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. 2019:1982:243-258.
doi: 10.1007/978-1-4939-9424-3_14.

HPLC-Based Monitoring of Oxidation of Hydroethidine for the Detection of NADPH Oxidase-Derived Superoxide Radical Anion

Jacek Zielonka  1   2   3 , Monika Zielonka  4   5 , Balaraman Kalyanaraman  4   5   6
Affiliations

HPLC-Based Monitoring of Oxidation of Hydroethidine for the Detection of NADPH Oxidase-Derived Superoxide Radical Anion

Jacek Zielonka et al. Methods Mol Biol. 2019.

Abstract

Hydroethidine is a fluorogenic probe that in the presence of the superoxide radical anion is oxidized to a red fluorescent product, 2-hydroxyethidium. In cells, hydroethidine is also oxidized to other products, including red fluorescent ethidium. Thus, selective monitoring of 2-hydroxyethidium is required for specific detection of the superoxide radical anion. Here, we provide protocols for HPLC- and LC-MS-based quantitation of 2-hydroxyethidium, among other oxidation products. Also, a protocol for continuous sampling for real-time monitoring of superoxide production using rapid HPLC measurements of 2-hydroxyethidium is described.

Keywords: 2-hydroxyethidium; HPLC; Hydroethidine; LC-MS; Superoxide radical anion.

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Figures

Fig. 1
Fig. 1
HE oxidation scheme
Fig. 2
Fig. 2
Scheme showing collection of cells and media from cell experiment
Fig. 3
Fig. 3
Scheme showing preparation of the cell samples for HPLC analysis
Fig. 4
Fig. 4
Scheme showing preparation of the media samples for HPLC analysis
Fig. 5
Fig. 5
HPLC-Abs/Fl chromatograms of standards of HE (2 μM, upper traces) and its oxidation products (2-OH-E+, E+, and E+-E+; 1 μM each, lower traces). Both samples contained DAPP (1 μM) as the internal standard. Samples were analyzed using the gradient elution method, as described in Subheading 3.3
Fig. 6
Fig. 6
HPLC-Abs/Fl chromatograms of the extracts from RAW 264.7 cells. The upper traces correspond to untreated (control) cells, and the lower traces correspond to cells stimulated with a phorbol ester (PMA) to activate superoxide production. Samples were analyzed using the gradient elution method, as described in Subheading 3.3
Fig. 7
Fig. 7
LC-MS/MS chromatograms of standards of HE (2 μM, upper traces) and its oxidation products (2-OH-E+, E+, and E+-E+; 1 μM each, lower traces). Samples were analyzed using the gradient elution method, as described in Subheading 3.4
Fig. 8
Fig. 8
LC-MS/MS chromatograms of the extracts from RAW 264.7 cells. The upper traces correspond to untreated (control) cells, and the lower traces correspond to cells stimulated with a phorbol ester (PMA) so as to induce superoxide production. Samples were analyzed using the gradient elution method, as described in Subheading 3.4
Fig. 9
Fig. 9
Scheme showing sampling of the cell media samples for real-time monitoring of O2•−formation
Fig. 10
Fig. 10
HPLC-Abs/Fl chromatograms of standards of HE (1 μM) and its oxidation products (2-OH-E+, E+, and E+-E+; 1 μM each, lower traces). Both samples contained DAPP (1 μM) as the internal standard. Samples were analyzed using the rapid HPLC method with isocratic elution, as described in Subheading 3.6
Fig. 11
Fig. 11
Real-time monitoring of superoxide production by differentiated HL60 (dHL60) cells stimulated with PMA. (A) HPLC peak of 2-OH-E+ as a function of incubation time of dHL60 cells with the HE probe. (B) Effect of PMA, SOD, and CAT on the HPLC peak of 2-OH-E+ detected at 60 min of incubation time of dHL60 cells with the HE probe. (C) Effect of PMA, SOD, and CAT on the dynamics of 2-OH-E+ formation during incubation of dHL60 cells with the HE probe. Samples were analyzed using the isocratic elution method, as described in Subheading 3.6, but using the column and mobile phase composition as detailed in Note 12
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