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

Abstract

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), phosphatidylserine (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 36:5 and 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 STD3.

Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry

“…Of interest is increasing evidence that suggests that the phosphorylation of ceramide to ceramide-1-phosphate regulates cell proliferation and apoptosis ( 53 ), Generally, lipid characterization by MALDI IMS in ocular tissue has been limited to mass spectra that extend to roughly m/z 900, eliminating the possibility of detecting and identifying the VLC-PUFA-containing PC lipids, which occur at very low concentrations in photoreceptors. Electrospray mass spectrometry analysis of retinas [salamander ( 29 ), rat ( 11 ), and human ( 10,24 )] has revealed the presence of VLC-PUFA-containing PC lipids, but localization of these lipids by MALDI imaging has not been presented. It is now understood that 22:6 is required for a healthy retina (6)(7)(8).…”

ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3

“…The normal ELOVL4 protein is responsible for elongation of LC-PUFA to VLC-PUFA found in retinal photoreceptor cells and in several other tissues ( 15,18,19,42 ). Reduced levels of C32-36 acyl PC have been found in the retina of genetic mouse models of autosomal dominant Stargardt-like macular dystrophy (STGD3) ( 43 ).…”

“…Also, the Atlantic salmon ELOVL4 protein has been shown to effectively elongate C24-C26 polyunsaturated FAs to polyenoic products up to C36 ( 49 ). All of these fi ndings collectively in testis and sperm, or esterifi ed to the sn-1 position of phosphatidylcholine (PC) in the retina (15)(16)(17)(18)(19).…”

Characterization of Phospholipid Molecular Species by Means of HPLC-Tandem Mass Spectrometry

“…Electrospray ionization also leads to the formation of negative ions derived from PC lipids in spite of the quaternary nitrogen atom with its permanent positive charge. Under negative Anyway, this discrepancy of abundance is not so visible when the PC molecular species contain a long-chain fatty acid linked to the sn-2 position (Berdeaux et al 2010). Kerwin, Tuininga, & Ericsson (1994) intensity are low as well as those of the fragment at 454.1 m/z arising from the loss of docohexanoyl acyl moieties.…”

“…MS signals of PL are influenced by different effects related to acyl chain length, the degree of acyl chain unsaturation and concentration. Several authors (Berdeaux et al;Ahn et al 2007) have shown that the ESI-MS response increases with increasing degree of insaturation of the fatty acid side chains whereas it decreases with an increase in chain length when the phospholipids concentration range in the samples is higher than picomolar. Only the picomolar range concentration results, in fact, in a linear response.…”