The reduction of nitroblue tetrazolium by Red Blood Cells: a measure of red cell membrane antioxidant capacity and hemoglobin-membrane binding sites

The reduction of nitroblue tetrazolium (NBT) with intact Red Blood Cells (R BCs) is biphasic with an initial rapid reduction followed by a slower secon d phase. This biphasic kinetics has been explained with the initial rapid p hase attributed to antioxidants in the red cell which reduce membrane bound NBT and the slower phase associated with the reaction of NBT with membrane bound hemoglobin. This model has been confirmed by a utilization of a numb er of red cell modifications which either increase the red cell antioxidant s (vitamin C and vitamin E) or damage the red cell membrane (cumene hydrope roxide and N-ethylmaleimide). The utilization of this assay for human blood samples was investigated by studying a series of 20 human subjects ranging between 34 and 87 years of age. It was possible to fit all of these sample s with two adjustable parameters which reflect the red cell membrane antiox idant capacity (x) and the hemoglobin membrane interactions (m). The antiox idant capacity shows a significant (p <.002; R = -.67) decrease with age. T his finding is consistent with a decrease in the level of antioxidants in a ged subjects. In addition, the number of hemoglobin membrane sites are nega tively correlated with the antioxidant capacity (p <.02; R = -.52) suggesti ng that the oxidative stress associated with reduced antioxidants results i n increased hemoglobin-membrane interactions.