Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems

Abstract

Dihydroethidium (DHE) is a widely used sensitive superoxide (O2(*-)) probe. However, DHE oxidation yields at least two fluorescent products, 2-hydroxyethidium (EOH), known to be more specific for O2(*-), and the less-specific product ethidium. We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree peroxynitrite/carbon dioxide system led to EOH and ethidium formation. Peroxidase/H2O2 but not H2O2 alone yielded ethidium as the main product. In vascular smooth muscle cells incubated with ANG II (100 nM, 4 h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in vascular smooth muscle cell membrane fractions, showing that EOH was uniquely increased after ANG II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was approximately 1.5- to 2-fold increase in EOH/DHE and ethidium/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of ethidium product and EOH/ethidium ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in ethidium generation. Thus HPLC analysis of DHE-derived oxidation products can improve assessment of O2(*-) production or NADPH oxidase activity in many vascular experimental studies.

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

“…These findings are very similar to those observed with HE [3]. Using large amounts of iron (0.5 mM) and hydrogen peroxide (0.5 mM), investigators claimed that hydroxyl radicals also could oxidize HE to form 2-OH-E + [20]. The effect of SOD on 2-OH-E + formed under these conditions was not, however, determined [20].…”

“…Using large amounts of iron (0.5 mM) and hydrogen peroxide (0.5 mM), investigators claimed that hydroxyl radicals also could oxidize HE to form 2-OH-E + [20]. The effect of SOD on 2-OH-E + formed under these conditions was not, however, determined [20]. By reacting HE with Fenton's reagent, we detected only low levels of 2-OH-E + (that was inhibited by SOD) formed from the reaction between HE and superoxide radicals [3].…”

Park7 interacts with p47phox to direct NADPH oxidase-dependent ROS production and protect against sepsis

“….− than ethidium, we used HPLC analysis to measure EOH levels to assess O 2 .− production or NADPH oxidase activity [32]. As shown in Figure 4G, under the PMA treatment, WT macrophages generated more superoxide anion than Park7 −/− macrophages.…”

“…The conditions of HPLC experiment were as described previously [32]. The optimal emission wavelength for both EOH and ethidium detection is 595 nm.…”

Nox4 NADPH Oxidase Mediates Peroxynitrite-dependent Uncoupling of Endothelial Nitric-oxide Synthase and Fibronectin Expression in Response to Angiotensin II

“…Detection of Intracellular Superoxide in MCs Using HPLCCellular superoxide production in MCs was assessed by HPLC analysis of dihydroethidium (DHE)-derived oxidation products, as described previously (42). The HPLC-based assay allowed the separation of the superoxide-specific 2-hydroxyethidium (EOH) from the nonspecific ethidium.…”