HYDROGEN-PEROXIDE PRODUCTION BY RED-BLOOD-CELLS

Red blood cells are frequently employed in studies of oxidative stress . Technical difficulties have previously prevented the measurement of H2O2 production by red blood cells, except during exposure to certain drugs or toxicants. We now show that a combination of glutathione depl etion and 3-amino-1.2,4-triazole (aminotriazole) treatment can be used to measure the endogenous generation of H2O2 by red blood cells. In o ur studies, aminotriazole was used as an H2O2 dependent (irreversible) catalase inhibitor, and catalase inhibition was used as an indirect m easure of H2O2 production. Our results indicate that H2O2 is generated at a rate of 1.36 +/- 0.2 muM/h (3.9 +/- 0.6 nmol . h-1. g Hb-1), and that the steady-state red blood cell concentration of H2O2 is approxi mately 2 x 10(-10) M. Kinetic comparisons of H2O2 production and oxyhe moglobin autooxidation (which generates O2.- that dismutates to H2O2) indicate that the latter is probably the main source of H2O2 in red bl ood cells.