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 μM) 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.

Introduction

Reactive oxygen species (ROS) are produced in metabolic and physiological processes, and harmful oxidative reactions may occur in organisms, which remove them via enzymatic and non-enzymatic antioxidative mechanisms. Under some conditions, the increase in oxidants and decrease in antioxidants cannot be prevented, and the oxidative/antioxidative balance shifts toward the oxidative status. Consequently, oxidative stress, which has been implicated in over 100 disorders, develops [1].

Hydroxyl radical (OH) and its subsequent radicals are the most harmful ROS and they are mainly responsible for the oxidative injury of biomolecules. Alone hydrogen peroxide and superoxide molecules cannot directly oxidize lipids, nucleic acids and sugars. These species can lead to oxidative injury in the biomolecules indirectly by producing OH via Fenton reaction and/or iron-catalyzed Haber–Weiss reaction [2]. Oxidized molecules generally form new radicals leading to radical chain reactions or they are neutralized by antioxidants.

Antioxidant molecules prevent and/or inhibit these harmful reactions [3]. Serum (or plasma) concentrations of different antioxidants can be measured in laboratories separately, but the measurements are time-consuming, labor-intensive, costly and they require complicated techniques. Because the measurement of different antioxidant molecules separately, is not practical and antioxidant effects of them are additive, total antioxidant response (TAR) of a sample is measured and this is named as total antioxidant capacity [4], total antioxidant activity [5], total antioxidant power [6], [7], total antioxidant status [8], TAR or their other synonyms.

Various methods have been developed to measure total antioxidant activity [4], [5], [6], [7], [8], [9], [10], [11], [12], [13] and none of them is an ideal reference method. In general, a radical is generated in the assay, and the antioxidant response of the sample against the radical is measured. However, the oxidative potentials of the generated radicals [4], [9], [10] and the used Fe³⁺–TPTZ complex [6], [7] are generally weaker than those of OH and its subsequent radicals, which occur in biological reactions.

Recently, Koracevic et al. [5] developed a manual measurement method in which OH is generated via Fenton reaction. In this method, a standardized solution of Fe–EDTA complex reacts with H₂O₂ by a Fenton reaction, leading to the formation of OH. These ROS degrade benzoate, resulting in the release of thiobarbituric acid reactive substances (TBARS). Antioxidants present in the added sample cause suppression of the production of TBARS. However, in this method, vitamin C and bilirubin, two of the most important antioxidants, are also degraded to TBARS. Any substance the oxidized product of which is degraded to TBARS cannot be measured by this method.

In the novel method, o-dianisidine was used instead of benzoate and the suppression of oxidation reaction by the sample was monitored by following the change of absorbance of the dianisidyl radical instead of the measurement of TBARS which are released from the oxidized benzoate. In this way, the steps in the process were decreased, the assay period was shortened, the requirement of boiling of sample was eliminated, and fully automated measurement was easily performed by using an automated analyzer.