Erythrocyte defense against hydrogen peroxide: preeminent importance of catalase

To investigate the relative importance of catalase and glutathione in erythrocyte oxidant defense, human and mouse (normal and acatalasemic) erythrocytes were reversibly lysed and resealed in the presence of exogenous catalase or glutathione. This resulted in an increase in intracellular catalase activity or glutathione concentration in the resealed erythrocytes while normal cellular structure, hemoglobin concentration, cell volume, cellular deformability, and adenosine triphosphate concentration were maintained. Resealing alone had no effect on oxidant sensitivity. In human cells, a threefold increase in catalase activity resulted in the maintenance of glutathione levels in response to hydrogen peroxide (H2O2) challenge. Reconstitution of congenitally acatalasemic mouse erythrocytes, which were extremely sensitive to even micromolar concentrations of H2O2 with purified catalase resulted in complete protection against H2O2. Indeed, the catalase-reconstituted acatalasemic cells were less sensitive to H2O2-mediated damage than were normal, catalase-replete mouse cells. In contrast, alteration of the glutathione status of human and mouse (normal and acatalasemic) cells had no significant effect on oxidant sensitivity. Even a five-fold increase in intracellular glutathione concentration (greater than 30 micromoles glutathione per gram of hemoglobin) in normal or catalase-deficient (azide-treated or acatalasemic) red blood cells had no protective effect against H2O2-mediated lipid peroxidation or methemoglobin generation. Similarly, depletion of glutathione by 1-chloro-2,4-dinitrobenzene also had no effect on erythrocyte H2O2 sensitivity. These results suggest an important role for catalase in protection against H2O2-mediated damage at physiologic levels and that catalase is as at least as important as glutathione in cellular defense against H2O2.