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.

Keywords: advanced glycation end-products; diabetes; glomerular filtration; microangiopathy.

Figure 1

An overview of a process of AGE10 ELISA development; FPLC—the fast protein liquid chromatography, LC-MS—liquid chromatography mass spectrometer, LMW-MAGE—low molecular mass MAGE, MAGE—melibiose-derived AGE, MB-MELs—products of reaction of myoglobin with melibiose, NAc^αLys-MEL—products of reaction of N^α-acetyllysine with melibiose.

Figure 2

Glycation of myoglobin with melibiose and selection of MAGE with the highest reactivity towards anti-MAGE mouse monoclonal antibodies: (a) FPLC chromatogram with marked three peaks denoted 1(A), 1(B), and 1(C); (b) SDS-PAGE image of selected FPLC fractions representative for main peaks; (c) graphical presentation of competitive ELISA of selected FPLC fractions representative for main peaks. Different detection wavelengths for the analysis of elution profile were marked by red (225 nm), pink (297 nm), and blue (280 nm). M, molecular mass marker.

Figure 2

Glycation of myoglobin with melibiose and selection of MAGE with the highest reactivity towards anti-MAGE mouse monoclonal antibodies: (a) FPLC chromatogram with marked three peaks denoted 1(A), 1(B), and 1(C); (b) SDS-PAGE image of selected FPLC fractions representative for main peaks; (c) graphical presentation of competitive ELISA of selected FPLC fractions representative for main peaks. Different detection wavelengths for the analysis of elution profile were marked by red (225 nm), pink (297 nm), and blue (280 nm). M, molecular mass marker.

Figure 3

Purification and characterization of low molecular weight glycation products obtained in the synthesis of N-acetyl lysine with melibiose (NAc_αLys-MEL) in a microwave reactor: (a) The elution profile of NAc_αLys-MEL separated by FPLC chromatography (HW40-S column); (b) elution profile of “1(B)” peak separated by FPLC chromatography (Bio-Gel P-2 column); (c) effectiveness of NAc_αLys-MEL fractions 1(A), 1(B0), 1(B1) and 1(B2) in inhibiting MAGE/anti-MAGE reaction; (d) mass chromatograms for ion 513.23 m/z for NAc_αLys-MEL peaks 1(A), 1(B0), 1(B1) and 1(B2).

Figure 3

Purification and characterization of low molecular weight glycation products obtained in the synthesis of N-acetyl lysine with melibiose (NAc_αLys-MEL) in a microwave reactor: (a) The elution profile of NAc_αLys-MEL separated by FPLC chromatography (HW40-S column); (b) elution profile of “1(B)” peak separated by FPLC chromatography (Bio-Gel P-2 column); (c) effectiveness of NAc_αLys-MEL fractions 1(A), 1(B0), 1(B1) and 1(B2) in inhibiting MAGE/anti-MAGE reaction; (d) mass chromatograms for ion 513.23 m/z for NAc_αLys-MEL peaks 1(A), 1(B0), 1(B1) and 1(B2).

Figure 4

The reactivity of antibodies from Clone Nos. 10, 19 and 49 with BSA-MAGE (bovine serum albumin-MAGE), MB-MAGE (myoglobin-MAGE), LYS-MAGE (lysozyme-MAGE) and with BSA, MB, LYS as respective controls.

Figure 5

Scheme of AGE10 ELISA. Ab, antibodies; LMW-MAGE, low molecular mass advanced glycation end-products obtained by NAcαLys modification with melibiose (analogue of AGE10 epitope) depicted as red circles; MAGE, advanced glycation end-products obtained by myoglobin modification by melibiose depicted as blue rectangles.

Figure 6

The association of AGE10 with microangiopathy in patients stratified based on aspirin (ASA) treatment. Data presented as means with 95% confidence interval (closed squares with whiskers and figures below the dot-plots) and analyzed using two-way ANOVA. Connectors with asterisks indicate significant (p < 0.05) between-group differences.

Figure 7

AGE10 as biomarker of: (a) microangiopathy; (b) stage ≥3 chronic kidney disease. Data presented as receiver operating characteristics (ROC) curves (solid blue line) with 95% confidence interval (dotted green lines). The performance of a chance marker devoid of discriminating power (AUC = 0.5) is represented by diagonal red line and optimal cut-off value is indicated as closed blue circle. AUC, area under ROC curve.