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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 T I S S U E  CARNITINE LEVELS IN THE RAT  by  MARY JANE B S c H E c . . Mount S a i n t  MUISE  TAYLOR  Vincent  University, 1 9 7 7  A THESIS SUBMITTED IN PARTIAL POLFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY Division  OF GRADUATE STUDIES o f Human N u t r i t i o n  S c h o o l o f Home E c o n o m i c s  We a c c e p t to  this  t h e s i s as conforming  the required  THE UNIVERSITY  standard  OF BRITISH COLUMBIA  April 1 9 8 0  Mary M u i s e  Taylor, 1 9 8 0  >E-6  In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I f u r t h e r agree that permission for extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by his representatives.  It i s understood that copying or p u b l i c a t i o n  of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department of ,  Home Economics  The U n i v e r s i t y of B r i t i s h Columbia 2075 wesbrook Place Vancouver, Canada V6T 1W5  BP  75-51 1 E  ABSTRACT The  effect  carnitine  of a  levels  dams, f e t u s e s and were  fed  diet to 20%  and  either  ad  o i l and  Dams  fed  food  pregnancy  intake, and  high-lysine gained  dams the  than  lactation  weight  lactation.  Litter deficiency  size or  gestation^ and  by  of  the  On  day  15  turn  suggesting the  weighed a  Liver  and  the  weight  en  by  gluten,  restricted  less  food  their  pair-fed  to  controls  pregnancy  either  in  during  When compared  offspring  and  lost  dams  gained  a dietary  lysine  intake  i n the  during  lew-lysine  significantly  the  the  lactation  tissue day  21  high-lysine  high-lysine  lower  performance f o r  diet  and the  pups  restricted  mere t h a n t h e  t h o s e dams c o n s u m i n g  offspring  high-lysine  wheat  high-lysine  g r o u p s was  control  heart  animals  pair-fed  i n t o t a l food  of  significantly  high-lysine  in  diet  or  dams.  during  affected  lactation  superior  levels  than  controls. .  more than  performance f o r  their  of  in lysine  dams and  small reduction  restricted  20%  significantly  weight  not  high-lysine  significantly in  the  contained  Ion  whereas  However, b i r t h  high-lysine  that  was  high-lysine  high-lysine  less  weight d u r i n g during  either  low-lysine  significantly  the  milk  carnitine.  consumed  lactation  or  on  Experimental  (0.27% l y s i n e ) * a  A l l diets  diet,  deficiency  liver carnitine  studied. .  libitum,  negligible a  lysine  p l a s m a and  a low-lysine diet  l o w - l y s i n e group.  corn  total  on  n e o n a t e s was  (1.07% l y s i n e )  the  maternal d i e t a r y  the  pups,  low-lysine those  poorest  low-lysine  s a m p l e s were o b t a i n e d of  p r e g n a n c y and  day  weighed  15  dams  which pups, fed  lactation  diet.  from of  dams  and  lactation.  iv  When l i v e r of  weight or h e a r t  weight  were e x p r e s s e d  t o t a l body w e i g h t f o r dams o r p u p s , no s i g n i f i c a n t  between d i e t a r y that  liver The  g r o u p s was  and h e a r t  on  fetal  carnitine  liver  showed  a  results  w e i g h t s were p r o p o r t i o n a l  t o body  had no s i g n i f i c a n t e f f e c t ,  maternal  small  detected^  plasma o r l i v e r  levels,  whereas  but s i g n i f i c a n t  g r o u p , . On day 15 o f l a c t a t i o n  levels  were s i g n i f i c a n t l y h i g h e r  low-lysine  increase  diet,  in plasna  a lowered  intake since  the  low-lysine  levels  plasma  to the high-  respective levels  was  r e s p o n s e as d i d a n i m a l s f e d t h e l o w - l y s i n e  high-lysine  group.  those  group  group and l o w e s t  The carnitine  results levels  of t h i s r e s e a r c h  diet  same  lcw-lysine  indicate  i n b o t h dams and o f f s p r i n g by t h e  lysine  diet tissue  diet.  restricted levels  diet  restricted  that  due  were h i g h e s t i n  milk c a r n i t i n e  or the h i g h - l y s i n e  l e v e l s i n dams, a r e n o t l i m i t e d maternal  the  i n the h i g h - l y s i n e  h i g h e r i n dams f e d t h e  fed the high-lysine  showed  on day 2 o f l a c t a t i o n  On d a y s 8 and 15 o f l a c t a t i o n  significantly  This  animals fed the high-lysine  carnitine  the  fed  probably  to  levels  carnitine  controls.  pair-fed  Milk c a r n i t i n e  o r on  carnitine  p l a s m a and l i v e r  carnitine  food  weight.  i n b o t h dams and o f f s p r i n g  than i n t h e i r  and l i v e r  carnitine  fetal  indicate  on d a y 21>of  i n c r e a s e compared  lysine  the  difference  These  low-lysine diet  gestation,  to  as a p e r c e n t a g e  than  were in  diet, .  p l a s m a and l i v e r  and milk content  under t h e e x p e r i m e n t a l c o n d i t i o n s o f t h i s  carnitine of  the  study.  V  TABLE OF CONTENTS  Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L i s t o f Tables i............ L i s t of Figures Acknowledgement ..............  1..  Chapter I ................................................. Introduction  i i i vii ix xi 1 1  Chapter I I ... .. 4 Review o f L i t e r a t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 F u n c t i o n o f C a r n i t i n e . . . . . . . . . . . . . . . . . . . . . . . . . . .. ... 4 C a r n i t i n e Metabolism ................................ 9 M a t e r n a l - N e o n a t a l 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 Postnatal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 M a t e r n a l 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 Diets .. 26 Animal Experiments . . . . . . . . . . . . . . . . . . . 26 E x p e r i m e n t I ......^..............................31 E x p e r i m e n t I I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Experiment I I I 33 P l a s m a , L i v e r , and M i l k 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 o f Assay 35 Preparation of Plasma C a r n i t i n e E x t r a c t - P a r t A, Section 1 36 Preparation of Milk C a r n i t i n e Extracts - Part A, Section 2 37 P r e p a r a t i o n o f 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 A s s a y P r o c e d u r e - P a r t B . . . . . . . i . 39 D e t e r m i n a t i o n o f C a r n i t i n e C o n c e n t r a t i o n ......... 42 R e c o v e r y ................................... ^ ...... .43 Statistics 43 Chapter IV i . 45 R e s u l t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 E x p e r i m e n t I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 E x p e r i m e n t I I ........................................57 E x p e r i m e n t I I I . . . . . . . . . . . . . . . . . . . . . . . . . . ^ . . . . . . . . . . . 76 Chapter V Discussion Dietary Dietary  ....100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 L y s i n e and R e p r o d u c t i o n ... . . . . . . . . . . . . . 100 L y s i n e and L a c t a t i o n P e r f o r m a n c e ..102  D i e t and T i s s u e H e i g h t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 5 D i e t a r y L y s i n e and P l a s m a and L i v e r C a r n i t i n e . . . . . . . 1 0 5 Dams 105 F e t u s and Neonates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 7 Milk Carnitine .........110 Chapter  VI  -: ................................ 1 1 4  Summary o f R e s u l t s Bibliography  .....................................II * 4  .-. . . . . . . . . . . . . . . . . . . . ^ . . . . . . . . . . ; . - . . . . . . . - - . 1 1 7  vii  LIST  OF TABLES  I  Composition o f l o w - l y s i n e  II  Calculated essential diets  die~t  amino  acid  27 composition  of 28  III  Food intake and w e i g h t l y s i n e or l o w - l y s i n e d i e t  IV  H e a r t and l i v e r w e i g h t s o f dams, f e d a h i g h - l y s i n e o r 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 o f a h i g h - l y s i n e o r l c w - l y s i n e d i e t on t h e number of f e t u s e s , and f e t a l heart and l i v e r w e i g h t s cn day 21 o f 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 low-lysine diet on plasma and l i v e r c a r n i t i n e o f dams and f e t u s e s on d a y 21 o f 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 o f dams f e d a highl y s i n e , low-lysine, or high-lysine 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 E x p e r i m e n t I I ................. 58  VIII  Food i n t a k e and w e i g h t c h a n g e s c f dams f e d 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 high-lysine lactation  X  Effect lysine of  g a i n o f dams f e d a h i g h d u r i n g g e s t a t i o n ....... 46  dams f e d a h i g h - l y s i n e , restricted diet until  low-lysine,or day 15 of 63  of a h i g h - l y s i n e , lcw-lysine* o r highr 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  dams i n E x p e r i m e n t  II ..................i.......  65  XI  E f f e c t of a high-lysine, lcw-lysine, or highl y s i n e r e s t r i c t e d m a t e r n a l d i e t on w e i g h t g a i n s o f pups i n E x p e r i m e n t I I . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66  XII  Effect of a high-lysine, lcw-lysine, or highl y s i n e r e s t r i c t e d m a t e r n a l d i e t on h e a r t and l i v e r w e i g h t s c f 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 , low-lysine, or highlysine restricted diet on p l a s m a and liver c a r n i t i n e of dams and p u p s on day 15 o f l a c t a t i o n 71  XIV  Food i n t a k e and w e i g h t g a i n o f dams fed a highl y s i n e * low-lysine, 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 E x p e r i m e n t I I I ............... 79  XV  Food intake and w e i g h t c h a n g e s o f dams m i l k e d on day 15 o f l a c t a t i o n and f e d a h i g h - l y s i n e * l e w -  viii  lysine, or lactation  high-lysine  restricted  diet  during 83  XVI  F o o d i n t a k e and weight c h a n g e s c f dams milked on day 15 o f l a c t a t i o n and f e d a h i g h - l y s i n e , lowlysine or h i g h - l y s i n e restricted diet during lactation 85  XVII  Effect of a high-lysine, lcw-lysine, o r highl 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 o f dams i n E x p e r i m e n t I I I . . . . . . . . . . . . . . . . . . . . . . . . 87  XVIII E f f e c t of a high-lysine, lcw-lysine, or highl y s i n e r e s t r i c t e d m a t e r n a l d i e t on w e i g h t g a i n s o f pups i n E x p e r i m e n t I I I 88 XIX  Effect of a h i g h - l y s i n e , low-lysine, l y s i n e r e s t r i c t e d d i e t on m i l k c a r n i t i n e 2, 8, and 15 p o s t p a r t u m  or on  highdays ....94  ix  L I S T 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 oxidation . . .. . . . . . . . . . . . . . . . .  2  Biosynthesis  o f c a r n i t i n e f r o m l y s i n e and m e t h i o n i n e  3  Diagramatic representation III ...  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 g a i n o f dams d u r i n g g e s t a t i o n  6 10  o f e x p e r i m e n t s I , I I , and ...... 29 d i e t on w e i g h t 47  5  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 p l a s m a and l i v e r c a r n i t i n e i n dams on d a y 21 o f g e s t a t i o n .... 53  6  E f f e c t o f a h i g h - l y s i n e o r l o w - l y s i n e d i e t on p l a s m a and liver carnitine i n fetuses on d a y 21 of g e s t a t i o n ..................  55  Effect o f 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 restricted diet on weight gain o f dams during g e s t a t i o n i n Experiment I I . . . . . . . . . . . . . . . . . . . . . . . .  60  Effect of a high-lysine, lcw-lysine, or high-lysine r e s t r i c t e d m a t e r n a l d i e t on w e i g h t g a i n of pups i n Experiment I I .......................................  67  Effect of a high-lysine, lcw-lysine, or high-lysine r e s t r i c t e d d i e t on p l a s m a and l i v e r c a r n i t i n e of dams on day 15 o f l a c t a t i o n  72  7  8  9  10  Effect of a h i g h - l y s i n e , low-lysine, or high-lysine restricted maternal d i e t on plasma and liver c a r n i t i n e o f pups on day 15 o f l a c t a t i o n . ............ 74  11  Effect of a high-lysine, low-lysine* or high-lysine r e s t r i c t e d d i e t on w e i g h t changes o f dams during p r e g n a n c y and l a c t a t i o n i n E x p e r i m e n t I I . . . . . . . . . . . . 77  12  Effect of a high-lysine, low-lysine, or high-lysine restricted diet on w e i g h t gain o f dams during g e s t a t i o n i n E x p e r i m e n t I I I ...........^.............81  13  Effect of a high-lysine, low-lysine, or high-lysine r e s t r i c t e d m a t e r n a l d i e t on weight gains o f pups born t o t h o s e dams m i l k e d on day 15 o f l a c t a t i o n .... 89  14  Effect of a high-lysine, low-lysine, or high-lysine r e s t r i c t e d m a t e r n a l d i e t oh w e i g h t g a i n o f pups b o r n t o t h o s e dams m i l k e d on d a y 8 c f 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 high-lysine r e s t r i c t e d d i e t on m i l k c a r n i t i n e on d a y s 2, 8, a n d , 15 o f l a c t a t i o n  95  Effect of a high-lysine, low-lysine, or h i g h - l y s i n e restricted dist on maternal plasma and milk carnitine i n dams, and pup p l a s m a c a r n i t i n e on day 15 o f l a c t a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xi  ACKNOWLEDGEMENT I  would  Division his  like  suggestions  of  would l i k e of  are  the and  financial  C o l u m b i a and  Nadia  thank love  my  P.  of  V.  British  James,  Arts  Computing,  and  assistance  thesis.  Also  Hahn,  fcr  their  instructions  assay. .  support  Bursary  (1S77-1978)  a " h e l p i n g hand"  Finally,  Department  Dri  University  Lewis  of  the  o f Canada (1979),  t o whom I am  Natural  Sciences  (1978-1979),  the  and t h e U n i v e r s i t y  are g r a t e f u l l y  S p e c i a l t h a n k s a r e e x t e n d e d t o my providing  Hahn,  helpful  appreciated;.  Research C o u n c i l  Assistantship  The  members o f Dr.. Hahn's l a b , p a r t i c u l a r l y  the c a r n i t i n e  Student  P..  Stapleton,  Columbia, f o r  support.  programs used t o c o m p l e t e t h i s  concerning  Graduate  and  Columbia, f o r h i s s k i l l s  Smale  Engineering  D r . . P..  of B r i t i s h  Nutrition,  t o acknowledge  Freda-Ann  The  University  of B r i t i s h  Human  British  t h e computer  acknowledged  advisor  advise,  University  are gratefully  University  my  my c o m m i t t e e members, Br.  Division  Columbia,  with  guidance,  of  of P e d i a t r i c s ,  I  thank  o f Human N u t r i t i o n ,  invaluable  Gallo,  to  when i t was especially  husband, P a u l T a y l o r ,  and  Summer Teaching  acknowledged.  sister, needed  Audrey M u i s e , f o r most.. like  to  f o r h i s h e l p , encouragement,  and  throughout the c o u r s e o f t h i s  grateful,  s t u d y . '.  I would  To my Gerald for  a lifetime  and  parents Mary  of support  Muise and  encouragement  1  CHAPTER I INTRODUCTION  Carnitine role  in  the  oxidation  of  is  (B-hydroxy-ytrimethylaminobutyrate) plays a intramitochondrial lcng-chain  synthesized  essential Carnitine,  fatty acids  from l y s i n e  dietary  and m e t h i o n i n e ,  amino  acids  carnitine  one.  to lysine i s similiar  and  present  i n the diet, carnitine i n t h e body  carnitine that for  tryptophan.  It  is  carnitine  deficiency  known  much  which  lysine  of  i t s role an  i n tissues  i n fatty acid  impairment  in  and B r e s s l e r ,  lysine in  is  adequate  i s  required  for  (1971) have e s t i m a t e d crassa  carnitine whereas p l a n t  (Fraenkel, likely  oxidation, fat  1 9 6 4 ) , and  generally  result  between  1973; B r o g u i s t ,  i s used  i s foods  carnitine  human  obtained  1954; P a n t e r a n d Mudd,  196S).  Diets  Fat  (Isaacs  et  deficiencies.  poor  of c a r n i t i n e . .  carnitine  e t a l . , 1976),  usually  t o be d e f i c i e n t  are  a  utilization.  of the r a t (Tanphaichitr  Dietary  precursors  1975). i s not  exists  o f Neurospora  with  also  are  the r e l a t i o n s h i p of  synthesized  1976) h a s been a s s o c i a t e d  carnitine  vitamin  and E r o q u i s t ,  requirement  al*,.  i f sufficient  c a n be  how  which  synthesis..  pig (Wittles  foodstuffs,  may  not  may c a u s e  accumulation  are  that  Therefore  subseguent  of et  of a  s y n t h e s i s a l t h o u g h Horne e t a l .  Because  al.,  (Broguist  to  (Tanphaichitr  0.2% o f t h e l y s i n e  guinea  both  I t h a s been s u g g e s t e d t h a t  niacin  1976).  and  ( F r i t z and Yue, 1963) . . I t  although having the a t t r i b u t e s  considered  amounts  transport  vital  from  animal  sources Plant  of  foods  i n lysine  and/or  methionine, the  comprised  mainly  of plant  i n s i g n i f i c a n t l y lowered  plasma  carnitine  foods  levels i n  2  humans  (Mikhail  and Mansour,  Availability  of  1976; L a t i f a and B a m j i ,  carnitine  is  of  prime  physiological  conditions that  are associated  metabolisnu  Pregnancy  lactation  physiological  conditions, . Since the neonatal  of  i t s energy  impair  from  utilization  development  of  and  fat  are  the neonate.  and  examples  such  derives  most  of c a r n i t i n e  could  thus  adversely  carnitine  for  carnitine  synthesis  appears i n s i g n i f i c a n t , a t l e a s t  stages of lactation  contain  lower  mainly  from  levels  sources  carnitine  (Hahn  of  maternal  carnitine  In  relationship  the post-weanling heart  control  values. .  differently female  Broquist,  during the  Fetal  limited  Skala,  could  between  but  male  tissues  by  1975).  result  to  Female  dietary  in  placental Therefore a  a  dietary  muscle c a r n i t i n e  rats  of  the  a lysine deficiency.  was n o t a f f e c t e d 1977).  carnitine  levels  lower  in  to  tissue  or l a c t a t i n g r a t . lysine  deficiency  by 25% compared t o  same  age,  lysine  levels  the respective  were h i g h e r  and  responded  Heart muscle c a r n i t i n e o f  by d i e t a r y  Compared  lysine  i n t h e pregnant  rat, a  and s k e l e t a l  plasma c a r n i t i n e rats,  is  and  h a s n o t been s t u d i e d  reduced  the  i  neonatal  which a p p e a r s t o be d e r i v e d  and  deficiency  because  i n the offspring..  The carnitine  rat  e t a l . , 1978)  of c a r n i t i n e ,  maternal  transfer  deficiency  (Ferre  affect  p r o v i d e s t h e major  source of  early  suckling  in  of  rat  The dam's milk  the  importance  w i t h changes i n f a t  milk f a t , a d e f i c i e n c y  of d i e t a r y  1977),.  in  the  control  plasma  t h e p l a s m a o f male r a t s ,  (Borum  of  and  values, female  fed a low-lysine  diet. Stapleton  and  Hill  (1972,  1980)  previously  reported  a  3  significant  drop  i n plasma l y s i n e  gestation  i n dams f e d a d i e t  negligable  carnitine  lysine. lysine the  However, deficient  lysine  dietary the  maternal  status  of protein  deficient  a  dependency  supply  the  of  the  21  of b)  of  supplemented  that  adversely  levels  with i n the  neonate  milk.  and q u a l i t y o f  effected  The e f f e c t s  by  the  of feeding a  and c o n t a i n i n g  lactation,  and  her  such  a  on  negligible  the  offspring dietary  carnitine  i s n o t known.  condition evidence  does  suggests  on t h e mother r a t f o r i t s major  objectives  of  of a maternal l y s i n e  carnitine  in  p r o d u c t i o n was r e d u c e d , a s was  t o meet p h y s i o l o g i c a l  the e f f e c t s  tissue  diet  week o f  and c o n t a i n i n g  The q u a n t i t y  lysine  and  mother  T h e r e f o r e t h e major  a)  i n lysine  the n e o n a t e i s p r o p o s e d s i n c e  of c a r n i t i n e  determine  in  gestation  both  affect  the l a s t  i n plasma l y s i n e  animals.  i n their  However, t h e p o s s i b i l i t y adversely  during  a n i m a l s was s i g n i f i c a n t l y g r e a t e r t h a n t h a t  during  of  decrease  r e s t r i c t i o n . . Milk  diet  carnitine,  the  by t h e dams was a l s o  lysine  quantity  deficient  o r t h e same b a s a l  supplemented  milk produced  levels  requirements. this  thesis  deficiency  were  to  on:  levels  o f dams and t h e i r f e t u s e s on day  levels  o f dams and t h e i r  gestation. tissue  carnitine  pups on day 15  lactation. c)  milk  lactation.  carnitine  levels  o f dams on days 2,  8,  and  15  of  4  CHAPTER REVIEW OF  FUNCTION OF  existence  of  trimethylaminobutyrate  was  many  its  years  before  1957) .  mealworm T e n e b r i o Blewett,  1946)  mplitar,  the  and  extensive  the  the  rate  of  study of the  specifically  and  activated  One  acid of  by  1964;  i t  was  and  BT  (T s t a n d i n g  for  was  recognized  as  first  Fritz  observations in  liver  oxidation.  Short  chain  oxidation  B r e s s l e r and  intra-mitochondrial  Wittles,  1955  who  homcgenates  This  led to  an  metabolism,  fatty  (Fritz  enzymes  the  (Fraenkel  of the by  -  established  carnitine in fat  acids.  carnitine for their Bressler,  1946  c a r n i t i n e to r a t  fatty  fatty  finally  BT  c a r n i t i n e was of  IfJOO's b u t  Vitamin  vitamin  function  long-chain  require  Wittles  addition  was  i s o l a t e d in  years l a t e r  of  early  e s s e n t i a l growth f a c t o r f o r  t e n t a t i v e l y named  function  that  increased  not  structure  ( C a r t e r e t a l . , 1952).  regarding reported  known i n t h e  was  B -hydroxy  carnitine,  C a r n i t i n e , an  Tenebrio)« . S e v e r a l carnitine  LITERATURE  CARNITINE  The  (Fraenkel,  II  et  acids  al.,  1962;  and  are  1965)  (Rossi  do  and  Gibson,  1964 ; B r e m e r , 1 968) . Long-chain  fatty  a c i d s , the  metabolism  in  fatty  bound t o a l b u m i n  et  acids  al.,  important  1958;  tissues,  are  Fredrickson  r o l e i n the  principal  transported (Dole, et  transport  1956;  fuel i n the  Gordon,  a l . , 1958). of  the  for  oxidative  plasma as 1957;  Carnitine  activated  free  Gordon plays  an  long-chain  5  fatty  acyl  groups  from  mitochondrial matrix the  enzymes  Activation coenzyme  cf  occurs A  (Fritz  the  site  B-oxidation  are  takes  p l a c e i n both  activating endoplasmic  system  r e t i c u l u m with t h e (Norum  inner  i s acylated  (Rossi  1968).  extra-  and  Gibson,  The l o n g - c h a i n  i s localized remaining  mainly  fatty  (70S) i n  activity  in  e t a l . , 1966; F a r s t a d e t a l . , 1 967),.. i s found  in  the  to  outer  the Most  membrane  (Norum e t a l . , 1966).,  The mediated  mitochondria  i n the l i v e r  of t h e mitochondrial a c t i v i t y fraction  1968).  (Figure 1).. This process the  the where  (Eeattie,  acid  e t a l . , 1966; Bremer,  mitochondria  1975),  fatty  1964;  the  located  to  when t h e l o n g - c h a i n  s i t e s and  acid  activation  and Yue, 1963; Pande,  mitochondrial Norum  of  transport  of acyl-coenzyme  by t h e f o r m a t i o n  membrane  of a c y l c a r n i t i n e .  Garland,  1966) b u t i t i s i m p e r m e a b l e t o a c y l - c o e n z y m e  A and f r e e  coenzyme  A  1966),..  T r a n s a c y l a t i o n o f t h e coenyme  mediated  by two d i s t i n c t l y  ( Y a t e s and  One enzyme*  Garland,  carnitine  fatty  to  carnitine  mitochondrial ( Y a t e s and  Klingenberg  permeable  The  esters  long-chain  i s  A i n t o the mitochondria i s  1966;  separate  and  A and c a r n i t i n e  enzymes  A  Pfaff,  esters i s  (Figure 1).  palmityltransferase i s  acyl-coenzyme  and E f a f f  Klingenberg  (Fritz  specific  and  Yue,  for 1963;  1966).  palmitlytransferase palmityl-coenzyme  A •  (-)-carnitine  ( - ) - p a l m i t y l c a r n i t i n e + coenzyme  The e f f e c t  of carnitine  on f a t t y  ASH  acid  o x i d a t i o n i s m e d i a t e d by  >  6  Figure  1  The f u n c t i o n oxidation  of c a r n i t i n e i n l o n g - c h a i n  fatty  acids  Acylcarnitine CoA  Long-chain Fatty Acid a c t i v a t i n g system  Long-chain carnitine acyl transferase A  Long-chain carnitine acyl transferase B  CoA  Carnitine Acyl  'Acyl  CoA  Acetyl  CoA  CoA  Free F a t t y Acid  Inner carnitine I Lacetyltranferasei Carnitine-acyl c a r n i t i n e translocase  CoA  Ketone Bodies +  Acetyl Carnitine  Outer Mitochondrial  Space  M i t o c h o n d r i a l Membrane  Matrix  Co„  8  this  enzyme  carnitine. localized  which t r a n s f e r s  The on  the  Tomac, 1 9 7 2 ) . is  enzyme, cuter  The  converted  oxidized  acyl by  coenzyme A..  T h e s e two  b)  be  used  c)  or  be  second  acetyl  1962;  B-oxidation  enter  The  Fritz,  A  the  inner  crosses the  the  ( H o p p e l and within two  the  A  in the  (Hoppel  and  membrane  and  by  Tomac,  1972).  production  coenzyme A and  The  out  fragments  to  of  and  of  Ketone  the  bodies  mitochondria.  acetyltransferase, chain  a c y l coenzyme  Yue,  1963;  Norum  carnitine  and  specific  A  (Bremer,  Bremer,  1963).  acetyltransferase .  carnitine acetyltransferase Several  researchers  functions  between t h e i r s i t e  of f o r m a t i o n  space  >  interesting  that  as  has  has  a transporter by  been  B-oxidation, 1970)  .  or  no  outer  carnitine  of a c e t y l to  the  However,  been s u b s t a n t i a t e d ,  very l i t t l e  difficult  have p r o p o s e d t h a t  (Bressler,  carnitine-dependent transfer also  is  A  acetyltransferase  mitochondrial  acetyl  * acetyl-coenzyme A  of  understand._  of  be  either:  short  c a r n i t i n e «• coenzyme  function  the  mitochondria.may then  —  (-)-carnitine  of  Cycle  ,  (-) a c e t y l  carnitine  surface  carbon  enzyme, c a r n i t i n e  Fritz  is  inner  c a r b o n f r a g m e n t s can  Krebs  inner  A to  A,  membrane  acyl-ccenzyme  to  transported  1963;  palmityltransferase  the  l o c a l i z e d on  coenzyme  a)  of  to  m i t o c h o n d r i a l membrane The  for  surface  back B,  a c y l g r o u p f r o m coenzyme  carnitine  acyl-carnitine  palmityltransferase inner  the  and  groups outer  no  such i t  is  acetyltransferase  9  is  found  may  i n the mitochondria  instead  mitochondrial et  represent  a  Brener,  system  or  evidence  during  for  Brosman  A availablefor  periods  a t present  depot  1973;  1977) l e a v i n g coenzyme  ketogenesisi . Sufficient this  Acetylcarnitine  ( Kopec and F r i t z ,  acid oxidation, p a r t i c u l a r l y  support  1970) „ _  buffering  acetyl residues  a l . , 1973;  fatty  (Solberg,  of  increased  i s not a v a i l a b l e t o  hypothesise  CARNITINE METABOLISM  Carnitine  is  widely  distributed  concentrations  a r e found  Plant  generally contain  and  products Mudd,  1969).  i n animal  and  Lindstedt, The Figure  and  methionine  2.  low l e v e l s o f c a r n i t i n e  (Panter  i t s amino et  the r a t i s  acid  a l , 1974;  forcarnitine biosynthesis  are derived  Broguist,  adenosyl  from  Hoppel,  lysine  1973)..  methionine  precursors  Cederblad  is  an  and  represented  nitrogen  Similarly,  et  a l . ,  established  lysine  1976)..  precursor  of  t h e two c a r b o n f r a g m e n t  and  lost  Henderson  from  i s  by  S-  Thus  £Y -  t o y i e l d L-  1967; L i n d s t e d t and L i n d s t e d t ,  Holchalter  Home  £ -trimethyllysine  o f p r o t e i n bound  in  atom o f t h e  1973a;  which i n t u r n i s h y d r o x y l a t e d  (Lindstedt, 1973b).  is  {Cox and H o p p e l ,  (LaBadie  trimethylaminobutyrate carnitine  and q u a r t e n a r y  v i a the methylation  trimethyllysine  that  1954).  from  (Tsai  The carbon c h a i n  synthesized  and  (Fraenkel,  1976) .  pathway  carnitine  products  and i t s h i g h e s t  In addition t o dietary c a r n i t i n e ,  able t o synthesize c a r n i t i n e lysine  i n nature  (1976)  1965; Cox reported  t h e t r i m e t h y l l y s i n e was  10  Figure  2  Biosynthesis  of c a r n i t i n e  from  lysine  and  methionine  11  CO - R H N(CH ) 2  2  I  CH  4  Lysine  I  (protein-bound)  NH - R  H 0  S-Adenosyl M e t h i o n i n e  2  COO  +  I  E-Trimethyllysine  (CH,), N (CH„). - CH 5 5 2 4 | NH„  Ascorbate  • a - K e t o g l u t a r a t e +0„  Fe - • S u c c i n a t e +C0, COO  + (CH ) 3  3  l  N (CH ) 2  CHOH - CH  3  3-Hydroxy-£-Trimethyllysine  l  NH„  K  Pyridoxal phosphate  Glycine  + (CH ) 3  3  N (CH ) 2  CHO  3  s  NAD  Y-Trimethylaminobutyraldehyde  +  • NADH H  +  + + (CH ) 3  3  N (CH ) 2  y-Trimethylaminobutyrate  COO  3  Ascorbate  a - K e t o g l u t a r a t e .+.0, •• S u c c i n a t e +C0  (CH.). N CH_CHCH COO 5 5 I| z  Carnitine  0  (3-Hydroxy-Y-trimethylaminobutyrate)  OH  adapted  from H u l s e and E l l i s  r  (1978)  12  glycine  and  proposed  trimethyllysine succeeded to  the  t c c a r n i t i n e . . Hulse  amino a c i d s  in  addition  pyridoxine,  and  niacin)..  The  liver  a  et  limited  Erickson,  to  three  site  for  Tanphaichitr including  and  in  and E r o q u i s t ,  heart,  liver,  i n vivo  final  v i a the  conversion  Broquist,  muscle,  how  to s y n t h e s i z e estimated  is  of  used  body  1976).  for  100  per  by  100 g body  convert  of  i s required 1967;  1974;  tissues,  can  Yf o r the  Haigler  and  1974). by t h e r a t i s used  Hcrne e t a l i requirement  carnitine synthesis.  synthesis  Lindstedt  Tsai weight  of  (1976)  g body w e i g h t p e r d a y .  calculated  lysine  several  (1971)  of  have  Neurospora  The e s t i m a t e d  ( T s a i e t a l i , 1974; C e d e r b l a d  daily  and  the  Broquist,  total  o f c a r n i t i n e i n r a t s i s 0.33 u m o l e s p e r  weight  The  Cederblad  and B r o g u i s t ,  c a r n i t i n e i s n o t known.  t h a t 0.2% o f  and  Transport  (Lindstedt,  testes  (Tanphaichitr  and t e s t e s  much o f t h e l y s i n e i n g e s t e d  body c o n c e n t r a t i o n q  carnitine  the  and  and  plasma t o t h e l i v e r  1974; T a n p h a i c h i t r  Exactly  crassa  to  acid,  (Casillas  1974) i n d i c a t e t h a t  £-trimethyllysine t o Y - b u t y r o b e t a i n e . butyxobetaine  r e q u i r e s two  (ascorbic  synthesis  (Haigler  kidney,  recently  from t r i m e t h y l l y s i n e  1974), a l t h o u g h  such  vitro  of  of c a r n i t i n e biosynthesis i n  and B r o q u i s t ,  capacity  1974)  conversion  a l . , (1978)  vitamins  1975). . Reports of s t u d i e s  Broguist,  the  Thus c a r n i t i n e s y n t h e s i s  i s the primary  r a t (Tanphaichitr  have  for  i n e s t a b l i s h i n g t h e whole pathway  c a r n i t i n e i n the r a t .  the  pathway  et al. .  Thus  carnitine  would the  and  as  dietary  Lindstedt,  computed  approximate  100  by  2 u moles p e r  requirement  as  (1974) o f 1.5 u moles o f c a r n i t i n e  p e r day c a n be met by  daily  biosynthesis  13  of  carnitine  from  its  amino  acid  precursors  lysine  and  methionine*.  MA TE RN AL-NEON AT A l CARNITINE RELATIONSHIP  The rat  carnitine relationship that  and  her  o f f s p r i n g , both  aroused c o n s i d e r a b l e of  the  neonate  carnitine Skala,  to  1975;  attention*.  on  meet  pre-  the its  and  mother  rat  physiological  Robles-Valdes  al.,  in supplying  the  p c s t n a t a l l y , has  Evidence suggests a  et  as  the  c a r n i t i n e to her  mother recently  dependence  i t s major s u p p l i e r  requirements  a l . , 1976 ; F e r r e  Human s t u d i e s h a v e a l s o e m p h a s i z e d plays  e x i s t s between  important  (Hahn  et  of and  a l . , 1 978).  r o l e the  treast-fed infant  mother  (Ncvak  et  1979) .  Prenatal  The  mammalian  maternally is  well  acid  derived  synthesis  rather  with t h i s  addition,  oxidation  in  the  rat  fetal  of  Koldovsky*  from  1966).. I t  acid  oxidation.  fat utilization  by  the  rat  The fetus  concept. capacity liver  (Augenfeld  concentration  energy  fatty  oxidation the  its  than  palmityltransferase  and  (Hahn and  of  carbohydrate d i e t supports' f a t t y  carnitine  and  most  a high  findings regarding  i n keeping In  gains  carbohydrates  established that  biochemical are  fetus  for is  lew.  required  Fritz,  197C;  carnitine  long-chain The  for lonq  fatty  acid  concentration chain  Lockwood and  fatty  Bailey,  acetyltransferase  are  of  acid 1970) also  14  low  during  fetal  Correspondingly, Valdes  et  Valdes et  a l . , 1976)  of  carnitine sources  there  transfer  1975).  In  both  Skala,  1975)  be  with  rapid  ewe  led  effect four  be  result  mammals.  by  Hahn e t  limited  a l . , 1977)  are  to  1975)..  i n the  In  carnitine  low  the  f e t a l blood l e v e l s the  Skala and  amounts.. S i n c e t h e a  1975),  brown source  carnitine  deficiency  in  radioactive  fetal  low  as this  by  the  appears  and  fetus. pregnant  but  had  no  Twentyinto  carnitine  was  although  i n the  offspring.  and  carnitine  carnitine  deficiency the  there  1977).  labelled  adipose t i s s u e , of  are  maternal blood  of  skala,  (Hahn  i n t o the  (Hahn e t a l . ,  injections  though  carnitine  mother  the  rise  rat  for  maternal  even  both s p e c i e s , between  fetal  Apparently  of  in  transient  from  fetus  levels..  retention  that  (Hahn and  and  a  in a carnitine  rat  demands  derived  to  maternal,  (Bobles-  a l . , 1977),  is  blood  part  Skala,  liver  liver  Fetal  supplies  (Hahn e t  in  (Hahn and  Bobles-  indicate  L-carnitine  in fetal  mainly  evidence to  carnitine  exchange o f  after  1971).  1975;  i n the  of  mother r a t s  small  and  intravenous i n j e c t i o n  hours  found  met  maternal  due  (Hahn and  on  no  1975;  sheep  the  is  a slow  The  be  of  the  1978)  #  blood l e v e l s of c a r n i t i n e  restriction  to  is  Skala,  placental  al.  Fritz,  Skala,  concentration  occurs i n  instead  (Hahn and  placenta  and  (Hahn  and  values.  carnitine  may  compared  plasma  carnitine  postnatal  present,  synthesis  (Lee  a l . , 1976 ; Seccombe e t  compared t o At  fetal  development  only  in  appears  to  mother  may  15  Postnatal  At b i r t h , energy  and  is  a  sudden  y i e l d i n g substrates._  by a h i g h al. ,  there  f a t d i e t of milk  1954) o f which Mueller,  requires  Maternal  (Cox a n d  of  fatty  a d e q u a t e amount o f c a r n i t i n e formation  of  acyl  mitochondrial  suckling  ketone bodies Page  rates  period  et  of  after  coincides  rats..  birth  of  gradually Fritz,  increases  acid  lipid,  the mitochondria. necessary  An  f o r the  to cross the  utilization  ; Icckwood  an i n c r e a s e d  during  In v i t r o  the  1  liver  to  slices  The h i g h after  level which  values  i s maintained time  birth  the  (Taylor et a l . ,  rapid  palmityltransferase.. and  has  have  fatty  acid  for  **C02  diet of  by  increases  liver rapidly  i s achieved  at  weaning  (21  until  oxidation 1967;  1971;  rate  falls  Augenfeld  and  1970).  rapid increase  by a s i m i l i a r  sharply  the  of  studies  of t h e h i g h - f a t  of *C-palmitate and  and B a i l e y ,  capacity  the i n t a k e s  1970; Lockwood a n d B a i l e y ,  carnitine  dietary  so t h a t t h e maximum r a t e o f o x i d a t i o n  t o adult  accompanied  in  e t a l . , 1S78).  preparations  age),  After  et  1963)*.  fatty  with  Oxidation  2 d a y s o f agee days  Luckey  c a l o r i e s a r e f a t (Cox  therefore  (Drahota e t a l . , 1964  mitochondrial  i s replaced  1937;  o f energy from  is  of  a r e 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  shown more p r e c i s e l y t h a t  suckling  glucose  Mueller,  acids  (Fritz,  a l . , 1971; F e r r e  degradation  blood  availability  c a r n i t i n e s which a r e t h e n a b l e  memfcraie  Increased  in  69.5% o f t h e i n g e s t e d  1937).. The r e l e a s e  oxidation  change  i n palmitate  increase The  in  activity  i t s highest  the of  activity  oxidation i s activity this  of  enzyme  during  the  16  suckling  period  ((Augenfeld and  1970).  Likewise, carnitine  Valdes  et  liver  al-,  1976;  b i r t h , while the  decrease  when  pup  pup  The  levels  {Eorum,  primary  source  least  24  hours  (Ferre  e t a l . , 1978).  initially 1976)*  and  falls  concentrations decreased  lactation  immediate  precursor  nursing rats  liver  to  neonatal  heart  Cycling documented unable  by  and  the  liver  r a t can  secreted  i n her  to  diet. liver  content  is  very  (Robles-Valdes liver  the  high  day  carnitine  followed a f t e r  milk  hours a f t e r  of  levels.  and  back t o t h e  rats  finally.into  r a t has  infant  the  injected and  been  rats  empties  swallowing  mother  maternal  i t s injection.  mother a n i m a l  Thus substances  the  injection  from  infant  Since  r e g i o n and  When i n f a n t  carnitine  **C-butyrobetaine,  (1975),.  1956) .  high  et a l . ,  seventh  i n milk  was  the  at  mother r a t  maternal  own,  tissue,  the  between mother and  perineal  be p a s s e d milk*.  and  l a t e g e s t a t i o n t o be  of  w i t h i n 24  their  the  diet,  m i l k from  movement o f c a r n i t i n e  Skala  Jelinek*  suckling  during  fate  plasma  Hahn and  and  rapidly  i n plasma o r  reported  decrease  the  of c a r n i t i n e  licking  (1976)  of c a r n i t i n e ,  t o u r i n a t e on  b l a d d e r by (Capek  and  milk  neonatal  approximately  indicated  maternal  in  i s the  mother r a t s  which  increase  the  differences  carnitine  et a l . ,  t o normal a t  in  sex  nursing continues  accompanying  Experiments  into  in  Borum, 1978)  carnitine  Milk  Robles-Valdes  Seccombe e t a l . , 1978)  i s consuming  partum,  as  (Eobles-  1978)., of  post  plasma  Bailey,  i s weaned t o a h i g h c a r b o h y d r a t e  P r i o r t o w e a n i n g t h e r e a r e no carnitine  Lockwood and  c o n c e n t r a t i o n i n the  ( R o b l e s - V a l d e s e t a l . , 1976;  the  1970;  Borum, 1978;  following  but  Fritz,  are their urine  into  eventually  were i n j e c t e d  with  the be *»C-  17  carnitine, the  the label  mother  rat  and  pups o f t h e l i t t e r  i n the bleed  a l s o i n i t s milk  (Hahn and S k a l a ,  induced  deficiency  carnitine  status  of  appeared  of  oxidation..  of free fatty  starvation  indicates  i s oxidized  factor underlying  and d i a b e t e s  1971;  McGarry  ali _  the B-oxidation part,  by  source  during  the  fatty  acid  the  specifically  step  States  (Fritz,  1967;  (Fritz  which  that  A  high  i s the  McGarry  the  as  hepatic  and F o s t e r ,  require  is  such  t y an i n c r e a s e d  and 1974;  carnitine  a n d Yue, 1963).  (1975) s u g g e s t e d , on t h e o r e t i c a l g r o u n d s ,  of f a t t y  acids  concentration  might be g o v e r n e d ,  of c a r n i t i n e .  First,  i n the o x i d a t i o n  of  i f  only  They b a s e d  this  c a r n i t i n e i s necessary f o r long  chain  fatty  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  1 972)..  established  develops.  e t a l . , 1973; McGarry  on two c o n s i d e r a t i o n s . .  initial  Tomac,  the  carbohydrate  ketogenesis.  and k e t o g e n e s i s  system  et  of  and k e t o s i s  are accompanied  BcGarry  acyl transferase  notion  least  enhanced  availability  McGarry e t a l . , 1975), p r o c e s s e s  that  affect  a c i d uptake and o x i d a t i o n by the l i v e r  acid oxidation  Foster,  the  more l i p i d  most i m p o r t a n t  the  adversely  KETOSIS  ketogenesis Whenever  inadequate  fatty  maternally  stages of l a c t a t i o n * i s maternal*  Increased  rate  a  of  t c the  o f f s p r i n g , s i n c e t h e primary  RELATIONSHIP OF CARNITINE TO  in  may  Thus  c a r n i t i n e f o r the mother-infant c y c l e , a t  initial  organs  thereby r e t u r n i n g  1975).  carnitine  of s u c k l i n g  and o t h e r  Second,  studies  conducted  c a r n i t i n e was c a p a b l e  by  ( H o p p e l and  Fritz  of s t i m u l a t i n g  acids,  (1955)  fatty  acid  18  oxidation  when i n t r o d u c e d  Furthermore, s t a t e s , such as  McGarry  fasting  et  or  were a c c o m p a n i e d  by an  The  capacity  ketogenic  into rat liver  alloxan  increased of  carnitine  c o n c e n t r a t i o n s . . In  capacity  to  liver  from  directly  nonketotic  Carnitine during body  formation  1964; Fetal  liver  Valdes  suckling  a l . , 1976;  and  the  and  of k e t o n e  bodies  (Drahota  et  al.,  Robles-Valdes et  Bailey, almost  the  and  et  identical  concentrations e t a l . , 1978;  correlated  with  had  the  perfused  In  levels  (Drahota  and  et  plasma  contrast,  al.,  described  a  direct  fatty  acids.  increases and  Page as  result  (Roblesare  during  of k e t o n e b o d i e s  B a i l e y , 1971;  be  a l . , 1976 ; F e r r e  Borum, 1978)..  Ketone  carnitine concentrations  Lockwood  i n the neonate  a l s o been s t u d i e d .  1978)  k e t o s i s as  has  bodies  1976)  may  1971;  ketone  al.,  period.  blood  of  tissues  Lockwood and  profile  liver*  R o b l e s - V a l d e s e t a l . , 1976)..  o x i d a t i o n of  Eage,  rat  197C;  plasma  1964;  i n the  in isolated  production have  Borum,  s u c k l i n g p e r i o d and  1971;  tissue  weaned  a d d i t i o n , c a r n i t i n e alone  Fritz,  in the  i n the  c a r n i t i n e content  in fetal  neonate  state of  utilization  during  the  e t a l . , 1964;  physiological of  low  that k e t o t i c  rats.  the p r e n a t a l  period  (Drahota 1971)  and  during  diabetes,  hepatic  development  (Robles-Valdes  et  a l s o low  (fed)  i s very  Augenfeld  (1975) showed  a c t i v a t e ketogenesis  tissue levels  perinatal  ali  homogenates*.  are et  being of h i g h  The  the high al., in  a  rates  concentration  rapidly  after  B a i l e y , 1971;  birth  Page,  1971; high  et  al.,  1978),  remains  falls  after  weaning  (Lockwood  and  a l . , 1976).  An  Robles-Valdes been observed  et for  (Robles-Valdes  liver  carnitine  e t a l . , 1976;  Ferre  19  These  data  of c a r n i t i n e  and the  oxidation  is  are  than  higher  MATERNAL  health and  of  ketogenesis.  increased,  When  tissue  levels  fatty  carnitine  acid levels  EEFICIENCIES  of  a  nutritionally  sound  diet  during  and l a c t a t i o n f o r t h e m a i n t e n a n c e o f p r e g n a n c y and t h e  of  Lee,  review,  the  the  offspring  i s well  Niiyama e t a l . ,  1 9 6 4 ;  effects  maternal d i e t a r y are  rate  between h e p a t i c  normal*.  importance  gestation  a relationship  substantially  DIETARY  The  indicate  of  a  documented  1 9 7 0 ,  In this  1 9 7 3 ) » .  maternal  1 9 2 0 ; Chow  (Barry,  lysine  food r e s t r i c t i o n on dams and  particular  deficiency their  and/or  offspring,  discussed.  Pregnancy  The devoid (1970*  maintenance  of a s i n g l e  to control  size  (Niiyama  fetal  weight  for control deficient lysine  basis  was examined  fed a lysine-free  fed purified  by  diet  animals and maintained  Niiyama  diets  et a l .  showed f o o d  intake Litter  a l i , 1 9 7 0 , 1 9 7 3 ) was n o t a f f e c t e d * .  However,  animals.  and  i n rats  pregnancy.  (Niiyama  sparing  (Yamashita  et  diets  pregnancy  amino a c i d  Rats  1 9 7 3 ) . .  comparable  of  et a l * , 1 9 7 3 )  was s i g n i f i c a n t l y l o w e r  Niiyama suggested that  may  have  maintained  mechanism shown t o Ashida,  f o r t h e extensive  1 9 6 9 ;  exist  Canfield  preservation  animals fed  than  lysine  pregnancy because o f t h e in  non-pregnant  and C h y t i l ,  of lysine  1 9 7 8 ) .  i n rats  rats The  subjected  20  to  a lysine  the  d e f i c i e n c y , a p p e a r s t o be  first  Stapleton containing  and  0.42%  to  a  Hill  effect  1.12%  pups i n t h e l o w - l y s i n e group..  significantly 21  of  pregnancy,  weight  consuming gestation  a  food  has diet  (Barry,  g u a n t i t y and  that  litter  lysine concentration  intake  the  during  weight  revealed  on day  diet  resulted  restricted 192C;  of  7 and  a day  than i n those in  gestation  effect  dietary  been  when  a  reduced  the l a s t  week birth  group,.  reduction  also  diet  lower than i n the  analysis  This  a  size  average b i r t h  a v a i l a b l e t o the o f f s p r i n g during  reduced  weight  or  acid  lysine-  g e s t a t i o n , d i d not  significantly  amino  t o have a s i g n i f i c a n t  (1964)  during  i n dams f e d t h e l o w - l y s i n e  observed a s i g n i f i c a n t birth  was  reported  The  of  1976),..  intake  diet*.  of the l y s i n e d e f i c i e n t  reported  decline  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  Restricting  Lee  on f o o d  the high-lysine diet..  amount o f l y s i n e of  1 98C)  fed to rats  group  lower plasma  Hegsted,  (1972,  lysine  Plasma  pregnancy  consuming  (Chu and  lysine,  have a s i g n i f i c a n t  control  adaptive  enzyme i n t h e pathway o f l y s i n e d e g r a d a t i o n *  ketoglutarate reductase  compared  the  intake  reported  Niiyama  also  on r e p r o d u c t i o n .  in fetal  in  has  of  rats  weight.  by A  been  Chow  and  25%  and  decreased  i n the o f f s p r i n g of r a t s  total  food  intake  e t a l . , 1973).  q u a l i t y of the maternal d i e t  during  Thus both  affected  the  reproductive  performance..  Lactat ion  Imposing  a dietary d e f i c i e n c y during  lactation  affects  not  21  only  food  intake  offspring 1976; a  during  gluten  and  lactation  protein lysine the  lower  deficient  weight  Jansen intake  bread  diet  bread  during  loss  weaning  during  weight  of  imposed a t o t a l  food  Thus  some  deficiency  on t h e  decreased  weaning  less readily  fortification  the  of tread  offspring.  major  weights..  engorgement of than  dams  reduction i n  i n rats  fed a basal  and  flow. .  fortified  decreased  Chow  of  a  maternal increased  and L e e (1964) both  pregnancy  least  maternal  birth  A maternal l y s i n e  i n t a k e o f dams, a t  fed  i n the offspring.  decreased  decreases t h e food depresses milk  When  effects  are  lysine  t h a t dams f e d  significantly  stunting resulted  offspring  the  diet.  r e s t r i c t i o n on dams d u r i n g  the  contributed  in  a significant  and  and i t s  i n dams f e d t h e  dams consuming a l y s i n e  lactation*  growth of  rats  showed l i t t l e  milk  dams  as was t h e a v e r a g e  (1978) r e p o r t e d  o r ad l i b i t u j  with  the  during  deficient  probably  pregnancy and l a c t a t i o n  Lysine  lactation*  suckling  (1976) r e p o r t e d  when compared  diet. .  weight  restricted  to rats  production  on a i l k  lactation  a i d yielded  and Chase  food  of l y s i n e  s i g n i f i c a n t l y reduced  of  of  (1972, 1980) f e d  i n lysine*  Milk  This effect  d i e t during  a caloric  intake  C r n i c and C h a s e  mammary t i s s u e  either  and H i l l  deficient  gain  gain  1972, 1980; J a n s e n and C h a s e ,  offspring.  diet.  group.  a low p r o t e i n  weight  was s i g n i f i c a n t l y r e d u c e d ,  were a l s o  deficient  also  Stapleton  Food  of t h e i r  content  but  Hill,  based d i e t ,  and l a c t a t i o n .  weaning w e i g h t  and  dams  Chow and L e e , 1964).  wheat  the  the  (Stapleton  pregnancy  to  of  lysine  weight  deficiency  and also  during • l a c t a t i o n ,  22  hl§.UJ  DEFICIENCY AND  A  large  cereal  in  Consumption in  proportion  based d i e t s  limiting  of  with  (Tanphaichitr 1964), and  et  human  a  containing male  20%  may  a l . , 1976)  and  finding  may  carnitine i.e.,  diet, to  higher  pig  with  on  lower  of  the  The  control  have of  (Wittles  and  liver  and  effect lysine  carnitine  lysine  levels  rat  group  than  in  suggest  that  hepatic  tissue  fed  a  carnitine  in 0.8%  group.  the  lysine  this  latter  synthesis  food  were  compared  of  employed, intake.  laboratory  carnitine,  and  significantly  nutritional conditions  s i g n i f i c a n t l y reduced  a  of  skeletal  deficient  however, was  a h i g h e r l e v e l of  l e v e l s of  of  Bressler,  animals r e c e i v i n g  for increased  stringent  an  been  the  l e v e l s i n h e a r t and  i n the  authors  need  state  tissue  tissue  animals i n t h i s study  which c o n t a i n e d  have  33%  deficiency of  a  in  r e f l e c t the  A t h i r d group  such  limiting  group..  a lysine  oxidation  diet,  unsupplemented  the  acid  gluten  level  under  1977).  the  were g e n e r a l l y  supplemented  Bamji,  measured  When compared  the  result  (1973)  carnitine,  methionine.  f o o d s may  and  the  and  1976)..  muscle  in  and  deficiencies  in  supplemented d i e t , c a r n i t i n e  higher  plant  consumes  carnitine  Broguist  wheat  carnitine  in  lysine  fatty  , guinea al.,  low  r e s u l t from  lysine  The  of  population  (Latifa  in  accumulation  negligible  rats..  mainly  Carnitine  (Isaacs et  Tanphaichitr feeding  utilization  fat  he  precursors  role  deficiency. .  associated  to  i n humans  carnitine's  LEVELS  world's  likely  acid  deficiency  in fat  carnitine  amino  the  diets consisting  of  impairment  of  which a r e  its  a carnitine  Because  TISSUE CARNITINE  to  chow  reported animals  23  given  the  suggest  carnitine-free,  that  carnitine  is  The tissue  tissue  of c e r e a l  carnitine  l e v e l s and  (Tanphaichitr et  and  major p a r t content  of  partly  skeletal diet  of  was  as  wheat  increase fatty  and  well  in  The  results  when  dietary  liver  was  and  may  on  been 1979).  72%  rice  limiting  in  accumulation,  attained  lysine*  transporters  liver  fat  However,  until  the  diet  Therefore  occur  as  a  liver  a result  of  f o r removing t r i g l y c e r i d e s resulting  oxidation. . (1979)  total  lipid  reported content  fed  a carnitine  protein).  Oxidation  i n animals  in their with  oxidation,  a  Eeduction i n  not  rats  Bamji  as  Bamji,  rats  lipid  also  feeding c a r n i t i n e .  and  these  lysine,  has  Khan and  carnitine  by  and  tissue*  Supplementation  threonine  at  fed  pair-fed  0.2%  liver  of the  wheat muscle  heart,  lysine  fatty acids wheat  gluten  in and  depleted by  heart  diet  ad  Supplementation  produced a s i g n i f i c a n t  carnitine  of  l e v e l improved  and  controls,.  the  elevation  liver,  free  reduced the  marked of  carnitine  p l a s m a , m u s c l e , and  0.2%  1976;  in  c o u r s e , a carni±ine d e f i c i e n c y  impaired  gluten  acid  of  in rats  wheat  homogenate libitum,  and  muscle  (5%  in  deficient  oxidation  triglycerides.  with t h r e o n i n e  and  triglyceride  diet.  higher  male w e a n l i n g  condition  f a t t y acid  Khan  al.,  They o b s e r v e d  lipoprotein  liver,  impaired  fatty acid  achieved  accumulaticn  from t h e  are  diets,  detectable  of the  supplemented  insufficient  in  no  which was  was  based  et  lysine*.  complete r e l i e f  lipid  levels  a l . . (1976) f e d  containing  threonine  supplemented  available.  Tanphaichitr  was  carnitine  effect  observed  diet  lysine  levels,  triglyceride diet  with  carnitine  restored level  of  lysine  and  levels  and  24  palmitate  oxidation  tissues*  and  These data suggest  dietary  nutrient,  either  amino p r e c u r s o r s l y s i n e Sex  differences  observed and  in  humans  1976). the  both  Female  in  Broguist,  female r a t s  rats  1977  Borum,  ; the  fed  (Borum  a 20%  Male r a t s  subjected to  decreased  carnitine  carnitine  levels  differences  in  in  to  carnitine  higher  a  as or  of a its  lysine higher  levels  been  Borum,  1978)  and  cf  (Borum  and  Broguist  When  carnitine and  liver..  deficient  a dietary  diet  p l a s m a and  The  carnitine  (1977)  i n male  and  compared  to  in a l l  heart*  Only  had  lysine  of  h e a r t , but  rats,  deficiency,  higher.  moderately  mechanism u n d e r l y i n g levels  lysine  levels  h o w e v e r , were s i g n i f i c a n t l y  lysine  in and  supplemented d i e t , the tissue  al.,  liver  females  deficiency  diet* .  et  much c a r n i t i n e  content  than i n  carnitine  have  Maebashi  h a l f as  Eorum  gluten  1977;  1971;  i . e . , plasma, l i v e r ,  the  carnitine  levels  Broguist,  about  l e v e l s i n the  plasma  response  understood.  of  consuming a  plasma c a r n i t i n e  and  carnitine  wheat  analyzed,  carnitine  1979).  had  preformed  Lindstedt,  slightly  female rats  three tissues  opposite  tissue  effects  triglyceride levels  e s s e n t i a l i t y of  have o n l y  is  the  methionine.  male r a t s , and  females  deficient  the  in  males  investigated  control  and  rats  the  through  ( C e d e r b l a d and  plasma a s  heart  diminished  elevated the  and i s not  sex  their well  25  CHAPTER I I I METHODS AND  MATERIALS  ANIMALS  White Breeding  rats  of  Wistar  Laboratories,  Female r a t s ,  weighing  strain  Guelph)  were  approximately  plastic  b r e e d i n g cages  water  ad  libitum  less  t h a n 2 5 0 g were f e d a s i m i l a r  g were mated by cage.  until  (3 p e r c a g e ) mated.. M a l e  placing  overnight*„  (purchased  From  used 115  and  from  i n a l l experiments. g,  were  housed  f e d P u r i n a Rat  breeding  rats  d i e t . . Females  one  of gestation,  f o u n d i n a v a g i n a l smear, dams were  placed  on  in  Chow  and  weighing  no  averaging 2 2 5  1 male r a t w i t h 3 f e m a l e s i n a day  Woodlyn  t h e day their  breeding sperm  was  respective  diets* Once  mated  stainless of  steel  controlled  (24°C).  The  internal  transferred cages weekly  with  recorded. .  light  (lighting  were e q u i p p e d  unless  allowing  specified  wire  shavings  changes  as  during  individually floors  060C-1200 ad  hours)  libitum  otherwise.  housed  under and  w i t h e x t e r n a l water  1 8 days o f g e s t a t i o n  wood  weight  were  with r a i s e d  feeders  at  animals  cages  cages  cup  experiments  the  conditions temperature bottles  intake Pregnant  to larger  plastic  bedding.  Daily  pregnancy  and  in  in  and a l l  dams were breeding  food intake lactation  and were  2 6  DIETS  A on  diet  wheat g l u t e n  the  diet  diet  t o FAO  did were  not  the  Nutritional  detailed  lysine  supplementing  Basal diets  determined  than by  1964). of  based  composition  composition  of  of the gluten  ( 1 * 7 0 ) . . T h o s e amino  acids  diet  (the  the  diet  carnitine  essential  did  diet)  carnitine  the  1972) ,  zinc  and  was  diet,  prepared  by  HCl, e q u i v a l e n t and  20% f a t .  sole  protein  and B r o g u i s t ,  1973) t o  as  per  acetyltransferase diet  amino  meet  18% p r o t e i n  gluten  II  not  1. 036 I - L y s i n e  contained  and was  Table  ( T a b l e I ) . . The c o n t r o l  Thus, t h e l o w - l y s i n e carnitine  the  (NEC,  (Tanphaichitr g  1972).  also  rat  with  20% wheat  u  for  high-lysine  Both d i e t s  0.1  (NEC,  mixture  for  been r e p o r t e d  contain l e s s  amounts  the  mineral  containing  have  was  of wheat  calculations  the basal  0. 836 L - L y s i n e .  Fritz,  The  composition  Studies  were added t o t h e r a t i o n  adequate i n  source  to  requirements  potassium  diet)  meet r e g u i r e m e n t s f o r t h e p r e g n a n t and l a c t a t i n g  Because the  nutrient  to  f r o m amino a c i d  supplementsd  includes acids.  (the l c w - l y s i n e  as the s o u r c e o f p r o t e i n .  was c a l c u l a t e d  that  i n lysine  i s shown i n T a b l e I . . The amine a c i d  according  rat  deficient  the  gram assay  of  diet  as  ( M a r g u i s and  contained  negligible  adequate i n a l l n u t r i e n t s  except  lysine.  ANIMAL  EXPERIMENTS  The t h r e e in Figure  3.  animal experiments are diagrammatically  depicted  T a b l e 1.  C o m p o s i t i o n of Low-Lysine D i e t  Ingredient  P e r cent of D i e t 2  20.00  Wheat G l u t e n 3  2.20  Vitamin Mixture 4  5.00  Mineral Mixture Corn o i l ^  20.00  Cellulose  5.00  Zinc Carbonate  7  0.006  L - H i s t i d i n e HC1 DL - M e t h i o n i n e  7  0.32  7  0.40  L - Threonine^ L - Tryptophan  0.15 7  L - A r g i n i n e HCL  0.06 7  Corn S t a r c h  1 2  0.18 46.684  0.5% K„P0. was added t o t h e d i e t f o r t h e l a c t a t i o n s t u d i e s o n l y . 2 4 Wheat G l u t e n p u r c h a s e d from N o r t h American S c i e n t i f i c C h e m i c a l s , Vancouver, B r i t i s h Columbia.  3  ICN V i t a m i n D i e t F o r t i f i c a t i o n M i x t u r e purchased from B i o c h e m i c a l s , C l e v e l a n d , Ohio.  Nutritional  4  B e r n h a r t T o m a r e l l i M i n e r a l M i x t u r e purchased from B i o c h e m i c a l s , C l e v e l a n d , Ohio.  5  M a z o l a 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 b u l k , ICN, purchased from B i o c h e m i c a l s , C l e v e l a n d , Ohio.  7  ICN, purchased from N u t r i t i o n a l B i o c h e m i c a l s , C l e v e l a n d , Ohio.  Nutritional  Nutritional  28  Table I I .  Amino A c i d  C a l c u l a t e d E s s e n t i a l Amino A c i d C o m p o s i t i o n of  % Contributed Wheat  Gluten  by  Diets  % Added as  T o t a l g/lOOg  Supplement  Diet  Aspartic Acid  0.54  Threonine  0.41  Glutamic Acid  5.99  Methionine  0.26  Isoleucine  0.67  0.67  Leucine  1.11  1.11  Tyrosine  0.59  0.59  Phenylalanine  0.83  0.83  Lysine  0.27  0.80  Histidine  0.35  0.25  0.60  Arginine  0.69  0.14  0.83  Proline  2.12  1  0.54 0.15  0.56 5.99  0.20  0.46  1  1.07  2.12  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 t o as t h e 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 o f l y s i n e .  29  Figure  3  Diagramatic representation III  of experiments  I , I I , and  30  EXPERIMENT I  LOW  LYSINE  HIGH LYSINE  N=6  N=6  DAY 21 PREGNANCY - TISSUES (DAMS, FETUSES)  EXPERIMENT I I  LOW  LYSINE  N=6  HIGH LYSINE RESTRICTED N=6  HIGH LYSINE  N=6  DAY 15 LACTATION - TISSUES (DAMS, PUPS)  EXPERIMENT I I I  LOW LYSINE N=18  HIGH LYSINE RESTRICTED N=18  DAY 2 LACTATION - 6/GROUP) DAY 8 LACTATION - 6/GROUP) DAY 15LACTATION - 6/GROUP)  HIGH LYSINE N=18  MILK  31  Experiment I  Effect Two each, day  o f a m a t e r n a l l y s i n e d e f i c i e n c y durincj p r e g n a n c y on t i s s u e c a r n i t i n e l e v e l s groups  of  rats,  were f e d e i t h e c 21  of  the low-lysine  was t a k e n by h e a r t  s y r i n g e . . The dam's h e a r t  by  recorded*'  caesarian  section..  capillary  tubes.  samples  of  experiment,  from  because  attached  decapitated  and  fetuses  weights the  were  fetuses  liver  of  an a d e q u a t e amount  were removed  recorded*  Tissue  from  for  of  were i m m e d i a t e l y  frozen  and a  heparinized  were removed  noted.  using  Blood  heparinized  10  and  5  recorded the placenta  minutes  in  this  from t h e  interfering  f o r analysis. and  tissue  sample  and s t o r e d  with Heart  weights  two f e t u s e s w i t h i n a l i t t e r  combined t o p r o v i d e a l a r g e enough tissues  of blood  a l l fetuses  samples from  On  by c e n t r i f u g i n g t h e b l o o d  net  separation  were  would h a v e r e s u l t e d i n a l o s s o f b l o o d ,  collection  ether  a  placenta  sites  was s e p a r a t e d  dams  Fetal  with  diets.  with  using  animals  t h e n were removed and t i s s u e  .Resorption  Plasma  the  respectively.  and  or h i g h - l y s i n e  puncture  and l i v e r  Fetuses  s a m p l e s were o b t a i n e d  fetus  of s i x pregnant  p r e g n a n c y , dams were a n a e s t h e t i z e d  sample o f b l o o d  weights  consisting  f o r analysis.  were All  at -20°C. .  Experiment I I Effect  o f 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  After that animals food  examining  the r e s u l t s  c f experiment  i n t h e l o w - l y s i n e g r o u p consumed  than t h o s e  i n the h i g h - l y s i n e  group..  I , i t was  significantly Therefore  a  found less third  32  group  o f a n i m a l s , the h i g h - l y s i n e  the  remaining experiments  food  intake.  animals l y s i n e or  group.  high-lysine  high-lysine diet  Dams  res-tricted  obtained.  fed their  containing  of  eight  per  restriction  and  third  weight  lcw-  group,  the  the  high-lysine  basis.  Commencing  samples  until daily  tissue  at twelve  those  this  imposed analysis.  ZnS04.  less  offspring.. Ink under  culled The  to eight eight  the skin  than  due  tc  At day  5,  size  lactation sexed,  Preference  was  numbered  by  were  1,  This  sample 1 of  pups.  pups  days  two  experiment. .  s u r f a c e near  o f pups were r e c o r d e d on  for  litters  b e f o r e t w e l v e nocn) a l l pups were size  were  neon  mothers with  experiencing in  6  the  diets  used was  dams,  were f e d e i t h e r The  to  of decreased  with  and  statistical  litter  India  weights  size  completed  g i v e n t o male  Body  were  added  were s u p p l e m e n t e d  Only  c r more young  for  (parturition  injecting  newborns.  was  pregnant  were p a i r - f e d  on a body  respective  on l i t t e r  requirements  weighed  animals,  a l l diets  litter  of  l i b i t um. .  P r e g n a n t dams were c h e c k e d  presence  deaths  groups  of the groups  group  1 of l a c t a t i o n , were  3  d i e t ad  to the low-lysine  on d a y  the  Two  group,  to determine the e f f e c t s  T h e r e were t h u s  per  restricted  t h e appendages* 10,  and  15 p o s t  partum. On day and  a  sample  heparinized weights  15 o f l a c t a t i o n cf  syringe..  recorded.  by d e c a p i t a t i o n . collect and  blood  was  taken  and l i v e r  Heparinized  weights  The  were  capillary  h e a r t and  recorded.  liver The  with  by h e a r t p u n c t u r e  P u p s were s a c r i f i c e d  the blood.  tissue  Heart  dams were a n a e s t h e t i z e d  removed  and  on day  15 o f  tubes  were  of each plasma  pup was  ether using  a  tissue  lactation used  to  were removed s e p a r a t e d by  33  centrifuging 5 minutes  b l o o d samples o f t h e  respectively.  All  f r o z e n and  s t o r e d at -20°C..  Experiment  III  Effect  of a maternal  tissue  lysine  carnitine In  this  animals  per  l y s i n e or  g r o u p . . Two  animals,  low-lysine  group  lactation,  a l l diets  fed  their  Pregnant of  II)  dams with  more t h a n  two  experiment  of  at  litters to  deaths  eight  twelve eight  eight  per  litter,  young o f f o s t e r  eight  treatments)  throughout  on  day  1 of  lactation  Preference  pups were numbered by near  the  was  per l i t t e r ,  lactation ycung,  milk  flow  experiment  or e x p e r i e n c i n g for was  use less  maintain  period.  litter  were  presence  maintained  (parturition given  in  size  to  the 1 of  Dams  permitted  were used  to  were o b t a i n e d .  case  (dams b r e d and  injecting  appendages.„  the  size  For was  completed  in than  on  one  litter those culled before  t o male o f f s p r i n g . .  I n d i a Ink  Body w e i g h t s  18  lcw-  day  noon f o r t h e  where l i t t e r  the  on  for stimulating  were  c o n t a i n i n g more t h a n e i g h t  noon).^  surface  dams  the  diet  ZnSOU.  m i l k samples  a t twelve  young  dams,  high-lysine  Commencing  with  ( a s was  In t h o s e c a s e s  the three dietary  size  samples  l e s s than  III*  e i g h t , the  basis.  until  the  the h i g h - l y s i n e  S i n c e pups were r e g u i r e d  for tissue  milk  of pregnant  group,  were s u p p l e m e n t e d  daily  immediately  were f e d e i t h e r  third  weight  dams were c h e c k e d  newborns._  The  diets  on  and  leyeIs  pair-fed  on a body  pups f o r 10  were  deficiency  of the groups  were  respective  r a t h e r than  samples  s t u d y , t h e r e were t h r e e g r o u p s  high-lysine diet.  restricted  dams and t h e i r  under  the  The skin  o f pups, n u r s e d  by  34  those 1,  dams m i l k e d  5,  those  1 0 , and 15 p o s t dams m i l k e d  1, 5, and 8 p o s t Milk second,  from  sufficient  intraperitoneal 1959)J.  0.5 u n i t s  collected  prior  t o milk  and  from  of  days  o f p u p s , n u r s e d by on  days  6 dams p e r g r o u p on t h e  lactation.  10-12 h o u r s  Litters  (from a p p r o x i m a t e l y  c o l l e c t i o n t o allow  injection  of nembutol  E a c h dam was t h e n  injected  collected  with  intraperitoneally,  M i l k was m a n u a l l y a  pasteur  M i l k s a m p l e s were i m m e d i a t e l y  samples c o l l e c t e d  were a n a l y z e d  with  (45 m g / k g , G r o s v e n o r  milk flow  were 2300  accumulation of  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  of oxytocin t o f a c i l i t a t e  containers^ milk  were  Mena e t a l . , 1974).  nipple  on  partum..  t h e i r mothers  milk  Body w e i g h t s  on day 8 o f l a c t a t i o n , were r e c o r d e d  samples  t o 0900 h o u r s )  1959;  partum..  e i g h t h and f i f t e n t h days  removed  al.,  on day 15 o f l a c t a t i o n , were r e c o r d e d  (Grosvenor expressed  pipette  into  et with  eta l . , from t h e 0.5  f r o z e n a t -20°C. .  for carnitine  an  content*  ml All  35  PLASMA  f  L I VEEj. AND  Carnitine and  m i l k was  Foster  (total  assayed  11976)  Seccombe e t  as  according to modified  was  activity  then  extracted  by  from  the  method  Parvin  and  plasma,  of  liver,  McGarry  Pande  and  (1977)  and  Assay  L-carnitine  was  carnitine)  ANALYSIS  a l . „ (1978) . .  Principle of  specific  MILK TOTAL CARNITINE  incubated  and  with  carnitine  transformed  to the  (1- *C)acety1-CoA 1  of  acetyltransferase.  a known  Carnitine  labelled acetyl-L-carnitine  by  the  enzyme.  acetyltransferase . L-carnitine  (1 -!*C)  >  acetyl-CoA  (1-* *C) a c e t y l - L - c a c n i t i n e • CoASH  The  labelled acetyl-L-carnitine  ( 1 - *C) a c e t y l - C o A 1  centrifugation determined.  the  by  an  was  separated  activated  isotope  content  charcoal cf  the  from the  unreacted  slurry.. supernatant  After was  36  Preparation  o f plasma C a r n i t i n e  Extract - Part A  x  Section 2  (Seccombe e t a l . , 1978) 1) R e a g e n t s and  their concentrations:  Reagent  zinc  Concentration  sulfate  barium  hydroxide  2) S o l u t i o n s o f z i n c a g a i n s t each supernatant 3)  pH  of  from  into  0.083 M  sulfate  o t h e r such  Constriction  of plasma  0.087 M  that  barium  hydroxide  were  e q u a l volumes o f e a c h  titrated  resulted  in a  7*3. pipettes  separate  Amersham).  and  were u s e d  conical  t o measure 0.100  polystyrene  A l l extractions  ml  tubes  samples  (purchased  were dene i n d u p l i c a t e  and  kept  on i c e . . 4)  0. 400  ml  o f barium  c o n t e n t s m i x e d . . The for 5)  1 hour  tube  was  t o r e l e a s e bound  0.400 ml  mixed and  hydroxide  of  the  zinc s u l f a t e  was  then  added  covered  t o each and  tube  heated  and at  the 37°C  carnitine. was  added t o each  t u b e , the  contents  a t 2000 RPMs f o r 20  minutes.  t u b e c o o l e d on i c e . .  6)  The  precipitate  The  supernatant  for  carnitine.  was  was  centrifuged  removed and  frozen  at  -20°C  until  assayed  37  P r e p a r at i o n o f M i l k C a r n i t i n e E x t r a c t s (Robles-Valdes et al.,1976; 1) R e a g e n t s and  sulfate  a g a i n s t each  3)  f o r days  duplicate 4)  mixed  contents  7) The for  The  were d i l u t e d 8 and  barium  hydroxide  were  e q u a l volumes of e a c h  with  distilled  2 respectively.  titrated  resulted  in a  fill  water  1:1,  samples  1:2,  and  were done i n  k e p t on i c e . were  covered  o f barium The  and  of  heated  a t 60°C  t o d i s p e r s e any  hydroxide tube  t o r e l e a s e bound  0.400 ml  and  7.3*  mixed*  1 hour  mixed and  that  f o r 30 s e c o n d s  5) 0*400 ml  6)  of  15,  and  Samples  then  for  pH  C.083 M  sulfate  o t h e r such  Milk samples  1:4  1978)  0.087 M  hydroxide  Solutions of zinc  supernatant  Seccombe e t a l * ,  Concentration  barium  2)  Aj_ S e c t i o n 2  their concentrations:  Reagent  zinc  - Pa r t  zinc s u l f a t e  was  was  f o r 30  particulate  added t o e a c h  then  minutes  covered  and  and  material.  tube heated  and  the  at  37°C  carnitine. was  added t o e a c h  tube, the  contents  t h e t u b e c o o l e d on i c e .  precipitate  supernatant carnitine.  was was  centrifuged  a t 2C00 RPMs f o r 20  removed and  f r o z e n a t -20°C  until  minutes. assayed  38  Preparation  of l i v e r  Carnitine  E x t r a c t s - P a r t hj_ S e c t i o n 3  ( T s a i e t a l . , 1974) 1) R e a g e n t s  and t h e i r c o n c e n t r a t i o n s :  Reagent  Concentration  potassium  hydroxide  10%  perchloric acid  2)  0.2 g o f  hydroxide  tissue  at  combined f e t a l solution  Samples  72%  perchloric  8C°C  6)  acid  to  15  ml  of  potassium  was  (For  hydrolyzed.)  The  and a c i d i f i e d  with  hydrolysis. temperature,  a t 2000 RPMs f o r 15  was removed a n d t h e p e l l e t  minutes.  washed t w i c e  with  of water.. with' p o t a s s i u m  and t h e n c e n t r i f u g e d  hydroxide,  at  2000  RPMs  dried  and  then  minutes.  The  supernatant  fractions  t o a predetermined  s t e p was c o n d u c t e d  were  freeze  volume u s i n g  volumetric  to obtain a carnitine  pipettes.  concentration that  was c a p a b l e o f d e t e c t i n g .  Rediluted  assays.  2.0  release carnitine.  sample  and c e n t r i f u g e d  on i c e f o r 30 minutes,  the a s s a y 7)  total  in  Combined w a s h i n g s were n e u t r a l i z e d  rediluted This  the  hours  were c o o l e d t o room  1.0 ml a l i g u o t s  for  hydrolyzed  for 2  livers  4) T h e s u p e r n a t a n t  left  was  was mixed t w i c e d u r i n g  3)  5)  72%  supernatants  were  used  for duplicated  carnitine  39  T o t a l C a r n i t i n e Assay P r o c e d u r e - P a r t (McGarry a n d F o s t e r , et  E  1976 ; P a r v i n  1977; Secccmbe  a l . , 1978)  1) R e a g e n t s and t h e i r  concentrations:  Reagent  a)  Concentration  N-2-Hydroxyethylpiperazine ethanesuIfcnic  b)  acid  N-2-  acetic  N-ethyl  d)  (1-**C) a c e t y l - C o A ( h o t )  e)  S-acetyl-CoA  f)  sodium  g)  carnitine acetyltransferase  h)  activated  i)  ethanol'  j)  phosphoric  Malimide  (cold)  hydroxide  1 N  HEPES  7.6  with  100% acid  85%  of Solutions  and  was made by d i s s o l v i n g 9.532  g  0.666 g o f EDTA i n w a t e r and a d j u s t i n g t h e pH t o  sodium hydroxide..  a final  5 mg/ml  charcoal  2) HEPES/EDTA B u f f e r : The b u f f e r of  0.016 M  (EDTA)  c)  Preparation  0.4 M  (HEPES)  (Ethylenedinitrilo)-Tetra acid  to  and Pande  volume o f 100 ml.  The p r e p a r e d  solution  was  adjusted  ao  3)  1,0  ml  o f N-ethy 1 4)  Hot  and  acetyl  CoA  HEPES/EDTA b u f f e r  Cold  such  ml  Acetyl  at a  NEM  2.0  6)  Carnitine  specific  ml  and  of  ml  1-0  used  w i l l be  activity mix  of  from  step  referred  3. to  Acetyltransferase enzyme  HEPES/EDTA  ml  to  dissolve  a total  step The  as  4  5-0  mg  of  was  50  nanamoles  step  2)  of  nanamole. the  1.0  volume o f  the  mix  mix.  ml  (purchased  were  added t o  assay  0.05  (1-**C)  S-acetyl-CoA  final  the  Mix:  (see  of  40,000 DPMs per  solution  buffer  aliguots  plus c o l d  contained  acetyl-CoA  acetyltransferase ml  Mix:  s o l u t i o n from  was  1.050  CoA  that each  of the  ml b u f f e r  was  malimide.  acetyl-CoA 1-0  the  ( p u r c h a s e d f r o m Amersham)  prepared  5)  of  of from  were  carnitine Sigma)  plus  combined  and  mixed.  Assa y  7)  Constriction  pipettes  were u s e d t o  of  each  tissue  extract  into  from  Amersham) .  (purchased 8)  0.100  9)  the  ml  of  assay  the was  acetyltransferase proceed at 10) an  The  room  was  started  by  (see  step  temperature was  charcoal  stirrer.  separate  A l l samples  mix  mix  reaction  activated  a magnetic  assay  measure 0.100  added t o t h e  6)  f o r 60  terminated slurry  and  0.05 the  samples  polystyrene  were k e p t on  adding  ml  tissue ml  of  ice. extract.  the  reaction  tubes  carnitine  allowed  to  0.600 ml  of  minutes. by  the  which was  addition  continuously  of  stirred  with  Charcoal  Slurry  activated  Amount  charcoal  ethancl phosphoric distilled  11)  acid  were  placed  on  of the charcoal  ml ml  mixture,  the  tubes  i c e and t h e c o n t e n t s mixed a t a p p r o x i m a t e l y 10  i n t e r v a l s . . The t u b e s  for  20  minutes.  12)  A 0.5  ml p o r t i o n  scintillation  distilled  water  and  were t h e n c e n t r i f u g e d  a t 2000  RPMs  o f t h e c l e a r s u p e r n a t a n t was t r a n s f e r r e d t o  mini s i z e d  After  54.5  4,2 5 ml  minute  Amersham) .  9  1.25  water  Following the addition  4  5  vials ml  (17 x 55 mm)  containing  o f ACS S c i n t i l l a n t  m i x i n g , and o b t a i n i n g  phase s y s t e m ,  samples  were c o u n t e d  Scintillation  Counter f o r 5 minutes  in a  each. ,  ml o f  (purchased  a relatively Eeckman  0.4  stable  LS-230  from one-  liguid  42  Determination  A series and  100  from  healthy  batch  M)  of C a r n i t i n e Concentration  of  and  guality  Wistar  of t i s s u e s  determination  guality  t i s s u e s they Blanks,  were used  throughout  ranged  reproducibility The carnitine  x=  The  used  the c o n d u c t e d and  58.5-62.3 of  and use  Treatment  M,  assayed of  these  the  exception  each  permitted and  the  standards,  the  analyses  liver  standard (average  variability  of  hydrolyzed  determine  n mol/ml  M,  obtained with  of b l a n k s ,  controls  of  50  to that of the r e s p e c t i v e  with  to  linearity  (25  concentrations  identical  guality  were  The  carnitine  to a s s e s s ,  and  standards  were h y d r o l y z e d  was  r>0.989) ,  between  water)  (pooled plasma samples  results*  controls  extracts  correlation*  males)  of the  concentrationsgood  controls  tissue  standards  plasma  (distilled  t h a t were a n a l y z e d .  of  reproducibility and  blanks  of t h e  plasma  liver. with  the  carnitine  curves  remained  coefficient  of  guality controls indicating  good  results..  following  formula  was  used  to  determine  tissue  concentration:  100-dmpJblank) X dpmjsample) X dpm(100 M s t a n d a r d )  - dpm  100%  (blank)  =  n mol/ml o r  g  43  Recp ver y  A known q u a n t i t y sets  of p l a s m a ,  recovery 93%  milk and l i v e r  of carnitine  for  of DL-carnitine  plasma,  from  reco/ery  to  100%.  97  to  not,  tissues.  to  Recovery  1  Seccombe added  et  of  t o the a u t h o r s knowledge,  t o plasma  been  added  a l .  Percent r e c o v e r y of  duplicate  t o determine the percent  of * C - c a r n i t i n e  recovery of a c y l c a r n i t i n e of t h e o r e t i c a l .  samples  added  carnitine  9 1 % f o r m i l k , and 95% f o r l i v e r .  (1974) r e p o r t e d be  was  Tsai  to l i v e r  was  et a l . tissue  (1978) c a l c u l a t e d t h e t o be g r e a t e r t h a n 95%  carnitine  from  milk  has  reported i n the l i t e r a t u r e .  STATISTICS  All the  statistical  Statistical  al. ,  1975)  on  Terminal  System  fetuses  and  p r o c e d u r e s were p e r f o r m e d  Package  f o r the S o c i a l  the  University  (MTS)  computer  of British program.  These  c a l c u l a t e t h e standard deviations. the  problem  (SPSS,  Columbia's Data  This  measurements o f i n d i v i d u a l  of correlation  of observations  method  offspring within  et  Michigan  obtained  means o f l i t t e r s  from  Nie  pups were e x p r e s s e d a s t h e means o f l i t t e r s  than a s i n d i v i d u a l samples..  than  Sciences  ty programs  from rather  were u s e d t o  was used  rather  t o eliminate the  litters  (Abbey and  Howard 1973) . .  Experiment I The t - t e s t , there  a t 0.05 l e v e l *  was any s i g n i f i c a n t  was used  differences  to  determine  whether  between t r e a t m e n t g r o u p s .  The  a n a l y s i s of variance  level  was  used  to  with  test  d i f f e r e n c e s i n weight g a i n  repeated  whether and/or  measures,  there  food  was  intake  at  the  0.05  any s i g n i f i c a n t  during  pregnancy.  E x p e r i m e n t I I and I I I Unless and  III  analysis  The  of variance  was used  of the e f f e c t ,  there  pups and  lact ation.  to determine  d i f f e r e n c e s among t r e a t m e n t Duncan's m u l t i p l e  a n a l y s i s of v a r i a n c e  whether and  stated, a l l analyses  were c o n d u c t e d a t t h e 0.05 l e v e l  significant source  otherwise  with  repeated  was any s i g n i f i c a n t food  intake  of  for  experiments  of s i g n i f i c a n c e . i f  there  were  II The any  g r o u p s . . To e x p l o r e t h e range  test  was  measures was u s e d  used. to test  d i f f e r e n c e i n w e i g h t o f dams  dams,  during  pregnancy  and/or  45  CHAPTEE I V RESULTS  EXPERIMENT I  A  low-lysine  significantly liver  diet,  when f e d t o p r e g n a n t r a t s ,  l o w e r m a t e r n a l body  weights  when compared  weights,  food  resulted i n  intakes  and,  to animals fed t h e h i g h - l y s i n e  diet  ( T a b l e s I I I , I V , and F i g u r e 4 ) . Dams f e d t h e h i g h - l y s i n e during ate  gestation,  whereas  body  weight  intake  there  (Table  (83.8  lysine diet  significant  consumption  no  g  g) d u r i n g  affected difference  diet  gestation  pregnancy  significantly  was e x p r e s s e d  and day  group  in  food  a s g/100 g  of diet  III).  gained  on f o o d  The  weekly  weight  I I I , Figure  dietary  treatment  4)  gain was  (p<0.005).  of also A  i n body w e i g h t s between t h e two d i e t a r y  p r e g n a n c y . . Body w e i g h t s o f dams i n  low-lysine  less  t h a n dams c o n s u m i n g t h e h i g h -  (Table by  significantly  g r o u p s was o b s e r v e d a s e a r l y a s day 7 o f g e s t a t i o n and throughout  food/day  decrease  significant effect  low-lysine  (147.1 g . T a b l e  during  significant  17.3  III) .  Dams f e d t h e weight  was  consumed  the animals i n t h e l o w - l y s i n e  an a v e r a g e o f 14.9 g/day, a  i n t a k e . . However, when f o o d  dams  diet  the  continued  high-lysine  g r o u p s were 376,3 g and 308.3 g r e s p e c t i v e l y , on  21 o f g e s t a t i o n . The  effect  weight i s  of d i e t a r y treatment  presented  i n Table  IV.  on m a t e r n a l  Liver  weights  liver  and h e a r t  f o r the  low-  Table I I I .  Food Intake and Weight G a i n of Dams Fed a H i g h - L y s i n e or Low-Lysine D i e t d u r i n g G e s t a t i o n  Week of G e s t a t i o n Experimental Group  Feed (g/day)  Body Weight  B  Initial Body Weight  Daily Food Intake  High-Lysine  6.5±0.2  6.710.4  6.610.3  17.310.6  Low-Lysine  6.3±0.3  6.5±0.5  5.9±0.3  14.910.5  High-Lysine  229.2+7.9  262.916.7  300.817.2  376.3+9.5  Low-Lysine  224.6+5.2  239.014.9°  259.418.8°  308.3110.0  Total Weight Gain  147.1110.0  (g) E  A  Mean 1 SEM  f o r 6 dams  B  Food i n t a k e i s expressed as g/100  C  Mean v a l u e d i f f e r s 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)  D  Mean v a l u e s 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<0.01)  E  Mean v a l u e s 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<0.001)  g body weight  83.817.5  E  47  Figure  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 g a i n o f dams d u r i n g g e s t a t i o n  diet  on  weight  T a b l e IV. Heart and L i v e r Weights o f Dams Fed a H i g h - L y s i n e o r Low-Lysine D i e t u n t i l Day 21 o f Gestation  T i s s u e Weight g  Experimental  T i s s u e Weight % o f Body Weight  Group Heart  Liver  High-Lysine  0.7430±0.01  14.5071±0.48  Low-Lysine  0.7035±0.04  11.4096±0.71  Heart  Liver  0.20±0.01  B  0.22±0.03  3.86±0.09  C  A  Mean ± SEM f o r 6 dams  B  Mean v a l u e s 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 v a l u e s 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)  3.71±0.23  50  lysine  group  high-lysine  (11.4096 g) animals  were e x p r e s s e d significant deficiency  did  a  not  percent  intake  not  affected  between  affect  the  the  by  low-lysine  the  low-lysine  the  twc  weight,  there  dietary  groups.  heart  weights  f o r the  V).  weights no  Lysine  which  group  greater  in the  group  (0.0142 g ) * . Only one  were n o t e d The  f o r the  were  and  low-  high-lysine  plasna  5),.  mol/ml f o r  or  liver  for the  respectively,  fed Liver  the  group  were  site  significantly  than  the  was  identified  evidence of  d i d not  low  lysine  levels  and  levels  168.87 and  166, 56  (Table 43.97 and  were  (18,04 n mol/ml)  group  (Table  VI,  VI  and  37.70 n diets  much h i g h e r  than  n mol/g t i s s u e i n  p l a s m a 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 mol/ml)  effect  high-lysine  h i g h - l y s i n e groups r e s p e c t i v e l y .  n  for  resorptions  have a s i g n i f i c a n t  carnitine  carnitine  (22.85  high-lysine  group.  low-lysine  low-lysine  lcw-lysine  high-lysine  averaging  Fetal  fetuses  i n the  plasma l e v e l s , and  was  lower  (0.0150 g)  no  size  Number o f  Plasma c a r n i t i n e l e v e l s a v e r a g e d dams  decrease i n  were s i g n i f i c a n t l y  however,  d i e t , and  dietary lysine level  maternal  than  resorption  low-lysine  V).  11,8  weights  weights  lcw-lysine  dams f e d t h e  and  (0. 2033 g)  14%  group, l i t t e r  (Table  11.7,  Fetal liver  (0.2523 g ) . . F e t a l h e a r t  the  the  was  high-lysine  diet resulted in a  high-lysine  h i g h - l y s i n e g r o u p was  in  Figure  body  dietary treatment  (Table  on  in  However, when l i v e r  maternal  compared t o t h e  group  the  of  lower than  groups.  food  group  g) .  0.7035 g r e s p e c t i v e l y ,  Although  for  (14.5071  difference  0,7430 g and lysine  as  were s i g n i f i c a n t l y  group Figure  than 6),  the  higher  the  in  high-lysine  Fetal  liver  Table V.  E f f e c t o f a H i g h - L y s i n e o r Low-Lysine D i e t on t h e Number of F e t u s e s , and F e t a l Heart and L i v e r Weight on Day 21 of G e s t a t i o n  Experimental  Number o f  F e t a l Heart Weight  Group  Fetuses  g  High-Lysine  11.8±1.4  0.0142±0.000  Low-Lysine  11.7±0.9  0.0150±0.001  F e t a l L i v e r Weight g  0.2523±0.005  0.2033±0.014  B  A  Mean ± SEM f o r 6 l i t t e r means  B  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<0.005)  C  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<0.05)  Table VI.  E f f e c t o f a H i g h - L y s i n e o r Low-Lysine D i e t on Plasma and L i v e r C a r n i t i n e o f Dams and F e t u s e s on Day 21 o f G e s t a t i o n  Dams  Fetuses  B  Experimental  Plasma C a r n i t i n e  Liver Carnitine  Group  nmol/ml  nmol/g  High-Lysine  37.70±8.60  166.56110.80  Low-Lysine  43.97±2.40  168.8719.50  High-Lysine  18.0410.50  174.62116.70  Low-Lys i n e  22.8511.40  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 v a l u e s 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  Figure  5  E f f e c t o f 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 p l a s m a and l i v e r c a r n i t i n e i n dams on day 21 o f g e s t a t i o n  55  Figure  6  E f f e c t of a h i g h - l y s i n e or and liver carnitine in gestation  l c w - l y s i n e d i e t on p l a s m a fetuses on day 21 of  200  •  LOW-LYSINE  I  180 HIGH-LYSINE  160  140  120  100  80l  601  4CH  20  PLASMA  LIVER  57  carnitine  l e v e l s tended  n mol/g) g r o u p t h a n t h e however  the  carnitine liver  t o be h i g h e r high-lysine  difference  levels  was  not  were 52% l o w e r  carnitine  levels  (174.62  n  maternal  similar  in  (189.12  mol/g)  group,  Fetal  plasma  significant.  than  were  i n the low-lysine  levels*  both  whereas  the  dams and  fetuses. .  EXPERIMENT I I  In experiment I food significantly a  third  i f  deficiency  the  or  diet.  low-lysine parameters Food the  a restricted or  less  significantly  lysine  effects food  gained  lower l i v e r  (Table intake  VII,  or  Dams weight  dams  Therefore  was added t o  to  a  I n experiment  significantly  was  diets less  lysine I I dams consumed  w e i g h t , and  weights than animals f e d the highd i f f e r e n c e s between groups  the  i n any o f t h e s e  V I I I , I X , and F i g u r e 7 ) . .  during  gestation  (15.0  was s i g n i f i c a n t l y  g/day)  high-lysine  g body w e i g h t t h e r e intake  due  restricted  and t h e h i g h - l y s i n e r e s t r i c t e d  However, when f o o d c o n s u m p t i o n  food  were  T h e r e were no s i g n i f i c a n t  g/day)  g/100  low-lysine  intake.  high-lysine  food,  h i g h - l y s i n e females  (13.0  the  high-lysine restricted,  observed  the l o w - l y s i n e  significantly had  of  l e s s t h a n t h a t o f t h e h i g h - l y s i n e dams.  group o f animals,  determine  fed  intake  than  restricted during  in  greater i n  the  lcw-lysine  (13.2 g/day)  pregnancy  females.  was e x p r e s s e d  was no s i g n i f i c a n t e f f e c t  of  diet  as on  ( T a b l e VII) . . fed  the  high-lysine  (124.6 g) d u r i n g  diet  pregnancy  gained than  significantly  more  either the low-lysine  Table VII.  Food Intake and Weight G a i n o f Dams Fed a H i g h - L y s i n e , Low-Lysine or H i g h - L y s i n e  Restricted  D i e t d u r i n g G e s t a t i o n i n Experiment I I  Week o f G e s t a t i o n  Experimental Group  Feed (g/day)  Body Weight  B  Initial Body Weight  Daily Food Intake  High-Lysine  6.9+0.3  6.3+0.3  5.9±0.2  15.0±0.5  Low-Lysine  6.1±0.2  6.1±0.5  4.9±0.4  13.0±0.4  High-Lysine Restricted  5.810.2  5.9±0.3  5.0±0.4  13.2±0.7  Total Weight G a i n  C  High-Lysine  217.2±7.1  244.1±4.4  275.8+5.4  341.8±5.6  124.6±5.2  Low-Lysine  228.4±1.2  238.4±1.2  259.4±8.5  299.3±5.7  70.8±8.3  High-Lysine Restricted  231.3±5.3  238.8±4.4  268.8±5.0  317.3±7.4  86.0±9.2  (g)  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 a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05)  C  59  (70.8 g) o r h i g h - l y s i n e Weekly Figure  weight  of  greater lysine  intake in  dams  during  g/day)  or  Table VIII).  pregnancy  on  intake  high-lysine  was  lactation  (Table (Table  high-lysine  indicated  (p<0.001) *  restricted  greater  dams  during  V I I and  treatment.  t h a n i n t h e lowgroups  during  weight, a s i g n i f i c a n t  VII).  significantly  restricted  consumption  dams was s i g n i f i c a n t l y  high-lysine  was  (27.1 g/day) g r o u p  When f o o d  a s g/100 g body  food  during  the high-lysine  (14.2  r  of  expressed  and  dams  (86.0 g) g r o u p s  7) were n o t s i g n i f i c a n t l y a f f e c t e d b y d i e t a r y  Food  g/day  gains  restricted  lactation  effect  Food  than  (13.7  of  intake  the  weeks  was diet  of the  lcw-lysine  1  and  2  of  l a c t at i o n * Dams  fed  lactation  whereas  groups l o s t Figure  the  high-lysine the  low-lysine  47.0 g and 45.9  1 1 ) . . There  were  between t h e l o w - l y s i n e  g,  high-lysine  and  changes d u r i n g compared  to  group*  lactation  significantly on day 15 restricted respectively The  the  groups (Table  effect  differed  VIII  restricted  of d i e t  during  restricted  differences  significant  and  i n weight groups  of  from t h a t o f  on w e e k l y  weight  (p<0.001).  When  t h e a v e r a g e body  and h i g h - l y s i n e  restricted  weights o f diets  were  1, 7, and 14. . Body w e i g h t s o f dams  high-lysine, were  g  (Table  significantly  effect  group,  20.1  high-lysine  respectively  was a l s o  l o w e r on d a y s  for  and  high-lysine  The  the high-lysine  dams f e d t h e l o w - l y s i n e  gained  no s i g n i f i c a n t  a n i m a l s h o w e v e r , both g r o u p s the  diet  296.8  lcw-lysine, g,  190.2  and g,  high-lysine  and  202. 2  g  VIII).,  of dietary  t r e a t m e n t on m a t e r n a l l i v e r  w e i g h t i n t h e dams i s p r e s e n t e d i n T a b l e I X .  Livers  and  heart  o f t h e low-  60  Figure  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 high-lysine restricted diet on weight gain of dams during g e s t a t i o n i n Experiment I I  DAY OF GESTATION  T a b l e V I I I . Food I n t a k e and Weight Changes o f Dams Fed a H i g h - L y s i n e , Low-Lysine o r H i g h - L y s i n e R e s t r i c t e d Diet during  Lactation  • B Lactation  Week o f Experimental Group  Feed (g/day)  Initial Weight  1  Daily Food I n t a k e  Total Weight G a i n  C High-Lysine  7.4±0.5  Low-Lys i n e  5.4±0.7 5.1±0.4  High-Lysine Restricted Body Weight (g)  2  12.6±l.l  a  a  6.9±0.9  b  7.0+1.0  b  b  b  27.1±l.l  a  14.2±1.5  b  13.7±1.5  b  High-Lysine  276.6±ll.l  Low-Lysine  236.1+ 4.6  b  208.3± 3.9  b  190.2±5.7.  b  -47.0+ 8.8  b  High-Lysine Restricted  249.2± 4.9  b  221.4± 5 . 1  b  202.2±8.5  b  -45.9± 4.7  b  a  278.4±ll.l  a  296.8±5.6  a  +20.1±10.7  A  Mean ± SEM f o r 6 dams  B  Food i n t a k e i s e x p r e s s e d 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 a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05)  a  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  Tissue Weight % of Body Weight  g  Heart  Liver  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 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 different (p<0.05)  64  lysine  (8.6252 g)  weighed  the  expressed no  Heart  three  dietary  as  a  Effects litter  effect  and for  high-lysine  on  dietary birth  the  than  weight  tissue  heart  weights  were  treatment  had  weight. by  weight o f  pups, i s presented  i n Table  X.  reproduction  (11.0), low-lysine  (13.0)  groups  was  with o f f s p r i n g  control  restriction  between t h e  significantly  assessed  however, s i g n i f i c a n t l y l o w e r e d  birth  or  high-lysine  as  When compared  food  differ  dams  on  was  or  the  g)  treatment  the  g)  of  When  liver  on  (5-5  net  (8.0036  body w e i g h t , d i e t a r y  high-lysine  high-lysine  those  groups.  either  restricted  different*  restricted  weights d i d  percentage of  of  size size  less  dams.  significant  Litter  high-lysine  significantly  (14.2202 g) among  and  diet  (5.4  (6.0 by  of g),  birth a  l o w - l y s i n e and  maintained  weight  lysine  T h e r e was  and  significantly  animals  either  g).  not  (10.3),  no  of  pups  deficiency  difference  high-lysine  in  restricted  groups. The  lactation  growth o f effect was  of  which  maternal d i e t  in  turn  d a y s 5,  10,  and  and  g  17.8 The  dams,  Table  weight  (p<0.001) .  XI  gain  The  and  15  of  effect  of in  (0.9184 g)  on  day  15  maternal d i e t Table XII.  on  measured by  and  of  Figure  Pups o f  weighed s i g n i f i c a n t l y  of  pups  restricted  group  low-lysine  group  28-9  g,  low12-6  g,  lactation.  pups l i v e r  Livers  The  high-lysine,  dams weighed  the  lactation  group  mere t h a n  of  8. .  pups d u r i n g  high-lysine  lactation..  high-lysine restricted  as  high-lysine  weighed s i g n i f i c a n t l y  respectively,  presented  group  on  in  of  s i g n i f i c a n t l y more t h a n t h e  lysine,  is  i s presented  significant  weighed  on  pups*  performance  of  and  pups i n  heart  the  weight  high-lysine  greater than those of  the  Table X.  E f f e c t o f a H i g h - L y s i n e , Low-Lysine 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 R e p r o d u c t i v e Performance o f Dams i n Experiment I I  Experimental P  L i t t e r Size  B i r t h Weight o f Pups g  High-Lysine  11.011.2  6.0i0.2  a  Low-Lysine  10.3±0.4  5.5+0.2  b  High-Lysine Restricted  13.010.3  5.4l0.8  b  G r o u  B  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 a r e s i g n i f i c a n t l y d i f f e r e n t  (p<0.05)  Table X I .  E f f e c t o f a H i g h - L y s i n e , Low-Lysine o r H i g h - L y s i n e R e s t r i c t e d M a t e r n a l D i e t on Weight Gains of Pups i n Experiment I I  Experimental Groups  Body Weight (g)  Days Post Partum 10  15  High-Lysine  6.1±0.3  C  10.1±0.3  Low-Lysine  5.5±0.2  8.4±0.3  10.810.5  12.610.7  High-Lysine Restricted  5.510.1  9.210.3  14.110.8  17.811.3  C  18.8±0.5  C  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 a r e s i g n i f i c a n t l y d i f f e r e n t  28.9±0.9  C  (p<0.05)  67  Figure 8  E f f e c t of a h i g h - l y s i n e , lcw-lysine, restricted maternal d i e t on w e i g h t Experiment I I  or high-lysine g a i n o f pups i n  68  DAY OF LACTATION  Table X I I .  E f f e c t on a H i g h - L y s i n e , Low-Lysine or H i g h - L y s i n e R e s t r i c t e d M a t e r n a l D i e t on Heart and L i v e r Weights o f Pups on Day 15 of L a c t a t i o n  Experimental Group  T i s s u e Weight  T i s s u e Weight % of Body Weight  g  Heart  Liver  Heart  Liver  0.4010.02  2.9010.60  High-Lysine  0.123410.010  Low-Lysine  0.063910.005  0.352810.028  0.5110.03  2.8210.02  High-Lysine Restricted  0.114610.054'  0.5205+0.050  0.6010.10  3.2010.10  c  0.918410.042  c  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 a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05)  70  high-lysine  restricted  significantly g) .  Heart  weights  high-lysine greater  greater  of  these  tissue  weights  there  were  g)  which  of the low-lysine  groups  of  (0.1146  g)  the low-lysine  also (0.3528  (0. 1234 g) and  were  group  were group  pups i n t h e h i g h - l y s i n e  were e x p r e s s e d  no  (0.5205  than those  restricted  than  group  significantly  (0*0639 g ) .  as a p e r c e n t a g e o f  body  When  weights,  s i g n i f i c a n t d i f f e r e n c e s among t h e t h r e e  dietary  groups. When compared t o h i g h - l y s i n e liver  carnitine  lactation,  diet  (Table  XIII,  were s i g n i f i c a n t l y  low-lysine  levels  plasma  ad  libitum  Figures  ,  h i g h e r a t d a y 15 o f low-  or the r e s t r i c t e d high-lysine  diet  9 and 10) .  and h i g h - l y s i n e  low-lysine of  38.22  Heart  high-lysine  and l i v e r  carnitine  significantly  for  between t h e  r e s t r i c t e d groups.  diet resulted n  and  the  t h e pups and dams d i d n o t d i f f e r  The  values,  i n b o t h t h e pups and dams c o n s u m i n g e i t h e r  lysine  both  levels  control  mol/ml  i n maternal  compared  plasma  carnitine  t c 34;79 n mol/ml f o r t h e  r e s t r i c t e d dams and 19.22 n  mol/ml  for  the  high-  l y s i n e dams* . L i v e r c a r n i t i n e v a l u e s o f dams i n t h e h i g h - l y s i n e , high-lysine  restricted  and  low-lysine  groups  187.60, and 176.24 n mol/ml r e s p e c t i v e l y . levels  i n dams f e d t h e h i g h - l y s i n e  pregnancy  (37*70 n mol/ml, T a b l e  (19.22 n m o l / m l , T a b l e X I I I ) . _ fed  t h e same h i g h - l y s i n e  of g e s t a t i o n and  diet  The p l a s m a  120.98; carnitine  were h i g h e r a t d a y 21 o f  VI) t h a n  a t day 15 o f l a c t a t i o n  L i v e r c a r n i t i n e l e v e l s i n animals  d i e t averaged  and 120.98 n mol/g  were,  166*56 n mol/g a t t h e end  on day 15 o f l a c t a t i o n  ( T a b l e VI  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 o f a H i g h - L y s i n e , Low-Lysine o r H i g h - L y s i n e R e s t r i c t e d M a t e r n a l D i e t on Plasma and L i v e r C a r n i t i n e o f Dams and Pups on Day 15 o f L a c t a t i o n  Experimental Group  Plasma C a r n i t i n e nmol/ml  Liver  Carnitine  nmol/g  C  A Dams  Pups  High-Lysine  19.22±3.86  a  120.98132.39  a  Low-Lysine  38.22±7.56  b  176.24113.23  b  High-Lysine Restricted  34.79±5.14  b  187.601 9.84  High-Lysine  51.10±1.78  a  253.99111.68  Low-Lysine  61.02+4.13  b  370.71185.44  High-Lysine Restricted  62.07±3.75  b  360.78140.82  b  3  b  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 a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05)  C  Mean 1 SEM f o r 6 l i t t e r means  72  Figure  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 , r e s t r i c t e d d i e t on p l a s m a and liver dams on day 15 o f l a c t a t i o n  or h i g h - l y s i n e carnitine of  NMOL CARNITINE PER ML PLASMA OR G LIVER  O  4^ O  7777771  00  o  o o  o  o  o o  00  o  o  • 1—1 1 tr -<  t—i z  73 •X  m ien —i a: i I-H cr* n < H cn m o z tn  TR  en m i  r1 o i — t•< en I-H z tn  74  Figure  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 restricted maternal diet on plasma and liver c a r n i t i n e o f pups on day 15 of l a c t a t i o n  75  76  and  high-lysine restricted  significantly mol/ml) . .  higher  Likewise,  (62.07 n mol/ml)  than those liver  o f t h e h i g h - l y s i n e pups  carnitine levels  (370.71 n mol/g) and h i g h - l y s i n e  restricted  pups  than  pups  were  significantly  (253.99 n m o l / g ) . .  pups  higher  Plasma and  on day 15 c f l a c t a t i o n  tissues  Weight c h a n g e s o f dams d u r i n g  in  i n Figure  weight g a i n s  restricted low-lysine amount  11.  during  mol/g)  f o r the h i g h - l y s i n e  carnitine  levels  of  were a l m o s t d o u b l e t h e  pregnancy  and  pregnancy.  lactation  are  emphasizes the s i m i l a r i t y  i n the low-lysine  and h i g h - l y s i n e r e s t r i c t e d  o f weight  n  (Table V I ) .  The h i s t o g r a m  that occurred  groups  liver  (51.10 n  f o rthe low-lysine (360.78  those  (Table XIII)  l e v e l s of c a r n i t i n e i n f e t a l  depicted  g r o u p s o f pups were  During dams  and  high-lysine  lactation  lost  a  both t h e  significant  whereas t h e h i g h - l y s i n e c o n t r o l a n i m a l s  gained  weight.  EXPERIMENT I I I  The lysine gained  dams i n e x p e r i m e n t restricted  significantly  high-lysine The greater lysine  food in  (13.5  g/day. T a b l e lysine food  diet  diets  III  fed  consumed  low-lysine  significantly less  l e s s body w e i g h t t h a n t h o s e  or  high-  f o o d and  animals f e d the  ( T a b l e s XIV, XV, X V I , and F i g u r e 1 2 ) .  i n t a k e o f dams d u r i n g t h e h i g h - l y s i n e group g/day) XIV).  restricted  consumption  the  or  g e s t a t i o n was (16*5 g/day)  high-lysine  significantly  than i n t h e low-  restricted  groups  Food i n t a k e between the l o w - l y s i n e females  during  d i d not d i f f e r  and h i g h -  significantly.  p r e g n a n c y was e x p r e s s e d  (13.5  When  as g/100 g body  77  Figure  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 high-lysine restricted diet on weight c h a n g e s o f dams d u r i n g p r e g n a n c y and l a c t a t i o n i n E x p e r i m e n t I I  78  LACTATION  PREGNANCY  +140  r LOW-LYSINE  +120  •  HIGH-LYSINE RESTRICTED  HIGH-LYSINE  +100  +80  +60 I-H  Q O CQ  +40  UJ z  < u  +20  -20  -40  Table XIV. Food I n t a k e and Weight Gain o f Dams Fed a H i g h - L y s i n e , Low-Lysine 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 d u r i n g G e s t a t i o n i n Experiment I I I  Week o f G e s t a t i o n Experimental Group  Feed (g/day)  Body Weight (g)  Initial Body Weight  1  2  3  Daily Food I n t a k e  Total Weight G a i n  C High-Lysine  7.2±0.3  7.4±0.3  6.1±0.3  16.5±0. 4  Low-Lysine  6.4±0.2  5.9±0.2  5.2±0.3  13.5±0. 3  High-Lysine Restricted  6.1±0.2  5.6±0.1  5.0±0.2  13.5±0. 4  a  u b  b  High-Lysine  217. 5±5. 2  248.1±4. 8  276.6±4.3  336.3±5.4  Low-Lysine  232. 0±3. 3  239.1±4. 8  244.2±3.8  281.4±3.5  49 .5±1. 7  High-Lysine Restricted  232. 0±3. 4  244.0±3. 6  267.0±4.5  318.2±6.5  86 .8±4. 4°  A  Mean ± SEM f o r 18 dams  B  Food i n t a k e i s e x p r e s s e d a s 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 a r e s i g n i f i c a n t l y d i f f e r e n t  118 .8±4. l  (p<0.05)  a  b  80  weight, t h e r e (Table  was no s i g n i f i c a n t  XIV) .  experiment  These  results  (49.5 and  (118.8  Figure  g)  during  12).  and  experiment  gained  g e s t a t i o n than  These r e s u l t s  high-lysine II  experiment  whereas  I I I (Tables  Food  intake  are  presented  II  {Table  lower  differed  body  restricted of  i n Table  dams  VIII).  a  during  lysine  was  by t h e l c w -  significant  lactation, 15  intake  of  in  effect  than  lactation  lactation,  restricted  (29.0 g/day) g r o u p .  was e x p r e s s e d of d i e t  for the  those  significantly  (17,5 g/day) and h i g h - l y s i n e  lactation  fcr  i n experiment  o f dams was a g a i n  on f o o d  high-lysine  a s g/100  g  intake  was  dams  was  f o r t h e l c w - l y s i n e and h i g h - l y s i n e  Body w e i g h t c h a n g e s f o r t h e s e  Dams consuming  XIV  i n experiment  gain  As p r e v i o u s l y r e p o r t e d  Food i n t a k e  greater  (Table  dams was n o t s i g n i f i c a n t i n  difference  significant  groups.  g body  weight  groups than i n t h e h i g h - l y s i n e  t h e same d i r e c t i o n  17.1  XV.  food  (p<0.001)*  significantly  groups  from t h o s e  changes during  consumption d u r i n g  weight,  observed  the  more  either the low-lysine  c a r n i t i n e a n a l y s i s on day  VII) ,  g/day)  When f o o d  obtained i n  V I I a n d XIV r e s p e c t i v e l y ) . .  in the low-lysine  (20.3  intake  significantly  (86.8 g)  restricted  and w e i g h t  dams used f o r m i l k  in  diet  The d i f f e r e n c e between t h e a b s o l u t e  lysine  on f o o d  II- _  g) o r h i g h - l y s i n e r e s t r i c t e d  II.  of d i e t  were s i m i l a r t o t h o s e  Dams f e d t h e h i g h - l y s i n e weight  effect  were o f a s m a l l e r  as those  obtained  three  magnitude than, but  i n experiment I I  the h i g h - l y s i n e diet  groups  gained  (Table  an a v e r a g e o f  w e i g h t whereas, t h e dams f e d t h e l c w - l y s i n e o r h i g h -  restricted  respectively*  diets  lost  Weight change  an  average  between t h e  of 16.4 and 29.2 g  low-lysine  and  high-  81  Figure  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 high-lysine restricted diet on weight gain 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 DAY OF GESTATION  J 21  TABLE XV.  Food I n t a k e and Weight Changes o f Dams M i l k e d on Day 15 o f L a c t a t i o n and Fed a H i g h - L y s i n e , Low-Lysine 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 d u r i n g L a c t a t i o n  Week o f L a c t a t i o n Experimental Group  Initial Body Weight  1  2  Daily Food I n t a k e  Total Weight G a i n  C Feed (g/day)  Body Weight (g)  High-Lysine  8.2±0.3  12.6i0.2  Low-Lysine  7.6±0.5  h 9.210.4  b 17. 510.5  High-Lysine Restricted  7.010.2  9.0i0.1  20. 311.5  High-Lysine  280. 9±6.0  Low-Lysine  217. 615.4  ,High-Lysine Restricted  259. o±io.o  279.915.4  a  a  b  204.416.9 b  b  248.0110.7°  29.0 i l . 5  a  a  b  b  b  297.915.7  +17. 117.8  b 201.217.1  ,b -16. 415.4  229.8110.2°  -29. 2111.4  a  A  Mean 1 SEM f o r 6 dams  B  Food i n t a k e i s e x p r e s s e d i n g/lOOg body  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 a r e s i g n i f i c a n t l y d i f f e r e n t  weight (p<0.05)  a  b  84  lysine  restricted  differed of  diet  groups  significantly on w e e k l y  significant  were  from  weight  similar  t h e high  changes  (p<0.001).  lysine  during  The  lysine  on days 7 and 15 o f l a c t a t i o n *  low-lysine  15  were  high-lysine  and h i g h - l y s i n e  restricted  significantly  different,  also  group.  high-lysine  more than  the  both  values  The e f f e c t  lactation  significantly dams  the  however  was  dams  also  weighed  restricted  and  The b o d y  low-  weight o f  a n i m a l s on d a y s 7 as  was  the  and  case i n  experiment I I . Food dams  intake  used  (Table  and w e i g h t c h a n g e s  for  milk  carnitine  XVI) a r e s i m i l a r  carnitine  analysis  significantly  lower  lysine  restricted  (24. 2  g/day)  in  dams  When f o o d  g/100  g body weight, a s i g n i f i c a n t l y  found  i n those  the  low-lysine  high-lysine week  of  restricted  cr  diet  high-lysine  gained  lactation, females  respectively* .  Weight  restricted  dams d i f f e r e d  On  8  day  significantly in  turn  of  Food  an  (15.8 g/day) and  high-  in  gain  of  greater diet  of  food than  weighed s i g n i f i c a n t l y  g  the  high-lysine and  effects  10.8  high-lysine  restricted  group  g  on r e p r o d u c t i o n *  dams. weighed  group,  more t h a n t h e l o w - l y s i n e  Maternal d i e t a r y treatment  first  and h i g h - l y s i n e  high-lysine  more t h a n t h e h i g h - l y s i n e  Dams f e d t h e  and  12.1  was  dams c o n s u m i n g  during  the low-lysine  the  intake  diets*  from  high-lysine  was e x p r e s s e d as  low-lysine  significantly  lactation  the  consumption  average  f o r milk was  restricted  the  those  intake  an a v e r a g e o f 14.8 g  whereas lost  f o r dams u s e d  than  dams f e d t h e h i g h - l y s i n e  for  on day 8 o f l a c t a t i o n  lactation..  low-lysine  (17.7 g/day)  females*  obtained  of  the  lactation,  analysis  t o those  on day 15  during  which  group. as  Table XVI.  Food I n t a k e and Weight Changes o f Dams M i l k e d on Day 8 o f L a c t a t i o n and Fed a H i g h L y s i n e , Low-Lysine 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 d u r i n g L a c t a t i o n  Week o f Lactation Experimental Group  Feed (g/day)  Body Weight (g)  B  Initial Body Weight  Daily Food I n t a k e  High-Lysine  8.9±0.3  Low-Lysine  7.0±0.3  15.8±0.4  High-Lysine Restricted  7.0±0.2  17.7+0.9  High-Lysine  274.1±9.4  Low-Lysine  220.5±7.3  High-Lysine Restricted  254.7±11.6  288.9±6.9  C  C  24.2±1.4  C  Total Weight G a i n  C  C  +14.8±6.0  210.5±5.0  -12.1+6.5  243.9+8.2  -10.8±4.4  A  Mean ± SEM f o r 6 dams  B  Food i n t a k e i s e x p r e s s e d 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 a r e s i g n i f i c a n t l y d i f f e r e n t  (p<0.05)  C  86  assessed in  Table  by  XVII..  lysine  size  litter  (11.9),  similar. was  litter  and  Birth  birth  (5.5  g)  weights  low-lysine (Table  high-lysine  greater or  the  birth  mortality  restricted,  and  respectively.. (Table  lactation  growth  of  14)  similar  in  (10.7),  (1.9  low-  (12.3) g r o u p s  of g)  group  the  was  (6.1  g)  high-lysine  groups.  although  d i f f e r s i g n i f i c a n t l y ' between groups  in  the  experiment  pups d i d d i f f e r  II  significantly  III*.  o f pups i n t h e groups  high-lysine, was  0.7%,  high-lysine  0.7%,  d i f f e r e n c e between g r o u p s was  not  and  1.2%  significant  XVII) .  The  On  that  restricted  low-lysine  The  lysine  high-lysine  than  not  pups, i s p r e s e n t e d  restricted  weight of  rate  high  low-lysine  between g r o u p s i n e x p e r i m e n t The  weight of  pups i n t h e  high-lysine  the  birth  f o r the  o f pups d i d  and  X) ,  size  weight o f  significantly  restricted  and  was  to that  high-lysine  15  in  the  group  restricted  more t h a n t h e was  reported  (p<0.001) and  high-lysine  day  of  pups, i n experiment I I I  both days 8 the  performance  27.7  low-lysine  (Table  XVIII  and II  by  significantly  The  weighed  average  high-lysine  13,  (Table  XI).  the  pups  more t h a n  the  significantly  weight of  group and  the  Figures  (p<0.001) of l a c t a t i o n ,  high-lysine  and  measured  i n experiment  which i n t u r n  group.  g i n the  as  weighed  group  low-lysine  15  dams,  10.9  pups and  restricted  on  20.7  g  groups  respectively. The. lysine  obtained i n experiment  restricted  carnitine effect  data  of  levels diet  on  and than  low-lysine the  I I i n d i c a t e d the  animals  high-lysine  milk c a r n i t i n e  levels,  had  the  higher  hightissue  c o n t r o l animals. as  presented  in  The  Table  Table XVII.  E f f e c t o f a H i g h - L y s i n e , Low-Lysine o r H i g h - L y s i n e R e s t r i c t e d  Diet  on R e p r o d u c t i v e  Performance o f Dams i n Experiment I I I  Experimental Group  Litter Size  B i r t h Weight of Pups g  %.'.Mortality  B High-Lysine  10.7±0.9  6.1±0.2  a  0,7  Low-Lysine  11.9±0.6  4.9±0.2  b  1.2  High-Lysine  12.3±0.6  5.5±0,2°  0.7  Restricted  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  a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.0.5)  Table XVIII.  E f f e c t o f a H i g h - L y s i n e , Low-Lysine o r H i g h - L y s i n e R e s t r i c t e d M a t e r n a l D i e t on Weight G a i n s o f Pups i n Experiment I I I  Experimental Group  1  Days Post Partum 8  5  10  15  C Body Weight (g)  Body Weight^ (g)  High-Lysine  6.8±0.2  a  11.4±0.3  Low-Lysine  5.1±0.2  b  7.4±0.5  High-Lysine Restricted  5.8±0.2°  High-Lysine  6.4±0.3  Low-Lysine  5.0±0.3  High-Lysine Restricted  5.6±0.2  a  20.1±0.6  b  9.2±0.7  9.4±0.7°  a  ab  b  a  27. 7±1. 3  a  b  10. 9±1. l  b  20. 7±2. o  c  16.0+1.0°  ll.l±0.5  a  14.9±0.5  7.6±0.5  b  8.4±0.7  b  9.5±0.5°  12.7±0.5  C  a  A  Mean ± SEM f o r 6 l i t t e r means  B  Pups o f dams m i l k e d 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 a r e s i g n i f i c a n t l y  D  Pups o f dams m i l k e d on day 8  d i f f e r e n t (p<0.05)  89  Figure  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 high-lysine restricted maternal diet on weight g a i n s o f pups b o r n t o t h o s e dams m i l k e d on day 15 o f l a c t a t i o n  90  DAY OF LACTATION  91  Figure  14  E f f e c t o f a h i g h - l y s i n e , l o w - l y s i n e , or high-lysine r e s t r i c t e d m a t e r n a l d i e t on weight g a i n o f pups b o r n t o t h o s e dams m i l k e d on day 8 o f l a c t a t i o n  92  93  XIX  and  Figure  milk  carnitine  mol/ml),  were  mol/ml) the of  15* i s n o t c l e a r *  levels  s i g n i f i c a n t l y higher  high-lysine  milk  restricted  t h e r e was no  carnitine  high-lysine lysine  was  in  restricted  other  low-lysine  dietary  during  than  levels  high-lysine  restricted  dams and  with  carnitine  values  of  in  f o r dams and t h e i r  carnitine  with  their  concentrations  groups,  the  low-lysine  levels  on day 8  animals  did  not  Milk  were  high-lysine,  change  carnitine  significantly partum.  l o w - l y s i n e , and  offspring  Figure  in  values f o r the  on days 8 and 15 p o s t  are  compared  16.. Plasma a n d l i v e r  offspring  respective f o r the  carnitine  and h i g h - l y s i n e  of l a c t a t i o n .  their  high-  dietary  than  groups  i n both t h e l o w - l y s i n e and h i g h - l y s i n e  when compared  the  pattern  fed  diet,  the  i n milk  v a l u e s a r e f o r day 15 o f l a c t a t i o n .  concentrations  On day 8  The same  Milk carnitine  course  carnitine  higher  two  or  milk c a r n i t i n e  restricted  Plasma  carnitine  the  Dams  v a l u e s , f o r dams f e d t h e h i g h - l y s i n e  milk  diet.  (194. 20 n mol/ml)  g r e a t e r on d a y 2 o f l a c t a t i o n  than  between  libitum,  difference  groups.  the  females.  (107. 15 n mol/ml)  higher  and h i g h - l y s i n e  significantly  for  groups.  (277.46 n mol/ml) and day 15 two  ad  significant  significantly  higher  difference  mol/ml)  the h i g h - l y s i n e  (300.65 n  a n i m a l s consuming e i t h e r t h e  diet n  (134,19 n mol/ml)  had  n mol/ml)  those  (128.52  no  significantly  significant  on d a y 15 o f l a c t a t i o n  l e v e l s between  the  were  A  (426.04 n  than the l o w - l y s i n e  (151.21  (122.42 n mol/ml)  there  diet  levels  restricted  prevailed i.e*,  o f dams f e d t h e h i g h - l y s i n e d i e t  f e m a l e s , which i n t u r n  lactation  On t h e 2nd day o f l a c t a t i o n ,  control  lcw-lysine  Plasma were  significantly  restricted values. and  carnitine  groups, Plasma  high-lysine  T a b l e XIX.  E f f e c t of a H i g h - L y s i n e , Low-Lysine 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 M i l k C a r n i t i n e on Days 2, 8 and 15 P o s t Partum  Experimental Group  Days Post Partum 8  2  ,B Milk Carnitine (n mol/ml)  High-Lysine  426.04±29.50 '  Low-Lysine  300.65+43.21  High-Lysine Restricted  151.21±37.76 '  d  277.46±38.21 ' b  a  e  128.52±27.68 ' a  , 107.15±12.56 '  a  b,d  C  .C 122.42±16.23 '  a  15  e  194.20112.63  b ,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 a r e s i g n i f i c a n t l y d i f f e r e n t ( 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 a r e s i g n i f i c a n t l y d i f f e r e n t ( d , e - p<0.05)  95  Figure  15  Effect of a h i g h - l y s i n e , l c 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 on m i l k c a r n i t i n e on days 2, 8, and, 15 o f l a c t a t i o n  96  97  Figure  16  E f f e c t o f a h i g h - l y s i n e , l o w - l y s i n e , or high-lysine restricted diet 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 o f l a c t a t i o n  PLASMA DAMS  MILK DAY 15  PLASMA PUPS  DAY 15  99  restricted levels, lysine  on  groups d i d not d i f f e r day  15  of lactation,  and h i g h - l y s i n e  control  group.  In  different  between  groups,  on  differences  day  significantly*  restricted addition,  the 15  groups  milk  low-lysine of  were  observed..  higher  than  the  c a r n i t i n e was and  lactation,  between t h e h i g h - l y s i n e  c o n t r o l groups  were a l s o  Milk  restricted  i n t h e lowhigh-lysine  significantly  high-lysine whereas  carnitine  restricted  no  significant  and  high-lysine  100  CHAPTEE V DISCUSSION  DIETABY L Y S I N E ANE  The Hill had  results  of  no  significant the  (1970,1973) the  effect  birth  birth by  animals  fed  feeding a the  1968)  (Canfield Hill  last  weight of  a  decreased  of  gestat ion.  free  lysine  week of  lysine  to  diet. .  sparing  1978;  Chu  the  quantity of  or h i g h - l y s i n e born  significant  pregnancy  lysine  of f o o d  dams.  to  dams  difference  fed  have  diets the  diet  and  H e g s t e d , 1976).  that  maintained  adult  born  Stapleton levels  these a n i m a l s consuming  pups may fetus  the  have r e s u l t e d during the  t c dams f e d  high-lysine  last  of  from week  reproductive the  diet..  b i r t h weight  a  decreased  low-lysine  weighed s i g n i f i c a n t l y l e s s  between t h e  and rats  drop i n plasma l y s i n e  the  not  mechanism' ( Y a m a s h i t a  consumed fcy dams a f f e c t s  Offspring  restricted  may  ncn-pregnant  deficient to  free  in  for  -al.  suggested  authors suggested that  lysine  et  however was  They  diet  significantly  lysine  size  and  deficiency  Niiyama  of a  Litter  exist  Stapleton  but  offspring. .  deficient  d i e t * .. The  s u p p l y of  performance  those  lysine  the  size  adverse e f f e c t pups.  Chytil,  lysine deficient  The  litter of  those of  a maternal l y s i n e  (1972) r e p o r t e d a d r a m a t i c  during the  birth  on  of  shown  and  that  weight  weight  p r e g n a n c y b e c a u s e of Ashita,  study confirm  reported  documented an  affected  and  this  (1972,1980) , who  decreased  on  REPRODUCTION  There  than was  pups f r o m  no the  101  lysine the  deficient  birth  weight  restricted results  and c a l o r i e c f pups  for  groups d i f f e r e d  of  the  restriction  and  affect birth  restricted  third lysine  of o f f s p r i n g  The  experiment cnly,  suggest t h a t both  food  deficiency  r e p o r t s have  with decreased food  a reduction i n f e t a l in  Niiyama e t a l , ,  food  intake  the average d a i l y  respective  diet. is  by d i e t a r y  The  and  1973).  during  gestation  rats  pair-fed  than c o n t r o l  during  gestation have  with a  pregnancy.  25% These  by dams d u r i n g p r e g n a n c y  weight  groups  as  of gestation. .  gain  Lysine  during deficient  consumed  animals f e d  reduced food i n t a k e  weight  Chow and l e e (1964)  period  treatment..  by dams d u r i n g  weight a s s o c i a t e d  intake  f o r the t o t a l  consumption  (g/day)  dams  a decrease i n b i r t h  intake  body  by  i n v e s t i g a t o r s expressed food  food  of  associated  reported  their  high-lysine  i n experiment I I I ,  1920;  affected  and  significantly  (Barry,  Food  low-lysine  weight o f o f f s p r i n g .  Previous  decrease  the  groups i n e x p e r i m e n t I I ,  the  pregnancy  were  animals  and  significantly  lysine  less  supplemented  o f t h e l c w - l y s i n e g r o u p o f dams  t h o u g h t t o be due t o d e c r e a s e d a p p e t i t e a s a r e s u l t o f p l a s m a  amino a c i d and  Harper,  i m b a l a n c e c a u s e d by t h e l y s i n e 1963; Leung  Dams c o n s u m i n g pregnancy Although of  also there  weight  et a l . ,  significantly  was no s i g n i f i c a n t  less  food than c o n t r o l s less  weight t h a n  difference  by t h e l o w - l y s i n e  g r o u p s , dams f e d t h e h i g h - l y s i n e  and h i g h - l y s i n e  restricted  diet  the lcw-lysine  that  pregnancy  of dams d u r i n g  amount  restricted  gained  diet,  during  controls.  between t h e  w e i g h t t h a n t h o s e dams c o n s u m i n g weight g a i n  (Sanahuja  1 968).  gained s i g n i f i c a n t l y  gained  deficiency  mere  suggesting  was a f f e c t e d  by t o t a l  102  food  intake  previously  and d i e t a r y l y s i n e reported  dams d u r i n g  that  pregnancy  level.  t h e weight  Stapleton gain  was s i g n i f i c a n t l y  of  and H i l l  lysine  (1980)  deficient  l e s s than t h a t o f l y s i n e  supplemented c o n t r o l s . .  DIETARY L Y S I N E  AND LACTATION  Several authors increase  their  indicates supplies Munro  that are  and  controls lysine  used  for  weight  suggest  utilization  and  calorie  restricted in  body  milk  intake  body  than  study  l a c t a t i o n while  weight  groups  during  the  dietary  high-lysine  dams i n t h e l o w -  lost  weight,  and H i l l  lactation  consumed  was g r e a t e s t  such  (1980),  deficient  lactation,  appears  during  this  t o be period.  f o r the high-lysine controls  of Stapleton  and H i l l  when e x p r e s s e d  w e i g h t , was a l s o s i g n i f i c a n t l y  animals  weight  groups*  the r e s u l t s  during  than  o f body t i s s u e s by t h e l y s i n e  food  Food  should  (Kon and Cowie, 1961;  r e p o r t s by S t a p l e t o n  Absolute  confirming  this  restricted  on t h e amount o f f o o d  again  rather  production In  dependent  intake  tissues  during  earlier  and  Change  body  1964). _  high-lysine  confirm  rats  t h e same p e r i o d , s i n c e a l o s s i n body  maternal  gained  that nursing  f a t and w e i g h t by a s much a s n o n - l a c t a t i n g  Allison,  and  results  are of the opinion  body  control r a t s during  PERFORMANCE  greater  f o r the the low-lysine  ( 1972, 1980).  i n proportion to  f o r the  high-lysine  cr high-lysine  restricted  a significant  reduction  groups. Jansen in  food  and Chase  intake  during  (1976) a l s o n o t e d lactation  i n dams f e d a b a s a l  bread  diet,  103  deficient  i n lysine.  fortification decreased  intake  o f the b a s a l  maternal  Weight  loss  i s  It is  which  accumulated 10%  during  of t h e i r It  diets  i s  pregnancy.  same  highly unlikely  extent  may  food  as  during  be r e f l e c t e d  weighed  that  food  i n t a k e and  lactation,  when  due t o i n c r e a s e d that  dietary  utilization  lactating  p r o t e i n but l o s t  Eats  lysine  rats  fed  70% o f t h e f a t  f e d a low p r o t e i n d i e t  lost  body p r o t e i n . .  (accumulated This  note  lactation.  (1971) r e p o r t e d no body  to  increased  during  i s probably  lost  s t o r e s or a reduced the  diet  during  occurs  restricted,  protein  bread  weight l o s s  of b o d y f a t . . N a i s m i t h 25%  interesting  a  intake dam  a lactating  would  with  pregnancy)  large and  than  dam with  small f a t  be a b l e t o p r o d u c e body  stores  animals  which  milk t o of f a t  a s u f f i c i e n t food  i n the deficient  significantly less  1 of l a c t a t i o n  that  intake.  not  only  t h e h i g h - l y s i n e c o n t r o l s on day  b u t a l s o consumed  significantly less  food  during  lactation* . The end  lower  of g e s t a t i o n  deficient of  body  experiment  of  the  former  lactation*  able t o maintain  A  their  group.  by  Therefore,  l o w e r f o r one g r o u p o f l y s i n e  slightly  III  deficient  compared  However  b c t h g r o u p s were s i m i l a r .  body f a t s t o r e s compensated during  of the lysine  lysine  accumulation  growth  their  rates  deficient  animals,  food  these  performance  by  of  t h e lower intake  e v e n though e n e r g y r e s e r v e s  food i n t a k e during between  the  A n i m a l s with  increasing  t h e same l a c t a t i o n  relationship  dams a t t h e  to  dams i n e x p e r i m e n t I I , i n d i c a t e s a l o w e r  body f a t b y  offspring  in  weight  were  dams were increasing  lactation..  food  intake during  l a c t a t i o n and  104  successful  milk  impairment  was  production observed  was r e s t r i c t e d  during  Lee,  Lactating  1964) .  produced diet caused  lower  (1980)  and  production-  amount  the  milk,  pregnancy rats  less  Cox,  than  1946) .  lactation  on  a  those  When compared  o f milk of  with  diet,  milk  there  significantly the  low  fed a  fed  by  was a s i g n i f i c a n t  a low-lysine diet*.  lower than present  lysine  study,  significantly  reduced  less  third  i n the p r o t e i n content  group of  lactation  that  both  production  l e s s than  food  in this  high-lysine  was s i g n i f i c a n t l y  significantly  intake  Hill  diet  on m i l k  dams  fed a  reduction i n  of  pups  protein  in  weaning  diet  from  were  the  and H i l l  greater  produced  study.  o f t h e mother r a t . .  lysine  the  ( 1972, 1980).  milk  produced  by dams f e d t h e  on  day  the low-lysine  t h e h i g h - l y s i n e c o n t r o l pups, and  was  was  Body w e i g h t o f t h e  restricted, than  high-  Food i n t a k e  i n t h e amount o f  o f t h e milk  also occurred pups,  than  by S t a p l e t o n  Thus i t a p p e a r s t h a t a d e c r e a s e  diet  and  f o r t h e l o w - l y s i n e dams, a s  i n the studies reported  low-lysine  volume  pups c f mothers f e d t h e l o w - l y s i n e  c o n t r o l f e m a l e s on day 15 o f l a c t a t i o n .  and  protein  that of controls.  lysine  case  diet  deficient  weighed  significantly  protein  Similarly  rations  also  and  i n milk  deficient  produced  intake  (Chow  Stapleton  p r o d u c e d and i n t h e g u a n t i t y  dams  Growth  high  This decrease  w e i g h t s o f pups from dams f e d t h e  In  and  placed  milk  reported.  c f dams whose f o o d  the e f f e c t s o f a l y s i n e  supplemented  the  teen  weaning w e i g h t s i n o f f s p r i n g .  examined  lysine  often  i n offspring  both  significantly  (Mueller  has  deficiency  15  of  pups y e t  indicating  affects  milk  105  DIET  AND TISSUE HEIGHTS  Liver the  weight  i n dams, n e o n a t e s , and f e t u s e s  high-lysine  c o n t r o l group  lysine restricted  groups. .  deficiency  theix fetuses during weight  control  lysine  i n proportion  offspring, on h e a r t  pregnancy..  whereas,  by  expressed  d i d not retard  o f the l o w - l y s i n e  values  affected  there  were  was  no  results  was p r o p o r t i o n a l t o t h e  weight.  Lysine  heart  weight  weight  weights  d i f f e r e n c e among t h e d i e t a r y g r o u p s . . T h e s e the animal's l i v e r  body  liver  significant  t o t a l body  of  when  as  that  in  t h a n i n t h e l o w - l y s i n e and h i g h -  However,  percentage  higher  expressed  suggest  a  was  there  weight  heart  heart  of  dams  or  However, on day 15 o f l a c t a t i o n pups  was s i g n i f i c a n t l y  weights  deficiency. t o body  weight  of  When  mothers  heart  less  than  were  not  weights  were  w e i g h t , f o r b o t h dams a n d  was no s i g n i f i c a n t  effect  of dietary  their  treatment  cn day 15 o f l a c t a t i o n *  DIET Afijf L Y S I N E AND PLASMA AND  LIVEE  CARNITINE  Dams  The liver  diets  containing  carnitine  levels  0.27% l y s i n e  d i d not l i m i t  o f dams d u r i n g  under t h e e x p e r i m e n t a l c o n d i t i o n s o f t h i s pregnancy plasma  there  and l i v e r  low-lysine  diet.  was a s m a l l carnitine  gestation  The i n c r e a s e  i n plasma  and  or l a c t a t i o n ,  s t u d y . .. On day  but i n s i g n i f i c a n t  concentrations  plasma  increase  21  of  i n both  i n t h o s e dams f e d t h e and l i v e r  carnitine i n  106 V  the  lysine  deficient  of l a c t a t i o n  was  Plasma during the  carnitine  However,  carnitine  during  control  values  between t h e 15  of  levels. the  low-lysine  by  heart  hepatic  tissue  concentrations. carnitine  oxidation.  to  did  not  that  differ  total  Yue,  1963) has  been  (Khan and  (1975) r e p o r t e d  the  by  Roblesauthor's  intake  liver  shown  normal  day was  of  to  for  long-Chain oxidation  increase  Bamji, 1979).  a  carnitine  i s reguired  interestingly,  t h a t the  for  g r o u p s on  oxidation  and  of  significantly  food  plasma and  and  that  controls  this  a c y l t r a n s f e r a s e system  muscle  when  Furthermore,  ketogenic  capacity  of  of f a s t e d weaned r a t s c o r r e l a t e d w i t h c a r n i t i n e Therefore,  synthesis  carnitine.levels  reason  high-lysine restricted  intake i s restricted  McGarry e t a l . ,  half  plasma  w h i c h t c compare  rats.  transport  ( F r i t z and  p r e g n a n c y and  that reported  for  in  Robles-Valdes et a l .  The  with  r e g u l a t i o n of  carnitine  acids  of p a l m i t a t e  fat  source  suggesting  f a c t o r in the The  15  were  c a r n i t i n e l e v e l s of  work and  levels  and  intramitocondrial  fatty  food  carnitine  lactation,  dominant  day  carnitine  animals,  u n d e r s t o o d , and  other  liver  approximately  liver  tissue carnitine levels  Tissue  by  reported.  present  i s no  earlier  were  previously  the  and  c a r n i t i n e during  (1976) i s n o t  knowledge, t h e r e maternal  liver  i n plasma and  al.  pregnancy  reported  lactation,  between et  p a i r - f e d c o n t r o l s on  high-lysine control  those  (1976).  Valdes  during  i n the  same r a n g e a s  contradictions  their  significant.  lactation,  animals  dams and  may  by be  i f dietary lysine the  expected  liver, under  an  is  not  increase  conditions  of  limiting in  tissue  increased  1C7  Tanphaichitr increase  in  plasma  weaning  female  compared  with  levels  lysine  the  carnitine The  i n the  are  results  of  controls,  in  diet.  liver  male w e a n l i n g in  response  suggests that involved  body  oxidation  for  present  dams,  Animals  When  carnitine Only  rats  fed  low-  lysine  factors,  regulation  liver a  to d i e t a r y  other  i n the  in fed  at fat  study  i.e.,  of  tissue  diets  that  of  P l a s m a and  in fact  higher  in  these  that  were a  depressed  r e s t r i c t e d food  presumably  due  liver carnitine  animals than  g a i n e d weight d u r i n g  tissue  lactation,  m e t a b o l i s m c a u s e d by  t h i s period,  body f a t i  suggest  l e a s t during  weight during  significantly  F e t u s and  the  alterations  intake.  to  intake,  increased  levels  in control  were  animals  lactation.  Neonates  A maternal carnitine  lysine  deficiency  levels in either  not  limit  plasma o r  or  offspring.  In  were h i g h e r i n t h e  lcw-lysine  and  levels  restricted  groups than i n the  rather  did  fetuses  carnitine  carnitine  deficient  post  values.  of  intake  a significant  d e f i c i e n t females.  difference  animal,  in  a lysine  supplemented  female r a t s  reflection  who,  consuming  This  levels  lost  non-pregnant,  levels  were i n c r e a s e d  carnitine  food  carnitine  increased  levels  diet.  of  noted  lysine  between male and sex  a l . . (1976) p r e v i o u s l y  rats  were a l s o  carnitine  et  than  controls,  dietary  lysine  indicating levels  fact  liver tissue  high-lysine that  affects  food tissue  levels.  Although  fetal  plasma  and  liver  carnitine  levels  were  108  higher only  in  the  the  plasma  level  to  note t h a t  interesting are  low-lysine  a reflection  though  of  placental  (Hahn and  Skala,  There  group than  was  significantly  the  and  long  Fritz,  is  utilizing  control  chain  f a t as  on  to  fetal was  day  15  reported  during  and  et  a l . , 1976;  is  tissues  levels,  acids and  be  even rat  Bailey, in  p l a s m a and  plasma  for  1971). the  liver 5C%  and  rat  Ferre  et  One  fetus  fetus  ftugenfeld expect  than i n  animals  Therefore,  lower than These  of the  i t is  of  control  results  rat  a l . , 1978;  not  high-lysine  that  carnitine  to  would  carnitine in  liver  the  Fetal carnitine  f o r the  energy*  development  1978;  fuel  glucose*  lactation*  postnatal Borum,  metabolic  synthesis  ( D r a h o t a e t a l * , 1964;  lower  of  indicate fetal  capacity  approximately  previously pre-  i s the  fatty  to  derived  a major s o u r c e o f  that  animals  neonates  as  lockwood  levels  surprising,  carnitine  primary  maternally low,  1970;  carnitine  It  c a r n i t i n e i s l i m i t e d i n the  e v i d e n c e at p r e s e n t  requirements are oxidize  higher*  group,  1975)*  i s no  tissue  plasma  of  o f c a r n i t i n e i n mammals.. The fetal  high-lysine  carnitine levels in fetal  maternal  transfer  i n the  confirm  concentrations (Eobles-Valdes  Seccombe et a l . ,  1978). Immediately rich  in  fat,  after birth whicI  acts  the  as  neonatal rat  the  (Dymsza, 1964;  Luckey e t  14%  fat  and  a d e q u a t e amounts o f  for  fatty  acid  oxidation  period  of  increased  al. ,  to  fatty  r a i s e d plasma c o n c e n t r a t i o n s  main e n e r g y  1954).  Eat  yielding  milk  c a r n i t i n e are  proceed  at  normal  acid utilization of  receives  ketone bodies  a  substrate  contains needed  diet  to  rates.  about allow This  i s characterized (Drahota  et  by  al..  109  1964;  Lockwood  carnitine, Valdes  and  et  to  the  increased  carnitine when the  high  Seccombe e t In  the  between  a l . , 1978)  less can  than  levels acid  the  diet  utilize  a  (Lockwood  energy  demands  and  (RoblesPlasma  concentrations  high f a t and  in tissue  high-lysine  compared  oxidation*  milk  diet  Bailey,  1971;  carnitine  levels  on  the  which  day  15  high  of  higher  the  low-  significantly lactation.  pups were fat  were  suggested  pups f r o m  weighed  groups of  percentage of therefore  controls,  groups  pups  significantly  addition,  controls  deficient  restricted  had to  In  restricted  the  larger  1978)  a l . , 1978).  carnitine  differences  high-lysine that  et  i s weaned from the  and  high-lysine  speculate  liver  al.,  concentration  Ferre  However, b o t h g r o u p s  fatty  and  1978;  et  .  present study  carnitine  lysine  r a t pup  Ferre  carnitine  l e v e l s and  low-lysine  increased  1971;  liver  carbohydrate  insignificant. liver  Bailey,  a l * , 1976 ; Borum,  k e t o n e and decrease  and  diet  One  forced  for  increasing  their  to  carnitine  to  immediate carnitine  requirements. The  suckling  precursors  rat's  ability  reaches adult  values  (Hahn, u n p u b l i s h e d d a t a ) . milk  as  its  major  by  Until  source of  that  carnitine  higher  circulating  transportation carnitine  via  levels their  requirements are  results  synthesis  of milk  neonate (Hahn and  of  depends Skala,  lactation  fat  carnitine to  lactation  of t h i s study  during  utilization  from i t s  s e c o n d week o f  carnitine  a l . , 1976).. The  t h o s e dams h a v i n g i n c r e a s e d  the  t h e n , the  Robles-Valdes et increased  synthesize  nursing  1975;  suggest occurs  stores.  are  on  In  turn  available  offspring  g r e a t e r . . Plasma c a r n i t i n e  in  for whose  levels  in  110  lysine  deficient  similar  whereas,  lactation similar  during  plasma  trend  was  lactaticn  carnitine  were 5C% of t h o s e  t h e r e was l i t t l e lysine  dams  in  observed  deficient  dams  values  controls  but  pregnancy  carnitine  values  during  pregnancy, values,  p r e g n a n c y and l a c t a t i o n  control  were  i n controls  during  forliver  change between  and  dropped  A  i.e.,  i n the by 7 3 % i n  controls._  MILK CARNITINE  The least et  primary  24 h o u r s  postpartum  a l . , 1978)..  control by  source o f  animals  in  agreement  were h i g h e s t  carnitine  with  carnitine  Dietary carnitine,  The  was s m a l l Milk  the  15 were  levels  during  is  less  increased  With  Thus  during  higher  the exception restricted milk  levels  course  those  carnitine  were s i m i l a r  levels  appear  milk  synthesis. levels levels.  deficient of  dams  lactation.  g r o u p on d a y s 8 in  high-lysine  o f day 2, m i l k c a r n i t i n e dams  cn  carnitine  deficient  than  (1976) , and  dependent  i n lysine  the  is in  m a t e r n a l plasma  milk  This  lactation  carnitine  i n the l y s i n e  significantly  high-lysine  controls.  carnitine  and i n s i g n i f i c a n t  carnitine  controls.  pup  deficiency  i n milk  and d e c r e a s e d  of Bobles-Valdes e t a l .  aging  at  high-lysine  partum r e s p e c t i v e l y .  which a p p e a r s t o i n c r e a s e  decrease  i n the  on day 2 o f . l a c t a t i o n  because o f i n s i t u  lysine  tissue,  t h e mother r a t ( F e r r e  concentrations  concentration  the r e s u l t s  supplies  neonatal  i s t h e m i l k from  Milk c a r n i t i n e  may o c c u r b e c a u s e t h e  and  in  71% and 75% on d a y s 8 and 15 p o s t  decline  of  carnitine  values i n  t c those i nthe to  respond  to  111  dietary  lysine  However*  levels  on day 15  carnitine, responded  in  of  both  rather  than  lactation*  to  milk  total  food  carnitine  dams and o f f s p r i n g ,  within  i n t h e same way t o t h a t d i e t a r y  intake.  and  plasma  a dietary  group,  treatment  (see  Figure  16) . The  higher  probably their  milk  levels  contributed to the higher  offspring.  maternal liver  carnitine  plasma  plasma and  Movement o f c a r n i t i n e  into  maternal  h a s been r e p o r t e d  butyrobetaine,  i n lysine  milk  after  t h e immediate  and  from  deficient  liver  of  in  liver to  into  m o t h e r s were i n j e c t e d precursor  levels  maternal  finally  dams  neonatal  with  carnitine  o f *C 1  (Robles-  V a l d e s e t a l . , 1976) . . A in  problem  milk*.  The  quantities carnitine rats 15  of  when d e t e r m i n i n g  volume milk  of  may  milk  have  nutrient  obtained a  concentrations  varies  concentrating  and  the  t h e lowest  low-lysine  quantity  fed  The s i g n i f i c a n c e  e f f e c t on t h e  of milk,  animals  yielded  o f t h e low milk  whereas a t d a y s 8  and  t h e s m a l l e s t volume o f  carnitine  concentraticns  in  t h e h i g h - l y s i n e r e s t r i c t e d dams on day 2 c a n n o t be  at  this  explained  time.  The  results  of  this  deficiency  does not i m p a i r  the  fetus  dam,  actually  small  l e v e l s , f o r e x a m p l e , a t day 2 o f l a c t a t i o n t h e c o n t r o l  yielded  milk.  exists  higher  or  study  indicate  p l a s m a and l i v e r  offspring*  i n deficient  t h a t maternal  levels  in  P l a s m a and l i v e r c a r n i t i n e  was  animals,  carnitine  lysine  the higher  level  being  in  112  response  to  deficiency  per  alterations i n both  a general food se. .  deficiency  The  study  the  of dietary  several  newborn  study in  carnitine  reports  mammal.  (Hahn  carnitine  tissue  i n lysine of  and c o n t a i n i n g  carnitine  acetylcarnitine  formula  are  Soyabean  human b r e a s t  that  a  infant  in  the  formulas  carnitine  levels.  milk* .  i n infants reduced  et a l . ,  of this  deficiency  A deficiency  because  a carnitine  (Ncvak  of  the  t o human  plasma  carnitine  receiving  a soybean  based  a l . ,  1979)..  et  whereas  a s i g n i f i c a n t amount o f  1978).  It  milk  or  i s  probable  be i n d u c e d  The i m p l i c a t i o n  deficiency.  of  compared  i n the r a t could  feeding.  the  of animals f e d a diet  When  contain  a baby f e d human b r e a s t  against  from  have no d e t e c t a b l e c a r n i t i n e ,  deficiency  of a r t i f i c i a l  of carnitine  synthesized  milk o r ecu's milk,  levels  1975; R o b l e s -  of a maternal l y s i n e  m i l k and cow's m i l k  i s that  protected  is  carnitine  (Schmidt-Sommerfeld  conditions  lysine  and S k a l a ,  minimal c a r n i t i n e  significantly  based  carnitine  maternal  f o r human newborns i s  may n o t d e v e l o p i n t h e o f f s p r i n g  newborns f e d human b r e a s t and  levels  One o f t h e main o b j e c t i v e s  was t o examine t h e e f f e c t  supply  metabolic  carnitine  t h e importance  Carnitine  amino a c i d l y s i n e . .  t h e r a t on n e o n a t a l  low  during  the  i s warranted.  but  essential  than t o a l y s i n e  regarding  offspring  V a l d e s e t a l . , 1976), h a v e i n d i c a t e d in  rather  with changes i n t i s s u e  their  significance  unknown*  Further  associated  t h e dams and  restriction  under  f o r t h e human  cow's  milk  is  113  Since energy  fatty  acid  i s p r o d u c e d by  may  be  the  adult.  carnitine neonate, s t u d y may carnitine  the  more i m p o r t a n t Thus may  the  only  be  if artificial indicate  to  i s the  neonate, d i e t a r y  during  this  results an  of  not  main pathway by intake than  this  the  are  effect a  of  of  as  a  or  suggest  nutrient used.  the  Further  decreased  source of  in  that  for  detrimental effect  fatty acids  which  carnitine  prenatally  study  dietary  practices  n e o n a t e has  utilize  period  essential  feeding  whether o r  l e v e l s i n the  newborn's a b i l i t y  oxidation  tissue on  the  energy.  114  CHAPTER VI SUMMARY OF  1) A m a t e r n a l l y s i n e gain  and  daily  Weight g a i n  RESULTS  deficiency  food  significantly  intake  of r e s t r i c t e d  (g/day)  control  decreased  o f dams d u r i n g  dams  was  also  weight  pregnancy.  significantly  lower.  2)  A  during  maternal  lysine  pregnancy  did not affect  weight o f o f f s p r i n g  3)  A  of  dams  deficient  fetuses  significantly  A  lysine  plasma  pregnancy. .  5)  reduced f o o d whereas  However,  d i d not r e t a r d  pregnancy.  were  heart  birth  weight i n  However, l i v e r  significantly  deficiency  and l i v e r  However,  weights  lower i n the l y s i n e  during  carnitine  fetal  pregnancy  levels  plasma  tended  to  o f dams on d a y 21 o f  carnitine  levels  were  increased.  maternal 1  size.  restriction  group.  4) A m a t e r n a l increase  litter  deficiency  fetuses during  and  or maternal food  was s i g n i f i c a n t l y d e c r e a s e d . .  maternal lysine  dams o r t h e i r  deficiency  lysine  intake  lysine  (g/day)  restricted  during  resulted  and w e i g h t  supplemented  period.. High-lysine amount o f w e i g h t  deficiency  dams dams  lactation.  loss  gained also  in a significantly during weight  lost  a  lactation, during  this  significant  115  6)  A  maternal  resulted of  lactation-  significantly  7)  A  less  Livers  less  than  Heart  A  liver  liver  Liver  of  more  lysine  in  bcth  offspring  those  deficiency  had  than l y s i n e  A maternal  lysine  deficiency  increased  15 o f l a c t a t i o n  lactation,  supplemented  and  pups were  milk  during  significantly  deficient  restricted  carnitine  lysine  less  pups.  on day 15  restricted  higher  plasma  of dams and  controls.  and l a c t a t i o n  c e n c e n t r a t i o n s on d a y s  when compared t o m i l k o f l y s i n e  of  group.  p r e g n a n c y and l a c t a t i o n  d u r i n g pregnancy  restricted  but  significantly  supplemented  milk c a r n i t i n e  high-lysine  dams  restricted  controls,  of the l y s i n e  significantly  levels  significantly  and l a c t a t i o n  weight of  pups weighed  dams and pups. . H i g h - l y s i n e  carnitine  controls  d u r i n g pregnancy  plasma and l i v e r  also  but  reduced.  deficient  increased  weighed  controls,  weights of h i g h - l y s i n e  lysine  than  also  15  pups.  i n t h e h i g h - l y s i n e and h i g h - l y s i n e  significantly  their  supplemented  of high-lysine restricted those  pups  weight b u t net h e a r t  significantly  maternal  lactation  lysine  and l a c t a t i o n  weight o f pups on d a y  restricted  deficiency  weights of l y s i n e  than those  and  lysine  reduced  significantly  10)  than  d a y 15 o f l a c t a t i o n *  8)  d u r i n g pregnancy  more t h a n t h e l o w - l y s i n e  dams were a l s o  and  High-lysine  maternal  significantly  9)  deficiency  i n a s i g n i f i c a n t l y l o w e r body  significantly  on  lysine  animals*  supplemented  8  supplemented On  controls  day  2 of  contained  116  significantly which  in  high-lysine  more c a r n i t i n e  turn  had  restricted  than  a higher dams.  milk  milk  cf lysine  carnitine  deficient  dams,  c o n c e n t r a t i o n than  117  BIBLIOGRAPHY  Abbey, H.,  and Howard, £.. 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