Keywords: cyclooxygenase-2, hydroxy-eicosatetraenoic acid, lipoxygenase, macrophage, circular dichroism

Materials

Arachidonic acid was purchased from NuChek Prep, Inc. (Elysian, MN), lipopolysaccharide (LPS) (serotype 0111:B4) was from Calbiochem, and RAW264.7 and CT26 cells were obtained from ATCC (Manassas, VA). 5S-HETE was prepared by chemical synthesis from arachidonic acid as described (13). 15R-HETE and 11R-HETE were prepared through vitamin E-controlled autoxidation of arachidonic acid methyl ester and purified by consecutive RP-, straight phase-, and chiral phase HPLC [Chiralpak AD (14)], and a final step of mild hydrolysis of the methyl ester using KOH.

Cell culture

RAW264.7 cells were cultured in DMEM and grown at 37°C in an atmosphere of 5% CO₂. Cells of passages 5 and 6 only were used. Cells were stimulated by treatment with 100 ng/ml LPS and 10 units/ml of IFN-γ for 6 h to induce expression of COX-2. CT26 cells were cultured in RPMI 1640 medium. 5S-HETE, 5 μg dissolved in 1 μl of ethanol, was added to ∼70% confluent cells in 100 mm dishes, and after 10 min at 37°C, the culture medium was removed, acidified to pH 4, and extracted using a 30 mg Waters HLB cartridge. Products were eluted from the cartridge with methanol, evaporated, and dissolved in 50 μl of LC-MS solvent A. CT26 and RAW264.7 control cells were not treated with LPS. In some experiments, CT26 and activated RAW264.7 cells were treated with 2 mM aspirin, respectively (from a 40 mM stock solution in DMSO), or with 10 μM NS-398 (from a 10 mM stock solution in ethanol) 30 min prior to incubation with 5S-HETE.

Reaction of recombinant COX-2 with 5S-HETE

The reaction of 5S-HETE (120 μg total; containing 300,000 cpm of [1-¹⁴C]5S-HETE) with recombinant human COX-2 was performed in four separate 2 ml reactions with 30 μg substrate each as described (12). The products were extracted using a Waters HLB cartridge and analyzed by RP-HPLC using a Waters Symmetry C18 5-μm column (4.6 × 250 mm) eluted with a gradient of acetonitrile/water/acetic acid programmed from 20/80/0.01 (by vol) to 70/30/0.01 (by vol) within 20 min at 1 ml/min flow rate. The elution profile was monitored using an Agilent 1200 diode array detector coupled on-line to a Packard Radiomatic A100 Flo-one radioactive detector. The by-products eluting at 19.8 and 20.5 min retention time were collected, extracted from HPLC solvent, and stored in methanol at −20°C until further analysis.

Reaction of aspirin-acetylated COX-2 with 5S-HETE

Recombinant human COX-2 (0.5 μM final concentration) was diluted in 1 ml of 100 mM Tris-HCl buffer pH 8.0 and treated with 2 mM aspirin in a 37°C water bath for 30 min (15). A control reaction incubated with arachidonic acid and analyzed by LC-MS before and after treatment showed >95% inhibition of PG formation. The buffer was supplemented with hematin (1 μM) and phenol (500 μM), and 30 μg of 5S-HETE were added. After 5 min at room temperature, 25 μl of methanol were added, the mixture was acidified to pH 4 with glacial acetic acid, and loaded onto a preconditioned Waters Oasis HLB cartridge. After washing with water, the products were eluted with methanol. The 5,15-diHETE product was isolated using RP-HPLC as described above for the native enzyme.

Synthesis and isolation of diHETE reference compounds

5S,15S-diHETE was synthesized by reaction of 5S-HETE with the LOX-1 isozyme from soybean seeds (16). To 3 ml of 100 mM K₂HPO₄ buffer pH 10 were added 100 μg of 5S-HETE and 1 μl of soybean lipoxygenase solution (∼25,000 units; Sigma). 5R,15S-diHETE was synthesized using 5R-HETE (100 μg) as substrate and 3 μl of soybean lipoxygenase solution. After 1 min reaction time, the solution was acidified (pH 4) and extracted with methylene chloride. The organic extract was evaporated, dissolved in methanol, and treated with 200 μg of triphenylphosphine (TPP) for 15 min at room temperature. 5S,15S-DiHETE and 5R,15S-diHETE were isolated by RP-HPLC using a Waters Symmetry C18 column (4.6 × 250 mm) eluted with a solvent of methanol/water/acetic acid (80/20/0.01, by vol) at 1 ml/min flow rate and UV detection at 235 nm.

5S,15R-DiHETE was synthesized by reaction of 15R-HETE with recombinant human 5-LOX. For the enzymatic transformation, a pellet ofSf9 insect cells expressing 5-LOX (∼300 μl) was sonicated and transferred to 1 ml of PBS containing 2 mM CaCl₂ and 1 mM ATP. 15R-HETE (50 μg) was added and the reaction was allowed to proceed for 15 min at room temperature. The reaction was terminated by the addition of 250 μl of methanol and 10 mg of NaBH₄. After 15 min at room temperature, the pH was adjusted to 3 using 1 N HCl, and the products were extracted using methylene chloride. 5S,15R-DiHETE was purified by RP-HPLC as described above for the 5S,15S-diastereomer.

5S,11R-DiHETE was synthesized by reaction of 5S-HETE with the recombinant linoleic acid 9R-LOX fromAnabaena sp. PCC7120 expressed inEscherichia coli, a gift from Alan R. Brash at Vanderbilt University (17). The substrate, 5S-HETE (100 μg), was added to 3 ml of 100 mM Tris-HCl pH 7.5 containing 150 mM NaCl and 0.01% CHAPS. The reaction was initiated by addition of 1 μl of the purifiedAnabaena 9R-LOX. After 3 min reaction time, the solution was acidified to pH 4 using 1 N HCl, and the products were extracted using a 30 mg Waters HLB cartridge and eluted with methanol. After evaporation of the solvent, the residue was dissolved in 100 μl of methanol, and the products were reduced with 150 μg TPP at room temperature for 15 min. 5S,11R-DiHETE was isolated using RP-HPLC conditions as described above for 5S,15S-diHETE. HPLC-purified 5S,11R-diHETE was dissolved in CDCl₃ for NMR analysis using a Bruker AV-II 600 MHz spectrometer equipped with a cryoprobe. Chemical shifts are reported relative to the signal for residual CHCl₃ at δ 7.25 ppm.

A mixture of 5,11-diHETE diastereomers was synthesized by autoxidation of racemic 11-HETE. Three 200 μg aliquots of 11-HETE were evaporated in small plastic tubes and placed in an oven at 37°C. After 2 h, the samples were dissolved in 50 μl of methanol, treated with triphenylphosphine (TPP), and analyzed using RP-HPLC. The diastereomers eluted as a single peak and purification was performed as described for 5S,15S-diHETE.

SP-HPLC analysis of diHETEs

The 5,15-diHETE diastereomers were resolved using an Agilent Zorbax RX-SIL 5-μm column (4.6 × 250 mm) eluted with hexane/isopropanol/acetic acid (95/5/0.1, by vol) at 1 ml/min flow rate. The 5,11-diHETEs were analyzed using the same HPLC conditions after conversion to the methyl ester derivatives with diazomethane. Eluting peaks were monitored using an Agilent 1200 series diode array detector.

CD spectroscopy

Aliquots of ∼20 μg each of 5S-HETE, 15S-HETE, 15R-HETE, 11S-HETE, 11R-HETE, and the enzymatically synthesized standards of 5S,15S-diHETE, 5R,15S-diHETE, and 5S,11R-diHETE were treated with ethereal diazomethane for 30 s, evaporated, and dissolved in 50 μl of dry acetonitrile. To the solution was added 1 μl of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and a few grains each of 4-dimethylaminopyridine (DMAP) and 2-naphthoylchloride. The reaction was carried out at room temperature overnight, the solvent was evaporated, and the residue was dissolved in methylene chloride and washed with water twice. Purification of the methyl ester, 2-naphthoyl derivatives of the HETEs and diHETEs was achieved by RP-HPLC using a Waters Symmetry C18 column (4.6 × 250 mm) eluted with a solvent of methanol/water/acetic acid (95/5/0.01, by vol) at 1 ml/min flow rate and UV detection at 235 nm. Samples were extracted from HPLC solvent using methylene chloride and dissolved in acetonitrile to an optical density (OD) of 0.75 absorbance unit (AU) for HETE derivatives and OD 1.5 AU for diHETE derivatives (when possible), respectively. Circular dichroism (CD) spectra were recorded using an Aviv Model 215 CD spectrometer at room temperature in a 1 cm pathlength cuvette scanning from 350–200 nm. The¹H NMR spectrum (600 MHz) of the 2-naphthoate derivatized 5S,11R-diHETE methyl ester was recorded in CD₃CN, δ 1.93 ppm.

GC-MS and LC-MS analysis

For GC-MS analysis, 5,15-diHETE and 5,11-diHETE formed by reaction of COX-2 with 5S-HETE were purified using RP- and SP-HPLC and methylated using ethereal diazomethane. Hydrogenation was performed in 100 μl of ethanol in the presence of palladium/carbon and bubbling with hydrogen gas for 5 min. Trimethylsilyl ethers were prepared usingbis(trimethylsilyl)trifluoroacetamide at room temperature for 1 h. The reagents were evaporated and the samples were dissolved in hexane. GC-MS analysis was carried out in the EI mode (70 eV) using a ThermoFinnigan DSQ mass spectrometer equipped with a 5 m SPB-1 column (0.1 mm i.d., film thickness 0.25 μm) and a temperature program from 100°C, hold 2 min, and then increased to 260°C at 20°C/min.

LC-MS was performed using a ThermoFinnigan Quantum Access instrument equipped with an electrospray interface and operated in the negative ion mode. User modified parameters of sheath and auxiliary gas pressures, temperature, and voltage settings were optimized using direct infusion of a solution of PGD₂. A Waters Symmetry Shield C18 3.5 μm-column (2.1 × 150 mm) was eluted with a linear gradient of acetonitrile/water, 10 mM NH₄OAc (5/95, by vol; solvent A) to acetonitrile/water, 10 mM NH₄OAc (95/5, by vol) at a flow rate of 0.2 ml/min within 10 min. Negative ion collision-induced dissociation (CID) mass spectra of the standards of PGD₂, 5S-HETE, 5S,15S-diHETE, and 5S,11R-diHETE were obtained. The fragmentation patterns were used to establish ion transitions for analyses in the selected reaction monitoring (SRM) mode. The following transitions were monitored: for PGD₂ and PGE₂:m/z 351 → 271; 5-HETE:m/z 319 → 115; 5,15-diHETE:m/z 335 → 201; and 5,11-diHETE:m/z 335 → 183. Relative levels of prostaglandins and diHETEs between treatments were calculated using peak areas of the signals in the SRM chromatograms.

Reaction of native and acetylated COX-2 with 5S-HETE

RP-HPLC analysis of the transformation of [1-¹⁴C]5S-HETE by recombinant COX-2 shows one main product that was identified previously as a highly oxygenated di-endoperoxide (12), in addition to two minor, less polar peaks designated I and II representing by-products of the reaction (Fig. 1A). When COX-2 was treated with aspirin prior to incubation with 5S-HETE, one major product (III) was formed with retention time similar to peak I in the untreated enzyme (Fig. 1B). Both I and III had a characteristic UV spectrum with a λmax at 243 nm that was readily identified as 5,15-diHETE (Fig. 1C) (18). The UV spectrum of peak II had a maximum at 238 nm with shoulders around 228 nm and 247 nm (Fig. 1C). The retention time and UV spectrum of II implicated that the product also contained two hydroxy groups and conjugated diene moieties.

The products I and II were isolated using RP-HPLC and further purified as the methyl ester derivatives using SP-HPLC. GC-MS analysis in the EI mode (70 eV) of the hydrogenated, TMS-ether derivatives confirmed the identification of the first peak I as 5,15-diHETE. Characteristic α-cleavage fragments were found atm/z 203 (55% relative intensity) andm/z 311 [after loss of O-trimethylsilyl (OTMS); 9%] for the 5-hydroxy, and atm/z 173 and 341 (after loss of OTMS) (56% and 7%, respectively) for the 15-hydroxy group; the base peak was atm/z 73. Peak III from the aspirin-acetylated COX-2 reaction was identified as 5,15-diHETE based on identical UV spectra and retention times on RP-HPLC, and in addition to subsequent experimental evidence as described below.

Product II gave a very weak [M⁺] (m/z 502) and [M-CH₃⁺] (m/z 487) ion, with characteristic α-cleavage fragments atm/z 203 (42%) andm/z 311 (after loss of OTMS; 4%) indicating a 5-hydroxy group, and atm/z 229 (38% relative intensity) andm/z 285 (after loss of OTMS) (5%) indicative of a 11-hydroxy group. The LC-ESI mass spectrum confirmed the molecular weight as 336 and also gave a major fragment atm/z 183 and a minor fragment atm/z 115, compatible with two hydroxyls at carbons 5 and 11 (Fig. 1D). Based on UV, GC-MS, and LC-MS analyses, product II was identified as 5,11-diHETE.

¹H NMR and H,H COSY data for product II were recorded using a chromatographically and spectroscopically (UV, LC-MS/MS) identical standard of 5S,11R-diHETE that was prepared as described below. The¹H NMR spectrum showed eight signals in the double bond region that appeared as a pair of two similar motives of four protons each comprised of the two conjugatedcis,trans-dienes (H7: δ 6.57 ppm, dd,J = 15.1 Hz/11.0 Hz; H8: δ 6.13, dd,J = 11.0; H6: δ 5.70, dd,J = 14.9 Hz/6.3 Hz, H9: δ 5.55, m; and H13: δ 6.51, dd,J = 14.9 Hz/11.4 Hz; H14: 5.96, dd,J = 11.0 Hz; H12: δ 5.67, m; H15: δ 5.46, m). Two protons attached to carbons bearing a hydroxyl group were located at 4.25 ppm (H11: δ 4.25, dt,J = 6.3 Hz/6.1 Hz) and 4.17 ppm (H5: δ 4.17, dt,J = 6.2 Hz/6.0Hz). H4 was detected as a cross-peak from H5 in the H,H-COSY spectrum at 1.57 ppm, H3 was a multiplet (1.70 ppm) and was coupled to the triplet signal of H2 at 2.34 ppm (J = 7.4 Hz). Both protons of H10 were detected as a multiplet at 2.47 ppm, and H16 was a dt signal at 2.17 ppm (J = 7.6 Hz/7.2 Hz).

The configuration of C-15 in the 5,15-diHETE products (I and III) and of C-11 in the 5,11-diHETE (II) was established by coelution with corresponding diHETE diastereomers of known configuration. The configuration of the 5-hydroxy group in all diHETE products was expected to be unchanged from the starting substrate, 5S-HETE.

Synthesis of standards of diastereomeric diHETEs

Table 1 gives an overview of the diHETE standards prepared as reference compounds. Authentic 5S,15S-diHETE was prepared by reaction of soybean LOX-1 with 5S-HETE. Synthesis of 5S,15R-diHETE by reaction of 15R-HETE with the recombinant human 5-LOX gave only a minor yield of product, albeit it was sufficient to determine the retention times on RP- and SP-HPLC. In addition, the enantiomer 5R,15S-diHETE was prepared by reaction of 5R-HETE with the lipoxygenase from soybean seeds. 5S,15R-diHETE and 5R,15S-diHETE have indistinguishable retention times on RP- and SP-HPLC.

An authentic standard of 5S,11R-diHETE was prepared by reaction of 5S-HETE with the recombinant 9R-LOX fromAnabaena sp PCC7120. A mixture of the 5,11-diHETE diastereomers was prepared by thin-film autoxidation of racemic 11-HETE. Initial attempts to prepare 5S,11S- and 5S,11R-diHETEs by reaction of 11S-HETE and 11R-HETE, respectively, with the recombinant human 5-LOX did not yield a significant amount of either 5,11-diHETE diastereomer. The assignment of the absolute configuration of the hydroxy groups in the diHETE standards was confirmed using CD spectroscopy (see below).

Absolute configuration of 5,15-diHETEs from native and acetylated COX-2

The diastereomers of 5,15-diHETE do not resolve on RP-HPLC (18), but there is adequate separation on SP-HPLC to allow for secure assignment of the absolute configuration at C-15. Therefore, the 5,15-diHETEs were first isolated as a single peak using RP-HPLC and then resolved using SP-HPLC (Fig. 2). The 5,15-diHETE (peak I) isolated form the reaction of human COX-2 gave a %-ratio for 5S,15S-diHETE to 5S,15R-diHETE of 77:33, 80:20, and 75:25 in three separate experiments (Fig. 2A). The authentic standards of 5S,15S-diHETE and 5S,15R-diHETE eluted at 12.3 min and 12.8 min retention times, respectively (Fig. 2B, 2C). Peak identification was further confirmed by cochromatography with the authentic standards (Fig. 2D). Aspirin-treatment of human COX-2 resulted in a shift of the chiral distribution of 5,15-diHETE, and now the product (peak III) was 95% 5S,15R-diHETE (Fig. 3).

Absolute configuration of 5,11-diHETEs

Standards for the 5S,11R- and 5S,11S-diHETE diastereomers were prepared by thin-film autoxidation of racemic 11-HETE followed by reduction with triphenylphosphine. 5,11-DiHETE was the almost exclusive diHETE formed, and the diastereomers eluted as a single peak when analyzed by RP-HPLC. Using SP-HPLC, satisfactory resolution of the methyl ester derivatives was achieved (Fig. 4). The first peak comprised of the 5S,11S- and 5R,11R-diastereomers eluted at 17.8 min and the second peak (5S,11R- and 5R,11S-diastereomers) eluted at 18.3 min. The authentic standard of 5S,11R-diHETE prepared using theAnabaena LOX coeluted with the second peak on SP-HPLC and established the elution order. SP-HPLC analysis of 5,11-diHETE from recombinant human COX-2 showed that the configuration was >98% 5S,11R-diHETE.

CD-spectroscopy of diHETE standards

We used the exciton-coupled circular dichroism method in order to confirm assignment of the absolute configuration of the hydroxy groups in the diHETE standards. This method uses the coupling of two chromophores attached to the chiral center in circular polarized light in order to determine the absolute configuration from the sign of the Cotton effects (CEs). One of the chromophores in the (di)HETEs is present as the conjugated diene system, and in order to introduce the second chromophore, the hydroxy groups were derivatized to the 2-naphthoate ester (19). Mono- and di-2-naphthoate derivatives, of the following methyl ester fatty acids were prepared, respectively: 5S-HETE, 11S-HETE, 11R-HETE, 15S-HETE, 15R-HETE, 5S,15S-diHETE, 5R,15S-diHETE, and 5S,11R-diHETE.

As expected, pairs of derivatized enantiomers, e.g., 15S-HETE and 15R-HETE, gave mirror-image CD spectra (Fig. 5). Furthermore, the CD spectra of allS-configuration HETEs (5S-HETE, 11S-HETE, and 15S-HETE) gave a positive first Cotton effect (i.e., the CE at the higher wavelength) and a negative second CE. Due to the additive nature of the absorbance in the CD spectrum, the two chiral centers in the diHETEs were expected to result in a CD spectrum that represents the mathematical sum of the spectra obtained for the two individual chiral centers. The spectrum of 5S,15S-diHETE showed increased intensities for the two CEs, although the Δϵ intensities were not doubled when compared with 15S-HETE, which was likely due to saturation effects at the high concentration measured (1.5 AU in the UV) (Fig. 5). In contrast, for the 5R,15S-diHETE diastereomer, the CEs cancelled each other out and the resulting CD spectrum was an almost flat line. The CD spectra of the diastereomeric 5,15- diHETEs confirmed the assignment of the absolute configuration of the two chiral centers in the 5S,15S-diHETE standard.

The configuration of C11 in 5S,11R-diHETE formed by reaction of theAnabaena 9-LOX with 5S-HETE was confirmed using the same approach. The individual CD spectra of 2-naphthoate-derivatized 5S-HETE and 11R-HETE are mirror images of each other, but unexpectedly, the CD spectrum of 5S,11R-diHETE was not a flat line (Fig. 6). The spectrum showed CEs at 245 nm (Δϵ +7.6) and 228 nm (Δϵ –6.7), resembling a weaker version of the CD spectrum of 5S-HETE with slightly shifted maxima. We hypothesized that the transition moment of the chromophores at C5 gave a stronger spectrum due to more optimal alignment of the two chromophores, thereby overcompensating the spectrum for the chiral center at C11.¹H NMR determination of theJ_5,6 andJ_11,12 coupling constants that can be taken as a measure for the alignment of the chromophores within the conformer, however, gave essentially equivalent values, i.e, 6.7 Hz and 6.8 Hz, respectively. Because SP-HPLC has confirmed the relative configuration of the 5S,11R-diHETE (Fig. 4D), the question why the corresponding CD spectrum showed slight predominance of theS-configurated chiral center remains unexplained.

Formation of diHETEs in RAW264.7 and CT26 cells

RAW264.7 were treated in four different ways and incubated with 4 μM 5S-HETE. We used nonstimulated cells, cells stimulated with LPS only, and LPS-stimulated cells treated with NS-398 or aspirin, respectively. Formation of diHETEs was analyzed using negative ion LC-ESI-MS in the SRM mode (Fig. 7A). Both 5,15-diHETE and 5,11-diHETE were detected in RAW264.7 cells activated with LPS and IFN-γ (Fig. 7A, upper panel), and their concentration was reduced to 0.5% and 3%, respectively, by incubation of the cells with the COX-2 inhibitor NS-398 (10 μM) prior to the addition of 5S-HETE (Fig. 7A, middle panel). Treatment of RAW264.7 cells with 2 mM aspirin led to about 60% and 50% reduction in 5,15-diHETE and 5,11-diHETE, respectively (Fig. 7A, lower panel). Levels of PGD₂ and PGE₂ were reduced to 4% and 30% by NS-398 and aspirin, respectively. PGD₂ and PGE₂ are not completely absent in the inhibitor treated cells because a fraction was formed from (endogenous) arachidonic substrate already before addition of the drugs. Furthermore, aspirin showed only modest efficacy in reducing eicosanoid formation, consistent with previous findings that a high cellular redox state in activated RAW264.7 cells impedes aspirin's ability to covalently modify COX enzymes (20).

Formation of diHETEs was also analyzed in CT26 mouse colon carcinoma cells incubated with 5S-HETE. CT26 cells incubated with 5S-HETE showed robust formation of 5,15-diHETE and 5,11-diHETE, and the levels of both were reduced >100-fold by preincubation with 10 μM NS-398 (Fig. 7B). Pretreatment with 2 mM aspirin inhibited the formation of 5,11-diHETE by about 90%, and 5,15-diHETE was reduced to only 23% compared with the cells not treated with aspirin, reflecting enhanced formation of 5S,15R-diHETE.

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