Metabolic acidosis is an acid–base balance disorder caused by the lack of alkaline substances and/or the excess of acidic substances (except CO₂) in blood (1). During high-intensity exercise, H⁺ released by adenosine triphosphate (ATP) hydrolysis cannot be cleared out of cells in time, which is the main cause of metabolic acidosis (2, 3). Therefore, exercise-induced metabolic acidosis is due to a decrease in mitochondrial oxidative phosphorylation for energy supply under relative hypoxia induced by high-intensity exercise, which relies on anaerobic glycolysis for energy supply. This phenomenon leads to the accumulation of H⁺ and lactic acid in tissues and the reduction of the blood buffer substance HCO₃⁻ (3, 4). Previous studies have shown that high-intensity interval exercise (HIIE) can induce metabolic acidosis in a short time (5–7). High-intensity exercise can increase ATP demand rate by 1,000 times relative to that in the resting state. This process relies on glycolytic ATP hydrolysis for energy production, and the generated H⁺ cannot be completely removed during the relief interval. With the repetition of exercise bouts, metabolic acidosis is induced. Acidosis in this exercise mode is a unique form of metabolic change (8, 9). In addition, this type of exercise is an important constitute of many sports that require intermittent explosiveness (especially team sports). on the other hand, the inhibiting effects of low pH on Ca²⁺ sensitivity, velocity and peak power in HIIE support the conclusion that acidosis is an important fatiguing agent (10). As such, exploring the approach to relieve metabolic acidosis in HIIE has guiding significance for delaying exercise fatigue in competitive sports.

Pyruvate is a key intermediate in the body’s metabolic pathway. Furthermore, it can be used as a new alkaline buffer in clinical practice to exert superior biomedical properties through exogenous supplementation (11–13). In recent years, studies on clinical trials have verified the corrective effect of exogenous pyruvate on metabolic acidosis, which can relieve blood acidity by combining weak base anions with H⁺ (14, 15). However, the effect of pyruvate on metabolic acidosis is not only the result of its chemical buffering ability as an alkalizing agent but may also be mainly through its biological properties to consume excess H⁺, thus effectively avoiding or correcting metabolic acidosis (16). Notably, the lactate dehydrogenase (LDH) reduction reaction has a unique advantage in the pathway of pyruvate-correcting metabolic acidosis. First, pyruvate as a sole substrate, can be spontaneously converted into lactate by LDH, and the carboxylate anion of each exogenous pyruvate absorbs an H⁺ from the cytoplasmic hydrogen pool in this process. The reduction of pyruvate is a systemic alkalinizing reaction with H⁺ consumption, raising the pH in hypoxia and even in anoxia (3, 17). Another important function of the LDH reaction is the production of cytosolic nicotinamide adenine dinucleotide (NAD⁺), which supports the NAD⁺ substrate demand of the glyceraldehyde 3-phosphate dehydrogenase reaction. This process then improves the maintenance of the cytosolic redox potential (NAD⁺/reduced NAD⁺ [NADH]) and supports the continuous substrate flux through phase two of glycolysis, which allows constant ATP regeneration from glycolysis (3, 14) (see Supplementary Material).

At present, the effect of short-term pre-supplementation of pyruvate on exercise-induced metabolic acidosis has been scarcely reported. It was preliminarily confirmed in human experiments in our laboratory that pyruvate supplementation may accelerate restoration of the acid–base balance, but its internal mechanism remains unclear (18). Moreover, whether oral pyruvate can alleviate acidosis by improving the LDH reaction needs further verification. Available evidence has shown that exogenous pyruvate can treat pathological metabolic acidosis (e.g., hypoxic metabolic acidosis associated with lethal hemorrhagic shock) in rats, raise intracellular pH (pH_i) and improve glycolysis in hypoxic, ischemic or even anoxic conditions by sustaining the NAD⁺/NADH with the LDH reaction (14). Thus, we hypothesized that pyruvate pre-supplementation can effectively prevent HIIE-induced metabolic acidosis, mitigate acidification of blood and skeletal muscle in rats by accelerating H⁺ consumption in LDH reaction and provide beneficial effects in improving redox state and glycolysis metabolism. Acid metabolism variables, redox status, lactate dehydrogenase A (LDHA) enzyme activity and rate-limiting glycolytic enzyme activities are determined in rat blood or skeletal muscle.

The main finding of this study is that pyruvate taken orally for 7 days can promote the absorption of H⁺ in the LDH reaction, further preventing exercise-induced metabolic acidosis. Consistent with this finding, the decreased pH_i, pH_e, BE, and HCO₃⁻ after HIIE was reversed, and LDHA activity was significantly increased in the EP. In addition, pyruvate pre-treated skeletal muscle redox status and glycolytic enzyme activity were improved. These results provide compelling evidence that oral pyruvate can prevent HIIE-induced metabolic acidosis in rats by promoting the LDH response, while simultaneously improving the redox state and glycolysis rate.

Arterial blood gas analysis can assist in the assessment of the acid–base balance in clinical practice. Metabolic acidosis is diagnosed when the parameters most frequently used—pH (7.35–7.45), BE (−2 ± 2 mM) and HCO₃⁻ (22–26 mM)—fluctuate below the standard range (1). In current human experiments that performed different combinations of HIIE in 4–6 bouts, with each bout approaching or exceeding the maximum intensity exercise from 30 s to 1 min, the body is acidified to varying degrees, resulting in increased lactate and HCO₃⁻ levels and decreased pH_i (muscle tissue)/pH_e (blood) levels (5–7, 24). In animal experiments, it was also found that performing a six–bout high-intensity exercise for 2 min each at 80% peak speed with a 1-min interval, the pH_i has been decreased to 6.6 (6.54–6.66) (25). In our study, pH_e, pH_i, BE, HCO₃⁻ levels were significantly decreased, and LAC level was significantly increased after HIIE compared with the CC. Although the post-exercise blood pH (pH_e = 7.37 ± 0.04) slightly exceeded the lower limit of the clinical standard range (1), it was still lower than the normal range of blood pH in rats reported in previous studies (26), and other blood gas indexes all satisfied the criteria for metabolic acidosis. Skeletal muscle pH also decreased to approximately 6.5, which is consistent with the results reported by Hedges et al. (27). From the abovementioned findings, the exercise mode of HIIE in this study successfully induced metabolic acidosis.

Pyruvate can effectively correct metabolic acidosis, an effect which has been demonstrated in many preliminary experiments. As early as 1999, Mongan et al. first highlighted the “systemic alkalization” induced by intravenous pyruvate (28). It was found that pyruvate injection significantly increased pH, BE, and HCO₃⁻ levels and prolonged survival time in a swine hemorrhagic shock model. The phenomena in which pyruvate increased pH and BE values in swine and rats are then discovered one after the other on pathological metabolic acidosis (29–31); however, no additional studies have investigated the effect of oral pyruvate on exercise-induced metabolic acidosis, especially on skeletal muscle pH, in exercise rats. Our data showed that oral pyruvate significantly reversed the decline in pH_e, BE, HCO₃⁻, and pH_i values after HIIE in rats. This is explained by the more H⁺ consumption via the LDHA reaction following the pyruvate supplementation (as described below) and thus alkaline substances in blood were preserved. The preliminary results presented within this study suggest that oral pyruvate is effective in alleviating exercise-induced metabolic acidosis, similar to that found in models of pathological metabolic acidosis (19), and alkalizing both intracellular and extracellular environments.

Lactate dehydrogenase A activity in the current investigation was significantly elevated in the EP compared with that in the EC. To our knowledge, this report is the first to demonstrate the significant effect of pyruvate on up-regulating LDHA activity in skeletal muscle, aiming to explore the possible mechanism for acidosis alleviation. As a marker enzyme of anaerobic metabolism, LDHA can promote LDH reaction in the direction of consuming H⁺, and when the oxygen supply is limited, the regenerated NAD⁺ can further maintain glycolysis (32). Notably, NAD⁺, as a cofactor for several enzymatic activities, can provide the need to maintain continuous muscle activity, and the NAD⁺/NADH ratio is a key factor that reflects cellular redox status and regulates cellular metabolism (33). In this study, increased LDHA activity in pyruvate-treated exercised rats may contribute to the elevation of NAD⁺/NADH ratio. Pyruvate can convert pyruvate to lactate through the coupling of LDH to NADH oxidation, while elevating NAD⁺/NADH ratio and accelerating proton depletion (14, 34). However, there was no significant increase in blood LAC level in the current study, which could be attributed to lactate transport and further absorption and utilization by other tissues. As previous studies have concluded, when lactate flux increases, monocarboxylate transporters (MCTs) will promote lactate anion to co-cross the membrane barrier with H⁺(1,1 molecular ratio), and continue to act as metabolic substrates and key signaling molecules in skeletal muscle, heart, brain, and other tissues (35–37). Therefore, we hypothesize that pyruvate may further improve the lactate transport rate while accelerating the lactate production, so there was no accumulation of LAC, which provides the direction for future studies to further explore the effect of pyruvate on lactate transport. Furthermore, pyruvate pre-treatment reduces L/P ratio, and the stabilization of this ratio suggests that a large NAD⁺/NADH ratio is preserved with decreasing L/P ratio (38). The trend toward increased NAD⁺/NADH ratio and reduced L/P ratio in animal models of metabolic acidosis after pyruvate pre-treatment, as observed in the previous studies, may be indicative of pyruvate that can increase the redox potential in multiple tissues (38, 39). In the current study, no significant differences in NAD⁺/NADH and L/P were observed between the EP and EC. This may be partly explained by the fact that NAD⁺ is easily degraded and unstable in vitro due to the influence of temperature or acid–base environment, so it is easy to transform with NADH. Similarly, lactate concentration will change greatly with the delay of time, while pyruvate concentration remains relatively constant, and the L/P ratio will also fluctuate (40–42). However, compared with the quiet control group, EP showed a significant increase in NAD⁺/NADH ratio, and EC exhibited a significant increase in L/P ratio. Given that the increase in L/P ratio after exercise-induced acidosis indicated impaired redox status, which was mitigated by pyruvate pre-treatment with no difference from the quiet status, and accompanied by an increase in the NAD⁺/NADH ratio, the results of the current pilot study may provide a preliminary indication that the redox status may be improved following pyruvate pre-treatment. Further studies on the effects of a pyruvate pre-treatment on NAD⁺/NADH ratio and L/P ratio are required however, given the small sample size used in the current study. The present study adds to the current knowledge that the up-regulation of LDHA activity may be the key to promoting the LDH reaction after pyruvate supplementation, which contributes to the accelerated depletion of H⁺, alleviates the phenomenon of exercise-induced metabolic acidosis, and potentially improves the redox state.

The key rate-limiting enzymes in the glycolytic pathway include HK, PFK and PK. Previous studies have shown that HK (43), PFK (44), and PK (45) increase skeletal muscle activity after intense exercise in rats. Both HK and PFK activities showed similar responses after acute HIIE. However, our data showed no significant increase in PK activity after HIIE in the control group. This difference may be due to a decrease in pH of muscle tissue, inhibiting its activity or different movement patterns. Pyruvate enhances NAD⁺/NADH ratio simultaneously induced in the pyruvate reductive reaction, promoting the regular glycolysis pathway (34, 46). Increases in HK, PFK, and PK activities after pyruvate supplementation in exercised rats are found to be regulated by hypoxia-inducible factor-1α (HIF-1α). Some previous studies have reported that pyruvate inhibits HIF-1α degradation and enhances HIF-1α activity. HIF-1α is a key transcription factor that upregulates a series of downstream genes involved in glucose metabolism and promotes the expression of glucose metabolization-related transporters and enzymes. Such as the expression of glucose transporters, phosphoglycerate kinase 1 (PGK1), pyruvate dehydrogenase kinase 1 (PDK1), LDHA and glycolytic enzymes such as HK1, HK2, PFK1, PFK2 and PKM2 (16, 47, 48). The up-regulation of these proteins is conducive to the increase of glycolysis rate and the consumption of H⁺. In our study, oral pyruvate upregulated the activity of three key rate-limiting enzymes in the glycolytic pathway in HIIE rats, which may be partly responsible for the increased glycolytic rate. Given the limitations of the current study on interpretation at the protein or gene level, further research is warranted, especially on the regulatory mechanisms of HIF-1α.

The results of the study provide strong evidence that 7-day oral pyruvate can effectively attenuate HIIE-induced intracellular and extracellular acidification, which may be explained by increased LDHA activity, thus promoting the LDH reaction to absorb H⁺. The beneficial effect of pyruvate is also explained, in part, by the improvement of redox state and the increase of glycolysis rate in skeletal muscle. The findings of the study suggest that pyruvate can be used to design nutraceutical supplements aimed to preserve homeostasis against HIIE-induced metabolic acidosis, which may be important in preventing exercise fatigue and ensuring the energy supply during sports competitions and its rapid recovery afterwards.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Review Board of Beijing Sport University (BSU IRB) (No. 2020057H).

KC was responsible for the operation of the experiment, data collection, data interpretation, writing and revision of the manuscript, under the direction and assistance of JQ who assisted with each step and completion of the manuscript. YY, JZ, and YX assisted in the completion of the experiment. LF and QL assisted in the revision of the manuscript. All authors contributed to the article and approved the submitted version.

Funding was provided by the Herbalife Winter Sports Nutrition Research Fund (Nos. KBL2019003 and KBL2021007) and the Fundamental Research Funds for the Central Universities (No. 2021QN028).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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