Isolation and identification of glycated aminophospholipids from red cells and plasma of diabetic blood

Abstract

Glycosylation is a major pathway for posttranslational modification of tissue protein and begins with nonenzymatic addition of carbohydrate to the primary amino groups. Excessive glycation of tissue protein has been implicated in the pathogenesis of diabetes and ageing. While glycation of aminophospholipids has also been postulated, glycated aminophospholipids have not been isolated. Using normal phase HPLC with on-line electrospray mass spectrometry we found glycated ethanolamine phospholipids to make up 10-16% of the total phosphatidylethanolamine (PE) of the red blood cells and plasma of the diabetic subjects. The corresponding values for glycated PE of control subjects were 1-2%.Key words: Glucosylated aminophospholipid; Glucose; Phosphatidylethanolamine; Phosphatidylserine; Electrospray; Thin-layer chromatography; Liquid chromatography, mass spectrometry; Normal phase HPLC bovine brain phosphatidylinositol (PI) and sphingomyelin (SPH) were obtained from Sigma Chemical Co., St. Louis, MO. All chemicals were of reagent grade quality, while the solvents were of chromatographic purity and were obtained from local suppliers. The purity of the reference compounds was ascertained by thin-layer chromatography (TLC) [3,7]. Isolation of phospholipids from bloodBlood was obtained from six diabetic patients and six non-diabetic donors. The diabetics were selected for elevated blood glucose levels indicated by their content of glycosylated hemoglobin (9-15%). EDTA blood was centrifuged (2300xg for 10 min) in a swinging bucket rotor to separate the plasma from the red cells. The cells were washed three times with five volumes of phosphate buffered saline (150 mM NaC1, 50 mM sodium phosphate, pH 8.0) and centrifuged (2300xg for 10 min). The red blood cell phospholipids were extracted according to Rose and Oaklander [8]. The plasma phospholipids were extracted with chloroform-methanol 2:1 modified from Folch et al. [9]. Glucosylated PE could be stored at -20°C in neutral chloroform methanol for several days without decomposition. The Schiff base dissociated in dilute acetic acid.

Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes

“…However, in this study, glycated PS was undetectable in human plasma (data not shown). Similarly, Ravandi et al (25) were also unable to detect glycated PS in human plasma or red blood cell membranes.…”

“…Recent studies reported the detection of Amadori-PE in biological samples (i.e., blood plasma, red blood cells, and human atherosclerotic plaques) using LC-MS (25)(26)(27)(28). During LC-MS analysis, chromatographic peaks that showed mass increments of 162 Da (most likely C 6 H 10 O 5 ) relative to the molecular masses of nonglycated PE were tentatively ascribed to Amadori-PE.…”

“…When the QTRAP was used to perform neutral loss (303 Da) scanning, only Amadori-PE species were detectable. Normally, it is very difficult to detect Amadori-PE in biological samples because of overlapping background contaminants (25). In contrast, by means of neutral loss scanning, individual Amadori-PE species in plasma could be effectively identified without any preliminary LC separations.…”

“…In contrast, by means of neutral loss scanning, individual Amadori-PE species in plasma could be effectively identified without any preliminary LC separations. On the other hand, Ravandi et al (25) performed LC-MS analysis of Amadori-PE in diabetic plasma and compared the glycated species of the diacyl PE and alkenyl-acyl PE (plasmalogen). They suggested that only the diacyl PE species, not the alkenyl-acyl species, became glycated.…”

Glycated Phosphatidylethanolamine Promotes Macrophage Uptake of Low Density Lipoprotein and Accumulation of Cholesteryl Esters and Triacylglycerols

“…We have previously demonstrated that the amino phospholipids of plasma and red blood cells (RBC) from diabetic subjects show a 10-fold increase in glycated PtdEtn over control subjects (9,10) and that incorporation of Glc-PtdEtn into LDL (11) facilitates peroxidation of LDL lipids. The present study demonstrates that the presence of Glc-PtdEtn in LDL results in an increased uptake of LDL and a dramatic increase in neutral lipid accumulation in THP-1 macrophages.…”