5-lipoxygenase-dependent biosynthesis of novel 20:4 n-3 metabolites with anti-inflammatory activity

Abstract

5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) into pro-inflammatory leukotrienes. N-3 PUFA like eicosapentaenoic acid are subject to a similar metabolism and are precursors of pro-resolving mediators. Stearidonic acid (18:4 n-3, SDA) is a plant source of n-3 PUFA that is elongated to 20:4 n-3, an analogue of AA. However, no 5-LO metabolites of 20:4 n-3 have been reported. In this study, control and 5-LO-expressing HEK293 cells were stimulated in the presence of 20:4 n-3. Metabolites were characterized by LC-MS/MS and their anti-inflammatory properties assessed using AA-induced autocrine neutrophil stimulation and leukotriene B₄-mediated chemotaxis. 8‑hydroxy‑9,11,14,17-eicosatetraenoic acid (Δ¹⁷-8-HETE) and 8,15-dihydroxy-9,11,13,17-eicosatetraenoic acid (Δ¹⁷-8,15-diHETE) were identified as novel metabolites. Δ¹⁷-8,15-diHETE production was inhibited by the leukotriene A₄ hydrolase inhibitor SC 57461A. Autocrine neutrophil leukotriene stimulation and neutrophil chemotaxis, both BLT1-dependent processes, were inhibited by Δ¹⁷-8,15-diHETE at low nM concentrations. These data support an anti-inflammatory role for Δ¹⁷-8,15-diHETE, a novel 5-LO product.

Keywords: AA, arachidonic acid; ADA, adenosine deaminase; ALA, alpha-linolenic acid; Abbreviations: 19-OH-PGB(2), 19(R)-hydroxy-prostaglandin B(2); BLT1, leukotriene B(4) receptor 1; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; ETA, eicosatetraenoic acid; Eicosanoids; EtOH, ethanol; HpETE, hydroperoxyeicosatetraenoic acid; Inflammation; LO, lipoxygenase; LTA(4), leukotriene A(4); LTB(4), leukotriene B(4); LTC(4), leukotriene C(4); Leukotrienes; Lipid mediators; MeOH, methanol; NEM, N-ethylmaleimide; Neutrophils; RP-HPLC, reverse phase-HPLC; SDA, stearidonic acid.