The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical ((OH)-O-.) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the (OH)-O-. traps respectively. The (OH)-O-. adduct formation was inhibited by lactate up to 0.4 mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H2O2 dependence was examined, the DMPO-OH signal was increased linearly with H2O2 concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H2O2 concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe3+ at lactate/Fe3+ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H2O2 mediated Fe3+ reduction. When the Fe3+-lactate (1:2) complex was reacted with H2O2, the initial rate of hydroxylated 3-CCA formation was linearly increased with H2O2 concentrations. All the data obtained in the present experiments suggested that the Fe3+-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H2O2 to produce additional (OH)-O-. in the Fenton reaction by other mechanisms than lactate or lactate/Fe3+ mediated promotion of Fe3+/Fe2+ redox cycling.