Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration

2010; Elsevier BV; Volume: 51; Issue: 11 Linguagem: Inglês

10.1194/jlr.m007518

ISSN

1539-7262

Autores

Aihua Liu, Jen-Yuan Chang, Yanhua Lin, Zhengqing Shen, Paul S. Bernstein,

Tópico(s)

Cholesterol and Lipid Metabolism

Resumo

Retinal long-chain PUFAs (LC-PUFAs, C12-C22) play important roles in normal human retinal function and visual development, and some epidemiological studies of LC-PUFA intake suggest a protective role against the incidence of advanced age-related macular degeneration (AMD). On the other hand, retinal very long-chain PUFAs (VLC-PUFAs, Cn>22) have received much less attention since their identification decades ago, due to their minor abundance and more difficult assays, but recent discoveries that defects in VLC-PUFA synthetic enzymes are associated with rare forms of inherited macular degenerations have refocused attention on their potential roles in retinal health and disease. We thus developed improved GC-MS methods to detect LC-PUFAs and VLC-PUFAs, and we then applied them to the study of their changes in ocular aging and AMD. With ocular aging, some VLC-PUFAs in retina and retinal pigment epithelium (RPE)/choroid peaked in middle age. Compared with age-matched normal donors, docosahexaenoic acid, adrenic acid, and some VLC-PUFAs in AMD retina and RPE/choroid were significantly decreased, whereas the ratio of n-6/n-3 PUFAs was significantly increased. All these findings suggest that deficiency of LC-PUFAs and VLC-PUFAs, and/or an imbalance of n-6/n-3 PUFAs, may be involved in AMD pathology. Retinal long-chain PUFAs (LC-PUFAs, C12-C22) play important roles in normal human retinal function and visual development, and some epidemiological studies of LC-PUFA intake suggest a protective role against the incidence of advanced age-related macular degeneration (AMD). On the other hand, retinal very long-chain PUFAs (VLC-PUFAs, Cn>22) have received much less attention since their identification decades ago, due to their minor abundance and more difficult assays, but recent discoveries that defects in VLC-PUFA synthetic enzymes are associated with rare forms of inherited macular degenerations have refocused attention on their potential roles in retinal health and disease. We thus developed improved GC-MS methods to detect LC-PUFAs and VLC-PUFAs, and we then applied them to the study of their changes in ocular aging and AMD. With ocular aging, some VLC-PUFAs in retina and retinal pigment epithelium (RPE)/choroid peaked in middle age. Compared with age-matched normal donors, docosahexaenoic acid, adrenic acid, and some VLC-PUFAs in AMD retina and RPE/choroid were significantly decreased, whereas the ratio of n-6/n-3 PUFAs was significantly increased. All these findings suggest that deficiency of LC-PUFAs and VLC-PUFAs, and/or an imbalance of n-6/n-3 PUFAs, may be involved in AMD pathology. The biochemical profile of FAs in human retina was first reported over 45 years ago when Futterman and Andrews (1Futterman S. Andrews J.S. The fatty acid composition of human retinal vitamin A ester and the lipids of human retinal tissue.Invest. Ophthalmol. 1964; 3: 441-444PubMed Google Scholar) identified five major FAs in human retina, including docosahexaenoic acid (DHA, 22:6n-3), arachidonic acid (AA, 20:4n-6), stearic acid (SA, 18:0), oleic acid (OA, 18:1), and palmitic acid (PA, 16:0), all of which belong to the long-chain FA (LC-FA, C12-C22) family. Subsequent studies demonstrated that DHA and AA are the major components of human and vertebrate retinal long-chain PUFAs (LC-PUFAs), and the highest content of LC-PUFAs is found in human and vertebrate retinal rod outer segment membranes (2van Kuijk F.J. Buck P. Fatty acid composition of the human macula and peripheral retina.Invest. Ophthalmol. Vis. Sci. 1992; 33: 3493-3496PubMed Google Scholar, 3Martinez M. Ballabriga A. Gil-Gibernau J.J. Lipids of the developing human retina: I. Total fatty acids, plasmalogens, and fatty acid composition of ethanolamine and choline phosphoglycerides.J. Neurosci. Res. 1988; 20: 484-490Crossref PubMed Scopus (73) Google Scholar, 4Anderson R.E. Lipids of ocular tissues. IV. A comparison of the phospholipids from the retina of six mammalian species.Exp. Eye Res. 1970; 10: 339-344Crossref PubMed Scopus (163) Google Scholar). Their high content and specific tissue distribution suggest that LC-PUFAs have an important functional role in the retina. Human clinical studies and animal studies involving dietary manipulation in rats (5Benolken R.M. Anderson R.E. Wheeler T.G. Membrane fatty acids associated with the electrical response in visual excitation.Science. 1973; 182: 1253-1254Crossref PubMed Scopus (249) Google Scholar, 6Wheeler T.G. Benolken R.M. Anderson R.E. Visual membranes: specificity of fatty acid precursors for the electrical response to illumination.Science. 1975; 188: 1312-1314Crossref PubMed Scopus (290) Google Scholar) have shown that LC-PUFAs are important for the development and function of the central nervous system, particularly the visual system (7Greiner R.S. Moriguchi T. Hutton A. Slotnick B.M. Salem Jr, N. Rats with low levels of brain docosahexaenoic acid show impaired performance in olfactory-based and spatial learning tasks.Lipids. 1999; 34: S239-S243Crossref PubMed Google Scholar). DHA prevented photoreceptor apoptosis caused by oxidative damage (8Rotstein N.P. Politi L.E. German O.L. Girotti R. Protective effect of docosahexaenoic acid on oxidative stress-induced apoptosis of retina photoreceptors.Invest. Ophthalmol. Vis. Sci. 2003; 44: 2252-2259Crossref PubMed Scopus (139) Google Scholar) and promoted differentiation of developing photoreceptors in culture (9Rotstein N.P. Politi L.E. Aveldano M.I. Docosahexaenoic acid promotes differentiation of developing photoreceptors in culture.Invest. Ophthalmol. Vis. Sci. 1998; 39: 2750-2758PubMed Google Scholar). DHA also has significant effects on photoreceptor membranes and neurotransmitters involved in the signal transduction process, rhodopsin activation, rod and cone development, neuronal dendritic connectivity, and functional maturation of the central nervous system (10Uauy R. Hoffman D.R. Peirano P. Birch D.G. Birch E.E. Essential fatty acids in visual and brain development.Lipids. 2001; 36: 885-895Crossref PubMed Scopus (358) Google Scholar). Low plasma and blood cell lipid DHA have been linked to increased risk of poor visual and neural development in infants and children (11Hibbeln J.R. Davis J.M. Steer C. Emmett P. Rogers I. Williams C. Golding J. Maternal seafood consumption in pregnancy and neurodevelopmental outcomes in childhood (ALSPAC study): an observational cohort study.Lancet. 2007; 369: 578-585Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar, 12Innis S.M. Friesen R.W. Essential n-3 fatty acids in pregnant women and early visual acuity maturation in term infants.Am. J. Clin. Nutr. 2008; 87: 548-557Crossref PubMed Scopus (165) Google Scholar). Depletion of DHA, AA, or eicosapentaenoic acid (EPA) from the retina interferes with normal neurogenesis, neurological function, and visual signaling pathways, impairs vision and visual learning tasks, and leads to an abnormal electroretinogram (13Alessandri J.M. Guesnet P. Vancassel S. Astorg P. Denis I. Langelier B. Aid S. Poumes-Ballihaut C. Champeil-Potokar G. Lavialle M. Polyunsaturated fatty acids in the central nervous system: evolution of concepts and nutritional implications throughout life.Reprod. Nutr. Dev. 2004; 44: 509-538Crossref PubMed Scopus (230) Google Scholar, 14Weisinger H.S. Vingrys A.J. Sinclair A.J. The effect of docosahexaenoic acid on the electroretinogram of the guinea pig.Lipids. 1996; 31: 65-70Crossref PubMed Scopus (96) Google Scholar, 15Uauy R. Peirano P. Hoffman D. Mena P. Birch D. Birch E. Role of essential fatty acids in the function of the developing nervous system.Lipids. 1996; 31: S167-S176Crossref PubMed Google Scholar, 16Connor W.E. Neuringer M. Reisbick S. Essential fatty acids: the importance of n-3 fatty acids in the retina and brain.Nutr. Rev. 1992; 50: 21-29Crossref PubMed Scopus (211) Google Scholar, 17Neuringer 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: 272-276Crossref PubMed Scopus (505) Google Scholar, 18Neuringer M. Connor W.E. Lin D.S. Barstad L. Luck S. Biochemical and functional effects of prenatal and postnatal omega 3 fatty acid deficiency on retina and brain in rhesus monkeys.Proc. Natl. Acad. Sci. USA. 1986; 83: 4021-4025Crossref PubMed Scopus (757) Google Scholar, 19Neuringer M. Anderson G.J. Connor W.E. The essentiality of n-3 fatty acids for the development and function of the retina and brain.Annu. Rev. Nutr. 1988; 8: 517-541Crossref PubMed Scopus (669) Google Scholar, 20Tinoco J. Williams M.A. Hincenbergs I. Lyman R.L. Evidence for nonessentiality of linolenic acid in the diet of the rat.J. Nutr. 1971; 101: 937-945Crossref PubMed Scopus (51) Google Scholar). Thus, it is clear that LC-PUFAs play an essential role in the development of vision during human early life (16Connor W.E. Neuringer M. Reisbick S. Essential fatty acids: the importance of n-3 fatty acids in the retina and brain.Nutr. Rev. 1992; 50: 21-29Crossref PubMed Scopus (211) Google Scholar, 21Cho 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: 209-218Crossref PubMed Scopus (294) Google Scholar). In later human life, some epidemiological studies of LCn-3PUFA intake on the prevalence of advanced age-related macular degeneration (AMD) suggest a protective relationship (21Cho 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: 209-218Crossref PubMed Scopus (294) Google Scholar, 22Sangiovanni J.P. Agron E. Meleth A.D. Reed G.F. Sperduto R.D. Clemons T.E. Chew E.Y. {omega}-3 Long-chain polyunsaturated fatty acid intake and 12-y incidence of neovascular age-related macular degeneration and central geographic atrophy: AREDS report 30, a prospective cohort study from the Age-Related Eye Disease Study.Am. J. Clin. Nutr. 2009; 90: 1601-1607Crossref PubMed Scopus (129) Google Scholar, 23Parekh N. Voland R.P. Moeller S.M. Blodi B.A. Ritenbaugh C. Chappell R.J. Wallace R.B. Mares J.A. Association between dietary fat intake and age-related macular degeneration in the Carotenoids in Age-Related Eye Disease Study (CAREDS): an ancillary study of the Women's Health Initiative.Arch. Ophthalmol. 2009; 127: 1483-1493Crossref PubMed Scopus (64) Google Scholar, 24Tuo J. Ross R.J. Herzlich A.A. Shen D. Ding X. Zhou M. Coon S.L. Hussein N. Salem Jr., N. Chan C.C. A high omega-3 fatty acid diet reduces retinal lesions in a murine model of macular degeneration.Am. J. Pathol. 2009; 175: 799-807Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 25SanGiovanni 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: 87-138Crossref PubMed Scopus (609) Google Scholar, 26Seddon 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: 995-1001Crossref PubMed Scopus (310) Google Scholar, 27Hodge W.G. Barnes D. Schachter H.M. Pan Y.I. Lowcock E.C. Zhang L. Sampson M. Morrison A. Tran K. Miguelez M. et al.Evidence for the effect of omega-3 fatty acids on progression of age-related macular degeneration: a systematic review.Retina. 2007; 27: 216-221Crossref PubMed Scopus (25) Google Scholar), and Hubbard et al. (28Hubbard 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) showed a protective effect of DHA and EPA intake in autosomal dominant Stargardt disease. Their role in the prevention of inherited retinal degenerations such as retinitis pigmentosa is more ambiguous (29Li F. Marchette L.D. Brush R.S. Elliott M.H. Le Y.Z. Henry K.A. Anderson A.G. Zhao C. Sun X. Zhang K. et al.DHA does not protect ELOVL4 transgenic mice from retinal degeneration.Mol. Vis. 2009; 15: 1185-1193PubMed Google Scholar). Although retinal LC-PUFAs have received extensive attention over the past decades, very long-chain PUFAs (VLC-PUFAs, Cn>22) (30Hardy S.J. Ferrante A. Poulos A. Robinson B.S. Johnson D.W. Murray A.W. Effect of exogenous fatty acids with greater than 22 carbon atoms (very long chain fatty acids) on superoxide production by human neutrophils.J. Immunol. 1994; 153: 1754-1761PubMed Google Scholar, 31Suh 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: 959-968Crossref PubMed Scopus (41) Google Scholar) are also found in mammalian tissues, but they occur in only a restricted number of organs such as retina, testes, thymus, and brain in a low amount and their saturated analogs are essential parts of the skin moisture barrier (32Poulos A. Very long chain fatty acids in higher animals–a review.Lipids. 1995; 30: 1-14Crossref PubMed Scopus (152) Google Scholar, 33Aveldano 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). Unlike DHA, AA, EPA, and their precursors, VLC-PUFAs are not present in a normal human diet (34McMahon A. Kedzierski W. Polyunsaturated very-long-chain C28–C36 fatty acids and retinal physiology.Br J Ophthalmol. 2010; (Epub ahead of print. May 21, 2010)Crossref Scopus (28) Google Scholar), but they can be synthesized from precursors such as 22:4n-6 and 22:5n-3 via a biochemical pathway (supplementary Fig. I) featuring the enzymes of the elongation of very long-chain FAs family along with β-oxidases and desaturases (31Suh 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: 959-968Crossref PubMed Scopus (41) Google Scholar, 35Denic V. Weissman J.S. A molecular caliper mechanism for determining very long-chain fatty acid length.Cell. 2007; 130: 663-677Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 36Agbaga 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: 12843-12848Crossref PubMed Scopus (200) Google Scholar, 37Agbaga 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 (121) Google Scholar). Defects in the elongation of the very long-chain FAs-4 (ELOVL4) gene are associated with autosomal dominant Stargardt macular dystrophy in the heterozygous state and neonatal demise in transgenic mice due to loss of skin barrier function in the homozygous state. VLC-PUFAs are of particular interest, because they exhibit a unique hybrid structure combining a proximal end with typical saturated FA character and a distal end more characteristic of common PUFAs (35Denic V. Weissman J.S. A molecular caliper mechanism for determining very long-chain fatty acid length.Cell. 2007; 130: 663-677Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 36Agbaga 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: 12843-12848Crossref PubMed Scopus (200) Google Scholar). They may play important roles in biological systems that cannot be performed by the more common saturated and unsaturated LC-FAs (35Denic V. Weissman J.S. A molecular caliper mechanism for determining very long-chain fatty acid length.Cell. 2007; 130: 663-677Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 36Agbaga 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: 12843-12848Crossref PubMed Scopus (200) Google Scholar); however, their greater length and minor abundance make them unusually difficult to analyze (36Agbaga 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: 12843-12848Crossref PubMed Scopus (200) Google Scholar), which means that VLC-PUFAs have often been overlooked since their discovery (33Aveldano 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, 38Poulos A. Sharp P. Singh H. Johnson D. Fellenberg A. Pollard A. Detection of a homologous series of C26–C38 polyenoic fatty acids in the brain of patients without peroxisomes (Zellweger's syndrome).Biochem. J. 1986; 235: 607-610Crossref PubMed Scopus (54) Google Scholar). In this study, we optimized analytical methods to detect LC-PUFAs and VLC-PUFAs in human retina and retinal pigment epithelium (RPE)/choroid. We applied these methods to explore possible retinal functions of LC-PUFAs and VLC-PUFAs, and we assessed how their levels change in ocular aging and AMD. Human donor eyes were obtained from the Utah Lions Eye Bank within 26 h after death, and donor demographic data are shown in Table 1. All experimental procedures including tissue procurement and distribution were conducted according to the tenets of the Declaration of Helsinki. The time between donor death and enucleation was <4 h. Dissections of donor eyeballs were carried out 6–26 h [(mean ± SD) 17.4 ± 2.3 h] (Table 1) after donor death in a dim-light environment. The posterior pole of donor eyeballs was placed on a back-lit table and observed under a dissecting microscope to prepare the tissue needed in the present study. Normal donor eyes were inspected under magnification to assure that no significant macular pathology was present. Likewise, eyes from donors with a clinical history of AMD were inspected to confirm that their macular characteristics correlated with clinical examinations recorded prior to death. After removal of the vitreous body, the whole retina and the RPE/choroid layer were carefully separated with forceps. Wet sample weights were recorded for all collected tissues after blotting excess moisture. All samples were immediately blanketed with argon and stored at −80°C until further analysis.TABLE 1.The demographic information of all samplesSample no.GroupAverage AgeaAverage ± SD.Gender (M/F)Age (y)Delay of Collection after Death (h)Weight of Retina (g)Weight of RPE/Choroid (g)Eye Disease HistoryCause of Death1Young age group16.4 ± 3.6M17140.15470.0953NoHeart failure2F22210.08790.1208NoRespiratory collapse3F12160.11490.1223NoMultiple sclerosis4F15220.08730.1006NoAnoxic brain injury5M16220.11490.1223NoBlunt force head trauma6Middle age group38.2 ± 6.2M36260.09170.0867NoRespiratory failure7M38260.08460.0775NoAspiration pneumonia8M49130.11570.1181NoRespiratory arrest9M39160.10990.1090NoMyocardial infarction10M3760.07990.0990NoEnd-stage renal failure11M3080.10560.0976NoCongestive heart failure12Old age group74.0 ± 3.4M77150.11640.1363NoTrauma13F78230.13320.0992NoCerebrovascular accident14M72200.15500.1089NoCerebrovascular accident15F7070.12420.1378NoCerebrovascular accident16F73170.15420.1678NoAmyotrophic lateral sclerosis17Age-matched AMD group77.2 ± 6.4M78230.15380.1375AMDCancer18F70230.11230.1462AMDMyocardial infarction19M75220.15570.1562AMDLymphoma20M73170.09470.1008AMDRespiratory failure21M8180.13570.1313AMDbThese AMD patients had exudative AMD. All other AMD patients had early to intermediate dry AMD with drusen and pigmentary changes but no history of geographic atrophy or exudative disease.Circulatory collapse22F84220.25570.2562AMDRenal failure23F87180.16710.1395AMDbThese AMD patients had exudative AMD. All other AMD patients had early to intermediate dry AMD with drusen and pigmentary changes but no history of geographic atrophy or exudative disease.Respiratory failure, breast cancer24M7080.12220.0722AMDCirculatory collapsea Average ± SD.b These AMD patients had exudative AMD. All other AMD patients had early to intermediate dry AMD with drusen and pigmentary changes but no history of geographic atrophy or exudative disease. Open table in a new tab All analytical solutions such as methanol, hydrochloric acid, isopropanol, and n-hexane were GC-MS grade reagents and were purchased from Fisher Scientific (Pittsburgh, PA). All standards such as tridecanoic acid (C13) and hentriacontanoic acid (C31) were purchased from Sigma-Aldrich (St. Louis, MO). Silica gel glass-encased solid phase extraction cartridges (500 mg/6 ml) were purchased from Sorbent Technology (Atlanta, GA). Samples were removed from the −80°C freezer and placed on ice. Tridecanoic acid (20 μg) and hentriacontanoic acid (1 μg) were added as the internal standards. Total lipid was extracted from whole retina or RPE/choroid tissue based on a previously described method (39Hara A. Radin N.S. Lipid extraction of tissues with a low-toxicity solvent.Anal. Biochem. 1978; 90: 420-426Crossref PubMed Scopus (2049) Google Scholar). The samples were probe sonicated with 0.5 ml hexane:isopropanol (3:2) in a glass bottle. Then hexane:isopropanol (3:2) was added to a final volume equivalent to 40 times the sample weight (i.e., 1 g in 40 ml of solvent mixture). The sample bottles sealed under argon with Teflon-lined caps were bath sonicated for 20 min at room temperature. The extract was then centrifuged at 5,000 rpm for 5 min, and the upper layer was dried under vacuum. The dried film dissolved in 200 µl hexane and 2 ml 8% HCl/MeOH was sealed under argon with Teflon-lined caps and heated at 80°C for 4 h to form fatty acid methyl esters (FAMEs) (36Agbaga 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: 12843-12848Crossref PubMed Scopus (200) Google Scholar), then cooled on ice. The cool reacted solution was extracted three times with 1 ml distilled water and 2 ml hexane. The hexane layers were combined and dried under vacuum. Silica gel glass-encased solid-phase extraction cartridges were subsequently used to clean the FAME extracts. The cartridge was activated by 6 ml of hexane before loading samples. The crude FAME extract was dissolved in 200 µl of hexane and loaded onto the activated cartridge. Then 6 ml hexane was used to wash the cartridge, and the eluate was discarded. Then the FAMEs were eluted by 5 ml hexane:ether (8:2), and the eluate was evaporated to dryness under vacuum. 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. Then 1 µl of sample was injected into the GC-MS instrument for LC-PUFA analysis. The sample was dried under vacuum again and redissolved in 30 μl of hexane. Then 5 µl samples were injected into the GC-MS instrument for VLC-PUFA analysis. The Thermo Trace GC-DSQ system (ThermoFisher Scientific, Waltham, MA) consisted of an automatic sample injector (AI 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). For LC-FA analyses, we used the following MS conditions (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 (EI) mode; ion source temperature, 200°C; multiplier voltage, 1,182 V; solvent delay, 5 min. All data were obtained by collecting the full-scan mass spectra within the scan range of 50–650 amu. Compounds were identified by comparing their mass spectra with those in the National Institute of Standards and Technology (NIST) library. Authentic reference compounds were used to calculate the weight percentage of every peak. For VLC-PUFAs, we used the following MS conditions (Method B): 5 µl from 30 µl of sample was injected onto 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; 10 degrees/min to 240°C; 1 degree/min to 290°C; and 290°C for 5 min. Transfer line temperature was 290°C. Helium was used as the carrier gas at a flow rate of 1.5 ml/min. Both liquid chemical ionization (LCI) and EI modes were used to identify VLC-PUFAs, whereas only the EI mode was used for quantification. The MS conditions for LCI were as follows: ion source temperature, 180°C; multiplier voltage, 1,182 V; solvent delay, 22 min; selected ion monitoring (SIM) mode with molecular weights plus one ion; acetonitrile flow rate, 0.1 µl/min. The MS conditions under EI were as follows: ion source temperature, 200°C; multiplier voltage, 1,182 V; solvent delay, 22 min, SIM mode with m/z 79, 108, and 150. Comparison was made by normalizing the each peak area with the internal standard and the retina or RPE/choroid sample weight. The formula to calculate the values in TABLE 4., TABLE 5. and Fig. 5 is as follows: Value=PARF×W×105(Formula 1) RF=PAaPAb(Formula 2) In the first formula, PA, RF, and W are the peak area of target peak, response factor, and sample weight with grams as the unit. In the second formula, PAa is the peak area of IS in the sample, and PAb is the peak area of fresh IS solution at the same concentration.TABLE 4.VLC-PUFA composition from whole retinas collected from young, middle, and old age and age-matched AMD human donorsPeak Area Value Normalized with IS and Sample WeightdMean ± SEM; young age group: n = 5, middle age group: n = 6, old age group: n = 5, age-matched AMD group: n = 8.Peak no.aPeak no. was shown in chromatograms of Fig. 2.RT (min)bRetention time.FAscNumber of carbon atoms: number of double bonds, the position of first double bound.Young Age GroupMiddle Age GroupOld Age GroupAge-Matched AMD Group123.2724:5n-6185.24 ± 49.86193.23 ± 41.19147.60 ± 27.4390.45 ± 18.65223.4324:6n-3677.13 ± 237.78668.48 ± 93.48463.55 ± 55.82151.65 ± 35.28g∗ Significant differences (P < 0.05) between old and age-matched AMD age group.∗323.5824:4n-61199.82 ± 315.711188.55 ± 142.751153.45 ± 210.28684.20 ± 126.50423.7424:5n-31315.65 ± 537.161442.42 ± 246.561124.25 ± 199.82468.76 ± 111.35527.5526:5n-66.64 ± 2.4610.74 ± 2.504.07 ± 0.99f∗ Significant differences (P < 0.05) between middle and old age group.∗3.49 ± 0.92627.7226:6n-325.77 ± 8.7627.74 ± 4.0723.73 ± 5.2210.45 ± 1.78728.0026:4n-660.61 ± 14.5474.85 ± 4.8858.90 ± 17.9023.34 ± 4.20g∗ Significant differences (P < 0.05) between old and age-matched AMD age group.∗828.2226:5n-357.07 ± 15.6165.93 ± 6.2546.70 ± 11.2822.68 ± 4.36933.6128:6n-32.15 ± 0.612.09 ± 0.521.04 ± 0.450.13 ± 0.071033.9528:4n-62.54 ± 0.685.47 ± 0.72e∗ Significant differences (P < 0.05) between young and middle age group.∗2.63 ± 0.88f∗ Significant differences (P < 0.05) between middle and old age group.∗0.44 ± 0.24g∗ Significant differences (P < 0.05) between old and age-matched AMD age group.∗1134.4228:5n-38.71 ± 2.1610.25 ± 1.237.51 ± 3.250.93 ± 0.50g∗ Significant differences (P < 0.05) between old and age-matched AMD age group.∗1240.8230:6n-32.62 ± 0.954.94 ± 0.93e∗ Significant differences (P < 0.05) between young and middle age group.∗2.07 ± 0.73f∗ Significant differences (P < 0.05) between middle and old age group.∗0.66 ± 0.261341.4230:4n-63.61 ± 1.086.84 ± 1.464.29 ± 1.790.90 ± 0.34g∗ Significant differences (P < 0.05) between old and age-matched AMD age group.∗1441.8230:5n-35.57 ± 1.8212.98 ± 2.16e∗ Significant differences (P < 0.05) between young and middle age group.∗4.93 ± 1.00f∗ Significant differences (P < 0.05) between middle and old age group.∗2.20 ± 0.701548.8432:5n-68.45 ± 2.7614.30 ± 2.277.52 ± 2.40f∗ Significant differences (P < 0.05) between middle and old age group.∗1.51 ± 0.38g∗ Significant differences (P < 0.05) between old and age-matched AMD age group.∗1649.3232:6n-331.36 ± 9.6647.84 ± 5.4825.11 ± 5.78f∗ Significant differences (P < 0.05) between middle and old age group.∗3.97 ± 0.97g∗ Significant differences (P < 0.05) between old and age-matched AMD age group.∗1750.0032:4n-640.12 ± 11.3580.71 ± 15.27e∗ Significant differences (P < 0.05) between young and middle age group.∗33.26 ± 8.27f∗ Significant differences (P < 0.05) between middle and old age group.∗9.20 ± 3.211850.4632:5n-320.20 ± 5.9750.74 ± 9.02e∗ Significant differences (P < 0.05) between young and middle age group.∗21.53 ± 6.22f∗ Significant differences (P < 0.05) between middle and old age group.∗3.93 ± 1.35g∗ Significant differences (P < 0.05) between old and age-matched AMD age group.∗1958.4934:6n-320.61 ± 8.4325.94 ± 10.7715.31 ± 3.801.71 ± 0.422059.2134:4n-625.37 ± 8.4536.43 ± 5.7

Referência(s)