Open Access
Issue
OCL
Volume 18, Number 5, Septembre-Octobre 2011
Lipids and Brain II. Actes des Journées Chevreul 2011 (Deuxième partie)
Page(s) 284 - 290
Section PUFA and Ocular Pathologies
DOI https://doi.org/10.1051/ocl.2011.0406
Published online 15 September 2011

© John Libbey Eurotext 2011

The retina is one of the vertebrate tissues with the highest content of polyunsaturated fatty acids (PUFA). A large proportion of the retinal glycerophospholipids, especially those of photoreceptor membranes, consist of dipolyunsaturated molecular species. Studies have reported that dipolyunsaturated phosphatidylcholine (PC) molecular species present in both rod- and cone-dominant retinas contain C22:6n-3 as one of the acyl chains, the other one being very-long-chain (C24–C36) polyunsaturated fatty acids (four, five or six double bonds, VLC-PUFA) (Aveldano, 1987; Aveldano and Sprecher, 1987; Poulos, 1995). Several studies have reported that only PC contain C28-C36 VLC-PUFA (Aveldano, 1988; Suh et al., 1994; Suh and Clandinin, 2005; McMahon, Jackson et al., 2007). Docosahexaenoic acid (DHA, 22:6n-3) tends to be located at the sn-2 position of the glycerol backbone while VLC-PUFA tend to locate predominantly at the sn-1 position (figure 1) (Aveldano, 1988). The majority of these PC species containing VLC-PUFA (named in this presentation VLC-PC) are localized in photoreceptor outer segments where the phototransduction reactions take place (Rotstein and Aveldano, 1988). In bovine photoreceptor outer segments, VLC-PC species are significant components of lipid membranes since the C28-C36 VLC-PUFA represent 10 mol % of total fatty acids in PCs (Aveldano, 1987).

thumbnail Figure 1.

Structure of a phosphatidylcholine molecule containing C34:4n-3 and docosahexaenoic acid (C22:6n-3) in the sn-1 and sn-2 positions respectively.

Retinal C28-C36 VLC-PUFA belong to the n-3 and n-6 families (Aveldano and Sprecher, 1987) and they are synthesized in situ (Rotstein and Aveldano, 1988; Suh et al., 1994). The predominant n-3 polyunsaturated C28-C36 fatty acids in rat retina have been shown to be synthesized from EPA, and not from DHA (Suh and Clandinin, 2005). A protein called Elongation of very-long-chain fatty acids 4 (ELOVL4) is involved in their biosynthesis (Rotstein and Aveldano, 1988; Agbaga, Brush et al. 2008). Based on sequence homology with ELOVL1, 2, 3, and 5 proteins, which are implicated in the elongation of saturated, monounsaturated, or polyunsaturated fatty acids (PUFA) from 18 to 26 carbons, the ELOVL4 protein was predicted to have similar functions (Tvrdik et al., 2000; Zhang et al., 2001; Leonard et al., 2004; Meyer et al., 2004; Westerberg et al., 2004; Agbaga et al., 2010). The ELOVL4 protein was shown to synthesize VLC-PUFA with carbon chain length of C28 and certainly longer (C30-C36) in cultured cells expressing transgenic ELOVL4 gene (Agbaga, Brush et al. 2008) and in mouse retina (McMahon et al., 2007).

VLC-PUFA and Stargardt-like macular dystrophy type 3

Recent interest in the functions played by VLC-PUFA arose from findings showing that alterations in the ELOVL4 gene in patients suffering from Stargardt-like macular dystrophy type 3 (STGD3), which is a dominantly inherited juvenile macular degeneration. These patients are affected by a severe vision loss due to large areas of macular atrophy in their retina (Zhang et al., 2001). These findings are completed by other data obtained in monkeys and showing that the ELOVL4 mRNA is exclusively expressed in cone photoreceptors that are known to be concentrated in the macular region of the retina (Umeda et al., 2003). Since at least three different mutations in the ELOVL4 gene have been identified in STGD3 patients (Edwards et al., 2001; Zhang et al., 2001; Grayson and Molday, 2005; Agbaga et al., 2010), this suggests that retinal health is highly dependent on the presence of C28-C36 PUFA, in addition to that of DHA (SanGiovanni and Chew, 2005).

In parallel to these ophthalmologic, genetic, and biochemical studies, both ELOVL4 knockout and knockin mice have been generated in order to understand the molecular mechanisms by which mutations in the ELOVL4 gene contribute to this eye disease. Within knockout models, homozygous mice had normal prenatal retinal development but were unusable after birth since they died after several hours of living due to defective skin permeability. This is consistent with a depletion of epidermal ceramides that are known to be rich in VLC saturated and monounsaturated fatty acids (Cameron et al., 2007; Li et al., 2007). Mice that were heterozygous for ELOVL4 gene displayed reduced ELOVL4 mRNA levels in their tissues but developed very normally with regular retinal functionality and minimal morphological alterations in their retinal photoreceptors (Raz-Prag et al., 2006; Li et al., 2007). Other groups have generated and studied knockin mice expressing mutant forms of ELOVL4 gene, carrying for example the human pathogenic 5-bp deletion in the ELOVL4 gene (Karan et al., 2005; McMahon et al., 2007; Vasireddy et al., 2009). The animals were characterized by a retinal phenotype resembling that of human STGD3, including an early selective deficiency in retinal C28–C36 acyl PCs, followed by a reduced retinal functionality evaluated by electroretinography, an increased accumulation undigested phagosomes and lipofuscin containing toxic N-retinylidene-N-retinylethanolamine (A2E) and a degeneration of photoreceptor cells in the central retina.

According to these findings, the proposed pathogenesis of human STGD3 is based on the alteration of photoreceptor outer segments (corresponding to the distal part of retinal photoreceptor cells) composition in VLC-PUFA affecting phototransduction processes and leading to the accumulation of toxic A2E and further to photoreceptor death (McMahon and Kedzierski, 2010). However, and even if the relationship between VLC-PUFA and STGD3 is now well established, the exact functions of VLC-PUFA in retinal health remain unclear.

Analysis of VLC-PUFA in retina

In this context, dipolyunsaturated PC molecular species containing VLC-PUFA in retina must be precisely characterized to improve our understanding of the pathogenesis of STGD3. Several current approaches were used for the characterization and quantification of VLC-PUFA or VLC-PC in biological samples. Gas chromatography-mass spectrometry equipped with electronic ionization (GC-EI-MS) and Liquid-chromatography – tandem mass spectrometry equipped with an electrospray ionization interface (LC-ESI-MS/MS) have been shown to be the most reliable analytical approaches to characterize and quantify VLC-PUFA or VLC-PC in biological samples and in particular in the retina.

The VLC-PUFAs were initially characterized in PC from bovine retina by Aveldaño and co-workers (Aveldano, 1987; Aveldano and Sprecher, 1987). In their study, PC were purified by TLC, converted into acetyldiglycerides and resolved into groups of molecular species (fraction of similar unsaturation) by means of argentation thin layer chromatography (AgNO3-TLC). VLC-PUFA were purified and separated by successive chromatographic steps (TLC, AgNO3-TLC and HPLC), and converted into fatty acid methyl esters (FAME) derivatives. FAMEs were then characterized with precision using a combination of oxidative ozonolysis and GC-EI-MS analyses. Using oxidative ozonolysis for the localization of the double bond, it was shown that very long chain tetraenes belonged to the n-6 series, hexaenes to the n-3 series, and major pentaenes to the n-3 series of fatty acids but very long chain n-6 pentaenes also occurred. Molecular ions were obtained by GC-EI-SM of FAME (figure 2) which conclusively identified the major VLC-PUFA in bovine retina. Suh and co-workers developed a similar GC-EI-MS methodology to quantify VLC-PUFA. In their study, they aimed to determine the effect of altering diet fat composition on the long-chain-PUFA and VLC-PUFA content of individual phospholipids of the photoreceptor outer segments (ROS) in normal and diabetic animals (Suh et al., 1994). VLC-PUFA were isolated from the total lipid extract from ROS by successive chromatographic steps and converted to FAME derivatives. AgNO3-TLC was used to resolve FAMEs on the basis of degree of unsaturation and FAMEs of each band were injected in GC-EI-MS analyses. Fatty acid identifying was confirmed by molecular ion [M]+ identification. In addition, GC-EI-MS provided definitive identification of the series of PUFA containing diagnostic ion at m/z 79 as the base pic (figure 2). Moreover, mass spectra of PUFA containing a significant fragment at m/z 108 are from the n-3 series whereas those containing a significant ion at m/z 150 are from the n-6 series. More recently, Agbaga and co-worker used GC-EI-MS to study the role of ELOVL4 protein in the biosynthesis of VLC-PUFA (Agbaga et al., 2008). In this study, VLC-PUFA were first isolated from total lipids extract of bovine retina, converted to FAME derivatives and identified by GC-EI-MS as described above. In a second step, and by using mass spectra and relative retention time data, they were able to identify these fatty acids in samples containing low concentrations by the use of single ion monitoring (SIM) mass spectrometry. By monitoring the m/z ratios 79, 108 and 150, they have identified and measured the VLC-PUFA from ELOVL4-expressings cells for their study.

thumbnail Figure 2.

Electron impact mass spectra and total ion of 34:4 (n-6) and 32:6 (n-3) methyl esters (Suh et al., 1994; Agbaga et al., 2008). Reproduced by kind permission of Proceeding of the National Academy of Sciences and redrawn from the original publication.

However, for structural identification of PUFA and in particular of VLC-PUFA, mass spectra of FAMEs not contain ions indicating the position of double bonds on the aliphatic chain. In the most useful approach to structure determination, the carboxyl group is derivatized with a reagent containing a nitrogen atom. When the molecule is ionized in the mass spectrometer, the nitrogen atom but not the alkyl chain carries the charge, and double bond ionization and migration is minimized. 4,4-Dimethyloxazoline (DMOX) derivatives give excellent mass spectra of fatty acids with electron-impact ionization that frequently permit unequivocal identification (Zhang et al., 1988; Fay and Richli, 1991; Berdeaux and Wolff, 1996). Recently, we have used DMOX derivatives for structural identification of VLC-PUFA with precise localization of the double bonds along the carbon chain (Berdeaux et al., 2010). The VLC-PC fractions from bovine and human retinas were isolated by HPLC and hydrolyzed. VLC-PUFA were then converted into DMOX derivatives and analyzed using GC-ESI-MS. For illustration, the mass spectra of the DMOX derivative of C32:6 (n-3) are presented in figure 3. All VLC-PUFA from human retina were characterized with precision. In samples from bovine retinas, as in those from human retinas, C30-C34 VLC-PUFA of the n-6 family had only four double bonds, while C28-C36 VLC-PUFA of the n-3 family had five or six double bonds according to previous studies (Aveldano, 1987; Suh et al., 1994). As expected, C32 and C34 VLC-PUFA with four, five and six double bonds seemed to represent the prominent VLC-PUFA in bovine as well as in human retinas (Berdeaux et al., 2010).

thumbnail Figure 3.

Mass spectra of 4,4-dimethyloxazoline (DMOX) derivatives of 32:6n-3 (Berdeaux et al., 2010). Reproduced by kind permission of Journal of Chromatography A and redrawn from the original publication.

But most of these conventional approaches are time-consuming, requiring successive extraction, chromatographic steps (HPLC, TLC) and often a derivatization step before gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) analyses.

Electrospray ionization-mass spectrometry (ESI-MS) has been described as a soft ionization technology (Kerwin et al., 1994). It is the most sophisticated and easiest technique for assessing the phospholipid content of a biological sample because of its high sensitivity and unmatched specificity. It directly analyses phospholipids as intact molecules and preserves the information based on the relative position of acyl radicals on the glycerol backbone.

McMahon and coworker developed an ESI-MS/MS method for identification of VLC-PC in retinal extracts from STGD3-knockin mice carrying a human pathogenic mutation in the ELOVL4 gene (McMahon et al., 2007). Lipids were directly analyzed by ESI-MS in the positive mode using a Q-ToF mass spectrometer. When operated In the single stage MS mode in ESI+, each PC and VLC-PC molecular specie was detected as protonated molecular ions [M+H]+. Moreover, to gain more detailed information on the PC structures, authors further analyzed retinal lipids in presence of LiCl salt. Li-adducts of PC are more extensively fragmented than are the protonated PCs, thus yielding more pertinent structural information (Hsu et al., 1998). More than twenty mass peaks were detected. Among them, 3 VLC-PC, namely PC 32:6/22:6, PC34:6/22:6 and PC36:6/22:6 were clearly characterized. When the MS spectra of retinal lipids from STGD3-heterozygous mice were compared with the spectra of their wild-type littermates, most of the corresponding peaks were similar (figure 4). The only peaks that have had their heights reduced in mutant mouse sample belonged to C32-C36 VLC-PC. These MS analyses demonstrated that the STGD3 mutation causes selective deficiency of C32-C36 acyl PCs in mouse retina.

thumbnail Figure 4.

MS analysis of retinal lipids from (A) a 1-month-old wild-type and (B) STGD3-heterozygous mouse (McMahon et al., 2007). Reproduced by kind permission of FEBS Letters and redrawn from the original publication.

LC-ESI-MS was widely used for characterization and quantification of phospholipids in different types of tissues. The use of LC prior to the ESI-MS/MS analysis enhances the detection of the minor isobaric species in the mixture. Additionally, a suitable chromatographic separation may reduce any ESI suppression of non-isobaric and co-eluting species. Recently, we have developed a LC-ESI-MS/MS method for the structural characterization and the quantification of VLC-PC molecular species in total lipid extracts from bovine and human retinas (Berdeaux et al., 2010). The total lipid extract was directly analyzed by LC-ESI-MS/MS without purification or derivatization. A good baseline separation of phospholipid classes in bovine and human retinas was achieved using normal-HPLC conditions (figure 5). Moreover VLC-PC molecular species containing VLC-PUFA eluted separately just before the other PC molecular species. Hence, it was possible to analyze directly this fraction containing only VLC-PC for structure characterization in LC-MS or to collect this fraction in order to concentrate VLC-PC for further structure characterization using GC-MS.

thumbnail Figure 5.

LC-ESI-MS normal-phase chromatogram of the lipid extract from human retina. (Berdeaux et al., 2010). Reproduced by kind permission of Journal of Chromatography A and redrawn from the original publication.

When operated in the single stage MS mode in ESI-, each PC and VLC-PC molecular specie produced an abundant demethylated molecular ions [M – CH3] used for its structural characterization and its quantification. Then, a complete structural characterization of intact PC and VLC-PC species in retina was obtained by collision-induced dissociation (CID) in the negative mode. Indeed, the MS2 experiment of the selected demethylated molecular ions [M–CH3] of PC species gave a characteristic fragmentation. Fatty acid composition and distribution could be clearly assigned based on the intensity of sn-2/sn-1 fragment ions as illustrated in Figure 6 for PC 32:4/22:6 at m/z 1006. Thus, VLC-PC species characterised in bovine and human retina were dipolyunsaturated PC species containing one VLC-PUFA (C24–C36) with three to six double bonds. Moreover, VLC-PUFA was always in the sn-1 position of VLC-PC whilst PUFA at the sn-2 position was exclusively C22:6. Therefore, the quantitative analysis of the different PC and VLC-PC molecular species was performed (figure 7) (Berdeaux et al., 2010). 36 PC and choline plasmalogens (PlsC) species were quantified in retinas from human donors. Among them, 12 VLC-PC were detected and quantified. 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. This study showed that HPLC-ESI-MS/MS method is a valuable method for a direct and precise characterization of PC molecular species containing VLC-PUFA in retina and may be useful for a better understanding of the pathogenesis of STGD3.

thumbnail Figure 6.

Product-ion spectra of negative ions of the [M – CH3] of PC 32.4/22:6 at m/z 1006 showing two abundant ions at m/z 327 and m/z 471, corresponding to C22:6 and C32:4 carboxylate anions, and the product ions formed by neutral loss of either an FA or a ketene at m/z 678 ([M – CH3 – R2CH-COOH]), m/z 696 ([M – CH3 – R2CH=C=O]), and m/z 552 ([M – CH3 – R1CH=C=O]).

thumbnail Figure 7.

Positive-ion HPLC-ESI-MS mass spectra of total PC fraction collected from human neural retina by scanning for precursors at m/z 184 in the positive mode (Berdeaux et al., 2010). Reproduced by kind permission of Journal of Chromatography A and redrawn from the original publication.

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To cite this article: Berdeaux O, Acar N. Very-long-chain polyunsaturated fatty acids in the retina: analysis and clinical relevance in physiological and pathological conditions. OCL 2011; 18(5): 284–290. doi: 10.1051/ocl.2011.0406

All Figures

thumbnail Figure 1.

Structure of a phosphatidylcholine molecule containing C34:4n-3 and docosahexaenoic acid (C22:6n-3) in the sn-1 and sn-2 positions respectively.

In the text
thumbnail Figure 2.

Electron impact mass spectra and total ion of 34:4 (n-6) and 32:6 (n-3) methyl esters (Suh et al., 1994; Agbaga et al., 2008). Reproduced by kind permission of Proceeding of the National Academy of Sciences and redrawn from the original publication.

In the text
thumbnail Figure 3.

Mass spectra of 4,4-dimethyloxazoline (DMOX) derivatives of 32:6n-3 (Berdeaux et al., 2010). Reproduced by kind permission of Journal of Chromatography A and redrawn from the original publication.

In the text
thumbnail Figure 4.

MS analysis of retinal lipids from (A) a 1-month-old wild-type and (B) STGD3-heterozygous mouse (McMahon et al., 2007). Reproduced by kind permission of FEBS Letters and redrawn from the original publication.

In the text
thumbnail Figure 5.

LC-ESI-MS normal-phase chromatogram of the lipid extract from human retina. (Berdeaux et al., 2010). Reproduced by kind permission of Journal of Chromatography A and redrawn from the original publication.

In the text
thumbnail Figure 6.

Product-ion spectra of negative ions of the [M – CH3] of PC 32.4/22:6 at m/z 1006 showing two abundant ions at m/z 327 and m/z 471, corresponding to C22:6 and C32:4 carboxylate anions, and the product ions formed by neutral loss of either an FA or a ketene at m/z 678 ([M – CH3 – R2CH-COOH]), m/z 696 ([M – CH3 – R2CH=C=O]), and m/z 552 ([M – CH3 – R1CH=C=O]).

In the text
thumbnail Figure 7.

Positive-ion HPLC-ESI-MS mass spectra of total PC fraction collected from human neural retina by scanning for precursors at m/z 184 in the positive mode (Berdeaux et al., 2010). Reproduced by kind permission of Journal of Chromatography A and redrawn from the original publication.

In the text

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