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‐Fe2+‐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‐Fe2+‐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 N G‐monomethyl‐l‐arginine, an •NO synthase inhibitor, caused a marked reduction in the in vivo EPR signal of the [(MGD)2‐Fe2+‐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.
The effect of dietary Mg deficiency on nitric oxide (NO) production and its role in mediating oxidative depletion of red blood cell (RBC) glutathione in rats were investigated. Male Sprague-Dawley rats were placed on Mg-deficient or Mg-sufficient diets for up to 3 wk. Plasma nitrate plus nitrite levels, determined by the Escherichia coli reductase/Griess reagent procedures, increased 1.7-fold during the 1st wk and increased 2- to 2.4-fold during the 2nd and 3rd wk on the Mg-deficient diet. In association, substantial losses (approximately 50%) of RBC glutathione occurred during the 2nd and 3rd wk. Administration of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in drinking water (0.5 mg/ml) effectively blunted the increases in plasma nitrate/nitrite during Mg deficiency. Concomitantly, losses of RBC glutathione exhibited by Mg-deficient rats were significantly attenuated. Packed RBCs, obtained from Mg-deficient but not from Mg-sufficient animals, displayed a prominent nitrosyl hemoglobin signal detected by electron spin resonance spectroscopy; the signals of the samples from the L-NAME-treated Mg-deficient rats were greatly reduced. With isolated RBCs, losses of the glutathione could be induced directly by peroxynitrite or 3-morpholinosydnonimine, which generates NO + .O2-, but not by NO (from sodium nitroprusside) alone, in a concentration-dependent manner. The results clearly indicate that NO overproduction occurs and participates in RBC glutathione loss during Mg deficiency. Because neutrophil activation also occurs, we suggest that NO might interact with superoxide anions to form peroxynitrite, which then directly oxidizes RBC glutathione.
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 microM, 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 15N-arginine (10 mg per mouse), a composite EPR spectrum, consisting of a three-line spectrum of the [(MGD)2/Fe-14NO] complex and a two-line spectrum of the [(MGD)2/Fe-15NO] 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 pathophysiological conditions where .NO is overproduced.
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