Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O-2(center dot-)). 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 O-2(center dot-) 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 O-2(center dot-) 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 O-2(center dot-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O-2(center dot-). 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 O-2(center dot-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable reactive oxygen species for detecting intracellular O-2(center dot-).