Current knowledge about pyruvate supplementation: A brief review

Pyruvate is a three-carbon ketoacid that occurs naturally in cells. It is produced through enzymatic reactions in the glycolytic pathway and plays a crucial role in energy metabolism. Despite promising early results, later well-controlled studies of physically active people have shown that pyruvate supplementation lasting more than 1 week has no ergogenic effects. However, some data suggest that ingested pyruvate may be preferentially metabolized without accumulation in the bloodstream. Pyruvate exhibits antioxidant activity and can affect the cellular redox state, and exogenous pyruvate can influence metabolism by affecting the acid-base balance of the blood. This brief review focuses on the potential effects of pyruvate as a supplement for active people. The current state of understanding suggests that studies of the effects of pyruvate supplementation should prioritize investigating the timing of pyruvate intake.


Introduction
Pyruvate (PYR) plays an important role in the major mammalian metabolic pathways.PYR occupies a central position between the catabolic and anabolic pathways involved in the metabolism of carbohydrates, fats, and amino acids (for details see 1,2 ).A closer examination of these pathways indicates that, in most cases, PYR enters the mitochondria before further metabolism.Within mitochondria, PYR may be converted by the pyruvate dehydrogenase complex to acetyl-CoA, which can enter the tricarboxylic acid (TCA) cycle, or it may serve as the starting point for the synthesis of long chain fatty acids, steroids, and ketone bodies.PYR may be used to produce alanine (ALA) via the action of glutamate-pyruvate transaminase (GPT), and may thus play an important role in maintaining the levels of the TCA cycle intermediate, α-ketoglutarate.In the cytoplasm, PYR can be reduced to lactate (LA) via LA dehydrogenase (LDH) or to malate via decarboxylating malate dehydrogenase (malic enzyme: ME).In both cases, the reduced form of nicotinamide adenine dinucleotide (NADH) is oxidized to NAD þ .Therefore, PYR may affect the NADH/NAD þ ratio.

Effects of prolonged PYR supplementation on body weight and composition
The research findings from studies of PYR supplementation as a weight loss aid are summarized in Table 1.The first studies by Stanko et al., [3][4][5][6] showed that prolonged PYR supplementation decreased body weight by significantly reducing body fat mass.4][5][6] Subsequent studies have examined lower doses of PYR supplementation (2-10 g/d) and found conflicting results. 7,8The meta-analysis of six randomized clinical trials showed that PYR was not consistently effective in reducing body weight. 9Two of the studies that found PYR supplementation to be ineffective involved trained athletes who had low body fat composition at the start of the study (15.7% for American football players 10 and 8.8% for soccer players 11 ), and fat loss is unlikely to occur in such lean active people.After excluding the studies involving athletes, we observed inverse relationship between the changes in fat loss and the daily PYR dose (Fig. 1).Because PYR may accelerate fatty acid synthesis through acetyl-CoA, excessive PYR intake may be ineffective in promoting fat loss.

Supplementation and exercise performance
The first studies of PYR supplementation and exercise performance were also conducted by Stanko et al. 13,14 These authors found improvements in aerobic endurance capacity after PYR supplementation, possibly as a result of an increased rate of muscle glucose uptake and sparing of muscle glycogen.However, these studies involved untrained participants who consumed 25 g of PYR per day combined with 75 g dihydroxyacetone.Later studies reported different results. 10,15,16In one study, 5 weeks of PYR supplementation (0.22 g/kg/d) during the training program of American football players did not improve maximal strength, cycle ergometer peak power, and static vertical jump power output. 10imilarly, 2 weeks of PYR supplementation (8.1 g/d) did not improve the ability to maintain power output without fatigue, which is defined as critical power. 15In another study, 1 week of PYR supplementation (7 g/d) did not influence the time to exhaustion during exercise performed  16 These findings have led to a loss of interest in PYR supplementation by athletes. 17 recent study reported higher blood pH, bicarbonate level, and base excess, as well as improved performance during high-intensity interval exercise after PYR supplementation.The study involved male soccer players aged (20 AE 2) years (body fat 13.1% AE 3.5%) with at least 5 years of training experience and _ VO 2 max (55.9 AE 5.4) ml/kg/min of O 2 . 18The supplementation protocol lasted for 1 week and the dose was 0.1 g/kg/d, which provides about 7 g/d of PYR. 18One major difference in the supplementation protocol may explain the differences in results between this study and those mentioned above: on day 7 of the more recent study, the entire daily dose was ingested at least 60 min before the exercise test started. 18[15]

Single-dose PYR ingestion
Morrison et al. 16 measured whole-blood and plasma PYR levels for the 4-h period following various single-dose PYR consumption and found no effect of 7, 15, and 25 g of PYR.The inability to detect any elevation of PYR level in the blood, as well as increased borborygmus and flatulence in subjects consuming higher doses of PYR, led the authors to the speculation that PYR may be decarboxylated in the gastrointestinal tract or eliminated through the feces, and not delivered into muscle cells. 16upplemental PYR may be absorbed by the intestinal epithelium and transported via the portal vein to the liver, where hepatocytes may utilize PYR for gluconeogenesis, 19 which would not affect circulating PYR levels.Blood glucose level is also not significantly affected by various PYR doses. 16However, in another study, an increase in the resting respiratory exchange ratio 3 h after a single oral intake of PYR suggested greater carbohydrate oxidation. 20In addition, the increase in plasma free fatty acid level observed in placebo was attenuated 3 h after acute ingestion of 7 g of PYR 20 and 4 h after intake of 25 g of PYR compared with the 7 g dose. 16These findings suggest that PYR may be used as a preferential energy source in the human body without increasing blood PYR levels.
The rate-limiting step of citrate formation in the TCA cycle is the low concentration of oxaloacetate (OAA) in mitochondria, 21 and it is possible that PYR could be used to replenish mitochondrial OAA levels.In nonmuscle cells, PYR can be carboxylated to OAA, and may play a role in the muscles in supporting OAA formation by the generation of greater α-ketoglutarate concentration via GPT (Fig. 2). 1 Intravenous PYR infusion increases the levels of TCA cycle intermediates, mainly malate, in skeletal muscles. 22he uptake of PYR by cells depends on the monocarboxylate transporter system. 23This system is located in the plasma membrane and transports monocarboxylates together with H þ (Fig. 2), which may indirectly spare blood bicarbonate and increase blood pH. 24Because of sodium-coupled transport, the effects of sodium PYR (NaPYR) differ from those of calcium PYR (CaPYR) in terms of their alkalosis-inducing effects. 20The buffering property of NaPYR has been described in an intravenous infusion study, 25 although intravenous PYR infusion has been found to be ineffective in increasing muscle PYR content. 22Moreover, single oral PYR intake causes an increase in blood pH, bicarbonate level, and base excess. 20,26 recently reported PYR supplementation protocol did not modify aerobic energy contributions during high-intensity interval exercises, 18 a finding that agrees with previously published results. 16In the recent study, no changes were observed in the contributions of glycolytic energy after PYR supplementation despite modification of the blood buffering capacity.Interestingly, PYR supplementation improved phosphagen energy system regeneration during four sessions of 1-min cycling at 110%  Wmax, interspersed with 1 min recovery periods, and six sessions of 6 s maximal cycling sprints, interspersed with 24 s passive recovery periods. 18In addition, peak power output and mean power output increased.Although, supraphysiological PYR concentration in a perfusing solution increases phosphagen levels and improves contractile properties of stressed myocardium, 27 there is no evidence that oral PYR supplementation, even in the form of creatine-PYR, affects muscle creatine content and/or performance 10,15 (this topic has been reviewed in detail elsewhere 28,29 ).Therefore, it has been suggested that improved exercise performance may be achieved by increased phosphocreatine resynthesis and muscle contraction through a decrease in H þ concentration. 18However, PYR supplementation at a higher dose, and for a longer period does not improve explosive power, 10 or delay fatigue. 15he resulting differences between studies may be explained by alkalization induced by a PYR dose ingested 60 min before the exercise tests. 18lternatively, different forms of PYR may also explain resulting differences between studies.For example, NaPYR supplementation at a dose of 0.10 g/kg/d for 1 week is ergogenic, 18 whereas CaPYR at a dose of 0.22 g/kg/d for 5 weeks is not, 10 possibly because of the different alkalosis-inducing effects. 20

Redox state modulation
The nonenzymatic reaction of PYR with hydrogen peroxide was described more than 100 years ago, 30 as follows: Later studies confirmed the PYR antioxidant properties in various models [31][32][33][34] and indicated the direct scavenging potential toward peroxynitrite, 35 hydroxyl radical, 36 and superoxide anion radical. 37In addition to the antioxidant properties of PYR, other mechanisms of PYR stress-ameliorating effects have been proposed.These include an increase in sarcoplasmic reticular Ca 2þ transport, improvement in mitochondrial function, increase in ATP concentration, or support of NADPH production to maintain the glutathione/glutathione disulfide (GSH/GSSG) redox potential (for review, see 27,38 ).Another possible mechanism of PYR-induced protection is the modulation of cellular redox potential by decreasing the cytosolic NADH/NAD þ ratio. 39n our study, a single-dose PYR ingestion caused a greater increase in blood LA concentration after exercise at 90% _ VO 2 max compared with placebo. 26This result may reflect elevated blood bicarbonate concentration. 20,26It is possible that a higher LA concentration may also be caused by the reaction of PYR with accumulated NADH.In muscle cells, NADH is oxidized continuously by the malate-aspartate shuttle or by LDH. 40During exercise at a higher intensity, the rate of anaerobic glycolysis and the concentration of NADH in cytosol increase. 41Therefore, ingestion of PYR may not modify blood LA concentration at rest 16 but may affect the redox state of muscle cells and accelerate LA production during exercise at higher power output (Fig. 3). 26

Mechanisms of adaptation
The recognition of NAD þ as a multifunctional signaling molecule has been driven mainly by research in the field of exercise and nutrition.One of the key cell signaling candidates proposed is the NAD-dependent mitochondrial deacetylases familysirtuins (SIRTs).SIRTs modulate many cellular processes, including energy metabolism, mitochondrial biogenesis, and protection against oxidative stress (these topics have been reviewed in detail elsewhere [42][43][44] ).
Because NAD þ is a coenzyme of the reaction catalyzed by SIRTs, the activity of these enzymes increases when NAD þ concentration increases.By contrast, the expression follows a different pattern; that is, the enzyme expression is induced by an increase in the NADH/NAD þ ratio (predominantly a simultaneous decrease in NAD þ and increase in NADH concentrations). 45Physical exercise modulates the redox state of muscle cells according to the exercise intensity.During low or moderate exercise intensity, cytosolic NADH is continuously oxidized by the malate-aspartate shuttle, which controls the NADH/NAD þ ratio. 40At higher exercise intensity, the accumulation of cytosolic NADH can constrain glycolysis and thus limit performance. 40Therefore, a higher turnover via LDH is necessary to restore the NADH/NAD þ ratio.The modulation of the NADH/NAD þ ratio by exogenous PYR may impact histone deacetylase activity but only during exercise performed at intensities higher than the LA threshold.

PYR and neuroprotection
The brain is a highly energy-demanding organ, and its proper functioning depends on an adequate supply of energy substrates. 46,479][50] A diet enriched with PYR and β-hydroxybutyrate for 5 weeks, averaging ~26 mg daily intake of substrates, improved cerebral energy metabolism in transgenic mice.This involved mitigating glycogen depletion and NAD(P)H autofluorescence. 48Subsequent study on PYR alone confirmed increased brain glycogen storages, and indicated elevation of energy metabolites, such as creatine, LA, and glutamate. 49oivisto and colleagues 49 demonstrated that long-term NaPYR supplementation (~800 mg/kg/d for 2-6 months) increased exploratory behavior in both wild-type and AD transgenic mice, highlighting its effects on cognitive function.
In addition to its well-recognized function in energy metabolism, PYR may be an effective neuroprotector to reduce the rate of cognitive decline. 51A recent study reported that the intraperitoneal injection of ethyl pyruvate (EtPYR) promoted the expression of brain-derived neurotrophic factor (BDNF) by astrocytes and astrocyte transdifferentiation into oligodendrocytes, which suggested that PYR treatment may help to facilitate myelin sheath regeneration. 52Given that triggering BDNF expression in the brain is recognized as having neuroprotective effects, 53 BDNF stimulation by PYR may have neuroprotective properties and improve cognitive performance.
PYR acts as both an antioxidant and anti-inflammatory compound, and may protect neurons from oxidative stress and neuroinflammation, both of which are associated with cognitive decline and neurodegenerative disorders. 49,54Systemic administration of PYR has been reported to have neuroprotective effects in animal models of brain injury, 55 hypoglycemic cognitive impairment, 56 ethanol-induced neurodegeneration, 57 and age-dependent cognitive deficits in a mouse model of AD. 50The results of in vivo studies showing a neuroprotective effect of PYR are summarized in Table 2.In addition, in vitro studies have shown that PYR has protective effects against glutamate neurotoxicity, 58 neuronal cell death induced by hydrogen peroxide, 59 oxygen-glucose deprivation, 60 and zinc-induced cortical neuronal death. 61

Future directions
Further research is warranted to elucidate the optimal timing, dosage, and form of PYR supplementation for maximizing its potential benefits on exercise performance, body composition, and metabolic health.
Deeper mechanistic studies are needed to unravel the molecular pathways underlying PYR's diverse effects on metabolism, cellular redox state, and exercise physiology.This includes investigating its interactions with key enzymes, signaling molecules, and metabolic pathways involved in energy production, antioxidant defense, and cellular signaling.
Conducting well-designed studies in specific populations, such as individuals with obesity, metabolic disorders, neurodegenerative diseases, or athletes, could provide valuable insights into the efficacy of PYR supplementation in diverse contexts.
Given the emerging role of gut microbiota in modulating host metabolism and health, investigating the impact of PYR supplementation on gut microbiota composition and function could provide novel insights into its metabolic effects and potential mechanisms of action.

Conclusions
PYR is an important compound in aerobic and anaerobic energy metabolism.PYR ingested before high-intensity exercise can affect power output.Prolonged supplementation, especially in combination with interval training, may induce adaptive changes in skeletal muscle metabolism and/or affect cognitive function through neuroprotective effects.It may be of interest to study the effects of PYR supplementation by focusing on the optimal timing, dosage, and form of PYR intake.

Submission statement
The manuscript has not been published previously, is not under consideration for publication elsewhere, and is approved by all authors.If accepted, it will not be published elsewhere, including electronically in the same form, in English or any other language, without the written consent of the copyright holder.

Fig. 1 .
Fig. 1.Daily dose of PYR supplementation effect on body fat loss.( Â ) studies on athletes with low body fat content excluded from the analysis; dotted lines represent 95% confidence interval.

Table 1
Main results of PYR supplementation on weight loss studies.

Table 2
Main results of PYR neuroprotective studies.