Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages
2019; Elsevier BV; Volume: 60; Issue: 11 Linguagem: Inglês
10.1194/jlr.m094235
ISSN1539-7262
AutoresIan de Bus, Han Zuilhof, Renger F. Witkamp, Michiel G.J. Balvers, Bauke Albada,
Tópico(s)Cannabis and Cannabinoid Research
ResumoCyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE3-ethanolamide (PGE3-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13- and 16-hydroxy-DHEA (13- and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites. Cyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE3-ethanolamide (PGE3-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13- and 16-hydroxy-DHEA (13- and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites. Cyclooxygenase 2 (COX-2) is a nonconstitutional enzyme that is upregulated upon inflammation in order to generate inflammatory regulators (1Ricciotti E. FitzGerald G.A. Prostaglandins and inflammation.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 986-1000Crossref PubMed Scopus (2298) Google Scholar, 2Rouzer C.A. Marnett L.J. Endocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: cross-talk between the eicosanoid and endocannabinoid signaling pathways.Chem. Rev. 2011; 111: 5899-5921Crossref PubMed Scopus (233) Google Scholar, 3Smith W.L. DeWitt D.L. Garavito R.M. Cyclooxygenases: structural, cellular, and molecular biology.Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2457) Google Scholar). It is known to convert arachidonic acid (AA) into prostaglandin (PG)H2, the precursor of various inflammation-regulating PGs and thromboxanes (1Ricciotti E. FitzGerald G.A. Prostaglandins and inflammation.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 986-1000Crossref PubMed Scopus (2298) Google Scholar, 2Rouzer C.A. Marnett L.J. Endocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: cross-talk between the eicosanoid and endocannabinoid signaling pathways.Chem. Rev. 2011; 111: 5899-5921Crossref PubMed Scopus (233) Google Scholar, 4Serhan C.N. Chiang N. Van Dyke T.E. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators.Nat. Rev. Immunol. 2008; 8: 349-361Crossref PubMed Scopus (2207) Google Scholar). Although AA is considered the prototypical COX-2 substrate, the enzyme has a broad substrate specificity that includes other PUFAs and their derivatives, such as the endocannabinoid, arachidonoylethanolamide (AEA; also known as anandamide) (2Rouzer C.A. Marnett L.J. Endocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: cross-talk between the eicosanoid and endocannabinoid signaling pathways.Chem. Rev. 2011; 111: 5899-5921Crossref PubMed Scopus (233) Google Scholar, 5Alhouayek M. Muccioli G.G. COX-2-derived endocannabinoid metabolites as novel inflammatory mediators.Trends Pharmacol. Sci. 2014; 35: 284-292Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 6Fowler C.J. The contribution of cyclooxygenase-2 to endocannabinoid metabolism and action.Br. J. Pharmacol. 2007; 152: 594-601Crossref PubMed Scopus (74) Google Scholar, 7Kozak K.R. Prusakiewicz J.J. Rowlinson S.W. Prudhomme D.R. Marnett L.J. Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide.Biochemistry. 2003; 42: 9041-9049Crossref PubMed Scopus (58) Google Scholar, 8Prusakiewicz J.J. Kingsley P.J. Kozak K.R. Marnett L.J. Selective oxygenation of N-arachidonylglycine by cyclooxygenase-2.Biochem. Biophys. Res. Commun. 2002; 296: 612-617Crossref PubMed Scopus (63) Google Scholar, 9Prusakiewicz J.J. Turman M.V. Vila A. Ball H.L. Al-Mestarihi A.H. Marzo V.D. Marnett L.J. Oxidative metabolism of lipoamino acids and vanilloids by lipoxygenases and cyclooxygenases.Arch. Biochem. Biophys. 2007; 464: 260-268Crossref PubMed Scopus (30) Google Scholar, 10Rouzer C.A. Marnett L.J. Non-redundant functions of cyclooxygenases: oxygenation of endocannabinoids.J. Biol. Chem. 2008; 283: 8065-8069Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 11Urquhart P. Nicolaou A. Woodward D.F. Endocannabinoids and their oxygenation by cyclo-oxygenases, lipoxygenases and other oxygenases.Biochim. Biophys. Acta. 2015; 1851: 366-376Crossref PubMed Scopus (87) Google Scholar). Previous studies revealed that COX-2 converts AEA into PG ethanolamides (EAs), also called prostamides, and potent anti-inflammatory monohydroxylated AEAs (5Alhouayek M. Muccioli G.G. COX-2-derived endocannabinoid metabolites as novel inflammatory mediators.Trends Pharmacol. Sci. 2014; 35: 284-292Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 6Fowler C.J. The contribution of cyclooxygenase-2 to endocannabinoid metabolism and action.Br. J. Pharmacol. 2007; 152: 594-601Crossref PubMed Scopus (74) Google Scholar, 11Urquhart P. Nicolaou A. Woodward D.F. Endocannabinoids and their oxygenation by cyclo-oxygenases, lipoxygenases and other oxygenases.Biochim. Biophys. Acta. 2015; 1851: 366-376Crossref PubMed Scopus (87) Google Scholar, 12Alhouayek M. Masquelier J. Cani P.D. Lambert D.M. Muccioli G.G. Implication of the anti-inflammatory bioactive lipid prostaglandin D2-glycerol ester in the control of macrophage activation and inflammation by ABHD6.Proc. Natl. Acad. Sci. USA. 2013; 110: 17558-17563Crossref PubMed Scopus (108) Google Scholar, 13Urquhart P. Wang J. Woodward D.F. Nicolaou A. Identification of prostamides, fatty acyl ethanolamines, and their biosynthetic precursors in rabbit cornea.J. Lipid Res. 2015; 56: 1419-1433Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 14Yu M. Ives D. Ramesha C.S. Synthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2.J. Biol. Chem. 1997; 272: 21181-21186Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Although various amide- and ester-bound derivatives of AA are converted by COX-2 (5Alhouayek M. Muccioli G.G. COX-2-derived endocannabinoid metabolites as novel inflammatory mediators.Trends Pharmacol. Sci. 2014; 35: 284-292Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 6Fowler C.J. The contribution of cyclooxygenase-2 to endocannabinoid metabolism and action.Br. J. Pharmacol. 2007; 152: 594-601Crossref PubMed Scopus (74) Google Scholar, 13Urquhart P. Wang J. Woodward D.F. Nicolaou A. Identification of prostamides, fatty acyl ethanolamines, and their biosynthetic precursors in rabbit cornea.J. Lipid Res. 2015; 56: 1419-1433Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 14Yu M. Ives D. Ramesha C.S. Synthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2.J. Biol. Chem. 1997; 272: 21181-21186Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 15Kozak K.R. Crews B.C. Morrow J.D. Wang L-H. Ma Y.H. Weinander R. Jakobsson P-J. Marnett L.J. Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide, into prostaglandin, thromboxane, and prostacyclin glycerol esters and ethanolamides.J. Biol. Chem. 2002; 277: 44877-44885Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 16Kozak K.R. Crews B.C. Ray J.L. Tai H-H. Morrow J.D. Marnett L.J. Metabolism of prostaglandin glycerol esters and prostaglandin ethanolamides in vitro and in vivo.J. Biol. Chem. 2001; 276: 36993-36998Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 17Kozak K.R. Rowlinson S.W. Marnett L.J. Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2.J. Biol. Chem. 2000; 275: 33744-33749Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), the interaction between COX-2 and endocannabinoid-like molecules derived from PUFAs other than AA is barely investigated. Previous work revealed that docosahexaenoylethanolamide (DHEA), an n-3 PUFA endocannabinoid-like metabolite, has potent anti-inflammatory properties in vitro (18Meijerink J. Poland M. Balvers M.G.J. Plastina P. Lute C. Dwarkasing J. van Norren K. Witkamp R.F. Inhibition of COX-2-mediated eicosanoid production plays a major role in the anti-inflammatory effects of the endocannabinoid N-docosahexaenoylethanolamine (DHEA) in macrophages.Br. J. Pharmacol. 2015; 172: 24-37Crossref PubMed Scopus (45) Google Scholar). DHEA is present in human blood and a variety of animal tissues in a concentration that depends on the dietary intake of n-3 PUFAs as present in fish oil (19Balvers M.G.J. Verhoeckx K.C.M. Bijlsma S. Rubingh C.M. Meijerink J. Wortelboer H.M. Witkamp R.F. Fish oil and inflammatory status alter the n-3 to n-6 balance of the endocannabinoid and oxylipin metabolomes in mouse plasma and tissues.Metabolomics. 2012; 8: 1130-1147Crossref PubMed Scopus (90) Google Scholar, 20Balvers M.G.J. Wortelboer H.M. Witkamp R.F. Verhoeckx K.C.M. Liquid chromatography–tandem mass spectrometry analysis of free and esterified fatty acid N-acyl ethanolamines in plasma and blood cells.Anal. Biochem. 2013; 434: 275-283Crossref PubMed Scopus (36) Google Scholar, 21Meijerink J. Balvers M.G.J. Witkamp R.F. N-acyl amines of docosahexaenoic acid and other n–3 polyunsatured fatty acids – from fishy endocannabinoids to potential leads.Br. J. Pharmacol. 2013; 169: 772-783Crossref PubMed Scopus (72) Google Scholar, 22de Bus I. Witkamp R. Zuilhof H. Albada B. Balvers M. The role of n-3 PUFA-derived fatty acid derivatives and their oxygenated metabolites in the modulation of inflammation.Prostaglandins Other Lipid Mediat. 2019; 144: 106351Crossref PubMed Scopus (58) Google Scholar). Several studies showed interesting biological properties of DHEA. For example, DHEA is able to inhibit head and neck squamous cell carcinoma proliferation, the formation of pro-inflammatory cytokines, such as interleukin (IL)-6, and to induce the production of the anti-inflammatory cytokine, IL-10, which all results in strong anti-inflammatory effects (23Yang R. Fredman G. Krishnamoorthy S. Agrawal N. Irimia D. Piomelli D. Serhan C.N. Decoding functional metabolomics with docosahexaenoyl ethanolamide (DHEA) identifies novel bioactive signals.J. Biol. Chem. 2011; 286: 31532-31541Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24McDougle D.R. Watson J.E. Abdeen A.A. Adili R. Caputo M.P. Krapf J.E. Johnson R.W. Kilian K.A. Holinstat M. Das A. Anti-inflammatory ω-3 endocannabinoid epoxides.Proc. Natl. Acad. Sci. USA. 2017; 114: E6034-E6043Crossref PubMed Scopus (114) Google Scholar, 25Park S-W. Hah J.H. Oh S-M. Jeong W-J. Sung M-W. 5-lipoxygenase mediates docosahexaenoyl ethanolamide and N-arachidonoyl-L-alanine-induced reactive oxygen species production and inhibition of proliferation of head and neck squamous cell carcinoma cells.BMC Cancer. 2016; 16: 458Crossref PubMed Scopus (18) Google Scholar, 26Alhouayek M. Bottemanne P. Makriyannis A. Muccioli G.G. N-acylethanolamine-hydrolyzing acid amidase and fatty acid amide hydrolase inhibition differentially affect N-acylethanolamine levels and macrophage activation.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2017; 1862: 474-484Crossref PubMed Scopus (27) Google Scholar). In addition, DHEA stimulates neurite growth, hippocampal development, and synaptogenesis in developing neurons in the central nervous system (27Kim H-Y. Moon H-S. Cao D. Lee J. Kevala K. Jun S.B. Lovinger D.M. Akbar M. Huang B.X. N-docosahexaenoylethanolamide promotes development of hippocampal neurons.Biochem. J. 2011; 435: 327-336Crossref PubMed Scopus (88) Google Scholar, 28Kim H-Y. Spector A.A. N-docosahexaenoylethanolamine: a neurotrophic and neuroprotective metabolite of docosahexaenoic acid.Mol. Aspects Med. 2018; 64: 34-44Crossref PubMed Scopus (62) Google Scholar, 29Kim H-Y. Spector A.A. Xiong Z-M. A synaptogenic amide N-docosahexaenoylethanolamide promotes hippocampal development.Prostaglandins Other Lipid Mediat. 2011; 96: 114-120Crossref PubMed Scopus (75) Google Scholar, 30Lee J-W. Huang B.X. Kwon H. Rashid M.A. Kharebava G. Desai A. Patnaik S. Marugan J. Kim H-Y. Orphan GPR110 (ADGRF1) targeted by N-docosahexaenoylethanolamine in development of neurons and cognitive function.Nat. Commun. 2016; 7: 13123Crossref PubMed Scopus (95) Google Scholar, 31Park T. Chen H. Kevala K. Lee J-W. Kim H-Y. N-docosahexaenoylethanolamine ameliorates LPS-induced neuroinflammation via cAMP/PKA-dependent signaling.J. Neuroinflammation. 2016; 13: 284Crossref PubMed Scopus (70) Google Scholar), hence the alternative name synaptamide for DHEA (29Kim H-Y. Spector A.A. Xiong Z-M. A synaptogenic amide N-docosahexaenoylethanolamide promotes hippocampal development.Prostaglandins Other Lipid Mediat. 2011; 96: 114-120Crossref PubMed Scopus (75) Google Scholar, 32Kim H-Y. Spector A.A. Synaptamide, endocannabinoid-like derivative of docosahexaenoic acid with cannabinoid-independent function.Prostaglandins Leukot. Essent. Fatty Acids. 2013; 88: 121-125Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Recently, it was suggested that the neural and hippocampal stimulating roles are at least partly caused by the interaction of DHEA with the G protein-coupled receptor 110, stimulating a cAMP-dependent signal transduction pathway (28Kim H-Y. Spector A.A. N-docosahexaenoylethanolamine: a neurotrophic and neuroprotective metabolite of docosahexaenoic acid.Mol. Aspects Med. 2018; 64: 34-44Crossref PubMed Scopus (62) Google Scholar, 30Lee J-W. Huang B.X. Kwon H. Rashid M.A. Kharebava G. Desai A. Patnaik S. Marugan J. Kim H-Y. Orphan GPR110 (ADGRF1) targeted by N-docosahexaenoylethanolamine in development of neurons and cognitive function.Nat. Commun. 2016; 7: 13123Crossref PubMed Scopus (95) Google Scholar). Previously, our group has demonstrated that DHEA reduces the production of the pro-inflammatory mediator PGE2 in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, which could not be fully explained by the modest reduction of COX-2 protein expression (18Meijerink J. Poland M. Balvers M.G.J. Plastina P. Lute C. Dwarkasing J. van Norren K. Witkamp R.F. Inhibition of COX-2-mediated eicosanoid production plays a major role in the anti-inflammatory effects of the endocannabinoid N-docosahexaenoylethanolamine (DHEA) in macrophages.Br. J. Pharmacol. 2015; 172: 24-37Crossref PubMed Scopus (45) Google Scholar). This observation suggests that DHEA may act as a COX-2 substrate, which reduces PG formation through competitive inhibition. Several studies aimed at unraveling the metabolic fate of DHEA. Although the interactions of DHEA and 15-lipooxygenase (15-LOX) or cytochrome P450 (CYP450) were reported (22de Bus I. Witkamp R. Zuilhof H. Albada B. Balvers M. The role of n-3 PUFA-derived fatty acid derivatives and their oxygenated metabolites in the modulation of inflammation.Prostaglandins Other Lipid Mediat. 2019; 144: 106351Crossref PubMed Scopus (58) Google Scholar, 23Yang R. Fredman G. Krishnamoorthy S. Agrawal N. Irimia D. Piomelli D. Serhan C.N. Decoding functional metabolomics with docosahexaenoyl ethanolamide (DHEA) identifies novel bioactive signals.J. Biol. Chem. 2011; 286: 31532-31541Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24McDougle D.R. Watson J.E. Abdeen A.A. Adili R. Caputo M.P. Krapf J.E. Johnson R.W. Kilian K.A. Holinstat M. Das A. Anti-inflammatory ω-3 endocannabinoid epoxides.Proc. Natl. Acad. Sci. USA. 2017; 114: E6034-E6043Crossref PubMed Scopus (114) Google Scholar, 28Kim H-Y. Spector A.A. N-docosahexaenoylethanolamine: a neurotrophic and neuroprotective metabolite of docosahexaenoic acid.Mol. Aspects Med. 2018; 64: 34-44Crossref PubMed Scopus (62) Google Scholar), the possible interaction between COX-2 and DHEA is poorly understood. For instance, Serhan and coworkers reported that 15-LOX yields di-hydroxylated and epoxidated DHEA products. Two of these, i.e., 10,17-diHDHEA and 15-HEDPEA, were found to reduce platelet leukocyte aggregation, and 15-HEDPEA was even shown to be protective in vivo in murine hind limb ischemia and second organ reperfusion injury (23Yang R. Fredman G. Krishnamoorthy S. Agrawal N. Irimia D. Piomelli D. Serhan C.N. Decoding functional metabolomics with docosahexaenoyl ethanolamide (DHEA) identifies novel bioactive signals.J. Biol. Chem. 2011; 286: 31532-31541Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Similarly, several CYP450-derived epoxide products of DHEA increased the formation of anti-inflammatory cytokines and exerted anti-angiogenic effects in human microvascular endothelial cells, vasodilatory actions on bovine coronary arteries, and reciprocally regulated platelet aggregation in washed human platelets (24McDougle D.R. Watson J.E. Abdeen A.A. Adili R. Caputo M.P. Krapf J.E. Johnson R.W. Kilian K.A. Holinstat M. Das A. Anti-inflammatory ω-3 endocannabinoid epoxides.Proc. Natl. Acad. Sci. USA. 2017; 114: E6034-E6043Crossref PubMed Scopus (114) Google Scholar). In view of the prominent role of COX-2 in inflammation regulation, we studied the interaction between DHEA and COX-2. To this end, n-3 PUFA-ethanolamine-conjugates were applied in a cell-free enzymatic human recombinant (h)COX-2 assay, and a LC separation coupled to a high-resolution (HR) MS (LC-HRMS) method was used to identify yet unidentified COX-2 metabolites of eicosapentaenoylethanolamide (EPEA), i.e., PGE3-EA and 11-HEPE-EA (and the postulated products 14- and 18-HEPE-EA), and DHEA, i.e., 13- and 16-HDHEA. Then, we developed an ultrahigh-performance (UP)LC-MS/MS-based method to quantify the products and studied the role of COX-2 metabolism of DHEA in cells by measuring the COX-2-related production of 13- and 16-HDHEA in LPS-stimulated RAW264.7 macrophages. We show that formation of these products is inhibited upon addition of the selective COX-2 inhibitor, celecoxib, proving a COX-2-dependent metabolism. COX-2 (hCOX-2), AA (≥98% purity), EPA (≥98% purity), DHA (≥98% purity), EPEA (≥98% purity), DHEA (≥98% purity), DHEA-d4 [≥99% purity deuterated forms (d1–d4)], PGE2-EA-d4 [≥99% purity deuterated forms (d1–d4)], PGE3 (≥98% purity), (±)11-HEPE (≥98% purity), (±)13-hydroxydocosahexaenoic acid (HDHA) (≥98% purity), (±)16-HDHA (≥98% purity), and celecoxib (≥98% purity) were purchased at Cayman Chemicals (supplied by Sanbio B.V., Uden, The Netherlands). The lactate dehydrogenase (LDH) cytotoxicity kit was purchased at Roche Applied Science (Almere, The Netherlands). Anandamide (100% purity from HPLC) was obtained from Tocris Chemicals (Abingdon, UK). DMEM was purchased from Corning (supplied by VWR Chemicals, Amsterdam, The Netherlands); FBS, streptomycin, and penicillin were obtained from Lonza (Verviers, Belgium). Hematin porcine, Triton™ X-100, LPS, butylated hydroxytoluene (BHT) (99%), isobutyl chloroformate (98%), triethylamine (99.5%), ethanolamine (≥98%), ethanol-1,1,2,2-d4-amine (98 atom % D), and ethanol (EtOH; absolute for analysis) were purchased at Sigma-Aldrich (Zwijndrecht, The Netherlands). Ethyl acetate and n-heptane were acquired from VWR Chemicals. Trizma base (99%), phenol (>99%, for biochemistry), DMSO (>99.7%), and methanol (MeOH) (99.99%, LC/MS grade) were obtained from Fisher Scientific (Landsmeer, The Netherlands). Formic acid (99%, ULC/MS-CC/SFC) and acetonitrile (ACN) (ULC/MS-CC/SFC) were purchased at Biosolve Chemicals (Valkenswaard, The Netherlands). The HLB solid phase extraction columns (Oasis; 60 mg, 3cc) were obtained from Waters (Etten-Leur, The Netherlands). Ultrapure water was filtered by a MilliQ Integral 3 system from Millipore (Molsheim, France). A cell-free enzymatic hCOX-2 assay was performed similar to previously reported protocols (33Cao H. Yu R. Tao Y. Nikolic D. van Breemen R.B. Measurement of cyclooxygenase inhibition using liquid chromatography–tandem mass spectrometry.J. Pharm. Biomed. Anal. 2011; 54: 230-235Crossref PubMed Scopus (37) Google Scholar, 34Reininger E.A. Bauer R. Prostaglandin-H-synthase (PGHS)-1 and -2 microtiter assays for the testing of herbal drugs and in vitro inhibition of PGHS-isoenzymes by polyunsaturated fatty acids from Platycodi radix.Phytomedicine. 2006; 13: 164-169Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 35Sharma N.P. Dong L. Yuan C. Noon K.R. Smith W.L. Asymmetric acetylation of the cyclooxygenase-2 homodimer by aspirin and its effects on the oxygenation of arachidonic, eicosapentaenoic, and docosahexaenoic acids.Mol. Pharmacol. 2010; 77: 979-986Crossref PubMed Scopus (48) Google Scholar). A 0.1 M Tris/HCl buffer (pH 8.0) was prepared containing 50 μM of Na2EDTA to stabilize the enzyme in solution. For the enzymatic incubations, 179 μl of buffer were mixed with 2 μl of 100 μM hematin solution in DMSO, 10 μl of 25 mM phenol solution in buffer and 5 μl of hCOX-2 in buffer giving a total amount of 1.0 μg of enzyme per reaction. In the negative control, 5 μl of buffer were added instead of 5 μl of hCOX-2 solution. The mixtures were preincubated for 2 min at room temperature before adding 4 μl of a 1 mM substrate solution in EtOH, giving a final substrate concentration of 20 μM. The reaction mixture was heated at 37°C for 20 min before quenching the reaction with 5 μl of formic acid. Eight hundred microliters of ethyl acetate:heptane (1:1, v/v) were used to extract the substrates and metabolites, after which the organic solvent was evaporated under reduced pressure at 40°C. The dried extract was reconstituted with 200 μl of EtOH and analyzed on the LC-HRMS system (see below). For the UPLC-MS/MS-based quantification of the EPEA and DHEA metabolites, the reactions were quenched with 5 μl of formic acid followed by the addition of 2 ml of MeOH containing 806 pg/mL product (11-HEPE-EA-d4 or PGE2-EA-d4 or 13-HDHEA-d4) and 1,000 pg/mL starting material (DHEA-d4) as internal standards. Thereafter, the metabolites were collected by SPE using HLB columns (see below). LC-HRMS analyses were performed using a Finnigan Surveyor Plus HPLC from Thermo Fischer Scientific (Breda, The Netherlands) coupled to a Q-Exactive Focus Quadrupole Orbitrap high-resolution mass spectrometer equipped with a higher-energy collisional dissociation chamber, also from Thermo Fischer Scientific. The machine was operated at a mass resolution of 70,000 to allow the chemical characterization of the products. Ionization was performed using a heated electrospray ionization source applying a spray voltage of 3.2 kV and a capillary temperature of 300°C using polarity switching (1 s for a cycle of one positive and one negative scan). For the fragmentation analysis, the collision energies were optimized per ion. Chromatography was performed on a Reprosil Gold 120 C8, 3 μm column of 250 × 3 mm (Dr. Maisch GmbH, Germany), using the following eluents and gradient. Eluent A consisted of water/ACN (95/5) with 0.1% formic acid; eluent B consisted of 100% ACN with 0.1% formic acid. For elution of the various lipids and oxylipins, the program started isocratically with 50% B in A. From 3 to 8 min, the concentration was linearly increased to 100% B, and the run was continued with 100% B until 15 min. Then the gradient was decreased to 50% B in A in 2 min, and run with 50% B in A until 21 min. During the run, column temperature was kept at 30°C and the sample tray was cooled to 4°C to limit auto-oxidation. For the runs, 25 μl or 10 μl of sample were injected. Data analysis of the mass spectra and the extracted ion chromatograms were performed using Thermo Xcalibur software version 3.0 from Thermo Fischer Scientific. All extracted ion chromatograms displayed are within a range of 0.03 Da. All chromatograms were processed with peak smoothening using boxcar smoothening to give representative peaks, unless stated otherwise. The displayed fragmentation spectra were obtained after background subtraction and were reproducible in multiple experiments. To verify whether DHEA is also metabolized in COX-2-expressing cells, RAW264.7 macrophages (American Type Culture Collection, Teddington, UK) were cultured in DMEM containing 10% FCS and 1% penicillin and streptomycin at 37°C in a 5% CO2 humidified incubator. For the in vitro experiments, 2 ml of 250,000 cells/ml were seeded and incubated for 24 h in 6-well plates. The medium of the adherent cells was discarded and replaced by new fresh medium, and the various compounds were subsequently added for the experiments. To investigate COX-2-mediated conversion of DHEA, RAW macrophages were preincubated for 30 min with 10 μM of DHEA, followed by stimulation with 1.0 μg/ml LPS. In the celecoxib control experiments, the RAW macrophages were preincubated with 0.3 μM of celecoxib before DHEA incubation. After 24 h, the lipids were extracted for metabolite identification (see below). To assess the viability of the macrophages after the various treatments, microscopic evaluation of the wells was performed after incubation. Pictures were taken with a LEICA DFS450C microscope from Leica Microsystems (Amsterdam, The Netherlands), and white balancing was applied to optimize the color brightness of each picture. Cell cytotoxicity was tested in the medium based on the LDH concentration. The LDH assay was performed according to the manufacturer's instructions. Quantification of PGE2 was done by performing PGE2 EIA assays. A PGE2 ELISA kit (monoclonal) was purchased at Cayman Chemical (supplied by Sanbio B.V.) and the ELISA was performed according to the supplied instructions. Extraction of intracellular lipids from incubated RAW264.7 macrophages was performed using an adapted version of a previously described oxylipin extraction protocol, which was optimized for adherent cells. (19Balvers M.G.J. Verhoeckx K.C.M. Bijlsma S. Rubingh C.M. Meijerink J. Wortelboer H.M. Witkamp R.F. Fish oil and inflammatory status alter the n-3 to n-6 balance of the endocannabinoid and oxylipin metabolomes in mouse plasma and tissues.Metabolomics. 2012; 8: 1130-1147Crossref PubMed Scopus (90) Google Scholar). In short, the medium was replaced by 1 ml of fresh medium and the macrophages were scraped from the culture plates in order to suspend them in the fresh medium. The cells were centrifuged (330 g at 4°C) for 5 min and the supernatant was discarded. Subsequently, the macrophages were treated with 1 ml of MeOH containing 806 pg/ml 13-HDHEA-d4 as an internal standard. This suspension was sonicated for 5 min and centrifuged (330 g at 4°C) for 5 min, after which the supernatant was stored in a clean 15 ml falcon tube. The pellet was treated a second time with 1 ml of fresh MeOH containing 806 pg/ml 13-HDHEA-d4. After 5 min sonication and 5 min centrifugation (330 g at 4°C), this supernatant was also transferred to the 15 ml falcon tube containing the supernatant of the first extraction. The combined extracts were diluted with 8 ml of ultrapure water containing 0.125% formic acid before solid-phase extraction of the metabolites on HLB columns. The columns were preactivated using 2 ml of MeOH and 2 ml of ultrapure water containing 0.1% formic acid, respectively. Then the columns were loaded with the extract and rinsed using 2 ml of 20% MeOH in ultrapure water containing 0.1% formic acid. The lipids were eluted using 1 ml of MeOH containing 0.1% formic acid and collected in tubes filled with 20 μl of 10% glycerol and 500 μM of BHT in EtOH (BHT was present to prevent auto-oxidation of the products). The samples were evaporated to dryness using a speedyvac concentrator (Salm and Kipp, Breukelen, The Netherlands) and were redissolved in 100 μl of EtOH. The samples were directly analyzed by UPLC-MS/MS or stored at −80°C and analyzed at a later time point. For improved chromatographic separation and sensitivity, an UPLC-MS/MS method was developed to quantify the hydroxylated fatty acid-ethanolamine-conjugates. The standards used for the quantification were either commercially available
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