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Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine

Nature Protocols volume 3, pages 821 (2008) | Download Citation



Various detection methods of the specific product of reaction of superoxide (O2•−) with hydroethidine (HE), namely 2-hydroxyethidium (2-OH-E+), and with its mitochondria-targeted analog are described. The detailed protocol for quantification of 2-OH-E+, the unique product of HE/O2•− in cellular systems, is presented. The procedure includes cell lysis, protein precipitation using acidified methanol and HPLC analysis of the lysate. Using this protocol, we determined the intracellular levels of 2-OH-E+ and E+ in the range of 10 and 100 pmol per mg protein in unstimulated macrophage-like RAW 264.7 cells. In addition to HE, 2-OH-E+ and E+, we detected several dimeric products of HE oxidation in cell lysates. As several oxidation products of HE are formed, the superoxide-specific product, 2-OH-E+ needs to be separated from other HE-derived products for unequivocal quantification.

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  1. 1.

    Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95 (2002).

  2. 2.

    & Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R1014–R1030 (2004).

  3. 3.

    , & Measurement of reactive oxygen species in cardiovascular studies. Hypertension 49, 717–727 (2007).

  4. 4.

    et al. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. USA 103, 15038–15043 (2006).

  5. 5.

    et al. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic. Biol. Med. 34, 1359–1368 (2003).

  6. 6.

    et al. Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc. Natl. Acad. Sci. USA 102, 5727–5732 (2005).

  7. 7.

    , , & Mechanistic similarities between oxidation of hydroethidine by Fremy's salt and superoxide: stopped-flow optical and EPR studies. Free Radic. Biol. Med. 39, 853–863 (2005).

  8. 8.

    , , , & Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants. Arch. Biochem. Biophys. 456, 39–47 (2006).

  9. 9.

    , & Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radic. Biol. Med. 25, 826–831 (1998).

  10. 10.

    , & The confounding effects of light, sonication, and Mn(III)TBAP on quantification of superoxide using hydroethidine. Free Radic. Biol. Med. 41, 1050–1057 (2006).

  11. 11.

    et al. Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems. Am. J. Physiol. Cell Physiol. 292, C413–C422 (2007).

  12. 12.

    , & Interference of non-specific peroxidases in the fluorescence detection of superoxide radical by hydroethidine oxidation: a new assay for H2O2. Anal. Bioanal. Chem. 381, 1065–1072 (2005).

  13. 13.

    & Improved analysis of hydroethidine and 2-hydroxyethidium by HPLC and electrochemical detection. Free Radic. Biol. Med. 43, 1095–1096 (2007).

  14. 14.

    , & The fluorescence detection of superoxide radical using hydroethidine could be complicated by the presence of heme proteins. Anal. Biochem. 332, 290–298 (2004).

  15. 15.

    & Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescin. J. Leukoc. Biol. 47, 440–448 (1990).

  16. 16.

    , & Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J. Leukoc. Biol. 55, 253–258 (1994).

  17. 17.

    et al. Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am. J. Physiol. Cell Physiol. 287, C895–C902 (2004).

  18. 18.

    , , , & An ultrasensitive fluorescent assay for the in vivo quantification of superoxide radical in organisms. Anal. Biochem. 347, 144–151 (2005).

  19. 19.

    , & Polyunsaturated fatty acids modulate NOX 4 anion superoxide production in human fibroblasts. Biochem. J. 406, 77–83 (2007).

  20. 20.

    et al. Endothelial cell superoxide anion radical generation is not dependent on endothelial nitric oxide synthase-serine 1179 phosphorylation and endothelial nitric oxide synthase dimer/monomer distribution. Free Radic. Biol. Med. 40, 2056–2068 (2006).

  21. 21.

    , , , & Definitive evidence for the nonmitochondrial production of superoxide anion by human spermatozoa. J. Clin. Endocrinol. Metab. 91, 1968–1975 (2006).

  22. 22.

    , , , & Superoxide released into the mitochondrial matrix. Free Radic. Biol. Med. 41, 950–959 (2006).

  23. 23.

    , & Simultaneously monitoring the superoxide in the mitochondrial matrix and extramitochondrial space by micellar electrokinetic chromatography with laser-induced fluorescence. Anal. Chem. 79, 4588–4594 (2007).

  24. 24.

    et al. Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers. Free Radic. Biol. Med. (2007), doi: 10.1016/j.freeradbiomed.2007.11.013.

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This work was supported by National Institutes of Health grants HL073056, NS039958 and R01HL067244. We thank Jennifer Whitsett (Department of Biophysics, Medical College of Wisconsin) for her help in cell culture experiments and Daniel Brody (Department of Pharmacology and Toxicology, Medical College of Wisconsin) for performing the HPLC-MS analysis. We also thank all present and past members of the Free Radical Research Center, whose names are given in the references, for their various contributions to the development of the HPLC-based assay for 2-hydroxyethidium.

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  1. Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.

    • Jacek Zielonka
    • , Jeannette Vasquez-Vivar
    •  & Balaraman Kalyanaraman
  2. Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

    • Jacek Zielonka
    • , Jeannette Vasquez-Vivar
    •  & Balaraman Kalyanaraman


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Correspondence to Balaraman Kalyanaraman.

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