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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

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Abstract

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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Acknowledgements

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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https://doi.org/10.1038/nprot.2007.473

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