Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence
Authors: Zhao Hongtao , Joseph Joy , Fales Henry M. , Sokoloski Edward A. , Levine Rodney L. , Vasquez-Vivar Jeannette , Kalyanaraman B. 
Published: April 2005

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (

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). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E
+
). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and

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formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-
tert
-butoxycarbonyl-5-methyl-1-pyrroline
N
-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the

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that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E
+
. However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of

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with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of

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. Analysis of the fluorescence characteristics of ethidium (E
+
) and 2-OH-E
+
strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular

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. We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable reactive oxygen species for detecting intracellular

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.