A peroxidase-independent method for the quantitation of extracellular hydrogen peroxide generated by activated...

A peroxidase-independent method for the determination of phagocyte-derived hydrogen peroxide in vitro is described. The method is based on the catalase-inhibitable oxidation of added cysteine. Human blood neutrophils (1 x 10(6)/ml) were coincubated with the myeloperoxidase (MPO) inhibitor, sodium azide (10 micrograms/ml), for 30 min at 37 degrees C followed by addition of phorbol 12-myristate 13-acetate (PMA, 10 ng/ml), a potent activator of membrane-associated oxidative metabolism. After incubation at 37 degrees C the cells were removed and the supernatants aliquoted and incubated with and without catalase (500 U/ml) for 10 min at room temperature followed by addition of cysteine (50 microM). Thereafter the catalase-inhibitable, H2O2-mediated oxidation of added cysteine was assayed spectrophotometrically following the addition of the thiol-reactive agent 5,5'-dithiobis-(2-nitrobenzoic acid), and the H2O2 concentration determined using a standard curve constructed from difference data based on the oxidation of cysteine by H2O2 (0-200 nmol). The average amount of H2O2 produced by 10(6) PMA-activated neutrophils was 47 +/- 7 nmol/30 min. This sensitive assay procedure is likely to be particularly useful for investigating the effects of pharmacological agents and anti-oxidant nutrients on H2O2 production by activated phagocytes, since these agents are generally unsuited for use in peroxidase-based assays.