Association of Novel Advanced Glycation End-Product (AGE10) with Complications of Diabetes as Measured by Enzyme-Linked Immunosorbent Assay

Abstract

Advanced glycation end-products (AGEs) contribute to vascular complications and organ damage in diabetes. The unique AGE epitope (AGE10) has recently been identified in human serum using synthetic melibiose-derived AGE (MAGE). We aimed at developing ELISA for AGE10 quantification, determining whether AGE10 is present in diabetic patients (n = 82), and evaluating its association with diabetic complications. In a competitive ELISA developed, the reaction of synthetic MAGE with anti-MAGE was inhibited by physiological AGE10 present in serum. In this assay, new murine IgE anti-MAGE monoclonal antibodies, which do not recognize conventional AGEs, a synthetic MAGE used to coat the plate, and LMW-MAGE (low molecular mass MAGE) necessary to plot a standard curve were used. AGE10 was significantly higher in patients with microangiopathy, in whom it depended on treatment, being lower in patients treated with aspirin. AGE10 levels were positively correlated with estimated glomerular filtration rate (eGFR) and negatively with creatinine. As a marker of stage ≥3 chronic kidney disease or microangiopathy, AGE10 displayed moderate overall accuracy (respectively, 69% and 71%) and good sensitivity (82.6% and 83.3%) but poor specificity (58.1% and 57.8%). In conclusion, newly developed immunoassay allows for AGE10 quantification. AGE10 elevation is associated with microangiopathy while its decrease accompanies stage ≥3 chronic kidney disease.

Proteins in human body fluids contain in vivo antigen analog of the melibiose-derived glycation product: MAGE

“…The analogue of MAGE present naturally in human blood was extracted from the samples collected in the Military Center for Blood Donation and Haemotherapy in Wroclaw (honorary blood donors) and the Department of Angiology, Diabetology and Hypertension in Wroclaw (diabetic patients). The presence of the MAGE antigen in the tested diabetic sera was previously confirmed by competitive ELISA 5 . MAGE extraction was performed using the purified murine anti-MAGE monoclonal antibody (described in Supplementary data) immobilized on Sepharose resin using the Pierce Direct IP Kit (Thermo Scientific, Waltham, MA, USA).…”

“…In addition, MAGE has been detected in the extracellular matrix of various animal tissues 4 , including various cancer tumors (manuscript in preparation). MAGE has also been associated with diabetic complications and atherosclerosis 5 , 6 . Interestingly, in other studies MAGE showed genotoxicity on human peripheral blood lymphocytes, melanoma, lung cancer, and colorectal cancer cells in vitro, while protein-free adducts prevented cells from this effect 7 .…”

Analysis of the Site-Specific Myoglobin Modifications in the Melibiose-Derived Novel Advanced Glycation End-Product

“…In many scientific studies on glycation, synthetic structural analogs of AGEs corresponding to the compounds present in vivo are used [ 36 , 37 , 38 ]. Model AGEs have been used to resolve the structure and properties of the formed compounds [ 39 , 40 ], to obtain specific antibodies [ 41 , 42 , 43 ], and to develop standards needed in clinical and diagnostic tests [ 26 ]. To understand the nature of the unconventional MAGE product found in various human and animal tissues [ 23 , 24 ], we characterized the extent of glycation and spatial amino acid modifications on the model protein.…”

“…The identified biological properties of MAGE show genotoxicity studied in vitro on human peripheral blood lymphocytes, melanoma, lung cancer, and colorectal cancer cells, while protein-free adducts prevent cells from this effect [ 25 ]. It has also been associated with diabetic complications and atherosclerosis [ 26 , 27 ]. It should be noted that melibiose is produced during food fermentation by bacteria of the genus Bifidobacterium [ 28 , 29 ], Lactobacillus, Lactococcus, Leuconostoc [ 30 ], and yeast [ 31 ], and it is also found in cocoa beans, honey, and processed soybeans [ 32 ].…”

Techniques for advanced glycation end product measurements for diabetic bone disease: pitfalls and future directions

“…Serum measurement of PEN and CML is preferred in clinical setting as collection of blood samples from human subjects is far less invasive than bone biopsies. However, quantitation of the two aforementioned AGEs as well as other AGEs of interest by ELISA depends on the availability of ELISA kits [51 ▪ ,52,53]. In contrast to the measurement of the CML content in serum and from different soft tissues using ELISA kits [54], PEN is typically quantified using chromatography techniques.…”