Go to research.

Methods

Endogenous generation of electrophilic omega-3 PUFAs and their hydroxy precursors was evaluated by mass spectrometry in neutrophils isolated from healthy subjects, both at baseline and upon stimulation with calcium ionophore. For the clinical trial, participants were healthy adults 30–55 years of age with a reported EPA+DHA consumption of ≤300 mg/day randomly assigned to parallel groups receiving daily oil capsule supplements for a period of 4 months containing either 1.4 g of EPA+DHA (active condition, n = 24) or identical appearing soybean oil (control condition, n = 21). Participants and laboratory technicians remained blinded to treatment assignments.

Results

5-lypoxygenase-dependent endogenous generation of 7-oxo-DHA, 7-oxo-DPA and 5-oxo-EPA and their hydroxy precursors is reported in human neutrophils stimulated with calcium ionophore and phorbol 12-myristate 13-acetate (PMA). Dietary EPA+DHA supplementation significantly increased the formation of 7-oxo-DHA and 5-oxo-EPA, with no significant modulation of arachidonic acid (AA) metabolite levels.

Introduction

Herein, the endogenous formation of electrophilic α,β-unsaturated ketone derivatives of DHA, EPA and DPA is reported in neutrophils isolated from healthy subjects. Specifically, 5-LO-dependent generation of 7-oxo-DHA, 7-oxo-DPA, 5-oxoEPA and their hydroxy precursors is reported. Moreover, the effects of EPA and DHA consumption at a clinically-relevant dose (1.4 g/day) on the production of these ω-3 PUFA ketone derivatives was evaluated in healthy human subjects participating in a randomized, placebo-controlled clinical trial. There was a significant increase in levels of electrophilic ω-3 PUFA ketone derivatives in neutrophils under both basal and activated conditions, with no significant change in AA-derived metabolites. It is proposed that these reactive products transduce the salutary actions of ω-3 PUFAs by modifying the activity of electrophile-sensitive transcription factors that regulate the expression of inflammatory and metabolic-related genes. This report follows CONSORT guidelines for clinical trials .

Abstract

Dietary ω-3 polyunsaturated fatty acids (PUFAs) decrease cardiovascular risk via suppression of inflammation. The generation of electrophilic α,β-unsaturated ketone derivatives of the ω-3 PUFAs docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) in activated human macrophages is catalyzed by cyclooxygenase-2 (Cox-2). These derivatives are potent pleiotropic anti-inflammatory signaling mediators that act via mechanisms including the activation of Nrf2-dependent phase 2 gene expression and suppression of pro-inflammatory NF-κB-driven gene expression. Herein, the endogenous generation of ω-3 PUFAs electrophilic ketone derivatives and their hydroxy precursors was evaluated in human neutrophils. In addition, their dietary modulation was assessed through a randomized clinical trial.

Introduction

Among oxygenated PUFAs, electrophilic α,β-unsaturated ketone derivatives are of interest because they a) are end-products of hydroxylated fatty acid metabolism, b) display reversible electrophilic reactivity with nucleophilic protein targets via Michael addition reaction and c) modulate the activity of critical inflammatory and host defense signaling pathways . In this regard, electrophilic PUFAs activate the Nrf2-dependent anti-oxidant response, are partial agonists of the nuclear lipid receptor PPARγ, and suppress pro-inflammatory NF-κB signaling at multiple levels , –. Recently, a method has been developed for the mass spectrometric detection and quantification of electrophilic fatty acids, based on the use of the β-mercaptoethanol (BME) as nucleophilic bait . Its use in profiling electrophilic fatty acid generation by activated macrophages led to the discovery of Cox-2-dependent generation of 13- and 17-oxo-derivatives of docosahexaenoic (DHA) and docosapentaenoic acid (DPA). These electrophilic ketone derivatives mediate pleitropic anti-inflammatory actions via modulation of transcriptional regulatory protein function thus representing a new class of endogenous lipid mediators.