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

Overview

Journal Ann Biomed Eng

Specialty Biomedical Engineering

Date 2012 Sep 12

PMID 22965641

Citations 33

Authors

Juan Chen

Steven C Rogers

Mahendra Kavdia

Affiliations

Soon will be listed here.

Abstract

Superoxide (O(2) (-)) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O(2) (-) 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 O(2) (-) generation and a O(2) (-) sensitive fluorescent dye dihydroethidium (DHE) for O(2) (-) measurement. To quantify O(2) (-) and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE-O(2) (-) interaction to understand the effect of O(2) (-) self-dismutation and to quantify DHE-O(2) (-) reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O(2) (-) interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169 ± 0.059 × 10(3) M(-1) s(-1) for DHE-O(2) (-) reaction that is ~100× slower than the reported value of 2.6 ± 0.6 × 10(5) M(-1) s(-1). In addition, the O(2) (-) self-dismutation has significant effect on DHE-O(2) (-) interaction. A slower reaction rate of DHE with O(2) (-) is more reasonable for O(2) (-) measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O(2) (-). Results suggest that an accurate measurement of O(2) (-) production rate may be difficult due to competitive interference for many factors; however O(2) (-) concentration may be quantified.

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