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.
Highlights
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
Reported analogous examples in 12/15-LOX metabolism of DHA are the conversions of leukotriene A (LTA)-related 16,17-trans-epoxy-DHA to protectin D1 (PD1), a 10R,17-diol with 11trans,13trans,15cis triene (Scheme 1B) [17], and more recently, the production of maresin R1 (MaR1), formed from the analogous 13,14epoxy intermediate as a 7R,14S-diol with a trans,trans,cis triene chromophore [18, 19]
From the biphasic reaction used for preparation of LTArelated epoxides, aliquots of the methylated hexane phase were analyzed by reversed-phase HPLC (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)
Summary
DHA and EPA were purchased from NuChek Prep, Inc (Elysian, MN). Soybean LOX-1 (lipoxidase, type V) was purchased from Sigma. From the biphasic reaction used for preparation of LTArelated 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). 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. 1,024 scans were acquired for a 1D spectrum on ~20 μg of LTA-related epoxide methyl ester
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