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

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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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

Keywords

  • DHE
  • Reaction rate constant
  • Kinetic analysis
  • Oxidative stress
  • Cytochrome c