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A Microplate-Based Colorimetric Assay of the Total Peroxyl Radical Trapping Capability of Human Plasma

1999; Elsevier BV; Volume: 269; Issue: 1 Linguagem: Inglês

10.1006/abio.1999.4010

1096-0309

S. Lussignoli, Marta Fraccaroli, G. Andrioli, Giorgio Brocco, Paolo Bellavite,

Antioxidant Activity and Oxidative Stress

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 μl of plasma is required in a complete dose–inhibition curve. The IC50of normal human plasma resulted of 2.70 μl of plasma/250 μl assay volume. The total antioxidant capability (TAC) of plasma was defined as the reciprocal of IC50and 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.