More than ten years after our initial report, there appears to be no consensus about the true physiological levels of S-nitrosothiols in human plasma, which range from 10 ^M [8,87] to 10 nM [88,89]. The differences have their origin in the diverse methodological approaches used to detect them [90]. Challenges about the physiological importance of S-nitrosothiols [91,92] arise from the difficulties and technical limitations of detecting them [93]. In particular, S-nitrosothiols formed under physiological conditions by either endogenous NO production or exogenous NO donors has remained an analytical morass, largely owing to the variable stability of the protein — S—N bond. S-nitrosothiols can form from thiols in the presence of nitrite at pH less than 7.4. In the presence of ascorbate, glutathione, and transition metals in biological fluids or buffer, S-nitrosothiols decompose rapidly and trans-S-nitrosation between protein S-nitrosothiols and low-molecular-weight thiols can occur. Endogenous S-nitrosothiols in biological fluid and tissue have not been detected directly in intact form, but only after conversion to stable derivatives [93]. An ideal assay for S-nitrosothiols would be rapid, specific, and effective in situ, avoiding transfer or loss of the S-nitrosothiol signal in the process.

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