Anne Barden, Ph.D. and Trevor Mori, Ph.D.

School of Medicine and Pharmacology, University of Western Australia

Epidemiological, clinical, and animal studies provide substantial support that the long chain ω-3 fatty acids from fish and fish oils, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are cardioprotective, particularly in the setting of secondary prevention.¹ Population studies have shown that ω-3 fatty acids have anti-arrhythmic effects that protect against sudden cardiac death. ω-3 Fatty acids benefit multiple cardiometabolic risk factors including blood pressure, vascular reactivity, arterial compliance, cardiac function, and lipid metabolism, as well as having antithrombotic, anti-oxidative, and anti-inflammatory actions.¹

EPA and DHA improve outcomes in a number of disease states associated with inflammation including rheumatoid arthritis, acute myocardial infarction, and heart failure.^1,2 The mechanisms by which ω-3 fatty acids exert their protection are still emerging but likely include alterations in cell membrane composition and effects on gene expression and receptors regulating signaling. The anti-inflammatory actions of ω-3 fatty acids are, in part, related to reduced leukocyte-derived cytokine formation and modulation of eicosanoid synthesis. For example, ω-3 fatty acid-derived 3-series prostanoids and 5-series leukotrienes are substantially less potent than the analogous 2-series metabolites formed from arachidonic acid.

The emergence of a family of lipid mediators derived from EPA and DHA, which actively resolve inflammation, offers another avenue by which ω-3 fatty acids may protect against conditions associated with inflammation. Resolution of inflammation is completed by specialized pro-resolving lipid mediators (SPMs) that are synthesized by transcellular processes involving enzymes of epithelial cells and leukocytes.³ SPMs derived from EPA and DHA are known as E-series and D-series resolvins, respectively. The synthesis of the E-series resolvins (RvE1, RvE2, and RvE3) from EPA involves acetylated COX-2 or cytochrome P450 monooxygenases and occurs via formation of an unstable hydroperoxy compound (18-HpEPE) leading to the intermediate 18-hydroxyeicosapentaenoic acid (18-HEPE) that is converted by 5-lipoxygenase (5-LO) to RvE1 or RvE2.⁴ In humans, RvE3 can also be generated from EPA by 15-LO (Figure 1).⁵ The stereochemistry of the E-series resolvins depends on whether initial synthesis involves acetylated COX-2 that predominantly produces 18R-HpEPE rather than 18S-HpEPE, leading to the 18R-E-series resolvins that are also known as aspirin-triggered E-series resolvins.

Figure 1. The biosynthesis of E-series resolvins from EPA and D-series resolvins, PD1, and maresins from DHA.

The D-series resolvins (RvD1, RvD2, RvD3, RvD4, RvD5, and RvD6) are synthesized from DHA by acetylated COX-2 or 15-LO. D-Series resolvin synthesis results via formation of 17-hydroperoxydocosahexaenoic acid (17-HpDHA) and 17-hydroxydocosahexaenoic acid (17-HDHA). The stereochemistry of these products is also enzyme dependent with the products of 15-LO leading to mainly 17S-HpDHA while acetylated COX-2 yields 17R-HpDHA.⁶ The action of 5-LO on 17-HDHA leads to the D-series resolvins RvD1-RvD6 that can differ in their stereochemistry at the 17 carbon position.⁷ In the absence of 5-LO, protectin D1 (PD1) and 10S,17S-DiHDHA are formed from 17-HpDHA. In humans, DHA can be metabolized by macrophage 12-LO to 14-HpDHA that leads to formation of 14-HDHA and the maresins (Figure 1).^8,9

The majority of studies examining the effects of SPMs in the resolution of inflammation have utilized cell cultures or animal models of disease. The actions of SPMs in disease models have been extensively reviewed and include controlling inflammatory pain (RvE1, RvD1, 17R-RvD1, RvD2, and MaR1), accelerating wound healing in diabetes (RvD1), inhibiting secondary thrombosis and necrosis in burn injury (RvD2), preventing colitis (RvE1, RvD1, and RvD2), protection against reperfusion injury in the heart (RvE1), and inhibiting kidney fibrosis (RvE1 and RvD1).³ The precursor to the D-series resolvins, 17-HDHA, is also biologically active, modulating macrophage function and alleviating experimental colitis and mediating B-cell differentiation to antibody secreting cells.^10,11

In humans, plasma is the most readily available blood component for examination of SPM levels. Levels of SPMs in plasma can be measured using targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) that requires authentic SPM standards and an appropriate internal standard for identification and quantification. Our group was the first to describe in detail an LC-MS/MS method for measuring levels of 18-HEPE, 17-HDHA, RvD1, 17R-RvD1 RvD2, 10S,17S-DiHDHA, and PD1 in human plasma.¹² In this report, we examined the effect of different blood collection methods on levels of SPMs. The levels of 18-HEPE, 17-HDHA, RvD1, 17R-RvD1, and RvD2 were all measurable in plasma from healthy humans after three weeks of ω-3 fatty acid supplements and ranged between 20 and 200 pg/ml.¹²

In a follow-up study, healthy volunteers were given ω-3 fatty acid supplements for seven days and then randomized to receive aspirin or placebo in addition to ω-3 fatty acids during the last two days.¹³ A number of SPMs were measured at baseline, after five days of ω-3 fatty acids, and after seven days (i.e., after two days of aspirin in addition to ω-3 fatty acids). The SPMs measured included 18-HEPE, RvE1, RvE2, RvE3, and 18R-RvE3 derived from EPA and 17-HDHA, RvD1, 17R-RvD1, RvD2, PD1, 14-HDHA, and MaR1 that are derived from DHA. Chiral chromatography was used to separate and quantify the concentration of different epimers of 18-HEPE and 17-HDHA before and after aspirin. The study showed that, at baseline, the plasma concentration of 14-HDHA was 3-fold higher than the other SPMs. ω-3 Fatty acid supplementation for five days increased plasma levels of 18-HEPE, 17-HDHA, 14-HDHA, and RvE1. Aspirin taken in addition to ω-3 fatty acids did not differentially affect any SPM.¹³ However, the ratio of R- to S-isomers of 17-HDHA, but not 18-HEPE, was significantly reduced by aspirin.

The changes in SPMs with ω-3 fatty acids are not confined to healthy humans. We reported plasma SPMs in 74 patients with chronic renal disease randomized to ω-3 fatty acids or coenzyme Q10 (CoQ) for eight weeks.¹⁴ ω-3 Fatty acids significantly increased plasma levels of 18-HEPE, 17-HDHA, and RvD1 (Figure 2). CoQ had no effect on any plasma SPM. In regression analysis, the increase in 18-HEPE and 17-HDHA following ω-3 fatty acids was predicted by the change in platelet EPA and DHA, respectively.

Figure 2. Post-intervention concentrations of plasma 18-HEPE, 17-HDHA, and RvD1 in patients with chronic kidney disease who received no ω-3 fatty acids (NO ω-3 FA) or were supplemented with ω-3 fatty acids (ω-3 FA) for eight weeks. †p<0.001 or *p<0.05 for between group differences after adjusting for baseline SPMs

Our studies consistently show that ω-3 fatty acid supplementation can elevate plasma levels of the E- and D-series resolvins and their precursors 18-HEPE and 17-HDHA, respectively, as well as 14-HDHA, the upstream metabolite of the maresin family. After ω-3 fatty acid supplementation, many of the SPMs in plasma are present at concentrations shown to have biological activity. Given that SPMs are regarded as autocoids released at the sites of inflammation, one would anticipate the levels measured at the inflammatory site would be even higher than that in blood. The pro-resolving actions of ω-3 fatty acids after supplementation add further support to the overall attenuation of inflammation after an insult, and are likely important in the overall concept of the protective effects of ω-3 fatty acids.

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