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

Abstract

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.