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

Human red blood cells (HRBCs) were exposed to H(2)O(2) either as bolus or as a flux generated by a glucose-glucose oxidase system. H(2)O(2) 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 (13)CO(2) from [1-(13)C]glucose. Depending on the duration of exposure and H(2)O(2) concentration, the production of (13)CO(2) by HRBCs under a flux of H(2)O(2) was increased two- to eight-fold in comparison with that obtained under a bolus of H(2)O(2). 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 H(2)O(2) led to a two- to five-fold increases in (13)CO(2) production compared with controls, depending on H(2)O(2) concentration. In contrast, 3-AT-inhibited HRBCs exposed to a flux of H(2)O(2) did not present an increase in (13)CO(2) production. The present paper emphasizes the importance and role of NADPH production following a bolus or a flux stimulation of H(2)O(2). 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 H(2)O(2) exposure.