Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
JCR Impact Factor (IF) – 1.736
5-Year Impact Factor – 2.135
Index Copernicus  – 168.52
MEiN – 70 pts

ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
Periodicity – monthly

Download original text (EN)

Advances in Clinical and Experimental Medicine

2020, vol. 29, nr 1, January, p. 91–100

doi: 10.17219/acem/112612

Publication type: original article

Language: English

License: Creative Commons Attribution 3.0 Unported (CC BY 3.0)

Download citation:

  • BIBTEX (JabRef, Mendeley)
  • RIS (Papers, Reference Manager, RefWorks, Zotero)

Non-standard AGE4 epitopes that predict polyneuropathy independently of obesity can be detected by slot dot-blot immunoassay

Agnieszka Bronowicka-Szydełko1,A,D, Małgorzata Krzystek-Korpacka1,C,D, Aleksandra Kuzan1,C, Kinga Gostomska-Pampuch1,2,C, Małgorzata Gacka3,B, Urszula Jakobsche-Policht3,B, Rajmund Adamiec3,D,E, Andrzej Gamian1,2,D,F

1 Department of Medical Biochemistry, Wroclaw Medical University, Poland

2 Laboratory of Medical Microbiology, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland

3 Department of Angiology, Diabetes and Hypertension, Wroclaw Medical University, Poland


Background. Advanced glycation end-products (AGEs) are formed during cascade reactions between reducing sugars or reactive aldehydes and proteins, lipids or DNA molecules. They constitute a group of various stable compounds. Advanced glycation end-products are considered potential biomarkers of metabolic disorders. However, so far only a few methods to determine the level of individual AGEs have been developed.
Objectives. The aim of the study was to compare the efficiency of the slot-dot blot method and direct enzyme-linked immunosorbent assay (ELISA) in detecting non-standard epitopes of methylglyoxal (MGO)-modified proteins (AGE4) found in diabetes serum in trace amounts, and to assess AGE4 in diabetes and associated metabolic abnormalities.
Material and Methods. The presence of AGE4 was detected using 2 methods: direct ELISA and the slot-dot blot method – a newly developed immunoassay based on monoclonal, commercially available antibody detection of non-standard AGE epitopes. AGE4 quantification, expressed as median AGE4 in arbitrary units (AU) and AGE4 positivity (the percent of samples with detectable AGE4) was related to diabetes-associated metabolic abnormalities, complications and treatment.
Results. Slot-dot blot was significantly more efficient than ELISA in detecting non-standard AGE4 epitopes. AGE4 positivity was less frequent in patients with microangiopathy and in those with polyneuropathy. In patients with abnormal glucose metabolism, metformin treatment was associated with higher AGE4. AGE4 positivity was significantly lower in gliptin-treated patients. Multivariate analysis showed that polyneuropathy and obesity were independently associated with AGE4 positivity, with odds ratios (ORs) of 0.21 and 3.02, respectively. Moreover, logistic regression showed that AGE4 positivity and HbA1c are independent predictors of polyneuropathy. Considering both indicators allows correct classification of 70.4% of cases with a general accuracy of 76%.
Conclusion. The slot dot-blot method detects compounds found in serum in trace amounts. Accumulation of AGE4 was associated with glucose metabolism abnormalities. A tendency toward AGE4 positivity was less frequent in patients with microangiopathy and in non-treated and gliptin-treated diabetes patients.

Key words

ELISA, diabetes, methylglyoxal, advanced glycation end-products (AGEs), slot dot-blot

References (41)

  1. Singh VP, Bali A, Singh N, Jaggi AS. Advanced glycation end products and diabetic complications. Korean J Physiol Pharmacol. 2014;18(1):1–14.
  2. Ott C, Jacobs K, Haucke E, Navarrete Santos A, Grune T, Simm A. Role of advanced glycation end products in cellular signaling. Redox Biol. 2014;2:411–429.
  3. Singh R, Barden A, Mori T, Beilin L. Advanced glycation end-products. Diabetologia. 2001;44(2):129–146.
  4. Goh SY, Cooper ME. Clinical review: The role of advanced glycation end products in progression and complications of diabetes. J Clin Endocrinol Metab. 2008;93(4):1143–1152.
  5. Neelofar K, Arif Z, Ahmad J, Alam K. Non-enzymatic glucosylation induced neo-epitopes on human serum albumin: A concentration based study. PLoS One. 2017;13:2(2):e0172074.
  6. Kaur N, Kishore L, Singh R. Dillenia indica L. attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in STZ-nicotinamide induced diabetic rats. J Tradit Complement Med. 2017;8(1):226–238.
  7. Bär KJ, Franke S, Wenda B, et al. Pentosidine and N-ε carboxymethyl)-lysine in Alzheimer’s disease and vascular dementia. Neurobiol Aging. 2003;24(2):333–338.
  8. de Vos LC, Lefrandt JD, Dullaart RP, Zeebregts CJ, Smit AJ. Advanced glycation end products: An emerging biomarker for adverse outcome in patients with peripheral artery disease. Atherosclerosis. 2016;254:291–299.
  9. Lopez-Clavijo AF, Duque-Daza CA, Soulby A, Canelon IR, Barrow M, O’Connor PB. Unexpected crosslinking and diglycation as advanced glycation end-products from glyoxal. J Am Soc Mass Spectrom. 2014;25(12):2125–2133.
  10. Haddad M, Knani I, Bouzidi H, Berriche O, Hammami M, Kerkeni M. Plasma levels of pentosidine, carboxymethyl-lysine, soluble receptor for advanced glycation end products, and metabolic syndrome: The metformin effect. Dis Markers. 2016;2016:6248264.
  11. Hipkiss AR. On the relationship between energy metabolism, proteostasis, aging and Parkinson’s disease: Possible causative role of methylglyoxal and alleviative potential of carnosine. Aging Dis. 2017;8(3):334–345.
  12. Lin CC, Chan CM, Huang YP, Hsu SH, Huang CL, Tsai SJ. Methylglyoxal activates NF-κB nuclear translocation and induces COX-2 expression via a p38-dependent pathway in synovial cells. Life Sci. 2016;149:25–33.
  13. Jyot, Mir AR, Habib S, Siddiqui SS, Ali A, Moinuddin. Neo-epitopes on methylglyoxal modified human serum albumin lead to aggressive autoimmune response in diabetes. Int J Biol Macromol. 2016;86:799–809
  14. Mir AR, Moinuddin, Habib S, Khan F, Alam K, Ali A. Structural changes in histone H2A by methylglyoxal generate highly immunogenic amorphous aggregates with implications in auto-immune response in cancer. Glycobiology. 2016;26(2):129–141.
  15. Wetzels S, Wouters K, Schalkwijk CG, Vanmierlo T, Hendriks J. Methyl­glyoxal-derived advanced glycation endproducts in multiple sclerosis. Int J Mol Sci. 2017;18(2). doi:10.3390/ijms18020421
  16. Brunvand L, Heier M, Brunborg C, et al. Advanced glycation end products in children with type 1 diabetes and early reduced diastolic heart function. BMC Cardiovasc Disord. 2017;17(1):133.
  17. WHO. Global report on diabetes. Geneva, Switzerland: World Health Organization; 2017.
  18. WHO. Obesity: Preventing and managing the global epidemic. Report of a World Health Organization Consultation. WHO Technical Report Series 894. Geneva, Switzerland: World Health Organization; 2000.
  19. Peralta C, Hamid P, Batool H, Al Achkar Z, Maximus P. Psoriasis and Metabolic Syndrome: Comorbidities and Environmental and Thera­peutic Implications. Cureus. 2019;11(12):e6369. doi: 10.7759/cureus.6369
  20. Catapano AL, Graham I, De Backer G, et al. 2016 ESC/EAS Guidelines for the Management of Dyslipidaemias. Eur Heart J. 2016;37(39):2999–3058.
  21. Centers for Disease Control and Prevention. The Third National Health and Nutrition Examination Survey (NHANES III 1988-94) reference manuals and reports. Hyattsville, MD: National Center for Health Statistics; 1996.
  22. Takeuchi M, Yanase Y, Matsuura N, et al. Immunological detection of a novel advanced glycation end-product. Mol Med. 2001;7(11):783–791.
  23. Heier M, Margeirsdottir HD, Torjesen PA, Seljeflot I, Stensaeth KH, Gaarder M. The advanced glycation end product methylglyoxal­derivedhydroimidazolone-1 and early signs of atherosclerosis in childhood diabetes. Diab Vasc Dis Res. 2015;12(2):139–145.
  24. Adisakwattana S, Thilavech T, Chusak C. Mesona chinensis Benth extract prevents AGE formation and protein oxidation against fructose-induced protein glycation in vitro. BMC Complement Altern Med. 2014;14:130.
  25. Stone W, Grabias B, Lanke K, et al. A comparison of Plasmodium falciparum circumsporozoite protein-based slot blot and ELISA immuno-assays for oocyst detection in mosquito homogenates. Malar J. 2015;14(1):451.
  26. Kumar S, Zheng H, Sangweme DT, et al. A chemiluminescent-western blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein. J Immunol Methods. 2013;390(1–2):99–105.
  27. Khuhawar MY, Zardari LA, Laghari AJ. Capillary gas chromatographic determination of methylglyoxal from serum of diabetic patients by precolumn derivatization with 1,2-diamonopropane. J Chromatogr B Analyt Technol Biomed Life Sci. 2008;873(1):15–19.
  28. van der Heijden RA, Bijzet J, Meijers WC, et al. Obesity-induced chronic inflammation in high fat diet challenged C57BL/6J mice is associated with acceleration of age-dependent renal amyloidosis. Sci Rep. 2015;5:16474. doi:10.1038/srep16474
  29. Mirza MA, Kandhro AJ, Memon SQ, Khuhawar MY, Arain R. Determination of glyoxal and methylglyoxal in the serum of diabetic patients by MEKC using stilbenediamine as derivatizing reagent. Electro­phoresis. 2007;28(21):3940–3947.
  30. Kandhro AJ, Mirza MA, Khuhawar MY. Capillary gas chromatographic determination of methylglyoxal from serum of diabetic patients by precolumn derivatization using meso-stilbenediamine as derivatizing reagent. J Chromatogr Sci. 2008;46(6):539–543.
  31. Hanssen NMJ, Scheijen JLJM, Jorsal A, et al. Higher plasma methylglyoxal levels are associated with incident cardiovascular disease in individuals with type 1 diabetes: A 12-year follow-up study. Diabetes. 2017;66(8):2278–2283.
  32. Aikawa T, Matsubara H, Ugaji S, et al. Contribution of methylglyoxal to delayed healing of bone injury in diabetes. Mol Med Rep. 2017;16(1):403–409.
  33. Cantero AV, Portero-Otín M, Ayala V, et al. Methylglyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor-beta: Implications for diabetic atherosclerosis. FASEB J. 2007;21(12):3096–3106.
  34. Morioka Y, Teshigawara K, Tomono Y, et al. The specific localization of advanced glycation end-products (AGEs) in rat pancreatic islets. J Pharmacol Sci. 2017;134(4):218–224.
  35. Peters AS, Wortmann M, Fleming TH, et al. Effect of metformin treatment in patients with type 2 diabetes with respect to glyoxalase 1 activity in atherosclerotic lesions. Vasa. 2019;48(2):186–192.
  36. Beisswenger PJ, Howell SK, Touchette AD, Lal S, Szwergold BS. Metformin reduces systemic methylglyoxal levels in type 2 diabetes. Diabetes. 1999;48(1):198–202.
  37. Kiho T, Kato M, Usui S, Hirano K. Effect of buformin and metformin on formation of advanced glycation end products by methylglyoxal. Clin Chim Acta. 2005;358(1–2):139–145.
  38. Nagai T, Doi S, Nakashima A, et al. Linagliptin ameliorates methylglyoxal-induced peritoneal fibrosis in mice. PLoS One. 2016;11(8):e0160993
  39. Jung E, Kim J, Kim SH, Kim S, Cho MH. Gemigliptin, a novel dipeptidyl peptidase-4 inhibitor, exhibits potent anti-glycation properties in vitro and in vivo. Eur J Pharmacol. 2014;744:98–102.
  40. Prázný M, Škrha J, Šoupal J, Škrha J Jr. Glycemic variability and microvascular complications of diabetes. Cas Lek Cesk. 2017;156(6):308–313.
  41. Beisswenger PJ, Howell SK, Russell GB, Miller ME, Rich SS, Mauer M. Early progression of diabetic nephropathy correlates with methylglyoxal-derived advanced glycation end products. Diabetes Care. 2013;36(10):3234–3239.