title: Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence.
abstract: Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O(2)(.-)). 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)(.-) 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)(.-) 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)(.-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O(2)(.-). 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)(.-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable [corrected] for detecting intracellular O(2)(.-).
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Zhao
authors.forename: Joy
authors.initials: J
authors.lastname: Joseph
authors.forename: Henry M
authors.initials: HM
authors.lastname: Fales
authors.forename: Edward A
authors.initials: EA
authors.lastname: Sokoloski
authors.forename: Rodney L
authors.initials: RL
authors.lastname: Levine
authors.forename: Jeannette
authors.initials: J
authors.lastname: Vasquez-Vivar
authors.forename: B
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D002518:Ceramides / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003497:Cyclic N-Oxides / Q000737:chemistry; D042783:Endothelial Cells / Q000737:chemistry / Q000187:drug effects; D005456:Fluorescent Dyes / Q000737:chemistry*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010617:Phenanthridines / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000737:chemistry; D019097:Spin Trapping; D013481:Superoxides / Q000737:chemistry*; D024483:Vitamin K 3 / Q000737:chemistry
chemical_list: C441196:5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide; D002518:Ceramides; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D010617:Phenanthridines; D017382:Reactive Oxygen Species; D013481:Superoxides; C058428:hydroethidine; D024483:Vitamin K 3