Superoxide Detection by Chemiluminescence, Fluorescence, and Spectrophotometry Methods

Pathology of vessel wall endothelium has been attributed to the presence of reactive oxygen species (ROS) and its effects during the onset of oxidative stress. The root of all ROS produced is superoxide (O₂*^-). Hence, its detection is imperative in filtering out appropriate antioxidants and obtaining deeper knowledge on its role. The present study used lucigenin-enhanced chemiluminescence, hydroethidine fluorescence, and cytochrome c spectrophotometric assays to measure O₂*^-. The aim was to compare and contrast among the methods using human embryonic kidney cells (HEK). Superoxide production was induced by NADH via NAD(P)H oxidase system. The study showed that lucigenin enhanced chemiluminescence assay was the most sensitive method whilst hydroethidine and cytochrome c assays were proficient as well.