Identification of intact oxidation products of glycerophospholipids in vitro and in vivo using negative ion electrospray iontrap mass spectrometry

Abstract

Free radical-induced oxidation products of polyunsaturated fatty acids esterified to phospholipids have been implicated in a number of human diseases including atherosclerosis and neurodegenerative diseases. Some of these phospholipid oxidation products have potent biological activities and likely contribute to human pathophysiological conditions. Oxidation products have also been used as markers of oxidative stress in vivo. Identification and quantification of phospholipid oxidation products are often performed by analyzing the oxidized free fatty acid moieties after hydrolysis from the phospholipids head groups by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS). We now describe the definitive identification of intact oxidized products of glycerophospholipids including glycerophosphatidylcholine (GPC), glycerophosphatidylethanolamine (GPE), and glycerophosphatidylserine (GPS) in vitro and in vivo using iontrap MS. For these analyses, the negative ions of the oxidation products of phospholipids are fragmented to MS n and unequivocal structural characterization is obtained based on collision-induced dissociation (CID) of the sn-2 carboxylate ion. This technique overcomes the need to hydrolyze fatty acids from phospholipids in the analysis. The method has been used to identify a number of oxidation products of glycerophospholipids including hydroxyeicosatetraenoates (HETEs) and isoprostanes (IsoPs) esterified to different classes of glycerophospholipids in vitro and in vivo. These studies thus provide a new approach to identify the intact oxidation products of glycerolphospholipids.

A simplified procedure for semi-targeted lipidomic analysis of oxidized phosphatidylcholines induced by UVA irradiation

“…An advantage of the HPLC procedure that was used in this study is compatibility with mass spectrometry detection in the negative mode, which allows characterization of oxidized and unoxidized acyl residues in PCs (45)(46)(47). As a proof of principle, we identifi ed endogenous fragmented OxPCs, such as POVPC, PGPC, PONPC, and PAzPC, using LC-ESI-MS/MS in negative mode using MS/MS parameters as described in Nakanishi et al ( 48 ) (Supplementary Fig.…”

“…This opens the possibility to identify novel OxPLs that may have detrimental effects in photoageing and UV-induced pathologies as well as photoproducts that mediate benefi cial effects of UVA radiation, which is used for therapy of infl ammatory skin diseases. This task, which was outside of the scope of this work, can be pursued using other modes of HPLC-MS/MS ( 47,48,50 ), high-resolution mass spectrometry ( 51 ), group-specifi c reagents ( 27,31 ), and other approaches demonstrated here identifi cation by MS/MS fragmentation studies in the negative ion mode of four molecular species of endogenously formed OxPCs accumulating in UVA-irradiated fi broblasts.…”

A Common CD36 Variant Influences Endothelial Function and Response to Treatment with Phosphodiesterase 5 Inhibition

“…Free FA was measured using NEFA-HR (2) (Wako, Sopachem BV, Ochten, the Netherlands), total cholesterol (Diagnostics Chemicals, Oxford, CT, USA) and triglycerides (Roche Diagnostics Corporation, Indianapolis, IN, USA) by enzymatic colorimetry. F2-isoprostanes were assayed in plasma using negative ion gas chromatography mass spectroscopy as previously described (30). Sildenafil levels were assessed by liquid chromatography-tandem mass spectrometry on silica column with aqueous-organic mobile phase as previously reported (31).…”

Oxidative Lipidomics Coming of Age: Advances in Analysis of Oxidized Phospholipids in Physiology and Pathology

“…It has been used to identify novel lipid oxidation products such as isolevuglandins as well as to determine mechanisms of oxidative fragmentation (68,104). In ion trap instruments, multiple sequential fragmentations (MS n ) can be carried out to provide more detailed analysis of the structure in question (149). Product ion scanning also provides information on diagnostic fragment ions for individual oxidized products, which can subsequently be used in the targeted scanning approaches.…”