Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 102, Issue 16, Pages 5727-5732Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0501719102
Keywords
free radical; reactive oxygen species; 2-hydroxyethidium; EPR; spin trapping
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Funding
- NCI NIH HHS [5R01 CA 77822, R01 CA077822] Funding Source: Medline
- NHLBI NIH HHS [5R01 HL 067244, P01 HL068769, R01 HL067244, 5P01 HL 68769-01] Funding Source: Medline
- NINDS NIH HHS [2R01 NS 39958, R01 NS039958] Funding Source: Medline
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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-).
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