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Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay

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Abstract

Superoxide (O2 −) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O2 − is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O2 − generation and a O2 − sensitive fluorescent dye dihydroethidium (DHE) for O2 − measurement. To quantify O2 − and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE–O2 − interaction to understand the effect of O2 − self-dismutation and to quantify DHE–O2 − reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O2 − interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169 ± 0.059 × 103 M−1 s−1 for DHE–O2 − reaction that is ~100× slower than the reported value of 2.6 ± 0.6 × 105 M−1 s−1. In addition, the O2 − self-dismutation has significant effect on DHE–O2 − interaction. A slower reaction rate of DHE with O2 − is more reasonable for O2 − measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O2 −. Results suggest that an accurate measurement of O2 − production rate may be difficult due to competitive interference for many factors; however O2 − concentration may be quantified.

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Abbreviations

DHE:

Dihydroethidium

E+ :

Ethidium

EOH:

2-Hydroethidium

GSH:

Glutathione

H2DCF-DA:

Dichlorodihydrofluorescein-diacetate

H2O2 :

Hydrogen peroxide

HPLC:

High performance liquid chromatography

HX:

Hypoxanthine

MS:

Mass spectrometry

NO:

Nitric oxide

O2 − :

Superoxide

·OH:

Hydroxyl radical

ONOO− :

Peroxynitrite

PBS:

Phosphate buffer solution

ROS:

Reactive oxygen species

RNS:

Reactive nitrogen species

SOD:

Superoxide dismutase

XOD:

Xanthine oxidase

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Acknowledgments

This study was supported by NIH grant # R01 HL084337.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Wayne State University, 2152 Engineering Bldg, 5050 Anthony Wayne Dr., Detroit, MI, 48202, USA

    Juan Chen & Mahendra Kavdia

  2. Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, 72701, USA

    Steven C. Rogers

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  1. Juan Chen
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Correspondence to Mahendra Kavdia.

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Associate Editor Gerald Saidel oversaw the review of this article.

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Chen, J., Rogers, S.C. & Kavdia, M. Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay. Ann Biomed Eng 41, 327–337 (2013). https://doi.org/10.1007/s10439-012-0653-x

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  • DOI: https://doi.org/10.1007/s10439-012-0653-x

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