Skip to Content
Merck

Nitrosothiol quantification in human plasma.

Analytical biochemistry (1998-05-30)
R K Goldman, A A Vlessis, D D Trunkey
ABSTRACT

A high-pressure liquid chromatography (HPLC) assay for measuring picomole quantities of nitrosothiol in biological samples was developed. The assay utilizes the catalytic reduction of nitrosothiol by mercuric cation (Hg2+). Released nitrogen oxide reacts with sulfanilamide (SA) and N-(1-napthyl)ethylenediamine (NNED) to form a stable azo dye. The azo dye is then separated from N-(1-napthyl)ethylenediamine and quantified 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 reference sample, which includes the nitrosothiol sample and all reagents except Hg2+, is utilized for the subtraction of nitrite and atmospheric nitrogen oxides which "contaminate" the nitrosothiol sample and reagents. This method is a sensitive (approximately 3 pmol; approximately 10(-1) microM) and accurate means to measure nitrosothiol concentration in biologic samples.

MATERIALS
Product Number
Brand
Product Description

Supelco
N-(1-Naphthyl)ethylenediamine dihydrochloride, for determination of sulfonamide and nitrite, ACS reagent, ≥98%
Sigma-Aldrich
N-(1-Naphthyl)ethylenediamine dihydrochloride, ACS reagent, >98%
Sigma-Aldrich
N-(1-Naphthyl)ethylenediamine dihydrochloride, ≥98%
Supelco
N-(1-Naphthyl)ethylenediamine dihydrochloride monomethanolate, for spectrophotometric det. of nitrate and nitrite, ≥99.0%