HPLC analysis of human erythrocytic glutathione forms using OPA and N-acetyl-cysteine ethyl ester: evidence for nitrite-induced GSH oxidation to GSSG.

Glutathione exists in biological samples in the reduced form (GSH), as its disulfide (GSSG) and as a mixed disulfide (GSSR) with thiols (RSH). GSH is the most abundant low-molecular-mass thiol and plays important roles as a cofactor and as a main constituent of the intracellular redox status. Due to its own sulfhydryl (SH) group, GSH reacts readily with o-phthaldialdehyde (OPA) to form a highly stable and fluorescent isoindole derivative (GSH-OPA), which allows for sensitive and specific quantitative determination of GSH in biological systems by HPLC with fluorescence (FL) detection.

GC-MS analysis of S-nitrosothiols after conversion to S-nitroso-N-acetyl cysteine ethyl ester and in-injector nitrosation of ethyl acetate.

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK(L) 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC-MS method for the analysis of S-nitrosothiols and their (15)N- or (2)H-(15)N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S(15)NACET or d(3)-S(15)NACET derivatives, respectively, after their extraction with ethyl acetate.