Specific S-nitrosothiol (thionitrite) quantification as solution nitrite after vanadium(III) reduction and ozone-chemiluminescent detection.

Abstract

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, and selective micromethod has been developed and validated for quantification of S-nitrosothiols as their mercury-displaceable nitrogen monoxide content. In this method, brief (5-min), room-temperature pretreatment of S-nitrosothiol with a molar excess of aqueous mercuric chloride was used to liberate into solution, quantitatively, the nitrogen monoxide moiety, which rapidly and quantitatively converted to its stable solution end-product, nitrite. Solution nitrite was reduced back to nitric oxide with vanadium(III), and the nitric oxide was detected by gas-phase chemiluminescence after reaction with ozone in a commercial nitric oxide analyzer. A linear relationship was observed between S-nitrosothiol-bound nitrogen monoxide and ozone-chemiluminescent detector response over a wide range (16.3-3500 pmol) of nitric oxide, as generated by reaction of vanadium(III) with either nitrite standard or mercury-treated S-nitrosothiol. Assay response was quantitatively identical for equivalent amounts of nitrite and S-nitrosothiol-bound nitrogen monoxide. The method displayed 96% selectivity for nitrite vs. nitrate and negligible (<2%) interference by nitrosated compounds bearing nitrogen monoxide moieties bound to either nitrogen or carbon. The lower limits of quantitative sensitivity and qualitative detection were below 50 and 20 pmol S-nitrosothiol-bound nitrogen monoxide-equivalents, respectively. The intraday and interday coefficients of variation did not exceed 8%. This technique has been applied to quantify structurally diverse natural and synthetic S-nitrosothiols with quantitative recovery from complex biological samples such as culture media and plasma at levels of nitrogen monoxide-equivalents undetectable by the popular Saville colorimetric method.