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The effect of a maternal dietary lysine deficiency on tissue carnitine levels in the rat Taylor, Mary Jane Muise 1980

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THE EFFECT OF A MATERNAL DIETARY LYSINE DEFICIENCY ON TISSUE CARNITINE LEVELS IN THE RAT by MARY JANE MUISE TAYLOR B S c H E c . . Mount S a i n t V i n c e n t U n i v e r s i t y , 1 9 7 7 A THESIS SUBMITTED IN PARTIAL POLFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Human N u t r i t i o n School of Home Economics We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1 9 8 0 Mary Muise T a y l o r , 1 9 8 0 In presenting th i s thesis in par t i a l fu l f i lment of the requirements for an advanced degree at the Univers ity of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly avai lable for reference and study. I further agree that permission for extensive copying of th i s thesis for scholar ly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publ icat ion of th i s thesis for f inanc ia l gain shal l not be allowed without my written permission. Department of , The Univers ity of B r i t i s h Columbia 2075 wesbrook Place Vancouver, Canada V6T 1W5 Home Economics >E-6 B P 75-51 1 E ABSTRACT The e f f e c t of a maternal d i e t a r y l y s i n e d e f i c i e n c y on milk c a r n i t i n e l e v e l s and on plasma and l i v e r c a r n i t i n e l e v e l s i n dams, f e t u s e s and neonates was s t u d i e d . . Experimental animals were fed e i t h e r a l o w - l y s i n e d i e t (0.27% l y s i n e ) * a h i g h - l y s i n e d i e t (1.07% l y s i n e ) ad l i b i t u m , or the h i g h - l y s i n e d i e t p a i r - f e d to the l o w - l y s i n e group. A l l d i e t s contained 20% wheat g l u t e n , 20% corn o i l and n e g l i g i b l e c a r n i t i n e . Dams fed a d i e t , e i t h e r Ion i n l y s i n e or r e s t r i c t e d i n t o t a l food i n t a k e , consumed s i g n i f i c a n t l y l e s s food d u r i n g pregnancy and l a c t a t i o n than h i g h - l y s i n e dams. When compared to h i g h - l y s i n e dams the l o w - l y s i n e dams and t h e i r p a i r - f e d c o n t r o l s gained s i g n i f i c a n t l y l e s s weight during pregnancy and l o s t weight d u r i n g l a c t a t i o n whereas the h i g h - l y s i n e dams gained weight d u r i n g l a c t a t i o n . L i t t e r s i z e was not a f f e c t e d by e i t h e r a d i e t a r y l y s i n e d e f i c i e n c y or by the s m a l l r e d u c t i o n i n t o t a l food i n t a k e during g e s t a t i o n ^ However, b i r t h weight of o f f s p r i n g i n the l e w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups was s i g n i f i c a n t l y lower than th a t of the h i g h - l y s i n e c o n t r o l s . . On day 15 of l a c t a t i o n the h i g h - l y s i n e pups weighed s i g n i f i c a n t l y more than the h i g h - l y s i n e r e s t r i c t e d pups, which i n t u r n weighed s i g n i f i c a n t l y mere than the l o w - l y s i n e pups, suggesting a s u p e r i o r l a c t a t i o n performance f o r those dams fe d the h i g h - l y s i n e c o n t r o l d i e t and the poorest l a c t a t i o n performance f o r those dams consuming the l o w - l y s i n e d i e t . L i v e r and heart t i s s u e samples were obtained from dams and t h e i r o f f s p r i n g en day 21 of pregnancy and day 15 of l a c t a t i o n . i v When l i v e r weight or heart weight were expressed as a percentage of t o t a l body weight f o r dams or pups, no s i g n i f i c a n t d i f f e r e n c e between d i e t a r y groups was d e t e c t e d ^ These r e s u l t s i n d i c a t e that l i v e r and heart weights were p r o p o r t i o n a l t o body weight. The l o w - l y s i n e d i e t had no s i g n i f i c a n t e f f e c t , on day 21>of g e s t a t i o n , on maternal plasma or l i v e r c a r n i t i n e l e v e l s or on f e t a l l i v e r c a r n i t i n e l e v e l s , whereas f e t a l plasma c a r n i t i n e showed a s m a l l but s i g n i f i c a n t i n c r e a s e compared to the h i g h -l y s i n e group,. On day 15 of l a c t a t i o n plasma and l i v e r c a r n i t i n e l e v e l s were s i g n i f i c a n t l y higher i n both dams and o f f s p r i n g f e d the l o w - l y s i n e d i e t , than i n t h e i r r e s p e c t i v e c o n t r o l s . T h i s i n c r e a s e in plasn a and l i v e r c a r n i t i n e l e v e l s was probably due to a lowered food i n t a k e s i n c e animals fed the h i g h - l y s i n e d i e t p a i r - f e d to the l o w - l y s i n e group showed the same t i s s u e c a r n i t i n e response as d i d animals f ed the l o w - l y s i n e d i e t . Milk c a r n i t i n e l e v e l s on day 2 o f l a c t a t i o n were h i g h e s t i n the h i g h - l y s i n e group and lowest i n the h i g h - l y s i n e r e s t r i c t e d group. On days 8 and 15 of l a c t a t i o n milk c a r n i t i n e l e v e l s were s i g n i f i c a n t l y higher i n dams f e d the l c w - l y s i n e d i e t than i n those fed t h e h i g h - l y s i n e or the h i g h - l y s i n e r e s t r i c t e d d i e t , . The r e s u l t s of t h i s r e s e a r c h i n d i c a t e t h a t plasma and l i v e r c a r n i t i n e l e v e l s i n both dams and o f f s p r i n g and milk c a r n i t i n e l e v e l s i n dams, are not l i m i t e d by the l y s i n e content of the maternal d i e t under the exp e r i m e n t a l c o n d i t i o n s of t h i s study. V TABLE OF CONTENTS Ab s t r a c t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i i L i s t o f Ta b l e s 1.. i............ v i i L i s t o f F i g u r e s i x Acknowledgement .............. x i Chapter I ................................................. 1 I n t r o d u c t i o n 1 Chapter I I ... . . 4 Review of L i t e r a t u r e ................................... 4 Fu n c t i o n of C a r n i t i n e .......................... .. ... 4 C a r n i t i n e Metabolism ................................ 9 Maternal-Neonatal C a r n i t i n e R e l a t i o n s h i p ............ 13 P r e n a t a l .......................................... 13 P o s t n a t a l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 R e l a t i o n s h i p of C a r n i t i n e t o K e t o s i s ................ 17 Maternal D i e t a r y D e f i c i e n c i e s w»^.................... 19 Pregnancy 19 L a c t a t i o n .........................^............^.20 L y s i n e D e f i c i e n c y and T i s s u e C a r n i t i n e L e v e l s ....... 22 Chapter I I I . 25 Methods and M a t e r i a l s ............... i . . . . . . . . . . . . . . . . . . 25 Animals 25 Di e t s .. 26 Animal Experiments ................... 26 Experiment I ......^..............................31 Experiment I I .................................... 31 Experiment I I I 33 Plasma, L i v e r , and Milk T o t a l C a r n i t i n e A n a l y s i s . ... 35 P r i n c i p l e of Assay 35 Pr e p a r a t i o n of Plasma C a r n i t i n e E x t r a c t - P a r t A, S e c t i o n 1 36 Pre p a r a t i o n of Milk C a r n i t i n e E x t r a c t s - P a r t A, S e c t i o n 2 37 Pr e p a r a t i o n of L i v e r C a r n i t i n e E x t r a c t s - P a r t A, S e c t i o n 3 .. 38 T o t a l C a r n i t i n e Assay Procedure - Part B . . . . . . . i . 39 Determination of C a r n i t i n e C o n c e n t r a t i o n ......... 42 Recovery ................................... ^ ...... .43 S t a t i s t i c s 43 Chapter IV i . 45 R e s u l t s ................................................ 45 Experiment I ........................................ 45 Experiment I I ........................................57 Experiment I I I ..........................^........... 76 Chapter V ....100 D i s c u s s i o n ............................................. 100 D i e t a r y Lysine and Reproduction ... . . .......... . - 100 Di e t a r y L y s i n e and L a c t a t i o n Performance ..102 D i e t and Tissue Heights ............................. 1 0 5 D i e t a r y L y s i n e and Plasma and L i v e r C a r n i t i n e ....... 1 0 5 Dams 1 0 5 Fetus and Neonates ............................... 1 0 7 Mil k C a r n i t i n e . . . . . . . . . 1 1 0 Chapter V I -: ................................ 1 1 4 Summary o f Results .....................................II4* B i b l i o g r a p h y .-. . . . . . . . . . . . . . . . . . . . ^ . . . . . . . . . . ; . - . . . . . . . - - . 1 1 7 v i i LIST OF TABLES I Composition of l o w - l y s i n e die~t 27 I I C a l c u l a t e d e s s e n t i a l amino a c i d composition of d i e t s 28 I I I Food i n t a k e and weight gain of dams fed a high-l y s i n e or l o w - l y s i n e d i e t d u r i n g g e s t a t i o n ....... 46 IV Heart and l i v e r weights of dams, fed a h i g h - l y s i n e or l o w - l y s i n e d i e t u n t i l day 21 o f g e s t a t i o n 49 V E f f e c t of a h i g h - l y s i n e or l c w - l y s i n e d i e t on the number of f e t u s e s , and f e t a l heart and l i v e r weights cn day 21 of g e s t a t i o n 51 VI E f f e c t of a h i g h - l y s i n e or l o w - l y s i n e d i e t on plasma and l i v e r c a r n i t i n e of dams and f e t u s e s on day 21 of g e s t a t i o n . . . . . . . . . . . . . . . . . . . . . i , . . . . 52 VII Food i n t a k e and weight g a i n of dams f e d a high-l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t during g e s t a t i o n i n Experiment I I ................. 58 V I I I Food intake and weight changes cf dams fed a h i g h -l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t d u r i n g l a c t a t i o n .................................. 62 IX Weights of dams f e d a h i g h - l y s i n e , l o w - l y s i n e , o r h i g h - l y s i n e r e s t r i c t e d d i e t u n t i l day 15 of l a c t a t i o n 63 X E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e * or high-l y s i n e r e s t r i c t e d d i e t on r e p r o d u c t i v e performance of dams i n Experiment I I . . . . . . . . . . . . . . . . . . i . . . . . . . 65 XI E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e , or high-l y s i n e r e s t r i c t e d maternal d i e t on weight g a i n s of pups i n Experiment I I ............................ 66 XII E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e , or h i g h -l y s i n e r e s t r i c t e d maternal d i e t on he a r t and l i v e r weights cf pups, on day 15 o f . l a c t a t i o n «* .69 XIII E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h -l y s i n e r e s t r i c t e d d i e t on plasma and l i v e r c a r n i t i n e of dams and pups on day 15 of l a c t a t i o n 71 XIV Food i n t a k e and weight g a i n o f dams fed a high-l y s i n e * l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t d u r i n g g e s t a t i o n i n Experiment I I I ............... 79 XV Food intake and weight changes of dams milked on day 15 of l a c t a t i o n and f e d a h i g h - l y s i n e * lew-v i i i l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t during l a c t a t i o n 83 XVI Food i n t a k e and weight changes cf dams milked on day 15 of l a c t a t i o n and f e d a h i g h - l y s i n e , low-l y s i n e or h i g h - l y s i n e r e s t r i c t e d d i e t during l a c t a t i o n 85 XVII E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e , or high-l y s i n e r e s t r i c t e d d i e t on r e p r o d u c t i v e performance of dams i n Experiment I I I ........................ 87 XVIII E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e , or h i g h -l y s i n e r e s t r i c t e d maternal d i e t on weight gains of pups i n Experiment I I I 88 XIX E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or hi g h -l y s i n e r e s t r i c t e d d i e t on milk c a r n i t i n e on days 2, 8, and 15 post partum ....94 i x LIST OF FIGURES 1 The f u n c t i o n of c a r n i t i n e i n l o n g - c h a i n f a t t y a c i d s o x i d a t i o n . . .. . . . . . . . . . . . . . . . . 6 2 B i o s y n t h e s i s of c a r n i t i n e from l y s i n e and methionine 10 3 Diagramatic r e p r e s e n t a t i o n of experiments I , I I , and I I I ... ...... 29 4 E f f e c t o f a h i g h - l y s i n e or l o w - l y s i n e d i e t on weight gain of dams during g e s t a t i o n 47 5 E f f e c t of a h i g h - l y s i n e or l o w - l y s i n e d i e t on plasma and l i v e r c a r n i t i n e i n dams on day 21 of g e s t a t i o n .... 53 6 E f f e c t of a h i g h - l y s i n e or l o w - l y s i n e d i e t on plasma and l i v e r c a r n i t i n e i n f e t u s e s on day 21 of g e s t a t i o n .................. 55 7 E f f e c t of a h i g h - l y s i n e , l o w — l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t on weight gain of dams durin g g e s t a t i o n i n Experiment I I .... . . . . . . . . . . . . . . . . . . . . 60 8 E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d maternal d i e t on weight g a i n of pups i n Experiment I I ....................................... 67 9 E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t on plasma and l i v e r c a r n i t i n e of dams on day 15 of l a c t a t i o n 72 10 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d maternal d i e t on plasma and l i v e r c a r n i t i n e of pups on day 15 of l a c t a t i o n . ............ 74 11 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e * or h i g h - l y s i n e r e s t r i c t e d d i e t on weight changes of dams durin g pregnancy and l a c t a t i o n i n Experiment I I ............ 77 12 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t on weight g a i n of dams durin g g e s t a t i o n i n Experiment I I I ...........^.............81 13 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d maternal d i e t on weight gains of pups born t o those dams milked on day 15 of l a c t a t i o n .... 89 14 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d maternal d i e t oh weight gain of pups born to those dams milked on day 8 cf l a c t a t i o n .......... 91 15 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t on milk c a r n i t i n e on days 2, 8, and, 15 of l a c t a t i o n 95 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i s t on maternal plasma and milk c a r n i t i n e i n dams, and pup plasma c a r n i t i n e on day 15 of l a c t a t i o n .................................... x i ACKNOWLEDGEMENT I would l i k e to thank my a d v i s o r Dr.. P.. S t a p l e t o n , D i v i s i o n of Human N u t r i t i o n , U n i v e r s i t y of B r i t i s h Columbia, f o r h i s i n v a l u a b l e guidance, a d v i s e , and support. The h e l p f u l s uggestions of my committee members, Br. P.. Hahn, Department of P e d i a t r i c s , U n i v e r s i t y of B r i t i s h Columbia and D r i P. V. G a l l o , D i v i s i o n of Human N u t r i t i o n , U n i v e r s i t y of B r i t i s h Columbia, are g r a t e f u l l y appreciated;. I would l i k e to acknowledge Lewis James, Arts Computing, U n i v e r s i t y o f B r i t i s h Columbia, f o r h i s s k i l l s and a s s i s t a n c e with the computer programs used t o complete t h i s t h e s i s . Also acknowledged are the members of Dr.. Hahn's l a b , p a r t i c u l a r l y Freda-Ann Smale and Nadia Hahn, f c r t h e i r i n s t r u c t i o n s concerning the c a r n i t i n e assay. . The f i n a n c i a l support of the N a t u r a l Sciences and E n g i n e e r i n g Research C o u n c i l of Canada (1978-1979), the Summer Graduate Student Bursary (1979), and the U n i v e r s i t y Teaching A s s i s t a n t s h i p (1S77-1978) are g r a t e f u l l y acknowledged. S p e c i a l thanks are extended t o my s i s t e r , Audrey Muise, f o r p r o v i d i n g a " h e l p i n g hand" when i t was needed most.. F i n a l l y , to whom I am e s p e c i a l l y g r a t e f u l , I would l i k e t o thank my husband, Paul T a y l o r , f o r h i s help, encouragement, and love throughout the course of t h i s study. '. To my p a r e n t s Gerald and Mary Muise f o r a l i f e t i m e of support and encouragement 1 CHAPTER I INTRODUCTION C a r n i t i n e (B-hydroxy-ytrimethylaminobutyrate) p l a y s a v i t a l r o l e i n the i n t r a m i t o c h o n d r i a l t r a n s p o r t and subseguent o x i d a t i o n of l c n g - c h a i n f a t t y a c i d s ( F r i t z and Yue, 1963) . . I t i s s y n t h e s i z e d from l y s i n e and methionine, both of which are e s s e n t i a l d i e t a r y amino a c i d s (Broguist et a l * , . 1975). C a r n i t i n e , although having the a t t r i b u t e s of a vitamin i s not considered one. I t has been suggested t h a t the r e l a t i o n s h i p of c a r n i t i n e t o l y s i n e i s s i m i l i a r to th a t which e x i s t s between n i a c i n and tryptophan. T h e r e f o r e i f s u f f i c i e n t l y s i n e i s present i n the d i e t , c a r n i t i n e can be synthesized i n adequate amounts i n the body ( T a n p h a i c h i t r and E r o q u i s t , 1973; B r o g u i s t , 1976). I t i s not known how much l y s i n e i s r e q u i r e d f o r c a r n i t i n e s y n t h e s i s although Horne e t a l . (1971) have estimated that 0.2% of the l y s i n e requirement of Neurospora c r a s s a i s used f o r c a r n i t i n e s y n t h e s i s . . Because of i t s r o l e i n f a t t y a c i d o x i d a t i o n , a c a r n i t i n e d e f i c i e n c y may cause an impairment i n f a t u t i l i z a t i o n . F a t accumulation i n t i s s u e s of the r a t ( T a n p h a i c h i t r et a l . , 1976), guinea pig ( W i t t l e s and B r e s s l e r , 1964), and human (Isaacs et a l . , 1976) has been a s s o c i a t e d with c a r n i t i n e d e f i c i e n c i e s . D i e t a r y c a r n i t i n e i s g e n e r a l l y obtained from animal f o o d s t u f f s , whereas p l a n t foods are u s u a l l y poor sources of c a r n i t i n e (Fraenkel, 1954; Panter and Mudd, 196S). Pl a n t foods are a l s o l i k e l y to be d e f i c i e n t i n l y s i n e and/or methionine, the p r e c u r s o r s of c a r n i t i n e . . D i e t s comprised mainly of p l a n t foods may r e s u l t i n s i g n i f i c a n t l y lowered plasma c a r n i t i n e l e v e l s i n 2 humans ( M i k h a i l and Mansour, 1976; L a t i f a and Bamji, 1977),. A v a i l a b i l i t y of c a r n i t i n e i s of prime importance i n p h y s i o l o g i c a l c o n d i t i o n s t h a t are a s s o c i a t e d with changes i n f a t metabolisnu Pregnancy and l a c t a t i o n are examples of such p h y s i o l o g i c a l c o n d i t i o n s , . S ince the neonatal r a t d e r i v e s most of i t s energy from milk f a t , a d e f i c i e n c y of c a r n i t i n e c o u l d impair u t i l i z a t i o n of d i e t a r y f a t and thus a d v e r s e l y a f f e c t development o f the neonate. The dam's milk p r o v i d e s the major source of c a r n i t i n e f o r the s u c k l i n g r a t because neonatal c a r n i t i n e s y n t h e s i s appears i n s i g n i f i c a n t , a t l e a s t d u r i n g the e a r l y stages of l a c t a t i o n (Ferre et a l . , 1978) i F e t a l t i s s u e s c o n t a i n lower l e v e l s of c a r n i t i n e , which appears to be d e r i v e d mainly from maternal sources and i s l i m i t e d by p l a c e n t a l t r a n s f e r of c a r n i t i n e (Hahn and Skala, 1975). T h e r e f o r e a maternal c a r n i t i n e d e f i c i e n c y c o u l d r e s u l t i n a c a r n i t i n e d e f i c i e n c y i n the o f f s p r i n g . . The r e l a t i o n s h i p between d i e t a r y l y s i n e and t i s s u e c a r n i t i n e has not been s t u d i e d i n the pregnant or l a c t a t i n g r a t . In the post-weanling male r a t , a d i e t a r y l y s i n e d e f i c i e n c y reduced h e a r t and s k e l e t a l muscle c a r n i t i n e by 25% compared to c o n t r o l values. . Female r a t s o f the same age, responded d i f f e r e n t l y t o a l y s i n e d e f i c i e n c y . Heart muscle c a r n i t i n e of the female was not a f f e c t e d by d i e t a r y l y s i n e l e v e l s (Borum and Broq u i s t , 1977). Compared to the r e s p e c t i v e c o n t r o l v a l u e s , plasma c a r n i t i n e l e v e l s were h i g h e r i n the plasma of female r a t s , but lower i n the plasma o f male r a t s , f e d a l o w - l y s i n e d i e t . S t a p l e t o n and H i l l (1972, 1980) p r e v i o u s l y r e p o r t e d a 3 s i g n i f i c a n t drop i n plasma l y s i n e l e v e l s during the l a s t week of g e s t a t i o n i n dams fed a d i e t d e f i c i e n t i n l y s i n e and c o n t a i n i n g n e g l i g a b l e c a r n i t i n e or the same b a s a l d i e t supplemented with l y s i n e . However, the decrease i n plasma l y s i n e l e v e l s i n the l y s i n e d e f i c i e n t animals was s i g n i f i c a n t l y g r e a t e r than t h a t i n the l y s i n e supplemented animals. The q u a n t i t y and q u a l i t y of milk produced by the dams was a l s o a d v e r s e l y e f f e c t e d by the d i e t a r y l y s i n e r e s t r i c t i o n . . M i l k p r o d u c t i o n was reduced, as was the q u a n t i t y of p r o t e i n i n t h e i r m i l k . The e f f e c t s of f e e d i n g a maternal d i e t d e f i c i e n t i n l y s i n e and c o n t a i n i n g n e g l i g i b l e c a r n i t i n e , d u r i n g g e s t a t i o n and l a c t a t i o n , on the c a r n i t i n e s t a t u s of both the mother and her o f f s p r i n g i s not known. However, the p o s s i b i l i t y t h a t such a d i e t a r y c o n d i t i o n does a d v e r s e l y a f f e c t the neonate i s proposed s i n c e evidence suggests a dependency of the neonate on the mother r a t f o r i t s major supply of c a r n i t i n e to meet p h y s i o l o g i c a l requirements. T h e r e f o r e the major o b j e c t i v e s of t h i s t h e s i s were t o determine the e f f e c t s of a maternal l y s i n e d e f i c i e n c y on: a) t i s s u e c a r n i t i n e l e v e l s of dams and t h e i r f e t u s e s on day 21 of g e s t a t i o n . b) t i s s u e c a r n i t i n e l e v e l s of dams and t h e i r pups on day 15 of l a c t a t i o n . c) milk c a r n i t i n e l e v e l s of dams on days 2, 8, and 15 of l a c t a t i o n . 4 CHAPTER I I REVIEW OF LITERATURE FUNCTION OF CARNITINE The e x i s t e n c e of c a r n i t i n e , B -hydroxy -t r i m e t h y l a m i n o b u t y r a t e was known i n the e a r l y IfJOO's but i t was many years before i t s s t r u c t u r e was f i n a l l y e s t a b l i s h e d (Fraenkel, 1957) . C a r n i t i n e , an e s s e n t i a l growth f a c t o r f o r the mealworm Tenebrio m p l i t a r , was i s o l a t e d i n 1946 (Fraenkel and Blewett, 1946) and t e n t a t i v e l y named Vitamin BT (T s t a n d i n g f o r Tenebrio)« . S e v e r a l years l a t e r v i t a m i n BT was r e c o g n i z e d as c a r n i t i n e (Carter et a l . , 1952). One of the f i r s t o b s e r v a t i o n s r e g a r d i n g the f u n c t i o n of c a r n i t i n e was by F r i t z i n 1955 who reported t h a t the a d d i t i o n of c a r n i t i n e to r a t l i v e r homcgenates i n c r e a s e d the r a t e of f a t t y a c i d o x i d a t i o n . T h i s l e d t o an e x t e n s i v e study of the f u n c t i o n of c a r n i t i n e i n f a t metabolism, s p e c i f i c a l l y l o n g - c h a i n f a t t y a c i d s . Short c h a i n f a t t y a c i d s do not r e q u i r e c a r n i t i n e f o r t h e i r o x i d a t i o n ( F r i t z et a l . , 1962; W i t t l e s and B r e s s l e r , 1964; B r e s s l e r and W i t t l e s , 1965) and are a c t i v a t e d by i n t r a - m i t o c h o n d r i a l enzymes (Rossi and Gibson, 1964 ; Bremer, 1 968) . Long-chain f a t t y a c i d s , the p r i n c i p a l f u e l f o r o x i d a t i v e metabolism i n t i s s u e s , are t r a n s p o r t e d i n the plasma as f r e e f a t t y a c i d s bound to albumin (Dole, 1956; Gordon, 1957; Gordon et a l . , 1958; F r e d r i c k s o n et a l . , 1958). C a r n i t i n e p l a y s an important r o l e i n the t r a n s p o r t o f the a c t i v a t e d l o n g - c h a i n 5 f a t t y a c y l groups from the s i t e of a c t i v a t i o n to the m i t o c h o n d r i a l matrix ( F r i t z and Yue, 1963; Pande, 1975), where the enzymes cf B - o x i d a t i o n are lo c a t e d ( E e a t t i e , 1968). A c t i v a t i o n occurs when the l o n g - c h a i n f a t t y a c i d i s a c y l a t e d to coenzyme A (Figure 1).. T h i s p r o c e s s takes p l a c e i n both e x t r a -m i t o c h o n d r i a l s i t e s and the mitochondria (Rossi and Gibson, 1964; Norum e t a l . , 1966; Bremer, 1968). The lo n g - c h a i n f a t t y a c i d a c t i v a t i n g system i n the l i v e r i s l o c a l i z e d mainly (70S) i n the endoplasmic r e t i c u l u m with the remaining a c t i v i t y i n the mitochondria (Norum e t a l . , 1966; F a r s t a d et a l . , 1 967),.. Most of the m i t o c h o n d r i a l a c t i v i t y i s found i n the outer membrane f r a c t i o n (Norum e t a l . , 1966)., The t r a n s p o r t of acyl-coenzyme A i n t o the mitochondria i s mediated by the formation of a c y l c a r n i t i n e . The m i t o c h o n d r i a l i n n e r membrane i s permeable t o c a r n i t i n e e s t e r s (Yates and Garland, 1966) but i t i s impermeable t o acyl-coenzyme A and f r e e coenzyme A (Yates and Garland, 1966; Klingenberg and P f a f f , 1966),.. T r a n s a c y l a t i o n of the coenyme A and c a r n i t i n e e s t e r s i s mediated by two d i s t i n c t l y s eparate enzymes (Figure 1). One enzyme* c a r n i t i n e p a l m i t y l t r a n s f e r a s e i s s p e c i f i c f o r lo n g - c h a i n f a t t y acyl-coenzyme A ( F r i t z and Yue, 1963; Klingenberg and E f a f f 1966). p a l m i t l y t r a n s f e r a s e > palmityl-coenzyme A • ( - ) - c a r n i t i n e ( - ) - p a l m i t y l c a r n i t i n e + coenzyme ASH The e f f e c t o f c a r n i t i n e on f a t t y a c i d o x i d a t i o n i s mediated by 6 Figure 1 The f u n c t i o n of c a r n i t i n e i n l o n g - c h a i n f a t t y a c i d s o x i d a t i o n CoA Long-chain F a t t y Acid a c t i v a t i n g system A c y l CoA Free F a t t y Acid A c e t y l C a r n i t i n e Outer M i t o c h o n d r i a l Space A c y l c a r n i t i n e Long-chain c a r n i t i n e a c y l t r a n s f e r a s e A Long-chain c a r n i t i n e a c y l t r a n s f e r a s e B CoA C a r n i t i n e 'Acyl CoA C a r n i t i n e - a c y l c a r n i t i n e translocase Inner c a r n i t i n e I L a c e t y l t r a n f e r a s e i CoA A c e t y l CoA Ketone Bodies + Co„ M i t o c h o n d r i a l Membrane M a t r i x 8 t h i s enzyme which t r a n s f e r s the a c y l group from coenzyme A t o c a r n i t i n e . The enzyme, c a r n i t i n e p a l m i t y l t r a n s f e r a s e A, i s l o c a l i z e d on the cuter s u r f a c e of the i n n e r membrane (Hoppel and Tomac, 1972). The a c y l - c a r n i t i n e c r o s s e s the inner membrane and i s converted back to acyl-ccenzyme A by c a r n i t i n e p a l m i t y l t r a n s f e r a s e B, l o c a l i z e d on the i n n e r surface of the i n n e r m i t o c h o n d r i a l membrane (Hoppel and Tomac, 1972). The a c y l coenzyme A w i t h i n the mitochondria.may then be o x i d i z e d by B - o x i d a t i o n to two carbon fragments of a c e t y l coenzyme A.. These two carbon fragments can e i t h e r : a) e n t e r the Krebs C y c l e b) be used in the p r o d u c t i o n of Ketone bodies c) or be t r a n s p o r t e d out o f the mitochondria. The second enzyme, c a r n i t i n e a c e t y l t r a n s f e r a s e , i s s p e c i f i c f o r a c e t y l coenzyme A and s h o r t c h a i n a c y l coenzyme A (Bremer, 1962; F r i t z , 1963; F r i t z and Yue, 1963; Norum and Bremer, 1963). , c a r n i t i n e a c e t y l t r a n s f e r a s e — . > (-) a c e t y l c a r n i t i n e «• coenzyme A ( - ) - c a r n i t i n e * acetyl-coenzyme A The f u n c t i o n of c a r n i t i n e a c e t y l t r a n s f e r a s e has been d i f f i c u l t to understand._ Several r e s e a r c h e r s have proposed t h a t c a r n i t i n e a c e t y l t r a n s f e r a s e f u n c t i o n s as a t r a n s p o r t e r of a c e t y l groups between t h e i r s i t e of formation by B - o x i d a t i o n , t o the outer m i t o c h o n d r i a l space ( B r e s s l e r , 1970) . However, no such c a r n i t i n e - d e p e n d e n t t r a n s f e r has been s u b s t a n t i a t e d , and i t i s a l s o i n t e r e s t i n g that very l i t t l e or no outer a c e t y l t r a n s f e r a s e 9 i s found i n the mitochondria (Solberg, 1970) „ _ A c e t y l c a r n i t i n e may i n s t e a d represent a b u f f e r i n g system or depot f o r m i t o c h o n d r i a l a c e t y l r e s i d u e s ( Kopec and F r i t z , 1973; Brosman et a l . , 1973; Brener, 1977) l e a v i n g coenzyme A a v a i l a b l e f o r f a t t y a c i d o x i d a t i o n , p a r t i c u l a r l y during p e r i o d s of i n c r e a s e d k e t o g e n e s i s i . S u f f i c i e n t evidence at present i s not a v a i l a b l e t o support t h i s hypothesise CARNITINE METABOLISM C a r n i t i n e i s widely d i s t r i b u t e d i n nature and i t s h i g h e s t c o n c e n t r a t i o n s are found i n animal products (Fraenkel, 1954). Pl a n t products g e n e r a l l y c o n t a i n low l e v e l s of c a r n i t i n e (Panter and Mudd, 1969). In a d d i t i o n t o d i e t a r y c a r n i t i n e , the r a t i s able t o s y n t h e s i z e c a r n i t i n e from i t s amino a c i d p r e c u r s o r s l y s i n e and methionine ( T s a i e t a l , 1974; Cederblad and L i n d s t e d t , 1976) . The pathway f o r c a r n i t i n e b i o s y n t h e s i s i s represented i n Fi g u r e 2. The carbon chain and quartenary n i t r o g e n atom of the c a r n i t i n e are derived from l y s i n e {Cox and Hoppel, 1973a; Home and B r o g u i s t , 1973).. S i m i l a r l y , £ - t r i m e t h y l l y s i n e i s synth e s i z e d v i a the me t h y l a t i o n of p r o t e i n bound l y s i n e by S-adenosyl methionine (LaBadie et a l . , 1976).. Thus £ -t r i m e t h y l l y s i n e i s an e s t a b l i s h e d p r e c u r s o r of Y -trimethylaminobutyrate which i n t u r n i s hydroxylated t o y i e l d L-c a r n i t i n e (Lindstedt, 1967; L i n d s t e d t and L i n d s t e d t , 1965; Cox and Hoppel, 1973b). H o l c h a l t e r and Henderson (1976) repo r t e d that the two carbon fragment l o s t from the t r i m e t h y l l y s i n e was 10 F i g u r e 2 B i o s y n t h e s i s of c a r n i t i n e from l y s i n e and methionine 11 H 2N(CH 2) 4 H 20 CO I CH I NH - R - R Lysine (protein-bound) + S-Adenosyl Methionine COO I (CH,), N (CH„). - CH 5 5 2 4 | NH„ E - T r i m e t h y l l y s i n e Ascorbate Fe + •a-Ketoglutarate +0„ -•Succinate +C0, COO l ( C H 3 ) 3 N ( C H 2 ) 3 CHOH - CH Pyridoxal phosphate + l NH„ 3-Hydroxy-£-Trimethyllysine K Glycine ( C H 3 ) 3 N ( C H 2 ) 3 CHO Y-Trimethylaminobutyraldehyde s NAD+ • NADH H + + + ( C H 3 ) 3 N ( C H 2 ) 3 COO y-Trimethylaminobutyrate Ascorbate a-Ketoglutarate .+.0, •• Succinate +C0r (CH.). N CH_CHCH0 COO 5 5 I | z OH Carnitine (3-Hydroxy-Y-trimethylaminobutyrate) adapted from Hulse and E l l i s (1978) 12 g l y c i n e and proposed the pathway f o r the co n v e r s i o n of t r i m e t h y l l y s i n e t c c a r n i t i n e . . Hulse et a l . , (1978) r e c e n t l y succeeded i n e s t a b l i s h i n g the whole pathway from t r i m e t h y l l y s i n e to c a r n i t i n e i n the r a t . Thus c a r n i t i n e s y n t h e s i s r e q u i r e s two amino a c i d s i n a d d i t i o n to t h r e e vitamins ( a s c o r b i c a c i d , p y r i d o x i n e , and n i a c i n ) . . The l i v e r i s the primary s i t e of c a r n i t i n e b i o s y n t h e s i s i n the r a t ( T a n p h a i c h i t r and B r o q u i s t , 1974), although the t e s t e s have a l i m i t e d c a p a c i t y f o r such s y n t h e s i s ( C a s i l l a s and E r i c k s o n , 1975). . Reports of s t u d i e s i n v i v o ( T a n p h a i c h i t r and B r o g u i s t , 1974) and i n v i t r o ( H a i g l e r and B r o q u i s t , 1974; T a n p h a i c h i t r and E r o q u i s t , 1974) i n d i c a t e t h a t s e v e r a l t i s s u e s , i n c l u d i n g h e a r t , l i v e r , kidney, muscle, and t e s t e s can convert £-trimethyllysine to Y - b u t y r o b e t a i n e . Transport of Y-butyxobetaine v i a the plasma t o the l i v e r i s r e q u i r e d f o r the f i n a l c o n v e r s i o n to c a r n i t i n e ( L i n d s t e d t , 1967; H a i g l e r and B r o q u i s t , 1974; Ta n p h a i c h i t r and B r o g u i s t , 1974). E x a c t l y how much of the l y s i n e i n g e s t e d by the r a t i s used to s y n t h e s i z e c a r n i t i n e i s not known. Hcrne e t a l i (1971) have estimated t h a t 0.2% of the l y s i n e requirement of Neurospora c r a s s a i s used f o r c a r n i t i n e s y n t h e s i s . The est i m a t e d t o t a l body c o n c e n t r a t i o n of c a r n i t i n e i n r a t s i s 0.33 u moles per 100 q o f body weight ( T s a i et a l i , 1974; Cederblad and L i n d s t e d t , 1976). The d a i l y s y n t h e s i s o f c a r n i t i n e as computed by Cederblad and L i n d s t e d t (1976) would approximate 2 u moles per 100 g body weight per day. Thus the d i e t a r y requirement as c a l c u l a t e d by Tsai e t a l . . (1974) o f 1.5 u moles of c a r n i t i n e per 100 g body weight per day can be met by d a i l y b i o s y n t h e s i s 13 of c a r n i t i n e from i t s amino a c i d p r e c u r s o r s l y s i n e and methionine*. MA TE RN AL-NEON AT A l CARNITINE RELATIONSHIP The c a r n i t i n e r e l a t i o n s h i p t h a t e x i s t s between the mother r a t and her o f f s p r i n g , both pre- and p c s t n a t a l l y , has r e c e n t l y aroused c o n s i d e r a b l e a t t e n t i o n * . Evidence suggests a dependence of the neonate on the mother r a t as i t s major s u p p l i e r o f c a r n i t i n e t o meet i t s p h y s i o l o g i c a l requirements (Hahn and Skala, 1975; Robles-Valdes et a l . , 1976 ; F e r r e et a l . , 1 978). Human s t u d i e s have a l s o emphasized the important r o l e the mother p l a y s i n s u p p l y i n g c a r n i t i n e to her t r e a s t - f e d i n f a n t (Ncvak e t a l . , 1979) . P r e n a t a l The mammalian f e t u s g a i n s most of i t s energy from maternally d e r i v e d carbohydrates (Hahn and Koldovsky* 1966).. I t i s w e l l e s t a b l i s h e d t h a t a high carbohydrate d i e t supports' f a t t y a c i d s y n t h e s i s r a t h e r than f a t t y a c i d o x i d a t i o n . The b i o c h e m i c a l f i n d i n g s r e g a r d i n g f a t u t i l i z a t i o n by the r a t f e t u s are i n keeping with t h i s concept. In a d d i t i o n , the c a p a c i t y f o r l o n g - c h a i n f a t t y a c i d o x i d a t i o n i n r a t f e t a l l i v e r i s lew. The c o n c e n t r a t i o n of c a r n i t i n e p a l m i t y l t r a n s f e r a s e r e q u i r e d f o r lonq chain f a t t y a c i d o x i d a t i o n (Augenfeld and F r i t z , 197C; Lockwood and B a i l e y , 1970) and the c o n c e n t r a t i o n of c a r n i t i n e a c e t y l t r a n s f e r a s e are a l s o 14 low d u r i n g f e t a l development (Lee and F r i t z , 1971). Correspondingly, f e t a l plasma (Hahn and S k a l a , 1975; Bobles-Valdes e t a l . , 1976 ; Seccombe et a l . # 1978) and l i v e r (Bobles-Valdes et a l . , 1976) c a r n i t i n e c o n c e n t r a t i o n i n the r a t are low compared to p o s t n a t a l v a l u e s . At p r e s e n t , there i s no evidence t o i n d i c a t e t h a t f e t a l s y n t h e s i s of c a r n i t i n e occurs i n mammals. F e t a l demands f o r c a r n i t i n e may i n s t e a d be met by s u p p l i e s d e r i v e d from maternal sources (Hahn and Skala, 1975; Hahn et a l . , 1977), even though p l a c e n t a l t r a n s f e r of c a r n i t i n e i s l i m i t e d (Hahn and s k a l a , 1975). I n both the sheep (Hahn et a l . , 1977) and r a t (Hahn and S k a l a , 1975) blood l e v e l s of c a r n i t i n e i n the f e t u s are low as compared with the maternal b l o o d l e v e l s . . Apparently t h i s r e s t r i c t i o n i s due i n p a r t to r e t e n t i o n of c a r n i t i n e by the p l a c e n t a (Hahn and S k a l a , 1975).. In both s p e c i e s , t h e r e appears to be a slow exchange of c a r n i t i n e between mother and f e t u s . The rapid i n t r a v e n o u s i n j e c t i o n of L - c a r n i t i n e i n t o the pregnant ewe l e d t o a t r a n s i e n t r i s e i n the maternal blood but had no e f f e c t on t h e f e t a l blood l e v e l s (Hahn e t a l . , 1977). Twenty-f o u r hours a f t e r the i n j e c t i o n s of l a b e l l e d c a r n i t i n e i n t o mother r a t s (Hahn and Skala 1975), r a d i o a c t i v e c a r n i t i n e was found i n f e t a l l i v e r and brown adipose t i s s u e , although only i n s m a l l amounts.. Since the source of f e t a l c a r n i t i n e appears to be mainly maternal, a c a r n i t i n e d e f i c i e n c y i n the mother may r e s u l t i n a c a r n i t i n e d e f i c i e n c y i n the o f f s p r i n g . 15 P o s t n a t a l At b i r t h , t h e r e i s a sudden change i n a v a i l a b i l i t y of energy y i e l d i n g substrates._ Maternal blood glucose i s r e p l a c e d by a high f a t d i e t of milk (Cox and Mueller, 1937; Luckey et a l . , 1954) of which 69.5% of the i n g e s t e d c a l o r i e s a r e f a t (Cox and M u e l l e r , 1937).. The r e l e a s e o f energy from d i e t a r y l i p i d , r e q u i r e s o x i d a t i o n of f a t t y a c i d s i n the mitochondria. An adequate amount of c a r n i t i n e i s t h e r e f o r e necessary f o r the formation of a c y l c a r n i t i n e s which are then a b l e t o c r o s s the m i t o c h o n d r i a l memfcraie ( F r i t z , 1963)*. Increased r a t e s of f a t t y a c i d u t i l i z a t i o n during the s u c k l i n g p e r i o d are i n d i c a t e d by r a i s e d plasma c o n c e n t r a t i o n s of ketone bodies (Drahota e t a l . , 1964 ; Icckwood and B a i l e y , 1971; Page e t a l . , 1971; F e r r e et a l . , 1S78). In v i t r o s t u d i e s have shown more p r e c i s e l y t h a t an i n c r e a s e d c a p a c i t y f o r f a t t y a c i d degradation c o i n c i d e s with the i n t a k e s of the h i g h - f a t d i e t of s u c k l i n g r a t s . . Oxidation of 1 * C - p a l m i t a t e t o **C02 by l i v e r m i t o c h o n d r i a l p r e p a r a t i o n s and l i v e r s l i c e s i n c r e a s e s r a p i d l y a f t e r b i r t h so that the maximum r a t e o f o x i d a t i o n i s achieved a t 2 days of agee The high l e v e l i s maintained u n t i l weaning (21 days of a g e ) , a f t e r which time the o x i d a t i o n r a t e f a l l s g r a d u a l l y t o a d u l t values ( T a y l o r e t a l . , 1967; Augenfeld and F r i t z , 1970; Lockwood and B a i l e y , 1970). A f t e r b i r t h the r a p i d i n c r e a s e i n p a l m i t a t e o x i d a t i o n i s accompanied by a s i m i l i a r r a p i d i n c r e a s e i n the a c t i v i t y o f c a r n i t i n e p a l m i t y l t r a n s f e r a s e . . The a c t i v i t y of t h i s enzyme i n c r e a s e s s h a r p l y and has i t s hi g h e s t a c t i v i t y d u r i n g the 16 s u c k l i n g p e r i o d ((Augenfeld and F r i t z , 1970; Lockwood and B a i l e y , 1970). L i k e w i s e , c a r n i t i n e c o n c e n t r a t i o n i n the plasma (Eobles-Valdes e t a l - , 1976; Borum, 1978; Seccombe et a l . , 1978) and l i v e r (Robles-Valdes e t a l . , 1976; Borum, 1978) i n c r e a s e r a p i d l y f o l l o w i n g b i r t h , while the pup i s consuming the milk d i e t , and decrease when the pup i s weaned to a high carbohydrate d i e t . P r i o r t o weaning there are no sex d i f f e r e n c e s i n plasma or l i v e r c a r n i t i n e l e v e l s {Eorum, 1978)., The primary source of c a r n i t i n e i n neonatal t i s s u e , at l e a s t 24 hours post partum, i s the milk from the mother r a t (Ferre et a l . , 1978). Milk c a r n i t i n e content i s very high i n i t i a l l y and f a l l s as nursing c o n t i n u e s (Robles-Valdes et a l . , 1976)* Robles-Valdes e t a l . , (1976) rep o r t e d l i v e r c a r n i t i n e c o n c e n t r a t i o n s i n mother r a t s d u r i n g l a t e g e s t a t i o n to be high but decreased to normal at approximately the seventh day of l a c t a t i o n accompanying the decrease i n milk c a r n i t i n e l e v e l s . Experiments i n which the f a t e of **C-butyrobetaine, the immediate p r e c u r s o r of c a r n i t i n e , was followed a f t e r i n j e c t i o n i n t o n u r s i n g r a t s i n d i c a t e d movement of c a r n i t i n e from maternal l i v e r t o maternal plasma to maternal milk and f i n a l l y . i n t o n eonatal heart and l i v e r w i t h i n 24 hours a f t e r i t s i n j e c t i o n . C y c l i n g of c a r n i t i n e between mother and i n f a n t r a t has been documented by Hahn and Skala (1975),. Since i n f a n t r a t s are unable t o u r i n a t e on t h e i r own, the mother animal empties t h e i r bladder by l i c k i n g the p e r i n e a l r e g i o n and swallowing the u r i n e (Capek and J e l i n e k * 1956) . Thus substances i n j e c t e d i n t o the s u c k l i n g r a t can be passed back t o the mother and e v e n t u a l l y be s e c r e t e d i n her milk*. When i n f a n t r a t s were i n j e c t e d with *»C-17 c a r n i t i n e , the l a b e l appeared i n the bleed and other organs of the mother r a t and a l s o i n i t s milk thereby r e t u r n i n g t c the pups o f t h e l i t t e r (Hahn and S k a l a , 1975). Thus a ma t e r n a l l y induced d e f i c i e n c y of c a r n i t i n e may adversely a f f e c t the c a r n i t i n e s t a t u s of s u c k l i n g o f f s p r i n g , s i n c e the primary source of c a r n i t i n e f o r the mother-infant c y c l e , a t l e a s t d u r i n g the i n i t i a l s tages of l a c t a t i o n * i s maternal* RELATIONSHIP OF CARNITINE TO KETOSIS Increase d ketogenesis i n d i c a t e s enhanced f a t t y a c i d o x i d a t i o n . . Whenever the a v a i l a b i l i t y of carbohydrate i s inadequate more l i p i d i s o x i d i z e d and k e t o s i s develops. A high r a t e o f f r e e f a t t y a c i d uptake and o x i d a t i o n by the l i v e r i s the most important f a c t o r u n d e r l y i n g ketogenesis. S t a t e s such as s t a r v a t i o n and diabetes are accompanied ty an i n c r e a s e d h e p a t i c f a t t y a c i d o x i d a t i o n and k e t o g e n e s i s ( F r i t z , 1967; McGarry and F o s t e r , 1971; BcGarry et a l . , 1973; McGarry and F o s t e r , 1974; McGarry et a l . , 1975), processes which r e q u i r e the c a r n i t i n e a c y l t r a n s f e r a s e system ( F r i t z and Yue, 1963). McGarry e t a l i _ (1975) suggested, on t h e o r e t i c a l grounds, that the B - o x i d a t i o n of f a t t y a c i d s might be governed, i f o n l y i n p art, by the c o n c e n t r a t i o n of c a r n i t i n e . They based t h i s n o t i o n on two c o n s i d e r a t i o n s . . F i r s t , c a r n i t i n e i s necessary f o r the i n i t i a l s tep i n the o x i d a t i o n of long c h a i n f a t t y a c i d s , s p e c i f i c a l l y the c a r n i t i n e a c y l t r a n s f e r a s e r e a c t i o n (Hoppel and Tomac, 1 972).. Second, s t u d i e s conducted by F r i t z (1955) e s t a b l i s h e d t h a t c a r n i t i n e was capable of s t i m u l a t i n g f a t t y a c i d 18 o x i d a t i o n when i n t r o d u c e d i n t o r a t l i v e r homogenates*. Furthermore, McGarry e t a l i (1975) showed t h a t k e t o t i c s t a t e s , such as f a s t i n g or a l l o x a n d i a b e t e s , i n the weaned r a t were accompanied by an i n c r e a s e d c a r n i t i n e content i n the l i v e r * The k e t o g e n i c c a p a c i t y of h e p a t i c t i s s u e c o r r e l a t e d with c a r n i t i n e c o n c e n t r a t i o n s . . In a d d i t i o n , c a r n i t i n e alone had the c a p a c i t y t o d i r e c t l y a c t i v a t e k e t o g e n e s i s i n i s o l a t e d perfused l i v e r from no n k e t o t i c (fed) r a t s . C a r n i t i n e t i s s u e l e v e l s and p r o d u c t i o n of ketone bodies during p e r i n a t a l development have a l s o been s t u d i e d . Ketone body formation i s very low i n f e t a l t i s s u e s (Drahota et a l . , 1964; Augenfeld and F r i t z , 197C; Robles-Valdes et a l . , 1976).. F e t a l l i v e r (Robles-Valdes e t a l . , 1976) and plasma (Robles-Valdes et a l . , 1976; Borum, 1978) c a r n i t i n e c o n c e n t r a t i o n s are a l s o low d u r i n g the p r e n a t a l p e r i o d . In c o n t r a s t , during the s u c k l i n g p e r i o d the plasma l e v e l s of ketone bodies are high (Drahota et a l . , 1964; Lockwood and B a i l e y , 1971; Page et a l . , 1971) and the neonate may be d e s c r i b e d as being i n a p h y s i o l o g i c a l s t a t e of k e t o s i s as a d i r e c t r e s u l t of high r a t e s of u t i l i z a t i o n and o x i d a t i o n of f a t t y a c i d s . The c o n c e n t r a t i o n of ketone bodies i n the blood i n c r e a s e s r a p i d l y a f t e r b i r t h (Drahota e t a l . , 1964; Lockwood and B a i l e y , 1971; Page, 1971; Robles-Valdes e t a l . , 1976 ; F e r r e et a l . , 1978), remains high during t h e s u c k l i n g p e r i o d and f a l l s a f t e r weaning (Lockwood and B a i l e y , 1971; Eage, 1971; Robles-Valdes et a l . , 1976). An almost i d e n t i c a l p r o f i l e has been observed f o r l i v e r c a r n i t i n e c o n c e n t r a t i o n s i n the neonate (Robles-Valdes e t a l . , 1976; F e r r e e t a l . , 1978; Borum, 1978).. 1 9 These data i n d i c a t e a r e l a t i o n s h i p between h e p a t i c l e v e l s of c a r n i t i n e and the r a t e of k e t o g e n e s i s . When f a t t y a c i d o x i d a t i o n i s s u b s t a n t i a l l y i n c r e a s e d , t i s s u e c a r n i t i n e l e v e l s are higher than normal*. MATERNAL DIETARY EEFICIENCIES The importance of a n u t r i t i o n a l l y sound d i e t d u r i n g g e s t a t i o n and l a c t a t i o n f o r the maintenance of pregnancy and the he a l t h of the o f f s p r i n g i s w e l l documented (Barry, 1 9 2 0 ; Chow and Lee, 1 9 6 4 ; Niiyama e t a l . , 1 9 7 0 , 1 9 7 3 ) » . I n t h i s p a r t i c u l a r review, the e f f e c t s of a maternal l y s i n e d e f i c i e n c y and/or maternal d i e t a r y food r e s t r i c t i o n on dams and t h e i r o f f s p r i n g , are d i s c u s s e d . Pregnancy The maintenance of pregnancy i n r a t s f e d p u r i f i e d d i e t s devoid of a s i n g l e amino a c i d was examined by Niiyama et a l . ( 1 9 7 0 * 1 9 7 3 ) . . Rats f e d a l y s i n e - f r e e d i e t showed food i n t a k e comparable to c o n t r o l animals and maintained pregnancy. L i t t e r s i z e (Niiyama e t a l i , 1 9 7 0 , 1 9 7 3 ) was not a f f e c t e d * . However, f e t a l weight (Niiyama et a l * , 1 9 7 3 ) was s i g n i f i c a n t l y lower than f o r c o n t r o l animals. Niiyama suggested that animals fed l y s i n e d e f i c i e n t d i e t s may have maintained pregnancy because of the l y s i n e s p a r i n g mechanism shown t o e x i s t i n non-pregnant r a t s (Yamashita and Ashida, 1 9 6 9 ; C a n f i e l d and C h y t i l , 1 9 7 8 ) . The ba s i s f o r t h e extensive p r e s e r v a t i o n of l y s i n e i n r a t s s u b j e c t e d 20 to a l y s i n e d e f i c i e n c y , appears to be the a d a p t i v e d e c l i n e of the f i r s t enzyme i n the pathway o f l y s i n e degradation* l y s i n e -k e t o g l u t a r a t e reductase (Chu and Hegsted, 1976),.. S t a p l e t o n and H i l l (1972, 1 98C) r e p o r t e d t h a t a d i e t c o n t a i n i n g 0.42% l y s i n e , f e d t o r a t s during g e s t a t i o n , d i d not have a s i g n i f i c a n t e f f e c t on food i n t a k e or l i t t e r s i z e when compared to a 1.12% l y s i n e d i e t * . The average b i r t h weight of pups i n t h e l o w - l y s i n e group was s i g n i f i c a n t l y lower than i n the c o n t r o l group.. Plasma amino a c i d a n a l y s i s r e v e a l e d a s i g n i f i c a n t l y lower plasma l y s i n e c o n c e n t r a t i o n on day 7 and day 21 of pregnancy i n dams f e d the l o w - l y s i n e d i e t than i n those consuming the h i g h - l y s i n e d i e t . . T h i s r e s u l t e d i n a reduced amount of l y s i n e a v a i l a b l e t o the o f f s p r i n g d u r i n g the l a s t week of pregnancy, a f a c t o r p o s s i b l y c o n t r i b u t i n g t o the lower b i r t h weight of the l y s i n e d e f i c i e n t group,. R e s t r i c t i n g food i n t a k e d u r i n g g e s t a t i o n has a l s o been r e p o r t e d to have a s i g n i f i c a n t e f f e c t on r e p r o d u c t i o n . Chow and Lee (1964) reduced the d i e t a r y i n t a k e of r a t s by 25% and observed a s i g n i f i c a n t r e d u c t i o n i n f e t a l weight. A decreased b i r t h weight has a l s o been r e p o r t e d i n the o f f s p r i n g of r a t s consuming a d i e t r e s t r i c t e d i n t o t a l food i n t a k e d u r i n g g e s t a t i o n (Barry, 192C; Niiyama et a l . , 1973). Thus both the g u a n t i t y and q u a l i t y of the maternal d i e t a f f e c t e d r e p r o d u c t i v e performance.. L a c t a t ion Imposing a d i e t a r y d e f i c i e n c y during l a c t a t i o n a f f e c t s not 21 only food i n t a k e of the dams but a l s o weight g a i n of the o f f s p r i n g (Stapleton and H i l l , 1972, 1980; Jansen and Chase, 1976; Chow and Lee, 1964). S t a p l e t o n and H i l l (1972, 1980) fed a wheat g l u t e n based d i e t , d e f i c i e n t i n l y s i n e * t o r a t s d u r i n g pregnancy and l a c t a t i o n . Food i n t a k e of l y s i n e d e f i c i e n t dams during l a c t a t i o n was s i g n i f i c a n t l y reduced, as was the average weaning weight of t h e i r o f f s p r i n g . Milk p r o d u c t i o n and i t s p r o t e i n c o n t e n t were a l s o s i g n i f i c a n t l y reduced i n dams f e d the l y s i n e d e f i c i e n t d i e t . T h i s e f f e c t on a i l k probably c o n t r i b u t e d to the lower weight gain of s u c k l i n g r a t s i n the l y s i n e d e f i c i e n t group. C r n i c and Chase (1978) reported t h a t dams f e d a low p r o t e i n d i e t during l a c t a t i o n showed l i t t l e engorgement of the mammary t i s s u e a i d y i e l d e d milk l e s s r e a d i l y than dams fed e i t h e r a c a l o r i c r e s t r i c t e d or ad l i b i t u j d i e t . Jansen and Chase (1976) r e p o r t e d a s i g n i f i c a n t r e d u c t i o n i n food intake during pregnancy and l a c t a t i o n i n r a t s fed a b a s a l bread d i e t when compared with dams consuming a l y s i n e f o r t i f i e d bread d i e t . . Lysine f o r t i f i c a t i o n of tread decreased maternal weight l o s s during l a c t a t i o n * and s i g n i f i c a n t l y i n c r e a s e d weaning weight of the o f f s p r i n g . When Chow and Lee (1964) imposed a t o t a l food r e s t r i c t i o n on dams during both pregnancy and l a c t a t i o n * growth s t u n t i n g r e s u l t e d i n the o f f s p r i n g . Thus some of the major e f f e c t s of a maternal l y s i n e d e f i c i e n c y on the o f f s p r i n g a r e decreased b i r t h weight and decreased weaning weights.. A maternal l y s i n e d e f i c i e n c y a l s o decreases t h e food i n t a k e of dams, at l e a s t during • l a c t a t i o n , and depresses milk flow. . 22 hl§.UJ DEFICIENCY AND TISSUE CARNITINE LEVELS A l a r g e p r o p o r t i o n of the world's p o p u l a t i o n consumes c e r e a l based d i e t s which are l i k e l y t o he low i n c a r n i t i n e and l i m i t i n g i n i t s amino a c i d p r e c u r s o r s l y s i n e and methionine. Consumption of d i e t s c o n s i s t i n g mainly of p l a n t foods may r e s u l t i n a c a r n i t i n e d e f i c i e n c y i n humans ( L a t i f a and Bamji, 1977). Because of c a r n i t i n e ' s r o l e i n f a t t y a c i d o x i d a t i o n an impairment i n f a t u t i l i z a t i o n may r e s u l t from such a s t a t e of c a r n i t i n e d e f i c i e n c y . . C a r n i t i n e d e f i c i e n c i e s have been a s s o c i a t e d with f a t accumulation i n the t i s s u e of the r a t ( T a n p h a i c h i t r e t a l . , 1976) , guinea p i g ( W i t t l e s and B r e s s l e r , 1964), and human (Isaacs et a l . , 1976).. T a n p h a i c h i t r and B r o g u i s t (1973) measured the e f f e c t o f f e e d i n g a 20% wheat g l u t e n d i e t , l i m i t i n g i n l y s i n e and c o n t a i n i n g n e g l i g i b l e c a r n i t i n e , on t i s s u e c a r n i t i n e l e v e l s i n male r a t s . . When compared with c o n t r o l animals r e c e i v i n g a 0.8% l y s i n e supplemented d i e t , c a r n i t i n e l e v e l s i n h e a r t and s k e l e t a l muscle were g e n e r a l l y 33% lower i n the l y s i n e d e f i c i e n t group. The c a r n i t i n e l e v e l of the l i v e r however, was s i g n i f i c a n t l y higher i n the unsupplemented group than i n the l y s i n e supplemented group.. The authors suggest t h a t t h i s l a t t e r f i n d i n g may r e f l e c t the need f o r i n c r e a s e d h e p a t i c s y n t h e s i s of c a r n i t i n e under the s t r i n g e n t n u t r i t i o n a l c o n d i t i o n s employed, i . e . , a l y s i n e d e f i c i e n c y and s i g n i f i c a n t l y reduced food i n t a k e . A t h i r d group of animals i n t h i s study fed a l a b o r a t o r y chow d i e t , which contained a higher l e v e l of c a r n i t i n e , were re p o r t e d to have h i g h e r l e v e l s of t i s s u e c a r n i t i n e compared to animals 23 given the c a r n i t i n e - f r e e , l y s i n e supplemented d i e t . The r e s u l t s suggest t h a t t i s s u e c a r n i t i n e l e v e l s are higher when d i e t a r y c a r n i t i n e i s a v a i l a b l e . The e f f e c t of c e r e a l based d i e t s , d e f i c i e n t i n l y s i n e , on t i s s u e c a r n i t i n e l e v e l s and f a t t y a c i d o x i d a t i o n has a l s o been observed ( T a n p h a i c h i t r et a l . , 1976; Khan and Bamji, 1979). T a n p h a i c h i t r et a l . . (1976) f e d male weanling r a t s a 72% r i c e d i e t c o n t a i n i n g no d e t e c t a b l e c a r n i t i n e and l i m i t i n g i n threonine and l y s i n e * . They observed l i v e r l i p i d accumulation, a major p a r t o f which was t r i g l y c e r i d e s . Eeduction i n l i v e r f a t content was p a r t l y achieved by f e e d i n g c a r n i t i n e . However, complete r e l i e f of the c o n d i t i o n was not a t t a i n e d u n t i l the d i e t was supplemented with t h r e o n i n e and l y s i n e * T h e r e f o r e l i v e r l i p i d a ccumulaticn i n these r a t s may occur as a r e s u l t o f i n s u f f i c i e n t l i p o p r o t e i n t r a n s p o r t e r s f o r removing t r i g l y c e r i d e s from the l i v e r , and of course, a carni±ine d e f i c i e n c y r e s u l t i n g i n impaired f a t t y a c i d o x i d a t i o n . . Khan and Bamji (1979) r e p o r t e d marked e l e v a t i o n i n t r i g l y c e r i d e and t o t a l l i p i d content of l i v e r , h e a r t , and s k e l e t a l muscle i n r a t s fed a c a r n i t i n e f r e e and l y s i n e depleted d i e t (5% wheat p r o t e i n ) . O x i d a t i o n of f a t t y a c i d s by heart homogenate was impaired i n animals fed the wheat g l u t e n d i e t ad l i b i t u m , as w e l l as i n t h e i r p a i r - f e d c o n t r o l s , . Supplementation of wheat g l u t e n with 0.2% c a r n i t i n e produced a s i g n i f i c a n t i n c r e a s e i n plasma, muscle, and l i v e r c a r n i t i n e l e v e l s , r e s t o r e d f a t t y a c i d o x i d a t i o n , and reduced th e t r i g l y c e r i d e l e v e l of t i s s u e * Supplementation of the wheat d i e t with l y s i n e and threonine a t 0.2% l e v e l improved muscle c a r n i t i n e l e v e l s and 24 p a l m i t a t e o x i d a t i o n and d i m i n i s h e d the t r i g l y c e r i d e l e v e l s o f t i s s u e s * These data suggest the e s s e n t i a l i t y of c a r n i t i n e as a d i e t a r y n u t r i e n t , e i t h e r through preformed c a r n i t i n e or i t s amino p r e c u r s o r s l y s i n e and methionine. Sex d i f f e r e n c e s i n t i s s u e c a r n i t i n e l e v e l s have been observed i n both r a t s (Borum and B r oguist, 1977; Borum, 1978) and humans (Cederblad and L i n d s t e d t , 1971; Maebashi et a l . , 1976). Female r a t s have only about h a l f as much c a r n i t i n e i n the plasma as male r a t s , and c a r n i t i n e content c f l i v e r and heart i n males i s s l i g h t l y h i g h e r than i n females (Borum and B r o g u i s t , 1977 ; Borum, 1979). Eorum and B r o g u i s t (1977) i n v e s t i g a t e d the e f f e c t s of a c a r n i t i n e d e f i c i e n c y i n male and female r a t s f e d a 20% wheat g l u t e n d i e t * . When compared to c o n t r o l females consuming a l y s i n e supplemented d i e t , the l y s i n e d e f i c i e n t female r a t s had h i g h e r t i s s u e c a r n i t i n e l e v e l s i n a l l t h r e e t i s s u e s analyzed, i . e . , plasma, l i v e r , and h e a r t * Only the plasma c a r n i t i n e l e v e l s however, were s i g n i f i c a n t l y higher. Male r a t s s u b j e c t e d t o the l y s i n e d e f i c i e n t d i e t had moderately decreased c a r n i t i n e l e v e l s i n the plasma and h e a r t , but e l e v a t e d c a r n i t i n e l e v e l s i n l i v e r . . The mechanism u n d e r l y i n g the sex d i f f e r e n c e s i n plasma c a r n i t i n e l e v e l s of r a t s , and t h e i r opposite response to a d i e t a r y l y s i n e d e f i c i e n c y , i s not w e l l understood. 2 5 CHAPTER I I I METHODS AND MATERIALS ANIMALS White r a t s of Wistar s t r a i n (purchased from Woodlyn Breeding L a b o r a t o r i e s , Guelph) were used i n a l l experiments. Female r a t s , weighing approximately 115 g, were housed i n p l a s t i c b r e e d i n g cages (3 per cage) and fed Purina Rat Chow and water ad l i b i t u m u n t i l mated.. Male breeding r a t s weighing no l e s s than 2 5 0 g were f e d a s i m i l a r d i e t . . Females averaging 2 2 5 g were mated by p l a c i n g 1 male r a t with 3 females i n a breeding cage. overnight*„ From day one of g e s t a t i o n , the day sperm was found i n a v a g i n a l smear, dams were placed on t h e i r r e s p e c t i v e d i e t s * Once mated the animals were i n d i v i d u a l l y housed i n s t a i n l e s s s t e e l cages with r a i s e d wire f l o o r s under c o n d i t i o n s of c o n t r o l l e d l i g h t ( l i g h t i n g 0 6 0 C - 1 2 0 0 hours) and temperature ( 2 4 ° C ) . The cages were equipped with e x t e r n a l water b o t t l e s and i n t e r n a l cup f e e d e r s a l l o w i n g ad l i b i t u m i n t a k e i n a l l experiments u n l e s s s p e c i f i e d otherwise. Pregnant dams were t r a n s f e r r e d at 18 days of g e s t a t i o n t o l a r g e r p l a s t i c breeding cages with wood shavings as bedding. D a i l y food i n t a k e and weekly weight changes during pregnancy and l a c t a t i o n were recorded. . 2 6 DIETS A d i e t d e f i c i e n t i n l y s i n e (the l c w - l y s i n e d i e t ) was based on wheat g l u t e n as the source of p r o t e i n . The composition of the d i e t i s shown i n Tab l e I . . The amine a c i d composition o f the d i e t was c a l c u l a t e d from amino a c i d composition of wheat g l u t e n a c c o r d i n g t o FAO N u t r i t i o n a l S t u d i e s (1*70).. Those amino a c i d s that did not meet reguirements f o r the pregnant and l a c t a t i n g r a t were supplementsd to the d i e t (NEC, 1972). Table I I i n c l u d e s the d e t a i l e d c a l c u l a t i o n s f o r the e s s e n t i a l amino a c i d s . Because the mineral mixture a l s o d i d not meet the n u t r i e n t requirements f o r the r a t (NEC, 1972) , z i n c and potassium were added to the r a t i o n (Table I ) . . The c o n t r o l d i e t , adequate i n l y s i n e (the h i g h - l y s i n e diet) was prepared by supplementing the basal d i e t with 1. 036 I - L y s i n e HCl, e q u i v a l e n t to 0. 836 L - L y s i n e . Both d i e t s c o n t a i n e d 18% p r o t e i n and 20% f a t . B a s a l d i e t s c o n t a i n i n g 20% wheat g l u t e n as the s o l e p r o t e i n source have been r e p o r t e d ( T a n p h a i c h i t r and B r o g u i s t , 1973) t o c o n t a i n l e s s than 0.1 u g c a r n i t i n e per gram of d i e t as determined by c a r n i t i n e a c e t y l t r a n s f e r a s e assay (Marguis and F r i t z , 1964). Thus, the l o w - l y s i n e d i e t contained n e g l i g i b l e amounts of c a r n i t i n e and was adequate i n a l l n u t r i e n t s except l y s i n e . ANIMAL EXPERIMENTS The t h r e e animal experiments are diagrammatically d e p i c t e d i n F i g u r e 3. Table 1. Composition of Low-Lysine Diet Ingredient Per cent of Diet 2 Wheat Gluten 20.00 3 Vitamin Mixture 2.20 4 M i n e r a l M i x t u r e 5.00 Corn o i l ^ 20.00 C e l l u l o s e 5.00 Zinc Carbonate 7 0.006 L - H i s t i d i n e HC1 7 0.32 DL - Me t h i o n i n e 7 0.40 L - Threonine^ 0.15 L - Tryptophan 7 0.06 L - A r g i n i n e HCL 7 0.18 Corn Starch 46.684 1 0.5% K„P0. was added to the d i e t f o r the l a c t a t i o n s t u d i e s only. 2 4 2 Wheat Gluten purchased from North American S c i e n t i f i c Chemicals, Vancouver, B r i t i s h Columbia. 3 ICN Vitamin Diet F o r t i f i c a t i o n Mixture purchased from N u t r i t i o n a l Biochemicals, Cleveland, Ohio. 4 Bernhart T o m a r e l l i M i n e r a l Mixture purchased from N u t r i t i o n a l Biochemicals, Cleveland, Ohio. 5 Mazola Corn O i l . 6 A l f a c e l l N o n - n u t r i t i v e bulk, ICN, purchased from N u t r i t i o n a l Biochemicals, Cleveland, Ohio. 7 ICN, purchased from N u t r i t i o n a l Biochemicals, Cleveland, Ohio. 28 Table I I . Ca l c u l a t e d E s s e n t i a l Amino A c i d Composition of D i e t s Amino Ac i d % Contributed by Wheat Gluten % Added as Supplement T o t a l g/lOOg Diet A s p a r t i c A c i d 0.54 0.54 Threonine 0.41 0.15 0.56 Glutamic A c i d 5.99 5.99 Methionine 0.26 0.20 0.46 I s o l e u c i n e 0.67 0.67 Leucine 1.11 1.11 Tyrosine 0.59 0.59 Phenylalanine 0.83 0.83 Lysine 0.27 0.80 1 1.07 H i s t i d i n e 0.35 0.25 0.60 A r g i n i n e 0.69 0.14 0.83 P r o l i n e 2.12 2.12 1 D i e t s w i t h 0.8% l y s i n e were r e f e r r e d to as the h i g h - l y s i n e d i e t . The l o w - l y s i n e d i e t s r e c e i v e d no supplement of l y s i n e . 29 F i g u r e 3 Diagramatic r e p r e s e n t a t i o n of experiments I , I I , and I I I EXPERIMENT I LOW LYSINE N=6 HIGH LYSINE N=6 DAY 21 PREGNANCY - TISSUES (DAMS, FETUSES) EXPERIMENT II LOW LYSINE N=6 HIGH LYSINE RESTRICTED N=6 HIGH LYSINE N=6 DAY 15 LACTATION - TISSUES (DAMS, PUPS) EXPERIMENT I I I 30 LOW LYSINE N=18 HIGH LYSINE RESTRICTED N=18 HIGH LYSINE N=18 DAY 2 LACTATION - 6/GROUP) DAY 8 LACTATION - 6/GROUP) MILK DAY 15LACTATION - 6/GROUP) 31 Experiment I E f f e c t o f a maternal l y s i n e d e f i c i e n c y durincj pregnancy on t i s s u e c a r n i t i n e l e v e l s Two groups of r a t s , c o n s i s t i n g of s i x pregnant animals each, were fed eithec the l o w - l y s i n e or h i g h - l y s i n e d i e t s . On day 21 of pregnancy, dams were a n a e s t h e t i z e d with ether and a sample of blood was taken by heart puncture using a h e p a r i n i z e d s y r i n g e . . The dam's heart and l i v e r then were removed and t i s s u e weights recorded*' F e t u s e s with attached placenta were removed by c a e s a r i a n s e c t i o n . . .Resorption s i t e s were noted. Blood samples were obtained from d e c a p i t a t e d f e t u s e s using h e p a r i n i z e d c a p i l l a r y tubes. Plasma was sepa r a t e d by c e n t r i f u g i n g the blood samples of the dams and f e t u s e s f o r 10 and 5 minutes r e s p e c t i v e l y . F e t a l weights were net recorded i n t h i s experiment, because the s e p a r a t i o n of the p l a c e n t a from the f e t u s would have r e s u l t e d i n a l o s s of blood, i n t e r f e r i n g with c o l l e c t i o n of an adequate amount of blood f o r a n a l y s i s . Heart and l i v e r were removed from a l l f e t u s e s and t i s s u e weights recorded* T i s s u e samples from two fe t u s e s w i t h i n a l i t t e r were combined t o provide a l a r g e enough sample f o r a n a l y s i s . A l l t i s s u e s were immediately f r o z e n and s t o r e d at -20°C. . Experiment I I E f f e c t of a maternal l y s i n e d e f i c i e n c y d u r i n g pregnancy a n d . l a c t a t i o n on t i s s u e c a r n i t i n e l e v e l s A f t e r examining the r e s u l t s cf experiment I, i t was found t h a t animals i n the l o w - l y s i n e group consumed s i g n i f i c a n t l y l e s s food than those i n the h i g h - l y s i n e group.. T h e r e f o r e a t h i r d 32 group of animals, the h i g h - l y s i n e r e s t r i c t e d group, was added to the remaining experiments to determine the e f f e c t s of decreased food i n t a k e . There were thus 3 groups of pregnant dams, 6 animals per group. Two of the groups were f e d e i t h e r the lcw-l y s i n e or h i g h - l y s i n e d i e t ad l i b i t um. . The t h i r d group, the h i g h - l y s i n e r e s - t r i c t e d animals, were p a i r - f e d the h i g h - l y s i n e d i e t t o the l o w - l y s i n e group on a body weight b a s i s . Commencing on day 1 of l a c t a t i o n , a l l d i e t s were supplemented with ZnS04. Dams were fed t h e i r r e s p e c t i v e d i e t s u n t i l t i s s u e samples were obtained. Pregnant dams were checked d a i l y at twelve neon f o r the presence of newborns. Only those mothers with l i t t e r s c o n t a i n i n g e i g h t cr more young and e x p e r i e n c i n g l e s s than two deaths per l i t t e r were used i n t h i s experiment. . T h i s r e s t r i c t i o n on l i t t e r s i z e was imposed due t c sample s i z e requirements f o r s t a t i s t i c a l a n a l y s i s . At day 1 of l a c t a t i o n ( p a r t u r i t i o n completed before twelve nocn) a l l pups were sexed, weighed and l i t t e r s i z e c u l l e d t o e i g h t pups. P r e f e r e n c e was given t o male o f f s p r i n g . . The e i g h t pups were numbered by i n j e c t i n g I n d i a Ink under the s k i n s u r f a c e near the appendages* Body weights of pups were r e c o r d e d on days 1, 5, 10, and 15 post partum. On day 15 of l a c t a t i o n dams were an a e s t h e t i z e d with ether and a sample cf blood was taken by h e a r t puncture using a h e p a r i n i z e d s y r i n g e . . Heart and l i v e r were removed and t i s s u e weights r e c o r d e d . Pups were s a c r i f i c e d on day 15 of l a c t a t i o n by d e c a p i t a t i o n . H e p a r i n i z e d c a p i l l a r y tubes were used to c o l l e c t t h e blood. The heart and l i v e r of each pup were removed and t i s s u e weights recorded. The plasma was separated by 33 c e n t r i f u g i n g blood samples of the dams and t h e i r pups f o r 10 and 5 minutes r e s p e c t i v e l y . A l l t i s s u e samples were immediately f r o z e n and s t o r e d at -20°C.. Experiment I I I E f f e c t of a maternal l y s i n e d e f i c i e n c y on milk c a r n i t i n e l e y e I s In t h i s study, t h e r e were t h r e e groups of pregnant dams, 18 animals per group. . Two of the groups were f e d e i t h e r the lcw-l y s i n e or h i g h - l y s i n e d i e t . The t h i r d group, the h i g h - l y s i n e r e s t r i c t e d animals, were p a i r - f e d the h i g h - l y s i n e d i e t t o the l o w - l y s i n e group on a body weight b a s i s . Commencing on day 1 o f l a c t a t i o n , a l l d i e t s were supplemented with ZnSOU. Dams were fed t h e i r r e s p e c t i v e d i e t s u n t i l milk samples were o b t a i n e d . Pregnant dams were checked d a i l y a t twelve noon f o r the presence of newborns._ Since pups were r e g u i r e d f o r s t i m u l a t i n g milk flow r a t h e r than f o r t i s s u e samples (as was the case i n experiment II) dams with l e s s than e i g h t young per l i t t e r , or e x p e r i e n c i n g more than two deaths per l i t t e r , were permitted f o r use i n experiment I I I * In those cases where l i t t e r s i z e was l e s s than e i g h t , the young of f o s t e r dams (dams bred and maintained on one of the t h r e e d i e t a r y treatments) were used t o maintain l i t t e r s i z e a t e i g h t throughout the l a c t a t i o n p e r i o d . For those l i t t e r s c o n t a i n i n g more than e i g h t ycung, l i t t e r s i z e was c u l l e d to e i g h t on day 1 of l a c t a t i o n ( p a r t u r i t i o n completed before twelve noon).^ Preference was given t o male o f f s p r i n g . . The e i g h t pups were numbered by i n j e c t i n g I n d i a Ink under the s k i n s u r f a c e near the appendages.„ Body weights of pups, nursed by 34 those dams milked on day 15 of l a c t a t i o n , were recorded on days 1, 5, 10, and 15 post partum.. Body weights of pups, nursed by those dams milked on day 8 of l a c t a t i o n , were recorded on days 1, 5, and 8 post partum.. Mil k samples were c o l l e c t e d from 6 dams per group on the second, e i g h t h and f i f t e nth days o f l a c t a t i o n . L i t t e r s were removed from t h e i r mothers 10-12 hours (from approximately 2300 to 0900 hours) p r i o r to milk c o l l e c t i o n t o allow accumulation of s u f f i c i e n t milk f o r a n a l y s i s . . Dams were a n a e s t h e t i z e d with an i n t r a p e r i t o n e a l i n j e c t i o n of nembutol (45 m g/kg, Grosvenor et a l . , 1959)J. Each dam was then i n j e c t e d i n t r a p e r i t o n e a l l y , with 0.5 u n i t s o f ox y t o c i n t o f a c i l i t a t e milk flow (Grosvenor et a l . , 1959; Mena e t a l . , 1974). M i l k was manually expressed from the n i p p l e and c o l l e c t e d with a pasteur p i p e t t e i n t o 0.5 ml co n t a i n e r s ^ M i l k samples were immediately f r o z e n a t -20°C. . A l l milk samples c o l l e c t e d were analyzed f o r c a r n i t i n e content* 35 PLASMA f LI VEEj. AND MILK TOTAL CARNITINE ANALYSIS C a r n i t i n e ( t o t a l c a r n i t i n e ) e x t r a c t e d from plasma, l i v e r , and milk was assayed a c c o r d i n g t o the method of McGarry and F o s t e r 11976) as m o d i f i e d by P a r v i n and Pande (1977) and Seccombe e t a l . „ (1978) . . P r i n c i p l e o f Assay L - c a r n i t i n e was i n c u b a t e d with (1- 1*C)acety1-CoA of a known s p e c i f i c a c t i v i t y and c a r n i t i n e a c e t y l t r a n s f e r a s e . C a r n i t i n e was then transformed to the l a b e l l e d a c e t y l - L - c a r n i t i n e by the enzyme. a c e t y l t r a n s f e r a s e . > L - c a r n i t i n e (1 -!*C) acetyl-CoA (1-* *C) a c e t y l - L - c a c n i t i n e • CoASH The l a b e l l e d a c e t y l - L - c a r n i t i n e was separated from the unreacted (1- 1 *C) a c e t y l - C o A by an a c t i v a t e d c h a r c o a l s l u r r y . . A f t e r c e n t r i f u g a t i o n the i s o t o p e c o n t e n t cf the supernatant was determined. 36 P r e p a r a t i o n of plasma C a r n i t i n e E x t r a c t - Part A x S e c t i o n 2 (Seccombe e t a l . , 1978) 1) Reagents and t h e i r c o n c e n t r a t i o n s : Reagent C o n c e n t r a t i o n z i n c s u l f a t e 0.087 M barium hydroxide 0.083 M 2) S o l u t i o n s of z i n c s u l f a t e and barium hydroxide were t i t r a t e d a g a i n s t each other such that equal volumes of each r e s u l t e d i n a supernatant pH of 7*3. 3) C o n s t r i c t i o n p i p e t t e s were used t o measure 0.100 ml samples of plasma i n t o separate c o n i c a l p o l y s t y r e n e tubes (purchased from Amersham). A l l e x t r a c t i o n s were dene i n d u p l i c a t e and kept on i c e . . 4) 0. 400 ml of barium hydroxide was added to each tube and the contents mixed.. The tube was then covered and heated at 37°C for 1 hour to r e l e a s e bound c a r n i t i n e . 5) 0.400 ml of z i n c s u l f a t e was added t o each tube, the contents mixed and the tube cooled on i c e . . 6) The p r e c i p i t a t e was c e n t r i f u g e d a t 2000 RPMs f o r 20 minutes. The supernatant was removed and f r o z e n at -20°C u n t i l assayed for c a r n i t i n e . 37 Prepar at i o n of M i l k C a r n i t i n e E x t r a c t s - Pa r t Aj_ S e c t i o n 2 (Robles-Valdes et al.,1976; Seccombe et a l * , 1978) 1) Reagents and t h e i r c o n c e n t r a t i o n s : Reagent C o n c e n t r a t i o n z i n c s u l f a t e 0.087 M barium hydroxide C.083 M 2) S o l u t i o n s of z i n c s u l f a t e and barium hydroxide were t i t r a t e d a g a i n s t each other such that e q u a l volumes of each r e s u l t e d i n a supernatant pH of 7.3* 3) Milk samples were d i l u t e d with d i s t i l l e d water 1:1, 1:2, and 1:4 f o r days 15, 8 and 2 r e s p e c t i v e l y . fill samples were done i n d u p l i c a t e and kept on i c e . 4) Samples were covered and heated at 60°C f o r 30 minutes and then mixed f o r 30 seconds t o d i s p e r s e any p a r t i c u l a t e m a t e r i a l . 5) 0*400 ml of barium hydroxide was added t o each tube and the contents mixed* The tube was then covered and heated at 37°C for 1 hour t o r e l e a s e bound c a r n i t i n e . 6) 0.400 ml of z i n c s u l f a t e was added to each tube, the contents mixed and the tube c o o l e d on i c e . 7) The p r e c i p i t a t e was c e n t r i f u g e d at 2C00 RPMs f o r 20 minutes. The supernatant was removed and f r o z e n a t -20°C u n t i l assayed f o r c a r n i t i n e . 38 P r e p a r a t i o n of l i v e r C a r n i t i n e E x t r a c t s - Part hj_ S e c t i o n 3 (Ts a i et a l . , 1974) 1) Reagents and t h e i r c o n c e n t r a t i o n s : Reagent C o n c e n t r a t i o n potassium hydroxide 10% p e r c h l o r i c a c i d 72% 2) 0.2 g of t i s s u e was h y d r o l y z e d i n 2.0 ml of potassium hydroxide at 8C°C f o r 2 hours to r e l e a s e c a r n i t i n e . (For combined f e t a l l i v e r s the t o t a l sample was hydrolyzed.) The s o l u t i o n was mixed twice during h y d r o l y s i s . 3) Samples were cooled to room temperature, and a c i d i f i e d with 72% p e r c h l o r i c a c i d and c e n t r i f u g e d at 2000 RPMs f o r 15 minutes. 4) The supernatant was removed and the p e l l e t washed twice with 1.0 ml a l i g u o t s of water.. 5) Combined washings were n e u t r a l i z e d with' potassium hydroxide, l e f t on i c e f o r 30 minutes, and then c e n t r i f u g e d at 2000 RPMs for 15 minutes. 6) The supernatant f r a c t i o n s were fre e z e d r i e d and then r e d i l u t e d to a predetermined volume using v o l u m e t r i c p i p e t t e s . T h i s step was conducted t o o b t a i n a c a r n i t i n e c o n c e n t r a t i o n that the assay was capable of d e t e c t i n g . 7) R e d i l u t e d supernatants were used f o r d u p l i c a t e d c a r n i t i n e a s s a ys. 39 T o t a l C a r n i t i n e Assay Procedure - Part E (McGarry and F o s t e r , 1976 ; P a r v i n and Pande 1977; Secccmbe et a l . , 1978) 1) Reagents and t h e i r c o n c e n t r a t i o n s : Reagent Co n c e n t r a t i o n a) N-2-Hydroxyethylpiperazine N-2- 0.4 M e t h a n e s u I f c n i c a c i d (HEPES) b) ( E t h y l e n e d i n i t r i l o ) - T e t r a a c e t i c 0.016 M a c i d (EDTA) c) N - e t h y l Malimide d) (1-**C) acetyl-CoA(hot) e) S-acetyl-CoA (cold) f) sodium hydroxide 1 N g) c a r n i t i n e a c e t y l t r a n s f e r a s e 5 mg/ml h) a c t i v a t e d c h a r c o a l i ) e t h a n o l ' 100% j) phosphoric acid 85% P r e p a r a t i o n o f S o l u t i o n s 2) HEPES/EDTA B u f f e r : The b u f f e r was made by d i s s o l v i n g 9.532 g of HEPES and 0.666 g of EDTA i n water and a d j u s t i n g the pH to 7.6 with sodium hydroxide.. The prepared s o l u t i o n was adjusted to a f i n a l volume of 100 ml. ao 3) 1,0 ml of the HEPES/EDTA b u f f e r was used to d i s s o l v e 5-0 mg of N-ethy 1 malimide. 4) Hot and Cold Acetyl CoA Mix: 1-0 ml a l i g u o t s of (1-**C) a c e t y l CoA (purchased from Amersham) plus c o l d S-acetyl-CoA were prepared such that each ml c o n t a i n e d a t o t a l of 50 nanamoles of acetyl-CoA a t a s p e c i f i c a c t i v i t y o f 40,000 DPMs per nanamole. 5) 1-0 ml of the acetyl-CoA mix from step 4 was added to the 1.0 ml b u f f e r NEM s o l u t i o n from step 3. The f i n a l volume of the mix was 2.0 ml and w i l l be r e f e r r e d to as the assay mix. 6) C a r n i t i n e A c e t y l t r a n s f e r a s e Mix: 0.05 ml of c a r n i t i n e a c e t y l t r a n s f e r a s e enzyme s o l u t i o n (purchased from Sigma) plus 1.050 ml of HEPES/EDTA b u f f e r (see step 2) were combined and mixed. Assa y 7) C o n s t r i c t i o n p i p e t t e s were used t o measure 0.100 ml samples of each t i s s u e e x t r a c t i n t o separate p o l y s t y r e n e tubes (purchased from Amersham) . A l l samples were kept on i c e . 8) 0.100 ml of the assay mix was added to the t i s s u e e x t r a c t . 9) the assay was s t a r t e d by adding 0.05 ml of the c a r n i t i n e a c e t y l t r a n s f e r a s e mix (see step 6) and the r e a c t i o n allowed to proceed at room temperature f o r 60 minutes. 10) The r e a c t i o n was terminated by the a d d i t i o n of 0.600 ml of an a c t i v a t e d c h a r c o a l s l u r r y which was c o n t i n u o u s l y s t i r r e d with a magnetic s t i r r e r . Charcoal S l u r r y Amount a c t i v a t e d c h a r c o a l 4 9 e t h a n c l 54.5 ml phosphoric a c i d 1.25 ml d i s t i l l e d water 4,2 5 ml 11) F o l l o w i n g the a d d i t i o n of the c h a r c o a l mixture, the tubes were placed on i c e and the contents mixed at approximately 10 minute i n t e r v a l s . . The tubes were then c e n t r i f u g e d a t 2000 RPMs f o r 20 minutes. 12) A 0.5 ml p o r t i o n of the c l e a r supernatant was t r a n s f e r r e d t o mini s i z e d s c i n t i l l a t i o n v i a l s (17 x 55 mm) c o n t a i n i n g 0.4 ml o f d i s t i l l e d water and 5 ml of ACS S c i n t i l l a n t (purchased from Amersham) . A f t e r mixing, and o b t a i n i n g a r e l a t i v e l y s t a b l e one-phase system, samples were counted i n a Eeckman LS-230 l i g u i d S c i n t i l l a t i o n Counter f o r 5 minutes each. , 42 Determination of C a r n i t i n e C o n c e n t r a t i o n A s e r i e s of blanks ( d i s t i l l e d water) standards (25 M, 50 M, and 100 M) and g u a l i t y c o n t r o l s (pooled plasma samples obtained from healthy Wistar males) were h y d r o l y z e d and assayed with each batch of t i s s u e s that were a n a l y z e d . The use of these permitted d e t e r m i n a t i o n of t i s s u e c a r n i t i n e c o n c e n t r a t i o n s and the r e p r o d u c i b i l i t y of the r e s u l t s * Treatment of b l a n k s , standards, and g u a l i t y c o n t r o l s was i d e n t i c a l to t h a t of the r e s p e c t i v e t i s s u e s they were used to a s s e s s , with the e x c e p t i o n of l i v e r . Blanks, standards and g u a l i t y c o n t r o l s hydrolyzed with the plasma e x t r a c t s were used to determine l i v e r c a r n i t i n e c o n c e n t r a t i o n s - The l i n e a r i t y of the standard curves remained good throughout the conducted a n a l y s e s (average c o e f f i c i e n t of c o r r e l a t i o n * r>0.989) , and v a r i a b i l i t y of the g u a l i t y c o n t r o l s ranged between 58.5-62.3 n mol/ml plasma i n d i c a t i n g good r e p r o d u c i b i l i t y of r e s u l t s . . The f o l l o w i n g formula was used to determine t i s s u e c a r n i t i n e c o n c e n t r a t i o n : x= 100-dmpJblank) X dpmjsample) X 100% = n mol/ml or g dpm(100 M standard) - dpm (blank) 43 Recp ver y A known quantity of D L - c a r n i t i n e was added to d u p l i c a t e s e t s of plasma, milk and l i v e r samples to determine the percent recovery o f c a r n i t i n e from t i s s u e s . Recovery of c a r n i t i n e was 93% f o r plasma, 91% f o r milk, and 95% f o r l i v e r . T s a i e t a l . (1974) r e p o r t e d reco/ery of 1 * C - c a r n i t i n e added to l i v e r t i s s u e to be 97 to 100%. Seccombe e t a l . (1978) c a l c u l a t e d the recovery of a c y l c a r n i t i n e added to plasma t o be grea t e r than 95% of t h e o r e t i c a l . Percent r e c o v e r y of c a r n i t i n e from milk has not, to the authors knowledge, been r e p o r t e d i n the l i t e r a t u r e . STATISTICS A l l s t a t i s t i c a l procedures were performed ty programs from the S t a t i s t i c a l Package f o r the S o c i a l Sciences (SPSS, Nie et a l . , 1975) on the U n i v e r s i t y o f B r i t i s h Columbia's Michigan Terminal System (MTS) computer program. Data o b t a i n e d from f e t u s e s and pups were expressed as the means of l i t t e r s r a t h e r than as i n d i v i d u a l samples.. These means of l i t t e r s were used t o c a l c u l a t e t h e standard d e v i a t i o n s . T h i s method was used r a t h e r than the measurements of i n d i v i d u a l o f f s p r i n g t o e l i m i n a t e the problem of c o r r e l a t i o n of o b s e r v a t i o n s within l i t t e r s (Abbey and Howard 1973) . . Experiment I The t - t e s t , at 0.05 l e v e l * was used t o determine whether t h e r e was any s i g n i f i c a n t d i f f e r e n c e s between treatment groups. The a n a l y s i s of variance with r e p e a t e d measures, at the 0.05 l e v e l was used to t e s t whether there was any s i g n i f i c a n t d i f f e r e n c e s i n weight gain and/or food i n t a k e during pregnancy. Experiment I I and I I I Unless otherwise s t a t e d , a l l analyses f o r experiments I I and I I I were conducted at the 0.05 l e v e l of s i g n i f i c a n c e . The a n a l y s i s of variance was used to determine i f there were any s i g n i f i c a n t d i f f e r e n c e s among treatment groups.. To e x p l o r e the source of the e f f e c t , Duncan's m u l t i p l e range t e s t was used. The a n a l y s i s of variance with r e p e a t e d measures was used t o t e s t whether t h e r e was any s i g n i f i c a n t d i f f e r e n c e i n weight of dams and pups and food i n t a k e of dams, during pregnancy and/or l a c t a t i o n . 45 CHAPTEE IV RESULTS EXPERIMENT I A l o w - l y s i n e d i e t , when f e d to pregnant r a t s , r e s u l t e d i n s i g n i f i c a n t l y lower maternal body weights, food i n t a k e s and, l i v e r weights when compared to animals fed the h i g h - l y s i n e d i e t (Tables I I I , IV, and F i g u r e 4). Dams fed the h i g h - l y s i n e d i e t consumed 17.3 g food/day during g e s t a t i o n , whereas the animals i n the l o w - l y s i n e group ate an average of 14.9 g/day, a s i g n i f i c a n t decrease i n food i n t a k e . . However, when food consumption was expressed as g/100 g body weight t h e r e was no s i g n i f i c a n t e f f e c t of d i e t on food i n t a k e (Table III) . Dams f e d the l o w - l y s i n e d i e t gained s i g n i f i c a n t l y l e s s weight (83.8 g) during g e s t a t i o n than dams consuming the h i g h -l y s i n e d i e t (147.1 g. Table I I I ) . The weekly weight gain of dams d u r i n g pregnancy (Table I I I , Fig u r e 4) was a l s o s i g n i f i c a n t l y a f f e c t e d by d i e t a r y treatment (p<0.005). A s i g n i f i c a n t d i f f e r e n c e i n body weights between the two d i e t a r y groups was observed as e a r l y as day 7 of g e s t a t i o n and con t i n u e d throughout pregnancy. . Body weights of dams i n the h i g h - l y s i n e and l o w - l y s i n e groups were 376,3 g and 308.3 g r e s p e c t i v e l y , on day 21 of g e s t a t i o n . The e f f e c t of d i e t a r y treatment on maternal l i v e r and heart weight i s presented i n Table IV. L i v e r weights f o r the low-Table I I I . Food Intake and Weight Gain of Dams Fed a High-Lysine or Low-Lysine Diet during Gestation Week of Gestation B Experimental I n i t i a l Group Body Weight Daily Food Intake Total Weight Gain Feed (g/day) High-Lysine Low-Lysine 6.5±0.2 6.3±0.3 6.710.4 6.5±0.5 6.610.3 5.9±0.3 17.310.6 14.910.5 Body Weight High-Lysine (g) Low-Lysine 229.2+7.9 262.916.7 300.817.2 376.3+9.5 224.6+5.2 239.014.9° 259.418.8° 308.3110.0E 147.1110.0 83.817.5 E A Mean 1 SEM for 6 dams B Food intake i s expressed as g/100 g body weight C Mean value d i f f e r s s i g n i f i c a n t l y from high-lysine group (p<0.05) D Mean values d i f f e r s i g n i f i c a n t l y from high-lysine group (p<0.01) E Mean values d i f f e r s i g n i f i c a n t l y from high-lysine group (p<0.001) 47 F i g u r e 4 E f f e c t of a h i g h - l y s i n e or l o w - l y s i n e d i e t on weight gain of dams during g e s t a t i o n Table IV. Heart and L i v e r Weights of Dams Fed a High-Lysine or Low-Lysine Diet u n t i l Day 21 of Ge s t a t i o n Experimental Group Tissue Weight g Tissue Weight % of Body Weight Heart L i v e r Heart L i v e r High-Lysine 0.7430±0.01 14.5071±0.48 0.20±0.01 3.86±0.09 Low-Lysine 0.7035±0.04 11.4096±0.71B 0.22±0.03C 3.71±0.23 A Mean ± SEM f o r 6 dams B Mean values d i f f e r s i g n i f i c a n t l y from h i g h - l y s i n e group (p<0.005) C Mean values d i f f e r s i g n i f i c a n t l y from h i g h - l y s i n e group (p<0.05) 50 l y s i n e group (11.4096 g) were s i g n i f i c a n t l y lower than i n the h i g h - l y s i n e animals (14.5071 g) . However, when l i v e r weights were expressed as a percent of body weight, t h e r e was no s i g n i f i c a n t d i f f e r e n c e between the twc d i e t a r y groups. L y s i n e d e f i c i e n c y d i d not a f f e c t maternal heart weights which were 0,7430 g and 0.7035 g r e s p e c t i v e l y , f o r the h i g h - l y s i n e and low-l y s i n e groups. Although the l o w - l y s i n e d i e t r e s u l t e d i n a 14% decrease i n food i n t a k e compared to the h i g h - l y s i n e group, l i t t e r s i z e was not a f f e c t e d by d i e t a r y treatment (Table V). Number of f e t u s e s f o r the h i g h - l y s i n e group was 11.7, and 11,8 f o r the l c w - l y s i n e group (Table V ) . F e t a l l i v e r weights were s i g n i f i c a n t l y lower i n the l o w - l y s i n e group (0. 2033 g) than i n the h i g h - l y s i n e group (0.2523 g ) . . F e t a l heart weights however, were s i g n i f i c a n t l y g r e a t e r i n t h e l c w - l y s i n e group (0.0150 g) than the h i g h - l y s i n e group (0.0142 g)* . Only one r e s o r p t i o n s i t e was i d e n t i f i e d f o r the dams fe d the l o w - l y s i n e d i e t , and no evidence of r e s o r p t i o n s were noted f o r the h i g h - l y s i n e group. The d i e t a r y l y s i n e l e v e l d i d not have a s i g n i f i c a n t e f f e c t on maternal plasna or l i v e r c a r n i t i n e l e v e l s (Table VI and F i g u r e 5 ) , . Plasma c a r n i t i n e l e v e l s averaged 43.97 and 37.70 n mol/ml f o r dams fed the low l y s i n e and h i g h - l y s i n e d i e t s r e s p e c t i v e l y , L i v e r c a r n i t i n e l e v e l s were much higher than plasma l e v e l s , averaging 168.87 and 166, 56 n mol/g t i s s u e i n the l o w - l y s i n e and h i g h - l y s i n e groups r e s p e c t i v e l y . F e t a l plasma c a r n i t i n e l e v e l s were s i g n i f i c a n t l y higher i n the l o w - l y s i n e (22.85 n mol/ml) group than the h i g h - l y s i n e (18,04 n mol/ml) group (Table VI, F i g u r e 6 ) , F e t a l l i v e r Table V. E f f e c t of a High-Lysine or Low-Lysine Diet on the Number of Fetuses, and F e t a l Heart and L i v e r Weight on Day 21 of Ge s t a t i o n Experimental Number of F e t a l Heart Weight F e t a l L i v e r Weight Group Fetuses g g High-Lysine 11.8±1.4 0.0142±0.000 0.2523±0.005 Low-Lysine 11.7±0.9 0.0150±0.001B 0.2033±0.014 A Mean ± SEM for 6 l i t t e r means B Mean values differ significantly from high-lysine group (p<0.005) C Mean values differ significantly from high-lysine group (p<0.05) Table VI. E f f e c t of a High-Lysine or Low-Lysine Diet on Plasma and L i v e r C a r n i t i n e of Dams and Fetuses on Day 21 of Gest a t i o n Experimental Plasma C a r n i t i n e L i v e r C a r n i t i n e Group nmol/ml nmol/g Dams High-Lysine Low-Lysine Fetuses B High-Lysine Low-Lys ine 37.70±8.60 43.97±2.40 166.56110.80 168.8719.50 18.0410.50 22.8511.40 174.62116.70 189.12119.90 A Mean 1 SEM f o r 6 dams B Mean 1 SEM f o r 6 l i t t e r means C Mean values d i f f e r s i g n i f i c a n t l y from h i g h - l y s i n e group (p<0.05) 53 F i g u r e 5 E f f e c t of a h i g h - l y s i n e or l c w - l y s i n e d i e t on plasma and l i v e r c a r n i t i n e i n dams on day 21 of g e s t a t i o n 5 5 F i g u r e 6 E f f e c t of a h i g h - l y s i n e or l c w - l y s i n e d i e t on plasma and l i v e r c a r n i t i n e i n fetuses on day 21 of g e s t a t i o n 200 I • L O W - L Y S I N E 180 160 H I G H - L Y S I N E 140 120 100 80l 601 4CH 20 PLASMA LIVER 57 c a r n i t i n e l e v e l s tended to be high e r i n the l o w - l y s i n e (189.12 n mol/g) group than the h i g h - l y s i n e (174.62 n mol/g) group, however the d i f f e r e n c e was not s i g n i f i c a n t . F e t a l plasma c a r n i t i n e l e v e l s were 52% lower than maternal l e v e l s * whereas l i v e r c a r n i t i n e l e v e l s were s i m i l a r i n both the dams and f e t u s e s . . EXPERIMENT I I In experiment I food i n t a k e o f the l o w - l y s i n e dams was s i g n i f i c a n t l y l e s s than t h a t of the h i g h - l y s i n e dams. Th e r e f o r e a t h i r d group of animals, h i g h - l y s i n e r e s t r i c t e d , was added t o determine i f the observed e f f e c t s were due to a l y s i n e d e f i c i e n c y or a r e s t r i c t e d food i n t a k e . I n experiment I I dams fed the l o w - l y s i n e or h i g h - l y s i n e r e s t r i c t e d d i e t s consumed s i g n i f i c a n t l y l e s s food, gained s i g n i f i c a n t l y l e s s weight, and had s i g n i f i c a n t l y lower l i v e r weights than animals f e d the high-l y s i n e d i e t . There were no s i g n i f i c a n t d i f f e r e n c e s between the l o w - l y s i n e and the h i g h - l y s i n e r e s t r i c t e d groups i n any o f these parameters (Table VII, V I I I , IX, and F i g u r e 7).. Food i n t a k e during g e s t a t i o n was s i g n i f i c a n t l y g r e a t e r i n the h i g h - l y s i n e females (15.0 g/day) than i n the l c w - l y s i n e (13.0 g/day) or h i g h - l y s i n e r e s t r i c t e d (13.2 g/day) females. However, when food consumption d u r i n g pregnancy was expressed as g/100 g body weight t h e r e was no s i g n i f i c a n t e f f e c t of d i e t on food i n t a k e (Table VII) . . Dams f e d the h i g h - l y s i n e d i e t gained s i g n i f i c a n t l y more weight (124.6 g) during pregnancy than e i t h e r the l o w - l y s i n e Table VII. Food Intake and Weight Gain of Dams Fed a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Diet during Gestation i n Experiment II Week of Gestation B Experimental I n i t i a l Group Body Weight Dail y Total Food Intake Weight Gain Feed (g/day) High-Lysine Low-Lysine High-Lysine Restricted 6.9+0.3 6.1±0.2 5.810.2 6.3+0.3 6.1±0.5 5.9±0.3 5.9±0.2 4.9±0.4 5.0±0.4 15.0±0.5 C 13.0±0.4 13.2±0.7 Body Weight (g) High-Lysine Low-Lysine High-Lysine Restr i c t e d 217.2±7.1 244.1±4.4 275.8+5.4 341.8±5.6 228.4±1.2 238.4±1.2 259.4±8.5 299.3±5.7 231.3±5.3 238.8±4.4 268.8±5.0 317.3±7.4 124.6±5.2 C 70.8±8.3 86.0±9.2 A Mean ± SEM for 6 dams B Food intake i s expressed as g/100 g body weight C Means i n a column with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) 59 (70.8 g) or h i g h - l y s i n e r e s t r i c t e d (86.0 g) groups (Table V I I ) . Weekly weight gains of dams d u r i n g pregnancy (Table VII and Fi g u r e 7) were not s i g n i f i c a n t l y a f f e c t e d by d i e t a r y treatment. Food i n t a k e of dams du r i n g l a c t a t i o n was s i g n i f i c a n t l y g r e a t e r i n the h i g h - l y s i n e (27.1 g/day) group than i n the low-l y s i n e (14.2 g/day) or h i g h - l y s i n e r e s t r i c t e d groups (13.7 g/day r Table V I I I ) . When food consumption duri n g l a c t a t i o n was expressed as g/100 g body weight, a s i g n i f i c a n t e f f e c t of d i e t on food i n t a k e was i n d i c a t e d (p<0.001) * Food i n t a k e of the h i g h - l y s i n e dams was s i g n i f i c a n t l y g r e a t e r than the l c w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d dams during weeks 1 and 2 of l a c t at ion* Dams f e d the h i g h - l y s i n e d i e t gained 20.1 g durin g l a c t a t i o n whereas the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups l o s t 47.0 g and 45.9 g, r e s p e c t i v e l y (Table V I I I and Fig u r e 11).. There were no s i g n i f i c a n t d i f f e r e n c e s i n weight between t h e l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups of animals however, both groups d i f f e r e d s i g n i f i c a n t l y from t h a t of the h i g h - l y s i n e group* The e f f e c t of d i e t on weekly weight changes d u r i n g l a c t a t i o n was a l s o s i g n i f i c a n t (p<0.001). When compared t o the h i g h - l y s i n e group, the average body weights o f dams f e d the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d d i e t s were s i g n i f i c a n t l y lower on days 1, 7, and 14. . Body weights of dams on day 15 f o r the h i g h - l y s i n e , l c w - l y s i n e , and h i g h - l y s i n e r e s t r i c t e d groups were 296.8 g, 190.2 g, and 202. 2 g r e s p e c t i v e l y (Table V I I I ) . , The e f f e c t of d i e t a r y treatment on maternal l i v e r and heart weight i n the dams i s presented i n Table IX. L i v e r s of the low-60 F i g u r e 7 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t on weight g a i n of dams durin g g e s t a t i o n i n Experiment I I DAY OF GESTATION Table V I I I . Food Intake and Weight Changes of Dams Fed a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Diet during L a c t a t i o n Week of • B L a c t a t i o n Experimental Group I n i t i a l Weight 1 2 D a i l y T o t a l Food Intake Weight Gain Feed (g/day) High-Lysine Low-Lys ine C 7.4±0.5a 5.4±0.7b 12.6±l.la 6.9±0.9b 27.1±l.la 14.2±1.5b High-Lysine R e s t r i c t e d 5.1±0.4b 7.0+1.0b 13.7±1.5b Body Weight (g) High-Lysine Low-Lysine 276.6±ll.la 236.1+ 4.6 b 278.4±ll.la 208.3± 3.9 b 296.8±5.6a 190.2±5.7.b +20.1±10.7a -47.0+ 8.8 b High-Lysine R e s t r i c t e d 249.2± 4.9 b 221.4± 5.1 b 202.2±8.5b -45.9± 4.7 b A Mean ± SEM f o r 6 dams B Food i n t a k e i s expressed as g/100 g body weight C Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) Table IX. Heart and Liver Weights of Dams Fed a High-Lysine, Low-Lysine or High-Lysine Restricted Diet u n t i l Day 15 of Lactation Experimental Group Tissue Weight g Heart Liver Tissue Weight % of Body Weight Heart Liver High-Lysine 4.914810.30 14.220210.62 1.7010.10 4.8010.24 Low-Lys ine 3.933610.30 8.625210.93 2.0710.10 4.5510.22 High-Lysine Restricted 4.856010.30 8.003611.02 2.4310.20 3.9010.32 A Mean 1 SEM for 6 dams B Means in a column with different superscripts are significantly different (p<0.05) 64 l y s i n e (8.6252 g) and h i g h - l y s i n e r e s t r i c t e d (8.0036 g) dams weighed s i g n i f i c a n t l y l e s s than those o f the h i g h - l y s i n e (14.2202 g) dams. Heart weights d i d net d i f f e r s i g n i f i c a n t l y among the three d i e t a r y groups. When t i s s u e weights were expressed as a percentage of body weight, d i e t a r y treatment had no s i g n i f i c a n t e f f e c t on e i t h e r l i v e r or heart weight. E f f e c t s of d i e t a r y treatment on r e p r o d u c t i o n as assessed by l i t t e r s i z e and b i r t h weight of pups, i s presented i n Table X. L i t t e r s i z e f o r the h i g h - l y s i n e (11.0), l o w - l y s i n e (10.3), and h i g h - l y s i n e r e s t r i c t e d (13.0) groups was not s i g n i f i c a n t l y d i f f e r e n t * When compared with o f f s p r i n g of animals maintained on the h i g h - l y s i n e c o n t r o l d i e t (6.0 g), b i r t h weight of pups was however, s i g n i f i c a n t l y lowered by e i t h e r a l y s i n e d e f i c i e n c y (5-5 g) or food r e s t r i c t i o n (5.4 g ) . There was no d i f f e r e n c e i n b i r t h weight between the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups. The l a c t a t i o n performance o f dams, as measured by the growth of pups* i s presented i n Table XI and F i g u r e 8. . The e f f e c t of maternal d i e t on weight gain of pups during l a c t a t i o n was s i g n i f i c a n t (p<0.001) . The h i g h - l y s i n e group of pups weighed s i g n i f i c a n t l y more than th e h i g h - l y s i n e r e s t r i c t e d group which i n t u r n weighed s i g n i f i c a n t l y mere than l o w - l y s i n e group on days 5, 10, and 15 of l a c t a t i o n . . Pups o f h i g h - l y s i n e , low-l y s i n e , and h i g h - l y s i n e r e s t r i c t e d dams weighed 28-9 g, 12-6 g, and 17.8 g r e s p e c t i v e l y , on day 15 of l a c t a t i o n . The e f f e c t of maternal d i e t on pups l i v e r and heart weight i s presented i n Table XII. L i v e r s of pups i n the h i g h - l y s i n e group (0.9184 g) weighed s i g n i f i c a n t l y greater than those of the Table X. E f f e c t of a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Diet on Reproductive Performance of Dams i n Experiment I I Experimental L i t t e r Size B i r t h Weight G r o u P of Pups g B High-Lysine 11.011.2 6.0i0.2 a Low-Lysine 10.3±0.4 5.5+0.2b High-Lysine 13.010.3 5.4l0.8 b R e s t r i c t e d A Mean 1 SEM f o r 6 l i t t e r means B Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) Table X I . E f f e c t of a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Maternal Diet on Weight Gains of Pups i n Experiment I I Experimental Groups Days Post Partum 10 15 Body Weight High-Lysine (g) 6.1±0.3C 10.1±0.3C 18.8±0.5C 28.9±0.9C Low-Lysine 5.5±0.2 8.4±0.3 10.810.5 12.610.7 High-Lysine R e s t r i c t e d 5.510.1 9.210.3 14.110.8 17.811.3 A Mean 1 SEM f o r 6 l i t t e r means B Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) 67 F i g u r e 8 E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d maternal d i e t on weight gain of pups i n Experiment I I 68 DAY OF LACTATION Table X I I . E f f e c t on a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Maternal Diet on Heart and L i v e r Weights of Pups on Day 15 of L a c t a t i o n Experimental Group Tissue Weight g Heart L i v e r Tissue Weight % of Body Weight Heart L i v e r High-Lysine Low-Lysine 0.123410.010c 0.063910.005 0.918410.042c 0.352810.028 0.4010.02 0.5110.03 2.9010.60 2.8210.02 High-Lysine R e s t r i c t e d 0.114610.054' 0.5205+0.050 0.6010.10 3.2010.10 A Mean 1 SEM f o r 6 l i t t e r means B Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) 70 h i g h - l y s i n e r e s t r i c t e d group (0.5205 g) which were a l s o s i g n i f i c a n t l y greater than those of the l o w - l y s i n e group (0.3528 g) . Heart weights of pups i n the h i g h - l y s i n e (0. 1234 g) and h i g h - l y s i n e r e s t r i c t e d groups (0.1146 g) were s i g n i f i c a n t l y g reater than these of the l o w - l y s i n e group (0*0639 g ) . When t i s s u e weights were expressed as a percentage of body weights, t h e r e were no s i g n i f i c a n t d i f f e r e n c e s among the t h r e e d i e t a r y groups. When compared t o h i g h - l y s i n e c o n t r o l v a l u e s , plasma and l i v e r c a r n i t i n e l e v e l s were s i g n i f i c a n t l y h i g h e r a t day 15 of l a c t a t i o n , i n both the pups and dams consuming e i t h e r the low-l y s i n e d i e t ad l i b i t u m , or the r e s t r i c t e d h i g h - l y s i n e d i e t (Table X I I I , F i g u r e s 9 and 10) . Heart and l i v e r c a r n i t i n e f o r both the pups and dams d i d not d i f f e r s i g n i f i c a n t l y between the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups. The l o w - l y s i n e d i e t r e s u l t e d i n maternal plasma c a r n i t i n e l e v e l s o f 38.22 n mol/ml compared t c 34;79 n mol/ml f o r the h i g h - l y s i n e r e s t r i c t e d dams and 19.22 n mol/ml f o r the high-l y s i n e dams* . L i v e r c a r n i t i n e values o f dams i n the h i g h - l y s i n e , h i g h - l y s i n e r e s t r i c t e d and l o w - l y s i n e groups were, 120.98; 187.60, and 176.24 n mol/ml r e s p e c t i v e l y . The plasma c a r n i t i n e l e v e l s i n dams fed the h i g h - l y s i n e d i e t were higher a t day 21 of pregnancy (37*70 n mol/ml, Table VI) than at day 15 of l a c t a t i o n (19.22 n mol/ml, Table X I I I ) . _ L i v e r c a r n i t i n e l e v e l s i n animals f e d the same h i g h - l y s i n e d i e t averaged 166*56 n mol/g a t the end of g e s t a t i o n and 120.98 n mol/g on day 15 o f l a c t a t i o n (Table VI and XIII r e s p e c t i v e l y ) . . Plasma c a r n i t i n e l e v e l s f o r t h e l o w - l y s i n e (61.02 n mol/ml) Table X I I I . E f f e c t of a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Maternal Diet on Plasma and L i v e r C a r n i t i n e of Dams and Pups on Day 15 of L a c t a t i o n Experimental Plasma C a r n i t i n e L i v e r C a r n i t i n e Group nmol/ml nmol/g A Dams High-Lysine C 19.22±3.86a 120.98132.39 a Low-Lysine 38.22±7.56b 176.24113.23 b High-Lysine R e s t r i c t e d 34.79±5.14b 187.601 9.84 b Pups High-Lysine 51.10±1.78a 253.99111.68 3 Low-Lysine 61.02+4.13b 370.71185.44 b High-Lysine R e s t r i c t e d 62.07±3.75b 360.78140.82 b A Mean 1 SEM f o r 6 dams B Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) C Mean 1 SEM f o r 6 l i t t e r means 72 F i g u r e 9 E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t on plasma and l i v e r c a r n i t i n e of dams on day 15 of l a c t a t i o n NMOL CARNITINE PER ML PLASMA OR G LIVER O 4^  O 00 o o o o o o 00 o o o 7777771 • 73 •X r 1 1—1 m i—i o en en m i TR a: i i t— tr1 I-H cr* •< -< n -< en H cn I-H t—i m z z o z tn tn 74 F i g u r e 10 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d maternal d i e t on plasma and l i v e r c a r n i t i n e of pups on day 15 of l a c t a t i o n 75 76 and h i g h - l y s i n e r e s t r i c t e d (62.07 n mol/ml) groups of pups were s i g n i f i c a n t l y higher than those of the h i g h - l y s i n e pups (51.10 n mol/ml) . . Likewise, l i v e r c a r n i t i n e l e v e l s f o r the l o w - l y s i n e (370.71 n mol/g) and h i g h - l y s i n e r e s t r i c t e d (360.78 n mol/g) pups were s i g n i f i c a n t l y h i g h e r than those f o r the h i g h - l y s i n e pups (253.99 n mol/g).. Plasma and l i v e r c a r n i t i n e l e v e l s of pups on day 15 cf l a c t a t i o n (Table XIII) were almost double the l e v e l s of c a r n i t i n e i n f e t a l t i s s u e s (Table V I). Weight changes of dams durin g pregnancy and l a c t a t i o n are de p i c t e d i n F i g u r e 11. The histogram emphasizes the s i m i l a r i t y i n weight g a i n s t h a t occurred i n t h e l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups duri n g pregnancy. During l a c t a t i o n both the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d dams l o s t a s i g n i f i c a n t amount o f weight whereas the h i g h - l y s i n e c o n t r o l animals gained weight. EXPERIMENT I I I The dams i n experiment I I I f e d the l o w - l y s i n e or high-l y s i n e r e s t r i c t e d d i e t s consumed s i g n i f i c a n t l y l e s s food and gained s i g n i f i c a n t l y l e s s body weight than those animals f e d the h i g h - l y s i n e d i e t (Tables XIV, XV, XVI, and F i g u r e 12). The food i n t a k e of dams d u r i n g g e s t a t i o n was s i g n i f i c a n t l y g r e a t e r i n t h e h i g h - l y s i n e group (16*5 g/day) than i n the low-l y s i n e (13.5 g/day) or h i g h - l y s i n e r e s t r i c t e d groups (13.5 g/day. Table XIV). Food i n t a k e between the l o w - l y s i n e and high-l y s i n e r e s t r i c t e d females d i d not d i f f e r s i g n i f i c a n t l y . When food consumption during pregnancy was expressed as g/100 g body 77 F i g u r e 11. E f f e c t of a h i g h - l y s i n e * l o w - l y s i n e * or h i g h - l y s i n e r e s t r i c t e d d i e t on weight changes of dams during pregnancy and l a c t a t i o n i n Experiment I I 78 PREGNANCY LACTATION +140 r +120 +100 • L O W - L Y S I N E H I G H - L Y S I N E R E S T R I C T E D H I G H - L Y S I N E I-H Q O CQ +80 +60 +40 UJ z < u +20 -20 -40 Table XIV. Food Intake and Weight Gain of Dams Fed a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Diet during G e s t a t i o n i n Experiment I I I Week of Gestation Experimental I n i t i a l 1 2 3 D a i l y T o t a l Group Body Weight Food Intake Weight Gain Feed High-Lysine 7.2±0.3 7.4±0.3 6.1±0.3 16.5±0. C 4 a (g/day) u Low-Lysine 6.4±0.2 5.9±0.2 5.2±0.3 13.5±0. 3 b High-Lysine 6.1±0.2 5.6±0.1 5.0±0.2 13.5±0. 4 b R e s t r i c t e d Body Weight High-Lysine 217. 5±5. 2 248.1±4. 8 276.6±4.3 336.3±5.4 118 .8±4. l a (g) Low-Lysine 232. 0±3. 3 239.1±4. 8 244.2±3.8 281.4±3.5 49 .5±1. 7 b High-Lysine 232. 0±3. 4 244.0±3. 6 267.0±4.5 318.2±6.5 86 .8±4. 4° R e s t r i c t e d A B C Mean ± SEM f o r 18 dams Food i n t a k e i s expressed as g/100 g body weight Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) 80 weight, t h e r e was no s i g n i f i c a n t e f f e c t of d i e t on food i n t a k e (Table XIV) . These r e s u l t s were s i m i l a r t o those obtained i n experiment I I - _ Dams f e d the h i g h - l y s i n e d i e t gained s i g n i f i c a n t l y more weight (118.8 g) durin g g e s t a t i o n than e i t h e r the l o w - l y s i n e (49.5 g) or h i g h - l y s i n e r e s t r i c t e d (86.8 g) groups (Table XIV and F i g u r e 12). These r e s u l t s d i f f e r e d from those i n experiment I I . The d i f f e r e n c e between the abs o l u t e weight gain by the lcw-l y s i n e and h i g h - l y s i n e r e s t r i c t e d dams was not s i g n i f i c a n t i n experiment I I whereas the d i f f e r e n c e was s i g n i f i c a n t i n experiment I I I (Tables VII and XIV r e s p e c t i v e l y ) . . Food i n t a k e and weight changes during l a c t a t i o n , f c r those dams used f o r milk c a r n i t i n e a n a l y s i s on day 15 of l a c t a t i o n , are presented i n Table XV. As p r e v i o u s l y reported i n experiment I I {Table VII) , food i n t a k e o f dams was again s i g n i f i c a n t l y lower i n the l o w - l y s i n e (17,5 g/day) and h i g h - l y s i n e r e s t r i c t e d (20.3 g/day) groups than i n the h i g h - l y s i n e (29.0 g/day) group. When food consumption duri n g l a c t a t i o n was expressed as g/100 g body weight, a s i g n i f i c a n t e f f e c t of d i e t on food i n t a k e was observed (p<0.001)* Food i n t a k e f o r the h i g h - l y s i n e dams was s i g n i f i c a n t l y greater than f o r the l c w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups. Body weight changes f o r these three groups of dams d u r i n g l a c t a t i o n were of a s m a l l e r magnitude than, but i n the same d i r e c t i o n as those o b t a i n e d i n experiment I I (Table V I I I ) . Dams consuming the h i g h - l y s i n e d i e t gained an average of 17.1 g body weight whereas, the dams fed the l c w - l y s i n e or high-l y s i n e r e s t r i c t e d d i e t s l o s t an average of 16.4 and 29.2 g r e s p e c t i v e l y * Weight change between the l o w - l y s i n e and hi g h -81 F i g u r e 12 E f f e c t of a h i g h - l y s i n e * l o w - l y s i n e * or h i g h - l y s i n e r e s t r i c t e d d i e t on weight g a i n of dams during g e s t a t i o n i n Experiment I I I 82 380 LOW-LYSINE 360 HIGH-LYSINE RESTRICTED 340 HIGH-LYSINE 320 300 280 260 240 220 200 180 14 J 21 DAY OF GESTATION TABLE XV. Food Intake and Weight Changes of Dams Mil k e d on Day 15 of L a c t a t i o n and Fed a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Diet during L a c t a t i o n Week of L a c t a t i o n Experimental I n i t i a l 1 2 D a i l y T o t a l Group Body Weight Food Intake Weight Gain C Feed High-Lysine 8.2±0.3 12.6i0.2a 29. 0 i l . 5 a (g/day) h b Low-Lysine 7.6±0.5 9.210.4 17. 510.5b High-Lysine 7.010.2 9.0i0.1b 20. 311.5b R e s t r i c t e d Body Weight High-Lysine 280. 9±6.0 a 279.915.4a 297.915.7a +17. 117.8a (g) b b b ,b Low-Lysine 217. 615.4 204.416.9 201.217.1 -16. 415.4 ,High-Lysine 259. o±io.ob 248.0110.7° 229.8110.2° -29. 2111.4b R e s t r i c t e d A Mean 1 SEM f o r 6 dams B Food intake i s expressed i n g/lOOg body weight C Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) 84 l y s i n e r e s t r i c t e d groups were s i m i l a r however both values d i f f e r e d s i g n i f i c a n t l y from the high l y s i n e group. The e f f e c t of d i e t on weekly weight changes during l a c t a t i o n was al s o s i g n i f i c a n t (p<0.001). The h i g h - l y s i n e dams weighed s i g n i f i c a n t l y more than the h i g h - l y s i n e r e s t r i c t e d and low-l y s i n e dams on days 7 and 15 o f l a c t a t i o n * The body weight of the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d animals on days 7 and 15 were a l s o s i g n i f i c a n t l y d i f f e r e n t , as was the case i n experiment I I . Food i n t a k e and weight changes during l a c t a t i o n , f o r those dams used f o r milk c a r n i t i n e a n a l y s i s on day 8 of l a c t a t i o n (Table XVI) are s i m i l a r to those obtained f o r dams used f o r milk c a r n i t i n e a n a l y s i s on day 15 of l a c t a t i o n . . Food i n t a k e was s i g n i f i c a n t l y lower i n the l o w - l y s i n e (15.8 g/day) and hi g h -l y s i n e r e s t r i c t e d (17.7 g/day) dams than i n the h i g h - l y s i n e (24. 2 g/day) females* When food consumption was expressed as g/100 g body weight, a s i g n i f i c a n t l y greater food i n t a k e was found i n those dams f e d the h i g h - l y s i n e d i e t than dams consuming the l o w - l y s i n e cr h i g h - l y s i n e r e s t r i c t e d d i e t s * Dams f e d the h i g h - l y s i n e d i e t gained an average of 14.8 g during the f i r s t week of l a c t a t i o n , whereas the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d females l o s t an average of 12.1 g and 10.8 g r e s p e c t i v e l y * . Weight gain of the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d dams d i f f e r e d s i g n i f i c a n t l y from h i g h - l y s i n e dams. On day 8 of l a c t a t i o n t h e h i g h - l y s i n e group weighed s i g n i f i c a n t l y more than the h i g h - l y s i n e r e s t r i c t e d group, which i n t u r n weighed s i g n i f i c a n t l y more than the l o w - l y s i n e group. Maternal d i e t a r y treatment e f f e c t s on r e p r o d u c t i o n * as Table XVI. Food Intake and Weight Changes of Dams Milked on Day 8 of L a c t a t i o n and Fed a High-L y s i n e , Low-Lysine or High-Lys ine R e s t r i c t e d Diet during L a c t a t i o n Week of L a c t a t i o n B Experimental Group I n i t i a l Body Weight D a i l y Food Intake T o t a l Weight Gain Feed (g/day) High-Lysine Low-Lysine High-Lysine R e s t r i c t e d 8.9±0.3C 7.0±0.3 7.0±0.2 24.2±1.4C 15.8±0.4 17.7+0.9 Body Weight (g) High-Lysine Low-Lysine High-Lysine R e s t r i c t e d 274.1±9.4C 220.5±7.3 254.7±11.6C 288.9±6.9C 210.5±5.0 243.9+8.2 +14.8±6.0C -12.1+6.5 -10.8±4.4 A Mean ± SEM f o r 6 dams B Food i n t a k e i s expressed as g/lOOg body weight C Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) 86 assessed by l i t t e r s i z e and b i r t h weight of pups, i s presented i n Table XVII.. l i t t e r s i z e f o r the high l y s i n e (10.7), low-l y s i n e (11.9), and h i g h - l y s i n e r e s t r i c t e d (12.3) groups was s i m i l a r . B i r t h weight of pups i n the h i g h - l y s i n e group (6.1 g) was s i g n i f i c a n t l y g r e a t e r than t h a t of the h i g h - l y s i n e r e s t r i c t e d (5.5 g) or the l o w - l y s i n e (1.9 g) groups. although b i r t h weights of pups did not d i f f e r s i g n i f i c a n t l y ' between the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups i n experiment I I (Table X) , the b i r t h weight of pups did d i f f e r s i g n i f i c a n t l y between groups i n experiment I I I * . The m o r t a l i t y r a t e of pups i n the h i g h - l y s i n e , h i g h - l y s i n e r e s t r i c t e d , and l o w - l y s i n e groups was 0.7%, 0.7%, and 1.2% r e s p e c t i v e l y . . The d i f f e r e n c e between groups was not s i g n i f i c a n t (Table XVII) . The l a c t a t i o n performance of dams, as measured by the growth o f pups, i n experiment I I I (Table XVIII and F i g u r e s 13, 14) was s i m i l a r to that r e p o r t e d i n experiment I I (Table XI). On both days 8 (p<0.001) and 15 (p<0.001) of l a c t a t i o n , the pups i n the h i g h - l y s i n e group weighed s i g n i f i c a n t l y more than the h i g h - l y s i n e r e s t r i c t e d group which i n t u r n weighed s i g n i f i c a n t l y more than th e l o w - l y s i n e group. The average weight of pups on day 15 was 27.7 g i n the h i g h - l y s i n e group and 10.9 and 20.7 g i n the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups r e s p e c t i v e l y . The. data obtained i n experiment I I i n d i c a t e d the the high-l y s i n e r e s t r i c t e d and l o w - l y s i n e animals had h i g h e r t i s s u e c a r n i t i n e l e v e l s than the h i g h - l y s i n e c o n t r o l animals. The e f f e c t of d i e t on milk c a r n i t i n e l e v e l s , as presented i n Table Table XVII. E f f e c t of a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Diet on Reproductive Performance of Dams i n Experiment I I I Experimental Group L i t t e r S i z e B i r t h Weight of Pups g %.'.Mortality B High-Lysine 10.7±0.9 6.1±0.2a 0,7 Low-Lysine 11.9±0.6 4.9±0.2b 1.2 High-Lysine 12.3±0.6 5.5±0,2° 0.7 R e s t r i c t e d A Mean ± SEM f o r 16 l i t t e r means B Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.0.5) Table X V I I I . E f f e c t of a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Maternal Diet on Weight Gains of Pups i n Experiment I I I Experimental Days Post Partum Group 1 5 8 10 15 Body Weight High-Lysine C 6.8±0.2a 11.4±0.3a 20.1±0.6a 27. 7±1. 3 a (g) Low-Lysine 5.1±0.2b 7.4±0.5b 9.2±0.7b 10. 9±1. l b High-Lysine 5.8±0.2° 9.4±0.7° 16.0+1.0° 20. 7±2. o c R e s t r i c t e d Body Weight^ High-Lysine 6.4±0.3a ll.l±0.5a 14.9±0.5a (g) Low-Lysine 5.0±0.3 a b 7.6±0.5b 8.4±0.7b High-Lysine 5.6±0.2b 9.5±0.5° 12.7±0.5C R e s t r i c t e d A Mean ± SEM f o r 6 l i t t e r means B Pups of dams milked on day 15 C Means i n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) D Pups of dams milked on day 8 89 F i g u r e 13 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e * or h i g h - l y s i n e r e s t r i c t e d maternal d i e t on weight g a i n s of pups born to those dams milked on day 15 of l a c t a t i o n 9 0 DAY OF LACTATION 91 F i g u r e 14 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d maternal d i e t on weight g a i n of pups born t o those dams milked on day 8 of l a c t a t i o n 92 93 XIX and F i g u r e 15* i s not c l e a r * On the 2nd day of l a c t a t i o n , A milk c a r n i t i n e l e v e l s of dams f e d the h i g h - l y s i n e d i e t (426.04 n mol/ml), were s i g n i f i c a n t l y h i g h e r than the l o w - l y s i n e (300.65 n mol/ml) females, which i n t u r n were s i g n i f i c a n t l y higher than the h i g h - l y s i n e r e s t r i c t e d (151.21 n mol/ml) females. On day 8 of l a c t a t i o n t h e r e was no s i g n i f i c a n t d i f f e r e n c e between the milk c a r n i t i n e l e v e l s i n those animals consuming e i t h e r the h i g h - l y s i n e (122.42 n mol/ml) d i e t ad l i b i t u m , or the high-l y s i n e r e s t r i c t e d (128.52 n mol/ml) d i e t . The same p a t t e r n p r e v a i l e d on day 15 of l a c t a t i o n f o r the two d i e t a r y groups, i . e * , t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n milk c a r n i t i n e l e v e l s between the h i g h - l y s i n e (107. 15 n mol/ml) and h i g h - l y s i n e r e s t r i c t e d (134,19 n mol/ml) groups. Dams f e d the l o w - l y s i n e d i e t had s i g n i f i c a n t l y higher milk c a r n i t i n e l e v e l s on day 8 (277.46 n mol/ml) and day 15 (194. 20 n mol/ml) than animals i n the two other d i e t a r y groups. M i l k c a r n i t i n e values f o r the lo w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups d i d not change s i g n i f i c a n t l y during the course of l a c t a t i o n . Milk c a r n i t i n e v a l u e s , f o r dams fed the h i g h - l y s i n e d i e t , were s i g n i f i c a n t l y g r e a t e r on day 2 of l a c t a t i o n than on days 8 and 15 post partum. Plasma c a r n i t i n e l e v e l s of h i g h - l y s i n e , l o w - l y s i n e , and h i g h - l y s i n e r e s t r i c t e d dams and t h e i r o f f s p r i n g are compared with milk c a r n i t i n e values i n F i g u r e 16.. Plasma and l i v e r c a r n i t i n e values are f o r day 15 of l a c t a t i o n . Plasma c a r n i t i n e c o n c e n t r a t i o n s f o r dams and t h e i r o f f s p r i n g were s i g n i f i c a n t l y higher i n both the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups, when compared with t h e i r r e s p e c t i v e c o n t r o l values. Plasma c a r n i t i n e c o n c e n t r a t i o n s f o r the l c w - l y s i n e and h i g h - l y s i n e Table XIX. E f f e c t of a High-Lysine, Low-Lysine or High-Lysine R e s t r i c t e d Diet on M i l k C a r n i t i n e on Days 2, 8 and 15 Post Partum Experimental Days Post Partum  Group 2 8 15 M i l k C a r n i t i n e (n mol/ml) High-Lysine Low-Lysine ,B .C , 426.04±29.50a' 122.42±16.23a' 107.15±12.56a' 300.65+43.21 b,d 277.46±38.21b'e 194.20112.63 b ,e High-Lysine R e s t r i c t e d 151.21±37.76C'd 128.52±27.68a'e 134.19±22.43 a,e A Mean ± SEM f o r 6 dams B Means I n a column w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (a, b, c - p<0.05) C Means i n a row w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (d, e - p<0.05) 95 F i g u r e 15 E f f e c t of a h i g h - l y s i n e , l c w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t on milk c a r n i t i n e on days 2, 8, and, 15 of l a c t a t i o n 96 97 F i g u r e 1 6 E f f e c t of a h i g h - l y s i n e , l o w - l y s i n e , or h i g h - l y s i n e r e s t r i c t e d d i e t on maternal plasma and milk c a r n i t i n e i n dams, and pup plasma c a r n i t i n e on day 15 of l a c t a t i o n PLASMA MILK PLASMA DAMS DAY 15 PUPS DAY 15 99 r e s t r i c t e d groups d i d not d i f f e r s i g n i f i c a n t l y * Milk c a r n i t i n e l e v e l s , on day 15 of l a c t a t i o n , were a l s o higher i n the low-l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups than the h i g h - l y s i n e c o n t r o l group. In a d d i t i o n , milk c a r n i t i n e was s i g n i f i c a n t l y d i f f e r e n t between the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups, on day 15 of l a c t a t i o n , whereas no s i g n i f i c a n t d i f f e r e n c e s between the h i g h - l y s i n e r e s t r i c t e d and h i g h - l y s i n e c o n t r o l groups were observed.. 100 CHAPTEE V DISCUSSION DIETABY LYSINE ANE REPRODUCTION The r e s u l t s of t h i s study c o n f i r m those of S t a p l e t o n and H i l l (1972,1980) , who r e p o r t e d t h a t a maternal l y s i n e d e f i c i e n c y had no s i g n i f i c a n t e f f e c t on l i t t e r s i z e but s i g n i f i c a n t l y decreased the b i r t h weight o f o f f s p r i n g . . Niiyama et - a l . (1970,1973) documented an adverse e f f e c t of a l y s i n e f r e e d i e t on the b i r t h weight of pups. L i t t e r s i z e however was not a f f e c t e d by f e e d i n g a l y s i n e f r e e d i e t . . They suggested t h a t animals f e d the l y s i n e d e f i c i e n t d i e t may have maintained pregnancy because of the l y s i n e s p a r i n g mechanism' (Yamashita and A s h i t a , 1968) shown to e x i s t i n ncn-pregnant a d u l t r a t s ( C a n f i e l d and C h y t i l , 1978; Chu and Hegsted, 1976). S t a p l e t o n and H i l l (1972) repo r t e d a dramatic drop i n plasma l y s i n e l e v e l s d u r i n g th e l a s t week of pregnancy f o r these animals consuming a l y s i n e d e f i c i e n t d i e t * .. The authors suggested t h a t t h e decreased b i r t h weight of the l y s i n e d e f i c i e n t pups may have r e s u l t e d from a decreased supply of l y s i n e t o the f e t u s d u r i n g the l a s t week of gestat i o n . The q u a n t i t y of food consumed fcy dams a f f e c t s r e p r o d u c t i v e performance of dams. O f f s p r i n g born t c dams fed the l o w - l y s i n e or h i g h - l y s i n e r e s t r i c t e d d i e t s weighed s i g n i f i c a n t l y l e s s than those born t o dams fed the h i g h - l y s i n e d i e t . . There was no s i g n i f i c a n t d i f f e r e n c e between the b i r t h weight of pups from the 101 l y s i n e d e f i c i e n t and c a l o r i e r e s t r i c t e d groups i n experiment I I , the b i r t h weight c f pups f o r the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups d i f f e r e d s i g n i f i c a n t l y i n experiment I I I , The r e s u l t s of the t h i r d experiment c n l y , suggest t h a t both food r e s t r i c t i o n and l y s i n e d e f i c i e n c y of dams during g e s t a t i o n a f f e c t b i r t h weight of o f f s p r i n g . P r e v i o u s r e p o r t s have a s s o c i a t e d a decrease i n b i r t h weight of o f f s p r i n g with decreased food i n t a k e by dams during g e s t a t i o n (Barry, 1920; Niiyama e t a l , , 1973). Chow and lee (1964) have reported a r e d u c t i o n i n f e t a l body weight a s s o c i a t e d with a 25% decrease i n food i n t a k e by r a t s during pregnancy. These i n v e s t i g a t o r s expressed food i n t a k e by dams du r i n g pregnancy as the average d a i l y f o r the t o t a l p e r i o d of g e s t a t i o n . . Food consumption and weight gain d u r i n g pregnancy were a f f e c t e d by d i e t a r y treatment.. L y s i n e d e f i c i e n t animals and t h e i r r e s p e c t i v e p a i r - f e d groups consumed s i g n i f i c a n t l y l e s s food (g/day) than c o n t r o l animals f e d the l y s i n e supplemented d i e t . The reduced food i n t a k e of the l c w - l y s i n e group of dams i s thought t o be due to decreased a p p e t i t e as a r e s u l t of plasma amino a c i d imbalance caused by t h e l y s i n e d e f i c i e n c y (Sanahuja and Harper, 1963; Leung et a l . , 1 968). Dams consuming s i g n i f i c a n t l y l e s s food than c o n t r o l s d u r i n g pregnancy a l s o gained s i g n i f i c a n t l y l e s s weight than c o n t r o l s . Although there was no s i g n i f i c a n t d i f f e r e n c e between the amount of weight gained by the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups, dams f e d the h i g h - l y s i n e r e s t r i c t e d d i e t gained mere weight than those dams consuming the l c w - l y s i n e d i e t , suggesting t h a t weight g a i n of dams during pregnancy was a f f e c t e d by t o t a l 102 food i n t a k e and d i e t a r y l y s i n e l e v e l . Stapleton and H i l l (1980) p r e v i o u s l y r e p o r t e d that the weight gain of l y s i n e d e f i c i e n t dams during pregnancy was s i g n i f i c a n t l y l e s s than t h a t of l y s i n e supplemented c o n t r o l s . . DIETARY LYSINE AND LACTATION PERFORMANCE S e v e r a l authors are of the o p i n i o n that n u r s i n g r a t s should i n c r e a s e t h e i r body f a t and weight by as much as n o n - l a c t a t i n g c o n t r o l r a t s d u r i n g the same p e r i o d , s i n c e a l o s s i n body weight i n d i c a t e s t h a t maternal body t i s s u e s r a t h e r than d i e t a r y s u p p l i e s are used f o r milk p r o d u c t i o n (Kon and Cowie, 1961; Munro and A l l i s o n , 1964). _ I n t h i s study the h i g h - l y s i n e c o n t r o l s gained weight during l a c t a t i o n while dams i n the low-l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups l o s t weight, such r e s u l t s c o n f i r m e a r l i e r r e p o r t s by Sta p l e t o n and H i l l (1980), and suggest u t i l i z a t i o n of body t i s s u e s by the l y s i n e d e f i c i e n t and c a l o r i e r e s t r i c t e d groups* Change i n body weight d u r i n g l a c t a t i o n appears t o be dependent on the amount of food consumed d u r i n g t h i s p e r i o d . Absolute food i n t a k e was g r e a t e s t f o r the h i g h - l y s i n e c o n t r o l s again c o n f i r m i n g the r e s u l t s of S t a p l e t o n and H i l l ( 1972, 1980). Food i n t a k e during l a c t a t i o n , when expressed i n p r o p o r t i o n t o body weight, was also s i g n i f i c a n t l y g r e a t e r f o r the h i g h - l y s i n e animals than f o r the the l o w - l y s i n e c r h i g h - l y s i n e r e s t r i c t e d groups. Jansen and Chase (1976) a l s o noted a s i g n i f i c a n t r e d u c t i o n i n food i n t a k e during l a c t a t i o n i n dams fed a b a s a l bread d i e t , 103 d e f i c i e n t i n l y s i n e . I t i s i n t e r e s t i n g to note t h a t l y s i n e f o r t i f i c a t i o n of the basal bread d i e t i n c r e a s e d food i n t a k e and decreased maternal weight l o s s d u r i n g l a c t a t i o n . Weight l o s s which occurs d u r i n g l a c t a t i o n , when d i e t a r y i n t a k e i s r e s t r i c t e d , i s probably due to i n c r e a s e d u t i l i z a t i o n of body f a t . . Naismith (1971) r e p o r t e d that l a c t a t i n g r a t s fed 25% p r o t e i n d i e t s l o s t no body p r o t e i n but l o s t 70% of the f a t accumulated d u r i n g pregnancy. Eats f e d a low p r o t e i n d i e t l o s t 10% of t h e i r body p r o t e i n . . I t i s h i g h l y u n l i k e l y t h a t a l a c t a t i n g dam with small f a t s t o r e s or a reduced food i n t a k e would be able t o produce milk t o the same extent as a dam with l a r g e body s t o r e s of f a t (accumulated during pregnancy) and a s u f f i c i e n t food i n t a k e . This may be r e f l e c t e d i n the d e f i c i e n t animals which not onl y weighed s i g n i f i c a n t l y l e s s than the h i g h - l y s i n e c o n t r o l s on day 1 of l a c t a t i o n but a l s o consumed s i g n i f i c a n t l y l e s s food during l a c t a t i o n * . The lower body weight of the l y s i n e d e f i c i e n t dams at the end of g e s t a t i o n i n experiment I I I compared t o the l y s i n e d e f i c i e n t dams i n experiment I I , i n d i c a t e s a lower accumulation of body f a t by the former group. However growth r a t e s of o f f s p r i n g of bcth groups were s i m i l a r . Animals with the lower body f a t s t o r e s compensated by i n c r e a s i n g t h e i r food i n t a k e during l a c t a t i o n * T h e r e f o r e , even though energy r e s e r v e s were lower f o r one group of l y s i n e d e f i c i e n t animals, these dams were able t o ma i n t a i n the same l a c t a t i o n performance by i n c r e a s i n g s l i g h t l y t h e i r food i n t a k e d u r i n g l a c t a t i o n . . A r e l a t i o n s h i p between food i n t a k e d u r i n g l a c t a t i o n and 104 s u c c e s s f u l milk production has o f t e n teen r e p o r t e d . Growth impairment was observed i n o f f s p r i n g cf dams whose food i n t a k e was r e s t r i c t e d during both pregnancy and l a c t a t i o n (Chow and Lee, 1964) . L a c t a t i n g r a t s placed on a low p r o t e i n d i e t produced s i g n i f i c a n t l y l e s s milk than those f e d a high p r o t e i n d i e t (Mueller and Cox, 1946) . T h i s decrease i n milk volume caused lower weaning weights i n o f f s p r i n g . S t a p l e t o n and H i l l (1980) examined the e f f e c t s of a l y s i n e d e f i c i e n t d i e t on milk p r o d u c t i o n - When compared with milk produced by dams f e d a l y s i n e supplemented d i e t , t h e r e was a s i g n i f i c a n t r e d u c t i o n i n the amount of milk produced and i n the g u a n t i t y of p r o t e i n i n the milk, of dams f e d a l o w - l y s i n e d i e t * . S i m i l a r l y weaning weights o f pups from dams fed the l y s i n e d e f i c i e n t d i e t were s i g n i f i c a n t l y lower than t h a t o f c o n t r o l s . In the present study, pups c f mothers f e d the l o w - l y s i n e r a t i o n s weighed s i g n i f i c a n t l y l e s s than pups from the high-l y s i n e c o n t r o l females on day 15 of l a c t a t i o n . Food i n t a k e was a l s o s i g n i f i c a n t l y reduced f o r the l o w - l y s i n e dams, as was the case i n the s t u d i e s r e p o r t e d by S t a p l e t o n and H i l l ( 1972, 1980). Thus i t appears that a decrease i n the amount of milk produced and i n the p r o t e i n content of t h e milk produced by dams fed the l o w - l y s i n e d i e t a l s o occurred i n t h i s study. Body weight of the t h i r d group of pups, h i g h - l y s i n e r e s t r i c t e d , on day 15 of l a c t a t i o n was s i g n i f i c a n t l y g r e a t e r than the l o w - l y s i n e pups yet s i g n i f i c a n t l y l e s s than the h i g h - l y s i n e c o n t r o l pups, i n d i c a t i n g that both food i n t a k e and l y s i n e d e f i c i e n c y a f f e c t s milk production of the mother r a t . . 105 DIET AND TISSUE HEIGHTS L i v e r weight i n dams, neonates, and f e t u s e s was higher i n the h i g h - l y s i n e c o n t r o l group than i n the l o w - l y s i n e and high-l y s i n e r e s t r i c t e d groups. . However, when l i v e r weights were expressed as a percentage of body weight t h e r e was no s i g n i f i c a n t d i f f e r e n c e among the d i e t a r y groups.. These r e s u l t s suggest t h a t the animal's l i v e r weight was p r o p o r t i o n a l t o the t o t a l body weight. L y s i n e d e f i c i e n c y d i d not r e t a r d heart weight of dams or t h e i x f e t u s e s during pregnancy.. However, on day 15 of l a c t a t i o n heart weight of the l o w - l y s i n e pups was s i g n i f i c a n t l y l e s s than c o n t r o l values whereas, heart weights of mothers were not a f f e c t e d by l y s i n e d e f i c i e n c y . When heart weights were expressed i n p r o p o r t i o n to body weight, f o r both dams and t h e i r o f f s p r i n g , there was no s i g n i f i c a n t e f f e c t o f d i e t a r y treatment on heart weight cn day 15 of l a c t a t i o n * DIET Afijf LYSINE AND PLASMA AND LIVEE CARNITINE Dams The d i e t s c o n t a i n i n g 0.27% l y s i n e did not l i m i t plasma and l i v e r c a r n i t i n e l e v e l s of dams du r i n g g e s t a t i o n or l a c t a t i o n , under the experimental c o n d i t i o n s of t h i s study. .. On day 21 of pregnancy there was a smal l but i n s i g n i f i c a n t i n c r e a s e i n both plasma and l i v e r c a r n i t i n e c o n c e n t r a t i o n s i n those dams fed the l o w - l y s i n e d i e t . The i n c r e a s e i n plasma and l i v e r c a r n i t i n e i n 106 V the l y s i n e d e f i c i e n t dams and t h e i r p a i r - f e d c o n t r o l s on day 15 of l a c t a t i o n was s i g n i f i c a n t . Plasma c a r n i t i n e during pregnancy and l i v e r c a r n i t i n e during l a c t a t i o n , i n the h i g h - l y s i n e c o n t r o l animals, were i n the same range as those r e p o r t e d e a r l i e r by Robles-Valdes e t a l . (1976). However, l i v e r c a r n i t i n e during pregnancy and plasma c a r n i t i n e d u r i n g l a c t a t i o n , were approximately h a l f t h a t o f c o n t r o l values p r e v i o u s l y r e p o r t e d . The reason f o r the c o n t r a d i c t i o n s i n plasma and l i v e r c a r n i t i n e l e v e l s of c o n t r o l s animals between the present work and t h a t r e p o r t e d by Robles-Valdes et a l . (1976) i s not understood, and to t h i s author's knowledge, there i s no other source with which t c compare normal maternal t i s s u e c a r n i t i n e l e v e l s f o r r a t s . Tissue c a r n i t i n e l e v e l s d i d not d i f f e r s i g n i f i c a n t l y between the l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups on day 15 of l a c t a t i o n , suggesting t h a t t o t a l food i n t a k e was a dominant f a c t o r i n the r e g u l a t i o n of plasma and l i v e r c a r n i t i n e l e v e l s . The c a r n i t i n e a c y l t r a n s f e r a s e system i s r e g u i r e d f o r the i n t r a m i t o c o n d r i a l t r a n s p o r t and o x i d a t i o n of long-Chain f a t t y a c i d s ( F r i t z and Yue, 1963) and i n t e r e s t i n g l y , o x i d a t i o n of p a l m i t a t e by heart muscle has been shown to i n c r e a s e when food i n t a k e i s r e s t r i c t e d (Khan and Bamji, 1979). Furthermore, McGarry e t a l . , (1975) r e p o r t e d t h a t the ketogenic c a p a c i t y of h e p a t i c t i s s u e of f a s t e d weaned r a t s c o r r e l a t e d with c a r n i t i n e c o n c e n t r a t i o n s . Therefore, i f d i e t a r y l y s i n e i s not l i m i t i n g c a r n i t i n e s y n t h e s i s by the l i v e r , an i n c r e a s e i n t i s s u e c a r n i t i n e . l e v e l s may be expected under c o n d i t i o n s of i n c r e a s e d f a t o x i d a t i o n . 1C7 T a n p h a i c h i t r et a l . . (1976) p r e v i o u s l y noted a s i g n i f i c a n t i n c r e a s e i n plasma c a r n i t i n e l e v e l s i n non-pregnant, post weaning female r a t s consuming a l y s i n e d e f i c i e n t d i e t . When compared with l y s i n e supplemented c o n t r o l s , l i v e r c a r n i t i n e l e v e l s were a l s o i n c r e a s e d i n the d e f i c i e n t females. Only l i v e r c a r n i t i n e l e v e l s were i n c r e a s e d i n male weanling r a t s fed a low-l y s i n e d i e t . This d i f f e r e n c e i n response to d i e t a r y l y s i n e between male and female r a t s suggests that other f a c t o r s , i . e . , sex of the animal, are i n v o l v e d i n the r e g u l a t i o n of t i s s u e c a r n i t i n e v a l u e s . The r e s u l t s of the present study suggest t h a t t i s s u e c a r n i t i n e l e v e l s f o r dams, a t l e a s t during l a c t a t i o n , were a r e f l e c t i o n of a l t e r a t i o n s i n f a t metabolism caused by depressed food i n t a k e . Animals f e d d i e t s t h a t r e s t r i c t e d food i n t a k e , l o s t body weight during t h i s p e r i o d , presumably due t o i n c r e a s e d o x i d a t i o n of body f a t i Plasma and l i v e r c a r n i t i n e l e v e l s were s i g n i f i c a n t l y higher i n these animals than i n c o n t r o l animals who, i n f a c t gained weight d u r i n g l a c t a t i o n . Fetus and Neonates A maternal l y s i n e d e f i c i e n c y d i d not l i m i t plasma or l i v e r c a r n i t i n e l e v e l s i n e i t h e r f e t u s e s or o f f s p r i n g . In f a c t t i s s u e c a r n i t i n e l e v e l s were higher i n the l c w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups than i n the c o n t r o l s , i n d i c a t i n g t h a t food i n t a k e r a t h e r than d i e t a r y l y s i n e l e v e l s a f f e c t s t i s s u e c a r n i t i n e l e v e l s . Although f e t a l plasma and l i v e r c a r n i t i n e l e v e l s were 108 higher i n the l o w - l y s i n e group than i n the h i g h - l y s i n e group, only the plasma l e v e l was s i g n i f i c a n t l y higher* I t i s i n t e r e s t i n g t o note that the c a r n i t i n e l e v e l s i n f e t a l t i s s u e s are a r e f l e c t i o n of maternal plasma c a r n i t i n e l e v e l s , even though p l a c e n t a l t r a n s f e r of c a r n i t i n e i s l i m i t e d i n the r a t (Hahn and S k a l a , 1975)* There i s no evidence at p r e s e n t t o i n d i c a t e f e t a l s y n t h e s i s of c a r n i t i n e i n mammals.. The primary metabolic f u e l f o r the f e t a l t i s s u e i s maternally d e r i v e d glucose* F e t a l c a r n i t i n e requirements a r e low, as i s the c a p a c i t y f o r the r a t f e t u s to o x i d i z e long c h a i n f a t t y a c i d s (Drahota e t a l * , 1964; ftugenfeld and F r i t z , 1970; lockwood and B a i l e y , 1971). One would expect c a r n i t i n e l e v e l s to be lower i n the f e t u s than i n animals u t i l i z i n g f a t as a major source of energy* T h e r e f o r e , i t i s not s u r p r i s i n g , t h a t f e t a l plasma and l i v e r c a r n i t i n e i n h i g h - l y s i n e c o n t r o l animals was approximately 5C% lower than that of c o n t r o l neonates on day 15 of l a c t a t i o n * These r e s u l t s confirm p r e v i o u s l y r e p o r t e d plasma and l i v e r c a r n i t i n e c o n c e n t r a t i o n s d u r i n g pre- and p o s t n a t a l development of the r a t (Eobles-Valdes et a l . , 1976; Borum, 1978; F e r r e e t a l . , 1978; Seccombe et a l . , 1978). Immediately a f t e r b i r t h the n e o n a t a l r a t r e c e i v e s a d i e t r i c h i n f a t , whic I a c t s as the main energy y i e l d i n g s u b s t r a t e (Dymsza, 1964; Luckey et a l . , 1954). Eat milk c o n t a i n s about 14% f a t and adequate amounts of c a r n i t i n e are needed to allow f o r f a t t y a c i d o x i d a t i o n to proceed at normal r a t e s . T h i s period of i n c r e a s e d f a t t y a c i d u t i l i z a t i o n i s c h a r a c t e r i z e d by r a i s e d plasma c o n c e n t r a t i o n s of ketone bodies (Drahota e t a l . . 109 1964; Lockwood and B a i l e y , 1971; F erre et a l . , 1978) and c a r n i t i n e , and i n c r e a s e d l i v e r c a r n i t i n e c o n c e n t r a t i o n (Robles-Valdes e t a l * , 1976 ; Borum, 1978; F erre et a l . , 1978). Plasma ketone and c a r n i t i n e l e v e l s and l i v e r c a r n i t i n e c o n c e n t r a t i o n s decrease when the r a t pup i s weaned from the high f a t milk d i e t to the h i g h carbohydrate d i e t (Lockwood and B a i l e y , 1971; Seccombe et a l . , 1978) . In the present study d i f f e r e n c e s i n t i s s u e c a r n i t i n e l e v e l s between l o w - l y s i n e and h i g h - l y s i n e r e s t r i c t e d pups were i n s i g n i f i c a n t . However, both groups had s i g n i f i c a n t l y higher l i v e r c a r n i t i n e l e v e l s compared to c o n t r o l s , which suggested i n c r e a s e d f a t t y a c i d o x i d a t i o n * In a d d i t i o n , pups from the low-l y s i n e and h i g h - l y s i n e r e s t r i c t e d groups weighed s i g n i f i c a n t l y l e s s than the h i g h - l y s i n e c o n t r o l s on day 15 of l a c t a t i o n . One can speculate t h a t the d e f i c i e n t groups of pups were f o r c e d t o u t i l i z e a l a r g e r percentage of t h e high f a t d i e t f o r immediate energy demands t h e r e f o r e i n c r e a s i n g t h e i r c a r n i t i n e requirements. The s u c k l i n g r a t ' s a b i l i t y t o synthesize c a r n i t i n e from i t s p r e c u r s o r s reaches a d u l t values by the second week of l a c t a t i o n (Hahn, unpublished d a t a ) . U n t i l then, the neonate depends on milk as i t s major source o f c a r n i t i n e (Hahn and S k a l a , 1975; Robles-Valdes e t a l . , 1976).. The r e s u l t s of t h i s study suggest that i n c r e a s e d c a r n i t i n e s y n t h e s i s during l a c t a t i o n o ccurs i n those dams having i n c r e a s e d u t i l i z a t i o n of f a t s t o r e s . In t u r n higher c i r c u l a t i n g l e v e l s of c a r n i t i n e are a v a i l a b l e f o r t r a n s p o r t a t i o n v i a t h e i r milk to nursing o f f s p r i n g whose c a r n i t i n e requirements are g r e a t e r . . Plasma c a r n i t i n e l e v e l s i n 110 l y s i n e d e f i c i e n t dams during l a c t a t i c n and pregnancy were s i m i l a r whereas, plasma c a r n i t i n e values i n c o n t r o l s d u r i n g l a c t a t i o n were 5C% of those i n c o n t r o l s during pregnancy, A s i m i l a r t rend was observed f o r l i v e r c a r n i t i n e v a l u e s , i . e . , t h e r e was l i t t l e change between pregnancy and l a c t a t i o n i n the l y s i n e d e f i c i e n t dams but c o n t r o l values dropped by 73% i n c o n t r o l s . _ MILK CARNITINE The primary source of c a r n i t i n e i n neona t a l t i s s u e , a t l e a s t 24 hours postpartum i s the milk from the mother r a t (Ferre e t a l . , 1978).. Milk c a r n i t i n e c o n c e n t r a t i o n s i n the h i g h - l y s i n e c o n t r o l animals were hi g h e s t on day 2 o f . l a c t a t i o n and decreased by 71% and 75% on days 8 and 15 post partum r e s p e c t i v e l y . T h i s d e c l i n e i n c a r n i t i n e c o n c e n t r a t i o n during l a c t a t i o n i s i n agreement with the r e s u l t s of Bobles-Valdes e t a l . (1976) , and may occur because the aging pup i s l e s s dependent cn milk c a r n i t i n e s u p p l i e s because of i n s i t u c a r n i t i n e s y n t h e s i s . D i e t a r y l y s i n e d e f i c i e n c y i n c r e a s e d maternal plasma l e v e l s of c a r n i t i n e , which appears to i n c r e a s e milk c a r n i t i n e l e v e l s . The decrease i n milk c a r n i t i n e l e v e l s i n l y s i n e d e f i c i e n t dams was small and i n s i g n i f i c a n t d u r i n g the course of l a c t a t i o n . Milk c a r n i t i n e l e v e l s i n the l y s i n e d e f i c i e n t group on days 8 and 15 were s i g n i f i c a n t l y higher than those i n h i g h - l y s i n e c o n t r o l s . With the exce p t i o n of day 2, milk c a r n i t i n e values i n the h i g h - l y s i n e r e s t r i c t e d dams were s i m i l a r t c those i n the c o n t r o l s . Thus milk c a r n i t i n e l e v e l s appear to respond t o 111 d i e t a r y l y s i n e l e v e l s r a t h e r than to t o t a l food i n t a k e . However* on day 15 of l a c t a t i o n * milk c a r n i t i n e and plasma c a r n i t i n e , i n both dams and o f f s p r i n g , w i t h i n a d i e t a r y group, responded i n the same way to t h a t d i e t a r y treatment (see F i g u r e 16) . The higher milk c a r n i t i n e l e v e l s i n l y s i n e d e f i c i e n t dams probably c o n t r i b u t e d to the higher plasma and l i v e r l e v e l s i n t h e i r o f f s p r i n g . Movement of c a r n i t i n e from maternal l i v e r t o maternal plasma i n t o maternal milk and f i n a l l y i n t o neonatal l i v e r has been reported a f t e r mothers were i n j e c t e d with of 1 *C butyr o b e t a i n e , the immediate p r e c u r s o r of c a r n i t i n e (Robles-Valdes e t a l . , 1976) . . A problem e x i s t s when determining n u t r i e n t c o n c e n t r a t i o n s i n milk*. The volume of milk obtained v a r i e s and smal l q u a n t i t i e s of milk may have a c o n c e n t r a t i n g e f f e c t on the c a r n i t i n e l e v e l s , f o r example, at day 2 of l a c t a t i o n the c o n t r o l r a t s y i e l d e d the lowest q u a n t i t y of milk, whereas a t days 8 and 15 the l o w - l y s i n e f e d animals y i e l d e d the s m a l l e s t volume of milk. The s i g n i f i c a n c e of the low milk c a r n i t i n e c o n c e n t r a t i c n s in t he h i g h - l y s i n e r e s t r i c t e d dams on day 2 cannot be e x p l a i n e d a t t h i s time. The r e s u l t s of t h i s study i n d i c a t e t h a t maternal l y s i n e d e f i c i e n c y does not impair plasma and l i v e r c a r n i t i n e l e v e l s i n the dam, f e t u s or o f f s p r i n g * Plasma and l i v e r c a r n i t i n e was a c t u a l l y higher i n d e f i c i e n t animals, the higher l e v e l being i n 112 response t o a general food r e s t r i c t i o n r a t h e r than to a l y s i n e d e f i c i e n c y per se. . Further study r e g a r d i n g the metabolic a l t e r a t i o n s a s s o c i a t e d with changes i n t i s s u e c a r n i t i n e l e v e l s i n both the dams and t h e i r o f f s p r i n g during maternal l y s i n e d e f i c i e n c y i s warranted. The s i g n i f i c a n c e of d i e t a r y c a r n i t i n e f o r human newborns i s unknown* but s e v e r a l r e p o r t s (Hahn and S k a l a , 1975; Robles-Valdes et a l . , 1976), have i n d i c a t e d the importance of c a r n i t i n e i n the newborn mammal. C a r n i t i n e i s syn t h e s i z e d from the e s s e n t i a l amino a c i d l y s i n e . . One o f the main o b j e c t i v e s of t h i s study was t o examine the e f f e c t of a maternal l y s i n e d e f i c i e n c y i n the r a t on neonatal t i s s u e c a r n i t i n e l e v e l s . A d e f i c i e n c y of c a r n i t i n e may not develop i n the o f f s p r i n g of animals f e d a d i e t low i n l y s i n e and c o n t a i n i n g minimal c a r n i t i n e because of the supply of c a r n i t i n e i n the milk* . When compared to human newborns f e d human breast milk o r ecu's milk, plasma c a r n i t i n e and a c e t y l c a r n i t i n e l e v e l s i n i n f a n t s r e c e i v i n g a soybean based formula are s i g n i f i c a n t l y reduced (Ncvak e t a l . , 1979).. Soyabean based formulas have no d e t e c t a b l e c a r n i t i n e , whereas human b r e a s t milk and cow's milk c o n t a i n a s i g n i f i c a n t amount of c a r n i t i n e (Schmidt-Sommerfeld et a l . , 1978). I t i s probable that a c a r n i t i n e d e f i c i e n c y i n the r a t co u l d be induced under c o n d i t i o n s of a r t i f i c i a l f e e d i n g . The i m p l i c a t i o n f o r the human i n f a n t i s t h a t a baby f e d human b r e a s t milk or cow's milk i s p r o t e c t e d a g a i n s t a c a r n i t i n e d e f i c i e n c y . 113 Since f a t t y a c i d o x i d a t i o n i s the main pathway by which energy i s produced by the neonate, d i e t a r y i n t a k e of c a r n i t i n e may be more important du r i n g t h i s p e r i o d than p r e n a t a l l y or i n the a d u l t . Thus the r e s u l t s of t h i s study suggest t h a t c a r n i t i n e may only be an e s s e n t i a l d i e t a r y n u t r i e n t f o r the neonate, i f a r t i f i c i a l f e e d i n g p r a c t i c e s are used. Further study may i n d i c a t e whether or not the e f f e c t o f decreased t i s s u e c a r n i t i n e l e v e l s i n the neonate has a d e t r i m e n t a l e f f e c t on the newborn's a b i l i t y t o u t i l i z e f a t t y a c i d s as a source of energy. 114 CHAPTER VI SUMMARY OF RESULTS 1) A maternal l y s i n e d e f i c i e n c y s i g n i f i c a n t l y decreased weight gain and d a i l y food i n t a k e (g/day) of dams durin g pregnancy. Weight gain of r e s t r i c t e d c o n t r o l dams was a l s o s i g n i f i c a n t l y lower. 2) A maternal l y s i n e d e f i c i e n c y or maternal food r e s t r i c t i o n d u r i n g pregnancy did not a f f e c t l i t t e r s i z e . However, b i r t h weight of o f f s p r i n g was s i g n i f i c a n t l y decreased.. 3) A maternal l y s i n e d e f i c i e n c y d i d not r e t a r d h e a r t weight i n dams or t h e i r f e t u s e s during pregnancy. However, l i v e r weights of dams and fe t u s e s were s i g n i f i c a n t l y lower i n the l y s i n e d e f i c i e n t group. 4) A maternal l y s i n e d e f i c i e n c y d u r i n g pregnancy tended to i n c r e a s e plasma and l i v e r c a r n i t i n e l e v e l s of dams on day 21 of pregnancy. . However, f e t a l plasma c a r n i t i n e l e v e l s were s i g n i f i c a n t l y i n c r e a s e d . 5) A maternal l y s i n e d e f i c i e n c y r e s u l t e d i n a s i g n i f i c a n t l y reduced 1 f o o d i n t a k e (g/day) and weight l o s s d u r i n g l a c t a t i o n , whereas l y s i n e supplemented dams gained weight during t h i s p e r i o d . . H i g h - l y s i n e r e s t r i c t e d dams al s o l o s t a s i g n i f i c a n t amount of weight during l a c t a t i o n . 115 6) A maternal l y s i n e d e f i c i e n c y d u r i n g pregnancy and l a c t a t i o n r e s u l t e d i n a s i g n i f i c a n t l y lower body weight of pups on day 15 of l a c t a t i o n - H i g h - l y s i n e r e s t r i c t e d pups a l s o weighed s i g n i f i c a n t l y l e s s than l y s i n e supplemented c o n t r o l s , but s i g n i f i c a n t l y more than the l o w - l y s i n e pups. 7) A maternal l y s i n e d e f i c i e n c y d u r i n g pregnancy and l a c t a t i o n s i g n i f i c a n t l y reduced l i v e r weight but net heart weight of dams on day 15 of l a c t a t i o n * L i v e r weights of h i g h - l y s i n e r e s t r i c t e d dams were a l s o s i g n i f i c a n t l y reduced. 8) L i v e r s of h i g h - l y s i n e r e s t r i c t e d pups weighed s i g n i f i c a n t l y l e s s than those of l y s i n e supplemented c o n t r o l s , but s i g n i f i c a n t l y more than those of the l y s i n e d e f i c i e n t group. Heart weights of l y s i n e d e f i c i e n t pups were s i g n i f i c a n t l y l e s s than those i n the h i g h - l y s i n e and h i g h - l y s i n e r e s t r i c t e d pups. 9) A maternal l y s i n e d e f i c i e n c y d u r i n g pregnancy and l a c t a t i o n s i g n i f i c a n t l y i n c r e a s e d plasma and l i v e r c a r n i t i n e on day 15 of l a c t a t i o n i n bcth dams and pups. . H i g h - l y s i n e r e s t r i c t e d dams and t h e i r o f f s p r i n g a l s o had s i g n i f i c a n t l y h i g h e r plasma and l i v e r c a r n i t i n e l e v e l s than l y s i n e supplemented c o n t r o l s . 10) A maternal l y s i n e d e f i c i e n c y d u r i n g pregnancy and l a c t a t i o n s i g n i f i c a n t l y i n c r e a s e d milk c a r n i t i n e c e n c e n t r a t i o n s on days 8 and 15 of l a c t a t i o n when compared t o milk of l y s i n e supplemented c o n t r o l s and h i g h - l y s i n e r e s t r i c t e d animals* On day 2 o f l a c t a t i o n , milk of l y s i n e supplemented c o n t r o l s c o n t a i n e d 116 s i g n i f i c a n t l y more c a r n i t i n e than milk cf l y s i n e d e f i c i e n t dams, which i n t u r n had a higher milk c a r n i t i n e c o n c e n t r a t i o n than h i g h - l y s i n e r e s t r i c t e d dams. 117 BIBLIOGRAPHY Abbey, H., and Howard, £.. (1973). 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