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UBC Theses and Dissertations

Glycogen metabolism in meal-fed pyridoxine-deficient rats Mellor, Ruth Marie 1973

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GLYCOGEN METABOLISM IN MEAL-FED PYRIDOXINE-DEFICIENT RATS by RUTH MARIE MELLOR B.H.Sc., U n i v e r s i t y o f G u e l p h , 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e d i v i s i o n o f HUMAN NUTRITION SCHOOL OF HOME ECONOMICS We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1973 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada i i ABSTRACT P y r i d o x i n e - d e f i c i e n t r a t s a r e known t o e x h i b i t l i t t l e , i f a n y , w e i g h t g a i n ; t h e y a l s o h a v e d e c r e a s e d f a t s t o r e s i n c o m p a r i s o n w i t h t h e i r p a i r - f e d c o n t r o l s . The d e f e c t i n e n e r g y m e t a b o l i s m r e s p o n s i b l e f o r t h i s phenomenon i s n o t w e l l u n d e r s t o o d a t p r e s e n t . T h i s s t u d y was u n d e r -t a k e n t o i n v e s t i g a t e some a s p e c t s o f g l y c o g e n e s i s a nd g l y c o g e n o l y s i s i n o r d e r t o a d d t o t h e p r e s e n t i n f o r m a t i o n on e n e r g y m e t a b o l i s m i n t h e p y r i d o x i n e d e f i c i e n c y s t a t e . M e a l - f e d a n i m a l s w e r e u s e d , i n o r d e r t o e l i m i n a t e d i f f e r -e n c e s due t o t h e mode o f f e e d i n g b e t w e e n t h e e x p e r i m e n t a l a n d t h e p a i r - f e d c o n t r o l a n i m a l s . M a l e w e a n l i n g r a t s w e r e f e d a p y r i d o x i n e - d e f i c i e n t d i e t i n one 2 - h o u r d a i l y m e a l , w h i l e t h e c o n t r o l s w e r e p a i r - f e d . T h i s e l i m i n a t e d d i f f e r e n c e s due t o f e e d i n g f r e q u e n c y when t h e s e g r o u p s w e r e c o m p a r e d w i t h e a c h o t h e r . A s p a r t a t e a m i n o - t r a n s f e r a s e a nd a l a n i n e a m i n o -t r a n s f e r a s e a c t i v i t i e s w e r e a s s a y e d i n l i v e r and e r y t h r o -c y t e s i n o r d e r t o v e r i f y t h e p r e s e n c e o f a p y r i d o x i n e d e f i c i e n c y s t a t e u n d e r t h e c o n d i t i o n s u s e d i n t h i s l a b o r a -t o r y . The a c t i v i t i e s o f g l y c o g e n p h o s p h o r y l a s e , t h e r a t e -l i m i t i n g enxyme i n g l y c o g e n o l y s i s , and g l y c o g e n UDP— g l u c o s y l t r a n s f e r a s e w e r e a s s a y e d i n l i v e r and m u s c l e . G l y c o g e n s t o r a g e i n t h e s e t i s s u e s was a l s o m e a s u r e d . F i n a l l y , the i n c o r p o r a t i o n o f l a b e l l e d c arbon atoms i n t o b l o o d g l u c o s e and l i v e r g l y c o g e n f o l l o w i n g i n t r a p e r i t o n e a l 14 i n j e c t i o n o f L - a l a n i n e - C was assayed. Glycogen p h o s p h o r y l a s e a c t i v i t y was reduced i n p y r i d o x i n e - d e f i c i e n t a n i m a l s . T h i s d e f e c t was n o t accompanied by a c o n c o m i t a n t i n c r e a s e i n the d e p o s i t i o n o f g l y c o g e n . There was, t h e r e f o r e , the p o s s i b i l i t y o f a d e c r e a s e d a b i l i t y t o form g l y c o g e n . Glycogen U D P - g l u c o s y l t r a n s f e r a s e a c t i v i t y was normal i n muscle and e l e v a t e d i n l i v e r i n d i c a t i n g , i f any-t h i n g , an u n i m p a i r e d a b i l i t y t o s y n e t h e s i z e g l y c o g e n from UDPG. A t r e n d towards a l e s s e r i n c o r p o r a t i o n o f l a b e l l e d carbon atoms i n t o the b l o o d g l u c o s e by the p y r i d o x i n e -d e f i c i e n t group appeared when the r e s u l t s were e x p r e s s e d as a p e r c e n t o f a d m i n i s t e r e d dose p e r ml. T h i s became s t a t i s t i c a l l y s i g n i f i c a n t when the d a t a was e x p r e s s e d i n terms o f the c i r c u l a t i n g g l u c o s e p o o l . A l t h o u g h n o t a t a s t a t i s t i c a l l y s i g n i f i c a n t l e v e l , t h e r e was a g r e a t e r i n c o r p o r a t i o n o f l a b e l l e d carbon atoms i n t o the l i v e r g l y c o g e n o f the p y r i d o x i n e - d e f i c i e n t group. I t appeared from t h e s e f i n d i n g s t h a t the d e f e c t i n energy m e t a b o l i s m i n p y r i d o x i n e d e f i c i e n c y may be the r e s u l t o f a reduced a v a i l a b i l i t y o f carbon s k e l e t o n s and o c c u r r e d p r i o r t o the f o r m a t i o n o f g l y c o g e n . F u r t h e r s t u d y i n t h i s a r e a i s n e c e s s a r y t o r e v e a l the e x a c t p o i n t a t w h i c h energy l o s s o c c u r r e d . i v TABLE OF CONTENTS Page 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 2 3 MATERIALS AND METHODS 23 4 EXPERIMENTAL AND RESULTS 32 5 DISCUSSION 45 6 LITERATURE CITED 51 7 APPENDIX 59 V L I S T OF TABLES Page T a b l e I C o m p o s i t i o n o f t h e d i e t 24 T a b l e I I C o m p o s i t i o n o f v i t a m i n m i x f o r t h e p y r i d o x i n e - d e f i c i e n t t e s t d i e t 25 T a b l e I I I C o m p o s i t i o n o f m i n e r a l m i x f o r a l l d i e t s .. 26 T a b l e I V L i v e r a nd e r y t h r o c y t e a s p a r t a t e a m i n o t r a n s -f e r a s e (GOT) and a l a n i n e a m i n o t r a n s f e r a s e (GPT) i n m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s 35 T a b l e V L i v e r and g a s t r o c n e m i u s m u s c l e g l y c o g e n p h o s p h o r y l a s e a c t i v i t i e s o f m e a l - f e d p y r i -d o x i n e - d e f i c i e n t r a t s 37 T a b l e V I L i v e r w e i g h t s and l i v e r a n d g a s t r o c n e m i u s m u s c l e g l y c o g e n l e v e l s o f m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s 3 8 T a b l e V I I L i v e r a n d g a s t r o c n e m i u s m u s c l e g l y c o g e n p h o s p h o r y l a s e a c t i v i t i e s o f m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s 40 T a b l e V I I I L i v e r and g a s t r o c n e m i u s m u s c l e g l y c o g e n -UDP g l u c o s y l t r a n s f e r a s e a c t i v i t i e s o f m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s 41 T a b l e I X I n c o r p o r a t i o n o f r a d i o a c t i v i t y f r o m a l a n i n e - U - 1 4 c i n t o b l o o d g l u c o s e a n d l i v e r g l y c o g e n i n m e a l - f e d p y r i d o x i n e -d e f i c i e n t r a t s 43 v i LIST OF FIGURES Page Figure 1. Composition of p y r i d o x o l , p y r i d o x i n e and p y r i d o x a l 3 Figure 2. S c h i f f base formed between p y r i d o x a l - 5 -phosphate and amino a c i d 4 Figure 3. Role of glycogen-UDP g l u c o s y l t r a n s f e r a s e and glycogen phosphorylase i n glycogen synthesis and g l y c o g e n o l y s i s , r e s p e c t i v e l y .. 13 Figure 4. B i n d i n g of p y r i d o x a l phosphate to phosphorylase 16 Figure 5. Food consumption of meal-fed p y r i d o x i n e -deprived r a t s 33 Figure 6. Weight gain of meal-fed p y r i d o x i n e - d e p r i v e d r a t s . 34 v i i ACKNOWLEDGEMENT I w o u l d l i k e t o t h a n k D r . J . F . A n g e l , D i v i s i o n o f Human N u t r i t i o n , U . B . C , f o r h i s knowledge and a s s i s t a n c e t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y . I w o u l d a l s o l i k e t o t h a n k D r . M. L e e , D i v i s i o n o f Human N u t r i t i o n , U . B . C , and D r . T. P e r k s , D e p a r t m e n t o f Z o o l o g y , U . B . C , f o r s e r v i n g on my c o m m i t t e e . I w o u l d l i k e t o a c k n o w l e d g e t h e a s s i s t a n c e o f o t h e r members o f t h e D i v i s i o n o f Human N u t r i t i o n , e s p e c i a l l y Ms. I . B o r g e n . F i n a l l y I w i s h t o t h a n k my h u s b a n d , D r . G a r y M e l l o r , f o r p a t i e n c e , p r a c t i c a l a d v i c e and p e t r i d i s h e s . 1. 1 INTRODUCTION P y r i d o x i n e - d e f i c i e n t r a t s g a i n l e s s w e i g h t and s t o r e l e s s f a t t h a n t h e i r p a i r - f e d c o n t r o l s , b u t t h e c a u s e o f t h i s d e f e c t has n o t been c l a r i f i e d t o d a t e . The e x c e s s e n e r g y l o s s i n t h e p y r i d o x i n e d e f i c i e n c y s t a t e c o u l d n o t be a c c o u n t e d f o r by i n c r e a s e s i n h e a t p r o d u c t i o n , p h y s i c a l e x e r c i s e o r l o s s t h r o u g h e x c r e t a . An i m p a i r m e n t i n f a t s y n t h e s i s c o u l d a c c o u n t f o r l o w e r e d body f a t s t o r e s . However, i t has b e e n o b s e r v e d t h a t t h e l i v e r and t h e a d i p o s e t i s s u e h a ve a n o r m a l c a p a c i t y t o s y n t h e s i z e f a t . The o t h e r m a j o r d i e t a r y s o u r c e o f e n e r g y i s c a r b o -h y d r a t e . W i t h t h e i n c l u s i o n o f p y r i d o x a l p h o s p h a t e i n t h e s t r u c t u r e o f g l y c o g e n p h o s p h o r y l a s e , t h e i m p a c t o f p y r i d o x i n e d e f i c i e n c y on t h i s enzyme c o u l d m a n i f e s t i t s e l f as an o v e r a l l change i n e n e r g y m e t a b o l i s m . I n s t u d i e s o f e n e r g y u t i l i z a t i o n by a n i m a l s s u b j e c t t o d i f f e r e n t n u t r i t i o n a l t r e a t m e n t s , i t i s i m p o r t a n t t o s t a n d a r d i z e n o t o n l y t h e amounts e a t e n by t h e e x p e r i m e n t a l and c o n t r o l g r o u p s , b u t a l s o t h e mode o f f e e d i n g . The p r e s e n t s t u d y was u n d e r t a k e n t o l o o k a t t h e c h a n g e s , i f a n y , t h a t o c c u r d u r i n g t h e s y n t h e s i s , s t o r a g e and s u b s e q u e n t breakdown o f g l y c o g e n i n p y r i d o x i n e d e f i c i e n t r a t s u n d e r a m e a l - f e e d i n g r e g i m e u t i l i z e d t o e l i m i n a t e d i f f e r e n c e s due t o mode o f f e e d i n g r a t h e r t h a n t h e d e f i c i e n c y i t s e l f . 2. 2 REVIEW OF LITERATURE 2.1 The R o l e o f P y r i d o x i n e i n M e t a b o l i s m I n 1934, G y o r g y d i s c o v e r e d a B - v i t a m i n t h a t c u r e d a c r o d y n i a i n r a t s ( G y o r g y , 1 9 3 4 ) . T h i s v i t a m i n was i s o l a t e d and i d e n t i f i e d as p y r i d o x o l i n 1938 ( G y o r g y , 1938; K e r e z t e s y and S t e v e n s , 1 9 3 8 ) . Much o f t h e e a r l y work on t h e r o l e o f t h i s f a c t o r i n n u t r i t i o n and m e t a b o l i s m was r e v i e w e d by S n e l l ( S n e l l , 1958; S n e l l , 1 9 6 1 ) . I n i t i a l l y , t h e t e r m " v i t a m i n Bg" was e m p l o y e d t o d e s i g n a t e p y r i d o x o l ( F i g . l a ) . However, S n e l l (1944) f o u n d t h a t a m i n a t i o n o r o x i d a t i o n p r o d u c e d p y r i d o x a m i n e ( F i g . l b ) o r p y r i d o x a l ( F i g . l c ) , r e s p e c t i v e l y .1 E i t h e r o f t h e s e d e r i v a t i v e s p o s s e s s e d a g r e a t e r g r o w t h - p r o m o t i n g a c t i v i t y f o r l a c t i c a c i d b a c t e r i a t h a n p y r i d o x o l i t s e l f ( S n e l l , 1944) . 2.1.1 R o l e o f P y r i d o x i n e i n M e t a b o l i c Pathways Much o f t h e i n f o r m a t i o n on t h e mechanism o f a c t i o n o f p y r i d o x a l p h o s p h a t e i n enzymes r e l a t e d t o amino a c i d m e t a b o l i s m has b een b a s e d on s t u d i e s w i t h n o n e n z y m a t i c model s y s t e m s . I t was s u g g e s t e d t h a t t h e n o n e n z y m a t i c r e a c t i o n s i n v o l v e t h e f o r m a t i o n o f S c h i f f - b a s e - m e t a l The t e r m i n o l o g y f o r v i t a m i n Bg i s as f o l l o w s : c o l l e c t i v e name i s p y r i d o x i n e ; v i t a m i n B , a l c o h o l i s p y r i d o x o l ; v i t a m i n B , a l d e h y d e i s p y r i d o x a l ; v i t a m i n B amine i s p y r i d o x a m i n e . 3. F i g u r e 1. C o m p o s i t i o n o f p y r i d o x o l , p y r i d o x a m i n e and p y r i d o x a l . P y r i d o x a m i n e F i g . l b P y r i d o x a l F i g . l c 4. Figure 2. S c h i f f base formed between pyridoxal-5-phosphate and amino a c i d . 5 . c h e l a t e complexes o f p y r i d o x a l ( F i g . 2 ) , i n whi c h t h e aldehyde group i s combined w i t h the amino group o f an amino a c i d ( S e b r e l l and H a r r i s , 1 9 6 8 ; S n e l l , 1 9 5 8 ; S n e l l , 1 9 6 1 ) . A l t h o u g h a b i - o r t r i v a l e n t m e t a l i o n p a r t i c i p a t e s i n S c h i f f -base f o r m a t i o n i n model systems, t h i s r e q u i r e m e n t appears t o be f u l f i l l e d i n the e n z y m a t i c r e a c t i o n by i n t e r a c t i o n s between p y r i d o x a l phosphate and the apoenzyme i t s e l f ( S e b r e l l and H a r r i s , 1 9 6 8 ) . The p h o s p h o r y l a t e d form o f p y r i d o x a l , p y r i d o x a l - 5 1 -p h o s p h a t e , was shown t o be a h i g h l y s p e c i f i c coenzyme w h i l e p y r i d o x a m i n e - 5 - p h o s p h a t e has l i m i t e d coenzyme a c t i v i t y ( Roberts and F r a n k e l , 1 9 5 1 ) . P y r i d o x a l phosphate-dependent enzymes are i n v o l v e d i n a wide v a r i e t y o f r e a c t i o n s , a l m o s t e n t i r e l y r e l a t e d t o amino a c i d m e t a b o l i s m . These i n c l u d e : t r a n s a m i n a t i o n s , d e c a r b o x y l a t i o n s , amine o x i d a t i o n s , a l d o l c o n d e n s a t i o n s , c l e a v a g e r e a c t i o n s , and d e a m i n a t i n g d e h y d r a -t i o n s ( S e b r e l l and H a r r i s , 1 9 6 8 ) . Some 5 0 p y r i d o x a l phosphate-dependent enzymes have been l i s t e d by S a u b e r l i c h ( S e b r e l l and H a r r i s , 1 9 6 8 ) . A r o l e f o r p y r i d o x a l - 5 - p h o s p h a t e i n c a r b o h y d r a t e m e t a b o l i s m i s s u g g e s t e d by i t s i n v o l v e m e n t w i t h p o l y s a c c h a r -i d e p h o s p h o r y l a s e s . The p o l y s a c c h a r i d e p h o s p h o r y l a s e s d i f f e r from most o f the p y r i d o x a l phosphate enzymes i n t h a t they do n o t r e q u i r e a f r e e a ldehyde group f o r t h e i r a c t i v i t y . T h i s i s borne o ut o f the f a c t t h a t r e d u c t i o n o f g l y c o g e n p h o s p h o r y l a s e w i t h NaBH. has no i n f l u e n c e on the a c t i v i t y 6 . o f t h e enzyme ( F i s c h e r e t a_l . , 1 9 5 8 ) . I t was shown t h a t t h e r o l e o f p y r i d o x a l p h o s p h a t e i n t h e a c t i v a t i o n o f t h i s enzyme i s s t r u c t u r a l : i n m a i n t a i n i n g i t s t e t r a m e r i c f o r m i n t a c t ( F i s c h e r a n d K r e b s , 1 9 6 6 ) . P y r i d o x i n e h a s a l s o b e e n i m p l i c a t e d i n l i p i d m e t a -b o l i s m , b u t i t s r o l e h a s n o t b e e n f u l l y c h a r a c t e r i z e d . T h u s , s e v e r a l r e p o r t s h a v e s u g g e s t e d i t s i n v o l v e m e n t w i t h f a t t y a c i d e l o n g a t i o n , c h o l e s t e r o l m e t a b o l i s m , b o d y a n d l i v e r f a t s t o r a g e and u n s a t u r a t e d f a t t y a c i d i n t e r c o n v e r -s i o n s ( S e b r e l l a n d H a r r i s , 1 9 6 8 ) . H o w e v e r , no p y r i d o x a l p h o s p h a t e - d e p e n d e n t enzyme s y s t e m s i n v o l v e d i n l i p i d m e t a b o l i s m h a v e b e e n i s o l a t e d . 2.2 E n e r g y U t i l i z a t i o n i n P y r i d o x i n e D e f i c i e n c y P y r i d o x i n e d e f i c i e n c y h a s l o n g b e e n known t o r e s u l t i n a l t e r a t i o n s i n c a r b o h y d r a t e a n d f a t m e t a b o l i s m , b u t t h e e x a c t n a t u r e a n d s p e c i f i c i t y o f t h i s r e l a t i o n s h i p h a s n o t y e t b e e n a d e q u a t e l y c l a r i f i e d . The m a i n a l t e r a t i o n i s a r e d u c t i o n i n t h e a b i l i t y o f a n i m a l s t o s t o r e e n e r g y as bo d y f a t i n t h e d e f i c i e n c y s t a t e ( M u e l l e r , 1 9 6 4 ; S a k u r a g i , 1 9 5 9 ; S h e r m a n , 1 9 5 0 ) . I n a d d i t i o n , s e v e r a l i n t e r f e r e n c e s w i t h c a r b o h y d r a t e m e t a b o l i s m h a v e b e e n r e p o r t e d ( K r e b s a n d F i s c h e r , 1 9 6 4 ) . One o f t h e e a r l i e s t r e s p o n s e s t o n u t r i t i o n a l d e f i c -i e n c y i s i n a n i t i o n . A c c o r d i n g l y , i t i s i m p o r t a n t i n s t u d i e s c o n c e r n e d w i t h t h e e f f e c t s o f d e f i c i e n c y on e n e r g y s t o r a g e 7. to e q u a l i z e the c a l o r i c i ntakes of the experimental and the c o n t r o l animals. Since i n most st u d i e s reported i n the l i t e r a t u r e comparison has been made between p y r i d o x i n e -d e f i c i e n t and p a i r - f e d c o n t r o l animals, the discrepancy i n energy storage i n v i t a m i n d e f i c i e n c y must be r e l a t e d to energy u t i l i z a t i o n . The amount of energy stored by animals under any c o n d i t i o n i s the balance between int a k e and output. Out-put represents the f r a c t i o n of energy spent f o r maintaining b a s a l metabolism, f o r heat pr o d u c t i o n , and f o r p h y s i c a l a c t i v i t y . /Any excess would be s t o r e d . 2.2.1 Basal Metabolic Rate O r s i n i e t a l . (1942) observed a lowered metabolic r a t e i n p y r i d o x i n e - d e p r i v e d r a t s as compared to ad l i b i t u m -fed c o n t r o l s . J.R. Beaton e t a l . (1956) found no d i f f e r e n c e i n the b a s a l metabolic r a t e between py r i d o x i n e - d e p r i v e d and p a i r - f e d r a t s . Paul (1968) confirmed that the b a s a l metabolic rates of p y r i d o x i n e - d e f i c i e n t r a t s and t h e i r p a i r - f e d c o n t r o l s were s i m i l a r . However, the l e v e l of oxygen consumption of both groups was greater than t h a t a t t a i n e d by the ad l i b i t u m - f e d group. 2.2.2 Heat Production D i r e c t q u a n t i t a t i v e measurements on heat production i n p y r i d o x i n e d e f i c i e n c y are l a c k i n g . However, Yeh and Weiss (1963) found t h a t when pyr i d o x i n e - d e p r i v e d r a t s were 8. e x p o s e d t o a c o l d e n v i r o n m e n t , t h e i r t e m p e r a t u r e d e c r e a s e d more r a p i d l y t h a n t h a t o f p a i r - f e d c o n t r o l s . When t r a i n e d t o p r e s s a l e v e r t o o b t a i n h e a t , t h e p y r i d o x i n e - d e f i c i e n t r a t s d i d s o more o f t e n t h a n t h e p a i r - f e d g r o u p . The a u t h o r s c o n c l u d e d t h a t t h i s r e f l e c t e d e i t h e r i n c r e a s e d h e a t l o s s o r i n a b i l i t y t o m a i n t a i n b o d y t e m p e r a t u r e u n d e r c o l d s t r e s s . 2.2.3 P h y s i c a l A c t i v i t y T h e r e a r e no d a t a i n t h e l i t e r a t u r e on t h e p h y s i c a l a c t i v i t y l e v e l s o f p y r i d o x i n e - d e f i c i e n t a n i m a l s . T h e r e f o r e , i t i s n o t p o s s i b l e t o e v a l u a t e t h i s a s p e c t o f e n e r g y e x p e n -d i t u r e . 2.2.4 E n e r g y L o s s i n E x c r e t a C a r t e r a n d P h i z a c k e r l e y (1951) f o u n d t h a t t h e a b s o r p t i o n o f c a r b o h y d r a t e , f a t and t h e p r o d u c t s o f p r o t e i n d i g e s t i o n was n o t i m p a i r e d i n p y r i d o x i n e d e f i c i e n c y . I t was m e n t i o n e d i n t h e s t u d i e s o f H u b e r e t a l . (1964) t h a t u n r e p o r t e d d a t a on u r i n a r y e x c r e t i o n i n p y r i d o x i n e d e f i c i e n c y showed no i n c r e a s e d l o s s o f c a l o r i e s . P a u l (196 8) c o n f i r m e d t h a t t h e l o s s o f e n e r g y i n t h e f a e c e s o f p y r i d o x i n e - d e f i c i e n t r a t s was u n a l t e r e d . H o w e v e r , t h e a n i m a l s showed e n e r g y l o s s e s i n t h e u r i n e t h a t w o u l d a c c o u n t f o r a p p r o x i m a t e l y h a l f t h e c a l c u l a t e d d i s c r e p a n c y i n e n e r g y s t o r a g e b e t w e e n t h e e x p e r i m e n t a l a n d t h e p a i r - f e d c o n t r o l r a t s . 9. 2.2.5 Body Fat Stores As e a r l y as 1949, Sure and E a s t e r l i n g (1949) demon-s t r a t e d that p y r i d o x i n e - d e p r i v e d r a t s s t o r e l e s s carcass f a t than t h e i r p a i r - f e d c o n t r o l s . This was confirmed subsequently by Beare e t al_. (1953) . While the lowering of body f a t l e v e l s i n p y r i d o x i n e d e f i c i e n c y has been demonstrated repeatedly, the observa-t i o n s on l i v e r l i p i d s have not been c o n s i s t e n t . Guggenheim and Diamant (1957) reported increased d e p o s i t i o n of f a t i n the l i v e r of py r i d o x i n e - d e p r i v e d r a t s . This was i n d i r e c t o p p o s i t i o n to the f i n d i n g s of Ca r t e r and P h i z a c k e r l y (1951) who found no a l t e r a t i o n i n l i v e r f a t i n pyr i d o x i n e - d e p r i v e d r a t s , even when a d i e t c o n t a i n i n g 20% f a t was used. An impairment i n f a t s y n t h e s i s could account f o r lowering body f a t s t o r e s . Desikachar and McHenry (1954) demonstrated decreased f a t d e p o s i t i o n i n p y r i d o x i n e - d e f i c i e n t 14 r a t s as compared to p a i r - f e d c o n t r o l s . When C - l a b e l l e d glucose was given o r a l l y , the rates of r a d i o a c t i v i t y i n c o r -p o r a t i o n i n t o carcass f a t t y acids were s i m i l a r i n both groups p r e c l u d i n g any impairment i n f a t synthesis under those c o n d i t i o n s . The authors suggested that increased f a t o x i d a t i o n i s a p l a u s i b l e mechanism of lowering body f a t l e v e l s . This was i n disagreement w i t h the suggestion of C a r t e r and P h i z a c k e r l y (1951), t h a t p y r i d o x i n e was r e -qu i r e d f o r the conversion of carbohydrate to f a t . 10. 14 Sabo e_t a l . (1971) using glucose-1- C and acetate-14 1- C, s t u d i e d f a t synt h e s i s by l i v e r s l i c e s from animals deprived of p y r i d o x i n e f o r 3-4 weeks. There was no d i f f e r e n c e i n l i p i d s y nthesis from acetate or glucose i n fa s t e d or fed d e f i c i e n t and p a i r - f e d c o n t r o l r a t s • These authors concluded t h a t the l i v e r and the adipose t i s s u e of the deprived r a t s had a normal ca p a c i t y to synthesize f a t when glucose was m e t a b o l i c a l l y a v a i l a b l e . 2.3 Carbohydrate Metabolism i n P y r i d o x i n e D e f i c i e n c y The only known p y r i d o x a l phosphate-dependent enzyme th a t could p o s s i b l y a f f e c t energy metabolism d i r e c t l y i s glycogen phosphorylase. Information on t h i s area of carbo-hydrate metabolism should be u s e f u l i n the study of energy metabolism i n py r i d o x i n e d e f i c i e n c y . P y r i d o x i n e has long been known t o a f f e c t carbo-hydrate metabolism, but the exact mechanism i s not c l e a r . J.R. Beaton and Goodwin (1954) found t h a t there was a s i g n i f i c a n t decrease i n the l e v e l s of blood sugar and l i v e r glycogen i n p y r i d o x i n e - d e f i c i e n t r a t s as compared to p a i r -fed c o n t r o l s a f t e r only 5 days of d e p r i v a t i o n . A f t e r 2 l days, there was a l s o a decrease i n f a s t i n g l e v e l s of p y r u v i c and l a c t i c acids suggesting t h a t carbohydrate s t o r e s were e i t h e r r a p i d l y depleted or not formed i n normal amounts. Other work by J.R. Beaton (1955) revealed a decrease i n a p y r u v i c a c i d s y n t h e s i z i n g enzyme ( i . e . aldolase) i n the d e f i c i e n c y s t a t e . Huber e t a l . (41) reported hypoglycemia i n p y r i d o x i n e -deprived r a t s . This was i n agreement w i t h the r e s u l t s of Lyon and P o r t e r (1962). Guggenheim and Diamant (1957) showed no d i f f e r e n c e s i n l i v e r glycogen syn t h e s i s of p y r i d o x i n e -def i c i e n t r a t s as compared to ad l i b i t u m - f e d r a t s ; however, the p a i r - f e d c o n t r o l s had elevated blood glucose and l i v e r glycogen l e v e l s . This explained previous f i n d i n g s from experiments i n which only p a i r - f e d c o n t r o l s were used. The e l e v a t e d l e v e l s of l i v e r glycogen and blood sugar i n p a i r -fed animals p o s s i b l y r e f l e c t e d the meal-eating p a t t e r n of food consumption. This e f f e c t has been well-documented by many workers ( F u l l e r and D i l l e r , 19 70; L e v e i l l e , 19 66; L e v e i l l e and Chakrabarty, 1967). Muscle glycogen l e v e l s have a l s o been s t u d i e d . I l l i n g w o r t h e t a l . (1960) found no s i g n i f i c a n t d i f f e r e n c e i n muscle glycogen l e v e l s between py r i d o x i n e - d e p r i v e d r a t s and ad l i b i t u m - f e d c o n t r o l s . Lyon and P o r t e r (19 62) reported decreased l e v e l s of muscle glycogen i n two s t r a i n s of p y r i -doxine-deprived mice compared to ad l i b i t u m - f e d c o n t r o l s . Angel (1968) found that meal-fed p y r i d o x i n e - d e f i c i e n t r a t s deposited l e s s glycogen f o l l o w i n g the meal than t h e i r u n r e s t r i c t e d but meal-fed c o n t r o l s . The subcutaneous admin-i s t r a t i o n of 300 Kg p y r i d o x o l HCl/rat/day f o r 2 days increased -glycogen d e p o s i t i o n over t h a t of the c o n t r o l s as long as 12. t h e r e p l e t e d a n i m a l s had u n r e s t r i c t e d a c c e s s t o f o o d d u r i n g t h e d a i l y m e a l . T h u s , i t a p p e a r s t h a t t h e e f f e c t o f p y r i -d o x i n e on g l y c o g e n d e p o s i t i o n may be m e d i a t e d t h r o u g h i t s e f f e c t on f o o d i n t a k e . As shown i n F i g . 3 ( F i s c h e r and K r e b s , 1966) , g l y c o g e n - U D P g l u c o s y l t r a n s f e r a s e ( G l y c o g e n s y n t h e t a s e ) and g l y c o g e n p h o s p h o r y l a s e a r e t h e two enzymes i n v o l v e d d i r e c t l y i n g l y c o g e n s y n t h e s i s and b r e a k d o w n , r e s p e c t i v e l y . T h e s e enzymes p r o b a b l y a c t t o c o n t r o l t h e r a t e o f g l y c o g e n d e p o s i t i o n and m o b i l i z a t i o n . . G l i n s m a n e t a l . (19 70) n o t e d t h a t h y p e r g l y c e m i a c a u s e d t h e i n a c t i v a t i o n o f p h o s p h o r y l a s e i . e . l i v e r p h o s p h o r y l a s e > dephospho p h o s p h o r y l a s e and t h e a c t i v a t i o n o f g l y c o g e n s y n t h e t a s e i . e . g l u c o s e - 6 -p h o s p h a t e d e p e n d e n t (D) > g l u c o s e - 6 - p h o s p h a t e i n d e p e n d -e n t (I) i n p e r f u s e d r a t l i v e r . B i s h o p and L a r n e r (1967) and L a r n e r (1967-68) f o u n d t h a t g l u c a g o n had an e f f e c t on t h e s e enzymes s i m i l a r t o t h a t o f g l u c o s e and i n s u l i n r e v e r s e d t h i s e f f e c t . 2.3.1 G l y c o g e n P h o s p h o r y l a s e P y r i d o x a l - 5 - p h o s p h a t e has b een i s o l a t e d f r o m p h o s -p h o r y l a s e p r e p a r a t i o n s o f l o b s t e r m u s c l e , c a t m u s c l e , human s t r i a t e d and h e a r t m u s c l e , p i g l i v e r and p o t a t o e s ( B a r a n o w s k i e t a l . , 1957; F i s c h e r e t a l _ . 1 9 6 3 ) . P h o s p h o r y l a s e f r o m m u s c l e t i s s u e has been t h o r o u g h l y s t u d i e d and e x t e n s i v e r e v i e w s a r e a v a i l a b l e ( F i s c h e r e t a l . , 1963; K r e b s and g u r e 3. R o l e o f g l y c o g e n - U D P g l u c o s y l t r a n s f e r a s e and g l y c o g e n p h o s p h o r y l a s e i n g l y c o g e n s y n t h e s i s and g l y c o g e n o l y s i s , r e s p e c t i v e l y . UDP UTP GLUCOSE * GLUCOSE-6-P UTILIZATION 1 4 . F i s c h e r , 1 9 6 4 ) . The g l y c o g e n p h o s p h o r y l a s e o f m u s c l e e x i s t s i n two f o r m s : ' a ' and 'b', t h e f o r m e r b e i n g t h e a c t i v e o n e . The r e -l a t i o n s h i p between t h e two forms i s as f o l l o w s : 2 p h o s p h o r y l a s e 'b' + 4 ATP 2YSii2 _ d ? ? ^ p h o s p h o r y l a s e ' a ' + 4 ADP p h o s p h o r y l a s e ' a ' + 4 R^O > 2 p h o s p h o r y l a s e ' b_' + 4 P i The 'b' f o r m i s i n a c t i v e w i t h o u t AMP w h i l e t h e ' a ' f o r m i s s t i m u l a t e d by AMP b u t does n o t r e q u i r e i t f o r a c t i v i t y . E a c h m o l e c u l e o f p h o s p h o r y l a s e ' b_' c o n s i s t s o f two s u b u n i t s o f a m o l e c u l a r w e i g h t o f 1 2 5 , 0 0 0 e a c h , w h i l e p h o s p h o r y l a s e 'a' i s composed o f f o u r o f t h e s e s u b u n i t s . L i v e r p h o s p h o r y l a s e i s a d i f f e r e n t t y p e o f p r o t e i n t h a n t h a t o f m u s c l e . U n l i k e m u s c l e p h o s p h o r y l a s e , l i v e r p h o s p h o r y l a s e has t h e same m o l e c u l a r w e i g h t i . e . 2 3 7 , 0 0 0 i n b o t h t h e a c t i v e and i n a c t i v e f orms c a l l e d l i v e r p h o s p h o r -y l a s e and dephospho p h o s p h o r y l a s e r e s p e c t i v e l y and b o t h c o n s i s t o f two s u b u n i t s . A p r o b l e m e x i s t s i n a s s a y i n g l i v e r p h o s p h o r y l a s e i n t h a t , u n l i k e t h a t o f m u s c l e , t h e i n a c t i v e o r dephospho p h o s p h o r y l a s e f o r m i s o n l y s l i g h t l y a c t i v a t e d by 7AMP and t h e r e f o r e u s u a l l y o n l y t h e a c t i v e f o r m i s i n c l u d e d i n t h e a s s a y f o r t o t a l p h o s p h o r y l a s e ( K r e b s and F i s c h e r , 1 9 6 4 ) . P y r i d o x a l p h o s p h a t e i s bound t o p h o s p h o r y l a s e as a s u b s t i t u t e d a l d a m i n e d e r i v a t i v e i n v o l v i n g an amine g r o u p and a n o t h e r u n i d e n t i f i e d g r o u p X f r o m t h e p r o t e i n ( F i s c h e r e t a l . , 1 9 6 3 ) . I n a c i d o r b a s e , f o r m I ( F i g . 4) i s c o n v e r t e d t o y e l l o w S c h i f f b a s e ( I I ) w h i c h can h y d r o l y z e t o g i v e f r e e p y r i d o x a l p h o s p h a t e and a p o p h o s p h o r y l a s e ( I I I ) . The amino g r o u p t o w h i c h p y r i d o x a l p h o s p h a t e i s bound was shown t o be t h e e p s i l o n - a m i n o g r o u p o f a l y s i n e r e s i d u e i n p h o s p h o r -y l a s e . T h e r e have been v a r y i n g r e p o r t s on t h e e f f e c t o f p y r i d o x i n e d e f i c i e n c y on p h o s p h o r y l a s e a c t i v i t y . E i s e n -s t e i n (1962) and L y o n and P o r t e r (1962) f o u n d a d e c r e a s e i n m u s c l e p h o s p h o r y l a s e 'a' w h i l e I l l i n g w o r t h e t a l . (1960) f o u n d no change. T o t a l m u s c l e p h o s p h o r y l a s e ( E i s e n s t e i n , 1962; I l l i n g w o r t h e t a l . , 1960; L y o n and P o r t e r , 1962) and t o t a l l i v e r p h o s p h o r y l a s e ( E i s e n s t e i n , 1962; L y o n and P o r t e r , 1962) a c t i v i t i e s were d e c r e a s e d i n p y r i d o x i n e d e f i -c i e n c y . I l l i n g w o r t h p o s t u l a t e d t h a t i n c r e a s e d 'a' t o 'b' c o n v e r s i o n may o c c u r as a p r o t e c t i v e measure t o m a i n t a i n t h e enzyme a c t i v i t y l e v e l s n e e d e d t o s u p p l y e n e r g y f o r m u s c l e c o n t r a c t i o n s . T akami e t a l . (19 6 8) f o u n d t h a t p y r i d o x a l p h o s p h a t e l e v e l i n t h e l i v e r was d e c r e a s e d by 30% a f t e r s i x t y days on a p y r i d o x i n e - f r e e d i e t , and t h e n i t r e m a i n e d c o n s t a n t . I t was s u g g e s t e d t h a t t h e s o u r c e o f p y r i d o x a l p h o s p h a t e t o m a i n t a i n t h i s l e v e l was m u s c l e p h o s p h o r y l a s e . K r e b s and F i s c h e r (1964) have s t a t e d t h e b e l i e f t h a t t h e r e i s a l a r g e r e s e r v o i r o f p y r i d o x a l p h o s p h a t e i n m u s c l e 17. phosphorylase (perhaps one-half of a l l p y r i d o x i n e i n the body). About 60% of a l l p y r i d o x i n e i n muscle i s a s s o c i a t e d w i t h phosphorylase; i n l i v e r , much of the p y r i d o x i n e i s not a s s o c i a t e d w i t h phosphorylase. The importance of t h i s i s unknown. P y r i d o x i n e i n muscle e x i s t s i n a p y r i d o x a l r a t h e r than pyridoxamine form (Krebs and F i s c h e r , 1964) and thus does not need f u r t h e r conversion before use. 2.3.2 Glycogen-UDP g l u c o s y l t r a n s f e r a s e (Glycogen Synthetase) Glycogen synthetase does not contain p y r i d o x a l phos-phate i n i t s s t r u c t u r e ; n e v e r t h e l e s s , i t may be important as a p o s s i b l e r e g u l a t o r of the r a t e of glycogen s y n t h e s i s . 14 The r a d i o a c t i v e glycogen formed from UDPG- C i n d i c a t e s t h a t the glucose residue becomes attached to an o<(l-4) l i n k a g e , the same l i n k hydrolyzed by glycogen phosphorylase ( L e l o i r e t a l . , 1959) . L e l o i r e t a l . (1959) found that the i n v i t r o r e a c t i o n r e q u i r e d the presence of a polysaccharide primer and t h a t i t i s a c t i v a t e d by hexose-6-phosphates. J u s t as glycogen phosphorylase e x i s t s i n two forms, so does glycogen synthetase. These are known as glucose-6-phosphate dependent (D) and glucose-6-phosphate independent (I) forms (Glinsmann e t a l . , 1970). Gold (1968) disagreed w i t h the i m p l i c a t i o n s of t h i s terminology, s t a t i n g t h a t the EDTA used i n the assay medium masked glucose-6-phosphate a c t i v a t i o n of the s o - c a l l e d independent form. Gold found t h a t i n v i t r o a c t i v a t i o n r e s u l t s i n the appearance of a 18. f o r m w i t h i n c r e a s e d s e n s i t i v i t y t o g l u c o s e - 6 - p h o s p h a t e and an i n c r e a s e d a f f i n i t y t o M g + + . The o n l y p u b l i s h e d s t u d y on t h e e f f e c t o f p y r i d o x i n e d e f i c i e n c y on g l y c o g e n s y n t h e t a s e a c t i v i t y i s t h a t o f I l l i n g w o r t h e t a l _ . (1960) . The a c t i v i t y o f t h e m u s c l e enzyme r e m a i n e d u n a l t e r e d a f t e r s e v e n t o e i g h t weeks on a p y r i d o x i n e - f r e e d i e t , as c o m p a r i s o n w i t h t h e c o n t r o l s r e -v e a l e d . A n o t h e r means by w h i c h p y r i d o x i n e may p l a y a r o l e i n c a r b o h y d r a t e m e t a b o l i s m was s u g g e s t e d by t h e r e s u l t s o f G l a z e r and Weber ( 1 9 7 1 ) . T h e s e a u t h o r s f o u n d t h a t p y r i d o x a l p h o s p h a t e i n h i b i t e d g l u c o s e c o n v e r s i o n t o l a c t a t e i n r a t l i v e r homogenate, b u t h a d l i t t l e e f f e c t on t h e c o n v e r s i o n o f g l u c o s e - 6 - p h o s p h a t e t o l a c t a t e . T h e r e f o r e , i t was s u g g e s t e d t h a t p y r i d o x a l p h o s p h a t e i n h i b i t s g l u c o s e - 6 -p h o s p h a t e f o r m a t i o n , p o s s i b l y b y d e p r e s s i n g h e x o k i n a s e . a c t i v i t y . On t h e b a s i s o f t h i s , i t w o u l d f o l l o w t h a t t h e i n h i b i t i o n w o u l d be r e d u c e d i n p y r i d o x i n e d e f i c i e n c y , t h u s i n c r e a s i n g g l u c o s e - 6 - p h o s p h a t e a v a i l a b i l i t y f o r u t i l i -z a t i o n . 2.4 E f f e c t o f Mode o f F e e d i n g To o b t a i n r e s u l t s d i r e c t l y c a u s e d by p y r i d o x i n e d e f i c i e n c y , t h e e f f e c t s o f t h e a c c o m p a n y i n g i n a n i t i o n must be e l i m i n a t e d . T h i s i s b e s t a c c o m p l i s h e d u s i n g p a i r - f e d c o n t r o l s . G.H. B e a t o n e t a l . (195 6) t r i e d t o overcome i n a n i t i o n by a d m i n i s t e r i n g i n s u l i n t o p y r i d o x i n e - d e p r i v e d r a t s and f o u n d t h a t f a t s t o r e s were i n c r e a s e d t o t h e l e v e l o f t h e c o n t r o l s . The a u t h o r s c o n c l u d e d t h a t i n a n i t i o n c o u l d , t h e r e f o r e , be t h e c a u s e o f d e c r e a s e d f a t s t o r e s . Huber e t a l (1964) a l s o f o u n d t h a t i n s u l i n r e v e r s e d many o f t h e e f f e c t s o f p y r i d o x i n e d e f i c i e n c y , b u t c o n c l u d e d t h a t more t h a n j u s t i n a n i t i o n was i n v o l v e d , as i n s u l i n s e n s i t i v i t y o f a d i p o s e t i s s u e segments was a l s o i n c r e a s e d . I n p a i r - f e e d i n g t r i a l s , t h e d e f i c i e n t r a t s a r e a l l o w e d f o o d ad l i b i t u m w h i l e t h e p a i r - f e d a n i m a l s a r e f o r c e d t o consume l e s s t h a n t h e y w o u l d e a t i f f e d ad l i b i t u m . U n der t h e s e c o n d i t i o n s t h e p a i r - f e d c o n t r o l s consume t h e i r f o o d q u i c k l y , and become ' m e a l - e a t e r s ' r a t h e r t h a n ' n i b b l e r s ' T h u s , t h e y u n d e r g o l o n g p e r i o d s o f f a s t i n g , t h e e f f e c t s o f w h i c h i n c l u d e h y p e r p l a s i a o f t h e g a s t r o i n t e s t i n a l t r a c t and h y p e r l i p o g e n e s i s (Cohn and J o s e p h , 19 59; Cohn and J o s e p h , 1960; Cohn and J o s e p h , 1968; Cohn and J o s e p h , 1970; F a b r y and Brown, 1967; H o l l i f i e l d and P a r s o n , 1962; L e v e i l l e and H a n s o n , 1965; Tepperman and T epperman, 1 9 6 4 ) . L e v e i l l e and O'Hea (1967) r e p o r t e d a d e c r e a s e d a c t i v i t y l e v e l o f m e a l - f e d a n i m a l s s u c h t h a t t h e a n i m a l s were a b l e t o m a i n t a i n n o r m a l w e i g h t g a i n on 70% o f t h e f o o d c o n s u m p t i o n o f t h e a d l i b i t u m - f e d a n i m a l s . T h e r e f o r e i t does n o t seem r e a s o n a b l e t o compare t h e ad. l i b i t u m - f e d i . e . n i b b l i n g , d e p r i v e d r a t t o e i t h e r a 20. p a i r - f e d o r a p a i r - w e i g h e d c o n t r o l . A l o g i c a l a l t e r n a t i v e w o u l d be t o a d a p t t h e g r o u p t o be d e p r i v e d o f p y r i d o x i n e t o a m e a l - f e e d i n g s c h e d u l e . T h i s w o u l d e l i m i n a t e n o t o n l y d i f f e r e n c e s due t o c a l o r i c i n t a k e , b u t a l s o d i f f e r e n c e s due t o f e e d i n g f r e q u e n c y . E v i d e n c e i n s u p p o r t o f t h e v a l u e o f m e a l - f e e d i n g as s u g g e s t e d c a n be o b t a i n e d f r o m t h e w o r k o f E m e r s o n e_t a l . (1959) a n d C o c k b u r n a n d V a n B r u g g e n ( 1 9 5 9 ) . They d e m o n s t r a t e d t h a t t h e l e n g t h o f t i m e a f t e r e a t i n g m u s t be known i n w o r k m e a s u r i n g l i p i d - l a b e l l i n g b e c a u s e o f t h e r a p i d i n c r e a s e and s u b s e q u e n t d r o p t h a t o c c u r s a f t e r t h e i n g e s t i o n o f f o o d . F u l l e r and D i l l e r (1970) p o i n t o u t t h e d a n g e r o f c o m p a r i n g r e s u l t s o f l i v e r g l y c o g e n d e p o s i t i o n i n m e a l -f e d and a d l i b i t u m - f e d r a t s , i n t h a t one may c o n c l u d e m e a l -f e d h a d g r e a t e r , l e s s e r o r t h e same d e g r e e o f d e p o s i t i o n d e p e n d i n g on w h e t h e r t h e m e a s u r e m e n t was t a k e n t w e l v e h o u r s a f t e r t h e s t a r t o f t h e m e a l , j u s t b e f o r e t h e m e a l , o r h a l f w a y i n b e t w e e n , r e s p e c t i v e l y . T h i s was s u g g e s t e d b e a r i n g i n m i n d t h a t ad l i b i t u m - f e d r a t s u s u a l l y e a t m o s t l y a t n i g h t a n d m e a l - f e d a n i m a l s a r e u s u a l l y f e d i n t h e m o r n i n g . B e f o r e s a c r i f i c e b o t h g r o u p s a r e commonly f a s t e d , a ' n o r m a l ' c o n d i t i o n f o r t h e r a t a d a p t e d t o m e a l -f e e d i n g b u t n o t f o r t h e n i b b l i n g r a t . Thus t h e two g r o u p s m u s t be a t a s i m i l a r p o i n t a f t e r f o o d i n g e s t i o n . Using p y r i d o x i n e - d e p r i v e d r a t s adapted to a two-hour d a i l y feeding schedule, Paul (1968) showed t h a t , a f t e r 60 days of d e p r i v a t i o n , the d e f i c i e n t r a t s weighed s i g n i f i -c a n t l y l e s s than e i t h e r the ad l i b i t u m - f e d or p a i r - f e d c o n t r o l s . Of i n t e r e s t i s the f a c t that although body f a t l e v e l s were s i g n i f i c a n t l y l e s s i n the p y r i d o x i n e - d e f i c i e n t than i n the ad l i b i t u m - f e d c o n t r o l s , they were only s l i g h t l y l e s s than those of the p a i r - f e d group. Thus, i t appears t h a t the e f f e c t of py r i d o x i n e d e f i c i e n c y on body f a t (cf. Sect i o n 2.2.5) may be a t t r i b u t e d t o d i f f e r e n c e s i n the mode of feeding between the experimental and the c o n t r o l r a t s . 2.5 Summary Pyr i d o x i n e d e f i c i e n c y leads t o a l t e r a t i o n s i n over-a l l energy pr o d u c t i o n , u t i l i z a t i o n and storage. As the pyri d o x i n e - d e p r i v e d animal stores l e s s energy as f a t , i t must be expending more energy. This increased energy expenditure of the animal should be r e f l e c t e d i n i t s b a s a l metabolic r a t e , i t s l e v e l of p h y s i c a l a c t i v i t y and heat production and/or the balance of energy between in t a k e and e x c r e t a . However, the reason f o r the excess energy l o s s i n p y r i d o x i n e d e f i c i e n c y cannot be explained i n these terms. Studies on f a t metabolism revealed t h a t adipose and l i v e r t i s s u e s from py r i d o x i n e - d e p r i v e d r a t s have, i f anything, a normal a b i l i t y to form l i p i d s from l a b e l l e d acetate or glucose. This i n d i c a t e s that lack of f a t precursors may be r e s p o n s i b l e f o r the observed r e d u c t i o n i n body f a t s t o r e s . The most immediate source of energy i s carbohydrate. With the i n c l u s i o n of p y r i d o x a l phosphate i n the s t r u c t u r e of glycogen phosphorylase, the impact of p y r i d o x i n e d e f i -ciency on energy metabolism i n general i s important. A la r g e gap i n inf o r m a t i o n e x i s t s at present between the expected decrease i n carbon skeletons a v a i l a b l e because of f a u l t y transamination and the well-documented e f f e c t s of p y r i d o x i n e d e f i c i e n c y on glycogen phosphorylase i n ad l i b i t u m - f e d deprived r a t s . The l i t e r a t u r e contains many c o n f l i c t i n g r e p o r t s as to the e f f e c t of p y r i d o x i n e d e f i c i e n c y on the various aspects of carbohydrate metabolism. One p o s s i b l e reason f o r t h i s i s the d i f f e r e n c e i n the modes of feeding between the c o n t r o l and the deprived r a t s . In order t o ob t a i n a c l e a r e r p i c t u r e of energy metabolism d u r i n g p y r i d o x i n e d e f i c i e n c y i t seemed necessary to observe some aspects of carbohydrate metabolism using comparable c o n d i t i o n s , by adapting the d e f i c i e n t group to meal-feeding. 23. 3 MATERIALS AND METHODS 3.1 Materials Male weanling Wistar rats (40-50 g i n i t i a l body-weight) purchased from Woodlyn Laboratories, Guelph, Ontario, were used throughout th i s i n v e s t i g a t i o n . The composition of the d i e t i s shown i n Table I. The vitamin mixture (Table II) and the mineral mixture (Table III) were prepared by General Biochemicals, Inc., Chagrin F a l l s , Ohio, U.S.A. Corn o i l (Mazola brand) and sucrose were purchased l o c a l l y . Chemicals were Fisher-brand reagent grade and were obtained from Fisher S c i e n t i f i c Ltd., Vancouver, B.C. Amberlite MB-3 ion-exchange r e s i n was purchased from Rohm and Haas, Philadelphia, Pennsylvania, U.S.A. Biochemicals were supplied by Sigma Chemical Co., St. Louis, Missouri, U.S.A. Radiochemicals were obtained from Amersham/Searle Corporation, Don M i l l s , Ontario. Rat l i v e r glycogen, used i n the glycogen phosphorylase assay mixture, was prepared by the method of Cowgill and Pardee (1957). 3.2 Methods 3.2.1 Handling of Animals The animals were housed i n d i v i d u a l l y i n screen-bottomed cages i n an air-conditioned room, maintained at TABLE I. Composition of the di e t . Ingredients Amount gm/kg die t Sucrose 667 Casein (92% protein) 170 Corn o i l 100 2 Vitamin mix 25 3 Mineral mix 38 The d i e t was mixed i n 4 kg lo t s and stored at 4°. The composition of the vitamin mix i s shown i n Table I I . The composition of the mineral mix i s shown i n Table I I I . TABLE I I . C o m p o s i t i o n o f v i t a m i n m i x f o r t h e p y r i d o x i n e - d e f i c i e n t t e s t d i e t . I n g r e d i e n t s Amount gm/kg d i e t T h i a m i n HC1 0.005 R i b o f l a v i n 0.01 d - C a l c i u m p a n t o t h e n a t e 0.02 N i a c i n a m i d e ( n i c o t i n a m i d e ) 0.05 B i o t i n 0.0003 F o l i c a c i d 0.001 V i t a m i n B ^ w i t h m a n n i t o l (0.1%) 0.05 M e n a d i o n e 0.0002 C h o l i n e d i h y d r o g e n c i t r a t e 5.0 V i t a m i n E a c e t a t e 110.0 I U / k g V i t a m i n A p a l m i t a t e 10,000.0 I U / k g V i t a m i n D-2 1,000.0 I U / k g N o n - n u t r i t i v e f i b r e ( c e l l u l o s e ) " ' " 19.4009 The same f o r m u l a t i o n was u s e d f o r p y r i d o x i n e c o n t r o l d i e t e x c e p t p y r i d o x o l HC1 was a d d e d a t 0.01 gm/kg d i e t a t e x p e n s e o f n o n - n u t r i t i v e f i b r e . TABLE I I I . Composition of mineral mix for a l l d i e t s . Ingredients Amount • gm/kg d i e t Calcium carbonate (CaCO^) 7.260698 Calcium phosphate dibasic (CaHPO^) 14.31973 Sodium phosphate dibasic (Na^PO^) 6.00875 Potassium chloride (KC1) 7.310174 Magnesium sulphate (MgSO^) 2.302268 F e r r i c c i t r a t e 0.589836 Manganese sulphate (MnSO^.H^O) 0.152216 Zinc carbonate (ZnCO^) 0.0532756 Potassium Iodate (KIO^) 0.0030443 24° s u b j e c t e d t o a l i g h t / d a r k c y c l e ( l i g h t : 6:00 am - 6:00 pm). W a t e r was p r o v i d e d a d l i b i t u m . The p y r i d o x i n e -d e p r i v e d r a t s w e r e g i v e n a c c e s s t o f o o d f o r 2 h o u r s d a i l y , b e t w e e n 0900 and 1100 h o u r s . The p a i r - f e d c o n t r o l s w e r e o f f e r e d , e a c h d a y , an amount o f t h e p y r i d o x o l - s u p p l e m e n t e d d i e t i s o c a l o r i c t o t h a t j u s t consumed by t h e d e p r i v e d g r o u p d u r i n g t h e m e a l . P a i r - f e e d i n g was p e r f o r m e d a t a p p r o x i m a t e l y 1130 h o u r s . F o o d c o n s u m p t i o n r e c o r d s w e r e k e p t a nd w e r e c o r r e c t e d f o r s p i l l a g e . The a n i m a l s w e r e h a n d l e d f r e q u e n t l y a n d w e r e w e i g h e d upon a r r i v a l a n d a t w e e k l y i n t e r v a l s t h e r e a f t e r . The r a t s w e r e f e d t h e e x p e r i m e n t a l a nd t h e c o n t r o l d i e t s f o r 45 d a y s . S u b s e q u e n t l y t h e y w e r e k i l l e d b y d e c a p i t a t i o n 4 h o u r s a f t e r t h e i n i t i a t i o n o f t h e l a s t f e e d i n g . I n e x p e r i m e n t I I I , t h e r a t s w e r e g i v e n an i n j e c t i o n 14 o f L - a l a n m e - U - C, 45 m i n u t e s b e f o r e s a c r i f i c e . I t was d i l u t e d t o a s p e c i f i c a c t i v i t y o f 2.3 X C i / m m o l e . R a d i o -a c t i v i t y , was a d m i n i s t e r e d i n t e r p e r i t o n e a l l y a t t h e r a t e o f m m o l e / 1 0 0 g b o d y w e i g h t i n 1.0 m l s a l i n e . 3.2.2 Enzyme A s s a y s 3.2.2.1 G l y c o g e n P h o s p h o r y l a s e ( o < - l , 4 - g l u c a n : o r t h o -p h o s p h a t e g l u c o s y l t r a n s f e r a s e , EC 2.4.1.1) T h i s a s s a y was c a r r i e d o u t on b o t h l i v e r a n d w h i t e l e g m u s c l e ( g a s t r o c n e -m i u s ) , u s i n g t h e m e t h o d o f N i e m e y e r e t a l _ . (1961) . 28. A p p r o x i m a t e l y 1 g o f l i v e r was w e i g h e d and a 1:10 h o mogenate i n 0.15 M KF was p r e p a r e d i n 30 s e c u s i n g a g l a s s h o m o g e n i z e r e q u i p p e d w i t h a T e f l o n p e s t l e . The a s s a y medium c o n t a i n e d 0.0125 A m o l e s g l u c o s e - l - p h o s p h a t e , 0.0335 X m o l e s c i t r a t e b u f f e r , pH 6.0, 0.0375 / t m o l e s K F , 0 . 0 1 2 5 x m o l e s AMP, a n d 5.0 mg g l y c o g e n i n a v o l u m e o f 0.5 m l . I n c u b a t i o n s w e r e c a r r i e d o u t a t 37°. I n o r g a n i c p h o s p h a t e was d e t e r m i n e d o n a s a m p l e f r o m t h e f i n a l a s s a y m i x t u r e by t h e m e t h o d o f T a u s s k y a n d S h o r r ( 1 9 5 3 ) . The m u s c l e enzyme was a s s a y e d i n t h e same manner as t h a t o f t h e l i v e r , e x c e p t t h a t a 1:20 h omogenate was p r e -p a r e d a n d s t r a i n e d t h r o u g h c h e e s e c l o t h p r i o r t o u s e . 3.2.2.2 G l y c o g e n - U D P g l u c o s y l t r a n s f e r a s e ( u r i d i n e d i p h o s p h a t e g l u c o s e : g l y c o g e n °<-4-glucosyltransferase, EC 2.4.1.11) G l y c o g e n - U D P g l u c o s y l t r a n s f e r a s e a c t i v i t y was d e t e r m i n e d by m e a s u r i n g t h e amount o f r a d i o a c t i v i t y 14 i n c o r p o r a t e d i n t o g l y c o g e n f r o m C - l a b e l l e d U D P - g l u c o s e f o l l o w i n g t h e m e t h o d o f G o l d a n d S e g a l (1967) as m o d i f i e d by W i l e y and L e v e i l l e (1970) . A c t i v i t y i n b o t h l i v e r a n d w h i t e l e g m u s c l e ( g a s t r o c n e m i u s ) was a s s a y e d . A p p r o x i m a t e l y 1 g o f e a c h t i s s u e was h o m o g e n i z e d i n 0.1 M g l y c y l g l y c i n e b u f f e r , pH 7.4. The a s s a y m i x t u r e 14 c o n t a i n e d 2.5 mg g l y c o g e n , 2 . 5 X m o l e s U D P - g l u c o s e - U - C ( O . l x c i / x m o l e ) , 3 . 5 x.moles g l u c o s e - 6 - p h o s p h a t e , and 3.3/tmoles g l y c y l g l y c i n e , pH 7.4, i n a t o t a l volume of 0.5 ml. The p u r i f i e d glycogen p e l l e t s were hydrolyzed by heating with 2 ml 1 N I^SO^. One ml of hydrolyzed sample was added to 10 ml s c i n t i l l a t i o n solvent (Patterson and Greene, 1965) and counted i n a Picker Nuclear Liquimat 220. 3.2.2.3 As par t at e ami no-trans feras e (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) This method employs the reaction between aspartic acid and «=<-ketoglu-tarate, and measures the dinitrophenylhydrazone of pyruvate formed from the chemical decarboxylation of oxalacetate. Both l i v e r and erythrocytes were used. A 1:10 l i v e r homogenate i n 0.25 M sucrose was pre-pared. The blood was c o l l e c t e d i n heparinized tubes. Afte r the buffy coat and the plasma were discarded, the c e l l s were washed with saline and then hemolyzed i n 2 volumes of d i s -t i l l e d water. The assay was ca r r i e d out as described by Umbreit et a l . (1957). 3.2.2.4 Alanine amino-transferase (L-alanine: 2-oxoglutarate aminotransferase, EC 2.6.1.2) Both l i v e r and erythrocytes were prepared as described above. The assay was performed according to Wroblewski and Cabaud (1957). 30. 3.2.3 Other Methods 3.2.3.1 Glycogen Glycogen deposition was determined on l i v e r and white leg muscle (gastrocnemius) by the method of Hassid and Abraham (1957). As the glycogen content of muscle was low, i t was co-precipitated with Na2SO^ and had to be p u r i f i e d for assay purposes i n order to avoid interference. The modified method described by Hassid and Abraham (1957) was used. However, rather than hydrolyzing the extracted glycogen p e l l e t , i t was dissolved i n 5 ml of water for assay by the anthrone reagent, as for l i v e r . The amount of r a d i o a c t i v i t y incorporated into l i v e r glycogen was determined as follows: the glycogen was ex-tracted as described for muscle (Hassid and Abraham, 1957) except that more washings were applied. To eliminate any l a b e l l e d protein that may have remained, 2 ml of 10% t r i c h l o r o a c e t i c acid (TCA) were added to 2 ml glycogen solution and the clear supernatant a f t e r centrifugation was used to form the f i n a l p e l l e t . One ml of hydrolyzed glycogen and 10 ml s c i n t i l l a t i o n solvent (Patterson and Greene, 1970) were then counted. 3.2.3.2 Blood Glucose Blood was c o l l e c t e d i n heparin-ized v i a l s containing KF and was deproteinized by the method of Somogyi (1945). For the determination of r a d i o a c t i v i t y incorporated into c i r c u l a t i n g glucose, 2 ml of the protein-free f i l t r a t e were passed through a 0.8x20 cm Amberlite MB-3 column (Friedmann e_t a l . , 19 65) . The column was washed with H^ O at a flow rate of 1 ml/min u n t i l 40 ml eluate were colle c t e d (Deodhar and Mistry, 1969). Preliminary tests 14 14 with l a b e l l e d L-alanine-U- C or glucose-U- C established that a negligable amount of alanine was passed through the column (>0.2%) , while the glucose passed through with approximately 92% e f f i c i e n c y . A 1 ml sample of the eluted l a b e l l e d glucose and 10 ml s c i n t i l l a t i o n solvent (Patterson and Greene, 1965) were mixed and counted at an e f f i c i e n c y of 78%. Glucose determination was performed on the protein-free f i l t r a t e by the glucose oxidase method recommended by Sigma B u l l e t i n No. 510. 3.2.3.3 Protein Protein content of enzyme solutions was determined using the Folin-Ciocalteu phenol reagent, following the method of Lowry et a l . (19 51). 3.2.4 S t a t i s t i c s The animals were randomly assigned to groups using a table of random numbers (Huntzberger, 1967). The selection for s a c r i f i c e on s p e c i f i c days was done i n a s i m i l a r manner. Student's t-tests were carr i e d out to determine differences between two group means. 4 EXPERIMENTAL AND RESULTS 4.1 E x p e r i m e n t I T h i s s t u d y was c o n d u c t e d t o e v a l u a t e t h e e f f e c t s o f p y r i d o x i n e d e p r i v a t i o n i n m e a l - f e d r a t s on t h e a c t i v i t i e s o f l i v e r and m u s c l e g l y c o g e n p h o s p h o r y l a s e s and on g l y c o g e n s t o r a g e i n t h e s e t i s s u e s . One g r o u p o f w e a n l i n g r a t s was m e a l - f e d t h e p y r i d o x i n e - f r e e d i e t i n t h e manner d e s c r i b e d i n S e c t i o n 3.2.1, w h i l e t h e o t h e r g r o u p was g i v e n t h e p y r i d o x i n e - s u p p l e m e n t e d d i e t i n q u a n t i t i e s e q u a l t o t h o s e consumed by t h e d e p r i v e d g r o u p d a i l y . The e x p e r i m e n t a l p e r i o d was 45 d a y s , a t w h i c h t i m e t h e a n i m a l s were s a c r i -f i c e d . The t e r m i n a l p r o c e d u r e s were a r r a n g e d s o t h a t t h e a n i m a l s i n e a c h g r o u p were k i l l e d 4 h o u r s a f t e r t h e i r l a s t m e a l . The f o o d c o n s u m p t i o n and w e i g h t g a i n d a t a a r e p r e s e n t e d i n F i g s . 5 and 6, r e s p e c t i v e l y . A l t h o u g h t h e d e p r i v e d and t h e p a i r - f e d c o n t r o l s consumed e q u a l amounts o f f o o d , t h e l a t t e r a n i m a l s e x h i b i t e d a g r e a t e r w e i g h t g a i n o v e r t h e 45-day e x p e r i m e n t a l p e r i o d ( P < 0 . 0 0 5 ) . I n o r d e r t o v e r i f y t h e s t a t e o f d e f i c i e n c y i n t h e d e p r i v e d r a t s , t h e a c t i v i t i e s o f l i v e r and e r y t h r o c y t e a s p a r t a t e and a l a n i n e a m i n o t r a n s f e r a s e s were a s s a y e d . As shown i n T a b l e IV, p y r i d o x i n e d e p r i v a t i o n i n m e a l - f e d r a t s was a s s o c i a t e d w i t h d e c r e a s e s i n t h e a c t i v i t i e s o f b o t h o f t h e l i v e r enzymes, as c o m p a r i s o n w i t h t h e p a i r - f e d 33. Figure 5 Food Consumption of Meal-fed P y r i d o x i n e - d e p r i v e d Rats 3 2 0 h T I M E ( w e e k s ) 34. F i g u r e 6 W e i g h t G a i n o f M e a l ^ f e d P y r i d o x i n e - d e p r i v e d R a t s T I M E ( w e e k s ) T a b l e I V . L i v e r and e r y t h r o c y t e a s p a r t a t e a m i n o t r a n s f e r a s e (GOT) and a l a n i n e a m i n o -t r a n s f e r a s e (GPT) i n m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s . Group L i v e r 1 E r y t h r o c y t e GOT GPT GOT GPT N m o l e s k e t o a c i d f o r m e d / g / m i n u t e x m o l e s k e t o a c i d formed/ml h aemoly z a t e / m i n u t e P y r i d o x i n e - d e f i c i e n t 4 P a i r - f e d c o n t r o l 2,3 + 5 + 5 51 - 5 . 7° 14 - 1 . 7° 0.45 - 0.0515 0.35 - 0.043 10.2 50 - 6.3 0.95 - 0.059 0.37 - 0.051 + 1. Mean - s t a n d a r d e r r o r o f t h e mean f o r 12 r a t s . 2. F e d t h e p y r i d o x i n e - f r e e d i e t f o r 45 days a f t e r w e a n i n g . 3. Had a c c e s s t o f o o d f r o m 9:00 t o 11:00 a.m. d a i l y . 4. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t p l u s 10 mg p y r i d o x o l . H C l / k g d i e t . 5. S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p a t P < 0.001. OJ c o n t r o l s r e v e a l e d (P< 0.001 i n b o t h c a s e s ) . I n e r y t h r o c y t e s , o n l y a s p a r t a t e a m i n o t r a n s f e r a s e was d e c r e a s e d i n t h e d e -f i c i e n c y s t a t e (P < 0 . 0 0 1 ) , w h i l e a l a n i n e a m i n o t r a n s f e r a s e a c t i v i t y r e m a i n e d u n a l t e r e d . The a c t i v i t i e s o f l i v e r and m u s c l e g l y c o g e n p h o s p h o -r y l a s e s a r e summarized i n T a b l e V. P y r i d o x i n e d e f i c i e n c y was a s s o c i a t e d w i t h s i g n i f i c a n t d e c r e a s e s i n t h e a c t i v i t i e s o f b o t h t h e l i v e r ( P < 0 . 0 5 ) and t h e m u s c l e (P < 0.001) en z y m e s , as shown by c o m p a r i s o n w i t h t h e p a i r - f e d c o n t r o l s . T h i s was t r u e w h e t h e r t h e a c t i v i t i e s were e x p r e s s e d i n terms o f f r e s h t i s s u e w e i g h t o r i n terms o f t i s s u e p r o t e i n c o n t e n t . I t i s n o t e w o r t h y t h a t t h e d e f i c i e n c y d i d n o t a f f e c t t h e l i v e r and t h e m u s c l e p h o s p h o r y l a s e s t o t h e same d e g r e e i.?e. whereas t h e a c t i v i t y o f t h e l i v e r enzyme (p e r mg p r o t e i n ) was 6 8% o f t h a t o b s e r v e d i n t h e c o n t r o l g r o u p , t h a t o f t h e m u s c l e enzyme was 40% as much. The e f f e c t s o f p y r i d o x i n e d e f i c i e n c y on g l y c o g e n d e p o s i t i o n a r e p r e s e n t e d i n T a b l e V I . As c o m p a r i s o n w i t h t h e p a i r - f e d c o n t r o l s r e v e a l e d , t h e a c c u m u l a t i o n o f g l y c o g e n i n b o t h t h e l i v e r and t h e s k e l e t a l m u s c l e d u r i n g t h e 4-hour p e r i o d a f t e r t h e i n i t i a t i o n o f f e e d i n g was c l e a r l y i m p a i r e d ( P < 0 . 0 5 ) . I t a p p e a r s t h a t a t l e a s t i n t h e l i v e r , d i f f e r e n c e s i n g l y c o g e n d e p o s i t i o n were n o t due t o d i f f e r e n c e s i n t i s s u e s i z e , as b o t h t h e d e p r i v e d and t h e c o n t r o l g r o u p s had c o m p a r a b l e l i v e r w e i g h t s . T a b l e V. L i v e r and g a s t r o c n e m i u s m u s c l e g l y c o g e n p h o s p h o r y l a s e a c t i v i t i e s o f m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s . Group 1 L i v e r M u s c l e 1 X moles P/g/min x m o l e s P/mg Mmoles P/g/min A m o l e s P/mg p r o t e i n / m i n p r o t e i n / m i n 2 3 P y r i d o x i n e - d e f i c i e n t ' + 5 70 - 8.3 0.62 - 0.0745 228 - 2 8 . 46 4.02 - 0.7366 4 P a i r - f e d c o n t r o l s 101 - 12.3 0.95 - 0.122 559 - 42.0 10.07 - 1.415 1. Mean - s t a n d a r d e r r o r o f t h e mean f o r 12 r a t s . 2. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t f o r 45 d a ys a f t e r w e a n i n g . 3. A c c e s s t o f o o d f r o m 9:00 - 11:00 a.m. d a i l y . 4. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t p l u s 10 mg p y r i d o x o l . H C l / k g d i e t . 5. S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p a t P < 0 . 0 5 . 6. S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p a t P < 0 . 0 0 1 . T a b l e V I . L i v e r w e i g h t s and l i v e r and g a s t r o c n e m i u s m u s c l e g l y c o g e n l e v e l s o f m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s . Group L i v e r w e i g h t L L i v e r g l y c o g e n x M u s c l e g l y c o g e n g/lOOg body wt mg/g t i s s u e mg/g t i s s u e 2 P y r i d o x i n e - d e f i c i e n t ' 3 + 3.6 - .33 26.7 i 1.69 5 0.17 - 0.013 5 4 P a i r - f e d c o n t r o l s 3.4 - .20 38.5 - 4.79 0.21 - 0.011 1. Mean - s t a n d a r d e r r o r o f t h e mean f o r 12 r a t s « 2. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t f o r 45 days a f t e r w e a n i n g . 3. Had a c c e s s t o f o o d f r o m 9:00 - 11:00 a.m. d a i l y . 4. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t p l u s 10 mg p y r i d o x o l . H C l / k g d i e t . 5. S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p a t P 0.05. 4.2 E x p e r i m e n t I I Glycogen-UDP g l u c o s y 1 t r a n s f e r a s e ( g l y c o g e n s y n t h e t -ase) i s t h e o t h e r enzyme b e s i d e p h o s p h o r y l a s e w h i c h c o u l d a f f e c t g l y c o g e n l e v e l s i n mammalian t i s s u e s . T h e r e f o r e , t h e p r e s e n t e x p e r i m e n t was c o n d u c t e d i n o r d e r t o c o r r e l a t e t h e e f f e c t s o f p y r i d o x i n e d e f i c i e n c y on g l y c o g e n p h o s p h o r y l a s w i t h t h e a c t i v i t i e s o f l i v e r and m u s c l e g l y c o g e n s y n t h e t a s e s . The g e n e r a l e x p e r i m e n t a l p r o t o c o l was s i m i l a r t o t h a t d e s c r i b e d p r e v i o u s l y ( c f . S e c t i o n 3.2.1 and S u b h e a d i n g 4.1) . As shown i n T a b l e V I I , t h e r e s p o n s e s o f g l y c o g e n p h o s p h o r y l a s e t o p y r i d o x i n e d e p r i v a t i o n were s i m i l a r t o t h o s e o b s e r v e d i n E x p e r i m e n t I ( T a b l e V ) . T h i s was t r u e f o r b o t h t h e l i v e r and t h e m u s c l e enzymes. The a c t i v i t i e s o f l i v e r and m u s c l e g l y c o g e n s y n t h e t a s e s a r e shown i n T a b l e V I I I . I n t h e f o r m e r t i s s u e , p y r i d o x i n e d e p r i v a t i o n l e d t o a r i s e i n t h e a c t i v i t y o f g l y c o g e n s y n t h e t a s e t o l e v e l s s i g n i f i c a n t l y g r e a t e r t h a n t h o s e a t t a i n e d by t h e p a i r - f e d c o n t r o l g r o u p , b o t h when a c t i v i t y was e x p r e s s e d i n terms o f f r e s h t i s s u e w e i g h t (P < 0.001) and t i s s u e p r o t e i n ( P < 0 . 0 1 ) . I n c o n t r a s t , t h e a c t i v i t y o f m u s c l e g l y c o g e n s y n t h e t a s e r e m a i n e d u n a l t e r e d i n r e l a t i o n t o t h e c o n t r o l g r o u p , r e g a r d l e s s o f t h e manner i n w h i c h r e s u l t s were e x p r e s s e d . T a b l e V I I . L i v e r and g a s t r o c n e m i u s m u s c l e g l y c o g e n p h o s p h o r y l a s e a c t i v i t i e s o f m e a l -f e d p y r i d o x i n e - d e f i c i e n t r a t s . Group L i v e r 1 M u s c l e 1 Kmoles P/g/min x m o l e s P/mg x m o l e s P/g/min x m o l e s P/mg p r o t e i n / m i n p r o t e i n / m i n P y r i d o x i n e - d e f i c i e n t2'3 130 - 10.05 1.23 - 0.07475 256 - 2 8 . 06 3.74 - 0.6676 P a i r - f e d c o n t r o l s4 170 - 9.1 1.56 - 0.0795 897 - 35.7 11.00 - 0.607 1. Mean - s t a n d a r d e r r o r o f t h e mean f o r 12 r a t s . 2. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t f o r 45 days a f t e r w e a n i n g . 3. Had a c c e s s t o f o o d f r o m 9:00 - 11:00 a.m. d a i l y . 4. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t p l u s 10 mg p y r i d o x o l . H C l / k g d i e t . 5. S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p a t P < 0 . 0 1 . 6. S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p a t P< 0.001. T a b l e V I I I . L i v e r and g a s t r o c n e m i u s m u s c l e glycogen-UDP g l u c o s y l t r a n s f e r a s e a c t i v i t i e s o f m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s . Group L i v e r 1 M u s c l e 1 (dpm/g/min) (dpm/mg p r o t e i n / (dpm/g/min) (dpm/mg p r o t e i n / x I O- 3 min) x 1 0 ~3 x I O- 3 min) x 1 0 ~3 P y r i d o x i n e - d e f i c i e n t2 , 3 616 - 24.8 5.01 - 0.2005 828 - 75.8 15.64 - 1.546 P a i r - f e d c o n t r o l s4 482 - 22.6 4.20 * 0.207 9 8 6 - 2 6 . 8 18.46 - 1.033 1. Mean - s t a n d a r d e r r o r o f t h e mean f o r 12 r a t s . 2. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t f o r 45 days a f t e r w e a n i n g . 3. Had a c c e s s t o f o o d f r o m 9:00 - 11:00 a.m. d a i l y . 4. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t p l u s 10 mg p y r i d o x o l . H C l / k g d i e t , 5. S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p a t P< 0.01. 6. S i g n i f i c a n t l y d i f f e r e n t f rom t h e c o n t r o l g r o u p a t P< 0.001. 4.3 E x p e r i m e n t I I I The p o s s i b i l i t y e x i s t s t h a t t h e e f f e c t s o f p y r i d o x i n d e f i c i e n c y on t i s s u e g l y c o g e n d e p o s i t i o n ( E x p e r i m e n t I) may r e f l e c t a d e c r e a s e i n t h e a b i l i t y o f t h e a n i m a l t o p r o d u c e g l y c o g e n f r o m s o u r c e s o t h e r t h a n c a r b o h y d r a t e , v i z . , g l u c o -n e o g e n e s i s f r o m amino a c i d s . T h e r e f o r e , E x p e r i m e n t I I I was 14 c o n d u c t e d t o t e s t t h i s h y p o t h e s i s , u s i n g a l a n m e - U - C as a t r a c e r . The a n i m a l s were f e d t h e p y r i d o x i n e - f r e e and t h e c o n t r o l d i e t s i n t h e same manner d e s c r i b e d p r e v i o u s l y ( c f . S e c t i o n 3 . 2 . 1 ) . A t t h e end o f d e p l e t i o n , t h e r a t s were 14 g i v e n 1 mmole a l a n i n e - U - C/ 100 g body w e i g h t i n t r a p e r i -t o n e a l l y , 4 h o u r s a f t e r t h e i n i t i a t i o n o f t h e i r l a s t m e a l . A l l a n i m a l s were k i l l e d 45 m i n u t e s a f t e r i n j e c t i o n and t h e a p p e a r a n c e o f r a d i o a c t i v i t y i n l i v e r g l y c o g e n and b l o o d g l u c o s e was d e t e r m i n e d . As shown i n T a b l e I X , t h e r e were no d i f f e r e n c e s i n b l o o d g l u c o s e l e v e l s between t h e d e f i c i e n t and t h e p a i r - f e d c o n t r o l g r o u p s a t t h e t i m e o f s a c r i f i c e . T h i s was a l s o 14 t r u e w i t h r e g a r d t o t h e i n c o r p o r a t i o n o f C f r o m a l a n m e -14 U- C i n t o b l o o d g l u c o s e , when t h e d a t a were e x p r e s s e d as a p e r c e n t a g e o f a d m i n i s t e r e d r a d i o a c t i v i t y / 1 0 0 ml b l o o d . However, t h e amount o f r a d i o a c t i v i t y p r e s e n t i n t h e c i r c u -l a t i n g g l u c o s e s p a c e (assumed t o be 30% o f body w e i g h t ) ( F r i e d m a n n e t a l . , 1965) was c o n s i d e r a b l y s m a l l e r i n t h e T a b l e I X . I n c o r p o r a t i o n o r r a d i o a c t i v i t y f r o m a l a n i n e - U - C i n t o b l o o d g l u c o s e and l i v e r g l y c o g e n i n m e a l - f e d p y r i d o x i n e - d e f i c i e n t rats-^-. Group B l o o d g l u c o s e L i v e r g l y c o g e n P y r i d o x i n e - d e f i c i e n t P a i r - f e d c o n t r o l 4,5 mg/100 ml 114 - 5.2 114 - 5.3 R a d i o a c t i v i t y _ ~ (dpm/ml) x 10 4.17 - 0.524 5.16 - 0.348 R a d i o a c t i v i t y (dpm/g) dpm i n c i r c u l a t i n g ^ x 10~3 g l u c o s e s p a c e x 10 143.06 - 4.7257 1.32 - 0.475 189.78 - 2.021 0.63 - 0.142 14 1. L - a l a n i n e - U C (2.3 /(Ci/mmole) was a d m i n i s t e r e d i n t r a p e r i t o n e a l l y a t t h e r a t e o f 1 mmole/100 g r a t i n 1 ml p h y s i o l o g i c a l s a l i n e 45 m i n u t e s b e f o r e s a c r i f i c e . 2. Mean - s t a n d a r d e r r o r o f t h e mean f o r 10 r a t s . 3. T h i s v a l u e r e p r e s e n t s t h e r a d i o a c t i v i t y p r e s e n t i n t h e c i r c u l a t i n g g l u c o s e p o o l w h i c h i s assumed t o be 30% o f body w e i g h t ( F r i e d m a n n e t a l . , 1 9 6 5 ) . 4. Fed t h e p y r i d o x i n e - f r e e d i e t f o r 45 days a f t e r w e a n i n g . 5. Had a c c e s s t o f o o d f r o m 9:00 - 11:00 a.m. d a i l y . 6. Fed t h e p y r i d o x i n e - d e f i c i e n t d i e t p l u s 10 mg p y r i d o x o l . H C l / k g d i e t . 7. S i g n i f i c a n t l y d i f f e r e n t f r o m the c o n t r o l g r o u p a t P< 0.001. d e f i c i e n t group than i n the c o n t r o l s (P< 0.001). The amount of r a d i o a c t i v i t y i n c o r p o r a t e d i n t o l i v e r glycogen, expressed/g t i s s u e , was somewhat gr e a t e r i n the p y r i d o x i n e - d e f i c i e n t r a t s than i n the c o n t r o l s , but the d i f f e r e n c e between the two groups d i d not reach the l e v e l of s t a t i s t i c a l s i g n i f i c a n c e . As l i v e weight i n both groups was comparable ( c f . Appendix), the same statements may be made regarding t o t a l glycogen i n t h i s t i s s u e . In any case, t h i s amount of r a d i o a c t i v i t y was small compared to th a t present i n c i r c u l a t i n g glucose. 5 DISCUSSION A d e c r e a s e i n v o l u n t a r y f o o d c o n s u m p t i o n r e s u l t s f r o m many t r e a t m e n t s i n n u t r i t i o n a l s t u d i e s . The d i f f i c u l t y e n c o u n t e r e d i n i n v e s t i g a t i n g e n e r g y u t i l i z a t i o n u n d e r t h e s e c o n d i t i o n s i s t h e n e c e s s i t y o f d i s t i n g u i s h i n g t h e c o n s e q u e n c e s o f n u t r i t i o n a l a l t e r a t i o n i t s e l f f r o m t h o s e o f t h e accompany-i n g i n a n i t i o n . The u s u a l a p p r o a c h i s t o e q u a l i z e t h e c a l o r i c i n t a k e s o f t h e e x p e r i m e n t a l and t h e c o n t r o l a n i m a l s , by e m p l o y i n g p a i r - f e e d i n g t e c h n i q u e s . However, when a p p e t i t e d e p r e s s i o n i s s e v e r e , t h e d a i l y a l l o t m e n t o f f o o d o f f e r e d t o t h e p a i r - f e d a n i m a l w i l l be c o n s i d e r a b l y l e s s t h a n t h a t r e q u i r e d t o s a t i s f y i t s a p p e t i t e and w i l l be e a t e n w i t h i n a r e l a t i v e l y s h o r t p e r i o d o f t i m e . T h u s , t h e p a i r - f e d a n i m a l becomes an i n t e r m i t t a n t e a t e r a n a l o g o u s t o t h e m e a l - f e d r a t . M e a l - f e e d i n g , t h e r e s t r i c t i o n o f f o o d a v a i l a b i l i t y t o a s h o r t d a i l y p e r i o d , l e a d s t o s e v e r a l a d a p t a t i o n s r e l a t e d t o e n e r g y s t o r a g e and u t i l i z a t i o n by t h e r a t ( L e v e i l l e , 1 9 7 0 ) . T h e s e r e s p o n s e s i n c l u d e i n c r e a s e s i n t h e a c t i v i t i e s o f enzymes i n v o l v e d w i t h f a t t y a c i d s y n t h e s i s ( C h a k r a b a r t y and L e v e i l l e , 1969; L e v e i l l e and H a n s o n , 1 9 6 6 ) , i n c r e a s e d l i p o g e n i c p o t e n t i a l s o f t h e l i v e r and t h e a d i p o s e t i s s u e ( B r a u n and F a b r y , 1969; Cohn and J o s e p h , 1960; F a b r y and B r a u n , 1967; H o l l i f i e l d and P a r s o n , 1962 a,b; L e v e i l l e , 1 9 66, 1967 a-d; L e v e i l l e and H a n s o n , 1965; Tepperman and Tepperman, 1958), as w e l l as enhanced glycogen d e p o s i t i o n i n l i v e r , muscle and adipose t i s s u e ( L e v e i l l e and Chakra-b a r t y , 1967). Because of the foregoing c o n s i d e r a t i o n s , i t i s important i n studi e s of energy u t i l i z a t i o n i n n u t r i t i o n a l d e f i c i e n c y to standardize not only the l e v e l of food i n t a k e , but a l s o the mode of feeding of the experimental and the c o n t r o l groups. In the st u d i e s reported h e r e i n , the p y r i d o x i n e - d e p r i v e d r a t s were adapted to meal-feeding, w h i l e the c o n t r o l s were p a i r - f e d . In t h i s manner, d i f f e r -ences i n both c a l o r i c i n t a k e and feeding frequency between the two groups of r a t s were minimized. I t appears that p y r i d o x i n e d e f i c i e n c y i n meal-fed r a t s i s as s o c i a t e d w i t h a decrease i n feed e f f i c i e n c y (growth i n r e l a t i o n to food i n t a k e ) , s i n c e the deprived r a t s showed lower weight gain than the p a i r - f e d c o n t r o l s . This was i n agreement w i t h the f i n d i n g s of Paul (1968) under the same experimental c o n d i t i o n s . Paul (1968) a l s o showed t h a t the magnitude of the d i f f e r e n c e i n weight gain between the meal-fed d e f i c i e n t and p a i r - f e d c o n t r o l groups was considerably smaller than t h a t observed when ad l i b i t u m -fed ( n i b b l i n g ) d e f i c i e n t r a t s were compared w i t h t h e i r r e s p e c t i v e c o n t r o l s . Thus, the e f f e c t s of p y r i d o x i n e d e f i c i e n c y on growth as reported i n the l i t e r a t u r e (Sure and E a s t e r l i n g , 1949) may be a t t r i b u t e d l a r g e l y to d i f f e r -e n c e s i n t h e mode o f f e e d i n g between t h e e x p e r i m e n t a l and c o n t r o l a n i m a l s . I t has b een s u g g e s t e d t h a t t h e a c t i v i t i e s o f a s p a r t a t e and a l a n i n e a m i n o t r a n s f e r a s e s i n t h e l i v e r ( R a d h a k r i s h n a m u r t y e t a _ l . , 1968) and e r y t h r o c y t e s ( B r i n e t a l . / 1960; Cheney et. a l . 1967; R a d h a k r i s h n a m u r t y e t a l . , 1968) r e f l e c t t h e p y r i d o x i n e s t a t u s o f a n i m a l s . In t h e p r e s e n t s t u d i e s , t h e a c t i v i t i e s o f l i v e r and e r y t h r o -c y t e a s p a r t a t e a m i n o t r a n s f e r a s e s and l i v e r a l a n i n e a mino-t r a n s f e r a s e were s i g n i f i c a n t l y r e d u c e d t o l e v e l s b e l o w t h o s e a t t a i n e d by t h e p a i r - f e d c o n t r o l s . However, t h e a c t i v i t y o f e r y t h r o c y t e a l a n i n e ' a m i n o t r a n s f e r a s e r e m a i n e d a p p a r e n t l y u n a l t e r e d . T h i s may have been a r e f l e c t i o n o f f o o d r e s t r i c t i o n i n t h e c o n t r o l g r o u p , w h i c h i s known t o e l e v a t e t h e a c t i v i t y o f t h i s enzyme i n r e d b l o o d c e l l s t o a p o i n t t h a t c o u l d mask t h e e f f e c t s o f d e f i c i e n c y on t h e e x p e r i m e n t a l g r o u p (Cheney e t a l . , 1965). The r e s u l t s o f E x p e r i m e n t s I and I I show t h a t t h e m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s p o s s e s s l e s s g l y c o g e n p h o s p h o r y l a s e a c t i v i t y i n t h e l i v e r , and p a r t i c u l a r l y i n t h e m u s c l e , t h a n t h e i r p a i r - f e d c o n t r o l s . T h e s e r e s p o n s e s were c o n s i s t e n t w i t h t h o s e o b s e r v e d by E i s e n s t e i n (1962) , u s i n g n i b b l i n g r a t s w i t h p a i r - w e i g h e d c o n t r o l s . M i c e d e -p r i v e d o f p y r i d o x i n e h a ve b e e n a l s o r e p o r t e d t o h a ve l e s s 48. p h o s p h o r y l a s e a c t i v i t y i n t h e i r t i s s u e s t h a n a n i m a l s f e d a c o m p l e t e d i e t ad l i b i t u m (Lyon and P o r t e r , 1 9 6 2 ) . I t s h o u l d be n o t e d t h a t o n l y ' t o t a l ' p h o s p h o r y l a s e a c t i v i t y was m e a s u r e d i n t h e s t u d i e s r e p o r t e d h e r e i n . T h i s r e p r e s e n t s t h e enzyme a c t i v e i n t h e p r e s e n c e o f AMP i n t h e a s s a y medium (K r e b s and F i s c h e r , 1 9 6 2 ) . G l y c o g e n p h o s p h o r y l a s e c a t a l y z e s t h e r a t e - l i m i t i n g s t e p i n g l y c o g e n u t i l i z a t i o n ( K r e b s and F i s c h e r , 1 9 6 4 ) . A c c o r d i n g l y , a r e d u c t i o n i n t h e a c t i v i t y o f t h i s enzyme m i g h t be e x p e c t e d t o i m p a i r g l y c o g e n o l y s i s and t h e r e b y i n c r e a s e t i s s u e g l y c o g e n l e v e l s . However, t h e r e s u l t s o f E x p e r i m e n t I showed t h a t l i v e r and m u s c l e g l y c o g e n s t o r e s i n t h e d e f i c i e n t r a t s were d e c r e a s e d b e l o w t h o s e o b s e r v e d i n t h e c o n t r o l s . T h a t t i s s u e g l y c o g e n l e v e l s a r e d e c r e a s e d i n p y r i d o x i n e d e f i c i e n c y has b een a l s o d e m o n s t r a t e d i n n i b b l i n g r a t s ( B e a t o n and G o odwin, 1954; B e a t o n , 1955; Guggenheim and D i a m a n t , 1957; E i s e n s t e i n , 1 9 6 2 ) , and m i c e (Lyon and P o r t e r , 1 9 6 2 ) . A c c o r d i n g l y , i t a p p e a r s t h a t t h e p h o s p h o r y l a s e a c t i v i t y r e m a i n i n g i n t h e t i s s u e s o f t h e d e f i c i e n t a n i m a l may be s u f f i c i e n t t o m a i n t a i n a ' n o r m a l ' a b i l i t y t o m o b i l i z e g l y c o g e n . The r e s u l t s o f E x p e r i m e n t I I d e m o n s t r a t e d t h a t t h e t o t a l a c t i v i t i e s o f l i v e r and m u s c l e g l y c o g e n s y n t h e t a s e were n o t i m p a i r e d i n m e a l - f e d p y r i d o x i n e - d e f i c i e n t r a t s . I l l i n g w o r t h et a l . (1960) reported s i m i l a r f i n d i n g s i n n i b b l i n g r a t s w i t h ad l i b i t u m - f e d c o n t r o l s . However, the data of these authors are open to q u e s t i o n , because the deprived r a t s were given a semi-synthetic d i e t w h i l e the c o n t r o l s were maintained on " l a b o r a t o r y chow". In e i t h e r case, the r e d u c t i o n of t i s s u e glycogen l e v e l s i n p y r i d o x i n e d e f i c i e n c y does not appear to be a r e f l e c t i o n of impaired glucose t r a n s f e r from UDPG to glycogen. This does not r u l e out the p o s s i b i l i t y that the a c t i v i t i e s of other enzymes r e l a t e d to glycogen synthesis may be decreased. Unfortunate-l y , t h i s cannot be a s c e r t a i n e d at the present time because of the lack of published data. The glucose u t i l i z e d i n glycogenesis i s s u p p l i e d by the d i e t and by gluconeogenesis from non-carbohydrate sources such as l a c t a t e and amino a c i d s . Since p y r i d o x i n e d e f i c i e n c y decreases the a c t i v i t i e s of l i v e r aminotransfer-ases (Experiment I ) , i t would be reasonable to expect that the a v a i l a b i l i t y of amino a c i d carbon f o r glucose s y n t h e s i s i s impaired under these c o n d i t i o n s . The r e s u l t s of Experiment I I I support t h i s c o n t e n t i o n , s i n c e the conversion of l a b e l l e d alanine to glucose was lower i n the meal-fed d e f i c i e n t r a t s than i n the p a i r - f e d c o n t r o l s , p a r t i c u l a r l y when the r a d i o a c t i v i t y of blood glucose was c a l c u l a t e d i n terms of the c i r c u l a t i n g p o o l . However, i t was not p o s s i b l e t o demonstrate t h i s response i n l i v e r glycogen, probably 50. because of the r e l a t i v e l y small amount of r a d i o a c t i v i t y i ncorporated and the high v a r i a b i l i t y of the values obtained. Judged by the r e s u l t s obtained i n Experiment I , the redu c t i o n i n t i s s u e glycogen s t o r e s i n p y r i d o x i n e - d e f i c i e n t animals appears to be 20-30% of those a t t a i n e d by the p a i r -fed c o n t r o l s . Although the p r o p o r t i o n of t o t a l body glycogen deri v e d from amino a c i d carbon i s not known, the o v e r a l l f i n d i n g s of the present i n v e s t i g a t i o n suggest that decreased t i s s u e glycogen l e v e l s may be a t t r i b u t e d , at l e a s t p a r t l y , to the l i m i t e d supply of substrates f o r gluconeogenesis r e s u l t i n g from impaired transamination. 6 LITERATURE CITED A n g e l , J . F . and Z.K. S a b r y . 1968. I n v i t r o a c e t a t e i n c o r -p o r a t i o n i n t o l i v e r l i p i d s i n p y r i d o x i n e - r e p l e t e d r a t s . P r o c . C a n . F e d . B i o l . S o c . 11:132. B a r a n o w s k i , T. , B. I l l i n g w o r t h , D.H. Brown and C F . C o r i . 1957. The i s o l a t i o n o f p y r i d o x a l - 5 - p h o s p h a t e f r o m c r y s t a l l i n e m u s c l e p h o s p h o r y l a s e . B i o c h i m . B i o p h y s . A c t a 25:16-21. B e a r e , J . L . , J.R. B e a t o n and E.W. McHenry. 1953. S t u d i e s on v i t a m i n Bfi I I I . C a r c a s s c o m p o s i t i o n o f t h e v i t a m i n B , - d e f i c i e n t r a t . J . B i o l . Chem. 202:589-595. 6 B e a t o n , G.H., A.H. H a u f s c h i l d and E.W. McHenry. 1956. P r e v e n t i o n o f i n a n i t i o n i n v i t a m i n Bg d e p r i v e d r a t s by i n s u l i n t r e a t m e n t . J . N u t r . 60:455-462. B e a t o n , J.R., J . L . B e a r e , G.H. B e a t o n , J.M. W h i t e and E.W. McHenry. 1953. The b a s a l m e t a b o l i c r a t e and t h e e f f e c t s o f t h i o u r a c i l a d m i n i s t r a t i o n and o f t h y r o i -dectomy on c o n t r o l and v i t a m i n B f i - d e f i c i e n t r a t s . J . N u t r . 51:599-608. B e a t o n , J.R. and M.E. Goodwin. 19 54. S t u d i e s on t h e e f f e c t o f v i t a m i n Bg d e p r i v a t i o n on c a r b o h y d r a t e m e t a b o l i s m i n t h e r a t . C a n . J . B i o c h e m . P h y s i o l . 32:684-688. B e a t o n , J.R. 1955. F u r t h e r s t u d i e s on c a r b o h y d r a t e m e t a b o l i s m i n t h e v i t a m i n - B g - d e p r i v e d r a t . C a n . J . B i o c h e m . P h y s i o l . 33:161-166. B i s h o p , J . S . and J . L a m e r . 19 67. R a p i d a c t i v a t i o n and i n a c t i v a t i o n o f l i v e r u r i d i n e d i p h o s p h a t e g l u c o s e -g l y c o g e n t r a n s f e r a s e and p h o s p h o r y l a s e by i n s u l i n and g l u c a g o n i n v i v o . J . B i o l . Chem. 24.2:1354-1356. B r a u n , T. and P. F a b r y . 1969. A d a p t a t i o n t o t h e p a t t e r n o f f o o d i n t a k e : Changes i n a d i p o s e t i s s u e . A d v a n c e s E n z . Reg. 7:47-55. B r i n , M., M. T a i , A.S. O s t a s h e v e r and H. K a l i n s k y . 1960. The r e l a t i v e e f f e c t s o f p y r i d o x i n e d e f i c i e n c y on two p l a s m a t r a n s a m i n a s e s i n t h e g r o w i n g and i n t h e a d u l t r a t . J . N u t r . 71:416-420. 52. C a r t e r , C.W. and P.J.R. P h i z a c k e r l e y . 1951. The i n f l u e n c e o f p y r i d o x i n e on f a t m e t a b o l i s m i n t h e r a t . B i o c h e m . J . 49:227-232. C h a k r a b a r t y , K. and G.A. L e v e i l l e . 1969. A c e t y l CoA c a r b o x -y l a s e and f a t t y a c i d s y n t h e t a s e a c t i v i t i e s i n l i v e r and a d i p o s e t i s s u e o f m e a l - f e d r a t s . P r o c . S o c . E x p . B i o l . Med. 131:1051-1054. C h e n e y , M.C., D.M. C u r r y and G.H. B e a t o n . 1965. B l o o d t r a n s a m i n a s e a c t i v i t i e s i n v i t a m i n B - d e f i c i e n c y : s p e c i f i c i t y and s e n s i t i v i t y . C a n . J . P h y s i o l . Pharm. 43:579-589. C h e n e y , M . C , Z . I . S a b r y and G.H. B e a t o n . 1967. B l o o d t r a n s a m i n a s e a c t i v i t i e s i n v i t a m i n B g - d e f i c i e n c y : E f f e c t o f d e p l e t i o n and r e p l e t i o n i n e r y t h r o c y t e enxymes. C a n . J . P h y s i o l . Pharm. 45:343-351. C o c k b u r n , R.M. and J . T . Van B r u g g e n . 1959. A c e t a t e m e t a -b o l i s m i n v i v o : E f f e c t o f r e f e e d i n g . J . B i o l . Chem. 234:431-434. C o h n , C. and D. J o s e p h . 19 59. E f f e c t o f r a t e o f i n g e s t i o n o f d i e t on hexosemonophosphate s h u n t a c t i v i t y . Am. J . P h y s i o l 197:1347-1349. C o h n , C. and D. J o s e p h . 1960. R o l e o f r a t e o f i n g e s t i o n o f d i e t on r e g u l a t i o n o f i n t e r m e d i a r y m e t a b o l i s m ("meal e a t i n g " v s . " n i b b l i n g " ) . M e t a b o l i s m 9:492-500. C o h n , C. and D. J o s e p h . 1968. C o l o r i c i n t a k e , w e i g h t l o s s , and c h a nges i n body c o m p o s i t i o n o f r a t s as i n f l u e n c e d by f e e d f r e q u e n c y . J . N u t r . 96:94-100. C o h n , C. and D. J o s e p h . 1970. E f f e c t s o f c a l o r i c i n t a k e and f e e d i n g f r e q u e n c y on c a r b o h y d r a t e m e t a b o l i s m o f t h e r a t . J . N u t r . 100:78-84. C o w g i l l , R.W. and A.B. P a r d e e . 1957. E x p e r i m e n t s i n B i o c h e m i c a l R e s e a r c h T e c h n i q u e s . John W i l e y & Sons I n c . , New Y o r k , pp 158-159. D e o d h a r , A.D. and S.P. M i s t r y . 1969. R e s t o r a t i o n o f g l u c o n e o g e n e s i s i n b i o t i n - d e f i c i e n t r a t s . A r c h . B i o -chem. B i o p h y s . 131:507-512. D e s i k a c h a r , H.S.R. and E.W. McHenry. 1954. Some e f f e c t s o f v i t a m i n B6 d e f i c i e n c y on f a t m e t a b o l i s m i n r a t s . B i o c h e m . J . 56:544-547. E i s e n s t e i n , A.B. 1962. The e f f e c t o f p y r i d o x i n e d e f i c i e n c y on l i v e r and m u s c l e p h o s p h o r y l a s e . B i o c h i m . B i o p h y s . A c t a 58:244-247. E m e r s o n , R . J . , W.C. B e r n a r d s and J . T . Van B r u g g e n . 1959. A c e t a t e m e t a b o l i s m i n v i t r o : E f f e c t o f r e f e e d i n g . J . B i o l . Chem. 234:435-437. F a b r y , P. and T. Brown. 1967. A d a p t a t i o n t o t h e p a t t e r n o f f o o d i n t a k e : some mechanisms and c o n s e q u e n c e s . Symposium P r o c e e d i n g s 26:144-151. F i s c h e r , E.H., A.B. K e n t , E.R. S n y d e r and E.G. K r e b s . 1958. The r e a c t i o n o f s o d i u m b o r o h y d r i d e w i t h m u s c l e p h o s p h o r y l a s e . J . Am. Chem. S o c . 80:2906-2907. F i s c h e r , E.H., A.W. F o r r e y , J . L . H e d r i c k , R.G. H u g h e s , A.B. K e n t and E.G. K r e b s . 1963. P y r i d o x a l - 5 ' - p h o s p h a t e i n t h e s t r u c t u r e and f u n c t i o n o f p h o s p h o r y l a s e . I n C h e m i c a l and B i o l o g i c a l A s p e c t s o f P y r i d o x a l C a t a l y s i s . M a c m i l l a n C o . , New Y o r k , pp 543-562. F i s c h e r , E.H. and E.G. K r e b s . 1966. R e l a t i o n s h i p o f s t r u c t u r e t o f u n c t i o n o f m u s c l e p h o s p h o r y l a s e . F e d . P r o c . 25:1511-1520. F r i e d m a n n , B., E.H. Goodman and S. W e i n h o u s e . 1965. D i e t a r y and h o r m o n a l e f f e c t s on g l u c o n e o g e n e s i s i n t h e r a t . J . B i o l . Chem. 240:3729-3735. F u l l e r , R.W. and E.R. D i l l e r . 1970. D i u r n a l v a r i a t i o n o f l i v e r g l y c o g e n and p l a s m a - f r e e f a t t y a c i d s i n r a t s f e d ad l i b i t u m o r s i n g l e d a i l y m e a l . M e t a b o l i s m 19 :226-229. G l a z e r , R . I . and G. Weber. 1971. P y r i d o x a l 5 ' - p h o s p h a t e i n h i b i t i o n o f l a c t a t e p r o d u c t i o n i n r a t l i v e r s u p e r -n a t e n t f l u i d . B i o c h i m . B i o p h y s . A c t a 237:11-13. G l i n s m a n n , W., G. Pauk and E . H e r n . 1970. C o n t r o l o f r a t l i v e r g l y c o g e n s y n t h e t a s e and p h o s p h o r y l a s e a c t i v i t i e s by g l u c o s e . B i o c h e m . B i o p h y s . R e s . Comm. 39:774-782. G o l d , A.H. and H.L. S e g a l . 1967. T i m e - d e p e n d e n t i n c r e a s e i n r a t l i v e r g l y c o g e n s y n t h e a s e a c t i v i t y i n v i t r o . A r c h . B i o c h e m . B i o p h y s . 120:359-364. G o l d , A.H. 1968. A c o m p a r i s o n o f n o n a c t i v a t e d and a c t i v a t e d l i v e r g l y c o g e n s y n t h e t a s e . B i o c h e m . B i o p h y s . R e s . Comm. 31:361-367. Guggenheim, K. and E . J . D i a m a n t . 1957. C a r b o h y d r a t e m e t a -b o l i s m i n p y r i d o x i n e - d e f i c i e n t r a t s . J . B i o l . Chem. 224:861 - 8 6 9 . G y o r g y , P. 19 34. V i t a m i n B 2 and t h e p e l l e g r a - l i k e derm-a t i t i s i n r a t s . N a t u r e 133:498-499. G y o r g y , P. 1938. C r y s t a l l i n e v i t a m i n B,. Am.(Chem. S o c . J . 60:983 - 9 8 4 . H a s s i d , W.Z. and S. Abraham. 1957. C h e m i c a l P r o c e d u r e s f o r A n a l y s i s o f P o l y s a c c h a r i d e s . I D e t e r m i n a t i o n o f g l y c o g e n and s t a r c h . I_n Methods i n E n z y m o l o g y . V o l . I I I . S.P. C o l o w i c k and V.O. K a p l a n e d s . A c a d e m i c P r e s s , New Y o r k , pp 34-37. H o l l i f i e l d , G. and W. P a r s o n . 1 9 6 2 a . M e t a b o l i c a d a p t a t i o n s t o a ' s t u f f and s t a r v e ' f e e d i n g p r o g r a m . 1. S t u d i e s o f a d i p o s e t i s s u e and l i v e r g l y c o g e n i n r a t s l i m i t e d t o a s h o r t d a i l y f e e d i n g p e r i o d . J . C l i n . I n v e s t . 41:245-249. H o l l i f i e l d , G. and W. P a r s o n . 1962 b . M e t a b o l i c a d a p t a t i o n s t o a ' s t u f f and s t a r v e1 f e e d i n g p r o g r a m . I I . O b e s i t y and t h e p e r s i s t e n c e o f a d a p t a i v e c h a nges i n a d i p o s e t i s s u e and l i v e r o c c u r r i n g i n r a t s l i m i t e d t o a s h o r t d a i l y f e e d i n g p e r i o d . J . C l i n . I n v e s . 41:250-253. H u b e r , A.M., S.N. G e r s h o f f and D.M. H e g s t e d . 1964. C a r b o -h y d r a t e and f a t m e t a b o l i s m and r e s p o n s e t o i n s u l i n i n v i t a m i n B _ - d e f i c i e n t r a t s . J . N u t r . 82:371-378. 6 H u n t z b e r g e r , D.V. 1967. E l e m e n t s o f S t a t i s t i c a l I n f e r e n c e . 2nd e d . A l l y n and Baco n I n c . , B o s t o n , M a s s . I l l i n g w o r t h , B., R. K o r n f i e l d and D.H. Brown. 1960. P h o s -p h o r y l a s e and u r i d i n e d i p h o s p h o g l u c o s e - g l y c o g e n t r a n s f e r a s e i n p y r i d o x i n e d e f i c i e n c y . B i o c h i m . B i o p h y s . A c t a 42:486-489. K e r e s z t e s y , J . C . and J.R. S t e v e n s . 1938. C r y s t a l l i n e v i t a m i n B g . P r o c . S o c . E x p . B i o l . Med. 38:64-65. K r e b s , E.G. and E.H. F i s c h e r . 1962. M o l e c u l a r p r o p e r t i e s and t r a n s f o r m a t i o n s o f g l y c o g e n p h o s p h o r y l a s e i n a n i m a l t i s s u e s . A d v a n c e s i n E n z y m o l o g y 24:263-290. K r e b s , E.G. and E.H. F i s c h e r . 1964. P h o s p h o r y l a s e and r e l a t e d enzymes o f g l y c o g e n m e t a b o l i s m . V i t . and Horm. 22:399-409. L a r n e r , J . 1967-68. Hormonal and n o n h o r m o n a l c o n t r o l o f g l y c o g e n m e t a b o l i s m . T r a n s . N.Y. A c a d . S c i . S e r . 2. 29:192-209. L e l o i r , L.G., J.M. O l i v a r r i a , S.H. G o l d e m b e r g and H. C a r m i n a t t i . 19 59. B i o s y n t h e s i s o f g l y c o g e n f r o m u r i d i n e d i p h o s p h a t e g l u c o s e . A r c h . B i o c h e m . B i o p h y s . 81:508-520. L e v e i l l e , G.A. and R.H. H a n s o n . 1965. I n f l u e n c e o f p e r i o d i c i t y o f e a t i n g on a d i p o s e t i s s u e m e t a b o l i s m i n t h e r a t . C a n . J . P h y s i o l . Pharm. 43:857-868. L e v e i l l e , G.A. 1966. G l y c o g e n m e t a b o l i s m i n m e a l - f e d r a t s and c h i c k s and t h e t i m e s e q u e n c e o f l i p o g e n i c and e n z y m a t i c a d a p t i v e c h a n g e s . J . N u t r . 90:449-460. L e v e i l l e , G.A. and R.W. H a n s o n . 1966. A d a p t i v e changes i n enzyme a c t i v i t y and m e t a b o l i c pathways i n a d i p o s e t i s s u e f r o m m e a l - f e d r a t s . J . L i p i d R e s . 7:46-55. L e v e i l l e , G.A. 1967 a . I n f l u e n c e o f d i e t a r y f a t and p r o t e i n on m e t a b o l i c and e n z y m a t i c a c t i v i t i e s i n a d i p o s e t i s s u e o f m e a l - f e d r a t s . J . N u t r . 91:25-34. L e v e i l l e , G.A. 1967 b . I n f l u e n c e o f d i e t a r y f a t l e v e l on t h e e n z y m a t i c and l i p o g e n i c a d a p t a t i o n i n a d i p o s e t i s s u e o f m e a l - f e d r a t s . J . N u t r . 91:267-274. L e v e i l l e , G.A. 1967 c . C o n t r o l o f l i p o g e n e s i s i n a d i p o s e t i s s u e o f f a s t e d and f e d m e a l - e a t i n g r a t s . J . N u t r . 92 :460-466. L e v e i l l e , G.A. 1967 d . I n v i v o f a t t y a c i d s y n t h e s i s i n a d i p o s e t i s s u e and l i v e r o f m e a l - f e d r a t s . P r o c . S o c . E x p . B i o l . Med. 125:85-88. L e v e i l l e , G.A. and K. C h a k r a b a r t y . 1967. D i u r n a l v a r i a t i o n i n t i s s u e g l y c o g e n and l i v e r w e i g h t o f m e a l - f e d r a t s . J . N u t r . 93:546-554. L e v e i l l e , G.A. and E.K. O'Hea. 1967. I n f l u e n c e o f p e r i o d i c i t y o f e a t i n g on e n e r g y m e t a b o l i s m i n t h e r a t . J . N u t r . 93 :541-545. L e v e i l l e , G.A. 1970. A d i p o s e t i s s u e m e t a b o l i s m : I n f l u e n c e o f p e r i o d i c i t y o f e a t i n g and d i e t c o m p o s i t i o n . F e d e r -a t i o n P r o c . 29:1294-1301. 56. L o w r y , O.H., N . J . R o s e b r o u g h , A . L . F a r r and R . J . R a n d a l l . 1951. P r o t e i n measurement w i t h t h e f o l i n p h e n o l r e a g e n t . J . B i o l . Chem. 193:265-275. L y o n , J . B . and J . P o r t e r . 1962. The e f f e c t o f p y r i d o x i n e d e f i c i e n c y on m u s c l e and l i v e r p h o s p h o r y l a s e o f i n b r e d s t r a i n s o f m i c e . B i o c h i m . B i o p h y s . A c t a 58:248-254. M u e l l e r , J . F . 1964. V i t a m i n B.fi i n f a t m e t a b o l i s m . V i t a m i n s and Hormones 8:55-6 8. M u i r u r i , K.L. and G.A. L e v e i l l e . 1970. M e t a b o l i c a d a p t a t i o n i n m e a l - f e d r a t s : e f f e c t s o f i n c r e a s e d m e a l f r e q u e n c y o r ad l i b i t u m f e e d i n g on r a t s p r e v i o u s l y a d a p t e d t o a s i n g l e d a i l y m e a l . J . N u t r . 100:450-460. N i e m e y e r , H., C. G o n z a l e s and R. R o z z i . 1961. The i n f l u e n c e o f d i e t on l i v e r p h o s p h o r y l a s e 1. E f f e c t s o f f a s t i n g and r e f e e d i n g . J . B i o l . Chem. 236:610-613. O r s i n i , D., H.A. Warsman and C.A. E l v e j e m . 1942. E f f e c t o f v i t a m i n d e f i c i e n c i e s on b a s a l m e t a b o l i s m and r e s p i r a t o r y q u o t i e n t i n r a t s . P r o c . S o c . E x p . B i o l . Med. 51:99-102. P a t t e r s o n , M.S. and R.C. G r e e n e . 19 65. Measurement o f low e n e r g y b e t a - e m i t t e r s i n aqueous s o l u t i o n by l i q u i d s c i n t i l l a t i o n c o u n t i n g o f e m u l s i o n s . A n a l . Chem. 37: 854-857. P a u l , R. 1968. E n e r g y u t i l i z a t i o n i n p y r i d o x i n e d e f i c i e n c y . M.Sc. t h e s i s , U n i v . o f T o r o n t o . R a d h a k r i s h n a m u r t y , R., J . F . A n g e l and Z . I . S a b r y . 1968. Re s p o n s e o f l i p o g e n e s i s t o r e p l e t i o n i n t h e p y r i d o x i n e -d e f i c i e n t r a t . J . N u t r . 95:341-348. R o b e r t s , E . and S. F r a n k e l . 1951. F u r t h e r s t u d i e s o f g l u t a m i c a c i d d e c a r b o x y l a s e i n b r a i n . J . B i o l . Chem. 190:505-512. S a b o , D . J . , R.P. F r a n c e s c o n i and S.N. G e r s h o f f . 1971. E f f e c t o f v i t a m i n B^ d e f i c i e n c y on t i s s u e d e h y d r o g e n a s e s and f a t s y n t h e s i s i n r a t s . J . N u t r . 101:29-34. S a k u r a g i , T. 19 59. The f u n c t i o n o f v i t a m i n B g i n f a t m e t a b o -l i s m . J . Am. O i l Chem. S o c . 36:251-253. S e b r e l l , W.H. and R.S. H a r r i s . 1968. V i t a m i n B G r o u p . I n The V i t a m i n s . 2nd e d . V o l I I . A c a d e m i c P r e s s , New Y o r k , NY. C h a p t . 3, pp 1-117. Sherman, H. 1950. P y r i d o x i n e and f a t m e t a b o l i s m . V i t a m i n s and Hormones 22:787-794. S n e l l , E . E . , B.M. B u i r a r d and R . J . W i l l i a m s . 1944. The v i t a m i n a c t i v i t i e s o f p y r i d o x a l and p y r i d o x a m i n e . J . B i o l . Chem. 154:313-314. S n e l l , E . E . 195 8. C h e m i c a l s t r u c t u r e i n r e l a t i o n t o b i o l o g i c a l a c t i v i t i e s o f v i t a m i n B,. V i t . and Horm. 16:77-125. b S n e l l , E . E . 1961. The r o l e o f v i t a m i n Bg i n c a t a l y s i s o f r e a c t i o n s o f amino a c i d s . I n The Mechanism o f A c t i o n o f W a t e r - s o l u b l e V i t a m i n s . C i b a F o u n d a t i o n S t u d y Group No. 11. A.V.S. de Reuck and M. O'Connor e d s . J . & A. C h u r c h i l l L t d . , L o n d o n , pp 18-37. S o m o g y i , M. 1945. D e t e r m i n a t i o n o f b l o o d s u g a r . J . B i o l . Chem. 160:69-73. S u r e , B. and L . E a s t e r l i n g . 1949. The r o l e o f p y r i d o x i n e i n economy o f f o o d u t i l i z a t i o n . J . N u t r . 39:393-396. T a k a m i , M., M. F u j i o k a , H. Wada and T. T a g u c h i . 1968. S t u d i e s on p y r i d o x i n e d e f i c i e n c y i n r a t s . P r o c . S o c . E x p . B i o l . Med. 129:110-117. T a u s s k y , H.H. and E . S h o r r . 1953. A m i c r o c o l o r i m e t r i c method f o r t h e d e t e r m i n a t i o n o f i n o r g a n i c p h o s p h o r u s . J . B i o l . Chem. 236:610-613. T e p p e r m a n , J . and H.M. Tepperman. 19 58. E f f e c t o f a n t e c e d e n t f o o d i n t a k e p a t t e r n on h e p a t i c l i p o g e n e s i s . Am. J . P h y s i o l . 193:55-64. Tepperman, H.M. and J . Tepperman. 19 64. A d a p t i v e h y p e r -l i p o g e n e s i s . F e d . P r o c . 23:73-75. U m b r e i t , W.W., N.T. Rahway, C.R. K i n g s l e y , R.R. S c h a f f e r t and H. S i p l e t . 1957. A c o l o r i m e t r i c method f o r t r a n s a -m i n a s e i n serum o r p l a s m a . J . L a b . C l i n . Med. 49:454-459. W i l e y , J . H . and G.A. L e v e i l l e . 1970. I n f l u e n c e o f p e r i o d i c i t y o f e a t i n g on t h e a c t i v i t y o f a d i p o s e t i s s u e and m u s c l e g l y c o g e n s y n t h e s i z i n g enzymes i n t h e r a t . J . N u t r . 100: 85-93. 58. W r o b l e w s k i , F . and P. C a b a u d . 1957. C o l o r i m e t r i c measurement o f serum g l u t a m i n p y r u v i c t r a n s a m i n a s e . Am. J . C l i n . P a t h . 27:235-239. Y e h , S.D. and B. W e i s s . 19 63. B e h a v i o u r a l t h e r m o r e g u l a t i o n d u r i n g v i t a m i n B , d e f i c i e n c y . 7Am. J . P h y s i o l . 205:857-862. b APPENDIX Body wt L i v e r wt (g) (g) E x p e r i m e n t I P y r i d o x i n e - d e f i c i e n t3'4 116 - 7.2 1 3.95 - 0.0371 1 P a i r - f e d c o n t r o l s5 124 - 1 . 3 4.38 - 0.320 E x p e r i m e n t I I P y r i d o x i n e - d e f i c i e n t3'4 105 - 4 . 91'6 4.19 - 0.004951 P a i r - f e d c o n t r o l s5 112 - 1.1 4.21 - 0.0660 E x p e r i m e n t I I I + 2.7 P y r i d o x i n e - d e f i c i e n t3'4 110 - 6 . 72'6 3.75 - 0.130 * P a i r - f e d c o n t r o l s5 122 - 1 . 4 3.33 - 0.0650 1. Mean - s t a n d a r d e r r o r f o r 12 r a t s . 2. Mean - s t a n d a r d e r r o r f o r 10 r a t s . 3. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t f o r 45 days a f t e r w e a n i n g . 4. Had a c c e s s t o f o o d f r o m 9:00 - 11:00 a.m. d a i l y . 5. F e d t h e p y r i d o x i n e - d e f i c i e n t d i e t p l u s 10 mg p y r i d o x o l . H C 1 / k g d i e t . 6. S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p a t P < 0 . 0 1 . 7. S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l g r o u p a t P< 0.001. 

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