Europ. J. appl. Physiol. 34, ~69--172 C1975) 9 by Springer-Verlag ~1975

Ammonia Production Following Maximal Exercise: Treadmill vs. Bicycle Testing* J. E. W i l k e r s o n , D. L. B a t t e r t o n , a n d S. M. H o r v a t h Institute of Environmental Stress, University of California, Santa Barbara, California 93106 Received ~areh 10, ~975

Abstract. From a population of 20 healthy male volun~ers, half performed constant speed, incremental toad maximal aerobic capacity (!?o2,~x) tests on a motor-driven treadmill, while the other half performed similar ]'{)2..... tests on a bicycle ergometer. The two groups, matched for size and age, showed no significant differences in Vo~m~x,maximum heart rate, or in postexercise (4 rain) peripheral venous blood concentrations of lactate or pyruvate. However, postexercise peripheral venous blood ammonia levels were significantly higher in tile group tested on the bicycle ergometer than in the treadmill group. Key words:

goamax --

Maximum Aerobic Capacity Tests -- Lactate -- Pyruvate -- Venous

Blood Ammonia.

Introduction Blood a m m o n i a c o n c e n t r a t i o n h a s b e e n shown to increase e x p o n e n t i a l l y with increasing w o r k l o a d (oxygen consumption) in h u m a n s p e r f o r m i n g t r e a d m i l l exercise 1. H o w e v e r , r e p o r t s concerning t h e m a g n i t u d e a n d n a t u r e of t h e changes in blood a m m o n i a levels following different t y p e s of exercise h a v e been inconsistent (Luck et al., t925; E m b d c n et al., t928; P u m a s , 1929; A l l e n a n d Corm, t960; Lowenstein, 1972). Allen a n d Corm (t960), utilizing a r m exercise, f o u n d increasing levels of a m m o n i a in b o t h a r t e r i a l a n d v e n o u s blood w i t h increasing w o r k load. A l t h o u g h r e p o r t i n g similar findings, P a r n a s a n d coworkers (t927) f o u n d large v a r i a b i l i t y in t h e i r p o p u l a t i o n . L u c k et al. (t925) d i d n o t find a n y increase in b l o o d a m m o n i a levels w i t h " l m e e s - u p " or " s t a i r r u n n i n g " exercise. L o w e n s t e i n (t972), in reviewing this area, r e p o r t e d similar results. I n o r d e r to m o r e e x a c t l y d e l i m i t t h e possible differences in t h e p r o d u c t i o n of a m m o n i a in response to v a r i o u s t y p e s of exercise, a n d to gain insight into t h e physiological role of a m m o n i a in t h e exercising h u m a n , t h i s s t u d y was u n d e r t a k e n .

Methods 20 adult male volunteers were assigned to two equally numbered groups in order to obtain groups matched as to physical size and age. The subjects' pertinent physical characteristics are presented in Table 1. The individuals in one group each performed a constant speed, incremental slope maximal aerobic capacity (17o2~) test on a motor-driven treadmill (Drinkwater and Horvath, 1971). Those assigned t.o the second group each performed a constant pedalling speed, incremental load 17o2.... test on a bicycle ergometer (Michael and Horvath, 1965). Measurements of heart rate and oxygen uptake were made as previously observed 2. * This work was supported in part by the Air Force Office of Scientific Research, Air Force Systems Command, Grant AFOSB 73-2455. Manuscript pending publication (Wilkerson, J. E., Batterton, D. L., Horvath, S. M.: Exercise-induced changes in blood ammonia levels in humans).

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Table I. Physical characteristics of the subjects. Values are means ( • SEM) and ranges (n = t0 for both groups) Age Bicycle testing Treadmill testing

Mean(+_SEM) ]%ange ~[ean ( _+ SEI~) Range

t scores

(yr)

Ht (cm)

27(+_4) 20--54 32 ( _+ 8) 21--44 --1.114

179(+_2) 83.15(• t69--t89 73.27--98.59 t77 ( + 2) 78.25 ( • 2.33) t65--183 72.92--96.95 0.819 1.491

DuBois body surface area.

Wt (kg)

BSA (m2)9 2.02(_+0.03) t.87--2.20 1.94 ( • 0.02) 1.82--2.06 2.139 b

0.025 < P < 0.01.

Blood samples were drawn without stasis from an antecubital vein during the last minute of a 5-min seated rest period preceding each exercise bout, and at the 4th rain of a seated, resting recovery period following the maximal exercise tests. Blood samples were analyzed for hemoglobin (cyanmethemoglobin technique), hematocrit (microhematoerit: uncorrected for trapped plasma), total plasma protein (plasma refractive index), lactate (enzymatic), pyruvate (enzymatic), and ammonia (Hyland Laboratories phenate hypoehlorite method). Ammonia assays were always initiated in duplicate within 1/~ hr of the drawing of the blood samples. The enzymatic analyses were performed with reagent kits purchased from the Boehringer Mannhelm Corporation. Protein-free extracts to be used in these enzymatic assays were prepared within 5 rain of drawing of a blood sample. The data obtained was analyzed for differences between bicycle and treadmill experimental treatment groups using a Student's t-test (Winer, 1971). Where pre-exercise resting control values were found to be different, an omega-squared (~oe) test was performed to determine the extent of the variance in the post-exercise samples accounted for by the pre-values (Winer, 197t). Analyses were performed on both raw data and difference scores (post-value minus prevalue) for all parameters. Correlations reported are least-squares linear regression equations and Pearson product-moment correlation coefficients (Wiaer, 1971).

Results T h e resting v a l u e s o b s e r v e d for all p a r a m e t e r s in all s u b j e c t s were w i t h i n t h e e x p e c t e d r a n g e for m a l e s u b j e c t s of t h i s age d i s t r i b u t i o n ( A l t m a n a n d D i t t m e r , 1971). T h e r e were no significant differences b e t w e e n t h e t w o groups for a n y preexercise c o n t r o l v a l u e s e x c e p t for h e a r t r a t e ( t - - - - 2 A 0 , P < 0.05). P o s t - e x e r c i s e p e r i p h e r a l v e n o u s b l o o d a m m o n i a levels were significantly g r e a t e r t h a n preexercise m e a s u r e m e n t s ( t = - - 2 . 3 7 , P < 0.02). T h e difference v a l u e s (postexercise m i n u s pre-exercise) for a m m o n i a (t = - - 2.78, P < 0.02) a n d h e a r t r a t e (t = - - 3 . 0 7 , P < 0.01) were f o u n d to be significantly different b e t w e e n t r e a t m e n t groups. T a b l e 2 p r e s e n t s t h e m e a n ( • SEM) d a t a for all p a r a m e t e r s m e a s u r e d for b o t h g r o u p s for pre-exercise a n d p o s t - e x e r c i s e (or m a x i m a l ) m e a s u r e m e n t periods. N o significant correlations were o b s e r v e d for e i t h e r t r e a d m i l l or bicycle e r g o m e t e r t e s t i n g in t h i s s t u d y b e t w e e n p e r i p h e r a l v e n o u s b l o o d levels of a m m o n i a a n d o t h e r b l o o d m e t a b o l i t e s , or b e t w e e n a m m o n i a levels a n d l~o~maxor m a x i m u m h e a r t r a t e . T h e r e was a significant c o r r e l a t i o n b e t w e e n t h e difference scores for h e a r t r a t e a n d b l o o d a m m o n i a c o n c e n t r a t i o n s ( r = - - 0 . 5 1 5 , P < 0.02) for all s u b j e c t s (n = 20). See footnote l on p. 169.

Ammonia Production and Maximal Exercise

171

Table 2. Mean ( + SEM) blood metabolite levels, oxygen uptakes (Vow)and heart rates before and after incremental maximal aerobic capacity tests. Post-exercise heart rate and l?o2 are maximal values observed during the exercise. Post-exercise blood samples were drawn at the 4th min of a seated, resting recovery period following the maximal exercise tests Treadmill 1?o2 (1/min) Heart rate (beats/min) Hemoglobin (g %) Hematocrit (%) Plasma proteins (g %) Ammonia (~zg%) Lactate (raM) Pyruvate (~zM)

Bicycle

Pre 0.357, + 0.045) 0.425 ( • 0.043) Post 3:681 _+0.254) 3.624 ( _+0.229) 77 (_+4)~ Pre 7t _+3)~ 181 ( _+3) Post 186 _+ 3) t5.5 ( _+0.2) Pre t5.7 _+0.3) Post 17.0 + 0.2) 16.7 ( _+0.3) 4s.5 ( _+ 0.7) Ire 48.4 _+ 0.7) 52.3 ( • o,s) Post 52.2 _+ 1.0) ])re 7.3 + OA) 7.2 ( _+0.1) Post 8A +_ o.t) s.2 ( _+ o.t) 64 ( _+ 6) Pre 62 • 4) t54 ( _+14)b Post tt2 _+12)b Pre 1.24 _+ 0.25) t.23 ( • 0.15) Post 11,00 + 1.31) ~t.t7 ( + 0.60) 95 ( + l i ) Pre 78 _+ 9) 222 ( _+28) Post 264 _+24)

"Pre" bicycle value significantly different from "pre" treadmill value (P < 0.05). b "Post" bicycle value significantly different from "post" treadmill value (P < 0.05).

Discussion E v e n t h o u g h m a x i m u m oxygen uptakes and m a x i m u m heart rates for the two groups were not statistically different, peripheral venous blood a m m o n i a concentrations were significantly higher following m a x i m a l bicycle exercise t h a n after m a x i m a l treadmill work. All individuals in the bicycle testing group h a d post-exercise peripheral venous blood a m m o n i a values equal to or greater t h a n the m e a n observed for the treadmill group ( t t 2 fag%). E v e n when the effect of different degrees of h e m o c o n c e n t r a t i o n was statistically partialled out (through the expression of a m m o n i a levels as fag a m m o n i a per g hemoglobin), the groups were still different. This is in contrast to our earlier findings t h a t would suggest t h a t groups having equal FO2max (treadmill work) would exhibit similar blood a m m o n i a values a. The lack of significant correlation of blood a m m o n i a concentration with other blood metabolite (lactate and p y r u v a t e ) levels, or with ~2max or m a x i m u m heart rate, is in agreement with our earlier report a, in which no correlation was seen between m a x i m u m heart rate or m a x i m u m o x y g e n consumption and the blood levels of ammonia, lactate, or p y r u v a t e determined at the 4th rain of recovery following m a x i m u m treadmill exercise. Thus, in contrast to the situation observed with blood lactate (or pyruvate), the use of the 4-rain post-exercise blood level of a m m o n i a as an index of a t t a i n m e n t of l?O~max does n o t appear to be warranted. The explanation for this p r o b a b l y lies in the widely divergent rates of diffusion of these energy substrates from active muscles into the blood. The differences observed in the present s t u d y in blood a m m o n i a levels following treadmill and bicycle ergometer exercise, therefore, m a y be a reflection of the test procedures 3 See footnote I on p. 169.

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J.E. Wilkerson et al.

themselves, or of differences in the leg blood flow following these two types of exercise. The bicycle ergometer maximal tests were of shorter duration (approximately i2 rain) than were the treadmill bouts (approximately 22 rain). Thus, the onset of anaerobiosis would be expected to occur sooner, and to be possibly more severe with the bicycle protocols. This would account for the higher blood ammonia values observed following maximal bicycle exercise tha~ following similar treadmill bouts if ammoma produetior~ and liberation is via art "overload" mechanism coupled to anaerobic metabolic pathways 4. However, the lack of correlation between blood ammonia levels and either lactate or pyruvate argues against this hypothesis. Similarly, since the groups did not differ in post-maximal exercise values for pyruvate or lactate, this explanation must be questioned. Thus, our earlier findings that blood ammonia levels are highly correlated with 1)o2, lactate, and pyruvate m a y have been a function of the type of exercise employed and not indicative of the metabolic pathway interrelationships. A logical explanation for these differences in blood ammonia levels may be that splar~ctmie amd/or hepatic blood flow was different during the contrasting types of maximal exercise. These could be differences in either total flow to the splanchnic bed or in the distribution of flow within that bed. I f either (or both) occur, then hepatic ammonia clearance via the urea cycle would be reduced, resulting in higher observed blood ammonia values. Thus, one would predict that hepatic blood flow would be significantly less during and following maximal bicycle ergometer work than during similar tests on a treadmill. However, further experimentation is required to furnish the data necessary to provide a more concrete evaluation of the physiological role of ammonia in the exercising human. Acknowledgements. The authors thank Dr. J. A. Gliner and Dr. B. L. Drinkwater from the Institute staff for their assistance in the statistical analyses of the data.

References Allen, S. I., Corm, It. O. : Observations on the effect of exercise on blood ammonia concentration in man. Yale J. Biol. Hed. 88, 133--144 (1960) Altman, P. L., Dittmer, D. S. (Eds) : Blood and other body fluids. Bethesda, Md. : Fed. Amer. Soc. Exp. Biol. t971 Drinkwater, B. L., Horvath, S. M. : Responses of young female track athletes to exercise. Med. Sci. Sports 8, 56--62 (1971) Embden, G., Wassermeyer, tI. : ~ber die Bedeutung der Adenylsiiure fiir die Muskelfunktion. 3. Bus Verhalten der Ammoniakbildung bei der Huskelarbeit unter verschiedcnen biologischen Bedingungen. Z. physiol. Chem. 179, 161--185 (1928) Lowenstein, J. M. : Ammonia production in muscle and other tissues: the purine nucleotide cycle. Physiol. Rev. 52, 382--4t3 (1972) Luck, J. H,, Thacker, G., Marrack, J.: Ammonia in the blood of epileptics. Brit. J. exp. Path. 6,276--279 (1925) Michael, E. D., Jr., tIorvath, S. M. : Physical work capacity of college women. J. ~ppl. Physiol. 20, 263--266 (1965) Parnas, J. K.: Ammonia formation in muscle and its source. Amer. J. Physiol. 90, 467 (1929) Parnas, J. K., Mozalowski, W., Lewinski, W.: ~ber den Ammoniakgehalt und die Ammoniakbildung im Blur. IX. Der Zusammenhang des Blutammoniaks mit der Huskelarbeit. Biochem. Z. 188, 15--23 (1927) Winer, B. J. : Statistical principles in experimental design, 2nd ed. New York: McGraw-Hill 1971 Prof. Dr. Steven l~. Horvath, Institute of Environmental Stress, University of California, Santa Barbar~, Caliornia 93106, USA 4 See footnote I on p. t69