A novel automated method to measure total antioxidant response against potent free radical reactions

Abstract

Objectives: Oxidative damage of biomolecules occurs as a result of potent free radical reactions. In this study, a novel, colorimetric and fully automated method for measuring total antioxidant response (TAR) against potent free radical reactions is described.

Design and methods: Potent free radical reactions were initiated with the production of hydroxyl radical (OH(*)) via Fenton reaction, and the rate of the reactions was monitored by following the absorbance of colored dianisidyl radicals. Ortho-dianisidine (10 mM) and ferrous ammonium sulfate (45 microM) were dissolved in KCl/HCl solution (75 mM, pH 1.8). This mixture was named as Reagent 1 and hydrogen peroxide solution (7.5 mM) as Reagent 2. The OH(*), produced by mixing of R1 and R2, oxidized o-dianisidine molecules into dianisidyl radicals, leading to a bright yellow-brown color development within seconds. Antioxidants, present in the sample, suppressed the color formation to a degree that is proportional to their concentrations. The method was applied to an automated analyzer and analytical performance characteristics of the assay were determined.

Results: Vitamin C and Trolox, reduced glutathione, bilirubin, uric acid and (+/-)-catechin solutions suppressed the color formation depending on their concentrations. Serum TAR against potent free radical reactions was lower in patients with chronic renal failure (1.13 +/- 0.21 mmol Trolox equiv./l) and was higher in the individuals with neonatal icterus (2.82 +/- 1.18 mmol Trolox equiv./l) than in healthy subjects (1.54 +/- 0.15 mmol Trolox equiv./l).

Conclusions: The easy, inexpensive and fully automated method described can be used to measure TAR of samples against potent free radical reactions.