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


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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Figure 1: Structures of hydroethidine (HE), Mito-HE and their oxidation products.
Figure 2
Figure 3: Mechanism of the reaction of hydroethidine (HE) with nitrosodisulfonate radical dianion (NDS) adapted from ref. 7.
Figure 4: UV-visible absorption spectra.
Figure 5: Fluorescence properties of hydroethidine (HE) and its oxidation products.
Figure 6: HPLC of the extract from RAW 264.7 cells.
Figure 7: HPLC-EC chromatogram of the extract from RAW 264.7 cells.
Figure 8: HPLC-MS chromatogram of the cell extract from RAW 264.7 cells after incubation with HE as described in the Figure 6 caption.


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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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Correspondence to Balaraman Kalyanaraman.

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Zielonka, J., Vasquez-Vivar, J. & Kalyanaraman, B. Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine. Nat Protoc 3, 8–21 (2008).

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