Divergent Shifts in Lipid Mediator Profile Following Supplementation with N-3 Docosapentaenoic Acid and Eicosapentaenoic Acid † † ‡ § James F

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Divergent shifts in lipid mediator profile following supplementation with n-3 docosapentaenoic acid and eicosapentaenoic acid † † ‡ § James F. Markworth,* Gunveen Kaur, Eliza G. Miller, Amy E. Larsen, Andrew J. Sinclair, { k Krishna Rao Maddipati, , and David Cameron-Smith* † *Liggins Institute, University of Auckland, Auckland, New Zealand; Centre for Physical Activity and Nutrition Research, School of Exercise ‡ and Nutrition Sciences, Deakin University, Melbourne, Victoria, Australia; Department of Physiology, Anatomy and Microbiology, La Trobe { University Melbourne, Victoria, Australia; §School of Medicine, Deakin University, Geelong, Victoria, Australia; and Department of k Pathology, Lipidomics Core Facility, and Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, USA

ABSTRACT: In contrast to the well-characterized effects of specialized proresolving lipid mediators (SPMs) derived from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), little is known about the metabolic fate of the intermediary long-chain (LC) n-3 polyunsaturated fatty acid (PUFA) docosapentaenoic acid (DPA). In this double blind crossover study, shifts in circulating levels of n-3 and n-6 PUFA-derived bioactive lipid mediators were quantified by an unbiased liquid chromatography-tandem mass spectrometry lipidomic approach. Plasma was obtained from human subjects before and after 7 d of supplementation with pure n-3 DPA, n-3 EPA or placebo (olive oil). DPA supplementation increased the SPM resolvin D5n-3DPA (RvD5n-3DPA) and maresin (MaR)-1, the DHA vicinal diol 19,20-dihydroxy-DPA and n-6 PUFA derived 15-keto-PG E2 (15-keto-PGE2). EPA supplementation had no effect on any plasma DPA or DHA derived mediators, but markedly elevated monohydroxy-eicosapentaenoic acids (HEPEs), including the E-series resolvin (RvE) precursor 18-HEPE; effects not observed with DPA supplementation. These data show that dietary n-3 DPA and EPA have highly divergent effects on human lipid mediator profile, with no overlap in PUFA metabolites formed. The recently uncovered biologic activity of n-3 DPA docosanoids and their marked modulation by dietary DPA intake reveals a unique and specific role of n-3 DPA in human physiology.—Markworth, J. F., Kaur, G., Miller, E. G., Larsen, A. E., Sinclair, A. J., Maddipati, K. R., Cameron-Smith, D. Divergent shifts in lipid mediator profile following supplementation with n-3 docosapentaenoic acid and eicosapentaenoic acid. FASEB J. 30, 3714–3725 (2016). www.fasebj.org

KEY WORDS: eicosanoids • fish oil • inflammation • lipidomics • omega-3 fatty acids

a-Linolenic acid (ALA; 18:3, n-3) is the essential fatty acid 22:6, n-3), and docosapentaenoic acid (DPA; 22:5, n-3) precursor to the long-chain (LC) n-3 PUFAs, including (Fig. 1). Dietary LC n-3 PUFA ingestion from marine eicosapentaenoic (EPA; 20:5, n-3), docosahexanoic (DHA; oil consumption contributes to anti-inflammatory properties and cardiovascular, metabolic, and neural – ABBREVIATIONS: COX, cyclooxygenase; CYP, cytochrome p450; DHA, health (1 4). The n-6 and -3 PUFAs exert their biologic docosahexanoic acid; DiHETE, hydroperoxyeicosatetraenoic acid; DiHETre, activities mainly via formation of bioactive lipid media- dihydroxyeicsatrienoic acid; DiHOME, dihydroxy-octadecenoic acid; DiHoPE, tors, termed eicosanoids and docosanoids (5). For ex- dihydroxy DPA; DPA, docosapentanoic acid; EPA, 5Z,8Z,11Z,14Z,17Z- eicosapentanoic acid; EpDPE, epoxydocosapentaenoic acid; EpETE, ample, arachidonic acid (AA; 20:4, n-6) is the major epoxyeicosatetraenoic acid; EpOME, epoxyoctadecenoic acid; HDoHE, mono- physiologic precursor to proinflammatory eicosanoids; hydroxy DHA; HDoPE, monohydroxy DPA; HEPE, hydroxy-eicosapentaenoic acid; HODE, hydroxyoctadecadienoic acid; HpDoHE, monohydroperoxy DHA; prostaglandins (PGs; e.g., PGE2), thromboxanes (TXs; e. LC,longchain;LC–MS, liquid chromatography–mass spectrometry; LOX, g., TXB2), and leukotrienes (LTs; e.g., LTB4). Compara- lipoxygenase; LT, leukotriene; MaR, maresin; MRM, multireaction monitor- tively, the EPA-derived eicosanoids (e.g., PGE3,TXB3, ing; OO, olive oil; PD, protectin; PG, prostaglandin; Rv, resolvin; RvD, D-series resolvin; RvE, E-series resolvin; SPM, specialized proresolving and LTB5) possess less potency, and competition be- mediator;TAG,triacylglycerol;TX,thromboxane tween EPA and AA has long been proposed to contrib- 1 Correspondence: The Liggins Institute, University of Auckland, Private ute to anti-inflammatory LC n-3 PUFA bioactivity (6). Bag 92019, 85 Park Rd., Grafton, Auckland 1142 New Zealand. E-mail: More recently, LC n-3 PUFAs were discovered to be [email protected] precursors to distinct lipid mediators with dual anti- doi: 10.1096/fj.201600360R This article includes supplemental data. Please visit http://www.fasebj.org to inflammatory and proresolving bioactivity, including obtain this information. the resolvins (Rvs), protectins (PDs), and MaRs, collectively

3714 0892-6638/16/0030-3714 © FASEB Downloaded from www.fasebj.org by (2001:388:608c:4800:fdd6:e532:12c5:d389) on November 26, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3714-3725. pathway (25), and recent reports show that CYP metabolites may also contribute to LC n-3 PUFA bioactivity (26). CYP preferentially targets the n-3 double bond resulting in the formation of 17,18-epoxyeicosatetraenoic acid (EpETE) from EPA and 19,20-epoxydocosapentaenoic acid (EpDPE) from DHA (27). Subsequent action of soluble epoxide hydrolase on these epoxides forms the downstream vicinal diols [e.g., 17,18-hydroperoxyeicosatetraenoic acid (DiHETE) from EPA and 19,20-dihydroxy DPA (DiHDoPE) from DHA (28)]. Compared with EPA and DHA, very little is known re- garding the metabolic fate and bioactivity of the third intermediary dietary n-3 PUFA DPA (22:5, n-3) (29, 30). n-3 DPA contributes a considerable proportion of the LC n-3 PUFA found in marine sources. For example, n-3 DPA levels in common fish oils vary from 1 to 5% of total fatty acids, in which the total LC n-3 PUFA content (EPA+DPA+DHA) varies from 11 to 27% (31). We have shown that dietary supplementation of humans with pure n-3 DPA or EPA (8 g total over 7 d) had distinct and specific incorporation patterns into plasma lipid fractions and red blood cell phospholipids (32). DPA supplementation increased the levels of both EPA and DHA, in addition to n-3 DPA, in the blood triacylglycerol (TAG) fraction (32). Consistently, DPA supplementation in rats increased liver concentrations of not only n-3 DPA, but also of both DHA and EPA (33). Thus, DPA may act as an LC n-3 PUFA intermediate reservoir contributing to the biosynthesis of both EPA- and DHA-derived bioactive lipid mediators. n-3 DPA itself is also a potential direct substrate for enzymes in the lipid mediator biosynthetic pathways. During inflammation resolution in Figure 1. Biosynthetic pathway from EPA to DHA via DPA. 24:5, mice, n-3 DPA is converted to novel docosanoid medi- n-3, tetracosapentaenoic acid; 24:6, n-3, tetracosahexaenoic acid. ators congenerous to the previously established SPMs derived from DHA (34). These newly identified n-3 termed specialized proresolving lipid mediators (SPMs) DPA docosanoid families termed RvDn-3DPA,PDn-3DPA, (7, 8). and MaRn-3DPA were shown to possess potent anti- SPM biosynthesis involves sequential metabolism of inflammatory and proresolving bioactivity similar to the n-3 PUFA substrates by multiple cell types that express the earlier described SPMs derived from EPA and DHA (34). required enzymatic machinery in a compartmentalized Whether such DPA specific docosanoids are detectable manner [e.g., neutrophil 5-lipoxygenase (LOX), platelet in human biologic fluids/tissue, the potential of dietary 12-LOX, and monocyte 15-LOX]. The LOX pathways gen- PUFA intake to modulate levels of these compounds in erate specific positional monohydroperoxy/monohydroxy vivo, and their physiologic relevance in human physiology EPA (HpEPE/HEPE) and DHA [monohydroperoxy DHA and nutrition currently remain unknown. (HpDoHE)/monohydroxy DHA (HDoHE)] isomers that The purpose of the current study wasto characterize the may have inherent bioactivity, in addition to functioning as changes in human plasma lipid mediator profile in re- intermediates in transcellular SPM biosynthetic pathways. sponse to 1 wk of dietary supplementation with either For example, metabolism of DHA via 15-LOX and 12-LOX pure n-3 DPA or pure EPA. We hypothesized on the basis pathways generates 17S-HpDoHE and 14S-HpDoHE, key of our previous studies (32, 33), that dietary DPA may intermediates in the PD (9) and MaR (10, 11) biosynthetic influence plasma EPA- and DHA-derived bioactive lipid pathways, respectively. Following reduction from 17- mediators, as well as modulate abundance of novel DPA- HpDoHE, 17-hydroxydocosahexanoic acid (HDoHE) is specific docosanoids in human plasma. also a precursor to the D-series resolvins (RvDs) 1–6 (12–15). Similarly, EPA is converted to 18R-hydroxy-EPA (18R-HEPE) by acetylated COX-2 (in the presence of as- MATERIALS AND METHODS pirin) (16) or endogenous cytochrome P450 (in the absence of aspirin) (17). 18R-HEPE is a key intermediate in the Study population biosynthesis of the E-series resolvins (RvEs) including Ten healthy female subjects (age, 25.5 6 3.3 yr; BMI, 22.3 6 RvE1 (5S,12R,18R-TriHEPE) (16, 18, 19), RvE2 (5S,18R- 2 – 1.6 kg/m ) were recruited to participate in the study. Participants DiHEPE) (20, 21), and RvE3 (17R,18R-DiHEPE) (22 24). provided written informed consent and completed a medical LC n-3 PUFAs are also effective substrates for the less questionnaire. Participants were excluded if they were at high well-characterized cytochrome P450 (CYP) epoxygenase risk of any form of cardiovascular disease (based on family

n-3 DPA MODULATES PLASMA LIPID MEDIATOR PROFILE 3715 Downloaded from www.fasebj.org by (2001:388:608c:4800:fdd6:e532:12c5:d389) on November 26, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3714-3725. history), or were overweight ($26 kg/m2). Ethics approval was and to consume a single 2-g dose of the oil with 200 ml of standard obtained from the Deakin University Human Research Ethics commercial orange juice each morning for the following 6 d. The Committee (EC2011-023). dose of DPA and EPA administered was thus 2 g on the first day followed by a dose of 1 g/d for 6 additional days (8 g total/wk). Although evidence of the tolerable upper intake of DPA in iso- Subject habitual PUFA intake lation is lacking, supplemental intakes of DHA or EPA alone up to ;1 g/d do not appear raise safety concerns for the general All participants completed a PUFA Food Frequency Question- population (30). On the morning of d 7, participants attended the naire(FFQ) duringscreening to determine habitual dietaryPUFA clinical facility following an overnight fast to provide a post- intake and study eligibility. Subjects were excluded from par- supplementation period fasting blood sample. Participants re- ticipation if they consumed more than 500 mg of LC n-3 PUFA ceived daily reminders to consume their supplement and were per day [based on results of the PUFA FFQ (35–37)]. The habitual asked to return the vials after consuming the supplements, to dietary intake of the subjects deemed eligible for the study was monitor compliance. found to be 102 6 66 mg of LC n-3 PUFA/d. Participants were also requested to refrain from consuming high LC n-3 PUFA products during the study, including fish, red meat, and LC n-3 Sample preparation and liquid PUFA-fortified products (2 marine or 2 red meat meals per chromatography–mass spectometry analysis week and 2 LC n-3 PUFA-fortified products/wk). Finally, the of lipid mediators participants were asked to provide a recall of their diet 24 h before blood sampling. As previously reported, it was found Fastingvenousbloodsamplesond0and7ofeachofthe3dietary that the participants did not consume any fish during the supplementation trials were collected into EDTA-coated Vacu- study period, and consumption of red meat was #2servings tainers (BD Biosciences, Franklin Lakes, NJ, USA) and were im- per week (32). mediately centrifuged for 15 min at 591 g at 15°C. The plasma was immediately collected from the red blood cell pellet and stored in aliquots at 280°C until further analysis (,1 yr storage). Aliquots DPA and EPA supplements of plasma samples (100 ml) were thawed once, spiked with 5 ng each of PG E1-d4 (PGE1-d4), leukotriene B4-d4 (LTB4-d4), and 15 Purified EPA (Maxomega EPA 98 FFA, 99.8% PUFA containing (S)-HETE-d8 as internal standards for analyte recovery and $98% EPA; w/w) and n-3 DPA (Maxomega DPA 97 FFA: 99.8% quantitation and mixed thoroughly. The samples were then PUFA containing $97% n-3 DPA; w/w) oils, both as free fatty extracted for fatty acyl lipid metabolites with C18 extraction acids, were sourced from Equateq, Ltd. (Breasclete, United columns, as described earlier with minor modifications (38, 39). Kingdom). The fatty acid profiles of the oils (area% FAMES) were In brief, the internal standard spiked samples were applied to testedandapprovedbythemanufacturerandmetcompany conditioned C18 cartridges, washed with 15% methanol in water release specifications of $97% pure n-3 DPA (22:5, n-3) for followed by hexane, and dried in a vacuum. The cartridges were Maxomega DPA 97 FFA and $98% pure n-3 EPA (20:5 n-3) for eluted with 0.5 ml methanol containing 0.1% formic acid. The Maxomega EPA 98 FFA. All supplements were used within eluate was dried under a gentle stream of nitrogen. The residue 1 year of the date of manufacture before the indicated expiration was dissolved in 50 ml methanol-25 mM aqueous ammonium date. For dietary supplementation trials the LC n-3 PUFA oils acetate (1:1) and subjected to liquid chromatography–mass were diluted 1:1 in olive oil (OO). Pure OO alone in the absence of spectrometry (LC–MS) analysis. LC n-3 PUFA served as the control supplement. HPLC was performed on a Prominence XR system (Shimadzu, Somerset, NJ, USA) equipped with a Luna C18 (3 mm, 2.1 3 150 mm) column. The mobile phase consisted of a gradient Study design between A: methanol-water-acetonitrile (10:85:5 v/v) and B methanol-water-acetonitrile (90:5:5 v/v), both containing 0.1% This study was a double-blind, placebo-controlled trial in which ammonium acetate. The gradient program with respect to the each subject received each of 3 dietary supplements (OO, n-3 EPA compositionofBwasasfollows:0–1min,50%;1–8min, or n-3 DPA) in a crossover manner. All participants received the 50–80%; 8–15 min, 80–95%; and 15–17 min, 95%. The flow rate OO placebo supplement on their first visit and were then ran- was 0.2 ml/min. The HPLC eluate was directly introduced to domized to receive either the DPA or EPA supplement on the the ESI source of QTrap5500 mass spectrometer (AB Sciex, subsequent 2 visits. Each supplementation period lasted 7 d, and Singapore) in the negative ion mode with following condi- a 2 wk washout period was included between each trial before tions:curtaingas:35psi,GS1:35psi,GS2:65psi;temperature: crossover administration of the next intervention. 600°C, ion spray voltage: 21500 V; collision gas: low; The night before each of the 3 experimental supplementation declustering potential, 260 V, and entrance potential, 27V. trials, participants were provided with a standardized meal (200 g The eluate was monitored by multireaction monitoring dry pasta and 70 g tomato sauce). The subjects arrived at the (MRM) method to detect unique molecular ion–daughter ion laboratory the following morning in a fasted state, and a resting combinations for each of 125 transitions (to monitor a total of venous blood sample was drawn (d 0). They were provided with a 144 lipid mediators and 3 internal standards) with 8 ms dwell meal consisting of 180 g of instant mashed potato (Continental time for each transition. Optimized collisional energies Deb; Unilever Australasia, Sydney, NSW, Australia) containing: (18–35 eV) and collision cell exit potentials (7–10 V) were used the placebo (20 ml OO), 2 g of purified EPA mixed with 18 ml of for each MRM transition. The data were collected with Ana- OO, or 2 g of purified DPA mixed with 18 ml of OO. The partic- lyst 1.6 software and the MRM transition chromatograms ipants were required to consume each of these breakfasts in 15 were quantitated by MultiQuant software (both from AB min. They were then provided with six 2-g aliquots of a 1:1 Sciex). The internal standard signals in each chromatogram mixture of either 1 g purified n-3 DPA or 1 g purified n-3 EPA were used for normalization for recovery as well as relative mixed with 1 g OO. During the placebo period, the participants quantitation of each analyte [PGE1-d4 for prostaglandins, received six 2-g aliquots containing OO alone as a placebo. The LTB4-d4 for di- and trihydroxy fatty acids, and 15(S)-HETE-d8 oils were packaged in individual 2-ml cryovials within an opaque for monohydroxy and epoxy fatty acids]. All lipid mediators box (to protect the oils from exposure to light). The participants quantified were positively identified by comparing HPLC re- were instructed to keep the supplements in a refrigerator at 4°C tention times with authentic standards (Cayman Chemicals,

3716 Vol. 30 November 2016 The FASEB Journal x www.fasebj.org MARKWORTH ET AL. Downloaded from www.fasebj.org by (2001:388:608c:4800:fdd6:e532:12c5:d389) on November 26, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3714-3725. Ann Arbor, MI, USA) and specific parent–daughter ion combi- human plasma. These included monohydroxy DPA nations as well as mass spectra obtained from information de- (HDoPE) and dihydroxy DPA (DiHDoPE) products pendent acquisition (IDA) method. (Fig. 3). Of these, 13-, 14-, 16-, and 20-HDoPE (but not 7-, Detection and quantitation limits (expressed as picograms 10-, 11-, or 17-HDoPE) were present at detectable con- injected into the column) of the method for each class of lipid centrations (Fig. 3A). Among the monohydroxylated mediators were determined by injecting serial dilutions of representative standards, and relative response ratios of analytes to DPA products detected, only 13-HDoPE was signifi- internal standards were determined, as described before (38). cantly influenced by dietary DPA supplementation Minimum signal–noise ratios used for detection and quantitation (mass spectrum in Fig. 3C). We also identified a dihy- were 3 and 5, respectively. The detection and quantitation limits, droxylated n-3 DPA mediator 7,17-dihydroxy-DPA respectively, were 2 and 10 pg for monohydroxy fatty acids (e.g., (7,17-DiHDoPE) in human plasma (mass spectrum in HETEs), 1 and 10 pg for dihydroxy fatty acids (e.g., LTB4), 1 and 8 Fig. 3D). After DPA supplementation there was a robust pg for trihydroxy fatty acids (e.g., RvD1), and 8 and 25 pg for PGs ∼ (e.g., PGE1). increase ( 20-fold) in plasma levels of 7,17-DiHDoPE (Fig. 3B). Notably, 7,17-DiHDoPE is a structural analog of the 17-dihydroxy-DHA SPM RvD5, which Dalli et al. (34) Statistical analysis recently termed RvD5n-3DPA following its discovery and elucidation in mice and cultured human neutrophils/ Statistical analysis was performed using SigmaPlot 12.0. Data macrophages. were analyzed using 2-way ANOVA with both supplement (OO vs. EPA vs. DPA) and time (d 0 vs. 7) as within-subject effects. When main or interactive effects were observed, pair-wise com- EPA metabolites parisons were made with Student-Newman-Keuls post hoc test. 6 , Results are presented as means SEM.AvalueofP 0.05 was EPA metabolites detected in human plasma included nu- considered statistically significant. merous monohydroxy-EPA regioisomers (HEPEs), EPA epoxides/vicinal diols (e.g., EpETEs/DiHETEs), and series-3 PGs (e.g., PGD3) (Supplemental Table 1). Peaks RESULTS corresponding to di- and trihydroxylated EPA mediators (e.g., LTB5,LXA5, and RvE1) were also observed but were Effect of DPA (22:5, n-3) supplementation on generally #LOQ of the assay used in most of the samples. plasma n-3 PUFA lipid mediators Of the numerous EPA metabolites detected in human plasma, none was significantly influenced by DPA DHA metabolites supplementation. DPA supplementation increased plasma levels of the DHA metabolite 13-HDoHE (1.9-fold) (Supplemental Ta- Effect of DPA (22:5, n-3) supplementation on ble 1), but did not influence any other monohydroxy-DHA plasma n-6 PUFA lipid mediators regioisomers or DHA epoxides (EpDPEs) (Fig. 2A and Supplemental Table 1). Nevertheless, the DHA epoxide AA (20:4, n6) metabolites vicinal diol metabolite 19,20-DiHDoPE was markedly elevated (22-fold) after DPA supplementation (Fig. 2A). We found unexpectedly that plasma concentrations of Peaks corresponding to several di- and trihydroxylated several AA-derived PGs and their downstream degradation DHA mediators (the D-series SPMs) were detected, in- products were increased by n-3 DPA supplementation cluding RvD1 (7S,8R,17S-TriHDoHE), RvD2 (7S,16R, (Fig. 4A). Most notably, 15-keto-PGE2 (a PG 15-hydroxy 17S-TriHDoHE), RvD6 (4S,17S-DiHDoHE), MaR1 (7R, dehydrogenase metabolite of PGE2) was markedly ele- 14S-DiHDoHE), and PD1 isomer 10S,17S-DiHDoHE vated (;40-fold) in the DPA trial (Fig. 4A). This response (PDX) (Supplemental Table 1). Basal plasma levels of DHA was also evident, albeit to a lesser extent, for PGA2, SPMs were low and generally around the quantitative limits PGD2, PGF2a, and the downstream circulating PG me- (#LOQ) of the assay used. Plasma MaR1 was significantly tabolites 15-keto-PGF2a, and bicyclo-PGE2 (Fig. 4A). ; ; elevated ( 3 fold), to within the quantifiable range ( 150 Furthermore, the AA-derived isoprostane F2a-VI (Fig. pg/ml) after 7 d of DPA supplementation (Fig. 2B). Fur- 4B) and 5S,6S-DiHETE (Fig. 4C), a nonenzymatic hy- thermore, plasma RvD1 decreased from d 0 to 7 of the drolysis product of leukotriene A4 (LTA4) were similarly placebo OO supplementation trial (Fig. 2C); a response not found to be elevated only after DPA supplementation. observed in either the EPA or DPA trials. Transitions cor- Plasma TXB2 and PGE2 themselves were not influenced responding to PD1 and RvD5 were monitored, but peaks by DPA supplementation (Supplemental Table 1). corresponding to these analytes were not detectable in most of the samples. Other detected DHA SPMs were not influenced by DPA supplementation (Supplemental Table 1). Dihomo-g-linolenic acid (20:3, n6) metabolites

PlasmaPG metabolites of dihomo-g-linolenic acid (DGLA, DPA metabolites 20:3, n-6) including 13,14-dihydro-15-keto-PGE1 (13,14dh- 15k-PGE1), bicyclo-PGE1, 2,3-dinor PGE1, were also sig- In addition to known DHA-derived lipid mediators, we nificantly increased after the n-3 DPA supplementation detected several related metabolites of n-3 DPA itself in trial (Fig. 4D).

n-3 DPA MODULATES PLASMA LIPID MEDIATOR PROFILE 3717 Downloaded from www.fasebj.org by (2001:388:608c:4800:fdd6:e532:12c5:d389) on November 26, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3714-3725. Figure 2. Plasma DHA metabolites inﬂuenced by DPA supplementation. A) Plasma concentration of CYP-pathway–derived DHA epoxides (EpDPE) and downstream vicinal diols (DiHDoPE) in response to DPA supplementation. B) Plasma concentration of MaR1 in response to supplementation with OO, EPA, or DPA. C) Plasma concentrations of RvD1 in response to supplementation # with OO, EPA, or DPA. Value are means 6 SEM.*P , 0.05 vs. d 0 within respective supplementation period; P , 0.05 vs. placebo OO supplementation at the same time point.

Effect of EPA (20:5, n-3) supplementation on (255%) and 12-HETE (260%), enzymatic products of plasma PUFA lipid mediators 5-LOX and 12-LOX, respectively, were most notably suppressed. In contrast, 20-HETE, a CYP (g-hydroxylase EPA metabolites pathway) metabolite of AA was not influenced by EPA supplementation. CYP pathway AA epoxides including Unlike DPA, EPA supplementation increased plasma 5,6-, 8,9-, 11,12-, and 14,15-epoxy-eicosatrienoic acid levels of the monohydroxylated EPA (HEPE) metabo- (EpETrE) were also unaffected, whereas select down- lites (see Fig. 5A). Most notably, 8-HEPE (20-fold) and the stream vicinal diols 5,6-, 8,9-dihydroxy-eicosatrienoic RvE series precursor 18-HEPE (13-fold) were markedly acid (DiHETrE) were significantly reduced (Supple- increased by EPA supplementation. The EPA-derived mental Table 1). Further notable changes in n-6 PUFA CYP pathway epoxide 11,12-epoxy-eicosatetraenoic acid lipid mediator profile in response to EPA supplementa- (11,12-EpETE; but not related to 8,9-, 14,15-, or 17,18-EpETE tion included a decrease in the PGE2 metabolite, tetranor- regioisomers) was also increased (;3-fold) by EPA supple- 2 PGEM ( 70%) and an increase (2.7-fold) in the PGD2 mentation (Supplemental Table 1). Plasma RvE1 concentra- metabolite, 13,14-dihydro-15-keto-PGD2. tions were generally #LOQ of our assay, but qualitatively corresponded to ;150 pM (;50 pg/ml). RvE1 remained #LOQ after EPA supplementation, despite marked in- DHA metabolites creases in the abundance of the RvE precursor 18-HEPE. Several hydroxy-DHA species were found to be suppressed in human plasma after EPA supplementation AA metabolites (Fig. 5C). Most notably, 14-HDoHE, the most abundant hydroxy-DHA derivative detected in human plasma and EPA supplementation reduced numerous plasma mono- a key activation marker of the MaR biosynthesis path- hydroxylated metabolites of AA (HETEs; Fig. 5B). 5-HETE way was reduced by 55% after the EPA trial. 17-HDoHE

3718 Vol. 30 November 2016 The FASEB Journal x www.fasebj.org MARKWORTH ET AL. Downloaded from www.fasebj.org by (2001:388:608c:4800:fdd6:e532:12c5:d389) on November 26, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3714-3725. Figure 3. Plasma n-3 DPA metabolites inﬂuenced by DPA supplementation. A) HDoPE metabolites in human plasma in response to dietary DPA supplementation. B) Plasma concentrations of 7,17-DiHDoPE (RvD5n-3DPA) in response to supplementation with OO, EPA, or DPA. C, D) Representative tandem mass spectra and fragment assignments (insets) used for the identiﬁcation of 13HDoPE (C) and 7,17-DiHDoPE (or RvD5n-3DPA)(D). The mass spectra shown are for a representative plasma sample obtained on d 7 of the n-3 DPA supplementation trial. Value are means 6 SEM.*P , 0.05 vs. d 0 within respective supplementation period; #P , 0.05 vs. placebo OO supplementation at the same time point.

a key intermediate in the biosynthesis of D-series resol- HOTrE and 9-OxoOTrE], dihomo-g-linolenic acid (DGLA) vins also tended to be more modestly reduced by EPA (20:3, n-6) [8(S)-HETrE] and Mead acid (20:3, n-9) [5(S)- (226%; P = 0.063). DHA metabolites of CYP epoxygenase HETrE] were reduced in response to OO supplementation including 10,11- and 13,14-epoxy-docosapentaenoic acid (Supplemental Table 1). A subset of AA metabolites de- (EpDPE) were also decreased (Supplemental Table 1). rived from the CYP pathway (both epoxygenase and Despite these changes in several primary DHA metabo- g-hydroxylase) also appeared to be influenced by the OO lites, raw DHA-derived SPMs themselves were not influ- placebo (Fig. 6B). These analytes included the AA vicinal enced by EPA supplementation. diols 5,6-, 8,9-, 11,12-, and 14,15-DiHETrE (epoxygenase pathway), as well as 20-hydroxy-eicosatetraenoic acid Effect of OO placebo on plasma (20-HETE) g-hydroxylase pathway, all of which were de- lipid mediators creased from d 0 to 7 of the OO supplementation trial. Similar patterns of a reduction in these OO-sensitive We found that a specific subset of lipid mediators was metabolites of LA (HODEs/OxoODE, EpOMEs/DiHOME), significantly influenced by our OO placebo condition ALA [9(S)-HOTrE/9-OxoOTrE], DGLA [8(S)-HETrE], (Fig. 6). These analytes included metabolites of lino- Mead acid [5(S)-HETrE], and AA (DiHETrE regioisomers) leic acid (LA) (18:2, n-6) encompassing both hydroxy-/ were generally observed in response to the subsequent oxo-octadecadienoic acids (9-HODE/9-OxoODE, 13- EPA and DPA supplementation trials (Fig. 6 and Sup- HODE/13-OxoODE) and epoxy-/dihydroxy-octadecanoic plemental Table 1). However, as n-3 PUFA supplements acids[9(10)EpOME/9(10)DiHOME, 12(13)EpOME/12 were administered as 1:1 mixtures of OO:EPA or :DPA, (13)DiHOME], all of which decreased by ;30–60% from these changes cannot be true effects of EPA or DPA d 0 to 7 of the initial OO trial (Fig. 6A). Similarly, plasma supplementation and were likely a direct result of the monohydroxylated metabolites of ALA (18:3, n-3) [9(S)- OO component.

n-3 DPA MODULATES PLASMA LIPID MEDIATOR PROFILE 3719 Downloaded from www.fasebj.org by (2001:388:608c:4800:fdd6:e532:12c5:d389) on November 26, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3714-3725. Figure 4. Plasma n-6 PUFA metabolites inﬂuenced by n-3 DPA supplementation. A) Plasma concentrations of AA (20:4, n-6)- derived (series 2) PGs in response to 7-d DPA supplementation. B) Plasma isoprostane F2aVI levels in response to 7 d supplementation with OO, EPA, or DPA. C) Plasma levels of 5(S),6(S)-DiHETE, a nonenzymatic degradation product of LTA4 in response to 7 d supplementation with OO, EPA, or DPA. D) Plasma concentrations of DGLA (20:3, n-6)-derived (series 1) PGs in # response to 7 d DPA supplementation. Value are means 6 SEM.*P , 0.05 vs. d 0 within the respective supplementation period; P , 0.05 vs. placebo OO supplementation at the same time point.

DISCUSSION in circulating DHA metabolites (54–59). However, many previous human PUFA supplementation studies have ei- A considerable amount of research has focused on the ther not included the D-seriesSPMsthemselvesintheir potential roles of EPA and DHA in the beneficial effects of analysis (56–58, 60), or were unable to detect them (54, marine oils. In contrast, the third intermediary LC-n-3 61–64). Nevertheless, recent studies have detected D-series PUFA DPA has rarely been investigated. Limited studies resolvins in human blood (55, 59, 65–68), and plasma on n-3 DPA have reported effects on platelet aggregation RvD1 concentrations have been reported to be increased (40, 41), modulation of endothelial cell function (42, 43), after fish oil supplementation in 2 recent studies (55, 59). In inhibition of tumor growth (44), reduction in liver lipo- the present study, we detected several DHA-derived genic gene expression (45), lowering of circulating lipids SPMs at low pictogram permilliliter concentrations in (46–48), and improvements in age-associated cognitive human plasma, including RvD1, RvD2, RvD6, MaR1, and decline (49). Although interconversion of EPA, DPA, and 10S,17S-DiHDoHE (PD1 isomer or PDX). On the other DHA via retroconversion and elongation pathways may hand, PD1 itself and RvD5 were not detected. Both RvD1 occur (Fig. 1), important differences appear evident in the and MaR-1 were found to be modestly increased after the bioactivity of individual LC n-3 PUFAs. After short-term n-3 DPA supplementation trial. Furthermore, the DHA supplementation in humans, pure n-3 DPA and EPA vicinal diol 19,20-DiHDoPE was markedly increased by possess specific incorporation patterns into plasma and DPA supplementation. Unlike DPA, supplementation with red blood cell lipids (32). In comparison to the other LC n-3 pure EPA actually decreased several plasma DHA me- PUFAs, DPA is also more effective in inhibiting platelet tabolites, most notably the MaR pathway intermediate aggregation (40), stimulating endothelial cell migration 14-HDoHE. The collective data suggest that one impor- (42), and has more potent antiproliferative and proa- tant metabolic fate of dietary n-3 DPA is a contribution to poptotic effects on cancer cells (44). Associations between biosynthesis of DHA-derived docosanoids. In contrast, higher blood DPA levels and reduced human cardiovas- dietary EPA has no substantial contribution to docosa- cular disease risk further suggest beneficial properties of noid biosynthesis in healthy humans and EPA supple- this little-studied dietary fatty acid that are distinct from mentation in isolation has opposing modulatory effect other LC n-3 PUFAs (50–53). Despite these observations, on D-series SPM pathway intermediates, likely via com- the metabolic fate and mechanisms of action of n-3 DPA petition with endogenous DHA as the substrate. have remained poorly understood. In addition to established DHA docosanoids, we de- Dietary supplementation with fish oils containing a tected several specific metabolites of n-3 DPA itself mixture of LC n-3 PUFAs have generally showed increases in human plasma, including HDoPE isomers and

3720 Vol. 30 November 2016 The FASEB Journal x www.fasebj.org MARKWORTH ET AL. Downloaded from www.fasebj.org by (2001:388:608c:4800:fdd6:e532:12c5:d389) on November 26, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3714-3725. Figure 5. Plasma PUFA metabolites inﬂuenced by EPA supplementation. A) HEPE metabolites in human plasma in response to EPA supplementation. B) HETE metabolites in human plasma in response to EPA supplementation. C) HDoHE metabolites in human plasma in response to EPA supplementation. Value and means 6 SEM *P , 0.05 vs. d 0 within the respective supplementation period; #P , 0.05 vs. placebo OO supplementation at the same time point.

the dihydroxylated DPA metabolite 7,17-DiHDoPE of EPA as well as DPA in blood TAG fraction, suggesting (RvD5n-3DPA). In the absence of supplemental n-3 PUFA that dietary DPA may undergo some degree of retro- intake, circulating levels of DPA-derived monohydroxy conversion to EPA in humans (32) (Fig. 1). Nevertheless, fatty acids were generally lower (20–60 pg/ml) when DPA supplementation failed to influence any of the nu- compared to levels of monohydroxylated EPA and DHA merous detected plasma EPA metabolites here, sug- products (100–300 pg/ml) (Supplemental Table 1). How- gesting that the metabolic fate of dietary DPA and EPA ever, plasma concentrations of the dihydroxylated n-3 are highly distinct. Supplementation with pure EPA DPA metabolite RvD5n-3DPA weresimilartothatofSPMs was, on the other hand, highly effective in elevating derived from EPA (e.g., RvE1) and DHA (e.g., MaR1; plasma levels of numerous EPA metabolites. Of partic- ; 50 pg/ml). RvD5n-3 DPA is a recently discovered n-3 DPA ular interest, the E-series resolvin precursor 18-HEPE analog of the 17-dihydroxy-DHA-derived SPM RvD5 was markedly increased by EPA supplementation (;13- which was shown to possess potent anti-inflammatory/ fold). We detected RvE1 itself in basal plasma at con- ; ; proresolving bioactivity (34). In our study, RvD5n-3DPA centrations of 150 pM ( 50 pg/ml), but despite the was detected at low picogram/milliliter concentrations marked increases in plasma concentrations of the RvE (;50 pg/ml) in basal plasma, but was markedly elevated precursor 18-HEPE, RvE1 levels were unaffected. A ($20 fold) to concentrations of $1 ng/ml after DPA sup- number of recent studies have reported elevated plasma plementation. Although RvD5n-3DPA has been shown to be 18-HEPE in human subjects consuming fish oil (54, 59, produced in vitro by human neutrophils and macro- 66), and RvE1 has been measured in human blood at phages, to our knowledge, this is the first report of the comparable concentrations to that reported here, rang- detection and modulation of n-3 DPA series resolvins in ing between ;10–200 pg/ml (19, 65, 66, 68). Barden et al. human blood in vivo. Unlike DPA, supplementation with (66) also found that fish oil supplementation could in- pure EPA had no effect on plasma levels of RvD5n-3DPA or crease plasma levels of RvE1 in healthy human volun- the DPA content of plasma lipids [previously reported in teers. On the other hand, others have questioned the Miller et al. (32)], at least in the timeframe of a week, and relevance of circulating RvEs in humans consuming fish ingestion of DPA substrate itself is needed to modulate oil supplements (55, 58, 62, 63, 69). The reason for these circulating levels of DPA docosanoids. The biologic fate of discrepancies between studies remains unclear, but dietary DPA to form unique lipid mediators with anti- may relate in part to analyte stability, sample handling, inflammatory and proresolving properties, and lack of and methodological and analytical differences (65). redundancy by EPA, provides a novel mechanism that A notable finding of the current study is that several may explain bioactivity of n-3 DPA independent of other additional metabolites of EPA, the in vivo enzymatic bio- LC n-3 PUFAs. synthesis of which is not well characterized, were mark- In our previous report, we found that in these same edly elevated with EPA supplementation. For example, subjects n-3 DPA supplementation increased the proportion 8-HEPE was one of the most responsive analytes to EPA

n-3 DPA MODULATES PLASMA LIPID MEDIATOR PROFILE 3721 Downloaded from www.fasebj.org by (2001:388:608c:4800:fdd6:e532:12c5:d389) on November 26, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3714-3725. Figure 6. Plasma PUFA metabolites inﬂuenced by placebo (OO) supplementation. A) LA (18:2, n-6) derived HODEs/ oxo-octadecadienoic acids (OxoODEs) and epoxy-octadecenoic acids (EpOMEs)/ DiHOMEs). B) AA (20:4, n-6) derived COX-1 – and 2 pathway prostanoids (PGE2 and TXB2) and CYP-pathway derived epoxide vicinal diols (5,6-, 8,9-, 11,12-, and 14,15- DiHETrE). Value are means 6 SEM.*P , 0.05 vs. d 0 within the respective supplementation period.

feeding, a finding that is consistent with several studies in and bicyclo-PGE2) were also markedly elevated after DPA which human subjects were fed fish oil (57, 60, 70). Al- supplementation. Other AA- and DGLA-derived PG spe- though the physiologic significance of these analytes in cies were found to be more modestly increased by DPA. human blood remains to be determined, 8-HEPE and Although the conversion of PGE2 to 15-keto-PGE2 has 9-HEPE in krill extracts possess potent bioactivity as PPAR been classically viewed as an inactivating step, recent agonists, inducing metabolic responses including stimu- studies have suggested that 15-keto-PGE2 possesses ac- lation of fatty acid oxidation, adipogenesis, and glucose tivity distinct from PGE2. For example, it was recently re- uptake in vitro (71). ported that 15-keto-PGE2,servesasanendogenous We were surprised to find that some n-6 PUFA me- PPARg ligand, inducing potential beneficial physiologic tabolites were elevated in the plasma of subjects receiving effects (72–73). The mechanism by which n-3 DPA may n-3 DPA supplementation. Most notably, 15-keto-PGE2 modulate n-6 PUFA metabolite profile in human blood was markedly increased ($40-fold) to concentrations of and the physiologic ramifications of this apparent effect ; ; 250 nM ( 100 ng/ml). 15-keto-PGE2 is generally known currently remain unclear. as an inactive metabolite of PGE2 produced via the PG We found that supplementation with pure EPA re- 15-hydroxy dehydrogenase pathway. Therefore, an increase duced plasma levels of the AA-derived LOX pathway in 15-keto-PGE2/PGE2 ratio could be seen to be indicative products5-,12-,and15-HETE.Thisfindingisconsistent that DPA clears active PGE2 from circulation and thus tones with previous studies reporting reduced hydroxy- and – down inflammatory events. However, total PGE2 levels epoxy-AA metabolites in humans receiving fish oil (54 58, (sum of PGE2, 15-keto-PGE2, 13,14-dihydro-15-keto-PGE2, 60). DPA, however, had no effect on plasma hydroxy- and

3722 Vol. 30 November 2016 The FASEB Journal x www.fasebj.org MARKWORTH ET AL. Downloaded from www.fasebj.org by (2001:388:608c:4800:fdd6:e532:12c5:d389) on November 26, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3714-3725. epoxy-AA metabolites, further confirming the divergent University). Equateq, Ltd. (Breasclete, United Kingdom) gener- activity of EPA and DPA. Few previous studies have re- ously provided the pure supplements and the National Center ported the effect of dietary PUFAs on metabolites of the for Research Resources, U.S. National Institutes of Health, National Center for Research Resources Grant S10RR027926 (to COX pathways, probably because of the instability of K.R.M.). The authors declare no conflicts of interest. some of these analytes during the sample saponification used in many studies to liberate esterified oxylipins (54, 55, 58, 60). We found that EPA supplementation elevated AUTHOR CONTRIBUTIONS plasma levels of EPA-derived LTB5, but had little or no influence on any of the detected AA-derived PGs (e.g., A.J.Sinclair,D.Cameron-Smith,G.Kaur,A.E.Larson,and PGE2)orLTs(e.g., LTB4). Fish oil supplementation has also E. G. Miller conceived and designed the study; J. F. been reported to increase PGE3 and LTB5 (54), but has little Markworth, G. Kaur, E. G. Miller, A. E. Larson, and K. R. if any effect on AA eicosanoids (54, 56, 58). One exception Maddipati collected and analyzed the data; J. F. Markworth, in the current study was 13,14dh-15k-PGD2,adown- D.C.Sinclair,K.R.Maddipati,G.Kaurinterpretedthedata; J. F. Markworth, K. R. Maddipati, G. Kaur, A. J. Sinclair, and stream plasma metabolite of PGD2, which was elevated after EPA supplementation (2.6-fold). Notably, PGD and D. Cameron-Smith drafted the manuscript; and all authors 2 fi its downstream metabolic products have been posited to helped to revise and approved the nal version of the possess anti-inflammatory properties and may thus po- manuscript. tentially contribute to the bioactivity of EPA. Although this study involved a small number of sub- REFERENCES jects, one of its strengths was that participants were of a single gender and of a similar age and body mass index. 1. Flachs, P., Rossmeisl, M and Kopecky, J. 2014. The effect of n-3 fatty This is supported by a recent review which indicated that acids on glucose homeostasis and insulin sensitivity. Physiol. Res. 63 – because women and men have different proportions of (Suppl 1):S93 S118. 2. Gerber, P. A., Gouni-Berthold, I., and Berneis, K. (2013) Omega-3 DHA in blood and tissues and because they convert ALA fatty acids: role in metabolism and cardiovascular disease. 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