NITROSOTHIOL QUANTIFICATION IN HUMAN PLASMA

A high-pressure liquid chromatography (HPLC) assay for measuring picom ole quantities of nitrosothiol in biological samples was developed. Th e assay utilizes the catalytic reduction of nitrosothiol by mercuric c ation (Hg2+). Released nitrogen oxide reacts with sulfanilamide (SA) a nd N-(1-napthyl)ethylenediamine (NNED) to form a stable azo dye. The a zo dye is then separated from N-(1-napthyl) ethylene diamine and quant ified by reversed-phase HPLC. In addition to nitrosothiol, nitrite and atmospheric nitrogen oxides are sources of nitrogen oxide that react with the reagents, SA and NNED, to form the azo dye. Therefore, a refe rence sample, which includes the nitrosothiol sample and all reagents except Hg2+, is utilized for the subtraction of nitrite and atmospheri c nitrogen oxides which ''contaminate'' the nitrosothiol sample and re agents. This method is a sensitive (similar to 3 pmol; similar to 10(- 1) mu M) and accurate means to measure nitrosothiol concentration in b iologic samples. (C) 1998 Academic Press.