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Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors

Open AccessPublished:July 09, 2018DOI:https://doi.org/10.1194/jlr.M082495
      Long-chain PUFAs (LC-PUFAs; C20–C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C ≥28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods.
      DHA (22:6n3) and arachidonic acid (AA; 20:4n6) are the major long-chain PUFAs (LC-PUFAs) in the retina and rod outer segment (ROS) membranes of all vertebrate species examined thus far (
      • Fliesler S.J.
      • Anderson R.E.
      Chemistry and metabolism of lipids in the vertebrate retina.
      ,
      • SanGiovanni J.P.
      • Chew E.Y.
      The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina.
      ). DHA is also highly enriched in some, but not all, invertebrate retinas (
      • Anderson R.E.
      • Benolken R.M.
      • Kelleher P.A.
      • Maude M.B.
      • Wiegand R.D.
      Chemistry of photoreceptor membrane preparations from squid retinas.
      ,
      • Benolken R.M.
      • Anderson R.E.
      • Maude M.B.
      Lipid composition of Limulus photoreceptor membranes.
      ). Both DHA and AA are obtained either from the diet or synthesized from dietary essential fatty acids [α-linolenic acid (18:3n3) and linoleic acid (18:2n6), respectively]. DHA is essential for normal development of rod function in the rat retina, first demonstrated by our laboratory in 1973 and 1975 (
      • Benolken R.M.
      • Anderson R.E.
      • Wheeler T.G.
      Membrane fatty acids associated with the electrical response in visual excitation.
      ,
      • Wheeler T.G.
      • Benolken R.M.
      • Anderson R.E.
      Visual membranes: specificity of fatty acid precursors for the electrical response to illumination.
      ). In these studies, rats deficient in PUFAs had reduced electroretinographic responses (
      • Benolken R.M.
      • Anderson R.E.
      • Wheeler T.G.
      Membrane fatty acids associated with the electrical response in visual excitation.
      ). Feeding diets containing only n3 or n6 PUFAs restored visual function, with the greatest effect coming from n3 PUFA supplementation (
      • Wheeler T.G.
      • Benolken R.M.
      • Anderson R.E.
      Visual membranes: specificity of fatty acid precursors for the electrical response to illumination.
      ). Later studies in primates by Neuringer and colleagues confirmed the essentiality of DHA for development of rod visual function (
      • Neuringer M.
      • Connor W.E.
      • Van Petten C.
      • Barstad L.
      Dietary omega-3 fatty acid deficiency and visual loss in infant rhesus monkeys.
      ,
      • Neuringer M.
      • Reisbick S.
      • Janowsky J.
      The role of n-3 fatty acids in visual and cognitive development: current evidence and methods of assessment.
      ). It was subsequently discovered that visual function in preterm human infants was significantly improved in the early developmental state by supplementing their diets with fish oil (
      • O'Connor D.L.
      • Hall R.
      • Adamkin D.
      • Auestad N.
      • Castillo M.
      • Connor W.E.
      • Connor S.L.
      • Fitzgerald K.
      • Groh-Wargo S.
      • Hartmann E.E.
      • et al.
      Growth and development in preterm infants fed long-chain polyunsaturated fatty acids: a prospective, randomized controlled trial.
      ), which contains DHA and its precursor, EPA (20:5n3). Thus, it is now accepted that DHA and AA are essential for normal brain and retina development. Both are components of breast milk and both are now included in most infant formulas and prenatal supplements.
      In 1987, Avendaño and Sprecher reported the presence in bovine ROSs of n3 and n6 PUFAs containing up to 38 carbons, esterified exclusively to the sn-1 position of phosphatidylcholine, with DHA on the sn-2 position (
      • Aveldaño M.I.
      • Sprecher H.
      Very long chain (C24 to C36) polyenoic fatty acids of the n-3 and n-6 series in dipolyunsaturated phosphatidylcholines from bovine retina.
      ,
      • Aveldaño M.I.
      A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina.
      ). No specific function or importance was assigned to these very-long-chain PUFAs (VLC-PUFAs; ≥C28). In 2001, three groups reported that mutations in exon 6 of a gene named elongation of very-long-chain fatty acids-4 (ELOVL4) are the cause of autosomal dominant Stargardt-like macular dystrophy (STGD3) (
      • Bernstein P.S.
      • Tammur J.
      • Singh N.
      • Hutchinson A.
      • Dixon M.
      • Pappas C.M.
      • Zabriskie N.A.
      • Zhang K.
      • Petrukhin K.
      • Leppert M.
      • et al.
      Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.
      ,
      • Edwards A.O.
      • Donoso L.A.
      • Ritter III, R.
      A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
      ,
      • Zhang X.M.
      • Yang Z.
      • Karan G.
      • Hashimoto T.
      • Baehr W.
      • Yang X.J.
      • Zhang K.
      Elovl4 mRNA distribution in the developing mouse retina and phylogenetic conservation of Elovl4 genes.
      ). The gene was so named because it shared sequence homology with a family of yeast enzymes named ELOVL, which catalyze the rate-limiting condensation reaction in the addition of 2-carbon units to existing fatty acids to produce even longer chain saturated fatty acids and PUFAs (
      • Dittrich F.
      • Zajonc D.
      • Huhne K.
      • Hoja U.
      • Ekici A.
      • Greiner E.
      • Klein H.
      • Hofmann J.
      • Bessoule J.J.
      • Sperling P.
      • et al.
      Fatty acid elongation in yeast–biochemical characteristics of the enzyme system and isolation of elongation-defective mutants.
      ,
      • Leonard A.E.
      • Pereira S.L.
      • Sprecher H.
      • Huang Y.S.
      Elongation of long-chain fatty acids.
      ). Our laboratory cloned and expressed ELOVL4 and identified its products as saturated fatty acids and PUFAs of 28 carbons or greater in length (
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Henry K.
      • Elliott M.H.
      • Anderson R.E.
      Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
      ,
      • Yu M.
      • Benham A.
      • Logan S.
      • Brush R.S.
      • Mandal M.N.
      • Anderson R.E.
      • Agbaga M.P.
      ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3.
      ). We subsequently showed that the STGD3 mutant protein did not have enzymatic activity and exerted a dominant negative effect on the activity of the WT enzyme in vitro (
      • Logan S.
      • Agbaga M.P.
      • Chan M.D.
      • Brush R.S.
      • Anderson R.E.
      Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity.
      ,
      • Logan S.
      • Agbaga M.P.
      • Chan M.D.
      • Kabir N.
      • Mandal N.A.
      • Brush R.S.
      • Anderson R.E.
      Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.
      ), consistent with reports that the mutant formed dimers with the WT protein (
      • Grayson C.
      • Molday R.S.
      Dominant negative mechanism underlies autosomal dominant Stargardt-like macular dystrophy linked to mutations in ELOVL4.
      ,
      • Ambasudhan R.
      • Wang X.
      • Jablonski M.M.
      • Thompson D.A.
      • Lagali P.S.
      • Wong P.W.
      • Sieving P.A.
      • Ayyagari R.
      Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein.
      ,
      • Karan G.
      • Yang Z.
      • Howes K.
      • Zhao Y.
      • Chen Y.
      • Cameron D.J.
      • Lin Y.
      • Pearson E.
      • Zhang K.
      Loss of ER retention and sequestration of the wild-type ELOVL4 by Stargardt disease dominant negative mutants.
      ).
      STGD3 is a juvenile-onset macular dystrophy that primarily affects the cones in the macula of the retina, starting as early as the first decade of life (
      • Donoso L.A.
      • Edwards A.O.
      • Frost A.
      • Vrabec T.
      • Stone E.M.
      • Hageman G.S.
      • Perski T.
      Autosomal dominant Stargardt-like macular dystrophy.
      ,
      • Edwards A.O.
      • Miedziak A.
      • Vrabec T.
      • Verhoeven J.
      • Acott T.S.
      • Weleber R.G.
      • Donoso L.A.
      Autosomal dominant Stargardt-like macular dystrophy: I. Clinical characterization, longitudinal follow-up, and evidence for a common ancestry in families linked to chromosome 6q14.
      ). Because the mutation is in an enzyme that utilizes LC-PUFA precursors to make VLC-PUFAs, there is considerable interest in knowing the size and nature of the precursor pool in human cones and the VLC-PUFA products generated from them. Van Kuijk and Buck (
      • van Kuijk F.J.
      • Buck P.
      Fatty acid composition of the human macula and peripheral retina.
      ) were the first to compare the fatty acid compositions of peripheral and macular regions of the human retina, and found that the macula had less DHA than the periphery. Their analyses did not include VLC-PUFAs. More recently, Bernstein's group showed that while there were no age-related changes in DHA in total lipids from whole human retinas, there was a significant reduction in DHA in retinas from age-matched patients with age-related macular degeneration (AMD) (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ). Interestingly, there was no change in AA in these same retinas. The Bernstein group also reported significantly lower VLC-PUFAs in the whole retina lipids compared with age-matched control values (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ). In a later study, using GC-MS technology to analyze the lipids in 4 mm trephine retinal punches, the Bernstein group found that human macula lipids contained less VLC-PUFAs than peripheral lipids, although the levels of DHA were the same (
      • Liu A.
      • Terry R.
      • Lin Y.
      • Nelson K.
      • Bernstein P.S.
      Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina.
      ). An interesting finding from the Bernstein study was that mouse retinas contain almost 10 times the level of VLC-PUFAs and almost twice the level of DHA as do normal human retinas.
      Given the differences in LC-PUFAs and VLC-PUFAs reported for peripheral versus central normal human retinas (
      • van Kuijk F.J.
      • Buck P.
      Fatty acid composition of the human macula and peripheral retina.
      ,
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ), we undertook a systematic comparative analysis of lipids from several species of rodents whose retinas either contained a preponderance of cone photoreceptors or had been genetically manipulated to express cone-like photoreceptors. These rodents included mice with a global knockout of the neural leucine zipper transcription (Nrl), which results in a nocturnal retina containing entirely cone-like photoreceptors and no rod photoreceptors, as previously characterized (
      • Mears A.J.
      • Kondo M.
      • Swain P.K.
      • Takada Y.
      • Bush R.A.
      • Saunders T.L.
      • Sieving P.A.
      • Swaroop A.
      Nrl is required for rod photoreceptor development.
      ,
      • Zhu X.
      • Brown B.
      • Li A.
      • Mears A.J.
      • Swaroop A.
      • Craft C.M.
      GRK1-dependent phosphorylation of S and M opsins and their binding to cone arrestin during cone phototransduction in the mouse retina.
      ,
      • Daniele L.L.
      • Lillo C.
      • Lyubarsky A.L.
      • Nikonov S.S.
      • Philp N.
      • Mears A.J.
      • Swaroop A.
      • Williams D.S.
      • Pugh Jr., E.N.
      Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse.
      ,
      • Nikonov S.S.
      • Daniele L.L.
      • Zhu X.
      • Craft C.M.
      • Swaroop A.
      • Pugh Jr., E.N.
      Photoreceptors of Nrl −/− mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.
      ). Thirteen-lined ground squirrels (TLGSs) (Ictidomys tridecemlineatus), tree squirrels (Sciurus niger), and tree shrews (Tupaia belangeri) are visually guided cone-dominant diurnal mammals with photoreceptor distributions similar to the cone-rich foveal region within the primate macula (
      • Wässle H.
      • Riemann H.J.
      The mosaic of nerve cells in the mammalian retina.
      ). We performed comprehensive glycerophospholipid analyses on these rod- and cone-dominant retinas to define and compare their lipid compositions, which may account for the differences in their function. Here, we show that outer segment (OS) membranes from cone-dominant retinas have much lower levels of PUFAs and VLC-PUFAs than do rod-dominant retinas.

      MATERIALS AND METHODS

      Animal care and preparation of OS membranes

      Nrl−/− mice were generously provided by Anand Swaroop, PhD (National Eye Institute, Bethesda, MD) and were housed and bred at the Dean McGee Eye Institute and the University of Oklahoma Animal Research Facility vivarium on 12 h light:12 h dark cyclic light. The Nrl−/− mice were bred with C57BL6/J mice purchased from Jackson Laboratories (Bar Harbor, ME) to generate Nrl+/− and WT C57BL6/J controls. Because the retinas of Nrl−/− mice undergo age-related retinal structural changes by about three months of age (
      • Mears A.J.
      • Kondo M.
      • Swain P.K.
      • Takada Y.
      • Bush R.A.
      • Saunders T.L.
      • Sieving P.A.
      • Swaroop A.
      Nrl is required for rod photoreceptor development.
      ,
      • Stuck M.W.
      • Conley S.M.
      • Naash M.I.
      Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina.
      ), our studies were carried out in 1-month-old WT, Nrl+/−, and Nrl−/− animals. Sprague Dawley rats were raised at the Dean McGee Eye Institute vivarium and TLGSs (I. tridecemlineatus) were raised at the University of Wisconsin Oshkosh (Oshkosh, WI). Tree squirrels (S. niger) were obtained from local licensed trappers. Tree shrews were bred and raised at the Max Planck Florida Institute, Jupiter, FL, by Rebekah Corlew, PhD, who generously provided us with retinal tissues. All animals were handled and euthanized according to the guidelines outlined in the Statement for the Use of Animals in Ophthalmic and Vision Research, and the protocols were approved by the University of Oklahoma Health Sciences Center IACUC.
      Prior to preparation of OS membranes, animals were dark-adapted overnight. Retinas were dissected under dim red light, flash-frozen in liquid nitrogen, and stored at −80°C until they were used. Cone OSs (COSs) or ROSs, collectively referred to as photoreceptor OSs, were prepared by discontinuous sucrose gradient ultracentrifugation according to protocols previously published (
      • Martin R.E.
      • Elliott M.H.
      • Brush R.S.
      • Anderson R.E.
      Detailed characterization of the lipid composition of detergent-resistant membranes from photoreceptor rod outer segment membranes.
      ,
      • Martin R.E.
      • Fliesler S.J.
      • Brush R.S.
      • Richards M.J.
      • Hopkins S.A.
      • Anderson R.E.
      Lipid differences in rod outer segment membranes of rats with P23H and S334ter opsin mutations.
      ). Two interfaces were collected: the top band containing OS-enriched membranes (band I), the second band (band II) containing mostly broken rods and mitochondria, and the pellet at the bottom of the tube containing nuclei, melanin granules, and other non-OS membranes. The three preparations were washed with Krebs-Ringer phosphate buffer (pH 7.4) and the membranes were pelleted by centrifugation at 45,145 g for 10 min (
      • Bicknell I.R.
      • Darrow R.
      • Barsalou L.
      • Fliesler S.J.
      • Organisciak D.T.
      Alterations in retinal rod outer segment fatty acids and light-damage susceptibility in P23H rats.
      ). All procedures were carried out at 4°C under ambient room light.

      Retinal OS membrane preparation and validation

      An aliquot of membranes from each fraction was taken for protein determination by quantitative BCA assay in order to use equal amounts of membrane fractions for lipid and fatty acid analyses. For Western blotting, the membranes were lysed in a lysis buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, EDTA-free protease inhibitor tablets (Roche Applied Sciences, Indianapolis, IN), and 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO). After brief sonication on ice, the lysates were incubated with gentle rocking at 4°C for 1 h, and centrifuged at 27,000 g for 30 min at 4°C. Supernatants were collected and protein concentrations determined using the Pierce BCA assay (Thermo Fisher Scientific, Rockford, IL). Equal amounts of protein (5 μg) were separated by SDS/PAGE on 12% polyacrylamide gels, followed by either silver staining or electro-transfer to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk and incubated with rhodopsin antibodies 1D4 (1:3,000), a gift from Dr. James McGinnis (Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK). Membranes were stripped and reprobed with M- and S-opsin antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX) at 1:1,000, and then with monoclonal antibody against β-actin (Sigma-Aldrich). Immunoreaction of horseradish peroxidase-conjugated donkey anti-rabbit or goat anti-mouse IgG secondary antibodies (GE Healthcare Bio-Sciences, Pittsburgh, PA) was detected using Super-Signal West Dura extended duration substrate (Thermo Fisher Scientific).

      Lipid extraction and fatty acid analysis

      Total lipids were extracted from 100 μg protein of the three retinal membrane fractions, 100 μl of plasma, or whole retinas following the method of Bligh and Dyer (
      • Bligh E.G.
      • Dyer W.J.
      A rapid method of total lipid extraction and purification.
      ) with minor modifications (
      • Martin R.E.
      • Elliott M.H.
      • Brush R.S.
      • Anderson R.E.
      Detailed characterization of the lipid composition of detergent-resistant membranes from photoreceptor rod outer segment membranes.
      ). The purified lipid extracts were stored under nitrogen until use. To these lipid extracts, 50 nmol each of 15:0, 17:0, and 23:0, and 4 nmol of 30:3n6 were added as internal standards. One milliliter of 16.6% concentrated HCl in methanol was then added. The tubes were sealed under N2 with Teflon-lined caps and heated at 100°C overnight. The tubes were cooled on ice and fatty acid methyl esters (FAMEs) were extracted and processed as previously described (
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Henry K.
      • Elliott M.H.
      • Anderson R.E.
      Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
      ). All reagents for fatty acid extraction and derivatization were of the highest quality available from Sigma-Aldrich.
      FAMEs were identified using an Agilent Technologies 7890A gas chromatograph with a 5975C inert XL mass spectrometer detector (Agilent Technologies, Lexington, MA). The gas chromatograph-mass spectrometer was operated in the electron impact total ion and single ion monitoring modes. The injection volume was 1 μl and the inlet, held at 280°C, was set to pulsed splitless mode. An Agilent Technologies DB-23 column (60 m × 0.32 mm × 0.25 μm) was used with a helium carrier gas flow rate of 1.9 ml/min. The oven temperature began at 130°C for 1.0 min, was ramped to 170°C at 6.8°C/min, and was then ramped to 215°C at 2.9°C/min. After holding at 215°C for 11.4 min, the oven was ramped to 230°C at 42°C/min and held for 9.6 min. The oven was then ramped to 290°C at 10°C/min and held for 14.4 min. The mass spectrometer transfer line, ion source, and quadrupole temperatures were 290°C, 230°C, and 150°C, respectively. The PUFAs were identified by using the m/z 79.1, 108.1, and 150.1 in single ion monitoring mode and the full scan mass spectra in total ion mode.
      FAMEs were quantified using an Agilent Technologies 6890N gas chromatograph with flame ionization detector. Sample concentrations were determined by comparison to internal standards 15:0, 17:0, 23:0, and 30:3n6. The injection volume was 1 μl and the inlet, held at 280°C, was set to pulsed split mode (10:1 ratio). An Agilent Technologies DB-23 column (60 m × 0.32 mm × 0.25 μm) was used with a hydrogen carrier gas constant pressure of 13.1 psi. The oven temperature began at 130°C for 0.8 min, was ramped to 170°C at 8.2°C/min, and was then ramped to 215°C at 3.5°C/min. After holding at 215°C for 9.5 min, the oven was ramped to 230°C at 50°C/min, and was then held for 8 min. The oven was then ramped to 290°C at 12.0°C/min and was held for 12 min. The detector was held at 290°C.

      Lipid extraction and glycerophospholipid analysis

      The method used for MS/MS analysis of retinal lipids has been described previously (
      • Busik J.V.
      • Reid G.E.
      • Lydic T.A.
      Global analysis of retina lipids by complementary precursor ion and neutral loss mode tandem mass spectrometry.
      ) and was used with slight modification. Briefly, tissue was homogenized in 40% aqueous methanol and diluted 1:40 with 2-propanol/methanol/chloroform (4:2:1 v/v/v) containing 20 mM ammonium formate and 1.0 μmol each of glycerophosphatidylcholine (PC) 14:0/14:0 and glycerophosphatidylethanolamine (PE) 14:0/14:0, 0.33 μmol of glycerophos­phatidylserine (PS) 14:0/14:0, and 12.5 nmol of d18:1/12:0 ceramide as internal standards. Samples were introduced into an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) operating at a resolution of 100,000 (at m/z 400) using a chip-based nano-ESI source (Advion NanoMate, Ithaca, NY) operating in infusion mode. PC, PE, and PS molecular species were determined by high-resolution MS and MS/MS using accurate mass measurements in positive ion mode (
      • Lydic T.A.
      • Busik J.V.
      • Reid G.E.
      A monophasic extraction strategy for the simultaneous lipidome analysis of polar and nonpolar retina lipids.
      ). Quantification of lipid molecular species was performed using the Lipid Mass Spectrum Analysis (LIMSA) software's peak model fit algorithm (
      • Haimi P.
      • Chaithanya K.
      • Kainu V.
      • Hermansson M.
      • Somerharju P.
      Instrument-independent software tools for the analysis of MS-MS and LC-MS lipidomics data.
      ,
      • Haimi P.
      • Uphoff A.
      • Hermansson M.
      • Somerharju P.
      Software tools for analysis of mass spectrometric lipidome data.
      ,
      • Song H.
      • Ladenson J.
      • Turk J.
      Algorithms for automatic processing of data from mass spectrometric analyses of lipids.
      ).

      Statistical analysis

      Statistical analyses were performed using GraphPad Prism 5 software. Results are expressed as the mean ± SD. All relative mole percentages of n3 and n6 LC-PUFAs and VLC-PUFAs were from total lipids extracted from sample homogenates equivalent to 100 µg or 2.0 mg of protein. For all figures, significant differences are indicated by P < 0.05, while n.s. indicates no significant difference at P > 0.05, using a multivariate ANOVA with Neuman-Keuls posthoc test or Bonferroni's multiple comparisons test.

      RESULTS

      Enrichment and purity of OS membrane preparations

      To determine the enrichment and purity of OS membrane preparations, equal amounts of protein of OS membranes from WT mouse (C57B6/J), Nrl+/− mouse, Nrl−/− mouse, rat, and TLGS retinas were resolved on SDS-PAGE, as described by Laemmli (
      • Laemmli U.K.
      Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
      ), and either stained with silver nitrate or immunoblotted with anti-opsin, anti-rod transducin, anti-M-opsin, or anti-actin antibodies (Fig. 1A, B). Band I, representing enriched OS membranes from WT and Nrl+/− retinas, showed prominent staining in the regions generally accepted for rhodopsin and rhodopsin polymers. This staining was absent in the Nrl−/− OS membranes. Similar staining patterns were found for band II, which represented broken OS and inner segment membranes, although the amounts of rhodopsin were less. There was very little staining for rhodopsin in the pellet membranes. M-opsin was present in the OS membranes from the three groups of mice, showing that all three had the visual pigment found in mouse cones. Rhodopsin and rod transducin were found only in OS preparations from WT and Nrl+/− retinas, indicating that the Nrl−/− OS membranes did not contain these two prominent components of ROSs. OS preparations from TLGS retinas were also examined by Western blot (Fig. 1B). We did not detect significant rhodopsin labeling from the all-cone TLGS OS membranes compared with the rat ROS membranes, which was expected due to the cone-dominant nature of the TLGS retina. However, it is possible that the 1D4 antibody does not recognize TLGS rhodopsin. Rat ROSs also contained a small amount of M-opsin, which was present along with S-opsin in OS membranes from TLGS.
      Figure thumbnail gr1
      Fig. 1Determination of purity of prepared retinal OS membranes by immunoblotting. A, B: Retinal OS membranes were prepared by discontinuous sucrose gradient centrifugation and analyzed by silver nitrate staining and Western blotting for OS purity using photoreceptor-specific antibodies. Opsin and transducin immunoblotting assays confirmed enrichment of OS membranes in band I OS membranes relative to band II and pellet fractions.

      Characterization and quantification of fatty acids in cone-dominant versus rod-dominant retinas

      We determined the fatty acid composition of whole retina lipids from rod-dominant WT and Nrl+/− mice, relative to cone-dominant retinas of Nrl−/− mice and TLGSs. To our surprise, whole retinal fatty acids from Nrl−/− mice and TLGS yielded 23.6 and 15.2 relative mole percent of DHA (22:6n3), respectively, which is significantly lower than the 34% 22:6n3 content of WT and Nrl+/− retinas (Fig. 2). The reduced 22:6n3 in cone-dominant retinas was accompanied by a relative increase in stearic acid (18:0), oleic acid (18:1), and AA (20:4n6) (Fig. 2). ROS membranes are known to be enriched in 22:6n3, which has been shown to modulate rod phototransduction processes. Hence, we analyzed FAMEs prepared from equal amounts of retinal OS membranes from WT, Nrl+/−, Nrl−/−, and TLGSs (Fig. 3). Consistent with the whole-retina results (Fig. 2), 22:6n3 was the most abundant fatty acid in WT and Nrl+/− OS membranes (Fig. 3), which showed no statistically significant differences in their fatty acid composition. However, OS membranes from the Nrl−/− retinas had significantly lower levels of 22:6n3, with compensatory increases in 18:0, 18:1, and 20:4n6. The OS membranes from the cone-dominant TLGS retinas had even lower 22:6n3 levels than did the Nrl−/− OS membranes (Fig. 3). Compared with the rod-dominant retinas, the cone-dominant retinas also had slightly more adrenic acid (22:4n6) without any significant enrichment in n6 docosapentaenoic acid (22:5n6), which is usually increased in pathological n3 fatty acid deficiency (
      • Salem Jr., N.
      • Loewke J.
      • Catalan J.N.
      • Majchrzak S.
      • Moriguchi T.
      Incomplete replacement of docosahexaenoic acid by n-6 docosapentaenoic acid in the rat retina after an n-3 fatty acid deficient diet.
      ,
      • Levant B.
      • Radel J.D.
      • Carlson S.E.
      Reduced brain DHA content after a single reproductive cycle in female rats fed a diet deficient in N-3 polyunsaturated fatty acids.
      ,
      • Carlson S.E.
      • Carver J.D.
      • House S.G.
      High fat diets varying in ratios of polyunsaturated to saturated fatty acid and linoleic to linolenic acid: a comparison of rat neural and red cell membrane phospholipids.
      ,
      • Wiegand R.D.
      • Koutz C.A.
      • Stinson A.M.
      • Anderson R.E.
      Conservation of docosahexaenoic acid in rod outer segments of rat retina during n-3 and n-6 fatty acid deficiency.
      ,
      • Delton-Vandenbroucke I.
      • Maude M.B.
      • Chen H.
      • Aguirre G.D.
      • Acland G.M.
      • Anderson R.E.
      Effect of diet on the fatty acid and molecular species composition of dog retina phospholipids.
      ). This further confirmed our findings that cone photoreceptors indeed have a different lipid profile from rod photoreceptors, and this difference is not an artifact of captivity.
      Figure thumbnail gr2
      Fig. 2Quantification of PUFAs in cone-dominant Nrl−/− and TLGS retinas compared with rod-dominant Nrl+/− and WT mouse retinas. Cone-dominant Nrl−/− and TLGS whole retinas have significantly reduced 22:6n3 compared with rod-dominant Nrl+/− and WT mice. The decreased 22:6n3 is associated with increased 18:0, 18:1, and 20:4n6 in the cone-dominant retinas. However, there is no significant increase in 22:5n6 as occurs in n3 PUFA deficiency. Statistically significant differences are indicated as: BWT versus Nrl−/−; CWT versus TLGS; DNrl+/− versus Nrl−/−; ENrl+/− versus TLGS; and FNrl−/− versus TLGS for P < 0.05 to P < 0.001. Error bars represent mean ± SD.
      Figure thumbnail gr3
      Fig. 3The 22:6n3 in Nrl−/− and TLGS OS membranes compared with Nrl+/− and WT OS membranes. Relative mole percent of fatty acids from Nrl−/− and TLGS OS membranes show significantly reduced 22:6n3 levels, but increased 18:0, 18:1, and 20:4n6 without significantly increased 22:5n6 levels, which is consistent with decreased 22:6n3 in whole-retina fatty acids. Statistically significant differences are indicated as: BWT versus Nrl−/−; CWT versus TLGS; DNrl+/− versus Nrl−/−; ENrl+/− versus TLGS; and FNrl−/− versus TLGS; P < 0.05 to P < 0.001; mean ± SD.

      Composition of retinal VLC-PUFAs in rod-dominant versus cone-dominant retinas

      Having identified differences in LC-PUFAs, we next compared the relative composition of VLC-PUFAs in rod-dominant retinas to that of cone-dominant retinas. In total, the sum of VLC-PUFAs was significantly less in Nrl−/− retinas when compared with whole retinas from WT and Nrl+/− mice (Fig. 4). The major VLC-PUFAs present in the mouse retina membranes included 32:6n3, 34:5n3, 34:6n3, and 36:6n3, all of which were similar in WT and Nrl+/− retinas, but significantly lower in Nrl−/− retinas, with the exception of 36:6n3 and 36:5n3 (Fig. 4). Because VLC-PUFAs are elongated products of C20–C26 n3 and n6 PUFAs, reduced levels of these PUFAs in the retina are thus consistent with lower levels of VLC-PUFAs. To determine whether reduced levels of 22:6n3 in the TLGSs are intrinsic to cone photoreceptors, or whether they are due to influence from laboratory diets, we also analyzed the fatty acid composition of total retinas and OS membrane preparations collected directly from wild TLGSs caught near the University of Wisconsin, Oshkosh. We found that the relative mole percent of 22:6n3 in the TLGS retinas, whether captive-bred or wild-caught, was 12–14% compared with 33–35% of 22:6n3 in Nrl+/− and WT mouse retinas. This suggests that reduced levels of retinal 22:6n3 and VLC-PUFAs in cone-dominant retinas are a function of cone retinal physiology and metabolism, rather than reduced amounts of plasma VLC-PUFA precursors.
      Figure thumbnail gr4
      Fig. 4Quantification of retinal VLC-PUFAs in Nrl−/− retina compared with Nrl+/− and WT retina. Relative mole percent of whole retinal VLC-PUFAs is significantly decreased in Nrl−/− mice compared with Nrl+/− and WT mice. The major VLC-PUFAs in the Nrl+/− and WT retina include 32:6n3, 34:5n3, and 34:6n3, which are reduced in the Nrl−/− retina. However, the Nrl−/− retina is enriched in 36:5n3 and 36:6n3. Statistically significant differences are indicated as: BWT versus Nrl−/−; and DNrl+/− versus Nrl−/−; P < 0.05 to P < 0.001; mean ± SD.

      Glycerophospholipid analysis and fatty acid composition of cone-dominant retinas

      We next determined the molecular species composition of the three major phospholipids, PC, PE, and PS, in rod- and cone-dominant retinas. We compared nocturnal rod-dominant mouse and rat retinas with those of diurnal cone-dominant TLGSs, tree squirrels, and tree shrews using MS/MS. Within the mouse strains (WT, Nrl+/−, and Nrl−/−), there was a significant reduction between WT and Nrl−/− mice in PC, PE, and PS species containing two molecules of 22:6n3 (PC-, PE-, PS-22:6/22:6), while there was no difference between the WT and Nrl+/− retinas (Figs. 5, 6A, B; 7A, B). These findings support the fatty acid data that indicate reduced levels of 22:6n3 in cone-dominant Nrl−/− mice. Within retinal dipoly-22:6n3-PC and VLC-PUFA-PC species, we observed a significant reduction in PC-44:12 (22:6/22:6), PC-54:12 (32:6/22:6), PC-56:12 (34:6/22:6), and PC-56:11 (34:5/22:6) in Nrl−/− retinas compared with WT and Nrl+/− retinas (Fig. 5). PC-56:12 (34:6/22:6) is the most abundant VLC-PUFA-PC in the mouse retina, followed by PC-54:12 (32:6/22:6), PC-58:12 (36:6/22:6), and PC-56:11(34:5/22:6). We further confirmed these results in OS preparations in which we found a significant reduction in 32:6/22:6, 34:5/22:6, and 34:6/22:6 PC in Nrl−/− versus WT and Nrl+/−. Interestingly, there was no difference in PC-58:12 (36:6/22:6) between the three genotypes. Collectively, however, there was a significant reduction in the sum of retinal VLC-PUFA-PC in Nrl−/− mice compared with WT and Nrl+/− mice (Fig. 5). To determine whether the decreased 22:6n3/22:6n3 in PC could be accounted for in the other glycerophospholipid species, we analyzed the composition of retinal PE (Fig. 6A, B) and PS (Fig. 7A, B). Similar to the PC data, both PE and PS species had decreased levels of 22:6/22:6 in the cone-dominant retina compared with the WT and Nrl+/− retinas (Figs. 6A, B; 7A, B). The PE and PS species did not contain any VLC-PUFAs.
      Figure thumbnail gr5
      Fig. 5Comparison of di-poly-DHA-PC and VLC-PUFA-PC in Nrl−/−, Nrl+/−, and WT retina. Consistent with decreased retinal DHA and VLC-PUFAs, di-poly-DHA-PC and VLC-PUFA-PC and the sum of retinal VLC-PUFA-PC are significantly decreased in Nrl−/− retina relative to Nrl+/− and WT retinal VLC-PUFA-PC. The most abundant VLC-PUFA-PC species in the Nrl+/− and WT retina include 32:6/22:6, 34:5/22:6, 34:6/22:6, and 36:6/22:6. Statistically significant differences are indicated as: AWT versus Nrl+/−; BWT versus Nrl−/−; and DNrl+/− versus Nrl−/−; P < 0.05 to P < 0.001; mean ± SD.
      Figure thumbnail gr6
      Fig. 6Retinal LC-PUFA-PE levels in rod-dominant retinas compared with cone-dominant retinas. The most abundant LC-PUFA-PE in both rod- and cone-rich retinas was 18:0/22:6 (A, B). The 22:6/22:6-PUFA-PE is significantly decreased in cone-dominant retinas compared with rod-dominant WT and Nrl+/− retinas (A). However, among the cone-rich retinas, there was no significant difference in 22:6/22:6-PUFA-PE levels. No VLC-PUFA-PEs were detected. Statistically significant differences are indicated as: AWT versus Nrl+/−; BWT versus Nrl−/− DNrl+/− versus Nrl−/− FNrl−/− versus TLGS; GNrl−/− versus tree squirrel; HNrl−/− versus tree shrew; Itree squirrel versus TLGS; Jtree squirrel versus tree shrew; and KTLGS versus tree shrew; P < 0.05 to P < 0.001; mean ± SD.
      Figure thumbnail gr7
      Fig. 7Retinal PUFA-PS species in rod-dominant retinas compared with cone-dominant retinas. The 18:0/22:6-PS presents the major PS in both rod- and cone-rich retinas (A, B). Consistent with PC and PE data, 22:6/22:6-PS is significantly decreased in the cone-dominant retinas compared with rod-dominant WT and Nrl+/− retinas, without any differences detected in the cone-rich retina, except for Nrl−/− and TLGS that were significantly different at P < 0.05. Statistically significant differences are indicated as: BWT versus Nrl−/−; DNrl+/− versus Nrl−/−; FNrl−/− versus TLGS; GNrl−/− versus tree squirrel; HNrl−/− versus tree shrew; Itree squirrel versus TLGS; Jtree squirrel versus tree shrew; and KTLGS versus tree shrew; P < 0.05 to P < 0.001; mean ± SD.
      Based on the finding that cone-dominant Nrl−/− retinas had significantly lower levels of PC-44:12, PE-44:12, PS-44:12, PS-46:12 (Figs. 5, 6, 7), and VLC-PUFA-PC (Fig. 5) compared with WT and Nrl+/− retinas, we determined the levels of these molecular species in retinas from Sprague-Dawley rats, TLGSs, tree squirrels, and tree shrews. Between these four species, as well as Nrl−/−, there were no significant differences in levels of PC-32:0 (16:0/16:0), PC-34:01 (16:0/18:1), PC-36:04 (16:0/20:4), PC-36:01 (18:0/18:1), and PC-38:04 (18:0/20:4) (data not shown). This finding suggests that, biochemically, cone-dominant retinas have reduced levels of 22:6n3 diacyl-glycerophospholipids. Similarly, there was a significant decrease in PE-44:12 and PS-44:12 in the TLGS, tree squirrel, and tree shrew retinas (Figs. 6B, 7B) when compared with rod-dominant rat retinas (Figs. 6A, 7A). Furthermore, within the cone-dominant retinas, VLC-PUFA-PC classes were significantly reduced compared with rat retina (Fig. 8). While PC-54:12 and PC-56:12 were enriched in the rat retinas, they were significantly reduced in the squirrel retinas, with the tree shrew retinas having higher levels than the squirrel retinas (Fig. 8). Similarly, PC-54:11, PC-54:10, and PC-56:11 were enriched in rat retinas compared with cone-dominant retinas (Fig. 8). Consistent with the mouse retina data, PC-58:12 was enriched in the cone-dominant retinas (Fig. 8).
      Figure thumbnail gr8
      Fig. 8Comparison of retinal VLC-PUFA-PC in cone-dominant versus rod-dominant retinas. Compared with the rod-dominant rat retinas (n = 4), the cone-dominant retinas of Nrl−/− mice (n = 5), tree squirrels (n = 3), TLGSs (n = 4), and tree shrews (n = 6) all have significantly less VLC-PUFA-PC. Tree shrew retinas have much higher 34:6/22:6 and 36:6/22:6 compared with the other cone-dominant species. Statistically significant differences are indicated as: FNrl−/− versus TLGS; GNrl−/− versus tree squirrel; HNrl−/− versus tree shrew; Itree squirrel versus TLGS; Jtree squirrel versus tree shrew; KTLGS versus tree shrew; and LSprague-Dawley rats (SD rats) versus (Nrl−/−, tree squirrel, TLGS, tree shrew); P < 0.05 to P < 0.001; mean ± SD.

      DISCUSSION

      Increasing life expectancy has brought in its wake increasing numbers of people with various aging diseases, including vision loss due to AMD, diabetic retinopathy, glaucoma, and cataracts. Multiple experimental, epidemiological, and clinical trial results have indicated that dietary n3 PUFAs might attenuate AMD progression (
      • Cho E.
      • Hung S.
      • Willett W.C.
      • Spiegelman D.
      • Rimm E.B.
      • Seddon J.M.
      • Colditz G.A.
      • Hankinson S.E.
      Prospective study of dietary fat and the risk of age-related macular degeneration.
      ,
      • Seddon J.M.
      • Cote J.
      • Rosner B.
      Progression of age-related macular degeneration: association with dietary fat, transunsaturated fat, nuts, and fish intake.
      ,
      • Seddon J.M.
      • George S.
      • Rosner B.
      Cigarette smoking, fish consumption, omega-3 fatty acid intake, and associations with age-related macular degeneration: the US Twin Study of Age-Related Macular Degeneration.
      ,
      • Seddon J.M.
      • Rosner B.
      • Sperduto R.D.
      • Yannuzzi L.
      • Haller J.A.
      • Blair N.P.
      • Willett W.
      Dietary fat and risk for advanced age-related macular degeneration.
      ,
      • SanGiovanni J.P.
      • Chew E.Y.
      • Agron E.
      • Clemons T.E.
      • Ferris III, F.L.
      • Gensler G.
      • Lindblad A.S.
      • Milton R.C.
      • Seddon J.M.
      • Klein R.
      • Age-Related Eye Disease Study Research Group
      • et al.
      The relationship of dietary omega-3 long-chain polyunsaturated fatty acid intake with incident age-related macular degeneration: AREDS report no. 23.
      ,

      SanGiovanni, J. P., ChewE. Y., ClemonsT. E., DavisM. D., FerrisF. L. III, GenslerG. R., KurinijN., LindbladA. S., MiltonR. C., SeddonJ. M., et al.; Age-Related Eye Disease Study Research Group. 2007. The relationship of dietary lipid intake and age-related macular degeneration in a case-control study: AREDS report no. 20. Arch. Ophthalmol. 125: 671–679.

      ,
      • Simonelli F.
      • Manna C.
      • Romano N.
      • Nunziata G.
      • Voto O.
      • Rinaldi E.
      Evaluation of fatty acids in membrane phospholipids of erythrocytes in retinitis pigmentosa patients.
      ). Reports from experimental studies have shown that n3 PUFAs reduce the risk of diabetic retinopathy through inhibition of retinal vascular damage, enhancing the reparative function of endothelial progenitor cells and reducing the expression of inflammatory markers (
      • Tikhonenko M.
      • Lydic T.A.
      • Opreanu M.
      • Li Calzi S.
      • Bozack S.
      • McSorley K.M.
      • Sochacki A.L.
      • Faber M.S.
      • Hazra S.
      • Duclos S.
      • et al.
      N-3 polyunsaturated Fatty acids prevent diabetic retinopathy by inhibition of retinal vascular damage and enhanced endothelial progenitor cell reparative function.
      ,
      • Tikhonenko M.
      • Lydic T.A.
      • Wang Y.
      • Chen W.
      • Opreanu M.
      • Sochacki A.
      • McSorley K.M.
      • Renis R.L.
      • Kern T.
      • Jump D.B.
      • et al.
      Remodeling of retinal Fatty acids in an animal model of diabetes: a decrease in long-chain polyunsaturated fatty acids is associated with a decrease in fatty acid elongases Elovl2 and Elovl4.
      ,
      • Chen W.
      • Esselman W.J.
      • Jump D.B.
      • Busik J.V.
      Anti-inflammatory effect of docosahexaenoic acid on cytokine-induced adhesion molecule expression in human retinal vascular endothelial cells.
      ,
      • Chen W.
      • Jump D.B.
      • Esselman W.J.
      • Busik J.V.
      Inhibition of cytokine signaling in human retinal endothelial cells through modification of caveolae/lipid rafts by docosahexaenoic acid.
      ,
      • Sapieha P.
      • Stahl A.
      • Chen J.
      • Seaward M.R.
      • Willett K.L.
      • Krah N.M.
      • Dennison R.J.
      • Connor K.M.
      • Aderman C.M.
      • Liclican E.
      • et al.
      5-Lipoxygenase metabolite 4-HDHA is a mediator of the antiangiogenic effect of omega-3 polyunsaturated fatty acids.
      ). The Blue Mountains Eye Study reported an association between reduced incidence of nuclear cataracts with higher dietary intake of n3 PUFAs (
      • Townend B.S.
      • Townend M.E.
      • Flood V.
      • Burlutsky G.
      • Rochtchina E.
      • Wang J.J.
      • Mitchell P.
      Dietary macronutrient intake and five-year incident cataract: the Blue Mountains Eye Study.
      ). However, the recent AREDS2 and the Nutritional AMD Treatments-2 studies surprisingly reported that dietary n3 PUFAs had no beneficial effect on the outcome of AMD (
      • Age-Related Eye Disease Study 2 Research Group
      Lutein + zeaxanthin and omega-3 fatty acids for age-related macular degeneration: the Age-Related Eye Disease Study 2 (AREDS2) randomized clinical trial.
      ). To gain a better understanding of a possible biochemical basis for these confounding results, we compared the fatty acid and molecular species compositions of lipids from whole retinas and OS membranes from rod- and cone-rich retinas.
      The most significant findings of the present study are that cone-dominant retinas and OS membranes have about 2-fold less 22:6n3 than do rod-dominant retinas and that PC molecular species containing VLC-PUFAs are significantly less abundant in cone-rich retinas and their OS membranes. Our data thus provide support for previous studies that reported less 22:6n3 and VLC-PUFAs in human macula than in the peripheral retina (
      • van Kuijk F.J.
      • Buck P.
      Fatty acid composition of the human macula and peripheral retina.
      ,
      • Liu A.
      • Terry R.
      • Lin Y.
      • Nelson K.
      • Bernstein P.S.
      Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina.
      ). Interestingly, our results contrast with those reported in Nile rats (Arvicanthis ansorgei, 33% cones), which had 14% higher levels of 22:6n3 compared with Norway rats (Rattus norvegicus, 2–3% cones) (
      • Boudard D.L.
      • Acar N.
      • Bretillon L.
      • Hicks D.
      Retinas of the diurnal rodent Arvicanthis ansorgei are highly resistant to experimentally induced stress and degeneration.
      ). Our data, however, are consistent with the reported average 15.9 relative mole percent of 22:6n3 found in human macula (
      • van Kuijk F.J.
      • Buck P.
      Fatty acid composition of the human macula and peripheral retina.
      ), as well as low 22:6n3 levels in cone-rich chicken retinas (
      • Johnston D.
      • Hudson R.A.
      Phospholipids of the cone-rich chicken retina and its photoreceptor outer segments membranes.
      ). Our findings are also different from a previous report that found high percentages of DHA in PC and PS (55% and 63%, respectively) in the P521 cone-rich retinas of Tokay geckos (Gekko gecko) (
      • Yuan C.
      • Chen H.
      • Anderson R.E.
      • Kuwata O.
      • Ebrey T.G.
      The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes.
      ). Nevertheless, for mammals, it seems safe to conclude that COS membranes contain lower levels of DHA than do ROS membranes. We also report significantly lower levels of retinal and OS VLC-PUFAs in cone-dominant retinas than in rod-dominant retinas, which is consistent with recent reports by Liu et al. (
      • Liu A.
      • Terry R.
      • Lin Y.
      • Nelson K.
      • Bernstein P.S.
      Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina.
      ) that the total VLC-PUFAs and the n3/n6 ratio of VLC-PUFAs in human retinas were significantly higher in rod-enriched peripheral retina, compared with the cone-rich macula. While Liu et al. (
      • Liu A.
      • Terry R.
      • Lin Y.
      • Nelson K.
      • Bernstein P.S.
      Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina.
      ) attributed these differences to the possibility of lipids within the relatively thick inner retina overwhelming detection of VLC-PUFAs within the macula, our studies suggest that cone photoreceptors intrinsically have less 22:6n3 and VLC-PUFAs than do rods.
      DHA plays essential roles in phototransduction and protection of photoreceptor cells against cellular insults, such that DHA depletion affects retinal function. Within the retina, G protein-coupled signal transduction is essential for sensory processing and visual perception, which are compromised by 22:6n3 deficiency, which occurs in rats raised on an n3-deficient diet (
      • Salem Jr., N.
      • Loewke J.
      • Catalan J.N.
      • Majchrzak S.
      • Moriguchi T.
      Incomplete replacement of docosahexaenoic acid by n-6 docosapentaenoic acid in the rat retina after an n-3 fatty acid deficient diet.
      ,
      • Wiegand R.D.
      • Koutz C.A.
      • Stinson A.M.
      • Anderson R.E.
      Conservation of docosahexaenoic acid in rod outer segments of rat retina during n-3 and n-6 fatty acid deficiency.
      ). Previous studies demonstrate that rats raised on an n3-deficient diet have an almost stoichiometric replacement of 22:6n3 by 22:5n6 in the retina (
      • Salem Jr., N.
      • Loewke J.
      • Catalan J.N.
      • Majchrzak S.
      • Moriguchi T.
      Incomplete replacement of docosahexaenoic acid by n-6 docosapentaenoic acid in the rat retina after an n-3 fatty acid deficient diet.
      ,
      • Wiegand R.D.
      • Koutz C.A.
      • Stinson A.M.
      • Anderson R.E.
      Conservation of docosahexaenoic acid in rod outer segments of rat retina during n-3 and n-6 fatty acid deficiency.
      ,
      • Lin D.S.
      • Anderson G.J.
      • Connor W.E.
      • Neuringer M.
      Effect of dietary N-3 fatty acids upon the phospholipid molecular species of the monkey retina.
      ). However, the replacement of 22:6n3 with 22:5n6 in ROS membranes of rats results in functional deficiencies in the photoreceptor signal transduction pathway (
      • Niu S.L.
      • Mitchell D.C.
      • Lim S.Y.
      • Wen Z.M.
      • Kim H.Y.
      • Salem Jr., N.
      • Litman B.J.
      Reduced G protein-coupled signaling efficiency in retinal rod outer segments in response to n-3 fatty acid deficiency.
      ). In contrast, DHA-rich phospholipids optimize photoreceptor signaling, which underscores the importance of DHA in neural G protein-coupled receptor signaling. The cone-dominant retinas do not have increased 22:5n6, which demonstrates that reduction of DHA in COSs is not the result of a limited supply of 22:6n3 to these cells. Rather, the reduced amounts of DHA and VLC-PUFAs suggest that the biophysical lipid requirement for these fatty acids in visual function in cone photoreceptor cells is intrinsically different from rod photoreceptors.
      The daily shedding of DHA- and VLC-PUFA-enriched OS membranes and subsequent phagocytic activity of retinal pigment epithelium (RPE) cells result in recycling and conservation of 22:6n3 between photoreceptor cells and the RPE cells (
      • Gordon W.C.
      • Bazan N.G.
      Docosahexaenoic acid utilization during rod photoreceptor cell renewal.
      ,
      • Bazan N.G.
      Homeostatic regulation of photoreceptor cell integrity: significance of the potent mediator neuroprotectin D1 biosynthesized from docosahexaenoic acid: the Proctor Lecture.
      ,
      • Mukherjee P.K.
      • Marcheselli V.L.
      • de Rivero Vaccari J.C.
      • Gordon W.C.
      • Jackson F.E.
      • Bazan N.G.
      Photoreceptor outer segment phagocytosis attenuates oxidative stress-induced apoptosis with concomitant neuroprotectin D1 synthesis.
      ,
      • Chen H.
      • Wiegand R.D.
      • Koutz C.A.
      • Anderson R.E.
      Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding.
      ,
      • Anderson R.E.
      • O'Brien P.J.
      • Wiegand R.D.
      • Koutz C.A.
      • Stinson A.M.
      Conservation of docosahexaenoic acid in the retina.
      ). Phospholipase activity on membrane phospholipids of photoreceptor and RPE cells leads to release of free fatty acids, including DHA, AA, and VLC-PUFAs, some of which are converted to bioactive lipids that mediate further intracellular signal transduction and cytoprotection of photoreceptors from cellular stress (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • de Rivero Vaccari J.C.
      • Gordon W.C.
      • Jackson F.E.
      • Bazan N.G.
      Photoreceptor outer segment phagocytosis attenuates oxidative stress-induced apoptosis with concomitant neuroprotectin D1 synthesis.
      ,
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      Neuroprotectin D1: a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress.
      ). Bazan's laboratory has reported that 22:6n3 can be converted by the RPE into the bioactive neuroprotective docosanoid, neuroprotectin D1 (NPD1; 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) in response to oxidative and other stress (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      Neuroprotectin D1: a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress.
      ). NPD1 and other bioactive docosanoids synthesized in RPE and photoreceptor cells on demand act as potent anti-apoptotic and anti-inflammatory molecules that preserve retinal integrity in response to cellular damage (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Barreiro S.
      • Hu J.
      • Bok D.
      • Bazan N.G.
      Neurotrophins enhance retinal pigment epithelial cell survival through neuroprotectin D1 signaling.
      ,
      • Bazan N.G.
      • Calandria J.M.
      • Gordon W.C.
      Docosahexaenoic acid and its derivative neuroprotectin D1 display neuroprotective properties in the retina, brain and central nervous system.
      ). Apart from acting as signaling molecules, DHA-derived docosanoids, such as the d-series resolvins (RvD1 and RvD2) and maresins, as well as NPD1, play essential homeostasis roles by promoting cellular repair and phagocytosis, which are essential for maintaining the structure and integrity of postmitotic cells such as retinal neurons. More recently, the Bazan laboratory also showed that ELOVL4-derived VLC-PUFAs 32:5n3 and 34:6n3 (
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Henry K.
      • Elliott M.H.
      • Anderson R.E.
      Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
      ,
      • Yu M.
      • Benham A.
      • Logan S.
      • Brush R.S.
      • Mandal M.N.
      • Anderson R.E.
      • Agbaga M.P.
      ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3.
      ,
      • Suh M.
      • Clandinin M.T.
      20:5n-3 but not 22:6n-3 is a preferred substrate for synthesis of n-3 very-long- chain fatty acids (C24–C36) in retina.
      ) can be converted by the RPE into neuroprotective molecules coined “elovanoids” (
      • Jun B.
      • Mukherjee P.K.
      • Asatryan A.
      • Kautzmann M.A.
      • Heap J.
      • Gordon W.C.
      • Bhattacharjee S.
      • Yang R.
      • Petasis N.A.
      • Bazan N.G.
      Elovanoids are novel cell-specific lipid mediators necessary for neuroprotective signaling for photoreceptor cell integrity.
      ). Because these precursors of neuroprotective lipids are less abundant in the OSs of cone-dominant animals and in the macula of the human retina (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ,
      • Liu A.
      • Terry R.
      • Lin Y.
      • Nelson K.
      • Bernstein P.S.
      Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina.
      ,
      • Liu A.
      • Chang J.
      • Shen Z.
      • Bernstein P.S.
      Enhanced methods for analysis of very long chain polyunsaturated fatty acids from retina and RPE.
      ), and because their reduction in the human macula is age-dependent (
      • van Kuijk F.J.
      • Buck P.
      Fatty acid composition of the human macula and peripheral retina.
      ,
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ), we suggest that with age there may be a compromised supply of docosanoids and elovanoids in the central retina, which makes the RPE and, subsequently, the underlying photoreceptors more susceptible to stress-induced cell death. Our earlier studies (
      • De La Paz M.
      • Anderson R.E.
      Region and age-dependent variation in susceptibility of the human retina to lipid peroxidation.
      ) demonstrated an age-dependent increase in the susceptibility of the human macula to oxidative stress and an increase in lipid peroxidation compared with peripheral retina.
      The higher n6/n3 ratios in cone photoreceptor cells could, with age, potentially tilt the balance toward pro-inflammatory AA-derived eicosanoids within macular RPE cells, thereby increasing inflammation-induced AMD progression. This reasoning is supported by studies by Liu et al. (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ) that showed that DHA and VLC-PUFA abundance in the retina and RPE/choroid peaks in middle age and decreases with age and that, when compared with normal age-matched human donors, there is a significant decrease in DHA and VLC-PUFAs in the AMD retina and RPE/choroid accompanied by increased n6/n3 PUFA ratios. Thus, it is possible that aging and epigenetic and inherited genetic factors that affect DHA and VLC-PUFA biosynthesis, such as in STGD3 (
      • Bernstein P.S.
      • Tammur J.
      • Singh N.
      • Hutchinson A.
      • Dixon M.
      • Pappas C.M.
      • Zabriskie N.A.
      • Zhang K.
      • Petrukhin K.
      • Leppert M.
      • et al.
      Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.
      ,
      • Edwards A.O.
      • Donoso L.A.
      • Ritter III, R.
      A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
      ,
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ,
      • McMahon A.
      • Butovich I.A.
      • Mata N.L.
      • Klein M.
      • Ritter 3rd, R.
      • Richardson J.
      • Birch D.G.
      • Edwards A.O.
      • Kedzierski W.
      Retinal pathology and skin barrier defect in mice carrying a Stargardt disease-3 mutation in elongase of very long chain fatty acids-4.
      ), contribute to the exacerbation of macular degeneration.

      CONCLUSIONS

      Compared with well-characterized rod-dominant retinas, cone-dominant retinas have significantly less 22:6n3 and VLC-PUFAs in whole retinas and OS membranes. Because 22:6n3 is necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones and cone signaling may not have the same lipid requirements as rods. Moreover, our studies confirm the differences in LC-PUFAs found in human macula relative to the peripheral retina. While this study was done on rodents, the data support decreased DHA and VLC-PUFAs found in human macula. AREDS2 results showed that DHA supplementation is not able to attenuate progression of macular degeneration. Our data show that cone photoreceptors intrinsically have low levels of DHA, which suggests that perhaps increasing cone DHA levels after onset of retinal degeneration will not provide therapeutic benefits. On the other hand, it might also suggest that cone photoreceptors do not readily take up and retain DHA as do rod photoreceptors, so dietary DHA is not able to influence cone photoreceptor DHA levels. We propose that genetic and epigenetic modification coupled with age-related reduction in macular 22:6n3 and VLC-PUFAs (which are precursors of neuroprotective bioactive lipids that are synthesized in the RPE) increase the susceptibility of the macula to age-related RPE and photoreceptor cell degeneration. Hence, an increase in the dietary intake of LC-PUFAs and VLC-PUFAs early in life would maintain retinal PUFA and PUFA-derived neuroprotective bioactive lipid levels that are beneficial for visual function.

      REFERENCES

        • Fliesler S.J.
        • Anderson R.E.
        Chemistry and metabolism of lipids in the vertebrate retina.
        Prog. Lipid Res. 1983; 22: 79-131
        • SanGiovanni J.P.
        • Chew E.Y.
        The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina.
        Prog. Retin. Eye Res. 2005; 24: 87-138
        • Anderson R.E.
        • Benolken R.M.
        • Kelleher P.A.
        • Maude M.B.
        • Wiegand R.D.
        Chemistry of photoreceptor membrane preparations from squid retinas.
        Biochim. Biophys. Acta. 1978; 510: 316-326
        • Benolken R.M.
        • Anderson R.E.
        • Maude M.B.
        Lipid composition of Limulus photoreceptor membranes.
        Biochim. Biophys. Acta. 1975; 413: 234-242
        • Benolken R.M.
        • Anderson R.E.
        • Wheeler T.G.
        Membrane fatty acids associated with the electrical response in visual excitation.
        Science. 1973; 182: 1253-1254
        • Wheeler T.G.
        • Benolken R.M.
        • Anderson R.E.
        Visual membranes: specificity of fatty acid precursors for the electrical response to illumination.
        Science. 1975; 188: 1312-1314
        • Neuringer M.
        • Connor W.E.
        • Van Petten C.
        • Barstad L.
        Dietary omega-3 fatty acid deficiency and visual loss in infant rhesus monkeys.
        J. Clin. Invest. 1984; 73: 272-276
        • Neuringer M.
        • Reisbick S.
        • Janowsky J.
        The role of n-3 fatty acids in visual and cognitive development: current evidence and methods of assessment.
        J. Pediatr. 1994; 125: S39-S47
        • O'Connor D.L.
        • Hall R.
        • Adamkin D.
        • Auestad N.
        • Castillo M.
        • Connor W.E.
        • Connor S.L.
        • Fitzgerald K.
        • Groh-Wargo S.
        • Hartmann E.E.
        • et al.
        Growth and development in preterm infants fed long-chain polyunsaturated fatty acids: a prospective, randomized controlled trial.
        Pediatrics. 2001; 108: 359-371
        • Aveldaño M.I.
        • Sprecher H.
        Very long chain (C24 to C36) polyenoic fatty acids of the n-3 and n-6 series in dipolyunsaturated phosphatidylcholines from bovine retina.
        J. Biol. Chem. 1987; 262: 1180-1186
        • Aveldaño M.I.
        A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina.
        J. Biol. Chem. 1987; 262: 1172-1179
        • Bernstein P.S.
        • Tammur J.
        • Singh N.
        • Hutchinson A.
        • Dixon M.
        • Pappas C.M.
        • Zabriskie N.A.
        • Zhang K.
        • Petrukhin K.
        • Leppert M.
        • et al.
        Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.
        Invest. Ophthalmol. Vis. Sci. 2001; 42: 3331-3336
        • Edwards A.O.
        • Donoso L.A.
        • Ritter III, R.
        A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
        Invest. Ophthalmol. Vis. Sci. 2001; 42: 2652-2663
        • Zhang X.M.
        • Yang Z.
        • Karan G.
        • Hashimoto T.
        • Baehr W.
        • Yang X.J.
        • Zhang K.
        Elovl4 mRNA distribution in the developing mouse retina and phylogenetic conservation of Elovl4 genes.
        Mol. Vis. 2003; 9: 301-307
        • Dittrich F.
        • Zajonc D.
        • Huhne K.
        • Hoja U.
        • Ekici A.
        • Greiner E.
        • Klein H.
        • Hofmann J.
        • Bessoule J.J.
        • Sperling P.
        • et al.
        Fatty acid elongation in yeast–biochemical characteristics of the enzyme system and isolation of elongation-defective mutants.
        Eur. J. Biochem. 1998; 252: 477-485
        • Leonard A.E.
        • Pereira S.L.
        • Sprecher H.
        • Huang Y.S.
        Elongation of long-chain fatty acids.
        Prog. Lipid Res. 2004; 43: 36-54
        • Agbaga M.P.
        • Brush R.S.
        • Mandal M.N.
        • Henry K.
        • Elliott M.H.
        • Anderson R.E.
        Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
        Proc. Natl. Acad. Sci. USA. 2008; 105: 12843-12848
        • Yu M.
        • Benham A.
        • Logan S.
        • Brush R.S.
        • Mandal M.N.
        • Anderson R.E.
        • Agbaga M.P.
        ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3.
        J. Lipid Res. 2012; 53: 494-504
        • Logan S.
        • Agbaga M.P.
        • Chan M.D.
        • Brush R.S.
        • Anderson R.E.
        Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity.
        J. Lipid Res. 2014; 55: 698-708
        • Logan S.
        • Agbaga M.P.
        • Chan M.D.
        • Kabir N.
        • Mandal N.A.
        • Brush R.S.
        • Anderson R.E.
        Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.
        Proc. Natl. Acad. Sci. USA. 2013; 110: 5446-5451
        • Grayson C.
        • Molday R.S.
        Dominant negative mechanism underlies autosomal dominant Stargardt-like macular dystrophy linked to mutations in ELOVL4.
        J. Biol. Chem. 2005; 280: 32521-32530
        • Ambasudhan R.
        • Wang X.
        • Jablonski M.M.
        • Thompson D.A.
        • Lagali P.S.
        • Wong P.W.
        • Sieving P.A.
        • Ayyagari R.
        Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein.
        Genomics. 2004; 83: 615-625
        • Karan G.
        • Yang Z.
        • Howes K.
        • Zhao Y.
        • Chen Y.
        • Cameron D.J.
        • Lin Y.
        • Pearson E.
        • Zhang K.
        Loss of ER retention and sequestration of the wild-type ELOVL4 by Stargardt disease dominant negative mutants.
        Mol. Vis. 2005; 11: 657-664
        • Donoso L.A.
        • Edwards A.O.
        • Frost A.
        • Vrabec T.
        • Stone E.M.
        • Hageman G.S.
        • Perski T.
        Autosomal dominant Stargardt-like macular dystrophy.
        Surv. Ophthalmol. 2001; 46: 149-163
        • Edwards A.O.
        • Miedziak A.
        • Vrabec T.
        • Verhoeven J.
        • Acott T.S.
        • Weleber R.G.
        • Donoso L.A.
        Autosomal dominant Stargardt-like macular dystrophy: I. Clinical characterization, longitudinal follow-up, and evidence for a common ancestry in families linked to chromosome 6q14.
        Am. J. Ophthalmol. 1999; 127: 426-435
        • van Kuijk F.J.
        • Buck P.
        Fatty acid composition of the human macula and peripheral retina.
        Invest. Ophthalmol. Vis. Sci. 1992; 33: 3493-3496
        • Liu A.
        • Chang J.
        • Lin Y.
        • Shen Z.
        • Bernstein P.S.
        Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
        J. Lipid Res. 2010; 51: 3217-3229
        • Liu A.
        • Terry R.
        • Lin Y.
        • Nelson K.
        • Bernstein P.S.
        Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina.
        J. Chromatogr. A. 2013; 1307: 191-200
        • Mears A.J.
        • Kondo M.
        • Swain P.K.
        • Takada Y.
        • Bush R.A.
        • Saunders T.L.
        • Sieving P.A.
        • Swaroop A.
        Nrl is required for rod photoreceptor development.
        Nat. Genet. 2001; 29: 447-452
        • Zhu X.
        • Brown B.
        • Li A.
        • Mears A.J.
        • Swaroop A.
        • Craft C.M.
        GRK1-dependent phosphorylation of S and M opsins and their binding to cone arrestin during cone phototransduction in the mouse retina.
        J. Neurosci. 2003; 23: 6152-6160
        • Daniele L.L.
        • Lillo C.
        • Lyubarsky A.L.
        • Nikonov S.S.
        • Philp N.
        • Mears A.J.
        • Swaroop A.
        • Williams D.S.
        • Pugh Jr., E.N.
        Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse.
        Invest. Ophthalmol. Vis. Sci. 2005; 46: 2156-2167
        • Nikonov S.S.
        • Daniele L.L.
        • Zhu X.
        • Craft C.M.
        • Swaroop A.
        • Pugh Jr., E.N.
        Photoreceptors of Nrl −/− mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.
        J. Gen. Physiol. 2005; 125: 287-304
        • Wässle H.
        • Riemann H.J.
        The mosaic of nerve cells in the mammalian retina.
        Proc. R. Soc. Lond. B Biol. Sci. 1978; 200: 441-461
        • Stuck M.W.
        • Conley S.M.
        • Naash M.I.
        Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina.
        PLoS One. 2012; 7: e32484
        • Martin R.E.
        • Elliott M.H.
        • Brush R.S.
        • Anderson R.E.
        Detailed characterization of the lipid composition of detergent-resistant membranes from photoreceptor rod outer segment membranes.
        Invest. Ophthalmol. Vis. Sci. 2005; 46: 1147-1154
        • Martin R.E.
        • Fliesler S.J.
        • Brush R.S.
        • Richards M.J.
        • Hopkins S.A.
        • Anderson R.E.
        Lipid differences in rod outer segment membranes of rats with P23H and S334ter opsin mutations.
        Mol. Vis. 2005; 11: 338-346
        • Bicknell I.R.
        • Darrow R.
        • Barsalou L.
        • Fliesler S.J.
        • Organisciak D.T.
        Alterations in retinal rod outer segment fatty acids and light-damage susceptibility in P23H rats.
        Mol. Vis. 2002; 8: 333-340
        • Bligh E.G.
        • Dyer W.J.
        A rapid method of total lipid extraction and purification.
        Can. J. Biochem. Physiol. 1959; 37: 911-917
        • Busik J.V.
        • Reid G.E.
        • Lydic T.A.
        Global analysis of retina lipids by complementary precursor ion and neutral loss mode tandem mass spectrometry.
        Methods Mol. Biol. 2009; 579: 33-70
        • Lydic T.A.
        • Busik J.V.
        • Reid G.E.
        A monophasic extraction strategy for the simultaneous lipidome analysis of polar and nonpolar retina lipids.
        J. Lipid Res. 2014; 55: 1797-1809
        • Haimi P.
        • Chaithanya K.
        • Kainu V.
        • Hermansson M.
        • Somerharju P.
        Instrument-independent software tools for the analysis of MS-MS and LC-MS lipidomics data.
        Methods Mol. Biol. 2009; 580: 285-294
        • Haimi P.
        • Uphoff A.
        • Hermansson M.
        • Somerharju P.
        Software tools for analysis of mass spectrometric lipidome data.
        Anal. Chem. 2006; 78: 8324-8331
        • Song H.
        • Ladenson J.
        • Turk J.
        Algorithms for automatic processing of data from mass spectrometric analyses of lipids.
        J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2847-2854
        • Laemmli U.K.
        Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
        Nature. 1970; 227: 680-685
        • Salem Jr., N.
        • Loewke J.
        • Catalan J.N.
        • Majchrzak S.
        • Moriguchi T.
        Incomplete replacement of docosahexaenoic acid by n-6 docosapentaenoic acid in the rat retina after an n-3 fatty acid deficient diet.
        Exp. Eye Res. 2005; 81: 655-663
        • Levant B.
        • Radel J.D.
        • Carlson S.E.
        Reduced brain DHA content after a single reproductive cycle in female rats fed a diet deficient in N-3 polyunsaturated fatty acids.
        Biol. Psychiatry. 2006; 60: 987-990
        • Carlson S.E.
        • Carver J.D.
        • House S.G.
        High fat diets varying in ratios of polyunsaturated to saturated fatty acid and linoleic to linolenic acid: a comparison of rat neural and red cell membrane phospholipids.
        J. Nutr. 1986; 116: 718-725
        • Wiegand R.D.
        • Koutz C.A.
        • Stinson A.M.
        • Anderson R.E.
        Conservation of docosahexaenoic acid in rod outer segments of rat retina during n-3 and n-6 fatty acid deficiency.
        J. Neurochem. 1991; 57: 1690-1699
        • Delton-Vandenbroucke I.
        • Maude M.B.
        • Chen H.
        • Aguirre G.D.
        • Acland G.M.
        • Anderson R.E.
        Effect of diet on the fatty acid and molecular species composition of dog retina phospholipids.
        Lipids. 1998; 33: 1187-1193
        • Cho E.
        • Hung S.
        • Willett W.C.
        • Spiegelman D.
        • Rimm E.B.
        • Seddon J.M.
        • Colditz G.A.
        • Hankinson S.E.
        Prospective study of dietary fat and the risk of age-related macular degeneration.
        Am. J. Clin. Nutr. 2001; 73: 209-218
        • Seddon J.M.
        • Cote J.
        • Rosner B.
        Progression of age-related macular degeneration: association with dietary fat, transunsaturated fat, nuts, and fish intake.
        Arch. Ophthalmol. 2003; 121: 1728-1737
        • Seddon J.M.
        • George S.
        • Rosner B.
        Cigarette smoking, fish consumption, omega-3 fatty acid intake, and associations with age-related macular degeneration: the US Twin Study of Age-Related Macular Degeneration.
        Arch. Ophthalmol. 2006; 124: 995-1001
        • Seddon J.M.
        • Rosner B.
        • Sperduto R.D.
        • Yannuzzi L.
        • Haller J.A.
        • Blair N.P.
        • Willett W.
        Dietary fat and risk for advanced age-related macular degeneration.
        Arch. Ophthalmol. 2001; 119: 1191-1199
        • SanGiovanni J.P.
        • Chew E.Y.
        • Agron E.
        • Clemons T.E.
        • Ferris III, F.L.
        • Gensler G.
        • Lindblad A.S.
        • Milton R.C.
        • Seddon J.M.
        • Klein R.
        • Age-Related Eye Disease Study Research Group
        • et al.
        The relationship of dietary omega-3 long-chain polyunsaturated fatty acid intake with incident age-related macular degeneration: AREDS report no. 23.
        Arch. Ophthalmol. 2008; 126: 1274-1279
      1. SanGiovanni, J. P., ChewE. Y., ClemonsT. E., DavisM. D., FerrisF. L. III, GenslerG. R., KurinijN., LindbladA. S., MiltonR. C., SeddonJ. M., et al.; Age-Related Eye Disease Study Research Group. 2007. The relationship of dietary lipid intake and age-related macular degeneration in a case-control study: AREDS report no. 20. Arch. Ophthalmol. 125: 671–679.

        • Simonelli F.
        • Manna C.
        • Romano N.
        • Nunziata G.
        • Voto O.
        • Rinaldi E.
        Evaluation of fatty acids in membrane phospholipids of erythrocytes in retinitis pigmentosa patients.
        Ophthalmic Res. 1996; 28: 93-98
        • Tikhonenko M.
        • Lydic T.A.
        • Opreanu M.
        • Li Calzi S.
        • Bozack S.
        • McSorley K.M.
        • Sochacki A.L.
        • Faber M.S.
        • Hazra S.
        • Duclos S.
        • et al.
        N-3 polyunsaturated Fatty acids prevent diabetic retinopathy by inhibition of retinal vascular damage and enhanced endothelial progenitor cell reparative function.
        PLoS One. 2013; 8: e55177
        • Tikhonenko M.
        • Lydic T.A.
        • Wang Y.
        • Chen W.
        • Opreanu M.
        • Sochacki A.
        • McSorley K.M.
        • Renis R.L.
        • Kern T.
        • Jump D.B.
        • et al.
        Remodeling of retinal Fatty acids in an animal model of diabetes: a decrease in long-chain polyunsaturated fatty acids is associated with a decrease in fatty acid elongases Elovl2 and Elovl4.
        Diabetes. 2010; 59: 219-227
        • Chen W.
        • Esselman W.J.
        • Jump D.B.
        • Busik J.V.
        Anti-inflammatory effect of docosahexaenoic acid on cytokine-induced adhesion molecule expression in human retinal vascular endothelial cells.
        Invest. Ophthalmol. Vis. Sci. 2005; 46: 4342-4347
        • Chen W.
        • Jump D.B.
        • Esselman W.J.
        • Busik J.V.
        Inhibition of cytokine signaling in human retinal endothelial cells through modification of caveolae/lipid rafts by docosahexaenoic acid.
        Invest. Ophthalmol. Vis. Sci. 2007; 48: 18-26
        • Sapieha P.
        • Stahl A.
        • Chen J.
        • Seaward M.R.
        • Willett K.L.
        • Krah N.M.
        • Dennison R.J.
        • Connor K.M.
        • Aderman C.M.
        • Liclican E.
        • et al.
        5-Lipoxygenase metabolite 4-HDHA is a mediator of the antiangiogenic effect of omega-3 polyunsaturated fatty acids.
        Sci. Transl. Med. 2011; 3: 69ra12
        • Townend B.S.
        • Townend M.E.
        • Flood V.
        • Burlutsky G.
        • Rochtchina E.
        • Wang J.J.
        • Mitchell P.
        Dietary macronutrient intake and five-year incident cataract: the Blue Mountains Eye Study.
        Am. J. Ophthalmol. 2007; 143: 932-939
        • Age-Related Eye Disease Study 2 Research Group
        Lutein + zeaxanthin and omega-3 fatty acids for age-related macular degeneration: the Age-Related Eye Disease Study 2 (AREDS2) randomized clinical trial.
        JAMA. 2013; 309: 2005-2015
        • Boudard D.L.
        • Acar N.
        • Bretillon L.
        • Hicks D.
        Retinas of the diurnal rodent Arvicanthis ansorgei are highly resistant to experimentally induced stress and degeneration.
        Invest. Ophthalmol. Vis. Sci. 2011; 52: 8686-8700
        • Johnston D.
        • Hudson R.A.
        Phospholipids of the cone-rich chicken retina and its photoreceptor outer segments membranes.
        Biochim. Biophys. Acta. 1974; 369: 269-277
        • Yuan C.
        • Chen H.
        • Anderson R.E.
        • Kuwata O.
        • Ebrey T.G.
        The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes.
        Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1998; 120: 785-789
        • Lin D.S.
        • Anderson G.J.
        • Connor W.E.
        • Neuringer M.
        Effect of dietary N-3 fatty acids upon the phospholipid molecular species of the monkey retina.
        Invest. Ophthalmol. Vis. Sci. 1994; 35: 794-803
        • Niu S.L.
        • Mitchell D.C.
        • Lim S.Y.
        • Wen Z.M.
        • Kim H.Y.
        • Salem Jr., N.
        • Litman B.J.
        Reduced G protein-coupled signaling efficiency in retinal rod outer segments in response to n-3 fatty acid deficiency.
        J. Biol. Chem. 2004; 279: 31098-31104
        • Gordon W.C.
        • Bazan N.G.
        Docosahexaenoic acid utilization during rod photoreceptor cell renewal.
        J. Neurosci. 1990; 10: 2190-2202
        • Bazan N.G.
        Homeostatic regulation of photoreceptor cell integrity: significance of the potent mediator neuroprotectin D1 biosynthesized from docosahexaenoic acid: the Proctor Lecture.
        Invest. Ophthalmol. Vis. Sci. 2007; 48: 4866-4881
        • Mukherjee P.K.
        • Marcheselli V.L.
        • de Rivero Vaccari J.C.
        • Gordon W.C.
        • Jackson F.E.
        • Bazan N.G.
        Photoreceptor outer segment phagocytosis attenuates oxidative stress-induced apoptosis with concomitant neuroprotectin D1 synthesis.
        Proc. Natl. Acad. Sci. USA. 2007; 104: 13158-13163
        • Chen H.
        • Wiegand R.D.
        • Koutz C.A.
        • Anderson R.E.
        Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding.
        Exp. Eye Res. 1992; 55: 93-100
        • Anderson R.E.
        • O'Brien P.J.
        • Wiegand R.D.
        • Koutz C.A.
        • Stinson A.M.
        Conservation of docosahexaenoic acid in the retina.
        Adv. Exp. Med. Biol. 1992; 318: 285-294
        • Mukherjee P.K.
        • Marcheselli V.L.
        • Serhan C.N.
        • Bazan N.G.
        Neuroprotectin D1: a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress.
        Proc. Natl. Acad. Sci. USA. 2004; 101: 8491-8496
        • Mukherjee P.K.
        • Marcheselli V.L.
        • Barreiro S.
        • Hu J.
        • Bok D.
        • Bazan N.G.
        Neurotrophins enhance retinal pigment epithelial cell survival through neuroprotectin D1 signaling.
        Proc. Natl. Acad. Sci. USA. 2007; 104: 13152-13157
        • Bazan N.G.
        • Calandria J.M.
        • Gordon W.C.
        Docosahexaenoic acid and its derivative neuroprotectin D1 display neuroprotective properties in the retina, brain and central nervous system.
        Nestle Nutr. Inst. Workshop Ser. 2013; 77: 121-131
        • Suh M.
        • Clandinin M.T.
        20:5n-3 but not 22:6n-3 is a preferred substrate for synthesis of n-3 very-long- chain fatty acids (C24–C36) in retina.
        Curr. Eye Res. 2005; 30: 959-968
        • Jun B.
        • Mukherjee P.K.
        • Asatryan A.
        • Kautzmann M.A.
        • Heap J.
        • Gordon W.C.
        • Bhattacharjee S.
        • Yang R.
        • Petasis N.A.
        • Bazan N.G.
        Elovanoids are novel cell-specific lipid mediators necessary for neuroprotective signaling for photoreceptor cell integrity.
        Sci. Rep. 2017; 7: 5279
        • Liu A.
        • Chang J.
        • Shen Z.
        • Bernstein P.S.
        Enhanced methods for analysis of very long chain polyunsaturated fatty acids from retina and RPE.
        Invest. Ophthalmol. Vis. Sci. 2009; 50: 3410
        • De La Paz M.
        • Anderson R.E.
        Region and age-dependent variation in susceptibility of the human retina to lipid peroxidation.
        Invest. Ophthalmol. Vis. Sci. 1992; 33: 3497-3499
        • Zhang K.
        • Kniazeva M.
        • Han M.
        • Li W.
        • Yu Z.
        • Yang Z.
        • Li Y.
        • Metzker M.L.
        • Allikmets R.
        • Zack D.J.
        • et al.
        A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
        Nat. Genet. 2001; 27: 89-93
        • McMahon A.
        • Butovich I.A.
        • Mata N.L.
        • Klein M.
        • Ritter 3rd, R.
        • Richardson J.
        • Birch D.G.
        • Edwards A.O.
        • Kedzierski W.
        Retinal pathology and skin barrier defect in mice carrying a Stargardt disease-3 mutation in elongase of very long chain fatty acids-4.
        Mol. Vis. 2007; 13: 258-272