Nitric oxide and superoxide in biological systems

Oxygen-derived free radicals play a major role in the physiology and pathology of the vessel wall [1]. To assess superoxide anion production (O2) from endothelium, four techniques are mainly used: reduced cytochrome c UV-visible absorption [2,3], ESR spin trapping [4,5], hydroethydine fluorescence [6] and lucigenin-enhanced chemiluminescence [7,8]. The simplest technique which has been used for the longest time consists in following the reduction of cytochrome c by O2, but the nature and the type of the cytochrome c used is not always mentioned. A few studies employing ESR and hydroethidine fluorescence have been reported. The most sensitive method commonly used appears to be lucigenin-enhanced chemiluminescence, but its reliability was recently questioned because reduced lucigenin can itself generate O2 [9,10]. In the literature, generally only one of these techniques is used for any publication and sometimes results diverge. In the present review we compare these four methods [5,11,12] to measure the O2 released from A23187-stimulated BAEC using the electron donors NADH and NADPH.