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UBC Theses and Dissertations

Relationships between plasma amino acid concentration and milk protein production Shelford, James Arthur 1974

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c / R E L A T I O N S H I P S BETWEEN PLASMA AMINO ACID CONCENTRATION AND MILK PROTEIN PRODUCTION b y JAMES ARTHUR SHELFORD B . S . A . , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1966 M . S c . , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1969 A T H E S I S SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e D e p a r t m e n t o f ANIMAL S C I E N C E We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y O F B R I T I S H COLUMBIA J a n u a r y , 1974 In presenting th is thesis in par t ia l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thesis for f inanc ia l gain sha l l not be allowed without my writ ten pe rm i ss i on . Depa rtment The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada A B S T R A C T I n r u m i n a n t s t h e a e r o b i c h o s t a n i m a l i s d e p e n d e n t on t h e a n a e r o b i c f e r m e n t a t i o n w i t h i n t h e rumen t o s u p p l y p r o t e i n a n d e n e r g y . T h i s a n a e r o b i c f e r m e n t a t i o n r e s u l t s i n a r a t h e r c o n -s t a n t r a t i o b e t w e e n p r o t e i n and e n e r g y . In y o u n g g r o w i n g a n i m a l s a n d l a c t a t i n g a n i m a l s t h e demand f o r p r o t e i n i s g r e a t e r t h a n t h e demand f o r e n e r g y . P l a s m a a m i n o a c i d d a t a i n d i c a t e t h a t p r o t e i n c o u l d be l i m i t i n g i n t h e s e two s i t u a t i o n s . In t h e f i r s t p h a s e o f t h e s t u d y , p l a s m a f r e e a m i n o a c i d s , m i l k v o l u m e a n d m i l k c o m p o n e n t s w e r e d e t e r m i n e d a t two week i n t e r v a l s t h r o u g h o u t t h e l a c t a t i o n s o f two m a t u r e A y r s h i r e c o w s . T h e l a r g e a m o u n t s o f p r o t e i n t h a t w e r e s e c r e t e d d a i l y d u r i n g t h e f i r s t t h i r d o f l a c t a t i o n p l a c e d h e a v y demands o n t h e a n i m a l s f o r p r o t e i n s u p p l i e s . T h e s e demands w e r e r e f l e c t e d i n c h a n g e s i n p l a s m a f r e e a m i n o a c i d s d u r i n g t h e l a c t a t i o n c y c l e . L y s i n e , l e u c i n e , i s o l e u c i n e a n d m e t h i o n i n e e x h i b i t e d t h e g r e a t e s t v a r i a -t i o n i n r e s p o n s e t o t h e demands o f l a c t a t i o n . A s e r i e s o f a b o m a s a l i n f u s i o n s o f t h e a b o v e a m i n o a c i d s a n d o t h e r s was u n d e r t a k e n t o d e t e r m i n e t h e i m p o r t a n c e o f a m i n o a c i d s f o r t h e s y n t h e s i s o f m i l k p r o t e i n . T h e i n f u s i o n s w e r e g r o u p e d i n t o t h r e e l o t s ; t h o s e c o n t a i n i n g m e t h i o n i n e , t h o s e c o n t a i n i n g i s o l e u c i n e a n d t h o s e c o n t a i n i n g l y s i n e . E f f e c t s o b s e r v e d d u r i n g i n f u s i o n s c o n t a i n i n g m e t h i o n i n e s u g g e s t e d t h a t m e t h i o n i n e i t s e l f h a d l i t t l e e f f e c t on- p r o t e i n p r o d u c t i o n . M e t h i o n i n e d i d n o t a l t e r t h e e f f e c t s o f o t h e r a m i n o i i i a c i d s o n m i l k p r o t e i n s y n t h e s i s . T h e r e s p o n s e s n o t e d when m e t h i o n i n e was a c c o m p a n i e d b y b r a n c h e d c h a i n a m i n o a c i d s o r b y l y s i n e w e r e t y p i c a l o f r e s p o n s e s o f b r a n c h e d c h a i n a m i n o a c i d s o r l y s i n e a l o n e . I n f u s i o n s c o n t a i n i n g i s o l e u c i n e and o t h e r b r a n c h e d c h a i n a m i n o a c i d s c a u s e d an i n c r e a s e i n m i l k p r o t e i n c o n c e n t r a t i o n , a d e c r e a s e i n m i l k p r o d u c t i o n a n d no i n c r e a s e i n t o t a l p r o t e i n p r o d u c t i o n . P l a s m a a m i n o a c i d d a t a r e v e a l e d t h a t i n f u s i o n o f b r a n c h e d c h a i n a m i n o a c i d s r e s u l t e d i n d e c r e a s e s i n c o n c e n t r a -t i o n s o f t h e o t h e r p l a s m a a m i n o a c i d s . T h e d e c r e a s e i n p l a s m a a m i n o a c i d c o n c e n t r a t i o n , t h e l a c k o f r e s p o n s e i n m i l k p r o t e i n p r o d u c t i o n a n d t h e known e f f e c t s o f b r a n c h e d c h a i n a m i n o a c i d s o n i n s u l i n s e c r e t i o n s u g g e s t t h a t i n s u l i n m i g h t be a f f e c t i n g t h e i n t a k e o f a m i n o a c i d s b y m u s c l e t i s s u e . When l y s i n e - c o n t a i n i n g i n f u s i o n s w e r e e x a m i n e d m i l k p r o -d u c t i o n was f o u n d t o i n c r e a s e . M i l k p r o t e i n c o n c e n t r a t i o n r e m a i n e d c o n s t a n t r e s u l t i n g i n an i n c r e a s e i n t o t a l m i l k p r o -t e i n p r o d u c t i o n . T h e m o s t d r a m a t i c i n c r e a s e i n m i l k a n d p r o t e i n p r o d u c t i o n o c c u r r e d when l y s i n e was i n f u s e d a l o n e . I n f u s i o n s c o n t a i n i n g l y s i n e d i d n o t r e s u l t i n d e c r e a s e s i n p l a s m a f r e e a m i n o a c i d s . T h e r e was a g e n e r a l t r e n d f o r a l l t h e e s s e n t i a l a m i n o a c i d s t o i n c r e a s e o r r e m a i n a t t h e same l e v e l d u r i n g t h e i n f u s i o n . L y s i n e a p p e a r s t o be t h e l i m i t i n g a m i n o a c i d f o r m i l k p r o t e i n s y n t h e s i s . I t i s p o s s i b l e t h a t t h e i n c r e a s e d s u p p l y i v o f l y s i n e f r o m t h e a b o m a s a l i n f u s i o n a f f e c t e d t h e a m i n o a c i d m e t a b o l i s m i n m u s c l e i n s u c h a way a s t o f r e e a m i n o a c i d s f o r m i l k p r o t e i n s y n t h e s i s a n d e n e r g y m e t a b o l i s m . V T A B L E OF CONTENTS P a g e ABSTRACT i i T A B L E OF CONTENTS V L I S T OF T A B L E S v i i i L I S T OF F IGURES x i i ACKNOWLEDGEMENTS x i i i A . INTRODUCTION 1 B . L I T E R A T U R E SURVEY 3 1. D e g r a d a t i o n o f n i t r o g e n o u s m a t e r i a l s i n t h e rumen 3 a) Ammonia p r o d u c t i o n 3 b) O t h e r p r o d u c t s 7 2 . E x t e n t o f f o r m a t i o n o f m i c r o b i a l c e l l s i n t h e rumen 8 a) A s s i m i l a t i o n o f n i t r o g e n b y t h e m i c r o b e s 8 b) L i m i t a t i o n o f m i c r o b i a l p r o t e i n s y n t h e s i s b y a v a i l a b l e e n e r g y 13 3 . Q u a l i t y o f rumen m i c r o b i a l c e l l s 18 4 . D i g e s t i o n a n d A b s o r p t i o n o f p r o t e i n 20 a) D i g e s t i o n 20 b) A b s o r p t i o n 24 5 . P l a s m a f r e e a m i n o a c i d s 25 a) P l a s m a f r e e a m i n o a c i d s a s a p r o p o r -t i o n o f t h e t o t a l b o d y a m i n o a c i d s 2 6 b) U t i l i z a t i o n o f f r e e a m i n o a c i d s f o r p r o t e i n s y n t h e s i s 27 c) R o l e o f a m i n o a c i d s u p p l y o n p r o t e i n s y n t h e s i s 28 v i P a g e d) S u p p l y o f f r e e a m i n o a c i d s f o r p r o -t e i n s y n t h e s i s 30. (1) S o u r c e 30 (2) F o o d r e s t r i c t i o n o n a m i n o a c i d s u p p l y a n d p r o t e i n s y n t h e s i s 30 (3) D i e t a r y e f f e c t s 33 (4) T r a n s p o r t 35 e) U s e o f p l a s m a f r e e a m i n o a c i d c o n c e n -t r a t i o n s t o d e t e r m i n e t h e a d e q u a c y o f d i e t a r y p r o t e i n 37 (1) M o n o g a s t r i c s 37 (2) R u m i n a n t s 4 0 6 . Summary o f l i t e r a t u r e s u r v e y 47 B . EXPERIMENTS 4 9 1 . E x p e r i m e n t I. M i l k , m i l k c o m p o s i t i o n a n d p l a s m a f r e e a m i n o a c i d s o v e r t h e c o m p l e t e l a c t a t i o n 49 a) M a t e r i a l s a n d m e t h o d s 49 b) R e s u l t s 52 c) D i s c u s s i o n 64 2 . E x p e r i m e n t I I . A b o m a s a l i n f u s i o n o f c a s e i n a n d a m i n o a c i d s i n t h e l a c t a t i n g cow and t h e e f f e c t s o n p l a s m a a m i n o a c i d s and m i l k c o m p o s i t i o n 77 a) M e t h o d s 77 b) M e t h i o n i n e i n f u s i o n s 80 (1) R e s u l t s 80 (2) D i s c u s s i o n 100 c) I s o l e u c i n e i n f u s i o n s I l l v i i P a g e (1) R e s u l t s I l l (2) D i s c u s s i o n 128 d) L y s i n e i n f u s i o n s 137 (1) R e s u l t s 137 (2) D i s c u s s i o n 154 D. SUMMARY AND CONCLUSIONS 164 E . L I T E R A T U R E C I T E D 168 F . APPENDIX 18 2 v i i i L I S T OF T A B L E S T a b l e P a g e I - 1 A M i l k a n d m i l k c o m p o s i t i o n - Cow e f f e c t s 53 I - 1 B M i l k a n d m i l k c o m p o s i t i o n - P e r i o d e f f e c t s . . . 54 I - 2 A E s s e n t i a l a m i n o a c i d s y g / m l b l o o d p l a s m a - Cow e f f e c t s 56 I - 2 B E s s e n t i a l a m i n o a c i d s y g / m l b l o o d p l a s m a - P e r i o d e f f e c t s 57 I - 3 A N o n - e s s e n t i a l a m i n o a c i d s y g / m l b l o o d p l a s m a - Cow e f f e c t s 59 I - 3 B N o n - e s s e n t i a l a m i n o a c i d s y g / m l b l o o d p l a s m a - P e r i o d e f f e c t s 59 I - 3 C A m i n o a c i d d a t a - y g / m l b l o o d p l a s m a - S i g n i f i c a n t cow x p e r i o d i n t e r a c t i o n t e r m s 6 0 I - 4 A E s s e n t i a l a m i n o a c i d s a s a p e r c e n t a g e o f t h e t o t a l e s s e n t i a l a m i n o a c i d s - Cow e f f e c t s 62 I - 4 B E s s e n t i a l a m i n o a c i d s a s a p e r c e n t a g e o f t h e t o t a l e s s e n t i a l a m i n o a c i d s - P e r i o d e f f e c t s 63 I - 5 A N o n - e s s e n t i a l a m i n o a c i d s a s a p e r c e n t a g e o f t h e t o t a l n o n - e s s e n t i a l a m i n o a c i d s -Cow e f f e c t s 65 I - 5 B N o n - e s s e n t i a l a m i n o a c i d s a s a p e r c e n t a g e o f t h e t o t a l n o n - e s s e n t i a l a m i n o a c i d s -P e r i o d e f f e c t s 66 1-6 M i l k a n d i n d i v i d u a l m i l k p r o t e i n s f o r cow 65002 - P e r i o d e f f e c t s . 67 I I - l I n f u s i o n s c h e d u l e - Gms o f m a t e r i a l i n f u s e d p e r 24 h o u r s 7 8 I I I - 1 A M e t h i o n i n e i n f u s i o n s - M i l k a n d m i l k c o m -p o s i t i o n - I n f u s i o n e f f e c t s 81 I l l - I B M e t h i o n i n e i n f u s i o n s - M i l k a n d m i l k c o m -p o s i t i o n - P e r i o d e f f e c t s 82 i x Table Page I I I - 1 C Meth ion ine i n f u s i o n s - M i l k and m i l k com-p o s i t i o n - S i g n i f i c a n t i n f u s i o n x p e r i o d i n t e r a c t i o n terms 8 3 I I I - 2 A Meth ion ine i n f u s i o n s - E s s e n t i a l amino ac id s - y g/ml b lood plasma - I n fu s ion e f f e c t s 86 I I I - 2 B Meth ionine i n f u s i o n s - E s s e n t i a l amino a c i d s y g/ml b lood plasma - P e r i o d e f f e c t s . . . 87 I I I - 3 A Methionine i n f u s i o n s - N o n - e s s e n t i a l amino a c i d s y g/ml b lood plasma - I n fu s ion e f f e c t s 89 I I I - 3 B Meth ion ine i n f u s i o n s - N o n - e s s e n t i a l amino ac id s y g/ml b lood plasma - P e r i o d e f f e c t s . . . 91 I I I - 3 C Meth ion ine i n f u s i o n s - Amino ac id s y g/ml b lood plasma - S i g n i f i c a n t i n f u s i o n x p e r i o d i n t e r a c t i o n terms 92 I I I - 4 A Methionine i n f u s i o n - Amino a c i d data -In fu s ion e f f e c t s 94 I I I - 4 B Meth ion ine i n f u s i o n s - Amino a c i d data -P e r i o d e f f e c t s 95 I I I - 5 A Meth ion ine i n f u s i o n s - E s s e n t i a l amino ac id s as a percentage of the t o t a l e s s e n t i a l amino a c i d s - I n fus ion e f f e c t s 96 I I I - 5 B Meth ion ine i n f u s i o n s - E s s e n t i a l amino a c i d s as a percentage of the t o t a l e s s e n t i a l amino ac id s - P e r i o d e f f e c t s 98 I I I - 5 C Methionine i n f u s i o n s - E s s e n t i a l amino a c i d s as a percentage of the t o t a l e s s e n t i a l amino a c i d s - S i g n i f i c a n t i n f u s i o n x p e r i o d i n t e r -a c t i o n terms 99 I I I - 6 A Methionine i n f u s i o n s - N o n - e s s e n t i a l amino a c i d s as a percentage of the t o t a l non-e s s e n t i a l amino ac id s - I n fu s ion e f f e c t s . . . . 101 I I I - 6 B Meth ionine i n f u s i o n s - N o n - e s s e n t i a l amino a c i d s as a percentage of the t o t a l non-e s s e n t i a l amino a c i d s - P e r i o d e f f e c t s 102 X T a b l e P a g e I V - 1 A I s o l e u c i n e i n f u s i o n s - M i l k a n d m i l k c o m -p o s i t i o n - I n f u s i o n e f f e c t s 112 I V - 1 B I s o l e u c i n e i n f u s i o n s - M i l k a n d m i l k c o m -p o s i t i o n - P e r i o d e f f e c t s 114 I V - 2 A I s o l e u c i n e i n f u s i o n s - E s s e n t i a l a m i n o a c i d s y g / m l b l o o d p l a s m a - I n f u s i o n e f f e c t s . . 116 I V - 2 B I s o l e u c i n e i n f u s i o n s - E s s e n t i a l a m i n o a c i d s y g / m l b l o o d p l a s m a - P e r i o d e f f e c t s . . . 117 I V - 3 A I s o l e u c i n e i n f u s i o n s - N o n - e s s e n t i a l a m i n o a c i d s y g / m l b l o o d p l a s m a - I n f u s i o n e f f e c t s . . 118 I V - 3 B I s o l e u c i n e i n f u s i o n s - N o n - e s s e n t i a l a m i n o a c i d s y g / m l b l o o d p l a s m a - P e r i o d e f f e c t s . . . 120 I V - 4 A I s o l e u c i n e i n f u s i o n s - E s s e n t i a l a m i n o a c i d s a s a p e r c e n t a g e o f t h e t o t a l e s s e n t i a l a m i n o a c i d s - I n f u s i o n e f f e c t s 121 I V - 4 B I s o l e u c i n e i n f u s i o n s - E s s e n t i a l a m i n o a c i d s a s a p e r c e n t a g e o f t o t a l e s s e n t i a l a m i n o a c i d s - P e r i o d e f f e c t s 123 I V - 5 A I s o l e u c i n e i n f u s i o n s - N o n - e s s e n t i a l a m i n o a c i d s a s a p e r c e n t a g e o f t h e t o t a l n o n -e s s e n t i a l a m i n o a c i d s - I n f u s i o n e f f e c t s . . . . 125 I V - 5 B I s o l e u c i n e i n f u s i o n s - N o n - e s s e n t i a l a m i n o a c i d s a s a p e r c e n t a g e o f t o t a l n o n - e s s e n t i a l a m i n o a c i d s - P e r i o d e f f e c t s 126 I V - 5 C I s o l e u c i n e i n f u s i o n s - A m i n o a c i d s e x p r e s s e d a s a p r o p o r t i o n o f t h e t o t a l e s s e n t i a l o r n o n - e s s e n t i a l p l a s m a a m i n o a c i d s - S i g n i f i c a n t i n f u s i o n x p e r i o d i n t e r a c t i o n t e r m s 127 V - 1 A L y s i n e i n f u s i o n s - M i l k a n d m i l k c o m p o s i t i o n - I n f u s i o n e f f e c t s 138 V - 1 B L y s i n e i n f u s i o n s - M i l k and m i l k c o m p o s i t i o n - P e r i o d e f f e c t s 139 V - 1 C L y s i n e i n f u s i o n s - M i l k and m i l k c o m p o s i t i o n - S i g n i f i c a n t i n f u s i o n x p e r i o d i n t e r a c t i o n t e r m s 14 0 x i Table Page V-2A L y s i n e i n f u s i o n s - E s s e n t i a l amino ac id s y g/ml b lood plasma - In fus ion e f f e c t s . . . . . 143 V-2B L y s i n e i n f u s i o n s - E s s e n t i a l amino ac id s y g /ml b lood plasma - P e r i o d e f f e c t s 144 V-3A L y s i n e i n f u s i o n s - N o n - e s s e n t i a l amino ac id s y g/ml b lood plasma - I n fu s ion e f f e c t s 146 V-3B L y s i n e i n f u s i o n s - N o n - e s s e n t i a l amino ac id s y g/ml b lood plasma - P e r i o d e f f e c t s . . . 148 V-4A L y s i n e i n f u s i o n s - E s s e n t i a l amino ac id s as a percentage of the t o t a l e s s e n t i a l amino a c i d s - I n f u s i o n e f f e c t s 149 V-4B L y s i n e i n f u s i o n s - E s s e n t i a l amino ac id s as a percentage of the t o t a l e s s e n t i a l amino a c i d s - P e r i o d e f f e c t s 151 V-5A L y s i n e i n f u s i o n s - N o n - e s s e n t i a l amino ac id s as a percentage of the t o t a l n o n - e s s e n t i a l amino a c i d s - I n fu s ion e f f e c t s 152 V-5B L y s i n e i n f u s i o n s - N o n - e s s e n t i a l amino ac id s as a percentage of the t o t a l n o n - e s s e n t i a l amino a c i d s - P e r i o d e f f e c t s 153 V-5C L y s i n e i n f u s i o n s - Amino a c i d data - S i g n i f -i c a n t i n f u s i o n x p e r i o d i n t e r a c t i o n terms . . . 155 V I - 1 Standard d e v i a t i o n s fo r 80 d u p l i c a t e amino a c i d a n a l y s i s 187 x i i L I S T OF F IGURES F i g u r e P a g e 1. P e r i o d 1 v a l u e s f o r e s s e n t i a l a n d n o n -e s s e n t i a l a m i n o a c i d s (y g / m l b l o o d p l a s m a ) a n d m i l k p r o t e i n (%) 184 2 . P e r i o d 1 e s s e n t i a l o r n o n - e s s e n t i a l a m i n o a c i d s a s a p e r c e n t a g e o f t h e t o t a l e s s e n t i a l o r n o n - e s s e n t i a l a m i n o a c i d s 185 3 . P e r i o d 1 v a l u e s f o r t o t a l e s s e n t i a l (TESS) a n d t o t a l n o n - e s s e n t i a l (TNESS) a m i n o a c i d s . . . 186 x i i i ACKNOWLEDGMENTS T h r o u g h o u t t h i s p r o j e c t I h a v e b e e n f o r t u n a t e t o h a v e t h e c o u n s e l o f many h e l p f u l p e o p l e . D r . W . D . K i t t s , t h e s u p e r v i s o r o f t h i s p r o j e c t , h a s b e e n a c o n s t a n t s o u r c e o f a s s i s t a n c e a n d e n c o u r a g e m e n t . D r . C R . K r i s h n a m u r t i g a v e i n v a l u a b l e a d v i c e d u r i n g t h e d e s i g n a n d p r o g r e s s o f t h e s t u d y . D r . R . G . P e t e r s o n was e s p e c i a l l y h e l p f u l d u r i n g t h e a n a l y s i s a n d i n t e r p r e t a t i o n o f t h e d a t a . I w o u l d l i k e t o a c k n o w l e d g e t h e g e n e r o u s h e l p o f M r . J . C . M a c G r e g o r when t h e e x p e r i m e n t a l a n i m a l s w e r e s e l e c t e d a n d h a n d l e d . M r . H . P e t e r s e n a n d M r . A . J a n z e n w e r e m o s t w i l l i n g w i t h t h e i r h e l p i n c o l l e c t i o n o f t h e d a t a . I s i n c e r e l y t h a n k a l l o f t h e s e p e o p l e and t h e many o t h e r s f o r t h e i r h e l p a n d e n c o u r a g e m e n t . I w o u l d l i k e t o t h a n k my w i f e , H e l e n , f o r h e r p a t i e n c e a n d e n c o u r a g e m e n t t h r o u g h o u t t h e p e r i o d o f my s t u d i e s . 1 A . INTRODUCTION T h e s u p p l y o f a m i n o a c i d s t o t h e mammary g l a n d o f t h e l a c t a t i n g cow i s o f p r i m e i m p o r t a n c e i n t h e s y n t h e s i s o f m i l k p r o t e i n . T h e s y n t h e s i s o f t h e p r o t e i n s i n m i l k f o l l o w s t h e g e n e r a l b i o l o g i c a l m e c h a n i s m o f p r o t e i n s y n t h e s i s ( L a r s o n , 1 9 6 9 ) . P r o t e i n s y n t h e s i s i s u n d e r a v a r i e t y o f b i o l o g i c a l c o n t r o l s . One o f t h e m a i n c o n t r o l s i s t h e s u p p l y o f s u b s t r a t e , n a m e l y a m i n o a c i d s . Due t o t h e u n i q u e s o u r c e o f a m i n o a c i d s i n r u m i -n a n t s , t h e a i m o f t h i s s t u d y i s t o d e t e r m i n e t h e r e l a t i o n s h i p b e t w e e n p l a s m a a m i n o a c i d s a n d m i l k p r o t e i n p r o d u c t i o n . In a l l a n i m a l s t h e s u p p l y o f a m i n o a c i d s f o r v a r i o u s f u n c -t i o n s i s c o m p l i c a t e d b y i n t e r a c t i o n s w i t h e n e r g y , m i n e r a l s a n d v i t a m i n s . In r u m i n a n t s t h e s i t u a t i o n i s c o m p l i c a t e d e v e n f u r t h e r t h r o u g h t h e a c t i o n o f v e r y e x t e n s i v e m i c r o f l o r a a n d m i c r o f a u n a i n t h e r u m i n o r e t i c u l a r s e c t i o n o f t h e c o m p l e x s t o m a c h . A m i n o a c i d s u p p l y i n r u m i n a n t s i s t h e n d e p e n d e n t n o t o n l y o n i n t e r -a c t i o n s w i t h o t h e r n u t r i e n t s b u t a l s o w i t h t h e m i c r o b i a l p o p u l a -t i o n i n t h e r u m e n . 1 . O b j e c t i v e s A t t e m p t s w i l l be made i n t h i s s t u d y t o d e t e r m i n e some o f t h e l i m i t i n g f a c t o r s i n v o l v e d i n t h e s y n t h e s i s o f m i l k p r o t e i n i n t h e d a i r y c o w . T h e f i r s t f a c e t i n v o l v e s a s t u d y o f t h e p r o t e i n p r o d u c t i o n o v e r t h e l a c t a t i o n c y c l e a n d t h e e n s u i n g r e l a t i o n -s h i p o f p r o t e i n p r o d u c t i o n w i t h p l a s m a a m i n o a c i d c o n c e n t r a t i o n s . T h e s e c o n d p a r t i n c l u d e s t h e s u p p l e m e n t a t i o n o f d i e t a r y p r o t e i n w i t h p r o t e i n or amino ac id s p o s t e r i o r to the rumen. M i l k protein p roduc t ion and i n t e r r e l a t i o n s h i p s among the plasma amino a c i d s , w i l l be observed. The e f f e c t of abomasal i n f u s i o n of d i f f e r e n t combinat ions of amino a c i d s should have some e f f e c t on m i l k p r o -t e i n syn thes i s and p r o d u c t i o n . These p o s i t i v e or nega t ive e f f e c t s on p r o t e i n p roduc t ion can then be r e l a t e d to the i n d i -v i d u a l plasma amino a c i d s . I t i s hoped tha t from the r e a c t i o n of the i n d i v i d u a l amino a c i d s to abomasal amino a c i d i n f u s i o n s some c o n c l u s i o n s may be drawn about the p o s s i b l e l i m i t i n g amino ac id s fo r m i l k p r o t e i n p r o d u c t i o n . 3 B. LITERATURE SURVEY This discussion i s divided into several sections to study the supply of amino acids and the effects on protein synthesis i n the mammary glands of ruminants. The supply of amino acids i n ruminants i s unique i n that dietary protein i s degraded extensively to ammonia and b u i l t back into, microbial protein by the rumen microbes. The various factors involved i n the e f f i -cient conversion of dietary protein into microbial protein along with subsequent digestion and absorption w i l l be discussed. The role of plasma free amino acids i n mammalian protein synthesizing systems w i l l be explored. F i n a l l y the l i t e r a t u r e on ruminant plasma free amino acid levels w i l l be compared with the other mammalian systems to give some expected patterns of response i n the lac t a t i n g bovine. 1. Degradation of nitrogenous materials i n the rumen The majority of the nitrogenous compounds entering the rumen undergo some degradation. The degree of degradation depends on factors such as s o l u b i l i t y and retention time i n the rumen. The end product of t h i s degradation by rumen microbes i s usually ammonia and short chain f a t t y acids. a) Ammonia production A report published by L o o s l i e_t aJL. (1949) on the a b i l i t y of rumen microorganisms to synthesize a l l the ess e n t i a l amino acids from non-protein nitrogen aroused much i n t e r e s t i n ruminant 4 protein n u t r i t i o n and requirements. Chalmers et a l . (1954), i n studying the pr o t e o l y t i c a c t i v i t y and ammonia production from rumen microbes, stated that the extent of ammonia formation from dietary nitrogen may be an important factor i n determining the usefulness of the dietary nitrogen to the host. The evaluation of protein by d i g e s t i b i l i t y alone was questioned as some protein with a high d i g e s t i b i l i t y was poorly u t i l i z e d by the host. This poor u t i l i z a t i o n was accompanied by an increased ammonia produc-t i o n . Chalmers and Synge (1954) i n comparing herring meal with casein showed that herring meal produced a lower l e v e l of ammonia in the rumen but had a greater capacity for growth and nitrogen retention than casein. This implication of ammonia i n poor nitrogen u t i l i z a t i o n led to studies on the extent of degradation of protein i n the rumen and passage to the duodenum. Gray et a l . (1958) found that with diets low i n nitrogen, 100% or more of the dietary nitrogen was passed from the rumen into the duodenum. Diets with high le v e l s of nitrogen were found to pass only 48 to 65% of the dietary nitrogen from the rumen into the duodenum. Blackburn and Hobson (1960) also noted a loss of nitrogen from the rumen. In t h e i r studies they noted that soluble proteins were rapidly degraded to ammonia; however, peptides and amino acids were degraded at a slower rate. The concentration of ammonia i n the rumen appeared to be dependent on the s o l u b i l i t y of the casein in the d i e t . The less soluble casein supplied a more constant supply of peptides, amino acids and ammonia to the rumen microflora. 5 The a s s i m i l a t i o n of n i t r o g e n by rumen b a c t e r i a was s t u d i e d by P h i l l i p s o n et a l . (1962) u s i n g an i n v i t r o technique. There was a decrease i n "^N d u r i n g -,the i n c u b a t i o n which was f o l l o w e d 15 by an uptake of N by the m i c r o b i a l c e l l s . S t a r c h and glucose fermenting b a c t e r i a were found to u t i l i z e ammonia even i n the presence of peptone. E a r l i e r , Annison e t a_l. (1954) had noted t h a t ammonia c o n c e n t r a t i o n i n rumen f l u i d decreased when the amount of s t a r c h or c e r e a l i n the d i e t accompanying the p r o t e i n was i n c r e a s e d . The importance of s o l u b i l i t y i n c o n t r o l l i n g the breakdown of p r o t e i n i n t o ammonia was s t r e s s e d by Waldo (1968). He observed t h a t as the r a t e of breakdown was decreased so was the accumu-l a t i o n of ammonia. Heating had been e f f e c t i v e when used by Blackburn and Hobson (1960) i n d e c r e a s i n g s o l u b i l i t y . The more gradu a l breakdown of the p r o t e i n r e s u l t e d i n l e s s ammonia absorp-t i o n and a lower u r i n a r y N l o s s . T i l l m a n and Sidhu (1969) summarized t h e i r s t u d i e s by s t a t i n g t h a t ammonia i n the rumen was the r e s u l t of metabolism of p r o-t e i n s , p e p t i d e s , amino a c i d s , amides, urea, and n i t r a t e . They found t h a t the c o n c e n t r a t i o n of ammonia was dependent on i ) the uptake of ammonia by b a c t e r i a i i ) metabolism by the rumen w a l l i i i ) a b s o r p t i o n i n t o the p o r t a l v e i n iv) passage to the omasum B a c t e r i a u t i l i z e d ammonia, carbon s k e l e t o n s and energy to form amino a c i d s and m i c r o b i a l p r o t e i n . The l o s s of ammonia from 6 t h e rumen b y a b s o r p t i o n was d e p e n d e n t o n t h e c o n c e n t r a t i o n o f ammonia a n d H - i o n c o n c e n t r a t i o n . T h e p r o b l e m o f l o s s o f n i t r o g e n f r o m t h e rumen b y a n i m a l s o n a h i g h p r o t e i n d i e t was n o t e d b y G r a y e t a l . (1958) a n d B l a c k b u r n a n d H o b s o n ( 1 9 6 0 ) . P i l g r i m e t a l . (1969) s t u d i e d the d i s a p p e a r a n c e o f " ^ N - a m m o n i a a n d c o n c l u d e d t h a t t h i s l o s s was v e r y e x t e n s i v e a n d c o u l d a c c o u n t f o r 55 t o 66% o f t h e ammonia i n t h e r u m e n . C h a l m e r s e t ajL. (1971) s u m m a r i z e d t h e e v i d e n c e t o d a t e f o r ammonia a b s o r p t i o n a n d t r a n s p o r t f r o m t h e rumen a s f o l l o w s : i ) T h e r e i s i n c r e a s e d t r a n s p o r t o f ammonia t o t h e r u m i n a l v e i n s w i t h i n c r e a s i n g c o n c e n t r a t i o n o f r u m i n a l a m m o n i a , i i ) T h e movement o f ammonia i s a c c e l e r a t e d f r o m t h e rumen a t pH a b o v e 7 . 3 due t o t h e i n c r e a s e d p r o p o r t i o n o f N H ^ . f a n d i i i ) Ammonia t o x i c i t y i s t h e d i r e c t r e s u l t o f an i n c r e a s e d ammonia c o n c e n t r a t i o n i n p o r t a l v e i n b l o o d . T h e i n c r e a s e d amount o f ammonia o v e r l o a d s t h e l i v e r a n d r e s u l t s i n an i n c r e a s e o f ammonia c o n c e n t r a t i o n i n p e r i p h e r a l c i r c u l a t i o n . T h e a u t h o r f e l t t h a t t h e l i v e r was n o t n e c e s s a r i l y o v e r -l o a d e d a n d p o s t u l a t e d t h a t t h e j u g u l a r v e i n s d e r i v e d some ammonia f r o m a s o u r c e o t h e r t h a n t h e l i v e r , s u c h a s t h e p e r i t o n e a l f l u i d . T h e p e r i t o n e a l f l u i d o b t a i n e d ammonia f r o m t h e rumen t h r o u g h d i f f u s i o n . T h e r e s u l t s o b t a i n e d w e r e c o n s i s t e n t w i t h t h i s h y -p o t h e s i s . I t h a s b e e n s u g g e s t e d t h a t b a c t e r i a a r e more e f f i c i e n t i n t h e u t i l i z a t i o n o f r u m i n a l ammonia t h a n p r o t o z o a ( M a l e s a n d P u r s e r , 1 9 7 0 ; K l o p f e n s t e i n e t a l . , 1 9 6 6 ) . I n c r e a s e d n u m b e r s 7 o f b a c t e r i a a n d l o w e r e d l e v e l s o f rumen ammonia w e r e n o t e d i n d e f a u n a t e d a n i m a l s . Mangan (1972) s t u d i e d t h e p r o t e o l y s i s o f c a s e i n a n d o v a l b u -m i n i n t h e r u m e n . T h e p r o t e i n s w e r e f i r s t d e g r a d e d t o p e p t i d e s , t h e n t o a m i n o a c i d s a n d f i n a l l y t o a m m o n i a . He e s t i m a t e d t h a t 10% o r l e s s o f t h e c a s e i n w o u l d r e a c h t h e duodenum u n d e g r a d e d . Up t o 43% o f t h e c a s e i n n i t r o g e n was f o u n d a s e x c e s s a m m o n i a . A l - R a b b a t e_t a l . (1971) e s t i m a t e d u s i n g a n i n v i t r o s y s t e m t h a t 61% o f m i c r o b i a l N s y n t h e s i z e d was f r o m ammonia w i t h t h e r e m a i n -i n g 39% c o m i n g f r o m p e p t i d e s a n d amino a c i d s . Mangan (1972) n o t e d t h a t f r e e a m i n o a c i d s w e r e r a p i d l y u t i l i z e d i n t h e rumen b u t i t was p o s s i b l e t o p i c k o u t some d i f f e r e n c e s i n r a t e s o f m e t a b o l i s m . A r g i n i n e a n d m e t h i o n i n e w e r e m e t a b o l i z e d v e r y r a p i d l y . T h e b r a n c h c h a i n a m i n o a c i d s w e r e m e t a b o l i z e d m o s t s l o w l y w i t h t h e r e m a i n d e r o f t h e a m i n o a c i d s b e i n g i n t e r m e d i a t e . T h e i m p o r t a n c e o f ammonia p r o d u c t i o n f r o m d i e t a r y n i t r o g e n s o u r c e s m u s t be s t r e s s e d n o t o n l y f o r t h e c e n t r a l r o l e o f ammonia i n m i c r o b i a l p r o t e i n s y n t h e s i s b u t a l s o f o r t h e l o s s f r o m t h e rumen when e x c e s s ammonia i s p r o d u c e d . T h i s c e n t r a l r o l e o f ammonia e m p h a s i z e s t h e i m p o r t a n c e o f c o n t r o l l e d n i t r o g e n r e l e a s e f r o m t h e rumen t o e n s u r e e f f i c i e n t u s a g e by t h e m i c r o b i a l p o p u l a -t i o n . b) O t h e r p r o d u c t s A l t h o u g h ammonia i s t h e m o s t i m p o r t a n t e n d p r o d u c t o f n i t r o g e n d e g r a d a t i o n i n t h e r u m e n , o t h e r s , e s p e c i a l l y t h e i n t e r -m e d i a t e s o f ammonia p r o d u c t i o n , p l a y a r o l e i n t h e n i t r o g e n 8 m e t a b o l i s m o f t h e r u m i n a n t i n t h e i n t e r v a l f o l l o w i n g i n g e s t i o n . I t h a s b e e n m e n t i o n e d e a r l i e r t h a t p e p t i d e s a n d a m i n o a c i d s a r e u t i l i z e d b y b a c t e r i a a n d p r o t o z o a a s n i t r o g e n s o u r c e s ( A l - R a b b a t e t a l . , 1 9 7 1 ; M a n g a n , 1 9 7 2 ) . L e i b h o l z (1971) e s t i m a t e d t h a t 6% o f t h e n i t r o g e n a b s o r b e d f r o m t h e rumen c o u l d be a - a m i n o n i t r o g e n , w i t h t h e u p t a k e o f t h e b r a n c h e d c h a i n a m i n o a c i d s b e i n g g r e a t e r t h a n t h e o t h e r s . S m i t h (1969) i n h i s r e v i e w f e l t t h a t o f t h e n i t r o g e n o u s c o m p o u n d s o t h e r t h a n ammonia r e l e a s e d i n t h e rumen d u r i n g m i c r o b i a l h y d r o l y s i s , o n l y t h e a m i n o a c i d s a p p r o a c h n u t r i t i o n a l s i g n i f i c a n c e . T h e a m i n o a c i d s c o u l d a c c o u n t f o r a p p r e c i a b l e a m o u n t s o f n i t r o g e n a b s o r b e d f r o m t h e rumen a s w e l l a s s e r v e a s a n i t r o g e n s o u r c e f o r m i c r o o r g a n i s m s . 2 . E x t e n t o f f o r m a t i o n o f m i c r o b i a l c e l l s i n t h e rumen T h e f o r m a t i o n o f m i c r o b i a l p r o t e i n i n t h e rumen h a s b e e n i n v e s t i g a t e d u s i n g two d i f f e r e n t a p p r o a c h e s . T h e f i r s t a p p r o a c h i n v e s t i g a t e s t h e n i t r o g e n o u s c o m p o n e n t s o f t h e d i e t a n d d i g e s t a , and g i v e s an e s t i m a t e o f t h e e x t e n t o f a s s i m i l a t i o n o f n i t r o g e n by t h e m i c r o b e s . T h e s e c o n d a p p r o a c h a s s u m e s t h a t p r o t e i n p r o d -u c t i o n b y m i c r o b e s s h o u l d be r e l a t e d t o t h e amount o f e n e r g y a v a i l a b l e i n t h e rumen f o r m i c r o b i a l c e l l s y n t h e s i s . E s t i m a t e s o f m i c r o b i a l p r o t e i n p r o d u c t i o n a r e made f r o m t h e v a r i o u s e n e r g y a n d n i t r o g e n p a r a m e t e r s w h i c h c a n be s t u d i e d i n t h e r u m e n . a) A s s i m i l a t i o n o f n i t r o g e n b y t h e m i c r o b e s A v a r i e t y o f t e c h n i q u e s h a v e b e e n u s e d t o d e t e r m i n e t h e c o n v e r s i o n o f d i e t a r y n i t r o g e n i n t o m i c r o b i a l p r o t e i n . M c D o n a l d 9 (1954) used the s o l u b i l i t y of zein i n alcohol to estimate that 40% of the zein i n a p a r t i a l l y p u r i f i e d d i e t was u t i l i z e d by the microbes for synthesis of t h e i r own proteins. The phosphorus content of casein was used by McDonald and Hall (19 57) to estim-ate that 90% of the casein was degraded i n the rumen and u t i l i z e d for microbial growth. The author suggested that there might be an upper l i m i t to the capacity for growth of microorganisms i n the rumen. Diaminopimelic acid, an amino acid found only i n bacteria, was used as a b a c t e r i a l marker by Weller et a l . (1958) to estimate the production of microbial protein from plant nitrogen. He found that 63 to 82% of the t o t a l nitrogen i n the rumen was mi-c r o b i a l nitrogen, 11 to 27% was plant nitrogen, and 5 to 10% was soluble nitrogen. It was reported by Blackburn and Hobson (1960) that the pro t e o l y t i c a c t i v i t y of rumen contents varied from d i e t to d i e t . They noted also that nitrogen disappeared from the rumen at a faster rate than polyethylene g l y c o l , i n d i c a t i n g absorption from the rumen. The numbers of bacteria and protozoa remained f a i r l y constant indicating a balance between growth and loss of c e l l s to the abomasum. Total VFA (Vo l a t i l e Fatty Acids) concentration was maximal at three hours and some of these VFA appeared to be from deaminated amino acids (vis v a l e r i c a c i d ) . These data again point out the p o s s i b i l i t y of an upper l i m i t on the extent of microbial growth and also indicate that excess nitrogen i s l o s t from the rumen and the carbon chains of the amino acids may be used as an energy source by the microbes. 10 T h e u t i l i z a t i o n o f p r o t e i n a s a s o u r c e o f e n e r g y was s tud ied b y B l a x t e r a n d M a r t i n (1962) . C a s e i n was a d m i n i s t e r e d b y a c o n s t a n t i n f u s i o n t o e i t h e r t h e rumen o r abomasum. D i g e s t i o n c o e f f i c i e n t s w e r e e s s e n t i a l l y s i m i l a r w i t h s l i g h t l y g r e a t e r d i g e s t i o n o c c u r r i n g i n t h e abomasum. T h e r e t e n t i o n o f n i t r o g e n was s i m i l a r b u t t h e e x c r e t i o n o f n i t r o g e n i n t h e f e c e s a n d u r i n e was d i s s i m i l a r . T h e i n c r e a s e d N i n t h e f e c e s was e x p l a i n e d a s b e i n g due t o t h e l o w e r d i g e s t i b i l i t y o f b a c t e r i a l c e l l w a l l s . T h e g r e a t e r c o n t e n t o f ammonia i n t h e u r i n e was due t o t h e i n -c r e a s e d a b s o r p t i o n o f ammonia f r o m t h e rumen when c a s e i n was i n f u s e d i n t o t h e r u m e n . H o g a n (1965) o b s e r v e d t h a t t h e l o s s o f n i t r o g e n f r o m t h e rumen o f g r a z i n g s h e e p i n c r e a s e d a s t h e i n t a k e o f c r u d e p r o t e i n i n c r e a s e d . T h e s e n i t r o g e n l o s s e s w e r e a l s o a s s o c i a t e d w i t h i n c r e a s e s i n ammonia c o n c e n t r a t i o n . I t was s u g g e s t e d t h a t t h i s l o s s o f n i t r o g e n r e p r e s e n t e d a p r o p o r t i o n o f t h e i n g e s t e d n i t r o -g e n . T h e h y p o t h e s i s o f an u p p e r l i m i t o n t h e amount o f d i e t a r y p r o t e i n t h a t c o u l d be i n c o r p o r a t e d i n t o m i c r o b i a l c e l l s was f u r t h e r d e v e l o p e d by t h e o b s e r v a t i o n o f H u n g a t e ( 1 9 6 5 ) . He n o t e d t h a t i n b a t c h c u l t u r e s o f rumen m i c r o b e s , 8 t o 14% o f t h e s u b s t r a t e was i n c o r p o r a t e d i n t o t h e c e l l s . 15 V i r t a n e n (1966) u s e d N t o d e m o n s t r a t e t h e i m p o r t a n c e o f ammonia a s an i n t e r m e d i a t e i n m i c r o b i a l p r o d u c t i o n . In d i e t s w h e r e ammonia s a l t s p r o v i d e d 99.5% o f t h e n i t r o g e n , a l l t h e a m i n o a c i d s w e r e l a b e l l e d , a l t h o u g h a t f i r s t t h e n o n - e s s e n t i a l a m i n o a c i d s w e r e l a b e l l e d more s t r o n g l y t h a n t h e e s s e n t i a l a m i n o a c i d s . 11 L a b e l l e d s u l p h u r was u s e d b y C o n r a d et. aul. (1967) t o e s t i m -a t e m e t h i o n i n e s y n t h e s i s i n t h e rumen a n d t h e n t o e s t i m a t e p r o t e i n d e g r a d a t i o n i n t h e r u m e n . F i s h m e a l p r o t e i n was o n l y 9% d e g r a d e d i n t h e r u m e n . F r o m t h e r e s u l t s o f t h e s e w o r k e r s c i t e d a b o v e i t c o u l d be c o n c l u d e d t h a t t h e d e g r e e o f c o n v e r s i o n o f d i e t a r y n i t r o g e n i s d e p e n d e n t o n a number o f f a c t o r s w h i c h i n c l u d e s l e v e l o f n i t r o g e n i n t h e d i e t , s o l u b i l i t y o f t h e n i t r o g e n i n t h e rumen f l u i d a n d some c o n t r o l l i n g f a c t o r w h i c h i m p o s e s a n u p p e r l i m i t o n m i c r o b i a l p r o t e i n s y n t h e s i s . T i l l m a n and S i d h u (1969) n o t e d t h a t f o r o p t i m u m s y n t h e s i s o f a m i n o a c i d s f r o m a m m o n i a , a d e q u a t e e n e r g y a n d a s o u r c e o f c a r b o n s k e l e t o n s i s n e c e s s a r y . He s p e c u l a t e d t h a t t h e r e i s a r e q u i r e m e n t f o r s p e c i a l c a r b o n s k e l e t o n s f o r t h e s y n t h e s i s o f v a l i n e , i s o l e u c i n e , l e u c i n e , p h e n y l a l a n i n e a n d t y r o s i n e . A l l i s o n (1969) r e p o r t e d t h a t rumen m i c r o o r g a n i s m s i n c o r p o r a t e o n l y a s m a l l p r o p o r t i o n o f d i e t a r y a m i n o a c i d s i n t o m i c r o b i a l p r o t e i n , w i t h ammonia a p p e a r i n g t o be t h e m o s t i m p o r t a n t s o u r c e o f n i t r o g e n . F e e d b a c k i n h i b i t i o n f r o m s y n t h e s i z e d a m i n o a c i d s d o e s n o t a p p e a r t o be a p r o b l e m i n t h e s e m i c r o o r g a n i s m s . T h e e f f e c t o f b a c t e r i a a n d p r o t o z o a i n m i c r o b i a l p r o d u c t i o n i s q u i t e m a r k e d . K l o p f e n s t e i n e t a l . (1966) o b s e r v e d t h a t t h e n u m b e r s o f b a c t e r i a w e r e r e d u c e d i n f a u n a t e d a n i m a l s . T h i s r e d u c t i o n i n b a c t e r i a l n u m b e r s was a c c o m p a n i e d by an i n c r e a s e i n rumen ammonia c o n c e n t r a t i o n . B a c t e r i a a p p e a r t o be much more e f f i c i e n t a t u t i l i z i n g ammonia a s a n i t r o g e n s o u r c e t h a n p r o t o z o a . 12 H o w e v e r , t h e o v e r a l l b e n e f i t t o t h e a n i m a l m u s t be j u d g e d d i f f e r e n t l y a s t h e q u a l i t y o f b a c t e r i a l a n d p r o t o z o a l p r o t e i n i s n o t t h e s a m e . T h e p r o t o z o a 1 i n c o r p o r a t e b a c t e r i a l a m i n o a c i d s by f i r s t k i l l i n g t h e b a c t e r i a a n d t h e n u t i l i z i n g t h e a m i n o a c i d s d i r e c t l y f o r p r o t o z o a l p r o t e i n ( A l l i s o n , 1 9 6 9 ) . T h e b a c t e r i a l a m i n o a c i d s do n o t a p p e a r t o be m e t a b o l i z e d t o a g r e a t e x t e n t w i t h i n t h e p r o t o z o a a l t h o u g h some may be c a t a b o l i z e d t o V F A . T h i s d i f f e r e n c e i n p r e f e r e n c e s f o r n i t r o g e n s u b s t r a t e s was a l s o p o i n t e d o u t by P i l g r i m e t a l . (1970) who n o t e d t h a t ammonia was t h e s t a r t i n g p o i n t f o r o v e r 80% o f b a c t e r i a l n i t r o g e n f o r m e d . H o w e v e r , w i t h t h e t o t a l m i c r o b i a l p o p u l a t i o n ammonia was t h e s t a r t i n g p o i n t f o r o n l y 55 t o 68% o f t h e t o t a l m i c r o b i a l p r o t e i n . A min imum v a l u e o f 50 t o 65% o f b a c t e r i a l p r o t e i n n i t r o g e n b e i n g d e r i v e d f r o m rumen ammonia was e s t i m a t e d b y M a t h i s o n a n d M i l l i g a n ( 1 9 7 1 ) . W i t h p r o t o z o a t h e c o n t r i b u t i o n o f ammonia t o t h e t o t a l n i t r o g e n i n c o r p o r a t e d was much s m a l l e r . O n l y 10% o f t h e c a r b o n f r o m a m i n o a c i d s was r e c o v e r e d i n m i c r o b i a l c e l l s w h e r e a s o v e r 80% o f t h e d i e t a r y p l a n t n i t r o g e n was i n c o r p o r a t e d i n t o m i c r o b i a l c e l l s . A s ammonia c o n c e n t r a t i o n i n c r e a s e s t h e c o n c e n t r a t i o n o f p e p t i d e s a n d a m i n o a c i d s s h o u l d a l s o i n c r e a s e . T h e d a t a a l s o s u p p o r t t h e h y p o t h e s i s t h a t a s t h e c o n c e n t r a t i o n o f a m i n o a c i d s a n d p e p t i d e s i n c r e a s e t h e i r r a t e o f i n c o r p o r a t i o n i n t o m i c r o b i a l p r o t e i n a l s o i n c r e a s e s . T h e r e f o r e , a s ammonia c o n c e n t r a t i o n i n c r e a s e s t h e p r o p o r t i o n c o n v e r t e d i n t o m i c r o b i a l p r o t e i n d e c r e a s e s due t o i n c r e a s e d c o n c e n t r a t i o n o f p e p t i d e s and a m i n o a c i d s a n d a l s o i n c r e a s e d l o s s o f ammonia f r o m t h e r u m e n . When a s s i m i l a t i o n o f n i t r o g e n b y m i c r o b e s was u s e d t o assess t h e e x t e n t o f f o r m a t i o n o f m i c r o b i a l c e l l s i n t h e rumen a l o s s o f n i t r o g e n f r o m t h e g a s t r o - i n t e s t i n a l t r a c t was o b s e r v e d w i t h h i g h p r o t e i n d i e t s . Ammonia p l a y e d a n i m p o r t a n t r o l e a s t h e m a i n s o u r c e o f n i t r o g e n f o r m i c r o b i a l g r o w t h a n d a l s o t h e m a i n n i t r o g -e n o u s p r o d u c t a b s o r b e d f r o m t h e rumen when p r e s e n t i n e x c e s s a m o u n t s . B a c t e r i a a p p e a r t o be much m o r e e f f i c i e n t t h a n protozoa i n u t i l i z i n g ammonia a s t h e i r m a i n n i t r o g e n s o u r c e . Ammonia i s t h e s t a r t i n g p o i n t o f 50 t o 65% o f m i c r o b i a l p r o t e i n . I t was a l s o a p p a r e n t t h a t t h e r e was an u p p e r l i m i t o n t h e amount o f m i c r o b i a l p r o t e i n w h i c h c o u l d be s y n t h e s i z e d i n t h e rumen t h a t c o u l d n o t b e a t t r i b u t e d t o t h e n i t r o g e n s o u r c e s a v a i l a b l e t o t h e m i c r o o r g a n i s m s . b) L i m i t a t i o n o f m i c r o b i a l p r o t e i n s y n t h e s i s b y a v a i l a b l e e n e r g y  T h e s e c o n d m a j o r a p p r o a c h i n e s t i m a t i n g t h e e x t e n t o f a s s i m -i l a t i o n o f d i e t a r y n i t r o g e n b y m i c r o b i a l c e l l s i s b a s e d o n t h e a s s u m p t i o n t h a t p r o t e i n a n d e n e r g y m e t a b o l i s m a r e c l o s e l y l i n k e d . B a u c h o p a n d E l s d e n (1960) n o t e d t h a t m i c r o o r g a n i s m s i n an e n e r g y l i m i t i n g e n v i r o n m e n t a p p e a r t o p r o d u c e 1 0 . 5 g r a m s o f c e l l s p e r m o l e o f A T P . A p p r o x i m a t e ATP p r o d u c t i o n s w e r e c a l c u l a t e d f r o m a v a r i e t y o f e n e r g y s o u r c e s . W a l k e r (1965) e x t r a p o l a t e d t h e s e f i n d i n g s , t o t h e a n a e r o b i c f e r m e n t a t i o n i n t h e r u m i n a n t . T h e v a l u e s f o r ATP p r o d u c t i o n f r o m h e x o s e a n d p e n t o s e f o r m i c r o b i a l c e l l s y n t h e s i s w e r e c a l c u l a t e d f r o m known b i o c h e m i c a l p a t h w a y s . T h e a m o u n t s and p r o p o r t i o n o f V F A p r o d u c e d w i l l a l s o g i v e a n 14 e s t i m a t e o f ATP p r o d u c t i o n . F a c t o r s o t h e r t h a n a n a e r o b i o s i s w h i c h c o u l d l i m i t t h e e n e r g y a v a i l a b l e f o r c e l l s y n t h e s i s s u c h a s c o s t o f m a i n t a i n i n g a l a r g e m i c r o b i a l p o p u l a t i o n , d e g r a d a t i o n o f c a r b o h y d r a t e t o V F A a n d s y n t h e s i s o f c a r b o h y d r a t e f r o m V F A w e r e n o t e d . A l s o t h e l o s s o f e x c e s s n i t r o g e n f r o m t h e rumen m u s t be i n c l u d e d a s a p o s s i b l e f a c t o r i n l i m i t i n g c e l l s y n t h e s i s . T h e d e p e n d e n c e o f t h e r u m i n a n t o n an a n a e r o b i c f e r m e n t a t i o n t o s u p p l y t h e p r o t e i n a n d e n e r g y n e e d s p u t s a l i m i t on t h e extent o f f e e d u t i l i z a t i o n . O n l y 4 t o 5 m o l e s o f ATP a r e a v a i l a b l e f o r m i c r o b i a l s y n t h e s i s p e r m o l e o f h e x o s e f e r m e n t e d . H u n g a t e ( 1 9 6 5 ) , (1966) f e l t t h a t a t t e n t i o n s h o u l d b e f o c u s e d o n two a r e a s , v i z i ) i n c r e a s i n g t h e p e r c e n t a g e o f f e e d s y n t h e s i z e d i n t o m i c r o b i a l p r o t e i n , a n d i i ) s h u n t i n g p r o t e i n d i r e c t l y t o t h e abomasum. He f e l t t h a t e m p h a s i s s h o u l d b e p l a c e d i n t h e s e two a r e a s d u e t o t h e r u m i n a n t b e i n g more e f f i c i e n t a t c o n v e r t i n g d i e t a r y energy i n t o p r o d u c t i v e e n e r g y t h a n d i e t a r y p r o t e i n i n t o p r o d u c t i v e p r o -t e i n . T h e m a j o r s o u r c e o f p r o t e i n a v a i l a b l e t o t h e a n i m a l i s m i c r o b i a l p r o t e i n b u t o n l y 10% o f t h e e n e r g y i s a v a i l a b l e f o r m i c r o b i a l c e l l s y n t h e s i s . In a e r o b i c s y s t e m s 60 t o 70% o f t h e e n e r g y i s u t i l i z e d i n t h e s y n t h e s i s o f c e l l m a t e r i a l . A s t h e h o s t a n i m a l i s a e r o b i c i t s e n e r g y s u p p l y g i v e s i t t h e p o t e n t i a l f o r g r e a t e r p r o t e i n s y n t h e s i s b u t i t s d i g e s t i v e s y s t e m makes i t d e p e n d e n t o n t h e p r o t e i n s y n t h e s i z e d i n t h e a n a e r o b i c m i c r o b i a l s y s t e m . H o g a n (19 67) o b s e r v e d t h a t t h e amount o f m i c r o b i a l p r o t e i n e n t e r i n g t h e abomasum was more r e l a t e d t o t h e o r g a n i c m a t t e r 15 d i g e s t e d t h a n t h e p r o t e i n l e v e l o f t h e d i e t . He c o n c l u d e d t h a t t h e e n e r g y r e l e a s e d d u r i n g o r g a n i c m a t t e r d i g e s t i o n i n t h e rumen p l a c e s an u p p e r l i m i t o n t h e e x t e n t o f n i t r o g e n c o n v e r t e d t o p r o t e i n b y t h e m i c r o b e s . I t h a s b e e n f o u n d t h a t m i c r o o r g a n i s m s u t i l i z e t h e e n e r g y made a v a i l a b l e f r o m c a r b o h y d r a t e f e r m e n t a t i o n w i t h a h i g h d e g r e e o f e f f i c i e n c y . W a l k e r (1968) f o u n d t h a t i n l i m i t i n g c o n d i t i o n s m i c r o o r g a n i s m s c a n s t o r e e x c e s s e n e r g y a s r e s e r v e p o l y s a c c h a r i d e . T h i s s t o r a g e may r e p r e s e n t up t o 8 0% o f t h e a v a i l a b l e e n e r g y when c o n d i t i o n s o t h e r t h a n e n e r g y a r e l i m i t i n g . Much e m p h a s i s m u s t be p l a c e d on m a x i m i z i n g c e l l s y n t h e s i s f r o m t h e e n e r g y a v a i l a b l e . O p t i m a l c o n d i t i o n s f o r m i c r o b i a l c e l l s y n t h e s i s w e r e s o u g h t b y Hume e t a l . (1970) i n a s e r i e s o f f o u r e x p e r i m e n t s w i t h s h e e p . In t h e f i r s t e x p e r i m e n t t h e r e l a t i o n b e t w e e n l e v e l o f i n t a k e o f n i t r o g e n a n d m i c r o b i a l p r o t e i n , p r o d u c t i o n was l i n e a r up t o a n i n t a k e o f 9 g r a m s N p e r d a y (Hume e t a l . , 1 9 7 0 ) . A b o v e t h a t l e v e l o f i n t a k e t h e p r o d u c t i o n o f m i c r o b i a l p r o t e i n d e c r e a s e d p e r u n i t o f n i t r o g e n i n t a k e . D a t a f r o m o r g a n i c m a t t e r d i g e s t i o n a n d V F A p r o d u c t i o n s u g g e s t t h a t ATP p r o d u c t i o n was s i m i l a r a t a l l l e v e l s o f n i t r o g e n i n t a k e . T h i s i n d i c a t e s t h a t ATP was n o t u t i l i z e d w i t h e q u a l e f f i c i e n c y i n t h e s y n t h e s i s o f m i c r o b i a l p r o t e i n . In an a t t e m p t t o d e t e r m i n e t h e l i m i t i n g f a c t o r s i n m i c r o b i a l c e l l s y n t h e s i s , Hume (1970a) s t u d i e d t h e e f f e c t s o f an a d d i t i o n o f b r a n c h e d c h a i n V F A i n t h e s e c o n d e x p e r i m e n t . T h e r e a p p e a r e d a d d i t i o n o f b r a n c h e d c h a i n V F A i n t h e s e c o n d e x p e r i m e n t . T h e r e a p p e a r e d t o be l i t t l e e f f e c t i n c o n t r a s t w i t h t h e r e s u l t s o b s e r v e d b y O l t j e n e t a l . (1971) w i t h b r a n c h e d c h a i n V F A a d d i -t i o n s t o p u r i f i e d d i e t s . In t h e t h i r d e x p e r i m e n t t h e e f f e c t o f a d d i t i o n o f s p e c i f i c p r o t e i n s t o a u r e a b a s e d d i e t was s t u d i e d (Hume, 1 9 7 0 b ) . T h e g r e a t e s t i n c r e a s e i n n i t r o g e n r e t e n t i o n was b r o u g h t a b o u t by z e i n w h i c h was o n l y 13% d e g r a d e d i n t h e r u m e n . T h e z e i n t h a t was u n d e g r a d e d i n t h e rumen was h i g h l y d i g e s t i b l e i n t h e duodenum a n d a d d e d t o t h e a n i m a l s n i t r o g e n e c o n o m y . T h e r e q u i r e m e n t m u s t be q u i t e s p e c i f i c a s t h e 13% o f t h e z e i n d i g e s t e d i n t h e rumen g a v e t h e same r e s p o n s e i n m i c r o b i a l g r o w t h a s t h e c a s e i n w h i c h was 100% d i g e s t e d i n t h e r u m e n . I t a p p e a r s t h a t a m i n o a c i d s a n d p e p t i d e s n o t a v a i l a b l e i n g e l a t i n may be l i m i t i n g t h e e x t e n t o f m i c r o b i a l s y n t h e s i s . T h e a d d i t i o n o f s u l p h u r t o t h e d i e t s was s t u d i e d i n t h e f o u r t h e x p e r i m e n t (Hume a n d B i r d , 1 9 7 0 ) . A s i g n i f i c a n t i n c r e a s e i n m i c r o b i a l c e l l s y n t h e s i s was o b s e r v e d , h o w e v e r , t h e a m o u n t s o f m i c r o b i a l p r o t e i n s y n t h e s i z e d w e r e n o t a s h i g h a s t h o s e o b t a i n e d w i t h z e i n o r c a s e i n . M i c r o b i a l g r o w t h i s t h e n d e p e n d e n t n o t o n l y o n t h e ATP a v a i l a b l e f o r m i c r o b i a l g r o w t h b u t a l s o t h e e f f i c i e n c y o f u t i l i -z a t i o n o f t h a t ATP f o r g r o w t h . A T P i s u s e d f o r a v a r i e t y o f s y n t h e t i c r e a c t i o n s d u r i n g g r o w t h . T h e r e q u i r e m e n t s f o r t h e s e p r o c e s s e s h a v e b e e n s t u d i e d a n d t h e t h e o r e t i c a l y i e l d o f m i c r o -b i a l p r o t o p l a s m h a s b e e n c a l c u l a t e d t o be 23 g rams d r y w e i g h t p e r m o l e o f ATP ( B a l d w i n , 1 9 7 0 ) . T h i s c o m p a r e s w i t h t h e o b s e r v e d r e s u l t s o f B a u c h o p a n d E l s d e n (1960) a n d H u n g a t e (1966) o f a p p r o x -i m a t e l y 1 0 . 5 g rams d r y w e i g h t p e r m o l e o f ATP o b s e r v e d w i t h p u r e c u l t u r e s o f rumen b a c t e r i a . W i t h m i x e d c u l t u r e s t h e y i e l d h a s b e e n c l o s e r t o 1 6 . 5 ( B a l d w i n , 1 9 7 0 ) . P u r s e r (1970) c a l c u l a t e d t h a t t h e t h e o r e t i c a l g rams o f p r o t e i n p e r m e g a c a l o r i e o f d i g e s t i b l e e n e r g y was 1 8 . 3 : 1 . T h e m a i n t e n a n c e r e q u i r e m e n t o f m o n o g a s t r i c s i s 2 0 : 1 . T h e a u t h o r d o e s n o t e t h a t s e v e r a l w o r k e r s u s i n g a v a r i e t y o f d i e t s h a v e r e s u l t s i n w h i c h t h e p r o t e i n t o e n e r g y r a t i o e x c e e d s t h e t h e o -r e t i c a l maximum. I t i s s u g g e s t e d t h a t p e r h a p s e m p h a s i s s h o u l d be p l a c e d o n m a x i m a l m i c r o b i a l p r o d u c t i o n r a t h e r t h a n o n e f f i -c i e n t c o n v e r s i o n o f d i e t a r y n i t r o g e n i n t o m i c r o b i a l n i t r o g e n . B l a c k (1971) u s e d t h e o r e t i c a l c o n s i d e r a t i o n s t o c a l c u l a t e t h e e f f e c t o f a n a e r o b i c f e r m e n t a t i o n o n t h e n i t r o g e n a n d e n e r g y economy o f t h e r u m i n a n t . In t h e n o n - r u m i n a n t 39 t o 54% more n e t e n e r g y i s a v a i l a b l e f o r m a i n t e n a n c e a n d 22 t o 61% m o r e n e t e n e r g y a v a i l a b l e f o r p r o d u c t i o n t h a n i n t h e r u m i n a n t a s s u m i n g t h e same a m o u n t s o f d i e t a n d t h e same d i g e s t i b i l i t y . When d i e t a r y p r o t e i n was c o m p l e t e l y d e g r a d e d i n t h e rumen b y m i c r o o r g a n i s m s , a p p r o x i m a t e l y 50% l e s s p r o t e i n was a b s o r b e d f r o m t h e s m a l l i n t e s -t i n e i n t h e r u m i n a n t . I t was c a l c u l a t e d t h a t t h e f i b r e l e v e l o f t h e d i e t m u s t e x c e e d 22 t o 35% b e f o r e t h e r u m i n a n t d i g e s t i v e s y s t e m r e s u l t s i n b e t t e r u t i l i z a t i o n o f e n e r g y t h a n t h e n o n -18 ruminant . U t i l i z a t i o n of non -p ro t e in n i t r o g e n (NPN), minimal amounts of d i e t a r y p r o t e i n to s t i m u l a t e m i c r o b i a l s y n t h e s i s , and rumen by-pass of p r o t e i n are needed to inc rease the a v a i l -able p r o t e i n fo r ruminants . The ruminant appears to be most e f f i c i e n t i n p r o t e i n u t i l i z a t i o n when d i e t a r y p r o t e i n i s l i m i t i n g . However, fo r maximum p roduc t ion of the host t h i s c o n s t r a i n t must be removed. Even a t maximal l e v e l s of, m i c r o b i a l p r o t e i n p r o d u c t i o n , p r o t e i n appears to be l i m i t i n g to the hos t . A number of workers have repor ted tha t s i g n i f i c a n t ga ins can be made i n n i t r o g e n r e t e n -t i o n and p roduc t ion by post rumina l a d m i n i s t r a t i o n of h igh q u a l i t y p r o t e i n (Reis and Downes, 1971; L i t t l e and M i t c h e l l , 1967). 3. Q u a l i t y of rumen m i c r o b i a l c e l l s The q u a l i t y and q u a n t i t y of the p r o t e i n pass ing i n t o the i n t e s t i n e i s impor tan t . Rumen microorganisms tend to upgrade low q u a l i t y p r o t e i n and downgrade h igh q u a l i t y p r o t e i n sources . The p r o p o r t i o n of undegraded d i e t a r y p r o t e i n e n t e r i n g the i n t e s -t i n e s can be important a l though m i c r o b i a l p r o t e i n i n most cases i s the major s u p p l i e r of n i t r o g e n i n the o v e r a l l n i t r o g e n metab-o l i s m . The amino a c i d compos i t ion of rumen microorganisms has been w e l l documented. Bulk rumen microorganisms from a v a r i e t y of donors on d i f f e r e n t d i e t s appear to have remarkably s i m i l a r amino a c i d p a t t e r n s . We l l e r (1957), V i r t a n e n (1966), Bergen et a l . (1968a), and Purser and Buechler (1966) s tud i ed 22 pure s t r a i n s 19 o f rumen m i c r o o r g a n i s m s a n d c o u l d d e t e c t v e r y l i t t l e d i f f e r e n c e i n a m i n o a c i d c o m p o s i t i o n . S m a l l d i f f e r e n c e s w e r e n o t e d by W e l l e r (1957) a n d P u r s e r a n d B u e c h l e r (1966) i n t h e a m i n o a c i d p a t t e r n s o f b a c t e r i a a n d p r o t o z o a . C a l c u l a t i o n o f b i o l o g i c a l v a l u e , b a s e d o n e s s e n t i a l a m i n o a c i d c o n t e n t w i t h e g g p r o t e i n h a v i n g a v a l u e o f 1 0 0 , r e s u l t e d i n v a l u e s o f 8 l and 8 0 , r e s p e c -t i v e l y , f o r b a c t e r i a a n d p r o t o z o a ( P u r s e r and B u e c h l e r , 1 9 6 6 ) . P u r s e r (1970) commented t h a t c h a n g e s i n p l a s m a a m i n o a c i d s which c a n be d e t e c t e d u n d e r d i f f e r e n t d i e t a r y c o n d i t i o n s m u s t be c a u s e d by some e f f e c t o t h e r t h a n b u l k a m i n o a c i d c o m p o s i t i o n o f t h e m i c r o b i a l p r o t e i n . A f a c t o r w h i c h d o e s p l a y a r o l e i n d e t e r m i n i n g t h e q u a l i t y o f t h e m i c r o b i a l p r o t e i n i s t h e d i g e s t i b i l i t y o f t h e v a r i o u s m i c r o b i a l s p e c i e s . M c N a u g h t e t a l . (1954) d e t e r m i n e d t h e d i g e s t -i b i l i t y o f d r i e d p r e p a r a t i o n s o f rumen m i c r o b e s a n d n o t e d t h a t b a c t e r i a h a d a much l o w e r d i g e s t i b i l i t y t h a n p r o t o z o a . O t h e r w o r k e r s h a v e v e r i f i e d t h e d i f f e r e n c e i n d i g e s t i o n o f p r o t o z o a and b a c t e r i a b y u s i n g r a t s ( B e r g a n e t a l . , 1 9 6 8 b ; P u r s e r , 1970). S m i t h (1969) s t a t e s t h a t i n g e n e r a l r u m i n a n t s e x h i b i t a g r e a t e r l o s s o f n i t r o g e n i n t h e f e c e s t h a n m o n o g a s t r i c s p e c i e s . I t was f e l t t h a t t h i s was due to . t h e l o w d i g e s t i b i l i t y o f t h e rumen b a c t e r i a . M a s o n a n d P a l m e r (1971) l o o k e d a t t h e d i g e s t i o n o f v a r i o u s f r a c t i o n s o f t h e b a c t e r i a l p r o t e i n . A t l e a s t 20% o f the t o t a l n i t r o g e n , m a i n l y i n c o r p o r a t e d i n t h e c e l l w a l l s o f b a c t e r i a i s c o m p l e t e l y i n d i g e s t i b l e . M o s t o f t h e d a t a o n q u a l i t y o f rumen m i c r o b i a l p r o t e i n h a s 20 been determined using i n v i t r o r a t or chick s t u d i e s . The question remains as to the proper e v a l u a t i o n of these data. The f o l l o w i n g f a c t o r s were l i s t e d by Purser (1970) which may cause d i f f e r e n c e s between ruminants and monogastrics: i ) d i f f e r e n t regions of absorption of energy, i n the form of VFA and p r o t e i n , and amino acids i i ) r a t e of d i g e s t i o n of s p e c i f i c amino acids from micro-b i a l p r o t e i n i i i ) e f f e c t of amino a c i d composition on absorption i v ) s e l e c t i v e absorption or d i g e s t i o n of e s s e n t i a l amino acids as compared to n o n - e s s e n t i a l amino a c i d s . The source of amino acids from m i c r o b i a l p r o t e i n f o r the ruminant i s very important. This importance i s not due to the d i f f e r e n c e i n amino a c i d composition as protozoan and b a c t e r i a l species are remarkably s i m i l a r , but due to the d i f f e r e n c e i n a v a i l a b i l i t y of amino a c i d s . In p a r t i c u l a r the amino acid s i n the c e l l w a l l s of b a c t e r i a appear to have a very low d i g e s t i b i l i t y . 4. D i g e s t i o n and absorption of p r o t e i n a) D i g e s t i o n In ruminants i t appears that there may be a few d e v i a t i o n s from the general scheme of p r o t e i n d i g e s t i o n . Kay (1969) l i s t e d a few suggestions as to why the d i g e s t i o n of p r o t e i n i n ruminants might be d i s t i n c t i v e . i ) The food i s r e t a i n e d by the rumen and flows to the lower gut at a slow and constant r a t e . i i ) M i c r o b i a l a c t i v i t y transforms the d i e t a r y p r o t e i n i n t o 21 a u n i f o r m p r o d u c t . i i i ) V e r y l i t t l e s u g a r i s a b s o r b e d f r o m t h e i n t e s t i n e , i v ) T h e f l o w t o a n d f r o m t h e abomasum i s l a r g e , c o n t i n u o u s a n d u n i f o r m i n c o n s i s t e n c y . P a n c r e a t i c s e c r e t i o n s a r e a l s o c o n t i n u o u s . v) A b o m a s a l s e c r e t i o n o f a c i d i s c o n t i n u o u s a n d t h e d i g e s t a r e m a i n s a c i d i n t h e u p p e r p o r t i o n o f t h e s m a l l i n t e s t i n e . v i ) L a r g e a m o u n t s o f s a l t s a n d w a t e r a r e s e c r e t e d i n t o t h e g u t a n d m u s t be e f f i c i e n t l y r e a b s o r b e d i n t h e s m a l l a n d l a r g e i n t e s t i n e . A l l o f t h e s e p o i n t s a b o v e a r e d e v i a t i o n s f r o m t h e g e n e r a l scheme o f p r o t e i n d i g e s t i o n a s s o c i a t e d w i t h m o n o g a s t r i c s . T h e d e g r e e o f h y d r o l y s i s o f p r o t e i n o c c u r r i n g i n t h e abomasum i s a f u n c t i o n o f t h e t i m e a n d p a s s a g e o f d i g e s t a . K n i g h t e t a l . (1972) n o t e d t h a t pH d r o p p e d s u d d e n l y a f t e r f e e d -i n g and g r a d u a l l y i n c r e a s e d t o n o r m a l o v e r t h e s u b s e q u e n t s e v e n h o u r s . W i t h f a s t e r p a s s a g e t h r o u g h t h e abomasum t h e p r o p o r t i o n o f p r o t e i n e n t e r i n g t h e duodenum i n c r e a s e d a s d i d t h e s i z e o f t h e p e p t i d e s . G o r r i l l e t a l . (1968) s t u d i e d t h e p r o t e o l y t i c a c t i v i t y and i n v i t r o enzyme s t a b i l i t y o f t h e s m a l l i n t e s t i n e o f a v a r i e t y o f s p e c i e s . He f o u n d t h a t p r o t e o l y t i c d i g e s t i v e f u n c t i o n o f the c a l f i n c r e a s e d w i t h b o t h age a n d rumen f u n c t i o n , p a r t i c u l a r l y f o r t r y p s i n a n d c h y m o t r y p s i n a c t i v i t y . A m o u n t s o f p r o t e i n d i g e s t e d a n d e f f i c i e n c y o f p r o t e i n d i g e s t i o n w e r e g r e a t e r i n t h e u p p e r p o r t i o n o f t h e s m a l l i n t e s t i n e t h a n t h e l o w e r s e g m e n t s . 22 When c o m p a r e d o n t h e b a s i s o f m e t a b o l i c b o d y w e i g h t r u m i n a n t s h a d h i g h e r a c t i v i t i e s f o r t r y p s i n a n d c h y m o t r y p s i n t h a n n o n -r u m i n a n t s . Kay (1969) s u g g e s t e d t h a t h y d r o l y s i s a n d a b s o r p t i o n o f p r o t e i n i n t h e s h e e p may be a r e l a t i v e l y s l o w p r o c e s s . N o t o n l y a r e t h e p l a n t a n d m i c r o b i a l p r o t e i n s r e s i s t a n t t o a t t a c k b u t t h e d i g e s t a may n o t be n e u t r a l i z e d s u f f i c i e n t l y f o r o p t i m u m p r o t e o l y t i c a c t i v i t y . I t was f o u n d t h a t t h e pH i n c r e a s e s v e r y s l o w l y i n t h e d u o d e n a l d i g e s t a o f s h e e p , i n s h a r p c o n t r a s t t o m o n o g a s t r i c s . N e u t r a l i t y was o n l y r e a c h e d i n t h e l o w e r s e g m e n t s o f t h e j e j u n u m . T h e l o w pH was due t o t h e c o p i o u s s e c r e t i o n s o f HC1 i n t o t h e abomasum a n d t h e weak a l k a l i n i t y o f t h e b i l e a n d p a n c r e a t i c s e c r e t i o n s . T h i s h i g h a c i d i t y may a f f e c t p r o t e i n d i g e s t i o n t h e f o l l o w i n g w a y s : i ) a c i d c o n d i t i o n s i n t h e duodenum i n f l u e n c e a b o m a s a l a n d p a n c r e a t i c s e c r e t i o n , i i ) a c t i v i t y o f a b o m a s a l p e p s i n may be e x t e n d e d i n t o t h e duodenum b u t d e l a y t h e o n s e t o f a c t i v i t y o f p a n c r e a t i c e n z y m e s , o r i i i ) a c i d i t y may a f f e c t a b s o r p t i o n a n d i n t e s t i n a l p e p t o s e s . T h e i n c r e a s e d a c i d i t y o f t h e c o n t e n t s o f t h e s m a l l i n t e s t i n e t e n d s t o s p r e a d p r o t e i n d i g e s t i o n more e v e n l y o v e r t h e e n t i r e s m a l l i n t e s t i n e . T h i s s l o w e r d i g e s t i o n a n d more c o n s t a n t f l o w t o t h e duodenum may l e a d t o more e f f i c i e n t u s e o f p r o t e i n t h r o u g h e l i m i n a t i o n o f a s u r g e o f a m i n o a c i d s e n t e r i n g t h e p o r t a l s y s t e m a f t e r a m e a l w h i c h m i g h t r e s u l t i n w a s t e f u l d e a m i n a t i o n . H o o g e n r a a d and H i n d (1970) s t u d i e d t h e e f f e c t o f v a r i o u s f a c t o r s o n t h e l y s i s o f b a c t e r i a i n t h e a l i m e n t a r y t r a c t o f s h e e p . I t a p p e a r s t h a t a c o m b i n a t i o n o f l y t i c p r o c e s s e s f r o m b a c t e r i a , rumen d i g e s t a , a n d t h e n o r m a l d i g e s t i v e p r o c e s s e s o f t h e a n i m a l a r e i n v o l v e d . A t pH 3 . 0 , e n z y m e s i n t h e c e l l w a l l s o f b a c t e r i a r e l e a s e 26% o f t h e c e l l mass and 13% o f t h e a m i n o a c i d s . A t pH 8 . 0 , t h e y l i b e r a t e 40% o f t h e c e l l m a s s , 35% o f t h e a m i n o a c i d s a n d 54% o f t h e c a r b o h y d r a t e . P e p s i n a t pH 3 . 0 , r e l e a s e d 15% o f t h e w a l l m a s s a n d 45% o f t h e a m i n o a c i d s a b o v e t h a t a l r e a d y r e l e a s e d b y t h e c e l l w a l l e n z y m e s t h e m s e l v e s . A t t h i s s t a g e t h e r e a p p e a r e d t o be no p r e f e r e n t i a l r e l e a s e o f amino a c i d s . T r y p s i n a n d c h y m o t r y p s i n a t pH 8 . 0 r e l e a s e d 10% o f t h e c e l l w a l l and 35% o f t h e t o t a l a m i n o a c i d s a b o v e t h a t a l r e a d y r e l e a s e d b y t h e c o n t r o l ( b a c t e r i a l c e l l w a l l e n z y m e s ) . W i t h t h e t r y p t i c d i g e s t a t h e r e a p p e a r e d t o b e a p r e f e r e n t i a l r e l e a s e o f l y s i n e . P u r s e r (1970) r e p o r t e d a p r e f e r e n t i a l r e l e a s e o f a m i n o a c i d s when c o m p a r i n g b a c t e r i a l , p r o t o z o a l a n d e g g p r o t e i n d i g e s t i o n i n v i t r o . P r o t e o l y t i c e n z y m e s w i t h i n b a c t e r i a p l a y a n i m p o r t a n t r o l e a l o n g w i t h c u s t o m a r y p r o t e o l y t i c p r o c e s s e s i n s u p p l y i n g a m i n o a c i d s a n d p e p t i d e s f o r a b s o r p t i o n t h r o u g h o u t t h e s m a l l i n t e s t i n e . I t i s a p p a r e n t i n r u m i n a n t s t h a t t h e r e a r e d e v i a t i o n s f r o m m o n o g a s t r i c s i n p r o t e i n d i g e s t i o n . T h e g r e a t e r a c i d i t y o f t h e d i g e s t a t e n d s t o d e c r e a s e t h e e f f e c t i v e n e s s o f t h e p a n c r e a t i c a n d p e r h a p s i n t e s t i n a l e n z y m e s . However t h e a c t i o n o f t h e p r o -t e o l y t i c e n z y m e s o f t h e b a c t e r i a t h e m s e l v e s a n d t h e i r c o n t r i b u -t i o n to proteolysis must be considered. The constant supply of amino acids for absorption into the hepatic system may increase the e f f i c i e n c y of amino acid use through elimination of a surge of amino acids and wasteful deamination. b) Absorption The absorption of amino acids by ruminants has not been exten-s i v e l y studied and i t has been assumed that, i n general, absorption w i l l be sim i l a r to that i n other species. Crane (1969) noted the close functional r e l a t i o n s h i p between digestion and absorption of proteins. I t has been observed that amino acids are absorbed faster from the inte s t i n e i n the form of peptides than as free amino acids (Porter and Roll s , 1971; L i s et a l . , 1971). As there i s no evidence of these peptides i n portal blood the s i t e of peptide hydrolysis i s s t i l l being inves-tigated. There appear to be at least three transport systems for the absorption of amino acids. There are d i s t i n c t systems for a c i d i c , neutral and basic amino acids. These systems may be sub-divided even further (Schultz and Markscheid-kaspi, 1971; Reiser and Christiansen, 1971). Most of these systems have been studied by comparing the effects of competition or i n h i b i t i o n between amino acids on t h e i r absorption. I t must be kept i n mind that an e f f e c t of one amino acid on the transport of another does not mean they share the same c a r r i e r . The in t e r a c t i o n may be either a l l o s t e r i c or competitive for energy supply. In general, the rate of absorption of an amino acid diminishes with increasing 25 e l e c t r i c a l c h a r g e o f t h a t a m i n o a c i d . The p a t t e r n o f a m i n o a c i d s o b s e r v e d i n p o r t a l a n d p e r i p h e r a l b l o o d d u r i n g p r o t e i n a b s o r p t i o n w i l l b e a r some r e s e m b l a n c e t o t h e c o m p o s i t i o n o f i n g e s t e d p r o t e i n . The d i g e s t e d m a t e r i a l h a s b e e n m o d i f i e d b y many f a c t o r s i n c l u d i n g i ) r a t e s o f r e l e a s e o f i n d i v i d u a l a m i n o a c i d s d u r i n g d i g e s t i o n , i i ) r a t e s a t w h i c h t h e compounds a r e t a k e n up b y t h e i n t e s -t i n a l m u c o s a s , i i i ) t h e s i m u l t a n e o u s d i g e s t i o n and a b s o r p t i o n o f e n d o g e n o u s p r o t e i n , i v ) m e t a b o l i c t r a n s f o r m a t i o n d u r i n g a b s o r p t i o n , a n d v ) r a t e s o f u p t a k e a n d r e l e a s e o f a m i n o a c i d s b y t h e l i v e r and o t h e r t i s s u e s . 5 . P l a s m a f r e e a m i n o a c i d s The r o l e o f p l a s m a a m i n o a c i d s i n m i l k p r o t e i n s y n t h e s i s was n o t f u l l y r e c o g n i z e d u n t i l m e t h o d s f o r s e p a r a t i o n a n d i d e n t i f i c a -t i o n o f m i l k and b l o o d p r o t e i n s , t h e u s e o f s p e c i f i c i s o t o p e s and mo re k n o w l e d g e o f t h e g e n e r a l m e c h a n i s m s o f p r o t e i n s y n t h e s i s became a v a i l a b l e ( B a r r y , 1 9 5 8 ; A s k o n a s e t a l . , 1 9 5 5 ) . E v i d e n c e o f s u f f i c i e n t u p t a k e o f p l a s m a a m i n o a c i d s b y t h e mammary g l a n d t o a c c o u n t f o r t h e a m i n o a c i d r e s i d u e s p r o d u c e d i n m i l k p r o t e i n was shown b y V e r b e k e a n d P e e t e r s (1965) a n d Mepham a n d L i n z e l l ( 1 9 6 6 ) . S c h i n g o e t h e e t a l . , (1967) s t u d i e d w h i c h a m i n o a c i d s w e r e e s s e n t i a l f o r m i l k p r o t e i n s y n t h e s i s u s i n g i n . v i t r o s e c r e -t o r y c e l l s . The u s u a l a m i n o a c i d s w e r e f o u n d e s s e n t i a l p l u s 26 a requirement f o r c y s t i n e i n the bovine system and t y r o s i n e i n the r a t system. B e i t z et a l . (1967) u t i l i z e d a c e l l f r e e system i s o l a t e d from bovine mammary t i s s u e to s y n t h e s i z e s p e c i f i c m i l k p r o t e i n s . The study demonstrated t h a t the s y n t h e s i s of milk p r o t e i n s conforms to the mechanism of p r o t e i n s y n t h e s i s found i n other b i o l o g i c a l systems. Larson (1969) and Jones (1969) i n t h e i r review a r t i c l e s , d i s c u s s e d the s i m i l a r i t y between m i l k p r o t e i n s y n t h e s i s and g e n e r a l p r o t e i n s y n t h e s i s . Because of these s i m i l a r i t i e s the r o l e of amino a c i d s i n p r o t e i n s y n t h e s i s i n d i f f e r e n t t i s s u e s w i l l be d i s c u s s e d here. a) Plasma f r e e amino a c i d s as a p r o p o r t i o n of the t o t a l body amino a c i d s Munro (197 0) suggested t h a t f r e e amino a c i d s are the currency through which p r o t e i n metabolism op e r a t e s . I t i s important to have some id e a of the p r o p o r t i o n of amino a c i d s t h a t are f r e e and a l s o what p r o p o r t i o n of the f r e e amino a c i d s are found i n plasma. Herbert e t a_l. (1966) u s i n g f a s t e d r a t s determined t h a t approximately 20% of the c a r c a s s was amino a c i d s ( p r o t e i n ) . I t was estimated t h a t 0.5% of these amino a c i d s were i n the f r e e form. Of these f r e e amino a c i d s over 80% were rep r e s e n t e d by fo u r non-e s s e n t i a l amino a c i d s ; a l a n i n e , glutamine, glutamic a c i d and g l y c i n e . S k e l e t a l muscle was by f a r the b i g g e s t r e s e r v o i r of each f r e e amino a c i d accounting f o r between 50 to 80% of the t o t a l f r e e amino a c i d s . Plasma co n t a i n e d a very s m a l l p r o p o r t i o n , v a r y i n g from 0.5 to 6% of the t o t a l f r e e amino a c i d s , depending 27 o n t h e a m i n o a c i d . T h e c o n c e n t r a t i o n o f f r e e a m i n o a c i d s i n t h e p l a s m a t e n d s t o be l o w e r t h a n i n t h e t i s s u e s ( M u n r o , 1 9 7 0 ) . T h e p a t t e r n s b e t w e e n t h e d i f f e r e n t t i s s u e s and p l a s m a a r e u s u a l l y s i m i l a r . T h e p l a s m a f r e e a m i n o a c i d s r e p r e s e n t a b a l a n c e b e t w e e n i n p u t f r o m i n t e s t i n a l a b s o r p t i o n a n d p r o t e i n t u r n o v e r a n d o u t p u t i n t e r m s o f p r o t e i n s y n t h e s i s a n d a m i n o a c i d c a t a b o l i s m . I n v i e w o f t h e l a r g e a m o u n t s o f a m i n o a c i d s a d d e d t o t h e f r e e p o o l f r o m b o t h a b s o r p t i o n f r o m t h e i n t e s t i n e s a n d p r o t e i n t u r n o v e r , t h e h a l f - l i f e o f f r e e a m i n o a c i d s i n t h e p l a s m a v a r i e s b e t w e e n two a n d 15 m i n u t e s ( M u n r o , 1 9 7 0 ) . In o r d e r t o m a i n t a i n a r e a s o n -a b l e e q u i l i b r i u m t h e p l a s m a f r e e a m i n o a c i d s a r e u n d e r a v a r i e t y o f c o n t r o l s . B e c a u s e o f t h e m u l t i p l i c i t y o f t h e s e c o n t r o l s l a r g e c h a n g e s i n c e r t a i n p o o l s o f f r e e a m i n o a c i d s may o n l y g i v e a v e r y moderate r e s p o n s e i n t h e p l a s m a f r e e a m i n o a c i d s . P a w l a k a n d P i o n (1968) showed t h a t i n c r e a s i n g i n t a k e s o f d i e t a r y l y s i n e r e s u l t e d i n a 28 f o l d i n c r e a s e i n m u s c l e l y s i n e w h e r e a s p l a s m a l y s i n e was i n c r e a s e d o n l y 7 f o l d . I t m i g h t be s u g g e s t e d t h a t c h a n g e s i n t h e p l a s m a a r e i n d i c a t i o n s o f e v e n g r e a t e r c h a n g e s e l s e w h e r e i n a m i n o a c i d m e t a b o l i s m . b) U t i l i z a t i o n o f f r e e a m i n o a c i d s f o r p r o t e i n s y n t h e s i s E v e n t h o u g h t h e f r e e p l a s m a a m i n o a c i d s r e p r e s e n t a s m a l l p r o p o r t i o n o f t h e f r e e a m i n o a c i d s t h e i r r o l e i n p r o t e i n s y n t h e s i s i s i m p o r t a n t . H i d e r e t a l . (1969) c o n c l u d e d t h a t a m i n o a c i d s i n m u s c l e p r o t e i n w e r e i n c o r p o r a t e d d i r e c t l y f r o m t h e e x t r a c e l l u l a r 28 p o o l . T h e r e was no a p p a r e n t d i r e c t r e l a t i o n s h i p b e t w e e n t h e i n t r a c e l l u l a r p o o l a n d p r o t e i n s y n t h e s i s . I t was f e l t t h a t t h e i n t r a c e l l u l a r p o o l a c t e d a s a r e s e r v o i r o f a m i n o a c i d s . S a m l i e t a l . (1971) f e l t t h a t t h e c l o s e r e l a t i o n s h i p d e m o n s t r a t e d b e t w e e n e x t r a c e l l u l a r a m i n o a c i d l e v e l s , a m i n o a c i d u p t a k e and i n c o r p o r a t i o n i n t o p r o t e i n i n d i c a t e s t h a t a t l e a s t i n t h e p i t u i -t a r y t h e a v a i l a b i l i t y o f a m i n o a c i d s may be l i m i t i n g t h e l e v e l o f i n c o r p o r a t i o n i n t o p r o t e i n . I t was n o t e d t h a t t h e r e was a b o u t a f i v e m i n u t e l a g b e f o r e e x t r a c e l l u l a r a m i n o a c i d s w e r e i n c o r -p o r a t e d i n t o p r o t e i n . R e s u l t s o b t a i n e d b y W i n k l e r (1972) i n d i c a t e t h a t e x t r a -c e l l u l a r a m i n o a c i d s a r e u s e d p r e f e r e n t i a l l y t o i n t e r n a l p o o l amino a c i d s i n p r o t e i n s y n t h e s i s i n l e u c o c y t e s . When e x t r a -c e l l u l a r a m i n o a c i d s become l i m i t i n g , p r o t e i n s y n t h e s i s was s u p p o r t e d b y t h e i n t e r n a l p o o l . T h e r e was no a p p a r e n t l a g p h a s e i n t h e u t i l i z a t i o n o f e x t r a c e l l u l a r a m i n o a c i d s . T h e r e s u l t s d e m o n s t r a t e t h a t i n c o r p o r a t i o n o f a m i n o a c i d s f r o m t h e i n c u b a t i o n medium i s d e p e n d e n t o n i ) p r o t e i n s y n t h e s i s r a t e a n d i i ) e x t r a -c e l l u l a r a m i n o a c i d c o n c e n t r a t i o n . W i n k l e r (1972) e x p e c t s t h a t i n c o r p o r a t i o n o f a m i n o a c i d s i s a l s o d e p e n d e n t o n t h e t r a n s p o r t a c t i v i t y o f t h e membrane f o r a m i n o a c i d s . U n d e r n o r m a l c o n d i t i o n s e x t r a c e l l u l a r f r e e a m i n o a c i d s , w h i c h i n c l u d e p l a s m a f r e e amino a c i d s , a r e u t i l i z e d p r e f e r e n t i a l l y f o r i n c o r p o r a t i o n i n t o p r o t e i n . c) R o l e o f a m i n o a c i d s u p p l y o n p r o t e i n s y n t h e s i s B a l i g a e t a l . (1968) r e p o r t e d on t h e r e g u l a t i o n o f p o l y s o m e 29 a g g r e g a t i o n i n c e l l - f r e e s y s t e m s t h r o u g h a m i n o a c i d s u p p l y . I t was c o n c l u d e d t h a t a m i n o a c i d s u p p l y c o u l d c o n t r o l t h e d e g r e e o f a s s o c i a t i o n o r d i s s o c i a t i o n o f r i b o s o m e s w i t h mRNA. S i m i l a r i l y , J e f f e r s o n a n d K o r n e r (1969) s u g g e s t e d t h a t a m i n o a c i d s d i r e c t l y o r i n d i r e c t l y c o u l d s t i m u l a t e t h e a b i l i t y o f r i b o s o m e s t o become a t t a c h e d t o t h e m e s s e n g e r . W h i l e t h e s e a u t h o r s w e r e w o r k i n g on c o m p l e t e m i x e s o f a m i n o a c i d s t o s t i m u l a t e p r o t e i n o t h e r w o r k e r s w e r e s t u d y i n g t h e s t i m u l a t i o n o f p r o t e i n s y n t h e s i s by e l e v a t e d l e v e l s o f s i n g l e a m i n o a c i d s . H a n k i n g a n d R o b e r t s (19 65) c o n c l u d e d t h a t e l e v a t e d l e v e l s o f s i n g l e a m i n o a c i d s r e s u l t e d i n i n c r e a s e s i n g r o s s p r o t e i n s y n t h e s i s b u t t h a t n e t p r o t e i n s y n t h e s i s was d e c r e a s e d . P o s s i b l e e x p l a n a t i o n s o f t h e s e r e s u l t s w e r e t h a t c o m p l e t e s a t u r a -t i o n o f a c t i v a t i n g e n z y m e s may n o t o c c u r a t n o r m a l a m i n o a c i d c o n c e n t r a t i o n s , p r o t e i n s y n t h e s i s may be a c t i v a t e d i f e l e v a t e d l e v e l s o f one a m i n o a c i d s t i m u l a t e d e g r a d a t i o n o f p r o t e i n a n d t h u s p r o v i d e a f u l l c o m p l e m e n t o f a m i n o a c i d s , o r new RNA m o i e t i e s w e r e f o r m e d . R o t h s c h i l d e t a l . (19 69) i n s t u d y i n g a l b u m i n s y n t h e s i s i n l i v e r , f o u n d t h a t e l e v a t e d t r y p t o p h a n l e v e l s increased o v e r a l l s y n t h e s i s . T h e s e r e s u l t s w e r e c o m p a t i b l e w i t h t h e c o n -c e p t t h a t t r y p t o p h a n s t i m u l a t e s r i b o s o m a l r e a g g r e g a t i o n a n d e n h a n c e s a l b u m i n p r o d u c t i o n . I t i s a p p a r e n t t h a t t r y p t o p h a n p l a y s a more i m p o r t a n t r o l e i n a g g r e g a t i o n o f p o l y s o m e p r o f i l e s i n t h e w h o l e a n i m a l t h a n i n c e l l f r e e s y s t e m s ( M u n r o , 1 9 7 0 ) . W i t h c e l l - f r e e s t u d i e s e a c h a m i n o a c i d m u s t be s u p p l i e d ; h o w e v e r , i n t h e i n v i v o s t a t e t h e 30 t u r n o v e r and c a t a b o l i s m o f p r o t e i n s c a n s u p p l y m o s t o f t h e a m i n o a c i d s e x c e p t p e r h a p s t r y p t o p h a n i n a l l b u t e x t r e m e c a s e s . T h i s m i g h t be a t t r i b u t e d t o t h e l o w c o n c e n t r a t i o n o f t r y p t o p h a n i n t h e f r e e a m i n o a c i d p o o l a n d p r o t e i n s . d) S u p p l y o f f r e e a m i n o a c i d s f o r p r o t e i n s y n t h e s i s I t h a s b e e n p o i n t e d o u t t h a t s u p p l y o f f r e e a m i n o a c i d s p l a y s an i m p o r t a n t r o l e i n c o n t r o l o f p r o t e i n s y n t h e s i s . T h e r e a r e many d i f f e r e n t f a c t o r s w h i c h a f f e c t t h e s u p p l y o f f r e e a m i n o a c i d s . A number o f t h e s e f a c t o r s a r e d i s c u s s e d b e l o w . (1) S o u r c e P i c o u and T a y l o r - R o b e r t s (1969) r e p o r t e d t h a t a m i n o a c i d s f r o m f o o d and f r o m c a t a b o l i s m o f t i s s u e a r e i n d i s c r i m i n a n t l y u s e d by t h e b o d y . K l e v e c z (1971) s u g g e s t e d t h a t c a t a b o l i s m o f p r o t e i n i n mammal ian c e l l s i s m a s k e d b y r a p i d r e u t i l i z a t i o n o f a m i n o a c i d s . T h e s e r e p o r t s s u g g e s t t h a t i n t i m e s o f n e e d s p e c i f i c p r o t e i n s may be s a c r i f i c e d t o a l l o w f o r s y n t h e s i s o f more i m p o r -t a n t o r d e s i r a b l e p r o t e i n s a t t h a t t i m e , e s p e c i a l l y i f d i e t a r y s u p p l y o f a m i n o a c i d s i s l i m i t i n g . (2) F o o d r e s t r i c t i o n o n a m i n o a c i d , s u p p l y a n d p r o t e i n s y n t h e s i s  R a t e o f m u s c l e s y n t h e s i s a p p e a r s t o be r e l a t e d t o b o t h t h e amount a n d a c t i v i t y o f RNA i n m u s c l e . H o w a r t h and B a l d w i n (1971a) d e m o n s t r a t e d t h a t f o o d r e s t r i c t i o n i n h i b i t e d s y n t h e s i s and a c c u m u l a t i o n o f n u c l e i c a c i d s a n d RNA. In a f u r t h e r s t u d y , H o w a r t h a n d B a l d w i n (1971b) r e p o r t e d r e s u l t s w h i c h i n d i c a t e t h a t i n t h e i n h i b i t i o n o f m u s c l e g r o w t h t h e a v a i l a b i l i t y o f e n e r g y 31 y i e l d i n g me tabo l i t e s and a c t i v i t i e s of o x i d a t i v e enzymes were not key f a c t o r s . Enwonwu e t a l . (1971) r epor ted r e s u l t s i n d i c a t i n g tha t syn thes i s of l i v e r RNA i s not depressed by f a s t i n g u n t i l s i g n i f i c a n t l o s se s of RNA and polysomes have o c c u r r e d . However, r ibosomal RNA turnover r a t e i s r egu la t ed i n p a r t by changes i n the p o p u l a t i o n of membrane-free ribosomes which i n t u r n are e f f ec t ed by food d e p r i v a t i o n . A r a p i d decrease i n t o t a l RNA was noted i n r a t s fed a p r o t e i n - f r e e d i e t (Howarth, 1972). The r e s u l t s support the c o n c l u s i o n tha t a v a i l a b i l i t y of r ibosomal RNA i s a major f a c t o r i n the long term r e g u l a t i o n of muscle p r o t e i n s y n t h e s i s . I t i s apparent tha t the depress ion of r ibosomal RNA dur ing food r e s t r i c t i o n and p r o t e i n d e f i c i e n c y causes a depress ion i n muscle p r o t e i n s y n t h e s i s . As y e t , supply of amino ac id s fo r format ion of r ibosomal p r o t e i n fo r format ion of ribosomes has not been i m p l i c a t e d . I t i s a l s o apparent tha t the depress ion i n RNA and p r o t e i n syn thes i s i s not due to l a c k of energy y i e l d i n g s u b s t r a t e s . In malnour ished r a t s i t was noted by Young et a_l. (1971) tha t the r a t e of degrada t ion of muscle p r o t e i n was inc reased s l i g h t l y w h i l e the r a t e of syn thes i s was depressed r e s u l t i n g i n a net decrease i n p r o t e i n p r o d u c t i o n . A f t e r r e f e e d i n g , the degradat ion of p r o t e i n was lower than tha t of the c o n t r o l s and the syn thes i s inc reased to g rea te r than the c o n t r o l s . Another p r o t e c t i v e mechanism was r epor t ed by Pe te rs and Pe te r s (1972). A drop i n albumin syn thes i s was noted f o l l o w i n g a shor t f a s t . L i v e r p r o t e i n syn thes i s was main ta ined , p o s s i b l y due to adapt ive 32 c h a n g e s i n c o m p o s i t i o n o f h e p a t i c e n z y m e s . F e l i g e t a l_ . (1969) i n s t u d i e s on a m i n o a c i d m e t a b o l i s m d u r i n g p r o l o n g e d f a s t i n g n o t e d t h a t many o f t h e p l a s m a a m i n o a c i d s d e c r e a s e d i n c o n c e n t r a t i o n . T h e g r e a t e s t d e c r e a s e was n o t e d i n p l a s m a a l a n i n e . I t was c o n c l u d e d t h a t t h e a l a n i n e r e s p o n s e was due t o p r e f e r e n t i a l s p l a n c h n i c u t i l i z a t i o n o f t h i s a m i n o a c i d i n e a r l y s t a r v a t i o n r e s u l t i n g i n s u b s t r a t e d e p l e t i o n . T h i s was p o s t u l a t e d t o be an i m p o r t a n t m e c h a n i s m w h e r e h e p a t i c g l u c o n e o g e n e s i s was d i m i n i s h e d and p r o t e i n c a t a b o l i s m m i n i m i z e d i n f a s t i n g . F e l i g e t a l . (1970) s u m m a r i z e d t h e i r f i n d i n g s b y s t a t i n g t h a t i n p o s t a b s o r p t i v e man t h e r e i s a n e t f l u x o f a m i n o a c i d s f r o m m u s c l e t o t h e l i v e r . A l a n i n e i s q u a n t i t a t i v e l y t h e p r i m a r y g l u c o n e o g e n i c a m i n o a c i d . In p r o l o n g e d f a s t i n g l i m i t a -t i o n o f g l u c o n e o g e n e s i s i s b r o u g h t a b o u t b y d e c r e a s e d s u b s t r a t e p r e s e n t a t i o n t o t h e l i v e r , c o n s e r v i n g b o d y p r o t e i n . P r e g n a n c y was f o u n d t o d e c r e a s e t h e t i m e i n t e r v a l i n w h i c h t h e d r o p i n a l a n i n e c o n c e n t r a t i o n o c c u r r e d ( F e l i g e_t a l . 1 9 7 2 ) . T h e s e s t u d i e s show t h a t d u r i n g f a s t i n g c o n d i t i o n s , t h e b o d y t e n d s t o s l o w down m u s c l e m e t a b o l i s m . T o t a l m u s c l e RNA i s d e c r e a s e d , d e g r a d a t i o n o f m u s c l e p r o t e i n i s s l i g h t l y i n c r e a s e d a n d m u s c l e p r o t e i n s y n t h e s i s i s r e d u c e d . T h e s e s t e p s i n d i c a t e a l o s s o f a m i n o a c i d s f r o m m u s c l e . L o s s d o e s o c c u r b u t i t i s r e d u c e d b y t h e d e c r e a s i n g p r o d u c t i o n o f a l a n i n e b y m u s c l e w h i c h i s u s e d p r e f e r e n t i a l l y f o r g l u c o n e o g e n e s i s . T h e c o m b i n a t i o n o f t h e s e c o n t r o l s r e s u l t s i n an o r d e r e d g r a d u a l l o s s o f a m i n o a c i d s f r o m m u s c l e t i s s u e a n d a d e c r e a s e i n p r o t e i n s y n t h e s i s d u r i n g f a s t i n g c o n d i t i o n s . (3) Dietary e f f e c t s The supply of nutrients, i n p a r t i c u l a r energy and amino acids, i s an important factor i n c o n t r o l l i n g protein synthesis. Omstedt and Von der Decken (1972) found that the amino acid incorporating a c t i v i t y of s k e l e t a l muscle ribosomes was dependent on the amino acid make-up of the dietary protein fed to the animals over as short a period as six days. The response i s understandably not rapid due to the modifying action of digestion, absorption, transport and hepatic metabolism on the dietary pro-t e i n . The response i s further modified by the large amount of endogenous protein also digested and absorbed as well as the entrance of the dietary amino acids into an ever evolving pool of free amino acids. James (1972) states that dietary protein in the human adult may make a small contribution to the d a i l y flux of free amino acids i n the body. It was also noted that low protein feeding did not bring about as great a f a l l i n pro-t e i n synthesis as starvation. It was f e l t that t h i s might be due to the presence of i n s u l i n . I t i s apparent that decreases in dietary protein do not bring about an immediate response in protein synthesis, but a more gradual response than observed i n starvation. 14 The incorporation of C from a lab e l l e d amino acid mix into l i v e r and carcass protein by rats was constant when compar-ing incorporation i n a protein free or a 25% casein d i e t (Kirnura and Ashida, 1968). However, incorporation of the la b e l l e d com-ponent into blood sugar, l i v e r glycogen and l i p i d s i n the l i v e r 34 a n d c a r c a s s was g r e a t l y i n c r e a s e d i n t h e 25% c a s e i n d i e t . The s l i g h t l y i n c r e a s e d i n c o r p o r a t i o n i n t o p r o t e i n s i n t h e p r o t e i n f r e e d i e t was b a l a n c e d b y much l o w e r i n c o r p o r a t i o n i n t o g l y c o g e n a n d l i p i d s . I t i s a p p a r e n t t h a t t h e b o d y i s e f f i c i e n t i n c o n -s e r v i n g a m i n o a c i d s e s p e c i a l l y when p r o t e i n i s l i m i t i n g . E s s e n -t i a l a m i n o a c i d s p r o d u c e d b y b r e a k d o w n o f b o d y p r o t e i n s a r e r e u t i l i z e d . Two o t h e r f a c t o r s w h i c h f a v o u r t h e r e u t i l i z a t i o n o f a m i n o a c i d s i n t i m e s o f r e s t r i c t e d s u p p l y a r e i n c r e a s e s i n a m i n o a c i d a c t i v a t i n g e n z y m e s a n d d e c r e a s e s i n u r e a c y c l e enzymes. T h e s e r e s u l t s s u g g e s t e x i s t e n c e o f a d a p t i v e m e c h a n i s m s t h a t f u n c t i o n t o c o n s e r v e i n d i s p e n s a b l e c a r b o n s k e l e t o n s . W h i l e t h e s e f a c t o r s h a v e b e e n p r i m a r i l y i n v o l v e d w i t h p r o t e i n s y n t h e s i s , t h e e n z y m e s c a t a b o l i z i n g a m i n o a c i d s a r e a l s o a f f e c t e d i n t i m e s o f p r o t e i n d e p r i v a t i o n . N a k a n o e t a l . (1970) s t u d i e d t h e e f f e c t s o f d i e t a r y a m i n o a c i d s o n t h e a m i n o a c i d c a t a b o l i c e n z y m e s i n r a t l i v e r * T h e o n l y a m i n o a c i d s t o g i v e c o n s i s t e n t i n c r e a s e s w e r e t r y p t o p h a n a n d m e t h i o n i n e . O m i s s i o n o f t h e s e two a m i n o a c i d s r e s u l t e d i n no i n d u c t i o n . M a t s u t a k a e t c^L. (1971) f o u n d t h a t a d m i n i s t r a t i o n o f g l u c o n e o g e n i c p r e c u r s o r s c a u s e d t h e a c t i v i t y o f a m i n o a c i d c a t a b o l i c e n z y m e s t o d r o p . T h i s e f f e c t was m o s t p r o n o u n c e d i n f a s t i n g a n i m a l s . F e e d i n g o f g l u c o n e o g e n i c a m i n o a c i d s d i d n o t d i f f e r e n t i a l l y d i s p o s e t h e enzyme a c t i v i t i e s , a n d i n some c a s e s e v e n i n c r e a s e d t h e m . I t m u s t b e k e p t i n m i n d t h a t n o t a l l t h e a m i n o a c i d c a t a b o l i c e n z y m e s w e r e a s s a y e d . I t i s i n d i c a t e d t h a t i f t h e b o d y i s p r o v i d e d w i t h a d e q u a t e e n e r g y , a m i n o a c i d c o n s e r v a t i o n b e c o m e s v e r y e f f i c i e n t when p r o t e i n i s l i m i t i n g . (4) T r a n s p o r t A n i m p o r t a n t a r e a f o r c o n t r o l o f s u p p l y o f a m i n o a c i d s f o r p r o t e i n s y n t h e s i s i s t h e t r a n s p o r t o f a m i n o a c i d s f r o m t h e e x t r a -c e l l u l a r p o o l i n t o t h e c e l l . W i n k l e r e t a l . (1972) n o t e d t h a t i n c o r p o r a t i o n o f a m i n o a c i d s i n t o c e l l u l a r p r o t e i n s o f l e u c o c y t e s was i n h i b i t e d when f u r t h e r a m i n o a c i d s w e r e a d d e d t o t h e m e d i u m . T h e y c o n c l u d e d t h a t t h e i n h i b i t i o n o f i n c o r p o r a t i o n was c a u s e d n o t by r e d u c e d p r o t e i n s y n t h e s i s b u t b y a n i n h i b i t i o n o f membrance t r a n s p o r t . A m i n o a c i d s w e r e c l a s s i f i e d i n t o c e r t a i n g r o u p s a c c o r d i n g t o t h e i r a b i l i t y t o i n h i b i t one a n o t h e r . T h e d e g r e e o f i n h i b i t i o n o f t r a n s p o r t t h a t w o u l d be e x h i b i t e d i n t h e i n  v i v o s t a t e when a m i n o a c i d i m b a l a n c e s o c c u r h a s n o t b e e n d e t e r -m i n e d . T h e a c t i o n o f many h o r m o n e s a p p e a r s t o be r e l a t e d t o e f f e c t s o n a m i n o a c i d t r a n s p o r t and p r o t e i n s y n t h e s i z i n g a c t i v i t y . I t i s q u i t e d i f f i c u l t t o s e p a r a t e t h e s e two r e s p o n s e s . T h e d i s c u s s i o n t h a t f o l l o w s w i l l be c o n c e r n e d m a i n l y w i t h g e n e r a l a n a b o l i c a n d / o r c a t a b o l i c e f f e c t s o f h o r m o n e s a n d t h e i r i n t e r a c t i o n s w i t h p l a s m a a m i n o a c i d c o n c e n t r a t i o n s . G r o w t h hormone a l t e r s movement o f s e v e r a l a m i n o a c i d s i n t o m u s c l e t i s s u e , b u t d o e s n o t a f f e c t o t h e r s ( R i g g s , 1 9 7 0 ) . T h i s i n c r e a s e d u p t a k e o f a m i n o a c i d s i s n e c e s s a r y f o r t h e g r o w t h hormone t o a c t a s a p r o t e i n a n a b o l i c a g e n t . T h e d e g r e e o f a s s o -c i a t i o n b e t w e e n t h e two p r o c e s s e s h a s n o t b e e n f u l l y d e t e r m i n e d a l t h o u g h i t a p p e a r s a s i f t h e y a r e more o r l e s s i n d e p e n d e n t . S i r e k e t a_l. (1969) n o t e d a d e p r e s s i o n i n p l a s m a a m i n o a c i d s when g r o w t h hormone was i n j e c t e d . F r e e a m i n o a c i d s a l s o a p p e a r 36 t o p l a y a r o l e i n c o n t r o l o f s e c r e t i o n o f g r o w t h h o r m o n e . A r g i n i n e was f o u n d b y H a r t e l a n d e t a_l. (1970) a n d a r g i n i n e p l u s l e u c i n e b y D a v i s (1972) t o c a u s e i n c r e a s e s i n p l a s m a g r o w t h hormone l e v e l s . I n s u l i n i s r e c o g n i z e d a s a p r o t e i n a n a b o l i c h o r m o n e . Riggs (1970) s t a t e s t h a t t h e a c t i o n o f i n s u l i n o n t h e t r a n s p o r t o f n e a r l y e v e r y n a t u r a l a m i n o a c i d h a s b e e n s t u d i e d . T h e r e i s some d i s c u s s i o n o v e r w h e t h e r t h e i n c r e a s e i n t r a n s p o r t i s b e c a u s e o f i n c r e a s e s i n p r o t e i n s y n t h e s i s o r w h e t h e r t h e two p r o c e s s e s a r e i n d e p e n d e n t . I n s u l i n i s c a p a b l e o f c a u s i n g r e d u c t i o n i n p l a s m a a m i n o a c i d s i n s h e e p , p r e s u m a b l y t h r o u g h t h e same r e s p o n s e s a s m e n t i o n e d a b o v e ( C a l l e t a l . , 1 9 7 2 ) . A m i n o a c i d s a p p e a r t o h a v e a g r e a t e r e f f e c t o n t h e s e c r e t i o n o f i n s u l i n t h a n g r o w t h h o r m o n e . W h i l e i t was n o t e d by C a l l e t a l . (1972) t h a t i n s u l i n i n j e c t i o n s c a u s e d d e p r e s s i o n s i n c e r t a i n p l a s m a a m i n o a c i d s , many o f t h e s e same a m i n o a c i d s a r e known t o c a u s e i n c r e a s e d i n s u l i n s e c r e t i o n ( D a v i s , 1 9 7 2 ) . G l u c a g o n a n d i n s u l i n a r e d e s c r i b e d b y K e n n e y (1970) a s b e i n g p h y s i o l o g i c a l a n t a g o n i s t s . W h i l e i n s u l i n i s c o n s i d e r e d a p r o t e i n a n a b o l i c h o r m o n e , g l u c a g o n m i g h t b e c o n s i d e r e d a p r o -t e i n c a t a b o l i c h o r m o n e . G l u c a g o n h a s b e e n f o u n d t o i n c r e a s e t h e p r o t e o l y t i c a c t i v i t y , i n c r e a s i n g t h e a c t i o n o f a m i n o a c i d c a t a b o l i c e n z y m e s ( M a t s u t a k a e t a l . , 1 9 7 1 ) . G l u c a g o n a p p e a r s t o p l a y a r o l e o n l y i n t i m e s o f p l e n t y . G l u c a g o n s e c r e t i o n i s s t i m u l a t e d b y e l e v a t e d l e v e l s o f a m i n o a c i d s , e s p e c i a l l y t h o s e w h i c h e n t e r t h e TCA c y c l e v i a p y r u v a t e ( R o c h a e t a l . 1972) . 37 R i g g s (1970) r e p o r t s t h a t c h a n g e s i n a m i n o a c i d t r a n s p o r t a p p e a r t o be a n e s s e n t i a l p a r t o f t h e a c t i o n o f g l u c o c o r t i c o i d s i n i n c r e a s i n g a m i n o a c i d a n d p r o t e i n c a t a b o l i s m . T r a n s p o r t i n t o t h e l i v e r i s f a c i l i t a t e d w h i l e t r a n s p o r t i n t o m u s c l e i s decreased. G l u c o c o r t i c o i d s a p p e a r t o e n h a n c e t h e a c t i v i t y o f t h e enzyme s y s t e m s w h i c h c a u s e m o b i l i z a t i o n o f e x t r a h e p a t i c p r o t e i n a n d c a p t u r e o f t h e a m i n o a c i d s i n t h e l i v e r . E s t r o g e n s m o d i f y t r a n s p o r t o f a m i n o a c i d s i n t o t i s s u e s w h i c h g e n e r a l l y show t h e m o s t d r a m a t i c r e s p o n s e s t o t h e h o r m o n e . T h e two m a i n e f f e c t s o f h o r m o n e s o n p r o t e i n s y n t h e s i s a r e t r a n s p o r t o f a m i n o a c i d s i n t o c e l l s a n d a c t i v i t y o f t h e p r o t e i n s y n t h e s i z i n g m a c h i n e r y . B o t h w i l l p l a y a r o l e i n d e t e r m i n i n g t h e c o n c e n t r a t i o n o f i n d i v i d u a l a m i n o a c i d s i n t h e f r e e a m i n o a c i d p o o l o f p l a s m a . I t m u s t a l s o b e c o n s i d e r e d t h a t e l e v a t e d l e v e l s o f c e r t a i n a m i n o a c i d s a p p e a r t o p l a y a r o l e i n t h e c o n t r o l o f t h e s e c r e t i o n o f c e r t a i n h o r m o n e s . e) U s e o f p l a s m a f r e e a m i n o a c i d c o n c e n t r a t i o n s t o d e t e r -m i n e t h e a d e q u a c y o f d i e t a r y p r o t e i n  In t h i s s e c t i o n t h e i m p o r t a n c e o f f r e e a m i n o a c i d s i n p r o -t e i n s y n t h e s i s i s e s t a b l i s h e d . Some g e n e r a l f a c t s r e l a t i n g t o f r e e a m i n o a c i d p o o l s i n t h e b o d y , t h e i m p o r t a n c e o f f r e e a m i n o a c i d s i n m a i n t a i n i n g p r o t e i n s y n t h e s i s , and some o f t h e f a c t o r s a f f e c t i n g t r a n s p o r t o f f r e e a m i n o a c i d s i n t o t h e c e l l a r e d i s -c u s s e d . (1) M o n o g a s t r i c s Many a t t e m p t s h a v e b e e n made t o u s e p l a s m a - f r e e a m i n o a c i d 38 c o n c e n t r a t i o n s t o d e t e r m i n e t h e q u a l i t y o f d i e t a r y p r o t e i n f o r d i f f e r e n t s p e c i e s . O b s e r v a t i o n s b y H e w i t t a n d L e w i s (1966) t h a t p l a s m a l y s i n e c o n c e n t r a t i o n s d i d n o t r i s e u n t i l r e q u i r e m e n t s w e r e m e t , a s d e t e r m i n e d b y g r o w t h s t u d i e s , s u g g e s t e d t h e u s e f u l n e s s o f p l a s m a a m i n o a c i d d a t a i n e s t i m a t i n g d i e t a r y n e e d s . J a c o b s a n d C r a n d a l l (1972) r e p o r t e d t h a t t h e r a t i o o f p l a s m a -f r e e e s s e n t i a l t o n o n - e s s e n t i a l a m i n o a c i d s was n o t a s s e n s i t i v e a s o t h e r i n d i c e s s u c h a s g r o w t h a n d p r o t e i n e f f i c i e n c y f i g u r e s . T h e m a i n p r o b l e m was d u e t o i n v e r s e r e l a t i o n s h i p among t h e e s s e n -t i a l a m i n o a c i d s . F a s t i n g p l a s m a a m i n o a c i d l e v e l s h a v e b e e n u s e d t o f o r m u l a t e a d e q u a t e d i e t s f o r r a t s ( J a r o w s k i e t a l . , 1 9 7 1 ) . Rao ejt a l . (1968) c o m p a r e d t h e f a s t i n g p l a s m a f r e e a m i n o a c i d c o n c e n t r a t i o n s o f c o n t r o l s w i t h t h e s i x h o u r p o s t p r a n d i a l f r e e a m i n o a c i d c o n c e n t r a t i o n s o f t e s t g r o u p s a n d f o u n d t h a t t h e r e s u l t s w e r e n o t s e n s i t i v e e n o u g h t o p i c k o u t m u l t i p l e a m i n o a c i d d e f i -c i e n c i e s . T h e s e m e t h o d s do a p p e a r a d e q u a t e i n p i c k i n g o u t t h e o f i r s t o n e o r two l i m i t i n g a m i n o a c i d s . " B o o m g a r d t a n d M a c D o n a l d (1969) f o u n d t h a t t h e r e was a c o n s i d e r a b l e d i f f e r e n c e b e t w e e n a v i a n a n d m a m m a l i a n s p e c i e s i n t h e i r f a s t i n g a m i n o a c i d m e t a b o l i s m . T h e s e f i n d i n g s l e d t o a d i s c u s s i o n o f t h e u s e f u l n e s s o f f a s t i n g p l a s m a a m i n o a c i d c o n c e n t r a t i o n s . Graham e t a l . (1972) f e l t t h a t p o s t p r a n d i a l p l a s m a a m i n o a c i d c o n c e n t r a t i o n s w e r e more u s e f u l i n p r e d i c t i n g l i m i t i n g a m i n o a c i d s i n c h i l d r e n ' s d i e t s t h a n f a s t i n g l e v e l s o r e s s e n t i a l t o n o n - e s s e n t i a l r a t i o s . A n o t h e r m e t h o d o f d e t e r m i n i n g l i m i t i n g a m i n o a c i d s i s t o s t u d y t h e d e p r e s s e d a m i n o a c i d c o n c e n t r a t i o n s i n d u c e d b y g l u c o s e 39 i n f u s i o n s or a p r o t e i n - f r e e meal (Nassett and Ju, 1969) . The p a t t e r n of removal i s expected to g ive some idea of the r e q u i r e -ments. K n i p f e l e t aJL. (1969) i n commenting on t h i s method f e l t t ha t the uptake of amino a c i d s was i n s u l i n - i n d u c e d and d i d not n e c e s s a r i l y r e l a t e to p r o t e i n syn thes i s or requi rements . A f a c t o r which can a l t e r the response of plasma amino a c i d s i s the amount of the p r o t e i n supplement and how long the animal has been adapted to i t . A sudden inc rease i n p r o t e i n i n t ake was found by Anderson et a l . (1968) to inc rease g r e a t l y the plasma c o n c e n t r a t i o n of most amino a c i d s . Feed i n t ake was a l s o depressed u n t i l there was a b u i l d - u p of c a t a b o l i c enzymes to decrease the plasma concen t r a t i ons of amino a c i d s . I t was a l s o shown by Anderson et a l . (1969) tha t r a t s fed a h igh p r o t e i n d i e t were more able to cope w i t h a sudden change to an amino a c i d imbalanced d i e t than r a t s fed a low p r o t e i n d i e t . The d i f f e r e n c e between the two groups was a t t r i b u t e d to the g rea te r amino a c i d degrading a b i l i t y of the r a t s on the h igh p r o t e i n d i e t . With time the r a t s on the low p r o t e i n d i e t i nc reased t h e i r a b i l i t y to degrade excess amino a c i d s . Sudden changes i n p r o t e i n n u t r i t i o n are then ev iden t i n plasma amino a c i d s . With time the changes i n plasma amino a c i d s grow l e s s as the body adapts to changing amino a c i d supp ly . The r e l a t i o n s h i p between energy and p r o t e i n i s a l s o r e f l e c t e d i n plasma amino a c i d c o n c e n t r a t i o n s . Heard e t a l . (1969) suggested tha t the r a t i o of n o n - e s s e n t i a l to e s s e n t i a l amino ac id s r e f l e c t s the a v a i l a b i l i t y of carbohydrate and p r o t e i n . Th i s r a t i o r e f l e c t s the r e l a t i o n s h i p s between 1) carbohydrate and gluconeogenic amino a c i d s and 2) p r o t e i n and source of 40 e s s e n t i a l a m i n o a c i d s . A r r o y a v e (1970) r e p o r t e d t h a t r a t i o s b e t w e e n s e l e c t e d a m i n o a c i d s w e r e much more p r o m i s i n g a s a n i n d e x o f p r o t e i n n u t r i t i o n t h a n e s s e n t i a l t o n o n - e s s e n t i a l r a t i o s . G r i m b l e e t a l . (1969) r e p o r t e d t h a t t h e r a t i o o f n o n -e s s e n t i a l s t o e s s e n t i a l s o n l y became e l e v a t e d when t h e a n i m a l s w e r e p r o t e i n d e f i c i e n t . T h i s r i s e o c c u r r e d when p r o t e i n was l o s t f r o m t h e l i v e r . In u n d e r n o u r i s h e d r a t s t h i s l o s s o f p r o -t e i n f r o m t h e l i v e r was n o t o b s e r v e d . In l a t e r s t u d i e s G r i m b l e and W h i t e h e a d (1970) f o u n d t h a t w i t h p i g s o n a l o w p r o t e i n h i g h c a r b o h y d r a t e d i e t t h e c o n c e n t r a t i o n o f some o f t h e n o n -e s s e n t i a l a m i n o a c i d s i n p l a s m a b e g a n t o r i s e . T h i s was a t t r i b u t e d t o r e d u c e d g l u c o n e o g e n e s i s f r o m a m i n o a c i d s . T h e r e d u c e d l e v e l o f c e r t a i n e s s e n t i a l a m i n o a c i d s was a t t r i b u t e d t o an i n s u l i n e f f e c t o n u p t a k e o f a m i n o a c i d s . T h e r e s u l t s f r o m v a r i o u s w o r k e r s show t h a t b a l a n c e o f e n e r g y t o p r o t e i n i n t a k e w i l l h a v e a m a r k e d e f f e c t o n p l a s m a a m i n o a c i d c o n c e n t r a t i o n s a n d s h o u l d be c o n s i d -e r e d when i n t e r p r e t i n g a m i n o a c i d d a t a . In m o n o g a s t r i c s t h e p l a s m a a m i n o a c i d s w i l l r e f l e c t , u n d e r t h e p r o p e r c o n d i t i o n s , t h e q u a l i t y o f t h e p r o t e i n b e i n g f e d . M e t h o d s o f i n t e r p r e t a t i o n a n d a k n o w l e d g e o f t h e b a l a n c e o f p r o -t e i n and e n e r g y m u s t b e r e c o g n i z e d i n o r d e r t o o b t a i n m e a n i n g f u l r e s u l t s . (2) R u m i n a n t s T h e u s e o f p l a s m a a m i n o a c i d s t o d e t e r m i n e t h e a d e q u a c y o f d i e t a r y p r o t e i n i n r u m i n a n t s i s c o m p l i c a t e d b y a c t i o n o f rumen m i c r o b e s o n t h e d i e t a r y p r o t e i n . P l a s m a a m i n o a c i d s may r e f l e c t 41 d i e t a r y p r o t e i n q u a l i t y d e p e n d i n g o n t h e e x t e n t o f b r e a k d o w n i n t h e r u m e n . In t h e c a s e o f v e r y s o l u b l e p r o t e i n s w h i c h a r e e x t e n s i v e l y b r o k e n down i n t h e r u m e n , t h e q u a l i t y o f t h e m i c r o -b i a l p r o t e i n w i l l be r e f l e c t e d i n t h e p l a s m a a m i n o a c i d s . T h e p r o t e i n b e i n g d i g e s t e d i n t h e i n t e s t i n e s i s a m i x t u r e o f d i e t a r y , m i c r o b i a l a n d e n d o g e n o u s p r o t e i n . D i e t a r y p r o t e i n q u a l i t y e f f e c t s o n p l a s m a a m i n o a c i d s i n r u m i n a n t s h a v e o n l y b e e n f o u n d i n e x t r e m e c a s e s . O l t j e n a n d Putnam (1966) f o u n d a l t e r e d p l a s m a a m i n o a c i d c o n c e n t r a t i o n s when u r e a s u p p l i e d a l l t h e d i e t a r y n i t r o g e n . R e i s a n d T u n k s (1970) f o u n d t h a t t r e a t i n g c a s e i n w i t h f o r m a l d e h y d e t o make i t r e s i s t a n t t o m i c r o b i a l a t t a c k b u t s t i l l d i g e s t i b l e i n t h e i n t e s t i n e s c a u s e d an i n c r e a s e i n c e r t a i n o f t h e a m i n o a c i d s . R u m i n a n t s a l s o d e p e n d e x t e n s i v e l y o n g l u c o n e o g e n e s i s f o r g l u c o s e . P r o p i o n a t e a n d a m i n o a c i d s a r e t h e two m a i n p r e c u r s o r s . A t t i m e s o f p e a k p r o d u c t i o n , e s p e c i a l l y l a c t a t i o n , t h e demands f o r g l u c o s e a n d p r o t e i n s y n t h e s i s r e s u l t i n e x t e n s i v e c o m p e t i t i o n f o r a m i n o a c i d s . B l a c k et_ a l . (1968) e s t i m a t e d t h a t b e t w e e n 33 t o 50% o f t h e c a r b o n r e q u i r e d f o r g l u c o s e s y n t h e s i s may be p r o -v i d e d b y a m i n o a c i d s . W o l f and B e r g m a n (1972) e s t i m a t e d t h a t t h e amount o f g l u c o s e d e r i v e d f r o m a m i n o a c i d s w o u l d be b e t w e e n 11 a n d 30%. I t was p o i n t e d o u t b y B l a c k e t a_l. (1970) t h a t w i t h p h e n y l a l a n i n e , t h e m o d i f i c a t i o n o f m e t a b o l i c p r o c e s s e s t o r e d u c e o x i d a t i o n w o u l d h a v e l i t t l e e f f e c t o n e n e r g y m e t a b o l i s m and t h e d e f i c i t c o u l d be r e p l a c e d b y o x i d a t i o n o f a r e l a t i v e l y s m a l l amount o f o t h e r s u b s t a n c e s . T h e amount o f p h e n y l a l a n i n e c o n s e r v e d 42 f o r p r o t e i n s y n t h e s i s w o u l d be much more s i g n i f i c a n t . G l u c o n e o -g e n e s i s f r o m a m i n o a c i d i s v e r y i m p o r t a n t , n o t o n l y f o r t h e s u p p l y o f g l u c o s e f o r v i t a l p r o c e s s e s b u t f o r t h e l a r g e amount o f a m i n o a c i d s r e m o v e d t h a t w o u l d h a v e b e e n a v a i l a b l e f o r p r o t e i n s y n t h e s i s . D i e t a r y s u p p l e m e n t a t i o n o f a m i n o a c i d s i n r u m i n a n t s h a s n o t met w i t h much s u c c e s s . S c h e l l i n g e t a_l. (1967) a n d P a p a s e t a l . (1972) c o u l d d e t e c t no e f f e c t o f o r a l s u p p l e m e n t a t i o n o f a m i n o a c i d s o n p l a s m a a m i n o a c i d c o n c e n t r a t i o n s . T h e a m i n o a c i d s a r e d e g r a d e d b y t h e rumen m i c r o o r g a n i s m s b e f o r e t h e y h a v e a c h a n c e t o b e a b s o r b e d a n d u t i l i z e d b y t h e a n i m a l . I t was n o t e d b y H o g a n e t a_l. (1968) t h a t a s t h e amount o f p r o t e i n d i g e s t e d i n t h e i n t e s t i n e s o f s h e e p f e d r y e g r a s s d i e t s i n c r e a s e d , t h e l e v e l s o f m o s t o f t h e e s s e n t i a l a m i n o a c i d s a l s o r o s e . T h e r e s p o n s e was s i m i l a r t o t h a t o b s e r v e d w i t h c a s e i n i n f u s i o n s i n t o t h e abomasum, e x c e p t t h a t w i t h t h e i n f u s i o n s t h e r e s p o n s e was more m a r k e d . A s a p r o p o r t i o n o f t h e e s s e n t i a l a m i n o a c i d s o n l y t h e v a l i n e , l e u c i n e a n d p h e n y l a l a n i n e i n c r e a s e d . I t was a l s o n o t e d t h a t t h e r a t i o o f g l y c i n e t o t o t a l e s s e n t i a l a m i n o a c i d s f e l l a s t h e amount o f p r o t e i n d i g e s t e d i n t h e i n t e s t i n e s i n c r e a s e d . I t was s u g g e s t e d t h a t t h i s r a t i o m i g h t be u s e d f o r p r e d i c t i n g t h e amount o f p r o t e i n d i g e s t e d i n t h e i n t e s t i n e s . R e i s a n d T u n k s (1970) n o t e d s i m i l a r e f f e c t s when f o r m a l d e h y d e t r e a t e d c a s e i n was f e d o r c a s e i n was i n f u s e d i n t o t h e abomasum. C a s e i n f e d o r a l l y h a d no e f f e c t . S i m i l a r l y , H a l e e t a l . (1972) r e p o r t e d t h a t a b o m a s a l i n f u s i o n o f c a s e i n i n t h e d a i r y cow a l s o r e s u l t e d i n i n c r e a s e d p l a s m a v a l i n e , s e r i n e , p h e n y l a l a n i n e and 43 a l a n i n e . T h e s e r e s u l t s i n d i c a t e t h a t p r o t e i n s u p p l e m e n t s t h a t a v o i d r u m i n a l d e g r a d a t i o n r e s u l t i n s i m i l a r r e s p o n s e s i n m o n o -g a s t r i c s and r u m i n a n t s . The m e d i a t i n g i n f l u e n c e o f t h e rumen m u s t be s t r e s s e d . I n f u s i o n o f i n d i v i d u a l a m i n o a c i d s o r m i x t u r e s i n t o t h e abomasum h a s a l s o b e e n e f f e c t i v e i n i n c r e a s i n g n i t r o g e n r e t e n t i o n i n s t e e r s . O l t j e n e_t a l . (1970) f o u n d t h a t t h e i n f u s i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s p l u s p h e n y l a l a n i n e i n u r e a f e d s t e e r s r e s u l t e d i n i m p r o v e d n i t r o g e n r e t e n t i o n and a l o w e r i n g o f p l a s m a g l y c i n e a n d s e r i n e . E l e v a t e d g l y c i n e a n d s e r i n e h a v e b e e n a s s o c i a t e d w i t h l o w e r e d n i t r o g e n r e t e n t i o n o f u r e a f e d a n i m a l s . I n f u s i o n o f s e r i n e a n d g l y c i n e i n t o s o y - f e d s t e e r s r e s u l t e d i n l o w e r e d n i t r o g e n r e t e n t i o n . Hume (1971) n o t e d t h a t a b o m a s a l l y i n f u s e d l e u c i n e r e s u l t e d i n s i g n i f i c a n t i n c r e a s e s i n l e u c i n e a b s o r p t i o n o n l y a t t h e h i g h e r l e v e l s o f i n f u s i o n . A t t h e h i g h e r l e v e l s o f i n f u s i o n t h e a b s o r p t i o n o f l y s i n e a p p e a r e d t o b e i n h i b -i t e d . T h e e f f e c t i v e n e s s o f L - m e t h i o n i n e o r m e t h i o n i n e h y d r o x y a n a l o g (MHA) i n i n c r e a s i n g t h e p l a s m a c o n c e n t r a t i o n o f m e t h i o n i n e was s t u d i e d b y P a p a s e t a l . (1972) . L - m e t h i o n i n e i n f u s i o n r e s u l t e d i n i n c r e a s e d p l a s m a m e t h i o n i n e t o v a l i n e r a t i o s . T h e a b o v e r e s u l t s i n d i c a t e t h e e f f e c t i v e n e s s o f i n f u s i o n o f f r e e a m i n o a c i d s i n t o t h e abomasum i n s u p p l e m e n t i n g a m i n o a c i d s u p p l y . H o w e v e r , p o s s i b l e p r o b l e m s d u e t o c o m p e t i t i o n o n i n h i b i t i o n o f a m i n o a c i d a b s o r p t i o n a n d t r a n s p o r t s i t e s m u s t be k e p t i n m i n d . F i s h e r (1972) o b s e r v e d c h a n g e s i n p r o t e i n m e t a b o l i s m i n t h e l a c t a t i n g d a i r y cow w i t h i n t r a v e n o u s i n f u s i o n o f s e v e r a l a m i n o a c i d s . I n g e n e r a l , h i s r e s u l t s w e r e s i m i l a r t o t h o s e o b t a i n e d 44 by w o r k e r s s t u d y i n g i n f u s i o n v i a t h e abomasum o r d u o d e n u m . T h e s e r e s u l t s w o u l d be e x p e c t e d e v e n w i t h t h e m e d i a t i n g e f f e c t o f t h e l i v e r . T h e u r e r e t a l . (1966) n o t e d t h a t i n g e n e r a l t h e c o n c e n t r a t i o n o f j u g u l a r p l a s m a a m i n o a c i d s p a r a l l e l e d t h e c o n -c e n t r a t i o n s f o u n d i n p o r t a l p l a s m a . W o l f e t a l . (1972) repor ted t h a t t h e l i v e r r e m o v e d m o s t o f t h e a m i n o a c i d s f r o m t h e p o r t a l s y s t e m . U p t a k e o f v a l i n e , i s o l e u c i n e a n d l e u c i n e was f o u n d t o be l e s s t h a n g u t o u t p u t . I t was c o n c l u d e d t h a t m e t a b o l i s m by t h e g u t and l i v e r r e s u l t e d i n a n e t s p l a n c h n i c o u t p u t o f e a c h a m i n o a c i d t h a t was much d i f f e r e n t f r o m i t s a b s o r p t i o n . T h e j u g u l a r i n f u s i o n o f a m i n o a c i d s w o u l d be e x p e c t e d t o h a v e a g r e a t e r e f f e c t o n m i l k p r o t e i n s y n t h e s i s b e c a u s e t h e m e d i a t i n g e f f e c t o f t h e l i v e r w o u l d n o t p l a y a r o l e u n t i l a f t e r p a s s a g e o f t h e i n f u s a t e t h r o u g h t h e mammary g l a n d . L e i b h o l z (1970) r e p o r t e d t h a t s t a r v a t i o n i n s h e e p r e s u l t e d i n an i n c r e a s e d r a t i o o f p l a s m a e s s e n t i a l t o n o n - e s s e n t i a l a m i n o a c i d s . T h i s a l t e r e d r a t i o was c a u s e d p r i m a r i l y b y t h e d e c r e a s e i n c o n c e n t r a t i o n o f s e r i n e , g l y c i n e , a l a n i n e , a n d g l u t a m i c a c i d a n d t h e i n c r e a s e i n c o n c e n t r a t i o n o f l y s i n e a n d i s o l e u c i n e . When s h e e p w e r e f e d a l o w n i t r o g e n d i e t b u t w i t h a d e q u a t e e n e r g y , t h e r a t i o o f e s s e n t i a l t o n o n - e s s e n t i a l p l a s m a a m i n o a c i d s was f o u n d t o d e c r e a s e . T h e c o n c e n t r a t i o n s o f g l u t a m i c a c i d , g l y c i n e , g l u t a m i n e , i s o l e u c i n e a n d l e u c i n e w e r e f o u n d t o i n c r e a s e . T h e s e r e s u l t s p o i n t o u t t h e d i f f e r e n c e s t h a t m i g h t b e e x p e c t e d i n t h e l a c t a t i n g d a i r y cow t h a t m i g h t be b o t h e n e r g y and p r o t e i n d e f i -c i e n t o r j u s t p r o t e i n d e f i c i e n t . 45 R u m i n a l o r i n t r a v e n o u s i n f u s i o n o f e n e r g y s o u r c e s h a s b e e n f o u n d t o h a v e a d e p r e s s i n g e f f e c t o n p l a s m a e s s e n t i a l a m i n o a c i d c o n c e n t r a t i o n s b y P o t t e r e t a_l. ( 1 9 6 8 ) a n d E s k e l a n d e t a l . ( 1 9 7 1 ) . Rook a n d L i n e ( 1 9 6 1 ) f o u n d t h a t w i t h i n c r e a s e d p l a n e o f e n e r g y n u t r i t i o n t h e s y n t h e s i s o f m i l k p r o t e i n i n c r e a s e d . H a l f p e n n y e t a l . ( 1 9 6 9 ) n o t e d t h a t w i t h i n c r e a s i n g e n e r g y i n t a k e t h e c o n c e n t r a t i o n o f e s s e n t i a l a m i n o a c i d s was f o u n d t o d e c r e a s e . T h e s e r e s u l t s s u g g e s t t h a t i n p a r t e n e r g y i s l i m i t i n g p r o t e i n s y n t h e s i s . Y o u s e f e_t al_. ( 1 9 6 9 ) s u g g e s t e d t h a t b o t h p r o t e i n a n d e n e r g y w e r e l i m i t i n g . I n c r e a s e s c o u l d be f o u n d i n m i l k p r o t e i n s y n t h e s i s w i t h i n t r a v e n o u s s u p p l e m e n t a t i o n o f c a s e i n h y d r o l y z a t e p l u s g l u c o s e o v e r g l u c o s e i t s e l f . A t p a r t u r i t i o n a p r o f o u n d c h a n g e i n p l a s m a a m i n o a c i d c o n -c e n t r a t i o n s o c c u r s i n t h e d a i r y c o w . V e r b e k e e t a l . ( 1 9 7 2 ) r e p o r t e d t h a t a m i n o a c i d s c o u l d be d i v i d e d i n t o s e v e r a l g r o u p s due t o t h e i r b e h a v i o r a t p a r t u r i t i o n . T h e s e s i m i l a r i t i e s c o u l d be d u e t o t h e s h a r i n g o f common m e t a b o l i c p a t h w a y s . T h e r e l a t i o n -s h i p b e t w e e n p l a s m a a m i n o a c i d s , p r o g e s t e r o n e a n d p r o l a c t i n i n d i c a t e a n i m p o r t a n t i n f l u e n c e o f b o t h h o r m o n e s o n t h e i n i t i a -t i o n o f l a c t a t i o n . T h e i n c r e a s e i n p l a s m a a l a n i n e a n d g l y c i n e a f t e r c a l v i n g was a s s o c i a t e d w i t h t h e m o b i l i z a t i o n o f m u s c l e p r o t e i n . H a l f p e n n y e t a l . ( 1 9 6 9 ) i n c o m p a r i n g t h e p l a s m a a m i n o a c i d c o n c e n t r a t i o n s a t e i g h t m o n t h s o f p r e g n a n c y a n d a t two m o n t h s p o s t p a r t u m s u g g e s t e d t h a t t h e o b s e r v e d r i s e i n g l y c i n e was d u e t o t i s s u e m o b i l i z a t i o n . L y s i n e , t h r e o n i n e , h i s t i d i n e , a r g i n i n e , and l e u c i n e w e r e a l l l o w e r e d a t two m o n t h s l a c t a t i o n . T h e r e s u l t s w e r e i n t e r p r e t e d a s s h o w i n g a l o w e r i n g o f c o n c e n t r a -46 t i o n o f p l a s m a e s s e n t i a l a m i n o a c i d s due t o t h e i n c r e a s e d o u t p u t o f m i l k p r o t e i n . T h e demand f o r a m i n o a c i d s i s v e r y g r e a t a t the o n s e t o f l a c t a t i o n a n d e a r l y l a c t a t i o n . T h e d a i r y cow c o m p e n -s a t e s f o r t h e i n a d e q u a t e d i e t a r y s u p p l y b y m o b i l i z i n g t i s s u e p r o t e i n s . V a r i o u s p r o c e d u r e s h a v e b e e n u s e d t o d e t e r m i n e t h e l i m i t i n g a m i n o a c i d s i n r u m i n a n t s . E s s e n t i a l t o n o n - e s s e n t i a l r a t i o s h a v e b e e n shown t o be i n s e n s i t i v e . P o t t e r e t a l . (1972) s t u d i e d t h e p l a s m a a m i n o a c i d (PAA) i n d e x u s i n g g l u c o s e i n f u s i o n s t o d e t e r m i n e t h e l i m i t i n g a m i n o a c i d s a n d f o u n d t h a t i t was n o t s e n s i t i v e e n o u g h . A s e c o n d m e t h o d c o m p a r e d t e s t p r o t e i n a m i n o a c i d c o n c e n t r a t i o n s w i t h p l a s m a a m i n o a c i d c o n c e n t r a t i o n s d e t e r -m i n e d f o r a r e f e r e n c e d i e t w h i c h i n c l u d e d d u o d e n a l i n f u s i o n s o f h i g h q u a l i t y p r o t e i n . T h i s m e t h o d c o r r e c t l y i d e n t i f i e d t h e l i m i t i n g a m i n o a c i d s . T h e m a i n d i f f i c u l t y i s t h a t t h i s m e t h o d r e q u i r e s a r e f e r e n c e d i e t f o r e a c h s p e c i e s a n d f o r e a c h p h y s -i o l o g i c a l c o n d i t i o n i n w h i c h t h a t a n i m a l i s s t u d i e d . C h a n d l e r a n d P o l a n (1972) a t t e m p t e d t o d e t e r m i n e l i m i t i n g a m i n o a c i d s by s t u d y i n g p l a s m a a m i n o a c i d s a n d p r o t e i n p r o d u c -t i o n . W i t h e s t i m a t e s o f b l o o d f l o w t h r o u g h t h e mammary g l a n d p r e d i c t i o n s w e r e made a s t o t h e l i m i t i n g a m i n o a c i d s . M e t h i o n i n e , l y s i n e , p h e n y l a l a n i n e , t y r o s i n e and t h r e o n i n e w e r e p r e d i c t e d a s b e i n g t h e l i m i t i n g a m i n o a c i d s . I n r u m i n a n t s , p l a s m a f r e e a m i n o a c i d s a r e n o t a g o o d i n d i -c a t o r o f t h e q u a l i t y o f t h e p r o t e i n b e i n g c o n s u m e d b y t h e a n i m a l due t o t h e e x t e n s i v e a c t i o n o f t h e m i c r o b i a l p o p u l a t i o n i n t h e r u m e n . H o w e v e r , p l a s m a f r e e a m i n o a c i d l e v e l s i n r u m i n a n t s may 47 be used to determine protein status of the animal when other parameters such as energy balance and protein outputs are known. When these conditions are known many of the r e s u l t s obtained from ruminants appear si m i l a r to those obtained from monogastrics under si m i l a r conditions. The amounts of free amino acids present i n the body as a proportion of the t o t a l amino acids i s small. The free amino acids present i n blood plasma make up a small proportion of the t o t a l free amino acids. Changes i n tissue free amino acids are usually evident i n plasma free amino acid l e v e l s . Free amino acids are supplied by the d i e t and by the catabolism of body pro-tein s . R e s t r i c t i o n of dietary protein and energy has an e f f e c t on free amino acid l e v e l s . Free amino acids appear to play an important role i n protein synthesis. Transport mechanisms may also play an important role i n c o n t r o l l i n g the supply of free amino acids to the protein synthesizing machinery. Plasma free amino acid concentrations have been used successfully under con-t r o l l e d conditions to determine the adequacy of protein supply in monogastrics. In ruminants the problem i s compounded through the extensive action of the microbial population i n the rumen. In the l a c t a t i n g cow where large amounts of protein are secreted each day,it appears as i f there i s a rela t i o n s h i p between t o t a l protein supply and plasma free amino acid l e v e l s . 6. Summary of l i t e r a t u r e survey In ruminants dietary protein i s extensively degraded to ammonia within the rumen. The supply of amino acids for protein 48 s y n t h e s i s w i t h i n t h e r u m i n a n t a n i m a l i s d e p e n d e n t o n t h e c a p t u r e a n d s u b s e q u e n t s y n t h e s i s o f ammonia i n t o m i c r o b i a l p r o t e i n b y t h e rumen m i c r o o r g a n i s m s . However a p p r o x i m a t e l y 10% o f t h e e n e r g y d e r i v e d f r o m t h e a n a e r o b i c f e r m e n t a t i o n o f f e e d s t u f f s t h a t t a k e s p l a c e w i t h i n t h e rumen i s a v a i l a b l e f o r m i c r o b i a l c e l l s y n t h e s i s . A v a i l a b l e e n e r g y t h e n p l a y s a v e r y i m p o r t a n t r o l e i n t h e e f f i c i e n c y o f ammonia c a p t u r e b y t h e m i c r o o r g a n i s m s . T h e f r e e a m i n o a c i d s w h i c h r e s u l t f r o m t h e d i g e s t i o n a n d a b s o r p t i o n o f t h e m i c r o b i a l p r o t e i n a r e s y n t h e s i z e d i n t o p r o t e i n i n a s i m i l a r f a s h i o n t o t h a t i n m o n o g a s t r i c s . F r e e a m i n o a c i d s a p p e a r t o p l a y an i m p o r t a n t r o l e i n t h e c o n t r o l o f p r o t e i n s y n t h e s i s . F r e e a m i n o a c i d l e v e l s h a v e b e e n u s e d t o d e t e r m i n e t h e a d e q u a c y o f d i e t a r y p r o t e i n s u p p l y . In r u m i n a n t s t h e s i t u a t i o n o c c u r s w h e r e t h e a e r o b i c h o s t a n i m a l i s d e p e n d e n t o n t h e a n a e r o b i c f e r m a n t a t i o n w i t h i n t h e rumen t o s u p p l y p r o t e i n . T h i s a n a e r o b i c f e r m e n t a t i o n r e s u l t s i n a r a t h e r c o n s t a n t p r o t e i n t o e n e r g y r a t i o . T h e s u p p l y o f p r o t e i n i s a d e q u a t e a s l o n g a s t h e demands f o r e n e r g y a n d p r o -t e i n r e m a i n i n t h e same p r o p o r t i o n s . However i n t h e y o u n g g r o w i n g a n i m a l s a n d t h e l a c t a t i n g a n i m a l s i t a p p e a r s a s i f t h e demand f o r p r o t e i n i s g r e a t e r t h a n t h e demand f o r e n e r g y . P l a s m a a m i n o a c i d d a t a i n d i c a t e s t h a t p r o t e i n c o u l d be l i m i t i n g i n t h e s e two s i t u a t i o n s . C . EXPERIMENTS 49 1 . E x p e r i m e n t I - M i l k , m i l k c o m p o s i t i o n , and p l a s m a f r e e a m i n o a c i d s o v e r t h e c o m p l e t e l a c t a t i o n  a) M a t e r i a l s a n d m e t h o d s (1) A n i m a l s Two A y r s h i r e c o w s , #65002 (Cow #1) a n d #67133 (Cow # 2 ) , i n t h e i r 4 t h and 2nd l a c t a t i o n , r e s p e c t i v e l y , w e r e u s e d . T h e a n i m a l s w e r e f e d a c c o r d i n g t o N a t i o n a l R e s e a r c h C o u n c i l S t a n d a r d s f o r D a i r y C a t t l e (1971) o v e r t h e c o m p l e t e l a c t a t i o n . T h e d i e t was c o m p o s e d o f a c o m m e r c i a l 16% p r o t e i n c o n c e n t r a t e s u p p l e m e n t e d w i t h b e e t p u l p a n d a l f a l f a h a y . B e e t p u l p and c o n c e n t r a t e w e r e f e d a t a 1:1 r a t i o a n d t h e l e v e l o f h a y was n o t a l l o w e d t o d r o p b e l o w 40% o f t h e d r y m a t t e r i n t a k e . (2) S a m p l i n g T h e cows w e r e s a m p l e d f o r m i l k and j u g u l a r b l o o d on t h e f i r s t week a f t e r p a r t u r i t i o n , t h e f o l l o w i n g week a n d t h e n a t two week i n t e r v a l s u n t i l t h e e n d o f t h e l a c t a t i o n p e r i o d . M i l k s a m p l e s w e r e t a k e n a t e a c h m i l k i n g , p r i o r t o a n d f o l l o w i n g t h e b l o o d s a m p l e . T h e j u g u l a r b l o o d s a m p l e was t a k e n a t 2 : 0 0 p . m . , p r i o r t o t h e a f t e r n o o n m i l k i n g a n d f e e d i n g , a p p r o x i m a t e l y s e v e n h o u r s a f t e r t h e l a s t f e e d i n g . M i l k p r o d u c t i o n was r e c o r d e d a t e a c h m i l k i n g t h r o u g h o u t t h e l a c t a t i o n . 50 (3) A n a l y s e s (a) M i l k F a t , p r o t e i n a n d l a c t o s e w e r e d e t e r m i n e d u s i n g I n f r a r e d M i l k A n a l y z e r ( I R M A ) . 1 (b) I n d i v i d u a l m i l k p r o t e i n s F o r cow #65002, i n d i v i d u a l m i l k p r o t e i n s w e r e d e t e r m i n e d u s i n g d i s c g e l e l e c t r o p h o r e s i s a c c o r d i n g t o t h e p r o c e d u r e d e s -c r i b e d by D a v i s (1964) . i ) C a s e i n s A 7% s e p a r a t i n g g e l t h a t was 4.5 M i n u r e a was u s e d . S k i m m i l k , 0.05 m l , was a d d e d t o 1.44 m l 9 M u r e a p l u s 0.01 m l m e r -c a p t o e t h a n o l . T e n y 1 o f t h i s s o l u t i o n was p l a c e d o n t o p o f t h e 0 s t a c k i n g g e l a n d r u n a t 4° C a t 2 m i l l i a m p s / t u b e u n t i l t h e t r a c k -i n g d y e ( b r o m p h e n o l b l u e ) p a s s e d i n t o t h e s e p a r a t i o n g e l a n d t h e a m p e r a g e was i n c r e a s e d t o 3 m i l l i a m p s / t u b e . T h e r u n was t e r m i n -a t e d when t h e t r a c k i n g d y e was 0.5 cm f r o m t h e b o t t o m o f t h e t u b e . i i ) Whey p r o t e i n s A 9% s e p a r a t i o n g e l was u s e d . Two m l o f s k i m m i l k w e r e a d d e d t o 2 m l o f 40% s u c r o s e s o l u t i o n and m i x e d . D r o p w i s e a d d i -t i o n o f 0.13 m l o f 10% a c e t i c a c i d was u s e d t o p r e c i p i t a t e t h e c a s e i n s . T h e f i n a l pH r a n g e d b e t w e e n 4.5 a n d 4.6. T h e s o l u t i o n 1 M i l k a n a l y s i s d o n e by t h e B r i t i s h C o l u m b i a D e p a r t m e n t o f A g r i c u l t u r e , B u r n a b y , B . C . was t h e n f i l t e r e d and 1 m l o f t h e f i l t r a t e was a d d e d t o 1 m l o f t h e t r i s - g l y c i n e b u f f e r t o b r i n g t h e s o l u t i o n b a c k t o n e u t r a l -i t y . T e n vi 1 o f t h i s s o l u t i o n was a l s o u s e d f o r t h e s e p a r a t i o n w h i c h was r u n t h e same a s t h e c a s e i n s e x c e p t t h e r u n was s t o p p e d when t h e t r a c k i n g d y e was 1 cm f r o m t h e b o t t o m o f t h e t u b e . G e l s w e r e r e m o v e d a n d s t a i n e d f o r 30 m i n u t e s i n a 0.1% a m i d o b l a c k s t a i n made up o f 1 gm a m i d o b l a c k , 100 m l g l a c i a l a c e t i c a c i d , 700 m l m e t h a n o l , a n d 200 m l w a t e r . T h e g e l s w e r e d e - s t a i n e d i n r e p e a t e d r i n s i n g o f 7% a c e t i c a c i d . T h e m i l k p r o -t e i n s w e r e q u a n t i t a t e d b y s c a n n i n g t h e g e l s i n a m o d i f i e d P h o t o v o l t M o d e l #52 C d e n s i t o m e t e r . T h e c o n c e n t r a t i o n s o f e a c h p r o t e i n a r e r e p o r t e d i n t e r m s o f p e a k h e i g h t . (c) P l a s m a a m i n o a c i d s E i g h t m l o f p l a s m a f r o m j u g u l a r b l o o d t h a t h a d b e e n c e n t r i -f u g e d a t 1 2 , 0 0 0 G f o r 10 m i n u t e s a t 0 ° C was m i x e d w i t h 2 m l o f 15% s u l p h o s a l i c y l i c a c i d . T h e m i x t u r e was c e n t r i f u g e d a t 12,000 G f o r 15 m i n u t e s . T h e s u p e r n a t a n t was f r o z e n u n t i l a n a l y z e d . One m l o f t h i s s o l u t i o n was a p p l i e d d i r e c t l y t o t h e c o l u m n f o r t h e a n a l y s i s o f t h e a c i d i c and n e u t r a l , a n d b a s i c a m i n o a c i d s . T h e two c o l u m n p r o c e d u r e a s d e s c r i b e d b y Spackman e t a l . (1958) f o r p h y s i o l o g i c a l a m i n o a c i d s was u s e d . Two a m i n o a c i d a n a l y z e r s w e r e u s e d ; a P h o e n i x M7800 a n d a H i t a c h i K L A 3 B . T h e s t a t i s t i c a l m o d e l u s e d was a f a c t o r i a l d e s i g n w i t h c o w , p e r i o d a n d t h e i r i n t e r a c t i o n e f f e c t s b e i n g a n a l y z e d . T h e m o d e l was y . . . = y + c . + p . + (cp) . . + e . . , Where y . . , = t h e k*1*1 o b s e r v a t i o n i n t h e j * " * 1 p e r i o d a n d cow = t h e o v e r a l l mean t h c . = e f f e c t o f t h e i cow 1 t h P j = e f f e c t o f t h e j p e r i o d t h ( c p ) . . = e f f e c t o f t h e i j c p s u b c l a s s a f t e r t h e a v e r a g e 1 - ' e f f e c t s o f c a n d p h a v e b e e n r e m o v e d e . = r a n d o m e r r o r s a s s u m e d t o be i n d e p e n d e n t and - i j k n o r m a l l y d i s t r i b u t e d , T h e d a t a were a n a l y z e d u s i n g a L e a s t s q u a r e s a n a l y s i s p r o g r a m o u t l i n e d b y H a r v e y (1960) a n d a d a p t e d b y D r . R . G . P e t e r s o n o f t h e D e p a r t m e n t o f A n i m a l S c i e n c e , U . B . C . S t a t i s t i c a l d i f f e r e n c e s b e t w e e n means w e r e d e t e r m i n e d b y D u n c a n ' s M u l t i p l e Range T e s t . O n l y t h o s e i n t e r a c t i o n s t h a t w e r e s t a t i s t i c a l l y s i g n i f i c a n t w e r e e x p a n d e d a n d p r e s e n t e d i n t a b u l a r f o r m . b) R e s u l t s (1) M i l k a n d m i l k c o m p o s i t i o n T h e p r o d u c t i o n o f m i l k a n d m i l k c o m p o n e n t s was s i m i l a r f o r t h e two cows o v e r t h e t h r e e p e r i o d s s t u d i e d ( T a b l e ' I - 1 A ) . When t h e l a c t a t i o n was d i v i d e d i n t o t h r e e e q u a l s e g m e n t s t h e p r o d u c -t i o n a n d c o n c e n t r a t i o n o f a l l c o m p o n e n t s e x c e p t f a t a n d l a c t o s e p e r c e n t a g e d i f f e r e d b e t w e e n e a c h p e r i o d ( T a b l e I - 1 B ) . F a t p e r c e n t a g e r e m a i n e d s i m i l a r o v e r t h e e n t i r e l a c t a t i o n . T o t a l p r o t e i n p r o d u c t i o n d e c l i n e d a s m i l k p r o d u c t i o n d e c l i n e d . However, p r o t e i n p e r c e n t a g e i n c r e a s e d a s m i l k p r o d u c t i o n d e c l i n e d . Lactose 53 TABLE I -1A MILK AND MILK COMPOSITION - COW EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS MEAN , 0 ^ , 0 c x? Cow 1 Cow 2 S.E. MILK (kg) 19.324 .317 - .317 .427 FAT (%) 4.594 - .012 .012 .079 (gm) 877.20 6.32 - 6.32 24.585 PROTEIN (%) 3.707 .025 - .025 .040 (gm) 699.26 13.03 -13 .03 16.64 LACTOSE (%) 4.891 .004 - .004 .033 (gm) 959.98 16.04 -16 .04 23.92 TABLE I-IB MILK AND MILK COMPOSITION - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER MEAN S.E. 2 S.E. 3 S.E. MILK (kg) 19.324 ** 6.862 a .595 .460 b .581 - 7.322c .636 FAT (%) 4.594 - .190 .110 .056 .108 .133 .118 (gm) 877.20 **274.50a 34.20 41.72 b 33.44 -316.21 c 36.59 PROTEIN (%) 3.707 **- .290 a .055 - .083 b .054 .374° .059 (gm) 699.26 **195.27a 23.15 16.24b 22.63 -211.50° 24.76 LACTOSE (%) 4.891 ** .221 a .046 .097S .045 - .319b .049 (gm) 959.98 **379.02a 33.27 28.39b 32.54 -407.42 c 35.60 ** P <.01. a,b,c Constant within rows with the same superscripts are not significantly different. 55 p e r c e n t a g e d e c r e a s e d i n t h e l a s t t h i r d o f t h e l a c t a t i o n . (2) P l a s m a f r e e a m i n o a c i d s (a) P l a s m a a m i n o a c i d s e x p r e s s e d a s m i c r o g r a m s p e r m l o f p l a s m a When a m i n o a c i d s w e r e e x p r e s s e d a s m i c r o g r a m s p e r m l o f b l o o d p l a s m a d i f f e r e n c e s w e r e n o t e d b e t w e e n cows f o r s e v e r a l a m i n o a c i d s ( T a b l e I - 2 A ) . T h e e s s e n t i a l a m i n o a c i d s v a l i n e , l e u c i n e , a n d h i s t i d i n e v a r i e d s i g n i f i c a n t l y b e t w e e n c o w s . T h e n o n - e s s e n t i a l a m i n o a c i d s ( p l u s n i n h y d r i n p o s i t i v e compounds ) s e r i n e , g l u t a m i c a c i d , t y r o s i n e and o r n i t h i n e v a r i e d s i g n i f i c a n t l y b e t w e e n cows ( T a b l e I - 3 A ) . T h e r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s t o t o t a l n o n - e s s e n t i a l a m i n o ac ids showed a d i f f e r e n t r e s p o n s e i n e a c h c o w . T h e r a t i o o f g l y c i n e t o t h e b r a n c h e d c h a i n a m i n o a c i d s , v a l i n e , i s o l e u c i n e and l e u c i n e a l s o showed a s i g n i f i c a n t d i f f e r e n c e b e t w e e n c o w s . When e f f e c t s due t o p e r i o d o f l a c t a t i o n w e r e s t u d i e d t h e r e w e r e s i g n i f i c a n t d i f f e r e n c e s n o t e d w i t h l y s i n e , g l y c i n e a n d o r n i t h i n e ( T a b l e s I - 2 B a n d I - 3 B ) . T h e r a t i o o f e s s e n t i a l , t o n o n - e s s e n t i a l a m i n o a c i d s a n d t h e r a t i o o f g l y c i n e t o t o t a l b r a n c h e d c h a i n a m i n o a c i d s showed a s i g n i f i c a n t d i f f e r e n c e . Cows showed s i g n i f i c a n t cow x p e r i o d i n t e r a c t i o n t e r m s f o r t h r e o n i n e , m e t h i o n i n e , p h e n y l a l a n i n e , u r e a , a s p a r a g i n e a n d g l u t a m i n e , a n d t o t a l n o n - e s s e n t i a l a m i n o a c i d s ( T a b l e I - 3 C ) . D u r i n g p e r i o d 1 t h e c o n c e n t r a t i o n o f t h r e o n i n e was l o w e r i n cow #1 t h a n i n cow #2. M e t h i o n i n e i n t e r a c t i o n r e s u l t s showed cow #1 h a d l o w e r p l a s m a m e t h i o n i n e c o n c e n t r a t i o n s d u r i n g p e r i o d 56 TABLE I-2A ESSENTIAL AMINO ACIDS u g/ml BLOOD PLASMA COW EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS MEAN COW NUMBER Cow 1 Cow 2 S.E. THREONINE 13. 859 - , .449 .449 .537 VALINE 41. 740 **-4, .663 4, .663 1.410 METHIONINE 5. 106 - , .138 .138 .118 ISOLEUCINE 24. 960 - , .621 .621 .906 LEUCINE 24. 288 **-3. .092 3, .092 .832 PHENYIALANINE 8. 771 .185 - , .185 .257 LYSINE 22. 799 - .472 .472 .997 HISTIDINE 13. 963 .786 -1 .786 .457 ARGININE 18. 055 - .198 .198 .863 TESS1 173. 539 -7 .661 7 .661 4.965 TNESS2 180. 623 - .178 .178 4.150 RATIO3 1. 967 .052 .052 .025 TOTAL4 354. 162 -7 .838 7 .838 8.346 GLY/BRANCHED5 ,305 * .062 — .062 .025 ** P <.05. P <.01. TESS 2TNESS "RATIO T^OTAL Total essential amino acids y g/ml blood plasma. Total non-essential amino acids (not including urea) u g/ml blood plasma. Tess/Tness. Tess + Tness. ^GLY/BRANCHED Glycine/Valine + Isoleucine + Leucine. 57 TABLE I-2B ESSENTIAL AMINO ACIDS p g/ml BLOOD PLASMA - PERIOD EFFECTS LEAST SQUARES MEAN LEAST SQUARES CONSTANTS - PERIOD NUMBER S.E. 2 S.E. 3 S.E. THREONINE 13.859 - . 546 .747 • 512 • 731 • 033 • 799 VALINE 41.740 - . 699 1 .961 • 025 1. 918 • 674 2. 098 METHIONINE 5.106 • 181 .164 - . 145 • 160 • 036 • 176 ISOIEUCINE '24.960 • 094 1 .261 -1. 047 1. 233 • 952 1. 349 LEUCINE 24.288 - 2. 842 1 .158 1. 164 1. 132 1. 677 1. 238 PIIENYLALANINE 8.771 - . 035 .358 - . 113 • 350 • 148 • 383 LYSINE 22.799 *- 4. 072a 1 .387 1. 694b 1. 356 2. 378b 1. 484 HISTIDINE 13.963 - 1. 583 .636 • 164 621 1. 418 • 680 ARfJTNTNF. 18.055 - 2. 476 1 .200 1, 625 1. 174 • 851 1. 284 TFSS1 173.539 -11. 977 6 .907 3. 878 6. 753 8. 098 7. 389 TNESS2 180.623 2. 891 5 .774 -3. 926 5. 646 1. 035 6. 177 RATIO3 .967 *- 096a .034 063b ,033 034b 037 4 TOfALaa 354.162 **- 9. 086 11 .610 - . 047 11. ,353 9. ,133 12. 420 GLY/BRA1\1CKED5 .305 ,125a .034 - . 04 8 b ,033 - . ,07^ 037 * P <.05. ** P <.01. a,b Constants v/ithin rows with the same superscripts are not significantly different. ^TESS Total essential amino acids u g/ml blood plasma. 2 TNESS Total non-essential amino acids (not including urea) v g/ml blood plasma. 3RATIO Tess/Tness. Tess + Tness. TOTALaa GLY/BRANQ1ED GlyeineA'aline + Isoleucine + Leucine. 58 TABLE I-3A NON-ESSENTIAL AMINO ACIDS yg/ml BLOOD PLASMA -COW EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS MEAN COW NUMBER Cow 1 Cow 2 S.E. TAURINE 4.981 - .220 .220 .259 UREA 235.454 -18.261 18.261 13.568 SERINE 11.943 * 1.222 - 1.222 .525 ASPARAGINE1 47.394 - .168 .168 1.754 PROLINE 12.529 - .724 .724 .684 GLUTAMIC ACID 12.396 * 1.729 - 1.729 .657 CITRULLINE 11.751 .480 - .480 .473 GLYCINE 25.697 1.871 - 1.871 1.030 ALANINE 32.400 - 1.907 1.907 .992 a-AMINO-N-BUTYRIC ACID 4.113 - .936 .936 .363 TYROSINE 10.207 ** .155 - .155 .454 ORNITHINE 7.213 **- 1.681 1.681 .407 * P <.05. ** P <.01. ^Asparagine + Glutamine. TABLE I-3B NON-ESSENTIAL AMINO ACIDS yg/ml BLOOD PLASMA - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER TAURINE 4. 981 - 185 .360 - , .072 352 .257 .386 UREA 235. 454 -15. 23 18, .874 2, .58 18. 455 12, .65 20 .191 SERINE 11. 943 • 671 .731 - , .609 • 715 - .062 .782 ASPARAGINE1 47. 394 - 2. 820 2. .440 -2, .927 2. 386 5 .747 2 .610 PROLINE 12. 529 - . 218 .952 .801 • 931 - .582 1 .018 GLUTAMIC ACID 12. 396 1. 226 .914 - .620 • 894 - .606 .978 CITRULLINE 11. 751 • 440 .658 .121 • 644 - .560 .704 GLYCINE 25. 697 ** 6. 997a 1, .432 -2 .418b 1. 401 - 4 .579b 1 .533 ALANINE 32. 400 - 1. 530 1 .381 1 .057 1. 350 .473 1 .477 a-AMINO-N-BUTRIC ACLD 4. 113 • 207 .505 - .427 • 494 .221 .540 TYROSINE 10. 207 - . 453 .632 .229 • 618 .225 .676 ORNITHINE 7. 213 *- 1. 444a .566 .941b 554 .503^ .606 * P <.05. ** P <.01. ''"Asparagine + Glutsmine. a ,b Constants w i t h i n rows wi th the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t . TABLE I-3C AMINO ACID DATA - y g /ml BLOOD PLASMA - SIGNIFICANT COW X PERIOD INTERACTION TERMS LEAST SQUARES CONSTANTS VARIABLE THREONINE METHIONINE • PHENYJJ^LANINE UREA ASPARAGINE TNESS LEAST SQUARES MEAN 13.859 5.106 8.771 235.54 47.394 180.62 Cow 1 P l 3 S . E . - 2 .783 a 1.199 - . 398 a .248 - . 3 4 1 * .540 - 9 1 . 0 5 a 28.46 - 5 . 1 8 1 * 3.680 - 3 . 0 8 * 8.71 Cow 1 P 2 S . E . 1 .955 b .996 .440 b .225 1.029 b .491 17 .73 b 25.88 be 4 . 9 9 4 ° ° 3.346 12 .57 a 7.92 Cow 1 P 3 S . E . - . 5 2 0 * 1.032 - . 780 a .234 - . 1 3 4 * .509 18 .54 b 26.84 - . 3 1 7 * ° 3.470 - 1 0 . 0 2 * 8.21 Cow 2 P 1 S . E . 1 .691 b 1.177 . 760 b .266 . 2 7 1 * .580 60 .59 b 30.58 - . 4 5 9 * ° 3.954 8 . 8 7 * 9.36 Cow 2 P 2 S . E . - . 9 3 1 a b 1.116 - . 730 a .252 - 1 . 2 5 4 a .550 - 1 2 . 5 7 * 29.01 -10 .848 a 3.750 - 2 0 . 4 2 b 8.87 Cow 2 P 3 S . E . . 5 8 7 * 1.460 . 0 6 0 * .330 - . 5 0 3 * .720 6 . 7 6 * 37.96 1 1 . 8 1 3 ° 4.908 1 2 . 0 9 * 11.61 a , b , c Constants w i t h i n columns w i th the same superscr ip ts are not s i g n i f i c a n t l y d i f f e r e n t . Asparagine + glutairiine. TNESS T o t a l n o n - e s s e n t i a l amino a c i d s . Cow 1 P 1 Cow number and p e r i o d number. 61 1 t h a n cow #2 b u t h i g h e r c o n c e n t r a t i o n s d u r i n g p e r i o d 2 . P h e n -y l a l a n i n e was p r e s e n t i n h i g h e r c o n c e n t r a t i o n s d u r i n g p e r i o d 2 i n cow #1 t h a n i n cow #2. U r e a r e s u l t s d i f f e r e d d u r i n g p e r i o d 1 i n w h i c h t h e c o n c e n t r a t i o n s w e r e l o w e r w i t h cow #1 t h a n w i t h cow #2. A s p a r a g i n e a n d g l u t a m i n e c o n c e n t r a t i o n s w e r e h i g h e r d u r i n g p e r i o d 2 w i t h cow #1 t h a n cow #2. T o t a l n o n - e s s e n t i a l a m i n o a c i d s f o l l o w e d a s i m i l a r p a t t e r n w i t h h i g h e r v a l u e s expressed d u r i n g p e r i o d 2 w i t h cow #1 t h a n cow #2. (b) P l a s m a a m i n o a c i d s e x p r e s s e d a s a p e r c e n t a g e o f t h e t o t a l e s s e n t i a l o r n o n - e s s e n t i a l a m i n o a c i d s E x p r e s s i o n o f a m i n o a c i d s a s a p r o p o r t i o n o f e i t h e r t h e t o t a l e s s e n t i a l o r t o t a l n o n - e s s e n t i a l a m i n o a c i d s g a v e q u i t e d i f f e r -e n t r e s u l t s . W i t h t h e e s s e n t i a l a m i n o a c i d s t h e d i f f e r e n c e s b e t w e e n cows r e m a i n e d t h e same a s when e x p r e s s e d a s m i c r o g r a m s p e r m l e x c e p t t h a t p h e n y l a l a n i n e was a l s o s i g n i f i c a n t l y d i f f e r e n t ( T a b l e I - 4 A ) . However when t h e e f f e c t s d u e t o p e r i o d w e r e a n a l y z e d i t was n o t e d t h a t m e t h i o n i n e , i s o l e u c i n e , l e u c i n e and l y s i n e v a r i e d s i g n i f i c a n t l y b e t w e e n p e r i o d s ( T a b l e I - 4 B ) . A t t h e p e r i o d s o f p e a k m i l k o r m i l k p r o t e i n p r o d u c t i o n t h e p r o p o r -t i o n o f l e u c i n e a n d l y s i n e was s i g n i f i c a n t l y r e d u c e d w h i l e t h e p r o p o r t i o n o f m e t h i o n i n e a n d i s o l e u c i n e was e l e v a t e d . T h e e x p r e s s i o n o f t h e n o n - e s s e n t i a l a m i n o a c i d s a s a p r o -p o r t i o n o f t h e t o t a l n o n - e s s e n t i a l a m i n o a c i d s ( n o t i n c l u d i n g u r e a ) r e s u l t e d i n many d i f f e r e n c e s b e t w e e n t h e two cows ( T a b l e I - 5 A ) . S e r i n e , g l u t a m i c a c i d , g l y c i n e , a l a n i n e , a - a m i n o - n -b u t y r i c a c i d a n d o r n i t h i n e w e r e a l l s i g n i f i c a n t l y d i f f e r e n t . T h e b e t w e e n p e r i o d v a r i a t i o n f o r many o f t h e a m i n o a c i d s a l s o showed TABLE I-4A ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL ESSENTIAL AMINO ACIDS - COW EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS MEAN COW NUMBER Cow 1 Cow 2 S.E. THREONINE 7. ,844 • .012 ,012 .204 VALINE 23. ,740 •1.805 1, .805 .494 METHIONINE 2. ,968 .059 - , .059 .102 ISOLEUCINE 14. .242 .188 - , .188 .236 LEUCINE 13. .710 -1.357 1, .357 .215 PHENYLALAt-IINE 5, .077 * .327 - .327 .134 LYSINE 12, .833 .038 - .038 .383 HISTIDINE 8 .162 ** 1.407 -1 .407 .392 ARGININE 10 .163 .100 - .100 .343 * P <.05. ** P <.01. TABLE I-4B ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL ESSENTIAL AMINO AC IDS PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER MEAN 1 S.E. 2 S.E. 3 S.E. THREONINE 7.844 .187 .283 .055 .277 -.243 .303 VALINE 23.740 1.127 .687 -.507 .672 -.620 .735 METHIONINE 2.968 * .430 a .142 - .223 b .138 -.207b .151 ISOLEUCINE 14.242 ** 1.112a .329 - .951 b .321 - .161b .352 LEUCINE 13.710 *- .809 a .300 .471 b .293 .338b .321 PHENYLALANINE 5.077 .476 .187 -.246 .183 -.230 .200 LYSINE 12.833 *-1.613 a .533 .781 b .521 .833b .570 HISTIDINE 8.162 .107 .545 -.364 .533 .257 .583 ARGININE 10.163 - .857 .478 .777 .461 .080 .511 * P <.05. ** P <.01. a,b Constants within rows with the same superscripts are not significantly different. s i g n i f i c a n t d i f f e r e n c e s . T h e s e i n c l u d e d a s p a r a g i n e , g l y c i n e , a l a n i n e a n d o r n i t h i n e ( T a b l e I - 5 B ) . When e x p r e s s e d a s a p r o p o r t i o n o f t h e t o t a l n o n - e s s e n t i a l a m i n o a c i d s , a l a n i n e was t h e o n l y a m i n o a c i d t h a t h a d a s i g n i f -i c a n t cow x p e r i o d i n t e r a c t i o n t e r m ( T a b l e I - 3 C ) . The p e r c e n t -a g e o f a l a n i n e was s i g n i f i c a n t l y h i g h e r i n p e r i o d 2 w i t h cow #2 t h a n cow #1. A l l t h e o t h e r p e r i o d v a l u e s f o r a l a n i n e w e r e s i m i l a r f o r e a c h c o w . (3) C o n c e n t r a t i o n o f t h e i n d i v i d u a l m i l k p r o t e i n s o f cow #65002  When t h e l a c t a t i o n was d i v i d e d i n t o t h r e e e q u a l p e r i o d s no d i f f e r e n c e s c o u l d be f o u n d b e t w e e n p e r i o d s f o r a - l a c t a l b u m i n o r 6 - l a c t o g l o b u l i n A a n d B ( T a b l e 1 - 6 ) . B o t h a S ^ - c a s e i n a n d 3-c a s e i n w e r e f o u n d t o i n c r e a s e a s t h e l a c t a t i o n p r o g r e s s e d . T h e m i l k a n d m i l k c o m p o n e n t p r o d u c t i o n w e r e a l s o i n c l u d e d i n T a b l e r f o r c o m p a r i s o n . c ) D i s c u s s i o n (1) M i l k a n d m i l k c o m p o n e n t s A s e x p e c t e d t h e p r o d u c t i o n o f m i l k v a r i e d s i g n i f i c a n t l y b e t w e e n p e r i o d s . T h e d a i r y a n i m a l n o r m a l l y r e a c h e s p e a k m i l k p r o d u c t i o n w i t h i n t h e f i r s t s i x weeks o f l a c t a t i o n . In t h i s s t u d y b o t h cows r e a c h e d p e a k p r o d u c t i o n w i t h i n t h i s i n t e r v a l , #65002 a t f i v e weeks a n d #67133 a t two w e e k s . T h e p e a k o f l a c -t a t i o n f o r b o t h cows was d e f i n i t e l y w i t h i n t h i s f i r s t p e r i o d . T h e s e c o n d p e r i o d o r m i d d l e t h i r d o f l a c t a t i o n r e p r e s e n t s i n t e r -TABLE I-5A NON-ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL NON-ESSENTIAL AMINO ACIDS - COW EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS MEAN COT NUMBER Cow 1 Cow 2 S.E. TAURINE 2.780 - .121 .121 .143 SERINE 6.612 * .718 - .718 .271 ASPARAGINE1 26.030 - .071 .071 .625 PROLINE 6.861 - .447 .447 .292 GLUTAMIC 6.936 * 1.020 -1.020 .376 CITRULLINE 6.512 .261 - .261 .228 GLYCINE 14.257 * 1.189 -1.189 .563 ALANINE 18.045 *-1.036 1.036 .354 a-AMINO-N-BUTYRIC ACID 2.254 *- .504 .504 .184 TYROSINE 5.627 .037 - .037 .195 ORNITHINE 4.082 *-1.045 1.045 .275 * P <.05. ** P <.01. ^Asparagine + Glutamine. TABLE I-5B NON-ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL NON-ESSTENTIAL AMINO ACIDS PERIOD EFFECTS LEAST SQUARES MEAN 1 LEAST SQUARES CONSTANTS -S.E. 2 S.E. PERIOD NUMBER 3 S.E. TAURINE 2.780 - .153 .198 .0345 .194 .118 .212 SERINE 6.612 .305 .378 - .237 .369 - .068 .404 ASPARAGINE1 26.030 *-1.849a .870 -1.250a .851 3.099b .931 PROLINE 6.861 - .207 .406 .600 .398 - .394 .435 GLUTAMIC ACID 6.936 .551 .523 - .220 .512 - .330 .560 CITRULLINE 6.512 .076 .318 .202 .310 - .278 .340 GLYCINE 14.257 ** 3.748a .784 -1.198b .766 -2.549b .838 ALANINE 18.045 *-1.161a .492 1.086b .481 .075^ .526 a-AMINO-N-BUTYRIC ACID 2.254 .090 .256 - .153 .250 .063 .274 TYROSINE 5.627 .378 .272 .268 .266 .110 .291 ORNITHINE 4.082 *-1.018a .383 .865b .374 .153^ .410 * P <.05. ** P <.01. ^Asparagine + Glutamine. a,b Constants within rows with the same superscripts are not significantly different. TABLE 1-6 MILK AND INDIVIDUAL MILK PROTEINS FOR COW 65002 - PERIOD EFFECTS COMPONENT LEAST SQUARES MEAN 1 LEAST SQUARES CONSTANTS S.E. 2 - PERIOD S.E. NUMBER 3 S.E. MILK (kg) 19.68 ** 7.17 a .626 - .41b .626 - 6.76C .602 PROTEIN (%) 3.733 **_ .381C .075 - .098b .075 .479 a .072 PROTEIN (gm) 712.34 ** 184.50a 23.98 -13.73b 23.98 -170.78C 23.08 LACTOSE (%) 4.894 ** .210 a .054 .145a .054 - .356 b .052 LACTOSE (gm) 975.99 ** 392.73a 36.34 - 5.34b 36.34 -387.39C 34.99 a-LACTALBUMIN 3.451 .187 .351 - .204 .351 .017 .338 B-LACTOGLOBULIN A 5.036 .172 .654 - .128 .654 .045 .630 B-LACTOGLOBULIN B 4.145 .176 .552 - .095 .552 .081 .531 aS-j-CASEIN 6.619 *. .509 b .201 - .101* .201 .610a .194 B-CASEIN 6.503 **. .698b .208 .061* .208 .637a .200 * P <.05 ** P <.01 a,b,c Constants within rows with the same superscripts are not significantly different. 68 m e d i a t e p r o d u c t i o n a n d t h e t h i r d p e r i o d o r l a s t t h i r d o f l a c t a -t i o n r e p r e s e n t s l o w p r o d u c t i o n . T h e d a i l y p r o d u c t i o n o f f a t , p r o t e i n and l a c t o s e when e x p r e s s e d i n g r a m s was s i g n i f i c a n t l y d i f f e r e n t b e t w e e n p e r i o d s d u e m a i n l y t o t h e d i f f e r e n c e i n m i l k p r o d u c t i o n a l t h o u g h t h e p e r c e n t l a c t o s e i n m i l k d r o p p e d d u r i n g t h e l a s t t h i r d o f l a c t a -t i o n . T h e l a c t o s e c o n t e n t o f t h e m i l k o f many s p e c i e s h a s b e e n f o u n d t o v a r y i n v e r s e l y w i t h p r o t e i n a n d f a t c o n t e n t o f m i l k ( E b n e r , 1 9 7 1 ) . In t h i s s t u d y t h e r e s u l t s r e p o r t e d h e r e i n d i c a t e t h a t t h e r e was a n o n - s i g n i f i c a n t i n c r e a s e i n f a t c o n t e n t a n d a s i g n i f i c a n t i n c r e a s e i n m i l k p r o t e i n c o n t e n t i n e a c h p e r i o d a s l a c t a t i o n p r o g r e s s e d . T h i s i n c r e a s e d c o n c e n t r a t i o n o f p r o t e i n i n m i l k i n l a t e l a c t a t i o n i s e x p e c t e d ( L a r s o n e t a l . , 1 9 5 6 ) . Rook (1961) s u g g e s t e d t h a t t h e o b s e r v e d c h a n g e s i n p r o t e i n c o n -t e n t o f m i l k i n l a t e l a c t a t i o n m i g h t be d u e t o p r e g n a n c y . M i l k p r o t e i n p e r c e n t was f o u n d t o i n c r e a s e much more i n p r e g n a n t cows t h a n t h a t i n b a r r e n cows a f t e r t h e s i x t h m o n t h o f l a c t a t i o n . D u r i n g p e a k p r o d u c t i o n o f m i l k t h e a n i m a l i s o f t e n i n n e g a t i v e e n e r g y b a l a n c e w h i l e i n t h e l a t e r s t a g e s o f l a c t a t i o n t h e r e i s a t e n d e n c y t o w a r d s o v e r f e e d i n g . S u c h d i f f e r e n c e s i n r e l a t i v e e n e r g y s t a t u s d u e t o l o w e r e d p r o d u c t i o n w i l l a f f e c t t h e s u p p l y o f a m i n o a c i d s f o r p r o t e i n s y n t h e s i s . S m a l l e r q u a n t i t i e s o f a m i n o a c i d s w i l l be r e q u i r e d f o r g l u c o n e o g e n i c s u b s t r a t e s a n d t o t a l p r o t e i n s y n t h e s i s ; c o n s e q u e n t l y t h e r e w i l l be an i n c r e a s e d m i l k p r o t e i n c o n t e n t i n l a t e l a c t a t i o n . T h e m a i n i m p o r t a n c e o f t h e s e f i n d i n g s i s t h a t t h e demands p l a c e d o n t h e a n i m a l f o r a m i n o a c i d s f o r p r o d u c t i o n o f m i l k a n d 69 m i l k p r o t e i n d i f f e r e d s i g n i f i c a n t l y b e t w e e n t h e t h r e e p e r i o d s o f l a c t a t i o n . I t i s a l s o i n t e r e s t i n g t o n o t e t h a t a s t h e demands f o r m i l k s y n t h e s i s a r e d e c r e a s e d due t o t h e l o w e r e d t o t a l p r o -d u c t i o n , t h e c o n c e n t r a t i o n o f p r o t e i n i n t h e m i l k shows a s i g n i f i c a n t i n c r e a s e . (2) P l a s m a a m i n o a c i d s (a) P l a s m a a m i n o a c i d s e x p r e s s e d a s m i c r o g r a m s p e r m l o f b l o o d p l a s m a  D i f f e r e n c e s w e r e f o u n d b e t w e e n cows i n t h e c o n c e n t r a t i o n o f f r e e a m i n o a c i d s . T h e s e d i f f e r e n c e s do n o t a p p e a r t o be e x p l a i n e d i n t e r m s o f p r o d u c t i o n a s p r o d u c t i o n r e s p o n s e s o f b o t h cows w e r e s i m i l a r . S i g n i f i c a n t d i f f e r e n c e s b e t w e e n p e r i o d s w e r e n o t e d f o r l y s i n e , g l y c i n e a n d o r n i t h i n e . T h e s i g n i f i c a n t l y l o w e r c o n c e n t r a t i o n o f l y s i n e a t h i g h l e v e l s o f m i l k p r o t e i n p r o d u c t i o n h a s a l s o b e e n n o t e d b y W i l l i e r a n d K e s l e r ( 1 9 7 0 ) . I t i s p o s s i b l e t o s p e c u l a t e t h a t l y s i n e i s t h e l i m i t i n g a m i n o a c i d f o r m i l k p r o t e i n s y n t h e s i s and t h a t a s t h e amount o f m i l k p r o t e i n s y n t h e s i z e d d e c r e a s e s t h e amount o f l y s i n e r e q u i r e d a l s o d e c r e a s e s and p l a s m a l y s i n e l e v e l s r i s e . I t was s u g g e s t e d b y C h a n d l e r a n d P o l a n (1972) t h a t l y s i n e c o u l d be one o f t h e l i m i t i n g a m i n o a c i d s i n m i l k p r o t e i n s y n t h e s i s . T h e i n c r e a s e i n g l y c i n e c o n c e n t r a t i o n i n p l a s m a d u r i n g p e a k m i l k p r o t e i n p r o d u c t i o n h a s b e e n a t t r i b u t e d t o i n c r e a s e d t i s s u e m o b i l i z a t i o n t o m e e t t h e a m i n o a c i d demands o f t h e a n i m a l ( H a l f -p e n n y , 1 9 6 9 ) . T h i s phenomenon o f i n c r e a s e d g l y c i n e l e v e l s when a m i n o a c i d s a r e l i m i t i n g i s common i n m o s t s p e c i e s ( A d i b i , 1 9 7 1 ; 70 Reis and Tunks, 1970; Leibholz, 1970). The decreased concentration of ornithine i n early lactation i s consistent with the reports of s i g n i f i c a n t uptake of ornithine by the mammary glands of l a c t a t i n g animals (Verbeke and Peeters, 1965; Mepham and L i n z e l l , 1966; Yip and Knox, 1972). Ornithine i t s e l f does not appear i n milk protein but does act as a precursor for proline and other non-essential amino acids. The general trend of increased concentration of esse n t i a l amino acids in the l a t e r part of l a c t a t i o n was shown by a s i g n i f -icant increase i n the r a t i o of t o t a l e ssential amino acids to t o t a l non-essential amino acids as l a c t a t i o n progressed. These results indicate a reduced demand by the animal for es s e n t i a l amino acids as production declined i n late l a c t a t i o n (Halfpenny et a l . , 1969) . The r a t i o of glycine to the branched chain amino acids (valine, isoleucine and leucine) was found to decrease s i g n i f -i c a n t l y as l a c t a t i o n progressed. Halfpenny et a l . (19 69) suggested that the increase i n glycine i n early l a c t a t i o n was due to increased tissue mobilization. The level s of valine, isoleucine and leucine were lowered at t h i s time due to th e i r high concen-t r a t i o n i n milk protein. This r a t i o of glycine to one or more of the branched chain amino acids has been suggested to serve as an index of protein n u t r i t i o n (Reis and Tunks, 1970; Arroyave, 1970). The elevated glycine le v e l s found i n the present experi-ment during early l a c t a t i o n are in d i c a t i v e of tissue mobilization The s i g n i f i c a n t i n t e r a c t i o n terms for threonine, methionine, phenylalanine, urea, asparagine and glutamine, and t o t a l non-e s s e n t i a l amino a c i d s are d i f f i c u l t to e x p l a i n (Table I-3C). The on ly f a c t o r which might he lp e x p l a i n some of the r e s u l t s i s the c o n c e n t r a t i o n of t o t a l n o n - e s s e n t i a l amino a c i d s . The t rend for h igher c o n c e n t r a t i o n of t o t a l n o n - e s s e n t i a l amino ac id s d u r i n g p e r i o d 2, cow #1, i s a l s o r e f l e c t e d i n the asparag ine , pheny la l an ine and methionine d a t a . There i s no obvious e x p l a n -a t i o n fo r t h i s t r end du r ing p e r i o d 2 as p roduc t ion was s i m i l a r . There were seasonal d i f f e r e n c e s . P e r i o d 2 fo r cow #1 f e l l between November and mid-January , fo r cow #2, mid-February and the end of A p r i l . Seasonal e f f e c t s may be confounded w i t h animal d i f f e r e n c e s . The decreased urea c o n c e n t r a t i o n dur ing p e r i o d 1 i n d i c a t e s lower urea c y c l e a c t i v i t y o r i n o ther words a decrease i n c a t -abo l i sm of amino a c i d s . Th i s response would be expected i n both cows dur ing p e r i o d 1 as t h i s i s the i n t e r v a l of g r ea t e s t produc-t i o n when amino a c i d s would most l i k e l y be l i m i t i n g . Th i s d i f f e r e n t response i n p e r i o d 1 i s a l s o noted w i t h methionine and t h r eon ine , but not w i t h p h e n y l a l a n i n e . (b) Plasma amino ac id s expressed as a p r o p o r t i o n of the t o t a l e s s e n t i a l or n o n - e s s e n t i a l amino ac id s  I t was f e l t tha t the amino a c i d c o n c e n t r a t i o n expressed as a p r o p o r t i o n o f the t o t a l e s s e n t i a l or n o n - e s s e n t i a l amino a c i d s p rov ides a s e n s i t i v e assessment of the amino a c i d s ta tus of the animal (Hogan e_t a l . , 1968). Whi le t a k i n g i n t o account the change i n c o n c e n t r a t i o n of a s p e c i f i c amino a c i d the change r e l a t i v e to the o ther amino ac id s i s r ecorded . 72 S i g n i f i c a n t d i f f e r e n c e s were noted between cows for v a l i n e , l e u c i n e , h i s t i d i n e and p h e n y l a l a n i n e . These r e s u l t s were s i m i l a r to those repor ted when the amino ac id s were expressed i n terms of micrograms per ml of b lood plasma except tha t pheny la l an ine was s i g n i f i c a n t l y lower i n cow #2. More s i g n i f i c a n t d i f f e r e n c e s between cows were noted w i t h the n o n - e s s e n t i a l amino a c i d s . Besides s e r i n e , g lu tamic a c i d and o r n i t h i n e which were s i m i l a r f o r both methods, g l y c i n e , a l a n i n e and a -amino-n -bu ty r i c e x h i b i t e d s i g n i f i c a n t d i f f e r e n c e s . These r e s u l t s re-emphasize the d i f f e r e n t response o f the two animals to the demands p laced on them. While d i f f e r e n c e s were noted between cows when amino a c i d concen t r a t i ons were expressed as a p r o p o r t i o n of the t o t a l essen-t i a l or n o n - e s s e n t i a l amino a c i d s , more d i f f e r e n c e s were noted between the pe r iods of l a c t a t i o n . The p r o p o r t i o n of methionine and i s o l e u c i n e decreased s i g n i f i c a n t l y over l a c t a t i o n w h i l e the p r o p o r t i o n of l e u c i n e and l y s i n e inc reased s i g n i f i c a n t l y . I t has been shown by s e v e r a l workers tha t the c o n c e n t r a t i o n of a l i m i t i n g amino a c i d i n plasma w i l l remain r e l a t i v e l y cons tant at a lov; l e v e l u n t i l the requirements have been met (Hewitt and Lewis , 1966; S tock land e t aJL., 1970). The l e v e l i n plasma w i l l then i nc rea se markedly . L y s i n e then appears as a l i k e l y suspect as a l i m i t i n g amino a c i d due to i t s low c o n c e n t r a t i o n du r ing the f i r s t t h i r d o f l a c t a t i o n (Table I-2A) and a l s o due to i t s lower p r o p o r t i o n i n plasma du r ing t h i s f i r s t t h i r d of l a c t a t i o n (Table I - 4 A ) . The c o n c e n t r a t i o n and p r o p o r t i o n of l y s i n e inc reases as l a c t a t i o n progresses not because of inc reased supply but because of d imin i shed demand for l y s i n e (Halfpenny e t al_. , 1969). Leucine d o e s r e s p o n d i n a somewhat s i m i l a r manner b u t t h e c o n c e n t r a t i o n o f l e u c i n e i s n o t s i g n i f i c a n t l y , l o w e r i n t h e f i r s t t h i r d o f l a c t a t i o n a l t h o u g h t h e p r o p o r t i o n o f l e u c i n e i n p l a s m a i s s i g n i f -i c a n t l y l o w e r e d i n e a r l y l a c t a t i o n . T h e s i g n i f i c a n t d e c r e a s e i n p r o p o r t i o n o f m e t h i o n i n e a n d i s o l e u c i n e i s a r e f l e c t i o n o f t h e i r l a c k o f c h a n g e i n c o n c e n t r a -t i o n o v e r t h e l a c t a t i o n p e r i o d w h i l e t h e c o n c e n t r a t i o n o f m o s t o f t h e o t h e r e s s e n t i a l a m i n o a c i d s i s i n c r e a s i n g . T h e s e r e s u l t s i n d i c a t e t h a t i n f u t u r e s t u d i e s e m p h a s i s m i g h t be p l a c e d on t h e demands o r r e q u i r e m e n t s o n t h e a n i m a l b y l a c t a t i o n f o r m e t h i o n i n e , i s o l e u c i n e , l e u c i n e and l y s i n e . E x p r e s s i o n o f t h e c o n c e n t r a t i o n o f t h e n o n - e s s e n t i a l a m i n o a c i d s i n p l a s m a a s a p r o p o r t i o n o f t h e t o t a l n o n - e s s e n t i a l a m i n o a c i d s showed s i g n i f i c a n t d i f f e r e n c e s b e t w e e n c o w s . E v e n w i t h t h e s e d i f f e r e n c e s b e t w e e n cows t h e r e w e r e s e v e r a l s i g n i f i c a n t d i f f e r e n c e s b e t w e e n p e r i o d s t h a t w e r e s i m i l a r f o r b o t h c o w s . T h e p r o p o r t i o n s o f a s p a r a g i n e a n d g l u t a m i n e , a l a n i n e a n d o r n i t h i n e w e r e f o u n d t o i n c r e a s e a s l a c t a t i o n p r o g r e s s e d . D u r i n g e a r l y l a c t a t i o n t h e r e i s s t r e s s p l a c e d u p o n t h e a n i m a l f o r s u p p l y o f g l u c o s e a s w e l l a s a m i n o a c i d s . Up t o a maximum o f 60% o f t h e g l u c o s e c a n be d e r i v e d f r o m p r o p i o n a t e ( W i l t r o u t a n d S a t t e r , 1972) a m i n o a c i d s a l s o c o n t r i b u t e up t o 30% o f t h e g l u c o s e ( B l a c k , e t a l . , 1 9 6 8 ) . T h e i m p o r t a n c e o f a l a n i n e a s a g l u c o n e o g e n i c p r e -c u r s o r i n man h a s b e e n e x t e n s i v e l y s t u d i e d b y F e l i g e t a l_ . (1970) . A l a n i n e a p p e a r s t o a c t a s a c a r r i e r o f c a r b o n f r o m t h e m u s c l e mass t o t h e l i v e r . In o r d e r t o p r e v e n t e x c e s s i v e l o s s o f a m i n o "74 a c i d s to g luconeogenes is , a l a n i n e l e v e l s i n plasma drop q u i t e d r a s t i c a l l y i n f a s t i n g an ima l s . I t i s p o s s i b l e tha t a s i m i l a r response causes the depress ion i n a l a n i n e i n e a r l y l a c t a t i o n i n d a i r y cows. Th i s p reven t ion of exces s ive l o s s of amino a c i d s fo r g lucose p roduc t ion has important r a m i f i c a t i o n s . B lack e t a]^. (1970) showed tha t o x i d a t i o n of pheny la l an ine s u p p l i e d 0.4 - 0.9% of the carbon d i o x i d e i n sheep. However tha t amount of carbon d i o x i d e represented 8 - 14% of the pheny la l an ine t u rnove r . Therefore , the r e l a t i v e l y sma l l c o n t r i b u t i o n to g lucose or energy supply may represent dramatic changes i n p recur so r supply for p r o t e i n s y n t h e s i s . The lowered o r n i t h i n e p r o p o r t i o n du r ing p e r i o d 1 r e f l e c t s the lower c o n c e n t r a t i o n of o r n i t h i n e i n the f i r s t t h i r d of l a c t a -t i o n which was a t t r i b u t e d to the l a r g e uptake of o r n i t h i n e by the mammary g l a n d . The lower c o n c e n t r a t i o n of asparagine and glutamine cannot be exp l a ined i n the same terms as Verbeke and Peeters (1965) and Mepham and L i n z e l l (1966); they repor ted sma l l uptakes of these two components by the mammary g l a n d . I t i s most l i k e l y tha t the inc reased g lucose demand p lays a r o l e i n t h e i r lower p r o p o r t i o n of the t o t a l n o n - e s s e n t i a l amino ac id s i n the f i r s t t h i r d of l a c t a t i o n . The inc reased p r o p o r t i o n of g l y c i n e i n the f i r s t t h i r d of l a c t a t i o n aga in i n d i c a t e d inc reased t i s s u e m o b i l i z a t i o n as was d i scussed under expres s ion of amino a c i d s i n terms of mg per m l . 7 5 (3) C o n c e n t r a t i o n o f t h e i n d i v i d u a l m i l k p r o t e i n s o f cow #65002  T h e r e s u l t s o b t a i n e d i n t h i s e x p e r i m e n t a g r e e w i t h t h o s e o f L a r s o n a n d K e n d a l (1957) w h e r e t h e c o n c e n t r a t i o n o f t h e whey-p r o t e i n s was f o u n d t o d e c r e a s e a s l a c t a t i o n p r o g r e s s e d w h i l e t h o s e o f t h e c a s e i n s i n c r e a s e d . T h e i n c r e a s e d c o n c e n t r a t i o n o f m i l k p r o t e i n n o t e d i n t h e l a t e r s t a g e s o f l a c t a t i o n c a n be a t t r i b u t e d t o i n c r e a s e d c o n c e n t r a t i o n o f t h e c a s e i n s . To s u m m a r i z e t h i s p h a s e o f t h e s t u d y , b o t h cows h a d s i m i l a r l a c t a t i o n s when c o n s i d e r i n g y i e l d o f m i l k a n d m i l k c o m p o n e n t s . When t h e l a c t a t i o n p e r i o d was d i v i d e d i n t o t h r e e e q u a l p e r i o d s t h e y i e l d o f m i l k , p r o t e i n a n d l a c t o s e d e c r e a s e d a s t h e l a c t a t i o n p r o g r e s s e d . T h e c o n c e n t r a t i o n o f p r o t e i n i n c r e a s e d a n d t h e c o n c e n t r a t i o n o f l a c t o s e t e n d e d t o d e c r e a s e f r o m p e r i o d 1 t o p e r i o d 3 . T h e a b o v e d i f f e r e n c e s i n p r o d u c t i o n w e r e r e f l e c t e d i n a l t e r a -t i o n s i n t h e c o n c e n t r a t i o n o f s e v e r a l p l a s m a f r e e a m i n o a c i d s . T h e i n c r e a s e d c o n c e n t r a t i o n o f g l y c i n e i n p l a s m a d u r i n g t h e p e r i o d o f g r e a t e s t p r o d u c t i o n i s i n d i c a t i v e o f t h e m o b i l i z a t i o n o f t i s s u e p r o t e i n s t o s u p p l y t h e r e q u i r e m e n t s f o r m i l k p r o t e i n s y n t h e s i s . A s was m e n t i o n e d i n t h e l i t e r a t u r e r e v i e w , t h e r u m i n a n t w i t h i t s u n i q u e m e t h o d o f r e l y i n g o n rumen m i c r o b i a l p r o t e i n t o m e e t i t s p r o t e i n n e e d s c o u l d p o s s i b l y b e p r o t e i n d e f i c i e n t a t t i m e s o f m a x i m a l p r o t e i n s y n t h e s i s . T h e i n a d e q u a c y o f t h e r u m i n a n t system i n s u p p l y i n g s u f f i c i e n t p r o t e i n i s a l s o s u g g e s t e d b y t h e r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s t o n o n - e s s e n t i a l a m i n o a c i d s . T h i s r a t i o i s f o u n d t o i n c r e a s e o v e r l a c t a t i o n i n d i c a t i n g t h a t s u p p l y 76 o f a m i n o a c i d s i s m o r e a b l e t o m e e t t h e r e q u i r e m e n t s o f t h e a n i m a l a s p r o d u c t i o n d r o p s . C e r t a i n a m i n o a c i d s a p p e a r t o be i n s h o r t e r s u p p l y o r g r e a t e r demand t h a n o t h e r a m i n o a c i d s . T h e p e r c e n t a g e o f t h e t o t a l e s s e n t i a l a m i n o a c i d s made up by l y s i n e and l e u c i n e i n -c r e a s e d a s l a c t a t i o n p r o g r e s s e d i n d i c a t i n g a g r e a t e r u t i l i z a -t i o n o f t h e s e two a m i n o a c i d s d u r i n g p e a k p r o d u c t i o n . T h e d r o p i n m e t h i o n i n e a n d i s o l e u c i n e d u r i n g t h i s same i n t e r v a l m i g h t s u g g e s t t h a t t h e i r i m p o r t a n c e i s n o t a s g r e a t d u r i n g p e a k p r o -d u c t i o n . 77 2 . E x p e r i m e n t I I - A b o m a s a l i n f u s i o n o f c a s e i n a n d a m i n o a c i d s i n t h e l a c t a t i n g cow a n d t h e e f f e c t s o n p l a s m a a m i n o a c i d s a n d m i l k c o m p o s i t i o n  a) M a t e r i a l s and m e t h o d s (1) A n i m a l A H o l s t e i n cow #68127, i n h e r s e c o n d l a c t a t i o n was s u r g i c a l l y f i t t e d w i t h an a b o m a s a l c a n n u l a d u r i n g t h e s i x t h week o f h e r l a c t a t i o n . T h e c a n n u l a was made up o f 2 mm i d s i l a s t i c t u b i n g . T h e c a n n u l a f i x e d w i t h a p p r o p r i a t e c o l l a r s was s u t u r e d i n t o t h e abomasum. T h e t u b i n g was p a s s e d o u t t h r o u g h t h e a b d o m i n a l w a l l . T h e abomasum was t h e n s u t u r e d t o t h e abdomen i n t h e r e g i o n s u r -r o u n d i n g t h e c a n n u l a . T h e c a n n u l a was p a s s e d u n d e r t h e s k i n t o t h e m i d - b a c k l i n e . Two weeks w e r e a l l o w e d f o r r e c o v e r y a l t h o u g h i t was n o t e d t h a t t h e r e was o n l y a s l i g h t d r o p i n m i l k p r o d u c -t i o n o n t h e d a y o f t h e o p e r a t i o n , w h i c h f o l l o w e d a 2 4 - h o u r f a s t . M i l k p r o d u c t i o n was b a c k t o n o r m a l w i t h i n 24 h o u r s . T h e a n i m a l was f e d a c c o r d i n g t o N a t i o n a l R e s e a r c h C o u n c i l S t a n d a r d s f o r D a i r y C a t t l e ( 1 9 7 1 ) . T h e r a t i o n i n c l u d e d a l f a l f a h a y , b e e t p u l p a n d a c o m m e r c i a l d a i r y c o n c e n t r a t e (16%). D i e t a r y a l l o w a n c e s w e r e a d j u s t e d a t t h e b e g i n n i n g o f e a c h 1 0 - d a y r e c o v e r y p e r i o d . (2) S a m p l i n g T h e a n i m a l was i n f u s e d w i t h . c a s e i n and a v a r i e t y o f a m i n o a c i d s o l u t i o n s ( s e e T a b l e I I - l ) . E a c h t r i a l c o n s i s t e d o f one d a y p r e - i n f u s i o n a n d s i x d a y s c o n s t a n t i n f u s i o n . T e n d a y s w e r e a l l o w e d f o r a r e c o v e r y p e r i o d b e f o r e t h e n e x t t r i a l b e g a n . E a c h TABLE II-l INFUSION SCHEDULE - GMS OF MATERIAL INFUSED PER 24 HOURS. INFUSION NUMBER 4 5 6 7 8 9 10 SODIUM CASEINATE 400 THREONINE 11.8 11.8 VALINE 20.6 20.6 20.6 METHIONINE 13.0 10 13.0 13.0 13.0 40.0 ISOLEUCINE 26.4 26.4 26.4 26.4 26.4 LEUCINE 33.6 33.6 33.6 33.6 PHENYLALANINE 18.3 18.3 LYSINE - HCl 48.9 48.9 48.9 HISTIDINE 14•2 ARGININE - HCl 19.3 TRYPTOPHAN 8.4 co d a y f r e s h i n f u s a t e was made u p . T h e a m o u n t s o f a m i n o a c i d s u s e d w e r e d e s i g n e d t o s u p p l y t h e same amount o f t h a t a m i n o a c i d a s w o u l d be p r e s e n t i n 400 g r a m s o f a-S.^ - C a s e i n . T h e m a t e r i a l s w e r e d i s s o l v e d i n 4 l i t r e s o f d i s t i l l e d w a t e r a n d s u b s e q u e n t l y i n f u s e d i n t o t h e cow u s i n g a p e r i s t a l t i c pump. M i l k s a m p l e s w e r e t a k e n a t e a c h m i l k i n g . J u g u l a r b l o o d s a m p l e s w e r e t a k e n a t 2 : 0 0 p . m . o n e a c h d a y o f t h e t r i a l . T h e b l o o d s a m p l e was t a k e n a p p r o x i m a t e l y 7 h o u r s a f t e r t h e l a s t f e e d i n g . (3) A n a l y s e s A n a l y s i s o f m i l k f o r f a t , p r o t e i n a n d l a c t o s e was d o n e a s d e s c r i b e d i n E x p e r i m e n t I. P l a s m a f r e e a m i n o a c i d s w e r e d e t e r m i n e d a s i n E x p e r i m e n t I e x c e p t t h a t a l l a n a l y s e s e m p l o y e d t h e H i t a c h i KLA3B a m i n o a c i d a n a l y z e r . P l a s m a g l u c o s e was d e t e r m i n e d u s i n g t h e g l u c o s e o x i d a s e m e t h o d . ^ T h e s t a t i s t i c a l m o d e l u s e d was a f a c t o r i a l d e s i g n w i t h i n f u s i o n , p e r i o d a n d t h e i r i n t e r a c t i o n e f f e c t s b e i n g a n a l y z e d . T h e m o d e l was y . . . = y + x. + P . + (XP) . . + e . J i } k I j iO l j k W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n , F r e e h o l d , N . J . G l u c o s t a t ; p r e p a r e d r e a g e n t s f o r t h e e n z y m a t i c d e t e r m i n a t i o n o f g l u c o s e . 80 Where: y ^ . j , = the k f c ^ obse rva t i on of the p e r i o d and the 1 - ] i t n i n f u s i o n y = the o v e r a l l mean th = the e f f e c t of the i i n f u s i o n t h Pj = the e f f e c t of the j p e r i o d ( X P ) . . = the e f f e c t of the i j XP subc lass a f t e r the i i J average e f f e c t s of X and P have been removed e —^ = random e r r o r . Assumed to be independent and -1 normal ly d i s t r i b u t e d . S t a t i s t i c a l methods were s i m i l a r to those used i n E x p e r i -ment I . b) Meth ion ine i n f u s i o n s (1) Resu l t s (a) M i l k and m i l k compos i t ion Methionine was in fused alone or i n combinat ion w i t h o ther e s s e n t i a l amino ac id s i n s i x separate i n f u s i o n s (see Table I I - l ) . Each i n f u s i o n was d i v i d e d i n t o three pe r iods 1) p r e i n f u s i o n 2) the f i r s t 2 days of i n f u s i o n and 3) the l a s t 2 days of i n f u s i o n . Dur ing i n f u s i o n #10 data was u n a v a i l a b l e fo r p e r i o d 3. There were s i g n i f i c a n t d i f f e r e n c e s between i n f u s i o n s i n each category of m i l k compos i t ion or p roduc t ion measured (Table I I I -1A) . T o t a l m i l k p roduc t ion decreased s i g n i f i c a n t l y as l a c t a t i o n progressed . T o t a l p r o t e i n p roduc t ion fo l l owed the same p a t t e r n a l though there were some s i g n i f i c a n t d e v i a t i o n s which might be TABLE I I I - 1 A METHIONINE INFUSIONS MILK AND MILK COMPOSITION - INFUSION EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 1 2 5 6 7 10 MILK (kg) S.E. 21.507 ** 2.529 a .102 1.546 b .102 .450° .102 - 1.402 d .102 - 1.631 d .102 - 1.493 d .146 PROTEIN (gm) S.E. 675.00 ** 69.85 a 3.64 69.38 a 3.64 24.65 b 3.64 -61.04 d 3.64 -61.68 d 3.64 -41.17° 5.22 PROTEIN (%) S.E. 3.136 **_ ab • .037 .018 .094° .018 .049° .018 - .082 b .018 ab - .050 .018 .026 a° .025 LACTOSE (gm) S.E. 1087.66 ** 1 3 8 . l l a 5.26 72.74 b 5.26 31.11° 5.26 -83.22 d 5.26 -79.52 d 5.26 -79.21 d 7.55 LACTOSE (%) S.E. 5.054 * .044 a .017 - .022° .017 . 0 3 9 * .017 - .059° .017 .014* .017 - .Olff** .025 * P <.05. ** P <.01. a,b,c,d Constants w i t h i n rows w i t h the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t . TABLE I l l - I B METHIONINE INFUSIONS MILK AND MILK COMPOSITION - PERIOD EFFECTS LEAST SQUARES MEAN 1 S.E. PERIOD NUMBER 2 S.E. 3 S.E. MILK (kg) 21.507 ** .072 a .073 .26 l a .062 - .332b .065 PROTEIN (gm) 675.00 **_ -7.74 a 2.61 10.78b 2.21 - 3.05a 2.31 PROTEIN (%) 3.136 *_ • .044 a .013 .o io b .011 .034b .011 LACTOSE (gm) 1087.66 ** 8.04 a 3.77 8.11 a 3.20 -16.15 b 3.33 LACTOSE (%) 5.054 .018 .012 - .023 .010 .006 .011 * P < .05. ** P < .01. a,b = Constants within rows with the same superscripts are not significantly different. 83 TABLE III-1C METHIONINE INFUSIONS MILK AND MILK COMPOSITION - SIGNIFICANT INFUSION X PERIOD INTERACTION TERMS LEAST SQUARE CONSTANTS MILK (kg) S . E . PROTEIN (gm) S . E . LACTOSE (gm) S . E . LEAST SQUARES MEAN 21.507 675.00 1087.66 IN 1 IN 1 IN 1 P P P I 1 2 3 2.983a, 2.418f° 2.188 .209 .160 .184 69.627* 75.443?° 64.480 7.44 5.72 6.54 177.54a 127.59° 109.19 10.763 8.264 9.46 IN 2 IN 2 IN 2 P P P 1 2 3 be 2 - 0 7 3 c d 1.508°a 1.058° .209 .160 .184. 53.840^ 86.826 . 67.488aD 7.44 5.72 6.54 97.59° 63.76 58.87° 10.763 8.264 9.46 IN 5 IN 5 IN 5 P P P 1 ' 2 3 .033^ 1.168a .148e 1.209 .160 .184 - 6.961? 56.349° 24.574C 7.44 5.72 6.54 cd 29.42 C Q 62.83° 1.09 10.763 8.264 9.46 IN 6 IN 6 IN 6 P P P 1 2 3 -1.327^g - .872^ -2.007n .209 .160 .184 -56.361^fg -59.94l r g -69.821g 7.44 5.72 6.54 ef - 86.57e - 61.73^ -101.38 10.763 8.264 9.46 IN 7 IN 7 IN 7 P P P 1 2 3 -2.236$ -1.102*? -1.557 g h .209 .160 .184 -77.451g -46.802^ 60.871° 7.44 5.72 6.54 -115.12f - 56.16e - 67.29e 10.763 8.264 9.46 IN 10 IN 10 P P 1 2 -1.097^ -1.557 f g h .217 .217 -29.121^fg -50.172 e 7.74 7.74 - 54.63e - 95.76et 11.20 11.20 a , b , c , d , e , f , g , h Constants w i t h i n columns wi th the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t . 1 I N 1 P 1 In fus ion number and p e r i o d number. 84 a t t r i b u t e d t o e f f e c t s o f t h e i n f u s i o n s . T h i s e f f e c t o f i n f u s i o n i s m o r e p r o n o u n c e d when t h e p e r c e n t a g e p r o t e i n i n m i l k i s s t u d i e d . The g r e a t e s t c o n c e n t r a t i o n o f p r o t e i n o c c u r r e d when c a s e i n , a l l t h e e s s e n t i a l amino a c i d s , o r l a r g e a m o u n t s o f m e t h i o n i n e a r e i n f u s e d . P e r i o d s ( p r e - i n f u s i o n , f i r s t h a l f o f i n f u s i o n , l a s t h a l f o f i n f u s i o n ) showed s i g n i f i c a n t e f f e c t s f o r a l l c o m p o n e n t s e x c e p t l a c t o s e c o n c e n t r a t i o n i n m i l k ( T a b l e I l l - I B ) . M i l k p r o t e i n p e r -c e n t i n c r e a s e d a s e a c h i n f u s i o n p r o g r e s s e d . M i l k p r o d u c t i o n ( k g ) , m i l k p r o t e i n p r o d u c t i o n (gm) a n d l a c t o s e p r o d u c t i o n (gm) h a d s i g n i f i c a n t i n t e r a c t i o n t e r m s s u g g e s t i n g t h a t t h e p e r i o d r e s p o n s e f o r e a c h i n f u s i o n was n o t s i m i l a r ( T a b l e I I I-1C). When t h e s i g n i f i c a n t i n t e r a c t i o n t e r m f o r k g m i l k p r o d u c e d was s t u d i e d t h e r e a p p e a r e d t o be an e v e n d e c l i n e f o r e a c h p e r i o d o v e r t h e r a n g e o f i n f u s i o n e x c e p t f o r i n f u s i o n #7 p e r i o d 1, i n w h i c h t h e r e was a s i g n i f i c a n t d r o p b e l o w t h e p e r i o d 1 p r o d u c t i o n f o r i n f u s i o n s #6 a n d #10. I n g e n e r a l t h e r e was a d e c r e a s e i n m i l k p r o d u c t i o n f r o m p e r i o d 1 t o p e r i o d 3 . However w i t h i n f u s i o n #5 and #7 t h e p r o d u c t i o n d u r i n g p e r i o d 3 was s i m i l a r t o p r e -i n f u s i o n p r o d u c t i o n . P e r i o d 2 p r o d u c t i o n was e l e v a t e d o v e r p r e -i n f u s i o n p r o d u c t i o n i n b o t h c a s e s . When t h e p r o t e i n p r o d u c t i o n was b r o k e n down i n t o i n f u s i o n by p e r i o d s t h e r e s u l t s w e r e q u i t e d i f f e r e n t f r o m t h e m i l k d a t a . When j u s t t h e p r e - i n f u s i o n p e r i o d was s t u d i e d t h e m i l k p r o t e i n p r o d u c t i o n d e c l i n e d f r o m i n f u s i o n #1 t o i n f u s i o n #10. However i n f u s i o n e f f e c t s c o u l d be s e e n when p e r i o d 2 and p e r i o d 3 r e s u l t s w e r e l o o k e d a t f o r a l l i n f u s i o n s . In b o t h p e r i o d 2 a n d 3 t h e 85 lowest p r o d u c t i o n of p r o t e i n o c c u r r e d w i t h i n f u s i o n #6. Infusions #1 and #2 showed the h i g h e s t p r o d u c t i o n i n p e r i o d 2 w h i l e i n f u s i o n s #1, #2 and #7 showed the g r e a t e s t p r o t e i n p r o d u c t i o n i n p e r i o d 3. In a l l i n f u s i o n s , p r o t e i n p r o d u c t i o n was equal to or h i g h e r than p r e - i n f u s i o n p r o d u c t i o n . With i n f u s i o n #5 and #7 there was a s i g n i f i c a n t i n c r e a s e i n p r o t e i n p r o d u c t i o n d u r i n g p e r i o d 2 and 3 above p r e - i n f u s i o n p r o d u c t i o n . I n f u s i o n #2 r e s u l t e d i n increased p r o t e i n p r o d u c t i o n i n p e r i o d 2, but a d e c l i n e to an i n t e r m e d i a t e n o n - s i g n i f i c a n t l e v e l of p r o d u c t i o n i n p e r i o d 3. There was a g e n e r a l d e c l i n e i n l a c t o s e p r o d u c t i o n as the experiment progressed from i n f u s i o n #1 to #10. However d u r i n g p e r i o d 1 and p e r i o d 3 the lowest p r o d u c t i o n of l a c t o s e d i d not occur d u r i n g i n f u s i o n #10 but d u r i n g #7 and #6 r e s p e c t i v e l y . In g e n e r a l the p r o d u c t i o n of l a c t o s e decreased as each i n f u s i o n progressed. During i n f u s i o n #7 the p r o d u c t i o n i n c r e a s e d s i g n i f -i c a n t l y above p r e - i n f u s i o n l e v e l s d u r i n g p e r i o d 2 and 3. (b) Plasma amino a c i d s i ) Plasma amino a c i d s expressed as micrograms per ml of plasma There were s i g n i f i c a n t d i f f e r e n c e s between i n f u s i o n s f o r the e s s e n t i a l amino a c i d s v a l i n e , methionine, i s o l e u c i n e and l e u c i n e (Table I I I - 2 A ) . V a l i n e c o n c e n t r a t i o n s were e l e v a t e d d u r i n g the i n f u s i o n s o f the branched c h a i n amino a c i d s and methionine (#6). Intermediate l e v e l s were observed when #2 (casein) and #7 (threonine, methionine) were i n f u s e d . The c o n c e n t r a t i o n of methionine i n TABLE ILT-2A METHIONINE INFUSIONS ESSENTIAL AMINO ACIDS - u g/ml BLOOD PLASMA - INFUSION EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 1 2 5 6 7 10 THREONINE 11.371 - .226 -1.409 - .509 - .058 1.540 .662 S.E. .632 .632 .632 .632 .632 .907 VALINE 43.731 **-14.083a -1.106b 9.655C cd 8.193 1.603" -4.263* S.E. 2.245 2.245 2.245 2.245 2.245 3.223 METHIONINE 8.123 **- 1.254a -1.739a - .774a - .353a - .626a 4.747b S.E. .556 .556 .556 .556 .556 .798 ISOLEUCINE 19.415 **- 5.382a - .600b 3.15^°° 4.555C - .016b -1.710* S.E. 1.278 1.278 1.278 1.278 1.278 i.835 LEUCINE 23.966 **- 8.944a -1.40^ 4.870cd 6.365° bd .874DQ -1.755b S.E. 1.374 1.374 1.374 1.374 1.374 1.973 PHF2TYLALANINE 9.204 - 1.419 .499 .516 - .001 .794 - .388 S.E. .474 .474 .474 .474 .474 .680 LYSINE 18.025 - 2.485 .745 1.753 1.183 3.973 -5.170 S.E. 1.693 1.693 1.693 1.693 1.693 2.430 HISTIDINE 13.897 .368 - .737 .723 .980 .358 -1.692 S.E. .758 .758 .758 .758 .758 1.088 ARGININE 16.900 - .337 -1.150 - .128 2.884 2.647 -3.917 S.E. 1.225 1.225 1.225 1.225 1.225 1.758 * P <.05. ** P <.0L a,b,c,d Constant within rows with the same superscripts are not significantly different. ca CT) TABLE III-2B METHIONINE INFUSIONS ESSENTIAL AMINO ACIDS u g/ml BLOOD PLASMA - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER ^ 1 S.E. 2 S.E. 3 S.E. THREONINE 11.371 - .296 .452 .388 .384 - .093 .400 VALINE 43.781 *-4.746a 1.608 2.199b 1.364 2.547b . 1.422 METHIONINE 8.123 **-2.53l a .397 1.881° .338 .650° .352 ISOLEUCINE 19.415 -2.070 .915 .961 .777 1.109 .810 LEUCINE 23.966 -2.383 .984 .951 .835 1.432 .871 PHENYLALANINE 9.204 - .243 .339 .690 .288 - .447 .300 LYSINE 18.025 - .188 1.212 1.018 1.029 - .830 1.073 HISTIDINE 13.897 .136 .543 - .465 .480 .329 .460 ARGININE 16.900 .767 .877 - .073 .744 - .693 .776 * P < .05. ** P <.01. a,b = Constants within rows with the same superscripts are not signficantly different. 88 plasma remained r e l a t i v e l y c o n s t a n t except when methionine was i n f u s e d a t three times the u s u a l amount as i n i n f u s i o n #10. I s o l e u c i n e and l e u c i n e c o n c e n t r a t i o n s f o l l o w e d a p a t t e r n s i m i l a r to t h a t of v a l i n e . There were s i g n i f i c a n t d i f f e r e n c e s between p e r i o d s f o r v a l i n e and methionine (Table I I I - 2 B ) . In both cases the p r e -i n f u s i o n l e v e l was s i g n i f i c a n t l y lower than the l e v e l s d u r i n g the two i n f u s i o n p e r i o d s . In g e n e r a l there was an i n c r e a s e i n methionine c o n c e n t r a -t i o n from the p r e - i n f u s i o n l e v e l as each i n f u s i o n progressed (Table I I I - 3 C ) . However o n l y d u r i n g the two i n f u s i o n s where methionine alone was i n f u s e d d i d the d i f f e r e n c e s become s i g n i f -i c a n t . I n f u s i o n of t h r e o n i n e , methionine, p h e n y l a l a n i n e and l y s i n e r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e i n methionine i n the second p e r i o d . However d u r i n g the t h i r d p e r i o d methionine c o n c e n t r a t i o n s dropped t o p r e - i n f u s i o n l e v e l s . There were s e v e r a l s i g n i f i c a n t d i f f e r e n c e s between i n f u s i o n s when the c o n c e n t r a t i o n s o f the n o n - e s s e n t i a l amino a c i d s i n blood plasma were s t u d i e d (Table I I I - 3 A ) . T a u r i n e , c i t r u l l i n e , g l y c i n e , a l a n i n e and a-amino-n-butyric a c i d were found to vary s i g n i f i c a n t l y between i n f u s i o n s . Taurine c o n c e n t r a t i o n s i n plasma were i n c r e a s e d s i g n i f i c a n t l y i n the l a t e r stages of the experiment except f o r i n f u s i o n #7 where a n o n - s i g n i f i c a n t i n t e r -mediate l e v e l was observed. C i t r u l l i n e a l s o appeared to f o l l o w the same p a t t e r n except t h a t i n i n f u s i o n #10 there was an i n t e r -mediate n o n - s i g n i f i c a n t v a l u e . The s i g n i f i c a n t l y lowest value was observed during i n f u s i o n #2 ( c a s e i n ) . I t should be noted 89 TABLE III-3A METIIIONINK INFUSIONS NON-ESSENTIAL AMINO ACIDS - u g/ml BLOOD PLASMA - INFUSION EFFECTS LEAST SQUARES MEAN LEAPT SQUARES CONSTANTS - INFUSION NUMBERS 2 5 6 7 10 TAURINE S.E. 7.172 *- 1.505 .531 - 1.444 .531 .710 .'531 .58 4 . 531 .122 .531 ab 1.777 .7C3 UREA S.E. 164.21 -46.18 15.36 31. 21 15.36 18.64 15. 36 7.55 15.36 - 2.01 15.36 - 9.21 22.05 SERINE S.E. 11.110 .745 .400 1.156 .400 . 075 .400 . 062 .400 . 588 . 400 .315 . 575 ASPARAGINE S.E. 50.035 4 . 391 2.757 . 043 2.757 2.418 2.757 1.834 2.757 4 .433 2 . 757 4 . 254 3 .958 PROLINE S.E. 10.484 1.444 .827 . 604 .827 .24 5 .827 1.149 .827 1.082 .827 .427 1.187 GLUTAMIC ACID S.E. 6.969 *- .669 .462 . 222 .462 .394 .462 .798 .462 .424 .462 2.508 . 663 CITRULLINE S.E. 16.979 **- 2.466 .819 - 5.512 . 819 3.694 . 819 3 .14 9 .819 .978 .819 GLYCINE S.E. 29.091 ** 6.937 .957 2.049 .957 cd 3.721 .957 .154 .957 .933 .957 c d ALANINE S.E. 19.284 * 1.816° .771 .002 .771 ab 2.622 .771 . 964 .771 ab 1.082 .771 n-AMINO-M-BUTYRIC ACID S.E. .992 . 041 .047 ab .111" . 047 .135 .047 . 078 .047 be .052 .047 abc CYSTINE S.E. 4.708 .072 . 314 .258 .314 .473 .314 .025 .314 .145 .314 TYROSINE S.E. 8.866 .779 .494 .793 .494 .093 .494 .211 .494 . 118 .494 ORNITHINE S.E. 6.883 .591 .564 .634 .564 .602 .564 .999 .564 1.052 .564 * P < . 05. ** P <.01. a,b,c,d C o n s t a n t s w i t h i n rows w i t h t h e same s u p e r s c r i p t s a r e n o t s i g n i f i c a n t l y d i f f e r e n t . 90 t h a t t h e two m e t h i o n i n e i n f u s i o n s #1, a n d #10 r e s u l t e d i n t h e n e x t l o w e s t c o n c e n t r a t i o n s o f c i t r u l l i n e . G l y c i n e c o n c e n t r a t i o n s w e r e h i g h e s t w i t h l o w l e v e l m e t h i o n i n e i n f u s i o n (#1). I n f u s i o n #5 ( a l l t h e e s s e n t i a l a m i n o ac ids) r e s u l t e d i n s i g n i f i c a n t l y l o w e r g l y c i n e l e v e l s t h a n when #1 ( low l e v e l m e t h i o n i n e ) o r #6 (branched c h a i n a m i n o a c i d s a n d m e t h i o n i n e ) w e r e i n f u s e d . A l a n i n e c o n c e n t r a t i o n i n p l a s m a was l o w e s t when a l l t h e e s s e n t i a l a m i n o a c i d s w e r e i n f u s e d . T h e h i g h e s t a l a n i n e v a l u e s w e r e o b s e r v e d w i t h i n f u s i o n #1 ( low l e v e l m e t h i o n i n e ) ' a n d #7 ( t h r e o n i n e , m e t h i o n i n e , p h e n y l a l a n i n e a n d l y s i n e ) . T h e r e w e r e b e t w e e n p e r i o d d i f f e r e n c e s f o r t a u r i n e , s e r i n e a n d g l y c i n e ( T a b l e I I I-3B) . T a u r i n e c o n c e n t r a t i o n was f o u n d t o i n c r e a s e a s e a c h i n f u s i o n p r o c e e d e d w h e r e a s s e r i n e a n d g l y c i n e c o n c e n t r a t i o n s i n p l a s m a d e c r e a s e d f r o m t h e p r e - i n f u s i o n l e v e l s . S i g n i f i c a n t i n t e r a c t i o n t e r m s w e r e n o t e d f o r a - a m i n o - n -b u t y r i c a c i d a n d c y s t i n e ( T a b l e I I I-3C) . a - a m i n o - n - b u t y r i c a c i d g e n e r a l l y i n c r e a s e d a s e a c h i n f u s i o n p r o g r e s s e d w i t h t h e h i g h e s t c o n c e n t r a t i o n b e i n g f o u n d i n p e r i o d t h r e e . When m e t h i o n i n e was i n f u s e d a t a l o w l e v e l (#1) t h e c o n c e n t r a t i o n o f a - a m i n o - n - b u t y r i c a c i d r e t u r n e d t o p r e - i n f u s i o n l e v e l s i n p e r i o d t h r e e . I n i n f u s i o n #7 ( t h r e o n i n e , m e t h i o n i n e , p h e n y l a l a n i n e a n d l y s i n e ) t h e c o n c e n -t r a t i o n i n c r e a s e s o f a - a m i n o - n - b u t y r i c w e r e m o r e p r o n o u n c e d i n e a c h p e r i o d . C y s t i n e i n g e n e r a l d i d n o t v a r y o v e r the. t h r e e p e r i o d s o f e a c h i n f u s i o n . In i n f u s i o n #1 ( low l e v e l m e t h i o n i n e ) c y s t i n e l e v e l s w e r e s i g n i f i c a n t l y d i f f e r e n t b e t w e e n p e r i o d s two a n d t h r e e . T h e t o t a l c o n c e n t r a t i o n o f e s s e n t i a l a m i n o a c i d s g e n e r a l l y 91 TABLE III-3B METHIONINE INFUSIONS NON-ESSENTIAL AMINO ACIDS \i g/ml BLOOD PLASMA - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS MEAN PERIOD NUMBERS 1 2 3 TAURINE S.E. 7.172 *- .859 a .380 - .390 a .322 1.248b .337 UREA S.E. 164.21 - 6.38 11.00 4.92 9.33 1.46 9.73 SERINE S.E. 11.110 * .737 a .286 - .036* .254 - .701 b .243 ASPARAGINE S.E. 50.035 - .016 1.973 -1.312 1.675 1.329 1.747 PROLINE S.E. 10.484 .594 .592 .781 .502 -1.375 .524 GLUTAMIC ACID S.E. 6.969 .279 .330 - .013 .280 - .266 .292 CITRULLINE S.E. 16.979 - .516 .586 - .315 .497 .831 .519 GLYCINE S.E. 29.091 ** 3.069 a .684 -2.222 b .581 - .847 b .606 ALANINE S.E. 19.284 .569 .552 .578 .418 -1.147 .488 a-AMINO-N-BUTYRIC ACID S.E. • .992 - .089 .034 .039 .029 .050 .299 CYSTINE S.E. 4.708 - .315 .225 .034 .191 .281 .199 TYROSINE S.E. 8.866 - .222 .354 .762 .300 - .540 .313 ORNITHINE S.E. 6.883 - .196 .404 .186 .342 .011 .358 * P <.05. ** P <.01. a,b Constants within rows with the same superscripts are not significantly different. 9 2 TABLE III -3C METHIONINE INFUSIONS AMINO ACIDS u g/ml BLOOD PLASMA - SIGNIFICANT INFUSION X PERIOD INTERACTION TERMS LEAST SQUARES CONSTANTS METHIONINE ct-AMINO-N-BUTYRIC ACID CYSTINE LEAST SQUARES 8 . 1 2 3 MEAN S.E. . 9 9 2 S.E. 4 . 7 0 8 S.E. IN 1 P r IN 1 P 2 IN 1 P 3 IN 2 P 1 IN 2 P 2 IN 2 P 3 IN 5 P 1 IN 5 P 2 IN 5 P 3 IN 6 P 1 IN 6 P 2 IN 6 P 3 IN 7 P 1 IN 7 P 2 IN 7 P 3 IN 10 P 1 IN 10 P 2 - 3 . 7 2 3 - . 3 6 3 . 3 2 2 - 2 .903 ! - . 178 - 2 . 1 3 8 Ibcde |bcd de bcde cde 2 ' 4 8 3 S e .oi2r~r > 1 4 ^ c d - 1 . 9 7 3 - .188; 1 .102 bcde bcde be 2 . 5 2 2 p d e . 9 3 2 D C a e - 1 .583 1 0 . 4 2 7 c bcde 1 .137 . 8 7 3 . 999 1 .137 . 8 7 3 . 999 1 .137 . 8 7 3 . 999 1 .137 . 8 7 3 . 999 1 .137 .873 . 9 9 9 1 .183 1 .183 " .441? • 1 6 3 e f - . 3 9 5 .096 .074 . 0 8 5 - 1 1 be . 708 . 1 3 8 C . 6 3 2 a . 6 4 3 . 4 9 3 . 5 6 5 . 1 8 8 ° . 0 2 8 ° ° . 096 . 074 . 0 8 5 1 • 4 0 8 S C . 0 3 2 ^ . 643 . 4 9 3 . 5 6 5 - .037P1 . 0 7 3 ° ° . 096 .074 . 0 8 5 - 1 - 1 . 2 2 8 ° . 0 1 8 ° . 917 . 6 4 3 . 4 9 3 . 5 6 5 - ' 8 8 9 rS - .022^ - . 1 2 2 O T . 096 . 074 . 0 8 5 • 1 8 1 £ • 8 5 7 a D C . 9 6 3 C . 643 . 4 9 3 . 5 6 5 -1.2075 - . 2 9 7 d e . 5 5 1 a . 096 .074 . 0 8 5 4 5 2 ^ . 3 2 8 a D C . 6 4 3 . 4 9 3 . 5 6 5 " -Q59t .066°° . 1 0 0 . 1 0 0 - . 668 .668 a,b,c,d,e,f,g,h Constants within columns with the same superscripts are not significantly different. "^IN 1 P I Infusion number and period number. showed an i n c r e a s e a s t h e e x p e r i m e n t p r o c e e d e d ( T a b l e I I I - 4 A ) . T h e r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s t o t o t a l n o n - e s s e n t i a l a m i n o a c i d s d e c l i n e d d u r i n g t h e i n f u s i o n s o f m e t h i o n i n e a l o n e ( i n f u s i o n s #1 a n d # 1 0 ) . T h e r a t i o a l s o showed a p e r i o d r e s p o n s e by i n c r e a s i n g a b o v e p e r i o d 1 l e v e l s d u r i n g p e r i o d s 2 a n d 3 ( T a b l e I I I - 4 B ) . T h e r a t i o o f g l y c i n e t o t h e b r a n c h e d c h a i n a m i n o a c i d s was h i g h e s t d u r i n g t h e two m e t h i o n i n e i n f u s i o n s . However t h e p e r i o d r e s p o n s e showed a d e c l i n e d u r i n g p e r i o d s 2 and 3 o f e a c h m e t h i o n i n e i n f u s i o n . i i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s a p e r -c e n t a g e o f t o t a l e s s e n t i a l o r n o n -e s s e n t i a l a m i n o a c i d s E s s e n t i a l a m i n o a c i d s e x p r e s s e d a s a p e r c e n t a g e o f t h e t o t a l e s s e n t i a l a m i n o a c i d s w e r e s i g n i f i c a n t l y d i f f e r e n t b e t w e e n i n f u s i o n s f o r a l l t h e e s s e n t i a l a m i n o a c i d s e x c e p t l y s i n e ( T a b l e I I I - 5 A ) . T h r e o n i n e , m e t h i o n i n e , p h e n y l a l a n i n e a n d a r g i n i n e h a d s i g n i f i c a n t i n f u s i o n x p e r i o d i n t e r a c t i o n t e r m s a n d w i l l be d i s c u s s e d l a t e r . V a l i n e was p r e s e n t i n t h e h i g h e s t p r o p o r t i o n s when c a s e i n ( i n f u s i o n # 2 ) , a l l t h e e s s e n t i a l s ( i n f u s i o n # 5 ) , m e t h i o n i n e and t h e b r a n c h e d c h a i n a m i n o a c i d s ( i n f u s i o n #6) o r m e t h i o n i n e a t t h r e e t i m e s t h e n o r m a l d o s e ( i n f u s i o n #10 ) w e r e i n f u s e d . T h e l o w e s t p e r c e n t a g e o f v a l i n e was o b s e r v e d when m e t h i o n i n e was i n f u s e d a t l o w d o s a g e s ( i n f u s i o n # 1 ) . I n f u s i o n e f f e c t s f o r i s o l e u c i n e and l e u c i n e w e r e s i m i l a r t o t h o s e o b s e r v e d f o r v a l i n e . H i g h e s t p r o p o r t i o n s w e r e o b s e r v e d d u r i n g #5 and #6 w h i l e t h e l o w e s t v a l u e s w e r e o b s e r v e d w i t h i n f u -s i o n # 1 . TABLE III-4A METHIONINE INFUSION AMINO ACID DATA - INFUSION EFFECTS LEAST SQUARES MEAN 1 LEAST SQUARES CONSTANTS 2 5 - INFUSION 6 NUMBER 7 10 TESS1 164.68 *-33.77 a - 6 .91* 19.26 b 23.75b 11.15 b -13.48* S.E. 8.29 8.29 8.29 8.29 8.29 12.67 TNESS2 172.58 - 2.38 -10.50 - 4.35 2.35 7.95 6.93 S.E. 6.34 6.34 6.34 6.34 6.34 9.11 RATIO3 .957 **- .187 a .021 b c .133° .123C .021 b C - .110* S.E. .033 .033 .033 .033 .033 .048 TOTAL4 337.26 -36.14 -17.42 14.91 26.10 19.10 - 6.55 S.E. 14.26 14.26 14.26 14.26 14.26 20.48 RGLY5 .330 ** .230 a - .018 b - .099d -- .074G d - .036 b c - .002 b S.E. .014 .014 .014 .014 .014 .014 GLUCOSE6 59.458 3.541 - 1.417 1.908 3.850 3.025 -10.908 S.E. 2.210 2.210 2.210 2.210 2.210 3.173 * P <.05. ** P <.01. a,b,c, Constants within rows with the same superscripts are not significantly different. -^ TESS Total essential amino acids yg/ml blood plasma. 2TNESS Total non-essential amino acids (not including urea) yg/ml blood plasma. 3RATIO Tess/Tness. ^TOTAL Tess + Tness . 5RGLY Glycine/Valine + Isoleucine + Leucine. 6GLUCOSE mg/100 ml plasma. ^ TABLE III-4B METHIONINE INFUSIONS AMINO ACID DATA - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER MEAN 1 S.E. 2 S.E. 3 S.E. TESS1 164.68 -11.55 6.32 7.55 5.36 4.00 5.59 TNESS2 172.58 3.036 4.54 -1.91 3.86 -1.12 4.02 RATIO3 .957 *- .084 a .024 .050 b .020 .034b .021 4 TOTAL 337.26 - 8.52 10.21 5 . 6 4 8.67 2.88 9.04 RGLY5 .330 ** .079 a .010 - .046 b .009 - .034b .009 GLUCOSE6 59.458 1.075 1.582 - .875 1.343 - .200 1.400 * P <.05. ** P <.01. a,b Constants within rows with the same superscripts are not significantly different. ^YESS Total essential amino acids yg/ml blood plasma. 2TNESS Total non-essential amino acids (not including urea) yg/ml blood plasma. 3RATIO Tess/Tness. 4TOTAL Tess + Tness. 5RGLY Glycine/Valine + Isoelucine + Leucine. 6GLUCOSE mg/100 ml plasma. TABLE I I I -5A METHIONINE INFUSIONS ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL ESSENTIAL AMINO ACIDS - INFUSION EFFECTS LEAST SQUARES MEAN 1 LEAST SQUARES CONSTANTS -2 5 INFUSION NUMBER 6 7 10 THREONINE 7.010 ** 1.486 a - . 7 0 8 ° - 1 . 0 6 0 ° - . 9 9 5 ° .275 1.002 a S . E . .137 .137 .137 .137 .137 .196 VALINE 26.329 **. -3 .634 a . 6 3 2 b G 2 . 7 1 4 ° be 1 . 2 3 6 ^ - .658 b be - .290 S . E . .700 .700 .700 .700 .700 1.005 METHIONINE 4.990 ** . 246 a - . 9 6 4 b - 1 . 0 1 7 b - .878 b - .818 b 3 . 4 3 1 ° S . E . .271 .271 .271 .271 .271 .389 ISOLEUCINE 11.691 **. - . 986 a be . 2 0 4 ° ° . 5 3 1 ° 1.004 C - . 7 1 4 * - . 0 4 0 * ° S . E . .268 .268 .268 .268 .268 .384 LEUCINE 14.362 **. -2 .891 3 - . 0 8 3 b 1.295°°- 1.713 d - .297 b be . 2 6 3 ° ° S . E . .269 .269 .269 .269 .269 .386 PHENYLALANINE 5.640 ** .325 s . 513 a - . 2 9 7 ° - . 743 d be - . 0 0 3 ^ . 2 0 5 * ° S . E . .126 .126 .126 .126 .126 .181 LYSINE 10.921 .930 .980 - .255 - .690 1.284 -2.250 S . E . .608 .608 .608 .608 .608 .873 HISTIDINE 8.575 ** 2 .400 a - . 1 5 3 b - .618 b - .682 b - .465 b - .482 b S . E . .354 .354 .354 .354 .354 .509 ARGININE 10.349 ** 2 .260 a - . 2 9 4 b C - 1 . 1 5 3 C .166 b .642 b - 1 . 6 2 0 ° S . E . .352 .352 .352 .352 .352 .505 * P <.05. * * P <.01. a , b , c , d Constants w i t h i n rows wi th the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t . 97 H i s t i d i n e was f o u n d i n t h e h i g h e s t p e r c e n t a g e when m e t h i o -n i n e was i n f u s e d a l o n e ( i n f u s i o n #1 ) . A l l t h e o t h e r h i s t i d i n e v a l u e s w e r e s i m i l a r . S i g n i f i c a n t p e r i o d e f f e c t s w e r e n o t e d f o r p e r c e n t a g e s o f t h r e o n i n e , m e t h i o n i n e , l e u c i n e , p h e n y l a l a n i n e , h i s t i d i n e and a r g i n i n e ( T a b l e I I I - 5 B ) . L e u c i n e v a l u e s i n c r e a s e d a s e a c h i n f u s i o n p r o c e e d e d . H i s t i d i n e p e r c e n t a g e d e c r e a s e d s i g n i f i c a n t l y d u r i n g p e r i o d 2 b u t r o s e t o an i n t e r m e d i a t e v a l u e d u r i n g p e r i o d 3 . When t h e i n t e r a c t i o n t e r m s w e r e e x p a n d e d ( T a b l e I I I - 5 C ) i t c o u l d be s e e n t h a t f o r t h r e o n i n e f o r p e r i o d s 1 a n d 3 t h e r e s u l t s f o r t h e d i f f e r e n t i n f u s i o n s w e r e s i m i l a r . H i g h e s t p e r c e n t a g e s o f t h r e o n i n e w e r e p r e s e n t d u r i n g i n f u s i o n #1 and l o w e s t d u r i n g i n f u s i o n #6. P e r i o d e f f e c t s w e r e o b s e r v e d o n l y f o r i n f u s i o n #1, #2, a n d #5. T h e p e r c e n t a g e o f p h e n y l a l a n i n e d e c r e a s e d a s t h e s e i n f u s i o n s p r o c e e d e d . P r e - i n f u s i o n p e r c e n t a g e s o f a r g i n i n e w e r e h i g h e s t d u r i n g i n f u s i o n #1. F o r p e r i o d s 2 a n d 3 t h e p e r c e n t a g e a r g i n i n e was h i g h e s t d u r i n g i n f u s i o n #1 a n d #7. T h e r e w e r e p e r i o d r e s p o n s e s f o r i n f u s i o n s #1, #2, #6 a n d #10. T h e p e r c e n t a g e a r g i n i n e d e c r e a s e d a s e a c h o f t h e a b o v e i n f u s i o n s p r o c e e d e d . D u r i n g i n f u s i o n #2 t h e a r g i n i n e v a l u e s i n c r e a s e d t o an i n t e r m e d i a t e v a l u e i n p e r i o d 3 . I n f u s i o n s #1, #6 w e r e s i g n i f i c a n t l y l o w e r i n p e r i o d 3 t h a n p e r i o d 1 . S e r i n e , a s p a r a g i n e a n d g l u t a m i n e , g l u t a m i c a c i d , c i t r u l l i n e , g l y c i n e , a l a n i n e a n d a - a m i n o - n - b u t y r i c a c i d e x h i b i t e d s i g n i f i c a n t d i f f e r e n c e s b e t w e e n i n f u s i o n s when e x p r e s s e d a s a p e r c e n t a g e o f t h e t o t a l n o n - e s s e n t i a l a m i n o a c i d s ( T a b l e I I I - 6 A ) . TABLE III-5B METHIONINE INFUSIONS ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL ESSENTIAL AMINO ACIDS PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER M E M > ] 1 S.E. 2 S.E. 3 S.E. THREONINE 7.010 * .293 a .098 - .128° .083 - .166° .087 VALINE 26.329 - .982 .502 .095 .426 .887 .443 METHIONINE 4.990 **-1.315 a .194 1.012° .165 .303° .172 ISOLEUCINE 11.691 - .412 .192 .040 .162 .372 .170 LEUCINE 14.362 *- .397 a .193 - .093* .164 .490° .170 PHENYLALANINE 5.640 ** .255 a .090 .155* .077 - .410° .080 LYSINE 10.921 .768 .435 - .132 .369 - .636 .385 HISTIDINE 8.575 * .723 a .254 - .640° .215 - .083* .224 ARGININE 10.349 ** 1.200a .252 - .618° .214 - .583° .223 * P <.05. ** P <.01. a,b,c = Constants within rows with the same superscripts are not significantly different. TABLE III-5C METHIONINE INFUSIONS ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL ESSENTIAL AMINO ACIDS INFUSION X PERIOD INTERACTION TERMS - SIGNIFICANT LEAST SQUARES CONSTANTS THREONINE METHIONINE PHENYLALANINE ARGININE LEAST SQUARES 7.010 4.990 5.640 10.349 MEAN S.E. S.E. S.E. S.E. IN 1 P i 1 2.040* .790°° .280 -1.430? .860°° .554 • 2 3 0 ^ .258 4' 5 3 2Sc .720 IN 1 P 2 .215 .426 - . 1 3 5 ° ^ .198 .553 IN 1 P 3 1.630a .246 .488 .227 .900 .633 IN 2 P 1 - . 9 5 0 f a - 5 7 5 d e f - : 6 o o d e f .280 -1.370 d -1.030 a .554 1- 5 0 0bc .620°; - .580 e r .258 1.290^ .720 IN IN 2 2 P P 2 3 .215 .246 .426 .488 .198 .227 -1.659~L - . 514 c d e .553 .633 IN 5 P 1 - . 5 3 0 ? f - 1 .130 f g -1 .520 g h .280 -1.330 d .554 - . 365? f .258 ,_-bcde ' i 2?def .720 IN 5 P 2 .215 - .780°: .426 .198 -1.3142 -2.264 e .553 IN 5 P 3 .246 - .970 d .488 - .925 .227 .633 IN 6 P 1 " -13°S^ - ' 9 7 0 h 9 .280 -1.680 d .554 - .460 f .258 2.400* - 1 9 4 ° d e ef .720 IN 6 P 2 .215 - . 8 0 5 ° , - . 1 5 0 ^ .426 - .895^ - .875 .198 .553 IN 6 P 3 -1.885 .246 .488 .227 -1.709 .633 IN 7 P 1 . 2 3 0 ^ .050^ .545°° .280 -1.640 d - . 1 6 0 ^ .554 .260^ .258 ^„„bcde • 0 0 0bc .720 IN 7 P 2 .215 .426 .198 ^ * ^bcd .553 IN 7 P 3 .246 - .665 d .488 .227 .740 .633 IN 10 P 1 1.070* .291 - . 4 4 0 ^ .577 . 6 9 0 * ° .268 I.OOoJ 0* .749 IN 10 P 2 .291 7.000 .577 .268 -3.659 .749 a,b,c,d,e,f,g,h Constants within columns with the same superscripts are not significantly different. "^IN 1 P 1 Infusion number and period number 100 S e r i n e p e r c e n t a g e s w e r e h i g h e s t d u r i n g i n f u s i o n #1 a n d l o w e s t d u r i n g i n f u s i o n s #2 a n d #10. G l y c i n e p e r c e n t a g e s w e r e h i g h e s t d u r i n g i n f u s i o n #1 b u t w e r e s i m i l a r f o r t h e r e s t o f t h e i n f u s i o n s . A l a n i n e f o l l o w e d t h e same p a t t e r n w i t h h i g h e s t p r o -p o r t i o n s d u r i n g i n f u s i o n #1; l o w e s t a l a n i n e v a l u e s w e r e f o u n d d u r i n g i n f u s i o n s #5, #6 and #10. A s p a r a g i n e a n d g l u t a m i n e p e r c e n t a g e s w e r e h i g h e s t d u r i n g i n f u s i o n s #2 a n d #7. L o w e s t v a l u e s w e r e o b s e r v e d d u r i n g i n f u s i o n #1. c t - a m i n o - n - b u t y r i c a c i d v a l u e s w e r e h i g h e s t d u r i n g i n f u s i o n #2, i n t e r m e d i a t e f o r i n f u s i o n s #1 and #10 a n d l o w e r d u r i n g i n f u s i o n s #5, #6 a n d #7. G l u t a m i c a c i d p e r c e n t a g e s w e r e s i m i l a r e x c e p t f o r i n f u s i o n #10 w h e r e t h e y w e r e s i g n i f i c a n t l y i n c r e a s e d . C i t r u l l i n e p e r -c e n t a g e s w e r e h i g h e s t w i t h i n f u s i o n #5 a n d l o w e s t w i t h i n f u s i o n #2. P e r i o d e f f e c t s w e r e n o t e d f o r t a u r i n e , s e r i n e , p r o l i n e , g l y c i n e , a l a n i n e a n d t y r o s i n e ( T a b l e I I I - 6 B ) . E x c e p t f o r t a u r i n e a n d t y r o s i n e t h e p e r c e n t a g e s o f t h e a b o v e a m i n o a c i d s d e c l i n e d a s e a c h i n f u s i o n p r o c e e d e d a n d t y r o s i n e i n c r e a s e d d u r i n g p e r i o d 2 b u t d r o p p e d t o p e r i o d 1 l e v e l s d u r i n g p e r i o d 3 . (1) D i s c u s s i o n (a) M i l k a n d m i l k c o m p o s i t i o n T h e e f f e c t s o f i n f u s i o n o f a m i n o a c i d s on m i l k p r o d u c t i o n a r e d i f f i c u l t t o d e t e r m i n e b e c a u s e t h e i n f u s i o n s a r e s u p e r i m p o s e d o n t h e l a c t a t i o n c y c l e . In t h i s c a s e t h e i n f u s i o n s w e r e p e r -f o r m e d o v e r a 150 d a y s p a n s o t h a t l a c t a t i o n e f f e c t s w i l l be p r e s e n t a l t h o u g h m i l k p r o d u c t i o n d r o p p e d o n l y 4 k g o v e r t h e w h o l e 101 TABLE J I J - 6 A METHIONINE TI.'FUSTONS HON -ESSENTIAL AMIIJO ACTUS AS A rKRCKHTAr.E OF TIIK TOTAL KON—ESSENTIAL AMINO ACIDS INFUSION EFFECTS LEAST SQUARES MEAN LEAST SQUARES CONSTANTS - INFUSION NUMBERS 2 5 6 7 10 TAURINE S.E. SERINE S. F.. ASPARAGIME S. F.. 4.101 6.432 28.904 .834 .356 .546° .130 *-2.289 .707 . 593 . 356 .316 .130 1.774 .707 . 509 . 356 .218 .130 ab . 292 .356 .074 .130 be . 7 4 1 n b C - 1 . 4 1 9 3 C .707 .707 .209 .356 .083 .130 1.429 . 707 be .839 . 511 . 4 57 . 187 1.247 1.015 be PROLINE S.E. 6.075 .730 . 203 .004 . 283 .305 . 283 . 572 . 283 . 390 .283 .482 .406 GLUTAMIC ACID S.E. CITRULLINE S.E. 4.041 9 . 861 .340 . 204 * * - l . 3 1 1 . 503 ab .132 .204 -2.721 . 503 .131 . 204 2.456 .503 . 510 . 204 1.629 . 503 cd .380 . 204 .257 . 50 ad 1 . 228 ' . 293 . 310 .722 ad GLYCINE S.E. ALANINE S.E. 16 . 896 11.197 ** 4.283 .486 ** 1.256 a .273 - .214 .486 . 674 .27 3 ab -1.804 .486 -1.267 .273 - .341" .486 .739 . 273 cd -1.148 .486 .191 . 273 be .775 . 697 .116 . 392 bed a-AMIHO-IJ-BUTYRIC ACID . 578 .039 .035 ab .104 .035 .071 .035 .053 .035 .056 b . 035 .038 . 050 CYSTINE S.E. 2.737 .012 . 221 .343 . 221 .232 .221 .032 .221 .025 .221 . 041 .318 TYROSINE S.E. 5.161 .404 .241 .797 .241 .166 . 241 .024 .241 .107 .241 .475 .347 ORNITHINE S.E. 3.843 .150 .480 .027 .480 .590 .480 .648 .480 .418 .480 .697 .689 * P <.05. ** P <.01. a,b,c,d C o n s t a n t s w i t h i n rows w i t h t h e same s u p e r s c r i p t s a r e n o t s i g n i f i c a n t l y d i f f e r e n t . ^ A s p a r a c j i n e + G l u t a m i n e . 102 TABLE I I I -6B METHIONINE INFUSIONS NON-ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL NON-ESSENTIAL AMINO ACIDS - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS MEAN PERIOD NUMBER 1 2 3 TAURINE 4.161 *_ . 590 a - . 159 a . 750 b S . E . .255 .216 .225 SERINE 6.452 ** . 298 a .092 a - . 390 b S . E . .093 .079 .083 ASPARAGINE 28.994 — .491 - .432 .923 S . E . .506 .429 .448 PROLINE 6.075 ** . 246 a . 498 a - . 745 b S . E . .202 .172 .172 GLUTAMIC ACID 4.041 .085 .055 - .141 S . E . .146 .124 .129 CITRULLINE 9.861 — .498 - .034 .532 S . E . .360 .306 .319 GLYCINE 16.896 ** 1.409 a - . 9 7 1 ° - . 439 b S . E . .348 .295 .308 ALANINE 11.197 ** . 108 a . 540 a - . 648 b S . E . .195 .166 .173 a-AMINO-BUTYRIC ACID .578 • .065 .033 .032 S . E . .025 .021 .022 CYSTINE 2.737 • .240 .059 .182 S . E . .158 .134 .140. TYROSINE 5.161 *_ • . 216 a . 514 b - . 298a S . E . .173 .147 .153 ORNITHINE 3.843 • .045 - .198 .243 S . E . .344 .292 .304 * P <.05. * * P <.01. a,b Constants within rows with the same superscripts are not significantly different. 103 t r i a l . The drop was quite even which indicates l i t t l e e f f e c t of infusions. However, the s i g n i f i c a n t infusion x period i n t e r -action term indicates that period response to the d i f f e r e n t infusions was not the same. The increase i n production during period 2 for infusions #5 and #7 does not appear to be related to protein composition. For a l l infusions studied protein content of milk increased as each infusion progressed. However t o t a l protein production did not follow t h i s pattern. Except for infusion #7 there was a decline from period 2 production to period 3 production. The production of protein i s a function of milk production and milk protein concentration. In most cases the drop i n milk production countered the e f f e c t of increased milk protein concentration r e s u l t i n g i n decreases i n milk protein concentration. Infusion #7 not only maintained milk production but also increased protein production. Except for infusion #7 the production of milk appears to be negatively correlated with milk protein production. The greatest increases during period 2 above pre-infusion milk protein production were shown for infusions #2, #5, and #7. It might be speculated that these amino acids have the greatest e f f e c t on milk protein production. That infusion #7 was the only one of the three infusions to show a further increase i n protein production during period 3 must be kept i n mind when plasma amino acids are discussed. There were no period e f f e c t s for lactose concentration as would be expected as lactose i s present i n milk at rather con-stant values changing gradually over the l a c t a t i o n . Lactose con-104 c e n t r a t i o n a p p e a r s t o d e t e r m i n e m i l k v o l u m e b y some o s m o t i c c o n t r o l ( P a l m i t e r , 1 9 6 9 ) . T h e s i g n i f i c a n t i n t e r a c t i o n t e r m f o r l a c t o s e c a n t h e n b e e x p l a i n e d i n t e r m s o f m i l k p r o d u c t i o n . T h e r e s u l t s ( T a b l e I I - 4 ) show s i m i l a r p e r i o d a n d i n f u s i o n e f f e c t s f o r l a c t o s e p r o d u c t i o n and m i l k p r o d u c t i o n . (b) P l a s m a a m i n o a c i d s i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s m i c r o -g r a m s p e r m l o f p l a s m a I n f u s i o n o f v a l i n e , i s o l e u c i n e a n d l e u c i n e i n t h e f r e e f o r m r e s u l t e d i n h i g h e r c o n c e n t r a t i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s i n b l o o d p l a s m a ( i n f u s i o n s #5 a n d # 6 ) . I n f u s i o n a s w h o l e p r o t e i n ( c a s e i n ) r e s u l t e d i n i n t e r m e d i a t e l e v e l s . M e t h i o n i n e i n f u s i o n a l o n e r e s u l t e d i n t h e l o w e s t l e v e l s . T h e o n l y d i s c r e p -a n c y was i n f u s i o n #7 i n w h i c h n o n e o f t h e b r a n c h e d c h a i n a m i n o a c i d s w e r e i n f u s e d , m i l k p r o d u c t i o n a n d m i l k p r o t e i n p r o d u c t i o n w e r e m a x i m a l a n d y e t t h e c o n c e n t r a t i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s was i n t e r m e d i a t e . F r o m t h e s e r e s u l t s i t w o u l d a p p e a r a s i f m e t h i o n i n e p l a y s a r o l e i n c o n t r o l l i n g t h e d e g r a d a t i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s . An increased r a t i o o f m e t h i o n i n e t o c y s t i n e i n t h e d i e t was f o u n d t o r e s u l t i n i n c r e a s e d c o n c e n t r a -t i o n s o f p l a s m a i s o l e u c i n e and v a l i n e i n r a t s ( B y i n g t o n and Howe, 1 9 7 2 ) . M e t h i o n i n e h a s b e e n i m p l i c a t e d i n c o n t r o l o f a m i n o a c i d d e g r a d a t i v e e n z y m e s i n t h e l i v e r , a l o n g w i t h t r y p t o p h a n (Nakano e t a l . , 1 9 7 0 ) ; h o w e v e r , t h e b r a n c h e d c h a i n a m i n o a c i d s a r e m e t a b -o l i z e d i n t h e p e r i p h e r a l t i s s u e s . I f m e t h i o n i n e i s c a u s i n g t h e a b o v e e f f e c t t h e d e c l i n e o b s e r v e d d u r i n g i n f u s i o n #7 i n t h e c o n -105 c e n t r a t i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s m i g h t r e f l e c t i n c r e a s e d u p t a k e o f s u b s t r a t e f o r p r o t e i n s y n t h e s i s . M e t h i o n i n e was i n c l u d e d i n a l l i n f u s i o n s a t t h e same l e v e l e x c e p t f o r i n f u s i o n #10 w h e r e t h e l e v e l was i n c r e a s e d t h r e e t i m e s . T h i s i n c r e a s e was r e f l e c t e d i n i n c r e a s e d p l a s m a m e t h i o -n i n e ( T a b l e I I I - 3 C ) . T h e i n c r e a s e i n v a l i n e c o n c e n t r a t i o n a s e a c h i n f u s i o n p r o g r e s s e d i s d i f f i c u l t t o e x p l a i n . V a l i n e was i n c l u d e d i n t h r e e o f t h e s i x i n f u s i o n s w h i c h w o u l d e x p l a i n t h e r e s p o n s e d u r i n g i n f u s i o n s #2, #5 a n d #6. T h e d r o p i n m i l k and p r o t e i n p r o d u c t i o n d u r i n g i n f u s i o n #1 a n d #10 m i g h t h e l p e x p l a i n t h e i n c r e a s e i n v a l i n e d u r i n g t h o s e i n f u s i o n s . T h e l a r g e i n c r e a s e s o b s e r v e d d u r i n g i n f u s i o n #7 i n m i l k and p r o t e i n p r o d u c t i o n w o u l d be e x p e c t e d t o d r i v e down t h e c o n c e n t r a t i o n o f m o s t o f t h e e s s e n t i a l a m i n o a c i d s . However s u c h a d e c r e a s e w o u l d h a v e r e s u l t e d i n a s i g n i f i c a n t i n t e r a c t i o n t e r m . T h e s i g n i f i c a n t i n t e r a c t i o n t e r m f o r m e t h i o n i n e i s e x p l a i n e d t h r o u g h t h e d i f f e r e n t r e s p o n s e o f p l a s m a m e t h i o n i n e when m e t h i o -n i n e was i n f u s e d a l o n e ( i n f u s i o n s #1 and #10) . W h i l e t h e r e was a g e n e r a l t r e n d f o r i n c r e a s e d m e t h i o n i n e l e v e l s a b o v e p r e - i n f u s i o n l e v e l s f o r a l l i n f u s i o n s t h e s e r e s u l t s w e r e s i g n i f i c a n t o n l y f o r i n f u s i o n s #1 and #10. T h e u r e r e_t a l . (1966) f o u n d t h a t t h e c o n c e n t r a t i o n o f f r e e m e t h i o n i n e i n j u g u l a r p l a s m a was c o n s i s t e n t l y l o w e r t h a n p o r t a l c o n c e n t r a t i o n s . I t i s p o s s i b l e t h a t i n a l l i n f u s i o n s t h e c o n -c e n t r a t i o n o f m e t h i o n i n e i n p o r t a l b l o o d w o u l d h a v e shown a s i g n i f i c a n t i n c r e a s e . 106 T h e t a u r i n e r e s p o n s e t o t h e d i f f e r e n t i n f u s i o n s a p p e a r s t o r e s e m b l e t h e m e t h i o n i n e r e s p o n s e . T a u r i n e i s a m e t a b o l i t e o f c y s t e i n e m e t a b o l i s m . E x t r a m e t h i o n i n e c o u l d p l a y a r o l e i n s p a r -i n g c y s t e i n e w h i c h m i g h t r e s u l t i n g r e a t e r t a u r i n e p r o d u c t i o n e v e n t h o u g h t no s u c h r e s p o n s e was n o t e d f o r c y s t i n e . T a u r i n e a g a i n r e s e m b l e s t h e m e t h i o n i n e r e s p o n s e . B y i n g t o n a n d Howe (1972) f o u n d i n r a t s t h a t i n c r e a s e d o r g a n i c s u l p h u r ( m e t h i o n i n e a n d c y s t i n e a t 70 : 30 r a t i o ) r e s u l t e d i n i n c r e a s e d t a u r i n e c o n c e n -t r a t i o n i n p l a s m a . C i t r u l l i n e was p r e s e n t i n s i g n i f i c a n t l y l o w e r c o n c e n t r a t i o n s d u r i n g i n f u s i o n #2 ( c a s e i n ) . A r g i n i n e , a m e t a b o l i c p r e c u r s o r o f c i t r u l l i n e was a l s o p r e s e n t i n l o w e r amounts d u r i n g t h i s i n f u s i o n . A c r o s s a l l i n f u s i o n s t h e a r g i n i n e a n d c i t r u l l i n e r e s u l t s a p p e a r s i m i l a r . T h e h i g h g l y c i n e c o n c e n t r a t i o n s d u r i n g i n f u s i o n #1 m i g h t r e f l e c t t h e g r e a t e r m i l k p r o d u c t i o n a t t h e t i m e . A s t h e g l y c i n e c o n c e n t r a t i o n s d r o p p e d w i t h e a c h i n f u s i o n t h e a r g u m e n t c a n n o t b e u s e d t h a t m e t h i o n i n e s t i m u l a t e d p r o t e i n s y n t h e s i s d u r i n g i n f u s i o n #1 c a u s i n g i n c r e a s e d t i s s u e m o b i l i z a t i o n a n d g l y c i n e p r o d u c t i o n . I t i s a p p a r e n t t h a t t i s s u e m o b i l i z a t i o n a c t u a l l y d e c r e a s e d d u r -i n g e a c h i n f u s i o n i n t h i s s e r i e s o f i n f u s i o n s . T h a t t h r e o n i n e may h a v e b e e n p r e s e n t i n e x c e s s a m o u n t s d u r -i n g i n f u s i o n #7 i s i n d i c a t e d b y t h e i n c r e a s e d c o n c e n t r a t i o n o f a - a m i n o - n - b u t y r i c a c i d d u r i n g t h i s i n f u s i o n . a - a m i n o - n - b u t y r i c a c i d r e p r e s e n t s one o f t h e e n d p r o d u c t s o f t h r e o n i n e d e g r a d a t i o n . O n l y d u r i n g i n f u s i o n #1 was t h e r e a s i g n i f i c a n t c y s t i n e e f f e c t . D u r i n g t h e t h i r d p e r i o d t h e l e v e l o f c y s t i n e was s i g n i f -107 i c a n t l y e l e v a t e d w h i c h c o u l d i n d i c a t e t h a t e x c e s s m e t h i o n i n e h a s h a d a s p a r i n g e f f e c t on c y s t i n e . U n f o r t u n a t e l y t h e r e a r e no d a t a f o r p e r i o d 3 o f i n f u s i o n #10 t o f u r t h e r s u b s t a n t i a t e t h i s i d e a . T h e i n f u s i o n e f f e c t s o f p l a s m a a m i n o a c i d s do r e f l e c t t h e n a t u r e o f t h e m a t e r i a l i n f u s e d ( T a b l e I I - l ) . T h e d e c r e a s e d c o n c e n t r a t i o n o f t o t a l e s s e n t i a l a m i n o a c i d s d u r i n g t h e i n f u s i o n s o f m e t h i o n i n e a l o n e ( i n f u s i o n #1 a n d #10) r e f l e c t s t h i s t r e n d ( T a b l e I I I - 4 A ) . T h e r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s t o t o t a l n o n - e s s e n t i a l a m i n o a c i d s shows t h e same t r e n d o f i n c r e a s e d e s s e n t i a l a m i n o a c i d s d u r i n g i n f u s i o n s #2, #5, #6 a n d #7 when more t h a n one a m i n o a c i d was i n f u s e d . However t h e r e was a l s o a s i g n i f i c a n t p e r i o d r e s p o n s e w h i c h i n d i c a t e d t h a t d u r i n g a l l i n f u s i o n s t h e r a t i o i n c r e a s e d d u r i n g p e r i o d s 2 a n d 3 ( T a b l e I I I -4 B ) . E v e n d u r i n g t h e m e t h i o n i n e i n f u s i o n s s u c h i n c r e a s e s w e r e o b s e r v e d . D u r i n g i n f u s i o n s #1 a n d #10 t h e t o t a l p r o d u c t i o n o f m i l k p r o t e i n d i d n o t i n c r e a s e a b o v e p e r i o d 1 p r o d u c t i o n ( T a b l e I I I -1 C ) . T h e l o w e r e d demand f o r a m i n o a c i d s d u r i n g t h e s e two i n f u s i o n s c o u l d be r e f l e c t e d i n p l a s m a a m i n o a c i d c o n c e n t r a t i o n s . T h e r a t i o o f g l y c i n e t o t h e b r a n c h e d c h a i n a m i n o a c i d s showed r e s u l t s w h i c h o p p o s e t h o s e o f t h e r a t i o o f t h e t o t a l e s s e n -t i a l a m i n o a c i d s t o t o t a l n o n - e s s e n t i a l a m i n o a c i d s . I f b o t h o f t h e s e r a t i o s a r e r e l a t e d t o p r o t e i n p r o d u c t i o n and i n f u s i o n s o f e s s e n t i a l a m i n o a c i d s t h i s o p p o s i t e r e s u l t i s e x p e c t e d (Hogan e t a l . , 1 9 6 8 ) . 108 i i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s a p r o -p o r t i o n o f t h e t o t a l e s s e n t i a l o r n o n -e s s e n t i a l a m i n o a c i d s T h e b r a n c h e d c h a i n a m i n o a c i d s b e h a v e much t h e same u n d e r t h e d i f f e r e n t i n f u s i o n s . T h e i n c r e a s e i n p e r c e n t a g e o f t h e b r a n c h e d c h a i n a m i n o a c i d s a s e a c h i n f u s i o n p r o g r e s s e d was s i m -i l a r t o t h e r e s p o n s e n o t e d e a r l i e r when t h e c o n c e n t r a t i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s a l s o i n c r e a s e d . T h e r e w e r e no s i g n i f i c a n t i n t e r a c t i o n t e r m s f o r a n y o f t h e b r a n c h e d c h a i n a m i n o a c i d s , w h i c h r u l e s o u t t h e s u g g e s t i o n o f d i f f e r e n t p e r i o d r e s p o n s e s f o r t h e d i f f e r e n t i n f u s i o n s . T h e s i g n i f i c a n t d e c r e a s e i n t h r e o n i n e d u r i n g i n f u s i o n #6 m i g h t be e x p l a i n e d i n t h a t t h r e o n i n e was n o t i n c l u d e d i n t h i s i n f u s i o n m i x a n d t h a t t h e i n c r e a s e d p r o t e i n s y n t h e s i s c a u s e d a d e c r e a s e i n p e r c e n t a g e o f p l a s m a t h r e o n i n e . E v e n t h o u g h t h e r e was a r e l a t i v e l y s m a l l i n c r e a s e i n m i l k p r o t e i n p r o d u c t i o n t h e i n s u l i n o t r o p i c e f f e c t o f t h e b r a n c h e d c h a i n a m i n o a c i d s c o u l d c a u s e an i n c r e a s e i n a m i n o a c i d u p t a k e by t h e t i s s u e s . A s i m -i l a r e f f e c t c o u l d e x p l a i n some o f t h e d e c r e a s e i n p e r c e n t a g e t h r e o n i n e d u r i n g i n f u s i o n #5. T h e s i m i l a r i t y o f t h e p r e - i n f u s i o n v a l u e s f o r m e t h i o n i n e p e r c e n t a g e was u n e x p e c t e d b u t d o e s i n d i c a t e t h a t f l u c t u a t i o n s i n m e t h i o n i n e c o n c e n t r a t i o n s w e r e s i m i l a r t o t h e g e n e r a l p a t t e r n ( T a b l e I I I - 5 C ) . T h e m a i n r e a s o n f o r t h e s i g n i f i c a n t i n t e r a c t i o n t e r m i s d u e t o t h e i n c r e a s e d p r o p o r t i o n o f m e t h i o n i n e d u r i n g i n f u s i o n s #1 a n d #10, when m e t h i o n i n e a l o n e was i n f u s e d . T h e p e r c e n t a g e o f p h e n y l a l a n i n e d e c l i n e d f r o m p r e - i n f u s i o n l e v e l s d u r i n g i n f u s i o n s #2 a n d #5. The f l u c t u a t i o n s i n t o t a l 109 e s s e n t i a l a m i n o a c i d s may be more r e s p o n s i b l e f o r t h e a b o v e e f f e c t t h a n t h e d e v i a t i o n s n o t e d i n p h e n y l a l a n i n e c o n c e n t r a t i o n . H i s t i d i n e p e r c e n t a g e d e c l i n e d i n i t i a l l y - d u r i n g p e r i o d 2 o f e a c h i n f u s i o n a n d t h e n r o s e t o i n t e r m e d i a t e l e v e l s d u r i n g p e r i o d 3 . H i s t i d a s e a n d u r o c a n a s e , e n z y m e s i n v o l v e d i n t h e d e g r a d a t i o n o f h i s t i d i n e , a p p e a r t o be c o n t r o l l e d b y d i e t a r y p r o t e i n (Lee and H a r p e r , 1 9 7 1 ) . T h e f a c t o r s o r a m i n o a c i d s w h i c h t r i g g e r t h e i n d u c t i o n o f t h o s e c a t a b o l i c e n z y m e s a r e n o t m e n t i o n e d . I t i s p o s s i b l e t h a t some o f t h e i n f u s e d a m i n o a c i d s o t h e r t h a n h i s t i d i n e a r e i n v o l v e d i n t h e a b o v e c o n t r o l . When d i e t a r y h i s t i d i n e r e a c h e s s u c h l o w l e v e l s o t h e r c o n t r o l m e c h a n -i s m s w i l l b l o c k t h e c a t a b o l i s m o f h i s t i d i n e . H o g a n e t a l . (1968) n o t e d a d e c r e a s e i n t h e p r o p o r t i o n o f a r g i n i n e i n p l a s m a when c a s e i n was i n f u s e d i n t o t h e abomasum o f s h e e p . A r g i n i n e c o n c e n t r a t i o n s i n p l a s m a a r e d e p r e s s e d b y s t a r v a t i o n ( L e i b h o l z , 1 9 7 0 ) , b y u r e a d i e t s ( P r i o r e t a l . , 1972) a n d b y i n f u s i o n s o f g lucose ( E s k e l a n d e t a l . , 1 9 7 1 ) . T h e d e p r e s -s i o n n o t e d d u r i n g i n f u s i o n #2 ( c a s e i n ) s h o u l d be d u e t o a n e f f e c t s i m i l a r t o t h a t o b s e r v e d b y H o g a n e t a_l. (1968) w h i c h was a t t r i b -u t e d t o e f f e c t s o f e n e r g y a n d p r o t e i n i n t a k e o n u p t a k e o f a m i n o a c i d s . Due t o t h e s i m i l a r i t i e s i n e n e r g y i n t a k e i n a l l p e r i o d s o f e a c h i n f u s i o n , t h e f l u c t u a t i o n s i n a r g i n i n e m i g h t b e due t o a l t e r e d p r o t e i n m e t a b o l i s m d u e t o m e t h i o n i n e i n f u s i o n s o r m e t h i o n i n e i n c o n j u n c t i o n w i t h t h e b r a n c h e d c h a i n a m i n o a c i d s . When t h e n o n - e s s e n t i a l p l a s m a a m i n o a c i d s w e r e e x p r e s s e d a s a p e r c e n t a g e o f t h e t o t a l p l a s m a n o n - e s s e n t i a l a m i n o a c i d s 110 t h e r e w e r e s i g n i f i c a n t p e r i o d a n d i n f u s i o n e f f e c t s f o r s e r i n e , g l y c i n e a n d a l a n i n e . W h i l e l e v e l s w e r e d i f f e r e n t b e t w e e n e x p e r i -m e n t s t h e r e s p o n s e d u r i n g e a c h i n f u s i o n was s i m i l a r . F o r t h e s e t h r e e a m i n o a c i d s t h e p r o p o r t i o n o f t h e t o t a l d e c l i n e d a s e a c h i n f u s i o n p r o g r e s s e d . A d e c l i n e i n t h e s e a m i n o a c i d s i s i n d i c a -t i v e o f p r o t e i n a n d e n e r g y d e f i c i e n c i e s ( L e i b h o l z , 1 9 7 0 ) . B y i n g t o n a n d Howe (1972) f o u n d t h a t t h e c o n c e n t r a t i o n o f t a u r i n e i n p l a s m a i n c r e a s e d a s t h e d a i l y i n t a k e o f o r g a n i c s u l p h u r ( m e t h i o n i n e a n d c y s t i n e ) i n c r e a s e d . T h e p e r i o d i n c r e a s e s i n m e t h i o n i n e ( T a b l e I I I - 5 C ) i n d i c a t e t h a t t h e r e was a n i n c r e a s e d u p t a k e o f m e t h i o n i n e f r o m t h e i n t e s t i n e s d u r i n g e a c h i n f u s i o n . T h a t t a u r i n e l e v e l s a l s o r o s e i s a f u r t h e r i n d i c a t i o n o f t h i s g r e a t e r amount o f c i r c u l a t i n g m e t h i o n i n e . F r a n c e s c o n i e t a l . , (1972) f o u n d t h a t p l a s m a l e v e l s o f t y r o s i n e d r o p p e d d u r i n g a 12 h o u r f a s t . I f t h e f a s t was c o n t i n u e d f o r 48 h o u r s t h e l e v e l s o f t y r o s i n e i n c r e a s e d p r o b a b l y due t o t i s s u e m o b i l i z a t i o n . To s u m m a r i z e , t h e d i f f e r e n t i n f u s i o n s u s e d w e r e n o t a l l e q u a l i n t h e i r a b i l i t y t o a f f e c t t h e y i e l d o f m i l k and m i l k c o m -p o n e n t s . A s m e t h i o n i n e was i n c l u d e d i n e a c h i n f u s i o n a t t h e same c o n c e n t r a t i o n ( e x c e p t i n f u s i o n #10) i t i s a s s u m e d t h a t m e t h i o n i n e d i d n o t c h a n g e t h e y i e l d s o f m i l k a n d m i l k c o m p o n e n t s . T h r e o n i n e , p h e n y l a l a n i n e o r l y s i n e a p p e a r t o h a v e an e f f e c t o n m i l k y i e l d . M e t h i o n i n e i n f u s i o n may e f f e c t m i l k p r o t e i n s y n t h e s i s a s p r o t e i n c o n c e n t r a t i o n i n c r e a s e d f r o m p e r i o d 1 l e v e l s i n a l l i n f u s i o n s . T o t a l p r o t e i n p r o d u c t i o n s a l s o a p p e a r e d t o be i n -f l u e n c e d by t h r e o n i n e , p h e n y l a l a n i n e a n d l y s i n e . M i l k p r o t e i n p r o d u c t i o n i n c r e a s e d a b o v e p e r i o d 1 p r o d u c t i o n o n l y when t h e s e t h r e e a m i n o a c i d s w e r e i n c l u d e d i n t h e i n f u s a t e . I l l T h e a m i n o a c i d s i s o l e u c i n e , l e u c i n e , l y s i n e a n d m e t h i o n i n e , w h i c h a p p e a r e d t o b e m o s t i m p o r t a n t d u r i n g E x p e r i m e n t I, d i d n o t r e f l e c t a g r e a t amount o f c h a n g e d u r i n g m e t h i o n i n e i n f u s i o n s . N e i t h e r t h e c o n c e n t r a t i o n o r p r o p o r t i o n o f l y s i n e showed a n y c h a n g e b e t w e e n i n f u s i o n s o r w i t h i n i n f u s i o n s . I s o l e u c i n e a n d l e u c i n e c o n c e n t r a t i o n s a p p e a r e d e l e v a t e d d u r i n g t h o s e i n f u s i o n s i n w h i c h t h e y w e r e i n c l u d e d . T h e r e w e r e no p e r i o d e f f e c t s o b s e r v e d f o r i s o l e u c i n e o r l e u c i n e c o n c e n t r a t i o n s . When a m i n o a c i d s w e r e e x p r e s s e d a s a p e r c e n t a g e o f t h e t o t a l a m i n o a c i d s t h e p e r c e n t a g e o f l e u c i n e i n c r e a s e d a s e a c h i n f u s i o n p r o g r e s s e d . M e t h i o n i n e c o n c e n t r a t i o n a n d p e r c e n t a g e w e r e f o u n d t o i n c r e a s e a b o v e p e r i o d 1 v a l u e s o n l y d u r i n g t h o s e i n f u s i o n s i n w h i c h m e t h i o -n i n e a l o n e was i n f u s e d . P r o t e i n c o n c e n t r a t i o n i n c r e a s e d d u r i n g a l l i n f u s i o n s b u t t o t a l p r o t e i n p r o d u c t i o n i n c r e a s e d o n l y d u r i n g t h o s e i n f u s i o n s w h i c h i n c l u d e d t h r e o n i n e , p h e n y l a l a n i n e a n d l y s i n e . c) I s o l e u c i n e i n f u s i o n s (1) R e s u l t s (a) M i l k a n d m i l k c o m p o s i t i o n I s o l e u c i n e was i n f u s e d a l o n e o r i n c o m b i n a t i o n w i t h o t h e r e s s e n t i a l a m i n o a c i d s i n s i x s e p a r a t e i n f u s i o n s ( s e e T a b l e I I - l ) . E a c h i n f u s i o n was d i v i d e d i n t o t h r e e p e r i o d s , 1) p r e - i n f u s i o n , 2) t h e f i r s t 2 d a y s o f t h e i n f u s i o n a n d 3) t h e l a s t 2 d a y s o f t h e i n f u s i o n . T h e d e c l i n e i n m i l k p r o d u c t i o n a s t h e e x p e r i m e n t p r o c e e d e d was s i g n i f i c a n t and a l s o q u i t e r e g u l a r ( T a b l e I V - 1 A ) . TABLE IV-1A ISOLEUCINE INFUSIONS MILK AND MILK COMPOSITION - INFUSION EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 2 4 5 6 8 9 S.E. MILK (kg) 21.383 ** 1.6703 .724° .574b - 1.278C - .903° - .788° .243 PROTEIN (gm) 676.95 ** 67.44 a be 2.68°° 22.70b -62.99 e -23.90 d - 5 .93^ 7.26 PROTEIN (%) 3.164 ** .066 a - .089 b . 0 2 l a - .110 b .022a .091 a .022 LACTOSE (gm) 1089.56 ** 70.84 a 51.96 a 29 .2 l a -85.12° 33.40b 33.54 b 12.97 LACTOSE (%) 5.094 **. - .062* .068° ~«ibc - .001 - .099 a .063° .030° .019 * P <.05. ** P <.01. a,b,c,d,e Constants within rows with the same superscripts are not significantly different. 113 T h e r e w e r e no o b v i o u s e f f e c t s o f i n f u s i o n o n m i l k p r o d u c t i o n . T h e c o n c e n t r a t i o n o f p r o t e i n i n m i l k was s i g n i f i c a n t l y l o w e r w i t h i n f u s i o n s #4 ( i s o l e u c i n e a l o n e ) a n d #6 ( v a l i n e , m e t h i o n i n e , i s o l e u c i n e , l e u c i n e ) , t h a n w i t h i n f u s i o n s #2, #5, #8 a n d #9. T o t a l p r o t e i n p r o d u c t i o n f o l l o w e d t h e same p a t t e r n a s m i l k p r o d u c t i o n e x c e p t t h a t w i t h i n f u s i o n #6 t h e p r o d u c t i o n o f p r o t e i n was s i g n i f i c a n t l y l o w e r t h a n i n a n y o t h e r i n f u s i o n . T h e l a c t o s e c o n c e n t r a t i o n i n m i l k was s i g n i f i c a n t l y h i g h e r i n i n f u s i o n s #4, #5, #8 a n d #9 t h a n i n #2 a n d #6. T h e l o w e s t c o n c e n t r a t i o n o f l a c t o s e was o b s e r v e d w i t h i n f u s i o n #6 ( v a l i n e , m e t h i o n i n e , i s o l e u c i n e , l e u c i n e ) . L a c t o s e p r o d u c t i o n d e c r e a s e d a s t h e e x p e r i m e n t p r o g r e s s e d w i t h t h e e x c e p t i o n o f i n f u s i o n #6 when t h e c o n c e n t r a t i o n was s i g n i f i c a n t l y l o w e r t h a n f o r a n y o t h e r i n f u s i o n . P e r i o d e f f e c t s ( p r e - i n f u s i o n , f i r s t h a l f o f i n f u s i o n a n d s e c o n d h a l f o f i n f u s i o n ) w e r e o b s e r v e d f o r m i l k , p r o t e i n (gm a n d %) a n d l a c t o s e (gm) ( T a b l e I V - 1 B ) . M i l k p r o d u c t i o n d e c r e a s e d a s e a c h i n f u s i o n p r o g r e s s e d . P r o t e i n c o n c e n t r a t i o n i n m i l k was f o u n d t o i n c r e a s e s i g n i f i c a n t l y . T o t a l p r o t e i n p r o d u c t i o n (gm) was s i g n i f i c a n t l y h i g h e r i n p e r i o d 2 b u t d e c r e a s e d t o p r e - i n f u s i o n l e v e l s i n p e r i o d 3 . T h e r e was no p e r i o d e f f e c t o n l a c t o s e c o n c e n t r a t i o n . L a c t o s e p r o d u c t i o n d e c r e a s e d d u r i n g e a c h i n f u s i o n , s i m i l a r t o m i l k p r o -d u c t i o n . (b) P l a s m a a m i n o a c i d s TABLE IV-IB ISOLEUCINE INFUSIONS MILK AND MILK COMPOSITION - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER 1 S.E. 2 S.E. ' 3 S.E. MILK (kg) 21.383 ** ,119a .172 .479a .144 - .598b .144 PROTEIN (gm) 676.95 **-4.74a 5.13 15.90b 4.30 -11.16a 4.30 PROTEIN (%) 3.164 *- .038a .016 .002* .013 .036b .013 LACTOSE (gm) 1089.56 ** 8.58a .013 21.40a .011 -29.98b .011 LACTOSE (%) 5.094 .010 .014 - .013 .011 .004 .011 * P <.05. ** P <.0L a,b,c,d,e Constants within rows with the same superscripts are not significantly 115 i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s m i c r o -g r a m s p e r m l o f b l o o d p l a s m a Of t h e e s s e n t i a l a m i n o a c i d s s i g n i f i c a n t i n f u s i o n e f f e c t s w e r e n o t e d f o r v a l i n e , m e t h i o n i n e a n d l e u c i n e ( T a b l e I V - 2 A ) . V a l i n e c o n c e n t r a t i o n s w e r e s i g n i f i c a n t l y h i g h e r i n i n f u s i o n s #5, #6 a n d #9, when v a l i n e was i n c l u d e d i n t h e i n f u s i o n m i x t u r e t h a n i n i n f u s i o n s #2, #4 a n d #8. M e t h i o n i n e c o n c e n t r a t i o n s i n p l a s m a w e r e s i g n i f i c a n t l y l o w e r when i s o l e u c i n e was i n f u s e d a l o n e (#4) t h a n when i n f u s e d i n c o m b i n a t i o n w i t h o t h e r a m i n o a c i d s (#5, #6, #8 a n d #9 ) . L e u c i n e c o n c e n t r a t i o n s w e r e h i g h e r i n i n f u s i o n #6 t h a n i n #2 ( c a s e i n ) o r #4 ( i s o l e u c i n e ) . L e u c i n e l e v e l s w e r e t h e l o w e s t when c a s e i n was i n f u s e d . P e r i o d e f f e c t s w e r e o b s e r v e d f o r v a l i n e , i s o l e u c i n e a n d l e u c i n e ( T a b l e I V - 2 B ) . T h e c o n c e n t r a t i o n o f e a c h o f t h e s e a m i n o a c i d s was f o u n d t o i n c r e a s e a s e a c h i n f u s i o n p r o g r e s s e d . T h e r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s t o t o t a l n o n - e s s e n t i a l a m i n o a c i d s was f o u n d t o i n c r e a s e a s e a c h i n f u s i o n p r o g r e s s e d . T h e r a t i o o f g l y c i n e t o t h e b r a n c h e d c h a i n a m i n o a c i d s ( v a l i n e , i s o l e u c i n e , l e u c i n e ) d e c r e a s e d a s e a c h i n f u s i o n p r o g r e s s e d . I n f u s i o n e f f e c t s o n t h e n o n - e s s e n t i a l a m i n o a c i d s w e r e n o t e d f o r s e r i n e , a s p a r a g i n e , g l u t a m i n e , g l u t a m i c a c i d , c i t r u l l i n e and a l a n i n e ( T a b l e I V - 3 A ) . T h e c o n c e n t r a t i o n o f s e r i n e was s i g n i f i c a n t l y h i g h e r i n i n f u s i o n #4 ( i s o l e u c i n e ) t h a n i n a l l t h e o t h e r i n f u s i o n s . S i m -i l a r l y a s p a r a g i n e and g l u t a m i n e c o n c e n t r a t i o n i n p l a s m a was h i g h e s t w i t h i n f u s i o n #4 a n d #8 ( i s o l e u c i n e a n d l e u c i n e ) . The l e v e l o f a s p a r a g i n e a n d g l u t a m i n e was s i g n i f i c a n t l y l o w e r w i t h TABLE IV-2A ISOLEUCINE INFUSIONS ESSENTIAL AMINO ACIDS u g/ml BLOOD PLASMA - INFUSION EFFECTS LEAST'SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 2 4 5 6 8 9 S.E. THREONINE 10.733 - .771 .347 .129 .580 .203 - .081 .458 VALINE 43.586 *- 5.910a - 1.719* 4.351 b 3.389b -4.844 a 4.233b 2.319 METHIONINE 6.867 *- .433* ' - 1.443b .482a .903a • 557a .015 a .403 ISOLEUCINE 22.307 - 3.492 1.193 .261 1.663 .351 1.39^° .019 1.281 LEUCINE 27.205 *- 4.649a - 2.570* 1.63ihc 3.126c .465^ " 1.420 PHENYLALANINE 9.345 .359 • - .135 .375 - .141 .210 - .668 .442 LYSINE 18.437 .283 1.810 1.292 .722 -1.657 -2.500 1.223 HISTIDINE 14.940 - 1.779 .602 - .319 - .062 1.064 .494 .706 ARGININE 18.154 - 2.404 2.314 -1.383 1.630 1.282 -1.439 1.071 TESS 1 176.626 -18.856 .403 7.321 11.811 -1.240 .561 7.71 TNESS2 175.352 -13.278 12.612 -7.128 - .429 9.762 -1.541 5.266 RATIO3 1.011 - .033 - .071 .079 .069 - .061 - .019 .037 4 RGLY .272 .040 .040 - .040 - .015 - .005 - .020 .019 TOTAL5 351.979 -32.133 13.016 .194 11.381 .8.521 - .979 11.490 GLUCOSE6 59.997 ** 1.956b 4.169a 1.369* 3.311* 1.419* -8.314 1.519 * P <.05. ** P <.01. a,b,c Constants within rows with the same superscripts are not s i g n i f i c a n t l y d i f f e r e n t . " ' T E S S Total e s s e n t i a l amino acids y g/ml blood plasma. TNESS Total non-essential amino acids (not including urea) y g/ml blood plasma. 3 RATIO Tess/Tness. 4RGLY Glycine/Valine + Isoleucine + Leucine. 5 TOTAL Tess + Tness. ^GLUCOSE mg/100 ml plasma. 117 TABLE IV-2B ISOLEUCINE INFUSIONS ESSENTIAL AMINO ACIDS u g/ml BLOOD PLASMA - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER M 1 3^ ! S.E. 2 S.E. 3 S.E. THREONINE 10.733 .157 .324 .244 .271 - .401 • 271 VALINE 48.586 **_ 6.443a 1.639 3.089b 1.372 3.355b 1. 372 METHIONINE 6.867 - .718 .285 .532 .238 .187 • 238 ISOLEUCINE 22.307 3.919a .906 1.870b .758 2.049b 758 LEUCINE 27.205 **_ 4.0253 1.004 1.63^ .840 2.388b ,840 PIEMYLAIANINE 9.345 .009 .312 .236 .262 - .226 ,262 LYSINE 18.487 .317 .864 .815 .723 -1.132 .723 HISTIDINE 14.940 .019 .499 .195 .418 - .214 .418 ARGININE 18.154 1.016 .757 .231 .634 -1.247 .634 TESS 1 176.626 13.607 5.455 8.849 4.564 4.759 4 .564 2 TNESS 175.352 1.578 3.723 3.875 3.115 -5.453 3 .115 RATIO3 1.011 *_ • .090a .026 .031b .022 .059b .022 RGLY4 .272 ** .060a .013 - .021b .011 - ,039b .011 TOTAL5 351.979 •12.030 8.124 12.724 6.797 - .694 6 .797 GLUCOSE6 59.997 1.127 1.074 - .501 .899 - .626 .899 * P <.05. ** P <.01. a,b Constants within rows with the same superscripts are not s i g n i f i c a n t l y d i f f e r e n t . ^TESS Total essential amino acids y g/ml blcod plasma. 2TNESS Total non-essential amino acids (not including urea) u g/ml blcod plasma. RATIO Tess/Tness. 4RGLY Glycine/Valine + Isoleucine + Leucine. 5TOTAL Tess + Tness. 6GLUCOSE nrj/100 uil plasma. TABLE IV-3A ISOLEUCINE INFUSIONS NON-ESSENTIAL AMINO ACIDS u g/ml BLOOD PLASMA - INFUSION EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 2 4 5 6 8 9 S.E. TAURINE 7.644 - 1.916 .859 .237 .112 .599 .109 .931 UREA 184.03 11.388 -2.505 -1.180 -12.269 14.541 -9.975 15.396 SERINE 11.119 **- 1.1663 1.586b .066a .053a - .424a - .116a .342 ASPARAGINE1 50.778 *- .787* 7.357° -3.16^ - 2.578* 4.125b° -4.955a 2.200 PROLINE 10.393 - .513 - .215 .337 1.240 - .138 - .710 .730 GLUTAMIC ACID 8.032 **- 1.286a -2.240a -1.457a - 1.860a 3.632b 3.211b .749 CITRULLINE 18.412 **- 6.945a .279b 2.261b 1.716b 1.834b .854b .910 GLYCINE 27.714 - .672 2.695 -2.344 1.222 .001 - .902 1.394 ALANINE 18.879 * .407* 1.397a -2.218° - .559°° .404* .569* .530 a-AMINO-N- .965 .138 - .010 - .109 - .052 .035 - .002 .053 BUTYRIC ACID CYSTINE 4.774 .193 - .587 - .539 - .040 .088 .885 .401 TYROSINE 9.118 .541 .544 - .159 - .041 - .454 - .431 .492 ORNITHINE 7.524 - 1.276 .954 - .039 .357 .057 - .053 .456 * P <.05. ** P <.01. i -co a ,b,c Constants within rows with the same superscripts are not significantly different. "^Asparagine + Glutamine. #4 a n d #8 t h a n i n f u s i o n #9 ( v a l i n e , i s o l e u c i n e , l e u c i n e ) . G l u t a m i c a c i d c o n c e n t r a t i o n i n p l a s m a was e l e v a t e d d u r i n g . . i n f u s i o n s #8 ( i s o l e u c i n e , l e u c i n e ) a n d #9 ( i s o l e u c i n e , l e u c i n e , v a l i n e ) a b o v e l e v e l s o b s e r v e d d u r i n g t h e o t h e r i n f u s i o n s . C i t -r u l l i n e l e v e l s w e r e s i m i l a r f o r a l l i n f u s i o n s e x c e p t f o r i n f u -s i o n #2 ( c a s e i n ) when i t was s i g n i f i c a n t l y l o w e r . A l a n i n e c o n c e n t r a t i o n was h i g h e s t w i t h i n f u s i o n #4 ( i s o l e u c i n e ) a n d l o w e s t w i t h #5 ( a l l e s s e n t i a l a m i n o a c i d s ) and #6 ( v a l i n e , m e t h i o n i n e , i s o l e u c i n e , l e u c i n e ) . T h e r e w e r e s i g n i f i c a n t p e r i o d e f f e c t s f o r s e r i n e , p r o l i n e and a l a n i n e ( T a b l e I V - 3 B ) . T h e c o n c e n t r a t i o n o f e a c h a m i n o a c i d d e c r e a s e d a s e a c h i n f u s i o n p r o g r e s s e d . i i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s a p r o -p o r t i o n o f t h e t o t a l e s s e n t i a l o r n o n -e s s e n t i a l a m i n o a c i d s When i n f u s i o n e f f e c t s o n t h e p r o p o r t i o n o f e s s e n t i a l a m i n o a c i d s w e r e s t u d i e d ( T a b l e I V-4A) , t h e v a l u e s f o r v a l i n e and l e u c i n e showed s i m i l a r r e a c t i o n s t o t h e d i f f e r e n t i n f u s i o n s . T h e p r o p o r t i o n s o f b o t h w e r e i n c r e a s e d i n i n f u s i o n s i n w h i c h t h e y w e r e i n c l u d e d i n t h e p u r e f o r m . T h e v a l u e s w e r e a l s o h i g h e r when t h e r e w e r e f e w e r a m i n o a c i d s i n c l u d e d i n t h e i n f u -s i o n m i x t u r e . F o r e x a m p l e t h e p r o p o r t i o n o f l e u c i n e was h i g h e r when i s o l e u c i n e a n d l e u c i n e w e r e i n f u s e d t h a n when v a l i n e , i s o l e u c i n e a n d l e u c i n e w e r e i n f u s e d . M e t h i o n i n e v a l u e s w e r e l o w e r when i s o l e u c i n e was i n f u s e d a l o n e t h a n when i s o l e u c i n e was i n c o m b i n a t i o n w i t h a n y o f t h e o t h e r a m i n o a c i d s . P h e n y l a l a n i n e v a l u e s w e r e h i g h e r w i t h t h e TABLE IV-3B ISOLEUCINE INFUSIONS NON-ESSENTIAL AMINO ACIDS y g/ml BLOOD PLASMA - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER MEAN 1 S.E. 2 S.E. '3 S.E. TAURINE 7. ,644 .584 .658 - .213 .551 .797 .551 UREA 184. ,03 -13, .550 10.886 12.615 9.108 .935 9.108 SERINE 11. .119 * .639 a .242 . 0 2 2 * .202 - , .661 b .202 ASPARAGINE 1 50. ,778 - , .587 1.555 2.120 1.302 -1, .533 1.302 PROLINE 10. .393 * .930 a .516 .303 a .432 -1, .233° .432 GLUTAMIC ACID 8, .032- .004 .529 - .057 .443 .053 .443 CITPLJLLINE 18. .412 - 1. .356 .643 .849 .538 .506 .538 GLYCINE 27, .714 1 .810 .985 - .042 .834 -1 .769 .824 ALANINE 18, .879 ** .825 a .374 .376 a .313 -1 .202° .313 a-AMINO-N-BUTYRIC ACID .965 - .078 .037 .031 .031 .048 .031 CYSTINE 4, .774 - .090 .283 - .098 .237 .188 .237 TYROSINE 9, .118 .226 .348 .368 .291 - .594 .291 ORNITHINE 7 .524 - .164 .322 .216 .270 .052 .270 * P <.05. ** P <.01. ^"Asparagine + Glutamine. a,b Constants within rows with the same superscripts are not significantly different. TABLE TV-4A ISOLEUCINE INFUSIONS ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL ESSENTIAL AMINO ACIDS - INFUSION EFFECTS LEAST SQUARES MEAN 2 LEAST SQUARES CONSTANTS - INFUSION NUMBER 4 5 6 8 9 S.E. THREONINE 6.105 .197 .214 - .155 -.090 - .105 - .061 .161 VALINE 27.449 **- .487*° 1.069* 1.594°° .116^ -2.449a 2.294d .668 METHIONINE 3.882 * .144a - .811b .09l a .229a .352a .006a .175 ISOLEUCINE 12.564 - .669 .565 - .342 .131 .313 .003 .286 LEUCINE 15.338 **-1.060a -1.498a .31$ .737^ 1.267° .237b .251 PHENYLALANINE 5.327 ** .826a - .124b .016b -.430b .123b - .412b .172 LYSINE 10.507 ** 1.394a 1.058* .160*° „_,-bcd - . Z ID - .905°d -1.432d .402 HISTIDINE 8.500 - .079 .363 - .544 -.607 .634 .233 .331 ARGININE 10.327 **- .272* 1.302° -1.130a .188*° .767^ - .855a .379 ** P <.01. a,b,c,d Constant within rows with the same superscripts are not significantly different. 122 c a s e i n i n f u s i o n t h a n w i t h a n y o t h e r i n f u s i o n . T h e p r o p o r t i o n o f l y s i n e d e c r e a s e d i n a v e r y r e g u l a r f a s h i o n a s t h e e x p e r i m e n t p r o g r e s s e d . T h e r e d i d n o t a p p e a r t o be a n y p a t t e r n t o t h e f l u c t u a t i o n i n a r g i n i n e p r o p o r t i o n s . V a l u e s w e r e l o w e s t w i t h i n f u s i o n #5 ( a l l e s s e n t i a l a m i n o a c i d s ) a n d #6 ( v a l i n e , m e t h i o -n i n e , i s o l e u c i n e , l e u c i n e ) and h i g h e s t w i t h i n f u s i o n #4 ( i s o -l e u c i n e a l o n e ) . T h e p e r i o d e f f e c t s f o r t h e p r o p o r t i o n o f t h e e s s e n t i a l a m i n o a c i d s w e r e much more s t r a i g h t f o r w a r d . The b r a n c h e d c h a i n a m i n o a c i d s ( v a l i n e , i s o l e u c i n e , l e u c i n e ) a l l i n c r e a s e d s i g n i f -i c a n t l y a s e a c h i n f u s i o n p r o g r e s s e d ( T a b l e I V - 4 B ) . T h e p r o p o r t i o n s o f l y s i n e , h i s t i d i n e a n d a r g i n i n e d e c r e a s e d s i g n i f i c a n t l y f r o m p e r i o d 1 v a l u e s d u r i n g p e r i o d s 2 and 3 . T h r e o n i n e and p h e n y l a l a n i n e showed a s i g n i f i c a n t i n t e r a c t i o n t e r m a n d w i l l be d i s c u s s e d l a t e r . M e t h i o n i n e was t h e o n l y a m i n o a c i d w h i c h d i d n o t show a n y s i g n i f i c a n t d e v i a t i o n s b e t w e e n p e r i o d s . B o t h t h r e o n i n e a n d p h e n y l a l a n i n e e x h i b i t e d a s i g n i f i c a n t i n t e r a c t i o n t e r m i n d i c a t i n g a d i f f e r e n t r e s p o n s e b e t w e e n p e r i o d s f o r t h e d i f f e r e n t i n f u s i o n s ( T a b l e I V - 5 C ) . O n l y i n i n f u s i o n #4, #6 a n d #9 w e r e t h e r e s i g n i f i c a n t d i f f e r e n c e s b e t w e e n p e r i o d s i n t h r e o n i n e . When e x p r e s s e d a s a p r o p o r t i o n o f t h e t o t a l e s s e n t i a l a m i n o a c i d s t h e p e r c e n t a g e t h r e o n i n e was s i g n i f i c a n t l y l o w e r i n p e r i o d 3 t h a n i n p e r i o d 1 d u r i n g i n f u s i o n s #6 a n d #9. D u r i n g i n f u s i o n #4 t h e p e r c e n t a g e t h r e o n i n e was s i g n i f i c a n t l y d e c r e a s e d d u r i n g p e r i o d 2 b u t i n c r e a s e d t o a n i n t e r m e d i a t e v a l u e d u r i n g p e r i o d 3 . T h e p r o p o r t i o n o f p h e n y l a l a n i n e i n p l a s m a showed TABLE IV-4B ISOLEUCINE INFUSIONS ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF TOTAL ESSENTIAL AMINO ACIDS PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER MEANS 1 S.E. 2 S.E. 3 S.E. THREONINE 5. ,994 ** ,554a .114 - .178b .095 - .376b .095 VALINE 27. ,449 *. •1. ,599a .472 .447b .395 1.15^ .395 METHIONINE 3. ,882 .117 .124 .113 .104 .004 .104 ISOLEUCINE 12. .564 **. -1. .292a .202 .465b .169 .827b .169 LEUCINE 15. .338 -1. .140a .178 .222b .149 .917C .149 PHENYLALANINE 5. .327 * .440a .122 - .158b .102 - .282b .102 LYSINE 10. .507 ** 1. .055a .284 - .134b .238 - .920b .238 HISTIDINE 8, .500 * .691a .234 - .33813 .196 - .353b .196 ARGININE 10, .327 ** 1, .408a .268 - .440b .224 - .968b .224 * P <.05. * * P <.01. a,b = Constants within rows with the same superscripts are not significantly different. 124 s i g n i f i c a n t d i f f e r e n c e s b e t w e e n p e r i o d s f o r i n f u s i o n s #2, #5 a n d #9. D u r i n g i n f u s i o n #2 s i g n i f i c a n t l y l o w e r p r o p o r t i o n s o f p h e n y l a l a n i n e w e r e p r e s e n t i n t h e p l a s m a d u r i n g p e r i o d 3 t h a n i n p e r i o d 1 o r 2 . T h e r e s u l t s w e r e s i m i l a r f o r i n f u s i o n #5 e x c e p t t h a t t h e v a l u e f o r p e r i o d 2 was i n t e r m e d i a t e a n d n o t d i f f e r e n t f r o m t h e f i r s t o r t h i r d p e r i o d s . . D u r i n g i n f u s i o n #9 t h e p r o p o r t i o n o f p h e n y l a l a n i n e d e c r e a s e d s i g n i f i c a n t l y i n t h e m i d d l e p e r i o d and r o s e t o an i n t e r m e d i a t e v a l u e i n t h e t h i r d p e r i o d . T h e r e d i d n o t a p p e a r t o be a n y p a t t e r n b e t w e e n t h e p r o p o r -t i o n s o f t h e n o n - e s s e n t i a l a n d e s s e n t i a l a m i n o a c i d s ( T a b l e I V -5 A ) . S e r i n e , a s p a r a g i n e a n d g l u t a m i n e d i d e x h i b i t h i g h e s t v a l u e s w i t h i n f u s i o n #4 ( i s o l e u c i n e a l o n e ) . T h e l o w e s t p r o p o r -t i o n f o r s e r i n e was w i t h i n f u s i o n #8 ( i s o l e u c i n e a n d v a l i n e ) . The p r o p o r t i o n o f a s p a r a g i n e a n d g l u t a m i n e was l o w e s t w i t h i n f u s i o n #9 ( v a l i n e , i s o l e u c i n e , l e u c i n e ) . G l u t a m i c a c i d v a l u e s w e r e d e p r e s s e d when c a s e i n ( i n f u s i o n #1) a n d i s o l e u c i n e a l o n e ( i n f u s i o n #4) w e r e i n f u s e d . T h e p r o p o r t i o n o f a l a n i n e was h i g h e s t when c a s e i n was i n f u s e d a n d l o w e s t when a l l o f t h e e s s e n t i a l s ( i n f u s i o n #5) w e r e i n f u s e d . When p e r i o d e f f e c t s f o r t h e p r o p o r t i o n s o f t h e n o n - e s s e n t i a l a m i n o a c i d s w e r e s t u d i e d t h e r e s u l t s w e r e much more s t r a i g h t f o r w a r d . T h e r e w e r e s i g n i f i c a n t d e c l i n e s i n t h e p r o p o r t i o n s o f s e r i n e , p r o l i n e and a l a n i n e a s e a c h i n f u s i o n p r o g r e s s e d ( T a b l e I V -5B) . A s i g n i f i c a n t t r e a t m e n t b y p e r i o d i n t e r a c t i o n t e r m was f o u n d f o r c i t r u l l i n e ( T a b l e I V - 5 C ) . D u r i n g i n f u s i o n #2 and #9 TABLE IV-5A ISOLEUCINE INFUSIONS NON-ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL NON-ESSENTIAL AMINO ACIDS INFUSION EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 2 4 5 6 8 9 S.E. TAURINE 4. 358 - .795 .198 .312 .095 .053 .137 .498 SERINE 6. 338 - . 2 0 1 * .442 d cd .332 . 0 4 1b o d - .561 a - .054 b C .132 ASPARAGINE 1 28. 940 * 1.829 3 1.945 a - .686*° -1.36^ . 7 4 2 * -2.465° .869 PROLINE 5. 910 .161 - .520 .470 .736 - .374 - .474 .291 GLUTAMIC ACID 4. 587 - .409 a -1.490 a - .672 a -1.050 a 1.714 b 1.908 .338 CITRULLINE 10. 505 **. -3.365 a - .597 b 1.811 C .985° .4 2 1 b G .745 b G .424 GLYCINE 15. 797 .884 .383 - .706 .758 - .830 - .489 .602 ALANINE 10. 775 ** 1.097 a be .020 - .845d - .317°* - .340 C d .385 b .193 a-AMINO-N- 554 .128 - .044 - .048 - .029 - .012 .006 .036 BUTYRIC ACID CYSTINE 2. 737 .343 - .494 - .232 - .032 - .105 .520 .260 TYROSINE 5. 216 .743 - .072 .111 - .030 - .522 - .229 .246 ORNITHINE 4. 286 - .416 .231 .148 .206 - .186 .016 .198 * P <.05. ** P <.01. Asparagine + Glutamine. a,b,c,d Constants w i t h i n rows w i t h the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t . TABLE IV-5B ISOLEUCINE INFUSIONS NON-ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF TOTAL NON-ESSENTIAL AMINO ACIDS PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER MEANS 1 S.E. 2 S.E. 3 S.E. TAURINE 4. 358 - .402 .352 - .185 .295 .587 .295 SERINE 6. 338 * .302a .093 - .112b .078 -.190b .078 ASPARAGINE1 28. 940 - .481 .614 .476 .514 .005 .514 PROLINE 5. 910 * .483a .206 .039* .172 -.522b .172 GLUTAMIC ACID 4. 582 - .070 .239 - .096 .200 .167 .200 CITRULLINE 10. 505 *_ .859a .299 .225b .250 .634b .250 GLYCINE 15. 797 .898 .426 - .412 .356 -.486 .356 ALANINE 10. 775 * .357a .136 .007* .114 -.364b .114 a-AMI!]C>-N-BUTYRIC ACID ,554 - .052 .025 .008 .021 .045 .021 CYSTINE 2. 737 - .109 .184 .- .094 .154 .203 .154 TYROSINE 5. ,216 .079 .174 .111 .146 -.190 .146 ORNITHINE 4. ,286 — .146 .140 .033 .117 .113 .117 * P <.05. •^Asparagine + Glutamine. a,b Constants within rows with the same superscripts are not significantly different. 127 TABLE IV-5C ISOLEUCINE INFUSIONS AMINO ACIDS EXPRESSED AS A PROPORTION OF THE TOTAL ESSENTIAL OR NON-ESSENTIAL PLASMA AMINO ACIDS - SIGNIFICANT INFUSION X PERIOD INTERACTION TERMS LEAST SQUARES CONSTANTS THREONINE PHENYLALANINE CITRULLINE LEAST SQUARES MEAN 5.994 S . E . 5.327 S . E . 10.505 S . E . IN 2 IN 2 IN 2 P P P I 1 2 3 - 0 4 4 b G d e • 3 3 0 * ? . 3 0 5 ^ .360 .260 .260 1.813 a , 9 3 2 * - ! 2 6 7 c d e .384 .277 .277 de -4.138 - 4 . 7 8 5 s - 1 . 0 9 5 ^ .947 .683 .683 IN 4 IN 4 IN 4 P P P 1 2 3 . 9 4 6 * - 2 5 9 C d S bcde .360 .260 .260 • 0 8 3 ^ d .247*°* - . 7 0 2 d S .384 .277 .277 -2.536°? . 3 2 0 * ° .947 .683 .683 IN 5 IN 5 IN 5 P P P 1 2 3 ^^abcd - '.225fe - . 6 1 5 d e .360 .260 .260 . 7 1 3 * * ; - -053°? - . 6 1 2 d e .384 .277 .277 1 . 7 4 4 * 3 .020 a . 6 6 9 * .947 .683 .683 IN 6 IN 6 IN 6 P P P 1 2 3 • 7 7 5 S e - .oes^^ - . 980 e .360 .260 .260 .146*=° " - 5 8 2 d e - . 5 6 2 d e .384 .277 .277 . 6 0 0 * 1 . 1 1 0 * .947 .683 .683 IN 8 IN 8 IN 8 P P P 1 2 3 , -i rbcde . 0 8 5 ^ - . 2 8 5 G d e .360 .260 .260 . 0 0 3 ^ " . 3 1 2 ° ^ . 6 7 8 * ° .384 .277 .277 . 2 5 4 * ° .059°° .947 .683 .683 IN 9 IN 9 IN 9 P P P 1 2 3 1 .386 a - . 6 3 5 d e .360 .260 .260 . 1 7 3 ^ ^ - 1 . 1 8 2 8 , - , 2 2 7 C d e .384 .277 .277 -1.645 , 1 . 1 4 0 * 2 .740 a .947 .683 .683 a,b,c,d,e Constants within columns with the same superscripts are not sig-nificantly different. IN 1 P I In fus ion number and p e r i o d number. 128 t h e p r o p o r t i o n o f c i t r u l l i n e was l o w e s t i n p e r i o d 1 a n d i n c r e a s e d d u r i n g p e r i o d s 2 a n d 3 . W i t h i n f u s i o n #4 t h e p r o p o r t i o n o f c i t r u l l i n e i n c r e a s e d d u r i n g t h e s e c o n d p e r i o d b u t d e c r e a s e d t o a n i n t e r m e d i a t e v a l u e i n t h e t h i r d p e r i o d . (2) D i s c u s s i o n (a) M i l k a n d m i l k c o m p o s i t i o n T h i s s e r i e s o f i n f u s i o n s t o o k p l a c e o v e r a 134 d a y p e r i o d . T h e m i l k p r o d u c t i o n d r o p p e d 2.46 k g f r o m t h e b e g i n n i n g t o t h e e n d o f t h e e x p e r i m e n t . T h i s was n o t a l a r g e d r o p a n d t h e f a c t t h a t t h e r e w e r e t h r e e s i g n i f i c a n t l y d i f f e r e n t r a n g e s i n t h i s d r o p g i v e s some i n d i c a t i o n o f t h e s t a b i l i t y o f m i l k p r o d u c t i o n o v e r t h e e n t i r e p e r i o d . I n f u s i o n d i d n o t a p p e a r t o a f f e c t t h e n o r m a l d e c l i n e i n m i l k p r o d u c t i o n . T h e c o n c e n t r a t i o n o f p r o t e i n i n m i l k d i d a p p e a r t o show some e f f e c t s o f i n f u s i o n . D u r i n g a n o r m a l l a c t a t i o n t h e p r o t e i n c o n t e n t o f m i l k i s e x p e c t e d t o i n c r e a s e a s t h e l a c t a t i o n p r o -c e e d s . However i n t h i s s t u d y t h e p r o t e i n c o n t e n t o f m i l k was s t a b l e e x c e p t f o r two i n f u s i o n s , #4 ( i s o l e u c i n e ) a n d #6 ( v a l i n e , m e t h i o n i n e , i s o l e u c i n e , l e u c i n e ) . T h e p o s s i b l e e f f e c t s o f t h e v a r i o u s a m i n o a c i d l e v e l s w i l l b e d i s c u s s e d l a t e r u n d e r a m i n o a c i d s . T h e t o t a l p r o d u c t i o n o f p r o t e i n f o l l o w e d t h e r e s p o n s e o f m i l k p r o d u c t i o n w i t h t h e e x c e p t i o n o f i n f u s i o n #6 w h e r e t h e p r o d u c t i o n o f p r o t e i n was s i g n i f i c a n t l y l o w e r . T h i s r e s u l t i s e x p e c t e d f r o m t h e l o w e r e d m i l k p r o t e i n c o n t e n t and t h e n o n -s i g n i f i c a n t l o w e r e d m i l k p r o d u c t i o n o b s e r v e d d u r i n g i n f u s i o n #6. 129 L a c t o s e c o n c e n t r a t i o n i n m i l k was s i g n i f i c a n t l y l o w e r d u r -i n g i n f u s i o n s #2 a n d #6. D u r i n g a n o r m a l l a c t a t i o n l a c t o s e c o n c e n t r a t i o n t e n d e d t o d e c r e a s e when p r o t e i n c o n c e n t r a t i o n s a r e i n c r e a s i n g . ( E b n e r , 1 9 7 1 ) . In t h i s c a s e t h e c o n c e n t r a t i o n o f l a c t o s e b e a r s l i t t l e r e s e m b l a n c e t o t h e c o n c e n t r a t i o n o f p r o t e i n i n m i l k . T o t a l l a c t o s e p r o d u c t i o n was s i m i l a r t o m i l k p r o d u c t i o n e x c e p t t h a t d u r i n g i n f u s i o n #6 l a c t o s e p r o d u c t i o n was s i g n i f i c a n t l y l o w e r . T h e s i g n i f i c a n t d e c r e a s e i n m i l k p r o d u c t i o n i n t h e t h i r d p e r i o d o f e a c h i n f u s i o n m u s t be a t t r i b u t e d t o t h e i n f u s i o n m a t e r i a l . T h i s d e c r e a s e d m i l k p r o d u c t i o n d o e s c o i n c i d e w i t h i n c r e a s e d m i l k p r o t e i n c o n c e n t r a t i o n ( T a b l e I V - 1 B ) . T h e d e c r e a s e i n t o t a l p r o t e i n p r o d u c t i o n t o p r e - i n f u s i o n v a l u e s d u r i n g t h e l a s t p e r i o d o f t h e i n f u s i o n c a n be a t t r i b u t e d t o t h e d e c r e a s e d m i l k p r o d u c t i o n . I t a p p e a r s t h a t e v e n w i t h t h e a d d e d a m i n o a c i d s and i n c r e a s e d p r o t e i n c o n c e n t r a t i o n i n m i l k t h e a n i m a l i s a b l e t o s e c r e t e o n l y s o much p r o t e i n p e r d a y . The s t i m u l a -t i o n o f p r o t e i n s y n t h e s i s b y t h e b r a n c h e d c h a i n a m i n o a c i d s may be b r o u g h t a b o u t b y t h e r e m o v a l o f some o t h e r f a c t o r n e c e s -s a r y t o m a i n t a i n p r o t e i n s y n t h e s i s , i . e . g l u c o s e . (b) P l a s m a a m i n o a c i d s i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s m i c r o -g rams p e r m l o f b l o o d p l a s m a When i n f u s i o n e f f e c t s f o r a m i n o a c i d s w e r e s t u d i e d t h e l e v e l s o f v a l i n e i n p l a s m a w e r e e l e v a t e d when v a l i n e was i n c l u d e d i n t h e i n f u s i o n m i x t u r e ( i n f u s i o n s #5, #6 a n d #9) . I f i s o l e u c i n e 130 i s t h e f i r s t l i m i t i n g a m i n o a c i d , a d d i t i o n o f i s o l e u c i n e t o t h e m e t a b o l i c p o o l s s h o u l d r e s u l t i n a d e c l i n e i n t h e c o n c e n t r a t i o n o f o t h e r a m i n o a c i d s ( S n y d e r m a n e t a l . , 1 9 6 8 ) . T h e d e p r e s s i o n o f m e t h i o n i n e c o n c e n t r a t i o n s u p p o r t s t h i s h y p o t h e s i s . I f i s o -l e u c i n e was n o t l i m i t i n g , t h e l e v e l s o f v a l i n e a n d l e u c i n e i n p l a s m a w o u l d be e x p e c t e d t o d r o p d u r i n g p e r i o d s 2 and 3 when t h e y w e r e n o t i n c l u d e d i n t h e i n f u s i o n m i x t u r e . T h i s e x p e c t e d d r o p i s due t o t h e f a c t t h a t t h e f i r s t t h r e e e n z y m e s i n v o l v e d i n t h e b r e a k d o w n o f i s o l e u c i n e a r e a l s o t h e same o n e s u s e d i n t h e f i r s t t h r e e s t e p s i n t h e b r e a k d o w n o f v a l i n e and l e u c i n e ( K a p l a n a n d P i t o t , 1 9 7 0 ) . E x c e s s i s o l e u c i n e w o u l d c a u s e an i n c r e a s e i n t h e s e enzyme s y s t e m s a n d r e s u l t s i n l o w e r e d l e v e l s o f t h e two a m i n o a c i d s i n t h e l a s t p e r i o d o f i n f u s i o n ( P h a n -s a l k a r e t a_l. , 1 9 7 0 ) . T h e f a c t t h a t t h i s d e c r e a s e i n v a l i n e , and l e u c i n e was n o t o b s e r v e d a l s o s u g g e s t s t h a t i s o l e u c i n e was n o t p r e s e n t i n e x c e s s a m o u n t s . T h e i n c r e a s e d r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s t o t o t a l n o n - e s s e n t i a l a m i n o a c i d s c a n be p a r t l y e x p l a i n e d b y t h e s i g n i f -i c a n t i n c r e a s e s o b s e r v e d f o r t h e b r a n c h e d c h a i n a m i n o a c i d s a s e a c h i n f u s i o n p r o g r e s s e d . In g e n e r a l t h e n o n - e s s e n t i a l a m i n o a c i d s , m o s t n o t a b l y s e r i n e , p r o l i n e a n d a l a n i n e , d e c r e a s e d s i g -n i f i c a n t l y o v e r e a c h i n f u s i o n . H a l f p e n n y ejt a l_ . (1969) f o u n d a d e c l i n e i n p l a s m a e s s e n t i a l a m i n o a c i d s w i t h i n c r e a s i n g p r o t e i n p r o d u c t i o n . T h e a b o v e i n c r e a s e s h o w e v e r w e r e due t o i n c r e a s e d e n e r g y i n t a k e . W h i l e t h e s u p p l y o f p r o p i o n a t e w o u l d be i n c r e a s e d t h e s u p p l y o f m i c r o b i a l p r o t e i n w o u l d a l s o m o s t l i k e l y i n c r e a s e due t o t h e r e l a t i o n s h i p b e t w e e n d i g e s t i b l e d i e t a r y e n e r g y and m i c r o b i a l c e l l s y n t h e s i s ( W a l k e r , 1 9 6 5 ) . T h e i n c r e a s e d p r o d u c -t i o n o f m i l k p r o t e i n a n d p e r h a p s a n i n s u l i n e f f e c t ( K n i p f e l e t a l . 1 9 6 9 ; G r i m b l e and W h i t e h e a d , 1970) c o u l d be r e s p o n s i b l e f o r t h e r e d u c e d p l a s m a a m i n o a c i d l e v e l s . T h e d e c r e a s e d g l y c i n e t o b r a n c h e d c h a i n a m i n o a c i d r a t i o i s a l s o p a r t l y e x p l a i n e d b y a n o n - s i g n i f i c a n t d e c r e a s e i n g l y c i n e o v e r e a c h i n f u s i o n a n d b y a s i g n i f i c a n t i n c r e a s e i n b r a n c h e d c h a i n a m i n o a c i d s o v e r t h e same p e r i o d . T h e d i f f e r e n c e s b e t w e e n n o n - e s s e n t i a l a m i n o a c i d s f o r e a c h i n f u s i o n a r e d i f f i c u l t t o e x p l a i n d u e t o t h e c o n f o u n d i n g e f f e c t o f l a c t a t i o n c y c l e . I t i s i n t e r e s t i n g t o n o t e t h a t s e r i n e , a s p a r a g i n e and g l u t a m i n e , a n d a l a n i n e c o n c e n t r a t i o n s w e r e e l e v a v/hen i s o l e u c i n e a l o n e was i n f u s e d ( i n f u s i o n #4) . T h e s i g n i f i c a n t d i f f e r e n c e s n o t e d v/hen t h e b e t w e e n p e r i o d e f f e c t s w e r e s t u d i e d a r e much more i m p o r t a n t . T h e d e c r e a s e s i n s e r i n e , p r o l i n e a n d a l a n i n e t h a t o c c u r r e d d u r i n g e a c h i n f u s i o n a r e i n d i c a t i v e o f i n c r e a s e d p r o t e i n s y n t h e s i s . H e a r d e t a l . (1969) s u g g e s t e d t h a t n o n - e s s e n t i a l a m i n o a c i d l e v e l s i n p l a s m a a r e i n d i c a t i v e o f t h e c a r b o h y d r a t e s t a t u s o f t h e a n i m a l w h i l e t h e e s s e n t i a l a m i n o a c i d s i n p l a s m a a r e i n d i c a t i v e o f t h e p r o -t e i n s t a t u s o f t h e a n i m a l . In t h e a b o v e c a s e t h e l o w e r i n g o f t h e c o n c e n t r a t i o n o f c e r t a i n o f t h e n o n - e s s e n t i a l a m i n o a c i d s when e s s e n t i a l a m i n o a c i d s a r e i n f u s e d c o u l d i n d i c a t e 1) g r e a t e r u t i l i z a t i o n o f t h e n o n - e s s e n t i a l a m i n o a c i d s f o r p r o t e i n s y n t h e s i s , a n d 2) g r e a t e r s t r e s s p u t o n t h e g l u c o n e o g e n i c a m i n o a c i d s t o s u p p l y g l u c o s e f o r t h e i n c r e a s e d m e t a b o l i c a c t i v i t y . 132 i i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s a p r o -p o r t i o n o f t h e t o t a l e s s e n t i a l o r n o n -e s s e n t i a l a m i n o a c i d s T h e p r o p o r t i o n s o f v a l i n e and l e u c i n e w e r e e x p e c t e d t o i n c r e a s e when t h e y w e r e i n f u s e d . T h a t t h e i n c r e a s e s w e r e n o t a s g r e a t when t h e y w e r e a c c o m p a n i e d by o t h e r a m i n o a c i d s m i g h t r e f l e c t a n i n c r e a s e d u t i l i z a t i o n o f a m i n o a c i d s f o r p r o t e i n s y n t h e s i s . T h e p r o p o r t i o n o f t h e t o t a l e s s e n t i a l a m i n o a c i d s c o n t r i b u t e d b y v a l i n e a n d l e u c i n e c o u l d be d e c r e a s e d b y an i n c r e a s e i n t h e c o n c e n t r a t i o n o f t h e o t h e r e s s e n t i a l a m i n o a c i d s . H o w e v e r , t h e p r o p o r t i o n s o f t h e a m i n o a c i d s o t h e r t h a n v a l i n e a n d l e u c i n e ( T a b l e I V - 4 A ) d i d n o t i n c r e a s e when t h e y w e r e i n f u s e d w i t h v a l i n e a n d l e u c i n e . T h e i n f u s i o n o f c a s e i n may h a v e r e s u l t e d i n a s p a r i n g a c t i o n on p h e n y l a l a n i n e by s u p p l y i n g t y r o s i n e . When t y r o s i n e was n o t a v a i l a b l e i n t h e i n f u s i o n m i x t h e i n c r e a s e d p r o t e i n s y n t h e s i s c o u l d h a v e i n c r e a s e d t h e demand o n p h e n y l a l a n i n e . T h e r e g u l a r d e c l i n e i n t h e p r o p o r t i o n o f l y s i n e i s d i f f i c u l t t o e x p l a i n . In E x p e r i m e n t I i t was n o t e d t h a t t h e p r o p o r t i o n o f l y s i n e i n c r e a s e d a s t h e l a c t a t i o n p r o g r e s s e d . T h e c o n c e n t r a t i o n o f l y s i n e was d e c r e a s i n g ( T a b l e I V - 4 A ) w h i l e t h e c o n c e n t r a t i o n o f t o t a l e s s e n t i a l a m i n o a c i d s was i n c r e a s i n g o r a t l e a s t r e m a i n -i n g s t e a d y ( T a b l e I V - 2 A ) . T h e p r o p o r t i o n o f a r g i n i n e t e n d e d t o be l o w e r when t h e i n f u -s i o n c o n t a i n e d a more c o m p l e t e m i x o f a m i n o a c i d s . K a p l a n and P i t o t (1970) s t a t e d t h a t a r g i n a s e , one o f t h e e n z y m e s i n v o l v e d i n t h e d e g r a d a t i o n o f a r g i n i n e , i s i n d u c e d b y d i e t a r y p r o t e i n . T h e more c o m p l e t e m i x o f a m i n o a c i d s i s p e r h a p s more l i k e l y t o 133 d u p l i c a t e t h e e f f e c t s o f d i e t a r y p r o t e i n . H i g h e r v a l u e s w e r e o b t a i n e d when i s o l e u c i n e a l o n e ( i n f u s i o n #4) o r i s o l e u c i n e a n d v a l i n e ( i n f u s i o n #8) w e r e i n f u s e d . When p e r i o d e f f e c t s w e r e s t u d i e d d i f f e r e n c e s w e r e n o t e d f o r a l l e s s e n t i a l a m i n o a c i d s e x c e p t m e t h i o n i n e . S i g n i f i c a n t i n t e r a c t i o n t e r m s w e r e o b s e r v e d f o r t h r e o n i n e a n d p h e n y l a l a n i n e and t h e y w i l l be d i s c u s s e d l a t e r . T h e p r o p o r t i o n s o f t h e b r a n c h e d c h a i n a m i n o a c i d s i n c r e a s e d s i g n i f i c a n t l y f r o m p r e -i n f u s i o n l e v e l s . T h e i n c r e a s e d v a l u e s w o u l d be e x p e c t e d f o r i s o l e u c i n e a s i t was i n c l u d e d i n a l l i n f u s i o n s . V a l i n e a n d l e u c i n e p r o p o r t i o n s w o u l d a l s o b e e x p e c t e d t o i n c r e a s e when t h e y w e r e i n c l u d e d i n t h e i n f u s i o n m i x , b u t a d e c l i n e o r a t l e a s t no i n c r e a s e w o u l d b e n o t e d when t h e y w e r e n o t i n c l u d e d . T h e i n t e r a c t i o n t e r m s f o r v a l i n e and l e u c i n e w e r e n o t s i g n i f -i c a n t w h i c h i n d i c a t e s t h a t t h e p e r i o d r e s p o n s e was t h e same f o r e a c h i n f u s i o n . T h e d e c r e a s e i n t h e p r o p o r t i o n o f t h e r e m a i n i n g e s s e n t i a l a m i n o a c i d s f r o m p r e - i n f u s i o n l e v e l s c a n be e x p l a i n e d i n t e r m s o f i n c r e a s e d p r o t e i n s y n t h e s i s . A m a j o r p o r t i o n o f t h i s i n c r e a s e w o u l d o c c u r i n t h e mammary g l a n d , h o w e v e r , t h e i n s u l i n o t r o p h i c e f f e c t o f c e r t a i n o f t h e b r a n c h e d c h a i n amino a c i d s c o u l d s t i m -u l a t e u p t a k e o f a m i n o a c i d s by m u s c l e a n d r e s u l t i n i n c r e a s e d m u s c l e p r o t e i n s y n t h e s i s ( K n i p f e l e t a l_ . , 1 9 6 9 ) . T h e i n t e r a c t i o n v a l u e s f o r t h r e o n i n e ( T a b l e I V - 5 C ) i n d i c a t e t h a t t h e p r o p o r t i o n o f t h r e o n i n e g r a d u a l l y d e c r e a s e d a s t i m e p r o g r e s s e d , s i m i l a r t o l y s i n e b u t i n d i s a g r e e m e n t w i t h E x p e r i -ment I w h e r e v a l u e s d i d n o t c h a n g e s i g n i f i c a n t l y . When t h e 134 b r a n c h e d c h a i n a m i n o a c i d s p l u s m e t h i o n i n e ( i n f u s i o n #6) o r j u s t t h e b r a n c h e d c h a i n a m i n o a c i d s ( i n f u s i o n #9) w e r e i n f u s e d , t h e p r o p o r t i o n o f t h r e o n i n e was s i g n i f i c a n t l y l o w e r i n t h e l a s t p e r i o d o f t h e i n f u s i o n t h a n i n t h e p r e - i n f u s i o n p e r i o d . W i t h t h e o t h e r i n f u s i o n s t h e r e was no c h a n g e o v e r t h e i n f u s i o n p e r i o d . T h i s d e c r e a s e d p r o p o r t i o n o f t h r e o n i n e a s s o c i a t e d w i t h i n f u s i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s i n d i c a t e s i n c r e a s e d u t i l i z a -t i o n o f t h r e o n i n e . I n f u s i o n o f c a s e i n o r a l l o f t h e e s s e n t i a l s d i d n o t c a u s e a r e d u c t i o n i n t h e p r o p o r t i o n o f t h r e o n i n e due t o i t s i n c l u s i o n i n t h e i n f u s a t e . I s o l e u c i n e a l o n e ( i n f u s i o n #4) o r i s o l e u c i n e and l e u c i n e ( i n f u s i o n #8) a p p a r e n t l y h a d no e f f e c t o n t h r e o n i n e u t i l i z a t i o n . P h e n y l a l a n i n e e x p r e s s e d a s a p r o p o r t i o n o f t h e t o t a l e s s e n -t i a l a m i n o a c i d s t e n d e d t o d e c r e a s e a s t h e i n f u s i o n s p r o g r e s s e d . T h i s c a n be s e e n b y l o o k i n g a t t h e p r e - i n f u s i o n p r o p o r t i o n s o f p h e n y l a l a n i n e f o r t h e v a r i o u s i n f u s i o n s ( T a b l e 1 1 - 2 7 ) . T h e s e r e s u l t s a r e s i m i l a r t o t h o s e o b s e r v e d i n E x p e r i m e n t I ( T a b l e I-4 B ) . T h e r e w e r e s i g n i f i c a n t d i f f e r e n c e s b e t w e e n p e r i o d s f o r i n f u s i o n s #2, #5, a n d #9. D u r i n g i n f u s i o n s #2 a n d #5 t h e p r o -p o r t i o n o f p h e n y l a l a n i n e d e c r e a s e d a s t h e i n f u s i o n p r o g r e s s e d . T h e s e w e r e a l s o t h e i n f u s i o n s i n w h i c h p h e n y l a l a n i n e was i n c l u d e d i n t h e i n f u s i o n m i x . T h e s e r e s u l t s i n d i c a t e t h a t p h e n y l a l a n i n e was b e i n g r e m o v e d f r o m p l a s m a a t a g r e a t e r r a t e d u r i n g t h e i n f u -s i o n p e r i o d t h a n i t was b e i n g a d d e d t o t h e p l a s m a . T h i s f a l l i n p h e n y l a l a n i n e c o n c e n t r a t i o n a l s o i n d i c a t e s t h a t p h e n y l a l a n i n e was n o t l i m i t i n g p r o t e i n s y n t h e s i s . T h e t r a n s i t o r y d e c r e a s e i n t h e p r o p o r t i o n o f p h e n y l a l a n i n e d u r i n g p e r i o d 2 i n i n f u s i o n #9 135 i s d i f f i c u l t t o e x p l a i n . T h e r e was s l i g h t l y more p r o t e i n p r o -d u c e d d u r i n g i n f u s i o n #9 t h a n d u r i n g t h e p r e v i o u s two i n f u s i o n s . No d e f i n i t e p a t t e r n e m e r g e s when t h e p e r c e n t a g e s o f t h e n o n - e s s e n t i a l a m i n o a c i d s a r e s t u d i e d . T h e p r o p o r t i o n o f a l a -n i n e i s h i g h e s t when n o n - e s s e n t i a l a m i n o a c i d s w e r e i n c l u d e d i n t h e i n f u s a t e ( i n f u s i o n #2) a n d l o w e s t when a l l t h e e s s e n t i a l a m i n o a c i d s w e r e i n f u s e d ( i n f u s i o n #5 ) . S e r i n e a n d a s p a r a g i n e a n d g l u t a m i n e a p p e a r t o r e s p o n d t o i s o l e u c i n e i n f u s i o n a l o n e ( i n f u s i o n #4) w i t h i n c r e a s e d p r o p o r t i o n s i n d i c a t i n g a d e c r e a s e d u t i l i z a t i o n o f n o n - e s s e n t i a l a m i n o a c i d s . T h e p e r i o d e f f e c t s f o r t h e p e r c e n t a g e s o f t h e n o n - e s s e n t i a l s a r e much more s t r a i g h t f o r w a r d . A s e a c h i n f u s i o n p r o g r e s s e d t h e p r o p o r t i o n s o f s e r i n e , p r o l i n e , and a l a n i n e d e c r e a s e d . T h e s e r e s u l t s c o i n c i d e w i t h t h e p r o t e i n c o n c e n t r a t i o n i n m i l k i n d i c a t -i n g i n c r e a s e d s y n t h e s i s o f p r o t e i n . W i t h i n c r e a s e d p r o t e i n s y n t h e s i s a d e c r e a s e i n p r o p o r t i o n o f t h e n o n - e s s e n t i a l s i s e x p e c t e d ( H a l f p e n n y e t a l . , 1 9 6 9 ) . When t h e p r e - i n f u s i o n e f f e c t s f o r e a c h i n f u s i o n were s t u d i e d w i t h r e g a r d s t o c i t r u l l i n e t h e p r o p o r t i o n o f c i t r u l l i n e i n c r e a s e d a s t h e e x p e r i m e n t p r o g r e s s e d . T h e r e was no r e s p o n s e i n E x p e r i -ment I w i t h r e g a r d t o p r o p o r t i o n o f c i t r u l l i n e . C i t r u l l i n e i s f o r m e d f r o m o r n i t h i n e by t h e a c t i o n o f t h e enzyme o r n i t h i n e c a r b a -m o y l t r a n s f e r a s e . K a p l a n and P i t o t (1970) s t a t e d t h a t t h e a c t i v i t y o f t h e enzyme i s i n c r e a s e d b y d i e t a r y p r o t e i n a n d c o r t i c o s t e r o i d s . A s t h e e x p e r i m e n t p r o g r e s s e d t h e r e w e r e s i g n i f i c a n t d e c l i n e s i n p r o t e i n p r o d u c t i o n w h i c h s h o u l d be r e f l e c t e d i n i m p r o v e d p r o t e i n s t a t u s o f t h e a n i m a l . T h e r e w e r e s i g n i f i c a n t p e r i o d r e s p o n s e s 136 f o r t h e p r o p o r t i o n o f c i t r u l l i n e d u r i n g i n f u s i o n s #2, #4, a n d #9. T h e p e r i o d r e s p o n s e o f c i t r u l l i n e d o e s n o t a p p e a r t o be r e l a t e d t o p l a s m a v a l u e s o f a r g i n i n e , o r n i t h i n e o r u r e a . T o s u m m a r i z e t h e e f f e c t s o f e a c h i n f u s i o n , t h e r e was a n i n c r e a s e i n t h e c o n c e n t r a t i o n o f p r o t e i n i n m i l k a b o v e p r e -i n f u s i o n l e v e l s . However m i l k p r o d u c t i o n d e c l i n e d d u r i n g p e r -i o d 3 s o t h a t p r o d u c t i o n o f p r o t e i n d u r i n g p e r i o d 3 was s i m i l a r t o p r o d u c t i o n d u r i n g p r e - i n f u s i o n . T h e r e was no d i f f e r e n c e b e t w e e n i n f u s i o n s i n m i l k a n d m i l k c o m p o n e n t p e r i o d r e s p o n s e . T h e c o n c e n t r a t i o n s o f i s o l e u c i n e , l e u c i n e , a n d v a l i n e i n c r e a s e d a b o v e p r e - i n f u s i o n l e v e l s d u r i n g e a c h i n f u s i o n . T h e c o n c e n t r a t i o n s o f s e r i n e , a l a n i n e a n d p r o l i n e d r o p p e d a s e a c h i n f u s i o n p r o g r e s s e d . T h e s e r e s u l t s t e n d t o i n d i c a t e a g r e a t e r r e m o v a l o f a m i n o a c i d s f r o m t h e p l a s m a d u r i n g e a c h i n f u s i o n . E x p r e s s i o n o f a m i n o a c i d s a s a p e r c e n t a g e o f t h e t o t a l a m i n o a c i d s f u r t h e r e m p h a s i z e d t h e a b o v e i n c r e a s e i n r e m o v a l o f a m i n o a c i d s f r o m p l a s m a . A l l o f t h e a m i n o a c i d s e x c e p t t h e b r a n c h e d c h a i n a m i n o a c i d s a n d m e t h i o n i n e w e r e f o u n d t o make up a s m a l l e r p e r c e n t a g e o f t h e t o t a l e s s e n t i a l a m i n o a c i d s a s e a c h i n f u s i o n p r o c e e d e d . M e t h i o n i n e p e r c e n t a g e d i d n o t c h a n g e b u t t h e p e r c e n t a g e o f v a l i n e , i s o l e u c i n e a n d l e u c i n e i n c r e a s e d . I t m u s t a l s o be p o i n t e d o u t t h a t v a l i n e , i s o l e u c i n e and l e u c i n e w e r e i n c l u d e d i n m o s t o f t h e d i f f e r e n t i n f u s i o n s . W h i l e t h e c o n c e n t r a t i o n o f p r o t e i n i n m i l k was i n c r e a s e d d u r i n g t h e s e i n f u s i o n s i t m u s t a l s o b e n o t e d t h a t t h e t o t a l s y n t h e s i s o f m i l k p r o t e i n was n o t i n c r e a s e d . I t a p p e a r s t h e r e -f o r e t h a t i f t h e i n f u s i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s d i d 137 s t i m u l a t e p r o t e i n s y n t h e s i s t h i s s t i m u l a t i o n r e m o v e d some f a c t o r w h i c h was n e c e s s a r y t o m a i n t a i n t o t a l m i l k p r o d u c t i o n . d) L y s i n e i n f u s i o n s (1) R e s u l t s (a) M i l k and m i l k c o m p o s i t i o n L y s i n e was i n f u s e d a l o n e o r i n c o m b i n a t i o n w i t h o t h e r e s s e n -t i a l a m i n o a c i d s i n f o u r s e p a r a t e i n f u s i o n s ( s e e T a b l e I I - l ) . E a c h i n f u s i o n was d i v i d e d i n t o t h r e e p e r i o d s , 1) p r e - i n f u s i o n , 2) t h e f i r s t 2 d a y s o f i n f u s i o n , a n d 3) t h e l a s t 2 d a y s o f i n f u s i o n . T h e r e were s i g n i f i c a n t i n t e r a c t i o n t e r m s f o r a l l o f t h e m i l k f a c t o r s e x c e p t t h e c o n c e n t r a t i o n o f p r o t e i n i n m i l k . V7hen i n f u s i o n e f f e c t s w e r e s t u d i e d ( T a b l e V - 1 A ) t h e c o n c e n t r a t i o n o f p r o t e i n i n m i l k was s i g n i f i c a n t l y l o w e r i n t h e l y s i n e i n f u s i o n ( i n f u s i o n #3) t h a n i n any o f t h e o t h e r i n f u s i o n s . I n f u s i o n #7 r e s u l t e d i n i n t e r m e d i a t e m i l k p r o t e i n c o n c e n t r a t i o n w h i l e i n f u s i o n o f a l l t h e e s s e n t i a l a m i n o a c i d s i n i n f u s i o n s #2 and #5 g a v e t h e g r e a t e s t c o n c e n t r a t i o n o f p r o t e i n i n m i l k . T h e r e was a l s o a s i g n i f i c a n t p e r i o d e f f e c t f o r m i l k p r o t e i n c o n c e n t r a t i o n ( T a b l e V -1 B ) . The v a r i o u s i n f u s i o n s r e s u l t e d i n s i g n i f i c a n t i n c r e a s e s i n m i l k p r o t e i n c o n c e n t r a t i o n i n t h e s e c o n d p e r i o d . H o w e v e r , m i l k p r o t e i n c o n c e n t r a t i o n f e l l t o an i n t e r m e d i a t e l e v e l i n p e r -i o d 3 w h i c h was n o t d i f f e r e n t f r o m t h e p e r i o d 1 ( p r e - i n f u s i o n ) l e v e l o r t h e l e v e l i n p e r i o d 2 . T a b l e V - 1 C i l l u s t r a t e s t h e i n t e r a c t i o n t e r m s f o r t h e r e m a i n -d e r o f t h e m i l k d a t a f o r t h e f o u r i n f u s i o n s . When k g m i l k w e r e 138 TABLE V-1A LYSINE INFUSIONS MILK AND MILK COMPOSITION - INFUSION EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MILK (kg) 21.904 ** 1.153a .816 a .056 b - 2.025C .160 PROTEIN (%) 3.118 ** .112 a - .145C .067 a - .033b .018 PROTEIN (gm) 683.157 **61.229a -7.892C 16.497b -69.834d 3.199 LACTOSE (%) 5.116 **- .034 a .155° - .023 b - .048* .013 LACTOSE (gm) 1121.344 **39.054a 76.729b -2.573° -113.210d 7.035 ** P <.01. a,b,c,d Constants within rows with the same superscripts are not significantly different. TABLE V-1B LYSINE INFUSIONS MILK AND MILK COMPOSITION - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER MEAN 1 S.E. 2 S.E. 3 S.E. MILK (kg) 21.904 *- .473a .146 .293* .122 .ISO13 .122 PROTEIN (%) 3.118 *- .048a .016 .039b .014 .009* .014 PROTEIN (gm) 683.157 **-25.190a 2.920 18.178° 2.443 7.012b 2.443 LACTOSE (%) 5.116 * .011 .012 - .016 .010 .005 .010 LACTOSE (gm) 1121.34 *-21.480a 6.422 10.903b 5.373 10.57* 5.373 * P <.05. ** P <.01. a,b,c, Constants within rows with the same superscripts are not significantly different. TABLE V-1C LYSINE INFUSIONS MILK AND MILK COMPOSITION - SIGNIFICANT INFUSION X PERIOD INTERACTION TERMS LEAST SQUARES CONSTANTS MILK (kg) PROTEIN (gm) LACTOSE (%) LACTOSE (gm) LEAST SQUARES MEAN 21.904 S.E. 683.157 S.E. 5.116 S.E. 1121.344 S.E. IN.2 IN 2 IN 2 P P P I 1 2 3 1.680* .665°° .369 .269 .269 45.682 78.673* 59.331° 7.39 5.38 5.38 -.096? -.115d -.041°d .300 .219 .219 63.904**: 30.076°? 23.181"* 16.24 11.84 11.84 IN 3 IN 3 IN 3 P P P 1 2 3 .779^ 2.250a .369 .269 .269 -45.217*; .804de 21.239° 7.39 5.38 5.38 .153a .153a .158a .300 .219 .219 3.245°^ 74.241 152.696a 16.24 11.84 11.84 IN 5 IN 5 IN 5. P P P 1 2 3 - - 3 6 i S e 774 - -.Ill* .369 .269 .269 -15.118S 48.193-, 16.416°d 7.39 5.38 5.38 -.091°* .300 .219 .219 29.145°d - 32.596e 16.24 11.84 11.84 IN 7 IN 7 IN 7 P P P 1 2 3 -2.630* -1.495?* -1.950 .369 .269 .269 -85.6071? -54.95612 -68.937g 7.39 5.38 5.38 -.061? -.006°° .300 .219 .219 -148.810f - 89.847^ -100.975 g 16.24 11.84 11.84 a,b,c,d,e,f,g,h ^ 1 P I Constants within columns with the same superscripts are not significantly different. Infusion number and period number. s t u d i e d a g e n e r a l d e c r e a s e c o u l d be s e e n i n t h e p r e - i n f u s i o n m i l k p r o d u c t i o n v a l u e s a s t h e e x p e r i m e n t p r o c e e d e d . I n g e n e r a l t h e r e was no p e r i o d e f f e c t o n m i l k p r o d u c t i o n e x c e p t when l y s i n e ( i n f u s i o n #3) was i n f u s e d . M i l k p r o d u c t i o n i n c r e a s e d s i g n i f -i c a n t l y a b o v e p r e - i n f u s i o n l e v e l s d u r i n g p e r i o d 2 a n d t h e r e was a f u r t h e r s i g n i f i c a n t i n c r e a s e i n p e r i o d 3 o f t h e l y s i n e i n f u -s i o n . I n f u s i o n #7 h a d t h e l o w e s t m i l k p r o t e i n p r o d u c t i o n . When p e r i o d r e s p o n s e s w e r e l o o k e d a t t h e g r e a t e s t r e s p o n s e i n p r o t e i n p r o d u c t i o n g e n e r a l l y o c c u r r e d d u r i n g p e r i o d 2 . However w i t h l y s i n e i n f u s i o n ( i n f u s i o n #3) t h e t o t a l p r o t e i n p r o d u c t i o n i n p e r i o d 2 was s i g n i f i c a n t l y h i g h e r t h a n p e r i o d 1 ( p r e - i n f u s i o n ) l e v e l s a n d t h e p r o d u c t i o n i n p e r i o d 3 was s i g n i f i c a n t l y h i g h e r t h a n i n p e r i o d 2 . I n g e n e r a l l a c t o s e c o n c e n t r a t i o n was h i g h e r d u r i n g i n f u s i o n #3 i n a l l t h r e e p e r i o d s . T h e r e was no p e r i o d e f f e c t f o r a n y o f t h e i n f u s i o n s e x c e p t #5 i n w h i c h t h e c o n c e n t r a t i o n o f l a c t o s e i n m i l k d r o p p e d s i g n i f i c a n t l y b e l o w p e r i o d 1 l e v e l s i n b o t h p e r -i o d s 2 a n d 3 . When p r e - i n f u s i o n v a l u e s f o r l a c t o s e p r o d u c t i o n (gm) w e r e s t u d i e d t h e l e v e l o f p r o d u c t i o n d e c l i n e d a s t h e e x p e r i m e n t p r o g r e s s e d . T h e p e r i o d e f f e c t s w e r e d i f f e r e n t f o r e a c h i n f u s i o n W i t h c a s e i n i n f u s i o n (#2) t h e r e was no d i f f e r e n c e b e t w e e n p e r i o d W i t h l y s i n e i n f u s i o n (#3) t h e r e was a s t e a d y a n d s i g n i f i c a n t i n c r e a s e f o r e a c h p e r i o d a b o v e t h e p r e - i n f u s i o n l e v e l . I n f u s i o n o f a l l t h e e s s e n t i a l a m i n o a c i d s (#5) r e s u l t e d i n s i g n i f i c a n t l y g r e a t e r l a c t o s e p r o d u c t i o n i n p e r i o d 2 t h a n p e r i o d 3 b u t n e i t h e r v a l u e s w e r e d i f f e r e n t t h a n p e r i o d 1. I n f u s i o n #7 ( t h r e o n i n e , m e t h i o n i n e , p h e n y l a l a n i n e , l y s i n e ) showed a s i g n i f i c a n t i n c r e a s e i n p e r i o d 2 a b o v e p r e - i n f u s i o n l e v e l s o f l a c t o s e p r o d u c t i o n . P e r i o d 3 p r o d u c t i o n was i n t e r m e d i a t e a n d n o t s i g n i f i c a n t l y d i f f e r e n t f r o m p e r i o d 1 o r 2 . (b) P l a s m a a m i n o a c i d s i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s m i c r o -g r a m s p e r m l o f b l o o d p l a s m a S i g n i f i c a n t i n f u s i o n e f f e c t s w e r e o b s e r v e d f o r v a l i n e , m e t h i o n i n e , i s o l e u c i n e , l e u c i n e a n d p h e n y l a l a n i n e ( T a b l e V - 2 A ) . In g e n e r a l t h e c o n c e n t r a t i o n s o f t h e a m i n o a c i d s w e r e h i g h e s t d u r i n g i n f u s i o n #5 ( a l l o f t h e e s s e n t i a l a m i n o a c i d s ) and l o w e s t d u r i n g #3 ( l y s i n e a l o n e ) . When p e r i o d e f f e c t s w e r e s t u d i e d ( T a b l e V - 2 B ) s i g n i f i c a n t r e s u l t s w e r e o b t a i n e d w i t h v a l i n e , m e t h i o n i n e , i s o l e u c i n e , l e u c i n e and p h e n y l a l a n i n e . In g e n e r a l l o w e s t l e v e l s o f a m i n o a c i d s w e r e o b s e r v e d i n p e r i o d 1 ( p r e - i n f u s i o n ) . H i g h e s t l e v e l s w e r e f o u n d i n p e r i o d 2 w i t h p e r i o d 3 r e s u l t s b e i n g s i m i l a r t o p e r i o d 2 e x c e p t w i t h p h e n y l a l a n i n e w h e r e t h e l e v e l s d r o p p e d b a c k t o p r e - i n f u s i o n l e v e l s . S i g n i f i c a n t i n f u s i o n e f f e c t s w e r e o b s e r v e d f o r t h e r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s o v e r t o t a l n o n - e s s e n t i a l a m i n o a c i d s a n d f o r t h e r a t i o o f g l y c i n e t o t h e b r a n c h e d c h a i n a m i n o a c i d s ( T a b l e V - 2 A ) . The r a t i o o f e s s e n t i a l s t o n o n - e s s e n t i a l s was s i g n i f i c a n t l y l o w e r w i t h i n f u s i o n #3 ( l y s i n e ) t h a n i n f u s i o n #5 ( a l l o f t h e e s s e n t i a l a m i n o a c i d s ) . T h e r a t i o o f g l y c i n e t o 143 TABLE V-2A LYSINE INFUSIONS ESSENTIAL AMINO ACIDS y g/ml BLOOD PLASMA - INFUSION EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 2 3 5 7 S.E. THREONINE VALINE 11.306 45.127 **_ 1.344 2.452 .182_ - 6.117a " '4 4 4 b 8.310 1.606a .258a .663 1.815 METHIONINE ISOLEUCINE 6.430 19.405 *_ *_ .047* .590^ - 1.938b - 2.567° .918a 3.163a 1.067a - .007^ .506 1.007 LEUCINE PHENYLALANINE 24.016 9.204 **_ * 1.459* .499a - 4.186a - 1.808b 4.821° .515a .824b .794a 1.039 .410 LYSINE HISTIDINE 20.492 • 13.741 -1.722 .581 .931 - .812 - .714 . .879 1.506 .514 1.971 .712 ARGININE TESS1 17.360 167.082 -1.610 9.312 .010 -16.302 - .588 16.864 2.187 8.750 1.050 7.446 TNESS RATIO3 171.166 .977 * 9.091 .001^ 2.674^ - .114b - 2.941 .112a 9.359 .OOl3*5 5.472 .033 RGLY4 TOTAL GLUCOSE .315 338.249 61.875 **_ .004a 18.404 3.833 .108b -13.629 3.733 - .084° 13.924 - .508 - .020a 18.109 .608 .014 11.785 2.135 * P <.05. ** P <.01. a,b,c Constants within rows with the same superscripts are not significantly different. ^TESS Total essential amino acids y g/ml blood plasma. TNESS Total non-essential amino acids (not including urea) y g/ml blood plasma. •3 RATIO Tess/Tness. 4 RGLY Glycine/Valine + Isoleucine + Leucine. ^TOTAL Tess + Tness. 6GLUCOSE mg/100 ml plasma. 144 TABLE V-2B LYSINE INFUSIONS ESSENTIAL AMINO ACIDS y g/ml BLOOD PLASMA - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER MEAN 1 S.E. 2 S.E. 3 S.E. THREONINE 11.306 _ 1.491 .605 1.152 .506 .338 .506 VALINE 45.127 *_ 6.449a 1.657 3.800b 1.386 2.648u 1.386 METHIONINE 6.430 *_ 1.3053 .462 1.257b .386 .048* .386 ISOLEUCINE 19.405 *_ 2.852a .919 1.668b .769 1.185u .769 LEUCINE 24.016 *_ 2.9913 .948 1.544b .794 1.447b .794 PHENYLALANINE 9.204 *_ .530a .374 1.027b - .313 - ,497a .313 LYSINE 20.492 - 4.280 1.800 3.424 1.505 .856 1.505 HISTIDINE 13.741 - .813 .650 .160 .544 .653 .544 ARGININE 17.360 - 1.480 .959 1.607 .802 - .127 .802 TESS1 167.082 *_ 22.1873 6.797 15.637b 5.687 6.550 5.687 TNESS2 171.166 _ 2.951 4.995 3.846 4.180 - .895 4.180 RATIO3 .977 *_ .117a .030 .071b .025 .046b .025 A RGLY' .315 ** .072a .013 - .037b .011 - .035b .011 TOTAL0 338.249 25.139 10.758 19.483 9.001 5.656 9.001 GLUCOSE6 61.875 • .300 1.949 - 1.050 1.631 1.350 1.630 * P < .05. ** P < .01. a,b Constants within rows with the same superscripts are not significantly < " 4 E S S Total essential amino acids y g/ml blood plasma. 2TNESS Total non-essential amino acids (not including urea) y g/ml blood plasm 3 RATIO Tess/Tness. 4RGLY GlycineA'aline + Isoleucine + Leucine. 5 TOTAL Tess + Tness. 6GLUCOSE mg/100 ml plasma. 145 b r a n c h e d c h a i n a m i n o a c i d s was f o u n d t o h a v e a s i g n i f i c a n t i n t e r a c t i o n w i t h p e r i o d s a n d w i l l be d i s c u s s e d l a t e r . T h e l e v e l o f t o t a l e s s e n t i a l a m i n o a c i d s , t h e r a t i o o f e s s e n t i a l s t o n o n - e s s e n t i a l s a n d t h e r a t i o o f g l y c i n e t o t h e b r a n c h e d c h a i n a m i n o a c i d s w e r e f o u n d t o h a v e s i g n i f i c a n t p e r -i o d e f f e c t s . B o t h t h e l e v e l o f e s s e n t i a l a m i n o a c i d s and t h e r a t i o o f e s s e n t i a l s t o n o n - e s s e n t i a l s w e r e s i g n i f i c a n t l y h i g h e r i n p e r i o d s 2 a n d 3 t h a n i n p e r i o d 1 ( p r e - i n f u s i o n ) ( T a b l e V - 2 B ) . T h e r a t i o o f g l y c i n e t o t h e b r a n c h e d c h a i n a m i n o a c i d s showed ( T a b l e V - 5 C ) s i g n i f i c a n t d i f f e r e n c e s i n p e r i o d 1 ( p r e -i n f u s i o n ) . V a l u e s w e r e h i g h e r i n t h e l y s i n e p r e - i n f u s i o n ( i n f u s i o n #3) t h a n i n t h e a l l e s s e n t i a l a m i n o a c i d p r e - i n f u s i o n ( i n f u s i o n #5 ) . In p e r i o d 3 , i n f u s i o n #5 h a d t h e l o w e s t v a l u e f o r t h e g l y c i n e t o b r a n c h e d c h a i n a m i n o a c i d r a t i o n w h i l e i n f u s i o n #3 h a d a n i n t e r m e d i a t e v a l u e . T h e r e w e r e no p e r i o d e f f e c t s f o r i n f u s i o n #5 a n d #7. I n f u s i o n s #2 a n d #3 showed a d e c l i n e i n t h e r a t i o o f g l y c i n e t o t h e b r a n c h e d c h a i n a m i n o a c i d s a s t h e i n f u -s i o n p r o c e e d e d . S i g n i f i c a n t i n f u s i o n e f f e c t s w e r e n o t e d f o r t h e c o n c e n t r a -t i o n s o f c i t r u l l i n e , g l y c i n e , a l a n i n e , c x - a m i n o - n - b u t y r i c and t y r o s i n e ( T a b l e V - 3 A ) . C i t r u l l i n e l e v e l s w e r e l o w e s t w i t h i n f u -s i o n #2 a n d h i g h e s t w i t h i n f u s i o n #5. L e v e l s d u r i n g i n f u s i o n s #3 a n d #7 w e r e i n t e r m e d i a t e a n d s i g n i f i c a n t l y d i f f e r e n t . G l y c i n e l e v e l s w e r e e l e v a t e d d u r i n g l y s i n e i n f u s i o n (#3) . S i m i l a r l y a l a n i n e c o n c e n t r a t i o n s w e r e h i g h e r d u r i n g i n f u s i o n s #3 a n d #7 t h a n d u r i n g #5. T y r o s i n e v a l u e s w e r e s i g n i f i c a n t l y 146 TABLE V-3A LYSINE INFUSIONS NON-ESSENTIAL AMINO ACIDS u g/ml BLOOD PLASMA ~ INFUSION EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 2 3 5 7 S.E. TAURINE UREA 6.931 173.155 - 1.202 22.265 .132 -21.008 .951 9.697 .119 -10.953 .591 15.016 SERINE ASPARAGINE 11.081 50.738 - 1.128 - .747 .406 .138 .104 -3.122 .618 3.730 .541 2.775 PROLINE GLUTAMIC ACID 10.219 6.416 - .339 .331 - 1.519 - .619 .511 .159 1.348 .129 .840 .309 CITRULLINE GLYCINE 16.935 28.360 **- 5.469* *- 1.319 .710£ 4.511 3.738° -2.990 1.021 - .202^ .632 1.388 ALANINE a-AMINO-N-BUTYRIC ACID 19.302 .965 *- .016^ * .138 a 1.592a - .004 a b -2.641 b - .109 b 1.064a - .025^ .793 .044 CYSTINE TYROSINE 4.533 8.646 .433 1.012a - .455 - 1.662b - .298 .312 a .320 .338 a .295 .368 ORNITHINE 7.036 - .788 - .560 .449 .899 .408 * P <.05. ** P <.01. a,b,c Constants within rows with the same superscripts are not significantly different. "'"Asparagine + Glutamine. 147 l o w e r d u r i n g i n f u s i o n #3 ( l y s i n e ) t h a n d u r i n g t h e o t h e r i n f u -s i o n s , a - a m i n o - n - b u t y r i c a c i d showed a s i g n i f i c a n t i n f u s i o n x p e r i o d i n t e r a c t i o n a n d w i l l be d i s c u s s e d l a t e r . P e r i o d e f f e c t s w e r e n o t e d f o r o n l y t y r o s i n e a n d o r n i t h i n e ( T a b l e V - 3 B ) . T y r o s i n e c o n c e n t r a t i o n s w e r e e l e v a t e d d u r i n g t h e s e c o n d p e r i o d o f a l l i n f u s i o n s b u t f e l l t o p r e - i n f u s i o n l e v e l s d u r i n g p e r i o d 3 . O r n i t h i n e c o n c e n t r a t i o n s w e r e s i g n i f i c a n t l y h i g h e r d u r i n g p e r i o d s 2 a n d 3 t h a n p e r i o d 1. When t h e p e r i o d 3 d a t a f o r e a c h i n f u s i o n was o b s e r v e d t h e r e was no d i f f e r e n c e i n a - a m i n o - n - b u t y r i c a c i d c o n c e n t r a t i o n ( T a b l e V - 5 C ) . T h e c o n c e n t r a t i o n was l o w e r f o r i n f u s i o n #7 d u r -i n g p e r i o d 2 a n d i n i n f u s i o n #5 d u r i n g p e r i o d 1 . I n f u s i o n #5 showed a s i g n i f i c a n t p e r i o d e f f e c t w i t h t h e c o n c e n t r a t i o n o f a - a m i n o - n - b u t y r i c a c i d b e i n g s i g n i f i c a n t l y l o w e r i n t h e p r e -i n f u s i o n p e r i o d t h a n i n p e r i o d s 2 a n d 3 . D u r i n g i n f u s i o n #7 t h e c o n c e n t r a t i o n was e l e v a t e d d u r i n g p e r i o d 2 b u t f e l l t o p r e -i n f u s i o n l e v e l s d u r i n g p e r i o d 3 . i i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s a p e r -c e n t a g e o f t h e t o t a l e s s e n t i a l o r t o t a l n o n - e s s e n t i a l a m i n o a c i d s S i g n i f i c a n t i n f u s i o n e f f e c t s w e r e n o t e d f o r t h r e o n i n e , l e u c i n e , p h e n y l a l a n i n e a n d a r g i n i n e when e x p r e s s e d a s a p e r c e n t -age o f t h e t o t a l e s s e n t i a l a m i n o a c i d s ( T a b l e V - 4 A ) . T h r e o n i n e p r o p o r t i o n s w e r e l o w e r d u r i n g i n f u s i o n #3 a n d #7 t h a n d u r i n g i n f u s i o n s #2 a n d #5. T h e p r o p o r t i o n o f l e u c i n e was l o w e r i n i n f u s i o n #5 t h a n i n any o f t h e o t h e r t h r e e i n f u s i o n s . A r g i n i n e TABLE V-3B LYSINE INFUSIONS NON-ESSENTIAL AMINO ACIDS pg/ml BLOOD PLASMA - PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANT - PERIOD NUMBER MEAN 1 S.E. 2 S.E. 3 S.E. TAURINE UREA 6.931 173.155 - 1.251 -16.350 .540 13.708 .382 14.096 .451 11.469 .869 2.254 .452 11.469 SERINE , ASPARAGINE 11.081 50.738 .344 - .358 .494 2.533 .070 - .231 .413 2.119 - .414 .589 .413 2.119 PROLINE GLUTAMIC ACID 10.219 6.416 .166 - .333 .767 .282 .848 .379 .642 .236 -1.014 - .046 .642 .236 CITRULLINE GLYCINE 16.935 28.360 - .918 1.705 .577 1.267 .392 - .629 .482 1.060 .526 -1.075 .482 1.060 ALANINE . a-AMlT^ICKN-PIJTYRIC-ACID 19.302 .965 - .080 - .060 .724 .040 .619 - .008 .606 .033 - .539 .068 .606 .033 CYSTINE TYROSINE 4.533 8.646 - .281 *- .713 a .269 .335 .221 1.045 D .225 .281 .059 - .332 s .225 .281 ORNITHINE 7.036 *- 1.174 a .373 . 7 5 ^ .312 .312 * P <.05. Asparagine + Glutamine. a,b Constants w i t h i n rows w i t h the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t . 149 TABLE V-4A LYSINE INFUSIONS ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL ESSENTIAL AMINO ACIDS " INFUSION EFFECTS . . LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 1 3 5 7 S.E. THREONINE 6. ,780 *_ -.478a .802 b - . 830a . 5 0 ^ .198 VALINE 26. ,900 .062 - .978 2. 143 -1.228 .751 METHIONINE 3. .804 .222 - .758 • 169 .367 .245 ISOLEUCINE 11. .560 .335 - .413 • 662 - .583 .302 LEUCINE 14. .304 *. -.026a -1.087 a 1. - .239 a .318 PffiNYLALANTNE 5. .25 ** .628 a - .558° - . be 182 .112b .117 LYSINE 12. .158 -.256 1.699 - 1 . 490 .047 .780 HISTIDINE 8. .304 .118 .423 - . 347 - .194 .334 ARGININE 10, .444 -.390* 1.090° - 1 . 248 .547 b ° .331 * P <.05. ** P <.01. a,b,c Constants within rows with the same superscripts are not significantly different. 150 p e r c e n t a g e was h i g h e s t w i t h i n f u s i o n #3 a n d was l o w e s t i n i n f u s i o n #5. P h e n y l a l a n i n e p e r c e n t a g e showed a s i g n i f i c a n t i n f u s i o n x p e r i o d i n t e r a c t i o n . T h e r e w e r e no s i g n i f i c a n t p e r i o d e f f e c t s e x c e p t f o r t h e p e r c e n t a g e o f p h e n y l a l a n i n e ( T a b l e V - 4 B ) . P h e n y l a l a n i n e p e r -c e n t a g e s w e r e h i g h e r d u r i n g t h e i n f u s i o n #2 p r e - i n f u s i o n p e r i o d . T h e same h e l d t r u e f o r p e r i o d 2 . H o w e v e r , i n p e r i o d 3 p h e n y l a l a n i n e was h i g h e s t d u r i n g i n f u s i o n #7. O v e r e a c h i n f u s i o n t h e p e r c e n t a g e p h e n y l a l a n i n e t e n d e d t o d e c r e a s e w i t h t h e e x c e p t i o n b e i n g i n f u s i o n #7 i n w h i c h t h e p e r c e n t a g e o f p h e n y l a l a n i n e r e m a i n e d c o n s t a n t . When t h e n o n - e s s e n t i a l a m i n o a c i d s w e r e e x p r e s s e d a s a p e r c e n t a g e o f t h e t o t a l n o n - e s s e n t i a l a m i n o a c i d s s i g n i f i c a n t i n f u s i o n e f f e c t s w e r e n o t e d f o r g l u t a m i c a c i d , c i t r u l l i n e , g l y c i n e , a l a n i n e a n d t y r o s i n e ( T a b l e V - 5 A ) . G l u t a m i c a c i d p e r c e n t a g e was l o w e r d u r i n g i n f u s i o n #3 ( l y s i n e a l o n e ) t h a n i n i n f u s i o n #2 ( c a s e i n ) . C i t r u l l i n e p e r -c e n t a g e s w e r e l o w e s t d u r i n g i n f u s i o n #2, i n c r e a s e d s i g n i f i c a n t l y d u r i n g i n f u s i o n #3 a n d #7 ( t h r e o n i n e , m e t h i o n i n e , p h e n y l a l a n i n e and l y s i n e ) a n d showed a f u r t h e r s i g n i f i c a n t i n c r e a s e d u r i n g i n f u s i o n #5 ( a l l e s s e n t i a l a m i n o a c i d s ) . G l y c i n e p e r c e n t a g e s were e l e v a t e d d u r i n g i n f u s i o n #3. A l a n i n e p e r c e n t a g e was d e c r e a s e d d u r i n g i n f u s i o n #5. T y r o s i n e e x h i b i t e d a s i g n i f i c a n t i n t e r a c t i o n t e r m . T h e r e w e r e no s i g n i f i c a n t p e r i o d e f f e c t s e x c e p t f o r t y r o s i n e ( T a b l e V - 5 B ) . T y r o s i n e p e r c e n t a g e s w e r e l o w e s t d u r i n g p e r i o d s 1 and 2 f o r i n f u s i o n s #3 when a l l i n f u s i o n s TABLE V-4B LYSINE INFUSIONS ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL ESSENTIAL AMINO ACIDS PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - PERIOD NUMBER MEAN 1 S.E. 2 S.E. 3 S.E. THREONINE 6. 780 .015 .181 .007 .151 -.022 .151 VALINE 26. 900 -.310 .686 -.222 .574 .532 .574 METHIONINE 3. 804 -.260 .223 .334 .187 -.074 .187 ISOLEUCINE 11. 560 -.182 .275 -.086 .230 .269 .230 LEUCINE 14. 304 .126 .290 -.412 .243 .286 .243 PHENYLALANINE 5. 525 ** .452a .107 .036b .089 -.489° .089 LYSINE 12. 158 -.905 .712 .758 .596 .147 .596 HISTIDINE 8. 304 .704 .305 -.716 .255 .012 .255 ARGININE 10. 444 .580 .302 -.141 .253 -.440 .253 ** P <.01. a,b,c Constants w i t h i n rows w i t h the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t . 152 TABLE V-5A LYSINE INFUSIONS NON-ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL NON-ESSENTIAL AMINO ACIDS - INFUSION EFFECTS LEAST SQUARES LEAST SQUARES CONSTANTS - INFUSION NUMBER MEAN 2 3 5 7 S.E. TAURINE 4.055 • .492 - .019 .614 -.104 .381 SERINE , ASPARAGINE 6.489 29.707 - .352 1.061 .124 - .324 .181 -1.454 .046 .716 .190 1.033 PROLINE GLUTAMIC ACID 5.976 3.768 * .095 .406a - .988 - .442b .404 . .142 a b .489 -.106 a b .294 .150 CITRULLINE GLYCINE 9.933 16.600 * -2.793a .082a .224? 2.278° 2.384C -1.508a .18^ -.853a .522 .573 ALANINE a-AMINO-N-BUTYRIC ACID 11.300 .566 ** .572a .115 .709a - .011 -1.370b - .060 .089a -.044 .250 .034 CYSTINE TYROSINE 2.661 5.085 ** .419 .873a - .314 -1.083c - .156 .242 a b .051 -.032b .184 .199 ORNITHINE 3.858 .012 - .154' .575 -.433 .463 * P <.05. ** P <.01. ^Asparagine + Glutamine. a,b,c Constants within rows with the same superscripts are not significantly-different. TABLE V-5B LYSINE INFUSIONS NON-ESSENTIAL AMINO ACIDS AS A PERCENTAGE OF THE TOTAL NON-ESSENTIAL AMINO ACIDS -PERIOD EFFECTS LEAST SQUARES LEAST SQUARES CONSTANT - PERIOD NUMBER MEAN 1 S.E. 2 S.E. 3 S.E. TAURINE 4.055 - .685 .348 .178 .291 .507 .291 SERINE 6.489 .311 .173 -.076 .145 -.235 .145 ASPARAGINE1 29.707 .223 .944 -.632 .790 .409 .790 PROLINE 5.976 .189 .268 .378 .225 -.566 .225 GLUTAMIC ACID 3.768 - .155 .137 .166 .115 -.011 .115 CITPJJLLINE 9.933 - .395 .477 .083 .399 .312 .399 GLYCINE 16.600 1.305 .523 -.733 .438 -.572 .438 ALANINE 11.300 .115 .228 .164 .191 -.280 .191 a-AMINO-N-BUTYRIC ACID .566 - .031 .031 -.012 .026 .044 .026 CYSTINE 2.661 - .143 .168 .097 .140 .047 .140 TYROSINE 5.085 *- .358a .182 .536b .152 -.179a .152 ORNITHINE 3.858 - .378 .423 -.150 .354 .528 .354 * P <.05. '''Asparagine + Glutamine. a,b Constants within rows with the same superscripts are not significantly different. 154 and pe r iods were compared (Table V - 5 C ) . P e r i o d 3 p ropo r t i ons of t y r o s i n e were s i m i l a r fo r a l l i n f u s i o n s . During i n f u s i o n s #2 and #7 there was a s i g n i f i c a n t inc rease i n the p r o p o r t i o n of t y r o s i n e dur ing p e r i o d 3 the percentage of t y r o s i n e had f a l l e n to p r e - i n f u s i o n l e v e l s . (2) D i s c u s s i o n (a) M i l k and m i l k compos i t ion The lower p r o t e i n c o n c e n t r a t i o n found i n m i l k du r ing i n f u -s i o n of l y s i n e alone ( i n f u s i o n #3) (Table V-1A) should not be due to excess ive l y s i n e c r e a t i n g an imbalance of amino a c i d s . Plasma l y s i n e r e f l e c t s excess l y s i n e (Hewitt and Lewi s , 1966; S tock land et a_l. , 1970). I n d i v i d u a l amino ac id s d i d not r eac t d i f f e r e n t l y to i n f u s i o n #3 than the o ther i n f u s i o n s . A t r a n s i -t o r y inc rease i n m i l k p r o t e i n c o n c e n t r a t i o n du r ing p e r i o d 2 (Table V-1B) appears to be r e l a t e d to inc reases i n c o n c e n t r a t i o n of many of the e s s e n t i a l amino a c i d s dur ing p e r i o d 2 (Table V -2B) . The e f f e c t of l y s i n e i n f u s i o n on m i l k p roduc t ion was q u i t e marked. The sharp inc rease i n p roduc t ion du r ing p e r i o d 3 of i n f u s i o n #3 was i n c o n t r a s t to the even p roduc t i on over the three pe r iods fo r the o ther i n f u s i o n s ( i n fus ions #1, #5, #7). When t o t a l p r o t e i n p roduc t ion i s compared w i t h p r o t e i n c o n c e n t r a t i o n i t can be seen tha t i n genera l they both fo l lowed the same pa t t e rns w i t h h ighes t va lues du r ing p e r i o d 2 (Table V -1C) . However w i t h i n f u s i o n #3 ( l y s i n e ) p roduc t ion of p r o t e i n du r ing p e r i o d 3 was s i g n i f i c a n t l y h igher than p r e - i n f u s i o n l e v e l s . TABLE V-5C LYSINE INFUSIONS AMINO ACID DATA - SIOSILFICANT INFUSION X PERIOD INTERACTION TERMS RGly a-^lINO-N-BUTYRIC ACID PHENYLALANINE- TYROSINE' LEAST SQUARES MEAN .315 .965 5.25 5.085 IN 2 IN 2 IN 2 P I 5 P 2 P 3 . 1 1 4 * - . 0 9 1 ° - . • 0 3 5 ° * .033 .024 .024 .144 a . 215 a .054 a .101 .074 .074 1.615? .735^ . - . 4 6 5 d e f • .270 .197 .197 . 3 0 5 ^ 2 . 0 7 0 ? , . 2 4 5 ^ .460 .336 .336 IN 3 IN 3 IN 3 P 1 P 2 P 3 . 2 2 5 a - . 0 0 5 ^ .033 .024 .024 - . 0 8 5 * . 035 a . 040 a .101 .074 .074 - . 4 5 5 d e f -1 .055 .270 .197 .197 -1.925*? - . 4 6 0 ° ^ .460 .336 .336 IN 5 IN 5 IN 5 P 1 P 2 P 3 - . 0 9 6 ° - .no d .033 .024 .024 - . 4 1 5 b - . 1 0 4 a .iooa .101 .074 .074 be 515 - * 2 5 0 C d e ef - .810 .270 .197 .197 •805?**: - . 0 8 ? ^ .460 .336 .336 IN 7 IN 7 IN 7 P 1 P 2 P 3 - . 0 0 5 ^ - . 0 6 5 ^ . 0 1 0 ° ° .033 .024 .024 .115? - .270 .080 a .101 .074 .074 . 1 1 ? ? . 3 7 5 ° ° .270 .197 .197 . 9 3 5 * - . 4 1 5 ° * .460 .336 .336 a , b , c , d , e •hRGly 2a-AMIM>N-BUTYRIC ACID 3PHENYIALANINE ^TYROSINE r I N l P I Constants w i t h i n columns wi th the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t . G l y c i n e / V a l i n e + Iso leuc ine + Leucine , v g / m l . Phenyla lanine as a percentage of the t o t a l e s s e n t i a l amino a c i d s . Tyros ine as a percentage of the t o t a l n o n - e s s e n t i a l amino a c i d s . In fus ion number and per iod number. Ul Ul 156 T h i s i n c r e a s e i n m i l k p r o t e i n p r o d u c t i o n i s due m a i n l y t o t h e i n c r e a s e d m i l k p r o d u c t i o n a s t h e c o n c e n t r a t i o n o f p r o t e i n i n m i l k f o l l o w e d t h e g e n e r a l p a t t e r n a n d f e l l d u r i n g p e r i o d 3 t o i n t e r m e d i a t e l e v e l s t h a t w e r e n o t s i g n i f i c a n t l y d i f f e r e n t . I n f u s i o n o f a l l t h e e s s e n t i a l a m i n o a c i d s ( i n f u s i o n #5) r e s u l t e d i n a s i g n i f i c a n t d e c r e a s e i n l a c t o s e c o n c e n t r a t i o n d u r i n g p e r i o d s 2 and 3 . T h e o t h e r i n f u s i o n s h a d no p e r i o d e f f e c t s . I t c a n be s u g g e s t e d t h a t i n f u s i o n o f e s s e n t i a l a m i n o a c i d s c o u l d c a u s e a n i n c r e a s e i n g e n e r a l p r o t e i n s y n t h e s i s ( i n c l u d i n g m i l k ) a n d t h u s h a v e an e f f e c t o n l a c t o s e s y n t h e s i s b y r e m o v i n g s u b s t r a t e n e c e s s a r y f o r g l u c o s e p r o d u c t i o n . T h e r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s t o t o t a l n o n - e s s e n t i a l a m i n o a c i d s d i d d e c r e a s e a s e a c h i n f u s i o n p r o g r e s s e d w h i c h H a l f p e n n y e t al. (1969) c o n c l u d e d was d u e t o i n c r e a s e d p r o t e i n s y n t h e s i s . T h e i n c r e a s e d l a c t o s e p r o d u c t i o n d u r i n g i n f u s i o n #3 i s due t o i n c r e a s e d m i l k p r o d u c t i o n w h i l e l a c t o s e c o n c e n t r a t i o n r e m a i n -e d e v e n . T h e d e c r e a s e d l a c t o s e c o n c e n t r a t i o n s d u r i n g p e r i o d s 2 a n d 3 o f i n f u s i o n #5 w e r e r e f l e c t e d i n d e c r e a s e d l a c t o s e p r o d u c -t i o n d u r i n g p e r i o d 3 . (b) P l a s m a a m i n o a c i d s i ) P l a s m a a m i n o a c i d s e x p r e s s e d a s m i c r o -g rams p e r m l o f b l o o d p l a s m a T h e i n f u s i o n e f f e c t s r e f l e c t i n g e n e r a l t h e a m i n o a c i d s w h i c h w e r e i n f u s e d d u r i n g t h a t t i m e . T h e m o s t n o t a b l e e x c e p t i o n i s l y s i n e w h i c h d o e s n o t show a n y i n f u s i o n e f f e c t a l t h o u g h t h i s 157 c o u l d be e x p l a i n e d b y l y s i n e b e i n g p r e s e n t i n e a c h i n f u s i o n (as was n o t e d f o r i s o l e u c i n e ) . T h e p e r i o d e f f e c t s show t h a t when s i g n i f i c a n t d i f f e r e n c e s w e r e n o t e d t h e s e a m i n o a c i d s a l l i n c r e a s e d i n c o n c e n t r a t i o n a s e a c h i n f u s i o n p r o c e e d e d . I t m u s t be p o i n t e d o u t t h a t when l y s i n e a l o n e was i n f u s e d t h i s same g e n e r a l t r e n d was o b s e r v e d . B e f o r e t h e m i l k data were a n a l y z e d i t was f e l t t h a t p e r h a p s t h e r e was an a m i n o a c i d i m b a l a n c e c a u s i n g a n e l e v a t i o n o f c e r t a i n o f t h e a m i n o a c i d s . D u r i n g p e r i o d 3 m e t h i o n i n e a n d p h e n y l a l a n i n e d i d r e t u r n t o p r e - i n f u s i o n l e v e l s . However m i l k d a t a d i d n o t s u b s t a n t i a t e t h i s i m b a l a n c e , i n f a c t r a t h e r t h e o p p o s i t e o c c u r r e d . M i l k p r o d u c t i o n and m i l k p r o t e i n p r o d u c t i o n w e r e e l e v a t e d d u r i n g p e r i o d 3 . I t i s i m p o r t a n t t o n o t e t h a t t h e e l e v a t i o n o f t h o s e a m i n o a c i d s ( T a b l e V - 2 B ) d i d r e s u l t i n i n c r e a s e d m i l k a n d m i l k p r o t e i n s y n t h e s i s . T h e m e c h a n i s m f o r t h a t e l e v a t i o n i n u n k n o w n . L y s i n e i t s e l f d i d n o t show a n y s i g n i f i c a n t p e r i o d e f f e c t s . T h e mean c h a n g e s i n l y s i n e c o n c e n t r a t i o n w e r e q u i t e l a r g e , s h o w i n g an i n c r e a s e d u r i n g p e r i o d 2 a n d a somewhat l e s s e r i n c r e a s e d u r -i n g p e r i o d 3 . T h e s t a n d a r d e r r o r s h o w e v e r w e r e a l s o l a r g e w h i c h r e f l e c t s d i f f e r e n c e s w i t h i n p e r i o d X i n f u s i o n g r o u p s . T h e r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s t o t o t a l n o n - e s s e n -t i a l a m i n o a c i d s showed s i g n i f i c a n t d i f f e r e n c e s b e t w e e n i n f u s i o n s f o r o n l y ( l y s i n e ) i n f u s i o n #3, a n d ( a l l t h e e s s e n t i a l a m i n o a c i d s ) i n f u s i o n #5. T h e s e r e s u l t s a r e d u e t o a n o n - s i g n i f i c a n t d e c r e a s e i n e s s e n t i a l a m i n o a c i d s d u r i n g i n f u s i o n #3 w h i l e t h e e s s e n t i a l a m i n o a c i d s d u r i n g i n f u s i o n #5 w e r e i n c r e a s i n g ( n o n - s i g n i f i c a n t l y ) . 158 T h e i n c r e a s e o b s e r v e d i n t o t a l e s s e n t i a l a m i n o a c i d s a s e a c h i n f u s i o n p r o g r e s s e d i s e x p e c t e d d u e t o t h e i n c r e a s e s i n i n d i v i d u a l a m i n o a c i d s a t t h i s t i m e ( T a b l e V - 2 B ) . T h e r e i s a c a r r y - o v e r e f f e c t t o t h e r a t i o o f t o t a l e s s e n t i a l a m i n o a c i d s t o t o t a l n o n - e s s e n t i a l a m i n o a c i d s i n c r e a s i n g a s e a c h i n f u s i o n p r o g r e s s e s . T h e r a t i o o f g l y c i n e t o t h e b r a n c h e d c h a i n a m i n o a c i d s showed a s i g n i f i c a n t i n t e r a c t i o n t e r m i n d i c a t i n g a d i f f e r e n t r e s p o n s e d u r i n g the' t h r e e p e r i o d s f o r t h e d i f f e r e n t r e s p o n s e d u r i n g t h e t h r e e p e r i o d s f o r t h e d i f f e r e n t i n f u s i o n s . A s t h e l e v e l s o f t h e b r a n c h e d c h a i n a m i n o a c i d s i n c r e a s e d s i g n i f i c a n t l y f o r e a c h i n f u s i o n t h e s i g n i f i c a n t t e r m m u s t b e d u e t o e i t h e r f l u c t u a t i o n s i n g l y c i n e c o n c e n t r a t i o n o r t o d i f f e r e n t l e v e l s b e t w e e n i n f u s i o n s . G l y c i n e c o n c e n t r a t i o n s w e r e h i g h d u r i n g i n f u s i o n s #2 a n d #3 ( T a b l e V - 3 A ) . V a l i n e , i s o l e u c i n e and l e u -c i n e w e r e a l s o l o w e r d u r i n g t h e s e two i n f u s i o n s . One w o u l d s p e c u l a t e t h a t t h e a b o v e r e s u l t s i n d i c a t e a p o o r e r s u p p l y o r a g r e a t e r u p t a k e o f a m i n o a c i d s t h a n d u r i n g i n f u s i o n s #5 a n d #7. T h e m i l k p r o t e i n p r o d u c t i o n d a t a d o e s s u p p o r t t h i s h y p o t h -e s i s , i n d i c a t i n g t h a t t h e i n c r e a s e d s y n t h e s i s o f p r o t e i n c a u s e d a r e d u c t i o n i n p l a s m a a m i n o a c i d s . T h e b e t w e e n i n f u s i o n d i f f e r e n c e s f o r t h e n o n - e s s e n t i a l a m i n o a c i d s a p p e a r t o f a l l i n t o two g r o u p s d e p e n d i n g o n t h e i r r e s p o n s e t o l y s i n e i n f u s i o n . G l y c i n e a n d a l a n i n e a p p e a r t o b e i n g r e a t e r c o n c e n t r a t i o n i n b l o o d p l a s m a when l y s i n e a l o n e i s i n f u s e d . I f t h e b r a n c h e d 159 chain amino acid s are present i n s u f f i c i e n t amounts when they are infused to e l i c i t an i n s u l i n response then d e l e t i o n of the branched chain amino a c i d s from the i n f u s a t e could r e s u l t i n an increased c o n c e n t r a t i o n of a l a n i n e . C a l l et a l . (1972) have demonstrated that i n s u l i n i s e f f e c t i v e i n reducing plasma a l a -nine c o n c e n t r a t i o n s . Tyrosine c o n c e n t r a t i o n was depressed when l y s i n e alone was i n f u s e d . This could be a r e f l e c t i o n of the reduced conce n t r a t i o n of phenylalanine observed at the same time. The increase observed f o r c i t r u l l i n e as the experiment progresses f i t s i n w e l l w i t h the f a c t t h a t c i t r u l l i n e i s formed from o r n i t h i n e . The enzyme r e s p o n s i b l e f o r t h i s a l t e r a t i o n i s induced by d i e t a r y p r o t e i n (Kaplan and P i t o t , 1970). While d i e t a r y i n t a k e d i d not a l t e r much over t h i s p e r i o d of time there was a d e c l i n e i n production and t o t a l amino acids d i d show a s l i g h t increase (Table V-2A). The p e r i o d e f f e c t s observed f o r t y r o s i n e again r e f l e c t those of phenylalanine. The s i g n i f i c a n t increase above pre-i n f u s i o n l e v e l s f o r o r n i t h i n e i n d i c a t e s increased breakdown of a r g i n i n e by arginase (Kaplan and P i t o t , 1970) . This r e a c t i o n i s induced by increases i n d i e t a r y p r o t e i n i n r a t s . Again the increase i n e s s e n t i a l amino acids as each i n f u s i o n progressed might a l s o induce the enzyme arginase. These r e s u l t s a l s o suggest that plasma l y s i n e was not i n excess. Excess l y s i n e i s supposed to decrease the a c t i v i t y of arginase (Munro, 1970). Rapid degradation of threonine could r e s u l t i n an increase i n a-amino-n-butyric a c i d . a-Amino-n-butyric a c i d i s a metab-160 o l i t e of a-keto b u t y r a t e . a-keto b u t y r a t e i s formed from threonine ( v i a threonine dehydratase) and homoserine ( v i a homo-s e r i n e dehydratase) (Ishimoto, e t a l . , 1971). The c o n c e n t r a t i o n of t h r e o n i n e d i d not vary i n any of the i n f u s i o n s or p e r i o d s even though th r e o n i n e was p r e s e n t i n three of the four i n f u s a t e s . However, onl y d u r i n g i n f u s i o n #5 was there an i n c r e a s e i n a-amino-n - b u t y r i c a c i d as the i n f u s i o n proceeded. During i n f u s i o n #7 the l e v e l a c t u a l l y dropped be f o r e r e t u r n i n g to p r e - i n f u s i o n l e v e l s d u r i n g p e r i o d 3. i i ) Plasma amino a c i d s expressed as a per-centage of the t o t a l e s s e n t i a l or non-e s s e n t i a l amino a c i d s Threonine was p r e s e n t i n a l l i n f u s i o n s except #3 but y e t the p r o p o r t i o n of t h r e o n i n e i n plasma was h i g h e s t d u r i n g i n f u -s i o n s #3 and #5. The i n c r e a s e d percentage d u r i n g i n f u s i o n #7 might be expected due to i n f u s i o n of a small number of amino a c i d s i n the f r e e form, but there i s no apparent reason f o r the i n c r e a s e d u r i n g i n f u s i o n #3. The i n c r e a s e d p r o p o r t i o n of l e u c i n e d u r i n g i n f u s i o n #5 appears to be due to the f a c t t h a t t h i s i s the o n l y i n f u s i o n i n which l e u c i n e was i n c l u d e d i n the i n f u s i o n mix. A r g i n i n e p r o p o r t i o n s showed a decrease d u r i n g the i n f u s i o n s where a r g i n i n e was i n c l u d e d (#2, #5). These might r e f l e c t the i n d u c t i o n of a r g i n a s e by the i n c r e a s e d plasma e s s e n t i a l amino a c i d values d u r i n g these two i n f u s i o n s (Table V-4A) (Kaplan and P i t o t , 1970). An i n c r e a s e i n c i t r u l l i n e was noted d u r i n g i n f u -s i o n #5 (Table V-5A) but no e f f e c t d u r i n g i n f u s i o n #2. O r n i t h i n e d i d not show any response. 161 T h e r e s p o n s e o f p h e n y l a l a n i n e t o t h e v a r i o u s i n f u s i o n s a p p e a r s t o be r e l a t e d t o t h e i n c l u s i o n o f p h e n y l a l n i n e i n t h e i n f u s i o n m i x and t h e number o f a m i n o a c i d s a c c o m p a n y i n g i t . O n l y d u r i n g i n f u s i o n #7 d i d t h e p r o p o r t i o n o f p h e n y l a l a n i n e n o t d e c r e a s e a s t h e i n f u s i o n p r o c e e d e d . I t m u s t a l s o be p o i n t -ed o u t t h a t when a l l t h e p e r i o d 3 p r o t e i n p r o d u c t i o n v a l u e s a r e c o m p a r e d ( T a b l e V - 1 C ) t h a t t h e l o w e s t p r o d u c t i o n a l s o o c c u r r e d d u r i n g t h i s p e r i o d . T h e e l e v a t e d p r o p o r t i o n o f p h e n -y l a l a n i n e c o u l d be d u e t o a c o m b i n a t i o n o f t h e two f a c t o r s , n a m e l y a n i n c r e a s e i n p h e n y l a l a n i n e s u p p l y p l u s a d e c r e a s e i n p h e n y l a l a n i n e u p t a k e . T h e p r o p o r t i o n o f g l u t a m i c a c i d m i g h t be e x p e c t e d t o be h i g h e r d u r i n g i n f u s i o n o f c a s e i n a s g l u t a m i c a c i d i s o n e o f t h e m a j o r a m i n o a c i d s o f m i l k p r o t e i n (19.8%) (Egan a n d B l a c k , 1968) . A s m e n t i o n e d b e f o r e t h e i n c r e a s e i n c i t r u l l i n e d u r i n g i n f u s i o n #5 c o r r e s p o n d s w i t h a r g i n i n e v a l u e s . However t h e low p e r c e n t a g e d u r i n g t h e c a s e i n i n f u s i o n i s d i f f i c u l t t o e x p l a i n . T h a t g l y -c i n e p e r c e n t a g e s w e r e e l e v a t e d d u r i n g i n f u s i o n #3 i s e x p e c t e d . D u r i n g t h i s i n f u s i o n t h e r e was a g r e a t e r o u t p u t o f m i l k p r o t e i n b u t a much l e s s e r i n p u t i n t h e f o r m o f i n f u s e d a m i n o a c i d s . T h e e x t r a s u p p l y w o u l d h a v e t o come f r o m t i s s u e s w h i c h w o u l d g i v e t h e a b o v e g l y c i n e r e s p o n s e ( L e i b h o l z , 1 9 7 0 ) . T h e d e c r e a s e i n p l a s m a a l a n i n e d u r i n g i n f u s i o n #5 w o u l d s u g g e s t i n c r e a s e d u p t a k e o f c i r c u l a t i n g a l a n i n e . S u p p l y o f a l a n i n e d u r i n g t h i s i n f u s i o n i n c o m p a r i s o n w i t h #3 and #7 s h o u l d n o t be d i f f e r e n t . M i l k p r o t e i n p r o d u c t i o n was a t an i n t e r m e d i a t e l e v e l d u r i n g t h i s i n f u s i o n ; l a c t o s e p r o d u c t i o n 162 was a l s o r e d u c e d . The l e v e l s o f p r o d u c t i o n o f t h e s e two c o m -p o n e n t s w o u l d s u g g e s t t h a t t h e r e was no i n c r e a s e d u p t a k e o f s u b s t r a t e due t o i n c r e a s e d p r o d u c t i o n . T h e b r a n c h e d c h a i n amino a c i d s w e r e p r e s e n t d u r i n g i n f u s i o n #5 a n d l e u c i n e i s known t o h a v e an i n s u l i n o t r o p i c e f f e c t ( D a v i s , 1 9 7 2 ) . I n s u l i n i s known t o c a u s e a n i n c r e a s e d u p t a k e o f a m i n o a c i d s b y t h e t i s s u e s . However n o n e o f t h e o t h e r a m i n o a c i d s showed s u c h an e f f e c t , u n l e s s t h e i n s u l i n e f f e c t i s m a s k i n g some o t h e r c o n -t r o l m e c h a n i s m . T h e t y r o s i n e r e s p o n s e t o t h e v a r i o u s i n f u s i o n s d o e s n o t a p p e a r t o h a v e a n y r e l a t i o n s h i p w i t h t h e r e s p o n s e o f p h e n y l a l a n i n e . T o s u m m a r i z e t h e i n f u s i o n o f s o l u t i o n s c o n t a i n i n g l y s i n e a l o n e o r i n c o m b i n a t i o n w i t h o t h e r a m i n o a c i d s a p p e a r s t o h a v e e l i c i t e d d i f f e r e n t r e s p o n s e s f o r t h e v a r i o u s m i l k c o m p o n e n t s . T h e i n f u s i o n o f l y s i n e a l o n e c a u s e d an i n c r e a s e i n m i l k p r o -d u c t i o n a b o v e t h a t o f t h e p r e - i n f u s i o n l e v e l . M i l k p r o d u c t i o n r e m a i n e d c o n s t a n t o v e r t h e t h r e e p e r i o d s o f e a c h i n f u s i o n f o r t h e o t h e r t h r e e i n f u s i o n s . T h e c o n c e n t r a t i o n o f p r o t e i n i n m i l k i n c r e a s e d d u r i n g p e r i o d 2 a n d t h e n d r o p p e d d u r i n g p e r i o d 3 t o an i n t e r m e d i a t e l e v e l . I n a l l i n f u s i o n s t h e p r o d u c t i o n o f p r o t e i n i n c r e a s e d d u r i n g p e r i o d 2 . D u r i n g p e r i o d 3 t h e r e was a d e c r e a s e i n p r o t e i n p r o d u c t i o n w i t h t h e e x c e p t i o n o f i n f u s i o n o f l y s i n e a l o n e w h e r e p e r i o d 3 p r o d u c t i o n o f p r o t e i n 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 p e r i o d 2 p r o d u c t i o n . L a c t o s e p r o d u c t i o n was a l s o i n c r e a s e d d u r i n g l y s i n e i n f u s i o n . T h e c o n c e n t r a t i o n o f t h e e s s e n t i a l a m i n o a c i d s i n c r e a s e d a s e a c h i n f u s i o n p r o g r e s s e d . T h e s e i n c r e a s e s w e r e s i g n i f i c a n t 163 f o r v a l i n e , methionine and i s o l e u c i n e . The percentages of the e s s e n t i a l amino a c i d s remained r e l a t i v e l y c onstant when each e s s e n t i a l amino a c i d was expressed as a percentage of the t o t a l e s s e n t i a l amino a c i d s . Only p h e n y l a l a n i n e showed an a l t e r a t i o n . The percentage of p h e n y l a l a n i n e decreased d u r i n g each i n f u s i o n except f o r i n f u s i o n #7. T y r o s i n e and o r n i t h i n e were the onl y n o n - e s s e n t i a l amino a c i d s to show any s i g n i f i c a n t e f f e c t s . These r e s u l t s i m p l i c a t e l y s i n e as a c o n t r o l l i n g or l i m i t -ing f a c t o r i n the p r o d u c t i o n of m i l k p r o t e i n . The g e n e r a l i n c r e a s e i n c o n c e n t r a t i o n of e s s e n t i a l amino a c i d s d u r i n g l y s i n e i n f u s i o n i s d i f f i c u l t to e x p l a i n at t h i s time. I t i s i n t e r -e s t i n g t o note t h a t t h i s g e n e r a l i n c r e a s e i n plasma amino a c i d s was r e f l e c t e d by an i n c r e a s e i n milk p r o t e i n p r o d u c t i o n . 164 D. GENERAL SUMMARY AND CONCLUSIONS One o f t h e m a j o r f a c t o r s i n v o l v e d i n t h e c o n t r o l o f p r o -t e i n s y n t h e s i s i s t h e s u p p l y o f a m i n o a c i d s . In r u m i n a n t s t h i s c o m p l e x s y s t e m i s f u r t h e r c o m p l i c a t e d b y t h e e x t e n s i v e a c t i o n o f t h e rumen m i c r o f l o r a on d i e t a r y p r o t e i n s . T h e a n a e r o b i c f e r m e n t a t i o n w i t h i n t h e rumen p l a c e s e n e r g e t i c r e s t r i c t i o n s o n t h e a m o u n t s o f m i c r o b i a l c e l l s f o r m e d t o s u p p l y t h e h o s t a n i m a l w i t h p r o t e i n . U n f o r t u n a t e l y , e x c e s s d i e t a r y p r o t e i n i s d e a m i n a t e d and t r e a t e d a s an e n e r g y s o u r c e w h i l e t h e e x c e s s n i t r o g e n i s a b s o r b e d a s ammonia and may be l o s t . In t h e d a i r y a n i m a l , t h e l a r g e a m o u n t s o f p r o t e i n t h a t a r e s e c r e t e d d a i l y i n t h e m i l k p l a c e h e a v y demands o n p r o t e i n s u p p l y . T h e s e h e a v y demands a r e r e f l e c t e d i n t h e c h a n g e s i n p l a s m a f r e e a m i n o a c i d s d u r i n g t h e l a c t a t i o n c y c l e . E x p e r i m e n t I d a t a i n d i c a t e t h a t d u r i n g t h e p e r i o d o f p e a k p r o d u c t i o n t h e . a n i m a l i s i n n e g a t i v e n i t r o g e n b a l a n c e . P l a s m a c o n c e n t r a t i o n s o f l y s i n e , l e u c i n e a n d m e t h i o n i n e w e r e a f f e c t e d b y t h e demands o f l a c t a t i o n . To a c e r t a i n t h e i m p o r t a n c e o f t h e v a r i o u s a m i n o a c i d s f o r t h e s y n t h e s i s o f m i l k p r o t e i n , a s e r i e s o f i n f u s i o n s o f t h e a b o v e a m i n o a c i d s and o t h e r s i n t o t h e abomasum o f a l a c t a t i n g cow was u n d e r t a k e n . A s m i c r o b i a l a c t i o n i s i n h i b i t e d by t h e a c i d c o n d i t i o n s o f t h e abomasum, i n t r o d u c t i o n o f a m i n o a c i d s i n t o t h e r u m i n a n t m e t a b o l i c s y s t e m a t t h i s r e g i o n o f t h e d i g e s -t i v e t r a c t was t h e m o s t s a t i s f a c t o r y m e t h o d a v a i l a b l e . 165 When t h e i n f u s i o n s w e r e g r o u p e d i n t o t h r e e l o t s , t h o s e c o n t a i n i n g m e t h i o n i n e , t h o s e c o n t a i n i n g i s o l e u c i n e and t h o s e c o n t a i n i n g l y s i n e , s e v e r a l g e n e r a l t r e n d s w e r e o b s e r v e d . I n f u -s i o n s c o n t a i n i n g i s o l e u c i n e and t h e o t h e r b r a n c h e d c h a i n a m i n o a c i d s p r o m o t e d an i n c r e a s e i n t h e p r o t e i n c o n t e n t o f m i l k , a n d a d e c r e a s e i n m i l k s y n t h e s i s , b u t d i d n o t a f f e c t t o t a l p r o t e i n p r o d u c t i o n . R e s p o n s e s s i m i l a r t o t h o s e o b s e r v e d a b o v e w e r e n o t e d when i n f u s i o n s c o n t a i n i n g m e t h i o n i n e w e r e c o n s i d e r e d , a l t h o u g h t h e r e s p o n s e s t o i n f u s i o n s c o n t a i n i n g m e t h i o n i n e a p p e a r e d t o r e f l e c t t h e a m i n o a c i d s a c c o m p a n y i n g m e t h i o n i n e m o r e t h a n m e t h i o n i n e i t s e l f . When t h e b r a n c h e d c h a i n a m i n o a c i d s w e r e i n c l u d e d a l o n g w i t h m e t h i o n i n e t h e r e s p o n s e was s i m i l a r t o t h e r e s p o n s e t o t h e b r a n c h e d c h a i n a m i n o a c i d s a l o n e . However when m e t h i o n i n e was a c c o m p a n i e d b y t h r e o n i n e , p h e n y l a l a n i n e a n d l y s i n e t h e r e s p o n s e i n m i l k a n d m i l k p r o t e i n p r o d u c t i o n was q u i t e d i f f e r e n t f r o m t h o s e n o t e d a b o v e . W h i l e t h e r e was a g e n e r a l i n c r e a s e i n m i l k p r o t e i n c o n t e n t d u r i n g a l l i n f u s i o n s c o n t a i n i n g m e t h i o n i n e , o n l y d u r i n g t h e l a t t e r i n f u s i o n t h e r e was no d e c l i n e i n m i l k v o l u m e . In t h i s c a s e t h e r e was a s i g n i f i c a n t i n c r e a s e i n t o t a l m i l k p r o t e i n p r o d u c t i o n . T h e d e c l i n e s i n m i l k p r o d u c t i o n n o t e d f o r t h e o t h e r i n f u -s i o n s w e r e r e v e r s e d w i t h i n f u s i o n s c o n t a i n i n g l y s i n e . T h e m o s t d r a m a t i c i n c r e a s e i n p r o d u c t i o n o f m i l k a n d m i l k p r o t e i n o c c u r r e d when l y s i n e a l o n e was i n f u s e d . 166 I n f u s i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s r e s u l t e d i n a d e c r e a s e i n t h e o t h e r a m i n o a c i d s . T h i s d e c r e a s e t e n d s t o i n d i c a t e t h a t t h e b r a n c h e d c h a i n a m i n o a c i d s w e r e l i m i t i n g . I n c r e a s i n g t h e s u p p l y o f b r a n c h e d c h a i n a m i n o a c i d s r e s u l t e d i n r e d u c t i o n s i n t h e c o n c e n t r a t i o n s o f t h e o t h e r a m i n o a c i d s i n d i c a t i n g t h e i r i n c r e a s e d u t i l i z a t i o n f o r p r o t e i n s y n t h e s i s . T h e i n c r e a s e i n m i l k p r o t e i n c o n c e n t r a t i o n a l s o s u g g e s t e d i n c r e a s e d p r o t e i n s y n t h e s i s . However t o t a l m i l k p r o t e i n p r o d u c -t i o n was n o t a l t e r e d i n d i c a t i n g t h a t t h e o u t p u t o f a m i n o a c i d s i n m i l k was n o t r e s p o n s i b l e f o r t h e d e p r e s s e d c o n c e n t r a t i o n s o f a m i n o a c i d s i n p l a s m a . T h e i n c r e a s e d s u p p l y o f b r a n c h e d c h a i n a m i n o a c i d s d u r i n g t h e i r i n f u s i o n may h a v e i n d u c e d a n i n c r e a s e i n i n s u l i n s e c r e -t i o n . U p t a k e o f a m i n o a c i d s b y t i s s u e s and p l a s m a g l u c o s e c o n -c e n t r a t i o n s w o u l d b e a f f e c t e d b y i n c r e a s e d p l a s m a i n s u l i n . D u r i n g i n f u s i o n o f t h e b r a n c h e d c h a i n a m i n o a c i d s t h e c o n c e n -t r a t i o n o f s e v e r a l o f t h e p l a s m a f r e e e s s e n t i a l a m i n o a c i d s was l o w e r t h a n d u r i n g p r e - i n f u s i o n p e r i o d s . T h e r e was a l s o a t r e n d t o w a r d s l o w e r p l a s m a g l u c o s e c o n c e n t r a t i o n s d u r i n g t h e i n f u s i o n s . In f u s i o n o f l y s i n e d i d n o t r e s u l t i n s u c h d e c r e a s e s i n p l a s m a f r e e a m i n o a c i d s . T h e r e was a g e n e r a l t r e n d f o r t h e e s s e n t i a l a m i n o a c i d s t o i n c r e a s e o r r e m a i n a t t h e same l e v e l d u r i n g t h e i n f u s i o n . D u r i n g l y s i n e i n f u s i o n s t h e r e was a t r e n d t o w a r d h i g h e r p l a s m a g l u c o s e c o n c e n t r a t i o n s . T h e s e r e s u l t s u n d e r l i n e t h e i m p o r t a n c e o f a m i n o a c i d s , n o t o n l y a s p r e c u r s o r s 167 f o r p r o t e i n s y n t h e s i s , a n d a s p r e c u r s o r s f o r g l u c o s e s y n t h e s i s b u t a l s o a s m e d i a t o r s o f m e t a b o l i c c o n t r o l . T h r o u g h some a c t i o n l y s i n e was a b l e t o s t i m u l a t e p r o t e i n s y n t h e s i s w i t h o u t e l i c i t i n g an i n s u l i n r e s p o n s e . L y s i n e a p p e a r s t o be t h e l i m i t i n g a m i n o a c i d f o r m i l k p r o -t e i n s y n t h e s i s . 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The amino a c i d composition of h y d r o l y s a t e s of m i c r o b i a l p r e p a r a t i o n s from the rumen of sheep. Aust. J . B i o l . S c i . 10: 384-389. We l l e r , R.A., F.V. Gray and A.F. P i l g r i m . 1958. The c o n v e r s i o n of p l a n t n i t r o g e n to m i c r o b i a l n i t r o g e n i n the rumen of sheep. B r i t . J . Nutr. 12: 421-429. W i l l i e r , G.A. and E.M. K e s l e r . 1970. V a r i a t i o n s i n blood f r e e amino a c i d s i n l a c t a t i n g d a i r y cows. J . D a i r y S c i . 53: 647. A b s t r . W i l t r o u t , D.W. and L.D. S a t t e r . 1972. C o n t r i b u t i o n of p r o p i o n -ate t o glucose s y n t h e s i s i n the l a c t a t i n g and n o n - l a c t a t i n g cow. J . D a i r y S c i . 55: 307-317. Winkler, K. 1972. P r o t e i n s y n t h e s i s i n human l e u c o c y t e s . I I I . K i n e t i c s of the flow of amino a c i d s from the e x t r a c e l l u l a r space and the i n t r a c e l l u l a r pools r e s u l t i n g i n p r o t e i n s y n t h e s i s . Hoppe-Seyler's Z. P h y s i o l . Chem. 353: 782-786. Winkler, K., G. He l l e r - S c h o c h and R. Neth. 1972. P r o t e i n s y n t h e s i s i n human l e u c o c y t e s . IV. Mutual i n h i b i t i o n of amino a c i d i n c o r p o r a t i o n by amino a c i d s i n c e l l suspensions and c e l l - f r e e systems. Hoppe-Seyler's Z. P h y s i o l . Chem. 353: 787-792. 181 Wolff, J.E. and E.N. Bergman. 1972. Gluconeogenesis from plasma amino a c i d s i n fed sheep. Amer. J . P h y s i o l . 223: 455-460. Wolff, J.E., E.N. Bergman and H.H. W i l l i a m s . 1972. Net metabolism of plasma amino a c i d s by l i v e r and p o r t a l -d r a i n e d v i s c e r a of fed sheep. Amer. J . P h y s i o l . 223: 438-446 Yip , M.C.M. and W.E. Knox. 1972. F u n c t i o n of a r g i n a s e i n l a c t a t i n g mammary gla n d . Biochem. J . 127: 893-899. Young, V.R., S.C. S t o t h e r s and G. V i l a i r e . 1971. S y n t h e s i s and d e g r a d a t i o n of mixed p r o t e i n s and composition changes i n s k e l e t a l muscle of malnourished and r e f e d r a t s . J . Nutr. 101: 1379-1390. Yousef, I.M., J.T. Huber and R.S. Emery. 1969. A c t i o n of h i g h energy r a t i o n s on m i l k p r o t e i n s y n t h e s i s . J . D a i r y S c i . 52: 943. 182 F. APPENDIX The s i g n i f i c a n t l i n e a r r e l a t i o n s h i p , between p e r i o d 1 v a l u e s over the d u r a t i o n of Experiment II f o r s e v e r a l of the milk and plasma amino a c i d parameters measured, supports the use of p e r i o d 1 v a l u e s as a c o n t r o l f o r each i n f u s i o n . The f o l l o w i n g are the l e a s t squares equations determined f o r each s i g n i f i c a n t l i n e a r r e l a t i o n s h i p found, along w i t h the R 2. P r o t e i n (%) 2. 82 + .0017 (days) R 2 = .68 Glutamic A c i d (pg/ml) 2. 85 + • . 032 (days) R 2 - .59 C i t r u l l i n e (yg/ml) = 8. 74 + .048 (days) R 2 - .47 V a l i n e (yg/ml) = 22. 75 + .101 (days) R 2 .50 C y s t i n e (yg/ml) = 2. 91 + .011 (days) R 2 .49 Methionine (yg/ml) - 2. 36 + .021 (days) R 2 = .80 I s o l e u c i n e (yg/ml) = 10. 47 + .040 (days) R 2 = .41 Leucine (yg/ml) 11. 06 + .063 (days) R 2 = .48 O r n i t h i n e (yg/ml) - 4. 03 + .017 (days) R 2 = .37 H i s t i d i n e (yg/ml) 11. 04 + .021 (days) R 2 .38 Glutamic A c i d (%) 1 = 2. 33 + .014 (days) R 2 - .42 G l y c i n e (%) = 26. 12 - .049 (days) R 2 = .50 A l a n i n e (%) = 13. 18 - . 012 (days) R 2 = .46 C y s t i n e (%) = 2. 037 + .004 (days) R 2 = .39 Methionine (%) - 2. 891 + .006 (days) R 2 = .44 TESS 2 (yg/ml) = 96. 84 + .339 (days) R 2 - .48 TNESS 3 (yg/ml) = 14. 95 + .165 (days) R 2 = .45 183 R G l y ,792 - 002 (days] .47 TOTAL (yg/ml) 246.3 + .504 ( d a y s ) R' .52 (%) E s s e n t i a l o r n o n - e s s e n t i a l amino a c i d e x p r e s s e d as a p e r c e n t a g e o f t h e t o t a l e s s e n t i a l o r n o n - e s s e n t i a l a mino a c i d s . 'TESS = T o t a l e s s e n t i a l a m ino a c i d s , TNESS T o t a l n o n - e s s e n t i a l a m ino a c i d s , R G l y G l y c i n e ( y g / m l ) / V a l i n e + I s o l e u c i n e + L e u c i n e ( y g / m l ) . TOTAL = TESS + TNESS G r a p h i c a l r e p r e s e n t a t i o n o f s e v e r a l o f t h e p a r a m e t e r s i s g i v e n i n F i g u r e s I , I I and I I I . Figure 2 Period 1 Essential or Non-Essential Amino Acids as a Percentage of the Total Essential or Non-Essential Amino Acids. 25 20 Percentage 15! 10 -X X Glycine Alalanine Glutamic Acid Methionine Glycajie/Valine Isoleucine + Leucine xlO K A r— 75 100 ~ 1 2 5 1 5 0 Time - Days 175 lb~0 225 Figure 3 Period 1 Values for Total Essential (TEES) and Total Non-Essential (TNESS) Amino Acids. TABLE VT-1 STANDARD DEVIATIONS FOR 80 DUPLICATE AMINO ACID ANALYSIS MEAN STANDARD DEVIATION TAURINE 6. 551 .0053 UREA 189. 689 1.618 THREONINE 12. 108 .0412 SERINE 11. 531 .055 ASPARAGINE1 48. 827 .414 PROLINE 10. 691 .138 GLUTAMIC ACID 9. 182 .165 CITRULLINE 15. 930 .087 GLYCINE 28. 212 .073 ALANINE 23. 505 .094 a-AMINO-N-BUTYRIC ACID 1. 899 .023 VALINE 43. 975 .106 METHIONINE 6. 435 .025 ISOLEUCINE 21. 934 .052 LEUCINE 24. 533 .085 TYROSINE 9. 139 .036 PHENYLALANINE 7. 092 .049 LYSINE 19. ,621 .068 HISTIDINE 14. ,206 .115 ARGININE 17. ,406 .094 'Asparagine + Glutamine. 

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