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

Clinical biochemistry (2004-01-17)
Ozcan Erel
ABSTRACT

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

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ammonium iron(II) sulfate hexahydrate, ACS reagent, 99%
Sigma-Aldrich
Ammonium iron(II) sulfate hexahydrate, 99.997% trace metals basis
Sigma-Aldrich
Ammonium iron(II) sulfate hexahydrate, ReagentPlus®, ≥98%
Sigma-Aldrich
Ammonium iron(II) sulfate hexahydrate, BioUltra, ≥99.0% (RT)
Supelco
o-Dianisidine, for spectrophotometric det. of Au, NO2-, Ce(IV), for the detection of Au, Co, Cu, SCN-, V, ≥97.0%
Sigma-Aldrich
o-Dianisidine dihydrochloride, Suitable for use in glucose determination
Sigma-Aldrich
Ammonium iron(II) sulfate hexahydrate, BioXtra, ≥98%