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Am J Physiol Cell Physiol (August 11, 2004). doi:10.1152/ajpcell.00028.2004
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Submitted on January 16, 2004
Accepted on May 21, 2004

Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay

Bruno Fink, Karine Laude, Louise McCann, Abdul Doughan, David Harrison, and Sergey Dikalov*

* To whom correspondence should be addressed. E-mail: dikalov{at}emory.edu.

Recently it has been demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxy-ethidium) that differs from ethidium by the presence of an additional oxygen atom in its molecular structure (Zhao H. et al. Free Radical Biology & Medicine 2003; 34:1359-1368). We have adapted this new HPLC-based assay to quantify this product as a tool to estimate intracellular superoxide in intact tissues. Ethidium and oxy-ethidium were separated using a C-18 column and quantified using fluorescence detection. Initial cell-free experiments with potassium superoxide and xanthine oxidase confirmed formation of oxy-ethidium from dihydroethidium. Formation of oxy-ethidium was inhibited by superoxide dismutase but not catalase and did not occur upon addition of H2O2, peroxynitrite or hypochlorous acid. In bovine aortic endothelial cells (BAECs) and mouse aortas the redox cycling drug menadione increased formation of oxy-ethidium from dihydroethidium 9-fold (0.4 nmol/mg in control vs. 3.6 nmol/mg with 20 µM menadione), and polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) significantly inhibited this. Treatment of BAECs with Ang II caused 2-fold increase in oxy-ethidium formation, and this was reduced by PEG-SOD (0.5 nmol/mg). In addition, in aortas of mice with angiotensin II-induced hypertension and DOCA-salt hypertension, formation of oxy-ethidium was increased in a manner corresponding superoxide production as estimated with cytochrome c reduction. Detection of oxy-ethidium by HPLC represents a new convenient and quantitative method for detection of superoxide in intact cells and tissues.




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