p h e r o l was e f f i c i e n t l y as was  1968).  Gloor et a l .  a-tocopherol.  Since y ^ t o c o p h e r o l i s the predominant  s i g n i f i c a n t l y underestimate  Poukka E v a r t s ,  have  absorbed from the i n t e s t i n e almost as  t o c o p h e r o l i n the N o r t h American d i e t , c a l c u l a t i o n s based on a-tocopherol  (1966)  only  vitamin E intakes ( B i e r i  and  1973).  There have been numerous s t u d i e s a t t e m p t i n g  t o determine the  q u a n t i t a t i v e 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 h i g h e r l e v e l s o f v i t a m i n E v e r s u s plasma and l i v e r t o c o p h e r o l l e v e l s .  Losowsky e t a l .  (1972)  examined the e f f i c i e n c y of a b s o r p t i o n of d i e t a r y t o c o p h e r o l i n both and a n i m a l s .  Over a narrow d i e t a r y range of i n t a k e the percentage  t i o n f a l l s o f f as the dose i s i n c r e a s e d .  intake have man  absorp-  E x c r e t i o n measurements w i t h r a t s  18  i n d i c a t e d a marked decrease i n t o c o p h e r o l a b s o r p t i o n e f f i c i e n c y a s t h e dose was i n c r e a s e d from t h e microgram t o m i l l i g r a m range (Losowsky e t a l . , B o l l i g e r and B o l l i g e r - Q u a i f e  (1956)  1972).  i n experiments w i t h r a t s have  r e p o r t e d t h a t the r e l a t i o n s h i p between t h e dose o f t o c o p h e r o l and i t s storage  i n l i v e r i s l i n e a r when b o t h a r e e x p r e s s e d  as logarithms.  They  a l s o suggested a l i n e a r r e l a t i o n s h i p between plasma t o c o p h e r o l l e v e l and the l o g o f t h e dose o f v i t a m i n E i n t a k e . Wiss e t a l .  (1972)  (1962)  I n experiments w i t h t h e c h i c k ,  r e p o r t e d s i m i l a r r e s u l t s a s the former study.  a l s o r e p o r t e d a l i n e a r r e l a t i o n s h i p between plasma  Bieri  tocopherol  c o n c e n t r a t i o n and t h e l o g o f t h e d i e t a r y v i t a m i n E i n experiments w i t h rats.  Gray  (i960) d i s a g r e e d  t h a t such a r e l a t i o n s h i p e x i s t e d .  I n a a 28 week study o f h i g h v i t a m i n E i n t a k e i n r a t s , et a l .  (1972)  found t h a t t h e plasma t o c o p h e r o l 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. administered.  Alfin-Slater  The plasma l e v e l s were n o t p r o p o r t i o n a l t o t h e dose  A l s o t o c o p h e r o l l e v e l s i n f e m a l e s were almost two-times  g r e a t e r t h a n i n male r a t s .  Awad e t a l .  (1975)  r a b b i t s r e p o r t e d t h a t supplementation with  ina  5i000 IU  4 week  study  with  v i t a m i n E/kg d i e t  i n c r e a s e d t h e plasma and l i v e r t o c o p h e r o l l e v e l s , but o n l y t h e l a t t e r was significantly  increased.  These r e s u l t s suggest t h a t t h e r e l a t i o n s h i p between t h e d i e t a r y l e v e l o f v i t a m i n E and t i s s u e s t o r a g e may be v a r i a b l e , depending on t h e animal s p e c i e s , growth r a t e o f t h e animal, dosage l e v e l and t h e t i s s u e b e i n g  6.  length of the t e s t p e r i o d ,  examined.  T i s s u e Storage o f V i t a m i n A I t has been r e c o g n i z e d f o r many y e a r s t h a t t h e r e 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 v i t a m i n E and v i t a m i n A.  Many i n v e s t i g a t o r s have  19  Moore e t a l . (1940)  found a " s p a r i n g " e f f e c t of v i t a m i n E on v i t a m i n A.  o r i g i n a l l y reported t h a t vitamin E increased l i v e r storage i n r a t s o v e r a p e r i o d o f 8 t o 12 this finding.  months.  Hichman e t a l . (1944)  Other e a r l y workers though f o u n d no  a simple  The  c o n t r a d i c t o r y evidence  r e l a t i o n s h i p between the two  Herbert  vitamins  and  c o n t r a d i c t o r y r e s u l t s r e p o r t e d i n the e a r l y s t u d i e s ,  t i s s u e l e v e l s of v i t a m i n A (Cawthorne e t a l . , 1968; Prodouz and N a v a r i ,  19755  R o e l s e t a l . , 1964).  increases  J e n k i n s and  Mitchell,  Prodouz and  (1975) chose d i e t a r y l e v e l s of v i t a m i n E r a n g i n g from 0 . 0 0  Navari  iu/week t o  iu/week and examined the e f f e c t of v i t a m i n E on v i t a m i n A s t o r a g e  rats.  not  v i t a m i n s , but i n s t e a d a complex  more r e c e n t i n v e s t i g a t i o n s i n d i c a t e d t h a t d i e t a r y v i t a m i n E  3«5  and  experiment.  In s p i t e of the  1975i  of  demonstrated t h a t t h e r e was  e f f e c t , dependent on d i e t , the dosage regimen of the two the l e n g t h o f the  confirmed  "sparing" effect  v i t a m i n E i n experiments l i m i t e d t o 4 weeks (Lemley, 1947; Morgan, 1 9 5 3 ) '  of v i t a m i n A  They found a much l a r g e r i n c r e a s e i n l i v e r v i t a m i n A p e r  IU  v i t a m i n E f e d than p e r IU o f v i t a m i n A i n the d i e t .  I n examining  d e p l e t i o n of l i v e r s t o r e s of v i t a m i n A Cawthorne and  colleagues  r e p o r t e d t h a t supplementary v i t a m i n E s i g n i f i c a n t l y d e c r e a s e d  in  the  (1968)  the r a t e of  d e p l e t i o n o f v i t a m i n A r e s e r v e s i n the r a t , t h u s c o n f i r m i n g the r e s u l t s of Moore e t a l . ( 1 9 4 0 ) . i n t a k e s ; even 1 mg  was  T h i s v i t a m i n E e f f e c t was  shown a t remarkable  low  s u f f i c i e n t t o produce a t h r e e - f o l d d i f f e r e n c e on  v i t a m i n A storage w i t h i n 6 weeks i n r a t s .  T h i s e f f e c t was  demonstrable  though o n l y when the i n i t i a l r e s e r v e s were h i g h , about 3 0 , 0 0 0 IU v i t a m i n 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  liver  r e s e r v e s of v i t a m i n A were o n l y 3 , 0 0 0 IU, which suggests a r o l e f o r v i t a m i n E i n a l t e r i n g the c a p a c i t y of the l i v e r t o b i n d v i t a m i n  A.  20  The  e f f e c t of supplementation with h i g h l e v e l s of v i t a m i n E  t i s s u e v i t a m i n A s t o r a g e has been examined by two  (1964) (50  t o 500  or  Roels e t a l .  reported that a t e n - f o l d increase i n dietary vitamin E iu/kg d i e t ) r e s u l t e d i n a 11  vitamin A storage.  600  groups.  6,000 IU  percent  v i t a m i n E:/kg d i e t J e n k i n s and M i t c h e l l  found t h a t the plasma v i t a m i n A was  intake  increase i n l i v e r  In examining the e f f e c t of supplementation  t h a t v i t a m i n E i n c r e a s e d the s t o r a g e  on  (19750  with confirmed  of v i t a m i n A i n the l i v e r .  They a l s o  s i g n i f i c a n t l y i n c r e a s e d when h i g h  l e v e l s of v i t a m i n E were f e d . Green and Bunyan  (1969)  suggested t h a t v i t a m i n E may  "spare"  v i t a m i n A by p r o t e c t i o n from o x i d a t i o n i n t h e gut, by i n c r e a s i n g v i t a m i n A a b s o r p t i o n , by i n c r e a s i n g v i t a m i n A e f f e c i e n c y , and/or by i n c r e a s i n g the s t o r a g e  of v i t a m i n A.  p r o p e r t i e s of v i t a m i n E may Roel e t a l .  (1964)  and  o r may  They noted t h a t the  not be  antioxidant  s i g n i f i c a n t i n the mechanism.  J e n k i n s and M i t c h e l l ' s  (1975) !  e x p l a i n e d by the a n t i o x i d a n t e f f e c t of v i t a m i n E .  f i n d i n g s cannot  be  Even the supplementary  ^~~) J6  l e v e l i n the experiment of Roel was  et a l .  (1964), 50  IU v i t a m i n E/kg  diet  more than adequate f o r the r a t s needs, y e t e x c e s s i v e l y l a r g e r doses  of v i t a m i n E a c c e n t u a t e d T h i s supports  (1972b)  the  " s p a r i n g " e f f e c t o f v i t a m i n E on v i t a m i n  the p r o p o s a l of T a p p e l  t h a t v i t a m i n E may  (1973). D i L u z i o (1973)  and  A.  Green  have a more s p e c i f i c i n v i v o b i o c h e m i c a l  role  i n a d d i t i o n t o i t s suggested i n v i v o and/or i n v i t r o a n t i o x i d a n t properties. 7«  Liver Lipid According  Levels  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  l i p i d l e v e l s i n c r e a s e d p r o g r e s s i v e l y as the d i e t a r y v i t a m i n E i n t a k e  total was  '  X  21  increased. for  T h i s e f f e c t was observed i n r a t s f e d h i g h l e v e l s o f v i t a m i n E  a 28-week p e r i o d . Other workers have examined the simultaneous e f f e c t s o f v a r i o u s  l e v e l s o f v i t a m i n E and v i t a m i n A ( H a r r i l l e t a l . , Mitchell,  19755 Prodouz and N a v a r i ,  1975). o r v i t a m i n  methionine ( H a r r i l l and G i f f o r d , 1966) total  19655  J e n k i n s and  E and a r g i n i n e o r  on l e v e l s o f l i v e r c h o l e s t e r o l and  lipids. Contrary t o t h e r e s u l t s o f A l f i n - S l a t e r e t a l .  Gifford  (1966)  decreased  (1972), H a r r i l l  and  found that i n c r e a s i n g the d i e t a r y l e v e l of vitamin E  the l e v e l o f 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 a r e n o t c o n s i s t e n t l y seen though when examining the simultaneous e f f e c t s o f v i t a m i n E and A on t i s s u e l i p i d l e v e l s .  (1975)  and H a r r i l l e t a l .  (1965)  vitamin E s i g n i f i c a n t l y decreased cholesterol.  Prodouz and N a v a r i  reported that increasing dietary l i v e r t o t a l l i p i d s and i n c r e a s e d  However, J e n k i n s and M i t c h e l l  (1975)  liver  reported that increasing  d i e t a r y v i t a m i n E s i g n f i c a n t l y i n c r e a s e d t o t a l l i p i d s and d e c r e a s e d cholesterol i n rat l i v e r .  The reason f o r t h e d i s c r e p a n c y  t h i s a r e a remains obscure. to  I t might w e l l be t h a t t h e r a t i o o f v i t a m i n E  vitamin A i s the d e c i s i v e f a c t o r determining  v i t a m i n s on t i s s u e l i p i d l e v e l s i n these  8.  Blood L i p i d  i n results i n  the e f f e c t o f t h e s e  experiments.  Levels  The r e l a t i o n s h i p between h i g h d i e t a r y v i t a m i n E and plasma l e v e l i s not y e t c<hear.  lipid  Most i n v e s t i g a t i o n s i n t h i s a r e a have examined  the a b i l i t y o f supplemental v i t a m i n E t o a l t e r plasma c h o l e s t e r o l l e v e l s . Some s t u d i e s have r e p o r t e d 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 v i t a m i n 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 N a v a r i ,  1975)-  Chen e t a l .  (1972)  showed t h a t r a i s i n g  the  d i e t a r y v i t a m i n E i n t a k e 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 , p r o p o r t i o n a l t o the amount supplemented.  The r e g r e s s i o n curves  c h o l e s t e r o l l e v e l t o v i t a m i n E i n t a k e (up t o 50  of  iu/kg d i e t ) were not  l i n e a r though. However, s e v e r a l workers have r e p o r t e d t h a t h i g h d i e t a r y v i t a m i n E i n t a k e s 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  1975;  Gilbreath,  Horn e t a l . ,  1962), c h i c k s  r a t s ( J e n k i n s and M i t c h e l l ,  1975).  d i e t s formulated  5»000 IU  to contain  serum c h o l e s t e r o l i n r a b b i t s . l e v e l s of v i t a m i n E  et a l . ,  Awad and G i l b r e a t h v i t a m i n E/kg  1966),  (1975)  and  found  that  d i e t had no e f f e c t  (1975)  J e n k i n s and M i t c h e l l  (6,000 iu/kg  c h o l e s t e r o l l e v e l s were not  (Koyangi  d i e t ) t o r a t s and  observed  on  a l s o f e d high t h a t plasma  significantly affected.  Some i n v e s t i g a t o r s have r e p o r t e d t h a t h i g h doses of v i t a m i n E a c t u a l l y caused h y p e r c h o l e s t e r o l e m i a Little  evidence  (Bruger,  194-5; Campbell, 1952).  o f the e f f e c t of l a r g e d o s e s of v i t a m i n E on b l o o d  l e v e l s can be g a i n e d though from e i t h e r of these two the u n n a t u r a l e x p e r i m e n t a l  c o n d i t i o n s employed.  l e v e l o f v i t a m i n E supplementation,  s t u d i e s because of  Both s t u d i e s f e d  a t h e r o s c l e r o t i c d i e t s t o r a b b i t s and v i t a m i n E was The  lipid  injected intramuscularly.  the l e n g t h of t r e a t m e n t  the d i e t a r y i n g r e d i e n t s v a r y w i d e l y i n the experiments r e p o r t e d above. Whether one  o r more of these  c o n d i t i o n s 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 r e p o r t e d i n the l i t e r a t u r e i s n o t y e t known.  and  22  CHAPTER I I I MATERIALS AND METHODS  A.  Animal Care N i n e t y female weanling W i s t a r r a t s , 45 - 55 g i n weight,  from B i o b r e e d i n g L a b o r a t o r i e s , Ottawa, O n t a r i o .  were o b t a i n e d  Upon a r r i v a l t h e y were  randomly d i v i d e d i n t o s i x groups o f f i f t e e n animals each.  F o r the i n i t i a l  two-week p e r i o d 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 room m a i n t a i n e d a t  s t a i n l e s s s t e e l cages k e p t i n an a i r - c o n d i t i o n e d  23-25°C.  L i g h t i n g was r e g u l a t e d a u t o m a t i c a l l y t o  p r o v i d e a l t e r n a t e 12-hour p e r i o d s o f l i g h t and darkness 6:00  a.m. t o 6:00  p.m.).  Food and water were g i v e n a d l i b i t u m  t h e e x p e r i m e n t a l p e r i o d o f s i x t e e n months (December  B.  ( l i g h t on from throughout  1973 t o A p r i l 1975)•  Experimental D i e t s S i x e x p e r i m e n t a l d i e t s were used: a t o c o p h e r o l - f r e e d i e t , and t h e  same d i e t supplemented w i t h e i t h e r  25, 250, 2,500, 10,000 o r 25,000  vitamin E (dl-a-tocopherol acetate) per kg d i e t . modified Draper's  (1964) Standard  These were based  V i t a m i n E-Free d i e t .  IU  on a  The c o m p o s i t i o n  of t h e d i e t s and t h a t o f t h e m i n e r a l and v i t a m i n mixes used a r e shown i n T a b l e 2.  D i e t a r y i n g r e d i e n t s were o b t a i n e d from T e x l a b M i l l s , Madison,  W i s c o n s i n , U.S.A.  C.  E x p e r i m e n t a l Groups The  s i x e x p e r i m e n t a l groups were d e s i g n a t e d a s shown below. Group A  :  Vitamin E - f r e e d i e t - Basal  Group B  :  B a s a l d i e t p l u s 25 IU v i t a m i n E/kg  Group C  :  B a s a l d i e t p l u s 250 IU v i t a m i n E / k g  Group D  :  Basal d i e t plus  2,500 IU v i t a m i n  E/kg  TABLE 2  Composition  of the Basal Diet  Ingredient  f  D-Dextrose  64.9  Vitamin-free casein  20.0  Corn o i l , tocopherol stripped Salt mix (no. 4l64)  0  10.0  4.0  2  Vitamin mix  0.6  Choline chloride  0.5  Modified from Draper, H.H., e t a l ,  (1964)  J . Nutr. 84, 395-400.  Trovided the following as g/kg d i e t : CaCO^,  6-54;  CaHPO^  s  2H 0, 14.2; 2  NaCl, 4.3; K HP0^, 3.09; K (C R" 0;p -H 0, 9-46; MgCO^, 1.64; F e t C g H ^ ) • 2  3  5  0.055; ZnCO^,  3H 0, 0.64; MnS0^-H 0, 2  6  2  2  0.018; CuSO^ • 5H 0, 2  0.007i  KI, 0.0018. 'Provided the following amount per kg d i e t ( i n III): 25.000 vitamin A as r e t i n y l palmitate; 2,000 e r g o c a l c i f e r o l ; (inmg): menadione, 1; b i o t i n , 0.1; vitamin B^ , 0.1; calcium pantothenate, 2  10; f o l i c acid, 1;  n i a c i n , 25» pyridoxine HC1, 5*0; r i b o f l a v i n , 5-0; thiamine HC1, 10.  2k  D.  Group E  :  Basal diet plus 10,000 IU vitamin E/kg  Group F  :  Basal diet plus 25i000 IU vitamin E/kg  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 treatment.  and 16 months of  Blood samples were taken by t a i l cutting a f t e r anesthetizing  the r a t s with anhydrous d i e t h y l ether (Fisher S c i e n t i f i c ) f o r the determination of hemoglobin, hematocrit and erythrocyte hemolysis.  Blood  was  d i r e c t l y drawn from the t a i l into a sodium oxalate coated Miale prothrombin pipet f o r estimation of prothrombin time. At 11 months, a 24-hour urine sample was c o l l e c t e d f o r the determination of urinary creatine and creatinine. The samples were stored i n p l a s t i c b o t t l e s without preservative at -20°G u n t i l analysis. Four r a t s from each group were k i l l e d a f t e r 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 d i e t h y l ether.  Blood was drawn from the i n f e r i o r vena cava using a heparinized  syringe.  Plasma was obtained by centrifugation and placed i n t o small  p l a s t i c tubes and frozen at -20°G u n t i l further processing.  The  storage  of the i n d i v i d u a l plasma a l i q u o t s permitted avoidance of repeated thawing and re-freezing. and t o t a l l i p i d s .  Plasma samples were analyzed f o r vitamin E, cholesterol Plasma alkaline phosphatase a c t i v i t y and vitamin A  ( r e t i n o l ) were also measured i n r a t s treated f o r 16 months. Immediately a f t e r exsanguination, the l i v e r , spleen, heart, kidney and uterus were rapidly removed.  They were trimmed f o r extraneous  25  t i s s u e s , washed i n c o l d p h y s i o l o g i c a l s a l i n e s o l u t i o n and then weighed. The l i v e r was f r o z e n a t l i p i d s and  -20°C  f o r a n a l y s i s o f v i t a m i n A, v i t a m i n E ,  cholesterol.  The l e f t femur was removed and s t r i p p e d o f s o f t t i s s u e . frozen at  total  -20°C  I t was  f o r a n a l y s i s o f hone a s h , c a l c i u m and i n o r g a n i c  E..  Biochemical Determinations  1.  Hemoglobin and H e m a t o c r i t  then  phosphate.  Hemoglobin was determined by the s p e c t r o p h o t o m e t r i c method d e s c r i b e d by E i l e r s  (1967).  A  0.02  ml a l i q u o t o f b l o o d was d i l u t e d w i t h  5 ml  of  cyanmethemoglobin reagent (Hyland D i v i s i o n , T r a v e n o l L a b o r a t o r i e s I n c . , C a s t a Mesa, C a l i f . , U.S.A.) and then r e a d a t 5^0  nm  spectrophotometer.  c a l c u l a t e d by  Hemoglobin c o n c e n t r a t i o n was  o p t i c a l d e n s i t y (0D) a t 5^0  u s i n g the Beckman  nm w i t h a f a c t o r determined on the  c a l i b r a t i o n curve as shown i n F i g u r e  DU-2  multiplying hemoglobin  2.  H e m a t o c r i t was r e a d from a h e p a r i n i z e d m i c r o - h e m a t o c r i t tube (Fisher S c i e n t i f i c ) after centrifugation at (Eilers,  2.  11,500 x  G for  5  minutes  1967).  Prothrombin Time The prothrombin time was determined by a micromethod o f the s t a n d a r d  one-stage prothrombin time method d e s c r i b e d by M i a l e and Winningham  (1967).  T h i s procedure used a s i l i c o n i z e d M i a l e Prothrombin P i p e t (Dade, Miami, F l . ) f o r the c o l l e c t i o n o f c a p i l l a r y b l o o d . o f sodium plasma.  o x a l a t e s o l u t i o n (100  mM)  I t was mixed w i t h a measured amount  and c e n t r i f u g e d t o o b t a i n o x a l a t e d  The t e s t was t h e n performed by blowing the o x a l a t e d plasma i n t o a  t e s t tube o f t h r o m b o p l a s t i n - C a C l m i x t u r e (Dade) a t 2  time was  noted.  37°C  and the  clotting  FIGURE 2  Standard Curve F o r Hemoglobin  g Hb/lOO ml  27 3•  Erythrocyte  Hemolysis  The hemolysis procedure was that described by Draper and (1966).  Gsallany  I t i s based on the degree of spontaneous hemolysis of erythrocytes  i n a buffered i s o t o n i c saline s o l u t i o n .  Following incubation of the  erythrocyte a l i q u o t s , 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.  A  b  % hemolysis =  x 100 c  where = absorbance of buffer solution at 415 nm A  c  4.  = absorbance of H^O 2  solution at 415 nm  Urinary Creatine and Creatinine The urinary creatine and creatinine l e v e l s were determined by a  method based on the Jaffe reaction as described by Henry et a l . ( 1 9 7 4 ) . Creatinine was determined by quantitating the red pigment, a l k a l i n e creatinine p i c r a t e .  The o p t i c a l density was measured with a Beckman DU-2  spectrophotometer at 500  nm.  The urinary creatinine l e v e l 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 r a t s was then expressed as follows, mg creatinine/kg body weight/24 hours  28  The  u r i n a r y c r e a t i n e l e v e l was  creatinine before The  c r e a t i n i n e (mg  p l u s mg  mg  The  of creatine to c r e a t i n i n e .  t h e n c a l c u l a t e d as  follows.  reformed c r e a t i n i n e  x  c r e a t i n e as c r e a t i n i n e / m l u r i n e )  ^s  c r e a t i n e as  creatinine/ml  urine = t o t a l  u r i n a r y c r e a t i n e o f the r a t s was  mg 5.  and a f t e r the d e h y d r a t i o n  u r i n a r y c r e a t i n e l e v e l was  total  determined by the d i f f e r e n c e i n  c r e a t i n i n e - preformed creatinine  then e x p r e s s e d as  follows,  c r e a t i n e / k g body weight/24 h o u r s  Plasma V i t a m i n A Plasma v i t a m i n A l e v e l s were determined a c c o r d i n g  described  by Neeld and  Pearson  c l a s s i c Carr-Price technique.  (1963)>  method  which i s a m o d i f i c a t i o n of  the  blue  chromophore produced by  the  i n t e r a c t i o n o f 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 a t 620  nm  The  t o the  on a Beckman DU-2  spectrophotometer and  i n d i c a t i o n o f the amount o f v i t a m i n A p r e s e n t Standard curves f o r mg  An  The  average s l o p e  plasma.  (Hoffmann-La Roche Inc., N u t l e y ,  of the  curve a t  620  i l l u s t r a t i o n of t h i s curve i s shown i n F i g u r e  nm  was  6.  and  e x p r e s s e d as u,g p e r 100  ml  N.J.,  found t o be  7«53•  3-  Plasma v i t a m i n A l e v e l s were c a l c u l a t e d from the s t a n d a r d ( F i g . 3)  an  v i t a m i n A/lOO ml plasma were e s t a b l i s h e d  using a l l trans r e t i n y l acetate U.S.A.).  i n the  gave  curve  of plasma.  Plasma V i t a m i n E Plasma v i t a m i n E l e v e l s were determined a c c o r d i n g  described  by Fabianek e t a l .  (1968), which  t o the method  i s a m o d i f i c a t i o n o f the  classic  FIGURE 3 STANDARD CURVE FOR PLASMA VITAMIN A  0.5  Llg R e t i n o l / t u b e  (2.0  ml c h l o r o f o r m )  30  Emmerie-Engel t e c h n i q u e . ion  The a n a l y s i s i s based on a r e d u c t i o n o f f e r r i c  t o the f e r r o u s form by t o c o p h e r o l s , w i t h the r e s u l t a n t f o r m a t i o n o f a  p i n k complex o f f e r r o u s i o n s w i t h 4,7-diphenyl-10,10-phenanthroline.  The  use o f p h o s p h o r i c a c i d p r e v e n t s the photochemical r e d u c t i o n o f f e r r i c c h l o r i d e and a l s o r e d u c e s i n t e r f e r e n c e o f carotene t o a minimum.  The  complex was  nm.  measured w i t h a Beckman DU-2  spectrophotometer a t 536  Standard curves f o r mg d l - a - t o c o p h e r o l / l O O ml plasma were e s t a b l i s h e d u s i n g d l - a - t o c o p h e r o l (Hoffmann-La Roche I n c . , N u t l e y , N.J., average  s l o p e o f the curve a t 536  nm  was  found t o be 2.54  The plasma t o c o p h e r o l c o n c e n t r a t i o n was s t a n d a r d curve ( F i g . 4)  U.S.A.).  The  ( F i g . 4).  t h e n c a l c u l a t e d from the  and e x p r e s s e d as mg t o c o p h e r o l p e r 100  ml o f  plasma•  7.  Plasma C h o l e s t e r o l Plasma c h o l e s t e r o l was  d e s c r i b e d by R o s c h l a u e t a l . mixed w i t h 5  m  assayed by an enzymatic  (1974).  M ammonium phosphate  0.1%  hydroxypolyethoxydodecane; mU/ml).  The  b u f f e r , pH 7>  The  d e n s i t y was  procedure  ml a l i q u o t o f plasma (1.7  M  was  methanol;  M acetylacetone5  0.02  c a t a l a s e > 670  u/ml;  c h o l e s t e r o l * - .,  c o n t e n t s o f the t e s t t u b e s were mixed w e l l  u s i n g a V o r t e x mixer and 0.02 added.  0.02  l of c h o l e s t e r o l r e a g e n t mixture  0.57  e s t e r a s e > 26  A  color  ml o f c h o l e s t e r o l o x y d a s e (4 u/ml)  samples were i n c u b a t e d a t  37°C  for  60  was  minutes and the  r e a d a t 410 nm a g a i n s t a sample blank on a Beckman  optical  DU-2  spectrophotometer. Standard curves f o r mg  c h o l e s t e r o l / l O O ml plasma were determined  u s i n g pure c h o l e s t e r o l ( P r e c i s e t C h o l e s t e r o l  B o e h r i n g e r Mannheim GmbH, Mannheim, W.  ).  Germany  The average  s l o p e o f the  FIGURE 4 STANDARD CURVE FOR PLASMA VITAMIN E  0.40  0.80  1.20  mg d l - a - t o c o p h e r o l / l O O ml e t h a n o l  1.60  2.00  32  curve a t 4 1 0  nm was f o u n d t o be 8 1 8 . 1  ( i l l u s t r a t e d i n Figure  5)-  The plasma c h o l e s t e r o l l e v e l s were c a l c u l a t e d as shown below, c h o l e s t e r o l / l O O ml plasma = 0 D ^ ^ Q  mg  8.  Plasma T o t a l  ^  x  818.1  Lipids  T o t a l l i p i d s i n plasma were measured by the method o f Amenta  (1970).  L i p i d s were e x t r a c t e d from the plasma i n t o a chloroform-methanol s o l u t i o n 1.5*1  (v/v)  and n o n - l i p i d i m p u r i t i e s  w i t h an aqueous GaClg s o l u t i o n  and methanol were removed by a wash  (0.5%)•  An a l i q u o t o f the l i p i d - c o n t a i n i n g  c h l o r o f o r m phase was e v a p o r a t e d and the t o t a l l i p i d measured by w i t h an a c i d dichromate r e a g e n t  (0.5%)'  The amount of dichromate reduced  was determined by the change i n a b s o r p t i o n measured a t 430 Beckman  DU=2  s p e c t r o p h o t o m e t e r which was d i r e c t l y  l i p i d present. palmitic acid  nm on a  proportional  t o the  (0.1%)  The s t a n d a r d f o r t o t a l l i p i d s was l e c i t h i n  (0.15%), d i s s o l v e d  reacting  and  i n chloroform.  T o t a l l i p i d s i n plasma were t h e n determined a c c o r d i n g t o the f o r m u l a shown below. A:x mg t o t a l l i p i d s / l O O ml plasma =  x A  Z  s  where A A  x s  = 0D,,OA  430  nm  method b l a n k — 0 D , , o „  430  nm  sample *  = 0D,lOO reagent b l a n k — 0 D , . . o A standard 4 3 0 nm ^° (00 nm  Z  = c o n c e n t r a t i o n o f the s t a n d a r d x d i l u t i o n  9•  Plasma A l k a l i n e Phosphatase  factor  Plasma a l k a l i n e phosphatase was assayed by a procedure d e s c r i b e d by Henry e t a l . A 0.1  (1974).  ml a l i q u o t o f plasma was mixed with 1 ml o f 0.02  M phenol  33  FIGURE 5  STANDARD CURVE FOR PLASMA CHOLESTEROL  O.50 4-  34  phosphate.  The h y d r o l y s i s p r o d u c t , p h e n o l , was condensed w i t h  4-aminoanti-  p y r i n e and t h e n o x i d i z e d w i t h a l k a l i n e f e r r i c y a n i d e t o g i v e a r e d complex which was measured a t 5°0 0  nm on a Beckman DU-2  spectrophotometer.  One u n i t o f a l k a l i n e phosphatase a c t i v i t y was d e f i n e d a s the amount  of enzyme i n 100  ml o f plasma which l i b e r a t e d 1 mg phenol i n 15 minutes  a t 37°C. The amount o f a l k a l i n e phosphatase i n the plasma was then c a l c u l a t e d as f o l l o w s . A  x  — A  u n i t s a l k a l i n e phosphatase/lOO ml plasma =  c x  A  Z  s  where A  x  = absorbance o f unknown a t 500  nm  A  c  = absorbance o f c o n t r o l a t 500  nm  A  s  = absorbance o f s t a n d a r d a t 500  nm  Z.  = c o n c e n t r a t i o n o f the s t a n d a r d x d i l u t i o n f a c t o r  10.  Liver Lipid Extraction The c o n c e n t r a t i o n s o f v i t a m i n A, 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  M v e r m o f s r a t s were measured i n the c h l o r o f o r m - e x t r a c t o f l i v e r , p r e p a r e d by a m o d i f i c a t i o n of t h e methods o f F o l c h e t a l .  (1957)  and Amenta  (1970).  The l i p i d e x t r a c t i o n procedure was c a r r i e d out as f o l l o w s . One h a l f g o f 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 w i t h a S o r v a l l micro-homogenizer a t t a t c h e d t o a S o r v a l l omni-mixer and t h e n w i t h a P o t t e r - E l v e h j e m g l a s s and t e f l o n p l u n g e r type o f homogenizer.  One h a l f m i l l i l i t e r o f crude homogenate  was e x t r a c t e d w i t h 3 ml o f chloroform-methanol 1.521  (v/v)  i n a glass  stoppered c e n t r i f u g e tube by a g i t a t i n g v i g o r o u s l y f o r 3 minutes w i t h a  35  V o r t e x mixer.  The tubes were then c e n t r i f u g e d a t 1 , 2 0 0  x G for 5  The upper c h l o r o f o r m phase was p i p e t t e d o f f and r e t a i n e d . phase was  e x t r a c t e d w i t h 3 ml o f chloroform-methanol  and r e c e n t r i f u g e d .  The l i q u i d phase was  phase from the f i r s t e x t r a c t i o n . of  aqueous CaClg s o l u t i o n ( 6 7 - 5  and then c e n t r i f u g e d a t 1 , 2 0 0  x G.  1.5*1  (v/v)  supernatant as b e f o r e  combined w i t h the c h l o r o f o r m  The mixture was mM)  The  minutes.  t h e n washed w i t h 3 ml  by shaking v i g o r o u s l y f o r 3  minutes  A l i q u o t s o f the l i p i d - c o n t a i n i n g  c h l o r o f o r m phase were t h e n ready f o r the v i t a m i n A, c h o l e s t e r o l  and  t o t a l l i p i d analyses.  11.  L i v e r Vitamin A The l e v e l o f v i t a m i n A i n l i v e r was determined a c c o r d i n g t o the  method o f N e e l d and Pearson  (1973)'  c h l o r o f o r m phase was d i l u t e d 1 : 3 used f o r the v i t a m i n A a n a l y s i s .  An a l i q u o t o f the l i p i d - c o n t a i n i n g  w i t h c h l o r o f o r m , from which 0 . 2 ml  was  The b l u e chromophore produced by the  i n t e r a c t i o n o f t r i f l u o r o a c e t i c a c i d and v i t a m i n A i n c h l o r o f o r m e x t r a c t was measured a t 6 2 0 nm  on a Beckman D U - 2  spectrophotometer.  Standard curves f o r r e t i n o l e q u i v a l e n t s p e r tube were e s t a b l i s h e d u s i n g a l l t r a n s r e t i n y l a c e t a t e (Hoffmann-La Roche I n c . ) . s l o p e o f the curve a t 6 2 0 nm was f o u n d t o be 7 ' 1 9 .  The  average  An i l l u s t r a t i o n o f  t h i s c a l i b r a t i o n curve i s shown i n F i g u r e 6 . In  the p r e l i m i n a r y l a b o r a t o r y work, known amounts o f a l l t r a n s  r e t i n y l a c e t a t e were added t o l i v e r b e f o r e the l i p i d e x t r a c t i o n A n a l y s i s was  c a r r i e d out a c c o r d i n g t o the method d i s c u s s e d above and the  p e r cent r e c o v e r y was cent was  procedure.  calculated.  I t was found t h a t r e c o v e r y o f 1 0 3  per  attained.  The v i t a m i n A c o n c e n t r a t i o n i n l i v e r was  t h e n c a l c u l a t e d from the  36  s t a n d a r d curve  12.  ( F i g . 6 ) and e x p r e s s e d as fig p e r g of l i v e r .  L i v e r Vitamin K The l e v e l of a - t o c o p h e r o l i n l i v e r was  determined  t h i n - l a y e r chromatography (TLC) method o f B i e r i Two-dimensional a n a l y s i s was  a c c o r d i n g t o the  (1969).  c a r r i e d out on p r e c o a t e d  silica  g e l G TLC p l a t e s ( R e d i / P l a t e , F i s h e r S c i e n t i f i c ) u s i n g benzene-elthanol ( 9 9 l ) and hexane-ethanol  ( 9 l ) m i x t u r e s as s o l v e n t s .  J  !  had evaporated from the second dimension  A f t e r the s o l v e n t  r u n the chromatograms were  sprayed w i t h 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. a i d e d 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 - t o c o p h e r o l F o l l o w i n g e l u t i o n , a c o l o r i m e t r i c d e t e r m i n a t i o n of the i n the e t h a n o l e l u a t e was  c a r r i e d out.  This  spot. a-tocopherol  The method e s s e n t i a l l y c o n s i s t e d  of e x t r a c t i n g the e t h a n o l e l u a t e w i t h x y l e n e , f o l l o w e d by the a d d i t i o n o f 0.4%  4,7-diphenyl-10,10-phenanthroline,  8 5 % orthophosphoric Standard  0 . 6 % f e r r i c chloride  acid.  curves f o r fig a - t o c o p h e r o l p e r tube were e s t a b l i s h e d u s i n g  d l - a - t o c o p h e r o l (Hoffmann-La Roche Inc.).^ a t 5 3 6 nm was  and  found t o be  10.2  The  average  (illustrated in Fig.  s l o p e o f the  curve  7)'  In the p r e l i m i n a r y l a b o r a t o r y work, known amounts o f d l - a - t o c o p h e r o l were added t o l i v e r from v i t a m i n E - f r e e t r e a t e d r a t s p r i o r t o s a p o n i f i c a tion.  A n a l y s i s was  c a r r i e d out and the per cent r e c o v e r y was  I t was found t h a t up t o 8 4 . 6 Consequently this  per cent r e c o v e r y c o u l d be  a c o r r e c t i o n f a c t o r of 1.18  was  determined.  obtained.  employed t o compensate f o r  loss. The a - t o c o p h e r o l c o n c e n t r a t i o n i n l i v e r was  s t a n d a r d curve  (Fig. 7)  fig p e r whole l i v e r .  then c a l c u l a t e d from  and e x p r e s s e d as fig p e r g o f l i v e r and a l s o  as  the  FIGURE 6  STANDARD CURVE FOR LIVER VITAMIN A  0.94  |lg R e t i n o l / t u b e  (2.0 ml c h l o r o f o r m )  FIGURE 7 STANDARD CURVE FOR LIVER VITAMIN E  39  13•  Liver Total Lipids T o t a l l i p i d s i n l i v e r were determined by the method d e s c r i b e d by  Amenta ( 1 9 7 0 ) . phase was  One  half m i l l i l i t e r  mixed with 1 . 5  o f the l i p i d - c o n t a i n i n g c h l o r o f o r m  ml c h l o r o f o r m , 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 w i t h an a c i d dichromate The  amount o f dichromate  when measured a t 4 3 0 nm  reduced was  In  determined by the change i n a b s o r p t i o n  on a Beckman DU-2  p r o p o r t i o n a l t o the amount o f l i p i d  reagent.  spectrophotometer  and was  directly  present.  the p r e l i m i n a r y l a b o r a t o r y work, known amounts o f l i p i d  added t o l i v e r b e f o r e the l i p i d e x t r a c t i o n p r o c e d u r e .  Analysis  were was  c a r r i e d out a c c o r d i n g t o the method d i s c u s s e d above and the p e r cent r e c o v e r y was was  calculated.  I t was  f o u n d t h a t the r e c o v e r y o f 1 0 1  per cent  attainable. T o t a l l i p i d s i n l i v e r were c a l c u l a t e d by t h e same f o r m u l a used  for  d e t e r m i n i n g plasma t o t a l l i p i d s  (See S e c t i o n 8.)  and were e x p r e s s e d  as f o l l o w s . mg  14.  Liver  total lipid/g  liver  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 t e s t of Roschlau et a l . ( 1 9 7 4 ) . c h l o r o f o r m phase was  determined by the enzymatic  A 1.5  nil a l i q u o t o f the  lipid-containing  e v a p o r a t e d t o d r y n e s s by f l u s h i n g w i t h n i t r o g e n i n  a t e s t tube p l a c e d i n a h e a t i n g b l o c k s e t a t 5 0 ° C r e s i d u e was  The  cholesterol-  d i s s o l v e d i n 5 ml o f c h o l e s t e r o l r e a g e n t mixture  (See S e c t i o n 7>  Plasma C h o l e s t e r o l f o r a d e s c r i p t i o n o f the r e a g e n t m i x t u r e ) by treatment a t O^C.  color  The  sonic  c o n t e n t s o f t h e t e s t tube were mixed w e l l u s i n g  a Vortex mixer and 0 . 0 2 ml o f c h o l e s t e r o l o x y d a s e ( 4 u/ml) samples were i n c u b a t e d a t 3 7 ° C f o r 6 0 minutes  was  added.  The  and the o p t i c a l d e n s i t y 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. 368.1  The c a l i b r a t i o n f a c t o r of the curve at 410 nm was found to be  ( i l l u s t r a t e d 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. out  Analysis was carried  according to the method discussed above and per cent recovery was  calculated.  I t 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 ( F i g . 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) f o r 24 hours.  was dried i n an isothermal oven at  105°C f o r 24 hours, weighed and then  ashed i n a furnace at  The defatted bone  650°C f o r 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. per cent ash = 16.  weight of ash (g) weight of defatted dry bone (g)  x 100  Femoral Calcium The calcium i n 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 i n 3 N HC1 was d i l u t e d with lanthanum chloride solution so that the calcium concentration l a y 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 Spectrophotometer. Calibration curves f o r mg per cent calcium were determined using AnalaR calcium carbonate (Canadina Lab. Supplies Ltd.) i n AnalaR hydrochloric a c i d . found to be 6.67-  The c a l i b r a t i o n f a c t o r of the curve at 422.7 mu. was 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 9' The t o t a l calcium i n the femur was calculated from the c a l i b r a t i o n curve ( F i g . 9) and expressed as g calcium per femur.  The per cent calcium  i n the dry and defatted femur was calculated as shown below.  per cent calcium „ i n femur 17-  =  weight of calcium i n femur (g) v—rz— T; 7—v~ x weight of defatted dry femur (g) ^  100  Femoral Inorganic Phosphate Inorganic phosphate i n the femur was determined by a modified method  of Fiske and Subbarrow  (1970).  The inorganic phosphate analysis of the d i l u t e d bone-HCl solution involved photometric determination of the molybdenum blue formed by reduction of the molybdenum diphosphate, using aminonaphtholsulfonic a c i d as the reducing agent. C a l i b r a t i o n curves f o r Llg phosphate per tube were determined using a phosphorus standard (5 |ig/ml).  660  nm was found to be  The c a l i b r a t i o n f a c t o r of the curve at  35*8 ( i l l u s t r a t e d  in Fig.  10).  The t o t a l inorganic phosphate i n the femur was calculated from the c a l i b r a t i o n curve ( F i g . 10) and expressed as g inorganic phosphate per femur.  The per cent phosphate i n the dry defatted femur was  as shown below.  calculated  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  p e r cent phosphate = i n femur  F.  weight o f phosphate i n femur ( g ) weight o f d e f a t t e d d r y femur ( g )  x  100  S t a t i s t i c a l A n a l y s i s o f the D a t a The raw d a t a were a n a l y z e d s t a t i s t i c a l l y by computer a t t h e  Computing Centre o f the U n i v e r s i t y o f B r i t i s h Columbia. computer program package ( K i t a , 1976)  The SPSS  was employed t o draw up a program  f o r the d e s i r e d a n a l y s e s . Experimental variance.  d a t a were t e s t e d by a p p l y i n g one-way a n a l y s i s o f  The homogeneity o f v a r i a n c e s was t e s t e d by Cochrans C t e s t .  A l o g t r a n s f o r m a t i o n was used f o r d a t a with heterogeneous v a r i a n c e . Statistical  comparisons were made u s i n g r e g r e s s i o n a n a l y s i s and  Duncan's new m u l t i p l e - r a n g e  t e s t f o r d a t a c o n t a i n i n g equal number o f  samples among groups o r by S c h e f f e ' s range t e s t f o r d a t a c o n t a i n i n g unequal number o f samples on a p r o b a b i l i t y cent f o r a l l measurements.  l e v e l o f a t l e a s t 95 p e r  46  CHAPTER IV RESULTS  A.  Body and Organ Weights R e s u l t s o f the e f f e c t o f d i f f e r e n t l e v e l s o f d i e t a r y v i t a m i n E on  growth i n r a t s t r e a t e d f o r 8 and 16 months are p r e s e n t e d respectively.  i n T a b l e s 3 and 4  As can be seen i n T a b l e 3 , h i g h l e v e l s o f v i t a m i n E had a  s i g n i f i c a n t d e p r e s s i n g e f f e c t ( P < 0 . 0 5 ) on growth of r a t s t r e a t e d f o r 8 months.  V i t a m i n E d e f i c i e n c y r e s u l t e d i n a g r e a t e r growth  t h a n excess d i e t a r y v i t a m i n E supplementation.  depression  The r e s u l t s i n Table  a l s o show t h a t growth r a t e was s i g n i f i c a n t l y reduced  4  i n r a t s t r e a t e d with  h i g h l e v e l s o f d i e t a r y v i t a m i n E f o r 16 months. The  organ weights (expressed i n mg/lOO g body weight) o f r a t s t r e a t e d  w i t h d i f f e r e n t l e v e l s o f v i t a m i n E f o r 8 and 16 months are a l s o shown i n T a b l e s 3 and 4 .  Treatment f o r 8 months a t a l l l e v e l s o f v i t a m i n 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 , u t e r u s , s p l e e n o r  k i d n e y weight.  However, i n c r e a s i n g t h e d i e t a r y l e v e l of v i t a m i n E was  found t o s i g n i f i c a n t l y i n c r e a s e ( P < - 0 . 0 5 ) the r e l a t i v e h e a r t weights i n r a t s t r e a t e d 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  h e a r t weights (Y) and l o g d i e t a r y v i t a m i n E (x) was X= 232.316 + 1 1 . 3 4 7 X , the c o r r e l a t i o n c o e f f e c i e n t (R) b e i n g 0.465 ( P < 0 . 0 5 ) -  R e s u l t s f o r the  r e l a t i v e organ weights o f t h e v i t a m i n E d e f i c i e n t r a t s show t h a t a l l organs, except  u t e r u s , were s i g n i f i c a n t l y l a r g e r ( P < 0 . 0 l ) t h a n  receiving vitamin E  those  treatment.  Table 4 shows t h a t treatment v i t a m i n E supplementation  f o r 16 months a t a l l l e v e l s o f  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 o f l i v e r , u t e r u s o r k i d n e y .  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 L e v e l s o f D i e t a r y V i t a m i n E F o r 8 Months Dietary  Body  vitamin E  weight  iu/kg d i e t  0 25  Liver  2,500 10,000 25,000  Heart  Spleen  Kidney  mg/lOOg; body weight  g  I66 (13) a  313 (26)  l,159  (27)  416 (24)  2,790 (123)  189  248  536  (20)  (16)  I60 (4)  2,470  204  I6l  b  (70)  (25)  269 (12)  560 (30)  I64  b  496  5,215 (292)  183  a  366  M  (3D  250  Uterus  332 (12)  cd  b  b  a  a  b2  b2  b  (8)  a  (32) b  (42) b  356 (16)  2,469 (204)  198  2 l  (13)  (10)  (5)  (32)  3l3  2,540  176 (19)  282  159  (13)  (14)  576 (29)  197  289  (16)  (10)  I67 (6)  547 (19)  M  cd  b  b  (8)  (102)  301  2,451 (157)  c  (16)  b  b 2  5  b2  b2  b  b  b  b  b  V a l u e s a r e means o f f o u r r a t s w i t h t h e i r SEM g i v e n i n p a r e n t h e s e s . V a l u e s w i t h i n each column not s h a r i n g a common s u p e r s c r i p t 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) u s i n g Duncan's new m u l t i p l e - r a n g e t e s t . L i n e a r response s i g n i f i c a n t (P<0.05) a g a i n s t d i e t a r y v i t a m i n E u s i n g r e g r e s s i o n 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 h e a r t weights (Y) and l o g d i e t a r y v i t a m i n 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) b e i n g 0.465 (P<0.05). 2  48  TABLE 4 Body and Organ Weights o f R a t s on D i f f e r e n t L e v e l s o f D i e t a r y V i t a m i n E F o r 16 Months  Dietary  Number  Body  vitamin E  rats  weight  iu/kg d i e t  Liver  g  Uterus  Heart  Spleen-'  Kidney  mg/lOOg body weight  0 25  4  1,957  165  268  167  599  (109)  (25)  (8)  (14)  (25)  2,702  217  229  168  501  (20)  (166)  (20)  (9)  (6)  (20)  4l4  3,088  214  263  184  586  (175)  (14)  (7)  (11)  (41)  3,065  202  268  218  583  (-1'07)  (19)  (6)  (14)  2,873 '  248  294  240  627  (135)  (22)  (6)  (34)  (21)  408  a  (12) 250  2,500  9  6  457  a  a  (17) 10,000  7  398 (21)  25,000  5  358  b  (10)  a b  a b  a  a b  a b  ,(21) b  V a l u e s a r e means w i t h t h e i r SEM g i v e n i n p a r e n t h e s e s . Values w i t h i n each column not s h a r i n g a common s u p e r s c r i p t l e t t e r a r e 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 ) u s i n g S c h e f f e ' s range t e s t . x  Q u a d r a t i c response s i g n i f i c a n t ( P O O . O l ) a g a i n s t d i e t a r y v i t a m i n E . 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 h e a r t weight (Y) and l o g d i e t a r y v i t a m i n E (x) was Y = 355-603-85.825 X + 16.219 X , the m u l t i p l e c o r r e l a t i o n c o e f f i c i e n t (R) b e i n g 0.64-9 ( P < 0 . 0 l ) . 2  L i n e a r response s i g n i f i c a n t (P<0.008) a g a i n s t d i e t a r y v i t a m i n E u s i n g r e g r e s s i o n 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 s p l e e n weight (Y) and l o g d i e t a r y v i t a m i n E (x) was Y= 2.127 + 0.051 X, where R = 0 - 5 8 0 ( P < 0.008).  49  (P<O.Ol) i n r e l a t i v e heart weight was shown i n r a t s fed high l e v e l s of vitamin E f o r 1 6 months.  The functional 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 = 3 5 5 - 6 0 3 - 8 5 . 8 2 5 X + 16.219 X , the multiple correlation c o e f f i c i e n t (R) being 0.649 2  (P<0.0l).  The r e l a t i v e spleen weights were also s i g n i f i c a n t l y  increased (P< 0.008) i n r a t s f e d high l e v e l s of vitamin E f o r 16 months. The l i n e a r relationship between r e l a t i v e spleen weight (Y) and l o g d i e t 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 d i f f e r e n t l e v e l s of dietary  vitamin E f o r 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 s i g n i f i c a n t difference i n hemoglobin l e v e l s (Table 5 ) when the r a t s were fed d i f f e r e n t dietary l e v e l s of vitamin E f o r 9 , 12 and 1 6 months. The hematocrit 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 6 .  There were no s i g n i f i c a n t differences i n  the hematocrit values of r a t s treated f o r 9 or 12 months.  However, the  hematocrit values were s i g n i f i c a n t l y increased (P<0.04) by treatment with excess vitamin E f o r 1 6 months.  The functional r e l a t i o n s h i p  between hematocrit value (Y) and l o g dietary vitamin E (x) was Y = 41.018 + 1.242X, where R= 0.482 (P<0.04). 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 the spontaneous hemolysis of the erythrocytes i n a buffered i s o t o n i c saline solution i n r a t s treated f o r 9, 12 and 1 6 months are presented i n  TABLE 5 Hemoglobin V a l u e s  1  o f R a t s Fed D i f f e r e n t  L e v e l s of D i e t a r y Vitamin E  Dietary vitamin E  iu/kg d i e t  Feeding period 9  (4)  (months) 12  16  (3)  (4)  0  15.9±  0.4  25  15.3±  0.1  15.2±  0.3  14.8±  0.5  250  15.9±  0.1  15.5±  0.7  15.5±  0.4  15.0±  0.4  14.8±  0.3  2,500  15.6+  0.2  10,000  15.6±  0.1  14.5±  1.2  15.1±  0.5  25,000  14.6±  0.3  15.7+  0.4  12.4±  2.2  Each v a l u e r e p r e s e n t s mean i SFJM f o r t h e number o f r a t s g i v e n i n parentheses  above each column.  51  TABLE 6 1 Hematocrit Values  of Rats Fed D i f f e r e n t  Levels of Dietary Vitamin E  Dietary vitamin E  FeedingI period (months) 12  9  iu/kg d i e t  Hematocrit (4)  16  2  (%)  (3)  (4)  0  45.6± 0.9  25  45.5± 0 . 5  44.7±  1.2  42.61 0 . 8  250  45.1± 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  4 3 . 4 ± 3-1  46.61  25,000  44.1±  46.21 0 . 7  47.2+ 2 . 0  1.1  1.0  Each value represents mean 1 SEM f o r the number of rats given in parentheses above each column. Linear response s i g n i f i c a n t (P<0.04) against dietary vitamin E using regression analysis.  The functional relationship between  hematocrit value (Y) and l o g dietary vitamin E was Y= 41.018 + 1.242X, where R= 0.482 (P<0.04).  52  TABLE  7  E r y t h r o c y t e Hemolysis  of R a t s Fed  D i f f e r e n t L e v e l s of D i e t a r y V i t a m i n  Feeding I p e r i o d  Dietary vitamin  iu/kg  E  E  (months)  12  9  diet  Hemolysis  (4)  16  (%)  (4)  (3)  86.8±  2.4  a  25  2.4±  0.5  b  2.2±  0.3  3-7±  0.6  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  0  Each value  i s the mean ± SEM  f o r the number o f r a t s g i v e n  p a r e n t h e s e s above each column.  V a l u e s w i t h i n each column n o t  s h a r i n g a common s u p e r s c r i p t l e t t e r are s i g n i f i c a n t l y ( P < 0 . 0 5 ) u s i n g Duncan's new  in  multiple-range  test.  different  a  TABLE 8  Prothrombin Times of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E  Dietary  Feeding period (months)  vitamin E  9  iu/kg diet  122  Prothrombin Time (sec)  (4) 0  16"  (4)  (3)  13.1±  0.2  14.9±  0.8  14.6±  0.1  13.0±  12.9±  0.4  15.6±  0.5  14.5± 0 . 2  2.500  13.8±  0.5  14.3±  0.7  13.6±  0.6  10,000  13.9±  0.5  11.3±  0.3  12.8±  0.5  25,000  14.2+  0.6  11.3±  0.4  12.7±  0.3  25 250  0.4  Each value i s the mean ± SEM f o r the number of r a t s give i n parentheses above each column. 1  Quadratic response s i g n i f i c a n t (P<0.000l) against d i e t a r y vitamin E. The functional 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= 10.409 + 4 . 4 3 9 - 0 . 9 9 1 X , where R= 0 . 8 9 4 (P<0.000l). Quadratic response s i g n i f i c a n t (P<0.02) against d i e t a r y vitamin E using regression a n a l y s i s . The functional r e l a t i o n s h i p between prothrombin time ( Y l and l o g 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  T a b l e 7»  V i t a m i n E supplementation d i d n o t 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 o f the e r y t h r o c y t e a t any p e r i o d of t r e a t m e n t .  Vitamin E  d e 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 i n c r e a s e d the f r a g i l i t y o f the e r y t h r o c y t e membrane. The prothrombin time v a l u e s o f r a t s t r e a t e d w i t h d i f f e r e n t of v i t a m i n E a r e shown i n T a b l e 8 .  levels  Treatment f o r 9 months a t a l l l e v e l s  o f v i t a m i n E supplementation had no s i g n i f i c a n t e f f e c t on prothrombin time.  I n r a t s t r e a t e d w i t h h i g h d i e t a r y l e v e l s o f v i t a m i n 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 s h o r t e r .  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 d i e t a r y v i t a m i n E (x) was Y= 10.409 + 4 . 4 3 9 X - 0-991 X , where 2  R= 0.894 ( P < 0 . 0 0 0 1 ) .  I n r a t s t r e a t e d 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 v i t a m i n E  (x)  was Y = 9.819 + 3 - 2 0 8 X - - 0 . 5 9 7 X , where R= 0.609 (P<0.02). 2  C.  Femoral  Parameters  The i n f l u e n c e of e x c e s s v i t a m i n E a d m i n i s t r a t i o n f o r 8 and 16 months on ash content and c a l c i u m and phosphate i n T a b l e s 9 and 10. phosphatase  c o n c e n t r a t i o n o f bone a r e shown  I n c l u d e d i n T a b l e 10 i s the plasma a l k a l i n e  a c t i v i t y which was measured a t 16 months as an a d d i t i o n a l  parameter o f bone Treatment  calcification.  w i t h d i f f e r e n t l e v e l s o f v i t a m i n 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 o f the femur.(Table 9).  However,  a f t e r 16 months o f h i g h d i e t a r y v i t a m i n E supplementation ( T a b l e 10) the ash content o f bone d e c r e a s e d 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 o f bones (Y) and l o g d i e t a r y v i t a m i n E (x) was Y= 68.970 - i.20?X, where R = 0.703  (P<0.0005).  55  TABLE 9  Femoral Parameters o f R a t s F e d D i f f e r e n t  Levels  o f D i e t a r y V i t a m i n E f o r 8 Months  Feeding p e r i o d  Dietary  vitamin E  : 8 months  Ash  Calcium  Phosphate  %  %  %  iu/kg d i e t 0  65.3±  0.7  25  65.5±  0.2  250  66.2+  0.3  23.61  2,500  65.71  0.7  22.81 0 . 5  A  10,000  66.01  0.9  2 3 . 9 1 0.4  b  25,000  66.31 0 . 5  23-41  0.3  22.91  0.3  A B  ah 0.3  A B  ah 23.01  'Each v a l u e i s t h e mean 1 SEM f o r 4 r a t s .  0.2  12.61  0.4  12.01  0.3  A B  11.81  0.1  A B  11.31 0 . 2 11.91  0.2  11.51  o.i  Values within  a  B  A B  b  each  column n o t s h a r i n g a common s u p e r s c r i p t l e t t e r a r e s i g n i f i c a n t l y different  (P<0.05)  u s i n g Duncan's new m u l t i p l e - r a n g e t e s t .  TABLE 1 0 Femoral Parameters of Rats Fed D i f f e r e n t Levels of Dietary Vitamin E For 1 6 Months  Feeding period : 1 6 months  Dietary 2  vitamin E  Ash  Calcium  Phosphate  Plasma a l k a l i n e phosphatase3 »4  iu/kg diet  %  %  %  units/100 ml  0 25  67.41 0.5  23-1±  0.5  11.9±  o.i  250  65.9± 0.4  21.9+  0.8  11.6±  0.6  2,500  65.0± 0.6  23.6±  1.1  12.4±  0.6  B  10,000  64.21 1.1  22.5±  0.5  10.9±  0.3  A  25,000  63.6± 1.0  21.0±  0-3  11.4±  0.3  a b  A B  A B  17-9±  1.1  14.11  1.8  15.71  0.1  22.11  4.6  24.61  4.3  """Each value i s the mean 1 SEM f o r four 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 . ^Linear response s i g n i f i c a n t ( P < 0 . 0 0 0 5 ) against dietary vitamin E. 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 dietary vitamin E (x) was Y= 68.970 - 1.207X, where R= 0 . 7 0 3 ( P < 0 . 0 0 0 5 ) . Quadratic response s i g n i f i c a n t (P<0.04) against d i e t a r y vitamin E using regression a n a l y s i s . The functional r e l a t i o n s h i p between plasma a l k a l i n e phosphatase^activity (Y) and l o g dietary vitamin E (x) was Y = 13-789 + 0.468X , where R= 0 . 4 6 3 (P<0.04). 4  One unit 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 1 5 minutes at 3 7 C.  57  Femoral calcium content was not s i g n i f i c a n t l y affected by vitamin E supplementation  excess  f o r either 8 or 16 months.  There were some s l i g h t , but s i g n i f i c a n t differences ( P < 0 . 0 5 ) i n femoral phosphate concentration i n 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.  Regression analysis though was unable to  show any s i g n i f i c a n t r e l a t i o n s h i p between the phosphate concentration and the dietary l e v e l of vitamin E supplemented. As can be seen i n Table 10, excess vitamin E supplementation,  from  2 5 0 to 25,000 Ill/kg d i e t increased plasma alkaline phosphatase a c t i v i t y a f t e r 16 months treatment.  The f u n c t i o n a l r e l a t i o n s h i p between plasma  a l k a l i n e phosphatase a c t i v i t y (Y) and l o g dietary vitamin E (x)  was  Y= 1 3 - 7 8 9 + 0.469 X , where R = 0.463 (P<0.04). 2  D.  Urinary Creatine and Creatinine Data from the analysis of urinary creatine and creatinine 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 11 months are presented i n Table 11.  Vitamin E supplementation  at a l l l e v e l s d i d not influence t  urinary l e v e l s of either creatine or creatinine.  Vitamin E d e f i c i e n c y  s i g n i f i c a n t l y increased (P<0.0l) the urinary creatine excretion, while the urinary creatinine excretion decreased s i g n i f i c a n t l y  E.  Fat Soluble Vitamins  1. b  L i v e r and Plasma Vitamin E  (P<0.0l).  The influence of high l e v e l s of dietary vitamin E on l i v e r storage of a-tocopherol a f t e r 8 and 16 months of treatment  i s shown i n Table  12.  The r e s u l t s 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 t o t a l a-tocopherol content of the l i v e r (|lg/whole l i v e r ).  58  TABLE 11  Urinary creatine and creatinine of rats on d i f f e r e n t l e v e l s of dietary vitamin E f o r 11 months  Dietary  1  Creatine/Creatinine  Creatine  Creatinine  mg/kg/24 hr  mg/kg/24 hr  vitamin E iu/kg diet 0  54.07±19.90A  ratio  14.501  2.31  A  5.041  2.86  A  25  3-141  0.62  B  27-501  1.95B  o.lll  o.03B  250  1.441  0.20  B  27-121  l-32  B  0.051  0.01  B  i.92± 0 . 3 9  B  27-831  1.28  b  0.071  0.02  B  b  25.72±  0.60  B  0.121  0.02  B  27-ll± 1 . 4 9 B  0.071  0.02  B  2,500  10,000  3.20±  0.46  25,000  1.941  0.38  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<0.0l) using Duncan's new multiple-range  test.  59 As  can be seen i n T a b l e 12,  i n c r e a s i n g the l e v e l of d i e t a r y vitamin E  up t o 1 0 , 0 0 0 i u / k g 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 (P<0.000l) the v i t a m i n E c o n c e n t r a t i o n of t h e l i v e r .  increased  Additional vitamin E  supplementation, above 1 0 , 0 0 0 I l l / k g d i e t , d i d not s i g n i f i c a n t l y the l i v e r v i t a m i n E c o n c e n t r a t i o n any f u r t h e r .  increase  A n a l y s i s o f the d a t a  r e v e a l e d 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 o f v i t a m i n E and the v i t a m i n E c o n c e n t r a t i o n i n l i v e r when both were e x p r e s s e d a s logarithms  (Fig. 11).  The l o g a r i t h m i c r e l a t i o n s h i p between l i v e r v i t a m i n E  and d i e t a r y v i t a m i n E was 9 4 % l i n e a r f o r r a t s t r e a t e d f o r 8 months and 98% l i n e a r f o r r a t s t r e a t e d 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 v i t a m i n E (Y) and l o g d i e t a r y v i t a m i n E ( x ) i n r a t s t r e a t e d f o r 8 months was Y= - 0 . 1 0 6  +  (P<0.0001); and i n r a t s t r e a t e d f o r  1 . 2 5 2 X - 0.10?X , where R 2  1 6 months was  Y= 0-762  =0.980  0.652X,  *  where R= 0-988 ( P < 0 . 0 0 0 l ) . Shown i n F i g u r e 12 a r e the c o n c e n t r a t i o n s o f v i t a m i n E i n plasma o f 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 o f v i t a m i n E f o r 8 and 1 6 months. In r a t s t r e a t e d f o r 8 months plasma l e v e l s o f v i t a m i n E were increased ( P < 0 . 0 0 5 ) The  significantly  with i n c r e a s i n g l e v e l s of d i e t a r y vitamin E i n t a k e .  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 v i t a m i n E (Y) and l o g  d i e t a r y v i t a m i n E ( x ) i n r a t s t r e a t e d f o r 8 months was Y = — 1 . 4 1 9  *  0.949X- 0.132X , where R = 0 . 8 4 ( P < 0 . 0 0 5 ) .  levels  2  The plasma v i t a m i n E  were over 2 - f o l d h i g h e r i n r a t s f e d v a r i o u s l e v e l s o f v i t a m i n 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 v i t a m i n E (Y) and l o g d i e t a r y v i t a m i n E ( x ) i n r a t s t r e a t e d f o r 1 6 months was Y= - 0 . 6 2 2  2.  +  0-595X - 0 . 0 6 4 X , where R = 0 - 9 6 2  (P<0.0000l).  L i v e r and Plasma V i t a m i n A The  e f f e c t o f d i f f e r e n t l e v e l s o f d i e t a r y v i t a m i n E on the  TABLE 1 2 The C o n c e n t r a t i o n s o f ot-Tocopherol i n L i v e r s o f Rats F e d D i f f e r e n t L e v e l s o f D i e t a r y V i t a m i n E F o r 8 and 1 6 Months  Dietary  Liver  8 months  vitamin E  iu/kg d i e t  0 25  a-tocopherol  / T fj.g/g l x v e r  2  fj.g/whole  0.5± 0.1 27.1±  16 months  liver  4.61  1.8  (ig/g  liver  |lg/whole  liver  1.1  277.51  33.5  182.0  43.71 1-4 214.2+ 1 7 . 1  577-61 29.7  250  204.9+ 15-5  1,691.91  2,500  703.o± 8 8 . 7  6,104.41 882.3  1,172.61181.7  14,323.8+2,150.2  10,000  1,952.3±203.1  15,403.511,456.7  2'?822.61283-1  30,994.715,826.5  25,000  2,214.7±587.8  17,137.316,572.0  3,411.11513.1  37,236.616,268.2  2,535-71  296.4  y Each v a l u e r e p r e s e n t s mean 1 SEM f o r f o u r r a t s . Q u a d r a t i c response s i g n i f i c a n t L i n e a r response s i g n i f i c a n t  (P<0.0001)  (P<0.0001)  a g a i n s t d i e t a r y v i t a m i n E i n supplemented  against dietary vitamin E .  rats.  FIGURE 11  P l o t o f the l o g a r i t h m o f l i v e r a - t o c o p h e r o l  concentration  v e r s u s the l o g a r i t h m o f d i e t a r y v i t a m i n E , i n r a t s t r e a t e d f o r 8 and 16 months.  FIGURE 12  Plasma a - t o c o p h e r o l c o n c e n t r a t i o n o f 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 o f v i t a m i n E f o r 8 months (open b a r s ) and 16 months ( c l o s e d b a r s ) . Each p o i n t r e p r e s e n t s mean ± SEM o f f o u r r a t s . In r a t s t r e a t e d f o r 8 months plasma t o c o p h e r o l i n group A i s s i g n i f i c a n t l y l o w e r than o t h e r groups ( P < 0 . 0 1 ) u s i n g Duncan's m u l t i p l e - r a n g e t e s t . In r a t s t r e a t e d f o r 8 months, q u a d r a t i c 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 t r e a t e d f o r 16 months,, q u a d r a t i c response i s s i g n i f i c a n t ( P < 0 . 0 0 0 l ) against d i e t a r y vitamin E.  Plasma N b  oc-Tocopherol CO b  ( mg /100 b  ml )  o  to 01 as 3  to en o  2°  o o ca  a 5'  o b o o to b o o ON  FIGURE 1 3  L i v e r vitamin A 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 o f v i t a m i n E f o r 8 months (open b a r s ) and 16 months ( c l o s e d b a r s ) . Each p o i n t r e p r e s e n t s mean ± SEM o f f o u r r a t s . In r a t s t r e a t e d 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 o t h e r groups (P<0.0l). In r a t s t r e a t e d f o r 16 months group G i s s i g n i f i c a n t l y h i g h e r than groups D, E and F (P<0.05)«  Vitamin E  (lU/kg  Diet)  FIGURE 14  Plasma v i t a m i n A c o n c e n t r a t i o n o f 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 o f v i t a m i n E f o r 16 months. Each p o i n t r e p r e s e n t s mean ± SEM o f f o u r r a t s .  65  concentration of vitamin A i n l i v e r i s shown i n Figure 13•  Supplementation  with high l e v e l s of vitamin E f o r 8 and 16 months had no s i g n i f i c a n t influence on l i v e r vitamin A storage.  However, a f t e r 8 months, vitamin E  deficiency was found to s i g n i f i c a n t l y decrease  (P<0.01) the l i v e r  vitamin A storage. The influence of high l e v e l s of vitamin E on plasma vitamin A l e v e l s was examined only a f t e r 16 months treatment treatment  ( F i g . 14). Long-term  with excess vitamin E had no s i g n i f i c a n t e f f e c t on plasma  vitamin A l e v e l s . F.  Lipids  1.  L i v e r Total L i p i d s and Cholesterol" The influence of high l e v e l s of vitamin E on l i v e r concentrationso  of t o t a l l i p i d s and cholesterol are presented i n Figures 1 5 and 16 respectively. The l i v e r t o t a l l i p i d s were s i g n i f i c a n t l y increased (P<0.000l) a f t e r 8 months treatment  with high l e v e l s of dietary vitamin E ( F i g . 1 5 ) -  The l i v e r t o t a l l i p i d s were increased from 4 . 5 % of the l i v e r weight i n r a t s f e d 2 5 I U vitamin E/kg diet to 14.0% i n r a t s f e d 25,000 I U vitamin E/kg diet.  The l i n e a r r e l a t i o n s h i p between l o g l i v e r l i p i d s (Y) and l o g d i e t -  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 i n t e r e s t i n g to observe that there was no s i g n i f i c a n t difference i n l i v e r t o t a l l i p i d s at d i f f e r e n t l e v e l s of vitamin E supplementation.  A l l groups treated f o r  16 months, except those f e d 2 5 I U vitamin E/kg d i e t , had lower l i p i d l e v e l s than those groups treated f o r 8 months at comparable l e v e l s of vitamin E  supplementation.  FIGURE  15  Total l i p i d s i n l i v e r of rats f e d d i f f e r e n t dietary l e v e l s o f v i t a m i n E f o r 8 months (open b a r s ) and 16 months ( c l o s e d b a r s ) . Each p o i n t r e p r e s e n t s mean + SEM o f f o u r r a t s . Group A i s s i g n i f i c a n t l y l o w e r than o t h e r groups ( P < 0 . 0 1 ) u s i n g Duncan's new m u l t i p l e - r a n g e t e s t i n r a t s f e d 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<0.0001) against dietary vitamin E.  Vitamin E  ( lU/kg  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 o f v i t a m i n E f o r 8 months (open b a r s ) and 16 months ( c l o s e d b a r s ) . Each p o i n t r e p r e s e n t s mean ± SEM o f f o u r  rats.  Group G i s s i g n i f i c a n t l y h i g h e r than o t h e r groups ( P < 0 . 0 5 ) except group F r a t s f e d f o r 8 months.  68  As shown i n Figure  1 6 , l i v e r cholesterol was unaffected  of vitamin E a f t e r 8 and 1 6 months of treatment.  by high l e v e l s  In contrast to the r e s u l t s  of l i v e r t o t a l l i p i d s , rats treated f o r 1 6 months had generally higher l i v e r cholesterol l e v e l s than those treated f o r 8 months. 2.  Plasma Total L i p i d s and Cholesterol 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  plasma t o t a l l i p i d s and cholesterol are shown i n Figure 1 7 and 18 respectively. Treatment with high l e v e l s of dietary vitamin E f o r 8 months had no s i g n i f i c a n t e f f e c t on plasma t o t a l l i p i d s .  However, the plasma t o t a l  l i p i d s of rats f e d 2 5 or 2 5 0 IU vitamin E/kg diet f o r 8 months were higher than i n r a t s fed more than 2 , 5 0 0 iu/kg d i e t . observed i n the rats treated f o r 16 months.  This was also  Regression analysis  revealed  that increasing the dietary l e v e l of vitamin E s i g n i f i c a n t l y decreased (P<0.024) the plasma t o t a l l i p i d s i n r a t s treated f o r 1 6 months.  The  l i n e a r r e l a t i o n s h i p between l o g plasma l i p i d s (Y) and l o g dietary vitamin E  (x) i n rats treated f o r  1 6 months was Y = 3 - 0 3 0 6 - 0.00814X, where R = 0.4l  (P<0.024). Vitamin E supplementation f o r 8 months had no s i g n i f i c a n t e f f e c t on plasma cholesterol ( F i g . 18). The plasma cholesterol l e v e l s of vitamin E d e f i c i e n t rats was s i g n i f i c a n t l y lower ( P < 0 . 0 5 ) than those supplemented with 2 , 5 0 0 IU vitamin E/kg d i e t f o r 8 months. Treatment with excess l e v e l s of dietary vitamin E, 2 , 5 0 0 iu/kg diet or higher f o r 1 6 months s i g n i f i c a n t l y lowered ( P < 0 . 0 5 ) the plasma cholesterol l e v e l ( F i g . 18).  FIGURE 17  Total l i p i d s i n plasma of r a t s fed d i f f e r e n t dietary 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 number of rats shown i n parentheses. In r a t s treated f o r 16 months, l i n e a r response i s s i g n i f i c a n t (P<0.024) against dietary vitamin E.  69;  lOOOf  800+  6004  400  200  25  250 Vitamin E  2,500  (iu/kg  10,000  Diet)  25,000  FIGURE 18  Cholesterol concentration i n plasma of r a t s fed d i f f e r e n t dietary l e v e l s of vitamin E f o r 8 months (open bars) and 1 6 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 D i s s i g n i f i c a n t l y higher ( P < 0 . 0 5 ) than group A. In r a t s treated f o r 1 6 months, groups B and C are s i g n i f i c a n t l y higher ( P < 0 . 0 5 ) than groups D, E and F.  E  O  25  250  2.500  Vitamin E  (iu/kg  10.000 Diet)  25,000  71  CHAPTER V DISCUSSION A.  Body and The  (10,000  body weights o f r a t s t r e a t e d with h i g h l e v e l s o f v i t a m i n  and  (P<0.05). 25,000 25  Organ Weights  25,000  E  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 d e p r e s s e d  Body weights were a l s o s i g n i f i c a n t l y reduced i n r a t s f e d d i e t f o r 1 6 months compared t o those r e c e i v i n g  IU v i t a m i n E/kg  to 2 , 5 0 0  iu/kg d i e t .  From t h e s e  r e s u l t s i t appears t h a t e x c e s s d i e t a r y  v i t a m i n E f e d t o r a t s over an extended p e r i o d of time d e p r e s s e d  their  body w e i g h t s . The  r e s u l t s of r e s e a r c h  on the e f f e c t of excess d i e t a r y v i t a m i n March e t a l . ( 1 9 7 3 )  on the growth r a t e i n a n i m a l s v a r y w i d e l y .  E  concluded  t h a t growth r a t e i n c h i c k s appeared t o be r e l a t i v e l y i n s e n s i t i v e t o e x c e s s dietary vitamin E 2,200  (1,000  IU v i t a m i n E/kg  iu/kg d i e t ) , a l t h o u g h a d e p r e s s i o n o c c u r r e d  d i e t i n t h e i r s h o r t term study.  6 4 , 0 0 0 iu/kg d i e t ) s i g n i f i c a n t l y r e d u c e d the  c h i c k body weight.  McCuaig and Motzok ( 1 9 7 0 ) f e d a 1 0 , 0 0 0 IU v i t a m i n E/kg r e p o r t e d t h a t growth r a t e was  unaffected.  r e p o r t e d i n the r a b b i t (Awad and  Nockels et a l . ( 1 9 7 5 )  supplementation ( 8 , 0 0 0  a l s o r e p o r t e d t h a t h i g h l e v e l s of v i t a m i n E  However, and  S i m i l a r r e s u l t s have a l s o been  Gilbreath, 1 9 7 5 ) *  E f f e c t s of excess In a 28 week  A l f i n - S l a t e r e t a l . ( 1 9 7 2 ) f o u n d t h e r e were no d i f f e r e n c e s i n  weight g a i n s of r a t s f e d 1 0 0 3 0 mg  and  diet to chicks  v i t a m i n E on growth r a t e of r a t s have a l s o been s t u d i e d . study,  with  v i t a m i n E/day.  mg  v i t a m i n E/day compared t o t h o s e f e d  However, J e n k i n s and M i t c h e l l  i n c r e a s e i n body weight of r a t s f e d 6 0 0 and f o r 2 months.  6,000  ( 1 9 7 5 ) reported  IU v i t a m i n E/kg  Treatment with d i e t a r y v i t a m i n E a t 6 , 0 0 0  ( J e n k i n s and M i t c h e l l ,  1975)  an  diet  iu/kg d i e t  i s a comparable l e v e l t o an o r a l  supplement  72  of  100  mg  v i t a m i n E/day ( A l f i n - S l a t e r e t a l . , 1972)  the r a t consumes 15  g diet/day.  The  i f i t s assumed t h a t  r e a s o n f o r the wide d i s c r e p a n c y i n  r e s u l t s i n t h i s a r e a remains t o be i n v e s t i g a t e d . 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 v i t a m i n E f o r 8 and 3 and k r e s p e c t i v e l y .  16 months are shown i n T a b l e s  F o r r a t s g i v e n d i e t a r y v i t a m i n E from 25  25»000 iu/kg d i e t f o r 8 months, t h e r e were no  to  significant differences  among groups with r e s p e c t t o weights of l i v e r ,  uterus, kidney  and  spleen.  However, h i g h l e v e l s of v i t a m i n E s i g n i f i c a n t l y i n c r e a s e d the groups r e l a t i v e h e a r t weight a f t e r 8 months treatment.  The r e g r e s s i o n a n a l y s i s  a l s o r e v e a l e d that a f t e r extending  t o 16 months, the groups  fed  excess  vitamin E  the treatment  c o n t i n u e d t o have r e l a t i v e h e a r t t w e i g h t s  than those f e d moderate l e v e l s of v i t a m i n E .  larger  A l s o a t t h i s time the  r e l a t i v e s p l e e n weights were s i g n i f i c a n t l y i n c r e a s e d .  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 f e d d i f f e r e n t l e v e l s of v i t a m i n E w i t h r e s p e c t t o weights o f l i v e r , k i d n e y and u t e r u s .  Hypervitaminosis  i n r a t s has been r e p o r t e d t o i n c r e a s e r e l a t i v e a d r e n a l weight, but e f f e c t r e l a t i v e weight of l i v e r , k i d n e y  o r s p l e e n ( J e n k i n s and  E  not  Mitchell,  1975). With the e x c e p t i o n of the u t e r u s , the r e l a t i v e organ weights i n the v i t a m i n E - f r e e 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 v i t a m i n E supplements f o r 8 months.  T h i s may  be due  t o the d e p r e s s i o n i n  body weight a f t e r 3 t o k months on the v i t a m i n E - f r e e d i e t . l o s s r e p r e s e n t s massive muscle atrophy  The  weight  i n vitamin E - f r e e r a t s , with  organs not b e i n g a f f e c t e d as much d u r i n g the  same p e r i o d o f time.  the As  consequence, the r e l a t i v e s i z e s of the organs appear t o be l a r g e r i n v i t a m i n E - f r e e r a t s with the e x c e p t i o n of t h e u t e r u s .  a  73  B.  H e m a t o l o g i c a l Parameters There was no evidence i n t h i s study t o suggest t h a t excess v i t a m i n E  would l e n g t h e n the prothrombin time o f r a t s .  I n s t e a d , a t the l a t t e r  two  t e s t p e r i o d s , 12 and 16 months, excess v i t a m i n E a c t u a l l y r e s u l t e d i n d e c r e a s e d prothrombin t i m e s . adequate  These f i n d i n g s i n d i c a t e a t h a t r a t s r e c e i v i n g  d i e t a r y v i t a m i n K do not develop p r o l o n g e d prothrombin time  even  when they are f e d a v e r y h i g h l e v e l o f v i t a m i n E . A c c o r d i n g t o March e t a l .  (1973)>  c h i c k s f e d excess amount of v i t a m i n E .  prothrombin timeswas l e n g t h e n e d i n The prothrombin time was  rapidly  r e v e r s e d by i n j e c t i o n of v i t a m i n K, which i n d i c a t e d an i n c r e a s e i n the d i e t a r y requirement f o r v i t a m i n K i n the presense o f e x c e s s d i e t a r y vitamin E.  One  p r e v i o u s study a l s o r e p o r t e d t h a t i n some s t r a i n s o f r a t s  the prothrombin l e v e l d e c l i n e d as h i g h e r doses of v i t a m i n E were a d m i n i s t e r e d ( M e l l e t t e and Leone,  i960).  I t i s d i f f i c u l t t o compare the f i n d i n g s of March e t a l . those o f t h i s study, however, because  (D973)  with  o f the d i f f e r e n c e s i n the d i e t a r y  requirement of v i t a m i n K i n r a t s and c h i c k e n s .  The i n d u c t i o n o f v i t a m i n K  d e f i c i e n c y i s a l s o a f f e c t e d by o t h e r p h y s i o l o g i c a l f a c t o r s , such a s the s t r a i n , age and sex of. the e x p e r i m e n t a l a n i m a l .  M e l l e t t e and Leone  have shown c l e a r - c u t 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  (i960)  susceptibility  t o p r o l o n g e d prothrombin time, a s an i n d i c a t i o n of v i t a m i n K d e f i c i e n c y . A l s o the female r a t , a s used i n t h i s study, i s more r e s i s t a n t t o v i t a m i n K d e f i c i e n c y than the male r a t (Johnson e t a l . ,  i960).  The r e s u l t s o f t h i s study, shown i n T a b l e 5»  indicate that high l e v e l s  of v i t a m i n E f o r p r o l o n g e d p e r i o d s d i d not a f f e c t hemoglobin significantly.  levels  These f i n d i n g s are i n agreement w i t h the o b s e r v a t i o n of  J e n k i n s and M i t c h e l l s '  (1975)  In a s h o r t term experiment w i t h r a t s .  74  Hematocrit v a l u e s were n o t i n f l u e n c e d by h i g h d i e t a r y l e v e l s o f v i t a m i n E a f t e r 9 and 1 2 months o f treatment ( T a b l e 6 ) . 1 6 months o f treatment h e m a t o c r i t  However,  v a l u e s were s i g n i f i c a n t l y  after  increased  when v i t a m i n E was f e d a t a l e v e l o f 1 0 , 0 0 0 iu/kg d i e t o r h i g h e r . March e t a l . ( 1 9 7 3 ) have r e p o r t e d t h a t h e m a t o c r i t chicks f e d 2 , 2 0 0  IU v i t a m i n E/kg d i e t f o r 5 0 days.  the r e d u c t i o n was more severe At p r e s e n t poietic has  v a l u e s were r e d u c e d i n They observed t h a t  when the c h i c k s were younger.  i t i s not p o s s i b l e t o a s c e r t a i n whether the hemotoe  system i s i n f l u e n c e d by an e x c e s s o f v i t a m i n E .  a t t e n t i o n been c e n t e r e d  Only r e c e n t l y  on a p o s s i b l e r o l e o f v i t a m i n E i n heme and  hemeprotein s y n t h e s i s (Murty e t a l . , 1 9 7 0 ;  Gaasi e t a l . , 1 9 7 2 ;  Nair,  1972).  Other i n v e s t i g a t o r s , however, have not been a b l e t o show any i n v o l v e m e n t of v i t a m i n E i n heme s y n t h e s i s ( C a r p e n t e r , The  1972;  Diplock,  1974).  r e s u l t s o f the spontaneous h e m o l y s i s o f e r y t h r o c y t e i n a  b u f f e r e d i s o t o n i c s l a i n e s o l u t i o n showed t h a t o n l y r e d b l o o d c e l l s o f v i t a m i n E d e f i c i e n t r a t s were s u s c e p t i b l e t o h e m o l y s i s .  Excess d i e t a r y  v i t a m i n E d i d not a l t e r the s t a b i l i t y o f t h e e r y t h r o c y t e membrane t o in vitro  C.  hemolysis.  Femoral Parameters Bone composition  o f the r a t s i n t h i s experimet were n o t a f f e c t e d by  h i g h l e v e l s o f v i t a m i n E f e d f o r 8 months.  However, the presence o f  excess v i t a m i n E i n t h e d i e t f o r 1 6 months s i g n i f i c a n t l y reduced ( P < 0 . 0 0 0 5 )  t h e ash c o n t e n t  o f bones.  10)  Treatment w i t h i n c r e a s i n g  l e v e l s o f d i e t a r y v i t a m i n E , r a n g i n g from 2 5 0 t o 2 5 , 0 0 0 i n c r e a s e d t h e plasma a l k a l i n e phosphatase a c t i v i t y  iu/kg d i e t ,  significantly.  a l 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 a s h content may i n d i c a t e i n c r e a s e d t u r n o v e r  (Table  The  a f t e r 1 6 months  i n bones o f r a t s f e d h i g h l e v e l s o f  75  vitamin E f o r prolonged  periods.  March e t a l . ( 1 9 7 3 ) r e p o r t e d t h a t hone c a l c i f i c a t i o n was when e x c e s s v i t a m i n E  iu/kg d i e t ) was a d m i n i s t e r e d  (2,200  e i t h e r c a l c i u m - d e f i c i e n t or v i t a m i n D - d e f i c i e n t d i e t s .  depressed  to chicks f e d  They  concluded  t h a t e x c e s s v i t a m i n E i n c r e a s e d the requirement f o r v i t a m i n D.  There i s  c o n s i d e r a b l e s p e c i e s v a r i a b i l i t y i n the d i e t a r y r e q u i r e m e n t s of v i t a m i n c a l c i u m and  phosphorus.  Animal performance depends on the  amounts of each n u t r i e n t as w e l l as the r e l a t i v e amounts.  absolute Chickens  r e q u i r e a h i g h e r calcium:phosphorus r a t i o than r a t s do f o r o p t i m a l In r a t s , t h e r e i s no e x t e n s i v e evidence  and adequate ( N a t i o n a l Research C o u n c i l , 1 9 7 2 ) .  Research C o u n c i l ( 1 9 7 2 ) has recommended t h a t approximately v i t a m i n D/kg  d i e t be f e d t o growing r a t s .  experimental  d i e t was  iu/kg d i e t .  2,000  growth.  to indicate that vitamin E i s  r e q u i r e d 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 c a l c i u m and are b a l a n c e d  D,  The  phosphorus The  1,000  v i t a m i n D content  National IU i n the  Thus, even i f e x c e s s v i t a m i n  E  has i n c r e a s e d the requirement f o r v i t a m i n D as March e t a l . ( 1 9 7 3 ) have suggested i n t h e i r work with c h i c k e n s , t h i s would not have been observed i n t h i s experiment s i n c e the a n i m a l s r e c e i v e d adequate l e v e l s of v i t a m i n D with balanced  D.  l e v e l s of c a l c i u m and  U r i n a r y C r e a t i n e and  phosphorus.  Creatinine  U r i n a r y e x c r e t i o n of c r e a t i n e and  c r e a t i n i n e were a p p a r e n t l y  i n a l l r a t s r e c e i v i n g h i g h l e v e l s of d i e t a r y v i t a m i n E f o r 1 1 V i t a m i n 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 h i g h e r c r e a t i n e and  c r e a t i n i n e l e v e l s i n urine©.. with v i t a m i n E d e f i c i e n c y .  C r e a t i n u r i a i s a recognized  Hillman  normal  months. lower  symptom a s s o c i a t e d  ( 1 9 5 7 ) and B r i g g s ( 1 9 7 4 ) have d e s c r i b e d  c r e a t i n u r i a i n t h r e e human s u b j e c t s r e c e i v i n g l a r g e doses of v i t a m i n  E.  76  Briggs reported that an elevated serum creatine kinase accompanied the creatinuria.  There was no i n d i c a t i o n i n t h i s study that excess vitamin E  induces damage to s k e l e t a l muscle. E.  Fat Soluble  Vitamins  1.  L i v e r and Plasma Vitamin E The r e l a t i o n s h i p between d i e t a r y l e v e l s of vitamin E and the  storage  of t h i s vitamin i n l i v e r a f t e r 8 and 16 months treatment was l i n e a r when both values were expressed as logarithms (Figure 11).  There was  a  s i g n i f i c a n t deviation from the r e l a t i o n s h i p between logarithm of vitamin E intake and l o g l i v e r tocopherol concentration when the d i e t a r y l e v e l increased beyond 10,000 IU vitamin E/kg d i e t .  As shown i n Figure 11,  further increases i n dietary vitamin E had no s i g n i f i c a n t e f f e c t on increasing the storage of t h i s vitamin i n l i v e r .  The t o t a l vitamin E  content i n the l i v e r (Table 12) was approximately  two-fold greater i n  rats_treated f o r 16 months than those treated f o r 8 months. accumulation  This  of vitamin E was a r e s u l t 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 l i n e a r r e l a t i o n s h i p between increasing l e v e l s of vitamin E intake and l i v e r l e v e l s when both were expressed i n logarithmic units i s supported by the work of B o l l i g e r and Bolliger-Quaife (1956) and Wiss et a l . (1962). In t h i s study the plasma tocopherol l e v e l increased s i g n i f i c a n t l y as the dietary vitamin E l e v e l was r a i s e d (Figure 12).  The plasma tocopherol  l e v e l s were not proportional to the vitamin E intake at a l l dietary l e v e l s , though.  In addition, the plasma tocopherol l e v e l s were at l e a s t two-fold  higher i n the r a t s fed f o r 16 months, than those treated f o r 8 months.  77  According and B i e r i  t o B o l l i g e r and B o l l i g e r - Q u a i f e ( 1 9 5 6 ) ,  (1972),  Wiss e t a l . ( 1 9 6 2 )  t h e r e i s a l i n e a r r e l a t i o n s h i p between plasma  and the l o g a r i t h m of the dose f e d .  The  f o r m e r two  term experiments, w h i l e the r e p o r t by B i e r i  tocopherol  s t u d i e s were s h o r t  ( 1 9 7 2 ) was  2 5 weeks l o n g  and  examined the e f f e c t of f e e d i n g a low l e v e l of v i t a m i n E , 3 2 iu/kg d i e t . In the o n l y l o n g term study examining the e f f e c t of l a r g e doses of v i t a m i n E on plasma t o c o p h e r o l l e v e l s i n r a t s , A l f i n - S l a t e r e t a l . ( 1 9 7 2 ) r e p o r t e d t h a t plasma t o c o p h e r o l l e v e l s r e f l e c t e d the t o c o p h e r o l  level  supplemented, but were not p r o p o r t i o n a l t o the dose.  2.  L i v e r and Plasma V i t a m i n  A  a f t e r 8 months treatment, the l i v e r v i t a m i n A  I n t h i s study,  of a l l v i t a m i n E supplemented groups was  s i g n i f i c a n t l y h i g h e r than of.  However, i n the r a t s t r e a t e d f o r 8 and  v i t a m i n E - f r e e groups.  the change i n d i e t a r y v i t a m i n E r a n g i n g from 2 5 t o 2 5 , 0 0 0 showed no  f o r 8 months was  be  Ill/kg d i e t  concluded  The  of r a t s f e d v a r i o u s d i e t a r y l e v e l s of v i t a m i n  not t e s t e d , but those f o r 1 6 months were measured and  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. may  1 6 months,  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 v i t a m i n A s t o r a g e .  plasma v i t a m i n A c o n t e n t  storage  t h a t t h e r e was  d i e t a r y v i t a m i n E and  no  i n t e r a c t i o n between h i g h l e v e l s  v i t a m i n A i n l i v e r o r plasma i n t h i s  Workers have confirmed  Therefore,  of v i t a m i n A i n the l i v e r  and N a v a r i ,  1975)*  This vitamin E  no it  :  of  study.  t h a t i n c r e a s e d i n t a k e s of v i t a m i n E  the s t o r a g e  E  (Cawthorne e t a l . , I 9 6 8 ;  increase  Prodouz  " s p a r i n g " e f f e c t on v i t a m i n A has  been  shown a t w i d e l y v a r y i n g l e v e l s of v i t a m i n E i n t a k e , f o r example, from 1 Ill/week (Cawthorne e t a l . , 1 9 6 8 ) Mitchell, 1975)  up t o 6 , 0 0 0  Ill/kg d i e t  (Jenkins  have been r e p o r t e d t o i n c r e a s e the l i v e r v i t a m i n A  and storage  78  in rats.  J e n k i n s and M i t c h e l l  ( 1 9 7 5 ) a l s o r e p o r t e d t h a t t h e r e was a  s i g n i f i c a n t i n c r e a s e i n plasma v i t a m i n A w i t h i n c r e a s i n g l e v e l s o f v i t a m i n E i n the d i e t . vitamins  i sstill  The mechanism o f a c t i o n between t h e s e two  unknown, b u t a c c o r d i n g t o Cawthorne e t a l . , ( 1 9 6 8 )  the r e l a t i o n s h i p between v i t a m i n E and v i t a m i n A i n v i v o cannot be regarded  a s t h a t between an a n t i o x i d a n t and a p e r o x i d i z a b l e s u b s t r a t e .  F.  Lipids  1.  L i v e r T o t a l L i p i d s and C h o l e s t e r o l 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 i n c r e a s e d by e x c e s s  v i t a m i n E s u p p l e m e n t a t i o n (from 2 5 0 t o 2 5 , 0 0 0 f o r 8 months.  Contrary  iu/kg d i e t ) i n r a t s t r e a t e d  t o t h e r e s u l t s f o u n d a f t e r 8 months t r e a t m e n t ,  l i v e r t o t a l l i p i d l e v e l s were n o t 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 t r e a t e d f o r 1 6 months with d i f f e r e n t l e v e l s o f v i t a m i n E .  No mechanism  has been proposed t o e x p l a i n why e x c e s s v i t a m i n E s h o u l d i n c r e a s e t o t a l l i p i d s o n l y i n younger r a t s .  liver  T h i s cannot be a c c o u n t e d f o r by an  i n c r e a s e i n the l e v e l o f l i v e r c h o l e s t e r o l , because a t b o t h 8 and 1 6 months e x c e s s v i t a m i n E d i d n o t 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. H i g h d i e t a r y l e v e l s o f v i t a m i n E have been r e p o r t e d t o i n c r e a s e 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 Mitchell,  1975)'  ( A l f i n - S l a t e r e t a l . , 1 9 7 2 ; J e n k i n s and  I n c r e a s i n g d i e t a r y v i t a m i n E i n t a k e a l s o has been  shown t o enhance t h e development o f a l c o h o l induced f a t t y l i v e r et a l . , 1 9 7 3 ) •  Contrary  (Levander  t o the above f i n d i n g s , o t h e r workers have r e p o r t -  ed t h a t i n c r e a s i n g d i e t a r y l e v e l s o f v i t a m i n E w i l l d e c r e a s e 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 1966;  ( H a r r i l l e t a l . , 1 9 6 5 ; H a r r i l l and G i f f o r d ,  Prodouz and N a v a r i , 1 9 7 5 ) -  The l e v e l s o f v i t a m i n E used were much  lower and the l e n g t h o f treatment was much s h o r t e r i n t h e s e  latter  79  i n v e s t i g a t i o n s compared t o the r e p o r t s  showing i n c r e a s e s  i n the  level  of t o t a l l i p i d s i n l i v e r .  2.  Plasma T o t a l L i p i d s and The  Cholesterol  r e s u l t s of t h i s study, shown i n F i g u r e s  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  17  and  18,  indicate  significantly altered  f o l l o w i n g 8 months t r e a t m e n t w i t h t h i g h l e v e l s o f v i t a m i n E.  However,  those r a t s t r e a t e d f o r 1 6 months w i t h h i g h l e v e l s of d i e t a r y v i t a m i n (over 2 , 5 0 0 Ill/kg d i e t ) had cholesterol.  The  s i g n i f i c a n t l y l o w e r plasma t o t a l l i p i d s  regression  E and  c u r v e s of plasma t o t a l l i p i d s on v i t a m i n  were l i n e a r , while those on plasma c h o l e s t e r o l were not more, the  decrease i n plasma t o t a l l i p i d s was  greater  c h o l e s t e r o l i n r a t s f e d h i g h l e v e l s of v i t a m i n  linear.  Further-  E suggesting that  other affected.  I t i s d i f f i c u l t t o compare the r e s u l t s of plasma t o t a l l l i p i d s c h o l e s t e r o l i n t h i s study w i t h those o f o t h e r workers, s i n c e the of v i t a m i n E s u p p l e m e n t a t i o n , the l e n g t h v a r y w i d e l y i n the  i n a l t e r i n g plasma t o t a l l i p i d and i n the l i t e r a t u r e are short  o f treatment and  experiments.  V i t a m i n E may  c h o l e s t e r o l , hut  inconsistent.  I t has  h i g h d i e t a r y v i t a m i n E had (Jenkins  level  dietary  reported  i n numerous lower  H a r r i l l et a l . ,  1965;  However, o t h e r workers have r e p o r t e d no  and M i t c h e l l , 1 9 7 5 ) ,  (Koyangi e t a l . , 1 9 6 6 ) .  that  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 rabbits  (Awad and  Gilbreath,  and  play a role  term s t u d i e s t h a t h i g h l e v e l s of d i e t a r y v i t a m i n E w i l l  Prodouz and N a v a r i , 1 9 7 5 ) -  chicks  the  the r e s u l t s  been r e p o r t e d  plasma c h o l e s t e r o l i n r a t s (Chen e t a l . , 1 9 7 2 ;  E  t h a n t h a t of plasma  components, such as t r i g l y c e r i d e s or p h o s p h o l i p i d s might a l s o he  ingredients  that  1975)  and  80  CHAPTER V I SUMMARY  The  purpose o f t h i s study was t o i n v e s t i g a t e t h e long-term  effect  of h i g h l e v e l s o f d i e t a r y v i t a m i n E on v a r i o u s m e t a b o l i c parameters i n the r a t .  S i x groups of female r a t s were f e d f o r a s l o n g a s 1 6 months  the b a s a l v i t a m i n E - f r e e d i e t w i t h supplements r a n g i n g from 0 t o 25,000  IU v i t a m i n E ( d l - a - t o c o p h e r y l a c e t a t e ) p e r k i l o g r a m d i e t .  l e v e l s o f v i t a m i n E chosen were 0 , diet.  25,  250,  2,500,  A l l n u t r i e n t s i n the b a s a l d i e t except  10,000  The  and 2 5 , 0 0 0  iu/kg  v i t a m i n E were adequate.  The m e t a b o l i c parameters s t u d i e d i n the r a t s f e d excess v i t a m i n E were compared s t a t i s t i c a l l y with t h e same parameters i n r a t s r e c e i v i n g a moderate o r normal l e v e l of d i e t a r y v i t a m i n E . T h e e f i n d i n g s o f t h i s study on the long-term  e f f e c t o f excess  intake  of v i t a m i n E i n t h e r a t were a s f o l l o w s : (1)  Body weights were depressed and 2 5 , 0 0 0  (2)  i n the groups f e d 1 0 , 0 0 0  IU v i t a m i n E/kg d i e t f o r 8 and 1 6 months.  High l e v e l s o f d i e t a r y v i t a m i n E i n c r e a s e d the r e l a t i v e h e a r t weight a f t e r 8 months and r e l a t i v e s p l e e n weight a f t e r 1 6 months.  (3)  Hemoglobin v a l u e s and spontaneous e r y t h r o c y t e  hemolysis  were n o t i n f l u e n c e d by e x c e s s i v e amounts o f v i t a m i n E . The  prothrombin time was reduced  while e l e v a t e d h e m a t o c r i t  a f t e r 1 2 months,  v a l u e was observed  after  1 6 months.of treatment. (4)  The a s h content o f bone d e c r e a s e d  with  concurrent  i n c r e a s e 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 o f  treatment.  (5)  U r i n a r y l e v e l s o f c r e a t i n e and c r e a t i n i n e were n o t a f f e c t e d by h i g h l e v e l s o f d i e t a r y v i t a m i n E .  (6) A l o g a r i t h m i c r e l a t i o n s h i p was observed  between  d i e t a r y l e v e l s o f v i t a m i n E and the c o n c e n t r a t i o n s of t h i s v i t a m i n i n l i v e r and plasma. (7) The c o n c e n t r a t i o n s o f v i t a m i n A i n l i v e r and plasma were n o t a f f e c t e d by h i g h l e v e l s o f d i e t a r y v i t a m i n E . (8) 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 by excess  v i t a m i n E supplementation  increased  i n rats fed  f o r 8 months, but n o t i n r a t s f e d f o r 16 months. 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