Methylglyoxal-derivative advanced glycation endproducts: detection by competitive immunofluorometric assay and...

Advanced glycation endproducts (AGE) are a family of heterogeneous chemical structures formed on the host protein in the conditions of carbonyl or oxidative stress. Among AGE precursors, methylglyoxal (MG) is considered one of the key intermediates.

In the current study, we describe and evaluate a solid phase time-resolved fluoroimmunoassay (DELFIA) based on the competitive reaction between MG-AGE antibody and competitive antigen for detecting MG-adducts in serum and urine. The fluorometry assay was validated by comparison with previously established competitive ELISA.

The concentration of MG-adducts assayed by competitive DELFIA in the sera of diabetic patients (DM, n=66) were higher in comparison to non-diabetic controls (C, n=28); DM median=294 mgEq/L (10th and 90th percentile 158-564) vs. C median=224 mgEq/L (10th and 90th percentile 124-290) (p=0.0022). In diabetic subjects, urinary excretion of MG-adducts exceeded the mean level measured in controls; DM median=36.0 mgEq/L (10th and 90th percentile 1-210) vs. C median=11.5 mgEq/L (10th and 90th percentile 1-49) p=0.0036. MG-adducts in urine were low and undetectable in 8 of 28 control subjects and 2 of 66 diabetics. The percentage recovery of MG-adduct added in the same concentration to control and diabetic pool sera showed under-recovery in the latter. Comparison of total AGE level and the amount of MG-adducts revealed MG-derivatives to account for 37% of the heterogeneous structure commonly termed AGE.

The fluoroimmunoassay for MG-derivative AGE evaluated can be utilized on biomonitoring of MG-adducts in serum and its urinary excretion. The competitive DELFIA assay was found to be substantially more sensitive than the standard ELISA having a wider dynamic range, while sharing similar quenching attributes.