The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA). A large proportion of the retinal glycerophospholipids, especially those found in photoreceptor membranes, are dipolyunsaturated molecular species. Among them, dipolyunsaturated phosphatidylcholine molecular species are known to contain very long chain polyunsaturated fatty acids (VLC-PUFA) from the n-3 and n-6 series and having 24 to 36 carbon atoms (C24 to C36) and four to six double bonds. Recent interest in the role of VLC-PUFA arose from the findings that a protein named ELOngation of Very Long chain fatty acids 4 (ELOVL4) is involved in their biosynthesis and that mutations in ELOVL4 gene are associated with Stargardt-like macular dystrophy (STD3), a dominantly inherited junevile macular degeneration leading to vision loss. The aim of the present work was to develop an HPLC-ESI-MS/MS method for the structural characterization and the quantification of dipolyunsaturated phosphatidylcholine (PC) molecular species containing VLC-PUFA and validated this methodology on retinas from bovines and human donors. Successful separation of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), lyso-phosphatidylcholine (LPC) and sphingomyeline (SM) was achieved by using a silica gel column and a gradient of hexane/isopropanol/water containing ammonium formate as a mobile phase. A complete structural characterization 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 characterization of PC species was done on bovine retina. Among them, 28 were dipolyunsaturated PC species containing one VLC-PUFA (C24 to C36) with three to six double bonds. VLC-PUFA was always in the sn-1 position whilst PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA, C22:6n-3). Most of these VLC-PUFA-containing dipolyunsaturated PC 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 represented compounds were those having VLC-PUFA of 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 PC with 36:5 and 36:6 were detected but at lower quantities. In conclusion, this new HPLC-ESI-MS/MS method is sensitive and specific enough to structurally characterize and quantify all molecular species of PC, including those esterified with VLC-PUFA. This technique is valuable for a precise characterization of PC molecular species containing VLC-PUFA in retina and may be useful for better understanding the pathogenesis of STD3.
Identification and quantification of phosphatidylcholines containing very long chain polyunsaturated fatty acid (VLC-PUFA) in bovine and human retina by liquid chromatography/tandem mass spectrometry