Abstract

A proposed beneficial impact of highly unsaturated “fish oil” fatty acids is their conversion by lipoxygenase (LOX) enzymes to specialized proresolving lipid mediators, including 12/15-LOX products from EPA and DHA. The transformations of DHA include formation of docosatrienes, named for the distinctive conjugated triene of the double bonds. To further the understanding of biosynthetic pathways and mechanisms, herein we meld together biosynthesis and NMR characterization of the unstable leukotriene A (LTA)-related epoxide intermediates formed by recombinant human 15-LOX-1, along with identification of the stable enzymatic products, and extend the findings into the 12/15-LOX metabolism in resident murine peritoneal macrophages. Oxygenation of EPA by 15-LOX-1 converts the initial 15S-hydroperoxide to 14S,15S-trans-epoxy-5Z,8Z,10E,12E,17Z-EPA (appearing as its 8,15-diol hydrolysis products) and mixtures of dihydroperoxy fatty acids, while mainly the epoxide hydrolysis products are evident in the murine cells. DHA also undergoes transformations to epoxides and dihydroperoxides by 15-LOX-1, resulting in a mixture of 10,17-dihydro(pero)xy derivatives (docosatrienes) and minor 7S,17S- and 14,17S-dihydroperoxides. The 10,17S-dihydroxy hydrolysis products of the LTA-related epoxide intermediate dominate the product profile in mouse macrophages, whereas (neuro)protectin D1, the leukotriene B4-related derivative with trans,trans,cis conjugated triene, was undetectable. In this study, we emphasize the utility of UV spectral characteristics for product identification, being diagnostic of the different double bond configurations and hydroxy fatty acid functionality versus hydroperoxide. LC-MS is not definitive for configurational isomers. Secure identification is based on chromatographic retention times, comparison with authentic standards, and the highly distinctive UV spectra.

Supplementary key wordsAbbreviations: 14S-HPDHA (14S-hydroperoxide-DHA), 15S-HPEPE (15S-hydroperoxide of EPA), 16,17-DTA6 (16,17-docosatriene A6 (analogue of LTA4), 16S,17S-trans-epoxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid), 17S-HPDHA (17S-hydroperoxide-DHA), LOX (lipoxygenase), LTA (leukotriene A), LTA4 (leukotriene A4), LTB4 (leukotriene B4), MaR1 (maresin R1), MeOH (methanol), NPD1 (neuroprotectin D1), PD1 (protectin D1), RP-HPLC (reversed-phase HPLC), SP-HPLC (straight-phase HPLC)The long-standing interest in the therapeutic benefits of dietary fish oil fatty acids—EPA and DHA—is extended in more recent times to the potential anti-inflammatory/proresolving effects of their lipoxygenase (LOX)-derived metabolites (1Treating inflammation and infection in the 21st century: new hints from decoding resolution mediators and mechanisms., 2Resolvins in inflammation: emergence of the pro-resolving superfamily of mediators.). Aside from direct effects of the omega-3 fatty acids themselves, the daily intake of high doses might act partly by diverting in vivo fatty acid metabolism away from the omega-6 arachidonic acid, thus decreasing production of proinflammatory prostaglandins and leukotrienes, the “bad guys” in controlling inflammation (3Consequences of essential fatty acids., 4Historical perspectives on the impact of n-3 and n-6 nutrients on health.). On the other hand, many studies appearing in the past two decades support the important roles played directly by potent local bioactive mediators derived from omega-3 PUFA. The series of molecules tagged as resolvins and protectins and “specialized proresolution mediators” has sparked great interest on account of their potent biological activities documented in the fields of inflammation, neuroscience, diabetes, and cancer (e.g., Refs. (1Treating inflammation and infection in the 21st century: new hints from decoding resolution mediators and mechanisms., 2Resolvins in inflammation: emergence of the pro-resolving superfamily of mediators., 5Bannenberg G. Serhan C.N. Specialized pro-resolving lipid mediators in the inflammatory response: an update., 6Claria J. Lopez-Vicario C. Rius B. Titos E. Pro-resolving actions of SPM in adipose tissue biology., 7Immunoresolvents signaling molecules at intersection between the brain and immune system., 8Fishbein A. Hammock B.D. Serhan C.N. Panigrahy D. Carcinogenesis: failure of resolution of inflammation.)).Modeled on the 5-LOX pathway of leukotriene biosynthesis and central to the production of the spectrum of the EPA- and DHA-derived metabolites is the production of an unstable leukotriene A (LTA)-related epoxide as a biosynthetic intermediate (e.g., Ref. (9Resolvins and protectins in inflammation resolution.)). On account of the extreme instability of LTA-related epoxides in neutral aqueous media, their biosynthesis is established primarily by analysis of their hydrolysis products; in aqueous media, the production of dihydroxy derivatives, and in “trapping” studies using an excess of alcohol to terminate reaction, the production of hydroxymethoxy derivatives (10Arachidonic acid metabolism in polymorphonuclear leukocytes: unstable intermediate in formation of dihydroxy acids.). Notwithstanding the technical challenges, leukotriene A4 (LTA4) itself has been isolated from leukocyte incubations (11Rådmark O. Malmsten C. Samuelsson B. Goto G. Marfat A. Corey E.J. Leukotriene A: isolation from human polymorphonuclear leukocytes.), and later, we prepared LTA4 and its 5,6-cis-epoxide analogue using recombinant 5-LOX enzyme (12Jin J. Zheng Y. Boeglin W.E. Brash A.R. Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration.). Herein, we applied the methodology using recombinant human 15-LOX-1 to preparation of the LTA-related epoxides from n-6 hydroperoxides of EPA and DHA, the latter and 22:5ω3 analogue also prepared by total chemical synthesis (13Aursnes M. Tungen J.E. Colas R.A. Vlasakov I. Dalli J. Serhan C.N. Hansen T.V. Synthesis of the 16S,17S-epoxyprotectin intermediate in the biosynthesis of protectins by human macrophages., 14Total synthesis of pro-resolving and tissue-regenerative protectin sulfido-conjugates., 15Vlasenko N. Glynn S. DeAngelo C.M. Lam T.F. Petasis N.A. Stereocontrolled total synthesis of the DHA-derived protectin-related epoxide and sulfido-conjugates., 16Primdahl K.G. Tungen J.E. De Souza P.R.S. Colas R.A. Dalli J. Hansen T.V. Vik A. Stereocontrolled synthesis and investigation of the biosynthetic transformations of 16(S), 17(S)-epoxy-PDn-3 (DPA).).Also modeled on the 5-LOX pathway is the reported hydrolysis of LTA-related epoxides in 15-LOX/DHA metabolism to a dihydroxy derivative exhibiting a cis,trans,trans conjugated triene chromophore (9Resolvins and protectins in inflammation resolution.). This is a special type of transformation that does not occur through nonenzymatic hydrolysis of LTA-related epoxides. In the leukotriene pathway, the epoxide LTA4 is enzymatically hydrolyzed to leukotriene B4 (LTB4), the prototypical lipid mediator with the unusual 5S,12R-dihydroxy configuration along with a cis,trans,trans conjugated triene (Scheme 1A). Reported analogous examples in 12/15-LOX metabolism of DHA are the conversions of LTA-related 16,17-trans-epoxy-DHA (designated herein as docosatriene A6) to protectin D1 (PD1), a 10R,17-diol with 11trans,13trans,15cis triene (Scheme 1B) (17Serhan C.N. Gotlinger K. Hong S. Lu Y. Siegelman J. Baer T. Yang R. Colgan S.P. Petasis N.A. Anti-inflammatory actions of neuroprotectin D1/protectin D1 and its natural stereoisomers: assignments of dihydroxy-containing docosatrienes.), and more recently, the production of maresin R1 (MaR1), formed from the analogous 13,14-epoxy intermediate as a 7R,14S-diol also with a trans,trans,cis triene chromophore (18Serhan C.N. Yang R. Martinod K. Kasuga K. Pillai P.S. Porter T.F. Oh S.F. Spite M. Maresins: novel macrophage mediators with potent antiinflammatory and proresolving actions., 19Serhan C.N. Dalli J. Karamnov S. Choi A. Park C.K. Xu Z.Z. Ji R.R. Zhu M. Petasis N.A. Macrophage proresolving mediator maresin 1 stimulates tissue regeneration and controls pain.).

Scheme 1Parallels in (A) LTB4 and (B) protectin D1 biosynthesis. LTB4, leukotriene B4.

From arachidonic acid, multiple 5,12-dihydroxy isomers of LTB4 can be formed, and by which criteria do we identify these biosynthetic products? We rely on distinctive HPLC retention times associated with the mass ions for 5,12-diols and on the characteristic UV spectra. Indeed, 40 years ago, the distinctive UV spectra of different isomers were established as central to the analysis (20Metabolism of arachidonic acid in polymorphonuclear leukocytes. Structural analysis of novel hydroxylated compounds., 21Borgeat P. Picard S. Vallerand P. Sirois P. Transformation of arachidonic acid in leukocytes. Isolation and structural analysis of a novel dihydroxy derivative.). With the EPA- and DHA-derived LOX products, quality UV spectra are reproducible and distinctive, and this criterion is among those used to identify products in the studies presented here. Herein, we meld together biosynthesis and NMR characterization of the LTA-related unstable epoxide intermediates, with analysis of their biosynthesis along with other enzymatic products by recombinant 15-LOX-1, and extend the findings into the metabolism in inflammatory cells. Most of these studies were completed as a PhD thesis (22Biosynthetic Mechanisms of LTA-Type Epoxides and Novel Bioactive Lipid Mediators.) and augmented more recently with the availability of synthetic PD1 and MaR1 as authentic standards.Materials and methods Materials

DHA and EPA were purchased from NuChek Prep, Inc (Elysian, MN). Soybean LOX-1 (lipoxidase, type V) was purchased from Sigma. C57BL/6NCrl mice (6–8 weeks) were purchased from Charles River Laboratories. Zymosan A was purchased from Sigma. We thank Professor Trond Hansen for providing a sample of synthetic PD1. PD1 and MaR1 were also purchased from Cayman Chemical.

 Synthesis and purification of fatty acid hydroperoxides

The 15S-hydroperoxide of EPA (15S-HPEPE) and 17S-hydroperoxide-DHA (17S-HPDHA) were prepared by the reaction of the fatty acids with the commercially available soybean LOX-1. 14S-hydroperoxide-DHA (14S-HPDHA) was prepared by the reaction of DHA with mouse platelet-type 12S-LOX. The purification of fatty acid hydroperoxides was achieved by straight-phase HPLC (SP-HPLC) using a solvent of hexane/isopropyl alcohol/glacial acetic acid, 100/1/0.02, by volume, and they were quantified by UV spectroscopy using an extinction coefficient of 25,000 M−1 cm−1 for the conjugated diene chromophore.

 Bacterial expression and purification of human 15-LOX-1

The cDNA of human 15-LOX-1 was subcloned into the pET3a vector (with an N-terminal His6 tag), and the protein was expressed in BL21 cells. A typical preparation of a 100 ml culture was carried out as follows: 100 ml of 2xYT medium containing 100 μg/ml ampicillin was inoculated with a single colony of human 15-LOX-1-His in BL21 cells and grown at 37°C at 250 rpm till an absorbance at 600 nm reached 0.8. IPTG (0.5 mM) was then added to the culture, which was grown at 16°C, 220 rpm for 4 days. On day 4, the cells were spun down at 5,000 g for 20 min in a Beckman Avanti J-25I centrifuge, washed with 40 ml of 50 mM Tris, pH 7.9, pelleted again at 5,000 g for 20 min, and resuspended in 10 ml of 50 mM Tris, pH 8.0, 500 mM NaCl, 20% glycerol, and 100 μM PMSF. The spheroplasts were then sonicated five times for 10 s using a model 50 Sonic Dismembrator (Fisher Scientific) at a setting of 5. CHAPS detergent was added at a final concentration of 1% (w/w), and the sample was kept on ice for 20 min. The resulting membranes were spun down at 5,000 g for 20 min at 4°C. The human 15-LOX-1 activity was present in the supernatant. The supernatant was loaded on a nickel-nitrilotriacetic acid column (0.5 ml bed volume; Qiagen) equilibrated with 50 mM Tris buffer, pH 8.0, and 500 mM NaCl. The column was then washed with the equilibration buffer, and the nonspecific bound proteins were eluted with 50 mM Tris buffer, pH 8.0, 500 mM NaCl, and 50 mM imidazole. The human 15-LOX-1 was then eluted with 50 mM Tris buffer, pH 8.0, 500 mM NaCl, and 250 mM imidazole. Fractions of 0.5 ml were collected and assayed for the LOX activity. The positive fractions were dialyzed against 50 mM Tris buffer, pH 7.5, and 150 mM NaCl. The purity of the enzyme preparations was determined by SDS-PAGE and Coomassie blue staining; the prominent band of h15-LOX-1 accounted for about 80% of the total protein.

 Biphasic reaction conditions for preparation of LTA-related epoxides

Enzyme reactions were performed at 0°C, with the fatty acid hydroperoxide substrate initially in hexane (5 ml, bubbled for 30 min prior to use with argon to decrease the O2 concentration, and containing ~200 μM substrate) layered over recombinant 15-LOX-1 enzyme (1–2 mg, ~20 nmol) in 400 μl of Tris buffer (pH 7.5 for h15-LOX-1). The reaction was initiated by vigorous vortex mixing of the two phases. After 1.5 min, the hexane phase was collected and scanned from 200 to 350 nm using a PerkinElmer Lambda-35 spectrophotometer. Then the hexane phase was evaporated to about 2 ml under a stream of nitrogen, treated with ethanol (20 μl) and ethereal diazomethane for 10 s at 0°C, and then rapidly blown to dryness and kept in hexane at −80°C until further analysis.

 Reactions of fatty acids and fatty acid hydroperoxides with recombinant 15-LOX-1

For analysis of polar products, incubations with recombinant 15-LOX-1 and fatty acids or fatty acid hydroperoxides (10 or 20 μg/ml) were conducted at room temperature in UV cuvettes in 1 ml 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl, with monitoring of the extent of the reaction by repetitive scanning (350–200 nm) in a Lambda 35 UV/Vis spectrometer (PerkinElmer). After 10–20 min and appearance of conjugated triene chromophores around 270 nm, reaction was halted by addition of 100 μl 1 M KH2PO4 buffer and 40 μl 1 N HCl to give approximately pH 3–4 and extracted using a 100 mg C18 Bond-Elut cartridge (Agilent), which was washed with water and eluted with methanol (MeOH).

 Mouse peritoneal resident macrophage incubationsMouse peritoneal resident macrophages were collected as described (23Heydeck D. Thomas L. Schnurr K. Trebus F. Thierfelder W.E. Ihle J.N. Kuhn H. Interleukin-4 and -13 induce upregulation of the murine macrophage 12/15-lipoxygenase activity: Evidence for the involvement of transcription factor STAT6.) by lavage from naive mice (6–8 week old C57BL/6NCrl mice; Charles River Laboratories). All animal studies were approved and performed in accordance with guidelines provided by the Vanderbilt Medical Standing Committee on Animals.

After centrifugation at 400 g and addition of DMEM + 10% FBS, macrophages (5 × 106 cells/ml) were incubated with zymosan A (200 μg/ml) and fatty acids or fatty acid hydroperoxides at 37°C for 30 min; the substrates were added in 2 μl ethanol per 0.5 ml cell incubation to give 20 μg/ml final concentration (equivalent to 60 μM 15S-HPEPE, 61 μM DHA, and 56 μM 17S-HPDHA). Incubations were stopped with two volumes of cold MeOH. After lysing with MeOH, the cell debris was removed by centrifugation at 10,000 rpm, the supernatant was mixed with three volumes of water, and the apparent pH was adjusted to approximately 3. The products were extracted on a 30 mg Oasis cartridge (Waters), eluted with MeOH, and analyzed by reversed-phase HPLC (RP-HPLC).

 HPLC analyses

From the biphasic reaction used for preparation of LTA-related epoxides, aliquots of the methylated hexane phase were analyzed by RP-HPLC using a Waters Symmetry column (25 × 0.46 cm), using a solvent of MeOH/20 mM triethylamine at pH 8.0 (90/10 by volume), at a flow rate of 1 ml/min, with on-line UV detection (Agilent 1100 series diode array detector with all 205, 220, 235, and 270 nm channels recorded at the same sensitivity). Further purification was achieved by SP-HPLC using a Beckmann Ultrasphere 5 μ silica column (25 × 0.46 cm) using a solvent of hexane/triethylamine (100/0.5) run at 1 ml/min. Aliquots of the room temperature incubation of recombinant human 15-LOX-1 with omega-3 fatty acid hydroperoxides were analyzed by RP-HPLC using a Waters Symmetry column (25 × 0.46 cm), using a solvent of acetonitrile (CH3CN)/H2O/HAc (45/55/0.01 by volume), at a flow rate of 1 ml/min. Aliquots of mouse macrophage incubations were analyzed by RP-HPLC using a Waters Symmetry column (15 × 0.21 cm), using a solvent of CH3CN/H2O/HAc (50/50/0.01 by volume), at a flow rate of 0.2 ml/min.

 LC-MS and GC-MS analyses

HPLC profiles were analyzed using a Thermo TSQ Vantage Triple Quadrapole MS instrument (Thermo Fisher Scientific, Waltham, MA). RP-HPLC analysis was performed with electrospray ionization in the negative ion mode. A Phenomenex Kinetex C18 2.6 μ column (100 × 3 mm) was eluted isocratically with CH3CN/water/glacial acetic acid (45:55:0.01 by volume) at a flow rate of 0.4 ml/min. The electrospray voltage was set at 4.0 kV: vaporizer temperature at 300°C; sheath and auxiliary gas pressure at 50 and 5 ψ, respectively; and capillary temperature at 300°C.

GC-MS was performed on hydroperoxy fatty acids by reduction with triphenylphosphine, SP-HPLC purification, methylation (diazomethane), hydrogenation (Pd/H2), silylation (N,O-bis(trimethylsilyl)trifluoroacetamide), and analysis on a Thermo-Finnigan DSQ mass spectrometer in the positive ion electron impact mode (70 eV).

 NMR analysis

1H NMR and 1H,1H COrrelated SpectroscopY NMR spectra were recorded on a Bruker AV-III 600 MHz spectrometer at 283 K. The parts/million values are reported relative to residual nondeuterated solvent (δ = 7.16 ppm for C6H6). Typically, 1,024 scans were acquired for a 1D spectrum on ~20 μg of LTA-related epoxide methyl ester.

Results Preparation, purification, and characterization of LTA-related epoxidesThe methodology, outlined in Fig. 1A, was optimized from biphasic reaction conditions originally developed for isolation of fatty acid allene oxides (24Gao B. Boeglin W.E. Zheng Y. Schneider C. Brash A.R. Evidence for an ionic intermediate in the transformation of fatty acid hydroperoxide by a catalase-related allene oxide synthase from the cyanobacterium Acaryochloris marina., 25Brash A.R. Boeglin W.E. Stec D.F. Voehler M. Schneider C. Cha J.K. Isolation and characterization of two geometric allene oxide isomers synthesized from 9S-hydroperoxylinoleic acid by cytochrome P450 CYP74C3: stereochemical assignment of natural fatty acid allene oxides.) and subsequently applied to LTA4 and its 5,6-cis-epoxy analogue (12Jin J. Zheng Y. Boeglin W.E. Brash A.R. Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration.). After vortex mixing of aqueous human 15-LOX-1 with a hexane solution of 15S-HPEPE or 17S-HPDHA at 0°C, UV spectroscopy of the hexane phase showed a decrease in substrate and appearance of a new chromophore with λmax at 280 nm characteristic of an LTA-type epoxide (Fig. 1B, C). Subsequent handling of the easily hydrolyzed epoxides was facilitated by prompt esterification to the methyl ester derivative using ethereal diazomethane and a trace of ethanol added to the hexane solvent. HPLC at room temperature with triethylamine in the running solvent allowed purification of the intact LTA-related methyl esters (see Materials and methods section and Fig. 2A–C).

Fig. 1Preparation and isolation of LTA-related epoxides. A: Overview of the biphasic synthesis and simultaneous extraction methodology. B: UV spectra of the hexane phase of 15S-HPEPE reaction with 15-LOX-1 before and after mixing with the enzyme at 0°C. C: The UV profiles before and after reaction of 17S-HPDHA with 15-LOX-1. 15-LOX-1, 15-lipoxygenase-1; 15S-HPEPE, 15S-hydroperoxide of EPA; 17S-HPDHA, 17S-hydroperoxide-DHA; LTA, leukotriene A.

Fig. 2HPLC purification of LTA-related methyl esters. A: SP-HPLC analysis of the products of 15-LOX-1 with 15S-HPEPE using a silica guard column (0.46 × 4.5 cm), a flow rate of 0.5 ml/min, and a solvent system of hexane/triethylamine (100/0.5, by volume) with UV detection at 270 nm. B: RP-HPLC analysis of 15-LOX-1 products with 17S-HPDHA. RP-HPLC analysis used a Waters Symmetry C18 column (0.46 × 25 cm), a flow rate of 1 ml/min, and a solvent system of methanol/20 mM triethylamine at pH 8.0 (90/10, by volume) with UV detection at 270 nm. C: Overlay of the UV spectra of the two epoxides in hexane/0.5% triethylamine solvent. The spectra are essentially indistinguishable, as expected from their identical molecular environments (26Characterization of HETEs and related hydroxy-dienes by UV spectroscopy.), which includes the nonconjugated cis double bond separated by a methylene group on the carboxyl side of the cis,trans,trans conjugated triene, and the epoxide functionality and then a methylene and extra cis double bond toward the fatty acid tail as illustrated at the top of panel C. Spectra were recorded online on HPLC using an Agilent 1200 series diode array detector with hexane/triethylamine 100/0.5 by volume as solvent. 15-LOX-1, 15-lipoxygenase-1; 15S-HPEPE, 15S-hydroperoxide of EPA; 17S-HPDHA, 17S-hydroperoxide-DHA; LTA, leukotriene A; RP-HPLC, reversed-phase HPLC; SP-HPLC, straight-phase HPLC.Proton NMR with COrrelated SpectroscopY spectra of the epoxide methyl esters in C6D6 permitted direct assignment of the structures of the enzymatic products (Fig. 3, Fig. 4). Key features established through these analyses include the cis,trans,trans configuration of the conjugated triene, confirmed from the coupling constants of the 8,10,12 double bonds of 14S,15S-trans-epoxy-5Z,8Z,10E,12E,17Z-EPA (14,15-LTA5) (from EPA) and the 10,12,14 bonds of 16,17-docosatriene A6 (analogue of LTA4), 16S,17S-trans-epoxy-4Z,7Z,10Z,12E,14E,19Z-DHA (16,17-DTA6) (from DHA). The trans-epoxide configurations are clearly indicated by the 2 Hz coupling constants between the epoxide protons (Figs. 3, 4), with the full 1H-NMR spectra summarized in supplemental Tables S1 and S2. These data directly establish the structures of these 15-LOX-1-derived epoxides: 14,15-LTA5 as 14S,15S-trans-epoxy-5Z,8Z,10E,12E,17Z-EPA, and 16,17-DTA6 as 16S,17S-trans-epoxy-4Z,7Z,10Z,12E,14E,19Z-DHA, with the data for the latter in accord with the synthetic epoxide (13Aursnes M. Tungen J.E. Colas R.A. Vlasakov I. Dalli J. Serhan C.N. Hansen T.V. Synthesis of the 16S,17S-epoxyprotectin intermediate in the biosynthesis of protectins by human macrophages., 14Total synthesis of pro-resolving and tissue-regenerative protectin sulfido-conjugates.).Fig. 3Proton NMR spectrum and COSY 2D spectrum of 14,15-LTA5 methyl ester. The COSY correlation spectrum allows assignment of the individual protons, and based on coupling constants, the expanded sections of the spectrum immediately below the structure establish the conjugated double bonds as 8cis,10trans,12trans and the epoxide configuration as 14,15-trans-epoxy (supplemental Table S1). 14,15-LTA5, 14S,15S-trans-epoxy-5Z,8Z,10E,12E,17Z-EPA; COSY, COrrelated SpectroscopY.Fig. 4Proton NMR spectrum and COSY 2D spectrum of 16,17-DTA6 methyl ester. The COSY correlation spectrum allows assignment of the individual protons, and based on coupling constants, the expanded sections of the spectrum immediately below the structure establish the conjugated double bonds as 10cis,12trans,14trans and the epoxide configuration as 16,17-trans-epoxy (supplemental Table S2). 16,17-DTA6, 16,17-docosatriene A6 (analogue of LTA4), 16S,17S-trans-epoxy-4Z,7Z,10Z,12E,14E,19Z-DHA; COSY, COrrelated SpectroscopY. RP-HPLC analysis of products from recombinant 15-LOX-1 and 15S-HPEPE

The biphasic reactions described previously recover the nonpolar LTA-related epoxides in hexane, a solvent that will not efficiently extract more polar products such as dihydroxy derivatives. These more polar and stable products were prepared in a conventional incubation of 15S-HPEPE with 15-LOX-1 in pH 7.4 buffer at room temperature and extracted on an Oasis cartridge and analyzed by RP-HPLC with diode array UV detection; wavelength profiles were recorded at 205, 220, 235, and 270 nm along with full UV spectra on all chromatographic peaks.

Figure 5 shows the profiles at 235 and 270 nm and the associated product identifications. The two equal-sized peaks eluting at approximately 11 min in the 270 nm channel represent the C-8 diastereomers of 8,15S-dihydroxy-5Z,9E,11E,13E,17Z-EPA, the major hydrolysis products of 14,15-LTA5 with their characteristic all-trans conjugated triene chromophore (20Metabolism of arachidonic acid in polymorphonuclear leukocytes. Structural analysis of novel hydroxylated compounds.). The other prominent product, eluting at 19 min, is an exact match in retention time and in its trans,cis,trans conjugated triene chromophore and 271 nm λmax with 8S,15S-dihydroperoxy-5Z,9E,11Z,13E,17Z-EPA prepared by double dioxygenation of EPA using soybean LOX-1 (cf. e.g., (27Van Os C.P.A. Rijke-Schilder G.P.M. Van Halbeek H. Verhagen J. Vliegenthart J.F.G. Double dioxygenation of arachidonic acid by soybean lipoxygenase-1. Kinetics and regio-stereo specificities of the reaction steps., 28Dobson E.P. Barrow C.J. Kralovec J.A. Adcock J.L. Controlled formation of mono- and dihydroxy-resolvins from EPA and DHA using soybean 15-lipoxygenase.)). In the CH3CN/water/acetic acid solvent system, hydroperoxy derivatives are well retained compared with their reduced hydroxy counterparts, and subsequent reanalysis of a reduced sample is associated with elution of the now-reduced products at shorter retention times (illustrated later for DHA dihydro(pero)xides).

Fig. 5Reversed-phase HPLC analysis of the reaction of 15S-HPEPE with human 15-LOX-1. Column: Waters Symmetry C18, 25 × 0.46 cm; solvent, CH3CN/H2O/HAc (45/55/0.01, by volume); flow rate, 1 ml/min, UV detection at 270 and 235 nm. 15-LOX-1, 15-lipoxygenase-1; 15S-HPEPE, 15S-hydroperoxide of EPA.

Minor products were identified as 12,15S-dihydroperoxy-5Z,8Z,10E,13E,17Z-EPA (with an unusual UV spectrum with the same profile as a standard of the 20:4 analogue, depicted in the supplement of Ref. (29Jin J. Zheng Y. Brash A.R. Demonstration of HNE-related aldehyde formation via lipoxygenase-catalyzed synthesis of a bis-allylic dihydroperoxide intermediate.)), and identification confirmed by GC-MS of the triphenylphosphine-reduced hydrogenated methyl ester TMS ether derivative with the expected α-cleavage ions m/z 341 (C1–C