DETECTION OF NITRIC-OXIDE PRODUCTION IN MICE BY SPIN-TRAPPING ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY

We describe here a spin-trapping method combined with X-band electron paramagnetic resonance (EPR) spectroscopy for ex vivo measurement of nitric oxide (. NO) levels in the urine of both normal and lipopolysaccharide (LPS)-induced shock mice. Normal or LPS-treated mice were injected subcutaneously with a metal-chelator complex, N-methyl-D-glucamine dithiocarbamate-ferrous iron, [(MGD)(2)/Fe], which binds to . NO and forms a water-soluble [(MGD)(2)/Fe-NO] complex. At 2 h after injection of the [(MGD)(2)/Fe] complex, a three-line EPR signal characteristic of the [(MGD)(2)/Fe-NO] complex was detected in the urine of either normal or LPS-treated mice. It is estimated that the concentrations of the [(MGD)(2)/Fe-NO] complex in normal and LPS-treated mouse urine were 1.3 and 35 mu M, respectively. This 25-fold increase in . NO levels in the LPS-treated mouse urine provides the direct evidence that LPS challenge induces the overproduction of . NO in mice. Administration of N-monomethyl-L-arginine (NMMA; 50 mg/kg) inhibited the ex vivo signal intensities of the [(MGD)(2)/Fe-NO] complex in the urine of either normal or LPS-treated mouse urine. Furthermore, after injection of N-15-arginine (10 mg per mouse), a composite EPR spectrum, consisting of a three-line spectrum of the [(MGD)(2)/Fe-(NO)-N-14] complex and a two-line spectrum of the [(MGD)(2)/Fe-(NO)-N-15] complex, was detected in the urine. These isotopic tracer experiments further confirm that the detected . NO levels in the mouse urine are produced via the arginine-nitric oxide pathway. This ex vivo spin-trapping method should readily be adapted to experiments on larger animals and provide a noninvasive way of measuring both constitutive and inducible . NO synthase activities in living animals under physiological as well as pathophyiological conditions where . NO is overproduced.