SPIN-TRAPPING ISOTOPICALLY-LABELED NITRIC-OXIDE PRODUCED FROM [N-15]L-ARGININE AND [O-17]DIOXYGEN BY ACTIVATED MACROPHAGES USING A WATER-SOLUBLE FE++-DITHIOCARBAMATE SPIN TRAP

The unique capabilities of EPR spin trapping of nitric oxide based on a ferrous-dithiocarbamate spin trap have been demonstrated in a study verifying the source of the nitrogen and oxygen atoms in nitric oxide produced from activated macrophages. Spin trapping experiments were performed during nitric oxide generation from activated mouse peritoneal macrophages using the ferrous complex of N-methyl D-glucamine dithiocarbamate as a spin trap. When N-15-substituted arginine was given to the activated macrophages in the presence of the spin trap a characteristic EPR spectrum of the nitric oxide spin adduct was obtained, which indicates the presence of the N-15 atom in the nitric oxide molecule. The hyperfine splitting (hfs) constant of the N-15 nucleus was 17.6 gauss. When O-17-containing dioxygen (55%) was supplied to the medium, an EPR spectrum consistent with the O-17-substituted nitric oxide spin adduct was observed in the composite spectrum. The hfs of O-17 was estimated to be 2.5 gauss. The (NO)-N-14 spin adduct observed after prolonged incubation in the medium which contains [N-15]L-arginine as the only extracellular source of arginine demonstrates that arginine is recycled through its metabolite in activated macrophages.