Dietary lysophosphatidylcholine-EPA enriches both EPA and DHA in the brain: potential treatment for depression
2018; Elsevier BV; Volume: 60; Issue: 3 Linguagem: Inglês
10.1194/jlr.m090464
ISSN1539-7262
AutoresY. Poorna Chandra Rao, Sugasini Dhavamani, Sridevi Dasarathi, Kalipada Pahan, Papasani V. Subbaiah,
Tópico(s)Adipose Tissue and Metabolism
ResumoEPA and DHA protect against multiple metabolic and neurologic disorders. Although DHA appears more effective for neuroinflammatory conditions, EPA is more beneficial for depression. However, the brain contains negligible amounts of EPA, and dietary supplements fail to increase it appreciably. We tested the hypothesis that this failure is due to absorption of EPA as triacylglycerol, whereas the transporter at the blood-brain barrier requires EPA as lysophosphatidylcholine (LPC). We compared tissue uptake in normal mice gavaged with equal amounts (3.3 μmol/day) of either LPC-EPA or free EPA (surrogate for current supplements) for 15 days and also measured target gene expression. Compared with the no-EPA control, LPC-EPA increased brain EPA >100-fold (from 0.03 to 4 μmol/g); free EPA had little effect. Furthermore, LPC-EPA, but not free EPA, increased brain DHA 2-fold. Free EPA increased EPA in adipose tissue, and both supplements increased EPA and DHA in the liver and heart. Only LPC-EPA increased EPA and DHA in the retina, and expression of brain-derived neurotrophic factor, cyclic AMP response element binding protein, and 5-hydroxy tryptamine (serotonin) receptor 1A in the brain. These novel results show that brain EPA can be increased through diet. Because LPC-EPA increased both EPA and DHA in the brain, it may help in the treatment of depression as well as neuroinflammatory diseases, such as Alzheimer's disease. EPA and DHA protect against multiple metabolic and neurologic disorders. Although DHA appears more effective for neuroinflammatory conditions, EPA is more beneficial for depression. However, the brain contains negligible amounts of EPA, and dietary supplements fail to increase it appreciably. We tested the hypothesis that this failure is due to absorption of EPA as triacylglycerol, whereas the transporter at the blood-brain barrier requires EPA as lysophosphatidylcholine (LPC). We compared tissue uptake in normal mice gavaged with equal amounts (3.3 μmol/day) of either LPC-EPA or free EPA (surrogate for current supplements) for 15 days and also measured target gene expression. Compared with the no-EPA control, LPC-EPA increased brain EPA >100-fold (from 0.03 to 4 μmol/g); free EPA had little effect. Furthermore, LPC-EPA, but not free EPA, increased brain DHA 2-fold. Free EPA increased EPA in adipose tissue, and both supplements increased EPA and DHA in the liver and heart. Only LPC-EPA increased EPA and DHA in the retina, and expression of brain-derived neurotrophic factor, cyclic AMP response element binding protein, and 5-hydroxy tryptamine (serotonin) receptor 1A in the brain. These novel results show that brain EPA can be increased through diet. Because LPC-EPA increased both EPA and DHA in the brain, it may help in the treatment of depression as well as neuroinflammatory diseases, such as Alzheimer's disease. The two major long-chain omega 3 FAs in animal tissues are EPA and DHA. Both of these FAs are known to have beneficial effects as anti-inflammatory agents and protect against various metabolic and neurologic diseases. Although the DHA concentration is higher than EPA in most tissues, the brain and retina are unique in having very high levels of DHA but virtually no EPA (1.Dyall S.C. Long-chain omega-3 fatty acids and the brain: a review of the independent and shared effects of EPA, DPA and DHA.Front. Aging Neurosci. 2015; 7: 52Crossref PubMed Scopus (496) Google Scholar). The major dietary sources of EPA and DHA are fish, fish oil, and krill oil, all of which usually contain more EPA than DHA (2.Kleiner A.C. Cladis D.P. Santerre C.R. A comparison of actual versus stated label amounts of EPA and DHA in commercial omega-3 dietary supplements in the United States.J. Sci. Food Agric. 2015; 95: 1260-1267Crossref PubMed Scopus (62) Google Scholar). However, the EPA levels in the brain are not increased significantly following the feeding of fish oil, krill oil, or even ethyl ester of EPA, although other tissues are enriched in both EPA and DHA (3.Rodrigues P.O. Lopes P.A. Ramos C. Miguueis S. Alfaia C.M. Pinto R.M.A. Rolo E.A. Bispo P. Batista I. Bandarra N.M. et al.Influence of feeding graded levels of canned sardines on the inflammatory markers and tissue fatty acid composition of Wistar rats.Br. J. Nutr. 2014; 112: 309-319Crossref PubMed Scopus (19) Google Scholar, 4.Kaur G. Begg D.P. Barr D. Garg M. Cameron-Smith D. Sinclair A.J. Short-term docosapentaenoic acid (22:5 n-3) supplementation increases tissue docosapentaenoic acid, DHA and EPA concentrations in rats.Br. J. Nutr. 2010; 103: 32-37Crossref PubMed Scopus (72) Google Scholar, 5.Tou J.C. Altman S.N. Gigliotti J.C. Benedito V.A. Cordonier E.L. Different sources of omega-3 polyunsaturated fatty acids affects apparent digestibility, tissue deposition, and tissue oxidative stability in growing female rats.Lipids Health Dis. 2011; 10: 179Crossref PubMed Scopus (46) Google Scholar, 6.Cruz-Hernandez C. Thakkar S.K. Moulin J. Oliveira M. Masserey-Elmelegy I. Dionisi F. Destaillats F. Benefits of structured and free monoacylglycerols to deliver eicosapentaenoic (EPA) in a model of lipid malabsorption.Nutrients. 2012; 4: 1781-1793Crossref PubMed Scopus (27) Google Scholar). Interestingly, several clinical and preclinical studies showed that dietary EPA is superior to dietary DHA in the prevention and treatment of depression (7.Martins J.G. EPA but not DHA appears to be responsible for the efficacy of omega-3 long chain polyunsaturated fatty acid supplementation in depression: evidence from a meta-analysis of randomized controlled trials.J. Am. Coll. Nutr. 2009; 28: 525-542Crossref PubMed Scopus (266) Google Scholar, 9.Ross B.M. The emerging role of eicosapentaenoic acid as an important psychoactive natural product: some answers but a lot more questions.Lipid Insights. 2008; 2: 89-97Crossref Google Scholar). It is therefore puzzling how EPA protects against depression without being incorporated appreciably into the brain. To explain this paradox, it has been proposed that the beneficial effects of EPA may result from the suppression of peripheral inflammation or from its hepatic conversion to DHA, rather than from a direct effect on the brain (10.Igarashi M. Chang L. Ma K. Rapoport S.I. Kinetics of eicosapentaenoic acid in brain, heart and liver of conscious rats fed a high n-3 PUFA containing diet.Prostaglandins Leukot. Essent. Fatty Acids. 2013; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). However, the conversion of EPA to DHA cannot explain why dietary DHA does not have similar effects. The lack of enrichment of brain EPA by the dietary EPA has been explained by proposing that EPA is rapidly oxidized by the brain, in contrast to DHA (11.Chen C.T. Bazinet R.P. β-Oxidation and rapid metabolism, but not uptake regulate brain eicosapentaenoic acid levels.Prostaglandins Leukot. Essent. Fatty Acids. 2015; 92: 33-40Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12.Kaur G. Molero J.C. Weisinger H.S. Sinclair A.J. Orally administered [14C]DPA and [14C]DHA are metabolised differently to [14C]EPA in rats.Br. J. Nutr. 2013; 109: 441-448Crossref PubMed Scopus (23) Google Scholar). This mechanism is supported by kinetic studies with labeled FAs showing the generation of more water-soluble degradation products from EPA, compared with DHA in the brain (10.Igarashi M. Chang L. Ma K. Rapoport S.I. Kinetics of eicosapentaenoic acid in brain, heart and liver of conscious rats fed a high n-3 PUFA containing diet.Prostaglandins Leukot. Essent. Fatty Acids. 2013; 89: 403-412Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 11.Chen C.T. Bazinet R.P. β-Oxidation and rapid metabolism, but not uptake regulate brain eicosapentaenoic acid levels.Prostaglandins Leukot. Essent. Fatty Acids. 2015; 92: 33-40Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). An alternative explanation that has not been explored is that the omega 3 FAs taken up into the brain do not accumulate in the brain if they are not taken up by the physiological transporter at the blood-brain barrier, namely major facilitator superfamily domain-containing protein 2a (Mfsd2a), which is specific for the lysophosphatidylcholine (LPC) form of the FA (13.Nguyen L.N. Ma D. Shui G. Wong P. Cazenave-Gassiot A. Zhang X. Wenk M.R. Goh E.L.K. Silver D.L. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid.Nature. 2014; 509: 503-506Crossref PubMed Scopus (585) Google Scholar). This sodium-dependent symporter has been shown to be critical for the uptake and accumulation of DHA by the brain, and loss of its activity leads to a deficiency of brain DHA, microcephaly, and mental retardation (13.Nguyen L.N. Ma D. Shui G. Wong P. Cazenave-Gassiot A. Zhang X. Wenk M.R. Goh E.L.K. Silver D.L. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid.Nature. 2014; 509: 503-506Crossref PubMed Scopus (585) Google Scholar, 14.Guemez-Gamboa A. Nguyen L.N. Yang H. Zaki M.S. Kara M. Ben-Omran T. Akizu N. Rosti R.O. Rosti B. Scott E. et al.Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome.Nat. Genet. 2015; 47: 809-813Crossref PubMed Scopus (147) Google Scholar). Although it is expressed in several tissues (15.Markhus M.W. Skotheim S. Graff I.E. Frøyland L. Braarud H.C. Stormark K.M. Malde M.K. Low omega-3 index in pregnancy is a possible biological risk factor for postpartum depression.PLoS One. 2013; 8: e67617Crossref PubMed Scopus (78) Google Scholar), it is functionally more important in the endothelial cells of the blood-brain barrier (13.Nguyen L.N. Ma D. Shui G. Wong P. Cazenave-Gassiot A. Zhang X. Wenk M.R. Goh E.L.K. Silver D.L. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid.Nature. 2014; 509: 503-506Crossref PubMed Scopus (585) Google Scholar), retina (16.Wong B.H. Chan J.P. Cazenave-Gassiot A. Poh R.W. Foo J.C. Galam D.L.A. Ghosh S. Nguyen L.N. Barathi V.A. Yeo S.W. et al.Mfsd2a is a transporter for the essential omega 3 fatty acid docosahexaenoic acid (DHA) in eye and is important for photoreceptor cell development.J. Biol. Chem. 2016; 291: 10501-10514Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and placenta (17.Naninck E.F.G. Hoeijmakers L. Kakava-Georgiadou N. Meesters A. Lazic S.E. Lucassen P.J. Korosi A. Chronic early life stress alters developmental and adult neurogenesis and impairs cognitive function in mice.Hippocampus. 2015; 25: 309-328Crossref PubMed Scopus (191) Google Scholar). Mfsd2a has been shown to transport most long-chain FAs in the form of lysophospholipids, as well as acylcarnitines (18.Quek D.Q.Y. Nguyen L.N. Fan H. Silver D.L. Structural insights into the transport mechanism of the human sodium-dependent lysophosphatidylcholine transporter MFSD2A.J. Biol. Chem. 2016; 291: 9383-9394Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). However, the potential role of this transporter in the accrual of EPA by the brain has not been investigated. Interestingly, recent studies show that the concentration of LPC-EPA is significantly lower than that of LPC-DHA in the plasma (19.Okudaira M. Inoue A. Shuto A. Nakanaga K. Kano K. Makide K. Saigusa D. Tomioka Y. Aoki J. Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS.J. Lipid Res. 2014; 55: 2178-2192Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), suggesting that the formation of LPC-EPA (presumably in the liver) may be a limiting factor in the accumulation of brain EPA. If so, increasing the plasma LPC-EPA concentration should result in its increased uptake and retention by the brain. We recently showed that feeding LPC-DHA (1 mg DHA per day or ∼40 mg/kg body weight) to normal mice for 30 days not only increased the plasma LPC-DHA concentration, but also increased the brain DHA content by 100% in normal adult mice and improved their memory function, as determined by the Morris water maze test (20.Sugasini D. Thomas R. Yalagala P.C.R. Tai L.M. Subbaiah P.V. Dietary docosahexaenoic acid (DHA) as lysophosphatidylcholine, but not as free acid, enriches brain DHA and improves memory in adult mice.Sci. Rep. 2017; 7: 11263Crossref PubMed Scopus (108) Google Scholar). The same amount of unesterified (free) DHA, a surrogate for fish oil, krill oil, and other current supplements, neither increased brain DHA nor improved the memory. In the present study, we tested the hypothesis that the brain EPA levels can be similarly increased if it is provided in the form of LPC-EPA in the diet by comparing the uptake and accretion of dietary LPC-DHA and free EPA by the brain in normal adult mice. The results show, for the first time, that the brain EPA can indeed be increased several-fold using a physiological dose of dietary LPC-EPA, but not free EPA. Furthermore, the amount of DHA in the brain increased even more than that of EPA by dietary LPC-EPA, indicating that EPA taken up as LPC is efficiently converted to DHA. However, there was no change in brain DHA by dietary free EPA at the same concentration. The expression of the neurotrophic factor, brain-derived neurotrophic factor (BDNF), and its downstream targets, cyclic AMP response element binding protein (CREB) and 5-hydroxy tryptamine (serotonin) receptor 1A (5-HT1A), as well as the phosphorylation of CREB were significantly elevated in the brain following treatment with dietary LPC-EPA, but not free EPA, showing that the dietary LPC-EPA is functionally effective in the brain. Because of the known beneficial effects of EPA in patients with depression, the enrichment of brain EPA by this strategy might be potentially useful in the prevention and treatment of depression. Furthermore, because LPC-EPA increases both EPA and DHA in the brain, it may be more beneficial than dietary DHA for the treatment of depression as well as various neuroinflammatory diseases, including Alzheimer's disease, which are more responsive to DHA. Free FAs (15:0, 17:0, 22:3, and 20:5) and tri-15:0 triacylglycerol (TAG) were purchased from Nu-Chek Prep Inc. (Elysian, MN). Di-17:0 phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were obtained from Avanti Polar Lipids (Alabaster, AL). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). All solvents (MS grade) were obtained from Thermo Fisher Scientific (Waltham, MA). The sn-1 EPA-LPC was prepared by the treatment of di-EPA PC with snake venom PLA2. di-EPA PC was synthesized by esterification of celite-bound glycerophosphorylcholine (265 mg) with EPA (1.48 g) in the presence of dimethylaminopyridine (0.31 g) and dicyclohexylcarbodiimide (1.01 g), essentially as described by Ichihara et al. (21.Ichihara K. Iwasaki H. Ueda K. Takizawa R. Naito H. Tomosugi M. Synthesis of phosphatidylcholine: an improved method without using the cadmium chloride complex of sn-glycero-3-phosphocholine.Chem. Phys. Lipids. 2005; 137: 94-99Crossref PubMed Scopus (40) Google Scholar). The PC was purified by silicic acid chromatography, dissolved in 20 ml of diethyl ether, and reacted with 10 mg Crotalus adamenteus venom dissolved in 0.5 ml Tris-HCl buffer (pH 7.4) containing 10 mM CaCl2. After 5 h of reaction on a metabolic shaker at room temperature, the precipitated LPC-EPA was washed with 5 ml of hexane to remove free EPA and extracted with chloroform:methanol (2:1 v/v) and used without further purification. The preparation gave a single spot on TLC plates corresponding to authentic LPC and contained only EPA by GC analysis. All protocols were approved by the University of Illinois at Chicago Institutional Animal Care and Use Committee, and the procedures were performed in adherence to the institutional guidance and regulations. Male C57BL/6 mice (age, 2 months) were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were fed a standard rodent chow (Teklad LM 485; Envigo, Indianapolis, IN) and water ad libitum throughout the experiment. All mice were housed in rooms with a 12 h light/dark cycle at controlled temperature (22°C ± 2°C). After 1 week of acclimatization, the mice were randomly divided into three groups (n = 6 each) and gavaged daily for 15 days with 80 μl of corn oil containing no EPA (control) or 1 mg EPA (3.3 μmol) either as free FA or as LPC. The standard rodent chow, which contained 5.8% total fat, did not contain any EPA or DHA, but had 0.3% α-linolenic acid (18:3, n-3) and 2.6% linoleic acid (18:2, n-6). The food intake and the body weights were not different between the three groups of mice. After 15 days of treatment, the mice were fasted overnight and anesthetized with 2% isoflurane. Blood was drawn by cardiac puncture into a heparinized syringe, and plasma was separated by centrifugation at 1,500 g for 15 min at 4°C. The mice were then perfused transcardially with ice-cold 100 mM PBS (pH 7.4), and liver, heart, brain, retina, and adipose tissues were harvested. All samples were flash-frozen in liquid nitrogen and stored at −80°C until analysis. The lipids were extracted from the fractions or tissues by the modified Bligh and Dyer procedure, as described by Ivanova et al. (22.Ivanova P.T. Milne S.B. Byrne M.O. Xiang Y. Brown H.A. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 21-57Crossref PubMed Scopus (137) Google Scholar), after adding tri-15:0 TAG and di-17:0 PC as internal standards. Methylation of FAs of total lipids and GC/MS analysis of the methyl esters was performed as described previously (20.Sugasini D. Thomas R. Yalagala P.C.R. Tai L.M. Subbaiah P.V. Dietary docosahexaenoic acid (DHA) as lysophosphatidylcholine, but not as free acid, enriches brain DHA and improves memory in adult mice.Sci. Rep. 2017; 7: 11263Crossref PubMed Scopus (108) Google Scholar, 23.Subbaiah P.V. Dammanahalli K.J. Yang P. Bi J. O'Donnell J.M. Enhanced incorporation of dietary DHA into lymph phospholipids by altering its molecular carrier.Biochim. Biophys. Acta. 2016; 1861: 723-729Crossref PubMed Scopus (23) Google Scholar). LC/MS analysis of molecular species of lipids was performed on an ABSciex 6500 QTRAP mass spectrometer coupled with an Agilent 2600 UPLC system, as described previously (20.Sugasini D. Thomas R. Yalagala P.C.R. Tai L.M. Subbaiah P.V. Dietary docosahexaenoic acid (DHA) as lysophosphatidylcholine, but not as free acid, enriches brain DHA and improves memory in adult mice.Sci. Rep. 2017; 7: 11263Crossref PubMed Scopus (108) Google Scholar). Quantification of EPA-, docosapentaenoic acid (DPA)-, and DHA-containing molecular species of PC, LPC, PE, and TAG in plasma and tissues was performed from the relative intensities of the various species and corresponding internal standards [17:0-17:0 PC, 17:0-LPC, 15:0-15:0 PE, 17:0-17:0 phosphatidylserine (PS), 14:1-17:0 phosphatidylinositol (PI), and 15:0-15:0-15:0 TAG]. The data processing was carried out using Analyst 1.6.2 (ABSciex, Redwood City, CA). The brain tissue was homogenized in lysis buffer [50 mM Tris (pH 8.0), 25 mM KCl, 0.5 mM EDTA, Nonidet P-40, 0.1 mM EGTA, 10 μl/ml aprotinin, 1 mM MgCl2, 1 mM CaCl2, 1 mM Na4P2O7, 1 mM Na4MO4, 1% protease inhibitor cocktail, 1 mM phenyl sulfonyl fluoride, 10 mM sodium flouoride, and 1 mM NaV] and the suspension was left on ice for 30 min and centrifuged at 13,000 g at 4°C for 10 min. The supernatant (30 μg protein) was separated on 10% SDS-PAGE and the separated proteins were transferred on to methanol-rinsed polyvinylidene difluoride membranes (Millipore) The membranes were blocked with 5% (w/v) nonfat dried milk in TBS containing 0.1% Tween-20 (TBST) for 1 h at 4°C and probed with primary antibodies in TBST overnight at 4°C. The membranes were then washed with TBST three times and incubated with appropriate horseradish peroxide-conjugated secondary antibodies at room temperature for 1 h. Finally, the membranes were washed with TBST three times and developed with ECL detection kits (Bio-Rad, Hercules, CA) for 1 min, respective proteins were quantified in a Bio-Rad ChemiDoc MP imaging system, and the results were expressed as mean relative densitometric units. Brain tissue was homogenized in the lysis buffer. The homogenates were centrifuged at 10,000 g for 20 min, the supernatants were collected, and total protein concentration was determined by MicroBCA procedure (Pierce, Rockford, IL) using BSA as standard. Endogenous concentrations of BDNF were quantified using an ELISA kit (BDNF Emax ImmunoAssay System kit, (Promega Inc., Madison, WI) according to manufacturer's protocol. Total RNA was isolated from the brain using Trizol reagent (Sigma) according to the manufacturer's protocol. The resulting RNA was reverse transcribed to cDNA using dNTP, oligo(dT)12-18 as primer and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Thermo Fisher) in a 20 μl reaction mixture. The mRNA quantification was performed using the ABI-Prism7700 sequence detection system (Applied Biosystems, Foster City, CA) using SYBR Green super mix (Quantabio, Beverly, MA) and the following primers for murine genes (Invitrogen): BDNF [forward (F): 5′-ATGGGACTCTGGAGAGCCTGAA-3′, reverse (R): 5′-CGCCAGCCAATTCTCTTTTTGC-3′]; CREB (F: 5′-TCAGCCGGGTACTACCATTC-3′, R: 5′-TTCAGCAGGCTGTGTAGGAA-3′); 5-HT1A (F: 5′-CTGTTTATCGCCCTGGATG-3′, R: 5′-ATGAGCCAAGTGAGCGAGAT-3′); TNFα (F: 5′-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC3′, R: 5′-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3′); GAPDH (F: 5′-GGTGAAGGTCGGTGTGAACG3′, R: 5′-TTGGCTCCACCCTTCAAGTG-3′). The mRNA expression of the targeted genes was normalized to the level of GAPDH mRNA. Data were processed by the ABI Sequence Detection system 1.6 software and analyzed by ANOVA. Unless otherwise indicated, all statistical analyses were performed using GraphPad Prism 7.0 software (La Jolla, CA). Analysis of significance was determined by one-way ANOVA with post hoc Tukey multiple comparison test. Figure 1A shows the composition of long-chain omega 3 FA levels in the plasma following the gavage of corn oil carrier alone (no-EPA control) or with free EPA or LPC-EPA at a dose of 1 mg EPA (3.3 μmol/day for 15 days). The total FA composition of plasma is shown in supplemental Table S1. The EPA concentration was very low in control plasma (0.15% of total FAs), but was increased significantly by free EPA (to 4.3% of total FAs) and by LPC-EPA (to 3.9% of total FAs) to a comparable extent. However, the increase in the elongation products, namely DPA (22:5, n-3) and DHA (22:6, n-3), was greater with free EPA than with LPC-EPA. The DHA content increased from 2.7% of total FAs (in control) to 8.4% of total by free EPA and to 4.5% of total by LPC-EPA. The DPA content rose from 0.53% of total (in control) to 1.06% of total in the free EPA group, and to 0.86% of total in the LPC-EPA group. The total omega 3 FA content (20:5 + 22:5 + 22:6) was thus significantly higher with free EPA (13.7% of total FAs) compared with LPC-EPA (9.3% of total FAs). The concentration of 20:4 decreased in both groups to a similar extent (supplemental Table S1).There were also significant decreases in 18:1 (n-9), 18:1 (n-7), and 16:1 (n-7) to a roughly equal extent by both treatments. These results show that both molecular forms of EPA are absorbed efficiently, and that EPA is converted to DPA and DHA in the liver and other tissues and secreted into plasma in the lipoproteins. A significant increase in the net amount of plasma LPC-EPA (expressed as nanomoles per milliliter) occurred after feeding LPC-EPA compared with free EPA (Fig. 1B). There was also an increase in the net amounts of LPC-DHA and LPC-DPA (nanomoles per milliliter) in the mice fed LPC-EPA. These results show that part of EPA absorbed through the LPC-EPA pathway is converted to DPA and DHA in the liver and secreted into plasma in the form of LPC. It is possible that LPC-EPA in the plasma was derived from direct absorption from the gut as well as secretion from the liver. However, the isomer composition of LPC shows that over 75% of LPC-EPA and LPC-DHA were sn-2 acyl isomers. Because we fed only sn-1 EPA isomer, and the isomerization does not favor the formation of sn-2 acyl isomer (24.Kiełbowicz G. Smuga D. Gładkowski W. Chojnacka A. Wawrzeńczyk C. An LC method for the analysis of phosphatidylcholine hydrolysis products and its application to the monitoring of the acyl migration process.Talanta. 2012; 94: 22-29Crossref PubMed Scopus (26) Google Scholar), these results show that most of the omega 3 FA LPC in plasma was secreted by the liver, which is known to secrete predominantly sn-2 acyl (unsaturated) isomers (25.Croset M. Brossard N. Polette A. Lagarde M. Characterization of plasma unsaturated lysophosphatidylcholines in human and rat.Biochem. J. 2000; 345: 61-67Crossref PubMed Scopus (0) Google Scholar, 26.Graham A. Zammit V.A. Christie W.W. Brindley D.N. Sexual dimorphism in the preferential secretion of unsaturated lysophosphatidylcholine by rat hepatocytes but no secretion by sheep hepatocytes.Biochim. Biophys. Acta. 1991; 1081: 151-158Crossref PubMed Scopus (12) Google Scholar, 27.Scagnelli G.P. Cooper P.S. VandenBroek J.M. Berman W.F. Schwartz C.C. Plasma 1-palmitoyl-2-linoleoyl phosphatidylcholine. Evidence for extensive phospholipase A1 hydrolysis and hepatic metabolism of the products.J. Biol. Chem. 1991; 266: 18002-18011Abstract Full Text PDF PubMed Google Scholar). It may also be noted that the animals were fasted overnight before collection of the plasma, and therefore it is unlikely that the plasma LPC-EPA measured here is derived from the recently absorbed LPC. The molecular species of PC, PE, and TAG containing the omega 3 FA were analyzed by LC/MS/MS to determine whether the metabolic fates of dietary free EPA and LPC-EPA differed after their absorption. Most of the lipid species containing EPA, DPA, and DHA were elevated in the plasma of mice fed LPC-EPA (Fig. 1C), supporting an efficient assimilation of the absorbed LPC-EPA by the liver, elongation to DHA, and subsequent secretion into plasma lipoproteins. In contrast to LPC-EPA, which increased the omega 3 FAs of both phospholipids and TAG, free EPA increased mostly the omega 3 FAs of TAG. The results also show that the majority of the omega 3 FA was present in plasma TAG in both the free EPA and LPC-EPA groups. However, a significantly higher fraction of plasma omega 3 FAs was incorporated into the phospholipids in the LPC-EPA group compared with the free EPA group. Figure 2 shows the percentage of omega 3 FAs in the brain lipids, as well as the concentration in nanomoles per gram of tissue in the three groups of mice. The total FA composition is shown in supplemental Table S2. The EPA concentration in the control group is very low (0.04% of total FAs), as reported by several previous studies (3.Rodrigues P.O. Lopes P.A. Ramos C. Miguueis S. Alfaia C.M. Pinto R.M.A. Rolo E.A. Bispo P. Batista I. Bandarra N.M. et al.Influence of feeding graded levels of canned sardines on the inflammatory markers and tissue fatty acid composition of Wistar rats.Br. J. Nutr. 2014; 112: 309-319Crossref PubMed Scopus (19) Google Scholar, 4.Kaur G. Begg D.P. Barr D. Garg M. Cameron-Smith D. Sinclair A.J. Short-term docosapentaenoic acid (22:5 n-3) supplementation increases tissue docosapentaenoic acid, DHA and EPA concentrations in rats.Br. J. Nutr. 2010; 103: 32-37Crossref PubMed Scopus (72) Google Scholar). Although this percentage was increased by both free EPA and LPC-EPA, the brain EPA content was 7-fold greater in the LPC-EPA group (4.14% of total FAs) compared with the free EPA group (0.56% of the total). Furthermore, there was a marked increase in DPA (22:5, n-3) and DHA (22:6) in the LPC-EPA group, but not in the free EPA group, whether expressed as percent of total FAs or as nanomoles per gram of tissue. Thus feeding LPC-EPA increased the net amount (nanomoles per gram) of DPA by 18-fold over the control and increased DHA by 2.5-fold over the control group. The total omega 3 FA content (nanomoles per gram) of the brain was increased by 2.8-fold over the no-EPA control by LPC-EPA, whereas it was not affected by the same dose of free EPA. These studies therefore show that LPC-EPA is efficiently transported into the brain and converted to DPA and DHA in addition to increasing the EPA content by >100-fold. It is likely that part of the increase in brain DHA is due to the uptake of LPC-DHA from the plasma, because the plasma LPC-DHA is also increased by oral LPC-EPA (Fig. 1B). We calculated that about 12% of the administered EPA (5.9 μmol out of 49.5 μmol) was incorporated into brain lipids over the period of 15 days, as indicated by the increase in omega 3 FAs of the brain. This is a much higher incorporation compared with the previous studies, which reported that less than 1% of the oral dose is deposited in the brain after ingestion of either TAG-DHA or PC-DHA (28.Graf B.A. Duchateau G.S.M.J. Patterson A.B. Mitchell E.S. van Bruggen P. Koek J.H. Melville S. Verkade H.J. Age dependent incorporation of 14C-DHA into rat brain and body tissues after dosing various 14C-DHA-esters.Prostaglandins Leukot. Essent. Fatty Acids. 2010; 83: 89-96Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 29.Kitson A.P. Metherel A.H. Chen C.T. Domenichiello A.F. Trepanier M.O. Berger A. Bazinet R.P. Effect of dietary docosahexaenoic acid (DHA) in phospholipids or triglycerides on brain DHA uptake and accretion.J. Nutr. Biochem. 2016; 33: 91-102Crossref PubMed Scopus (62) Google Scholar). The increase in the net amount of each omega 3 FA (nanomoles per gram) in various brain phospholipid classes was analyzed by LC/MS/MS and is shown in Fig. 3. In all cases, the increase in omega 3 FA was greater in the LPC-EPA group compared with the free EPA gro
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