Estimation of antioxidant capacity against pathophysiologically relevant oxidants using Pyrogallol Red

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Abstract

Peroxynitrite and hypochlorite are oxidants relevant in many pathological situations. We propose a simple spectrophotometric assay to determine antioxidant capacity against hypochlorite and peroxynitrite based on protection against Pyrogallol Red decolorization. The assay can be performed on a microplate and requires minute amounts of material. Standard antioxidants show different reactivities for both oxidants. Antioxidant capacity of blood plasma (anticoagulated with EDTA) of healthy persons was found to be 559 ± 49 μmol/l and 11.6 ± 1.2 mmol/l of ascorbic acid equivalents for peroxynitrite and hypochlorite, respectively.

Introduction

The “Total antioxidant capacity” assays have been widely used in several field of research including analysis of food and beverages, analysis of plant material and analysis of body fluids, especially in pathologies [1], [2], [3]. The concept of measurements of “total antioxidant capacity” has been lately criticized. It has been pointed out correctly that neither the term “total” nor the term “capacity” are justified since (i) the assays measure non-enzymatic antioxidant activity while the antioxidant defense of the body is mainly enzymatic, and (ii) the samples assayed are removed from their biological context, which is characterized by enzymatic maintenance of the steady state of antioxidant concentrations [4]. While this criticism is justified, one could nevertheless notice that there are reactive oxygen species occurring in vitro which are not dealt with by enzymes, such as peroxyl radicals, peroxynitrite and hypochlorite. Their formation is dependent on, or contributed by enzymes (microsomal lipid peroxidation, formation of hypochlorite by myeloperoxidase) and no specific enzymatic means of their detoxification is known, although peroxyredoxins, heme proteins and glutathione peroxidases may be of considerable importance in the detoxification of peroxynitrite [5], [6]. In some situations it is of importance to know how the damage by these species can be counteracted by components of the body, especially blood plasma and other extracellular fluids. It is of course, true that assays allowing for such estimates are made in vitro, on samples withdrawn from their biological environment. Nevertheless, they provide an estimate of the ability of a biological fluid to withstand acute exposure to an oxidant.

With this arguments in mind, we propose a simple assay allowing to evaluate the antioxidant capacity of biological fluids against two oxidants of physiological and pathological relevance for which no enzymatic defense is known, peroxynitrite and hypochlorite.

Section snippets

Materials and methods

All reagents were from Sigma/Aldrich (Poznań, Poland). Peroxynitrite was synthesized from sodium azide and ozone according to the method of Pryor et al. [7] with small modifications [8]. Blood, anticoagulated with EDTA, was collected from the antecubital vein of 22 healthy volunteers of both sexes, centrifuged (5000g, 15 min, 4 °C) and supernatant was used at once for the measurement (kept on ice until putting on a plate). All subjects gave informed consent for participating in the research. The

Results and discussion

Pyrogallol Red has been proposed as a convenient indicator for kinetic oxygen radical absorbance capacity assay [9], [10], [11]. Its bleaching induced by various oxidants (mainly peroxyl radicals generated during AAPH decomposition) can be prevented by antioxidants. In that assay, the initial rate of absorbance decrease or area under curve of absorbance vs time or lag time of the oxidation are taken as a measure of oxidation of the probe and of its protection. Because of the suitability of

Acknowledgment

We are indebted to Prof. Agata Karowicz-Bilińska for the help with collection of blood samples.

References (12)

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