High-throughput spectrophotometric assay of reactive oxygen species in serum

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Abstract

The derivatives of reactive oxygen metabolites (D-ROM) test has been developed to determine the amount of oxygen-centered free radicals in a blood sample as a marker of oxidative stress. This study aims to improve the D-ROM test and develop an automated assay system by use of a clinical chemistry analyzer. Five microliters of serum was added to 1 well of a 96-well microtiter plate for a total 240 μl of reaction solution containing alkylamine and metals. This was followed by automatic mixing, incubation and measurement of reactive oxygen species (ROS) levels as a color development at 505 nm using a spectrophotometer with catalytic capability for transition metals. This assay system was used to measure serum levels of ROS in cigarette smokers and never-smokers, by way of example. The levels of serum ROS determined by this system correlate with the amounts of free radicals and peroxides, which reacted with various molecules in the body and formed stable metabolites. This test can use frozen sera as well as fresh ones. The inter- and intra-deviation of this system was within 5% and showed consistent linearity in the range between 4 and 500 mg/l of hydrogen peroxides. Serum ROS levels among smokers increased with the number of cigarettes smoked per day (36.5% increment per pack per day; P < 0.0001). This assay system will be a simple, inexpensive, and reliable tool for assessing oxidative stress in human populations. Our preliminary results on cigarette smoking imply that this assay system has potential for application in various epidemiological and clinical settings.

Introduction

There is increasing concern about the deleterious effects that endogenously produced free radicals (i.e., reactive oxygen species, ROS, and nitrogen oxides) may exert on health. Excessive production of free radicals, specifically ROS, has been reported to have an association with a wide variety of clinical disorders, including cardiovascular disease, diabetes, and cancer [1], [2], [3]. ROS is produced in normal biological processes in the cells by means of either enzymatic or non-enzymatic mechanisms. Biologically relevant ROS include superoxide radical anion (O2radical dot), hydroxyl radicals (radical dotOH), hydrogen peroxide (H2O2), alkoxy radicals (ROradical dot), and peroxy radicals (ROOradical dot), as well as singlet oxygen (1O2). It is widely accepted that ROS play both beneficial and adverse roles in an organism.

Free radicals are highly reactive molecules with a very short half-life. They can be measured directly by an electron spin resonance (ESR) spectrometer [4], or indirectly by an ESR spectrometer coupled with a spin-trapping method [5], [6], [7], [8]. However, the technique of ESR spectroscopy requires extremely intricate and costly instruments. For these reasons, a simple method was recently developed to determine serum ROS levels: the derivatives of reactive oxygen metabolites (D-ROM) test [9], which is now recognized as an efficient method for evaluating oxidative stress in the body [10], [11], [12], [13], [14], [15]. It has been reported that serum ROS levels in patients with ROS-related diseases, such as cancer, cardiovascular disease, and diabetes are higher than those in healthy people [16], [17], [18]. The microplate assay system was also developed for measurement of oxidative stress levels in pigs and birds [19], [20], [21]. However, these systems still need to be improved for routine measurements of ROS levels in a large number of serum samples at clinical laboratories. Precise measurement of ROS levels requires a series of complicated procedures that are often difficult to achieve at laboratories, especially when measuring a large number of samples. On the basis of the principle of the D-ROM test, we have developed an improved assay system that allows high-throughput and automated analysis of numerous serum samples with high reproducibility, consistent accuracy, and much smaller amounts of sera and reagents than with the conventional D-ROM test.

The reaction of this system:Rsingle bondOOH + Fe2+  Rsingle bondOradical dot + Fe3+ + OH or Rsingle bondOOH + Fe3+  Rsingle bondOOradical dot + Fe2+ + H+ or Rsingle bondOradical dot + [Fedouble bondO]2+ + H+.Rsingle bondOradical dot or Rsingle bondOOradical dot + Asingle bondNH2  Rsingle bondO or Rsingle bondOO + [Asingle bondNH2radical dot]+ (coloured)

Rsingle bondOOH, Rsingle bondOradical dot, Rsingle bondOOradical dot, and Asingle bondNH2 are generic peroxide, the alkoxyl radical of a generic peroxide, the peroxyl radical of a generic peroxide, and N,N-diethyl-para-phenylendiamine (chromogenic substrate), respectively. [Asingle bondNH2radical dot]+ is the coloured radical cation of the chromogenic substrate.

In this paper, the usefulness of the new assay system is demonstrated by assessing the effects of cigarette smoking on oxidative status in terms of serum ROS levels.

Section snippets

Reagents

N,N-diethyl-para-phenylendiamine (DEPPD) sulfate and ferrous sulfate were purchased from Sigma Chemicals (St. Louis, MO, USA). Hydrogen peroxide was obtained from Wako Chemicals (Osaka, Japan). All other chemicals were commercially available. DEPPD was dissolved in 0.1 M sodium acetate buffer (pH 4.8) to attain a final concentration of 100 μg/ml (R1 solution as a chromogen), and ferrous sulfate was dissolved in 0.1 M sodium acetate buffer (pH 4.8) to attain a final concentration of 4.37 μM (R2

Results

In 1 well (5 μl serum) of a 96-well microtiter plate, instead of the cuvette (20 μl serum, 1.2 ml R2 reagent, and 20 μl R1 reagent) used in the conventional D-ROM test, the reaction was automatically processed and monitored using a spectrophotometric plate reader. Absorbance at 505 nm was measured at 15-s intervals with 10 different concentrations of hydrogen peroxide standard solution (50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 mg/l) and 1 blank solution. As shown in Fig. 1A, optical

Discussion

Free radicals react with various molecules in the body, resulting in the production of stable ROS metabolites (e.g., lipid peroxide, DNA adduct). This assay system measured the total amount of the ROS in sera, a figure derived from various ROS metabolites formed recently and therefore thought to be a more stable marker for oxidative stress than other markers for momentary oxidative stress. Although the D-ROM test and microplate assay system for measurement of oxidative stress have been promoted

Acknowledgements

The Radiation Effects Research Foundation (RERF), Hiroshima and Nagasaki, Japan is a private, non-profit foundation funded by the Japanese Ministry of Health, Labour and Welfare (MHLW) and the U.S. Department of Energy (DOE) the latter through the National Academy of Sciences. This publication was supported by RERF Research Protocols RP# 1-93 and RP# 2-00 and in part by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and

References (35)

  • E. Nishio et al.

    Cigarette smoke extract is a modulator of mitogenic action in vascular smooth muscle cells

    Life Sci.

    (1998)
  • F. Galli et al.

    Oxidative stress and reactive oxygen species

    Contrib. Nephrol.

    (2005)
  • J.K. Willcox et al.

    Antioxidants and prevention of chronic disease

    Crit. Rev. Food Sci. Nutr.

    (2004)
  • E.G. Janzen et al.

    Alpha-2,6-difluorophenyl-N-tert-butylnitrone: a spin trap for distinguishing different types of alkyl radicals based on long-range fluorine hyperfine splitting

    Free Radic. Res. Commun.

    (1990)
  • J.C. Barreto et al.

    Terephthalic acid: a dosimeter for the detection of hydroxyl radicals in vitro

    Life Sci.

    (1995)
  • B. Halliwell et al.

    Methods for the measurement of hydroxyl radicals in biomedical systems: deoxyribose degradation and aromatic hydroxylation

    Methods Biochem. Anal.

    (1988)
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