Annibal Andrea, Fedorova Maria, Schiller Jürgen, Hoffmann Ralf
Institute of Bioanalytical Chemistry (Center for Biotechnology and Biomedicine, LIFE-Leipzig Research Center for Civilization Diseases), Faculty of Chemistry and Mineralogy, University Leipzig, Germany.
Institute of Bioanalytical Chemistry (Center for Biotechnology and Biomedicine), Faculty of Chemistry and Mineralogy, University Leipzig,, Germany.
Free Radic Biol Med. 2014 Oct;75 Suppl 1:S21-2. doi: 10.1016/j.freeradbiomed.2014.10.734. Epub 2014 Dec 10.
Formation and accumulation of advanced glycation end products (AGEs) appear to correlate with many human diseases, such as atherosclerosis and inflammation. Whereas AGE-modified proteins relatively well studied, aminophospholipid AGE-adducts have been less intensively investigated. At elevated concentrations, glucose can react with amino groups of phosphatidylethanolamines (PE) to form Schiff bases or Amadori products, which can be further converted, under oxidative conditions, to various AGEs. Many of these products such as carboxymethylamine (CMA) and carboxyethylamine (CEA) can be formed by oxidative degradation of Amadori-products. The aim of this work was to investigate the different glycation and glycoxidation PE-adducts and to elucidate the most prominent mechanisms of AGE-PE formation. Four different aminophospholipids,[dipalmitoyl-(DPPE), palmitoyl-oleoyl-(POPE), palmitoyl-linoleoyl-(PLPE) or palmitoyl-arachidonoyl-phosphatidylethanolamine(PAPE); all 1mmol/L] were glycated in vitro (5mmol/L glucose) and oxidized by the Fenton reaction (80µmol/L FeSO4, 50mmol/L H2O2) or using electrochemical oxidation (Roxy EC System, Antec, Leiden, Netherlands). High resolution mass spectrometry (MS) in combination with MS(n) fragmentation allowed the identification of major products, such as CMA, CEA and oxo-glucuronic acid. The mechanism of CMA/CME formation by oxidative degradation of the Amadori product was confirmed by co-oxidation (Fenton reaction) of purified glycated (gPOPE) with nonglycated DPPE (Fenton reaction) and by electrochemical oxidation of purified gPOPE, whereas the reaction with glyoxal/methylglyoxal formed by glucose oxidation was excluded. In both cases CMA/CEA-POPE adducts were detected. When glycated with 1,2-(13)C glucose, DPPE-CMA/CME adducts contained two (13)C atoms further confirming the proposed mechanism. Finally, the ability of glycated PE when co-incubated with cells (HeLa) to induce Nrf2 activation was also addressed.
晚期糖基化终产物（AGEs）的形成和积累似乎与许多人类疾病相关，如动脉粥样硬化和炎症。虽然AGE修饰的蛋白质得到了较好的研究，但氨基磷脂AGE加合物的研究相对较少。在高浓度下，葡萄糖可与磷脂酰乙醇胺（PE）的氨基反应形成席夫碱或阿马多里产物，在氧化条件下，这些产物可进一步转化为各种AGEs。许多此类产物，如羧甲基胺（CMA）和羧乙基胺（CEA），可通过阿马多里产物的氧化降解形成。这项工作的目的是研究不同的糖基化和糖氧化PE加合物，并阐明AGE-PE形成的最主要机制。四种不同的氨基磷脂，[二棕榈酰-（DPPE）、棕榈酰-油酰-（POPE）、棕榈酰-亚油酰-（PLPE）或棕榈酰-花生四烯酰-磷脂酰乙醇胺（PAPE）；均为1mmol/L]在体外进行糖基化（5mmol/L葡萄糖），并通过芬顿反应（80µmol/L硫酸亚铁，50mmol/L过氧化氢）或使用电化学氧化（Roxy EC系统，Antec，荷兰莱顿）进行氧化。高分辨率质谱（MS）结合MS(n)碎片化技术能够鉴定主要产物，如CMA、CEA和氧代葡萄糖醛酸。通过将纯化的糖基化（gPOPE）与未糖基化的DPPE进行共氧化（芬顿反应）以及对纯化的gPOPE进行电化学氧化，证实了阿马多里产物氧化降解形成CMA/CME的机制，而排除了与葡萄糖氧化形成的乙二醛/甲基乙二醛的反应。在这两种情况下均检测到了CMA/CEA-POPE加合物。当用1,2-(13)C葡萄糖进行糖基化时，DPPE-CMA/CME加合物含有两个(13)C原子，进一步证实了所提出的机制。最后，还探讨了糖基化PE与细胞（HeLa）共孵育时诱导Nrf2激活的能力。
