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 imida-zolone 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 (N(epsilon)-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 intra-assay 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.
Zhang
Xiaohong
X
Institute of Pharmacy and Food Chemistry, University Erlangen-Nuremberg, Germany.
Frischmann
Matthias
M
Kientsch-Engel
Rose
R
Steinmann
Katharina
K
Stopper
Helga
H
Niwa
Toshimitsu
T
Pischetsrieder
Monika
M
D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal; D001774:Blood Chemical Analysis / Q000379:methods; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D017127:Glycation End Products, Advanced / Q000097:blood* / Q000276:immunology; D006801:Humans; D007093:Imidazoles / Q000097:blood / Q000276:immunology; D008239:Lysine / Q000031:analogs & derivatives* / Q000097:blood / Q000276:immunology; D008875:Middle Aged; D018384:Oxidative Stress; D006435:Renal Dialysis
D000911:Antibodies, Monoclonal; D017127:Glycation End Products, Advanced; D007093:Imidazoles; C117197:imidazolone; C048496:N(6)-carboxymethyllysine; D008239:Lysine