Free Radical Biology and Medicine

Running title: Metabolism of cyclic nitrone superoxide adducts Manuscript Click here to view linked References 2 Abstract Reactive oxygen species (ROS) are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, superoxide radical is of special interest as the origin of several damaging species such as H 2 O 2 , hydroxyl radical, or peroxynitrite (ONOO-). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In the present manuscript, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original set up combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes.