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O. Berdeaux, L. Martine, S. Cabaret, G. Thuret, S. Gregoire, P. Gain, C. P. Garcher, A. M. Bron, N. Acar, L. Bretillon; A New HPLC-ESI-MS/MS Method to Characterize and Quantify Phosphatidyl-Choline With VLC-PUFA: Application to Human Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5954.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the ELOVL4 gene have been found in Stargardt-like macular dystrophy or STD3. Previous studies have shown that ELOVL4 is involved in the biosynthesis of very long chain polyunsaturated fatty acids (VLC-PUFA). The aim of this work was to develop a HPLC-ESI-MS/MS method of characterization and quantification of dipolyunsaturated phosphatidyl-choline (PC) molecular species containing VLC-PUFA and to apply it on retinas from human donors.
Eyeballs were collected from calf as well as from nine human donors (body donation to Science). The neural retina was dissected from the RPE/choroid. Following lipid extraction, phosphorus content of total phospholipids was determined.Using a triple quadrupole MS instrument, PC molecular species were structurally characterized by collision-induced dissociation in the negative mode with a method based on normal-HPLC-ESI-MS/MS. PC molecular species were then quantified using precursor ion scanning of m/z 184amu in the positive mode.
The characterization of PC species was done on bovine retinas. Among them, 28 were dipolyunsaturated PC species containing one VLC-PUFA (C24 to C36) with three to six double bonds. VLC-PUFA were always in the sn-1 position whilst PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA, C22:6.n-3). Most of these VLC-PUFA-containing dipolyunsaturated PC were detected and quantified in human retinas. 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 in lower quantities.
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 containing VLC-PUFA in retina and may be useful for better understanding their implication in the pathogenesis of STD3.
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