HPLC-Based Monitoring of Oxidation of Hydroethidine for the Detection of NADPH Oxidase-Derived Superoxide Radical Anion
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.
Key wordsSuperoxide radical anion Hydroethidine 2-hydroxyethidium HPLC LC-MS
This work was supported by NIH grants NCI U01 CA178960 and R01 AA022986 to B.K.
- 6.Zielonka J, Kalyanaraman B (2018) Small-molecule luminescent probes for the detection of cellular oxidizing and nitrating species. Free Radic Biol Med 128:3–22. https://doi.org/10.1016/j.freeradbiomed.2018.03.032 CrossRefPubMedGoogle Scholar
- 11.Fernandes DC, Wosniak J Jr, Pescatore LA, Bertoline MA, Liberman M, Laurindo FR, Santos CX (2007) 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–C422CrossRefGoogle Scholar