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UBC Theses and Dissertations

Effect of starvation on femoral arterial blood flow determined by the Doppler flowmeter and substrate… Pratt, Robert John 1978

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E F F E C T OF S T A R V A T I O N ON FEMORAL A R T E R I A L BLOOD FLOW DETERMINED BY THE D O P P L E R FLOWMETER AND S U B S T R A T E M E T A B O L I S M I N THE SHEEP by ROBERT JOHN PRATT B . S c . , S i m o n F r a s e r U n i v e r s i t y , 1969 A T H E S I S SUBMITTED I N P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF THE F A C U L T Y OF GRADUATE S T U D I E S DEPARTMENT OF A N I M A L S C I E N C E We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMBIA D e c e m b e r , 197 8 Cc] R o b e r t J o h n P r a t t MASTER OF S C I E N C E i n \ In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of this thes is fo r f inanc ia l gain sha l l not be allowed without my written permission. Department of Animal Science The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date iQofVsrtA&U. /tfyg i i A B S T R A C T T h e f e m o r a l a r t e r i a l b l o o d f l o w i n s h e e p was d e t e r m i n e d b y i m p l a n t i n g D o p p l e r c u f f s a r o u n d t h e v e s s e l w h i c h f a c i l i t a t e d m e a s u r e m e n t s o v e r a p r o l o n g e d p e r i o d o f t i m e . S t a n d a r d i z a t i o n o f t h e c u f f s was a c c o m p l i s h e d w i t h a n e l e c t r o n i c i n t e g r a t o r -d i g i t a l t i m e c o u n t e r w h i c h c o n v e r t e d D o p p l e r f r e q u e n c y s h i f t s t o c o u n t s p e r m V / h r f r o m w h i c h b l o o d f l o w was e s t i m a t e d . M e t a -b o l i c c h a n g e s o c c u r r i n g i n t h e h i n d l i m b o f s h e e p f o l l o w i n g s t a r v a t i o n w e r e s t u d i e d u s i n g f e m o r a l 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 d i f f e r e n c e s o f m e t a b o l i t e s c o u p l e d w i t h b l o o d f l o w . I n d w e l l i n g c a t h e t e r s w e r e i n t r o d u c e d i n t o t h e f e m o r a l a r t e r y a n d v e i n t o e n a b l e b l o o d s a m p l i n g . Two s e r i e s o f e x p e r i m e n t s w e r e c a r r i e d o u t i n t h i s s t u d y . E x p e r i m e n t I i n v o l v e d a f o u r d a y s t a r v a t i o n o f t w o e w e s . T h e r e w a s n o s t a t i s t i c a l d i f f e r e n c e i n t h e f e m o r a l a r t e r i a l b l o o d f l o w b e t w e e n f e d a n d s t a r v e d a n i m a l s . I n E x p e r i m e n t I I a n o t h e r a n i m a l w a s u t i l i z e d f o r f o u r t r i a l s w i t h t h e d u r a t i o n o f s t a r v a t i o n i n c r e a s e d f r o m f o u r t o s i x d a y s . The a d j u s t e d mean f e d b l o o d f l o w o f 78 + 2 m l / m i n d e c r e a s e d t o a m i n i m u m o f 45 + 3 m l / m i n o n t h e s i x t h d a y o f s t a r v a t i o n . T h e a v e r a g e b l o o d f l o w d u r i n g t h e s i x d a y s o f s t a r v a t i o n w a s 2 3 . 5 p e r c e n t l o w e r t h a n t h e p r e - s t a r v a t i o n l e v e l (P 0 . 0 5 ) . T h e D o p p l e r F l o w m e t e r o f f e r s d e f i n i t e a d v a n t a g e s o v e r o t h e r m e t h o d s o f b l o o d f l o w d e t e r m i n a t i o n s b y f a c i l i t a t i n g t h e m e a s u r e m e n t o v e r p r o l o n g e d p e r i o d s w i t h o u t c a u s i n g e x c i t e m e n t o f t h e a n i m a l s . I n t h i s s t u d y t h e u s e o f t h e Doppler Flowmeter and the continuous m o n i t o r i n g of blood flow through a p e r i o d o f 11 days has demonstrated t h a t h i n d limb b l o o d flow i s reduced d u r i n g s t a r v a t i o n . The u t i l i z a t i o n o f glucose and l a c t a t e was reduced d u r i n g s t a r v a t i o n by 62 and 39% r e s p e c t i v e l y . There was a net p r o d u c t i o n of 25 ± 1.5 and 20 ± 1 pg/min of a l a n i n e on the f i f t h and s i x t h days of s t a r -v a t i o n versus a net u t i l i z a t i o n of 23 ± 2 pig/min p r i o r t o s t a r v a t i o n . The net a l a n i n e p r o d u c t i o n d u r i n g s t a r v a t i o n p r o v i d e s a p r e c u r s o r f o r gluconeogenesis. i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES . v i LIST OF FIGURES v i i LIST OF APPENDIX TABLES v i i i LIST OF APPENDIX FIGURES AND ILLUSTRATIONS i x LIST OF APPENDIX PLATES x ACKNOWLEDGEMENTS x i INTRODUCTION 1 LITERATURE REVIEW 5 Glucose Metabolism i n Ruminants . . . 5 Glucose Requirements ' 5 1. Nervous t i s s u e 6 2. Muscle t i s s u e 6 3. Adipose t i s s u e 6 4. Fetus 8 5. Mammary gland 8 Glucose P r o d u c t i o n i n the Ruminant 9 1. D i e t a r y source 9 2. He patic gluconeogenesis 10 3. Renal gluconeogenesis 10 P r e c u r s o r s f o r Gluconeogenesis 11 1. V o l a t i l e f a t t y a c i d s 11 2. G l y c e r o l 14 3. L a c t a t e 15 4. Amino a c i d s . . . . . . . 16 A l a n i n e - G l u c o s e I n t e r r e l a t i o n s h i p s 21 Blood Flow 24 I n d i c a t o r D i l u t i o n Techniques 25 Microspheres 2 8 Thermal D i l u t i o n Techniques 30 Flowmeters 31 1. E l e c t r o m a g n e t i c flowmeters 31 2. Doppler flowmeter 33 V EXPERIMENTAL 37 EXPERIMENT I • • 37 Introduction 37 Materials and Methods 38 1. Animals and N u t r i t i o n a l Regime 38 2. Implantation of Catheters 39 3. Cuff placement 41 4. Experimental Period 42 5. Chemical Determinations 42 6. Blood Flow Measurements 45 7. S t a t i s t i c a l Analysis 45 Results 46 1. Femoral Artery Blood Flow 46 2. U t i l i z a t i o n or Production of Sub-strates i n the hind limb 49 3. Feed Analysis 51 Discussion 56 Femoral A r t e r i a l Blood Flow 56 Substrate Metabolism 5 8 Conclusions 60 EXPERIMENT II 62 Introduction 62 Materials and Methods 6 2 1. Animals and Feeding Regime 62 2. Surgical Procedure 6 3 3; Blood Flow 6 4 4. Plasma Metabolites 6 4 5. Chemical Analysis. . 64 6. S t a t i s t i c a l Analysis 65 Results 66 1. Femoral A r t e r i a l Blood Flow. . . . . . . . 66 2. U t i l i z a t i o n or Production of Sub-strates i n the hind limb 71 Discussion . . " 78 Femoral A r t e r i a l Blood Flow 7 8 Substrate Metabolism 80 GENERAL CONCLUSION AND SUMMARY . 84 Femoral A r t e r i a l Blood Flow 84 Substrate Metabolism 84 BIBLIOGRAPHY 86 APPENDIX 96 v i LIST OF TABLES Table Page 1 Femoral A r t e r i a l Blood Flow in Sheep 2 42 and 39 under Fed and Starved Conditions using the Doppler Flowmeter and Analogue Integrator-Counter (Experiment I) 4 8 2 Concentration of Glucose and Lactate i n A r t e r i a l and Venous Plasma of Sheep 242 (Experiment I) . . . . . 52 3 Concentration of Glucose and Lactate i n Femoral Artery and vein of sheep 39 (Experiment I) 53 4 Analysis of Covariance and Test of Common Slope for Femoral A r t e r i a l Blood Flow i n Sheep 239 (Experiment II) '. 69 5 Adjusted Mean Femoral A r t e r i a l Blood Flow for Sheep 2 39 during Different Phases of Starvation (Experiment II) 70 6 Individual Regression of Femoral A r t e r i a l Blood Flow versus time in sheep 2 39 during d i f f e r e n t phases of starvation (Experiment I I ) . 72 7 Mean Femoral Arteriovenous Glucose Concen-t r a t i o n and Net U t i l i z a t i o n per min for Sheep 239 (Experiment II) 75 8 Mean Femoral Arteriovenous Lactate Concen-t r a t i o n and Net U t i l i z a t i o n per min for Sheep 2 39 (Experiment II) 76 9 Mean Femoral Arteriovenous Alanine Concen-t r a t i o n and Net Utilization/Production per min for Sheep 2 39 (Experiment II) 77 v i i LIST OF FIGURES Figure Page 1 Femoral A r t e r i a l Blood Flow using a Doppler Flowmeter and 3 mm Cuffs in Sheep 242 and 39 47 2 Femoral A r t e r i a l and Jugular Vein Glucose Concentration i n Sheep 2 42 (Experiment I ) . . . . 5 0 3 Femoral A r t e r i a l and Venous Glucose Con-centration i n Sheep 39 (Experiment I) 51 4 Femoral A r t e r i a l and Jugular Vein Lactate Concentration i n Sheep 242 (Experiment I ) . . . . 54 5 Femoral A r t e r i a l and Venous Lactate Concen-t r a t i o n i n Sheep 39 55 6 Femoral A r t e r i a l Blood Flow i n Sheep 239 ob-tained in Four T r i a l s A, B, C, and D . . . . . . 67 7 Linear Regression and Confidence Limits on Femoral A r t e r i a l Blood Flow in Sheep 2 39 during Different Phases of Starvation (Experiment II) 68 8 Carotid A r t e r i a l Blood Flow i n Sheep S-3 during Different Phases of Starvation 7 3 v i i i LIST OF APPENDIX TABLES Table Page 1 Femoral A r t e r i a l Blood Flow i n Sheep 2 39 and Carotid A r t e r i a l Blood Flow i n Sheep S-3 (Experiment II) 97 2 Femoral Arteriovenous Metabolic Parameters for Sheep 2 39 98 ix LIST OF APPENDIX FIGURES Figure Page 1 Ca l i b r a t i o n Curve for Blood Flow Determina-tions using the Doppler Flowmeter and Analogue Integrator-Counter for a 3 mm Cuff 100 2 C a l i b r a t i o n Curve for Blood Flow Determina-tions using the Doppler Flowmeter and Analogue Integrator-Counter for a 4 mm Cuff 101 3 C a l i b r a t i o n Curve for Glucose Determinations by the Glucose Oxidase Method 102 4 C a l i b r a t i o n Curve for Lactate Determinations . . 102 5 Femoral A r t e r i a l Blood Flow i n Sheep 2 39 (A, B, C, and D) 104 6 The Cori Cycle 108 7 The Glucose Alanine Cycle 108 8 Ruminant Gluconeogenesis 109 9 Alanine and Glutamine Cycles 110 10 Schematic Diagram of Electromagnetic Flowmeter . 111 11 Schematic Diagram of Doppler Flowmeter . . . . . 111 12 Schematic Diagram of Cuff Placement i n Hind Limb of Sheep (A) S u p e r f i c i a l View, (B) Cut Away View) 112 i X LIST OF APPENDIX PLATES Plate Page 1 Doppler Cuff i n Place around Femoral Artery. 114 x i ACKNOWLEDGEMENTS I wish to express ray sincere appreciation to Dr. CR. Krishnamurti for his guidance, assistance and patience over the past three years. I would further l i k e to acknowledge Mr. G. Galzy for his expertise in the design of the analogue integrator-counter; Mr. D. K i t t s and Mr. G. Smith for t h e i r s u r g i c a l assistance and Mr. D. Thompson for his experimental assistance. I would also l i k e to thank Mr. D. J e f f e r i e s and Dr. R. Peterson for t h e i r assistance i n s t a t i s t i c a l analysis. I would l i k e to pay special t r i b u t e to my wife, Susan, for her patience and t o t a l support i n the completion of t h i s thesis. 1 INTRODUCTION M o n o g a s t r i c a n i m a l s , i n c o n t r a s t t o r u m i n a n t s a r e g e n -e r a l l y f e d p r i m a r i l y f o r a g e d i e t s . T h e e x t e n s i v e f e r m e n t a -t i o n o f f e e d i n t h e rumen p r o f o u n d l y i n f l u e n c e s t h e n a t u r e a n d p r o p o r t i o n s o f n u t r i e n t s s u p p l i e d t o r u m i n a n t t i s s u e s . D i e t a r y c a r b o h y d r a t e s a r e l a r g e l y f e r m e n t e d t o v o l a t i l e f a t t y a c i d s w h i c h s u p p l y 60-80% o f t h e m e t a b o l i z a b l e e n e r g y i n r u m i -n a n t s ( K r o n f e l d a n d V a n S o e s t , 19 7 6 ) . Due t o t h e l i m i t e d s u p p l y o f a l i m e n t a r y g l u c o s e , g l u c o n e o g e n e s i s i s a m a j o r m e t a b o l i c a c t i v i t y i n t h e f e d a s w e l l a s f a s t e d r u m i n a n t . A s e c o n d m a j o r p r o c e s s , l i p o g e n e s i s , shows m a r k e d d i f f e r e n c e s f r o m m o n o g a s t r i c s i n t h a t a c e t a t e r a t h e r t h a n g l u c o s e , i s t h e m a j o r p r e c u r s o r ( B a l l a r d e t a l . 1 9 6 9 ) . T h e g l u c o s e s u p p l y o f t h e r u m i n a n t i s j u s t a d e q u a t e t o mee t i t s r e q u i r e m e n t s . S t r e s s c o n d i t i o n s r e a d i l y u p s e t t h i s b a l a n c e a n d may l e a d t o h y p o g l y c e m i a a n d , u l t i m a t e l y , i n k e t o s i s . I n d a i r y c o w s h y p o g l y c e m i a a n d k e t o s i s a r e r e -f e r r e d t o a s a c e t o n e m i a a n d o c c u r d u r i n g p e r i o d s o f h e a v y l a c -t a t i o n . H o w e v e r , i n s h e e p t h e s e symptoms o c c u r d u r i n g l a t e p r e g n a n c y a n d a r e r e f e r r e d t o a s p r e g n a n c y t o x e m i a . T h e s e d i s o r d e r s a r e n o t i d e n t i c a l b u t t h e y h a v e t h e common c h a r a c -t e r i s t i c s o f n e g a t i v e e n e r g y b a l a n c e , a r e d u c t i o n i n b l o o d g l u c o s e a n d i n c r e a s e d f a t m e t a b o l i s m . M e t a b o l i c c h a n g e s i n r u m i n a n t s o c c u r r i n g d u r i n g s t a r v a t i o n a r e s i m i l a r t o a c e t o n e m i a a n d p r e g n a n c y t o x e m i a . S t a r v a t i o n h a s t h e r e f o r e b e e n u s e d a s 2 a model i n t h i s s tudy to moni tor the mechanism o f g l u c o s e homeos tas i s . The t h r e e major v o l a t i l e f a t t y a c i d s , p r o p i o n i c , a c e t i c and b u t y r i c , are produced i n the rumen i n v a r y i n g amounts depending upon the n u t r i t i o n a l reg ime . Anni son e t a l . (1967) u s i n g r a d i o a c t i v e l y l a b e l l e d a c e t a t e and b u t y r a t e showed t h a t these end p r o d u c t s o f c a r b o h y d r a t e f e r m e n t a t i o n c o n t r i -buted l i t t l e i f any to g l u c o s e p r o d u c t i o n i n sheep. P r o -p i o n a t e has been shown to p r o v i d e 27-54% o f the g l u c o s e s u p p l y i n ruminants (Bergman, 1973). Amino a c i d s d e r i v e d from exogenous o r endogenous sources s erve as the o t h e r major p r e c u r s o r s f o r de novo s y n t h e s i s o f g l u c o s e . S t u d i e s on man ( F e l i g e t a l . 19 70) , r a t s ( S n e l l , 1976) and sheep (Cross e t a l . 1974) have p r o v i d e d ev idence t h a t a l a n i n e and g lutamine are the p r i n c i p a l amino a c i d s r e -l e a s e d by s k e l e t a l m u s c l e . W o l f f e t a l . (1972a) showed t h a t these amino a c i d s are the p r i n c i p a l amino acids removed by h e p a t i c t i s s u e s i n sheep f o r the purpose o f g l u c o n e o g e n e s i s . These f i n d i n g s l e d F e l i g e t al_. (1969) and M a r l i s s e t a l . (1971) to propose the g l u c o s e - a l a n i n e c y c l e and the g l u a t m i n e -a l a n i n e c y c l e r e s p e c t i v e l y (Appendix F i g u r e s 7 and 9 ) . Amino a c i d s have been e s t i m a t e d to s u p p l y from 11-30% o f the g l u c o s e p o o l i n sheep (Wolf f and Bergman, 1972b) to 46% o f the g l u -cose i n s t a r v e d cows ( B a i r d e t a l . 1977). Other s u b s t r a t e s which serve as g l u c o n e o g e n i c p r e c u r -sors i n c l u d e g l y c e r o l , l a c t a t e and p y r u v a t e . G l y c e r o l d e r i v e d 3 from the hydrolysis of t r i g l y c e r i d e s contributes 2-5% of the glucose i n fed sheep (Bergman, 196 8 and R e i l l y and Ford, 1971). The proportion of glucose derived from g l y c e r o l during starvation increased to 2 7% i n sheep (Bergman et a l . 1968; R e i l l y and Ford, 1971) and 16% i n l a c t a t i n g dairy cows (Baird et a_l. 19 77) . Lactate and pyruvate together c o n t r i -bute 7% and 19% to the glucose pool i n the fed and starved conditions respectively (Annison et a l . 1963). Quantitative measurements of amino acids and other glu-cogenic precursors released by s k e l e t a l muscle have been ham-pered by d i f f i c u l t i e s inherent i n the measurement of blood flow without causing major physiological disturbances. The methods for determining regional blood flow to organs and tissues of ruminants include thermodilution (Bensadoun et a l . 1962), dye d i l u t i o n (Katz and Bergman, 1969a and J a r r e t t et a l . 1976), microspheres (Hales, 1973 and B e l l et a l . 1976), electromagnetic flowmeters (Dobson et al_. 1966) and Doppler Flowmeters (Carr and Jacobson, 1968). In t h i s study a technique based on the Doppler s h i f t has been standardized for the determination of blood flow i n the femoral artery of sheep under normal phy s i o l o g i c a l conditions. Catheters were also implanted i n the femoral artery and vein of non-pregnant, non-lactating ewes to measure the a r t e r i o -venous differences i n the concentration of glucose, lactate and alanine under fed and starved conditions. The hind limb was chosen because of the ease with which blood flow can be 4 measured and because i t represents predominantly s k e l e t a l muscle metabolism. The study has provided information on the net release or uptake of metabolites from s k e l e t a l muscle during starvation. 5 LITERATURE REVIEW Glucose Metabolism i n Ruminants Glucose plays an es s e n t i a l role i n c e l l u l a r metabolism by providing energy necessary for endergonic processes and by supplying - carbon skeletons for the synthesis of complex molecules. Glucose metabolism i n ruminants d i f f e r s markedly from that i n monogastrics. Monogastrics or simple stomached animals degrade dietary carbohydrate to glucose, other hexoses and pentoses which are absorbed from the g a s t r o i n t e s t i n a l t r a c t into the portal c i r c u l a t i o n . On the other hand rumi-nants have a very large microbial population i n the rumeno-reticulum where the dietary carbohydrates are fermented to v o l a t i l e f a t t y acids. Even when high concentrate rations are fed v i r t u a l l y no glucose reaches the small int e s t i n e i n ruminants (Bergman, 1963). Ruminants therefore r e l y on endo-genous production of glucose from non-carbohydrate precur-sors to meet t h e i r glucose requirements. Glucose Requirements Though c i r c u l a t i n g levels of glucose i n ruminants (40-60 mg%) are lower than monogastrics (80-100 mg%) glucose i s no less important i n a ruminant than i n a monogastric (Kronfeld and Van Soest, 19 76). In both ruminant and mono-gast r i c animals glucose serves as the major metabolic f u e l 6 in the nervous system, musculature, adipose tis s u e , fetuses and mammary glands (Annison and White, 1961). In adult ruminants very l i t t l e glucose i s u t i l i z e d by erythrocytes since i n s u l i n has a reduced e f f e c t on glucose uptake by red blood c e l l s (Scharrer and Hunteman, 1977). 1. Nervous System The brain i s almost t o t a l l y dependent on glucose for i t s energy source. In man the brain u t i l i z e s as much as 80% of the glucose which i s released into the blood ( C a h i l l et a l . 19 70). In re s t i n g non-pregnant non-lactating sheep the brain and nervous tissue account for more than half of the glucose u t i l i z e d by the whole body (Kronfeld and Raggi, 1966). During prolonged starvation i n man and sheep the brain metabolizes ketones and f a t t y acids to meet some of the energy require-ments thereby reducing glucose oxidation (Owen et al_. 1967; Raju et a l . 1972). The nervous system s t i l l needs consider-able glucose but can adapt to ketone metabolism during periods of starvation (Raju et a l . 1972). 2. Muscle Tissue Though the glycogen i n the l i v e r (6-8%) i s higher than i n muscles (4%) the t o t a l quantity of glucose stored i n the muscles as glycogen i s greater than that of the l i v e r . The glycogen stores i n muscles are u t i l i z e d during anaerobic meta-bolism, exercise or when oxygen supplies are low. Starvation 7 depletes the glycogen stores i n muscles gradually whereas the l i v e r glycogen i s u t i l i z e d immediately. In the muscles glucose i s metabolized v i a g l y c o l y s i s to pyruvate which may then follow one of several pathways. It may be converted to lactate which i s released into the blood stream and returned to the l i v e r where i t i s reconverted to glucose. This re-cy c l i n g of glucose between the muscle and l i v e r i s c a l l e d the Cori Cycle (Cori, 1931). Pyruvate may also be trans-aminated to alanine and recycled to the l i v e r v i a the Glucose-Alanine Cycle (Felig et a l . 1969, Appendix Figure 9). Pyru-vate may also be oxidized to C0 2 and water v i a the T.C.A. Cycle producing the energy needed for muscular work. 3. Adipose Tissue Glucose i s the precursor of both body fat and milk f a t i n monogastrics. In ruminants glycer o l for t r i g l y c e r i d e f o r -mation arises from glucose since the enzyme gl y c e r o l kinase i s lacking i n both adipose tissue and mammary gland (Vaughn, 1961). Glucose also provides the reducing agent, NADPH, v i a the pentose phosphate shunt, which i s required for the syn-thesis of long chain fatty acids (Ballard et a l . 1969). In ruminants'acetate serves as the precursor of fa t t y acid synthesis since they lack the enzyme of the c i t r a t e cleavage pathway (Ballard et a l . 1969). Acetate i s produced i n large quantities i n the rumen and i s readi l y available i n the cytoplasm to be converted to acetoacetate and long chain f a t t y acids for t r i g l y c e r i d e formation. 8 4. Fetus The p r i n c i p l e metabolic f u e l of the fetus i s carbohy-drate (Dawes, 1968). Fetal glucose i s obtained p r i n c i p a l l y by placental transfer from the mother and the f e t a l glucose concentration i s dependent on the maternal glucose levels (Reid, 1968). Simmons et a l . (1974) reported that when pregnant sheep were starved there was a 35% drop i n f e t a l blood glucose concentration. James et a l . (1972) reported that glucose catabolism accounted for less than 50% of the oxygen consumption i n the fetus, Setchell et a l . (1972) re-ported that 70% of the f e t a l glucose was derived from the ewe. I t i s apparent that the fetus places a great s t r a i n on the maternal glucose supply. It has been demonstrated that maternal gluoconeogenesis equals or exceeds the energy require-ments of the pregnant ewe (Kronfeld and Van Soest, 1976). If the ewe f a i l s to synthesize adequate amounts of glucose she develops hypoglycemia and pregnancy toxemia. Pregnancy toxemia usually occurs i n underfed pregnant sheep suggesting the lack of exogenous glucose precursors as the root of the problem (Kronfeld and Van Soest, 1976). 5. Mammary Gland Lactation requirements for glucose are very high since lactose, the main milk sugar, i s derived from glucose. Milk contains approximately 90 times the t o t a l sugar content of 9 b l o o d a n d t h u s r e q u i r e s a n i n c r e a s e d p r o d u c t i o n o f g l u c o s e t o m e e t t h e demands o f l a c t a t i o n ( B e r g m a n , 1 9 7 3 ) . K e t o s i s , a m e t a b o l i c d i s e a s e o f l a c t a t i o n , m a n i f e s t s i t s e l f a s h y p o g l y -c e m i a b u t m i l k s u p p l y c a n b e r e d u c e d o r c u t o f f t o c o n s e r v e g l u c o s e . I n c o w s a b o u t 80% o f t h e c a r b o n i n m i l k l a c t o s e i s d e r i v e d f r o m g l u c o s e a n d a b o u t 5% o f t h e m i l k f a t i s s y n t h e -s i z e d f r o m g l u c o s e ( B a i r d e t a l . 1 9 7 4 ; K r o n f e l d a n d V a n S o e s t , 1 9 7 6 ) . The r e q u i r e m e n t o f g l u c o s e d e s c r i b e d a b o v e , i n d i c a t e s t h a t r u m i n a n t s m u s t d e p e n d h e a v i l y u p o n g l u c o n e o g e n e s i s t o m e e t t h e demands o f t h e a n i m a l . G l u c o s e P r o d u c t i o n i n t h e R u m i n a n t 1. D i e t a r y S o u r c e The a m o u n t o f g l u c o s e w h i c h e s c a p e s d e g r a d a t i o n i n t h e r u m e n a n d r e a c h e s t h e i n t e s t i n e d e p e n d s u p o n t h e t y p e o f f e e d , i n t e r v a l o f f e e d i n g , q u a n t i t y , a n d h e a l t h o f t h e a n i m a l ( B e r g m a n , 19 7 3 ) . T h e g l u c o s e r e a c h i n g t h e s m a l l i n t e s t i n e w o u l d b e i n t h e f o r m o f m i c r o b i a l s t a r c h o r s t a r c h e s c a p i n g m i c r o b i a l d i g e s t i o n . T h e a c t u a l a m o u n t o f g l u c o s e r e a c h i n g t h e s m a l l i n t e s t i n e w o u l d s t i l l be i n a d e q u a t e t o m e e t t h e r e q u i r e m e n t s o f t h e a n i m a l u n d e r n o r m a l c o n d i t i o n s ( S u t t o n , 1976) . 10 2. Hepatic GTucorieogens i s The l i v e r i n ruminants i s the major s i t e where gluconeo-genesis takes place. Bergman et a l . (1970) estimated that under steady state conditions 85% of the t o t a l endogenous glu-cose i s formed i n the l i v e r . The main exogenous precursors are absorbed from the digestive t r a c t and include amino acids, g l y c e r o l , and propionate. Another precursor, lac t a t e , may be absorbed from the g a s t r o i n t e s t i n a l t r a c t , synthesized i n the rumen epithelium, or derived i n i t i a l l y from glucose. Alanine and g l y c e r o l become more important as endogenous precursors of glucose during starvation. The pathways of glucose syn-thesis from these precursors are given i n Appendix Figure 9. 3. Renal Gluconeogenesis Renal gluconeogenesis accounts for approximately 8-10% of the t o t a l endogenous glucose production i n normal fed sheep and approximately 15% during fasting (Kaufman and Bergman, 1971, 1978). During prolonged starvation i n man renal gluconeogenesis can increase to 2 0% or more at the expense of hepatic gluconeogenesis (Exton, 1972). The renal increase i n gluconeogenesis i s the r e s u l t of p r e f e r e n t i a l removal of glutamine, which has been reported as the second most glucogenic amino acid, and which i s u t i l i z e d to reduce the acidosis which occurs during starvation (Snell and Duff, 1977). Since sheep suffer from similar metabolic problems 11 during starvation the kidneys probably play an important role i n maintaining an acid-base balance and increase gluco-neogenesis . The sum of glucose production from the l i v e r and kidney averages approximately 98% or v i r t u a l l y a l l of the body's glucose production. Precursors for Gluconeogenesis 1. V o l a t i l e Fatty Acids Many studies have shown that of the three v o l a t i l e fatty acids produced i n the rumen only propionic can contribute s i g n i f i c a n t l y to glucose synthesis (Annison et a l . 1963; Bergman et a l . 1966). There i s uncertainty as to the amount of glucose synthesis from propionate. At least part of the discrepancy may be attributed to the d i f f e r e n t sampling s i t e s chosen by d i f f e r e n t researchers. Bergman et a l . (1966) used an infusion of l a b e l l e d propionate into the rumen vein of non-pregnant, non-lactating sheep and reported that normal sheep absorb 29 mM of propionate per hour. These figures estimate that 50% of the propionate entering the portal bed i s converted to glucose accounting for 2 7% of the glucose entry rate. Leng 14 (1970) infused C-propionate into the rumen and estimated that 54% of the glucose arose from propionate. Following Pennington's early observations (1954) that propionate was converted to lactate i n the rumen epithelium, Leng (1967) reported that t h i s conversion was as high as 70%. 12 The two pathways t h a t have been proposed f o r the conver-s i o n of p ropionate are as f o l l o w s : A) p r o p i o n a t e i s absorbed a c r o s s the rumen e p i t h e l i u m i n t o the p o r t a l bed and i s converted to glucose i n the l i v e r v i a gluconeogenesis; B) propionate i s converted i n the rumen e p i t h e l i u m to l a c t a t e which then e n t e r s the p o r t a l v e i n and l i v e r where i t i s converted to g l u c o s e v i a gluconeogenesis. Low l e v e l s of l a c t a t e measured i n the p o r t a l v e i n of sheep i n d i c a t e t h a t o n l y n e g l i g i b l e amounts of propionate f o l l o w the second path-way (Roe e t a l . 1966). U t i l i z i n g d i f f e r e n t l y l a b e l l e d i s o t o p e s Leng e t a l . (1967) estimated t h a t 54% of the glucose e n t r y r a t e i s d e r i v e d from propionate i n the rumen whereas o n l y 32% of the propionate i s converted d i r e c t l y i n the l i v e r . The remainder o f the pro-p i o n a t e i s metabolized by the rumen e p i t h e l i u m . In summary the magnitude o f glucose d e r i v e d from pro-p i o n a t e (2 7-54%) appears to depend on whether the i n f u s i o n s are made i n the rumen (Leng et a_l. 1967) or i n the p o r t a l v e i n (Bergman e t a l . 1966). Recently Young (1977) has concluded propionate i s not converted to l a c t a t e i n the rumen e p i -t h e l i u m to any extent and t h a t 61% of the glucose s u p p l i e s c o u l d a r i s e from propionate i n cows. The amount of pro-p i o n a t e converted t o g l u c o s e i s f u r t h e r compounded by v a r i a -t i o n s i n d i e t w i t h h i g h g r a i n d i e t s i n c r e a s i n g both l a c t a t e and p r o p i o n a t e p r o d u c t i o n and a b s o r p t i o n (Sutton, 1976). 13 T h e o t h e r t w o v o l a t i l e f a t t y a c i d s h a v e b e e n e s t a b l i s h e d t o p l a y a r o l e i n l i p o g e n e s i s a n d s e r v e a s e n e r g y s o u r c e s i n s k e l e t a l m u s c l e s ( A n n i s o n e t a l . 1 9 6 7 ; J a r r e t t e t a l . 1 9 7 6 ) . A c e t a t e i s t a k e n up b y t h e l i v e r i n l i m i t e d a m o u n t s a n d a b o u t o n e h a l f i s o x i d i z e d f a i r l y r a p i d l y i n e x t r a h e p a t i c t i s s u e ( K r o n f e l d , 1 9 6 9 ) . M u c h o f t h e r e m a i n d e r i s u t i l i z e d f o r s y n t h e s i s o f l o n g c h a i n f a t t y a c i d s i n a d i p o s e t i s s u e a n d mammary g l a n d s . T h e r o l e o f a c e t a t e i n l i p o g e n e s i s i s c r u c i a l i n r u m i n a n t s f o r t h e y l a c k t h e e n z y m e s o f t h e c i t r a t e c l e a v a g e p a t h w a y s , e s s e n t i a l f o r l i p o g e n e s i s f r o m g l u c o s e ( B a l l a r d e t a l . 1 9 6 9 ) . B u t y r i c a c i d i s m e t a b o l i z e d a l m o s t c o m p l e t e l y d u r i n g i t s p a s s a g e t h r o u g h t h e r u m e n a n d o m a s a l e p i t h e l i u m y i e l d i n g m a i n l y b e t a - h y d r o x y b u t y r a t e (BOIIB) ( P e n n i n g t o n , 1952 , 1 9 5 4 ) . A n y b u t y r i c a c i d w h i c h e n t e r s t h e p o r t a l b l o o d i s e x t r a c t e d b y t h e l i v e r . BOHB i s o x i d i z e d e s p e c i a l l y i n c a r d i a c a n d s k e l e t a l m u s c l e s a n d i t i s u s e d f o r f a t t y a c i d s y n t h e s i s i n a d i p o s e t i s s u e a n d mammary g l a n d s ( K r o n f e l d a n d V a n S o e s t , 1 9 7 6 ) . B u t y r i c a c i d h a s b e e n d e s c r i b e d a s b e i n g g l u c o g e n i c b u t r e c e n t w o r k b y C o o k (1970) d e m o n s t r a t e d t h a t r a t h e r t h a n b e i n g g l u c o g e n i c BOHB a c t s t o s p a r e t h e u t i l i z a t i o n o f p y r u v a t e v i a t h e T . C . A . c y c l e . BOHB w h i c h c i r c u l a t e s , h a s b e e n s h o w n t o e n h a n c e p y r u v a t e c a r b o x y l a s e a n d h e n c e i n -c r e a s e t h e r a t e o f g l u c o n e o g e n e s i s ( P h i l l i p s e t a l . 1 9 6 9 ) . T h e BOHB i s a l s o o x i d i z e d b y m u s c l e t i s s u e w i t h t h e r e s u l t i t s p a r e s t h e o x i d a t i o n o f p y r u v a t e . B a i r d e t a l . ( 1977) 14 and J a r r e t t et a_l. (1976) have reported that during food deprivation BOHB and acetoacetate play important roles i n re-ducing glucose u t i l i z a t i o n and supplying energy to cardiac and s k e l e t a l muscles. The "Glucose Fatty Acid Cycle" was proposed by Randell et a l . (1963) who described that there was an inverse r e l a t i o n s h i p between fat t y oxidation and carbohy-drate metabolism i n cardiac muscle. Rennie and Holloszy (1977) reported that f a t t y acids cause a decrease i n peripheral glucose u t i l i z a t i o n . Fatty acids were shown to i n h i b i t glu-cose uptake, g l y c o l y s i s , glycogenolysis and pyruvate oxida-t i o n and lactate production i n peripheral s k e l e t a l muscles. In summary v o l a t i l e f a t t y acids, a c e t i c , butyric and propionic provide 6 0-80% of the metabolic energy i n ruminants (Kronfeld and Van Soest, 1976). They are either d i r e c t pre-cursors to glucose or supply the carbon skeleton for lipogenesis. One of the most important roles of BOHB and fatty acids i s one of sparing glucose u t i l i z a t i o n i n peripheral tissues e s p e c i a l l y during starvation, so that glucose i s available for obligatory organs. 2. Glycerol Glycerol i s released during the dynamic turnover of adipose tissue at a l l times, but e s p e c i a l l y during starvation when f a t supplies are being u t i l i z e d as energy. The free g l y c e r o l enters the gluconeogenic pathway at the Triose phosphate stage as i l l u s t r a t e d i n Appendix Figure 8. These findings 15 along with the high glycerokinase a c t i v i t y i n the l i v e r and kidney of ruminants indicate that g l y c e r o l may play an impor-tant role i n gluconeogenesis (Bergman, 1973). Bergman et a l . (1968) and R e i l l y and Ford (1971) using an infusion of 1 4C-g l y c e r o l demonstrated that fasted, ketotic and hypoglycemic sheep could provide up to 40% of t h e i r glucose requirements from the turnover of adipose tissue. On an average ketotic sheep derive about 28% of t h e i r glucose from g l y c e r o l . In the well fed ruminant however only 5% of the t o t a l glucose pool i s contributed by g l y c e r o l (Bergman et a l . 1968). 3. Lactate Lactate i s produced from anerobic metabolism of glucose, catabolism of amino acids, and the conversion of propionate i n the rumen epithelium. Under normal feeding very l i t t l e l actate enters or i s absorbed from the small i n t e s t i n e (Annison et a l . 1963). Leng (1970), Bergman (1973) and Dunlop (1972) have re-ported that 40% of the lactat e synthesized i s derived from g l y c o l y s i s , but only 4-10% of the glucose formed i n the l i v e r i s derived from la c t a t e . The production of glucose from lactate v i a the Cori Cycle does not cause a net increase i n glucose i f the lactate originated from glucose. The energy to drive t h i s cycle i s derived from amino acid and fatty, acid metabolism (Exton et a l . 1972). The conversion of lactate to pyruvate i n the l i v e r provides the NADK necessary for 16 gluconeogenesis. The lactate produced from propionate meta-bolism or absorbed through the rumen wall i n ruminants i s variable and d i f f i c u l t to quantify (Leng et a_l. 1967). Star-vation may cause an i n i t i a l decrease i n lactate production i n s k e l e t a l muscle due to the reduction i n glucose (Lindsay and Fle a t , 1974). Glucose produced from lactate may increase to 15% during starvation but not a l l of the lactate i s from glucose due to reduced g l y c o l y s i s (Exton, 1972). The kidneys play an important role i n lactate metabolism. Forty to sixty per cent of the renal glucose output i s derived from lactate (Kaufman et a l . 1974). Katz.and Bergman (1969b) reported that 15% of the t o t a l glucose was derived from the kidneys. This would mean that approximately 9% of the t o t a l glucose would come from lactate v i a kidney gluconeogenesis. Bergman et a l . (1970) reported lactate provided 4-10% of the l i v e r glucose production thus a t o t a l of 13-19% of glucose i s derived from la c t a t e . This agrees with the work of Annison et a l . (196 3) who reported using isotope d i l u t i o n techniques that lacta t e supplies approximately 19% of the t o t a l glucose pool. 4. Amino Acids When amino acids are used as fuels they undergo loss of th e i r amino groups; t h e i r remaining carbon skeletons then have two major fates: (1) conversion into glucose v i a the 17 process of gluconeogenesis, or (2) oxidation to C0 2 v i a the T.C.A. cycle. Amino acids converted into glucose f i r s t undergo enzymatic transformation to intermediates on the di r e c t pathway to glucose synthesis, such as pyruvate or the dicarboxylic intermediates of the T.C.A. cycle (Lehninger, 1974). Vertebrates a c t i v e l y oxidize both exogenous and endo-genous amino acids that are released from muscle. Almost a l l amino acids are glucogenic and ketogenic with the exception of leucine which i s ketogenic only. Leucine yields only acetyl CoA which can be converted to ketones, but not pyru-vate, whereas a l l other amino acids are metabolized to T.C.A. intermediates which are glucogenic and ketogenic. The rate and proportion of amino acid u t i l i z a t i o n depend on many fac-tors including the a v a i l a b i l i t y of other fuels as well as the n u t r i t i o n a l supply of esse n t i a l amino acids and the need for precursors for protein synthesis (Lehninger, 1974). Amino acids are converted to glucose i n the l i v e r and the cortex of the kidney. The l i v e r accounts for approximately 85% and the kidney 15% of the net glucose production from amino acids respectively (Bergman et a l . 1970). The incorporation of amino acids into the gluconeo-genic pathway i s given i n Appendix Figure 8. The rate of u t i l i z a t i o n i s dependent on the factors discussed previously. The assessment of the contribution of exogenous amino acids to glucose production i n ruminants i s d i f f i c u l t to quantify. 18 Assuming that 55g of glucose can be synthesized from 100 g of protein, Leng (1970) estimated that t h i s could provide 70% of the animals glucose requirements. This assumes amino acids are not metabolized i n the g a s t r o i n t e s t i n a l wall and therefore probably over estimates the contribution of dietary amino acids to gluconeogenesis. Ford and R e i l l y (1969) 1 4 u t i l i z i n g C-labelled amino acids estimated that 28% of the t o t a l glucose turnover was derived from amino acids i n sheep. Wolff et a_l. (1972a) employed two techniques to study the gluco-genicity of i n d i v i d u a l amino acids. The f i r s t method involved the net uptake of amino acids by the portal drained viscera and the l i v e r using the basic procedure of Katz and Bergman (1969c). Catheters were implanted i n the aorta and the po r t a l and hepatic veins for infusion and sampling. Blood flow i n the two veins was determined to follow net turnover of amino acids. P o s i t i v e values obtained from the po r t a l vein-a r t e r i a l differences indicate a net production whereas nega-t i v e values indicate u t i l i z a t i o n . Alanine was produced i n the greatest quantity accounting for 19% of the t o t a l net appearance of amino nitrogen i n the portal vein. The hepatic vein-portal vein concentration differences indicated i n a l -most a l l cases a removal of amino acids by the l i v e r . Alanine, glutamine and glycine accounted for 50% of the t o t a l amino nitrogen removed by the l i v e r . When hepatic uptake exceeded the gut output, Wolff and Bergman (1972a) postulated a net movement of these amino acids from peripheral tissues to the 19 l i v e r . The appearance of amino acids i n the portal blood i s the r e s u l t of dietary absorption and peripheral release of amino acids. The res u l t s from the previous study indicate a large hepatic uptake of alanine (3.2 mM/hr) and glutamine (2.1 mM/hr) which are consistent with the glucose-alanine cycle proposed by F e l i g et a l . (1970) and F e l i g (1973). The second approach to study the glucogenicity of amino acids i n ruminants u t i l i z e d r a d i o a c t i v e l y l a b e l l e d amino acids. 14 Wolff and Bergman (1972b) infused U- C-labelled alanine, aspartate, glutamate, glycine and serine into the vena cava for 6 hours. U t i l i z i n g the method of Katz and Bergman (1969c) samples were taken from the aorta and the por t a l and hepatic veins i n conjunction with blood flow measurements. Alanine contributed 5.5% of glucose turnover, aspartate 0.6%, gluta-mate 3.4%, glycine 0.9% and serine 0.7% giving a t o t a l of 11%. The res u l t s were not corrected for cross over i n the T.C.A. cycle which could a l t e r t h e i r respective contributions. 14 14 Heitemann et a l . (1973) u t i l i z e d U- C-glutamate and U- C-serine for the estimation of gluconeogenesis. The ov e r a l l results showed that the fi v e amino acids could supply 11% of the blood glucose while a maximum of 30% of the t o t a l glucose could be provided by other plasma amino acids. The conversion of alanine and glutamine to glucose accounted for 40% of the glucogenicity of a l l the amino acids. Alanine alone accounted for 8% of the t o t a l glucose turnover i n sheep under normal conditions. 20 F e l i g (1973) examined the ro l e of alanine as i t relates to glucose homeostasis i n man. In view of the fact that alanine was the p r i n c i p a l amino acid released by muscles (Felig et a l . 1970) a Glucose-Alanine Cycle was proposed. The de novo synthesis of alanine occurs i n the peripheral muscles by the transmination of pyruvate. The glucose-alanine cycle not only provides the carbon skeleton for gluconeogenesis but also permits the removal of ammonia from peripheral muscle - tissue to the l i v e r and kidney where i t can be disposed of as urea or ammonium s a l t (Appendix Figure 9). Further research has been car r i e d out to determine the sources of pyruvate for transamination to alanine and to quanti-fy the release of alanine. Many workers have studied meta-bolism i n in d i v i d u a l muscles or groups of muscles such as di a -phragms, hind limbs or legs and arms of man, ra t s , and sheep (Felig and Wahren, 1974; Owen and Reichard, 1971; Ruderman et a l . 1977; Cross et a l . 1974; and Ballard et a l . 1976). Since alanine i s highly glucogenic, studies have been performed on fed and starved animals to determine the extent of de novo synthesis of alanine when the animal r e l i e s on increased gluconeogenesis to meet the glucose requirements. The use of alanine i s es p e c i a l l y important i n ruminants where gluco-neogenesis supplies almost a l l of the glucose requirements. 21 Alanine-Glucose Interrelationships The previous discussion on amino acid metabolism dealt with the establishment of the glucose-alanine cycle. Quanti-t a t i v e techniques used for estimating the uptake or release of glucose and alanine from ske l e t a l muscle under varying n u t r i -t i o n a l conditions are discussed i n the following section. The i s o l a t e d hind limb of rats has been used extensively to assess the uptake or output of metabolites c h a r a c t e r i s t i c of peripheral muscle metabolism (Adibi et a l . 1976; Goldstein and Newsholme, 1976; S n e l l , 1976; Grubb et a l . 1976; Steel and Duff, 1977). Using the perfused rat hind limb Goldstein and Newsholme (1976) and Snell (1976) reported that alanine accounts for 50% of the amino nitrogen released by s k e l e t a l muscle. The transmination of pyruvate in muscle i s only possible to any extent with glutamate v i a alanine amino-trans-ferase. The glutamate then undergoes oxidative deamination and the ammonia moiety i s converted to the amide portion of glutamine. This accounts for the increase i n glutamine release i n conjunction with alanine. The kidneys normally release glutamine but can take up glutamine during pregnancy toxemia and metabolic acidosis (Bergman, 1973). The amide portion of glutamine i s removed by hydrolysis i n the kidneys and i s u t i l i z e d to increase the release of acid by the animal + + by combining with H and forming NH^ . The carbon skeleton i s then u t i l i z e d for gluconeogenesis. Gluconeogenesis from 22 the kidneys increases during starvation, while hepatic gluconeogenesis decreases (Exton, 1972). Odessey et a l . (1974) and Snell and Duff (1977) reported that the hind limb of rats p r e f e r e n t i a l l y catabolized branched chain amino acids and the l i v e r p r e f e r e n t i a l l y releases these amino acids. The flow of leucine, isoleucine and valine from the l i v e r to muscles i s i l l u s t r a t e d i n Appendix Figure 8. The catabolism of these three amino acids provides 8 ATP from the glucose-alanine cycle and i f the remaining carbon skeletons are f u l l y oxidized i n the muscle they could provide an additional 42, 43 and 32 moles of ATP respectively. This cycle could therefore provide up to 14% of the energy supplies of s k e l e t a l muscle (Odessey et a_l. 1974). Ruminants may d i f f e r s i g n i f i -cantly i n t h e i r a b i l i t y to use branched chain amino acids i n s k e l e t a l muscle. Making use of the i s o l a t e d hind limb of sheep, Ballard et a l . (1976) reported that there was an output of branched chain amino acids during starvation. They suggested that sheep muscle may lack the a b i l i t y of oxidize branched chain amino acids. C o n t r a r i l y , Cross et a l . (1974) using i s o l a t e d perfused sheep hind limbs reported a decrease i n the release of branched-chain amino acid and an increase i n that of alanine from muscle. Apparently the a b i l i t y of sheep muscles to oxidize branched chain amino acids needs further investigation. F e l i g et a l . (1969) and F e l i g and Wahren (1974) reported a reduction i n muscle amino acid metabolism during an extended 23 starvation i n man. This was attributed to a reduction i n pro-t e i n catabolism which would minimize protein wasting. Con-comitantly an increase i n ketone and f a t t y acid metabolism occurred to meet the energy requirements. Steel and Leng (1973) using sheep measured fa s t i n g energy metabolism a f t e r 4 to 6 days of starvation and showed that 75% of the energy was derived from oxidation of fat whereas 25% of the energy arose from the oxidation of amino acids. The increase i n fa t t y acid metabolism i n peripheral tissues as an energy source during starvation not only decreases muscle tissue cata-bolism but also reduces peripheral glucose u t i l i z a t i o n and t h i s i s referred to as the glucose-fatty acid cycle (Randell et a_l. 1963). Alanine i s the p r i n c i p a l amino acid released by peripheral muscle during starvation and i s most e f f e c t i v e l y extracted by the l i v e r (Felig, 1973). Using starved rats Ruderman et a l . (1977) , "' MacDonald et a l . (1976), and Grubb and Snaer (1976) reported that alanine accounted for 22-25% of the amino acids released by peripheral muscles. Sheep starved for four days released from peripheral muscles 24% of t h e i r amino acids as alanine (Lindsay et a l . 1977a). These two studies indicate that under short term starvation rats and sheep release alanine from peripheral muscles i n greater quan-t i t i e s than any other amino acid. The alanine i s used for gluconeogenesis which provides approximately 26% of the t o t a l glucose pool during starvation i n man (Fe l i g , 1973) whereas i n sheep alanine provides approximately 10 to 15% of the glu-cose pool (Lindsay et a l . 1977b; Wolff and Bergman, 1972b). 24 In summary the amino acid contributions to gluconeo-genesis depend on many factors. In ruminants gluconeogenesis occurs almost continuously i n contrast to non-ruminants. Both in ruminants and non-ruminants starvation increases gluconeo-genesis to meet the demands of obligatory organs. Variable figures have been quoted i n the l i t e r a t u r e on the contribu-tion of amino acids to glucose production. Bergman (1973), Baird et a l . (1972, 1977) and Leng (1970) have reported that 30-50% of the glucose turnover i s accounted for by amino acids in ruminants. According to Wolff and Bergman (1972c), Brockman and Bergman (1975), Cross et a_l. (1975), Lindsay et a l . (1977b) alanine supplies 6 - 8% of the glucose turnover on a maintenance di e t and 10 - 15% during starvation. The production of a,lanine decreases as starvation i s continued over a prolonged period to prevent protein wasting i n muscles (Sherwin et a l . 1975). In ruminants amino acids, s p e c i f i c a l l y alanine, there-fore play an important role i n the maintenance of glucose v i a gluconeogenesis under varying dietary conditions. These f i n d -ings agree with the work done i n man and rats as reviewed by F e l i g (1973) and Exton (1972). Blood Flow The net u t i l i z a t i o n and production of metabolites reviewed i n the previous section have been obtained by the determination of blood flow using d i f f e r e n t methods. In the following section 25 the methods u t i l i z e d by several workers for the determination of blood flow are reviewed. A d i s t i n c t i o n can be made between indicator d i l u t i o n and indicator transport techniques. The d i l u t i o n techniques are based on indicators which remain i n the blood stream but are d i l u t e d by mixing. Transport.tech-niques depend on the c a l c u l a t i o n of the blood volume required to d e l i v e r to or remove the indicator from a p a r t i c u l a r organ or tissue (Woodcock, 1975). Indicator D i l u t i o n Techniques Dye d i l u t i o n i s a general term applied to the use of many b i o l o g i c a l l y i n e r t dyes (Evans blue and Indocyanine green) which can c i r c u l a t e i n the blood stream. The system i s based on a single input and single output with very l i t t l e loss of the dye into the tissue over the sampling period. The system contains a s p e c i f i c volume of dye which enters and r e l i e s on a constant flow rate. Such a system depends on a number of assumptions: (1) the p a r t i c l e s entering the system at any time are dispersed when they e x i t i n exactly the same manner as p a r t i c l e s entering at any other time; t h i s property i s referred to as s t a t i o n a r i t y of flow; (2) the flow of the i n -dicator p a r t i c l e s i s representative of the flow of t o t a l f l u i d ; and (3) r e c i r c u l a t i o n of the indicator i s not present (Meier et a l . 1954). Further c r i t e r i a for dye d i l u t i o n have been put forward by Schenk and Race (1968) as described below. 26 (1) there be an instantaneous i n j e c t i o n of the dye; (2) ' there be a complete mixing of the dye with a l l the blood flowing past the i n j e c t i o n s i t e ; (3) that there i s no contamination at sampling s i t e with undyed blood; and (4) a steady state condition exists between the time of i n j e c t i o n and the end of the concentration curve. Though these c r i t e r i a can be met while measuring blood flow i n the cardiac chambers and associated large vessels they cannot be s a t i s f i e d i n the case of peripheral vessels leading to inaccuracy. The dye does not completely mix with blood by the f i r s t branching point i n the vessel and the consequential lower recovery of the dye indicates a higher flow rate (Schenk and Race, 1968). A comparison between indocyanine green and an electromagnetic cuff (discussed l a t e r i n the text) to deter-mine blood flow i n the femoral and carotid a r t e r i e s i n large dogs resulted i n a considerable v a r i a t i o n between the two systems. There was found to be no co r r e l a t i o n between the two measurements and the indocyanine green estimates were 50 to 100% above the electromagnetic method. J a r r e t t et a l . (1976) employed the indocyanine green procedure and found blood flow i n the femoral artery of sheep to be 195 ml/min. Using electromagnetic cuffs Kung Ming and Chien (1977) and Schenk and Race (1968) found the blood flow i n the femoral artery of dogs to be approximately 112 ml/min. 27 The major error i n u t i l i z i n g the dye d i l u t i o n method i s that i t does not account for the various branchings within the peripheral tissue and that i t r e l i e s on a steady state blood flow which does not e x i s t . Indicator Transport The indicator transport systems are primarily used for determining blood flow through an organ or t i s s u e . The a b i l i t y of an organ to remove indicators s e l e c t i v e l y forms the basis of the determination of blood flow to the kidney using para amino-hippuric acid (PAH) and to the l i v e r using both bromo-sulphalein (BSP) and Rose Bengal. Radioactive compounds such 135 85 32 as iodine, krypton and phosphate have also been employed to determine blood flow to s p e c i f i c organs or tissue. The use of PAH by Katz and Bergman (1969a) involved a continuous infusion u n t i l a constant concentration was reached i n the e x t r a - c e l l u l a r f l u i d . A plateau l e v e l was attained since PAH was rapidly and t o t a l l y excreted by the kidneys provided that the infusion rate was less than the maximal a b i l i t y of the kidney to excrete PAH. The major advantages of t h i s method include (1) the timing of the sample c o l l e c -t i o n i s no longer c r i t i c a l , (2) a greater s e n s i t i v i t y i s achieved due to a greater venoarterial concentration d i f f e r -ences (Katz and Bergman, 1969a). These same workers used PAH along with indwelling catheters i n the hepatic and po r t a l veins and c a r o t i d artery to measure blood flows i n 28 these vessels. Kaufman and Bergman (1971, 1978) made use of PAH to determine blood flow i n the renal artery under d i f f e r e n t conditions of feeding, f a s t i n g and pregnancy. The methods permitted the measurement of turnover rates of several meta-b o l i t e s . The r e s u l t s also indicated that during starvation there was a 28% reduction i n p o r t a l blood flow and a 20-30% reduction i n renal blood flow. Carr and Jacobson (1968) using a Doppler Flowmeter on the po r t a l vein i n calves reported a blood flow of 41 ml/min/kg body weight. Katz and Bergman (1969a) reported a blood flow of 45 ml/min/kg body weight i n the portal vein of sheep using the PAH method. It appears that the PAH method agrees favourably with Doppler Flowmeters i n the measurement of p o r t a l blood flow. A major disadvantage of PAH as an indicator of blood flow i s that i t does not take into consideration the psychological excitement and resultant increased blood flow due to sampling. I t r e l i e s on a steady state condition and cannot be used continuously to determine blood flow over a prolonged period. Since muscles regulate blood flow l o c a l l y accurate measurement of peripheral blood flow becomes very d i f f i c u l t (Scott et a l . 1965) . Microspheres Radioactive microspheres u t i l i z e carbonized microspheres 85 which are 15 ± 5 urn i n diameter l a b e l l e d with Strontium, 1.41 Cerium or other radioactive compounds. The same p r i n c i p l e 29 as r a d i o a c t i v e compounds used alone a p p l i e s t o measurement of b l o o d flow u s i n g microspheres. A c c o r d i n g to the F i c k p r i n c i p l e the amount Qi of any substance c a r r i e d i n the blood stream t r a n s p o r t e d i n t o a p a r t i c u l a r r e g i o n i n time Dt must be equal to the q u a n t i t y s t o r e d , Qs i n Dt, p l u s the q u a n t i t y converted, Qm i n Dt, plus the amount t r a n s p o r t e d away from the r e g i o n , Qo i n Dt: Q i _ Qs Qm Qo Dt Dt Dt Dt I f the a r t e r i a l and venous flows are equal and c o n s t a n t the flow can be c a l c u l a t e d from a r t e r i a l c o n c e n t r a t i o n (Ca) or r a d i o a c t i v i t y and venous c o n c e n t r a t i o n (Cv) or r a d i o a c t i v i t y . For example the blood flow (F) to the kidney may be determined by knowing Qu, amount of i n d i c a t o r i n u r i n e i n Dt, Ca and Cv i n Dt, i n the r e n a l a r t e r y and v e i n (Woodcock, 19 75). T h e r e f o r e Qu F(Ca-Cv) Dt B e l l e t a l . (1975) employed r a d i o a c t i v e microspheres to d e t e r -mine blo o d flow to i n d i v i d u a l muscles i n the hind limb of the young ox. The animals i n t h i s experiment were s a c r i f i c e d and the i n d i v i d u a l muscle uptake of microspheres was determined to y i e l d b l o o d flow to each muscle u s i n g a s i m i l a r formula. The use o f r a d i o a c t i v e microspheres or r a d i o a c t i v e com-pounds r e s u l t s i n extremely accurate measurements.of blo o d flow. The disadvantages l i e i n the f a c t t h a t the animals have to be s a c r i f i c e d b e f o r e samples can be c o l l e c t e d . The o t h e r 30 major problem i s the i n a b i l i t y to repeat blood flow measure-ments on the same animal u n t i l the r a d i o a c t i v i t y i s eliminated. Thermal D i l u t i o n Techniques The p r i n c i p l e behind the use of thermal techniques i s based on the measurement of heat conduction. It i s assumed that any condition leading to a loss or gain of heat i n the blood stream depends on, among other things, i t s volume flow. As an example i f a heat source i s placed i n the blood stream i n the form of an e l e c t r i c heating element d i s s i p a t i n g heat at a constant rate, the temperature change down stream w i l l depend on the rate of flow (Leraand, 1968). The major advantages of t h i s method are the small size of the heating element and the a b i l i t y to c a l i b r a t e i n v i t r o , using the blood of the animal to be investigated. The major disadvantage i s that the blood temperature must be known accurately before c a l i b r a t i n g the increase i n temperature. Furthermore c l o t t i n g occurs at slow flow rates owing to heat production. The thermal d i l u t i o n techniques of introducing cold saline into a vein and measuring the temperature change i n an artery with a small temperature sensor has also been u t i l i z e d to determine blood flow (Webster, 1973, 1974; Leraand, 1968). The advantages are si m i l a r to the previous system without the problems of blood c l o t t i n g . The errors that a r i s e from using t h i s system are: (1) the s p e c i f i c heat of the blood/ saline solution may be altered because of actual i n j e c t i o n ; 31 (2) long term h e m o d i l u t i o n may occur (hemotocrit d r o p s ) ; (3) a change i n h e a r t r a t e and i n c r e a s e i n b l o o d p r e s s u r e may a r i s e from the s a l i n e i n j e c t i o n ; and (4) an e r r o r may occur i n the amount o f s a l i n e i n j e c t e d and the temperature due t o warming o f the c a t h e t e r i n the b l o o d (Woodcock, 19 75). Flowmeters The i n c r e a s i n g demand f o r continuous i n v i v o b l o o d flow m o n i t o r i n g r e s u l t e d i n the development of two types of flow-meters wi t h s e v e r a l v a r i a t i o n s . The design of each system attempted to s a t i s f y the f o l l o w i n g c r i t e r i a : ease o f o p e r a t i o n , i n s e n s i t i v i t y o f environment, s e n s i t i v i t y w i t h i n the range o f measurement, accuracy o f c a l i b r a t i o n , l i n e a r i t y , and a low n o i s e so as not to confound the s i g n a l (Cappelen, 1968; F r a n k l i n e t a l . 1964). The systems evolved are the E l e c t r o -magnetic Flowmeter and the Doppler Flowmeter. These two methods of determining b l o o d flow w i l l be d i s c u s s e d s e p a r a t e l y w i t h emphasis g i v e n to the Doppler Flowmeter s i n c e t h i s system was u t i l i z e d i n these experiments. 1 . E l e ctroma gne t i c F1owme te r s The e l e c t r o m a g n e t i c flowmeter operates under the p r i n c i p l e t h a t i f a magnetic f i e l d i s a p p l i e d at r i g h t angles t o the d i r e c t i o n of motion of a conducting f l u i d a p o t e n t i a l d i f f e r e n c e w i l l be s e t up at r i g h t angles t o both flow and magnetic f i e l d (Woodcock, 1976) (Appendix F i g u r e 10). The s i g n a l p i c k e d up 32 by the electrode i s an in d i c a t i o n of the d i r e c t i o n and rate of flow. Electromagnetic flowmeters have been used since 1930 i n measuring blood flow i n animals. They have been de-signed i n many shapes and at present the A.C. current e l e c t r o -magnetic cuff probes are u t i l i z e d to a greater extent in i n vivo experiments. Dobson et a l . (1966) calibr a t e d electro-magnetic cuffs i n vivo using a T-piece cannula inserted into the carotid artery of sheep. The cannula was connected to. a known volume of blood which was injected into the cannula and measured by the flowmeter. On large vessels such as the aorta the c a l i b r a t i o n i s carried out by comparison with other techniques such as indicator d i l u t i o n . In v i t r o c a l i b r a t i o n s were carr i e d out by Beck et a l . (1965) using polyethylene tubing with flow rates ranging from 0-500 ml/min. Schenk and Race (1968) compared electromagnetic flowmeters with dye d i l u t i o n for peripheral blood flow and reported that dye dilution over estimated electromagnetic blood flow by 50 to 100%. The advantages of the electromagnetic flowmeters are that they can be calibrated i n vivo and i n v i t r o . They give true flow with a r e p r o d u c i b i l i t y within ±5-6% which results i n a l i n e a r c a l i b r a t i o n curve. The d i f f i c u l t i e s which follow the use of electromagnetic flowmeters are not considered insurmountable. Tissue growth may cause d i s t o r t i o n of the magnetic f i e l d and f l u i d between the cuff and vessel may produce an additional shunt which a l t e r s the output. To prevent t h i s from happening a s l i g h t l y smaller cuff i s placed on the vessel 33 which may i n turn cause some alter a t i o n s i n blood flow. Elec-t r i c a l equipment near the cuff may also r e s u l t i n interference. The most inconvenient problem i s that the cuffs are generally large and d i f f i c u l t to implant. In summary the electromagnetic flowmeter i s an accurate device for measuring blood flow i n both large and small vessels. The results are reproducible and l i n e a r . The use of el e c t r o -magnetic flowmeters enables investigators to monitor blood flow over a prolonged period of time while the animal i s calm and not affected by sampling. 2. Doppler Flowmeter A Doppler flowmeter i s a v e l o c i t y device which u t i l i z e s the frequency of ultrasound backscattering from c e l l s moving within blood as a measure of the blood v e l o c i t y . I f the diameter of the vessel remains the same the v e l o c i t y i s proportional to the blood flow (Vatner, 1970). The change i n frequency between the i n i t i a l s i g n a l from the flow cuff and the returning frequency which has been changed due to blood c e l l s i s referred to as the Doppler S h i f t , described by the equation: (F -F ) = F = 2F VCOS a/c e r e . ' F = absolute difference i n frequency between emitted signal frequency F g and received frequency F r . V = v e l o c i t y scattering medium. 34 a = angle between accoustical axis and blood v e l o c i t y . c = Veloci t y of sound i n blood. This formula i s the basis for a l l Doppler Flowmeters (Franklin et a l . 1963). Carr and Jacobson (1968) s i m p l i f i e d the equa-tio n to where blood flow Q i s : Q = FO ( d 2 ) (1.414)(10- 2) FO = frequency s h i f t i n cycles/sec. d = diameter of vessel. This i s applicable when using a cuff that i s secure and surrounds the vessel snugly. There are surface or skin mounted flowmeters that take into consideration the equation by Franklin et a l . (1963) . The advantages of the Doppler Flowmeter are that (1) i t can be cal i b r a t e d i n v i t r o using blood from the same species of animal; (2) i t i s small and e a s i l y implanted around the vessel causing l i t t l e obstruction; (3) i t gives continuous readings under various conditions; (4) the standardization of the cuffs r e s u l t s i n a li n e a r response; and (5) the choice of frequency r e s u l t s i n a very low noise l e v e l . Johnston et a l . (1977) describes several d i f f i c u l t i e s that arise with the use of a Doppler Flowmeter. These d i f f i c u l t i e s are (1) the v e l o c i t y p r o f i l e across the artery i s complex from f l a t to parabolic; (2) noise may be s i g n i f i c a n t on the output of the si g n a l ; (3) artery wall movements may cause a r t i f a c t s ; (4) the output trace i s usually smoothed by a low band pass f i l t e r which may prevent the recorder from r e g i s t e r i n g transient changes; and (5) back flow cannot be distinguished from forward flow. Johnston et a l . (1977) recommended the following procedures to eliminate these problems; (1) f a s t f o u r i e r transformation; (2) time compression analysis; (3) continuous p a r a l l e l f i l t e r s (4) analogue processing of o r i g i n a l Doppler s i g n a l ; and (5) a phase-locked loop technique. Doppler Flowmeters have been u t i l i z e d by several workers i n d i f f e r e n t areas of research with minor modifications from the basic system to f i t t h e i r needs. Franklin et a l . (1964, 19 66) used telemetry and a Doppler cuff to examine the changes in v e l o c i t y of blood flow i n unrestrained animals rather than determining actual blood flow. At present, i n cardiovascular surgery the Doppler Flowmeter i s used to determine i f there are a r t e r i a l or venous co n s t r i c t i o n s . It has been e s p e c i a l l y useful for lower limb and carot i d artery occlusions. Instead of placing a cuff around the vessel, which would be impracti-c a l i n human studies, a skin mounted electrode i s u t i l i z e d . The problem exists i n achieving the appropriate angle of the electrode to the vessel and the p o s s i b i l i t y of vessels below causing interference with the vessel i n question (Johnston et a l . 1977). The use of a cuff mounted Doppler Flowmeter to determine v e l o c i t y changes as an indicator of blood flow has been explored by Vatner (1970), Kayser (1966) and Sullivan and Tucker (1975). Actual blood flow measurements using a Doppler Flowmeter and telemetry were determined by Carr and Jacobson (1968) 36 using cuffs on the portal vein of unrestrained calves. This method resulted i n -blood flow determinations of 41 ml/min/ kg. Berman et a l . (1975) using a cuff on the umbilical vein of f e t a l lambs determined that 199 ml/min/kg passed through t h i s vessel. Sullivan and Tucker (1975) used anesthetized rabbits and followed uterine blood flow during various stages of pregnancy. Due to the accuracy of the Doppler Flowmeters and the l i m i t e d l i t e r a t u r e available on femoral blood flow measure-ments in sheep over prolonged periods the Doppler system was standardized and u t i l i z e d i n t h i s study. EXPERIMENTAL EXPERIMENT I  INTRODUCTION Changes i n the femoral arterio-venous differences i n the concentration of metabolites represent the metabolism of substrates mostly by the sk e l e t a l muscles i n the hind limb. The techniques for the implantation of catheters i n d i f f e r e n t blood vessels of sheep by Katz and Bergman (1969c) have f a c i l i t a t e d the c o l l e c t i o n of blood without stress i n meta-b o l i c studies i n selected regions of the animal. These studies coupled with the determination of blood flow yielded valuable information on the turnover of amino acids, glucose, and l a c t a t e , under fed and starved conditions. Glucose has been studied extensively because of i t s r o l e as an energy source and a precursor to many products. With 14 the advent of the C l a b e l l e d glucose the products of glucose metabolism have been studied by many authors. The estimation of lactate and alanine production has given r i s e to quantita-t i v e measurements of rec y c l i n g through the Cori Cycle and glucose-alanine cycle, respectively. As described e a r l i e r various methods have been u t i l i z e d for the determination of peripheral blood flow. However the 38 d i f f i c u l t y w i t h most methods i s t h a t they assume o n l y a con-s t a n t blood flow and do not take i n t o c o n s i d e r a t i o n f l u c t u a -t i o n s due t o n u t r i t i o n a l or environmental f a c t o r s over a prolonged p e r i o d . The o b j e c t i v e o f t h i s experiment was to develop a s u i t -a ble technique which may be u s e f u l i n m o n i t o r i n g changes i n the blood flow to the h i n d limb o f sheep over a prolonged p e r i o d . Simultaneously the uptake or r e l e a s e of m e t a b o l i t e s f o l l o w i n g s t a r v a t i o n was a l s o s t u d i e d to q u a n t i t a t e the meta-b o l i c processes i n v o l v e d i n the maintenance o f glucose homeo-s t a s i s . MATERIALS AND METHODS 1. Animals and N u t r i t i o n a l Regime Two Dorset ewes weighing 61 kg and 50 kg were used i n these experiments. They were maintained i n metal pens and fed 600 g o f a l f a l f a cubes twice d a i l y a t 0800 and 1500 h r s . Water was a v a i l a b l e ad l i b i t u m . Proximate analyses of feed and feces were c a r r i e d out Using a m i c r o - K j e l d a h l b l o c k d i g e s t e r * . T h i s method o f a n a l y s i s was compared t o the a s s o c i a t i o n o f o f f i c i a l a g r i c u l t u r a l chemists (1970) and proved t o be a s a t i s f a c t o r y method. N i t r o g e n and phosphorus were determined on a Technicon auto a n a l y z e r I I u s i n g the i n d u s t r i a l •Courtesy o f Dr. J.A. S h e l f o r d 39 methods No. 327-74W and No. 321-74A, respectively. Calcium was determined using a Unicam SP-90 atomic absorption spectro-photometer. 2. Implantation of Catheters Catheters were introduced into the femoral artery.and vein using the surgical procedure described below. The sheep were transferred from the pens to metabolism cages four to six days p r i o r to surgery to allow them to become adjusted to the environment. Feed was withheld from the animals for 4 8 hr p r i o r to surgery but water was available ad libitum. The sheep were shorn leaving a length of about 1 to 2 cm of wool on t h e i r body on the day pr i o r to surgery. T h i r t y minutes before the s t a r t of surgery A t r a v e t 1 (1 ml-25 mg/ml) was injected intramuscularly to calm the animals. At thi s time a subcutaneous i n j e c t i o n of Atropine 2 s u l f a t e (5 ml, 0.6 mg/ml) was also given to reduce s a l i v a r y secretion during surgery. The ventral abdominal wall and the medial portions of both legs were washed with iodine soap. The sheep were 3 anesthetized with a 10% solution of Pentothal Sodium at A t r a v e t (Acepromazine maleate), Ayerst Laboratories Ltd. 2Atropine, Clarke-Rogers Ltd., Abbotsford, B.C. 3Pentothal Sodium (Sodium Thiopental), Abbott Laboratories Ltd. 40 25 m g / k g b o d y w e i g h t v i a a n i n d w e l l i n g j u g u l a r c a n n u l a . F o u r m l o f t h e d r u g w e r e a d m i n i s t e r e d q u i c k l y f o l l o w e d b y o n e m l e v e r y t w e n t y s e c o n d s o r u n t i l t h e a n i m a l h a d n o e y e r e f l e x a n d t h e t e n s i o n o f t h e j a w m u s c l e s was m i n i m a l . T h e s h e e p w e r e p u t r n a p r o n e p o s i t i o n a n d a n u m b e r 8 e n d o t r a c h e a l t u b e was i n t r o d u c e d w h i c h was c o n n e c t e d t o a h a l o t h a n e 1 g a s a n d o x y g e n c l o s e d c i r c u i t a n e s t h e t i c m a c h i n e . T h i s p e r m i t t e d t h e c a r e f u l c o n t r o l o f t h e r e s p i r a t i o n r a t e a n d d e p t h o f a n e t h e s i a . T h e a n i m a l s w e r e t h e n p l a c e d i n a s u p i n e p o s i t i o n a n d t h e l e g s o f t h e a n i m a l w e r e s e c u r e d t o t h e o p e r a t i n g t a b l e . T h e t w o l e g s w e r e a g a i n d i s i n f e c t e d w i t h a t i n c t u r e o f i o d i n e s o a p a n d f i n a l l y w i t h Z e p h r i n e 2 . T h e t w o c r a n i a l h e a d s o f t h e S a r t o r i u s m u s c l e on t h e l e f t l e g w e r e p a l p a t e d a n d a 10 cm i n c i s i o n was made t h r o u g h t h e s k i n p a r a l l e l t o t h e m u s c l e . T h e m u s c l e s w e r e s e p a r a t e d w i t h f o r c e p s a n d t h e f e m o r a l a r t e r y a n d v e i n w e r e i d e n t i f i e d . T h e f a s c i a a r o u n d t h e a r t e r y was d i s s e c t e d a t a b o u t 4 cm f r o m t h e b r a n c h p o i n t o f t h e d e e p f e m o r a l a r t e r y . To p r e v e n t b l e e d i n g t h e f e m o r a l a r t e r y a n d v e i n w e r e l i g a t e d p o s t e r i o r t o t h e p o i n t o f c a n n u l a i m -p l a n t a t i o n . P o l y e t h y l e n e c a n n u l a s ( 0 . 1 2 7 cm O . D . x 0 . 0 8 6 cm I . D . ) w e r e i n s e r t e d i n t o e a c h v e s s e l t o a d i s t a n c e o f 10 c m . T h e t i p s o f t h e c a n n u l a e w e r e i n t h e i l i a c a r t e r y a n d v e i n a s shown i n A p p e n d i x F i g u r e 12 A , B . E a c h c a n n u l a was s e c u r e d t o t h e s u r r o u n d i n g f a s c i a w i t h s i z e 0 s i l k s u t u r e s . T h e s e p a r a t e d m u s c l e s w e r e t h e n s u t u r e d w i t h s i z e 2 - 0 c h r o m i c i H a l o t h a n e , A y e r s t L a b o r a t o r i e s . 2 Z e p h r i n e , U n i v e r s i t y o f B r i t i s h C o l u m b i a , H e a l t h S e r v i c e s P h a r m a c y . 41 catgut sutures. The cannulae were then ex t e r i o r i z e d under the skin approximately 2 0 cm posterior to the l a s t r i b on the dor-sa l l e f t side of the animal. 3. Cuff Placement Blood flow to the hind limb was determined by placing a Doppler 1 transducer cuff securely around the femoral artery i n the r i g h t leg. This was done to avoid possible d i s t o r t i o n of the blood flow because of the presence of the cannula i n the l e f t leg (Appendix Plate 1). The procedure,to expose the r i g h t femoral artery was i d e n t i c a l to the previous procedure. A Doppler transducer blood flow cuff of suitable s i z e , 3 mm or 4 mm c u f f , was placed around the artery and t i e d i n place, approximately at the same location as the cannula on the l e f t leg. The leads of the cuff were exteriorized to the r i g h t side of the animal under the skin p a r a l l e l to the cannula. Both the cannula and cuff leads were placed i n separate canvas pouches sutured to the animals with size 2 s i l k . The cannulae were kept patent during the recovery period by periodic flushing with heparinized saline (1,000 units/ml). Penalong-S 2, an a n t i b i o t i c , was injected (5 ml) intramuscularly for four days to reduce i n f e c t i o n . The sheep were kept i n ^•Parks E l e c t r o n i c s , Portland, Oregon. 2Penalong-S, Rogar/STB D i v i s i o n of BTI Products, Inc. 42 metabolic cages during the recovery period and a minimum of four days after surgery elapsed before the experiments were started. 4. Experimental Period In t h i s study the effects of starvation on the blood flow to the hind limb and metabolism of substrates were investigated. Two ewes (Nos. 2 42 and 39) were starved for four days and then returned to the prestarvation ra t i o n . Blood samples were ob-tained four to f i v e times on each day of the experiment at 0730, 0930, 1130, 1430 and 1630 hrs. Dopper blood flow read-ings were taken before each blood sample was taken. 5. Chemical Determinations Glucose and alanine determinations were done on depro-tein i z e d plasma samples. Whole blood was f i r s t centrifuged and the plasma was deproteinized by the addition of 10% ZnSO^ and 0.5N NaOH i n the following proportions: 1.7 ml H20 0.1 ml 0.5N NaOH 0.1 ml 10% ZnSO. 4 0.1 ml plasma. The ZnSO^ and NaOH were t i t r a t e d against each other i n order to ensure n e u t r a l i t y . A f t e r thorough mixing, the above preparation was centrifuged at 4°C for ten minutes at 3,000 rpm. 4 3 a) Glucose The glucose concentration was determined enzymatically by the glucose oxi d i z a t i o n method (Worthington Biochemicals). This test makes use of the coupled enzyme reactions. Glucose + 0, + H-0 ^ l u*: o s e> H 20, + gluconic acid 2 2 Oxidase 2  ^ P G i r o x i d s s © H 20 2 + reduced chromogen oxidized chromogen (color) A micro method of analysis was employed to determine the glucose concentration. One hundred yl of the glucostat rea-gent were combined with 600 y l of phosphate buffer (pH 7.0) and 300 pi of deproteinized plasma samples. The mixture was then placed i n a water bath for 30 min at 37°C. The reaction was stopped with two drops of 4N HC1 and read i n the LKB spectrophotometer at 40 8 nm. These readings were converted to mg/100 ml plasma by comparison with a standard curve (Appendix Figure 1) prepared from known glucose concentrations. b) Alanine The concentration of alanine was determined by an en-zymatic technique described by Grassl (1970) i n which alanine i s converted to pyruvate by glutamate pyruvate transaminase (GPT) : GPT* eCKetoglutarate + L-alanine >• L-glutamate + pyruvate This reaction i s coupled to a second one i n which l a c t i c dehydrogenase (LDH) reduces pyruvate i n the presence of NADH to la c t a t e . *Sigma Chemicals 44 Pyruvate + NADH + H L D H % L-lactate + NAD+. The disappearance of NADH was followed i n a Unicam SP 800 spectrophotometer at a wave length of 340 nm. Since there i s an excess of both enzymes, o<. ketoglutarate and NADH, the rate of the coupled reaction with l i m i t e d alanine concentrations i s s t r i c t l y proportional to the amount of alanine present. The measurement of the reaction rate per-mits the determination of alanine by the formation of NAD measured by the decrease i n extinction at 340 nm. c) Lactate Plasma lactate concentration was determined by the enzymatic oxidation of lactate to pyruvate catalysed by l a c -tate dehydrogenase (LDH) i n the presence of NAD+ (Gutmann et a l . 1970). L-lactate + NAD+ H^-y pyruvate + NADH + H + Plasma (0.4 ml) was added to 1.ON perchloric acid (PCA) - and centrifuged for 10 min at 3,000 rpm at 4°C. After the addition of 0.02 ml of LDK the formation of NADH was measured by the increase i n ext i n c t i o n of 340 nm on a Unicam SP 800 spectro-photometer . The concentration of the lactate i n the samples was obtained from a standard curve prepared from known lactate standards (Appendix Figure 2). *Sigma Chemicals 45 6 . B l o o d F l o w M e a s u r e m e n t s A D o p p l e r F l o w m e t e r m o d e l 80 3 ( P a r k s E l e c t r o n i c s L a b o r a -t o r y ) was u t i l i z e d t o d e t e r m i n e b l o o d f l o w i n t h e f e m o r a l a r t e r y . C u f f s o f 4 mm a n d 3 mm w e r e s t a n d a r d i z e d u s i n g a H a r v a r d A p p a r a t u s P e r i s t a l i c pump w i t h s h e e p b l o o d c o l l e c t e d f r o m t h e a b a t t o i r . B l o o d was warmed t o 3 7 ° C a n d pumped a t d i f f e r e n t r a t e s t h r o u g h p o l y e t h y l e n e t u b i n g (PE 2 8 0 , 2 . 1 5 mm I . D . x 3 . 2 5 mm O . D . a n d PE 3 5 0 , 3 . 1 6 mm I . D . x 3 . 9 9 O . D . ) . T h e b l o o d was c o l l e c t e d i n a g r a d u a t e d c y l i n d e r a n d t h e e x a c t v o l u m e o v e r 30 s e c o n d s was r e c o r d e d . T h e D o p p l e r F l o w m e t e r was c o n n e c t e d t o an i n t e g r a t e d d i g i t a l c o u n t e r * w h i c h p r o d u c e d a n u m e r i c a l d i s p l a y o f t h e D o p p l e r f r e q u e n c y s h i f t s . S t a n d a r d c u r v e s f o r t h e t w o c u f f s w e r e u t i l i z e d t o d e t e r m i n e t h e b l o o d f l o w i n t h e a r t e r i e s ( A p p e n d i x F i g u r e s 1 a n d 2 ) . 7 . S t a t i s t i c a l A n a l y s i s , T h e U . B . C . TRP c o m p u t e r p r o g r a m (Le a n d T e n i s c i , 1 9 7 7 ) was u s e d t o f o r m u l a t e t h e s t a n d a r d l i n e a r r e g r e s s i o n c u r v e s w i t h 95% c o n f i d e n c e l i m i t s u s e d i n t h i s e x p e r i m e n t t o d e t e r m i n e b l o o d f l o w i n t h e h i n d l i m b o f t h e s h e e p . The r e s u l t a n t b l o o d f l o w d a t a was s u b j e c t e d t o a s t u d e n t t - t e s t t o d e t e r m i n e w h e t h e r t h e r e was a s i g n i f i c a n t d i f f e r e n c e i n b l o o d f l o w b e t w e e n f e d a n d s t a r v e d c o n d i t i o n s . • D e s i g n e d b y M r . G . G a l z y , U n i v e r s i t y o f B r i t i s h C o l u m b i a , D e p a r t m e n t o f A n i m a l S c i e n c e . 46 R E S U L T S 1) F e m o r a l A r t e r y B l o o d F l o w T h e a n a l o g u e - i n t e g r a t o r t i m e c o u n t e r u s e d i n c o n j u n c -t i o n w i t h t h e D o p p l e r F l o w m e t e r p r o v i d e d a d i g i t a l d i s p l a y o f t h e s i g n a l w h i c h was p r o p o r t i o n a l t o t h e f r e q u e n c y c h a n g e s i n t h e D o p p l e r F l o w m e t e r . T h e a c t u a l b l o o d f l ow was then e s t i -m a t e d f r o m t h e c a l i b r a t i o n c u r v e . T h e l i n e a r r e g r e s s i o n f o r b l o o d f l o w c a l i b r a t i o n u s i n g a 3 mm c u f f r e s u l t e d i n an i n t e r c e p t o f 1 2 . 8 2 , a s l o p e o f 2 . 8 8 5 a n d an R 2 o f 0 . 9 9 . The 95% c o n f i d e n c e l i m i t s w e r e X = 0 a n d Y = 1 2 . 8 5 ± 5 . 7 8 , a t t h e a v e r a g e X = 116 m l / m i n a n d Y = 347 ± 3 . 0 6 a n d a t X = 2 40 m l / m i n a n d Y = 705 ± 6 . 0 7 . T h e l i n e a r r e g r e s s i o n o f t h e 4 mm c u f f r e s u l t e d i n an i n t e r c e p t o f 1 6 . 8 2 , a s l o p e o f 2 . 3 0 a n d an R 2 o f 0 . 9 9 . T h e 95% c o n f i d e n c e w e r e a t X = 0 a n d Y = 1 6 . 8 2 ± 5 . 9 7 ; a n d a t X = 149 m l / m i n a n d Y = 359 + 2 . 7 2 a n d a t X = 260 m l / m i n a n d Y = 598 ± 4 . 7 8 ( A p p e n d i x F i g u r e s 1 a n d 2 ) . T h e f e m o r a l a r t e r i a l b l o o d f l o w o v e r t h e e n t i r e e x p e r i -m e n t f o r b o t h t h e s h e e p i s g i v e n i n F i g u r e 1 a n d T a b l e 1. T h r o u g h o u t t h e e x p e r i m e n t a l p e r i o d t h e l a r g e r o f t h e t w o s h e e p , 2 4 2 , h a d t h e g r e a t e r b l o o d f l o w . T h e a v e r a g e b l o o d f l o w o f 93 m l / m i n f o r s h e e p 2 4 2 , a n d 81 m l / m i n f o r s h e e p 39 d u r i n g f e e d i n g i s c o n s i d e r a b l y l o w e r 125 25 -t • ' • 1 1 2 3 A 5 6 DAYS FED M STARVED H FED FIGURE 1. FEMORAL ARTERIAL BLOOD FLOW USING A DOPPLER FLOWMETER AND 3 mm CUFFS IN SHEEP 242 AND SHEEP 39 (EXPERIMENT I ) . 4* TABLE 1. FEMORAL ARTERIAL BLOOD FLOW IN SHEEP 242 AND UNDER FED AND STARVED CONDITIONS USING THE DOPPER FLOWMETER AND ANALOGUE INTEGRATOR-COUNTER (EXPERIMENT I ) . N u t r i t i o n a l Sheep 242 Sheep 3 9 State Day Time X ±S.E. X ±S. E. Fed 1 0730 98 0.4 97 3.1 1200 92 0.4 78 3.2 1600 90 1 . 3 70 2.2 Starved 2 0800 94 3.5 70 1.2 1200 68 2.2 74 1 .9 1600 78 2.2 74 2.7 Starved 3 1800 84 2.8 60 3.1 1200 78 2.2 64 3.5 1600 84 7.3 70 2.2 Starved 4 0800 90 3.5 60 1. 9 1200 90 3.1 1 58 3.8 1600 - - 69 2.7 Starved 5 0800 90 5.9 60 1.9 1200 94 5.9 68 1.2 1600 96 3.5 69 1.7 Re fed 6 0730 98 3.5 73 2.2 1200 99 5.9 7 3 3.5 1600 - - 76 3.1 Refed 7 0730 106 2.1 78 3.5 1200 106 3.1 78 3.1 1600 99 2.8 92 2.2 Time of feeding: 0800 and 1500 hrs. x Average of 5 readings. 49 than the 195 ml/min reported by J a r r e t t et a l . (1976). The use of the dye d i l u t i o n method by these workers could con-ceivably be influenced by stress and excitement giving higher values. Even i n the present study when the animal was mildly excited blood flows of 180-210 ml/min were obtained. After four days of starvation the blood flow appeared to i n -crease i n sheep 242, whereas a 20% decrease occurred i n sheep 39. There was however no s t a t i s t i c a l difference between fed and starved blood flows when res u l t s of both sheep were combined. Refeeding resulted i n blood flow returning to normal or pre-starvation le v e l s . 2. U t i l i z a t i o n or Production of Substrates i n the Hind Limb a) Glucose The a r t e r i a l and venous plasma glucose levels are pre-sented i n Tables 2 and 3 and plotted for each animal i n Figures 2 and 3. A 31% decrease i n glucose concentration occurred i n sheep No. 39. A reduction i n the u t i l i z a t i o n of glucose during starvation i s apparent from the smaller d i f f e r -ence i n arteriovenous concentration as the starvation pro-gressed (Figure 3). The femoral vein catheter did not remain patent i n sheep 242 so a l t e r n a t i v e l y the jugular vein was used for the c o l l e c t i o n of blood samples. The glucose arteriovenous difference i n sheep 242 i s therefore d i f f i c u l t to compare with that of sheep 39. At times the jugular venous glucose lev e l s i n sheep 242 was the same as the femoral FIGURE 3. FEMORAL ARTERIAL AND VENOUS GLUCOSE CONCENTRATION IN SHEEP 39 (EXPERIMENT I ) . 5 1 a a r t e r y w h i c h may b e due t o g l u c o s e p r o d u c t i o n i n t h e l i v e r . T h e h i g h l e v e l s i n t h e j u g u l a r v e i n o f s h e e p 242 may a l s o b e due t o p o s s i b l e s a m p l i n g i n t h e v e n a c a v a a s t h e c a t h e t e r w as l o n g a n d c o u l d h a v e b e e n p l a c e d i n t h a t v e s s e l . b) L a c t a t e T h e l a c t a t e c o n c e n t r a t i o n s i n s h e e p 242 w e r e c o n s i s t e n t l y h i g h e r t h a n t h a t o f s h e e p 39 a s r e p o r t e d i n T a b l e s 2 a n d 3 a n d p l o t t e d i n F i g u r e s 4 a n d 5 . L a c t a t e c o n c e n t r a t i o n s i n t h e f e m o r a l a r t e r y a n d v e i n r a n g e d f r o m 10 t o 20 m g / 1 0 0 m l a n d a g r e e w i t h t h e l e v e l s r e p o r t e d b y J a r r e t t e t a l . (19 76) a n d A n n i s o n e t a l . ( 1 9 6 3 ) . I n s h e e p 39 t h e a v e r a g e l a c t a t e a r t e r i a l - v e n o u s d i f f e r -e n c e d u r i n g f e e d i n g was - 1 . 8 mg%, w h e r e a s t h e a v e r a g e v e n o u s -a r t e r i a l d i f f e r e n c e a f t e r f o u r d a y s o f s t a r v a t i o n was - 1 . 3 7 mg% i n d i c a t i n g a 19 .5% d e c r e a s e i n l a c t a t e p r o d u c t i o n . T h e a v e r a g e c o n c e n t r a t i o n o f l a c t a t e d u r i n g f e e d i n g i n t h e f e m o r a l a r t e r y a n d v e i n was 1 1 . 1 a n d 1 2 . 9 mg% r e s p e c t i v e l y , w h i l e t h e a v e r a g e c o n c e n t r a t i o n a f t e r f o u r d a y s o f s t a r v a t i o n was 1 1 . 6 a n d 1 3 . 0 mg% r e s p e c t i v e l y , i n d i c a t i n g v e r y l i t t l e c h a n g e s d u r i n g t h e f o u r d a y s o f s t a r v a t i o n . 3 . F e e d A n a l y s i s T h e d r y m a t t e r a n a l y s i s o f t h e a l f a l f a c u b e s f e d a l l t h e s h e e p c o n t a i n e d o n t h e a v e r a g e 1 7 . 6 7% p r o t e i n , 1.6% c a l c i u m , a n d 0 .24% p h o s p h o r u s . T h e a p p a r e n t d i g e s t i b l e 52 TABLE 2. CONCENTRATION OF GLUCOSE .AND LACTATE IN ARTERIAL AND VENOUS ' ' PLASMA OF SHEEP 242 (EXPERIMENT I). GLUCOSE (mg/lOOml) LACTATE (mg/lOOml) Nutritional State Day Time Femoral Jugular A-V Femoral Jugular A-V Artery Vein Artery Vein Fed 1 0730 34.0 *26.5- 7.5 Not Analyzed  .0930 28.5 *25. .0 3.5 1130 50.0 *36. .0 14 1430 50.0 *30. .0 20 1630 34.5 *25. .0 9.5 Starved 2 0900 41.0 37, .0 4.0 18. .0 22. .0 -4. .0 1100 40.0 37, .0 3.0 15, .0 12. .0 3. .0 1400 43.5 41, .0 2.5 22, .0 18. .0 4. .0 1600 43.5 43, .0 0.5 18. .0 18. .0 0. .0 Starved 3 0900 42, .5 41. .0 1. 5 16, .0 15. .0 1. .0 1100 44, .0 44. .0 0. 0 15. .0 18, .0 -3. .0 1400 39, .0 38. .0 1. 0 18. .0 20. ,0 -2. .0 1600 41. .0 34. .0 7. 0 18. .0 13, .5 4. .5 Starved 4 0900 38. .0 36. .0 2. .0 18, .0 20, .0 -2, .0 1100 41. .0 38, .0 3, .0 16. .5 24, .0 -7, .5 1400 46. .0 38. .0 8, .0 18. .0 21, .0 -3, .0 1600 36. .0 36. .0 0. .0 22, .0 22, .0 0, .0 Starved 5 0900 36.0 36.0 0.0 10.5 20.0 -9, .5 1100 35.0 35.0 0.0 18.0 19.0 -1. ,0 1400 40.0 35.0 5.0 20.0 21.0 1. .0 1600 40.0 32.0 8.0 22.0 21.0 1. .0 Refed 6 0730 34.5 28.5 6. .0 20.0 14.0 6.0 0930 32.0 24.0 8. .0 16.5 15.0 1.5 1130 34.0 30.0 4. .0 20.0 15.0 5.0 1430 34.0 34.0 0. .0 20.0 20.0 . 0.0 1630 32.0 31.0 1. .0 15.0 15.0 0.0 0730 29.0 22, .0 7.0 16. .5 21.0 -4.5 0930 34.0 27. .0 7.0 22. .0 20.0 2.0 1130 34.0 30. .0 4.0 21. .0 16.5 4.5 1430 33.0 28. .0 5.0 18. .0 16.5 1.5 1630 30.0 26. .0 4.0 16. ,5 14.0 . 2.5 + Time Of Feeding * Femoral vein samples ,53 TABLE 3. CONCENTRATION OF GLUCOSE Al© LACTATE IN FEMORAL ARTERY AND VEIN OF SHEEP 39 (EXPERIMENT I). Nutritional State Day Time Fed l 0730 0930 1130 5 1430 1630 Starved 2 0900 1100 1400 1600 Starved 3 0900 1100 1400 1600 Starved 4 0900 1100 1400 1600 Starved 5 0900 1100 1400 1600 Refed 6 0730 0930 1130 1430 1630 Refed 7 0730 0930 1130 1430 1630 GLUCOSE (mg/lOOml) Femoral Femoral A-V Artery Vein 34.0 29.0 5.0 35.0 31.0 4.0 38.0 32.0 4.0 34.0 28.0 4.0 33.0 29.0 4.0 30. .0 28. .0 2, .0 31. .0 26, .0 5, ,0 32. .0 27, .0 5, ,0 30. .0 28, .0 2. ,0 28, ,0 27. .0 1.0 29. ,0 24. .0 5.0 30, .0 26 .0 4.0 32, .0 25 .0 7.0 26.0 24, .0 2.0 27.0 25, ,0 2.0 27.0 26. .0 1.0 26.0 25. .0 1.0 24, .0 23. ,0 1. ,0 25, .0 22, .0 3, .0 24, .0 23, .0 3. .0 25, .0 23, .0 2, .0 30.0 27.0 3.0 31.0 26.0 5.0 32.0 28.0 4.0 33.0 28.0 5.0 30.0 27.0 3.0 27.0 26.0 1.0 28.0 25.0 3.0 28.0 24.0 4.0 30.0 25.0 5.0 30.0 24.0 4.0 LACTATE (mg/lOOml) Femoral Femoral A-V Artery Vein 11.0 13.0 -2.0 10.5 13.5 -3.5 12.0 13.0 -1.0 11.5 12.5 -1.0 11.5 12.5 -1.0 11.0 11.5 -0.5 12.0 12.0 0.0 13.0 13.5 -0.5 11.0 11.5 -0.5 11.5 13.0 -1.5 12.0 13.5 -1.5 13.0 14.0 -1.0 13.0 14.0 -1.0 10.0 11.0 -1.0 11.5 13.5 -2.0 10.5 11.0 -0.5 9.0 10.0 -1.0 12.0 13.0 -1.0 10.0 12.5 -2.5 12.5 13.5 -1.0 12.0 13.0 -1.0 12.0 13.5 -1.5 11.0 13.0 -2.0 13.0 14.0 -1.0 12.0 13.0 -1.0 11.5 13.5 -2.0 11.0 12.0 -1.0 12.0 13.0 -1.0 11.0 13.0 -2.0 9.5 11.0 -1.5 10.5 11.0 -0.5 Time of Feeding 0800 and 1500 hrs. FIGURE 4. FEMDRAL ARTERIAL AND JUGULAR VEIN LACTATE CONCENTRATION IN SHEEP 242. (EXPERIMENT I). FIGURE 5. FEMORAL ARTERIAL AND VENOUS LACTATE CONCENTRATION IN SHEEP 39 (EXPERIMENT I ) . 56 energy was 2.003 to 2.546 kcal/g. These nutrients meet or exceed the N.R.C. requirements for sheep (NRC, 1975). DISCUSSION  Femoral A r t e r i a l Blood Flow The Doppler Flowmeter was chosen for these experiments over other methods for several reasons. The Doppler cuff allowed for continuous monitoring for several days throughout the experiment so that blood flow changes could be followed p r e c i s e l y . This system provided the most suitable and r e l a -t i v e l y accurate means to determine blood flow i n the hind limb of sheep. The placement of the cannulae i n blood vessels on one limb and the Doppler transducer on the other was done to avoid interference of the back scattering of the u l t r a -sound by the presence of the cannulae inside the vessel. Since the determination of blood flow by the Doppler method i s based primarily on the extent of back scattering i t was decided to minimize the errors due to t h i s factor by placing the cannulae on the opposite limb and assuming blood flow to the two limbs was equal. Other possible errors i n the use of the Doppler Flowmeter include movement of the cuffs and back flow. The back flow 57 was d e t e r m i n e d b y V a t n e r (1970) t o a c c o u n t f o r a p p r o x i m a t e l y 5% a n d t h e r e f o r e d i d n o t 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 t h e a c t u a l b l o o d f l o w . T h e movement o f t h e c u f f i n t h i s s t u d y i s n o t a p p l i c a b l e s i n c e an a u t o p s y r e v e a l e d t i s s u e g r o w t h a r o u n d t h e v e s s e l w h i c h w o u l d p r e v e n t m o v e m e n t . B l o o d f l o w r e a d i n g s v a r y l i t t l e b e t w e e n p u l s e d o r c o n s t a n t f l o w e x c e p t a t e x t r e m e l y l o w f l o w s ( V a t n e r , 1 9 7 0 ) . T h e f e m o r a l a r t e r i a l b l o o d f l o w was a u d i b l e w i t h a h e a d s e t e n a b l i n g o n e t o d e t e r m i n e w h e n t h e a n i m a l h a d c a l m e d down f r o m t h e i n i t i a l e x c i t e m e n t o f s e e i n g p e o p l e s t a n d i n g b e s i d e p r e p a r i n g f o r t h e t e s t s . The a v e r a g e t i m e f o r t h e d i g i t a l o u t p u t a n d t h e b l o o d f l o w t o r e a c h a s t e a d y s t a t e was b e t w e e n f i v e t o t e n m i n u t e s . T h u s t h i s p r o c e d u r e i s s u p e r i o r t o a n y o f t h e p r e v i o u s t e c h n i q u e s d e s c r i b e d as i t g i v e s b l o o d f l o w i n a c a l m c o n d i t i o n . T h e f e m o r a l a r t e r i a l b l o o d f l o w o f 93 ± 6 m l / m i n a n d 81 ± 5 m l / m i n , f o r s h e e p 2 42 a n d 39 r e s p e c t i v e l y , a p p e a r s t o b e l o w when c o m p a r e d t o t h e 195 ± m l / m i n r e p o r t e d b y J a r r e t t e t a l . ( 1 9 7 6 ) . T h e w o r k o f S c h e n k a n d R a c e (1968) i n d i c a t e s t h a t t h e d y e d i l u t i o n m e t h o d f o r b l o o d f l o w d e t e r m i n a t i o n s i s n o t a p p r o p r i a t e f o r p e r i p h e r a l b l o o d f l o w m e a s u r e m e n t s a n d may b e a n o v e r e s t i m a t e b y 5 0 - 1 0 0 % . T h e a c c o u s t i c a l h e a d s e t a t t a c h e d t o t h e D o p p l e r F l o w m e t e r i n c o n j u n c t i o n w i t h t h e i n t e g r a t o r - d i g i t a l c o u n t e r was h e l p f u l i n e s t a b l i s h i n g w h e n t h e a n i m a l r e a c h e d a s t e a d y b a s a l s t a t e . M i n g e t a l _ . ( 1977 ) a n d S c h e n k a n d R a c e (196 8) u s i n g D o p p l e r a n d e l e c t r o m a g n e t i c 58 c u f f s w i t h l a r g e d o g s r e p o r t e d f e m o r a l a r t e r i a l b l o o d f l o w s r a n g i n g f r o m 5 0 - 1 5 0 m l / m i n . A f t e r f o u r d a y s o f s t a r v a t i o n t h e f e m o r a l a r t e r y b l o o d f l o w r e m a i n e d r e l a t i v e l y u n c h a n g e d a t 93 ± 2 m l / m i n i n s h e e p 2 4 2 . T h e b l o o d f l o w i n s h e e p 39 d r o p p e d f r o m 81 ± 5 m l / m i n d u r i n g t h e p r e - s t a r v a t i o n t o a n a v e r a g e o f 65 ± 5 m l / m i n on t h e l a s t d a y o f s t a r v a t i o n . T h i s i s a 20% d e c r e a s e i n b l o o d f l o w , w h i c h i s i d e n t i c a l t o t h e d e c r e a s e i n r e n a l b l o o d f l o w r e p o r t e d b y K a u f m a n a n d B e r g m a n ( 1 9 7 8 ) . B l o o d f l o w r e t u r n e d t o p r e - s t a r v a t i o n l e v e l s f o r s h e e p 39 o r m o d e r a t e l y h i g h e r f o r s h e e p 2 4 2 , d u r i n g t h e r e -f e e d i n g p e r i o d . S u b s t r a t e M e t a b o l i s m T h e a r t e r i a l g l u c o s e c o n c e n t r a t i o n i n T a b l e 3 f o r s h e e p 39 d e c r e a s e d 31%, f r o m a n a v e r a g e o f 3 5 . 5 m g / 1 0 0 m l d u r i n g f e e d i n g t o a n a v e r a g e o f 2 4 . 5 m g / 1 0 0 m l o n t h e l a s t d a y o f s t a r v a t i o n . T h e g l u c o s e 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 d i f f e r -e n c e s d e c r e a s e d f r o m a n a v e r a g e o f 4 . 2 5 m g / 1 0 0 m l t o 2 . 2 5 m g / 1 0 0 m l w h i c h r e p r e s e n t s a 50% d e c r e a s e i n g l u c o s e u t i l i z a -t i o n d u r i n g s t a r v a t i o n ( T a b l e 3 ) . B e r g m a n (1973) h a s r e -p o r t e d a 33% d e c r e a s e i n g l u c o s e l e v e l s d u r i n g s t a r v a t i o n . T h e r e d u c t i o n i n g l u c o s e u t i l i z a t i o n i n t h e p e r i p h e r a l t i s s u e s may b e i n r e s p o n s e t o t h e a b i l i t y o f m u s c l e t o r e g u l a t e l o c a l l y , b l o o d f l o w a n d m e t a b o l i t e u t i l i z a t i o n b y t h e h i n d l i m b ( S c o t t e t a l . 1 9 6 5 , O r r e t a l . 1 9 7 2 ) . B l o o d f l o w , g l u c o s e a r t e r i a l c o n c e n t r a t i o n a n d h i n d l i m b u t i l i z a t i o n o f 59 g l u c o s e s t a r t e d t o r e t u r n t o n o r m a l when t h e a n i m a l was r e f e d o n d a y s i x a n d s e v e n o f t h e e x p e r i m e n t . I n s h e e p 242 d u r i n g t h e f i r s t d a y o f t h e t r i a l t h e a v e r -a g e a r t e r i a l g l u c o s e c o n c e n t r a t i o n ( T a b l e 2) was 39 mg%. T h e i n i t i a l i n c r e a s e i n b l o o d g l u c o s e (4 2 mg%) d u r i n g t h e f i r s t d a y o f s t a r v a t i o n i s s i m i l a r t o w o r k i n c o w s r e p o r t e d b y B a i r d e t a l . ( 1 9 7 2 ) . T h i s may be a t t r i b u t e d t o h o r m o n a l a n d s t r e s s r e a c t i o n s a t b e i n g s t a r v e d . T h e b l o o d g l u c o s e c o n c e n t r a t i o n d i d n o t f o l l o w t h e same t r e n d a s s h e e p 39 o v e r t h e e n t i r e e x p e r i m e n t . T h e l a c t a t e 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 d i f f e r e n c e s f o r s h e e p 39 ( T a b l e 3) a g r e e w i t h t h o s e r e p o r t e d b y J a r r e t t e t a l . ( 1976 ) a n d A n n i s o n e t a l . ( 1 9 6 3 ) . The h i g h e r v e n o u s l a c t a t e c o n c e n t r a t i o n s may b e t h e r e s u l t o f g l y c o l y s i s a n d a m i n o a c i d c a t a b o l i s m i n t h e h i n d l i m b o f t h e s h e e p w h i l e t h e l o w e r a r t e r i a l l a c t a t e c o n c e n t r a t i o n s may b e a t t r i b u t e d t o g l u c o n e o -g e n e s i s v i a t h e C o r i C y c l e . L e n g (1970) a n d A n n i s o n e t a l . (196 3) h a v e r e p o r t e d t h a t 39 t o 43% o f t h e l a c t a t e a r i s e s f r o m g l u c o s e . T h e o b s e r v e d r e d u c t i o n o f 1 9 . 5 % i n l a c t a t e p r o -d u c t i o n may t h e r e f o r e be a t t r i b u t e d t o t h e l o w e r g l u c o s e u t i l i z a t i o n b y t h e h i n d l i m b o f s h e e p d u r i n g s t a r v a t i o n . 60 CONCLUSIONS Blood flow measurements of sheep 242 and 39 did not show s t a t i s t i c a l differences between fed and starvation con-d i t i o n s . In one of the sheep (No. 2 42) the hind limb blood flow remained r e l a t i v e l y constant while i n the other i t de-creased by 20%. During starvation, i t i s possible that the period of starvation was not adequate to observe d e f i n i t e changes i n blood flow to the hind limb of sheep. The arteriovenous differences of metabolites i n the two sheep were not treated as r e p l i c a t e s for s t a t i s t i c a l purposes. Jugular vein samples were taken from sheep 2 42 while femoral vein samples were used i n sheep 39. The 4 days of starva-tion had l i t t l e e f f e c t on glucose a r t e r i a l concentration i n sheep 242 whereas a 31% drop occurred i n sheep 39. An i n t e r -esting observation i s that sheep 242 had a 15% and 19% decrease i n a r t e r i a l glucose concentration following refeeding on the sixth and seventh days of the experiment respectively. I t appears that sheep 2 42 required more than four days of starvation to put i n motion energy conserving mechanisms. This agrees with Baird et a l . (1972) and J a r r e t t et a l . (1976) who have reported that the g a s t r o i n t e s t i n a l t r a c t of the ruminant i s not empty u n t i l the f i f t h or sixth days of starvation. Lactate production by the hind limb of sheep i s the r e s u l t of g l y c o l y s i s and amino acid catabolism (Annison et a l . 196 3). The net e f f e c t of starvation causes a reduction i n 61 lactate production due to a reduced glucose a v a i l a b i l i t y . The recycling of lactate to glucose v i a the Cori cycle i s one way of maintaining glucose supplies for obligatory organs. 62 EXPERIMENT II INTRODUCTION In Experiment I no perceptible differences were observed in the blood flow or u t i l i z a t i o n or production of metabolites by the hind limb of sheep after four days of starvation. In t h i s experiment the period of starvation was extended from 4 to 6 days i n an e f f o r t to study changes i n blood flow when the metabolic processes of the sheep were altered severely. The procedure was also modified by repeating the experiment four times of the same sheep to minimize v a r i a b i l i t y . MATERIALS AND METHODS 1. Animals and Feeding Regime A mature Dorset ewe (No. 239) weighing 61 kg was u t i l i z e d for four i d e n t i c a l t r i a l s . It was fed 600 g of a l f a l f a cubes twice d a i l y . The experimental period consisted of 2 days of feeding, 6 days of starvation and 3 days of re-feeding. There was an i n t e r v a l of at least 10 days between t r i a l s . 6 3 2 . S u r g i c a l P r o c e d u r e T h e s u r g i c a l i m p l a n t a t i o n o f c a t h e t e r s was m o d i f i e d t o a v o i d l i g a t i n g t h e f e m o r a l a r t e r y a n d v e i n a n d t h u s g i v e a b e t t e r r e p r e s e n t a t i o n o f t h e t o t a l l i m b m e t a b o l i s m w i t h a s l i t t l e c h a n g e i n c i r c u l a t i o n as p o s s i b l e . A n i n c i s i o n was made o n t h e l a t e r a l a s p e c t o f t h e l e f t l e g a p p r o x i m a t e l y 4 cm a b o v e t h e t u b e r c a l c i s t o e x p o s e t h e e x t e r n a l s a p h e n o u s v e i n . A p o l y e t h y l e n e c a t h e t e r ( 0 . 1 2 7 cm O . D . x 0 . 0 8 6 cm I . D . ) was i n s e r t e d t o a t o t a l o f 30 cm i n t o t h e v e i n a n d a n c h o r e d t o t h e s u r r o u n d i n g f a s c i a . T h e t i p o f t h e c a t h e t e r was a p p r o x i -m a t e l y 5 cm i n t o t h e i l i a c v e i n . T h e c a t h e t e r was t h e n e x -t e r i o r i z e d o n t h e l e f t d o r s o - l a t e r a l a s p e c t o f t h e h i n d l i m b . A n 8 cm i n c i s i o n was made i n t h e t h i g h o v e r t h e t w o h e a d s o f t h e s a r t o r i u s m u s c l e t o e x p o s e t h e f e m o r a l a r t e r y . The f a s c i a a r o u n d t h e f e m o r a l a r t e r y was r e m o v e d a n d a s m a l l p u r s e s t r i n g s u t u r e a b o u t 1 cm i n d i a m e t e r was made o n t h e a r t e r y u s i n g 6 - 0 P r o l e n e * ( c o d e 8 6 0 1 ) . A s m a l l n i c k w i t h i n t h e p u r s e s t r i n g was made a n d a c a t h e t e r ( 0 . 1 2 7 cm O . D . x 0 . 0 8 6 I . D . ) was i n s e r t e d t o a l e n g t h o f 10 c m . T h e p u r s e s t r i n g was p u l l e d t i g h t t o p r e v e n t l e a k a g e , y e t a l l o w b l o o d f l o w t h r o u g h t h e v e s s e l . The c a t h e t e r was a n c h o r e d a n d e x -t e r i o r i z e d a s i n E x p e r i m e n t I . A D o p p l e r 4 mm c u f f was p l a c e d a r o u n d t h e r i g h t f e m o r a l a r t e r y a n d e x t e r i o r i z e d a s * P r o l e n e E t h i c o n S u t u r e s L t d . 64 i n E x p e r i m e n t I . To d e t e r m i n e w h e t h e r c h a n g e s a r e o b s e r v e d i n t h e b l o o d f l o w o f o t h e r a r t e r i e s i n t h e b o d y f o l l o w i n g s t a r v a t i o n a 4 mm c u f f was p l a c e d o n t h e r i g h t c a r o t i d a r t e r y o f a D o r s e t ewe ( S - 3 ) u s i n g t h e s u r g i c a l p r o c e d u r e a s d e s -c r i b e d e a r l i e r . 3 . B l o o d F l o w D o p p l e r r e a d i n g s w e r e t a k e n a t 0 8 0 0 , 1 2 0 0 , 1500 h r s a n d p r e c a u t i o n s w e r e t a k e n t o m i n i m i z e e x c i t i n g t h e a n i m a l . A t o t a l o f 5 r e a d i n g s was o b t a i n e d d u r i n g e a c h t i m e b l o o d f l o w was m e a s u r e d . T h e mean o f t h e f i f t e e n m e a s u r e m e n t s was t h e n t a k e n a s t h e b l o o d f l o w f o r t h a t d a y . T o p r e v e n t e x c i t e m e n t b l o o d f l o w m e a s u r e m e n t s a l w a y s p r e c e e d e d t h e c o l l e c t i o n o f b l o o d f o r d e t e r m i n a t i o n o f m e t a b o l i t e s . 4 . P l a s m a M e t a b o l i t e s S i m i l t a n e o u s b l o o d s a m p l e s w e r e o b t a i n e d f r o m t h e f e m o r a l a r t e r i a l a n d v e n o u s c a n n u l a e f o r t h e d e t e r m i n a t i o n o f p l a s m a m e t a b o l i t e s . B l o o d s a m p l e s w e r e t a k e n o n t h e d a y s i n d i c a t e d i n T a b l e s 7 - 9 a t 1200 h r s . 5 . C h e m i c a l A n a l y s i s P l a s m a g l u c o s e , l a c t a t e a n d a l a n i n e w e r e d e t e r m i n e d b y t h e p r o c e d u r e s d e s c r i b e d i n E x p e r i m e n t I . 65 6. S t a t i s t i c a l Analysis Each blood flow value represents an average of fiv e readings that were taken one after another. Since each of the f i v e readings was within the standard error of the mean, the means of the f i v e readings were used for subsequent s t a t i s t i c a l analysis. In order to examine the e f f e c t s of starvation on blood flow the measurements were divided into four phases: Phase I (2 days of prefeeding), Phase II (6 days of starvation), Phase III ( f i r s t day of refeeding), and Phase IV (Next 2 days of refeeding and recovery). The data from 4 experiments were combined to estimate an average blood flow and metabolite turnover during the four phases. Regression analysis was performed on each phase r e s u l t i n g i n four separate regression l i n e s (Le and T e n i s c i , 1977). Blood flow data i n the four phases were further analyzed by the analysis of covariance, with the test of common slope and the student Newman-Keul's test (Halm and Le, 1975) for mean comparisons. The student T-test was used to determine whether the mean fed a r t e r i a l l e v e l s of glucose, lactate and alanine were s i g n i f i c a n t l y d i f f e r e n t from the starved a r t e r i a l l e v e l s of the same metabolites. 66 R E S U L T S 1. F e m o r a l A r t e r i a l B l o o d F l o w T h e f e m o r a l a r t e r i a l b l o o d f l o w i n s h e e p 2 39 i n t h e f o u r t r i a l s i s i l l u s t r a t e d i n F i g u r e 6 . T h e r e s u l t s o f t h e i n d i v i d u a l t r i a l s a r e r e p o r t e d i n A p p e n d i x T a b l e 1 a n d A p p e n -d i x F i g u r e s 5 , A , B , C , a n d D . T h e b l o o d f l o w v a l u e s o f s h e e p 2 39 i n e a c h o f t h e f o u r t r i a l s w e r e c o m b i n e d t o g e t h e r t o y i e l d r e g r e s s i o n l i n e s i l l u s t r a t e d i n F i g u r e 7 a l o n g w i t h 95% c o n f i d e n c e l i m i t s . T h e t e s t o f common s l o p e i n t h e a n a l y s i s o f c o v a r i a n c e (MFAV) ( T a b l e 4) i n d i c a t e d t h a t s t a t i s t i c a l l y t h e r e was n o t a common s l o p e f o r a l l t h e d a t a (P ^ 0 . 0 5 ) . T h e s l o p e o f t h e r e f e e d i n g P h a s e I I I was d i f f e r e n t f r o m P h a s e s I , I I a n d I V a s s h o w n i n T a b l e 5 . A t e s t f o r common s l o p e f o r P h a s e I , I I a n d I V r e s u l t e d i n n o s i g n i f i c a n t d i f f e r e n c e ( P ^ - 0 . 0 5 ) among t h e m . T h e a n a l y s i s o f c o v a r i a n c e t o t e s t t h e h y p o t h e s i s o f no d i f f e r e n c e i n a d j u s t e d means f o r P h a s e I , I I a n d I V ( T a b l e 5) s h o w e d t h e r e was a s i g n i f i c a n t d i f f e r e n c e ( P 4 0 . 0 5 ) . T h e s t u d e n t N e w m a n - K e u l * s t e s t d e t e r m i n e d t h a t e a c h o f t h e a d j u s t e d means f o r P h a s e I , I I a n d I V was s i g n i f i c a n t l y d i f f e r e n t f r o m o n e a n o t h e r ( P ^ O . 0 5 ) . T h e a v e r a g e f e m o r a l b l o o d f l o w d u r i n g s t a r v a t i o n was 2 3 . 5 % l o w e r t h a n t h e a v e r a g e f l o w d u r i n g t h e f e d p e r i o d ( T a b l e 5 ) . T h e d e c l i n e i n b l o o d f l o w was d r a m a t i c d u r i n g t h e 5 t h FIGURE 6. FEMORAL ARTERIAL BLOOD FLOW IN SHEEP 2 39 OBTAINED IN FOUR TRIALS A = X, B =•, C D = 1 (EXPERIMENT I I ) . FIGURE 7. LINEAR REGRESSION AND 95% CONFIDENCE LIMITS ON FEMORAL ARTERY-\ BLOOD FLOW IN SHEEP 2 39 DURING DIFFERENT PHASES OF STARVATION (EXPERIMENT I I ) . ' CM 00 TABLE 4. ANALYSIS OF COVARIANCE AND TEST OF COMMON SLOPE FOR FEMORAL ARTERIAL BLOOD FLOW IN SHEEP 239 (EXPERIMENT I I ) . SUM . MEAN SQUARES SQUARES F-VALUE PROB. Common Slope (Phases I - IV) Slope -Error -3 107 3. 34 0.021 Common Slope (Phases I, I I , Slope -IV) 2 0.317 0. 72 8 Analyses of Covariance (Phases I, I I , Phase -Error -2 99 3895 1 1241 1948 113 17.15 (0.40 E 0.000 IV) 70 TABLE 5 . ADJUSTED MEAN FEMORAL ARTERIAL BLOOD FLOW FOR SHEEP 2 39 DURING DIFFERENT PHASES OF STAR-VATION (EXPERIMENT I I ) . / N u t r i t i o n a l State Recovery PHASE I PHASE II PHASE I I I * PHASE IV Mean Blood Flow (ml/min) 7 8 . 5 6 0 . 0 5 5 . 5 6 8 . 5 S.E. 2 . 5 1.5 3.5 2 . 5 •Arithmetic mean not u t i l i z e d i n analysis. Student Newman-Keul's test P = 0 . 0 5 . Means I, II and IV were s i g n i f i c a n t l y d i f f e r e n t from each other. 71 a n d 6 t h d a y s o f s t a r v a t i o n , a m o u n t i n g t o a r e d u c t i o n o f 30 -45% f r o m t h e a v e r a g e f e d b l o o d f l o w . T h e c a r o t i d a r t e r i a l b l o o d f l o w o f s h e e p S - 3 d u r i n g d i f f e r e n t p h a s e s o f s t a r v a t i o n i s g i v e n i n F i g u r e 8 a n d r e -p o r t e d i n A p p e n d i x T a b l e 1. T h e r e was a 2 8% r e d u c t i o n i n c a r o t i d a r t e r i a l b l o o d f l o w f r o m t h e mean o f p h a s e I t o t h e l a s t d a y o f s t a r v a t i o n . T h i s s h e e p became i l l d u e t o a n i n f e c t i o n a n d f u r t h e r t r i a l s w e r e n o t c o n t i n u e d . 2 . U t i l i z a t i o n o r P r o d u c t i o n o f S u b s t r a t e s i n t h e H i n d L i m b a) G l u c o s e T h e mean f e m o r a l a r t e r i o v e n o u s g l u c o s e c o n c e n t r a t i o n s a n d n e t u t i l i z a t i o n p e r m i n f o r s h e e p 2 39 a r e g i v e n i n T a b l e 7 . I n d i v i d u a l v a l u e s a r e r e p r e s e n t e d i n A p p e n d i x T a b l e 2 . T h e mean a r t e r i a l g l u c o s e c o n c e n t r a t i o n i n t h e f e d s t a t e (32 mg%) was s i g n i f i c a n t l y d i f f e r e n t f r o m t h e mean (26 mg%) o f t h e l a s t t w o d a y s o f s t a r v a t i o n ( P ^ 0 . 0 5 ) . T h e a t e r i o -v e n o u s c o n c e n t r a t i o n d i f f e r e n c e was r e d u c e d b y 21% a n d t h e g l u c o s e u t i l i z a t i o n b y t h e h i n d l i m b o f t h e s h e e p was r e d u c e d b y 62% d u r i n g s t a r v a t i o n ( T a b l e 7 ) . T h e i n c r e a s e i n a r t e r i a l g l u c o s e c o n c e n t r a t i o n i n P h a s e s I I I ( r e f e e d i n g ) a n d I V ( r e c o v e r y ) may b e due t o g l u c o -n e o g e n e s i s o c c u r r i n g a f t e r f e e d i n g . On t h e l a s t d a y o f t h e t r i a l t h e g l u c o s e l e v e l s a n d a r t e r i o v e n o u s d i f f e r e n c e r e t u r n e d t o t h o s e o b s e r v e d o n t h e f i r s t d a y o f t h e t r i a l . 72 TABLE 6. INDIVIDUAL REGRESSIONS OF FEMORAL ARTERIAL BLOOD FLOW VERSUS TIME IN SHEEP 2 39 DURING DIFFERENT PHASES OF STARVATION (EXPERIMENT I I ) . Phase Nutritional State I n t e r c e p t ml/min ± S.E. Slope + S.E. F D i f f I Fed 86.02 5.116 -0.1337 .2639 .2568 I I Starved 68.01 2.208 -0.1540 0.0233 43.37* I I I Refed 50.42 6.118 3.8333 1.58 5.888* IV Recovery 73.75 3.148 0.0108 0.1626 0.0044 * S i g n i f i c a n t (P<0.05) 150 FIGURE 8. CAROTID ARTERIAL BLOOD FLOW IN SHEEP S-3 DURING DIFFERENT PHASES OF STARVATION (EXPERIMENT I I ) . 74 b) L a c t a t e T h e mean f e m o r a l a r t e r i o v e n o u s l a c t a t e c o n c e n t r a t i o n d i f f e r e n c e a n d n e t u t i l i z a t i o n p e r m i n f o r s h e e p 2 39 a r e g i v e n i n T a b l e 8 . I n d i v i d u a l v a l u e s f o r e a c h t r i a l a r e p r e -s e n t e d i n A p p e n d i x T a b l e 2 . T h e s t a t i s t i c a l a n a l y s i s o f t h e means o f t h e f e m o r a l a r t e r i a l l a c t a t e c o n c e n t r a t i o n r e s u l t e d i n no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e f e d a n d s t a r v e d c o n d i t i o n s . S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h t h e means o f t h e f e m o r a l v e i n c o n c e n t r a t i o n o f l a c t a t e . T h e 70% r e d u c -t i o n i n l a c t a t e p r o d u c t i o n f r o m t h e f e d c o n d i t i o n t o t h e l a s t t w o d a y s o f s t a r v a t i o n was s t a t i s t i c a l l y s i g n i f i c a n t (P 0 . 0 5 ) ( T a b l e 8 ) . c ) A l a n i n e T h e mean f e m o r a l 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 a n d n e t u t i l i z a t i o n a n d p r o d u c t i o n p e r m i n f o r s h e e p 239 a r e p r e s e n t e d i n T a b l e 9 . I n d i v i d u a l v a l u e s f o r e a c h t r i a l a r e p r e s e n t e d i n A p p e n d i x T a b l e 2 . T h e 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 o n t h e l a s t t w o d a y s o f s t a r v a t i o n w e r e - 2 . 0 a n d - 1 . 8 v i g / m l , r e s p e c t i v e l y . S t a r -v a t i o n r e s u l t e d i n an i n c r e a s e o f a l a n i n e i n t h e v e n o u s b l o o d f r o m 1 2 . 7 t o 1 5 . 2 u g / m l ( T a b l e 9 ) . T h i s i n c r e a s e r e s u l t e d i n a c h a n g e f r o m a u t i l i z a t i o n r a t e o f 2 3 u g / m i n d u r i n g f e e d i n g t o a p r o d u c t i o n o f 25 a n d 20 u g / m i n o n t h e f i f t h a n d s i x t h d a y s o f s t a r v a t i o n r e s p e c t i v e l y . 75 TABLE 7. MEAN FEMORAL ARTERIOVENOUS GLUCOSE CONCENTRATIONS AND NET UTILIZATION PER MIN FOR SHEEP 2 39 (EXPERIMENT I I ) . NUTRITIONAL ARTERY VEIN A - V NET UTILIZATION STATE mg% + S.E. mg% ± S.E. mg% mg/min Fed (Day 2) a 33 .0 + 4. 7 31. 6 + 2. 5 1 .8 + 0. 08 0. 35 + 0 .09a Starved (Day 5) b 26 .0 + 3. 4 29. 9 + 2. 6 1 .0 + 0. 09 0. 14 + 0 .1b Starved (Day 6) c 26 .0 + 3. 4 24. 0 + 3. 8 1 .2 + 0. 04 0. 13 + 0 .08b Refed (Day 1) d 29 .0 + 11. 0 25. 6 + 5. 9 3 .7 + 0. 8 0. 56 + .02a Refed (Day 2) e 31 .5 + 10. 3 29. 1 + 6. 7 2 .7 + 0. 11 0. 52 + 0 .01a Refed (Day 3) f 31 .1 + 4. 9 29. 3 + 3. 7 1 .8 + 0. 04 0. 47 + 0 .05a Values with different superscripts are statistically different (P 0.05) 76 TABLE 8. MEAN FEMORAL ARTERIOVENOUS LACTATE CONCENTRATIONS AND NET UTILIZATION PER MIN FOR SHEEP 2 39 (EXPERIMENT II) NUTRITIONAL ARTERY VEIN A - V NET UTILIZATION STATE mg% ± S.E. mg% ± S.E. mg% mg/min Fed (Day 2) a 14. 8 + 2 .9 13 .5 + 2.9 2.4 + 0.09 0.62 + 0.053 Starved (Day 5) b 13. 5 + 1 .3 12 .5 + 1.0 1.0 + 0.02 0.13 + 0.02b Starved (Day 6) c 14. 3 + 5 .7 12 .7 + 2.5 1.6 + 0.03 0.19 + 0.01b Refed (Day 1) d 12. 5 + 1 .3 11 .4 + 1.5 1.1 + 0.05 0.17 + 0.03b Refed (Day 2) e 14. 8 + 5 .0 13 .4 + 2.5 2.2 + 0.07 0.61 + 0.04a Refed (Day 3) f 12. 3 + 1 .7 11 .3 + 1.1 1.2 + 0.02 0.27 + 0.01C Values with different superscripts are statistically different (P 0.05) 77 TABLE 9 . MEAN FEMORAL ARTERIOVENOUS ALANINE CONCENTRATION AND NET UTILIZATION/PRODUCTION PER MIN FOR SHEEP 2 39 (EXPERIMENT I I ) . NUTRITIONAL ARTERY VEIN A - V NET UTILIZATION/ STATE (ug/ml)±S.E. (pg/ml)±S.E. (ug/ml)±S.E. PRODUCTION (ug/Tnin)±S.E. Fed (Day 2) a 13 .5 + 3.0 12.7 + 6.6 0.95 ± 0.01 23 + 1. . 5 a Starved (Day 5) b 11 .9 + 2.2 13.9 + 2.1 - 2 . 0 ± 0.07 -25 + 0, .9b Starved (Day 6) c 13 .4 + 2.9 15.2 + 3.4 - 1 . 8 ± 0.05 -20 + 0, . 9b Refed (Day 1) d 12 .8 + 2.1 13.9 + 3.6 -1 .1 ± 0.01 -12 + 2, . 0 b Refed (Day 2) e 12 .8 + 2.2 11.9 + 3.2 0.9 ± 0.09 19 + 1, . o a Refed (Day 3) f 12 .6 + 2.3 11.5 + 2.5 0.75 ± 0.10 17 + 1. . 6 a Values with different superscripts are statistically different (P 0.05) Positive and negative A-V differences indicate utilization and produc-tion respectively. 78 DISCUSSION  Femoral A r t e r i a l Blood Flow The mean femoral a r t e r i a l blood flow under fed conditions i n sheep 239 used i n experiment II was 78.5 ± 2.5 ml/min. The adjusted mean blood flow i s considerably lower than the 195 ml/min reported by J a r r e t t et a l . (1976). As previously ex-plained the dye d i l u t i o n method may over estimate blood flow by 50-100%. Kung and Chien (1977) and Schenk and Race (1968) estimated the femoral artery blood flow i n large dogs by using Doppler and electromagnetic c u f f s . Their values ranged from 60 - 150 ml/min which i s similar to res u l t s with sheep 2 39. The lin e a r regression analysis of the four t r i a l s on sheep 2 39, shown i n Figure 8, indicates that blood flow i s s i g n i f i c a n t l y reduced during starvation (Table 6). The s t a t i s t i c a l analysis demonstrated that there was no common slope among the four phases (P«$ 0.05). Further analysis of the data revealed that the slope of Phase III (refeeding) was d i f f e r e n t from the slopes of Phase I, II and IV. The analysis for common slope for the remaining three phases indicated that there was no s i g n i f i c a n t difference between each phase (P^0.05). However i n comparison with Phase II (Starvation) the duration of Phase I and IV was too short rendering s t a t i s t i c a l analysis d i f f i c u l t . The slope of the f i r s t phase was not s i g n i f i c a n t l y d i f f e r e n t from zero 79 indicating a constant blood flow with n e g l i g i b l e changes (P<-0.05, Table 4 ) . The slope of Phase II (starvation) was s i g n i f i c a n t l y d i f f e r e n t from zero (P^0.05) i n d i c a t i n g an actual decrease i n blood flow. There was a 23.5% drop (Table 5) in the adjusted mean fed blood flow as compared to the ad-justed mean starved blood flow. The student Newman-Keul1s te s t that was ca r r i e d out demonstrated that each of the ad-justed mean blood flows was s i g n i f i c a n t l y d i f f e r e n t (Table 5). In the post-recovery period (Phase IV) blood flow did not return to the adjusted mean l e v e l of Phase I. This implies that i t takes more than 3 days from commencement of refeeding before metabolic processes i n the hind limb reach stable l e v e l s . The 2 3.5% average reduction i n blood flow agrees clos e l y with the 2 0% reduction i n renal blood flow (Kaufman and Bergman, 1978) and 28% reduction i n portal blood flow (Katz and Bergman, 1969) that occurred during a si m i l a r period of starvation. The 44 ml/min blood flow on the sixth day of starvation was 44% lower than the i n i t i a l blood flow. Scott et a l . (1965) and Orr et a l . (1972) reported that blood flow under times of stress can be l o c a l l y regulated i n muscle to conserve energy. The reduction in blood flow coupled with the reduction i n metabolite u t i l i z a t i o n i n sheep 2 39 agrees with these observations. The immediate r i s e i n blood flow from 44 ml/min on the l a s t day of starvation to a mean of 55 ± 3 ml/min following 80 r e f e e d i n g i s a r e s u l t o f f e e d i n g , p s y c h o l o g i c a l a n d h o r m o n a l c h a n g e s w h i c h i n c r e a s e h e a r t r a t e a n d c a r d i a c o u t p u t ( S e l l a r s e t a l . 1 9 6 4 ; C a r r a n d J a c o b s o n , 1 9 6 8 ) . H o w e v e r , e v e n i n t h e r e c o v e r y p e r i o d ( P h a s e I V ) , b l o o d f l o w was o n l y 6 8 + 2 m l / m i n w h i c h was s t i l l l o w e r t h a n t h e f l o w d u r i n g P h a s e I ( f e d ) i n d i c a t i n g t h a t t h e r e was a l a g b e f o r e b l o o d f l o w r e t u r n s t o n o r m a l . T h i s may be a t t r i b u t e d t o t h e s l o w r e t u r n o f r u m e n f e r m e n t a t i o n a n d g l u c o n e o g e n e s i s t o n o r m a l l e v e l s . T h e c a r o t i d a r t e r i a l b l o o d f l o w i n s h e e p S - 3 f o l l o w e d s i m i l a r p a t t e r n s t o t h e f e m o r a l a r t e r y i n s h e e p 2 39 ( F i g u r e 7 ) . T h e a v e r a g e p r e - s t a r v a t i o n l e v e l s o f 121 ± 11 m l / m i n f e l l t o a n a v e r a g e o f 86 ± 5 m l / m i n o n t h e 5 t h a n d 6 t h d a y s o f s t a r v a -t i o n . T h i s r e p r e s e n t e d a 29% r e d u c t i o n i n b l o o d f l o w w h i c h c o r r e s p o n d s t o t h e p r e v i o u s l y r e p o r t e d d a t a o f K a u f m a n a n d B e r g m a n (1978) a n d K a t z a n d B e r g m a n (1969b ) d u r i n g s t a r v a t i o n . A n a v e r a g e b l o o d f l o w o f 109 + m l / m i n d u r i n g r e f e e d i n g i s s l i g h t l y l o w e r t h a n t h e p r e - s t a r v a t i o n l e v e l s . T h i s i s s i m i l a r t o t h e c h a n g e s i n t h e f e m o r a l b l o o d f l o w i n s h e e p 2 3 9 . S u b s t r a t e M e t a b o l i s m T h e g l u c o s e c o n c e n t r a t i o n s a n d a r t e r i o v e n o u s d i f f e r e n c e s i n T a b l e s 7 r e p r e s e n t t h e l o w e r r a n g e r e p o r t e d i n a d u l t , n o n -p r e g n a n t , n o n - l a c t a t i n g s h e e p b y L i n d s a y a n d F l e a t (1974) a n d B e r g m a n ( 1 9 6 3 , 1 9 7 3 ) . S t e e l a n d L e n g (1973) a n d L i n d s a y a n d F l e a t (1974) h a v e r e p o r t e d t h a t a r t e r i a l g l u c o s e l e v e l s r e a c h 81 a basal l e v e l after the fourth day of starvation. The mean a r t e r i a l glucose concentration on the f i f t h and si x t h days of starvation i n Experiment II was 26 ± 3 mg/100 ml indica -t i n g that a basal glucose c i r c u l a t i n g l e v e l had been reached (Table 7). During starvation glucose u t i l i z a t i o n i s reduced to conserve energy for the brain and heart. If thi s did not occur gluconeogenesis would have to be maintained at a higher rate and protein wasting would be excessive (Berger et a l . 1976). The 62% reduction i n glucose u t i l i z a t i o n by the hind limb during starvation supports t h i s concept. Bergman (1973) obtained samples from the portal vein but only reported a 3 3% reduction i n glucose u t i l i z a t i o n . The differences between these two values may be attributed to sample s i t e and the greater reduction i n blood flow to the hind limb. In support of t h i s , Grubb et a l . (1976) reported that i n rats an increase or de-crease i n blood flow results i n a corresponding change i n the rate of glucose uptake by the hind limb over a wide range of glucose concentrations. Blood flow increased by 25% during refeeding while glucose concentration and u t i l i z a t i o n only increased as the precursors for gluconeogenesis are replenished. In the l a s t two days of Experiment II there was a return to level s close to pre-starvation period. Lactate arises primarily from g l y c o l y s i s , amino acid catabolism, and the conversion of propionate to lactate i n the rumen epithelium and possibly the l i v e r (Weigand et a l . 82 1 9 7 2 ; Y o u n g , 1 9 7 7 ) . G e n e r a l l y , a n e g a t i v e a r t e r i o v e n o u s d i f f e r e n c e h a s b e e n r e p o r t e d d u r i n g f e e d i n g i n a c c o r d a n c e w i t h t h e o p e r a t i o n o f t h e C o r i c y c l e ( B a l l a r d e t a l . 1 9 7 6 ; A n n i s o n e t a l . 1 9 6 3 ) . D u n l o p (1972) f o u n d a n e g a t i v e d i f f e r -e n c e d u r i n g s t a r v a t i o n o n l y . T h r o u g h o u t t h e e x p e r i m e n t o n s h e e p 239 t h e a r t e r i a l l e v e l o f l a c t a t e was g r e a t e r t h a n t h e v e n o u s l e v e l ( T a b l e 7 ) . T h i s w o u l d i m p l y t h a t t h e r e was e i t h e r a l o w u p t a k e o f l a c t a t e b y t h e l i v e r o r a l o w l e v e l o f g l y c o l y s i s i n t h e h i n d l i m b . T o m a i n t a i n t h e e n e r g y r e q u i r e m e n t s o f t h e h i n d l i m b d u r i n g t h e f e d a n d s t a r v e d c o n -d i t i o n s , f r e e f a t t y a c i d s a n d k e t o n e s w o u l d b e u t i l i z e d r e s p e c t i v e l y f o r e n e r g y s o u r c e s ( J a r r e t t e t a l . 1 9 7 6 ) . T h e 70% r e d u c t i o n i n t h e n e t p r o d u c t i o n o f l a c t a t e d u r i n g s t a r -v a t i o n may b e due t o t h e d e c r e a s e o f t h e t w o m a i n s o u r c e s o f l a c t a t e , g l y c o l y s i s a n d r u m e n e p i t h e l i a l m e t a b o l i s m . T h e p r o d u c t i o n o f l a c t a t e r o s e t o p r e - s t a r v a t i o n l e v e l s a s g l u -c o s e u t i l i z a t i o n , f e r m e n t a t i o n a n d b l o o d f l o w i n c r e a s e d d u r i n g t h e p e r i o d o f r e f e e d i n g . T h e a r t e r i a l a l a n i n e c o n c e n t r a t i o n s i n t h e h i n d l i m b d u r i n g f e e d i n g was 1 3 . 5 ± 3 . 0 u g / m l : T h i s i s s i m i l a r t o t h e l e v e l s r e p o r t e d b y B a l l a r d e t a l . (1976) a n d C r o s s e t a l . ( 1 9 7 6 ) . The n e t p r o d u c t i o n o f a l a n i n e d u r i n g s t a r v a t i o n i n d i c a t e s d e n o v o s y n t h e s i s ( T a b l e 9) i n t h e m u s c l e s o f t h e h i n d l i m b p r o b a b l y v i a t h e g l u c o s e - a l a n i n e c y c l e ( A p p e n d i x F i g u r e 9 ; F e l i g , 1 9 7 3 ) . W h i l e t h e r e w as a n e t s y n t h e s i s o f 2 5 a n d 20 y g / m l o f a l a n i n e o n t h e 5 t h a n d 6 t h d a y s o f 83 starvation respectively, there was a net u t i l i z a t i o n of 2 3 ug/ml by the hind limb during the fed condition. The 62% reduction i n glucose u t i l i z a t i o n during starvation would re-duce the products of g l y c o l y s i s as precursors of alanine. The c i r c u l a t i n g lactate may also have been u t i l i z e d i n a l a -nine synthesis. The major carbon skeleton for alanine f o r -mation arises from the catabolism of amino acids, s p e c i f i c a l l y branched chain amino acids (Bergman, 1973; Cross et a l . 1974; Wolff and Bergman, 1972b). On the sixth day of starvation there was a 20% reduction i n alanine production as compared to the f i f t h day. I t has been reported that during starvation alanine may account for 5 0% of the amino acids removed by the l i v e r (Adibi et a l . 1976; Grubb et a l . 1976; Bergman, 1973). The production of alanine by the hind limb therefore may not have kept up with the extraction by the l i v e r . A reduction i n protein catabolism to prevent muscle wasting and the u t i l i z a t i o n of ketones and fatty acids as sources of energy may be responsible for the decrease i n alanine production ( F e l i g , 1973; Sherwin et a l . 1975; Ballard et a l . 1976; Annison, 1976). During refeeding there was a return to normal alanine levels as blood flow and glucose levels increased with feeding. 8 4 GENERAL CONCLUSIONS AND SUMMARY Femoral A r t e r i a l Blood Flow The use of chronically implanted Doppler Flowmeter cuffs permitted accurate measurements of blood flow over prolonged periods of time. During starvation there was an average reduction of 2 3.5% i n blood flow to the hind limb. The re-duction may indicate that muscle can regulate blood flow during times of stress to conserve energy as has been reported by Scott et a l . (1965), Katz and Bergman (1969b) and Orr et a l . (1972) . Substrate Metabolism The uptake or release of any substrate by an organ i s equal to the product of blood flow and the changes i n concen-t r a t i o n of the substrate i n question as i t passes through that organ. Bycannulating the main blood vessel entering and leaving the hind limb i t became possible to study the u t i l i z a -t i o n or production of metabolites by the hind limb which i s predominantly s k e l e t a l muscle. The a b i l i t y of the muscles to regulate blood flow l o c a l l y would decrease glucose u t i l i z a t i o n during times of stress and ensure that glucose i s available f o r obligatory organs. During starvation the net production of lactate was reduced by approximately 70%. This may be due to the 85 concomitant decrease i n glucose u t i l i z a t i o n and a v a i l a b i l i t y . There was a negative arteriovenous concentration d i f f e r -ence of alanine during starvation i n d i c a t i n g production. A d i r e c t comparison of net alanine production by the hind limb obtained i n t h i s study with that of Ballard et a_l. (1976) was not possible since bloodflow during starvation was not re-ported by the above authors. Through the use of isotope studies, Bergman (1973), F e l i g (1973), Ruderman and Berger (1974) and Wolff and Bergman (1972a) i n sheep and rats have reported that alanine provides between 5 and 10% of the glucose requirements v i a gluconeogenesis during starvation. I t may be concluded that alanine plays an important role i n maintaining glucose lev e l s during short term starvation i n sheep through gluconeogenesis. 86 BIBLIOGRAPHY Adi b i , S. 19 76. Metabolism of branched chain amino acids i n altered n u t r i t i o n . 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Prentice-Hall, Inc., New Jersey. 96 APPENDIX TABLE I (APPENDIX): CALCULATED FEMORAL ARTERY BLOOD FLOW IN SHEEP 239 AND CAROTID ARTERY BLOOD FLOW IN SHEEP S-3 Experiment II, A Experiment II, B Experiment II, C Experiment II, D Experiment II, S-3 X X X X x Day Time (ml/min) +S. E.M. (ml/min) iS.E.M. (ml/min) iS.E.M. (ml/min) iS.E.M. (ml/min) iS.E.M. 1 900 82 0. 9± 86 2.5 70 2.5 140 4.2 1200 75 4. ,8+ 89 2.8 112 2.6 96 1.4 116 3.2 1500 - 84 2.8 103 1.9 82 0.7 -2 900 60 5. 9± 62 2.6 74 2.9 78 1.8 108 1.9 1200 - 76 2.8 115 1.4 98 1.4 117 1.5 1300 77 7. 3± 72 1.8 99 0.9 90 1.0 123 2.9 3 900 71 2. 8+ 62 1.5 73 1.9 82 2.2 96 1.0 1200 - 79 3.8 - 117 3.6 1500 71 1. ,1+ 58 1.5 56 2.2 76 1.6 101 1.1 4 900 74 1. .1+ 56 3.5 57 4.8 52 2.3 94 1.8 1200 - - 60 3.6 - 112 3.9 1500 75 1. ,5± 64 1.9 56 1.8 53 1.8 92 1.8 5 900 - 72 3.9 62 1.4 50 1.4 98 0.9 1200 - - 60 3.4 - 107 0.0 1500 . - 64 3.9 48 1.9 47 1.0 102 3.2 6 900 80 3. .6 67 3.7 45 1.8 48 1.9 89 1.8 1200 - 47 2.4 - 86 1.9 1300 84 3. 2 63 1.9 53 1.4 43 1.9 85 1.6 7 ' 900 45 2. ,1± 45 3.9 48 2.2 44 1.9 86 1.8 1200 - 47 1.7 48 2.9 49 2.6 -1500 52 3. 2+ 45 0.9 44 0.0 51 2.8 86 1.8 8 900 48 1. ,8± 40 1.4 c 51 3.6 40 0.9 103 1.4 1200 52 1. ,4 40 0.9 45 0.9 42 1.6 89 1.2 1500 59 1. .8 40 1.0 50 3.1 44 1.5 -9 900 , 56 1. ,8± 42 1.9 53 3.6 48 1.8 92 2.5 1200 - 50 0.9 56 2.7 93 2.9 -1500 63 4. ,2± 63 2.6 88 2.9 76 1.9 118 0.9 10 900 76 4. ,5+ 80 0.7 70 1.5 60 0.9 119 1.0 1200 62 1. ,8+ 76 1.8 86 0.9 68 0.6 123 1.2 1500 77 2. .2± 76 1.8 86 2.2 68 1.9 103 1.8 11 900 66 3. ,9 + 56 1.5 83 2.8 72 1.0 102 2.2 1200 62 1. ,8+ 96 3.6 73 2.1 77 0.9 103 2.9 1500 65 1. ,8± 80 1.4 86 1.2 76 0.9 103 1.9 x = Mean iS.E.M. = Standard Error of Mean T = 0.05 (2) n-1, N = 5 TABLE 2. FEMORAL ARTERIOVENOUS METABOLIC PARAMETERS FOR SHEEP 239 N u t r i t i o n a l ALANINE (ug/ml) GLUCOSE (mg/100 ml) LACTATE (mg/100 ml) S t a te A V A-V A V A-V A V A-V EXPERIMENT I I A Day Fed 2 15.5 14.2 1.3 31.0 30.5 0.5 13.8 12.7 1.1 S ta rved 5 13.0 14.2 -1.2 27.5 25.0 2.0 13.8 12.7 1.1 6 14.2 15.0 -0 .8 25.2 24.0 1.2 13.8 14.2 -0.4 Refed 1 13.0 14.0 1.0 25.5 24.0 1.5 13.5 11.1 2.4 2 12.0 11.0 1.- 24.2 24.2 0.0 12.7 11.4 1.3 3 11.0 12.7 -1.7 28.0 27.0 1.0 12.0 11.1 0.9 EXPERIMENT I I B Day Fed 2 12.0 13.0 - 1 .0 37.0 33.0 4.0 12.7 15.0 2.2 Starved 5 13.0 13.5 -0.5 27.5 24.5 3.0 14.2 12.7 1.5 6 14.2 15.5 -1.2 29.0 27.5 1.5 19.5 13.8 5.7 Refed 1 13.0 14.0 -1 .0 28.0 27.0 1.0 11.7 10.5 1.2 2 13.0 9.5 3.5 33.5 31.0 2.5 13.5 12.0 1.5 3 13.0 9.5 3.5 33.5 31.5 2.0 11.2 10.5 0.7 EXPERIMENT I I C Day Fed 2 12.0 6.5 5.5 30.5 30.0 0.0 16.2 15.0 1.2 S tarved 5 9.5 12.5 -3 .0 26.0 27.0 -1 .0 13.5 12.3 1.2 6 13.0 16.0 -3 .0 24.0 22.0 2.0 12.0 11.4 0.6 Refed 1 11.0 15.5 -4.5 23.5 21.5 2.0 12.7 12.7 0.0 2 13.0 14.0 -1 29.0 27.0 2.0 19.5 15.0 4.5 3 12.0 12.0 0 29.0 27.5 1.5 12.7 11.2 1.5 Table 2 (Continued). Nutritional ALANINE (ug/ml) GLUCOSE (mg/100 ml) LACTATE (mg/100 ml) State A v A _ v A V A-V A V A-V EXPERIMENT II D Day Fed 2 X4.2 16.5 -2. 33.5 33.0 2.5 16.5 11.2 5.3 Starved 5 12.0 15.5 -3.5 23.0 23.0 0.0 12.4 12.0 0.4 6 12.0 14.2 -2.2 26.0 26.0 0.0 12.0 11.2 0.8 Refed 1 14.2 14.2 0.0 39.0 30.0 9.0 12.0 11.2 0.8 2 13.0 13.0 0.0 39.5 34.0 5.5 13.5 12.0 1.5 3 14.2 13.0 1.2 34.0 31.0 3.0 13.8 12.1 1.7 A = V = A-V 1 Femoral Artery Femoral Vein Arteriovenous Difference -I 1 i 1 1 r~ 50 100 150 200 250 300 BLOOD FLOW (ml/min) FIGURE 1. CALIBRATION CURVE FOR BLOOD FLOW DETERMINATIONS USING THE DOPPLER FLOWMETER AND ANALOGUE-INTEGRATOR COUNTER FOR 3 mm CUFF. 1 1 1 i i r -50 100 150 200 250 300 BLOOD FLOW (ml/min) FIGURE 2. CALIBRATION CURVE FOR BLOOD FLOW DETERMINATIONS USING THE DOPPLER FLOWMETER AND ANALOGUE-INTEGRATOR COUNTER FOR A 4 mm CUFF I I > I 1 10 20 30 40 50 GLUCOSE (Mg/100 ml) FIGURE 3. CALIBRATION CURVE FOR GLUCOSE DETERMINATIONS BY THE GLUCOSE OXIDASE METHOD o to J 1 1 I I 5 10 15 20 LACTIC ACID (ug/ml) FIGURE 4. CALIBRATION CURVE FOR LACTATE DETERMINATIONS Blood Sample 100 FED STARVED FED FIGURE 5(A): CALCULATED FEMORAL ARTERY BLOOD FLOW IN SHEEP 239 100 125 FIGURE 5 (C ) . CALCULATED FEMORAL ARTERY BLOOD FLOW IN SHEEP 239. 108 F I G U R E 6 THE C O R I C Y C L E ( C O R I , 1931) V I G l u c o s e "« f L i v e r G l u c o s e G l y c o g e n P y r u v a t e L a c t a t e B l o o d L a c t a t e P y r u v a t e 1 M u s c l e G l y c o g e n * * G l u c o s e 6 - P L a c t a t e P y r u v a t e > F I G U R E 7 THE GLUCOSE A L A N I N E C Y C L E ( F E L I G , 1 9 6 9 , p . 1 0 0 3 ) L i v e r G l u c o s e A l a n i n e A m i n o A c i d s 109 FIGURE 8 RUMINANT GLUCONEOGENESIS (KRONFELD 1974 p- 44) Glucose Glucose * t Glucose-6-P 1 t Fructose-6-P 4 t Fructose 1,6-di P t g l y c e r o l Triose-P T r i g l y c e r i d e s GDP Oxa lacerate NADH P.E.P. CO, Alanine, NADH Propionic Lactate Fatty Acids Aspartate Aspartate % Oxa lace tat" Pyruvate ^ C ° 2 Ketone Acetyl-CoA^ Bodie, Ma late Fatty Acids ~7 Mitochondrion Ketone Bodies Malate •Glutamine Propionic 110 FIGURE .9 ALANINE AND GLUTAMINE CYCLES (BERGMAN, 1 9 7 3 , p . 3 6 4 ) 111 FIGURE 10 SCHEMATIC ELECTROMAGNETIC FLOWMETER (CAPPELEN, 1968, p.33) M I C R O V O L T S FIGURE 11 SCHEMATIC DOPPLER FLOWMETER (WOODCOCK, 1975, p.86) F R E Q U E N C Y FIGURE 12 (A) . SCHEMATIC DIAGRAM OF HIND LIMB (SUPERFICIAL VIEW) 112 113 FIGURE 12(B). SCHEMATIC DIAGRAM OF HIND LIMB AND CUFF PLACEMENT (CUTAWAY VIEW) 114 PLATE I. Doppler Cuff i n Place around Femoral Artery. 

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