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Measurement of methylglyoxal by stable isotopic dilution analysis LC-MS/MS with corroborative prediction in physiological samples

Nature Protocols volume 9, pages 1969–1979 (2014)Cite this article

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Abstract

This protocol describes a method for the detection and quantification of methylglyoxal (MG), the major physiological substrate of the cytosolic glyoxalase system. Accumulation of MG, also called dicarbonyl stress, is implicated in tissue damage in aging and disease. Measurement of MG is important in physiological studies, in the development of glyoxalase 1 (Glo1) inducer and inhibitor therapeutics, and in the characterization of medical products, especially dialysis fluids, and of thermally processed foods and beverages. MG can be derivatized with 1,2-diaminobenzene (DB), resulting in an adduct that can be detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantification is achieved by stable isotopic dilution analysis with [13C3]MG. Pre-analytic processing at ambient temperature, under acidic conditions with peroxidase inhibition, avoids artifactual overestimation of MG. Estimates obtained from physiological samples can be validated by kinetic modeling of in situ rates of protein glycation by MG for confirmation of the results. This procedure was developed for the analysis of cultured cells, plasma and animal tissue samples, and it can also be used to analyze plant material. Experimental measurement requires 4.5 h for sample batch pre-analytic processing and 30 min per sample for LC-MS/MS analysis.

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Figure 1: Estimation and prediction of MG concentration.

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References

  1. Rabbani, N. & Thornalley, P.J. Methylglyoxal, glyoxalase 1 and the dicarbonyl proteome. Amino Acids 42, 1133–1142 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Phillips, S.A. & Thornalley, P.J. The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal. Eur. J. Biochem. 212, 101–105 (1993).

    Article  CAS  PubMed  Google Scholar 

  3. Reichard, G.A., Skutches, C.L., Hoeldtke, R.D. & Owen, O.E. Acetone metabolism in humans during diabetic ketoacidosis. Diabetes 35, 668–674 (1986).

    Article  PubMed  Google Scholar 

  4. Lyles, G.A. & Chalmers, J. The metabolism of aminoacetone to methylglyoxal by semicarbazide-sensitive amino oxidase in human umbilical artery. Biochem. Pharmacol. 43, 1409–1414 (1992).

    Article  CAS  PubMed  Google Scholar 

  5. Thornalley, P.J., Langborg, A. & Minhas, H.S. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem. J. 344, 109–116 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Thornalley, P.J. Modification of the glyoxalase system in human red blood cells by glucose in vitro. Biochem. J. 254, 751–755 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Cooper, R.A. Metabolism of methylglyoxal in microorganisms. Ann. Rev. Microbiol. 38, 49–68 (1984).

    Article  CAS  Google Scholar 

  8. Thornalley, P.J. Dicarbonyl intermediates in the Maillard reaction. Ann. NY Acad. Sci. 1043, 111–117 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Rabbani, N. & Thornalley, P.J. in Uremic Toxins pp. 177–192 (John Wiley & Sons, 2012).

  10. Thornalley, P.J. & Rabbani, N. in Maillard Reaction: Interface Between Aging, Nutrition and Metabolism (eds. M.C. Thomas & J. Forbes) 158–163 RSC Publishing, 2010).

  11. Thornalley, P.J. et al. Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry. Biochem. J. 375, 581–592 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Thornalley, P.J. et al. Imidazopurinones are markers of physiological genomic damage linked to DNA instability and glyoxalase 1-associated tumour multidrug resistance. Nucleic Acids Res. 138, 5432–5442 (2010).

    Article  CAS  Google Scholar 

  13. Murata-Kamiya, N., Kamiya, H., Kaji, H. & Kasai, H. Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells. Mutat. Res. 468, 173–182 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Morcos, M. et al. Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans. Aging Cell 7, 260–269 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Dobler, D., Ahmed, N., Song, L.J., Eboigbodin, K.E. & Thornalley, P.J. Increased dicarbonyl metabolism in endothelial cells in hyperglycemia induces anoikis and impairs angiogenesis by RGD and GFOGER motif modification. Diabetes 55, 1961–1969 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Sejersen, H. & Rattan, S. Dicarbonyl-induced accelerated aging in vitro in human skin fibroblasts. Biogerontology 10, 203–211 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Xue, M., Rabbani, N. & Thornalley, P.J. Glyoxalase in ageing. Sem. Cell Dev. Biol. 22, 293–301 (2011).

    Article  CAS  Google Scholar 

  18. Baba, S.P. et al. Reductive metabolism of AGE precursors: a metabolic route for preventing AGE accumulation in cardiovascular tissue. Diabetes 58, 2486–2497 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Beisswenger, P.J., Howell, S.K., Touchette, A., Lal, S. & Szwergold, B.S. Metformin reduces systemic methylglyoxal levels in type 2 diabetes. Diabetes 48, 198–202 (1999).

    Article  CAS  PubMed  Google Scholar 

  20. Lapolla, A. et al. Evaluation of advanced glycation end products and carbonyl compounds in patients with different conditions of oxidative stress. Mol. Nutr. Food Res. 49, 685–690 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Scheijen, J. & Schalkwijk, C.G. Quantification of glyoxal, methylglyoxal and 3-deoxyglucosone in blood and plasma by ultra performance liquid chromatography tandem mass spectrometry: evaluation of blood specimen. Clin. Chem. Lab. Med. 52, 85–91 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Kurz, A. et al. α-synuclein deficiency leads to increased glyoxalase I expression and glycation stress. Cell Mol. Life Sci. 68, 721–733 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Hambsch, B. et al. Methylglyoxal-mediated anxiolysis involves increased protein modification and elevated expression of glyoxalase 1 in the brain. J. Neurochem. 113, 1240–1251 (2010).

    CAS  PubMed  Google Scholar 

  24. Rabbani, N. & Thornalley, P.J. Glyoxalase in diabetes, obesity and related disorders. Sem. Cell Dev. Biol. 22, 309–317 (2011).

    Article  CAS  Google Scholar 

  25. McLellan, A.C., Phillips, S.A. & Thornalley, P.J. The assay of methylglyoxal in biological systems by derivatization with 1,2-diamino-4,5-dimethoxybenzene. Anal. Biochem. 206, 17–23 (1992).

    Article  CAS  PubMed  Google Scholar 

  26. Wang, X.X., Desai, K., Chang, T.J. & Wu, L.Y. Vascular methylglyoxal metabolism and the development of hypertension. J. Hypertens. 23, 1565–1573 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Zeng, S. et al. Opposing roles of RAGE and Myd88 signaling in extensive liver resection. FASEB J. 26, 882–893 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lapolla, A. et al. Evaluation of glyoxal and methylglyoxal levels in uremic patients under peritoneal dialysis. Ann. NY Acad. Sci. 1043, 217–224 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. McLellan, A.C., Thornalley, P.J., Benn, J. & Sonksen, P.H. The glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications. Clin. Sci. 87, 21–29 (1994).

    Article  CAS  Google Scholar 

  30. Degen, J., Hellwig, M. & Henle, T. 1,2-Dicarbonyl compounds in commonly consumed foods. J. Agric. Food Chem. 60, 7071–7079 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Sakugawa, H., Kaplan, I.R. & Shepard, L.S. Measurements of H2O2, aldehydes and organic-acids in Los Angeles rainwater—their sources and deposition rates. Atmos. Environ. Part B Urban Atmos. 27, 203–219 (1993).

    Article  Google Scholar 

  32. McLellan, A.C. & Thornalley, P.J. Synthesis and chromatography of 1,2-diamino-4,5-dimethoxybenzene, 6,7-dimethoxy-2-methylquinoxaline and 6,7-dimethoxy-2,3-dimethylquinoxaline for use in a liquid chromatographic fluorimetric assay of methylglyoxal. Anal. Chim. Acta 263, 137–142 (1992).

    Article  CAS  Google Scholar 

  33. Rae, C., Berners-Price, S.J., Bulliman, B.T. & Kuchel, P.W. Kinetic analysis of the human erythrocyte glyoxalase system using 1 NMR and computer model. Eur. J. Biochem. 193, 83–90 (1990).

    Article  CAS  PubMed  Google Scholar 

  34. Nemet, I., Vikic-Topic, D. & Varga-Defterdarovic, L. Spectroscopic studies of methylglyoxal in water and dimethylsulfoxide. Bioorg. Chem. 32, 560–570 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Lo, T.W.C., Westwood, M.E., McLellan, A.C., Selwood, T. & Thornalley, P.J. Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with Nα-acetylarginine, Nα-acetylcysteine, Nα-acetyl-lysine, and bovine serum albumin. J. Biol. Chem. 269, 32299–32305 (1994).

    CAS  PubMed  Google Scholar 

  36. Thornalley, P.J., Yurek-George, A. & Argirov, O.K. Kinetics and mechanism of the reaction of aminoguanidine with the α-oxoaldehydes, glyoxal, methylglyoxal and 3-deoxyglucosone under physiological conditions. Biochem. Pharmacol. 60, 55–65 (2000).

    Article  CAS  PubMed  Google Scholar 

  37. Xue, M. et al. Transcriptional control of glyoxalase 1 by Nrf2 provides a stress responsive defence against dicarbonyl glycation. Biochem. J. 443, 213–222 (2012).

    Article  CAS  PubMed  Google Scholar 

  38. Bierhaus, A. et al. Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy. Nat. Med. 18, 926–933 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Brill, A.S. & Weinryb, I. Reactions of horseradish peroxidase with azide. Evidence for a methionine residue at the active site. Biochemistry 6, 3528–3535 (1967).

    Article  CAS  PubMed  Google Scholar 

  40. McMartin, D.N. & Schedlbauer, L.M. Phenylenediamine peroxidase-activity in brains of young and old mice. Gerontologist 15, 23–23 (1975).

    Google Scholar 

  41. Thornalley, P.J. & Rabbani, N. Assay of methylglyoxal and glyoxal and control of peroxidase interference. Biochem. Soc. Trans. 42, 504–510 (2014).

    Article  CAS  PubMed  Google Scholar 

  42. Latruffe, N., Elkebbaj, M.S., Moussard, C. & Gaudemer, Y. Permeability of inner mitochondrial-membrane to arginine reagents. FEBS Letters 144, 273–278 (1982).

    Article  CAS  PubMed  Google Scholar 

  43. Phillips, S.A. & Thornalley, P.J. Formation of methylglyoxal and D-lactate in human red blood cells in vitro. Biochem. Soc. Trans. 21, 163–163 (1993).

    Article  Google Scholar 

  44. Clelland, J.D. & Thornalley, P.J. Synthesis of 14C-labelled methylglyoxal and S-D-lactoylglutathione. J. Label. Comp. Radiopharm. 28, 1455–1464 (1990).

    Article  CAS  Google Scholar 

  45. Heller, D. Guidance for Industry: Mass Spectrometry for Confirmation of the Identity of Animal Drug Residues. (US Department of Health and Human Services, Food and Drug Administration (FDA), Center for Veterinary Medicine, 2003).

  46. Nicolay, J.P. et al. Stimulation of suicidal erythrocyte death by methylglyoxal. Cell. Physiol. Biochem. 18, 223–232 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Nemet, I., Turk, Z., Duvnjak, L., Car, N. & Varga-Defterdarovic, L. Humoral methylglyoxal level reflects glycemic fluctuation. Clin. Biochem. 38, 379–383 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Han, Y. et al. Plasma methylglyoxal and glyoxal are elevated and related to early membrane alteration in young, complication-free patients with type 1 diabetes. Mol. Cell. Biochem. 305, 123–131 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Babaei-Jadidi, R., Karachalias, N., Ahmed, N., Battah, S. & Thornalley, P.J. Prevention of incipient diabetic nephropathy by high-dose thiamine and benfotiamine. Diabetes 52, 2110–2120 (2003).

    Article  CAS  PubMed  Google Scholar 

  50. Rabbani, N., Sebekova, K., Sebekova, K. Jr., Heidland, A. & Thornalley, P.J. Protein glycation, oxidation and nitration free adduct accumulation after bilateral nephrectomy and ureteral ligation. Kidney Int. 72, 1113–1121 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Jia, X. & Wu, L. Accumulation of endogenous methylglyoxal impaired insulin signaling in adipose tissue of fructose-fed rats. Mol. Cell. Biochem. 306, 133–139 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Distler, M.G. et al. Glyoxalase 1 increases anxiety by reducing GABAA receptor agonist methylglyoxal. J. Clin. Invest. 122, 2306–2315 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Yadav, S.K., Singla-Pareek, S.L., Reddy, M.K. & Sopory, S.K. Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress. FEBS Lett. 579, 6265–6271 (2005).

    Article  CAS  PubMed  Google Scholar 

  54. Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. & Sopory, S.K. Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61–67 (2005).

    Article  CAS  PubMed  Google Scholar 

  55. Liu, J. et al. Aldolase B knockdown prevents high glucose-induced methylglyoxal overproduction and cellular dysfunction in endothelial cells. PLoS ONE 7, e41495 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Chen, B.-G., Lin, C., Chen, C., Hambsch, B. & Chern, C.-L. Quantitation by GC-MS of methylglyoxal as a marker in anxiety-related studies. Chromatographia 76, 571–576 (2013).

    Article  CAS  Google Scholar 

  57. Phillips, S.A., Mirrlees, D. & Thornalley, P.J. Modification of the glyoxalase system in streptozotocin-induced diabetic rats. Effect of the aldose reductase inhibitor Statil. Biochem. Pharmacol. 46, 805–811 (1993).

    Article  CAS  PubMed  Google Scholar 

  58. Seger, C. & Vogeser, M. in LC-MS in Drug Bioanalysis (eds. Q.A. Xu & T.L. Madden) Ch. 5, 109–126 (Springer, 2012).

  59. Ahmed, N., Babaei-Jadidi, R., Howell, S.K., Beisswenger, P.J. & Thornalley, P.J. Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes. Diabetologia 48, 1590–1603 (2005).

    Article  CAS  PubMed  Google Scholar 

  60. Peters, T. All About Albumin. (Academic Press, 1996)

  61. Price, J.C., Guan, S., Burlingame, A., Prusiner, S.B. & Ghaemmaghami, S. Analysis of proteome dynamics in the mouse brain. Proc. Natl. Acad. Sci. USA 107, 14508–14513 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Sampugna, J., Clements, J., Carter, T.P. & Campagnoni, A.T. Comparison of lipids in total brain-tissue from five mouse genotypes. J. Neurobiol. 6, 259–266 (1975).

    Article  CAS  PubMed  Google Scholar 

  63. Lohmann, K.M.O. Untersuchungen zur konstitution der adenylpyrophosphorsaure. Biochem. Z. 254, 386 (1933).

    Google Scholar 

  64. Hayashi, T., Mase, S. & Namiki, M. Formation of three-carbon sugar fragment at an early stage of the browning reaction of sugar with amines or amino-acids. Agric. Biol. Chem. 50, 1959–1964 (1986).

    CAS  Google Scholar 

  65. Nilsson-Thorell, C., Muscalu, N., Andren, A.H.G., Kjellstrand, P.T.T. & Wieslander, A.P. Heat sterilzation of fluids for peritoneal dialysis gives rise to aldehydes. Perit. Dial. Int. 13, 208–213 (1993).

    CAS  PubMed  Google Scholar 

  66. Yoshino, K. et al. Formation of methylglyoxal in reduced nicotinamide adenine dinucleotide phosphate-dependent lipid peroxidation of rat liver microsomes. Jpn. J. Toxicol. Environ. Health 42, 236–240 (1996).

    Article  CAS  Google Scholar 

  67. Pourmotabbed, T. & Creighton, D.J. Substrate specificity of bovine liver formaldehyde dehydrogenase. J. Biol. Chem. 261, 14240–14244 (1986).

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank the Biotechnology and Biological Sciences Research Council (UK), the Medical Research Council (UK), the Wellcome Trust (UK) and the British Heart Foundation (UK) for funding for our methylglyoxal-related research.

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  1. Clinical Sciences Research Laboratories, Warwick Medical School, University of Warwick, University Hospital, Coventry, UK

    Naila Rabbani & Paul J Thornalley

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N.R. and P.J.T. performed the experiments, analyzed the data and wrote the manuscript.

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Correspondence to Paul J Thornalley.

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Rabbani, N., Thornalley, P. Measurement of methylglyoxal by stable isotopic dilution analysis LC-MS/MS with corroborative prediction in physiological samples. Nat Protoc 9, 1969–1979 (2014). https://doi.org/10.1038/nprot.2014.129

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