Factors Affecting the Ascorbate- and Phenolic-dependent Generation of Hydrogen Peroxide in Dulbecco's Modified Eagles Medium

Abstract

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H₂0₂), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H₂0₂ production. H₂0₂ gene ration from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% C0₂ was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% C0₂. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H₂0₂ generated by ascorbate and phenolic compounds, but there was still substantial H₂0₂ generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H₂0₂ generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H₂0₂ generation is discussed.