Bronowicka-Szydełko Agnieszka, Krzystek-Korpacka Małgorzata, Kuzan Aleksandra, Gostomska-Pampuch Kinga, Gacka Małgorzata, Jakobsche-Policht Urszula, Adamiec Rajmund, Gamian Andrzej
Department of Medical Biochemistry, Wroclaw Medical University, Poland.
Laboratory of Medical Microbiology, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.
Adv Clin Exp Med. 2020 Jan;29(1):91-100. doi: 10.17219/acem/112612.
Advanced glycation end-products (AGEs) are formed during cascade reactions between reducing sugars or reactive aldehydes and proteins, lipids or DNA molecules. They constitute a group of various stable compounds. Advanced glycation end-products are considered potential biomarkers of metabolic disorders. However, so far only a few methods to determine the level of individual AGEs have been developed.
The aim of the study was to compare the efficiency of the slot-dot blot method and direct enzyme-linked immunosorbent assay (ELISA) in detecting non-standard epitopes of methylglyoxal (MGO)-modified proteins (AGE4) found in diabetes serum in trace amounts, and to assess AGE4 in diabetes and associated metabolic abnormalities.
The presence of AGE4 was detected using 2 methods: direct ELISA and the slot-dot blot method - a newly developed immunoassay based on monoclonal, commercially available antibody detection of non-standard AGE epitopes. AGE4 quantification, expressed as median AGE4 in arbitrary units (AU) and AGE4 positivity (the percent of samples with detectable AGE4) was related to diabetes-associated metabolic abnormalities, complications and treatment.
Slot-dot blot was significantly more efficient than ELISA in detecting non-standard AGE4 epitopes. AGE4 positivity was less frequent in patients with microangiopathy and in those with polyneuropathy. In patients with abnormal glucose metabolism, metformin treatment was associated with higher AGE4. AGE4 positivity was significantly lower in gliptin-treated patients. Multivariate analysis showed that polyneuropathy and obesity were independently associated with AGE4 positivity, with odds ratios (ORs) of 0.21 and 3.02, respectively. Moreover, logistic regression showed that AGE4 positivity and HbA1c are independent predictors of polyneuropathy. Considering both indicators allows correct classification of 70.4% of cases with a general accuracy of 76%.
The slot dot-blot method detects compounds found in serum in trace amounts. Accumulation of AGE4 was associated with glucose metabolism abnormalities. A tendency toward AGE4 positivity was less frequent in patients with microangiopathy and in non-treated and gliptin-treated diabetes patients.
晚期糖基化终产物（AGEs）是在还原糖或活性醛与蛋白质、脂质或DNA分子之间的级联反应过程中形成的。它们构成了一组各种稳定的化合物。晚期糖基化终产物被认为是代谢紊乱的潜在生物标志物。然而，到目前为止，仅开发了几种测定单个AGEs水平的方法。
本研究的目的是比较斑点印迹法和直接酶联免疫吸附测定（ELISA）检测糖尿病血清中痕量甲基乙二醛（MGO）修饰蛋白（AGE4）非标准表位的效率，并评估糖尿病患者中的AGE4及相关代谢异常情况。
使用两种方法检测AGE4的存在：直接ELISA和斑点印迹法——一种基于单克隆、市售抗体检测非标准AGE表位的新开发免疫测定法。AGE4定量以任意单位（AU）的中位数AGE4和AGE4阳性率（可检测到AGE4的样本百分比）表示，与糖尿病相关的代谢异常、并发症及治疗情况相关。
在检测非标准AGE4表位方面，斑点印迹法比ELISA显著更有效。微血管病变患者和多发性神经病患者中AGE4阳性率较低。在葡萄糖代谢异常的患者中，二甲双胍治疗与较高的AGE4相关。格列汀治疗的患者中AGE4阳性率显著较低。多变量分析显示，多发性神经病和肥胖与AGE4阳性独立相关，比值比（OR）分别为0.21和3.02。此外，逻辑回归显示AGE4阳性和糖化血红蛋白（HbA1c）是多发性神经病的独立预测指标。综合考虑这两个指标可正确分类70.4%的病例，总体准确率为76%。
斑点印迹法可检测血清中的痕量化合物。AGE4的积累与葡萄糖代谢异常相关。微血管病变患者以及未治疗和格列汀治疗的糖尿病患者中AGE4阳性的倾向较少见。
