s-nitroglutathione and peroxynitric-acid

The endogenous potent vasodilators and inhibitors of platelet aggregation S-nitrosoglutathione (GSNO) and S-nitroglutathione (GSNO2) are frequently analyzed by high-performance liquid chromatography (HPLC) using mobile phases of acidic pH. These systems are associated with problems stemming from rapid and considerable artifactual formation of GSNO from glutathione (GSH) and ubiquitous nitrite. We describe a novel ion-pairing HPLC method with UV absorbance detection at 334 nm for the highly specific and interference-free analysis of GSNO and GSNO2 in the presence of high GSH and nitrite concentrations. Complete avoidance of artifactual formation of GSNO was accomplished by using the anion-pairing agent tetrabutylammoniumhydrogen sulphate in the mobile phase that enables analysis of GSNO at neutral pH, at which GSH and nitrite do not react to form GSNO. This HPLC system was used to study formation of GSNO2 from GSH and peroxynitrite under physiological conditions. We found by this HPLC system that peroxynitrite (0-300 microM) reacts with GSH (0-5 mM) to form GSNO2 at a mean yield of 2%. Analysis of the same samples by a cation-pairing HPLC system with acidic mobile phase (pH 2.0) revealed, however, GSNO plus GSNO2 formation of the order of 20% due to on column reaction of GSH with peroxynitrite-derived nitrite to form GSNO. Ammonium sulfamate is frequently used to remove nitrite from thiol-containing solutions under acidic conditions. By means of the anion-pairing HPLC system it is demonstrated that nitrite removal by this method is incomplete even when ammonium sulfamate is used at high concentrations. These findings underscore the absolute requirement of neutral pH conditions for the analysis of GSNO. The novel anion-pairing HPLC method should be useful to provide reliable data on formation, reaction and metabolism of GSNO and GSNO2 in biological fluids using various detectors including mass spectrometers.

Topics: Anions; Artifacts; Chromatography, High Pressure Liquid; Glutathione; Hydrogen-Ion Concentration; Nitrates; Nitro Compounds; Nitroso Compounds; S-Nitrosoglutathione; Spectrophotometry, Ultraviolet

Peroxynitrite (ONOO-) has been shown in studies on vascular relaxation and guanylate cyclase activation to react with glutathione (GSH), generating an intermediate product that promotes a time-dependent production of nitric oxide (NO). In this study, reactions of ONOO- with GSH produced a new substance, which was characterized by liquid chromatography, ultraviolet spectroscopy, and electrospray tandem mass spectrometry. The mass spectrometric data provided evidence that the product of this reaction was S-nitroglutathione (GSNO2) and that S-nitrosoglutathione (GSNO) was not a detectable product of this reaction. Further evidence was obtained by comparison of the spectral and chromatographic properties with synthetic standards prepared by reaction of GSH with nitrosonium or nitronium borofluorates. Both the synthetic and ONOO-/GSH-derived GSNO2 generated a protonated ion, GSNO2H+, at m/z 353, which was unusually resistant to decomposition under collision activation, and no fragmentation was observed at collision energy of 25 eV. In contrast, an ion at m/z 337 (GSNOH+), generated from the synthetic GSNO, readily fragmented with the abundant loss of NO at 9 eV. Reactions of ONOO- with GSH resulted in the generation of NO, which was detected by the head space/NO-chemiluminescence analyzer method. The generation of NO was inhibited by the presence of glucose and/or CO2 in the buffers employed. Synthetic GSNO2 spontaneously generated NO in a manner that was not significantly altered by glucose or CO2. Thus, ONOO- reacts with GSH to form GSNO2, and GSNO2 decomposes in a manner that generates NO.

Topics: Chromatography, High Pressure Liquid; Glucose; Glutathione; Mass Spectrometry; Nitrates; Nitric Oxide; Nitro Compounds; Oxidants; Spectrophotometry, Ultraviolet