Analysis of 12/15-lipoxygenase metabolism of EPA and DHA with special attention to authentication of docosatrienes

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 15 S -hydroperoxide to 14 S ,15 S - trans -epoxy-5 Z ,8 Z ,10 E ,12 E ,17 Z -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 7 S ,17 S - and 14,17 S -dihydroperoxides. The 10,17 S -dihydroxy hydrolysis products of the LTA-related epoxide intermediate dominate the product profile in mouse macrophages, whereas (neuro)protectin D1, the leukotriene B 4 -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.

Response to Incremental Replacement of Palm Oil with Fish Oil in Starter Diet on Growth Performance, Plasma Metabolites, Ruminal Fermentation, and Behavior of Dairy Calves

“…In line with this, the present study hypothesizes that reducing the n-6/n-3 ratio in SF by incremental levels of n-3 FAs will enhance energy intake, metabolic status, and growth performance in young calves. To achieve the reduction of the n-6/n-3 ratio, fish oil was employed, as it is not only a rich source of n-3 FAs but also contains eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), with immune-modulating effects that are absent in plant oils [18]. The increased availability of eicosanoids with inflammation-resolving properties is expected to enhance the anti-inflammatory capacity [1].…”

Effect of fish oil and canola oil supplementation on immunological parameters, feed intake, and growth of Holstein calves

“…Animal tissues contain high amounts of arachidonic acid in plasma membrane phospholipids and low amounts of EPA and DHA because diets generally include higher concentrations of n-6 and n-9 than n-3 FA. Both EPA and DHA are found in high concentrations in feeds of marine origin; consequently, fish oil (FO) is an excellent source of n-3 FA (Jin et al, 2021). This may lead to greater availability of eicosanoids with proresolving inflammatory properties (e.g.…”