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Evaluation of techniques employed in the study of alanine metabolism in sheep Cooper, Donald Arthur 1974

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E v a l u a t i o n o f Techniques Employed i n t h e Study o f A l a n i n e Metabolism i n Sheep  by DONALD ARTHUR COOPER  BSc,  U n i v e r s i t y o f B r i t i s h Columbia, 1972  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department 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 as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1974  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l  f u l f i l m e n t o f the requirements  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree the L i b r a r y  s h a l l make i t f r e e l y  available for  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e  r e f e r e n c e and copying o f t h i s  It  i s understood that copying or  thesis  permission.  Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  or  publication  o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my written  that  study.  f o r s c h o l a r l y purposes may be granted by the Head of my Department by h i s r e p r e s e n t a t i v e s .  for  ABSTRACT I n view of the importance o f precursor  i n ruminants,  alanine  metabolic  a  gluconeogenic  t h e o b j e c t i v e o f t h e p r e s e n t study was  t o a s s e s s the e f f e c t i v e n e s s o f t h r e e the  as  parameters  techniques  i n estimating  s u r r o u n d i n g a l a n i n e i n wethers f e d a  maintenance d i e t of a l f a l f a hay. A p r e l i m i n a r y experiment u t i l i z e d a blood f l o w t e c h n i q u e t o study t h e net p r o d u c t i o n and/or u t i l i z a t i o n o f both a l a n i n e glucose  by  evaluating  and  the p o r t a l d r a i n e d v i s c e r a . Such a method i n v o l v e d the arterio-venous  concentration  differences  a l a n i n e and g l u c o s e , i n c o n j u n c t i o n w i t h d e t e r m i n i n g  of  the r a t e of  p o r t a l v e i n blood f l o w . Radioactively  l a b e l l e d **C - a l a n i n e was a d m i n i s t e r e d a s a  s i n g l e i n j e c t i o n i n t h e second s e r i e s o f e x p e r i m e n t s t o e s t i m a t e the m e t a b o l i c parameters o f a l a n i n e as w e l l as i t s c o n t r i b u t i o n to  glucose  synthesis.  The  L-0- *C-alanine l  i n t r a v e n o u s l y through p r e v i o u s l y i m p l a n t e d  determined.  given  j u g u l a r c a t h e t e r s and  the f a l l i n the s p e c i f i c a c t i v i t y o f plasma was  was  alanine  with  time  The l i n e o f b e s t f i t f o r the decay curve o f t h e  s p e c i f i c a c t i v i t y o f plasma a l a n i n e was c o n s t r u c t e d by means a computer u s i n g a m u l t i - t e r m e x p o n e n t i a l f u n c t i o n which  of  enables  the e s t i m a t i o n o f such parameters as t h e p o o l s i z e , s p a c e , t o t a l entry  r a t e , i r r e v e r s i b l e l o s s and r e c y c l i n g o f a l a n i n e . The p e r  cent c o n v e r s i o n o f a l a n i n e t o corresponding  peak  of  glucose  determined  by t h e  glucose s p e c i f i c a c t i v i t y f o l l o w i n g the  s i n g l e i n j e c t i o n o f *C - a l a n i n e . 4  was  ii  The t u r n o v e r o f a l a n i n e was a l s o s t u d i e d u s i n g a c o n t i n u o u s i n f u s i o n of L-U- *C - a l a n i n e w i t h o u t 1  a priming  injection.  The  s p e c i f i c a c t i v i t y o f p l a s n a a l a n i n e reached a p l a t e a u f i v e hours after  the  beginning  of t h e i n f u s i o n . I t was from these p l a t e a u  l e v e l s t h a t the r a t e o f i r r e v e r s i b l e l o s s c f a l a n i n e as w e l l i t s percent The  conversion  results  t o glucose  than a  ruminants, infusion  total  entry  rate  of  technique  alanine  into  l o s s and r e c y c l i n g and thus prove more i n f o r m a t i v e  continuous  suggested  estimated.  i n d i c a t e d t h a t the s i n g l e i n j e c t i o n  was a b l e t o p a r t i t i o n t h e irreversible  was  as  infusion  method.  The  present  study  also  t h a t under c e r t a i n p h y s i o l o g i c a l s t r e s s c o n d i t i o n s i n where  recycling  approach  may  becomes  overestimate  i r r e v e r s i b l e loss of alanine.  prominent, the  a  actual  continuous rate  of  iii  Tabl§_of_Conten_s_ Abstract  i  Table of C o n t e n t s  ••••  i i i  L i s t of Figures  v  L i s t of T a b l e s  v i i  Acknowledgements  ...........................................viii  Introduction  1  L i t e r a t u r e Survey  4  Glucose Metabolism i n t h e Ruminants A. ) Glucose r e q u i r e m e n t s B. ) Glucose P r o d u c t i o n i n t h e Ruminant ................. Techniques Used f o r Measurement o f A l a n i n e and Glucose Metabolism A. ) E a r l y D i e t a r y S t u d i e s .............................. B. ) I n d i c a t o r D i l u t i o n Techniques ...................... C. ) I s o t o p e D i l u t i o n Methods ........................... Experimental  4 5 9 27 28 30 35 46  I Metabolism of Glucose and A l a n i n e by the Portal-Drained Introduction  Viscera  46  ...........................................  46  Experiment A.1 M a t e r i a l s and Method Calculations R e s u l t s and D i s c u s s i o n Conclusion I I S i n g l e I n j e c t i o n of L a b e l l e d  48 48 54 56 58 C - A l a n i n e ................  61  I n t r o d u c t i o n ..........................................  61  Experiment B.1. M a t e r i a l s and Methods Calculations R e s u l t s and D i s c u s s i o n Experiment B.2........................................ M a t e r i a l s and Methods Calculations R e s u l t s and D i s c u s s i o n  63 63 69 71 74 74 77 77  1 4  iv Experiment B.3 M a t e r i a l s and Methods Calculation R e s u l t s and D i s c u s s i o n  ................................  Conclusion  88  I I I Continuous I n f u s i o n of L a b e l l e d Alanine:  Without a P r i m i n g  **C-labelled  Dose  92  Introduction Experiment C.1. M a t e r i a l s and Methods Calculations R e s u l t s and D i s c u s s i o n Experiment C.2. M a t e r i a l s and Methods Calculations R e s u l t s and D i s c u s s i o n G e n e r a l Summary and C o n c l u s i o n s Glossary  o f Terms  References C i t e d Appendix  81 81 83 83  92  ..  95 95 97 99 .101 101 103 .....104 106 112 116 ...125  V  List_of_Fi2ures  Figure  1. ) S t a n d a r d Curve f o r Glucose D e t e r m i n a t i o n  126  2. ) Standard Curve f o r A l a n i n e D e t e r m i n a t i o n  127  3. ) Standard Curve f o r P a r a - a m i n o h i p p u r i c A c i d (PAH) d e t e r m i n a t i o n  ...128  4. ) Quench C o r r e c t i o n Curve f o r C  129  1 4  5. ) Paper Chromatography S e p a r a t i o n o f C - A l a n i n e w i t h Dse o f t h e A c t i g r a p h Scanner 6. ) A c t i v i t y Curve f o r * C - G l u c o s e i n J u g u l a r V e i n B l o o d (J.V.) Experiment B.1. 7. ) A c t i v i t y Curve f o r »*C-Alanine i n J u g u l a r Vein (J.V.) Experiment B. 1 1 4  130  1  131 132  8. ) A c t i v i t y Curve f o r »*C-Glucose i n J u g u l a r V e i n (J.V.) Experiment B.2 ............133 9. ) A c t i v i t y Curve f o r »*C-Alanine i n J u g u l a r V e i n (J.V.) Experiment B.2 .............134 10a.) 10b.) 11a.) 11b.)  A c t i v i t y Curve f o r i*C-Glucose i n C a r o t i d A r t e r y (C.A.) Experiment B.3.  135  A c t i v i t y Curve f o r **C-Glucose i n J u g u l a r Vein (J.V.) Experiment B.3.  136  A c t i v i t y Curve f o r * * C - A l a n i n e i n C a r o t i d A r t e r y (C.A.) Experiment B.3  137  A c t i v i t y Curve f o r * * C - A l a n i n e i n J u g u l a r V e i n (J.V.) Experiment B.3.  ...138  12. ) A c t i v i t y Curves f o r i * C - A l a n i n e and i * C - G l u c o s e Experiment C . I . 13. ) A c t i v i t y Curves f o r C - A l a n i n e and i*C-Glucose Experiment C.2.  • 139  1 4  14. ) Major M e t a b o l i c Pathways i n The L i v e r and Kidneys of Ruminants  .....140 141  vi 15. ) Schematic Diagram  Illustrating  the P o s i t i o n  of the Sampling and I n f u s i o n C a t h e t e r s  142  16. ) The Glucose a l a n i n e C y c l e 17. ) Model f o r S i n g l e I n j e c t i o n and Continuous of a Dye 18. ) Model f o r Glucose Metabolism i n Sheep  143 Infusion 144 145  vii  List_of_Tables Table 1(a). 1(b).  133® P a c k e d C e l l Volume V a l u e s , G l u c o s e , A l a n i n e , and PAH C o n c e n t r a t i o n s i n P o r t a l and C a r o t i d A r t e r i e s C a l c u l a t i o n s f o r Experiment  A.1. ( B l o o d  .... 146  Flow)  117  2. P r e l i m i n a r y E x p e r i m e n t t o T e s t E f f i c i e n c y o f I o n Exchange C h r o m a t o g r a p h y f o r S e p a r a t i o n o f P l a s m a Components 3.  Paper Chromatography S e p a r a t i o n  4. Data and M e t a b o l i c a Single Injection E x p e r i m e n t B.1. 5.  of  1 4  C-Alanine  .......  148 149  Parameters o f A l a n i n e f o l l o w i n g o f 0- C - A l a n i n e : 1 4  150  Data M e t a b o l i c P a r a m e t e r s o f A l a n i n e F o l l o w i n g a Single Injection of D- C-Alanine: E x p e r i m e n t B.2 1 4  151  6. Data and M e t a b o l i c P a r a m e t e r s o f A l a n i n e Following a S i n g l e I n j e c t i o n of U- *C-Alanine E x p e r i m e n t B.3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  153  7. Summary o f M e t a b o l i c P a r a m e t e r s S i n g l e I n j e c t i o n Experiments  156  1  f o r the  8. Data and M e t a b o l i c P a r a m e t e r s o f A l a n i n e a Continuous I n f u s i o n of * C - A l a n i n e : E x p e r i m e n t C.1.  Following  9. Data and M e t a b o l i c P a r a m e t e r s o f A l a n i n e a Continuous I n f u s i o n of **C-Alanine: E x p e r i m e n t C. 2.  Following  l  10.  157  Summary o f P a r a m e t e r s f r o m S i n g l e I n j e c t i o n and Continuous I n f u s i o n Experiments  158 160  viii  ACKNOWLEDGEMENTS  The  author  University  wishes  t o express  h i s gratitude  to  the  o f B r i t i s h Columbia Research Committee f o r f i n a n c i a l  s u p p o r t , i n t h e form o f a K i l l a m P r e d o c t o r a l F e l l o w s h i p . I would l i k e t o extend a f u l l measure  of  appreciation to  the members o f my t h e s i s Committee: Dr. W. D. K i t t s  (Chairman)  Chairman Animal  Departments Science  of  and P o u l t r y  Science Dr. R. M. Beames  Department o f Animal S c i e n c e  Dr. D. B. Bragg  Department o f P o u l t r y S c i e n c e  Dr. R.M. T a i t  Department o f Animal  Dr. Animal  Science  C. R. K r i s h n a m u r t i ( A s s o c i a t e P r o f e s s o r , Department o f Science)  supervision,  must  receive  special  mention  for his  i n t e r e s t and encouragement d u r i n g t h e two y e a r s of  t h i s study. S i n c e r e thanks a r e extended PhD s t u d e n t i n t h e Department  t o Dr. B r i a n D. Mason who, as a  of  Animal  Science,  contributed  g r e a t l y t o my academic development. L a s t l y , I would l i k e t o thank Miss L y n e t t e F. L l o y d f o r her p a t i e n c e and h e l p i n p r o d u c i n g t h i s  dissertation.  1  IHTRODDCTION I n c o n t r a s t to m o n o g a s t r i c a n i m a l s r u m i n a n t s , f e d p r i m a r i l y a roughage  d i e t absorb n e g l i g i b l e amounts of g l u c o s e t h r o u g h the  gastro-intestinal  tract.  The  carbohydrates  are  rumen m i c r o o r g a n i s m s t o v o l a t i l e f a t t y a c i d s , of b u t y r i c , and p r o p i o n i c a c i d s predominate 1970,  Bergman  et  al.,  fermented by which  (Annison and  acetic,  Armstrong,  1965). Thus t h e ruminant a n i m a l depends  upon g l u c o n e o g e n e s i s or the endogenous s y n t h e s i s of g l u c o s e from non-carbohydrate s o u r c e s , as a means of  satisfying  i t s energy  demands. Ruminants  possess  a  glucose  m a i n t a i n e d under normal d i e t a r y However  this  under s t r e s s reduction  critical  environmental  Such  traumas  blood  rise  (ketosis).  whereas  in  disease.  whose  sheep  during  hypoglycemia  manifested  by  a  concentration  in  periods and  of  heavy  ketosis arise twin  While t h e s e d i s t u r b a n c e s o f d a i r y cows and  sheep  both which  disorders include  a  have  a  negative  r e d u c t i o n of g l u c o s e i n t h e b l o o d and l i v e r , fat  disrupted  or  are not i d e n t i c a l , characteristics,  conditions.  (hypoglycemia) and an  d u r i n g l a t e pregnancy and i s c a l l e d pregnancy lamb  i s just  I n the d a i r y cow t h i s c o n d i t i o n i s  r e f e r r e d t o as acetonemia and o c c u r s lactation,  are  blood glucose concentration  e x c e s s i v e p r o d u c t i o n o f ketone b o d i e s , the  which  energy b a l a n c e can be r e a d i l y  conditions.  in  and  requirement  toxemia  number  of  common  energy b a l a n c e , a and  an  increased  metabolism. The heavy d r a i n of g l u c o s e i n the ruminant a l s o  becomes a c u t e under c o n d i t i o n s of s t a r v a t i o n . Of  the  three  major  end  products  of  rumen  microbial  2  fermentation  it  has been shown t h a t between 27 and 54 per  of the g l u c o s e may propionate,  be  either  produced  before  or  from upon  compounds  arising  from  a b s o r p t i o n i n t o the b l o o d  d r a i n i n g the rumen (Bergman, 1973). F u r t h e r work by al.  cent  ftnnison  et  (1963a) c o n f i r m e d the g l u c o g e n i c r o l e of p r o p i o n a t e and,  employing  **C-labelled butyrate  these endproducts  and  of c a r b o h y d r a t e  acetate,  indicated  by  that  fermentation contribute l i t t l e  i f any t o the p r o d u c t i o n o f g l u c o s e . G l u c o g e n i c p r o p e r t i e s have been a s s i g n e d to such as  the  precursors  g l y c e r o l moiety of t r i g l y c e r i d e s as w e l l as l a c t a t e  pyruvate.  The  conditions  former  of  becomes  underfeeding  a c i d s are m o b i l i l i z e d from  a  or the  major  contributor  and  under  s t a r v a t i o n when the f r e e f a t t y adipose  tissue.  The  glycerol  which i s r e l e a s e d t o the b l o o d i s t r a n s p o r t e d to the l i v e r where it  readily  lactate,  gives  and  to  gluconeogenesis c o n t r o v e r s y . The unknown  rumen this  a in  &  potential  following  glucose  lesser  (Bergman, 1963). The  degree  ruminants  which  the degree of  wall,  to  has  pyruvate, undergone  s i t u a t i o n r e s u l t s because of the  amounts  t r a c t and  rise  a r e absorbed metabolism  i n t o the which  play  precursor  variable  and  gastro-intestinal  occurs  is  in  considerable  through  d e t a i l e d review o f the p r e s e n t u n d e r s t a n d i n g glucogenic  role  presented  in  the of the  section.  Other  than  propionate,  the  second  most  important  c o n t r i b u t o r to endogenous g l u c o s e p r o c u c t i o n i s t h e amino a c i d s . Host o f t h e s e a r e g l u c o g e n i c , leucine  and  with  the  exception  of  lysine,  t a u r i n e (Krebs, 1964). Recent r e s e a r c h w i t h humans  3  ( F e l i g et a l . , 1970), r a t s (Holff  (Aikawa  et  a l . , 1972)  ana  sheep  and Bergman 1972a) has p r o v i d e d c o n v i n c i n g e v i d e n c e t h a t  a l a n i n e and g l u t a m i n e are the p r i n c i p l e amino a c i d s e x t r a c t e d by the l i v e r  f o r g l u c o n e o g e n e s i s . I n a d d i t i o n these amino a c i d s are  the main amino a c i d s r e l e a s e d from  the  skeletal  t h i s f i n d i n g a l a n i n e ( F e l i g e t a l . , 1970) El  li» i  1971)  linking  amino  gluconeogenesis.  and g l u t a m i n e  c y c l e s have been proposed acid The  metabolism nature  and  effect  precursor  in  ruminants  of  the  e f f e c t i v e n e s s of v a r i o u s t e c h n i q u e s metabolic  parameters  of  the of  which  control  these c y c l e s are  alanine  as  present  study  employed  a  to  gluconeogenic assessed  the  estimate  the  maintenance  diet.  The  employed were a m o d i f i c a t i o n o f t h e s i n g l e  without  a  of  three  injection  **C-labelled  p r i m i n g dose and a p o r t a l b l o o d f l o w s t u d y ,  i n c l u d e d an a r t e r i o - v e n o u s c o n c e n t r a t i o n  g l u c o s e and a l a n i n e .  of  review.  * * C - l a b e l l e d a l a n i n e , a continuous i n f u s i o n  alanine  (Marliss  of a l a n i n e as w e l l as i t s c o n t r i b u t i o n t o  g l u c o s e s y n t h e s i s i n sheep f e d a techniques  From  as i m p o r t a n t means of  with  d i s c u s s e d f u l l y i n the f o l l o w i n g l i t e r a t u r e I n view of the importance  muscle.  assay  for  both  tt  Slucosa  plays  s e r v e s as a b a s i c macromolecules and l i p i d s . of  energy  an  essential  component  of  role  in cell  metabolism. I t  i n the c o n s t r u c t i o n of  complex  the c e l l , i n c l u d i n g n u c l e i c a c i d s , p r o t e i n s  In a d d i t i o n , g l u c o s e i s u t i l i z e d f o r t h e which  i s necessary  f o r the  production  various  endergonic  p r o c e s s e s w i t h i n t h e c e l l . Thus t h e c a l o r i c needs  of  are  Other  almost  entirely  satisfied  by  t h i s hexose.  t h e body sources  i n c l u d e the c a t a b o l i s m o f f a t t y a c i d s , however t h i s mechanism of energy p r o d u c t i o n becomes p r e v e l e n t o n l y when t h e former  source  i s depleted. The  importance  of  g l u c o s e i n m a i n t a i n i n g an a n i m a l i n an  adequate energy b a l a n c e i s p a r a l l e l e d m o n o g a s t r i c s . However, t h e metabolism many  respects  which  in  of  and  of carbohydrates v a r i e s i n  the  presence of a f a r g r e a t e d  ruminant  carbohydrates i n the simple degradation  i n ruminants  r e f l e c t t h e a n a t o m i c a l v a r i a t i o n s between  t h e two, and t h e subsequent population  both  dietrary  species.  stomached  carbohydrates  The  animals to  microbial  metabolism  of  i n v o l v e s the  glucose  and o t h e r  s i m p l e s u g a r s , which a r e then absorbed by t h e p o r t a l c i r c u l a t o r y system and u t i l i z e d ruminants,  the  by  the animal.  carbohydrates  p r e s e n t i n the rumen,  to  On  the o t h e r  a r e fermented  volatile  fatty  by  acids.  hand,  in  microorganisms Consequently  5  negligible  amounts  of  glucose  i n t e s t i n a l t r a c t . Thus an noncarbohydrate  are  endogenous  p r e c u r s o r s , or  absorbed  v i a the g a s t r o -  glucose  production  from  g l u c o n e o g e n e s i s i s r e l i e d upon  h e a v i l y t o meet the a n i m a l s g l u c o s e r e q u i r e m e n t s .  A.)  Glucose_ReG|uirements The r e q u i r e m e n t s f o r g l u c o s e a r e e q u a l l y i m p o r t a n t both  ruminants  and  monogastrics,  however  the  blood  glucose  c o n c e n t r a t i o n s d i f f e r c o n s i d e r a b l y . The l e v e l i n r u m i n a n t s 60 mg/100 ml)  i s l o w e r than t h a t o c c u r r i n g i n new  s i m p l e stomached Glucose  is  animals  (80-100 mg/100 ml)  (40-  born o r a d u l t  (Bergman,  1973).  needed by f i v e major a r e a s of the body, t h e nervous  system, t u r n o v e r and s y n t h e s i s of f a t , muscle, f e t u s e s mammary  in  gland.  and  the  The amount u t i l i z e d by o t h e r c e l l s of ruminants  such as e r y t h r o c y t e s has been shown t o be n e g l i g i b l e  (Leng  and  A n n i s o n , 1962) .  1  • 2£iii25i£i2£_fe2_£k6_S§Ey.2S§-§Is tern Work  by  Cahill  et  al.,  (1970)  on  metabolism  p o s t a b s o r t i v e humans has i n d i c a t e d t h a t the human nervous  system  u t i l i z e s as much as 80 p e r c e n t of t h e g l u c o s e r e l e a s e d i n t o blood.  Such  a  study  has  yet  to  in  the  be performed i n r u m i n a n t s ,  however from the a r t e r i a l - j u g u l a r c o n c e n t r a t i o n d i f f e r e n c e s i n sheep, c o n s i d e r a b l e g l u c o s e system  must  be  removed  by  the  nervous  and t h e b r a i n i n p a r t i c u l a r , of these s p e c i e s (McClyaont  6  and  Setchell,  nervous  1956);  system  absolutely  with  dependent  o x i d a t i v e metabolism. glucose  utilized  (Setchell, the brain upon  1961). Thus t h e c e l l s o f t h e being  most  important,  a r e g u l a r s u p p l y of g l u c o s e f o r i t s  An i n t e r e s t i n g v a r i a t i o n t o the amount  1967 E a j u  et  a l . , 1972 and Weiner e t a l . ,  a d a p t a t i o n i s p r e s e n t i n a l l t i s s u e s o f t h e body strikingly  2  (Owen e t  1971). T h i s  b u t i s most  e v i d e n t f o r t h e b r a i n and c o n s i s t s o f a c o n s e r v a t i o n  of g l u c o s e and a u t i l i z a t i o n o f ketone bodies f o r energy  of  d u r i n g p r o l o n g e d s t a r v a t i o n i n t h e sheep, dog  and man has been r e f e r r e d t o as a " g l u c o s e e x c l u s i o n " . al.,  are  purposes  and  fatty  acids  d u r i n g p e r i o d s of s t a r v a t i o n .  • y^JLlJZJf J-°8-£9g-f3JLJ!f£ai3gligB Glucose  p l a y s a d u a l r o s e i n t h e t u r n o v e r and s y n t h e s i s of  both m i l k and body f a t . I t i s the p r e c u r s o r glycerophosphate,  which  of  glycerol,  and  are u t i l i z e d i n the e s t e r i f i c a t i o n of  f a t t y a c i d s t o form t r i g l y c e r i d e . Vaughn,  (1961) has shewn  that  t h e enzyme g l y c e r o l k i n a s e i s l a c k i n g i n both a d i p o s e t i s s u e and mammary  gland.  Thus t h e g l y c e r o l r e q u i r e m e n t s o f these  must be met by g l u c o s e The second provision  of  metabolism.  r o l e o f g l u c o s e i n f a t metabolism  i s i n the  adequate s u p p l i e s o f NADPH which i s r e q u i r e d a s a  r e d u c i n g agent i n the s y n t h e s i s (Ballard  tissues  of  long-chain  fatty  acids.  e t a l . , 1969; K r e b s , 1966). I n a d d i t i o n t o t h e above  two, g l u c o s e can a l s o s e r v e as a carbon s u b s t r a t e f o r f a t t y  acid  7  synthesis.  However, i n t h e ruminant  synthesis  are supplied  Honruminants  rely  to  on  a  a n i m a l carbon atoms f o r f a t  greater  glucose  extent  rather  by  than  acetate.  acetate f o r  lipogenesis.  3.  Utiiization_by _Muscle_Tissue -  H u s c l e g l y c o g e n s y n t h e s i s depends quantity  of  that  1970).  present This  that i s involved. essential  glucose.  The  total  g l u c o s e s t o r e d i n t h e muscles remains c o n s t a n t and  i s g r e a t e r than (Lehninger,  on  The  function  or  body  fluids  i s due t o t h e much l a r g e r t i s s u e mass glycogen  stores  of  muscle  by p r o v i d i n g an a n a e r o b i c energy  the muscle d u r i n g  exercise  (Bergman,  The  1973).  i n the l i v e r  or  when  oxygen  serve  supply f o r  becomes  limiting  m a j o r i t y of t h e glycogen i s c o n v e r t e d t o  l a c t a t e and p y r u v a t e which i s r e t u r n e d t o the b l o o d and then the  an  to  l i v e r f o r r e s y n t h e s i s back i n t o g l u c o s e . T h i s g l u c o s e , when  l i b e r a t e d i n t h e b l o o d , may a g a i n r e t u r n t o t h e  muscles  to  be  c o n v e r t e d t o g l y c o g e n . Such a p r o c e s s i s r e f e r r e d t o a s t h e C o r i cycle  (Cori,  1931).  8  4. Ut i l i z a t i o n _ b y _ _ t h e _ F e t u s One lata  o f t h e g r e a t e s t g l u c o s e demands i n ruminants i s d u r i n g  pregnancy  pregnancy  and  toxaemia  time. S i n c e  the  lactation.  Such  metabolic  disorders  as  i n sheep and k e t o s i s i n cows o c c u r a t t h i s  principal  metabolic  fuel  of  the  fetus i s  c a r b o h y d r a t e , i t must m a i n t a i n a c o n s t a n t s u p p l y o f g l u c o s e from the  mother.  In  this  regard  work  by  Huggett  (1961)  firmly  e s t a b l i s h e d t h a t t h e p l a c e n t a s of ruminants c o n v e r t a p o r t i o n of t h e m a t e r n a l g l u c o s e t o f r u c t o s e so t h a t the both  sugars  reach even h i g h e r l e v e l s  concentrations  of  i n the body f l u i d s of the  f e t u s than t h e mother. I n a d d i t i o n , l a t e  i n fetal  life  large  g l y c o g e n r e s e r v e s a r e b u i l t up i n t h e p l a c e n t a , and i n t h e f e t a l liver,  lung  and  skeletal  muscle.  In  a  review  of  fetal  p h y s i o l o g y , Dawes (1968) s t a t e d g l y c o g e n c o n c e n t r a t i o n s t o be  8  to  10 p e r c e n t i n l i v e r and about 4 p e r c e n t i n muscles, which a r e  2  to  8  times  t h e c o r r e s p o n d i n g v a l u e s f o r a d u l t s o f t h e same  species.  5• D t i l i z a t i o n d u r i n g L a c t a t i o n L a c t a t i o n a l s o makes a l a r g e demand f o r g l u c o s e animal  upon  the  s i n c e m i l k c o n t a i n s a p p r o x i m a t e l y 90 t i m e s as much t o t a l  sugar as does  blood  (Bergman,  1970).  Of  the  two  metabolic  d i s o r d e r s i n v o l v i n g g l u c o s e , pregnancy toxaemia and k e t o s i s , the former  i s manifested  w i t h a f a r more severe hypoglycemia  the l a t t e r s i n c e the g l u c o s e  supply  f o r milk  production  than can  9  readily  be  reduced or c u t o f f c o m p l e t e l y . Such i s not t h e case  f o r a pregnant a n i m a l where the g l u c o s e  demands  of  the  fetus  must be s u s t a i n e d . From  this  of g l u c o s e by animals  b r i e f s u r v e y of t h e metabolism the  depend  ruminant,  heavily  i t  appears  and  evident  requirements that  these  upon g l u c o n e o g e n e s i s f o r t h e i r g l u c o s e  supply.  B.)  Glu cose_Product i o n ^ i ^ t h e ^ u m i n a n t  1. Di e t a r y__Sour ce The s i t e s o f g l u c o s e p r o d u c t i o n i n the body of a l l mammals are  the  gut  (by  absorption),  mentioned  earlier  stomached  species i n that l i t t l e  the  gastro-intestinal  controversy ruminants this  ruminant  animals  tract.  regarding  the  simple  of  still  exists  f e d g r a i n or h i g h c o n c e n t r a t e d i e t s . One  approach  to  to  quantity  there  from  in  d e t e c t the amount o f s t a r c h which small  intestines.  rumen  Further research the  only in  little this  grain  starch  area may  has  and  approach  t h a t b a r l e y and oat d i e t s are r e a d i l y fermented  and  of  escapes  HacBae  (1966) and Topps e t a l . , (1968) employed t h i s  and i n d i c a t e d  grinding  from  or no g l u c o s e i s absorbed  However,  f e r m e n t a t i o n and f l o w s i n t o the  the  differ  As  absorbed  is  the  l i v e r and the k i d n e y s .  glucose  problem  Armstrong  the  in  appears i n t h e i n t e s t i n e . postulated  that  a  fine  r e s u l t i n a f a s t e r r a t e of passage  through t h e rumen and t h u s enable more g l u c o s e  to  be  absorbed  10  (Sutton  and  N i c h o l s o n , 1968). , another method used t o s t u d y the  g l u c o s e a b s o r p t i o n under c o n c e n t r a t e d i e t s glucose  concentrations  technique  a  absorption  number into  et  the  i n p o r t a l and a r t e r i a l b l o o d . With  this  workers  detected  to  no  actual  glucose  b l o o d of a n i m a l s f e d a maintenance d i e t of hay  or a 50 p e r c e n t hay (Bergman,  measure  of  is  -  grain  a l . , 1970;  (wheat,  Katz  corn  or  oats)  mixture  and Bergman,, 1969; Roe e t a l . ,  1966). I n a r e v i e w a r t i c l e Bergman  (1973)  suggested  that  the  k i n d o f f e e d , as w e l l as the amount and f r e q u e n c y of f e e d e a t e n , may determine  whether or not g l u c o s e i s abosrbed.  2. R i n a l _ G l u c o n e o q e n e s i s The  kidneys  have  been suspected of p l a y i n g a r o l e i n the  p r o d u c t i o n of g l u c o s e s i n c e no a p p r e c i a b l e amounts a r e from  the  gastro-intestinal  tract  and  the  absorbed  liver  has  been  e s t i m a t e d t o c o n t r i b u t e 85 p e r c e n t o f t h e t o t a l g l u c o s e t u r n o v e r i n nonpregnant f e d sheep (Bergman e t Kaufman  and  t h e two  kidneys  pregnant  a l . , 1970)..  Research  Bergman (1971) r e p o r t e d t h a t g l u c o s e p r o d u c t i o n by  than  averaged in  0.4  nonpregnant  -0.8  g/hr  sheep.  and Renal  was  higher  in  normal  in  gluconeogenesis  appears t o account f o r 8 - 10 p e r c e n t o f the t o t a l body turnover  by  glucose  f e d sheep and f o r a p p r o x i m a t e l y 15 p e r c e n t  d u r i n g f a s t i n g . To f u r t h e r emphasize t h e s i g n i f i c a n c e  of  renal  g l u c o n e o g e n e s i s i n r u m i n a n t s , t h e s e workers p o i n t e d out t h a t the sum  of  averaged  the  g l u c o s e p r o d u c t i o n r a t e s of both l i v e r and  98 p e r c e n t , which accounts f o r  body's t o t a l g l u c o s e p r o d u c t i o n .  virtually  kidneys  a l l of  the  11  3.  Hagatic_Gluconeoaenesis The  animals total  liver  i s the  major  contributor  o f g l u c o s e f o r the  energy demands, w i t h an e s t i m a t e of 85 p e r c e n t glucose  turnover  arising  from  the  hepatic  of  source i n  r u m i n a n t s under a s t e a d y s t a t e c o n d i t i o n (Bergman e t a l . , Thus t h e s o u r c e s of h e p a t i c g l u c o s e p r o d u c t i o n c o n s i s t major  precursors,  which  include  propionate,  the  1970).  of  four  glycerol,  and  l a c t a t e and amino a c i d s .  A. From_Pror>inate The s h o r t c h a i n f a t t y a c i d s a r i s i n g from of  dietary  carbohydrates  per cent o f t h e a n i m a l ' s  the  fermentation  i n t h e rumen s u p p l y a p p r o x i m a t e l y  c a l o r i c requirements  (Bergman  et  70 al.,  1965)., Of t h e s e o n l y p r o p i o n a t e can c o n t r i b u t e s i g n i f i c a n t l y t o glucose synthesis (Annision e t a l . , 1971;  Black e t a l . ,  1963;  Bergman  and  1966 and 1972; Leng and Annison,  net c o n t r i b u t i o n of p r o p i o n a t e t o t h e s y n t h e s i s o f  Wolff,  1963). The  glucose  has  been s u b j e c t t o some c o n t r o v e r s y . U n c e r t a i n t y s t i l l e x i s t s as t o whether  the  rate  of  p r o p i o n a t e p r o d u c t i o n i n the ruminant i s  s u f f i c e n t t o meet t h e demands f o r g l u c o n e o g e n e s i s . Early propionate  work  in  absorbed  measuring,  quantitatively,  the  from t h e rumen has been c o m p l i c a t e d  l a c k of complete c o n t r o l i n t i m i n g t h e e x p e r i m e n t s w i t h to  feeding  metabolized  and  also  by  the  by  the  rumen  fact  that  epithelium  some during  amount by t h e respect  propionate i s absorption  12  (Pennington,  1954).  Thus  the gluconeogenic  remained obscure. The f i r s t advance i n t h i s Annision  et  al.,  (1957),  r o l e of p r o p i o n a t e area  who, by measuring  concluded  a  by  volatile  fatty  t h a t n e a r l y a l l of the p r o p i o n a t e was removed  by t h e l i v e r . Bergman e t a l . , using  made  the p o r t a l v e i n -  a r t e r i a l blood c o n c e n t r a t i o n d i f f e r e n c e s o f t h e acids,  was  constant  (1966),  extended  the  study  by  i n f u s i o n o f l a b e l l e d p r o p i o n a t e i n t o a rumen  v e i n of nonpregnant, n o n l a c t a t i n g ewes and found t h a t the amount of  p r o p i o n a t e absorbed  quality  of  depends g r e a t l y  upon  the  quantity  and  the f e e d b e i n g d i g e s t e d . The r e s u l t s i n d i c a t e d t h a t  normal sheep absorbed  about 24 mM  propionate  per  hour.  These  f i g u r e s e s t i m a t e t h a t a p p r o x i m a t e l y 50 p e r c e n t o f t h e p r o p i o n a t e entering for  the  p o r t a l bed i s c o n v e r t e d t o g l u c o s e which  accounts  27 per cent o f t h e g l u c o s e e n t r y r a t e s . The h i g h e r  infusion  absorption  rate  figures  under  direct  of **C - p r o p i o n a t e i n t o t h e rumen a r e j u s t i f i e d s i n c e  up t o 70 p e r c e n t o f p r o p i o n a t e produced converted  found  to  i n the  is  first  l a c t a t e i n t h e rumen e p i t h e l i u m d u r i n g a b s o r p t i o n  (Leng e t a l . , 1967). T h i s p a r c t i c u l a r  study  the  glucose  conversion  rumen  of  propionate  into  demonstrated may  that  undergo two  pathways, e i t h e r d i r e c t o r i n d i r e c t . The f o l l o w i n g r e a c t i o n s a r e a mere o u t l i n e of t h e s e pathways:  13  DIRECT - p r o p i o n a t e +CO. s u c c i n a t e -CO, phosphoenolpyruvate > + INDIRECT - P r o p i o n a t e +C0 s u c c i n a t e -CO^ l a c t a t e +C0 Z  >  glucose  X  >  —>  o x a l o a c e t a t e s u c c i n a t e -CO phosphoenolpyruvate  The f o r m a t i o n pathways  of  occurs  lactate  from  i n t h e rumen  propionate  v i a the  indirect  epethelium,  whereas  both t h e  i n d i r e c t and d i r e c t pathways t a k e p l a c e i n t h e al.,(1967)  compared  propionate  into  the i n c o r p o r a t i o n of  glucose  to  that  from  liver,  »*C  (2-»*C)  p r o p i o n a t e and e s t i m a t e d t h e r e l a t i v e importance from  a  knowledge  glucose  Leng  et  from  (1- *C)  or  (3-^C)  l  o f each pathway  o f t h e pathways i n v o l v e d . I f o n l y the d i r e c t  pathway i s o p e r a t i v e then 50 p e r c e n t as much C-1 as C-2  or  C-3  of p r o p i o n a t e would appear i n g l u c o s e . I f g l u c o s e i s s y n t h e s i z e d from  p r o p i o n a t e v i a t h e i n d i r e c t pathway then t h e i n c o r p o r a t i o n  of C-1 of p r o p i o n a t e i n t o g l u c o s e would be 25  percent  of  that  i n c o r p o r a t e d from C-2 o r C-3. S i n c e t h e i n c o r p o r a t i o n o f (1-»*C) p r o p i o n a t e t o t h a t from less  than  (2-**C) o r (3-**C) - p r o p i o n a t e was much  50 per c e n t . Leng e t a l . , ( 1 9 6 7 ) c o n c l u d e d t h a t t h e r e  i s probably extensive i n c o r p o r a t i o n of  propionate  carbon  into  l a c t a t e b e f o r e c o n v e r s i o n i n t o g l u c o s e . Thus they e s t i m a t e d t h a t approximately  54  per c e n t o f t h e g l u c o s e e n t r y r a t e i s d e r i v e d  from p r o p i o n a t e i n t h e rumen, w i t h o n l y  32  per  cent  of  this  propionate being converted t o glucose. In  summary  the variations  i n glucose production derived  from p r o p i o n a t e (27 t o 54 per cent) appear t o depend on two main f a c t o r s , t h e d i e t and whether t h e p r o p i o n a t e i s measured i n t h e  14  rumen  (Leng  et  a l . , 1967) o r i n p r o t a l blood  1966). The r a t e of p r o p i o n a t e not c l e a r . C o n t r a r y obtained  blood  of  to  these  conditions  lactate  by  differences,  of  1966). However high (Dunlop  and  (1972)  t h a t o n l y 1.0-4.6 p e r c e n t o f rumen d e r i v e d  normal  grain  Hammond,  1965)  epithelium.  be s t a t e d t h a t the blood  fermentation  will  and  rumen  i n portal  rumen  diets  the  i t can  amount of l a c t a t e a c t u a l l y appearing under  al.,  from t h o r a c i c a o r t a and p o r t a l v e i n o f H o l s t e i n  i s converted  Regardless  by rumen e p i t h e l i u m i s  t o Leng»s f i n d i n g s , Weigand e t  c a l v e s and e s t i m a t e d propionate  metabolised  (Bergman e t a l . ,  small  (Roe e t a l . ,  increase  propionate  is  both  lactate  (Lindsay,  1959)  p r o d u c t i o n and a b s o r p t i o n . Of t h e r e m a i n i n g  primary v o l a t i l e f a t t y a c i d s , a c e t a t e  has  been e s t a b l i s h e d t o p l a y a dominant r o l e i n ruminant l i p o g e n e s i s since  ATP- c i t r a t a l y a s e i s d e f f i c i e n t i n r u m i n a n t s and g l u c o s e  can s u p p l y  only l i m i t e d amounts of a c e t y l - COA f o r f a t t y  synthesis  (Ballard  et a l . ,  1969). B u t y r a t e , on t h e o t h e r hand,  has been a s c r i b e d a g l y c o g e n i c r o l e by a number of several  years.  For  example,  Potter  workers f o r  (1952) demonstrated t h a t  b u t y r a t e was more e f e c t i v e t h a n p r o p i o n a t e  in  increasing  sugar l e v e l s and r e l i e v i n g h y p o g l y c e m i c c o n v u l s i o n s sheep  t r e a t e d with i n s u l i n . K l e i b e r et a l . ,  u n l i k e a c e t a t e , b u t y r a t e was markedly cows  and  that  t h e *C i  from  (1954) showed t h a t ,  glucogenic  butyrate  studies  blood  i n lambs and  in  lactating  was u t i l i z e d more f o r  s y n t h e s i s of l a c t o s e than f o r s y n t h e s i s o f m i l k f a t . other  acid  These  and  on t h e u t i l i z a t i o n of b u t y r a t e i n r u m i n a n t s f a i l  t o demonstrate a g l u c o g e n i c  pathway  ( B l a c k e t a l . , 1966) o r i n sheep  i n either  lactating  (Leng and A n n i s o n , 1963).  cows  15  The  metabolic  role  of  i n v e s t i g a t e d by B l a c k et a l . , injections  of  pyruvate  butyrate  i n ruminants  (1966). These workers gave  2- *C  and  l  propionate  was  single  2- C i n t o t h e 14  j u g u l a r v e i n o f l a c t a t i n g cows and u t i l i z e d the i n t r a - m o l e c u l a r labelling  patterns  of  g l u t a m i c a c i d t o a s s e s s t h e pathway f o r  metabolism  o f these two g l u c o g e n i c compounds. The  results  t h i s s t u d y demonstrated t h a t when b u t y r a t e was i n j e c t e d with  pyruvate  2- C 14  i t caused  preferential  from  together  utilization  of  p y r u v a t e t o form o x a l o a c e t a t e and c o n s e q u e n t l y l e s s p y r u v a t e was d e c a r b o x y l a t e d t o form a c e t y l - COA. T h e r e f o r e they have suggested contribute  t h e r e a r e two  which  t o t h e g l u c o g e n i c e f f e c t o f b u t y r a t e i n t h e cow, t h e  f i r s t i s the s p a r i n g e f f e c t o f b u t y r a t e on and  efects  pyruvate  oxidation,  t h e second i s t h e g r e a t e r a c t i v i t y o f p y r u v a t e  carboxylase  and hence an i n c r e a s e d r a t e o f g l u c o n e o g e n e s i s . contrary  t o the r e s u l t s  acids  administering butyrate  work i s  o f Cook (1970), who i n v e s t i g a t e d t h e  e f f e c t of route of a d m i n i s t r a t i o n on t r a n s f e r fatty  This  of  **C  volatile  t o l i v e r glycogen and v a r i o u s b l o o d s u b s t r a t e s by (1-**C) a c e t a t e , ( 1 - C ) 14  v i a the j u g u l a r  propionate  and  (1-**C)  v e i n , p o r t a l v e i n o r added d i r e c t l y  i n t o t h e rumen o f sheep and g o a t s . The work demonstrated t h a t tracer  dose  of  ( C) 1 4  butyrate injected into the jugular vein  l a b e l e d blood g l u c o s e more than higher  specific  b u t y r a t e was butyrate  activity  infused  a  along  ( C) 14  of blood with  a  acetate  because  of the  b u t y r a t e . When u n l a b e l l e d tracer  dose  of  (1- C) 14  t h e s p e c i f i c a c t i v i t y of blood g l u c o s e was s i m i l a r t o  t h a t observed  when a c e t a t e was a d m i n i s t e r e d . Thus t h e g l u c o g e n i c  16  e f f e c t of b u t y r a t e t h a t has been observed Misconception  and  may  be  in  the  past  (1-**C) b u t y r a t e i n j e c t e d i n t o the j u g u l a r v e i n  diluted  w i t h u n l a b e l l e d b u t y r a t e s i n c e b u t y r a t e i s not  p r e s e n t i n p e r i p h e r a l b l o o d . Cook(1970) p r o v i d e d for  the  stimulation  of  »*C  p y r u v a t e t o g l u c o s e by b u t y r a t e observed in  that  (0.60  uM per kg body weight)  tissues.  the  level  Therefore,  o x i d a t i o n of  (**C)  Despite  the  of  has  a  p y r u v a t e t o *CO,and l  controversy  aternative  t r a n s f e r from  butyrate a  whether  »*C  labelled  not  normally  by B l a c k e t a l . ,  provided  butyrate  an  is  ( C) 14  (1966)  administered  substrate  sparing  effect  for  the  on  the  HO.  r e g a r d i n g the g l u c o g e n i c r o l e of  b u t y r a t e , the s t u d y of Cook (1970) r e v e a l e d t h a t r e s u l t s on metabolism o f  a  e x p l a i n e d by the f a c t t h a t a t r a c e r  dose of  explanation  was  volatile  fatty  acids  depend  the on  t h e a c i d s are a d m i n i s t e r e d v i a the j u g u l a r v e i n , p o r t a l  v e i n or added d i r e c t l y t o the rumen.  B.  From_Gly_cerol G l y c e r o l i s c o n s i d e r e d t o be a potent  and  is  released  glucogenic  from t h e adipose t i s s u e a l o n g w i t h f r e e f a t t y  a c i d s d u r i n g s t a r v a t i o n , k e t o s i s or o t h e r p e r i o d s mobilization  (Steinberg  and  it  increases  of  body  fat  Vaughn, 1965). Bergman, (1968) has  shown t h a t w h i l e the t u r n o v e r r a t e of g l y c e r o l sheep,  compound  markedly  during  d u r i n g hypoglycemic k e t o s i s . In the case  is  fasting of  rats  low and  in  fed  especially  and  humans,  17  a p p r o x i m a t e l y 80-90 per c e n t of t h e g l y c e r o l i s removed from t h e blood by the l i v e r and t h e k i d n e y s a p p a r e n t l y remove most of the remainder  ( B o r c h g r e v i n k , and  Havel,  1963;  L a r s e n , 1963;  L u n d q u i s t e t a l . , 1965). The f r e e g l y c e r o l t h u s the  gluconeogenic  pathway  at  the  triose  i l l u s t r a t e d i n F i g u r e 1, which summarizes pathways  enters  phosphate s t a g e as  the  major  metabolic  i n t h e l i v e r and k i d n e y s of r u m i n a n t s (Bergman, 1973).  These f i n d i n g s a l o n g w i t h activity of  removed  and  in  established  high  glycerokinase  the l i v e r and k i d n e y s suggest t h e major importance  glycerol  metabolism  the  for seams  gluconeogenesis. similar  to  In  addition,  glycerol  t h a t of p r o p i o n a t e metabolism i n  t h a t most of the former i s a l s o removed by t h e  liver  (ftnnision  et a l , 1957). Though, g l y c e r o l i s known t o be g l u c o g e n i c p r e c i s e estimates  of  its  actual  glucogenicity  s t a n d p o i n t seem t o be l a c k i n g . The f i r s t problem  quantitatively  came  c a l c u l a t e d , on t h e b a s i s o f  from the  work  Cahill  gross  from to  et  energy  quantitative assess  this  a l . , (1970)  who  metabolism  and  r e s p i r a t o r y q u o t i e n t , t h e m o b i l i z a t i o n of g l y c e r o l i n the f a s t e d human.  They  t h e o r i z e d t h a t t h i s g l y c e r o l c o u l d account f o r the  f o r m a t i o n o f a p p r o x i m a t e l y 20 g g l u c o s e / d a y f o r the average  man.  S t u d i e s on the g l u c o g e n i c i t y o f g l y c e r o l i n r u m i n a n t s by Bergman I * 1.1 • # (1968) u s i n g glycerol,  a  continuous  was  high  **C  labelled  of  release  from  adipose  and as a maximum, c o u l d account f o r n e a r l y 40  per cent o f the a n i m a l ' s average  of  demonstrated t h a t i n f a s t e d , k e t o t i c and hypoglycemic  sheep, t h e g l y c e r o l t u r n o v e r or r a t e tissue  infusion  glucose  production.  However,  on  an  pregnant k e t o t i c sheep d e r i v e d about 28 per cent of t h e  g l u c o s e from g l y c e r o l . The r e s u l t s a l s o i l l u s t r a t e d  that  about  18  30  per  cent  differences  of  the g l y c e r o l was o x i d i z e d t o CO and no marked  seemed  hypoglycemic  to  states*  occur  during  Thus  glycerol  the  fed,  becomes  fasted an  important  g l u c o g e n i c p r e c u r s o r and n e a r l y r e p l a c e s p r o p i o n a t e o n l y periods  of  during  u n d e r n u t r i t i o n , s t a r v a t i o n o r o t h e r p e r i o d s of body  f a t metabolism. Bergman e t a l . , by  or  stating  that  in  (1968) summarize t h e i r  findings  t h e w e l l f e d ruminant the c o n t r i b u t i o n of  g l y c e r o l t o g l u c o n e o g e n e s i s i s s m a l l and p r o b a b l y  accounts  for  l e s s than 5 p e r c e n t of the t o t a l g l u c o s e produced.  C.  From_Lactate_^nd_Px£uvate Lactate  and  to  attributed  with  monogastric  animals  i t 1.1 • # 1972) however,  a  lesser  gluconeogenic such as r a t s  and humans (Katz and  evaluation  of  degree  properties,  tract.  have  primarily  (Exton, 1972), dogs Dunn,  1967).  In  been in  (Issekutz ruminants  l a c t a t e metabolism i s d i f f i c u l t due t o  t h e v a r i a b l e and unknown amounts which digestive  pyruvate  are  absorbed  from  the  T h i s s i t u a t i o n r e s u l t s because l a c t a t e can be  produced by rumen f e r m e n t a a t i o n o r from p r o p i o n a t e metabolism i n the rumen w a l l d u r i n g a b s o r p t i o n (Leng et a l . , 1967; al.,  Bergand  et  1972) . Lactate  is  synthesized  from  the a n a e r o b i c metabolism of  g l u c o s e and i s c a r r i e d t o o t h e r areas o f t h e body aerobic  oxidation.  for  complete  The major r o u t e f o r l a c t a t e d i s p o s a l i s t h e  s y n t h e s i s of g l u c o s e i n l i v e r and k i d n e y s . Thus t h e  lactate  is  19  utilized  f o r t h e r e s y n t h e s i s o f g l u c o s e and c o n s t i t u t e s what i s  known as the C o r i c y c l e ( C o r i ,  1931).  The  operation  of  this  c y c l e does not r e s u l t i n a net i n c r e a s e i n g l u c o s e f o r m a t i o n f o r the  body  since  lactate  o r i g i n a t e s from  however, t r a n s f e r energy between t h e l i v e r The  energy  to  drive  glucose. and  It  other  does,  tissues.  such a system i s u l t i m a t e l y d e r i v e d from  o x i d a t i o n o f f a t t y a c i d s and o t h e r non-glucongenic The s t u d i e s p r e v i o u s l y mentioned monogastrics  compounds. estimated the  C o r i c y c l e c o n t r i b u t i n g between 10 and 33 per cent o f the glucose  turnover  underfeeding with  with  higher  determining  4  to  occurrring  o r s t a r v a t i o n . C o n s i d e r i n g t h e problems lactate  r e s e a r c h e r s have suggested than  rates  10  par  cent  metabolism  i n ruminants,  total during  associated a number of  t h a t t h i s c y c l e c o n s t i t u t e s no  more  o f t h e g l u c o s e t u r n o v e r i n f e d sheep  (Bergman e t a l . , 1970); Annison  e t a l . , 1963).  D. Fcom_Amino_Acids Almost a l l amino a c i d s a r e g l u c o g e n i c t o  varying  degrees,  w i t h t h e e x c e p t i o n o f l y s i n e , l e u c i n e and t a u r i n e (Krebs, 1964). The  m a j o r i t y of amino a c i d s a r e s t o r e d i n muscle and a l o n g w i t h  d i e t a r y amino a c i d s , a r e c o n v e r t e d t o g l u c o s e i n both l i v e r kidney c o r t e x w i t h t h e l i v e r a c c o u n t i n g f o r a p p r o x i m a t e l y  and  85 per  c e n t of the net g l u c o s e p r o d u c t i o n from t h i s s o u r c e i n f e d sheep (Bergman of  et  conversion  a l . , 1970; Kaufman and Bergman, of  the  various  amino  acids  1971). The pathway to  glucose  is  20  illustrated  in  Figure  14  (Appendix). When the amino a c i d s are  u t i l i z e d f o r g l u c o s e p r o d u c t i o n by nitrogen  is  quickly  these  sites,  the  urea  can  enter  the  t r a c t and the n i t r o g e n r e i n c o r p o r a t e d i n t o a d d i t i o n a l  amino a c i d s (Nolan and Leng  1972).  S i m i l a r t o the p r e v i o u s amino  of  m e t a b o l i z e d to urea and e x c r e t e d i n t o the  u r i n e . In r u m i n a n t s , however, some of the digestive  most  precursors,  gluconeogenesis  from  a c i d s w i l l v a r y w i d e l y depending upon t h e n u t r i t i o n a l  p h y s i o l o g i c a l s t a t e of t h e proportion  of  the  animal.  Attempts  to  estimate  and the  t o t a l g l u c o s e p r o d u c t i o n a r i s i n g from amino  a c i d s have been l i m i t e d i n  number  and  the  interpretation  of  r e s u l t s i s a s s o c i a t e d w i t h c o n s i d e r a b l e d i f f i c u l t y . For example, calculations  of  the  quantity  through the abomasum (Clarke  of  et a l . , 1966;  55 grams o f protein,  glucose  sheep  of  total  per  day  amino have  acids passing been  performed  Hogan and Weston, 1967). By assuming t h a t can  be  synthesized  from  100  grams  of  Leng (1970) has e s t i m a t e d t h a t t h i s c o u l d s u p p l y up t o  70 per c e n t of the g l u c o s e p r o d u c t i o n i n nonpregnant sheep. T h i s approach  i s indirect  and  probably  grossly  overestimates  c o n t r i b u t i o n of d i e t a r y amino a c i d s t o g l u c o n e o g e n e s i s actual  absorption  may  be  c o n s i d e r a b l y l e s s than i t s d i s a p p e a r a n c e from the rumen  due  to  assess  by  the  tissues.  contribution  measurements 1966)  gut  amino  of  urinary  of  acids  Attempts amino  nitrogen  or u r e a p r o d u c t i o n r a t e s (Nolan  estimates  are  into  the  s i n c e the  blood  metabolism  of  the  have  acids  a l s o been made to to  excretion and  glucose  from  (Bergman e t a l . ,  Leng,  1970).  These  a l s o o f l i m i t e d v a l u e s i n c e c o n s i d e r a b l e amounts  of ammonia are absorbed  from the rumen t o be c o n v e r t e d  to  urea  21  i n t h e l i v e r and u r e a i t s e l f i s r e c y c l e d through s e c r e t i o n s i n t o the d i g e s t i v e t r a c t (Bergman, 1973). Recently  the g l u c o g e n i c i t y o f amino a c i d s i n r u m i n a n t s has  been determined by u s i n g 0- C  amino  14  acids  l  * C - l a b e l l e d amino a c i d s . ft m i x t u r e  i s o l a t e d from c h l o r e l l a p r o t e i n was used by  R e i l l y and Ford  (1971) t o e s t i m a t e  total  turnover  glucose  Their  t h a t 28.2  vein  cent  was  s a m p l i n g . In a p r e v i o u s  used  contributed  f o r both  between  gluconeogenesis. intravenous acids,  et  and  estimated  14.7  al.,  per  (1968)  amino cent  employed  may  arise  each r e p r e s e n t  cows  amino  acid  into  plasma  Hunter  glucose  t h a t 30 t o 50 per c e n t of t h e g l u c o s e  from  c e n t of m i l k l a c t o s e  amino  acids  of  which  alanine  and  6 t o 8 per cent. By i n f u s i n g a m i x t u r e the  and M i l l s o n , (1964) r e p o r t e d was  derived  from  protein  jugular  t h a t 12 per  i n lactating  (Hunter and M i l l s e n , 1964). Techniques such as t h e s e ,  which use a m i x t u r e o f **C amino a c i d s ,  tend  the  amino  true  single  t o e v a l u a t e t h e i r r o l e as  of **C amino a c i d s d e r i v e d from a l g a l p r o t e i n i n t o  ruminants  towards  The t e c h n i q u e s t u d i e d the r a t e and e x t e n t of  t i m e . They e s t i m a t e d  of  acids  i n j e c t i o n s of s e v e r a l d i f f e r e n t U - * * C - l a b e l l e d  i n c o r p o r a t i o n of **C from t h e amino  glutamate  than  study i n which t h e j u g u l a r  i n t o l a c t a t i n g cows.and g o a t s ,  turnover  the  i n f u s i o n of l a b e l l e d amino a c i d s and  12.8  Black  glucose precursors.  vein  of  was d e r i v e d from amino a c i d s i n sheep.  c o l l e c t i o n of b l o o d , these same workers  with  per  work a l s o s t r e s s e d the i m p o r t a n c e o f a r t e r i a l r a t h e r  j u g u l a r blood  of  glycogenic  contribution  crossing  over  of of  the  to  underestimate  acids  label  due  to a  between  the  randomization  or  tricarboxylic  a c i d c y c l e and t h e g l u c o n e o g e i c pathway  (Krebs e t  22  a l . , 1966). Thus due t o these d i f f i c u l t i e s i t becomes to  measure  the  1 4  necessary  C - l a b e l l e d amino a c i d s s e p a r a t e l y r a t h e r t h a n  c o l l e c t i v e l y and, i n a d d i t i o n , the measurements s h o u l d on  portal  rather  than  on  jugular  p o i n t e d out by R e i l l y and Ford  (1971) s i n c e t h e g l u c o g e n i c i t y of  a c t u a l a n t i - g l u c o g a n i c e f f e c t o f another  confounded  by  r e c a n t s t u d i e s have employed two approaches t o a s s e s s  The  alfalfa  f i r s t method c o n s i s t e d o f measuring t h e q u a n t i t i e s o f  amino a c i d s added t o t h e plasma by (mainly  an  (e.g. l e u c i n e ) .  t h e c o n t r i b u t i o n of amino a c i d s t o g l u c o s e i n sheep f e d hay.  made  blood. These f a c t o r s were  i n d i v i d u a l amino a c i d s (e.g. a l a n i n e ) can be  More  be  tissues  juantities  of  the  metabolized  maintenance  the  portal-drained  gastrointestinal  by  the  liver  viscera  t r a c t ) and a l s o the  of  sheep  fed  a  near  d i e t ( W o l f f e t a l . , 1972). T h i s was a c c o m p l i s h e d by  c o l l e c t i n g blood samples from c a t h e t e r s , p r e v i o u s l y i m p l a n t e d i n the a o r t a and the p o r t a l and h e p a t i c v e i n s , f o r plasma  amino  acid  concentrations.  measurement  Figure  15  i l l u s t r a t e s the placement o f c a t h e t e r s f o r i n f u s i o n collection. flow  in  In  the  conjunction portal  and  with  of  (Appendix) and  blood  t h e s e measurement, t h e b l o o d  hepatic  veins  was  determined  s i m u l t a n e o u s l y by t h e method o f K a t z and Bergman (1969). The net metabolism  o f each amino a c i d was c a l c u l a t e d by m u l t i p l y i n g the  v e n o - a r t e r i a l c o n c e n t r a t i o n d i f f e r e n c e by the f l o w of plasma. In each case n e t o u t p u t or p r o d u c t i o n was a s s i g n e d a p o s i t i v e v a l u e and net uptake o r u t i l i z a t i o n a values  obtained  f o r the  negative  portal  value.  vein-arterial  The  concentration  d i f f e r e n c e s i n d i c a t e a n e t output by t h e p o r t a l d r a i n e d with  alanine  being  the  amino  aicd  positive  viscera  produced i n t h e g r e a t e s t  23  quantities.  The  mean  o u t p u t o f 2.3 mM/hr accounted f c r 19 per  cent o f t h e t o t a l n e t appearance o f -amino plasma. was  The  concentration  by  measuring  differences  gut o u t p u t was  removed  accounted  and  by  exceeded  the  hepatic  f o r about  Glycine,  one-half  of  (18.4 mM/hr).  t h e g u t output,  a  n e t movement from  v i s c e r a . T h i s concept values  alanine  the t o t a l  When  and  -amino  the  hepatic  i n t h e case f o r these t h r e e  amino a c i d s , Wolff and Bergman (1972) p o s t u l a t e d of  vein-portal  i n most c a s e s a l m o s t a l l of t h e  the l i v e r .  n i t r o g e n removed by t h e l i v e r uptake  i n portal  n e t h e p a t i c metabolism o f t h e v a r i o u s amine a c i d s  determined  glutamine  nitrogen  the occurance  p e r i p h e r a l t i s s u e s t o the s p l a n c h n i c  i s further  indicated  by  the  negative  f o r t h e n e t metabolism by t h e t o t a l s p l a n c h n i c v i s c e r a  ( l i v e r p l u s v i s c e r a ) , which were c a l c u l a t e d by t h e d i f f e r e n c e i i i c o n c e n t r a t i o n between t h e h e p a t i c v e i n and a r t e r i a l  source.  The v a l i d i t y o f t h i s s t u d y r e s t s on t h e assumption t h a t t h e net appearance o f amino a c i d s i n the p o r t a l b l o o d  reflects  the  amino a c i d p a t t e r n o f t h e 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 . The net o u t p u t o f amino a c i d s from g u t t i s s u e s i s t h e r e s u l t of both a b s o r p t i o n o f d i e t a r y amino a c i d s by the g a s t r o i n t e s t i n a l  tract  as w e l l a s v i s c e r a l t i s s u e m o b i l i z a t i o n . With t h i s i n l i n d  other  workers  (Elwyn,  1970) have  proposed  q u a n t i t y o f endogenous p r o t e i n change  over  the period  of  that,  so  long  i n the g u t t i s s u e s time  that  as the  does not  measurements a r e made  ( i . e . steady s t a t e c o n d i t i o n s ) , t h e n net g u t o u t p u t w i l l measure m a i n l y a b s o r p t i o n o f amino a c i d s  from  considering  continuous  the conditions  of  dietary  protein.  Thus,  f e e d i n g as used i n  t h e s e e x p e r i m e n t s , the t o t a l p r o t e i n s t a t u s o f t h e g u t t i s s u e s  24  i s assumed t o have endogenous  remained  protein  would  constant have  so  that  compenstated  f o r amino  removal from the a r t e r i a l s u p p l y . The r e s u l t s indicated  large  hepatic  uptakes  of  absorption  from  alanine  this  of acid  study  (3.2 mM/hr) and  g l u t a m i n e (2.1 mM/hr), which a r e c o n s i s t a n t w i t h t h e i r  proposed  r o l e i n the t r a n s p o r t of n i t r o g e n from p e r i p h e r a l t i s s u e s t o t h e liver, al.,  where  the n i t r o g e n can be c o n v e r t e d t o urea  1971; and P o l e l s k y e t a l . ,  acids  removed  by  the l i v e r  1969). play  (Marliss et  In addition an  t h e amino  important  r o l e i n the  s y n t h e s i s o f g l u c o s e , as w i l l be d i s c u s s e d l a t e r . However one o f the most s i g n i f i c a n t c o n t r i b u t i o n s made by W o l f f e t a l . , is  the confirmation  that  few  conclusions  n u t r i t i o n of the ruminant can be o b t a i n e d from amino  acid  concentrations  on t h e amino a c i d measurements  i n p e r i p h e r a l plasma  1968; L e i b h o l t z , 1969; and R e i s and Tunks,  (1972)  of  (Hogan e t a l . ,  1970).  The second major approach t o the s t u d y o f t h e g l u c o g e n i c i t y of amino a c i d s i n r u m i n a n t s has u t i l i z e d r a d i o a c t i v e l y **C-amino  acids.  This  method  estimate f o r gluconeogenesis  labelled  i s expected t o y i e l d a m i n i m a l  i n t h e whole  body  (liver  plus  k i d n e y s ) , whereas t h e p r e v i o u s t e c h n i q u e i s a measure o f maximal g l u c o n e o g e n e s i s from amino a c i d s i n t h e l i v e r . S u b s e q u e n t l y W o l f f and Bergman (1972) employed a c o n t i n u o u s infusion  of  each  of  f i v e g l u c o g e n i c * C - l a b e l l e d amino a c i d s l  i n t o t h e vena cava t o l a b e l t h e plasma p o o l and t o determine t h e t r a n s f e r of carbon from each amino a c i d t o g l u c o s e . The C d a t a 1 4  obtained  were  compared  with  t h e maximal  glucose  synthesis  possible  from t h e net h e p a t i c uptake of a l l plasma amino a c i d s .  25  Blood  was  vein  f o r measurement of blood f l o w v a l u e s as w e l l as the plasma  amino  sampled from the a o r t a , t h e p o r t a l  acid  activity  specific  levels  for  activities. both  the  amino  hepatic  plateau  specific  a c i d and g l u c o s e , a  overall  study  when sheep are f e d a near maintenance r a t i o n ,  they d e r i v e a t l e a s t 11 per c e n t of five  plasma  uptake  from  plasma  amino a c i d s i n whole-body g l u c o s e  have  made  to  hepatic  glucose  would l i e somewhere between these two acids  studied,  conversion with  a  from  plasma  alanine  r a t e of 2.45  was 0.82  corresponding  the  of  the  production  role  whereas  amino  acids  production. A true  value  f i g u r e s . Of the f i v e amino  most  glucogenic,  mM/hr, and g l u t a m i n e was  rate  was  30 per cent of  former f i g u r e i s a m i n i m a l . v a l u e f o r  the l a t t e r i s t h e maximal c o n t r i b u t i o n t h a t plasma could  this  glucose  the  t o produce a maximum of a p p r o x i m a t e l y  the g l u c o s e . The of  blood  of  amino a c i d s ( g l y c i n e , a l a n i n e , s e r i n e , a s p a r a t a t e ,  and glutamate) w h i l e net h e p a t i c sufficient  their  results  per  was  that  The  the  and  centage c o n v e r s i o n show  assessed.  From  vein  1.48  0.40  with  a  the second  mM/hr. Together they  account f o r at l e a s t 40 per c e n t of the g l u c o g e n i c i t y of a l l the amino a c i d s . The amino  f i n d i n g t h a t a l a n i n e and g l u t a m i n e  acids  confirmed  removed  the  principal  by the l i v e r f o r g l u c o n e o g e n e s i s has  i n r e c e n t i n v e s t i g a t i o n s on humans ( F e l i g , 1970;  §.£ al« r 1970  Following  these  acids  studies,  released  linking  Felig  both  amino  acid  metabolism  with  two  from  skeletal  a l a n i n e and  glutamine  c y c l e s ( F i g u r e 16, Appendix) were proposed as an i m p o r t a n t of  been  and Ruderman and Lund, 1972) . In a d d i t i o n t h e s e  were shown t o be the main amino muscle.  are  the  control  means of  26  gluconeogenesis.  In  a  recent  review  article,  Felig  (1973)  examined i n d e t a i l the metabolism of a l a n i n e , p a r t i c u l a r l y as i t r e l a t e s to glucose relationship  h o m e o s t a s i s i n man.  between  both p r e c u r s o r and "glucose-alanine  the  Considering  the  intimate  metabolism of a l a n i n e and g l u c o s e  product,  t h e c y c l e i s now  cycle".  described  as  acid  output  from  proteins  in  muscle  in  skeletal  per cent of t h e  (Kominz e t a l . ,  (Katz and C a r s t e n ,  p r o t e i n i n f a c t present  i n s m a l l and u n d e t e c t a b l e constant  1954)  preformed  acid  and c a r d i a c muscle  1963); second, a s p e c i f i c p o l y a l a n y l  i n man,  t h i r d , were  such  under s t e a d y s t a t e c o n d i t i o n ,  amounts, i t would not account f o r  i t is  apparent  that  amino  acid  to  the  Consequently F e l i g et a l . , alanine  the  a l a n i n e from muscle p r o t e i n or from t h e  amino a c i d p o o l cannot e x p l a i n the predominant  of  amino  the  a l a n i n e o u t p u t a f t e r 5 to 6 weeks of s t a r v a t i o n ( F e l i g  1.1 • i 1970). T h e r e f o r e  this  amino  a steady s t a t e c o n d i t i o n : f i r s t ,  p r o t e i n has not been i d e n t i f i e d i n muscle; and a  points  i n o r d e r t o account f o r the p a t t e r n of  a l a n i n e c o m p r i s e s no more than 7-10 residues  the  A l t h o u g h a l a n i n e i s the p r i m a r y amino  a c i d r e l e a s e d from p e r i p h e r a l p r o t e i n s t o r e s , a number of must be c o n s i d e r e d  as  from  total  release  of  intracellular  contribution  of  n i t r o g e n r e l e a s e from muscle.  (1970) suggested a de novo  the t r a n s a m i n a t i o n  of p y r u v a t e .  synthesis  I n s u p p o r t of  t h i s c o n c l u s i o n i s the f i n d i n g t h a t t h e r e e x i s t s a d i r e c t l i n e a r c o r r e l a t i o n between c i r c u l a t i n g c o n c e n t r a t i o n s p y r u v a t e i n man  of  under b a s a l c o n d i t i o n s ( F e l i g and  alanine  and  Wahren, 1971).  I n summary, t h e r e f o r e , t h e s e s t u d i e s i n d i c a t e the e x i s t e n c e of a glucose-alanina transamination  c y c l e i n which a l a n i n e i s formed p e r i p h e r a l l y by of glucose  d e r i v e d pyruvate and i s t r a n s p o r t e d  to  27  the l i v e r where i t s carbon s k e l e t o n i s r e c o n v e r t e d t o g l u c o s e . Although  attention  has  been  focused  on t h e r e l a t i o n of  a l a n i n e t o glucose homeostasis, the g l u c o s e - a l a n i n e c y c l e i s of importance  i n nitrogen  metabolism  as  well.  In  addition to  p r o v i d i n g carbon s k e l e t o n s f o r g l u c o n e o g e n e s i s , t h e of  p e r i p h e r a l s y n t h e s i s o f a l a n i n e and i t s subsequent  net  effect  uptake by  the l i v e r i s the t r a n s f e r of amino groups from muscle t o h e p a t i c t i s s a e s , where they may be d i s p o s e d o f as urea. C a r l s t e n e t a l . , (1967) suggested t h a t a l a n i n e p r o v i d e s a n o n t o x i c a l t e r n a t i v e t o ammonia i n t h e t r a n s p o r t o f n i t r o g e n from p e r i p h e r a l t i s s u e s  to  liver. The  importance  of  alanine  as a g l u c o g e n i c p r e c u r s o r has  been w e l l e s t a b l i s h e d i n m o n o g a s t r i c s a s w e l l as  ruminants.  is  due  of  special  dependence on  interest endogenous  i n the  latter  case  glucose  synthesis  to  It  to  their  supply  their  energy demands.  S§gfaS^g"^g y§g§-IS£_5g§§y£§i§Bt,2^ Alanine„and^Glucose m  Metabolism Throughout  the  previous  discussion  experimental  t e c h n i q u e s employed i n t h e s t u d i e s  of  g l u c o n e o g e n e s i s have been mentioned  b r i e f l y . The p r e s e n t s e c t i o n  comprises  a  summary  of  these  glucose  the  various  metabolism  and  methods w i t h emphasis  p l a c e d on a l a n i n e and i t s c o n t r i b u t i o n t o g l u c o s e s y n t h e s i s . The majority of the  research  involving  metabolic  parameters  has  28  centered to  around g l u c o s e . The o b j e c t i v e o f the p r e s e n t study  u t i l i z e a number of these t e c h n i q u e s t o measure the  parameters of a l a n i n e , as w e l l as  assess  was  metabolic  i t s contribution  to  a r e a o f g l u c o s e metabolism  in  glucose synthesis.  A.) E a r l y _ D i e t a r y _ S t u d i e g The  first  ruminants  is  problem the  in  the  absorption  of  glucose  from  the  gastro-  i n t e s t i n a l t r a c t . I t i s w e l l e s t a b l i s h e d t h a t n e g l i g i b l e amounts of  glucose  are  absorbed  However the  quantity  concentrate  or  of  under  d i e t a r y regimes of roughages.  glucose  absorbed,  in  ruminants  fed  h i g h g r a i n d i e t s i s s t i l l under i n v e s t i g a t i o n .  Topps et a l . , (1968a and b) measured the f l o w of d i g e s t a t o abomasum  in  sheep  and  young  s t e e r s on c o n c e n t r a t e d i e t s .  a c c o m p l i s h t h i s the a n i m a l s were f i t t e d w i t h rumen and cannulas.  Paper  impregnated  with  chromium  a d m i n i s t e r e d t o the rumen and samples were abomasum  the  abomasal  sesquioxide  collected  To  from  was the  as w e l l as from the f e c e s . By measuring -the amounts of  d r y m a t t e r , s t a r c h , c e l l u l o s e , t o t a l n i t r o g e n and energy p a s s i n g through t h e duodenum and the amounts e x c r e t e d i n the estimate  of the amount o f s t a r c h e s c a p i n g f e r m e n t a t i o n and  i n t o t h e s m a l l i n t e s t i n e s was that  feces,  under  dietary  made.  The  results  an flow  demonstrated  regimes o f c o n c e n t r a t e s very l i t t l e s t a r c h  escapes rumen f e r m e n t a t i o n . By t a k i n g j u g u l a r v e i n b l o o d samples they  showed  concentrations  that of  diets  had  little  or  no  effect  on  the  g l u c o s e . The major o b j e c t i o n t o t h i s type of  29  study  i s t h e use o f j u g u l a r v e i n blood  g l u c o s e f l u c t u a t i o n under pointed  out  the d i f f e r e n t  dietary  regimes.  As  p r e v i o u s l y , p e r i p h e r a l blood cannot be used as t h e  sole estimate al.,  as an i n d i c a t o r of any  o f f l u c t u a t i o n s i n blood  metabolites  (Wolff  et  1972). A  number  conclusions studies  of  based  using  investigators, on  jugular  realizing  vein  blood,  glucose concentration  and a r t e r i a l blood  (Schambye,  t h e i n a c c u r a c y of conducted  similar  d i f f e r e n c e s between p o r t a l  1951, Roe  et , a l . ,  1966).  The  r e s u l t s s t i l l showed t h a t no g l u c o s e was absorbed i n t o t h e blood of a n i m a l s on a d i e t o f hay o r one supplemented w i t h 50% g r a i n . The estimated  gluconeogenic  of  amino  acids  using t e c h n i q u e s which a s s e s s t h e q u a n t i t y  amino a c i d s p a s s i n g and  effects  Weston,  overestimate  have  been  of  total  through t h e abomasum o f sheep per day (Hogan  1967).  These  studies  the true glucogenic  are i n d i r e c t  and tend t o  c o n t r i b u t i o n of amino a c i d s  in  t h e r u m i n a n t . Measurement of u r i n a r y n i t r o g e n e x c r e t i o n has been used  as  an  index  u t i l i z e d as an glucose  of  estimate  synthesis  synthesized  not be e x c r e t e d storage  cf  i n sheep  r e s u l t s can be m i s l e a d i n g urea  protein  d e a m i n a t i o n which, i n t u r n , i s  the  amino  acid  contribution  (Bergman, Roe and Kon, 1966). The  because,  under  some  circumstances,  as a r e s u l t o f deamination of amino a c i d s may  i n the u r i n e . I n sheep.that  i n t h e body p o o l o f urea i n c r e a s e s  are  starved,  urea synthesized  urea  (Packett and Groves,  1965; Cocimano and Leng, 1967) and on low p r o t e i n - d i e t s some the  to  of  may be r e c y c l e d t o t h e d i g e s t i v e t r a c t and  u t i l i z e d f o r m i c r o b i a l growth (Schmidt - Nelson e t  a l . , 1957).  30  Nolan and Leng (1970) u t i l i z e d the  body  the r a t e of e n t r y  of  urea  p o o l of urea t o i n d i c a t e t h e upper l i m i t of net amino  a c i d d e a m i n a t i o n , and from t h i s d e r i v e d the . p o t e n t i a l glucose  synthesis  from  into  the  of  labelled  l  *C-  j u g u l a r v e i n and the urea e n t r y r a t e , p o o l s i z e  and urea space were c a l c u l a t e d radioactivity  rate  deaminated carbon r e s i d u e s . The method  c o n s i s t e d o f a s i n g l e i n j e c t i o n of r a d i o a c t i v e l y urea  into  of  from  the  decline  of  specific  urea i n t h e b l o o d . The e s t i m a t e s based on the  r a t e of e n t r y of urea a r e o f l i m i t e d v a l u e , and  would  tend  to  o v e r e s t i m a t e due to the c o n s i d e r a b l e amounts o f ammonia absorbed from  rumen  which  would  be  converted  to  urea i n the l i v e r .  ( W o l f f , Bergman and W i l l i a m s , 1972).  B.)  I n d i c a t o r D i l u t i o n Techniques Indicator dilution  referred  to  a  a  general  for  presently  range  use  a  specific from  which  such  as  pool  nutrient.  simple  r a d i o a c t i v e i s o t o p e s and measure a parameters  term  originally  method of measuring the a b s o r p t i v e a b i l i t y o f a  t i s s u e or an organ in  is  size,  large pool  i r r e v e r s i b l e l o s s , conversion rate,  dyes  as  The  indicators  (Evan's  number space, well  of  blue) to metabolic  turnover as  the  rate,  standard  a b s o r p t i o n and p r o d u c t i o n r a t e s . Heier  and  Zieler  (1954)  postulated  a working model f o r  i n d i c a t o r d i l u t i o n t e c h n i q u e s which i s based on system  with  a  single  input  and  a  closed  a s i n g l e o u t p u t . The  flow system  31  c o n t a i n s a s p e c i f i c volume o f f l u i d constant  rate  of  flow.  interlacings  of  entering  t h e same  at  blood  Due  vessels  which e n t e r s and e x i t s a t  to  the  within  many  branchings  t h e system  a  and  particles  i n s t a n t w i l l r e q u i r e v a r y i n g amounts o f  time t o reach t h e s i n g l e o u t p u t . The time r e q u i r e d i s dependent upon  t h e path  Thus  there  taken  i s no  distribution  of  and t h e v e l o c i t y w i t h which they  single  traversal  traversal times.  time,  Such  but  rather  system  a  a system depends on a  number o f assumptions which i n c l u d e ; (a) t h e p a r t i c l e s the  travel.  entering  a t any time a r e d i s p e r s e d when they e x i t i n e x a c t l y  t h e same manner as p a r t i c l e s e n t e r i n g a t any  other  time;  this  p r o p e r t y i s r e f e r r e d t o as s t a t i o n a r i t y o f f l o w ; (b) t h e f l o w o f indicator fluid;  particles  i s representative  of  the flow of t o t a l  and (c) r e c i r c u l a t i o n o f i n d i c a t o r i s n o t p r e s e n t .  In  d e v e l o p i n g t h i s model, Meier and Z i e r l e r , (1954) employed  both a s i n g l e i n j e c t i o n as w e l l as a c o n t i n u o u s i n f u s i o n indicator.  d e c l i n e s ( F i g u r e 17, Appendix).  a  maximum  function  quickly  circulation.  i n d i c a t o r passes through t h e pulmonary c i r c u l a t o r y t r e e and  t h e h e a r t and i s sampled from a s y s t e m i c a r t e r y . The s m a l l in  the curve  at  18  seconds  infusion  method,  the  i t s concentration  rise  i s i n t e r p r e t e d t o represent the  appearance o f d e t e c t a b l e r e c i r c u l a t i n g dye.  and  and  Curve A i s the r e s u l t o f a  s i n g l e i n j e c t i o n o f a dye i n t o t h e s y s t e m i c venous The  a  t i m e . The i n s t a n t a n e o u s i n j e c t i o n t e c h n i q u e i s c h a r a c t e r i z e d  by a c o n c e n t r a t i o n curve which reaches then  an  The i n d i c a t o r was a d m i n i s t e r e d a t t h e p o i n t o f e n t r y  and i t s c o n c e n t r a t i o n was measured a t t h e output as of  of  In  the  continuous  i n d i c a t o r i s i n j e c t e d a t a constant r a t e will  rise  to  a  plateau  level  as  32  illustrated  in  Figure  proposed by Meier and One  potential  17,  (Appendix)  Curve  Z i e r l e r i s v a l i d on  B.  This  theoretical  grounds.  weakness l i e s i n the c o n d i t i o n t h a t i t be  w i t h a s i n g l e i n p u t and a s i n g l e o u t p u t .  Such  a  model  closed  situation  is  v e r y d i f f i c u l t t o i s o l a t e i n an i n t a c t a i m a l . T h e r e f o r e f o r t h i s model t o h o l d f u r t h e r assumptions are n e c e s s a r y , one that  the c o n c e n t r a t i o n  vs time c u r v e i s e s s e n t i a l l y the same i n  a l l o u t p u t b r a n c h e s , and many  inputs,  t h u s the s i t u a t i o n r e d u c e s t o  that  of  s i n g l e output. A second i s t h a t the f l o w from  the  s i t e of i n j e c t i o n becomes mixed i n the sense t h a t of  i t  of which i s  the  fraction  l e a v i n g through any i n p u t c h a n n e l i s p r o p o r t i o n a l to  the  flow i n that channel. The  above  discussion  presents  c o n s i d e r a t i o n s behind i n d i c a t o r d i l u t i o n  the  theoretical  techniques.  1. Blood_Flow_Mgasurgments  can  The  net m e t a b o l i t e  c o n t r i b u t i o n of an organ or organ system  be  estimated  measurements  concentration blood  with  researchers of  amino  by  differences, i n reference  to  of  the v e n o a r t e r i a l  coordination  the  with  Bergman, 1972  to  rate  the  contribution  g l u c o s e s y n t h e s i s i n ruminants (Wolff  a,b,c; Bergman  et  of  system of i n t e r e s t . A number of  have used t h i s approach t o a s s e s s acidds  the  (V-A)  a l . , 1974;  and  Kaufman  and and  Bergman, 1974) . Q u a n t i t a t i v e e s t i m a t i o n of t h e a b s o r p t i o n  of p a r t i c u l a r end  33  p r o d u c t s o f d i g e s t i o n must e n t a i l measurement of the p o r t a l v e i n blood  f l o w ( F r i e s and Conner, 1961). Of t h e i n d i c a t o r s used f o r  p o r t a l v e i n blood f l o w , the method i n v o l v i n g acid  (PAH)  as  developed  by Roe,  para-aminchippuric  Bergman and  Kon  (1966) has a  number of advantages. The p r i m a r y c o n s i d e r a t i o n f o r t h e PAH  is  that  samples  can  be  taken  once  use  of  the plasma PAH  has  a t t a i n e d a constant concentration a f t e r a period infusion  in  which  the  PAH  of  continuous  i s a l l o w e d t o e g u i b r i a t e w i t h the  extra c e l l u l a r f l u i d . This plateau l e v e l i s atttained since PAH  the  i s r a p i d l y and t o t a l l y e x c r e t e d by the k i d n e y s p r o v i d e d t h a t  t h e i n f u s i o n r a t e i s l e s s than the maximal a b i l i t y of the t o e x c r e t e PAH.  The  kidney  two major advantages o f t h i s method i n c l u d e ;  1) t h e t i m i n g o f sample c o l l e c t i o n i s no l o n g e r as c r i t i c a l 2)  a  greater  sensitivity  is  achieved  due  v e n o a r t e r e a l c o n c e n t r a t i o n d i f f e r e n c e . K a t z and developed  a  method  to  to  a  and,  greater  Bergman  (1969)  measure both p o r t a l and h e p a t i c venous  blood f l o w s s i m u l t a n e o u s l y by u t i l i z i n g PAH  as  The  s h a l l be d e a l t w i t h  calculations  extensively  in  assumptions  made  for the  these  blood  experimental  are  that  the  ;  and  that  through  the i n f u s e d PAH  p o r t a l v e i n , Both o f these c r i t e r i a indicated  that  the  section  asumptions  indicator.  following.  The  PAH s h o u l d not be e x c r e t e d or  c h e m i c a l l y a l t e r e d i n i t s passage liver  flows  the  the  portal  bed  or  s h o u l d be w e l l mixed i n the were t e s t e d and the  were  valid  results  (Katz and Bergman  1969) . In an accompanying paper, Katz and Bergman (1969b) employed t h i s technique pregnancy  to measure the e f f e c t s o f  feeding,  on the h a p a t i c and p o r t a l metabolism  fasting  and  of glucose, f r e e  34  f a t t y and  ketone b o d i e s  in  sheep.  The  average net h e p a t i c g l u c o s e p r o d u c t i o n pregnant  sheep  is  0.13  hours a f t e r f e e d i n g nonpregnant and hepatic after  and 0.19  sheep  glucose production which  proves  gluconeogenesis increases The  figures  g/hr  kg  and  0.43  g/hr  Thus  kg  the  kidney  (Kaufman and infused  Bergman,  into  the  n e a r l y 50 per c e n t 3 hours  that,  unlike  mentioned  monogastrics,  on the e x t e n t of  1971,  1974).  posterior  venoarterial  vena  differences  (FFA), ketone b o d i e s ,  renal  studies  p a r a - a m i n o h i p p u r i c a c i d as the i n d i c a t o r The  and  PAH.  PAH  cava and  samples were taken from the a o r t a and r e n a l renal  mean  a f t e r feeding i n ruminants.  previously  using  and  in  the  g l u c o n e o g e n e s i s were a r r i v e d a t through b l o o d p e r f u s i o n on  the  r e s p e c t i v e l y . Three  respectively.  increased  indicate  by f a s t e d nonpregnant  t h e s e v a l u e s were 0.28  pregnant  feeding  results  for  constantly  simultaneous blood vein  glucose,  In  was  addition  to  determine  free fatty a  acids  catheter  was  i n s e r t e d i n t o the b l a d d e r f o r c o n t i n u o u s u r i n e c o l l e c t i o n  during  the  minute  period  of  blood  collection.  e g u a l s the u r i n a r y e x c r e t i o n minute,  divided  by  between t h e a o r t a and  rate  Renal b l o o d f l o w per of  PAH  in  milligrams  the d i f f e r e n c e i n blood PAH  per  concentrations  the r e n a l v e i n .  W o l f f et a l . , (1972) i n v e s t i g a t e d  the  quantity  a c i d s added t o the plasma by the p o r t a l d r a i n e d  of  amino  v i s c e r a and  that  m e t a b o l i z e d by the l i v e r o f sheep f e d a near-maintenance d i e t by employing  the  b l o o d f l o w t e c h n i q u e as d e s c r i b e d  as the i n d i c a t o r . The  c a t h e t e r s f o r i n f u s i o n and  above w i t h blood  PAH  sampling  were s u r g i c a l l y i m p l a n t e d i n t o the a o r t a , c a u d a l vena cava,  left  35  hepatic  vein,  conclusions previously  p r o t a l v e i n and m e s e n t e r i c v e i n . The r e s u l t s and  of  this  study  have  been  discussed  in  detail  (page 2 5 ) .  C) Isoto£e_Dilution_Me In t h e f i e l d o f ruminant metabolism, s p e c i f i c a l l y the  s t u d y of t h e body p o o l k i n e t i c s o f v a r i o u s  groups o f i n v e s t i g a t o r s c a n be d i s t i n g u i s h e d ; single  injection  method  and  those  metabolites, those  employing  continuous  radiocative  employed  to  a g r e a t e x t e n t i n measuring t h e k i n e t i c parameters  techniques  have  metabolism. in  relation  The to  techniques  the  of  glucose  Both  a  using  two  infusion  surrounding  tracer.  involving  application  disscusion  section  .ft list  these  t h e assessement  c o n t r i b u t i o n amino a c i d s t o g l u c o n e o g e n e s i s , the  have  been  two  o f the  i s emphasized  in  o f terms and t h e i r d e f i n i t i o n s  i s p r e s e n t e d i n a g l o s s a r y a t t h e end o f t h i s t h e s i s .  36  1.)  Single_Injection_Te^ The  single  intravenous specific  activity)  a  involves  ^C-labelled  the  rapid  of  work  t h e s i z e and  with  turnover  ( F e l l e r e t a l . , 1950;  (Searle  et  substrate  and  a l . , 1954)  this  method  Baker and  and  man  Incefy,  g l u c o s e gave v a l u e s high.  al.,  single  the  1955),  (Baker e t a l . , (1954)  body g l u c o s e p o o l s i z e which seemed t o be too discussed  involved  r a t e o f t h e body g l u c o s e  i n d i c a t e d t h a t a s i n g l e i n j e c t i o n o f **C  (1956)  the  of the d i l u t i o n r a t e of the r a d i o a c t i v e  (Cook, 1966). E a r l y  pool i n the r a t dog  of  measurement  measurements  the  technique  i n j e c t i o n of a t r a c e r dose ( n e g l i g i b l e w e i g h t , h i g h  subsequent material  injection  l i m i t a t i o n s of  Steele  dogs.  They  concluded  that  straightforward  of the c u r v e showing t h e d e c l i n e i n s p e c i f i c  of plasma  glucose  seriously  overestimated  the  injection  large  differences  in  glucose  and  White  oversimplified  and  glucose  lacked  a  (1961)  function,  i n f u s i o n . These l a r g e d i f f e r e n c e s are due,  t h e f a c t t h a t t h e a n a l y s i s of the d a t a from a was  labelled  and  technique. technique  in  sheep  further  Considering when  applying  e n t i r e decay c u r v e ,  the a  by  by  i n p a r t , to  single  injection  m u l t i c o m p a r t m e n t a l approach  (Baker, 1969). K r o n f e l d and Simesen (1961) i n v e s t i g a t e d kinetics  who  e n t r y r a t e measured by a  s i n g l e i n j e c t i o n , using a s i n g l e exponential constant  activity  g l u c o s e u t i l i z a t i o n r a t e s . I n agreement  w i t h t h i s c o n c l u s i o n i s work by Annison found  of  rates  mathematical  treatment  after  et  injection  p r o c e d u r e s i n s t u d i e s on g l u c o s e p o o l s i z e and u t i l i z a t i o n in  for  the  problems  glucose  use of a s i n g l e i n j e c t i o n associated  single exponential  these workers e s t i m a t e d  the  with  this  f u n c t i o n to  the  glucose  entry  37  rate  and p o o l s i z e by assuming the p o r t i o n of the c u r v e from 40  t o 160 minutes t o be l i n e a r on a s e m i l o g a r i t h m i c p l o t , and first  order  kinetics.  Thus  they  obey  d e r i v e d a d e f i n i t i o n of the  g l u c o s e p o o l t o i m p l y t h e group of carbon atoms  in  equilibrium  w i t h plasma g l u c o s e carbon from 40 t o 160 minutes a f t e r a s i n g l e intravenous  injection  of  a  tracer  o f * * C - g l u c o s e . The model  which f o l l o w s i s t h a t t h e p o o l comprised g l u c o s e m o l e c u l e s which a r e s i t u a t e d i n the e x t r a c e l l u l a r f l u i d equilibrium  with  glucose  molecules  which  Simesen  (1961)  proposed  In  by  this  Kronfeld  is  achieved  c e r t a i n l y i n l e s s than 40 minutes a f t e r t h e i n j e c t i o n .  addition  metabolic  dynamic  assumes t h a t thorough m i x i n g of the t r a c e r  m e c h a n i c a l l y and by d i f f u s i o n t h r o u g h o u t the space rapidly,  in  e n t e r i n g and l e a v i n g  space, t o and from the c e l l s . T h i s model and  are  re-entry  mixing  of  with  **C-glucose,  intermediates,  which is  has  undergone  assumed  not  to be  a p p r e c i a b l e b e f o r e 160 m i n u t e s . The t u r n o v e r r a t e r e p r e s e n t s the f l o w o f g l u c o s e i n t o and o u t of t h i s e x t r a c e l l u l a r Kronfeld  and  Simesen  pool,  which  (1961) r e g a r d as an i n d e x of t h e o v e r a l l  r a t e of g l u c o s e metabolism t h a t i s , an e s t i m a t e o f  the  glucose  t a k e n up by c e l l s and u t i l i z e d f o r both energy and s y n t h e s i s . An i n t e r e s t i n g p o i n t brought out by t h e s e workers i s t h a t r e c y c l i n g of  l  *C  from g l u c o s e through i n t e r m e d i a r i e s and back i n t o  as g l u c o s e - * * C would become prominent sooner i n than  in  cows  and  sheep.  This  is  rats  plasma  and  dogs  due t o the lower l e v e l of  g l u c o n k i n a s e and g l u c o s e - 6 - phosphatase a c t i v i t i e s of v a r i o u s t i s s u e s o f r u m i n a n t s t h a n i n nonruminants. An the  major  recycling  of  **C  example  is  between plasma g l u c o s e and  g l y c o g e n , which markedly a f f e c t s t h e  plasma  glucose  that liver  level  of  38  nonruminants,  is  insignificant  glucokinase a c t i v i t y  in  ( G a l l a g h e r and  sheep  which  Butterg,  lack hepatic  1959).  Therefore  r e c y c l i n g i n ruminants would be e x p e c t e d t o t a k e l o n g e r , and glucose  specific  activity  comparitively less (1961) ,  have  than  in  re-entering nonruminants.  time  plasma  would  Wrenshall  be  et a l . ,  a l s o p r e s e n t e d e v i d e n c e s u b s t a n t i a t i n g the use of  t h e s i n g l e - i n j e c t i o n method w i t h chosen  the  the  interval  e x t r a p o l a t i o n of  a  properly  t o e s t i m a t e the e n t r y r a t e of g l u c o s e i n  dogs. A f u r t h e r advance i n the s i n g l e i n j e c t i o n t e c h n i q u e was use o f m u l t i c o m p a r t m e n t a l  a n a l y s e s of the decay curve  §.£ a l . , (1969)., T h i s approach  was  that  a  recycling  of  "^C  is  and  major p r o c e s s i n ruminants.  well  as  the  fixation  of  J  *C0  likely  than  the  exponential  disappearance injection  to  of  term  in  radioactivity  of **C-glucose  in  acid  cycle,  which i s r e t u r n e d t o the  g l u c o s e p o o l . T h e r e f o r e t h e most one  glucose  The  i t s i n c o r p o r a t i o n w i t h i n a l a r g e p o o l of p o t e n t i a l  g l u c o s e p r e c u r s o r s or e n t r y i n t o t h e t r i c a r b o x y l i c as  White  i n i t i a t e d upon the r e a l i z a t i o n  e v i d e n c e s u p p o r t i n g t h i s i n c l u d e s t h e c a t a b o l i s m of lactate  by  the  explanation  equation plasma  for  more  describing  glucose  after  the an  i s t h a t carbon o r i g i n a l l y d e r i v e d from  g l u c o s e i s r e t u r n e d t o t h e sampled p o o l a t two d i f f e r e n t  rates.  The  (1961)  f i r s t o r d e r k i n e t i c approach  by K r o n f e l d and Simesen  was d e s c r i b e d as an o v e r s i m p l i f i c a t i o n and t h a t t h e whole of the isotope dilution parameters  of  curve glucose  should  be  metabolism  When t h e method employed by  Kronfeld  used  in  calculating  (Bescigno and Segre, and  Simesen  such 1966).  (1961)  was  a p p l i e d t o t h e s i n g l e i n j e c t i o n r e s u l t s o b t a i n e d i n t h e study of  39  White  et  a l . , (1969), the e s t i m a t e o f t u r n o v e r r a t e was 3 per  c e n t h i g h e r and the g l u c o s e p o o l s i z e was  14% h i g h e r  than  that  c a l c u l a t e d by a m u l i t c o m p a r t m e n t a l approach. Another drawback t o this  s i m p l i f i e d a n a l y s i s i s t h a t t h i s approach would not d e t e c t  t h e changes i n t h e i s o t o p e varying  physiological  dilution  states.  curve  The  which  occur  under  m u l i t c o m p a r t m e n t a l method,  however i s designed t o i n t e r p r e t the e n t i r e c u r v e and thus d e t e c t any o f t h e s e The will  described  following. of  changes.  mathematical  be  treatment  in  Briefly,  detail  time  of the i s o t o p e d i l u t i o n in  the  experimental  curve  section  t h e r e f o r e , the c u r v e i s d e s c r i b e d as a  e x p o n e n t i a l terms as f o l l o w s  where SR  will  (White et a l . ,  sum  1969):  e q u a l s the s p e c i f i c r a d i o a c t i v i t y of plasma g l u c o s e at  t  (muc/mg.C).  component  A^ e q u a l s  the  zero-time  (muc/mg.C), -m£, e q u a l s  the  intercept  of  each  constant  of  each  rate  component (min ) , n e q u a l s the number of e x p o n e n t i a l components, I e q u a l s the e x p o n e n t i a l component (min.).  The  number  of  number  and  t  equals  e x p o n e n t i a l terms i s determined by the  shape of the observed s p e c i f i c r a d i o a c t i v i t y time c u r v e the  basis  (Appendix) arbitrary  time  and  on  of a p o s t u l a t e d model which i s d e p i c t e d i n F i g u r e 18 (Leng  1970).  constants  of  The the  parameters  of  the  model  f u n c t i o n s or e g u a t i o n s and can  e s t i m a t e d w i t h the use of computer programs (Baker, 1969).  are be  no Most o f t h e work  i n body  pool  i n f u s i o n s e i t h e r s i n g l e or continuous measure  has  been  described  However,  previously  Black  individual  o f * * C - l a b e l l e d glucose to  et  amino  (Beilly  a l . , (1968),  1  *C-labelled  and  Ford,  amino  1970  investigated  the  acids  and 1971).  the r o l e  advantage  use of a s i n g l e i n j e c t i o n method f o r t h i s type o f study  i s t h a t one can a s s e s s time r e l a t i o n s h i p s among t h e amino as  glucose  precursors  which  would  not  constant  infusion  (1968) s t a t e d  acids  i n plasma  1955).  The  turnover  has been s t u d i e d p r e v i o u s l y u s i n g  t e c h n i q u e i n dogs (Elwyn e t a l . , §t  that  o f amino a c i d s i s l i m i t e d due t o t h e much  g r e a t e r inhomogeneity of t h e amino a c i d p o o l s . The amino  acids  be apparent d u r i n g a  constant i n f u s i o n . In a d d i t i o n Black e t a l . , a  of  acids i n glucose synthesis i n l a c t a t i n g d a i r y  cows u s i n g a s i n g l e i n j e c t i o n t e c h n i q u e . A p a r t i c u l a r in  with  t h e parameters o f g l u c o s e metabolism. The g l u c o g e n i c i t y  o f amino a c i d s has been s t u d i e d u s i n g as  kinetics  latter  1968) and i n r a b b i t s  attempted  a  of this  (Henriques  multicompartmental  a n a l y s i s of t h e d a t a and found t h a t t h r e e e x p o n e n t i a l terms were r e q u i r e d t o f i t t h e s p e c i f i c a c t i v i t y time Therefore technique employed  curve.  i t i s w i t h t h i s i n mind t h a t a s i n g l e  combined  with  a  multicompartmental  injection  approach  was  t o a s s e s s , q u a n t i t a t i v e l y , t h e m e t a b o l i c parameters o f  a l a n i n e a s w e l l as i t s c o n t r i b u t i o n  to  glucose  synthesis  r u m i n a n t s under steady s t a t e o r maintenance c o n d i t i o n s .  in  in  3•) Continuous I n f u s i o n Under  the heading o f c o n t i n u o u s i n f u s i o n i s o t o p e d i l u t i o n  methods t h e r e a r e two d i s t i n c t approaches which d i f f e r The f i r s t procedure infusion  t o be developed i s t h e p r i m i n g  technique,  and  t h e second  involves  slightly.  dose-constant a  continuous  i n f u s i o n o f t h e t r a c e r w i t h o u t a p r i m i n g dose. The the  priming  assumtpion  d o s e - c o n s t a n t i n f u s i o n t e c h n i q u e , i s based on that  instantaneously  mixed  t h e body and  pool  that  of  a  substrate  is  a  steady  s t a t e of s u b s t r a t e  A  single  injection  o  replacement tracer  exists  (Cook,  i s immediately  1966). followed  t r a c e r . The r a t i o o f p r i m i n g determined  o f the  by a c o n s t a n t i n f u s i o n of t h e  dose  to infusion  rate  must  be  such t h a t a r e l a t i v e l y c o n s t a n t s p e c i f i c a c t i v i t y of  t h e t e s t s u b s t r a t e i s maintained a f t e r the i n i t i a l mixing o f t h e p r i m i n g dose i s complete. With g l u c o s e as an example, of  change  of  total  **C  i n blood  d i f f e r e n c e between t h e r a t e a t which glucose  p o o l t h e r a t e a t which  on t h e t u r n o v e r radioactivity  rate  of  and  glucose  the r a t e  w i t h time i s t h e  **C e n t e r s and  leaves  the  * C l e a v e s t h e p o o l i s dependent 4  size  of  the p o o l .  The  specific  g l u c o s e carbon i n plasma a t any time  (SR ) i s  then p r e d i c t e d by t h e e x p r e s s i o n : SR  where  X  t  -  X  equals  radioactivity  of  -  Ye"  White e t a l . ,  bt  the asymptotic plasma  value  glucose  intercept specific radioactivity  for  (1969)  the  specific  (muC/mg o f C),X-Y e q u a l s t h e  (muC/mg o f  C)  at  t=0  and  b  42  equals  the  rate constant  between 60 and 180 X,  X-Y  and  b,  r e c y c l i n g of **C White e t a l . ,  (min * ) . The s p e c i f i c a c t i v i t y  minutes were used t o e s t i m a t e the due  to  a f t e r a l o n g time p e r i o d ( S t e e l e et a l . ,  (1969) determined  estimates.  obtained  by  Irreversible  dividing  i n t e r c e p t a t zero time  loss  from the was  the  metabolism  Bergman,  1963)  animals  and i n ruminants  due  were  to  (muC)  by size  by  the  kinetics  of 1956; 1961;  the s i g n i f i c a n t l y h i g h e r g l u c o s e e n t r y  analysis  high.  Pool  (Annison and White,  of  However,  the s i n g l e i n j e c t i o n  had y e t t o be a p p l i e d at t h i s t i m e , c o n s e q u e n t l y values  X.  (Searle et a l . ,  r a t e s o b t a i n e d by t h e s i n g l e i n j e c t i o n t e c h n i q u e . multicompartmental  original  (X-Y) o b t a i n e d by e x t r a p o l a t i o n .  i n monogastric  S t e e l e et a l . , 1956)  to  1956).  calculated  priming i n j e c t i o n  T h i s t e c h n i q u e has been employed t o study the glucose  of  t h e s e v a l u e s by u s i n g a computer  d i v i d i n g the r a t e o f i n f u s i o n of r a d i o i s o t o p e by was  values  the i n i t i a l mixing p r o c e s s and due  program which performed a i t e r a t i v e procedure parameter  values  the  the  technique parameter  Bergman and Hogue (1967) u t i l i z e d a p r i m i n g  dose c o n s t a n t i n f u s i o n t e c h n i q u e to measure the g l u c o s e t u r n o v e r and o x i d a t i o n r a t e s i n sheep d u r i n g v a r i o u s s t a g e s of l a c t a t i o n . The et  primed i n f u s i o n t e c h n i q u e , which was e v o l v e d by  al.,  (1956),  i s based on e v i d e n c e t h a t the g l u c o s e p o o l of  t h e dog i s d i s t r i b u t e d i n two compartments i n the fluid.  No  Steele  account  is  taken  of  recycling  between these  pools.  results  a l . , (1969) i n d i c a t e t h a t , i n  by  White  et  single  two  compartments and o t h e r s u b s t r a t e obtained  The  extracellular  injection  sheep, g l u c o s e i n both plasma and i n t e r s t i t i a l f l u i d c o n s t i t u t e s  43  a s i n g l e e n t i t y and t h a t r e c y c l i n g o f g l u c o s e results  in  a  carbon  from  peripheral  pool  mulitexponential  Consequently  the use o f a primed i n f u s i o n t o c a l c u l a t e t h e  the  curve. pool  s i z e o f any m e t a b o l i t e , g l u c o s e o r a l a n i n e appears t o be unsound on t h e o r e t i c a l grounds. (Leng.,1970) The  continuous  r e l i e s on a  infusion  simple  t e c h n i q u e w i t h o u t a p r i m i n g dose  mathematical  treatment  (Leng,  1970)., I n  s t u d i e s conducted  t o measure parameters o f g l u c o s e metabolism i n  sheep  a l . , 1967 and White e t a l . , 1969) t h e s p e c i f i c  (Leng  et  r a d i o a c t i v i t y of the i n f u s e d  glucose  p l a t e a u a f t e r 180 - 240 minutes. loss  i s deterimined  radioisotope  by  was  found  to  reach  a  An e s t i m a t e o f t h e i r r e v e r s i b l e  simply  d i v i d i n g the i n f u s i o n r a t e of  (muc/mg o f C) by t h e p l a t e a u s p e c i f i c  radioactivity  (muc/mg o f C) . P r e v i o u s work d e a l i n g w i t h amino a c i d t u r n o v e r i n sheep has involved a constant i n f u s i o n of a amino  acids  of  **C  will  the r a d i o a c t i v i t y  v a r y between themselves  of  **C  -labelled  was  a  mixture  of  individual  amino  employed by Wolff and Bergman (1972) t o e v a l u a t e t h e  portal-drained  infusion  amino  amino a c i d s must be i n t e r p r e t e d w i t h extreme  i n t e r c o n v e r s i o n s and metabolism  sheep.  with  and d i f f e r e n t b l o o d v e s s e l s  c a u t i o n . The use o f a c o n t i n u o u s i n f u s i o n acids  labelled  individual  o f t h e body. Thus d a t a o b t a i n e d from the i n f u s i o n o f of  -  ( R e i l l y and F o r d , 1971). The major d i f f i c u l t y  t h i s approach i s t h a t acids  mixture  Each of  viscera, experiment a  1 4  o f plasma  liver, involved  and  amino  acids  by t h e  p e r i p h e r a l t i s s u e s of f e d  the continuous  intravenous  C - l a b e l l e d amino a c i d , and s a m p l i n g o f b l o o d  an  from the a o r t a , the p o r t a l v e i n and h e p a t i c equations in  were d e s c r i b e d  the  separate  to assess  the r a t e s of  assess  the  contributions  g l u c o s e . As d e s c r i b e d indicated  that  of  the  the use o f  p r e v i o u s l y , the r e s u l t s the  this  technique  from  this  study  f i v e amino a c i d s examined, a l a n i n e  R e c y c l i n g of l a b e l has in  interconversions  of v a r i o u s plasma amino a c i d s t o  q u a n t i t a t i v e l y the most i m p o r t a n t  problem  Simultaneous  groups of t i s s u e s . Subsequently t h e s e a u t h o r s  (Wolff and Bergman, 1972b) d e s c r i b e d to  vein.  been  f o r blood g l u c o s e suggested  to  was  synthesis.  be  the  major  i n t e r p r e t a t i o n of i s o t o p e e x p e r i m e n t s , whether  these be s i n g l e i n j e c t i o n or c o n t i n u o u s i n f u s i o n (Bergman, and  Kaufman,  and L i n d s a y , was  used  1970,  Bergman e t a l . , 1965,  1970). During a s i x hour  in  the  Gurpide e t a l . , 1963,  continuous  study by W o l f f and  acid,  2)  synthesis  metabolically related organic  acids  and  and  compounds glucose.  infusion,  Bergman, (1972) the  c o u l d have r e c y c l e d t h r o u g h 1) the i n t r a c e l l u l a r amino  pools  such  Wolff  as and  of  amino  the  since  the  rates  t u r n o v e r r a t e ) a s s e s s the m e t a b o l i c  3)  acids,  Bergman (1972)  intracellular  of  pools  other  as  label  c a t a b o l i s m of p r o t e i n s , and  r e c y c l i n g through (entry  Katz  ignore  utilization processes  which l e d t o an i r r e v e r s i b l e l o s s of l a b e l from the plasma p o o l , w h i l e p r o d u c t i o n r a t e s determine the r a t e a t which the p o o l replenished  from u n l a b e l l e d s o u r c e s .  p r o t e i n s i s probably Doyle,  1970),  small  acid  R e c y c l i n g of l a b e l through  most  enzymes  (Schmike  a l l plasma p r o t e i n s , (McFarlane, 1964)  s t r u c t u r a l p r o t e i n s have Amino  since  half-lives  was  greater  than  and  six  i n t e r c o n v e r s i o n s are expected t o be i m p o r t a n t  and most  hours. as  an  avenue f o r r e c y c l i n g . However l a b e l l e d a l a n i n e was found t o have  l i t t l e a c t i v i t y i n any o t h e r 1972a).  amino  affected  amino a c i d s . However, t h e r e c e n t  g l u t a m i n e * c y c l e s ' , which a r e i m p o r t a n t nitrogen 1970,  (Wolff  and  Bergman,  Thus i t would appear t h a t e x p e r i m e n t s i n v o l v i n g the use  of **C - l a b e l l e d a l a n i n e a r e not as other  acid  from  Marlins  contributors  peripheral et to  tissue  a l . , 1971) the  discovery for  the  to the l i v e r ,  may  recycling  by  prove of  these  to  recycling  as  of a l a n i n e and transport  of  (Felig et a l . , be  labels,  q u a n t i t a t i v e measurements have not y e t been d e t e r m i n e d .  definite although  46  EXPERIMENTAL  I NET_METABOLISM_OF_GL VISCERA  Ii^roduction The  measurement of p o r t a l  venous  concentration  gained  importance  utilization  of  vein  differences  in  the  blood of  flow  and  arterio-  s p e c i f i c metabolites  quantitative estimation  of  end p r o d u c t s o f d i g e s t i o n by a n i m a l s  the  ( F r i e s and  Conner, 1960). By an a n a l y s i s of the p o r t a l b l o o d the amount material  absorbed  i n t o the v e i n s d r a i n i n g the  be determined.  I n o r d e r t o a c c o m p l i s h both these  the  liver  estimates,  K a t z and Bergman (1969a) proposed a method by which t h e and  of  gastrointestinal  t r a c t as w e l l as the degree o f metabolism o c c u r i n g i n the may  has  hepatic  p o r t a l venous blood f l o w s a r e measured s i m u l t a n e o u s l y .  Consequently, distinguish  such  a  technique  between the p r o d u c t i o n  enabled  researchers  ( a b s o r p t i o n ) and  utilization  o f i n d i v i d u a l components by the l i v e r from t h a t  by  bed  Utilizing  under  varying  physiologic  approach, Katz and  Bergman  portal  of  metabolism  conditions.  (1969b)  studied  the  to  the  portal  hepatic  this and  g l u c o s e i n both f e d and f a s t e d , pregnant  and nonpregnant sheep. Work by Bergman et a l . , (1970) u s i n g t h i s method demonstrated significant fed,  amounts  that  the  portal  bed  of  sheep  utilizes  of g l u c o s e , r e g a r d l e s s o f whether they  s t a r v e d or hypoglycemic.,Thus  the  portal-drained  are  viscera  U7  (gastrointestinal important  tract,  pancreas,  and  i n terms of g l u c o s e u t i l i z a t i o n  recognizee!. , However  this  discovery  spleen)  than  was  only  was  are  previously  possible  d i s t i n c t i o n c o u l d be made between p o r t a l u t i l i z a t i o n and p r o d u c t i o n , because i n  terms  of  total  splanchnic  ( h e p a t i c p l u s p o r t a l ) v a l u e s show a net p r o d u c t i o n of Alanine al.,  was  investigated  more  once  hepatic  metabolism glucose.  i n a s i m i l a r manner by W o l f f  (1972) t o determine the q u a n t i t y added t o t h e plasma by  p o r t a l - d r a i n e d v i s c e r a and t h a t  of  sheep f e d a near-maintenance r a t i o n . As a measurement of the  net  output,  alanine  was  absorbed  g r e a t e r degree than the o t h e r removed  large  amino  the  i n t o the p o r t a l blood to a acids  tested.  liver  i n t e r p r e t e d t o i n d i c a t e a net  movement of a l a n i n e from p e r i p h e r a l t i s s u e s  to  the  splanchnic  ( l i v e r p l u s p o r t a l bed).  Considering  these  findings,  the o b j e c t i v e of the  experiment was t o e v a l u a t e the use of t h i s t e c h n i q u e its  The  q u a n t i t e s of a l a n i n e . In f a c t the h e p a t i c uptake  exceeded t h e gut o u t p u t , which was  viscera  by  the  liver  gut  metabolized  et  relative  merit  i n the d e t e r m i n a t i o n  p e r t a i n i n g t o g l u c o s e and a l a n i n e i n  and  present study  of v a r i o u s parameters  ruminants.  48  Experimental  a.1  m  &  Haterials_and_Methods_  1  •) §.iiEa.i£.S.i_PE2£sd. ures A 2 year o l d wether weighing 42  e x p e r i m e n t . Feed was to  surgery.  withheld  Thirty  atropine  sulfate  shaved  and  anesthetized  before  given a  washed with a  ventral with  ^%  the  mg/ml))  abdominal  soap  pentathol  and  sodium  r e f l e x as w e l l as the t e n s i o n o f the jaw Following  the a n i m a l was table.  An  closed  delivered  mixed with oxygen. m o n i t o r e d and For  The  surgical  circuit a  gas  decrease  wall  was  The  injection  muscles were  the  was of the  closely  sheep  was  into  the  of the drug the  eye  frequently  ml of the p e n t h a t h o l sodium  was  the  operating  introduced  anaesthetic  which  was  machine.  The  c o n t r o l a b l e amount of t h e gas  Therefore  this  respiration  rate  halothane could  be  t h e s t a t e of a n e s t h e s i a c o n t r o l l e d c a r e f u l l y .  portal  cannulation,  catheter  injection  to  t o a prone p o s i t i o n on  endotracheal  connected t o a apparatus  i n f u s i o n of 15-18  transfered  in  anesthetic  water.  j u g u l a r v e i n . During the slow a d m i n i s t r a t i o n  tested.  employed  subcutaneous  (5 ml-dose of 0.6  s a l i v a r y s e c r e t i o n s . The  was  from the a n i m a l f o r 72 hours p r i o r  minutes  a d m i n i s t e r e d , the sheep was  kg  and  m e s e n t e r i c v e i n as w e l l as c a r o t i d a r t e r y  the sheep was area was  placed i n  left  lateral  f u r t h e r c l i p p e d , washed, and  w i t h weak t i n c t u r e of i o d i n e . An i n c i s i o n 25  to 30 cm  recumbency. disinfected long  was  49  made a p p r o x i m a t e l y 3 t o 5 cm behind and p a r a l l e l t o t h e l a s t r i b on  t h e r i g h t s i d e o f t h e sheep, which p e n e t r a t e d through  f a s c i a , and r e c t u s abdominis c a v i t y . The peritoneum The  left  muscle  and  into  skin,  the p e r i t o n e a l  was s e c u r e d f i r m l y w i t h hemostats.  l o b e o f t h e l i v e r was l o c a t e d and moved c e p h a l a d  (towards head)  exposing  abomasum  identified  were  the  portal  by  vein.  The  duodenum  f o l l o w i n g t h e s e s t r u c t u r e s i n an  a n t e r i o r d i r e c t i o n , t h e omasum was found and brought toward incision  s i t e . . The  cannulation. inserted  omasal  Silastic  with  fine  vein  was  tubing  readily  (.30 i n I.p. x .065 i n O.D.)  piano w i r e , was a d m i n i s t e r e d i n t o t h e v e i n  was c a r e f u l l y manipulated  the  a l o n g t h e omasal v e i n u n t i l i t  was f e l t i n the p o r t a l t r u n k . The w i r e was the  the  accessible f o r  through a 13 gauge needle. While p a l p a t i n g t h e p o r t a l v e i n , tubing  and  slowly  removed  and  c a t h e t e r was secured by s i l k s u t u r e s a t t h e p o i n t of e n t r y .  The c a n n u l a was f u r t h e r ancohored by a d d i t i o n a l s u t u r e s neighbouring  f a s c i a and e x t e r i o r i z e d a t t h e d o r s a l e x t r e m i t y of  the i n c i s i o n . A Deerfield,  t o the  4-way  plastic  stopcock  (Travenol  Laboratory  111. 60015) was f i x e d t o t h e end o f t h e c a t h e t e r and  s u t u r e d onto the back o f t h e a n i m a l . A second c a n n u l a t i o n was using  t h e same  performed  on  a  reticular  vein  m a t e r i a l and t e c h n i q u e s a s d e s c r i b e d above. I t  was e x t e r i o r i z e d i n t h e same p o s i t i o n and s e c u r e d t o t h e back o f t h e a n i m a l i n a s i m i l a r manner. T h i s c a t h e t e r was  intended  for  i n f u s i o n purposes o n l y . In  order  to  implant  t h e c a r o t i d a r t e r y c a n n u l a a 5-6 cm  i n c i s i o n was made a l o n g t h e m i d - l i n e o f t h e t r a c h e a . The  artery  50  on  the  side  surrounding  of  the  fascia.  introduced  via  neck The  was  l o c a t e d and i s o l a t e d from t h e  polyethylene  catheter  a 13 gauge needle and secured  of a p u r s e s t r i n g s u t u r e cannula  was  through  the  (P.E.90)  was  i n p l a c e by means  tunica  adventitia.  e x t e r i o r i z e d and f i x e d w i t h a 4-way stopcock  The  which  was s u t u r e d t o the back of the sheep's neck. The  abdominal i n c i s i o n  was  closed  in  three  p e r i t o n e u m , the muscle l a y e r and t h e s k i n . The  parts;  the  l a t t e r employed a  heavy grade s i l k s u t u r e , whereas the former two  used c a t  gut.  By the t i m e t h e i n c i s i o n s were b e i n g s u t u r e d the animal b r e a t h i n g s t r a i g h t oxygen from the h a l o t h a n e recovery  p r o c e s s and prevent  continued  strong.  preparation  was  administered  by an i n t r a m u s c u l a r i n j e c t i o n o f 5 ml o f D e r a f o r t ,  p e n i c i l l i n - strepytomycin  tube was  machine. To a i d the  i n f e c t i o n , an i n t r a v e n o u s i n f u s i o n  of 20 mis o f 20% s t e r i l e g l u c o s e followed  for  p r e p a r a t i o n . The  Derafort  3 days f o l l o w i n g the o p e r a t i o n . The  removed once the chewing r e f l e x This  procedure  was  reduces  the  of  the  chance  1  a  injections Endotrachial  sheep  became  the  animal  of  a s p i r a t i n g rumen c o n t e n t s . When t h e sheep began showing s i g n s of v o l u n t a r y motion, i t was c a r e f u l l y t r a n s f e r r e d t o a and  kept under c l o s e o b s e r v a t i o n . The a n i m a l  e q u i l i b r i u m w i t h i n 4 hours and  returned  to  was  padded  pen  a b l e to r e g a i n  normal  within  24  hours of the o p e r a t i o n . During  the  f i v e day r e c o v e r y  1. D e r a f o r t , A y e r s t ,  Montreal.  p e r i o d between the  operation  51  and  the  blood  (100 u n i t s / m l ) ensure t h e i r  2  flow was  experiment,  sterile  heparinized  saline  f l u s h e d through each c a n n u l a t w i c e d a i l y t o  patency.  •) N u t r i t i o n a l _ H e a i m e The a n i m a l  dehydrated  was  grass  placed  pellets  on  a  maintenance  diet  of  1 kg  per day. The p e l l e t s were f e d t o the  a n i m a l i n 200 gm p o r t i o n s e v e r y two hours s t a r t i n g a t 0900 hours and f i n i s h i n g a t 1700 hours. T h i s regime was m a i n t a i n e d f o r f o u r days p r i o r t o the experiment  day. The  infusion  began  at  1000  hours. The a n i m a l was not f e d , thus the r e s u l t s are based upon a 17 hour f a s t  3•)  (overnight).  5lucose_Determination The  glucose  concentration  colorimetric  glucose  Biochemicals).  T h i s mechanism  was  reagent,  determined glucostat  by t h e s t a n d a r d (Worthintion  (Hashko and B i c e , 1961)  makes use  of the c o u p l e d enzyme r e a c t i o n s :  Glucose + 0, + H,0 * 1  HjO^ + Reduced Chromogen For  this,  as  well  as  Glucose. H.0, + G l u c o n i c A c i d Oxidase> * 1  Peroxidase the  other  O x i d i z e d Chromogen concentration  (color) assays.  52  deproteinized first  plasma  centrifuged  deproteinized  by  was and  employed. The whole b l o o d plasma was the  resulting  supernatant  t h e use o f  10% ZnSO^  was  and . 5N NaOH i n t h e  following proportions:  1 ml plasma o r serum + 8 ml d i s t i l l e d  H^O  + 0.5 ml 10% ZnSO^. + 0.5 ml 0.5N NaOH  The ZnSO^. and NaOH were t i t r a t e d t o g e t h e r neutrality.  After  throughly  mixing  i n order  c l e a r s u p e r n a t a n t was employed f o r t h e subsequent ml  of  a t which  again  and t h e  assays.  t h e d e p r o t e i n i z e d plasma was added t o 4 ml o f  g l u c o s t a t p r e p a r a t i o n and t h e r e a c t i o n proceeded minutes  time  f o r exactly  20  spectrophometer  on  a  a t a wavelength o f 420 nm. These  r e a d i n g s were c o n v e r t e d t o mg/100 ml g l u c o s e by comparison standard  20  i t was stopped by a d d i n g 2 drops o f 4N  H C l . The o p t i c a l d e n s i t y o f each sample r e a c t i o n was r e a d Spectronic  ensure  t h e above p r e p a r a t i o n and  w a i t i n g 10 minutes, each sample was c e n t r i f u g e d  One  to  with  curve (Appendix, F i g u r e 1) prepared from known g l u c o s e  concentrations.  53  4.)  Alanine_Determination The  c o n c e n t r a t i o n o f a l a n i n e was determined by an e n z y m a t i c  technique  d e s c r i b e d by Bergmeyer  converted and  t o pyruvate  (1965)  by g l u t a m a t e pyruvate  alanine i s  t r a n s a m i n a s e (GPT)  oxoglutarate:  2-oxoglutarate  This  reaction  • L-alanine  i s coupled  GPT  to a  dehydrogenase (LDH) r e d u c e s pyruvate lactic  V  L-glutamate + pyruvate  second  in  which  • NADH+H*  disappearance  i n t h e presence o f NADH t o  of  KDH  NADH  L - l a c t a t e + HAD  was  followed  on a ONICAM SP 800  s p e c t r o p h o t o m e t e r a t a wavelength o f 340 nm. S i n c e t h e r e excess o f both enzymes, o x o g l u t a r a t e and NADH coupled  lactic  acid:  Pyruvate  The  i n which  i s an  , the rate of the  r e a c t i o n with l i m i t e d a l a n i n e c o n c e n t r a t i o n s i s s t r i c t l y  proportional  t o t h e amount o f a l a n i n e p r e s e n t . The measurement  of t h e r e a c t i o n r a t e p e r m i t s t h e d e t e r m i n a t i o n o f a l a n i n e w i t h i n each sample by use o f alanine concentration  a  standard  curve  (Appendix, F i g u r e 2) .  prepared  with  known  54  5 •) £i°2iL.Ii2£^eter mi n a t i o n The  p o r t a l v e i n b l o o d f l o w was measured with t h e use o f t h e  i n d i c a t o r para- aminohippuric of  a  1.5%  PAH  solution  acid  was a d m i n i s t e r e d i n t o t h e c a n n u l a t e d  r e t i c u l a r vein at t h e beginning immediately solution  followed  by  (PAH). A p r i m i n g dose (15 ml)  of  a continuous  This  was  i n f u s i o n o f t h e same PAH  (1.5%) i n t o t h e r e t i c u l a r v e i n by means o f a p c l y s t a t i c  i n f u s i o n pump ( B u c h l e r Instruments) A f t e r a one hour samples  the experiment.  a t a r a t e of 0.79 ml/min.  equlibration period,  heparinized  blood  were withdrawn from t h e p o r t a l v e i n , and c a r o t i d a r t e r y  at 15 minute i n t e r v a l s f o r one hour t o glucose  and  assessed  by the method o f Smith  densities  PAH  the a l a n i n e ,  c o n c e n t r a t i o n s . The plasma l e v e l s o f PAH were  (0. D.)  spectrophotometer  determine  of  the  et  al.,  (1945).  The  optical  samples were measured on a Spec 20  a t a wavelength of 540 nm. 1  The  corresponding  PAH c o n c e n t r a t i o n s were determined by comparison w i t h a s t a n d a r d curve o f known PAH c o n c e n t r a t i o n s  (Appendix,  Figure 3 ) .  Calculations The  portal  vein  plasma  flow  b l o o d flow  (PVBF) were c a l c u l a t e d  equations;  (Katz and Bergman, 1969a)  PVPF= CIxIR C  PV- * C  (PVPF) and t h e p o r t a l v e i n  according  to  the f o l l o w i n g  55  PVBF= PVPF (1-PCV) Where:  CI= c o n c e n t r a t i o n o f PAH  i n the i n f u s i o n s o l u t i o n  (mg/100 ml)  IR= i n f u s i o n r a t e (mls/min) PCV= the b l o o d packed c e l l volume C p ^ a n d C^= arterial  Plasma  blood  PAH  (hematocrit)  concentration  in  portal  vein  and  respectively.  The net p o r t a l p r o d u c t i o n r a t e s o f both g l u c o s e and a l a n i n e were d e t e r m i n e d as f o l l o w s :  (Katz and Bergman 1969b)  P=  where P r e p r e s e n t s metabolite; v e i n ; and C  F ^ p  p v  F  the  PV  { C  PV  ~ A> C  portal  for  and  indicates  the  production  i s the whole b l o o d f l o w  production  rates  of  (ml/min) i n the  a r e the c o n c e n t r a t i o n s o f  i n t h e p o r t a l v e i n and a r t e r i a l value  net  the  the  portal  metabolite  vessels respectively. A negative  rates  o f e i t h e r a l a n i n e or glucose  utilization.  The r e s u l t s were s t a t i s t i c a l l y a n a l y z e d u s i n g the s t u d e n t T t e s t t o denote s i g n i f i c a n t glucose  concentrations  differences  between  v e i n plasma. Values preceded by a e r r o r of the mean.  the  for  both  alanine  c a r o t i d a r t e r y and s i g n i n d i c a t e the  and  portal  standard  56  2£§fii£2_!£d_Discussion  A  summary  of  the  mean  blood concentrations of glucose,  a l a n i n e and PAH t h a t were o b t a i n e d by sampling from vein  and  carotid  artery,  the  portal  as w e l l as t h e c o r r e s p o n d i n g  packed  c e l l volume f i g u r e s a r e presented i n T a b l e  1(a) ( A p p e n d i x ) .  1.) P o r t a l Blood Flow To c a l c u l a t e t h e p o r t a l v e i n portal  vein  plasma  flow  blood  (PVPF),  flow  (PVBF)  t h e PAH  was  and t h e  allowed  to  e q u i l i b r a t e w i t h e x t r a c e l l u l a r f l u i d s and t h u s t h e c o n c e n t r a t i o n f i g u r e s employed a r e those a t t h e p l a t e a u l e v e l 1(a)).  In  this  concentrations attained  experiment  the  plateau  (Appendix,  Table  constant  PAH  or  f o r both t h e c a r o t i d a r t e r y and p o r t a l v e i n were  between t h e 1 hour 25 minute and 1 hour 45 minute b l o o d  c o l l e c t i o n s . Thus t h e average o f t h e s e two c o n c e n t r a t i o n were  used  summary  values  f o r t h e d e t e r m i n a t i o n o f t h e blood f l o w f i g u r e s . A  of  the calculations  and  results  i s presented  in  Appendix, Table 1 ( b ) . A  portal  vein 3/4  blood  flow  (47.85 ml/mm/kg/B. W.) was determined The  result  estimate 1800  agrees  the  mean  with portal  previously vein  value  1969a)  2010 mls/min  i n the p r e s e n t published  blood  ml/min (Bergman and Wolff 1971) t o  Bergman,  of  flow  experiment.  results  i n a range from  2493 ml/min  (Katz  f o r f e d , nonpregnant sheep. The l a t t e r  employed a t e c h n i q u e which e n a b l e d a  which  measurement  of  both  and study the  57  hepatic  and  p o r t a l b l o o d f l o w s . Such a r e s u l t was  i n the p r e s e n t experiment s i n c e c a t h e t e r s the  portal  vein  and  carotid  h e p a t i c v e i n . The v a l u e present  study  for  artery  portal  were only  vein  not p o s s i b l e  implanted ,and  blood  not i n t o flow  Bergman  v a l u e s from 1440 diet.  in  the  and W i l l i a m s (1972) who  work  by  quote p o r t a l blood f l o w  t o 2240 ml/min i n sheep f e d a  near-maintenance  These r e s u l t s are more r e l e v a n t t o the p r e s e n t experiment  s i n c e the d i e t a r y regime as w e l l as t h e p h y s i o l o g i c a l s t a t u s t h e sheep  2  the  appears t o f a l l w e l l w i t h i n range of e s t a b l i s h e d  r e s u l t s . A d d i t i o n a l s u p p o r t i s p r o v i d e d by more r e c e n t Wolff,  into  (nonpregnant and n o n l a c t a t i n g ) are  of  similar.  •) Sg.t-Slu£2gs,Metabolism The  negative  production  v a l u e (-.142 g/hr/kg  i n d i c a t e s a net u t i l i z a t i o n o f g l u c o s e viscera.  by  the  portal  always  obtained  regardless  sheep were hypoglycemic or Bergman  et  is slightly fed  )  drained  T h i s f i n d i n g i s i n agreement w i t h p u b l i s h e d work which  showed t h a t a net u t i l i z a t i o n of g l u c o s e i n the p o r t a l almost  B.W  fasted  bed  was  o f t h e d i e t or whether the (Katz  and  Bergman,  1969b;  a l , 1971). The f i g u r e o b t a i n e d i n the p r e s e n t  study  l a r g e r than t h e mean p o r t a l bed u t i l i z a t i o n r a t e  of  sheep and s m a l l e r than t h a t i n sheep f a s t e d f o r t h r e e days. BU  T h i s appears a p p r o p r i a t e s i n c e the v a l u e of was  determined  with  a  sheep  fasted  for  u t i l i z a t i o n of g l u c o s e , as shown here as works,  is  slight.  The  actual  .142  well  17 as  g/hr/kg  B.W.  hours. The  net  n  in  previous  portal-arterial  glucose  58  c o n c e n t r a t i o n d i f f e r e n c e s c a l c u l a t e d i n t h i s experiment statistically significant who have observed  3 •)  (P < 0.05 l e v e l )  were n o t  which s u p p o r t s  others  t h e same (Roe e t a l ., 1966; Schambye, 1951).  Stf_Alanine_Meta^olism The  figure  demonstrated  a  f o r alanine  metabolism  by  the portal  n e t p r o d u c t i o n ( a b s o r p t i o n by t h e p o r t a l  of 1.49 mM/hr. The p u b l i s h e d v a l u e s i n d i c a t e a mean production  bed  blood)  portal  bed  r a t e of 2.29 ±.29 mM/hr (Wolff Bergman and W i l l i a m s ,  1972), which was determined  on t e n s e p a r a t e experiments  whereas  the f i g u r e o b t a i n e d i n t h e p r e s e n t s t u d y was based upon a s i n g l e experiment.  Considering  this  fact,  1.49 mM/hr a r r i v e d a t here cannot be estimate  b u t undoubtedly  the single  regarded  as  value  an  of  absolute  i s w i t h i n t h e range r e p o r t e d by W o l f f  §t §i«'• * (1972). I t i s i m p o r t a n t  t o note,  however,  that the  a l a n i n e c o n c e n t r a t i o n s between t h e c a r o t i d a r t e r y and t h e p o r t a l vein  proved  t o be  significantly  different  i n the  present  experiment.  Conclusions  The r e s u l t s from t h e p r e s e n t d e t e r m i n a t i o n s o f p o r t a l v e i n b l o o d f l o w , a s w e l l a s t h e net metabolism the  portal  drained  viscera  of alanine  a i d more  and g l u c o s e  i n evaluation  by  of the  59  e f f e c t i v e n e s s of t h i s method r a t h e r than as which  absolute  would be r e p r e s e n t a t i v e of a l l sheep. The purpose o f t h i s  experiment flow  was t o a s s e s s t h e v a l u e and a p p l i c a t i o n of the  technique  using para-aminohippuric  blood  a c i d as t h e i n d i c a t o r .  Therefore the r e s u l t s f o r p o r t a l vein blood flow net  estimates  (2010 ml/min),  g l u c o s e u t i l i z a t i o n by t h e p o r t a l bed (.142 g/hr/kg  and n e t a l a n i n e p r o d u c t i o n by t h e p o r t a l within  the  range  of  previously  bed  B. W. ) ,  (1.49 mM/hr)  published  are  figures  consequently c o n t r i b u t e a d d i t o n a l support f o r the  use  and  of  this  approach i n m e t a b o l i c s t u d i e s . The  sophisticated surgical  procedures  method a r e the l i m i t i n g f a c t o r upon which study  is  based.  Thus  in  order  or  tissue  system,  the  the  catheters  success  represent  catheters  must  p r e c i s e l y . This process r e q u i r e s a great deal the  by t h i s of  the  to state accurately that the  arterio-venous concentration differences organ  required  of  a be  specific implanted  skill.  Often  become d i s l o d g e d o r b l o c k e d and c o n s e q u e n t l y the  e n t i r e experiment  i s d e s t r o y e d . The c h e m i c a l a n a l y s e s as w e l l as  t h e c a l c u l a t i o n s a r e s t r a i g h t f o r w a r d and r e q u i r e a  minimum  of  interpretation. The t e c h n i q u e of b l o o d f l o w measurement a l o n g w i t h a r t e r i o venous  concentration  differences  proved  to  be  approach i n d e t e r m i n i n g , q u a n t i t a t i v e l y , the net specific  compounds  by  individual  emphasized through t h e d e m o n s t r a t i o n  organs  is  divided  into  that  metabolism  of  or t i s s u e s . This i s  by K a t z and Bergman (1969b)  o f a method whereby t h e n e t metabolism region  an e f f e c t i v e  of the  total  splanchnic  of the l i v e r and p o r t a l - d r a i n e d  60  viscera.  Although  instrumental  highly  demanding,  their  technique  was  i n r e c o g n i z i n g t h a t there i s a net u t i l i z a t i o n of  g l u c o s e by the p o r t a l bed. The  results  of  the  present  study  c o n f i r m e d t h e i r f i n d i n g t h a t no n e t g l u c o s e a b s o r p t i o n o c c u r s i n sheep  f e d a maintenance d i e t , but a p o r t a l g l u c o s e  utilization  appears t o be p r e s e n t a t a l l t i m e s . A l t h o u g h t h e a l a n i n e data f o r n e t agree  as  substantiates  B.W.  i s s t i l l of  i t s established  role  the  to  obtained  determine  the  in this liver's  experiment  i t was  the  s u c c e s s f u l l y , thus the  a r e based on t h e p o r t a l bed and i t s r e l a t i o n t o a l a n i n e  this  study  t h a t t h e t e c h n i q u e employed i s e f f e c t i v e and a c c u r a t e  t o be a p p l i e d " i n v i v o " m e t a b o l i c s t u d i e s . The method  major  consequently  the r e s u l t s obtained are d i r e c t l y  t o t h e f u n c t i o n i n q o f t h a t system. technique  advantage  i s t h a t once t h e c a t h e t e r s have been p r o p e r l y  i m p l a n t e d , an organ or t i s s u e system can be c o m p l e t e l y  this  of  glucose.  indicated  and  not  cannulating  In summary t h e r e f o r e , t h e r e s u l t s o b t a i n e d from t h i s  to  which  r o l e i n t h e metabolism  h e p a t i c v e i n . T h i s was never accomplished  and  magnitude  a s the p r i m a r y amino a c i d  these two m e t a b o l i t e s due t o t h e d i f f i c u l t y i n  results  d i d not  by the p o r t a l c i r c u l a t i o n and d e l i v e r e d t o t h e l i v e r .  From the d a t a possible  output  c l o s e l y t o p u b l i s h e d r e s u l t s as t h a t f o r g l u c o s e , the  v a l u e o f 1.49 mM/hr/kg  absorbed  portal  The  primary  isolated  attributable  limitation  to  i s t h e complex s u r g i c a l s k i l l s r e q u i r e d around  which the e n t i r e study r e v o l v e s .  61  11 S i n g_ l e _ I n j e c t i o n _ o f _ £a be 1 led__ £*C-Alanine  IHifS'l uc t i o n The s i n g l e i n j e c t i o n t e c h n i q u e  of  a  labelled  tracer  is  regaining  acceptance  as an e f f e c t i v e and a c c u r a t e procedure i n  measuring  body  kinetics  pool  for  various  metabolites.  The  m a j o r i t y of the work done has i n v o l v e d l a b e l l e d **c - g l u c o s e to s t u d y g l u c o s e metabolism. Numerous models have been developed t o d e s c r i b e t h e movement of g l u c o s e carbon i n the body, with a 2 or 3 compartmental model as t h e most w i d e l y e s t a b l i s h e d t o f i t glucose  isotope  dilution  S t e e l e , 1964) and i n sheep 1959;  Hescigne  and  data  in  rats  (Baker e t a l . ,  **C1959;  (White e t a l . , 1969; S k i n n e r e t a l . ,  Segre,  1966). The f i t t i n g  of d a t a t o such  models has i n v o l v e d a number of d i f f i c u l t i e s which, i n the e a r l y s t a g e s , had persuaded many workers t o doubt the v a l i d i t y o f t h i s approach. Annison and White  (1961) s t a t e d  governing  o u t f l o w o f g l u c o s e from t h e proposed  the  entry  and  multi-compartment system cannot mathematical  treatment  be  of  that  adequately  the  parameters  defined  single-injection  data.  d i f f i c u l t i e s i n v o l v e d w i t h d e f i n i n g components suggest of  curve-fitting  computer  techniques  component s l o p e s w i t h i n a complex defined number  (Berman, of  metabolism  1963).  investigators in  This in  monogastrics  the  a The use  i n which the i n d i v i d u a l  curve  need  not  be  clearly  approach has been u t i l i z e d by a  defining  parameters  (Baker et a l . ,  1961). Work with r u m i n a n t s u s i n g a combined  for  single  of  glucose  1959; S e g a l e t a l . , injection  technique  w i t h a m u l t i c o m p a r t m e n t a l a n a l y s i s has been s p a r s e and  the r e s u l t s v a r i a b l e  (White  et  al. ,  1969;  Leng,  1970).  The  62  latter  author  attributes  the  variability  i n s u f f i c i e n t b l o o d samples were t a k e n  to  over  the f a c t t h a t  too  short  a  time  p e r i o d i n many experiments f o r a c c u r a t e a n a l y s i s o f t h e d a t a . The  multicompartmental  approach  t o the a n a l y s i s of s i n g l e  i n j e c t i o n data has been used t o a l i m i t e d with  t h e major  the f i e l d  degree  emphasis upon g l u c o s e metabolism  of amino a c i d r e s e a r c h , s p e c i f i c a l l y  parameters  and  contribution  to  glucose  Black,  1968).  I t i s only  d e t e r m i n a t i o n o f such parameters amino  acids  been conducted  as  well  recently  ruminants  s t u d i e s . Thus  their  metabolic  synthesis,  s p a r s e l y touched by t h e use o f t h i s technique and  in  has been  ( B l a c k , 1968; Egan that  work  on t h e  as t u r n o v e r r a t e s o f i n d i v i d u a l  as t h e i r r e l a t i o n t o g l u c o n e o g e n e s i s has  ( W o l f f , Bergman and  Williams,  1972, Wolff  and  Bergman, 1972a and b ) . The methods employed by t h e s e r e s e a r c h e r s involve  blood  flow  s t u d i e s , as d e s c r i b e d i n Experiment  continuous i n f u s i o n of discussed  i n the f o l l o w i n g  compartmental injection for  labelled  of  amino  acids,  will  be  s e c t i o n . I n one s t u d y r e p o r t e d , a  a n a l y s i s was attempted  on the d a t a  of  a  single  g l y c i n e and t h e e x i s t e n c e o f a t l e a s t t h r e e p o o l s  t h e metabolism  o f g l y c i n e r e v e a l e d (Henriques e t a l . ,  The o b j e c t i v e of t h e f o l l o w i n g experiments the  which  A, and  was  to  1955). assess  value of t h e s i n g l e i n j e c t i o n of * C - l a b e l l e d a l a n i n e along 1  with a multicompartmental  a n a l y s i s o f t h e data  as  a  means  of  q u a n t i t a t i v e l y e s t i m a t i n g t h e m e t a b o l i c parameters o f t h i s amino a c i d a s w e l l as i t s c o n t r i b u t i o n t o g l u c o s e . I n c o r p o r a t e d w i t h i n t h i s assessment a r e t h e r e l a t i v e advantages this  and d i s a d v a n t a g e s of  t e c h n i q u e . Such f a c t o r s a s p h y s i o l o g i c a l a s s u m p t i o n s ,  ease  63  of  application  and  c a l c u l a t i o n o f r e s u l t s as w e l l as time  expense are i n c l u d e d  i n evaluating  the  effectiveness  of  and this  method. The  study  consists  of  three  c o m p r i s i n g of a s i n g l e i n j e c t i o n of  s e p a r a t e e x p e r i m e n t s , each 1 4  C - a l a n i n e i n t o the  jugular  v e i n . Blood samples f o r a n a l y s i s were c o l l e c t e d from the v e i n , except i n the blood  was  alanine with  of  fall  in  arterial  a n a l y s i s for a l l three  comprised  specific  activity  of  plasma  parameters f o r a l a n i n e metabolism were determined from  best  f u n c t i o n a p p l i e d t o the data  be  to  generate  a  f i t f o r the decay c u r v e . T h i s procedure i n v o l v e d  the use of a computer. shall  carotid  the  where  time.  a multiexponential line  experiment  a l s o withdrawn. The  i n s p e c t i o n of the  The  third  jugular  discussed  The in  details  the  surrounding  following  the  materials  analysis  and  methods  section. P periment_B.1. x  M a t e r i a l s and  Methods  A t h r e e year o l d wether weighing 35 kg was experiment.  The  surgical  p o l y v i n y l catheter hours  prior  to  (P.E. the  maintained operative heparinized The  90)  preparation into  the  used  consisted right  s a l i n e (100  this  of i n s e r t i n g a  jugular  s t a r t of the e x p e r i m e n t . The by f r e q u e n t f l u s h i n g s w i t h  for  a  vein  24  catheter  was  solution  of  units/ml).  n u t r i t i o n a l regime c o n s i s t e d  of a roughage d i e t of 1 kg  64  a l f a l f a hay f e d t w i c e d a i l y a t 0800 hours and 1600 hours, on t h e day o f t h e experiment t h e a n i m a l was g i v e n i t s morning f e e d the  and  i n j e c t i o n began 4 hours l a t e r a t 1200 hours. Thus the sheep  was i n a f e d (4 hr f a s t ) c o n d i t i o n . The experiment was performed w i t h t h e a n i m a l i n a r a i s e d metabolism cage  which  enabled  the  c o l l e c t i o n of u r i n e and f e c e s f o r t h e proper waste d i s p o s a l . The building  was  well  ventilated  thus  adding  a further control  measure t o t h e use o f i s o t o p e s i n l i v i n g a n i m a l s  1 •)  Jsoto£e_and__Blood_Collection L-Alanine  Radioisotope  0-L- *C  was  J  division  obtained  with  the  from  ICN  following  s p e c i f i c a c t i v i t y 100-120 mC/mm, packaged  Chemical  and  specifications;  i n 0.01N  HC1  at  a  c o n c e n t r a t i o n o f 0.1 mC/ml. A  single  injection  o f .05 ml (50 uCi) o f t h e i s o t o p e was  a d m i n i s t e r e d i n t o t h e j u g u l a r v e i n and 10 ml o f b l o o d plasma was c o l l e c t e d from t h e same  jugular  vein  at  the f o l l o w i n g  time  intervals: Sample No. 1 2 3 4 5 6 7 8 9 10 The  C o l l e c t i o n Time C o n t r o l (before i n f u s i o n ) 5 min 10 min 20 min 30 min 52 min 60 min 120 min 180 min 1110 min (0830 hours f o l l o w i n g day)  t i m e r e q u i r e d f o r c o l l e c t i o n o f plasma was h e l d c o n s t a n t a t  between 80-100 seconds f o r each  sample.  65  2  •) Chemical_Methods  a) S e p a r a t j o n ^ q f ^Plasma^Coffiponents The  separation  accomplished  by  of  the  plasma  constituents  i o n exchange chromatography. The a d s o r p t i o n  and r e t e n t i o n o f t h e charged m o l e c u l e s by t h e r e s i n , p o s i t i v e ( c a t i o n exchange column) o r n e g a t i v e column),  permit  the  i n t o a basic f r a c t i o n acidic  fraction  neutral fraction  was  separation  (anion exchange  o f t h e plasma c o n s t i t u e n t s  ( p o s i t i v e l y charged  (negatively  either  charged  amino  organic  acids),  an  a c i d s ) and a  (sugars).  i ) C a t i o n Exchange Resin The from  c a t i o n r e s i n (DOWEX 50W-X8)  t h e Bio-Rad  employed  was  obtained  L a b o r a t o r i e s . I t was i n t h e hydrogen form  (200-400 mesh) w i t h a t o t a l c a p a c i t y o f 5.1 meg/dry  gram  or  1.7 meg/ml wet r e s i n . A p p r o x i m a t e l y 2 dry grams o f r e s i n were used  f o r s e p a r a t i o n . To ensure t h e r e s i n was i n t h e hydrogen  form 40 ml 2N H C l was passed t h r o u g h t h e column f o l l o w e d by a wash w i t h d a i o n i z e d water u n t i l t h e e l u e n t i s n e u t r a l .  Three  ml o f t h e d e p r o t e i n i z e d plasma sample were a p p l i e d t o t h e t o p of  t h e column  and  allowed  t o s t a n d f o r 15 minutes  being passed t h r o u g h . The sample was r u n s l o w l y  before  through  the  column and a l l non-adhering compounds (sugars and a c i d s ) were washed  through  with  50-60  ml  water. The b a s i c s u b s t a n c e s  (amino a c i d s ) which a r e adsorbed t o t h e r e s i n were e l u t e d passing  60-70  by  ml 2N H C l through t h e column. The amino a c i d s  66  retain  their  charge  concentration  and  o f hydrogen. The  r e s i n was r e g e n e r a t e d  ii)  were  eluted  by  the  greater  r e s i n was d i s c a r d e d and f r e s h  f o r the next sample.  *nionJ|xchanae_Resin The  a n i o n r e s i n was  Company  and  o b t a i n e d from J . T.  was i n the c h l o r i d e form  Baker  Chemical  (200-400 mesh). I t i s a  s t r o n g b a s i c r e s i n w i t h a t o t a l exchange c a p a c i t y of per  dry  gram.  Approximately  2.5  4.4  meg  dry grams of the r e s i n  was  u t i l i z e d f o r each plasma sample. The r e s i n was the  formate  formate.  by  with  through  60 ml  distilled  water  column was (sugars)  was  of 1M  added and  until  administered were  washed  the  column  neutral. wand  The  was  the  washed  column  substances  with water.  The  ( a c i d s ) were e l u t e d by p a s s i n g 40 ml of 4N f o r m i c a c i d  the  regenerated  resin  the  was  discarded  with  a  fresh  each f r a c t i o n was  evaporated to  liquid  scintillation  The c o u n t i n g e f f i c i e n c y was standards  quantity  dryness  use o f a B u c h i Rotovapor. These samples as w e l l as  the o r i g i n a l u n d i l u t e d plasma samples were counted ISOCAP/300  Once  f o r each subsequent sample.  Once s e p a r a t e d , with  with  sample from the c a t i o n  f o l l o w e d by 30 ml of 8N f o r m i c a c i d t h r o u g h t h e column. again  when  20-30 ml of  a l l non-adhering  through  to  solium  negative  AgNO^ F o l l o w i n g t h e sodium f o r m a t e ,  f o r m i c a c i d was  anions  passing  At t h i s stage the c h l o r i d e t e s t  tested 0.IN  form  converted  ratio  (ESR).  counter  d e t e c t e d by  Figure  4  (Nuclear  using  the  using  an  Chicago). external  (Appendix) i s the quench  67  c o r r e c t i o n curve u s i n g the ESR quenched **C  standards  method i n c o n j u n c t i o n w i t h  provided  by  Nuclear  Chicago.  It  the is  from t h i s F i g u r e t h a t t h e c o u n t i n g e f f i c i e n c y f o r each sample was  d e t e c t e d . In a l l of the c o u n t i n g throughout t h i s p r o j e c t ,  Phase  Combining  System  (PCS)  solubilizer  f l u o r s o l u t i o n . The s t a n d a r d m i x t u r e was combination In  w i t h 10 ml  was  used as  the  200 u l o f sample  in  PCS.,  o r d e r t o e v a l u a t e the e f f i c i e n c y of the i o n exchange  chromatography preliminary  run  in  separating  was  plasma  constituents,  undertaken u s i n g a prepared  mixture  a of  the f o l l o w i n g :  0.01  ml L-Alanine-OL-**C  1 ml plasma 0.1  mg l a c t a t e  0.1  mg l a c t a t e  T h i s s o l u t i o n was anion  exchange  passed s u c c e s s i v e l y through the c a t i o n column  as d e s c r i b e d above and the f r a c t i o n s  counted y i e l d i n g the f i g u r e s l i s t e d i n  Table  4  These  that  ion  recovery  figures  indicate  (Appendix) . exchange  chromatography can be used e f f e c t i v e l y f o r the s e p a r a t i o n plasma i n t o a c i d i c , b a s i c and To  confirm  and  of  neutral fractions.  f u r t h e r t h a t the r a d i o a c t i v i t y c o r r e s p o n d e d  to the m e t a b o l i t e s under i n v e s t i g a t i o n , t h e b a s i c and f r a c t i o n s were s p o t t e d  neutral  (50 ul) on a paper chromatogram. A  d i r e c t i o n a l paper chromatography was  used f o r the  two  separation  68  of  alanine  i n which  the solvent  d i r e c t i o n c o n s i s t e d of n-butanol, water  in a  second  ratio  direction  proportions  of  The  was  phenol:H 0  solvent  employed  system  b u t a n o l : w a t e r i n a 7:1:2 (v/v) sugar  f o r the acetic  first  acid  and  r e s p e c t i v e l y . For t h e was  (v/v) r e s p e c t i v e l y .  paper chromatography fraction.  glacial  80:20:20 (v/v)  liquified  94:28  system  used  Single  to  in  the  directional  separate  the  sugar  c o n s i s t e d of i s o - p r o p a n o l : n ratio.  The  amino  acid  and  chromatograms were compared t o s t a n d a r d s c o n s i s t i n g of  L-Alanine  UL-**C  standards  and  and  glucose-OL- *C  respectively.  l  t h e sample  chromatograms  i d e n t i c a l s t r i p s 3 cm wide. The s t r i p s radio-chromatography I I ) . By comparing  scanner  (Neuclear  procedure  the  location  provides  radioactivity  were  cut  put  through  Chicago,  into  Actigraph  of  evalulation  activity,  the information  as  can  be  made  consequently  this  to  the  whether  d e t e c t e d i n t h e b a s i c and n e u t r a l f r a c t i o n s i s  due t o a l a n i n e and g l u c o s e r e s p e c t i v e l y . The r e s u l t s o f t e c h n i q u e a r e i l l u s t r a t e d i n F i g u r e 5 (Appendix) The  approximately above  20  for alanine.  an  counter  was  developed  vials  maximum  of  utilizing  the  liquid  (ISOCAP 300). T h i s method c o n s i s t s of  c u t t i n g t h e above s t r i p s i n t o s m a l l e r into  efficiency  per c e n t . T h e r e f o r e a s l i g h t v a r i a t i o n o f  technique  scintillation  them  this  a c t i v i t y o f g l u c o s e was not s t r o n g enough f o r the use o f  the A c t i g r a p h s c a n n e r , which has  the  a  t h e peaks o f r a d i o a c t i v i t y o f t h e sample t o  t h a t o f t h e s t a n d a r d , an a c c u r a t e regarding  were  The  sections  and  placing  immersed i n t h e l i q u i d s c i n t i l l a t i o n  fluid  (PCS). The i n d i v i d u a l v i a l s were counted and by comparison t o  69  t h e s t a n d a r d U - L ^ O G l u c o s e chromatogram s t r i p , the peaks radioactivity  were  located.  Table 3  (Appendix)  of  illustrates  t h e r e s u l t s f o r a n e u t r a l f r a c t i o n sample and a s t a n d a r d .  3 •) B l o o d _ An a l j s i s Whole blood samples were c o l l e c t e d i n tubes  and  refrigerated deproteinized s i m i l a r manner assays  were  centrifuged  on  centrifuge.  The  with  10  per  described performed  a  Sorvall resulting  cent  ZnSO  previously. on  heparinized BC  2-B  test  automatic  supernatant and 0.5N  Glucose  NaOH i n a  and  alanine  the d e p r o t e i n i z e d supernatant  methods o u t l i n e d i n t h e p r e c e d i n g  was  by  experiment.  Sslsslations S p e c i f i c a c t i v i t y decay c u r v e s were f i t t e d w i t h a  function  which was t h e sum o f e x p o n e n t i a l terms having the form:  where:  SA=  s p e c i f i c a c t i v i t y of plasma g l u c o s e a t time t  (nC/mg C)  A.= z e r o - t i m e i n t e r c e p t of each component (nC/mg C)  -m-  r a t e c o n s t a n t o f each component (min -*)  70  n= number o f e x p o n e n t i a l components  i= exponential-component number  t= time (min) Fitting  was  a c c o m p l i s h e d by i t e r a t i v e m i n i m i z a t i o n of t h e  e r r o r sum o f s q u a r e s f o r t h e f i t t e d Fortran  IV  subprogram  function.  w r i t t e n by R. F l e t c h e r  The number of e x p o n e n t i a l terms i s determined by the  In  practice,  a  (1972) was used. the  shape  of  observed s p e c i f i c r a d i o a c t i v i t y decay c u r v e . On t h e p r e s e n t  experiment a two term f u n c t i o n was f i t t e d t o t h e c u r v e . P o o l s i z e and m e t a b o l i c t u r n o v e r e s t i m a t e s were from the  calculated  t h e parameters o f t h e l e a s t squares f i t t e d f u n c t i o n s u s i n g f o r m u l a e of Leng  P o o l s i z e , Q (mg C) =  (1970): P —p^-  where P i s the i n j e c t e d dose o f r a d i o a c t i v i t y .  I r r e v e r s i b l e Loss (mg C/min) =  Q  rv  T o t a l e n t r y r a t e (mg o f C/min)  where A*  t  are f r a c t i o n a l zero-time i n t e r c e p t s ; therefore  71  space i s d e f i n e d a s ;  P o o l S i z e , Q (mg) x  100 x Body H e i g h t (kg) plasma A l a n i n e c o n c e n t r a t i o n (mg/1)  R e c y c l i n g r a t e i s d e f i n e d as t o t a l e n t r y r a t e minus i r r e v e r s i b l e l o s s . The p e r c e n t  conversion  of  i*C-Alanine  to  glucose  was  e s t i m a t e d by t h e method o f K r e i s b u r g e t a l . , (1972) by u s i n g t h e equation;  % g l u c o s e from A l a n i n e = G x SA x G  S  x  100  where  I  G = g l u c o s e c o n c e n t r a t i o n (umoles/ml) SA = s p e c i f i c r a d i o a c t i v i t y o f g l u c o s e  (dpm/umole)  G = t h e g l u c o s e space (ml) S  I = injected  dosage o f D - * C - a l a n i n e (dpm) 1  The v a l u e o f a l a n i n e r e c o v e r e d maximum  glucose  specific  time i n t e r v a l f o l l o w i n g  i n g l u c o s e i s determined  activity  the infusion  purpose o f t h i s c a l c u l a t i o n ,  from t h e  a c h i e v e d d u r i n g t h e 3 hour of  **C-alanine.  For the  t h e g l u c o s e space was assumed t o be  e q u i v a l e n t t o 0.3 x body weight i n kg (Monugian e t a l . , 1964).  SS§Sli5_S£^«fiiscussion Before  proceeding  into  the s p e c i f i c  parameter e s t i m a t e s , i t was c o n f i r m e d that  the r a d i o a c t i v i t y  by  paper  values  f o r the  chromatography  i n t h e b a s i c and n e u t r a l f r a c t i o n s  was  72  due  to  alanine  proximity  of  and  the  glucose  peak  respectively.  From  the  o f r a d i o a c t i v i t i e s f o r t h e a l a n i n e and  g l u c o s e samples as compared t o the s t a n d a r d s o f each, Table  3,  close  (Figure  5  Appendix) i t i s o b v i o u s t h a t t h e r a d i o a c t i v i t y i n t h e  b a s i c ana n e u t r a l f r a c t i o n s i s a r e s u l t o f l a b e l l e d  **C-alanine  and i * C - g l u c o s e r e s p e c t i v e l y . In  addition  to  i n d i v i d u a l values irreversible  f o r pool  loss  from t h e change o f after  an  and  data.  size,  Table 4 (Appendix)  space,  total  shows  entry  rate,  r e c y c l i n g o f a l a n i n e i n sheep e s t i m a t e d  specific  injection  (Appendix)  t h e raw  of  50  radioactivity uCi of  of  plasma  U- C-alanine.  Figure  1 4  i l l u s t r a t e s the curve f o r *C-glucose  alanine  activity.  l  6  The  l i n e o f best f i t f o r t h e * * - a l a n i n e data i s p i c t u r e d i n F i g u r e 7 (Appendix).  I t i s from t h e l a t t e r t h a t t h e c a l c u l a t i o n s f o r t h e  m e t a b o l i c parameters a r e made. The p e r alanine  appearing  i n glucose  cent  i s also  of  injected  included  **C-  i n Table 6  (Appendix) . The m e t a b o l i c parameters o f show  a  total  entry  irreversible loss of rate  of  rate  of  alanine  8.61 mg C/min  4.96 mg C/min  Appendix)  (5.80 mM/hr), an a  recycling  3.65 mg C/min (2.46 mM/hr) and a c o n v e r s i o n  percentage  alanine  The t e c h n i q u e continuous  6,  (3.34 mM/hr),  t o g l u c o s e o f 3.57%. W o l f f ana Bergman plasma  (Table  turnover used  infusion  to  (1972a)  quote  a  total  r a t e o f between 8.3 and 11.3 mM/hour. measure  of  L-U-**C  these  latter  alanine  i n j e c t i o n . The t o t a l e n t r y r a t e and t h e  figures  without  irreversible  lower than t h e v a l u e s r e p o r t e d by Wolff and Bergman  a  was  a  priming loss  (1972a).  are  73  It  s h o u l d be emphasized t h a t t h e term t o t a l t u r n o v e r r a t e ,  as used by W o l f f and Bergman, titles  has  also  given  by v a r i o u s w o r k e r s , such a s , t r a n s f e r r a t e  Simesen, entry  1961), u t i l i z a t i o n r a t e  rate  (Leng  et  al.,  t h e sampled  different  ( K r o n f e l d and  (Annisoh and White,  1961) and  1967). A l l o f t h e s e terms i m p l y t h e  same c o n c e p t , namely t h e r a t e o f e n t r y into  been  of  a l l alanine  carbon  compartment. As p o i n t e d o u t by White e t a l . ,  (1969), t h i s i n d i v i d u a l parameter, when measured by a primed continuous  i n f u s i o n r e p r e s e n t s t h e i r r e v e r s i b l e l o s s concept as  d e f i n e d by t h e s i n g l e does  or  injection  technique.  Irreversible  loss  not i n c l u d e a l a n i n e carbon which has r e c y c l e d between t h e  sampled  compartment  difference  between  and total  any  peripheral  entry  compartment.  The  r a t e and i r r e v e r s i b l e l o s s i s  regarded a s the r a t e o f r e c y c l i n g o f a l a n i n e between t h e sampled and  peripheral  difference  compartments.  between  the  Based  on  irreversible  this  loss  p r e s e n t experiment and p u b l i s h e d v a l u e s  of  concept  determined entry  the by t h e  or turnover  r a t e f o r a l a n i n e would appear t o be even g r e a t e r . The  per c e n t c o n v e r s i o n o f a l a n i n e t o g l u c o s e o f 3.57% i s ,  a g a i n , l o w e r than t h e r e l a t i v e l y e s t a b l i s h e d v a l u e s o f between 6 and 8% (Black e t a l , 1968)., W o l f f estimated  that  and  Bergman  (1972b)  o f t h e e x i s t i n g g l u c o s e 5.4% comes from a l a n i n e  by t h e use of the c o n t i n u o u s i n f u s i o n approach, which said  to  account  has  been  f o r t h e assumptions and i n a c c u r a c i e s of t h e  s i n g l e i n j e c t i o n method. K r e i s b u r g e t a l . , per  have  (1972)  estimated  a  cent t r a n s f e r o f a l a n i n e t o g l u c o s e of 12.5%. These workers  along w i t h o t h e r s (Annison and White,  1961)  suggest  that  the  74  problems  of r a p i d metabolism  o f t h e l a b e l l e d m e t a b o l i t e as w e l l  as r e c y c l i n g y i e l d o v e r e s t i m a t e s o f metabolism  v a l u e s a r r i v e d at  by the s i n g l e i n j e c t i o n t e c h n i q u e . Thus they recommend  the  use  of a c o n t i n u o u s i n f u s i o n which e l i m i n a t e s t h e s e s o u r c e s of e r r o r and p r o v i d e s an a c c u r a t e assessment o f m e t a b o l i c parameters.  The  p r e s e n t r e s u l t s i l l u s t r a t e a c o n c l u s i v e u n d e r e s t i m a t i o n of these figures  when  experiments.  compared  the  values  of c o n t i n u o u s i n f u s i o n  However a p a r t i a l answer t o  p r o v i d e d by Leng single  to  (1970) who  the  is  s t a t e d t h a t f o r a c c u r a t e a n a l y s i s of  i n j e c t i o n d a t a , an adequate number of samples have t o be  taken i n the i n i t i a l 5 activity  of  amino  to  10  acids,  minutes  and  since  especially  Table 6 and F i g u r e 7 and 8 (Appendix) t h i s experiment.  the  decline  alanine,  dramatic within t h i s time i n t e r v a l . C l e a r l y ,  10  discrepancies  by  is  most  inspection  t h i s was not adhered  in  of  to i n  There a r e not enough p o i n t s w i t h i n the f i r s t  5-  minutes o f the decay c u r v e t o p e r m i t a c c u r a t e i n t e r p r e t a t i o n  by the computer f o r the c a l c u l a t i o n o f t h e  parameters.  H a t e r i a l s _ a n d Methods The  e x p e r i m e n t a l c o n d i t i o n s as w e l l as the  were i n d e n t i c a l t o t h a t d e s c r i b e d i n Experiment The catheters  employed  B.1.  s u r g i c a l p r e p a r a t i o n c o n s i s t e d of i m p l a n t i n g p o l y v i n y l (P.E.90) i n t o both  of  the  jugular  employed f o r i n f u s i o n and the o t h e r f o r blood The  sheep  nutritional  regime  was  also  the  veins.  One  was  collection. same  as o u t l i n e d  75  previously.  The  0800 hours and  sheep 1600  was  fed  hours. The  1 kg a l f a l f a hay infusion  t h e r e f o r e s i m i l a r t o Experiment b.1 conditions  1  •)  I s o t o p e and The  and  (3.5  hr  at  the r e s u l t s are  1130  hours  based on  fed  o b t a i n e d from  ICN  Collection  used, L - A l a n i n e U-I-**C was  adheres t o the s p e c i f i c a t i o n s s t a t e d  previously.  The  s o u r c e of v a r i a t i o n between the p r e s e n t experiment and one  at  fast).  Blood  isotope  began  twice d a i l y  major  the  i s i n the amount of i s o t o p e a d m i n i s t e r e d as w e l l as t h e  first time  periods f o r blood c o l l e c t i o n . 0.085 mCi  (85 uCi)  single  injection  plasma  was  following  of 1-alanine-O- **C  i n t o the l e f t j u g u l a r  collected  from  the  right  was  a d m i n i s t e r e d as a  v e i n and jugular  time i n t e r v a l s :  Sample no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17  C o l l e c t i o n Time  (min.)  CONTBOL (Before I n f u s i o n ) 3.0 3.5 4.0 4.5 5.0 10.0 15.0 20.0 30.0 45.0 60.0 75.0 90.0 105.0 120.0 135.0  10 ml o f vein  at  blood the  76  In  order t o accomplish the c o l l e c t i o n  period  5  of  of  blood  f o r the  minutes f o l l o w i n g i n f u s i o n , a c o n t i n u o u s  t e c h n i q u e was d e v i s e d . T h i s  method  utilized  t h e two  time  sampling jugular  v e i n s , such t h a t a t t h e e x a c t time when t h e l a b e l l e d a l a n i n e was injected  into  the  left  j u g u l a r , b l o o d was drawn c o n t i n u o u s l y  from the r i g h t j u g u l a r by means o f h e p a r i n i z e d s y r i n g e s . As soon as 10 ml of b l o o d syringe,  a  second  was  withdrawn  following  of  over  this  continued  for 5  time  interval.  The  t h e b l o o d samples were withdrawn a t t h e s p e c i f i e d  i n t e r v a l s d e s c r i b e d above. T h i s approach developed  first  t h e i n f u s i o n o f t h e i s o t o p e and r e p r e s e n t s a  continuous c o l l e c t i o n of blood remainder  the  was i m m e d i a t e l y a f f i x e d t o t h e c a t h e t e r and  t h e n e x t sample was c o l l e c t e d . T h i s procedure minutes  into  t o blood  sampling  was  i n an attempt t o a s s e s s t h e i n i t i a l d e c l i n e o f a l a n i n e  s p e c i f i c r a d i o a c t i v i t y more a c c u r a t e l y s i n c e t h e m a j o r i t y of the decay  curve  i s completed  w i t h i n the f i r s t  10 minutes f o l l o w i n g  injection.  2  •) Chemical_Methods The  glucose  procedure and  for fractionation  of  plasma  components,  a l a n i n e a n a l y s i s have a l l been d e s c r i b e d i n d e t a i l  previously. Another chromatography experiment that  was  performed  to  test  the a c t i v i t y of t h e n e u t r a l and b a s i c f r a c t i o n s was due t o  g l u c o s e and a l a n i n e r e s p e c t i v e l y .  77  The s o l v e n t system f o r t h e t w o - d i r e c t i o n a l  separation  of  a l a n i n e and t h e f o r t h e s i n g l e - d i r e c t i o n a l s e p a r a t i o n o f g l u c o s e were the same a s s t a t e d i n t h e p r e c e d i n g experiment. from  this  study  are a l s o  included  The r e s u l t s  i n F i g u r e 5 and Table 3  (Appendix).  Calculations A two term e x p o n e n t i a l f u n c t i o n was employed t o produce t h e l i n e of b e s t  f i t f o r t h e decay  activity.  detailed  A  determining  curve  description  the m e t a b o l i c  of  alanine  specific  of t h e c a l c u l a t i o n s used i n  parameters  c a l c u l a t i o n s section of the previous  i s provided  in  the  experiment.  E§§Sits_and_Discussion I n Table 5 (Appendix) alanine  and  glucose  i n d i v i d u a l values irreversible  loss  at  of  each  f o r pool and  time  size,  recycling  t r a n s f e r o f a l a n i n e carbon results  t h e s p e c i f i c a c t i v i t y v a l u e s f o r both  the p r e s e n t  to  interval, space,  total  entry  are presented.  glucose  experiment  s p e c i f i c r a d i o a c t i v i t y o f plasma  as w e l l as t h e  i s also  rate,  The p e r c e n t defined.  The  a r e based on t h e change of  alanine  with  time  after  an  i n j e c t i o n of 85 u C i o f 0 - * * C - a l a n i n e . Figure  8  (Appendix)  o u t l i n e s t h e l i n e a r curve f o r glucose  s p e c i f i c a c t i v i t y . The l i n e o f b e s t f i t  estimated  by  t h e two  78  term e x p o n e n t i a l f u n c t i o n i s i l l u s t r a t e d i n F i g u r e 9 The Table  parameters of 5  14.52  (Appendix)  mg C/min  alanine  metabolism  demonstrated  (9.77 mM/hr), a  total  enty  loss  4.65  mM/hr) and a percent c o n v e r s i o n t o glucose o f  much c l o s e r  i n the D i s c u s s i o n  to  t h e published  irreversible  11.3  mM/hr)  of  and Bergman  Experiment  loss  determined  i s comparatively  B.1,  It  of  (1972a). As  total  plasma i s the  by the s i n g l e  injection  7.60 mg C/min  c l o s e t o the f i g u r e s o f 8.3 and  mM/hr f o r t o t a l plasma turnover  Bergman,  When  estimates  method. T h e r e f o r e the value f o r i r r e v e r s i b l e l o s s (5.12  of  5.0055.  turnover as measured by a continuous i n f u s i o n technique as  of  r e c y c l i n g r a t e of 6.91 mg C/min  t o t a l a l a n i n e turnover r e p o r t e d by.Wolff  same  in  the r e s u l t s of the p r e v i o u s experiment, the above  values correspond  stated  rate  irreversible  mg C/min  to  mM/hr),  illustrated  7.60  compared  (5.12  an  a  as  (Appendix).  as  stated  by  Wolff  and  (1972a). i s s i g n i f i c a n t t h a t the r e s u l t s of t h i s experiment are  much c l o s e r to reported values than t h e f i g u r e s of the preceding experiment. T h i s f a c t i n d i c a t e s t h a t the changes made, first  specifically  the continuous  blood  other  words  the computer  r e l i a b l e s i n c e t h e r e were more initial  rapid  points,  accuracy  of  this  a n a l y s i s proved  more  especially  during  the  d e c l i n e o f a l a n i n e s p e c i f i c a c t i v i t y , t o compute  the l i n e o f best f i t . Thus the degree o f points  techniques  c o l l e c t i o n over the  5.minutes, improved the e f f i c i e n c y and  approach.. In  in  i n Figure  interpolation  9 (Appendix) i s reduced  experiment a s compared t o the p r e v i o u s one.  o f the  considerably i n this  79  The which  glucogenicity  is  in  determined  close  by  of  alanine  agreement  Wolff  and  was e s t i m a t e d t o be 5.00%,  with  Bergman  the (1972b)  i n f u s i o n approach on sheep. The 5.00%  of  5.4%  1968) and 12.5% i n man As  using a continuous  figure i s also  less  additional  support  to  the  validity  a  three  term  of  specific  injection  radioactivity  in  the  present  curve  of  to  alanine  experiment. S i n c e the  r e s u l t s o u t l i n e d i n T a b l e 5 (Appendix) a r e based on a  two  term  an approach o f t h i s n a t u r e would i n d i c a t e whether i n f a c t ,  the number of e x p o n e n t i a l terms i s determined by the  this  e x p o n e n t i a l f u n c t i o n was a l s o employed  d e t e r m i n e t h e l i n e of b e s t f i t f o r the decay  fit,  than  ( K r e i s b e r g e t a l . , 1972).  m u l t i c o m p a r t m e n t a l approach f o r the a n a l y s i s of s i n g l e data,  as  e s t i m a t e s of between 6 and 8% i n l a c t a t i n g cows (Black  previous §.£  values  the  shape  of  o b s e r v e d s p e c i f i c r a d i o a c t i v i t y decay c u r v e . The r e s u l t s of  a p p l y i n g a t h r e e term f u n c t i o n t o the  decay  which  a  has  already  been  fitted  to  two  p r e s e n t e d i n T a b l e 5 (Appendix). The v a l u e s parameters function shape  of  are  identical  to  those  decay  curve  is  what  of  alanine  term a n a l y s i s a r e for  the  metabolic  of a two term e x p o n e n t i a l  (Table 5 ) , which demonstrates t h a t the  curve  the  data,  or  the  d e t e r m i n e s t h e number of  e x p o n e n t i a l s t h a t can be used. The r e s u l t s alanine  and  of  glucose  the are  paper  chromatography  presented  in  Figure  separation  of  5 and Table 3  (Appendix) r e s p e c t i v e l y . S i m i l a r t o Experiment B.1, the peaks of r a d i o a c t i v i t y of the plasma f r a c t i o n s corresponded t o the s t a n d a r d s f o r a l a n i n e and g l u c o s e .  those  of  80  The  overall  significance  of  t h i s experiment i s t h a t  improvements made i n the p r e s e n t t e c h n i q u e s over previous  experiment  the  method  of  of  the  have y i e l d e d r e s u l t s which c o r r e s p o n d w e l l  t o the e s t i m a t e s determined by a c o n t i n u o u s Thus  those  the  infusion  a s i n g l e i n j e c t i o n of an i s o t o p e  approach. combined  w i t h a m u l t i c o m p a r t m e n t a l a n a l y s i s has improved i t s s t a t u s as means t o s t u d y the k i n e t i c s o f a l a n i n e  metabolism  i n ruminants.  a  81  H a t e r i a l s _ a n d _ Methods A final  1.5  year  old  w e i g h i n g 37 kg  was  used f o r t h i s  s i n g l e i n j e c t i o n experiment. The  the  s u r g e r y performed on the a n i m a l  previous  two  experiments  (P.E.90) were i n s e r t e d addition The  wether  one  left  jugular  collected were  was  The  right  patent  heparinized saline  (100  nutritional  by  used f o r  once  animal  is  and  flushings  left  catheters  j u g u l a r , but  and  a  in  artery.  blood  c a r o t i d . The with  from  was  catheters  solution  of  units/ml) twice d a i l y . regime  was  a t 0800 hours and under  polyvinyl  infusion  jugular  s i m i l a r t h a t of the  e x p e r i m e n t s i n t h a t 2 kg a l f a l f a hay daily;  slightly  a l s o i m p l a n t e d i n t o the r i g h t c a r o t i d  both  maintained  that  i n t o both r i g h t and  c a n n u l a was  from  in  varied  fed  was  f e d t o the sheep  once a t 1300  conditions  (5  first  hour  hours and fast)  two  twice  again  during  the the  experiment.  1  •)  Isptope_ and L-Alanine  left  jugular  10 ml jugular  of  Collection  U-L-**C  obtained  from ICN  v e i n as a s i n g l e i n j e c t i o n of  blood  v e i n and  intervals:  Blood  plasma  samples  was  infused  the  (95  uCi).  were withdrawn from the  right  the r i g h t c a r o t i d a r t e r y  0.095 mCi  into  at the  following  time  82  CAROTID ARTERY Sample no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19  C o l l e c t i o n Time (min) CONTROL CONTROL .67 1.33 1.66 2.25 2.83 3.83 4.83 5.00 10 15 25 35 45 60 75 90 120  JUGULAR VEIN Sample No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 The  C o l l e c t i o n Time (min) CONTROL CONTROL .33 .58 .83 1.95 2.17 2.45 2.75 10 15 25 35 45 60 75 90 120  continuous  blood  sampling  technique  Experiment B.2 was employed i n t h e p r e s e n t  developed  experiment.  in  83  2.)  Chemical_Wethods The procedures  determination  of  f o r f r a c t i o n a t i o n of b l o o d plasma,  and  the  plasma g l u c o s e and a l a n i n e have been o u t l i n e d  i n d e t a i l p r e v i o u s l y . Due  t o the p o s i t i v e r e s u l t s  the paper chromatographic  s e p a r a t i o n o f * C - l a b e l l e d a l a n i n e and  glucose  in  conducted  Experiment  obtained  for  4  B.1  and  B.2,  no  i n t h i s the f i n a l s i n g l e i n j e c t i o n  such  analysis  was  experiment.  Calculations The c a l c u l a t i o n s those  used  by  for  the  the  first  present  two.  experiment  duplicates  However i n s t e a d of a two  term  e x p o n e n t i a l f u n c t i o n , t h e decay c u r v e o f * C - a l a n i n e r e q u i r e d 1  three  term  function  to  produce  a  line  of  best  a  f i t which  i n c o r p o r a t e s a l l of the p o i n t s . Results and,Discussions Table 6  (Appendix)  outlines  the  data  for  glucose  a l a n i n e s p e c i f i c a c t i v i t i e s i n both the j u g u l a r v e i n and artery.  In  metabolism  addition  the  v a l u e s f o r t h e parameters  which i n c l u d e p o o l s i z e ,  irreversible  loss,  recycling  space,  total  carotid  of a l a n i n e  entry  with 1  time  rate,  and the c o n v e r s i o n percentage  a l a n i n e t o g l u c o s e are a l s o found i n t h i s t a b l e . T h e r e f o r e r e s u l t s a r e based on the change  and  of  alanine  specific  of  these  activity  a f t e r a s i n g l e i n t r a v e n o u s i n j e c t i o n of 95 u C i of  U-  *C-alanine. The s p e c i f i c a c t i v i t y c u r v e s of g l u c o s e i n the j u g u l a r v e i n  and c a r o t i d a r t e r y r e s p e c t i v e l y , a r e diagramed i n F i g u r e 10a b  (Appendix). F i g u r e 11a and b (Appendix)  i l l u s t r a t e the  and  fitted  84  line  t o the l o g a r i t h m i c decay d a t a o f a l a n i n e by t h e t h r e e  exponential  function i n the jugular  vein  and  carotid  term  artery  respectively. The  results  o f t h e g l u c o s e and a l a n i n e c o n c e n t r a t i o n f o r  t h e c a r o t i d a r t e r y and j u g u l a r v e i n were compared by  using  the s t u d e n t s  t  1  test  statistically  f o r significance at a l e v e l  P>0.05. The v a l u e s (Appendix)  f o r t h e parameters  show  total  entry  (33.52 mM/hr) and 61.37 mg/min vein  and  described  rates  of  (41.32 mM/hr)  mM/hr)  and  and  carotid  9.33 mg C/min  39.75 mg C/min  (26.77 mM/hr)  52.04 mg C/min  (35.04) mM/hr)  estimates  of  j u g u l a r vein  irreversible  jugular  artery  a r e 10,01 mg C/min  (6.28 mM/hr) r e s p e c t i v e l y , and  relatively  turnover  of  rates  Bergman  experiment  the  jugular  f o r the carotid  loss  amounts t o vein  artery.  f o r the c a r o t i d  indicate  entry  b  The  a r t e r y and  well  to the estimates  of  The that yields  rates.  This  recycling this  values  of  the  process i s occurring t o a high  correspondingly factor  t h e number o f e x p o n e n t i a l  three  total  present  plays  high  values f o r  t h e major  role i n  terms r e q u i r e d t o a s s i g n a  l i n e o f b e s t f i t t o t h e decay c u r v e . By i n s p e c t i n g F i g u r e and  and  between 8 and 11 mM/hr a s r e p o r t e d by W o l f f  (19 72a).  degree and o b v i o u s l y  determining  for  which  (6.28 mM/hr and 6.75 mM/hr) a r e e x t r e m e l y c l o s e and  both correspond  total  6  49.78 mg C/min  f o r the  r e c y c l i n g was determined from the d i f f e r e n c e  and  i n Table  c a r o t i d a r t e r y r e s p e c t i v e l y . The r a t e o f i r r e v e r s i b l e  l o s s f o r jugular vein (6.75  as  distinct  slopes  may  11  a  be noted. An i n i t i a l r a p i d  85  d e c l i n e , f o l l o w e d by a second more l i n e a r phase and a  elongated  curvilinear  function,  with  s e c t i o n . Thus by v i s u a l i n s p e c t i o n of  the l o g a r i t h m i c decay c u r v e exponential  ending  it  which  is  obvious  was  used  that  in  a  the  two  two  term  previous  e x p e r i m e n t s , i s not adequate t o account f o r the t h i r d component. T h e r e f o r e a t h r e e term f i t i s n e c e s a r y i n o r d e r t o the  large  amount  of  r e c y c l i n g which o c c u r r e d .  account  An i n t e r e s t i n g  p o i n t t o note i s t h a t the v a l u e s f o r i r r e v e r s i b l e l o s s influenced  by  the  high  degree  of  is  are  not  Although  the  parameters,  it  recycling.  c a l c u l a t i o n s are designed t o s e p a r a t e these two  for  e n c o u r a g i n g t o note t h a t the system i s f u n c t i o n i n g  according  t o the p a t t e r n s e t . The  glucose  concentration  in  the  carotid  artery  is  s i g n i f i c a n t l y h i g h e r t h a n t h a t i n the j u g u l a r v e i n (P<0.05). corresponding  values  for  alanine concentration  i n these  The  blood  v e s s e l s proved t o be not s i g n i f i c a n t l y d i f f e r e n t . Thus from t h i s statistical utilized  by  analysis  one  can  The the  of blood f l o w was  per c e n t c o n v e r s i o n  jugular  that  the b r a i n t i s s u e . No e s t i m a t e  s i n c e t h e p r e s e n t experiment was no e s t i m a t e  state  vein  glucose  (Wolff and  being  of q u a n t i t y i s v a l i d  performed on one  sheep o n l y  and  made. of a l a n i n e carbon  to  glucose  for  (6.78%) and c a r o t i d a r t e r y (6.74%) agree w e l l  w i t h each o t h e r as w e l l as w i t h t h e p u b l i s h e d sheep  is  Bergman 1972a), and  v a l u e s o f 5.4%  6 t o 8% f o r l a c t a t i n g  for cows  (Black et a l . , 1968). The sampling  d i f f e r e n c e s between the c a r o t i d a r t e r y and in  terms of i r r e v e r s i b l e l o s s and  jugular vein  the g l u c o g e n i c  role  86  of a l a n i n e are collection  insignificant.  appears  determination  of  parameters.  to  total  However  be  Therefore of  body  the  the  minimal or  place  importance  total  total  entry  rate  blood  for  plasma  and  the  metabolic  v a r i a t i o n between a r t e r i a l and  plasma becomes more s i g n i f i c a n t when c o n s i d e r i n g the  of  venous  such v a l u e s  the r a t e of r e c y c l i n g which  as  deviate  depending upon the v e s s e l from which b l o o d i s c o l l e c t e d . Such result  has  been  1972a&b) and estimates  demonstrated  i s also evident  before  and  (Wolff and  Bergman  i n the p r e s e n t experiment w i t h  of t o t a l e n t r y r a t e  (61.3  mg C/hr)  and  (39.74 mg C/min)  (49.78 mg C/min)  determined  in  the  and  jugular  t h e s e blood v e s s e l s  ( F i g u r e 11 a and  b  r  vein.  This for  i n s p e c i f i c r a d i o a c t i v i t y of  a r t e r i a l plasma l e v e l s o f f and it  rapidly  as  does  in  1 4  rate is each  A p p e n d i x ) . By v i s u a l  i n s p e c t i o n the t h i r d component or s e c t i o n of the c u r v e decline  larger  recycling  r e a s o n a b l e by e x a m i n a t i o n of the a l a n i n e decay c u r v e s of  the  r e c y c l i n g rate  (52.04 mg C/min) of the c a r o t i d a r t e r y b e i n g c o n s i d e r a b l y t h a n the t o t a l enty r a t e  a  for  the  C - a l a n i n e i n the c a r o t i d  does not f a l l  to the a b s c i s s a  as  the j u g u l a r v e i n . By the shape of  the  decay c u r v e s i t appears t h a t r e c y c l i n g i s more pronounced i n the carotid  artery  postulation parameter third  is  plasma given  than  the  support  jugular  from  (A), which r e p r e s e n t s  the  vein  plasma.  f a c t t h a t the l i n e a r  the z e r o - t i m e component, f o r  term i s l a r g e r i n the c a r o t i d a r t e r y than i n  vein.  So  the  vein  the  the  jugular  l e v e l of s p e c i f i c r a d i o a c t i v i t y r e m a i n i n g i n the  blood of t h e c a r o t i d a r t e r y i s more e x t e n s i v e jugular  This  which  implies  a  higher  Although i t i s d i f f i c u l t t o speculate  than t h a t  degree  of  in  the  recycling.  upon the e x a c t reason  why  87  recycling  is  more  prominent  i n the c a r o t i d a r t e r y , a p o s s i b l e  e x p l a n a t i o n a r i s e s from c o n s i d e r a t i o n of the anatomy o f t h e a r e a i n question. Blood samples are withdrawn from jugular  vein  simultaneously  and  the  by  p l a c e the t r a c e r dose of * C - a l a n i n e traversed  through  labelled  virtually  bas  metabolize  this  amino  utilization  are the l i v e r and the k i d n e y s  been  However  of  re-entry  been  taken  a c i d . , Two  up  by  the  mixed  and  tissues  for  the  incorporated  into  the  which  purpose  glucose  alanine  in  synthesized  de  novo  the  glucose  from  the  1970)  muscle  transamination of glucose derived pyruvate. Blood  tissues carrying  process  by the  l i v e r i s t r a n s p o r t e d t o the muscle 1  this  the  where  t i s s u e s where t h e c o n v e r s i o n t o * C - l a b e l l e d a l a n i n e o c c u r s . time  of  pool.  t h e l a b e l l e d carbon can occur through ( F e l i g et a l . ,  the  the  p o t e n t i a l s o u r c e s of a l a n i n e  established "glucose-alanine cycle"  labelled  takes  Thus t h e r a d i o a c t i v e t r a c e r has l e f t the amino  a c i d p o o l and has  is  and  t h e e n t i r e c i r c u l a t o r y system. T h e r e f o r e  alanine  gluconeogenesis.  artery  the time r e c y c l i n g  has  l  carotid  becomes  By  pronounced b l o o d e n t e r i n g the  c a r o t i d a r t e r y a f t e r m i x i n g i n t h e h e a r t i s t h e best e s t i m a t e of t h e r e c y c l i n g e f f e c t s i n c e i t has y e t t o p e r f u s e any  additional  t i s s u e s . However the j u g u l a r v e i n b l o o d c o l l e c t e d i n the p r e s e n t experiment the  brain  i s i n f l u e n c e d by t h e degree of metabolism tissue.  Although  the  statistical  occuring i n  a n a l y s i s of the  a r t e r i o - v e n o u s a l a n i n e c o n c e n t r a t i o n d i f f e r e n c e s have  shown  s i g n i f i c a n t d i f f e r e n c e a d i l u t i o n e f f e c t o f t h e t r a c e r may work.  That  no  be a t  i s the b r a i n t i s s u e i s i n a dynamic s t a t e where t h e  i n p u t e q u a l i z e s the output i n terms o f a l a n i n e . Thus t h e  tracer  88  may  be  taken  up  or  diluted  by  c o m b i n a t i o n of b o t h . Consequently t h e brain  tissue  may  be  the  addition  of  dilution  nonlabel or a effect  of  the  i n f l u e n c i n g f a c t o r which causes the  r e c y c l i n g v a l u e t o be l a r g e r i n the c a r o t i d a r t e r y than  in  the  jugular vein. This  f i n a l s i n g l e i n j e c t i o n experiment c o n t r i b u t e s f u r t h e r  e v i d e n c e t o t h e use of t h i s parameters  of  alanine.  approach  in  evaluating  metabolic  The s l i g h t m o d i f i c a t i o n s i n t r o d u c e d i n  t h i s experiment such as u s i n g a t h r e e term e x p o n e n t i a l f u n c t i o n , d i d not a f f e c t t h e v a l u e s of conversion  to  glucose  t e c h n i q u e o f employing adjusted  irreversible  observed  a  loss  previously.  multicompartmental  and  per  cent  Therefore  analysis  the  can  be  t o any changes o c c u r r i n g w i t h i n the a n i m a l which  might  i n f l u e n c e the r e s u l t s . Consequently t h e t e c h n i q u e employed  here  and i n t h e p r e v i o u s two e x p e r i m e n t s i s f l e x i b l e .  The  metabolic  parameters  o f a l a n i n e as determined i n the  above t h r e e e x p e r i m e n t s a r e summarized i n The  results  transfer  for  the  rate  of  the  irreversible  7  (Appendix).  l o s s and p e r c e n t  ( g l u c o g e n i c i t y ) o f a l a n i n e conform r e a s o n a b l y w e l l w i t h  t h e p u b l i s h e d v a l u e s as mentioned for  Table  three  studies  v a l u e s as compared experiments  to  (Wolff  is the  and  a  p r e v i o u s l y . The o v e r a l l  trend  s l i g h t underestimation of these  results Bergman,  from  continuous  1972a).  i n t e r e s t i n g s i n c e the major c o m p l a i n t o f  the  This single  infusion  finding  is  injection  89  technique  is  i t s proposed o v e r e s t i m a t i o n of t u r n o v e r r a t e s o f  the m e t a b o l i t e under i n v e s t i g a t i o n  (White e t a l . ,  The m u l t i - e x p o n e n t i a l approach i n best  f i t to  the  decay  curve  1969).  determining  a  line  of  of a l a n i n e a l s o c a l c u l a t e d  the  parameters o f a l a n i n e p o o l s i z e and space. The former  represents  the q u a n t i t y of body a l a n i n e w i t h which t h e i n j e c t e d mixes and the l a t t e r i m p l i e s the volume o f f l u i d the  alanine  pool  and  comparable  cent for  body it  i n d i v i d u a l v a r i a t i o n i n body weight. article  presents  of  body  through  weight  is  incorporates  Leng  (1970)  weight)  glucose  t h e sheep's  in  space  food  intake,  f e e d i n g regime of the sheep. On the average t h e h i g h e r of  glucose  most  a  review  v a l u e s of (as  per  ranged from 18 t o 35 per c e n t depending  upon the t e c h n i q u e used as w e l l as t h e  values  which  the  a summary o f t h e g l u c o s e metabolism  nonpregnant and n o n l a c t a t i n g sheep. The cent  C-alanine  i s d i s t r i b u t e d . Of the two measurements, the  a l a n i n e space i n terms o f per informative  1 4  space  were determined  diet  and  estimated  through the use o f a  s i n g l e i n j e c t i o n t e c h n i q u e . However none o f the workers employed a multicompartmental  approach to a n a l y z e the decay c u r v e of  g l u c o s e . A l t h o u g h t h e s e r e s u l t s do not bear d i r e c t r e l e v a n c e  1*Cto  the p r e s e n t work which i n v e s t i g a t e s t h e s e parameters of a l a n i n e , they  give  some i n s i g h t i n t o the o v e r a l l p o o l s i z e and space of  g l u c o s e w i t h i n sheep. T h i s significance experiments  of  knowledge  helps  the v a l u e s determined  s i n c e the reported  work  on  in  assessing  the  i n the p r e s e n t s e r i e s of alanine  is  extremely  sparce. The  figures  for  alanine  space as a per cent o f t h e body  90  weight range from 13.27% f o r t h e f i r s t experiment down t o 3.71% and 4.44% f o r t h e second and t h i r d e x p e r i m e n t s r e s p e c t i v e l y , a l l of  these  v a l u e s a r e determined on j u g u l a r o r p e r i p h e r a l venous  blood which i s the o n l y t r u e e s t i m a t e o f size  or  space.  the  total  body  pool  Thus t h e a l a n i n e space o f 10.88% as determined  from the c a r o t i d a r t e r y plasma i n t h e t h i r d experiment i s not valid  estimate  of  a  t h i s parameter i n terms o f t h e e n t i r e body.  The v a l u e f o r t h e f i r s t experiment i s h i g h r e l a t i v e t o those f o r the  second and t h i r d  because  of  the  lack  experiment of  may  blood  be  samples  minutes post i n j e c t i o n . As mentioned depends  on  frequent  sampling  explained  of  i n t h e f i r s t 5 t o 10  previously blood  adequately  this  approach  f o l l o w i n g the s i n g l e  i n f u s i o n . T h e r e f o r e t h e v a l u e s of 3.71 and 4.44% r e f l e c t  closely  the  dietary  a c t u a l a l a n i n e pool space,  conditions.  The  i n sheep  under  these  j u s t i f i c a t i o n o f t h i s statement stems from the  f a c t t h a t t h e a l a n i n e p o o l i s r e g a r d e d a s one of compartments the  which i s connected t o t h e l a r g e g l u c o s e p o o l . From  v a l u e s o f t h e p e r c e n t c o n t r i b u t i o n of  synthesis  (6-8%)  i n nonpregnant,  alanine  nonlactating,  sheep i t appears t h a t t h e r e l a t i o n s h i p (mean o f 4.07%) t o g l u c o s e p o o l space to  the p r e c u r s o r  of  to  glucose  steady s t a t e  alanine  pool  space  (range o f 18-35%) i s c l o s e  what would be e x p e c t e d . The  multicompartmental  c u r v e s o f i s o t o p e s was metabolism.  Besides  first the  approach used  numerous  t o t h e a n a l y s i s o f decay  i n the  study  parameters  of  isolated,  method a s c e r t a i n e d the e x i s t e n c e o f t h r e e compartments; body  glucose  a  this large  p o o l o f g l u c o s e which i s i n t e r c o n n e c t e d w i t h two p r e c u r s o r  p o o l s (Leng 1970) . The  proposed  model  (Figure  18,  Appendix)  91  d e p i c t s t h e r e l a t i o n s h i p s of the g l u c o s e p o o l s . The r e s u l t s the  preceding  s e r i e s o f experiments  i n d i c a t e a two compartment  and p o s s i b l y a t h r e e compartment model f o r a l a n i n e It  is  from  metabolism.  not the purpose o f t h e p r e s e n t s t u d y t o s p e c u l a t e on the  e x a c t number of compartments w i t h r e f e r e n c e t o a l a n i n e ,  nor  to  p r e d i c t what t h e s e c o n s i s t o f . Answers t o these q u e s t i o n s can be formulated  through  more  e x t e n s i v e work w i t h t h i s t e c h n i q u e i n  the f u t u r e . The primary o b j e c t i v e o f the p r e s e n t s e r i e s o f  experiments  was t o study t h e e f f e c t i v e n e s s of t h e s i n g l e i n j e c t i o n coupled decay  with  a  curve,  parameters  multicompartmental  as  of  a  means  alanine  of  analysis  quantitatively  metabolism  above  discussions  indicate  of the r e s u l t i n g assessing  the  i n nonpregnant, n o n l a c t a t i n g  sheep. The r e s u l t s presented i n T a b l e 7 the  technique  that  (Appendix) this  along  method  has  with the  p o t e n t i a l t o be used e x t e n s i v e l y f o r i n _ y i y o m e t a b o l i c s t u d i e s .  92  III  CONTINUOUS_INF0SIO ISlSOfiT_A_PRIMING_DOSE  Introduction The c o n t i n u o u s i n f u s i o n o f a r a d i o a c t i v e l y l a b e l l e d has  been  widely  used f o r metabolism  s t u d i e s i n a n i m a l s . There  appears t o be two s c h o o l s o f thought g o v e r n i n g t h e use technique,  (Steele, et a l . ,  et  a l . , 1969).  intensively (Bergman, Leng  i n the  The  study  1956  and  Steele  (Leng  et  a l . , 1967  l a t t e r t e c h n i g u e has been employed of  glucose  metabolism  in  (1970)  has  indicated the  that  due  glucose  to  pool  the  sheep  extensive  and  peripheral  s u b s t r a t e p o o l s i n sheep, the use o f a primed i n f u s i o n the  and  1973).  r e c y c l i n g o c c u r r i n g between  for  this  and t h e second c o m p r i s e s t h e use o f a c o n t i n u o u s i n f u s i o n  of l a b e l w i t h o u t a p r i m i n g i n j e c t i o n White  of  t h e f i r s t i n v o l v e s a c o n t i n u o u s i n f u s i o n preceded by  a p r i m i n g dose of t h e t r a c e r 1964)  tracer  determination  of  glucose  technique  p o o l s i z e i s u n r e l i a b l e on  t h e o r e t i c a l grounds.  The l a r g e amount of r e c y c l i n g o f a l a n i n e as  determined  single  indicate  by that  the a  injection  primed-infusion  experiment  approach  e f f i c i e n t method t o measure these parameters The c o n t i n u o u s i n f u s i o n t e c h n i q u e (1967  and White e t a l . ,  a  simple  would  not  be  an  of a l a n i n e . by  Leng  et  al.,  (1969) has a d i s t i n c t advantage i n t h a t  the c a l c u l a t i o n of i r r e v e r s i b l e l o s s on  used  previously,  mathematical  (total entry  treatment.  The  rate)  infusion  relies rate  of  93  radioactivity  d i v i d e d by t h e  radioactivity  of  the  mean  plateau  metabolite,  which  between 3 and 4 hours f o r g l u c o s e , g i v e s  level  of  specific  i s normally a t t a i n e d an  estimate  of  this  parameter. T h i s i s indeed a s i m p l i f i c a t i o n o f t h e a c t u a l i s o t o p e dilution  curve  which  i s described  by the f o l l o w i n g e q u a t i o n  ( S t e e l e e t a l . , 1956; S t e e l e , 1964):  Where F i s the i n f u s i o n r a t e (nCi/min) , Q i s t h e p o o l s i z e , is  the  fractional  and  SR  i s the  (nC/mg C ) .  As  1  zero-time i n t e r c e p t , - m c i s the r a t e constant plasma  time  specific  approaches  radioactivity  infinity  during  infusion of i s o t o p i c a l l y labelled materials a radioactivity  A^  at a  plateau  time  t  continuous specific  i s o b t a i n e d which i s d e s c r i b e d a s ;  SR i=l  Therefore  i f the s p e c i f i c r a d i o a c t i v i t y o f t h e m e t a b o l i t e a t t h e  plateau  level  irreversible loss  (as d e s c r i b e d then  above)  i s used  to c a l c u l a t e  94  i r r e v e r s i b l e l o s s = i n f u s i o n r a t e (nci/mjn) i>R (nCi/mg C) Though the c o n t i n u o u s i n f u s i o n o f l a b e l l e d t r a c e r has used  extensively  in  the  measurement  s u r r o u n d i n g g l u c o s e metabolism, this  the  i t i s relatively  investigated  the  (Wolff e t a l . ,  turnover  of  c o n t r i b u t i o n t o gluconeogenesis.  recently  1972a,b,c).  plasma One  amino  of the  above s t u d i e s was t o a s s e s s t h e metabolism portal-drained  viscera,  liver  and  These acids  continuous  hepatic  Blood  of  the  peripheral  t i s s u e s of  consisted  of  portal  vein,  and  s i m u l t a n e o u s l y . The c a l c u l a t i o n s d e v i s e d by  each  a  f l o w i n both the p o r t a l and h e p a t i c v e i n s  researchers f o r determining  later  their  1  determined  by  and  i n t r a v e n o u s i n f u s i o n of a * C - l a b e l l e d amino a c i d  vein.  turnover  in  of plasma amino a c i d s  and b l o o d was sampled from the a o r t a , the  was  that  workers  objectives  sheep. I n o r d e r to a c c o m p l i s h t h i s each experiment a  parameters  approach has been used to measure amino a c i d metabolism  sheep f e d a l f a l f a hay  by  of  been  tissue  the  rates  of  plasma  amino  these acid  s h a l l be o u t l i n e d and d i s c u s s e d i n a  section. This  work  continuous  is  based  infusion  upon  the  assumption  that  when  of l a b e l l e d amino a c i d i s g i v e n , w h i l e t h e  a n i m a l i s i n a s t e a d y s t a t e , the plasma and t i s s u e p o o l s plateau  s p e c i f i c a c t i v i t i e s . The  t h e r e f o r e depends upon t h e r a t e unlabelled  precursors  v a l u e of the s p e c i f i c of  renewal  or  attain activity  turnover  from  (Gan and J e f f a y , 1967). These p r e c u r s o r s  can be amino a c i d s absorbed  from  compounds  metabolism,  of  a  intermediary  the  gastrointestinal  tract,  or t h e p r o t e i n s of body  t i s s u e s . With t h i s i n mind Wolff and Bergman  (1972a)  estimated  95  the  turnover  rates  and s e r i n e i n t h e tissues  as  f o r a l a n i n e , a s p a r t a t e , glutamate, portal-drained  viscera,  liver,  glycine  peripheral  w e l l as t h e t o t a l plasma t u r n o v e r f o r t h e body p o o l  of each amino a c i d . The  p r e s e n t sequence o f e x p e r i m e n t s u t i l i z e s t h e c o n t i n u o u s  infusion  technique  as  described  above  to  evaluate  its  e f f e c t i v e n e s s f o r t h e s t u d y o f a l a n i n e metabolism and t o compare it  w i t h t h e two p r e v i o u s l y d e s c r i b e d methods. The parameters o f  a l a n i n e c e n t e r around i t s r a t e contribution  to  glucose  of  irreversible  an  estimation  and i t s  s y n t h e s i s i n sheep under steady  c o n d i t i o n s . By u t i l i z i n g t h e b l o o d experiment  loss  of  flow  the  data  portal  from  the  metabolism  state first  o f both  a l a n i n e and g l u c o s e has been be made.  Materials_and_Methods  1.)  Surgical_Procedures The  experiment was conducted  weighing  45 kg.  Surgery  on  a  3.5  year  o l d wether  was performed on t h e a n i m a l one week  p r i o r t o t h e commencement of t h e experiment. The p r e p a r a t i o n the  sheep  of  f o r s u r g e r y , t h e method o f i m p l a n t a t i o n o f c a t h e t e r s  as w e l l as the p o s t - o p e r a t i v e c a r e a r e d e s c r i b e d i n d e t a i l under the  surgical  involving  procedures  blood  flow  section  studies.  of  the  first  experiment  C a t h e t e r s were p l a c e d i n t o t h e  96  portal vein,  mesenteric  vein  and  carotid  artery,  m a i n t a i n e d open by i n f u s i n g h e p a r i n i z e d s a l i n e  and  (100 u n i t s / m l ) .  U n f o r t u n a t e l y the day b e f o r e t h e experiment t h e p o r t a l catheter Thus  the  the  mesenteric  vein  and  carotid  artery  cannula  patent.„The experiment was c a r r i e d out as planned w i t h  continuous  infusion  of  labelled  1 4  C-alanine  into  m e s e n t e r i c v e i n and blood c o l l e c t i o n from t h e c a r o t i d The  vein  was caught on an unknown o b j e c t and had t o be removed.  only  remained  were  nutritional  regime c o n s i s t e d  the  artery.  o f a maintenance d i e t of  200 gm dehydrated g r a s s p e l l e t s f e d e v e r y two hours b e g i n n i n g a t 0900 hours and e n d i n g a t 1700 h o u r s . The i n f u s i o n began a t hours  and  1100  the a n i m a l was l a s t f e d t h e p r e v i o u s e v e n i n g a t 1700  hours t h u s t h e r e s u l t s a r e based on an o v e r n i g h t f a s t  (18 h r ) .  2•) l§ot0£e_and_Blood_Collection 200 u C i o f L - U - * C - a l a n i n e o b t a i n e d from ICN conforming l  the  s p e c i f i c a t i o n s described previously  s t e r i l e s a l i n e and i n f u s e d  i n 400 ml  i n t o t h e m e s e n t e r i c v e i n a t a r a t e of  0.475 u C i / m i n . B l o o d (10 ml) a r t e r y a t the f o l l o w i n g  was d i s s o l v e d  to  was  collected  from  time i n t e r v a l s :  Sample No.  C o l l e c t i o n Time (min)  1 2 3 4 5 6 7 8 9  CONTROL (Before I n f u s i o n ) 5 15 30 60 90 120 150 180  the  carotid  97  10 11 12 13 14 15 •)  210 240 270 300 330 360  Qk®iisii_S§ibods  3  The  s e p a r a t i o n o f plasma components, as w e l l as the plasma  a n a l y s i s f o r a l a n i n e and g l u c o s e c o n c e n t r a t i o n s were t h e same as o u t l i n e d i n the previous  experiments.  Calculations Due t o t h e l o s s o f t h e p o r t a l v e i n c a t h e t e r , the p r o d u c t i o n and u t i l i z a t i o n r a t e s o f drained  alanine  and  glucose  by  the portal  v i s c e r a c o u l d not be determined. T h e r e f o r e  t h e apparent  t o t a l plasma t u r n o v e r o f a l a n i n e was t h e o n l y f i g u r e c a l c u l a t e d . The  apparent t u r n o v e r o f  similar  this  amino  manner t o t h a t f o r g l u c o s e  acid  i s defined,  in a  (Bergman, 1963), as t h e r a t e  a t which t h e a r t e r i a l c o n c e n t r a t i o n o f »*C-alanine i s d i l u t e d by u n l a b e l l e d a l a n i n e and t h u s :  a p p a r e n t t u r n o v e r , mmoles/hr Where I is  =  i s the i n f u s i o n r a t e (uCi/hr) o f * * C - a l a n i n e  the specific activity  rate  to  account  modified  f o r the continual  u n l a b e l l e d amino a c i d s from t h e g u t , some immediately  removes.  and  SA  (uCi/mmole) of a l a n i n e i n t h e a r t e r i a l  plasma. Wolff and Bergman (1972a) have turnover  1_ SA  Therefore  of  this  apparent  absorption  which  the  of  liver  a correction factor i s required  t o f i n d t h e t r u e t u r n o v e r o f amino a c i d . The term  was  obtained  98  by m u l t i p l y i n g the p r o d u c t i o n o f t h e amino a c i d from t h e p o r t a l drained  viscera  by  t h e f r a c t i o n a l uptake of i^C-amino a c i d by  the l i v e r . Thus the c o r r e c t e d t u r n o v e r i s c a l c u l a t e d as f o l l o w s :  Corrected Turnover  If  the  =  Apparent Turnover  arterial  corrected  +  Portal Production  concentrations  turnover  provides  x  F r a c t i o n a l Uptake amino a c i d by L i v e r  remain  an  constant,  then  the  e s t i m a t e o f plasma amino a c i d  u t i l i z a t i o n by a l l t i s s u e s of the body and i t f o l l o w s t h a t :  P e r i p h e r a l U t i l i z a t i o n = Corrected - (Portal • Hepatic Turnover Utilization)  P e r i p h e r a l P r o d u c t i o n = P e r i p h e r a l + net P e r i p h e r a l P r o d u c t i o n Utilization  A value f o r corrected present  experiment  turnover  i s not  and t h e r e f o r e o n l y t h e apparent  a l a n i n e can be a s s e s s e d . Table 8 (Appendix) for  this  continuous  infusion  p i c t u r e d i n F i g u r e 12 addition  to  alanine  activity  of  and  the  the  data  specific  glucose  are  (Appendix). the  turnover  rates  p r o d u c t i o n from a l a n i n e was determined specific  and  i n the  t u r n o v e r of  presents  experiment  a c t i v i t y v e r s u s time c u r v e s f o r both  In  possible  a  percent  by d i v i d i n g  the  glucose plateau  g l u c o s e i n the c a r o t i d a r t e r y by t h a t of  a l a n i n e i n the same v e s s e l . The s t a t i s t i c a l  treatment  of  the  date  consisted  of  a  99  c o r r e l a t i o n c o e f f i c i e n t a n a l y s i s of both the g l u c o s e and a l a n i n e plateau  levels  to  detect  i f the x  and y v a r i a b l e s a r e n o t  c o r r e l a t e d over t h i s t i m e i n t e r v a l .  I§§2^f J>_and^Discussion As i l l u s t r a t e d i n F i g u r e 12, (Appendix), alanine  the glucose  and  s p e c i f i c a c t i v i t y c u r v e s reached p l a t e a u l e v e l s between  5 and 6 hours a f t e r t h e s t a r t of the experiment. The s t a t i s t i c a l a n a l y s i s proved t h a t t h e r e i s no s i g n i f i c a n t s l o p e i n t h i s period  f o r either  alanine  or  g l u c o s e . The apparent t u r n o v e r  v a l u e f o r a l a n i n e o f 7.23 mH/hr  (10.75 mg/min),  equated  as  to i r r e v e r s i b l e  i n j e c t i o n experiments r e p o r t e d by  Wolff  loss  time  determined  which by  can be  the  single  (White e t a l . , 1969) agrees w e l l w i t h t h a t  and  Bergman  (1972b)  of  between  8.3  and  11.3 mH/hr f o r two sheep weighing 45 and 59 kg r e s p e c t i v e l y . The former  value  i s a much c l o s e r comparison t o t h e p r e s e n t r e s u l t  s i n c e t h e sheep used by weight  both  experiments  are of  equal  body  (45 kg) . The per c e n t o f g l u c o s e p r o d u c t i o n from a l a n i n e  i n t h e p r e s e n t experiment was a s s e s s e d t o comparable  to  be  5.07%,  which  is  t h e v a l u e o f 5.4% r e p o r t e d by W o l f f and Bergman  (1972b) . The major s i g n i f i c a n c e o f t h e p r e s e n t the  continuous  infusion  o f *'C- l a b e l l e d 1  experiment alanine  p r i m i n g dose y i e l d e d a s t a t i s t i c a l l y s i g n i f i c a n t of  specific  activity  for  both  alanine  i s that without  plateau and  a  level  glucose  at  100  a p p r o x i m a t e l y f i v e hours a f t e r t h e s t a r t of t h e Additional  calculations  such as t u r n o v e r time and p o o l s i z e  were not p o s s i b l e i n the p r e s e n t experiment was  terminated  during  since  the  infusion  the p l a t e a u i n t e r v a l and thus the  l i f e o f * C - a l a n i n e c o u l d not be l  infusion.  determined.  half-  101  l2£eriffient_C.2.„  Materials_and_Methods_ T h i s f i n a l c o n t i n u o u s i n f u s i o n experiment was conducted a  2  on  year o l d wether w e i g h i n g 42 kg. Surgery was performed i n a  s i m i l a r f a s h i o n as i n t h e p r e c e d i n g catheters artery  experiment  with  polyvinyl  (P.E.90) b e i n g i m p l a n t e d i n t o t h e p o r t a l v e i n , c a r o t i d  and  m e s e n t e r i c v e i n . The c a t h e t e r s were m a i n t a i n e d i n a  f u n c t i o n a l s t a t e by f r e q u e n t heparinized  saline  flushings  with  the s o l u t i o n  of  (100 u n i t s / m l ) . T h i s time a l l t h e c a t h e t e r s  remained p a t e n t d u r i n g  the  post-operative  recovery  phase  (5  days) and d u r i n g t h e experiment. The a n i m a l was p l a c e d on a roughage d i e t s i m i l a r t o t h e one employed  i n the  p r e v i o u s experiment. The sheep was not f e d i n  t h e morning o f t h e e x p e r i m e n t , thus t h e r e s u l t s a r e a l s o on 1045  an  overnight  fast  based  (17 hr) w i t h t h e i n f u s i o n commencing a t  hours.  1.) I s o t o p e _ a n d _ B l o o d _ C o l l e c t i o n L-O *C l  infused  alanine  into  (.44 u C i / m i n ) . simultaneously  the  at  concentration of  mesenteric  Blood from  a  plasma  vein  at  a  samples  0.40 uCi/ml  rate were  was  o f 1.1 ml/min collected  t h e c a r o t i d a r t e r y and p o r t a l v e i n a t t h e  f o l l o w i n g time i n t e r v a l s :  102  Sample No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32  2  -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.A. -P.V. -C.V.  C o l l e c t i o n Time (min) CONTBOL-1 CONTROL-1 CONTROL-2 CONTROL-2 30 30 60 60 90 90 120 120 150 150 180 180 210 210 270 270 300 300 330 330 345 345 360 360 INFUSION STOPPED 375 375 390 390  • ) Chemical_Mathods Plasma s p e c i f i c a c t i v i t i e s  determined  from t h e t e c h n i q u e s  of  alanine  and  glucose  d e s c r i b e d e a r l i e r which i n v o l v e d  s e p a r a t i o n o f plasma c o n s t i t u e n t s as w e l l as a s s a y s f o r and g l u c o s e  concentrations.  were  alanine  103  Calculations, The  techniques  described  employed t o measure apparent again  i n the p r e v i o u s experiment were  turnover  rate  alanine.  t h e c o r r e c t e d t u r n o v e r r a t e of a l a n i n e cannot be  s i n c e t h e h e p a t i c v e i n b l o o d f l o w as w e l l as of  of  this  amino  acid  in  the  t h e h e p a t i c v e i n were not determined.  by t h e p o r t a l - d r a i n e d v i s c e r a was assessed  where  P  py/  p v  and  p v /  -C^ )  represents  metabolite, F vein,  (C  p  and  using the e g u a t i o n :  portal  net  production  rates  (ml/min) i n t h e  v e s s e l s r e s p e c t i v e l y . The  for  of  vein  of  the  portal  are the c o n c e n t r a t i o n s of the metabolite  i n the p o r t a l v e i n and a r t e r i a l portal  glucose  (Katz and Bergman 1968)  i s the whole blood f l o w  C y and C^  assessed  concentration  However a v a l u e f o r t h e metabolism of both a l a n i n e  P = F  Once  blood  flow  2010 ml/min  value  as e s t i m a t e d i n  Experiment I on b l o o d f l o w , was used f o r the c a l c u l a t i o n s . The g l u c o g e n i c i t y o f a l a n i n e was determined manner a s i n t h e p r e c e d i n g A  statistical  coefficient)  and  continuous  in  concentration  c a r o t i d a r t e r y and p o r t a l v e i n  similar  i n f u s i o n experiment.  a n a l y s i s of t h e p l a t e a u l e v e l s the  a  differences  (correlation between  the  ( s t u d e n t s ' t t e s t ) was conducted.  104  Sesults_and_Discussion_ The  data  as  well  as t h e r e s u l t s o f t h e c a l c u l a t i o n s a r e  p r e s e n t e d i n Table 9 (Appendix). F i g u r e 13, (appendix) the  specific  activity  curves  of alanine  diagrams  and g l u c o s e i n t h e  c a r o t i d a r t e r y and p o r t a l v e i n r e s p e c t i v e l y . The p l a t e a u l e v e l s f o r both a l a n i n e activities interval  (Figure  13, Appendix)  as i n the preceding  infusion).  and  were  glucose  reached  experiment  specific  a t t h e same  (5-6 hours  post  From t h e mean v a l u e f o r a l a n i n e s p e c i f i c a c t i v i t y a t  t h i s l e v e l , t h e apparent  plasma t u r n o v e r r a t e  was  assessed  at  8.5% mM/hr (12.76 mg/min). The f i g u r e s agree e x t r e m e l y w e l l w i t h that  determined  by t h e work o f W o l f f and Bergman (1972b)  which  e s t i m a t e d t h i s r a t e t o be i n t h e range of 8.3 and 11.3 mM/hr reported The  earlier. statistical  o f both a l a n i n e and portal  as  vein  analysis of the concentration differences glucose  proved  between  significant  the c a r o t i d  variations  T h e r e f o r e i t was decided t o i n c o r p o r a t e  artery  i n both  and  cases.  another  arterio-venous  c o n c e n t r a t i o n study i n t o t h e p r e s e n t experiment.  Thus t h e p o r t a l  vein  blood  flow  used t o determine the  portal  value  o f 2010 ml/min from Experiment  t h e net metabolism  drained  viscera.  Employing  above (Katz and Bergman, 1969) a (.190 g/hr/kg )  was  again  o f a l a n i n e and  A.1 was  glucose  by  the e q u a t i o n d e s c r i b e d  net u t i l i z a t i o n  calculated.  The  of  value  glucose derived  p r e s e n t l y conforms r e a s o n a b l y t o t h e p r e v i o u s l y e s t i m a t e d f i g u r e of 0,142 g/hr/kg and  Bergman  and t o t h e range o f v a l u e s determined by  (1969), The e s t i m a t e f o r a l a n i n e metabolism  Katz  by t h e  105  p o r t a l bed demonstrated a net p r o d u c t i o n r a t e o f 2.29 (Wolff,  Bergman  determined result  and W i l l i a m s ,  i n the present  of  1972),  experiment  the Experiment  A.1  which i s c l o s e t o t h a t  (2.02 mM/hr).  dealing  .29 mM/hr  From t h e  w i t h blood flow a n e t  a l a n i n e p r o d u c t i o n r a t e o f 1.49 mM/hr was o b t a i n e d . Both o f t h e e s t i m a t e s d e r i v e d i n t h e p r e s e n t s e r i e s o f experiments 2.02 mM/hr)  concur  and  f l o w f i g u r e was determined  a r e v a l i d assessments s i n c e t h e b l o o d on t h e same sheep as was used i n t h i s  second c o n t i n o u s i n f u s i o n experiment. Experiment  A.1  The b l o o d f l o w v a l u e  conduct t h e experiment percent  estimated  t o be  previously.  This  from  can be employed i n t h e p r e s e n t experiment  both s t u d i e s m a i n t a i n t h e sheep on a s i m i l a r roughage  The  (1.49 and  since  diet  and  f o l l o w i n g an o v e r n i g h t f a s t .  conversion  of  7.20% by  using  the  technique  i s again  well  w i t h i n the p r e v i o u s l y  value  a l a n i n e carbon t o g l u c o s e was described  s t a t e d v a l u e s o f 5.4% (Wolff and Bergman, 1972) and 6-8%  (Black  e t a l . , 1968) The  preceding  figures  a l a n i n e and g l u c o s e metabolism assess the value relation  of  f o r t h e parameters  surrounding  s e r v e a s a guide i n an attempt t o  the continuous  infusion  experiment  t o t h e two p r e v i o u s l y mentioned p r o c e d u r e s ,  f l o w study and t h e s i n g l e i n j e c t i o n  technique.  in  the blood  106  The are  t h r e e e x p e r i m e n t a l t e c h n i q u e s employed  designed  metabolism  to  of  primary  specific  aim  of  effectiveness parameters  measure  of  of  by  plasma  constituents  the  present  each  method  research in  liver  sheep (Black e t a l . , Bergman  in  was  studying  1972b),  1968;  the  study  for  the  animals.  The  assess  the  to the  a l a n i n e and i t s r e l a t i o n s h i p t o  the  this  v a r i o u s parameters p e r t a i n i n g t o the  S i n c e a l a n i n e has been e s t a b l i s h e d as extracted  in  metabolic  gluconeogenesis.  primary  amino  acid  endogenous g l u c o s e p r o d u c t i o n i n  R e i l l y and F o r d , 1971  results  from  such  and  Wolff  and  a comparison s e r v e a  u s e f u l purpose i n ruminant p h y s i o l o g y . The blood  f i r s t method used was the  flow  study  combined  concentration  differences  investigation.  As  with of  previously  traditonal  the  metabolites  reported  their  close  approximation  The to  of  a  measuring the a r t e r i o - v e n o u s  this  a c c u r a t e means of e s t i m a t i n g the metabolism by an organ o r t i s s u e system.  technique  approach  is  an  o f a blood component  results  reported  under  obtained  here  and  values a t t e s t to t h i s  statement. The major drawback t o the use sophisticated  s u r g i c a l procedures  of  this  technique  the  which are mandatory. Thus i t s  e f f e c t i v e n e s s r e l i e s upon a c c u r a t e i m p l a n t a t i o n and of  is  maintenance  c a t h e t e r s i n s p e c i f i c v e i n s and a r t e r i e s . An example o f t h i s  i s i l l u s t r a t e d i n the portal  present  study.  The  metabolism  of  the  d r a i n e d v i s c e r a was e s t i m a t e d through c a t h e t e r i z a t i o n of  107  the c a r o t i d a r t e r y and t h e p o r t a l v e i n . However the a c t i v i t y  of  the  be  liver  in  relation  a s s e s s e d s i n c e no h e p a t i c required this  from  p o l y v i n y l catheter accesible  in  the  hepatic  become obscured due and  does  p r o c e d u r e s are takes  a  and  vein catheter  glucose could  was  i m p l a n t e d , which  Bergman (1968). The  extreme  difficulty  i n t o the h e p a t i c  not  of  v e i n which  is  reason f o r  introducing is  not  a  readily  the abdominal c a v i t y , a l t h o u g h a t t e m p t s were made  t h a t the i n i t i a l  can  the  numerous  successful is  alanine  as i n d i c a t e d by K a t z and  arose  during  to  operations  performed  vein cannulation o b j e c t i v e s of  in  this  project^  remained e l u s i v e . The  such  blood  flow  point  study  may  t o t h e outcome of the s u r g e r y . T h i s approach  prove  effective  conducted  only  when  successfully.  g r e a t d e a l of p r a c t i c e and  may  accurate  Such  surgical  technical  not always be  skill  available  under normal l a b o r a t o r y c o n d i t i o n s , a d i s t i n c t advantage t o t h i s t e c h n i q u e i s the s i m p l e mathematical a n a l y s i s which f o l l o w s . addition  In  the number of assumptions as w e l l as the t o t a l expense  involved  have l i m i t e d e f f e c t s upon the a p p l i c a t i o n of blood  flow  techniques to i n _ v i v o metabolic s t u d i e s . The  second t e c h n i q u e attempted i n t h i s t h e s i s p r o j e c t was  s i n g l e i n j e c t i o n of review  a  **C-labelled  alanine.  h i s t o r y of the v a r i o u s  In  the  presented analysis  and is  procedure, exponential iterative  the the  in  conclusion most  brief,  function minimization  was  efficient consists to  the  that and  of  determine of  literature  approaches used t o a n a l y z e  r e s u l t i n g decay c u r v e of s p e c i f i c r a d i o a c t i v i t y  error  with  time  the was  a multicompartmental  accurate  utilizing a  a  line  method. a  of  2 best  The  or 3 term fit  sums of s q u a r e s f o r  by the  108  f i t t e d function. This  complicated  accomplished  the  through  use  c a l c u l a t i o n s o u t l i n e d by Leng parameters  mathematical  treatment  o f a computer program. From the (1970)  the  following  metabolic  o f a l a n i n e were a s s e s s e d ; p o o l s i z e , space  (% o f body  w e i g h t ) , t o t a l e n t r y r a t e , i r r e v e r s i b l e l o s s and The  was  majority  of  applications  recycling.  using  the  m u l t i e x p o n e n t i a l a n a l y s i s have been i n v o l v i n g g l u c o s e  above  metabolism  i n a n i m a l s , with s p a r s e work conducted on r u m i n a n t s . The  present  study  alanine  investigated  metabolism  the  use  of  this  approach  i n sheep and c o n s e q u e n t l y the r e s u l t s have l i t t l e i n  the l i t e r a t u r e t o compare w i t h . However single this  injection technique  described  experiments is  here.  valid  One  the  results  and  accurate  for  o f the major advantages  the  turnover,  recycling.  rates  loss  and  conditions  parameters  i n d i c a t e d r e c y c l i n g and c o n s e q u e n t l y  As  of  total  the  third  total  turnover  were h i g h , but t h e e s t i m a t e of i r r e v e r s i b l e l o s s  comparable t o the p r e v i o u s two experiments as literature  three  o f t h i s method i s  the  irreversible  of  present evidence suggesting t h a t  i t s a b i l i t y t o s e p a r a t e and i d e n t i f y  experiment  with  well  as  remained to  the  v a l u e s . I n a d d i t i o n t o t h i s , but i n a more p r a c t i c a l  sense, the s i n g l e i n j e c t i o n t e c h n i q u e r e q u i r e s  simple  procedures  vein  which  consist  of  two  jugular  T h e r e f o r e such a s t u d y c o u l d be conducted  surgical  catheters.  i n a l a b o r a t o r y or out  i n the f i e l d . The expense i n v o l v e d i s c e r t a i n l y  less  than  the  c o n t i n u o u s i n f u s i o n of r a d i o a c t i v e i s o t o p e . Previous  workers  i n j e c t i o n approach  have  doubted  the v a l i d i t y of the s i n g l e  due t o the numerous assumptions  which must be  109  made  which  i n c l u d e , r a p i d metabolism  mixing, r e c y c l i n g  These  p o i n t s are not o v e r l o o k e d or i g n o r e d but become accounted  f o r by  use  the  above  well  instantaneous  of a m u l t i - c o m p a r t m e n t a l a n a l y s i s . I n c o n j u n c t i o n w i t h a  possible  mathematical  analyses  drawback which  However once d e r i v e d the apply  as  incomplete  infusion.  the  as  o f the t r a c e r ,  and  is  the  extremely  accompanies  analysis  this  becomes  a  type c f study.  straightforward  to  can be a d j u s t e d t o s u i t the decay c u r v e i n q u e s t i o n .  The t h i r d s e r i e s of experiments r e p o r t e d on of  complex  continuous  initial  priming  approach  was  infusion dose.  not  A  of  consist  l a b e l l e d »*C-alanine without an  priming  considered  previously  dose-continuous  here  due  to  the  infusion theoretical  o b j e c t i o n s t o t h e t e c h n i q u e as p o i n t e d out p r e v i o u s l y . . A p l a t e a u or c o n s t a n t s p e c i f i c a c t i v i t y l e v e l f o r both g l u c o s e and a l a n i n e was a c h i e v e d some 5 hours a f t e r the s t a r t of the i n f u s i o n .  From  t h i s l e v e l the c a l c u l a t i o n s o f t o t a l t u r n o v e r r a t e of a l a n i n e as well  as t h e per c e n t c o n t r i b u t i o n of t h i s amino a c i d t o g l u c o s e  s y n t h e s i s were made w i t h t h e r e s u l t s being comparable cited  in  the  literature.  p r e s e n t experiments  Thus  to  those  the procedure employed i n t h e  y i e l d r e s u l t s which  conform  to  previously  p u b l i s h e d d a t a and c o n s e q u e n t l y t h e s e methods proved a c c u r a t e i n representing continuous i n f u s i o n techniques i n g e n e r a l . Of  the  drawbacks  a f f e c t i n g t h i s type of an approach,  s k i l l e d s u r g i c a l procedures i n f l u e n c e t h e outcome i n fashion  a  the  similar  as i n d i c a t e d f o r blood f l o w s t u d i e s . T h i s i s e s p e c i a l l y  t r u e i f f o l l o w i n g Wolff and Bergmans's (1972a)  modification  of  u s i n g the d i f f e r e n c e i n r a d i o a c t i v i t y o f a substance between two vessels  to  determine  an  estimate  of  tissue  utilization  or  110  production.  An a d d i t i o n a l n e g a t i v e f e a t u r e of t h i s t e c h n i q u e  the expense  required  to  obtain  the  necessary  is  radioactivily  l a b e l l e d compounds. However the major problem i s r e c y c l i n g . In  theory  the continuous  i n f u s i o n of a l a b e l l e d  a l l o w s enough time f o r the t r a c e r t o e q u i l i b r i a t e the  body p o o l s and thus r e c y c l i n g has l i t t l e  i s e s p e c i a l l y t r u e when the r e c y c l i n g of **C fact  that  substrate  there and  originally  is  the  a  dilution  »*c  labelled  is  of  loss  (White e t a l . ,  as  i s due  Consequently  defined  by  all  significance.  converted  e n t r y or t u r n o v e r r a t e has been c o n s i d e r e d irreversible  with  of  This  o n l y to  the  t h i s carbon i n a p o o l of  randomly  tracer.  substrate  a  back  to  the  the v a l u e f o r t o t a l synonymous  with  the  s i n g l e i n j e c t i o n system.  1969). However under c e r t a i n  circumstances  this  r e c y c l i n g becomes more prominent, s p e c i f i c a l l y i n s t a r v a t i o n and other  physiological stress  c o n d i t i o n s such as, k e t o s i s i n the  b o v i n e and pregnancy toxemia i n the o v i n e .  In  terms  of  amino  a c i d metabolism and s p e c i f i c a l l y a l a n i n e i n r u m i n a n t s , r e c y c l i n g of  label  through  amino  a c i d i n t e r c o n v e r s i o n s was  p r o v i d e n e g l i g i b l e e r r o r due available  for  the  to  the  utilization  of  Bergman 1972a). Such assumptions have scrutiny (Felig,  s i n c e the e s t a b l i s h m e n t 1973).  Under  this  and  thus  the  r a t e of i r r e v e r s i b l e overestimation  of  which i n the p r e s e n t  alanine come  of  carbon under  pathways (Wolff  and  considerable  of the " g l u c o s e - a l a n i n e c y c l e "  conditions  pronounced the c o n t i n u o u s  multiplicity  expected t o  where  recycling  i n f u s i o n technique  becomes  i s unable t o d e t e c t  t u r n o v e r r a t e no l o n g e r c o r r e s p o n d s to the loss. the  The  actual  result  of  this  will  t u r n o v e r r a t e of the  investigation i s alanine.  be  an  metabolite  111  Of the t h r e e t e c h n i q u e s project,  the  single  a n a l y s i s appears to have  investigated  injection the  with  greatest  r e s e a r c h i n ruminant p h y s i o l o g y and  during a  present  multi-compartmental  potential  metabolic  the  for  studies.  further  112  GLOSSARY.OF_TERRS  1) S p_ e c i f i c _ A c t i v i t ^  The s p e c i f i c a c t i v i t y of a substance c o n t a i n i n g is  the  often  amount  a labelled  atom  of r a c i o a c t i v i t y per u n i t of s u b s t a n c e . T h i s i s  expressed as the number of r a d i o a c t i v e u n i t s  (microcuries,  counts per -minute, d i s i n t e g r a t i o n s per minute) per m i l l i g r a m  or  mi H i mole of the s u b s t a n c e .  2) Steady  In  a  State  situation  synthesis  where  the  rate  of  entry  of  or t r a n s p o r t e q u a l s the r a t e of e x i t by  transport,  the  concentration  a molecule by breakdown  or  o f t h e m o l e c u l e remains c o n s t a n t  and a steady s t a t e e x i s t s whenever the i n f l u x of  material  does  not b a l a n c e the o u t f l u x .  3) Model  k  model  represents  any  set  of  equations  or f u n c t i o n s  d e s c r i b e t h e b e h a v i o r of a t r a c e r i n the system. The of  a  model  are  the  arbitrary  constants  that  parameters  of the f u n c t i o n or  equations.  4)  Turnover  Turnover r e f e r s t o the p r o c e s s o f renewal o f a s u b s t a n c e i n  the  113  body o r i n a g i v e n t i s s u e .  5) T u r n o v e r _ K a t e  The  Turnover Rate i s t h e r a t e a t which a  over  in a  substance  is  turning  g i v e n compartment or m e t a b o l i c p o o l . The meaning of  t u r n o v e r r a t e i s e x p l i c i t o n l y when a steady s t a t e e x i s t s , is,  that  when t h e r a t e o f s y n t h e s i s and t r a n s p o r t i n t o a compartment  e q u a l s the r a t e o f breakdown and e x i t .  6) Entry__Rate  The  rate  continuous  parameters  of  turnover  infusions  or  single  estimated infections  turnover r a t e , transfer  rate,  utilization  inflow-outflow  flux,  renewal  rate,  i r r e v e r s i b l e loss or disposal  have rate,  rate,  d i s p o s a l . These measurements a r e p r o b a b l y  from  primed  been termed entry  or  or  rate,  irreversible  a l l synonymous  with  (White e t a l . , 1969).  7) Tot al_Entry__Rate  The  total  e n t r y r a t e i s t h e r a t e a t which a m e t a b o l i t e  appears  i n t h e sampled p o o l .  8)  l£reversible_Loss  I r r e v e r s i b l e l o s s i s t h e r a t e a t which a m e t a b o l i t e , not  return  to  t h e sampled  pool  during  the  which  course  does of t h e  1 14  experiment, leaves t h i s pool.  9)  Recycling  R e c y c l i n g i s the r a t e a t which the l a b e l l e d **C plasma  metabolite  pool,  returns  to  the  which has p r e v i o u s l y l e f t the sampled  pool.  10)  Alanine_Pool_Size  A l a n i n e p o o l s i z e i s the q u a n t i t y of injected  11)  l 4  body  alanine  with  which  C - l a b e l l e d a l a n i n e mixes.  Alanine_Sp_ace  Alanine  space  i s the volume o f f l u i d t h r o u g h which t h e a l a n i n e  pool i s d i s t r i b u t e d .  12) T urnover__Time  The t u r n o v e r time o f a s u b s t a n c e i s the time for  the  present  13)  turnover  of  a  quantity  of  that  is  required  substance equal to that  i n t h e compartment.  Precursor  The term p r e c u r s o r r e f e r s t o a another  by  chemical  compound  transformation  or  which  gives  rise  to  by t r a n s p o r t from  one  organ t o a n o t h e r .  116  REFERENCES CITED 1. A n n i s o n , E. F. , K. J . H i l l and D. L e w i s . (1957). S t u d i e s on P o r t a l B l o o d o f Sheep: A b s o r p t i o n of V o l a t i l e F a t t y A c i d s from the Rumen of Sheep. Biochem. J . 66: 592 2. A n n i s o n , E. F., R. A. Leng, D. B. L i n d s a y , and R. R. White. (1963a) . The Metabolism o f A c e t i c A c i d , P r o p i o n i c A c i d and B u t y r i c A c i d i n Sheep. Biochem. J . 88: 248 3. 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S u t t o n , J . D. and J . W. G. N i c h o l s o n (1968). The D i g e s t i o n o f Energy and S t a r c h a l o n g the G a s t r o - l n t e s t i n a l T r a c t o f Sheep. P r o c . N u t r . Soc. 27: 49 107. Topps, J . H., R. N. B. Kay, and E. D. G o d a l l (1968a). D i g e s t i o n of C o n c e n t r a t e and o f Hay D i e t s i n the Stomach and I n t e s t i n e s of Ruminants. 1) Sheep. B r i t . J . o f Nutr. 22: 261 108. Topps. J . H., R. N. B. Kay, E. D. G o o d a l l , F. G. Whitelaw, and R. S. R e i d (1968b). D i g e s t i o n of C o n c e n t r a t e and of Hay d i e t s i n t h e Stomach and I n t e s t i n e s o f Ruminants. 2) Young S t e e r s . B r i t . J . of N u t r . 22: 281 109. Vaughn, M. (1961). The Metabolism of Adipose T i s s u e i n v i t r o . J . L i p i d Res. 2: 2930 110. Washko, M. E. and E. W. R i c e (1961). D e t e r m i n a t i o n of Glucose by an Improved " G l u c o s t a t " Procedure. C l i n . Chem. 7: 542 111. Weigand, E., J . W. Young, and A. D. M c G i l l i a r d (1972). E x t e n t o f P r o p i o n a t e Metabolism d u r i n g A b o s r p t i o n from t h e Bovine Ruminoreticulum. Biochem. J . J 2 6 : 201 112. Weiner, R. , H. J . H i r s c h , and J . J . S p i t z e r (1971). C e r e b r a l E x t r a c t i o n o f Ketones and t h e i r P e n e t r a t i o n CSF i n the Dog. Am. J . o f P h y s i o l . 223: 447  into  113. White, R. G., J . W. S t e e l , R. A. Leng, and J . R. L u i c k (1969). E v a l u a t i o n of t h r e e I s o t o p e - D i l u t i o n Techniques f o r S t u d y i n g t h e K i n e t i c s o f Glucose Metabolism i n Sheep. Biochem. J . 114: 203  124  114.  W o l f f , J . E., E. N. Bergman, ana H. H. W i l l i a m s (1972). Net Metabolism of Plasms Amino A c i a s by L i v e r and P o r t a l D r a i n e a V i s c e r a o f Fed Sheep. Am. J . of P h y s i o l . 223: 438  115. W o l f f , J . E. and E. N. Bergman (1972a). Metabolism and I n t e r c o n v e r s i o n s of F i v e Plasma Amino A c i d s by T i s s u e s of t h e Sheep. Am. J . of P h y s i o l . 223: 447 116. W o l f f , J . E. and E. N. Bergman (1972b), Gluconeogenesis from Plasma Amino A c i d s i n Fed Sheep. Am. J . of P h y s i o l . 223: 455 117. W r e n s h a l l , G. A., G. H e n t e n y i , and C. A. Best (1961). The V a l i d i t y o f Bates o f Glucose Appearance i n the Dog C a l c u l a t e d by the Method of S u c c e s s i v e T r a c e r I n j e c t i o n s I I . The I n f l u e n c e o f I n t e r m i x i n g Time F o l l o w i n g T r a c e r I n j e c t i o n s . Can. J . of Biochem. S P h y s i o l . 39: 267  APPEND 1X  126 Figure  1.)  Standard  Curve  for Glucose  Absorbance  CO  o  Determination.  12 7  F i g u r e 2.)  Standard  Curve  for Alanine  Absorbance  0  Determination  128  Figure  3.)  Standard Curve for Determi n a t i o n  Para-aminhippuric A c i d  Absorbance  (PAH)  0  1  Lxternal  Standards  Ratio  130  Figure  5.)  Paper Chromatography with  Separation  Use o f t h e A c t i g r a p h  of  Scanner.  C-Alanine  LOG  SPECIFIC ACTIVITY  (NCl/UG) fD  133  Figure  8.)  Activity  Curve  for C-Glucose l 4  in Jugular  txperiment:  B.2.  Vein  (J,V.)  LOG  ro  SPECIFIC ACTIVITY (NCl/UG)  to  >  O  c < ro  o  rr  I  TD  >  X  ro  m  O CO CO CO  3  ro r t  3 ro  c c < ro  C-  <  Figure 10a)  Activity  Curve for ^ - G l u c o s e in C a r o t i d A r t e r y hxper iment  P.3.  (C.  136  F i pure  T I M E (MIN)  138 Figure  lib)  Activity  Curve  f o r l' C-Alanine l  i n Jugular  Vein  t x p e r imen t B.3.  0  .3  o R  =  0.999  > o  <  o LL  o  LU CL CO  (3  O —I  i  1  1  1  1  1  TIME  1  1  1  (MIN)  1  r  (J.V.)  Figure  13.)  Activity  Curves  for  1 ! |  C - A l a n i n e and hxperiment  1!  *C-G 1 u c o s e .  C.2.  TIME '(MIN)  Ihl  Figure  Ik.  Major Of  M e t a b o l i c Pathways  Ruminants.  (Bergman,  Glycogen Blood  Glucoses  In The  Liver  And  Kidneys  1973)  Stores  ~-^=*,G 1 u c o s e - 6 - P  t r F r u c t o s e - 1 , 6-P II Tr|ose-P<.  P-Enol-Pyruvate  2  . -,  ..^G1 y c e r o 1  142 F i g u r e 15.)  Schematic Diagram  Illustrating  and I n f u s i o n C a t h e t e r s .  Posterior Vena Cava  the P o s i t i o n o f t h e Sampling  (Katz and Bergman, 1969,  pg.  948).  Ih 3  Figure  16.  The  Glucose Alanine  Cycle.  ( F e l i g . , 1973)  Figure  17.  Model  for Single  Continuous Meier  I n j e c t i o n ( C u r v e A) and  Infusion  and Z i e r l e r ,  Time  ( C u r v e B) o f a Dye. 1954,pg 732 .  (Seconds)  .U5 Figure  18.  Model  f o r G1ucose  (Leng,1970,  *Pools  B  and  C  pg.  are  Me t a b o 1 i s m  probably  made  i . e . ,glucogenic  propionate,  and  et  a_L,  Sheep.  216)  precursors,  (Whi t e  in  glycerol,  1969 )  and  up  amino CO2  of  a l l  acids, and  glucogenic lactate,  glycogen.  Table 1 ( a ) .  A l a n i n e and  Packed C e l l Volume V a l u e s , G l u c o s e ,  PAH C o n c e n t r a t i o n s  i n P o r t a l and C a r o t i d Arteries„  Sample Time  Collection Site  Mean PCV {% RBC) 19.25  Glucose (mg/100 n l )  Alanine (ug/ml)  • PAH (mg/ml)  76.5  10.8  80.5  10.6  0.006  Control  Carotid Artery  1 hr  Carotid Artery  1 h r 15 rnln  Carotid Artery  19.8  73.5  10.2  0/0085  1 h r 25 min  Carotid Artery  23.5  77.0  10.4  0.0080  1 h r 45 w i n  Carotid Artery  29.3  81.0  10.8  0.0080  mean  - 19*75  22.32  SE  1.90  78.70  1.28  0.1  0.0080  Control  Portal Vein  24.0  74.0  1 hr  Portal Vein  23.25  75o0  11.6  0.040  1 h r 15 min  Portal Vein  24.0  79.5  11.5  0.068  1 h r 25 min  Portal Vein  28.0  76.0  12.0  0.0155  1 h r 45 min  Portal. Vein  27.6  79.3  11.8  0.0165  mean i SE  Portal  -25.37  ±1.00  76.76 ±  .  10.6  1.12  11.4  11.71 ± 0 . 1 2  0.016  Table K b ) .  Blood Flow  Calculations  : PVPF = CJ. C  for txperiment A . l . (Blood Flow)  ~  P V  C  A  where  PCV CI  25.37% 15.0 mg/ml  IR C  PV  0.8 ml/min 0.016 mg/ml 0.008 mg/ml  P o r t a l V e i n Plasma  Flow  12 mg/ml 0.0160 - 0.008 mg/ml  (PVPF)  150 0 ml/min 35.01 ml/m in/kg  Portal  Vein Blood Flow  BW  3/!+  1500 ml/min 1 - 0.2537  (PVBF)  2010 ml/min «*8 . 08 ml/min/kg  Net P o r t a l  Glucose  3/U  P = Fpv^ Cpy -C^ )  Metabolism a)  BW  Utilization  P = 2010 ml/min(  0.7676 - 0.7870 mg/ml)  =-38.99 mg/min = -0.142g/hr/kg BW * 3/!  b)  Alanine  Production  P = 2010ml/min( =  0.0117 - 0.0106 mg/ml)  +2.21 mg/min  =  +1.49 mMoles/hr  Table  Preliminary  2.  Exchange  Experiment  to  Chromatography  Test  for  Efficiency  Separation  of  of  Ion  Plasma  Components.  Sample U-  Fraction  C-Alanine (control) 1 4  Basic  (amino  DPM  % Alanine  Recovered  167,971  158,657  94.5  Neutral (sugars)  712  0.4  Acidic  261  0.2  acids)  (acids)  Ik9 Table  3.  Paper  Chromatography  Separation  of  14,  C-Glucose  DPM Control C-Glucose  Strip Number  Section Number  4  1  4  2  4  3  4  4  3.5  4  5  50.0  4  6  4  14  Plasma Sample E x p . S.l  Plasma Sample Exp.B2.  •4.0  3.0  1850.0  65.0  58.0  7  520„0  2.5  1.0  4  8  2 5 .0  4  9  2.0  4  10  4  11  4 0 e  150 T a b l e 4.  D a t a and M e t a b o l i c Parameters o f A l a n i n e f o l l o w i n g a S i n g l e of U-^C-Alanine:  Experiment  Injection  B l, e  A) Data  Collection Time (min a f t e r injection)  Glucose Concentration  (rag/100 rid)  54 38  control  5 10 20 30 52 60 120 180 mean ±  -  1.970  10.4  0.256 0.211  0.4759 0.4702  62.5  0.5152 0.3218  11.4 11.3 11.7 11.0  0.1864  11.7  0.4086 0.4321  11.26 ±0.17  B) M e t a b o l i c P a r a m e t e r s :  P o o l S i z e (mg o f C)  z . 52.-83  (ml)  4,645.16  Space (g-'of.BW)  13.27  Space  C/rdn.)  8.61  C/inin)  4.96  (mg C/min)  3.65 3.57 50.0  T o t a l E n t r y Rate (mg I r r e v e r s i b l e Loss (mg  P e r c e n t G l u c o s e from A l a n i n e I s o t o p e Dose ( u C i ) Body Weight  (kg)  35.0  -  10.5 12.2  54 57 58 56  ±2.40  Recycling  Alanine Specific ConcenActivity tration (nC/mg)" (ug/ml)  11.2  1.5921  55 51.5 SE  Specific Activity (nC/mg)  0.410  0.142. 0.130 0.081 0.047  Table 5 . Data and M e t a b o l i c Parameters o f A l a n i n e A Single Injection  Following  o f U - C - A l a i i i n e : Experiment g. 2 . 1 4  A) Data Collection Time (min a f t e r injection) control  Cone en-tration  Glucose Specific Activity  (mg/100ml)  (nC/mg)  Alanine ConcenSpecific tration Activity (ug/ml) (nC/mg) 13.6  3 9 . 8  1 2 . 7  1 . 6 6 8  0.25  1 3 . 6  0  5 2 „ 0  0 . 2 9  1 3 . 3  0 c 2 8 9  4.5  4 8 . 0  0 . 4 1  1 4 . 6  0 . 1 9 5  5  4 2  0  0 „ 6 1  1 3 . 0  0  o  1 8 5  1 0 * 0  5 3 . 0  0 . 8 5  1 4 . 2  0  o  1 0 6  1 5  0  5 3 . 3  1.00  1 2  0  o  1 1 2  2 0 . 0  6 1 . 8  1.10  1 3 . 8  0 . 0 7 5  3 0  6 1 . 0  1.20  1 4 . 0  0 o 0 5 0  4 5 . 0  6 0  l  1 3 o 4  0 . 0 4 4  6 0  0  6 4 . 8  1.20  12.9  0 . 0 3 1  7 5 . 0  6 5 . 0  1.04  1 3 . 0  0 . 0 2 7  9 0 . 0  6 2 . 5  0.97  1 4 . 2  0 . 0 2 1  1 0 5 . 0  6 5 . 0  0.92  1 4 . 6  0  1 2 0 . 0  6 0  0  0.78  13.9  0 . 0 1 4  1 3 5 . 0  6 6 . 5  0 . 5 0  1 3 o 7  0 . 0 1 2  1 5 0 . 0  6 2  0 . 3 9  1 3 . 5  0  3.0  2 6 . 5  3  4 5 . 8  4.6  5  0  0  0  mean i  o  o  o  0  SE  o  o  o  0  O  8  5 4 . 9 4 i 2.52  -  0 . 3 9  o  3 0  0  6  13.59 0 . 9 5  ±  3 5 4  o  o  o  0 1 7  0 0 7  152 Table  B)  5.  cont'd  Metabolic  Parameters  Pool  :  Two  S i z e (mg  Space Total  L n t r y Rate  Irreversible  Per  cent  Term b x p o n e n t i a 1 F u n c t i o n  C)  =  17.69  Space (ml)  =  1296.85  =  3.71  C/min) =  14.52  U  of  of  (mg  BW)  Loss  (mg  C/min)  =  7.60  Recycling  (mg  C/min)  =  6.91  =  5.00  G l u c o s e From A l a n i n e Isotope  Dose ( u C i ) =  Body W e i g h t  (kg) ' *  50.0 35.0  T h r e e Term b x p o n e n t i a l  Pool  Total  cent  o f C)  =  17.69  Space (ml)  =  1296.85  Space U  of  =  3.71  R a t e (mg  C/min) =  14.52  Loss  (mg  C/min)  =  7.60  Recycling  (mg  C/min) =  6.91  tntry  Irreversible  Per  S i z e (mg  Glucose  BW)  From A l a n i n e  Isotope  =  Dose ( u C i ) =  Body W e i g h t  (kg)  =  5.00 .50.0 35.0  Function  Table 6 .  D a t a and M e t a b o l i c Parameters o f A l a n i n e F o l l o w i n g A Single Injection  A)  C - A l a n i n e s Experiment 8.3.  Carotid Artery  Data  Collection Time (min a f t e r injection)  o f U-  Glucose ConcenSpecific tration Activity (mg/100ml) (nC/mg)  control  64.0  control  62 5 0  -  Alanine ConcenSpecific tration Activity (ug/ml) (nC/mg)  10.8 10.5  —  -  0.33  61.5  0.193  9.5  1.324  0.67  71.0  0.190  10.6  1.177  0.83  66.0  0.197  10.8  0.533  1.13  62.5  0.476  10.6  0.512  1.42  66.0  0.569  12.0  0.361  1.92  64.0  0.565  11.4  0.302  2.17  69.0  0.746  11.6  0.277  5.00  69.0  0.710  9.5  0.259  10c0  59.0  1.124  10.3  0.133  15.0  69.0  1.178  10.4  0.115  25.0  67.0  1.394  10.8  0.065  35.0  71.0  1.382  10.6  0.052  45.0  69.0  1.454  10.6  0.040  60.0  71.0  1.538  10.6  0.033  75.0  71.5  1.467  10.0  O.027  90.0  73.0  1.330  10.3  0.023  120.0  77.0  1.124  10.6  0.020  ean ± SE ±  67.52 1.04  .  10.59 ±0.14  Table  A)  6.  cont'd  Data  Collection Time (min after injection)  .  •  Concentration (mg/100ml)  •  J u g u l a r Vein  G l u c o ;e Specific Activity (nC/mg)  control  51.0  -  control  54.0  -  Alanine ConcenSpecific tration Activity (ug/ral) (nC/mg)  -  11.6  •  -  11.3 .  0.702  0.33  52.0  0.138  10.8  0.58  59.0  0.216  10.0"  1.184  0.83  62.0  0.233  10.9  0.699  1.95  60.0  0.392  10.3  0.278  2.17  61.0  0.490  10.6  0.264  2.45  59.0  0.532  10.1  0.257  2 75  62.0  0.593  10.3  0.229  lOcO  59.0  0.930  9.5  .0.181  15.0  54.0  1.018  11.3  0.103  25.0  61.0  1.521  10.3  0.058  35.0  61.0  1.534  • 10„8  0.042  45.0  61.0 '  1.800  10.3  60.0  66.0  1.465  75.0  .67.0  1.319  11.8  0.023  90.0  66.0  1.316  11.6  0.021  120.0  74.0  1.244  10.3  0.018  0  mean ± SE  60.28 ±1.36  •  11.3  10.73 ± 0 . 1 5  . '  0.035 0.030  Table  B)  6„  cont'd  Metabolic  Parameters  Carotid  Pool  S i z e (mg o f C) = Space Space  Total  Entry Rate  cent  (ral)=  (% o f BW)  4026.53  =  Loss  (mg C/min) =  9.32  Recycling  (mg C/min) =  5.20  From A l a n i n e ) =  6.74  Glucose  Dose ( u C i ) =  Body W e i g h t  (kg)  Pool  Size  Entry Rate  Vein  17.49  (ral)=  1640.91  (% O f BW).=  4.44.  (mg C/min) =  49.78  Loss  (mg C/min) =  10.03  Recycling  (mg C/min) =  39 75  Irreversible  cent  37.0  (mg o f C ) = Space  Space  95.0  =  Jugular  Per  10.8 61.37  Isotope  Total  42.69  (mg C/min) =  Irreversible  Per  Artery  Glucose  From A l a n i n e  Isotope  =  Dose ( u C i ) =  Body W e i g h t  (kg) =  0  6.78 95.0 37.0  to LTi  Table 7. ' Summary of Metabolic Parameters for the Single Injection Experiments.  Sheep Experiment Blood number Source Weight and (kg) type  Plasma Alanine concentration (ug/ml)  Space Pool {% of Size BW) (mg)  Irreversible Loss (mM/hr)  Recycling ' % Conversion Total •to Glucose Rate Entry (mM/hr) Rate (mM/hr)  S.I.-l  Jugular Vein  35  11.26  .17 52.9  13.27  3.34  5.80  2.46  3.57  S.I.-2  Jugular Vein  35  13.59  .95 17.7  3.71  5.12  9.77  4.65  5.00  'S.I.-3  Jugular Vein  37  10.73  .15 17^5  4.44  6.75  33.52  26.77  6.78  Carotid Artery  37  10.60  .14 42.70 10.88  9.33  :61.37  52.04  6.74  32.70 8.07 i17.92 ±4.73  •6.13 ±2.54  21.48 27.61 ±25.61 ±23.14  5.52 ±1.54  mean - SE  Table 8.  Data and Metabolic Parameters of Alanine Following a Continuous Infusion of "^C-Alanine;  A)  Carotid Artery-  Data  Collection Time (min a f t e r s t a r t of infusion)  Experiment C. 1.  Glucose ConcenSpecific tration Activity (mg/lOOml) (nC/mg)  Alanine ConcenSpecific tration Activity (ugM) (nC/mg)  control  83.0  -  13.2  5  68.0  0.151  12.8  8.4  15  68.0  0.205  14.8  9.6  30  67.0  0.255  14.2  14.4  60  64.0  0.289  15.5  18.3  . 90  61.0  ' 0.310  L4.0  . 26.7  120  62.0  0.577  16.7  21.0  150  62.0  0.824.  13.3  31.1  180  90.0  0.703  14.0  33.7  210  46.0  •• 1.74  14.3  36.8  240-  62.0  1.26  14.6  34.6  270  55.0  1.69  15.9  43.1  300  55.0  2.24  15.7  44.0  330  56.0  2.27  15.3  46.2  360 '.'  60,0  2.20  16.2  43.4  >an ± SE  63.96 ± 2.81  B)  14*7 ±0.29  Metabolic Parameters I r r e v e r s i b l e Loss (mM/hr)  7.23  % Conversion t o Glucose  5.07  158  Table  9.  Data and M e t a b o l i c  Parameters  A Continuous Infusion o f  A)  C~Alanine.  Carotid  Data  Collection Time (min a f t e r injection)  1 4  Concentration (mg/100ml)  of Alanine  Glucose Specific Activity (nC/mg)  Following  Experiment  C.2  Artery  Alanine ConcenSpecific tration Activity (ug/ml) (nC/mg)  control  55.0  -  13.8  30  62.0  0.11  13.0  0.615  60  48.0  0.335  12.0 •  2.50  53.5  0.323  15.4  8.57  120  53.5  0.33  14.4  17.92  150  53.5  0.38  14.9  15.37  180  53.5  0.47  14.7  31.97  240  50.0  0.86  13.4  39 70  300  46.5  1.67  14.3  34.75  360  51.0  2.43  15.2  34.56  390  56.O  2.46  14.5  34.21  405  48.0  2.37'  .14.8  34.41  420  46.4  1.76  14.0  28.70  435  53.0  1.33  14.2  9.36  450  51.0  0.66  14.0  5.57  90  •  mean ± S E  52.06 ±1.06  14.40 ±0.26  —  o  Table 9 .  A)  cont'd  Portal  Data  Collection Time (min a f t e r injection)  Control  Vein  Glucose Concen~ Specific tration Activity (mg/lOOml) (nC/rag)  -  53.0  Alanine ConcenSpecific Activity tration (nC/mg) (ug/ml)  14.4  -  30  46.5  0.097  14.0  3.07  60  48 0  0.30  13.8  2.61  90  52.0  0 54  15.3  15.7  120  5 0 oO  0.41  15.8  10.0  150  52 0  0 40  14.5  14.6  180  48.0  0.913  15.5  24.7'  240  47.0  l 52  15.0  31.8  300  46.5  1„75  14.9  35.2  360  49.0  2.41  16.7  32.9  390  44.0  2.55  20.5  34.9  405  54.0  2.44  17.2  24.4  420  52.0  1.51  14.9  16.6  435  48 .0  1.18  450  52 0  0.727  mean ~ S E  B)  o  o  o  o  c  0  6.2 5.7  14.6  49o46  15.89  ± 0 . 7 7  ± 0 . 5 3  M e t a b o l i c Parameters  Irreversible  L o s s (mM/hr) = 8 . 5 9  % Conversion' to Glucose  =  5 . 0 7  Table 1 0 .  Summary of Parameters from S i n g l e I n j e c t i o n and Continuous I n f u s i o n Experiments. Parameters of Alanine Metabolism i n Sheep  F&periment  Sheep  number* and type  WT (kg)  Plasma Alanine Concentration (ug/ml)  Pool Space S i z e . (% o f (mg) B W )  Irre-versible Loss (mM/hr)  Total Entry Rate (mM/hr)  Recycling Rate (mK/nr)  £ .Conversion t o Glucose  35  11.26  17  52,9  13.27  3.34  . 5.80  2.46  3.57  . 35 '  13.59  .95  7.7  3.71  5.12  9.77  4.65  5.00  s t, X«—3  37  10.73  .15  7.49  4.44  6.75  33.52  26.77  6.78  C.I.-4  45  14.70  .29  7.23  5.07  C.I.-5  42  14.40  .26  8.59 •.  -  "5  T  -2  mean ± SE  :  31.69  i14.10  7.14  ±3.07  6.21 ±0.91  -  16.36  i8.65  11.27 ±7.74  7.20 5.52 ±0.66  

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