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Associations of human retinal very long-chain polyunsaturated fatty acids with dietary lipid biomarkers

2016; Elsevier BV; Volume: 57; Issue: 3 Linguagem: Inglês

10.1194/jlr.p065540

1539-7262

Aruna Gorusupudi, Aihua Liu, Gregory S. Hageman, Paul S. Bernstein,

Fatty Acid Research and Health

The human retina is well-known to have unique lipid profiles enriched in long-chain polyunsaturated fatty acids (LC-PUFAs) and very long-chain polyunsaturated fatty acids (VLC-PUFAs) that appear to promote normal retinal structure and function, but the influence of diet on retinal lipid profiles in health and disease remains controversial. In this study, we examined two independent cohorts of donor eyes and related their retinal lipid profiles with systemic biomarkers of lipid intake. We found that serum and red blood cell lipids, and to a lesser extent orbital fat, are indeed excellent biomarkers of retinal lipid content and n-3/n-6 ratios in both the LC-PUFA and VLC-PUFA series. Eyes from age-related macular degeneration (AMD) donors have significantly decreased levels of VLC-PUFAs and low n-3/n-6 ratios. These results are consistent with the protective role of dietary n-3 LC-PUFAs against AMD and emphasize the importance of monitoring systemic biomarkers of lipid intake when undertaking clinical trials of lipid supplements for prevention and treatment of retinal disease. The human retina is well-known to have unique lipid profiles enriched in long-chain polyunsaturated fatty acids (LC-PUFAs) and very long-chain polyunsaturated fatty acids (VLC-PUFAs) that appear to promote normal retinal structure and function, but the influence of diet on retinal lipid profiles in health and disease remains controversial. In this study, we examined two independent cohorts of donor eyes and related their retinal lipid profiles with systemic biomarkers of lipid intake. We found that serum and red blood cell lipids, and to a lesser extent orbital fat, are indeed excellent biomarkers of retinal lipid content and n-3/n-6 ratios in both the LC-PUFA and VLC-PUFA series. Eyes from age-related macular degeneration (AMD) donors have significantly decreased levels of VLC-PUFAs and low n-3/n-6 ratios. These results are consistent with the protective role of dietary n-3 LC-PUFAs against AMD and emphasize the importance of monitoring systemic biomarkers of lipid intake when undertaking clinical trials of lipid supplements for prevention and treatment of retinal disease. The vertebrate retina has long been known to contain unusual lipid profiles that appear to be essential for normal visual function. The photoreceptor cell rod outer segments have extraordinarily high levels of n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) such as DHA (22:6n-3) (1Fliesler A.J. Anderson R.E. Chemistry and metabolism of lipids in the vertebrate retina.Prog. Lipid Res. 1983; 22: 79-131Crossref PubMed Scopus (824) Google Scholar) that far exceed concentrations encountered in the serum and other tissues. More recently, a new class of nondietary polyunsaturated fatty acids with chain lengths greater than 24 carbons, the very long-chain polyunsaturated acids (VLC-PUFAs), were identified in the vertebrate retina and just a few other tissues (2Agbaga M.P. Mandal M.N. Anderson R.E. Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein.J. Lipid Res. 2010; 51: 1624-1642Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 3Aveldaño M.I. A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina.J. Biol. Chem. 1987; 262: 1172-1179Abstract Full Text PDF PubMed Google Scholar). Enhanced membrane fluidity contributed by LC-PUFAs and VLC-PUFAs is thought to be essential for the maintenance of the highly curved membrane disks of the photoreceptor outer segments, but their high degree of unsaturation renders these lipids susceptible to oxidative damage (4Suh M. Wierzbicki A.A. Lien E.L. Clandinin M.T. Dietary 20:4n-6 and 22:6n-3 modulates the profile of long- and very-long-chain fatty acids, rhodopsin content, and kinetics in developing photoreceptor cells.Pediatr. Res. 2000; 48: 524-530Crossref PubMed Scopus (35) Google Scholar, 5McMahon A. Kedzierski W. Polyunsaturated very-long-chain C28–C36 fatty acids and retinal physiology.Br. J. Ophthalmol. 2010; 94: 1127-1132Crossref PubMed Scopus (28) Google Scholar). Retinal n-3 and n-6 LC-PUFAs cannot be synthesized de novo in vertebrates and must be consumed either intact or from a select group of precursors such as α-linolenic acid (18:3n-3, typically from vegetable sources), EPA (20:5n-3, commonly from marine sources), linoleic acid (18:2n-6), and arachidonic acid (AA, 20:4n-6). The influence of dietary intake on retinal levels and visual health remains controversial, however, especially in humans (6Makrides M. Neumann M.A. Byard R.W. Simmer K. Gibson R.A. 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Lipid Res. 2009; 50: S400-S405Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), while inflammation is promoted by n-6 LC-PUFAs such as AA (13Dwyer J.H. Allayee H. Dwyer K.M. Fan J. Wu H. Mar R. Lusis A.J. Mehrabian M. Arachidonate 5-lipoxygenase promoter genotype, dietary arachidonic acid, and atherosclerosis.N. Engl. J. Med. 2004; 350: 29-37Crossref PubMed Scopus (553) Google Scholar). A balance between dietary n-3 and n-6 LC-PUFAs is essential for optimal function of cell membranes, enzyme activity, gene expression, and cell-to-cell communication. Multiple epidemiological studies have indicated that diets rich in n-3 LC-PUFAs are associated with lower risk of age-related macular degeneration (AMD) (14Cho 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. 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Oral docosahexaenoic acid in the prevention of exudative age-related macular degeneration: the Nutritional AMD Treatment 2 study.Ophthalmology. 2013; 120: 1619-1631Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 18Merle B.M.J. Benlian P. Puche N. Bassols A. Delcourt C. Souied E.H. Nutritional AMD Treatment 2 Study Group Circulating omega-3 fatty acids and neovascular age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2014; 55: 2010-2019Crossref PubMed Scopus (72) Google Scholar) or the Age-Related Eye Disease Study 2 (AREDS2) randomized, placebo-controlled clinical trials (19The Age-Related Eye Disease Study 2 (AREDS2) 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.J. Am. Med. Assoc. 2013; 309: 2005-2015Crossref PubMed Scopus (839) Google Scholar). The n-3 and n-6 VLC-PUFAs are not normally consumed in the human diet, and EPA and AA appear to be their major precursors, respectively, in a process mediated by the action of the elongation of very long-chain fatty acids elongase 4 (ELOVL4) enzyme (Fig. 1) (20Agbaga M-P. Logan S. Brush R.S. Anderson R.E. Biosynthesis of very long-chain polyunsaturated fatty acids in hepatocytes expressing ELOVL4.in: Ash J.D. Grimm C. Hollyfield J.G. Anderson R.E. LaVail M.M. Rickman C.B. In Retinal degenerative diseases. Springer, New York2014: 631-636Crossref Scopus (7) Google Scholar). Autosomal dominant mutations in this enzyme lead to a rare form of Stargardt macular dystrophy (STGD), STGD3 (21Zhang 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: 89-93Crossref PubMed Scopus (375) Google Scholar, 22Bernstein P.S. Tammur J. Singh N. 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Alkuraya H. Bedell M. Sun W. Wang X. Hsu Y.H. Esteve-Rudd J. Hughes G. et al.Essential role of ELOVL4 protein in very long chain fatty acid synthesis and retinal function.J. Biol. Chem. 2012; 287: 11469-11480Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Our study of dietary biomarkers of lipid consumption has shown that members of a Utah family with STGD3 who consume large amounts of fish have a milder phenotype than those who rarely consume fish (26Hubbard A.F. Askew E.W. Singh N. Leppert M. Bernstein P.S. Association of adipose and red blood cell lipids with severity of dominant Stargardt macular dystrophy (STGD3) secondary to an ELOVL4 mutation.Arch. Ophthalmol. 2006; 124: 257-263Crossref PubMed Scopus (25) Google Scholar), and an open-label clinical intervention trial with fish oil supplementation is in progress for this family (ClinicalTrials.gov, #NCT00420602). In order to clarify the roles of LC-PUFAs and VLC-PUFAs in retinal health and disease, we previously undertook a study of human donor eyes to determine whether abnormalities in lipid profiles are present in AMD eyes. We found that DHA and many VLC-PUFA levels are significantly lower in AMD eyes relative to age-matched controls and that n-3/n-6 ratios are significantly lower for both LC-PUFAs and VLC-PUFAs (27Liu 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: 3217-3229Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Because ELOVL4 genetic variants are not associated with AMD risk (28DeAngelis M.M. Ji F. Kim I.K. Adams S. Capone Jr., A. Ott J. Miller J.W. Dryja T.P. Cigarette smoking, CFH, APOE, ELOVL4, and risk of neovascular age-related macular degeneration.Arch. Ophthalmol. 2007; 125: 49-54Crossref PubMed Scopus (97) Google Scholar), these findings suggested that a retinal deficiency of LC-PUFAs and VLC-PUFAs influenced by diet and/or a dietary imbalance of n-3/n-6 LC-PUFA ratios may be involved in AMD pathology, but our study was limited by the low number of AMD eyes (n = 8) and lack of availability of biomarkers of dietary lipid intake. We now report follow-up studies showing that dietary intake of LC-PUFA and VLC-PUFA precursors clearly influence retinal lipid profiles in retina, and they confirm that AMD eyes have numerous abnormalities in these profiles. The studies reported here were conducted on two distinct collections of human donor eye tissues. All experimental procedures including tissue procurement were conducted according to the tenets of the Declaration of Helsinki. In the first-phase study, human donor eyes with no history of eye disease were obtained from the Utah Lions Eye Bank. The time between donor death and enucleation was 63 µm and ≤125 µm); 3, soft drusen (>125 µm) with or without pigmentary irregularities; 4A, geographic atrophy; 4B, choroidal neovascular membrane; 4C, both 4A and 4B].ELOVL4 (rs3812153)AdipoR1 (rs10753929)Control subjects0166-9883F0TTCC0171-9874F0TTCT0020-9876F0TTCC0248-9876M0TTCC0010-0282M0TTCT0151-0283F0TTCC0148-9974F0TTCC0171-9984F0TTCC0317-9979M0TTCT0059-0088M0TTCC0088-0078M0TTCC0312-0074M0TTCT0508-0183F0TTCC0101-1176F0TTCT0206-1186M0TTCC0815-1177M0CTCT0742-1183F0TTCC0549-1187F0TTCC0307-1382M0CTCC>1522-1286M0TTCC0487-1281F0TTCCAMD subjects0459-9983M2TTCC0002-0085M2CTCC0373-0180F2TTCC0457-0185M2TTCC0498-0174F4BCTCC0149-0273M2TTCT0034-0287F2TTCC1278-1184F2TTCC0564-1182F4BCTCC0596-1185F4CTTCC0754-1389F1BCTCC0896-1191M3TTCC0919-1277M1BTTCC0258-1183F2TTCC0020-1285F4ATTCCCC, the lower risk allele of AdipoR1; CT, the higher risk allele of AdipoR1 and ELOVL4; TT, the lower risk allele of ELOVL4.a AMD grading based on a modified Rotterdam scale (31van Leeuwen R. Klaver C.C. Vingerling J.R. Hofman A. de Jong P.T. The risk and natural course of age-related maculopathy: follow-up at 6 1/2 years in the Rotterdam study.Arch. Ophthalmol. 2003; 121: 519-526Crossref PubMed Scopus (279) Google Scholar) [0, no signs of maculopathy; 1A, distinct drusen (≤63 µm); 1B, pigmentary irregularities only; 2, soft drusen (>63 µm and ≤125 µm); 3, soft drusen (>125 µm) with or without pigmentary irregularities; 4A, geographic atrophy; 4B, choroidal neovascular membrane; 4C, both 4A and 4B]. Open table in a new tab CC, the lower risk allele of AdipoR1; CT, the higher risk allele of AdipoR1 and ELOVL4; TT, the lower risk allele of ELOVL4. All chemical reagents, such as methanol, hydrochloric acid, isopropanol, n-hexane, n-nonane, and diethyl ether, were of GC/MS grade and purchased from Fisher Scientific (Pittsburgh, PA). All standards including the internal standards such as tridecanoic acid (13:0), hentriacontanoic acid (34:0), and all fatty acids methyl esters (FAMEs) such as methyl eicosanoate, methyl linolenate, methyl melissate, and Supelco-37 (a commercial mixture of FAMEs) were purchased from Sigma-Aldrich (St. Louis, MO) and Matreya (Pleasant Gap, PA). Silica gel, glass-encased, solid-phase extraction cartridges (500 mg/6 ml) were purchased from Sorbent Technology (Atlanta, GA). The internal standards, tridecanoic acid and hentriacontanoic acid, were dissolved in nonane at concentrations of 1.0 mg/ml and 0.023 mg/ml, respectively. Serum, RBCs, orbital adipose tissue, and retina punches were extracted using the procedure previously adopted in our laboratory (30Liu 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: 191-200Crossref PubMed Scopus (28) Google Scholar). The samples and internal standards (50 µg of tridecanoic acid and 1.15 µg of hentriacontanoic acid) were added in 2 ml stainless steel vials and then homogenized with 0.7 ml silica beads and 1 ml hexane-isopropanol (3:2 v/v) by a Mini Bead Beater-16 (BioSpec Products Inc., Bartlesville, OK) and a Sonic Dismembrator Model 100 (Fisher Scientific). The homogenized samples were bath sonicated for 5 min in an ice water bath. After centrifugation at 10,000 rpm for 5 min, the extracted solution supernatant was transferred to a clean vial and then dried under a stream of nitrogen. The dried film was dissolved in 200 µl hexane, and 2 ml of 4% HCl in methanol was added. The tubes were flushed with argon and incubated at 80°C for 4 h to form FAMEs (30Liu 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: 191-200Crossref PubMed Scopus (28) Google Scholar) and then allowed to cool. The FAME mixture was extracted three times with 1 ml distilled water and 2 ml hexane. The hexane layers were combined and dried under nitrogen gas. Silica gel, glass-encased, solid-phase extraction cartridges were subsequently used to clean the FAME extracts. The cartridge was activated with 6 ml of hexane before loading samples. The crude FAME extract was dissolved in 200 µl of hexane and loaded onto the activated cartridge. The cartridge was washed with 6 ml hexane, and the eluate was discarded. FAMEs were eluted from the cartridge with 5 ml hexane-ether (4:1), and the eluate was evaporated under nitrogen gas. The dry film was dissolved in 200 µl of hexane and centrifuged for 3 min at 14,000 rpm to remove particles prior to GC/MS analysis. One microliter of sample was injected into the GC/MS instrument for LC-PUFA analysis. For VLC-PUFA analysis, the sample was dried with nitrogen again and redissolved in 20 μl of n-nonane, and 5 µl samples were injected into the GC/MS instrument. The Thermo Trace GC-DSQ II system (ThermoFisher Scientific, Waltham, MA) consists of an automatic sample injector (AS 3000), gas chromatograph, single quadrupole mass detector, and an analytical workstation. The chromatographic separation was carried out with an Rxi-5MS-coated 5% diphenyl/95% dimethyl polysiloxane capillary column (30 m × 0.25 mm inner diameter, 0.25 µm film thickness) (Restek, Bellefonte, PA). Two methods (A and B) were used for detection and quantitation of LC-PUFAs and VLC-PUFAs, respectively. For LC-PUFA analyses, the following MS conditions were used (method A): 1 µl from a 200 µl sample was injected into the GC/MS using a splitless mode, the septum purge was on, and the injector temperature was set at 200°C. The column temperature was programmed as follows: initial temperature 60°C, 5 degrees/min to 170°C, 1 degree/min to 180°C, 2 degrees/min to 240°C, 4 degrees/min to 290°C, and a hold at 290°C for 5 min. Transfer line temperature was 290°C. Helium was used as the carrier gas at a flow rate of 1.0 ml/min. MS conditions were as follows: electron ionization mode with full scan and selected ion monitoring (SIM, m/z 79, 108, and 150) because m/z 79, 108, and 150 are typical ions of PUFAs, and n-3 and n-6 PUFAs can be distinguished by comparing the ratio of ions of m/z 108 and 150; ion source temperature, 200°C; multiplier voltage, 1,182 V; and detector delay, 10 min. For peak identification, the data were obtained by collecting the full-scan mass spectra within the scan range of 50–650 amu, and these peaks were identified by comparing their mass spectra with those in the standard solution and the National Institute of Standards and Technology library. For the quantification of LC-PUFAs, the data were obtained by SIM. Authentic reference compounds were used to calculate the mol percentage of each peak. VLC-PUFAs analyses (method B) were conducted as described previously (30Liu 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: 191-200Crossref PubMed Scopus (28) Google Scholar). Bovine retina VLC-PUFAs were extracted and used as VLC-PUFA standards to establish retention times because commercial standards are not available, and identification of each VLC-PUFA in retinal samples was achieved as described in prior work from our laboratory (27Liu 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: 3217-3229Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). For the quantification of VLC-PUFAs, we used MS conditions similar to the LC-PUFA method but with a larger injection volume of 5 µl from a 20 µl sample that had been concentrated from a 200 µl original volume. The column temperature program was similar to the LC-PUFA method but held at 290°C for 35 min. MS conditions were similar to the LC-PUFA method, but the detector delay time was 20 min. The low amounts of VLC-PUFAs that are present in the mammalian retina elute very late from the GC/MS, and standards are not available commercially, which means that their quantitation can be particularly challenging. To achieve this, two separate GC/MS runs linked by common C-24 PUFAs were necessary. With method A, the complete set of long-chain fatty acids (LC-FAs) up to 22 carbons in length and two C-24 FAs (24:1n-9 and 24:0) can be quantified under full scan mode, and when the chromatogram is reanalyzed under selective ion mode (m/z 79, 108, and 150), we can identify and quantify all n-3 and n-6 LC-PUFAs, and even the C-24 VLC-PUFAs become detectable and can be quantified by comparing their mole percentages relative to the C-22 PUFAs. The C-24 VLC-PUFAs can then be used as the common link between method A and method B because they are present in both GC/MS chromatograms. All the VLC-PUFAs could be subsequently quantified relative to the total LC-FAs determined by method A after correcting for the effects of carbon chain length and the degree of unsaturation on the response of the mass spectrometer (30Liu 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: 191-200Crossref PubMed Scopus (28) Google Scholar). Genotyping was done using the TaqMan platform (Applied Biosystems, Grand Island, NY). Amplification and genotype assignments were conducted using the 7900HT and SDS 2.4 software. The SNPs, rs381253 and rs10753929, used in this study were selected based on their association with ELOVL4 and adiponectin receptor 1 (AdipoR1) as reported in earlier literature (32Yi J. Li S. Jia X. Xiao X. Wang P. Guo X. Zhang Q. Evaluation of the ELOVL4, PRPH2 and ABCA4 genes in patients with Stargardt macular degeneration.Mol. Med. Rep. 2012; 6: 1045-1049Crossref PubMed Scopus (23) Google Scholar, 33Kaarniranta K. Paananen J. Nevalainen T. Sorri I. Seitsonen S. Immonen I. Salminen A. Pulkkinen L. Uusitupa M. Adiponectin receptor 1 gene (ADIPOR1) variant is associated with advanced age-related macular degeneration in Finnish population.Neurosci. Lett. 2012; 513: 233-237Crossref PubMed Scopus (28) Google Scholar). Statistical analyses were performed using ANOVA, linear regressions, Chi-square tests, and t-tests on Prism software (GraphPad Software Inc., La Jolla, CA). Data are represented as the mean ± SD. Significance is indicated by P value measurements, with P < 0.05 considered significant. Forty-four normal patient samples were collected (average age ± SD = 71.4 ± 13.4 years), and their serum, RBCs, orbital fat, and retinal punches were analyzed using GC/MS techniques. We studied the correlations between the retinal lipid profile and those of serum, RBCs, and fat, which are validated biomarkers of short-term (weeks), medium-term (months), and long-term (years) dietary fat intake (26Hubbard A.F. Askew E.W. Singh N. Leppert M. Bernstein P.S. Association of adipose and red blood cell lipids with severity of dominant Stargardt macular dystrophy (STGD3) secondary to an ELOVL4 mutation.Arch. Ophthalmol. 2006; 124: 257-263Crossref PubMed Scopus (25) Google Scholar, 34Sarkkinen E.S. Agren J.J. Ahola I. Ovaskainen M.L. Uusitupa M.I. Fatty acid composition of serum cholesterol esters, and erythrocyte and platelet membranes as indicators of long-term adherence to fat-modified diets.Am. J. Clin. Nutr. 1994; 59: 364-370Crossref PubMed Scopus (99) Google Scholar, 35Baylin A. Kabagambe E.K. Siles X. Campos H. Adipose tissue biomarkers of fatty acid intake.Am. J. Clin. Nutr. 2002; 76: 750-757Crossref PubMed Scopus (264) Google Scholar, 36Baylin A. Campos H. The use of fatty acid biomarkers to reflect dietary intake.Curr. Opin. Lipidol. 2006; 17: 22-27Crossref PubMed Scopus (183) Google Scholar) (Table 2). Individual key LC-PUFAs (EPA, DHA, and AA) in serum, RBCs, and fat generally showed significant positive correlations with corresponding levels in retinal lipids. We observed strong positive correlations between EPA levels in serum, RBCs, and fat with EPA levels in retina (all P < 0.001), while retinal DHA levels correlate relatively weakly only with DHA levels of RBCs (P < 0.05). The levels of the major n-6 fatty acid in retina, AA, were also in positive correlation with AA levels in serum and RBCs (P < 0.001). Likewise, serum and RBC total LC-PUFAs positively correlated with retinal total LC-PUFAs (P < 0.001), but fat had a weaker negative correlation (P < 0.05) (Table 2).TABLE 2Correlations of LC-PUFA levels in serum, RBCs, and fat with retina lipidsaP values: *** P < 0.001; ** P < 0.01; * P < 0.05. Values are based on regression analysis.Systemic LipidsSerumRBCsFatRetinal LipidsEPA0.78***0.60***0.55***EPADHA0.080.29*−0.17DHAAA0.62***0.60***−0.26AATotal LC-PUFAs0.44***0.51***−0.30*Total LC PUFAsn-3 LC-PUFAsbn-3 LC-PUFAs: 18:3n-3, 18:4n-3, 20:4n-3, 20:5n-3, 22:6n-3, 22:5n-3.0.160.26**−0.20n-3 LC-PUFAsn-6 LC-PUFAscn-6 VLC-PUFAs: 18:2n-6, 18:3n-6, 20:3n-6, 20:4n-6, 22:4n-6.0.50***0.37***0.39***n-6 LC-PUFAsa P values: *** P < 0.001; ** P < 0.01; * P < 0.05. Values are based on regression analysis.b n-3 LC-PUFAs: 18:3n-3, 18:4n-3, 20:4n-3, 20:5n-3, 22:6n-3, 22:5n-3.c n-6 VLC-PUFAs: 18:2n-6, 18:3n-6, 20:3n-6, 20:4n-6, 22:4n-6.