Skeletal muscle metabolism is unaffected by DCA infusion and hyperoxia after onset of intense aerobic exercise

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1 Am J Physiol Endocrinol Metab 283: E108 E115, 2002; /ajpendo Skeletal muscle metabolism is unaffected by DCA infusion and hyperoxia after onset of intense aerobic exercise INGRID SAVASI, 1 MELISSA K. EVANS, 1 GEORGE J. F. HEIGENHAUSER, 2 AND LAWRENCE L. SPRIET 1 1 Department of Human Biology & Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; and 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Received 26 July 2001; accepted in final form 12 February 2002 Savasi, Ingrid, Melissa K. Evans, George J. F. Heigenhauser, and Lawrence L. Spriet. Skeletal muscle metabolism is unaffected by DCA infusion and hyperoxia after the onset of intense aerobic exercise. Am J Physiol Endocrinol Metab 283: E108 E115, 2002; /ajpendo This study investigated whether hyperoxic breathing (100% O 2 ) or increasing oxidative substrate supply [dichloroacetate (DCA) infusion] would increase oxidative phosphorylation and reduce the reliance on substrate phosphorylation at the onset of high-intensity aerobic exercise. Eight male subjects cycled at 90% maximal O 2 uptake (V O 2 max ) for 90 s in three randomized conditions: 1) normoxic breathing and saline infusion over 1 h immediately before exercise (CON), 2) normoxic breathing and saline infusion with DCA (100 mg/kg body wt), and 3) hyperoxic breathing for 20 min at rest and during exercise and saline infusion (HYP). Muscle biopsies from the vastus lateralis were sampled at rest and after 30 and 90 s of exercise. DCA infusion increased pyruvate dehydrogenase (PDH) activation above CON and HYP ( , , mmol kg wet muscle 1 min 1, respectively) and significantly increased both acetyl-coa and acetylcarnitine ( , , mmol/kg dry muscle, respectively) at rest. However, DCA and HYP did not alter phosphocreatine degradation and lactate accumulation and, therefore, the reliance on substrate phosphorylation during 30 s (CON, ; DCA, ; HYP, mmol ATP/kg dry muscle) and 90 s of exercise (CON, ; DCA, ; HYP, mmol ATP/kg dry muscle). These data suggest that the rate of oxidative phosphorylation at the onset of exercise at 90% V O 2 max is not limited by oxygen availability to the active muscle or by substrate availability (metabolic inertia) at the level of PDH in aerobically trained subjects. pyruvate dehydrogenase activity; oxidative phosphorylation; substrate phosphorylation; 100% oxygen; dichloroacetate; acetylcarnitine THERE IS STILL CONSIDERABLE DEBATE over what limits the rate of oxidative phosphorylation during the transition from rest to exercise. The initial rate of ATP hydrolysis exceeds the rate of ATP production from oxidative phosphorylation (2, 20, 33). This transient shortfall in Address for reprint requests and other correspondence: L. L. Spriet, Dept. of Human Biology and Nutritional Sciences, Univ. of Guelph, Guelph, ON, N1G 2W1 Canada (lspriet@uoguelph.ca). oxidative ATP supply, often termed the O 2 deficit, is provided by substrate phosphorylation. Glycolytic ATP production with lactate formation and the breakdown of phosphocreatine (PCr) are the major sources of substrate phosphorylation. Two explanations for the apparent lag in aerobic energy production at the onset of exercise have been proposed (for review, see Ref. 37). The first suggests that a metabolic inertia, including lags in enzyme activation or substrate availability, requires a certain amount of time to produce the reducing equivalents needed to drive the electron transport chain. The second theory suggests that a suboptimal oxygen supply limits the production of ATP in the mitochondria of some muscle fibers. The possibility that metabolic inertia may exist at the level of pyruvate dehydrogenase (PDH) has been investigated by examining the effects of dichloroacetate (DCA) infusion on skeletal muscle metabolism during exercise (12, 18, 19, 26, 35, 36). DCA infusion at rest causes an inhibition of PDH kinase and increased transformation of PDH to its nonphosphorylated, active form (PDHa) (39). This elevated PDH activation increases the provision of acetyl-coa during the onset of low- and moderate-intensity exercise and decreases the reliance on substrate level ATP production, implying an increased rate of oxidative phosphorylation (18, 26, 35, 36). Therefore, these studies concluded that metabolic inertia limits the rate of oxidative phosphorylation during the onset of light- and moderate-intensity exercise (19, 35). However, the effects of DCA infusion on human muscle metabolism during high-intensity aerobic exercise have not been investigated and may be of interest for two reasons. First, the requirement for substrate phosphorylation is greater after the onset of exercise at higher intensities [i.e., 90 vs. 65% maximal O 2 uptake (V O 2 max )] (20), suggesting that DCA may have a greater effect at intense power ouputs. Second, DCA caused only a 35% reduction in substrate phosphorylation after the onset of exercise at 65% V O 2 max, suggesting that other factors, such as oxygen availability, The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. E /02 $5.00 Copyright 2002 the American Physiological Society

2 E109 may be limiting the rate of oxidative phosphorylation in some muscle fibers. This effect may be exacerbated at high power outputs, as hyperoxia has been shown to accelerate V O 2 kinetics at the onset of exercise above the ventilatory threshold ( 78 82% V O 2 max ) while having no effect at moderate (below ventilatory threshold) power outputs (24, 25). Most investigations examining the existence of an O 2 limitation at the onset of intense exercise have relied on estimations of energy demand and whole body V O 2 kinetics measured at the mouth, an indirect estimation of muscle O 2 phosphorylation. By use of another approach that directly measured muscle substrate phosphorylation, hyperoxia decreased PCr use and lactate accumulation after 4 5 min of exercise (22, 23). However, similar measurments were not made during the onset of intense aerobic exercise in these studies. This study was designed to investigate whether activating PDH before exercise (DCA infusion) or breathing 100% oxygen would increase oxidative phosphorylation more rapidly during the onset of exercise at 90% V O 2 max, as indicated by a decrease in skeletal muscle substrate phosphorylation. We hypothesized that both DCA infusion and hyperoxia would reduce substrate phosphorylation (O 2 deficit) at the onset of intense aerobic exercise. In both cases, we directly measured muscle substrate phosphorylation (PCr degradation and lactate accumulation) and assumed a reciprocal relationship with oxidative phosphorylation. METHODS Subjects. Eight healthy, aerobically trained males, who regularly engaged in at least three aerobic training sessions per week (mainly running and cycling exercise), volunteered to participate in this study. Their mean ( SE) age, height, weight, and V O 2 max were yr, cm, kg, and ml kg 1 min 1, respectively. Before participating, subjects completed health histories, the experimental procedures and potential risks of the study were explained, and written informed consent was received. The study was approved by the human ethics committees of the University of Guelph and McMaster University. DCA. DCA (monosodium salt) was obtained from TCI America (Portland, OR). It was prepared under sterile conditions at a concentration of 1 mg/ml (ph 7.0), and the concentration and purity were verified by HPLC. It was delivered intravenously to subjects in the dose of 100 mg/kg body wt using 500 ml of normal saline solution over the course of 1 h immediately before exercise. Preexperimental protocol. Subjects underwent a continuous incremental exercise test on an electonically braked cycle ergometer (Excalibur, manufactured by Lode and distributed by Quinton Instruments, Seattle, WA) to determine their V O 2 max using a metabolic cart (SensorMedics model 2900, Yorba Linda, CA). From these values, the power output required to elicit 90% V O 2 max was calculated (273 5 W). To familiarize the subjects with the experimental protocol, they reported to the laboratory on a separate day, rested for 20 min while breathing through a headgear-supported mouthpiece, and cycled with the headgear for 5 min to confirm the 90% V O 2 max power output. Subjects were instructed to refrain from strenuous physical activity on the day before and the day of the experiments and to consume the same diet (no alcohol) before each trial. Experimental protocol. On 3 experimental days, each separated by 1 wk, subjects arrived at the laboratory having consumed the same meal 2 4 h before the the start of the study. The three experimental conditions were 1) breathing room air with a saline infusion (control trial, CON), 2) breathing room air with a DCA infusion (DCA), and 3) breathing 100% oxygen with a saline infusion (hyperoxia trial, HYP). The order of the trials was randomized, and the subjects were blind to the treatments. On each experimental day, a catheter was inserted into the antecubital vein 1 h before exercise, and a 500-ml infusion of saline or DCA with saline was started while the subjects rested on a bed (Fig. 1). During this hour, subjects had one leg prepared for needle biopsies, with three incisions made through the skin superficial to the vastus lateralis muscle under local anesthesia (2% lidocaine without epinephrine) as previously described (4). Beginning 20 min before the start of exercise and lasting until the end of exercise, subjects inspired either room air or hyperoxic (100% O 2) gas. Immediately before exercise, the infusion was stopped, the venous catheter was removed, and a resting muscle biopsy was taken. The subject then moved to the cycle ergometer and began pedaling at 90% V O 2 max, leading with the biopsied leg from an initial 90 angle to the ground. Exercise biopsies were taken at 30 and 90 s while the subject remained on the cycle ergometer. The stop time to take the 30-s biopsy was fixed at 30 s in all trials. Muscle biopsies were immediately frozen in the needle in liquid N 2, removed, and stored in liquid N 2 until analysis. Analyses. A small piece of frozen muscle (8 15 mg) was removed under liquid N 2 for the determination of PDH activity in its active form (PDHa), as described by Putman et al. (29). Total creatine (Cr) contents were measured for each muscle homogenate, and PDHa values were corrected to the Fig. 1. Schematic diagram of experimental protocol. DCA, dichloroacetate; V O 2max, maximal O 2 uptake.

3 E110 highest total Cr value in the nine biopsies from a given subject. The remainder of the biopsy sample was freezedried, dissected of all visible blood, connective tissue, and fat, powdered, and stored at 80 C for subsequent analysis. One aliquot of freeze-dried muscle (8 10 mg) was extracted with 0.5 M HClO 4 and 1 mm EDTA, neutralized with 2.2 M KHCO 3, and used for determination of Cr, PCr, ATP, glycerol 3-phosphate (G-3-P), glucose 6-phosphate (G-6-P), and lactate by spectrophotometric assays (3, 17). Pyruvate was measured using enzymatic methods modified for fluorometry (27), and acetyl-coa and acetylcarnitine were determined by radiometric assays (6). Muscle glycogen content was determined on a second aliquot of freeze-dried muscle (17). All muscle metabolites were corrected for the highest total Cr content measured in the nine biopsies from a given subject. Calculations. Free ADP and AMP concentrations were calculated assuming equilibrium of the creatine kinase and adenylate kinase reactions (8). Free ADP was calculated using the measured ATP, PCr, and Cr contents, a H concentration estimated from the muscle lactate content (31), and the creatine kinase equilibrium constant of Free AMP was calculated from the measured ATP, the estimated free ADP, and the adenylate kinase equilibrium of Free inorganic phosphate (P i) was calculated by adding the estimated resting P i of 10.8 mmol/kg dry muscle (8) to the difference in PCr ( PCr) minus the accumulation of glycolytic intermediates G-6-P ( G-6-P) and G-3-P ( G-3-P) between rest and each exercise time point. Substrate phosphorylation (mmol ATP/kg dry muscle) was determined for each treatment during 0 30 and s by adding the PCr degradation and 1.5 times the lactate accumulation (34). Statistics. All data are presented as means SE. For metabolite contents, a 2-way ANOVA (time trial) with repeated measures was used to test for significance. For glycogen content and substrate phosphorylation, a one-way ANOVA with repeated measures was used. Significance was set at P 0.05, and Tukey s post hoc test identified where significant differences occurred. RESULTS PDH activation. In the CON trial, PDHa increased dramatically from rest during the initial 30 s of exercise at 90% V O 2 max and to a lesser extent during the 30- to 90-s period (Fig. 2). Resting and exercise PDHa values were unaffected by breathing 100% oxygen. However, DCA infusion markedly increased resting PDHa activity in the hour preceding exercise compared with the CON and HYP trials, as expected (Fig. 2). In the DCA trial, PDHa remained higher than the CON and HYP trials at 30 s. In the final 60 s of exercise, PDHa decreased significantly from the resting value in the DCA trial, such that it was not different from CON and HYP at 90 s (Fig. 2). Muscle metabolites. There were no significant differences in ATP content among the trials or during exercise (Table 1). PCr decreased significantly from mmol/kg dry muscle at rest to mmol/kg dry muscle at 30 s and 40 mmol/kg dry muscle at 90 s of cycling at 90% V O 2 max in all three trials (Fig. 3). Free ADP, AMP, and P i accumulations were not significantly different among trials at any time (Table 1). Free ADP content increased significantly by 30 s of exercise and continued to increase at 90 s in all trials. Fig. 2. Pyruvate dehydrogenase (PDH) activation during control, DCA infusion, and hyperoxia trials at the onset of cycling at 90% V O 2max. *Significantly different from rest for the same trial; significantly different from 30 s for the same trial; significantly different from control and hyperoxia for the same time point. Free AMP increased during exercise and was significantly higher by 90 s in all trials. The 30-s free P i values were markedly higher than the arbitrary resting value in all trials and accumulated further in the CON and DCA trials at 90 s (Table 1). Resting muscle glycogen content was similar among trials (CON, ; DCA, ; HYP, ). Glycogen was not measured during exercise, because the expected use during 90 s of cycling was small. Resting G-6-P, G-3-P, pyruvate, and lactate contents were not significantly different among trials (Table 1; Fig. 4). G-3-P and G-6-P contents increased during exercise and reached significantly higher levels than at rest by 90 s in all three trials (Table 1). Muscle pyruvate was unchanged at 30 s of exercise in all trials and increased at 90 s in the DCA trial (Table 1). Lactate levels increased significantly at all time points during exercise, but there were no differences among trials (Fig. 4). DCA infusion markedly increased muscle acetyl-coa and acetylcarnitine contents at rest and throughout exercise compared with the CON and HYP trials (Fig. 5). The acetyl-coa content increased during cycling in the CON and HYP trials, reaching significance at 90 s. During exercise in the DCA trial, there was no significant change in acetyl-coa, such that the 30- and 90-s values were higher than in the CON and HYP trials (Fig. 5). The acetylcarnitine content increased after 90 s of cycling in all three trials, reaching values significantly higher than at rest. Substrate phosphorylation. There was no significant difference in the calculated substrate phosphorylation during the 0- to 30-, 30- to 90-, and 0- to 90-s exercise periods between the trials (Table 2). DISCUSSION At the onset of submaximal exercise, a transient mismatch between the required ATP and oxidative

4 E111 Table 1. Muscle metabolite contents at rest and during the onset of cycling at 90% V O 2max CON, DCA infusion, and HYP trials Time, s CON ATP DCA HYP CON * * G-3-P DCA * * HYP * CON * G-6-P DCA * HYP * CON Pyruvate DCA * HYP CON * * Free ADP DCA * * HYP * * CON * Free AMP DCA * HYP * CON * * Free P i DCA * * HYP * * All values are means SE in mmol/kg dry muscle, except free ADP and free AMP, which are in mol/kg dry muscle. G-3-P, glycerol 3-phosphate; G-6-P, glucose 6-phosphate; P i, inorganic phosphate; V O 2max, maximal oxygen uptake; CON, control; DCA, dichloroacetate; HYP, hyperoxia. * Significantly different from rest for the same trial. Significantly different from 30 s for the same trial. Significantly different from CON and HYP for the same time point. Significantly different from CON for the same time point. Fig. 3. Phosphocreatine degradation during control, DCA infusion, and hyperoxia trials at the onset of cycling at 90% V O 2max. *Significantly different from rest for the same trial; significantly different from 30 s for the same trial. Fig. 4. Intramuscular lactate accumulation during control, DCA infusion, and hyperoxia trials at the onset of cycling at 90% V O 2max. * Significantly different from rest for the same trial; significantly different from 30 s for the same trial. phosphorylation necessitates that skeletal muscle rely on substrate phosphorylation to supplement oxidative ATP synthesis. It has been proposed that either a metabolic limitation, including lags in enzyme activation or substrate availability, and/or an oxygen availability limitation explains why oxidative phosphorylation does not turn on quicker at the onset of aerobic exercise. To investigate these possibilities, this study examined the response of human skeletal muscle metabolism to both enhanced oxidative substrate supply (DCA infusion) and O 2 delivery (hyperoxia) at the onset of exercise at 90% V O 2 max. It was hypothesized that both perturbations would increase oxidative ATP production and consequently result in a decreased reliance on substrate phosphorylation. However, the contribution of ATP from substrate phosphorylation at the onset of exercise, as measured from PCr degradation and lactate accumulation, was unaffected by DCA infusion or hyperoxia. This implies that oxidative phosphorylation is not limited by metabolic inertia at the level of PDH or by O 2 delivery to muscle during the transition from rest to exercise at 90% V O 2 max. DCA infusion. DCA inhibits the activity of PDH kinase and therefore increases the amount of PDHa (39). In the present study, DCA infusion increased resting muscle PDHa to levels previously reported during intense aerobic exercise (11, 20) and increased the muscle contents of acetyl-coa and acetylcarnitine well above the CON condition. PDHa remained significantly higher for the initial 30 s of exercise at 90% V O 2 max in the DCA trial and decreased slightly to match the CON value at 90 s. We also reported this finding at 65% V O 2 max (19) and believe it is a function of powerful exercise-related regulators overriding the effect of DCA on the control of PDH activation during exercise, resulting in PDHa levels that match the control condition. The key finding of the DCA infusion was that, despite the potential for increased acetyl-coa availability early in exercise, no difference in the reliance on substrate phosphorylation and, by inference, oxidative phosphorylation was found. In fact, acetyl- CoA remained at the elevated preexercise level, and

5 E112 Fig. 5. Acetyl-CoA (A) and acetylcarnitine (B) accumulation during control, DCA infusion, and hyperoxia trials at the onset of cycling at 90% V O 2max. *Significantly different from rest for the same trial; significantly different from 30 s for the same trial; significantly different from control and hyperoxia for the same time point. acetylcarnitine accumulated during the 90 s of cycling in the DCA trial. These findings conflict with the results of studies examining the effect of DCA infusion on human muscle metabolism at the onset of low- to moderate-intensity exercise ( 45 70% V O 2 max )(12, 19, 26, 35, 36). These authors reported that the increased provision of oxidative substrate from increased flux through PDHa (9, 19, 26) and/or the acetylcarnitine store (34, 35) after DCA infusion decreased the reliance on substrate level phosphorylation. A recent study from our laboratory reported that the increased substrate provision with DCA is a result of increased flux through PDH and not from acetylcarnitine (9). The collective results implied that the rate of oxidative phosphorylation at the onset of low- to moderate-intensity exercise is limited by inertia associated with PDH activation. It is not clear why skeletal muscle was unable to use the extra substrate provided at the onset of exercise at 90% V O 2 max, given that substrate phosphorylation (O 2 deficit) increases as a function of the power output (20). However, the present results are consistent with recent reports also demonstrating that DCA infusion did not alter oxidative and substrate phosphorylation at the onset of sprint exercise (18) and knee extensor exercise at 110% of the thigh V O 2 peak (1). One possibility for the lack of effect during intense exercise could be that another site of metabolic inertia, downstream of PDH, is responsible for limiting the provision of acetyl-coa, and ultimately NADH, at this exercise intensity. The tricarboxylic (TCA) cycle is a likely candidate, as it provides a large portion of the NADH required for oxidative ATP production and is controlled by three regulatory enzymes and possibly by the provision of TCA cycle intermediates (TCAI). To our knowledge, there has been no work examining the activation of the TCA cycle regulatory enzymes, including citrate synthase, 2-oxoglutarate dehydrogenase, and isocitrate dehydrogenase, at the onset of exercise in human skeletal muscle. However, it has been suggested that the accumulation of the TCAI may be important for controlling the flux through the TCA pathway (32) and that an increase in TCAI pool size at the onset of exercise is important for maximal TCA cycle flux and optimal oxidative energy production (12). The TCAI pool increases severalfold during exercise, and the majority of this increase occurs within the first minute of contraction (10, 12). However, an alternate hypothesis is that the accumulation is merely a consequence of the mismatch between glycolytic flux and oxidative disposal of pyruvate and represents a sink for pyruvate when production exceeds oxidation (7, 12). In this scheme, TCAI accumulation occurs as a function of mass action events and would not play a regulatory role in the flux through the TCA cycle. Gibala and colleagues have performed a series of studies to determine the importance of TCAI accumulation during leg-kicking exercise (10, 11, 12) and whole body cycling (13). In one study, they infused DCA and observed a 50% reduction in the TCAI pool at rest (12). However, the increase in TCAI during the onset of exercise at 70% of the leg V O 2 max was highest in the DCA trial such that, by 1 min, there was no difference in the TCAI pool size between the DCA and control trials. The general conclusion from these studies is that the accumulation of TCAI does not limit flux through the TCA cycle at the onset of aerobic exercise. An alternate explanation for the disparate effects of DCA infusion at the onset of exercise at 65 and 90% V O 2 max, at least in our hands, may be related to the subjects training status. The subjects in this study regularly engaged in aerobic exercise and had higher Table 2. Substrate phosphorylation during the onset of cycling at 90% V O 2max in CON, DCA infusion, and HYP trials Trial Time Interval, s CON DCA HYP All values are means SE in mmol ATP/kg dry muscle. See METHODS for calculation details.

6 E113 V O 2 max values than in our previous study that employed inactive subjects (19). The differing training status is also apparent when the measured substrate phosphorylation is examined during the initial 30 s of exercise at the two power outputs. Although the requirement for substrate phosphorylation increases as a function of exercise intensity in a given set of subjects (20), the subjects in the present study required only 50 mmol ATP/kg dry muscle in the control trial at 90% V O 2 max, whereas the requirement was 70 mmol ATP/kg dry muscle at 65% V O 2 max in the previous study. It is therefore possible that the trained subjects in the current study were already adapted to maximize oxidative phosphorylation and minimize substrate phosphorylation at the onset of intense aerobic exercise. This suggests that aerobic training speeds up the V O 2 kinetics at the onset of exercise, as has been shown at the mouth during exercise at 60% V O 2 max (28). Training minimizes the potential for metabolic inertia, such that the provision of extra oxidative substrate during DCA infusion is unused. However, we realize that this suggestion is tentative, given the overlap of oxidative capapcity in the two groups and the lack of within-subject day-to-day repeatability of the estimation of substrate level phosphorylation. To fully test this suggestion, a longitudinal study is needed to examine the effects of DCA on substrate phosphorylation during the onset of exercise at both 65 and 90% V O 2 max before and after aerobic training. Two other possibilities for the lack of a DCA effect exist. A marked reliance on substrate phosphorylation was still present at the onset of intense exercise in the CON trial in these aerobically trained subjects. Therefore, it is possible that a certain level of substrate phosphorylation is obligatory at this high intensity. Because the provision of acetyl-coa derived from fat is likely minimal early in exercise, a certain amount of PCr breakdown, and the consequent build-up of free ADP, AMP, and P i, may be important for activating glycogenolysis and oxidative phosphorylation to the required levels. This implies that increasing the available NADH at the onset of intense aerobic exercise did not permit decreases in the energy status of the cell or increase the rate of oxidative phosphorylation (as occurred at moderate-intensity exercise). Last, to test whether a potential metabolic limitation was masked by a greater limitation at the level of oxygen delivery to the active muscle, we examined substrate phosphorylation while subjects were breathing 100% oxygen during the onset of intense exercise. Hyperoxic gas breathing. We hypothesized that the rate of increase in oxidative phosphorylation at the onset of intense aerobic exercise may be limited in some muscle fibers by the supply of oxygen. Breathing 100% O 2 has the potential to increase both the convective and diffusive delivery of oxygen to the active muscle. Increased O 2 provision may increase the rate of muscle oxidative phosphorylation and subsequently reduce the reliance on substrate phosphorylation. However, substrate phosphorylation was not attentuated in the HYP trial, and we concluded that O 2 availability does not limit O 2 uptake during the initial 90 s of exercise at 90% V O 2 max. Many studies have examined whether the provision of oxygen limits the V O 2 rate during steady-state exercise, but few have done so during the onset of intense exercise using direct measurements of skeletal muscle V O 2 or substrate phosphorylation in humans. The present findings conflict with two previous metabolic studies, which reported significant reductions in substrate phosphorylation (PCr degradation and lactate accumulation) during exhaustive exercise lasting 4 5 min while breathing either room air at 1.4 atmospheric pressure (22) or 60% O 2 (23). However, these studies based their conclusions on data obtained before and after exercise, with no time course measurements. Because there was no accounting for the lactate that escaped the muscle during the 4 5 min of exercise, the substrate phosphorylation estimates are not representative of the events occurring early in exercise. In a more relevant study, Bangsbo et al. (2) measured O 2 delivery and uptake in the working muscles during the initial seconds of leg kicking exercise at 120% of leg V O 2 peak while subjects breathed room air and concluded that O 2 delivery was not limiting. Richardson et al. (30) used magnetic resonance spectroscopy to measure myoglobin-associated PO 2 during maximal legkicking exercise in humans and reported that breathing 100% O 2 increased the intracellular PO 2 and the directly measured leg V O 2 max. Although these authors did not measure V O 2 kinetics during exercise, their findings suggest that hyperoxia should have increased O 2 availability to the mitochondria in the present study. In a related study using V O 2 measures at the mouth, MacDonald et al. (24) reported that breathing 70% O 2 increased the V O 2 kinetics at the onset of exercise above the ventilatory threshold ( 80% V O 2 max ) but not below the ventilatory threshold ( 50% V O 2 max ). Grassi et al. (16) used a canine model and examined the response of isolated gastrocnemius muscle V O 2 kinetics to increased blood flow during the onset of isometric tetanic contractions corresponding to V O 2 peak. The increased O 2 delivery resulted in faster V O 2 kinetics in the initial minute of exercise. Similar experiments with increased blood flow and pharmacologically increased O 2 dissociation plus hyperoxia had no effect on V O 2 kinetics during contractions corresponding to 60% V O 2 peak (14, 15). On the basis of these studies, it is not clear why hyperoxic breathing did not result in increased oxidative and reduced substrate phosphorylation during the onset of exercise at 90% V O 2 max. It may be that hyperoxia simply did not increase the convective O 2 delivery to the mitochondria or that oxidative phosphorylation is not limited by O 2 availability at the onset of cycling exercise at 90% V O 2 max. The ability of hyperoxia to increase convective O 2 delivery to the active muscle has been questioned. Despite measurable increases in the arterial O 2 content, hyperoxia has been shown to decrease blood flow in canine studies (5, 40) and one human study (37), effectively negating any potential

7 E114 increase in O 2 delivery. However, other human studies have reported no difference in blood flow during hyperoxia compared with normoxia (21, 25, 30). Complicating this issue is the fact that some studies used moderate power outputs (25, 38), blood flow measurements were made during steady-state exercise and not during the rest-to-exercise transition (30, 38), and 70% O 2 (not 100%) was occasionally used (25). In addition, Mac- Donald et al. (25) reported no significant decrease in leg exercise blood flow during hyperoxia, although there was a small absolute decrease that negated the increase in arterial O 2 content such that O 2 delivery was unchanged. However, from the bulk of the exercise blood flow data and the solid assumption of a high arterial PO 2 with hyperoxia (30), it seems likely that extra O 2 was available to the working muscles in the HYP trial of the present study. Because muscle substrate phosphorylation was not decreased, we conclude that O 2 availability was not limiting during the onset of intense exercise in aerobically trained subjects. In summary, this study examined the response of human skeletal muscle metabolism to both enhanced oxidative substrate supply (DCA infusion) and O 2 delivery (hyperoxia) at the onset of exercise at 90% V O 2 max. It was hypothesized that both conditions would increase oxidative ATP production and decrease the reliance on substrate phosphorylation. However, the contribution of ATP from substrate phosphorylation at the onset of exercise, as measured from PCr degradation and lactate accumulation, was unaffected by DCA infusion or hyperoxia. This suggests that oxidative phosphorylation during the transition from rest to exercise at 90% V O 2 max is not limited by metabolic inertia at the level of PDH or by O 2 delivery to muscle. 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