Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS.

Studies in the last decade have revealed that lysophospholipids (LysoGPs) are bioactive lipids with a number of biological activities, most of which are mediated by specific receptors belonging to the class A G protein-coupled receptor (GPCR) family (1). To date, 16 such GPCRs have been identified, including 6 lysophosphatidic acid (LPA) receptors, 5 sphingosine 1-phosphate receptors, 4 lysophosphatidylserine (LysoPS) receptors, and 1 lysophosphatidylinositol (LPI) receptor (1–4). Recent studies of gene-targeting mice have clearly shown that these receptors function individually in various pathological and physiological conditions (5). Glycero-LysoGPs have one acyl chain that is linked to either the sn-1 or sn-2 position of glycerol. Phospholipase A (PLA)1 and PLA2 cleave diacyl-phospholipids (GPs) and produce 2-acyl-1-lysophospholipids (2-acyl-1-LysoGPs) and 1-acyl-2-LysoGPs, respectively. Recent studies have suggested that some LysoGP receptors discriminate 1-acyl-2-LysoGPs from 2-acyl-1-LysoGPs. For example, LPA3/Edg7 (6, 7) and LPA6/P2Y5 (8) prefer 2-acyl-1-LPA to 1-acyl-2-LPA. In addition, they prefer LPA with unsaturated fatty acids, which are usually attached at the sn-2 position of GPs. LPS1/GPR34 also shows preference for 2-acyl-1-LysoPS (9). These findings indicate that 2-acyl-1-LysoGPs exert their activities through specific receptors. Because of their different activities, 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs should be quantified separately. However, it has been difficult to detect and quantify 2-acyl-1-LysoGPs in biological samples because 2-acyl-1-LysoGPs are unstable and are quickly converted to the corresponding 1-acyl-2-LysoGPs by a spontaneously occurring intra-molecular acyl migration reaction (10), yielding a mixture of 1-acyl-2-LysoGPs and 2-acyl-1-LysoGPs. Pluckthun and Dennis (10) demonstrated that 1-palmitoyl-2-lysophosphatidylcholine (LPC) prepared by PLA2 is actually the equilibrium mixture consisting of approximately 90% of the 1-acyl-2-lyso isomers and 10% of the 2-acyl-1-lyso isomers. They also showed that the rate of the acyl migration was pH dependent, with a minimum around pH 4 to pH 5. Their technique was based on 31P NMR and thus could be used only on pure samples. In the present study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-lyso isomers using LC-MS/MS under conditions that completely eliminate the acyl migration reaction. Using this method, we determined the distribution of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs with various head groups in biological samples. To the best of our knowledge, this is the first report to precisely determine the distribution of acyl chains between the sn-1 and sn-2 positions.