No abstract

Because it has been reported that hypoxia in rats may promote lipid peroxidation and other free radical reactions that could modify membrane lipids and proteins, the effect of acute hypobaric hypoxia on human erythrocyte membranes was investigated. 12-(1-Pyrene)dodecanoic acid fluorescent probe was used to assess short-range lateral diffusion status in the membrane bilayer. Membrane protein modification was detected by SDS-PAGE. Healthy young men were exposed for 20 min to the hypobaric hypoxia, simulating an altitude of 4,500 m. Under this condition, erythrocyte membrane lipids reached a state of higher lateral diffusivity with respect to normobaric conditions and membrane band 3 protein was modified, becoming more susceptible to membrane-bound proteinases. These observations suggest that acute hypobaric hypoxia may promote an oxidative stress condition in the erythrocyte membrane.

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2'-Azobis(2-amidinopropane) (AAPH) and 4,4'-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively. AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin. Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.

We have previously shown that subjects exposed to acute hypobaric hypoxia display an erythrocyte membrane protein band 3 with an increased susceptibility to proteolytic degradation. We suggested it was due to an oxidative damage of band 3. We now report that exposure to hypobaric hypoxia followed by reoxygenation affects protein band 3 functions such as anion transport and binding of glyceraldehyde-3P-dehydrogenase. Transport capacity was assessed with the fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] ethanesulfonate (NBD-taurine). Binding capacity was evaluated from the activity of the membrane-associated enzyme. Healthy young men were exposed for 20 min to hypobaric hypoxia, simulating an altitude of 4,500 m above sea level and after recompression band 3 function was assessed. An inhibition of band 3 anion transport function and a decrease in the binding of glyceraldehyde-3P-dehydrogenase to band 3 were observed. Evidence is given supporting the hypothesis that functional alteration of band 3 is due to its oxidative modification originated as a consequence of the exposure to hypobaric hypoxia and further reoxygenation.