Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols

While nitrosothiol compounds have been hypothesized to be important in the transport and function of nitric oxide (NO) in biological systems many important questions regarding their mechanism of formation and functional importance remain. In view of these fundamental questions there has been a great need for simple, sensitive, and specific methods for quantitation of nitrosothiols in biological samples. We report the development of two methods, for the measurement of nitrosothiol compounds using a chemiluminescence nitric oxide analyzer with a standard purging vessel. The first method is based on treatment with acidified solutions of potassium iodide in the presence or absence of dissolved free iodine. Quantitative release of NO occurs either from both nitrite and nitrosothiols or from nitrite alone, respectively. Subtraction of the amount of NO released without iodine from NO released in the presence of iodine allows estimation of the nitrosothiol concentration. To selectively measure nitrosothiols, we developed a redox quinone-hydroquinone alkaline reactant that selectively releases NO from nitrosothiols. This reactant quantitatively converts nitrosothiols to NO at elevated temperature, > 60 degrees C. Both methods were shown to detect nitrosothiols in biological buffers or blood plasma down to 10 nM concentration with high accuracy and reproducibility, variability less than 5%. These assays should be a useful addition to techniques used to characterize the biochemistry of NO.