Oxidation of glycated phosphatidylethanolamines: evidence of oxidation in glycated polar head identified by LC-MS/MS

Abstract

Phosphatidylethanolamine glycation occurs in diabetic patients and was found to be related with oxidative stress and with diabetic complications. Glycated phosphatidylethanolamines seem to increase oxidation of other molecules; however, the reason why is not understood. In this work, we have studied the oxidation of glycated phosphatidylethanolamines (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylethanolamine (PLPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (dPPE)) using a Fenton system. Liquid chromatography-electrospray ionization (ESI)-mass spectrometry and ESI-tandem mass spectrometry in both positive and negative modes were used for detecting and identifying the oxidation products. We were able to identify several oxidation products with oxidation in unsaturated sn-2 acyl chain of PLPE, as long- and short-chain products with main oxidation sites on C-7, C-8, C-9, and C-12 carbons. Other products were identified in both glycated PLPE and glycated dPPE, revealing that oxidation also occurs in the glycated polar head. This fact has not been reported before. These products may be generated from oxidation of glycated phosphatidylethanolamines (PE) as Schiff base, leading to short-chain product without the amine moiety, due to cleavage of glycated polar head and long-chain product with two keto groups linked to the glycated polar head or from glycated PE as Amadori product, short-chain products with -NHCHO and -NHCHOHCHO terminal in polar head. Oxidation of glycated phosphatidylethanolamines occurred more quickly than the oxidation of non-glycated phosphatidylethanolamines probably because of the existence of more oxidation sites derived from glycation of polar head group. Monitoring glycated polar head oxidation could be important to evaluate oxidative stress modifications that occur in diabetic patients.

Photooxidation of glycated and non‐glycated phosphatidylethanolamines monitored by mass spectrometry

“…Ions with higher m/z value correspond to the glucPE+14Da that can be identified as a keto and/or epoxy derivative or a gluconic acid derivative due to oxidation in C6 (Scheme , structure 8). The formation of this oxidation product was previously reported by Simões and co‐authors during oxidation of glycated PLPE . The other ions were observed at lower m/z values and the majority of them correspond to oxidation products formed by oxidative cleavage of glucose moiety.…”

“…The photooxidation products with terminal ‐NHCHO (Scheme , structure 1), due to oxidative cleavage between C1and C2 of glucose, were identified for all glycated PE after UV irradiation. A similar product was observed during Fenton reaction of PLPE (ion at m/z 742.3 [M‐H] − ) . Tandem mass spectra of these ions in the negative mode (Fig.…”

“…It was not possible to detect these ions in the positive ion mode, probably due to the presence of the carbonyl group. In this case, the deprotonation of the phosphate group can favour the detection of these species in the negative ion mode, as reported by Simões and co‐authors, which also identified this oxidation product during oxidation of glycated PLPE …”

“…Interestingly, the relative abundance of this isomer gradually decreases after the first day of oxidation and eventually disappears. In fact, this oxidation product was reported to be an unstable oxidation product while the keto and/or epoxy derivative of linoleoyl fatty acyl chain was considered to be a stable oxidation product …”

“…Oxidation was monitored by ESI‐MS and products structurally characterized by MS/MS both in positive and in negative mode. MS has been widely used for identification and characterization of oxidized and glycated phospholipids . To evaluate the extent of oxidation of PE and glycated‐PE, the total hydroperoxides, as important products of lipid oxidation, were quantified by ferrous iron/xylenol orange (FOX 2) assay.…”