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J Appl Physiol 94: 1145-1152, 2003. First published November 1, 2002; doi:10.1152/japplphysiol.01024.2000
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Vol. 94, Issue 3, 1145-1152, March 2003

Metabolic determinants of the onset of acidosis in exercising human muscle: a 31P-MRS study

M. Roussel1, J. P. Mattei1,2, Y. Le Fur1, B. Ghattas3, P. J. Cozzone1, and D. Bendahan1

1 Centre de Résonance Magnétique Biologique et Médicale, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6612, and Faculté de Médecine de Marseille, 2 Service de Rhumatologie, Hôpital de La Conception, 13005 Marseille; and 3 Département de Statistiques, Faculté des Sciences de Luminy, 13288 Marseille cedex 09, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Onset of intracellular acidosis during muscular exercise has been generally attributed to activation or hyperactivation of nonoxidative ATP production but has not been analyzed quantitatively in terms of H+ balance, i.e., production and removal mechanisms. To address this issue, we have analyzed the relation of intracellular acidosis to H+ balance during exercise bouts in seven healthy subjects. Each subject performed a 6-min ramp rhythmic exercise (finger flexions) at low frequency (LF, 0.47 Hz), leading to slight acidosis, and at high frequency (HF, 0.85 Hz), inducing a larger acidosis. Metabolic changes were recorded using 31P-magnetic resonance spectroscopy. Onset of intracellular acidosis was statistically identified after 3 and 4 min of exercise for HF and LF protocols, respectively. A detailed investigation of H+ balance indicated that, for both protocols, nonoxidative ATP production preceded a change in pH. For HF and LF protocols, H+ consumption through the creatine kinase equilibrium was constant in the face of increasing H+ generation and efflux. For both protocols, changes in pH were not recorded as long as sources and sinks for H+ approximately balanced. In contrast, a significant acidosis occurred after 4 min of LF exercise and 3 min of HF exercise, whereas the rise in H+ generation exceeded the rise in H+ efflux at a nearly constant H+ uptake associated with phosphocreatine breakdown. We have clearly demonstrated that intracellular acidosis in exercising muscle does not occur exclusively as a result of nonoxidative ATP production but, rather, reflects changes in overall H+ balance.

human skeletal muscle; exercise intensity; anaerobic metabolic threshold; pH; phosphorus-31-magnetic resonance spectroscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PHOSPHORUS MAGNETIC RESONANCE spectroscopy (31P-MRS) has been widely used to study biochemical reactions involved in energy supply during muscular contraction in vivo. This noninvasive technique offers the possibility to record intracellular pH and concentrations of high-energy phosphate metabolites at rest and during and after muscular exercise. Depending on exercise intensity, various levels of phosphocreatine (PCr) consumption and intracellular acidosis can be reached. Low exercise intensity results in moderate PCr breakdown associated with no change in pH, and this kind of protocol has been widely used to analyze mitochondrial function through the relation of the Pi-to-PCr ratio {considered an index of ADP concentration ([ADP]) as long as pH does not change} to work output (work-cost relation) during exercise in control subjects (8, 9) and patients with oxidative disorders (2) and to illustrate the metabolic effects of training (5, 32, 40, 41). It has been postulated that the absence of acidosis during exercise could be considered an index of exclusive aerobic ATP contribution with no involvement of nonoxidative glycogenolysis, although this issue has not been clearly addressed (7).

As expected, more intense exercise induces larger metabolic changes, usually associated with an accumulation of lactate and pH changes. Interestingly, pH changes do not occur simultaneously with the start of exercise, and the exact meaning of this mechanism has not been properly addressed. An anaerobic threshold (AT) has been defined with respect to blood lactate accumulation (6, 19, 21, 48) or respiratory gas exchange (18, 48, 54-56), but it is widely accepted that this so-called AT does not properly reflect anaerobic metabolism. In support of this statement, muscle biopsy studies (23, 34) have shown an increase in muscle lactate production immediately after moderate exercise (50-60% of maximal O2 uptake), whereas Connett et al. (16) reported lactate accumulation in dog gracilis muscle for very-low-intensity (10% of maximal O2 uptake) exercise. Muscle lactate accumulation has also been observed beyond the AT during exercise (19). Interestingly, the largest blood lactate concentration is usually measured several minutes after the end of exercise (48). On the basis of exercise-induced pH changes, a few groups have addressed the issue of defining an intracellular AT within the muscle as an index of anaerobic ATP production. With the use of the time-dependent changes of proton concentration ([H+]) and Pi-to-PCr concentration ratio ([Pi]/[PCr]) during exercise, intracellular AT has been proposed to be associated with an activation (3, 38, 49, 50) or hyperactivation (22, 33, 37, 53) of the nonoxidative glycogenolytic pathway (leading to lactate production). Overall, if H+ balance with consuming (PCr hydrolysis, buffering capacity, and H+ efflux) and producing processes is considered, it seems unlikely that 1) the absence of acidosis is associated with a negligible contribution of anaerobic glycolysis and 2) anaerobic glycolysis activation accounts for the onset of intracellular acidosis, but these two hypotheses have not been addressed.

In the present study, we have analyzed these two hypotheses through a quantitative analysis considering all the factors involved in H+ balance. The aim of the study was to test whether the onset of intracellular acidosis could be explained by a change in H+ balance, rather than an activation or a sharp increase in glycogenolysis, and to identify which component of the balance mechanism, if any, is associated with the onset of intracellular acidosis.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

The study was conducted on the dominant forearm of seven healthy volunteers [age 31 ± 3 (SE) yr]. Each subject was right handed; none of the subjects was involved in any regular physical activity. All subjects provided informed consent to participate in the study, which was approved by the Timone Hospital Committee on Ethics (Marseille, France).

31P-MRS

Each subject sat on a chair close to the magnet and inserted the belly of the forearm horizontally into the magnet bore. The forearm was placed approximately at the same height as the shoulder to ensure a good venous return. Finger flexor muscles were examined by 31P-MRS with a 5-cm, double-tuned (1H and 31P) surface coil in a 4.7-T superconducting horizontal magnet (30 cm bore diameter) interfaced to a spectrometer (Bruker 47/30, Biospec) operating at 81.15 and 200.14 MHz for 31P and 1H, respectively. The homogeneity of the magnetic field was adjusted by monitoring the 200.14-MHz signal from the muscle water and fat protons. Data were acquired after 120-µs radio frequency pulses applied at 1.8-s intervals and with a sweep width of 10 kHz. 31P magnetic resonance signals were time averaged over 1 min (32 scans/spectrum) and sequentially recorded throughout the experimental protocols.

Experimental Protocol

Each subject performed two different rest-exercise-recovery protocols on separate days. Each experimental protocol encompassed 3 min of rest, 6 min of exercise, and 20 min of recovery. Muscular exercise consisted of finger flexions lifting a weight that was gradually increased by 1 kg/min from 1 to 6 kg. Exercise frequency was imposed by a metronome and was set to 0.47 Hz [low-frequency (LF) protocol] and 0.85 Hz [high-frequency (HF) protocol]. Our aim was to test the relationship between the onset of acidosis and the activation or hyperactivation of glycolysis. The utilization of these two contraction frequencies offers the possibility to test two experimental conditions: early onset (for the HF protocol) and late onset (for the LF protocol) of acidosis. In addition, a progressive exercise type was selected, because intracellular acidosis is delayed with incremental loads (22). So we compared two exercise protocols showing a striking difference. The sliding of the weight was recorded using a home-built displacement transducer connected to a personal computer, and results were expressed as power output measured in watts (ATS Sysma France).

Spectra Analysis

31P magnetic resonance signals were transferred to an IBM RISC 6000 workstation and processed using the NMR1 spectroscopy processing software (New Methods Research, Syracuse, NY). After deconvolution of free induction decays (corresponding to a line broadening of 15 Hz) and Fourier transformation, baseline correction was performed as previously described with baseline deconvolution using computer-estimated filter size and flattening factor (39). Peak areas of PCr, Pi, ATP, and phosphomonoesters were measured by curve fitting of the spectrum signals to a Lorentzian shape (39).

Quantitation of MRS Data

Absolute concentrations of all phosphorylated metabolites were calculated after correction for partial saturation (correction factors are 2.02, 2.47, and 1.58 for PCr, Pi, and ATP, respectively) and with the assumption that ATP concentration ([ATP]) is 8.2 mM at rest (51). Intracellular pH was calculated as previously described according to the chemical shift of Pi relative to PCr (51). The free cytosolic [ADP] was calculated from [PCr] and pH using the creatine kinase equilibrium constant (KCK = 1.66 × 109 M-1) and assuming that total creatine (Cr) content ([PCr] + [Cr], where [Cr] is Cr concentration) is 42.5 mM (4).

Timing

Data from each spectrum were assigned to its midpoint, as previously described (51). Thus the first exercise spectrum was 0.5 min, and subsequent spectra were assigned at intervals of 1 min.

H+ Balance Throughout Dynamic Exercise

To elucidate the relation of the onset of intracellular acidosis to H+ balance, each component of the H+ balance, i.e., overall H+ production associated with nonoxidative ATP production, H+ efflux, and H+ uptake associated with PCr breakdown, was quantified as previously described (14, 24, 35).

H+ uptake associated with PCr consumption. H+ consumed by net PCr breakdown ([H+]PCr) was calculated as follows
[H<SUP>+</SUP>]<SUB>PCr</SUB><IT>=&ggr; · V</IT><SUB>PCr</SUB> (1)
where gamma  is the proton stoichiometric coefficient of the coupled Lohmann reaction, as described originally by Kushmerick (35), and VPCr refers to the kinetics of PCr consumption calculated for each minute of exercise.

H+ efflux. As previously described, the rate of H+ efflux (vE proton) cannot be directly estimated during exercise. The first-order rate constant linking the rate of H+ efflux to the extent of pH changes was computed first from PCr and pH changes recorded during the recovery period, as previously described (24, 26)
v<SUB>E proton</SUB>(mM/min)<IT>=&bgr; · V</IT><SUB>pH</SUB> + [H<SUP>+</SUP>]<SUB>PCr</SUB> (2)
where beta  represents the buffer capacity of muscle cytosol (Eq. 4), VpH is the rate of pH change in the initial period of recovery, and [H+]PCr is the rate of H+ production associated with PCr resynthesis (defined in Eq. 1). We previously determined a linear relationship between vE proton and the extent of intracellular acidosis measured at the end of exercise (Delta pH) (52). If, in agreement with others (27), we consider that this pH dependence of H+ efflux remains valid during exercise, H+ efflux has been calculated for each minute of exercise on the basis of the Delta pH calculated at the corresponding time of exercise.

Protons produced in exercising muscle are buffered by metabolites (mainly PCr and Pi) and bicarbonate (beta bicarbonate) and nonbicarbonate (beta nonbicarbonate non-Pi) compounds. This composite buffering capacity can be estimated from initial changes in PCr and pH at the onset of exercise as far as an alkalinization, resulting from H+ consumption through the creatine kinase reaction, is recorded. The buffering capacity is then calculated from the ratio of positive changes in pH to the amount of H+ consumed during PCr breakdown (1, 11, 14). When the initial alkalinization is not recorded at the onset of exercise because of the exercise intensity or a low time resolution, the overall buffering capacity can be estimated as previously described (24, 30, 31, 42) taking into account the buffer capacity due to Pi (beta Pi), a variable component, and the buffer capacity mainly due to imidazole groups of histidine (beta nonbicarbonate non-Pi), a constant component, which has been estimated as 20 slykes from several studies in vitro (1) and in vivo (29, 31, 36). In an "open" muscle system, the buffer capacity due to bicarbonate is between 30 slykes at resting pH and 3 slykes at pH close to 6 (26, 45). In the present study, the muscle was assumed to be a "closed" system, in agreement with previous studies in humans (26) and rat leg muscle (1, 28), and buffer capacity due to bicarbonate was set to zero
&bgr; = &bgr;<SUB>nonbicarbonate non-P<SUB>i</SUB></SUB> + &bgr;<SUB>P<SUB>i</SUB></SUB> + &bgr;<SUB>bicarbonate</SUB> (3)
where beta Pi has been calculated from the typical Henderson-Hasselbalch equation based on the dissociation constant of the buffer (K) according to the standard formula (11)
&bgr;<SUB>P<SUB>i</SUB></SUB> = 2.303[H<SUP>+</SUP>] · <IT>K</IT> · [P<SUB>i</SUB>])/(<IT>K+</IT>[H<SUP>+</SUP>])<SUP>2</SUP> (4)
where K = 1.77 × 10-7.

Rate of H+ production from anaerobic glycogenolysis. The overall H+ production associated with nonoxidative ATP production from glycogen breakdown was calculated taking into account H+ uptake associated with PCr breakdown (Eq. 1), H+ efflux (Eq. 2), and pH changes with the buffering power of cytosol taken into account as follows
[H<SUP>+</SUP>]<SUB>&bgr;</SUB> = &bgr; · &Dgr;pH (5)
where beta  is the overall buffering capacity.

Statistical Analysis

ANOVA was performed using the General Linear Models procedure (SAS Institute; options REPEATED, LSMEANS, and CONTRAST).

A one-way ANOVA with repeated measures (repeated factor being time) was used to analyze pH time-dependent changes. F tests were performed to determine the overall effect of time on pH changes. Then multiple comparison procedures (Scheffé's contrasts) were used to compare each value recorded during exercise with the value measured at rest. The onset of intracellular acidosis was determined as the first time point at which pH was significantly different from the resting value.

Two-way ANOVA with repeated measures (repeated factors being time and protocol) was used to characterize the time-dependent changes of each component of the H+ balance. Wilk's lambda  tests were performed to analyze the effects of time and the interaction between time and protocol. Post hoc repeated comparisons (Scheffé's contrasts) were performed to compare values recorded for similar time points and for different exercise protocols.

P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dynamics of Metabolite Content and Intracellular Acidosis

Figure 1 shows a typical series of spectra recorded from the finger flexor muscles during a rest-exercise-recovery experiment. Absolute quantification of phosphorylated metabolites was performed using 8.2 mM ATP as an internal standard according to the values reported from muscle biopsies (20). Metabolic parameters measured at rest (Table 1) did not differ between LF and HF protocols.


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  		Fig. 1.   Typical series of 31P magnetic resonance spectra recorded on finger flexor muscles during rest (1 min), exercise (6 min), and recovery (minutes 1, 2, 5, 10, 15, and 20) of the high-frequency (HF) protocol. Ref, reference compound (phenylphosphonic acid); PME, phosphomonoesters; PCr, phosphocreatine. PCr signal is at -2.45 parts/million (ppm) relative to phosphoric acid on the chemical shift scale (x-axis).


                              
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  		Table 1.   Metabolic parameters measured throughout rest period and at the end of HF and LF protocols

Exercise

Analysis of mechanical and metabolic parameters. As expected, power output measured during exercise increased linearly with respect to time, reaching significantly higher values for the HF exercise (Table 1). Muscular exercise was associated with PCr consumption (Fig. 2B) and intracellular acidosis (Fig. 2A), whereas an accumulation of Pi and ADP was simultaneously observed. Exercise-induced metabolic changes were significantly larger throughout HF than throughout LF exercise (Table 1). However, the increase in [ADP] was similar in the two protocols.


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  		Fig. 2.   Time-dependent changes in pH (A) and PCr concentration ([PCr], B) during transition from rest to exercise. black-lozenge , LF exercise; , HF exercise; arrows, onset of intracellular acidosis for LF (solid) and HF (open) exercise protocol. Values are means ± SE. * Significantly different from LF protocol (P < 0.05).

Onset of intracellular acidosis. A significant pH drop was measured at different times of exercise as a function of the exercise intensity. This net acidosis was not simultaneous with the beginning of exercise but, rather, appeared after the 4th min of exercise for the LF protocol at mean pH 6.95 (P = 0.03) and at the 3rd min of exercise for the HF protocol at mean pH 6.93 (P = 0.001; Fig. 2). The corresponding power output was significantly greater for the HF than for the LF protocol (1.1 ± 0.1 vs. 0.8 ± 0.1 W). [PCr] and [ADP] were similar in the two protocols at the onset of intracellular acidosis (Table 2).

                              
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  		Table 2.   Metabolic parameters measured at onset of intracellular acidosis for LF and HF protocols

Quantitative Analysis of Factors Involved in H+ Balance

The second step of our analysis involved quantifying all the factors interfering with H+ balance during exercise, i.e., the rate of H+ efflux, the rate of H+ consumption through the creatine kinase equilibrium, and the rate of H+ production associated with nonoxidative ATP production (see METHODS). All these factors, except nonoxidative ATP production, are associated with a limitation of pH changes. It could be expected that the onset of intracellular acidosis is associated with a decrease of a factor leading to H+ consumption or an increase in the contribution of glycogenolytic ATP to energy production. Time courses of these factors are presented in Figs. 3 and 4 for HF and LF exercise protocols. Given the striking difference between the pH time-dependent changes for both experimental protocols, we focused on a comparative analysis of both protocols.


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  		Fig. 3.   Time course of rate of H+ changes during LF (black-lozenge ) and HF () exercise protocols. A: kinetics of H+ changes related to H+ efflux. [H+], H+ concentration. B: kinetics of H+ changes related to PCr breakdown. CK, creatine kinase. C: overall kinetics of H+ consumption ([H+]cons). Arrows, onset of intracellular acidosis for LF (solid) and HF (open) exercise protocol. Values are means ± SE. * Significantly different from LF protocol (P < 0.05).



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  		Fig. 4.   Kinetics of H+ changes during LF (A) and HF (B) exercise protocols. black-lozenge , Kinetics of H+ generation; , kinetics of H+ consumption; arrows, onset of intracellular acidosis for each exercise protocol. Values are means ± SE. * Significant difference between sources and sinks of protons for a given protocol (P < 0.05).

Rate of H+ efflux. A linear increase of H+ efflux with respect to time was recorded for both exercise protocols (Fig. 3A). Overall, the rate of H+ efflux was significantly higher for the HF exercise, and contrast analyses disclosed significant differences for the second and third time points of exercise. No acute changes were recorded at times corresponding to significant intracellular acidosis, demonstrating no significant contribution of H+ efflux to the net decrease in pH.

Rate of H+ consumption through creatine kinase. The time-dependent changes in H+ consumption linked to PCr consumption were similar for both exercise protocols, with an initial decrease (Fig. 3B). Thereafter, increasing exercise intensity resulted in rather constant kinetics of PCr breakdown and, consequently, constant kinetics of H+ uptake.

Overall, as shown in Fig. 3B, H+ uptake through the creatine kinase equilibrium during both exercise protocols was rather constant in the face of increasing efflux. To test our hypotheses, we compared H+ generation resulting from nonoxidative ATP production with the processes of H+ consumption and efflux, limiting pH changes by definition and, therefore, for both experimental protocols. Corresponding results are presented in Fig. 4.

For both experimental protocols, the rate of H+ production progressively increased with respect to time, indicating no activation or hyperactivation when a significant pH change was measured. Similarly, a progressive increase in the total rate of H+ production was measured. Interestingly, a significant difference between both processes occurred at the 4th min of exercise for the LF protocol and at the 3rd min of exercise for the HF exercise (Fig. 4), along with the onset of acidosis under both experimental conditions (Fig. 2). This phenomenon clearly indicates that a change in H+ balance, rather than a hyperactivation of nonoxidative ATP-producing processes, underlies the intracellular acidosis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main result of the present study is that the onset of intracellular acidosis recorded during a graded exercise in humans is likely to be the consequence of a change in H+ balance, rather than an activation or hyperactivation of glycogenolysis leading to lactate production.

To demonstrate this cause-effect relation, we have quantified the various components affecting [H+] according to Kemp and Radda (24). This approach is based on several hypotheses, including the theory that the relation of H+ efflux to pH changes previously demonstrated during the recovery period (25, 26, 52) is still valid during the exercise period. To independently check the validity of this assumption, the rate of aerobic ATP production (the value of H+ efflux is taken into account in this calculation) calculated at end of exercise can be compared with the rate of PCr resynthesis at the onset of recovery, which relies exclusively on oxidative metabolism (4, 26, 44, 49). In the present work, the rate of aerobic ATP production calculated from the initial rate of PCr recovery or indirectly during exercise provided similar values, further demonstrating the validity of this quantitative approach. As widely described in the literature, absolute concentrations of phosphorus metabolites were calculated from the ratio of their peak areas to beta -ATP, and the values for PCr/ATP presented in Table 1 are similar to those previously reported in a number of studies related to the same muscle (11-14, 49). We have chosen two different exercise protocols leading to slight and larger exercise-induced intracellular acidosis. In both cases, the onset of intracellular acidosis was delayed to coincide with the start of exercise. This protocol allowed us to obtain two different experimental conditions in which intracellular acidosis occurred at different times of exercise and test the cause-effect relation between intracellular acidosis and H+ balance. In addition, this ramp-type exercise was selected because intracellular acidosis is known to be delayed with incremental loads (22).

Two nonexclusive hypotheses have been tested in the present quantitative work. The first is related to the absence of nonoxidative ATP production as a result of no change in pH. Our results clearly indicate that nonoxidative ATP production occur long before pH is affected significantly. Therefore, our results are not consistent with previous speculations regarding the association between intracellular acidosis and nonoxidative ATP production. In a large number of studies, nonoxidative ATP production has not been consistently related to pH changes (2, 5, 32, 40, 41). In addition, intracellular thresholds have tentatively been defined on the basis of the association between the onset of intracellular acidosis and nonoxidative ATP production without consideration of H+ balance, i.e., production and removal mechanisms (3, 22, 33, 37, 38, 49, 50, 53). In other words, such studies concluding that the onset of intracellular acidosis indicates an activation or a hyperactivation of nonoxidative ATP production have oversimplified the situation, ignoring the mechanisms of production and removal of H+ in exercising muscle. Mechanisms of glycolysis activation have been analyzed in detail, and it is generally accepted that, at the onset of contraction, phosphorylase b-to-a transformation occurs rapidly as a result of increased cytoplasmic calcium concentration. Also, the immediate degradation of PCr rapidly increases Pi concentration, which enables glycogenolysis to proceed at a very high rate (10). Original studies from Conley et al. (11, 14) clearly indicate that glycolytic ATP production is tightly coupled to muscle activation and likely uncoupled from muscle oxygenation and metabolic feedback. They showed in both studies that muscle stimulation did not immediately activate glycogenolysis and glycolysis and that the onset and subsequent rates were dependent on stimulation frequency. In the present study, we reported an immediate nonoxidative glycolytic ATP production that is, considering our time scale, compatible with the results of Conley et al., although a rapid onset of glycogenolysis could reflect a higher exercise intensity, as previously described in dog gastrocnemius muscle (15).

In addition to testing this first hypothesis linking intracellular acidosis with nonoxidative ATP production, we have performed, in the present work, a quantitative analysis of H+ balance in exercising muscle to compare H+ generation resulting from nonoxidative ATP production with the sum of H+ uptake and efflux and to determine the factors that could be associated with the onset of intracellular acidosis. We have clearly shown that the change in H+ generation with increasing exercise intensity exceeds the change in H+ efflux and that the resulting net H+ accumulation accounts for the onset of acidosis. In agreement with the present results, it has been previously shown that H+ generation increases with increasing exercise intensity (11, 14). However, it is, to our knowledge, the first time that the onset of intracellular acidosis is explained in terms of H+ balance. For the LF and HF protocols, H+ consumption through the creatine kinase equilibrium was constant in the face of increasing H+ generation and efflux. For both protocols, changes in pH were not recorded as long as sources and sinks for H+ approximately balanced. In contrast, a significant acidosis occurred after 4 min of LF exercise and 3 min of HF exercise, whereas the rise in H+ generation exceeded the rise in H+ efflux at a nearly constant H+ uptake associated with PCr breakdown. A total dissociation between the onset of intracellular acidosis and nonoxidative ATP production is implicit from these findings. Such a dissociation can be deduced from a careful analysis of several studies performed in healthy subjects and patients. An increased glycogenolytic rate of ATP production during exercise has been calculated in patients with obstructive lung disease, whereas no pH changes were recorded (36). Similar results have been recorded in patients with peripheral vascular disease for whom muscle contraction is ischemic because of the pathological restriction of blood flow (43). Also, in vivo analyses of glycolytic control during ischemic contraction have clearly shown a substantial glycolytic energy production not related to any changes affecting pH (11, 14). More recently, Crowther et al. (17) reported a significant H+ production from glycolysis after 27 s of voluntary ischemic contraction, whereas pH was higher than the resting value. A total dissociation between nonoxidative glycolysis activity and pH changes can also be found from biospy data in humans. Glycolysis and glycogenolytic activities have been estimated from accumulation of glycolytic intermediates on biopsy samples (46, 47). During electrical stimulation in men, no pH changes were recorded in the middle of the contraction period, whereas the glycolysis rate was significant. In addition, a substantial pH decrease was reported as neither glycolytic nor glycogenolytic activity changed (46, 47).

Taking into account the present quantitative analysis and the various processes affecting [H+] during exercise, we have provided additional evidence of a complete dissociation between nonoxidative ATP production and pH changes. Because of the striking difference in acidosis between the experimental protocols, analysis of the underlying H+ balance clearly highlights the acidosis onset mechanism. Whatever the activity level of nonoxidative glycolysis, no pH change occurs as long as sources and sinks for H+ approximately balance throughout the exercise. In contrast, when the rise in H+ generation exceeds the rise in H+ uptake, a significant acidosis can be measured.


    ACKNOWLEDGEMENTS

This work was supported by Centre National de la Recherche Scientifique Grant UMR 6612, the Association Française Contre les Myopathies, Ministère de la Santé Grant PHRC 1997, and the Institut Universitaire de France and the Association pour le Développement des Recherches Biologiques et Médicales au Centre Hospitalier Régional de Marseille.


    FOOTNOTES

Address for reprint requests and other correspondence: D. Bendaham, Centre de Résonance Magnétique Biologique et Médicale (CRMBM), UMR CNRS 6612, Faculté de Médecine de Marseille, 27 Blvd. Jean-Moulin, 13005 Marseille, France (E-mail: david.bendahan{at}medecine.univ-mrs.fr).

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.

First published November 1, 2002;10.1152/japplphysiol.01024.2000

Received 2 November 2000; accepted in final form 20 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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