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Buffer capacity of human skeletal muscle : relationships to fiber composition and anaerobic performance 1982

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B u f f e r c a p a c i t y of human s k e l e t a l muscle; r e l a t i o n s h i p s to f i b e r composition and anaerobic performance by Wade Stephen Parkhouse B.P.E. The U n i v e r s i t y of A l b e r t a , 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF PHYSICAL EDUCATION in THE FACULTY OF GRADUATE STUDIES Department of Sport Science School of P h y s i c a l Education We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May 1982 (S) Wade Stephen Parkhouse, 1982 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department O f P h y s i c a l Education The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 August 26, 1982 II A b s t r a c t Twenty male v o l u n t e e r s , comprising four d i s t i n c t sub- samples (S=800m runners; R=varsity oarsmen; M=marathon runners; UT=untrained c o n t r o l s ) , p a r t i c i p a t e d i n t h i s study. They were made aware of the p o t e n t i a l r i s k s i n v o l v e d and informed consent was o b t a i n e d . Anthropometric ( h y d r o s t a t i c weighing), p h y s i c a l c h a r a c t e r i s t i c and pulmonary f u n c t i o n ( C o l l i n s Respirometer) were assessed by standard t e c h n i q u e s . Maximal oxygen consumption was determined on a p r o g r e s s i v e t r e a d m i l l run (0.22 m.s~* every minute; i n i t i a l speed 2.22 m.s"*) to f a t i g u e . R e s p i r a t o r y gases were monitered every 15 seconds (Beckman M e t a b o l i c Measurement Cart) with the four h i g h e s t c o n s e c u t i v e oxygen uptake values being averaged f o r det e r m i n a t i o n of maximal oxygen uptake. Anaerobic performance (AST) was assessed as the time to f a t i g u e a the constant workload t r e a d m i l l run at 3.52 m.s"', 20 percent i n c l i n e . Post e x e r c i s e blood l a c t a t e l e v e l s (HLa) were determined as an a d d i t i o n a l v a r i a b l e i n assessment of anaerobic c a p a c i t y . The M were s i g n i f i c a n t l y o l d e r than the other 3 groups while no s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between the t r a i n e d groups f o r maximal oxygen uptake v a l u e s . The S and R demonstrated s i g n i f i c a n t l y e l e v a t e d AST (p<.0l) and post-AST HLa (p<.05) l e v e l s above the M, whose values were s i m i l i a r to the UT. T h i s enhanced anaerobic performance c o u l d not be a t t r i b u t e d to p h y s i c a l c h a r a c t e r i s t i c , pulmonary f u n c t i o n or a e r o b i c c a p a c i t y d i f f e r e n c e s of the t r a i n e d a t h l e t e s . Post-AST HLa d i s p l a y e d a s i g n i f i c a n t r e l a t i o n s h i p to anaerobic performance (r=.90). An enhanced l a c t a t e e f f l u x mechanism was shown by the I l l t r a i n e d groups, which was not a l t e r e d b y ' t r a i n i n g s p e c i f i c i t y . Muscle b i o p s i e s o b tained at r e s t from the vastus l a t e r a l i s muscle, were examined f o r f i b e r composition, pH, h i s t i d i n e and car n o s i n e l e v e l s and b u f f e r c a p a c i t y (B). B was found to be e l e v a t e d i n the a n a e r o b i c l y t r a i n e d groups(p<.01) demonstrating a s i g n i f i c a n t r e l a t i o n s h i p to AST (r=.5l) and f a s t - t w i t c h f i b e r percentage (FT%;r=.51), which i m p l i e d a r e l a t i o n s h i p to muscle g l y c o l y t i c c a p a c i t y . W i t h i n the S and R, c a r n o s i n e l e v e l s were found to be s i g n i f i c a n t l y e l e v a t e d (p<.0l), i l l u s t r a t i n g a s i g n i f i c a n t c o r r e l a t i o n to B (r=.64) and FT% (r=.46) , which emphasized the importance of ca r n o s i n e as a p h y s i o l o g i c a l b u f f e r and i t s p o s s i b l e r e l a t i o n s h i p to the g l y c o l y t i c c a p a c i t y of the t i s s u e . No d i f f e r e n c e s i n h i s t i d i n e l e v e l s or r e s t i n g i n t r a m u s c u l a r pH were demonstrated with t r a i n i n g s p e c i f i c i t y . These r e s u l t s suggest that the enhanced anaerobic performance c o u l d p a r t i a l l y be a t t r i b u t e d to e l e v a t e d B and c a r n o s i n e l e v e l s demonstrated w i t h i n s k e l e t a l muscle su b j e c t e d to anaerobic t r a i n i n g . T h i s may be due to the t i s s u e s enhanced c a p a c i t i e s to seqestor the protons which accumulate d u r i n g anaerobic g l y c o l y s i s . IV Table of Contents Page L i s t of symbols . .. . VI L i s t of f i g u r e s VII L i s t of t a b l e s VIII Acknowledgements IX V i t a , P u b l i c a t i o n s and F i e l d s of Study X I n t r o d u c t i o n 1 Methodology 4 R e s u l t s 8 D i s c u s s i o n 19 Summary of F i n d i n g s 28 References . 30 Appendices. 46 Review of L i t e r a t u r e 47 A. Acid-base s t a t u s and performance 47 1 . Metabolism 48 2. Processes of l a c t a t e d i s p o s a l and proton r e l e a s e .... 49 3. Enzymatic c o n t r o l of anaerobic g l y c o l y s i s 51 4. E x e r c i s e p h y s i o l o g y 52 B. I n t r a c e l l u l a r pH 55 1. T o t a l muscle pH i n r e l a t i o n to e x e r c i s e ... 57 2. pH changes and muscular work ......... 59 3. Mechanisms of a c t i o n 60 V C. B u f f e r c a p a c i t y 62 1. S k e l e t a l muscle b u f f e r c a p a c i t y 64 2. S k e l e t a l muscle b u f f e r c o n s t i t u e n t s 66 3. Carnosine and a n s e r i n e 68 D. Summary 72 Appendix A. Repeated b u f f e r c a p a c i t y d e t e r m i n a t i o n s 74 Appendix B. Blood l a c t a t e l e v e l s pre- and post-a n a e r o b i c performance 75 Appendix C. B u f f e r c a p a c i t y c o n v e r s i o n s 76 Appendix D. S e r i a l s e c t i o n s of UT vastus l a t e r a l i s muscle s t a i n e d f o r NADH-TR and Myosin ATPase... 77 Appendix E. Regression analyses 79 L i s t of Symbols. ATP adenosine t r i p h o s p h a t e NADH reduced n i c o t i n a m i d e adenine d i n u c l e o t i d e SI S l y k e , standard u n i t f o r B (mmol.pH .1 IC H 0) PFK phosphofructokinase G6P glucose-6-phosphate LDH l a c t a t e dehydrogenase CP c r e a t i n e phosphate FT f a s t - t w i t c h (type II) s k e l e t a l muscle f i b e r s ST slow-twitch (type I) s k e l e t a l muscle f i b e r s PH. i n t r a c e l l u l a r pH IAA i o d o a c e t i c a c i d HCO; b i c a r b o n a t e F6P fructose-6-phosphate B b u f f e r c a p a c i t y P. phosphate HIS h i s t i d i n e HLa blood l a c t a t e S s p r i n t e r s R rowers M marathon runners UT u n t r a i n e d c o n t r o l s VO^max maximal oxygen uptake AST anaerobic speed t e s t DMO 5,5 dimethyl-2,4-oxazolidedione VII L i s t of F i g u r e s F i g u r e Page 1. Anaerobic performance versus post-AST HLa l e v e l s ........ 11 2. R a t i o AST/Post-AST HLa versus HLa 12 3. B u f f e r c a p a c i t y versus t r a i n i n g s p e c i f i c i t y 16 4. B u f f e r c a p a c i t y versus c a r n o s i n e c o n c e n t r a t i o n determined by t i t r a t i o n with 0.01 HCl 17 VIII L i s t of Tables Table Page 1. P h y s i c a l c h a r a c t e r i s t i c s , anthropometric and pulmonary f u n c t i o n p r o f i l e s 9 2. P h y s i o l o g i c a l assessment and h i s t o l o g i c a l a n a l y s i s . of the vastus l a t e r a l i s muscle 10 3. B u f f e r c a p a c i t y , pH, h i s t i d i n e and c a r n o s i n e l e v e l s of the r e s t i n g vastus l a t e r a l i s muscle 14 4. C o r r e l a t i o n matrix f o r the b i o c h e m i c a l , h i s t o l o g i c a l and anaerobic performance v a r i a b l e s 15 5. Average non-bicarbonate b u f f e r values f o r s k e l e t a l muscle determined by homogenate t i t r a t i o n with HC1 or NaOH 65 IX Acknowledgements . To a l l my v o l u n t e e r s who through t h e i r e n t h u s i a s t i c p a r t i c i p a t i o n , made t h i s i n v e s t i g a t i o n p o s s i b l e , 1 express my g r a t i t u d e . 1 would s i n c e r e l y l i k e to thank my committee members: Drs. Peter Hochachka, B i l l O v a l l e , Ken Coutts and e s p e c i a l l y Don Mckenzie, who through t h e i r knowledge and guidance, made t h i s i n v e s t i g a t i o n a rewarding e x p e r i a n c e . S p e c i a l a p p r e c i a t i o n i s extended to Dr. Tom Mommsen and Susan Shinn f o r t h e i r e x p e r t i s e and p a t i e n c e i n te a c h i n g me the necessary t e c h n i q u e s . To a l l those i n d i v i d u a l s who helped me c o l l e c t my data and to Miss Diane Barnett f o r her support, I extend my thanks. X ; Vita.- . • • August 28, 1955. - Born - Hamilton, O n t a r i o . 1974 Secondary School Honours Graduation Diploma, Ancaster High and V o c a t i o n a l School, Ancaster, O n t a r i o . 1975-1980 Bachelor of P h y s i c a l E d u c a t i o n , U n i v e r s i t y of A l b e r t a , Edmonton, A l b e r t a . 1980-1982 Masters of P h y s i c a l E d u c a t i o n , Department of Sport Science School of P h y s i c a l Education and R e c r e a t i o n U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. P u b l i c a t i o n s Parkhouse, W.S., D.C. McKenzie, E.C. Rhodes, D. Dunwoody and P. Wiley. E x e r c i s e p r e s c r i p t i o n i n r e l a t i o n to anaerobic t h r e s h o l d . Can,. J . Appl .Sp.Sci . , 6 ( 4) : 1 35 ,1 981 . Parkhouse, W.S., D.C. McKenzie, E.C. Rhodes, D. Dunwoody and P. Wiley. Cardiac frequency and anaerobic t h r e s h o l d : I m p l i c a t i o n s f o r p r e s c r i p t i v e e x e r c i s e programs. Eur. J . Appl. P h y s i o l . , 1982 ( i n p r e s s ) . Rhodes, E . C , D.C. McKenzie, P. Wiley, D. Dunwoody and W.S. Parkhouse. Anaerobic t h r e s h o l d and p r e d i c t e d marathon performance. Can.J.Appl.Sp.Sci.,6(4):156,1981. Parkhouse, W.S., D.C. Mckenzie, P.W. Hochachka, W.K. O v a l l e , T.P. Mommsen and S.L. Shinn. The r e l a t i o n s h i p between c a r n o s i n e l e v e l s , b u f f e r i n g c a p a c i t y , f i b e r type and anaerobic c a p a c i t y i n e l i t e a t h l e t e s . Int'1.J.Sp.Med., 1982 ( i n p r e s s ) . XI Mckenzie, D.C, W.S. Parkhouse, E.C. Rhodes, P.W. Hochachka, W.K. O v a l l e , T.P. Mommsen and S.L. Shinn. S k e l e t a l muscle b u f f e r i n g c a p a c i t y i n e l i t e a t h l e t e s . Int'1.J.Sp.Med., 1982 (i n p r e s s ) . Parkhouse, W.S., D.C. Mckenzie, P.W. Hochachka, T.P. Mommsen, W.K. O v a l l e , S.L. Shinn and E.C. Rhodes. Muscle b u f f e r i n g c a p a c i t y , f i b e r composition and anaerobic c a p a c i t y of e l i t e a t h l e t e s . Med.Sci.Sp. , 14:132, 1982. Mckenzie, D.C, W.S. Parkhouse, E.C. Rhodes, W.K. O v a l l e and S. Shinn. Anaerobic c a p a c i t y and muscle f i b e r type. Med.Sci. Sp., 14:132, 1982. F i e l d s of Study Major f i e l d : E x e r c i s e p h y s i o l o g y Dr. D.C. Mckenzie S t u d i e s i n metabolism and bi o c h e m i s t r y Dr. P.W. Hochachka S t u d i e s i n h i s t o l o g y Dr. W.K. O v a l l e 1 I n t r o d u c t i o n S p r i n t t r a i n e d a t h l e t e s demonstrate a remarkable a b i l i t y to perform h i g h • i n t e n s i t y , short" d u r a t i o n work, with the energy requirements being met p r i n c i p a l l y by anaerobic g l y c o l y s i s . Most anaerobic s t u d i e s have c o n c e n t r a t e d on changes i n s u b s t r a t e l e v e l s or, enzyme a c t i v i t i e s and m e t a b o l i t e c o n c e n t r a t i o n s which c o u l d be capable of g e n e r a t i n g ATP, and m a i n t a i n i n g redox balance (Hochachka 1980). A l t e r a t i o n s i n s u b s t r a t e l e v e l s (Knuttgen and S a l t i n 1972; G o l l n i c k and Hermansen 1973), g l y c o l y t i c enzymes (Baldwin et a l . , 1972; G o l l n i c k et a l . , 1972; Hickson et a l . , 1975; C o s t i l l et a l . , 1976) and f i b e r composition (Thorstenson 1976; C o s t i l l et a l . , 1976,1979; Roberts et a l . , 1981) a s s o c i a t e d with anaerobic t r a i n i n g have been i n s u f f i c i e n t to account f o r the enhanced anaerobic performances of s p r i n t t r a i n e d a t h l e t e s . Anaerobic g l y c o l y s i s r e s u l t s i n the rapid, p r o d u c t i o n of ATP which i s a s s o c i a t e d with e l e v a t e d muscle and blood l a c t a t e l e v e l s , which have been i m p l i c a t e d i n reduced performances ( K a r l s s o n et a l . , 1975; Klausen et a l . , 1972). Proton accumulation a s s o c i a t e d with the e l e v a t e d l a c t a t e l e v e l s r e s u l t s i n pH decrements w i t h i n muscle and blood (Roos and Boron 1981; S a h l i n 1978). Reduced r a t e s of g l y c o l y s i s (Toews et a l . , 1970; Sutten et a l . , 1981; Roos and Boron 1981), c o r r e l a t i o n s between pH and f a t i g u e ( F i t t s and H o l l o s z y 1976; Stevens 1980), i n v e r s e r e l a t i o n s h i p s between f o r c e g e n e r a t i o n and proton c o n c e n t r a t i o n s (Dawson et a l . , 1978; F a b i a t o and F a b i a t o 1978) and proton i n h i b i t i o n of the e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g mechanisns 2 (Nocker et a l . , 1964; Katz 1970) have been demonstrated when the pH decrement was of s u f f i c i e n t magnitude. Thus i t i s important to b u f f e r the protons which . accumulate, thereby a l t e r i n g the r a t e of pH decrease, which subsequently may a d v e r s l y e f f e c t anaerobic performance. The a b i l i t y of a t i s s u e to r e s i s t changes i n pH upon a d d i t i o n of a strong a c i d or base, has been termed by Van Slyke (1922) as i t s b u f f e r c a p a c i t y ( B ) , r e f l e c t i n g the t i s s u e ' s a b i l i t y to sequester e i t h e r protons or hydroxide i o n s . Numerous i n v e s t i g a t i o n s by a v a r i e t y of techniques have attempted to i d e n t i f y the B of v a r i o u s t i s s u e s (Roos and Boron 1981). Though v a r i a t i o n s i n B e x i s t between the methods, i t appears that s k e l e t a l muscle c o n t r i b u t e s s i g n i f i c a n t l y to o v e r a l l pH homeostasis of the organism (Clancy and Brown 1966; S i e s j o and Messeter 1971; H e i s l e r and P i i p e r 1971; L a i et a l . , 1973). Recently, s k e l e t a l muscle was examined i n r e l a t i o n to g l y c o l y t i c c a p a c i t y w i t h i n a number of t e r r e s t e r i a l mammals and f i s h s p e c i e s by the the homogenate technique. Corresponding e l e v a t e d B and g l y c o l y t i c c a p a c i t y values v a l u e s were obtained ( C a s t e l l i n i and Somero 1981). The d e t e r m i n a t i o n of B by the homogenate technique c o n s i s t s of simply the phys i c o - c h e m i c a l b u f f e r i n g component, which comprises the b u f f e r i n g w i t h i n a c e l l merely as a consequence of proton a s s o c i a t i o n with bases (Roos and Boron 1981). Burton (1978) and Somero (1981) have i d e n t i f i e d the major b u f f e r i n g components of s k e l e t a l muscle to be the i m i d a z o l e - c o n t a i n i n g compounds: f r e e h i s t i d i n e , h i s t i d i n e - c o n t a i n i n g d i p e p t i d e s and protein-bound h i s t i d i n e r e s i d u e s . The 3 d i p e p t i d e c a r nosine ( B - a l a n y l h i s t i d i n e ) has been found to occur i n g r e a t e r c o n c e n t r a t i o n s predominantly w i t h i n muscles c l a s s i f i e d as white as opposed red (Tamaki et a l . , 1976). W i t h i n human s k e l e t a l muscle c a r n o s i n e c o n c e n t r a t i o n s demonstrate a wide v a r i a t i o n (Christman 1976; Bergstrom et a l . , 1978). Carnosine's b u f f e r i n g a b i l i t y has been suggested to c o n t r i b u t e up to 40 percent of the t o t a l b u f f e r i n g w i t h i n p re- and post r i g o r s k e l e t a l muscle (Bate-Smith 1938; Davey 1960b). Ther e f o r e the purpose of the present i n v e s t i g a t i o n was to examine the i n t e r - r e l a t i o n s h i p s between B, pH and f i b e r composition of human r e s t i n g vastus l a t e r a l i s muscle i n r e l a t i o n to t r a i n i n g s p e c i f i c i t y . Since the i m i d a z o l e - c o n t a i n i n g compounds, c a r n o s i n e and h i s t i d i n e , have been suggested to c o n t r i b u t e s i g n i f i c a n t l y to b u f f e r i n g of in, v i t r o p r e p a r a t i o n s (Somero 19.81; Burton 1978); t h e i r r e l a t i o n s h i p s to B, f i b e r composition and t r a i n i n g s p e c i f i c i t y were examined as p o s s i b l e f a c t o r s i n f l u e n c i n g anaerobic performance. 4 Methodology Twenty male v o l u n t e e r s served as s u b j e c t s i n t h i s study. They were made aware of the p o t e n t i a l r i s k s and informed consent was ob t a i n e d . Four equal groups of f i v e s u b j e c t s , c o n s i s t i n g of s p r i n t e r s (S=800m runne r s ) , rowers (R=varsity oarsmen), marathon runners (M) and u n t r a i n e d c o n t r o l s (UT) p a r t i c i p a t e d i n t h i s i n v e s t i g a t i o n . The s p r i n t e r s r e g u l a r l y ran the 800m d i s t a n c e i n l e s s than one minute 55 seconds which would make them a h i g h l y a n a e r o b i c a l l y t r a i n e d group. The marathon runners had to have been a c t i v e l y engaged i n endurance t r a i n i n g (>40 m i l e s per week f o r p r e v i o u s 6 months). As w e l l they must have completed a marathon run (26 mi l e s 385 yards) i n 2:30 to 2:50 (hoursrminutes). The u n t r a i n e d c o n t r o l s only p a r t i c i p a t e d i n r e c r e a t i o n a l a c t i v i t y . Anthropometric and p h y s i c a l c h a r a c t e r i s t i c data were recorded on each s u b j e c t ; percentage body f a t was determined by h y d r o s t a t i c weighing. Standard spirometry was performed at r e s t ( C o l l i n s Respirometer). The s u b j e c t s performed a continuous t r e a d m i l l t e s t c o n s i s t i n g of a ten minute warm-up at 1.56 m.s"' , immediately f o l l o w e d by the t e s t with a s t a r t i n g v e l o c i t y of 2.22 m.s"*, which was i n c r e a s e d by 0.22 m.s"1 each minute u n t i l f a t i g u e . E x p i r e d gases were c o n t i n u o u s l y sampled and analyzed (Beckman Me t a b o l i c Measurement C a r t ) ; measurements were t a b u l a t e d by a data a c q u i s i t i o n system (Hewlett Packard 3052A), which determined r e s p i r a t o r y gas exchange v a r i a b l e s every 15 seconds. Maximal oxygen consumption was determined by averaging the four h i g h e s t c o n s e c u t i v e 15 second oxygen uptake v a l u e s . 5 Anaerobic performance was assessed by the Anaerobic Speed Test (AST) of Cunningham and Faulkner. (i969) employing time, i n seconds to f a t i g u e as the performance index. The s u b j e c t s performed an e l e v a t e d t r e a d m i l l run c o n s i s t i n g of a 30 second warm-up at 2.66 m.s"', 10 degree i n c l i n e immediately f o l l o w e d by the t e s t at 3.52 m.s"1, 20 degree i n c l i n e u n t i l f a t i g u e . R e s t i n g and two minute p o s t - e x e r c i s e blood samples were obtained by venous puncture f o r det e r m i n a t i o n of blood l a c t a t e l e v e l s . A n a l y s i s of blood l a c t a t e s (HLa) was v i a the enzymatic c o n v e r s i o n of l a c t a t e to pyruvate i n the presence of LDH and NAD. (Hohorst 1962). Needle b i o p s i e s were obtained at r e s t from the vastus l a t e r a l i s muscle by the technique of Bergstrom et a l . , (1962) w i t h i n one week of the e x e r c i s e t e s t s . The s u b j e c t s had been informed not to have p a r t i c i p a t e d i n any p h y s i c a l a c t i v i t y p r i o r to the bio p s y . The sampling s i t e was 20 cm. above the l a t e r a l f e m o r a l - t i b i a l j o i n t l i n e . Samples being u t i l i z e d f o r h i s t o c h e m i c a l a n a l y s i s were o r i e n t e d under a d i s s e c t i n g microscope and mounted i n gum tra g a c a n t h compound. The samples were then f r o z e n i n isopentane c o o l e d to the temperature of l i q u i d n i t r o g e n . S e r i a l s e c t i o n s 1Ou t h i c k were cut i n a c r y o s t a t a f t e r warming to -20°C. The s e c t i o n s were mounted on cover s l i p s and e q u i l i b r a t e d at room temperature. Samples f o r bioc h e m i c a l d e t e r m i n a t i o n s were immediately immersed i n l i q u i d n i t r o g e n . S k e l e t a l muscle f i b e r s were s t a i n e d f o r myosin ATPase at d i f f e r e n t p r e - i n c u b a t i o n pH's (4.3, 4.6, 9.4) and f o r NADH 6 T e t r a z o l i u m Reductase(Dubowitz and Brooke . 1973). S e r i a l s e c t i o n s were obtained f o r p o s i t i v e i d e n t i f i c a t i o n of f i b e r types. F i b e r s were c l a s s i f i e d on the b a s i s of t h e i r s t a i n i n g i n t e n s i t y f o r myosin ATPase at pH 4.6. A 0.01 sq. cm. c r o s s - s e c t i o n a l area of muscle t i s s u e was employed f o r d e t e r m i n a t i o n of percent f a s t - t w i t c h (FT) or slow-twitch (ST) per sample. Mean f i b e r type diameters were c a l c u l a t e d by p r o j e c t i o n of the s l i d e s on to a screen (300x m a g n i f i c a t i o n ) with 10 f i b e r s of each f i b e r type being measured. M a g n i f i c a t i o n was checked by the use of a 1um micrometer p r o j e c t e d on the s c r e e n . The d e t e r m i n a t i o n of r e s t i n g muscle pH was by the method of S a h l i n et a l . , (1976) i n v o l v i n g homogenization of the sample at 25*C i n 10 volumes of a s a l t s o l u t i o n c o n t a i n i n g 145 mmol.l KC1, 10 mmol.l NaCl and 5 mmol.l i o d o a c e t i c a c i d (IAA). Homogenate pH measurements were made at 38*C with a m i c r o e l e c t r o d e (MI 410, M i c r o e l e c t r o d e s Inc.) f o l l o w i n g a 10 minute p r e - i n c u b a t i o n at 38* C. I n h i b i t i o n of g l y c o l y s i s was achieved by the a d d i t i o n of IAA. The remaining volume was d e p r o t e i n i z e d with the a d d i t i o n of 3 percent s o l i d s u l f o s a l i s y l i c a c i d and c e n t r i f u g e d f o r a n a l y s i s of b u f f e r c a p a c i t y . B u f f e r c a p a c i t y was determined by a m o d i f i c a t i o n of the method of Davey (1960b). Supernatant e x t r a c t s OOOul) were a d j u s t e d to pH 7.00+0.05 with 0.1 N NaOH. The 100U1 a l i q u o t s were t i t r a t e d to pH 6.00+0.05 with 0.01N HC1. R e l i a b i l i t y was a s c e r t a i n e d by r e - t i t r a t i o n of the e x t r a c t s f o l l o w i n g pH readjustment to 7.00+0.05. B u f f e r c a p a c i t y was determined as the number of moles per gram t i s s u e (w/w) of H* r e q u i r e d to change the pH one u n i t over the pH range 7.0 to 6.0. 7 The remaining supernatant demonstrating a pH of l e s s than 2.2 was used f o r f r e e amino a c i d d e t e r m i n a t i o n . Free amino a c i d l e v e l s were determined on an amino a c i d a utoanalyzer (Beckman 118C) u s i n g a s i n g l e column l i t h i u m hydroxide b u f f e r system. AA- 20 r e s i n (Beckman) was used on a 510 mm column with a diameter of 6 mm employing d i r e c t a p p l i c a t i o n of the sample. Standards were run at the beginning and end of each new n i n h y d r i n s o l u t i o n . The amino a c i d c o n c e n t r a t i o n s were determined by manually i n t e g r a t i n g the area under the curve. By t h i s method h i s t i d i n e immediately preceded c a r n o s i n e i n r e t e n t i o n time. To determine the r e l a t i v e c o n t r i b u t i o n of ca r n o s i n e to t o t a l b u f f e r c a p a c i t y , d i f f e r e n t c o n c e n t r a t i o n s of c a r n o s i n e (Sigma L- ca r n o s i n e reagent grade) i n 100U1 a l i q u o t s were t i t r a t e d and r e - t i t r a t e d between pH 7.00+0.05 and 6.00+0.05 with 0.01 N HCl. U n i v a r i a t e comparisons of groups were performed on the p h y s i c a l c h a r a c t e r i s t i c , pulmonary f u n c t i o n , h i s t o c h e m i c a l and amino a c i d data. A n a l y s i s of v a r i a n c e was u t i l i z e d to eva l u a t e p o s s i b l e i n t e r g r o u p d i f f e r e n c e s . The S c h e f f e t e s t of s i g n i f i c a n c e was performed, on the v a r i a b l e s demonstrating s i g n i f i c a n t omnibus F r a t i o s , to i d e n t i f y where group d i f f e r e n c e s e x i s t e d . M u l t i v a r i a t e comparisons of groups with pre-planned orthogonal c o n t r a s t s were performed on the p h y s i o l o g i c a l , post HLa, percent FT and B v a r i a b l e s to i d e n t i f y p o s s i b l e i n t e r g r o u p d i f f e r e n c e s . Regression equations were c a l c u l a t e d f o r the v a r i a b l e s demonstrating s i g n i f i c a n t c o r r e l a t i o n s with B, as w e l l as those v a r i a b l e s demonstrating r e l a t i o n s h i p s to ca r n o s i n e c o n c e n t r a t i o n s . 8 R e s u l t s A n t h r o p o m e t r i c , p h y s i c a l c h a r a c t e r i s t i c and pulmonary f u n c t i o n d a t a a r e p r e s e n t e d i n T a b l e 1. S i g n i f i c a n t d i f f e r e n c e s e x i s t e d between the groups f o r the v a r i a b l e s weight and p e r c e n t body f a t (p<.05) due t o the u n t r a i n e d group. The marathon r u n n e r s were s i g n i f i c a n t l y (p<.05) o l d e r than t h e o t h e r t h r e e groups. No s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between the groups f o r pulmonary f u n c t i o n . The low i n t r a - v a r i a b i l i t i e s of the t r a i n e d groups w i t h r e s p e c t t o t h e i r pulmonary f u n c t i o n and a n t h r o p o m e t r i c d a t a suggest t h a t the sub-samples were r e l a t i v e l y homogeneous. The p h y s i o l o g i c a l assessment and h i s t o c h e m i c a l a n a l y s i s r e s u l t s a r e c o n t a i n e d i n Table 2. The i n c l u s i o n of the u n t r a i n e d group r e s u l t e d i n s i g n i f i c a n t l y d i f f e r e n t (p<.05) maximal oxygen uptake v a l u e s but no d i f f e r e n c e s e x i s t e d between the t r a i n e d g roups. W h i l e no s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between the M and UT groups i n AST performance (p=.084), s i g n i f i c a n t d i f f e r e n c e s d i d e x i s t between groups S and R ( p < . 0 l ) . Both the S and R groups demonstrated a s i g n i f i c a n t i n c r e a s e (p<.0l) i n AST t i m e s w i t h r e s p e c t t o each of the o t h e r groups. Post-AST HLa v a l u e 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 o r the S and R groups (p<.01> and w i t h r e s p e c t t o each of the o t h e r g r o u p s . No p o s t - AST HLa d i f f e r e n c e s e x i s t e d between the M and UT g r o u p s . A n a e r o b i c performance was h i g h l y c o r r e l a t e d w i t h post-AST HLa v a l u e s ( r = . 9 0 ; F i g u r e 1 ) . The r a t i o of AST/Post-AST HLa v e r s u s Post-AST HLa r e v e a l e d a s u p e r i o r r a t i o f o r t h e t r a i n e d groups as compared t o the UT group b u t , no d i f f e r e n c e s between the t r a i n e d Table 1. P h y s i c a l c h a r a c t e r i s t i c , anthropometric and pulmonary f u n c t i o n p r o f i l e s (Mean + SD) . Percent Forced Forced FEV^ Group Age Height Weight Body V i t a l E x p i r a t o r y '__ Fat Capacity Volume FVC (yrs) (cm) (kg) •(%) (1) (1) (%) S p r i n t e r s , 20.6 180;6 68.6 6.5 6.17. 4.79 79 +2.3 +4.9 +3.4 +3.4 +0.79 +0.22 +8 Rowers 20.6 177.2 70.0 7.2 5.58 4.79 84 +1.8 +3.4 +2.8 +3.6 +0.57 +0.67 +8 Marathoners . 37.8 a 176.5 69.0 10.6 5.26 4.22 81 +9.3 +4.2 +4.4 +4.0 +0.63 +0.59 +5 Untrained 22.6 182.4 81.7 a 21.1 a 5.89 4.34 . 74 +0.9 +4.3 +5.1 +4.9 +0.86 +0.75 +5 a p<0.05 s i g n i f i c a n t l y d i f f e r e n t from other 3 groups Table 2. P h y s i o l o g i c a l assessment and h i s t o l o g i c a l a n a l y s i s of the vastus l a t e r a l i s muscle (Mean + SD.) ' Maximal Anaerobic Pre Post Fast Fast Slow Oxygen Speed Blood Blood Twitch Twitch Twitch Group Uptake Test Lactate Lactate Percentage . Mean Mean (ml.kgl'min l) (sec) (mmol • r 1 ) (%) Diameter (urn) Diameter (um) S p r i n t e r s 63.2 115 c 1.1 21.9 c 56.6 94.7 90.0 +3.1 +18 +0.3. + 1.5 +7.0 +26.2 +22.8 Rowers 62.4 76 b 1.0 13.9 b 50.4 96.0 95.0 +1.7 +9 +0.2 +0.9 + 12.3 +14.4 +18.7 Marathoners 60.1 53 1.0 10.1 33.0 a 74.2 79.5 +4.2 +15 +0.2 +3.1 +12.2 +9.5 +16.4 Untrained 46.9 a 38 0.8 10.1 50.6 79.1 75.0 +3.3 +9 +0.2 +2.6 +9.9 +12.2 +15.2 a p<0.05 s i g n i f i c a n t l y d i f f e r e n t from other 3 groups b p<0.01 s i g n i f i c a n t l y > M and UT groups c p<0.01 s i g n i f i c a n t l y > a l l other groups 11 Figure 1. Anaerobic performance versus post-AST blood l a c t a t e l e v e l s . Post-AST Blood L a c t a t e Levels (mmol.1 ) i 12 Post-AST HLa Figure 2. R a t i o AST/Post-AST HLa versus Post-AST HLa (Mean + SD) ' AST 4 t M UT 10 15 T 20 25 Post-AST HLa Levels (mmol.l ^) 13 groups e x i s t e d (Figure 2 ) . S i g n i f i c a n t d i f f e r e n c e s i n f i b e r composition e x i s t e d between the groups ( p < . 0 5 ) . T h i s c o u l d be a t t r i b u t e d to the low FT f i b e r percentage of the M group. Though the d i f f e r e n c e s i n f i b e r diameters were not s i g n i f i c a n t ( p > . 0 5 ) , when FT f i b e r diameters were expressed r e l a t i v e t o ST f i b e r diameters the S appeared to demonstrate enl a r g e d FT f i b e r s and the M appeared to d i s p l a y enlarged ST f i b e r s . R e s t i n g intramuscular pH and B values are c o n t a i n e d i n Table 3. No s i g n i f i c a n t d i f f e r e n c e s e x i s t e d f o r r e s t i n g pH ( p > . 0 5 ) . B r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e s between the S and R or between the M and UT groups but s i g n i f i c a n t d i f f e r e n c e s ( p < . 0 l ) d i d e x i s t between these sub-samples. The B of the S and R group were almost 50 percent g r e a t e r than the B of the M or. UT groups. M u l t i v a r i a t e a n a l y s i s r e v e a l e d that the S group was s i g n i f i c a n t l y d i f f e r e n t ( p < . 0 l ) from the R group and t h i s was due to s i g n i f i c a n t d i f f e r e n c e s ( p < . 0 l ) i n AST performance and post HLa. The M group was s i g n i f i c a n t l y d i f f e r e n t from the UT group i n r e s p e c t to f i b e r composition ( p < . 0 2 ) , VO max ( p < . 0 l ) , percent body f a t ( p < . 0 l ) , and age ( p < . 0 l ) . Comparison of the S and R groups versus the M group r e v e a l e d s i g n i f i c a n t d i f f e r e n c e s to e x i s t f o r AST performance ( p < . 0 l ) , post HLa v a l u e s ( p < . 0 l ) , percent FT (p<.025) and B ( p < . 0 l ) . Muscle c a r n o s i n e and h i s t i d i n e c o n c e n t r a t i o n s are a l s o c o n t a i n e d i n Table 3. Between group comparisons r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e s i n h i s t i d i n e l e v e l s but c a r n o s i n e l e v e l s Table 3. B u f f e r c a p a c i t y , pH, h i s t i d i n e and carnosine l e v e l s of the r e s t i n g vastus l a t e r a l i s muscle (Mean + SD). Bu f f e r H i s t i d i n e Carnosine Group Capacity , pH Levels Levels (umbl.'g^'.pH *) (umol.g^) (umol.g^) S p r i n t e r s 30.03 a 6.99 0.64 4.93 a +5.6 +0.13 +0.06 +0.76 Rowers 31.74a 6.97 0.71 5.04 a +7.2 +0.11 +0.10 +0.72 Marathoners 20.83 7.11 ' 0.63 2.80 +4.4 +0.11 +0.14 +0.74 Untrained 21.25 6.91 0.89 3.75 +5.0 +0.17 +0.29 +0.86 a p ̂ 0.0.1 s i g n i f i c a n t l y > M and UT groups 15 Figure 3. B u f f e r c a p a c i t y (umol.g w/w.pH )versus t r a i n i n g s p e c i f i c i t y (Mean + SD). 40 -r 35 + 30 4- 25 + o — h ~ — L _ l — J — J — \ UT M R S Group p<0.01 s i g n i f i c a n t l y ) M and UT groups Carnosine Concentration 17 Table 4. C o r r e l a t i o n matrix f o r the bi o c h e m i c a l , h i s t o c h e m i c a l and anaerobic performance v a r i a b l e s ; Carn .64"* AST .51* .61** FT% .51* .46* .37 Carn AST * p<0.05 ** p <0.01 18 v a r i e d with t r a i n i n g s p e c i f i c i t y as only the S and R demonstrated s i g n i f i c a n t d i f f e r e n c e s (p<.0l) from the UT group. No s i g n i f i c a n t d i f f e r e n c e s i n c a r n o s i n e l e v e l s e x i s t e d between the M and UT groups. E m p i r i c a l t i t r a t i o n of c a r n o s i n e (Figure 4) r e v e a l e d t h a t the i n c r e a s e d B of the s p r i n t t r a i n e d a t h l e t e s ( F i g u r e 3) c o u l d be s i g n i f i c a n t l y accounted f o r by the int r a m u s c u l a r c a r n o s i n e l e v e l s . C o r r e l a t i o n s are c o n t a i n e d i n Table 4. B was e l e v a t e d i n the S and R groups ( F i g u r e s 3 ). S i g n i f i c a n t r e l a t i o n s h i p s were demonstrated between anaerobic performance and B (p<.05;Table 4). A f u r t h e r r e l a t i o n s h i p e x i s t e d between FT% and B ( r = . 5 l ) . Carnosine l e v e l s were found to demonstrate s i g n i f i c a n t (p<.0l) r e l a t i o n s h i p s to B and FT%. 19 D i s c u s s i o n The major f i n d i n g s of the present i n v e s t i g a t i o n a r e : f i r s t , t h a t the a n a e r o b i c a l l y t r a i n e d groups e x h i b i t e d an enhanced anaerobic performance which was s i g n i f i c a n t l y r e l a t e d to B; second, t h a t B d i s p l a y e d a s i g n i f i c a n t r e l a t i o n s h i p t o f a s t - t w i t c h f i b e r percentage and t h i r d , t h a t c a r n o s i n e l e v e l s were s i g n i f i c a n t l y r e l a t e d to B, demonstrating e l e v a t e d l e v e l s w i t h i n the a n a e r o b i c l y t r a i n e d groups. Although these are d e s c r i p t i v e data and i t i s not p o s s i b l e to s t a t e that the b i o c h e m i c a l parameters B and c a r n o s i n e l e v e l s , are a f u n c t i o n of anaerobic t r a i n i n g , i t i s tempting to s p e c u l a t e that a d a p t a t i o n may occur on the b a s i s of r e p e t i t i v e anaerobic work. S k e l e t a l muscle c o n s i s t s of two d i s t i n c t f i b e r types ( f a s t - t w i t c h and slow- t w i t c h ) , each f i b e r type p o s s e s s i n g d i f f e r e n t c o n t r a c t i l e and metabolic p r o p e r t i e s ( G o l l n i c k and Hermansen 1973). F a s t - t w i t c h f i b e r s demonstrate a high c a p a c i t y f o r anaerobic g l y c o l y s i s (Lowry et a l . , 1978; Essen et a l . , 1975). The enhanced B which was a s s o c i a t e d with an e l e v a t e d f a s t - t w i t c h f i b e r percentage, suggested that B may be r e l a t e d to the g l y c o l y t i c c a p a c i t y of s k e l e t a l muscle. Man performing short d u r a t i o n , high i n t e n s i t y work has a requirement to maintain redox balance, while f u l f i l l i n g the need fo r c o ntinued muscle energy metabolism, predominantly based upon intr a m u s c u l a r s u b s t r a t e s and a c a p a c i t y to b u f f e r the i n h i b i t o r y a c t i o n of the proton accumulation. Hermansen et a l . , (1971) has r e p o r t e d blood l a c t a t e v a l u e s of up to 32 mM f o r short d u r a t i o n work. Proton accumulation a s s o c i a t e d with the e l e v a t e d l a c t a t e 20 l e v e l s a l t e r s the acid-base balance of the c y t o s o l r e s u l t i n g i n pH decrements w i t h i n muscle and blood ( S a h l i n et . a l . , 1978; Hultman and S a h l i n 1980; Roos and Boron 1981). The importance of pH to the r e g u l a t i o n of anaerobic performance has been demonstrated by Sutton et a l . , (1981) who r e p o r t e d pH decrements to be a s s o c i a t e d with decreased anaerobic performances. R e l a t i o n s h i p s between pH and f a t i g u e ( F i t t s and H o l l o s z y 1976; Stevens 1980), proton accumulation and f o r c e g e n e r a t i o n (Dawson et a l . , 1977), reduced r a t e s of g l y c o l y s i s with pH decrements (Toews et a l . , 1970; Sutton et a l . , 1981; Roos and Boron 1981) and decreased maximal t e n s i o n development ( F a b i a t o and F a b i a t o 1978), p o s s i b l y due to proton c o m p e t i t i o n with Ca H f o r the b i n d i n g s i t e s (Katz 1970) have suggested the importance of b u f f e r i n g the protons which accumulate d u r i n g anaerobic g l y c o l y s i s . The present r e s u l t s imply a b i o c h e m i c a l phenomenon w i t h i n a n a e r o b i c a l l y t r a i n e d a t h l e t e s , which would f o s t o r an enhanced c a p a c i t y f o r a muscle to f u n c t i o n under anaerobic c o n d i t i o n s . When the anaerobic g l y c o l y t i c machinery i s f u n c t i o n i n g , the high b u f f e r i n g c a p a c i t i e s of the s k e l e t a l muscle may n e u t r a l i z e the accumulated protons. Thus the r a t e at which pH would normally decrease would be reduced due to the enhanced proton s e q u e s t e r i n g c a p a c i t i e s . I t i s important to emphasize that the g l y c o l y t i c enzyme a c t i v i t i e s examined pre- and post s p r i n t t r a i n i n g have r e p o r t e d with minor ex c e p t i o n s no s i g n i f i c a n t d i f f e r e n c e s ( G o l l n i c k et a l . , 1972; H o l l o s z y et a l . , 1973; Hickson et a l . , 1975; C o s t i l l et a l . , 1976). T r a i n i n g s p e c i f i c i t y appears to be a s s o c i a t e d with a f i b e r type 21 predominance and a r e l a t i o n s h i p to f i b e r type hypertrophy ( G o l l n i c k et a l . , 1972, 1973; C o s t i l l et a l . , 1976; S a h l i n et a l . , 1976; Andersen and Hennriksen 1977). In agreement with the p r e v i o u s i n v e s t i g a t i o n s , s p r i n t e r s were found to have an e l e v a t e d f a s t - t w i t c h f i b e r composition while the marathon runners demonstrated a slow-twitch f i b e r predominance but no s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between the s p r i n t e r s , rowers or u n t r a i n e d c o n t r o l s . The i n t e r c o n v e r s i o n of s k e l e t a l muscle f a s t - t w i t c h sub-group f i b e r s does appear to occur as a r e s u l t of t r a i n i n g (Janssen and K a i j s e r 1977) but no i n t e r c o n v e r s i o n of f a s t - t w i t c h and slow-twitch f i b e r s has been i d e n t i f i e d with t r a i n i n g ( G o l l n i c k et a l . , 1982). F i b e r hypertrophy appeared to demonstrate a r e l a t i o n s h i p to t r a i n i n g s p e c i f i c i t y but no d i f f e r e n c e s i n f i b e r diameters e x i s t e d between the s p r i n t e r s and rowers. Thus though f i b e r composition appears to be r e l a t e d to anaerobic c a p a c i t y , i t alone c o u l d not account f o r the enhanced anaerobic performance of the s p r i n t e r s . T h e r e f o r e i t appears that the enhanced anaerobic c a p a c i t y a s s o c i a t e d with the a n a e r o b i c a l l y t r a i n e d a t h l e t e s i s more a f u n c t i o n of s k e l e t a l muscle c a p a c i t y to b u f f e r the protons i n a s s o c i a t i o n with f i b e r composition, than to a change i n g l y c o l y t i c enzyme a c t i v i t i e s . The present i n v e s t i g a t i o n r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e s between the t r a i n e d groups with respect to anthropometric, pulmonary f u n c t i o n and maximal oxygen uptake data suggesting the subsamples were r e l a t i v e l y homogeneous. The maximal oxygen uptakes demonstrated f o r each of the groups were comparable to the values r e p o r t e d f o r s i m i l a r s u b j e c t s (Carey et 22 a l . , 1974; Hagerman and Mickelson 1980; Roberts et a l . , 1980; C o s t i l l et a l . , (1976). Although the marathon runners were h i g h l y t r a i n e d competetive a t h l e t e s , there was no s i g n i f i c a n t d i f f e r e n c e between t h i s group and the UT c o n t r o l s i n measures of anaerobic performance. Anaerobic performance was found to be h i g h l y r e l a t e d to t r a i n i n g s p e c i f i c i t y with the run times of the marathon runners and u n t r a i n e d s u b j e c t s being comparable to the p r e - t r a i n e d t e s t values of Cunningham and Faulkner (1969). Thus anaerobic performance appears to be a . f u n c t i o n of t r a i n i n g s p e c i f i c i t y and the d i f f e r e n c e s observed c o u l d not be a t t r i b u t e d to the p h y s i c a l c h a r a c t e r i s t i c , pulmonary f u n c t i o n , a e r o b i c c o n d i t i o n i n g or f i b e r composition. Post anaerobic performance blood l a c t a t e values were s i g n i f i c a n t l y e l e v a t e d i n a l l the groups, demonstrating maximal va l u e s which are comparable to those of K a r l s s o n (1971) f o r short term, i n t e n s i v e e x e r c i s e bouts of e l i t e a t h l e t e s . The removal of muscle l a c t a t e was p r e v i o u s l y assumed to be a simple process of d i f f u s i o n down c o n c e n t r a t i o n g r a d i e n t s (Hirche et a l . , 1971; J o r f e l d t 1978) but, r e c e n t l y a c a r r i e r mediated, pH dependent l a c t a t e t r a n s f e r mechanism has been i d e n t i f i e d i n a few t i s s u e s (Barac-Nieto et a l . , 1978; Dubinsky and Racker 1978; Spencer and Lehninger 1976; Johnson et a l . , 1980; Monson et a l . , 1981). Koch et a l . , (1981) examining mouse diaphragm muscle have suggested that at l e a s t t h r e e - q u a r t e r s of the l a c t a t e t r a n s f e r was c a r r i e r mediated. S i n c e protons are e f f l u x e d with l a c t a t e anions (Mainwood and Brown 1975), an enhanced l a c t a t e t r a n s f e r mechanism a s s o c i a t e d with s p r i n t t r a i n i n g may f a c i l i t a t e the 23 n e u t r a l i z a t i o n of the protons w i t h i n the c y t o s o l . Post blood l a c t a t e v a l u e s demonstrated a h i g h l y s i g n i f i c a n t c o r r e l a t i o n with anaerobic performance which was even stronger f o r the t r a i n e d s u b j e c t s . T r a i n i n g appears to enhance the l a c t a t e t r a n s p o r t mechanism producing a s i m i l i a r r a t e of l a c t a t e r e l e a s e f o r the t r a i n e d groups, r e g a r d l e s s of t r a i n i n g s p e c i f i c i t y ( F i gure 2). Thus the g r e a t e r anaerobic performances of the t r a i n e d a t h l e t e s may p a r t i a l l y be a t t r i b u t e d to t h i s enhanced l a c t a t e r e l e a s e , which would f a c i l i t a t e a reduced a c i d i f i c a t i o n of the c y t o s o l . Muscle pH determinations by m i c r o e l e c t r o d e we're f i r s t performed by Furusawa and K e r r i d g e i n 1927 on cat s k e l e t a l , c a r d i a c and u t e r i n e muscle. More r e c e n t l y a s e r i e s of experiments examining pH . were conducted on human quadricep muscle homogenates o b t a i n e d pre- and p o s t - e x e r c i s e (Hermansen and Osnes 1972; S a h l i n , H a r r i s and Hultman 1975; S a h l i n et a l . , 1976). A muscle pH of 6.50 to 6.60 was i d e n t i f i e d as a c r i t i c a l value where f a t i g u e caused c e s s a t i o n of the e x e r c i s e bout ( S a h l i n e t a l . , 1978). Decreased muscle pH has been i d e n t i f i e d to have an i n h i b i t o r y e f f e c t on the c o n t r a c t i l e mechanism (Nocker 1964; Campion 1974; Katz 1970; F a b i a t o and F a b i a t o 1978) and the g l y c o l y t i c r e g u l a t o r y enzymes (Danforth 1965; T r i v e d i and Danforth 1966; Toews et a l . , 1970; Sutton et a l . , 1981; Roos and Boron 1981). R e s t i n g i n t r a m u s c u l a r pH was found to e x h i b i t no s i g n i f i c a n t d i f f e r e n c e s a c r o s s the groups, demonstrating a pH comparable to those r e p o r t e d i n the l i t e r a t u r e . Thus achievement of an enhanced anaerobic performance due to a l a r g e r 24 intramuscular pH gr a d i e n t (pre - post e x e r c i s e pH) due to e l e v a t e d i n i t i a l pH l e v e l s a s s o c i a t e d with t r a i n i n g s p e c i f i c i t y was unfounded. H e i s l e r and P i i p e r (1971) s t a t e d that s k e l e t a l muscle must be s u b j e c t to l a r g e v a r i a t i o n s i n a c i d p r o d u c t i o n . Thus the a b i l i t y of a t i s s u e to b u f f e r the protons a s s o c i a t e d with anaerobic g l y c o l y t i c energy p r o d u c t i o n may a f f e c t performance. The s u p e r i o r b u f f e r c a p a c i t i e s demonstrated by the s p r i n t e r s and rowers suggest that s k e l e t a l muscle b u f f e r c a p a c i t y may be a f u n c t i o n of t r a i n i n g s p e c i f i c i t y . C a s t e l l i n i and Somero (1981) found i n both mammals and f i s h e s , high b u f f e r i n g c a p a c i t i e s i n the locomotory muscles of s p e c i e s e x h i b i t i n g pronounced a b i l i t i e s f o r burs t locomotion. The b u f f e r c a p a c i t y v a l u e s found in the present i n v e s t i g a t i o n appear to be comparable to the r e s u l t s of Davey (1960b) on d e p r o t e i n i z e d p r e - r i g o r r a b b i t psoas muscle. The values are somewhat lower than those r e p o r t e d by C a s t e l l i n i and Somero (1981) f o r t e r r e s t e r i a l mammals. T h i s d i s c r e p a n c y may be accounted f o r by the lack of d e p r o t e i n i z a t i o n i n the C a s t e l l i n i and Somero i n v e s t i g a t i o n . S a h l i n e t a l . , (1978) c a l c u l a t e d p r o t e i n s to account f o r an a d d i t i o n a l 22 percent of b u f f e r i n g i n r e s t i n g samples. The c o n t r i b u t i o n of p r o t e i n s to t o t a l b u f f e r c a p a c i t y was determined by Bate-Smith (1938), Hultman and S a h l i n (1980) to be approxiamently 50 percent of the physic o - c h e m i c a l b u f f e r i n g . Reeves and Malan (1976) found the c o n t r i b u t i o n of p r o t e i n s to b u f f e r i n g i n f r o g s k e l e t a l muscle to be between 40 and 50 per c e n t . T h e r e f o r e p r o t e i n s c o n s t i t u t e a s i g n i f i c a n t b u f f e r i n g component w i t h i n 25 s k e l e t a l muscle. B u f f e r c a p a c i t y determined by the homogenate technique d i f f e r s from i n t a c t p r e p a r a t i o n s due to the transmembrane f l u x e s of H*and/or HCO^, which do not occur i n a c l o s e d system such as the muscle homogenate technique ( H e i s l e r and Pi.iper 1971, 1972; Brown 1971). In agreement with C a s t e l l i n i and Somero (1981) b u f f e r c a p a c i t y was found to be r e l a t e d to the muscles anaerobic g l y c o l y t i c p o t e n t i a l as evidenced by the r e l a t i o n s h i p to f a s t - t w i t c h percentage. B u f f e r c a p a c i t y was a l s o found to be r e l a t e d to anaerobic performance which suggests that the inherent a b i l i t y of s k e l e t a l m u s c l e . b u f f e r s to f u n c t i o n as proton sequesters i n the p h y s i o l o g i c a l pH range may augment performance. B u f f e r c a p a c i t y determined by the homogenate technique i n v o l v e s e s s e n t i a l l y the non-bicarbonate b u f f e r i n g i n the c y t o s o l , due p r i n c i p a l l y to phosphate compounds and i m i d a z o l e - c o n t a i n i n g compounds (Burton 1978; Somero 1981). Imidazole- c o n t a i n i n g compounds c o n s i s t of protein-bound h i s t i d y l r e s i d u e s , h i s t i d i n e - c o n t a i n i n g d i p e p t i d e s (eg. ca r n o s i n e and ans e r i n e ) and f r e e h i s t i d i n e (Somero 1981). Free h i s t i d i n e occurs i n much lower c o n c e n t r a t i o n s than the other two types of i m i d a z o l e - c o n t a i n i n g compounds (Burton 1978). Davey (1960b) suggested that the h i s t i d i n e - c o n t a i n i n g d i p e p t i d e s c o u l d c o n t r i b u t e as much as 40 percent of the t o t a l b u f f e r i n g of pre- and post r i g o r muscle.. C a s t e l l i n i and Somero (1981) suggested that the d i f f e r e n c e s i n b u f f e r c a p a c i t i e s , of the f i s h s p e c i e s observed, c o u l d be due to d i f f e r e n c e s i n t o t a l p r o t e i n b u f f e r i n g or f r e e h i s t i d i n e c o ntent. Within human s k e l e t a l muscle a f u r t h e r p o s s i b i l i t y to 26 account f o r the d i f f e r e n t b u f f e r c a p a c i t i e s may be v a r i a t i o n s i n the h i s t i d i n e - c o n t a i n i n g d i p e p t i d e c o n c e n t r a t i o n s . Large v a r i a t i o n s i n c a r n o s i n e content of the human vastus l a t e r a l i s muscle have been r e p o r t e d (Christman 1976). Free h i s t i d i n e c o n c e n t r a t i o n s were found to be comparable to those r e p o r t e d by Bergstrom et a l . , (1974) e x h i b i t i n g no s i g n i f i c a n t d i f f e r e n c e s between groups. Thus a l t e r a t i o n s i n h i s t i d i n e content can not account f o r the d i f f e r e n c e s observed i n b u f f e r c a p a c i t i e s . Carnosine l e v e l s were s i m i l a r to those r e p o r t e d by Zachmann et a l . , (1966), Christman (1976) and Bergstrom et a l . , (1978), d i s p l a y i n g s u p e r i o r v a l u e s f o r the a n a e r o b i c a l l y t r a i n e d groups. Thus ca r n o s i n e l e v e l s may p o s s i b l y be e l e v a t e d by anaerobic t r a i n i n g . In agreement with animal i n v e s t i g a t i o n s (Zapp and Wilson 1938; Tamaki et a l . , 1976) c a r n o s i n e l e v e l s demonstrated a r e l a t i o n s h i p to muscle g l y c o l y t i c c a p a c i t y . The present i n v e s t i g a t i o n r e v e a l e d that c a r n o s i n e c o u l d c o n t r i b u t e s i g n i f i c a n t l y to t o t a l muscle b u f f e r i n g , accounting f o r 41 percent of the v a r i a n c e in B. E m p i r i c a l t i t r a t i o n s of c a r n o s i n e p r e d i c t e d a s u p e r i o r B of approxiamently 9 umoles.g*.pH*for the s p r i n t t r a i n e d a t h l e t e s . T h e r e f o r e c a r n o s i n e l e v e l s c o u l d be r e s p o n s i b l e f o r the observed d i f f e r e n c e s i n B (approxiamently 10 umoles.g' .pH*' ). T h i s f u r t h e r emphasizes the the importance of c a r n o s i n e as a p h y s i o l o g i c a l b u f f e r w i t h i n a muscle homogenate. T h e r e f o r e the present i n v e s t i g a t i o n suggested that c a r n o s i n e by v i r t u e of i t s a b i l i t y to act as a b u f f e r i n the p h y s i o l o g i c a l pH range c o u l d c o n t r i b u t e s i g n i f i c a n t l y to the observed d i f f e r e n c e s i n t o t a l 27 b u f f e r c a p a c i t y . In c o n c l u s i o n , b u f f e r c a p a c i t y was e l e v a t e d i n the a n a e r o b i c a l l y t r a i n e d groups and appears to be r e l a t e d to the anaerobic g l y c o l y t i c p o t e n t i a l of muscle. In short d u r a t i o n , high i n t e n s i t y work, the requirement f o r e l e v a t e d b u f f e r f u n c t i o n s , r e s u l t s from the proton accumulation a s s o c i a t e d with the maintenance of redox balance, while c o n t i n u i n g to produce energy m e t a b o l i c a l l y . Thus the a b i l i t y of s k e l e t a l muscle to sequester protons and t h e r e f o r e b u f f e r the protons which accumulated, may enhance anaerobic c a p a c i t y . Carnosine by v i r t u e of i t s s i g n i f i c a n t c o n t r i b u t i o n to b u f f e r c a p a c i t y may p o s s i b l y c o n t r i b u t e to the enhanced anaerobic performance of s p r i n t t r a i n e d a t h l e t e s . Future i n v e s t i g a t i o n s must attempt to q u a n t i f y the d i f f e r e n c e s i n B and the c o n t r b u t i o n of c a r n o s i n e to b u f f e r i n g w i t h i n human s k e l e t a l muscle which c o u l d be a t t r i b u t e d to t r a i n i n g regime. Furthermore the B of i n v i v o human s k e l e t a l muscle and the b u f f e r i n g c o n s t i t u e n t s c o n t r i b u t i o n to b u f f e r i n g d u r i n g dynamic e x e r c i s e must be determined. 28 Summary of F i n d i n g s 1. Anaerobic performance as assessed by the anaerobic speed t e s t •appears to be h i g h l y t r a i n i n g s p e c i f i c such that s p r i n t t r a i n e d a t h l e t e s demonstrate s i g n i f i c a n t l y e l e v a t e d performances. 2. Enhanced anaerobic performances were s i g n i f i c a n t l y r e l a t e d to e l e v a t e d s k e l e t a l muscle homogenate b u f f e r c a p a c i t e s . T h i s suggests that the a b i l i t y of s k e l e t a l muscle to sequester the protons which accumulated d u r i n g anaerobic g l y c o l y t i c energy p r o d u c t i o n may augment anaerobic performance. Anaerobic t r a i n i n g may p o s s i b l y be a necessary pre- r e q u i s i t e f o r enhanced b u f f e r c a p a c i t i e s . 3. The r e l a t i o n s h i p between f a s t - t w i t c h f i b e r percentage and b u f f e r c a p a c i t y suggested that enhanced b u f f e r c a p a c i t i e s may be r e l a t e d to e l e v a t e d g l y c o l y t i c c a p a c i t i e s of s k e l e t a l muscle. 4. Intramuscular c a r n o s i n e l e v e l s were e l e v a t e d only w i t h i n the a n a e r o b i c l y t r a i n e d groups, demonstrating s i g n i f i c a n t c o r r e l a t i o n s with b u f f e r c a p a c i t y . Carnosine c o n t r i b u t e d s i g n i f i c a n t l y to B, p o s s i b l y due to i t s a b i l i t y to act as a b u f f e r i n the p h y s i o l o g i c a l pH range. 5. W ithin animal i n v e s t i g a t i o n s , c a r n o s i n e has been found t 29 predominantly w i t h i n f a s t - t w i t c h f i b e r s . The r e l a t i o n s h i p between carnosine l e v e l s and f a s t - t w i t c h f i b e r percentage i n the present i n v e s t i g a t i o n , suggests a l i n k between c a r n o s i n e and the g l y c o l y t i c p o t e n t i a l of the muscle. Though v a r i a n c e s i n h i s t i d i n e l e v e l s have been suggested as a p o s s i b l e c o n t r i b u t o r to d i f f e r e n c e s i n b u f f e r c a p a c i t i e s a c r o s s many s p e c i e s , the present i n v e s t i g a t i o n , r e v e a l e d no d i f f e r e n c e s i n h i s t i d i n e c o n c e n t r a t i o n s with t r a i n i n g s p e c i f i c i t y . E l e v a t e d blood l a c t a t e l e v e l s were s i g n i f i c a n t l y r e l a t e d to a naerobic performance, demonstrating an enhanced l a c t a t e t r a n s p o r t mechanism w i t h i n t r a i n e d a t h l e t e s , which was not a l t e r e d by t r a i n i n g s p e c i f i c i t y . 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Co n c e n t r a t i o n s of amino a c i d s i n plasma and muscle. Amer.J.Dis.Child., 112:283-289, 1966. Zapp, J.A. and D. Wright Wilson. Q u a n t i t a t i v e s t u d i e s of c a r n o s i n e and a n s e r i n e i n mammalian muscle. J.Biol.Chem. 126:9-18, 1938. 46 APPENDICES 47 Review of L i t e r a t u r e A. Acid-Base Status and Performance. S p r i n t t r a i n e d a t h l e t e s , demonstrate a remarkable a b i l i t y to perform h i g h i n t e n s i t y , short d u r a t i o n work, with the energy requirements being met p r i n c i p a l l y by anaerobic g l y c o l y s i s . T h i s metabolic pathway r e s u l t s i n the r a p i d p r o d u c t i o n of ATP, the energy source f o r muscular c o n t r a c t i o n , with an a s s o c i a t e d muscle and blood l a c t a t e l e v e l i n c r e a s e , which has been demonstrated to i n h i b i t a t h l e t i c performance (Klausen et a l . , 1972; K a r l s s o n et a l . , 1975). Proton accumulation a s s o c i a t e d with the e l e v a t e d l a c t a t e l e v e l s a l t e r s the acid-base balance of t i s s u e r e s u l t i n g i n pH decrements w i t h i n muscle and blood (Hochachka and Mommsen 1982; S a h l i n 1978; Roos and Boron 1981). Reduced r a t e s of g l y c o l y s i s (Toews et a l . , 1970; Sutton et a l . , 1981; Roos and Boron 1 9 8 1 ) , c o r r e l a t i o n s between pH and f a t i g u e ( F i t t s and H o l l o s z y 1976; Stevens 1980) and i n v e r s e r e l a t i o n s h i p s between f o r c e g e n e r a t i o n of i s o l a t e d muscle p r e p a r a t i o n s and ^ c o n c e n t r a t i o n (Dawson et a l . , 1978) have been demonstrated when pH drops to too great a degree. Anaerobic g l y c o l y s i s which i s u t i l i z e d when energy expenditure i s too high to be met through the a e r o b i c combustion of f u e l s , r e s u l t s i n the u l t i m a t e p r o d u c t i o n of 2 moles of H*and 2 l a c t a t e anions with the concominant p r o d u c t i o n of ATP per g l u c o s y l u n i t . G l y c o l y s i s and ATP h y d r o l y s i s occur d u r i n g anaerobic energy p r o d u c t i o n demonstrating opposing pH dependencies of H*production. Thus the r e l a t i v e c o n t r i b u t i o n of e i t h e r g l y c o l y s i s or ATP h y d r o l y s i s to the 2 moles of H*produced 48 per g l u c o s y l u n i t i s pH dependent (Hochachka and Mommsen 1982). L a c t i c a c i d was f i r s t i d e n t i f i e d i n muscle by J . J . B e r z e l i u s (1807) with the r e l a t i o n s h i p between l a c t a t e formation and glucose degradation being i n i t i a l l y d e s c r i b e d by C. Bernard (1877). That l a c t a t e was produced w i t h i n muscle d u r i n g e x e r c i s e was f i r s t r e p o r t e d by F l e t c h e r and Hopkins (1906), H i l l (1926) and Meyerhof (1930). I t i s now g e n e r a l l y accepted that l a c t i c a c i d accumulates when there i s an imbalance i n the ra t e of energy r e q u i r e d and that which can be achieved by pyruvate o x i d a t i o n w i t h i n the mit o c h o n d r i a . Hochachka and Storey (1975) s t a t e d t h at l a c t a t e accumulation was the best i n d i c a t o r of a n a e r o b i o s i s . Within a t h l e t e s performing continuous intense short d u r a t i o n e x e r c i s e , l a c t a t e values of 32mM have been r e p o r t e d (Hermansen et a l . , 1971).. Thus the performance of short d u r a t i o n , high i n t e n s i t y e x e r c i s e p l a c e s enormous demands on anaerobic g l y c o l y s i s f o r energy p r o d u c t i o n and on the mechanisms fo r h a n d l i n g the accumulation of end products. 1. Metabolism. The biochem i c a l parameters necessary to allow humans to perform at i n c r e a s e d r a t e s or d u r i n g p e r i o d s of hypoxia can be c a t e g o r i z e d . F i r s t , energy p r o d u c t i o n i n the form of ATP s y n t h e s i s must be performed at a f a s t r a t e . Second, redox balance must be maintained so that i n t e r m e d i a t e m e t a b o l i t e s and c o - f a c t o r s do not accumulate i n t h e i r reduced forms ( C a s s t e l l i n i 1981). T h i r d , mechanisms f o r the b u f f e r i n g of the accumulated protons must be adequate. Under normal c o n d i t i o n s the need f o r a hig h energy y i e l d exceeds the need f o r a high energy r a t e . Fat demonstrating a 49 r e s p i r a t r y q u o t i e n t of 0.7 (Mathews and Fox 1976) and y i e l d i n g 36 ATP per mole of s u b s t r a t e (Lehninger 1980) i s predominantly u t i l i z e d under these c o n d i t i o n s . T h i s pathway bypasses g l y c o l y s i s e n t e r i n g the Krebs C y c l e from A c e t y l CoA. Within the o x i d a t i v e p h o s p h o r y l a t i o n pathway there i s an abs o l u t e requirement f o r oxygen at the cytochrome oxidase l e v e l . Anaerobic g l y c o l y s i s produces only 2 or 3 ATP per mole of s u b s t r a t e depending upon whether glucose or glycogen i s used. Pyruvate a c t s as a s u b s t r a t e f o r both a e r o b i c and anaerobic metabolism with the purpose of i t s r e d u c t i o n being that of o x i d i z i n g NADH to maintain redox balance. When the ra t e of energy demand i s high pyruvate i s converted to l a c t a t e , NADH i s o x i d i z e d , redox balance i s maintained and ATP p r o d u c t i o n per g l u c o s y l u n i t i s low (Newsholme and S t a r t 1973). 2. Processes of L a c t a t e D i s p o s a l and Proton Release. The removal of l a c t a t e has g e n e r a l l y been assumed to be a simple process of d i f f u s i o n down c o n c e n t r a t i o n g r a d i e n t s (Hirche et a l . , 1971; J o r f e l d t , 1978; Mainwood and Brown 1975). Recently i t has become apparent that l a c t a t e t r a n s f e r w i t h i n a few t i s s u e s i s c a r r i e r mediated. Within the proximal tubule of the kidney i t appears to be Na* t r a n s p o r t l i n k e d (Barac-Nieto et a l . , 1978) while i n s e v e r a l tumour l i n e s (Spencer and Lehninger 1976; Johnson et a l . , 1980) red blood c e l l s , (Dubinsky and Racker 1978) and l i v e r (Monson et a l . , 1981), the c a r r i e r mediated t r a n s f e r of l a c t a t e i s pH dependent appearing to be an a n t i p o r t system, l a c t a t e anions exchanging f o r OH* ions (Hochachka and Mommsen 1982). Within mouse diaphragm muscle Koch et a l . , (1981) 50 suggested that at. l e a s t three q u a r t e r s of the l a c t a t e t r a n s f e r was c a r r i e r mediated. Whatever the mechanism of t r a n s f e r , l a c t a t e has many d i s p o s a l s i t e s . I t can be s t o r e d w i t h i n the producing muscle (K a r l s s o n 1971),.or d i l u t e d i n . b l o o d and other body f l u i d s (Hirche et a l . , 1971; J o r f e l d t 1978; Mainwood and Brown 1975). From blood, l a c t a t e may be taken up by l i v e r , heart muscle, kidney, b r a i n ( B e l c a s t r o and Bonen 1975), a c t i v e ( J o r f e l d t 1970) and i n a c t i v e (Poortmans 1978) s k e l e t a l muscle. The u l t i m a t e f a t e i s re c o n v e r s i o n t o pyruvate where i t i s e i t h e r u t i l i z e d w i t h i n the Krebs Cycle or converted i n t o glycogen or glucose (Poortmans 1978). Hermansen and Vaage (1977) found the a l a n i n e c y c l e to be of minor s i g n i f i c a n c e i n the disappearance of l a c t a t e from s k e l e t a l muscle. Protons a s s o c i a t e d with l a c t a t e formation accumulate w i t h i n muscle and are r e l e a s e d to blood and e x t r a c e l l u l a r f l u i d s . I t has been r e p o r t e d that the r a t e of H*release was dependent upon e x t e r n a l b i c a r b o n a t e c o n c e n t r a t i o n (Mainwood and Brown 1975). Benade and H e i s l e r (1978) r e p o r t e d that H* r e l e a s e i n i t i a l l y exceeded l a c t a t e r e l e a s e while Hermansen and Osnes (1972) found the H r e l e a s e to occur to a much g r e a t e r extent than would be expected from t h e i r l a c t a t e e f f l u x d a t a . The i d e n t i f i c a t i o n of a maximal r a t e of l a c t a t e r e l e a s e by J o r f e l d t et a l . , (1978) may account f o r t h i s f i n d i n g . Anaerobic g l y c o l y s i s r e s u l t i n g i n the pr o d u c t i o n of l a c t a t e s a t i s f i e s the requirements necessary f o r high i n t e n s i t y , short d u r a t i o n work: f i r s t , ATP i s generated; second, redox balance i s optim i z e d such that the r e d u c t i o n of pyruvate to l a c t a t e 51 p r o v i d e s the c o - f a c t o r NAD necessary to maintain g l y c o l y t i c f u n c t i o n i n g and t h i r d , mechanisms f o r the removal of the i n h i b i t i n g end products e x i s t . The l i m i t a t i o n of anaerobic g l y c o l y s i s occurs i n the second and t h i r d areas. The accumulation and removal of end products can not occur i n d e f i n i t e l y i n a t i s s u e s i n c e at h i g h energy demands , the NADH /NAD r a t i o i s i n c r e a s e d , a l t e r i n g redox balance and r e s u l t i n g i n a proton accumulation, u l t i m a t e l y lowering pH to v a l u e s too low f o r g l y c o l y s i s to c o n t i n u e . 3. Enzymatic C o n t r o l of Anaerobic G l y c o l y s i s . Carbohydrate metabolism from glucose to pyruvate i n v o l v e s nine separate chemical r e a c t i o n s which are c a t a l y z e d by s p e c i f i c enzymes. Enzymes can be c l a s s i f i e d as e i t h e r r e g u l a t o r y or non-regulatory depending upon t h e i r d i s t a n c e from thermodynamic e q u i l i b r i u m . Non-regulatory enzymes i n v o l v e thermodynamically e q u i v a l e n t r e a c t i o n s which can proceed e s s e n t i a l l y i n e i t h e r d i r e c t i o n . Regulatory enzymes s i t u a t e d f a r from thermodynamic e q u i l i b r i u m have a l a r g e f r e e energy change and proceed e s s e n t i a l l y i n one d i r e c t i o n . Regulatory enzymes u s u a l l y a d j u s t the r a t e of carbon flow by responding s e n s i t i v e l y to changes i n m e t a b o l i t e modulators, c o - f a c t o r s , co-enzymes and s u b s t r a t e s . Non- r e g u l a t o r y enzymes f u n c t i o n to t r a n s p o r t carbon along the chain as f a s t as p o s s i b l e (Hochachka 1980). S e v e r a l r e g u l a t o r y enzymes have been i d e n t i f i e d i n anaerobic g l y c o l y s i s . The enzymes hexokinase (HK), phosphorylase, phosphofructokinase (PFK) and pyruvate kinase (PK) are a l l s i t u a t e d f a r from e q u i l i b r i u m and a c t as r e g u l a t o r y enzymes. The 52 enzyme HK c a t a l y z e s the r e a c t i o n glucose to glucose-6-phosphate (G6P) t h e r e f o r e being r e s p o n s i b l e f o r the pathways a b i l i t y to u t i l i z e blood g l u c o s e . Phosphorylase e x i s t s i n both an a c t i v e and i n a c t i v e form being modulated by both c o n t r a c t i l e and hormonal i n f l u e n c e s . PFK c a t a l y z e s an intermediate step i n the pathway with i t s a c t i v i t y being m o d i f i e d by many f a c t o r s : adenylate c o u p l i n g , energy charge of the c e l l and s u b s t r a t e l e v e l s . PK enhances the p r o d u c t i o n of pyruvate which can be metabolized i n a v a r i e t y of ways (Newsholme and S t a r t 1973). The enzyme l a c t a t e dehydrogenase (LDH) c a t a l y z e s the pyruvate to l a c t a t e r e a c t i o n and i s s t r o n g l y p o i s e d thermodynamically i n the l a c t a t e d i r e c t i o n (Everse and Kaplan 1973). Within s k e l e t a l muscle the M4-LDH isoenzyme form predominates. T h i s isoenzyme demonstrates a high Km f o r pyruvate and NADH while being i n s e n s i t i v e to pyruvate or l a c t a t e i n h i b i t i o n and has a low a f f i n i t y f o r l a c t a t e (Hochachka 1980). Thus M4-LDH a c t s e s s e n t i a l l y as a pyruvate reductase whose a c t i v i t y i s e s s e n t i a l to maintain redox balance. 4. E x e r c i s e P h y s i o l o g y . S p e c i f i c a l l y i n anaerobic work the i n i t i a l source of energy f o r in t e n s e muscular c o n t r a c t i o n comes from creatine-phosphate (CP) and ATP s t o r e s which are approximately 16 and 4 mmol.kg"1 wet muscle ( K a r l s s o n et a l . , 1971; Knuttgen and S a l t i n 1972). In humans, these sources can only p r o v i d e enough energy f o r the f i r s t ten to t h i r t y seconds of e x e r c i s e a f t e r which anaerobic g l y c o l y s i s p r o v i d e s the necessary energy r e q u i r e d f o r continued muscular c o n t r a c t i o n . Thus f o r improved anaerobic performances t r a i n e d a t h l e t e s must 53 have e i t h e r : e l e v a t e d l e v e l s of s t o r e d CP, ATP and/or glycogen, i n c r e a s e d a c t i v i t y of the r a t e l i m i t i n g r e g u l a t o r y enzymes, and/or an enhanced mechanism f o r b u f f e r i n g the i n h i b i t i n g e f f e c t s of accumulated anaerobic end p r o d u c t s . During high i n t e n s i t y , short d u r a t i o n worik, glycogen l e v e l s are never d e p l e t e d before f a t i g u e causes c e a s a t i o n of the e x e r c i s e ( G o l l n i c k and Hermansen 1973). Changes i n i n t r a c e l l u l a r s t o r e s of ATP and CP f o l l o w i n g t r a i n i n g are of such low magnitude as not to s i g n i f i c a n t l y enhance performance on anaerobic workloads (Knuttgen and S a l t i n 1972). Thus enhanced performance as a r e s u l t of i n c r e a s e d s t o r e d s u b s t r a t e l e v e l s does not seem f e a s i b l e . The advent of the needle biopsy technique (Bergstrom et a l . , 1962) and the subsequent i d e n t i f i c a t i o n of two d i s t i n c t muscle f i b e r types demonstrating d i s t i n g u i s h i n g enzymatic and c o n t r a c t i l e p r o p e r t i e s p r o v i d e d the impetus f o r much of the i n v e s t i g a t i o n surrounding enhanced anaerobic performance. F i b e r s were o r i g i n a l l y c l a s s i f i e d as e i t h e r f a s t - t w i t c h or slow-twitch based on t h e i r c o n t r a c t i l e p r o p e r t i e s and s t a i n i n g i n t e n s i t i e s f o r o x i d a t i v e enzyme a c t i v i t y (Dubowitz and Brooke 1973). Many of the problems a s s o c i a t e d with s k e l e t a l muscle f i b e r composition can be a t t r i b u t e d to the nomenclature u t i l i z e d f o r h i s t o c h e m i c a l c l a s s i f i c a t i o n . B r i e f l y f o r non-human s k e l e t a l muscle, f i b e r s are p r e s e n t l y being c l a s s i f i e d on the b a s i s of t h e i r r e a c t i o n s to a c o n t r a c t i l e c h a r a c t e r i s t i c (myosin ATPase) and to t h e i r o x i d a t i v e c a p a c i t y (NADH-TR). U t i l i z i n g these s t a i n s 3 f i b e r types have been i d e n t i f i e d : f a s t - t w i t c h high 54 g l y c o l y t i c (FG), fast-twi.tch high g l y c o l y t i c - h i g h o x i d a t i v e (FOG) and slow-twitch o x i d a t i v e (SO) (Houston 1978). With human s k e l e t a l muscle most i n v e s t i g a t i o n s . have u t i l i z e d a two f i b e r c l a s s i f i c a t i o n based on the h i s t o c h e m i c a l m y o f i b r i l l a r ATPase r e a c t i o n at pH 9.4 (Houston 1978). The l i g h t s t a i n i n g f i b e r s were c l a s s i f i e d as type I and the dark s t a i n i n g f i b e r s as type I I . These f i b e r s represent slow and f a s t c o n t r a c t i o n v e l o c i t i e s r e s p e c t i v e l y . An a l t e r n a t e c l a s s i f i c a t i o n of slow-twitch (ST) and f a s t - t w i t c h (FT) has been used e x t e n s i v e l y ( G o l l n i c k et a l . , 1972; C o s t i l l et a l . , 1976). With the improvement i n s t a i n i n g techniques f u r t h e r s u b d i v i s i o n s of f a s t - t w i t c h (type II) f i b e r s have been i d e n t i f i e d on the b a s i s of t h e i r s t a i n i n g i n t e n s i t i e s f o r myosin ATPase at d i f f e r e n t p r e - i n c u b a t i o n pH's. Thus the type II (FT) f i b e r s have f u r t h e r been c l a s s i f i e d as types IIA, IIB, IIC. U t i l i z i n g a two f i b e r c l a s s i f i c a t i o n scheme f o r human s k e l e t a l muscle slow-twitch r e f e r s to type I and f a s t - t w i t c h corresponds to type II which i n c l u d e s a l l the type II s u b d i v i s i o n s . F i b e r composition of s p r i n t (Edstrom and Ekblom 1972; Thorstenson 1975, 1976; C o s t i l l et a l . , 1976, 1979; P r i n c e et a l . , 1976; Thomson et a l . , 1979, Roberts et a l . , 1981) and endurance ( C o s t i l l et a l . , 1971, 1976, 1979; G o l l n i c k et a l . , 1973, 1974; E r i k s s o n et a l . , 1973; P r i n c e et a l . , 1976, 1977; Hennrikson 1977; Jansen and K a i j s e r 1977; S a l t i n et a l . , 1977; Essen et a l . , 1978; L i t h i l l e t a l . , 1979) t r a i n e d a t h l e t e s have been i n v e s t i g a t e d r e v e a l i n g a higher p r o p o r t i o n of f a s t - t w i t c h f i b e r s w i t h i n s p r i n t a t h l e t e s and a higher p r o p o r t i o n of slow-twitch f i b e r s w i t h i n endurance 55 a t h l e t e s . The i n t e r c o n v e r s i o n of f a s t - t w i t c h to slow-twitch and v i c e v e r s a does not appear.to occur as a r e s u l t of t r a i n i n g though the r e l a t i v e g l y c o l y t i c or o x i d a t i v e c a p a c i t i e s w i t h i n each f i b e r type are augmented ( G o l l n i c k 1982). Hypertrophy of f i b e r s appears to be s e l e c t i v e l y chosen as to the type of t r a i n i n g performed ( G o l l n i c k et a l . , 1972, 1973; C o s t i l l et a l . , 1976; P r i n c e et a l . , 1976; S a l t i n et a l . 1976, Andersen and Hennriksen 1977). Increased s i z e and number of f a s t - t w i t c h f i b e r s w i t h i n the s p r i n t t r a i n e d p o p u l a t i o n can not t o t a l l y account f o r t h e i r enhanced performances. F a s t - t w i t c h f i b e r s have a much higher g l y c o l y t i c p o t e n t i a l than slow-twitch (Lowry et a l . , 1978; Essen et a l . , 1975). Enzyme a c t i v i t i e s (LDH, PFK, phosphorylase, HK) w i t h i n a t h l e t e s and animals f o l l o w i n g s p r i n t t r a i n i n g have demonstrated with minor e x c e p t i o n s no s i g n i f i c a n t d i f f e r e n c e s (Baldwin et a l . , 1972; G o l l n i c k et a l . , 1972; H o l l o s z y et a l . , 1971; Hickson et a l . , 1975). C o s t i l l et a l . , (1976) examined d i f f e r e n t t r a c k a t h l e t e s and found s p r i n t t r a i n e d a t h l e t e s to have e l e v a t e d LDH and phosphorylase a c t i v i t i e s as compared to endurance t r a i n e d a t h l e t e s . T h e r e f o r e though some enhancement of g l y c o l y t i c enzyme a c t i v i t y appears to accompany s p r i n t t r a i n i n g , the q u a n t i t a t i v e changes are of i n s u f f i c i e n t magnitude to s o l e l y account f o r the d i f f e r e n c e s demonstrated i n anaerobic performance. Accumulation of anaerobic end products has been a s s o c i a t e d with the f a t i g u e process probably through the proton .action on i n t r a c e l l u l a r pH. S t u d i e s on e x e r c i s i n g man have found i n t r a m u s c u l a r pH to be as low as 6.4 to 6.6 d u r i n g a short 56 i n t e n s i v e e x e r c i s e bout (Osnes and Hermansen 1972; S a h l i n et a l . , 1976). A s s o c i a t e d with the pH decrement are reduced r a t e s of g l y c o l y s i s (Tpews et a l . , 1970; Sutton et a l . , 1981; Roos and Boron 1981) and c o r r e l a t i o n s with f a t i g u e ( F i t t s and H o l l o s z y 1976; Stevens 1980). Thus anaerobic performance may be enhanced by reducing the r a t e of pHi decrement which accompanies the proton accumulation dur i n g high i n t e n s i t y , short d u r a t i o n work. B. I n t r a c e l l u l a r pH. The pH value of a s o l u t i o n i s a measure of the r e l a t i v e chemical p o t e n t i a l s of the protons i n that s o l u t i o n (Waddell 1971). S e v e r a l techniques e x i s t f o r the dete r m i n a t i o n of i n t r a c e l l u l a r pH ( p H i ) : homogenate, d i s t r i b u t i o n of weak a c i d s and bases (DMO), c a l o r i m e t r y and fluo r o m e t r y , m i c r o e l e c t r o d e and 31P nu c l e a r magnetic resonance spectroscopy. Each method has i t s r e s p e c t i v e advantages and disadvantages d i s p l a y i n g l a r g e v a r i a t i o n s i n i n t r a c e l l u l a r pH de t e r m i n a t i o n of human s k e l e t a l muscle (Roos and Boron 1981). Measurement of pH on a homogenate was f i r s t employed by M i c h a e l i s and Dav i d o f f (1912) on red blood c e l l s . The homogenate technique possess s e v e r a l problems: l a c t i c a c i d and CO pro d u c t i o n continue a f t e r c e l l u l a r d e s t r u c t i o n l e a d i n g to a f a l l i n pH\. (Waddell and Bates 1969), mixing of 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 can l e a d to pH changes i f the s o l u t i o n s are of d i s s i m i l a r pH, the d i l u t i o n of the e x t r a - and i n t r a c e l l u l a r b u f f e r s (Bates 1973), and d i s r u p t i o n of i n t r a c e l l u l a r o r g a n e l l e s with t h e i r r e s p e c t i v e i n t e r n a l pH (Cohen and l i e s 1975). 57 Furusawa and K e r r i d g e (1927) examined cat s k e l e t a l , c a r d i a c and u t e r i n e muscle, e l i m i n a t e d the CO^and l a c t a t e problems, by immediate submersion of the sample in l i q u i d . a i r , ;with subsequent mincing and pH d e t e r m i n a t i o n by g l a s s m i c r o e l e c t r o d e at 0°C. A s e r i e s of s t u d i e s were conducted examining both r e s t i n g and p o s t - e x e r c i s e i n t r a c e l l u l a r pH d e t e r m i n a t i o n s on human q u a d r i c e p muscle homogenates (Hermansen and Osnes 1972; S a h l i n , H a r r i s and Hultman 1975; S a h l i n 1976). Reported r e s t i n g pH v a l u e s were 6.92+.10 (Hermansen and Osnes 1972) and 7.08+.03 ( S a h l i n , 1976) r e s p e c t i v e l y . The i n c l u s i o n of i o d o a c e t i c a c i d (IAA) i n the p r e p a r a t i o n prevented the continuous decrease i n pH d u r i n g the measurement. S a h l i n (1976) estimated that the mixing of the i n t r a - with the e x t r a c e l l u l a r compartments would i n c r e a s e the r e s t i n g i n t r a c e l l u l a r pH of the samples by about 0.03 u n i t s . A pH of 6.8 to 7.1 has been obtained i n most animal s t u d i e s on s k e l e t a l muscle by a l a r g e number of techniques (Furusawa and K e r r i d g e 1927; M i l l a r , Tyson and Relman 1963; Hault et a l . , 1974; Waddell and Bates 1969; A i c k i n and Thomas 1977). 1. T o t a l muscle pH i n r e l a t i o n to e x e r c i s e . Furusawa and K e r r i d g e (1927) studying e l e c t r i c a l l y , s t i m u l a t e d cat gastrocnemius muscle found pH to decrease from 7.04 at r e s t to 6.26 at f a t i g u e . The r e s u l t s from more recent s t u d i e s are i n agreement with these f i g u r e s . Steinhagen et a l . , (1976) examined i n t e r s t i t i a l pH of dog working gastrocnemius muscle with implanted g l a s s m i n i e l e c t r o d e s and found a proton c o n c e n t r a t i o n g r a d i e n t to always e x i s t between i n t e r s t i t i a l f l u i d and venous blood. I n t e r s t i t i a l pH w i t h i n muscle has shown an i n i t i a l 58 a l k a l i n i z a t i o n f o l l o wed by an i n c r e a s e d a c i d i f i c a t i o n to occur d u r i n g c o n t r a c t i o n (Gebert and Sydney 1973; Steinhagen et a l . , 1976). The time course of pH changes are i n agreement with the metabolic changes w i t h i n muscle (Danforth et a l . , 1965). The i n t r a c e l l u l a r pH of r a t t h i g h muscle measured by :Rooth (1966) using the DMO method was found to decrease only from 6.64 at r e s t to 6.57 a f t e r exhaustive e x e r c i s e . S i m i l a r l y , Hermansen (1969) examined one s u b j e c t running i n t e r m i t t e n t l y f o r 20 minutes, with pH determined by the DMO technique. He found the pH to decrease from only 6.88 at r e s t to 6.73 a f t e r e x e r c i s e . R e l i a b l e pH valu e s determined by the DMO method r e q u i r e at l e a s t one hour of e q u i l i b r a t i o n time between the i n t r a - and e x t r a c e l l u l a r compartments (Waddell and B u t l e r 1959). T h i s c o n d i t i o n was not met i n e i t h e r the study by Rooth (1966) or Hermansen (1969). In a study on maximum b i c y c l e e x e r c i s e of short d u r a t i o n , t o t a l muscle pH of the musculus quadreceps femoris was determined by the homogenate technique. Muscle pH was found to decrease from 6.92 at r e s t to 6.41 a f t e r exhaustive e x e r c i s e (Hermansen and Osnes 1972). Muscle samples of the musculus quadriceps femoris were obtained p r i o r and post i s o m e t r i c e x e r c i s e to f a t i g u e with muscle pH dete r m i n a t i o n s being made by the homogenate technique ( S a h l i n , H a r r i s and Hultman, 1975). Muscle pH was found to decrease from 7.09 at r e s t to 6.56 at f a t i g u e . In a subsequent i n v e s t i g a t i o n employing dynamic e x e r c i s e , muscle pH was found to decrease from 7.08 at r e s t to 6.60 at exhaustion ( S a h l i n eta.l., 1976). I t thus appears that 59 human quadricep t o t a l muscle pH determined by the homogenate technique i s roughly 7.0 and that i n t e n s e muscular e x e r c i s e to f a t i q u e r e s u l t s i n a r e d u c t i o n i n intramuscular pH to approximatly 6.5. 2.. pH changes and muscular work of s k e l e t a l muscle. The e f f e c t s of acid-base changes on s k e l e t a l muscle were f i r s t demonstrated by Creese (1950) on i s o l a t e d r e p e t i t i v e l y s t i m u l a t e d r a t diaphragm muscle. The t r a n s i e n t s which o c c u r r e d i n t w i t c h t e n s i o n c o u l d be a s c r i b e d to the pH changes r e s u l t i n g from removal and readmission of CO t. Foulks and Perry (1977) found e x t r a c e l l u l a r pH changes from 5 to 9 at constant PCO t to a f f e c t t w i t c h t e n s i o n of f r o g muscle very l i t t l e . S t i m u l a t i n g f r o g muscle to f a t i g u e , Mainwood and Brown (1975) found t w i t c h t e n s i o n decreased to 20 percent of c o n t r o l . In an e a r l i e r i n v e s t i g a t i o n , Mainwood et a l . , (1972) found e x t e r n a l HCO^ to modulate i n t r a c e l l u l a r proton balance and to l i m i t l a c t a t e e f f l u x . I n c r e a s i n g e x t r a c e l l u l a r pH, thus r a i s i n g i n t e r n a l pH, i n c r e a s e d l a c t a t e e f f l u x l e a d i n g to n e a r l y 100 percent recovery of t e n s i o n i n f a t i g u e d muscle. In experiments where pH was monitored by 31P-nuclear magnetic resonance Dawson et a l . , (1978) found i n v e r s e r e l a t i o n s h i p s between i s o l a t e d f r o g muscle p r e p a r a t i o n s f o r c e g e n e r a t i o n c a p a b i l i t i e s and proton c o n c e n t r a t i o n . Stevens (1980) found i s o l a t e d f r o g s a r t o r i u s muscle p r e p a r a t i o n s to demonstrate c o r r e l a t i o n s between pH and f a t i g u e . S i m i l a r r e l a t i o n s h i p s were demonstrated by F i t t s and H o l l o s z y (1976) on f r o g muscle p r e p a r a t i o n s between l a c t a t e l e v e l s and f a t i g u e . 60 3. Mechanisms of A c t i o n . I t has been suggested by Nocker (1964) that decreased pH may a f f e c t the membrane p e r m e a b i l i t y to Na*and K* r e s u l t i n g i n a h y p e r p o l a r i z e d s t a t e . T h i s e f f e c t may be even more important i n f a s t - t w i t c h f i b e r s than slow-twitch f i b e r s s i n c e they have a lower r e s t i n g membrane p o t e n t i a l (Campion 1974). Muscle c o n t r a c t i o n would be impaired s i n c e membrane p e r m e a b i l i t y i s v i t a l to the e l i c i t a t i o n of an a c t i o n p o t e n t i a l . A decreased a c t i v e c r o s s b r i d g e formation due to proton c o m p e t i t i o n with Ca f o r the actomyosin b i n d i n g s i t e s , may reduce work c a p a c i t y (Katz 1970). F a b i a t o and F a b i a t o (1978) examined the e f f e c t s of pH on the myofilaments and sarcoplasmic r e t i c u l u m of skinned f r o g s k e l e t a l muscle c e l l s . They found s k e l e t a l muscle i n c r e a s e d i t s r e l e a s e of Ca*at moderate a c i d i f i c a t i o n i n an attempt to compensate f o r the decreased s e n s i t i v i t y of the myofilaments to Ca*. Th e r e f o r e they i d e n t i f i e d the only e f f e c t of pH v a r i a t i o n w i t h i n s k e l e t a l muscle, was the decreased maximum t e n s i o n development c a p a b i l i t y d u r i n g a c i d o s i s . I n t r a c e l l u l a r pH has many i n t e r a c t i o n s with metabolic t r a n s f o r m a t i o n s . I o n i z a b l e groups of a c t i v e s i t e s on enzymes, may a f f e c t the enzymes conformation and thus i t s s u b s t r a t e b i n d i n g and c a t a l y t i c p r o p e r t i e s through t h e i r s t a t e of i o n i z a t i o n . S p e c i f i c groups on s u b s t r a t e s or c o f a c t o r s through t h e i r degree of i o n i z a t i o n may a f f e c t t h e i r a b i l i t y to bind to the enzyme. The d i r e c t uptake or r e l e a s e of protons or CG\by the metabolic t r a n s f o r m a t i o n s themselves may produce pH changes (Roos and Boron 1981). Within the g l y c o l y t i c pathway the co n v e r s i o n of i n a c t i v e 61 phosphorylase b i n t o a c t i v e phosphorylase a i s pH sensitive.. Danforth (1965) examined i n t a c t f r o g muscle and demonstrated a l a g p e r i o d before appearance of phosphorylase a i n response t o muscle s t i m u l a t i o n as C0Z c o n c e n t r a t i o n was r a i s e d . In v i t r o p r e p a r a t i o n s s i m i l a r l y depressed phosphorylase b to a c o n v e r s i o n as pH decreased. T r i v e d i and Danforth (1966) i d e n t i f i e d a marked pH s e n s i t i v i t y of PFK, the enzyme that phosphorylates f r u c t o s e - 6 - phosphate (F6P). U t i l i z i n g an i n v i t r o p r e p a r a t i o n , a 10 to 20 f o l d r e d u c t i o n i n enzyme a c t i v i t y o c c u r r e d when pH was reduced by 0.1 u n i t s . The.actual pH range that produced the markedly decreased enzyme a c t i v i t y was dependent upon F6P c o n c e n t r a t i o n . The i n c r e a s e d c o n c e n t r a t i o n s of G6P as a r e s u l t of e l e v a t e d F6P a c t i v i t y tends to i n h i b i t r e a c t i o n s higher up i n the g l y c o l y t i c pathway ( T r i v e d i and Danforth 1966). The- c o n c e n t r a t i o n s of muscle g l y c o l y t i c i n t e r m e d i a t e s determined at exhaustion, were u t i l i z e d to determine at which p o i n t i n the g l y c o l y t i c pathway a c i d o s i s produced i t s i n h i b i t o r y a c t i o n (Toews et a l . , 1970). PFK was i d e n t i f i e d as the enzyme whose a c t i o n was i n h i b i t e d by a c i d o s i s . The enzyme a c t i o n s of PFK and pyruvate kinase (PK) are l i n k e d by adenylate c o u p l i n g , thus i n h i b i t i n g the p r o d u c t i o n of pyruvate. Decreases i n pH l e a d to l a r g e changes i n ATP and CP s e n s i t i v i t y as w e l l as l a r g e i n c r e a s e s i n a f f i n i t y f o r both s u b s t r a t e s (Hochachka 1980). Sutton et a l . , (1981) examined the e f f e c t of pH on muscle g l y c o l y s i s d u r i n g e x e r c i s e , c o n c l u d i n g that i n agreement with the i n v i t r o s t u d i e s , e l e v a t e d proton c o n c e n t r a t i o n s e v e n t u a l l y 62 i n h i b i t g l y c o l y s i s reducing the supply of ATP necessary f o r continued muscular c o n t r a c t i o n . These a c t i o n s appear to be e l i c i t e d by pH dependent i n h i b i t i o n of the c o n t r a c t i l e process and of the g l y c o l y t i c r e g u l a t o r y enzymes phosphorylase and phosphofructokinase. Thus the importance of n e u t r a l i z i n g the proton accumulation and l i m i t i n g the decrement i n pH a s s o c i a t e d with muscular work may i n f l u e n c e anaerobic performance. C. B u f f e r C a p a c i t y Most anaerobic s t u d i e s have c o n c e n t r a t e d on changes i n s u b s t r a t e l e v e l s or enzyme a c t i v i t y and m e t a b o l i t e c o n c e n t r a t i o n s which c o u l d be capable of g e n e r a t i n g ATP and m a i n t a i n i n g redox balance (Hochachka 1980). Few i n v e s t i g a t i o n s have c e n t r e d on the c a p a c i t y of i n t r a c e l l u l a r f l u i d s to b u f f e r the a c i d i c end products of anaerobic g l y c o l y s i s . H e i s l e r and P i i p e r (1971) s t a t e t h at s k e l e t a l muscle by v i r t u e of i t s h i g h mass, i t s abrupt and l a r g e changes i n metabolic a c t i v i t y , and i t s h i g h anaerobic c a p a c i t y , show that i t must be s u b j e c t to l a r g e l o c a l , v a r i a t i o n s i n a c i d p r o d u c t i o n . T h e r e f o r e they suggest that s k e l e t a l muscle must be the most important determinant of o v e r a l l b u f f e r i n g a b i l i t y of the organism. Although B of s k e l e t a l muscle had been reco g n i z e d as an important f a c t o r i n pH r e g u l a t i o n , i t s involvement to pH homeostasis w i t h i n t r a i n e d a t h l e t e s has not been i n v e s t i g a t e d . Homeostasis of pH r e l i e s on the c e l l s a b i l i t y to extrude H* and/or accumulate HCO^ or OH~ (Roos and Boron 1981). I t i s g e n e r a l l y accepted that an a c i d i s a proton doner and a base i s 63 a hydrogen acc e p t o r , while a b u f f e r i s something that r e s i s t s change. A pH b u f f e r i s a substance, or mixture of substances, that permit s o l u t i o n s to r e s i s t l a r g e changes i n pH upon the a d d i t i o n of small amounts of H*or OH*ions (Segal 1976). B u f f e r c a p a c i t y r e f e r s to the a b i l i t y of a b u f f e r to r e s i s t changes i n pH. B can be d e f i n e d as the number of moles per l i t e r of H*or OH r e q u i r e d to cause a given change i n pH of 1 u n i t . E s s e n t i a l l y B i s the r e c i p r o c a l of the slope of the t i t r a t i o n curve at any p o i n t (Segal 1976). Larsen and B u r n e l l (1978) s t a t e d that B was a f u n c t i o n of b u f f e r c o n c e n t r a t i o n and the proton s e q u e s t e r i n g c a p a b i l i t i e s of the b u f f e r s i n the s p e c i f i c pH range. In 1908 Henderson and Washburn working s e p a r a t e l y r e p o r t e d that a weak a c i d e x e r t e d i t s maximum b u f f e r i n g when . i t s d i s s o c i a t i o n constant e q u a l l e d the H * c o n c e n t r a t i o n . Koppel and S p i r o (1914) were the f i r s t to demonstrate that t o t a l b u f f e r a c t i o n was the sum of the i n d i v i d u a l b u f f e r a c t i o n s . They demonstrated that the maximal b u f f e r a c t i o n of a l l monovalent weak a c i d s at e q u i v a l e n t t o t a l c o n c e n t r a t i o n s were the same. M i c h a e l i s i n 1922 m o d i f i e d the d e f i n i t i o n of b u f f e r i n g power to B =dB/dpH. Van Slyke (1922) d e f i n e d b u f f e r i n g power i n the same way as M i c h a e l i s . T h i s d e f i n i t i o n i n v o l v e s the so c a l l e d s e l f b u f f e r i n g of water, which only becomes apparent at extreme pH v a l u e s and i s n e g l i g i b l e i n the p h y s i o l o g i c a l pH range (Roos and Boron 1981). T h i s d e f i n i t i o n of b u f f e r i n g power given by M i c h a e l i s and Van Slyke i s now r e a d i l y accepted. 64 - U S k e l e t a l Muscle B u f f e r i n g C a p a c i t y . S e v e r a l methods employing the a d d i t i o n of a c i d or base loads while m o n i t o r i n g pH changes by a v a r i e t y of means have been u t i l i z e d f o r the d e t e r m i n a t i o n of B w i t h i n s k e l e t a l muscle. T i t r a t i o n of c e l l u l a r homogenates was f i r s t used by Furusawa and K e r r i d g e (1927) on c a t s k e l e t a l , c a r d i a c and smooth muscle at 0*C over the pH range 6.4 to 7.4. D i l u t i o n d i d not s u b s t a n t i a l l y a l t e r the pH. T i t r a t i o n of i n t r a c e l l u l a r f l u i d i n s i t u by the i n j e c t i o n of a c i d or a l k a l i or by exposure to a weak a c i d or base was another technique employed. T i t r a t i o n of i n t a c t c e l l s impaled with a pH s e n s i t i v e m i c r o e l e c t r o d e i s a t h i r d technique which has been used. I n t r a c e l l u l a r b u f f e r c a p a c i t y of s k e l e t a l muscle has been determined i n a v a r i e t y of s p e c i e s and a summary of the r e s u l t s are c o n t a i n e d i n t a b l e 5. Values range from 40 to l00(mmol.pH .1 IC H tO) and appear to be r e l a t e d to the muscles c a p a c i t y f o r h i g h g l y c o l y t i c f u n c t i o n ( C a s t e l l i n i and Somero 1981). H e i s l e r and P i i p e r (1971, 1972) demonstrated t h a t these v a l u e s d i f f e r from the B of i n t a c t p r e p a r a t i o n s and that these d i s c r e p a n c i e s are due to the transmembrane f l u x e s of H*"and/or HCO a i n i n t a c t muscle. Protons appear to be t r a n s p o r t e d i n t o s k e l e t a l muscle and out of heart and b r a i n t i s s u e d u r i n g severe r e s p i r a t o r y a c i d o s i s (Clancy and Brown 1966; S i e s j o and Messeter 1971; L a i et a l . , 1973). Thus the l a r g e b u f f e r i n g a b i l i t y of muscle appears to be u t i l i z e d f o r the p r o t e c t i o n of more c r i t i c a l t i s s u e s . Hultman and S a h l i n (1980) examining a c i d base balance d u r i n g e x e r c i s e c a l c u l a t e d an apparent B of 73.5 (mmol.pH .1 H^O) based on proton r e l e a s e c a l c u l a t e d from l a c t a t e p r o d u c t i o n and Table 5. Average non-bicarbonate b u f f e r values f o r s k e l e t a l muscle determined by homogenate t i t r a t i o n w i t h HC1 or NaOH (mmol.kg .1 HO I C ) . Reference Tissue B u f f e r Capacity Furusawa and Kerridge 1927 Bate-Smith 1938 Ecke l et a l . 1959 Davey 1960b Cat gastrocnemius s e v e r a l i n r i g o r s r a t mouse soleus p r e - r i g o r Larsen-and B u r n e l l 1978 C a s t e l l i n i and Somero 1981 t e r r e s t e r i a l mammals 43- 74-97 61 40 66 85 66 the change i n pH with e x e r c i s e . A s i m i l a r value of 68.5(S1) was e a r l i e r c a l c u l a t e d by S a h l i n (1978) f o r B by the v a r i o u s b u f f e r i n g c o n s t i t u e n t s d u r i n g dynamic e x e r c i s e when intra m u s c u l a r pH decreases from 7.0 to 6.4. .2. S k e l e t a l Muscle B u f f e r i n g C o n s t i t u e n t s . S i e s j o and Messeter (1971) have c l a s s i f i e d the major b u f f e r i n g c o n s t i t u e n t s i n t o three components: f i r s t , p h y s i c o - c h e m i c a l b u f f e r i n g ; second, consumption or p r o d u c t i o n of n o n - v o l a t i l e a c i d s and t h i r d , transmembrane f l u x e s of H and HCO a . B of i n v i t r o p r e p a r a t i o n s c o n s i s t s of simply the physi c o - c h e m i c a l b u f f e r i n g component which comprises the b u f f e r i n g w i t h i n a c e l l merely as a consequence of H * a s s o c i a t i o n with bases (Roos and Boron 1981). Burton (1978) suggested that the h i s t i d i n e r e l a t e d compounds and in o r g a n i c phosphate (P^) were the major b u f f e r i n g components of s k e l e t a l muscle. T i t r a t i o n of muscle homogenates p r o v i d e s a c l o s e d system whereby the HCO b u f f e r i n g mechanism can be ne g l e c t e d (Larsen and B u r n e l l 1978). S a h l i n (1978) suggested that HCO c o u l d c o n t r i b u t e as much as 15 to 18 percent of t o t a l B i n v i v o d u r i n g exhaustive e x e r c i s e . Davey (1960b) suggested that at l e a s t 90 percent of the B on d e p r o t e i n i z e d homogenates of pre- and post r i g o r s k e l e t a l muscle c o u l d be accounted f o r by ATP, i n o r g a n i c phosphate, c a r n o s i n e , a n s e r i n e and u n i d e n t i f i e d phosphate. ATP has an a p p r o p r i a t e pKa (7.0) but, occurs complex bound to Mg and p r o t e i n s , r e n d e r i n g i t s b u f f e r i n g power n e g l i g i b l e (Burton 1978; Hultman and S a h l i n 1980). ADP, demonstrating a pKa of 6.7, occurs i n too low c o n c e n t r a t i o n s to c o n t r i b u t e to b u f f e r i n g 67 (Dawson et' a l . , 1977; Burton 1978; Hultman and S a h l i n 1 980).. Burton ( 1978) suggested that P{ c o n t r i b u t e d r e l a t i v e l y . l i t t l e to b u f f e r i n g , u n t i l muscle c o n t r a c t i o n i s a c t i v a t e d and CP i s h y d r o l y z e d . Somero (1981) suggested that f r e e P{ was the second most important b u f f e r i n b i o l o g i c a l f l u i d s . I t appeared that P( was p o o r l y s u i t e d f o r s t a b i l i z i n g pH as i t s pk was i n s e n s i t i v e to temperature. Imidazole type b u f f e r s , e s p e c i a l l y a n s e r i n e , c a r n o s i n e and ophidine which occur predominantly i n h i g h l y g l y c o l y t i c t i s s u e , have been suggested to dominate b u f f e r i n g of b i o l o g i c a l f l u i d s due to t h e i r a b i l i t y to maintain t h e i r B with e l e v a t e d temperatures (Somero 1981). T h i s b u f f e r type c o n s i s t s of f r e e h i s t i d i n e , the h i s t i d i n e d i p e p t i d e s and p r o t e i n bound h i s t i d i n e r e s i d u e s . P r o t e i n s are recognized as a major b u f f e r w i t h i n s k e l e t a l muscle (Bate-Smith 1938; Woodbury 1965; S a h l i n 1978). Woodbury (1965) c a l c u l a t e d the b u f f e r . v a l u e of muscle p r o t e i n based on h i s t i d i n e content to be about 15 SI while Bate- Smith (1938) examining t i t r a t i o n s of muscle e x t r a c t s with and without p r o t e i n s found p r o t e i n c o n t r i b u t i o n v a l u e s of 17 to 37 SI which corresponds to approxiamently 40 to 50 percent of B. Hultman and S a h l i n (1980) c a l c u l a t e d p r o t e i n s to c o n t i b u t e up to 50 percent of the t o t a l p h y s i c o - c h e m i c a l b u f f e r i n g . The c o n t r i b u t i o n of the f r e e amino a c i d s t o B i n the p h y s i o l o g i c a l pH range i s l i m i t e d to those demonstrating a pKa f o r t h e i r i o n i z a b l e R groups i n that range (Hultman and S a h l i n 1980). Somero (1981) found the f r e e H i s t i d i n e c o n t r i b u t i o n to t o t a l B to be of minor importance, while S a h l i n (1976) c a l c u l a t e d the f r e e h i s t i d i n e c o n t r i b u t i o n to b u f f e r i n g to be only 0.1 SI. 68 Since amino a c i d s demonstrating these pK c h a r a c t e r i s t i c s occur i n such minor c o n c e n t r a t i o n s w i t h i n s k e l e t a l muscle, t h e i r r e l a t i v e c o n t r i b u t i o n t o t o t a l B i s n e g l i g i b l e . D i f f e r e n c e s i n b u f f e r c a p a c i t y a c r o s s f i s h s p e c i e s were a t t r i b u t e d to v a r i a t i o n s i n p r o t e i n content or f r e e h i s t i d i n e c o n c e n t r a t i o n s ( C a s t e l l i n i and Somero 1981). Within humans the c o n c e n t r a t i o n of f r e e h i s t i d i n e i s s u b s t a n t i a l l y l e s s than that of the h i s t i d i n e - c o n t a i n i n g d i p e p t i d e s (Bergstrom et a l . , 1974; Rennie et a l . , 1981).. Thus the p o s s i b i l i t y e x i s t s that w i t h i n human s k e l e t a l muscle, a l t e r a t i o n s i n content of the h i s t i d i n e - c o n t a i n i n g d i p e p t i d e s may account f o r the v a r i a n c e s observed i n b u f f e r c a p a c i t y . T h e r e f o r e the r e l a t i v e r o l e of . the d i p e p t i d e s c a r n o s i n e and ans e r i n e i n r e l a t i o n to b u f f e r c a p a c i t y of human s k e l e t a l muscle must be i n v e s t i g a t e d . 3. Carnosine and An s e r i n e . The d i p e p t i d e s a n s e r i n e (B- a l a n y l - N - m e t h y l h i s t i d i n e ) and c a r n o s i n e ( B - a l a n y l h i s t i d i n e ) are found i n the s k e l e t a l muscles of many s p e c i e s of animals (Crush et a l . , 1970; Christman 1976). Carnosine was f i r s t d i s c o v e r e d by G u l e v i c h i n 1900. The b i o s y n t h e s i s of a n s e r i n e and ca r n o s i n e i n ra t s k e l e t a l muscle has been demonstrated by Aonuma et a l . , (1969, 1970) to i n v o l v e the c o n v e r s i o n of a n s e r i n e to ca r n o s i n e preceded by s y n t h e s i s of a n s e r i n e from B-alanine and N- m e t h y l h i s t i d i n e . Carnosine and i t s methylated analogues p l a y some p h y s i o l o g i c a l r o l e i n the s p e c i a l i z e d t i s s u e s where they are found but, no u n i f i e d e x p l a n a t i o n of t h e i r r o l e e x i s t s . A nserine and ca r n o s i n e were demonstrated to a c t as b u f f e r s to n e u t r a l i z e the a c i d o s i s which o c c u r r e d d u r i n g anaerobic 69 g l y c o l y s i s (Shertsner 1958; Davey 1960a,b; Quershi and Wood 1962; Meshkova 1965). The pK c h a r a c t e r i s t i c s of both c a r n o s i n e (pk = 6.83) and a n s e r i n e (pk = 7.04) were i d e n t i f i e d by Bate- Smith (1938) and Eggleton and Eggleton (1938) who suggested that they were i d e a l l y s u i t e d f o r the r o l e of b u f f e r s i n ;the p h y s i o l o g i c a l pH range. B a t e _ S m i t h (1938) and Davey (1960a) suggested that as much as 40 percent of the t o t a l b u f f e r i n g of pre- and post r i g o r muscle c o u l d be a t t r i b u t e d to the a c t i o n of these d i p e p t i d e s . S e v e r i n (1963) found that f r o g muscle immersed i n a s o l u t i o n c o n t a i n i n g c a r n o s i n e c o u l d c o n t r a c t longer and with g r e a t e r amplitude. Meshkova (1965) c o u l d not account f o r the i n c r e a s e d g l y c o l y t i c a c t i v i t y demonstrated when ca r n o s i n e was added to the medium s o l e l y to i t s b u f f e r a c t i o n which suggests that c a r n o s i n e may augment g l y c o l y s i s by more than one mechanism. The d i p e p t i d e s were found to occur o t o g e n e t i c a l l y at the onset of muscle f u n c t i o n (Skvortsova 1953). In a s e r i e s of i n v e s t i g a t i o n s , S e v e r i n (1962, 1963, 1966) found the c o n c e n t r a t i o n of c a r n o s i n e to be g r e a t e s t at nerve endings and that the d i p e p t i d e i n c r e a s e d the work a b i l i t y of exhausted f r o g muscle. Bowen in 1965 demonstrated c a r n o s i n e and h i s t i d i n e to be powerful p o t e n t i a t o r s of ATP induced muscular c o n t r a c t i o n of r a b b i t psoas. The d i p e p t i d e s were l a t e r i d e n t i f i e d as myosin ATPase a c t i v a t o r s (Avena and Brown 1969; Parker and Ring 1970). Boldyrev i n a s e r i e s of i n v e s t i g a t i o n s (I97la,b, 1978; Lopina and Boldyrev 1974) suggested that the s p e c i f i c a c t i v a t i n g e f f e c t of c a r n o s i n e was on the sarcolemma Na* , k* - ATPase. Ikeda et a l . , 70 (1979) found the a c t i v i t y of f r u c t o s e 1,6 bisphosphatase to be s t i m u l a t e d by c a r n o s i n e and a n s e r i n e . T h i s enzyme i s i n v o l v e d i n the s u b s t r a t e c y c l e between f r u c t o s e 6 phosphate (F6P) and f r u c t o s e 1,6 diphosphate which i n v o l v e s continuous h y d r o l y s i s of ATP. T h i s c y c l e i s necessary i n muscle whose energy u t i l i z a t i o n v a r i e s widely i n order to augment the r a t e of F6P ph o s p h o r y l a t i o n and changes i n AMP c o n c e n t r a t i o n (Newsholme and S t a r t 1973). Brown (1981) found c a r n o s i n e and ans e r i n e to be l o c a t e d w i t h i n the s k e l e t a l muscle of r a t e x h i b i t i n g a c t i v e o x i d a t i v e metabolism and/or g l y c o l y s i s . Brown suggested that the d i p e p t i d e ' s r o l e may be i n t r a c e l l u l a r t r a n s p o r t of copper f o r a c t i v a t i o n of cytochrome oxidase at the end of the e l e c t r o n t r a n s p o r t c h a i n and i n r e g u l a t i o n of anaerobic g l y c o l y s i s . I t - was hypothesized that c a r n o s i n e c o u l d reverse the i n h i b i t i o n of g l y c o l y s i s w i t h i n s k e l e t a l muscle by c h e l a t i n g copper. Though many p h y s i o l o g i c a l r o l e s f o r the d i p e p t i d e s have been proposed and a s i n g l e f u n c t i o n i s u n l i k e l y , the only r o l e u n i v e r s a l l y accepted i s t h a t of a that of p h y s i o l o g i c a l b u f f e r (Boldyrev 1978). More recent i n v e s t i g a t i o n s on pH r e g u l a t i o n and B, have d i s c u s s e d the r e l a t i v e c o n t r i b u t i o n of the imidazole c o n t a i n i n g d i p e p t i d e s (Burton 1978; Hultman and S a h l i n 1980; Somero 1981). I t appears though that these d i p e p t i d e s have many f u n c t i o n a l r o l e s u l t i m a t e l y l i n k e d to r e g u l a t i o n of a e r o b i c and anaerobic metabolism. I t has been demonstrated t h a t c a r n o s i n e and a n s e r i n e l e v e l s w i t h i n many s p e c i e s are higher i n s k e l e t a l muscle denoted as 71 being white as opposed red under normal c o n d i t i o n s (Christman 1976; Tamaki et a l . , 1976), while i n humans a n s e r i n e l e v e l s appear to be e i t h e r n o n e x i s t a n t or i n s i g n i f i c a n t (Christman 1976). Tamaki et a l . , (1976) r e p o r t e d that d e n e r v a t i o n r e s u l t e d in decreased c a r n o s i n e l e v e l s and i n c r e a s e d c a r n o s i n a s e a c t i v i t y . Determinations made f o l l o w i n g an acute bout of swimming e x e r c i s e or e l e c t r i c a l s t i m u l a t i o n r e s u l t e d i n no s i g n i f i c a n t change i n r a t c a r n o s i n e l e v e l s (Eggleton and Eggleton 1933). Hunter (1924, 1925) demonstrated reduced c a r n o s i n e l e v e l s upon s t a r v a t i o n and e l e v a t e d l e v e l s upon a p r o t e i n d i e t i n r a t s . Christman (1976) r e p o r t e d an i n v e r s e r e l a t i o n s h i p between age and c a r n o s i n e l e v e l s i n human s k e l e t a l muscles, c a r n o s i n e l e v e l s d e c r e a s i n g as age i n c r e a s e d . Values i n mmol.l HjO IC ranged from 1.5 i n 60+ year o l d i n d i v i d u a l s (n=2) to 7.2 i n the 15 to 19 year o l d age range (n=5), f o r the l i m i t e d number of samples i n v e s t i g a t e d (Christman 1976). A mean i n t r a c e l l u l a r c a r n o s i n e value of 6.15 (mmol.l H^O) was r e p o r t e d by Bergstrom et a l . , (1978) f o r normal h e a l t h y a d u l t s . Carnosine occurs i n l a r g e c o n c e n t r a t i o n s w i t h i n human s k e l e t a l muscle and has been found to occur p r i m a r i l y w i t h i n s k e l e t a l muscle denoted as being white of a v a r i e t y of s p e c i e s . Thus due to i t s high c o n c e n t r a t i o n , optimal pK c h a r a c t e r i s t i c s and p o s s i b l e predominance w i t h i n f a s t g l y c o l y t i c f i b e r s , c a r n o s i n e may p l a y a s i g n i f i c a n t r o l e i n b u f f e r i n g the protons which accumulate d u r i n g i n t e n s e muscular e x e r c i s e . 72 P.. Summary. L a c t i c a c i d accumulation w i t h i n muscle and blood appears to be one of the f a c t o r s i n v o l v e d i n the complicated process of f a t i g u e as i t r e l a t e s to performance (Bagby et a l . , 1978, Klausen et a l . 1972, K a r l s s o n et a l . , 1975). Fast t w i t c h (FT) f i b e r s demonstrate the hig h e s t degree of f a t i g u a b i l i t y and have higher c o n c e n t r a t i o n s of the M-LDH isozyme (Sjoden 1976) suggesti n g t h a t f i b e r composition of s k e l e t a l muscle may be r e l a t e d to performance (Essen and Haggmark 1975; J o r d f e l t 1970). Ivy et a l . , (1980) found that both the p r o p o r t i o n of slow t w i t c h f i b e r s and the muscle r e s p i r a t o r y c a p a c i t y p l a y a r o l e i n de t e r m i n a t i o n of l a c t a t e t h r e s h o l d s . T r a i n e d s u b j e c t s demonstrate s u p e r i o r c a p a b i l i t i e s than u n t r a i n e d s u b j e c t s to t o l e r a t e h i g h blood l a c t a t e l e v e l s . I t has been suggested that i n t r a c e l l u l a r b u f f e r i n g c a p a c i t y may pl a y a r o l e i n the r e g u l a t i o n of i n t r a c e l l u l a r pH. During a high i n t e n s i t y , short d u r a t i o n workload, the B of t i s s u e may be of importance to reduce the accumulation of protons which would u l t i m a t e l y decrease pH r e s u l t i n g i n a decrement i n performance. The major b u f f e r i n g components w i t h i n human s k e l e t a l muscle, i f comparable to animal t i s s u e , are i n o r g a n i c phosphate, p r o t e i n bound h i s t i d i n e r e s i d u e s and the d i p e p t i d e c a r n o s i n e . Carnosine l e v e l s w i t h i n animal t i s s u e s appear to be h i g h l y r e l a t e d to the g l y c o l y t i c c a p a c i t y of s k e l e t a l muscle and may play a r o l e i n anaerobic performance. B u f f e r i n g i_n v i t r o c o n s i s t s merely of proton a s s o c i a t i o n with bases. There appears t o be a s p e c i f i c i n t r a m u s c u l a r pH at which f a t i g u e f o r c e d c e s s a t i o n of the e x e r c i s e ( S a h l i n et a l . , 1975, 1976; Hermansen and Osnes 1972). 73 An e l e v a t e d r e s t i n g pH may prolong one's a b i l i t y to perform by i n c r e a s i n g the amount of protons which must accumulate before the c r i t i c a l pH l e v e l i s a t t a i n e d . Thus the c a p a c i t y of s k e l e t a l muscle to b u f f e r the r e s u l t a n t pH decrement, a s s o c i a t e d with the proton accumulation which accompanies high i n t e n s i t y , short d u r a t i o n work, may enhance anaerobic performance, p o s s i b l y through a l t e r a t i o n s i n b u f f e r c a p a c i t y and c a r n o s i n e with t r a i n i n g s p e c i f i c i t y . Appendix A. Repeated buffer capacity determinations (umol.g .pi I ). Buffer Capacity Group • Subject T i t r a t i o n 1 T i t r a t i o n 2 WF 31.98 32.29 IG 24.27 22.67 Sprinters KB 32.30 31.98 BS 39.64 35.61 SH 25.67 GB 32.68 31 .93 K\7 26.95 Rowers GS 26.91 27.32 AH 28.90 29.41 SB . 45.45 42.86 BP 1 5.84 lr>.72 UJ) 17.72 1 7.02 Marathoners JC 25.30 22.88 i)S - 20.92 20.43 NW 27. 2'J 25.25 BA 17.20 RW .14.56 15.18 Untrained RW 24.60 2-3.00 BF 26.46 27.14 KM 23.02 23.26 R e l i a b i l i t y r=0.99 Appendix B. Blood lactate levels^pre- and post-anaerobic performance (mmol.l ). • Blood Laci;;ilc Concentration Group Subject Pre-AST Post-AST WF 1.4 22.7 IG . 1.1 22.8 Sprinters KB r.4 22.6 BS 0.8 22.3 SH N 0.9 19.3 GB 1.4 L3.3 KW 0.9 13.9 Rowers GS 1.0 14.1 AH 0.8 13.0 SB 1.0 15.2 BP •0.8 11.1 BB 1.3 10.8 Marathoners JC 0.9 7.2 DS 1.3 6.9 NW 0.9 14.5 BA 0.6 N 9.0 RW 0.7 6.5 Untrained RW 0.9 13.6 BF 1.0 10.8 KM 0.6 10.4 Appendix C. Bu f f e r capac i t y convers ions 1 mmol.pH !"Kg 1.29 mmol.pH ! l * t i s s u e water 1.29 m m o l . p H - . I - 1 t i s s u e water = 1.548 mmol.pH -^1 1 IC H 20 Group umoles.pH lg * mmol.pH } l * IC Î O Sp r i n t e r s 3 0 . 0 3 + 5 . 6 4 6 . 4 9 + 8 . 7 Rowers 31.74 + 7.2 49.13 + 11.2 Marathoners 2 0 . 8 3 + 4 . 4 3 2 . 2 5 + 6 . 8 Unt ra ined 2 1 . 2 5 + 5 . 0 3 2 . 9 0 + 7 . 7 77 Appendix D. S e r i a l s e c t i o n s of UT vastus l a t e r a l i s muscle s t a i n e d f o r Myosin ATPase and NADH-TR Fi b e r TyP e S t a i n i n g I n t e n s i t y a NADH-TR Type I dark Type 1.1 l i g h t b Myosin ATPase . Type I l i g h t pll 9.4 Type TI dark c Myosin ATPase Type T. ilnrk pll 4.6 Tpye T I A l i g h t Type TI B moderate d Myosin ATPase Type I dark pH 4.3 Tpye I I l i g h t 78 Appendix E. Regression analyses Buffer capacity (umol.gw/w.pĤ ) versus anaerobic performance. 50-r r=0.51 p=0.0217 y=0.115 AST + 17.87 40- 30 + 20- 10 M M U M R R . R M 25 50 — r — 75 AST (sec) 100 125 150 CO o B u f f e r c a p a c i t y (umol.gw/w.pH ) versus carnosine c o n c e n t r a t i o n . B u f f e r c a p a c i t y (uraol.gw/w.pH ) versus f a s t - t w i t c h f i b e r percentage. F a s t - t w i t c h f i b e r percentage oo tsj Carnosine concentration

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