Advertisement

Nitric Oxide pp 113-129 | Cite as

Measurement of S -Nitrosoglutathione in Plasma by Liquid Chromatography–Tandem Mass Spectrometry

  • Dimitrios Tsikas
  • Erik Hanff
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)

Abstract

This chapter describes an ultraperformance liquid chromatographic–tandem mass spectrometric (UPLC-MS/MS) method for the quantitative determination of S-nitrosoglutathione (GSNO) in human plasma. S-[15N]Nitrosoglutathione (GS15NO) serves as the internal standard. The protocol involves inactivation of plasma γ-glutamyltransferase activity by serine-borate, stabilization of GSNO with EDTA, and avoidance of S-transnitrosylation reactions by blocking SH groups with N-ethylmaleimidide (NEM). Fresh blood is treated with NEM/serine-borate/EDTA, plasma is spiked with GS15NO (50 nM), ultrafiltered (cutoff 10 kDa) and 10-μL aliquots of ultrafiltrate are analyzed by UPLC-MS/MS in the positive electrospray ionization (ESI+) mode. LC is performed on a Nucleoshell column using isocratic (0.5 mL/min) elution with acetonitrile-20 mM ammonium formate (70:30, v/v), pH 7. Quantification is performed by selected-reaction monitoring the mass transition m/z 337 ([M+H]+) → m/z 307 ([M+H−14NO]+●) for GSNO and m/z 338 ([M+H]+) → m/z 307 ([M+H−15NO]+●) for GS15NO. Matrix effects are outweighed by the internal standard GS15NO. The lower limit of quantitation (LOQ) is 2.8 nM.

Key words

Carbonic anhydrase Electrospray ionization GSNO LC-MS/MS Nitric oxide Nitrite Plasma Stable isotopes 

References

  1. 1.
    Stamler JS (2004) S-nitrosothiols in the blood: roles, amounts, and methods of analysis. Circ Res 94:414–417CrossRefPubMedGoogle Scholar
  2. 2.
    Giustarini D, Milzani A, Colombo R, Dalle-Donne I, Rossi R (2004) Nitric oxide, S-nitrosothiols and hemoglobin: is methodology the key? Trends Pharmacol Sci 25:311–316CrossRefPubMedGoogle Scholar
  3. 3.
    Lipton AJ, Johnson MA, Macdonald T, Lieberman MW, Gozal D, Gaston B (2001) S-nitrosothiols signal the ventilatory response to hypoxia. Nature 413:171–174CrossRefPubMedGoogle Scholar
  4. 4.
    Tsikas D, Sandmann J, Rossa S, Gutzki FM, Frölich JC (1999) Investigations of S-transnitrosylation reactions between low- and high-molecular-weight S-nitroso compounds and their thiols by high-performance liquid chromatography and gas chromatography-mass spectrometry. Anal Biochem 272:231–241CrossRefGoogle Scholar
  5. 5.
    Giustarini D, Tsikas D, Rossi R (2011) Study of the effect of thiols on the vasodilatory potency of S-nitrosothiols by using a modified aortic ring assay. Toxicol Appl Pharmacol 256(2):95–102CrossRefPubMedGoogle Scholar
  6. 6.
    Hogg N, Singh RJ, Konorev E, Joseph J, Kalyanaraman B (1997) S-Nitrosoglutathione as a substrate for gamma-glutamyl transpeptidase. Biochem J 323(Pt 2):477–481CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bramanti E, Angeli V, Mester Z, Pompella A, Paolicchi A, D’Ulivo A (2010) Determination of S-nitrosoglutathione in plasma: comparison of two methods. Talanta 81(4–5):1295–1299CrossRefPubMedGoogle Scholar
  8. 8.
    Tsikas D, Sandmann J, Gutzki FM, Stichtenoth DO, Frölich JC (1999) Measurement of S-nitrosoalbumin by gas chromatography-mass spectrometry. II. Quantitative determination of S-nitrosoalbumin in human plasma using S-[15N]nitrosoalbumin as internal standard. J Chromatogr B 726(1–2):13–24CrossRefGoogle Scholar
  9. 9.
    Taubert D, Roesen R, Lehmann C, Jung N, Schömig E (2007) Effects of low habitual cocoa intake on blood pressure and bioactive nitric oxide: a randomized controlled trial. JAMA 298(1):49–60CrossRefPubMedGoogle Scholar
  10. 10.
    Tsikas D, Rossi R (2007) Cocoa intake and blood pressure. JAMA 298(16):1862–1863. author reply 1863–1864CrossRefPubMedGoogle Scholar
  11. 11.
    Tsikas D, Schmidt M, Böhmer A, Zoerner AA, Gutzki FM, Jordan J (2013) UPLC-MS/MS measurement of S-nitrosoglutathione (GSNO) in human plasma solves the S-nitrosothiol concentration enigma. J Chromatogr B 927:147–157CrossRefGoogle Scholar
  12. 12.
    Mansoor MA, Svardal AM, Ueland PM (1992) Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma. Anal Biochem 200(2):218–129CrossRefPubMedGoogle Scholar
  13. 13.
    Tsikas D (2008) A critical review and discussion of analytical methods in the l-arginine/nitric oxide area of basic and clinical research. Anal Biochem 379:139–163CrossRefPubMedGoogle Scholar
  14. 14.
    Giustarini D, Milzani A, Dalle-Donne I, Rossi R (2007) Detection of S-nitrosothiols in biological fluids: a comparison among the most widely applied methodologies. J Chromatogr B 851:124–139CrossRefGoogle Scholar
  15. 15.
    Chobanyan-Jürgens K, Schwarz A, Böhmer A, Beckmann B, Gutzki FM, Michaelsen JT, Stichtenoth DO, Tsikas D (2012) Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite. Nitric Oxide 26(2):126–131CrossRefPubMedGoogle Scholar
  16. 16.
    Tsikas D, Ikic M, Tewes KS, Raida M, Frölich JC (1999) Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets. FEBS Lett 442(2–3):162–166CrossRefPubMedGoogle Scholar
  17. 17.
    de Belder AJ, MacAllister R, Radomski MW, Moncada S, Vallance PJ (1994) Effects of S-nitroso-glutathione in the human forearm circulation: evidence for selective inhibition of platelet activation. Cardiovasc Res 28(5):691–694CrossRefPubMedGoogle Scholar
  18. 18.
    Sandmann J, Schwedhelm KS, Tsikas D (2005) Specific transport of S-nitrosocysteine in human red blood cells: implications for formation of S-nitrosothiols and transport of NO bioactivity within the vasculature. FEBS Lett 579(19):4119–4124CrossRefPubMedGoogle Scholar
  19. 19.
    Hanff E, Böhmer A, Zinke M, Gambaryan S, Schwarz A, Supuran CT, Tsikas D (2016) Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets. Amino Acids 48(7):1695–1706CrossRefPubMedGoogle Scholar
  20. 20.
    Tsikas D, Frölich JC (2004) Trouble with the analysis of nitrite, nitrate, S-nitrosothiols and 3-nitrotyrosine: freezing-induced artifacts? Nitric Oxide 11:209–213. author reply 214–215CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Toxicology, Core Unit ProteomicsHannover Medical SchoolHannoverGermany

Personalised recommendations