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 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.
Komarov
A M
AM
Biophysics Research Institute, Medical College of Wisconsin, Milwaukee 53226, USA.
Lai
C S
CS
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D002614:Chelating Agents / Q000493:pharmacokinetics; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005260:Female; D005296:Ferrous Compounds; D007668:Kidney / Q000737:chemistry; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000737:chemistry; D051379:Mice; D008813:Mice, Inbred ICR; D009566:Nitrates / Q000097:blood; D009569:Nitric Oxide / Q000032:analysis / Q000096:biosynthesis / Q000652:urine*; D009573:Nitrites / Q000097:blood; D009587:Nitrogen Isotopes; D012772:Shock, Septic / Q000139:chemically induced; D013012:Sorbitol / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D013113:Spin Labels; D013859:Thiocarbamates / Q000493:pharmacokinetics; D019323:omega-N-Methylarginine
D002614:Chelating Agents; D005296:Ferrous Compounds; D008070:Lipopolysaccharides; D009566:Nitrates; D009573:Nitrites; D009587:Nitrogen Isotopes; D013113:Spin Labels; D013859:Thiocarbamates; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D013012:Sorbitol; C041262:N-methyl-D-glucamine dithiocarbamate; D001120:Arginine