Login | Register

Development of a LC-MS method for analysis of thiol ratios as an indicator of oxidative stress


Development of a LC-MS method for analysis of thiol ratios as an indicator of oxidative stress

de Sa Tavares Russo, Mariana (2017) Development of a LC-MS method for analysis of thiol ratios as an indicator of oxidative stress. Masters thesis, Concordia University.

Text (application/pdf)
deSaTavaresRusso_MSc_F2017.pdf - Accepted Version
Restricted to Repository staff only until 1 September 2019.
Available under License Spectrum Terms of Access.


Reactive oxygen species are free radicals capable of damaging the cellular components in a process called oxidative stress. Among the different biomarkers that are used to determine level of oxidative stress is the ratio between reduced and oxidized thiols, such as glutathione and oxidized glutathione. The use of glutathione ratio as a biomarker of oxidative stress is possible because the thiols are responsible for reducing the oxidizing species in a process that oxidizes the thiols into their disulfides. Under normal conditions, the cells can regenerate the reduced thiols by the action of reductases, which keeps the ratio constant. However, under oxidative stress, the cell cannot regenerate the reduced thiols rapidly enough. This in turn increases the concentration of the disulfide, and the ratio decreases. The ratio can also be inadvertently altered during sample manipulation because thiols can autoxidize. Therefore, for their accurate determination, thiols should be derivatized prior to analysis. The existing protocols using liquid chromatography-mass spectrometry (LC-MS) for thiol analysis largely focus on urine or plasma analysis, and do not consider exposure to oxidation during sample handling, while the few studies on intracellular thiol concentrations employ derivatization after cell lysis. The main objective of this thesis was to develop a LC-MS method to accurately measure individual thiols and disulfides, and their ratios in Jurkat cells.
To achieve this goal, the selectivity and efficiency of two different derivatizing agents that are able to permeate the cell membrane were first compared in detail: N-ethyl maleimide (NEM) and N-phenyl ethyl maleimide (NPEM). They were compared in terms of their derivatization efficiency, electrospray ionization enhancement, stability and selectivity/side product formation with focus on four abundant intracellular thiols: cysteine (CYS), homocysteine (HCY), N-acetyl cysteine (NAC), glutathione (GSH) and their corresponding disulfides. While NPEM provided greater ionization efficiency than NEM (NPEM/NEM varies from 2.1x for GSH to 5.7x for CYS), it was also more unstable, forming more side-products. The instability of its maleimide ring led to reaction with amines, as well as double derivatization and cyclization reactions, which corresponded to about 10% of the signal of CYS. NEM showed only minor contribution of side reactions (about 1.5% of the signal of CYS), so it was chosen as the derivatizing reagent for the protocol. The derivatizing conditions with NEM were further optimized to minimize side product formation, and pH 7.0 was selected for further assay development while being compatible with cell handling.
In the next step, a full cell extraction protocol was developed to quantify the thiol ratios in Jurkat T cells. Briefly, the optimized protocol required 1 × 106 cells and combined NEM derivatization prior to cell lysis, cell lysis and extraction using 20% methanol (v/v) and protein precipitation by methanol. The thiols were then chromatographically separated using a biphenyl, reversed-phase, separation in combination with Quadrupole Time of Flight Mass Spectrometry (QToF-MS) analysis. Protocol optimization included evaluation of different lysis solvents, recovery, matrix effects, and evaluation of the number of washes required to ensure as complete removal of extracellular metabolites as possible without compromising cellular integrity. The final method was tested for its capacity to evaluate oxidative stress in cells stimulated by hydrogen peroxide, a known inducer of oxidative damage. The results show that the method was capable of differentiating between the control, mild and intense oxidative stress conditions.
To the best of my knowledge, this is the first cellular protocol that combines NEM derivatization prior to cell lysis with LC-MS determination of individual thiol ratios. An innovative aspect of this procedure is the protection of reduced thiols prior to lysis, which minimizes changes in the ratio caused by sample manipulation, as opposed to the typical procedure which has the derivatization after extraction. This work is also the first systematic comparison of NEM versus NPEM derivatization for LC-MS analysis and shows clearly the propensity of NPEM for side-product formation under conditions commonly used for maleimide derivatization. In summary, this research contributes towards more accurate measurement of thiol ratios as readouts of oxidative stress.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:de Sa Tavares Russo, Mariana
Institution:Concordia University
Degree Name:M. Sc.
Date:31 July 2017
Thesis Supervisor(s):Vuckovic, Dajana
ID Code:982934
Deposited On:16 Nov 2017 15:51
Last Modified:18 Jan 2018 17:56


1. Sentellas, S., Morales-Ibanez, O., Zanuy, M. & Albertí, J. J. GSSG/GSH ratios in cryopreserved rat and human hepatocytes as a biomarker for drug induced oxidative stress. Toxicol. Vitr. 28, 1006–1015 (2014).
2. Betteridge, D. J. What is oxidative stress? Metabolism. 49, 3–8 (2000).
3. Pizzimenti, S. et al. Interaction of aldehydes derived from lipid peroxidation and membrane proteins. Front. Physiol. 4 SEP, 1–17 (2013).
4. Wood, L. G., Gibson, P. G. & Garg, M. L. Biomarkers of lipid peroxidation, airway inflammation and asthma. Eur. Respir. J. 21, 177–186 (2003).
5. Rossi, R., Dalle-Donne, I., Milzani, A. & Giustarini, D. Oxidized forms of glutathione in peripheral blood as biomarkers of oxidative stress. Clin. Chem. 52, 1406–1414 (2006).
6. Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D. & Milzani, A. Biomarkers of oxidative damage in human disease. Clin. Chem. 52, 601–623 (2006).
7. Lokireddy, M. Measurement of Lipid Peroxidation in biology models using gas-chromatography-mass spectrometry. (Eastern Michigan University, 2005).
8. D’Agostino, L. A., Lam, K. P., Lee, R. & Britz-McKibbin, P. Comprehensive plasma thiol redox status determination for metabolomics. J. Proteome Res. 10, 592–603 (2011).
9. Winnik, W. M. & Kitchin, K. T. Measurement of oxidative stress parameters using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Toxicol. Appl. Pharmacol. 233, 100–106 (2008).
10. Strimbu, K. & Tavel, J. a. What are Biomarkers? Curr Opin HIV AIDS 5, 463–466 (2011).
11. Halliwell, B. & Whiteman, M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br. J. Pharmacol. 142, 231–255 (2004).
12. Labuschagne, C. F., Van Den Broek, N. J. F., Postma, P., Berger, R. & Brenkman, A. B. A protocol for quantifying lipid peroxidation in cellular systems by F2-isoprostane analysis. PLoS One 8, 1–12 (2013).
13. Janicka, M., Kot-Wasik, A., Kot, J. & Namieśnik, J. Isoprostanes-biomarkers of lipid peroxidation: Their utility in evaluating oxidative stress and analysis. Int. J. Mol. Sci. 11, 4631–4659 (2010).
14. Dalle-Donne, I., Rossi, R., Giustarini, D., Milzani, A. & Colombo, R. Protein carbonyl groups as biomarkers of oxidative stress. Clin. Chim. Acta 329, 23–38 (2003).
15. Poynton, R. A. & Hampton, M. B. Peroxiredoxins as biomarkers of oxidative stress. Biochim. Biophys. Acta - Gen. Subj. 1840, 906–912 (2014).
16. Giustarini, D. et al. Protein glutathionylation in erythrocytes. Clin. Chem. 49, 327–330 (2003).
17. Giustarini, D. et al. Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures. Free Radic. Biol. Med. 89, 972–981 (2015).
18. Mandal, A. K. et al. Quantitation and characterization of glutathionyl haemoglobin as an oxidative stress marker in chronic renal failure by mass spectrometry. Clin. Biochem. 40, 986–994 (2007).
19. Toyo’oka, T. Recent advances in separation and detection methods for thiol compounds in biological samples. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877, 3318–3330 (2009).
20. Hansen, R. E. & Winther, J. R. An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations. Anal. Biochem. 394, 147–158 (2009).
21. Dalle-Donne, I. & Rossi, R. Analysis of thiols. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877, 3271–3273 (2009).
22. Hansen, R. E., Roth, D. & Winther, J. R. Quantifying the global cellular thiol-disulfide status. Proc. Natl. Acad. Sci. 106, 422–427 (2009).
23. Oka, O. B. V & Bulleid, N. J. Forming disulfides in the endoplasmic reticulum. Biochim. Biophys. Acta - Mol. Cell Res. 1833, 2425–2429 (2013).
24. Poole, L. B. The basics of thiols and cysteines in redox biology and chemistry. Free Radic. Biol. Med. 80, 148–157 (2015).
25. Bechtel, T. J. & Weerapana, E. From structure to redox: The diverse functional roles of disulfides and implications in disease. Proteomics 17, 1600391 (2017).
26. Gupta, V. & Carroll, K. S. Sulfenic acid chemistry, detection and cellular lifetime. Biochim. Biophys. Acta - Gen. Subj. 1840, 847–875 (2014).
27. Kurilova, L. S., Krutetskaya, Z. I., Lebedev, O. E. & Antonov, V. G. The effect of oxidized glutathione and its pharmacological analogue glutoxim on intracellular Ca2+ concentration in macrophages Ca2+. Cell tissue biol. 2, 322–332 (2008).
28. Van Laer, K., Hamilton, C. J. & Messens, J. Low-Molecular-Weight Thiols in Thiol-Disulfide Exchange. Antioxid. Redox Signal. 18, 1642–1653 (2013).
29. Ortmayr, K., Schwaiger, M., Hann, S. & Koellensperger, G. An integrated metabolomics workflow for the quantification of sulfur pathway intermediates employing thiol protection with N-ethyl maleimide and hydrophilic interaction liquid chromatography tandem mass spectrometry. Analyst 140, 7687–7695 (2015).
30. Tomaiuolo, M. et al. A new method for determination of plasma homocystine by isotope dilution and electrospray tandem mass spectrometry. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 842, 64–69 (2006).
31. Innis, S. M. & Hasman, D. Evidence of choline depletion and reduced betaine and dimethylglycine with increased homocysteine in plasma of children with cystic fibrosis. J. Nutr. 136, 2226–31 (2006).
32. Rao, Y., McCooeye, M. & Mester, Z. Mapping of sulfur metabolic pathway by LC Orbitrap mass spectrometry. Anal. Chim. Acta 721, 129–136 (2012).
33. Whillier, S., Raftos, J. E., Chapman, B. & Kuchel, P. W. Role of N-acetylcysteine and cystine in glutathione synthesis in human erythrocytes. Redox Rep. 14, 115–124 (2009).
34. Osman, L. P., Mitchell, S. C. & Waring, R. H. Cysteine, its Metabolism and Toxicity. Sulfur Rep. 20, 155–172 (1997).
35. Wang, W. et al. Detection of homocysteine and cysteine. J. Am. Chem. Soc. 127, 15949–15958 (2005).
36. Pastore, A., Federici, G., Bertini, E. & Piemonte, F. Analysis of glutathione: Implication in redox and detoxification. Clin. Chim. Acta 333, 19–39 (2003).
37. Rafii, M. et al. Measurement of homocysteine and related metabolites in human plasma and urine by liquid chromatography electrospray tandem mass spectrometry. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877, 3282–3291 (2009).
38. Guan, X., Hoffman, B., Dwivedi, C. & Matthees, D. P. A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples. J. Pharm. Biomed. Anal. 31, 251–261 (2003).
39. Finamor, I. et al. Chronic aspartame intake causes changes in the trans-sulphuration pathway, glutathione depletion and liver damage in mice. Redox Biol. 11, 701–707 (2017).
40. Monostori, P., Wittmann, G., Karg, E. & Túri, S. Determination of glutathione and glutathione disulfide in biological samples: An in-depth review. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877, 3331–3346 (2009).
41. Hansen, R. E., Østergaard, H., Nørgaard, P. & Winther, J. R. Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4’-dithiodipyridine. Anal. Biochem. 363, 77–82 (2007).
42. Reeve, J., Kuhlenkamp, J. & Kalowitz, N. Estimation of glutathione in rat liver by reversed-phase high-performance liquid chromatography: separation from cysteine and γ-glutamylcysteine. J. Chromatogr. 194, 424–428 (1980).
43. Katrusiak, A. E., Paterson, P. G., Kamencic, H., Shoker, A. & Lyon, A. W. Pre-column derivatization high-performance liquid chromatographic method for determination of cysteine, cysteinyl-glycine, homocysteine and glutathione in plasma and cell extracts. J. Chromatogr. B Biomed. Sci. Appl. 758, 207–212 (2001).
44. Winther, J. R. & Thorpe, C. Quantification of thiols and disulfides. Biochim. Biophys. Acta - Gen. Subj. 1840, 838–846 (2014).
45. Owens, C. W. I. & Belcher, R. V. A Colorimetric Micromethod for the Determination of Glutathione. Biochem. J. 94, 705–711 (1965).
46. Tietze, F. Enzymic Method for Quantitative Determination of Nanogram Amounts of Total and Oxidized Glutathione Applications to Mammalian Blood and Other Tissues. Anal. Biochem. 27, 502–522 (1969).
47. Noctor, G. & Foyer, C. H. Simultaneous measurement of foliar glutathione, gamma-glutamylcysteine, and amino acids by high-performance liquid chromatography: comparison with two other assay methods for glutathione. Anal. Biochem. 264, 98–110 (1998).
48. Giustarini, D. et al. Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room. J. Chromatogr. B 1019, 21–28 (2016).
49. Reed, D. J. et al. High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and selected thiols and disulfides. Anal. Biochem. 106, 55–62 (1980).
50. Imai, K. & Toyo’oka, T. Fluorometric assay of thiols with fluorobenzoxadiazoles. Methods Enzymol. 143, 67–75 (1987).
51. Svardal, A. M., Mansoor, M. A. & Ueland, P. M. Determination of reduced, oxidized, and protein-bound glutathione in human plasma with precolumn derivation with monobromobimane and liquid chromatography. Anal. Biochem. 184, 338–346 (2000).
52. Abukhalaf, I. K. et al. High performance liquid chromatographic assay for the quantitation of total glutathione in plasma. J. Pharm. Biomed. Anal. 28, 637–643 (2002).
53. Baeyens, W., Van Der Weken, G., Ling, B. L. & De Moerloose, P. HPLC Determination of N-Acetylcysteine in Pharmaceutical Preparations After Pre-Column Derivatization With Thiolyte R MB Using Fluorescence Detection. Anal. Lett. 21, 741–757 (1988).
54. Oe, T., Ohyagi, T. & Naganuma, A. Determination of γ-glutamylglutathione and other low-molecular-mass biological thiol compounds by isocratic high-performance liquid chromatography with fluorimetric detection. J. Chromatogr. B Biomed. Appl. 708, 285–289 (1998).
55. Husted, L. B., Sørensen, E. S. & Sottrup-Jensen, L. 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole is not specific for labeling of sulfhydryl groups in proteins as it may also react with phenolic hydroxyl groups and amino groups. Anal. Biochem. 314, 166–168 (2003).
56. Michaelsen, J. T., Dehnert, S., Giustarini, D., Beckmann, B. & Tsikas, D. HPLC analysis of human erythrocytic glutathione forms using OPA and N-acetyl-cysteine ethyl ester: Evidence for nitrite-induced GSH oxidation to GSSG. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877, 3405–3417 (2009).
57. Zuman, P. Reactions of orthophthalaldehyde with nucleophiles. Chem. Rev. 104, 3217–3238 (2004).
58. Jacobs, W. A., Leburg, M. W. & Madaj, E. J. Stability of o-phthalaldehyde-derived isoindoles. Anal. Biochem. 156, 334–340 (1986).
59. Willig, S., Hunter, D. L., Dass, P. & Padilla, S. Validation of the use of 6,6’-dithiodinicotinic acid as a chromogen in the Ellman method for cholinesterase determinations. Vet. Hum. Toxicol. 38, 249–53 (1996).
60. Jemal, M. & Hawthorne, D. High performance liquid chromatography/ionspray mass spectrometry of N-ethylmaleimide and acrylic acid ester derivatives for bioanalysis of thiol compounds. Rapid Commun. Mass Spectrom. 8, 854–857 (1994).
61. Seiwert, B. & Karst, U. Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides. Anal. Chem. 79, 7131–7138 (2007).
62. Majima, E. et al. Stabilities of the fluorescent SH-reagent eosin-5-maleimide and its adducts with sulfhydryl compounds. BBA - Gen. Subj. 1243, 336–342 (1995).
63. Giustarini, D., Dalle-Donne, I., Milzani, A., Fanti, P. & Rossi, R. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat. Protoc. 8, 1660–1669 (2013).
64. Camera, E. & Picardo, M. Analytical methods to investigate glutathione and related compounds in biological and pathological processes. J. Chromatogr. B 781, 181–206 (2002).
65. Hammermeister, D. E., Serrano, J., Schmieder, P. & Kuehl, D. W. Characterization of dansylated glutathione, glutathione disulfide, cysteine and cystine by narrow bore liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 14, 503–508 (2000).
66. Bouligand, J., Deroussent, A., Paci, A., Morizet, J. & Vassal, G. Liquid chromatography-tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 832, 67–74 (2006).
67. Iwasaki, Y., Hoshi, M., Ito, R., Saito, K. & Nakazawa, H. Analysis of glutathione and glutathione disulfide in human saliva using hydrophilic interaction chromatography with mass spectrometry. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 839, 74–79 (2006).
68. Wang, J. et al. Simultaneous Quantification of Amino Metabolites in Multiple Metabolic Pathways Using Ultra-High Performance Liquid Chromatography with Tandem-mass Spectrometry. Sci. Rep. 7, 1423 (2017).
69. Kindt, T. J., Goldsby, R. A., Osborne, B. A. & Kuby, J. Kuby Immunology. (W. H. Freeman, 2007).
70. Chkhikvishvili, I. et al. Constituents of French Marigold (Tagetes patula L.) Flowers Protect Jurkat T-Cells against Oxidative Stress. Oxid. Med. Cell. Longev. 2016, (2016).
71. Vuckovic, D. Current trends and challenges in sample preparation for global metabolomics using liquid chromatography-mass spectrometry. Anal. Bioanal. Chem. 403, 1523–1548 (2012).
72. Dettmer, K. et al. Metabolite extraction from adherently growing mammalian cells for metabolomics studies: Optimization of harvesting and extraction protocols. Anal. Bioanal. Chem. 399, 1127–1139 (2011).
73. Dhakshinamoorthy, S., Dinh, N.-T., Skolnick, J. & Styczynski, M. P. Metabolomics identifies the intersection of phosphoethanolamine with menaquinone-triggered apoptosis in an in vitro model of leukemia. Mol. BioSyst. 11, 2406–2416 (2015).
74. Chen, W., Zhao, Y., Seefeldt, T. & Guan, X. Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid. J. Pharm. Biomed. Anal. 48, 1375–1380 (2008).
75. Baty, J. W., Hampton, M. B. & Winterbourn, C. C. Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells. Biochem. J. 389, 785–795 (2005).
76. Kand’ár, R., Žáková, P., Lotková, H., Kučera, O. & Červinková, Z. Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection. J. Pharm. Biomed. Anal. 43, 1382–1387 (2007).
77. Ramanathan, A. & Schreiber, S. L. Direct control of mitochondrial function by mTOR. Proc. Natl. Acad. Sci. U. S. A. 106, 22229–22232 (2009).
78. Lewis, G. D. et al. Metabolite profiling of blood from individuals undergoing planned myocardial infarction reveals early markers of myocardial injury. J. Clin. Invest. 118, 3503–3512 (2008).
79. Baumann, S. Starvation of Jurkat T cells causes metabolic switch from glycolysis to lipolysis as revealed by comprehensive GC-qMS. J. Integr. OMICS 3, 70–74 (2013).
80. Harwood, D. T., Kettle, A. J., Brennan, S. & Winterbourn, C. C. Simultaneous determination of reduced glutathione, glutathione disulphide and glutathione sulphonamide in cells and physiological fluids by isotope dilution liquid chromatography-tandem mass spectrometry. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877, 3393–3399 (2009).
81. Cereser, C. et al. Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts. J. Chromatogr. B. Biomed. Sci. Appl. 752, 123–132 (2001).
82. Camera, E., Rinaldi, M., Briganti, S., Picardo, M. & Fanali, S. Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry. J. Chromatogr. B. Biomed. Sci. Appl. 757, 69–78 (2001).
83. Sitnikov, D. G. G., Monnin, C. S. S. & Vuckovic, D. Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS. Sci. Rep. 6, 38885 (2016).
84. Liao, W. et al. Combined Metabonomic and Quantitative Real-Time PCR Analyses Reveal Systems Metabolic Changes in Jurkat T ‑ Cells Treated with HIV ‑ 1 Tat Protein. J. Proteome Res. 11, 5109–5123 (2012).
85. Matuszewski, B. K. K., Constanzer, M. L. L. & Chavez-Eng, C. M. M. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem. 75, 3019–3030 (2003).
86. Scopus. Scopus search for Oxidative Stress articles published in the United States in 2016. Available at: https://www.scopus.com/results/results.uri?sort=plf-f&src=s&sid=f1f2708d2a781de6e4b73abd3719c558&sot=a&sdt=a&cluster=scoaffilctry%2C%22United+States++%22%2Ct%2Bscopubyr%2C%222016+%22%2Ct&sl=35&s=%28TITLE-ABS-KEY%28%22oxidative+stress%22%29%29&origin=searc. (Accessed: 16th August 2017)
87. Scopus. Scopus search for Oxidative Stress and Glutathione articles published in the United States in 2016. Available at: https://www.scopus.com/results/results.uri?sort=plf-f&src=s&sid=6800a12fb2451e368cd7084ff3d00e89&sot=a&sdt=a&cluster=scoaffilctry%2C%22United+States++%22%2Ct%2Bscopubyr%2C%222016+%22%2Ct&sl=53&s=%28TITLE-ABS-KEY%28%22oxidative+stress%22%29%29+AND+%28gluta. (Accessed: 15th August 2017)
88. Iwasaki, Y. et al. Chromatographic and mass spectrometric analysis of glutathione in biological samples. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877, 3309–3317 (2009).
89. Winters, R. A., Zukowski, J., Ercal, N., Matthews, R. H. & Spitz, D. R. Analysis of Glutathione, Glutathione Disulfide, Cysteine, Homocysteine, and Other Biological Thiols by High-Performance Liquid Chromatography Following Derivatization by N-(1-Pyrenyl)maleimide. Analytical Biochemistry 227, 14–21 (1995).
90. Paroni, R. et al. HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue. Clin. Chem. 41, 448–454 (1995).
91. Asensi, M., Sastre, J., Pallardo, F. V., Estrela, J. M. & Vina, J. Determination of oxidized glutathione in blood: High-performance liquid chromatography. Methods Enzymol. 234, 367–371 (1994).
92. Smyth, D. G., Nagamatsu, A. & Fruton, J. S. Some Reactions of N-Ethylmaleimide. J. Am. Chem. Soc. 82, 4600–4604 (1960).
93. Ates, B., Ercal, B. C., Manda, K., Abraham, L. & Ercal, N. Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol. Biomed. Chromatogr. 23, 119–123 (2009).
94. Fontana, A. & Toniolo, C. in The Chemistry of the Thiol Group (ed. Patai, S.) 294–298 (John Wiley & Sons, Ltd., 1974). doi:10.1002/9780470771310.ch5
95. Giustarini, D., Dalle-Donne, I., Milzani, A. & Rossi, R. Detection of glutathione in whole blood after stabilization with N-ethylmaleimide. Anal. Biochem. 415, 81–83 (2011).
96. Asensi, M. et al. A High-Performance Liquid Chromatography Method for Measurement of Oxidized Glutathione in Biological Samples. Analytical Biochemistry 217, 323–328 (1994).
97. Santori, G. et al. Different efficacy of iodoacetic acid and N-ethylmaleimide in high-performance liquid chromatographic measurement of liver glutathione. J. Chromatogr. B Biomed. Appl. 695, 427–433 (1997).
98. Halliwell, B. & Chirico, S. Lipid peroxidation: its mechanism, measurement and significance. Am. J. Clin. Nutr. 57, 715S–725S (1993).
99. Carretero, A. et al. In vitro/in vivo screening of oxidative homeostasis and damage to DNA, protein, and lipids using UPLC/MS-MS. Anal. Bioanal. Chem. 406, 5465–5476 (2014).
100. Cotgreave, I. A. & Moldéus, P. Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components of biological systems. J. Biochem. Biophys. Methods 13, 231–249 (1986).
101. Suh, J. H. et al. Clinical assay of four thiol amino acid redox couples by LC-MS/MS: Utility in thalassemia. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877, 3418–3427 (2009).
102. Rellán-Álvarez, R., Hernández, L. E., Abadía, J. & Álvarez-Fernández, A. Direct and simultaneous determination of reduced and oxidized glutathione and homoglutathione by liquid chromatography-electrospray/mass spectrometry in plant tissue extracts. Anal. Biochem. 356, 254–264 (2006).
103. Steghens, J. P., Flourié, F., Arab, K. & Collombel, C. Fast liquid chromatography-mass spectrometry glutathione measurement in whole blood: Micromolar GSSG is a sample preparation artifact. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 798, 343–349 (2003).
104. Alwael, H., Connolly, D., Barron, L. & Paull, B. Development of a rapid and sensitive method for determination of cysteine/cystine ratio in chemically defined media. J. Chromatogr. A 1217, 3863–3870 (2010).
105. Kosower, N. S. & Kosower, E. M. Thiol labeling with bromobimanes. Methods Enzymol. 143, 76–84 (1987).
106. Bakirdere, S., Bramanti, E., D’ulivo, A., Ataman, O. Y. & Mester, Z. Speciation and determination of thiols in biological samples using high performance liquid chromatography-inductively coupled plasma-mass spectrometry and high performance liquid chromatography-Orbitrap MS. Anal. Chim. Acta 680, 41–47 (2010).
107. Dudman, N. P. B., Guo, X. W., Crooks, R., Xie, L. & Silberberg, J. S. Assay of plasma homocysteine: Light sensitivity of the fluorescent 7- benzo-2-oxa-1,3-diazole-4-sulfonic acid derivative, and use of appropriate calibrators. Clin. Chem. 42, 2028–2032 (1996).
108. Nolin, T. D., McMenamin, M. E. & Himmelfarb, J. Simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma by high-performance liquid chromatography: Application to studies of oxidative stress. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 852, 554–561 (2007).
109. Nielsen, J. W., Jensen, K. S., Hansen, R. E., Gotfredsen, C. H. & Winther, J. R. A fluorescent probe which allows highly specific thiol labeling at low pH. Anal. Biochem. 421, 115–120 (2012).
110. Brigelius-Flohé, R. Mixed results with mixed disulfides. Arch. Biochem. Biophys. 595, 81–87 (2016).
111. Han, J. C. & Han, G. Y. A Procedure for Quantitative Determination of Tris(2-carboxyethyl)phospine, an Odorless Reducing Agent More Stable and Effective Than Dithiothreitol. Anal. Biochem. 220, 5–10 (1994).
112. Zhang, W. et al. Simultaneous determination of glutathione, cysteine, homocysteine, and cysteinylglycine in biological fluids by ion-pairing high-performance liquid chromatography coupled with precolumn derivatization. J. Agric. Food Chem. 62, 5845–5852 (2014).
113. Mosharov, E., Cranford, M. R. & Banerjee, R. The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes. Biochemistry 39, 13005–13011 (2000).
114. Food and Drug Administration. Draft Guidance for Industry: Bioanalytical method validation. U.S. Department of Health and Human Services (2013).
115. Wishart, D. S., Jewison, T., Guo, A. C., Wilson, M. & Knox, C. L-Cysteine. Available at: http://www.hmdb.ca/metabolites/HMDB00574. (Accessed: 15th July 2017)
116. Wishart, D. S., Jewison, T., Guo, A. C., Wilson, M. & Knox, C. Homocysteine. Available at: http://www.hmdb.ca/metabolites/HMDB00742. (Accessed: 15th July 2017)
117. Wishart, D. S., Jewison, T., Guo, A. C., Wilson, M. & Knox, C. Glutathione. Available at: http://www.hmdb.ca/metabolites/HMDB00125. (Accessed: 15th July 2017)
118. Wishart, D. S., Jewison, T., Guo, A. C., Wilson, M. & Knox, C. Acetylcysteine. Available at: http://www.hmdb.ca/metabolites/HMDB01890. (Accessed: 15th July 2017)
119. Winterbourn, C. C. C. & Hampton, M. B. B. Thiol chemistry and specificity in redox signaling. Free Radic. Biol. Med. 45, 549–561 (2008).
120. Iciek, M., Chwatko, G., Lorenc-Koci, E., Bald, E. & Włodek, L. Plasma levels of total, free and protein bound thiols as well as sulfane sulfur in different age groups of rats. Acta Biochim. Pol. 51, 815–824 (2004).
All items in Spectrum are protected by copyright, with all rights reserved. The use of items is governed by Spectrum's terms of access.

Repository Staff Only: item control page

Downloads per month over past year

Back to top Back to top