A microplate-based colorimetric assay of the total peroxyl radical trapping capability of human plasma

We developed a colorimetric assay estimating the radical-scavenging activity of human plasma. The test is based on a measure, in 96-well microplates at 450 nm, of the bleaching of carotenoid crocin by peroxyl radicals generated during thermal decomposition of 2,2'-azobis-(2-amidinopopane) dihydrochloride (ABAP). The inhibition of this bleaching is a function of the antioxidant power of substances added to incubation mixture. We determined the optimal conditions for a sensitive, rapid, and reproducible assay of 50% inhibitory capacity (IC50) of a range of antioxidant substances and of plasma. Only a total of 200 mu l of plasma is required in a complete dose-inhibition curve. The IC50 of normal human plasma resulted of 2.70 mu l of plasma/250 mu l assay volume. The total antioxidant capability (TAC) of plasma was defined as the reciprocal of IC50 and its value in a group of 19 healthy adults resulted in 0.369 +/- 0.06. Intraassay and interassay coefficients of variation of plasma TAC were 6.13 and 4.80%, respectively. Measurement of samples with different uric acid concentration showed that antioxidant activity of uric acid accounts for approximately two-thirds of TAC. (C) 1999 Academic Press.