Measurement of superoxide dismutase in erythrocytes and whole blood using iodonitrotetrazolium violet

simple analytical method for superoxide dismutase (SOD) is described. It is based on the competitive reduction of a superoxide anion (O-2(-)) by p-iodonitrotetrazolium (2-[4-iodophenyl-3-[4-nitrophenyl]-5-phenyltetrazolium chloride, INT). The magnitude of inhibition of INT reduction by SOD was dependent on the rate of O-2(-) generation by xanthine oxidase: within the range of xanthine oxidase we have examined (2.2 - 4.4 mu g/mL or 1.1 - 2.2 mU), the inhibition ranged from 49 to 64% and was inversely related to the xanthine oxidase activity. With 4.4 mu g/mL of xanthine oxidase, as high as 1.0 units (0.32 mu g/mL) of SOD inhibited about 50% of INT reduction, and the calibration curve derived from the INT assay correlates quite well with that of the cytochrome c method (r = 0.991). Analysis of the patient samples (n = 19) for erythrocytes SOD by both the INT and the cytochrome c methods compares reasonably well (r = 0.901). Student's t-test on the results shows no significant difference between the values obtained by these methods. Considering convenience, simplicity, and cost effectiveness of the reagents for the INT method, this assay has the potential of being useful in routine analysis of the red blood cells and serum samples for SOD.