Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1974_A6_7 C66.pdf [ 7.52MB ]
Metadata
JSON: 831-1.0099870.json
JSON-LD: 831-1.0099870-ld.json
RDF/XML (Pretty): 831-1.0099870-rdf.xml
RDF/JSON: 831-1.0099870-rdf.json
Turtle: 831-1.0099870-turtle.txt
N-Triples: 831-1.0099870-rdf-ntriples.txt
Original Record: 831-1.0099870-source.json
Full Text
831-1.0099870-fulltext.txt
Citation
831-1.0099870.ris

Full Text

Evaluation of Techniques Employed i n the Study of Alanine Metabolism i n Sheep by DONALD ARTHUR COOPER B S c , U n i v e r s i t y of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Animal Science. We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1974 In p resent ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree tha t permiss ion for e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT In view of the importance of alanin e as a gluconeogenic precursor i n ruminants, the o b j e c t i v e of the present study was to assess the e f f e c t i v e n e s s of three techniques i n estimating the metabolic parameters surrounding a l a n i n e i n wethers fed a maintenance d i e t of a l f a l f a hay. A p r e l i m i n a r y experiment u t i l i z e d a blood flow technique to study the net production and/or u t i l i z a t i o n of both a l a n i n e and glucose by the p o r t a l drained v i s c e r a . Such a method in v o l v e d e v a l u a t i n g the arterio-venous concentration d i f f e r e n c e s of al a n i n e and glucose, i n conjunction with determining the r a t e of p o r t a l vein blood flow. R a d i o a c t i v e l y l a b e l l e d **C - alanin e was administered as a s i n g l e i n j e c t i o n i n the second s e r i e s of experiments to estimate the metabolic parameters of a l a n i n e as w e l l as i t s c o n t r i b u t i o n to glucose s y n t h e s i s . The L-0- l*C-alanine was given i n t r a v e n o u s l y through p r e v i o u s l y implanted j u g u l a r catheters and the f a l l i n the s p e c i f i c a c t i v i t y of plasma a l a n i n e with time was determined. The l i n e of best f i t f o r the decay curve of the s p e c i f i c a c t i v i t y of plasma a l a n i n e was constructed by means of a computer using a multi-term exponential f u n c t i o n which enables the e s t i m a t i o n of such parameters as the pool s i z e , space, t o t a l entry r a t e , i r r e v e r s i b l e l o s s and r e c y c l i n g of a l a n i n e . The per cent conversion of alanin e to glucose was determined by the corresponding peak of glucose s p e c i f i c a c t i v i t y f o l l o w i n g the s i n g l e i n j e c t i o n of 4*C - al a n i n e . i i The turnover of a l a n i n e was a l s o studied using a continuous i n f u s i o n of L-U-1*C - al a n i n e without a priming i n j e c t i o n . The s p e c i f i c a c t i v i t y of plasna a l a n i n e reached a plateau f i v e hours a f t e r the beginning of the i n f u s i o n . I t was from these plateau l e v e l s that the r a t e of i r r e v e r s i b l e l o s s cf al a n i n e as well as i t s percent conversion to glucose was estimated. The r e s u l t s i n d i c a t e d that the s i n g l e i n j e c t i o n technique was able to p a r t i t i o n the t o t a l entry rate of al a n i n e i n t o i r r e v e r s i b l e l o s s and r e c y c l i n g and thus prove more i n f o r m a t i v e than a continuous i n f u s i o n method. The present study a l s o suggested that under c e r t a i n p h y s i o l o g i c a l s t r e s s c o n d i t i o n s i n ruminants, where r e c y c l i n g becomes prominent, a continuous i n f u s i o n approach may overestimate the a c t u a l r a t e of i r r e v e r s i b l e l o s s of a l a n i n e . i i i Tabl§_of_Conten_s_ Abstr a c t i Table of Contents •••• i i i L i s t of Figures v L i s t of Tables v i i Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i i i I n t r o d u c t i o n 1 L i t e r a t u r e Survey 4 Glucose Metabolism i n the Ruminants 4 A. ) Glucose requirements 5 B. ) Glucose Production i n the Ruminant ................. 9 Techniques Used f o r Measurement of Alanine and Glucose Metabolism 27 A. ) E a r l y D i e t a r y Studies .............................. 28 B. ) I n d i c a t o r D i l u t i o n Techniques ...................... 30 C. ) Isotope D i l u t i o n Methods ........................... 35 Experimental 46 I Metabolism of Glucose and Alanine by the Portal-Drained V i s c e r a 46 I n t r o d u c t i o n ........................................... 46 Experiment A.1 48 M a t e r i a l s and Method 48 C a l c u l a t i o n s 54 Re s u l t s and Discussion 56 Conclusion 58 I I S i n g l e I n j e c t i o n of L a b e l l e d 1 4 C - A l a n i n e ................ 61 Int r o d u c t i o n .......................................... 61 Experiment B.1. 63 M a t e r i a l s and Methods 63 C a l c u l a t i o n s 69 Results and Discussion 71 Experiment B.2........................................ 74 M a t e r i a l s and Methods 74 C a l c u l a t i o n s 77 Results and Discussion 77 i v Experiment B.3 81 M a t e r i a l s and Methods 81 C a l c u l a t i o n 83 Results and Discussion ................................ 83 Conclusion 88 I I I Continuous I n f u s i o n of L a b e l l e d * * C - l a b e l l e d A l a n i n e : Without a Priming Dose 92 Int r o d u c t i o n 92 Experiment C.1. 95 M a t e r i a l s and Methods 95 C a l c u l a t i o n s . . 97 Results and Discussion 99 Experiment C.2. .101 M a t e r i a l s and Methods 101 C a l c u l a t i o n s 103 Results and Discussion .....104 General Summary and Conclusions 106 Glossary of Terms 112 References C i t e d 116 Appendix ...125 V L i s t _ o f _ F i 2 u r e s Figure 1. ) Standard Curve f o r Glucose Determination 126 2. ) Standard Curve f o r Alanine Determination 127 3. ) Standard Curve f o r Para-aminohippuric Acid (PAH) determination ...128 4. ) Quench C o r r e c t i o n Curve f o r 1 4C 129 5. ) Paper Chromatography Separation of 1 4 C - A l a n i n e with Dse of the Actigraph Scanner 130 6. ) A c t i v i t y Curve f o r 1*C-Glucose i n Jugular Vein Blood (J.V.) Experiment B.1. 131 7. ) A c t i v i t y Curve f o r »*C-Alanine i n Jugular Vein (J.V.) Experiment B. 1 132 8. ) A c t i v i t y Curve f o r »*C-Glucose i n Jugular Vein (J.V.) Experiment B.2 ............133 9. ) A c t i v i t y Curve f o r »*C-Alanine i n Jugular Vein (J.V.) Experiment B.2 .............134 10a.) A c t i v i t y Curve f o r i*C-Glucose i n C a r o t i d Artery (C.A.) Experiment B.3. 135 10b.) A c t i v i t y Curve f o r **C-Glucose i n Jugular Vein (J.V.) Experiment B.3. 136 11a.) A c t i v i t y Curve f o r **C-Alanine i n C a r o t i d A r t e r y (C.A.) Experiment B.3 137 11b.) A c t i v i t y Curve f o r **C-Alanine i n Jugular Vein (J.V.) Experiment B.3. ...138 12. ) A c t i v i t y Curves f o r i*C-Alanine and i*C-Glucose Experiment C . I . • 139 13. ) A c t i v i t y Curves f o r 1 4 C - A l a n i n e and i*C-Glucose Experiment C.2. .....140 14. ) Major Metabolic Pathways i n The L i v e r and Kidneys of Ruminants 141 vi 15. ) Schematic Diagram I l l u s t r a t i n g the P o s i t i o n of the Sampling and I n f u s i o n Catheters 142 16. ) The Glucose alanine Cycle 143 17. ) Model f o r S i n g l e I n j e c t i o n and Continuous Infusion of a Dye 144 18. ) Model f o r Glucose Metabolism i n Sheep 145 v i i L i s t _ o f _ T a b l e s Table 133® 1(a). Packed C e l l Volume Values, Glucose, A l a n i n e , and PAH Co n c e n t r a t i o n s i n P o r t a l and C a r o t i d A r t e r i e s .... 146 1(b). C a l c u l a t i o n s f o r Experiment A.1. (Blood Flow) 117 2. P r e l i m i n a r y Experiment to Test E f f i c i e n c y of Ion Exchange Chromatography f o r S e p a r a t i o n of Plasma Components 148 3. Paper Chromatography S e p a r a t i o n of 1 4 C - A l a n i n e ....... 149 4. Data and Metabolic Parameters of A l a n i n e f o l l o w i n g a S i n g l e I n j e c t i o n of 0- 1 4 C - A l a n i n e : Experiment B.1. 150 5. Data M e t a b o l i c Parameters of Alanine F o l l o w i n g a S i n g l e I n j e c t i o n of D - 1 4 C - A l a n i n e : Experiment B.2 151 6. Data and Metabolic Parameters of Alanine F o l l o w i n g a S i n g l e I n j e c t i o n of U- 1*C-Alanine Experiment B.3. ...................................... 153 7. Summary of Metabolic Parameters f o r the S i n g l e I n j e c t i o n Experiments 156 8. Data and Metabolic Parameters of Alanine F o l l o w i n g a Continuous I n f u s i o n of l * C - A l a n i n e : Experiment C.1. 157 9. Data and Metabolic Parameters o f A l a n i n e F o l l o w i n g a Continuous I n f u s i o n of **C-Alanine: Experiment C. 2. 158 10. Summary of Parameters from S i n g l e I n j e c t i o n and Continuous I n f u s i o n Experiments 160 v i i i ACKNOWLEDGEMENTS The author wishes to express h i s g r a t i t u d e to the U n i v e r s i t y of B r i t i s h Columbia Research Committee f o r f i n a n c i a l support, i n the form of a K i l l a m P r e d o c t o r a l F e l l o w s h i p . I would l i k e to extend a f u l l measure of a p p r e c i a t i o n to the members of my t h e s i s Committee: Dr. W. D. K i t t s (Chairman) Chairman Departments of Animal Science and P o u l t r y Science Dr. R. M. Beames Department of Animal Science Dr. D. B. Bragg Department of P o u l t r y Science Dr. R.M. T a i t Department of Animal Science Dr. C. R. Krishnamurti (Associate P r o f e s s o r , Department of Animal Science) must r e c e i v e s p e c i a l mention f o r h i s s u p e r v i s i o n , i n t e r e s t and encouragement during the two years of t h i s study. Sincere thanks are extended to Dr. Brian D. Mason who, as a PhD student i n the Department of Animal Science, c o n t r i b u t e d g r e a t l y to my academic development. L a s t l y , I would l i k e to thank Miss Lynette F. Lloyd f o r her patience and help i n producing t h i s d i s s e r t a t i o n . 1 IHTRODDCTION In c o n t r a s t to monogastric animals ruminants, fed p r i m a r i l y a roughage d i e t absorb n e g l i g i b l e amounts of glucose through the g a s t r o - i n t e s t i n a l t r a c t . The carbohydrates are fermented by rumen microorganisms t o v o l a t i l e f a t t y a c i d s , of which a c e t i c , b u t y r i c , and p r o p i o n i c a c i d s predominate (Annison and Armstrong, 1970, Bergman et a l . , 1965). Thus the ruminant animal depends upon gluconeogenesis or the endogenous synthesis of glucose from non-carbohydrate sources, as a means of s a t i s f y i n g i t s energy demands. Ruminants possess a glucose requirement which i s j u s t maintained under normal d i e t a r y and environmental c o n d i t i o n s . However t h i s c r i t i c a l energy balance can be r e a d i l y disrupted under s t r e s s c o n d i t i o n s . Such traumas are manifested by a r e d u c t i o n i n blood glucose concentration (hypoglycemia) and an excessive production of ketone bodies, whose concent r a t i o n i n the blood r i s e ( k e t o s i s ) . In the d a i r y cow t h i s c o n d i t i o n i s r e f e r r e d to as acetonemia and occurs during periods of heavy l a c t a t i o n , whereas i n sheep hypoglycemia and k e t o s i s a r i s e during l a t e pregnancy and i s c a l l e d pregnancy toxemia or twin lamb disease. While these disturbances of d a i r y cows and sheep are not i d e n t i c a l , both d i s o r d e r s have a number of common c h a r a c t e r i s t i c s , which i n c l u d e a negative energy balance, a reduction of glucose i n the blood and l i v e r , and an increased f a t metabolism. The heavy d r a i n of glucose i n the ruminant a l s o becomes acute under c o n d i t i o n s of s t a r v a t i o n . Of the three major end products of rumen m i c r o b i a l 2 fermentation i t has been shown th a t between 27 and 54 per cent of the glucose may be produced from compounds a r i s i n g from propionate, e i t h e r before or upon absorption i n t o the blood d r a i n i n g the rumen (Bergman, 1973). Further work by ftnnison et a l . (1963a) confirmed the glucogenic r o l e of propionate and, by employing * * C - l a b e l l e d butyrate and a c e t a t e , i n d i c a t e d that these endproducts of carbohydrate fermentation c o n t r i b u t e l i t t l e i f any to the production of glucose. Glucogenic p r o p e r t i e s have been assigned to such precursors as the g l y c e r o l moiety of t r i g l y c e r i d e s as w e l l as l a c t a t e and pyruvate. The former becomes a major c o n t r i b u t o r under c o n d i t i o n s of underfeeding or s t a r v a t i o n when the f r e e f a t t y acids are m o b i l i l i z e d from the adipose t i s s u e . The g l y c e r o l which i s released t o the blood i s transported to the l i v e r where i t r e a d i l y g ives r i s e to glucose (Bergman, 1963). The r o l e l a c t a t e , and to a l e s s e r degree pyruvate, play i n gluconeogenesis i n ruminants has undergone considerable controversy. The s i t u a t i o n r e s u l t s because of the v a r i a b l e and unknown amounts which are absorbed i n t o the g a s t r o - i n t e s t i n a l t r a c t and the degree of metabolism which occurs through the rumen w a l l , & d e t a i l e d review of the present understanding of t h i s p o t e n t i a l glucogenic precursor i s presented i n the f o l l o w i n g s e c t i o n . Other than propionate, the second most important c o n t r i b u t o r to endogenous glucose procuction i s the amino a c i d s . Host of these are glucogenic, with the exception of l y s i n e , l e u c i n e and t a u r i n e (Krebs, 1964). Recent research with humans 3 ( F e l i g et a l . , 1970), r a t s (Aikawa et a l . , 1972) ana sheep (H o l f f and Bergman 1972a) has provided convincing evidence t h a t a l a n i n e and glutamine are the p r i n c i p l e amino aci d s extracted by the l i v e r f o r gluconeogenesis. In a d d i t i o n these amino aci d s are the main amino a c i d s released from the s k e l e t a l muscle. From t h i s f i n d i n g a l a n i n e ( F e l i g e t a l . , 1970) and glutamine ( M a r l i s s E l l i » i 1971) c y c l e s have been proposed as important means of l i n k i n g amino a c i d metabolism with the c o n t r o l of gluconeogenesis. The nature and e f f e c t of these c y c l e s are discussed f u l l y i n the f o l l o w i n g l i t e r a t u r e review. In view of the importance of a l a n i n e as a gluconeogenic precursor i n ruminants the present study assessed the e f f e c t i v e n e s s of various techniques employed t o estimate the metabolic parameters of alanine as we l l as i t s c o n t r i b u t i o n to glucose s y n t h e s i s i n sheep fed a maintenance d i e t . The three techniques employed were a m o d i f i c a t i o n of the s i n g l e i n j e c t i o n of * * C - l a b e l l e d a l a n i n e , a continuous i n f u s i o n of * * C - l a b e l l e d a l a n i n e without a priming dose and a p o r t a l blood flow study, which i n c l u d e d an arterio-venous concentration assay f o r both glucose and a l a n i n e . tt Slucosa plays an e s s e n t i a l r o l e i n c e l l metabolism. I t serves as a b a s i c component i n the c o n s t r u c t i o n of complex macromolecules of the c e l l , i n c l u d i n g n u c l e i c a c i d s , p r o t e i n s and l i p i d s . In a d d i t i o n , glucose i s u t i l i z e d f o r the production of energy which i s necessary f o r the various endergonic processes wit h i n the c e l l . Thus the c a l o r i c needs of the body are almost e n t i r e l y s a t i s f i e d by t h i s hexose. Other sources i n c l u d e the catabolism of f a t t y a c i d s , however t h i s mechanism of energy production becomes prevelent only when the former source i s depleted. The importance of glucose i n maintaining an animal i n an adequate energy balance i s p a r a l l e l e d both i n ruminants and monogastrics. However, the metabolism of carbohydrates v a r i e s i n many respects which r e f l e c t the anatomical v a r i a t i o n s between the two, and the subsequent presence of a f a r greated m i c r o b i a l population i n the ruminant s p e c i e s . The metabolism of carbohydrates i n the simple stomached animals i n v o l v e s the degradation of d i e t r a r y carbohydrates to glucose and other simple sugars, which are then absorbed by the p o r t a l c i r c u l a t o r y system and u t i l i z e d by the animal. On the other hand, i n ruminants, the carbohydrates are fermented by microorganisms present i n the rumen, to v o l a t i l e f a t t y a c i d s . Consequently 5 n e g l i g i b l e amounts of glucose are absorbed v i a the g a s t r o -i n t e s t i n a l t r a c t . Thus an endogenous glucose production from noncarbohydrate p r e c u r s o r s , or gluconeogenesis i s r e l i e d upon h e a v i l y to meet the animals glucose requirements. A.) Glucose_ReG|uirements The requirements f o r glucose are e q u a l l y important both i n ruminants and monogastrics, however the blood glucose concentrations d i f f e r considerably. The l e v e l i n ruminants (40-60 mg/100 ml) i s lower than that o c c u r r i n g i n new born or adult simple stomached animals (80-100 mg/100 ml) (Bergman, 1973). Glucose i s needed by f i v e major areas of the body, the nervous system, turnover and s y n t h e s i s of f a t , muscle, f e t u s e s and the mammary gland. The amount u t i l i z e d by other c e l l s of ruminants such as e r y t h r o c y t e s has been shown to be n e g l i g i b l e (Leng and Annison, 1962) . 1 • 2£iii25i£i2£_fe2_£k6_S§Ey.2S§-§Is tern Work by C a h i l l et a l . , (1970) on metabolism i n postabsortive humans has i n d i c a t e d that the human nervous system u t i l i z e s as much as 80 percent of the glucose released i n t o the blood. Such a study has yet to be performed i n ruminants, however from the a r t e r i a l - j u g u l a r c oncentration d i f f e r e n c e s i n sheep, considerable glucose must be removed by the nervous system and the b r a i n i n p a r t i c u l a r , of these species (McClyaont 6 and S e t c h e l l , 1956); ( S e t c h e l l , 1961). Thus the c e l l s of the nervous system with the bra i n being most important, are ab s o l u t e l y dependent upon a reg u l a r supply of glucose f o r i t s o x i d a t i v e metabolism. An i n t e r e s t i n g v a r i a t i o n to the amount of glucose u t i l i z e d during prolonged s t a r v a t i o n i n the sheep, dog and man has been r e f e r r e d to as a "glucose e x c l u s i o n " . (Owen et a l . , 1967 Eaju et a l . , 1972 and Weiner et a l . , 1971). This adaptation i s present i n a l l t i s s u e s of the body but i s most s t r i k i n g l y evident f o r the b r a i n and c o n s i s t s of a conservation of glucose and a u t i l i z a t i o n of ketone bodies and f a t t y a c i d s f o r energy purposes during periods of s t a r v a t i o n . 2 • y^JLlJZJf J-°8-£9g-f3JLJ!f£ai3gligB Glucose plays a dual rose i n the turnover and synt h e s i s of both milk and body f a t . I t i s the precursor of g l y c e r o l , and glycerophosphate, which are u t i l i z e d i n the e s t e r i f i c a t i o n of f a t t y a c i d s to form t r i g l y c e r i d e . Vaughn, (1961) has shewn th a t the enzyme g l y c e r o l kinase i s l a c k i n g i n both adipose t i s s u e and mammary gland. Thus the g l y c e r o l requirements of these t i s s u e s must be met by glucose metabolism. The second r o l e of glucose i n f a t metabolism i s i n the p r o v i s i o n of adequate s u p p l i e s of NADPH which i s required as a reducing agent i n the sy n t h e s i s of long-chain f a t t y a c i d s . ( B a l l a r d et a l . , 1969; Krebs, 1966). In a d d i t i o n to the above two, glucose can also serve as a carbon substrate f o r f a t t y a c i d 7 sy n t h e s i s . However, i n the ruminant animal carbon atoms f o r f a t syn t h e s i s are s u p p l i e d to a greater extent by acetate. Honruminants r e l y on glucose r a t h e r than acetate f o r l i p o g e n e s i s . 3. U t i i i z a t i o n _ b y - _ M u s c l e _ T i s s u e Huscle glycogen s y n t h e s i s depends on glucose. The t o t a l q u a n t i t y of glucose stored i n the muscles remains constant and i s greater than that present i n the l i v e r or body f l u i d s (Lehninger, 1970). This i s due to the much l a r g e r t i s s u e mass that i s in v o l v e d . The glycogen s t o r e s of muscle serve an e s s e n t i a l f u n c t i o n by providing an anaerobic energy supply f o r the muscle during e x e r c i s e or when oxygen becomes l i m i t i n g (Bergman, 1973). The majority of the glycogen i s converted to l a c t a t e and pyruvate which i s returned to the blood and then to the l i v e r f o r r e s y n t h e s i s back i n t o glucose. This glucose, when l i b e r a t e d i n the blood, may again r e t u r n to the muscles to be converted to glycogen. Such a process i s r e f e r r e d to as the C o r i c y c l e ( C o r i , 1931). 8 4. Ut i l i z a t i o n _ b y _ _ t h e _ F e t u s One of the greatest glucose demands i n ruminants i s during l a t a pregnancy and l a c t a t i o n . Such metabolic d i s o r d e r s as pregnancy toxaemia i n sheep and k e t o s i s i n cows occur at t h i s time. Since the p r i n c i p a l metabolic f u e l of the f e t u s i s carbohydrate, i t must maintain a constant supply of glucose from the mother. In t h i s regard work by Huggett (1961) f i r m l y e s t a b l i s h e d that the placentas of ruminants convert a p o r t i o n of the maternal glucose to f r u c t o s e so that the concentrations of both sugars reach even higher l e v e l s i n the body f l u i d s of the fetus than the mother. In a d d i t i o n , l a t e i n f e t a l l i f e l a r g e glycogen reserves are b u i l t up i n the p l a c e n t a , and i n the f e t a l l i v e r , lung and s k e l e t a l muscle. In a review of f e t a l physiology, Dawes (1968) s t a t e d glycogen concentrations to be 8 to 10 percent i n l i v e r and about 4 percent i n muscles, which are 2 to 8 times the corresponding values f o r a d u l t s of the same spe c i e s . 5• D t i l i z a t i o n during L a c t a t i o n L a c t a t i o n also makes a l a r g e demand f o r glucose upon the animal s i n c e milk contains approximately 90 times as much t o t a l sugar as does blood (Bergman, 1970). Of the two metabolic d i s o r d e r s i n v o l v i n g glucose, pregnancy toxaemia and k e t o s i s , the former i s manifested with a f a r more severe hypoglycemia than the l a t t e r since the glucose supply f o r milk production can 9 r e a d i l y be reduced or cut o f f completely. Such i s not the case f o r a pregnant animal where the glucose demands of the f e t u s must be s u s t a i n e d . From t h i s b r i e f survey of the metabolism and requirements of glucose by the ruminant, i t appears evident that these animals depend h e a v i l y upon gluconeogenesis f o r t h e i r glucose supply. B.) Glu cose_Product i o n ^ i ^ t h e ^ u m i n a n t 1. Di etar y__Sour ce The s i t e s of glucose production i n the body of a l l mammals are the gut (by a b s o r p t i o n ) , the l i v e r and the kidneys. As mentioned e a r l i e r ruminant animals d i f f e r from the simple stomached species i n t h a t l i t t l e or no glucose i s absorbed from the g a s t r o - i n t e s t i n a l t r a c t . However, there s t i l l e x i s t s controversy regarding the q u a n t i t y of glucose absorbed i n ruminants fed g r a i n or high concentrate d i e t s . One approach to t h i s problem i s to detect the amount of s t a r c h which escapes fermentation and flows i n t o the s m a l l i n t e s t i n e s . HacBae and Armstrong (1966) and Topps et a l . , (1968) employed t h i s approach and i n d i c a t e d that b a r l e y and oat d i e t s are r e a d i l y fermented i n the rumen and only l i t t l e s t a r c h appears i n the i n t e s t i n e . Further research i n t h i s area has postulated t h a t a f i n e g r i n d i n g of the g r a i n may r e s u l t i n a f a s t e r r a t e of passage through the rumen and thus enable more glucose t o be absorbed 10 (Sutton and Nicholson, 1968). , another method used to study the glucose absorption under concentrate d i e t s i s to measure the glucose concentrations i n p o r t a l and a r t e r i a l blood. With t h i s technique a number of workers detected no a c t u a l glucose absorption i n t o blood of animals fed a maintenance d i e t of hay or a 50 percent hay - g r a i n (wheat, corn or oats) mixture (Bergman, et a l . , 1970; Katz and Bergman,, 1969; Roe et a l . , 1966). In a review a r t i c l e Bergman (1973) suggested that the kind of feed, as w e l l as the amount and frequency of feed eaten, may determine whether or not glucose i s abosrbed. 2. Rinal_Gluconeoqenesis The kidneys have been suspected of p l a y i n g a r o l e i n the production of glucose s i n c e no appreciable amounts 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 and the l i v e r has been estimated to c o n t r i b u t e 85 percent of the t o t a l glucose turnover i n nonpregnant fed sheep (Bergman et a l . , 1970).. Research by Kaufman and Bergman (1971) reported t h a t glucose production by the two kidneys averaged 0.4 -0.8 g/hr and was higher i n pregnant than i n nonpregnant sheep. Renal gluconeogenesis appears to account f o r 8 - 10 percent of the t o t a l body glucose turnover i n normal fed sheep and f o r approximately 15 percent during f a s t i n g . To f u r t h e r emphasize the s i g n i f i c a n c e of r e n a l gluconeogenesis i n ruminants, these workers pointed out t h a t the sum of the glucose production r a t e s of both l i v e r and kidneys averaged 98 percent, which accounts f o r v i r t u a l l y a l l of the body's t o t a l glucose production. 11 3. Hagatic_Gluconeoaenesis The l i v e r i s the major c o n t r i b u t o r of glucose f o r the animals energy demands, with an estimate of 85 per cent of the t o t a l glucose turnover a r i s i n g from the hepatic source i n ruminants under a steady s t a t e c o n d i t i o n (Bergman et a l . , 1970). Thus the sources of hepatic glucose production c o n s i s t of four major precursors, which i n c l u d e propionate, g l y c e r o l , and l a c t a t e and amino a c i d s . A. From_Pror>inate The short chain f a t t y a c i d s a r i s i n g from the fermentation of d i e t a r y carbohydrates i n the rumen supply approximately 70 per cent of the animal's c a l o r i c requirements (Bergman et a l . , 1965)., Of these only propionate can c o n t r i b u t e s i g n i f i c a n t l y to glucose s y n t h e s i s (Annision et a l . , 1963; Bergman and Wolff, 1971; Black et a l . , 1966 and 1972; Leng and Annison, 1963). The net c o n t r i b u t i o n of propionate to the s y n t h e s i s of glucose has been subject to some controversy. Uncertainty s t i l l e x i s t s as to whether the r a t e of propionate production i n the ruminant i s s u f f i c e n t to meet the demands f o r gluconeogenesis. E a r l y work i n measuring, q u a n t i t a t i v e l y , the amount propionate absorbed from the rumen has been complicated by the l a c k of complete c o n t r o l i n timing the experiments with respect to f e e d i n g and a l s o by the f a c t that some propionate i s metabolized by the rumen epith e l i u m during absorption 12 (Pennington, 1954). Thus the gluconeogenic r o l e of propionate remained obscure. The f i r s t advance i n t h i s area was made by Annision et a l . , (1957), who, by measuring the p o r t a l v ein -a r t e r i a l blood c o n c e n t r a t i o n d i f f e r e n c e s of the v o l a t i l e f a t t y a c i d s , concluded that nearly a l l of the propionate was removed by the l i v e r . Bergman et a l . , (1966), extended the study by using a constant i n f u s i o n of l a b e l l e d propionate i n t o a rumen vein of nonpregnant, n o n l a c t a t i n g ewes and found that the amount of propionate absorbed depends g r e a t l y upon the q u a n t i t y and q u a l i t y of the feed being d i g e s t e d . The r e s u l t s i n d i c a t e d that normal sheep absorbed about 24 mM propionate per hour. These f i g u r e s estimate that approximately 50 percent of the propionate entering the p o r t a l bed i s converted to glucose which accounts f o r 27 per cent of the glucose entry r a t e s . The higher absorption rate f i g u r e s found under d i r e c t i n f u s i o n of **C - propionate i n t o the rumen are j u s t i f i e d s ince up to 70 percent of propionate produced i n the rumen i s f i r s t converted to l a c t a t e i n the rumen epithelium during absorption (Leng et a l . , 1967). This p a r c t i c u l a r study demonstrated that the conversion of propionate i n t o glucose may undergo two pathways, e i t h e r d i r e c t or i n d i r e c t . The f o l l o w i n g r e a c t i o n s are a mere o u t l i n e of these pathways: 13 DIRECT - propionate +CO. succinate -CO, phosphoenolpyruvate glucose > + INDIRECT - Propionate +C0Z succinate -CO^ l a c t a t e +C0X > — > > oxaloacetate succinate -CO phosphoenolpyruvate glucose The formation of l a c t a t e from propionate v i a the i n d i r e c t pathways occurs i n the rumen epethelium, whereas both the i n d i r e c t and d i r e c t pathways take place i n the l i v e r , Leng et al.,(1967) compared the i n c o r p o r a t i o n of »*C from (1- l*C) propionate i n t o glucose to that from (2-»*C) or (3-^C) propionate and estimated the r e l a t i v e importance of each pathway from a knowledge of the pathways i n v o l v e d . I f only the d i r e c t pathway i s operative then 50 percent as much C-1 as C-2 or C-3 of propionate would appear i n glucose. I f glucose i s synthesized from propionate v i a the i n d i r e c t pathway then the i n c o r p o r a t i o n of C-1 of propionate i n t o glucose would be 25 percent of t h a t i n c o r p o r a t e d from C-2 or C-3. Since the i n c o r p o r a t i o n of (1-»*C) propionate to t h a t from (2-**C) or (3-**C) - propionate was much l e s s than 50 per cent. Leng et al.,(1967) concluded that there i s probably extensive i n c o r p o r a t i o n of propionate carbon i n t o l a c t a t e before conversion i n t o glucose. Thus they estimated that approximately 54 per cent of the glucose entry rate i s derived from propionate i n the rumen, with only 32 per cent of t h i s propionate being converted to glucose. In summary the v a r i a t i o n s i n glucose production derived from propionate (27 t o 54 per cent) appear to depend on two main f a c t o r s , the d i e t and whether the propionate i s measured i n the 14 rumen (Leng et a l . , 1967) or i n p r o t a l blood (Bergman et a l . , 1966). The rate of propionate metabolised by rumen epithelium i s not c l e a r . Contrary to Leng»s f i n d i n g s , Weigand et a l . , (1972) obtained blood from t h o r a c i c aorta and p o r t a l v e i n of H o l s t e i n c a l v e s and estimated that only 1.0-4.6 per cent of rumen derived propionate i s converted t o l a c t a t e by the rumen e p i t h e l i u m . Regardless of these d i f f e r e n c e s , i t can be s t a t e d t h a t the amount of l a c t a t e a c t u a l l y appearing i n p o r t a l blood i s s m a l l under c o n d i t i o n s of normal rumen fermentation (Roe et a l . , 1966). However high g r a i n d i e t s w i l l increase both l a c t a t e (Dunlop and Hammond, 1965) and propionate (Lindsay, 1959) production and absorption. Of the remaining primary v o l a t i l e f a t t y a c i d s , acetate has been e s t a b l i s h e d to play a dominant r o l e i n ruminant l i p o g e n e s i s since ATP- c i t r a t a l y a s e i s d e f f i c i e n t i n ruminants and glucose can supply only l i m i t e d amounts of a c e t y l - COA f o r f a t t y a c i d s y n t h e s i s ( B a l l a r d et a l . , 1969). Butyrate, on the other hand, has been as c r i b e d a glycogenic r o l e by a number of workers f o r s e v e r a l years. For example, P o t t e r (1952) demonstrated t h a t butyrate was more e f e c t i v e than propionate i n i n c r e a s i n g blood sugar l e v e l s and r e l i e v i n g hypoglycemic convulsions i n lambs and sheep t r e a t e d with i n s u l i n . K l e i b e r et a l . , (1954) showed t h a t , u n l i k e acetate, butyrate was markedly glucogenic i n l a c t a t i n g cows and that the i*C from butyrate was u t i l i z e d more f o r s y n t h e s i s of l a c t o s e than f o r s y n t h e s i s of milk f a t . These and other s t u d i e s on the u t i l i z a t i o n of butyrate i n ruminants f a i l to demonstrate a glucogenic pathway i n e i t h e r l a c t a t i n g cows (Black et a l . , 1966) or i n sheep (Leng and Annison, 1963). 15 The metabolic r o l e of butyrate i n ruminants was i n v e s t i g a t e d by Black et a l . , (1966). These workers gave s i n g l e i n j e c t i o n s of pyruvate 2-l*C and propionate 2- 1 4C i n t o the ju g u l a r v e i n of l a c t a t i n g cows and u t i l i z e d the i n t r a - m o l e c u l a r l a b e l l i n g patterns of glutamic a c i d to assess the pathway f o r metabolism of these two glucogenic compounds. The r e s u l t s from t h i s study demonstrated that when butyrate was i n j e c t e d together with pyruvate 2- 1 4C i t caused p r e f e r e n t i a l u t i l i z a t i o n of pyruvate to form oxaloacetate and consequently l e s s pyruvate was decarboxylated to form a c e t y l - COA. Therefore they have suggested there are two e f e c t s which c o n t r i b u t e to the glucogenic e f f e c t of butyrate i n the cow, the f i r s t i s the sparing e f f e c t of butyrate on pyruvate o x i d a t i o n , and the second i s the greater a c t i v i t y of pyruvate carboxylase and hence an increased r a t e of gluconeogenesis. This work i s contr a r y to the r e s u l t s of Cook (1970), who i n v e s t i g a t e d the e f f e c t of route of a d m i n i s t r a t i o n on t r a n s f e r of **C v o l a t i l e f a t t y acids t o l i v e r glycogen and various blood substrates by administering (1-**C) acetate, (1- 1 4C) propionate and (1-**C) butyrate v i a the j u g u l a r v e i n , p o r t a l v ein or added d i r e c t l y i n t o the rumen of sheep and goats. The work demonstrated that a t r a c e r dose of ( 1 4C) butyrate i n j e c t e d i n t o the j u g u l a r vein l a b e l e d blood glucose more than ( 1 4C) acetate because of the higher s p e c i f i c a c t i v i t y of blood butyrate. When u n l a b e l l e d butyrate was infused along with a t r a c e r dose of (1- 1 4C) butyrate the s p e c i f i c a c t i v i t y of blood glucose was s i m i l a r to that observed when acetate was administered. Thus the glucogenic 16 e f f e c t of butyrate that has been observed i n the past was a Misconception and may be explained by the f a c t t h a t a t r a c e r dose of (1-**C) butyrate i n j e c t e d i n t o the j u g u l a r v e i n i s not d i l u t e d with u n l a b e l l e d butyrate since butyrate i s not normally present i n p e r i p h e r a l blood. Cook(1970) provided an a t e r n a t i v e explanation f o r the s t i m u l a t i o n of »*C t r a n s f e r from ( 1 4C) pyruvate t o glucose by butyrate observed by Black et a l . , (1966) i n that the l e v e l of butyrate administered (0.60 uM per kg body weight) provided a substrate f o r the t i s s u e s . Therefore, butyrate has a s p a r i n g e f f e c t on the o x i d a t i o n of (**C) pyruvate t o l*CO,and HO. Despite the controversy regarding the glucogenic r o l e of butyrate , the study of Cook (1970) revealed that r e s u l t s on the metabolism of »*C l a b e l l e d v o l a t i l e f a t t y a c i d s depend on whether the a c i d s are administered v i a the j u g u l a r v e i n , p o r t a l v e i n or added d i r e c t l y to the rumen. B. From_Gly_cerol G l y c e r o l i s considered to be a potent glucogenic compound and i s released from the adipose t i s s u e along with free f a t t y acids during s t a r v a t i o n , k e t o s i s or other periods of body f a t m o b i l i z a t i o n (Steinberg and Vaughn, 1965). Bergman, (1968) has shown that while the turnover r a t e of g l y c e r o l i s low i n fed sheep, i t increases markedly during f a s t i n g and e s p e c i a l l y during hypoglycemic k e t o s i s . In the case of r a t s and humans, 17 approximately 80-90 per cent of the g l y c e r o l i s removed from the blood by the l i v e r and the kidneys apparently remove most of the remainder (Borchgrevink, and Havel, 1963; Larsen, 1963; and Lundquist et a l . , 1965). The f r e e g l y c e r o l thus removed enters the gluconeogenic pathway at the t r i o s e phosphate stage as i l l u s t r a t e d i n Figure 1, which summarizes the major metabolic pathways i n the l i v e r and kidneys of ruminants (Bergman, 1973). These f i n d i n g s along with the e s t a b l i s h e d high g l y c e r o k i n a s e a c t i v i t y i n the l i v e r and kidneys suggest the major importance of g l y c e r o l f o r gluconeogenesis. In a d d i t i o n , g l y c e r o l metabolism seams s i m i l a r to that of propionate metabolism i n th a t most of the former i s also removed by the l i v e r (ftnnision et a l , 1957). Though, g l y c e r o l i s known to be glucogenic p r e c i s e estimates of i t s a c t u a l g l u c o g e n i c i t y from q u a n t i t a t i v e standpoint seem to be l a c k i n g . The f i r s t work to assess t h i s problem q u a n t i t a t i v e l y came from C a h i l l et a l . , (1970) who c a l c u l a t e d , on the basis of the gross energy metabolism and r e s p i r a t o r y q u o t i e n t , the m o b i l i z a t i o n of g l y c e r o l i n the f a s t e d human. They th e o r i z e d that t h i s g l y c e r o l could account f o r the formation of approximately 20 g glucose/day f o r the average man. Studies on the g l u c o g e n i c i t y of g l y c e r o l i n ruminants by Bergman I * 1.1 • # (1968) using a continuous i n f u s i o n of **C l a b e l l e d g l y c e r o l , demonstrated that i n f a s t e d , k e t o t i c and hypoglycemic sheep, the g l y c e r o l turnover or r a t e of release from adipose t i s s u e was high and as a maximum, could account f o r nearly 40 per cent of the animal's glucose production. However, on an average pregnant k e t o t i c sheep derived about 28 per cent of the glucose from g l y c e r o l . The r e s u l t s a l s o i l l u s t r a t e d that about 18 30 per cent of the g l y c e r o l was o x i d i z e d to CO and no marked d i f f e r e n c e s seemed to occur during the f e d , fa s t e d or hypoglycemic s t a t e s * Thus g l y c e r o l becomes an important glucogenic precursor and nearly r e p l a c e s propionate only during periods of u n d e r n u t r i t i o n , s t a r v a t i o n or other periods of body f a t metabolism. Bergman et a l . , (1968) summarize t h e i r f i n d i n g s by s t a t i n g t h a t i n the w e l l fed ruminant the c o n t r i b u t i o n of g l y c e r o l t o gluconeogenesis i s s m a l l and probably accounts f o r l e s s than 5 percent of the t o t a l glucose produced. C. From_Lactate_^nd_Px£uvate Lactate and to a l e s s e r degree pyruvate have been a t t r i b u t e d with gluconeogenic p r o p e r t i e s , p r i m a r i l y i n monogastric animals such as r a t s (Exton, 1972), dogs (Issekutz i t 1.1 • # 1972) and humans (Katz and Dunn, 1967). In ruminants however, e v a l u a t i o n of l a c t a t e metabolism i s d i f f i c u l t due to the v a r i a b l e and unknown amounts which are absorbed from the d i g e s t i v e t r a c t . This s i t u a t i o n r e s u l t s because l a c t a t e can be produced by rumen fermentaation or from propionate metabolism i n the rumen w a l l during absorption (Leng et a l . , 1967; Bergand et a l . , 1972) . L a c t a t e i s synthesized from the anaerobic metabolism of glucose and i s c a r r i e d to other areas of the body f o r complete aerobic o x i d a t i o n . The major route f o r l a c t a t e d i s p o s a l i s the s y n t h e s i s of glucose i n l i v e r and kidneys. Thus the l a c t a t e i s 19 u t i l i z e d f o r the r e s y n t h e s i s of glucose and c o n s t i t u t e s what i s known as the C o r i c y c l e ( C o r i , 1931). The operation of t h i s c y c l e does not r e s u l t i n a net i n c r e a s e i n glucose formation f o r the body s i n c e l a c t a t e o r i g i n a t e s from glucose. I t does, however, t r a n s f e r energy between the l i v e r and other t i s s u e s . The energy to d r i v e such a system i s u l t i m a t e l y derived from o x i d a t i o n of f a t t y acids and other non-glucongenic compounds. The s t u d i e s p r e v i o u s l y mentioned monogastrics estimated the C o r i c y c l e c o n t r i b u t i n g between 10 and 33 per cent of the t o t a l glucose turnover with higher r a t e s o c c u r r r i n g during underfeeding or s t a r v a t i o n . Considering the problems associated with determining l a c t a t e metabolism i n ruminants, a number of researchers have suggested that t h i s c y c l e c o n s t i t u t e s no more than 4 to 10 par cent of the glucose turnover i n fed sheep (Bergman et a l . , 1970); Annison et a l . , 1963). D. Fcom_Amino_Acids Almost a l l amino a c i d s are glucogenic to v a r y i n g degrees, with the exception of l y s i n e , l e u c i n e and t a u r i n e (Krebs, 1964). The majority of amino aci d s are stored i n muscle and along with d i e t a r y amino a c i d s , are converted to glucose i n both l i v e r and kidney cortex with the l i v e r accounting f o r approximately 85 per cent of the net glucose production from t h i s source i n fed sheep (Bergman et a l . , 1970; Kaufman and Bergman, 1971). The pathway of conversion of the v a r i o u s amino aci d s t o glucose i s 20 i l l u s t r a t e d i n Figure 14 (Appendix). When the amino acids are u t i l i z e d f o r glucose production by these s i t e s , most of the ni t r o g e n i s q u i c k l y metabolized to urea and excreted i n t o the u r i n e . In ruminants, however, some of the urea can enter the d i g e s t i v e t r a c t and the nitrogen r e i n c o r p o r a t e d i n t o a d d i t i o n a l amino acids (Nolan and Leng 1972). S i m i l a r to the previous precursors, gluconeogenesis from amino a c i d s w i l l vary widely depending upon the n u t r i t i o n a l and p h y s i o l o g i c a l s t a t e of the animal. Attempts to estimate the proportion of the t o t a l glucose production a r i s i n g from amino ac i d s have been l i m i t e d i n number and the i n t e r p r e t a t i o n of r e s u l t s i s a s s o c i a t e d with considerable d i f f i c u l t y . For example, c a l c u l a t i o n s of the q u a n t i t y of t o t a l amino a c i d s passing through the abomasum of sheep per day have been performed (Clarke et a l . , 1966; Hogan and Weston, 1967). By assuming that 55 grams of glucose can be synthesized from 100 grams of p r o t e i n , Leng (1970) has estimated that t h i s could supply up to 70 per cent of the glucose production i n nonpregnant sheep. This approach i s i n d i r e c t and probably g r o s s l y overestimates the c o n t r i b u t i o n of d i e t a r y amino a c i d s to gluconeogenesis since the a c t u a l absorption of amino acids i n t o the blood may be considerably l e s s than i t s disappearance from the rumen due to metabolism by gut t i s s u e s . Attempts have a l s o been made to assess the c o n t r i b u t i o n of amino acids to glucose from measurements of u r i n a r y n i t r o g e n e x c r e t i o n (Bergman et a l . , 1966) or urea production r a t e s (Nolan and Leng, 1970). These estimates are a l s o of l i m i t e d value since considerable amounts of ammonia are absorbed from the rumen to be converted t o urea 21 i n the l i v e r and urea i t s e l f i s r e c y c l e d through s e c r e t i o n s i n t o the d i g e s t i v e t r a c t (Bergman, 1973). Recently the g l u c o g e n i c i t y of amino acid s i n ruminants has been determined by using l * C - l a b e l l e d amino a c i d s . ft mixture of 0- 1 4C amino acid s i s o l a t e d from c h l o r e l l a p r o t e i n was used by R e i l l y and Ford (1971) to estimate that 28.2 per cent of the t o t a l glucose turnover was derived from amino a c i d s i n sheep. Their work a l s o stressed the importance of a r t e r i a l r a t h e r than j u g u l a r blood sampling. In a previous study i n which the j u g u l a r vein was used f o r both i n f u s i o n of l a b e l l e d amino acids and c o l l e c t i o n of blood, these same workers estimated amino a c i d s c o n t r i b u t e d between 12.8 and 14.7 per cent towards gluconeogenesis. Black et a l . , (1968) employed s i n g l e intravenous i n j e c t i o n s of s e v e r a l d i f f e r e n t U-**C-labelled amino a c i d s , i n t o l a c t a t i n g cows.and goats, to evaluate t h e i r r o l e as glucose precursors. The technique studied the rate and extent of i n c o r p o r a t i o n of **C from the amino a c i d i n t o plasma glucose with time. They estimated that 30 to 50 per cent of the glucose turnover may a r i s e from amino a c i d s of which a l a n i n e and glutamate each represent 6 to 8 per cent. By i n f u s i n g a mixture of **C amino acids derived from a l g a l p r o t e i n i n t o the jug u l a r vein of cows Hunter and M i l l s o n , (1964) reported that 12 per cent of milk l a c t o s e was derived from p r o t e i n i n l a c t a t i n g ruminants (Hunter and M i l l s e n , 1964). Techniques such as these, which use a mixture of **C amino a c i d s , tend to underestimate the true glycogenic c o n t r i b u t i o n of amino acid s due to a randomization or c r o s s i n g over of the l a b e l between the t r i c a r b o x y l i c acid c y c l e and the gluconeogeic pathway (Krebs et 22 a l . , 1966). Thus due to these d i f f i c u l t i e s i t becomes necessary to measure the 1 4 C - l a b e l l e d amino acids separately r a t h e r than c o l l e c t i v e l y and, i n a d d i t i o n , the measurements should be made on p o r t a l r a t h e r than on j u g u l a r blood. These f a c t o r s were pointed out by R e i l l y and Ford (1971) si n c e the g l u c o g e n i c i t y of i n d i v i d u a l amino acid s (e.g. alanine) can be confounded by an a c t u a l a n t i - g l u c o g a n i c e f f e c t of another (e.g. l e u c i n e ) . More recant s t u d i e s have employed two approaches to assess the c o n t r i b u t i o n of amino a c i d s t o glucose i n sheep fed a l f a l f a hay. The f i r s t method c o n s i s t e d of measuring the q u a n t i t i e s of amino acids added t o the plasma by the p o r t a l - d r a i n e d v i s c e r a (mainly t i s s u e s of the g a s t r o i n t e s t i n a l t r a c t ) and a l s o the j u a n t i t i e s metabolized by the l i v e r of sheep fed a near maintenance d i e t (Wolff et a l . , 1972). This was accomplished by c o l l e c t i n g blood samples from c a t h e t e r s , p r e v i o u s l y implanted i n the aorta and the p o r t a l and hepatic veins, f o r measurement of plasma amino a c i d concentrations. Figure 15 (Appendix) i l l u s t r a t e s the placement of catheters f o r i n f u s i o n and blood c o l l e c t i o n . In conjunction with these measurement, the blood flow i n the p o r t a l and hepatic veins was determined simultaneously by the method of Katz and Bergman (1969). The net metabolism of each amino a c i d was c a l c u l a t e d by m u l t i p l y i n g the v e n o - a r t e r i a l c o n c e n t r a t i o n d i f f e r e n c e by the flow of plasma. In each case net output or production was assigned a p o s i t i v e value and net uptake or u t i l i z a t i o n a negative value. The p o s i t i v e values obtained f o r the p o r t a l v e i n - a r t e r i a l concentration d i f f e r e n c e s i n d i c a t e a net output by the p o r t a l drained v i s c e r a with a l a n i n e being the amino a i c d produced i n the gr e a t e s t 23 q u a n t i t i e s . The mean output of 2.3 mM/hr accounted f c r 19 per cent of the t o t a l net appearance of -amino nitrogen i n p o r t a l plasma. The net hepatic metabolism of the various amine a c i d s was determined by measuring the hepatic v e i n - p o r t a l c o n c e n t r a t i o n d i f f e r e n c e s and i n most cases almost a l l of the gut output was removed by the l i v e r . G l y c i n e , a l a n i n e and glutamine accounted f o r about one-half of the t o t a l -amino nitrogen removed by the l i v e r (18.4 mM/hr). When the hepatic uptake exceeded the gut output, i n the case f o r these three amino a c i d s , Wolff and Bergman (1972) postulated the occurance of a net movement from p e r i p h e r a l t i s s u e s to the splanchnic v i s c e r a . This concept i s f u r t h e r i n d i c a t e d by the negative values f o r the net metabolism by the t o t a l splanchnic v i s c e r a ( l i v e r plus v i s c e r a ) , which were c a l c u l a t e d by the d i f f e r e n c e i i i c o n c e n t r a t i o n between the hepatic v e i n and a r t e r i a l source. The v a l i d i t y of t h i s study r e s t s on the assumption that the net appearance of amino a c i d s i n the p o r t a l blood r e f l e c t s the amino a c i d pattern of the p r o t e i n digested i n the i n t e s t i n e . The net output of amino acid s from gut t i s s u e s i s the r e s u l t of both absorption of d i e t a r y amino a c i d s by the gastro i n t e s t i n a l t r a c t as w e l l as v i s c e r a l t i s s u e m o b i l i z a t i o n . With t h i s i n l i n d other workers (Elwyn, 1970) have proposed t h a t , so long as the quant i t y of endogenous p r o t e i n i n the gut t i s s u e s does not change over the period of time that measurements are made ( i . e . steady s t a t e c o n d i t i o n s ) , then net gut output w i l l measure mainly absorption of amino a c i d s from d i e t a r y p r o t e i n . Thus, con s i d e r i n g the c o n d i t i o n s of continuous feeding as used i n these experiments, the t o t a l p r o t e i n s t a t u s of the gut t i s s u e s 24 i s assumed to have remained constant so that absorption of endogenous p r o t e i n would have compenstated f o r amino a c i d removal from the a r t e r i a l supply. The r e s u l t s from t h i s study i n d i c a t e d large hepatic uptakes of al a n i n e (3.2 mM/hr) and glutamine (2.1 mM/hr), which are c o n s i s t a n t with t h e i r proposed r o l e i n the tra n s p o r t of nitrogen from p e r i p h e r a l t i s s u e s to the l i v e r , where the nitrogen can be converted to urea (Marliss et a l . , 1971; and P o l e l s k y et a l . , 1969). In a d d i t i o n the amino acids removed by the l i v e r p lay an important r o l e i n the sy n t h e s i s of glucose, as w i l l be discussed l a t e r . However one of the most s i g n i f i c a n t c o n t r i b u t i o n s made by Wolff et a l . , (1972) i s the co n f i r m a t i o n t h a t few conclusions on the amino a c i d n u t r i t i o n of the ruminant can be obtained from measurements of amino a c i d concentrations i n p e r i p h e r a l plasma (Hogan et a l . , 1968; L e i b h o l t z , 1969; and Reis and Tunks, 1970). The second major approach to the study of the g l u c o g e n i c i t y of amino a c i d s i n ruminants has 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 **C-amino a c i d s . This method i s expected to y i e l d a minimal estimate f o r gluconeogenesis i n the whole body ( l i v e r p l u s k i d n e y s ) , whereas the previous technique i s a measure of maximal gluconeogenesis from amino a c i d s i n the l i v e r . Subsequently Wolff and Bergman (1972) employed a continuous i n f u s i o n of each of f i v e glucogenic l * C - l a b e l l e d amino a c i d s i n t o the vena cava to l a b e l the plasma pool and to determine the t r a n s f e r of carbon from each amino a c i d to glucose. The 1 4C data obtained were compared with the maximal glucose s y n t h e s i s p o s s i b l e from the net hepatic uptake of a l l plasma amino ac i d s . 25 Blood was sampled from the aorta, the p o r t a l v e i n and hepatic vein f o r measurement of blood flow values as w e l l as the plasma amino a c i d s p e c i f i c a c t i v i t i e s . From the plateau s p e c i f i c a c t i v i t y l e v e l s f o r both the amino a c i d and glucose, a per centage conversion was assessed. The o v e r a l l r e s u l t s of t h i s study show that when sheep are fed a near maintenance r a t i o n , they d e r i v e at l e a s t 11 per cent of t h e i r blood glucose from f i v e plasma amino a c i d s ( g l y c i n e , a l a n i n e , s e r i n e , asparatate, and glutamate) while net hepatic uptake from the plasma was s u f f i c i e n t t o produce a maximum of approximately 30 per cent of the glucose. The former f i g u r e i s a minimal.value f o r the r o l e of plasma amino a c i d s i n whole-body glucose production whereas the l a t t e r i s the maximal c o n t r i b u t i o n that plasma amino a c i d s could have made to hepatic glucose production. A true value would l i e somewhere between these two f i g u r e s . Of the f i v e amino acids s t u d i e d , a l a n i n e was the most glucogenic, with a conversion rate of 2.45 0.82 mM/hr, and glutamine was the second with a corresponding r a t e of 1.48 0.40 mM/hr. Together they account f o r at l e a s t 40 per cent of the g l u c o g e n i c i t y of a l l the amino a c i d s . The f i n d i n g that a l a n i n e and glutamine are the p r i n c i p a l amino acids removed by the l i v e r f o r gluconeogenesis has been confirmed i n recent i n v e s t i g a t i o n s on humans ( F e l i g , 1970; F e l i g §.£ al« r 1970 and Ruderman and Lund, 1972) . In a d d i t i o n these two were shown to be the main amino acids r e l e a s e d from s k e l e t a l muscle. Following these s t u d i e s , both a l a n i n e and glutamine c y c l e s (Figure 16, Appendix) were proposed as an important means of l i n k i n g amino a c i d metabolism with the c o n t r o l of 26 gluconeogenesis. In a recent review a r t i c l e , F e l i g (1973) examined i n d e t a i l the metabolism of a l a n i n e , p a r t i c u l a r l y as i t r e l a t e s to glucose homeostasis i n man. Considering the i n t i m a t e r e l a t i o n s h i p between the metabolism of al a n i n e and glucose as both precursor and product, the c y c l e i s now described as the "glucose-alanine c y c l e " . Although a l a n i n e i s the primary amino a c i d released from p e r i p h e r a l p r o t e i n s t o r e s , a number of points must be considered i n order to account f o r the p a t t e r n of amino a c i d output from muscle i n a steady sta t e c o n d i t i o n : f i r s t , a l a n i n e comprises no more than 7-10 per cent of the amino a c i d residues i n s k e l e t a l (Kominz et a l . , 1954) and cardiac muscle p r o t e i n s (Katz and Carsten, 1963); second, a s p e c i f i c p o l y a l a n y l p r o t e i n has not been i d e n t i f i e d i n muscle; and t h i r d , were such a p r o t e i n i n f a c t present i n man, under steady s t a t e c o n d i t i o n , i n s m a l l and undetectable amounts, i t would not account f o r the constant a l a n i n e output a f t e r 5 to 6 weeks of s t a r v a t i o n ( F e l i g 1.1 • i 1970). Therefore i t i s apparent that the r e l e a s e of preformed alanine from muscle p r o t e i n or from the i n t r a c e l l u l a r amino a c i d pool cannot e x p l a i n the predominant c o n t r i b u t i o n of t h i s amino a c i d to the t o t a l nitrogen r e l e a s e from muscle. Consequently F e l i g et a l . , (1970) suggested a de novo syn t h e s i s of a l a n i n e from the transamination of pyruvate. In support of t h i s conclusion i s the f i n d i n g that there e x i s t s a d i r e c t l i n e a r c o r r e l a t i o n between c i r c u l a t i n g concentrations of a l a n i n e and pyruvate i n man under bas a l c o n d i t i o n s ( F e l i g and Wahren, 1971). In summary, t h e r e f o r e , these s t u d i e s i n d i c a t e the existence of a glucose-alanina c y c l e i n which a l a n i n e i s formed p e r i p h e r a l l y by transamination of glucose derived pyruvate and i s transported to 27 the l i v e r where i t s carbon skeleton i s reconverted to glucose. Although a t t e n t i o n has been focused on the r e l a t i o n of al a n i n e t o glucose homeostasis, the glucose-alanine c y c l e i s of importance i n nitrogen metabolism as w e l l . In a d d i t i o n to p r o v i d i n g carbon skeletons f o r gluconeogenesis, the net e f f e c t of p e r i p h e r a l s y n t h e s i s of al a n i n e and i t s subsequent uptake by the l i v e r i s the t r a n s f e r of amino groups from muscle to hepatic t i s s a e s , where they may be disposed of as urea. C a r l s t e n et a l . , (1967) suggested that a l a n i n e provides a nontoxic a l t e r n a t i v e to ammonia i n the tr a n s p o r t of n i t r o g e n from p e r i p h e r a l t i s s u e s to l i v e r . The importance of alanine as a glucogenic precursor has been w e l l e s t a b l i s h e d i n monogastrics as well as ruminants. I t i s of s p e c i a l i n t e r e s t i n the l a t t e r case due to t h e i r dependence on endogenous glucose s y n t h e s i s to supply t h e i r energy demands. S§gfaS^g"^gmy§g§-IS£_5g§§y£§i§Bt,2^ Alanine„and^Glucose Metabolism Throughout the previous d i s c u s s i o n the experimental techniques employed i n the s t u d i e s of glucose metabolism and gluconeogenesis have been mentioned b r i e f l y . The present s e c t i o n comprises a summary of these various methods with emphasis placed on alanine and i t s c o n t r i b u t i o n to glucose s y n t h e s i s . The majority of the research i n v o l v i n g metabolic parameters has 28 centered around glucose. The o b j e c t i v e of the present study was to u t i l i z e a number of these techniques to measure the metabolic parameters of a l a n i n e , as w e l l as assess i t s c o n t r i b u t i o n to glucose s y n t h e s i s . A.) E a r l y _ D i e t a r y _ S t u d i e g The f i r s t problem i n the area of glucose metabolism i n ruminants i s the absorption of glucose from the gastro-i n t e s t i n a l t r a c t . I t i s w e l l e s t a b l i s h e d that n e g l i g i b l e amounts of glucose are absorbed under d i e t a r y regimes of roughages. However the q u a n t i t y of glucose absorbed, i n ruminants fed concentrate or high g r a i n d i e t s i s s t i l l under i n v e s t i g a t i o n . Topps et a l . , (1968a and b) measured the flow of d i g e s t a to the abomasum i n sheep and young s t e e r s on concentrate d i e t s . To accomplish t h i s the animals were f i t t e d with rumen and abomasal cannulas. Paper impregnated with chromium sesquioxide was administered to the rumen and samples were c o l l e c t e d from the abomasum as w e l l as from the f e c e s . By measuring -the amounts of dry matter, s t a r c h , c e l l u l o s e , t o t a l nitrogen and energy passing through the duodenum and the amounts excreted i n the f e c e s , an estimate of the amount of s t a r c h escaping fermentation and flow i n t o the s m a l l i n t e s t i n e s was made. The r e s u l t s demonstrated th a t under d i e t a r y regimes of concentrates very l i t t l e s t a r c h escapes rumen fermentation. By t a k i n g j u g u l a r v e i n blood samples they showed th a t d i e t s had l i t t l e or no e f f e c t on the concentrations of glucose. The major o b j e c t i o n t o t h i s type of 29 study i s the use of ju g u l a r vein blood as an i n d i c a t o r of any glucose f l u c t u a t i o n under the d i f f e r e n t d i e t a r y regimes. As pointed out p r e v i o u s l y , p e r i p h e r a l blood cannot be used as the sole estimate of f l u c t u a t i o n s i n blood metabolites (Wolff e t a l . , 1972). A number of i n v e s t i g a t o r s , r e a l i z i n g the inaccuracy of conc l u s i o n s based on ju g u l a r vein blood, conducted s i m i l a r s t u d i e s using glucose concentration d i f f e r e n c e s between p o r t a l and a r t e r i a l blood (Schambye, 1951, Roe e t , a l . , 1966). The r e s u l t s s t i l l showed that no glucose was absorbed i n t o the blood of animals on a d i e t of hay or one supplemented with 50% g r a i n . The gluconeogenic e f f e c t s of amino a c i d s have been estimated using techniques which assess the qua n t i t y of t o t a l amino acids passing through the abomasum of sheep per day (Hogan and Weston, 1967). These s t u d i e s are i n d i r e c t and tend to overestimate the true glucogenic c o n t r i b u t i o n of amino acid s i n the ruminant. Measurement of u r i n a r y nitrogen e x c r e t i o n has been used as an index of p r o t e i n deamination which, i n t u r n , i s u t i l i z e d as an estimate cf the amino a c i d c o n t r i b u t i o n to glucose s y n t h e s i s i n sheep (Bergman, Roe and Kon, 1966). The r e s u l t s can be misleading because, under some circumstances, urea synthesized as a r e s u l t of deamination of amino a c i d s may not be excreted i n the ur i n e . In sheep.that are star v e d , urea storage i n the body pool of urea incre a s e s (Packett and Groves, 1965; Cocimano and Leng, 1967) and on low p r o t e i n - d i e t s some of the urea synthesized may be re c y c l e d to the d i g e s t i v e t r a c t and u t i l i z e d f o r m i c r o b i a l growth (Schmidt - Nelson et a l . , 1957). 30 Nolan and Leng (1970) u t i l i z e d the r a t e of entry of urea i n t o the body pool of urea to i n d i c a t e the upper l i m i t of net amino a c i d deamination, and from t h i s derived the . p o t e n t i a l r a t e of glucose s y n t h e s i s from deaminated carbon r e s i d u e s . The method c o n s i s t e d of a s i n g l e i n j e c t i o n of r a d i o a c t i v e l y l a b e l l e d l*C-urea i n t o the jugular vein and the urea entry r a t e , pool s i z e and urea space were c a l c u l a t e d from the d e c l i n e of s p e c i f i c r a d i o a c t i v i t y of urea i n the blood. The estimates based on the rate of entry of urea are of l i m i t e d v a l u e , and would tend to overestimate due to the considerable amounts of ammonia absorbed from rumen which would be converted to urea i n the l i v e r . (Wolff, Bergman and W i l l i a m s , 1972). B.) I n d i c a t o r D i l u t i o n Techniques I n d i c a t o r d i l u t i o n i s a general term which o r i g i n a l l y r e f e r r e d t o a method of measuring the absorptive a b i l i t y of a t i s s u e or an organ f o r a s p e c i f i c n u t r i e n t . The i n d i c a t o r s p r e s e n t l y i n use range from simple dyes (Evan's blue) to r a d i o a c t i v e isotopes and measure a lar g e number of metabolic parameters such as pool s i z e , pool space, turnover r a t e , i r r e v e r s i b l e l o s s , conversion r a t e , as we l l as the standard absorption and production r a t e s . Heier and Z i e l e r (1954) postulated a working model f o r i n d i c a t o r d i l u t i o n techniques which i s based on a closed flow system with a s i n g l e input and a s i n g l e output. The system 31 contains a s p e c i f i c volume of f l u i d which enters and e x i t s a t a constant r a t e of flow. Due to the many branchings and i n t e r l a c i n g s of blood vessels w i t h i n the system p a r t i c l e s e n t e r i n g at the same i n s t a n t w i l l r e quire varying amounts of time to reach the s i n g l e output. The time r e q u i r e d i s dependent upon the path taken and the v e l o c i t y with which they t r a v e l . Thus there i s no s i n g l e t r a v e r s a l time, but rather a d i s t r i b u t i o n of t r a v e r s a l times. Such a system depends on a number of assumptions which i n c l u d e ; (a) the p a r t i c l e s e n t e r i n g the system at any time are dispersed when they e x i t i n e x a c t l y the same manner as p a r t i c l e s e n t e r i n g at any other time; t h i s property i s r e f e r r e d t o as s t a t i o n a r i t y of f l o w ; (b) the flow of i n d i c a t o r p a r t i c l e s i s r e p r e s e n t a t i v e of the flow of t o t a l f l u i d ; and (c) r e c i r c u l a t i o n of i n d i c a t o r i s not present. In developing t h i s model, Meier and Z i e r l e r , (1954) employed both a s i n g l e i n j e c t i o n as w e l l as a continuous i n f u s i o n of an i n d i c a t o r . The i n d i c a t o r was administered at the point of entry and i t s c o n c e n t r a t i o n was measured at the output as a f u n c t i o n of time. The instantaneous i n j e c t i o n technique i s c h a r a c t e r i z e d by a concentration curve which reaches a maximum q u i c k l y and then d e c l i n e s (Figure 17, Appendix). Curve A i s the r e s u l t of a s i n g l e i n j e c t i o n of a dye i n t o the systemic venous c i r c u l a t i o n . The i n d i c a t o r passes through the pulmonary c i r c u l a t o r y t r e e and the heart and i s sampled from a systemic a r t e r y . The small r i s e i n the curve at 18 seconds i s i n t e r p r e t e d to represent the appearance of detectable r e c i r c u l a t i n g dye. In the continuous i n f u s i o n method, the i n d i c a t o r i s i n j e c t e d at a constant r a t e and i t s concentration w i l l r i s e to a plateau l e v e l as 32 i l l u s t r a t e d i n Figure 17, (Appendix) Curve B. This model proposed by Meier and Z i e r l e r i s v a l i d on t h e o r e t i c a l grounds. One p o t e n t i a l weakness l i e s i n the c o n d i t i o n that i t be c l o s e d with a s i n g l e input and a s i n g l e output. Such a s i t u a t i o n i s very d i f f i c u l t to i s o l a t e i n an i n t a c t aimal. Therefore f o r t h i s model to hold f u r t h e r assumptions are necessary, one of which i s that the concentration vs time curve i s e s s e n t i a l l y the same i n a l l output branches, and thus the s i t u a t i o n reduces to t h a t of many i n p u t s , s i n g l e output. A second i s that the flow from the s i t e of i n j e c t i o n becomes mixed i n the sense that the f r a c t i o n of i t l e a v i n g through any input channel i s p r o p o r t i o n a l to the flow i n that channel. The above d i s c u s s i o n presents the t h e o r e t i c a l c o n s i d e r a t i o n s behind i n d i c a t o r d i l u t i o n techniques. 1. Blood_Flow_Mgasurgments The net metabolite c o n t r i b u t i o n of an organ or organ system can be estimated by measurements of the v e n o a r t e r i a l (V-A) concentration d i f f e r e n c e s , i n c o o r d i n a t i o n with the r a t e of blood with reference to the system of i n t e r e s t . A number of researchers have used t h i s approach to assess the c o n t r i b u t i o n of amino acidds to glucose s y n t h e s i s i n ruminants (Wolff and Bergman, 1972 a,b,c; Bergman et a l . , 1974; and Kaufman and Bergman, 1974) . Q u a n t i t a t i v e e s t i m a t i o n of the absorption of p a r t i c u l a r end 33 products of d i g e s t i o n must e n t a i l measurement of the p o r t a l vein blood flow ( F r i e s and Conner, 1961). Of the i n d i c a t o r s used f o r p o r t a l v e i n blood flow, the method i n v o l v i n g para-aminchippuric a c i d (PAH) as developed by Roe, Bergman and Kon (1966) has a number of advantages. The primary c o n s i d e r a t i o n f o r the use of PAH i s t h a t samples can be taken once the plasma PAH has a t t a i n e d a constant concentration a f t e r a period of continuous i n f u s i o n i n which the PAH i s allowed to e g u i b r i a t e with the e x t r a c e l l u l a r f l u i d . This plateau l e v e l i s a t t t a i n e d s i n c e the PAH i s r a p i d l y and t o t a l l y excreted by the kidneys provided that the i n f u s i o n r a t e i s l e s s than the maximal a b i l i t y of the kidney to excrete PAH. The two major advantages of t h i s method i n c l u d e ; 1) the t i m i n g of sample c o l l e c t i o n i s no longer as c r i t i c a l and, 2) a greater s e n s i t i v i t y i s achieved due to a greater venoartereal concentration d i f f e r e n c e . Katz and Bergman (1969) developed a method to measure both p o r t a l and hepatic venous blood flows simultaneously by u t i l i z i n g PAH as the i n d i c a t o r . The c a l c u l a t i o n s f o r these blood flows s h a l l be d e a l t with e x t e n s i v e l y i n the experimental s e c t i o n f o l l o w i n g . The assumptions made are that the PAH should not be excreted or chemically a l t e r e d i n i t s passage through the p o r t a l bed or l i v e r ; and that the i n f u s e d PAH should be w e l l mixed i n the p o r t a l v e i n , Both of these c r i t e r i a were tested and the r e s u l t s i n d i c a t e d that the asumptions were v a l i d (Katz and Bergman 1969) . In an accompanying paper, Katz and Bergman (1969b) employed t h i s technique to measure the e f f e c t s of feeding, f a s t i n g and pregnancy on the hapatic and p o r t a l metabolism of glucose, f r e e 34 f a t t y and ketone bodies i n sheep. The r e s u l t s i n d i c a t e the average net hepatic glucose production by f a s t e d nonpregnant and pregnant sheep i s 0.13 and 0.19 g/hr kg r e s p e c t i v e l y . Three hours a f t e r feeding these values were 0.28 and 0.43 g/hr kg i n nonpregnant and pregnant sheep r e s p e c t i v e l y . Thus the mean hepati c glucose production increased nearly 50 per cent 3 hours a f t e r feeding which proves t h a t , u n l i k e monogastrics, gluconeogenesis i n c r e a s e s a f t e r feeding i n ruminants. The f i g u r e s p r e v i o u s l y mentioned on the extent of r e n a l gluconeogenesis were a r r i v e d at through blood pe r f u s i o n s t u d i e s on the kidney using para-aminohippuric a c i d as the i n d i c a t o r (Kaufman and Bergman, 1971, 1974). The PAH was c o n s t a n t l y i n f u s e d i n t o the p o s t e r i o r vena cava and simultaneous blood samples were taken from the aorta and r e n a l v e i n to determine r e n a l v e n o a r t e r i a l d i f f e r e n c e s f o r glucose, f r e e f a t t y acids (FFA), ketone bodies, and PAH. In a d d i t i o n a catheter was i n s e r t e d i n t o the bladder f o r continuous u r i n e c o l l e c t i o n during the p e r i o d of blood c o l l e c t i o n . Renal blood flow per minute eguals the u r i n a r y e x c r e t i o n r a t e of PAH i n m i l l i g r a m s per minute, d i v i d e d by the d i f f e r e n c e i n blood PAH concentrations between the aorta and the r e n a l v e i n . Wolff et a l . , (1972) i n v e s t i g a t e d the q u a n t i t y of amino ac i d s added to the plasma by the p o r t a l drained v i s c e r a and t h a t metabolized by the l i v e r of sheep fed a near-maintenance d i e t by employing the blood flow technique as described above with PAH as the i n d i c a t o r . The c a t h e t e r s f o r i n f u s i o n and blood sampling were s u r g i c a l l y implanted i n t o the aorta, caudal vena cava, l e f t 35 hepatic v e i n , p r o t a l v e i n and mesenteric vein. The r e s u l t s and conc l u s i o n s of t h i s study have been discussed i n d e t a i l p r e v i o u s l y (page 25). C) Isoto£e_Dilution_Me In the f i e l d of ruminant metabolism, s p e c i f i c a l l y i n v o l v i n g the study of the body pool k i n e t i c s of va r i o u s metabolites, two groups of i n v e s t i g a t o r s can be d i s t i n g u i s h e d ; those using the s i n g l e i n j e c t i o n method and those employing a continuous i n f u s i o n of r a d i o c a t i v e t r a c e r . Both techniques have been employed to a great extent i n measuring the k i n e t i c parameters surrounding glucose metabolism. The a p p l i c a t i o n these two techniques have i n r e l a t i o n to the assessement of the c o n t r i b u t i o n amino acids t o gluconeogenesis, i s emphasized i n the d i s s c u s i o n s e c t i o n . f t l i s t of terms and t h e i r d e f i n i t i o n s i s presented i n a g l o s s a r y at the end of t h i s t h e s i s . 36 1.) S i n g l e _ I n j e c t i o n _ T e ^ The s i n g l e i n j e c t i o n technique i n v o l v e s the r a p i d intravenous i n j e c t i o n of a t r a c e r dose ( n e g l i g i b l e weight, high s p e c i f i c a c t i v i t y ) of a ^ C - l a b e l l e d substrate and the subsequent measurement of the d i l u t i o n rate of the r a d i o a c t i v e m a t e r i a l (Cook, 1966). E a r l y work with t h i s method i n v o l v e d measurements of the s i z e and turnover r a t e of the body glucose pool i n the r a t ( F e l l e r et a l . , 1950; Baker and I n c e f y , 1955), the dog (Searle et a l . , 1954) and man (Baker et a l . , (1954) i n d i c a t e d that a s i n g l e i n j e c t i o n of **C glucose gave values f o r body glucose pool s i z e which seemed t o be too high. Steele et a l . , (1956) discussed the l i m i t a t i o n s of s i n g l e i n j e c t i o n procedures i n s t u d i e s on glucose pool s i z e and u t i l i z a t i o n r a t e s i n dogs. They concluded that s t r a i g h t f o r w a r d mathematical treatment of the curve showing the d e c l i n e i n s p e c i f i c a c t i v i t y of plasma glucose a f t e r the i n j e c t i o n of l a b e l l e d glucose s e r i o u s l y overestimated glucose u t i l i z a t i o n r a t e s . In agreement with t h i s c o n c l u s i o n i s work by Annison and White (1961) who found l a r g e d i f f e r e n c e s i n glucose entry r a t e measured by a s i n g l e i n j e c t i o n , using a s i n g l e exponential f u n c t i o n , and by constant i n f u s i o n . These large d i f f e r e n c e s are due, i n part, to the f a c t that the a n a l y s i s of the data from a s i n g l e i n j e c t i o n was o v e r s i m p l i f i e d and lacked a multicompartmental approach (Baker, 1969). Kro n f e l d and Simesen (1961) i n v e s t i g a t e d glucose k i n e t i c s i n sheep f u r t h e r by the use of a s i n g l e i n j e c t i o n technique. Considering the problems a s s o c i a t e d with t h i s technique when applying a s i n g l e exponential f u n c t i o n to the e n t i r e decay curve, these workers estimated the glucose entry 37 r a t e and pool s i z e by assuming the p o r t i o n of the curve from 40 to 160 minutes to be l i n e a r on a semilogarithmic p l o t , and obey f i r s t order k i n e t i c s . Thus they derived a d e f i n i t i o n of the glucose pool to imply the group of carbon atoms i n e q u i l i b r i u m with plasma glucose carbon from 40 t o 160 minutes a f t e r a s i n g l e intravenous i n j e c t i o n of a t r a c e r of **C-glucose. The model which f o l l o w s i s that the pool comprised glucose molecules which are s i t u a t e d i n the e x t r a c e l l u l a r f l u i d which are i n dynamic e q u i l i b r i u m with glucose molecules e n t e r i n g and l e a v i n g t h i s space, to and from the c e l l s . This model proposed by Kronfeld and Simesen (1961) assumes that thorough mixing of the t r a c e r mechanically and by d i f f u s i o n throughout the space i s achieved r a p i d l y , c e r t a i n l y i n l e s s than 40 minutes a f t e r the i n j e c t i o n . In a d d i t i o n r e - e n t r y of **C-glucose, which has undergone metabolic mixing with i n t e r m e d i a t e s , i s assumed not to be appreciable before 160 minutes. The turnover r a t e represents the flow of glucose i n t o and out of t h i s e x t r a c e l l u l a r pool, which K r o n f e l d and Simesen (1961) regard as an index of the o v e r a l l r a t e of glucose metabolism that i s , an estimate of the glucose taken up by c e l l s and u t i l i z e d f o r both energy and s y n t h e s i s . An i n t e r e s t i n g p o i n t brought out by these workers i s that r e c y c l i n g of l*C from glucose through i n t e r m e d i a r i e s and back i n t o plasma as glucose-**C would become prominent sooner i n r a t s and dogs than i n cows and sheep. This i s due to the lower l e v e l of gluconkinase and glucose - 6 - phosphatase a c t i v i t i e s of various t i s s u e s of ruminants than i n nonruminants. An example i s t h a t the major r e c y c l i n g of **C between plasma glucose and l i v e r glycogen, which markedly a f f e c t s the plasma glucose l e v e l of 38 nonruminants, i s i n s i g n i f i c a n t i n sheep which l a c k h e p a t i c glucokinase a c t i v i t y (Gallagher and Butterg, 1959). Therefore r e c y c l i n g i n ruminants would be expected to take longer, and the glucose s p e c i f i c a c t i v i t y r e - e n t e r i n g the plasma would be c o m p a r i t i v e l y l e s s than i n nonruminants. Wrenshall et a l . , (1961) , have a l s o presented evidence s u b s t a n t i a t i n g the use of the s i n g l e - i n j e c t i o n method with e x t r a p o l a t i o n of a properly chosen time i n t e r v a l t o estimate the entry r a t e of glucose i n dogs. A f u r t h e r advance i n the s i n g l e i n j e c t i o n technique was the use of multicompartmental analyses of the decay curve by White §.£ a l . , (1969)., This approach was i n i t i a t e d upon the r e a l i z a t i o n t hat r e c y c l i n g of "^ C i s a major process i n ruminants. The evidence supporting t h i s i n cludes the catabolism of glucose to l a c t a t e and i t s i n c o r p o r a t i o n w i t h i n a large pool of p o t e n t i a l glucose precursors or entry i n t o the t r i c a r b o x y l i c a c i d c y c l e , as w e l l as the f i x a t i o n of J*C0 which i s returned to the glucose pool. Therefore the most l i k e l y e xplanation f o r more than one exponential term i n the equation d e s c r i b i n g the disappearance of r a d i o a c t i v i t y i n plasma glucose a f t e r an i n j e c t i o n of **C-glucose i s that carbon o r i g i n a l l y derived from glucose i s returned to the sampled pool a t two d i f f e r e n t r a t e s . The f i r s t order k i n e t i c approach by Kronfeld and Simesen (1961) was described as an o v e r s i m p l i f i c a t i o n and t h a t the whole of the i s o t o p e d i l u t i o n curve should be used i n c a l c u l a t i n g such parameters of glucose metabolism (Bescigno and Segre, 1966). When the method employed by K r o n f e l d and Simesen (1961) was a p p l i e d to the s i n g l e i n j e c t i o n r e s u l t s obtained i n the study of 39 White et a l . , (1969), the estimate of turnover r a t e was 3 per cent higher and the glucose pool s i z e was 14% higher than t h a t c a l c u l a t e d by a mulitcompartmental approach. Another drawback to t h i s s i m p l i f i e d a n a l y s i s i s that t h i s approach would not detect the changes i n the isotope d i l u t i o n curve which occur under varying p h y s i o l o g i c a l s t a t e s . The mulitcompartmental method, however i s designed to i n t e r p r e t the e n t i r e curve and thus w i l l detect any of these changes. The mathematical treatment of the isotope d i l u t i o n curve w i l l be described i n d e t a i l i n the experimental s e c t i o n f o l l o w i n g . B r i e f l y , t h e r e f o r e , the curve i s described as a sum of e x ponential terms as f o l l o w s (White et a l . , 1969): where SR t equals the s p e c i f i c r a d i o a c t i v i t y of plasma glucose at time (muc/mg.C). A^ equals the zero-time i n t e r c e p t of each component (muc/mg.C), -m£, equals the r a t e constant of each component (min ) , n equals the number of e x p o n e n t i a l components, I equals the exponential component number and t equals time (min.). The number of exponential terms i s determined by the shape of the observed s p e c i f i c r a d i o a c t i v i t y time curve and on the b a s i s of a postulated model which i s depicted i n Figure 18 (Appendix) (Leng 1970). The parameters of the model are a r b i t r a r y constants of the f u n c t i o n s or eguations and can be estimated with the use of computer programs (Baker, 1969). no Most of the work i n body pool k i n e t i c s has been with i n f u s i o n s e i t h e r s i n g l e or continuous of * * C - l a b e l l e d glucose to measure the parameters of glucose metabolism. The g l u c o g e n i c i t y of amino a c i d s has been s t u d i e d using 1 * C - l a b e l l e d amino acid s as described p r e v i o u s l y ( B e i l l y and Ford, 1970 and 1971). However, Black et a l . , (1968), i n v e s t i g a t e d the r o l e of i n d i v i d u a l amino a c i d s i n glucose synthesis i n l a c t a t i n g d a i r y cows using a s i n g l e i n j e c t i o n technique. A p a r t i c u l a r advantage i n the use of a s i n g l e i n j e c t i o n method f o r t h i s type of study i s t h a t one can assess time r e l a t i o n s h i p s among the amino acids as glucose precursors which would not be apparent during a constant i n f u s i o n . In a d d i t i o n Black et a l . , (1968) s t a t e d that a constant i n f u s i o n of amino a c i d s i s l i m i t e d due to the much greater inhomogeneity of the amino a c i d pools. The turnover of amino a c i d s i n plasma has been s t u d i e d p r e v i o u s l y using t h i s technique i n dogs (Elwyn et a l . , 1968) and i n r a b b i t s (Henriques §t 1955). The l a t t e r attempted a multicompartmental a n a l y s i s of the data and found t h a t three exponential terms were r e q u i r e d t o f i t the s p e c i f i c a c t i v i t y time curve. Therefore i t i s with t h i s i n mind th a t a s i n g l e i n j e c t i o n technique combined with a multicompartmental approach was employed t o assess, q u a n t i t a t i v e l y , the metabolic parameters of al a n i n e as well as i t s c o n t r i b u t i o n to glucose s y n t h e s i s i n ruminants under steady s t a t e or maintenance c o n d i t i o n s . in 3•) Continuous I n f u s i o n Under the heading of continuous i n f u s i o n isotope d i l u t i o n methods there are two d i s t i n c t approaches which d i f f e r s l i g h t l y . The f i r s t procedure to be developed i s the priming dose-constant i n f u s i o n technique, and the second i n v o l v e s a continuous i n f u s i o n of the t r a c e r without a priming dose. The priming dose-constant i n f u s i o n technique, i s based on the assumtpion that the body pool of a substrate i s instantaneously mixed and that a steady s t a t e of substrate o replacement e x i s t s (Cook, 1966). A s i n g l e i n j e c t i o n of the t r a c e r i s immediately f o l l o w e d by a constant i n f u s i o n of the t r a c e r . The r a t i o of priming dose to i n f u s i o n r a t e must be determined such that a r e l a t i v e l y constant s p e c i f i c a c t i v i t y of the t e s t s u b s t r a t e i s maintained a f t e r the i n i t i a l mixing of the priming dose i s complete. With glucose as an example, the rat e of change of t o t a l **C i n blood glucose with time i s the d i f f e r e n c e between the r a t e at which **C enters and leaves the glucose pool the r a t e at which * 4C leaves the pool i s dependent on the turnover r a t e and s i z e of the pool. The s p e c i f i c r a d i o a c t i v i t y of glucose carbon i n plasma at any time (SR ) i s then predicted by the expression: SR t - X - Ye" b t White et a l . , (1969) where X equals the asymptotic value f o r the s p e c i f i c r a d i o a c t i v i t y of plasma glucose (muC/mg of C),X-Y equals the i n t e r c e p t s p e c i f i c r a d i o a c t i v i t y (muC/mg of C) at t=0 and b 42 equals the rate constant (min * ) . The s p e c i f i c a c t i v i t y values between 60 and 180 minutes were used to estimate the values of X, X-Y and b, due t o the i n i t i a l mixing process and due to r e c y c l i n g of **C a f t e r a long time period (Steele et a l . , 1956). White et a l . , (1969) determined these values by using a computer program which performed a i t e r a t i v e procedure from the o r i g i n a l parameter estimates. I r r e v e r s i b l e l o s s was c a l c u l a t e d by d i v i d i n g the r a t e of i n f u s i o n of r a d i o i s o t o p e by X. Pool s i z e was obtained by d i v i d i n g the priming i n j e c t i o n (muC) by the i n t e r c e p t at zero time (X-Y) obtained by e x t r a p o l a t i o n . This technique has been employed to study the k i n e t i c s of glucose metabolism i n monogastric animals (Searle et a l . , 1956; S t e e l e et a l . , 1956) and i n ruminants (Annison and White, 1961; Bergman, 1963) due t o the s i g n i f i c a n t l y higher glucose entry r a t e s obtained by the s i n g l e i n j e c t i o n technique. However, the multicompartmental a n a l y s i s of the s i n g l e i n j e c t i o n technique had yet to be a p p l i e d at t h i s time, consequently the parameter values were high. Bergman and Hogue (1967) u t i l i z e d a priming dose constant i n f u s i o n technique to measure the glucose turnover and o x i d a t i o n r a t e s i n sheep during various stages of l a c t a t i o n . The primed i n f u s i o n technique, which was evolved by S t e e l e et a l . , (1956), i s based on evidence t h a t the glucose pool of the dog i s d i s t r i b u t e d i n two compartments i n the e x t r a c e l l u l a r f l u i d . No account i s taken of r e c y c l i n g between these two compartments and other substrate pools. The s i n g l e i n j e c t i o n r e s u l t s obtained by White e t a l . , (1969) i n d i c a t e t h a t , i n sheep, glucose i n both plasma and i n t e r s t i t i a l f l u i d c o n s t i t u t e s 43 a s i n g l e e n t i t y and that r e c y c l i n g of glucose carbon from the p e r i p h e r a l pool r e s u l t s i n a m u l i t e x p o n e n t i a l curve. Consequently the use of a primed i n f u s i o n to c a l c u l a t e the pool s i z e of any metabolite, glucose or alanin e appears t o be unsound on t h e o r e t i c a l grounds. (Leng.,1970) The continuous i n f u s i o n technique without a priming dose r e l i e s on a simple mathematical treatment (Leng, 1970)., In s t u d i e s conducted t o measure parameters of glucose metabolism i n sheep (Leng et a l . , 1967 and White e t a l . , 1969) the s p e c i f i c r a d i o a c t i v i t y of the i n f u s e d glucose was found to reach a plateau a f t e r 180 - 240 minutes. An estimate of the i r r e v e r s i b l e l o s s i s deterimined by simply d i v i d i n g the i n f u s i o n r a t e of rad i o i s o t o p e (muc/mg of C) by the plateau s p e c i f i c r a d i o a c t i v i t y (muc/mg of C) . Previous work de a l i n g with amino a c i d turnover i n sheep has inv o l v e d a constant i n f u s i o n of a mixture of **C - l a b e l l e d amino acids ( R e i l l y and Ford, 1971). The major d i f f i c u l t y with t h i s approach i s that the r a d i o a c t i v i t y of i n d i v i d u a l amino a c i d s w i l l vary between themselves and d i f f e r e n t blood v e s s e l s of the body. Thus data obtained from the i n f u s i o n of a mixture of **C - l a b e l l e d amino aci d s must be i n t e r p r e t e d with extreme c a u t i o n . The use of a continuous i n f u s i o n of i n d i v i d u a l amino acids was employed by Wolff and Bergman (1972) to evaluate the i n t e r c o n v e r s i o n s and metabolism of plasma amino acid s by the p o r t a l - d r a i n e d v i s c e r a , l i v e r , and p e r i p h e r a l t i s s u e s of fed sheep. Each experiment i n v o l v e d the continuous intravenous i n f u s i o n of a 1 4 C - l a b e l l e d amino a c i d , and sampling of blood an from the a o r t a , the p o r t a l vein and hepatic v e i n . Simultaneous equations were described to assess the r a t e s of i n t e r c o n v e r s i o n s i n the separate groups of t i s s u e s . Subsequently these authors (Wolff and Bergman, 1972b) described the use of t h i s technique to assess the c o n t r i b u t i o n s of various plasma amino a c i d s to glucose. As described p r e v i o u s l y , the r e s u l t s from t h i s study i n d i c a t e d that of the f i v e amino a c i d s examined, alani n e was q u a n t i t a t i v e l y the most important f o r blood glucose s y n t h e s i s . Recycling of l a b e l has been suggested to be the major problem i n the i n t e r p r e t a t i o n of isotope experiments, whether these be s i n g l e i n j e c t i o n or continuous i n f u s i o n (Bergman, Katz and Kaufman, 1970, Bergman et a l . , 1965, Gurpide et a l . , 1963, and Lindsay, 1970). During a s i x hour continuous i n f u s i o n , as was used i n the study by Wolff and Bergman, (1972) the l a b e l could have r e c y c l e d through 1) the i n t r a c e l l u l a r pools of the amino a c i d , 2) s y n t h e s i s and catabolism of p r o t e i n s , and 3) m e t a b o l i c a l l y r e l a t e d compounds such as other amino a c i d s , organic a c i d s and glucose. Wolff and Bergman (1972) ignore r e c y c l i n g through i n t r a c e l l u l a r pools since the u t i l i z a t i o n r a t e s (entry of turnover rate) assess the metabolic processes which l e d to an i r r e v e r s i b l e l o s s of l a b e l from the plasma p o o l , while production r a t e s determine the rate a t which the pool was replenished from u n l a b e l l e d sources. Recycling of l a b e l through p r o t e i n s i s probably s m a l l s i n c e most enzymes (Schmike and Doyle, 1970), a l l plasma p r o t e i n s , (McFarlane, 1964) and most s t r u c t u r a l p r o t e i n s have h a l f - l i v e s greater than s i x hours. Amino a c i d i n t e r c o n v e r s i o n s are expected to be important as an avenue f o r r e c y c l i n g . However l a b e l l e d a l a n i n e was found to have l i t t l e a c t i v i t y i n any other amino a c i d (Wolff and Bergman, 1972a). Thus i t would appear that experiments i n v o l v i n g the use of **C - l a b e l l e d a l a n i n e are not as a f f e c t e d by r e c y c l i n g as other amino ac i d s . However, the recent discovery of alanin e and glutamine * c y c l e s ' , which are important f o r the t r a n s p o r t of nitr o g e n from p e r i p h e r a l t i s s u e to the l i v e r , ( F e l i g et a l . , 1970, M a r l i n s e t a l . , 1971) may prove to be d e f i n i t e c o n t r i b u t o r s to the r e c y c l i n g of these l a b e l s , although q u a n t i t a t i v e measurements have not yet been determined. 46 EXPERIMENTAL I NET_METABOLISM_OF_GL VISCERA I i ^ r o d u c t i o n The measurement of p o r t a l v e i n blood flow and a r t e r i o -venous co n c e n t r a t i o n d i f f e r e n c e s of s p e c i f i c metabolites has gained importance i n the q u a n t i t a t i v e e s t i m a t i o n of the u t i l i z a t i o n of end products of d i g e s t i o n by animals (Fries and Conner, 1960). By an a n a l y s i s of the p o r t a l blood the amount of m a t e r i a l absorbed i n t o the veins d r a i n i n g the g a s t r o i n t e s t i n a l t r a c t as w e l l as the degree of metabolism occuring i n the l i v e r may be determined. In order to accomplish both these estimates, Katz and Bergman (1969a) proposed a method by which the hepatic and the p o r t a l venous blood flows are measured simultaneously. Consequently, such a technique enabled researchers to d i s t i n g u i s h between the production (absorption) and u t i l i z a t i o n of i n d i v i d u a l components by the l i v e r from that by the p o r t a l bed under varying p h y s i o l o g i c c o n d i t i o n s . U t i l i z i n g t h i s approach, Katz and Bergman (1969b) studied the hepatic and p o r t a l metabolism of glucose i n both fed and f a s t e d , pregnant and nonpregnant sheep. Work by Bergman et a l . , (1970) using t h i s method demonstrated that the p o r t a l bed of sheep u t i l i z e s s i g n i f i c a n t amounts of glucose, r e g a r d l e s s of whether they are fe d , s tarved or hypoglycemic.,Thus the p o r t a l - d r a i n e d v i s c e r a U7 ( g a s t r o i n t e s t i n a l t r a c t , pancreas, and spleen) are more important i n terms of glucose u t i l i z a t i o n than was p r e v i o u s l y recognizee!. , However t h i s discovery was only p o s s i b l e once d i s t i n c t i o n could be made between p o r t a l u t i l i z a t i o n and hepatic production, because i n terms of t o t a l splanchnic metabolism (hepatic plus p o r t a l ) values show a net production of glucose. Alanine was i n v e s t i g a t e d i n a s i m i l a r manner by Wolff et a l . , (1972) to determine the q u a n t i t y added to the plasma by the p o r t a l - d r a i n e d v i s c e r a and t h a t metabolized by the l i v e r of sheep fed a near-maintenance r a t i o n . As a measurement of the net gut output, a l a n i n e was absorbed i n t o the p o r t a l blood to a greater degree than the other amino acid s t e s t e d . The l i v e r removed l a r g e q u a n t i t e s of alanine. In f a c t the hepatic uptake exceeded the gut output, which was i n t e r p r e t e d to i n d i c a t e a net movement of alanine from p e r i p h e r a l t i s s u e s to the splanchnic v i s c e r a ( l i v e r plus p o r t a l bed). Considering these f i n d i n g s , the o b j e c t i v e of the present experiment was to evaluate the use of t h i s technique and study i t s r e l a t i v e merit i n the determination of various parameters p e r t a i n i n g to glucose and alanine i n ruminants. 48 Experimental m a.1 & Haterials_and_Methods_ 1 •) §.iiEa.i£.S.i_PE2£sd. ures A 2 year o l d wether weighing 42 kg was employed i n t h i s experiment. Feed was withheld from the animal f o r 72 hours p r i o r to surgery. T h i r t y minutes before the a n e s t h e t i c was administered, the sheep was given a subcutaneous i n j e c t i o n of a t r o p i n e s u l f a t e (5 ml-dose of 0.6 mg/ml)) to decrease the s a l i v a r y s e c r e t i o n s . The v e n t r a l abdominal w a l l was c l o s e l y shaved and washed with soap and water. The sheep was anesthetized with a ^% pentathol sodium i n j e c t i o n i n t o the j u g u l a r v e i n . During the slow a d m i n i s t r a t i o n of the drug the eye r e f l e x as w e l l as the tension of the jaw muscles were f r e q u e n t l y t e s t e d . Following i n f u s i o n of 15-18 ml of the penthathol sodium the animal was t r a n s f e r e d to a prone p o s i t i o n on the operating t a b l e . An endotracheal catheter was introduced which was connected to a closed c i r c u i t gas a naesthetic machine. The apparatus d e l i v e r e d a c o n t r o l a b l e amount of the gas halothane mixed with oxygen. Therefore the r e s p i r a t i o n r a t e could be monitored and the s t a t e of anesthesia c o n t r o l l e d c a r e f u l l y . For p o r t a l and mesenteric vein as w e l l as c a r o t i d a r t e r y c a n n u l a t i o n , the sheep was placed i n l e f t l a t e r a l recumbency. The s u r g i c a l area was f u r t h e r c l i p p e d , washed, and d i s i n f e c t e d with weak t i n c t u r e of i o d i n e . An i n c i s i o n 25 to 30 cm long was 49 made approximately 3 to 5 cm behind and p a r a l l e l t o the l a s t r i b on the r i g h t s i d e of the sheep, which penetrated through s k i n , f a s c i a , and rec t u s abdominis muscle and i n t o the p e r i t o n e a l c a v i t y . The peritoneum was secured f i r m l y with hemostats. The l e f t lobe of the l i v e r was located and moved cephalad (towards head) exposing the p o r t a l v e i n . The duodenum and abomasum were i d e n t i f i e d by f o l l o w i n g these s t r u c t u r e s i n an a n t e r i o r d i r e c t i o n , the omasum was found and brought toward the i n c i s i o n s i t e . . The omasal vein was r e a d i l y a c c e s s i b l e f o r can n u l a t i o n . S i l a s t i c tubing (.30 i n I.p. x .065 i n O.D.) i n s e r t e d with f i n e piano wire, was administered i n t o the vein through a 13 gauge needle. While p a l p a t i n g the p o r t a l v e i n , the tubing was c a r e f u l l y manipulated along the omasal vein u n t i l i t was f e l t i n the p o r t a l trunk. The wire was s l o w l y removed and the catheter was secured by s i l k sutures at the point of entry. The cannula was f u r t h e r ancohored by a d d i t i o n a l sutures to the neighbouring f a s c i a and e x t e r i o r i z e d at the d o r s a l extremity of the i n c i s i o n . A 4-way p l a s t i c stopcock (Travenol Laboratory D e e r f i e l d , 111. 60015) was f i x e d t o the end of the catheter and sutured onto the back of the animal. A second cannulation was performed on a r e t i c u l a r v e i n using the same m a t e r i a l and techniques as described above. I t was e x t e r i o r i z e d i n the same p o s i t i o n and secured to the back of the animal i n a s i m i l a r manner. T h i s catheter was intended f o r i n f u s i o n purposes on l y . In order to implant the c a r o t i d a r t e r y cannula a 5-6 cm i n c i s i o n was made along the mi d - l i n e of the trachea. The a r t e r y 50 on the s i d e of the neck was located and i s o l a t e d from the surrounding f a s c i a . The polyethylene catheter (P.E.90) was introduced v i a a 13 gauge needle and secured i n place by means of a purse s t r i n g suture through the t u n i c a a d v e n t i t i a . The cannula was e x t e r i o r i z e d and f i x e d with a 4-way stopcock which was sutured to the back of the sheep's neck. The abdominal i n c i s i o n was c l o s e d i n three p a r t s ; the peritoneum, the muscle l a y e r and the s k i n . The l a t t e r employed a heavy grade s i l k suture, whereas the former two used cat gut. By the time the i n c i s i o n s were being sutured the animal was breathing s t r a i g h t oxygen from the halothane machine. To a i d the recovery process and prevent i n f e c t i o n , an intravenous i n f u s i o n of 20 mis of 20% s t e r i l e glucose preparation was administered followed by an intramuscular i n j e c t i o n of 5 ml of D e r a f o r t , 1 a p e n i c i l l i n - strepytomycin preparation. The Derafort i n j e c t i o n s continued f o r 3 days f o l l o w i n g the operation. The E n d o t r a c h i a l tube was removed once the chewing r e f l e x of the sheep became st r o n g . This procedure reduces the chance of the animal a s p i r a t i n g rumen contents. When the sheep began showing signs of voluntary motion, i t was c a r e f u l l y t r a n s f e r r e d to a padded pen and kept under c l o s e observation. The animal was able to regain e q u i l i b r i u m w i t h i n 4 hours and returned to normal w i t h i n 24 hours of the operation. During the f i v e day recovery period between the operation 1. D e r a f o r t , Ayerst, Montreal. 51 and the blood flow experiment, s t e r i l e h e p a r i n i z e d s a l i n e (100 units/ml) was f l u s h e d through each cannula twice d a i l y to ensure t h e i r patency. 2•) Nutritional_Heaime The animal was placed on a maintenance d i e t of 1 kg dehydrated grass p e l l e t s per day. The p e l l e t s were fed to the animal i n 200 gm p o r t i o n s every two hours s t a r t i n g at 0900 hours and f i n i s h i n g at 1700 hours. This regime was maintained f o r four days p r i o r to the experiment day. The i n f u s i o n began at 1000 hours. The animal was not fed, thus the r e s u l t s are based upon a 17 hour f a s t ( o v e r n i g h t ) . 3•) 5lucose_Determination The glucose concentration was determined by the standard c o l o r i m e t r i c glucose reagent, g l u c o s t a t (Worthintion B i o c h e m i c a l s ) . This mechanism (Hashko and B i c e , 1961) makes use of the coupled enzyme r e a c t i o n s : Glucose + 0, + H,0 Glucose. H.0, + Gluconic Acid 1 * Oxidase> 1 * HjO^ + Reduced Chromogen Peroxidase Oxidized Chromogen (color) For t h i s , as w e l l as the other c o n c e n t r a t i o n assays. 52 de p r o t e i n i z e d plasma was employed. The whole blood plasma was f i r s t c e n t r i f u g e d and the r e s u l t i n g supernatant was de p r o t e i n i z e d by the use of 10% ZnSO^ and . 5N NaOH i n the f o l l o w i n g proportions: 1 ml plasma or serum + 8 ml d i s t i l l e d H^ O + 0.5 ml 10% ZnSO^. + 0.5 ml 0.5N NaOH The ZnSO^. and NaOH were t i t r a t e d together i n order to ensure n e u t r a l i t y . A f t e r throughly mixing the above preparation and wa i t i n g 10 minutes, each sample was c e n t r i f u g e d again and the c l e a r supernatant was employed f o r the subsequent assays. One ml of the d e p r o t e i n i z e d plasma was added to 4 ml of g l u c o s t a t preparation and the r e a c t i o n proceeded f o r e x a c t l y 20 minutes at which time i t was stopped by adding 2 drops of 4N HCl. The o p t i c a l d e n s i t y of each sample r e a c t i o n was read on a Spectronic 20 spectrophometer at a wavelength of 420 nm. These readings were converted t o mg/100 ml glucose by comparison with standard curve (Appendix, Figure 1) prepared from known glucose concentrations. 53 4.) Alanine_Determination The concentration of alanine was determined by an enzymatic technique described by Bergmeyer (1965) i n which a l a n i n e i s converted to pyruvate by glutamate pyruvate transaminase (GPT) and ox o g l u t a r a t e : 2-oxoglutarate • L-alanine GPTV L-glutamate + pyruvate This r e a c t i o n i s coupled to a second i n which l a c t i c dehydrogenase (LDH) reduces pyruvate i n the presence of NADH to l a c t i c a c i d : Pyruvate • NADH+H* KDH L - l a c t a t e + HAD The disappearance of NADH was followed on a ONICAM SP 800 spectrophotometer at a wavelength of 340 nm. Since there i s an excess of both enzymes, oxoglutarate and NADH , the rat e of the coupled r e a c t i o n with l i m i t e d a l a n i n e concentrations i s s t r i c t l y p r o p o r t i o n a l to the amount of a l a n i n e present. The measurement of the r e a c t i o n r a t e permits the determination of alanine w i t h i n each sample by use of a standard curve prepared with known al a n i n e c o n c e n t r a t i o n (Appendix, Figure 2) . 54 5 •) £i°2iL.Ii2£^eter mi nation The p o r t a l v e i n blood flow was measured with the use of the i n d i c a t o r para- aminohippuric a c i d (PAH). A priming dose (15 ml) of a 1.5% PAH s o l u t i o n was administered i n t o the cannulated r e t i c u l a r v ein at the beginning of the experiment. This was immediately followed by a continuous i n f u s i o n of the same PAH s o l u t i o n (1.5%) i n t o the r e t i c u l a r vein by means of a p c l y s t a t i c i n f u s i o n pump (Buchler Instruments) a t a rate of 0.79 ml/min. A f t e r a one hour e q u l i b r a t i o n p e r i o d , heparinized blood samples were withdrawn from the p o r t a l v e i n , and c a r o t i d a r t e r y at 15 minute i n t e r v a l s f o r one hour to determine the a l a n i n e , glucose and PAH con c e n t r a t i o n s . The plasma l e v e l s of PAH were assessed by the method of Smith et a l . , (1945). The o p t i c a l d e n s i t i e s (0. D.) of the samples were measured on a Spec 20 spectrophotometer at a wavelength of 540 nm1. The corresponding PAH concentrations were determined by comparison with a standard curve of known PAH concentrations (Appendix, Figure 3 ) . C a l c u l a t i o n s The p o r t a l vein plasma flow (PVPF) and the p o r t a l v e i n blood flow (PVBF) were c a l c u l a t e d according to the f o l l o w i n g equations; (Katz and Bergman, 1969a) PVPF= CIxIR C P V - C * 55 PVBF= PVPF (1-PCV) Where: CI= concentration of PAH i n the i n f u s i o n s o l u t i o n (mg/100 ml) IR= i n f u s i o n r a t e (mls/min) PCV= the blood packed c e l l volume (hematocrit) Cp^and C^= Plasma PAH c o n c e n t r a t i o n i n p o r t a l vein and a r t e r i a l blood r e s p e c t i v e l y . The net p o r t a l production r a t e s of both glucose and a l a n i n e were determined as f o l l o w s : (Katz and Bergman 1969b) P= F P V { C P V ~ CA> where P represents the p o r t a l net production r a t e s of the metabolite; F p ^ i s the whole blood flow (ml/min) i n the p o r t a l v e i n ; and C p v and are the concentrations of the metabolite i n the p o r t a l v e i n and a r t e r i a l v e s s e l s r e s p e c t i v e l y . A negative value f o r the production r a t e s of e i t h e r a l a n i n e or glucose i n d i c a t e s u t i l i z a t i o n . The r e s u l t s were s t a t i s t i c a l l y analyzed using the student T t e s t to denote s i g n i f i c a n t d i f f e r e n c e s f o r both a l a n i n e and glucose concentrations between the c a r o t i d a r t e r y and p o r t a l v e i n plasma. Values preceded by a s i g n i n d i c a t e the standard error of the mean. 56 2£§fii£2_!£d_Discussion A summary of the mean blood concentrations of glucose, a l a n i n e and PAH that were obtained by sampling from the p o r t a l v ein and c a r o t i d a r t e r y , as w e l l as the corresponding packed c e l l volume f i g u r e s are presented i n Table 1(a) (Appendix). 1.) P o r t a l Blood Flow To c a l c u l a t e the p o r t a l vein blood flow (PVBF) and the p o r t a l vein plasma flow (PVPF), the PAH was allowed to e q u i l i b r a t e with e x t r a c e l l u l a r f l u i d s and thus the concent r a t i o n f i g u r e s employed are those a t the plateau l e v e l (Appendix, Table 1 ( a ) ) . In t h i s experiment the plateau or constant PAH concentrations f o r both the c a r o t i d a r t e r y and p o r t a l v e i n were a t t a i n e d between the 1 hour 25 minute and 1 hour 45 minute blood c o l l e c t i o n s . Thus the average of these two con c e n t r a t i o n values were used f o r the determination of the blood flow f i g u r e s . A summary of the c a l c u l a t i o n s and r e s u l t s i s presented i n Appendix, Table 1(b). A p o r t a l vein blood flow value of 2010 mls/min 3/4 (47.85 ml/mm/kg/B. W.) was determined i n the present experiment. The r e s u l t agrees with p r e v i o u s l y published r e s u l t s which estimate the mean p o r t a l vein blood flow i n a range from 1800 ml/min (Bergman and Wolff 1971) to 2493 ml/min (Katz and Bergman, 1969a) f o r f e d , nonpregnant sheep. The l a t t e r study employed a technique which enabled a measurement of both the 57 h e p a t i c and p o r t a l blood f l o w s . Such a r e s u l t was not p o s s i b l e i n the present experiment s i n c e c a t h e t e r s were implanted i n t o the p o r t a l v e i n and c a r o t i d a r t e r y only ,and not i n t o the hepatic v e i n . The value f o r p o r t a l v e i n blood flow i n the present study appears to f a l l w e l l w i t h i n range of e s t a b l i s h e d r e s u l t s . A d d i t i o n a l support i s provided by more recent work by Wolff, Bergman and Williams (1972) who quote p o r t a l blood flow values from 1440 to 2240 ml/min i n sheep fed a near-maintenance d i e t . These r e s u l t s are more r e l e v a n t to the present experiment since the d i e t a r y regime as w e l l as the p h y s i o l o g i c a l s t a t u s of the sheep (nonpregnant and nonlactating) are s i m i l a r . 2 •) Sg.t-Slu£2gs,Metabolism The negative production value (-.142 g/hr/kg B.W ) i n d i c a t e s a net u t i l i z a t i o n of glucose by the p o r t a l drained v i s c e r a . This f i n d i n g i s i n agreement with published work which showed that a net u t i l i z a t i o n of glucose i n the p o r t a l bed was almost always obtained r e g a r d l e s s of the d i e t or whether the sheep were hypoglycemic or f a s t e d (Katz and Bergman, 1969b; Bergman et a l , 1971). The f i g u r e obtained i n the present study i s s l i g h t l y l a r g e r than the mean p o r t a l bed u t i l i z a t i o n r a t e of fed sheep and smaller than t h a t i n sheep fasted f o r three days. BU This appears appropriate s i n c e the value of .142 g/hr/kg B.W.n was determined with a sheep f a s t e d f o r 17 hours. The net u t i l i z a t i o n of glucose, as shown here as w e l l as i n previous works, i s s l i g h t . The a c t u a l p o r t a l - a r t e r i a l glucose 58 concent r a t i o n d i f f e r e n c e s c a l c u l a t e d i n t h i s experiment were not 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 < 0.05 l e v e l ) which supports others who have observed the same (Roe et a l ., 1966; Schambye, 1951). 3 •) Stf_Alanine_Meta^olism The f i g u r e f o r al a n i n e metabolism by the p o r t a l bed demonstrated a net production (absorption by the p o r t a l blood) of 1.49 mM/hr. The published values i n d i c a t e a mean p o r t a l bed production rate of 2.29 ±.29 mM/hr (Wolff Bergman and Will i a m s , 1972), which was determined on ten separate experiments whereas the f i g u r e obtained i n the present study was based upon a s i n g l e experiment. Considering t h i s f a c t , the s i n g l e value of 1.49 mM/hr a r r i v e d at here cannot be regarded as an absolute estimate but undoubtedly i s w i t h i n the range reported by Wolff §t §i«'• * (1972). I t i s important to note, however, that the al a n i n e concentrations between the c a r o t i d a r t e r y and the p o r t a l v e i n proved to be s i g n i f i c a n t l y d i f f e r e n t i n the present experiment. Conclusions The r e s u l t s from the present determinations of p o r t a l vein blood flow, as we l l as the net metabolism of alanin e and glucose by the p o r t a l drained v i s c e r a a i d more i n e v a l u a t i o n of the 59 e f f e c t i v e n e s s of t h i s method ra t h e r than as absolute estimates which would be r e p r e s e n t a t i v e of a l l sheep. The purpose of t h i s experiment was to assess the value and a p p l i c a t i o n of the blood flow technique using para-aminohippuric a c i d as the i n d i c a t o r . Therefore the r e s u l t s f o r p o r t a l vein blood flow (2010 ml/min), net glucose u t i l i z a t i o n by the p o r t a l bed (.142 g/hr/kg B. W. ), and net alanine production by the p o r t a l bed (1.49 mM/hr) are w i t h i n the range of p r e v i o u s l y published f i g u r e s and consequently c o n t r i b u t e a d d i t o n a l support f o r the use of t h i s approach i n metabolic s t u d i e s . The s o p h i s t i c a t e d s u r g i c a l procedures r e q u i r e d by t h i s method are the l i m i t i n g f a c t o r upon which the success of the study i s based. Thus i n order to s t a t e a c c u r a t e l y t h a t the arterio-venous c o n c e n t r a t i o n d i f f e r e n c e s represent a s p e c i f i c organ or t i s s u e system, the c a t h e t e r s must be implanted p r e c i s e l y . This process r e q u i r e s a great deal of s k i l l . Often the catheters become dislodged or blocked and consequently the e n t i r e experiment i s destroyed. The chemical analyses as w e l l as the c a l c u l a t i o n s are s t r a i g h t forward and r e q u i r e a minimum of i n t e r p r e t a t i o n . The technique of blood flow measurement along with a r t e r i o -venous concen t r a t i o n d i f f e r e n c e s proved t o be an e f f e c t i v e approach i n determining, q u a n t i t a t i v e l y , the net metabolism of s p e c i f i c compounds by i n d i v i d u a l organs or t i s s u e s . This i s emphasized through the demonstration by Katz and Bergman (1969b) of a method whereby the net metabolism of the t o t a l splanchnic region i s d i v i d e d i n t o t h a t of the l i v e r and p o r t a l - d r a i n e d 60 v i s c e r a . Although h i g h l y demanding, t h e i r technique was inst r u m e n t a l i n r e c o g n i z i n g that there i s a net u t i l i z a t i o n of glucose by the p o r t a l bed. The r e s u l t s of the present study confirmed t h e i r f i n d i n g that no net glucose absorption occurs i n sheep fed a maintenance d i e t , but a p o r t a l glucose u t i l i z a t i o n appears to be present at a l l times. Although the a l a n i n e data f o r net p o r t a l output d i d not agree as c l o s e l y to published r e s u l t s as t h a t f o r glucose, the value of 1.49 mM/hr/kg B.W. i s s t i l l of the magnitude which s u b s t a n t i a t e s i t s e s t a b l i s h e d r o l e as the primary amino a c i d absorbed by the p o r t a l c i r c u l a t i o n and d e l i v e r e d t o the l i v e r . From the data obtained i n t h i s experiment i t was not p o s s i b l e to determine the l i v e r ' s r o l e i n the metabolism of these two metabolites due to the d i f f i c u l t y i n cannulating the he p a t i c v e i n . This was never accomplished s u c c e s s f u l l y , thus the r e s u l t s are based on the p o r t a l bed and i t s r e l a t i o n t o a l a n i n e and glucose. In summary t h e r e f o r e , the r e s u l t s obtained from t h i s study i n d i c a t e d t h a t the technique employed i s e f f e c t i v e and accurate to be a p p l i e d " i n v i v o " metabolic s t u d i e s . The major advantage to t h i s method i s th a t once the catheters have been properly implanted, an organ or t i s s u e system can be completely i s o l a t e d and consequently the r e s u l t s obtained are d i r e c t l y a t t r i b u t a b l e to the f u n c t i o n i n q of that system. The primary l i m i t a t i o n to t h i s technique i s the complex s u r g i c a l s k i l l s required around which the e n t i r e study revolves. 61 11 S i n g_ l e _ I n j e c t ion_of _ £a be 1 led__ £*C-Alanine IHifS'l uc t i o n The s i n g l e i n j e c t i o n technique of a l a b e l l e d t r a c e r i s r e g a i n i n g acceptance as an e f f e c t i v e and accurate procedure i n measuring body pool k i n e t i c s f o r various m e t a b o l i t e s . The majo r i t y of the work done has i n v o l v e d l a b e l l e d **c - glucose to study glucose metabolism. Numerous models have been developed to des c r i b e the movement of glucose carbon i n the body, with a 2 or 3 compartmental model as the most widely e s t a b l i s h e d to f i t **C-glucose isotope d i l u t i o n data i n r a t s (Baker et a l . , 1959; S t e e l e , 1964) and i n sheep (White et a l . , 1969; Skinner et a l . , 1959; Hescigne and Segre, 1966). The f i t t i n g of data to such models has involved a number of d i f f i c u l t i e s which, i n the e a r l y stages, had persuaded many workers to doubt the v a l i d i t y of t h i s approach. Annison and White (1961) s t a t e d t h a t the parameters governing the entry and outflow of glucose from the proposed multi-compartment system cannot be adequately defined f o r a mathematical treatment of s i n g l e - i n j e c t i o n data. The d i f f i c u l t i e s i n v o l v e d with d e f i n i n g components suggest the use of c u r v e - f i t t i n g computer techniques i n which the i n d i v i d u a l component slopes w i t h i n a complex curve need not be c l e a r l y defined (Berman, 1963). This approach has been u t i l i z e d by a number of i n v e s t i g a t o r s i n d e f i n i n g parameters of glucose metabolism i n monogastrics (Baker et a l . , 1959; Segal e t a l . , 1961). Work with ruminants using a s i n g l e i n j e c t i o n technique combined with a multicompartmental a n a l y s i s has been sparse and the r e s u l t s v a r i a b l e (White et a l . , 1969; Leng, 1970). The 62 l a t t e r author a t t r i b u t e s the v a r i a b i l i t y to the f a c t that i n s u f f i c i e n t blood samples were taken over too short a time p e r i o d i n many experiments f o r accurate a n a l y s i s of the data. The multicompartmental approach to the a n a l y s i s of s i n g l e i n j e c t i o n data has been used to a l i m i t e d degree i n ruminants with the major emphasis upon glucose metabolism s t u d i e s . Thus the f i e l d of amino a c i d r e s e a r c h , s p e c i f i c a l l y t h e i r metabolic parameters and c o n t r i b u t i o n to glucose s y n t h e s i s , has been s p a r s e l y touched by the use of t h i s technique (Black, 1968; Egan and Black, 1968). I t i s only r e c e n t l y that work on the determination of such parameters as turnover r a t e s of i n d i v i d u a l amino acids as w e l l as t h e i r r e l a t i o n to gluconeogenesis has been conducted (Wolff, Bergman and Will i a m s , 1972, Wolff and Bergman, 1972a and b). The methods employed by these researchers i n v o l v e blood flow s t u d i e s , as described i n Experiment A, and continuous i n f u s i o n of l a b e l l e d amino a c i d s , which w i l l be discussed i n the f o l l o w i n g s e c t i o n . In one study reported, a compartmental a n a l y s i s was attempted on the data of a s i n g l e i n j e c t i o n of g l y c i n e and the exist e n c e of at l e a s t three pools f o r the metabolism of g l y c i n e revealed (Henriques et a l . , 1955). The o b j e c t i v e of the f o l l o w i n g experiments was to assess the value of the s i n g l e i n j e c t i o n of 1 * C - l a b e l l e d a l a n i n e along with a multicompartmental a n a l y s i s of the data as a means of q u a n t i t a t i v e l y e s t i m a t i n g the metabolic parameters of t h i s amino a c i d as w e l l as i t s c o n t r i b u t i o n to glucose. Incorporated w i t h i n t h i s assessment are the r e l a t i v e advantages and disadvantages of t h i s technique. Such f a c t o r s as p h y s i o l o g i c a l assumptions, ease 63 of a p p l i c a t i o n and c a l c u l a t i o n of r e s u l t s as w e l l as time and expense are included i n e v a l u a t i n g the e f f e c t i v e n e s s of t h i s method. The study c o n s i s t s of three separate experiments, each comprising of a s i n g l e i n j e c t i o n of 1 4 C - a l a n i n e i n t o the j u g u l a r v e i n . Blood samples f o r a n a l y s i s were c o l l e c t e d from the j u g u l a r v e i n , except i n the t h i r d experiment where c a r o t i d a r t e r i a l blood was a l s o withdrawn. The a n a l y s i s f o r a l l three comprised i n s p e c t i o n of the f a l l i n the s p e c i f i c a c t i v i t y of plasma al a n i n e with time. The parameters f o r a l a n i n e metabolism were determined from a m u l t i e x p o n e n t i a l f u n c t i o n a p p l i e d to the data to generate a l i n e of best f i t f o r the decay curve. This procedure i n v o l v e d the use of a computer. The d e t a i l s surrounding the a n a l y s i s s h a l l be discussed i n the f o l l o w i n g m a t e r i a l s and methods s e c t i o n . P xperiment_B.1. M a t e r i a l s and Methods A three year o l d wether weighing 35 kg was used f o r t h i s experiment. The s u r g i c a l preparation c o n s i s t e d of i n s e r t i n g a p o l y v i n y l catheter (P.E. 90) i n t o the r i g h t j u g u l a r vein 24 hours p r i o r to the s t a r t of the experiment. The catheter was maintained operative by frequent f l u s h i n g s with a s o l u t i o n of h e p a r i n i z e d s a l i n e (100 u n i t s / m l ) . The n u t r i t i o n a l regime co n s i s t e d of a roughage d i e t of 1 kg 64 a l f a l f a hay fed twice d a i l y at 0800 hours and 1600 hours, on the day of the experiment the animal was given i t s morning feed and the i n j e c t i o n began 4 hours l a t e r at 1200 hours. Thus the sheep was i n a fed (4 hr f a s t ) c o n d i t i o n . The experiment was performed with the animal i n a r a i s e d metabolism cage which enabled the c o l l e c t i o n of urine and feces f o r the proper waste d i s p o s a l . The b u i l d i n g was w e l l v e n t i l a t e d thus adding a f u r t h e r c o n t r o l measure t o the use of iso t o p e s i n l i v i n g animals 1 •) Jsoto£e_and__Blood_Collection L-Alanine 0-L-J*C was obtained from ICN Chemical and Radioisotope d i v i s i o n with the f o l l o w i n g s p e c i f i c a t i o n s ; s p e c i f i c a c t i v i t y 100-120 mC/mm, packaged i n 0.01N HC1 at a concentr a t i o n of 0.1 mC/ml. A s i n g l e i n j e c t i o n of .05 ml (50 uCi) of the isotope was administered i n t o the ju g u l a r vein and 10 ml of blood plasma was c o l l e c t e d from the same ju g u l a r vein a t the f o l l o w i n g time i n t e r v a l s : Sample No. C o l l e c t i o n Time 1 C o n t r o l (before i n f u s i o n ) 2 5 min 3 10 min 4 20 min 5 30 min 6 52 min 7 60 min 8 120 min 9 180 min 10 1110 min (0830 hours f o l l o w i n g day) The time r e q u i r e d f o r c o l l e c t i o n of plasma was held constant at between 80-100 seconds f o r each sample. 65 2 •) Chemical_Methods a) Separatjon^qf ^Plasma^Coffiponents The s e p a r a t i o n of the plasma c o n s t i t u e n t s was accomplished by i o n exchange chromatography. The adsorption and r e t e n t i o n of the charged molecules by the r e s i n , e i t h e r p o s i t i v e ( c a t i o n exchange column) or negative (anion exchange column), permit the separation of the plasma c o n s t i t u e n t s i n t o a basic f r a c t i o n ( p o s i t i v e l y charged amino a c i d s ) , an a c i d i c f r a c t i o n (negatively charged organic acids) and a n e u t r a l f r a c t i o n (sugars). i ) Cation Exchange Resin The c a t i o n r e s i n (DOWEX 50W-X8) employed was obtained from the Bio-Rad L a b o r a t o r i e s . I t was i n the hydrogen form (200-400 mesh) with a t o t a l c a p a c i t y of 5.1 meg/dry gram or 1.7 meg/ml wet r e s i n . Approximately 2 dry grams of r e s i n were used f o r s e p a r a t i o n . To ensure the r e s i n was i n the hydrogen form 40 ml 2N HCl was passed through the column followed by a wash with daionized water u n t i l the eluent i s n e u t r a l . Three ml of the dep r o t e i n i z e d plasma sample were ap p l i e d to the top of the column and allowed t o stand f o r 15 minutes before being passed through. The sample was run sl o w l y through the column and a l l non-adhering compounds (sugars and acids) were washed through with 50-60 ml water. The basic substances (amino acids) which are adsorbed t o the r e s i n were eluted by passing 60-70 ml 2N HCl through the column. The amino acids 66 r e t a i n t h e i r charge and were el u t e d by the greater concentration of hydrogen. The r e s i n was discarded and f r e s h r e s i n was regenerated f o r the next sample. i i ) *nionJ|xchanae_Resin The anion r e s i n was obtained from J. T. Baker Chemical Company and was i n the c h l o r i d e form (200-400 mesh). I t i s a strong b a s i c r e s i n with a t o t a l exchange c a p a c i t y of 4.4 meg per dry gram. Approximately 2.5 dry grams of the r e s i n was u t i l i z e d f o r each plasma sample. The r e s i n was converted to the formate form by passing through 60 ml of 1M solium formate. At t h i s stage the c h l o r i d e t e s t was negative when tested with AgNO^ Following the sodium formate, 20-30 ml of 0.IN formic a c i d was added and the column was washed with d i s t i l l e d water u n t i l n e u t r a l . The sample from the c a t i o n column was administered wand a l l non-adhering substances (sugars) were washed through the column with water. The anions (acids) were eluted by passing 40 ml of 4N formic a c i d followed by 30 ml of 8N formic a c i d through the column. Once again the r e s i n was discarded with a f r e s h q u a n t i t y regenerated f o r each subsequent sample. Once separated, each f r a c t i o n was evaporated to dryness with the use of a Buchi Rotovapor. These samples as w e l l as the o r i g i n a l u n diluted plasma samples were counted using an ISOCAP/300 l i q u i d s c i n t i l l a t i o n counter (Nuclear Chicago). The counting e f f i c i e n c y was detected by using the e x t e r n a l standards r a t i o (ESR). Figure 4 (Appendix) i s the quench 67 c o r r e c t i o n curve using the ESR method i n conjunction with the quenched **C standards provided by Nuclear Chicago. I t i s from t h i s Figure that the counting e f f i c i e n c y f o r each sample was detected. In a l l of the counting throughout t h i s p r o j e c t , Phase Combining System (PCS) s o l u b i l i z e r was used as the f l u o r s o l u t i o n . The standard mixture was 200 u l of sample i n combination with 10 ml PCS., In order to evaluate the e f f i c i e n c y of the i o n exchange chromatography i n separating plasma c o n s t i t u e n t s , a p r e l i m i n a r y run was undertaken using a prepared mixture of the f o l l o w i n g : 0.01 ml L-Alanine-OL-**C 1 ml plasma 0.1 mg l a c t a t e 0.1 mg l a c t a t e This s o l u t i o n was passed s u c c e s s i v e l y through the c a t i o n and anion exchange column as described above and the f r a c t i o n s counted y i e l d i n g the f i g u r e s l i s t e d i n Table 4 (Appendix) . These recovery f i g u r e s i n d i c a t e t h a t i o n exchange chromatography can be used e f f e c t i v e l y f o r the separation of plasma i n t o a c i d i c , b a s i c and n e u t r a l f r a c t i o n s . To confirm f u r t h e r that the r a d i o a c t i v i t y corresponded to the metabolites under i n v e s t i g a t i o n , the basic and n e u t r a l f r a c t i o n s were spotted (50 ul) on a paper chromatogram. A two d i r e c t i o n a l paper chromatography was used f o r the separation 68 of a l a n i n e i n which the sol v e n t system f o r the f i r s t d i r e c t i o n c o n s i s t e d of n-butanol, g l a c i a l a c e t i c a c i d and water i n a r a t i o of 80:20:20 (v/v) r e s p e c t i v e l y . For the second d i r e c t i o n l i q u i f i e d phenol:H 0 was used i n the propo r t i o n s 94:28 (v/v) r e s p e c t i v e l y . S i n g l e d i r e c t i o n a l paper chromatography was employed to separate the sugar f r a c t i o n . The solvent system c o n s i s t e d of iso-propanol:n-butanol:water i n a 7:1:2 (v/v) r a t i o . The amino a c i d and sugar chromatograms were compared to standards c o n s i s t i n g of L-Alanine UL-**C and glucose-OL- l*C r e s p e c t i v e l y . The standards and the sample chromatograms were cut i n t o i d e n t i c a l s t r i p s 3 cm wide. The s t r i p s were put through a radio-chromatography scanner (Neuclear Chicago, Actigraph I I ) . By comparing the peaks of r a d i o a c t i v i t y of the sample to that of the standard, an accurate e v a l u l a t i o n can be made regarding the l o c a t i o n of a c t i v i t y , consequently t h i s procedure provides the information as to whether the r a d i o a c t i v i t y detected i n the bas i c and n e u t r a l f r a c t i o n s i s due to alanin e and glucose r e s p e c t i v e l y . The r e s u l t s of t h i s technique are i l l u s t r a t e d i n Figure 5 (Appendix) f o r a l a n i n e . The a c t i v i t y of glucose was not strong enough f o r the use of the Actigraph scanner, which has an e f f i c i e n c y maximum of approximately 2 0 per cent. Therefore a s l i g h t v a r i a t i o n of the above technique was developed u t i l i z i n g the l i q u i d s c i n t i l l a t i o n counter (ISOCAP 300). This method c o n s i s t s of c u t t i n g the above s t r i p s i n t o smaller s e c t i o n s and p l a c i n g them i n t o v i a l s immersed i n the l i q u i d s c i n t i l l a t i o n f l u i d (PCS). The i n d i v i d u a l v i a l s were counted and by comparison to 69 the standard U-L^OGlucose chromatogram s t r i p , the peaks of r a d i o a c t i v i t y were l o c a t e d . Table 3 (Appendix) i l l u s t r a t e s the r e s u l t s f o r a n e u t r a l f r a c t i o n sample and a standard. 3 •) Blood_ An a l j s i s Whole blood samples were c o l l e c t e d i n heparinized t e s t tubes and c e n t r i f u g e d on a S o r v a l l BC 2-B automatic r e f r i g e r a t e d c e n t r i f u g e . The r e s u l t i n g supernatant was d e p r o t e i n i z e d with 10 per cent ZnSO and 0.5N NaOH i n a s i m i l a r manner described p r e v i o u s l y . Glucose and a l a n i n e assays were performed on the d e p r o t e i n i z e d supernatant by methods o u t l i n e d i n the preceding experiment. S s l s s l a t i o n s S p e c i f i c a c t i v i t y decay curves were f i t t e d with a f u n c t i o n which was the sum of exponential terms having the form: SA= s p e c i f i c a c t i v i t y of plasma glucose a t time t (nC/mg C) A.= zero-time i n t e r c e p t of each component (nC/mg C) where: -m- r a t e constant of each component (min -*) 70 n= number of exponential components i= exponential-component number t= time (min) F i t t i n g was accomplished by i t e r a t i v e minimization of the err o r sum of squares f o r the f i t t e d f u n c t i o n . In p r a c t i c e , a Fo r t r a n IV subprogram w r i t t e n by R. F l e t c h e r (1972) was used. The number of exponential terms i s determined by the shape of the observed s p e c i f i c r a d i o a c t i v i t y decay curve. On the present experiment a two term f u n c t i o n was f i t t e d to the curve. Pool s i z e and metabolic turnover estimates were c a l c u l a t e d from the parameters of the l e a s t squares f i t t e d f u n c t i o n s using the formulae of Leng (1970): P Pool s i z e , Q (mg C) = —p^-where P i s the i n j e c t e d dose of r a d i o a c t i v i t y . I r r e v e r s i b l e Loss (mg C/min) = Q rv T o t a l entry r a t e (mg of C/min) where A*t are f r a c t i o n a l zero-time i n t e r c e p t s ; t h e r e f o r e 71 space i s defined as; Pool S i z e , Q (mg) x 100 x Body Height (kg) plasma Alanine concentration (mg/1) Rec y c l i n g r a t e i s defined as t o t a l entry rate minus i r r e v e r s i b l e l o s s . The percent conversion of i*C-Alanine to glucose was estimated by the method of Kreisburg et a l . , (1972) by using the equation; %glucose from Alanine = G x SA x G S x 100 where I G = glucose c o n c e n t r a t i o n (umoles/ml) SA = s p e c i f i c r a d i o a c t i v i t y of glucose (dpm/umole) GS= the glucose space (ml) I = i n j e c t e d dosage of D- 1*C-alanine (dpm) The value of alanine recovered i n glucose i s determined from the maximum glucose s p e c i f i c a c t i v i t y achieved during the 3 hour time i n t e r v a l f o l l o w i n g the i n f u s i o n of **C-alanine. For the purpose of t h i s c a l c u l a t i o n , the glucose space was assumed to be equiv a l e n t to 0.3 x body weight i n kg (Monugian et a l . , 1964). SS§Sli5_S£^«fiiscussion Before proceeding i n t o the s p e c i f i c values f o r the parameter estimates, i t was confirmed by paper chromatography t h a t the r a d i o a c t i v i t y i n the b a s i c and n e u t r a l f r a c t i o n s was 72 due to alanin e and glucose r e s p e c t i v e l y . From the c l o s e proximity of the peak of r a d i o a c t i v i t i e s f o r the alanine and glucose samples as compared to the standards of each, (Figure 5 Table 3, Appendix) i t i s obvious that the r a d i o a c t i v i t y i n the basic ana n e u t r a l f r a c t i o n s i s a r e s u l t of l a b e l l e d **C-alanine and i*C-glucose r e s p e c t i v e l y . In a d d i t i o n to the raw data. Table 4 (Appendix) shows i n d i v i d u a l values f o r pool s i z e , space, t o t a l entry r a t e , i r r e v e r s i b l e l o s s and r e c y c l i n g of alanine i n sheep estimated from the change of s p e c i f i c r a d i o a c t i v i t y of plasma a l a n i n e a f t e r an i n j e c t i o n of 50 uCi of U- 1 4C-alanine. Figure 6 (Appendix) i l l u s t r a t e s the curve f o r l*C-glucose a c t i v i t y . The l i n e of best f i t f o r the ** - a l a n i n e data i s p i c t u r e d i n Figure 7 (Appendix). I t i s from the l a t t e r t h a t the c a l c u l a t i o n s f o r the metabolic parameters are made. The per cent of i n j e c t e d **C-al a n i n e appearing i n glucose i s a l s o i n c l u d e d i n Table 6 (Appendix) . The metabolic parameters of alanin e (Table 6, Appendix) show a t o t a l entry r a t e of 8.61 mg C/min (5.80 mM/hr), an i r r e v e r s i b l e l o s s of 4.96 mg C/min (3.34 mM/hr), a r e c y c l i n g rate of 3.65 mg C/min (2.46 mM/hr) and a conversion percentage to glucose of 3.57%. Wolff ana Bergman (1972a) quote a t o t a l plasma a l a n i n e turnover r a t e of between 8.3 and 11.3 mM/hour. The technique used t o measure these l a t t e r f i g u r e s was a continuous i n f u s i o n of L-U-**C al a n i n e without a priming i n j e c t i o n . The t o t a l entry r a t e and the i r r e v e r s i b l e l o s s are lower than the values reported by Wolff and Bergman (1972a). 73 I t should be emphasized that the term t o t a l turnover r a t e , as used by Wolff and Bergman, has a l s o been given d i f f e r e n t t i t l e s by va r i o u s workers, such as, t r a n s f e r r a t e (Kronfeld and Simesen, 1961), u t i l i z a t i o n r a t e (Annisoh and White, 1961) and entry r a t e (Leng et a l . , 1967). A l l of these terms imply the same concept, namely the rate of entry of a l l alanin e carbon i n t o the sampled compartment. As pointed out by White e t a l . , (1969), t h i s i n d i v i d u a l parameter, when measured by a primed or continuous i n f u s i o n represents the i r r e v e r s i b l e l o s s concept as defined by the s i n g l e i n j e c t i o n technique. I r r e v e r s i b l e l o s s does not i n c l u d e a l a n i n e carbon which has r e c y c l e d between the sampled compartment and any p e r i p h e r a l compartment. The d i f f e r e n c e between t o t a l entry r a t e and i r r e v e r s i b l e l o s s i s regarded as the rat e of r e c y c l i n g of alanine between the sampled and p e r i p h e r a l compartments. Based on t h i s concept the d i f f e r e n c e between the i r r e v e r s i b l e l o s s determined by the present experiment and published values of entry or turnover r a t e f o r al a n i n e would appear to be even g r e a t e r . The per cent conversion of a l a n i n e to glucose of 3.57% i s , again, lower than the r e l a t i v e l y e s t a b l i s h e d values of between 6 and 8% (Black et a l , 1968)., Wolff and Bergman (1972b) have estimated that of the e x i s t i n g glucose 5.4% comes from alani n e by the use of the continuous i n f u s i o n approach, which has been s a i d to account f o r the assumptions and i n a c c u r a c i e s of the s i n g l e i n j e c t i o n method. Kreisburg et a l . , (1972) estimated a per cent t r a n s f e r of alanin e to glucose of 12.5%. These workers along with others (Annison and White, 1961) suggest that the 74 problems of r a p i d metabolism of the l a b e l l e d metabolite as w e l l as r e c y c l i n g y i e l d overestimates of metabolism values a r r i v e d at by the s i n g l e i n j e c t i o n technique. Thus they recommend the use of a continuous i n f u s i o n which e l i m i n a t e s these sources of e r r o r and provides an accurate assessment of metabolic parameters. The present r e s u l t s i l l u s t r a t e a c o n c l u s i v e underestimation of these f i g u r e s when compared to the values of continuous i n f u s i o n experiments. However a p a r t i a l answer to the d i s c r e p a n c i e s i s provided by Leng (1970) who s t a t e d t h a t f o r accurate a n a l y s i s of s i n g l e i n j e c t i o n data, an adequate number of samples have to be taken i n the i n i t i a l 5 to 10 minutes s i n c e the d e c l i n e i n a c t i v i t y of amino a c i d s , and e s p e c i a l l y a l a n i n e , i s most dramatic wit h i n t h i s time i n t e r v a l . C l e a r l y , by i n s p e c t i o n of Table 6 and Figure 7 and 8 (Appendix) t h i s was not adhered to i n t h i s experiment. There are not enough points w i t h i n the f i r s t 5-10 minutes of the decay curve to permit accurate i n t e r p r e t a t i o n by the computer f o r the c a l c u l a t i o n of the parameters. H a t e r i a l s _ a n d Methods The experimental c o n d i t i o n s as w e l l as the sheep employed were i n d e n t i c a l to that described i n Experiment B.1. The s u r g i c a l preparation c o n s i s t e d of implanting p o l y v i n y l c a t h e t e r s (P.E.90) i n t o both of the j u g u l a r veins. One was employed f o r i n f u s i o n and the other f o r blood c o l l e c t i o n . The n u t r i t i o n a l regime was a l s o the same as o u t l i n e d 75 p r e v i o u s l y . The sheep was fed 1 kg a l f a l f a hay twice d a i l y a t 0800 hours and 1600 hours. The i n f u s i o n began at 1130 hours the r e f o r e s i m i l a r to Experiment b.1 the r e s u l t s are based on fed c o n d i t i o n s (3.5 hr f a s t ) . 1•) Isotope and Blood C o l l e c t i o n The i s o t o p e used, L-Alanine U-I-**C was obtained from ICN and adheres to the s p e c i f i c a t i o n s s t a t e d p r e v i o u s l y . The major source of v a r i a t i o n between the present experiment and the f i r s t one i s i n the amount of isotope administered as w e l l as the time periods f o r blood c o l l e c t i o n . 0.085 mCi (85 uCi) of 1-alanine-O- **C was administered as a s i n g l e i n j e c t i o n i n t o the l e f t j u g u l a r vein and 10 ml of blood plasma was c o l l e c t e d from the r i g h t jugular vein a t the f o l l o w i n g time i n t e r v a l s : Sample no. C o l l e c t i o n Time (min.) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 CONTBOL (Before Infusion) 3.0 3.5 4.0 4.5 5.0 10.0 15.0 20.0 30.0 45.0 60.0 75.0 90.0 105.0 120.0 135.0 76 In order to accomplish the c o l l e c t i o n of blood f o r the time period of 5 minutes f o l l o w i n g i n f u s i o n , a continuous sampling technique was devised. This method u t i l i z e d the two j u g u l a r v e i n s , such that at the exact time when the l a b e l l e d alanine was i n j e c t e d i n t o the l e f t j u g u l a r , blood was drawn continuously from the r i g h t j u g u l a r by means of heparinized s y r i n g e s . As soon as 10 ml of blood was withdrawn i n t o the f i r s t s y r i n g e , a second was immediately a f f i x e d to the catheter and the next sample was c o l l e c t e d . This procedure continued f o r 5 minutes f o l l o w i n g the i n f u s i o n of the isotope and represents a continuous c o l l e c t i o n of blood over t h i s time i n t e r v a l . The remainder of the blood samples were withdrawn at the s p e c i f i e d i n t e r v a l s described above. This approach t o blood sampling was developed i n an attempt t o assess the i n i t i a l d e c l i n e of al a n i n e s p e c i f i c r a d i o a c t i v i t y more a c c u r a t e l y s i n c e the majority of the decay curve i s completed w i t h i n the f i r s t 10 minutes f o l l o w i n g i n j e c t i o n . 2•) Chemical_Methods The procedure f o r f r a c t i o n a t i o n of plasma components, glucose and al a n i n e a n a l y s i s have a l l been described i n d e t a i l p r e v i o u s l y . Another chromatography experiment was performed to t e s t that the a c t i v i t y of the n e u t r a l and basic f r a c t i o n s was due to glucose and alanin e r e s p e c t i v e l y . 77 The solvent system f o r the t w o - d i r e c t i o n a l separation of al a n i n e and the f o r the s i n g l e - d i r e c t i o n a l separation of glucose were the same as sta t e d i n the preceding experiment. The r e s u l t s from t h i s study are a l s o included i n Figure 5 and Table 3 (Appendix). C a l c u l a t i o n s A two term exponential f u n c t i o n was employed to produce the l i n e of best f i t f o r the decay curve of al a n i n e s p e c i f i c a c t i v i t y . A d e t a i l e d d e s c r i p t i o n of the c a l c u l a t i o n s used i n determining the metabolic parameters i s provided i n the c a l c u l a t i o n s s e c t i o n of the previous experiment. E§§Sits_and_Discussion In Table 5 (Appendix) the s p e c i f i c a c t i v i t y values f o r both a l a n i n e and glucose a t each time i n t e r v a l , as w e l l as the i n d i v i d u a l values f o r pool s i z e , space, t o t a l entry r a t e , i r r e v e r s i b l e l o s s and r e c y c l i n g are presented. The percent t r a n s f e r of alanine carbon t o glucose i s a l s o defined. The r e s u l t s of the present experiment are based on the change of s p e c i f i c r a d i o a c t i v i t y of plasma alani n e with time a f t e r an i n j e c t i o n of 85 u C i of 0-**C-alanine. Figure 8 (Appendix) o u t l i n e s the l i n e a r curve f o r glucose s p e c i f i c a c t i v i t y . The l i n e of best f i t estimated by the two 78 term exponential function i s i l l u s t r a t e d i n Figure 9 (Appendix). The parameters of alanine metabolism as i l l u s t r a t e d in Table 5 (Appendix) demonstrated a t o t a l enty rate of 14.52 mg C/min (9.77 mM/hr), an i r r e v e r s i b l e loss of 7.60 mg C/min (5.12 mM/hr), a rec y c l i n g rate of 6.91 mg C/min 4.65 mM/hr) and a percent conversion to glucose of 5.0055. When compared to the resu l t s of the previous experiment, the above values correspond much closer to the published estimates of t o t a l alanine turnover reported by.Wolff and Bergman (1972a). As stated i n the Discussion of Experiment B.1, t o t a l plasma turnover as measured by a continuous infusion technique i s the same as i r r e v e r s i b l e loss determined by the single i n j e c t i o n method. Therefore the value for i r r e v e r s i b l e loss 7.60 mg C/min (5.12 mM/hr) i s comparatively close to the figures of 8.3 and 11.3 mM/hr f o r t o t a l plasma turnover as stated by Wolff and Bergman, (1972a). It i s s i g n i f i c a n t that the results of thi s experiment are much closer to reported values than the figures of the preceding experiment. This fact indicates that the changes in techniques made, s p e c i f i c a l l y the continuous blood c o l l e c t i o n over the f i r s t 5.minutes, improved the e f f i c i e n c y and accuracy of t h i s approach.. In other words the computer analysis proved more r e l i a b l e since there were more points, e s p e c i a l l y during the i n i t i a l rapid decline of alanine s p e c i f i c a c t i v i t y , to compute the l i n e of best f i t . Thus the degree of inte r p o l a t i o n of the points i n Figure 9 (Appendix) i s reduced considerably in t h i s experiment as compared to the previous one. 79 The g l u c o g e n i c i t y of a l a n i n e was estimated t o be 5.00%, which i s i n c l o s e agreement with the values of 5.4% as determined by Wolff and Bergman (1972b) using a continuous i n f u s i o n approach on sheep. The 5.00% f i g u r e i s a l s o l e s s than previous estimates of between 6 and 8% i n l a c t a t i n g cows (Black §.£ 1968) and 12.5% i n man (Kreisberg et a l . , 1972). As a d d i t i o n a l support to the v a l i d i t y of t h i s multicompartmental approach f o r the a n a l y s i s of s i n g l e i n j e c t i o n data, a three term exponential f u n c t i o n was a l s o employed to determine the l i n e of best f i t f o r the decay curve of a l a n i n e s p e c i f i c r a d i o a c t i v i t y i n the present experiment. Since the r e s u l t s o u t l i n e d i n Table 5 (Appendix) are based on a two term f i t , an approach of t h i s nature would i n d i c a t e whether i n f a c t , the number of exponential terms i s determined by the shape of the observed s p e c i f i c r a d i o a c t i v i t y decay curve. The r e s u l t s of applying a three term f u n c t i o n to the decay curve of a l a n i n e which has already been f i t t e d to a two term a n a l y s i s are presented i n Table 5 (Appendix). The values f o r the metabolic parameters are i d e n t i c a l t o those of a two term exponential f u n c t i o n (Table 5), which demonstrates that the data, or the shape of the decay curve i s what determines the number of exponentials t h a t can be used. The r e s u l t s of the paper chromatography separation of a l a n i n e and glucose are presented i n Figure 5 and Table 3 (Appendix) r e s p e c t i v e l y . S i m i l a r to Experiment B.1, the peaks of r a d i o a c t i v i t y of the plasma f r a c t i o n s corresponded to those of the standards f o r a l a n i n e and glucose. 80 The o v e r a l l s i g n i f i c a n c e of t h i s experiment i s t h a t the improvements made i n the present techniques over those of the previous experiment have y i e l d e d r e s u l t s which correspond w e l l t o the estimates determined by a continuous i n f u s i o n approach. Thus the method of a s i n g l e i n j e c t i o n of an isotope combined with a multicompartmental a n a l y s i s has improved i t s s t a t u s as a means to study the k i n e t i c s of a l a n i n e metabolism i n ruminants. 81 H a t e r i a l s _ a n d _ Methods A 1.5 year o l d wether weighing 37 kg was used f o r t h i s f i n a l s i n g l e i n j e c t i o n experiment. The surgery performed on the animal varied s l i g h t l y from the previous two experiments i n that p o l y v i n y l c atheters (P.E.90) were i n s e r t e d i n t o both r i g h t and l e f t j u g u l a r , but i n a d d i t i o n one was a l s o implanted i n t o the r i g h t c a r o t i d a r t e r y . The l e f t j u g u l a r cannula was used f o r i n f u s i o n and blood was c o l l e c t e d from both r i g h t j u g u l a r and c a r o t i d . The c a t h e t e r s were maintained patent by f l u s h i n g s with a s o l u t i o n of h e p a r i n i z e d s a l i n e (100 units/ml) twice d a i l y . The n u t r i t i o n a l regime was s i m i l a r that of the f i r s t two experiments i n that 2 kg a l f a l f a hay was fed t o the sheep twice d a i l y ; once at 0800 hours and once at 1300 hours and again the animal i s under fed c o n d i t i o n s (5 hour f a s t ) during the experiment. 1•) Isptope_ and Blood C o l l e c t i o n L-Alanine U-L-**C obtained from ICN was i n f u s e d i n t o the l e f t j u g u l a r vein as a s i n g l e i n j e c t i o n of 0.095 mCi (95 u C i ) . 10 ml of blood plasma samples were withdrawn from the r i g h t j u g u l a r vein and the r i g h t c a r o t i d a r t e r y at the f o l l o w i n g time i n t e r v a l s : 82 CAROTID ARTERY Sample no. C o l l e c t i o n Time (min) 1 CONTROL 2 CONTROL 3 .67 4 1.33 5 1.66 6 2.25 7 2.83 8 3.83 9 4.83 10 5.00 11 10 12 15 13 25 14 35 15 45 16 60 17 75 18 90 19 120 JUGULAR VEIN Sample No. C o l l e c t i o n Time (min) 1 CONTROL 2 CONTROL 3 .33 4 .58 5 .83 6 1.95 7 2.17 8 2.45 9 2.75 10 10 11 15 12 25 13 35 14 45 15 60 16 75 17 90 18 120 The continuous blood sampling technique developed i n Experiment B.2 was employed i n the present experiment. 83 2.) Chemical_Wethods The procedures f o r f r a c t i o n a t i o n of blood plasma, and the determination of plasma glucose and alanine have been o u t l i n e d i n d e t a i l p r e v i o u s l y . Due to the p o s i t i v e r e s u l t s obtained f o r the paper chromatographic separation of 4 * C - l a b e l l e d a l a n i n e and glucose i n Experiment B.1 and B.2, no such a n a l y s i s was conducted i n t h i s the f i n a l s i n g l e i n j e c t i o n experiment. C a l c u l a t i o n s The c a l c u l a t i o n s f o r the present experiment d u p l i c a t e s those used by the f i r s t two. However i n s t e a d of a two term exponential f u n c t i o n , the decay curve of 1*C-alanine required a three term f u n c t i o n t o produce a l i n e of best f i t which i n c o r p o r a t e s a l l of the p o i n t s . Results and,Discussions Table 6 (Appendix) o u t l i n e s the data f o r glucose and a l a n i n e s p e c i f i c a c t i v i t i e s i n both the j u g u l a r vein and c a r o t i d a r t e r y . In a d d i t i o n the values f o r the parameters of a l a n i n e metabolism which i n c l u d e pool s i z e , space, t o t a l entry r a t e , i r r e v e r s i b l e l o s s , r e c y c l i n g and the conversion percentage of a l a n i n e to glucose are a l s o found i n t h i s t a b l e . Therefore these r e s u l t s are based on the change of alanine s p e c i f i c a c t i v i t y with time a f t e r a s i n g l e intravenous i n j e c t i o n of 95 uCi of U-1 * C - a l a n i n e . The s p e c i f i c a c t i v i t y curves of glucose i n the j u g u l a r vein and c a r o t i d a r t e r y r e s p e c t i v e l y , are diagramed i n Figure 10a and b (Appendix). Figure 11a and b (Appendix) i l l u s t r a t e the f i t t e d 84 l i n e to the l o g a r i t h m i c decay data of alanine by the three term exponential f u n c t i o n i n the j u g u l a r vein and c a r o t i d a r t e r y r e s p e c t i v e l y . The r e s u l t s of the glucose and ala n i n e concentration f o r the c a r o t i d a r t e r y and j u g u l a r vein were compared s t a t i s t i c a l l y by using the s t u d e n t s 1 t t e s t f o r s i g n i f i c a n c e at a l e v e l P>0.05. The values f o r the parameters as described i n Table 6 (Appendix) show t o t a l entry r a t e s of 49.78 mg C/min (33.52 mM/hr) and 61.37 mg/min (41.32 mM/hr) f o r the ju g u l a r vein and c a r o t i d a r t e r y r e s p e c t i v e l y . The rate of i r r e v e r s i b l e l o s s f o r j u g u l a r vein and c a r o t i d a r t e r y are 10,01 mg C/min (6.75 mM/hr) and 9.33 mg C/min (6.28 mM/hr) r e s p e c t i v e l y , and r e c y c l i n g was determined from the d i f f e r e n c e which amounts to 39.75 mg C/min (26.77 mM/hr) f o r the ju g u l a r vein and 52.04 mg C/min (35.04) mM/hr) f o r the c a r o t i d a r t e r y . The estimates of i r r e v e r s i b l e l o s s f o r the c a r o t i d a r t e r y and ju g u l a r vein (6.28 mM/hr and 6.75 mM/hr) are extremely c l o s e and both correspond r e l a t i v e l y w e l l to the estimates of t o t a l turnover r a t e s of between 8 and 11 mM/hr as reported by Wolff and Bergman (19 72a). The r e c y c l i n g values of the present experiment i n d i c a t e t h a t t h i s process i s o c c u r r i n g to a high degree and obviously y i e l d s correspondingly high values f o r t o t a l entry r a t e s . This f a c t o r plays the major r o l e i n determining the number of exponential terms required to assign a l i n e of best f i t to the decay curve. By i n s p e c t i n g Figure 11 a and b three d i s t i n c t slopes may be noted. An i n i t i a l r a p i d 85 d e c l i n e , followed by a second more l i n e a r phase and ending with a elongated c u r v i l i n e a r s e c t i o n . Thus by v i s u a l i n s p e c t i o n of the l o g a r i t h m i c decay curve i t i s obvious that a two term exponential f u n c t i o n , which was used i n the two previous experiments, i s not adequate to account f o r the t h i r d component. Therefore a three term f i t i s necesary i n order to account f o r the large amount of r e c y c l i n g which occurred. An i n t e r e s t i n g point to note i s t h a t the values f o r i r r e v e r s i b l e l o s s are not i n f l u e n c e d by the high degree of r e c y c l i n g . Although the c a l c u l a t i o n s are designed to separate these two parameters, i t i s encouraging to note t h a t the system i s f u n c t i o n i n g according t o the pattern set. The glucose concentration i n the c a r o t i d a r t e r y i s s i g n i f i c a n t l y higher than t h a t i n the j u g u l a r vein (P<0.05). The corresponding values f o r a l a n i n e concentration i n these blood v e s s e l s proved to be not s i g n i f i c a n t l y d i f f e r e n t . Thus from t h i s s t a t i s t i c a l a n a l y s i s one can s t a t e t h a t glucose i s being u t i l i z e d by the b r a i n t i s s u e . No estimate of q u a n t i t y i s v a l i d s i n c e the present experiment was performed on one sheep only and no estimate of blood flow was made. The per cent conversion of a l a n i n e carbon to glucose f o r the j u g u l a r v e i n (6.78%) and c a r o t i d a r t e r y (6.74%) agree w e l l with each other as w e l l as with the published values of 5.4% f o r sheep (Wolff and Bergman 1972a), and 6 to 8% f o r l a c t a t i n g cows (Black et a l . , 1968). The d i f f e r e n c e s between the c a r o t i d a r t e r y and j u g u l a r vein sampling i n terms of i r r e v e r s i b l e l o s s and the glucogenic r o l e 86 of a l a n i n e are i n s i g n i f i c a n t . Therefore the place of blood c o l l e c t i o n appears to be of minimal importance f o r the determination of t o t a l body or t o t a l plasma metabolic parameters. However the v a r i a t i o n between a r t e r i a l and venous plasma becomes more s i g n i f i c a n t when considering such values as the t o t a l entry r a t e and the r a t e of r e c y c l i n g which deviate depending upon the vessel from which blood i s c o l l e c t e d . Such a r e s u l t has been demonstrated before and (Wolff and Bergman 1972a&b) and i s a l s o evident i n the present experiment with the estimates of t o t a l entry r a t e (61.3 mg C/hr) and r e c y c l i n g rate (52.04 mg C/min) of the c a r o t i d a r t e r y being considerably l a r g e r than the t o t a l enty r a t e (49.78 mg C/min) and r e c y c l i n g r a t e (39.74 mg C/min) determined i n the j u g u l a r v e i n . This i s reasonable by examination of the a l a n i n e decay curves f o r each of these blood v e s s e l s (Figure 11 a and b r Appendix). By v i s u a l i n s p e c t i o n the t h i r d component or s e c t i o n of the curve f o r the d e c l i n e i n s p e c i f i c r a d i o a c t i v i t y of 1 4 C - a l a n i n e i n the c a r o t i d a r t e r i a l plasma l e v e l s o f f and does not f a l l to the a b s c i s s a as i t r a p i d l y as does i n the j u g u l a r v e i n . By the shape of the decay curves i t appears th a t r e c y c l i n g i s more pronounced i n the c a r o t i d a r t e r y plasma than the j u g u l a r vein plasma. This p o s t u l a t i o n i s given support from the f a c t that the l i n e a r parameter (A), which represents the zero-time component, f o r the t h i r d term i s l a r g e r i n the c a r o t i d a r t e r y than i n the j u g u l a r v e i n . So the l e v e l of s p e c i f i c r a d i o a c t i v i t y remaining i n the blood of the c a r o t i d a r t e r y i s more extensive than that i n the j u g u l a r vein which i m p l i e s a higher degree of r e c y c l i n g . Although i t i s d i f f i c u l t t o speculate upon the exact reason why 87 r e c y c l i n g i s more prominent i n the c a r o t i d a r t e r y , a p o s s i b l e explanation a r i s e s from c o n s i d e r a t i o n of the anatomy of the area i n question. Blood samples are withdrawn from the c a r o t i d a r t e r y and j u g u l a r v e i n simultaneously and by the time r e c y c l i n g takes place the t r a c e r dose of l*C-alanine has v i r t u a l l y mixed and t r a v e r s e d through the e n t i r e c i r c u l a t o r y system. Therefore the l a b e l l e d a l a n i n e bas been taken up by the t i s s u e s which metabolize t h i s amino a c i d . , Two p o t e n t i a l sources of a l a n i n e u t i l i z a t i o n are the l i v e r and the kidneys f o r the purpose of gluconeogenesis. Thus the r a d i o a c t i v e t r a c e r has l e f t the amino a c i d pool and has been incorporated i n t o the glucose p o o l . However re - e n t r y of the l a b e l l e d carbon can occur through the e s t a b l i s h e d "glucose-alanine c y c l e " ( F e l i g et a l . , 1970) where al a n i n e i s synthesized de novo i n the muscle t i s s u e s by transamination of glucose derived pyruvate. Blood c a r r y i n g the l a b e l l e d glucose from the l i v e r i s transported to the muscle t i s s u e s where the conversion to 1 * C - l a b e l l e d a l a n i n e occurs. By the time t h i s process becomes pronounced blood e n t e r i n g the c a r o t i d a r t e r y a f t e r mixing i n the heart i s the best estimate of the r e c y c l i n g e f f e c t s i n c e i t has yet to perfuse any a d d i t i o n a l t i s s u e s . However the j u g u l a r vein blood c o l l e c t e d i n the present experiment i s i n f l u e n c e d by the degree of metabolism occuring i n the b r a i n t i s s u e . Although the s t a t i s t i c a l a n a l y s i s of the arterio-venous a l a n i n e c o n c e n t r a t i o n d i f f e r e n c e s have shown no s i g n i f i c a n t d i f f e r e n c e a d i l u t i o n e f f e c t of the t r a c e r may be at work. That i s the b r a i n t i s s u e i s i n a dynamic s t a t e where the input e q u a l i z e s the output i n terms of a l a n i n e . Thus the t r a c e r 88 may be taken up or d i l u t e d by a d d i t i o n of nonlabel or a combination of both. Consequently the d i l u t i o n e f f e c t of the b r a i n t i s s u e may be the i n f l u e n c i n g f a c t o r which causes the r e c y c l i n g value to be l a r g e r i n the c a r o t i d a r t e r y than i n the j u g u l a r vein. This f i n a l s i n g l e i n j e c t i o n experiment c o n t r i b u t e s f u r t h e r evidence to the use of t h i s approach i n e v a l u a t i n g metabolic parameters of a l a n i n e . The s l i g h t m o d i f i c a t i o n s introduced i n t h i s experiment such as using a three term exponential f u n c t i o n , d i d not a f f e c t the values of i r r e v e r s i b l e l o s s and per cent conversion to glucose observed p r e v i o u s l y . Therefore the technique of employing a multicompartmental a n a l y s i s can be adjusted to any changes occ u r r i n g w i t h i n the animal which might i n f l u e n c e the r e s u l t s . Consequently the technique employed here and i n the previous two experiments i s f l e x i b l e . The metabolic parameters of a l a n i n e as determined i n the above three experiments are summarized i n Table 7 (Appendix). The r e s u l t s f o r the r a t e of i r r e v e r s i b l e l o s s and percent t r a n s f e r (glucogenicity) of a l a n i n e conform reasonably w e l l with the published values as mentioned p r e v i o u s l y . The o v e r a l l trend f o r the three s t u d i e s i s a s l i g h t underestimation of these values as compared to the r e s u l t s from continuous i n f u s i o n experiments (Wolff and Bergman, 1972a). This f i n d i n g i s i n t e r e s t i n g since the major complaint of the s i n g l e i n j e c t i o n 89 technique i s i t s proposed overestimation of turnover r a t e s of the metabolite under i n v e s t i g a t i o n (White et a l . , 1969). The m u l t i - e x p o n e n t i a l approach i n determining a l i n e of best f i t to the decay curve of alanine a l s o c a l c u l a t e d the parameters of a l a n i n e pool s i z e and space. The former represents the q u a n t i t y of body a l a n i n e with which the i n j e c t e d 1 4 C - a l a n i n e mixes and the l a t t e r i m p l i e s the volume of f l u i d through which the a l a n i n e pool i s d i s t r i b u t e d . Of the two measurements, the a l a n i n e space i n terms of per cent body weight i s the most in f o r m a t i v e and comparable f o r i t i n c o r p o r a t e s the sheep's i n d i v i d u a l v a r i a t i o n i n body weight. Leng (1970) i n a review a r t i c l e presents a summary of the glucose metabolism values of nonpregnant and n o n l a c t a t i n g sheep. The glucose space (as per cent of body weight) ranged from 18 to 35 per cent depending upon the technique used as w e l l as the food i n t a k e , d i e t and feeding regime of the sheep. On the average the higher estimated values of glucose space were determined through the use of a s i n g l e i n j e c t i o n technique. However none of the workers employed a multicompartmental approach to analyze the decay curve of 1*C-glucose. Although these r e s u l t s do not bear d i r e c t relevance to the present work which i n v e s t i g a t e s these parameters of a l a n i n e , they give some i n s i g h t i n t o the o v e r a l l pool s i z e and space of glucose w i t h i n sheep. This knowledge helps i n assessing the s i g n i f i c a n c e of the values determined i n the present s e r i e s of experiments s i n c e the reported work on al a n i n e i s extremely sparce. The f i g u r e s f o r a l a n i n e space as a per cent of the body 90 weight range from 13.27% f o r the f i r s t experiment down to 3.71% and 4.44% f o r the second and t h i r d experiments r e s p e c t i v e l y , a l l of these values are determined on j u g u l a r or p e r i p h e r a l venous blood which i s the only true estimate of the t o t a l body pool s i z e or space. Thus the al a n i n e space of 10.88% as determined from the c a r o t i d a r t e r y plasma i n the t h i r d experiment i s not a v a l i d estimate of t h i s parameter i n terms of the e n t i r e body. The value f o r the f i r s t experiment i s high r e l a t i v e to those f o r the second and t h i r d experiment may be explained adequately because of the la c k of blood samples i n the f i r s t 5 to 10 minutes post i n j e c t i o n . As mentioned p r e v i o u s l y t h i s approach depends on frequent sampling of blood f o l l o w i n g the s i n g l e i n f u s i o n . Therefore the values of 3.71 and 4.44% r e f l e c t c l o s e l y the a c t u a l a l a n i n e pool space, i n sheep under these d i e t a r y c o n d i t i o n s . The j u s t i f i c a t i o n of t h i s statement stems from the f a c t t h a t the al a n i n e pool i s regarded as one of the precursor compartments which i s connected t o the l a r g e glucose pool. From the values of the per cent c o n t r i b u t i o n of a l a n i n e to glucose s y n t h e s i s (6-8%) i n nonpregnant, n o n l a c t a t i n g , steady s t a t e sheep i t appears that the r e l a t i o n s h i p of a l a n i n e pool space (mean of 4.07%) to glucose pool space (range of 18-35%) i s c l o s e to what would be expected. The multicompartmental approach to the a n a l y s i s of decay curves of isotopes was f i r s t used i n the study of glucose metabolism. Besides the numerous parameters i s o l a t e d , t h i s method as c e r t a i n e d the existence of three compartments; a large body pool of glucose which i s interconnected with two precursor pools (Leng 1970) . The proposed model (Figure 18, Appendix) 91 d e p i c t s the r e l a t i o n s h i p s of the glucose pools. The r e s u l t s from the preceding s e r i e s of experiments i n d i c a t e a two compartment and p o s s i b l y a three compartment model f o r a l a n i n e metabolism. I t i s not the purpose of the present study to speculate on the exact number of compartments with reference to a l a n i n e , nor to p r e d i c t what these c o n s i s t of. Answers to these questions can be formulated through more extensive work with t h i s technique i n the f u t u r e . The primary o b j e c t i v e of the present s e r i e s of experiments was t o study the e f f e c t i v e n e s s of the s i n g l e i n j e c t i o n technique coupled with a multicompartmental a n a l y s i s of the r e s u l t i n g decay curve, as a means of q u a n t i t a t i v e l y assessing the parameters of alani n e metabolism i n nonpregnant, n o n l a c t a t i n g sheep. The r e s u l t s presented i n Table 7 (Appendix) along with the above d i s c u s s i o n s i n d i c a t e that t h i s method has the p o t e n t i a l to be used e x t e n s i v e l y f o r i n _ y i y o metabolic s t u d i e s . 92 I I I CONTINUOUS_INF0SIO ISlSOfiT_A_PRIMING_DOSE Introduction The continuous i n f u s i o n of a r a d i o a c t i v e l y l a b e l l e d t r a c e r has been widely used f o r metabolism s t u d i e s i n animals. There appears to be two schools of thought governing the use of t h i s technique, the f i r s t i n v o l v e s a continuous i n f u s i o n preceded by a priming dose of the t r a c e r (Steele, et a l . , 1956 and Steele 1964) and the second comprises the use of a continuous i n f u s i o n of l a b e l without a priming i n j e c t i o n (Leng et a l . , 1967 and White et a l . , 1969). The l a t t e r technigue has been employed i n t e n s i v e l y i n the study of glucose metabolism i n sheep (Bergman, 1973). Leng (1970) has i n d i c a t e d that due to the extensive r e c y c l i n g occurring between the glucose pool and p e r i p h e r a l s u b s t r a t e pools i n sheep, the use of a primed i n f u s i o n technique f o r the determination of glucose pool s i z e i s u n r e l i a b l e on t h e o r e t i c a l grounds. The larg e amount of r e c y c l i n g of alani n e as determined by the s i n g l e i n j e c t i o n experiment p r e v i o u s l y , i n d i c a t e that a primed-infusion approach would not be an e f f i c i e n t method to measure these parameters of a l a n i n e . The continuous i n f u s i o n technique used by Leng et a l . , (1967 and White et a l . , (1969) has a d i s t i n c t advantage i n that the c a l c u l a t i o n of i r r e v e r s i b l e l o s s ( t o t a l entry rate) r e l i e s on a simple mathematical treatment. The i n f u s i o n rate of 93 r a d i o a c t i v i t y d i v i d e d by the mean plateau l e v e l of s p e c i f i c r a d i o a c t i v i t y of the metabolite, which i s normally a t t a i n e d between 3 and 4 hours f o r glucose, gives an estimate of t h i s parameter. This i s indeed a s i m p l i f i c a t i o n of the a c t u a l isotope d i l u t i o n curve which i s described by the f o l l o w i n g equation (Steele et a l . , 1956; S t e e l e , 1964): Where F i s the i n f u s i o n r a t e (nCi/min) , Q i s the pool s i z e , A 1^ i s the f r a c t i o n a l zero-time intercept,-mc i s the r a t e constant and SR i s the plasma s p e c i f i c r a d i o a c t i v i t y at time t (nC/mg C). As time approaches i n f i n i t y during a continuous i n f u s i o n of i s o t o p i c a l l y l a b e l l e d m a t e r i a l s a plateau s p e c i f i c r a d i o a c t i v i t y i s obtained which i s described as; SR i = l Therefore i f the s p e c i f i c r a d i o a c t i v i t y of the metabolite at the plateau l e v e l (as described above) i s used to c a l c u l a t e i r r e v e r s i b l e l o s s then 94 i r r e v e r s i b l e loss= i n f u s i o n r a t e (nci/mjn) i>R (nCi/mg C) Though the continuous i n f u s i o n of l a b e l l e d t r a c e r has been used e x t e n s i v e l y i n the measurement of the parameters surrounding glucose metabolism, i t i s r e l a t i v e l y r e c e n t l y t h a t t h i s approach has been used to measure amino a c i d metabolism i n sheep fed a l f a l f a hay (Wolff et a l . , 1972a,b,c). These workers i n v e s t i g a t e d the turnover of plasma amino acids and t h e i r c o n t r i b u t i o n t o gluconeogenesis. One of the o b j e c t i v e s of the above s t u d i e s was to assess the metabolism of plasma amino a c i d s by p o r t a l - d r a i n e d v i s c e r a , l i v e r and p e r i p h e r a l t i s s u e s of sheep. I n order to accomplish t h i s each experiment c o n s i s t e d of a continuous intravenous i n f u s i o n of a 1 * C - l a b e l l e d amino a c i d and blood was sampled from the aorta, the p o r t a l v e i n , and a h e p a t i c v e i n . Blood flow i n both the p o r t a l and hepatic veins was determined simultaneously. The c a l c u l a t i o n s devised by these researchers f o r determining the r a t e s of plasma amino a c i d turnover by each t i s s u e s h a l l be o u t l i n e d and discussed i n a l a t e r s e c t i o n . This work i s based upon the assumption that when a continuous i n f u s i o n of l a b e l l e d amino a c i d i s g i v e n , while the animal i s i n a steady s t a t e , the plasma and t i s s u e pools a t t a i n plateau s p e c i f i c a c t i v i t i e s . The value of the s p e c i f i c a c t i v i t y t h e r e f o r e depends upon the r a t e of renewal or turnover from u n l a b e l l e d precursors (Gan and J e f f a y , 1967). These precursors can be amino acids absorbed from the g a s t r o i n t e s t i n a l t r a c t , compounds of intermediary metabolism, or the p r o t e i n s of body t i s s u e s . With t h i s i n mind Wolff and Bergman (1972a) estimated 95 the turnover r a t e s f o r a l a n i n e , aspartate, glutamate, g l y c i n e and s e r i n e i n the p o r t a l - d r a i n e d v i s c e r a , l i v e r , p e r i p h e r a l t i s s u e s as wel l as the t o t a l plasma turnover f o r the body pool of each amino a c i d . The present sequence of experiments u t i l i z e s the continuous i n f u s i o n technique as described above to evaluate i t s e f f e c t i v e n e s s f o r the study of al a n i n e metabolism and to compare i t with the two p r e v i o u s l y described methods. The parameters of ala n i n e center around i t s r a t e of i r r e v e r s i b l e l o s s and i t s c o n t r i b u t i o n to glucose s y n t h e s i s i n sheep under steady s t a t e c o n d i t i o n s . By u t i l i z i n g the blood flow data from the f i r s t experiment an es t i m a t i o n of the p o r t a l metabolism of both a l a n i n e and glucose has been be made. Materials_and_Methods 1.) Surgical_Procedures The experiment was conducted on a 3.5 year o l d wether weighing 45 kg. Surgery was performed on the animal one week p r i o r to the commencement of the experiment. The preparation of the sheep f o r surgery, the method of i m p l a n t a t i o n of cat h e t e r s as w e l l as the post-operative care are described i n d e t a i l under the s u r g i c a l procedures s e c t i o n of the f i r s t experiment i n v o l v i n g blood flow s t u d i e s . Catheters were placed i n t o the 96 p o r t a l v e i n , mesenteric vein and c a r o t i d a r t e r y , and were maintained open by i n f u s i n g h e p a r i n i z e d s a l i n e (100 u n i t s / m l ) . Unfortunately the day before the experiment the p o r t a l vein catheter was caught on an unknown object and had to be removed. Thus only the mesenteric vein and c a r o t i d a r t e r y cannula remained patent.„The experiment was c a r r i e d out as planned with the continuous i n f u s i o n of l a b e l l e d 1 4 C - a l a n i n e i n t o the mesenteric vein and blood c o l l e c t i o n from the c a r o t i d a r t e r y . The n u t r i t i o n a l regime c o n s i s t e d of a maintenance d i e t of 200 gm dehydrated grass p e l l e t s fed every two hours beginning at 0900 hours and ending at 1700 hours. The i n f u s i o n began at 1100 hours and the animal was l a s t f ed the previous evening at 1700 hours thus the r e s u l t s are based on an overnight f a s t (18 h r ) . 2•) l§ot0£e_and_Blood_Collection 200 uCi of L-U- l*C-alanine obtained from ICN conforming to the s p e c i f i c a t i o n s described p r e v i o u s l y was d i s s o l v e d i n 400 ml s t e r i l e s a l i n e and infused i n t o the mesenteric v e i n at a rat e of 0.475 uCi/min. Blood (10 ml) was c o l l e c t e d from the c a r o t i d a r t e r y a t the f o l l o w i n g time i n t e r v a l s : Sample No. C o l l e c t i o n Time (min) 1 2 3 4 5 6 7 8 9 CONTROL (Before Infusion) 5 15 30 60 90 120 150 180 97 10 11 12 13 14 15 210 240 270 300 330 360 3•) Qk®iisii_S§ibods The s e p a r a t i o n of plasma components, as w e l l as the plasma a n a l y s i s f o r a l a n i n e and glucose concentrations were the same as o u t l i n e d i n the previous experiments. Due t o the l o s s of the p o r t a l vein c a t h e t e r , the production and u t i l i z a t i o n r a t e s of alanine and glucose by the p o r t a l drained v i s c e r a could not be determined. Therefore the apparent t o t a l plasma turnover of al a n i n e was the only f i g u r e c a l c u l a t e d . The apparent turnover of t h i s amino a c i d i s defined, i n a s i m i l a r manner to that f o r glucose (Bergman, 1963), as the r a t e a t which the a r t e r i a l c o n c e n t r a t i o n of »*C-alanine i s d i l u t e d by un l a b e l l e d a l a n i n e and thus: apparent turnover, mmoles/hr = 1_ Where I i s the i n f u s i o n r a t e (uCi/hr) of **C-alanine and SA i s the s p e c i f i c a c t i v i t y (uCi/mmole) of al a n i n e i n the a r t e r i a l plasma. Wolff and Bergman (1972a) have modified t h i s apparent turnover rate to account f o r the c o n t i n u a l absorption of u n l a b e l l e d amino acids from the gut, some of which the l i v e r immediately removes. Therefore a c o r r e c t i o n f a c t o r i s req u i r e d to f i n d the t r u e turnover of amino a c i d . The term was obtained C a l c u l a t i o n s SA 98 by m u l t i p l y i n g the production of the amino a c i d from the p o r t a l -drained v i s c e r a by the f r a c t i o n a l uptake of i^C-amino a c i d by the l i v e r . Thus the correc t e d turnover i s c a l c u l a t e d as f o l l o w s : Corrected = Apparent + P o r t a l x F r a c t i o n a l Uptake Turnover Turnover Production amino a c i d by L i v e r I f the a r t e r i a l concentrations remain constant, then the corrected turnover provides an estimate of plasma amino a c i d u t i l i z a t i o n by a l l t i s s u e s of the body and i t f o l l o w s t h a t : P e r i p h e r a l U t i l i z a t i o n = Corrected - (P o r t a l • Hepatic Turnover U t i l i z a t i o n ) P e r i p h e r a l Production = P e r i p h e r a l + net P e r i p h e r a l Production U t i l i z a t i o n A value f o r corrected turnover i s not p o s s i b l e i n the present experiment and t h e r e f o r e only the apparent turnover of a l a n i n e can be assessed. Table 8 (Appendix) presents the data f o r t h i s continuous i n f u s i o n experiment and the s p e c i f i c a c t i v i t y versus time curves f o r both alanine and glucose are p i c t u r e d i n Figure 12 (Appendix). In a d d i t i o n to the turnover rates a percent glucose production from a l a n i n e was determined by d i v i d i n g the plateau s p e c i f i c a c t i v i t y of glucose i n the c a r o t i d a r t e r y by that of al a n i n e i n the same v e s s e l . The s t a t i s t i c a l treatment of the date consisted of a 99 c o r r e l a t i o n c o e f f i c i e n t a n a l y s i s of both the glucose and al a n i n e plateau l e v e l s to detect i f the x and y v a r i a b l e s are not c o r r e l a t e d over t h i s time i n t e r v a l . I§§2^f J>_and^Discussion As i l l u s t r a t e d i n Figure 12, (Appendix), the glucose and alanine s p e c i f i c a c t i v i t y curves reached plateau l e v e l s between 5 and 6 hours a f t e r the s t a r t of the experiment. The s t a t i s t i c a l a n a l y s i s proved that there i s no s i g n i f i c a n t slope i n t h i s time period f o r e i t h e r a l a n i n e or glucose. The apparent turnover value f o r alanin e of 7.23 mH/hr (10.75 mg/min), which can be equated to i r r e v e r s i b l e l o s s as determined by the s i n g l e i n j e c t i o n experiments (White et a l . , 1969) agrees w e l l with t h a t reported by Wolff and Bergman (1972b) of between 8.3 and 11.3 mH/hr f o r two sheep weighing 45 and 59 kg r e s p e c t i v e l y . The former value i s a much c l o s e r comparison to the present r e s u l t s i n c e the sheep used by both experiments are of equal body weight (45 kg) . The per cent of glucose production from a l a n i n e i n the present experiment was assessed to be 5.07%, which i s comparable t o the value of 5.4% reported by Wolff and Bergman (1972b) . The major s i g n i f i c a n c e of the present experiment i s that the continuous i n f u s i o n of 1*'C- l a b e l l e d a l a n i n e without a priming dose y i e l d e d a s t a t i s t i c a l l y s i g n i f i c a n t plateau l e v e l of s p e c i f i c a c t i v i t y f o r both alanine and glucose at 100 approximately f i v e hours a f t e r the s t a r t of the i n f u s i o n . A d d i t i o n a l c a l c u l a t i o n s such as turnover time and pool s i z e were not p o s s i b l e i n the present experiment since the i n f u s i o n was terminated during the plateau i n t e r v a l and thus the h a l f -l i f e of l*C-alanine could not be determined. 101 l2£eriffient_C.2.„ Materials_and_Methods_ This f i n a l continuous i n f u s i o n experiment was conducted on a 2 year o l d wether weighing 42 kg. Surgery was performed i n a s i m i l a r f a s h i o n as i n the preceding experiment with p o l y v i n y l c a t h e t e r s (P.E.90) being implanted i n t o the p o r t a l v e i n , c a r o t i d a r t e r y and mesenteric vein. The ca t h e t e r s were maintained i n a f u n c t i o n a l s t a t e by frequent f l u s h i n g s with the s o l u t i o n of heparinized s a l i n e (100 u n i t s / m l ) . This time a l l the c a t h e t e r s remained patent during the post-operative recovery phase (5 days) and during the experiment. The animal was placed on a roughage d i e t s i m i l a r to the one employed i n the previous experiment. The sheep was not fed i n the morning of the experiment, thus the r e s u l t s are a l s o based on an overnight f a s t (17 hr) with the i n f u s i o n commencing at 1045 hours. 1.) Isotope_and_Blood_Collection L-Ol*C alanine at a concen t r a t i o n of 0.40 uCi/ml was infu s e d i n t o the mesenteric v e i n at a r a t e of 1.1 ml/min (.44 uCi/min). Blood plasma samples were c o l l e c t e d simultaneously from the c a r o t i d a r t e r y and p o r t a l vein at the f o l l o w i n g time i n t e r v a l s : 102 Sample No. C o l l e c t i o n Time (min) 1 -P.V. CONTBOL-1 2 -C.A. CONTROL-1 3 -P.V. CONTROL-2 4 -C.A. CONTROL-2 5 -P.V. 30 6 -C.A. 30 7 -P.V. 60 8 -C.A. 60 9 -P.V. 90 10 -C.A. 90 11 -P.V. 120 12 -C.A. 120 13 -P.V. 150 14 -C.A. 150 15 -P.V. 180 16 -C.A. 180 17 -P.V. 210 18 -C.A. 210 19 -P.V. 270 20 -C.A. 270 21 -P.V. 300 22 -C.A. 300 23 -P.V. 330 24 -C.A. 330 25 -P.V. 345 26 -C.A. 345 27 -P.V. 360 28 -C.A. 360 INFUSION STOPPED 29 -P.V. 375 30 -C.A. 375 31 -P.V. 390 32 -C.V. 390 2•) Chemical_Mathods Plasma s p e c i f i c a c t i v i t i e s of alanine and glucose were determined from the techniques described e a r l i e r which i n v o l v e d s e p a r a t i o n of plasma c o n s t i t u e n t s as w e l l as assays f o r alanin e and glucose c o n c e n t r a t i o n s . 103 C a l c u l a t i o n s , The techniques described i n the previous experiment were employed to measure apparent turnover r a t e of alanine. Once again the c o r r e c t e d turnover r a t e of alani n e cannot be assessed s i n c e the hepatic v e i n blood flow as w e l l as the concentration of t h i s amino a c i d i n the hepa t i c vein were not determined. However a value for the metabolism of both alan i n e and glucose by the p o r t a l - d r a i n e d v i s c e r a was assessed using the eguation: P = F p y / (C p v / - C ^ ) (Katz and Bergman 1968) where P represents p o r t a l net production r a t e s of the metabolite, F p v i s the whole blood flow (ml/min) i n the p o r t a l v e i n , and C p y and C^ are the concentrations of the metabolite i n the p o r t a l vein and a r t e r i a l v e s s e l s r e s p e c t i v e l y . The value f o r p o r t a l v e i n blood flow of 2010 ml/min as estimated i n Experiment I on blood f l o w , was used f o r the c a l c u l a t i o n s . The g l u c o g e n i c i t y of alanine was determined i n a s i m i l a r manner as i n the preceding continuous i n f u s i o n experiment. A s t a t i s t i c a l a n a l y s i s of the plateau l e v e l s ( c o r r e l a t i o n c o e f f i c i e n t ) and the concen t r a t i o n d i f f e r e n c e s between the c a r o t i d a r t e r y and p o r t a l vein (students' t t e s t ) was conducted. 104 Sesults_and_Discussion_ The data as well as the r e s u l t s of the c a l c u l a t i o n s are presented i n Table 9 (Appendix). Figure 13, (appendix) diagrams the s p e c i f i c a c t i v i t y curves of alanin e and glucose i n the c a r o t i d a r t e r y and p o r t a l vein r e s p e c t i v e l y . The plateau l e v e l s f o r both a l a n i n e and glucose s p e c i f i c a c t i v i t i e s (Figure 13, Appendix) were reached at the same i n t e r v a l as i n the preceding experiment (5-6 hours post i n f u s i o n ) . From the mean value f o r a l a n i n e s p e c i f i c a c t i v i t y at t h i s l e v e l , the apparent plasma turnover rate was assessed at 8.5% mM/hr (12.76 mg/min). The f i g u r e s agree extremely w e l l with that determined by the work of Wolff and Bergman (1972b) which estimated t h i s r a t e to be i n the range of 8.3 and 11.3 mM/hr as reported e a r l i e r . The s t a t i s t i c a l a n a l y s i s of the concent r a t i o n d i f f e r e n c e s of both alanine and glucose between the c a r o t i d a r t e r y and p o r t a l vein proved s i g n i f i c a n t v a r i a t i o n s i n both cases. Therefore i t was decided t o incorporate another arterio-venous conce n t r a t i o n study i n t o the present experiment. Thus the p o r t a l v e i n blood flow value of 2010 ml/min from Experiment A.1 was used t o determine the net metabolism of al a n i n e and glucose by the p o r t a l drained v i s c e r a . Employing the equation described above (Katz and Bergman, 1969) a net u t i l i z a t i o n of glucose (.190 g/hr/kg ) was again c a l c u l a t e d . The value derived p r e s e n t l y conforms reasonably to the p r e v i o u s l y estimated f i g u r e of 0,142 g/hr/kg and to the range of values determined by Katz and Bergman (1969), The estimate f o r a l a n i n e metabolism by the 105 p o r t a l bed demonstrated a net production r a t e of 2.29 .29 mM/hr (Wolff, Bergman and W i l l i a m s , 1972), which i s c l o s e t o that determined i n the present experiment (2.02 mM/hr). From the r e s u l t of the Experiment A.1 d e a l i n g with blood flow a net al a n i n e production r a t e of 1.49 mM/hr was obtained. Both of the estimates derived i n the present s e r i e s of experiments (1.49 and 2.02 mM/hr) concur and are v a l i d assessments s i n c e the blood flow f i g u r e was determined on the same sheep as was used i n t h i s second continous i n f u s i o n experiment. The blood flow value from Experiment A.1 can be employed i n the present experiment since both s t u d i e s maintain the sheep on a s i m i l a r roughage d i e t and conduct the experiment f o l l o w i n g an overnight f a s t . The percent conversion of alanin e carbon to glucose was estimated to be 7.20% by using the technique described p r e v i o u s l y . This value i s again we l l w i t h i n the p r e v i o u s l y st a t e d values of 5.4% (Wolff and Bergman, 1972) and 6-8% (Black et a l . , 1968) The preceding f i g u r e s f o r the parameters surrounding a l a n i n e and glucose metabolism serve as a guide i n an attempt to assess the value of the continuous i n f u s i o n experiment i n r e l a t i o n to the two p r e v i o u s l y mentioned procedures, the blood flow study and the s i n g l e i n j e c t i o n technique. 106 The three experimental techniques employed i n t h i s study are designed to measure va r i o u s parameters p e r t a i n i n g to the metabolism of s p e c i f i c plasma c o n s t i t u e n t s i n animals. The primary aim of the present research was to assess the e f f e c t i v e n e s s of each method i n studying the metabolic parameters of a l a n i n e and i t s r e l a t i o n s h i p t o gluconeogenesis. Since alanine has been e s t a b l i s h e d as the primary amino a c i d e x t r a c t e d by the l i v e r f o r endogenous glucose production i n sheep (Black et a l . , 1968; R e i l l y and Ford, 1971 and Wolff and Bergman 1972b), the r e s u l t s from such a comparison serve a u s e f u l purpose i n ruminant physiology. The f i r s t method used was the t r a d i t o n a l technique of a blood flow study combined with measuring the arterio-venous concentration d i f f e r e n c e s of the metabolites under i n v e s t i g a t i o n . As p r e v i o u s l y reported t h i s approach i s an accurate means of e s t i m a t i n g the metabolism of a blood component by an organ or t i s s u e system. The r e s u l t s obtained here and t h e i r c l o s e approximation to reported values a t t e s t to t h i s statement. The major drawback to the use of t h i s technique i s the s o p h i s t i c a t e d s u r g i c a l procedures which are mandatory. Thus i t s e f f e c t i v e n e s s r e l i e s upon accurate i m p l a n t a t i o n and maintenance of c a t h e t e r s i n s p e c i f i c veins and a r t e r i e s . An example of t h i s i s i l l u s t r a t e d i n the present study. The metabolism of the p o r t a l drained v i s c e r a was estimated through c a t h e t e r i z a t i o n of 107 the c a r o t i d a r t e r y and the p o r t a l v e i n . However the a c t i v i t y of the l i v e r i n r e l a t i o n to a l a n i n e and glucose could not be assessed s i n c e no hepatic vein c a t h e t e r was implanted, which i s required as i n d i c a t e d by Katz and Bergman (1968). The reason f o r t h i s arose from the extreme d i f f i c u l t y of i n t r o d u c i n g a p o l y v i n y l catheter i n t o the hepatic vein which i s not r e a d i l y a c c e s i b l e i n the abdominal c a v i t y , although attempts were made during the numerous operations performed i n t h i s p r o j e c t ^ s u c c e s s f u l hepatic vein cannulation remained e l u s i v e . The point i s t hat the i n i t i a l o b j e c t i v e s of such blood flow study may become obscured due to the outcome of the surgery. This approach can and does prove e f f e c t i v e only when accurate s u r g i c a l procedures are conducted s u c c e s s f u l l y . Such t e c h n i c a l s k i l l takes a great deal of p r a c t i c e and may not always be a v a i l a b l e under normal l a b o r a t o r y c o n d i t i o n s , a d i s t i n c t advantage to t h i s technique i s the simple mathematical a n a l y s i s which f o l l o w s . In a d d i t i o n the number of assumptions as w e l l as the t o t a l expense inv o l v e d have l i m i t e d e f f e c t s upon the a p p l i c a t i o n of blood flow techniques to i n _ v i v o metabolic s t u d i e s . The second technique attempted i n t h i s t h e s i s p r o j e c t was a s i n g l e i n j e c t i o n of * * C - l a b e l l e d a l a n i n e . In the l i t e r a t u r e review a h i s t o r y of the various approaches used to analyze the r e s u l t i n g decay curve of s p e c i f i c r a d i o a c t i v i t y with time was presented and the c o n c l u s i o n was that a multicompartmental a n a l y s i s i s the most e f f i c i e n t and accurate method. The procedure, i n b r i e f , c o n s i s t s of u t i l i z i n g a 2 or 3 term exponential f u n c t i o n t o determine a l i n e of best f i t by i t e r a t i v e minimization of the e r r o r sums of squares f o r the 108 f i t t e d f u n c t i o n . This complicated mathematical treatment was accomplished through the use of a computer program. From the c a l c u l a t i o n s o u t l i n e d by Leng (1970) the f o l l o w i n g metabolic parameters of a l a n i n e were assessed; pool s i z e , space (% of body weight), t o t a l e n t r y r a t e , i r r e v e r s i b l e l o s s and r e c y c l i n g . The majority of a p p l i c a t i o n s using the above m u l t i e x p o n e n t i a l a n a l y s i s have been i n v o l v i n g glucose metabolism i n animals, with sparse work conducted on ruminants. The present study i n v e s t i g a t e d the use of t h i s approach with a l a n i n e metabolism i n sheep and consequently the r e s u l t s have l i t t l e i n the l i t e r a t u r e to compare wit h . However the r e s u l t s of three s i n g l e i n j e c t i o n experiments present evidence suggesting that t h i s technique i s v a l i d and accurate f o r the c o n d i t i o n s described here. One of the major advantages of t h i s method i s i t s a b i l i t y t o separate and i d e n t i f y the parameters of t o t a l turnover, i r r e v e r s i b l e l o s s and r e c y c l i n g . As the t h i r d experiment i n d i c a t e d r e c y c l i n g and consequently t o t a l turnover rates were high, but the estimate of i r r e v e r s i b l e l o s s remained comparable to the previous two experiments as w e l l as to the l i t e r a t u r e values. In a d d i t i o n to t h i s , but i n a more p r a c t i c a l sense, the s i n g l e i n j e c t i o n technique r e q u i r e s simple s u r g i c a l procedures which c o n s i s t of two j u g u l a r vein c a t h e t e r s . Therefore such a study could be conducted i n a l a b o r a t o r y or out i n the f i e l d . The expense i n v o l v e d i s c e r t a i n l y l e s s than the continuous i n f u s i o n of r a d i o a c t i v e i s o t o p e . Previous workers have doubted the v a l i d i t y of the s i n g l e i n j e c t i o n approach due to the numerous assumptions which must be 109 made which i n c l u d e , r a p i d metabolism of the t r a c e r , incomplete mixing, r e c y c l i n g as w e l l as instantaneous i n f u s i o n . These points are not overlooked or ignored but become accounted f o r by the use of a multi-compartmental a n a l y s i s . In conjunction with the above a p o s s i b l e drawback i s the extremely complex mathematical analyses which accompanies t h i s type cf study. However once derived the a n a l y s i s becomes s t r a i g h t f o r w a r d to apply and can be adjusted to s u i t the decay curve i n question. The t h i r d s e r i e s of experiments reported on p r e v i o u s l y c o n s i s t of a continuous i n f u s i o n of l a b e l l e d »*C-alanine without an i n i t i a l priming dose. A priming dose-continuous i n f u s i o n approach was not considered here due t o the t h e o r e t i c a l o b j e c t i o n s to the technique as pointed out previously.. A plateau or constant s p e c i f i c a c t i v i t y l e v e l f o r both glucose and a l a n i n e was achieved some 5 hours a f t e r the s t a r t of the i n f u s i o n . From t h i s l e v e l the c a l c u l a t i o n s of t o t a l turnover r a t e of alanine as w e l l as the per cent c o n t r i b u t i o n of t h i s amino a c i d to glucose s y n t h e s i s were made with the r e s u l t s being comparable to those c i t e d i n the l i t e r a t u r e . Thus the procedure employed i n the present experiments y i e l d r e s u l t s which conform to p r e v i o u s l y published data and consequently these methods proved accurate i n rep r e s e n t i n g continuous i n f u s i o n techniques i n g e n e r a l . Of the drawbacks a f f e c t i n g t h i s type of an approach, the s k i l l e d s u r g i c a l procedures i n f l u e n c e the outcome i n a s i m i l a r f a s h ion as i n d i c a t e d f o r blood flow s t u d i e s . This i s e s p e c i a l l y true i f f o l l o w i n g Wolff and Bergmans's (1972a) m o d i f i c a t i o n of using the d i f f e r e n c e i n r a d i o a c t i v i t y of a substance between two v e s s e l s t o determine an estimate of t i s s u e u t i l i z a t i o n or 110 production. An a d d i t i o n a l negative f e a t u r e of t h i s technique i s the expense required to obtain the necessary r a d i o a c t i v i l y l a b e l l e d compounds. However the major problem i s r e c y c l i n g . In theory the continuous i n f u s i o n of a l a b e l l e d s ubstrate a l l o w s enough time f o r the t r a c e r to e q u i l i b r i a t e with a l l of the body pools and thus r e c y c l i n g has l i t t l e s i g n i f i c a n c e . This i s e s p e c i a l l y true when the r e c y c l i n g of **C i s due only to the f a c t that there i s a d i l u t i o n of t h i s carbon i n a pool of s u b s t r a t e and the »*c i s randomly converted back to the o r i g i n a l l y l a b e l l e d t r a c e r . Consequently the value f o r t o t a l entry or turnover r a t e has been considered synonymous with the i r r e v e r s i b l e l o s s as defined by a s i n g l e i n j e c t i o n system. (White et a l . , 1969). However under c e r t a i n circumstances t h i s r e c y c l i n g becomes more prominent, s p e c i f i c a l l y i n s t a r v a t i o n and other p h y s i o l o g i c a l s t r e s s c o n d i t i o n s such as, k e t o s i s i n the bovine and pregnancy toxemia i n the ovine. In terms of amino a c i d metabolism and s p e c i f i c a l l y a l a n i n e i n ruminants, r e c y c l i n g of l a b e l through amino a c i d i n t e r c o n v e r s i o n s was expected to provide n e g l i g i b l e e r r o r due to the m u l t i p l i c i t y of pathways a v a i l a b l e f o r the u t i l i z a t i o n of alanine carbon (Wolff and Bergman 1972a). Such assumptions have come under considerable s c r u t i n y s i n c e the establishment of the "glucose-alanine c y c l e " ( F e l i g , 1973). Under c o n d i t i o n s where r e c y c l i n g becomes pronounced the continuous i n f u s i o n technique i s unable to detect t h i s and thus the turnover r a t e no longer corresponds to the r a t e of i r r e v e r s i b l e l o s s . The r e s u l t of t h i s w i l l be an overestimation of the a c t u a l turnover r a t e of the metabolite which i n the present i n v e s t i g a t i o n i s a l a n i n e . 111 Of the three techniques i n v e s t i g a t e d during the present p r o j e c t , the s i n g l e i n j e c t i o n with a multi-compartmental a n a l y s i s appears to have the greatest p o t e n t i a l f o r f u r t h e r research i n ruminant physiology and metabolic s t u d i e s . 112 GLOSSARY.OF_TERRS 1) S p_ e c i f i c _ A c t i v i t ^ The s p e c i f i c a c t i v i t y of a substance con t a i n i n g a l a b e l l e d atom i s the amount of r a c i o a c t i v i t y per u n i t of substance. This i s often expressed as the number of r a d i o a c t i v e u n i t s (microcuries, counts per -minute, d i s i n t e g r a t i o n s per minute) per m i l l i g r a m or mi H i mole of the substance. 2) Steady State I n a s i t u a t i o n where the r a t e of entry of a molecule by sy n t h e s i s or t r a n s p o r t equals the ra t e of e x i t by breakdown or t r a n s p o r t , the concentration of the molecule remains constant and a steady s t a t e e x i s t s whenever the i n f l u x of m a t e r i a l does not balance the o u t f l u x . 3) Model k model represents any set of equations or f u n c t i o n s that describe the behavior of a t r a c e r i n the system. The parameters of a model are the a r b i t r a r y constants of the f u n c t i o n or equations. 4) Turnover Turnover r e f e r s to the process of renewal of a substance i n the 113 body or i n a given t i s s u e . 5) Turnover_Kate The Turnover Rate i s the rat e a t which a substance i s t u r n i n g over i n a given compartment or metabolic po o l . The meaning of turnover r a t e i s e x p l i c i t only when a steady s t a t e e x i s t s , t h a t i s , when the r a t e of s y n t h e s i s and tr a n s p o r t i n t o a compartment equals the rate of breakdown and e x i t . 6) Entry__Rate The r a t e parameters of turnover estimated from primed or continuous i n f u s i o n s or s i n g l e i n f e c t i o n s have been termed turnover r a t e , t r a n s f e r r a t e , u t i l i z a t i o n r a t e , entry r a t e , i n f l o w - o u t f l o w r a t e , f l u x , renewal r a t e , or i r r e v e r s i b l e d i s p o s a l . These measurements are probably a l l synonymous with i r r e v e r s i b l e l o s s or d i s p o s a l (White et a l . , 1969). 7) Tot al_Entry__Rate The t o t a l entry r a t e i s the rate at which a metabolite appears i n the sampled pool. 8) l£reversible_Loss I r r e v e r s i b l e l o s s i s the r a t e at which a metabolite, which does not r e t u r n to the sampled pool during the course of the 1 14 experiment, leaves t h i s pool. 9) R e c y c l i n g R e c y c l i n g i s the r a t e at which the l a b e l l e d **C r e t u r n s to the plasma metabolite p o o l , which has p r e v i o u s l y l e f t the sampled pool. 10) A l a n i n e _ P o o l _ S i z e Alanine pool s i z e i s the quantity of body a l a n i n e with which i n j e c t e d l 4 C - l a b e l l e d a l a n i n e mixes. 11) Alanine_Sp_ace Alanine space i s the volume of f l u i d through which the a l a n i n e pool i s d i s t r i b u t e d . 12) T urnover__Time The turnover time of a substance i s the time that i s r e q u i r e d f o r the turnover of a q u a n t i t y of substance equal to that present i n the compartment. 13) Precursor The term precursor r e f e r s to a compound which gives r i s e to another by chemical transformation or by t r a n s p o r t from one organ to another. 116 REFERENCES CITED 1. Annison, E. F. , K. J . H i l l and D. Lewis. (1957). Studies on P o r t a l Blood of Sheep: Absorption of V o l a t i l e F a t t y Acids from the Rumen of Sheep. Biochem. J . 66: 592 2. Annison, E. F., R. A. Leng, D. B. Lindsay, and R. R. White. (1963a) . The Metabolism of A c e t i c A c i d , P r o p i o n i c Acid and B u t y r i c Acid i n Sheep. Biochem. J . 88: 248 3. Annison, E. F. , D.B. Lindsay, and R. R. White. (1963b). Metabolic I n t e r r e l a t i o n s of Glucose and Lactate i n Sheep. Biochem. J . 88: 243 4. Annison, E. F. and R. R. White. (1961) . Glucose U t i l i z a t i o n ,in Sheep. Biochem. J . 80: 162 5. Baker, N. (1969). The Use of Computers to Study Rates of L i p i d Metabolism. J . of L i p i d Research .10: 1 6. Baker, N. and G. Incefy. (1955). Glucose Pool S i z e i n Normal and D i a b e t i c Rats. Fed. Proc. J[4: 7 7. Baker, N., R. A. S h i p l e y , R. E. Clark and G. E. Inefy. (1959). C Studies i n Carbohydrate Metabolism: Glucose pool s i z e and turnover Rate i n the normal r a t . Am. J. of P h y s i o l . 1 9 6 : 245 8. B a l l a r d , F. J. , R. W. Hanson, and D. S. K r o n f e l d . (1969). Gluconeogenesis and Lipogenesis i n Tissue from Ruminant and Nonruminant Animals. Fed. Proc. 29: f218-231. 9. Bergman, E. N. (1963). Q u a n t i t a t i v e Aspects of Glucose Metabolism i n Pregnant and Nonpregnant Sheep. Am. J. of P h y s i o l . 204: 147 10. Bergman, E. N. (1968). G l y c e r o l Turnover i n the Nonpregnant and K e t o t i c Pregnant Sheep. Am. J. of P h y s i o l 21,5: 865 11. Bergman, E. N. (1970). Disorders of Carbohydrate and Fat Metabolism. I n : Duke's Physiology of Domestic Animals, ed. by M.J. Swenson. I t h a c a , N.Y.: C o r n e l l Univ. Press, p. 595 12. Bergman, E.N. (1973). Glucose Metabolism i n Ruminants As Related to Hypoglycemia and K e t o s i s . C o r n e l l Vet. 63: 341 13. Bergman, E. N. and D. E. Hogue (1967). Glucose Turnover and Oxidation Rates i n L a c t a t i n g Sheep. Am. J . of P h y s i o l . 213: 1378-1384 14. Bergman, E. N., M. L. Katz, and C. F. Kaufman (1970)., Q u a n t i t a t i v e Aspects of Hepatic and P o r t a l Glucose Metabolism and Turnover i n Sheep. Am. J. of P h y s i o l . 219: 785-793 117 15. Bergman, E. N. , C. F. Kaufman, J . E. Wolff, and H. H. Williams (1974). Renal Metabolism of Amino Acids and Ammonia i n Fed and Fasted Pregnant Sheep. Am. J . of P h y s i o l . 226: 833 16. Bergman, E. N., R. S. Reid, H. S. Murray, J. M. Brockway and F. S, Whitelow (1965). Interconversions and Production of V o l a t i l e F a t t y Acids i n the Sheep Rumen. Biochem. J. 97: 53 17. Bergman, E. N. , W. E. Roe, and K. Kon (1966). Q u a n t i t a t i v e Aspects of Propionate Metabolism and Gluconeogenesis i n Sheep. Am. J . of P h y s i o l . 21.1: 793 18. Bergman, E. N., D. J . S t a r r , and S. S. Reulein (1968). G l y c e r o l Metabolism and Glyconeogenesis i n the Normal and Hypoglycemic K e t o t i c Sheep. Am. J . of P h y s i o l . 21.5: 874 19. Bergmen, E. N. and J . E. Wolff (1971). Metabolism of V o l a t i l e Fatty Acids by L i v e r and P o r t a l Drained V i s c e r a i n Sheep. Am. J . of P h y s i o l . 222: 586 20. Bergmeyer, H. U. (1965). Methods of Enzymatic A n a l y s i s . Academic Press Neu York & London. 21. Berman, M. (1963). The Formulation and Testing of Models. Annals of the New York Academy of Sciences 108: 182 22. Black. A. L., Modern Techniques f o r Studying the Metabolism of Nitrogenous Compounds, e s p e c i a l l y Amino Acids. In: Isotope Studies on the Nitrogen Chain. Vienna: I n t e r n a t i o n a l Atomic Energy Agency, 1968, p 287 23. Black, A. L., A. R.Egan, R.S. Anand, and T. E. Chapman (1968). The Role of Amino Acids i n Gluconeogenesis i n L a c t a t i n g Ruminants, I n : Isotope Studies on the Nitrogen Chain. Vienna: I n t e r n a t i o n a l Atomic Energy Agency, p. 147 24. Black, A. L., M. K l e i b e r , and M. A. Brown (1961). Butyrate Metabolism i n the L a c t a t i n g Cow. J. of B i o l . Chem 236: 2399 25. Black, A. L., J. R. Luick, and K. Knox (1972). Glycogenic Pathway f o r Acetate Metabolism i n L a c t a t i n g Cow. Am. J. of P h y s i o l . 222: 1575 26. Black, A. L. , J.R. Luick, F. H o l l e r and R. S. Anand (1966). Pyruvate and Propionate Metabolism i n L a c t a t i n g Cows J. of B i o l . Chem. 241: 233 27. Borchgrevink, C. F. and R. j . Navel. (1963). Transport of G l y c e r o l i n Human Blood. Proc. Soc. E x p t l . B i o l . Med. 113: 946 28. C h i l l , G. F., C. B. M a r l i s s , and T. T Aoki (1970). Fat and Nitrogen Metabolism i n F a s t i n g Man. Hormone and Metabolic 118 Research. Suppl. 2, New York, Academic Press. 181 29. C a r l s t e n , B. B., H a l l g r e n , R. Jagenburg, A. Svanborg and L. Werdo (1967). A r t e r i o - H e p a t i c Venous Dif f e r e n c e s of Free F a t t y Acids and Amino Acids. Acta. Med. Scand. 1.81: 199 30. C l a r k e , E. M. W., G.M. E l l i n g e r , and A. T. P h i l l i p s o n (1966). The Influence of Diet on the Nitrogenous Components passing to the Duodenum and through the Lower Ileum of Sheep. Proc. Royal Soc. .166: 63 31. Cocimano, M. R. and R. A. Leng (1967). Metabolism of Urea i n Sheep. Br. J . of Nutr 21.: 353 32. Cook, R. M. (1966). The Use of »*C to Study U t i l i z a t i o n of Substrates i n Ruminants. J . of Dairy S c i . 49: 1018 33. Cook, R. M. (1970). U t i l i z a t i o n of V.E.A. In Ruminants I I . Comparison of the Metabolism of Acetate Propionate and Butyrate when I n j e c t e d i n the Jugular Vein, P o r t a l Vein or the Rumen. Biochenica et Biophysica Acta 201.: 91 34. C o r i , C. F. (1931). Mannalain Carbohydrate Metabolism P h y s i o l o g i c a l Reviews V I : 143 35. Dawes, G. S. (1968). F e t a l and Neonatal Physiology. Chicago: Yearbook Med. P u b l i s h e r s . 36. Dunlop, R. E. and P. B. Hammond (1965). D - l a c t i c A c i d o s i s of Ruminants Ann. N. Y. acad. S c i . _19: 1109 37. Elwyn, D.H. (1970). The Role of the L i v e r i n Regulation of Amino Acid and P r o t e i n Metabolism. I n : Mammalian P r o t e i n Metabolism ed. by H. N. Munro, Academic Press: New York, 1970, p. 523 38. Elwyn, D. H., H. C. Parkh, and W. C. Shoemaker. (1968). Amino Acid Movements between Gut, L i v e r , and Periphery i n Unanesthetized Dogs. Am. J . of P h y s i o l . 215: 1260 39. Exton, J. H. (1972). Gluconeogenesis. Metabolism 21: 945 40. F e l i g , P. (1972). I n t e r a c t i o n of I n s u l i n and Amino A c i d Metabolism i n the Regulation of Gluconeogenesis. I s r e a l J . Med. S c i . 8: 262 41. F e l i g , P. (1973). The Glucose-Alanine Cycle. Metabolism 22: 179 42. F e l i g , P . , T. Pozefsky, E. M a r l i s s , and G. F. C a h i l l . (1970). Alanine: Key Role i n Gluconeogenesis. Science 167: 1003 43. F e l i g , P. And J . Hahren. (1971). Amino Acid Metabolism i n E x e r c i s i n g Man. J. of C l i n . Invest. 50: 2703 119 44. F e l l e r , D. D., E.H. S t r i s o w e r , and I . L. Chaikoff. (1950). Turnover and Oxidation of Body Glucose i n Normal and A l l o x a n - D i a b e t i c Rats J . of B i o l . Chem. 1 8 7 : 571 45. F l e t c h e r , R. (1972). Fortran Subroutines f o r Minimization by Quasi-Newton Methods. United Kingdom Atomic Energy A u t h o r i t y . Research Troup Report AERE-R7125. 4 6. Ford, E. J . H. and P. E. B. R e i l l y (1970). The U t i l i z a t i o n of Free Amino Ac i d and Glucose Carbon by Sheep Pregnant with Twins. Res. Vet. S c i . j M : 575 47. F r i e s , G. F. and G. H. Conner (1961). Studies on Bovine P o r t a l Blood. I I Blood Flow Determinations with Observations on Hemodilution i n P r o t a l Vein. Am. J . of Vet. Res. 22: 487 48. Gallagher, G. and S. H Buttery (1959). Biochemistry of Sheep Tissues, Enzyme Systems of L i v e r , Brain and Kidney. Biochem J . 72: 575 49. Gurpide, E., P. C. MacDonald, R. L. Vande Wiele and S. Lieberman. (1963). Measurement of the Rates of S e c r e t i o n and of p e r i p h e r a l Metabolism of two I n t e r c o n v e r t i b l e Compounds: dehydroisoandrosterone-dehydroisoandrosterone s u l f a t e . J . C l i n . Endocrinol Metab. 23: 346 50. Henrigues, 0. B., S. B. Henrigues, and A. Neuberger. (1955). Q u a n t i t a t i v e Aspects of Glycine Metabolism i n the Rabbit. Biochem. J . 60: 409 51. Hogan, J . P. and R. H. Weston (1967). The D i g e s t i o n of Nitrogen and some carbohydrate F r a c t i o n s i n the Stomach and i n t e s t i n e s . Aust. J . Agric. Res. J 8 : 80 3 52. Hogan, J. P., R.H. Weston, and F. R. Lindsay (1968). Influence of P r o t e i n D i g e s t i o n on Plasma Amino Acid Levels i n Sheep. Aust. J . B i o l . S c i . 2.1: 1263 53. Huggett,A. St. G. (1961). Carbohydrate Metabolism i n the Placenta and Fetus. B r i t . Med. B u l l . V7: 122 54. Hunter, G. D. and B. C. M i l l s o n (1964). Gluconeogenesis i n the L a c t a t i n g Dairy Cow. Res. Vet. S c i , 5: 1 55. I s s e k u t z , B., M. A l l e n , and R. Borkow (1972), Estimation of Glucose Turnover i n the Dog with Glucose-2-T and Glucose-U-**C. Am. J . of P h y s i o l . 222: 710 56. Katz, M. L. and E. N. Bergman (1969a). Hepatic and P o r t a l Metabolism of Glucose, Free F a t t y A c i d s , and Ketone Bodies i n the Sheep. Am, J . of P h y s i o l . 2JM5: 953 57. Katz, M. A. and E. N. Bergman (1969b). Simultaneous Measurements of Hepatic and P o r t a l Venous Blood Flow i n the Sheep and the Dog. Am. J . of P h y s i o l . 2.16: 946 120 58. Katz, A. M. and M. E. Carsten (1963) . A c t i n from Heart Muscle: Studies on Amino Acid Composition. C i r . Bes. 13: 474 59. Katz, J . and A. Dunn (1967). Glucose-2-T as a Tracer for Glucose Metabolism. Biochem. 6: 1 60. Kaufman, C. F. and E. N. Bergman (1971). Renal Glucose, Free F a t t y A c i d , and Ketone Body Metabolism i n the Unanesthized Sheep. Am. J . of P h y s i o l . 22.1: 967 61. Kaufman, C. F. and E. N. Bergman (1971). Renal Ketone Body Metabolism and Gluconeogenesis i n Normal and Hypoglycemic Sheep. Am. J. of P h y s i o l . 226: 827 62. K l e i b e r , M. , A. L. Black, M. A. Brown, J . L u i c k , C. F. Baxter, and B. M. Tolbert (1951). Butyrate as a Precursor of Milk Constituents i n the I n t a c t Dairy Cow. J . of B i o l . Chem. 2jK>: 239 63. Kominz, D. R., A. Hough, P. Symond, and K. La k i (1954). The Amino Acid Composition of A c t i n , Myosin, Tropomyosin and the Meromyosins. Arch. Biochem. Biophys. 50: 148 64. Krebs, H. A. (1964), Gluconeogenesis Proc. Royal Sec. 159: 545 65. Krebs, H. A. (1966). Bovine Ketosis Vet. Record, 78: 187 66. Krebs, H. A., R. Hems, J . J. Weideman, and R, N. Speake (1966), The Fate of I s o t o p i c Carbon i n Kidney S y n t h e s i z i n g Glucose from L a c t a t e . Biochem. J. ±011 242 67. K r e i s b e r g , R. A., A. M. S i e g a l , and W. Crawford Owen, (1972). Alanine and Gluconeogenesis i n Man: E f f e c t of Ethanol, J . C l i n . Endo. and Metabolism 34: 876 68. K r o n f e l d , D. S. And M. G. Simesen (1961) Glucose B i o k i n e t i c s i n Normal and K e t o t i c Cows. J . of Appl. P h y s i o l . 1 4 : 1026 69. Larsen, J . A . (1963). E l i m i n a t i o n of G l y c e r o l as a Measure of Hepatic Blood Flow i n the Cat. Acta P h y s i o l . Scand. 57: 224 70. Lehinger, A. L. (1970). Biochemistry G 597 Worth P u b l i s h e r s Inc. New York. 71. L e i b h o l t z , J. (1969). E f f e c t of Diet on the Concentration of Free Amino Aci d s , Ammonia, and Orea i n Rumen Liquor and Blood Plasma of the Sheep. J . of An. S c i . 29: 628 72. Leng, R. A. (1970). Glucose Synthesis i n Ruminants. Adv. Vet. S c i . 14: 209 121 73. Leng, R. A. and E. F. Annison (1962). Metabolic A c t i v i t i e s of Sheep Erythro c y t e s . Aust. J . A g r i c . Res. V3: 31 74. Leng. R. A. and E. F. Annison (1963) Metabolism of Acetate, Propionate and Butyrate by Sheep L i v e r S l i c e s . Biochem. J . 86: 319 75. Leng, R. A., J . W. S t e e l , and J. R. Luick (1967). C o n t r i b u t i o n of Propionate to Glucose Synthesis i n Sheep. Biochem. J . 103: 785 76. Lindsay, D. B. (1970). Carbohydrate Metabolism i n Ruminants. In: Physiology of Di g e s t i o n and Metabolism i n the Ruminant, ed. by A. T. P h i l l i p s o n , O r i e l Press: Newcastle upon Tyne, p. 438 77. Lindsay, D. B. (1959). The S i g n i f i c a n c e of Carbohydrate i n Ruminant Metabolism. Vet. Rev. Annot. 5: 103 78. Lundquist, F., N. Tygstrup, K. Winkler and K. B. Jensen G l y c e r o l Metabolism i n the Human l i v e r : I n h i b i t i o n by Ethanol. Science 150: 616 79. McClymost, G, C. and B. P. S e t c h e l l (1956). Induced Hypoglycemia Encephalopathy i n Sheep and i t s I m p l i c a t i o n s as regards Pathogenesis of the Disease. Aust. Vet. J . 32: 97 80. McFarlane, A. S. (1964). Metabolism of plasma P r o t e i n s . In "Mammalian P r o t e i n Metabolism" ed. by H. N. Munro and J . B. A l l s i o n . New York: Academic, 4: 197 81. MacRae, J . C. and D. G. Armstrong (1966). I n v e s t i g a t i o n s of the Passage of L-Linked Glucose Polymers i n t o the Duodenum of Sheep. Proc. Nutr. Soc. 25: 33 82. M a r l i s s , E.B., T. T. A o k i , T. Pozefsky, A. S. Most and G. F. C a h i l l (1971). Muscle and Splanchnic Glutamine and Glutamate Metabolism i n Post Absorptive and Starved Man. J. C l i n . Invest. 50: 814 82. Meier, P. and K. L. Z i e r l e r (1954). On the theory of D i l u t i o n Method f o r Measurement of Blood Flow and Volume. J. of Applied P h y s i o l . 6: 731 84. Nolan, J . V. and R. A. Leng {1970) . Metabolism of Drea i n Late Pregnancy on the P o s s i b l e C o n t r i b u t i o n of Amino Acid Carbon to Glucose Synthesis i n Sheep. B r i t . J . Nutr. 24: 905 85. Nolan, J . V. and R. A. Leng (1972). Dynamic Aspects of Ammonia and Drea Metabolism i n Sheep. B r i t . J. Nutr. 27: 177 . 86. Owen, P. E., A. P. Morgan, H. G. Kemp, J . M. S u l l i v a n , M. G. Herrera, and G. F. C a h i l l (1967). B r a i n Metabolism 122 During F a s t i n g . J . of C l i n . Invest. 46: 1589 87. Packett, L. V. and T. D. D. Groves (1965). Urea Recycl i n g i n the Bovine. J. An. S c i 24: 341 88. Pennington, R. I. (1954). The Metabolism of Short Chain Fatty Acids in Sheep. Biochem. J. 56: 410 89. P o t t e r , B. J . (1952). R e l i e f of Hypoglycemic Convulsions with B u t y r i c A cid. Nature T70: 541 90. Pozefsky, T., P. F e l i g , J . D. Tobin, J . S. Soeldner, and G. F. C a h i l l (1969). Amino Acid Balance Across Tissues of the Forearm i n Postabsorptive Man. E f f e c t s of I n s u l i n at two Dose L e v e l s . J . C l i n . I n v e s t . 48: 2273 91. Raju, K. G., B. C. Fong. S. K. Srungaram, and W. C. Bowie (1972). E f f e c t of Experimentally Induced Hyperketcnemial on Glucose Metabolism of Ovine Brain i n Vivo. Fed. Proc. 31: 345 92. R e i l l y , P. E. B. and E. J . H. Ford (1971). The E f f e c t s of D i f f e r e n t Dietary Contents of P r o t e i n on Amino Ac i d and Glucose Production and on the C o n t r i b u t i o n of Amino Acids to Gluconeogenesis i n Sheep. B r i t . J . Nutr. 26: 249 93. R e i s , P. J . and D. A. Tunks (1970). Changes i n Plasma Amino Acid Patterns i n Sheep Associated With Supplements of Casein and Formaldehyde-treated Casein. Aust. J . B i o l . S c i . 23: 673 94. Rescigno, A. and G. Segre (1966). Drug and Tracer K i n e t i c s . Walthams; B l a i s d e l l 95. Roe, W. E., E. N. Bergmanand, K. Kan (1966). Absorption of Ketone Bodies and other M e t a b o l i t e s v i a the P o r t a l Blood of Sheep. Am. J. of Vet. Res. 27: 729 96. Ruderman, N. B. and p. Lund (1972). Amino A c i d Metabolism i n S k e l e t a l Muscle. Regulation of Glutamine and Alanine Release i n the Perfused Rat Hindquarter. I s r a e l J . Med. S c i . 8: 295 97. Sabine, J . R. and B. C. Johnson (1964). Acetate Metabolism i n the Ruminant. J. of B i o l . Chem. 239: 89 98. Schambye, P. (1951). V o l a t i l e Fatty Acids and Glucose i n P o r t a l Blood of Sheep. Nord. Veterinarmedicin 3: 748 99. Schimke, R. T. and D. Doyle (1970). C o n t r o l of Enzyme Levels i n Animal Tissues. Ann. Rev. Biochem. 39: 929 100. Schmidt-Nielsen, B., K. Schmidt-Nielsen, T. R. Houspt, and K. Jarnum (1957). Urea E x c r e t i o n . Am. J. of P h y s i o l . .188: 477 1 2 3 101. S e a r l e , G. L., E. H. S t r i s o w e r , ana I . L. C h a i k o f f (1954). Glucose Pool and Glucose Space i n the Normal and Di a b e t i c Dog. Am. J . of P h y s i o l . 1 7 6 : 190 102. Segal, S., M. Berman, and A. B l a i r (1961). The Metabolism of v a r i o u s l y * * C - l a b e l l e d Glucose i n Man and an Estimation of the Extent of Glucose Metabolism by the Hexose Monophosphate Pathway. J . of C l i n . Invest. 40: 1263 103. S e t c h e l l , B. P. (1961). C e r e b r a l Metabolism i n the Sheep. Biochem. J. 72: 265 104. Smith, H. W., N. F i n k e l s t e i n , L. Aliminosa, B. Crawford, and M. Graber (1945). The r e n a l Clearances of S u b s t i t u t e d Hippuric Acid D e r i v a t i v e s and Other Aromatic Acids i n Dog and Man. J . C l i n . I n v e s t . 24: 388 105. Steinberg, D. and M. Vaughn (1965). Release of F.F.A. from Adipose Tissue i n v i t r o i n B e l a t i o n t o Rates of T r i g l y c e r i d e Synthesis and Degradation. I n : Handbook of Physiology Adipose Tissue, Washington D. C. Am. P h y s i o l . S o c , 1965, s e c t . 5, chapt. 34 p 335 106. Sutton, J. D. and J . W. G. Nicholson (1968). The D i g e s t i o n of Energy and Starch along the G a s t r o - l n t e s t i n a l Tract of Sheep. Proc. Nutr. Soc. 27: 49 107. Topps, J . H., R. N. B. Kay, and E. D. Godall (1968a). Di g e s t i o n of Concentrate and of Hay Di e t s i n the Stomach and I n t e s t i n e s of Ruminants. 1) Sheep. B r i t . J. of Nutr. 22: 261 108. Topps. J . H., R. N. B. Kay, E. D. Goodall, F. G. Whitelaw, and R. S. Reid (1968b). D i g e s t i o n of Concentrate and of Hay d i e t s i n the Stomach and I n t e s t i n e s of Ruminants. 2) Young Steers. B r i t . J . of Nutr. 22: 281 109. Vaughn, M. (1961). The Metabolism of Adipose Tissue i n v i t r o . J. L i p i d Res. 2: 2930 110. Washko, M. E. and E. W. Rice (1961). Determination of Glucose by an Improved " G l u c o s t a t " Procedure. C l i n . Chem. 7: 542 111. Weigand, E., J . W. Young, and A. D. M c G i l l i a r d (1972). Extent of Propionate Metabolism during Abosrption from the Bovine Ruminoreticulum. Biochem. J . J 2 6 : 201 112. Weiner, R. , H. J. H i r s c h , and J . J . S p i t z e r (1971). Cerebral E x t r a c t i o n of Ketones and t h e i r Penetration i n t o CSF i n the Dog. Am. J . of P h y s i o l . 223: 447 113. White, R. G., J. W. S t e e l , R. A. Leng, and J . R. Luick (1969). Evalua t i o n of three I s o t o p e - D i l u t i o n Techniques f o r Studying the K i n e t i c s of Glucose Metabolism i n Sheep. Biochem. J . 114: 203 124 114. Wolff, J. E., E. N. Bergman, ana H. H. W i l l i a m s (1972). Net Metabolism of Plasms Amino Acias by L i v e r and P o r t a l Drainea Viscera of Fed Sheep. Am. J. of P h y s i o l . 223: 438 115. Wolff, J . E. and E. N. Bergman (1972a). Metabolism and Interconversions of Five Plasma Amino Acids by Tissues of the Sheep. Am. J . of P h y s i o l . 223: 447 116. Wolff, J . E. and E. N. Bergman (1972b), Gluconeogenesis from Plasma Amino Acids i n Fed Sheep. Am. J . of P h y s i o l . 223: 455 117. Wrenshall, G. A., G. Hentenyi, and C. A. Best (1961). The V a l i d i t y of Bates of Glucose Appearance i n the Dog C a l c u l a t e d by the Method of Successive Tracer I n j e c t i o n s I I . The Influence of Intermixing Time Following Tracer I n j e c t i o n s . Can. J . of Biochem. S P h y s i o l . 39: 267 APPEND 1X 126 F i g u r e 1.) S t a n d a r d Curve f o r G l u c o s e D e t e r m i n a t i o n . Absorbance CO o 12 7 F i g u r e 2.) S t a n d a r d Curve f o r A l a n i n e D e t e r m i n a t i o n Absorbance 0 128 Figure 3.) Standard Curve for Para-aminhippuric Acid (PAH) Determi nation Absorbance 0 1 L x t e r n a l S t a n d a r d s R a t i o 130 F i g u r e 5 . ) Paper Chromatography S e p a r a t i o n of C - A l a n i n e w i t h Use of the A c t i g r a p h Scanner. LOG SPECIFIC ACTIVITY (NCl/UG) fD 133 F i g u r e 8.) A c t i v i t y Curve f o r l 4 C - G l u c o s e i n J u g u l a r V e i n ( J , V . ) txperiment: B.2. LOG SPECIFIC ACTIVITY (NCl/UG) ro to m > O CO CO CO rr X TD ro 3 ro r t O c < ro o I > 3 ro c c < ro C-< Figure 10a) A c t i v i t y Curve for ^-G lucose in Carot id Artery (C. hxper iment P.3. 136 F i pure T I M E ( M I N ) 138 F i g u r e l i b ) A c t i v i t y Curve f o r l ' l C - A l a n i n e i n J u g u l a r V e i n ( J . V . ) t x p e r imen t B.3. 0 . 3 o > o < o L L o LU CL CO (3 O —I R = 0 . 9 9 9 i 1 1 1 1 1 1 1 1 1 r T I M E (MIN) F i g u r e 1 3 . ) A c t i v i t y Curves f o r 1 ! | C - A l a n i n e and 1!*C-G 1 ucose. h x p e r i m e n t C.2. TIME '(MIN) Ihl F i g u r e Ik. Major M e t a b o l i c Pathways In The L i v e r And Kidneys Of Ruminants. (Bergman, 1973) Glycogen S t o r e s B l o o d G l u c o s e s ~-^ =*,G 1 ucose-6-P t r F r u c t o s e - 1 , 6-P I I Tr|ose-P<. . -, ..^G1 y c e r o 1 P - E n o l - P y r u v a t e 2 142 Figure 15.) Schematic Diagram I l l u s t r a t i n g the P o s i t i o n of the Sampling and Infusion Catheters. (Katz and Bergman, 1969, pg. 948). Posterior Vena Cava Ih 3 F i g u r e 16. The G l u c o s e A l a n i n e C y c l e . ( F e l i g . , 1973) F i g u r e 17. Model f o r S i n g l e I n j e c t i o n C o n t i n u o u s I n f u s i o n (Curve M e i e r and Z i e r l e r , 1954,pg (Curve A) and B) of a Dye. 732 . Time (Seconds) .U5 F i g u r e 1 8 . M o d e l f o r G 1 u c o s e Me t a b o 1 i sm i n S h e e p . ( L e n g , 1 9 7 0 , p g . 2 1 6 ) * P o o l s B a n d C a r e p r o b a b l y made u p o f a l l g l u c o g e n i c p r e c u r s o r s , i . e . , g l u c o g e n i c a m i n o a c i d s , l a c t a t e , p r o p i o n a t e , a n d g l y c e r o l , a n d CO2 a n d g l y c o g e n . ( W h i t e e t a_L, 1 9 6 9 ) Table 1(a). Packed C e l l Volume Values, Glucose, Alanine i n P o r t a l and Car o t i d Arteries„ and PAH Concentrations Sample Time Control 1 hr 1 hr 15 rnln 1 hr 25 min 1 hr 45 win mean SE Control 1 hr 1 hr 15 min 1 hr 25 min 1 hr 45 min mean i SE C o l l e c t i o n S i t e Carotid Artery Carotid A r t e r y Carotid A r t e r y Carotid A r t e r y C a r o t i d A r t e r y P o r t a l Vein P o r t a l Vein P o r t a l V e i n P o r t a l Vein P o r t a l . Vein Mean PCV {% RBC) 19.25 - 19*75 Glucose (mg/100 n l ) 76.5 80.5 19.8 23.5 2 9 . 3 73.5 7 7 . 0 81.0 22.32 1.90 78.70 1.28 2 4 . 0 7 4 . 0 . 23.25 2 4 . 0 28.0 27.6 75o0 7 9 . 5 76.0 7 9 . 3 Alanine (ug/ml) 10.8 1 0 . 6 10.2 10.4 10.8 11 .6 11.5 12.0 11 .8 • PAH (mg/ml) 0.006 0/0085 0.0080 0.0080 10.6 0.1 0.0080 11.4 0 . 0 4 0 0.068 0.0155 0.0165 P o r t a l -25.37 ± 1 . 0 0 76.76 ± 1.12 11 .71 ± 0 . 1 2 0.016 Table K b ) . Ca lcu la t ions for txperiment A . l . (Blood Flow) Blood Flow : PVPF = CJ. where C P V ~ C A PCV CI IR CPV 25.37% 15.0 mg/ml 0.8 ml/min 0.016 mg/ml 0.008 mg/ml Portal Vein Plasma Flow (PVPF) 12 mg/ml 0.0160 - 0.008 mg/ml 150 0 ml/min 35.01 ml/m in/kg BW 3 / ! + Portal Vein Blood Flow (PVBF) 1500 ml/min 1 - 0.2537 2010 ml/min «*8 . 08 ml/min/kg BW 3 / U Net Portal Metabolism P = Fpv^ Cpy -C^ ) a) Glucose U t i l i z a t i o n P = 2010 ml/min( 0.7676 - 0.7870 mg/ml) =-38.99 mg/min = -0.142g/hr/kg BW3/!* b ) Alanine Production P = 2010ml/min( 0.0117 - 0.0106 mg/ml) = +2.21 mg/min = +1.49 mMoles/hr T a b l e 2 . P r e l i m i n a r y E x p e r i m e n t t o T e s t E f f i c i e n c y o f I o n E x c h a n g e C h r o m a t o g r a p h y f o r S e p a r a t i o n o f P l a s m a C o m p o n e n t s . S a m p l e F r a c t i o n DPM % A l a n i n e R e c o v e r e d U - 1 4 C - A l a n i n e 1 6 7 , 9 7 1 ( c o n t r o l ) B a s i c ( a m i n o 1 5 8 , 6 5 7 9 4 . 5 a c i d s ) 7 1 2 0 . 4 A c i d i c ( a c i d s ) 2 6 1 0 . 2 N e u t r a l ( s u g a r s ) Ik9 14, T a b l e 3. P a p e r C h r o m a t o g r a p h y S e p a r a t i o n o f C - G l u c o s e S t r i p Number S e c t i o n Number DPM 1 4 C o n t r o l P l a s m a S a m p l e P l a s m a S a m p l e C - G l u c o s e E x p . S.l Exp.B2. 4 4 4 4 4 4 4 4 4 4 4 1 2 3 4 5 6 7 8 9 10 11 3.5 50.0 1 8 5 0 . 0 520„0 25 .0 2.0 •4.0 6 5 . 0 2.5 3.0 5 8 . 0 1.0 4 e 0 150 Table 4. Data and Metabolic Parameters of Alanine f o l l o w i n g a S i n g l e I n j e c t i o n of U-^C-Alanine: Experiment B el, A) Data C o l l e c t i o n Glucose Alanine Time (min a f t e r i n j e c t i o n ) Concen-t r a t i o n (rag/100 rid) S p e c i f i c A c t i v i t y (nC/mg) Concen-t r a t i o n (ug/ml) S p e c i f i c A c t i v i t y (nC/mg)" c o n t r o l 54 - 11.2 -5 38 1.5921 10.5 1.970 10 54 0.4759 12.2 0.410 20 57 0.4702 10.4 0.256 30 58 0.4086 11.4 0.211 52 56 0.4321 11.3 0.142. 60 62.5 0.5152 11.7 0.130 120 55 0.3218 11.0 0.081 180 51.5 0.1864 11.7 0.047 mean ± SE ±2.40 11.26 ±0.17 B) Metabolic Parameters: P o o l Size (mg of C) z . 52.-83 Space (ml) 4,645.16 Space (g-'of.BW) 13.27 T o t a l Entry Rate (mg C/rdn.) 8.61 I r r e v e r s i b l e Loss (mg C/inin) 4.96 Recycling (mg C/min) 3.65 Per cent Glucose from Alanine 3.57 Isotope Dose (uCi) 50.0 Body Weight (kg) 35.0 Table 5 . Data and Metabolic Parameters of Alanine Following A Single Injection of U- 1 4C-Alaiiine : Experiment g. 2 . A) Data C o l l e c t i o n Time (min aft e r i n j e c t i o n ) c o n t r o l 3 . 0 3 0 5 4 . 6 4 . 5 5 0 0 1 0 * 0 1 5 o 0 2 0 . 0 3 0 o 0 4 5 . 0 6 0 o 0 7 5 . 0 9 0 . 0 1 0 5 . 0 1 2 0 . 0 1 3 5 . 0 1 5 0 . 0 mean i SE Glucose Cone en-- S p e c i f i c t r a t i o n A c t i v i t y (mg/100ml) (nC/mg) 3 9 . 8 2 6 . 5 - 0 . 3 9 4 5 . 8 0 . 2 5 5 2 „ 0 0 . 2 9 4 8 . 0 0 . 4 1 4 2 o 0 0 „ 6 1 5 3 . 0 0 . 8 5 5 3 . 3 1 . 0 0 6 1 . 8 1 . 1 0 6 1 . 0 1 . 2 0 6 0 o O l o 3 0 6 4 . 8 1 . 2 0 6 5 . 0 1 . 0 4 6 2 . 5 0 . 9 7 6 5 . 0 0 . 9 2 6 0 o 0 0 . 7 8 6 6 . 5 0 . 5 0 6 2 0 8 0 . 3 9 5 4 . 9 4 i 2 . 5 2 Alanine Concen- S p e c i f i c t r a t i o n A c t i v i t y (ug/ml) (nC/mg) 1 3 . 6 1 2 . 7 1 . 6 6 8 1 3 . 6 0 o 3 5 4 1 3 . 3 0 c 2 8 9 1 4 . 6 0 . 1 9 5 1 3 . 0 0 o 1 8 5 1 4 . 2 0 o 1 0 6 1 2 0 6 0 o 1 1 2 1 3 . 8 0 . 0 7 5 1 4 . 0 0 o 0 5 0 1 3 o 4 0 . 0 4 4 1 2 . 9 0 . 0 3 1 1 3 . 0 0 . 0 2 7 1 4 . 2 0 . 0 2 1 1 4 . 6 0 o 0 1 7 1 3 . 9 0 . 0 1 4 1 3 o 7 0 . 0 1 2 1 3 . 5 0 o 0 0 7 1 3 . 5 9 ± 0 . 9 5 152 T a b l e 5. c o n t ' d B) M e t a b o l i c Parameters : Two Term b x p o n e n t i a 1 F u n c t i o n Pool S i z e (mg of C) = 17.69 Space (ml) = 1296.85 Space U of BW) = 3.71 T o t a l L n t r y Rate (mg C/min) = 14.52 I r r e v e r s i b l e Loss (mg C/min) = 7.60 R e c y c l i n g (mg C/min) = 6.91 Per c e n t G l u c o s e From A l a n i n e = 5.00 I s o t o p e Dose ( u C i ) = 50.0 Body Weight (kg) ' * 35.0 Three Term b x p o n e n t i a l F u n c t i o n Pool S i z e (mg o f C) = 17.69 Space (ml) = 1296.85 Space U of BW) = 3.71 T o t a l t n t r y Rate (mg C/min) = 14.52 I r r e v e r s i b l e Loss (mg C/min) = 7.60 R e c y c l i n g (mg C/min) = 6.91 Per c e n t G l u c o s e From A l a n i n e = 5.00 Isotope Dose ( u C i ) = .50.0 Body Weight (kg) = 35.0 T a b l e 6 . Data and Metabolic Parameters of Alanine Following A Single Injection of U- C-Alanine s Experiment 8.3. A) Data Carotid Artery C o l l e c t i o n Time (min af t e r i n j e c t i o n ) Glucose Concen- S p e c i f i c t r a t i o n A c t i v i t y (mg/100ml) (nC/mg) Alanine Concen- S p e c i f i c t r a t i o n A c t i v i t y (ug/ml) (nC/mg) contr o l 6 4 . 0 - 1 0 . 8 — control 6 2 0 5 - 1 0 . 5 -0 . 3 3 6 1 . 5 0 . 1 9 3 9 . 5 1 . 3 2 4 0 . 6 7 7 1 . 0 0 . 1 9 0 1 0 . 6 1 . 1 7 7 0 . 8 3 6 6 . 0 0 . 1 9 7 1 0 . 8 0 . 5 3 3 1 . 1 3 6 2 . 5 0 . 4 7 6 1 0 . 6 0 . 5 1 2 1 . 4 2 6 6 . 0 0 . 5 6 9 1 2 . 0 0 . 3 6 1 1 . 9 2 6 4 . 0 0 . 5 6 5 1 1 . 4 0 . 3 0 2 2 . 1 7 6 9 . 0 0 . 7 4 6 1 1 . 6 0 . 2 7 7 5 . 0 0 6 9 . 0 0 . 7 1 0 9 . 5 0 . 2 5 9 1 0 c 0 5 9 . 0 1 . 1 2 4 1 0 . 3 0 . 1 3 3 1 5 . 0 6 9 . 0 1 . 1 7 8 1 0 . 4 0 . 1 1 5 2 5 . 0 6 7 . 0 1 . 3 9 4 . 1 0 . 8 0 . 0 6 5 3 5 . 0 7 1 . 0 1 . 3 8 2 1 0 . 6 0 . 0 5 2 4 5 . 0 6 9 . 0 1 . 4 5 4 1 0 . 6 0 . 0 4 0 6 0 . 0 7 1 . 0 1 . 5 3 8 1 0 . 6 0 . 0 3 3 7 5 . 0 7 1 . 5 1 . 4 6 7 1 0 . 0 O . 0 2 7 9 0 . 0 7 3 . 0 1 . 3 3 0 1 0 . 3 0 . 0 2 3 1 2 0 . 0 7 7 . 0 1 . 1 2 4 1 0 . 6 0 . 0 2 0 ean ± SE 6 7 . 5 2 ± 1 . 0 4 1 0 . 5 9 ± 0 . 1 4 T a b l e 6 . c o n t ' d A ) D a t a . • • C o l l e c t i o n G l u c o T i m e C o n c e n -( m i n a f t e r t r a t i o n i n j e c t i o n ) ( m g / 1 0 0 m l ) c o n t r o l 5 1 . 0 c o n t r o l 5 4 . 0 0 . 3 3 5 2 . 0 0 . 5 8 5 9 . 0 0 . 8 3 6 2 . 0 1 . 9 5 6 0 . 0 2 . 1 7 6 1 . 0 2 . 4 5 5 9 . 0 2 0 7 5 6 2 . 0 lOcO 5 9 . 0 1 5 . 0 5 4 . 0 2 5 . 0 6 1 . 0 3 5 . 0 6 1 . 0 4 5 . 0 6 1 . 0 ' 6 0 . 0 6 6 . 0 7 5 . 0 . 6 7 . 0 9 0 . 0 6 6 . 0 1 2 0 . 0 7 4 . 0 m e a n ± S E 6 0 . 2 8 ± 1 . 3 6 J u g u l a r Vein ;e A l a n i n e S p e c i f i c C o n c e n - S p e c i f i c A c t i v i t y t r a t i o n A c t i v i t y ( n C / m g ) ( u g / r a l ) ( n C / m g ) - • 1 1 . 6 -- 1 1 . 3 -0 . 1 3 8 1 0 . 8 . 0 . 7 0 2 0 . 2 1 6 1 0 . 0 " 1 . 1 8 4 0 . 2 3 3 1 0 . 9 0 . 6 9 9 0 . 3 9 2 1 0 . 3 0 . 2 7 8 0 . 4 9 0 1 0 . 6 0 . 2 6 4 0 . 5 3 2 1 0 . 1 0 . 2 5 7 0 . 5 9 3 1 0 . 3 0 . 2 2 9 0 . 9 3 0 9 . 5 . 0 . 1 8 1 1 . 0 1 8 1 1 . 3 0 . 1 0 3 1 . 5 2 1 1 0 . 3 0 . 0 5 8 1 . 5 3 4 • 1 0 „ 8 0 . 0 4 2 1 . 8 0 0 1 0 . 3 . 0 . 0 3 5 1 . 4 6 5 • 1 1 . 3 ' 0 . 0 3 0 1 . 3 1 9 1 1 . 8 0 . 0 2 3 1 . 3 1 6 1 1 . 6 0 . 0 2 1 1 . 2 4 4 1 0 . 3 1 0 . 7 3 ± 0 . 1 5 0 . 0 1 8 T a b l e 6„ co n t ' d B) M e t a b o l i c Parameters C a r o t i d A r t e r y P o o l S i z e (mg o f C) = 42.69 Space (ral)= 4026.53 Space (% o f BW) = 10.8 T o t a l E n t r y Rate (mg C/min) = 61.37 I r r e v e r s i b l e Loss (mg C/min) = 9.32 R e c y c l i n g (mg C/min) = 5.20 Per cent G l u c o s e From A l a n i n e ) = 6.74 Isotope Dose ( u C i ) = 95.0 Body Weight (kg) = 37.0 J u g u l a r V e i n P o o l S i z e (mg o f C) = 17.49 Space (ral)= 1640.91 Space (% Of BW).= 4.44. T o t a l E n t r y Rate (mg C/min) = 49.78 I r r e v e r s i b l e Loss (mg C/min) = 10.03 R e c y c l i n g (mg C/min) = 39 075 Per cent G l u c o s e From A l a n i n e = 6.78 Isotope Dose ( u C i ) = 95.0 Body Weight (kg) = 37.0 to LTi Table 7. ' Summary of Metabolic Parameters for the Single Injection Experiments. Experiment Blood number Source and type Sheep Weight (kg) Plasma Alanine concen-tration (ug/ml) Pool Size (mg) Space {% of BW) Irreversible Loss (mM/hr) Total Entry Rate (mM/hr) Recycling ' Rate (mM/hr) % Conversion •to Glucose S.I.-l Jugular Vein 35 11.26 .17 52.9 13.27 3.34 5.80 2.46 3.57 S.I .-2 Jugular Vein 35 13.59 .95 17.7 3.71 5.12 9.77 4.65 5.00 'S.I.-3 Jugular Vein 37 10.73 .15 17^5 4.44 6.75 33.52 26.77 6.78 Carotid 37 10.60 .14 42.70 10.88 9.33 :61.37 52.04 6.74 Artery mean - SE 32.70 i17.92 8.07 ±4.73 •6.13 ±2.54 27.61 ±25.61 21.48 ±23.14 5.52 ±1.54 Table 8. Data and Metabolic Parameters of Alanine Following a Continuous Infusion of "^C-Alanine; Experiment C. 1. A) Data Carotid Artery-Collection Time (min after start of infusion) Glucose Concen- Specific tration A c t i v i t y (mg/lOOml) (nC/mg) Alanine Concen- Specific tration Activity (ugM) (nC/mg) control 83.0 - 13.2 5 68.0 0.151 12.8 8 .4 15 68.0 0.205 14.8 9.6 30 67.0 0.255 14 .2 14.4 60 64.0 0.289 15.5 18 .3 . 90 61.0 ' 0.310 L4 .0 . 26.7 120 62.0 0.577 16.7 21.0 150 62.0 0.824. 13.3 31 .1 180 90.0 0.703 14.0 33.7 210 46.0 •• 1.74 14 .3 36.8 240- 62.0 1.26 1 4 . 6 34.6 270 55.0 1.69 15.9 43 .1 300 55.0 2.24 15.7 44.0 330 56.0 2.27 15.3 46.2 360 '.' 60,0 2.20 16.2 43 .4 >an ± SE 63.96 ± 2 .81 14*7 ± 0 . 2 9 B) Metabolic Parameters Irreversible Loss (mM/hr) % Conversion to Glucose 7.23 5.07 T a b l e 9 . Data and M e t a b o l i c Parameters o f A l a n i n e F o l l o w i n g A Continuous I n f u s i o n o f 1 4 C ~ A l a n i n e . Experiment C.2 A) Data C a r o t i d A r t e r y C o l l e c t i o n Time (min a f t e r i n j e c t i o n ) G l u c o s e Concen- S p e c i f i c t r a t i o n (mg/100ml) A c t i v i t y (nC/mg) A l a n i n e Concen- S p e c i f i c t r a t i o n A c t i v i t y (ug/ml) (nC/mg) 1 5 8 c o n t r o l 55.0 30 62.0 60 48.0 90 • 53.5 120 53.5 150 53.5 180 53.5 240 50.0 300 46.5 360 51.0 390 56.O 405 48.0 420 46.4 435 53.0 450 51.0 mean ± SE 52.06 ± 1 . 0 6 - 13.8 — 0.11 13.0 0.615 0.335 12.0 • 2.50 0.323 15.4 8.57 0.33 14.4 17.92 0.38 14.9 15.37 0.47 14.7 31.97 0.86 13.4 39 o70 1.67 14.3 34.75 2.43 15.2 34.56 2.46 14.5 34.21 2.37' .14.8 34.41 1.76 14.0 28.70 1.33 14.2 9.36 0.66 14.0 14.40 ± 0 . 2 6 5.57 T a b l e 9 . c o n t ' d A) Data P o r t a l V e i n C o l l e c t i o n Time (min a f t e r i n j e c t i o n ) Concen~ t r a t i o n (mg/lOOml) Glu c o s e S p e c i f i c A c t i v i t y (nC/rag) A l a n i n e Concen- S p e c i f i c t r a t i o n (ug/ml) A c t i v i t y (nC/mg) C o n t r o l 5 3 . 0 - 1 4 . 4 -3 0 4 6 . 5 0 . 0 9 7 1 4 . 0 3 . 0 7 6 0 4 8 o 0 0 . 3 0 1 3 . 8 2 . 6 1 9 0 5 2 . 0 0 o 5 4 1 5 . 3 1 5 . 7 1 2 0 5 0 oO 0 . 4 1 1 5 . 8 1 0 . 0 1 5 0 5 2 o 0 0 o 4 0 1 4 . 5 1 4 . 6 1 8 0 4 8 . 0 0 . 9 1 3 1 5 . 5 2 4 . 7 ' 2 4 0 4 7 . 0 l c 5 2 1 5 . 0 3 1 . 8 3 0 0 4 6 . 5 1 „ 7 5 1 4 . 9 3 5 . 2 3 6 0 4 9 . 0 2 . 4 1 1 6 . 7 3 2 . 9 3 9 0 4 4 . 0 2 . 5 5 2 0 . 5 3 4 . 9 4 0 5 5 4 . 0 2 . 4 4 1 7 . 2 2 4 . 4 4 2 0 5 2 . 0 1 . 5 1 1 4 . 9 1 6 . 6 4 3 5 4 8 . 0 1 . 1 8 6 . 2 4 5 0 5 2 0 0 0 . 7 2 7 1 4 . 6 5 . 7 mean ~ SE 4 9 o 4 6 ± 0 . 7 7 1 5 . 8 9 ± 0 . 5 3 B) M e t a b o l i c Parameters I r r e v e r s i b l e L o ss (mM/hr) = 8 . 5 9 % Conversion' t o G l u c o s e = 5 . 0 7 Table 1 0 . Summary of Parameters from Single Injection and Continuous Infusion Experiments. Parameters of Alanine Metabolism i n Sheep F&periment Sheep number* and type WT (kg) Plasma Pool Space Alanine Size . (% of Concen- (mg) B W ) t r a t i o n (ug/ml) Irre-- Total ver s i b l e Entry Loss Rate (mM/hr) (mM/hr) Recycling Rate (mK/nr) 35 1 1 . 2 6 "5 T - 2 . 35 ' 13.59 . 9 5 s t, X«—3 37 1 0 . 7 3 .15 C.I.-4 45 : 1 4 . 7 0 . 2 9 C.I.-5 42 14 .40 . 2 6 17 5 2 , 9 13.27 7.7 3.71 7.49 4.44 mean ± SE 3 1 . 6 9 7 . 1 4 i 1 4 . 1 0 ± 3 .07 £ .Conversion to Glucose 3 . 3 4 . 5.80 2 . 4 6 3 . 5 7 5 . 1 2 9 . 7 7 4 . 6 5 5 . 0 0 6 . 7 5 3 3 . 5 2 2 6 . 7 7 6.78 7 . 2 3 - - 5 . 0 7 8 . 5 9 •. - - 7 . 2 0 6 . 2 1 16.36 11 .27 5 . 5 2 ± 0 . 9 1 i 8 . 6 5 ± 7 . 7 4 ± 0 . 6 6 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0099870/manifest

Comment

Related Items