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Lactate turnover in fast-moving vertebrates : the control of plasma metabolite fluxes Weber, Jean-Michel 1987

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LACTATE TURNOVER IN FAST-MOVING VERTEBRATES: THE CONTROL OF PLASMA METABOLITE FLUXES. By JEAN-MICHEL WEBER L i e . es S c i e n c e s , U n i v e r s i t e de N e u c h a t e l ( S w i t z e r l a n d ) , 1978 M . S c , M c G i l l U n i v e r s i t y , 1982 A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILLEMENT OF FOR THE DEGREE OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department o f Z o o l o g y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J a n u a r y 1987 Q J e a n - M i c h e l Weber, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^— L-\J \J The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i ABSTRACT D u r i n g s u s t a i n e d e x e r c i s e , w o r k i n g m u s c l e s must be s u p p l i e d w i t h a d e q u a t e k i n d s and amounts o f e x o g e n o u s f u e l s , and t h e d e l i v e r y r a t e s o f o x y g e n and o x i d i z a b l e s u b s t r a t e s s h o u l d be m a t c h e d . The s t u d y o f m e t a b o l i t e f l u x e s and t h e i r r e g u l a t i o n i s t h e r e f o r e c r i t i c a l t o t h e u n d e r s t a n d i n g o f e x e r c i s e m e t a b o l i s m . L a c t a t e h a s r e c e i v e d renewed a t t e n t i o n f r o m p h y s i o l o g i s t s and b i o c h e m i s t s w i t h t h e r e a l i z a t i o n t h a t i t i s n o t o n l y an end p r o d u c t o f g l y c o l y s i s , b u t a l s o an i m p o r t a n t f u e l f o r a e r o b i c work. As an o x i d i z a b l e f u e l , t h i s s u b s t r a t e may p r o v i d e some p e r f o r m a n c e a d v a n t a g e o v e r o t h e r f u e l s s u c h as g l u c o s e and f r e e f a t t y a c i d s . The g o a l s o f t h i s t h e s i s were: 1) t o d e t e r m i n e w h e t h e r e n d u r a n c e - a d a p t e d a n i m a l s c a n s u p p o r t h i g h e r p l a s m a l a c t a t e t u r n o v e r r a t e s t h a n s e d e n t a r y a n i m a l s ; and 2) t o i n v e s t i g a t e t h e m a j o r f a c t o r s i n v o l v e d i n t h e r e g u l a t i o n o f p l a s m a m e t a b o l i t e t u r n o v e r a t t h e w h o l e - o r g a n i s m l e v e l - u s i n g l a c t a t e as a m o d e l . L a c t a t e t u r n o v e r r a t e s were measured by b o l u s i n j e c t i o n o f 14 [U- C ] l a c t a t e i n s k i p j a c k t u n a , Katsuwonus p e l a m i s , and i n t h o r o u g h b r e d r a c e h o r s e s , Equus c a b a l l u s . I n t u n a , t u r n o v e r r a t e s r a n g e d from 112 t o 431 umol min-"'" k g ~ \ and t h e y were p o s i t i v e l y c o r r e l a t e d w i t h l a c t a t e c o n c e n t r a t i o n ( s l o p e = 15.1, r = 0.92). T h i s t e l e o s t i s a b l e t o s u p p o r t h i g h e r p l a s m a l a c t a t e t u r n o v e r r a t e s t h a n e x p e c t e d f o r a mammalian s p e c i e s o f e q u i v a l e n t s i z e , e ven t h o u g h i t o p e r a t e s a t a much l o w e r t e m p e r a t u r e , and l a c t a t e i s p r o b a b l y an i m p o r t a n t o x i d i z a b l e f u e l i n t h i s s p e c i e s . F o r c o m p a r a t i v e p u r p o s e s , r e s t i n g t u r n o v e r r a t e s o f l a c t a t e and g l u c o s e were p l o t t e d v e r s u s body mass on a , l o g - l o g s c a l e f o r a wide r a n g e o f mammalian s p e c i e s . T h e s e p l o t s were l i n e a r , and t h e y showed t h e same s l o p e as t h e c l a s s i c body mass vs m e t a b o l i c r a t e r e l a t i o n s h i p . T h o r o u g h b r e d h o r s e s a r e l i k e l y t o h ave an a e r o b i c s c o p e o f 4 0 - f o l d o r more. One o f t h e i r m a i n p h y s i o l o g i c a l a d a p t a t i o n s t o e x e r c i s e i s t h e a b i l i t y t o i n c r e a s e h e m a t o c r i t by more t h a n one and a h a l f - f o l d i n r e s p o n s e t o e x e r c i s e . I n t h e p r e s e n t s t u d y , t h i s a d j u s t m e n t a l l o w e d them t o r e a c h an A-V d i f f e r e n c e i n 0^ c o n t e n t o f more t h a n 23 v o l % d u r i n g m a x i m a l e x e r c i s e , a much h i g h e r v a l u e t h a n o t h e r mammals. T h e i r l a c t a t e t u r n o v e r r a t e and c a r d i a c o u t p u t were m e a s u r e d a t r e s t and two l e v e l s o f s u b m a x i m a l e x e r c i s e (45 and 55 V ^ m a x ) t o i n v e s t i g a t e t h e r e l a t i o n s h i p between c a r d i o v a s c u l a r a d j u s t m e n t s on p l a s m a l a c t a t e t u r n o v e r r a t e . C a r d i a c o u t p u t r a n g e d from 106 t o 571 ml m i n - 1 k g - 1 , and mean l a c t a t e t u r n o v e r r a t e f r o m 9.3 a t r e s t , t o 75.9 umol . -1 -1 min kg a t 55% V0 2max. In c o n t r a s t w i t h t h e s i t u a t i o n f o u n d i n t u n a , t h e l a c t a t e t u r n o v e r r a t e s o f t h o r o u g h b r e d s were n o t e l e v a t e d compared w i t h o t h e r mammals, s h o w i n g t h a t t h e m e t a b o l i c a d a p t a t i o n s o f t h e s e o u t s t a n d i n g a t h l e t e s do n o t i n c l u d e t h e a b i l i t y t o s u s t a i n h i g h e r l a c t a t e f l u x e s t h a n s e d e n t a r y a n i m a l s . I n h o r s e s , t h e c o n t r i b u t i o n o f i v p l a s m a l a c t a t e o x i d a t i o n t o VO^ i s m i n i m a l , and t h i s s u b s t r a t e i s n o t an i m p o r t a n t o x i d a t i v e f u e l ; l i p i d o x i d a t i o n may r e p r e s e n t t h e i r m a j o r pathway f o r a e r o b i c e n e r g y p r o d u c t i o n d u r i n g e x e r c i s e . The a b i l i t y t o o x i d i z e p l a s m a l a c t a t e a t h i g h r a t e s i s t h e r e f o r e n o t n e c e s s a r i l y r e q u i r e d f o r t h e " e l i t e " p e r f o r m a n c e o f e n d u r a n c e e x e r c i s e . T h i s s t u d y a l s o shows t h a t b o t h , p l a s m a l a c t a t e c o n c e n t r a t i o n and c a r d i a c o u t p u t a r e p o s i t i v e l y c o r r e l a t e d w i t h t u r n o v e r r a t e . The c o r r e l a t i o n between c a r d i a c o u t p u t and l a c t a t e t u r n o v e r r a t e i s i n d e p e n d e n t o f t h e r e l a t i o n s h i p between p l a s m a l a c t a t e c o n c e n t r a t i o n and t u r n o v e r r a t e . Plasma m e t a b o l i t e c o n c e n t r a t i o n and c a r d i a c o u t p u t c a n be r e g u l a t o r s o f p l a s m a m e t a b o l i t e t u r n o v e r r a t e . I t i s p r o p o s e d t h a t t h e s e two v a r i a b l e s a r e , r e s p e c t i v e l y , t h e f i n e and c o a r s e c o n t r o l s f o r f l u x r a t e a d j u s t m e n t s d u r i n g e x e r c i s e . V TABLE OF CONTENTS A b s t r a c t i i L i s t o f T a b l e s i x L i s t o f F i g u r e s x Acknowledgements x i i CHAPTER 1; GENERAL INTRODUCTION 1.1 STATEMENT OF PROBLEM 1 1.2 LACTATE METABOLISM 5 1.3 METABOLITE TURNOVER 14 1.4 THESIS OUTLINE 16 CHAPTER 2: LACTATE AND GLUCOSE TURNOVER IN SKIPJACK TUNA 2.1 INTRODUCTION 21 2.2 MATERIALS AND METHODS 23 2.2.1 EXPERIMENTAL ANIMALS 23 2.2.2 CATHETERIZATION .. 24 2.2.3 INJECTION OF LABELED METABOLITES AND BLOOD SAMPLING 26 2.2.4 METABOLITE ASSAYS AND COUNTING 26 v i 2.2.5 TERMINOLOGY, CALCULATIONS, AND STATISTICS . 30 2.3 RESULTS 34 2.3.1 LACTATE TURNOVER 34 2.3.2 GLUCOSE TURNOVER 41 2.4 DISCUSSION 46 CHAPTER 3: ONSET OF SUBMAXIMAL EXERCISE IN THOROUGHBRED HORSES 3.1 INTRODUCTION 59 3.2 MATERIALS AND METHODS 62 3.2.1 EXPERIMENTAL ANIMALS AND CATHETERIZATION .. 62 3.2.2 EXERCISE PROTOCOLS 63 3.2.3 BLOOD AND TISSUE SAMPLING 63 3.2.4 METABOLITE ASSAYS 65 3.3 RESULTS 66 3.3.1 TROT PROTOCOL 66 3.3.2 CANTER PROTOCOL 7 0 3.3.3 MUSCLE METABOLITE CONCENTRATIONS 71 3.4 DISCUSSION 76 3.4.1 CHANGES IN HEMATOCRIT DURING EXERCISE 77 3.4.2 PLASMA LACTATE CONCENTRATION AT THE START OF EXERCISE 78 3.4.3 LACTATE FLUXES TO AND FROM THE PLASMA 79 3.4.4 MUSCLE METABOLITES 83 v i i CARDIAC OUTPUT AND OXYGEN CONSUMPTION OF EXERCISING CHAPTER 4; THOROUGHBRED HORSES 4.1 INTRODUCTION 86 4.2 MATERIALS AND METHODS 88 4.2.1 ANIMALS 88 4.2.2 CATHETERIZATIONS 88 4.2.3 EXERCISE PROTOCOLS 89 4.2.4 CARDIAC OUTPUT AND BLOOD OXYGEN CONTENT 90 4.2.5 CALCULATIONS AND STATISTICS 93 4.3 RESULTS 93 4.3.1 CARDIAC PARAMETERS 93 4.3.2 A-V DIFFERENCES IN OXYGEN CONTENT 99 4.3.3 OXYGEN CONSUMPTION 100 4.4 DISCUSSION 105 4.4.1 CARDIAC OUTPUT 105 4.4.2 A-V DIFFERENCE IN OXYGEN CONTENT 107 4.4.3 METABOLIC RATE 109 CHAPTER 5; LACTATE TURNOVER IN EXERCISING THOROUGHBRED HORSES: EFFECT OF CHANGES IN CARDIAC OUTPUT 5.1 INTRODUCTION 112 v i i i 5.2 MATERIALS AND METHODS 114 5.2.1 ANIMALS AND CATHETERIZATIONS 114 5.2.2 EXERCISE PROTOCOLS 115 5.2.3 MEASUREMENT OF LACTATE TURNOVER 116 5.2.4 CALCULATIONS AND STATISTICS 119 5.3 RESULTS 120 5.4 DISCUSSION 136 5.4.1 LACTATE TURNOVER RATE AND ROLE OF PLASMA LACTATE AS AN OXIDATIVE FUEL 137 5.4.2 LACTATE CLEARANCE RATE 141 5.4.3 EFFECT OF PLASMA METABOLITE CONCENTRATION ON TURNOVER RATE 14 2 5.4.4 EFFECT OF CHANGES IN CARDIAC OUTPUT 143 5.4.5 CONTROL OF EXOGENOUS FUEL SUPPLY 145 5.4.6 CONCLUSIONS 146 REFERENCES 149 i x L I S T OF TABLES 1. T i s s u e m e t a b o l i t e c o n c e n t r a t i o n s i n c o n t r o l g r o u p o f s k i p j a c k t u n a 35 2. B l o o d m e t a b o l i t e c o n c e n t r a t i o n s and l a c t a t e t u r n o v e r r a t e s i n s k i p j a c k t u n a 36 3. B l o o d g l u c o s e c o n c e n t r a t i o n and g l u c o s e t u r n o v e r r a t e i n s k i p j a c k t u n a 43 4. W e i g h t , age, and r e s t i n g v a l u e s f o r p l a s m a l a c t a t e c o n c e n t r a t i o n , h e a r t r a t e , and h e m a t o c r i t i n f o u r t r a i n e d t h o r o u g h b r e d h o r s e s , 67 5. S t a t i s t i c a l a n a l y s i s o f t h e d e c l i n e i n h e m a t o c r i t a f t e r 3-4 min o f t r e a d m i l l e x e r c i s e i n t h o r o u g h b r e d s 72 \ 6. M e t a b o l i t e c o n c e n t r a t i o n s b e f o r e and a f t e r a 15 min c a n t e r i n t h e m i d d l e g l u t e a l m u s c l e o f t h o r o u g h b r e d h o r s e s r u n n i n g on a t r e a d m i l l 75 7. L a c t a t e t u r n o v e r r a t e , l a c t a t e c l e a r a n c e r a t e , and c a r d i a c o u t p u t o f t h o r o u g h b r e d h o r s e s a t r e s t and d u r i n g e x e r c i s e 127 and 128 8. E f f e c t s o f p l a s m a l a c t a t e c o n c e n t r a t i o n and c a r d i a c o u t p u t on t h e r a t e o f l a c t a t e t u r n o v e r i n t h o r o u g h b r e d h o r s e s 134 X L I S T OF FIGURES 1. L a c t a t e t u r n o v e r measurement i n s k i p j a c k t u n a 38 2. R e l a t i o n s h i p between l a c t a t e t u r n o v e r r a t e and l a c t a t e c o n c e n t r a t i o n i n s k i p j a c k t u n a 40 3. G l u c o s e t u r n o v e r measurement i n s k i p j a c k t u n a 45 4. R e l a t i o n s h i p between body mass and l a c t a t e t u r n o v e r .52 5. R e l a t i o n s h i p between body mass and g l u c o s e t u r n o v e r .56 6. Plasma l a c t a t e c o n c e n t r a t i o n , h e a r t r a t e , and h e m a t o c r i t i n t h o r o u g h b r e d s d u r i n g a 40-min t r o t ....69 7. Plasma l a c t a t e c o n c e n t r a t i o n , h e a r t r a t e , and h e m a t o c r i t i n t h o r o u g h b r e d s d u r i n g a 15-min c a n t e r ..74 8. Model s h o w i n g c h a n g e s i n l a c t a t e R g and R^ d u r i n g t h e t r a n s i t i o n f r o m r e s t t o s u b m a x i m a l e x e r c i s e 82 9. C a r d i a c o u t p u t , h e a r t r a t e , and s t r o k e volume o f r e s t i n g and e x e r c i s i n g t h o r o u g h b r e d h o r s e s 96 10. B l o o d oxygen c o n t e n t i n t h o r o u g h b r e d h o r s e s , a t r e s t and d u r i n g e x e r c i s e 98 11. H e m a t o c r i t o f r e s t i n g and e x e r c i s i n g t h o r o u g h b r e d h o r s e s 102 12. Oxygen c o n s u m p t i o n o f r e s t i n g and e x e r c i s i n g t h o r o u g h b r e d h o r s e s 104 13. L a c t a t e s p e c i f i c a c t i v i t y d e c a y c u r v e i n t h e p l a s m a o f a r e s t i n g t h o r o u g h b r e d h o r s e 123 14. L a c t a t e s p e c i f i c a c t i v i t y d e c a y c u r v e o f a t h o r o u g h b r e d h o r s e a t r e s t and d u r i n g a c a n t e r .... 125 x i 15. L a c t a t e s p e c i f i c a c t i v i t y d e c a y c u r v e s o f a t h o r o u g h b r e d h o r s e a t t h e b e g i n n i n g and end o f t h e t r o t p r o t o c o l 126 16. L a c t a t e t u r n o v e r r a t e i n r e s t i n g and e x e r c i s i n g t h o r o u g h b r e d h o r s e s 131 17. R e l a t i o n s h i p between l a c t a t e t u r n o v e r r a t e and p l a s m a l a c t a t e c o n c e n t r a t i o n i n t h o r o u g h b r e d h o r s e s 133 ACKNOWLEDGEMENTS I t h a n k P e t e r and B r e n d a Hochachka f o r t h e i r c o n t i n u o u s i n t e l l e c t u a l and g a s t r o n o m i c e n c o u r a g e m e n t t h r o u g h o u t my t h e s i s . The t u n a p r o j e c t was c a r r i e d o u t w i t h t h e h e l p o f R i c h a r d W. B r i l l i n H o n o l u l u . S p e c i a l t h a n k s t o t h e Z o o l o g y D e p t . (UBC) f o r p r o v i d i n g a g r e a t e n v i r o n m e n t f o r r e s e a r c h and enough wine t o t r i g g e r t h e Newmarket e x p e d i t i o n . Wade P a r k h o u s e and G e o f f Dobson h e l p e d i n a l l t h e t h o r o u g h b r e d e x p e r i m e n t s and t h e whole e x p e d i t i o n w o u l d n o t h a v e been p o s s i b l e w i t h o u t t h e i r e f f o r t s and f r i e n d s h i p . I thank them f o r r e f u s i n g t o become b l i s t e r s on t h e h e e l o f p r o g r e s s . I n Newmarket, J o y c e Harman p e r f o r m e d a l l t h e h o r s e c a t h e t e r i z a t i o n s ; P a u l i n e F e r r i e , L o u i s e F o s t e r , and t h e e n t i r e s t a f f f r o m t h e A n i m a l H e a l t h T r u s t worked h a r d t o o e n s u r e t h e s u c c e s s o f t h e h o r s e e x p e r i m e n t s . D e r e c k Wheeldon t e m p o r a r i l y abandoned human h e a r t t r a n s p l a n t s f o r e x p e r t a s s i s t a n c e i n t h e measurement o f c a r d i a c o u t p u t i n much l a r g e r p a t i e n t s , and I t h a n k him f o r h i s h e l p . F i n a l l y , b u t most i m p o r t a n t l y , I a c c u s e H i r o k i Abe, G a y l e Brown, M i c h a e l C a s t e l l i n i , R i c h a r d Foreman, U l i Hoeger, Tom Mommsen, J e n n i f e r N e ner, Tom P e t e r s e n and R a u l S u a r e z o f p u r p o s e l y p r o v i d i n g a r g u m e n t s , e n d l e s s d i s c u s s i o n s , and u n t h i n k a b l e b i o l o g i c a l p r o b l e m s , i n a d e l i b e r a t e a t t e m p t t o g e t me o u t o f UBC as s o o n as p o s s i b l e . My t h e s i s w o u l d n e v e r h a v e been w r i t t e n w i t h o u t t h e i r e f f o r t s . T h i s work was s u p p o r t e d by an NSERC g r a n t t o P.W. H o c h a c h k a . 1 CHAPTER 1; GENERAL INTRODUCTION 1.1 STATEMENT OF PROBLEM. D u r i n g s u s t a i n e d e x e r c i s e , c o n t r a c t i n g m u s c l e s must be s u p p l i e d w i t h a d e q u a t e k i n d s and amounts o f exogenous f u e l s t o m i n i m i z e t h e u s e o f endogenous s u b s t r a t e s and d e l a y t h e o n s e t o f f a t i g u e . I n a d d i t i o n , t h e d e l i v e r y r a t e s o f o xygen and o x i d i z a b l e f u e l s t o t h e a c t i v e m u s c u l a t u r e s h o u l d be m a t c h e d . Oxygen f l u x e s h a v e been s t u d i e d t h o r o u g h l y , b u t o x i d a t i v e f u e l k i n e t i c s h a v e not,. The u n d e r s t a n d i n g o f e x e r c i s e m e t a b o l i s m depends c r i t i c a l l y on a l a r g e amount o f q u a n t i t a t i v e i n f o r m a t i o n c o n c e r n i n g t h e f l u x r a t e s o f c a r b o n f u e l s and t h e i r r e g u l a t i o n . T h i s i n f o r m a t i o n i s s t i l l q u i t e f r a g m e n t a r y b e c a u s e t h e measurement o f m e t a b o l i t e f l u x e s i s t e c h n i c a l l y d i f f i c u l t and l a b o r i n t e n s i v e . F o r t h e s e r e a s o n s , a d e c i s i o n had t o be made t o l i m i t t h i s s t u d y t o t h e i n v e s t i g a t i o n o f one m e t a b o l i t e ( l a c t a t e ) , and two e x p e r i m e n t a l s p e c i e s ( t u n a and h o r s e ) . The g o a l s o f t h i s t h e s i s a r e : 1) t o d e t e r m i n e l a c t a t e t u r n o v e r r a t e s i n two s p e c i e s a d a p t e d f o r f a s t l o c o m o t i o n , and t o compare t h e i r a b i l i t y t o move t h i s m e t a b o l i t e t h r o u g h t h e i r p l a s m a compartment ( f r o m s i t e s o f p r o d u c t i o n t o s i t e s o f u t i l i z a t i o n ) w i t h t h e c o r r e s p o n d i n g a b i l i t y o f more s e d e n t a r y 2 s p e c i e s , and 2) t o i n v e s t i g a t e t h e m a j o r f a c t o r s i n v o l v e d i n t h e r e g u l a t i o n o f w h o l e - o r g a n i s m p l a s m a m e t a b o l i t e t u r n o v e r -u s i n g l a c t a t e as a m o d e l . B e f o r e g i v i n g some b a c k g r o u n d i n f o r m a t i o n c o n c e r n i n g l a c t a t e m e t a b o l i s m s t u d i e s and t h e measurement o f l a c t a t e f l u x e s ir\ v i v o , a more d e t a i l e d e x p l a n a t i o n o f t h e aims o f t h i s work w i l l be p r e s e n t e d i n t h e n e x t few p a r a g r a p h s . The f i r s t g o a l o f t h i s t h e s i s (#1 c i t e d above) d e a l s w i t h t h e a b i l i t y o f a n i m a l s a d a p t e d f o r f a s t e n d u r a n c e l o c o m o t i o n t o s u p p o r t h i g h l a c t a t e f l u x e s i n t h e i r p l a s m a . Do s u c h a n i m a l s s u p p o r t h i g h e r p l a s m a l a c t a t e f l u x e s t h a n s e d e n t a r y o r g a n i s m s ? A n i m a l s a b l e t o s u p p o r t e n d u r a n c e - t y p e e x e r c i s e o v e r l o n g p e r i o d s o f t i m e , and a t m o d e r a t e l y h i g h s p e e d s , w o u l d be e x p e c t e d t o be a b l e t o u s e o x i d i z a b l e f u e l s f r o m t h e i r p l a s m a a t h i g h r a t e s b e c a u s e , u n d e r s u c h e x e r c i s e c o n d i t i o n s , t h e i r w o r k i n g m u s c l e s o p e r a t e as open s y s t e m s , and t h e s e a c t i v e m u s c l e s h a v e t h e o p p o r t u n i t y t o o x i d i z e b l o o d - b o r n e s u b s t r a t e s i n a d d i t i o n t o endogenous ( i n t r a m u s c u l a r ) f u e l s . The d e p l e t i o n o f endogenous f u e l s h as been shown t o be one o f t h e l i m i t i n g f a c t o r s f o r e n d u r a n c e e x e r c i s e , and "good" e n d u r a n c e p e r f o r m e r s c o u l d h a v e t h e a b i l i t y t o u t i l i z e exogenous f u e l s a t h i g h e r r a t e s t h a n s e d e n t a r y a n i m a l s t o s l o w down t h e u s e o f i n t r a m u s c u l a r f u e l s i n t h e i r o x i d a t i v e m u s c l e f i b e r s . B e c a u s e p l a s m a l a c t a t e c a n be a v e r y good o x i d i z a b l e f u e l f o r many t i s s u e s ( o x i d a t i v e m u s c l e f i b e r s i n p a r t i c u l a r ) , t h e l a c t a t e t u r n o v e r r a t e o f e n d u r a n c e p e r f o r m e r s c o u l d be h i g h e r t h a n i n s e d e n t a r y s p e c i e s t h a t r a r e l y e x e r c i s e l o n g enough t o d e p l e t e t h e i r 3 endogenous f u e l s t o r e s . R e c e n t l y , t h e i m p o r t a n c e o f l a c t a t e as an o x i d i z a b l e f u e l d u r i n g e x e r c i s e h a s been e m p h a s i z e d by a number o f r e s e a r c h e r s i n v e s t i g a t i n g w h o l e - b o d y m e t a b o l i s m o f r a t s , d o g s , and humans ( f o r example s e e B r o o k s , 1985; I s s e k u t z e t a l , 1976; O k a j i m a e t al, 1 9 8 1 ) . R e s u l t s from G e o r g e s B r o o k s l a b o r a t o r y , i n p a r t i c u l a r (see B r o o k s , 1985 f o r a b r i e f summary), show t h a t l a c t a t e c o u l d be an i d e a l o x i d i z a b l e f u e l f o r e x e r c i s i n g a n i m a l s b e c a u s e i t c a n be t r a n s p o r t e d a t h i g h r a t e s t o w o r k i n g m u s c l e s . I t c a n be m o b i l i z e d f a s t e r t h a n p l a s m a f r e e f a t t y a c i d s , and a n i m a l s c a n s u s t a i n h i g h e r t u r n o v e r r a t e s o f l a c t a t e t h a n g l u c o s e o r f r e e f a t t y a c i d s . B e c a u s e l a c t a t e c o u l d p r o v i d e some a d v a n t a g e s o v e r o t h e r o x i d i z a b l e f u e l s f o u n d i n p l a s m a , good e n d u r a n c e p e r f o r m e r s may u s e l a c t a t e a t a h i g h e r r a t e t h a n s e d e n t a r y a n i m a l s f o r a g i v e n l e v e l o f work, and s u c h " a n i m a l a t h l e t e s " w o u l d t h e r e f o r e h a v e t o be a b l e t o t r a n s p o r t t h i s s u b s t r a t e r a p i d l y t h r o u g h t h e i r p l a s m a compartment. Tuna and t h o r o u g h b r e d h o r s e s were c h o s e n as two e x a mples o f " e l i t e a n i m a l a t h l e t e s " r e p r e s e n t i n g two w i d e l y d i f f e r e n t g r o u p s o f o r g a n i s m s t o i n v e s t i g a t e t h i s a s p e c t o f t h e p r o b l e m . T h i s t h e s i s shows t h a t , as e x p e c t e d f o r an e n d u r a n c e a t h l e t e , t u n a c a n s u p p o r t h i g h l a c t a t e t u r n o v e r r a t e s . In c o n t r a s t , t h o r o u g h b r e d h o r s e s do n o t show s u c h an a b i l i t y , and t h e u s e o f l a c t a t e as an o x i d i z a b l e f u e l i n t h i s s p e c i e s i s n o t i m p o r t a n t . The s e c o n d g o a l o f t h i s s t u d y (#2 c i t e d above) i s t o c h a r a c t e r i z e t h e m a j o r ways u s e d by v e r t e b r a t e s t o a l t e r 4 t h e f l u x r a t e s o f o x i d i z a b l e f u e l s t o t h e i r w o r k i n g m u s c l e s i n r e s p o n s e t o submaximal e x e r c i s e a t t h e w h o l e - o r g a n i s m  l e v e l . The aim o f t h e s t u d y i s n e i t h e r t o d e t e r m i n e what l i m i t s m aximal l a c t a t e f l u x r a t e s i n p l a s m a , no r t o i n v e s t i g a t e d i r e c t l y t h e r e g u l a t i o n o f l a c t a t e p r o d u c t i o n and u t i l i z a t i o n a t t h e c e l l u l a r l e v e l . I n s t e a d , t h e f o l l o w i n g q u e s t i o n i s a s k e d : - How do a n i m a l s s u p p o r t d i f f e r e n t s t e a d y - s t a t e r a t e s o f p l a s m a s u b s t r a t e t u r n o v e r i n r e s p o n s e t o d i f f e r e n t l e v e l s o f s u s t a i n a b l e p h y s i c a l a c t i v i t y ? I t i s h y p o t h e s i z e d t h a t an i n c r e a s e i n p l a s m a l a c t a t e c o n c e n t r a t i o n s h o u l d a l l o w them t o augment t h e r a t e o f l a c t a t e s u p p l y t o o x i d a t i v e m u s c l e s , t h e r e f o r e , l a c t a t e c o n c e n t r a t i o n s h o u l d be c o r r e l a t e d w i t h t h e r a t e o f p l a s m a l a c t a t e t u r n o v e r . M a i n t a i n i n g d i f f e r e n t s t e a d y - s t a t e . l a c t a t e l e v e l s i n t h e i r p l a s m a s h o u l d a l l o w t h e a n i m a l t o d e t e r m i n e d i f f e r e n t r a t e s o f l a c t a t e d e l i v e r y r a t e t o t h e w o r k i n g m u s c l e s ( f o r a g i v e n r a t e o f m u s c l e p e r f u s i o n ) i n r e s p o n s e t o d i f f e r e n t r a t e s o f e x e r c i s e . The e f f e c t o f c h a n g e s i n p l a s m a c o n c e n t r a t i o n on l a c t a t e t u r n o v e r r a t e w o u l d be most c l e a r l y d e m o n s t r a t e d i n a s p e c i e s s h o w i n g a v e r y w i de r a n g e o f l a c t a t e c o n c e n t r a t i o n s u n d e r n a t u r a l c o n d i t i o n s . Tuna was c h o s e n f o r t h i s p u r p o s e (Chap. 2) b e c a u s e t h i s t e l e o s t h a s t h e a b i l i t y t o b r i n g i t s p l a s m a l a c t a t e c o n c e n t r a t i o n s t o v a l u e s e x c e e d i n g 30 mM (see C h a p t e r 2, and Guppy e t a l . 1 9 7 9 ) . An i n c r e a s e i n c a r d i a c o u t p u t s h o u l d a l s o a l l o w a n i m a l s t o augment t h e s u p p l y r a t e o f l a c t a t e t o t h e i r w o r k i n g m u s c l e s by i n c r e a s i n g p e r f u s i o n i n l a c t a t e - u t i l i z i n g t i s s u e s d u r i n g e x e r c i s e . A l m o s t a l l t h e i n c r e a s e i n c a r d i a c 5 o u t p u t e l i c i t e d by e x e r c i s e goes t o w o r k i n g m u s c l e s , and t h e o x i d a t i v e f i b e r s o f t h e s e m u s c l e s s h o u l d be b e t t e r s u p p l i e d w i t h o x i d i z a b l e s u b s t r a t e s ( i n c l u d i n g l a c t a t e ) when c a r d i a c o u t p u t i s augmented. T h e r e f o r e , c a r d i a c o u t p u t s h o u l d a l s o be c o r r e l a t e d w i t h l a c t a t e t u r n o v e r r a t e , and a change i n c a r d i a c o u t p u t s h o u l d m o d i f y t h e s l o p e o f t h e r e l a t i o n s h i p b etween l a c t a t e t u r n o v e r r a t e and p l a s m a l a c t a t e c o n c e n t r a t i o n . Tuna does n o t r e p r e s e n t a good e x p e r i m e n t a l a n i m a l t o i n v e s t i g a t e t h i s p r o b l e m b e c a u s e t h e c a r d i a c o u t p u t o f t h i s f i s h c a n n o t be m a n i p u l a t e d e x p e r i m e n t a l l y u n d e r c o n t r o l l e d c o n d i t i o n s , and i t i s d i f f i c u l t t o measure b l o o d f l o w a c c u r a t e l y i n t h i s s p e c i e s . A mammalian model i n w h i c h c a r d i a c o u t p u t c a n be a l t e r e d o v e r a wide r a n g e o f v a l u e s w o u l d be more a p p r o p r i a t e , and t h e t h o r o u g h b r e d r a c e h o r s e was c h o s e n as an e x p e r i m e n t a l a n i m a l t o i n v e s t i g a t e t h e e f f e c t o f c h a n g e s i n wh o l e - b o d y p e r f u s i o n r a t e ( i . e . c h a n g e s i n c a r d i a c o u t p u t ) on p l a s m a m e t a b o l i t e t u r n o v e r r a t e (Chap. 4 and 5 ) . More s p e c i f i c g o a l s f o r e a c h p a r t o f t h i s s t u d y w i l l be g i v e n i n t h e r e s p e c t i v e i n t r o d u c t i o n s t o t h e d i f f e r e n t c h a p t e r s . 1.2 LACTATE METABOLISM L a c t a t e has o c c u p i e d a p r o m i n e n t p o s i t i o n i n t h e s t u d y o f m u s c u l a r a c t i v i t y f o r a l o n g t i m e . I t s p r o d u c t i o n by w o r k i n g m u s c l e p r o v i d e s a means f o r ATP s y n t h e s i s t h r o u g h t h e g l y c o l y t i c pathway. T h i s c a n o c c u r w i t h o u t t h e p r e s e n c e of o x y g e n , w i t h t h e n e t f o r m a t i o n o f 2 ATP m o l e c u l e s p e r 6 g l u c o s e , and 3 ATP p e r g l y c o s y l u n i t u t i l i z e d (Mole, 1 9 8 3 ) . The p r i m a r y s u b s t r a t e f o r l a c t a t e p r o d u c t i o n i n m u s c l e i s g l u c o s y l u n i t s d e r i v e d f r o m i n t r a m u s c u l a r g l y c o g e n s t o r e s . The main o r i g i n o f l a c t a t e i s most c l e a r l y d e m o n s t r a t e d i n e x p e r i m e n t s d e a l i n g w i t h p a t i e n t s a f f l i c t e d w i t h a m e t a b o l i c d i s o r d e r c a l l e d M c A r d l e ' s d i s e a s e . T h e s e p a t i e n t s l a c k t h e enzyme g l y c o g e n p h o s p h o r y l a s e i n t h e i r s k e l e t a l m u s c l e s , and t h e y c a n n o t u t i l i z e m u s c l e g l y c o g e n as a s u b s t r a t e f o r e n e r g y m e t a b o l i s m d u r i n g work (Lewis and H a l l e r , 1986) . The m u s c l e s o f t h e s e p a t i e n t s do n o t p r o d u c e l a c t a t e , e v e n u n d e r maximal e x e r c i s e c o n d i t i o n s . U n t i l r e c e n t l y , t h e i n v e s t i g a t i o n o f l a c t a t e m e t a b o l i s m i_n v i v o d u r i n g e x e r c i s e r e l i e d s o l e l y on c o n c e n t r a t i o n measurements, and . i n f o r m a t i o n c o n c e r n i n g l a c t a t e f l u x e s was l a c k i n g . Over t h e l a s t d e c a d e , f l u x measurements have become more w i d e l y a v a i l a b l e v i a t r a c e r t e c h n i q u e s , and a new p i c t u r e o f l a c t a t e m e t a b o l i s m has emerged. I n p a r t i c u l a r , m e t a b o l i c t r a c e r s h a v e a l l o w e d t o q u a n t i f y t h e r a t e s o f l a c t a t e p r o d u c t i o n and u t i l i z a t i o n a t t h e whole o r g a n i s m l e v e l , s o m e t h i n g t h a t had n o t been p o s s i b l e w i t h t h e u s e o f l a c t a t e c o n c e n t r a t i o n measurements o n l y . M e t a b o l i c t r a c e r t e c h n i q u e s h a v e p e r m i t t e d t o c l e a r l y d e m o n s t r a t e t h a t l a c t a t e was not s i m p l y an e n d - p r o d u c t o f g l y c o l y s i s ( i t s u n i q u e and t r a d i t i o n a l r o l e f o r v e r y l o n g ) , b u t t h a t t h i s m e t a b o l i t e was a l s o a v e r y i m p o r t a n t o x i d i z a b l e f u e l f o r m u s c l e a t r e s t , and d u r i n g s ubmaximal e x e r c i s e . S e v e r a l r e c e n t s t u d i e s have e m p h a s i z e d t h i s s e c o n d r o l e o f l a c t a t e (see f o r example B r o o k s , 1985; E l d r i d g e , 1974; 7 I s s e k u t z e t a_l, 1976; J o r f e l d t , 1970) . The modern s t u d y o f l a c t a t e m e t a b o l i s m h a s been r e v i e w e d by s e v e r a l a u t h o r s ( t h e b e s t a c c o u n t s c a n be f o u n d i n D i P r a m p e r o , 1981; Hultman and S a h l i n , 1980; K r e b s e t a l , 1975; M i n a i r e , 1973; Mole, 1983; M o r e t e_t a l , 1980). However, no u p d a t e d d i s c u s s i o n o f t u r n o v e r d a t a i s p r e s e n t e d i n t h e s e p a p e r s . B e f o r e d e s c r i b i n g i n more d e t a i l t h e f i n d i n g s and t h e s i g n i f i c a n c e o f r e c e n t work, t h e h i s t o r y o f l a c t a t e m e t a b o l i s m s t u d i e s w i l l be r e v i e w e d . The s t u d y o f l a c t a t e s t a r t e d i n t h e 18th c e n t u r y when t h i s m e t a b o l i t e was f i r s t i s o l a t e d f r o m s o u r m i l k . The o r i g i n a l p r o c e d u r e u s e d a t t h a t t i m e was f i r s t d e s c r i b e d by S c h e e l e ( s e e S c h e e l e , 1786 f o r an e n g l i s h t r a n s l a t i o n o f t h e o r i g i n a l p a p e r p u b l i s h e d i n S w e d i s h i n 1 780). In 1807, B e r z e l i u s i d e n t i f i e d l a c t a t e as a p r o d u c t o f m u s c l e c o n t r a c t i o n (see Needham, 1 9 7 1 ) . However, B e r z e l i u s d i d n o t c o n t i n u e h i s s t u d y o f c h e m i c a l c h a n g e s i n m u s c l e t i s s u e , and t h e c a u s e o f l a c t a t e f o r m a t i o n i n m u s c l e s d u r i n g c o n t r a c t i o n c o u l d n o t be d e t e r m i n e d t h e n . E i g h t y y e a r s l a t e r , and i n d e p e n d e n t l y from e a c h o t h e r , G a g l i o ( 1 8 8 6 ) , and B e r l i n e r b l a u (1887) i s o l a t e d l a c t i c a c i d f r o m l i v i n g t i s s u e s as z i n c l a c t a t e , and t h e y m easured i t s c o n c e n t r a t i o n i n t h e b l o o d o f r e s t i n g dogs f o r t h e f i r s t t i m e . I n t h e l a t e 1800's, i t t o o k a minimum o f 85 ml o f b l o o d , b u t p r e f e r a b l y 100 t o 200 ml, t o make one l a c t a t e c o n c e n t r a t i o n measurement! S e v e r a l d a y s were n e c e s s a r y t o p e r f o r m one a n a l y s i s . Today, t h a n k s t o t h e a n a l y t i c a l use o f p u r e enzymes, 0.1 ml o f b l o o d or l e s s , and a few m i n u t e s , a r e s u f f i c i e n t t o g i v e a c c u r a t e 8 v a l u e s (Krebs e t a_l, 1 9 7 5 ) . A f t e r l a c t a t e was d i s c o v e r e d and i s o l a t e d , r e s e a r c h e r s s t a r t e d t o i n v e s t i g a t e t h e s p e c i f i c c o n d i t i o n s u n d e r w h i c h i t i s p r o d u c e d by m u s c l e . A r a k i was one o f t h e f i r s t p h y s i o l o g i s t s t o s t u d y t h e s e c o n d i t i o n s i n i n t a c t a n i m a l s . He showed t h a t l a c t a t e c o n c e n t r a t i o n was i n c r e a s e d i n d o g s , r a b b i t s , and hens s u b j e c t e d t o h y p o x i a ( A r a k i , 1 8 9 1 ) . I n c r e a s e d l a c t a t e f o r m a t i o n u n d e r i m p a i r e d o x y g e n s u p p l y c o n d i t i o n s was c o n f i r m e d by s e v e r a l o t h e r a u t h o r s i n d i f f e r e n t s p e c i e s . I n 1920, M e y e r h o f and H i l l p r o p o s e d t h e i r c o n c e p t o f t h e "oxygen d e b t " and d e m o n s t r a t e d t h a t s t i m u l a t e d f r o g m u s c l e shows a d e c r e a s e i n g l y c o g e n w h i l e l a c t a t e i s b e i n g p r o d u c e d ( M e y e r h o f , 1 9 2 0 ) . They d e d u c e d t h a t l a c t a t e f o r m a t i o n was an i n d i c a t i o n o f o x y g e n l a c k c a u s e d by e x e r c i s e , and t h a t t h e r e m o v a l o f l a c t a t e d u r i n g r e c o v e r y was t h e d i r e c t c a u s e o f p o s t - e x e r c i s e e x c e s s o x y g e n c o n s u m p t i o n . H i l l and M e y e r h o f ( s e e M e y e r h o f , 1930) showed a few y e a r s l a t e r t h a t l a c t a t e was t h e f i n a l p r o d u c t o f g l y c o l y s i s . U n t i l t h e 1930's, l a c t a t e f o r m a t i o n was assumed t o be n e c e s s a r y f o r t h e c o n v e r s i o n o f c h e m i c a l e n e r g y t o m e c h a n i c a l e n e r g y i n c o n t r a c t i n g m u s c l e s , and t h e r o l e o f ATP as immediate s o u r c e o f c h e m i c a l e n e r g y had n o t y e t been d i s c o v e r e d . A few y e a r s l a t e r , s k e l e t a l m u s c l e s were shown t o r e l e a s e l a c t a t e i n t h e r e s t i n g s t a t e (Kramer e_t a_l, 1939) . Some a t t e n t i o n was f o c u s s e d a r o u n d t h e p r o b l e m o f l a c t a t e movements between m u s c l e and b l o o d . H i l l , Long and L u p t o n (1924) h y p o t h e s i z e d t h a t m u s c l e and b l o o d l a c t a t e c o n c e n t r a t i o n s s h o u l d be e q u a l u n d e r s t e a d y - s t a t e e x e r c i s e c o n d i t i o n s . S e v e r a l r e s e a r c h e r s t r i e d t o measure l a c t a t e 9 c o n c e n t r a t i o n i n m u s c l e and b l o o d s i m u l t a n e o u s l y d u r i n g s u b m a x i m a l e x e r c i s e , b u t t h e y u s u a l l y f a i l e d t o f i n d s i m i l a r c o n c e n t r a t i o n s i n b o t h t i s s u e s (see f o r example S a c k s and S a c k s , 1 9 3 7 ) . Most s t u d i e s c o n c l u d e d t h a t l a c t a t e was n o t i n e q u i l i b r i u m between m u s c l e and b l o o d . However, i t was n o t c l e a r w h e t h e r t h e e l e v a t e d l a c t a t e c o n c e n t r a t i o n s f o u n d i n m u s c l e t i s s u e were n o t s i m p l y due t o t h e d i f f e r e n t s a m p l i n g p r o c e d u r e s u t i l i z e d f o r b l o o d and m u s c l e . In l i g h t o f t h e i r r e s u l t s , S a c k s and S a c k s (1937) c r i t i c i z e d t h e a p p r o a c h u s e d by M a r g a r i a ' s g r o u p . A l r e a d y t h e n , t h e y r e c o g n i z e d t h a t i t was i m p o s s i b l e t o q u a n t i f y t h e r a t e o f l a c t a t e p r o d u c t i o n f r o m c h a n g e s i n b l o o d l a c t a t e c o n c e n t r a t i o n as M a r g a r i a h a d done i n h i s s t u d i e s . Net r e l e a s e ( e s t i m a t e d f r o m A-V d i f f e r e n c e s i n l a c t a t e c o n c e n t r a t i o n ) a p p e a r e d t o i n c r e a s e a p p r o x i m a t e l y e x p o n e n t i a l l y w i t h e x e r c i s e i n t e n s i t y (Bang, 1936; Wahren, 1966; Wahren and H a g e n f e l d t , 1 9 6 8 ) . Bang (1936) showed t h a t l a c t a t e c o n c e n t r a t i o n went t h r o u g h a t r a n s i e n t i n c r e a s e a t t h e b e g i n n i n g o f s u s t a i n e d s ubmaximal e x e r c i s e b e f o r e d e c r e a s i n g b a c k t o a s t e a d y v a l u e . A l r e a d y a t t h e b e g i n n i n g o f t h e c e n t u r y , t h e r e was some i n d i c a t i o n s t h a t oxygen l a c k was n o t a r e q u i r e d c o n d i t i o n f o r t h e f o r m a t i o n o f l a c t a t e . F o r example, t h e o r a l a d m i n i s t r a t i o n o f s u c r o s e t o human s u b j e c t s was shown t o i n c r e a s e b l o o d l a c t a t e c o n c e n t r a t i o n ( C o l l a z o and L e w i c k i , 1 9 2 5 ) . The same phenomenon was o b s e r v e d a f t e r g l u c o s e i n f u s i o n i n r a t s ( C o r i and C o r i , 1 9 2 9). D i l l e t a l (1932) o b s e r v e d t h a t t h e a c c u m u l a t i o n o f l a c t a t e was n o t n e c e s s a r i l y c a u s e d by low oxygen a v a i l a b i l i t y . D e s p i t e t h e s e e a r l y 10 r e s u l t s , t h e p r o d u c t i o n o f l a c t a t e has been a s s o c i a t e d a l m o s t a u t o m a t i c a l l y w i t h o x y g e n l a c k by a l a r g e number o f p e o p l e i n c l u d i n g s e v e r a l c o n t e m p o r a r y r e s e a r c h e r s . L a c t a t e p r o d u c t i o n i s now known t o o c c u r i n t h e p r e s e n c e as w e l l as t h e a b s e n c e o f oxygen (see f o r example C o n n e t t e t a_l, 1984; J o b s i s and S t a i n s b y , 1 9 6 8 ) . R e c e n t r e s u l t s f r o m t r a c e r k i n e t i c s e x p e r i m e n t s h a ve c o n f i r m e d t h a t l a c t a t e c a n be p r o d u c e d a t h i g h r a t e s i n t h e p r e s e n c e o f o x y g e n ( B r o o k s and Donovan, 1983; Depocas e t a_l, 1969; Donovan and B r o o k s , 1983; E l d r i d g e e_t a l , 1974; F r e m i n e t e t al_, 1972; F r e m i n e t e t a l , 1974; I s s e k u t z e t a l , 1976; I s s e k u t z , 1 9 84). As i n d i c a t e d p r e v i o u s l y , r e c e n t t r a c e j : s t u d i e s a l l o w e d t o q u a n t i f y t h e r o l e o f l a c t a t e as an o x i d i z a b l e f u e l . However, t h i s r o l e had been r e c o g n i z e d e a r l i e r i n r e s t i n g m u s c l e by a number o f r e s e a r c h e r s who o b s e r v e d t h a t , d u r i n g e x e r c i s e , t h e m u s c l e s t h a t were n o t i n v o l v e d i n a c t i v e work had t h e a b i l i t y t o u t i l i z e l a c t a t e ( B a r r and Himwich, 1923; C a r l s o n and Pernow, 1959; M a r g a r i a e t a_l, 1933) . I t was l a t e r m e a s u r e d t h a t a c t i v e r e c o v e r y ( a t low e x e r c i s e i n t e n s i t i e s ) was more e f f e c t i v e i n r e m o v i n g l a c t a t e f r o m t h e b l o o d t h a n r e s t i n g r e c o v e r y ( A h l b o r g et. a_l, 1976; Bang, 1936; J o r f e l d t , 1970; M c G r a i l e t a l , 1 9 7 8 ) . A l s o , s t u d i e s c o n d u c t e d d u r i n g t h e s e v e n t i e s showed t h a t a l l m u s c l e s ( a c t i v e and r e s t i n g ) had an i n c r e a s e d a b i l i t y t o t a k e up l a c t a t e f r o m th e b l o o d when t h e r e c o v e r y p e r i o d was a c t i v e ( A h l b o r g e t a l , 1976; a l s o , s e e M c G r a i l e_t a_l_, 1978) . W i t h o u t u s i n g m e t a b o l i c t r a c e r s , , t h e s e e x p e r i m e n t s a l r e a d y s u p p o r t e d t h e i d e a t h a t l a c t a t e c a n be a good s u b s t r a t e f o r e n e r g y m e t a b o l i s m i n 11 s k e l e t a l m u s c l e . Over t h e y e a r s , a l o t o f a t t e n t i o n has been d e v o t e d t o t h e i n v e s t i g a t i o n o f l a c t a t e p r o d u c t i o n and r e m o v a l a t t h e o n s e t o f e x e r c i s e , d u r i n g e x e r c i s e , and d u r i n g t h e r e c o v e r y p r o c e s s f o l l o w i n g e x e r c i s e . Many f a c t o r s i n f l u e n c e t h e r a t e and m a g n i t u d e o f t h e a c c u m u l a t i o n o f l a c t a t e i n b l o o d and m u s c l e s . T h e s e f a c t o r s i n c l u d e d i e t , s t a t e o f p h y s i c a l f i t n e s s , and t h e t y p e and d u r a t i o n o f t h e e x e r c i s e ( G o l l n i c k e t a l , 1 9 8 6 ) . F o r v e r y l o n g , i t was t h o u g h t t h a t no l a c t a t e was p r o d u c e d a t r e s t o r d u r i n g s u b m a x i m a l e x e r c i s e b e c a u s e no l a c t a t e a c c u m u l a t i o n i n m u s c l e o r b l o o d c o u l d be m e a s u r e d u n d e r t h e s e c o n d i t i o n s . M a r g a r i a (1976) was s t i l l s u p p o r t i n g t h i s v i e w r e c e n t l y . Wasserman and c o - w o r k e r s p r o p o s e d t h e c o n c e p t o f " a n a e r o b i c t h r e s h o l d " (Wasserman e_t aul, 1973; Wasserman e_t a_l, 1981) t o e x p l a i n t h e sudden i n c r e a s e i n b l o o d l a c t a t e c o n c e n t r a t i o n o b s e r v e d when a u g m e n t i n g work beyond a b o u t 55 t o 60% maximal o x y g e n c o n s u m p t i o n . T h i s c o n c e p t h a s been c h a l l e n g e d o v e r t h e l a s t few y e a r s b e c a u s e t h e o b s e r v e d t h r e s h o l d does n o t i n d i c a t e t h e sudden o n s e t o f l a c t a t e p r o d u c t i o n due t o a n a e r o b i o s i s (see f o r example B r o o k s and Fahey, 1984). S e v e r a l l i n e s o f e v i d e n c e show t h a t l a c t a t e i s p r o d u c e d u n d e r a l l c o n d i t i o n s o f a c t i v i t y ( E l d r i d g e , 1975; I s s e k u t z e_t a l , 1976; J o r f e l d t , 1 9 7 0 ) . Even a t r e s t , t h e t i s s u e c o n c e n t r a t i o n s o f l a c t a t e a r e m a i n t a i n e d above z e r o by a b a l a n c e between p r o d u c t i o n and r e m o v a l . I n man, t h e m a j o r t i s s u e s c o n t r i b u t i n g t o l a c t a t e p r o d u c t i o n a t r e s t a r e : s k e l e t a l m u s c l e , e r y t h r o c y t e s , b r a i n , and l e u c o c y t e s (Mole, 1 9 8 3 ) . S k e l e t a l m u s c l e h a s been e s t i m a t e d 12 t o p r o d u c e a b o u t 35% o f t o t a l l a c t a t e a p p e a r i n g i n t h e b l o o d o f r e s t i n g humans (Hultman and S a h l i n , 1 9 8 0 ) . Thus, a t l e a s t some m u s c l e c e l l s do p r o d u c e l a c t a t e when t h e o r g a n i s m i s a t r e s t . D u r i n g s ubmaximal e x e r c i s e , t h e p r o d u c t i o n and r e l e a s e o f l a c t a t e f r o m t h e s e c e l l s i s g r e a t l y i n c r e a s e d , b u t l a c t a t e c o n c e n t r a t i o n s t a y s c o n s t a n t b e c a u s e o t h e r c e l l s t a k e up l a c t a t e and o x i d i z e i t f a s t e r d u r i n g work ( t h e r e b y m a i n t a i n i n g a s i t u a t i o n i n w h i c h n e t p r o d u c t i o n i n t h e whole m u s c l e i s n i l ) . I n t h i s way, i t i s p o s s i b l e t o have l a c t a t e b e i n g p r o d u c e d and r e l e a s e d i n some f i b e r s w h i l e o t h e r f i b e r s a r e t a k i n g up and o x i d i z i n g l a c t a t e i n an a c t i v e m i x e d m u s c l e , w i t h no change i n b l o o d l a c t a t e c o n c e n t r a t i o n ( see Mole, 1 9 8 3 ) . The f a t e o f l a c t a t e d u r i n g and a f t e r e x e r c i s e i s s t i l l an open q u e s t i o n , p a r t i c u l a r l y w i t h r e g a r d t o t h e q u a n t i f i c a t i o n o f t h e d i f f e r e n t p a t h ways u t i l i z e d f o r l a c t a t e d i s p o s a l . The r e m o v a l o f l a c t a t e f r o m t h e b l o o d compartment i s p e r f o r m e d m a i n l y v i a o x i d a t i o n , g l u c o n e o g e n e s i s ( l a c t a t e -g l u c o s e c o n v e r s i o n i n t h e l i v e r w i t h d i r e c t r e l e a s e o f g l u c o s e i n t o t h e b l o o d ) , and g l y c o n e o g e n e s i s ( g l y c o g e n s y n t h e s i s i n l i v e r and s k e l e t a l m u s c l e o c c u r i n g m a i n l y d u r i n g r e c o v e r y f r o m e x e r c i s e t o r e p l e n i s h t h e c a r b o h y d r a t e s t o r e s i n t h e s e t i s s u e s ) . The amount o f l a c t a t e l o s t v i a r e n a l e x c r e t i o n i s n e g l i g i b l e i n mammals ( D i e s e_t a_l, 1969) . The same i s t r u e i n f i s h f o r b o t h r e n a l and g i l l e x c r e t i o n (Cameron and Kormanik, 1982; K o b a y a s h i and Wood, 19 8 0 ) . The d e s i g n o f s y s t e m s a v o i d i n g t h e l o s s o f l a c t a t e t o t h e e n v i r o n m e n t makes s e n s e b e c a u s e t h i s m e t a b o l i t e r e p r e s e n t s a 13 v e r y v a l u a b l e s o u r c e o f e n e r g y . The r e s p e c t i v e c o n t r i b u t i o n s o f o x i d a t i o n , g l u c o n e o g e n e s i s , and g l y c o n e o g e n e s i s t o t o t a l l a c t a t e r e m o v a l a r e s t i l l u n d e r i n v e s t i g a t i o n , b u t i t i s now c l e a r t h a t t h e y depend upon s e v e r a l f a c t o r s i n c l u d i n g t h e a c t i v i t y l e v e l o f t h e a n i m a l , and i t s s t a t e o f t r a i n i n g . The f a t e o f l a c t a t e a t t h e whole o r g a n i s m l e v e l c a n o n l y be u n d e r s t o o d i f k i n e t i c p a r a m e t e r s a r e q u a n t i f i e d . Most o f t h e r e c e n t work on l a c t a t e k i n e t i c s r e l e v a n t t o t h i s t h e s i s has been p e r f o r m e d i n t h e l a b o r a t o r i e s o f G. B r o o k s , B. I s s e k u t z , and J . K a t z , e x c l u s i v e l y on mammalian s p e c i e s . L a c t a t e f l u x e s o f r e s t i n g r a t s h a v e been i n v e s t i g a t e d by O k a j i m a e_t a^ L ( 1 9 8 1 ) . Donovan and B r o o k s (1983) h a v e l o o k e d a t t h e e f f e c t s o f e x e r c i s e and e n d u r a n c e t r a i n i n g on t u r n o v e r r a t e i n t h e same a n i m a l . More r e c e n t l y , f l u x r a t e s h a v e been m e a s u r e d i n e x e r c i s i n g humans ( S t a n l e y e_t a_l, 1985; and Mazzeo e_t a l , 1 9 86), and I s s e k u t z (1984) h a s s t u d i e d t h e e f f e c t o f b e t a - a d r e n e r g i c b l o c k a d e on l a c t a t e t u r n o v e r o f e x e r c i s i n g d o g s . I n t e r s p e c i f i c c o m p a r i s o n s o f l a c t a t e k i n e t i c s a r e r e s t r i c t e d t o mammals, as no o t h e r g r o u p o f v e r t e b r a t e s has been s t u d i e d . To summarize i_n v i v o f l u x s t u d i e s : p l a s m a l a c t a t e f l u x e s a r e h i g h compared w i t h the f l u x e s o f o t h e r s u b s t r a t e s , e v e n i n r e s t i n g a n i m a l s , and t h e y i n c r e a s e d r a s t i c a l l y w i t h e x e r c i s e . A t l e a s t 50% o f t o t a l l a c t a t e p r o d u c e d i s o x i d i z e d a t r e s t , and t h i s p e r c e n t a g e i n c r e a s e s t o more t h a n 80% a t s u b m a x i m a l work i n t e n s i t i e s (Donovan and B r o o k s , 1983; I s s e k u t z e_t a_l, 1976; Mazzeo e_t a_l, 1986; S t a n l e y e t a ^ , 1 9 8 5 ) . T h e s e r e s u l t s e m p h a s i z e t h e f a c t t h a t l a c t a t e i s n o t o n l y an end p r o d u c t o f g l y c o l y s i s , b u t i t i s 14 a l s o an i m p o r t a n t o x i d i z a b l e s u b s t r a t e f o r s k e l e t a l m u s c l e , p a r t i c u l a r l y d u r i n g s ubmaximal e x e r c i s e i n r a t s , d o g s , and humans. L a c t a t e p r o d u c t i o n by g l y c o l y t i c m u s c l e f i b e r s c o u l d r e p r e s e n t an i m p o r t a n t means o f s u p p l y i n g c a r b o n s u b s t r a t e t o o x i d a t i v e m u s c l e s d u r i n g work ( B r o o k s , 1985) . I f o r g a n i s m s c a n d e r i v e a p e r f o r m a n c e a d v a n t a g e f r o m t h e i r a b i l i t y t o s u p p o r t h i g h p l a s m a l a c t a t e f l u x e s , t h e s t u d y o f t r a i n e d o r n a t u r a l a t h l e t e s s h o u l d be e s p e c i a l l y i n s t r u c t i v e ; m e t a b o l i c a d a p t a t i o n s t o e x e r c i s e i n v o l v i n g t h e p r o d u c t i o n , movement, o r u t i l i z a t i o n o f l a c t a t e s h o u l d be e m p h a s i z e d i n e l i t e a n i m a l p e r f o r m e r s compared w i t h s e d e n t a r y s p e c i e s . L a c t a t e was c h o s e n as a model m e t a b o l i t e f o r t h i s s t u d y b e c a u s e 1 ) : i t i s a good exogenous f u e l f o r o x i d a t i v e m u s c l e s , and 2 ) : b e c a u s e t h e t u r n o v e r r a t e o f l a c t a t e v a r i e s o v e r a v e r y wide r a n g e o f v a l u e s i n r e s p o n s e t o d i f f e r e n t l e v e l s o f a c t i v i t y . T h e r e f o r e , t h e t u r n o v e r r a t e o f l a c t a t e c a n be m a n i p u l a t e d o v e r a wide r a n g e u s i n g e x e r c i s e as an e x p e r i m e n t a l t o o l t o c o n t r o l t h i s r a t e . A l s o , t h e r e g u l a t o r y mechanisms i n v o l v e d i n t h e c o n t r o l o f p l a s m a m e t a b o l i t e t u r n o v e r r a t e ( a t t h e w h o l e - o r g a n i s m l e v e l ) s h o u l d be e x a g g e r a t e d i n t h e c a s e o f l a c t a t e . 1.3 METABOLITE TURNOVER A l l t h e body c o n s t i t u e n t s o f a l i v i n g o r g a n i s m a r e c o n t i n u a l l y p r o d u c e d and u t i l i z e d i n a p r o c e s s c a l l e d 15 t u r n o v e r (see H e t e n y i e t a_l, 1 9 83). L i k e a l l o t h e r compounds, p l a s m a m e t a b o l i t e s a r e t h e r e f o r e i n a d y n a mic s t a t e , u n d e r g o i n g c o n s t a n t r e p l a c e m e n t . F o r d e c a d e s however, r e s e a r c h e r s h a v e u s e d c h a n g e s i n t h e c o n c e n t r a t i o n o f s u b s t r a t e s i n p l a s m a t o draw c o n c l u s i o n s a b o u t t h e i r r a t e o f r e l e a s e i n t h e c i r c u l a t i o n and t h e i r r a t e o f u p t a k e t h e r e f r o m . Such c o n c l u s i o n s a r e o f t e n i n v a l i d b e c a u s e c o n c e n t r a t i o n c h a n g e s do n o t a l l o w t h e q u a n t i f i c a t i o n o f r a t e s o f p r o d u c t i o n and u t i l i z a t i o n by t h e o r g a n i s m ( W o l f e , 1 9 8 4 ) . M e t a b o l i t e f l u x r a t e s c a n be a l t e r e d d r a s t i c a l l y w i t h o u t n o t i c e a b l e c h a n g e s i n t h e i r p l a s m a c o n c e n t r a t i o n . I n a d d i t i o n , l a r g e c h a n g es i n c o n c e n t r a t i o n may o n l y r e f l e c t m i n o r i m b a l a n c e s between t h e r a t e s o f a p p e a r a n c e ( R a ) and d i s a p p e a r a n c e ( R a ) i n t o and f r o m t h e p l a s m a compartment, p a r t i c u l a r l y i f t h e s e r a t e s a r e h i g h . The u n d e r s t a n d i n g o f m u s c l e f u e l m e t a b o l i s m a t t h e w h o l e - o r g a n i s m l e v e l i s o n l y p o s s i b l e i f s u b s t r a t e f l u x e s a r e q u a n t i f i e d . The t u r n o v e r r a t e o f a p l a s m a m e t a b o l i t e shows how r a p i d l y t h i s p a r t i c u l a r compound i s e x c h a n g e d between c e l l s , t i s s u e s , or o r g a n s v i a t h e c i r c u l a t i o n . I t i s a measure o f i t s r a t e o f movement t h r o u g h t h e b l o o d compartment f r o m s i t e ( s ) o f p r o d u c t i o n t o s i t e ( s ) o f u t i l i z a t i o n o r e x c r e t i o n i f t h i s compound i s i n d e e d e x c r e t e d (which i s n o t t h e c a s e f o r a m e t a b o l i t e s u c h as l a c t a t e ) . The t u r n o v e r o f an o x i d i z a b l e s u b s t r a t e r e p r e s e n t s t h e m aximal r a t e a t w h i c h t h i s exogenous f u e l i s b e i n g s u p p l i e d t o o x i d a t i v e w o r k i n g m u s c l e s f r o m v a r i o u s s t o r a g e a r e a s s u c h as l i v e r , a d i p o s e t i s s u e , or o t h e r m u s c l e s . In t h e c a s e o f l a c t a t e , a l a r g e 16 f r a c t i o n o f t o t a l t u r n o v e r r a t e i s o x i d i z e d [50% t o more t h a n 80%: s e e Donovan and B r o o k s ( 1 9 8 3 ) , I s s e k u t z e t a l ( 1 9 7 6 ) , and Mazzeo e t al_ (1986)] and t h e i m p o r t a n c e o f l a c t a t e as an o x i d i z a b l e f u e l c a n be e s t i m a t e d f r o m t u r n o v e r r a t e m easurements (Mazzeo e_t a_l, 1986; S t a n l e y e t a^, 1985; W o l f e , 1984) . A good u n d e r s t a n d i n g o f e x o g e n o u s s u b s t r a t e s u p p l y i s p a r t i c u l a r l y r e l e v a n t t o t h e s t u d y o f s u s t a i n a b l e e x e r c i s e w h i c h c a n l a s t l o n g e r i f endogenous f u e l s a r e u s e d s p a r i n g l y . 1.4 THESIS OUTLINE T h i s t h e s i s i n v e s t i g a t e s p l a s m a l a c t a t e t u r n o v e r i n two o u t s t a n d i n g a n i m a l " a t h l e t e s " : t h e s k i p j a c k t u n a and t h e t h o r o u g h b r e d h o r s e . The p u r p o s e o f t h i s work i s n o t t o compare t h e two a n i m a l s b u t t o u s e e a c h one o f them t o s o l v e d i f f e r e n t p r o b l e m s c o n c e r n i n g t h e r e g u l a t i o n o f l a c t a t e f l u x e s as e x p l a i n e d i n t h e p r e v i o u s s e c t i o n s . I n b o t h c a s e s , l a c t a t e i s u s e d as a model t o s t u d y f l u x r e g u l a t i o n i n g e n e r a l . I n a d d i t i o n , l a c t a t e t u r n o v e r r a t e i s q u a n t i f i e d i n two s p e c i e s whose p l a s m a m e t a b o l i t e k i n e t i c s h a v e n e v e r been i n v e s t i g a t e d . C h a p t e r 2 examines l a c t a t e t u r n o v e r r a t e i n t h e s k i p j a c k t u n a . The p r i m a r y g o a l o f t h i s c h a p t e r i s t o s t u d y t h e r e l a t i o n s h i p between p l a s m a l a c t a t e c o n c e n t r a t i o n and t h e r a t e o f l a c t a t e t u r n o v e r i n t h i s t e l e o s t whose p l a s m a l a c t a t e c o n c e n t r a t i o n c a n r e a c h v a l u e s e x c e e d i n g 30 mM. A c l o s e e x a m i n a t i o n o f t h e r e s u l t s f r o m t h i s f i r s t s t u d y shows t h a t 17 c a r d i o v a s c u l a r a d j u s t m e n t s may a l s o i n f l u e n c e p l a s m a m e t a b o l i t e t u r n o v e r r a t e . The e f f e c t s o f c h a n g e s i n c a r d i a c o u t p u t on m e t a b o l i t e t u r n o v e r r a t e h a v e n e v e r been i n v e s t i g a t e d a t t h e whole o r g a n i s m l e v e l . T h e s e c h a n g e s may h a v e an i m p o r t a n t i m p a c t on t h e t r a n s f e r o f o x i d i z a b l e f u e l s f r o m t h e i r s t o r a g e s i t e s t o t h e i r u t i l i z a t i o n s i t e s : t h e w o r k i n g m u s c l e s . A l s o , t h e y may a l t e r t h e r e l a t i o n s h i p between l a c t a t e c o n c e n t r a t i o n and p l a s m a l a c t a t e t u r n o v e r r a t e . U n f o r t u n a t e l y , t h e s t u d y o f c a r d i o v a s c u l a r c h a n g e s , and t h e i r e f f e c t on l a c t a t e t u r n o v e r r a t e c o u l d n ot be p e r f o r m e d on t u n a , and a d i f f e r e n t e x p e r i m e n t a l a n i m a l was needed t o s t u d y t h i s p r o b l e m i n more d e t a i l ( m a i n l y b e c a u s e t u n a c o u l d n o t be e x e r c i s e d u n d e r c o n t r o l l e d c o n d i t i o n s ) . W i t h a mammalian model, e x e r c i s e c o u l d be .used as a t o o l t o e l i c i t c a r d i o v a s c u l a r a d j u s t m e n t s , and t o e x p e r i m e n t a l l y a l t e r m e t a b o l i t e f l u x r a t e s . The t h o r o u g h b r e d r a c e h o r s e was c h o s e n f o r t h e s e c o n d p a r t o f t h i s i n v e s t i g a t i o n ( C h a p t e r s 3 t o 5) b e c a u s e 1 ) : t h i s a n i m a l i s known t o u n d e r g o l a r g e c a r d i o v a s c u l a r c h a n g e s i n r e s p o n s e t o e x e r c i s e , 2 ) : l a c t a t e t u r n o v e r r a t e and c a r d i a c o u t p u t h a v e n e v e r been m e a s u r e d i n t h i s s p e c i e s , and 3 ) : t h e t h o r o u g h b r e d h o r s e i s a v e r y good example o f an " e l i t e " mammalian a t h l e t e . C h a p t e r 2 a l s o d e a l s w i t h t h e c o m p a r a t i v e a s p e c t s o f m e t a b o l i t e k i n e t i c s s t u d i e s , and i t d i s c u s s e s p r o b l e m s a s s o c i a t e d w i t h t h e c o m p a r i s o n o f k i n e t i c d a t a between s p e c i e s ( i n p a r t i c u l a r between h o m e o t h e r m i c and e c t o t h e r m i c o r g a n i s m s ) . I t i s shown t h a t t u n a h a v e t h e a b i l i t y t o move l a c t a t e a c r o s s t h e i r p l a s m a compartment a t r a t e s e q u a l o r e x c e e d i n g t h e r a t e s measured i n 18 r a t s , d o g s , and humans. I n t h e s e c o n d p a r t o f t h e t h e s i s ( C h a p t e r s 3 t o 5 ) , l a c t a t e t u r n o v e r i s m e a s u r e d i n t h e t h o r o u g h b r e d h o r s e , an a n i m a l b o t h g e n e t i c a l l y g e a r e d f o r h i g h p e r f o r m a n c e , and t r a i n e d f o r r a c i n g . C h a p t e r 3 c h a r a c t e r i z e s t h e r e s p o n s e o f t h o r o u g h b r e d h o r s e s t o submaximal e x e r c i s e on a t r e a d m i l l i n o r d e r t o d e t e r m i n e t h e n e c e s s a r y c o n d i t i o n s f o r the measurement o f l a c t a t e t u r n o v e r r a t e , and o f t h e c a r d i o v a s c u l a r p a r a m e t e r s r e l e v a n t t o t h i s work. I n l a t e r c h a p t e r s , l a c t a t e t u r n o v e r r a t e i s m e a s u r e d by use o f t h e b o l u s i n j e c t i o n t e c h n i q u e . B e c a u s e t h i s t e c h n i q u e r e q u i r e s p l a s m a l a c t a t e s t e a d y - s t a t e c o n d i t i o n s , C h a p t e r 3 e x a m i n e s when, and how s u c h c o n d i t i o n s a r e e s t a b l i s h e d . I t f o c u s s e s on t h e measurement o f t h e c a r d i o v a s c u l a r and m e t a b o l i c e v e n t s a s s o c i a t e d w i t h t h e o n s e t o f submaximal e x e r c i s e , p a r t i c u l a r l y w i t h r e s p e c t t o t h e i m p a c t t h e s e e v e n t s h a v e on p l a s m a l a c t a t e c o n c e n t r a t i o n . I n t h i s p a r t o f t h e t h e s i s , t h e s p e c i f i c r e s p o n s e o f t h e a n i m a l s u s e d f o r t h e l a c t a t e t u r n o v e r measurements i s a s s e s s e d . The d a t a from c h a p t e r 3 a l l o w e d t o d e t e r m i n e t h e t i m i n g o f i n j e c t i o n s and s a m p l i n g f o r a l l t h e measurements r e p o r t e d i n c h a p t e r 4 and 5. To c o m p l e t e t h i s s t u d y o f t h e g e n e r a l r e s p o n s e o f t h o r o u g h b r e d h o r s e s t o t r e a d m i l l e x e r c i s e , m u s c l e b i o p s i e s were t a k e n t o a s s e s s t h e m e t a b o l i c s t a t u s o f one o f t h e m a i n l o c o m o t o r y m u s c l e s . T h e s e b i o p s i e s were sample d t o s e e w h e t h e r th e l a c t a t e s t e a d y s t a t e o b s e r v e d i n p l a s m a a f t e r a few m i n u t e s o f s ubmaximal e x e r c i s e was a l s o p r e s e n t i n w o r k i n g m u s c l e . T h i s p r e l i m i n a r y work was n e c e s s a r y b e c a u s e , a t t h e t i m e 19 t h e s e e x p e r i m e n t s were c o n d u c t e d , no t h o r o u g h b r e d h o r s e had been e x e r c i s e d on a t r e a d m i l l , and t h e m e t a b o l i c r e s p o n s e o f t h e s e a n i m a l s t o t r e a d m i l l work was unknown. In C h a p t e r 4, t h e c a r d i a c o u t p u t o f r e s t i n g and e x e r c i s i n g t h o r o u g h b r e d h o r s e s i s d e t e r m i n e d by use o f t h e t h e r m o d i l u t i o n t e c h n i q u e . The c a r d i a c o u t p u t measurements made h e r e a r e u s e d i n t h e f o l l o w i n g c h a p t e r (5) t o d e t e r m i n e t h e i m p a c t o f c a r d i o v a s c u l a r c h a n g e s on p l a s m a l a c t a t e t u r n o v e r r a t e . C h a p t e r 4 p r e s e n t s t h e f i r s t c a r d i a c o u t p u t measurements p e r f o r m e d i n t h o r o u g h b r e d r a c e h o r s e s . I n a d d i t i o n , a r t e r i o - v e n o u s d i f f e r e n c e s i n o x y g e n c o n t e n t a r e me a s u r e d a t t h e d i f f e r e n t l e v e l s o f a c t i v i t y c h o s e n f o r t h e l a c t a t e t u r n o v e r s t u d y . T h e s e measurements, t o g e t h e r w i t h c a r d i a c o u t p u t v a l u e s , a l l o w t o c a l c u l a t e t h e m e t a b o l i c r a t e o f t h e a n i m a l s f o r t h e d i f f e r e n t e x e r c i s e p r o t o c o l s u s e d i n t h i s s t u d y . The q u a n t i t a t i v e a s s e s s m e n t o f t h e o x y g e n c o n s u m p t i o n o f t h e s e a n i m a l s was n e c e s s a r y t o be a b l e t o compare t h e e x e r c i s e i n t e n s i t i e s s e l e c t e d f o r t h e h o r s e s w i t h e x e r c i s e r e g i m e s u s e d i n o t h e r s t u d i e s done on d i f f e r e n t s p e c i e s i n c l u d i n g man. A v a l i d c o m p a r i s o n o f l a c t a t e t u r n o v e r r a t e s c o u l d o n l y be made i f m e t a b o l i c r a t e s were known. In C h a p t e r 5, p l a s m a l a c t a t e t u r n o v e r i s q u a n t i f i e d i n r e s t i n g and e x e r c i s i n g t h o r o u g h b r e d s by u s e o f t h e b o l u s i n j e c t i o n t e c h n i q u e . An i n t e g r a t e d a n a l y s i s o f t h e t u r n o v e r r a t e and c a r d i a c o u t p u t d a t a i s p r e s e n t e d i n t h i s c h a p t e r . The m a j o r f a c t o r s i n v o l v e d i n t h e r e g u l a t i o n o f p l a s m a m e t a b o l i t e t u r n o v e r r a t e a r e i d e n t i f i e d i n t h e f i n a l a n a l y s i s . The i m p a c t o f c h a n g e s i n p l a s m a l a c t a t e 20 c o n c e n t r a t i o n and i n c a r d i a c o u t p u t on t h e t u r n o v e r r a t e o f o x i d i z a b l e s u b s t r a t e s i s d i s c u s s e d . U n l i k e what i s f o u n d i n C h a p t e r 2 f o r t u n a , t h i s l a s t c h a p t e r shows t h a t l a c t a t e i s n o t an i m p o r t a n t m e t a b o l i c f u e l f o r m u s c l e i n e x e r c i s i n g t h o r o u g h b r e d h o r s e s . 21 CHAPTER 2: LACTATE AND GLUCOSE TURNOVER IN SKIPJACK TUNA. 2.1 INTRODUCTION Over t h e p a s t few y e a r s , e x e r c i s e p h y s i o l o g i s t s and b i o c h e m i s t s h a v e shown renewed i n t e r e s t i n t h e s t u d y o f l a c t a t e w i t h t h e r e a l i z a t i o n t h a t t h i s compound p l a y s a d o u b l e r o l e i n m u s c l e m e t a b o l i s m . I t i s n o t s i m p l y an end p r o d u c t o f g l y c o l y s i s b u t a l s o an i m p o r t a n t f u e l f o r a e r o b i c work (Hochachka e_t al, 1985) . I n mammals, m u s c l e l a c t a t e p r o d u c t i o n s t a r t s l o n g b e f o r e 0^ becomes l i m i t i n g ( C o n n e t t e t a l , 1984), and l a c t a t e t u r n o v e r r a t e i n c r e a s e s w i t h b l o o d l a c t a t e c o n c e n t r a t i o n ( B r o o k s e t a_l, 1984; Donovan and B r o o k s , 1983; E l d r i d g e e t a l , 1974; I s s e k u t z e t a_l, 1976; O k a j i m a e t a l , 1 9 8 1 ) . As e l i t e swimmers, t u n a a r e c h a r a c t e r i z e d by a c o m b i n a t i o n o f e n d u r a n c e and s p r i n t c a p a b i l i t i e s . Not o n l y c a n t h e y s u s t a i n m o d e r a t e swimming s p e e d s o v e r v e r y l o n g d i s t a n c e s , b u t t h e y c a n r e a c h t o p v e l o c i t i e s o f o v e r 20 body l e n g t h s / s (Waters and F i e r s t i n e , 1 9 6 4 ) . The m e t a b o l i c m a c h i n e r y s u p p o r t i n g t u n a l o c o m o t i o n must meet t h e c h a l l e n g e s imposed by t h i s l i f e s t y l e , and i t s h o u l d i n c l u d e : 1) f a s t f l u x e s o f f u e l s t o t h e w o r k i n g m u s c l e s d u r i n g a e r o b i c e n d u r a n c e swimming and 2) f a s t f l u x e s o f a n a e r o b i c end 22 p r o d u c t s t o t h e i r s i t e s o f c a t a b o l i s m d u r i n g r e c o v e r y f r o m b u r s t e x e r c i s e . W h i l e swimming a t h i g h s p e e d , s k i p j a c k t u n a c a n g e n e r a t e l a c t a t e v e r y r a p i d l y , up t o r e c o r d c o n c e n t r a t i o n s o f 90 umol/g wet w e i g h t i n w h i t e m u s c l e (Guppy et_ a_l, 1979) . D u r i n g r e c o v e r y , b l o o d l a c t a t e c l e a r a n c e o c c u r s much f a s t e r i n t u n a t h a n i n o t h e r t e l e o s t s . Minimum b l o o d l a c t a t e c o n c e n t r a t i o n s a r e r e a c h e d a f t e r l e s s t h a n 2 h o f r e c o v e r y i n e x h a u s t e d s k i p j a c k s ( B a r r e t t and C o n n o r , 1 9 6 4 ) . Even w i t h a much s m a l l e r l a c t a t e l o a d , c l e a r a n c e t a k e s 12 h i n r a i n b o w t r o u t ( B l a c k e t a l , 1 9 6 2 ) . P r e s e n t v i e w s o f e x e r c i s e and r e c o v e r y i n f i s h r e l y m a i n l y on t h e i n t e r p r e t a t i o n o f m e t a b o l i t e c o n c e n t r a t i o n m e asurements. E s t i m a t e s o f m e t a b o l i c f l u x e s w o u l d p r o v i d e a new d i m e n s i o n t o t h e u n d e r s t a n d i n g o f f u e l m e t a b o l i s m as a d y n a m ic p r o c e s s . However, g l u c o s e t u r n o v e r r a t e has o n l y been measured i n k e l p b a s s and c oho s a l m o n (Bever e_t a_l, 1977; L i n e t a l , 1978), and l a c t a t e f l u x e s h a v e n o t b een i n v e s t i g a t e d i n f i s h . The g o a l o f t h i s s t u d y , t h e r e f o r e , was t o measure l a c t a t e and g l u c o s e t u r n o v e r r a t e s i n s k i p j a c k t u n a t o : 1) e x t e n d t h e o b s e r v e d c o r r e l a t i o n between l a c t a t e t u r n o v e r r a t e and l a c t a t e c o n c e n t r a t i o n f o u n d i n mammals t o a much w i d e r r a n g e o f l a c t a t e c o n c e n t r a t i o n s and t o a nonmammalian s p e c i e s , 2) f i n d o u t w h e t h e r t h e h i g h r a t e o f l a c t a t e c l e a r a n c e r e p o r t e d i n t u n a d u r i n g r e c o v e r y c o u l d be a c c o u n t e d f o r by h i g h b l o o d l a c t a t e f l u x e s , 3) a s s e s s t h e i m p o r t a n c e o f l a c t a t e and g l u c o s e as m e t a b o l i c f u e l s i n a f a s t p e l a g i c f i s h 23 and compare t h e i r f l u x r a t e s w i t h v a l u e s m easured i n mammals, and 4) o b t a i n some i n s i g h t i n t o t h e p o s s i b l e o p e r a t i o n o f t h e C o r i c y c l e i n t u n a . 2.2 MATERIALS AND METHODS 2.2.1 EXPERIMENTAL ANIMALS. S k i p j a c k t u n a , Katsuwonus p e l a m i s , were c a u g h t w i t h b a r b l e s s hooks o f f t h e c o a s t o f Oahu, H a w a i i , f r o m May t o A u g u s t 1984. They were b r o u g h t back t o t h e Kewalo R e s e a r c h F a c i l i t y ( N a t i o n a l M a r i n e F i s h e r i e s S e r v i c e , H o n o l u l u l a b o r a t o r y ) i n t h e b a i t w e l l s o f c o m m e r c i a l f i s h i n g v e s s e l s and t r a n s f e r r e d t o 7 5 , 0 0 0 - l i t e r c i r c u l a r h o l d i n g t a n k s s u p p l i e d w i t h w e l l - a e r a t e d 25^C s e a w a t e r . The f i s h were n o t f e d i n c a p t i v i t y and were u s e d i n t h e f i r s t 4 d a y s a f t e r c a p t u r e . The s t a t u s o f t h e i r c a r b o h y d r a t e s t o r e s was d e t e r m i n e d i n a c o n t r o l g r o u p o f f i v e f i s h t o a s s e s s w h e t h e r c a p t u r e and c a p t i v i t y c a u s e d g l y c o g e n or g l u c o s e d e p l e t i o n . The c o n t r o l a n i m a l s were k e p t f o r 4 d a y s i n c a p t i v i t y u n d e r t h e same c o n d i t i o n s as t h e f i s h u s e d i n t u r n o v e r e x p e r i m e n t s , t h e r e f o r e , t h e i r c a r b o h y d r a t e s t o r e s were s i m i l a r t o t h e s t o r e s o f t h e f i s h u s e d f o r t h e measurement o f l a c t a t e t u r n o v e r . The c o n t r o l f i s h were c a u g h t w i t h a n e t and k i l l e d w i t h a s h a r p blow on t h e h e a d . A b l o o d sample was i m m e d i a t e l y t a k e n by c a r d i a c p u n c t u r e . A p p r o x i m a t e l y 1 g o f w h i t e m u s c l e , r e d m u s c l e , and l i v e r were f r e e z e c lamped i n l i q u i d N 2 and e x t r a c t e d i n p e r c h l o r i c a c i d by u s e o f p r e v i o u s l y d e s c r i b e d p r o c e d u r e s (Guppy e t a l , 24 1 9 7 9 ) . G l u c o s e , g l u c o s e 6 - p h o s p h a t e , and g l y c o g e n c o n c e n t r a t i o n s were d e t e r m i n e d i n t h e s e t i s s u e s . F i s h t o be u s e d i n t u r n o v e r e x p e r i m e n t s swam v i g o r o u s l y a r o u n d t h e i r h o l d i n g t a n k s ( f r o m 1 t o 10 min) b e f o r e b e i n g d i r e c t e d t o a l a r g e f u n n e l l e a d i n g t o a 1 0 - l i t e r p l a s t i c bag c o n t a i n i n g ( ^ - s u p e r s a t u r a t e d w a t e r w i t h MS-222 ( t r i c a i n e m e t h a n e s u l f o n a t e , 1:2,000, w t / v o l ) : an e f f e c t i v e f i s h a n e s t h e t i c . 2.2.2 CATHETERIZATION. As s o o n as t h e f i s h s t o p p e d m o v i n g , i t was b r o u g h t i n t o t h e l a b o r a t o r y and p l a c e d v e n t r a l s i d e up on an o p e r a t i n g t a b l e . The g i l l s were 0 i r r i g a t e d w i t h a e r a t e d 24 C r e c i r c u l a t i n g s e a w a t e r c o n t a i n i n g MS-222 ( 1 : 1 5 , 0 0 0 ) . A c a t h e t e r ( S u r f l o I.V. C a t h e t e r 20 g X 2 i n . , ID 0.80 X 51 mm, Terumo, Japan) was 0 i n s e r t e d a t a 45 a n g l e a p p r o x i m a t e l y 2 cm i n f r o n t o f t h e p e l v i c f i n s , d i r e c t e d t o w a r d s t h e head f o r t h e p e r c u t a n e o u s c a n n u l a t i o n o f t h e v e n t r a l a o r t a j u s t a n t e r i o r t o t h e h e a r t . The c a t h e t e r was c o n n e c t e d t o a p r e s s u r e t r a n s d u c e r f o r p r e s s u r e v e r i f i c a t i o n o f c a t h e t e r p o s i t i o n . A 40-cm p i e c e o f PE-160 t u b i n g was a t t a c h e d t o t h e c a t h e t e r and s u t u r e d t o t h e u n d e r s i d e o f t h e a n i m a l . D o u b l e c a n n u l a t i o n ( v e n t r a l and d o r s a l a o r t a ) was a t t e m p t e d on a few i n d i v i d u a l s . The d o r s a l a o r t a c a n n u l a t i o n was done t h r o u g h t h e mouth by u s e o f a t e c h n i q u e d e s c r i b e d i n J o n e s e_t a_l ( 1 9 8 6 ) . T h i s p r o c e d u r e was d i f f i c u l t t o p e r f o r m , and s u c c e s s r a t e was low (under 3 0 % ) . C a t h e t e r s , PE t u b i n g , and s y r i n g e s were a l w a y s r i n s e d w i t h h e p a r i n i z e d s a l i n e (10 U/ml) b e f o r e u s e . T o t a l 25 c a n n u l a t i o n t i m e n e v e r e x c e e d e d 20 and 35 min f o r s i n g l e and d o u b l e c a n n u l a t i o n s , r e s p e c t i v e l y . Once t h e c a t h e t e r (s) was i n p l a c e , h e a r t r a t e and b l o o d p r e s s u r e were m o n i t o r e d v i a t h e t r a n s d u c e r . The t u n a was t u r n e d u p r i g h t , s t r a p p e d t o a P l e x i g l a s s h o l d e r , .and submerged. The a n e s t h e t i c c o n c e n t r a t i o n was t h e n r e d u c e d t o a p p r o x i m a t e l y 1:30,000 and was a d j u s t e d i n d i v i d u a l l y f o r e a c h f i s h t h r o u g h o u t t h e t u r n o v e r e x p e r i m e n t s . The a n i m a l s were a l l o w e d t o r e c o v e r u n t i l t h e y s l o w l y moved t h e i r t a i l w i t h o u t t h r a s h i n g . They were m a i n t a i n e d i n t h i s s t a t e by c o n t i n u o u s l y a d j u s t i n g t h e a n a e s t h e t i c c o n c e n t r a t i o n . P r e i n j e c t i o n measurements o f h e a r t r a t e and b l o o d p r e s s u r e t o g e t h e r w i t h b l o o d g l u c o s e c o n c e n t r a t i o n were v a l u a b l e i n d i c a t o r s o f t h e s t a t e o f t h e a n i m a l a f t e r s u r g e r y . A n i m a l s s h o w i n g a b l o o d g l u c o s e c o n c e n t r a t i o n o f l e s s t h a n 1 mM were n o t u s e d i n t u r n o v e r e x p e r i m e n t s . The l i m i t was s e t a t t h i s v a l u e b e c a u s e no f i s h w i t h b l o o d g l u c o s e l e v e l s o f 1 mM or more showed s i g n s o f s t r e s s a t any o c c a s i o n . I n some c a s e s , b l o o d g l u c o s e c o n c e n t r a t i o n s t a r t e d t o d e c r e a s e f r o m i t s o r i g i n a l s t e a d y - s t a t e v a l u e a f t e r s e v e r a l h o u r s o f s a m p l i n g i n a t u r n o v e r e x p e r i m e n t . Whenever t h i s o c c u r r e d t h e t a i l ends o f d e c a y c u r v e s were n o t i n c l u d e d i n t h e a n a l y s i s . A f t e r t h i s o p e r a t i o n , s a m p l i n g t i m e s f o r a l l t u r n o v e r e x p e r i m e n t s r a n g e d from 2 t o 6 h ( i . e . a minimum o f 2 h o u r s o f s a m p l i n g was a l w a y s a v a i l a b l e t o d e r i v e a d e c a y c u r v e ) , and t h e a s s u m p t i o n s o f s t e a d y s t a t e k i n e t i c s r e q u i r e d by t h e b o l u s i n j e c t i o n t e c h n i q u e were met. 26 2.2.3 INJECTION OF LABELED METABOLITES AND BLOOD SAMPLING. A b o l u s o f 25-35 u C i [ U - 1 4 C ] l a c t a t e ( s p e c i f i c 14 a c t i v i t y > 100 mCi/mmol), 10 u C i [U- C J g l u c o s e (4 3 mCi/mmol) o r 25-30 u C i [6- H ] g l u c o s e (300 mCi/mmol) was i n j e c t e d v i a t h e c a t h e t e r a t t i m e 0. I n two s p e c i m e n s 14 3 [U- C ] g l u c o s e and [6- H ] g l u c o s e were i n j e c t e d s i m u l t a n e o u s l y t o e s t i m a t e g l u c o s e c a r b o n r e c y c l i n g . The l i n e was f l u s h e d w i t h 3 ml h e p a r i n i z e d s a l i n e i m m e d i a t e l y a f t e r i n j e c t i o n . B l o o d s a m p l e s (0.5 ml) were drawn s t a r t i n g 1 min a f t e r i n j e c t i o n t o a l l o w t h e b o l u s t o e q u i l i b r a t e i n t h e r a p i d l y m i x i n g p o o l . T h i s t i m e was s u f f i c i e n t t o a l l o w t o t a l b l o o d volume t o be c i r c u l a t e d a c r o s s t h e e n t i r e body, and t h e b o l u s was assumed t o be m i x e d a f t e r t h i s p e r i o d . Samples were drawn a b o u t e v e r y 40 s d u r i n g t h e f i r s t 5 min and t h e n a t a p p r o p r i a t e i n t e r v a l s . T h i s c h o i c e o f s a m p l i n g t i m e s was made i n o r d e r t o o b t a i n a good measurement o f l a c t a t e s p e c i f i c a c t i v i t y d e c a y w i t h o u t d r a w i n g e x c e s s i v e volumes o f b l o o d f r o m t h e a n i m a l . The c a t h e t e r was f l u s h e d w i t h 0.5 ml s a l i n e between s a m p l e s . The amount o f b l o o d drawn t h r o u g h o u t an e x p e r i m e n t n e v e r e x c e e d e d 10% o f t o t a l b l o o d volume. R a d i o c h e m i c a l s were p u r c h a s e d f r o m New E n g l a n d N u c l e a r ( B o s t o n , Mass.) and Amersham ( O a k v i l l e , O n t a r i o ) . 2.2.4 METABOLITE ASSAYS AND COUNTING. B l o o d samples were d e p r o t e i n i z e d i m m e d i a t e l y i n 1-2 p a r t s c o l d p e r c h l o r i c a c i d (8%) and s p u n down. The s u p e r n a t a n t was 0 s t o r e d a t -4 C. L a c t a t e and g l u c o s e c o n c e n t r a t i o n s were d e t e r m i n e d e n z y m a t i c a l l y a t 340 nm by u s e o f t h e p r o c e d u r e o f 27 Bergmeyer ( 1 9 7 4 ) . T i s s u e g l y c o g e n o f t h e c o n t r o l f i s h was d e t e r m i n e d w i t h t h e a m y l o g l u c o s i d a s e h y d r o l y s i s t e c h n i q u e (Bergmeyer, 1 9 7 4 ) . A l l m e t a b o l i t e a s s a y s were p e r f o r m e d i n d u p l i c a t e , a t t h e l a t e s t 7 d a y s a f t e r s a m p l i n g and t h e mean o f t h e s e measurements was u s e d i n t h e c a l c u l a t i o n s . F o r e a c h d e t e r m i n a t i o n o f m e t a b o l i t e c o n c e n t r a t i o n , a new s e t o f two measurements was made i f t h e f i r s t two v a l u e s were d i f f e r i n g by more t h a n 10%. G l u c o s e d e t e r m i n a t i o n s n e v e r showed s u c h v a r i a b i l i t y , b u t i t was n e c e s s a r y t o r e p e a t l a c t a t e d e t e r m i n a t i o n s (two a s s a y s ) i n a b o u t 5% o f t h e c a s e s . The s p e c i f i c a c t i v i t y o f l a c t a t e was e s t i m a t e d by s u b t r a c t i n g t h e a c t i v i t y f o u n d i n g l u c o s e f r o m t o t a l b l o o d a c t i v i t y . T h i s method i s a d e q u a t e when m e a s u r i n g l a c t a t e 14 t u r n o v e r w i t h C - l a c t a t e by u s e o f t h e b o l u s i n j e c t i o n t e c h n i q u e b e c a u s e o t h e r c a r b o n p o o l s t h a n l a c t a t e and g l u c o s e n e v e r r e a c h s i g n i f i c a n t s p e c i f i c a c t i v i t i e s ( i n a b o l u s i n j e c t i o n e x p e r i m e n t , t h e a n i m a l i s i n c o n t a c t w i t h h i g h i s o t o p i c a c t i v i t i e s f o r a v e r y s h o r t p e r i o d o n l y , compared w i t h a c o n t i n u o u s i n f u s i o n e x p e r i m e n t i n w h i c h a l o n g e q u i l i b r a t i o n p e r i o d i s n e c e s s a r y b e f o r e a measurement o f t u r n o v e r c a n be made). When u s i n g t h e c o n t i n u o u s i n f u s i o n method t h e s p e c i f i c a c t i v i t y o f s e v e r a l o t h e r c a r b o n p o o l s t h a n l a c t a t e or g l u c o s e h a v e t i m e t o e q u i l i b r a t e ( b e c a u s e t h e a n i m a l i s i n c o n t a c t w i t h h i g h i s o t o p i c a c t i v i t i e s f o r s e v e r a l h o u r s ) . However, e v e n u n d e r s u c h c o n d i t i o n s , more t h a n 90% o f t o t a l b l o o d a c t i v i t y i s s t i l l f o u n d i n l a c t a t e , and a b o u t 6-8% i s f o u n d i n g l u c o s e ( D a v i s , 1983; Donovan and B r o o k s , 1983; O k a j i m a e t a l , 1 9 81). The r e m a i n i n g a c t i v i t y 28 c a n be me a s u r e d i n o t h e r compounds s u c h as a l a n i n e , p y r u v a t e , and o t h e r s , b u t , when t a k e n t o g e t h e r , t h e s e s e c o n d a r y compounds o n l y r e p r e s e n t a v e r y s m a l l f r a c t i o n o f t o t a l a c t i v i t y ( i . e . a maximum o f 5% i n e x t r e m e c a s e s ) . I n t h e b o l u s i n j e c t i o n t e c h n i q u e , t h e c o n t r i b u t i o n o f t h e s e o t h e r compounds w i l l be e v e n s m a l l e r t h a n 5% f o r r e a s o n s e x p l a i n e d p r e v i o u s l y , and t h e i r e f f e c t c a n be c o n s i d e r e d as n e g l i g i b l e . I n b o l u s i n j e c t i o n e x p e r i m e n t s , t h e s p e c i f i c a c t i v i t y o f l a c t a t e c a n t h e r e f o r e be e s t i m a t e d by s u b t r a c t i n g t h e a c t i v i t y f o u n d i n g l u c o s e f r o m t o t a l a c t i v i t y . The maximal p o s s i b l e e r r o r c a u s e d by i g n o r i n g t h e s e s e c o n d a r y m e t a b o l i t e s w o u l d be a 2 t o 3 % o v e r e s t i m a t i o n o f l a c t a t e s p e c i f i c a c t i v i t y i n t h e t a i l ends o f t h e d e c a y c u r v e s . T h i s o v e r e s t i m a t i o n o f s p e c i f i c a c t i v i t y w o u l d c a u s e a m i n o r u n d e r e s t i m a t i o n o f l a c t a t e t u r n o v e r (when d i v i d i n g t h e i n j e c t e d d o s e by t h e s u r f a c e a r e a u n d e r t h e d e c a y c u r v e ) , and t h i s e f f e c t h a s been c o n s i d e r e d a s n e g l i g i b l e i n t h e p r e s e n t s t u d y . I n t h r e e r a n d o m l y c h o s e n d e c a y c u r v e s , a l l t h e measured s p e c i f i c a c t i v i t i e s o f l a c t a t e were d e c r e a s e d by 5%, and t u r n o v e r r a t e was r e c a l c u l a t e d w i t h t h e s e new c u r v e s ; t h e new " c o r r e c t e d " v a l u e s f o r t u r n o v e r r a t e were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e o r i g i n a l " u n c o r r e c t e d " t u r n o v e r r a t e s . As e x p l a i n e d b e f o r e , i t i s v e r y u n l i k e l y however, t h a t t h e s p e c i f i c a c t i v i t y o f a l a n i n e , p y r u v a t e , and o t h e r s e c o n d a r y compounds wo u l d r e p r e s e n t 5% o f t o t a l a c t i v i t y i n a b o l u s i n j e c t i o n e x p e r i m e n t . B l o o d g l u c o s e was s e p a r a t e d t o d e t e r m i n e i t s a c t i v i t y . B l o o d p e r c h l o r i c a c i d e x t r a c t (200 u l ) was 29 i n c u b a t e d w i t h 4 ml 1 M g l u c o s e and 0.6 g A m b e r l i t e MB-3 r e s i n (Sigma C h e m i c a l s ) . The m i x t u r e was s h a k e n f o r 2 h a t 0 25 C, a l l o w i n g a l l t h e c h a r g e d compounds t o b i n d t o t h e r e s i n b e f o r e i t was spun down and 1 ml o f t h e s u p e r n a t a n t was c o u n t e d . A f t e r s e p a r a t i o n 90.1+3.2% o f t h e t o t a l r a d i o a c t i v e 3 g l u c o s e was r e c o v e r e d as d e t e r m i n e d w i t h [6- H ] g l u c o s e s t a n d a r d s . L a c t a t e a c t i v i t y was m easured by c o u n t i n g 40 u l p e r c h l o r i c a c i d e x t r a c t and c o r r e c t i n g f o r t h e a c t i v i t y f o u n d i n g l u c o s e . S c i n t i l l a t i o n c o u n t i n g was p e r f o r m e d on a Beckman LS-9000 l i q u i d s c i n t i l l a t i o n c o u n t e r u s i n g e x t e r n a l s t a n d a r d q u ench c o r r e c t i o n . A l l s a m p l e s were m i x e d w i t h 10 ml aqueous c o u n t i n g s c i n t i l l a n t (ACS I I , Amersham) and l e f t i n t h e d a r k f o r a t l e a s t 12 h b e f o r e c o u n t i n g . S i n g l e - l a b e l c o u n t i n g 14 3 showed an e f f i c i e n c y o f 94% f o r C and 42% f o r H ( d u a l - l a b e l : 73 and 35%, r e s p e c t i v e l y ) . No a t t e m p t was made 14 t o measure t h e a p p e a r a n c e o f C i n c a r b o n d i o x i d e b e c a u s e no r e l i a b l e t e c h n i q u e i s a v a i l a b l e f o r t h e q u a n t i t a t i v e t r a p p i n g o f C 0 2 i n s a l t w a t e r . However, l i k e i n mammals, t h e a c t i v i t y f o u n d i n b l o o d and i n o t h e r t i s s u e s d e c a y s v e r y 14 r a p i d l y a f t e r a b o l u s i n j e c t i o n o f C - l a c t a t e , s h o w i n g t h a t t h e m a j o r f a t e o f p l a s m a l a c t a t e i n t u n a i s a l s o o x i d a t i o n ( t h i s compound i s n o t e x c r e t e d i n f i s h : s e e Cameron and K o r m a n i k , 1982; K o b a y a s h i and Wood, 1 9 8 0 ) . I n some p r e l i m i n a r y e x p e r i m e n t s , 6 s k i p j a c k t u n a were i n j e c t e d w i t h 14 C - l a c t a t e and t h e y were a l l o w e d t o q u i e t l y swim i n a l a r g e t a n k (1-2 body l e n g t h p e r s e c o n d ) f o r 6 h o u r s . G l y c o g e n was i s o l a t e d f r o m t h e i r m u s c l e s and l i v e r t o d e t e r m i n e t h e 30 a c t i v i t y i n t h i s compound. A l m o s t no l a c t a t e had been i n c o r p o r a t e d , and t h e s p e c i f i c a c t i v i t y o f g l y c o g e n was e x t r e m e l y low i n t h e s e t i s s u e s . F u r t h e r m o r e , t h e a c t i v i t y m e a s u r e d was t o o v a r i a b l e t o r e l i a b l y q u a n t i f y g l y c o g e n s y n t h e s i s f r o m l a c t a t e and t h i s e x p e r i m e n t a l a p p r o a c h had t o be abandonned. T h e s e r e s u l t s c l e a r l y showed, however, t h a t 14 . . . C was n o t a c c u m u l a t i n g i n an unknown c a r b o n p o o l i n t h i s s p e c i e s . 2.2.5 TERMINOLOGY, CALCULATIONS AND S T A T I S T I C S . The c o n c e p t o f m e t a b o l i t e t u r n o v e r c a n o n l y be a p p l i e d t o a s y s t e m i n s t e a d y s t a t e ( i . e . when t h e p l a s m a c o n c e n t r a t i o n o f t h i s compound do e s n o t c hange o v e r t i m e ) . Under t h e s e c o n d i t i o n s , t h e r a t e o f a p p e a r a n c e (R ) i s e q u a l t o t h e r a t e o f d i s a p p e a r a n c e ( R ^ ) , and c a n be c a l l e d t u r n o v e r r a t e or r e p l a c e m e n t r a t e . I f t h e a n i m a l comes o u t o f s t e a d y s t a t e , R a and R^ must be r e f e r r e d t o and m easured i n d e p e n d e n t l y b e c a u s e t h e y a r e no l o n g e r e q u a l . H e t e n y i e_t a_l (1983) g i v e a good summary o f t h e t h e o r y and t e r m i n o l o g y a s s o c i a t e d w i t h f l u x measurements i n t h e s t u d y o f m e t a b o l i s m i_n v i vo. Two m a j o r t e c h n i q u e s a r e a v a i l a b l e f o r t h e measurement o f t u r n o v e r r a t e : b o l u s i n j e c t i o n and c o n t i n u o u s i n f u s i o n (Katz e_t a_l, 1981; O k a j i m a e t a_l, 1981; W o l f e , 1 9 84). They l e a d t o t h e same r e s u l t s b u t d i f f e r i n t h e i r a s s u m p t i o n s and i n t h e i r u s e f u l n e s s f o r v a r i o u s e x p e r i m e n t a l s i t u a t i o n s . U n l i k e c o n t i n u o u s i n f u s i o n , b o l u s i n j e c t i o n r e q u i r e s s t e a d y - s t a t e m e t a b o l i t e c o n c e n t r a t i o n , and i t does n o t a l l o w t h e i n d e p e n d e n t mesurement o f r a t e s o f a p p e a r a n c e 31 and d i s a p p e a r a n c e . H e re, b o l u s i n j e c t i o n was c h o s e n o v e r c o n t i n u o u s i n f u s i o n b e c a u s e o f s u r g i c a l d i f f i c u l t i e s ; i m p l a n t i n g t h e s e c o n d c a t h e t e r r e q u i r e d by t h e i n f u s i o n t e c h n i q u e was t o o d i f f i c u l t i n t h e s p e c i e s i n v e s t i g a t e d . An e x c e l l e n t a n a l y s i s o f c u r r e n t methods u s e d f o r t h e _in_ v i v o measurement o f f l u x e s w i t h r a d i o - and s t a b l e i s o t o p e s c a n be f o u n d i n R.R. W o l f e ' s book: T r a c e r s i n M e t a b o l i c R e s e a r c h ( 1 9 8 4 ) . T h e s e modern methods a l l o w t h e i n v e s t i g a t i o n o f m e t a b o l i s m _in v i vo, and, o v e r t h e l a s t 10 y e a r s , t h e y h a v e been u s e d by a l a r g e number o f i n v e s t i g a t o r s ( r e v i e w s by H e t e n y i e t a l , 1983; K a t z , 1979; W o l f e , 1984). B o t h s t a b l e and r a d i o i s o t o p e t r a c e r t e c h n i q u e s h a v e been a p p l i e d t o t h e measurement o f m e t a b o l i t e t u r n o v e r i n p l a s m a and t h e y h a v e been v a l i d a t e d f o r a number o f common b l o o d m e t a b o l i t e s ( H e t e n y i e t a_l, 1983; K a t z , 1979; W o l f e , 1 9 8 4 ) . B e c a u s e t h e c o n t i n u o u s i n f u s i o n t e c h n i q u e h a d been d e v e l o p e d p u r e l y t h e o r e t i c a l l y , i t had t o be v a l i d a t e d e x p e r i m e n t a l l y . Dogs were a n a e s t h e t i z e d , and a l l s o u r c e s o f endogenous g l u c o s e ( l i v e r and k i d n e y ) were removed s u r g i c a l l y ( A l l s o p e_t a l , 1 9 7 8 ) . G l u c o s e was t h e n i n f u s e d a t a known r a t e , and t h e c o n t i n u o u s i n f u s i o n t e c h n i q u e was u s e d t o c a l c u l a t e t h e r a t e o f g l u c o s e a p p e a r a n c e . The maximum d i f f e r e n c e between t h e t r u e r a t e and t h e c a l c u l a t e d r a t e was 5% and t h e c o e f f i c i e n t o f v a r i a t i o n o f t h e t r a c e r c a l c u l a t e d r a t e a b o u t the a c t u a l r a t e was 3.4% (see W o l f e , 1 9 8 4 ) . S e v e r a l l a b o r a t o r i e s have q u a n t i f i e d t h e r a t e , o f p l a s m a l a c t a t e t u r n o v e r i n d i f f e r e n t s p e c i e s , and u nder v a r i o u s e x p e r i m e n t a l c o n d i t i o n s ( D a v i s , 1983; Donovan and B r o o k s , 1983; E l d r i d g e , 1974; E l d r i d g e e t 32 a l , 1974; F r e m i n e t and L e c l e r c , 1980; I s s e k u t z , 1984; I s s e k u t z e t a l , 1976; K a t z e t a_l, 1981; Mazzeo e t a l , 1982; Mazzeo e_t a_l, 1986; O k a j i m a e t a_l, 1981; R e i l l y and C h a n d r a s e n a , 1977; S t a n l e y e_t a_l, 1985) . The s p e c i f i c u s e o f t r a c e r t e c h n i q u e s f o r . t h e i n v e s t i g a t i o n o f l a c t a t e k i n e t i c s h a s b een r e v i e w e d i n B r o o k s and F a h e y ( 1 9 8 4 ) , F r e m i n e t and M i n a i r e (1984), K a t z ( 1 9 8 6 ) , and W o l f e ( 1 9 8 4 ) . The use o f b o l u s i n j e c t i o n f o r l a c t a t e t u r n o v e r measurements may seem q u e s t i o n a b l e b e c a u s e t h e l a b e l e d compound may n o t h a v e t i m e t o r e a c h t h e i n t r a c e l l u l a r f l u i d w h i c h c o n t a i n s a f r a c t i o n o f t h e t o t a l l a c t a t e p o o l ( i . e . i s o t o p i c e q u i l i b r i u m may n o t h a v e t i m e t o o c c u r ) . However, t h e b o l u s i n j e c t i o n t e c h n i q u e has been shown t o l e a d t o t h e same r e s u l t s as t h e c o n t i n u o u s i n f u s i o n t e c h n i q u e f o r w h i c h t h i s p r o b l e m i s a b s e n t ( i n c o n t i n u o u s i n f u s i o n , t h e l a b e l e q u i l i b r a t e s between e x t r a -and i n t r a c e l l u l a r f l u i d s o v e r s e v e r a l h o u r s ) . The t u r n o v e r r a t e s measured w i t h t h e two t e c h n i q u e s were f o u n d t o be i d e n t i c a l f o r s e v e r a l m e t a b o l i t e s i n c l u d i n g g l u c o s e ( K a t z , R o s t a m i , and Dunn, 1974; K a t z , 1979), and l a c t a t e ( K a t z e t a l , 1981; K a t z , 1986; O k a j i m a e t a l , 1 9 8 1 ) . T h e s e two t e c h n i q u e s g i v e i d e n t i c a l r e s u l t s b e c a u s e l a b e l e d l a c t a t e e q u i l i b r a t e s q u i c k l y between b l o o d and m u s c l e compartments ( W o l f e , p e r s . comm.; a l s o s e e Koch e_t a_l, 1981) , as i n d i c a t e d by t h e K^ v a l u e s r e p o r t e d f o r l a c t a t e t r a n s p o r t e r s i n v a r i o u s c e l l membranes ( D u b i n s k y and R a c k e r , 1978; Koch e t a l , 1981; Roos, 1975; S p e n c e r and L e h n i n g e r , 1 9 7 6 ) . In t h e p r e s e n t s t u d y , t u r n o v e r r a t e was c a l c u l a t e d as t h e d o s e i n j e c t e d ( i n dpm) d i v i d e d by t h e s u r f a c e a r e a 33 u n d e r t h e s p e c i f i c a c t i v i t y d e c a y c u r v e ( i n dpm umol min, see K a t z e_t a_l, 1981) . To c a l c u l a t e t h i s s u r f a c e a r e a t h e d e c a y c u r v e was f i r s t f i t t e d w i t h 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 by use o f t h e n o n l i n e a r r e g r e s s i o n p r o g r a m P3R from B i o M e d i c a l D a t a P r o c e s s i n g (BMDP; s e e W o l f e , 1984), which d e t e r m i n e s t h e l e a s t - s q u a r e s e s t i m a t e s o f t h e f u n c t i o n p a r a m e t e r s by a m o d i f i e d Gauss-Newton a l g o r i t h m . The f i t t e d f u n c t i o n was t h e n i n t e g r a t e d between t i m e 0 and t h e t i m e when 5% o f t h e maximum p o s s i b l e s p e c i f i c a c t i v i t y was r e a c h e d . The maximum p o s s i b l e a c t i v i t y was c a l c u l a t e d as t h e d o s e i n j e c t e d d i v i d e d by t h e volume o f t h e r a p i d l y m i x i n g p o o l e s t i m a t e d t o be 8% o f t h e body volume. M e t a b o l i c c l e a r a n c e r a t e (MCR) was c a l c u l a t e d as t u r n o v e r r a t e d i v i d e d by t h e s t e a d y s t a t e m e t a b o l i t e c o n c e n t r a t i o n ( K a t z e t a l , 1 9 81). 34 2.3 RESULTS G l u c o s e , g l u c o s e 6 - p h o s p h a t e , and g l y c o g e n c o n c e n t r a t i o n s i n t i s s u e s o f a n i m a l s f r o m t h e c o n t r o l g r o u p a r e g i v e n i n T a b l e 1. S k i p j a c k t u n a b l o o d g l u c o s e c o n c e n t r a t i o n a p p e a r e d t o be r e g u l a t e d b e c a u s e i t was r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e t u r n o v e r e x p e r i m e n t s ( T a b l e s 2 and 3, F i g . 3 B ) . However, t h e s e t p o i n t was d i f f e r e n t between i n d i v i d u a l s , and t h e mean b l o o d g l u c o s e c o n c e n t r a t i o n r a n g e d f r o m 1.2 t o 6.8 mM. I n e a c h t u r n o v e r e x p e r i m e n t a b l o o d sample was drawn b e f o r e i n j e c t i o n o f t h e r a d i o a c t i v e b o l u s . L a c t a t e and g l u c o s e c o n c e n t r a t i o n s o f t h e p r e i n j e c t i o n sample were a l w a y s w i t h i n 2% o f t h e means g i v e n i n T a b l e s 2 and 3. 2.3.1 LACTATE TURNOVER. A f t e r i n j e c t i o n o f 14 [U- C ] l a c t a t e t h e a c t i v i t y r e c o v e r e d i n g l u c o s e n e v e r e x c e e d e d 5% o f t o t a l b l o o d a c t i v i t y , e ven a f t e r 6 h . The l a c t a t e s p e c i f i c a c t i v i t y d e c a y c u r v e s were b e s t f i t t e d w i t h t h e sum o f t h r e e e x p o n e n t i a l f u n c t i o n s ( F i g . I A ) . I n a l l c a s e s t h e c a l c u l a t e d f u n c t i o n s f i t t e d t h e o b s e r v e d v a l u e s e x t r e m e l y w e l l . F i s h No. 4 h a d a r e l a t i v e l y low b l o o d l a c t a t e c o n c e n t r a t i o n and t h e l o w e s t t u r n o v e r r a t e . S p e c i f i c a c t i v i t y d e c r e a s e d s h a r p l y o v e r t h e f i r s t 10 min a f t e r i n j e c t i o n and t h e n more g r a d u a l l y ( F i g . I A ) . B l o o d l a c t a t e c o n c e n t r a t i o n was r e l a t i v e l y s t a b l e t h r o u g h o u t t h e e x p e r i m e n t ( F i g . I B ) . T u r n o v e r r a t e s and MCR a r e g i v e n f o r s e v e n i n d i v i d u a l s i n T a b l e 2. T h e s e a n i m a l s were s e l e c t e d b e c a u s e 35 TABLE 1. TISSUE METABOLITE CONCENTRATIONS IN THE CONTROL GROUP OF SKIPJACK TUNA. V a l u e s a r e means +_ SEM f o r 5 a n i m a l s . C o n c e n t r a t i o n s g i v e n i n umol/g wet w e i g h t f o r m u s c l e and l i v e r , and umol/ml f o r b l o o d . Tuna were h a n d l e d and k e p t i n c a p t i v i t y u n d e r t h e same c o n d i t i o n s as a n i m a l s u s e d i n t h e t u r n o v e r e x p e r i m e n t s . TISSUE GLUCOSE GLUCOSE GLYCOGEN 6-PHOSPHATE (GLUCOSYL UNITS) W h i t e m u s c l e Red m u s c l e L i v e r B l o o d 0.68 + 0.12 0.8 4 + 0.17 3.54 + 0.34 3.04 + 0.39 4.16 + 0.97 1.30 + 0.34 92.54 + 9.17 24.22 + 5.20 4.06 + 0.87 36 TABLE 2. BLOOD METABOLITE CONCENTRATIONS AND LACTATE TURNOVER RATES IN SKIPJACK TUNA. V a l u e s a r e means +_ SEM, number o f b l o o d s a m p l e s (N) i n p a r e n t h e s e s . 1 S a m p l i n g s i t e a l s o g i v e n : VA = V e n t r a l A o r t a , DA = D o r s a l A o r t a . F i s h No. 6 and 7 were sampled s i m u l t a n e o u s l y f r o m VA and DA. T u r n o v e r r a t e was d e t e r m i n e d 14 by b o l u s i n j e c t i o n o f [U- C ] l a c t a t e . FISH WEIGHT GLUCOSE 1 No. (g) mM LACTATE (N) mM LACTATE TURNOVER RATE (umol . -1 min k g " 1 ) METABOLIC CLEARANCE RATE (ml . -1 min k g " 1 ) 1 VA 1065 4. 3 5 + 0. 08 27.5+1.0 (11) 380. 9 13 .9 2 VA 1298 4. 26 + 0. 12 25.0+0.4 (13) 296. 0 11.8 3 VA 1440 2. 3 0 + 0. 07 19.1+0.5 (12) 299. 8 15.7 4 VA 1436 6. 31 + 0. 14 14.6+0.3 (15) 112. 6 7.7 5 VA 1880 1. 8 5 + 0. 03 12.0+0.5 (13) 199. 9 16.7 6 VA 1405 4. 10 + 0. 11 11.6+0.3 (ID 180. 8 15.6 6 DA 4 . 12 + 0. 09 10.2+0.4 (ID 131. 8 12.9 7 VA 1412 1. 23+0. 02 26.5+0.3 (10) 415. 2 15.7 7 DA 1. 2 2 + 0. 02 27.6+0.5 (11) 431. 4 15.6 37 FIG. 1 L a c t a t e t u r n o v e r r a t e measurement i n s k i p j a c k tuna ( f i s h No. 4 ) . R a d i o a c t i v e b o l u s i n j e c t e d and b l o o d sampled v i a the v e n t r a l a o r t a c a t h e t e r . A: b l o o d l a c t a t e s p e c i f i c a c t i v i t y decay c u r v e a f t e r i n j e c t i o n of 25 uCi 14 [U- C ] l a c t a t e a t time 0. Curve f i t t e d w i t h the sum o f 3 e x p o n e n t i a l f u n c t i o n s . B: b l o o d l a c t a t e c o n c e n t r a t i o n d u r i n g the s a m p l i n g p e r i o d . SP. ACTIVITY (DPM/umol )x10 3 _l 01 _l \\ ro. o o CD o \\ ro-o L A C T A T E ( m M ) o _j JO o CO 3 9 F I G . 2 R e l a t i o n s h i p between l a c t a t e t u r n o v e r r a t e and b l o o d l a c t a t e c o n c e n t r a t i o n i n s k i p j a c k t u n a . C i r c l e s : a n i m a l s w i t h s i n g l e v e n t r a l a o r t a c a t h e t e r ; t r i a n g l e s : 2 a n i m a l s ( f i s h No. 6 and 7) s a m p l e d s i m u l t a n e o u s l y f r o m v e n t r a l a o r t a (VA) and d o r s a l a o r t a (DA). The l i n e was f i t t e d by l i n e a r r e g r e s s i o n ( s l o p e = 15.1, r 2 = 0 . 8 5 ) . TURNOVER RATE (y mol min 1 kg ) -1 N> O O 4^  O O Ofr 41 t h e c o e f f i c i e n t o f v a r i a t i o n o f t h e i r mean b l o o d l a c t a t e c o n c e n t r a t i o n was l e s s t h a n 15%. T h e s e s e v e n i n d i v i d u a l s had s t a b l e enough l a c t a t e c o n c e n t r a t i o n s t o j u s t i f y t h e use o f s t e a d y s t a t e k i n e t i c s i n our c a l c u l a t i o n s (3 e x p t s . were r e j e c t e d ) . Mean b l o o d l a c t a t e c o n c e n t r a t i o n s i n i n d i v i d u a l f i s h r a n g e d from 10.2 t o 27.6 mM. T u r n o v e r r a t e was p o s i t i v e l y c o r r e l a t e d w i t h l a c t a t e c o n c e n t r a t i o n ( F i g . 2 ) , and t h e s l o p e o f t h e r e l a t i o n s h i p was h i g h l y s i g n i f i c a n t l y d i f f e r e n t f r o m z e r o (F = 40, P < 0.001, 1 d f / 7 d f , ANOVA). MCR was not a f f e c t e d by b l o o d l a c t a t e c o n c e n t r a t i o n . A l i n e a r r e g r e s s i o n f o r b l o o d l a c t a t e v s . MCR 2 (gr a p h n o t shown) had a s l o p e o f 0.044 (r = 0.0134), w h i c h i s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m z e r o (F = 0, P > 0.05, 1 d f / 7 d f , ANOVA). Mean MCR was 14.0 + 0.9 (SEM) ml m i n - 1 k g " 1 (N = 9 ) . S i m u l t a n e o u s s a m p l i n g f r o m t h e d o r s a l a o r t a and t h e v e n t r a l a o r t a was p e r f o r m e d on f i s h No. 6 and 7. T u r n o v e r r a t e s d e t e r m i n e d f r o m b o t h s a m p l i n g s i t e s were s i m i l a r ( T a b l e 2, F i g . 2 ) , i n d i c a t i n g t h a t t h e s e two s i t e s c o u l d be u s e d i n t e r c h a n g e a b l y . D i f f e r e n c e s between t h e d o r s a l a o r t a and v e n t r a l a o r t a l a c t a t e s p e c i f i c a c t i v i t y d e c a y c u r v e s were n o t s u f f i c i e n t t o q u a n t i f y l a c t a t e u t i l i z a t i o n by t h e g i l l s . 2.3.2 GLUCOSE TURNOVER. As i n mammalian s p e c i e s , t h e t u r n o v e r r a t e f o r g l u c o s e was much l o w e r t h a n f o r l a c t a t e . A t y p i c a l s p e c i f i c a c t i v i t y d e c a y c u r v e i s g i v e n f o r f i s h No. 9 ( F i g . 3A), w h i c h e x h i b i t e d an a v e r a g e t u r n o v e r r a t e . The d e c a y c u r v e s f o r g l u c o s e were b e s t f i t t e d w i t h the 42 sum o f two e x p o n e n t i a l f u n c t i o n s . T u r n o v e r r a t e and MCR f o r g l u c o s e a r e g i v e n i n T a b l e 3. T u r n o v e r r a t e was i n d e p e n d e n t o f g l u c o s e c o n c e n t r a t i o n (F = 1, P > 0.25, 1 d f / 5 d f , ANOVA) and a v e r a g e d 15.3 + 1.2 (SEM) umol min""'*" kg-'*" as 3 d e t e r m i n e d w i t h [6- H J g l u c o s e (N = 5 ) . MCR was 4.7 + 1.0 (SEM) ml m i n " 1 k g - 1 (N = 5 ) . F i s h No. 10 and 12 showed a h i g h b l o o d g l u c o s e c o n c e n t r a t i o n , b u t t h e i r t u r n o v e r r a t e was no t e l e v a t e d . C o n s e q u e n t l y , g l u c o s e MCR was l o w e r t h a n a v e r a g e f o r t h e s e two f i s h . 3 [6- H ] g l u c o s e i s c o n s i d e r e d an i r r e v e r s i b l e t r a c e r b e c a u s e i t s p r e d o m i n a n t end p r o d u c t i s t r i t i a t e d ^ 0 . The r e i n c o r p o r a t i o n o f l a b e l e d w a t e r i n t o g l u c o s e i s i n s i g n i f i c a n t b e c a u s e r a p i d e q u i l i b r a t i o n o c c u r s w i t h i n t h e 14 l a r g e amount o f body H 2 ° * C ] g l u c o s e i s a r e v e r s i b l e 14 t r a c e r b e c a u s e C i n c o r p o r a t e d i n t o o t h e r m e t a b o l i c i n t e r m e d i a t e s c a n be r e c y c l e d back t o g l u c o s e . T h i s r e c y c l i n g s l o w s down t h e d e c a y o f t h e s p e c i f i c a c t i v i t y c u r v e , c a u s i n g an u n d e r e s t i m a t i o n o f t h e t r u e r a t e o f g l u c o s e t u r n o v e r . I n 3 t h i s s t u d y , t u r n o v e r r a t e d e t e r m i n e d w i t h [6- H ] g l u c o s e was . 1 4 much h i g h e r t h a n w i t h [U- C J g l u c o s e ( T a b l e 3 ) , i n d i c a t i n g t h a t g l u c o s e c a r b o n r e c y c l i n g i s r e l a t i v e l y h i g h i n t u n a (28 and 16% f o r f i s h No. 11 and 12, r e s p e c t i v e l y ) . 43 TABLE 3. BLOOD GLUCOSE CONCENTRATION AND GLUCOSE TURNOVER RATE IN SKIPJACK TUNA. V a l u e s a r e means _+ SEM. Number o f b l o o d s a m p l e s (N) i n p a r e n t h e s e s . T u r n o v e r r a t e s were d e t e r m i n e d by b o l u s 3 14 i n j e c t i o n o f [6- H ] g l u c o s e o r [U- C ] g l u c o s e . F i s h No. 11 and 12 were i n j e c t e d w i t h b o t h i s o t o p e s . I n j e c t i o n o f t h e r a d i o a c t i v e b o l u s and b l o o d s a m p l i n g were done v i a v e n t r a l a o r t a c a t h e t e r s . F ISH WEIGHT LABEL No. (g) GLUCOSE mM GLUCOSE TURNOVER RATE (umol min k g - 1 ) -1 METABOLIC CLEARANCE RATE (ml min -1 kg 1 ) 8 1680 3 H 2.52 + 0.08 (11) 14.8 5.9 9 1497 3 H 2.83 + 0.09 (13) 15.2 5.4 10 1320 3 H 6.77 + 0.17 (13) 11.0 1.6 11 1671 3 H 2.46 + 0.03 (11) 17.3 7.0 11 1 4 c 12.5 5.1 12 15 97 3 H 5.19 + 0.10 (13) 18. 1 3.5 12 1 4 c 15.2 2.9 44 F I G . 3 G l u c o s e t u r n o v e r r a t e measurement i n s k i p j a c k t u n a ( f i s h No. 9 ) . I n j e c t i o n and b l o o d s a m p l i n g same as F i g . 1. A: b l o o d g l u c o s e s p e c i f i c a c t i v i t y d e c a y c u r v e a f t e r 3 i n j e c t i o n o f 28.2 u C i [6- H ] g l u c o s e a t t i m e 0. The c u r v e was f i t t e d w i t h t h e sum o f two e x p o n e n t i a l f u n c t i o n s . B: b l o o d g l u c o s e c o n c e n t r a t i o n d u r i n g t h e s a m p l i n g p e r i o d . SP. ACTIVITY (DPM/umol )xl0 3 o o o 01 o GLUCOSE (mM) o ro co A ui - i 1 I I 6-ro -j o 46 2.4 DISCUSSION The l a c t a t e turnover r a t e s r e p o r t e d here f o r s k i p j a c k tuna range from 112 to 431 umol m i n - 1 k g - 1 . These values are h i g h e r than the r a t e s r e p o r t e d by Okajima e_t i - 1 - 1 a l (1981) f o r r e s t i n g r a t s (70 umol min kg ) and are comparable to values obtained at r e s t (200 umol m i n - 1 kg 1) and at d i f f e r e n t l e v e l s of e x e r c i s e (up to 500 umol min 1 kg 1) by Donovan and Brooks (1983), a l s o i n r a t s . Because l a c t a t e i s not e x c r e t e d i n f i s h (Cameron and Kormanik, 1982; Kobayashi and Wood, 1980), and because i t i s t r a n s p o r t e d r a p i d l y a c r o s s c e l l membranes (Dubinsky and Racker, 1978; Koch et a_l, 1981; Roos, 1975; Spencer and Lehninger, 1976), l a c t a t e turnover r a t e can be used as an index of plasma l a c t a t e metabolism i_n v i vo. The animals used i n t h i s study were not o v e r l y s t r e s s e d by c a p t u r e , t r a n s p o r t a t i o n , and s h o r t - t e r m c a p t i v i t y without food. They were not exhausted because t h e i r carbohydrate s t o r e s were f a r from being d e p l e t e d . Red muscle glycogen c o n c e n t r a t i o n was o n l y 30% lower than i n the group of " r e s t i n g " s k i p j a c k tuna r e p o r t e d by Guppy et a J L (1979); white muscle c o n c e n t r a t i o n was 90% h i g h e r . Glucose and glucose 6-phosphate l e v e l s i n the two muscle types were s i m i l a r to the " r e s t i n g " f i s h of Guppy e t a_l_. S u r p r i s i n g l y , l i v e r glycogen c o n c e n t r a t i o n s were low compared with other t e l e o s t s , but no other values are a v a i l a b l e f o r tuna l i v e r . There i s no doubt that the experimental s i t u a t i o n used f o r the p r e s e n t measurements was very a r t i f i c i a l because the 47 a n i m a l s were r e s t r a i n e d and l i g h t l y a n e s t h e t i z e d . However, f i s h t r e a t e d w i t h MS-222 do n o t show any c h a n g e s i n b l o o d g l u c o s e and l a c t a t e c o n c e n t r a t i o n s compared w i t h u n t r e a t e d i n d i v i d u a l s ( W e l l s e_t a_l, 1984) . The same a n e s t h e t i c c a u s e s a r e d u c t i o n i n t h e l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s (Mazeaud e_t a^L, 1977) , s u g g e s t i n g a p o s s i b l e d e p r e s s i o n o f c a r b o h y d r a t e m e t a b o l i s m . U n t i l now, a l l a t t e m p t s t o c a r r y o u t t h i s t y p e o f e x p e r i m e n t on u n a n e s t h e t i z e d f r e e - s w i m m i n g t u n a have been u n s u c c e s s f u l . Even u n d e r t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s ( i . e . s e m i - a n a e s t h e t i z e d f i s h a r t i f i c i a l l y v e n t i l a t e d ) , i t i s t e c h n i c a l l y d i f f i c u l t t o i n v e s t i g a t e m e t a b o l i s m i n t h i s s p e c i e s . T h e r e f o r e , t h e number o f t u n a u s e d i n t h i s s t u d y was l i m i t e d t o 12 w i t h o u t c o u n t i n g t h e f i s h u s e d f o r p r e l i m i n a r y e x p e r i m e n t s and u n s u c c e s s f u l e x p e r i m e n t s ( f o r example when p l a s m a m e t a b o l i t e c o n c e n t r a t i o n was o u t o f s t e a d y s t a t e ) . A l s o , a c c e s s t o l i v e t u n a f o r e x p e r i m e n t a l p u r p o s e s i s e x t r e m e l y r e s t r i c t e d and c o s t l y . I n f a c t , Kewalo R e s e a r c h F a c i l i t y i n H o n o l u l u i s t h e o n l y l a b o r a t o r y i n t h e w o r l d where l i v e t u n a a r e k e p t i n c a p t i v i t y on a r e g u l a r b a s i s . B e c a u s e f i s h p o s s e s s a s e c o n d a r y c i r c u l a t i o n ( V o g e l , 1985) t h e b o l u s i n j e c t i o n t e c h n i q u e may be i n a d e q u a t e f o r t h e measurement o f p l a s m a m e t a b o l i t e t u r n o v e r r a t e i n t h i s g r o u p o f a n i m a l s . However, t h e p r e s e n c e o f t h e s e c o n d a r y c i r c u l a t i o n does n o t seem t o i n v a l i d a t e t h e u s e o f b o l u s i n j e c t i o n i n t u n a b e c a u s e r e c e n t measurements i n d i c a t e t h a t t h e f l o w r a t e o f b l o o d t h r o u g h t h e s e c o n d a r y v a s c u l a r s y s t e m may be e x t r e m e l y low. The h a l f - t i m e f o r m i x i n g between the 48 p r i m a r y and s e c o n d a r y c i r c u l a t i o n s was e s t i m a t e d between 1.2 and 1.7 h o u r s i n r a i n b o w t r o u t (D. R a n d a l l p e r s . comm.). I n t h e c a s e o f r a i n b o w t r o u t , t h e s e c o n d a r y s y s t e m c a n be t r e a t e d as a s i n g l e i n p u t (or o u t p u t ) o f l a c t a t e i n t o t h e p r i m a r y s y s t e m and i t p l a y s t h e same r o l e as any o r g a n u s i n g o r p r o d u c i n g l a c t a t e . The b o l u s i n j e c t i o n t e c h n i q u e i s t h e r e f o r e a v a l i d e x p e r i m e n t a l a p p r o a c h t o measure l a c t a t e t u r n o v e r r a t e i n r a i n b o w t r o u t . I n t h e p r e s e n t s t u d y , i t was assumed t h a t t h e m i x i n g o f b l o o d between t h e p r i m a r y and s e c o n d a r y c i r c u l a t i o n o f s k i p j a c k t u n a was a l s o v e r y s l o w , b u t t h i s a s s u m p t i o n h a s n o t b een v e r i f i e d e x p e r i m e n t a l l y . I t i s i m p o r t a n t t o r e a l i z e t h a t m e t a b o l i t e t u r n o v e r r a t e s g e n e r a l l y d e p e n d on s e v e r a l c r i t i c a l f a c t o r s t h a t s h o u l d - be t a k e n i n t o a c c o u n t i f m e a n i n g f u l c o m p a r i s o n s a r e t o be made, p a r t i c u l a r l y w i t h mammalian v a l u e s . To b e g i n w i t h , t h e e f f e c t s o f e x e r c i s e , body mass, and t e m p e r a t u r e w i l l be c o n s i d e r e d i n some d e t a i l . Then t h e p o s s i b l e mechanisms a l l o w i n g t u n a t o s u p p o r t s u c h h i g h t u r n o v e r r a t e s w i l l be d i s c u s s e d . L a c t a t e t u r n o v e r r a t e has been shown t o be p o s i t i v e l y c o r r e l a t e d w i t h b l o o d l a c t a t e c o n c e n t r a t i o n i n r e s t i n g r a t s ( O k a j i m a e t a_l, 1981) and i n r e s t i n g dogs i n f u s e d w i t h d i f f e r e n t l a c t a t e c o n c e n t r a t i o n s ( E l d r i d g e e_t a l , 1 9 7 4 ) . T h i s i s n o t an a u t o c o r r e l a t i o n b e c a u s e l a c t a t e c o n c e n t r a t i o n p e r se i s n o t u s e d i n t h e c a l c u l a t i o n o f t u r n o v e r r a t e . I n s t e a d , s p e c i f i c a c t i v i t y i s u s e d , and t h i s v a l u e i s a r a t i o w h i c h i s i n d e p e n d e n t o f l a c t a t e c o n c e n t r a t i o n ( b o t h , t h e n u m e r a t o r and d e n o m i n a t o r o f t h i s 49 r a t i o d e p e n d on l a c t a t e c o n c e n t r a t i o n ) . T h i s e x p l a i n s why i t i s p o s s i b l e t o f i n d c a s e s i n w h i c h m e t a b o l i t e c o n c e n t r a t i o n and t u r n o v e r r a t e a r e n o t c o r r e l a t e d , o r c a s e s i n w h i c h a n e g a t i v e c o r r e l a t i o n between t h e s e two v a r i a b l e s c a n be d e m o n s t r a t e d (see f o r example J e n k i n s e t a_l, 1986, f o r an example o f a n e g a t i v e c o r r e l a t i o n between p l a s m a g l u c o s e c o n c e n t r a t i o n and g l u c o s e t u r n o v e r r a t e ) . L a c t a t e t u r n o v e r r a t e a l s o i n c r e a s e s w i t h e x e r c i s e i n t e n s i t y ( B r o o k s e_t a_l, 1984; Donovan and B r o o k s , 1983; I s s e k u t z ejt a_l, 1976) , b u t t h e e f f e c t o f e x e r c i s e i s t w o f o l d b e c a u s e b o t h p e r f u s i o n and b l o o d l a c t a t e c o n c e n t r a t i o n i n c r e a s e as work r a t e goes up. The p r e s e n t d a t a show t h a t l a c t a t e t u r n o v e r r a t e i s a l s o c o r r e l a t e d w i t h b l o o d l a c t a t e c o n c e n t r a t i o n i n t u n a ( F i g . 2 ) , and t h i s i s t h e f i r s t t i m e s u c h a d e m o n s t r a t i o n i s made f o r a nonmammalian s p e c i e s . A l s o , t h e r a n g e o f p l a s m a l a c t a t e c o n c e n t r a t i o n s c o v e r e d by th e p r e s e n t c o r r e l a t i o n i s e x t e n d e d t o v a l u e s l a r g e r t h a n 30 mM. Measurements o f h e a r t r a t e and b l o o d p r e s s u r e ( r e s u l t s n o t shown) made b e f o r e t h e b o l u s i n j e c t i o n s i n d i c a t e t h a t p e r f u s i o n was p r o b a b l y s i m i l a r between i n d i v i d u a l s s h o w i n g d i f f e r e n t s t e a d y s t a t e b l o o d l a c t a t e c o n c e n t r a t i o n s . U n f o r t u n a t e l y , t h e s e measurements were n o t made t h r o u g h o u t t h e t u r n o v e r e x p e r i m e n t s , and c a r d i a c o u t p u t may have d i f f e r e d between i n d i v i d u a l s . However, MCR was not c o r r e l a t e d w i t h l a c t a t e c o n c e n t r a t i o n . When s t u d y i n g e x e r c i s i n g a n i m a l s a p o s i t i v e c o r r e l a t i o n between MCR and work r a t e w o u l d i n d i c a t e d i f f e r e n t p e r f u s i o n r a t e s between d i f f e r e n t l e v e l s o f e x e r c i s e . T h e r e f o r e , t h e c o n s t a n c y o f MCR o v e r t h e 50 o b s e r v e d r a n g e o f l a c t a t e c o n c e n t r a t i o n s a l s o s u g g e s t s t h a t t h e r e l a t i o n s h i p shown i n F i g . 2 p r o b a b l y r e p r e s e n t s t h e t r u e e f f e c t o f l a c t a t e c o n c e n t r a t i o n i n d e p e n d e n t o f p e r f u s i o n r a t e . The r e s p e c t i v e c o n t r i b u t i o n s o f l a c t a t e c o n c e n t r a t i o n and p e r f u s i o n r a t e on t u r n o v e r i n c r e a s e s h o u l d be i n v e s t i g a t e d s e p a r a t e l y . The r e l a t i o n s h i p between body mass and w h o le-body l a c t a t e t u r n o v e r ( R t , i n umol m i n - 1 ) i s p r e s e n t e d f o r a few mammalian s p e c i e s i n F i g . 4. A l i n e a r r e g r e s s i o n a c r o s s t h e t u r n o v e r r a t e s o f r e s t i n g mammals ( c i r c l e s ) has been 2 drawn ( s l o p e = 0.64, r = 0 . 8 5 ) . The c o r r e l a t i o n i s n o t b e t t e r b e c a u s e i t i s d i f f i c u l t t o s t a n d a r d i z e " r e s t i n g c o n d i t i o n s " f o r a w i de r a n g e o f body s h a p e s and s i z e s , k nowing t h a t l a c t a t e p r o d u c t i o n , and r e m o v a l r a t e s a r e i n f l u e n c e d by a c t i v i t y . F u r t h e r m o r e , l a c t a t e R measurements a r e n o t a v a i l a b l e f o r many s p e c i e s . The e f f e c t o f e x e r c i s e on t u r n o v e r r a t e i s a l s o i l l u s t r a t e d f o r r a t , dog, and man ( t r i a n g l e s i n F i g . 4 ) . The r a n g e o f v a l u e s 0 o b t a i n e d f o r s k i p j a c k t u n a i n 25 C w a t e r i s a l s o p l o t t e d . T h e s e v a l u e s f a l l w e l l w i t h i n mammalian r a t e s e v e n w i t h o u t c o n s i d e r i n g t h e e f f e c t o f t e m p e r a t u r e . 51 F I G . 4 R e l a t i o n s h i p between body mass and w h o l e - b o d y l a c t a t e t u r n o v e r (R f c) on l o g - l o g p l o t f o r mammals and s k i p j a c k t u n a . Mammals a t r e s t ( c i r c l e s ) and e x e r c i s i n g ( t r i a n g l e s ) . The l i n e was f i t t e d by l i n e a r r e g r e s s i o n u s i n g r e s t i n g v a l u e s f o r mammals o n l y . T u r n o v e r r a t e s were me a s u r e d w i t h 14 [ C ] l a c t a t e i n a l l c a s e s . R e f e r e n c e s : r a t (Donovan and B r o o k s , 1983; F r e m i n e t and L e c l e r c , 1980; O k a j i m a e_t a l , 1 9 8 1 ) / g u i n e a p i g ( F r e m i n e t and L e c l e r c , 1 9 8 0 ) / dog ( E l d r i d g e e t a l , 1974; I s s e k u t z e t a l ^ , 1 9 7 6 ) / s h e e p ( R e i l l y and C h a n d r a s e n a , 1 9 7 7 ) / man ( B r o o k s and Donovan, 1983; S t a n l e y e_t a l , 1 9 8 4 ) . BODY M A S S 53 O c e a n - c a u g h t s k i p j a c k t u n a c a n h a v e a c o r e 0 t e m p e r a t u r e 10 C h i g h e r t h a n a m b i e n t , b u t s u c h a h i g h d i f f e r e n c e i s o n l y o b s e r v e d a f t e r f e e d i n g f r e n z i e s a t s e a . C a p t i v e a n i m a l s s u b j e c t e d t o v i o l e n t e x e r c i s e do n o t show 0 more t h a n a 5 C e x c e s s c o r e t r e m p e r a t u r e ( S t e v e n s and N e i l l , 1 9 7 8 ) . I t i s v e r y u n l i k e l y t h a t t h e body t e m p e r a t u r e o f t h e f i s h u s e d i n t h i s s t u d y e x c e e d e d w a t e r t e m p e r a t u r e by more t h a n 2 o r 3 0 C . I f a o f 2 i s assumed, t h e l a c t a t e 0 t u r n o v e r r a t e s m e a s u r e d a t 25 C s h o u l d a p p r o x i m a t e l y d o u b l e 0 a t 3 7 W C . I t i s i n t e r e s t i n g t o n o t i c e t h a t , e v e n w i t h a h i g h l a c t a t e t u r n o v e r r a t e o f 400 umol min -" 1' k g - 1 , an e x h a u s t e d t u n a w i t h 1 kg o f w h i t e m u s c l e a t a l a c t a t e c o n c e n t r a t i o n o f 90 umol/g (Guppy e_t al _ , 1979) wou l d t a k e more t h a n 3 h t o m e t a b o l i z e i t s l a c t a t e l o a d o f 90 mmoles. However, c o m p l e t e r e c o v e r y a p p e a r s t o be a c h i e v e d i n l e s s t h a n 2 h ( B a r r e t t and Co n n o r , 1 9 6 4 ) . A l s o , one o f t h e c a n n u l a t e d a n i m a l s i n t h i s s t u d y showed a d r o p i n b l o o d l a c t a t e c o n c e n t r a t i o n f r o m 32 t o 12 mM i n l e s s t h a n 2 h. T h e s e o b s e r v a t i o n s s u g g e s t t h a t n o t a l l l a c t a t e p a s s e s v i a the b l o o d compartment d u r i n g r e c o v e r y b u t t h a t a s i g n i f i c a n t p o r t i o n i s m e t a b o l i z e d d i r e c t l y i n w h i t e m u s c l e . How c a n a t u n a t u r n o v e r l a c t a t e a t t h e same r a t e o r e v e n f a s t e r t h a n a 37^C mammal? P e r f u s i o n i s l o w e r i n a 2 kg t u n a [ c a r d i a c o u t p u t o f a p p r o x i m a t e l y 90 ml/min ( B r i l l e t a l , 1978)] t h a n i n a mammal o f s i m i l a r s i z e . B l o o d l a c t a t e c o n c e n t r a t i o n c a n be h i g h e r i n t u n a , a l l o w i n g f a s t e r d i f f u s i o n between t h e b l o o d and l a c t a t e - u t i l i z i n g t i s s u e s . 54 However, t h i s d i f f u s i o n a l a d v a n t a g e c a n n o t f u l l y c o m p e n s a t e 0 f o r t h e e f f e c t o f a 10 C d i f f e r e n c e i n body t e m p e r a t u r e . T h e r e f o r e , t u n a must have o t h e r a d a p t a t i o n s a l l o w i n g them t o c y c l e l a c t a t e as f a s t as t h e y do. The v e r y h i g h a e r o b i c p o t e n t i a l o f l a c t a t e - u t i l i z i n g t i s s u e s , s u c h as r e d m u s c l e (Guppy e t al, 1979), c o u l d a l l o w f a s t l a c t a t e d i f f u s i o n by m a i n t a i n i n g a s t e e p c o n c e n t r a t i o n g r a d i e n t between t h e b l o o d and t h i s " l a c t a t e s i n k " . A s e c o n d p o s s i b i l i t y i n v o l v e s u s e o f t h e c e n t r a l v a s c u l a r c o u n t e r c u r r e n t h e a t e x c h a n g e r t o a c c e l e r a t e movements between l a c t a t e - p r o d u c i n g w h i t e m u s c l e and l a c t a t e - o x i d i z i n g r e d m u s c l e (P.W. H o c h a c h k a , R.W. B r i l l , J.-M. Weber, B. Emmett, C. Daxboeck, S. P e r r y , and T.W. Moon, u n p u b l i s h e d o b s e r v a t i o n s ) . Tuna a p p e a r t o r e g u l a t e t h e i r b l o o d g l u c o s e c o n c e n t r a t i o n much more t i g h t l y t h a n o t h e r t e l e o s t s ( T a b l e s 2 and 3, F i g . 3 ) . C a n n u l a t e d k e l p b a s s (Bever e_t a l _ , 1977) and r a i n b o w t r o u t (Weber, u n p u b l i s h e d d a t a ) show wide f l u c t u a t i o n s i n b l o o d g l u c o s e l e v e l s . Tuna and mammalian i n s u l i n s d e m o n s t r a t e s i m i l a r p r o p e r t i e s ( K i n g and Kahn, 1981), w h i c h may a l l o w t h e s e f i s h t o a c h i e v e g l u c o s e h o m e o s t a s i s . E v e n t h o u g h e a c h t u n a m a i n t a i n e d a s t e a d y b l o o d g l u c o s e c o n c e n t r a t i o n , t h e s e t p o i n t s r a n g e d f r o m 1.2 t o 6.8 mM i n d i f f e r e n t i n d i v i d u a l s . The s i g n i f i c a n c e o f t h e s e o b s e r v e d d i f f e r e n c e s i s n o t c l e a r b u t may r e p r e s e n t v a r i o u s n u t r i t i o n a l s t a t e s . G l u c o s e t u r n o v e r r a t e has o n l y been measured i n two o t h e r f i s h s p e c i e s . B e v e r e_t a_l (1977) showed t h a t k e l p b a s s , P a r a l a b r a x s p . , has a r a t e o f a b o u t 2 umol m i n - 1 k g - 1 . A 55 F I G . 5 R e l a t i o n s h i p between body mass and wh o l e - b o d y g l u c o s e t u r n o v e r r a t e (R f c) on l o g - l o g p l o t f o r mammals and s k i p j a c k t u n a . Same symb o l s as i n F i g . 4. A l i n e a r r e g r e s s i o n was f i t t e d f o r r e s t i n g mammals o n l y . The t u r n o v e r r a t e s were 3 d e t e r m i n e d w i t h [ H ] g l u c o s e i n a l l c a s e s e x c e p t f o r t h e 14 h o r s e ( C ) . R e f e r e n c e s : r a t ( B r o o k s and Donovan, 1983; F r e m i n e t and L e c l e r c , 1980; K a t z e t a l , 1974; K e t t l e h u t e t a l , 1 9 8 0 ) / g u i n e a p i g ( F r e m i n e t and L e c l e r c , 1 9 8 0 ) / r a b b i t (Katz- e t a l , 1 9 7 4 ) / c a t ( K e t t l e h u t e t a l , 1 9 8 0 ) / monkey ( A r m s t r o n g e_t al, 1 9 7 9 ) / dog ( I s s e k u t z , 1977; I s s e k u t z e t a l , 1 9 7 6 ) / p i g ( T r a y h u r n e t a_l, 1 9 8 1 ) / s h e e p (Brockman e t a l , 1975), man ( H a l l e t a l , 1 9 7 9 ) / pony (Anwer e t a l , 1 9 7 6 ) / h o r s e ( E v a n s , 1 9 7 1 ) . BODY M A S S (9) 57 s i m i l a r v a l u e was f o u n d f o r coho s a l m o n , O n c o r h y n c h u s k i s u t c h ( L i n e t a_l, 1978) . The r a t e s measured i n f i s h s p e c i e s o t h e r t h a n t u n a a r e a p p r o x i m a t e l y 1/30 t h o s e f o u n d i n r e s t i n g r a t s ( K a t z e t a l , 1 9 7 4 ) . A l t h o u g h t h e d a t a b a s e i s v e r y r e s t r i c t e d f o r f i s h , mammalian v a l u e s a r e a v a i l a b l e f o r a wide r a n g e o f body s i z e s . The r e l a t i o n s h i p between body mass and w h o l e - b o d y g l u c o s e t u r n o v e r a t r e s t (R f c, i n umol m i n - 1 ) i s p r e s e n t e d 2 i n F i g . 5 on a d o u b l e l o g a r i t h m i c s c a l e ( s l o p e = 0.72, r = 0.99, l i n e a r r e g r e s s i o n ) . I n t e r e s t i n g l y , t h i s s l o p e i s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e s l o p e f o u n d f o r t h e c l a s s i c body mass v s . m e t a b o l i c r a t e r e l a t i o n s h i p . The same a n a l o g y c a n be drawn f o r t h e body mass vs m a s s - s p e c i f i c g l u c o s e t u r n o v e r r a t e ( i n umol m i n - 1 kg ^; g r a p h n o t shown; s l o p e 2 = -0.27, r = 0 . 9 4 ) . The c l o s e c o r r e l a t i o n s o b t a i n e d s u g g e s t t h a t i t i s o f m i n i m a l i m p o r t a n c e t o d i r e c t l y measure r e s t i n g g l u c o s e t u r n o v e r r a t e i n mammals u n l e s s t h e r e a r e good r e a s o n s t o p r e d i c t t h a t t h e s p e c i e s u n d e r s t u d y w i l l d e p a r t f r o m t h i s l i n e ( e x t r e m e l y s l u g g i s h o r g a n i s m o r e l i t e a n i m a l a t h l e t e ) . When c o r r e c t e d f o r t e m p e r a t u r e t h e mean g l u c o s e R f o u n d f o r t u n a f a l l s e x a c t l y on t h e mammalian l i n e . The e f f e c t o f e x e r c i s e on g l u c o s e R i n mammals ( t r i a n g l e s i n F i g . 5) i s much l e s s p r o n o u n c e d t h a n on l a c t a t e R t ( F i g . 4) . Under t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s v e r y l i t t l e l a b e l e d l a c t a t e was c o n v e r t e d t o b l o o d g l u c o s e , i n d i c a t i n g t h a t t h e r o l e p l a y e d by t h e C o r i c y c l e i n t u n a l a c t a t e m e t a b o l i s m i s n o t i m p o r t a n t . I t i s n o t c l e a r why 58 g l u c o s e r e c y c l i n g was so h i g h n o r w i t h what o t h e r compounds. g l u c o s e may have been e x c h a n g i n g . In c o n c l u s i o n , t h i s s t u d y shows t h a t s k i p j a c k t u n a c a n s u p p o r t l a c t a t e and g l u c o s e t u r n o v e r r a t e s a t l e a s t as h i g h as t h o s e r e p o r t e d f o r mammalian s p e c i e s . G l u c o s e i s t u r n e d o v e r much f a s t e r i n t u n a t h a n i n o t h e r t e l e o s t s , p r o b a b l y as a c o n s e q u e n c e o f t h e i r h i g h m e t a b o l i c r a t e . Tuna l a c t a t e t u r n o v e r r a t e s a r e h i g h e r t h a n v a l u e s m e a s u r e d i n mammals, and t h e y a r e c o r r e l a t e d w i t h l a c t a t e c o n c e n t r a t i o n . T h e s e h i g h t u r n o v e r r a t e s i n d i c a t e t h a t t h e s e a n i m a l s c a n m e t a b o l i z e l a c t a t e a t h i g h r a t e s . F u r t h e r m o r e , t h e m a j o r f a t e o f p l a s m a l a c t a t e i n t u n a a p p e a r s t o be o x i d a t i o n l i k e i n 14 mammalian s p e c i e s (even t h o u g h CC^ was n o t c o l l e c t e d i n t h i s s t u d y , t h e m a i n f a t e o f p l a s m a l a c t a t e i s p r o b a b l y o x i d a t i o n b e c a u s e t o t a l a c t i v i t y o f b l o o d d e c r e a s e d r a p i d l y , 14 and no i m p o r t a n t a c c u m u l a t i o n o f C c o u l d be m e a s u r e d i n 14 t i s s u e s a f t e r b o l u s i n j e c t i o n o f C - l a c t a t e ) . T h e r e f o r e , t u n a may h a v e t h e a b i l i t y t o o x i d i z e p l a s m a l a c t a t e a t h i g h r a t e s ( c o m p a r a b l e t o t h e r a t e s r e p o r t e d f o r r a t s , d o g s , and humans, when t h e a p p r o p r i a t e t e m p e r a t u r e and body s i z e c o r r e c t i o n a r e a p p l i e d ) . T h e s e f i s h may be a b l e t o a c h i e v e s u c h h i g h t u r n o v e r r a t e s b e c a u s e t h e y e v o l v e d p a r t i c u l a r a n a t o m i c and e n z y m a t i c a d a p t a t i o n s f o r h i g h - p e r f o r m a n c e swimming. T h e s e h i g h v a l u e s , however, c a n n o t f u l l y a c c o u n t f o r t h e r e p o r t e d r a t e s o f l a c t a t e c l e a r a n c e d u r i n g r e c o v e r y , s u g g e s t i n g t h a t p a r t o f t h e l a c t a t e p r o d u c e d i n w h i t e m u s c l e i s m e t a b o l i z e d i n s i t u . The C o r i c y c l e i s p r o b a b l y n o t an i m p o r t a n t pathway f o r l a c t a t e c l e a r a n c e i n t u n a . 59 CHAPTER 3: ONSET OF SUBMAXIMAL EXERCISE IN THOROUGHBRED HORSES. 3.1 INTRODUCTION T h i s c h a p t e r i s t h e f i r s t o f t h r e e d e a l i n g w i t h e x e r c i s e m e t a b o l i s m and p h y s i o l o g y o f t h o r o u g h b r e d h o r s e s . I t c h a r a c t e r i z e s t h e i r b a s i c c a r d i o v a s c u l a r and m e t a b o l i c r e s p o n s e s t o t r e a d m i l l e x e r c i s e , f o c u s s i n g on t h e e f f e c t o f e x e r c i s e on p l a s m a l a c t a t e c o n c e n t r a t i o n . I t was n e c e s s a r y t o d e t e r m i n e when p l a s m a l a c t a t e c o n c e n t r a t i o n r e a c h e s a s t e a d y s t a t e - b e c a u s e t h e b o l u s i n j e c t i o n t e c h n i q u e u s e d i n C h a p t e r 5 f o r t h e measurement o f l a c t a t e t u r n o v e r r a t e r e q u i r e s s t e a d y c o n c e n t r a t i o n . T h e r e f o r e , t h e b e h a v i o r o f l a c t a t e c o n c e n t r a t i o n had t o be known d u r i n g t h e e x e r c i s e p r o t o c o l s s e l e c t e d f o r t h i s s t u d y t o be a b l e t o d e t e r m i n e t h e i n j e c t i o n and s a m p l i n g t i m e s o f t h e k i n e t i c s e x p e r i m e n t s p e r f o r m e d i n l a t e r c h a p t e r s . The s e l e c t i o n o f low i n t e n s i t y (submaximal) e x e r c i s e was made b e c a u s e t u r n o v e r r a t e c a n o n l y be measured u n d e r s t e a d y - s t a t e c o n d i t i o n s when u s i n g t h e b o l u s i n j e c t i o n t e c h n i q u e (plasma l a c t a t e c o n c e n t r a t i o n c a n n o t be i n s t e a d y s t a t e a t h i g h e r e x e r c i s e i n t e n s i t i e s ) . As e x p l a i n e d i n t h e g e n e r a l i n t r o d u c t i o n ( C h a p t e r 1 ) , t h e n e x t two c h a p t e r s w i l l i n v e s t i g a t e c h a n g e s i n c a r d i a c o u t p u t ( C h a p t e r 4) and t h e i r e f f e c t on l a c t a t e t u r n o v e r r a t e i n t h e same a n i m a l s ( C h a p t e r 5) . 60 A t r a n s i e n t i n c r e a s e i n p l a s m a l a c t a t e c o n c e n t r a t i o n h a s been commonly o b s e r v e d i n humans a f t e r t h e i n i t i a t i o n o f submaximal e x e r c i s e (Bang, 1936; Hermansen and S t e n s v o l d , 1972; R o w e l l e t a l , 1966; S a i k i e t a l , 1 9 6 7 ) . A maximum c o n c e n t r a t i o n i s r e a c h e d a f t e r a few m i n u t e s o f work, f o l l o w e d by a d e c r e a s e b a c k t o a new s t e a d y s t a t e . R e m a r k a b l y l i t t l e a t t e n t i o n h a s been d e v o t e d t o t h e mechanisms r e s p o n s i b l e f o r t h e s e c h a n g e s . They a r e u s u a l l y i n t e r p r e t e d as t h e c o n s e q u e n c e o f a d e l a y i n t h e a c t i v a t i o n o f o x i d a t i v e m e t a b o l i s m ( S a i k i e_t a_l, 1967) w h i c h , i n t u r n , i s p r e s u m a b l y due t o t h e r e l a t i v e l y s l o w o n s e t o f t h e o x y g e n t r a n s p o r t s y s t e m . I n a d d i t i o n t o t h e o r d i n a r y p h y s i o l o g i c a l a d j u s t m e n t s t o e x e r c i s e shown by o t h e r mammals, h o r s e s h a v e t h e a b i l i t y t o g r e a t l y i n c r e a s e t h e i r h e m a t o c r i t by a c t i v e l y r e l e a s i n g r e d b l o o d c e l l s f r o m t h e i r s p l e e n ( P e r s s o n , 1 9 8 3 ) . A t t h e o n s e t o f submaximal e x e r c i s e , t h e i r o xygen t r a n s p o r t s y s t e m i s a c t i v a t e d much more r a p i d l y t h a n i n humans ( F r e g i n and Thomas, 1983; Pan e t a l _ , 1984; F o r s t e r e t a l , 1984) and t h e y show a h e a r t r a t e o v e r s h o o t w h i c h d o e s n o t o c c u r i n man (Pan e t a_l, 1 9 8 4 ) . F o r t h e s e r e a s o n s , t h e l a c t a t e o v e r s h o o t o b s e r v e d i n humans may be r e d u c e d o r a b s e n t i n h o r s e s . The aim o f t h i s s t u d y was t o c h a r a c t e r i z e t h e l a c t a t e r e s p o n s e t o s u b m a x i m a l e x e r c i s e i n an e l i t e p e r f o r m e r , t h e t h o r o u g h b r e d r a c e h o r s e , t o e s t a b l i s h i f and when a l a c t a t e s t e a d y s t a t e was r e a c h e d . As m a j o r i n d i c e s o f t h e o x y g e n t r a n s p o r t s y s t e m , h e a r t r a t e and h e m a t o c r i t were measured s i m u l t a n e o u s l y w i t h p l a s m a l a c t a t e c o n c e n t r a t i o n t o 61 d e t e r m i n e w h e t h e r the m e t a b o l i c and c a r d i o v a s c u l a r r e s p o n s e s c o i n c i d e and t o g e t some i n s i g h t s i n t o t h e p o s s i b l e c a u s e s f o r t h e o b s e r v e d p a t t e r n o f c h a n g e s i n l a c t a t e c o n c e n t r a t i o n . The e f f e c t o f e x e r c i s e i n t e n s i t y was a l s o i n v e s t i g a t e d b e c a u s e i t h a s been shown t o i n f l u e n c e t h e s t e a d y s t a t e l a c t a t e c o n c e n t r a t i o n o b t a i n e d d u r i n g work (Hermansen and S t e n s v o l d , 1 9 7 2 ) . A m odel i s p r e s e n t e d t o e x p l a i n t h e m e a sured c o n c e n t r a t i o n ' c h a n g e s i n t e r m s o f l a c t a t e f l u x e s t o and f r o m t h e p l a s m a . F i n a l l y , t h e m e t a b o l i c i n t e r m e d i a t e s o f t h e g l y c o g e n o l y t i c pathway were m e a s u r e d i n one o f t h e m a i n l o c o m o t o r y m u s c l e s t o a s s e s s w hether a s t e a d y s t a t e was a l s o e s t a b l i s h e d i n t h i s t i s s u e . M u s c l e b i o p s i e s were t a k e n d u r i n g t h e l a c t a t e s t e a d y s t a t e i n p l a s m a t o d e t e r m i n e w h e t h e r p l a s m a measurements r e f l e c t m u s c l e l a c t a t e b e h a v i o r . 62 3.2 MATERIALS AND METHODS 3.2.1 EXPERIMENTAL ANIMALS AND CATHETERIZATION. A l l e x p e r i m e n t s were c a r r i e d o u t i n t h e summer o f 1985 a t t h e e x e r c i s e u n i t o f the^ A n i m a l H e a l t h T r u s t , Newmarket, UK. T h r e e t h o r o u g h b r e d h o r s e s ( K J , SB, and JW) were e x e r c i s e d 6 d a y s a week f o r t h r e e months. T h e i r r e g u l a r t r a i n i n g i n t h e f i e l d c o n s i s t e d o f w a l k i n g and t r o t t i n g w i t h an o c c a s i o n a l c a n t e r o r g a l l o p , and t h i s was s u p p l e m e n t e d w i t h t r e a d m i l l work a p p r o x i m a t e l y t w i c e a week t o f a m i l i a r i z e them w i t h t h e e x p e r i m e n t a l s e t - u p . A f o u r t h t h o r o u g h b r e d (IN) was o n l y u s e d f o r r e s t i n g v a l u e s and f o r p r e - and p o s t - c a n t e r m u s c l e b i o p s i e s , o one month a f t e r h a v i n g b een i n t r o d u c e d t o t r e a d m i l l e x e r c i s e . A l l h o r s e s were g e l d i n g s and t h e y were m a i n t a i n e d on a d i e t o f d r y f o o d and h a y w i t h u n l i m i t e d a c c e s s t o w a t e r . T h e i r w e i g h t s and ages a r e g i v e n i n T a b l e 4. B e f o r e c a t h e t e r i z a t i o n , a s m a l l a r e a o f s k i n was s h a v e d and d i s i n f e c t e d a t t h e s i t e o f c a t h e t e r i n t r o d u c t i o n j u s t above t h e j u g u l a r v e i n . L o c a l a n a e s t h e t i c was i n j e c t e d s u b c u t a n e o u s l y (3 X 0.5 ml 2% l i g n o c a i n e ; 1:200,000 e p i n e p h r i n e ) . A s t a b i n c i s i o n was made and an I.V. c a t h e t e r ( B e c t o n - D i c k i n s o n 14 g w i t h 17 g X 8 i n . i n n e r n e e d l e ) was i n t r o d u c e d i n t o t h e v e i n . The c a t h e t e r was s u t u r e d t o t h e s k i n and c o n n e c t e d t o a 100-cm e x t e n s i o n l i n e ( L e c t r o f l e x Vygon PVC, 2 ml c a p a c i t y ) w h i c h was f i l l e d w i t h s a l i n e and t i g h t l y t a p e d a r o u n d t h e neck o f t h e a n i m a l . No h e p a r i n was added, b u t t h e e x t e n s i o n l i n e and t h e c a t h e t e r were f l u s h e d e v e r y 10-15 min t o keep them p a t e n t . 63 3.2.2 EXERCISE PROTOCOLS. A f t e r c a t h e t e r i z a t i o n , t h e h o r s e was f i t t e d w i t h a s a f e t y h a r n e s s , h e a r t r a t e m o n i t o r e l e c t r o d e s (EQB-HR7 h e a r t r a t e m e t e r ) , and ECG l e a d s . He was w a l k e d t o t h e t r e a d m i l l ( S a t o , BIAB I n d u s t r i a l , L u d v i k a , Sweden) where t h e h a r n e s s was s e c u r e d a bove him t o an emergency s w i t c h - o f f mechanism (which was n e v e r t r i g g e r e d i n t h e p r e s e n t e x p e r i m e n t s ) . A f a n was p l a c e d i n f r o n t o f t h e a n i m a l t o s i m u l a t e n o r m a l a i r c o o l i n g e x p e r i e n c e d d u r i n g o u t d o o r l o c o m o t i o n . A l l e x p e r i m e n t s were c a r r i e d o u t between 10 am and 3 pm, a t l e a s t 3 h a f t e r t h e m o r n i n g f e e d . T e m p e r a t u r e r a n g e d 0 f r o m 16 t o 20 C and r e l a t i v e h u m i d i t y f r o m 53 t o 73%. D i f f e r e n t c o m b i n a t i o n s o f s p e e d and i n c l i n e were t r i e d f o r e a c h h o r s e b e f o r e s e l e c t i n g two s u b m a x i m a l e x e r c i s e r e g i m e s e l i c i t i n g c o m f o r t a b l e p e r f o r m a n c e s a t b o t h t r o t t i n g and c a n t e r i n g g a i t s , ( i ) A 3-4 m/s t r o t a t 6% i n c l i n e s u s t a i n a b l e f o r 40 min, and ( i i ) a 6.5 m/s h o r i z o n t a l c a n t e r f o r 10-15 min, w h i c h r e p r e s e n t s l e s s t h a n 50% o f t h e i r maximal s p e e d on t h e t r e a d m i l l f o r t h i s i n c l i n e . A l l e x e r c i s e b o u t s were i n i t i a t e d w i t h a s h o r t warm-up p e r i o d c o n s i s t i n g o f a 4-min walk a t 1.6 m/s f o r t h e t r o t p r o t o c o l and a 4-min walk (1.6 m/s) f o l l o w e d by a 4-min t r o t (4 m/s) f o r t h e c a n t e r p r o t o c o 1 . 3.2.3 BLOOD AND TISSUE SAMPLING. B l o o d samples were c o l l e c t e d i n i c e - c o l d 7-ml v a c u t a i n e r s c o n t a i n i n g 0.07 ml e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) as a n t i c o a g u l a n t and p l a c e d on i c e . They were c a r e f u l l y m i x e d b e f o r e f i l l i n g 64 h e m a t o c r i t t u b e s t o a v o i d p r o b l e m s c a u s e d by t h e f a s t s e d i m e n t a t i o n o f h o r s e r e d c e l l s . A f t e r m e a s u r i n g h e m a t o c r i t on a m i c r o c e n t r i f u g e , t h e r e s t o f t h e sample was spun and t h e 0 p l a s m a was s e p a r a t e d . I t was p l a c e d a t -20 C b e f o r e f u r t h e r a n a l y s i s , and t h e e n t i r e p r o c e d u r e was c o m p l e t e d w i t h i n 10 min o f s a m p l i n g . A r e s t i n g b l o o d sample (7 ml) was t a k e n b e f o r e t h e h o r s e was b r o u g h t on t h e t r e a d m i l l . D u r i n g t h e t r o t p r o t o c o l , b l o o d was drawn e v e r y min f o r t h e f i r s t 10 min o f e x e r c i s e and e v e r y 5 min t h e r e a f t e r (each sample was drawn i n a b o u t 5 s ) . D u r i n g t h e c a n t e r p r o t o c o l , s a m p l e s were t a k e n e v e r y min t h r o u g h o u t t h e e x e r c i s e b o u t . The l i n e was a l w a y s f l u s h e d w i t h s a l i n e between s u c c e s s i v e s a m p l e s . B i o p s i e s f r o m t h e m i d d l e g l u t e a l m u s c l e were t a k e n f r o m t h e 4 h o r s e s b e f o r e and a f t e r t h e c a n t e r p r o t o c o l by t h e use o f p r e v i o u s l y d e s c r i b e d p r o c e d u r e s (Snow, 1 9 8 3 ) . B e f o r e t a k i n g t h e r e s t i n g b i o p s y ( p r e - c a n t e r ) , t h e a n i m a l was a l l o w e d t o s t a n d q u i e t l y i n a s t a l l f o r 15 m i n . S u c t i o n was a p p l i e d w h i l e s a m p l i n g and two m u s c l e s p e c i m e n s were p o o l e d e a c h t i m e t o o b t a i n t h e 200-300 mg o f t i s s u e n e c e s s a r y t o p e r f o r m a l l t h e m e t a b o l i t e a s s a y s . The b i o p s i e s were i m m e d i a t e l y f r o z e n i n l i q u i d N 2 » The p o s t - c a n t e r m u s c l e s a m p l e s were f r o z e n l e s s t h a n 1.5 min a f t e r the^ c e s s a t i o n of e x e r c i s e . The t i s s u e s a m p l e s were l a t e r g r o u n d i n l i q u i d N,, w i t h m o r t a r and p e s t l e . The f i n e powder o b t a i n e d was i m m e d i a t e l y mixed w i t h 6-7 p a r t s o f i c e - c o l d 8% p e r c h l o r i c a c i d and h o m o g e n i z e d f o r 2 X 15 s e c on an U l t r a - T u r r a x 65 h o m o g e n i z e r (Ika-Werk, West Germany). Two 1 0 0 - u l a l i q u o t s o f th e s u s p e n s i o n were f r o z e n i n l i q u i d f o r g l y c o g e n d e t e r m i n a t i o n s . The r e s t was spun a t 11,000 g f o r 3 min a t 0 4 C i n E p p e n d o r f t u b e s and t h e s u p e r n a t a n t s were k e p t i n l i q u i d N 2 u n t i l t h e m e t a b o l i t e a s s a y s were p e r f o r m e d . 3.2.4 METABOLITE ASSAYS. A l l t h e m e t a b o l i c i n t e r m e d i a t e s were a s s a y e d i n d u p l i c a t e on a Pye-Unicam s p e c t r o p h o t o m e t e r (SP6-500) by m e a s u r i n g t h e r e d u c t i o n o f NAD (or NADP) o r t h e o x i d a t i o n o f NADH (or NADPH) a t 340 nm, u s i n g t h e p r o c e d u r e s d e s c r i b e d i n Bergmeyer (1974) as m o d i f i e d f o r m i c r o - a s s a y s ( t o t a l c u v e t t e volume 0 . 5 - 0 . 7 m l ) . L a c t a t e c o n c e n t r a t i o n was me a s u r e d i n p l a s m a w i t h i n t h r e e d a y s o f s a m p l i n g . M u s c l e g l y c o g e n c o n c e n t r a t i o n was d e t e r m i n e d w i t h t h e a m y l o g l u c o s i d a s e t e c h n i q u e (Bergmeyer, 1974) and c o r r e c t e d f o r i n c o m p l e t e h y d r o l y s i s as measured w i t h r a b b i t m u s c l e g l y c o g e n s t a n d a r d s ( e f f i c i e n c y = 30.0%). G l u c o s e , g l u c o s e - 6 - p h o s p h a t e , f r u c t o s e - 6 - p h o s p h a t e , f r u c t o s e - 1 , 6 - b i s p h o s p h a t e , g l y c e r a l d e h y d e - 3 - p h o s p h a t e , d i h y d r o x y a c e t o n e - p h o s p h a t e , 3 - p h o s p h o g l y c e r a t e , 2 - p h o s p h o g l y c e r a t e , p h o s p h o e n o l p y r u v a t e , p y r u v a t e , and l a c t a t e were a l s o m e a s u r e d i n t h e m u s c l e s a m p l e s . 66 3.3 RESULTS. R e s t i n g v a l u e s f o r p l a s m a l a c t a t e c o n c e n t r a t i o n , h e a r t r a t e and h e m a t o c r i t o f t h e t h o r o u g h b r e d s u s e d i n t h i s s t u d y a r e p r e s e n t e d i n T a b l e 4 w i t h w e i g h t s and a g e s . 3.3.1 TROT PROTOCOL. R e s u l t s f r o m t h e 40-min t r o t e x p e r i m e n t s a r e summarized i n F i g . 6. KJ and SB were r u n n i n g a t 4 m/s, b u t a l o w e r s p e e d o f 3 m/s was c h o s e n f o r JW, t h e o l d e r h o r s e , who c o u l d n o t s u s t a i n 4 m/s o v e r 40 min w i t h o u t e x p e r i e n c i n g a r t i c u l a r p a i n c a u s e d by p o u n d i n g on t h e r e l a t i v e l y h a r d s u r f a c e o f t h e t r e a d m i l l b e l t . The c h o i c e o f t h i s l o w e r v e l o c i t y was n o t a c o n s e q u e n c e o f h i s s t a t e o f t r a i n i n g . Mean r e s t i n g l a c t a t e c o n c e n t r a t i o n was 0.45 mM. A t the end o f t h e 4 min warm-up walk (= t i m e 0 on F i g . 6 ) , t h i s c o n c e n t r a t i o n was unchanged (KJ) o r s l i g h t l y e l e v a t e d (KJ and S B ) . When t h e t r o t was s t a r t e d , l a c t a t e c o n c e n t r a t i o n i n c r e a s e d t o a maximum o f 1.5 t o 2 mM a f t e r 2 min o f r u n n i n g , d e c l i n e d t o l e v e l s a bove t h o s e a t r e s t a f t e r 6 t o 7 m i n , and s t a y e d c o n s t a n t and low f o r a b o u t 15 min t h e r e a f t e r . L a c t a t e s t a r t e d t o a c c u m u l a t e s l o w l y i n p l a s m a d u r i n g t h e l a s t 15-20 min o f t h e e x e r c i s e b o u t and r e a c h e d 2-3 mM a t t h e end o f t h e 40-min t r o t . R e s t i n g h e a r t r a t e a v e r a g e d 35.5 b e a t s / m i n . The p r e l i m i n a r y walk was s u f f i c i e n t t o d o u b l e (JW) o r e v e n t r i p l e t h a t r a t e (KJ and S B ) . An a d d i t i o n a l 40 b e a t s / m i n i n c r e a s e was s e e n a f t e r t h e f i r s t 2 min o f t r o t b e f o r e h e a r t r a t e 67 TABLE 4. WEIGHT, AGE, AND RESTING VALUES FOR PLASMA LACTATE CONCENTRATION, HEART RATE AND HEMATOCRIT IN FOUR TRAINED THOROUGHBRED HORSES. HORSE WEIGHT AGE LACTATE HEART RATE HEMATOCRIT kg y mM b e a t s / m i n % KJ SB JW IN 453 426 466 428 4 5 13 3 0.5 0.4 0.4 0.5 41 44 32 25 37 41 43 33 68 FIG. 6 Plasma l a c t a t e c o n c e n t r a t i o n , h e a r t r a t e , and h e m a t o c r i t o f 3 t r a i n e d t h o r o u g h b r e d h o r s e s d u r i n g a 40-min t r o t a t 6% i n c l i n e on a t r e a d m i l l (KJ and SB: 4 m/s; JW: 3 m/s). Time 0 when t r e a d m i l l speed s t a r t e d t o be i n c r e a s e d from a 4 min warm-up walk a t 1.6 m/s. T r a n s i t i o n from 1.6 t o 4 m/s (or 3 m/s f o r JW) was completed i n 20 s. Note t h a t on a l l g r a p h s , the time s c a l e changes a f t e r 10 min. T r i a n g l e s r e p r e s e n t r e s t i n g v a l u e s . HCT % HR beats/min LACTATE mM 69 70 d e c r e a s e d t o a s t a b l e v a l u e o f 120 b e a t s / m i n (JW) and 140 b e a t s / m i n (KJ and SB) w h i c h was s u s t a i n e d f o r a p p r o x i m a t e l y 15 m i n . D u r i n g t h e l a s t 15-20 min o f t h e t r o t , h e a r t r a t e s l o w l y i n c r e a s e d w i t h o u t r e a c h i n g t h e maximum o b t a i n e d a f t e r 2 m i n . Mean h e m a t o c r i t was 38.5% i n r e s t i n g a n i m a l s . The warm-up walk i n c r e a s e d t h i s v a l u e t o 52.0% and a maximum of 57.7% was r e a c h e d a f t e r 3 min o f t r o t t i n g . H e m a t o c r i t s t e a d i l y d e c r e a s e d t h e r e a f t e r a t a v e r y s l o w r a t e u n t i l t h e end o f t h e 40-min e x e r c i s e b o u t (bottom g r a p h s on F i g . 6 ) . The s l o p e s o f t h e s e d e c r e m e n t s and t h e i r s t a t i s t i c a l s i g n i f i c a n c e a r e p r e s e n t e d i n T a b l e 5. 3.3.2 CANTER PROTOCOL. A l l h o r s e s were r u n n i n g a t 6.5 m/s b u t t h e o l d e r h o r s e (JW) c o u l d n o t c o m f o r t a b l y s u s t a i n t h a t s p e e d f o r more t h a n 10 m i n . Plasma l a c t a t e c o n c e n t r a t i o n , h e a r t r a t e and h e m a t o c r i t d u r i n g t h e c a n t e r e x p e r i m e n t s a r e shown i n F i g . 7. L a c t a t e c o n c e n t r a t i o n was s l i g h t l y e l e v a t e d ( t o a b o u t 1 mM) compared w i t h r e s t i n g v a l u e s a f t e r t h e p r e l i m i n a r y walk and t r o t . The p a t t e r n s e e n i n t h e f i r s t few m i n u t e s o f c a n t e r was s i m i l a r t o what was o b s e r v e d f o r t h e t r o t . Maximum c o n c e n t r a t i o n s were a l s o r e a c h e d a t 3-4 min b u t t h e y were h i g h e r t h a n i n t h e t r o t (2-3 mM). The t i m e n e c e s s a r y t o r e a c h a new s t e a d y l a c t a t e c o n c e n t r a t i o n was g r e a t e r i n t h e c a n t e r p r o t o c o l (8-10 min vs 6-7 min i n t h e t r o t p r o t o c o l ) . H e a r t r a t e was a p p r o x i m a t e l y t r i p l e d d u r i n g the warm-up p e r i o d and r e a c h e d 170 b e a t s / m i n i n KJ and SB a f t e r 71 2-3 min o f c a n t e r i n g . E v e n t h o u g h JW was r u n n i n g a t t h e same s p e e d as t h e o t h e r h o r s e s , t h e h i g h e s t h e a r t r a t e he r e a c h e d was o n l y 135 b e a t s / m i n . F o r a l l a n i m a l s , h e a r t r a t e s s t a b i l i z e d a t some l o w e r v a l u e s between 120 and 140 b e a t s / m i n a f t e r 5-10 min a t 6.5 m/s. The h i g h e s t h e m a t o c r i t s s e e n i n t h i s s t u d y were m e a s u r e d a f t e r 3 min o f c a n t e r i n g . KJ r e a c h e d an a b s o l u t e v a l u e o f 61%, w h i c h r e p r e s e n t s a more t h a n 1.5 f o l d i n c r e a s e o v e r h i s r e s t i n g l e v e l o f 37%. JW and SB r e a c h e d 56 and 59% r e s p e c t i v e l y . A f t e r p e a k i n g a t 3 min, h e m a t o c r i t s i g n i f i c a n t l y d e c r e a s e d i n a l l h o r s e s u n t i l t h e end o f e x e r c i s e ( l i n e a r r e g r e s s i o n s and ANOVAS a r e g i v e n i n T a b l e 5) • 3.3.3 MUSCLE METABOLITE CONCENTRATIONS. The l e v e l s o f g l y c o g e n and o f t h e g l y c o g e n o l y t i c i n t e r m e d i a t e s i n t h e m i d d l e g l u t e a l m u s c l e b e f o r e and a f t e r t h e c a n t e r p r o t o c o l a r e g i v e n i n T a b l e 6. The v a l u e s o b t a i n e d f o r JW, who r a n o n l y f o r 10 min, were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e mean c o n c e n t r a t i o n s o b t a i n e d f o r t h e 3 o t h e r h o r s e s . T h e r e f o r e , r e s u l t s f r o m a l l 4 a n i m a l s were p o o l e d . E x e r c i s e d i d n o t c a u s e s i g n i f i c a n t c h a n g e s i n m e t a b o l i t e c o n c e n t r a t i o n s ( p a i r e d t - t e s t , p > 0.05) e x c e p t f o r g l y c o g e n w h i c h showed a 30% d e c r e a s e compared w i t h p r e - e x e r c i s e v a l u e s (p < 0.05). Even l a c t a t e c o n c e n t r a t i o n was n o t i n c r e a s e d a f t e r 15 min a t 6.5 m/s (p = 0 . 8 5 ) . 72 TABLE 5. S T A T I S T I C A L ANALYSIS OF THE DECLINE IN HEMATOCRIT AFTER 3-4 MIN OF TREADMILL EXERCISE IN THOROUGHBRED HORSES. L i n e a r r e g r e s s i o n s were p e r f o r m e d on t h e h e m a t o c r i t vs t i m e r e l a t i o n s h i p s o f F i g . 6 and F i g . 7 between t h e t i m e when maximum h e m a t o c r i t was r e a c h e d and t h e end o f t h e e x e r c i s e b o u t . ANOVA was u s e d t o d e t e r m i n e t h e p r o b a b i l i t y t h a t t h e s l o p e s a r e e q u a l t o 0. R e f e r t o methods f o r e x e r c i s e c ond i t i o n s . 0 N R HORSE SLOPE SLOPE KJ SB JW 0.22 < 0.001 0.18 < 0.001 0.06 0.06 0.61 < 0.001 0.31 < 0.01 0.18 < 0.01 73 F I G . 7 Same as F i g . 6, b u t d u r i n g a h o r i z o n t a l c a n t e r a t 6.5 m/s f o r a l l h o r s e s . Time 0 when t r e a d m i l l s p e e d s t a r t e d t o be i n c r e a s e d f r o m a 4 min warm-up t r o t a t 4 m/s. T r a n s i t i o n f r o m 4 t o 6.5 m/s was c o m p l e t e d i n 20 s. HCT % HR beats/min LACTATE mM 75 TABLE 6. METABOLITE CONCENTRATIONS BEFORE AND AFTER A 15 MIN CANTER (AT 6.5 m/s) IN THE MIDDLE GLUTEAL MUSCLE OF THOROUGHBRED HORSES RUNNING ON A TREADMILL. V a l u e s a r e means _+ SEM (n = 4) . C o n c e n t r a t i o n s a r e g i v e n i n umol/g wet w e i g h t . G l y c o g e n . g i v e n i n g l u c o s y l u n i t s . P r e - and p o s t - e x e r c i s e v a l u e s were compared w i t h a p a i r e d t - t e s t * ( i n d i c a t e s p < 0.05) METABOLITE PRE-EXERCISE POST-EXERCISE * G l y c o g e n 131 .7 + 8. .7 96 .3 + 17 .0 G l u c o s e 0. 31 + 0. .082 0. 30 + 0. 106 G l u c o s e 6 - p h o s p h a t e 0. 56 + 0. , 093 0. 56 + 0. 089 F r u c t o s e 6 -phosphate 0. 08 + 0. ,014 0. 06 + 0. 017 F r u c t o s e 1 , 6 - b i s p h o s p h a t e 0. 25 + 0. .050 0. 25 + 0. 023 G l y c e r a l d e h y d e 3 - p h o s p h a t e 0- 03 + 0. . 005 0. 04 + 0. 005 D i h y d r o x y a c e t o n e p h o s p h a t e 0. 06 + 0. .005 0. 07 + 0. 006 3 - P h o s p h o g l y c e r a t e 0. 17 + 0. .016 0. 20 + 0. 010 2 - P h o s p h o g l y c e r a t e 0. 03 + 0. .008 0. 03 + 0. 007 P h o s p h o e n o l p y r u v a t e 0. 11 + 0. .009 0. 11 + 0. 007 P y r u v a t e 0. 10 + 0. ,012 0. 11 + 0. 007 L a c t a t e 2. 73 + 0. . 13 2. 76 + 0. 04 76 3 . 4 DISCUSSION T h i s s t u d y a l l o w e d t h e s i m u l t a n e o u s measurement o f s h o r t - t e r m c h a n g e s i n p l a s m a l a c t a t e c o n c e n t r a t i o n , h e m a t o c r i t and h e a r t j a t e o f t h o r o u g h b r e d r a c e h o r s e s u n d e r w e l l c o n t r o l l e d e x e r c i s e c o n d i t i o n s . R a p i d d r a w i n g o f m u l t i p l e b l o o d s a m p l e s was made p o s s i b l e by t h e u s e o f c a t h e t e r i z e d a n i m a l s r u n n i n g on a l a r g e t r e a d m i l l . The t i m e c o u r s e o f t h e h e m a t o c r i t r e s p o n s e i n t h o r o u g h b r e d s i s p r e s e n t e d h e r e f o r t h e f i r s t t i m e . I n s p i t e o f t h e i r a b i l i t y t o m o b i l i z e t h e c a r d i o v a s c u l a r s y s t e m v e r y r a p i d l y , t h e y showed a marked p l a s m a l a c t a t e o v e r s h o o t w h i c h a p p e a r e d t o f o l l o w a p a t t e r n s i m i l a r t o what had been r e p o r t e d p r e v i o u s l y i n humans (Bang, 1936; Hermansen and S t e n s v o l d , 1972; R o w e l l e t a l , 1966; S a i k i ejt a_l, 1967) and i n dogs ( I s s e k u t z e_t a l , 1 9 7 6 ) . However, t h e low s a m p l i n g f r e q u e n c i e s o f t h e s e e a r l i e r s t u d i e s do n o t p r o v i d e t h e n e c e s s a r y t i m e r e s o l u t i o n t o make c a r e f u l c o m p a r i s o n s . The i n t e n s i t y o f e x e r c i s e had a marked e f f e c t on t h e p a t t e r n s o f c h a n g e s f o r a l l p a r a m e t e r s i n v e s t i g a t e d . When work i n t e n s i t y was i n c r e a s e d : 1) t h e maxima r e a c h e d d u r i n g t h e o n s e t o f e x e r c i s e were h i g h e r f o r p l a s m a l a c t a t e c o n c e n t r a t i o n , h e a r t r a t e and h e m a t o c r i t ; 2) l a c t a t e c o n c e n t r a t i o n and h e a r t r a t e t o o k l o n g e r t o r e a c h a s t e a d y s t a t e ; and 3) t h e s t e a d y s t a t e l a c t a t e c o n c e n t r a t i o n and s t e a d y s t a t e h e a r t r a t e e s t a b l i s h e d w h i l e e x e r c i s i n g were b o t h e l e v a t e d . 77 3.4.1 CHANGES IN HEMATOCRIT DURING EXERCISE. The a b i l i t y o f h o r s e s t o d r a s t i c a l l y i n c r e a s e h e m a t o c r i t when o x y g e n c o n s u m p t i o n goes up i s one o f t h e i r most s t r i k i n g a d a p t a t i o n s f o r h i g h - s p e e d l o c o m o t i o n . More t h a n one t h i r d o f t h e t o t a l number o f . e r y t h r o c y t e s c a n be l i b e r a t e d f r o m t h e s p l e e n v i a a d r e n e r g i c s t i m u l a t i o n ( P e r s s o n , 1 9 8 3 ) . The s i z e o f t h e s p l e n i c r e s e r v o i r and c o n s e q u e n t l y t h e m a g n i t u d e o f t h e h e m a t o c r i t r e s p o n s e t o e x e r c i s e a r e i n c r e a s e d by t r a i n i n g (Lykkeboe e_t a_l, 1977) . The t h o r o u g h b r e d s u s e d h e r e were a b l e t o b r i n g t h e i r h e m a t o c r i t f r o m a mean r e s t i n g v a l u e o f 38.5% t o 52.0% a f t e r w a l k i n g f o r 4 m i n . T h r e e a d d i t i o n a l m i n u t e s o f t r o t c a u s e d an i n c r e a s e t o 57.7% and v a l u e s above 60% were r e a c h e d a f t e r 3 min o f s u b s e q u e n t c a n t e r i n g . S t a n d a r d b r e d h o r s e s show s i m i l a r i n c r e a s e s t o t h o s e m e a s u r e d h e r e ( P e r s s o n e t a l , 1973; Pan e t a_l, 1984) and h e m a t o c r i t s as h i g h as 66% h a v e been r e p o r t e d f o r t h o r o u g h b r e d h o r s e s a f t e r r a c i n g (Snow e t a l , 1 9 8 3 ) . H e m a t o c r i t a d j u s t m e n t s a r e l i k e l y t o o c c u r e v e n more r a p i d l y d u r i n g maximal e x e r c i s e t h a n what was o b s e r v e d h e r e f o r submaximal work. A t r u e s t e a d y s t a t e was n o t e s t a b l i s h e d f o r h e m a t o c r i t . A f t e r r e a c h i n g maximum v a l u e s , a s l o w b u t s t e a d y d e c r e a s e was measured a t b o t h work i n t e n s i t i e s . T h i s s l o w d r i f t c o u l d be due t o f l u i d s h i f t i n t o t h e p l a s m a compartment o r t o a r e - s e q u e s t e r i n g o f t h e r e d c e l l s by t h e s p l e e n . P r e l i m i n a r y measurements show t h a t t o t a l p l a s m a p r o t e i n c o n c e n t r a t i o n and h e m a t o c r i t seem t o d e c r e a s e i n p a r a l l e l . T h i s o b s e r v a t i o n s u g g e s t s t h a t a s l o w i n f l u x o f w a t e r i n t o p l a s m a i s p r o b a b l y r e s p o n s i b l e f o r t h e d e c l i n e i n h e m a t o c r i t . 78 A t t h e end o f t h e t r o t p r o t o c o l , h e a r t r a t e s t a r t e d t o i n c r e a s e w h i l e h e m a t o c r i t d e c r e a s e d , s h o w i n g t h a t b o t h v a r i a b l e s c o u l d n o t be r e g u l a t e d by c i r c u l a t i n g c a t e c h o l a m i n e s a t t h a t s t a g e o f t h e e x e r c i s e b o u t . Humans c a n a l s o s l i g h t l y i n c r e a s e t h e i r h e m a t o c r i t i n r e s p o n s e t o e x e r c i s e ( H o l l m a n and K a s t n e r , 1969) b u t no r e s p o n s e was f o u n d i n r a t s ( B r o o k s and Donovan, 1983) . The l a r g e h e m a t o c r i t a d j u s t m e n t s e e n h e r e i n t h o r o u g h b r e d h o r s e s s h o u l d a l l o w them t o augment V0 2max by 50% i f t h e c o n s e q u e n t c h a n g e i n b l o o d v i s c o s i t y does n o t l o w e r t h e i r maximum c a r d i a c o u t p u t (see C r o w e l l and S m i t h , 1967) . H o r s e s may m a i n t a i n low h e m a t o c r i t s a t r e s t t o r e d u c e t h e work o f th e h e a r t , o n l y a l l o w i n g h i g h e r v i s c o s i t y when h i g h oxygen c a r r y i n g c a p a c i t y i s needed f o r a c t i v i t y . 3 . 4 . 2 PLASMA LACTATE CONCENTRATION AT THE START OF EXERCISE. D u r i n g t h e o n s e t o f work, t h e o v e r a l l p a t t e r n s o f c h a n g es o b s e r v e d f o r p l a s m a l a c t a t e c o n c e n t r a t i o n and h e a r t r a t e were t h e same. The t h r e e v a r i a b l e s e x a m i n e d p e a k e d s i m u l t a n e o u s l y a f t e r 2 t o 3 m i n u t e s o f submaximal e x e r c i s e . The r a p i d a c t i v a t i o n o f t h e c a r d i o v a s c u l a r s y s t e m f o u n d h e r e i n t h o r o u g h b r e d s ( s t r o n g h e m a t o c r i t r e s p o n s e and h e a r t r a t e o v e r s h o o t ) d i d n o t p r e v e n t t h e s e a n i m a l s f r o m r e l y i n g on a n a e r o b i c m e t a b o l i s m i n t h e e a r l y s t a g e s o f e x e r c i s e . They showed a l a c t a t e o v e r s h o o t e q u i v a l e n t t o what had been r e p o r t e d i n humans. A t t h e o n s e t o f work, oxygen t r a n s p o r t by 79 t h e b l o o d was p r o b a b l y n o t t h e l i m i t i n g f a c t o r f o r o x i d a t i v e m e t a b o l i s m o f m u s c l e c e l l s b e c a u s e t h e same p a t t e r n o f c h a n g e s i n l a c t a t e c o n c e n t r a t i o n c a n be e l i c i t e d by v e r y d i f f e r e n t c a r d i o v a s c u l a r r e s p o n s e s ( i . e . human vs h o r s e ) . F o r i n s t a n c e , oxygen s u p p l y c o u l d be l i m i t e d a t t h e c e l l u l a r l e v e l d u r i n g t h e t r a n s f e r o f 0^ f r o m c a p i l l a r i e s t o m u s c l e m i t o c h o n d r i a . A t t h e s t a r t o f s u b m a x i m a l e x e r c i s e , o x i d a t i v e ATP g e n e r a t i o n d o e s n o t f u l l y s u p p o r t t o t a l ATP u t i l i z a t i o n by t h e w o r k i n g m u s c l e s . The a c c u m u l a t i o n o f l a c t a t e i n p l a s m a c o u l d be c a u s e d by t h e p r e f e r e n t i a l r e c r u i t m e n t o f f a s t - t w i t c h g l y c o l y t i c f i b e r s o r t o t h e s l o w p e r f u s i o n o f l a c t a t e - u t i l i z i n g o x i d a t i v e f i b e r s . As e x e r c i s e i s s u s t a i n e d , t h e t y p e o f f i b e r s r e c r u i t e d o r t h e p a t t e r n o f b l o o d f l o w t o t h e d i f f e r e n t f i b e r t y p e s may be a l t e r e d , c a u s i n g p l a s m a l a c t a t e t o r e a c h a new s t e a d y s t a t e . 3.4.3 LACTATE FLUXES TO AND FROM THE PLASMA. B e c a u s e a n i m a l s c o n t i n u o u s l y p r o d u c e and u t i l i z e l a c t a t e a t h i g h r a t e s (see C h a p t e r 2; B r o o k s , 1985), t h e p l a s m a c o n c e n t r a t i o n o f t h i s m e t a b o l i t e i s d e t e r m i n e d b o t h by i t s r a t e o f a p p e a r a n c e i n t o t h e c i r c u l a t i o n (R ) and by i t s r a t e o f r e m o v a l from t h e c i r c u l a t i o n ( R ^ ) • A number o f i n s t r u c t i v e i n f e r e n c e s a b o u t l a c t a t e f l u x e s c a n be drawn from t h e p a t t e r n o f c o n c e n t r a t i o n c h a n g e s r e p o r t e d i n t h i s s t u d y . A model i s p r e s e n t e d i n F i g . 8 e x p l a i n i n g t h e c o n c e n t r a t i o n c h a n g e s e l i c i t e d by c o n s t a n t i n t e n s i t y s ubmaximal e x e r c i s e i n terms o f f l u x r a t e s . T h i s model p r e d i c t s t h e f l u c t u a t i o n s o f 80 R G and d u r i n g t h e t r a n s i t i o n f r o m r e s t t o s t e a d y s t a t e e x e r c i s e . I n a r e s t i n g o r g a n i s m , l a c t a t e R G and R ^ a r e e q u a l , and p l a s m a c o n c e n t r a t i o n t h e r e f o r e s t a y s c o n s t a n t ( H e t e n y i i e t a l 1983),. A t t h e o n s e t o f e x e r c i s e , t h i s e q u i l i b r i u m i s d i s t u r b e d and l a c t a t e s t a r t s a c c u m u l a t i n g i n t h e p l a s m a compartment. F o r t h e f i r s t few m i n u t e s o f work (phase [ A ] ) , R G i s g r e a t e r t h a n R ^ b e c a u s e c o n c e n t r a t i o n i n c r e a s e s (as s e e n i n t h e f i r s t 2-3 min o f e x e r c i s e i n F i g s . 6 and 7 ) . Between p h a s e [A] and p h a s e [ B ] , t h e R and R , c u r v e s c r o s s - o v e r and R ^ i s now g r e a t e r t h a n R A t h r o u g h o u t p h a s e [B] d u r i n g w h i c h c o n c e n t r a t i o n d e c r e a s e s ( F i g . 6 and 7 ) . I n t h e f i r s t h a l f o f p h a s e [ B ] , t h e r e i s an o v e r s h o o t i n R ^ c a u s i n g t h e d i s a p p e a r a n c e o f p a r t o f t h e l a c t a t e a c c u m u l a t e d i n p h a s e [ A ] . A t t h e end o f p h a s e [ B ] , a new s t e a d y s t a t e i s o b t a i n e d where R q i s a g a i n , l i k e a t r e s t , e q u i v a l e n t t o R ^ . The e x e r c i s e f l u x r a t e s a r e however much h i g h e r t h a n a t r e s t , as r e p o r t e d f o r a l l v e r t e b r a t e s s t u d i e d t o d a t e (see C h a p t e r 2 ) . The s t e a d y s t a t e l a c t a t e c o n c e n t r a t i o n s m e asured a t r e s t and a f t e r t h e o n s e t o f e x e r c i s e were s l i g h t l y d i f f e r e n t ( F i g s . 6 and 7 ) . The n e t amount o f l a c t a t e added t o t h e p l a s m a compartment a t t h e b e g i n n i n g o f e x e r c i s e ( p h a s e s [A] and [B]) c a n be c a l c u l a t e d by s u b t r a c t i n g t h e s u r f a c e a r e a d e l i m i t e d by t h e R G and R ^ c u r v e s i n p h a s e [B] f r o m t h e s u r f a c e a r e a d e l i m i t e d i n p h a s e [A] [ t h i s q u a n t i t y w i l l be i n umoles i f R G and R ^ a r e g i v e n i n umol min 1 and i f t h e t i m e s c a l e i s i n m i n u t e s ] . 81 F I G . 8 Model p r o p o s e d f o r c h a n g e s i n t h e r a t e s o f l a c t a t e a p p e a r a n c e (Ra) and r e m o v a l (Rd) d u r i n g t h e t r a n s i t i o n f r o m r e s t t o c o n s t a n t i n t e n s i t y s ubmaximal work. E x e r c i s e i s d i v i d e d i n t h r e e p h a s e s : (A) where Ra > Rd; (B) where Rd > Ra; and (C) where Ra = Rd. The s u r f a c e a r e a i n d i c a t e d i n (A) r e p r e s e n t s t h e n e t amount o f l a c t a t e added t o t h e r a p i d l y m i x i n g p o o l d u r i n g p h a s e ( A ) . S u r f a c e a r e a i n (B) i s e q u a l t o t h e n e t amount o f l a c t a t e removed f r o m t h e same p o o l d u r i n g p h a s e ( B ) . The d i f f e r e n c e between t h e s e 2 s u r f a c e a r e a s i s d i r e c t l y p r o p o r t i o n a l t o t h e d i f f e r e n c e i n p l a s m a l a c t a t e c o n c e n t r a t i o n s between r e s t and s t e a d y s t a t e e x e r c i s e [phase ( C ) ] . Ra \ Rd / W [A] [B] [C] REST— « S U B M A X I M A l F Y F R r | 5 E T I M E 83 D u r i n g t h e l a s t 15 t o 20 min o f t h e t r o t p r o t o c o l , p l a s m a l a c t a t e c o n c e n t r a t i o n s t a r t e d t o i n c r e a s e , i n d i c a t i n g t h a t a s l i g h t i m b a l a n c e between R g and was a g a i n p r e s e n t . T h i s c o u l d h a v e been c a u s e d by f a t i g u e o f t h e o x i d a t i v e f i b e r s w h i c h a r e now r e p l a c e d by f a s t - t w i t c h g l y c o l y t i c f i b e r s . 3.4.4 MUSCLE METABOLITES. The most i n t e r e s t i n g a s p e c t o f t h e m e t a b o l i c a n a l y s i s i n t h e m i d d l e g l u t e a l m u s c l e was t h e 30% r e d u c t i o n i n g l y c o g e n c a u s e d by t h e 15 min c a n t e r ( T a b l e 6 ) . As a f u e l , g l y c o g e n h a s two p o t e n t i a l f a t e s i n w o r k i n g s k e l e t a l m u s c l e . I t c a n e i t h e r be c o n v e r t e d a n a e r o b i c a l l y t o l a c t a t e w i t h t h e n e t p r o d u c t i o n o f 3 ATP p e r g l u c o s y l u n i t , o r i t c a n be c o m p l e t e l y o x i d i z e d i n t h e m i t o c h o n d r i a (Hochachka e t a_l, 1 9 8 3 ) . The s e c o n d pathway has t h e a d v a n t a g e o f y i e l d i n g 12.3 t i m e s more ATP t h a n t h e f i r s t f o r t h e same amount o f g l y c o g e n u t i l i z e d : an i m p o r t a n t f a c t t o c o n s i d e r d u r i n g s u s t a i n e d e x e r c i s e . The p r e s e n t r e s u l t s c o u l d be i n t e r p r e t e d i n 3 ways: i ) a n a e r o b i c g l y c o g e n o l y s i s w i t h r a p i d r e l e a s e o f l a c t a t e i n t o t h e c i r c u l a t i o n f o r i t s s u b s e q u e n t m e t a b o l i s m e l s e w h e r e ; i i ) a n a e r o b i c g l y c o g e n o l y s i s i n a s m a l l number o f f a s t - t w i t c h g l y c o l y t i c f i b e r s and t r a n s f e r o f l a c t a t e t o a d j a c e n t h i g h o x i d a t i v e f i b e r s f o r c o m p l e t e o x i d a t i o n ; o r i i i ) a e r o b i c g l y c o g e n o l y s i s e x c l u s i v e l y . I f t h e f i r s t mechanism was i m p o r t a n t , h i g h e r m u s c l e l a c t a t e l e v e l s w o u l d have been o b s e r v e d a f t e r e x e r c i s e . A e r o b i c g l y c o g e n o l y s i s was p r o b a b l y t h e main pathway f o r e n e r g y m e t a b o l i s m i n t h e m i d d l e g l u t e u s , 84 p a r t i c u l a r l y i n v i e w o f i t s h i g h p r o p o r t i o n o f o x i d a t i v e f i b e r s . I n t h o r o u g h b r e d h o r s e s , i t i s composed o f 57% f a s t - t w i t c h h i g h o x i d a t i v e f i b e r s and 11% s l o w o x i d a t i v e f i b e r s (Snow, 1 9 8 3 ) . The s e c o n d mechanism d i f f e r s f r o m th e t h i r d o n l y i n t h e f a c t t h a t i t r e q u i r e s t h e c o o p e r a t i v e m e t a b o l i s m o f two f i b e r t y p e s . L a c t a t e e x c h a n g e between g l y c o l y t i c and o x i d a t i v e f i b e r s h a s been p r o p o s e d l o n g ago i n f i s h ( W i t t e n b e r g e r and D i a c i u c , 1965) and i t has r e c e n t l y r e c e i v e d more s u p p o r t i n e x e r c i s i n g mammals ( B r o o k s , 1 9 8 5 ) . The n e t e f f e c t o f t h e s e c o n d and t h i r d mechanisms i s t h e a e r o b i c breakdown o f g l y c o g e n and b o t h mechanisms c o u l d h a v e been o p e r a t i n g i n t h e m i d d l e g l u t e u s . A s s u m i n g t h a t a e r o b i c g l y c o g e n o l y s i s was t h e o n l y pathway u t i l i z e d d u r i n g t h e 15 min c a n t e r , t h i s m u s c l e was t u r n i n g o v e r ATP a t a r a t e o f 85 umol/g p e r min, a v a l u e somewhat h i g h e r t h a n what h a s been o b s e r v e d i n o t h e r s p e c i e s o p e r a t i n g a t t h e i r maximum s u s t a i n e d l e v e l . No c h a n g e s i n t h e c o n c e n t r a t i o n s o f a s e l e c t e d number o f g l y c o l y t i c i n t e r m e d i a t e s were a p p a r e n t a f t e r t h e c a n t e r r e l a t i v e t o t h e p r e - e x e r c i s e s t a t e ( T a b l e 6 ) . L a r g e c h a n g e s were measured i n r a t m u s c l e a f t e r s ubmaximal e x e r c i s e u s i n g t h e same t i m e c o u r s e f o r f r e e z i n g (Dobson, 1 9 8 6 ) . Here, t h e l a c t a t e / p y r u v a t e r a t i o was t h e same b e f o r e and a f t e r e x e r c i s e , i n d i c a t i n g t h a t t h e c y t o s o l i c r e d o x p o t e n t i a l was m a i n t a i n e d a f t e r 15 min o f work. D u r i n g t h e c a n t e r p r o t o c o l , a s t e a d y s t a t e was e s t a b l i s h e d f o r p l a s m a l a c t a t e and e x e r c i s e was i n t e r r u p t e d w h i l e p l a s m a l a c t a t e c o n c e n t r a t i o n was c o n s t a n t . T h i s o b s e r v a t i o n , t o g e t h e r w i t h t h e 85 measurements o f g l y c o l y t i c i n t e r m e d i a t e s , c o n f i r m s t h a t a t r u e m e t a b o l i c s t e a d y s t a t e was r e a c h e d and m a i n t a i n e d u n t i l t h e end o f t h e e x e r c i s e b o u t . 86 CHAPTER 4: CARDIAC OUTPUT AND OXYGEN CONSUMPTION OF EXERCISING THOROUGHBRED HORSES. 4.1 INTRODUCTION The c a r d i o v a s c u l a r a d j u s t m e n t s e l i c i t e d by e x e r c i s e a r e u s u a l l y s i m p l y i n t e r p r e t e d a s t h e o r g a n i s m ' s way t o i n c r e a s e o xygen s u p p l y t o t h e a c t i v e m u s c u l a t u r e and t o f a c i l i t a t e c a r b o n d i o x i d e r e l e a s e i n t o t h e l u n g s . The p o t e n t i a l l y i m p o r t a n t r o l e p l a y e d by t h e s e a d j u s t m e n t s i n a u g m e n t i n g t h e p r o v i s i o n o f o x i d a t i v e f u e l s t o c o n t r a c t i n g m u s c l e s , and i n a c c e l e r a t i n g t h e t r a n s p o r t o f o t h e r m e t a b o l i c end p r o d u c t s t h a n C 0 2 and h e a t has n o t been i n v e s t i g a t e d q u a n t i t a t i v e l y a t t h e w h o l e - o r g a n i s m l e v e l . I n p a r t i c u l a r , i n c r e a s i n g c a r d i a c o u t p u t s h o u l d h a v e an e f f e c t on t h e t u r n o v e r r a t e o f p l a s m a m e t a b o l i t e s a s s u g g e s t e d i n C h a p t e r 2. L a c t a t e t u r n o v e r h a s been measured i n s e v e r a l s p e c i e s , and t h e f l u x r a t e s o f t h i s m e t a b o l i t e i n and o u t o f t h e p l a s m a compartment h a v e been shown t o v a r y c o n s i d e r a b l y as t h e a c t i v i t y s t a t e o f t h e a n i m a l c h a n g e s (see C h a p t e r 2 ) . E x e r c i s e c a u s e s an i n c r e a s e i n b o t h t h e p r o d u c t i o n o f l a c t a t e and i t s u t i l i z a t i o n as an o x i d a t i v e f u e l ( B r o o k s , 1985). B e c a u s e t h e t h o r o u g h b r e d r a c e h o r s e h a s been c a r e f u l l y s e l e c t e d f o r i t s a e r o b i c and a n a e r o b i c c a p a c i t y o v e r t h r e e 87 c e n t u r i e s , i t c o u l d p r o v i d e a good model f o r t h e i n v e s t i g a t i o n o f p l a s m a l a c t a t e f l u x e s d u r i n g e x e r c i s e . I n a d d i t i o n , h o r s e s h a v e a v e r y h i g h a e r o b i c s c o p e w i t h t h e i m p r e s s i v e a b i l i t y t o i n c r e a s e t h e i r o x y g e n c o n s u m p t i o n by 3 6 - f o l d between r e s t and h i g h - i n t e n s i t y e x e r c i s e (Thomas and F r e g i n , 1981; Manohar, 1 9 8 6 ) . F u n c t i o n a l a d a p t a t i o n s o f t h e i r c a r d i o v a s c u l a r s y s t e m a l l o w them t o a l t e r m e t a b o l i c r a t e o v e r s u c h a wide r a n g e , b u t t h e e f f e c t o f work r a t e on c a r d i a c o u t p u t has o n l y been m e a s u r e d i n s t a n d a r d b r e d h o r s e s (Thomas and F r e g i n , 1981; Thomas e_t a l , 1983) and i n p o n i e s (Pan e t a_l, 1984; F o r s t e r e t a_l, 1984; Manohar, 1 9 8 6 ) . The o v e r a l l g o a l o f t h i s s t u d y ( C h a p t e r s 4 and 5) was t h e r e f o r e t o i n v e s t i g a t e t h e e f f e c t o f c h a n g e s i n c a r d i a c o u t p u t on t h e r a t e o f p l a s m a l a c t a t e t u r n o v e r i n t h e t h o r o u g h b r e d r a c e h o r s e . The p r e s e n t c h a p t e r c h a r a c t e r i z e s t h e e f f e c t s o f e x e r c i s e on c a r d i a c o u t p u t and on t h e r a t e o f oxygen d e l i v e r y . The f o l l o w i n g c h a p t e r d e a l s w i t h t h e measurement o f p l a s m a l a c t a t e t u r n o v e r i n t h e same a n i m a l s u n d e r i d e n t i c a l e x e r c i s e c o n d i t i o n s . To c o m p l e t e t h e p r e s e n t i n v e s t i g a t i o n , t h e measurement o f maximal c a r d i a c o u t p u t and VC^max was a t t e m p t e d d u r i n g h i g h - i n t e n s i t y e x e r c i s e . D a t a f o r maximal c a r d i a c o u t p u t were n o t o b t a i n e d f o r t e c h n i c a l r e a s o n s , b u t a it l o w e s t p o s s i b l e v a l u e f o r VC^max and f o r t h e a e r o b i c s c o p e o f t h o r o u g h b r e d h o r s e s c o u l d be e s t i m a t e d f r o m h e a r t r a t e s and A-V d i f f e r e n c e s i n oxygen c o n t e n t . 88 4.2 MATERIALS AND METHODS 4.2.1 ANIMALS. The t h o r o u g h b r e d h o r s e s u s e d i n t h e p r e v i o u s s t u d y (JW, K J , and SB) were a l s o u s e d i n t h e p r e s e n t e x p e r i m e n t s . T h e i r body w e i g h t s , a g e s , d i e t , and s t a t e o f t r a i n i n g a r e g i v e n i n C h a p t e r 3. 4.2.2 CATHETERIZATIONS. (A) RIGHT ATRIUM. The s k i n was s h a v e d on a s m a l l a r e a above t h e l e f t j u g u l a r v e i n and d i s i n f e c t e d . B e f o r e c a t h e t e r i z a t i o n , l o c a l a n a e s t h e s i a and s t a b i n c i s i o n were p e r f o r m e d as i n C h a p t e r 3. The v e i n was t h e n p u n c t u r e d w i t h a M o n o j e c t 200 n e e d l e (16 g X 1.5 i n . ) . A m e t a l s p r i n g g u i d e - w i r e was i n t r o d u c e d and a c a t h e t e r i n t r o d u c e r (USCI, Hemaquet AV. p e r c u t a n e o u s c a t h e t e r i n t r o d u c e r ) was i n s e r t e d i n t h e v e s s e l . A f t e r r e m o v i n g t h e g u i d e - w i r e , t h e c a t h e t e r (USCI, woven D a c r o n , c l o s e d e nd, NIH c a r d i o v a s c u l a r c a t h e t e r , s i z e 8F X 100 cm) was p a s s e d t h r o u g h t h e i n t r o d u c e r i n t o t h e r i g h t v e n t r i c l e and c o n n e c t e d t o a p r e s s u r e t r a n s d u c e r f o r v e r i f i c a t i o n o f i t s l o c a t i o n . I t was t h e n s l o w l y w i t h d r a w n i n t o t h e r i g h t a t r i u m and s u t u r e d t o th e neck o f t h e a n i m a l t o s e c u r e i t s p o s i t i o n ( t h e p r e s s u r e wave o b s e r v e d on t h e t r a n s d u c e r w o u l d c l e a r l y i n d i c a t e w h e t h e r t h e c a t h e t e r t i p was p l a c e d i n t h e p u l m o n a r y a r t e r y , r i g h t v e n t r i c l e , r i g h t a t r i u m , o r vena c a v a ) . (B) PULMONARY ARTERY. A s e c o n d c a t h e t e r i n t r o d u c e r was p l a c e d i n t h e l e f t j u g u l a r v e i n and a c a r d i a c o u t p u t c a t h e t e r ( O x i m e t r i x , o p t i c a t h model P7110-E s i z e 7.5 F, f l o w d i r e c t e d t h e r m a l d i l u t i o n p u l m o n a r y a r t e r y c a t h e t e r 89 s e r i e s D) was p l a c e d s e v e r a l i n c h e s i n t o t h e p u l m o n a r y a r t e r y and s u t u r e d i n p o s i t i o n u s i n g t h e t e c h n i q u e d e s c r i b e d p r e v i o u s l y . (C) CAROTID ARTERY. S u r g i c a l t r a n s p o s i t i o n o f t h e r i g h t common c a r o t i d a r t e r y t o a s u b c u t a n e o u s l o c a t i o n had been p e r f o r m e d on a l l h o r s e s s e v e r a l months b e f o r e t h e e x p e r i m e n t s . P r i o r t o c a t h e t e r i z a t i o n , t h e s k i n was p r e p a r e d as d e s c r i b e d a b o v e . A s t a b i n c i s i o n was made b e f o r e i n t r o d u c i n g a g u i d e - w i r e , s l i d i n g t h e c a t h e t e r i n p o s i t i o n (Vygon XRO i n t r a v a s c u l a r c a t h e t e r 18 g X 46 cm) and s u t u r i n g i t t o t h e n e c k . The s a m p l i n g p o r t was l o c a t e d a b o u t 10 cm c r a n i a l t o t h e a o r t i c a r c h . The t h r e e c a t h e t e r s were c o n n e c t e d t o e x t e n s i o n l i n e s • ( L e c t r o f l e x Vygon PVC) w h i c h were s e c u r e l y t a p e d t o t h e neck and f i l l e d w i t h s a l i n e . No h e p a r i n was u s e d b u t l i n e s and c a t h e t e r s were f l u s h e d e v e r y 10-15 min t o keep them p a t e n t . 4.2.3 EXERCISE PROTOCOLS. A l l e x p e r i m e n t s were c a r r i e d o u t a t l e a s t 3 h a f t e r t h e m o r n i n g f e e d . T e m p e r a t u r e r a n g e d f r o m 18 t o 20°C and r e l a t i v e h u m i d i t y f r o m 61 t o 71%. The r e s t i n g measurements were p e r f o r m e d w h i l e t h e a n i m a l was s t a n d i n g i n a s t a l l b e s i d e t h e t r e a d m i l l a t l e a s t 30 min a f t e r c a t h e t e r i z a t i o n . The h o r s e was t h e n f i t t e d w i t h a h a r n e s s and b r o u g h t on t h e t r e a d m i l l ( see C h a p t e r 3 ) . Four e x e r c i s e p r o t o c o l s were u s e d : ( i ) a 1.6 m/s walk a t 6% i n c l i n e , ( i i ) a 3-4 m/s t r o t a t 6% i n c l i n e s u s t a i n a b l e f o r 40 min , ( i i i ) a 6.5 m/s h o r i z o n t a l c a n t e r s u s t a i n a b l e f o r 10 t o 90 15 min, and ( i v ) a 12.5 m/s g a l l o p a t 5% i n c l i n e s u s t a i n a b l e f o r 2 m i n . T r o t , c a n t e r and g a l l o p p r o t o c o l s were i n i t i a t e d w i t h a warm-up p e r i o d c o n s i s t i n g o f a 4 min walk a t 1.6 m/s f o r t h e t r o t , a 4 min walk (1.6 m/s) f o l l o w e d by a 4 min t r o t (4 m/s) f o r t h e c a n t e r , and a 4 min walk (1.6 m/s) f o l l o w e d by a 2 min t r o t (4 m/s) f o r t h e g a l l o p . The i n c r e a s e f r o m 4 m/s t o t h e g a l l o p i n g s p e e d o f 12.5 m/s was a c h i e v e d i n 45 s . D u r i n g t h e t r o t p r o t o c o l , KJ and SB were r u n n i n g a t 4 m/s, b u t a l o w e r s p e e d o f 3 m/s was c h o s e n f o r t h e o l d e r h o r s e (JW) b e c a u s e he c o u l d n o t s u s t a i n 4 m/s o v e r 40 min w i t h o u t e x p e r i e n c i n g a r t i c u l a r p a i n c a u s e d by p o u n d i n g on t h e t r e a d m i l l b e l t . When t h i s o c c u r r e d , t h e a n i m a l w o u l d s t o p h a v i n g a r e g u l a r g a i t and he w o u l d a t t e m p t t o b r e a k i n t o a v e r y s l o w c a n t e r on r e p e a t e d o c c a s i o n s ( p r e s u m a b l y t o change t h e p a t t e r n o f s t r e s s f o r c e s a p p l i e d t o t h e a r t i c u l a t i o n s ) . T h i s a l t e r a t i o n i n t h e l o c o m o t o r y p a t t e r n was n e v e r o b s e r v e d i f t h e a n i m a l was m a i n t a i n e d a t 3 m/s o v e r 40 m i n . The c h o s e n s u b m a x i m a l e x e r c i s e r e g i m e s were d e s i g n e d t o e l i c i t c o m f o r t a b l e , s t e a d y s t a t e p e r f o r m a n c e s s u s t a i n a b l e f o r l o n g enough t o a l l o w t h e measurement o f l a c t a t e t u r n o v e r . The i n v e s t i g a t i o n o f p l a s m a l a c t a t e f l u x e s i n t h e same a n i m a l s i s p r e s e n t e d i n C h a p t e r 5. I n t h e p r e s e n t s t u d y , no s i g n i f i c a n t d i f f e r e n c e s c o u l d be f o u n d between v a l u e s m e a s u r e d i n JW and i n t h e o t h e r h o r s e s . T h e r e f o r e , d a t a f o r a l l a n i m a l s were p o o l e d . 4.2.4 CARDIAC OUTPUT AND BLOOD OXYGEN CONTENT. C a r d i a c o u t p u t was measured by t h e t h e r m o d i l u t i o n t e c h n i q u e 91 u s i n g an A m e r i c a n Edwards L a b o r a t o r i e s C a r d i a c O u t p u t Computer (9520A) and O x i m e t r i x Shaw C a t h e t e r O x i m e t e r System (OS-1270A). The computer and t h e c a t h e t e r u s e d i n t h i s s t u d y were c a l i b r a t e d w i t h a pump and t h e r m o s t a t e d b a t h a t 3 7 ° C . The m e a s u r e d f l o w r a t e s u s i n g t h e t h e r m o d i l u t i o n t e c h n i q u e were a c c u r a t e w i t h i n 8% o f t h e t r u e v a l u e s . I n human s t u d i e s , t h e t h e r m o d i l u t i o n t e c h n i q u e g i v e s t h e most a c c u r a t e measurements o f c a r d i a c o u t p u t when compared w i t h dye d i l u t i o n o r n o n - i n v a s i v e t e c h n i q u e s s u c h as t h o r a c i c impedance measurements (D. Wheeldon p e r s . comm.). I n t h e p r e s e n t s t u d y , a b o l u s o f c o l d d e x t r o s e (5%) was i n j e c t e d i n t o t h e r i g h t a t r i u m and b l o o d t e m p e r a t u r e was r e c o r d e d f r o m a t h e r m i s t o r l o c a t e d a t t h e t i p o f t h e p u l m o n a r y a r t e r y c a t h e t e r . B e f o r e i n j e c t i o n , t h e t e m p e r a t u r e o f t h e d e x t r o s e s o l u t i o n was m o n i t o r e d w i t h a r e f e r e n c e t h e r m i s t o r c o n n e c t e d t o t h e c o m p u t e r . Volumes and t e m p e r a t u r e s o f t h e d e x t r o s e b o l u s e s r a n g e d f r o m 24 ml t o 28 ml and f r o m 4.2°C t o 13.0°C r e s p e c t i v e l y . B o l u s i n j e c t i o n was p e r f o r m e d a t l e a s t 3 t i m e s f o r e a c h a n i m a l and f o r e a c h a c t i v i t y l e v e l . A r t e r i a l and m i x e d venous b l o o d s a m p l e s were drawn s i m u l t a n e o u s l y f r o m t h e r i g h t c a r o t i d and f r o m t h e p u l m o n a r y a r t e r y t o d e t e r m i n e A-V d i f f e r e n c e s i n ox y g e n c o n t e n t . Samples were t a k e n b e f o r e (A) and a f t e r e a c h measurement o f c a r d i a c o u t p u t ( B ) . Oxygen c o n t e n t c o r r e s p o n d i n g t o a g i v e n c a r d i a c o u t p u t was c a l c u l a t e d as t h e mean between s a m p l e s (A) and ( B ) . B l o o d was c o l l e c t e d i n 5 ml h e p a r i n i z e d s y r i n g e s w h i c h were t i g h t l y s e a l e d and s t o r e d on i c e . Oxygen c o n t e n t 92 was d e t e r m i n e d a t 37°C on a M i c r o A u t o c a l pH and B l o o d Gas A n a l y z e r ( I n s t r u m e n t L a b o r a t o r i e s # 613) and a CO-Oximeter ( I n s t r u m e n t L a b o r a t o r i e s # 282) u s e d i n c o m b i n a t i o n . Measurements were p e r f o r m e d no l a t e r t h a n 3 h a f t e r s a m p l i n g . H e m o g l o b i n c o n c e n t r a t i o n , p 0 2 ' P C 0 2 ' a n c ^ P H w e r e measured d i r e c t l y . H e m o g l o b i n % s a t u r a t i o n was a u t o m a t i c a l l y back c a l c u l a t e d f r o m a human h e m o g l o b i n 0 2 s a t u r a t i o n c u r v e a d j u s t e d f o r pH and PCC^. Human and h o r s e h e m o g l o b i n s h a v e v e r y s i m i l a r and d i s p l a y s i m i l a r B o hr s h i f t s ( P r o s s e r , 1975); t h e e r r o r i n t r o d u c e d by u s i n g a human d i s s o c i a t i o n c u r v e s h o u l d t h e r e f o r e be m i n i m a l . I n a d d i t i o n , t h i s e r r o r w o u l d be e l i m i n a t e d o r a t l e a s t d e c r e a s e d f o r r e l a t i v e v a l u e s s u c h as A-V d i f f e r e n c e s o r c h a n g e s between r e s t and e x e r c i s e , w h i c h were more i m p o r t a n t t o t h i s s t u d y t h a n a b s o l u t e v a l u e s . Oxygen c o n t e n t was c a l c u l a t e d as [1.34 X h e m o g l o b i n c o n c e n t r a t i o n ( i n g/100 ml) X h e m o g l o b i n % s a t u r a t i o n ] + [0.003 X p 0 2 ( i n mm H g ) ] . H e m a t o c r i t was d e t e r m i n e d by s p i n n i n g s a m p l e s i n h e m a t o c r i t t u b e s f o r 10 min on a m i c r o c e n t r i f u g e . Oxygen c o n s u m p t i o n was c a l c u l a t e d f r o m c a r d i a c o u t p u t and A-V d i f f e r e n c e i n 0 2 c o n t e n t . Measurements were p e r f o r m e d a t r e s t ( R ) , 5 min a f t e r s t a r t i n g t h e walk p r o t o c o l (W), 1 min (ST) and 7 min (ET) a f t e r t h e a n i m a l had s t a r t e d t h e t r o t p r o t o c o l , 7 min a f t e r s t a r t i n g t h e c a n t e r p r o t o c o l ( C ) , and 1 min i n t o t h e g a l l o p ( G ) . The d e t e r m i n a t i o n o f c a r d i a c o u t p u t a t t h e h i g h e s t s p e e d was n o t s u c c e s s f u l b e c a u s e t h e neck movements o f g a l l o p i n g a n i m a l s c a u s e d t o o much m e c h a n i c a l i n t e r f e r e n c e w i t h o u r t e m p e r a t u r e measurements and t h e e x a c t p o s i t i o n o f t h e c a t h e t e r s c o u l d n o t be m a i n t a i n e d w i t h c e r t a i n t y . 93 T h e r e f o r e , o n l y A-V d i f f e r e n c e i n o x y g e n c o n t e n t , h e m a t o c r i t and h e a r t r a t e c o u l d be m easured i n t h e g a l l o p . 4.2.5 CALCULATIONS AND S T A T I S T I C S . C a r d i a c o u t p u t (CO) was c a l c u l a t e d as f o l l o w s : (T f a - T.) * V CO = J T d t o Where T b = b l o o d t e m p e r a t u r e , T ^ = t e m p e r a t u r e o f t h e b o l u s i n j e c t e d , and V=volume o f t h e b o l u s . The s u r f a c e a r e a u n d e r t h e t e m p e r a t u r e vs t i m e c u r v e ( i n t e g r a l T d t f r o m t i m e 0 t o i n f i n i t y ) was d e t e r m i n e d w i t h a d i g i t i z e r (GTCO C o r p . d i g i t i z i n g pad) and an IBM c o m p u t e r . Means were compared w i t h a oneway a n a l y s i s o f v a r i a n c e . The Student-Newman-Keuls t e s t (T-method f o r u n p l a n n e d c o m p a r i s o n s , s e e S o k a l and R o h l f , 1981) was u s e d as a p o s t h o c t e s t when t h e o v e r a l l F v a l u e i n d i c a t e d t h a t f u r t h e r a n a l y s i s was n e c e s s a r y . 4.3 RESULTS 4.3.1 CARDIAC PARAMETERS. Mean v a l u e s f o r c a r d i a c o u t p u t , h e a r t r a t e and s t r o k e volume a r e p r e s e n t e d i n F i g . 9. The c a r d i a c o u t p u t s measured a t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l (ST) and l a t e r i n t h e same p r o t o c o l (ET) were i d e n t i c a l , t h e r e f o r e o n l y one v a l u e (T) i s g i v e n i n F i g . 9. 94 E x e r c i s e had a s i g n i f i c a n t e f f e c t on c a r d i a c o u t p u t (3 d f / 8 d f , F=24.6, P<0.001), h e a r t r a t e (F=33.0, P<0.001), and s t r o k e volume (F=4.2, P<0.05). C a r d i a c o u t p u t went f r o m a mean r e s t i n g v a l u e o f 66.5 1/min t o 212.3 1/min d u r i n g t h e c a n t e r p r o t o c o l , w h i c h c o r r e s p o n d s t o a change f r o m 150 t o 475 ml m i n - 1 k g - 1 . I t was s i g n i f i c a n t l y l o w e r a t r e s t compared w i t h v a l u e s m e a s u r e d d u r i n g e x e r c i s e (P<0.01) b u t mean v a l u e s f o r t h e w a l k , t r o t , and c a n t e r p r o t o c o l s were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r . H e a r t r a t e went f r o m a mean r e s t i n g v a l u e o f 49 b e a t s / m i n t o 144 b e a t s / m i n i n t h e c a n t e r p r o t o c o l ( F i g . 9 ) . D u r i n g t h e g a l l o p p r o t o c o l , h e a r t r a t e r e a c h e d 197+10 b e a t s / m i n (mean +_ SEM) and v a l u e s o f 220 t o 230 b e a t s / m i n h a v e been measured i n t h e same h o r s e s d u r i n g t r e a d m i l l e x e r c i s e ( J o y c e Harman, u n p u b l i s h e d d a t a ) , c l e a r l y i n d i c a t i n g t h a t t h e c a n t e r p r o t o c o l r e p r e s e n t e d e a s y e x e r c i s e f o r t h e s e a n i m a l s . Mean h e a r t r a t e was l o w e r a t r e s t t h a n d u r i n g any e x e r c i s e p r o t o c o l (P<0.01), and i t was s i g n i f i c a n t l y l o w e r d u r i n g t h e walk t h a n d u r i n g t h e t r o t and c a n t e r p r o t o c o l s (P<0.05). No d i f f e r e n c e was f o u n d between t r o t and c a n t e r . H e a r t r a t e was h i g h e r f o r t h e g a l l o p t h a n f o r any o t h e r a c t i v i t y l e v e l (P<0.01). S t r o k e volume was 1.34 1 a t r e s t , and i t i n c r e a s e d t o a maximum o f 1.58 1 d u r i n g t h e walk p r o t o c o l . E v e n though t h e o v e r a l l ANOVA i n d i c a t e d a s i g n i f i c a n t e f f e c t o f e x e r c i s e on s t r o k e volume, no p a r t i c u l a r mean was f o u n d to be d i f f e r e n t f r o m any o t h e r mean u s i n g Student-Newman-Keuls as a p o s t h o c t e s t . 95 F I G . 9 C a r d i a c o u t p u t (CO), h e a r t r a t e (HR) and s t r o k e volume (SV) i n t r a i n e d t h o r o u g h b r e d h o r s e s a t r e s t and d u r i n g e x e r c i s e on a t r e a d m i l l . R = r e s t ; W = walk (1.6 m/s, 6% i n c l i n e ) ; T = t r o t (3-4 m/s, 6% i n c l i n e ) ; C = c a n t e r (6.5 m/s, 0% i n c l i n e ) . D u r i n g t h e c a n t e r p r o t o c o l h e a r t r a t e r e a c h e d 197 _+ 10 b e a t s / m i n ( n o t shown on F i g . 9) . See methods f o r d e t a i l s o f t h e e x e r c i s e p r o t o c o l s . V a l u e s g i v e n a r e means + SEM (N=3). \ o o \ DC X 200 100 -150 100 50 1.5 -_ ZP -I 1.0 > 0.5 -R W 97 F I G . 10 B l o o d o x y g e n c o n t e n t i n t r a i n e d t h o r o u g h b r e d h o r s e s a t r e s t and d u r i n g t r e a d m i l l e x e r c i s e . Open b a r s : a r t e r i a l b l o o d ; b l a c k b a r s : m i x e d v e n o u s b l o o d . R = r e s t ; W = walk;. ST = s t a r t o f t r o t p r o t o c o l ; ET = e n d o f t r o t p r o t o c o l ; C = c a n t e r ; G = g a l l o p (12.5 m/s, 5% i n c l i n e ) . V a l u e s g i v e n a r e means + SEM (N=3). 98 R W ST ET C G 99 4.3.2 A-V DIFFERENCES IN OXYGEN CONTENT. The o x y g e n c o n t e n t s o f a r t e r i a l and m i x e d venous b l o o d a r e g i v e n i n F i g . 10 f o r t h e d i f f e r e n t l e v e l s o f a c t i v i t y . Mean a r t e r i a l o x y g e n c o n t e n t went f r o m a minimum o f 19.8 v o l % a t r e s t t o a maximum o f 30.0 v o l % d u r i n g t h e c a n t e r p r o t o c o l . A t 15.8 v o l % , venous o x y g e n c o n t e n t was h i g h e s t d u r i n g t h e walk p r o t o c o l , and i t d e c r e a s e d t o a low v a l u e o f 5.9 v o l % i n g a l l o p i n g t h o r o u g h b r e d s . The mean A-V d i f f e r e n c e i n oxygen c o n t e n t i n c r e a s e d d r a s t i c a l l y f r o m 4.7 v o l % a t r e s t t o 23.4 v o l % d u r i n g t h e g a l l o p p r o t o c o l . The s t a t i s t i c a l a n a l y s i s was p e r f o r m e d on t h e mean A-V d i f f e r e n c e s . The o v e r a l l e f f e c t o f e x e r c i s e was h i g h l y s i g n i f i c a n t (5 d f / 1 2 d f , F=27.52, P<0.001). P o s t hoc t e s t s show t h a t : ( i ) t h e mean A-V d i f f e r e n c e was h i g h e r d u r i n g t h e walk p r o t o c o l t h a n a t r e s t (P<0.05), ( i i ) i t was h i g h e r a t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l (ST) t h a n d u r i n g t h e walk (P<0.01), and ( i i i ) i t was h i g h e r d u r i n g t h e g a l l o p t h a n d u r i n g t h e c a n t e r p r o t o c o l (P<0.01). No o t h e r d i f f e r e n c e s between a d j a c e n t means were s i g n i f i c a n t a t P<0.05. A r t e r i a l h e m a t o c r i t was o f t e n h i g h e r t h a n venous h e m a t o c r i t , b u t t h e d i f f e r e n c e was n o t s i g n i f i c a n t . O n l y a r t e r i a l l e v e l s a r e r e p o r t e d h e r e . Mean v a l u e s a t t h e d i f f e r e n t work i n t e n s i t i e s a r e g i v e n i n F i g . 11. H e m a t o c r i t g r e a t l y i n c r e a s e d f r o m a low r e s t i n g v a l u e o f 38.1% t o 56.2% d u r i n g the e x e r c i s e p r o t o c o l o f l o w e s t i n t e n s i t y ( w a l k ) . A maximum l e v e l o f 63.4% was r e a c h e d i n t h e h o r s e s g a l l o p i n g a t 12.5 m/s. The o v e r a l l e f f e c t o f e x e r c i s e on h e m a t o c r i t was h i g h l y s i g n i f i c a n t (5 d f / 1 2 d f , F=7.45, P<0.01). The mean 100 v a l u e was l o w e r a t r e s t t h a n a t any work i n t e n s i t y (P<0.01), b u t no s i g n i f i c a n t d i f f e r e n c e s c o u l d be f o u n d between t h e v a r i o u s l e v e l s o f e x e r c i s e . 4.3.3 OXYGEN CONSUMPTION. Mass s p e c i f i c r a t e s o f o x y g e n c o n s u m p t i o n a r e g i v e n i n F i g . 12 ( t o p g r a p h ) . The mean r e s t i n g r a t e was 7.2 ml min-"'" kg -" 1", and i t i n c r e a s e d t o 85.1 ml m i n - 1 k g - 1 d u r i n g t h e c a n t e r p r o t o c o l . Whole-body o x y g e n c o n s u m p t i o n r a t e s a r e p r e s e n t e d i n t h e b o t t o m g r a p h o f F i g . 12, and t h e s t a t i s t i c a l a n a l y s i s was p e r f o r m e d on t h e s e v a l u e s . Whole-body r a t e o f o x y g e n c o n s u m p t i o n went f r o m a r e s t i n g l e v e l o f 3.2 1/min t o 38.1 1/min i n t h e c a n t e r p r o t o c o l . The o v e r a l l e f f e c t o f e x e r c i s e was h i g h l y s i g n i f i c a n t (4 d f / 1 0 d f , F=12.83, P<0.001). P o s t hoc t e s t s show t h a t o x y g e n c o n s u m p t i o n was s i g n i f i c a n t l y h i g h e r d u r i n g t h e walk p r o t o c o l t h a n a t r e s t (P<0.05), and t h a t i t was a l s o h i g h e r a t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l (ST) compared w i t h t h e walk (P<0.05). Mean c o n s u m p t i o n r a t e s d u r i n g t h e t r o t (ST and ET) and c a n t e r (C) were n o t d i f f e r e n t . 101 F I G . 11 H e m a t o c r i t (HCT) o f t r a i n e d t h o r o u g h b r e d h o r s e s a t r e s t and d u r i n g t r e a d m i l l e x e r c i s e . Same l e g e n d as i n F i g . 10. V a l u e s g i v e n a r e means + SEM (N=3). 102 H 40 - nr 20 * i i i i i i R W S T E T C G 103 F I G . 12 Oxygen c o n s u m p t i o n o f t r a i n e d t h o r o u g h b r e d h o r s e s a t r e s t and d u r i n g t r e a d m i l l e x e r c i s e . Top g r a p h : mass s p e c i f i c o xygen c o n s u m p t i o n . B o t t o m g r a p h : w h o l e - b o d y oxygen c o n s u m p t i o n . Same l e g e n d as i n F i g . 10. V a l u e s g i v e n a r e means + SEM (N=3). 104 CM 50 -I CM 40 -20 -R W ST ET C 105 4.4 DISCUSSION The c a r d i a c o u t p u t o f t h o r o u g h b r e d r a c e h o r s e s was m e a s u r e d a t r e s t and a t d i f f e r e n t l e v e l s o f submaximal e x e r c i s e t o complement our i n v e s t i g a t i o n o f p l a s m a l a c t a t e f l u x e s p r e s e n t e d i n t h e f o l l o w i n g c h a p t e r . T h i s s t u d y shows t h a t t h o r o u g h b r e d h o r s e s h a v e t h e i m p r e s s i v e a b i l i t y t o i n c r e a s e t h e i r A-V d i f f e r e n c e i n o x y g e n c o n t e n t by a t l e a s t 5 - f o l d between r e s t and m a x i m a l e x e r c i s e . They have a h i g h e r m a x i m a l s t r o k e volume t h a n s t a n d a r d b r e d h o r s e s , and t h e i r m a ximal A-V d i f f e r e n c e i n 0^ c o n t e n t i s much h i g h e r t h a n i n p o n i e s (Manohar, 1 9 8 6 ) . T h o r o u g h b r e d s c a n b r i n g t h e i r a r t e r i a l c o n t e n t up t o 30 v o l % v i a a more t h a n 50% i n c r e a s e i n c i r c u l a t i n g h e m o g l o b i n c o n c e n t r a t i o n . T h i s r e s p o n s e o f t h e i r o x y g e n t r a n s p o r t s y s t e m r e p r e s e n t s one o f t h e most s t r i k i n g a d a p t a t i o n s shown by h o r s e s f o r a e r o b i c p e r f o r m a n c e . T h o r o u g h b r e d h o r s e s may be a b l e t o augment t h e i r o x y g e n c o n s u m p t i o n by more t h a n 4 0 - f o l d between r e s t and m a x i m a l e x e r c i s e : t h e h i g h e s t a e r o b i c s c o p e i n mammals. 4.4.1 CARDIAC OUTPUT. T h o r o u g h b r e d s showed a 3 - f o l d i n c r e a s e i n c a r d i a c o u t p u t between r e s t and t h e c a n t e r p r o t o c o l . The o b s e r v e d c hange was d i r e c t l y c a u s e d by an e q u i v a l e n t i n c r e m e n t i n h e a r t r a t e b e c a u s e s t r o k e volume d i d n o t v a r y s i g n i f i c a n t l y between t h e two a c t i v i t y s t a t e s ( F i g . 9 ) . The s t r o k e volumes measured h e r e were h i g h e r t h a n p r e v i o u s l y r e p o r t e d i n s t a n d a r d b r e d h o r s e s f o r w h i c h maximum v a l u e s o n l y r e a c h 1.27 1 i n u n t r a i n e d a n i m a l s (Thomas and 106 F r e g i n , 1981) and 1.36 1 a f t e r a s h o r t p e r i o d o f t r a i n i n g (Thomas e_t a_l, 1 9 8 3 ) . S t r o k e volumes o f t h o r o u g h b r e d s r a n g e d f r o m 1.34 1 a t r e s t t o 1.58 1 d u r i n g t h e walk p r o t o c o l . I n humans, maximum s t r o k e volume c a n i n c r e a s e by up t o 20% as a r e s u l t o f i n t e n s i v e ^ e n d u r a n c e t r a i n i n g , b u t g e n e t i c d i f f e r e n c e s i n h e a r t s i z e seem t o p l a y an i m p o r t a n t r o l e i n d e t e r m i n i n g t h e maximal p o s s i b l e c a r d i a c o u t p u t o f a g i v e n i n d i v i d u a l ( B r o o k s and F a h e y , 1 9 8 4 ) . The h i g h e r maximal s t r o k e volume f o u n d i n t h o r o u g h b r e d s compared w i t h t h e s t a n d a r d b r e d s o f Thomas e t a_l (1983) c a n t h e r e f o r e be a t t r i b u t e d t o a c o m b i n a t i o n o f t r a i n i n g and g e n e t i c d i f f e r e n c e s . The v a l u e s m e a s u r e d h e r e a t r e s t do n o t r e p r e s e n t t r u e r e s t i n g l e v e l s b e c a u s e mean h e a r t r a t e (49 b e a t s / m i n ) was 10 b e a t s / m i n h i g h e r t h a n m i n i m a l r a t e s measured i n t h e same a n i m a l s u n d e r d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s (see C h a p t e r 3 ) . The t r u e r e s t i n g h e a r t r a t e o f t h e s e h o r s e s may e v e n be u n d e r 30 b e a t s / m i n , b u t s u c h h i g h l y e x c i t a b l e a n i m a l s c o u l d n o t be k e p t p e r f e c t l y q u i e t i n t h e p r e s e n c e o f s e v e r a l p e o p l e m a n i p u l a t i n g t h e d i f f e r e n t c a t h e t e r s . T h e r e i s no d o u b t t h a t t h e c a r d i a c o u t p u t o f t h o r o u g h b r e d h o r s e s c a n v a r y o v e r a much w i d e r r a n g e o f v a l u e s t h a n were r e p o r t e d i n t h i s s t u d y . U n f o r t u n a t e l y , i t c o u l d n o t be m e a s u r e d i n g a l l o p i n g a n i m a l s , and maximal v a l u e s were f a r f r o m b e i n g a t t a i n e d d u r i n g t h e c a n t e r p r o t o c o l w h i c h r e p r e s e n t s l e s s t h a n h a l f t h e t o p s p e e d t h e s e h o r s e s c a n a c h i e v e on a t r e a d m i l l . A t t h i s s u b m a x i m a l work i n t e n s i t y , mean h e a r t r a t e o n l y r e a c h e d 144 b e a t s / m i n . The same a n i m a l s were a b l e t o i n c r e a s e t h i s 107 r a t e t o 197 b e a t s / m i n d u r i n g t h e g a l l o p p r o t o c o l , and t h e y h a v e been o b s e r v e d t o g a l l o p a t maximum h e a r t r a t e s o f 220-230 b e a t s / m i n ( J o y c e Harman, u n p u b l i s h e d d a t a ) . Thomas e_t a_l (1983) r e p o r t e d a 6 . 6 - f o l d i n c r e a s e i n c a r d i a c o u t p u t f o r s t a n d a r d b r e d h o r s e s between r e s t and e x e r c i s e , b u t t h e h i g h e s t work i n t e n s i t y u s e d i n t h e i r s t u d y d i d n o t e l i c i t VC^max, and t h e s c o p e o f t h e s e a n i m a l s i s p r o b a b l y h i g h e r . E n d u r a n c e - t r a i n e d human a t h l e t e s c a n s u p p o r t an e i g h t - f o l d i n c r e a s e i n c a r d i a c o u t p u t between r e s t and V0 2max ( B r o o k s and F a h e y , 1 9 8 4 ) . R e c e n t l y , Manohar d e m o n s t r a t e d t h a t u n t r a i n e d p o n i e s have t h e a b i l i t y t o augment t h e i r c a r d i a c o u t p u t by more t h a n e i g h t f o l d between r e s t and m a x i m a l e x e r c i s e (Manohar, 1 9 8 6 ) . I t i s p o s s i b l e t o e s t i m a t e maximal c a r d i a c o u t p u t .of t h e t h o r o u g h b r e d h o r s e s u s e d i n t h e p r e s e n t s t u d y i f we make t h e c o n s e r v a t i v e a s s u m p t i o n t h a t , when g a l l o p i n g a t VC^max, t h e i r s t r o k e volume i s e q u a l t o t h e l o w e s t v a l u e r e p o r t e d h e r e ( i . e . 1.34 1 i n r e s t i n g a n i m a l s ) . W i t h t h i s s t r o k e volume and a h e a r t r a t e o f 225 b e a t s / m i n , t h e i r c a r d i a c o u t p u t w o u l d r e a c h 300 1/min. In v i e w o f t h e above c a l c u l a t i o n , and when c o n s i d e r i n g t h e s c o p e f o u n d i n p o n i e s (Manohar, 1986) and t r a i n e d humans ( B r o o k s and F a h e y , 1984), i t i s v e r y l i k e l y t h a t t h o r o u g h b r e d h o r s e s c a n a l s o u n d e r g o an 8 - f o l d i n c r e a s e i n c a r d i a c o u t p u t between r e s t and V0 2max. 4.4.2 A-V DIFFERENCE IN OXYGEN CONTENT. R e s u l t s show t h a t t h o r o u g h b r e d h o r s e s h a v e t h e c a p a b i l i t y t o i n c r e a s e t h e i r A-V d i f f e r e n c e i n o x y g e n c o n t e n t by a t l e a s t 5 - f o l d 108 between r e s t and m a x i m a l e x e r c i s e . P o n i e s c l o s e l y a p p r o a c h t h i s s c o p e w i t h a 4 . 5 - f o l d i n c r e a s e (Manohar, 1986), b u t e n d u r a n c e - t r a i n e d human a t h l e t e s c a n o n l y a l t e r t h e i r A-V d i f f e r e n c e by 3 - f o l d ( B r o o k s and F a h e y , 1984; A s t r a n d and R o d a h l , 1 9 7 7 ) . The s c o p e o f t h o r o u g h b r e d h o r s e s i s p r o b a b l y e v e n g r e a t e r t h a n 5 - f o l d b e c a u s e t r u e r e s t i n g A-V d i f f e r e n c e s s h o u l d be l o w e r t h a n m e a s u r e d h e r e f o r r e a s o n s s t a t e d p r e v i o u s l y [ a l s o , l o w e r r e s t i n g v a l u e s o f 3.7% were f o u n d i n p o n i e s (Manohar, 1986) compared w i t h 4.7 v o l % i n t h i s s t u d y ] . I n a d d i t i o n , maximum A-V d i f f e r e n c e s c o u l d be h i g h e r t h a n m easured i n t h e g a l l o p p r o t o c o l i f h e a v y e x e r c i s e was s u s t a i n e d f o r a l o n g e r p e r i o d o f t i m e o r i f t h e a n i m a l s were o p e r a t i n g a t t h e i r maximal h e a r t r a t e o f 220-230 b e a t s / m i n . T h i s s t u d y shows t h a t t h o r o u g h b r e d h o r s e s c a n b r i n g t h e i r A-V d i f f e r e n c e i n ox y g e n c o n t e n t t o an a b s o l u t e v a l u e e x c e e d i n g 23 v o l % (see F i g . 1 0 ) . I n c o m p a r i s o n , t o p human a t h l e t e s and p o n i e s a r e o n l y a b l e t o r e a c h 17 v o l % when r u n n i n g a t V0 2max ( B r o o k s and Fahe y , 1984; Manohar, 1 9 8 6 ) . T h o r o u g h b r e d s h a v e t h e c a p a b i l i t y t o b o o s t t h e i r A-V d i f f e r e n c e t o s u c h a h i g h l e v e l b e c a u s e t h e i r a r t e r i a l o xygen c o n t e n t c a n a t t a i n much h i g h e r v a l u e s t h a n humans and p o n i e s ( B r o o k s and Fahe y , 1984; Manohar, 1986). The t h o r o u g h b r e d s o f t h i s s t u d y were a b l e t o b r i n g a r t e r i a l c o n t e n t t o 30.0 v o l % i n t h e c a n t e r p r o t o c o l , b u t p o n i e s c a n o n l y r e a c h maximal v a l u e s o f 22.4 v o l % (Manohar, 1 9 8 6 ) . No d i r e c t measurement o f a r t e r i a l o xygen c o n t e n t i s a v a i l a b l e f o r s t a n d a r d b r e d h o r s e s , b u t L y k k e boe e_t auL (1977) s a t u r a t e d venous b l o o d o f e x e r c i s i n g a n i m a l s w i t h o x y g e n and showed t h a t i t had an 0„ 109 c a p a c i t y o f 30 v o l % . S t a n d a r d b r e d h o r s e s may t h e r e f o r e show t h e same r a n g e o f A-V d i f f e r e n c e s i n o x y g e n c o n t e n t as r e p o r t e d h e r e f o r t h o r o u g h b r e d h o r s e s . I t i s c l e a r t h a t t h e l a r g e i n c r e a s e i n a r t e r i a l o x y g e n c o n t e n t e l i c i t e d by e x e r c i s e was a d i r e c t c o n s e q u e n c e o f a c o n c o m i t a n t change i n h e m a t o c r i t . I n d e e d , as h e m a t o c r i t went f r o m a b o u t 40% t o 60% ( F i g . 11) a r t e r i a l o x y g e n c o n t e n t i n c r e a s e d f r o m 20 t o 30 v o l % ( F i g . 1 0 ) . D u r i n g t h e r e s t t o work t r a n s i t i o n , a b o u t one t h i r d o f t h e t o t a l number o f e r y t h r o c y t e s was r e l e a s e d f r o m t h e s p l e e n (see P e r s s o n , 1 983), c a u s i n g a more t h a n 50% i n c r e a s e i n c i r c u l a t i n g h e m o g l o b i n c o n c e n t r a t i o n . H e m a t o c r i t c h a n g e s t h r o u g h o u t t h e t r o t and c a n t e r p r o t o c o l s i n t h e same h o r s e s h a v e been p r e s e n t e d i n C h a p t e r 3. 4.4.3 METABOLIC RATE. E n d u r a n c e - t r a i n e d humans c a n show a V 0 2 i n c r e a s e o f more t h a n 1 2 - f o l d i n r e s p o n s e t o e x e r c i s e ( B r o o k s and F a h e y , 1 9 8 4 ) . T h o r o u g h b r e d h o r s e s a l r e a d y showed a 1 2 - f o l d i n c r e a s e i n V 0 2 between r e s t and t h e c a n t e r p r o t o c o l , w h i c h o n l y r e p r e s e n t s low i n t e n s i t y e x e r c i s e f o r s u c h a n i m a l s . T h i s change i n t h e r a t e o f o xygen s u p p l y t o t h e w o r k i n g m u s c u l a t u r e was a c h i e v e d v i a a 3 . 2 - f o l d i n c r e a s e i n c a r d i a c o u t p u t a c c o m p a n i e d by a 3 . 8 - f o l d i n c r e a s e i n A-V 0 2 d i f f e r e n c e . Oxygen c o n s u m p t i o n went up s l i g h t l y b etween t h e b e g i n n i n g o f t h e t r o t p r o t o c o l (ST) and l a t e r i n t h e same p r o t o c o l (ET) b e c a u s e t h e A-V d i f f e r e n c e i n 0 2 c o n t e n t i n c r e a s e d somewhat (see F i g s . 10 and 1 2 ) . No c hange i n c a r d i a c o u t p u t c o u l d be m e asured between the two s t a g e s o f 110 t h e t r o t p r o t o c o l (ST and E T ) . M a x i m a l o x y g e n c o n s u m p t i o n o f t h o r o u g h b r e d s w o u l d r e a c h a p p r o x i m a t e l y 156 ml m i n - 1 k g - 1 (or 70 1/min) w i t h an A-V d i f f e r e n c e o f 23.4 v o l % and a maximal c a r d i a c o u t p u t o f 300 1/min. V0 2max_of t r a i n e d humans ( w i t h a body mass o f 70 kg) c a n e x c e e d 60 ml m i n - 1 k g - 1 i n t o p a t h l e t e s (more t h a n 41/min whole body c o n s u m p t i o n f o r 70 kg body mass; see R o w e l l , 1974; B r o o k s and F a h e y , 1 9 8 4 ) . W i t h a 6 - f o l d d i f f e r e n c e i n body mass h o r s e s w o u l d be e x p e c t e d t o h a v e a much l o w e r mass s p e c i f i c m e t a b o l i c r a t e t h a n humans (see S c h m i d t - N i e l s e n 1 9 7 9 ) . C l e a r l y , t h o r o u g h b r e d h o r s e s s u p p o r t an o u t s t a n d i n g a e r o b i c c a p a c i t y w i t h a mass s p e c i f i c VC^max two t o t h r e e t i m e s h i g h e r t h a n f o r t h e b e s t human a t h l e t e s . W i t h a m a x i m a l 0^ c o n s u m p t i o n o f 70 1/min, t h e t r o t p r o t o c o l w o u l d r e p r e s e n t 45 t o 50% V0 2max and t h e c a n t e r w o u l d be 55% V0 2max. O n l y a s m a l l d i f f e r e n c e i n V 0 2 was measured between t h e t r o t and t h e c a n t e r p r o t o c o l s . T h i s d i f f e r e n c e was low b e c a u s e t h e a n i m a l s s w i t c h e d g a i t , r e c r u i t i n g d i f f e r e n t m u s c l e g r o u p s . They have been shown t o change g a i t a t s p e c i f i c v e l o c i t i e s a l l o w i n g them t o m i n i m i z e e n e r g y e x p e n d i t u r e a t any g i v e n s p e e d (see Hoyt and T a y l o r , 1 9 8 1 ) . W i t h an 8 - f o l d i n c r e a s e i n c a r d i a c o u t p u t and a 5 - f o l d i n c r e a s e o f t h e i r A-V d i f f e r e n c e i n o x y g e n c o n t e n t , t h o r o u g h b r e d h o r s e s c a n augment t h e i r o xygen c o n s u m p t i o n by 4 0 - f o l d between r e s t and m a x i m a l e x e r c i s e . T h i s would r e p r e s e n t t h e h i g h e s t a e r o b i c s c o p e f o u n d i n mammals, i n c l u d i n g s t a n d a r d b r e d h o r s e s and p o n i e s . An e q u i v a l e n t s c o p e I l l i n t h e d e l i v e r y r a t e o f o x i d i z a b l e f u e l s t o t h e w o r k i n g m u s c l e s may be s u p p o r t e d by t h o r o u g h b r e d h o r s e s . The p r o b l e m o f how c h a n g e s i n c a r d i a c o u t p u t i n f l u e n c e p l a s m a m e t a b o l i t e t u r n o v e r r a t e d u r i n g e x e r c i s e i s a d d r e s s e d i n t h e l a s t c h a p t e r o f t h i s t h e s i s . 112 CHAPTER 5: LACTATE TURNOVER IN EXERCISING THOROUGHBRED HORSES: EFFECT OF CHANGES IN CARDIAC OUTPUT. 5.1 INTRODUCTION S u s t a i n e d m u s c l e work r e q u i r e s t h e a d e q u a t e s u p p l y o f e xogenous f u e l s t o m i n i m i z e t h e u s e o f endogenous s u b s t r a t e s and p o s t p o n e t h e i r d e p l e t i o n . O r g a n i s m s must be a b l e t o r e g u l a t e t h e f l u x r a t e s o f t h e i r p l a s m a m e t a b o l i t e s i f t h e r i g h t m i x t u r e o f o x i d i z a b l e f u e l s i s t o be p r o v i d e d v i a t h e b l o o d a t t h e a p p r o p r i a t e t i m e and s p e e d . To meet t h e e n e r g e t i c demands o f e x e r c i s e , s u b s t r a t e f l u x e s c a n be augmented v i a two m e c h a nisms: 1) by i n c r e a s i n g t h e i r c o n c e n t r a t i o n i n p l a s m a , and 2) by i n c r e a s i n g b l o o d f l o w t o t h e w o r k i n g m u s c l e s . A t t h e w h o l e - o r g a n i s m l e v e l , t h e f i r s t m echanism i s u s e d (as s e e n i n C h a p t e r 2 i n t h e c a s e o f l a c t a t e ) , b u t t h e s e c o n d one h a s n e v e r been i n v e s t i g a t e d . I n d e e d , t h e t u r n o v e r r a t e o f s e v e r a l s u b s t r a t e s i s p o s i t i v e l y c o r r e l a t e d w i t h t h e i r p l a s m a c o n c e n t r a t i o n ( C h a p t e r 2; V e r d o n k e t a^l, 1981; B o r t z e t a_l, 1972; H a g e n f e l d t , 1975), however, i t has n e v e r been shown w h e t h e r c h a n g e s i n c a r d i a c o u t p u t h a v e an e f f e c t on t h e t u r n o v e r r a t e o f p l a s m a m e t a b o l i t e s when t h e e n t i r e o r g a n i s m i s t a k e n i n t o a c c o u n t , o I f t h e s e c h a n g e s have an e f f e c t , t h e r e l a t i o n s h i p between p l a s m a l a c t a t e c o n c e n t r a t i o n and l a c t a t e t u r n o v e r r a t e would 113 a l s o d e p e n d on c a r d i a c o u p u t . T h i s e f f e c t h a s n e v e r been shown. L a c t a t e f l u x e s p r o v i d e an i d e a l model t o s t u d y t h e c o n t r o l o f m e t a b o l i t e t u r n o v e r b e c a u s e t h e y v a r y o v e r a much w i d e r r a n g e o f v a l u e s t h a n t h e f l u x e s o f o t h e r s u b s t r a t e s ( s e e C h a p t e r 2 ) , and t h e y c a n be m a n i p u l a t e d e a s i l y t h r o u g h e x e r c i s e . The s u p p l y o f l a c t a t e t o w o r k i n g m u s c l e s i s n o t d i f f u s i o n l i m i t e d b e c a u s e l a c t a t e t r a n s p o r t a c r o s s c e l l membranes i s r a p i d as i n d i c a t e d by t h e h i g h v a l u e s f o u n d f o r a l l t h e l a c t a t e c a r r i e r o f a l l c e l l s s t u d i e d t o d a t e ( D u b i n s k y and R a c k e r , 1978; Koch e t a l , 1981; Roos, 1975; S p e n c e r and L e h n i n g e r , 1 9 7 6 ) . T h e r e f o r e , l a c t a t e s u p p l y s h o u l d be p e r f u s i o n l i m i t e d i n a l l s i t u a t i o n s where c e l l u l a r l a c t a t e o x i d a t i o n c a n keep up w i t h l a c t a t e s u p p l y (as i t i s t h e c a s e d u r i n g s u b m a x i m a l e x e r c i s e ) , and c h a n g e s i n c a r d i a c o u t p u t s h o u l d h a v e an e f f e c t on t h e r a t e o f l a c t a t e t u r n o v e r i n p l a s m a ( n o t e t h a t , u n d e r s t e a d y - s t a t e c o n d i t i o n s , t u r n o v e r r a t e i s e q u a l t o t h e r a t e o f d i s a p p e a r a n c e and t o t h e r a t e o f a p p e a r a n c e f r o m and i n t o t h e r a p i d l y m i x i n g p o o l o f l a c t a t e ; s e e W o l f e , 1 9 8 4 ) . Plasma l a c t a t e c a n be an i m p o r t a n t o x i d i z a b l e s u b s t r a t e i n some, a n i m a l s (Donovan and B r o o k s , 1983; Mazzeo e t a l , 1986), and i t s r o l e d u r i n g s u s t a i n e d e x e r c i s e h a s been e m p h a s i z e d by B r o o k s ( 1 9 8 5 ) . I t i s n o t c l e a r w h ether e x e r c i s i n g o r g a n i s m s g a i n some p e r f o r m a n c e a d v a n t a g e by t r a n s p o r t i n g l a c t a t e a t h i g h r a t e s t h r o u g h t h e i r p l a s m a compartment. I f t h e y do, s p e c i e s g e n e t i c a l l y g e a r e d f o r h i g h p e r f o r m a n c e , l i k e t h e t h o r o u g h b r e d h o r s e , w o u l d be e x p e c t e d 114 t o h a v e t h e a b i l i t y t o s u s t a i n h i g h l a c t a t e t u r n o v e r r a t e s , and t r a i n i n g s h o u l d e n h a n c e t h i s a b i l i t y . I n e x e r c i s i n g r a t s , a l b e i t a s p e c i e s by no means n o t e d f o r i t s a t h l e t i c p r o w e s s , t r a i n i n g has no e f f e c t on t u r n o v e r r a t e (Donovan and B r o o k s , 1 9 8 3 ) . L a c t a t e t u r n o v e r ^ h a s n e v e r been m e a s u r e d i n h o r s e s . The g o a l o f t h i s s t u d y was t h e r e f o r e t o measure p l a s m a l a c t a t e f l u x r a t e s i n r e s t i n g and e x e r c i s i n g t h o r o u g h b r e d h o r s e s : 1) t o d e t e r m i n e t h e e f f e c t o f c h a n g e s i n c a r d i a c o u t p u t on l a c t a t e t u r n o v e r r a t e u s i n g ^ b l o o d f l o w d a t a f r o m t h e p r e v i o u s s t u d y ( C h a p t e r 4 ) , and 2) t o a s s e s s t h e i m p o r t a n c e o f l a c t a t e as a m e t a b o l i c f u e l i n a t r a i n e d a n i m a l a t h l e t e and t o compare i t s f l u x r a t e s w i t h v a l u e s m e a s u r e d i n o t h e r , more s e d e n t a r y s p e c i e s . I n a d d i t i o n , I a t t e m p t e d t o i n v e s t i g a t e w h e t h e r l a c t a t e t u r n o v e r r a t e d e c r e a s e s t h r o u g h o u t a b o u t o f s u b m a x i m a l e x e r c i s e , as w o u l d be p r e d i c t e d i f t h e c o n t r i b u t i o n o f f a t o x i d a t i o n t o t o t a l e n e r g y m e t a b o l i s m i n c r e a s e d . 5.2 MATERIALS AND METHODS 5.2.1 ANIMALS AND CATHETERIZATIONS. Measurements were p e r f o r m e d on t h e f o u r t h o r o u g h b r e d h o r s e s u s e d i n t h e p r e v i o u s s t u d i e s ( C h a p t e r 3 and C h a p t e r 4 ) . T h e i r w e i g h t s and ages a r e g i v e n i n T a b l e 4 (see C h a p t e r 3 ) . D u r i n g t h e p r e s e n t e x p e r i m e n t s , t h e h o r s e s were k e p t on t h e same d i e t and t h e i r r e g u l a r t r a i n i n g was i d e n t i c a l t o what has been d e s c r i b e d p r e v i o u s l y . The f o u r a n i m a l s had u n d e r g o n e a s u r g i c a l 115 t r a n s p o s i t i o n o f t h e r i g h t common c a r o t i d a r t e r y t o a s u b c u t a n e o u s p o s i t i o n s e v e r a l months b e f o r e t h e e x p e r i m e n t s t o f a c i l i t a t e t h e s u r g i c a l p r o c e d u r e s p e r f o r m e d f o r t h e measurement o f l a c t a t e t u r n o v e r r a t e . The t r a n s p o s i t i o n o f t h i s v e s s e l a l l o w e d a r e l a t i v e l y e a s y a c c e s s t o t h e a r t e r i a l s y s t e m whenever a r t e r i a l b l o o d had t o be c o l l e c t e d . B e f o r e a t u r n o v e r measurement, a Vygon XRO i n t r a v a s c u l a r c a t h e t e r (18 g X 46 cm) was i n t r o d u c e d i n t h e r i g h t c a r o t i d and a c a r d i a c c a t h e t e r (USCI, s i z e 8F X 125 cm) was p l a c e d i n t h e r i g h t a t r i u m u s i n g a K o n t r o n M e d i c a l p r e s s u r e t r a n s d u c e r (Model 1 0 8 ) . B o t h c a t h e t e r s were c o n n e c t e d t o e x t e n s i o n l i n e s (100 cm, 2-ml c a p a c i t y ) w h i c h were s e c u r e l y t a p e d t o t h e neck and f i l l e d w i t h s a l i n e . E x t e n s i o n l i n e s and c a t h e t e r s were f l u s h e d e v e r y 10-15 min t o keep them p a t e n t . D e t a i l e d c a t h e t e r i z a t i o n p r o c e d u r e s a r e d e s c r i b e d i n C h a p t e r 4. 5.2.2 EXERCISE PROTOCOLS. E x p e r i m e n t s were c a r r i e d o u t a t l e a s t 3 h a f t e r t h e m o r n i n g f e e d . T e m p e r a t u r e 0 r a n g e d f r o m 17 t o 20 C and r e l a t i v e h u m i d i t y f r o m 53 t o 71%. No measurement o f l a c t a t e t u r n o v e r r a t e was s t a r t e d e a r l i e r t h a n 30 min a f t e r c a t h e t e r i z a t i o n . The a n i m a l s were f i t t e d w i t h a h a r n e s s and a h e a r t r a t e m o n i t o r . Measurement o f r e s t i n g t u r n o v e r r a t e was p e r f o r m e d w h i l e t h e a n i m a l was q u i e t l y s t a n d i n g i n a s t a l l . F o r t h e d e t e r m i n a t i o n o f l a c t a t e f l u x e s d u r i n g work, t h e h o r s e was b r o u g h t on t h e t r e a d m i l l and t h e h a r n e s s was t i e d t o an emergency s w i t c h - o f f mechanism above him. A f a n s i m u l a t i n g a i r c o o l i n g d u r i n g n o r m a l 116 l o c o m o t i o n was p l a c e d i n f r o n t o f t h e a n i m a l . Two e x e r c i s e p r o t o c o l s were s e l e c t e d : 1) a 4 m/s. t r o t a t 6% i n c l i n e s u s t a i n a b l e f o r 40 min, and 2) a 6.5 m/s h o r i z o n t a l c a n t e r f o r 15 m i n . They were i d e n t i c a l t o t h e t r o t and c a n t e r p r o t o c o l s .. o f t h e p r e v i o u s s t u d y . I n two c a s e s , t h e s e p r o t o c o l s had t o be a d j u s t e d f o r i n d i v i d u a l h o r s e s . A lo w e r t r o t t i n g s p e e d o f 3 m/s h a d t o be u s e d f o r t h e o l d e r h o r s e (JW) b e c a u s e he c o u l d n o t s u s t a i n 4 m/s o v e r 40 min (see p r e v i o u s s t u d y f o r d e t a i l s ) , and a h i g h e r c a n t e r i n g s p e e d o f 8.3 m/s was c h o s e n f o r IN b e c a u s e he was a b l e t o t r o t a t 6.5 m/s and he h a d p r o b l e m s s w i t c h i n g t o a c a n t e r a t t h e l a t t e r v e l o c i t y . I n t h e s e 2 c a s e s , t h e v a l u e s m easured f o r JW and IN were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e measurements p e r f o r m e d on t h e o t h e r h o r s e s a t t h e r e s p e c t i v e g a i t s . T h e r e f o r e , d a t a f o r a l l a n i m a l s were p o o l e d i n t h e c a l c u l a t i o n s . A l l e x e r c i s e b o u t s were i n i t i a t e d w i t h a s h o r t warm-up p e r i o d c o n s i s t i n g o f a 4 min walk (1.6 m/s) b e f o r e t h e t r o t p r o t o c o l and a 4 min walk f o l l o w e d by a 4 min t r o t (4 m/s) b e f o r e t h e c a n t e r p r o t o c o l . H e a r t r a t e was r e c o r d e d e v e r y 30 s d u r i n g t h e f i r s t 10 min o f e a c h e x p e r i m e n t and a t l o n g e r i n t e r v a l s t h e r e a f t e r . The b a s i c m e t a b o l i c and c a r d i o v a s c u l a r r e s p o n s e s o f t h e same h o r s e s s u b j e c t e d t o i d e n t i c a l e x e r c i s e p r o t o c o l s h a v e been r e p o r t e d i n C h a p t e r 3. 5.2.3 MEASUREMENT OF LACTATE TURNOVER. The b o l u s i n j e c t i o n t e c h n i q u e was u s e d t o d e t e r m i n e t h e r a t e o f p l a s m a l a c t a t e t u r n o v e r (see C h a p t e r 1, and W o l f e , 1 9 8 4 ) . B o l u s i n j e c t i o n was s e l e c t e d o v e r c o n t i n u o u s i n f u s i o n b e c a u s e t h e 117 i n f u s i o n t e c h n i q u e r e q u i r e s much l a r g e r amounts o f i s o t o p e and i t w o u l d have made t h e e x p e r i m e n t s p r o h i b i t i v e l y e x p e n s i v e i n s u c h a l a r g e a n i m a l . E v e n when u s i n g b o l u s i n j e c t i o n (as was done h e r e ) , t h e h i g h c o s t o f a s i n g l e measurement p l a c e d some i m p o r t a n t c o n s t r a i n t s on t h e number o f t u r n o v e r r a t e s t h a t c o u l d be m e a s u r e d i n e a c h h o r s e . I n a d d i t i o n , b e c a u s e c o n t i n u o u s i n f u s i o n was t o o e x p e n s i v e t o p e r f o r m , t h e q u a n t i f i c a t i o n o f l a c t a t e o x i d a t i o n c o u l d n o t be done i n t h e p r e s e n t e x p e r i m e n t s . I n d e e d , i t i s n o t p o s s i b l e t o q u a n t i f y t h e c o n t r i b u t i o n o f l a c t a t e o x i d a t i o n t o t o t a l 14 t u r n o v e r r a t e (by f o l l o w i n g t h e p r o d u c t i o n r a t e o f C 0 2 ^ when t h e b o l u s i n j e c t i o n t e c h n i q u e i s u s e d . S e p a r a t e e x p e r i m e n t s were c a r r i e d o u t f o r e a c h h o r s e a t e a c h a c t i v i t y l e v e l . Measurements were p e r f o r m e d a t r e s t ( R), a t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l ( S T ) , l a t e r d u r i n g t h e t r o t p r o t o c o l ( E T ) , and d u r i n g t h e c a n t e r p r o t o c o l (C) . F o r e x e r c i s i n g a n i m a l s , t h e r a d i o a c t i v e b o l u s was i n j e c t e d 1 min a f t e r t h e b e g i n n i n g o f t h e t r o t p r o t o c o l ( S T ) , 7 min i n t o t h e same p r o t o c o l ( E T ) , and 7 min a f t e r s t a r t i n g t h e c a n t e r p r o t o c o l ( C ) . The same t i m i n g was u s e d f o r t h e measurement o f c a r d i a c o u t p u t i n t h e p r e v i o u s s t u d y ( C h a p t e r 14 4 ) . [U- C ] l a c t a t e ( s p e c i f i c a c t i v i t y = 150 mCi/mmol) was p u r c h a s e d f rom Amersham (UK), and d o s e s o f 120-150 u C i ( r e s t ) , 140-220 u C i ( t r o t ) and 230-260 u C i ( c a n t e r ) were i n j e c t e d i n t o t h e r i g h t a t r i u m . The l a b e l e d m e t a b o l i t e was a d m i n i s t e r e d i n a 13-20 ml s a l i n e s o l u t i o n , and t h e c a t h e t e r was f l u s h e d w i t h 50 ml s a l i n e . The t o t a l p r o c e d u r e ( b o l u s 118 i n j e c t i o n and f l u s h i n g ) was completed i n 6 s. The exact s i z e of the bolus was determined by weighing the s y r i n g e b e f o r e and a f t e r i n j e c t i o n . At the end of three randomly chosen experiments, the c a r d i a c c a t h e t e r was withdrawn and counted to determine whether .. p a r t of the bolus remained i n the l i n e a f t e r f l u s h i n g . The a c t i v i t y measured i n the c a t h e t e r s o n l y represented 0.1-0.6% of the t o t a l doses i n j e c t e d , and t h i s l o s s of a c t i v i t y was not taken i n t o account i n the c a l c u l a t i o n s . Blood samples (5 ml) were drawn from the c a r o t i d a r t e r y s t a r t i n g 10 s a f t e r i n j e c t i o n . They were taken approximately every 10 s f o r the f i r s t 3 min and a t longer i n t e r v a l s t h e r e a f t e r . T o t a l sampling time was 6 to 20 min d u r i n g e x e r c i s e and 2 h i n r e s t i n g animals. Samples were c o l l e c t e d i n i c e - c o l d 7-ml v a c u t a i n e r s c o n t a i n i n g 0.07 ml e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) as a n t i c o a g u l a n t and pla c e d on i c e . Hematocrit was measured immediately i n micro-hematocrit tubes (10 min s p i n ) . Plasma was separated 0 from the r e s t of the sample and s t o r e d a t -20 C bef o r e c o u n t i n g and m e t a b o l i t e assays. L a c t a t e and glucose c o n c e n t r a t i o n s were measured on a Pye-Unicam spectrophotometer (SP6-500) by f o l l o w i n g the r e d u c t i o n of NAD + or NADP + at 340 nm (Bergmeyer 1974). A l l the assays were performed i n d u p l i c a t e (see Chapter 2 f o r d e t a i l s ) w i t h i n 3 days of sampling with a t o t a l c u v e t t e volume of 1 ml. S c i n t i l l a t i o n c o u n t i n g was performed on an Isocap 300 s c i n t i l l a t i o n counter by use of i n t e r n a l standard quench c o r r e c t i o n . The a c t i v i t y of l a c t a t e was c a l c u l a t e d as 119 t o t a l p l a s m a a c t i v i t y minus t h e a c t i v i t y m e a sured i n p l a s m a g l u c o s e . G l u c o s e a c t i v i t y was d e t e r m i n e d by use o f p r e v i o u s l y d e s c r i b e d p r o c e d u r e s ( s e e Methods i n C h a p t e r 2 ) . Plasma s a m p l e s (0.5 ml) m i x e d w i t h 10 ml aqueous c o u n t i n g 0 s c i n t i l l a n t (ACS I I , a Amersham) were l e f t i n t h e d a r k a t 4 C f o r a t l e a s t 12 h b e f o r e b e i n g c o u n t e d f o r 20 m i n . E f f i c i e n c y r a n g e d f r o m 65 t o 82%. No a c t i v i t y c o u l d be m e a s u r e d i n g l u c o s e , e v e n 2 h a f t e r i n j e c t i o n o f t h e r a d i o a c t i v e b o l u s i n r e s t i n g a n i m a l s . S u c c e s s i v e e x p e r i m e n t s on t h e same h o r s e were a l w a y s s e p a r a t e d by a minimum o f 4 d a y s e v e n t h o u g h no r e s i d u a l a c t i v i t y c o u l d be m e a s u r e d i n p l a s m a o r u r i n e 24 h a f t e r i n j e c t i o n . 5.2.4 CALCULATIONS AND S T A T I S T I C S . T u r n o v e r r a t e was c a l c u l a t e d as t h e d o s e i n j e c t e d d i v i d e d by t h e s u r f a c e a r e a u n d e r t h e s p e c i f i c a c t i v i t y d e c a y c u r v e . To c a l c u l a t e t h i s s u r f a c e a r e a t h e d e c a y c u r v e was f i t t e d w i t h t h e sum o f two e x p o n e n t i a l f u n c t i o n by u s e o f t h e p r o c e d u r e d e s c r i b e d i n C h a p t e r 2 ( u s i n g t h e sum o f t h r e e e x p o n e n t i a l f u n c t i o n s d i d n o t i mprove t h e f i t o f t h e c a l c u l a t e d f u n c t i o n s , t h e r e f o r e , t h e sum o f o n l y two e x p o n e n t i a l s was u s e d h e r e ) , and t h i s f u n c t i o n was i n t e g r a t e d between t i m e 0 and t h e t i m e when 5% o f t h e maximum p o s s i b l e s p e c i f i c a c t i v i t y was r e a c h e d . The maximum a c t i v i t y was c a l c u l a t e d as t h e d o s e i n j e c t e d d i v i d e d by t h e t o t a l b l o o d volume [ u s i n g t h e mean v a l u e o f 142.1 ml o f b l o o d p e r kg body w e i g h t measured by P e r s s o n (1968) i n t h o r o u g h b r e d h o r s e s ] . M e t a b o l i c c l e a r a n c e r a t e (MCR) was c a l c u l a t e d as t u r n o v e r r a t e d i v i d e d by p l a s m a l a c t a t e 120 c o n c e n t r a t i o n ( W o l f e , 1 984). F o r e a c h e x p e r i m e n t , c a r d i a c o u t p u t was d e t e r m i n e d f r o m mean h e a r t r a t e by l i n e a r i n t e r p o l a t i o n o f t h e h e a r t r a t e vs c a r d i a c o u t p u t r e l a t i o n s h i p . T h i s r e l a t i o n s h i p was c a l c u l a t e d f o r e a c h h o r s e ( l i n e a r r e g r e s s i o n s ; r=0.98-0.99) u s i n g t h e r e s u l t s o f t h e p r e v i o u s s t u d y ( C h a p t e r 4 ) . D a t a f r o m JW, K J , and SB were p o o l e d t o c a l c u l a t e a r e g r e s s i o n e q u a t i o n f o r IN (r=0.98) whose c a r d i a c o u t p u t was n o t m e a s u r e d . Means were compared w i t h a oneway ANOVA, and t h e Student-Newman-Keuls p o s t h o c t e s t (T-Method f o r u n p l a n n e d c o m p a r i s o n s , s e e S o k a l and R o h l f 1981) was u s e d when t h e o v e r a l l F v a l u e i n d i c a t e d t h a t f u r t h e r a n a l y s i s was n e c e s s a r y . A m u l t i p l e r e g r e s s i o n a n a l y s i s was p e r f o r m e d t o e s t i m a t e t h e r e s p e c t i v e c o n t r i b u t i o n s o f c h a n g e s i n l a c t a t e c o n c e n t r a t i o n and c a r d i a c o u t p u t t o t h e t o t a l v a r i a b i l i t y i n l a c t a t e t u r n o v e r r a t e ( T a b l e 7 ) . ANOVA was u s e d t o d e t e r m i n e t h e s i g n i f i c a n c e o f t h e s l o p e s c a l c u l a t e d by l i n e a r r e g r e s s i o n and t o a n a l y z e t h e m u l t i p l e r e g r e s s i o n model (see S o k a l and R o h l f , 1 9 8 1 ) . 5.3 RESULTS The l a c t a t e s p e c i f i c a c t i v i t y d e c a y c u r v e s and t h e c o n c e n t r a t i o n o f l a c t a t e i n p l a s m a a f t e r b o l u s i n j e c t i o n o f 14 [U- C ] l a c t a t e a r e g i v e n i n F i g . 13 t o 15 f o r one o f t h e t h o r o u g h b r e d s ( K J ) , a t r e s t and d u r i n g e x e r c i s e . In a l l e x p e r i m e n t s , t h e c a l c u l a t e d m u l t i e x p o n e n t i a l f u n c t i o n s f i t t e d 121 t h e measured s p e c i f i c a c t i v i t i e s e x t r e m e l y w e l l . S p e c i f i c a c t i v i t y d e c r e a s e d much more s h a r p l y d u r i n g t h e c a n t e r t h a n a t r e s t ( F i g . 1 4 ) , p a r t i c u l a r l y when c o n s i d e r i n g t h a t t h e 14 d o s e o f [U- C ] l a c t a t e i n j e c t e d i n t h e r e s t i n g a n i m a l was l e s s t h a n h a l f t h e amount a d m i n i s t e r e d d u r i n g t h e c a n t e r . The l a c t a t e t u r n o v e r r a t e s m e a s u r e d a f t e r i n t e g r a t i n g t h e f i t t e d f u n c t i o n s were 11.2 umol m i n - 1 k g - 1 a t r e s t ( F i g . 13 and t o p g r a p h F i g . 14) and 42.3 umol m i n - 1 k g - 1 d u r i n g t h e c a n t e r (bottom g r a p h i n F i g . 1 4 ) . V a l u e s o f 51.3 and 45.2 umol m i n - 1 k g - 1 were measured a t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l and l a t e r d u r i n g t h e same p r o t o c o l ( F i g . 1 5 ) . P l a s m a l a c t a t e c o n c e n t r a t i o n was s t e a d y a t t h e d i f f e r e n t l e v e l s o f a c t i v i t y ( i n s e t s : F i g . 13 t o 15) and, f o r t h i s a n i m a l , mean c o n c e n t r a t i o n was 25% h i g h e r d u r i n g e x e r c i s e t h a n a t r e s t . L a c t a t e t u r n o v e r r a t e s f o r e a c h a n i m a l and e a c h a c t i v i t y s t a t e a r e g i v e n i n T a b l e 7, w h i c h summarizes t h e r e s u l t s o f t h i s s t u d y . F o r e a c h e x p e r i m e n t , T a b l e 7 a l s o shows t h e mean v a l u e s f o r h e a r t r a t e , h e m a t o c r i t and p l a s m a l a c t a t e c o n c e n t r a t i o n , as w e l l as c a r d i a c o u t p u t and m e t a b o l i c c l e a r a n c e r a t e f o r l a c t a t e . The a v e r a g e h e a r t r a t e o f t h e f o u r h o r s e s was 37.9 +_ 2.1 a t r e s t , 132.7 +_ 9.0 a t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l ( S T ) , 130.0 _+ 8.0 l a t e r i n t h e same p r o t o c o l (ET) , and 147.5 +_ 9.6 b e a t s / m i n i n t h e c a n t e r p r o t o c o l (means +_ SEM, N = 4) . F o r a g i v e n l e v e l o f e x e r c i s e , JW had a l o w e r h e a r t r a t e t h a n t h e o t h e r h o r s e s . Mean h e m a t o c r i t ( f o r t h e 4 a n i m a l s ) i n c r e a s e d f r o m a r e s t i n g v a l u e o f 36.2 + 2.7% t o a maximum o f 54.7 + 0.8% d u r i n g t h e c a n t e r 122 F I G . 13 L a c t a t e s p e c i f i c a c t i v i t y d e c a y c u r v e and l a c t a t e c o n c e n t r a t i o n ( i n s e t ) i n p l a s m a o f a r e s t i n g t h o r o u g h b r e d h o r s e (KJ) a f t e r i n j e c t i o n o f 122.14 u C i [ U - 1 4 C ] l a c t a t e a t t i m e 0 ( e x p e r i m e n t No. 1 1 ) . See F i g . 2 f o r a d e t a i l e d view o f t h e f i r s t 5 min a f t e r i n j e c t i o n . R a d i o a c t i v e b o l u s was i n j e c t e d i n t o t h e r i g h t a t r i u m a t t i m e 0 and b l o o d was s a m p l e d from t h e c a r o t i d a r t e r y . The d e c a y c u r v e was f i t t e d w i t h a d o u b l e e x p o n e n t i a l f u n c t i o n . fr0MpOIAIr//IAIdd] A11AIJL0V dS 124 F I G . 14 AND 15 L a c t a t e s p e c i f i c a c t i v i t y d e c a y c u r v e s and l a c t a t e c o n c e n t r a t i o n ( i n s e t s ) i n p l a s m a o f a t h o r o u g h b r e d h o r s e (KJ) a t r e s t and e x e r c i s i n g on a t r e a d m i l l d u r i n g t h e 14 f i r s t 5 min a f t e r b o l u s i n j e c t i o n o f [U- C ] l a c t a t e . Doses o f 122.14 u C i ( r e s t : R ) , 169.14 u C i ( s t a r t t r o t : S T ) , 144.27 u C i (end t r o t : ET) and 251.02 u C i ( c a n t e r : C) were i n j e c t e d a t t i m e 0 ( e x p e r i m e n t s No. 11 t o 14 r e s p e c t i v e l y ) . Same i n j e c t i o n and s a m p l i n g s i t e s a s i n F i g . 13. A l l d e c a y c u r v e s were f i t t e d w i t h d o u b l e e x p o n e n t i a l f u n c t i o n s . See methods f o r e x e r c i s e p r o t o c o l s and t h e t i m i n g o f i n j e c t i o n s . 126 CO O ZT o CL > > O < CL if) START TROT 10- 1.0 i r i r 4 V • • 4 10-i 1.0 5-\ END TROT 1 r-V. 2 - i r 4 MIN 127 TABLE 7. LACTATE TURNOVER RATE, LACTATE CLEARANCE RATE, AND CARDIAC OUTPUT OF THOROUGHBRED HORSES AT REST AND DURING EXERCISE (CONT'D ON NEXT PAGE). EXPT HORSE No. ACTIVITY STATE SPEED INC-m/s LINE % HEART RATE b/min HCT % PLASMA [LACTATE] mM 1 JW REST - - 38.8 44.0 0.80+0.02 2 JW TROT (ST) 4.0 6 118.8 59.0 2.07+0.03 3 JW TROT (ST) 3.0 6 118.0 55.3 1.47+0.03 4 JW TROT (ET) 3.0 6 108.1 57.6 0.74+0.02 5 JW CANTER 6.5 0 122.3 53.5 1.89+0.08 6 SB REST - - 43.3 35.1 0.83+0. 02 7 SB TROT (ST) 4. 0 6 157.5 58.0 1.99+0. 09 8 SB TROT (ET) 4. 0 6 138.4 57.9 0.98+0. 02 9 SB CANTER 6. 5 0 146.3 56.6 1.2 5+0. 02 10 SB CANTER 4. 0 6 175.3 58.2 3.54+0. 07 11 KJ REST — — 36.2 32.9 0.6 2 + 0. 02 12 KJ TROT (ST) 4. 0 6 134.2 54.1 0.84+0. 02 13 KJ TROT (ET) 4. 0 6 144.4 54.0 0.80+0. 03 14 KJ CANTER 6. 5 0 168.2 55.4 0.81+0. 02 15 IN REST — — 33.3 32.8 0.56+0. 02 16 IN TROT (ST) 4. 0 6 121.2 49.0 0.72+0. 02 17 IN TROT (ET) 4. 0 6 129.0 48.8 0.7 2 + 0. 02 18 IN CANTER 8. 3 0 153.1 53 .3 0.98+0. 02 128 TABLE 7. (CONT'D). N CARDIAC OUTPUT TURNOVER METABOLIC CLEARANCE ml min-1 kg-1 RATE (Rt) RATE (MCR) umol min-1 m l min-1 kg-1 kg-1 20 116.5 12.2 15.3 15 367.2 127.8 61.7 13 364.8 115.4 78.5 19 333.7 34.9 47.2 17 378.1 71.7 37.9 23 153.5 7.3 8.8 11 542.3 85.5 42.9 18 477.2 70.4 71.9 17 504.2 95.2 76.2 14 — 276.2 78.0 25 106.0 11.2 18.1 14 451.2 51.3 61.1 19 487.2 45.2 56.5 19 571.1 42.3 52.2 23 107.0 6.5 11.6 19 417.3 51.3 71.2 17 444.9 50.7 70.4 19 529.7 55.1 56.3 T u r n o v e r r a t e was m e a s u r e d by b o l u s i n j e c t i o n o f [U- C ] l a c t a t e . ST: s t a r t o f t r o t p r o t o c o l ; ET: l a t e r i n t r o t p r o t o c o l (see M e t h o d s ) . HCT = mean h e m a t o c r i t . L a c t a t e c o n c e n t r a t i o n g i v e n as mean + SEM; N = number o f b l o o d s a m p l e s . Note t h a t i n c a n t e r p r o t o c o l , s p e e d was h i g h e r f o r IN t h a n f o r t h e o t h e r h o r s e s ( e x p t . No. 1 8 ) . I n e x p e r i m e n t No. 10, SB was c a n t e r i n g a t a b n o r m a l l y low s p e e d and s t r u g g l i n g . C a r d i a c o u t p u t was c a l c u l a t e d by l i n e a r i n t e r p o l a t i o n u s i n g d a t a f r o m p r e v i o u s s t u d y ( C h a p t e r 4; see Methods f o r d e t a i l s ) . 129 p r o t o c o l . Plasma l a c t a t e c o n c e n t r a t i o n was s t e a d y t h r o u g h o u t a l l t h e e x p e r i m e n t s as shown by t h e low s t a n d a r d e r r o r s r e p o r t e d i n T a b l e 7 and by t h e i n s e t s i n F i g . 13 t o 15. The a s s u m p t i o n o f s t e a d y s t a t e r e q u i r e d by t h e b o l u s i n j e c t i o n t e c h n i q u e was t h e r e f o r e met i n e a c h e x p e r i m e n t . Mean l a c t a t e c o n c e n t r a t i o n r a n g e d from 0.56 t o 0.83 mM i n r e s t i n g a n i m a l s . A t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l , l a c t a t e l e v e l s were o n l y s l i g h t l y e l e v a t e d f o r two a n i m a l s (KJ and IN) and r e a c h e d a b o u t 2 mM f o r t h e o t h e r two (JW and S B ) . L a c t a t e c o n c e n t r a t i o n was l o w e r d u r i n g t h e s e c o n d p a r t o f t h e t r o t p r o t o c o l (ET) t h a n a t t h e b e g i n n i n g o f t h a t same p r o t o c o l ( S T ) , e x c e p t f o r IN where no d i f f e r e n c e was m e a s u r e d . D u r i n g t h e c a n t e r , mean l a c t a t e c o n c e n t r a t i o n s r a n g e d f r o m 0.81 t o 1.89 mM, w i t h t h e same i n d i v i d u a l s (JW and SB) s h o w i n g t h e h i g h e s t v a l u e s as i n t h e t r o t p r o t o c o l . A v e r a g e c a r d i a c o u t p u t f o r a l l a n i m a l s was 120.8 +_ 11.2 ml m i n - 1 k g - 1 a t r e s t , 443.9 _+ 37.3 a t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l ( S T ) , 435.8 + 35.2 l a t e r i n t h e same p r o t o c o l ( E T ) , and 495.8 + 41.6 d u r i n g t h e c a n t e r (means +_ SEM,' N = 4) . Mean MCR was 13.5 + 2.0 ml m i n - 1 k g - 1 a t r e s t , 63.4 + 7.7 (ST) and 61.5 +_ 5.9 (ET) d u r i n g t h e t r o t p r o t o c o l , and 55.7 + 7.9 i n the c a n t e r p r o t o c o l . Mean l a c t a t e t u r n o v e r r a t e s f o r t h e d i f f e r e n t a c t i v i t y s t a t e s a r e g i v e n i n F i g . 16. They r a n g e d from a low r e s t i n g v a l u e o f 9.3 umol m i n - 1 k g - 1 t o a maximum o f 75.9 a t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l . The o v e r a l l e f f e c t o f e x e r c i s e on t u r n o v e r r a t e was h i g h l y s i g n i f i c a n t (3 d f / 1 2 d f ; F=8.1; P<0.005). P o s t hoc t e s t s show t h a t l a c t a t e t u r n o v e r 130 F I G . 16 L a c t a t e t u r n o v e r r a t e i n r e s t i n g and e x e r c i s i n g t h o r o u g h b r e d h o r s e s . V a l u e s g i v e n a r e means + SEM (N = 4 ) . R: r e s t ; ST: s t a r t o f t r o t p r o t o c o l ; ET: l a t e r d u r i n g t h e t r o t p r o t o c o l ; C: d u r i n g t h e c a n t e r p r o t o c o l . See methods f o r d e t a i l s . 131 I 8 0 -I 6 0 -4 0 -2 0 -R S T E T C 1 13 2 F I G . 17 R e l a t i o n s h i p between l a c t a t e t u r n o v e r r a t e and mean p l a s m a l a c t a t e c o n c e n t r a t i o n i n r e s t i n g and e x e r c i s i n g t h o r o u g h b r e d h o r s e s . L i n e f i t t e d by l i n e a r r e g r e s s i o n ( s l o p e = 59.02, r = 0.80). D i f f e r e n t s y m b o l s r e p r e s e n t i n d i v i d u a l h o r s e s : s q u a r e s (JW); s o l i d c i r c l e s ( S B); open c i r c l e s ( K J ) ; and t r i a n g l e s ( I N ) . 133 150 H 2 L A C T A T E mM 134 TABLE 8. EFFECTS OF PLASMA LACTATE CONCENTRATION AND CARDIAC OUTPUT ON THE RATE OF LACTATE TURNOVER IN THOROUGHBRED HORSES REGRESSION MODEL % OF THE VARIABILITY IN R EXPLAINED BY MODEL Rfc = f ( [LAC] ) 0.80 64 26.5 < 0.001 Rfc = f(CO) 0.58 34 7.6 < 0.05 R t = f ( [ L A C ] , CO) 0.87 76 21.7 < 0.0001 L i n e a r r e g r e s s i o n s were p e r f o r m e d on t h e d a t a f r o m T a b l e 7 (N = 17; e x p e r i m e n t No. 10 was n o t i n c l u d e d ) . R = l a c t a t e t u r n o v e r r a t e ; [LAC] = p l a s m a l a c t a t e c o n c e n t r a t i o n ; CO = c a r d i a c o u t p u t . ANOVA was u s e d t o d e t e r m i n e w h e t h e r t h e s l o p e s o f t h e r e g r e s s i o n e q u a t i o n s were s i g n i f i c a n t l y d i f f e r e n t f r o m 0 [F v a l u e s and p r o b a b i l i t i e s (P) a r e g i v e n a b o v e ] . 135 r a t e was s i g n i f i c a n t l y l o w e r a t r e s t t h a n f o r t h e d i f f e r e n t e x e r c i s e p r o t o c o l s (P<0.05). Mean t u r n o v e r r a t e was n o t d i f f e r e n t between t h e c a n t e r (C) and t h e t r o t p r o t o c o l s (ST and E T ) . Mean l a c t a t e t u r n o v e r was h i g h e r a t t h e b e g i n n i n g o f t h e t r o t p r o t o c o l (ST) t h a n l a t e r d u r i n g t h e same p r o t o c o l ( E T ) , b u t t h e d i f f e r e n c e was n o t s i g n i f i c a n t a t P=0.05. I n e x p e r i m e n t 10, t h e a n i m a l was c a n t e r i n g a t an u n n a t u r a l l y low v e l o c i t y f o r t h i s g a i t (4 m / s ) . I n a d d i t i o n , he was t r y i n g t o go f a s t e r t h a n t h e t r e a d m i l l by c o n s t a n t l y p u s h i n g a g a i n s t a m e t a l r a i l p l a c e d i n f r o n t o f h i m . H i s mean h e a r t r a t e was much h i g h e r t h a n f o r h o r s e s c a n t e r i n g n o r m a l l y a t 6.5 m/s, and he m a i n t a i n e d a h i g h b u t s t e a d y p l a s m a l a c t a t e c o n c e n t r a t i o n o f 3.5 mM t h r o u g h o u t t h e e x p e r i m e n t . Under t h e s e c o n d i t i o n s , h i s r a t e o f l a c t a t e t u r n o v e r r e a c h e d 276.2 umol m i n - 1 k g - 1 . The r e l a t i o n s h i p between p l a s m a l a c t a t e c o n c e n t r a t i o n and l a c t a t e t u r n o v e r r a t e i s g i v e n i n F i g . 17. The l i n e was f i t t e d by l e a s t s q u a r e s ( l i n e a r r e g r e s s i o n : s l o p e = 5 9 . 0 , r = 0 . 8 0 ) . A c o m p l e t e r e g r e s s i o n a n a l y s i s o f t h e e f f e c t s o f c h a n g e s i n p l a s m a l a c t a t e c o n c e n t r a t i o n and c a r d i a c o u t p u t on l a c t a t e t u r n o v e r r a t e i s summarized i n T a b l e 8. The e f f e c t o f c h a n g e s i n p l a s m a l a c t a t e c o n c e n t r a t i o n was h i g h l y s i g n i f i c a n t and c o u l d e x p l a i n 64% o f t h e v a r i a b i l i t y i n t u r n o v e r r a t e . The e f f e c t o f c h a n g e s i n c a r d i a c o u t p u t was a l s o s i g n i f i c a n t b u t c o u l d o n l y a c c o u n t f o r 34% o f t h e v a r i a b i l i t y i n t u r n o v e r r a t e . When b o t h , l a c t a t e c o n c e n t r a t i o n and c a r d i a c o u t p u t , were u s e d i n a m u l t i p l e r e g r e s s i o n , t h e i r c ombined e f f e c t was h i g h l y 136 s i g n i f i c a n t and c o u l d e x p l a i n 76% o f t h e v a r i a b i l i t y i n l a c t a t e t u r n o v e r r a t e ( T a b l e 8 ) . The c o e f f i c i e n t o f m u l t i p l e 2 d e t e r m i n a t i o n (R ) was s i g n i f i c a n t l y i n c r e a s e d by t h e a d d i t i o n o f c a r d i a c o u t p u t as a s e c o n d i n d e p e n d e n t v a r i a b l e t o t h e s i m p l e r r e g r e s s i o n model o n l y u s i n g l a c t a t e c o n c e n t r a t i o n as a s i n g l e i n d e p e n d e n t v a r i a b l e (1 d f / 1 4 d f ; F=6.8; P<0.025; s e e S o k a l and R o h l f 1981, p. 6 3 4 ) . T h i s a n a l y s i s shows t h a t t h e i n c l u s i o n o f c a r d i a c o u t p u t i n t h e r e g r e s s i o n model c a u s e d a s i g n i f i c a n t i n c r e a s e i n t h e d e t e r m i n a t i o n o f t h e v a r i a b i l i t y i n l a c t a t e t u r n o v e r r a t e (see T a b l e 8 ) . 5.4 DISCUSSION. T h i s s t u d y shows t h a t c a r d i a c o u t p u t i s c o r r e l a t e d w i t h t h e r a t e o f l a c t a t e t u r n o v e r . I t a l s o p r o v i d e s t h e f i r s t m easurements o f p l a s m a l a c t a t e f l u x e s i n t h o r o u g h b r e d r a c e h o r s e s and c o n f i r m s t h e p o s i t i v e c o r r e l a t i o n between l a c t a t e c o n c e n t r a t i o n and t u r n o v e r r a t e o f p l a s m a l a c t a t e r e p o r t e d f o r t u n a i n C h a p t e r 2. B e c a u s e h o r s e s a r e v e r y e x p e n s i v e t o keep, and p a r t i c u l a r l y b e c a u s e t h o r o u g h b r e d r a c e h o r s e s a r e n o t commonly u s e d f o r i n v a s i v e e x p e r i m e n t s , t h e p r e s e n t s t u d y had t o be c a r r i e d o u t on f o u r a n i m a l s o n l y . I n a d d i t i o n , t h e h i g h c o s t o f t h e i s o t o p e r e q u i r e d f o r t h e measurement o f l a c t a t e t u r n o v e r r a t e l i m i t e d t h e t o t a l number o f e x p e r i m e n t s ; t u r n o v e r r a t e c o u l d o n l y be m e asured f o u r t i m e s i n e a c h 137 a n i m a l u n d e r d i f f e r e n t e x e r c i s e c o n d i t i o n s . F i n a l l y , v e r y few l a r g e a n i m a l t r e a d m i l l s a r e a v a i l a b l e f o r t h e t y p e o f s t u d i e s c a r r i e d o u t i n t h i s t h e s i s , and s u c h t r e a d m i l l s a r e e x p e n s i v e t o m a i n t a i n and t o o p e r a t e . T h e r e f o r e , t h e t o t a l amount o f t i m e a v a i l a b l e f o r t h e ^ p r e s e n t work was a l s o l i m i t i n g . 5.4.1 LACTATE TURNOVER RATE AND ROLE OF PLASMA LACTATE AS AN OXIDATIVE FUEL. I n t h o r o u g h b r e d h o r s e s , s ubmaximal e x e r c i s e o f up t o 55% V0 2max c a u s e d a 5 t o 7 - f o l d i n c r e a s e i n l a c t a t e t u r n o v e r r a t e o v e r t h e mean r e s t i n g f l u x r a t e o f 9.3 umol min 1 kg T h i s v a l u e i s l o w e r t h a n t h e r a t e r e p o r t e d by S t a n l e y e t a_l (1985) f o r r e s t i n g humans (15 umol min 1 kg "*") , b u t s u c h a d i s c r e p a n c y i s e x p e c t e d when . c o n s i d e r i n g t h e 6 - f o l d d i f f e r e n c e i n body mass (see C h a p t e r 2 ) . The t u r n o v e r r a t e s o f 50 umol min 1 kg 1 ( t r o t ) and 66 umol min 1 k g " 1 ( c a n t e r ) m e a s u r e d h e r e i n r u n n i n g h o r s e s a r e s i m i l a r t o v a l u e s f o u n d i n e x e r c i s i n g humans f o r t h e same work i n t e n s i t y (45-50 umol m i n - 1 k g - 1 ; s e e S t a n l e y e t a_l, 1985; and Mazzeo e_t a_l, 1986) . When t h e r a n g e o f t u r n o v e r r a t e s f o u n d i n t h o r o u g h b r e d s i s p r e s e n t e d a g a i n s t body mass on a l o g - l o g p l o t (see C h a p t e r 2) and compared w i t h o t h e r s p e c i e s , i t c l o s e l y m a t c h e s t h e s t a n d a r d mammalian p a t t e r n e x p e c t e d f o r t h e i r body s i z e . A t r e s t , a b o u t f i f t y p e r c e n t o f t h e l a c t a t e t u r n e d o v e r i s o x i d i z e d i n r a t s (Donovan and B r o o k s , 1983), i n dogs ( I s s e k u t z e t aj^, 1976) and i n humans (Mazzeo e_t a_l, 1986) . L a c t a t e o x i d a t i o n i s r e s p o n s i b l e f o r 13 t o 20% o f t h e o x y g e n 138 consumption of a r e s t i n g human [ c a l c u l a t e d from S t a n l e y e t aJL (1985) and Mazzeo e t a l (1986)] and for 39% of the r e s t i n g V 0 2 i n r a t s ( c a l c u l a t e d from Donovan and B r o o k s , 1983). I f a s i m i l a r f r a c t i o n of l a c t a t e t u r n o v e r r a t e i s o x i d i z e d i n thoroughbred h o r s e s , o n l y 7% of t h e i r r e s t i n g V 0 2 can be accounted f o r by l a c t a t e o x i d a t i o n (us ing a mean r e s t i n g . -1 -1 t u r n o v e r r a t e o f 9.3 umol min kg and a r e s t i n g V 0 2 of 5 ml m i n - 1 k g - 1 ) . Note t h a t even i f 100% of the r a t e of l a c t a t e t u r n o v e r was o x i d i z e d , the c o n t r i b u t i o n of l a c t a t e o x i d a t i o n to t o t a l oxygen consumption i n thoroughbreds would be 14% (the lowest e s t i m a t e found f o r humans, but a much lower va lue than i n r a t s ) . I n t e r s p e c i f i c d i f f e r e n c e s i n the r e l a t i v e use of l a c t a t e as an o x i d i z a b l e f u e l are even more s t r i k i n g d u r i n g e x e r c i s e . A t 50.% V0 2 max, 75 to 80% of l a c t a t e t u r n o v e r r a t e i s o x i d i z e d i n r a t s (Donovan and B r o o k s , 1983) and i n humans (Mazzeo e_t a_l, 1986) . I f the same percentage of the l a c t a t e f l u x i s o x i d i z e d i n t h o r o u g h b r e d s , the c o n t r i b u t i o n o f l a c t a t e o x i d a t i o n to V 0 2 decreases to o n l y 4.5% i n a c a n t e r i n g h o r s e . F o r the same e x e r c i s e i n t e n s i t y ( i . e . 50-60% V 0 2 m a x ) , humans use 12% ( c a l c u l a t e d from S t a n l e y e_t a_l, 1985, and Mazzeo e_t a_l, 1986) and r a t s use an a s t o u n d i n g 65% of t h e i r V 0 2 to o x i d i z e l a c t a t e a lone (from Donovan and B r o o k s , 1983) . U n f o r t u n a t e l y , i t was i m p o s s i b l e to q u a n t i f y l a c t a t e o x i d a t i o n d i r e c t l y i n the p r e s e n t s tudy because the bo lus i n j e c t i o n t e c h n i q u e was used (cont inuous i n f u s i o n i s n e c e s s a r y when o x i d a t i o n has to be 14 q u a n t i f i e d by c o l l e c t i o n of C 0 2 ' b u t ' a s e x p l a i n e d p r e v i o u s l y , i n f u s i o n would have been f a r too c o s t l y i n such 139 l a r g e a n i m a l s ) . However, e v e n i f 100% o f t h e l a c t a t e t u r n o v e r  m e a s u r e d h e r e was g o i n g t o o x i d a t i o n , t h i s pathway o f l a c t a t e  m e t a b o l i s m w o u l d o n l y a c c o u n t f o r l e s s t h a n 6% o f VO^ i n a  c a n t e r i n g t h o r o u g h b r e d h o r s e . E v e n t h o u g h t h e c o n t r i b u t i o n o f p l a s m a l a c t a t e as an o x i d i z a b l e f u e l i s s i g n i f i c a n t i n h o r s e s , t h e above c a l c u l a t i o n s c l e a r l y show t h a t t h i s s u b s t r a t e o n l y p l a y s a m i n o r r o l e d u r i n g s u b m a x i m a l e x e r c i s e compared w i t h o t h e r mammalian s p e c i e s , and i t i s n o t n e c e s s a r y t o have d i r e c t o x i d a t i o n measurements t o draw t h i s c o n c l u s i o n . R e s t i n g g l u c o s e t u r n o v e r r a t e s o f 7 umol min 1 k g - 1 h a v e been measured i n u n t r a i n e d p o n i e s and h o r s e s (Anwer e_t a^l, 1976; E v a n s , 1 9 7 1 ) . T r a i n e d h o r s e s c a n r e a c h 12 umol m i n - 1 k g - 1 a t r e s t ( E v a n s , 1971), b u t no d a t a a r e a v a i l a b l e d u r i n g work. E x e r c i s e c a u s e s g l u c o s e t u r n o v e r r a t e t o m a x i m a l l y i n c r e a s e by 2 t o 3 - f o l d i n o t h e r mammals and 50 t o 80% o f t h e t o t a l f l u x o f g l u c o s e i s o x i d i z e d d e p e n d i n g on work i n t e n s i t y ( B r o o k s , 1 9 8 5 ) . I f t h e g l u c o s e k i n e t i c s o f h o r s e s f o l l o w t h e s t a n d a r d mammalian p a t t e r n ( i . e . 50% and 80% o f g l u c o s e t u r n o v e r r a t e i s o x i d i z e d a t r e s t and a t 50% VC^max, r e s p e c t i v e l y ) , t h e combi n e d c o n t r i b u t i o n s o f exog e n o u s l a c t a t e and e x o g e n o u s g l u c o s e s u p p l i e s c a n o n l y a c c o u n t f o r 25% o f VC^ a t r e s t and f o r a b o u t 10% o f VC>2 d u r i n g t h e c a n t e r [ a s s u m i n g a g l u c o s e t u r n o v e r o f 36 umol min -' 1' k g - 1 : 3 t i m e s t h e r e s t i n g v a l u e r e p o r t e d by Evans (1971) i n t r a i n e d h o r s e s ] . A t r e s t and d u r i n g submaximal e x e r c i s e , i t i s c l e a r t h a t e xogenous f u e l s o t h e r t h a n c a r b o h y d r a t e s o_r_ endogenous s u b s t r a t e s must p l a y a major r o l e 140 i n o x i d a t i v e ATP p r o d u c t i o n . B e c a u s e t h e c o n t r i b u t i o n o f amino a c i d s i s l i k e l y t o be m i n o r , t h o r o u g h b r e d h o r s e s p r o b a b l y u s e f a t as t h e i r m a j o r o x i d i z a b l e s u b s t r a t e f o r m u s c l e work. The o f u n t r a i n e d s t a n d a r d b r e d h o r s e s i n c r e a s e s f r o m a r e s t i n g v a l u e o f 0.83 t o 0.98 d u r i n g h i g h - i n t e n s i t y e x e r c i s e (Thomas and F r e g i n , 1981) s u g g e s t i n g t h a t , i n t h e u n t r a i n e d s t a t e , c a r b o h y d r a t e o x i d a t i o n becomes more i m p o r t a n t when work r a t e i s e l e v a t e d . However, t h e same i n v e s t i g a t o r s h a v e shown t h a t t h i s i n c r e a s e i n R^ was s u p p r e s s e d by t r a i n i n g (Thomas e t a l , 1 983), i n d i c a t i n g t h a t f a t c o u l d r e m a i n an i m p o r t a n t o x i d i z a b l e f u e l d u r i n g h i g h - i n t e n s i t y e x e r c i s e i n t r a i n e d s t a n d a r d b r e d h o r s e s . A d i f f e r e n t k i n d o f e v i d e n c e a l s o s u g g e s t s t h a t f a t o x i d a t i o n may be an i m p o r t a n t s o u r c e o f e n e r g y i n e x e r c i s i n g h o r s e s . A v e r y l a r g e i n c r e a s e i n p l a s m a g l y c e r o l c o n c e n t r a t i o n h a s been o b s e r v e d d u r i n g and a f t e r e x e r c i s e i n t h o r o u g h b r e d h o r s e s (Snow e_t a_l_, 1985), i n d i c a t i n g t h a t t r i g l y c e r i d e u t i l i z a t i o n c o u l d be v e r y h i g h i n t h e s e a n i m a l s . H o r s e s may i n c r e a s e t h e i r r e l i a n c e on l a c t a t e o x i d a t i o n a t h i g h e r e x e r c i s e i n t e n s i t i e s t h a n u s e d i n t h e p r e s e n t s t u d y . The h i g h l a c t a t e t u r n o v e r r a t e o f 276.2 umol min 1 k g - 1 m e a s u r e d u n d e r u n u s u a l c o n d i t i o n s i n e x p e r i m e n t 10 (see R e s u l t s s e c t i o n ) i n d i c a t e s t h a t t h e y h a v e t h e a b i l i t y t o b o o s t l a c t a t e f l u x r a t e s much h i g h e r t h a n o b s e r v e d d u r i n g t h e t r o t and c a n t e r p r o t o c o l s . The h i g h e s t r a t e s o f l a c t a t e d i s a p p e a r a n c e measured i n humans by S t a n l e y ejt aJL (1985) o n l y r e a c h e d 230 umol mi n 1 kg 1 i n t r a i n e d s u b j e c t s . H e r e, SB was a l r e a d y a b l e t o r e a c h 276 umol m i n - 1 k g - 1 a t a r e l a t i v e l y low 141 p l a s m a l a c t a t e c o n c e n t r a t i o n o f 3.5 mM ( E x p t . 10) w h i l e t h e h i g h e s t human v a l u e s were m e a s u r e d a t c o n c e n t r a t i o n s o f 9 t o 11 mM. The p r e s e n t s t u d y s u g g e s t s t h a t l a c t a t e t u r n o v e r r a t e d e c r e a s e s t h r o u g h o u t a b o u t o f s u b m a x i m a l work. As e x e r c i s e i s s u s t a i n e d , r a t s , d o g s , and humans have been shown t o p r o g r e s s i v e l y i n c r e a s e t h e i r r e l i a n c e upon f a t as a s u b s t r a t e f o r e n e r g y m e t a b o l i s m ( H o l l o s z y e_t a_l, 1986) . P lasma f r e e f a t t y a c i d c o n c e n t r a t i o n i n c r e a s e s and goes down t h r o u g h o u t e x e r c i s e . A c o n c o m i t a n t d e c r e a s e i n p l a s m a c a r b o h y d r a t e t u r n o v e r w o u l d t h e r e f o r e be e x p e c t e d d u r i n g p r o l o n g e d e x e r c i s e . 5.4.2 LACTATE CLEARANCE,RATE. M e t a b o l i c c l e a r a n c e r a t e s (MCR) showed a 4 . 5 - f o l d i n c r e a s e between r e s t and t h e t r o t p r o t o c o l (13.5 t o 63.4 ml min-''" kg -'''). A t a h i g h e r e x e r c i s e i n t e n s i t y ( c a n t e r ) , t h i s r a t e d e c r e a s e d t o 55.7 ml min-"'" k g - 1 . The same p a t t e r n o f c h a n g e s i n MCR as a f u n c t i o n o f work i n t e n s i t y h a s been o b s e r v e d i n humans (Mazzeo e t a l _ , 1986; S t a n l e y e t a l , 1985) . The r e s t i n g MCR o f h o r s e s was a b o u t h a l f t h e v a l u e f o u n d i n humans, b u t t h e r a t e m e a sured d u r i n g t h e t r o t p r o t o c o l was 70% h i g h e r t h a n th e l a r g e s t MCR r e p o r t e d i n man ( S t a n l e y e t a_l, 1 9 8 5 ) . E x e r c i s i n g t h o r o u g h b r e d h o r s e s t h e r e f o r e h a v e t h e a b i l i t y t o s u p p o r t a g i v e n r a t e o f l a c t a t e t u r n o v e r w i t h a l o w e r p l a s m a l a c t a t e c o n c e n t r a t i o n t h a n humans. E n d u r a n c e t r a i n i n g h a s been shown t o l o w e r t h e p l a s m a c o n c e n t r a t i o n f o r a g i v e n t u r n o v e r r a t e i n r a t s , t h e r e b y i n c r e a s i n g MCR (Donovan and B r o o k s , 1983). 142 The h i g h e r MCR shown by t h o r o u g h b r e d s may t h e r e f o r e s i m p l y r e f l e c t t h e h i g h l y t r a i n e d c o n d i t i o n o f t h e s e a n i m a l s . 5.4.3 EFFECT OF PLASMA METABOLITE CONCENTRATION ON TURNOVER RATE. ' The t u r n o v e r r a t e and t h e p l a s m a c o n c e n t r a t i o n o f l a c t a t e a r e p o s i t i v e l y c o r r e l a t e d i n t h o r o u g h b r e d h o r s e s . The same r e l a t i o n s h i p has been o b s e r v e d i n a number o f a n i m a l s i n c l u d i n g r a t s ( O k a j i m a e_t a_l, 1981; Donovan and B r o o k s 1983), dogs ( E l d r i d g e e_t a_l, 1974; I s s e k u t z e t a_l, 1976), s e a l s ( D a v i s , 1983), humans (Mazzeo e t a l , 1 9 86), and e v e n t u n a ( C h a p t e r 2 ) . The f l u x r a t e s o f g l u c o s e (Verdonk e t a_l, 1981) , g l y c e r o l ( B o r t z e t a l , 1972) and f r e e f a t t y a c i d s ( P a u l and I s s e k u t z , 1967; H a g e n f e l d t , 1975)- h ave a l s o been shown t o . i n c r e a s e as t h e i r p l a s m a c o n c e n t r a t i o n goes up. A s i m p l e mass a c t i o n e f f e c t o f t h e s e m e t a b o l i t e s c o u l d e x p l a i n t h e o b s e r v e d r e l a t i o n s h i p s . The r a t e o f c e l l u l a r u p t a k e by w o r k i n g m u s c l e s s h o u l d be p r o p o r t i o n a l t o s u b s t r a t e c o n c e n t r a t i o n i n p l a s m a f o r any g i v e n r a t e o f o x i d a t i v e m e t a b o l i s m i n t h e s e m u s c l e s . I n d e e d , S t a n l e y e_t a_l_ (1985) o b s e r v e d t h a t t h e r a t e o f l a c t a t e d i s a p p e a r a n c e seems t o be d i r e c t l y c o n t r o l l e d by p l a s m a l a c t a t e c o n c e n t r a t i o n i n humans. Some c o n t r a d i c t o r y e v i d e n c e i s a v a i l a b l e f o r g l u c o s e however, where an i n v e r s e r e l a t i o n s h i p between c o n c e n t r a t i o n and t u r n o v e r r a t e has a l s o been r e p o r t e d r e c e n t l y ( J e n k i n s e_t a_l_, 1986) . T h e s e a u t h o r s showed t h a t i n s u l i n h a s an e f f e c t on t h e s l o p e o f t h e r e l a t i o n s h i p , and t h e i r r e s u l t s s u g g e s t t h a t h o r m o n a l i n f l u e n c e s c a n o v e r r i d e t h e mass a c t i o n e f f e c t o f g l u c o s e c o n c e n t r a t i o n . 143 Most o f t h e s t u d i e s i n v e s t i g a t i n g h o r m o n a l e f f e c t s on s u b s t r a t e f l u x e s d e a l w i t h g l u c o s e m e t a b o l i s m (see H e t e n y i e t a l , 1983 f o r a r e v i e w ) b u t l i m i t e d i n f o r m a t i o n i s a v a i l a b l e f o r l a c t a t e . C h a l l i s s e t a_l (1986) showed t h a t t h e r a t e o f l a c t a t e p r o d u c t i o n by t h e r a t h i n d l i m b d e pends on t h e c o n c e n t r a t i o n o f i n s u l i n i n p l a s m a , and I s s e k u t z (1984) r e p o r t e d t h a t t h e i n c r e a s e i n l a c t a t e t u r n o v e r r a t e e l i c i t e d by e x e r c i s e was a b o l i s h e d by b e t a - a d r e n e r g i c b l o c k a d e . I s s e k u t z ' s d a t a s u g g e s t t h a t e p i n e p h r i n e a f f e c t s l a c t a t e f l u x r a t e s i n d i r e c t l y v i a c h a n g e s i n p l a s m a l a c t a t e c o n c e n t r a t i o n . I n d e e d , t h e complex p a t t e r n o f c o n c e n t r a t i o n c h a n g e s c a u s e d by h i s e x p e r i m e n t a l p r o t o c o l i s e x a c t l y m a t ched by c o n c o m i t a n t f l u c t u a t i o n s i n f l u x r a t e s ( F i g . 2 i n I s s e k u t z , 1 9 8 4 ) . Hormonal e f f e c t s must b e . s t u d i e d more t h o r o u g h l y i f t h e c o n t r o l o f m e t a b o l i t e f l u x e s i s t o be f u l l y u n d e r s t o o d . 5.4.4 EFFECT OF CHANGES IN CARDIAC OUTPUT. T h i s s t u d y r e p r e s e n t s t h e f i r s t i n v e s t i g a t i o n o f t h e r o l e p l a y e d by t o t a l p e r f u s i o n r a t e on p l a s m a s u b s t r a t e t u r n o v e r a t t h e  whole o r g a n i s m l e v e l . I t shows t h a t c a r d i a c o u t p u t i s c o r r e l a t e d w i t h p l a s m a l a c t a t e f l u x e s , and t h a t t h i s c o r r e l a t i o n i s i n d e p e n d e n t o f t h e l a c t a t e c o n c e n t r a t i o n v e r s u s t u r n o v e r r a t e r e l a t i o n s h i p (see T a b l e 8 ) . T h e s e r e s u l t s a g r e e w i t h t h e k i n e t i c s d a t a o f o t h e r s t u d i e s (see n e x t p a r a g r a p h ) i n w h i c h t h e e f f e c t o f c h a n g e s i n c a r d i a c o u p u t was n o t s p e c i f i c a l l y i n v e s t i g a t e d ( i n p a r t i c u l a r I s s e k u t z e t a_l, 1976 i n t h e c a s e o f l a c t a t e t u r n o v e r ) . I t i s w e l l known t h a t t h e l a r g e i n c r e a s e i n c a r d i a c o u t p u t e l i c i t e d 144 by e x e r c i s e goes t o the w o r k i n g muscles (Brooks and Fahey, 1984). T h e r e f o r e , changes i n c a r d i a c o u t p u t must have an e f f e c t on the d e l i v e r y r a t e o f l a c t a t e t o t h e s e m u s c l e s . In f a c t , the t u r n o v e r r a t e s o f a l l plasma m e t a b o l i t e s s h o u l d a l s o be i n f l u e n c e d by changes i n c a r d i a c o u t p u t , and r e c e n t e v i d e n c e shows t h a t t h i s i s p r o b a b l y t r u e f o r g l u c o s e . C h a l l i s s e t a_l (1986) showed t h a t the r a t e of g l u c o s e u t i l i z a t i o n by the r a t h i n d l i m b depends on b l o o d f l o w a t r e s t and d u r i n g i s o m e t r i c c o n t r a c t i o n s . F u r t h e r m o r e , g l u c o s e t u r n o v e r r a t e i n c r e a s e s w i t h e x e r c i s e i n the absence of a s i g n i f i c a n t change i n plasma g l u c o s e c o n c e n t r a t i o n (Brooks and Donovan, 1983; J e n k i n s e_t a_l, 1986), and t h i s a d j u s tment i n f l u x r a t e c o u l d be m ediated by p e r f u s i o n changes. An i n c r e a s e i n c a r d i a c o u t p u t not o n l y p r o v i d e s more oxygen t o the c o n t r a c t i n g m u s c l e s , but i t a l s o a c c e l e r a t e s the s u p p l y of o x i d i z a b l e s u b s t r a t e s t o meet the e n e r g e t i c demands of e x e r c i s e . The s l o p e s r e p o r t e d f o r the r e l a t i o n s h i p between l a c t a t e c o n c e n t r a t i o n and t u r n o v e r r a t e v a r y over a v e r y wide range depending on the a n i m a l and on the e x p e r i m e n t a l c o n d i t i o n s . T h i s v a r i a b i l i t y can be p a r t l y a c c o u n t e d f o r by i n t e r s p e c i f i c d i f f e r e n c e s but a l s o , and more i m p o r t a n t l y , by the e f f e c t of c a r d i a c o u t p u t on l a c t a t e f l u x e s which has been i g n o r e d i n m e t a b o l i t e k i n e t i c s s t u d i e s . For i n s t a n c e , I s s e k u t z e_t a_l (1976) showed t h a t the s l o p e was much h i g h e r when the d i f f e r e n t l a c t a t e c o n c e n t r a t i o n s were e l i c i t e d by e x e r c i s e than when v a r y i n g amounts of N a - l a c t a t e were i n f u s e d i n r e s t i n g dogs ( F i g . 4 i n I s s e k u t z e_t a_l, 1976). T h e i r 145 r e s u l t s c a n be e x p l a i n e d e a s i l y when t h e e f f e c t o f p e r f u s i o n r a t e on t u r n o v e r i s c o n s i d e r e d . Why wou l d c h a n g e s i n c a r d i a c o u t p u t i n f l u e n c e l a c t a t e t u r n o v e r ? L a c t a t e t r a n s p o r t a c r o s s c e l l membranes i s r a p i d , and i t o c c u r s - b y d i f f u s i o n ( m a i n l y c a r r i e r - m e d i a t e d , b u t t o a l e s s e r e x t e n t by s i m p l e d i f f u s i o n : s e e D u b i n s k y and R a c k e r , 1978; Koch e t a_l, 1981; Roos, 1975; and S p e n c e r and L e h n i n g e r , 1 9 7 5 ) . The t r a n s f e r r a t e o f l a c t a t e t h e r e f o r e d epends on c o n c e n t r a t i o n g r a d i e n t s between e x t r a - and i n t r a c e l l u l a r f l u i d . D u r i n g e x e r c i s e , l o c a l d i s s i p a t i o n o f t h e s e g r a d i e n t s may o c c u r i f l a c t a t e i s o x i d i z e d f a s t e r i n t h e m u s c l e c e l l s t h a n i t i s s u p p l i e d by s u r r o u n d i n g c a p i l l a r i e s . A h i g h b l o o d f l o w i n t h e s e v e s s e l s w o u l d e n s u r e t h a t g r a d i e n t s a r e m a i n t a i n e d , t h e r e b y m a x i m i z i n g t h e r a t e o f l a c t a t e t r a n s l o c a t i o n . 5.4.5 CONTROL OF EXOGENOUS FUEL SUPPLY. B o t h c a r d i a c o u t p u t and p l a s m a l a c t a t e c o n c e n t r a t i o n i n f l u e n c e t h e r a t e o f l a c t a t e t u r n o v e r . B e c a u s e t h e f l u x e s o f a l l p l a s m a s u b s t r a t e s a r e p r o b a b l y e q u a l l y d e p e n d e n t on p e r f u s i o n c h a n g e s , c a r d i a c o u t p u t a d j u s t m e n t s c a n be v i e w e d as t h e c o a r s e c o n t r o l f o r p l a s m a m e t a b o l i t e f l u x r a t e s . T h e s e a d j u s t m e n t s do n o t a l l o w t h e t u r n o v e r o f an i n d i v i d u a l m e t a b o l i t e t o be a l t e r e d w i t h o u t a f f e c t i n g t h e f l u x e s o f a l l t h e o t h e r s . A u g m e n t i n g c a r d i a c o u t p u t t h e r e f o r e c a u s e s an o v e r a l l i n c r e a s e i n t h e s u p p l y o f o x i d a t i v e f u e l s as w e l l as o x y g e n . Changes i n p l a s m a m e t a b o l i t e c o n c e n t r a t i o n , on t h e o t h e r hand, a l l o w a f i n e r , s p e c i f i c a d j u s t m e n t f o r t h e f l u x 146 r a t e o f e a c h i n d i v i d u a l e x o g e n o u s f u e l . The a d j u s t m e n t o f c o n c e n t r a t i o n t h e r e f o r e r e p r e s e n t s t h e f i n e c o n t r o l f o r m e t a b o l i t e f l u x r a t e s b e c a u s e i t a l l o w s t h e s u p p l y r a t e o f e a c h o x i d a t i v e f u e l t o be a l t e r e d s e p a r a t e l y . The r e s p e c t i v e c o n t r i b u t i o n o f i n d i v i d u a l f u e l s t o o v e r a l l o x i d a t i v e e n e r g y p r o d u c t i o n c a n be f i n e l y r e g u l a t e d v i a t h i s mechanism t o s u p p l y t h e r i g h t c o m b i n a t i o n o f f u e l s a t t h e r i g h t t i m e . 5.4.6 CONCLUSIONS. I f l a c t a t e p r o v i d e d some p e r f o r m a n c e a d v a n t a g e o v e r o t h e r o x i d i z a b l e f u e l s s u c h as g l u c o s e or f r e e f a t t y a c i d s , a n i m a l s a d a p t e d f o r e n d u r a n c e e x e r c i s e w o u l d be e x p e c t e d t o s u p p o r t h i g h l a c t a t e t u r n o v e r r a t e s d u r i n g s t e a d y - s t a t e work. T h i s t h e s i s shows t h a t "good" e n d u r a n c e p e r f o r m e r s do n o t n e c e s s a r i l y s u p p o r t h i g h l a c t a t e t u r n o v e r r a t e s when o p e r a t i n g a t s u s t a i n a b l e e x e r c i s e i n t e n s i t i e s : s k i p j a c k t u n a h a v e h i g h e r t u r n o v e r r a t e s t h a n s e d e n t a r y mammals, b u t t h o r o u g h b r e d h o r s e s do n o t show an i n c r e a s e d a b i l i t y t o t r a n s f e r t h i s compound a c r o s s t h e i r p l a s m a compartment f r o m s i t e s o f p r o d u c t i o n t o s i t e s o f u t i l i z a t i o n compared w i t h o t h e r mammals. I n C h a p t e r 2, s k i p j a c k t u n a i s shown t o h a v e a r a n g e o f p l a s m a l a c t a t e f l u x r a t e s t h a t i s h i g h e r t h a n w o u l d be e x p e c t e d f o r a mammalian s p e c i e s o f e q u i v a l e n t s i z e , e v e n t h o u g h t h i s t e l e o s t o p e r a t e s a t a much l o w e r t e m p e r a t u r e ( a p p r o x i m a t e l y 1 0 ° C ) . B e c a u s e l a c t a t e t u r n o v e r r a t e s o f t u n a a r e h i g h , and b e c a u s e a l a r g e p r o p o r t i o n o f t h i s t u r n o v e r i s known t o be a c c o u n t e d f o r by l a c t a t e o x i d a t i o n i n s e v e r a l o t h e r s p e c i e s u n d e r d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s , t h e s k i p j a c k t u n a i s v e r y l i k e l y t o 147 be a b l e t o s u p p o r t h i g h r a t e s o f p l a s m a l a c t a t e o x i d a t i o n , and l a c t a t e i s p r o b a b l y an i m p o r t a n t o x i d i z a b l e f u e l i n t h i s s p e c i e s . T h e r e f o r e , r e s u l t s f r o m t h e t u n a s t u d y do n o t p r o v i d e e v i d e n c e a g a i n s t t h e i d e a t h a t " e l i t e a n i m a l a t h l e t e s " c o u l d p r e f e r l a c t a t e o v e r o t h e r o x i d i z a b l e f u e l s . I n c o n t r a s t , C h a p t e r 5 shows t h a t t h e l a c t a t e t u r n o v e r r a t e s o f t h o r o u g h b r e d h o r s e s f a l l w e l l w i t h i n t h e e x p e c t e d v a l u e s f o r s e d e n t a r y mammalian s p e c i e s . T h e r e f o r e , t h e m e t a b o l i c a d a p t a t i o n s o f t h o r o u g h b r e d h o r s e s f o r e x e r c i s e do n o t i n c l u d e t h e a b i l i t y . t o s u s t a i n h i g h e r l a c t a t e t u r n o v e r r a t e s t h a n s e d e n t a r y mammals. In a d d i t i o n , i t i s shown i n t h e l a s t c h a p t e r o f t h e p r e s e n t work t h a t p l a s m a l a c t a t e o n l y p l a y s a m i n o r r o l e as an o x i d i z a b l e f u e l f o r m u s c l e e n e r g y m e t a b o l i s m i n t h o r o u g h b r e d h o r s e s . The o x i d a t i o n o f t h i s m e t a b o l i t e r e p r e s e n t s l e s s t h a n 5% o f t o t a l o x y g e n c o n s u m p t i o n d u r i n g s u bmaximal work, and f a t c o u l d be t h e m a j o r o x i d a t i v e s u b s t r a t e i n e x e r c i s i n g h o r s e s . T h i s low u t i l i z a t i o n o f l a c t a t e as a f u e l i n h o r s e s i s i n s h a r p c o n t r a s t w i t h t h e h i g h e r r e l i a n c e on t h i s s u b s t r a t e shown by o t h e r s p e c i e s , b u t p a r t i c u l a r l y by e x e r c i s i n g r a t s . T h e s e r e s u l t s e m p h a s i z e t h e f a c t t h a t , e v e n among mammals, l a r g e i n t e r s p e c i f i c d i f f e r e n c e s i n o x i d i z a b l e f u e l p r e f e r e n c e a r e p r e s e n t . The l a c t a t e k i n e t i c s measurements o f C h a p t e r 5 show t h a t t h e a b i l i t y t o o x i d i z e p l a s m a l a c t a t e a t h i g h r a t e s i s n o t n e c e s s a r i l y r e q u i r e d f o r t h e " e l i t e " p e r f o r m a n c e o f e n d u r a n c e e x e r c i s e . 148 A p o s i t i v e c o r r e l a t i o n between p l a s m a l a c t a t e c o n c e n t r a t i o n and l a c t a t e t u r n o v e r r a t e i s d e m o n s t r a t e d i n b o t h s p e c i e s s t u d i e d i n t h i s t h e s i s . I n C h a p t e r 2, t h i s c o r r e l a t i o n i s shown f o r t h e f i r s t t i m e i n a non-mammalian s y s t e m , and t h e r e l a t i o n s h i p g i v e n f o r t u n a c o v e r s a much w i d e r r a n g e o f c o n c e n t r a t i o n s t h a n p r e v i o u s l y r e p o r t e d f o r mammals (a r a n g e o f more t h a n 20 mM). R e s u l t s f r o m C h a p t e r s 4 and 5 show t h a t c a r d i a c o u t p u t i s a l s o c o r r e l a t e d w i t h l a c t a t e t u r n o v e r r a t e , and t h a t t h i s c o r r e l a t i o n i s i n d e p e n d e n t o f t h e r e l a t i o n s h i p between p l a s m a l a c t a t e c o n c e n t r a t i o n and t u r n o v e r r a t e . Changes i n c a r d i a c o u t p u t h a v e an e f f e c t on t h e s l o p e o f t h i s r e l a t i o n s h i p , b u t t h i s e f f e c t had n o t been r e c o g n i z e d p r e v i o u s l y . D i f f e r e n c e s i n t h e s l o p e s o b t a i n e d f o r t h e c o n c e n t r a t i o n v e r s u s t u r n o v e r r e l a t i o n s h i p i n e a r l i e r s t u d i e s o f l a c t a t e k i n e t i c s f r o m t h e l i t e r a t u r e c a n be e x p l a i n e d e a s i l y i f t h e e f f e c t o f c a r d i a c o u t p u t c h a n g e s i s t a k e n i n t o a c c o u n t . B e c a u s e b o t h c a r d i a c o u t p u t and m e t a b o l i t e c o n c e n t r a t i o n i n f l u e n c e t h e r a t e o f m e t a b o l i t e t u r n o v e r i n p l a s m a , i t i s p r o p o s e d t h a t a d j u s t m e n t s i n c a r d i a c o u t p u t and i n m e t a b o l i t e c o n c e n t r a t i o n r e p r e s e n t t h e c o a r s e and f i n e c o n t r o l s f o r t h e r e g u l a t i o n o f p l a s m a m e t a b o l i t e t u r n o v e r . 149 REFERENCES AHLBORG, G., L. HAGENFELDT, AND J . WAHREN. 1975. S u b s t r a t e u t i l i z a t i o n by t h e i n a c t i v e l e d u r i n g one l e g o r arm e x e r c i s e . J . A p p l . P h y s i o l . 39: 718-723. AHLBORG, G., L. HAGENFELDT, AND J . WAHREN. 1976. I n f l u e n c e o f l a c t a t e i n f u s i o n on g l u c o s e and FFA m e t a b o l i s m i n man. Sc a n d . J . C l i n . L a b . I n v e s t . 36: 193-301. ALLSOP, J.R., R.R. WOLFE, AND J . F . BURKE. 1978. 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