Zhang Xiaohong, Frischmann Matthias, Kientsch-Engel Rose, Steinmann Katharina, Stopper Helga, Niwa Toshimitsu, Pischetsrieder Monika
Institute of Pharmacy and Food Chemistry, University Erlangen-Nuremberg, Germany.
Clin Chem Lab Med. 2005;43(5):503-11. doi: 10.1515/CCLM.2005.089.
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.
晚期糖基化终产物是尿毒症毒素，会在慢性肾衰竭患者的血清和组织中蓄积。在此，我们建立了两种用于检测N-ε-羧甲基赖氨酸和咪唑啉酮的酶联免疫吸附测定法（ELISA），以分析人血清中的晚期糖基化终产物。两种ELISA均以剂量依赖方式检测与人血清白蛋白结合的晚期糖基化终产物。虽然咪唑啉酮的形成与氧气的存在无关，但在氧化条件下，N-ε-羧甲基赖氨酸表位的浓度增加了20倍。N-ε-羧甲基赖氨酸ELISA对游离的、肽结合的和蛋白质结合的N-ε-羧甲基赖氨酸表现出相似的反应，而咪唑啉酮抗体对肽结合的咪唑啉酮的亲和力略高于蛋白质结合的咪唑啉酮。在人血清中，发现线性稀释范围为1:10至1:40（N-ε-羧甲基赖氨酸ELISA）和1:2至1:8（咪唑啉酮ELISA）。血清中N-ε-羧甲基赖氨酸的回收率分别为101±10%和94±12%，咪唑啉酮的回收率分别为93±15%和97±12%。批内变异系数为0.26 - 2.7%（N-ε-羧甲基赖氨酸）和0.1 - 2.4%（咪唑啉酮），批间变异系数为8.3 - 13.4%（N-ε-羧甲基赖氨酸）和7.8 - 12.5%（咪唑啉酮）。在血液透析患者（n = 20）和对照组（n = 20）的血清样本中，患者组检测到约两倍的增加（p < 0.001）。N-ε-羧甲基赖氨酸和咪唑啉酮ELISA的组合是用于临床研究测量血清晚期糖基化终产物浓度的有价值工具。
