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. 2002 May 15;364(Pt 1):1–14. doi: 10.1042/bj3640001

Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to Nepsilon-carboxymethyl-lysine- and Nepsilon-(1-carboxyethyl)lysine-modified albumin.

Naila Ahmed 1, Ognian K Argirov 1, Harjit S Minhas 1, Carlos A A Cordeiro 1, Paul J Thornalley 1
PMCID: PMC1222539  PMID: 11988070

Abstract

Glycation of proteins leads to the formation of early glycation adducts (fructosamine derivatives) and advanced glycation endproducts (AGEs). Formation of AGEs has been linked to the development of cataract, diabetic complications, uraemia, Alzheimer's disease and other disorders. AGEs are a group of compounds of diverse molecular structure and biological function. To characterize AGE-modified proteins used in studies of structural and functional effects of glycation, an assay was developed that surveys the content of early and advanced glycation adducts in proteins. The assay procedure involved enzymic hydrolysis of protein substrate, derivatization of the hydrolysate with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and HPLC of the resulting adducts with fluorimetric detection. Structural isomers of methylglyoxal-derived hydroimidazolone, glyoxal-derived hydroimidazolone, 3-deoxyglucosone-derived hydroimidazolone and N(delta)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)-ornithine (THP) were determined for the first time. AGEs with intrinsic fluorescence (argpyrimidine, pentosidine) were assayed without derivatization. Limits of detection were 2-17 pmol and levels of recovery were 50-99%, depending on the analyte. The AQC assay resolved structural and epimeric isomers of methylglyoxal-derived hydroimidazolones and THP. Hydroimidazolones, THP and argpyrimidine were AGEs of short-to-intermediate stability under physiological conditions, with half-lives of 1-2 weeks. Their measurement provides further insight into the glycation process. The assay was applied to the characterization of human serum albumin minimally and highly modified by N(epsilon)-carboxymethyl-lysine and N(epsilon)-(1-carboxyethyl)-lysine.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahmed M. U., Brinkmann Frye E., Degenhardt T. P., Thorpe S. R., Baynes J. W. N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem J. 1997 Jun 1;324(Pt 2):565–570. doi: 10.1042/bj3240565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahmed M. U., Thorpe S. R., Baynes J. W. Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein. J Biol Chem. 1986 Apr 15;261(11):4889–4894. [PubMed] [Google Scholar]
  3. Ahmed Naila, Thornalley Paul J. Chromatographic assay of glycation adducts in human serum albumin glycated in vitro by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and intrinsic fluorescence. Biochem J. 2002 May 15;364(Pt 1):15–24. doi: 10.1042/bj3640015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baynes J. W., Thorpe S. R., Murtiashaw M. H. Nonenzymatic glucosylation of lysine residues in albumin. Methods Enzymol. 1984;106:88–98. doi: 10.1016/0076-6879(84)06010-9. [DOI] [PubMed] [Google Scholar]
  5. Baynes J. W., Thorpe S. R. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes. 1999 Jan;48(1):1–9. doi: 10.2337/diabetes.48.1.1. [DOI] [PubMed] [Google Scholar]
  6. Berg T. J., Bangstad H. J., Torjesen P. A., Osterby R., Bucala R., Hanssen K. F. Advanced glycation end products in serum predict changes in the kidney morphology of patients with insulin-dependent diabetes mellitus. Metabolism. 1997 Jun;46(6):661–665. doi: 10.1016/s0026-0495(97)90010-x. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Chellan P., Nagaraj R. H. Protein crosslinking by the Maillard reaction: dicarbonyl-derived imidazolium crosslinks in aging and diabetes. Arch Biochem Biophys. 1999 Aug 1;368(1):98–104. doi: 10.1006/abbi.1999.1291. [DOI] [PubMed] [Google Scholar]
  9. Cohen S. A., Michaud D. P. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem. 1993 Jun;211(2):279–287. doi: 10.1006/abio.1993.1270. [DOI] [PubMed] [Google Scholar]
  10. Day J. F., Thorpe S. R., Baynes J. W. Nonenzymatically glucosylated albumin. In vitro preparation and isolation from normal human serum. J Biol Chem. 1979 Feb 10;254(3):595–597. [PubMed] [Google Scholar]
  11. Degenhardt T. P., Thorpe S. R., Baynes J. W. Chemical modification of proteins by methylglyoxal. Cell Mol Biol (Noisy-le-grand) 1998 Nov;44(7):1139–1145. [PubMed] [Google Scholar]
  12. Frye E. B., Degenhardt T. P., Thorpe S. R., Baynes J. W. Role of the Maillard reaction in aging of tissue proteins. Advanced glycation end product-dependent increase in imidazolium cross-links in human lens proteins. J Biol Chem. 1998 Jul 24;273(30):18714–18719. doi: 10.1074/jbc.273.30.18714. [DOI] [PubMed] [Google Scholar]
  13. Fu M. X., Requena J. R., Jenkins A. J., Lyons T. J., Baynes J. W., Thorpe S. R. The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem. 1996 Apr 26;271(17):9982–9986. doi: 10.1074/jbc.271.17.9982. [DOI] [PubMed] [Google Scholar]
  14. Glomb M. A., Monnier V. M. Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction. J Biol Chem. 1995 Apr 28;270(17):10017–10026. doi: 10.1074/jbc.270.17.10017. [DOI] [PubMed] [Google Scholar]
  15. Henle T., Bachmann A. Synthesis of pyrraline reference material. Z Lebensm Unters Forsch. 1996 Jan;202(1):72–74. doi: 10.1007/BF01229689. [DOI] [PubMed] [Google Scholar]
  16. Horiuchi S., Araki N., Morino Y. Immunochemical approach to characterize advanced glycation end products of the Maillard reaction. Evidence for the presence of a common structure. J Biol Chem. 1991 Apr 25;266(12):7329–7332. [PubMed] [Google Scholar]
  17. Iijima K., Murata M., Takahara H., Irie S., Fujimoto D. Identification of N(omega)-carboxymethylarginine as a novel acid-labileadvanced glycation end product in collagen. Biochem J. 2000 Apr 1;347(Pt 1):23–27. [PMC free article] [PubMed] [Google Scholar]
  18. Ikeda K., Nagai R., Sakamoto T., Sano H., Araki T., Sakata N., Nakayama H., Yoshida M., Ueda S., Horiuchi S. Immunochemical approaches to AGE-structures: characterization of anti-AGE antibodies. J Immunol Methods. 1998 Jun 1;215(1-2):95–104. doi: 10.1016/s0022-1759(98)00064-7. [DOI] [PubMed] [Google Scholar]
  19. Kislinger T., Fu C., Huber B., Qu W., Taguchi A., Du Yan S., Hofmann M., Yan S. F., Pischetsrieder M., Stern D. N(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression. J Biol Chem. 1999 Oct 29;274(44):31740–31749. doi: 10.1074/jbc.274.44.31740. [DOI] [PubMed] [Google Scholar]
  20. Lo T. W., 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 N alpha-acetylarginine, N alpha-acetylcysteine, and N alpha-acetyllysine, and bovine serum albumin. J Biol Chem. 1994 Dec 23;269(51):32299–32305. [PubMed] [Google Scholar]
  21. Makita Z., Vlassara H., Cerami A., Bucala R. Immunochemical detection of advanced glycosylation end products in vivo. J Biol Chem. 1992 Mar 15;267(8):5133–5138. [PubMed] [Google Scholar]
  22. McCance D. R., Dyer D. G., Dunn J. A., Bailie K. E., Thorpe S. R., Baynes J. W., Lyons T. J. Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus. J Clin Invest. 1993 Jun;91(6):2470–2478. doi: 10.1172/JCI116482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morioka T., Emoto M., Tabata T., Shoji T., Tahara H., Kishimoto H., Ishimura E., Nishizawa Y. Glycemic control is a predictor of survival for diabetic patients on hemodialysis. Diabetes Care. 2001 May;24(5):909–913. doi: 10.2337/diacare.24.5.909. [DOI] [PubMed] [Google Scholar]
  24. Murata-Kamiya N., Kaji H., Kasai H. Deficient nucleotide excision repair increases base-pair substitutions but decreases TGGC frameshifts induced by methylglyoxal in Escherichia coli. Mutat Res. 1999 Jun 7;442(1):19–28. doi: 10.1016/s1383-5718(99)00054-6. [DOI] [PubMed] [Google Scholar]
  25. Nagaraj R. H., Shipanova I. N., Faust F. M. Protein cross-linking by the Maillard reaction. Isolation, characterization, and in vivo detection of a lysine-lysine cross-link derived from methylglyoxal. J Biol Chem. 1996 Aug 9;271(32):19338–19345. doi: 10.1074/jbc.271.32.19338. [DOI] [PubMed] [Google Scholar]
  26. Neglia C. I., Cohen H. J., Garber A. R., Ellis P. D., Thorpe S. R., Baynes J. W. 13C NMR investigation of nonenzymatic glucosylation of protein. Model studies using RNase A. J Biol Chem. 1983 Dec 10;258(23):14279–14283. [PubMed] [Google Scholar]
  27. Niwa T. 3-Deoxyglucosone: metabolism, analysis, biological activity, and clinical implication. J Chromatogr B Biomed Sci Appl. 1999 Aug 6;731(1):23–36. doi: 10.1016/s0378-4347(99)00113-9. [DOI] [PubMed] [Google Scholar]
  28. Niwa T., Katsuzaki T., Ishizaki Y., Hayase F., Miyazaki T., Uematsu T., Tatemichi N., Takei Y. Imidazolone, a novel advanced glycation end product, is present at high levels in kidneys of rats with streptozotocin-induced diabetes. FEBS Lett. 1997 May 5;407(3):297–302. doi: 10.1016/s0014-5793(97)00362-1. [DOI] [PubMed] [Google Scholar]
  29. Oya T., Hattori N., Mizuno Y., Miyata S., Maeda S., Osawa T., Uchida K. Methylglyoxal modification of protein. Chemical and immunochemical characterization of methylglyoxal-arginine adducts. J Biol Chem. 1999 Jun 25;274(26):18492–18502. doi: 10.1074/jbc.274.26.18492. [DOI] [PubMed] [Google Scholar]
  30. Paul R. G., Avery N. C., Slatter D. A., Sims T. J., Bailey A. J. Isolation and characterization of advanced glycation end products derived from the in vitro reaction of ribose and collagen. Biochem J. 1998 Mar 15;330(Pt 3):1241–1248. doi: 10.1042/bj3301241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Portero-Otin M., Nagaraj R. H., Monnier V. M. Chromatographic evidence for pyrraline formation during protein glycation in vitro and in vivo. Biochim Biophys Acta. 1995 Feb 22;1247(1):74–80. doi: 10.1016/0167-4838(94)00209-y. [DOI] [PubMed] [Google Scholar]
  32. Sell D. R., Monnier V. M. Structure elucidation of a senescence cross-link from human extracellular matrix. Implication of pentoses in the aging process. J Biol Chem. 1989 Dec 25;264(36):21597–21602. [PubMed] [Google Scholar]
  33. Shipanova I. N., Glomb M. A., Nagaraj R. H. Protein modification by methylglyoxal: chemical nature and synthetic mechanism of a major fluorescent adduct. Arch Biochem Biophys. 1997 Aug 1;344(1):29–36. doi: 10.1006/abbi.1997.0195. [DOI] [PubMed] [Google Scholar]
  34. Skovsted I. C., Christensen M., Breinholt J., Mortensen S. B. Characterisation of a novel AGE-compound derived from lysine and 3-deoxyglucosone. Cell Mol Biol (Noisy-le-grand) 1998 Nov;44(7):1159–1163. [PubMed] [Google Scholar]
  35. Smith P. R., Thornalley P. J. Influence of pH and phosphate ions on the kinetics of enolisation and degradation of fructosamines. Studies with the model fructosamine, N epsilon-1-deoxy-D-fructos-1-yl-hippuryl-lysine. Biochem Int. 1992 Nov;28(3):429–439. [PubMed] [Google Scholar]
  36. Smith P. R., Thornalley P. J. Mechanism of the degradation of non-enzymatically glycated proteins under physiological conditions. Studies with the model fructosamine, N epsilon-(1-deoxy-D-fructos-1-yl)hippuryl-lysine. Eur J Biochem. 1992 Dec 15;210(3):729–739. doi: 10.1111/j.1432-1033.1992.tb17474.x. [DOI] [PubMed] [Google Scholar]
  37. Tajika T., Bando I., Furuta T., Moriya N., Koshino H., Uramoto M. Novel amino acid metabolite produced by Streptomyces sp.: I. Taxonomy, isolation, and structural elucidation. Biosci Biotechnol Biochem. 1997 Jun;61(6):1007–1010. doi: 10.1271/bbb.61.1007. [DOI] [PubMed] [Google Scholar]
  38. Takata K., Horiuchi S., Araki N., Shiga M., Saitoh M., Morino Y. Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins. J Biol Chem. 1988 Oct 15;263(29):14819–14825. [PubMed] [Google Scholar]
  39. Thornalley P. J. Cell activation by glycated proteins. AGE receptors, receptor recognition factors and functional classification of AGEs. Cell Mol Biol (Noisy-le-grand) 1998 Nov;44(7):1013–1023. [PubMed] [Google Scholar]
  40. Thornalley P. J., Langborg A., Minhas H. S. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem J. 1999 Nov 15;344(Pt 1):109–116. [PMC free article] [PubMed] [Google Scholar]
  41. Thornalley P., Wolff S., Crabbe J., Stern A. The autoxidation of glyceraldehyde and other simple monosaccharides under physiological conditions catalysed by buffer ions. Biochim Biophys Acta. 1984 Feb 14;797(2):276–287. doi: 10.1016/0304-4165(84)90131-4. [DOI] [PubMed] [Google Scholar]
  42. Verzijl N., DeGroot J., Thorpe S. R., Bank R. A., Shaw J. N., Lyons T. J., Bijlsma J. W., Lafeber F. P., Baynes J. W., TeKoppele J. M. Effect of collagen turnover on the accumulation of advanced glycation end products. J Biol Chem. 2000 Dec 15;275(50):39027–39031. doi: 10.1074/jbc.M006700200. [DOI] [PubMed] [Google Scholar]
  43. Wells-Knecht K. J., Zyzak D. V., Litchfield J. E., Thorpe S. R., Baynes J. W. Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose. Biochemistry. 1995 Mar 21;34(11):3702–3709. doi: 10.1021/bi00011a027. [DOI] [PubMed] [Google Scholar]
  44. Wells-Knecht M. C., Thorpe S. R., Baynes J. W. Pathways of formation of glycoxidation products during glycation of collagen. Biochemistry. 1995 Nov 21;34(46):15134–15141. doi: 10.1021/bi00046a020. [DOI] [PubMed] [Google Scholar]
  45. Westwood M. E., Thornalley P. J. Molecular characteristics of methylglyoxal-modified bovine and human serum albumins. Comparison with glucose-derived advanced glycation endproduct-modified serum albumins. J Protein Chem. 1995 Jul;14(5):359–372. doi: 10.1007/BF01886793. [DOI] [PubMed] [Google Scholar]
  46. Wilker S. C., Chellan P., Arnold B. M., Nagaraj R. H. Chromatographic quantification of argpyrimidine, a methylglyoxal-derived product in tissue proteins: comparison with pentosidine. Anal Biochem. 2001 Mar;290(2):353–358. doi: 10.1006/abio.2001.4992. [DOI] [PubMed] [Google Scholar]
  47. Yu P. H., Zuo D. M. Aminoguanidine inhibits semicarbazide-sensitive amine oxidase activity: implications for advanced glycation and diabetic complications. Diabetologia. 1997 Nov;40(11):1243–1250. doi: 10.1007/s001250050816. [DOI] [PubMed] [Google Scholar]

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