Nitrosothiol quantification in human plasma.
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.
Goldman
R K
RK
Department of Surgery, Oregon Health Sciences University, Portland 97201-3098, USA.
Vlessis
A A
AA
Trunkey
D D
DD
D001391:Azo Compounds; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005029:Ethylenediamines; D005978:Glutathione / Q000031:analogs & derivatives / Q000138:chemical synthesis; D006801:Humans; D007202:Indicators and Reagents; D008623:Mercaptoethanol; D008628:Mercury; D008832:Microchemistry / Q000379:methods; D009589:Nitrogen Oxides; D009603:Nitroso Compounds / Q000097:blood* / Q000138:chemical synthesis; D010949:Plasma / Q000737:chemistry; D015203:Reproducibility of Results; D026422:S-Nitrosoglutathione; D026403:S-Nitrosothiols; D012680:Sensitivity and Specificity; D000077145:Sulfanilamide; D013424:Sulfanilamides
D001391:Azo Compounds; D005029:Ethylenediamines; D007202:Indicators and Reagents; D009589:Nitrogen Oxides; D009603:Nitroso Compounds; D026403:S-Nitrosothiols; D013424:Sulfanilamides; C008588:N-(1-naphthyl)ethylenediamine; D000077145:Sulfanilamide; D026422:S-Nitrosoglutathione; D008623:Mercaptoethanol; C032635:S-nitrosomercaptoethanol; D008628:Mercury; D005978:Glutathione