Enzymatic formation of prostamide F2α from anandamide involves a newly identified intermediate metabolite, prostamide H2

Prostaglandin F2α 1-ethanolamide (prostamide F2α) is a potent ocular hypotensive agent in animals and represents a new class of fatty acid amide compounds. Accumulated evidence indicated that anandamide, an endogenous bioactive ligand for cannabinoid receptors, may serve as a common substrate to produce all prostamides, including prostamide F2α. After incubation of anandamide with cyclooxygenase 2 (COX-2), the reaction mixture was profiled by HPLC and an intermediate metabolite was discovered and characterized as a cyclic endoperoxide ethanolamide using HPLC-tandem mass spectrometry. Formation of prostamide F2α was also demonstrated when the intermediate metabolite was isolated and incubated with prostaglandin F synthase (PGF synthase).These results suggest that the biosynthesis of prostamide F2α proceeds in two consecutive steps: oxidation of anandamide to form an endoperoxide intermediate by COX-2, and reduction of the endoperoxide intermediate to form prostamide F2α by PGF synthase. This endoperoxide ethanolamide intermediate has been proposed as prostamide H2. Prostaglandin F2α 1-ethanolamide (prostamide F2α) is a potent ocular hypotensive agent in animals and represents a new class of fatty acid amide compounds. Accumulated evidence indicated that anandamide, an endogenous bioactive ligand for cannabinoid receptors, may serve as a common substrate to produce all prostamides, including prostamide F2α. After incubation of anandamide with cyclooxygenase 2 (COX-2), the reaction mixture was profiled by HPLC and an intermediate metabolite was discovered and characterized as a cyclic endoperoxide ethanolamide using HPLC-tandem mass spectrometry. Formation of prostamide F2α was also demonstrated when the intermediate metabolite was isolated and incubated with prostaglandin F synthase (PGF synthase). These results suggest that the biosynthesis of prostamide F2α proceeds in two consecutive steps: oxidation of anandamide to form an endoperoxide intermediate by COX-2, and reduction of the endoperoxide intermediate to form prostamide F2α by PGF synthase. This endoperoxide ethanolamide intermediate has been proposed as prostamide H2. Prostaglandins are unsaturated fatty acid metabolites with remarkably potent and diversified biological functions. They are generated from the oxidation of arachidonic acid by cyclooxygenases (1Bergstrom S. Carlson L.A. Weeks J.R. Prostaglandins: a family of biologically active lipids.Pharmacol. Rev. 1968; 20: 1-48Google Scholar). Prostaglandin H2, the hydroxy-endoperoxide, serves as a common intermediate product, through which prostaglandins are produced in specific tissues or cells (2Narumiya S. Sugimoto Y. Ushikubi F. Prostanoid receptors: structures, properties, and functions.Physiol. Rev. 1999; 79: 1193-1226Google Scholar). Because of the structural similarity, arachidonyl ethanolamide (anandamide), an endogenous agonist of cannabinoid receptors (3Devane W.A. Hanûs L. Breuer A. Pertwee R.G. Stevenson L.A. Griffin G. Gibson D. Mandelbaum A. Etinger A. Mechoulam R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor.Science. 1992; 258: 1946-1949Google Scholar), was indicated as a selective substrate for cyclooxygenase 2 (COX-2) (4Yu M. Ives D. Ramesha C.S. Synthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2.J. Biol. Chem. 1997; 272: 21181-21186Google Scholar). Recent evidence had demonstrated that anandamide was effectively oxygenated by COX-2 to form prostamides, a new class of prostaglandin analogs (5Kozak 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: 33744-33749Google Scholar, 6Kozak 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: 36993-36998Google Scholar, 7Weber A. Ni J. Ling K-H.J. Acheampong A. Tang-Liu D. D-S. Burk R. Cravatt B.F. Woodward D. Formation of prostamides from anandamide in FAAH knockout mice analyzed by HPLC with tandem mass spectrometry.J. Lipid Res. 2004; 45: 757-763Google Scholar), and the hydroxyl moiety of anandamide, as a critical determinant in the ability of COX-2 to effect robust endocannabinoid oxygenation (8Kozak K.R. Prusakiewicz P.J. Rowlinson S.W. Prudhomme D.R. Marnett L.J. Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide.Biochemistry. 2003; 42: 9041-9049Google Scholar). Although the physiological functions of the prostamides are not well defined, prostaglandin F2α 1-ethanolamide (prostamide F2α) was reported to be potent in the contraction of the cat iris sphincter (9Matias I. Chen L. Petrocellis L. De 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: 745-757Google Scholar) and to behave as an effective ocular hypotensive agent in monkeys (10Koda 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: 128-136Google Scholar). In addition, the antiglaucoma drug bimatoprost (Lumigan™) is similar to prostamide F2α in structure and behaves as a prostamide analog (9Matias I. Chen L. Petrocellis L. De 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: 745-757Google Scholar, 10Koda 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: 128-136Google Scholar). To identify the potential biosynthetic pathway of prostamide F2α, we conducted a series of metabolic studies of anandamide using recombinant human COX-2 and prostaglandin F synthase (PGF synthase) and analyzed the enzymatic metabolites using HPLC-radiometric detection (HPLC-RAD) and HPLC tandem mass spectrometry (HPLC-MS/MS). The results indicated that a prostamide congener of prostaglandin H2 serves as an intermediate metabolite of anandamide in prostamide F2α synthesis and demonstrated two consecutive enzymatic reactions in prostamide F2α formation. Prostamide F2α [N-(2-hydroxyethyl)-9α,11α,15S-trihydroxy-prosta-5Z,13E-dien-1-amide] was synthesized at Allergan, Inc. Anandamide and prostamides D2 and E2 were purchased from Cayman Chemical Co. (Ann Arbor, MI). [3H]anandamide (208 Ci/mmol, 99.7% purity), [3H]prostamide D2 (162 Ci/mmol, 98.1% purity), [3H]prostamide E2 (169 Ci/mmol, 97% purity), [3H]prostamide F2α (194 Ci/mmol, 99.7% purity), and [3H]prostamide 11β-F2α (20 Ci/mmol, 99.7% purity) were custom synthesized from Amersham Pharmacia Biotech (Piscataway, NJ). All chemicals and reagents were of reagent grade or better. Human recombinant COX-2 (rHCOX-2) was a gift from Dr. W. L. Smith at Michigan State University. The enzyme preparation contains 18,180 U/mg protein (1 unit of enzyme consumes 1 nmol of oxygen per minute at 37°C in a 0.1 M Tris-HCl buffer, pH 8.0, containing 100 μM arachidonate, 5 mM EDTA, 2 mM phenol, and 1 μM hematin). Human recombinant PGF synthase (rHPGF synthase) was a gift from Dr. K. Watanabe at The University of East Asia. The expressed enzyme was partially purified to yield a protein concentration of 15 mg/ml. The same expression vector carrying no PGF synthase DNA insert was prepared as a negative control. Twenty microliters of 0.5 mM anandamide solution (containing 0.43 μCi of [3H]anandamide) was added to 960 μl of rHCOX-2 reaction buffer (100 mM Tris-HCl, pH 8.0, containing 2 mM phenol, 5 μM hematin, and 1 mM EDTA) to result in a final anandamide concentration of 10 μM. One hundred units of rHCOX-2 in 20 μl volume was added to start the enzymatic reaction. After incubation at 37°C for 2 min, the reaction was stopped by adding 1 ml of dry ice-cooled solution (ether-methanol-1 M acetic acid at 30:4:1, v/v/v). The samples were extracted with 3 ml of ethyl acetate, and the organic phase was collected and dried at room temperature under nitrogen. The samples were reconstituted into 150 μl of HPLC mobile phase (acetonitrile-10 mM ammonium formate, pH 2.8, at 28:72, v/v) for HPLC-RAD profiling. A Hewlett-Packard (Palo Alto, CA) 1100 HPLC system coupled with a Packard (Meriden, CT) radiometric detector was used to profile the samples, and a 5 μm, 4.6 × 250 mm Inertsil ODS-2 column (GL Sciences, Inc.) was used in the analysis. Mobile phase A was 10 mM ammonium formate, pH 2.8, in water, and mobile phase B was acetonitrile. The injection volume was 50 μl, and the flow rate was set at 1 ml/min with the gradients listed in Table 1.TABLE 1HPLC gradient used for analysisTimeMobile Phase AMobile Phase BGradientmin%0 → 22.5 72 28Isocratic22.5 → 32 72 → 20 28 → 80Linear32 → 57 20 80Isocratic57 → 60 20 → 72 80 → 28Linear60 → 65 72 28IsocraticMobile phase A = 10 mM ammonium formate, pH 2.8, in water; mobile phase B = acetonitrile. Open table in a new tab Mobile phase A = 10 mM ammonium formate, pH 2.8, in water; mobile phase B = acetonitrile. The reconstituted sample was also used for structural elucidation by LC-MS/MS analysis. Reversed-phase HPLC-MS/MS and radiometric detection were used to characterize the reaction products. The HPLC conditions were the same as for profiling. The effluent from the HPLC column was split: one portion with a flow rate of ∼0.2 ml/min was introduced into a mass spectrometer, whereas the other portion with a flow rate of 0.3 ml/min was directed into the flow cell of a β-RAM (IN/US System, Tampa, FL). The β-RAM response was recorded in real time by the mass spectrometer computer, which provided simultaneous recording of radioactivity and mass spectral data. The delay in response between the two detectors was ∼0.4 min, with the mass spectrometric response being recorded first. The β-RAM was operated in homogeneous liquid scintillation counting mode with the addition of 3.2 ml/min Flo-scint III scintillation cocktail to the HPLC effluent. The radioactivity detector residence time was ∼16 s. Analysis of the metabolic products from the anandamide-rHCOX-2 reaction was carried out with a PE-Sciex API 3000 tandem mass spectrometer (Sciex, Toronto, Canada). The experiment was performed in positive ion turbo ion spray mode, and the LC effluent was sprayed into the mass spectrometer with a voltage of 5,000 V applied to the spray needle. The declustering voltage was 20 V, and the turbo ion spray temperature was set at 350°C. In the tandem mass spectrometric experiments, collision-activated dissociation in Q2 was induced by nitrogen as the collision gas at a collision energy of −30 eV. After incubation of anadamide with rHCOX-2 for 2 min, the enzymatic product with a retention time at 31.6 min from HPLC-RAD profiling was fractionally collected, dried under nitrogen, and stored at −80°C until use. The sample was used both for structural elucidation and for the PGF synthase reaction. In the PGF synthase reaction, the sample was reconstituted into 0.6 ml of PGF synthase reaction buffer (100 mM Tris-HCl, pH 8.0, 0.5 mM NADP, 5 mM glucose-6-phosphate, and 2 units/ml glucose-6-phosphate dehydrogenase). The concentration of the intermediate metabolite was determined by a radiolabeled standard calibration curve (range of 100–20,000 dpm/ml). Aliquots of 0, 10, and 100 μl of PGF synthase (1.5 μg protein/μl) were added to the reconstituted solution containing anandamide intermediate at a concentration of ∼4.5 μM to initiate the PGF synthase reaction. The reaction was carried out at 37°C for 2 and 10 min, separately, and stopped by adding 1 ml of dry ice-cooled stop solution (ether-methanol-1M acetic acid at 30:4:1, v/v/v). The sample was extracted with 3 ml of ethyl acetate, and the organic phase was collected and dried at room temperature under nitrogen. The residue was reconstituted using HPLC mobile phase (acetonitrile-10 mM ammonium formate, pH 2.8, at 28:72, v/v) and used for both HPLC-RAD profiling and LC-MS/MS analysis. After 2 min of incubation of anandamide with rHCOX-2, at least three major products with retention times of 31.6, 32.9, and 35.3 min were detected (Fig. 1A). Unlike the other two products, the peak with retention time at 31.6 min rapidly decreased with incubation time and completely disappeared after 20 min of incubation, suggesting that this product was a short-lived intermediate in anandamide metabolism. Reference standards of anandamide and its potential metabolites, including prostamide D2, prostamide E2, prostamide F2α, and 11β-prostamide F2α, were eluted at 40.9, 20.9, 16.9, 15.1, and 12.1 min, respectively (Fig. 1B). Kinetic study of this intermediate formation was conducted in the presence of 100 units of rHCOX-2 and increasing concentrations of anandamide from 0.5 to 400 μM after a 2 min incubation at room temperature. The observed Km and Vmax values for the conversion of anandamide to this intermediate were ∼15 μM and 1.3 μmol/min/mg protein, respectively. The product ion spectrum of anandamide produced a major fragment ion at m/z 62, which was interpreted as the protonated 2-amino ethanol moiety (Fig. 2). Because the protonated 2-amino ethanol ion was a characteristic fragment ion in the product ion spectra of ethanolamide-containing compounds such as anandamide and prostamides, a precursor ion scan of m/z 62 was conducted to monitor compounds with a prostamide structure. Prostamides E2 and D2 and the intermediate, in the precursor ion spectrum of m/z 62, all had the same molecular weight of 395 but different retention times (Fig. 1B). LC-MS/MS product ion spectra of m/z 396.4 for prostamides D2 and E2 and the intermediate metabolite showed different fragmentation patterns (Fig. 3). The product ion spectra of prostamides D2 and E2 were similar and displayed characteristic fragment ions at m/z 378, 360, and 342 via three consecutive water losses, at m/z 281 after further loss of neutral 2-amino ethanol from m/z 342, and at m/z 62 as protonated 2-amino ethanol ion (Fig. 3A, B). The product ion spectrum of the unique intermediate metabolite also showed a fragment ion at m/z 62, the protonated 2-amino ethanol ion. However, unlike those of prostamides D2 and E2, the product ion spectrum of this intermediate metabolite showed fragment ions at m/z 344 and 283 (Fig. 3C). The displayed characteristic fragment ion at m/z 344 could be interpreted as the loss of one water molecule and one neutral H2O2 moiety from the parent ion, and the fragment ion at m/z 283 could be interpreted as a further loss of neutral 2-amino ethanol from m/z 344.Fig. 3LC-MS/MS product ion spectrum of prostamide E2 (A), prostamide D2 (B), and the intermediate metabolite with retention time of 31.55 min (C).View Large Image Figure ViewerDownload (PPT) To investigate whether the intermediate metabolite is a precursor of prostamide F2α, the intermediate with retention time at 31.6 min was isolated via fraction collection. The sample was reconstituted in PGF synthase reaction buffer, and the purity of the intermediate metabolite was estimated to be >90% by HPLC-RAD. After incubation of 15 μg of rHPGF synthase with purified intermediate metabolite of anandamide at a concentration of 4.5 μM at 37°C for 2 min, 69% of the isolated intermediate was converted to prostamide F2α (Fig. 4C), whereas no conversion occurred after incubation of same amount of enzyme negative control with purified intermediate metabolite at the same concentration at 37°C for 2 min (Fig. 4B). The conversion of the intermediate to prostamide F2α was nearly completed after 10 min of incubation (Fig. 4D). The newly converted prostamide F2α (Fig. 5A)was further confirmed by comparing its retention time and fragmentation pattern with those of prostamide F2α standard using LC-MS/MS (Fig. 5B).Fig. 5LC-MS/MS product ion spectrum of the proposed prostamide F2α. Both the biosynthesized product from incubation of 4.5 μM of the intermediate metabolite and 15 μg of PGF synthase (A) and the standard prostamide F2α (B) were analyzed using the LC-MS/MS product ion spectrum.View Large Image Figure ViewerDownload (PPT) Prostamide F2α is a potent ocular hypotensive agent in animals and represents a potentially important metabolite of anandamide (11Chen, J., A. Kharlamb, R. M. Burk, A. H. Krauss, and D. F. Woodward. 2002. Studies on the intraocular pressure effects of prostamides in monkeys. ARVO 43 (E-Abstract): ABS # 4111. Accessed September 23, 2005, at http://www.arvo.org.Google Scholar). This study indicated that the biosynthesis of prostamide F2α from anandamide, one of the natural ligands for the cannabinoid receptors (3Devane W.A. Hanûs L. Breuer A. Pertwee R.G. Stevenson L.A. Griffin G. Gibson D. Mandelbaum A. Etinger A. Mechoulam R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor.Science. 1992; 258: 1946-1949Google Scholar), might include two consecutive steps, an oxidization of anandamide to form an endoperoxide intermediate by enzyme COX-2, and a reduction of the endoperoxide intermediate to form prostamide F2α by PGF synthase (Fig. 6, proposed metabolic scheme of prostamide F2α). The observed Km value of COX-2 oxidation of anandamide was 15 μM, and the observed Vmax value for COX-2 oxidation of anandamide was ∼1.3 μmol/mg protein/min. Our result for COX-2 oxygenation of anandamide is consistent with recent structure-activity studies by Kozak et al. (8Kozak K.R. Prusakiewicz P.J. Rowlinson S.W. Prudhomme D.R. Marnett L.J. Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide.Biochemistry. 2003; 42: 9041-9049Google Scholar), who had examined the structural requirements for COX-mediated anandamide oxygenation using a number of substrate analogs and site-directed mutants of COX-2. They concluded that the hydroxyl moiety of anandamide is a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation, whereas anandamide binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. LC-MS/MS analysis indicated that the intermediate metabolite of anandamide might be a new member of the prostaglandin 1-ethanolamide class of compounds. Although this intermediate metabolite shares the same molecular weight of 395 with prostamides D2 and E2, the product ion spectra of prostamides D2 and E2 indicated that there were three consecutive losses of water molecules, resulting in fragment ions at m/z 378, 360, and 342 (Fig. 3A, B), whereas the intermediate metabolite showed no direct loss of water molecules (Fig. 3C). The fragment ion at m/z 344 could be interpreted as a loss of one water and one H2O2 with a combined mass of 52. However, the intermediate metabolite, like prostamide D2 or E2, also had the characteristic m/z 62 fragment ion (Fig. 3), suggesting that it contained an ethanolamide group. In addition, a fragment ion at m/z 283 could be interpreted as a breakdown of the ion at m/z 344 by losing a neutral species of 2-amino ethanol (Fig. 3C). Based on the similarity of its molecular weight to that of prostamides D2 and E2, the unique mass spectral fragmentation pattern, including the characteristic ions at m/z 344, 283, and 62, as well as its instability during incubation, we proposed this unique intermediate metabolite as prostamide H2, with the chemical name 6-(6-hept-1-enyl-2,3,-dioxa-bicyclo[2.2.1]hept-5-yl)-hex-5-enoic acid (2-hydroxy-ethyl)-amide. According to the prostaglandin biosynthesis pathway, PGF synthase, an aldo-keto reductase, reduces prostaglandin H2 to PGF2α (12Watanabe K. Yoshida R. Shimizu T. Hayaishi O. Enzymatic formation of prostaglandin F2 alpha from prostaglandin H2 and D2. Purification and properties of prostaglandin F synthetase from bovine lung.J. Biol. Chem. 1985; 260: 7035-7041Google Scholar). Because of the structural similarity between prostamide F2α and prostaglandin F2α, the isolated intermediate metabolite was incubated with rHPGF synthase and the reaction mixture was profiled by HPLC-RAD. As demonstrated in Fig. 4, ∼70% of the intermediate metabolite was converted to prostamide F2α after 2 min of incubation at 37°C in the presence of 15 μg of rHPGF synthase (Fig. 4C), whereas no conversion occurred in the presence of the rHPGF synthase negative control (Fig. 4B). The conversion was time and enzyme concentration dependent. In the presence of the same amount of rHPGF for 10 min, >90% of the intermediate metabolite was converted to PGF2α (Fig. 4D). When a higher amount of rHPGF synthase (150 μg) was used, ∼90% of the intermediate metabolite was converted to PGF2α after 2 min of incubation and was completely converted to prostamide F2α after 10 min (data not shown). Finally, the converted prostamide F2α was confirmed using prostamide F2α standard by LC-MS/MS (Fig. 5). In conclusion, for the first time, we have demonstrated the biosynthesis of prostamide F2α from anandamide and proposed prostamide H2 as a possible intermediate of anandamide metabolism, similar to the role of prostaglandin H2 in the metabolism of arachidonic acid. The authors are grateful to Dr. W. L. Smith and Dr. K. Watanabe for the generous gift of human COX-2 and human PGF synthase preparations.

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