Formate oxidation as a measure of hydrogen peroxide production: effect of pH and involvement of superoxide anion.

Abstract The NADPH-dependent generation of hydrogen peroxide was investigated using 2 model systems: glucose-glucose oxidase as a source of H2O2 alone and xanthine-xanthine oxidase as a source of both superoxide anion and H2O2. With xanthine-xanthine oxidase, the generation of H2O2 (as measured by formate oxidation) was stimulated 2-fold in the presence of 0.40 mM NADPH and was optimal at acidic pH. With glucose-glucose oxidase, NADPH was without effect, although optimal activity was still observed at acid pH. This suggested that formate oxidation by intact cells might also exhibit a pH dependency. We observed that this was indeed the case, and the sensitivity of the assay with intact cells was markedly improved by lowering the pH. The role of superoxide anion in formate oxidation was examined with the same model systems. Xanthine-xanthine oxidase-induced formate oxidation was not affected by superoxide dismutase. However, if 0.40 mM NADPH was present, the degree of formate oxidation was stimulated 2-fold; this stimulated activity was reduced to the basal level by superoxide dismutase. The NADPH-induced stimulation of formate oxidation by xanthine oxidase was dose dependent from 0.20 to 0.80 mM NADPH and was completely sensitive to superoxide dismutase. In contrast, formate oxidation by glucose oxidase was unaffected by NADPH or sodium dismutase, either separately or in combination. These results suggest that formate oxidation is optimal at acid pH and that NADPH can amplify H2O2 production by a free radical chain reaction involving superoxide anion. These results are consistent with our previous observations employing a crude granule preparation for human neutrophils.