Two immunochemical assays to measure advanced glycation end-products in serum from dialysis patients

Advanced glycation end-products are uremic toxins that accumulate in the serum and tissues of patients with chronic renal failure. Here, we established two enzyme-linked immunosorbent assays (ELISAs) for N-epsilon-carboxymethyllysine and imidazolone to analyze advanced glycation end-products in human serum. Both ELISAs detected advanced glycation end-products bound to human serum albumin in a dose-dependent way. Whereas the formation of imidazolone was independent of the presence of oxygen, concentrations of N-epsilon-carboxymethyllysine epitopes increased 20-fold under oxidative conditions. The N-epsilon-carboxymethyllysine ELISA showed a similar response to free, peptide-bound and protein-bound N-epsilon-carboxymethyllysine, whereas the imidazolone antibody showed slightly higher affinity toward peptide-bound compared to protein-bound imidazolone. In human serum, linear dilution ranges from 1:10 to 1:40 (NI-carboxymethyllysine ELISA) and from 1:2 to 1:8 (imidazolone ELISA) were found. The recovery of N-epsilon-carboxymethyllysine from serum was 101 +/- 10% and 94 +/- 12%, respectively, and 93 +/- 15% and 97 +/- 12% for imidazolone. The coefficients of variation for intraassay variability were 0.26-2.7% (N-epsilon-carboxymethyllysine) and 0.1-2.4% (imidazolone), and 8.3-13.4% (N-epsilon-carboxymethyllysine) and 7.8-12.5% (imidazolone) for inter-assay variability. In serum samples from hemodialysis patients (n=20) and controls (n=20), an approximately two-fold increase was detected in the patient group (p<0.001). The combination of the N-epsilon-carboxymethyllysine and imidazolone ELISAs is a valuable tool to measure serum concentrations of advanced glycation end-products for clinical studies.