Analysis of Superoxide Anion Production in Tissue

Endothelial production of oxygen free radicals, especially superoxide anion (O₂ ), is an important mechanism of vascular dysfunction in hypertension. Overproduction of oxygen free radicals, mainly O₂ ^- occurs in human hypertension and in a wide variety of animal models. Thus, analysis of O₂ generation represents a useful tool for identifying oxidative stress in hypertension. Among the methods used for O₂ ^- detection, the chemiluminescent probe lucigenin has been widely shown to be a useful method for detecting and quantifying the O₂ ^- formation. On the other hand, staining by the oxidative fluorescent probe dihydroethidine, which is freely permeable to cell membranes, is suitable to monitor in situ production of O₂ ^- and to provide a reliable marker of its intracellular presence. Dihydroethidine is oxidized in the presence of O₂ ^- to a fluorescent marker product, which is rapidly intercalated into DNA. Thus, nuclei are the primary fluorescent structures labeled. By simply incubating experimental samples in the presence of dihydroethidine followed by analysis of fluorescence, this method allows rapid and specific detection of intracellular oxidative stress due to superoxide anion generation.