Identification of prostamides, fatty acyl ethanolamines, and their biosynthetic precursors in rabbit cornea
2015; Elsevier BV; Volume: 56; Issue: 8 Linguagem: Inglês
10.1194/jlr.m055772
ISSN1539-7262
AutoresPaula Urquhart, Jenny Wang, David F. Woodward, Anna Nicolaou,
Tópico(s)Fatty Acid Research and Health
ResumoArachidonoyl ethanolamine (anandamide) and prostaglandin ethanolamines (prostamides) are biologically active derivatives of arachidonic acid. Although available through different precursor phospholipids, there is considerable overlap between the biosynthetic pathways of arachidonic acid-derived eicosanoids and anandamide-derived prostamides. Prostamides exhibit physiological actions and are involved in ocular hypotension, smooth muscle contraction, and inflammatory pain. Although topical application of bimatoprost, a structural analog of prostaglandin F2α ethanolamide (PGF2α-EA), is currently a first-line treatment for ocular hypertension, the endogenous production of prostamides and their biochemical precursors in corneal tissue has not yet been reported. In this study, we report the presence of anandamide, palmitoyl-, stearoyl-, α-linolenoyl docosahexaenoyl-, linoleoyl-, and oleoyl-ethanolamines in rabbit cornea, and following treatment with anandamide, the formation of PGF2α-EA, PGE2-EA, PGD2-EA by corneal extracts (all analyzed by LC/ESI-MS/MS). A number of N-acyl phosphatidylethanolamines, precursors of anandamide and other fatty acyl ethanolamines, were also identified in corneal lipid extracts using ESI-MS/MS. These findings suggest that the prostamide and fatty acid ethanolamine pathways are operational in the cornea and may provide valuable insight into corneal physiology and their potential influence on adjacent tissues and the aqueous humor. Arachidonoyl ethanolamine (anandamide) and prostaglandin ethanolamines (prostamides) are biologically active derivatives of arachidonic acid. Although available through different precursor phospholipids, there is considerable overlap between the biosynthetic pathways of arachidonic acid-derived eicosanoids and anandamide-derived prostamides. Prostamides exhibit physiological actions and are involved in ocular hypotension, smooth muscle contraction, and inflammatory pain. Although topical application of bimatoprost, a structural analog of prostaglandin F2α ethanolamide (PGF2α-EA), is currently a first-line treatment for ocular hypertension, the endogenous production of prostamides and their biochemical precursors in corneal tissue has not yet been reported. In this study, we report the presence of anandamide, palmitoyl-, stearoyl-, α-linolenoyl docosahexaenoyl-, linoleoyl-, and oleoyl-ethanolamines in rabbit cornea, and following treatment with anandamide, the formation of PGF2α-EA, PGE2-EA, PGD2-EA by corneal extracts (all analyzed by LC/ESI-MS/MS). A number of N-acyl phosphatidylethanolamines, precursors of anandamide and other fatty acyl ethanolamines, were also identified in corneal lipid extracts using ESI-MS/MS. These findings suggest that the prostamide and fatty acid ethanolamine pathways are operational in the cornea and may provide valuable insight into corneal physiology and their potential influence on adjacent tissues and the aqueous humor. The cornea functions to refract light and protect the intraocular structures of the eye. While its outermost epithelial layer facilitates oxygen diffusion and acts to absorb UV radiation (UVR), the innermost endothelial layer contributes to corneal transparency that is essential for optimum vision and regulates ocular pressure (1.Srinivas S.P. Dynamic regulation of barrier integrity of the corneal endothelium.Optom. Vis. Sci. 2010; 87: E239-E254Crossref PubMed Scopus (86) Google Scholar). Although a healthy cornea is avascular of blood and lymph vessels, hypertension or glaucoma can cause injury through abrasion of the endothelial cell lining leading to neovascularization, which if uncontrolled causes scarring and can lead to blindness (2.Maddula S. Davis D.K. Burrow M.K. Ambati B.K. Horizons in therapy for corneal angiogenesis.Ophthalmology. 2011; 118: 591-599Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 3.Cursiefen C. Immune privilege and angiogenic privilege of the cornea.Chem. Immunol. Allergy. 2007; 92: 50-57Crossref PubMed Scopus (153) Google Scholar). Prostanoids are important regulators of corneal homeostasis with prostaglandin (PG) E2 and thromboxane (TX) A2 mediating corneal endothelial cell proliferation (4.Chen K.H. Hsu W.M. Chiang C.C. Li Y.S. Transforming growth factor-beta2 inhibition of corneal endothelial proliferation mediated by prostaglandin.Curr. Eye Res. 2003; 26: 363-370Crossref PubMed Scopus (21) Google Scholar, 5.Daniel T.O. Liu H. Morrow J.D. Crews B.C. Marnett L.J. Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis.Cancer Res. 1999; 59: 4574-4577PubMed Google Scholar, 6.Black A.T. Gordon M.K. Heck D.E. Gallo M.A. Laskin D.L. Laskin J.D. UVB light regulates expression of antioxidants and inflammatory mediators in human corneal epithelial cells.Biochem. Pharmacol. 2011; 81: 873-880Crossref PubMed Scopus (45) Google Scholar). While PGE2 is also involved in angiogenesis and polymorphonuclear monocyte recruitment (7.Liclican E.L. Nguyen V. Sullivan A.B. Gronert K. Selective activation of the prostaglandin E2 circuit in chronic injury-induced pathologic angiogenesis.Invest. Ophthalmol. Vis. Sci. 2010; 51: 6311-6320Crossref PubMed Scopus (36) Google Scholar), PGD2 can suppress corneal tumor growth and hyperpermeability (8.Murata T. Lin M.I. Aritake K. Matsumoto S. Narumiya S. Ozaki H. Urade Y. Hori M. Sessa W.C. Role of prostaglandin D2 receptor DP as a suppressor of tumor hyperpermeability and angiogenesis in vivo.Proc. Natl. Acad. Sci. USA. 2008; 105: 20009-20014Crossref PubMed Scopus (78) Google Scholar). Furthermore, analogs of PGs are used as ocular hypotensive agents with bimatoprost, a structural analog of PGF2α ethanolamide (PGF2α-EA), being widely used to treat glaucoma (9.Woodward D.F. Krauss A.H. Chen J. Liang Y. Li C. Protzman C.E. Bogardus A. Chen R. Kedzie K.M. Krauss H.A. et al.Pharmacological characterization of a novel antiglaucoma agent, Bimatoprost (AGN 192024).J. Pharmacol. Exp. Ther. 2003; 305: 772-785Crossref PubMed Scopus (119) Google Scholar). Prostaglandin ethanolamines (prostamides; PG-EAs) are sequentially biosynthesized from the endocannabinoid arachidonoyl ethanolamine (anandamide; A-EA) by cyclooxygenase (COX)-2 followed by various prostaglandin synthases (PGSs) (10.Yu 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 (349) Google Scholar, 11.Kozak 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, 12.Koda N. Tsutsui Y. Niwa H. Ito S. Woodward D.F. Watanabe K. Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme: new enzyme assay method using LC/ESI/MS.Arch. Biochem. Biophys. 2004; 424: 128-136Crossref PubMed Scopus (52) Google Scholar, 13.Yang W. Ni J. Woodward D.F. Tang-Liu D.D. Ling K.H. Enzymatic formation of prostamide F2alpha from anandamide involves a newly identified intermediate metabolite, prostamide H2.J. Lipid Res. 2005; 46: 2745-2751Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Anandamide is released from precursor phospholipids N-arachidonoyl phosphatidylethanolamines (NArPE) via N-acyl phosphatidylethanolamine (NAPE)-specific phospholipase (PL) D (NAPE-PLD), although recent findings have indicated the existence of other pathways mediated by either α,β-hydrolase 4 followed by cleavage of glycerophosphate to yield A-EA, or PLC and subsequent dephosphorylation of phosphoanandamide to A-EA [reviewed in (14.Ueda N. Tsuboi K. Uyama T. Metabolism of endocannabinoids and related N-acylethanolamines: canonical and alternative pathways.FEBS J. 2013; 280: 1874-1894Crossref PubMed Scopus (178) Google Scholar)]. The majority of studies investigating these pathways have been carried out in mice and rat tissues, and, interestingly, their prevalence appears to be time and cell specific (15.Liu J. Wang L. Harvey-White J. Huang B.X. Kim H.Y. Luquet S. Palmiter R.D. Krystal G. Rai R. Mahadevan A. et al.Multiple pathways involved in the biosynthesis of anandamide.Neuropharmacology. 2008; 54: 1-7Crossref PubMed Scopus (234) Google Scholar). Finally, A-EA may also be catabolized to arachidonic acid and ethanolamine by fatty acid amide hydrolase (FAAH; EC 3.5.1.99) or N-acylethanolamine-hydrolyzing acid amidase (N-AAA) (16.Cravatt B.F. Giang D.K. Mayfield S.P. Boger D.L. Lerner R.A. Gilula N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.Nature. 1996; 384: 83-87Crossref PubMed Scopus (1793) Google Scholar, 17.Tsuboi K. Takezaki N. Ueda N. The N-acylethanolamine-hydrolyzing acid amidase (NAAA).Chem. Biodivers. 2007; 4: 1914-1925Crossref PubMed Scopus (147) Google Scholar) (Fig. 1). Prostamides exhibit a range of activities in various systems. PGF2α-EA is involved in inflammatory pain and dorsal horn nociceptive neuron excitability, while PGE2-EA increases blood flow and reduces mean arterial pressure in the renal medulla, exhibits strong neuroprotective properties in cerebellar neurons, and along with PGD2-EA, induces apoptosis in an in vitro model of colorectal carcinoma (18.Ritter J.K. Li C. Xia M. Poklis J.L. Lichtman A.H. Abdullah R.A. Dewey W.L. Li P.L. Production and actions of the anandamide metabolite prostamide E2 in the renal medulla.J. Pharmacol. Exp. Ther. 2012; 342: 770-779Crossref PubMed Scopus (36) Google Scholar, 19.Patsos H.A. Hicks D.J. Dobson R.R. Greenhough A. Woodman N. Lane J.D. Williams A.C. Paraskeva C. The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2.Gut. 2005; 54: 1741-1750Crossref PubMed Scopus (94) Google Scholar, 20.Andrianova E.L. Genrikhs E.E. Bobrov M.Y. Lizhin A.A. Gretskaya N.M. Frumkina L.E. Khaspekov L.G. Bezuglov V.V. In vitro effects of anandamide and prostamide e2 on normal and transformed nerve cells.Bull. Exp. Biol. Med. 2011; 151: 30-32Crossref PubMed Scopus (2) Google Scholar, 21.Gatta L. Piscitelli F. Giordano C. Boccella S. Lichtman A. Maione S. Di Marzo V. Discovery of prostamide F2alpha and its role in inflammatory pain and dorsal horn nociceptive neuron hyperexcitability.PLoS ONE. 2012; 7: e31111Crossref PubMed Scopus (82) Google Scholar). Prostamides do not show potent interaction with prostanoid receptors, and studies using isolated feline iris cells have suggested the presence of prostamide-sensitive receptors different from the ones responding to PGs (22.Spada C.S. Krauss A.H. Woodward D.F. Chen J. Protzman C.E. Nieves A.L. Wheeler L.A. Scott D.F. Sachs G. Bimatoprost and prostaglandin F(2 alpha) selectively stimulate intracellular calcium signaling in different cat iris sphincter cells.Exp. Eye Res. 2005; 80: 135-145Crossref PubMed Scopus (39) Google Scholar, 23.Woodward D.F. Krauss A.H. Wang J.W. Protzman C.E. Nieves A.L. Liang Y. Donde Y. Burk R.M. Landsverk K. Struble C. Identification of an antagonist that selectively blocks the activity of prostamides (prostaglandin-ethanolamides) in the feline iris.Br. J. Pharmacol. 2007; 150: 342-352Crossref PubMed Scopus (62) Google Scholar, 24.Matias I. Chen J. De Petrocellis L. Bisogno T. Ligresti A. Fezza F. Krauss A.H. Shi L. Protzman C.E. Li C. et al.Prostaglandin ethanolamides (prostamides): in vitro pharmacology and metabolism.J. Pharmacol. Exp. Ther. 2004; 309: 745-757Crossref PubMed Scopus (127) Google Scholar). The prostamide precursor A-EA has also been shown to exhibit neuroprotective and analgesic roles in inflammation and pain models (25.Hernangómez M. Mestre L. Correa F.G. Loría F. Mecha M. Iñigo P.M. Docagne F. Williams R.O. Borrell J. Guaza C. CD200–CD200R1 interaction contributes to neuroprotective effects of anandamide on experimentally induced inflammation.Glia. 2012; 60: 1437-1450Crossref PubMed Scopus (92) Google Scholar, 26.Russo R. Loverme J. La Rana G. Compton T.R. Parrott J. Duranti A. Tontini A. Mor M. Tarzia G. Calignano A. et al.The fatty acid amide hydrolase inhibitor URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester) reduces neuropathic pain after oral administration in mice.J. Pharmacol. Exp. Ther. 2007; 322: 236-242Crossref PubMed Scopus (161) Google Scholar), while topical administration reduces intraocular pressure (27.Pate D.W. Järvinen K. Urtti A. Jarho P. Järvinen T. Ophthalmic arachidonylethanolamide decreases intraocular pressure in normotensive rabbits.Curr. Eye Res. 1995; 14: 791-797Crossref PubMed Scopus (71) Google Scholar). However, its mode of action is mediated through the CB1 and CB2 cannabinoid and vanilloid subtype-1 (TRPV1) receptors that are not activated by prostamides (24.Matias I. Chen J. De Petrocellis L. Bisogno T. Ligresti A. Fezza F. Krauss A.H. Shi L. Protzman C.E. Li C. et al.Prostaglandin ethanolamides (prostamides): in vitro pharmacology and metabolism.J. Pharmacol. Exp. Ther. 2004; 309: 745-757Crossref PubMed Scopus (127) Google Scholar, 28.Luchicchi A. Pistis M. Anandamide and 2-arachidonoylglycerol: pharmacological properties, functional features, and emerging specificities of the two major endocannabinoids.Mol. Neurobiol. 2012; 46: 374-392Crossref PubMed Scopus (79) Google Scholar). In addition, reports now indicate that NAPEs also have biological functions in their own right, such as membrane stabilization and inhibition of macrophage phagocytosis (29.Coulon D. Faure L. Salmon M. Wattelet V. Bessoule J.J. Occurrence, biosynthesis and functions of N-acylphosphatidylethanolamines (NAPE): not just precursors of N-acylethanolamines (NAE).Biochimie. 2012; 94: 75-85Crossref PubMed Scopus (43) Google Scholar). Consistently, NAPEs have been detected in low abundance in mammalian systems but then accumulate under conditions of cellular stress (e.g., ischemia and inflammation), leading to suggestions of putative protective roles [reviewed in (29.Coulon D. Faure L. Salmon M. Wattelet V. Bessoule J.J. Occurrence, biosynthesis and functions of N-acylphosphatidylethanolamines (NAPE): not just precursors of N-acylethanolamines (NAE).Biochimie. 2012; 94: 75-85Crossref PubMed Scopus (43) Google Scholar, 30.Wellner N. Diep T.A. Janfelt C. Hansen H.S. N-acylation of phosphatidylethanolamine and its biological functions in mammals.Biochim. Biophys. Acta. 2013; 1831: 652-662Crossref PubMed Scopus (71) Google Scholar)]. Although A-EA has been found in the cornea as a minor lipid (30.Wellner N. Diep T.A. Janfelt C. Hansen H.S. N-acylation of phosphatidylethanolamine and its biological functions in mammals.Biochim. Biophys. Acta. 2013; 1831: 652-662Crossref PubMed Scopus (71) Google Scholar), neither its metabolism through COX-2 to form prostamides nor the prevalence of its biochemical precursor NArPE have been investigated. In this study, we explored the endogenous production of PGF2α-EA, PGE2-EA, and PGD2-EA by the cornea and show its capability to form these prostamides when A-EA is added externally. We also present data detailing the levels of A-EA and its congeners, as well as NArPE and other fatty acyl NAPE species in rabbit corneal tissue. Given the pharmacological potency of prostamides in ocular health, detailed information on their profile and biochemical precursors in cornea could provide valuable insight into ocular physiology and potential therapeutics. PGE2, PGF2α, PGD2, 15-deoxy Δ12,14 PGJ2, PGJ2, Δ12PGJ2, PGE3, PGD3, PGE1, PGD1, 13,14 dihydro 15-keto PGE2, 13,14 dihydro 15-keto PGF2α, TXB2, 6-keto PGF1α PGB2-d4, A-EA, A-EA-d8, palmitoyl ethanolamine (P-EA), docosahexaenoyl ethanolamine (DH-EA), α-linolenoyl ethanolamine (AL-EA), oleoyl ethanolamine (O-EA), stearoyl ethanolamine (ST-EA), linoleoyl ethanolamine (L-EA), PGE2-EA, PGF2α-EA, PGD2-EA, and FAAH inhibitor PF3845 (31.Ahn K. Johnson D.S. Mileni M. Beidler D. Long J.Z. McKinney M.K. Weerapana E. Sadagopan N. Liimatta M. Smith S.E. et al.Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain.Chem. Biol. 2009; 16: 411-420Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) were purchased from Cayman Chemical Co. (Ann Arbor, MI). N-arachidonoyl dipalmitoyl phosphatidylethanolamine was purchased from Enzo Life Sciences (Exeter, UK). Security guard cartridges C18 (5 μm, 4 × 2.0 mm), C18-E solid phase extraction cartridges (SPE; 500 mg sorbent), amber glass vials (1.5 ml), insert glass vials (0.15 ml), Teflon septa and lids were from Phenomenex (Macclesfield, UK). Male white New Zealand rabbit corneas were provided by Sera Laboratories International Ltd. (Haywards Heath, UK). Chloroform, methanol, ethanol, acetonitrile, hexane (all HPLC grade), and methyl formate (97% spectroscopy grade) were from Fisher Scientific (Loughborough, UK). HPLC-grade glacial acetic acid, Trizma Base, indomethacin, and protease inhibitor cocktail [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (104 mM), aprotinin (80 μM), bestatin (4 mM), E-64 (1.4 mM), leupeptin (2 mM), pepstatin A (1.5 mM)] were purchased from Sigma-Aldridge (Dorset, UK). EDTA was sourced from BDH (Poole, UK). Ultrapure water was tapped by a MilliQ Gradient system (Millipore, Volketswil, Switzerland). Rabbit corneas (∼75–100 mg each) were individually homogenized, using a glass Dounce tissue grinder (1 ml) (Fisher Scientific) with a tightly fitting pestle, in 1 ml ice-cold Tris-hydrochloride buffer (100 mM, pH 8 adjusted with 1 M HCl) containing EDTA (1 mM), FAAH inhibitor PF3845 (100 nM), and a protease inhibitor cocktail (1:100 dilution). During homogenization the tissue grinder and homogenate were kept on ice. When endogenous production of prostamides was monitored, corneal tissue homogenates (eight corneas) were pooled. Subsequent ex vivo investigations of prostamide formation were carried out by incubating corneal tissue homogenates (two corneas in 3 ml Tris-hydrochloride buffer) for 10 min at 37°C with exogenously added a) A-EA (10 μM, 50 μM), b) A-EA (50 μM) with and without indomethacin (3 μM), and c) A-EA (50 μM) with and without the FAAH inhibitor PF3845. Prostamides and fatty acyl ethanolamines (FA-EA) were extracted using chloroform-methanol (2:1, v/v) (32.Astarita G. Piomelli D. Lipidomic analysis of endocannabinoid metabolism in biological samples.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2755-2767Crossref PubMed Scopus (76) Google Scholar, 33.Kingsley P.J. Marnett L.J. Analysis of endocannabinoids, their congeners and COX-2 metabolites.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2746-2754Crossref PubMed Scopus (24) Google Scholar). Specifically, ice-cold chloroform-methanol (9 ml) was added to each corneal tissue homogenate followed by internal standard (A-EA-d8, 5 μl as 1 ng/μl in ethanol). The resulting suspensions were kept on ice for 30 min with occasional vortexing. Each sample was vortexed and centrifuged at 4,000 rpm for 8 min to separate the organic and aqueous phases. The organic layer (bottom) from each sample was then removed into a clean wide-neck vial. The pooled supernatant was evaporated under a fine stream of nitrogen, and the remaining residue was reconstituted in 50–100 μl ethanol and stored at −20°C, for no more than 1 week, awaiting LC/ESI-MS/MS analysis. Analysis and characterization of PG-EA produced by corneal tissue was performed on an electrospray (ESI) tandem quadrupole Xevo TQ-S mass spectrometer (Waters, Elstree, Hertsfordshire, UK) coupled to an Acquity Ultrahigh Pressure Liquid Chromatography (UPLC) system. The system was controlled by MassLynx v4.1 Software. TargetLynx was used to construct calibration lines and calculate the concentration of analytes of interest. Optimized ESI-MS/MS conditions were achieved through use of Intellistart within MassLynx software. Individual standards (100 pg/μl) were introduced into the spectrometer by direct infusion via the Xevo TQ-S integrated syringe pump (flow rate 10 μl/min) combined with UPLC solvent flow (rate 0.2 ml/min). All analytes were monitored on the positive ionization mode. Capillary voltage was set at 2,000 V, source temperature at 100°C, desolvation temperature at 400°C, and the cone voltage at 35 V. The collision energy was optimized for each compound to obtain optimum sensitivity using argon as collision gas and was set at 14 eV for PGE2-EA and PGD2-EA, and 16 eV for PGF2α-EA. Chromatographic analysis of PG-EA was performed on an Acquity UPLC® BEH Phenyl C18 column (1.7 μm, 2.1 × 50 mm) (Waters) maintained at 25°C supported with Acquity UPLC® BEH Phenyl VanGuard precolumn (1.7 μm, 2.1 × 5 mm) (Waters). Sample injections were performed with the Acquity sample manager (Waters); the sample chamber temperature was set at 8°C, and the injection volume was 3 μl. Analytes were separated using a method comprising two solvents: solvent A, water-glacial acetic acid 99.5:0.5 (v/v); solvent B, acetonitrile-glacial acetic acid 99.5:0.5 (v/v). Prostamides were eluted using an isocratic method of 25.5% solvent B from 0 to 3 min with a flow rate of 0.4 ml/min. At 3.1 min, solvent B was increased to 80% and the flow rate to 0.6 ml/min to wash the column for a further 5 min before returning to the original conditions. Multiple reaction monitoring (MRM) assays were set up using the following transitions: PGF2α-EA: m/z 380 > 362, 380 > 344, 380 > 283, 380 > 62; PGE2-EA and PGD2-EA: m/z 378 > 360, 378 > 342, 378 > 299, 378 > 62. LC/ESI-MS/MS analysis of FA-EA was performed on an electrospray (ESI) triple quadrupole Quattro Ultima mass spectrometer (Waters) coupled to a Waters Alliance 2695 HPLC pump. Instrument control and data acquisition were performed using the MassLynx™ V4.0 software. For optimization of ESI/MS and ESI/MS/MS conditions, individual standards (10 ng/μl) were introduced into the spectrometer by direct infusion through a syringe pump (flow rate 10 μl/min) through the HPLC solvent flow (rate 0.2 ml/min). All analytes were monitored on the positive ionization mode. Capillary voltage was set at 3,500 V, source temperature at 100°C, desolvation temperature at 400°C, cone voltage at 35 V, while the collision energy was optimized for each compound using argon as collision gas and was set to the following: P-EA, 13 eV; AL-EA, 14 eV; L-EA, 15 eV; O-EA, 16 eV; ST-EA, 15 eV; eicosapentaenoyl ethanolamine (EP-EA), 15 eV; A-EA, 15 eV; DH-EA, 15 eV; A-EA-d8, 16 eV. Chromatographic analysis of FA-EA species was performed on a Luna C18(2) column (5 μm, 150 × 2.0 mm inner diameter) (Phenomenex, Macclesfield, UK) maintained at ambient temperature. Sample injections were performed with a Waters 2690 autosampler; the sample chamber temperature was set at 8°C. The injection volume was 10 μl, and the flow rate 0.2 ml/min. Analytes were separated using an acetonitrile-based gradient system comprising two solvents; solvent A, acetonitrile-water-glacial acetic acid 2:97.5:0.5 (v/v/v); solvent B, acetonitrile-water-glacial acetic acid 97.5:2:0.5 (v/v/v). The following gradient was used: 0.0–10.00 min, 30% solvent B increasing linearly to 70% solvent B; 10.00–40.00 min 70% solvent B decreasing linearly to 60% solvent B; 40.00–41.00 min 60% solvent B increasing linearly to 90% solvent B; 41.00–55.00 min 90% solvent B; 55.00–56.00 90% solvent B decreasing linearly to 30% solvent B; 56.00–69.00 min 30% solvent B. A shallow gradient was put in place between 10 and 40 min (70% to 60% solvent B) to improve the resolution of FA-EA. MRM assays were set up using the following transitions: P-EA, m/z 300 > 62; AL-EA, m/z 322 > 62; L-EA, m/z 324 > 62; O-EA, m/z 326 > 62; ST-EA, m/z 328 > 62; EP-EA, m/z 346 > 62; A-EA, m/z 348 > 62; DH-EA, m/z 372 > 62; A-EA-d8, m/z 356 > 63. Results are expressed as picograms metabolite per milligrams wet tissue, using calibration lines constructed with commercially available standards. Prostanoids were extracted and analyzed as previously described (34.Masoodi M. Nicolaou A. Lipidomic analysis of twenty-seven prostanoids and isoprostanes by liquid chromatography/electrospray tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2006; 20: 3023-3029Crossref PubMed Scopus (129) Google Scholar, 35.Masoodi M. Mir A.A. Petasis N.A. Serhan C.N. Nicolaou A. Simultaneous lipidomic analysis of three families of bioactive lipid mediators leukotrienes, resolvins, protectins and related hydroxy-fatty acids by liquid chromatography/electrospray ionisation tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2008; 22: 75-83Crossref PubMed Scopus (118) Google Scholar). Briefly, individual corneas were homogenized in 500 μl of ice-cold 15% methanol (v/v) using PGB2-d4 (40 μl of a 1 ng/μl ethanol solution) as internal standard. The homogenates were acidified to pH 3.0 with 1 M hydrochloric acid, semipurified using SPE and eluted with methyl formate. The solvent was then evaporated under nitrogen, and the lipid residue reconstituted in 100 μl ethanol and stored at −20°C. LC/ESI-MS/MS analysis of prostanoids was based on MRM assays using the following transitions: 15-deoxy Δ12,14 PGJ2, m/z 315 > 271; PGJ2, m/z 333 > 271; Δ12 PGJ2, m/z 333 > 271; PGE3, m/z 349 > 269; PGD3, m/z 349 > 269; PGE2, m/z 351 > 271; PGD2, m/z 351 > 271; 13,14-dihydro 15-keto PGE2, m/z 351 > 333; 13,14-dihydro 15-keto PGF2α, m/z 353 > 113; PGF2α, m/z 353 > 193; PGE1, m/z 353 > 317; PGD1, m/z 353 > 317; 6-keto PGF1α, m/z 369 > 163; TXB2, m/z 369 > 169; PGB2-d4, m/z 337 > 174. Results are expressed as picograms metabolite per milligrams wet tissue, using calibration lines constructed with commercially available prostanoid standards. Two corneas were homogenized individually, using a glass Dounce tissue grinder (1 ml) in ice-cold chloroform-methanol (2:1, v/v) (0.5 ml aliquots to a volume of 3 ml per cornea). The sample was then kept on ice for 90 min with occasional vortexing. Water (0.5 ml) was added to each sample and the vials vortexed before being centrifuged at 5,000 rpm for 8 min to separate the organic and aqueous phases. The organic layer (bottom) from each sample was then removed and pooled into a clean wide-neck vial, and the solvent evaporated under a fine stream of nitrogen. The lipid residue was reconstituted in 100 μl chloroform-methanol (1:4, v/v) and stored at −20°C awaiting ESI-MS/MS analysis (36.Astarita G. Ahmed F. Piomelli D. Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain.J. Lipid Res. 2008; 49: 48-57Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In order to optimize the ESI-MS and ESI-MS/MS conditions for NAPE analysis, commercially available N-arachidonoyl dipalmitoyl phosphatidyl ethanolamine was used. Using direct infusion (flow rate 10 μl/min), the optimum collision energy was found to be 40 eV, using argon as collision gas. The analyte was monitored on negative ionization mode and was found to fragment in a similar way to previously published data (36.Astarita G. Ahmed F. Piomelli D. Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain.J. Lipid Res. 2008; 49: 48-57Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The corneal extract was diluted 1:10 (v/v) with chloroform-methanol-water-acetic acid (2: 6.95:1:0.05, v/v/v/v) and analyzed through direct infusion. ESI-MS spectra were recorded over the range m/z 800–1,250. Ions with m/z [M-H]− corresponding to NArPE and NAPE were further analyzed by ESI-MS/MS to confirm their identity and obtain information on the sn-1 and sn-2 acyl chains. The ESI-MS, MS/MS spectra and fragmentation patterns of prostamides PGF2α-EA, PGE2-EA, and PGD2-EA were studied using commercially available standards (Fig. 2). All prostamide standards were found to form stable sodiated ions [M+Na]+m/z 420 for PGF2α-EA, and m/z 418 for both PGE2-EA and PGD2-EA, possibly reflecting their storage in glass vials. Notably, the relative abundance of [M+H]+ species (m/z 398 for PGF2α-EA, and m/z 396 for both PGE2-EA and PGD2-EA) was found to be very low (Fig. 2A, D, G, respectively), and, for all prostamides examined here, the predominant ions corresponded to [M+H-H2O]+ (m/z 380 for PGF2α-EA and m/z 378 for PGE2-EA and PGD2-EA). Further fragmentation of [M+H-H2O]+ ions resulted in the product ions [M+H-2H2O]+m/z 362, [M+H-3H2O]+m/z 344, and [M+H-3H2O-NH2CH2CH2OH] +m/z 283 for PGF2α-EA (Fig. 2C), and [M+H-2H2O]+m/z 360, [M+H-3H2O]+m/z 342, and [M+H-2H2O-NH2CH2CH2OH]+m/z 299 for PGE2-EA and PGD2-EA (Fig. 2F, I). The protonated 2-amino ethanol ion [NH3CH2CH2OH]+m/z 62, characteristic of ethanolamine metabolites, was also detected following fragmentation of [M+H]+ (PGE2-EA and PGD2-EA; Fig. 2E, H) and [M+H-H2O]+ (PGF2α-EA, PGE2-EA, and PGD2-EA; Fig. 2C, F, I). All these findings are in agreement with previously published data on the prostamide formation and identification in vitro and FAAH knockout mice (10.Yu 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 (349) Google Scholar, 12.Koda N. Tsutsui Y. Niwa H. Ito S. Woodward D.F. Watanabe K. Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme: new enzyme assay method using LC/ESI/MS.Arch. Biochem. Biophys. 2004; 424: 128-136Crossref PubMed Scopus (52) Google Scholar, 13.Yang W. Ni J. Woodward D.F. Tang-Liu D.D. Ling K.H. Enzymatic formation of prostamide F2alpha from anandamide involves a newly identified intermediate metabolite, prostamide H2.J. Lipid Res. 2005; 46: 2745-2751Abstract Full Text Full Text PDF PubMed
Referência(s)