Identification of Deoxy-D-Fructosyl Phosphatidylethanolamine as a non-enzymic glycation product of phosphatidylethanolamine and its occurrence in human blood plasma and red blood cells

The amino-carbonyl reaction (Maillard reaction), also known as glycation, of egg-yolk phosphatidylethanolamine (PE) was induced by incubating PE (50 mg/ml) with D-glucose (222 mM) in a methanol medium containing 2,6-di-tert-butyl-p-cresol as an antioxidant at 37°C for 4 days. The resultant product, glycated PE (gPE), was then isolated from the reaction mixture by two-step normal and reversed-phase high-performance liquid chromatography with UV diode array detection and was characterized as having a 1:1:1 elemental ratio of phosphorus:nitrogen:sugar. The Fourier transform-nuclear magnetic resonance spectrum and infrared absorbance spectrum indicate the isolated gPE to have been deoxy-D-fructosyl PE, which is an Amadori product of PE. The fast atom bombardment-mass spectrometric data for the glycation product of authentic dioleoyl PE (1,2-di-9-octadecenoyl-sn-glycero-3-phosphoethanolamine) show that the molecular weight of gPE corresponds to that of glucose-conjugated PE in the form of an Amadori product. This Amadori product formation was also confirmed in PE/phosphate buffer dispersions and in phosphatidylcholine-PE liposome/phosphate buffer suspensions in the presence of D-glucose at 37°C. gPE was degraded by phospholipase A₂, C and D. Freshly spiked blood plasma and red blood cells (RBC) from normal human volunteers contained substantial levels of gPE, the concentration corresponding to at least 9 mol% of PE. Remarkable formation of gPE, up to 15-45 mol% of PE in human blood plasma and RBC, was further confirmed by prolonged incubation with 5-45 mM D-glucose. The gPE formation in RBC was found to be proportional to the glycated hemoglobin formation.