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 microl of plasma is required in a complete dose-inhibition curve. The IC50 of normal human plasma resulted of 2.70 microl of plasma/250 microl 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.
Lussignoli
S
S
Istituto di Chimica e Microscopia Clinica, Università di Verona, Ospedale Policlinico, Verona, 37134, Italy.
Fraccaroli
M
M
Andrioli
G
G
Brocco
G
G
Bellavite
P
P
D000328:Adult; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism*; D002338:Carotenoids / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D010545:Peroxides / Q000737:chemistry / Q000378:metabolism*; D010949:Plasma / Q000378:metabolism*; D011786:Quality Control
D000975:Antioxidants; D010545:Peroxides; C049375:perhydroxyl radical; D002338:Carotenoids; C029036:crocin