The effect of γ-radiation on the hemoglobin of stored red blood cells: the involvement of oxidative stress in hemoglobin conformation

The aim of the present work was to evaluate the redox and oligomeric effects associated with the human hemoglobin of stored red blood cells that had been previously submitted to gamma radiation. Whole blood was collected from healthy donors and irradiated with 25 Gy of γ-radiation within 24 h of collection. At days 3, 5, 7, 9, 11, 14, and 28 postirradiation, fractions were removed and centrifuged, and the levels of methehemoglobin and oxyhemoglobin were measured. Hb was isolated to measure the denaturation and UV–vis spectra. The results from electrophoresis demonstrated that there was no fragmentation or cross-linking of the hemoglobin. However, ferrous center oxidation was identified as a very significant process. This mechanism is likely through an autoxidation process of the ferrous heme center, which has a maximal intensity between 5 and 7 days of storage. Interestingly, a subsequent reduction of the oxidized heme species was observed, and after 9 days of storage, the difference between the ferric species present in the control and irradiated samples was not representative. This interesting fact suggests a type of “protective action” by the blood to control the oxidative stress generated by the gamma irradiation. The UV–vis measurements demonstrated that the oxidized species was predominantly formed by hemichrome species (bis-histidine ferric heme species), which are usually associated with Heinz bodies. After 28 days of storage, evidence from the UV–vis measurements indicated that the oxidation of the irradiated sample was much higher than that observed in the control sample. These results demonstrate that despite the minimal polypeptide changes observed in the hemoglobin of stored red blood cells after gamma irradiation, the oxidation of the heme metallic center is not irrelevant and must be controlled to improve the hematological clinical procedures associated with the storage of red blood cells.