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 depletion and 3-amino-1.2,4-triazole (aminotriazole) treatment can be used to measure the endogenous generation of H2O2 by red blood cells. In our studies, aminotriazole was used as an H2O2 dependent (irreversible) catalase inhibitor, and catalase inhibition was used as an indirect measure 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 approximately 2 x 10(-10) M. Kinetic comparisons of H2O2 production and oxyhemoglobin autooxidation (which generates O2.- that dismutates to H2O2) indicate that the latter is probably the main source of H2O2 in red blood cells.