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. 2002 Jul 26;544(Pt 1):303–313. doi: 10.1113/jphysiol.2002.022764

Effects of respiratory alkalosis on human skeletal muscle metabolism at the onset of submaximal exercise

P J LeBlanc 1, M L Parolin 1, N L Jones 1, G J F Heigenhauser 1
PMCID: PMC2290561  PMID: 12356901

Abstract

The purpose of this study was to examine the effects of respiratory alkalosis on human skeletal muscle metabolism at rest and during submaximal exercise. Subjects exercised on two occasions for 15 min at 55 % of their maximal oxygen uptake while either hyperventilating (R-Alk) or breathing normally (Con). Muscle biopsies were taken at rest and after 1 and 15 min of exercise. At rest, no effects on muscle metabolism were observed in response to R-Alk. In the first minute of exercise, there was a delayed activation of pyruvate dehydrogenase (PDH) in R-Alk compared with Con, resulting in a reduced rate of pyruvate oxidation. Also, glycogenolysis was higher in R-Alk compared with Con, which was attributed to a higher availability of the monoprotonated form of inorganic phosphate (Pi), resulting in an elevated rate of pyruvate production. The mismatch between pyruvate production and its oxidation resulted in net lactate accumulation. These effects were not seen after 15 min of exercise, with no further differences in muscle metabolism between conditions. The results from the present study suggest that respiratory alkalosis may play an important role in lactate accumulation during the transition from rest to exercise in acute hypoxic conditions, but that other factors mediate lactate accumulation during steady-state exercise.

During acute hypoxia, submaximal exercise at a given absolute power output results in increases in both muscle and plasma lactate concentrations compared with normoxia (Katz & Sahlin, 1987; Brooks et al. 1992; Green et al. 1992; Brooks et al. 1998; Parolin et al. 2000a). These effects were thought to be due to a limitation in O2 supply to the muscle, resulting in anaerobic lactate accumulation. A recent study has shown that the intracellular PO2 of the exercising muscle is reduced during hypoxia, but to a level that is above the critical level at which mitochondrial respiration is compromised (Richardson et al. 1998). In addition, increased muscle blood flow helps to maintain whole body oxygen consumption rate (V̇O2) (Adams & Welch, 1980), and the V̇O2 of the exercising leg (Bender et al. 1988; Knight et al. 1993) at normoxic levels during hypoxia. Thus, it seems unlikely that hypoxia-induced lactate accumulation during submaximal exercise is solely the result of a limited O2 supply to the exercising muscle.

In contrast to steady-state exercise, the increase in V̇O2 during the transition from rest to exercise is slowed during hypoxia (Hughson & Kowalchuk, 1995). It has been suggested that lactate accumulation during this transition in hypoxia may be due to ‘metabolic inertia’ rather than limited O2 supply (Timmons et al. 1998). The term metabolic inertia refers to a delayed flux through metabolic pathways responsible for aerobic ATP production. Recent studies from our laboratory have shown that within the first minute of exercise, the increased lactate accumulation in hypoxia was a result of an enhanced glycogenolytic flux through glycogen phosphorylase (Phos) combined with a delayed activation (inertia) of pyruvate dehydrogenase (PDH; Parolin et al. 2000a). These results were partially reversed when PDH was activated by administration of dichloroacetate (DCA) before exercise (Parolin et al. 2000b). Decreased pyruvate oxidation, due to delayed activation of PDH, potentially reduces the availability of oxidative substrates for aerobic ATP production. As a result, there is a greater reliance on glycolysis and phosphocreatine degradation to regenerate ATP. The mismatch between pyruvate oxidation and its production results in a net-lactate accumulation, because any excess pyruvate is converted to lactate by the near-equilibrium enzyme lactate dehydrogenase.

Hyperventilation usually accompanies hypoxaemia, with ventilation rates rising by 150–200 % above normoxic values (Hughes et al. 1968; Lenfant & Sullivan, 1971). This results in arterial blood pH increasing by 0.1-0.3 units (Edwards & Clode, 1970; Zborowska-Sluis et al. 1970; Davies et al. 1986). Previous studies have shown an association between hyperventilation and lactate accumulation (Eldridge & Salzer, 1967; Edwards & Clode, 1970; Brice & Welch, 1985; Davies et al. 1986) and it has been suggested that the change in pH, rather than an oxygen limitation is the main contributing factor that results in an increased lactate accumulation (Davies et al. 1986). Thus, respiratory alkalosis may account, in part, for the increased lactate accumulation seen during exercise with acute hypoxia.

The present study was designed to determine the role of the rate-limiting enzymes, PDH and Phos, and their regulatory factors in lactate accumulation during respiratory alkalosis in human skeletal muscle during exercise. Lactate accumulation is a function of adjustments in the activation of the aerobic ATP producing metabolic pathways and/or alterations in glycogenolytic/glycolytic flux (see Spriet et al. 2000 for review). Thus, we hypothesized that respiratory alkalosis might result in increased glycogenolytic flux and a delayed activation of PDH with a concomitant increase in lactate accumulation. The effects of respiratory alkalosis were examined relative to control at rest, at 1 min during the transition from rest to exercise and after 15 min of submaximal exercise.

METHODS

Subjects

Eight healthy, active males were recruited to participate in the study (age 22 ± 1.2 (s.e.m.) years; height 181.3 ± 2.2 cm; weight 77.6 ± 3.4 kg). Individuals were asked to consume similar diets and refrain from caffeine, alcohol, and exercise for 48 h before each trial. Individuals served as their own control. Oral and written explanation of the experimental protocol and its attendant risks were provided and informed written consent was obtained from each subject. The study was approved by the McMaster University Ethics Committee.

Pre-experimental protocol

Individuals completed an initial incremental maximal exercise test on an electro-magnetically braked cycle ergometer (Lode Excalibur, Quinton Instruments, Seattle, WA, USA) to determine V̇O2,max and maximal work capacity using a metabolic measurement system (Quinton Q-Plex 2, Quinton Instruments). One week later, individuals returned to establish the degree of hyperventilation required to attain an end tidal PCO2 (PET,CO2) of approximately 20–25 mmHg, to mimic the PET,CO2 measured during hypoxia (Parolin et al. 2000a). PET,CO2 was monitored breath by breath and the subject ‘targeted’ the desired PET,CO2 displayed continuously on an oscilloscope during the equilibration period (20 min at rest prior to exercise) and the experimental protocol (15 min at 55 % V̇O2,max). This was repeated several times to familiarize the subject with the conditions of the study and ensure consistency of the experimental procedure. The workload required to obtain 55 % V̇O2,max was confirmed through these practice trials.

Experimental protocol

The experimental protocol was conducted during normoxia (Con) or normoxia with hyperventilation (R-Alk), on two occasions separated by 1 week. The order of the Con and R-Alk trials was randomized and took place at the same time of day for each subject. Prior to the beginning of the protocol, a venous catheter was inserted in the anticubital vein of the forearm for blood sampling and was maintained patent with saline. One thigh was prepared for needle biopsies of the vastus lateralis. Incisions were made through the skin to the deep fascia under local anaesthesia (2 % lignocaine (lidocaine) without adrenaline (epinephrine)) as described by Bergström (1975). Subjects exercised for 15 min at 55 % of their V̇O2,max. Blood samples were taken at pre-equilibration (R-Alk only), before exercise (time 0), and after 5, 10 and 14 min of exercise. Muscle biopsies were taken at pre-equilibration (R-Alk only), before exercise (time 0) and after 1 and 15 min of exercise.

Blood sampling and analysis

Venous blood samples (≈7 ml) were collected in heparinized syringes (Sarstedt, Germany) and placed on ice. Blood was centrifuged at 15 900 g for 2 min to isolate plasma. One portion of plasma was incubated with 5 m NaCl (4:1 plasma:NaCl) at 56 °C for 30 min and then frozen for later analysis of free fatty acids (Wako NEFA kit, WAKO Chemicals, Richmond, VA, USA). A second portion of plasma was deproteinized with 0.5 m perchloric acid (PCA) (1:2 plasma:PCA) and then frozen for later analysis for glucose, lactate and glycerol (Bergmeyer, 1983).

Muscle analysis

Muscle biopsies were immediately frozen in liquid N2. A small piece (4-26 mg) was chipped from each biopsy (under liquid N2) for determination of the fraction of PDH in the active form (PDHa) (Putman et al. 1993). The remainder of the sample was freeze dried, dissected free of blood and connective tissue, and powdered. One aliquot was analysed for total (a + b) and active Phos (Phosa+b and Phosa, respectively; Young et al. 1985) and the maximal velocity and mole fraction of Phos a + b and Phos a were calculated from the measured activities as described by Chasiotis et al. (1982). Phos measurements were made only on exercise samples because resting samples must be kept at room temperature for 30 s before freezing for accuracy, which would have required additional biopsies (Ren & Hultman, 1988). Previous studies have reported values of ≈10 % for the mole fraction of Phos a at rest (Chesley et al. 1996; Parolin et al. 1999). A second aliquot was extracted in 0.5 m PCA and 1 mm EDTA, neutralized to pH 7.0 with 2.2 m KHCO3 and analysed for acetyl-coenzymeA, free coenzymeA, acetylcarnitine, free carnitine, (Cederblad et al. 1990), ATP, pyruvate, lactate, phosphocreatine (PCr), creatine, glucose, glucose 6-phosphate (G-6-P), glucose 1-phosphate (G-1-P), fructose 6-phosphate (F-6-P), glycerol 3-phosphate (Gly-3-P; Bergmeyer, 1983), and glycogen (Harris et al. 1974). All muscle metabolites were normalized to the highest total creatine content for a given individual to correct for non-muscle contamination.

Calculations

Arterial CO2 pressure was estimated (ePa,CO2) from PET,CO2 and tidal volume (VT) according to Jones et al. (1979). PDHa flux was estimated from PDHa as measured in wet tissue and converted to dry tissue using the wet-to-dry muscle ratio of 4:1 at rest and 4.5:1 during exercise (Putman et al. 1998). Pyruvate production was calculated from increases in muscle lactate and pyruvate and in blood lactate concentrations, plus the flux of pyruvate through PDHa. Lactate accumulation was calculated from changes in muscle and blood lactate concentrations. The distribution volume of blood lactate was assumed to be 0.64 × body weight (Astrand et al. 1986) and the active muscle weight was assumed to be 9 kg wet weight. This calculation does not account for lactate oxidation by other tissues and would result in a slight underestimation of lactate accumulation.

Estimates of glycogenolytic rate during the first minute of exercise were derived from increases in muscle G-6-P, F-6-P, Gly-3-P plus pyruvate production from rest to 1 min of exercise. The average glycogenolytic rate during the subsequent 14 min of exercise was calculated from the reduction in muscle glycogen concentration during 15 min of exercise minus the estimated glycogen utilization in the 1st minute of exercise, divided by time.

The rate of ATP turnover from PCr was calculated from the decrease in PCr concentration. The rate of ATP turnover from glycolysis was calculated from pyruvate production (1 mmol glycosyl unit was equal to 3 mmol ATP). The rate of ATP turnover from oxidative phosphorylation originating from carbohydrate sources was calculated from total acetyl coenzyme A production by PDH (1 mmol of pyruvate oxidized was equal to 15 mmol ATP). These calculations were based on the mean data.

Statistical analysis

All data are presented as means ±s.e.m. A two-way analysis of variance (ANOVA; Steel & Torrie, 1980) with repeated measures was used to establish differences between conditions and time. Tukey's post hoc test was used to determine significance (P < 0.05). Assumption for normality were verified by generating appropriate residual plots. Data transformations (log, square root, and inverse square root) were used when appropriate to meet the above assumption.

RESULTS

Cardiorespiratory measurements

Mean V̇O2,max for the group was 3.4 ± 0.2 l min−1. Hyperventilation was attained by a 2.1- and 1.8-fold increase in expiratory ventilation (V̇E) at rest and exercise, respectively, and was accompanied by similar V̇O2, V̇CO2, and RER and lower PET,CO2 and ePa,CO2 in R-Alk compared with Con (Table 1).

Table 1.

Respiratory parameters at rest and during exercise in control and respiratory alkalosis

Rest Exercise
Con R-Alk Con R-Alk
V̇O2 (l min−1) 0.40 ± 0.03 0.39 ± 0.02 1.94 ± 0.08* 1.94 ± 0.07*
V̇CO2 (l min−1) 0.37 ± 0.03 0.40 ± 0.05 2.01 ± 0.08* 1.95 ± 0.11*
RER 0.99 ± 0.08 1.03 ± 0.13 1.04 ± 0.01 1.00 ± 0.02
PET,CO2 (mmHg) 33.4 ± 1.8 17.0 ± 0.9† 41.1 ± 1.7* 19.2 ± 0.5*†
ePa,CO2 (mmHg) 35.6 ± 1.6 20.8 ± 0.9† 42.5 ± 1.5* 22.8 ± 0.4*†
V̇E (BTPS) (l min−1) 15.3 ± 1.73 1.5 ± 2.6† 53.3 ± 2.4* 95.7 ± 5.2*†
VT (BTPS) (l) 0.85 ± 0.05 0.89 ± 0.24 2.26 ± 0.18* 2.10 ± 0.47*
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Values are means ±s.e.m.; V̇O2, rate of O2 uptake; V̇CO2, rate of CO2 production; RER, respiratory exchange ratio; PET,CO2, end-tidal partial pressure of CO2; ePa,CO2, estimated arterial partial pressure of CO2; V̇E, pulmonary ventilation; VT, tidal volume, BTPS, body temperature and pressure, saturated.

*

Significant from rest

†

significant from control. Parameters during exercise were obtained from the mean of the last 5 min of exercise for each subject.

Muscle measurements

After 20 min pre-equilibration in R-Alk, all values were similar to those of Con (Tables 2–5 and Fig. 1 and Fig. 3), with the exception of muscle [acetylcarnitine], which was higher in R-Alk compared with Con (Table 4).

Table 2.

Muscle contents of glycolytic intermediates at rest and during exercise in control and respiratory alkalosis

Exercise
Metabolite Condition Pre-equilibration Rest 1 min 15 min
Glycogen Con ND 439 ± 41 ND 333 ± 38*
R-Alk ND 538 ± 47 ND 346 ± 70*
G-1-P Con ND 0.17 ± 0.09 0.08 ± 0.02 0.30 ± 0.13
R-Alk 0.13 ± 0.11 0.09 ± 0.05 0.11 ± 0.07 0.12 ± 0.10
G-6-P Con ND 0.95 ± 0.14 2.03 ± 0.39 2.23 ± 0.42
R-Alk 1.01 ± 0.13 0.90 ± 0.12 1.18 ± 0.22 4.33 ± 0.82*
F-6-P Con ND 0.20 ± 0.05 0.63 ± 0.20* 0.30 ± 0.06
R-Alk 0.16 ± 0.05 0.05 ± 0.06 0.19 ± 0.07† 0.47 ± 0.09*
Gly-3-P Con ND 0.47 ± 0.01 0.53 ± 0.01 0.51 ± 0.01
R-Alk 0.51 ± 0.01 0.50 ± 0.01 0.61 ± 0.11* 0.53 ± 0.07*†
Pyruvate Con ND 0.49 ± 0.07 0.53 ± 0.06 0.43 ± 0.06
R-Alk 0.43 ± 0.05 0.58 ± 0.09 0.51 ± 0.10 0.49 ± 0.15
Lactate Con ND 6.4 ± 1.5 11.3 ± 1.8 13.9 ± 2.2*
R-Alk 3.6 ± 0.7 8.1 ± 1.6 20.1 ± 3.5*† 18.7 ± 4.3*
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Values are means ±s.e.m. and are expressed in mmol (kg dry wt)−1; G-1-P, glucose 1-phosphate; G-6-P, glucose 6-phosphate; F-6-P, fructose 6-phosphate; Gly-3-P, glycerol 3-phosphate; ND, not determined.

*

Significant from rest

†

significant from control.

Table 5.

Plasma lactate, glucose, glycerol and free fatty acid (FFA) concentration at rest and during exercise in control and respiratory alkalosis

Exercise
Metabolite Condition Pre-equilibration Rest 5 min 10 min 14 min
Lactate Con ND 1.2 ± 0.1 2.8 ± 0.3* 3.6 ± 0.3* 3.7 ± 0.4*
R-Alk 1.2 ± 0.1* 2.4 ± 0.4† 4.4 ± 0.6*† 5.1 ± 0.6*† 5.3 ± 0.5*†
Glucose Con ND 4.1 ± 0.2 3.8 ± 0.2 3.8 ± 0.1 3.9 ± 0.1
R-Alk 4.1 ± 0.2 4.3 ± 0.2 4.2 ± 0.3 4.2 ± 0.3 4.2 ± 0.3
Glycerol Con ND 38.0 ± 4.9 36.4 ± 2.3 42.6 ± 7.1 30.8 ± 4.9
R-Alk 27.8 ± 1.3 33.9 ± 6.0 47.1 ± 4.0 56.3 ± 4.7 49.7 ± 6.5
FFA Con ND 0.36 ± 0.07 0.38 ± 0.07 0.38 ± 0.07 0.40 ± 0.07
R-Alk 0.38 ± 0.07 0.50 ± 0.05 0.47 ± 0.06 0.47 ± 0.08 0.55 ± 0.09
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Values are means ±s.e.m.; glycerol is expressed in μm; all other measurements are expressed in mm. FFA, free fatty acid; ND, not determined.

*

Significant from rest

†

significant from control.

Figure 1. Muscle pyruvate dehydrogenase in its active form (PDHa) at rest and during exercise in control and respiratory alkalosis.

Figure 1

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Values are means ±s.e.m. * Significant from rest; † significant from control.

Figure 3. Muscle lactate concentration at rest and during exercise in control and respiratory alkalosis.

Figure 3

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Values are means ±s.e.m. * Significant from rest; † significant from control.

Table 4.

Muscle contents of CoA, carnitine, and their acetylated forms at rest and during exercise in control and respiratory alkalosis

Exercise
Metabolite Condition Pre-equilibration Rest 1 min 15 min
Acetyl CoA Con ND 8.0 ± 0.9 11.1 ± 2.2 15.8 ± 1.1*
R-Alk 8.3 ± 1.1 8.5 ± 0.7 11.2 ± 1.0 18.4 ± 2.3*
Free CoA Con ND 80.9 ± 7.3 74.7 ± 10.2 69.5 ± 3.5
R-Alk 82.3 ± 17.7 85.5 ± 9.1 66.9 ± 11.2 73.7 ± 12.4
Acetyl CoA/CoA Con ND 0.11 ± 0.02 0.16 ± 0.04 0.23 ± 0.03*
R-Alk 0.11 ± 0.02 0.11 ± 0.01 0.23 ± 0.07* 0.24 ± 0.03*
Acetylcarnitine Con ND 1.7 ± 0.2 4.1 ± 0.6 12.6 ± 1.3*
R-Alk 2.8 ± 0.6 4.3 ± 0.9† 5.4 ± 1.0 13.4 ± 2.2*
Free carnitine Con ND 17.2 ± 1.6 15.2 ± 1.1 8.3 ± 0.7*‡
R-Alk 19.1 ± 1.2 17.1 ± 1.8 13.3 ± 1.2* 9.1 ± 1.6*‡
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Values are means ±s.e.m.; acetyl-CoA and free CoA measures are expressed in μmol(kg dry wt)−1; all other measurements are expressed as mmol (kg dry wt)−1; acetyl CoA/CoA, ratio of acetyl CoA to CoA; ND, not determined.

*

Significant from rest

†

significant from control

‡

significant from 1 min.

After 1 min of exercise, PDHa increased during both conditions compared with rest but was lower in R-Alk (Fig. 1). After 15 min of exercise, there was no difference in PDHa between conditions. The mole fraction of Phos a was unaffected by R-Alk after 1 and 15 min of exercise (Fig. 2).

Figure 2. Muscle phosphorylase a mole fraction during exercise in control and respiratory alkalosis.

Figure 2

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Values are means ±s.e.m.

Resting muscle [glycogen] was similar between conditions (Table 2). At the end of 15 min of exercise, muscle [glycogen] decreased similarily by 24 and 36 % during both Con and R-Alk, respectively. Muscle [G-1-P] and [pyruvate] were unaltered by exercise and were similar between conditions. Muscle [G-6-P] was also similar between conditions; however muscle [G-6-P] in R-Alk increased above resting values after 15 min of exercise. Muscle [F-6-P] did not differ between conditions at rest but after 1 min of exercise, muscle [F-6-P] in Con increased above resting values and was higher than R-Alk. After 15 min of exercise, [F-6-P] in Con returned to levels similar to rest and did not differ from R-Alk, which was higher than resting levels. [Gly-3-P] was unaltered by exercise in Con but increased above resting levels in R-Alk during exercise and was higher than in Con after 15 min of exercise.

Resting muscle [lactate] was similar between conditions. After 1 min of exercise, muscle [lactate] increased in R-Alk and was higher than in Con (Table 2, Fig. 3). After 15 min of exercise, muscle [lactate] was higher than rest in both conditions, but was not different between conditions.

Muscle [ATP] was unaltered by exercise and similar in the two conditions (Table 3). Muscle [PCr] decreased after 1 and 15 min of exercise compared with rest in both conditions. Muscle [Cr] similarly increased after 1 min of exercise in both conditions and did not change after 15 min of exercise compared with the first minute.

Table 3.

Muscle contents of ATP, creatine, and phosphocreatineat rest and during exercise in control and respiratory alkalosis

Exercise
Metabolite Condition Pre-equilibration Rest 1 min 15 min
ATP Con ND 25.1 ± 2.2 24.6 ± 1.7 21.5 ± 1.8
R-Alk 26.6 ± 1.8 27.0 ± 1.7 25.5 ± 1.6 22.7 ± 1.8
Creatine Con ND 30.8 ± 3.0 44.0 ± 3.2* 52.9 ± 3.2*
R-Alk 38.0 ± 2.4 33.5 ± 2.2 45.3 ± 4.0* 56.4 ± 6.4*
Phosphocreatine Con ND 94.3 ± 2.0 75.3 ± 4.1* 71.9 ± 4.2*
R-Alk 98.3 ± 6.0 97.2 ± 4.5 80.9 ± 5.1* 63.9 ± 4.3*
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Values are means ±s.e.m. and are expressed as mmol (kg dry wt)−1; ND, not determined.

*

Significant from rest.

Muscle [free CoA] was similar between conditions and did not change with exercise (Table 4). In contrast, [acetyl-CoA] increased after 15 min of exercise in both conditions. Muscle [free carnitine] in Con decreased after 15 min of exercise compared with rest and 1 min of exercise. [Free carnitine] in R-Alk decreased after 1 min of exercise and decreased even further after 15 min of exercise. Muscle [acetylcarnitine] was similar between conditions and increased after 15 min of exercise.

Blood metabolites

Blood [glucose], [glycerol] and [free fatty acids] did not change with exercise and were similar between conditions (Table 5). Blood [lactate] increased with exercise and was higher in R-Alk compared with Con at rest and during exercise.

Glycogenolysis

During the first minute of exercise, the glycogenolytic rate was higher in R-Alk compared with Con (Fig. 4). During the subsequent 14 min of exercise, glycogenolytic rates were similar between conditions and did not differ from the first min.

Figure 4. Estimated rates of muscle glycogen use during exercise in control and respiratory alkalosis.

Figure 4

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Values are means ±s.e.m. * Significant from control.

Pyruvate production and oxidation and lactate accumulation

In Con during the first min of exercise there was a lower rate of pyruvate production compared with the subsequent 14 min of exercise, with a lower rate of pyruvate oxidation and no difference in lactate accumulation (Fig. 5). In contrast, a higher rate of pyruvate was produced in R-Alk during the first minute compared with the subsequent 14 min of exercise, with a lower rate of pyruvate oxidized and more lactate accumulation. During the first minute of exercise, there was a higher pyruvate production and lactate accumulation but lower pyruvate oxidation in R-Alk compared with Con. Between the first and fifteenth minute of exercise, there were no differences in pyruvate production and lactate accumulation between conditions and pyruvate oxidation was lower in R-Alk compared with Con.

Figure 5. A comparison of the rates of pyruvate production and oxidation and lactate accumulation during exercise in control and respiratory alkalosis.

Figure 5

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* Significant from 0–1 min; † significant from control.

ATP turnover at the onset of exercise

During the first minute of exercise in Con, 37 % of the total ATP turnover was attributed to glycolysis (26 %) and PCr degradation (11 %; Fig. 6). ATP regeneration through the TCA cycle/electron transport chain (oxidative phosphorylation) accounted for the remaining 63 % of total ATP turnover. In R-Alk, 48 % of the total ATP turnover was attributed to glycolysis (37 %) and PCr degradation (11 %). Oxidative phosphorylation accounted for the remaining 52 % of total ATP turnover.

Figure 6.

Figure 6

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ATP turnover rates from PCr degradation, glycolysis, and oxidative phosphorylation during the transition from rest to exercise in control and respiratory alkalosis.

DISCUSSION

The present study examined the acute effects of respiratory alkalosis, through voluntary hyperventilation, on the regulation of skeletal muscle metabolism at rest, during the transition from rest to exercise and during steady-state submaximal exercise. At rest, no effects on muscle metabolism were observed in response to R-Alk. During the first minute of exercise, R-Alk resulted in a greater pyruvate production, despite no difference in the mole fraction of Phos a or glycolytic intermediates. In addition to increased pyruvate production in R-Alk, a delayed activation of PDH resulted in lower pyruvate oxidation. This mismatch in pyruvate production and oxidation resulted in greater lactate accumulation in both muscle (Fig. 3) and plasma (Table 5) in R-Alk during the transition from rest to exercise. In contrast, there was no additional accumulation of muscle and plasma [lactate] during steady-state exercise. Hence, an elevated lactate accumulation associated with respiratory alkalosis during exercise was explained by the differing fluxes through the rate-limiting enzymes, Phos and PDH, at the onset of exercise.

Respiratory alkalosis

Voluntary hyperventilation resulted in a reduction in ePa,CO2 from 35.6 to 20.8 mmHg at rest (Table 1). A drop of approximately 15 mmHg in Pa,CO2 would translate to a rise in plasma pH from 7.42 to 7.59 (Siggard-Anderson, 1963). A change in extracellular pH (ΔpHe) induced by respiratory alkalosis has been associated with a 0.67 increase in intracellular pH (ΔpHe) of rat diaphragm (Heisler, 1975). Thus, assuming a muscle pH of 7 in Con and a ΔpHe of 0.17, intramuscular pH for R-Alk can be estimated at 7.11. This increase in intramuscular pH may be a contributing factor to the changes seen in muscle metabolism.

Oxidative phosphorylation

Mitochondrial oxidative phosphorylation is represented by the following equation:

graphic file with name tjp0544-0303-mu1.jpg

and the rate of ATP production is regulated by O2 availability, [NADH]/[NAD+], and [ADP][Pi]/[ATP] (Wilson, 1994). Changes in any of the parameters result in a compensatory change in the others to drive oxidative phosphorylation.

PDH is a mitochondrially bound rate-limiting enzyme that regulates the entrance of glycolytically produced acetyl units into oxidative metabolism. Two enzymes covalently modify PDH, PDH kinase (PDK) and PDH phosphatase (PDP). PDK catalyses the phosphorylation, and resulting inactivation of PDH, whereas PDP dephosphorylates the enzyme, thereby activating PDH (Denton et al. 1975; Stansbie, 1976). The amount of PDH in its active form (PDHa) determines its activity. In turn, both PDK and PDP are allosterically modulated by increased ratios of acetyl-coenzymeA to CoA and NADH to NAD+, with increased ratios activating PDK and inhibiting PDP (Denton et al. 1975; Pettit et al. 1975; Stansbie, 1976). Other modulators include pyruvate (Cate & Roche, 1978) and ATP to ADP ratio (Denton et al. 1975), which inhibits and activates PDK and Ca2+ and H+, which activate PDP (Chen et al. 1996).

During the transition from rest to exercise, Ca2+ release from the sarcoplasmic reticulum is probably the primary stimulus for the activation of PDH. Ca2+ release stimulates PDP and dephosphorylates PDH, rendering it more active. In the present study, PDH activation increased during the first minute of exercise in Con with no further increase in PDH activity after the subsequent 14 min of exercise, which is consistent with previous studies (Howlett et al. 1999; St Amand et al. 2000; Parolin et al. 2000a). In R-Alk there was a delayed activation of PDH during the first minute of exercise, similar to the effects of hypoxia (Parolin et al. 2000a). During the transition from rest to exercise, none of the typical allosteric modulators were affected by R-Alk, with the exception of [H+]. Both PDK and PDP have an optimum pH of 7.0-7.2 and 6.7-7.1, respectively (Hucho et al. 1972). The rate of activation of PDH is dependent on the ratio of active PDK to PDP, thus a slight change in the activity of either covalent modifier would alter the rate of activation or inactivation of PDH. An increase in intramuscular pH of 7 in Con to 7.11 in R-Alk may slow the activity of PDP and potentially enhance the activity of PDK, resulting in a delayed activation of PDH.

A delayed activation of PDH may cause a mismatch between ATP utilization and aerobic ATP production. Reduced flux through PDH during the transition from rest to exercise in R-Alk may result in metabolic inertia, delaying the availability of carbohydrate-derived substrate to the TCA cycle and the subsequent production of reducing equivalents for oxidative phosphorylation. In R-Alk, a similar [ATP] and O2 availability during the transition from rest to exercise and a lower [NADH] and [H+] may require higher free [ADP] and [Pi] to drive oxidative phosphorylation and maintain the same ATP turnover rate as Con. Free [ADP], calculated from the near-equilibrium reaction of creatine kinase (Dudley et al. 1987), was higher in R-Alk compared with Con (130 ± 29 and 92 ± 13 μmol (kg dry wt)−1, respectively). As a result, the increase in free [ADP] may stimulate glycogenolysis in R-Alk.

Previous studies have shown there is a delayed O2 uptake response during respiratory alkalosis during the transition from rest to exercise (Ward et al. 1983; Hayashi et al. 1999). It has been hypothesized that this delay may be due to impaired O2 off-loading from haemoglobin (Hayashi et al. 1999) or decreased blood flow (Brice & Welch, 1985; Gustafsson et al. 1993; Karlsson et al. 1994) through exercising muscles. We cannot disregard the fact that respiratory alkalosis in the present study may impair both diffusive and/or conductive delivery of oxygen to the exercising muscle. However, the present study demonstrated similar effects seen during hypoxia (Parolin et al. 2000a), suggesting the importance of metabolic inertia, at the level of PDH, on oxidative phosphorylation at the onset of exercise.

After 15 min of exercise, the activation of PDH in R-Alk was similar to Con and none of the typical allosteric modulators were affected by R-Alk. Previous studies of steady-state submaximal exercise during acute hypoxia (Parolin et al. 2000a) and metabolic alkalosis (Hollidge-Horvat et al. 2000) have shown an elevated PDHa and intramuscular [pyruvate], which exerted a feed-forward effect on PDHa. The present study did not show any differences in intramuscular [pyruvate] between conditions. Similar PDHa activities between conditions may be attributed to alterations in the acid-base status of the muscle. The alkalizing effects of hyperventilation are transient and lactate accumulation during the first minute of exercise in R-Alk may have reduced the estimated pH differences between conditions. However, the time course of pH changes in the muscle is difficult to determine during this transition from rest to exercise.

Phosphocreatine degradation and glycolysis

The regeneration of ATP is also possible through phosphocreatine (PCr) degradation and glycolysis. PCr degradation was similar between conditions during exercise and did not differ in regeneration of ATP during the transition from rest to exercise. However, glycogenolytic rate was higher in R-Alk compared with Con during the first minute of exercise and may have been the major contributor that maintained ATP turnover rates similar between conditions.

Phos is a flux-generating enzyme of glycogenolysis in skeletal muscle and is subject to both covalent and allosteric regulation. Phos a, the more active form, is active in the absence of AMP whereas Phos b requires AMP. Covalent transformation from Phos b to a is mediated by Phos kinase a, which in turn is allosterically regulated by adrenaline and cytosolic Ca2+. Post-transformational allosteric regulation of Phos b is mediated by AMP and inosine monophosphate (IMP), and inhibited by ATP and G-6-P. Of equal importance in regulating both Phos a and b is one of the substrates, inorganic phosphate (Pi) (Chasiotis et al. 1982). R-Alk compared with Con resulted in an elevated pyruvate production during the first minute of exercise, in the absence of any change in the percentage mole fraction of Phos a. This would suggest that enhanced glycogenolysis in R-Alk, with no change in Phos a transformation, is a result of post-transformational modulation.

The levels of intramuscular G-6-P did not differ between conditions and although not estimated in the present study, free [AMP] and free [Pi] have been shown to be similar during the first minute of exercise during acute hypoxia (Parolin et al. 2000a). However, an increase in pH, due to respiratory alkalosis, may influence the concentration of the monoprotonated form of Pi, HPO42-, over the diprotonated form, H2PO4−. The monoprotonated form is considered to be the only active substrate for Phos (Kasvinsky & Meyer, 1977). Estimated intramuscular free [Pi] at the onset of exercise in Con increased from the assumed resting concentration of 10.8 mmol (kg dry wt)−1 to a calculated level of 25.5 ± 5.2 after 1 min of exercise. The concentration of free Pi during the first minute of exercise was calculated as the difference between resting and exercise [PCr], less the accumulation of G-6-P, F-6-P and Gly-3-P, plus the assumed resting [Pi] of 10.8 mmol (kg dry wt)−1 (Dudley et al. 1987). A change in estimated intramuscular pH from 7 in Con to 7.11 in R-Alk prior to the onset of exercise would result in an increase of HPO42- from 15.4 to 16.9 mmol(kg dry wt)−1, assuming a negative dissociation acid constant (pKa) of 6.82 (Voet & Voet, 1995) and no change in free Pi. This 10 % increase in substrate availability for Phos may account for some of the increased pyruvate production, through increased glycogenolytic flux, seen during the transition from rest to exercise in R-Alk. Another factor contributing to increased glycogenolytic flux in R-Alk may be a result of the previously mentioned elevated intramuscular free [ADP], which may have led to an increased [IMP], which in turn may have activated Phos b.

The elevated pH in R-Alk would also affect another key enzyme that regulates glycolysis, phosphofructokinase (PFK). PFK catalyses the conversion of F-6-P to fructose 1, 6-bisphosphate with the use of ATP (Voet & Voet, 1995), and is subject to allosteric modulation by a number of metabolites. ATP and H+ inhibit whereas ADP, AMP, Pi, and F-6-P activate the enzyme complex. During the transition from rest to exercise in R-Alk compared with Con, elevated pH, [ADP], and [Pi] and lower F-6-P would stimulate PFK. As a consequence, a better match between PFK and glycogenolytically produced ATP would partially compensate for the decreased oxidative ATP production, due to the delayed activation of PDH.

After 15 min of exercise, the percentage mole fraction of Phos a, along with the glycogenolytic rate and pyruvate production, were all similar between conditions. This is contrary to what has been shown in previous studies of both acute hypoxia and metabolic alkalosis. During steady-state exercise, hypoxia (Parolin et al. 2000a) and metabolic alkalosis (Hollidge-Horvat et al. 2000) resulted in significant decreases in the mole fraction of Phos a and an increase in glycogen utilization and pyruvate production. The increased glycogenolytic rate was a result of post-transformational regulation of Phos through increases in intramuscular free AMP and free Pi (Hollidge-Horvat et al. 2000; Parolin et al. 2000a). The absence of these metabolic changes in the present study may be attributed to alterations in the acid-base status of the muscle. As previously mentioned, intramuscular lactate accumulation in R-Alk during exercise would have decreased the pH of the muscle, thereby potentially reversing the effects of respiratory alkalosis seen prior to the transition from rest to exercise.

Lactate accumulation

At rest, no differences were seen in intramuscular [lactate] between conditions. However, it is interesting to note that plasma [lactate] was higher in R-Alk after 20 min of hyperventilation compared with Con. This is consistent with previous studies examining the effects of hyperventilation on plasma lactate in humans (Eldridge & Salzer, 1967; Edwards & Clode, 1970; Davies et al. 1986). Plasma [lactate] represents the balance between lactate uptake and release from tissues. Thus, the elevated plasma lactate that accompanies hyperventilation may be related to increased lactate appearance and/or decreased lactate removal from the plasma. An increased lactate appearance may be due to increased lactate production of respiratory muscles due to increased work of breathing. However, it has been shown that isocapnic hyperventilation is not associated with a rise in blood lactate (Huckabee, 1957; Edwards & Clode, 1970; Martin et al. 1984; Engelen et al. 1995). Increased lactate appearance in plasma may also be due to glycolysis in erythrocytes, which is stimulated by low PCO2 or high pH (Murphy, 1960; Zborowska-Sluis & Dossetor, 1967) and may contribute to the elevated plasma lactate at rest.

Several studies have demonstrated that respiratory alkalosis decreases the clearance of lactate from plasma by active and inactive tissues (Eldridge et al. 1974; Druml et al. 1991). It has been demonstrated previously that inactive muscle during exercise responds in much the same way as active muscle, demonstrating an increase in PDHa (Putman et al. 1999). Thus, PDHa activity in inactive muscle during respiratory alkalosis may also be delayed, resulting in less lactate oxidation in inactive muscle and less lactate clearance from plasma.

Intramuscular lactate accumulation is also a function of the rate of efflux from the muscle. Lactate movement across the muscle occurs via a monocarboxylate lactate-proton cotransport protein (MCT), and as such is the rate-limiting step in lactate efflux (Bonen et al. 1997). The increased intramuscular [lactate] in R-Alk compared with Con at the onset of exercise may have resulted in a slight increase in the rate of lactate appearance in the blood from rest to 5 min of exercise. MCT is known to be sensitive to changes in concentration of the transport components, lactate and H+, demonstrating increased lactate movement out of the cell with increased lactate in the cell and increased pH outside the cell (Juel & Halestrap, 1999). The combined respiratory alkalosis and elevated intramuscular [lactate] may have resulted in an enhanced movement of lactate out of the muscle.

Summary and conclusions

This study demonstrates that respiratory alkalosis has a profound impact on muscle metabolism at the onset of exercise, resulting in increased lactate accumulation. This supports the claims from previous studies that hyperventilation-induced respiratory alkalosis during hypoxia plays an important role in increased lactate accumulation during exercise (Edwards & Clode, 1970; Adams & Welch, 1980; Davies et al. 1986; Parolin et al. 2000a).

In the present study, respiratory alkalosis resulted in metabolic effects that are found during the transition from rest to exercise and are similar to the effects seen during acute hypoxia (Parolin et al. 2000a). Hyperventilation prior to and during constant-load exercise resulted in a delayed activation of PDH during the transition from rest to exercise. This delayed activation of PDH was presumably a consequence of a hyperventilation-induced increase in muscle pH, as end-tidal PCO2 (and presumably Pa,CO2) was reduced from approximately 33 to 17 mmHg, and may be related to the pH sensitivity of the covalent modifiers PDK and PDP. A delayed activation of PDH would result in a concomitant decrease in carbohydrate-derived substrate availability for oxidative ATP production and increased lactate production. The cellular energetics dictate that to maintain a similar ATP production in R-Alk compared with Con, a higher [ADP] and [Pi] are needed to drive oxidative phosphorylation. As a result, a higher intramuscular [ADP] and/or [Pi] would stimulate glycogenolysis through Phos in the absence of covalent modification. Phos is further stimulated with an increased concentration in the monoprotonated form of Pi due to an increase in pH. The mismatch in pyruvate production and oxidation with respiratory alkalosis possibly explains the lactate accumulation seen in acute hypoxia during the transition from rest to exercise.

In contrast, the pH effects seen during the transition from rest to exercise are lost during steady-state exercise as a greater increase in lactate accumulation in R-Alk early in exercise presumably resulted in a greater metabolic acidosis to offset the effects of the respiratory alkalosis. This is contrary to what has been shown during acute hypoxia, where the rate of pyruvate production continued to exceed the rate of pyruvate oxidation, resulting in further lactate accumulation (Parolin et al. 2000a). The regulation of lactate accumulation is multifactorial and more work is required to ascertain what factors are responsible for lactate accumulation during steady-state exercise in acute hypoxia.

Acknowledgments

This study was funded by the Canadian Institutes of Health Research. Medical supervision of Drs K. J. Killian and G. L. Jones was greatly appreciated.

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