An assay method for nitric oxide-related compounds in whole blood

Analytical Biochemistry
M SonodaT Komoda

Abstract

Recent evidence suggests that nitric oxide (NO) generated in vivo will be converted into the forms of nitrite/nitrate, nitrosyl hemoproteins, nitrosyl metal complexes, and S-nitroso-compounds in the circulation. Nitrosothiols have also been reported to be relatively stable metabolites with micromolar levels in plasma. We hypothesized, therefore, that the determinations of all the NO-related compounds in blood would be of diagnostic significance. The assay method described here consists of the thermolysis of all the NO-related compounds in whole blood and the detection of resulting nitrate by fluorometry or chemiluminescence after an enzymatic reduction. S-Nitroso-albumin and nitrosyl hemoglobin can be easily thermolysed to nitrate, and relatively stable S-nitroso-glutathione is also degraded to nitrate in the presence of blood constituents with high molecular mass (above 30 kDa). Concentrations of NO-related compounds in blood from healthy human as well as control or lipopolysaccharide-stimulated rats were determined. We found that membrane-bound NO which showed the augmented levels under the pathophysiological states could also be detected. Together with electron spin resonance spectra, our data indicate that the fraction of N...Continue Reading

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