AN ASSAY-METHOD FOR NITRIC OXIDE-RELATED COMPOUNDS IN WHOLE-BLOOD

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 metabo lites with micromolar levels in plasma. We hypothesized, therefore, th at the determinations of all the NO-related compounds in blood would b e 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 chemiluminescenc e after an enzymatic reduction. S-itroso-albumin and nitrosyl hemoglob in can be easily thermolysed to nitrate, and relatively stable S-nitro so-glutathione is also degraded to nitrate in the presence of blood co nstituents 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 mem brane-bound NO which showed the augmented levels under the pathophysio logical states could also be detected. Together with electron spin res onance spectra, our data indicate that the fraction of NO diffused and metabolized within red cells and the other NO-metabolites in plasma s uch as nitrite/nitrate and S-nitroso-compounds, both of which can refl ect NO-production in vivo would be recovered and detected quantitative ly by this method. (C) 1997 Academic Press.