Detection Of Nitric Oxide Production Research Articles

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 μM 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 15 n-arginine (10 mg per mouse), a composite EPR spectrum, consisting of a three-line spectrum of the [(MGD) 2/Fe- 14 NO] complex and a two-line spectrum of the [(MGD) 2/Fe- 15 NO] 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.

Detection of nitric oxide production from a perfused organ by a luminol-H2O2 system.

Nitric oxide, thought to be the endothelium-derived relaxing factor (EDRF), is involved in intra- and intercellular signalling in various tissues. A system for the continuous detection of NO in the picomolar range from a perfused organ is described. The detection is based upon the chemiluminescence reaction between NO and the luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)-H2O2 system. The chemiluminescence is due to the formation of peroxynitrite from NO and H2O2. The luminol-H2O2 system is specifically reactive to NO, so that other nitrogen-containing compounds, (organic nitrite, organic nitrate, and thio-nitroso compounds) or endothelium-derived compounds do not interfere. The limit of determination was approximately 100 fM. This system has been used to measure continuous NO release from isolated perfused rat kidney and the simultaneous changes of perfusion pressure. In Wistar rats basal NO release was 85 +/- 11 fmol/min-1 (g of kidney weight)-1 (39 pM in the perfusate), and acetylcholine increased NO release dose dependently with a concomitant pressure reduction. The changes in NO release were always associated with mirror image changes in the perfusion pressure. Simple pressure reduction did not interfere with the chemiluminescence. Precise titration data as well as results of some preliminary experiments using this method are presented.

Detection of nitric oxide production in lipopolysaccharide-treated rats by ESR using carbon monoxide hemoglobin

Release of nitric oxide (NO), from macrophages activated with E. coli lipopolysaccharide (LPS) and endothelial cells, has been proposed using chemiluminescence and spectrophotometry. However these methods can not distinguish NO from NO 2 −. The present study was aimed to prove in vivo production of NO, by ESR using CO-hemoglobin (HbCO) as a trapping agent of NO in the peritoneal cavity of rats treated with LPS. We detected a broad signal in the recovered HbCO solution. Inositol hexaphosphate induced a three-line hyperfine structure, characteristic of NO-hemoglobin (HbNO). In the arterial blood, ESR signal of HbNO with faint hyperfine structure was detected. N G-Monomethyl-L-arginine inhibited the formation of HbNO. HbNO was not detected in the peritoneal cavity of the LPS-untreated rat given i.p. both NO 2 − and HbCO. HbNO was, therefore, derived from NO, not from NO 2 −. These results show that free NO is produced in vivo by the stimulation of LPS.