Abstract

Evidence is presented that the activity of the hexose monophosphate (HMP) pathway of red cells, assayed by r4C02 production from glucose-lJ4C, is regulated primarily by glutathione.Methemoglobin had little, if any, effect on the activity of this pathway.Increasing the ratio of oxidized to reduced glutathione, either by peroxidizing GSH to GSSG or by partially blocking GSH with NEM, increased the rate of HMP pathway metabolism.Complete blockage of cellular glutathione by NEM depressed the bulk of HMP pathway activity, despite little or no effect on the Embden-Meyerhof pathway.Sustained low levels of hydrogen peroxide, whether generated by aerobic oxidases or by the coupled oxidation of ascorbic acid with oxyhemoglobin, stimulated the HMP pathway of cells.This stimulation was potentiated by blocking catalase and was prevented by blocking GSH.The oxidation of NADPH by Hz02 in hemolysates was specifically dependent upon the presence of GSH.These results substantiate the existence in human red cells of the glutathione peroxidase mechanism proposed by Mills, whereby GSH protects cellular constituents such as hemoglobin from oxidative damage induced by HzOz.Oxidative denaturation of oxyhemoglobin to met-and sulfhemoglobin by HpOz-generating mechanisms is markedly potentiated in cells lacking sufficient GSH, and commences only after GSH levels approach zero.In contrast, oxidationreduction catalysts such as methylene blue and acetylphenylhydrazine catalyze the direct oxidation of NADPH and hemoglobin, as well as GSH, by molecular oxygen.Although GSH is partially protective against these agents, they appear to damage red cells by virtue of their ability to bypass the GSH peroxidase mechanism and to cause oxidative injury despite persisting GSH. Approximately10% of the glucose consumed by human red cells in serum in vitro is metabolized by way of the hexose mono-