Assessment of superoxide production and NADPH oxidase activity by HPLC analysis of dihydroethidium oxidation products.

Assessment of low-level superoxide in nonphagocytic cells is crucial for assessing redox-dependent signaling pathways and the role of enzymes such as the NADPH oxidase complex. However, most superoxide probes present inherent limitations. Particularly, assessment of dihydroethidium (DHE) fluorescence is limited regarding a lack of possible quantification and simultaneous detection of its two main products: 2-hydroxyethidium, more specific for superoxide, and ethidium, which reflects H2O2-dependent pathways involving metal proteins. HPLC separation and analysis of those two main products have been described. This chapter reports procedures used for the validation of superoxide measurements in vascular system. Superoxide assessment was performed for cultured cells and tissue fragments incubated with DHE, followed by acetonitrile extraction and HPLC run, with simultaneous fluorescence detection of 2-hydroxyethidium and ethidium and ultraviolet detection of remaining DHE. It also describes procedures for DHE-based NADPH oxidase activity assays using HPLC or fluorometry. Such methods can enhance accuracy and allow better quantitation of vascular superoxide measurements.