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

Abstract

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 (Fe 2+), 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‐Fe 2+] complex was injected subcutaneously 2 h before EPR measurement. No signal was detected in control groups. Administration of N G‐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.