I read with great interest the study by Dutka et al. (2011), investigating a possible role of S-nitrosoglutathione (GSNO) in muscle fatigue in certain conditions. Although the authors performed a well-conducted study and reported interesting observations, the applied GSNO concentrations of 0.1–10 mm and the complex chemistry of GSNO cause some concern from a physiological point of view. In this respect, one may wonder whether the possible role for GSNO emerging from the study by Dutka et al. (2011) would also have been revealed by the use of physiologically relevant GSNO concentrations, which are, however, highly contradictory (e.g. Giustarini et al. 2007). Accurate measurement of GSNO in biological fluids is a formidable analytical challenge and artefact-prone. Baseline concentration of GSNO in plasma and other biological fluids is considered to be on the threshold of the picomolar–nanomolar range (e.g. Giustarini et al. 2007; Tsikas, 2008). This is of the same order as the putative physiological concentration of the precursor of GSNO, nitric oxide (•NO) (e.g. Hall & Garthwaite, 2009). GSNO picomolar–nanomolar concentrations are two to three orders of magnitude lower than the concentration of nitrite (NO2−) (e.g. Tsikas, 2008). Nitrite is the final autoxidation product of •NO in aqueous systems and one possible and quite stable and more abundant storage form of •NO, but lacks the bioactivity of •NO. This issue demands a deep examination and more critical discussion.
Thiol reactions within cells are numerous; intermediate reactions in a redox cascade can be critical; and the final reactants are often enigmatic. Chemically, •NO in its own right is a relatively stable radical gas that is unable to nitrosate the sulfhydryl (SH) group of cysteine (Cys), and Cys-containing thiols (RSHs), such as glutathione (GSH), albumin (ALB) and haemoglobin (Hb), to form the corresponding S-nitrosothiols (RSNOs). Rather, higher oxides of •NO, notably dinitrogen trioxide (N2O3), an intermediate autoxidation product of •NO, are the actual strongly S-nitrosating species. In aqueous systems, N2O3 hydrolyses to nitrous acid (HONO), which further dissociates to nitrite (pKa 3.37). HONO can potently S-nitrosate RSH to RSNO in a wide pH range up to 6, but not at physiological plasma pH values around 7.4 (e.g. Tsikas, 2003).
Widely used experimental RSNOs from endogenous thiols include GSNO, S-nitrosocysteine (CysSNO), S-nitroso-N-acetylcysteine (SNAC), S-nitrosoalbumin (ALBSNO) and S-nitrosohaemoglobin (HbSNO). S-Nitroso-N-acetyl-penicillamine (SNAP) is one of the most frequently used exogenous RSNOs. In contrast to GSNO, CysSNO, SNAC, ALBSNO and HbSNO, SNAP is not an S-nitrosated Cys derivative but an S-nitrosated penicillamine derivative. With the exception of CysSNO, the other RSNOs (i.e. GSNO, CysSNO, SNAC, ALBSNO, HbSNO and SNAP) are very poor •NO donors in their own right, so that the term ‘•NO donor’ does not properly characterize the chemistry of these RSNOs. This has been confirmed by Dutka et al. (2011), who found that measured maximum •NO concentrations corresponded to only about 0.6% and 0.12% of the initial GSNO and SNAP concentrations, respectively.
A common feature of RSNOs is the S-transnitrosation reaction, which is a reversible chemical reaction and has an equilibrium constant close to unity for most of the investigated S-transnitrosation reactions. As a consequence, addition of GSNO or SNAP to biological samples such as muscle fibres at very high concentrations would result in formation of many different low and high molecular mass RSNOs, with concomitant depletion of the respective endogenous low and high molecular mass RSHs and stoichiometric formation of GSH and N-acetyl-penicillamine (NAP), respectively. It is obvious that such conditions will entirely change the physiology of the biological subject under study. Moreover, given the high RSNO concentration used in such experiments, additional pathways may be influenced, for example the activity of enzymes such as the cyclooxygenases may be increased or inhibited (Tsikas & Niemann, 2012). Consequently, the effects originally intended to be studied may be superimposed on secondary actually subject-independent mechanisms.
It is worth mentioning that the S-transnitrosation reaction per se is not a redox reaction. In particular, the reaction of GSNO with Cys moieties of proteins is an S-transnitrosation reaction but not an S-glutathionylation reaction. Thus, reactions of GSNO with proteinogenic SH groups of Cys moieties do not change the oxidative state of the S atom of the SH group of the proteinogenic Cys moiety or GSH, or the oxidative state of the N atom of the nitrosyl group (+NO) of RSNOs. In contrast to the S-transnitrosation reaction, the S-glutathionylation reaction is a redox reaction and cannot be carried out by authentic GSNO. S-Glutathionylation seen from the use of GSNO should be considered rather artefactual and secondary, and can be explained by the formation of glutathione disulfide (GSSG) and/or other minor species formed from decomposed GSNO originally applied in very high concentrations. In addition, nitrite, the major GSNO-derived nitrogen species (Tsikas 2008), may interact with enzymes and proteins including catalase (Bohmer et al. 2011), xanthine oxido reductase and myoglobin, thus potentially interfering with the actual actions of GSNO.
Considering the above-mentioned ramifications, and given the stronger S-glutathionylation potency of GSSG in association with its higher physiological concentration in biological systems compared to GSNO, studies on potential physiological roles of GSNO should involve careful use of GSNO at physiologically relevant concentrations and should be controlled by testing major decomposition products including GSSG and nitrite.