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

Abstract

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.

Figure 1

Mechanism of pyruvate and H₂O₂ reaction. Nucleophilic addition of H₂O₂ to the α-carbonyl group in pyruvate forms an unstable intermediate, 2-hydroperoxy-2-hydroxypropanoate, which subsequently undergoes rearrangement to produce CO₂, acetate, and water at neutral pH.

Figure 2

Reaction order with respect to pyruvate and H₂O₂. (A) The reaction order with respect to pyruvate was analyzed by reacting 20 µM H₂O₂ with increasing concentrations of pyruvate. The reaction was carried out for 5 min and the average reaction rate was calculated as the change of H₂O₂ concentration per min. (B) The reaction order of H₂O₂ was determined by reacting 200 µM pyruvate with increasing concentrations of H₂O₂. The reaction was carried out for 3 min and the rate was calculated as the change in pyruvate concentration per min. (C) and (D) HPLC chromatograms and the standard curve for pyruvate. (E) and (F) Standard curves of H₂O₂ measurement by a peroxidase-Amplex Red assay. The color product of the assay was read by absorption at OD₅₆₀ or by fluorescence at λ_ex 350 nm and λ_em 410 nm for the high or low concentration range of H₂O₂, respectively. Data were obtained from 3 separate experiments and presented as mean ± SD.

Figure 3

Rate constant of pyruvate and H₂O₂ reaction. Reactions were carried out with 300 µM each of pyruvate and H₂O₂ in DPBS at 37 °C. At the indicated timepoint, pyruvate concentration in the reaction solution was measured using the HPLC method. (A) Data are graphed as pyruvate concentration vs. time and fit with a second-degree polynomial equation (B) Data are graphed as inverse pyruvate concentration vs. time and fit with a linear equation. The slope of the linear line is the rate constant k of the reaction according to Eq. (3). Data were obtained from 6 separate experiments and presented as mean ± SD.

Figure 4

LC-MS measurement of pyruvate and acetate concentrations. Reactions were carried out with 300 µM each of pyruvate and H₂O₂ in DPBS at 37 °C. At the indicated time, aliquots of the reaction mixture were removed for LC-MS analysis. (A,B) LC-MS chromatograms showing the detection of pyruvate and acetate, respectively, in the samples at the indicated times. (C,D) Concentrations of pyruvate and acetate, respectively, were calculated according to standard curves, graphed vs. time, and fit to second-degree polynomial equations. (E) Data are graphed as the sum of pyruvate and acetate concentrations vs. time, showing an unchanged total concentration over time. (F) Data are graphed as the inverse concentration of pyruvate vs. time and fit to a linear equation. The slope of the line indicates the k of the reaction. Data were obtained from 3 separate experiments and presented as mean ± SD.

Figure 5

Calculated time course of H₂O₂ elimination by pyruvate. The time required for elimination of 25, 50, 75, or 95% of H₂O₂ of its initial concentration by pyruvate was calculated based on Eq. (5) and Table 1. (A) The initial concentration of pyruvate is 150 µM and of [H₂O₂]₀ is 50, 20, 5, 1, 0.1, or 0.01 µM. (B) The initial concentration of pyruvate is 1,000 µM and of [H₂O₂]₀ is 200, 100, 50, 10, or  5 µM. Data are graphed as [H₂O₂] vs. time, and fit to exponential decay equations. Inserts show the same graphs on a semilog scale.

Figure 6

Comparison of measured and calculated time course of pyruvate and H₂O₂ reaction. Reactions were carried out by reacting 1,000 µM (A) or 150 µM (B) pyruvate with 50 µM H₂O₂ in DPBS at 37 °C. Concentrations of H₂O₂ (A) or pyruvate (B) in the reaction solutions were measured at indicated timepoints. Calculated [H₂O₂] or [Pyr] values were obtained based on Eq. 6 and Table 2. Data were fitted to exponential decay (A) and to second-degree polynomial (B) equations as shown within the plots. Measured data were obtained from 3 separate experiments and are presented as mean ± SD.