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标题：Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence
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作者：Hongtao Zhao ; Joy Joseph ; Henry M. Fales 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2005
卷号：102
期号：16
页码：5727-5732
DOI：10.1073/pnas.0501719102
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide ([IMG]f1.gif" BORDER="0">). 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 [IMG]f1.gif" BORDER="0"> 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 [IMG]f1.gif" BORDER="0"> 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 [IMG]f1.gif" BORDER="0"> with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of [IMG]f1.gif" BORDER="0">. 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 [IMG]f1.gif" BORDER="0">. We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable reactive oxygen species for detecting intracellular [IMG]f1.gif" BORDER="0">.
关键词：free radical ; reactive oxygen species ; 2-hydroxyethidium ; EPR ; spin trapping