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• W1999224453 abstract "We recently uncovered two new families of potent docosahexaenoic acid-derived mediators, termed D series resolvins (Rv; resolution phase interaction products) and protectins. Here, we assign the stereochemistry of the conjugated double bonds and chirality of alcohols present in resolvin D1 (RvD1) and its aspirin-triggered 17R epimer (AT-RvD1) with compounds prepared by total organic synthesis. In addition, docosahexaenoic acid was converted by a single lipoxygenase in a “one-pot” reaction to RvD1 in vitro. The synthetic compounds matched the physical and biological properties of those enzymatically generated. RvD1 proved to be 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, AT-RvD1 matched 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, and they both stopped transendothelial migration of human neutrophils (EC50 ∼30 nm). In murine peritonitis in vivo, RvD1 and AT-RvD1 proved equipotent (at nanogram dosages), limiting polymorphonuclear leukocyte infiltration in a dose-dependent fashion. RvD1 was converted by eicosanoid oxidoreductase to novel 8-oxo- and 17-oxo-RvD1 that gave dramatically reduced bioactivity, whereas enzymatic conversion of AT-RvD1 was sharply reduced. These results establish the complete stereochemistry and actions of RvD1 and AT-RvD1 as well as demonstrate the stereoselective basis for their enzymatic inactivation. RvD1 regulates human polymorphonuclear leukocyte transendothelial migration and is anti-inflammatory. When its carbon 17S alcohol is enzymatically converted to 17-oxo-RvD1, it is essentially inactive, whereas the 17R alcohol configuration in its aspirin-triggered form (AT-RvD1) resists rapid inactivation. These results may contribute to the beneficial actions of aspirin and ω-3 fish oils in humans. We recently uncovered two new families of potent docosahexaenoic acid-derived mediators, termed D series resolvins (Rv; resolution phase interaction products) and protectins. Here, we assign the stereochemistry of the conjugated double bonds and chirality of alcohols present in resolvin D1 (RvD1) and its aspirin-triggered 17R epimer (AT-RvD1) with compounds prepared by total organic synthesis. In addition, docosahexaenoic acid was converted by a single lipoxygenase in a “one-pot” reaction to RvD1 in vitro. The synthetic compounds matched the physical and biological properties of those enzymatically generated. RvD1 proved to be 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, AT-RvD1 matched 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, and they both stopped transendothelial migration of human neutrophils (EC50 ∼30 nm). In murine peritonitis in vivo, RvD1 and AT-RvD1 proved equipotent (at nanogram dosages), limiting polymorphonuclear leukocyte infiltration in a dose-dependent fashion. RvD1 was converted by eicosanoid oxidoreductase to novel 8-oxo- and 17-oxo-RvD1 that gave dramatically reduced bioactivity, whereas enzymatic conversion of AT-RvD1 was sharply reduced. These results establish the complete stereochemistry and actions of RvD1 and AT-RvD1 as well as demonstrate the stereoselective basis for their enzymatic inactivation. RvD1 regulates human polymorphonuclear leukocyte transendothelial migration and is anti-inflammatory. When its carbon 17S alcohol is enzymatically converted to 17-oxo-RvD1, it is essentially inactive, whereas the 17R alcohol configuration in its aspirin-triggered form (AT-RvD1) resists rapid inactivation. These results may contribute to the beneficial actions of aspirin and ω-3 fish oils in humans. Lipids, like proteins and carbohydrates, are an essential component of the human diet. Observations first reported by Burr and Burr in 1929 demonstrated that exclusion of dietary lipids resulted in multiorgan pathology and premature death (1Burr G.O. Burr M.M. J. Biol. Chem. 1929; 82: 345-367Abstract Full Text PDF Google Scholar). Since these early studies, it has become clear that lipids and lipid-derived local chemical mediators play critical regulatory roles in a variety of cellular functions, including inflammation (2Gallin J.I. Snyderman R. Fearon D.T. Haynes B.F. Nathan C. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincott Williams & Wilkins, Philadelphia1999Google Scholar). Inflammation is a fundamental component of host defense, because the immune response is responsible for the clearance of injurious agents as well as the associated damaged tissue. In normal physiological states, the inflammatory response is cleared to allow for resolution back to the non-inflamed state to maintain tissue homeostasis (3Majno G. Joris I. Cells, Tissues, and Disease: Principles of General Pathology. 2nd Ed. Oxford University Press, New York2004Google Scholar). Recently, we identified molecular circuits involved in the promotion of this inflammatory resolution and uncovered novel families of ω-3 EPA 2The abbreviations used are: EPA, eicosapentaenoic acid; AT-RvD1, aspirin-triggered RvD1, 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid; DHA, docosahexaenoic acid; GC-MS, gas chromatography-mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LOX, lipoxygenase; PMN, polymorphonuclear leukocyte; RP-HPLC, reversedphase high-pressure liquid chromatography; Rv, resolvin, resolution phase interaction product; RvD1, 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid; RvD2, 7S,16R,17S-trihydroxydocosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid; EOR, 15-prostaglandin dehydrogenase/eicosanoid oxidoreductase; MPO, myeloperoxidase; HMEC, Human microvascular endothelial cell; HBSS, Hanks' balanced salt solution; fMLP, n-formylmethionylleucylphenylalanine; LX, lipoxin. - and DHA-derived local mediators, named resolvins and protectins (4Serhan C.N. Clish C.B. Brannon J. Colgan S.P. Chiang N. Gronert K. J. Exp. Med. 2000; 192: 1197-1204Crossref PubMed Scopus (954) Google Scholar, 5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar, 6Bannenberg G.L. Chiang N. Ariel A. Arita M. Tjonahen E. Gotlinger K.H. Hong S. Serhan C.N. J. Immunol. 2005; 174: 4345-4355Crossref PubMed Scopus (0) Google Scholar). Defects in these clearance mechanisms appear to be associated with persistent tissue inflammation and autoimmunity to cellular contents (for recent review, see Ref. 7Serhan C.N. Savill J. Nat. Immunol. 2005; 6: 1191-1197Crossref PubMed Scopus (1836) Google Scholar). It was first observed nearly 30 years ago that a diet enriched in fish oil (of which ω-3 fatty acids are the primary component) is associated with a lower risk of cardiovascular disease (8Bang H.O. Dyerberg J. Hjørne N. Acta Med. Scand. 1976; 200: 69-73Crossref PubMed Scopus (796) Google Scholar). Since that report, it is widely discussed that essential ω-3 fatty acids are critical to cellular function and human health (reviewed in Ref. 9Simopoulos A.P. Am. J. Clin. Nutr. 1999; 70: 560S-569SCrossref PubMed Scopus (1324) Google Scholar). Several studies demonstrated the beneficial effects of ω-3 supplementation in pathological states, including Crohn's disease (10Belluzzi A. Brignola C. Campieri M. Pera A. Boschi S. Miglioli M. N. Engl. J. Med. 1996; 334: 1557-1560Crossref PubMed Scopus (696) Google Scholar), coronary heart disease (11GISSI-Prevenzione InvestigatorsLancet. 1999; 354: 447-455Abstract Full Text Full Text PDF PubMed Scopus (3761) Google Scholar), and sudden cardiac death (12Albert C.M. Campos H. Stampfer M.J. Ridker P.M. Manson J.E. Willett W.C. Ma J. N. Engl. J. Med. 2002; 346: 1113-1118Crossref PubMed Scopus (1023) Google Scholar). Although the clinical findings suggest a benefit from ω-3 supplementation, the molecular mechanisms underlying this protective action have only begun to be uncovered. The two main ω-3 fatty acids present in fish oil are eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6). DHA is highly enriched in both the retina and neuronal synaptic membranes and is a critical component in neural development (reviewed in Ref. 13Bazan N.G. Wurtman R.J. Wurtman J.J. Nutrition and the Brain. Raven Press, New York1990: 1-22Google Scholar). The structures and potential bioactions of putative oxidative products of DHA have been discussed (13Bazan N.G. Wurtman R.J. Wurtman J.J. Nutrition and the Brain. Raven Press, New York1990: 1-22Google Scholar, 14Salem Jr., N. Litman B. Kim H.-Y. Gawrisch K. Lipids. 2001; 36: 945-959Crossref PubMed Scopus (772) Google Scholar) yet remain to be established. The resolvins (resolution phase interaction products) are a novel family of lipid mediators derived from both EPA and DHA (4Serhan C.N. Clish C.B. Brannon J. Colgan S.P. Chiang N. Gronert K. J. Exp. Med. 2000; 192: 1197-1204Crossref PubMed Scopus (954) Google Scholar, 5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar, 15Hong S. Gronert K. Devchand P. Moussignac R-L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). These potent lipid mediators are not only anti-inflammatory but also promote resolution back to the non-inflamed state (6Bannenberg G.L. Chiang N. Ariel A. Arita M. Tjonahen E. Gotlinger K.H. Hong S. Serhan C.N. J. Immunol. 2005; 174: 4345-4355Crossref PubMed Scopus (0) Google Scholar). The identification of the resolvins and protectins as well as their arachidonic acid (C20:4)-derived cousins, lipoxins (LXs), as endogenous stop signals in inflammation provides evidence that resolution is a biochemically active process (4Serhan C.N. Clish C.B. Brannon J. Colgan S.P. Chiang N. Gronert K. J. Exp. Med. 2000; 192: 1197-1204Crossref PubMed Scopus (954) Google Scholar) and not passive as was once believed (3Majno G. Joris I. Cells, Tissues, and Disease: Principles of General Pathology. 2nd Ed. Oxford University Press, New York2004Google Scholar). DHA is converted through a series of enzymatic oxygenations to both protectins and D series resolvins. Resolvin D1 (RvD1, 7S,8R,17S-trihydroxy-DHA) is produced in resolving exudates in vivo and is a product of transcellular biosynthesis with human leukocytes and endothelial cells (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar). RvD1 was also identified in activated human whole blood and murine brain (15Hong S. Gronert K. Devchand P. Moussignac R-L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar) as well as in fish (16Hong S. Tjonahen E. Morgan E.L. Yu L. Serhan C.N. Rowley A.F. Prostaglandins Other Lipid Mediat. 2005; 78: 107-116Crossref PubMed Scopus (79) Google Scholar). RvD1 biosynthesis involves sequential oxygenations by 15-lipoxygenase (LOX) and 5-LOX (Fig. 1A) (15Hong S. Gronert K. Devchand P. Moussignac R-L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). DHA is converted by 15-LOX to 17S-hydroxy-DHA (HDHA). In the case of aspirin treatment, aspirin-acetylated cyclooxygenase-2 generates 17R-HDHA, which following sequential oxygenation by 5-LOX results in production of 17-epi-RvD1, also known as aspirin-triggered RvD1 (AT-RvD1) (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar, 17Serhan, C. N. (2004) U. S. Patent Application Publication No. 2004/0116408 A1, published June 17, 2004Google Scholar, 18Serhan, C. N., and Clish, C. B. (2003) U.S. Patent No. 6,670,396 B2, December 30, 2003Google Scholar). Both the 17R and 17S D series resolvins exhibit potent anti-inflammatory action in vivo (15Hong S. Gronert K. Devchand P. Moussignac R-L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar), and the recent evidence that trout brain cells also produce RvD1 from endogenous stores of DHA (16Hong S. Tjonahen E. Morgan E.L. Yu L. Serhan C.N. Rowley A.F. Prostaglandins Other Lipid Mediat. 2005; 78: 107-116Crossref PubMed Scopus (79) Google Scholar) indicates that the RvD1 structure is evolutionarily conserved. These findings, along with the extensive literature surrounding the importance of DHA in neural development and function (14Salem Jr., N. Litman B. Kim H.-Y. Gawrisch K. Lipids. 2001; 36: 945-959Crossref PubMed Scopus (772) Google Scholar, 19Calder P.C. Lipids. 2001; 36: 1007-1024Crossref PubMed Scopus (628) Google Scholar), highlight the need for establishing the complete stereochemistry of both RvD1 and AT-RvD1. Here, we report the complete stereochemistry, anti-inflammatory properties, and enzymatic inactivation of RvD1 and its aspirin-triggered isomer (AT-RvD1). Mediator Lipidomics—LC-MS/MS-based mediator lipidomic analyses were performed as in previous studies (20Lu Y. Hong S. Gotlinger K. Serhan C.N. Scientific World-Journal. 2006; 6: 589-614Crossref Scopus (29) Google Scholar, 21Lu Y. Hong S. Tjonahen E. Serhan C.N. J. Lipid Res. 2005; 46: 790-802Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, sample analyses were carried out using a Finnigan LCQ LC ion trap tandem mass spectrometer equipped with a Thermoelectron BDS Hypersil C18 (100 mm × 2.1 mm × 5 μm) column (Fig. 2) or a Phenomenex C18 (150 mm × 2 mm × 5 μm) column equipped with a rapid spectra scanning UV diode array detector. For routine analyses, the mobile phase (methanol:water:acetic acid, 65:35:0.01) was eluted at a 0.2 ml/min flow rate, and UV spectra were recorded ∼0.1 min before samples entered MS/MS. For results in Table 1, spectra were recorded in methanol using a Hewlett-Packard 8453 UV spectrophotometer with accuracy ± 2 nm.TABLE 1Physical and chemical properties by HPLC, LC-MS/MS, and GC-MS analysesView Large Image Figure ViewerDownload Hi-res image Download (PPT)* See “Experimental Procedures” for further details. Open table in a new tab * See “Experimental Procedures” for further details. RP-HPLC—Liquid chromatographic analyses were performed using a Hewlett-Packard Series 1100 high-pressure liquid chromatography (HPLC) system equipped with a Phenomenex C18 (150 × 2 mm × 5 μm) column with a UV diode array detector. Matching of synthetic with biological and biogenic materials was carried out with the mobile phase (methanol:water:acetic acid, 70:30:0.01) at a 0.2 ml/min flow rate. RvD1 and AT-RvD1 as well as analysis of the oxo-containing products were performed using a mobile phase (methanol:water:acetic acid, 65:35:0.01) with a 0.2 ml/min flow rate. GC-MS Analysis—Samples were taken to dryness using a stream of N2, suspended in MeOH (10 μl), and treated with excess ethereal diazomethane (45 min at room temperature), followed by N,O-bis(trimethylsilyl)trifluoroacetamide) treatment (overnight at room temperature, obtained from Pierce) (22Serhan C.N. Methods Enzymol. 1990; 187: 167-175Crossref PubMed Scopus (11) Google Scholar). GC-MS analysis was performed with a Hewlett-Packard 5971A mass-selective quadrupole detector equipped with an HPG1030A workstation and HP6890 GC system. Samples were injected with hexane as the solvent and the temperature program was initiated at 150 °C and held for 2 min and reached 230 °C at 10 min (10 °C/min) and then 280 °C at 20 min (5 °C/min). Reference saturated fatty acid methyl esters carbons C14-C24 gave the following retention times (min, mean of n = 3): C14, 6.5; C16, 8.5; C18, 10.4; C20, 12.4; C22, 14.6; and C24, 17.0; these were used to calculate respective C values of fatty acid products. RvD1 Preparation—RvD1 was prepared using a one-pot reaction by incubating DHA and soybean LOX (type IV; from Sigma) as in previous studies (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar, 17Serhan, C. N. (2004) U. S. Patent Application Publication No. 2004/0116408 A1, published June 17, 2004Google Scholar). Briefly, DHA (2 mg) was incubated with soybean LOX (100 kilounits, 701 kilounits/mg of protein, 3.6 mg of protein/ml), in borate buffer (5 ml, pH 9.3) at 4 °C. The substrate DHA in micelle suspensions was vortexed (∼15 min, at room temperature), and the isolated soybean LOX (100 kilounits) was added to the micelle suspensions at 15- and 30-min intervals. These incubations were terminated at 40 min with addition of cold methanol (20 ml) followed by addition of NaBH4 for reduction. Next, they were taken for extraction using C18 solid phase (20Lu Y. Hong S. Gotlinger K. Serhan C.N. Scientific World-Journal. 2006; 6: 589-614Crossref Scopus (29) Google Scholar) and subsequent analyses. The product isolated by RP-HPLC matched RvD1 produced by isolated human PMN (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar). Total Organic Synthesis—Both RvD1 and AT-RvD1 were synthesized from chiral starting materials of known chirality in enantiomerically and geometrically pure form via total organic synthesis, which will be reported separately. 3N. A. Petasis, J. Uddin, and C. N. Serhan, manuscript in preparation. The structures of synthetic RvD1 and AT-RvD1 methyl esters were characterized by NMR spectroscopy. Methanol Trapping with Human PMNs—Human whole (venous) blood was collected with heparin from healthy volunteers that declined taking medication for 2 weeks prior to donation, according to Brigham and Women's Hospital protocol 88-02642. Briefly, PMNs were freshly isolated from whole blood by Ficoll gradient and enumerated. 17S-hydro(peroxy)-DHA (3 μg) was incubated with human PMN suspensions (30–50 × 106 cells/ml) and zymosan A (100 μg/ml) in Dulbecco's phosphate-buffered saline (with Mg2+ and Ca2+) at 37 °C for 5 min (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar). Acidified MeOH (apparent pH ∼ 3 after mixing, 4 °C) was added, and the mixture was incubated at 4 °C for 10 min. The samples were rapidly neutralized and taken for C18 solid phase extraction and analyses. Endothelial Cell Isolation and Culture—Human microvascular endothelial cells (HMEC-1) were a gift of Francisco Candal, Centers for Disease Control, Atlanta, GA (23Robinson K.A. Candal F.J. Scott N.A. Ades E.W. Angiology. 1995; 46: 107-113Crossref PubMed Scopus (19) Google Scholar) and were cultured by a modification of methods described previously (24Collard C.D. Park K.A. Montalto M.C. Alapati S. Buras J.A. Stahl G.L. Colgan S.P. J. Biol. Chem. 2002; 277: 14801-14811Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 25Lennon P.F. Taylor C.T. Stahl G.L. Colgan S.P. J. Exp. Med. 1998; 188: 1433-1443Crossref PubMed Scopus (193) Google Scholar). In brief, HMEC-1 were harvested with 0.1% trypsin and incubated at 37 °C in 95% air/5% CO2. Culture medium was supplemented with heat-inactivated fetal bovine serum, penicillin, streptomycin, l-glutamine, epidermal growth factor, and hydrocortisone. For preparation of experimental monolayers, confluent endothelial cells were seeded at ≈105 cells/cm2 onto permeable polycarbonate inserts. Human PMNs and Transmigration—PMNs for transmigration were isolated as above with acid citrate dextrose as the anticoagulant as described with minor modification (26Parkos C.A. Delp C. Arnaout M.A. Madara J.L. J. Clin. Invest. 1991; 88: 1605-1612Crossref PubMed Scopus (292) Google Scholar). Double density gradients of Histopaque 1077 and 1119 (Sigma) were prepared, and whole venous blood was applied to each gradient and centrifuged at 700 × g for 30 min at room temperature. PMNs localized to the buffy coat directly above the red blood cells were removed. Residual red blood cells were removed by lysis in ice-cold NH4Cl buffer. PMNs were >90% as determined by microscopic evaluation and suspended 1 × 108 cells/ml in HBSS– (with 10 mm Hepes, pH 7.4, and without Ca2+ or Mg2+, Sigma-Aldrich). PMNs were used within 2 h of isolation. PMNs were incubated with 0–1000 nm RvD1 or AT-RvD1 in HBSS– at room temperature for 15 min. Migration assays were performed (27Lawrence D.W. Bruyninckx W.J. Louis N.A. Lublin D.M. Stahl G.L. Parkos C.A. Colgan S.P. J. Exp. Med. 2003; 198: 999-1010Crossref PubMed Scopus (69) Google Scholar). Briefly, 106 PMNs were added to the upper chambers of Transwell inserts plated with HMECs. A chemotactic gradient was established by adding HBSS+ to the upper and 100 nm n-formylmethionylleucylphenylalanine (fMLP) in HBSS+ to the lower chambers. PMN transmigration was carried out at 37 °C for 30 min, after which transmigrated PMNs were quantified by monitoring myeloperoxidase (MPO) (27Lawrence D.W. Bruyninckx W.J. Louis N.A. Lublin D.M. Stahl G.L. Parkos C.A. Colgan S.P. J. Exp. Med. 2003; 198: 999-1010Crossref PubMed Scopus (69) Google Scholar). Acute Inflammation—Murine peritonitis was performed (28Perretti M. Getting S.J. Winyard P.G. Willoughby D.A. Inflammation Protocols. Humana, Totowa, NJ2003: 139-146Google Scholar) using 6- to 8-week-old FVB male mice (Charles River Laboratories, Wilmington, MA) that were fed laboratory Rodent Diet 5001 (Purina Mills, Richmond, IN). After anesthetization with isoflurane, compounds were administered in 100 μl of phosphate-buffered saline intravenously through a tail vein. Zymosan A (1 mg/1 ml in sterile saline, Sigma) was injected intraperitoneally immediately following compound administration. In accordance with the Harvard Medical Area Standing Committee on Animals protocol 02570, mice were sacrificed after 4 h and peritoneal lavages were rapidly collected in Dulbecco's phosphate-buffered saline (minus Mg2+ and Ca2+, 5 ml). Aliquots of the lavage were stained with trypan blue and enumerated by light microscopy. For differential leukocyte counts, 100 μl of the lavage was added to 300 μl of 15% bovine serum albumin and centrifuged onto microscope slides at 1600 rpm for 4 min using a Cytofuge (StatSpin, Norwood, MA). The slides were allowed to air dry, and cells were visualized using a modified Wright-Giemsa stain (Sigma). Recombinant Enzymes—Activity of 15-prostaglandin dehydrogenase/eicosanoid oxidoreductase (denoted here and throughout as EOR as in Ref. 29Clish C.B. Levy B.D. Chiang N. Tai H-H. Serhan C.N. J. Biol. Chem. 2000; 275: 25372-25380Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) was monitored spectrophotometrically by the formation of NADH from NAD+ at 340 nm. Substrates in ethanol were dried down under N2 stream and resuspended in buffer containing Tris-HCl (0.1 m, pH 9.0, Sigma) and NAD+ (1.0 mm, Sigma) to a final concentration of 20 μm (100-μl total volume). Reactions were initiated with the addition of partially purified EOR (0.05 μg/incubation), and absorptions were read every 30 s for 25 min at 37 °C. Oxo-RvD1 Products—RvD1-ME or RvD1 (10 μg) was dried down under N2 stream and suspended in buffer containing Tris-HCl (100 μl, 0.1 m, pH 7.4, Sigma) and NAD+ (1.0 mm, Sigma). Enzymatic conversion was initiated by the addition of partially purified EOR (5 μg) and allowed to proceed for 2 h at 37 °C. After 2 h, these reactions were stopped with 2 volumes of cold methanol, and samples were extracted with C18 solid phase extraction. Comparison of Synthetic RvD1 and AT-RvD1—To assign the complete stereochemistry of natural RvD1 and determine whether RvD1 and its aspirin-triggered form AT-RvD1 (Fig. 1) share biological properties, as well as match their reported properties and actions (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar, 15Hong S. Gronert K. Devchand P. Moussignac R-L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar), it was essential to first establish the spectroscopic and physical properties of the synthetic materials (Fig. 2). The structure and stereochemistry of synthetic RvD1 and AT-RvD1 were unambiguous on the basis of their total synthesis from chiral-starting materials of known stereochemistry. The R/S configurations of C-7, C-8, and C-17 were directly derived from starting materials of the same chirality. For example, the C-17 R alcohol chirality was retained from the starting material (S)-(–)-glycidol and the C-17S retained from (R)-(+)-glycidol. The Z/E configuration of the double bonds was determined and confirmed using 1H NMR (COSY). Both RvD1 and AT-RvD1 gave very similar spectra (Fig. 2), yet displayed different chromatographic behaviors with RP-HPLC (Fig. 3). Liquid chromatography analysis gave UV chromatograms when monitored at 301 nm with major peak AT-RvD1 at 13.9 min separating and eluting before RvD1 at 15.1 min (Fig. 3A). The column and mobile phase parameters used here permitted separation of these two diastereomers by ∼1.2 min. The carboxyl-methyl esters of RvD1 and AT-RvD1 also resolved using essentially identical conditions (not shown). Further analysis of both RvD1 and AT-RvD1 demonstrated the major anion for both MS spectra was at m/z 375 (see Fig. 3, B and C, cleavage site a), which represents [M-H] for both RvD1 and AT-RvD1. These were consistent with the original mass spectra and structural elucidation of these resolvins produced in vivo in mouse exudates and with isolated human leukocytes (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar). As expected, analysis of the MS/MS spectrum obtained for both RvD1 and AT-RvD1 gave essentially the same fragmentation patterns (Fig. 3). Prominent daughter ions were obtained for both compounds at m/z 357 [a-H2O]; 339 [a-2H2O]; 331 [375-CO2] (see Fig. 3, B and C, cleavage site b); 321 [a-3H2O]; 313 [b-H2O]; 295 [b-2H2O]; 277 [375-CHO-CH2-(CH)2-CH2-CH3] (see Fig. 3, B and C, cleavage site d); 259 [d-H2O]; 241 [d-2H2O]; 233 [375-CHOH-CH2-(CH)2-(CH2)2-CO2] (see cleavage site c); 215 [c-H2O]; 141 [CHO-CH2-(CH)2-(CH2)2-COO–] (see cleavage site c′) essentially matching the ions reported earlier for endogenous RvD1 and AT-RvD1 isolated from murine inflammatory exudates and human PMN (Fig. 3D and cf. Ref. 5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar). Of interest, as observed in the comparison of LXA4 and its endogenous aspirin-triggered form 15-epi-LXA4 (30Chiang N. Takano T. Clish C.B. Petasis N.A. Tai H-H. Serhan C.N. J. Pharmacol. Exp. Ther. 1998; 287: 779-790PubMed Google Scholar), successive MS/MS analysis gave subtle differences in the intensity of several of the main daughter ions between RvD1 and its 17 epimer, AT-RvD1. Table 1 reports a summary of the prominent ions (both LC-MS/MS and GC-MS) and chromatographic properties of synthetic RvD1 and AT-RvD1 using parameters obtained originally for the endogenous resolvins (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar, 15Hong S. Gronert K. Devchand P. Moussignac R-L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, 31Marcheselli V.L. Hong S. Lukiw W.J. Hua Tian X. Gronert K. Musto A. Hardy M. Gimenez J.M. Chiang N. Serhan C.N. Bazan N.G. J. Biol. Chem. 2003; 278: 43807-43817Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). The present results with both synthetic and endogenous RvD1 confirm the original assignments of its basic structure (Table 1). Matching of Enzymatically Generated RvD1 with Synthetic RvD1—Having established the physical properties of synthetic RvD1, we next sought evidence to determine whether it was identical to the biologically active enzymatically generated material as well as assign the geometry of its conjugated double bonds and chirality of the alcohol group at the carbon 8 position that remained to be established (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar, 15Hong S. Gronert K. Devchand P. Moussignac R-L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). It was essential to take this approach, because the nanogram amounts obtained from human cells and inflammatory exudates permitted assigning the basic structure and bioactions of RvD1 but precluded direct determination of the complete stereochemistry of the biologically derived RvD1 (5Serhan C.N. Hong S. Gronert K. Colgan S.P. Devchand P.R. Mirick G. Moussignac R-L. J. Exp. Med. 2002; 196: 1025-1037Crossref PubMed Scopus (1362) Google Scholar). To this end, biogenic RvD1 was prepared by incubating DHA with isolated 15-LOX type I (i.e. soybean LOX type IV, see “Experimental Procedures”) using a one-pot incubation procedure that employed micellar substrate presentation (15Hong S. Gronert K. Devchand P. Moussignac R-L. Serhan C.N. J. Biol. Chem. 2003; 278: 14677-14687Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, 17Serhan, C. N. (2004) U. S. Patent Application Publication No. 2004/0116408 A1, published Ju" @default.
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