Hexose monophosphate shunt activities in human erythrocytes during oxidative damage induced by hydrogen peroxide

Human red blood cells (HRBCs) were exposed to H2O2 either as bolus or as a flux generated by a glucose–glucose oxidase system. H2O2 concentrations were in the range 10−5–10−3 M and exposure times to the oxidative stress were 10 min and 60 min. The production of NADPH by the hexose monophosphate shunt (HMPS) was accurately measured by gas chromatography–isotope ratio mass spectrometry as the production of 13CO2 from [1-13C]glucose. Depending on the duration of exposure and H2O2 concentration, the production of 13CO2 by HRBCs under a flux of H2O2 was increased two- to eight-fold in comparison with that obtained under a bolus of H2O2. Under flux stimulation, spectral data show the formation of compound I, and a red shift caused by the presence of compounds II and III, whereas under a bolus stress no obvious spectra changes were observed. Inhibition of catalase by 3-amino-1,2,4-triazole (3-AT) or by sodium azide, followed by a bolus of H2O2 led to a two- to five-fold increases in 13CO2 production compared with controls, depending on H2O2 concentration. In contrast, 3-AT-inhibited HRBCs exposed to a flux of H2O2 did not present an increase in 13CO2 production. The present paper emphasizes the importance and role of NADPH production following a bolus or a flux stimulation of H2O2. The difference between responses in HMPS activities under the two types of stress could be related to a different balance of activity between 'catalatic' and 'peroxidatic' modes of catalase following H2O2 exposure.