Spin trapping isotopically-labelled nitric oxide produced from [15N]L-arginine and [17O]dioxygen by activated macrophages using a water soluble Fe(++)-dithiocarbamate spin trap.

Abstract

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 15N-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 15N atom in the nitric oxide molecule. The hyperfine splitting (hfs) constant of the 15N nucleus was 17.6 gauss. When 17O-containing dioxygen (55%) was supplied to the medium, an EPR spectrum consistent with the 17O-substituted nitric oxide spin adduct was observed in the composite spectrum. The hfs of 17O was estimated to be 2.5 gauss. The 14NO spin adduct observed after prolonged incubation in the medium which contains [15N]L-arginine as the only extracellular source of arginine demonstrates that arginine is recycled through its metabolite in activated macrophages.