Spin trapping of nitric oxide produced in vivo in septic-shock mice

A nitric oxide (.NO) spin-trapping technique combined with electron paramagnetic resonance (EPR) spectroscopy has been employed to measure the in vivo production of .NO in lipopolysaccharide (LPS)-treated mice. The in vivo spin-trapping of .NO was performed by injecting into mice a metal-chelator complex, consisting of N-methyl-D-glucamine dithiocarbamate (MGD) and reduced iron (Fe2+), that binds to .NO and forms a stable, water-soluble [(MGD)2-Fe(2+)-NO] complex, and by monitoring continuously the in vivo formation of the latter complex using an S-band EPR spectrometer. At 6 h after intravenous injection of LPS, a three-line EPR spectrum of the [(MGD)2-Fe(2+)-NO] complex, was observed in the blood circulation of the mouse tail; the [(MGD)2-Fe2+] complex was injected subcutaneously 2 h before EPR measurement. No signal was detected in control groups. Administration of NG-monomethyl-L-arginine, an .NO synthase inhibitor, caused a marked reduction in the in vivo EPR signal of the [(MGD)2-Fe(2+)-NO] complex, suggesting that the .NO detected is synthesized via the arginine-nitric oxide synthase pathway. The results presented here demonstrated, for the first time, the in vivo real time measurement of .NO in the blood circulation of conscious, LPS-treated animals.