P2 - In vitro glycation and glycoxidation of phosphatidylethanolamines

Abstract

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 MSn 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 1,2-13C glucose, DPPE-CMA/CME adducts contained two 13C 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.