Identification and quantification of phosphatidylcholines containing very-long-chain polyunsaturated fatty acid in bovine and human retina using liquid chromatography/tandem mass spectrometry

The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA) A large proportion of retinal phospholipids especially those found in photoreceptor membranes are dipolyunsaturated molecular species Among them dipolyunsaturated phosphatidylcholine (PC) molecular species are known to contain very-long-chain polyunsaturated fatty acids (VLC-PUFA) from the n-3 and n-6 series having 24-36 carbon atoms (C24-C36) and four to six double bonds Recent interest in the role played by VLC-PUFA arose from the findings that a protein called elongation of very-long-chain fatty acids 4 (ELOVL4) is Involved in their biosynthesis and that mutations in the ELOVL4 gene are associated with Stargardt-like macular dystrophy (STD3) a dominantly inherited Juvenile macular degeneration leading to vision loss The aim of the present study was to develop an HPLC-ESI-MS/MS method for the structural characterisation and the quantification of dipolyunsaturated PC molecular species containing VLC-PUFA and validate this methodology on retinas from bovines and human donors Successful separation of phosphatidylethanolamine (PE) phosphatidylinositol (PI) phosphandylserine (PS) PC lyso-phosphatidylcholine (LPC) and sphingomyelin (SM) was achieved using a silica gel column and a gradient of hexane/isopropanol/water containing ammonium formate as a mobile phase A complete structural characterisation of intact phosphatidylcholine species was obtained by collision-induced dissociation (CID) in the negative mode Fatty acid composition and distribution can be clearly assigned based on the intensity of sn-2/sn-1 fragment ions The PC species were characterised on bovine retina 28 of which were dipolyunsaturated PC species containing one VLC-PUFA (C24-C36) with three to six double bonds VLC-PUFA was always in the sn-1 position while PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA C22 6n-3) Most of these VLC-PUFA-containing dipolyunsaturated PCs were detected and quantified in human retinas The quantitative analysis of the different PC molecular species was performed in the positive mode using precursor ion scanning of m/z 184 and 14 0/14 0-PC and 24 0/24 0-PC as internal standards The relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration The main compounds represented were those having VLC-PUFA with 32 carbon atoms (C32 3 C32 4 C32 5 and C32 6) and 34 carbon atoms (C34 3 C34 4 C34 5 and C34 6) Dipolyunsaturated PCs with 36 Sand 36 6 were detected but in smaller quantities In conclusion this new HPLC-ESI-MS/MS method is sensitive and specific enough to structurally characterise and quantify all molecular PC species including those esterified with VLC-PUFA This technique is valuable for a precise characterisation of PC molecular species containing VLC-PUFA in retina and may be useful for a better understanding of the pathogenesis of STD