Elsevier

Clinical Biochemistry

Volume 38, Issue 12, December 2005, Pages 1103-1111
Clinical Biochemistry

A new automated colorimetric method for measuring total oxidant status

https://doi.org/10.1016/j.clinbiochem.2005.08.008Get rights and content

Abstract

Objectives:

To develop a new, colorimetric and automated method for measuring total oxidation status (TOS).

Design and methods:

The assay is based on the oxidation of ferrous ion to ferric ion in the presence of various oxidant species in acidic medium and the measurement of the ferric ion by xylenol orange. The oxidation reaction of the assay was enhanced and precipitation of proteins was prevented. In addition, autoxidation of ferrous ion present in the reagent was prevented during storage. The method was applied to an automated analyzer, which was calibrated with hydrogen peroxide and the analytical performance characteristics of the assay were determined.

Results:

There were important correlations with hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide solutions (r = 0.99, P < 0.001 for all). In addition, the new assay presented a typical sigmoidal reaction pattern in copper-induced lipoprotein autoxidation. The novel assay is linear up to 200 μmol H2O2 Equiv./L and its precision value is lower than 3%. The lower detection limit is 1.13 μmol H2O2 Equiv./L. The reagents are stable for at least 6 months on the automated analyzer. Serum TOS level was significantly higher in patients with osteoarthritis (21.23 ± 3.11 μmol H2O2 Equiv./L) than in healthy subjects (14.19 ± 3.16 μmol H2O2 Equiv./L, P < 0.001) and the results showed a significant negative correlation with total antioxidant capacity (TAC) (r = −0.66 P < 0.01).

Conclusions:

This easy, stable, reliable, sensitive, inexpensive and fully automated method that is described can be used to measure total oxidant status.

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 nonenzymatic antioxidative mechanisms. Under certain conditions, the increase in oxidants and decrease in antioxidants cannot be prevented, and the oxidative/antioxidative balance shifts towards the oxidative status. Consequently, oxidative stress, which has been implicated in over 100 disorders, develops [1], [2], [3].

Oxidant molecules are produced endogenously in organisms and they are also taken from the outer environment. The electron transport chain and a range of oxidase enzymes, including xanthine oxidase, glycollate and monoamine oxidases, make major endogenous ROS sources [4], [5]. It has been calculated that an adult at rest utilizes 3.5 mL O2/kg/min. If 1% makes superoxide radical anion (O2), this is 0.147 mol/day. During bodily exertion, this could increase (with O2 uptake) up to 10-fold, assuming that the 1% figure still applies [6]. In inflammation, NADPH oxidase and myeloperoxidase activities increase oxidant load [7], [8]. Furthermore, ultraviolet light and cigarettes produce considerable amounts of exogenous oxidants [9]. It has been shown that each puff of a cigarette contains 1015 oxidants in the gas phase [10].

Serum (or plasma) concentrations of different oxidant species can be measured in laboratories separately, but the measurements are time-consuming, labor-intensive and costly and require complicated techniques [11]. Since the measurement of different oxidant molecules separately is not practical and their oxidant effects are additive, the total oxidant status (TOS) of a sample is measured and this is named total peroxide (TP) [1], [2], [3], [9], [12], serum oxidation activity (SOA) [13], reactive oxygen metabolites (ROM) [14], [15], [16], [17], [18] or some other synonyms. Although a few different colorimetric methods have been developed to measure TOS [19], none of them is ideal and they have major analytical problems and technical restrictions. This study aimed to develop an easy, reliable, sensitive, automated and inexpensive method using reagents with long lifetimes for measuring total oxidant status (TOS).

Section snippets

Chemicals

Xylenol orange [o-cresosulfonphthalein-3,3-bis(sodium methyliminodiacetate)], horseradish peroxidase, 3,5,3′,5′-tetramethylbenzidine (TMB), ortho-dianisidine dihydrochloride (3-3′-dimethoxybenzidine), ferrous ammonium sulfate, ferric chloride, alchilamine (N-N-diethyl-para-phenylenediamine, DEPPD), sodium azide, hydrogen peroxide (H2O2), tert-butyl hydroperoxide, cumene hydroperoxide, sulfuric acid, hydrochloride acid, glycerol, butylated hydroxytoluene (BHT), thiobarbituric acid (TBA),

Type, concentration and pH of the solution used in the assay

The ferric-xylenol orange complex gave its maximal absorbance only within a narrow pH interval, 1.70–1.80. An appropriate buffer for this pH interval, namely Clark and Lubs solution, was tested but the results were not appropriate for serum samples. The sulfuric acid solution at 25 mM concentration was suitable for obtaining maximal absorbance.

Optimization of xylenol orange, glycerol, ferrous ion and o-dianisidine concentrations

The rate of color formation was fast and appropriate with 150 μM of the chromogen xylenol orange. The oxidation reaction rate was enhanced using the

Discussion

Various methods have been developed for measuring total oxidant status, but there is no accepted reference method. No final decisions concerning the standardizations, or the terms and units used have been made. This implies that this topic needs further study. Indeed, research efforts in this area have been accelerating over the last decade. The most widely used methods for measuring reactive oxygen species are colorimetric, fluorescence, chemiluminescence and electron spin resonance (ESR)

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