PYRUVATE DIMERIZATION BY DIVALENT METAL CATIONS

Purpose: Pyruvate enriched cardioplegia has been shown to reduce cardiopulmonary bypass induced inflammation and oxidative stress. Cardioplegia solutions are stored in the hospital pharmacy at room temperature. It has been argued that pyruvate is unstable in aqueous solutions containing divalent metal cations and undergoes an aldol condensation reaction to form parapyruvate. This substance is a potential inhibitor of a critical step in the TCA cycle, oxoglutarate dehydrogenase, and therefore may be toxic. Our pilot study is testing whether pyruvate remains stable in solution in the presence of a metal catalyst. Methods: Aqueous solutions of 5 mM pyruvate were prepared in 0.5 M Tris and aliquoted into test tubes. Zn2+ (ZnCl2) was then added to the test tubes at 0, 0.1, 0.3 and 1.0 mM concentrations. The solution was buffered to pH 4 to prevent precipitation of the Zn2+ which occurred at neutral pH. The solutions were stored in the dark at 20 and 4 ℃ to assess the effect of temperature on the aldol condensation. Absorbance of ultraviolet light (315 nm wavelength) by the pyruvate-Zn solution was measured by spectrophotometry at 0 and 10 days to detect formation of parapyruvate, which absorbs light at that wavelength. Results: The changes in absorbance of the solutions kept at 20 ℃ were as follows: 0 mM Zn2+ -0.002, 0.1 mM Zn2+ 0.007, 0.3 mM Zn2+ -0.011, 1.0 mM Zn2+ -0.0195. The corresponding absorbance changes in the solutions at 0 ℃ for 10 days were as follows: 0 mM Zn2+ -0.009, 0.1 mM Zn2+ 0.0015, 0.3 mM Zn2+ -0.005, 1.0 mM Zn2+ -0.0105. Thus, at both temperatures, only the highest Zn2+ concentration, 1 mM, produced a detectable, albeit modest, change in absorbance at 315 nm, and that change was in the direction opposite that which would indicate pyruvate dimerization. Conclusions: Over the course of 10 days, no appreciable pyruvate dimerization could be detected by spectrophotometry. Thus, pyruvate appeared to be stable in aqueous solution, even over 10 days at 20 ℃. To confirm these results, we are currently measuring pyruvate concentrations at 0, 1, 3, 7 and 14 days by a spectrophotometric assay at 340 nm utilizing the lactate dehydrogenase reaction, in which pyruvate reduction to lactate consumes NADH. This method will enable us to verify that the apparent absence of parapyruvate is accompanied by a stable concentration of pyruvate over time. Future studies will test the impact of other divalent metals, including Mg2+, an important component of many cardioplegia formulations.