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Olivier Berdeaux, Stéphanie Cabaret, Lucy Martine, Gilles Thuret, Stéphane Grégoire, Philippe Gain, Catherine P. Garcher, Alain M. Bron, Niyazi Acar, Lionel Bretillon; Quantitative Analysis Of Fatty Acids On Choline- And Ethanolamine-phospholipids In Human Ocular And Non-ocular Tissues By Liquid Chromatography/tandem Mass Spectrometry (LC-ESI-MS/MS). Invest. Ophthalmol. Vis. Sci. 2011;52(14):3352.
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Epidemiological studies have pointed out the role of polyunsaturated fatty acids (PUFAs) in the prevention of AMD. The surrounding mechanisms are poorly defined but may involve the conversion of PUFAs into active metabolites. The need for qualitative and quantitative data on the nature of the PUFAs-rich phospholipid species is crucial to delineate the signalling pathways. For this purpose, we have developed a quantitative method for determining the concentration of PUFAs in choline- and ethanolamine- phospholipids (PE, PC), and plasmalogens (PlsC, PlsE) using LC-ESI-MS/MS, and further tested this method on human ocular and non-ocular tissues.
Red blood cells, retinas and optic nerves were collected from nine human dead subjects. Following total lipid extraction, 3 internal standards were added. Using a triple quadrupole MS instrument, PE, PlsE, PC and PlsC molecular species were structurally characterized by collision-induced dissociation of [M-H]- in the negative mode with a method based on normal-HPLC-ESI-MS/MS. The individual species of PC and PlsC species were then quantified using precursor ion scanning at m/z 184 amu in the positive mode. PE species were quantified using neutral loss scan at 141amu in the positive mode. Finally PlsE species were quantified on multiple reactions monitoring of one parent/fragment transition for each PlsE species in the negative mode.
Individual species of PC, PlsC, PE, and PlsE species were fully characterized according to the PUFAs esterified to glycerol. Calibration curves for the quantification of PC and PE species were obtained using authentic standards of PC and PE containing PUFAs of various chain lengths. Using this method, intact phospholipids mixture from human red blood cells, retinas and optic nerves were analyzed and individual species of PC, PlsC, PE, and PlsE species were quantified accordingly.
Both quantitative and qualitative analysis of PE, PlsE, PC and PlsC species from human tissues can be achieved by normal-HPLC-ESI-MS/MS. Such data is a prerequisite for a better understanding of the mechanisms by which PUFAs, including omega 3 PUFAs, incorporate the retina and may participate in the prevention of AMD.
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