SPECIFIC S-NITROSOTHIOL (THIONITRITE) QUANTIFICATION AS SOLUTION NITRITE AFTER VANADIUM(III) REDUCTION AND OZONE-CHEMILUMINESCENT DETECTION

Increasing evidence suggests that S-nitrosothiols (thionitrites) might represent naturally occurring nitric oxide surrogates and function as intermediates in nitrogen monoxide metabolism. A facile, sensitive, a nd selective micromethod has been developed and validated for quantifi cation of S-nitrosothiols as their mercury-displaceable nitrogen monox ide content. In this method, brief (5-min), room-temperature pretreatm ent of S-nitrosothiol with a molar excess of aqueous mercuric chloride was used to liberate into solution, quantitatively, the nitrogen mono xide moiety, which rapidly and quantitatively converted to its stable solution end-product, nitrite. Solution nitrite was reduced back to ni tric oxide with vanadium(III), and the nitric oxide was detected by ga s-phase chemiluminescence after reaction with ozone in a commercial ni tric oxide analyzer. A linear relationship was observed between S-nitr osothiol-bound nitrogen monoxide and ozone-chemiluminescent detector r esponse over a wide range (16.3-3500 pmol) of nitric oxide, as generat ed by reaction of vanadium(III) with either nitrite standard or mercur y-treated S-nitrosothiol. Assay response was quantitatively identical for equivalent amounts of nitrite and S-nitrosothiol-bound nitrogen mo noxide. The method displayed 96% selectivity for nitrite vs, nitrate a nd negligible (<2%) interference by nitrosated compounds bearing nitro gen monoxide moieties bound to either nitrogen or carbon. The lower li mits of quantitative sensitivity and qualitative detection were below 50 and 20 pmol S-nitrosothiol-bound nitrogen monoxide-equivalents, res pectively. The intraday and interday coefficients of variation did not exceed 8%. This technique has been applied to quantify structurally d iverse natural and synthetic S-nitrosothiols with quantitative recover y from complex biological samples such as culture media and plasma at levels of nitrogen monoxide-equivalents undetectable by the popular Sa ville colorimetric method. (C) 1998 Elsevier Science Inc.