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Inhibition of lipoxygenases and cyclooxygenases by linoleyl hydroxamic acid: comparative in vitro studies

2008; Elsevier BV; Volume: 49; Issue: 6 Linguagem: Inglês

10.1194/jlr.m700602-jlr200

1539-7262

Igor A. Butovich, Svetlana M. Lukyanova,

Peroxisome Proliferator-Activated Receptors

In this first comparative in vitro study, linoleyl hydroxamic acid (LHA), a simple and stable derivative of linoleic acid, was tested as an inhibitor of several enzymes involved in arachidonic acid metabolism in mammals. The tested enzymes were human recombinant 5-lipoxygenase (h5-LO), porcine leukocyte 12-LO, rabbit reticulocyte 15-LO, ovine cyclooxygenases 1/2 (COX1/COX2), and human microsomal prostaglandin E synthase-1 (mPGES-1). Potato tuber and soybean lipoxygenases (ptLOX and sLOX, respectively) were studied for comparative purposes. LHA inhibited most of the tested enzymes with the exception of mPGES-1. The LHA inhibitory activity increased as follows: mPGES-1 (no inhibition)<<COX1 = COX2<h5-LO = sLOX = ptLOX<12-LO<<15-LO. The IC50 values for COX1/COX2, h5-LO, 12-LO, and 15-LO were 60, 7, 0.6, and 0.02 μM, respectively. sLOX was the only tested enzyme that was capable of aerobic oxygenation of LHA, producing 13-hydroperoxy-LHA. The enzyme rapidly inactivated during the reaction. Therefore, LHA could be used as an effective LO/LOX inhibitor without affecting COX1/COX2 and mPGES-1. Possible implications of this observation include treating diseases and pathological states that are caused by (or lead to) hyperproduction of LO-derived metabolites, e.g., inflammation, cardiovascular disorders, cancer, asthma, allergies, psoriasis, and stroke. In this first comparative in vitro study, linoleyl hydroxamic acid (LHA), a simple and stable derivative of linoleic acid, was tested as an inhibitor of several enzymes involved in arachidonic acid metabolism in mammals. The tested enzymes were human recombinant 5-lipoxygenase (h5-LO), porcine leukocyte 12-LO, rabbit reticulocyte 15-LO, ovine cyclooxygenases 1/2 (COX1/COX2), and human microsomal prostaglandin E synthase-1 (mPGES-1). Potato tuber and soybean lipoxygenases (ptLOX and sLOX, respectively) were studied for comparative purposes. LHA inhibited most of the tested enzymes with the exception of mPGES-1. The LHA inhibitory activity increased as follows: mPGES-1 (no inhibition)<<COX1 = COX2<h5-LO = sLOX = ptLOX<12-LO<<15-LO. The IC50 values for COX1/COX2, h5-LO, 12-LO, and 15-LO were 60, 7, 0.6, and 0.02 μM, respectively. sLOX was the only tested enzyme that was capable of aerobic oxygenation of LHA, producing 13-hydroperoxy-LHA. The enzyme rapidly inactivated during the reaction. Therefore, LHA could be used as an effective LO/LOX inhibitor without affecting COX1/COX2 and mPGES-1. Possible implications of this observation include treating diseases and pathological states that are caused by (or lead to) hyperproduction of LO-derived metabolites, e.g., inflammation, cardiovascular disorders, cancer, asthma, allergies, psoriasis, and stroke. arachidonic acid (eicosatetra-5Z,8Z,11Z,14Z-enoic acid) atmospheric pressure chemical ionization arbitrary unit (unitless) ovine cyclooxygenases 1 and 2 one-dimensional diode array detector hydroxyeicosatetraenoic acid electrospray ionization mass spectrometry hydroperoxyeicosatetraenoic acid hydroperoxyoctadecadienoic acid human recombinant 5-lipoxygenase linoleic acid (octadeca-9Z,12Z-dienoic acid) linoleyl hydroxamic acid lipoxygenase (mammalian) lipoxidase (plant) human microsomal prostaglandin E synthase-1 normal-phase HPLC nonsteroidal anti-inflammatory drug potato tuber lipoxygenase (lipoxidase) reverse-phase HPLC soybean lipoxygenase (lipoxidase) Lipoxygenase (LO) is the common name of a group of related enzymes that catalyze aerobic stereospecific oxidation of PUFAs with Z,Z-pentadienyl fragment in them (1.Peters-Golden M. Brock T.G. 5-Lipoxygenase and FLAP.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 69: 99-109Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The major primary product of such a reaction is a hydroperoxide of the corresponding substrate. Two typical natural substrates of LOs are linoleic acid (octadeca-9Z,12Z-dienoic acid, LA) and arachidonic acid (eicosatetra-5E,8E,11E,14E-enoic acid, AA). AA is considered a quintessential substrate for LOs of mammalian origin, whereas LA is a standard substrate for plant LOs, also known as lipoxidases (LOXs). There are several subgroups of LO and LOX that are classified in part on the basis of their substrate specificity, and in part on the basis of their origin.Depending on the point of insertion of molecular oxygen into AA, mammalian LOs are commonly classified into five major groups: 5-LO (EC 1.13.11.34), 8-LO (EC 1.13.11.40), 11-LO, (EC 1.13.11.45), 12-LO (EC 1.13.11.31), and 15-LO (EC 1.13.11.33). The primary products of corresponding reactions are hydroperoxides of AA (hydroperoxyeicosatetraenoic acids, or HPETEs). Thus, 5-LO produces mainly 5-HPETE, 15-LO, 15-HPETE, etc. No definite information on mammalian 9-LO is available at this time, although 9-hydroxyeicosatetraenoic acid (9-HETE), a product of 9-HPETE reduction, has been found in several tissues (2.Camp R.D. Mallet A.I. Woollard P.M. Brain S.D. Black A.K. Greaves M.W. The identification of hydroxy fatty acids in psoriatic skin.Prostaglandins. 1983; 26: 431-447Crossref PubMed Scopus (135) Google Scholar, 3.el Attar T.M. Lin H.S. Vanderhoek J.Y. Biosynthesis of prostaglandins and hydroxy fatty acids in primary squamous carcinomas of head and neck in humans.Cancer Lett. 1985; 27: 255-259Crossref PubMed Scopus (18) Google Scholar, 4.Kiss L. Schütte H. Mayer K. Grimm H. Padberg W. Seeger W. Grimminger F. Synthesis of arachidonic acid-derived lipoxygenase and cytochrome P450 products in the intact human lung vasculature.Am. J. Respir. Crit. Care Med. 2000; 161: 1917-1923Crossref PubMed Scopus (40) Google Scholar, 5.Yue H. Jansen S.A. Strauss K.I. Borenstein M.R. Barbe M.F. Rossi L.J. Murphy E. A liquid chromatography/mass spectrometric method for simultaneous analysis of arachidonic acid and its endogenous eicosanoid metabolites prostaglandins, dihydroxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and epoxyeicosatrienoic acids in rat brain tissue.J. Pharm. Biomed. Anal. 2007; 43: 1122-1134Crossref PubMed Scopus (82) Google Scholar).LOXs (EC 1.13.11.12) also differ in terms of their positional specificity toward their standard substrate, LA, producing either 9-, or 13-hydroperoxide of LA [9- or 13-hydroperoxyoctadecadienoic acid (HPODE)]. Complicating the classification is the fact that mammalian 15-LO oxidizes LA (6.Salzmann-Reinhardt U. Kühn H. Wiesner R. Rapoport S. Metabolism of polyunsaturated fatty acids by rabbit reticulocytes.Eur. J. Biochem. 1985; 153: 189-194Crossref PubMed Scopus (12) Google Scholar), and plant LOX can effectively oxidize AA (7.Wiseman J.S. Skoog M.T. Clapp C.H. Activity of soybean lipoxygenase in the absence of lipid hydroperoxide.Biochemistry. 1988; 27: 8810-8813Crossref PubMed Scopus (13) Google Scholar). For example, under standard conditions, soybean LOX (sLOX) utilizes LA to produce 13(S)-HPODE (8.Hamberg M. Steric analysis of hydroperoxides formed by lipoxygenase oxygenation of linoleic acid.Anal. Biochem. 1971; 43: 515-526Crossref PubMed Scopus (219) Google Scholar), while making almost exclusively 15(S)-HPETE from AA (9.Hamberg M. Samuelsson B. On the specificity of the oxygenation of unsaturated fatty acids catalyzed by soybean lipoxidase.J. Biol. Chem. 1967; 242: 5329-5335Abstract Full Text PDF PubMed Google Scholar). Potato tuber LOX (ptLOX), on the other hand, catalyzes the formation of various derivatives of LA (10.Galliard T. Phillips D.R. Lipoxygenase from potato tubers. Partial purification and properties of an enzyme that specifically oxygenates the 9-position of linoleic acid.Biochem. J. 1971; 124: 431-438Crossref PubMed Scopus (201) Google Scholar), docosahexaenoic acid (11.Butovich I.A. Hamberg M. Rådmark O. Novel oxylipins formed from docosahexaenoic acid by potato lipoxygenase—10(S)-hydroxydocosahexa-enoic acid and 10,20-dihydroxydocosahexaenoic acid.Lipids. 2005; 40: 249-257Crossref PubMed Scopus (17) Google Scholar, 12.Butovich I.A. On the structure and synthesis of neuroprotectin D1, a novel anti-inflammatory compound of the docosahexaenoic acid family.J. Lipid Res. 2005; 46: 2311-2314Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 13.Butovich I.A. Lukyanova S.M. Bachmann C. Dihydroxydocosahexaenoic acids of the neuroprotectin D family: synthesis, structure, and inhibition of human 5-lipoxygenase.J. Lipid Res. 2006; 47: 2462-2474Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), and AA (14.Shimizu T. Rådmark O. Samuelsson B. Enzyme with dual lipoxygenase activities catalyzes leukotriene A4 synthesis from arachidonic acid.Proc. Natl. Acad. Sci. USA. 1984; 81: 689-693Crossref PubMed Scopus (262) Google Scholar). (J. Whelan gathered and presented in his PhD dissertation (defended at Penn State University in 1988) a wealth of information on LOX-catalyzed oxidation of various polyunsaturated fatty acids.) A brief report by Hombeck, Pohnert, and Boland (15.Hombeck M. Pohnert G. Boland W. Biosynthesis of dictyopterene A: stereoselectivity of a lipoxygenase/hydroperoxide lyase from Gomphonema parvulum (Bacillariophyceae).Chem. Commun. 1999; 3: 243-244Crossref Scopus (28) Google Scholar) on AA metabolism by the freshwater diatom Gomphonema parvulum implicated a 9-LO in the formation of 9-HPETE as a major product. Importantly, sLOX is considered a quintessential 15-LOX (9.Hamberg M. Samuelsson B. On the specificity of the oxygenation of unsaturated fatty acids catalyzed by soybean lipoxidase.J. Biol. Chem. 1967; 242: 5329-5335Abstract Full Text PDF PubMed Google Scholar), whereas ptLOX is kinetically and mechanistically similar to human 5-LO (14.Shimizu T. Rådmark O. Samuelsson B. Enzyme with dual lipoxygenase activities catalyzes leukotriene A4 synthesis from arachidonic acid.Proc. Natl. Acad. Sci. USA. 1984; 81: 689-693Crossref PubMed Scopus (262) Google Scholar). Both of the enzymes are currently used in de facto standard LO inhibitor assays produced by Cayman Chemical Co. (Ann Arbor, MI). sLOX was the first LOX ever to crystallize and still is the enzyme whose three-dimensional structure is used to predict structures of other LOs that have so far eluded crystallization. Remarkably, the only other enzyme of the LO/LOX family that has been successfully crystallized is rabbit reticulocyte 15-LO (16.Sloane D.L. Browner M.F. Dauter Z. Wilson K. Fletterick R.J. Segal E. Purification and crystallization of 15-lipoxygenase from rabbit reticulocytes.Biochem. Biophys. Res. Commun. 1990; 173: 507-513Crossref PubMed Scopus (24) Google Scholar).As key players in the formation of various physiologically active compounds of the leukotriene, lipoxin, and oxylipin families, LOs are in the center of lipid signaling pathways that regulate cell metabolism and apoptosis (17.Phillis J.W. Horrocks L.A. Farooqui A.A. Cyclooxygenases, lipoxygenases, and epoxygenases in CNS: their role and involvement in neurological disorders.Brain Res. Brain Res. Rev. 2006; 52: 201-243Crossref PubMed Scopus (344) Google Scholar). Overproduction of certain LO metabolites has been shown to be associated with cancer development (18.Fürstenberger G. Krieg P. Müller-Decker K. Habenicht A.J. What are cyclooxygenases and lipoxygenases doing in the driver's seat of carcinogenesis?.Int. J. Cancer. 2006; 119: 2247-2254Crossref PubMed Scopus (126) Google Scholar), stroke (19.Jatana M. Giri S. 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Pharmacotherapy of diseases mediated by 5-lipoxygenase pathway eicosanoids.Prostaglandins Other Lipid Mediat. 2007; 83: 188-197Crossref PubMed Scopus (65) Google Scholar, 23.Chu L.S. Fang S.H. Zhou Y. Yu G.L. Wang M.L. Zhang W.P. Wei E.Q. Minocycline inhibits 5-lipoxygenase activation and brain inflammation after focal cerebral ischemia in rats.Acta Pharmacol. Sin. 2007; 28: 763-772Crossref PubMed Scopus (65) Google Scholar), arthritis (24.Klickstein L.B. Shapleigh C. Goetzl E.J. Lipoxygenation of arachidonic acid as a source of polymorphonuclear leukocyte chemotactic factors in synovial fluid and tissue in rheumatoid arthritis and spondyloarthritis.J. Clin. Invest. 1980; 66: 1166-1170Crossref PubMed Scopus (287) Google Scholar, 25.Araico A. Terencio M.C. Alcaraz M.J. Domínguez J.N. León C. Ferrándiz M.L. Evaluation of the anti-inflammatory and analgesic activity of Me-UCH9, a dual cyclooxygenase-2/5-lipoxygenase inhibitor.Life Sci. 2007; 80: 2108-2117Crossref PubMed Scopus (33) Google Scholar), asthma (26.Burka J.F. Paterson N.A. Evidence for lipoxygenase pathway involvement in allergic tracheal contraction.Prostaglandins. 1980; 19: 499-515Crossref PubMed Scopus (59) Google Scholar, 27.Berger W. De Chandt M.T. Cairns C.B. Zileuton: clinical implications of 5-lipoxygenase inhibition in severe airway disease.Int. J. Clin. Pract. 2007; 61: 663-676Crossref PubMed Scopus (151) Google Scholar), cancer (28.Steele V.E. Holmes C.A. Hawk E.T. Kopelovich L. Lubet R.A. Crowell J.A. Sigman C.C. Kelloff G.J. Lipoxygenase inhibitors as potential cancer chemopreventives.Cancer Epidemiol. Biomarkers Prev. 1999; 8: 467-483PubMed Google Scholar), inflammation (29.Steinhilber D. 5-Lipoxygenase: a target for antiinflammatory drugs revisited.Curr. Med. Chem. 1999; 6: 71-85PubMed Google Scholar, 30.Federico A. Morgillo F. Tuccillo C. Ciardiello F. Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis.Int. J. Cancer. 2007; 121: 2381-2386Crossref PubMed Scopus (715) Google Scholar), and many other diseases and pathological states. Therefore, LOs are justifiably among the most important targets for designing selective and safe inhibitors suitable for clinical use.Among the large number of LO inhibitors, linoleyl hydroxamic acid (LHA) stands out as a simple chemical derivative of the naturally occurring LA. Earlier, we reported results of our preliminary experiments with sLOX (31.Butovich I.A. Bridnya V.P. Kukhar V.P. Linoleyl-hydroxamic acid—a suicide inhibitor of lipoxygenase.Biochemistry–Moscow. 1990; 55: 908-912Google Scholar), ptLOX (32.Butovich I.A. Kharchenko O.V. Bondarenko L.B. Babenko V.M. Livarchuk L. Linoleyl hydroxamate as 5-lipoxygenase inhibitor.Biochemistry–Moscow. 1994; 59: 597-600Google Scholar, 33.Butovich I.A. Reddy C.C. Inhibition of potato lipoxygenase by linoleyl hydroxamic acid: kinetic and EPR spectral evidence for a two-step reaction.Biochem. J. 2002; 365: 865-871Crossref PubMed Google Scholar), and porcine leukocyte 12-LO (34.Kharchenko O.V. Cernjuk V.N. Butovich I.A. Inhibitory effect of linoleyl-hydroxamic acid on the oxidation of linoleic acid by 12-lipoxygenase from porcine leukocytes.Ukr. Biokhim. Zh. 1999; 71: 33-37Google Scholar). Inhibition of all the LOs was observed at micromolar concentrations of LHA, or below. In independent studies, LHA suppressed activity of a related enzyme, LA 8R-dioxygenase, albeit at rather high concentrations (35.Brodowsky I.D. Hamberg M. Oliw E.H. BW A4C and other hydroxamic acids are potent inhibitors of linoleic acid 8R-dioxygenase of the fungus Gaeumannomyces graminis.Eur. J. Pharmacol. 1994; 254: 43-47Crossref PubMed Scopus (10) Google Scholar). Importantly, no information is available on the ability of LHA to inhibit other PUFA-metabolizing enzymes, e.g., cyclooxygenases 1 and 2 (COX1/COX2).LHA was tested in vivo and was shown to decrease coronary perfusion pressure, systemic arterial pressure, and cardiac output in dogs under conditions of anaphylactic shock, presumably through downregulation of 5-LO-mediated leukotriene C4 biosynthesis (36.Moibenko A.A. Grabovskii L.A. Kotsiuruba V.N. Bulakh V.N. Tumanovskaia L.V. Azarov V.I. Butovich I.A. The mechanisms of the changes in coronary vascular resistance in anaphylactic shock.Fiziol. Zh. Im. I M Sechenova. 1994; 80: 77-82PubMed Google Scholar). LHA also decreased lipid peroxidation during acute hypoxic hypoxia (37.Kukoba T.V. Seredenko M.M. Butovych I.A. Moibenko O.O. The effect of linoleyl hydroxamic acid on lipid peroxidation processes and on the enzymatic activity of the antioxidant system in rats under hypoxia.Fiziol. Zh. 1998; 44: 43-48PubMed Google Scholar) and lessened the immune damage to the canine heart and coronary vessels in an experimentally induced coronary spasm of immune origin (36.Moibenko A.A. Grabovskii L.A. Kotsiuruba V.N. Bulakh V.N. Tumanovskaia L.V. Azarov V.I. Butovich I.A. The mechanisms of the changes in coronary vascular resistance in anaphylactic shock.Fiziol. Zh. Im. I M Sechenova. 1994; 80: 77-82PubMed Google Scholar, 38.Marchenko H.I. Kotsiuruba V.M. Butovych I.A. Sorochynskyi A.E. Zrazhevs'ka V.K. Tumanovs'ka L.V. The correction of disorders in arachidonic acid metabolism in coronary spasm of an immune origin.Fiziol. Zh. 1994; 40: 81-87PubMed Google Scholar). A decrease in free radical oxidation of lipoproteins and in formation of lipoid plaques was observed in rabbits once LHA was administered (39.Chobot'ko H.M. Effects of modifications of lipoproteins by water soluble forms of lineoleic-hydroxamic acid on biochemical markers of development of atherosclerosis.Ukr. Biokhim. Zh. 1998; 70: 64-68PubMed Google Scholar). For general information on hydroxamic acid, a review of Agrawal (40.Agrawal Y.K. Hydroxamic acids and their metal complexes.Russ. Chem. Rev. 1979; 48: 948-963Crossref Scopus (72) Google Scholar) is a good starting point.Here, we report the results of a first comparative study of enzyme inhibition by LHA for human recombinant 5-LO (h5-LO), porcine 12-LO, rabbit 15-LO, ovine COX1 and COX2, and human microsomal prostaglandin E synthase-1 (mPGES-1), all of which are involved in oxidative transformations of PUFAs relevant to lipid signaling in mammals and humans.MATERIALS AND METHODSReagents and equipmentFatty acids were purchased from Nu-Chek Prep, Inc. (Elysian, MN). COX1/COX2 inhibitor assay kits, ptLOX, h5-LO-1, and 12-LO from porcine leukocytes were purchased from Cayman Chemical Co. (Ann Arbor, MI). 15-LO from rabbits was a product of Biomol (Plymouth Meeting, PA). Another sample of h5-LO (h5-LO-2) overexpressed in Escherichia coli and purified on ATP agarose as described earlier (41.Hammarberg T. Zhang Y.Y. Lind B. Radmark O. Samuelsson B. Mutations at the C-terminal isoleucine and other potential iron ligands of 5-lipoxygenase.Eur. J. Biochem. 1995; 230: 401-407Crossref PubMed Scopus (56) Google Scholar) was received from Dr. Olof Rådmark (Karolinska Institute, Stockholm, Sweden). HPLC and MS-grade solvents were products of Burdick and Jackson (Muskegon, MI). Other reagents were from Sigma-Aldrich (St. Louis, MO). Ultraviolet (UV) and visible spectra were taken on a Beckman DU800 spectrophotometer (Beckman-Coulter; Fullerton, CA). Chromatographic experiments were conducted on a Waters Alliance 2695 HPLC separations module equipped with a Waters 2996 diode array detector (Waters; Milford, MA). Mass spectra were taken on an LCQ Deca XP Max MSn ion trap spectrometer using Xcalibur software (Thermo Electron; San Jose, CA). NMR spectra were recorded on a Varian 400 MHz spectrometer (Varian Inc.; Palo Alto, CA). Melting point of LHA was determined using an Optimelt MPA100 melting point apparatus (Stanford Research Systems; Sunnyvale, CA).Synthesis and analyses of LHABecause neither the analytical procedures for LHA characterization nor its properties have been described before in any detail, the following is a brief description of the methods utilized and the corresponding results. LHA was synthesized using procedures described earlier for polyfluorinated alcadiynoic hydroxamic fatty acids (42.Butovich I.A. Kukhar V.P. Bridnya V.P. Radchenko O.A. Kotlinskaya A. Polyfluorosubstituted alcadiynoic carboxylic and hydroxamic acids as new inhibitors of lipoxygenases.Proc. Natl. Acad. Sci. of Ukraine, ser. B. 1989; 5: 55-58Google Scholar). The crude product was purified by normal-phase open-column silica gel chromatography (melting point of the final product, 39 ± 1°C). The purity and structure of LHA was checked by electrospray ionization mass spectrometry (ESI-MS), atmospheric pressure chemical ionization MS (APCI-MS), reverse-phase HPLC (RP-HPLC), and normal-phase HPLC (NP-HPLC), with APCI-MS detection of the analytes, and one-dimensional (1D) and two-dimensional (2D) proton (1H) and carbon (13C) NMR analyses.ESI-MS analysis of LHAThe compound was dissolved in a MeOH-acetic acid (99:1; v/v) solvent mixture to a final concentration of 25 μM and was directly infused into the mass spectrometer using a syringe pump at a flow rate of 5 μl/min. The following settings were used: source voltage, 1.5 kV; sheath gas flow rate, ∼10 arbitrary units (au); capillary voltage, 40V; capillary temperature, 125°C.APCI-MS analysis of LHAA sample of 10 μM LHA in MeOH was directly infused at a flow rate of 7 μl/min using a syringe pump. The APCI-MS settings were as follows: source voltage, 4 kV; source current, 5 μA; vaporizer temperature, 350°C; sheath gas flow rate, 20 au; capillary voltage, 22V; capillary temperature, 300°C.HPLC analyses of LHAThe compound was analyzed chromatographically using two different methods, NP-HPLC and RP-HPLC.NP-HPLC analysis of LHA was conducted on a Phenomenex Diol HPLC column (3.2 mm × 150 mm, 5 μm) using isocratic elution with a solvent mixture composed of n-hexane, propan-2-ol, and glacial acetic acid (949:50:1; v/v/v). The effluent was continuously monitored using a tandem of a Waters 2996 diode array detector (DAD) and a mass spectrometer equipped with an APCI ion source. The DAD was set to record spectra in the range of 200 to 400 nm, and the mass spectrometer recorded spectra in either the positive- or the negative-ion modes in the range of m/z values between 200 and 1,000.RP-HPLC analysis of LHA was performed on a 2.1 × 150 mm, 5 μm Hypersil GOLD reverse-phase column (Thermo Electron Corp.; San Jose, CA) in methanol-5 mM ammonium formate in water (70:30; v/v) at a flow rate of 0.25 ml/min and a temperature of 30°C. Detection of the analytes was performed essentially as described above for NP-HPLC experiments.1D and 2D 1H and 13C NMR spectra1H spectra of LHA in CD3OD (10 mg/0.6 ml) were taken at 399.78 MHz at room temperature. The 13C spectra were recorded at the spectrometer frequency of 75.46 MHz.Compound stabilityA dry sample of LHA stored under nitrogen in the dark at −20°C to −70°C has been stable for at least 8 years. No decomposition/isomerization products have been detected by UV-visible light spectrometry, NMR, or MS.Kinetic and inhibitory studiesEnzymatic activities of COX1, COX2, 12-LO, and 15-LO were measured according to the manufacturers' recommendations with minor modifications (see below), whereas h5-LO, ptLOX, sLOX, and mPGES-1 were tested as described earlier (13.Butovich I.A. Lukyanova S.M. Bachmann C. Dihydroxydocosahexaenoic acids of the neuroprotectin D family: synthesis, structure, and inhibition of human 5-lipoxygenase.J. Lipid Res. 2006; 47: 2462-2474Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 41.Hammarberg T. Zhang Y.Y. Lind B. Radmark O. Samuelsson B. Mutations at the C-terminal isoleucine and other potential iron ligands of 5-lipoxygenase.Eur. J. Biochem. 1995; 230: 401-407Crossref PubMed Scopus (56) Google Scholar, 43.Thorén S. Weinander R. Saha S. Jegerschöld C. Pettersson P.L. Samuelsson B. Hebert H. Hamberg M. Morgenstern R. Jakobsson P.J. Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination.J. Biol. Chem. 2003; 278: 22199-22209Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 44.Pettersson P.L. Thorén S. Jakobsson P.J. Human microsomal prostaglandin E synthase 1: a member of the MAPEG protein superfamily.Methods Enzymol. 2005; 401: 147-161Crossref PubMed Google Scholar). To account for possible loss of enzymatic activity upon preincubation of the enzymes with LHA, control experiments were performed in which the enzymes were preincubated in the reaction buffers (without substrates) for the same period of time as in the experiments with the inhibitor(s). Then, fatty acid substrates were added and the remaining enzymatic activity was measured for each individual LO and LOX as described below. The calculated residual activity without inhibitor in the reaction media was used as control in the inhibition experiments.Inhibition of h5-LOEnzymatic activities of h-5LO-1 and h5-LO-2 were measured by using RP-HPLC with UV detection of the reaction products using an established procedure (13.Butovich I.A. Lukyanova S.M. Bachmann C. Dihydroxydocosahexaenoic acids of the neuroprotectin D family: synthesis, structure, and inhibition of human 5-lipoxygenase.J. Lipid Res. 2006; 47: 2462-2474Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Briefly, the reaction mixtures included 100 μM AA dissolved in 0.05 M Tris-HCl buffer, pH 7.6, 0.15 M NaCl, 1.2 mM EDTA × 2 Na+, 2 mM CaCl2, 2.3 mM ATP, 0.02 mM β-mercaptoethanol, 0.02 mg/ml phosphatidylcholine (from egg yolk), and 10 μM 13(S)-HPODE, in a final volume of 150 μl. The reactions were initiated by adding a concentrated stock solution of substrate (AA) and activator [13(S)-HPODE] to the mixture that already contained h5-LO. The reaction was carried out at 25°C ± 1°C for 20 min, then 350 μl of ice-cold stopping solution that contained acetonitrile and 2.5% glacial acetic acid (v/v) was added to the reaction mixture to stop the reaction and precipitate the proteins. The mixture was vortexed, left on ice for 10 min, and then centrifuged at 10,000 g at 4°C for another 10 min. The supernatant was transferred into a glass HPLC vial and stored at −20°C until the HPLC analysis. When LHA was being tested as 5-LO inhibitor, the compound was dissolved in the reaction buffer without AA and 13(S)-HPODE and was preincubated with the enzyme for 10 min. Then, the reaction was initiated by adding AA and 13(S)-HPODE and allowed to proceed for 20 min.To analyze the main products of AA oxidation, [5(S)-HPETE and 5(S)-HETE], the final product mixture was separated by RP-HPLC on a 4.6 mm × 125 mm, 5 μm C8 Zorbax Eclipse (Agilent Technologies, Wilmington, DE) XDB-C8 column. The separation was performed at 30°C at a flow rate of 0.5 ml/min. The compounds were eluted in a water-acetonitrile solvent mixture using a previously described protocol (13.Butovich I.A. Lukyanova S.M. Bachmann C. Dihydroxydocosahexaenoic acids of the neuroprotectin D family: synthesis, structure, and inhibition of human 5-lipoxygenase.J. Lipid Res. 2006; 47: 2462-2474Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The elution profile was monitored at 236 ± 2 nm [the maximum of adsorption of conjugated dienes; molar extinction coefficient, ϵm = 23,000 M−1 × cm−1 (45.Gibian M.J. Vandenberg P. Product yield in oxygenation of linoleate by soybean lipoxygenase: the value of the molar extinction coefficient in the spectrophotometric assay.Anal. Biochem. 1987; 163: 343-349Crossref PubMed Scopus (114) Google Scholar)].Inhibition of 12-LOThe enzymatic activity of 12-LO was measured with AA as suggested by the manufacturer, with two exceptions: use of Lubrol PX instead of Tween 20, and use of a UV-spectrophotometer set at 237 nm to monitor the product formation instead of an oxygen electrode. The control enzymatic activity was measured at 25°C in 1 ml of 0.1 M Tris-HCl buffer, pH 7.5, with 5 mM EDTA, 0.02% Lubrol PX, and 100 μM AA as substrate. First, the buffer (1 ml), placed in a 1 cm quartz spectrophotometric cuvette, was mixed with 5 μl of 20 mM stock solution of AA in ethanol. The reaction was started by adding an aliquot of the enzyme solution. The reaction mixture was briefly but vigorously mixed with a pipette, and then the product formation was recorded at 237 nm. In inhibitory studies, a concentrated stock solution of LHA (10 mM, in ethanol) or a blank (the same buffer) was added to the buffer first. Then, a 10 μl aliquot of 12-LO was added and incubated with LHA for 5 or 10 min. The remaining 12-LO activity was measured by adding the stock solution of AA, vigorous mixing, and recording the product formation at 237 nm.Inhibition of 15-LOThe control enzymatic activity (2 μl of the enzyme stock solution, as supplied by the manufacturer) was measured in 1 ml of 0.05 M sodium phosphate buffer, pH 7.6, with 100 μM LA as substrate at 25°C, as recommended by the manufacturer. The molar absorptivity of the product at 324 nm was assumed to be the same as for AA (see above). The inhibitory studies were performed as described above for 12-LO, with 1, 5, or 10 min preincubation of the enzyme with LHA, followed by the addition of 5 μl of 20 mM stock solution of LA (final substrate concentration, 100 μM).Inhibition of sLOXThe reactions were carried out in a 1 cm quartz spectrophotometric cuvette. The enzyme (final concentration 2 μg/ml) was preincubated for 5 min with the indicated concentrations of LHA dissolved in 1 ml of 50 mM sodium borate buffer, pH 9.0, at 25°C. Then, an aliquot of an AA stock solution was added to the sLOX/LHA mixture (final concentration of AA was 100 μM), and an increase in the optical density at 237 nm was continuously monitored for up to 10 min. The remaining activity of the LHA-inhibited enzyme