Reaction rate of pyruvate and hydrogen peroxide: assessing antioxidant capacity of pyruvate under biological conditions

Pyruvate, a pivotal glucose metabolite, is an α-ketoacid that reacts with hydrogen peroxide (H2O2). Its pharmacological precursor, ethyl pyruvate, has shown anti-inflammatory/anti-tissue injury effects in various animal models of disease, but failed in a multicenter clinical trial. Since rodents, but not humans, can convert ethyl pyruvate to pyruvate in blood plasma, this additional source of extracellular pyruvate may have contributed to the discrepancy between the species. To examine this possibility, we investigated the kinetics of the reaction under biological conditions and determined the second order rate constant k as 2.360 ± 0.198 M−1 s−1. We then calculated the time required for H2O2 elimination by pyruvate. The results show that, with an average intracellular concentration of pyruvate (150 µM), elimination of 95% H2O2 at normal to pathological concentrations (0.01–50 µM) requires 141–185 min (2.4–3 hour). With 1,000 µM pyruvate, a concentration that can only exist extracellularly or in cell culture media, 95% elimination of H2O2 at 5–200 µM requires 21–25 min. We conclude that intracellular pyruvate, or other α-ketoacids, whose endogenous concentration is controlled by metabolism, have little role in H2O2 clearance. An increased extracellular concentration of pyruvate, however, does have remarkable peroxide scavenging effects, considering minimal peroxidase activity in this space.

by glycolysis, pyruvate is decarboxylated to acetyl CoA, which can either enter the tricarboxylic acid (TCA) cycle and generate ATP via oxidation, or participate in fatty acid synthesis. Pyruvate can also be carboxylated to oxaloacetate, which replenishes the TCA cycle or enters the gluconeogenesis pathway to generate glucose. Transamination converts pyruvate to alanine, which participates in protein synthesis. Under oxygen-deficient conditions, pyruvate is reduced to lactate. These pathways comprise regulated enzymatic reactions, which result in maintaining a relatively constant concentration of pyruvate intracellularly.
In the present study, we first examined the reaction order of pyruvate and H 2 O 2 to confirm the concentration dependence of the reactants. The rate constant (k) was then determined, since there are no reports in the literature of the k obtained under physiological temperature, pH, and ionic strength. With this biomarker rate constant in hand and the biologically relevant concentrations of H 2 O 2 and pyruvate described above, the rates of H 2 O 2 elimination by pyruvate were calculated and confirmed by experimental measurements. To evaluate the reaction under biological conditions, experiments in this report were all carried out at 37 °C in Dulbecco's phosphate-buffered saline (DPBS), which has a pH of 7.3 ± 0.2 and ionic strength of 165 mM. The reaction order of pyruvate was estimated by reacting a fixed concentration of H 2 O 2 (20 µM) with increasing concentrations of pyruvate (40, 80, 160, and 320 µM) for 5 min. Hydrogen peroxide concentration in the solutions was subsequently measured by a peroxidase-Amplex Red colorimetric assay. As shown in Fig. 2A, the increase in the average reaction rate was approximately proportional to the increase in pyruvate concentration, indicating that the reaction was first order with respect to pyruvate, or a =1. To determine the reaction order of H 2 O 2 , 200 µM pyruvate was reacted with increasing concentrations of H 2 O 2 (400, 800, 1600, or 3200 µM) for 3 min. Pyruvate concentration was then measured using an HPLC method. The average reaction rate was found to increase proportionally to that of H 2 O 2 concentration, indicating a first order reaction with respect to H 2 O 2 , or b = 1 (Fig. 2B). Taken together, the overall reaction order is two. Note that the reaction rate was measured as an average rate rather than an instantaneous rate and is less accurate than the latter; this approach should not, however, affect the estimation of the reaction order. As a demonstration of the method used for the above measurements, Fig. 2C,D show the HPLC chromatograms of the pyruvate and its standard curve. Pyruvate was derivatized to become fluorescent prior to HPLC analysis. Hydrogen peroxide was measured by a peroxidase-Amplex Red assay. The color product of the assay exhibits both fluorescence and visible color. Hydrogen peroxide samples at low concentration range (0-2 µM) were read with fluorescence and high concentrations (0-20 µM) with absorbance (OD 560 ) in this assay. The standard curves in both ranges are linear (Fig. 2E,F

Reaction order with respect to pyruvate and H
The rate constant k was determined by reacting pyruvate with H 2 O 2 , at the initial concentration of 300 µM, for 0, 2, 4, 6, 8, 10, and 12 min; pyruvate concentration was then measured by the HPLC analysis. As shown in Fig. 3, pyruvate concentration decreased over time, which fits a second-degree polynomial equation (Fig. 3A); plotting the inverse concentration of pyruvate vs. time gives a linear relationship, with a slope of 2.360 and an intercept of 3285 (Fig. 3B). Compared to Eq. (3), the plot indicates that the reaction rate constant k is 2.360 M −1 s −1 , with [Pyr] 0 is 0.000304 M or 304 µM by this measurement. Figure 3 was plotted with data sets from 6 replicate   To confirm these HPLC measurements, a LC-MS method was used to measure the concentrations of pyruvate and acetate in the reaction solutions. As shown in Fig. 4A (4), in which x represents the amount of the reactants that had undergone transformation.
The integration of Eq. (4) from 0 to t and from 0 to x gives Eq. (5), which allows for the calculation of the time required for x amount of H 2 O 2 to be reacted. To confirm these calculations, experiments were carried out by reacting 150 µM pyruvate with 0.1, 1, 5, or 50 µM H 2 O 2 . After a 33 min incubation, the H 2 O 2 concentration in the reaction solution was measured. The H 2 O 2 concentrations were also calculated under these conditions using the method shown in Table 2. The measured values were consistent with what was calculated (Table 3). Similarly, 1,000 µM pyruvate was reacted with 5, 10, 50, 100, and 200 µM H 2 O 2 for 22 min, and the H 2 O 2 concentrations were subsequently measured and compared with calculated values (Table 3).
Confirmation was also carried out by reacting 50 µM H 2 O 2 with 1,000 µM (Fig. 6A) or 150 µM pyruvate (Fig. 6B) and measuring the concentrations of H 2 O 2 and pyruvate, respectively, after various incubation times. The corresponding time course was calculated according to Table 2. As shown in the figure, the measured and calculated time courses of the reaction aligned with each other. The data sets were fitted to exponential decay equations in Fig. 6A and second-degree polynomial equations in Fig. 6B.

Discussion
This study examined the reaction rate of pyruvate and H 2 O 2 under biological conditions. The reaction was determined to be first-order with respect to each reactant (Fig. 2), and the second order reaction rate constant k was 2.360 ± 0.198 M −1 s −1 or 0.000142 ± 0.000012 µM −1 m −1 (Figs. 3 and 4). Reaction rates were calculated with the determined k value using the concentrations of pyruvate and H 2 O 2 observed under various biological conditions. www.nature.com/scientificreports www.nature.com/scientificreports/ The analysis showed that in the presence of an average intracellular concentration of pyruvate (150 µM), 50% elimination of H 2 O 2 at normal to pathological concentrations (0.01-50 µM) requires 33-36 min, and 95% elimination takes 141-185 min (2.4-3 hr). In the presence of 1,000 µM pyruvate, 50% elimination of H 2 O 2 at 5-200 µM requires only 5 min (4.9-5.2 min), and 95% elimination takes 21-25 min (Fig. 5). Experimental measurements confirmed these calculations (Table 3 and Fig. 6).
In the extracellular space, the rate of H 2 O 2 elimination by 1,000 µM pyruvate (95%, 21-25 min) is rapid, considering minimal peroxidase activities present in this compartment and a 100-to 500-fold higher H 2 O 2 concentration in plasma compared to the intracellular environment. Inflammation-activated NOXs can result in an extracellular accumulation of H 2 O 2 . Removing this pool of extracellular H 2 O 2 by pyruvate would attenuate the oxidative stress/injury on the cells.
Ethyl pyruvate has been administered to mice and rats at doses of 40-100 mg/kg 1 , which would translate to blood concentrations of 4.4-12.3 mM using a blood-to-body weight ratio of 7% 22 . If ethyl pyruvate is activated by carboxylesterase in blood plasma, it will achieve an extracellular concentration close to 1,000 µM. At this concentration, pyruvate can markedly reduce the extracellular H 2 O 2 level before entering cells to be metabolized. By contrast, when ethyl pyruvate can only be activated intracellularly (due to lack of plasma carboxylesterase), the generated pyruvate would be rapidly metabolized, having little impact on H 2 O 2 levels. Thus, the conflicting results of the effects of ethyl pyruvate comparing rodent studies and the human trial may be related to the site of in vivo activation of the compound.
Based on the mechanism shown in Fig. 1, pyruvate could react with lipid peroxides and peroxynitrite in the same manner, forming an α-carbon adduct intermediate and releasing lipid hydroxide and nitrite as products, respectively. Thus, pharmacological concentrations of pyruvate could also scavenge these reactive oxygen species in the cell membrane, as well as other compartments. The same mechanism could also apply to other α-ketoacids, e.g., α-ketoglutarate and oxaloacetate. Nevertheless, the rate constant for these α-ketoacids would be lower than that of pyruvate owing to a bulkier alkyl chain 23 . The low intracellular concentrations of these metabolites, as with pyruvate, would, therefore, prevent them from having any practical "antioxidant" effects in that compartment.
It is also worthwhile noting that pyruvate is vital for primary cell culture and is added to various cell culture media at a concentration of 1,000 µM. Many oxidative stress studies add H 2 O 2 to culture medium as an oxidant stress for cells, usually with an extended incubation period. As the added H 2 O 2 is eliminated rapidly by pyruvate in these media, 50% in 5 min and 95% in 25 min, caution must be taken in the interpretation of the results, especially when comparing different studies.

Reaction of pyruvate and H 2 o 2 .
Pyruvate and H 2 O 2 reactions were carried out in DPBS at 37 °C in a total volume of 5 mL. Before the reaction, DPBS was incubated in a 37 °C water bath for at least 30 min to attain a stable temperature. Small aliquots of pyruvate and H 2 O 2 stock solutions (<0.2 mL) were added to the DPBS solution to achieve the final concentration. Reactions were carried out at 37 °C for the designated times, and aliquots were removed for subsequent analysis. Owing to the time-sensitivity of the measurements, mixing and removing the reaction solution was staggered in order. For the measurements of pyruvate and acetate concentrations (by HPLC or LC-MS methods, see below), the reactions were stopped by adding 20 units catalase (10 unit/µL) to a 150 µl reaction mixture. For measurement of H 2 O 2 concentration (by peroxidase-Amplex Red method, see below), aliquots of the reaction solution were directly loaded into a 96 well plate and immediately mixed with horseradish peroxidase and Amplex Red reagents to stop the reaction. Corresponding standards were included in each experiment and processed in the same way as samples.
HpLc measurement of pyruvate. The HPLC measurement of pyruvate followed a previously reported method with modifications 14 . Samples and pyruvate standards were derivatized by combining 1:1 by volume with O-phenylenediamine (OPD) dissolved at 25 mM in 2 M HCl, and incubated at 80 °C for 30 min. Derivatized pyruvate was quantified by HPLC with fluorescence detection using an Agilent 1260 Infinity II system with binary pump (G7112B) and fluorescence detector (G7121A). Samples were separated using an InfinityLab Poroshell 120 HPH C18 column (2.1 ×100 mm, 2.7 µm) with a guard column. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The injection volume was 5 µL. The mobile phase flow www.nature.com/scientificreports www.nature.com/scientificreports/ rate was 420 µL/min. The autosampler sample compartment was maintained at 4 °C, and the column oven was set at 40 °C. The mobile phase gradient (%B) was 0 min, 5%; 11 min, 60%; 11.1 min, 5%; 14.1 min, 5%. The fluorescence λ ex was 350 nm and λ em 410 nm with 100 LU attenuation. Peak areas were integrated using OpenLab CDS ChemStation (Agilent) and sample pyruvate concentrations calculated from a standard curve.

Lc-MS measurement of pyruvate and acetate concentrations. LC-MS analysis was performed
on a Vanquish ultra-high performance liquid chromatography system coupled to a Q Exactive mass spectrometer (Thermo) that was equipped with an Ion Max source and HESI II probe adapting previously described methods 24,25 . External mass calibration was performed every seven days. Metabolites were separated using a ZIC-pHILIC stationary phase column (2.1 ×150 mm, 5 µm; Merck) with a guard column. Mobile phase A was 20 mM ammonium carbonate and 0.1% ammonium hydroxide. Mobile phase B was acetonitrile. The injection volume was 1 µL, the mobile phase flow rate was 100 µL/min, the column compartment temperature was set at 25 °C, and the autosampler compartment was set at 4 °C. The mobile phase gradient (%B) was 0 min, 80%; 10 min, 50%; 10.5 min, 8%; 14 min, 8%; 14.5 min, 80%; 25 min, 80%. The column effluent was introduced to the mass spectrometer with the following ionization source settings: sheath gas 40, auxiliary gas 15, sweep gas 1, spray voltage -3.0 kV, capillary temperature 275 °C, S-lens RF level 40, probe temperature 350 °C. The mass spectrometer was operated in targeted selective ion monitoring mode for pyruvate (m/z 87.0088) and acetate (m/z 59.0135) with 2 m/z isolation window. The resolution was set to 140,000, and the AGC target was 3 × 10 6 ions. Data were acquired and analyzed using TraceFinder software (Thermo). Metabolite concentrations were calculated from the corresponding standard curve.
Amplex red assay. An Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit was used for the H 2 O 2 concentration measurement, following the manufacturer's instruction with modifications. Hydrogen peroxide standards and samples (10-50 µL) were loaded in triplicate into a 96 well black polystyrene microplate with a clear bottom (Corning). A working solution was immediately added for a final volume of 100 µL after each triplicate loading. The final concentration of horseradish peroxidase in the mixture was 0.1 U/mL, and Amplex Red was 0.05 mM. After 5-10 min incubation at room temperature in the dark, the plate was read with a microplate reader, SpectraMax MiniMax 300 Imaging Cytometer, equipped with SoftMax Pro 7 software (Molecular Devices). Fluorescence detection was set at λ ex 540 nm and λ em 580 nm and optical absorbance detection at 560 nm. Sample concentrations were calculated according to standard curves included in each experiment.
Statistics. Data were obtained from 3-6 replicate experiments, each of which was performed in duplicate or triplicate. Data are presented as mean ± standard deviation. Curve fitting was performed with nonlinear least-squares regression, and R 2 of the fitting is presented when necessary in figure plots.