Detection of omega-3 oxylipins in human plasma and response to treatment with omega-3 acid ethyl esters

• DOI: 10.1194/jlr.m900193-jlr200
• Publication Date: 2017-06-27T09:13:22Z
• Authors: Gregory C. Shearer; William S. Harris; Theresa L. Pedersen; John W. Newman
• Keywords: Ethyl ester

The long-chain omega-3 fatty acids (n-3 FA) eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) have beneficial health effects, but the molecular mediators of these effects are not well characterized. Oxygenated n-3 FAs (oxylipins) may be an important class mediators. Members this chemical include epoxides, alcohols, diols, ketones, many which bioactivity in vitro. Neither presence oxylipins human plasma nor effect FA ingestion on their levels has been documented. We measured derived from both n-6 classes healthy volunteers (n = 10) before after 4 weeks treatment with prescription ethyl esters (4 g/day). At baseline, EPA DHA were detected low (1–50 nM) range, alcohols > epoxides ≥ diols. Treatment increased oxylipin 2- to 5-fold reduced selected by ∼20%. This is first documentation that endogenous can modulated humans. extent cardiovascular mediated and/or remains explored. Omega-3 (1.Harris W.S. Miller M. Tighe A.P. Davidson M.H. 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There considerable speculation "eicosanoids" (oxygenated metabolites 20-carbon FAs; e.g., prostaglandins, leukotrienes, thromboxane) mediate some FAs, there little direct evidence humans support hypothesis. Other relating alterations membrane function (12.Shaikh S.R. Cherezov V. Caffrey Stillwell W. Wassall Interaction cholesterol acid-containing phosphatidylethanolamine: trigger microdomain/raft formation?.Biochemistry. 2003; 42: 12028-12037Crossref (79) or gene regulation (13.Deckelbaum R.J. Worgall Seo expression.Am. 1520S-1525SCrossref also involved. Eicosanoids belong larger molecules known oxylipins, subsets shown Figs. 2 supplemental Fig. I (5-LOX pathways). Many precursors either enzymatic reactions nonspecific autooxidative rearrangements. Oxylipins oxygenated unsaturated [e.g., α-linolenic (aLA), (EPA), (DHA)] linoleic (LA), dihomo-γ-linolenic (dgLA), arachidonic (AA)] series. Enzymatically generated prostaglandins thromboxanes (Tx) produced cyclooxygenase (COX)-derived metabolites, while leukotrienes 5-lipoxygenase (5-LOX) products. addition, various diols actions LOXs, cytochrome P-450s (CYP), epoxide hydrolases. Although reasonably described (14.Fleming I. Epoxyeicosatrienoic cell signaling angiogenesis.Prostaglandins Lipid Mediat. 82: 60-67Crossref (123) reports rare (15.Honstra van Houwelingen A.C. Kivits G.A. Fischer Uedelhoven Influence dietary fish eicosanoid metabolism man.Prostaglandins. 1990; 40: 311-329Crossref (24) focus only COX LOX metabolites; epoxy-n3 reported. Moreover, administration wide array remain unreported best our knowledge. To understand how biochemical cellular individual relate conditions affected fish-oil treatment, it critical establish normal concentration ranges document responsiveness parent FAs. study, we report intake impacts lipid 18-22 carbon blood small group subjects.Fig. 2Cytochrome P450 epoxygenase-associated (A), (B), (C), (D), (E) acids. Metabolites indicated circles enzymes rounded rectangles. Some multiple enzymes. color: black unchanged; blue decreased; orange increased; measured. Bolded arrows demarcate metabolic pathways evaluated gray unmeasured along pathways.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ten adults meeting inclusion exclusion criteria previously (16.Larson M.K. Ashmore J.H. Harris K.A. Vogelaar J.L. Pottala Sprehe Effects aspirin, alone combination, platelet subjects.Thromb. Haemost. 100: 634-641Crossref (62) participated. four six all Caucasian, median [interquartile range (IQR)] age 33 (24.Powell Gravel Gravelle Formation 5-oxo metabolite 5,8,11,14,17-eicosapentaenoic its neutrophils eosinophils.J. Res. 36: 2590-2598Abstract 51) years BMI 25.5 (21.Leaf Kang J.X. Xiao Y.F. Billman G.E. Clinical mechanism arrhythmias oils.Circulation. 107: 2646-2652Crossref 29.Xi Pham Ziboh V.A. Suppression proto-oncogene (AP-1) model skin epidermal hyperproliferation reversed topical application 13-hydroxyoctadecadienoic 15-hydroxyeicosatrienoic acid.Prostaglandins 2000; 62: 13-19Abstract (11) kg/m2. Oxylipin g/day (Lovaza, GlaxoSmithKline, Philadelphia, PA). study was approved University South Dakota Institutional Review Board, written informed consent obtained each subject. Blood drawn into sodium EDTA 10 h overnight fast, whole composition using gas chromatography erythrocyte extracts (17.Block R.C. Reid K.J. Sands S.A. Spertus J.A. membranes acute syndrome controls.Atherosclerosis. 821-828Abstract (165) Briefly, extracted 100 μl aliquots methylene chloride, methanol, water (2:2:1), followed 14% boron trifluoride methanol at 100°C min. resulting methyl analyzed GC2010 (Shimadzu, Columbia, MD) m SP2560 capillary column (Supelco, Bellefonte, isolation determination performed modifications reported procedures, included expansion previous analyte list (18.Newman J.W. Kaysen Hammock B.D. Shearer G.C. Proteinuria increases oxylipid concentrations VLDL HDL, LDL particles rat.J 48: 1792-1800Abstract (0) 19.Luria Weldon S.M. Kabcenell A.K. Ingraham R.H. Matera Jiang Gill Morisseau Newman Compensatory homeostatic pressure Ephx2 gene-disrupted mice.J. Biol. Chem. 282: 2891-2898Abstract (120) (see data more detail). (200 μl) spiked butylated hydroxytoluene/EDTA deuterated prostanoid, eicosanoid, octadecanoid surrogates Table II), subjected hydrolysis M methanolic hydroxide release ester linked lipids Samples 60 mg Oasis HLB solid phase extraction columns (Waters, Milford, MA), dried 30 min, wetted 0.5 ml eluted acetate 6 µL 30% glycerol, dryness under vacuum. confirmed stability than 5 months −80°C (unpublished observations). Residues reconstituted 75 400 nM solution 1-cyclohexyluriedo-3-dodecanoic (CUDA) 1-phenylurea-3-hexanoic (PUHA) vortexed, filtered 3 min centrifugation 0.1 µm Amicon Ultrafree-MC durapore PVDF filters (Millipore). Analytes within elutes separated reverse-phase ultra-performance liquid (UPLC) 1.7 μm Acquity BEH (Waters) 25 two-solvent gradient (solvent 0.1% acetic acid; solvent B 90:10 v/v acetonitrile/isopropanol; see I). Oxylipids negative mode electrospray ionization tandem quadrupole mass spectroscopy method (19.Luria Ionization fragmentation energies precursor-product ions optimized API 4000 QTrap (Applied Biosystems Inc., Foster City, CA). Collision induced dissociation transitions analytical target analytes Tables II–V. tested multiway ANOVA (ANOVA). Epoxides n3 DHA-derived diol 19,20-DiHDPA second sample 7; insufficient extract volumes subjects). Data subjects available other oxylipins. log-transformed achieved distributions equal variance. Tukey's honest significant difference test used posthoc analyses. least square mean (95% CI) regioisomers Results considered P < 0.05. Analyses JMP software (version 7.0.2). All assumptions verified fit confirmed. See data. baseline final (PUFA) given 1. As expected, ∼8- ∼3-fold, respectively. LA aLA altered P-OM3; however, decreases dgLA AA noted.TABLE 1Plasma content (% FAOxylipin Parent FAPre P-OM3Post P-OM3Fold ChangePLA34.2 (32, 36)33.5 35)–>0.05aLA0.558 (0.48, 0.65)0.551 (0.47, 0.64)–>0.05dgLA0.204 (0.19, 0.22)0.174 (0.16, 0.19)0.850.015AA7.24 (6.8, 7.8)6.34 (5.9, 6.8)0.880.012EPA0.425 (0.31, 0.59)3.6 (2.6, 5)8.460.0004DHA1.47 (1.2, 1.8)3.97 (3.3, 4.8)2.710.0001Before (P-OM3) 10). Abbreviations: AA, aLA, dgLA, dihommo-γ-linolenic DHA, EPA, FA, LA, linolenic P-OM3, esters. Open table new tab Before show, PUFAs present ranging ∼1–100 nM, except much greater (∼100–1000 nM). (except aLA) treatment. case 15-HETE highest concentration, progressively decreasing site oxygenation moves toward carboxyl group. exception trend 5-HETE, regioisomer single proximate double bond, same 15-HETE. 5-HEPE (with bond) 12- 15-HEPE two bonds) concentrations. 5.7- 2.1-fold, respectively, regardless ∼20% 34%. terminal alcohol 20-HETE P-OM3.TABLE 2Plasma octadecanoic ketone (nM)aSee 15-LOX analogous regioisomers.Alcohol OxylipinIsomer ComparisonbRegioisomers different letters differ significantly (P 0.05) Honest Significant Differences test.Pre ChangePcP-value concentrations.LA – HODE9-HODEA296 (250, 340)252 (220, 290)0.860.04713-HODEB741 (640, 860)641 (550, 750)aLA HOTE9-HOTE–8.07 (5.6, 12)7.65 (5, 12)–>0.0513-HOTE–6.82 8.2)5.84 (4.8, 7.1)LA KOTE9-KODEA88.2 (75, 100)74.9 (64, 88)–>0.0513-KODEB206 (180, 240)182 (160, 210)The 95% CI adjustment subject batch. HODE, hydroxyoctadecadienoic HOTE, hydroxyoctadecatrienoic KODE, ketooctadecadienoic KOTE, LOX, lipoxygenase; esters.a regioisomers.b Regioisomers test.c P-value TABLE 3Plasma eicosanoic docosanoic regioisomer.Alcohol ChangePcP value plasma.dgLA HETrE15-HETrE–13.7 (12, 16)8.98 (7.6, 11)0.660.003AA HETE5-HETEA81.9 (72, 93)66 (58, 75)0.8<0.00018-HETEB33.9 (30, 38)26.1 (23, 29)9-HETEC, B36.3 41)29.7 (26, 34)11-HETEC43.4 (38, 49)34.1 39)12-HETED52.6 (47, 59)43.4 49)15-HETEA84.7 96)67.4 (60, 76)ω-alcohol20-HETE–4.35 (3.6, 5.3)4.27 (3.5, 5.2)––EPA HEPE5-HEPEA12.4 (10, 15)71.1 88)5.7<0.000112-HEPEB3.54 (2.9, 4.4)20.1 (16, 25)15-HEPEB3.48 (2.8, 4.3)19.2 24)DHA HDoHE17-HDoHE–26.8 (21, 34)56 (44, 71)2.10.001AA KETE5-KETEA19.7 (17, 23)16.3 (14, 19)0.830.0315-KETEB63.1 (54, 74)53.8 (46, 63)Mean HDoHE, hydroxydocosahexaenoic HEPE, hydroxyeicosapentaenoic HETE, hydroxyeicosatetraenoic HETrE, hydroxyeicosatrienoic KETE, ketoeicosatetraenoic regioisomer.b plasma. Mean Ketones 10–200 nM. AA-derived ketones about 20% had no LA-derived ketones. sets most distal (15-KETE analog 13-KODE) 2–3 times abundant proximal (5-KETE 9-KODE). Standards EPA- commercially and, therefore, quantified. 4, PUFA decreased following order: epoxides. Of 11(12)-EpETrE others. DHA- 5- 2-fold, seen alcohols.TABLE 4Concentration vicinal nMaSee regioisomer.Epoxide OxylipinBaselinen-3 FAFold concentrations.Vicinal Diol EpOMELA DiHOME9(10)-EpOME83.4 97)79.5 (69, 92)–>0.059,10-DiHOMEA197 (170, 230)220 (190, 260)–>0.0512(13)-EpOME83.1 96)76.9 (66, 89)>0.0512,13-DiHOMEB18.1 21)16.7 19)aLA EpODEaLA DiHODE9(10)-EpODEAbRegioisomers test.5.5 (4.4, 6.9)5.32 (4.3, 6.6)–>0.059,10-DiHODEA1.38 (1.1, 1.7)1.6 (1.3, 2)–>0.0512(13)-EpODEB1.44 1.8)1.39 1.7)12,13-DiHODEdBelow limit detection.––15(16)-EpODEC9.19 (7.4, 11)8.09 (6.5, 10)15,16-DiHODEB14.5 18)16.9 (13, 21)AA EpETrEAA DiHETrE5(6)-EpETrEeSurrogate recovery failed quality control due lactone formation (See 2).––5,6-DiHETrEA20.1 (18, 23)15.1 17)0.81<0.00018(9)-EpETrEA16.4 19)14.7 17)–>0.058,9-DiHETrEB11.7 13)9.3 (8.2, 11)11(12)-EpETrEB61.7 71)56.4 (49, 64)11,12-DiHETrEC3.98 4.5)3.31 3.8)14(15)-EpETrEC28.5 (25, 33)26.1 30)14,15-DiHETrED2.57 (2.3, 2.9)2.28 (2, 2.6)EPA EpETEEPA DiHETE14(15)-EpETEA3.78 5.2)17.5 24)4.7<0.000114,15-DiHETEA2.14 (1.8, 2.6)8.6 (7.2, 10)4.1<0.000117(18)-EpETEB5.81 (4.2, 8)28 (20, 38)17,18-DiHETEB8.85 11)36.3 43)DHA EpDoPEDHA DiHDoPE16(17)-EpDoPEA11.1 (7.8, 16)24.6 35)2.10.000316,17-DiHDoPEfNo commercial standard available.––2.10.01719(20)-EpDoPEA11.9 (8.3, 17)24.2 35)19,20-DiHDoPE3.33 (2.4, 4.6)7.00 (5.1, 9.7)Means α-Linolenic DiHDoPE, dihydroxydocosapentaenoic DiHETE, dihydroxyeicosatetraenoic DiHETrE, dihydroxyeicosatrienoic DiHODE, dihydroxyoctadecadienoic DiHOME, dihydroxyoctadecamonoenoic EpDoPE, epoxyedocosapentaenoic EpETE, epoxyeicosatetraenoic EpETrE, epoxyeicosatrienoic EpOME, epoxyoctadecamonoenoic acid.a concentrations.d Below detection.e Surrogate 2).f No available. Means acid. Among EPA-derived DiHETEs DiDPA comparable those counterpart DiHETrEs. DiHETrEs, continuously (5,6-DiHETrE) (14,15-DiHETrE). 9,10-DiHOME, among three epoxide. number nonvicinal triols calibration standards, allowing quantitative compounds VI). Concentrations ranged 0.5–10 uniformly measurable (e.g., lipoxin A4) ∼35%. triol resolvin E1 above Plasma prostaglandin F2a (PGF2a) thromboxane (TxB2) ∼1–10 unaffected influence POM-3 PGE2 PGD2 could determined, unstable alkaline procedures liberate study. Levels LTB5, 6-keto PGF1α, 20-Hydroxy-LTB4, 20-carboxy-LTB4, 12,13-DiHODE below detection limits assay. facts point oxidation products components metabolome. These n3-FA biosynthetic pathways, promiscuous nature oxygenating enzymes, defined role n3-FAs homeostasis, mention reactive oxygen-dependent bulk pools. broad occurrence would suggest diet. While suggestive, information regarding suite potential sensitivity manipulation limited. For instance, knowledge, article demonstrate long chain EPA/DHA ingestion. One produce anti-inflammatory benefits