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Cited by 579Redox biology and oxidative stressNitric Oxide and Endothelin EffectsAntioxidant Activity and Oxidative Stress

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

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Zhao, Hongtao, Joseph, Joy, Fales, Henry M., Sokoloski, Edward A., Levine, Rodney L., Vasquez-Vivar, Jeannette, Kalyanaraman, B.·2005·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.0501719102PMC PMC556312
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Abstract

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} ). 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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} 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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} 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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} . 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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} . We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable reactive oxygen species for detecting intracellular \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} .

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Authors
Zhao, H., Joseph, J., Fales, H. M., Sokoloski, E. A., Levine, R. L., Vasquez-Vivar, J., & Kalyanaraman, B.
Affiliations
  • Medical College of Wisconsin (US)
  • University of Wisconsin–Madison (US)
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2005-04-11
Volume
102
Issue
16
Pages
5727-5732
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.0501719102
PMC
PMC556312
OpenAlex
W1989224728
Cited by
576
References
26
Citations by year
2026: 10 · 2025: 11 · 2024: 20 · 2023: 21 · 2022: 19 · 2021: 28 · 2020: 19 · 2019: 22 · 2018: 24 · 2017: 29 · 2016: 32 · 2015: 28 · 2014: 31 · 2013: 40 · 2012: 35
FWCI
13.05
Type
journal article
Language
en
Update notices
correction (2005-06-21)
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (5P01HL68769-01)
  • National Heart, Lung, and Blood Institute (5P01HL68769-01, P01HL068769, R01HL067244)
  • National Cancer Institute (5R01 CA 77822, R01CA077822)
  • National Institute of Diabetes and Digestive and Kidney Diseases
  • National Institute of Neurological Disorders and Stroke (R01NS039958, 2R01NS39958)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Health Sciences › Medicine › Biochemistry › Antioxidant Activity and Oxidative Stress
Keywords
hydroethidine, superoxide, reactive oxygen species, spin trapping, menadione, antimycin A, endothelial cells, HPLC determination, electron spin resonance, superoxide detection, membrane permeability, intracellular reactive oxygen species, extracellular superoxide, spin adducts
MeSH terms
Animals, Aorta, Biological Assay, Cattle, Ceramides, Chromatography, High Pressure Liquid, Cyclic N-Oxides, Fluorescent Dyes, Oxidation-Reduction, Phenanthridines, Superoxides, Molecular Structure, Reactive Oxygen Species, Spin Trapping, Vitamin K 3, Endothelial Cells

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